EPA 600/R-13/145 ( September 2013 | www.epa.gov/ord
United States
Environmental Protection
Agency
Technical Report
Field Study on Cleaning a Rendering
Plant Following a Foreign Animal
Disease (FAD) Outbreak
DARLIHG
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2 pooled samples
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8 pooled NTC
Office of Research and Development
National Homeland Security Research Center
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
Technical Report
U.S. Environmental Protection Agency
Field Study on Cleaning a Rendering Plant Following a
Foreign Animal Disease (FAD) Outbreak
U.S. Environmental Protection Agency
Office of Emergency Management
CBRN Consequence Management Advisory Team
Research Triangle Park, NC
National Homeland Security Research Center
Decontamination and Consequence Management Division
Research Triangle Park, NC
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
NOTICE
The U.S. Environmental Protection Agency (EPA), through its Office of Emergency
Management's (OEM's) Consequence Management Advisory Team (CMAT) and Office of
Research and Development's (ORD's) National Homeland Security Research Center (NHSRC),
directed and managed this work through Contract Number EP-W-12-026, Task Order TO-02-
011 with Dynamac Corporation. Funding for this work came through Interagency Agreement
RW-12-92306101 with the U.S. Department of Agriculture. This report has been subjected to
the Agency's administrative review and approved for publication. The views expressed in this
report are those of the authors and do not necessarily reflect the views or policies of the
Agency. Mention of trade names or commercial products does not constitute endorsement or
recommendation for use.
Questions concerning this document or its application should be addressed to:
Paul Lemieux
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Mail Code E343-06
Research Triangle Park, NC 27711
919-541-0962
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
EXECUTIVE SUMMARY
Test Objectives
A study was conducted to evaluate cleanup of a rendering plant after its use for disposal in
response to a Foreign Animal Disease (FAD) outbreak. The intent of this study was to develop
recommended procedures that could be used to aid in returning a rendering plant to normal
operation after use in support of an actual FAD incident.
This effort attempted to achieve three objectives:
• To generate data on fugitive emissions of a biological surrogate during the rendering
process;
• To determine the effectiveness of existing plant cleaning procedures for reducing the levels
of surrogate on the inside surfaces of the rendering facility; and
• To provide information that could be used to develop standard procedures for appropriately
clearing a rendering facility that had been used for "disposal rendering" after an FAD
outbreak, as part of returning the rendering facility back to its normal production use.
The Test Team conducted several sampling events at the Darling International (Darling)
Rendering Plant located in Des Moines, Iowa, which included:
• Acquiring a series of opportunistic swab samples at the first plant visit to gain an initial
insight into the culturable bacterial flora present in the plant;
• Acquiring a series of wipe samples at various locations in the plant to get a more detailed
evaluation of background culturable bacterial flora present in the plant;
• Performing an initial sampling effort to focus on potential biological surrogates to use for the
Cleaning/Inoculation study;
• Performing a series of laboratory spike tests involving potential biological surrogates in
idealized rendering plant sampling matrices and sampling media for air and wipe samples.
Based on the results of this and all previous testing, biological and nonbiological surrogates
were selected for the Cleaning/Inoculation study; and
• Performing a Cleaning/Inoculation study at the rendering plant to evaluate the movement of
the surrogates within the rendering process and subsequent plant cleaning procedures.
Although use of a thermophilic bacterium such as Geobacillus stearothermophilus as a
surrogate was originally desired, a lack of ability to positively identify G. stearothermophilus
using molecular microbiological techniques led the Test Team to select a nonthermophilic
organism. Based on the initial tests, an inoculum was selected for the Cleaning/Inoculation
study that was a mixture of 1E9 colony forming units (CFU) of Bacillus atrophaeus (aka Bacillus
globigii or Bg) spores and 1.47E9 beads of Polylactic-Co-Glycolic Acid (PLGA) fluorescent
microspheres, with an additional surfactant "Fluid D" per gallon of inoculum.
Over a series of weekends, the rendering plant was cleaned using cleaning procedures
normally utilized by the plant. Following the plant pre-cleaning, the Cleaning/Inoculation study
was then conducted in October of 2011.
Test Conclusions
The following conclusions were drawn from the Cleaning/Inoculation study:
• The results of the Cleaning/Inoculation study indicated that no Bg deoxyribonucleic acid
(DNA) was detected in any of the sample extracts from the post-inoculation or post-cleaning
surface wipes or from the air samples using various polymerase chain reaction (PCR)
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
techniques. A significant amount of additional effort was devoted to extracting Bg DNA from
the samples, including the use of alternate means to amplify the Bg DNA and achieve
detection. This additional effort was unsuccessful. Although Bg was possibly present in low
concentrations and below the limit of detection by quantitative PCR (qPCR), nondetection by
qPCR was very possibly due to inhibitors present in the sample matrices that carried over
during the extraction process. This hypothesis was formulated because putative Bg was
recoverable on brain heart infusion agar (BHIA) using nonmolecular microbiological
techniques and because Bg DNA could be extracted from, and detected in, spiked positive
controls of pristine gauze and filter matrices, as well as idealized materials similar to
rendering plant sample matrices (i.e., suet, grease, and deionized [Dl] water).
• Due to problems with extracting the PLGA microspheres from the sample matrix (both gauze
wipes and air filters), PLGA might not be a suitable synthetic surrogate, as the microspheres
become permanently immobilized in these sampling matrices. Extraction processes were
ineffective at removing PLGA microspheres for quantitation by fluorometer. In addition,
autofluorescence from the rendering plant sample matrices (e.g., grease, flesh, bone
materials) complicated detection of PLGA microspheres via direct microscopic observation.
Other PLGA microspheres with different colors that may not autofluoresce at the same
wavelength as the rendering sample matrices may be available. There were two issues with
the PLGA microspheres: immobilization on sampling materials and detection interference
caused by rendering materials. Other sampling matrices may possibly yield better results
with PLGA microspheres.
• Both PLGA and PCR analysis of rendering matrices proved to be difficult. Strides were
certainly made to help identify which analysis methods might work better to overcome
interferences such as hair, grease, and bone fragments. However, questions linger about
qualitative and quantitative analysis of rendering plant samples in the future. In addition,
this study raised questions concerning identification and use of a suitable surrogate and the
materials that would be necessary to acquire and analyze samples from an environment
containing considerable background biological microbes.
• Using nonmolecular microbiological culture techniques, viable bacteria very similar to the Bg
positive control colony morphology were recovered from eleven of the test sample extracts
(five contained putative Bg in quantities greater than the limit of quantitation [LOQ]).
• Based on results obtained from nonmolecular biological culture techniques, routine plant
cleaning procedures may potentially result in an approximately 1-log reduction in pathogen
loading within the potentially contaminated areas of the plant. This result is consistent with
results from previous systematic studies examining the effectiveness of different steps of a
multi-step cleaning/disinfection process that showed a 1-4 log reduction from individual
cleaning/disinfection steps. The plant cleaning procedures used in this study utilized hot
water and steam, a combination that would have been expected to remove contamination
from surfaces and transfer any removed contamination into the rinse water going into the
drains, as opposed to actually killing any surrogate organisms that would have existed in the
rinsate. Hot water would not have killed the surrogate spores used in these tests, but may
possibly kill some FAD viral agents.
• The cleaning process using the steam and hot water has the potential to spread the
contaminant throughout the plant, even if the cleaning process results in a reduction in the
overall levels of contamination. It is not entirely clear as to whether this dispersion of
contaminant is the result of plant personnel tracking the surrogate to various locations within
the plant or due to aerosol transport. High pressure spraying operations have been shown
to result in aerosol transport of spores. However, no air samples exhibited any Bg, either
through PCR analysis or by examination of colony morphology.
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
This study highlights the need for analytical methods that are compatible with the matrices
found in rendering facilities.
Recommendations for Future Rendering Plant Sampling/Analytical
Efforts
The information that was obtained from this study leads to many questions about the sampling
and analysis of the rendering plant matrices and air samples. The study revealed that more
work should be done to determine how to sample in a rendering facility environment and to
analyze the resulting extracts.
• Both wipe samples and swabs were used for sampling in this study because of the harsh
environment (i.e., rough, grimy surfaces) of a rendering plant. Swab samples were
negatively impacted by the rough surfaces in a rendering environment, and a single large
particle could potentially bias a swab sample. While wipe samples certainly could collect
more material, the amount of material collected by a wipe could require multiple dilutions
during the biological analysis portion of the study. Also, the materials used in wipe samples
interfered with the identification of the PLGA microspheres; i.e., PLGA microspheres
became permanently immobilized in the sampling matrices.
• Sample dilution might be a better alternative for these sample matrices, or a more desirable
solution for the end users, but testing would be necessary to determine the optimal dilution
to overcome PCR inhibition without significant loss of target DNA. However, dilution comes
at the expense of sensitivity; it is not clear whether a different/additional purification step
would be more advantageous than dilution of the inhibitor.
• Newer DNA extraction methods that have recently been developed have shown promise in
the ability to extract DNA from complex matrices and may be useful to test on rendering
samples.
• Due to the difficulty of extracting Bg DNA from the sample matrices coupled with the
success of using nonmolecular microbiological techniques to identify putative Bg colonies on
heat-shocked samples, the initial desire for a thermophilic bacterium (e.g., G.
stearothermophilus) to use as a potential biological surrogate for rendering plant studies
should be revisited. Results of this study as well as a subsequent literature review indicated
that further work on G. stearothermophilus may require construction of Geobacillus genus-
specific (GEOBAC) primers specific to the Geobacillus genus based on internal transcribed
spacer (ITS) sequences.
• Given that many FADs of interest are viral in nature, development of methods to extract viral
DNA from rendering plant matrices may be necessary to show that there is no residual viral
loading in the plant following cleaning procedures or at least that viral loading is below levels
pre-determined by the Incident Commander.
• The results of the analyses indicated that PLGA microspheres may not be a suitable
synthetic surrogate. The microspheres appear to become immobilized in the sampling
matrices, and the particles autofluoresce at a wavelength similar to hair and bone
fragments. This behavior makes it difficult to distinguish the PLGA spheres from
background. Also, the extraction processes were ineffective at removing PLGA
microspheres for quantitation by fluorometer, and autofluorescence from the sample
matrices complicated detection of PLGA microspheres via direct microscopic observation.
Other variants of the PLGA microspheres may exist that neither autofluoresce at the same
wavelengths as the sampling matrices nor become immobilized in the wipe gauze or air filter
materials.
Based on the results of the sampling and methods development work that has been done, an
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
ideal surrogate for use in the field test does not appear to exist. Tradeoffs must be taken into
account and a balance struck to pick the best available surrogate given the amount of
information that is currently available.
Recommendations for Developing Plant Cleaning Procedures
Following Use for Disposal Rendering as Part of an FAD Response
The results from this study suggest that the development of standard operating guidelines to
address the cleaning of a rendering plant following its use for disposal of animal carcasses as
part of an FAD response would include several distinct steps, with precautions being taken to
minimize movement of contamination. Due to the size of a typical rendering plant, the diversity
of process equipment in the plant, and the level of dirt and grime on many plant surfaces, it is
unlikely that fumigation would be recommended for the plant decontamination without data first
becoming available to assess decontamination efficacy and potential equipment damage in a
rendering plant environment. Until data on fumigation of a rendering plant scenario become
available, procedures using surface cleaning and subsequent disinfection may, therefore, be the
most appropriate means to restore a rendering plant to normal operation following its use in an
FAD response.
The purpose of this study was not specifically to develop the cleaning guidelines, but to develop
information that could be used by the rendering industry and agricultural emergency response
authorities to develop guidelines that could be used to restore a rendering plant to normal
operation following its use in an FAD response.
The following suggestions are offered for inclusion in plant cleaning guidelines:
• Due to the size and diversity of materials of construction in and around the rendering plant
and its various process units, as well as the nature of plant operations, there are abundant
opportunities to result in the buildup of a potentially significant quantity of dirt, grime, grease,
and organic matter on many plant surfaces. This buildup is likely to occur over a period of
time significantly longer than the time that the plant would likely be used for disposal
rendering. Subsequent cleaning operations following the use of the plant for disposal
rendering would be greatly facilitated if the plant were to be cleaned prior to being used for
disposal rendering. This prior cleaning may present a logistical challenge due to the lead
time associated with bringing in a commercial cleaning operation. However, removal of
accumulated grime, dirt, and organic matter prior to potentially contaminating the plant with
an FAD pathogen may greatly simplify later cleaning and decontamination operations.
• Due to the potential for transport of contamination throughout the plant due to the activity of
the plant personnel, establishing contaminant control procedures for plant workers prior to
delivery of any contaminated materials to the plant may be very important. Contaminant
control procedures may include such considerations as:
• Establishing egress pathways for workers to pass from areas of lower likelihood of
contamination to areas of higher likelihood of contamination;
• Dividing work duties and shift schedules so that workers performing activities in areas of
lower likelihood of contamination do not enter areas of higher likelihood of
contamination;
• Establishing procedures for donning and doffing clothing and personal protective
equipment (PPE) to minimize contaminant spread; and
• Using aerosol containment equipment (e.g., tent) at the grinding operation where the
most post-inoculation putative positive surrogate samples were observed.
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• Due to the potential for cleaning operations to spread contamination around the plant to
areas that may previously not have been contaminated, a multi-step (possibly three distinct
steps) cleaning/decontamination process, done in a systematic approach with runoff control,
appears to be the most effective way to clean the plant for restoration to normal operation.
Initial cleaning steps may include such activities as low pressure washing, steam cleaning,
and brushing. Minimization of the use of high pressure washing may minimize aerosol
transport of potential contaminants.
• The multi-step cleaning/decontamination process might be a three-step process that starts
with cleaning only the potentially most heavily contaminated portions of the plant, rather
than the entire plant. This initial cleaning might focus on removal of organic matter,
particularly on the tipping floor, in the feed hopper, the grinder, and on the auger ramps that
lead into the cooker, along with the walls and floors in those areas of the plant. This initial
cleaning should be staged to move the potentially contaminated materials eventually into the
cooker or the drains, such as by cleaning in the following sequence:
• Tipping floor area walls;
• Tipping floor;
• Feed hopper;
• Grinder; and
• Augers and ramps.
• During this initial cleaning operation, plant personnel movement from the areas being
cleaned to other plant areas that may not be as contaminated should be minimized.
• Utilizing the cooker where possible to process potentially contaminated materials may
minimize further contamination of the areas outside the plant.
• Where the cooker cannot be used to process potentially contaminated materials, the
remainder may be diverted into the drains, so that runoff can be collected and treated
separately.
• Once the heaviest loading of organic matter has been removed from the surfaces in the
areas of the plant that have the highest likelihood of contamination (i.e., tipping floor,
grinder, feed augers), subsequent cleaning operations should be initiated. These
subsequent cleaning steps may include a second pass through the entire plant using steam,
detergents, and low pressure spraying of water, with special attention being given to the
drain areas, where rendering material may accumulate. A final cleaning step that involves
the use of disinfectants that have been registered for use with the FAD organism of interest
would then be performed.
• Water and other runoff that is collected in the drains should be treated to kill the FAD
pathogen prior to discharge. This step is likely to vary significantly from rendering plant to
rendering plant and may require concurrence by permitting authorities who regulate water
discharges from the plant.
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
TABLE OF CONTENTS
NOTICE ii
EXECUTIVE SUMMARY iii
TABLE OF CONTENTS viii
ACRONYMS AND ABBREVIATIONS xi
ACKNOWLEDGMENTS xiv
1. INTRODUCTION 1
1.1 Introduction 1
1.2 Plant Description 2
2. EXPERIMENTAL PROCEDURES: SELECTION OF SURROGATE FOR
CLEANING/INOCULATION FIELD TEST 5
2.1 Initial Site Visit and Preliminary Scoping Samples 5
2.1.1 Purpose and Description 5
2.1.2 Results 5
2.2 Initial Plant Sampling 5
2.2.1 Purpose and Description 5
2.2.2 Results 8
2.3 Initial Surrogate Evaluation 8
2.3.1 Purpose and Description 8
2.3.2 Results 2
2.4 Preliminary Selection of Surrogates 4
2.5 Rendering Matrix Challenge Testing 4
2.5.1 Purpose and Description 4
2.5.2 Results 5
2.5.3 Significance of Challenge Test Results 5
2.6 Final Surrogate Selection 6
3. EXPERIMENTAL PROCEDURES: CLEANING/INOCULATION FIELD TEST 7
3.1 Test Design/Planned Approach 7
3.2 Sampling Procedures and Protocols 10
3.2.1 Background Sampling 11
3.2.2 Inoculation Phase Sampling 11
3.2.3 Post-Inoculation Phase Sampling 12
3.2.4 Post-Cleaning Phase Sampling 13
3.3 Inoculation of Incoming Raw Materials 13
3.4 Plant Cleaning 14
3.5 Analytical Procedures and Protocols 15
3.5.1 Bg Detection by Quantitative PCR 17
3.5.2 Detection of PLGA Microspheres 18
3.5.3 Enumeration of Putative Viable Bg in Archived Samples 19
3.5.4 Identification of Background Microflora by Sequence Analysis 20
4. RESULTS 25
4.1 Bg Detection by Quantitative PCR 25
4.2 Detection of PLGA Microspheres 32
4.3 Enumeration of Putative Viable Bg in Archived Samples 32
4.4 Identification of Background Microbial Flora by Sequence Analysis 39
4.4.1 Extraction of DNA 39
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
4.4.2 Amplification of 16S rRNA 39
4.4.3 Sequencing of 16S rRNA 40
4.5 Summary of Results 48
5. QUALITY ASSURANCE/QUALITY CONTROL 51
5.1 Experimental Approach 51
5.2 Sampling Approach 51
5.2.1 Wipe Sampling 51
5.2.2 Air Sampling 52
5.3 Timeline of Events for Study 52
5.3.1 Background Sampling 52
5.3.2 Carcasses Inoculated with PLGA and Bg 52
5.3.3 Process Sampling 53
5.3.4 Inoculation Phase and Process Sampling 53
5.3.5 Post-Inoculation and Process Sampling 53
5.3.6 Plant Cleaning After Inoculation and Process Sampling 53
5.3.7 Post-Cleaning Sampling 53
5.3.8 Grinder Study Sampling 54
5.4 Analytical Procedures 54
5.5 Results from Positive and Negative Control Samples 55
6. CONCLUSIONS 56
7. RECOMMENDATIONS 59
7.1 Recommendations for Future Rendering Plant Sampling/Analytical Efforts 59
7.2 Recommendations for Developing Plant Cleaning Procedures Following Use of
the Plant for Disposal Rendering as Part of an FAD Response 60
8. REFERENCES 62
APPENDIX A: Clemson Report from Initial Plant Sampling
APPENDIX B: Battelle Report
APPENDIX C: Photolog of Tests
APPENDIX D: Sample Chain of Custody Sheets
APPENDIX E: Formulation of Fluid D
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
List of Figures
Figure 1. Photograph of Rendering Plant Test Site 3
Figure 2. Conceptual Diagram of Dry Rendering Process 4
Figure 3. Sampling Map for the June 15, 2010, Initial Plant Sampling 6
Figure 4. Post-Inoculation Sampling Locations 9
Figure 5. Post-Cleaning Study Sampling Locations 10
Figure 6. Areas Cleaned at the Darling Des Moines plant 16
Figure 7. Gel Electrophoresis of AIR-10-21 -11 Samples Analyzed by PCR on the ABI 9700
Thermocycler 31
Figure 8. KRONA Visualization of BLAST® Results for Pool 2 45
Figure 9. KRONA Visualization of BLAST® Results for Pool 3 46
Figure 10. KRONA Visualization of BLAST® Results for Pool 4 47
Figure 11. KRONA Visualization of BLAST® Results for Pool 5 48
Figure 12. Locations of Putative Bg Colonies Before and After Cleaning 50
List of Tables
Table 1. Summary Table of Testing 2
Table 2. Samples Collected During Initial Plant Sampling Activities 7
Table 3. Results from First Isolation Attempt 8
Table 4. Results from Second Isolation Attempt 2
Table 5. Summary of the October 18-20, 2010, Environmental Surface Swab Sampling
Results from Darling International, Inc., Rendering Plant 2
Table 6. Species Identified during June and October 2010 Sampling Events 3
Table 7. Timeline of Events for the Cleaning/Inoculation Portion of Study 8
Table 8. Summary of Samples Collected in the Background, Inoculation, Post-Inoculation,
and Post-Cleaning Phases 11
Table 9. Weights of Inoculated Trucks 14
Table 10. Plant Cleaning Schedule 15
Table 11. Pooled Sample Extracts for Phire® Animal Tissue Direct PCR Kit 19
Table 12. 16S rRNA Primer Sequences 21
Table 13 Applied Biosystems 3130 Genetic Analyzer Run Parameters 22
Table 14. Results of Bg qPCR Analyses 26
Table 15. Microscopic Observations of PLGA Microspheres 33
Table 16. Enumeration of Putative Bg Colonies in Sample Extracts 41
Table 17. Samples Containing Colony Morphologies Similar to Bg 42
Table 18. Results of 16S rRNA Sequencing Based on BLAST® and QUEST™ Analysis 43
Table 19. Summary of the Sampling and Analytical Procedures 52
Table 20. Results of Bg qPCR Analyses of Positive Controls 55
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
ACRONYMS AND ABBREVIATIONS
°c
Degree(s) Celsius
°F
Degree(s) Fahrenheit
ABI
Applied Biosystems, Inc.
APHIS
(USDA) Animal and Plant Health Inspection Service
ATCC
American Type Culture Collection
Bg
Bacillus atrophaeus aka Bacillus globigii
BHI
Brain Heart Infusion
BHIA
Brain Heart Infusion Agar
BLASTn
Nucleotide Basic Local Alignment Search Tool
bp
Base Pair(s)
CBRN
Chemical, Biological, Radiological, and Nuclear
CFU
Colony Forming Unit(s)
cm
Centimeter(s)
CMAT
(EPA) Consequence Management Advisory Team
Ct
Cycle Threshold
DATS
(EPA) Decontamination Analytical and Technical Services contract
Dl
Deionized
DNA
Deoxyribonucleic Acid
dNTP
Deoxy ribonucleotide
EDTA
Ethylenediaminetetraacetic acid
EPA
U.S. Environmental Protection Agency
FAD
Foreign Animal Disease
ft
Foot/Feet
g
Gram(s)
gal
Gallon(s)
GC
Gene Copies
GEOBAC
Geobacillus genus-specific primers
HF
High Fidelity
hr
Hour(s)
hsDNA
Herring Sperm Carrier DNA
ID
Identification(s)
in
Inch(es)
IPC
Internal Positive Control
ISPs
Ion Sphere Particles
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ITS
Internal Transcribed Spacer
LOD
Limit of Detection
LOQ
Limit of Quantitation
Lpm
Liter(s) per minute
MC
Multicomponent
MCE
Mixed Cellulose Ester
mg
Milligram(s)
min
Minute(s)
mL
Milliliter(s)
mm
Millimeter(s)
M9
Microgram(s)
ml
Microliter(s)
|jm
Micrometer(s)
NCBI
National Institute of Health's National Center for Bioinformatics
ND
No Data
ng
Nanogram(s)
nk
Number of voltage ramp steps to reach Run Voltage
nm
Nanometer(s)
NTC
No Template Control
NHSRC
(EPA) National Homeland Security Research Center
NRF
National Response Framework
OEM
(EPA) Office of Emergency Management
ORD
(EPA) Office of Research and Development
PBS
Phosphate Buffered Saline
PC
Positive Control(s)
PCR
Polymerase Chain Reaction
PLGA
Polylactic-Co-Glycolic Acid
PPE
Personal Protective Equipment
PVA
Polyvinyl Alcohol
QAPP
Quality Assurance Project Plan
QA/QC
Quality Assurance/Quality Control
QN
Qiagen Neat
qPCR
Quantitative PCR
QV
Quality Value
R&D
Research and Development
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R'
Statistical correlation coefficient
rRNA
Ribosomal Ribonucleic Acid
rtp
Replication Termination Protein
RTP
Research Triangle Park
RT-PCR
Real-time Polymerase Chain Reaction
SDS
Sodium Dodecyl Sulfate
sec
Second(s)
TAE
Buffer solution containing a mixture of Tris base, acetic acid and EDTA
TBD
To Be Determined
TE
Tris ethylenediaminetetraacetic acid
TSA
Tryptic Soy Agar
USDA
U.S. Department of Agriculture
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ACKNOWLEDGMENTS
The author would like to acknowledge a number of people who played a key role in this
research effort. Test Team members Paul Lemieux, Joe Wood and M. Worth Calfee of
EPA/NHSRC and Leroy Mickelsen of EPA/CMAT helped develop the test protocols and
supported the testing and writing of this final report. This work was performed through Contract
Number EP-W-12-026, Task Order TO-02-011 with Dynamac Corporation. Funding for this
work came through Interagency Agreement RW-12-92306101 with the U.S. Department of
Agriculture. Lori Miller of USDA/Animal and Plant Health Inspection Service (APHIS) provided
the initial impetus to perform this study and provided much needed advice and support. Dr.
Annel Greene of Clemson University provided valuable advice on sampling and analytical
methods and provided laboratory support for analysis for the initial plant sampling phase of the
project. Some analytical work was performed by Battelle under a subcontract to Dynamac
Corporation. We would like to offer thanks to David Meeker of the National Renderer's
Association and Ross Hamilton and David Kirstein of Darling International for providing advice
and facilitating access to the rendering plant used for the field test. Mike Johnson, the plant
manager, deserves special acknowledgment for his hospitality in accommodating the Test
Team over the week of testing. Acknowledgments are also given to Anna Tschursin of EPA's
Office of Resource Conservation and Recovery, Wendy Davis-Hoover of EPA/ORD, and Lori
Miller of USDA/APHIS for providing review comments on the report and to Joan Bursey of the
Senior Environmental Employment (SEE) Program for her tenacity in doing the technical editing.
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1. INTRODUCTION
1.1 Introduction
Rendering is one of the technologies that could potentially be used to dispose of large numbers
of animal carcasses generated during a response to a foreign animal disease (FAD) outbreak
[1], There are currently approximately 300 rendering facilities in North America [2], However,
guidance is not available on restoring a rendering plant to normal operation following its use for
disposal as part of an FAD incident response. Therefore, in collaboration with the U.S.
Department of Agriculture's Animal and Plant Health Inspection Service (USDA/APHIS), Darling
International, Inc., and the National Renderer's Association, the U.S. Environmental Protection
Agency (EPA) conducted a study to evaluate fugitive emissions of a biological agent surrogate
released from a rendering process and subsequent cleanup procedures. For this project, the
Test Team (composed of personnel from EPA's National Homeland Security Research Center
(NHSRC), EPA's Office of Emergency Management's (OEM's ) Chemical, Biological,
Radiological, and Nuclear (CBRN) Consequence Management Advisory Team (CMAT) agreed
upon several objectives:
• To generate data on fugitive emissions of a biological surrogate from the rendering process;
• To determine the effectiveness of plant cleaning procedures for reducing the surrogate
levels on the inside surfaces of the rendering facility; and
• To provide information that could be used to develop standard procedures for appropriately
cleaning of a rendering facility that has been used for "disposal rendering" after an FAD
outbreak so that the rendering facility can be returned to normal production.
Note that at this point, cleanup goals were not identified; this initial effort was intended to identify
potential cleanup approaches and sampling strategies to use.
Environmental characterization, decontamination, and clearance are critical components of a
comprehensive public health recovery strategy in the aftermath of an FAD incident or intentional
release of a biological agent. Rendering plants could play a critical role in the nation's response
to an FAD event by assisting in the control of diseases and providing a mechanism to recycle
usable animal carcasses to safe and usable products. The National Response Framework
(NRF) [3] and the Food Safety Modernization Act [4] require multiagency participation and
identify USDA as the lead agency for carcass disposal with the EPA as a support agency.
As one step towards addressing the process for returning a rendering plant to normal operation,
the EPA, USDA/APHIS, and the rendering industry are working together to evaluate potential
cleanup approaches. The evaluation process includes characterizing the baseline biological
footprint of a rendering plant, determining a biological surrogate, performing pre-release and
post-release sampling, cleaning/decontaminating the rendering facility, and performing post-
decontamination sampling. The EPA's CMAT and NHSRC conducted a study to evaluate the
potential for cleaning a rendering plant following its potential use for disposal of contaminated
animal carcasses in response to an FAD outbreak. This study consisted of several distinct
components spread out over 2010 and 2011. To conduct this study, several test events
occurred at the Darling International (Darling) Rendering Plant located in Des Moines, Iowa.
Table 1 lists the various study-related events, dates on which they occurred, and the purpose of
that particular component of the study. Appendix C contains a photographic log of the activities
for these events.
1
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
Table 1. Summary Table of Testing
Study Event
Date(s)
Purpose
Selection of Surrogate for Field
Tests
Initial site visit and preliminary
scoping samples
January 6, 2010
To view the test site and to collect a limited
number of opportunistic surface swab samples
for the purpose of planning the tests
Initial Plant Sampling
June 15,2010
To collect background surface samples for the
purposes of identification of background flora
and initial surrogate candidates
Initial Surrogate Evaluation
October 18-20, 2010
To perform a systematic sampling effort to
identify appropriate surrogate(s) for field tests
Preliminary Selection of Surrogates
December 2010
Based on initial sampling, identify likely
surrogate(s) to use for later field tests
Rendering Matrix Challenge Testing
August 2011
Assess recovery of proposed surrogates
Bacillus atrophaeus (Bg) and polylactic-co-
glycolic acid (PLGA) from model rendering
plant matrices
Final Selection of Surrogates
August 2011
Make final decision on surrogates to use for
Cleaning/Inoculation Study
Cleaning/Inoculation Study
Plant pre-cleaning
September - October 2011
Remove bulk loading of organic material from
plant surfaces
Background Sampling
October 2011
Sample specific locations (surfaces and air) in
the rendering plant for initial concentrations of
the surrogates
Inoculation Phase Sampling
October 2011
Inoculate incoming trucks loaded with animal
carcasses with Bg spores and PLGA
microspheres. Air sampling occurred during
this stage of the test.
Post-Inoculation Phase Sampling
October 2011
Sample specific locations (surfaces and air) in
the rendering plant for the surrogates
Plant Cleaning
October 2011
Clean rendering plant using existing plant
cleaning procedures
Post-Cleaning Phase Sampling
October 2011
Sample specific locations (surfaces and air) in
the rendering plant for the surrogates
1.2 Plant Description
The rendering facility selected for this study was the Darling International, Inc., (Darling)
rendering plant located at 601 SE 18th Street, Des Moines, Iowa (Figure 1). The Darling
rendering plant processes "animal by-product materials for the production of tallow, grease, and
high-protein meat and bone meal" [5], Raw materials such as animal by-product materials,
animal carcasses, grease, feathers, offal, and blood are collected from a variety of commercial
locations including butcher shops, supermarkets, poultry processors, slaughterhouses, farms,
ranches, and feedlots. From these raw materials, the Darling rendering plant produces products
that are used in livestock and poultry feed, soap, inedible tallow, and grease.
2
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
DARLING
Figure 1. Photograph of Rendering Plant Test Site
The Darling rendering plant uses a dry rendering process to produce animal feed ingredients,
biodiesel feedstocks, and other non-food products [5] from animal carcasses and food animal
slaughter offal. The process involves the use of steam to cook the raw material and accomplish
separation of the fat (Figure 2). Dry rendering is a batch or continuous process that dehydrates
raw material to release fat. Following dehydration in batch or continuous cookers, the melted fat
and protein solids are separated as final products.
3
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
Meat &
Bone
Meal
Trucks
of Raw
Material
Wastewater
Discharge
Tallow
Air Emissions
Grinder
Cooker
Fan
Scrubbers
Separator
Raw
Material
Holding
Bin
Condenser
Figure 2. Conceptual Diagram of Dry Rendering Process
4
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
2. EXPERIMENTAL PROCEDURES: SELECTION OF SURROGATE
FOR CLEANING/INOCULATION FIELD TEST
2.1 Initial Site Visit and Preliminary Scoping Samples
2.1.1 Purpose and Description
On January 6, 2010, the Test Team toured the Darling International plant. Sampling was
initially not planned at the plant tour, and wetting solution was not available, but opportunistic
swab samples were acquired from plant surfaces to provide background information that could
be used for the Initial Plant Sampling effort. During the visit, a total of six opportunistic swab
samples were collected by EPA personnel. Each swab sample was collected from a 10
centimeter (cm) by 10 cm area with a dry, unsterilized swab and placed in a nonsterile
resealable plastic bag. Samples were logged on a facility map, pictures were taken of the
sample locations, and the time/date of sampling was recorded. The swabs were stored in the
swab container and placed in a Ziploc® bag. Test team members from Research Triangle Park,
NC (RTP), retained custody and carried the samples back to RTP with them. All samples were
streak-plated at the EPA Office of Research and Development's (ORD's) laboratory in RTP onto
two Tryptic Soy Agar (TSA) plates. One plate for each sample was incubated at 35 degrees
Celsius (°C) and the other at 55 °C for 24 hours (hr) to obtain an identification of bacteria that
were present.
2.1.2 Results
Results indicated no growth (zero colony forming units [CFU]) on all but two of the samples.
The two samples with growth were collected from the auger leaving the receiving floor and from
the carcass entry door (both incubated at 35 and 55 °C) near where the trucks of raw material
deposit their load prior to the carcasses being placed in the holding bin (see Figure 2).
2.2 Initial Plant Sampling
2.2.1 Purpose and Description
Because of the anticipation of seeing a rich collection of bacterial flora in all of the rendering
plant samples, prior to performing tests on effectiveness of plant cleanup activities prior to and
after inoculation, it was necessary to identify an appropriate surrogate organism or material to
use for the field testing. The surrogate(s) to be used should have the following characteristics:
• Not be present in the background flora of the plant;
• Be able to be identified in the matrices of interest in the rendering plant (dead animals, meat
and bone meal, tallow, wastewater); and
• Be able to be separated analytically from the probable high levels of background bacterial
flora in the rendering plant samples.
On June 15, 2010, after Test Team personnel gave plant personnel necessary sampling
supplies, materials, and directions on how to take the samples, rendering plant personnel
collected environmental surface samples at the Darling rendering plant. Figure 3 illustrates the
sample locations. The samples were collected using sterile swabs moistened with either Amies
(Liquid Amies, Single Swab, BD Diagnostics #220093; purchased from VWR Scientific,
Suwannee, GA, USA - VWR #90001-036) or Stuart's (Liquid Stuart, Single Swab, BD
Diagnostics #220099; purchased from VWR Scientific, Suwannee, GA - VWR #90001-040)
transport media. Odd-numbered samples were collected using Amies transfer media, and
even-numbered samples were collected using Stuart's transfer media to evaluate the efficacy of
each medium. Plant personnel shipped the samples on ice to Clemson University for analysis
5
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
(See Appendix A for the complete report from Clemson). Table 2 summarizes the samples
collected during the Initial Plant Sampling event.
LOCKER
& REST
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OFFICE
& LUNCH
ROOM
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, BOILERS
.BOILER
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TRAMS
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. lB1
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! LOAD-OUT
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O ©©
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FiESHUIS
ft TANKS
RESTR
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00 0
CONCRETE
SCALE (IN FEET)
0 25 50 75 100
l^LJ
legend
Swab sample •
Wastewater S«mpf<
t
VAPOR CONOEMSER
Figure 3. Sampling Map for the June 15, 2010, Initial Plant Sampling
6
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
Table 2. Samples Collected During Initial Plant Sampling Activities
Sample
Number
General Location
Description
Matrix
Measurement*
Total
Samples
1a, 1b
Raw receiving floor
area #1
Swab of surfaces
Polymerase chain reaction
(PCR)/deoxyribonucleic acid
(DNA) Sequencing (a) &
Culture/Enumeration (b)
Facility -
24
2a, 2b
Raw receiving floor
area #2
Swab of nonporous surfaces
PCR/DNA Sequencing (a) &
Culture/Enumeration (b)
3a, 3b
Pit area
Swab of nonporous surfaces
PCR/DNA Sequencing (a) &
Culture/Enumeration (b)
4a, 4b
Pit Area
Swab of nonporous surfaces
PCR/DNA Sequencing (a) &
Culture/Enumeration (b)
5a, 5b
Sump Area
Swab of nonporous surfaces
PCR/DNA Sequencing (a) &
Culture/Enumeration (b)
6a, 6b
Raw Material Incline
Area
Swab of nonporous surfaces
PCR/DNA Sequencing (a) &
Culture/Enumeration (b)
7a, 7b
Raw Grinder Area
Swab of nonporous surfaces
PCR/DNA Sequencing (a) &
Culture/Enumeration (b)
8a, 8b
Tallow Tanks/Dryer
Area
Swab of nonporous surfaces
PCR/DNA Sequencing (a) &
Culture/Enumeration (b)
9a, 9b
Load Out Screw (North
End)
Swab of nonporous surfaces
PCR/DNA Sequencing (a) &
Culture/Enumeration (b)
10a, 10b
Crax Grinder Area
Swab of nonporous surfaces
PCR/DNA Sequencing (a) &
Culture/Enumeration (b)
11a, 11b
Crax Storage Bin Area
Swab of nonporous surfaces
PCR/DNA Sequencing (a) &
Culture/Enumeration (b)
12a, 12b
Tailgate of Truck in
Receiving Bay
Swab of nonporous surfaces
PCR/DNA Sequencing (a) &
Culture/Enumeration (b)
13a, 13b
Wastewater from Raw
Pit Sump
Liquid
PCR/DNA Sequencing (a) &
Culture/Enumeration (b)
2
14a
through
16b
Laboratory Blanks
Agar blank, diluent blank, and
swab blank
PCR/DNA Sequencing (a) &
Culture/Enumeration (b)
6
17a, 17b
Field Blank
Swab prepared in field as a
sample
PCR/DNA Sequencing (a) &
Culture/Enumeration (b)
2
18
Positive Control (PCR /
DNA sequencing)
Pure culture of G.
stearothermophilus
PCR/Sequencing
1
19
Positive Control (swab
spike)
Swab spiked with 1E4 CFU G.
stearothermophilus
Culture/Enumeration
1
20
Positive Control
(extraction buffer spike)
Extraction buffer spiked with
1E4 CFU G. stearothermophilus
Culture/Enumeration
1
* See Appendix A for details on analytical procedures.
The swab samples were used to inoculate Brain Heart Infusion (BHI) broth tubes. The BHI
tubes were incubated overnight at 35 °C and 55 °C. The 55 °C pre-enrichment broth cultures
were streaked for isolation on BHI agar (BHIA) and incubated overnight at 55 °C. Growth was
detected at 55 °C on 28 of the 32 collected samples. The 35 °C pre-enrichment broth cultures
7
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
were streaked for isolation on BHIA and incubated overnight at 35 °C. At 35 °C, copious growth
was detected on all 32 samples. From the streak plates incubated at 55 °C, 32 pure cultures
were isolated on BHIA slants which were incubated at 55 °C. Five additional 55 °C plates
contained prolific spreader organisms which were not isolated during this study. PCR analysis
was conducted on the 32 isolated cultures to amplify the 16S ribosomal ribonucleic acid (rRNA)
gene from the bacterial isolates. Bacterial identity was selected from the top 25 BLASTn
database results with maximum identity greater than 90%. Gram reaction and morphological
characteristics were utilized to confirm the identity of bacterial isolates.
2.2.2 Results
In the initial experiment, only 14 isolates were successfully amplified and submitted for
sequencing. Results from this set of isolates are shown in Table 3.
Table 3. Results from First Isolation Attempt
11. Tepidiphilus sp. or
Petrobacter sp. 83%
12. Tepidiphilus margaritifer
99%
13. Aneurinibacillus
thermoaerophilus 91%
1.
B. licheniformis 90%
6.
No result
returned
2.
B. licheniformis 81%
7.
No result
returned
3.
B. licheniformis 88%
8.
No result
returned
4.
No result returned
9.
No result
returned
5.
No result returned
10.
No result
returned
14.
Aneurinibacillus thermoaerophilus 91%
In the second isolation attempt, 72 isolates were obtained. Many of these isolates were deemed likely
duplicates based on Gram stain and morphology. After amplifying, these 72 isolates were submitted
with four positive controls in duplicate (eight in total). The positive controls were American Type
Culture Collection (ATCC) 7953 Geobacillus stearothermophilus, ATCC 12980 G. stearothermophilus,
ATCC 12978 G. stearothermophilus, and SPORTROL* Spore Suspensions, NAMSA (VWR Scientific
Products, Inc., # 19872-024). Results from this set of isolates are shown in Table 4.
Bacterial identification results using PCR and amplicon sequencing indicated lack of sensitivity of the
procedure to identification of G. stearothermophilus. Only 37.5% of the positive controls were
successfully identified as G. stearothermophilus by the procedure. Results of this study as well as a
subsequent literature review indicated that further work on G. stearothermophilus may require
construction of Geobacillus genus-specific (GEOBAC) primers specific to the Geobacillus genus
based on Internal Transcribed Spacer (ITS) sequences [6],
2.3 Initial Surrogate Evaluation
2.3.1 Purpose and Description
A second more systematic sampling effort was then undertaken, using the information gathered
during the Initial Plant Sampling Event, in an effort to focus on an appropriate surrogate organism.
On October 18-20th, 2010, 26 samples were collected (twenty-four swab and two wastewater
samples) from 13 areas of the Darling plant. Two swab samples were collected from adjacent areas
at 12 sample locations that included the receiving floor, hard surfaces, grinders, and crax area. One
of the swabs was used for community characterization (PCR/DNA sequencing), and the other swab
was used for bacterial enumeration via dilution plating. The two wastewater samples were collected
from the wastewater (from scrubber discharge) collection sump near the equalizing tanks. Four
positive controls in duplicate (eight total) were also sent to the laboratory for analysis. See Section
3.5 for a description of the analytical procedures that were used.
8
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
Table 4. Results from Second Isolation Attempt
1.
No result returned
94%
54.
No result returned
2.
G. stearothermophilus
26.
Bacillus sp. 97%
55.
No result returned
77%
27.
No result returned
56.
No result returned
3.
No result returned
28.
No result returned
57.
No result returned
4.
No result returned
29.
No result returned
58.
B. licheniformis 94%
5.
Geobacillus sp. 96% or G.
30.
No result returned
59.
Klebsiella pneumonia
pallidus 94%
31.
Klebsiella pneumonia
93%
6.
B. coagulans 97%
93%
60.
No result returned
7.
*No result returned
32.
No result returned
61.
No result returned
8.
*G. stearothermophilus
33.
No result returned
62.
No result returned
92%
34.
No result returned
63.
B. licheniformis 77%
9.
fNo result returned
35.
No result returned
64.
No result returned
10.
fNo result returned
36.
No result returned
65.
No result returned
11.
§G. stearothermophilus
37.
No result returned
66.
No result returned
97%
38.
No result returned
67.
No result returned
12.
§No result returned
39.
No result returned
68.
No result returned
13.
fNo result returned
40.
No result returned
69.
No result returned
14.
JG. stearothermophilus
41.
No result returned
70.
No result returned
97%
42.
No result returned
71.
No result returned
15.
Klebsiella sp. 99%
43.
No result returned
72.
B. licheniformis 96%
16.
No result returned
44.
No result returned
73.
B. thermoamylovorans
17.
B. coagulans 97%
45.
Aneurinibacillus
97%
18.
G. pallidus 99%
thermoaerophilus 96%
74.
Brevibacillus sp 86%
19.
Klebsiella sp 97%
46.
No result returned
75.
Brevibacillus 84%
20.
No result returned
47.
No result returned
76.
No result returned
21.
No result returned
48.
No result returned
77.
B. thermoamylovorans
22.
No result returned
49.
No result returned
94%
23.
No result returned
50.
No result returned
78.
Bacillus sp. 90%
24.
Tepidiphilus sp. or
51.
No result returned
79.
B. licheniformis 95%
Petrobacter sp. 94%
52.
No result returned
80.
No result returned
25.
B. thermoamylovorans
53.
No result returned
* Positive Control = ATCC 7953 G. stearothermophilus
fPositive Control = ATCC 12980 G. stearothermophilus
§Positive Control = ATCC 12978 G. stearothermophilus
^Positive Control = SPORTROL* Spore Suspensions, NAMSA
2.3.2 Results
The results of the sampling activities are summarized in Table 5. Several Bacillus species were
identified as well as some potential positive identifications of Geobacillus species. Bg was not
identified in the background samples for these tests.
Because thermophilic bacterial enumeration results revealed wide variability between
duplicates, the experimental procedure on swab samples using BHI and both standard
phosphate (P04)/magnesium chloride (MgCI2) and lecithin buffer was repeated twice. The
problems with the variability of the results and the lack of the ability to identify the preferred
surrogate organism (G. stearothermophilus) successfully using PCR resulted in re-evaluation of
the surrogate to use for the Inoculation and Cleaning tests. Table 6, below, summarizes the
various species identified during both the June and October sampling events.
2
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
Table 5. Summary of the October 18-20, 2010, Environmental Surface Swab Sampling
Results from Darling International, Inc., Rendering Plant
Sample
Number
Sample Location
Predominant Bacteria Identity (>90% Identity Match, unless
stated)
1A
Raw receiving door jamb
B. licheniformis
"IB
Raw receiving door jamb
B. licheniformis
2
Raw receiving door jamb
indeterminate
3
Raw receiving door jamb
Brevibacillus thermoruber
4
Raw receiving door jamb
Dictyostelium discoideum (soil-living amoeba; only 72% match)
5
Concrete drive outside raw receiving bay
Geobacillus spp. (G. pallidus = top match 98%)
6
Concrete drive outside raw receiving bay
Geobaciiius spp. (G. pallidus = top match 84%)
7A
Concrete drive outside raw receiving bay
B. aestuarii
8
Concrete drive outside raw receiving bay
G. thermodenitrificans
9
Concrete drive outside raw receiving bay
B. aestuarii
10A
Concrete drive outside raw receiving bay
Brevibacillus brevis
10B
Concrete drive outside raw receiving bay
B. aestuarii
12A
Raw receiving floor
Ureibacillus thermosphaericus
12B
Raw receiving floor
Petrobacter spp.
13
Raw receiving floor
Aneurinibacillus thermoaerophilus
14
Raw receiving floor
Geobacillus spp.
15A
Raw receiving floor
Geobacillus spp. (G. toebii = top match 87%)
15B
Raw receiving floor
Tepidiphilus margaritifer
16
Raw receiving floor
Aneurinibacillus thermoaerophilus
17
Back of pit - dried material
G. pallidus
19A
Front face of pit - mixed material
B. coagulans
19B
Front face of pit - mixed material
indeterminate
20
Front face of pit - mixed material
B. thermoamylovorans
21
Front face of pit - dried material
B. coagulans
22
Front face of pit - dried material
B. coagulans
23
Raw material incline auger - dried material
Indeterminate
24
Raw material incline auger - dried material
B. licheniformis
25
Top cover - raw grinder
G. thermodenitrificans
27
Crax grinder housing
B. aestuarii
28
Crax grinder housing
Aneurinibacillus thermoaerophilus
2
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
Table 6. Species Identified during June and October 2010 Sampling Events
Sample Location
Result**
13
Aneurinibacillus thermoaerophilus
16
Aneurinibacillus thermoaerophilus
28
Aneurinibacillus thermoaerophilus
13
Aneurinibacillus thermoaerophilus 91 %
14
Aneurinibacillus thermoaerophilus 91 %
45
Aneurinibacillus thermoaerophilus 96%
7A
8. aestuani
9
B. aestuani
10B
B. aestuani
27
B. aestuani
19A
B. coaqulans
21
B. coaqulans
22
B. coapulans
6
B. coaqulans 97%
17
8. coaqulans 97%
1A
8. licheniformis
1B
B. licheniformis
24
B. licheniformis
63
B. licheniformis 77%
2
8. licheniformis 81%
3
8. licheniformis 88%
1
8. licheniformis 90%
58
8. licheniformis 94%
79
8. licheniformis 95%
72
8. licheniformis 96%
78
Bacillus sp. 90%
26
Bacillus sp. 97%
20
8. thermoamylovorans
25
B. thermoamylovorans 94%
77
8. thermoamylovorans 94%
73
8. thermoamylovorans 97%
75
Brevibacillus 84%
10A
Brevibacillus brevis
74
Brevibacillus sp 86%
3
Brevibacillus thermoruber
4
Dictyostelium discoideum (soil-living amoeba; only 72% match)
17
G. pallidus
18
G. pallidus 99%
5
Geobacillus sp. 96% or G. pallidus 94%
14
Geobacillus spp.
6
Geobacillus spp. (G. pallidus = top match 84%)
5
Geobacillus spp. (G. pallidus = top match 98%)
15A
Geobacillus spp. (G. toebii= top match 87%)
2
G. stearothermophilus 77%
8*
G. stearothermophilus 92%
11§
G. stearothermophilus 97%
14t
G. stearothermophilus 97%
8
G. thermodenitrificans
25
G. thermodenitrificans
2
indeterminate
19B
indeterminate
23
indeterminate
31
Klebsiella pneumonia 93%
59
Klebsiella pneumonia 93%
19
Klebsiella sp 97%
15
Klebsiella sp. 99%
12B
Petrobacter spp.
15B
Tepidiphilus margaritifer
12
Tepidiphilus marqaritifer99%
11
Tepidiphilus sp. or Petrobacter sp. 83%
24
Tepidiphilus sp. or Petrobacter sp. 94%
12A
Ureibacillus thermosphaencus
Notes:
* Positive Control = ATCC 7953 G. stearothermophilus
t Positive Control = ATCC 12980 G. stearothermophilus
§ Positive Control = ATCC 12978 G. stearothermophilus
X Positive Control = SPORTROL* Spore Suspensions, NAMSA
Identified in June sampling event
Identified in Oct sampling event; 14 isolates round
Identified in Oct sampling event; 72 duplicates round
Percentages reflect statistical confidence in identification of a specific organism
3
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
These results suggested the following considerations regarding selection of a biological
surrogate for the cleaning/inoculation study:
• Bg spores do not appear to exist in the background flora at this particular rendering plant;
and
• G. stearothermophilus spores, although thermophilic in nature and likely to simplify analyses
by allowing incubation at temperatures that would kill much of the background flora, cannot
be detected reliably using PCR in the positive controls, let alone when mixed with other
bacterial species.
2.4 Preliminary Selection of Surrogates
As previously mentioned, selection of an appropriate surrogate with the following characteristics
for plant inoculation tests was desired:
• Not to be present in the background flora of the plant;
• Able to be identified in the matrices of interest in the rendering plant (dead animals, meat
and bone meal, tallow, wastewater); and
• Able to be separated analytically from the probable high levels of background bacterial flora
in the rendering plant samples.
The Initial Plant Sampling Event did not observe any of the spore-forming Bacillus species
commonly used as surrogate organisms for decontamination studies, particularly G.
stearothermophilus and Bacillus atrophaeus (also known as Bacillus globigii, or Bg). A
thermophilic bacterium like G. stearothermophilus would likely be the best surrogate because
the high incubation temperature during culturing (55 °C) would preclude the growth of many of
the background microorganisms that could confound analysis. However, the Initial Plant
Sampling also observed that G. stearothermophilus was not able to be identified consistently
and positively using PCR even from the positive controls. This lack of Geobacillus-spec\f\c
primers is a significant obstacle to using G. stearothermophilus as a surrogate in a situation
where high concentrations of background flora would require positive identification using PCR.
G. stearothermophilus was, therefore, abandoned as a potential surrogate, and Bg was selected
as the biological surrogate to be used for the field tests.
Because of the uncertainties associated with using a nonthermophilic surrogate organism in the
field tests, a nonbiological surrogate was also chosen to use in the inoculum. This nonbiological
surrogate needed to be biodegradable and compatible with rendering plant products. To
maximize the ability to detect the surrogate utilized, food-grade Phosphorex, Inc.
DegraFluorex™ PLGA fluorescent microspheres (catalog #LGFG1000, lot# 101028-187) were
selected for inclusion in the inoculum as a second surrogate with the spore-forming bacterium
Bg.
2.5 Rendering Matrix Challenge Testing
2.5.1 Purpose and Description
Now that the proposed surrogates had been identified, verification that they could indeed be
recovered analytically from the likely environmental matrices found in a rendering plant was
desired.
Bg and PLGA were used to spike protein-based stock (i.e., suet) (1 gram (g) each), grease (1
milliliter (ml_) each), and deionized (Dl) water (1 ml_ each). These media were spiked with Bg
spores at a concentration of 1E8 CFU/sample (0.1 ml_ of a 1E9 CFU/mL culture). Separate
portions of meat and grease and Dl water were spiked with PLGA microspheres [1 micrometer
(|jm); green color; Ex/Em (nanometers [nm]) 460] at approximately 1E6 beads/g or mL (0.1 mL
4
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
of a 3 microgram (|jg)/ml_ solution was added to 1 g or 1 ml_ of meat or grease, respectively)
after nucleic acid extraction. The purpose of these samples was to assess the ability of the
laboratory to identify Bg through PCR and measure Bg and PLGA from matrices simulating the
matrices found at a meat-rendering facility.
Bg DNA was detected using a real-time polymerase chain reaction (RT-PCR) assay specific for
the sequence encoding the Replication Termination Protein (rtp) present on the Bg
chromosome, and PLGA microspheres were detected by direct microscopic count. The Bg rtp
RT-PCR assay was established using a standard curve prepared from Bg genomic DNA and
tested using spiked samples. Direct microscopic counts of PLGA were performed using
disposable hemacytometers (INCYTO, part number: DHC-N01-5) and a Zeiss epifluorescent
microscope (Carl Zeiss International, Jena, Germany).
2.5.2 Results
Preliminary tests were conducted in meat, grease, and water spiked with Bg spores and PLGA
microspheres. Detection was carried out via RT-PCR and fluorescent microscopy. The
laboratory provided their results and recommendations, which included the use of inocula >1E8
CFU/g or mL, and extraction using a commercial kit or dilution to overcome inhibition. Bg DNA
was detected in water samples but not in meat or grease samples when analyzed directly. After
extraction of nucleic acids using a commercial kit, Bg DNA was detected in all three matrices.
Recovery of Bg DNA signatures was detected in 6-7% of water samples and in less than 1% of
meat and grease samples.
PLGA microspheres were detected within the quantification range when visualized in water or
meat samples using microscopy. However, autofluorescence from the grease at the same
wavelength as the PLGA particles inhibited efficient detection and counting of PLGA
microspheres in grease samples.
2.5.3 Significance of Challenge Test Results
The results of the challenge tests showed that Bg DNA and PLGA microspheres could be
detected in water, but recovery percentages were not high. Bg DNA recoveries were <10% in
water and <1% in meat and grease samples. Additionally, Bg rtp was not detected in direct
analysis of meat or grease samples. Proteases and nucleases present in the meat and grease
matrices as well as other PCR-inhibitors may have prevented direct detection of target DNA.
Based on these challenge test results, the Test Team determined that to ensure efficient
distribution of the spike within the sample matrix and sufficient recovery of target signatures,
spikes should be prepared to contain approximately 1E8 CFU and 1E8 beads perg or mL of
crude protein.
PCR inhibition can be overcome by dilution or by extracting the nucleic acid samples from the
sample matrix. Also, extraction using a simple DNA purification kit may also result in detectable
signatures in the meat and grease samples, but at levels lower than the expected concentration
(recovery was less than 1%). Sample dilution might be a better alternative for these sample
matrices or a more desirable solution for the end users, but testing would be necessary to
determine the optimal dilution to overcome PCR inhibition without significant loss of target DNA.
However, dilution comes at the expense of sensitivity; it is not clear whether a
different/additional purification step would be better or if diluting the inhibitor is better. An
internal positive control (IPC) kit designed to test for the presence of inhibitors in PCR samples
by analysis of an exogenous target DNA could also possibly be used to test neat and diluted
samples prior to analysis to determine the optimal conditions for Bg rtp detection. The Test
Team decided that samples should undergo an extraction procedure, either using a
commercially-available kit for purification of DNA or other standardized method, prior to analysis
5
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
by RT-PCR.
In addition, grease samples were found not to be amenable to direct microscopic observation,
and PLGA microspheres are not distinguishable from the matrix due to background
autofluorescence. PLGA microspheres may possibly be washed or extracted from the grease,
or sample dilution could possibly overcome the interference, but further experimentation would
be required to develop a feasible method for visualizing PLGA microspheres from the grease
matrix. In addition, Bg cells may be visible in grease samples under phase contrast or in the
presence of an appropriate stain, but further research would be necessary to develop an
appropriate visualization method.
2.6 Final Surrogate Selection
Based on the results of the sampling and methods development work done, an ideal surrogate
for use in the field test did not appear to exist. Tradeoffs must be taken into account and a
balance struck to pick the best available surrogate given the amount of information that was
currently available.
Based on the results of the laboratory challenge samples, initial suggestions proposed that the
inoculum for the study would consist of an aqueous mixture containing 1E11 CFU of Bg spores
and 1.47E9 beads of PLGA microspheres dissolved in 1 gallon (gal) of distilled water, sprayed
over each truckload of raw material, to be sprayed evenly over the load (approximately 20 tons)
in each truck that arrived on site during the inoculation portion of the study (note - the proposed
inoculum would be only on the outside of the materials in the truck and would not be evenly
distributed within the 20 ton load). A surfactant would be added to the mixture to reduce
clumping. The Test Team also received information that 1E11 CFU of Bg spores tend to clump
together and produce a much lower level of contamination [7], By adding a surfactant to the
mixture, 1E9 Bg spores per mL could be utilized more effectively in the study. Clumping would
be reduced, and the estimated level of contamination would be greater than 1E9 of Bg per
truckload. In addition, significant cost savings could be realized. Thus, a surfactant, "Fluid D"
(see Appendix E), was added to the final mixture of 1E9 CFU of Bg spores and 1.47E9 beads of
PLGA/gal.
6
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
3. EXPERIMENTAL PROCEDURES: CLEANING/INOCULATION
FIELD TEST
3.1 Test Design/Planned Approach
The Cleaning/Inoculation Testing included the following elements in chronological order:
• Pre-clean the plant to remove the bulk of any built-up organic material that had accumulated
on various plant surfaces;
• Perform background sampling of surfaces and air at pre-identified locations within the plant
and in outside perimeter locations for Bg spores and PLGA microspheres;
• Inoculate each truckload of raw material (animal carcasses, offal) entering the plant over the
course of an eight-hour day with the solution containing Bg spores, PLGA microspheres,
and surfactant; perform air sampling for components of the inoculum during the inoculation
part of the study;
• Perform post-inoculation sampling of surfaces and air at pre-identified locations within the
plant and in outside perimeter locations for the same target analytes;
• Clean pre-determined areas of the plant with existing plant cleaning procedures, using hot
water and steam; and
• Perform post-cleaning sampling of surfaces and air at pre-identified locations within the
plant and in outside perimeter locations.
Table 7 lists the detailed timeline of events for the Cleaning/Inoculation Tests, including the
number of samples collected, types of samples collected, and other notations regarding the
procedures that were used.
A planned approach was developed that identified 124 sample locations throughout the
rendering plant, including the process room, grinders, and outside the cooker (Figures 4 and 5).
Thirty-four air sample locations were selected randomly inside and outside the plant while 90
wipe sample locations were pre-determined. Outside air samplers were to be positioned on all
sides of the plant, but the majority of the samples were to be collected downwind of plant
operations. Inside air sampler locations were to be concentrated in high dust areas or areas
where crushing and grinding could aerosolize the surrogates from the rendering process or from
fomites.
Unlike the Preliminary Scoping Samples that utilized swabs, four wipes were collected from
each of the 90 surface sampling locations. One wipe (designated as A) was collected for
community characterization by PCR, one wipe was collected for enumeration (designated as B),
and the third wipe was collected for PLGA identification (designated as C). A fourth sample was
collected and stored for archival purposes (designated as D). Wipe samples were chosen over
swabs because the wipes provided a slightly larger surface area and wipes are routinely used
by EPA to sample surfaces for biological agents. In addition, swabs were not an optimal
sampling medium for a rendering environment; the tip of the swab could be impacted by a single
large particle from the rendering process, and the characteristics of the material buildup on the
surfaces in the rendering plant made it difficult to establish the sample area from samples
utilizing swabs.
7
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
Table 7. Timeline of Events for the Cleaning/Inoculation Portion of Study
Day*
Day of
Week
Time
Primary Task
Additional Task
Samples | Notes/Assumptions
Wipe
Wipe
Blanks
Air
Air
Blanks
Sept.
24-25
Sat & Sun
Work Shift
Pre-Cleaning
(weekend 1)
Weekend 1: Cleaning conducted by plant (no oversight)
Oct.
1-2
Sat & Sun
Work Shift
Pre-Cleaning
(weekend 2)
Weekend 2: Cleaning conducted by plant (no oversight)
Oct.
8-9
Sat & Sun
Work Shift
Pre-Cleaning
(weekend 3)
Oversight / Documentation,
Finalize Sample locations
Weekend 3: Cleaning conducted by plant (Test Tearn oversight).
Oct.
17
Mon.
Work Shift
Background
Samples
Scoping and Prep,
Documentation, Package and
Ship samples
4
1
7
1
Collect background samples throughout entire facility.
Oct.
20-21
Thurs.,
Fri.
As trucks are
available
Inoculate loads of
carcasses
Documentation
5
Inoculate carcasses as loads arrive either off-site or in a containment area
to prevent spreading. 1) Spray the carcasses down; 2) Spike carcasses in
each load with the surrogate. 3) Continue inoculating for one 8-hr shift.
Oct.
21
Fri.
Work Shift (8
hours)
Process
Contaminated
Carcass
Sampling, Documentation
Inoculated material will be processed for eight hours.
Oct.
21
Fri.
Work Shift (8
hours)
Stage 1 -
Process
Sampling
Documentation, Package and
Ship samples
8
2
4
1
Sampling during processing of inoculated material. Eight hr air samples
will be initiated in the process area and throughout the building. Surface
wipe samples will be taken every two hr from grinder feed
Oct.
21
Fri.
Immediately
afterward
Post Inoculation
Sampling
Scoping and Prep,
Documentation, Package &
Ship samples
22
5
6
1
Immediately after all inoculated carcasses have been processed, Test
Team will collect samples throughout whole facility.
Oct.
21
Fri.
After Post
Dispersion
Sampling
Process Clean
Carcasses
Documentation
Plant will process clean material for eight hr
Oct.
21
Fri.
After process
runs for 2 hr
Stage 2 -
Process
Sampling
Documentation
8
2
Collect surface wipe samples from the grinder. Surface wipe samples will
be taken randomly every two hours from grinder feed
Oct.
22-23
Sat & Sun
All day
Plant Cleaning
Documentation
Cleaning conducted by the plant (Test Team oversight)
Oct.
23
Sun.
After cleaning is
complete
Post Cleaning
Sampling
Documentation, Package and
ship samples
40
8
13
3
Samples collected throughout whole facility
Total Samples
82
18
35
6
* - all dates are in 2011
8
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
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Legend
Post Dispersion Wipe
•
Sampling
Stage 1 and Stage 2
~
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9
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
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Post Cleaning Wipe samples #
Grinder Study Wipe samples A
Figure 5. Post-Cleaning Study Sampling Locations
3.2 Sampling Procedures and Protocols
Sampling procedures and protocols were developed to ensure sample viability, balance the
need to cover a very large plant adequately with a minimum number of representative samples,
and hopefully acquire sufficient sample so that analytical results would be not be constrained by
the detection limits. Twenty-eight additional Quality Assurance/Quality Control (QA/QC)
samples were collected, including 27 media blanks and three inoculation solution samples.
Wipes consisted of Versalon® synthetic gauze pads (Tyco Healthcare/Kendall, Versalon® All-
Synthetic Sterile Sponges, 2 inches (in) x 2 in - # 8042, Mansfield, MA, USA), and the sampled
areas were defined by a 10 cm x 10 cm paper template. The entire wipe was extracted. The
samples were acquired at the following time intervals:
• After initial cleaning of the plant, prior to inoculation with the surrogates;
• During inoculation of carcasses (air samples only);
• After processing inoculated carcasses for eight hours; and
• After the final cleaning of the plant.
Table 7 (above) provides an outline of the planned study events. Table 8 contains a summary
of samples that were collected in the background, inoculation, post-inoculation, and post-
10
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
cleaning phases. For a complete list of all sample identifications (IDs) and sample locations,
see Appendix D.
Table 8. Summary of Samples Collected in the Background, Inoculation, Post-
Inoculation, and Post-Cleaning Phases
Phase
Primary Task
Matrix
Measurement**
Sample Locations
QA/QC Sample Locations
Wipe
Air
Air
Blanks
Air
Spike
Wipe
Blanks
Pre-Cleaning
(weekend 1)
¦o
c
Pre-Cleaning
(weekend 2)
=5
2
O)
o
CD
CD
Pre-Cleaning
(weekend 3)
Background
Samples
Wipe of
nonporous
surfaces; MCE
filter* of air
Wipe: culture and counts of CFU; PCR;
PLGA
Air: PLGA; biologicals
4
7
1
1
c
O
Inoculate loads
of carcasses
Mixed Cellulose
Ester (MCE) Filter
of air
4
1
1
1
=5
O
o
c
Process
Contaminated
Carcass
Air: PLGA; biologicals
Stage 1 -
Process
Sampling
Wipe of
nonporous
surfaces; MCE
Filter of air
Wipe: Culture and counts of CFU; PCR;
PLGA
Air: PLGA; biologicals
8
4
1
2
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c
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-------
Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
(Product Code 225-3-01; SKC, Eighty Four, PA, USA). Biological agent extraction efficiencies
of 99% have been determined for this type of medium [8], The sample pumps were calibrated
to operate in the 2 liters/minute (Lpm) range and collect samples over a period of time ranging
from 1 to 8 hr, depending on sampling objectives and dust levels. The complete sampling
equipment and procedures are below.
MCE filter samples were collected in accordance with the following procedure:
1. Pre-calibrate the sampling pump to a flow rate between 3 and 4 Lpm;
2. Don a pair of sterile or clean sampling gloves;
3. Remove the top portion of the sampling cassette;
4. Attach the filter cassette to the sampling pump inlet using Tygon® tubing;
5. Set the sample pump and filter at the sample location and connect the cassette to the
sample stand;
6. Turn on the pump (sample is collected open-faced) and record start time;
7. Collect the sample for a run time of 120 minutes (min) or 2 hr;
8. Turn off the pump and record stop time;
9. Don a new pair of sterile sample gloves;
10. Disconnect the sample cassette from the sample pump;
11. Attach the top portion of the filter cassette to the sampling device;
12. Insert the end caps into the sampling cassette;
13. Label the sample;
14. Double-bag the sample;
15. Following decontamination of the samples, place the bags into a sample custody bag;
and
16. Change gloves.
During the inoculation activities, a total of six air samples were collected with personal sample
pumps (four test air samples and two QA/QC air samples). The purpose of these six air
samples was to measure air transport of spores from the inoculation area to the plant, if there
was any. All air samples were collected with SKC AirChek 2000 personal sampling pumps
(SKC, Eighty Four, PA, USA) and calibrated with a Bios® DC-Lite dry cell calibrator (Bios
International Corporation, Butler, NJ, USA) to 1.0 Lpm. The medium was 37-mm MCE filters in
three-stage pre-loaded cassettes. Air samples were collected in four areas during the
inoculation of the carcasses. Three air samples (to the east, west, and south) were collected in
the area adjacent to the detarping area. A fourth sample was collected by the large door near
the tipping floor.
3.2.3 Post-Inoculation Phase Sampling
During the Post-Inoculation Phase of the study, the plant processed inoculated carcasses for
eight hr. Two wipe samples were collected from the grinder every two hr during the eight hr of
processing (eight wipes total). Samples were collected from the grinder in the same location
each time during this stage of the study. The purpose of the grinder samples was to evaluate
the buildup and potential reduction of surrogate loading as the inoculated material began to be
fed, continued being fed, then after the inoculated material ceased being fed. In addition, a total
of four eight-hr air samples were initiated at the start of the eight-hr shift. The air samplers were
distributed in the dustiest areas of the plant, including the skimmer area, the cooker area, the
storage bin area, and near the plant exhaust vent. The purpose of the air samples was to
characterize movement of airborne contamination within the plant. Figure 4 shows the locations
of the post-inoculation samples. After the eight-hr shift was completed and all of the
contaminated carcasses were processed, the plant processed uninoculated (clean) carcasses
for an additional eight hr.
12
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
The plant then underwent the cleaning procedures described in Section 3.4.
3.2.4 Post-Cleaning Phase Sampling
After the cleaning had been performed by plant personnel, samples were collected from 53
locations. Thirteen air and 40 surface locations were sampled to determine the effectiveness of
the cleaning. The 40 surface locations were previously shown in Figure 5. Attempts were made
to acquire samples in the near vicinity of previous sample locations but not on exactly the same
spot.
3.3 Inoculation of Incoming Raw Materials
During the Inoculation Phase of the study, the PLGA and Bg solution was evenly sprayed over
the top of each truckload of raw material (i.e., animal carcasses and food animal slaughter offal)
intended for processing in the rendering plant for an eight-hr shift. The PLGA and Bg solution
was sprayed on the carcasses into and onto the load using a hand sprayer (D.B. Smith
Roundup Backpack® Sprayer) containing the surrogates within a phosphate buffer solution with
the added surfactant, filled to the one-gal level with distilled water. The surfactant was added to
the mixture to prevent the spores from clumping together. The estimated level of contamination
was >1E9 CFU of Bg and approximately 50 milligrams (mg) of PLGA (~1.47E9 spheres per mg)
per truckload. The inoculum mixture was prepared immediately before its use, in one-gal
batches, in the reservoir of the sprayer, and the reservoir from the sprayer was periodically
shaken as spraying occurred.
Once each truck arrived at the plant, the tractor number and trailer information were
documented as well as the weight of the load as measured by the plant's scales. The
carcasses loaded into the truck trailer were sprayed prior to entering the facility, outside in the
truck detarping area located on the south side of the plant. During one eight-hr shift, all arriving
loads (approximately 16) were inoculated, and carcasses were processed. After each truck
dumped its load of inoculated carcasses, the truck bed was washed out with water and dumped
inside the bay prior to leaving the dumping area. Prior to leaving the site, trucks were sprayed
with an amended bleach solution (1 part Clorox® bleach, 1 part white vinegar, 8 parts water) to
minimize potential for cross-contamination should that truck return to the site prior to the post-
cleaning sampling.
All doorways near the detarping area remained closed during the inoculations. To reduce
contamination, the individual performing inoculation did not enter the plant during this stage of
the study. After all of the trucks were inoculated, the person performing the inoculation was
sprayed off with water prior to leaving the area, their personal protective equipment (PPE) that
included Saranex® overalls and nitrile gloves was removed and disposed, and their boots were
removed and left outside the plant for later use.
Table 9 lists the inoculated trucks and the load weights along with the inoculation time for the
Inoculation Phase of the tests.
13
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
Table 9. Weights of Inoculated Trucks
Truck Number
Time
Truck ID
Weight
(pounds)
1
0831
3638
25,780
2
0852
4193
40,300
3
0955
4578
19,840
4
1101
3996
22,100
5
1201
4987
32,240
6
1248
3842
9,540
7
1320
4901
44,060
8
1332
4564
20,800
9
1441
3553
40,780
10
1454
3982
14,740
11
1507
4015
13,340
12
1528
4062
6,860
13
1541
4189
24,480
1622
***
14
1719
4749
15,160
15
1732
4943
37,780
Total
367,800
*** Positive control sample of inoculum acquired by spraying approximately 20 mL of
inoculation mixture into conical tube.
3.4 Plant Cleaning
Initial observations of the Darling plant that were made on the first site visit noted that many of
the surfaces of the plant had a significant bulk loading of organic material. To maximize the
probability of being able to detect any surrogates that were inoculated into the raw materials
entering the plant, cleaning as many of the plant surfaces as possible prior to testing was
desired. Therefore, prior to any sampling as part of the cleaning/inoculation portion of the study,
Darling plant workers cleaned parts of the plant over the course of several weekends to remove
buildup and bulk loading of organic material from plant surfaces. Plant personnel utilized
existing plant methods and external contract personnel to clean the plant. Water heated to
approximately 180 to 200 degrees Fahrenheit (°F) was used to wash loose particles from plant
surfaces. Plant personnel used brooms, shovels, scrapers and brushes to loosen gross
contamination. Materials loosened in this manner were pushed into the pit and fed into the
rendering process with the raw material. Heated water was then used to rinse the area. If
existing plant water lines did not reach an area of the plant, plant personnel utilized pressure
washers and a Steam Genie (Steam Genie, Inc., Compton, CA, USA) to clean those surfaces.
No detergents or disinfectants were used during these steps as per routine plant procedures.
Since this study was looking at the cleaning process and not the efficaciousness of
disinfectants, there was not an attempt to kill the surrogate Bg spores. In any event, sporicidal
conditions necessary to kill the Bg spores would have been unrealistically harsh relative to
disinfectants necessary to kill the viral agents that are of most concern from an FAD standpoint.
Table 10 outlines the areas (see Figure 6) to be cleaned and the cleaning methods to be
utilized.
14
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
Table 10. Plant Cleaning Schedule
Date (2011)
3m Party Cleaning Company
Cleaning by In-House Plant Staff
Sept 24-25
Raw Bay I
Raw Bay II
Scraping lower walls, floor, receiving
pits
Scraping lower walls, floor, receiving pits
Cleaning ceiling, walls, floor
Cleaning ceiling, walls, floor
Oct 1-2
Soapstock, fleshing areas and truck
bays
Duke/skimmer room
Cleaning ceiling, walls, floor,
nonelectrical equipment
Cleaning ceiling, walls, floor and nonelectrical
equipment
Oct 8-9
Cooker room, meal load-out
Work tank area
Cleaning ceiling, walls, floor,
nonelectrical equipment
Cleaning ceiling, walls, floor, nonelectrical
equipment
Oct 15-16
All other areas done on previous
weekends
All other areas done on previous weekends
Hot water washdown
Hot water washdown
Oct 22-23
All other areas done on previous
weekends
All other areas done on previous weekends
Hot water washdown
Hot water washdown
Following the Inoculation Phase sampling, a second cleaning was performed where plant
personnel cleaned the facility. Under the limited oversight by the Test Team, plant personnel
utilized existing plant methods and external contract personnel to clean the plant. Particular
attention was paid to the grinder area, tipping floor, pits, the processing area, and building
floors. As access allowed, plant personnel would attempt to clean any augers used in the
process by utilizing typical plant cleaning procedures, which took approximately 8 hr to complete
after inoculation had ceased.
3.5 Analytical Procedures and Protocols
The samples (air, wipe, and water) taken during the Cleaning/Inoculation Test were shipped
overnight in a chilled container to Battelle Memorial Institute in Columbus, OH, for analysis. For
more detail on the analytical report, please see Appendix B. All processing areas within the
analytical laboratory, including the biological safety cabinet and incubator, were thoroughly
decontaminated, and surfaces were sampled with swabs. Samples were plated onto BHIA to
ensure that working areas were sterile prior to processing of rendering plant samples. An
additional swab was taken and plated on BHIA on each day of sample extractions. The
samples were analyzed following the procedures specified in this section.
Sample processing occurred in five batches over the course of three weeks, and positive and
negative analytical controls were created for each batch as follows: a single negative control
and a single positive control for each matrix type were extracted in the batch. Negative controls
(Matrix Blank 1, 2, etc.) comprised a single pristine matrix, while positive controls (PCs) (Matrix
PC 1, 2, etc.) comprised a single pristine matrix spiked with Bg DNA at 1E7 gene copies
(GC)/sample and PLGA microspheres at 0.05 mg/sample. Control matrices were provided by
test personnel and were identical to sample matrices. Controls were processed in tandem with
the samples, and each control received treatment identical to the sample matrices.
15
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
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| | Cleaned on Oct. 1/5th
] Cleaned on Oct. 8/9th
Note: All shaded a reas to be
cleaned on Oct. 15,16, 22 & 23rd
Figure 6. Areas Cleaned at the Darling Des Moines plant.
Each sample or control was extracted according to a project-specific work instruction (DWI-01-
02, Work Instructions for the Extraction of Microorganisms, Nucleic Acids, and PLGA
Microspheres from Environmental Samples [Appendix B]). Briefly, samples were removed from
their original containers and placed into sterile 250 mL bottles, and phosphate buffered saline
(PBS) was added. Each sample was mixed by vortexing for approximately 30 seconds (sec)
and then incubated for 30 min at room temperature. An aliquot (1 mL) was removed to serve as
the microbiology extract, and the remaining sample was extracted for nucleic acids.
Microbiology extracts were plated onto BHIA and incubated at 36 ± 2 °C overnight to isolate
single colonies of bacteria. The remaining microbiology extracts were stored at 4 °C until being
processed further for sequence analysis. After addition of herring sperm carrier DNA (hsDNA)
and 1% sodium dodecyl sulfate (SDS), samples were incubated for 30 min at 65 C. Samples
were extracted in 12 to 15 mL PBS, and 1 mL was removed for microbiological analysis. The
hsDNA and 1% SDS were added to the remaining volume in the original sample extract after
removal of the microbiology aliquot (i.e., membrane filter with 12 mL PBS, 1 mL removed for
microanalysis, remaining volume is 11 mL). The original sample matrix was preserved in the
extraction vessel for detection of PLGA microspheres by microscopic analysis, and the aqueous
extract was transferred to a sterile Oakridge tube (Thermo Scientific [Nalgene], Rochester, NY,
16
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
USA). Nucleic acids were concentrated using method ABAT-V-012 (Battelle's Applied Biology
and Aerosol Technology - Concentration of Nucleic Acids by Isopropanol Precipitation). In this
method, nucleic acids were precipitated overnight with isopropanol, recovered by centrifugation,
washed with 70% ethanol and resuspended in IXTris ethylenediaminetetraacetic acid (EDTA)
(TE) buffer, pH 7. Spores and cells were lysed using SDS and incubated at 65 °C during the
extraction process outlined in DWI-01-02 (Appendix B).
3.5.1 Bg Detection by Quantitative PCR
Samples were extracted, and nucleic acids were purified with a final volume of 200 |jL. For
nucleic acid analysis, duplicate 5 microliter (|jL) aliquots of the 200 |jL sample extracts were
assayed via Quantitative PCR (qPCR) using an assay specific for the rtp gene of Bg on an ABI
7900HT platform. The limit of detection (LOD) was determined by running triplicate reactions of
a standard curve prepared with Bg genomic DNA; the lowest concentration of DNA that is
detected in the assay is considered the LOD, the lowest concentration detected in duplicate
reactions is the limit of quantitation (LOQ).
To determine how many GC were present in the standard curve preparations:
1) the mass of the genome in base pairs (bp) found in the published literature was converted
into pg/GCs using the conversion factor of 1.096e-21 g/bp [9];
2) genomic DNA was extracted and the amount of DNA was quantitated with a
spectrophotometer (Molecular Devices, Sunnyvale, CA, USA) in units of (nanograms [ng]/ml_);
3) using the pg/GC factor determined in step 1, extracted genomic DNA concentration was
converted to GC/mL; and
4) a standard curve was prepared in a tenfold serial dilution series.
The LOD and LOQ for this assay were determined to be 92.1 GC/5 |a,L. Prior to target analysis,
sample extracts were tested for inhibition using the Applied Biosystems (ABI) TaqMan®
Exogenous Internal Positive Control Reagents Kit (Life Technologies, Grand Island, NY, USA)
according to method ABAT-V-007 (TaqMan Inhibition Analysis on the 7900HT). Neat, 1:5, and
1:10 dilutions of each sample were initially assayed. In the event extracts did not pass IPC
testing at the 1:10 dilution, they were further purified using a Qiagen QIAQuick PCR Purification
kit (Qiagen, Germantown, MD, USA) according to the manufacturer's instructions. The Qiagen-
purified sample extracts were further diluted and tested by IPC analysis at Qiagen Neat (QN),
Qiagen 1:5 (Q5), Qiagen 1:10 (Q10) and Qiagen 1:20 (Q20) dilutions. Sample extracts that
passed IPC were analyzed for Bg DNA at the highest concentration passing the inhibition test
according to Method ABAT-V-008 "To Prepare a 96-Well Plate for DNA Quantitation on the
7900HT" (Appendix B). The Ct (cycle threshold) value and estimated nucleic acid quantity
based on the input standard curve were compiled, along with an amplification plot and a trace of
fluorescent signals (multicomponent plot) for each replicate sample. The multicomponent plot
was examined for each sample replicate to verify results; positive detections showed elevated
signal from the reporter fluorescent molecule. Assay acceptance criteria included the following:
• Valid standard curve with three or more duplicate points (assay acceptance requires a
standard curve with three or more duplicate points and an R2 > 0.95); and
• No amplification in No Template Control (NTC) wells.
A small subset of sample extracts (IRP-AIR-10-24-11-ABC-018 to IRP-AIR-10-24-11-ABC-025
and sample IRP-AIR-10-24-11-ABC-27) was not qPCR-analyzed. This set of sample extracts
was amplified on the ABI GeneAmp® 9700 PCR System (Life Technologies, Grand Island, NY,
USA) and analyzed by gel electrophoresis, with direct visualization of the ethidium bromide-
stained target amplicon. Positive and negative control reactions were prepared and analyzed
17
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
along with the sample extracts. Each sample was initially analyzed on a 2% agarose gel with
1X Tris base, acetic acid, and EDTA (TAE) running buffer (10 |a,L sample per well), and samples
were electrophoresed on a 1.2% gel to compare pooled sample extracts (5 |a,L each) against
pooled (No Template Controls) NTCs and the positive control reaction. Each gel contained an
appropriate molecular weight marker, either Quick-Load 2-log ladder 2% gels (New England
Biolabs, Ipswich, MA, USA) or 1Kb Plus Track It Ladder 1.2% gels (Life Technologies, Grand
Island, NY, USA).
Sample extracts that did not pass IPC were subject to PCR using the Phire® Animal Tissue
Direct PCR Kit (ThermoScientific, Waltham, MA, USA) (Table 11). Phire® PCR was conducted
according to the manufacturer's instructions using pooled DNA extracts. Samples were pooled
by combining 3 |a,L of each extract in groups of nine or ten. Reactions were created by
combining 5 |a,L of each pooled sample extract with 25 |a,L 2X Phire® Animal Tissue PCR Buffer,
10.875 |o,L RNase-Free water (ThermoScientific, Waltham, MA, USA), 2.5 |a,L of each forward
and reverse primer (10 |a,M), and 1 |a,L of Phire® Hot Start II DNA Polymerase. Reactions were
processed with the following cycling parameters: initial denaturation (5 min, 98° C); 40 cycles of
denaturation (98° C, 5 sec), annealing (65 °C, 5 sec), and extension (72°C, 20 sec); a final 1
min extension at 72°C. Each reaction was analyzed on 1.2% agarose gels; 25 |a,L of each PCR
reaction was combined with 5 |a,L 6X Track It Loading Dye (Life Technologies, Grand Island, NY,
USA) and run against the 1Kb Plus Track It Ladder.
3.5.2 Detection of PLGA Microspheres
During method development, a 96-well microtiter plate assay was developed for detection of
PLGA microspheres in an aqueous extract. PLGA microspheres were diluted in 1X PBS to
create a 10 mg/mL top concentration, which was then analyzed by dilution to extinction on two
platforms: 1) SpectraMax M2 Multi-Mode Microplate Reader (Molecular Devices, Sunnyvale,
CA, USA), and 2) Victor Fluorometer (PerkinElmer, Waltham, MA, USA) (0.1 and 1 sec
exposure times). PLGA microspheres were analyzed in concentrations that ranged from 10
mg/mL to 1.19E-6 mg/mL (diluted 1:2 in 1X PBS). The working range of the SpectraMax M2
was determined to be 10 to 0.02 mg/mL whereas the working range of the Victor was 10 to
0.001 mg/mL. Due to the lower limit of detection obtained using the Victor fluorometer, that
instrument was chosen for further assay development, and a standard curve was prepared and
validated from 10 to 0.001 mg/mL.
Once the assay was established, verification of the proposed extraction method was initiated.
Control sample matrices (sampling wipes, i.e., gauze and air filters) were spiked with 1 mg
PLGA microspheres, and a mock extraction was performed according to project-specific work
instructions (DWI-01-00; Appendix B). The PLGA microspheres were anticipated to be removed
from the gauze and filter matrices and suspended in the extract, whereupon they would be
recovered during the final filtration. However, the PLGA microspheres were discovered to be
adsorbed to the gauze and filter matrices, and all attempts to remove them were unsuccessful.
At the advice of the PLGA microsphere manufacturer, Phosphorex, Inc., 25 mL of a 2.5%
solution of polyvinyl alcohol (PVA) was added to each spiked filter and gauze sample, followed
by vortex agitation for 1 min. Room temperature incubation was continued up to 30 min with
intermittent agitation by vortex. As no change was observed after 30 min, a water bath
sonicator was used to agitate each sample for 5 min. Even after sonication in PVA, deposits of
PLGA microspheres visible to the naked eye remained on both types of sample.
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
fable 11. Pooled Sample Extracts for Phire Animal Tissue Direct PCR Kit
Pooled
Sample Extracts Combined
Pooled
Sample Extracts Combined
Sample
Sample
1
IRP-WIPE-10-19-11-ABC-B2 (QN)
4
IRP-WIPE-10-24-11-ABC-0072 (QN)
IRP-WIPE-10-21-11 -ABC-0015
QN)
IRP-WIPE-10-24-11-ABC-0073 (QN)
IRP-WIPE-10-21-11-ABC-0016
QN)
I RP-WIPE-10-24-11-ABC-0074 (QN)
IRP-WIPE-10-21-11 -ABC-0017
QN)
IRP-WIPE-10-24-11-ABC-0075 (QN)
IRP-WIPE-10-21-11-ABC-0030
QN)
IRP-WIPE-10-24-11-ABC-0076 (QN)
IRP-WIPE-10-21-11-ABC-0032
QN)
IRP-WIPE-10-24-11-ABC-0077 (QN)
IRP-WIPE-10-21-11-ABC-0035
QN)
IRP-WIPE-10-24-11-ABC-0078 (QN)
IRP-WIPE-10-21-11-ABC-0037
QN)
IRP-WIPE-10-24-11-ABC-0080 (QN)
IRP-WIPE-10-21-11-ABC-0042
QN)
IRP-WIPE-10-24-11-ABC-0081 (QN)
IRP-WIPE-10-24-11-ABC-0096 (QN)
2
IRP-WIPE-10-21-11-ABC-0044
QN)
5
IRP-WIPE-10-24-11-ABC-0083 (QN)
IRP-WIPE-10-21-11-ABC-0047
QN)
IRP-WIPE-10-24-11-ABC-0084 (QN)
IRP-WIPE-10-24-11-ABC-0051
QN)
IRP-WIPE-10-24-11-ABC-0086 (QN)
IRP-WIPE-10-24-11-ABC-0052
QN)
IRP-WIPE-10-24-11-ABC-0087 (QN)
IRP-WIPE-10-24-11-ABC-0055
QN)
IRP-WIPE-10-24-11-ABC-0088 (QN)
IRP-WIPE-10-24-11-ABC-0056
QN)
IRP-WIPE-10-24-11-ABC-0089 (QN)
IRP-WIPE-10-24-11-ABC-0057
QN)
IRP-WIPE-10-24-11-ABC-0090 (QN)
IRP-WIPE-10-24-11-ABC-0058
QN)
IRP-WIPE-10-24-11-ABC-0092 (QN)
IRP-WIPE-10-24-11-ABC-0059
QN)
IRP-WIPE-10-24-11-ABC-0093 (QN)
IRP-WIPE-10-24-11-ABC-0060
QN)
3
IRP-WIPE-10-24-11-ABC-0061
QN)
IRP-WIPE-10-24-11-ABC-0062
QN)
IRP-WIPE-10-24-11-ABC-0064
QN)
IRP-WIPE-10-24-11-ABC-0065
QN)
IRP-WIPE-10-24-11-ABC-0066
QN)
IRP-WIPE-10-24-11-ABC-0067
QN)
IRP-WIPE-10-24-11-ABC-0068
QN)
IRP-WIPE-10-24-11-ABC-0070
QN)
IRP-WIPE-10-24-11-ABC-0071
QN)
IRP-WIPE-10-20-11-ABC-001 (QN)
Due to the apparent irreversible immobilization of PLGA microspheres onto the filter and gauze
sample matrices, detection of PLGA microspheres was accomplished by direct microscopy
using a Zeiss Axioscope epifluorescent microscope (Carl Zeiss Microscopy GmbH, Jena,
Germany) equipped with a filter set with excitation at 495 and emission at 517 nm.
Representative images were captured using a Zeiss color camera. More detail on these
procedures can be found in Appendix B.
3.5.3 Enumeration of Putative Viable Bg in Archived Samples
Original air filter samples and archive gauze wipe samples were extracted according to the work
instructions, DWI-01-02 (Appendix B). Samples were pre-wetted with 1X PBS extraction buffer
(2 ml_ for filter samples, 5 ml_ for gauze samples) and mixed by vortexing for 30 seconds. An
additional 10 ml_ of 1X PBS was added to each sample, and samples were incubated at room
temperature (25+3 °C) for 30 min. Samples were mixed by vortexing for 0, 15, and 30 min.
Following incubation, 200 |a,L of each sample was spread-plated onto BHIA and incubated
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
overnight at 30° C. Plates were observed for microbiological growth, and colonies were
compared to an overnight positive control of Bg plated onto BHIA. Any samples containing
putative Bg were replated onto fresh BHIA for enumeration. The putative Bg samples were
diluted in 1X PBS and heat-shocked by incubation at 65° C for 30 min to kill any vegetative cells
that might out-compete the spore-forming Bg. Positive and negative controls were processed
along with the samples to ensure process efficacy. Negative controls were prepared by
transferring clean filter and gauze matrices into sterile sample reservoirs; positive controls were
prepared by transferring clean filters and gauze matrices into sterile sample reservoirs and
spiking with an aliquot of Bg.
3.5.4 Identification of Background Microflora by Sequence Analysis
3.5.4.1 Selection of Unknown Isolates and Pooled Samples
Microorganisms recovered on BHIA from wipe and filter samples were selected for follow-on
analysis using 16S rRNA sequencing. Thirty isolates that did not have morphology similar to Bg
were selected and streaked for isolation on BHIA, followed by incubation for 16 - 48 hr at 36 ± 2
°C. Appendix A lists the isolate morphology and the sample from which the isolate originated.
Bg (ATCC 9372) was included as a positive control.
Portions of the samples from each of the nucleic acid extract batches were combined to
generate five pooled samples for metagenomic 16S rRNA analysis using the Ion Torrent™
Personal Genome Machine™ (PGM™) Sequencer (Life Technologies, Grand Island, NY, USA).
3.5.4.2 Extraction of DNA
Three different extraction techniques were used to prepare DNA for 16S rRNA amplification.
Initially, each of the 30 isolates and five pooled samples was extracted following the DNeasy®
Gram-positive bacteria protocol (Qiagen, Germantown, MD, USA); however, the DNeasy®
extracts could not be used for PCR due to background 16S rRNA DNA that amplified in one of
the enzymatic lysis buffer components. A thermolysis technique was therefore used to amplify
the samples, but this technique too was unsuccessful at amplifying the 16S rRNA gene from the
five pooled samples. Finally, a OneStep™ PCR Inhibitor Removal Kit (Zymo Research
Corporation, Irvine, CA, USA) was used on the pooled samples prior to PCR amplification. Only
samples processed for sequencing were extracted using the OneStep™ PCR Inhibitor Removal
Kit (Zymo). Inhibition testing was not attempted on these samples, as this kit was specifically
designed for PCR and sequencing applications. Because all five pooled samples showed
amplification after treatment on the OneStep™ column, no inhibition was presumed.
For extraction using DNeasy® Blood and Tissue Kit, enzymatic lysis buffer was prepared as
follows: 2 ml_ of Tris-EDTA, 10X (Fisher Scientific, Pittsburgh, PA, USA), 120 |jL of Triton X-100
(Fisher Scientific, Pittsburgh, PA, USA), and 2 ml_ of 100 mg/ml_ lysozyme, egg white (Fisher
Scientific, Pittsburgh, PA, USA) was added to 5.88 ml_ of water. One to several colonies,
depending on size, were selected for extraction. After addition of the colonies to a tube
containing 180 |jL of the above enzymatic lysis buffer, extractions were completed following the
manufacturer's instructions for Gram-positive bacteria. To prepare pooled samples for
extraction, 1 ml_ of each pooled sample was centrifuged at 5,000 x g for 10 minutes, and the
pellet was suspended in 180 |jL of enzymatic lysis buffer and extracted according to the
manufacturer's instructions as stated above.
For extraction via thermolysis, DNA from the 30 isolates with distinct colony morphologies were
extracted by adding one to several colonies, depending on size, to a tube containing 250 |jL of
1X Tris-EDTA. The samples were autoclaved using a liquid cycle for 10 min at 121 °C.
Following autoclave treatment (121 °C for 10 min), the samples were cooled to room
temperature and stored at -80°C until ready for use. The autoclave treatment step is a historical
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
method used at Battelle to remove DNA from intact cells, and it is not referred to as a published
method. Five pooled samples were prepared by adding 10 |jL of each pooled sample to a
separate tube containing 250 |jL of 1X Tris-EDTA and treating them in the same manner as the
colony samples.
For extraction of PCR inhibitors using the OneStep™ column, fifty |jL of each pooled sample
was processed using the OneStep™ PCR Inhibitor Removal Kit following the manufacturer's
instructions.
3.5.4.3 Amplification of 16S rRNA
The 30 isolated colonies and five pooled samples were subject to PCR using 8F (isolated
colonies and pooled samples) or27F (pooled samples) and 1492R 16S rRNA primers (Table
12).
Table 12. 16S rRNA Primer Sequences
Primer ID
Sequence
8F
5'-AG AGTTT G ATCMTGGCT CAG-3'
27F
5, - AG AGTTT G ATCCTG GCT C AG -3'
1492R
5'-GGYTACCTT GTT ACGACTT-3'
A high-fidelity polymerase, Phusion™ (New England Biolabs, Ipswich, MA, USA) was used to
amplify the 16S rRNA gene from each of the 30 isolated colonies. PCR of the 30 isolated
colonies was carried out in a 50 |jL total volume containing: 1 X Phusion™ High-Fidelity (HF)
Buffer, 0.02 U/|jL of Phusion™ DNA Polymerase, 0.5 |jM of each primer, and 0.2 |jM of each
deoxyribonucleotide (dNTP) inoculated with 5 |jL of thermolyzed colonies. Cycling conditions
were carried out on an ABI 9700 thermocycler according to the following: an initial hold at 98°C
for 30 sec; 35 cycles of denaturation (98 °C for 10 sec), annealing (55 °C for 30 sec), and
extension (72 °C for 1 min); a final hold at 72 °C for 5 min. For samples amplified with primers
27F and 1492R, the annealing temperature was raised to 56 °C. PCR products were quantified
by UV-absorbance using the NanoDrop™ 2000 spectrophotometer (ThermoScientific, Waltham,
MA, USA).
Initially, pooled samples were subject to PCR using primers 8F and 1492R, and then amplified
using a polymerase with high resistance to many PCR inhibitors, Phire® (NEB) (New England
Biolabs, Ipswich, MA, USA). The Phire® PCR was carried out in 50 |jL total volume containing 1
X Phire® Animal Tissue PCR Buffer, 1 |jL of Phire® Hot Start II DNA Polymerase, and 0.5 |jM of
each primer, inoculated with 5 |jL of OneStep™ cleaned pooled sample. Cycling conditions
were carried out on an ABI 9700 thermocycler with an initial hold at 98 °C for 5 min; 40 cycles of
denaturation (98 °C for 5 sec), annealing (55 °C for 5 sec), and extension (72 °C for 40 sec); a
final hold at 72 °C for 1 min. Following amplification of the 16S rRNA gene, the size of the
amplified product was analyzed or visualized using 1.2 % Agarose E-Gel® (Life Technologies,
Grand Island, NY, USA) and an E-Gel® 1 Kb Plus DNA ladder (Life Technologies, Grand Island,
NY, USA).
A second amplification of the pooled samples was undertaken using the 27F and 1492R
primers; no further amplification was required for these PCR products prior to sequencing.
3.5.4.4 Sequencing of 16S rRNA genes
For sequencing of 16S rRNA from isolated colonies using an ABI 3130 Genetic Analyzer (Life
Technologies, Grand Island, NY, USA), the 16S rRNA PCR products generated from isolated
colonies were purified using the GenElute™ PCR Clean-up Kit (Sigma-Aldrich, St. Louis, MO,
USA), and the concentration of each PCR product was determined using the NanoDrop™ 2000
Spectrophotometer (ThermoScientific, Waltham, MA, USA). Forward and reverse cycle
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
sequencing reactions were set up using the same 8F and 1492R PCR primers that yielded the
original PCR product. Cycle sequencing was carried out using an ABI BigDye® Terminator v3.1
(Life Technologies, Grand Island, NY, USA) in 20 |jL total volume containing: 4 |jL of Ready
Reaction Mix, 2 |jL of BigDye Sequencing Buffer, 5 picomole primer, and 20 - 40 ng of 16S
rRNA PCR product. Cycling conditions were carried out on an ABI 9700 thermocycler with an
initial hold at 96 °C for 1 min; 25 cycles of denaturation (96 °C for 10 sec), annealing (50 °C for
5 sec), and extension (60 °C for 4 min). A positive control, pGEM®-3Zf(+), and NTC negative
controls were included. Cycle sequencing reactions were purified using the ABI BigDye®
XTerminator™ Purification Kit (Life Technologies, Grand Island, NY, USA) following the
manufacturer's instructions.
Capillary electrophoresis was conducted on each purified cycle sequencing reaction using the
ABI 3130 Genetic Analyzer with the run parameters shown in Table 13.
All raw sequencing files were imported into Sequencing Analysis Software v5.2 (Life
Technologies, Grand Island, NY, USA) and analyzed using the KB™ basecaller (Life
Technologies, Grand Island, NY, USA) to provide per-base quality value predictions. The KB
basecaller assigns a quality value (QV) for each basecall. A QV value of 20 or greater was
considered to be good quality, meaning that the probability that the base was miscalled is no
greater than 1%.
Table 13 Applied Biosystems 3130 Genetic Analyzer Run Parameters
Specific Parameters
Parameter
Setting
Template
BDx_StdSeq50_POP7
Oven Temperature
O
o
O
CD
Poly Fill Volume
5020 steps
Current Stability
5.0 Amps
Pre-Run Voltage
15.0 kVolts
Pre-Run Time
180 sec
Injection Voltage
1.6 kVolts
Injection Time
4 sec
Voltage Number of Steps
40 nk*
Voltage Step Interval
15 sec
Data Delay Time
480 sec
Run Voltage
8.5 kVolts
Run Time
6000 sec
* Number of voltage ramp steps to reach Run voltage
Six isolates were sequenced using the ABI 3130 Genetic Analyzer, and then the remaining
sequencing was completed on the Ion Torrent PGM™ (Life Technologies, Grand Island, NY,
USA). Sequencing on the traditional 3130 instrument requires an initial selection on solid
media, which can significantly limit the number of bacteria examined in a population as the
majority of environmental isolates are viable but nonculturable. Due to the massive parallel
sequencing capabilities, acquisition of genome sequences from several organisms at once was
possible without the bias of the primary culture. Additionally, sequence analysis on the Ion
Torrent PGM™ is less expensive than traditional sequencing methods.
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
For sequencing of 16S rRNA amplified with primers 8F and 1492R using Life Technologies Ion
Torrent Personal Genome Machine (PGM™), a total of fourteen 16S rRNA amplicons, including
a positive and negative control, 1550 base pairs (bp) in length, were initially processed to create
a sequencing library. Library preparation generated a pool of amplicons tagged with a specific
molecular barcode that allowed multiplexing of samples for analysis on a single PGM™
semiconductor chip. The Ion DNA Barcoding 1-16 kit (Life Technologies, Grand Island, NY,
USA) was used to prepare the library for the multiplexing experiment. Briefly, each of the 16S
DNA amplicons separately underwent enzymatic shearing to fractionate the 1550 bp products.
A purification step was performed using Agencourt® AMPure® magnetic particles (Beckman
Coulter, Brea, CA, USA) according to the manufacturer's instructions. Ion Barcode Adapters™
(Life Technologies, Grand Island, NY, USA) were ligated to the fragmented, purified DNA. An
additional purification step was performed using the Agencourt® AMPure® magnetic particles to
remove small molecular weight fragments. Following purification, additional PCR was
performed to incorporate unique molecular barcodes onto the adapter-modified, fragmented
DNA and further amplify each molecule. After PCR, a final purification step was performed
using the Agencourt® AMPure® magnetic particles. Each molecule in the final bar-coded library
preparation was approximately 180-210 bp in length, including amplicon sequence, adapter,
and barcode. Individual reactions were measured using the NanoDrop® instrument to quantify
DNA concentrations prior to pooling a portion of each reaction into a single mixed sample. The
concentration of the mixed sample was measured again using the NanoDrop® to determine the
library pool dilution required for sequencing.
To prepare the mixed barcoded sample for sequencing, clonal amplification was performed on
the Ion OneTouch™ instrument (Life Technologies, Grand Island, NY, USA). Briefly, the mixed,
barcoded library was combined with lonSphere Particles™ (ISPs) (Life Technologies, Grand
Island, NY, USA) followed by clonal amplification in an oil emulsion PCR, which binds a single
molecule to each particle and creates multiple copies of each particle-bound fragment.
Immediately following clonal amplification, the particle-bound fragments were enriched using the
Ion OneTouch ES™ instrument. This process removes unbound particles and unbound library
fragments to enrich particle-bound fragments. At this point, a quality control check was
performed, wherein a small amount of the enriched ISPs was quantitated using the Qubit® 2.0
Flourometer (Life Technologies, Grand Island, NY, USA) to determine the extent of enrichment.
After enrichment, ISPs were loaded into an Ion 316™ chip (a single ISP per well) (Life
Technologies, Grand Island, NY, USA), and sequencing was carried out according to
manufacturer's instructions.
Because sequencing could not be performed using the amplicons generated using the 8F and
1492R primer pair, a substitution was made for the forward primer. The primer substitution
resulted in amplification and high quality sequence data in all samples. For sequencing of 16S
rRNA amplified with primers 27F and 1492R using Life Technologies Ion Torrent PGM™,
qualitative and quantitative measurements of 16S amplicons were made using the Qubit dsDNA
BR Assay Kit on the Qubit 2.0 fluorometer followed by analysis on the Agilent Bioanalyzer
(Agilent Technologies, Santa Clara, CA, USA) using DNA High Sensitivity chips. Following this
procedure, 16S amplicon samples were fragmented using a Covaris S220 sonicator (Covaris,
Inc., Woburn, MA, USA) to generate approximately 300 bp fragments. Fragmentation quality
was assessed using an Agilent Bioanalyzer. Sequencing libraries were made using Ion Plus
Fragment Library kit (Life Technologies, Grand Island, NY, USA) for 200 bp sequencing.
Library quality was verified using the Agilent Bioanalyzer and the Qubit. Clonal amplification
was performed on an Ion One Touch instrument using the Ion Xpress™ Template 200 Kit (Life
Technologies, Grand Island, NY, USA). Enrichment for the ISPs was done on the Ion One
Touch ES, and quantification of the percent templated ISPs was performed on the Qubit
fluorometer. Sequencing was performed with 316 chips on an lonTorrent PGM sequencer using
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
the Ion Sequencing 200 kit. The lonTorrent Suite Server (1.5.1) performed base calling and
output raw sequence data in FASTQ format.
3.5.4.5 Sequence Analysis of 16S rRNA genes
Sequence reads from the ABI 3130 with a length of greater than 200 bps and high quality base
calls were subject to nucleotide-nucleotide Basic Local Alignment Search Tool (BLASTn) [10],
searching against the 16S microbial database. The BLASTn results with the highest maximum
identity percentage were reported.
FASTQ files were loaded into CLCBio Genomics Workbench software V 4.9 (CLCBio,
Cambridge, MA, USA). Trimming of sequence reads was performed to remove PCR primer
sequences and low quality reads (0.05 quality threshold). For trimming, the quality values were
used to establish a "clear range," bases from the ends of the sequence read were removed until
fewer than four bases out of 20 had a QV of less than 20. A final filtering of reads was
performed to select for reads of >175 bp. The National Institute of Health's National Center for
Bioinformatics (NCBI) 16S rRNA (v6/15/2102) sequence database was loaded into CLCBio as a
reference library. Two bioinformatics analyses were performed. First, read files were
processed using the Battelle Galileo high performance compute cluster and the Basic Local
Alignment Search Tool (BLAST®) (National Library of Medicine, Bethesda, MD, USA). Reads
were searched against the NCBI 16S rRNA gene database (v6/15/2102) (NCBI, Bethesda, MD),
which contained entries for 7,545 sequences. Search results were filtered for sequences with
>97% identity. The output from this search resulted in a list of taxonomic identifications,
associated organism names, and number of reads per taxiD for each sample. Krona v. 2.1 [11]
was used to create a comparative chart for viewing the relative abundance of organisms at the
genus level for each sample. A final filtering of results was performed to include only taxa
identified by numbers of hits greater than 0.1% (1:1000) of the total representation per sample.
The second analysis, the Battelle QUEST™ tool, a recent research and development (R&D)
using weighted probabilities based on genome coverage from reference aligned data, was used
to measure the amount of individual reads mapping to each 16S rRNA sequence with the
optimized parameters in CLCBio software and backend statistical analysis. The output was
reported as most probable species present in the sample.
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
4. RESULTS
4.1 Bg Detection by Quantitative PCR
The qPCR analysis for Bg signatures was complicated by the co-extraction of inhibitory
components from sample matrices. The extraction method (DWI-01-00; Appendix B) used for
this project is a slight modification of a method that has been used extensively to extract and
recover trace nucleic acids from environmental samples. Sample matrices successfully
processed using this method (DWI-01-00; Appendix B) include water, soil, cellulose, food, and
fabric compositions. Generally, any inhibitory components that are co-extracted with the DNA
can be counteracted by dilution (1:5 or 1:10) of the sample extract in 1XTE. In this case, only
approximately a third of the sample extracts could be analyzed Neat, 1:5, or 1:10. The
remaining two-thirds of the sample extracts required further purification using a Qiagen
QIAquick PCR purification kit. These samples were diluted and tested for inhibition at neat, 1:5,
1:10, and 1:20. Sample extracts that passed IPC were analyzed in duplicate for Bg rtp
signatures at the highest concentration that passed IPC. All controls performed as expected
(positive and negative), which demonstrate the validity of the methods used and that negative
results are not an artifact of method performance. Table 14 shows the results of qPCR,
including the analyzed dilution, the threshold cycle (Ct), and quantity in GC/5 |jL.
Bg DNA was not detected in any of the sample extracts. Sample number IRP-WIPE-10-21-11-
ABC-24 was first thought to be positive, but upon further investigation, the multicomponent plot
showed that the fluorescent signal in those wells was extremely high, and true amplification did
not occur. Samples that were inhibited at all tested dilutions (Neat, 1:5, 1:10, QN, Q5, Q10, and
Q20) were subject to PCR using the Phire® Animal Tissue Direct PCR Kit after pooling DNA
extracts into five composite samples comprised of nine or ten sample extracts (Table 9). Phire®
PCR was unsuccessful at amplification under these conditions; no amplification was observed in
any sample, including the positive control (1E4 GC/5 |jL amplified standard). Because the
positive control reaction did not amplify, the PCR conditions appeared to be sub-optimal, and it
was not possible to determine from this analysis whether these inhibited samples contained Bg
DNA. The results for a small subset of samples were inadvertently omitted from the Interim
Report. These samples were also analyzed by Phire® Animal Tissue Direct PCR Kit, in
duplicate reactions using the 7900HT, rather than in the sample pools as described above.
These samples did not amplify, also likely due to inhibition of the Phire® polymerase (results are
listed in Table 14). Note that the Sample identifications (IDs) listed in Table 14 include a
notation for the type of sample, date of sample acquisition, and a sampling location (see Figures
4 and 5). See Appendix B for a complete description of how positive and negative controls were
prepared (page 3 of Appendix B) and see page 4 of Appendix B for a description of how
controls were processed for RT-PCR. See page 9 of Appendix B for preparation of positive
controls for sequence analysis.
25
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
Table 14. Results of Bg qPCR Ana
yses
Sample ID
Dilution
Ct Value
GC/5 |iL*
Result"
IRP-AIR-10-19-11-ABC-B1
1:5
Undetermined
0
Negative
Undetermined
0
IRP-AIR-10-19-11-ABC-B2
1:5
Undetermined
0
Negative
Undetermined
0
IRP-AIR-10-19-11-ABC-B3
1:5
Undetermined
0
Negative
Undetermined
0
IRP-AIR-10-19-11-ABC-B4
1:5
Undetermined
0
Negative
Undetermined
0
IRP-AIR-10-19-11-ABC-B5
1:5
Undetermined
0
Negative
Undetermined
0
IRP-AIR-10-19-11-ABC-B6
1:5
Undetermined
0
Negative
Undetermined
0
IRP-AIR-10-19-11-ABC-B7
1:5
Undetermined
0
-------
Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
Table 14. Results of Bg qPCR Analyses (Continued)
Sample ID
Dilution
Ct Value
GC/5 |JL*
Result"
Undetermined
0
IRP-AIR-10-21 -11 -ABC-009
1:5
Undetermined
0
Negative
Undetermined
0
IRP-AIR-10-21 -11 -ABC-010
1:5
Undetermined
0
Negative
Undetermined
0
I RP-AI R-10-21-11-ABC-011
1:5
Undetermined
0
Negative
Undetermined
0
I RP-AI R-10-21-11-ABC-012
1:5
Undetermined
0
Negative
Undetermined
0
I RP-AI R-10-21-11-ABC-013
1:5
Undetermined
0
Negative
Undetermined
0
I RP-AI R-10-21-11-ABC-014
1:5
Undetermined
0
Negative
Undetermined
0
I RP-AI R-10-21-11-ABC-015
1:5
Undetermined
0
Negative
Undetermined
0
I RP-AI R-10-21-11-ABC-016
Neat
Undetermined
0
Negative
Undetermined
0
I RP-AI R-10-21-11-ABC-017
Neat
Undetermined
0
Negative
Undetermined
0
IRP-IW-10-20-11_ABC-001
Neat
Undetermined
0
-------
Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
Table 14. Results of Bg qPCR Analyses (Continued)
Sample ID
Dilution
Ct Value
GC/5 |JL*
Result"
Undetermined
0
IRP-WIPE-10-21-11 -ABC-0012
Qiagen 1:10
Undetermined
0
Negative
Undetermined
0
IRP-WIPE-10-21-11 -ABC-0013
Qiagen 1:5
Undetermined
0
Negative
Undetermined
0
I RP-WI PE-10-21-11 -ABC-0014
Qiagen 1:5
Undetermined
0
Negative
Undetermined
0
I RP-WI PE-10-21-11 -ABC-0018
Phire0
No Amplification
0
Undetermined
No Amplification
0
I RP-WI PE-10-21-11 -ABC-0019
Phire0
No Amplification
0
Undetermined
No Amplification
0
IRP-WIPE-10-20-11-ABC-020
Phire0
No Amplification
0
Undetermined
No Amplification
0
IRP-WIPE-10-20-11-ABC-021
Phire0
No Amplification
0
Undetermined
No Amplification
0
IRP-WIPE-10-20-11-ABC-022
Phire0
No Amplification
0
Undetermined
No Amplification
0
IRP-WIPE-10-20-11-ABC-023
Phire0
No Amplification
0
Undetermined
No Amplification
0
IRP-WIPE-10-21-11-ABC-0024
1:5
22.35
4.2E6
MC*™ Negative
19.84
1.45E7
IRP-WIPE-10-20-11-ABC-025
Phire0
No Amplification
0
Undetermined
No Amplification
0
IRP-WIPE-10-20-11-ABC-026
Phire0
No Amplification
0
Undetermined
No Amplification
0
IRP-WIPE-10-20-11-ABC-027
Phire0
No Amplification
0
Undetermined
No Amplification
0
IRP-WIPE-10-20-11-ABC-0028
Phire0
No Amplification
0
Undetermined
No Amplification
0
IRP-WIPE-10-21-11-ABC-0029
Phire0
No Amplification
0
Undetermined
No Amplification
0
IRP-WIPE-10-21-11-ABC-0031
Qiagen 1:5
Undetermined
0
Negative
Undetermined
0
IRP-WIPE-10-21-11-ABC-0033
Qiagen Neat
Undetermined
0
Negative
Undetermined
0
IRP-WIPE-10-21-11-ABC-0034
Qiagen Neat
Undetermined
0
Negative
Undetermined
0
IRP-WIPE-10-21-11-ABC-0036
Qiagen Neat
Undetermined
0
Negative
Undetermined
0
IRP-WIPE-10-21-11-ABC-0038
Qiagen 1:5
Undetermined
0
Negative
28
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
Table 14. Results of Bg qPCR Analyses (Continued)
Sample ID
Dilution
Ct Value
GC/5 |JL*
Result"
Undetermined
0
IRP-WIPE-10-21-11-ABC-0039
Qiagen 1:10
Undetermined
0
Negative
Undetermined
0
IRP-WIPE-10-21-11-ABC-0040
Qiagen Neat
Undetermined
0
Negative
Undetermined
0
IRP-WIPE-10-21-11-ABC-0041
Qiagen 1:20
Undetermined
0
Negative
Undetermined
0
IRP-WIPE-10-21-11-ABC-0043
Phire0
No Amplification
0
Undetermined
No Amplification
0
IRP-WIPE-10-21-11-ABC-0045
Qiagen 1:10
Undetermined
0
Negative
Undetermined
0
IRP-WIPE-10-21-11-ABC-0046
Qiagen 1:5
Undetermined
0
Negative
Undetermined
0
IRP-WIPE-10-21-11-ABC-0048
Qiagen 1:20
Undetermined
0
Negative
Undetermined
0
IRP-WIPE-10-21-11-ABC-0049
Qiagen 1:5
Undetermined
0
Negative
Undetermined
0
IRP-WIPE-10-24-11-ABC-050
Qiagen 1:5
Undetermined
0
Negative
Undetermined
0
IRP-WIPE-10-24-11-ABC-053
Qiagen 1:20
Undetermined
0
Negative
Undetermined
0
IRP-WIPE-10-24-11-ABC-054
Qiagen 1:10
Undetermined
0
Negative
Undetermined
0
IRP-WIPE-10-24-11-ABC-063
Phire0
No Amplification
0
Undetermined
No Amplification
0
IRP-WIPE-10-24-11-ABC-069
Qiagen 1:10
Undetermined
0
Negative
Undetermined
0
IRP-WIPE-10-24-11-ABC-079
Phire0
No Amplification
0
Undetermined
No Amplification
0
IRP-WIPE-10-24-11-ABC-082
Qiagen Neat
Undetermined
0
Negative
Undetermined
0
IRP-WIPE-10-24-11-ABC-085
Qiagen 1:10
Undetermined
0
Negative
Undetermined
0
IRP-WIPE-10-24-11-ABC-091
Qiagen 1:5
Undetermined
0
Negative
Undetermined
0
IRP-WIPE-10-24-11-ABC-094
Phire0
No Amplification
0
Undetermined
No Amplification
0
IRP-WIPE-10-24-11-ABC-095
Qiagen 1:5
Undetermined
0
Negative
Undetermined
0
IRP-WIPE-10-24-11-ABC-097
Qiagen 1:20
Undetermined
0
Negative
29
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
Table 14. Results of Bg qPCR Analyses (Continued)
Sample ID
Dilution
Ct Value
GC/5 |JL*
Result"
Undetermined
0
IRP-WIPE-10-24-11-ABC-098
Qiagen 1:10
Undetermined
0
Negative
Undetermined
0
IRP-WIPE-10-24-11-ABC-099
Qiagen 1:20
Undetermined
0
Negative
Undetermined
0
IRP-FPG-10-24-11-ABC-001
Neat
Undetermined
0
Negative
Undetermined
0
IRP-FPC-10-24-11 -ABC-001
1:5
Undetermined
0
Negative
Undetermined
0
Filter Blank 1
1:5
Undetermined
0
Negative
Undetermined
0
Filter Blank 2
1:5
Undetermined
0
Negative
Undetermined
0
Gauze Blank 1
1:5
Undetermined
0
Negative
Undetermined
0
Gauze Blank 2
Neat
Undetermined
0
Negative
Undetermined
0
Gauze Blank 3
Qiagen 1:5
Undetermined
0
Negative
Undetermined
0
Gauze Blank 4
1:5
Undetermined
0
Negative
Undetermined
0
Gauze Blank 5
Qiagen 1:5
Undetermined
0
Negative
Undetermined
0
Water Blank 1
Neat
Undetermined
0
Negative
Undetermined
0
Grease Blank 1
Neat
Undetermined
0
Negative
Undetermined
0
Filter PC 1
1:5
35.49
204.86
Positive
35.09
272.53
Filter PC 2
1:5
31.62
4821.90
Positive
31.06
6351.25
Gauze PC 1
1:5
32.65
1510.49
Positive
32.64
1523.35
Gauze PC 2
Neat
30.21
8087.46
Positive
30.04
8778.85
Gauze PC 3
Qiagen 1:5
32.21
3018.52
Positive
32.65
2425.59
Gauze PC 4
Qiagen 1:5
35.29
797.78
Positive
34.91
963.46
Gauze PC 5
Qiagen 1:5
38.43
171.44
Positive
30
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
Table 14. Results of Bg qPCR Analyses (Continued)
Sample ID
Dilution
Ct Value
GC/5 pL*
Result"
35.78
628.65
Water PC 1
1:5
31.37
3712.34
Positive
31.69
2976.29
Grease PC 1
Neat
32.93
1244.29
Positive
32.98
1200.10
Gene copies per 5 |jL of PCR reaction (after sample extraction, concentration by alcohol precipitation, re-suspension, etc.).
" Positive = > LOQ; LOQ was 92.1 GC/5 |jL; samples with mean <1 GC/5 |jL are considered Negative; samples with
multicomponent trace showing no amplification are considered Multicomponent Negative.
These sample extracts were inhibited Neat, 1:5, 1:10, and 1:20. They were further purified by Qiagen kit and diluted to overcome
inhibition.
MC = Multicomponent.
Sample extracts IRP-AIR-10-24-11-ABC-018 to IRP-AIR-10-24-11-ABC-025 and sample IRP-
AIR-10-24-11-ABC-27 were amplified on the ABI 9700 instrument and analyzed by gel
electrophoresis, with direct visualization of ethidium bromide-stained target amplicon (82 bp).
Positive control reactions containing 1000 GC/5 |jL standard control material and NTCs
containing 1X TE were prepared and analyzed along with the sample extracts. No Bg DNA was
detected in any of the samples or NTCs, but a faint band was observed in the 1000 GC/5 |jL
standard positive control well that could be consistent with the 82 bp amplicon (Figure 7). Note
that in Figure 7 the blue arrow denotes presence of a faint amplicon at -82 bp in the positive
control well. Bands are visible in the NTC and pooled sample wells but are migrating slightly
lower than the band in well 4 and may be primers. The amplification product in this figure was
generated using the primers developed for the quantitative PCR reaction against Bg rtp gene
(the Battelle assay), not the 8F and 1492R primers that were used for sequencing 16S rRNA.
The amplicon for the Bg Battelle assay generates an 82 bp amplicon and was used in an
attempt to detect amplification in samples processed using the Phire® Animal Tissue Direct PCR
kit — these samples had shown inhibition after extraction, dilution, and Qiagen purification with
subsequent dilution. The extensive inhibition was believed to be due to enzymatic and
proteolytic activity of the animal tissues present in/on the sample.
1 2 3 4 5 6 7 8
1 empty
2 pooled samples
3 empty
4 1000 GC/5mL
5 empty
6 1 Kb ladder
fc 7 empty
8 pooled NTC
Figure 7. Gel Electrophoresis of AIR-10-21-11 Samples Analyzed by PCR on the ABI
9700 Thermocycler
31
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
4.2 Detection of PLGA Microspheres
PLGA microspheres were observed in all positive control samples but in low quantities (i.e., less
than 20 microspheres per view). Sample autofluorescence prevented visualization of PLGA in
most samples; only samples IRP-WIPE-10-24-11-ABC-0089 and IRP-WIPE-10-24-11-ABC-
0099 contained fluorescent particles consistent with the PLGA microspheres. The gauze and
filter matrices are autofluorescent, creating a diffuse green background under the epifluorescent
conditions. Irregularly-shaped autofluorescent particulate matter in and on some sample
matrices made it impossible to discern PLGA microspheres, if present. Table 15 lists each
sample and the corresponding microscopic descriptions. Representative photos are also shown
in Table 15. Several of the samples had begun to support mold growth at the time of
microscopy, which also contributed to autofluorescence. Note that the Sample IDs listed in
Table 15 include a notation for the type of sample, date of sample acquisition, and a sampling
location (see Figures 4 and 5).
4.3 Enumeration of Putative Viable Bg in Archived Samples
After it was observed that the analyses of the original samples were not working well, the Test
Team decided to process the archive samples in a new, different manner in hopes of getting
better results. None of the air filter samples contained Bg, and no archival air samples were
collected. Ten gauze wipe samples had putative Bg colonies (Table 16). Putative Bg was
observed in six of these ten presumptive positives (i.e., putative) when plated for enumeration,
although one sample displayed quantities
-------
Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
Table 15. Microscopic Observations of PLGA Microsp
heres
Sample ID
Microscopic Observations
Image
IRP-AIR-10-19-11-ABC-B1
Dark field with diffuse green fluorescence; no PLGA
microspheres
N/A
IRP-AIR-10-19-11-ABC-B2
Diffuse green fluorescence, no PLGA microspheres
N/A
IRP-AIR-10-19-11-ABC-B3
Dark field with diffuse green fluorescence; no PLGA
microspheres
N/A
IRP-AIR-10-19-11-ABC-B4
Diffuse green fluorescence, no PLGA microspheres (Note -
photograph looks like a very dark green square; no features
observed)
IRP-AIR-10-19-11-ABC-B5
Dark field with diffuse green fluorescence; no PLGA
microspheres
IRP-AIR-10-19-11-ABC-B6
Diffuse green fluorescence, no PLGA microspheres
N/A
IRP-AIR-10-19-11-ABC-B7
Diffuse green fluorescence, no PLGA microspheres
N/A
IRP-AIR-10-19-11-ABC-B8
Diffuse green fluorescence, no PLGA microspheres
N/A
IRP-WIPE-10-19-11-ABC-B1
Two fluorescent particles observed, too large to be PLGA
microspheres
N/A
IRP-WIPE-10-19-11-ABC-B2
One-two fluorescent particles observed, too large to be PLGA
microspheres
N/A
IRP-WIPE-10-19-11-ABC-B3
No PLGA microspheres observed, background fluorescence
N/A
IRP-WIPE-10-19-11-ABC-B4
No PLGA microspheres observed, background fluorescence
N/A
IRP-WIPE-10-19-11-ABC-B5
No PLGA microspheres observed
N/A
IRP-AIR-10-20-11-ABC-001
Dark field with diffuse green fluorescence; no PLGA
microspheres
N/A
IRP-AIR-10-20-11 -ABC-002
Dark field with diffuse green fluorescence; no PLGA
microspheres
N/A
I RP-AIR-10-20-11-ABC-003
Dark field with diffuse green fluorescence; no PLGA
microspheres
N/A
I RP-AI R-10-20-11 -ABC-004
Dark field with diffuse green fluorescence; no PLGA
microspheres
N/A
I RP-AI R-10-20-11 -ABC-005
Dark field with diffuse green fluorescence; no PLGA
microspheres
N/A
I RP-AI R-10-20-11 -ABC-006
Dark field with diffuse green fluorescence; no PLGA
microspheres
N/A
I RP-AI R-10-20-11 -ABC-007
Dark field with diffuse green fluorescence; no PLGA
microspheres
N/A
I RP-AI R-10-20-11 -ABC-008
Dark field with diffuse green fluorescence; no PLGA
microspheres
N/A
I RP-AI R-10-21-11-ABC-009
Dark field with diffuse green fluorescence; no PLGA
microspheres
N/A
I RP-AI R-10-21-11-ABC-010
Dark field with diffuse green fluorescence; no PLGA
microspheres
N/A
I RP-AI R-10-21-11-ABC-011
Dark field with diffuse green fluorescence; no PLGA
microspheres
N/A
I RP-AI R-10-21-11 -ABC-012
Dark field with diffuse green fluorescence; no PLGA
microspheres
N/A
33
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
Table 15. Microscopic Observations of PLGA Microspheres
Sample ID
Microscopic Observations
Image
IRP-AIR-10-21-11 -ABC-013
Dark field with diffuse green fluorescence; no PLGA
microspheres
N/A
IRP-AIR-10-21-11 -ABC-014
Diffuse green background with no fluorescent particles
N/A
I RP-AI R-10-21-11-ABC-015
Diffuse green background with no fluorescent particles
N/A
I RP-AI R-10-21-11-ABC-016
Diffuse green background with no fluorescent particles
N/A
I RP-AI R-10-21-11-ABC-017
Diffuse green background with no fluorescent particles
N/A
IRP-IW-10-20-11-ABC-001
Observed crystalline-like fluorescent shards and spherical
fluorescent particles; none discernible as PLGA microspheres
N/A
IRP-WIPE-10-20-11 -ABC-001
Diffuse green background with many fluorescent particles of
irregular size and shape, indiscernible from PLGA microspheres
N/A
I RP-WIPE-10-20-11 -ABC-002
Diffuse green background with some fluorescent particles of
irregular size and shape, indiscernible from PLGA microspheres
N/A
I RP-WIPE-10-20-11 -ABC-003
Diffuse green background with some fluorescent particles of
irregular size and shape, indiscernible from PLGA microspheres
N/A
I RP-WIPE-10-20-11 -ABC-004
Dark field with diffuse some fluorescence; no PLGA
microspheres
N/A
I RP-WIPE-10-20-11 -ABC-005
Dark field with diffuse green fluorescence; no PLGA
microspheres
N/A
I RP-WIPE-10-20-11 -ABC-006
Dark field with diffuse green fluorescence; no PLGA
microspheres
N/A
I RP-WIPE-10-20-11 -ABC-007
Diffuse green background with some fluorescent particles of
irregular size and shape, indiscernible from PLGA microspheres
N/A
I RP-WIPE-10-20-11 -ABC-008
Dark field with diffuse green fluorescence; no PLGA
microspheres
N/A
I RP-WIPE-10-20-11 -ABC-009
Light field with diffuse green fluorescence; no PLGA
microspheres
N/A
IRP-WIPE-10-20-11 -ABC-0010
Light field with diffuse green fluorescence; no PLGA
microspheres
N/A
I RP-WIPE-10-21-11-ABC-0011
Dark field with diffuse green fluorescence; no PLGA
microspheres
N/A
I RP-WI PE-10-21-11 -ABC-0012
Dark field with diffuse green fluorescence; no PLGA
microspheres
N/A
I RP-WI PE-10-21-11 -ABC-0013
Diffuse green background with many fluorescent particles of
irregular size and shape, indiscernible from PLGA microspheres
N/A
I RP-WI PE-10-21-11 -ABC-0014
Dark field with diffuse green fluorescence; no PLGA
microspheres
N/A
I RP-WI PE-10-21-11 -ABC-0015
Dark field with diffuse green fluorescence; no PLGA
microspheres
N/A
I RP-WI PE-10-21-11 -ABC-0016
Dark field with diffuse green fluorescence; no PLGA
microspheres
N/A
I RP-WI PE-10-21-11 -ABC-0017
Dark field with diffuse green fluorescence; no PLGA
microspheres
N/A
I RP-WI PE-10-21-11 -ABC-0018
Dark field with diffuse green fluorescence; no PLGA
microspheres
N/A
I RP-WI PE-10-21-11 -ABC-0019
Light field with diffuse green fluorescence; no PLGA
microspheres
N/A
IRP-WIPE-10-21-11-ABC-0020
Light field with diffuse green fluorescence; no PLGA
microspheres
N/A
34
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
Table 15. Microscopic Observations of PLGA Microspheres
Sample ID
Microscopic Observations
Image
IRP-WIPE-10-21-11-ABC-0021
Dark field with diffuse green fluorescence; no PLGA
microspheres
N/A
IRP-WIPE-10-21-11-ABC-0022
Diffuse green background with many fluorescent particles of
irregular size and shape, indiscernible from PLGA microspheres
N/A
IRP-WIPE-10-21-11-ABC-0023
Dark field with diffuse green fluorescence; no PLGA
microspheres
N/A
IRP-WIPE-10-21-11-ABC-0024
Dark field with diffuse green fluorescence; no PLGA
microspheres
N/A
IRP-WIPE-10-21-11-ABC-0025
Dark field with diffuse green fluorescence; no PLGA
microspheres
N/A
IRP-WIPE-10-21-11-ABC-0026
Dark field with diffuse green fluorescence; no PLGA
microspheres
N/A
IRP-WIPE-10-21-11-ABC-0027
Diffuse green background with many fluorescent particles of
irregular size and shape, indiscernible from PLGA microspheres
N/A
IRP-WIPE-10-21-11-ABC-0028
Dark field with diffuse some fluorescence; no PLGA
microspheres
N/A
IRP-WIPE-10-21-11-ABC-0029
Dark field with diffuse green fluorescence; no PLGA
microspheres
N/A
IRP-WIPE-10-21-11-ABC-0030
Diffuse green background with many fluorescent particles of
irregular size and shape, indiscernible from PLGA microspheres
N/A
IRP-WIPE-10-21-11-ABC-0031
Bright green background with many fluorescent particles of
irregular size and shape, indiscernible from PLGA microspheres
N/A
IRP-WIPE-10-21-11-ABC-0032
Diffuse green background with few fluorescent particles of
irregular size and shape, indiscernible from PLGA microspheres
N/A
IRP-WIPE-10-21-11-ABC-0033
Bright green background with many fluorescent particles of
irregular size and shape, indiscernible from PLGA microspheres
N/A
IRP-WIPE-10-21-11-ABC-0034
Bright green background with many fluorescent particles of
irregular size and shape, indiscernible from PLGA microspheres
N/A
IRP-WIPE-10-21-11-ABC-0035
Bright green background with many fluorescent particles of
irregular size and shape, indiscernible from PLGA microspheres
N/A
IRP-WIPE-10-21-11-ABC-0036
Bright green background with many fluorescent particles of
irregular size and shape, indiscernible from PLGA microspheres
N/A
IRP-WIPE-10-21-11-ABC-0037
Very bright green background with few fluorescent particles of
irregular size and shape, indiscernible from PLGA microspheres
N/A
IRP-WIPE-10-21-11-ABC-0038
Very bright green background with few fluorescent particles of
irregular size and shape, indiscernible from PLGA microspheres
N/A
IRP-WIPE-10-21-11-ABC-0039
Diffuse green background with few fluorescent particles of
irregular size and shape, indiscernible from PLGA microspheres
N/A
IRP-WIPE-10-21-11-ABC-0040
Diffuse green background with few fluorescent particles of
irregular size and shape, indiscernible from PLGA microspheres
N/A
IRP-WIPE-10-21-11-ABC-0041
Diffuse green background with no fluorescent particles
N/A
IRP-WIPE-10-21-11-ABC-0042
Diffuse green background with no fluorescent particles
N/A
IRP-WIPE-10-21-11-ABC-0043
Diffuse green background with no fluorescent particles
N/A
IRP-WIPE-10-21-11-ABC-0044
Diffuse green background with no fluorescent particles
N/A
IRP-WIPE-10-21-11-ABC-0045
Diffuse green background with no fluorescent particles
N/A
IRP-WIPE-10-21-11-ABC-0046
Diffuse green background with no fluorescent particles
N/A
IRP-WIPE-10-21-11-ABC-0047
Diffuse green background with no fluorescent particles
N/A
IRP-WIPE-10-21-11-ABC-0048
Diffuse green background with no fluorescent particles
N/A
35
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
Table 15. Microscopic Observations of PLGA Microspheres
Sample ID
Microscopic Observations
Image
IRP-WIPE-10-21-11-ABC-0049
Diffuse green background with no fluorescent particles
N/A
IRP-WIPE-10-24-11 -ABC-050
Diffuse green background with no fluorescent particles
N/A
I RP-WIPE-10-24-11 -ABC-051
Diffuse green background with no fluorescent particles
N/A
I RP-WIPE-10-24-11 -ABC-052
Dark field with diffuse green fluorescence; no PLGA
microspheres
N/A
I RP-WIPE-10-24-11 -ABC-053
Dark field with diffuse green fluorescence; no PLGA
microspheres
N/A
I RP-WIPE-10-24-11 -ABC-054
Light field with diffuse green fluorescence; no PLGA
microspheres
N/A
I RP-WIPE-10-24-11 -ABC-055
Light field with diffuse green fluorescence; no PLGA
microspheres
N/A
I RP-WIPE-10-24-11 -ABC-056
Dark field with diffuse green fluorescence; no PLGA
microspheres
N/A
I RP-WIPE-10-24-11 -ABC-057
Dark field with diffuse green fluorescence; no PLGA
microspheres
N/A
I RP-WIPE-10-24-11 -ABC-058
Dark field with diffuse green fluorescence; no PLGA
microspheres
N/A
I RP-WIPE-10-24-11 -ABC-059
Diffuse green background with few fluorescent particles of
irregular size and shape, indiscernible from PLGA microspheres
N/A
I RP-WIPE-10-24-11 -ABC-060
Diffuse green background with many fluorescent particles of
irregular size and shape, indiscernible from PLGA microspheres
N/A
I RP-WIPE-10-24-11 -ABC-061
Diffuse green background with no fluorescent particles
N/A
I RP-WIPE-10-24-11 -ABC-062
Diffuse green background with few fluorescent particles of
irregular size and shape, indiscernible from PLGA microspheres
N/A
I RP-WIPE-10-24-11 -ABC-063
Diffuse green background with many fluorescent particles of
irregular size and shape, indiscernible from PLGA microspheres
N/A
I RP-WIPE-10-24-11 -ABC-064
Diffuse green background with many fluorescent particles of
irregular size and shape, indiscernible from PLGA microspheres
N/A
I RP-WIPE-10-24-11 -ABC-065
Diffuse green background with few fluorescent particles of
irregular size and shape, indiscernible from PLGA microspheres
N/A
I RP-WIPE-10-24-11 -ABC-066
Diffuse green background with few fluorescent particles of
irregular size and shape, indiscernible from PLGA microspheres
N/A
I RP-WIPE-10-24-11 -ABC-067
Diffuse green background with no fluorescent particles
N/A
I RP-WIPE-10-24-11 -ABC-068
Diffuse green background with many fluorescent particles of
irregular size and shape, indiscernible from PLGA microspheres
N/A
I RP-WIPE-10-24-11 -ABC-069
Diffuse green background with many fluorescent particles of
irregular size and shape, indiscernible from PLGA microspheres
N/A
I RP-WIPE-10-24-11 -ABC-070
Diffuse green background with many fluorescent particles of
irregular size and shape, indiscernible from PLGA microspheres
N/A
I RP-WIPE-10-24-11 -ABC-071
Diffuse green background with few fluorescent particles of
irregular size and shape, indiscernible from PLGA microspheres
N/A
I RP-WIPE-10-24-11 -ABC-072
Bright green background with many fluorescent particles of
irregular size and shape, indiscernible from PLGA microspheres
N/A
I RP-WIPE-10-24-11 -ABC-073
Diffuse green background with no fluorescent particles
N/A
I RP-WIPE-10-24-11 -ABC-074
Diffuse green background with few fluorescent particles of
irregular size and shape, indiscernible from PLGA microspheres
N/A
I RP-WIPE-10-24-11 -ABC-075
Diffuse green background with few fluorescent particles of
irregular size and shape, indiscernible from PLGA microspheres
N/A
36
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
Table 15. Microscopic Observations of PLGA Microspheres
Sample ID
Microscopic Observations
Image
IRP-WIPE-10-24-11 -ABC-076
Diffuse green background with many fluorescent particles of
irregular size and shape, indiscernible from PLGA microspheres
N/A
I RP-WIPE-10-24-11 -ABC-077
Diffuse green background with few fluorescent particles of
irregular size and shape, indiscernible from PLGA microspheres
N/A
I RP-WIPE-10-24-11 -ABC-078
Diffuse green background with few fluorescent particles of
irregular size and shape, indiscernible from PLGA microspheres
N/A
I RP-WIPE-10-24-11 -ABC-079
Diffuse green background with no fluorescent particles
N/A
I RP-WIPE-10-24-11 -ABC-080
Diffuse green background with many fluorescent particles of
irregular size and shape, indiscernible from PLGA microspheres
N/A
I RP-WIPE-10-24-11 -ABC-081
Diffuse green background with few fluorescent particles of
irregular size and shape, indiscernible from PLGA microspheres
N/A
I RP-WIPE-10-24-11 -ABC-082
Diffuse green background with few fluorescent particles of
irregular size and shape, indiscernible from PLGA microspheres
N/A
I RP-WIPE-10-24-11 -ABC-083
Bright green background with many fluorescent particles of
irregular size and shape, indiscernible from PLGA microspheres
N/A
I RP-WIPE-10-24-11 -ABC-084
Diffuse green background with no fluorescent particles
N/A
I RP-WIPE-10-24-11 -ABC-085
Diffuse green background with no fluorescent particles
N/A
I RP-WIPE-10-24-11 -ABC-086
Diffuse green background with no fluorescent particles
N/A
I RP-WIPE-10-24-11 -ABC-087
Diffuse green background with many fluorescent particles of
irregular size and shape, indiscernible from PLGA microspheres
N/A
I RP-WIPE-10-24-11 -ABC-088
Diffuse green background with few fluorescent particles of
irregular size and shape, indiscernible from PLGA microspheres
N/A
I RP-WIPE-10-24-11 -ABC-089
Dark background with diffuse green fluorescence, 1 fluorescent
particle observed consistent with PLGA microsphere
N/A
I RP-WIPE-10-24-11 -ABC-090
Diffuse green background with many fluorescent particles of
irregular size and shape, indiscernible from PLGA microspheres
N/A
I RP-WIPE-10-24-11 -ABC-091
Diffuse green background with no fluorescent particles
N/A
I RP-WIPE-10-24-11 -ABC-092
Diffuse green background with many fluorescent particles of
irregular size and shape, indiscernible from PLGA microspheres
N/A
I RP-WIPE-10-24-11 -ABC-093
Diffuse green background with many fluorescent particles of
irregular size and shape, indiscernible from PLGA microspheres
N/A
I RP-WIPE-10-24-11 -ABC-094
Dark background with green fluorescence, many fluorescent
particles of irregular size and shape, indiscernible from PLGA
microspheres
N/A
I RP-WIPE-10-24-11 -ABC-095
Diffuse green background with few fluorescent particles of
irregular size and shape, indiscernible from PLGA microspheres
N/A
I RP-WIPE-10-24-11 -ABC-096
Diffuse green background with few fluorescent particles of
irregular size and shape, indiscernible from PLGA microspheres
N/A
I RP-WIPE-10-24-11 -ABC-097
Diffuse green background with no fluorescent particles
N/A
I RP-WIPE-10-24-11 -ABC-098
Diffuse green background with no fluorescent particles
N/A
I RP-WIPE-10-24-11 -ABC-099
Very diffuse green background, ~5 fluorescent particles
observed consistent with PLGA microspheres
N/A
IRP-FPG-10-24-11 -ABC-001
N/A
N/A
IRP-FPC-10-24-11 -ABC-001
N/A
N/A
IRP-AIR-10-24-11 -ABC-018
Diffuse green background with no fluorescent particles
N/A
IRP-AIR-10-24-11 -ABC-019
Diffuse green background with no fluorescent particles
N/A
I RP-AI R-10-24-11 -ABC-020
Diffuse green background with no fluorescent particles
N/A
37
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
Table 15. Microscopic Observations of PLGA Microspheres
Sample ID
Microscopic Observations
Image
IRP-AIR-10-24-11-ABC-021
Diffuse green background with no fluorescent particles
N/A
IRP-AIR-10-24-11 -ABC-022
Diffuse green background with no fluorescent particles
N/A
I RP-AI R-10-24-11 -ABC-023
Diffuse green background with no fluorescent particles
N/A
I RP-AI R-10-24-11 -ABC-024
Diffuse green background with no fluorescent particles
N/A
I RP-AI R-10-24-11 -ABC-025
Diffuse green background with no fluorescent particles
N/A
I RP-AI R-10-24-11 -ABC-027
Diffuse green background with no fluorescent particles
N/A
Filter Blank 1
Diffuse green background with no fluorescent particles
N/A
Filter Blank 2
Diffuse green background with no fluorescent particles
N/A
Gauze Blank 1
Diffuse green background with no fluorescent particles
N/A
Gauze Blank 2
Green fluorescent background, no PLGA microspheres or
fluorescent particles
N/A
Gauze Blank 3
Very diffuse green background, no PLGA microspheres or
fluorescent particles
N/A
Gauze Blank 4
Diffuse green background, no PLGA microspheres or
fluorescent particles
N/A
Gauze Blank 5
Diffuse green background with no fluorescent particles
N/A
Water Blank 1
No fluorescent particles
N/A
Grease Blank 1
N/A
N/A
Filter PC 1
Many fluorescent PLGA microspheres observed in both
membrane and batting layer. (Note - photograph looks like a
very dark green square; no features observed)
Filter PC 2
Very bright green background with ~20 PLGA microspheres
visible on the membrane; no PLGA microspheres visible on the
batting
N/A
Gauze PC 1
Some fluorescent PLGA microspheres observed; fewer than on
Filter PC1
N/A
Gauze PC 2
Some fluorescent PLGA microspheres observed in background
of green autofluorescence
Gauze PC 3
Bright green background with one fluorescent particle
suspected to be PLGA microsphere
N/A
Gauze PC 4
Some fluorescent PLGA microspheres observed (~eight) in
diffuse green background
N/A
Gauze PC 5
Very bright green background, few PLGA microspheres
observed
N/A
Water PC 1
Five fluorescent PLGA microspheres
N/A
Grease PC 1
N/A
N/A
N/A - Not available
38
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
4.4 Identification of Background Microbial Flora by Sequence Analysis
4.4.1 Extraction of DNA
Initially, each of the 30 isolates and five pooled samples were extracted following the DNeasy®
Gram-positive bacteria protocol (Qiagen, Valencia, CA, USA). The DNeasy® extracts could not
be used for PCR due to background 16S rRNA DNA in one of the enzymatic lysis buffer
components. A thermolysis technique was used to reduce the number of reagents needed for
extraction, thus reducing the likelihood of contamination. All samples were heat-shocked. This
method worked well for five of the 30 isolates and resulted in a faint PCR product in eight other
isolates. These eight faint PCR products were purified and amplified again using the same 8F
and 1492R primers.
None of the pooled samples amplified when extracted using the thermolysis technique and
Phusion™ polymerase. The pooled samples that were extracted by thermolysis, as well as an
aliquot of each pooled sample that had not gone through an extraction method, were cleaned
using an OneStep™ PCR Inhibitor Removal Kit (Zymo) and amplified using Phire® polymerase.
Four of the five pooled samples that had been thermolyzed resulted in a PCR product after
cleaning, and all five pooled samples that had no prior extraction procedure amplified after
removing inhibitors.
4.4.2 Amplification of 16S rRNA
To isolate background microorganisms, 58 samples were plated onto BHIA and incubated
overnight at 36 ± 2 °C with a positive control of Bg. Colonies resembling the Bg positive control
were identified in 15 of the 58 samples (Table 17). Thirty colonies with variable morphologies
not resembling Bg were selected for sequence analysis. The 16S rRNA gene was successfully
amplified from only 13 of the 30 unknown isolates when using the 8F and 1492R primers. Of
those 13 samples, only five resulted in a clean PCR product with a concentration of at least 13
ng/|jL. All five of the isolates that resulted in a clean PCR product were able to be identified by
sequencing using the ABI 3130.
The majority of the organisms that were isolated either could not be extracted using the
thermolysis method or were not compatible with the 8F and 1492R primers. While 8F and
1492R primers are considered "universal primers", they are probably not ideal for all bacterial
species, and other "universal primers" that target the 16S rRNA gene could potentially be used
to amplify a portion of the gene.
Although not confirmed by PCR, if the putative CFU that were identified based on colony
morphologies (Table 17) are assumed to be Bg, there does appear to be a trend that the
number of positive samples (not the enumerated CFU, because those results were not available
for these samples) for the Post-Cleaning Phase samples (N=10) are significantly higher than the
number of positive samples for the Post-Inoculation Phase samples (N=4). In addition, the
locations within the plant where positive samples were found were much more widely dispersed
throughout the plant in the case of the Post-Cleaning Phase samples. This observation
suggests that the cleaning process using the steam and hot water has the potential to spread
the contaminant around the plant, even if the cleaning process results in a reduction in the
overall levels of contamination. The Sample IDs listed in Table 17 include a notation for the
type of sample, date of sample acquisition, and a sampling location (see Figures 4 and 5).
39
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
4.4.3 Sequencing of 16S rRNA
4.4.3.1 Sequencing of 16S rRNA from Isolated Colonies using Applied Biosystems 3130
Genetic Analyzer
The BLASTn result with the highest maximum identity percentage and the top 25 BLAST®
results, as well as the sequence information obtained, are described in Appendix B. Six of the
30 unknown isolates resulted in at least one high quality sequencing read. Isolates 4, 19, 22,
and 29 are likely Proteus species, isolate 15 is likely a Planomicrobium species, and isolate 16
is likely a Curtobacterium species.
4.4.3.2 Seguencing of 16S rRNA Amplified with Primers 8F and 1492R using Ion Torrent
PGM™
Initial sequencing on the PGM™ yielded poor results most likely due to failure of the library
preparation. Poor quality 16S DNA amplicons and/or the possibility of carryover inhibitory
components may have caused the library preparation to fail. The PGM™ functioned properly,
and a successful run occurred. After examination of the run, summary evidence pointed to the
likelihood that poor clonal amplification had occurred on the OneTouch™. The ISPs loaded
correctly into the micron-sized wells, and all fluidics and semiconductor functions operated
normally. However, template ISPs gave a reading of 8.23% on the Qubit® during the quality
analysis check prior to sequencing. The percentage recommended to proceed with sequencing
is >50%. Poor clonal amplification was potentially due to poor library construction in the
presence of inhibitors that interfered with ligation of the molecular barcodes and adapters. This
step is crucial for all other subsequent steps in the library preparation and sequencing.
Sequencing reads generated on the PGM™ were of low quality; a quality filtration was
performed on the reads using CLCGenomics Workbench software, but there were too few reads
post-filtration to perform accurate BLAST® analysis or assembly. The reads remaining after
filtration were not analogous to anything in the 16S database. No data were therefore obtained
from the PGM™ analysis.
4.4.3.3 Seguencing of 16S rRNA Amplified with Primers 27F and 1492R using Ion Torrent
PGM™
The 16S rRNA PCR strategy with 27F and 1492 primers was successful in producing amplicons
from all five pooled samples. Pools 2 through 5 gave high quality sequence data resulting from
lonTorrent sequencing. Pool 1 did not yield sufficiently high quality data, due either to the 16S
amplicon quality (source sample influence) or sequencing library and sequencer quality
(sequencing influence). Resequencing of pool 1 was not performed due to time and budget
constraints.
40
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
Table 16. Enumeration of Putative Bg Colonies in Sample Extracts
A. Prior to Heat-Shock
Sample Number
Plated Dilution
Average Plate Count
Final Enumeration
(CFU/mL)
IRP-WIPE-10-20-11-D-001
1 x 10-1
0
0
0
0
IRP-WIPE-10-20-11-D-004
1 x 10-1
0
0
0
0
IRP-WIPE-10-20-11-D-006**
1 x 10-1
<30
36
114
7.50E2
IRP-WIPE-10-20-11-D-007
1 x 10-1
0
<30
<30
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
Table 17. Samples Containing Colony Morphologies Similar to Bg
Bg-Containing Samples
Sample Description
IRP-WIPE-10-20-11 -ABC-002
IRP-WIPE-10-20-11 -ABC-003
I RP-WI PE-10-20-11 -ABC-006
I RP-WI PE-10-20-11 -ABC-008
IRP-WIPE-10-21-11-ABC-0011
IRP-WIPE-10-21-11-ABC-0012
IRP-WIPE-10-21-11-ABC-0014
IRP-WIPE-10-21-11 -ABC-0017
IRP-WIPE-10-21-11 -ABC-0018
IRP-WIPE-10-21-11 -ABC-0019
IRP-WIPE-10-21-11-ABC-0024
IRP-WIPE-10-21-11-ABC-0026
IRP-WIPE-10-21-11-ABC-0027
IRP-WIPE-10-21-11-ABC-0029
Post inoculation; Tipping floor, 12 feet (ft) from pit
Post inoculation; wall of auger
Post inoculation; Tipping floor, 12 ft from pit wall
Post inoculation; Tipping floor, 12 ft from pit wall
Post cleaning; Center-left of tipping floor near door
Post cleaning; Grinder wall; left side
Post cleaning; Between floor drains; in front of electrical panel
Post cleaning; End of railroad tracks
Post cleaning; Three ft south of western tallow tank
Post cleaning; Office door; cooker room
Post cleaning; two ft from small crax grinder control panel
Post cleaning; Center of doorway near M3 tallow tank
Post cleaning; walkway, four ft from stairs near SS5 tank
Post cleaning; six in from drain near maintenance roll-up door
Table 18 shows the dominant genera of bacteria identified by BLASTn search and the most
probable species identified by the Battelle QUEST™ method. Figures 8-11 present
hierarchically organized relative abundance data resulting from Ion Torrent PGM™ sequence
analysis using the KRONA tool. KRONA is an open source software built with HTML5 (web-
browser format) that may ingest BLAST® data and prepare visual results of the relative
abundances of the total top BLAST® hits. The KRONA maps in Figures 8-11 show resolution at
the genus level (outer ring of the circle) organized to lower sub-classifications (inner radii of the
circle). Percentages of BLAST® reads matching each group of bacteria is included in the figure
to assist in interpretation. In general, all pools had Pseudomonas as the most prevalent genus,
ranging from 31-87% of the total genetic sequences identified (Table 18). Pool five was the
least diverse sample with Pseudomonas and Shewanella species comprising 95% of the
sample. Other genera of bacteria discovered in the pools included Stenotrophomonas,
Xanthomonas, Comomonas, Herbaspirilium, Lactobacillus, Acinetobacter, and Yersinia. The
genus Bacillus was not observed in pools 2, 4 and 5 and was at a level near to the limit of
detection for the methods used in pooled sample 3 (0.04% of the genetic material identified).
Further, most of the species identified from pools 2-5 belonged to the phylum Proteobacteria,
with low observance (<5%) of the phyla Firmicutes, Bacteroidetes and Actinomycetales (Figures
8-11). In general, the pools had similar profiles of bacteria identified by 16S sequencing,
varying mostly by abundance of genera between pools.
Amplification of 16S rRNA genes was accomplished in only 13 of 30 attempted reactions from
the isolated colonies, and sequence analysis of the 16S rRNA genes was achieved for only six
of these 13 amplicons. The remaining seven amplicons were likely of poor quality and not
suitable for sequence analysis. Amplification of 16S rRNA genes is performed using 'universal'
primers that are generated to conserved regions in the 16S genes. However, there are several
sets of primers that can be used, and PCR conditions may favor certain amplicons over others.
If a different primer set is chosen, additional isolates may be identified.
Sequence analysis of the pooled sample extracts was improved using primers 27F and 1492R
(as compared to primers 8F and 1492R). Pseudomonas was the primary genus present in
sample pools 2 through 5. Sequence analysis could not be performed on pool 1; the 16S
amplicons were of insufficient quality.
42
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
Table 18. Results of 16S rRNA Sequencing Based on BLAST8 and QUEST™ Analysis
Sample
Dominant Genera by BLAST®
Dominant Organisms by QUEST™
(top 15 most probable species)
Pool 1
ND*
ND
Pool 2
Pseudomonas (48%)
Stenotrophomonas (18%)
Xanthomonas (5%)
Yersinia (4%)
Comamonas (4%)
Stenotrophomonas_rhizophila_strain_e-p10
Pseudomonas_fragi_strain_ATCC_4973
Acidaminococcus_intestini_strain_ADV_255.99
Stenotrophomonas_maltophilia_strain_IAM_12423
Acidaminococcus_fermentans_strain_VR4
Comamonas_kerstersii_strain_LMG_3475
Simplicispira_metamorpha_strain_DSM_1837
Comamonas_aquatica_strain_:_LMG_2370
Pseudomonas_psychrophila_strain_E-3
Microvirgula_aerodenitrificans_strain_Sgly2
Pseudomonas_lundensis_strain_ATCC_49968
Stenotrophomonas_koreensis_strain_TR6-01
Dysgonomonas_capnocytophagoides_strain_LMG
Pseudomonas_agarici_strain_71A
B revu n d i mo n as_te rrae_strai n_KS L-145
Pool 3
Pseudomonas (31%)
Shewanella (18%)
Acinetobacter (7%)
Herbaspirillium (6%)
Stenotrophomonas (4%)
Lactobacillus (3%)
Shewanella_baltica_strain_63
Stenotrophomonas_rhizophila_strain_e-p10
Pseudomonas_fragi_strain_ATCC_4973
Herbaspirillum_autotrophicum_strain_IAM_14942
Shewanella_morhuae_strain_U1417
Morganella_psychrotolerans_strain_U2/3
Herbaspirillum_rhizosphaerae_strain_UMS-37
Paucimonas_lemoignei_strain_LMG_2207
Acinetobacter_ursingii_strain_LUH
Arcobacter_nitrofigilis_strain_CI
Dysgonomonas_capnocytophagoides_strain_LMG
Lactobacillus_curvatus_strain_:DSM_20019
Shewanella_putrefaciens_strain_LMG_26268
Myroides_odoratimimus_strain_:_CCUG_39352
Acinetobacter_haemolyticus_strain_DSM_6962
43
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
Table 18. Results of 16S rRNA Sequencing Based on BLAST® and QUEST™ Analysis
Sample
Dominant Genera by BLAST®
Dominant Organisms by QUEST™
(top 15 most probable species)
Pool 4
Pseudomonas (34%)
Stenotrophomonas (42%)
Xanthomonas (10%)
Pseudoxanthomonas (3%)
Stenotrophomonas_rhizophila_strain_e-p10
Pseudomonas_fragi_strain_ATCC_4973
Stenotrophomonas_koreensis_strain_TR6-01
Stenotrophomonas_maltophilia_strain_IAM_12423
Pseudomonas_hibiscicola_strain_ATCC_19867
Pseudomonas_psychrophila_strain_E-3
Ste n otrop h o mo n as_n itriti red u ce n s_strai n_L2
Pseudomonas_geniculata_strain_ATCC_19374
Pseudomonas_mucidolens_strain_IAM 12406
Pseudoxanthomonas_spadix_strain_IMMIB_AFH-5
Mycoplana_bullata_strain_IAM_13153
Stenotrophomonas_terrae_strain_:_R-32768
Pseudomonas_extremorientalis_strain_KMM_3447
Pseudomonas_abietaniphila_strain_ATCC_700689
Pseudomonas_moraviensis_strain_CCM_7280
Pool 5
Pseudomonas (87%)
Shewanella (8%)
Pseudomonas_fragi_strain_ATCC_4973
Pseudomonas_agarici_strain_71A
Shewanella_putrefaciens_strain_LMG_26268
Pseudomonas_psychrophila_strain_E-3
Shewanella_baltica_strain_63
Pseudomonas_lundensis_strain_ATCC_49968
Pseudomonas_veronii_strain_CIP_104663
Pseudomonas_libanensis_strain_CIP_105460
Stenotrophomonas_rhizophila_strain_e-p10_
Pseudomonas_palleroniana_strain_CFBP_4389
Shewanella_hafniensis_strain_P010
Shewanella_oneidensis_strain_MR-1
Pseudomonas_mucidolens_strain_IAM 12406
Pseudomonas_caricapapayae_strain_Robbs_ENA-378
Pseudomonas_taetrolens_strain_l 11
*ND = no data
44
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
pf0teobacteria
Bacteria
Figure 8. KRONA Visualization of BLAST® Results for Pool 2
Count: 21658
Tax ID: 2
Rank: superlcingdom
Avg. log e-value: -89.3719
^2 rf*ofe
4% Yersinia
30 more
12 "lore
Pseudomonas I 48%
45
-------
Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
Bacteria
'asP'ri//um
ad-root.!ocal
Internet access
Figure 9. KRONA Visualization of BLAST® Results for Pool 3
46
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
Count: 3105
^proieobactena
Proteobacteria
,bacteria
-t erased
ad-root.!ocal
Internet access
frlphap'°*e0
Ca»to6ac'
Figure 10. KRONA Visualization of BLAST® Results for Pool 4
47
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
Gammaproteobacteria
Proteobacteria
Bacteria
Figure 11. KRONA Visualization of BLAST® Results for Pool 5
Bacteria
Count: 79754
Tax ID: 2
Rank: superkingdom
Avg. log e-value: -92.6326
4.5 Summary of Results
Quality control samples arid spiked samples were analyzed for Bg DNA. All the positive
controls came back positive. All the negative controls were negative (there was only one and it
was the distilled water). Bg DNA was not detected in any of the sample extracts collected from
the rendering plant from either the surface wipe samples or the air samples. However, viable
bacteria very similar to Bg positive control colony morphology were recovered from 15 of 58
samples that the original sample extracts were created from (air samples B1-B8, air samples
001-025 and -027, wipe samples 001-024), and from ten of the archived test samples (five
contained putative Bg in quantities greater than LOQ). Samples with one or more individual
plate counts <30 were reported as
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
grease, bone, hair, etc., that carried over during the extraction process because Bg was
recoverable on BHIA.
Detection of Bg DNA via PCR techniques might possibly be improved by dramatically increasing
the concentration of Bg in the inoculum. However, the concentration is likely to need to be
increased by one or more orders of magnitude, which may rapidly become cost-prohibitive for
field testing of full-scale facilities. Because the heat shocking techniques did appear to improve
detectability of Bg via conventional nonmolecular microbiological techniques, the use of heat-
shock should be standard procedure for enumeration of rendering plant samples when using Bg
as a surrogate.
The results of the analyses indicated that PLGA microspheres may not be a suitable synthetic
surrogate. The microspheres appear to become immobilized in the sampling matrices, and the
particles autofluoresce at a wavelength similar to hair and bone fragments, thus making the
PLGA spheres difficult to distinguish from background. Also, the extraction processes were
ineffective for removing PLGA microspheres for quantitation by fluorometer, and
autofluorescence from the sample matrices complicated detection of PLGA microspheres via
direct microscopic observation. Use of different-colored PLGA spheres, which may not
autofluoresce at the same wavelengths as the materials in the sampling matrices, may be
possible, although use of different-colored PLGA spheres would require additional methods
development work.
Although it is difficult to perform a purely quantitative complete assessment of these results
because the PCR was unsuccessful at reliably recovering Bg DNA from the samples,
conventional nonmolecular microbiological methods appeared to succeed. If the putatively
identified Bg CFU in Table 16, Section B are examined as a whole, the enumerated CFU from
the samples taken on October 20, 2011 (the Post-Inoculation Phase sampling) appear to be
approximately an order of magnitude or more higher than the enumerated CFU from the
samples taken on October 21, 2011 (the Post-Cleaning Phase sampling). This difference
suggests that routine plant cleaning procedures may potentially result in a 1-log reduction in
pathogen loading within the potentially contaminated areas of the plant. Sufficient data to
perform a statistical analysis on these results were lacking. This reduction in pathogen loading
is not inconsistent with results from previous systematic studies examining the effectiveness of
different steps of a multi-step cleaning/disinfection process that showed a 1-4 log reduction from
individual cleaning/disinfection steps [12], although this reduction should be treated only as
semi-quantitative in nature because the samples that showed putative Bg colonies were not
always found at collocated sampling locations, and recovery of Bg spores from the sample
media was poor. The plant cleaning procedures used in this study utilized hot water and steam,
which would have been expected to remove contamination from surfaces and transfer any
removed contamination into the rinse water going into the drains, as opposed to actually killing
any pathogens that may have existed in the rinsate.
Although not confirmed by PCR, if the putative CFU that were identified based on colony
morphologies (Table 17) are assumed to be Bg, there does appear to be a trend that the
number of positive samples (not the enumerated CFU because those results were not available
for these samples) for the Post-Cleaning Phase samples (N=10) are higher than the number of
positive samples for the Post-Inoculation Phase samples (N=4). In addition, the locations within
the plant where positive samples were found were much more diverse in the case of the Post-
Cleaning Phase samples. Figure 12 illustrates these observations. Although the most heavily
contaminated area (the Pit) did not show putative Bg colonies after cleaning, the cooker area
and other areas that are not typically directly exposed to the raw material showed putative Bg
colonies.
49
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
These observations suggest that the cleaning process using the steam and hot water has the
potential to spread the contaminant throughout the plant, even if the cleaning process results in
a reduction in the overall levels of contamination. It is not entirely clear whether this spread of
contamination is the result of plant personnel tracking the surrogate to various locations within
the plant or due to aerosol transport High pressure spraying operations have indeed been
shown to result in aerosol transport of spores [13]; however, in this study, no air samples
exhibited any Bg, either through PGR analysis or examination of colony morphology.
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50
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
5. QUALITY ASSURANCE/QUALITY CONTROL
This effort attempted to achieve three objectives to reach the overall stated goal of evaluating
cleanup of a rendering plant following its use for disposal following an FAD outbreak:
• To generate data on fugitive emissions of a biological surrogate during the rendering
process;
• To determine the effectiveness of plant cleaning procedures for reducing the surrogate
levels on the inside surfaces of the rendering facility; and
• To provide information that could be used to develop standard procedures for appropriately
clearing a rendering facility that has been used for "disposal rendering" after an FAD
outbreak so that the rendering facility can be returned to normal production.
An external review was not performed. However, an internal independent QC review was
performed by the laboratory prior to delivery of results to the EPA Test Team.
5.1 Experimental Approach
Environmental characterization, decontamination, and clearance are critical components of a
comprehensive public health recovery strategy in the aftermath of an FAD outbreak or a
biological agent terrorist incident. This study looked to investigate the unique environment of a
rendering plant and the rendering process that could play a critical role in the nation's response
to an FAD event by assisting in the control of diseases and providing a mechanism to recycle
usable animal carcasses into safe and usable products.
As one step toward addressing the clearance goals for returning a rendering plant back to
normal operation, the EPA, the U.S. Department of Agriculture, and private industry worked
together to evaluate fugitive emissions of a biological surrogate released from a rendering
process. The evaluation process included characterizing the native bacterial flora of a rendering
plant, determining a suitable biological surrogate, pre-release sampling, decontamination of the
rendering facility, and post-decontamination sampling.
5.2 Sampling Approach
Sampling activities were conducted according to the Quality Assurance Project Plan (QAPP)
that was approved prior to testing (available upon request). In several instances, sample
numbers and locations identified in the QAPP were adjusted to meet changes in the original
schedule or identifying valuable additional samples. Air samples (34) and surface and
equipment samples (90) were collected from 124 sample locations. An additional 26 (twenty
percent ratio) QA/QC samples were collected, including media blanks, duplicates, positive
controls and field blanks. The original planned approach (See Table 6) outlined a total of 120
samples from the air, surfaces, and equipment throughout the Darling rendering plant. The
additional samples were collected from air, grease (1) and crax (1). The grease and crax
samples were opportunistic samples that were collected from the final rendering products. The
additional air samples were collected to ensure that the inoculation process did not contaminate
the rendering plant. Table 19 lists a summary of the sampling and analytical procedures.
5.2.1 Wipe Sampling
A slight deviation was made from the QAPP in the number of wipes that were collected and sent
to the laboratory for analysis. The original plan called for three separate wipes from each of the
surface sampling locations. However, laboratory personnel reduced the number of wipes
because they could conduct Q-PCR, enumeration and PLGA identification from one wipe. The
reduction saved on costs, reduced analysis time, and allowed the sampling team to designate a
51
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
second wipe for archival purposes.
Table 19. Summary of the Sampling and Ana
Matrix
Measurement
Sampling/
Measurement
Method
Sample
Container/
Quantity of
Sample
Preservation/
Storage
Holding
Time(s)
Wipe
Culture and
counts of CFU
Colony Counts
per 100 cm2
25 mL
Conical Tube
Less than 25 °C
3 days
Wipe
PCR
Cycle time
25 mL
Conical Tube
Less than 25 °C
3 days
Wipe
Immunoassay
or
spectrography
for PLGA
Concentration
25 mL
Conical Tube
Less than 25 °C
3 days
MCE Filter
Culture and
counts of CFU
Colony Counts
per 100 cm2
37 mm filter
in plastic
cassette
Less than 25 °C
3 days
MCE Filter
PCR
Cycle time
37 millimeter
(mm) filter in
plastic
cassette
Less than 25 °C
3 days
MCE Filter
Immunoassay
or
spectrography
of PLGA
Concentration
25 mL
Conical Tube
Less than 25 °C
3 days
ytical Procedures
5.2.2 Air Sampling
Air sampling was conducted in accordance with the QAPP. A slight deviation was made in the
number of air samples collected. Additional air samples were collected to ensure that the
inoculation process did not contaminate the rendering plant. Also, positive controls were
collected that were not previously listed in the QAPP.
5.3 Timeline of Events for Study
The timeline of events was modified slightly from the QAPP due to budgetary concerns and
plant operations. Oversight was not conducted by the Test Team during pre-cleaning during
weekend three to reduce costs. Test personnel did tour the plant following the cleaning and
prior to sampling and collected photographs to confirm that background cleaning had occurred
properly. Also, a full grinder study that was planned for Monday, October 24th, could not be
conducted due to budgetary concerns and plant maintenance operations.
5.3.1 Background Sampling
Background sampling was conducted in accordance with the QAPP. Following pre-cleaning, a
total of 11 samples (four surface samples and seven air samples) were collected throughout the
plant as background samples prior to the inoculation of incoming loads with the PLGA and Bg.
5.3.2 Carcasses Inoculated with PLGA and Bg
PLGA and Bg were sprayed throughout each truckload of carcasses intended for processing in
the rendering plant for an eight-hr shift. The PLGA and Bg spores were sprayed on the
carcasses using a Roundup backpack-style hand sprayer. The carcasses were inoculated in
52
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
accordance with the QAPP, although a slight variation in the mixture was utilized after the
manufacturer of the Bg spores recommend using 1E9 Bg concentration and a surfactant to
prevent clumping. According to the manufacturer, 1E11 Bg concentration is prone to clumping
and using the lower (1E9) concentration with a surfactant is the optimum concentration to be
sprayed.
5.3.3 Process Sampling
Process sampling was conducted in accordance with the QAPP, though one of the sample
locations for the grinder was moved due to inaccessibility. During the processing of inoculated
material in the eight-hr shift, sampling personnel collected two grab samples every two hours
during the eight hr of processing (eight grab samples total). Originally, one of the samples was
to be collected from the bottom of the tipping floor pit, and a second sample was to be collected
from the incline screw leading from the pit. However, the pit was inaccessible from the top, so
the sample was moved to the outer edge of the tipping floor pit. This location was chosen as an
acceptable alternative because processing material was routinely pushed up against it with a
front end loader.
5.3.4 Inoculation Phase and Process Sampling
Inoculation Phase and process sampling were conducted in accordance with the QAPP.
Samples were collected from 28 sampling locations (six air sampling locations and 22 surface
sampling locations).
5.3.5 Post-Inoculation and Process Sampling
Post inoculation and process sampling were conducted in accordance with the QAPP, although
one of the sample locations for the grinder was moved due to inaccessibility. After the eight-hr
shift when all of the contaminated carcasses were processed, the plant processed uninoculated
carcasses for eight hr. During the Process Sampling, the sample location from the bottom of
the tipping floor pit was moved due to the inaccessibility of the outer edge of the tipping floor pit.
This same location was chosen for the Post-Process Sampling.
5.3.6 Plant Cleaning After Inoculation and Process Sampling
Following the sampling, plant personnel cleaned the facility according to the QAPP. Under the
oversight of EPA, plant personnel utilized existing plant methods to clean the plant. Particular
attention was paid to the grinder area, tipping floor, pits, the processing area, and building
floors.
5.3.7 Post-Cleaning Sampling
After the cleaning was performed by plant personnel, test personnel performed sampling in
accordance with the QAPP. Eighty wipe samples were collected from forty sample locations.
One wipe sample was collected for analysis and a second wipe sample was collected for
archiving. Sampling personnel collected eight air samples from areas where contamination and
dust would most likely occur. The previously mentioned opportunistic samples of grease and
crax were collected in this sampling phase to determine if the final rendering products showed
any signs of contamination. In addition, numerous QA/QC wipe and air samples were collected
in accordance with the QAPP.
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
5.3.8 Grinder Study Sampling
The full grinder study described in the QAPP was not conducted after final cleaning of the plant
due to budgetary concerns and plant maintenance. The grinder study was designed to
determine if new rendering material could possibly re-contaminate the plant from inoculum
potentially left inside the grinder. However, limited samples were collected from the grinder
during post-decontamination sampling. Data are therefore available for the grinder. In addition,
samples were collected in each stage of the study from the grinder.
5.4 Analytical Procedures
As discussed in Section 4 (Results) of this report, the analytical laboratories had difficulties with
the rendering matrix. Some modifications were therefore made to the analytical procedures
(Table 17). As previously stated, sampling personnel collected two wipe samples from each of
the surface sampling locations. One wipe (designated as ABC) was collected for community
characterization by qPCR, enumeration, and PLGA identification. The second sample was
collected and stored for archival purposes (designated as D). Sampling personnel collected
samples of air PLGA and Bg using MCE filters and a sample pump.
All samples, including a Bg spike, were submitted to Battelle for bacterial identification (PCR),
enumeration (counts), and analysis for PLGA microspheres (Victor fluorescence assay or
fluorescent microscopy). Bacterial identifications were conducted on all samples using qPCR to
determine the quantity of Bg and further processing, sequencing, and analysis utilizing 16S
(and/or 23S) rRNA sequencing to identify other community microorganisms. Each wipe or filter
sample was extracted using a standard procedure developed for recovery of viable
microorganisms and nucleic acids. See Appendix B for more discussion on the procedures
utilized during analysis.
A method for accurately enumerating PLGA fluorescent microspheres was developed in a 96-
well format for the Victor fluorescence plate reader. A standard curve was prepared and
evaluated in triplicate to determine the LOD and LOQ for the assay. Additionally, gauze and
filter samples were spiked with a known quantity of PLGA microspheres and extracted
according to the proposed method (filtration and recovery as described above) and enumerated
against the standard curve to verify the procedure.
Viable bacteria were compared to a control culture of Bg on BHI agar, and colonies that did not
have morphology similar to Bg were selected for follow-on analysis to amplify and sequence the
16S-23S rRNA gene. Up to thirty isolates with unknown colony morphologies were selected for
follow-on analysis. Briefly, a single colony was placed into a PCR reaction tube along with 50
|jL of Promega nuclease-free water (VWR, PAP1195, West Chester, PA, USA) with a sterile
inoculating loop and autoclaved for 15 min. at 121 °C. Each colony selected was analyzed by
sequencing the 16S rRNA gene. The 16S rRNA gene was sequenced by using the following
oligonucleotide primers (or primers from other conserved regions of the 16S rRNA gene) to
amplify the region of interest:
• forward oligonucleotide primer (8F, 5' AGAGTTTGATCMTGGCTCAG 3'), and
• reverse oligonucleotide primer (1492R, 5' GGYTACCTTGTTACGACTT 3').
Amplified 16S (and/or 23S) rRNA samples (-1500 bp) were cloned into the TOPO TA cloning
vector, sequenced using M13 forward and reverse primers, and then analyzed by the selected
laboratory using the BLASTn program from the NCBI website. A substitute identification
program may be utilized provided that similar results can be provided. Bacterial identity was
selected from the top 25 BLAST® nucleotide database results with maximum identity match
greater than 90%. In the event that cloning the 1500 bp fragment into TOPO TA is
54
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
unsuccessful, a smaller PCR may be closed using primers in alternate conserved regions of the
rRNA gene [14], If complete single coverage of the 16S rRNA gene was not complete using
M13 primers, the portion of high-quality sequence that was obtained was used for BLAST®
searches.
5.5 Results from Positive and Negative Control Samples
All the positive controls came back positive. All the negative controls were negative (there was
only one, and it was the distilled water). All of the field blanks came back negative except for
three. The following samples failed the internal positive control and had significant amounts of
growth: IRP-WIPE-10-21-11-ABC-0042; IRP-WIPE-10-21-11-ABC-0047; and IRP-WIPE-10-21-
11-ABC-055.
There was not a clear answer as to why these three field blanks had growth. The laboratory
performed extractions on blank matrix and spiked matrix concurrently with the samples
processed for the study—the gauze spiked with DNA showed variable inhibition (See Table 20).
One gauze PC was processed neat, one at 1:5, and three after Qiagen extraction and 1:5
dilution. This observation suggests that the gauze matrix itself may be inhibitory (because the
DNA was spiked in 1X TE, which is generally NOT inhibitory). Those three wipes combined
with the wetting solution used to process field blanks were even more inhibitory to PCR—
possibly the wetting agent released those inhibitory properties from the gauze prior to
extraction, which may have exacerbated the problem (particularly as there was no 'sample' to
act upon, and all enzymatic and physical actions were applied directly to the gauze itself, rather
than to cells and other cellular debris).
Table 20. Results of Bg qPCR Analyses of Positive Contro
S
Sample ID
Dilution
Ct Value
GC/5 mL
Result
Filter PC 1
1:5
35.49
204.86
Positive
35.09
272.53
Filter PC 2
1:5
31.62
4821.90
Positive
31.06
6351.25
Gauze PC 1
1:5
32.65
1510.49
Positive
32.64
1523.35
Gauze PC 2
Neat
30.21
8087.46
Positive
30.04
8778.85
Gauze PC 3
Qiagen 1:5
32.21
3018.52
Positive
32.65
2425.59
Gauze PC 4
Qiagen 1:5
35.29
797.78
Positive
34.91
963.46
Gauze PC 5
Qiagen 1:5
38.43
171.44
Positive
35.78
628.65
Water PC 1
1:5
31.37
3712.34
Positive
31.69
2976.29
Grease PC 1
Neat
32.93
1244.29
Positive
32.98
1200.10
- Gene copies per 5 mL of PCR reaction (after sample extraction, concentration by alcohol precipitation, re-
suspension, etc.)
** - Positive = > LOQ was 92.1 GC/5 mL; samples with mean <1 GC/5 mL are considered Negative; samples with
multicomponent trace showing no amplification are considered MC Negative.
55
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
6. CONCLUSIONS
A study was conducted to evaluate cleanup of a rendering plant after its use for disposal in
response to an FAD outbreak. The intent of this study was to develop recommended
procedures that could be used to aid in returning a rendering plant to normal operation after use
in support of an actual FAD incident.
This effort attempted to achieve three objectives:
• To generate data on fugitive emissions of a biological surrogate during the rendering
process;
• To determine the effectiveness of existing plant cleaning procedures for reducing the levels
of surrogate on the inside surfaces of the rendering facility; and
• To provide information that could be used to develop standard procedures for appropriately
clearing a rendering facility that had been used for "disposal rendering" after an FAD
outbreak, as part of returning the rendering facility back to its normal production use.
The Test Team conducted several sampling events at the Darling International (Darling)
Rendering Plant located in Des Moines, Iowa, which included:
• Acquiring a series of opportunistic swab samples at the first plant visit to gain an initial
insight into the culturable bacterial flora present in the plant;
• Acquiring a series of wipe samples at various locations in the plant to get a more detailed
evaluation of background culturable bacterial flora present in the plant;
• Performing an initial sampling effort to focus on potential biological surrogates to use for the
Cleaning/Inoculation study;
• Performing a series of laboratory spike tests involving potential biological surrogates in
idealized rendering plant sampling matrices and sampling media for air and wipe samples.
Based on the results of this and all previous testing, biological and nonbiological surrogates
were selected for the Cleaning/Inoculation study; and
• Performing a Cleaning/Inoculation study at the rendering plant to evaluate the movement of
the surrogates within the rendering process and subsequent plant cleaning activities.
Initially a thermophilic bacterium such as G. stearothermophilus was desired for use as the
biological surrogate because the analytical procedures for culturing G. stearothermophilus are
at temperatures that would inhibit the growth of most other bacterial species in the samples.
However, the results from the initial opportunistic samples and the background sampling
activities indicated that the PCR procedures used for sequencing the potential surrogate(s) were
not sensitive enough to identify G. stearothermophilus in the sampling matrices of interest. Only
37.5% of the positive controls (using typical rendering plant matrices) were identified
successfully as G. stearothermophilus by the procedure. Literature articles [6] further validated
the results and indicated that further work on G. stearothermophilus may require construction of
GEOBAC primers, which was beyond the scope of this study.
Based on those results, a series of challenge samples was evaluated in a bench-scale study
using Bg and PLGA fluorescent microspheres as potential surrogates in idealized rendering
matrix materials (i.e., suet, grease, and Dl water). Based on these challenge samples, the
inoculum that was selected for the Cleaning/Inoculation study was a mixture of 1E9 CFU of Bg
spores and 1.47E9 beads of PLGA, with an additional surfactant per gallon of inoculum to
prevent clumping of the Bg spores.
Over a series of weekends, the rendering plant was cleaned using cleaning methods normally
56
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
utilized by the plant. Following the plant pre-cleaning, a Cleaning/Inoculation study was then
conducted in October of 2011. The following conclusions were drawn from the
Cleaning/Inoculation study:
• The results of the Cleaning/Inoculation study indicated that no Bg DNA was detected in any
of the Post-Inoculation- or Post-Cleaning sample extracts from the surface wipes or from the
air samples using various PCR techniques. A significant amount of additional effort was
devoted to extracting Bg DNA from the samples, including sacrificing all of the archived
samples in an attempt to use alternate means to amplify the Bg DNA and achieve detection.
This additional effort was unsuccessful. Although Bg was possibly present in low
concentrations and below the limit of detection by quantitative PCR (qPCR), nondetection by
qPCR was very possibly due to inhibitors present in the sample matrices that carried over
during the extraction process. This hypothesis was formulated because putative Bg was
recoverable on brain heart infusion agar (BHIA) using nonmolecular microbiological
techniques and because Bg DNA could be extracted from, and detected in, spiked positive
controls of pristine gauze and filter matrices, as well as idealized materials similar to
rendering plant sample matrices (i.e., suet, grease, and Dl water).
• Due to problems with extracting the PLGA microspheres from the sample matrix (both gauze
wipes and air filters), PLGA might not be a suitable synthetic surrogate, as the microspheres
become permanently immobilized in these sampling matrices. Extraction processes were
ineffective at removing PLGA microspheres for quantitation by fluorometer. In addition,
autofluorescence from the rendering plant sample matrices (e.g., grease, flesh, bone
materials) complicated detection of PLGA microspheres via direct microscopic observation.
Other PLGA microspheres with different colors that may not autofluoresce at the same
wavelength as the rendering sample matrices may be available. There were two issues with
the PLGA microspheres: immobilization on sampling materials and detection interference
caused by rendering materials. Other sampling matrices may possibly yield better results
with PLGA microspheres.
• Both PLGA and PCR analysis of rendering matrices proved to be difficult. Strides were
certainly made to help identify which analysis methods might work better to overcome
interferences such as hair, grease, and bone fragments. However, questions linger about
qualitative and quantitative analysis of rendering plant samples in the future. In addition,
this study raised questions concerning identification and use of a suitable surrogate and the
materials that would be necessary to acquire and analyze samples from an environment
containing considerable background biological microbes.
• Using nonmolecular microbiological culture techniques, viable bacteria very similar to the Bg
positive control colony morphology were recovered from eleven of the test sample extracts
(five contained putative Bg in quantities greater than the LOQ).
• Based on results obtained from nonmolecular biological culture techniques, routine plant
cleaning procedures may potentially result in an approximately 1-log reduction in pathogen
loading within the potentially contaminated areas of the plant. This result is consistent with
results from previous systematic studies examining the effectiveness of different steps of a
multi-step cleaning/disinfection process that showed a 1-4 log reduction from individual
cleaning/disinfection steps. The plant cleaning procedures used in this study utilized hot
water and steam, a combination that would have been expected to remove contamination
from surfaces and transfer any removed contamination into the rinse water going into the
drains, as opposed to actually killing any surrogate organisms that would have existed in the
rinsate. Hot water would not have killed the surrogate spores used in these tests, but may
possibly kill some FAD viral agents.
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
• The cleaning process using the steam and hot water also has the potential to spread the
contaminant throughout the plant, even if the cleaning process results in a reduction in the
overall levels of contamination. The spread of contamination may be the result of plant
personnel tracking the surrogate to various locations within the plant or may be due to
aerosol transport. High pressure spraying operations have indeed been shown to result in
aerosol transport of spores [13], However, no air samples exhibited any Bg either through
PCR analysis or examination of colony morphology.
This study highlights the need for analytical methods that are compatible with the matrices
found in rendering facilities.
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
7. RECOMMENDATIONS
7.1 Recommendations for Future Rendering Plant Sampling/Analytical
Efforts
The information that was obtained from this study leads to many questions about the sampling
and analysis of the rendering plant matrices and air samples. The study revealed that more
work should be done to determine how to sample in a rendering facility environment and to
analyze the resulting extracts.
• Both wipe samples and swabs were used for sampling in this study because of the harsh
environment (i.e., rough, grimy surfaces) of a rendering plant. Swab samples were
negatively impacted by the rough surfaces in a rendering environment, and a single large
particle could potentially bias a swab sample. While wipe samples certainly could collect
more material, the amount of material collected by a wipe could require multiple dilutions
during the biological analysis portion of the study. Also, the materials used in wipe samples
interfered with the identification of the PLGA microspheres; i.e., PLGA microspheres
became permanently immobilized in the sampling matrices.
• Sample dilution might be a better alternative for these sample matrices or a more desirable
solution for the end users, but testing would be necessary to determine the optimal dilution
to overcome PCR inhibition without significant loss of target DNA. However, dilution comes
at the expense of sensitivity; it is not clear whether a different/additional purification step
would be more advantageous than dilution of the inhibitor.
• Newer DNA extraction methods that have been developed recently [15, 16] have shown
promise in the ability to extract DNA from complex matrices and may be useful to test on
rendering samples.
• Due to the difficulty of extracting Bg DNA from the sample matrices, coupled with the
success of using nonmolecular microbiological techniques to identify putative Bg colonies in
heat-shocked samples, the initial desire for a thermophilic bacterium (e.g., G.
stearothermophilus) to use as a potential biological surrogate for rendering plant studies
should be revisited. Results of this study as well as a subsequent literature review [6]
indicated that further work on G. stearothermophilus may require construction of GEOBAC
primers specific to the Geobacillus genus based on internal transcribed spacer (ITS)
sequences.
• Bench scale recovery tests (for biologicals, both CFU and DNA) using actual rendering plant
matrices (instead of idealized matrices) should be conducted to optimize recovery, minimize
interferences, and determine suitable surrogates for use in a rendering plant. Similar tests
should be conducted for nonbiological surrogates.
• Given that many FADs of interest are viral in nature, development of methods to extract
virions and viral DNA from rendering plant matrices may be necessary to show that there is
no residual viral loading in the plant following cleaning procedures, or at least that viral
loading is below levels pre-determined by the Incident Commander.
• The results of the analyses indicated that PLGA microspheres may not be a suitable
synthetic surrogate. The microspheres appear to become immobilized in the sampling
matrices, and the particles autofluoresce at a wavelength similar to hair and bone
fragments. This behavior makes it difficult to distinguish the PLGA spheres from
background. Also, the extraction processes were ineffective at removing PLGA
microspheres for quantitation by fluorometer, and autofluorescence from the sample
matrices complicated detection of PLGA microspheres via direct microscopic observation.
Other variants of the PLGA microspheres may exist that neither autofluoresce at the same
59
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Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
wavelengths as the sampling matrices nor become immobilized in the wipe gauze or air filter
materials.
Based on the results of the sampling and methods development work that has been done, an
ideal surrogate for use in the field test does not appear to exist. Tradeoffs must be taken into
account and a balance struck to pick the best available surrogate given the amount of
information that is currently available.
7.2 Recommendations for Developing Plant Cleaning Procedures
Following Use of the Plant for Disposal Rendering as Part of an FAD
Response
The results from this study suggest that the development of standard operating guidelines to
address the cleaning of a rendering plant following its use for disposal rendering as part of an
FAD response would include several distinct steps, with precautions being taken to minimize
movement of contamination. Due to the size of a typical rendering plant, the diversity of
process equipment in the plant, and the level of dirt and grime on many plant surfaces, it is
unlikely that fumigation would be recommended for the plant decontamination without first doing
extensive testing to verify decontamination efficacy and to assess potential equipment damage.
Procedures including surface cleaning and subsequent disinfection may, therefore, be the most
appropriate means to restore a rendering plant to normal operation following its use in an FAD
response.
The purpose of this study was not specifically to develop the cleaning guidelines, but to develop
information that could be used by the rendering industry and agricultural emergency response
authorities to develop guidelines that could be used to restore a rendering plant to normal
operation following its use in an FAD response.
The following suggestions are offered for inclusion in plant cleaning guidelines:
• Due to the size and diversity of materials of construction in and around the rendering plant
and its various process units, as well as the nature of plant operations, there are abundant
opportunities to result in the buildup of a potentially significant quantity of dirt, grime, grease,
and organic matter on many plant surfaces. This buildup is likely to occur over a period of
time significantly longer than the time that the plant would likely be used for disposal
rendering. This prior cleaning may present a logistical challenge due to the lead time
associated with bringing in a commercial cleaning operation. However, removal of
accumulated grime, dirt, and organic matter prior to potentially contaminating the plant with
an FAD pathogen may greatly simplify later cleaning and decontamination operations.
• Due to the potential for transport of contamination throughout the plant due to activity of the
plant personnel, establishing contaminant control procedures for plant workers prior to
delivery of any contaminated materials to the plant may be very important. Contaminant
control procedures may include such considerations as:
• Establishing egress pathways for workers to pass from areas of lower likelihood of
contamination to areas of higher likelihood of contamination;
• Dividing work duties and shift schedules so that workers performing activities in areas of
lower likelihood of contamination do not enter areas of higher likelihood of
contamination;
• Establishing procedures for donning and doffing clothing and PPE to minimize
contaminant spread; and
• Using aerosol containment equipment (e.g., tent) at the grinding operation where the
most post-inoculation putative positive surrogate samples were observed.
60
-------
Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
• Due to the potential for cleaning operations to spread contamination around the plant to
areas that may previously not have been contaminated, a multi-step (possibly three distinct
steps) cleaning/decontamination process, done in a systematic approach with runoff control,
appears to be the most effective way to clean the plant for restoration to normal operation.
Initial cleaning steps may include such activities as low pressure washing, steam cleaning,
and brushing. Minimization of the use of high pressure washing may minimize aerosol
transport of potential contaminants.
• The multi-step cleaning/decontamination process might be a three-step process that starts
with cleaning only the potentially most heavily contaminated portions of the plant rather than
the entire plant. This initial cleaning might focus on removal of organic matter, particularly
on the tipping floor, in the feed hopper, the grinder, and on the auger ramps that lead into
the cooker, along with the walls and floors in those areas of the plant. This initial cleaning
should be staged to move the potentially contaminated materials eventually into the cooker
or the drains, such as by cleaning in the following sequence:
• Tipping floor area walls;
• Tipping floor;
• Feed hopper;
• Grinder; and
• Augers and ramps.
• During this initial cleaning operation, plant personnel movement from the areas being
cleaned to other plant areas that may not be as contaminated should be minimized.
• Utilizing the cooker where possible to process potentially contaminated materials may
minimize further contamination of the areas outside the plant.
• Where the cooker cannot be used to process potentially contaminated materials, the
remainder may be diverted into the drains, so that runoff can be collected and treated
separately.
• Once the heaviest loading of organic matter has been removed from the surfaces in the
areas of the plant that have the highest likelihood of contamination (i.e., tipping floor,
grinder, feed augers), subsequent cleaning operations should be initiated. These
subsequent cleaning steps may include a second pass through the entire plant using steam,
detergents, and low pressure spraying of water, with special attention being given to the
drain areas, where rendering material may accumulate. A final cleaning step that involves
the use of disinfectants that have been registered for use with the FAD organism of interest
would then be performed.
• Water and other runoff that is collected in the drains should be treated to kill the FAD
pathogen prior to discharge. This step is likely to vary significantly from rendering plant to
rendering plant and may require concurrence by permitting authorities who regulate water
discharges from the plant.
61
-------
Field Study on Cleaning a Rendering Plant Following an FAD Outbreak
8. REFERENCES
1. Kansas State University. Carcass Disposal: A Comprehensive Review. 2004 [accessed
2013 May 29]; Available from: http://hdl.handle.net/2097/662.
2. National Renderers Association. 2012 [accessed 2012 May 4]; Available from:
http://nationalrenderers.org/about/process/.
3. U.S. Department of Homeland Security. National Response Framework. 2009
[accessed 2012 October 27]; Available from: http://www.fema.gov/pdf/emergency/nrf/nrf-
core.pdf.
4. U.S. Government Printing Office, Food Safety Modernization Act (FSMA), t. Congress,
Editor 2012.
5. U.S. EPA. Meat Rendering Plants. Final Section - Supplement A. AP 42, Fifth Edition,
Volume I Chapter 9: Food and Agricultural Industries 1995 [accessed 2010 July 22];
Available from: http://www.epa.gov/ttn/chief/ap42/ch09/final/c9s05-3.pdf.
6. Kuisiene, N., J. Raugalas, M. Stuknyte, and D. Chitavichius, Identification of the Genus
Geobacillus Using Genus-Specific Primers, Based on the 16S-23S rRNA Gene Internal
Transcribed Spacer FEMS Microbiological Letters, 2007. 277: p. 165-172.
7. Personal Communication, Decontamination Analytical and Technical Services contract
(DATS) Email and Phone Conversations with Mr Joe Dalmasso of Yakibou, Inc., 2011.
8. Burton, N.C., A. Adhikari, S.A. Grinshpun, R. Hornung, and T. Reponen, The Effect of
Filter Material on Bioaerosol Collection of Bacillus subtilis Spores Used as a Bacillus
anthracis Simulant. Journal of Environmental Monitoring, 2005. 7: p. 475-480.
9. Applied Biosystems. Creating Standard Curves with Genomic DNA orPlasmid DNA
Templates for Use in Quantitative PCR. 2003 [accessed 2013 August 5]; Available from:
http://www6.appliedbiosystems.com/support/tutorials/pdf/guant per.pdf.
10. U.S. National Institute of Health. National Center for Bioinformatics, Basic Local
Alignment Search Tool. 2013 [accessed 2013 July 25]; Available from:
http://blast.ncbi.nlm.nih.gov/.
11. Ondov, B.D., N.H. Bergman, and A.M. Phillippy, Interactive Metagenomic Visualization
in a Web Browser. BMC Bioinformatics, 2011. 12: p. 385.
12. U.S. EPA, Assessment of Liquid and Physical Decontamination Methods for
Environmental Surfaces Contaminated with Bacterial Spores: Development and
Evaluation of the Decontamination Procedural Steps, EPA/600/R-12/025, 2012.
Washington, D.C.
13. Calfee, M.W., S. Ryan, J. Wood, L. Mickelsen, C. Kempter, L. Miller, M. Colby, A. Touati,
M. Clayton, N. Griffin-Gatchalian, S. McDonald, and R. Delafield, Laboratory Evaluation
of Large-Scale Decontamination Approaches. Journal of Applied Microbiology, 2012.
112(5): p. 874-882.
14. Baker, G.C., J. Smith, and J. Cowan, Review and Re-analysis of Domain-specific 16S
Primers. Journal of Microbiological Methods, 2003. 55(3): p. 541-555.
15. Thomas, M.C., M.J. Shields, K.R. Hahn, T.W. Janzen, N. Goji, and K.K. Amoako,
Evaluation of DNA Extraction Methods for Bacillus anthracis Spores Isolated from
Spiked Food Samples. Journal of Applied Microbiology, 2013. 115: p. 156-162.
16. Amoako, K.K., K. Santiago-Mateo, M.J. Shields, and E. Rohonczy, Bacillus anthracis
Spore Decontamination in Food Grease. Journal of Food Protection, 2013. 76(4): p. 699-
701.
62
-------
APPENDICES
-------
APPENDIX A
CLEMSON DATA
REPORT
-------
RESULTS
Study of Fugitive Emissions of a Biological Surrogate Released During the
Rendering Process: Pre-Sampling of a Rendering Plant to Select an
Appropriate Surrogate Organism
conducted by:
Annel K. Greene, Ph.D., Paul L. Dawson, Ph.D. and Xiuping Jiang, Ph.D.
Co-Principal Investigators,
and
Inyee Han, Ph.D., M. Melissa Hayes, M.S., Chao Gong, M.S., Yubo Zhang, B.S. and
Jinkyung Kim, Ph.D.
Clemson University Animal Co-Products Research & Education Center (ACREC)
in response to:
REQUEST FOR QUOTATION FOR
Dynamac Corporation
September 21, 2010
DATS CONTRACT
ANALYTICAL SERVICES
to conduct laboratory services as specified in:
Quality Assurance Project Plan (QAPP) for the Study of Fugitive Emissions
Of a Biological Surrogate Released During the Rendering Process: Pre-sampling of a
Rendering Plant to Select an Appropriate
Surrogate Organism
QA Category III
July 28, 2010
U.S. ENVIRONMENTAL PROTECTION AGENCY
NATIONAL HOMELAND SECURITY RESEARCH CENTER
& NATIONAL DECONTAMINATION TEAM
DECONTAMINATION ANALYTICAL & TECHNICAL
SERVICE (DATS) CONTRACT
CONTRACT NUMBER: EP-W-06-089
TDD No. TO-02-10-03-0033
-------
1. PROJECT OBJECTIVES AND ORGANIZATION
1.1 Project Objectives
Dynamac Corporation under the Decontamination Analytical and Technical Services (DATS)
Contract will assist the U.S. EPA National Decontamination Team (NDT) and EPA/ National
Homeland Security Research Center (NHSRC) in conducting a study to evaluate fugitive
emissions of a biological surrogate released from a rendering process. To prepare for this study,
it is necessary to evaluate the background concentrations of the potential surrogate(s) selected in
a pre-study sampling event. The biological surrogate or surrogates selected to be evaluated in
the fugitive emissions study are to be selected based on the results from this pre-sampling effort.
1.2 Project
A total of twenty four (24) swab and two wastewater samples were collected from 13 areas of the Darling
International, Inc. Des Moines, IA plant by Anne Busher and Neil Daniell of Dynamac Corporation. One
wastewater source was sampled and various hard surfaces and pieces of equipment throughout the plant
were sampled. Two swab samples were collected from adjacent areas at each sample location - one for
enumeration and one for thermophilic bacterial identification via PCR/DNA sequencing.
Visual observation of the swabs upon receipt at the laboratory indicated varying degrees of slight
discoloration of the swab tips and the foam insert within the transport tube. Slight particulate matter was
observed on some swabs. The swabs were not significantly deformed across the entire swab surface; only
the tips of the swabs appeared flattened (Figures 1 -3).
Figure 1 - Swabs as received.
-------
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-------
Table 1 indicates sampling points and measurements that were determined on each sample.
Table 1 - Summary of samples
Sample Number
General Location
Description
Matrix
Measurement
Experimental
QC
Total
Samples
1a, 1b
Raw receiving floor area #1
Swab of surfaces
PCR/DNA Sequencing (a) &
Culture/Enumeration (b)
0
Facility -
24
2a, 2b
Raw receiving floor area #2
Swab of non-porous surfaces
PCR/DNA Sequencing (a) &
Culture/Enumeration (b)
0
3a, 3b
Pit area
Swab of non-porous surfaces
PCR/DNA Sequencing (a) &
Culture/Enumeration (b)
0
4a, 4b
Pit Area
Swab of non-porous surfaces
PCR/DNA Sequencing (a) &
Culture/Enumeration (b)
0
5a, 5b
Sump Area
Swab of non-porous surfaces
PCR/DNA Sequencing (a) &
Culture/Enumeration (b)
0
6a, 6b
Raw Material Incline Area
Swab of non-porous surfaces
PCR/DNA Sequencing (a) &
Culture/Enumeration (b)
0
7a, 7b
Raw Grinder Area
Swab of non-porous surfaces
PCR/DNA Sequencing (a) &
Culture/Enumeration (b)
0
8a, 8b
Tallow Tanks/Dryer Area
Swab of non-porous surfaces
PCR/DNA Sequencing (a) &
Culture/Enumeration (b)
0
9a, 9b
Load Out Screw (North
End)
Swab of non-porous surfaces
PCR/DNA Sequencing (a) &
Culture/Enumeration (b)
0
10a, 10b
Crax Grinder Area
Swab of non-porous surfaces
PCR/DNA Sequencing (a) &
Culture/Enumeration (b)
0
11a, 11b
Crax Storage Bin Area
Swab of non-porous surfaces
PCR/DNA Sequencing (a) &
Culture/Enumeration (b)
0
12a, 12b
Tailgate of Truck in
Receiving Bay
Swab of non-porous surfaces
PCR/DNA Sequencing (a) &
Culture/Enumeration (b)
0
13a, 13b
Wastewater from Raw Pit
Sump
Liquid
PCR/DNA Sequencing (a) &
Culture/Enumeration (b)
0
2
14a through 16b
Laboratory Blanks
Agar blank, diluent blank, and
swab blank
PCR/DNA Sequencing (a) &
Culture/Enumeration (b)
TBD
6
17a, 17b
Field Blank
Swab prepared in field as a
sample
PCR/DNA Sequencing (a) &
Culture/Enumeration (b)
0
2
18
Pos Control (PCR / DNA
sequencing)
Pure culture of 6.
stearothermophilus
PCR/Sequencing
0
1
19
Positive Control (swab
spike)
Swab spiked with 1E4 cfu 6.
stearothermophilus
Culture/Enumeration
0
1
20
Positive Control (extraction
buffer spike)
Extraction buffer spiked with 1E4
6. stearothermophilus
Culture/Enumeration
0
1
Enumeration procedures were conducted in duplicate using dilution rates typically used in
previous rendering plant swabbing experiments conducted in the various Clemson University
laboratories. Results indicated significantly lower counts than obtained in previous research
studies. Because of low thermophilic plate counts obtained in the study, the enumeration
procedures were repeated using lower dilutions. Results are reported in Tables 2-6. In addition
to standard plate count and thermophilic bacterial counts on BHI at 55°C, sample 6 was also
enumerated using TSA at 55°C. The template area was 9.62 cm2 for all sample areas.
-------
Table 2. Standard
P04/MgCI-SPC
CFU/sq cm
lb
1.89E+06
lb duplicate
2.41E+06
2b
1.25E+04
2b duplicate
1.46E+04
3b
1.87E+04
3b duplicate
4.16E+03
4b
2.08E+03
4b duplicate
4.16E+03
5b
2.08E+03
5b duplicate
0.00E+00
6b
4.54E+06
6b duplicate
7.19E+06
7b
0.00E+00
7b duplicate
0.00E+00
8b
0.00E+00
8b duplicate
0.00E+00
9b
2.08E+03
9b duplicate
0.00E+00
10b
0.00E+00
10b duplicate
0.00E+00
lib
0.00E+00
lib duplicate
0.00E+00
12b
0.00E+00
12b duplicate
0.00E+00
13b
1.83E+05
13b duplicate
5.00E+05
14b
0.00E+00
14b duplicate
0.00E+00
15b
0.00E+00
15b duplicate
0.00E+00
16b
0.00E+00
16b duplicate
0.00E+00
17b
0.00E+00
17b duplicate
0.00E+00
18b
18b duplicate
19b
0.00E+00
19b duplicate
0.00E+00
20b
0.00E+00
20 b duplicate
0.00E+00
-------
Tabic 3 Standard Plate Coiini using lecithin dilution buffer; incubated at 35°C.
Lecithin-SPC CFU/sq (
lb
3.95E+06
lb duplicate
2.49E+06
2b
2.29E+04
2b duplicate
1.04E+04
3b
1.04E+04
3b duplicate
8.32E+03
4b
2.08E+03
4b duplicate
0.00E+00
5b
0.00E+00
5b duplicate
0.00E+00
6b
9.36E+06
6b duplicate
9.15E+06
7b
0.00E+00
7b duplicate
0.00E+00
8b
0.00E+00
8b duplicate
0.00E+00
9b
0.00E+00
9b duplicate
0.00E+00
10b
0.00E+00
10b duplicate
0.00E+00
lib
0.00E+00
lib duplicate
0.00E+00
12b
0.00E+00
12b duplicate
0.00E+00
13b
6.57E+05
13b duplicate
5.20E+05
14b
0.00E+00
14b duplicate
0.00E+00
15b
0.00E+00
15b duplicate
0.00E+00
16b
0.00E+00
16b duplicate
0.00E+00
17b
0.00E+00
17b duplicate
0.00E+00
18b
18b duplicate
19b
1.00E+00
19b duplicate
0.00E+00
20b
4.16E+03
20 b duplicate
2.08E+03
-------
Table 4. Thermophilic Plate Count using standard P04/MgCl2 buffer and BHI agar: incubated at
55°C; Reps 1 and 2.
P04/MgCI-BHI
CFU/sq cm
P04/MgCI-BHI
CFU/sq cm
lb
4.16E+03
lb
3.12E+02
lb duplicate
1.66E+04
lb duplicate
2.08E+02
2b
0.00E+00
2b
1.04E+02
2b duplicate
1.04E+04
2b duplicate
2.08E+02
3b
0.00E+00
3b
1.04E+02
3b duplicate
4.16E+03
3b duplicate
1.04E+02
4b
6.24E+03
4b
7.28E+02
4b duplicate
0.00E+00
4b duplicate
3.12E+02
5b
0.00E+00
5b
0.00E+00
5b duplicate
0.00E+00
5b duplicate
0.00E+00
6b
0.00E+00
6b
3.12E+02
6b duplicate
0.00E+00
6b duplicate
5.20E+02
7b
2.08E+03
7b
0.00E+00
7b duplicate
0.00E+00
7b duplicate
1.04E+02
8b
0.00E+00
8b
0.00E+00
8b duplicate
0.00E+00
8b duplicate
0.00E+00
9b
0.00E+00
9b
0.00E+00
9b duplicate
0.00E+00
9b duplicate
0.00E+00
10b
0.00E+00
10b
1.04E+02
10b duplicate
0.00E+00
10b duplicate
0.00E+00
lib
0.00E+00
lib
4.37E+03
lib duplicate
0.00E+00
lib duplicate
3.43E+03
12b
0.00E+00
12b
0.00E+00
12b duplicate
0.00E+00
12b duplicate
0.00E+00
13b
0.00E+00
13b
4.16E+02
13b duplicate
0.00E+00
13b duplicate
2.08E+02
14b
0.00E+00
14b
0.00E+00
14b duplicate
0.00E+00
14b duplicate
0.00E+00
15b
0.00E+00
15b
0.00E+00
15b duplicate
0.00E+00
15b duplicate
0.00E+00
16b
0.00E+00
16b
0.00E+00
16b duplicate
0.00E+00
16b duplicate
0.00E+00
17b
0.00E+00
17b
0.00E+00
17b duplicate
0.00E+00
17b duplicate
0.00E+00
18b
18b
18b duplicate
18b duplicate
19b
0.00E+00
19b
1.98E+03
19b duplicate
4.16E+03
19b duplicate
TNTC
20b
1.01E+04
20b
Not measured
20 b duplicate
8.32E+03
20 b duplicate
Not measured
-------
Table 5. Thermophilic Plate Count using standard P04/MgCl2 buffer and TSA on sample 6:
incubated at 55°C
PQ4/MgCI-TSA CFU/sq cm
lb
lb duplicate
2b
2b duplicate
3b
3b duplicate
4b
4b duplicate
5b
5b duplicate
6b
0.00E+00
6b duplicate
0.00E+00
7b
7b duplicate
8b
8b duplicate
9b
9b duplicate
10b
10b duplicate
lib
lib duplicate
12b
12b duplicate
13b
13b duplicate
14b
14b duplicate
15b
15b duplicate
16b
16b duplicate
17b
17b duplicate
18b
18b duplicate
19b
19b duplicate
20b
20 b duplicate
-------
Tabic 6 Thermophilic Plate Count using lecithin buffer & BTTT incubated at 55°C; Rep 1 and 2
Lecithin-BHI
CFU/sq cm
Lecithin-BHI
CFU/sq cm
lb
8.32E+03
lb
0.00E+00
lb duplicate
4.16E+03
lb duplicate
1.04E+02
2b
0.00E+00
2b
2.08E+02
2b duplicate
1.04E+04
2b duplicate
2.08E+02
3b
1.25E+04
3b
0.00E+00
3b duplicate
0.00E+00
3b duplicate
1.04E+02
4b
4.16E+03
4b
0.00E+00
4b duplicate
2.08E+03
4b duplicate
2.08E+02
5b
0.00E+00
5b
2.08E+02
5b duplicate
4.16E+03
5b duplicate
4.16E+02
6b
0.00E+00
6b
6.24E+02
6b duplicate
4.16E+03
6b duplicate
8.32E+02
7b
0.00E+00
7b
1.04E+03
7b duplicate
4.16E+03
7b duplicate
1.46E+03
8b
6.24E+03
8b
7.28E+02
8b duplicate
0.00E+00
8b duplicate
5.20E+02
9b
0.00E+00
9b
0.00E+00
9b duplicate
0.00E+00
9b duplicate
0.00E+00
10b
4.16E+03
10b
0.00E+00
10b duplicate
2.08E+03
10b duplicate
0.00E+00
lib
0.00E+00
lib
0.00E+00
lib duplicate
0.00E+00
lib duplicate
0.00E+00
12b
0.00E+00
12b
0.00E+00
12b duplicate
0.00E+00
12b duplicate
0.00E+00
13b
0.00E+00
13b
1.66E+03
13b duplicate
0.00E+00
13b duplicate
1.14E+03
14b
0.00E+00
14b
0.00E+00
14b duplicate
0.00E+00
14b duplicate
0.00E+00
15b
0.00E+00
15b
0.00E+00
15b duplicate
0.00E+00
15b duplicate
0.00E+00
16b
0.00E+00
16b
0.00E+00
16b duplicate
0.00E+00
16b duplicate
0.00E+00
17b
0.00E+00
17b
0.00E+00
17b duplicate
0.00E+00
17b duplicate
0.00E+00
18b
18b
18b duplicate
18b duplicate
19b
2.08E+03
19b
7.90E+03
19b duplicate
2.08E+03
19b duplicate
8.11E+03
20b
4.16E+03
20b
Not measured
20 b duplicate
2.08E+03
20 b duplicate
Not measured
-------
Because thermophilic bacterial enumeration results revealed wide variability between duplicates, the
experimental procedure on swab samples using BHI and both standard POVMgCK and lecithin buffer was
repeated twice. Such variability in results has been noted in previous studies on rendering materials.
The second swab was used for identifying thermophilic bacterial strains from the samples. Swabs were
pre-enriched with BHI broth overnight at 55°C. The pre-enrichment broth cultures were streaked for
isolation on BHI agar and incubated overnight at 55°C. Pure cultures were isolated from the streak plates
and inoculated on BHI agar slants which will be incubated at 55°C. Results indicated few isolates
obtained from the swab samples. The initial round of plating swabs was conducted in duplicate and
results indicated very little growth from the swabs. Therefore, in order to ensure isolates for study, the
pre-enriched swabs were plated 10 times each to try to obtain thermophilic isolates. Isolates were
transferred to slants and Gram stained.
Colony polymerase chain reaction (PCR) was conducted on the isolates from the slants and the
wastewater to amplify the 16S rRNA gene from the bacterial isolates using the:
• forward oligonucleotide primer (8F, 5' AGAGTTTGATCMTGGCTCAG 3'), and
• the reverse oligonucleotide primer (1492R, 5' GGYTACCTTGTTACGACTT 3').
Amplified 16S rRNA samples will be sequenced and then analyzed using the BLASTn program
on the National Center for Bioinformatics (NCBI) website. Bacterial identity was selected from
the top twenty five BLAST nucleotide database results with max identity greater than 90%.
In the initial experiment only 14 isolates successfully amplified and submitted for sequencing.
Results of this set of isolates were as follows:
1
Bacillus licheniformis 90%
2
Bacillus licheniformis 81%
3
Bacillus licheniformis 88%
4
No result returned
5
No result returned
6
No result returned
7
No result returned
8
No result returned
9
No result returned
10
No result returned
11
Tepidiphilus sp. or Petrobacter sp. 83%
12
Tepidiphilus margaritifer 99%
13
Aneurinibacillus thermoaerophilus 91%
14
Aneurinibacillus thermoaerophilus 91%
In the second isolation attempt, 72 isolates were obtained. Many of these were deemed likely
duplicates based on Gram stain and morphology. After amplifying, these 72 isolates were
submitted along with 4 positive controls in duplicate (8 in total). The positive controls were
ATCC 7953 Geobacillus stearothermophilus, ATCC 12980 Geobacillus stearothermophilus,
ATCC 12978 Geobacillus stearothermophilus, and SPORTROL* Spore Suspensions, NAMSA
(VWR Scientific Products, Inc., # 19872-024). Results of this set of isolates were as follows:
-------
1 No result returned
2 Geobacillus stearothermophilus 77%
3 No result returned
4 No result returned
5 Geobacillus sp. 96% or Geobacillus pallidus 94%
6 Bacillus coagulans 97%
7* No result returned
8* Geobacillus stearothermophilus 92%
9| No result returned
10| No result returned
11 § Geobacillus stearothermophilus 97%
12§ No result returned
13t No result returned
14t Geobacillus stearothermophilus 97%
15 Klebsiella sp. 99%
16 No result returned
17 Bacillus coagulans 97%
18 Geobacillus pallidus 99%
19 Klebsiella sp 97%
20 No result returned
21 No result returned
22 No result returned
23 No result returned
24 Tepidiphilus sp. or Petrobacter sp. 94%
25 Bacillus thermoamylovorans 94%
26 Bacillus sp. 97%
27 No result returned
28 No result returned
29 No result returned
30 No result returned
31
Klebsiella
i pneumonia 93%
32
No
result
returned
33
No
result
returned
34
No
result
returned
35
No
result
returned
36
No
result
returned
37
No
result
returned
38
No
result
returned
39
No
result
returned
40
No
result
returned
41
No
result
returned
42
No
result
returned
43
No
result
returned
44
No
result
returned
-------
45
Aneurinibacillus thermoaerophilus
46
No result returned
47
No result returned
48
No result returned
49
No result returned
50
No result returned
51
No result returned
52
No result returned
53
No result returned
54
No result returned
55
No result returned
56
No result returned
57
No result returned
58
Bacillus licheniformis 94%
59
Klebsiella pneumonia 93%
60
No result returned
61
No result returned
62
No result returned
63
Bacillus licheniformis 77%
64
No result returned
65
No result returned
66
No result returned
67
No result returned
68
No result returned
69
No result returned
70
No result returned
71
No result returned
72
Bacillus licheniformis 96%
73
Bacillus thermoamylovorans 97%
74
Brevibacillus sp 86%
75
Brevibacillus 84%
76
No result returned
77
Bacillus thermoamylovorans 94%
78
Bacillus sp. 90%
79
Bacillus licheniformis 95%
80
No result returned
* Positive Control = ATCC 7953 Geobacillus stearothermophilus
tPositive Control = ATCC 12980 Geobacillus stearothermophilus
§Positive Control = ATCC 12978 Geobacillus stearothermophilus
^Positive Control = SPORTROL* Spore Suspensions, NAMSA
Bacterial identification results using PCR and amplicon sequencing indicated lack of sensitivity of the
procedure to identification of Geobacillus stearothermophilus. Only 37.5% of positive controls were
successfully identified as Geobacillus stearothermophilus by the procedure. Results of this study as well
as a literature review indicated that further work on Geobacillus stearothermophilus may require
construction of GEOBAC primers specific to the Geobacillus genus based on internal transcribed spacer
(ITS) sequences (Kuisiene, N., J.Raugalas, M. Stuknyte and D. Chitavichius. 2007. Identification of the
genus Geobacillus, using genus-specific primers, based on the 16S-23SrRNA gene internal transcribed
spacer. FEMS Microbiol Lett 277:165-172.)
-------
Appendix B. Battelle Report
-------
This Page Left Intentionally Blank
-------
FINAL REPORT
Study of Fugitive Emissions of a Biological Agent Surrogate
Released During the Rendering Process
Dynamac Corporation
July 20, 2012
This document contains information exempt
from mandatory disclosure under the FOIA.
Exemptions (a) 2, 5, and 7 apply.
"WARNING - This document may contain technical data whose export is restricted by U.S. law. Violators
of export control laws may be subject to severe legal penalties. Do not disseminate this document outside
the United States or disclose its contents to non-U.S. persons except in accordance with applicable laws and
regulations and after obtaining any required authorizations."
Battelle Columbus Operations
505 King Avenue
Columbus, Ohio 43201-2696
-------
TABLE OF CONTENTS Page
EXECUTIVE SUMMARY 1
1.0 BACKGROUND AND ASSUMPTIONS 2
2.0 OBJECTIVE 3
3.0 MATERIALS AND METHODS 3
4.0 RESULTS AND DISCUSSION 15
5.0 SUMMARY 39
6.0 RECOMMENDATIONS 40
APPENDIX A SAMPLE LIST, MORPHOLOGY, AND IDENTIFICATION A-1
APPENDIX B BLAST RESULTS B-l
APPENDIX C WORK INSTRUCTIONS: DWI-01 C-l
LIST OF FIGURES Page
Figure 1. Gel Electrophoresis of AIR-10-21-11 Samples Analyzed by PCR on the
ABI 9700 Thermocyler 17
Figure 2. Microscopic Images 28
Figure 3. KRONA Visualization of BLAST Results for Pool 2 35
Figure 4. KRONA Visualization of BLAST Results for Pool 3 36
Figure 5. KRONA Visualization of BLAST Results for Pool 4 37
Figure 6. KRONA Visualization of BLAST Results for Pool 5 38
LIST OF TABLES
Page
Table 1. Pooled Sample Extracts for Phire® Animal Tissue Direct PCR Kit 7
Table 2. 16S rRNA Primer Sequences 10
Table 3. Applied Biosystems 3130 Genetic Analyzer Parameters 12
Table 4. Results of Bacillus atrophaeus qPCR Analyses 16
Table 5. Microscopic Observations of PLGA Microspheres 18
Table 6. Enumeration of Putative B. atrophaeus Colonies in Sample Extracts 30
Table 7. Samples Containing Colony Morphologies Similar to B. atrophaeus 31
Table 8. Results of 16S rRNA Sequencing Based on BLAST and QUEST™ Analysis 33
i
-------
EXECUTIVE SUMMARY
This Final Report incorporates data presented in the Interim Report dated January 20, 2012 and data
obtained during follow-on microbiological and sequence analyses performed in May, 2012. The
objective of this project was to detect and enumerate Bacillus atrophaeus and poly(D,L-lactide-co-
glycolide) (PLGA) microspheres in gauze wipe and filter samples generated during a spiking and
decontamination exercise at a rendering facility. In addition, identification of background microbial flora
present in the rendering facility was undertaken using sequence analysis of 16S rRNA genes.
Putative B. atrophaeus (e.g. bacterial colonies resembling B. atrophaeus positive control colonies (2-
3mm, orange, entire, raised, smooth) on BHIA) was recovered from 1 of 60 sample extracts originally
tested for viable organisms. It was previously reported that 15 of these samples contained putative B.
atrophaeus, however, upon reassessment of the data it was determined that the majority of these had been
misreported due to misinterpretation of the raw data, and only one sample, IRP-IW-10-20-1 l-ABA-001
had colony morphologies similar to B. atrophaeus (Table Al, Appendix A). B. atrophaeus DNA was
not detected in any of the test samples due to the presence of compounds in the samples that caused
significant inhibition of quantitative polymerase chain reaction (qPCR) analysis. Follow-on
microbiological analysis, performed on archived samples from all test locations, showed that ten of the
archived gauze samples contained putative B. atrophaeus (as defined above by colony morphology).
Further attempts to enumerate B. atrophaeus from these presumptive positive samples yielded: four
samples with putative B. atrophaeus at or below 1.73 x 103 colony forming units (CFU)/mL, two samples
with putative B. atrophaeus detected below the limit of quantitation, and four samples with no detectable
B. atrophaeus. None of the filter samples contained putative B. atrophaeus.
Identification of six cultured isolates was obtained by sequence analysis of 16S rRNA genes. These
genera included Proteus, Planomicrobium, and Curtobacterium. Sequence analysis of pooled samples
showed that the most prevalent bacteria present in all of the samples are Pseudomonas species, other
genera included: Stenotrophomonas, Xanthomonas, Comomonas, Herbaspirilium, Lactobacillus,
Acinetobacter, and Yersinia.
PLGA microspheres were detected in only two samples; the microspheres became permanently
immobilized in the sample matrices, and autofluorescence from the matrices and particulate matter
occluded direct visualization of the microspheres by microscopy.
1.0 BACKGROUND AND ASSUMPTIONS
Dynamac Corporation is assisting the U. S. Environmental Protection Agency (EPA) Consequence
Management Advisory Team (CMAT) and EPA National Homeland Security Research Center (NHSRC)
to evaluate fugitive emissions of a biological agent surrogate released from a rendering process. The
ultimate objective was to develop standard procedures for clearing a rendering facility for normal
production after it has been used to process contaminated animals from a foreign animal disease outbreak.
In support of this larger program, the current project was developed to evaluate detection and
quantification of a spore-forming organism, Bacillus atrophaeus, and a synthetic surrogate for bacterial
agents, 1 |a,m poly(D,L-lactide-co-glycolide) fluorescent microspheres (PLGA), in gauze wipe and filter
Appendix B-l
-------
samples generated during rendering processes. An additional project goal was to identify background
microorganisms present in the wipe and filter samples through sequence analysis of 16S rRNA genes.
The ability to detect and quantify B. atrophaeus and PLGA microspheres in rendering facility samples
was dependent on several key assumptions:
B. atrophaeus and PLGA microspheres were spiked at levels high enough to withstand dilution of several
orders of magnitude and remain above limits of detection for the various analytical methods employed.
B. atrophaeus and PLGA microspheres would disperse uniformly throughout the animal carcasses,
mimicking a disease agent.
Sampling matrices would effectively capture dispersed surrogates, and capture would be reversible (i.e.,
the surrogates could be liberated from the matrices for analysis).
Sample matrices would not interfere with accurate detection of the surrogates.
This project was accomplished in two phases. Phase 1 consisted of method development for detection of
PLGA microspheres, and Phase 2 consisted of sample processing and analysis.
Samples were considered to contain target signatures (i.e., viable B. atrophaeus and PLGA microspheres)
at trace levels; therefore, measures to prevent cross-contamination between samples and/or inadvertent
introduction of target signature into a sample were employed, including: thorough decontamination and
pre-swab analysis of sample handling areas prior to study initiation and on each day of extraction;
thorough decontamination of hands and equipment between samples; and the addition of positive and
negative process controls to monitor effectiveness of trace-handling measures.
2.0 OBJECTIVE
The primary objective of this project was to evaluate surrogate dispersion in a rendering plant
during normal operations through detection of:
B. atrophaeus by heterotrophic plate count and qPCR,
PLGA microspheres by direct microscopic observation using a suitable filter set.
An additional project objective was to determine the background microbial flora present in
samples from the rendering facility through nucleic acid sequence analysis of amplified 16S rRNA genes;
elucidation of the background microflora would better enable selection of additional surrogate bacteria for
further study.
3.0 MATERIALS and METHODS
Appendix B-2
-------
Samples were generated by Dynamac Corporation in late October 2011. A truck load of animal carcasses
was spiked with viable B. atrophaeus and PLGA microspheres, and carcasses were subsequently handled
and processed in a rendering plant according to normal operations. Wipe and air samples were collected
before, during, and after processing and shipped to Battelle for analysis. Samples were received cold (wet
ice) in two separate shipments; each was logged and returned to cold storage at 2-8°C until processed.
All processing areas, including the biological safety cabinet and incubator, were thoroughly
decontaminated and swabs were taken and plated onto brain heart infusion agar (BHIA) to ensure that
working areas were sterile prior to sample processing. An additional swab was taken and plated on BHIA
on each day of sample extractions to serve as a laboratory blank for verification that the working surface
was free of B. atrophaeus each day of extraction. Sample processing occurred in five batches over the
course of three weeks, and analytical positive and negative controls were created for each batch as
follows: a single negative control and a single positive control for each matrix type was extracted in the
batch. Negative controls (Matrix Blank 1, 2, etc.) comprised a single pristine matrix, while positive
controls (Matrix PC 1, 2 etc.) comprised a single pristine matrix spiked with B. atrophaeus genomic DNA
(gDNA) at 1 x 107 gene copies (GC)/sample and PLGA microspheres at 0.05 mg/sample. Control
matrices were provided by Dynamac and were identical to sample matrices. Controls were processed in
tandem with the samples, and each received identical treatment to the sample matrices.
Each sample or control was extracted according to a project-specific work instruction (DWI-01, Work
Instructions for the Extraction of Microorganisms, Nucleic Acids, and PLGA Microspheres from
Environmental Samples) provided in Appendix C. Briefly, samples were removed from their original
containers and placed into sterile 250 mL bottles and phosphate buffered saline (PBS), Teknova,
Hollister, CA (Catalog Number PA205) was added (12 or 15 mL for filter and gauze wipe samples,
respectively). Each sample was mixed by vortexing (approximately 30 seconds) and incubated for 30
minutes at room temperature. At this point, 1 mL was removed to serve as the microbiology extract,
and the remaining sample was extracted for nucleic acids (DNA). Microbiology extracts from extraction
sets 1 and 2 (60 total samples) were plated onto BHIA (200 |o,L per plate) and incubated at 36 ± 2°C
overnight to isolate single colonies of bacteria; the remaining microbiology extracts were stored at 4°C
until being further processed for isolation of bacterial DNA. Briefly, 1 |o,L herring sperm carrier DNA
(hsDNA), Promega (Catalog Number )/mL sample and 1% (v:v) sodium dodecyl sulfate (SDS), Fisher
BioReagents, Pittsburgh, PA (Catalog Number BP 1311-200) were added to the remaining volume of
extracts and samples were incubated for 30 minutes at 65 °C. The original sample matrix (filter or gauze)
was preserved in the extraction vessel for detection of PLGA microspheres by microscopic analysis (from
method development testing, PLGA microspheres spiked onto gauze and filter matrices could not be
detected in aqueous solution, but could be visually observed on filters and gauze), and the aqueous extract
was transferred to a sterile Oakridge tube. DNA was concentrated using method ABAT-V-012
(Concentration of Nucleic Acids by Isopropanol Precipitation). In this method, DNA was precipitated
overnight with isopropanol, recovered by centrifugation, washed with 70% ethanol and resuspended in
IX Tris EDTA (TE) buffer (10 mM Tris and 1 mM EDTA), pH 7, Fisher BioReagents, Pittsburgh, PA
(Catalog Number BP2476-1). Pre-amplification analyses of the extracts were not conducted.
3.1 B. atrophaeus Detection by Quantitative PCR
Appendix B-3
-------
For DNA analysis, duplicate 5 |_iL aliquots of sample extracts were assayed via qPCR using an assay
specific for the rip gene of B. atrophaeus on an ABI 7900HT platform. The limit of detection (LOD) and
limit of quantification (LOQ) for this assay were determined to be 92.1 gene copies (GC)/5 |a,L. Prior to
target analysis, sample extracts were tested for inhibition using the Applied Biosystems (ABI) TaqMan®
Exogenous Internal Positive Control Reagents kit according to method ABAT-V-007 (TaqMan Inhibition
Analysis on the 7900HT). Neat, 1:5, and 1:10 dilutions of each sample were initially assayed. In the
event extracts did not pass internal positive control (IPC) testing at the 1:10 dilutions, they were further
purified using a Qiagen (Valencia, CA) QIAQuick PCR Purification kit according to the manufacturer's
instructions. The Qiagen-purified sample extracts were further diluted and tested by IPC analysis at
Qiagen Neat (QN), Qiagen 1:5 (Q5), Qiagen 1:10 (Q10) and Qiagen 1:20 (Q20) dilutions. Sample
extracts that passed IPC were analyzed for B. atrophaeus DNA at the highest concentration passing the
inhibition test according to method ABAT-V-008 (To Prepare a 96-Well Plate for DNA Quantitation on
the 7900HT). The Ct (threshold cycle) value and estimated nucleic acid quantity based on the input
standard curve were compiled, along with an amplification plot and a trace of fluorescent signals
(multicomponent plot) for each replicate sample. The multicomponent plot was examined for each
sample replicate to verify results; positive detections showed elevated signal from the reporter fluorescent
molecule (FAM). Assay acceptance criteria included the following:
Valid standard curve with three or more duplicate points and R2 value of >0.95.
No amplification ("Undetermined" at 45 cycles) in No Template Control (NTC) wells.
A small subset of sample extracts IRP-AIR-10-24-1 l-ABC-018 to IRP-AIR-10-24-1 l-ABC-025 and
sample IRP-AIR-10-24-1 l-ABC-27 was not analyzed by qPCR. This set of sample extracts was
amplified on the ABI 9700 thermocycler using B. atrophaeus rtp primers and analyzed by gel
electrophoresis, with direct visualization of ethidium bromide-stained target amplicon (B. atrophaeus rtp,
82 bp). Positive and negative control reactions were prepared and analyzed along with the sample
extracts. Each sample was initially analyzed on a 2% agarose gel with IX TAE running buffer (10 |a,L
sample per well), and an additional 1.2% gel was run to compare pooled sample extracts (5 |a,L each)
against pooled NTCs and the positive control reaction. Each gel contained an appropriate molecular
weight marker, either Quick-Load 2-log ladder (New England Biolabs, Ipswich, MA, 2% gels) or 1Kb
Plus Track It Ladder (Life Technologies™, Carlsbad, CA, 1.2% gels).
Sample extracts that did not pass IPC were subject to PCR using the Phire® Animal Tissue Direct
PCR Kit (ThermoScientific, Pittsburgh, PA). Phire® PCR was run according to the manufacturer's
instructions using pooled DNA extracts. Samples were pooled by combining 3 |o,L of each extract in
groups of nine or ten (Table 1). Reactions were created by combining 5 |o,L of each pooled sample
extract with 25 |o,L 2X Phire® Animal Tissue PCR Buffer, 10.875 |o,L RNase-Free water, 2.5 |o,L of each B.
atrophaeus rtp forward and reverse primer (10 |a,M), and 1 |o,L of Phire® Hot Start II DNA Polymerase.
Reactions were processed with the following cycling parameters: initial denaturation (5 minutes, 98 ° C);
40 cycles of denaturation (98 ° C, 5 seconds), annealing (65 ° C, 5 seconds), and extension (72 ° C, 20
seconds); a final 1 minute extension at 12° C. Each reaction was analyzed on 1.2% agarose gels; 25 |o,L of
Appendix B-4
-------
each PCR reaction was combined with 5 |a,L 6X Track It Loading Dye (Life Technologies™, Carlsbad,
CA) and run against the 1Kb Plus Track It Ladder (Invitrogen).
3.2 Detection of PLGA Microspheres
During method development, a 96-well microtiter plate assay was developed for detection of PLGA
microspheres (Phosphorex, Inc., St. Fall River, MA), Catalog Number LGFG1000, in an aqueous extract.
PLGA microspheres were diluted in IX PBS to create a 10 mg/mL top concentration, which was then
analyzed by dilution to extinction on two platforms: 1) SpectraMax M2 Multi-Mode Microplate Reader,
and 2) Victor Fluorometer (0.1 and 1 second exposure times). PLGA microspheres were analyzed in
concentrations that ranged from 10 mg/mL to 1.19 x 10"6 mg/mL (diluted 1:2 in IX PBS). The working
range of the SpectraMax M2 was determined to be 10 to 0.02 mg/mL whereas the working range of the
Victor was 10 to 0.001 mg/mL. Due to the lower limit of detection obtained using the Victor fluorometer,
that instrument was chosen for further assay development, and a standard curve was prepared and
validated from 10 to 0.001 mg/mL.
Table 1. Pooled Sample Extracts for Phire® Animal Tissue Direct PCR Kit
Pooled
Sample
Sample Extracts Combined
Pooled
Sample
Sample Extracts Combined
1
IRP-WIPE-10-19-11-ABC-B2 (QN)
IRP-WIPE-10-21-11 -ABC-0015 (QN)
IRP-WIPE-10-21-11 -ABC-0016 (QN)
IRP-WIPE -10-21-11 - AB C -0017 (QN)
IRP-WIPE -10-21-11 - AB C -0030 (QN)
IRP-WIPE -10-21-11 - AB C -0032 (QN)
IRP-WIPE -10-21-11 - AB C -003 5 (QN)
IRP-WIPE -10-21-11 - AB C -003 7 (QN)
IRP-WIPE-10-21-11 - AB C-0042 (QN)
4
IRP-WIPE-10-24-1 l-ABC-0072 (QN)
IRP-WIPE-10-24-1 l-ABC-0073 (QN)
IRP-WIPE-10-24-1 l-ABC-0074 (QN)
IRP-WIPE-10-24-1 l-ABC-0075 (QN)
IRP-WIPE-10-24-1 l-ABC-0076 (QN)
IRP-WIPE-10-24-1 l-ABC-0077 (QN)
IRP-WIPE-10-24-1 l-ABC-0078 (QN)
IRP-WIPE-10-24-1 l-ABC-0080 (QN)
IRP-WIPE-10-24-1 l-ABC-0081 (QN)
IRP-WIPE-10-24-1 l-ABC-0096 (QN)
2
IRP-WIPE-10-21-11 - AB C-0044 (QN)
IRP-WIPE -10-21-11 - AB C -0047 (QN)
IRP-WIPE -10-24-11 - AB C -0051 (QN)
IRP-WIPE-10-24-1 l-ABC-0052 (QN)
IRP-WIPE-10-24-1 l-ABC-0055 (QN)
IRP-WIPE-10-24-1 l-ABC-0056 (QN)
IRP-WIPE-10-24-1 l-ABC-0057 (QN)
IRP-WIPE-10-24-1 l-ABC-0058 (QN)
IRP-WIPE-10-24-1 l-ABC-0059 (QN)
IRP-WIPE-10-24-1 l-ABC-0060 (QN)
5
IRP-WIPE-10-24-1 l-ABC-0083 (QN)
IRP-WIPE-10-24-1 l-ABC-0084 (QN)
IRP-WIPE-10-24-1 l-ABC-0086 (QN)
IRP-WIPE-10-24-1 l-ABC-0087 (QN)
IRP-WIPE-10-24-1 l-ABC-0088 (QN)
IRP-WIPE-10-24-1 l-ABC-0089 (QN)
IRP-WIPE-10-24-1 l-ABC-0090 (QN)
IRP-WIPE-10-24-1 l-ABC-0092 (QN)
IRP-WIPE-10-24-1 l-ABC-0093 (QN)
3
IRP-WIPE-10-24-1 l-ABC-0061 (QN)
IRP-WIPE-10-24-1 l-ABC-0062 (QN)
IRP-WIPE-10-24-1 l-ABC-0064 (QN)
IRP-WIPE-10-24-1 l-ABC-0065 (QN)
IRP-WIPE-10-24-1 l-ABC-0066 (QN)
IRP-WIPE-10-24-1 l-ABC-0067 (QN)
Appendix B-5
-------
IRP-WIPE-10-24-1 l-ABC-0068 (QN)
IRP-WIPE-10-24-1 l-ABC-0070 (QN)
IRP-WIPE-10-24-1 l-ABC-0071 (QN)
IRP-WIPE-10-20-11-ABC-001 (QN)
Once the assay was established, verification of the proposed extraction method was initiated. Control
sample matrices (gauze and filters) were spiked with 1 mg PLGA microspheres, and a mock extraction
was performed according to DWI-01; it was anticipated that
the PLGA microspheres would be removed from the gauze and filter matrices and suspended in the
extract, whereupon they would be recovered during the final filtration. However, it was discovered that
the PLGA microspheres adsorbed to the gauze and filter matrices, and all attempts to remove them were
unsuccessful. At the advice of the PLGA microsphere manufacturer, Phosphorex, Inc., 25 mL of a 2.5%
solution of polyvinyl alcohol (PVA) was added to each spiked filter and gauze sample, followed by
vortex agitation for 1 minute. Room temperature incubation was continued up to 30 minutes with
intermittent agitation by vortex. As no change was observed after 30 minutes, a waterbath sonicator was
used to agitate each sample for 5 minutes. Even after sonication in PVA, deposits of PLGA
microspheres, visible to the naked eye, remained on both types of sample.
Due to the apparent irreversible immobilization of PLGA microspheres onto the filter and gauze sample
matrices, qualitative detection/non-detection of PLGA microspheres was accomplished by direct
microscopy using a Zeiss Axioscope epifluorescent microscope equipped with a filter set with excitation
at 495 and emission at 517 nm. Representative images were captured using a Zeiss color camera.
3.3 Enumeration of Putative Viable B. atrophaeus in Archived Samples
Original filter samples and archived gauze wipe samples were extracted according to the work
instruction DWI-01. Samples were pre-wet with IX PBS extraction buffer (2 mL for filter samples, 5 mL
for gauze samples) and mixed by vortexing for 30 seconds. An additional 10 mL of IX PBS was added
to each sample, and samples were incubated at room temperature (25+3 ° C) for 30 minutes. Samples
were mixed by vortexing at 0, 15, and 30 minutes. Following incubation, 200 |o,L of each sample was
spread-plated onto brain heart infusion agar (BHIA) and incubated overnight at 30° C. Plates were
observed for microbiological growth, and morphologies of resultant colonies were compared to that of an
overnight positive control of B. atrophaeus plated onto BHIA. Any samples containing putative B.
atrophaeus were re-plated onto fresh BHIA for enumeration. The putative B. atrophaeus samples were
diluted in IX PBS and heat-shocked by incubation at 65 ° C for 30 minutes to kill any vegetative cells that
might out-compete the spore-forming B. atrophaeus. Positive and negative controls were processed along
with the samples to ensure process efficacy. Negative controls were prepared by transferring clean filter
and gauze matrices into sterile sample reservoirs; positive controls were prepared by transferring clean
filters and gauze matrices into sterile sample reservoirs and spiking with an aliquot of B. atrophaeus.
3.4 Identification of Background Microflora by Sequence Analysis
Appendix B-6
-------
3.4.1 Selection of Unknown Isolates and Pooled Samples
Microorganisms recovered on BHIA from the 60 original analyzed wipe and filter samples were
selected for follow-on analysis by 16S rRNA sequencing. Thirty isolates that did not have similar
morphology to B. atrophaeus were selected and streaked for isolation on BHIA, followed by incubation
for 16 - 48 hours at 36 ± 2°C. Appendix A lists the isolate morphology and the sample from which the
isolate originated. B. atrophaeus ATCC 9372 was included as a positive control. A portion of the
samples from each of the nucleic acid extract batches were combined to generate five pooled samples for
metagenomic 16S rRNA analysis using the Ion Torrent™ Personal Genome Machine™ (PGM™)
Sequencer (Life Technologies).
3.4.2 Extraction of DNA
Three different extraction techniques were used to prepare DNA for 16S rRNA amplification.
Initially, each of the 30 isolates and aliquots of the five pooled samples (i.e. the remaining
microbiological extracts pooled according to extraction date) were extracted following the DNeasy®
Gram-positive bacteria protocol (Qiagen); however, the DNeasy® extracts could not be used directly for
PCR due to background 16S rRNA DNA that amplified in the enzymatic lysis buffer, which contained
lysozyme, triton X-100, and IX TE. As neither lysozyme nor triton X-100 is readily-available in a
certified DNA-free formulation, a thermolysis technique was attempted to circumvent the need for DNA-
free lysis buffer. However, subsequent attempts to amplify the 16S rRNA gene from the five pooled
samples were unsuccessful following thermolysis. Finally, a OneStep™ PCR Inhibitor Removal Kit
(Zymo) was used on the pooled samples prior to PCR amplification.
3.4.2.1 Extraction using DNeasy® Blood and Tissue Kit. Enzymatic lysis buffer was prepared as
follows: 2 mL of Tris-EDTA, 10X (Fisher), 120 (.iL of Triton X-100 (Fisher), and 2 mL of 100 mg/mL
lysozyme, egg white (Fisher) was added to 5.88 mL of MilliQ distilled water. One to several colonies,
depending on size, were selected for extraction; after addition of the colonies to a tube containing 180 (.iL
of the above enzymatic lysis buffer, extractions were completed following the manufacturer's instructions
for Gram-positive bacteria. To prepare pooled samples for extraction, 1 mL of each pooled sample was
centrifuged at 5,000 x g for 10 minutes, and the pellet was suspended in 180 (.iL of enzymatic lysis buffer
and extracted according to the manufacturer's instructions as stated above.
3.4.2.2 Extraction via Thermolysis. DNA from the 30 isolated colony morphologies were
extracted by adding one to several colonies, depending on size, to a tube containing 250 |_iL of IX Tris-
EDTA (Fisher). The samples were autoclaved using a liquid cycle for 10 minutes at 121°C. Following
autoclave treatment, the samples were cooled to room temperature and stored at -80°C until ready for use.
Pooled samples were treated in the same manner, after 10 |o,L of each pooled sample (described in 3.4.2)
was added to a separate tube containing 250 (.iL of IX Tris-EDTA.
3.4.2.3 Extraction of PCR inhibitors using OneStep™ column. Fifty microliters of each of the
original pooled samples (described in 3.4.2) was processed using the OneStep™ PCR Inhibitor Removal
Kit (Zymo) following the manufacturer's instructions.
Appendix B-7
-------
3.4.3 Amplification of 16S rRNA
The 30 isolated colonies and five pooled samples were subject to PCR using 8F (isolated colonies
and pooled samples) or 27F (pooled samples) and 1492R 16S rRNA primers (Table 2).
Table 2.16S rRNA Primer Sequences1
Primer ID
Sequence
8F
5 '-AGAGTTTGATCMTGGCTCAG-3'
27F
5,-AGAGTTTGATCCTGGCTCAG -3'
1492R
5 '-GGYTACCTTGTTACGACTT-3'
A high-fidelity polymerase, Phusion™ (New England Biolabs, Ipswich, MA) was used to amplify the
16S rRNA gene from each of the 30 isolated colonies. PCR amplification of the 30 isolated colonies was
carried out in 50 pL total volumes containing: 1 X Phusion™ HF Buffer, 0.02 U/pL of Phusion™ DNA
Polymerase, 0.5 pM of each 16S primer, and 0.2 pM of each dNTP inoculated with 5 |aL of thermolysed
colonies. Cycling conditions were carried out on a 9700 thermocycler (Applied Biosystems, Carlsbad,
CA) according to the following: an initial hold at 98°C for 30 seconds; 35 cycles of denaturation (98°C
for 10 seconds), annealing (55°C for 30 seconds), and extension (72°C for 1 minute); a final hold at 72°C
for 5 minutes. For samples amplified with primers 27F and 1492R, the annealing temperature was raised
to 56°C. PCR products were quantified by UV-absorbance using the NanoDrop™ 2000
spectrophotometer (Thermo Scientific).
Initially, pooled samples were subject to PCR using primers 8F and 1492R, and then amplified
using a polymerase with high resistance to many PCR inhibitors, Phire® (New England Biolabs, Ipswich,
MA). The Phire® PCR was carried out in 50 |_iL total volumes containing: 1 X Phire® Animal Tissue PCR
Buffer, 1 |_iL of Phire® Hot Start II DNA Polymerase, and 0.5 pM of each primer, inoculated with 5 |aL of
OneStep™ cleaned pooled sample. Cycling conditions were carried out on a 9700 thermocycler with an
initial hold at 98 °C for 5 minutes; 40 cycles of denaturation (98°C for 5 seconds), annealing (55°C for 5
seconds), and extension (72°C for 40 seconds); a final hold at 72 °C for 1 minute. Following
amplification of the 16S rRNA gene, the size of the amplified product was checked using 1.2 % Agarose
E-Gel® (Life Technologies) and an E-Gel® 1 Kb Plus DNA ladder (Life Technologies).
A second amplification of the pooled samples was undertaken using the 27F and 1492R primers;
no further amplification was required for these PCR products prior to sequencing.
3.4.4 Sequencing of 16S rRNA genes
1 Baker, G. C., Smith, J. J., Cowan. Review and re-analysis of domain-specific 16S primers. Journal of Micro.
Methods, 55 (3): 541-555. 2003.
Appendix B-8
-------
3.4.4.1 Sequencing of 16S rRNA from Isolated Colonies using Applied Biosystems 3130
Genetic Analyzer. The 16S rRNA PCR products generated from isolated colonies were purified using the
GenElute™ PCR Clean-up Kit (Sigma-Aldrich), and the concentration of each PCR product was
determined using the NanoDrop™ 2000 Spectrophotometer (Thermo Scientific, Pittsburgh, PA).
Forward and reverse cycle sequencing reactions were set up using the same 8F and 1492R PCR primers
that yielded the original PCR product. Cycle sequencing was carried out using BigDye® Terminator v3.1
(Life Technologies™, Carlsbad, CA) in 20 |_iL total volumes containing: 4 |_iL of Ready Reaction Mix, 2
|_iL of BigDye Sequencing Buffer, 5 pmol primer, and 20 - 40 ng of 16S rRNA PCR product. Cycling
conditions were carried out on a 9700 thermocycler (Applied Biosystems) with an initial hold at 96°C for
1 minute; 25 cycles of denaturation (96°C for 10 seconds), annealing (50°C for 5 seconds), and extension
(60°C for 4 minutes). A positive control, pGEM®-3Zf(+), and NTC negative controls were included.
Cycle sequencing reactions were purified using the BigDye® XTerminator™ Purification Kit (Applied
Biosystems) following the manufacturer's instructions.
Capillary electrophoresis was run on each purified cycle sequencing reaction using Applied
Biosystems 3130 Genetic Analyzer with the parameters shown in Table 3.
Table 3. Applied Biosystems 3130 Genetic Analyzer Parameters
Specific Parameters
Parameter
Setting
Template
BDx_StdSeq50_POP7
Oven Temperature
60 °C
Poly Fill Volume
5020 steps
Current Stability
5.0 Amps
Pre-Run Voltage
15.0 kVolts
Pre-Run Time
180 seconds
Injection Voltage
1.6 kVolts
Injection Time
4 seconds
Voltage Number of Steps
40 nk
Voltage Step Interval
15 seconds
Data Delay Time
480 seconds
Run Voltage
8.5 kVolts
Appendix B-9
-------
Run Time
6000 seconds
All raw sequencing files were imported into Sequencing Analysis Software v5.2 (Applied Biosystems)
and analyzed using the KB™ basecaller to provide per-base quality value predictions.
3.4.4.2 Sequencing of 16S rRNA Amplified with Primers 8F and 1492R using Life
Technologies Ion Torrent Personal Genome Machine (PGM™). A total of fourteen 16S rRNA
amplicons, including a positive and negative control, 1550 base pairs (bp) in length, were initially
processed to create a sequencing library. Library preparation generated a pool of amplicons tagged with a
specific molecular barcode that allowed multiplexing of samples for analysis on a single PGM™
semiconductor chip. The Ion DNA Barcoding 1-16 kit (Life Technologies™, Carlsbad, CA) was used to
prepare the library for the multiplexing experiment. Briefly, each of the 16S DNA amplicons separately
underwent enzymatic shearing to fractionate the 1550 bp products; a purification step was performed
using Agencourt® AMPure® magnetic particles (Beckman Coulter, Brea, CA) according to the
manufacturer's instructions; and Ion Barcode Adapters™ were ligated to the fragmented, purified DNA.
An additional purification step was performed using the Agencourt® AMPure® magnetic particles to
remove small molecular weight fragments. Following purification, an additional PCR was performed
incorporate unique molecular barcodes onto the adapter-modified, fragmented DNA and further amplify
each molecule. After PCR, a final purification step was performed using the Agencourt® AMPure®
magnetic particles. Each molecule in the final bar-coded library preparation was approximately 180-210
base pairs in length, including amplicon sequence, adapter, and barcode. Individual reactions were
measured using the NanoDrop® (Thermo Scientific, Pittsburgh, PA) to quantify DNA concentrations prior
to pooling a portion of each reaction into a single aggregate sample. The concentration of the aggregate
sample was measured again using the NanoDrop® to determine the library pool dilution required for
sequencing.
To prepare the aggregate, barcoded sample for sequencing, clonal amplification was performed
on the Ion OneTouch™ instrument. Briefly, the aggregate, barcoded library was combined with
IonSphere Particles™ (ISPs) followed by clonal amplification in an oil emulsion PCR, which binds a
single molecule to each particle and creates multiple copies of each particle-bound fragment.
Immediately following clonal amplification, the particle-bound fragments were enriched using the Ion
OneTouch ES™ instrument; this process removes unbound particles and unbound library fragments to
enrich for particle-bound fragments. At this point, a quality control check was performed, whereby a
small amount of the enriched ISPs was quantitated using the Qubit® 2.0 Flourometer (Life
Technologies™, Carlsbad, CA) to determine the extent of enrichment. After enrichment, ISPs were
loaded into an Ion 316™ chip (a single ISP per well) and sequencing was carried out according to
manufacturer's instructions.
3.4.4.3 Sequencing of 16S rRNA Amplified with Primers 27F and 1492R using Life
Technologies Ion Torrent PGM™. Qualitative and quantitative measurements of 16S amplicons were
made using the Qubit dsDNA BR Assay Kit on the Qubit 2.0 fluorometer followed by analysis on the
Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA) using DNA High Sensitivity chips.
Following this, 16S amplicon samples were fragmented using a Covaris S220 sonicator (Covaris, Inc,
Woburn, MA) to generate approximately 300bp fragments. Fragmentation quality was assessed using an
Appendix B-10
-------
Agilent Bioanalyzer. Sequencing libraries were made using Life Technologies™ Ion Plus Fragment
Library kit for 200bp sequencing. Library quality was verified using the Agilent Bioanalyzer and the
Qubit. Clonal amplification was performed on an Ion One Touch instrument using the Ion Xpress™
Template 200 Kit (Life Technologies™, Carlsbad, CA). Enrichment for the ISP's was done on the Ion
One Touch ES, and quantification of the percent templated ISP's was performed on the Qubit
fluorometer. Sequencing was performed with 316 chips on an IonTorrent PGM sequencer using the Ion
Sequencing 200 kit. The IonTorrent Suite Server (1.5.1) performed base calling and output raw sequence
data in FASTQ format.
3.4.5 Sequence Analysis of 16S rRNA genes
Sequence reads from the ABI 3130 with a length of greater than 200 base pairs and high quality
base calls were subject to BLASTn (GenBank, http://blast.ncbi.nlm.nih.gov/), searching against the 16S
microbial database. The BLAST nucleotide results with the highest maximum identity percentage were
reported.
3.4.5.1 Bioinformatics. FASTQ files were loaded into CLCBio Genomics Workbench software
V 4.9. Trimming of sequence reads was performed to remove PCR primer sequences and low quality
reads (0.05 quality threshold). A final filtering of reads was performed to select for reads of >175 bps.
The NCBI 16S rRNA (v6/15/2102) sequence database was loaded into CLCBio as a reference library.
Two bioinformatics analyses were performed. First, read files were processed using the Battelle Galileo
high performance compute cluster and the Basic Local Alignment Search Tool (BLAST®) (National
Library of Medicine, Bethesda, MD). Reads were searched against the NCBI 16S rRNA gene database
(v6/15/2102) (NCBI, Bethesda, MD), which contained entries for 7,545 sequences. Search results were
filtered for sequences with >97% identity. The output from this search resulted in a list of taxonomic IDs,
associated organism names, and number of reads per taxID for each sample. Krona2 v. 2.1 was used to
create a comparative chart for viewing the relative abundance of organisms at the genus level for each
sample. A final filtering of results was performed to include only taxa identified by numbers of hits
greater than 0.1% (1:1000) of the total representation per sample. The second analysis, the Battelle
QUEST™ tool, a recent R&D development using weighted probabilities based on genome coverage from
reference aligned data, was used to measure the amount of individual reads mapping to each 16S rRNA
sequence with the optimized parameters in CLCBio software and backend statistical analysis. The output
was reported as most probable species present in the sample.
4.0 RESULTS AND DISCUSSION
4.1 B. atrophaeus Detection by Quantitative PCR
2 Ondov BD, Bergman NH, and Phillippy AM. Interactive metagenomic visualization in a Web browser. BMC
Bioinformatics 2011. 12:385.
Appendix B-11
-------
Quantitative PCR (qPCR) analysis for B. atrophaeus signatures was complicated by the
co-extraction of inhibitory components from sample matrices. The extraction method used for this
project is a slight modification of a method that has been used by Battelle for over ten years to extract and
recover trace nucleic acids from environmental samples. Sample matrices successfully processed using
this method include aqueous, soil, cellulose, food, and fabric compositions. Generally, any inhibitory
components that are co-extracted with the DNA can be counteracted by dilution (1:5 or 1:10) of the
sample extract in IX TE containing 10 mM Tris and 1 mM EDTA. In this case, only about a third of the
sample extracts could be analyzed Neat, 1:5, or 1:10; the remaining two thirds of the sample extracts
required further purification using a Qiagen (Valencia, CA) QIAquick PCR purification kit. The Qiagen
samples were diluted and tested for inhibition at Neat, 1:5, 1:10, and 1:20.
Sample extracts that passed IPC were analyzed in duplicate for B. atrophaeus rtp signatures at the
highest concentration that passed IPC. Table 4 shows the results of qPCR, including the analyzed
dilution, the threshold cycle (Ct), and quantity in GC/5 |a,L for positive control samples. B. atrophaeus
DNA was not detected in any of the sample extracts; sample number IRP-WIPE-10-21-11-ABC-24 was
first thought to be positive, but upon further investigation, the multicomponent plot showed that the
fluorescent signal in those wells was extremely high, and true amplification did not occur. Samples that
were inhibited at all tested dilutions (Neat, 1:5, 1:10, QN, Q5, Q10, and Q20) were subject to PCR using
the Phire® Animal
Table 4. Results of Bacillus atrophaeus qPCR Analyses
Sample ID
Dilution
Ct Value
GC/5 (o,La
Resultb
Filter PC 1
1:5
35.49
204.86
Positive
35.09
272.53
Filter PC 2
1:5
31.62
4821.90
Positive
31.06
6351.25
Gauze PC 1
1:5
32.65
1510.49
Positive
32.64
1523.35
Gauze PC 2
Neat
30.21
8087.46
Positive
30.04
8778.85
Gauze PC 3
Qiagen 1:5
32.21
3018.52
Positive
32.65
2425.59
Gauze PC 4
Qiagen 1:5
35.29
797.78
Positive
34.91
963.46
Appendix B-12
-------
Sample ID
Dilution
Ct Value
GC/5 |aL"
Resultb
Gauze PC 5
Qiagen 1:5
38.43
171.44
Positive
35.78
628.65
Water PC 1
1:5
31.37
3712.34
Positive
31.69
2976.29
Grease PC 1
Neat
32.93
1244.29
Positive
32.98
1200.10
a Gene copies per 5 |_iL of PCR reaction (after sample extraction, concentration by alcohol precipitation, re-suspension, etc.);
bPositive = >Limit of Quantitation (LOQ); LOQ was 92.1 GC/5 |_iL; samples with mean <1 GC/5 |_iL are considered Negative;
samples with multicomponent trace showing no amplification are considered MC Negative.
c These sample extracts were inhibited Neat, 1:5, 1:10, and 1:20, they were further purified by Qiagenkit and diluted to overcome
inhibition
d Multicomponent
Tissue Direct PCR Kit after pooling DNA extracts into five composite samples comprised of nine or ten
sample extracts (Table 1). Phire® PCR was unsuccessful at amplification under these conditions; no
amplification was observed in any sample, including the positive control (1 x 104 GC/5 |a,L amplified
standard). Because the positive control reaction did not amplify, it appears that the PCR conditions were
sub-optimal, and it is not possible to determine from this analysis
whether these inhibited samples contain B. atrophaens DNA. The results for a small subset of
samples was inadvertently omitted from the Interim Report, these samples were also analyzed by Phire®
Animal Tissue Direct PCR Kit, in duplicate reactions using the 7900HT, rather than in the sample pools
as described above. These samples did not amplify, also likely due to inhibition of the Phire®)
polymerase.
Sample extracts IRP-AIR-10-24-11-ABC-018 to IRP-AIR-10-24-11-ABC-025 and sample IRP-
AIR-10-24-1 l-ABC-27 were amplified on the ABI 9700 instrument using the B. atrophaens rtp
primers and analyzed by gel electrophoresis (target amplicon 82 bp). Positive control reactions
containing 1000 GC/5 |a,L standard control material, and negative control reactions (NTC) containing IX
TE, were prepared and analyzed along with the sample extracts. No B. atrophaens DNA was detected in
the samples or NTCs, but a band was observed in the 1000 GC/5 |a,L standard positive control well
consistent with the expected 82 bp amplicon (Figure 1).
Appendix B-13
-------
1 empty
2 pooled samples
3 empty
4 1000 GC/5jiL
positive control
5 empty
6 1 Kb ladder
7 empty
8 pooled NTC
Figure 1. Gel Electrophoresis of AIR-10-21-11 Samples Analyzed by PCR on the ABI 9700
Thermocycler (blue arrow denotes an amplicon at -82 bp in the positive control well, consistent
with B. atrophaeus rtp. Bands are visible in the NTC and pooled sample wells, but are migrating
slightly lower than the band in well 4, and may be primers).
4.2 Detection of PLGA Microspheres
PLGA microspheres were observed in all positive control samples but at low quantities (i.e. less
than 20 microspheres per view). Sample autofluorescence prevented visualization of PLGA in most
samples; only samples IRP-WIPE-10-24-1 l-ABC-0089 and IRP-WIPE-10-24-1 l-ABC-0099 contained
fluorescent particles consistent with the PLGA micropsheres. The gauze and filter matrices are
autofluorescent, creating a diffuse green background under the epifluorescent conditions; moreover,
irregularly-shaped, autofluorescent particulate matter in and on some sample matrices made it impossible
to discern if PLGA microspheres were present. Table 5 lists each sample and the corresponding
microscopic descriptions. Representative photos are shown in Figure 2. Several of the samples had
begun to support mold growth at the time of microscopy, which also contributes to autofluorescence.
Table 5. Microscopic Observations of PLGA Microspheres
Sample ID
Microscopic Observations
Appendix B-14
-------
Sample ID
Microscopic Observations
IRP-AIR-10-19-11-ABC-B1
Dark field with diffuse green fluorescence; no
PLGA microspheres
IRP-AIR-10-19-11-ABC-B2
Diffuse green fluorescence, no PLGA
microspheres
IRP-AIR-10-19-11-ABC-B3
Dark field with diffuse green fluorescence; no
PLGA microspheres
IRP-AIR-10-19-11-ABC-B4
Diffuse green fluorescence, no PLGA
microspheres
IRP-AIR-10-19-11-ABC-B5
Dark field with diffuse green fluorescence; no
PLGA microspheres
IRP-AIR-10-19-11-ABC-B6
Diffuse green fluorescence, no PLGA
microspheres
IRP-AIR-10-19-11-ABC-B7
Diffuse green fluorescence, no PLGA
microspheres
IRP-AIR-10-19-11-ABC-B8
Diffuse green fluorescence, no PLGA
microspheres
IRP-WIPE-10-19-11-ABC-B1
2 fluorescent particles observed, too large to be
PLGA microspheres
IRP-WIPE-10-19-11-ABC-B2
1-2 fluorescent particles observed, too large to be
PLGA microspheres
Table 5. Microscopic Observations of PLGA Microspheres (Continued)
Sample ID
Microscopic Observations
IRP-WIPE-10-19-11-ABC-B3
No PLGA microspheres observed, background
fluorescence
IRP-WIPE-10-19-11-ABC-B4
No PLGA microspheres observed, background
fluorescence
IRP-WIPE-10-19-11-ABC-B5
No PLGA microspheres observed
IRP-AIR-10-20-1 l-ABC-001
Dark field with diffuse green fluorescence; no
PLGA microspheres
IRP-AIR-10-20-1 l-ABC-002
Dark field with diffuse green fluorescence; no
Appendix B-15
-------
Sample ID
Microscopic Observations
PLGA microspheres
IRP-AIR-10-20-1 l-ABC-003
Dark field with diffuse green fluorescence; no
PLGA microspheres
IRP-AIR-10-20-1 l-ABC-004
Dark field with diffuse green fluorescence; no
PLGA microspheres
IRP-AIR-10-20-1 l-ABC-005
Dark field with diffuse green fluorescence; no
PLGA microspheres
IRP-AIR-10-20-1 l-ABC-006
Dark field with diffuse green fluorescence; no
PLGA microspheres
IRP-AIR-10-20-1 l-ABC-007
Dark field with diffuse green fluorescence; no
PLGA microspheres
IRP-AIR-10-20-1 l-ABC-008
Dark field with diffuse green fluorescence; no
PLGA microspheres
IRP-AIR-10-21-11-ABC-009
Dark field with diffuse green fluorescence; no
PLGA microspheres
IRP-AIR-10-21-11-ABC-010
Dark field with diffuse green fluorescence; no
PLGA microspheres
IRP-AIR-10-21-11-ABC-011
Dark field with diffuse green fluorescence; no
PLGA microspheres
IRP-AIR-10-21-11-ABC-012
Dark field with diffuse green fluorescence; no
PLGA microspheres
IRP-AIR-10-21-11-ABC-013
Dark field with diffuse green fluorescence; no
PLGA microspheres
IRP-AIR-10-21-11-ABC-014
Diffuse green background with no fluorescent
particles
IRP-AIR-10-21-11-ABC-015
Diffuse green background with no fluorescent
particles
Appendix B-16
-------
Table 5. Microscopic Observations of PLGA Microspheres (Continued)
Sample ID
Microscopic Observations
IRP-AIR-10-21-11-ABC-016
Diffuse green background with no fluorescent
particles
IRP-AIR-10-21 -11 -ABC-017
Diffuse green background with no fluorescent
particles
IRP-IW -10-20-11 - AB C-001
Observed crystalline-like fluorescent shards and
spherical fluorescent particles; none discernible as
PLGA microspheres
IRP-WIPE-10-20-11-AB C-001
Diffuse green background with many fluorescent
particles of irregular size and shape, indiscernible
from PLGA microspheres
IRP-WIPE-10-20-1 l-ABC-002
Diffuse green background with some fluorescent
particles of irregular size and shape, indiscernible
from PLGA microspheres
IRP-WIPE-10-20-1 l-ABC-003
Diffuse green background with some fluorescent
particles of irregular size and shape, indiscernible
from PLGA microspheres
IRP-WIPE-10-20-1 l-ABC-004
Dark field with diffuse some fluorescence; no
PLGA microspheres
IRP-WIPE-10-20-1 l-ABC-005
Dark field with diffuse green fluorescence; no
PLGA microspheres
IRP-WIPE-10-20-1 l-ABC-006
Dark field with diffuse green fluorescence; no
PLGA microspheres
IRP-WIPE-10-20-1 l-ABC-007
Diffuse green background with some fluorescent
particles of irregular size and shape, indiscernible
from PLGA microspheres
IRP-WIPE-10-20-1 l-ABC-008
Dark field with diffuse green fluorescence; no
PLGA microspheres
IRP-WIPE-10-20-1 l-ABC-009
Light field with diffuse green fluorescence; no
PLGA microspheres
IRP-WIPE-10-20-1 l-ABC-0010
Light field with diffuse green fluorescence; no
PLGA microspheres
IRP-WIPE-10-21 -11 - AB C-0011
Dark field with diffuse green fluorescence; no
PLGA microspheres
Appendix B-17
-------
Sample ID
Microscopic Observations
IRP-WIPE-10-21-11 - AB C-0012
Dark field with diffuse green fluorescence; no
PLGA microspheres
IRP-WIPE-10-21 -11 - AB C-0013
Diffuse green background with many fluorescent
particles of irregular size and shape, indiscernible
from PLGA microspheres
Appendix B-18
-------
Table 5. Microscopic Observations of PLGA Microspheres (Continued)
Sample ID
Microscopic Observations
IRP-WIPE-10-21-11 - AB C-0014
Dark field with diffuse green fluorescence; no
PLGA microspheres
IRP-WIPE-10-21 -11 - AB C-0015
Dark field with diffuse green fluorescence; no
PLGA microspheres
IRP-WIPE-10-21-11 - AB C-0016
Dark field with diffuse green fluorescence; no
PLGA microspheres
IRP-WIPE-10-21 -11 - AB C-0017
Dark field with diffuse green fluorescence; no
PLGA microspheres
IRP-WIPE-10-21 -11 - AB C-0018
Dark field with diffuse green fluorescence; no
PLGA microspheres
IRP-WIPE-10-21 -11 - AB C-0019
Light field with diffuse green fluorescence; no
PLGA microspheres
IRP-WIPE-10-21 -11 - AB C-0020
Light field with diffuse green fluorescence; no
PLGA microspheres
IRP-WIPE-10-21 -11 - AB C-0021
Dark field with diffuse green fluorescence; no
PLGA microspheres
IRP-WIPE-10-21 -11 - AB C-0022
Diffuse green background with many fluorescent
particles of irregular size and shape, indiscernible
from PLGA microspheres
IRP-WIPE-10-21 -11-AB C-0023
Dark field with diffuse green fluorescence; no
PLGA microspheres
IRP-WIPE-10-21 -11 - AB C-0024
Dark field with diffuse green fluorescence; no
PLGA microspheres
IRP-WIPE-10-21 -11-AB C-0025
Dark field with diffuse green fluorescence; no
PLGA microspheres
IRP-WIPE-10-21 -11 - AB C-0026
Dark field with diffuse green fluorescence; no
PLGA microspheres
IRP-WIPE-10-21 -11-AB C-0027
Diffuse green background with many fluorescent
particles of irregular size and shape, indiscernible
from PLGA microspheres
IRP-WIPE-10-21 -11-AB C-0028
Dark field with diffuse some fluorescence; no
PLGA microspheres
Appendix B-19
-------
Sample ID
Microscopic Observations
IRP-WIPE-10-21 -11 -AB C-0029
Dark field with diffuse green fluorescence; no
PLGA microspheres
IRP-WIPE-10-21-11-ABC-0030
Diffuse green background with many fluorescent
particles of irregular size and shape, indiscernible
from PLGA microspheres
IRP-WIPE-10-21-1 l-ABC-0031
Bright green background with many fluorescent
particles of irregular size and shape, indiscernible
from PLGA microspheres
Table 5. Microscopic Observations of PLGA Microspheres (Continued)
Sample ID
Microscopic Observations
IRP-WIPE-10-21-1 l-ABC-0032
Diffuse green background with few fluorescent
particles of irregular size and shape, indiscernible
from PLGA microspheres
IRP-WIPE-10-21 -11 - AB C-003 3
Bright green background with many fluorescent
particles of irregular size and shape, indiscernible
from PLGA microspheres
IRP-WIPE-10-21-1 l-ABC-0034
Bright green background with many fluorescent
particles of irregular size and shape, indiscernible
from PLGA microspheres
IRP-WIPE-10-21 -11-AB C-003 5
Bright green background with many fluorescent
particles of irregular size and shape, indiscernible
from PLGA microspheres
IRP-WIPE-10-21-1 l-ABC-0036
Bright green background with many fluorescent
particles of irregular size and shape, indiscernible
from PLGA microspheres
IRP-WIPE-10-21 -11-AB C-003 7
Very bright green background with few fluorescent
particles of irregular size and shape, indiscernible
from PLGA microspheres
IRP-WIPE-10-21 -11-AB C-003 8
Very bright green background with few fluorescent
particles of irregular size and shape, indiscernible
from PLGA microspheres
IRP-WIPE-10-21 -11-AB C-003 9
Diffuse green background with few fluorescent
particles of irregular size and shape, indiscernible
Appendix B-20
-------
Sample ID
Microscopic Observations
from PLGA microspheres
IRP-WIPE-10-21 -11 - AB C-0040
Diffuse green background with few fluorescent
particles of irregular size and shape, indiscernible
from PLGA microspheres
IRP-WIPE-10-21 -11 - AB C-0041
Diffuse green background with no fluorescent
particles
IRP-WIPE-10-21 -11 - AB C-0042
Diffuse green background with no fluorescent
particles
IRP-WIPE-10-21 -11-AB C-0043
Diffuse green background with no fluorescent
particles
IRP-WIPE-10-21 -11 - AB C-0044
Diffuse green background with no fluorescent
particles
IRP-WIPE-10-21 -11-AB C-0045
Diffuse green background with no fluorescent
particles
Table 5. Microscopic Observations of PLGA Microspheres (Continued)
Sample ID
Microscopic Observations
IRP-WIPE-10-21 -11 - AB C-0046
Diffuse green background with no fluorescent
particles
IRP-WIPE-10-21 -11-AB C-0047
Diffuse green background with no fluorescent
particles
IRP-WIPE-10-21 -11-AB C-0048
Diffuse green background with no fluorescent
particles
IRP-WIPE-10-21 -11-AB C-0049
Diffuse green background with no fluorescent
particles
IRP-WIPE-10-24-1 l-ABC-050
Diffuse green background with no fluorescent
particles
IRP-WIPE-10-24-11-AB C-051
Diffuse green background with no fluorescent
particles
Appendix B-21
-------
Sample ID
Microscopic Observations
IRP-WIPE-10-24-1 l-ABC-052
Dark field with diffuse green fluorescence; no
PLGA microspheres
IRP-WIPE-10-24-1 l-ABC-053
Dark field with diffuse green fluorescence; no
PLGA microspheres
IRP-WIPE-10-24-1 l-ABC-054
Light field with diffuse green fluorescence; no
PLGA microspheres
IRP-WIPE-10-24-1 l-ABC-055
Light field with diffuse green fluorescence; no
PLGA microspheres
IRP-WIPE-10-24-1 l-ABC-056
Dark field with diffuse green fluorescence; no
PLGA microspheres
IRP-WIPE-10-24-1 l-ABC-057
Dark field with diffuse green fluorescence; no
PLGA microspheres
IRP-WIPE-10-24-1 l-ABC-058
Dark field with diffuse green fluorescence; no
PLGA microspheres
IRP-WIPE-10-24-1 l-ABC-059
Diffuse green background with few fluorescent
particles of irregular size and shape, indiscernible
from PLGA microspheres
IRP-WIPE-10-24-1 l-ABC-060
Diffuse green background with many fluorescent
particles of irregular size and shape, indiscernible
from PLGA microspheres
IRP-WIPE-10-24-11-AB C-061
Diffuse green background with no fluorescent
particles
IRP-WIPE-10-24-1 l-ABC-062
Diffuse green background with few fluorescent
particles of irregular size and shape, indiscernible
from PLGA microspheres
IRP-WIPE-10-24-1 l-ABC-063
Diffuse green background with many fluorescent
particles of irregular size and shape, indiscernible
from PLGA microspheres
Table 5. Microscopic Observations of PLGA Microspheres (Continued)
Sample ID
Microscopic Observations
IRP-WIPE-10-24-1 l-ABC-064
Diffuse green background with many fluorescent
particles of irregular size and shape, indiscernible
Appendix B-22
-------
Sample ID
Microscopic Observations
from PLGA microspheres
IRP-WIPE-10-24-1 l-ABC-065
Diffuse green background with few fluorescent
particles of irregular size and shape, indiscernible
from PLGA microspheres
IRP-WIPE-10-24-1 l-ABC-066
Diffuse green background with few fluorescent
particles of irregular size and shape, indiscernible
from PLGA microspheres
IRP-WIPE-10-24-1 l-ABC-067
Diffuse green background with no fluorescent
particles
IRP-WIPE-10-24-1 l-ABC-068
Diffuse green background with many fluorescent
particles of irregular size and shape, indiscernible
from PLGA microspheres
IRP-WIPE-10-24-1 l-ABC-069
Diffuse green background with many fluorescent
particles of irregular size and shape, indiscernible
from PLGA microspheres
IRP-WIPE-10-24-1 l-ABC-070
Diffuse green background with many fluorescent
particles of irregular size and shape, indiscernible
from PLGA microspheres
IRP-WIPE-10-24-1 l-ABC-071
Diffuse green background with few fluorescent
particles of irregular size and shape, indiscernible
from PLGA microspheres
IRP-WIPE-10-24-1 l-ABC-072
Bright green background with many fluorescent
particles of irregular size and shape, indiscernible
from PLGA microspheres
IRP-WIPE-10-24-1 l-ABC-073
Diffuse green background with no fluorescent
particles
IRP-WIPE-10-24-1 l-ABC-074
Diffuse green background with few fluorescent
particles of irregular size and shape, indiscernible
from PLGA microspheres
IRP-WIPE-10-24-1 l-ABC-075
Diffuse green background with few fluorescent
particles of irregular size and shape, indiscernible
from PLGA microspheres
IRP-WIPE-10-24-1 l-ABC-076
Diffuse green background with many fluorescent
particles of irregular size and shape, indiscernible
from PLGA microspheres
Appendix B-23
-------
Sample ID
Microscopic Observations
IRP-WIPE-10-24-1 l-ABC-077
Diffuse green background with few fluorescent
particles of irregular size and shape, indiscernible
from PLGA microspheres
IRP-WIPE-10-24-1 l-ABC-078
Diffuse green background with few fluorescent
particles of irregular size and shape, indiscernible
from PLGA microspheres
Table 5. Microscopic Observations of PLGA Microspheres (Continued)
Sample ID
Microscopic Observations
IRP-WIPE-10-24-1 l-ABC-079
Diffuse green background with no fluorescent
particles
IRP-WIPE -10-24-11 - AB C -0 80
Diffuse green background with many fluorescent
particles of irregular size and shape, indiscernible
from PLGA microspheres
IRP-WIPE-10-24-1 l-ABC-081
Diffuse green background with few fluorescent
particles of irregular size and shape, indiscernible
from PLGA microspheres
IRP-WIPE-10-24-1 l-ABC-082
Diffuse green background with few fluorescent
particles of irregular size and shape, indiscernible
from PLGA microspheres
IRP-WIPE-10-24-1 l-ABC-083
Bright green background with many fluorescent
particles of irregular size and shape, indiscernible
from PLGA microspheres
IRP-WIPE-10-24-1 l-ABC-084
Diffuse green background with no fluorescent
particles
IRP-WIPE-10-24-1 l-ABC-085
Diffuse green background with no fluorescent
particles
IRP-WIPE-10-24-1 l-ABC-086
Diffuse green background with no fluorescent
particles
IRP-WIPE-10-24-1 l-ABC-087
Diffuse green background with many fluorescent
particles of irregular size and shape, indiscernible
from PLGA microspheres
IRP-WIPE-10-24-1 l-ABC-088
Diffuse green background with few fluorescent
particles of irregular size and shape, indiscernible
Appendix B-24
-------
Sample ID
Microscopic Observations
from PLGA microspheres
IRP-WIPE-10-24-11-ABC-089
Dark background with diffuse green fluorescence, 1
fluorescent particle observed consistent with PLGA
microsphere
IRP-WIPE -10-24-11 - AB C -0 90
Diffuse green background with many fluorescent
particles of irregular size and shape, indiscernible
from PLGA microspheres
IRP-WIPE -10-24-11 - AB C -0 91
Diffuse green background with no fluorescent
particles
IRP-WIPE -10-24-11 - AB C -0 92
Diffuse green background with many fluorescent
particles of irregular size and shape, indiscernible
from PLGA microspheres
IRP-WIPE-10-24-11-ABC-093
Diffuse green background with many fluorescent
particles of irregular size and shape, indiscernible
from PLGA microspheres
IRP-WIPE -10-24-11 - AB C -0 94
Dark background with green fluorescence, many
fluorescent particles of irregular size and shape,
indiscernible from PLGA microspheres
Table 5. Microscopic Observations of PLGA Microspheres (Continued)
Sample ID
Microscopic Observations
IRP-WIPE-10-24-11-ABC-095
Diffuse green background with few fluorescent
particles of irregular size and shape, indiscernible
from PLGA microspheres
IRP-WIPE -10-24-11 - AB C -0 96
Diffuse green background with few fluorescent
particles of irregular size and shape, indiscernible
from PLGA microspheres
IRP-WIPE-10-24-11-ABC-097
Diffuse green background with no fluorescent
particles
IRP-WIPE-10-24-11-ABC-098
Diffuse green background with no fluorescent
particles
IRP-WIPE-10-24-11-ABC-099
Very diffuse green background, ~5 fluorescent
particles observed consistent with PLGA
microspheres
Appendix B-25
-------
Sample ID
Microscopic Observations
IRP-FPG-10-24-11-ABC-001
N/A
IRP-FPC -10-24-11 -AB C-001
N/A
IRP-AIR-10-24-11-ABC-018
Diffuse green background with no fluorescent
particles
IRP-AIR-10-24-11-ABC-019
Diffuse green background with no fluorescent
particles
IRP-AIR-10-24-11-ABC-020
Diffuse green background with no fluorescent
particles
IRP-AIR-10-24-11-ABC-021
Diffuse green background with no fluorescent
particles
IRP-AIR-10-24-11-ABC-022
Diffuse green background with no fluorescent
particles
IRP-AIR-10-24-11 -ABC-023
Diffuse green background with no fluorescent
particles
IRP-AIR-10-24-11-ABC-024
Diffuse green background with no fluorescent
particles
IRP-AIR-10-24-11-ABC-025
Diffuse green background with no fluorescent
particles
IRP-AIR-10-24-11-ABC-027
Diffuse green background with no fluorescent
particles
Filter Blank 1
Diffuse green background with no fluorescent
particles
Filter Blank 2
Diffuse green background with no fluorescent
particles
Table 5. Microscopic Observations of PLGA Microspheres (Continued)
Sample ID
Microscopic Observations
Gauze Blank 1
Diffuse green background with no fluorescent
particles
Gauze Blank 2
Green fluorescent background, no PLGA
microspheres or fluorescent particles
Appendix B-26
-------
Sample ID
Microscopic Observations
Gauze Blank 3
Very diffuse green background, no PLGA
microspheres or fluorescent particles
Gauze Blank 4
Diffuse green background, no PLGA microspheres
or fluorescent particles
Gauze Blank 5
Diffuse green background with no fluorescent
particles
Water Blank 1
No fluorescent particles
Grease Blank 1
N/A
Filter PC 1
Many fluorescent PLGA microspheres observed in
both membrane and batting layer
Filter PC 2
Very bright green background with ~20 PLGA
microspheres visible on the membrane; no PLGA
microspheres visible on the batting
Gauze PC 1
Some fluorescent PLGA microspheres observed;
fewer than on Filter PC 1
Gauze PC 2
Some fluorescent PLGA microspheres observed in
background of green autofluorescence
Gauze PC 3
Bright green background with 1 fluorescent
particle suspected to be PLGA microsphere
Gauze PC 4
Some fluorescent PLGA microspheres observed
(~8) in diffuse green background
Gauze PC 5
Very bright green background, few PLGA
microspheres observed
Water PC 1
5 fluorescent PLGA microspheres
Grease PC 1
N/A
Appendix B-27
-------
Figure 2. Microscopic Images. Top: IRP-AIR-10-19-11-ABC-B4 (no microspheres present, representative of negative samples), bottom
left: Filter PC 1 (two microspheres observed), bottom right: Gauze PC 2 (diffuse green
Appendix B-28
-------
background, several microspheres present, but not observed as discrete particles).
Appendix B-29
-------
4.3 Enumeration of Putative Viable B. atrophaeus in Archived Samples
None of the filter samples appeared to contain B. atrophaeus. Ten gauze samples had putative B.
atrophaeus colonies (Table 6), including five samples originally reported in the January 18, 2012 Interim
Report. Putative B. atrophaeus was observed in six of these ten presumptive positives when plated for
enumeration, although two samples displayed quantities less than the limit of quantification (
-------
IRP-WIPE-10-20-11 -D-004
1 x 101
0
0
0
0
IRP-WIPE-10-20-11 -D-006b
1 x 101
<30
36
114
-------
a Individual plate counts <30 are not statistically significant; these counts are reported as <30 and are not included in
the calculation for final enumeration.
b Originally reported in the January 18, 2012 Interim Report
0 13 ng/pL. All five of the isolates that resulted in a
clean PCR product were able to be identified by sequencing using the ABI 3130.
The majority of the organisms that were isolated either could not be extracted using the
thermolysis method, or could not be amplified with the 8F and 1492R primers. While 8F and 1492R
primers are considered "universal primers" they are likely not ideal for all bacterial species, and other
"universal primers" that target the 16S rRNA gene could potentially be used to amplify a portion of the
gene.
Table 7. Samples Containing Colony Morphologies Similar to B. atrophaeus
B. atrophai'm.v-Contain in<; Samples
Appendix B-32
-------
IRP-IW-10-20-11 -ABC-0011
IRP-WIPE-10-20-11-ABC002
IRP-WIPE-10-20-11-ABC003
IRP-WIPE-10-20-1 l-ABC-006
IRP-WIPE-10-20-1 l-ABC-008
IRP-WIPE-10-21-1 l-ABC-0011
IRP-WIPE-10-21-11-ABC-0012
IRP-WIPE-10-21-1 l-ABC-0014
IRP-WIPE-10-21-1 l-ABC-0017
IRP-WIPE-10-21-1 l-ABC-0018
IRP-WIPE-10-21-1 l-ABC-0019
IRP-WIPE-10-21 -1 l-ABC-0024
IRP-WIPE-10-21 -1 l-ABC-0026
IRP-WIPE-10-21 -1 l-ABC-0027
IRP-WIPE-10-21-1 l-ABC-0029
1 Upon additional data review, only one sample contained putative B. atrophaeus.
4.4.3 Sequencing of 16S rRNA
4.4.3.1 Sequencing of 16S rRNA from Isolated Colonies using Applied Biosystems 3130
Genetic Analyzer. The BLAST nucleotide result with the highest maximum identity percentage is listed
in Appendix A, and the top 25 BLAST results, as well as the sequence information obtained, are shown in
Appendix B. Six of the 30 unknown isolates resulted in at least one high quality sequencing read.
Isolates 4, 19, 22, and 29 are likely Proteus species, isolate 15 is likely a Planomicrobium species, and
isolate 16 is likely a Curtobacterium species.
4.4.3.2 Sequencing of 16S rRNA Amplified with Primers 8F and 1492R using Ion Torrent
PGM™ Initial sequencing on the PGM™ yielded poor results most likely due to failure of the library
preparation. Poor quality 16S DNA amplicons and/or the possibility of carry over inhibitory components
may have caused the library preparation to fail. The PGM™ functioned properly and a successful run
occurred. After examination of the run, summary evidence pointed to the likelihood that poor clonal
amplification had occurred on the OneTouch™. The ISPs loaded correctly into the micron sized wells,
and all fluidics and semiconductor functions operated normally; however, template ISPs gave a reading of
8.23% on the Qubit® during the quality analysis check prior to sequencing. The percentage recommended
to proceed with sequencing is >50%. Poor clonal amplification was potentially due to poor library
construction in the presence of inhibitors that interfered with ligation of the molecular barcodes and
adapters; this step is crucial for all other subsequent steps in the library preparation and sequencing.
Sequencing reads generated on the PGM™ were of low quality; a quality filtration was performed on the
reads using CLCGenomics Workbench software, but there were too few reads post-filtration to perform
accurate BLAST analysis or assembly. The reads remaining after filtration were not analogous to
anything in the 16S database. Therefore no data was obtained from the PGM™ analysis.
4.4.3.3 Sequencing of 16S rRNA Amplified with Primers 27F and 1492R using Ion Torrent
PGM™. The 16S rRNA PCR strategy used was successful in producing amplicons from all five pooled
samples. Pools 2-5 gave high quality sequence data resulting from IonTorrent sequencing. Pool 1 did not
Appendix B-33
-------
yield sufficient high quality data, which is either due to the 16S amplicon quality (source sample
influence) or sequencing library and sequencer quality (sequencing influence). Resequencing of pool 1
was not performed due to time and budget constraints.
Table 8 shows the dominant genera of bacteria identified by BLAST search and the most probable species
identified by the Battelle QUEST™ method. Figures 2-5 present hierarchically organized relative
abundance data resulting from Ion Torrent PGM™ sequence analysis using the KRONA tool. KRONA is
an opensource software built with HTML5 (web-browser format) that may ingest BLAST data and
prepare visual results of the relative abundances of the total top BLAST hits. The KRONA maps in
Figures 2-5 show resolution at the genus level (outer ring of the circle) organized to lower sub-
classifications (inner radii of the circle). Percentage of BLAST reads matching each group of bacteria are
included in the figure to assist in interpretation. In general, all pools had Pseudomonas as the most
prevalent genus, ranging from 31-87 % of the total genetics sequences identified (Table 8). Pool five was
the least diverse sample with Pseiidomoncts and Shewanellct species comprising 95% of the sample.
Other genera of bacteria discovered in the pools included Stenotrophomonas, Xanthomoncts, Comomonas,
Herbaspirilium, Lactobacillus, Acinetobacter, and Yersinia. The Genus Bacillus was not observed in
pools 2, 4 and 5 and was at a level near to the limit of detection for the methods used in pooled sample 3
(0.04% of the genetic material identified). Further, most of the species identified from pools 2-5
belonged to the phylum Proteobacteria, with low observance (<5%) of the phyla Firmicutes,
Bacteroidetes and Actinomvcetales (Figures 2-5). In general, the pools had similar profiles of bacteria
identified by 16S sequencing, varying mostly by abundance of genera between pools.
Table 8. Results of 16S rRNA Sequencing Based on BLAST and QUEST™ Analysis
Sample
Dominant Genera by
BLAST
Dominant Organisms by QUEST™
(top 15 most probable species)
Pooll
ND*
ND
Pool 2
Pseudomonas (48%)
Stenotrophomonas (18%)
Xanthomonas (5%)
Yersinia (4%)
Comamonas (4%)
Stenotrophomonas rhizophila strain e-plO
PseudomonasJragi strain ATCC 4973
Acidaminococcus intestini strain ADV 255.99
Stenotrophomonas maltophilia strain LAM 12423
Acidaminococcus_Jermentans strain VR4
Comamonas kerstersii strain LMG 3475
Simplicispira metamorpha strain DSM 1837
Comamonas aquatica strain : LMG 2370
Pseudomonas_psychrophila strain E-3
Microvirgula aerodenitrificans strain Sgly2
Pseudomonas lundensis strain ATCC 49968
Stenotrophomonas koreensis strain TR6-01
Dysgonomonas capnocytophagoides strain LMG
Pseudomonas agarici strain 71A
Bre\'imdimonas terrae strain KSL-145
Table 8. Results of 16S rRNA Sequencing Based on BLAST and QUEST™ Analysis (Continued)
Sample
Dominant Genera by
Dominant Organisms by QUEST™
Appendix B-34
-------
BLAST
(top 15 most probable species)
Pool 3
Pseudomonas (31%)
Shewanella (18%)
Acinetobacter (7%)
HerbaspiriUium (6%)
Stenotrophomonas (4%)
Lactobacillus (3%)
Shewanella baltica strain 63
Stenotrophomonas rhizophila strain e-plO
PseudomonasJragi strain ATCC 4973
Herbaspirillum autotrophicum strain LAM 14942
Shewanella morhuae strain U1417
Morganella_psychrotolerans strain U2/3
Herbaspirillum rhizosphaerae strain UMS-37
Paucimonas lemoignei strain LMG 2207
Acinetobacter ursingii strain LULL
Arcobacter nitrofigilis strain CL
Dvsgonomonas capnocytophagoides strain LMG
Lactobacillus cur\>atus strain :DSM 20019
Shewanella_putrefaciens strain LMG 26268
Mvroides odoratimimus strain : CCUG 39352
Acinetobacter haemolvticus strain DSM 6962
Pool 4
Pseudomonas (34%)
Stenotrophomonas (42%)
Xanthomonas (10%)
Pseudoxanthomonas (3%)
Stenotrophomonas rhizophila strain e-plO
PseudomonasJragi strain ATCC 4973
Stenotrophomonas koreensis strain TR6-01
Stenotrophomonas maltophilia strain LAM 12423
Pseudomonas hibiscicola strain ATCC 19867
Pseudomonas_psychrophila strain E-3
Stenotrophomonas nitritireducens strain L2
Pseudomonasj>eniculata strain ATCC 19374
Pseudomonas mucidolens strain IAM12406
Pseudoxanthomonas spadix strain LMMLB AFLL-5
Mvcoplana bullata strain LAM 13153
Stenotrophomonas terrae strain : R-32768
Pseudomonas extremorientalis strain KALM 3447
Pseudomonas abietaniphila strain .ATCC 700689
Pseudomonas moraviensis strain CCM 7280
Pool 5
Pseudomonas (87%)
Shewanella (8%)
PseudomonasJragi strain ATCC 4973
Pseudomonas agarici strain 71A
Shewanella_putrefaciens strain LMG 26268
Pseudomonas_psychrophila strain E-3
Shewanella baltica strain 63
Pseudomonas lundensis strain ATCC 49968
Pseudomonas veronii strain CLP 104663
Pseudomonas libanensis strain CLP 105460
Stenotrophomonas rhizophila strain e-plO
Pseudomonas_palleroniana strain CFBP 4389
Shewanella hafniensis strain P010
Shewanella oneidensis strain MR-1
Pseudomonas mucidolens strain LAAL12406
Pseudomonas caricapapavae strain Robbs ENA-378
Pseudomonas taetrolens strain 111
*ND = no data
Appendix B-35
-------
Bacteria
Count: 21658
Tax ID: 2
Rank: superkingdom
Avg. log e-value: -39.3719
Proteobactena
Pseudomonas
ad-root.local
Internet access
Figure 3. KRONA Visualization of BLAST Results for Pool 2
Appendix B-36
-------
Bacteria
ad-root.local
Internet access
Root —> ]
Count: 124177
Figure 4. KRONA Visualization of BLAST Results for Pool 3
Appendix B-37
-------
Root
)bacteria
.,eraceae
Wphap
CauWb®c
<
Root
Count: 3105
ad-root.local
Internet access
00
m
Bacteria
Ga^aproteobacter*
Proteobacteria
Figure 5. KRONA Visualization of BLAST Results for Pool 4
Appendix B-38
-------
Bacteria
Count:
Tax ID:
Rank: superkingdom
-92.6326
79754
Avg. log e-value:
Gammaproteobacteria
Proteobacteria
Bacteria
ad-root.local
Internet access
4 -fe
Figure 6. KRONA Visualization of BLAST Results for Pool 5
Appendix B-39
-------
5.0 SUMMARY
No B. atrophaeus DNA was detected in any of the sample extracts. However, viable bacteria very similar
to B. atrophaeus positive control colony morphology were recovered from 1 of 60 original sample
extracts, and from 10 of the archived test samples (five contained putative B. atrophaeus in quantities
greater than LOQ). Furthermore, B. atrophaeus DNA could be extracted from, and detected in, spiked
positive controls of pristine gauze and filter matrices prepared from the same lots of gauze and filters as
the samples. It is possible that B. atrophaeus was present in low quantities and below the limit of
detection by qPCR; however, it is more likely that non-detection by qPCR was due to inhibitors present in
the sample matrices that carried over during the extraction process, since putative B. atrophaeus was
recoverable on BHIA.
PLGA microspheres may not be a suitable synthetic surrogate, as they become permanently immobilized
in sampling matrices; extraction processes were ineffective at removing PLGA microspheres for
quantitation by fluorometer, and autofluorescence from the sample matrices complicated detection of
PLGA microspheres via direct microscopic observation.
Amplification of 16S rRNA genes was accomplished in only 13 of 30 attempted reactions from the
isolated colonies, and sequence analysis of the 16S rRNA genes was only achieved for six out of these 13
amplicons. The remaining seven amplicons were likely of poor quality and not suitable for sequence
analysis. Amplification of 16S rRNA genes is performed using 'universal' primers that are generated to
conserved regions in the 16S genes; however, there are several sets of primers that can be used, and PCR
conditions may favor certain amplicons over others. If a different primer set is chosen, additional isolates
may be identified.
Sequence analysis of the pooled sample extracts was improved using primers 27F and 1492R (as
compared to primers 8F and 1492R). Pseudomonas was the primary species present in sample pools 2-5.
Sequence analysis could not be performed on pool 1; the 16S amplicons were of insufficient quality.
6.0 RECOMMENDATIONS
Many sample extracts were highly inhibitory to qPCR. Inhibition was not attributable to the filter and
gauze wipe matrices, as sample extracts from negative and positive controls prepared using identical
pristine matrices were either not inhibitive or only slightly inhibitive to qPCR. Inhibition, therefore, must
be attributable to sample complexity derived from processing in the rendering facility; potential inhibitors
that may have been introduced onto the filter and gauze wipe matrices during sampling include: animal
tissues and fluids, particulate matter (e.g., soil, dirt, debris), industrial and mechanical fluids, and other
environmental contaminants. Furthermore, B. atrophaeus was likely present in trace quantities in many
of the samples; putative B. atrophaeus was recovered from one of the original sample extracts and 10 of
the archived samples, although the isolate identities were not independently confirmed using qPCR or
other genotypic or phenotypic assay. A laboratory spiking study in various tissues and in gauze wipes of
Appendix B-40
-------
tissue handling areas, combined with refined extraction processes would provide limit of detection
information and determine suitable extraction methods for mitigating carryover of inhibitory components
from the rendering facility.
PLGA microspheres appeared to be irreversibly bound to gauze and filter matrices, preventing efficient
extraction of the microspheres into an aqueous solution for detection in the 96-well microtiter plate assay
developed for this study. Furthermore, autofluorescence from matrices and other particulate matter in the
samples interfered with direct visualization of PLGA microspheres on the sample surfaces. Further
testing is warranted to determine if an alternate bead composition would prevent surface interaction and
irreversible binding to sample matrices. Alternate fluorophores could be integrated into the beads to
improve direct microscopic observations.
From metagenome analysis, Pseudomonas was the most prevalent genus present in all of the
samples and other genera included, Stenotrophomonas, Xanthomonas, Comomonas, Herbaspirilium,
Lactobacillus, Acinetobacter, and Yersinia. In general, the pools had similar profiles of bacteria
identified by 16S sequencing, varying mostly by abundance of genera between pools. In this study,
sequencing data from the pure isolates did not correlate with sequencing data from the pooled samples.
Because 16S rRNA products were amplified from both pooled extracts and isolated colonies prior to
sequencing, it was expected that metagenome sequencing of the pooled sample fractions should contain
all of the pure isolate sequences, thus obviating the need to isolate colonies prior to sequencing.
However, as previously stated, there are several 'universal' primer sets that can be used to amplify 16S
genes and PCR conditions may favor amplification from some targets over others. Primer set 8F and
1492R did not provide clean amplification products in the pooled samples, but it is possible the switch
from primer 8F (isolated colonies) to primer 27F (pooled samples) was sufficient to select for an entirely
different set of amplicons in the pooled samples. It is also possible that the isolated colonies may
represent a very small portion of the entire metagenome population, and while we could cultivate and
sequence these organisms, their sequence represents such a small portion of the entire metagenome that it
is occluded by the other organisms comprising the population majority.
If further identification of pure isolates is desired, there are several new library kits on the market which
could be evaluated for future use; New England Biolabs (Ipswich, MA) has released a new kit specifically
made for the PGM™ that can be used for single isolate chip runs and multiplexing runs, and Life
Technologies recently released a new library preparation kit that explicitly for bacterial amplicons from
environmental samples used for multiplexing. Future work could also include the development of Paired-
End sequencing (PES) on the PGM™ to obtain double coverage and crossover sequencing for more robust
sequence analysis.
Whole metagenome sequencing without prior amplification of 16S rRNA would definitely
increase the amount of information returned, as this is not biased by the amplification of 16S genes and
will provide identification of prokaryotic and eukaryotic communities. This process requires 0.5 to 1 jug
of DNA, and would be amenable to gauze and filter matrices. However, if the samples were heavily
burdened with animal tissues (meat, bone, hair, etc.) it is possible that the results would be biased with
mammalian sequences and background prokaryotic and eukaryotic community members may be
occluded. Thus, method development and validation using samples spiked with varying amounts of
mammalian tissues would be required prior to using whole metagenome sequencing in this setting.
Appendix B-41
-------
APPENDIX A
SAMPLE LIST, MORPHOLOGY, AND IDENTIFICATION
-------
PCR result
(thermolysis
method)
Forward
Reverse
Second
Isolate
number
Sample name
Colony
description
primer
sequencing
result
primer
sequencing
result
BLAST results
purification
and PCR
[yes or no]
1
IRP-AIR-102011-
ABC-002
~2 mm beige,
transparent,
circular, shiny
No amplification
NA
NA
NA
NA
2
IRP-IW-102011-
ABC-OOl-lOOuL
~2 mm circular,
orange, umbonate
No amplification
NA
NA
NA
NA
IRP-WIPE-102111-
ABC-15-100uL
-2-4 mm
3
colonies, wliite,
shiny, circular
No amplification
NA
NA
NA
NA
4
IRP-WIPE-102111-
ABC-0016-100uL
~4 mm colonies,
beige, dull, some
with spreading
irregular edge
Faint band, PCR
purified then
amplified again.
Poor
sequencing
quality
-682 quality
bases
Proteus
yes
IRP-WIPE-102111-
ABC-0025-100uL
~4 mm colonies.
5
light beige,
umbonate
No amplification
NA
NA
NA
NA
6
IRP-WIPE-102111-
ABC-0027-100uL
~4 mm beige,
circular
No amplification
NA
NA
NA
NA
7
IRP-AIR-102011-
ABC-001
~2 mm, orange,
circular, shiny
Faint band, PCR
purified then
amplified again.
Poor
sequencing
quality
Poor
sequencing
quality
NA
yes
8
IRP-AIR-102111-
ABC-0011
~4 mm,
translucent-beige,
circular
No amplification
NA
NA
NA
NA
9
IRP-AIR-102111-
ABC-0012
Pinpoint, beige
No amplification
NA
NA
NA
NA
10
IRP-WIPE-102111-
ABC-0013-100uL
Mold-like, slimy,
clear edge, with
center
Faint band, PCR
purified then
amplified again.
Poor
sequencing
quality
Poor
sequencing
quality
NA
yes
11
IRP-AIR-102111-
ABC-10
~2 mm, circular,
orange, shiny
No amplification
NA
NA
NA
NA
12
IRP-AIR-102011-
ABC-006
yellow pinpoint,
shiny
No amplification
NA
NA
NA
NA
13
IRP-AIR-102011-
ABC-006
6-8 mm, beige,
circular with
irregular edge
No amplification
NA
NA
NA
NA
14
IRP-AIR-102111-
ABC-15
~2 mm, white,
umbonate, shiny
No amplification
NA
NA
NA
NA
Battelle A-l
-------
Isolate
number
Sample name
Colony
description
PCR result
(thermolysis
method)
Forward
primer
sequencing
result
Reverse
primer
sequencing
result
BLAST results
Second
purification
and PCR
[yes or no]
15
IRP-AIR-102111-
ABC-16
2 mm, orange,
convex
Amplified
-573 quality
bases
-633 quality
bases
Planomicrobium
no
16
IRP-AIR-102111-
ABC-011
2 mm, convex,
beige, shiny
Amplified
-559 okay
quality bases
Poor
sequencing
quality
Curtobacterium
no
17
IRP-WIPE-102111-
ABC-0021
4-6 mm, white,
circular, convex
No amplification
NA
NA
NA
NA
18
IRP-WIPE-102111-
ABC-0024
mold-like, not
slimy, dull,
irregular
spreading edge
No amplification
NA
NA
NA
NA
19
IRP-WIPE-102011-
ABC-002-100uL
Swarm-like,
smooth lawn
Amplified
-642 quality
bases
-675 quality
bases
Proteus
no
20
IRP-WIPE-102111-
ABC-0029-100uL
4 mm colonies,
circular, beige,
shiny
No amplification
NA
NA
NA
NA
21
IRP-WIPE-102111-
ABC-0026-100uL
3-4 mm colonies,
light yellow,
convex
No amplification
NA
NA
NA
NA
22
IRP-WIPE-102011-
ABC-OOl-lOOuL
Smooth lawn,
beige
Amplified
-617 quality
bases
-675 quality
bases
Proteus
no
23
IRP-AIR-102011-
ABC-006
1 mm colonies,
light orange.
Faint band, PCR
purified then
amplified again.
Poor
sequencing
quality
Poor
sequencing
quality
NA
NA
24
IRP-AIR-102111-
ABC-013
1 mm colonies,
yellow, convex
Faint band, PCR
purified then
amplified again.
Poor
sequencing
quality
Poor
sequencing
quality
NA
NA
25
IRP-WIPE-101911-
ABC-B5
Pinpoint pink
colonies
Faint band, PCR
purified then
amplified again.
Poor
sequencing
quality
Poor
sequencing
quality
NA
NA
26
IRP-WIPE-101911-
ABC-B2
Branch-like
growth, light
spreading edge,
white, dull
No amplification
NA
NA
NA
NA
27
IRP-WIPE-102011-
ABC-004
Lawn thin,
smooth, beige
Faint band, PCR
purified then
amplified again.
Poor
sequencing
quality
Poor
sequencing
quality
NA
NA
Battelle A-2
-------
Isolate
number
Sample name
Colony
description
PCR result
(thermolysis
method)
Forward
primer
sequencing
result
Reverse
primer
sequencing
result
BLAST results
Second
purification
and PCR
[yes or no]
28
IRP-WIPE-102111-
ABC-OOll-lOOuL
lawn, light
brown, rippled
surface, shiny
No amplification
NA
NA
NA
NA
29
IRP-WIPE-102111-
ABC-0012-100 uL
lawn spread
throughout,
smaller beige
colonies where
streak lines are,
mixed.
Amplified
-244 quality
bases
-679 quality
Proteus
no
30
IRP-WIPE-102011-
ABC-006-100 uL
lawn spread
throughout,
smaller beige
colonies where
streak lines are,
mixed.
No amplification
NA
NA
NA
NA
B.
atrophaeus,
ATCC 9372
Positive control
Beige-orange,
~2mm circular
colonies.
Faint band, PCR
purified then
amplified again.
Poor
sequencing
quality
Poor
sequencing
quality
NA
NA
Battelle A-3
-------
APPENDIX B
BLAST RESULTS
-------
Isolate 4, Reverse Primer
#
Accession
Description
Max
Score
Total
Score
Query
Coverage
E
value
Max
ident
1
NR 043998.1
Proteus penneri strain NCTC 12737 16S ribosomal RNA, partial
sequence
1210
1210
100%
0.0
98%
2
NR 043997.1
Proteus inirabilis strain NCTC 11938 16S ribosomal RNA, partial
sequence
1210
1210
100%
0.0
98%
3
NR 025336.1
Proteus vulgaris strain DSM 30118 16S ribosomal RNA, partial
sequence
1199
1199
100%
0.0
98%
4
NR 043999.1
Proteus myxofaciens strain NCIMB 13273 16S ribosomal RNA, partial
sequence
1190
1190
100%
0.0
98%
5
NR 043648.1
Xenorhabdus hominickii strain KE01 16S ribosomal RNA, partial
sequence
1164
1164
100%
0.0
97%
6
NR 043646.1
Xenorhabdus kozodoii strain SaV 16S ribosomal RNA, partial
sequence
1153
1153
100%
0.0
97%
7
NR 043637.1
Xenorhabdus koppenhoeferi strain USNJ01 16S ribosomal RNA,
partial sequence
1149
1149
100%
0.0
97%
8
NR 042327.1
Xenorhabdus ehlersii strain :DSM 16337 16S ribosomal RNA, partial
sequence
1144
1144
100%
0.0
97%
9
NR 042326.1
Xenorhabdus budapestensis strain :DSM 16342 16S ribosomal RNA,
partial sequence
1138
1138
100%
0.0
96%
10
NR 042325.1
Xenorhabdus innexi strain :DSM 16336 16S ribosomal RNA, partial
sequence
1127
1127
100%
0.0
96%
11
NR 043634.1
Xenorhabdus stockiae strain TH01 16S ribosomal RNA, partial
sequence
1122
1122
100%
0.0
96%
12
NR 037074.1
Photorhabdus luminescens subsp. luminescens strain Hb 16S
ribosomal RNA, partial sequence
1122
1122
100%
0.0
96%
13
NR 027194.1
Xenorhabdus japonica strain SK-1T 16S ribosomal RNA, partial
sequence
1120
1120
100%
0.0
96%
14
NR 042822.1
Xenorhabdus beddingii strain DSM4764 16S ribosomal RNA, partial
sequence
1116
1116
100%
0.0
96%
15
NR 042820.1
Xenorhabdus bovienii strain DSM4766 16S ribosomal RNA, partial
sequence
1116
1116
100%
0.0
96%
16
NR 043642.1
Xenorhabdus doucetiae strain FRM16 16S ribosomal RNA, partial
sequence
1114
1114
100%
0.0
96%
17
NR 026538.1
Dickeya paradisiaca strain LMG 2542 16S ribosomal RNA, partial
sequence
1112
1112
100%
0.0
96%
Battelle B-l
-------
Isolate 4, Reverse Primer
#
Accession
Description
Max
Score
Total
Score
Query
Coverage
E
value
Max
ident
18
NR 025875.1
Xenorhabdus poinarii strain G1 16S ribosomal RNA, partial sequence
1109
1109
100%
0.0
96%
19
NR 042062.1
Serratia liquefaciens strain CIP 103238 16S ribosomal RNA, partial
sequence
1105
1105
100%
0.0
96%
20
NR 025340.1
Serratia grimesii strain DSM 30063 16S ribosomal RNA, partial
sequence
1105
1105
100%
0.0
96%
21
NR 042411.1
Providencia rustigianii strain : DSM 4541 16S ribosomal RNA,
complete sequence
1099
1099
100%
0.0
95%
22
NR 025334.1
Obesumbacterium proteus strain 42 16S ribosomal RNA, partial
sequence
1099
1099
100%
0.0
95%
23
NR 036851.1
Photorhabdus asymbiotica subsp. asymbiotica strain 3265-8 16S
ribosomal RNA, partial sequence
1099
1099
100%
0.0
95%
24
NR 025316.1
Pectobacterium carotovorum subsp. odoriferum strain LMG 17566 16S
ribosomal RNA, partial sequence
1098
1098
100%
0.0
95%
25
NR 029011.1
Photorhabdus luminescens subsp. kayaii strain 1121 16S ribosomal
RNA, complete sequence
1096
1096
100%
0.0
95%
Isolate 4, Reverse Primer, sequence
CGATTCCGACTTCATGGAGTCGAGTTGCANACTCCAATCCGGANTACGACAGACTTTATGAGTTCCGCTTGCTCTCGCGAGGNCNCTTCTCTTTGTATCTGNCATTGTAGC
ACGTGTGTAGCCCTACTCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTATCACCGGCAGTCTCCTTTGAGTTCCCNCCATTACGCGCTGGCAAC
AAAGGATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATTTCACAACACGAGCTGACGACAGCCATGCAGCACCTGTCTCAGAGTTCCCGAAGGCACTCCTCTATCT
CTAAAGGATTCTCTGGATGTCAAGAGTAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAAC
CTTGCGGCCGTACTCCCCAGGCGGTCGATTTAACGCGTTAGCTCCAGAAGCCACGGTTCAAGACCACAACCTCTAAATCGACATCGTTTACAGCGTGGACTACCAGGGT
ATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTGAGCGTCAGTCTTTGTCCAGGGGGCCGCCTTCGCCACCGGTATTCCTCCACATCTCTACGCATTTCACCGCTACA
CGTGGAATTCTACCCCCCTCT
Battelle B-2
-------
Isolate 15, Forward Primer
#
Accession
Description
Max
Score
Total
Score
Query
Coverage
E
value
Max
ident
1
NR 044384.1
Planomicrobium glaciei strain 0423 16S ribosomal RNA, partial
sequence
1044
1044
100%
0.0
99%
2
NR 025864.1
Planomicrobium okeanokoites strain IFO 12536 16S ribosomal RNA,
partial sequence
990
990
100%
0.0
98%
3
NR 025011.1
Planomicrobium koreense strain JG07 16S ribosomal RNA, partial
sequence
983
983
100%
0.0
98%
4
NR 025553.1
Planococcus rifietoensis strain M8 16S ribosomal RNA, partial
sequence
983
983
100%
0.0
98%
5
NR 025592.1
Planococcus maitriensis strain SI 16S ribosomal RNA, partial
sequence
977
977
100%
0.0
97%
6
NR 024881.1
Planomicrobium mcmeekinii strain S23F2 16S ribosomal RNA, partial
sequence
974
974
100%
0.0
97%
7
NR 044073.1
Planococcus donghaensis strain JH1 16S ribosomal RNA, partial
sequence
972
972
100%
0.0
97%
8
NR 025247.1
Planococcus maritimus strain TF-9 16S ribosomal RNA, partial
sequence
972
972
100%
0.0
97%
9
NR 042259.1
Planomicrobium chinense strain : DX3-12 16S ribosomal RNA, partial
sequence
970
970
100%
0.0
97%
10
NR 028950.1
Planomicrobium psychrophilum strain CMS 53or 16S ribosomal RNA,
partial sequence
961
961
100%
0.0
97%
11
NR 025469.1
Planococcus antarcticus strain CMS 26or 16S ribosomal RNA, partial
sequence
961
961
100%
0.0
97%
12
NR 026090.1
Planococcus citreus strain NCIMB 1493 16S ribosomal RNA, partial
sequence
957
957
100%
0.0
97%
13
NR 025781.1
Planococcus stackebrandtii strain K22-03 16S ribosomal RNA, partial
sequence
948
948
100%
0.0
97%
14
NR 024864.1
Planomicrobium alkanoclasticum strain MAE2 16S ribosomal RNA,
partial sequence
935
935
100%
0.0
96%
Battelle B-3
-------
Isolate 15, Forward Primer
#
Accession
Description
Max
Score
Total
Score
Query
Coverage
E
value
Max
ident
15
NR 026091.1
Planococcus kocurii strain NCIMB 629 16S ribosomal RNA, partial
sequence
935
935
100%
0.0
96%
16
NR 043267.1
Bacillus infantis strain SMC 4352-1 16S ribosomal RNA, partial
sequence
933
933
100%
0.0
96%
17
NR 041359.1
Sporosarcina saromensis strain HG645 16S ribosomal RNA, partial
sequence
917
917
100%
0.0
95%
18
NR 043527.1
Sporosarcina soli strain 180 16S ribosomal RNA, partial sequence
911
911
100%
0.0
95%
19
NR 043526.1
Sporosarcina koreensis strain F73 16S ribosomal RNA, partial
sequence
911
911
100%
0.0
95%
20
NR 043682.1
Bacillus kribbensis strain BT080 16S ribosomal RNA, partial sequence
909
909
100%
0.0
95%
21
NR 042395.1
Planococcus columbae strain : PgExll 16S ribosomal RNA, partial
sequence
907
907
100%
0.0
95%
22
NR 043084.1
Bacillus koreensis strain BR030 16S ribosomal RNA, partial sequence
904
904
99%
0.0
95%
23
NR 043268.1
Bacillus idriensis strain SMC 4352-2 16S ribosomal RNA, partial
sequence
900
900
100%
0.0
95%
24
NR 042274.1
Bacillus foraminis strain : CV53 16S ribosomal RNA, complete
sequence
900
900
100%
0.0
95%
25
NR 042726.1
Bacillus circulans 16S ribosomal RNA, partial sequence
900
900
100%
0.0
95%
Isolate 15, Forward Primer, sequence
GAAAGACGGTTTCGGCTGTCACTGCAGGATGGGCCCGCGGCGCATTAGCTAGTTGGTGGGGTAACGGCCCACCAAGGCCACGATGCGTAGCCGACCTGAGAGGGTGA
TCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGCAAGTCTGACGGAGCAACGCCGCGTGAGTGATG
AAGGTTTTCGGATCGTAAAACTCTGTTGCGAGGGAAGAAACCGTGCCAAGTAACTANTGGCACCTTGACGGTACCTCGCCAGAAAGCCACGGCTAACTACGTGCCAGCA
GCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTCCCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGG
GTCATTGGAAACTGGGGGACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTTTCT
GGTCT GTAACT GACGCT GAGGCGCGAAAGCGT G
Battelle B-4
-------
Isolate 15, Reverse Primer
#
Accession
Description
Max
Score
Total
Score
Query
Coverage
E
value
Max
ident
1
NR 044384.1
Planomicrobium glaciei strain 0423 16S ribosomal RNA, partial
sequence
1116
1116
100%
0.0
98%
2
NR 024881.1
Planomicrobium mcmeekinii strain S23F2 16S ribosomal RNA, partial
sequence
1094
1094
100%
0.0
98%
3
NR 025011.1
Planomicrobium koreense strain JG07 16S ribosomal RNA, partial
sequence
1092
1092
99%
0.0
98%
4
NR 042259.1
Planomicrobium chinense strain : DX3-12 16S ribosomal RNA, partial
sequence
1088
1088
100%
0.0
98%
5
NR 028950.1
Planomicrobium psychrophilum strain CMS 53or 16S ribosomal RNA,
partial sequence
1086
1086
99%
0.0
98%
6
NR 025864.1
Planomicrobium okeanokoites strain IFO 12536 16S ribosomal RNA,
partial sequence
1081
1081
100%
0.0
97%
7
NR 025553.1
Planococcus rifietoensis strain M8 16S ribosomal RNA, partial
sequence
1077
1077
100%
0.0
97%
8
NR 025247.1
Planococcus maritimus strain TF-9 16S ribosomal RNA, partial
sequence
1072
1072
100%
0.0
97%
9
NR 024864.1
Planomicrobium alkanoclasticum strain MAE2 16S ribosomal RNA,
partial sequence
1066
1066
99%
0.0
97%
10
NR 042395.1
Planococcus columbae strain : PgExll 16S ribosomal RNA, partial
sequence
1062
1062
100%
0.0
97%
11
NR 025592.1
Planococcus maitriensis strain SI 16S ribosomal RNA, partial
sequence
1059
1059
96%
0.0
98%
12
NR 025781.1
Planococcus stackebrandtii strain K22-03 16S ribosomal RNA, partial
sequence
1042
1042
99%
0.0
96%
13
NR 043526.1
Sporosarcina koreensis strain F73 16S ribosomal RNA, partial
sequence
1038
1038
100%
0.0
96%
Battelle B-5
-------
Isolate 15, Reverse Primer
#
Accession
Description
Max
Score
Total
Score
Query
Coverage
E
value
Max
ident
14
NR 025469.1
Planococcus antarcticus strain CMS 26or 16S ribosomal RNA, partial
sequence
1037
1037
100%
0.0
96%
15
NR 044073.1
Planococcus donghaensis strain JH1 16S ribosomal RNA, partial
sequence
1035
1035
100%
0.0
96%
16
NR 043527.1
Sporosarcina soli strain 180 16S ribosomal RNA, partial sequence
1033
1033
100%
0.0
96%
17
NR 026090.1
Planococcus citreus strain NCIMB 1493 16S ribosomal RNA, partial
sequence
1033
1033
96%
0.0
97%
18
NR 025049.1
Sporosarcina aquimarina strain SW28 16S ribosomal RNA, partial
sequence
1027
1027
100%
0.0
96%
19
NR 043720.1
Paenisporosarcina quisquiliarum strain SK 55 16S ribosomal RNA,
partial sequence
1026
1026
99%
0.0
96%
20
NR 044122.1
Sporosarcina sp. N-05 strain N-05 16S ribosomal RNA, partial
sequence
1022
1022
100%
0.0
96%
21
NR 025573.1
Sporosarcina macmurdoensis strain CMS 21w 16S ribosomal RNA,
partial sequence
1016
1016
99%
0.0
96%
22
NR 041359.1
Sporosarcina saromensis strain HG645 16S ribosomal RNA, partial
sequence
1011
1011
100%
0.0
95%
23
NR 044193.1
Bacillus ginsengi strain gel4 16S ribosomal RNA, partial sequence
1011
1011
100%
0.0
95%
24
NR 025409.1
Bacillus psychrodurans strain DSM 11713 16S ribosomal RNA, partial
sequence
1011
1011
100%
0.0
95%
25
NR 025408.1
Bacillus psychrotolerans strain DSM 11706 16S ribosomal RNA,
partial sequence
1011
1011
100%
0.0
95%
Isolate 15, Reverse Primer, sequence
GGGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGGCATGCTGAGCCAKGATCAAACTCT
NGANCCGGCTTCATGCAGGCGAGTTGCAGCCTGCAATCCGAACTGAGAACGGTTTTCTGGGATTGGCTCCCCCTCGCGGGTTGGCAACCCTTTGTACCGTCCATTGTAG
CACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACT
AAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACCGCTGTCCCCGAAGGGAAAGCCTTGTC
TCCAAGGCGGTCAGCGGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTC
AGCCTTGCGGCCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACACTTAGCACTC
Battelle B-6
-------
Isolate 16, Forward Primer
#
Accession
Description
Max
Score
Total
Score
Query
Coverage
E
value
Max
ident
1
NR 026156.1
Curtobacterium citreum strain DSM 20528 16S ribosomal RNA,
partial sequence
913
913
100%
0.0
94%
2
NR 026157.1
Curtobacterium luteum strain DSM 20542 16S ribosomal RNA, partial
sequence
911
911
100%
0.0
94%
3
NR 025467.1
Curtobacterium flaccumfaciens pv. flaccumfaciens strain LMG 3645
16S ribosomal RNA, partial sequence
907
907
100%
0.0
94%
4
NR 041495.1
Curtobacterium ammoniigenes strain NBRC 101786 16S ribosomal
RNA, partial sequence
907
907
100%
0.0
94%
5
NR 042315.1
Curtobacterium pusillum strain : DSM 20527 16S ribosomal RNA,
complete sequence
907
907
100%
0.0
94%
6
NR 036885.1
Curtobacterium albidum strain IFO 15078 16S ribosomal RNA, partial
sequence
887
887
98%
0.0
94%
7
NR 044240.1
Leifsonia kribbensis strain MSL-13 16S ribosomal RNA, partial
sequence
874
874
100%
0.0
93%
8
NR 043663.1
Leifsonia shinshuensis strain DB102; JCM10591 16S ribosomal RNA,
partial sequence
857
857
100%
0.0
92%
9
NR 043662.1
Leifsonia naganoensis strain DB 103; JCM10592 16S ribosomal RNA,
partial sequence
857
857
100%
0.0
92%
10
NR 041812.1
Corynebacterium bovis strain ATCC13722 16S ribosomal RNA,
partial sequence
857
857
100%
0.0
93%
11
NR 029264.1
Pseudoclavibacter helvolus strain DSM 20419 16S ribosomal RNA,
partial sequence
857
857
100%
0.0
93%
Battelle B-7
-------
Isolate 16, Forward Primer
#
Accession
Description
Max
Score
Total
Score
Query
Coverage
E
value
Max
ident
12
NR 027523.1
Leifsonia xyli subsp. cynodontis JCM 9733 16S ribosomal RNA,
partial sequence
857
857
100%
0.0
92%
13
NR 028739.1
Leifsonia poae strain VKM Ac-1401 16S ribosomal RNA, partial
sequence
852
852
100%
0.0
92%
14
NR 025461.1
Curtobacterium herbarum strain P 420/07 16S ribosomal RNA, partial
sequence
848
848
100%
0.0
92%
15
NR 043412.1
Leifsonia aquatica strain JCM 1368 16S ribosomal RNA, complete
sequence
848
848
99%
0.0
92%
16
NR 043982.1
Leucobacter chromiireducens subsp. solipictus strain TAN 31504 16S
ribosomal RNA, partial sequence
846
846
100%
0.0
92%
17
NR 041045.1
Plantibacter auratus strain IAM 18417 16S ribosomal RNA, partial
sequence
846
846
100%
0.0
92%
18
NR 025976.1
Clavibacter michiganensis subspecies tessellarius strain 78181 16S
ribosomal RNA, partial sequence
846
846
100%
0.0
92%
19
NR 036892.1
Clavibacter michiganensis strain DSM 46364 16S ribosomal RNA,
partial sequence
846
846
100%
0.0
92%
20
NR 042669.1
Leifsonia kafniensis strain : KFC-22 16S ribosomal RNA, partial
sequence
841
841
100%
0.0
92%
21
NR 042287.1
Leucobacter chromiireducens subsp. chromiireducens strain : L-l 16S
ribosomal RNA, partial sequence
841
841
100%
0.0
92%
22
NR 036947.1
Clavibacter michiganensis subsp. insidiosus strain Burkholder Pb 16S
ribosomal RNA, partial sequence
841
841
100%
0.0
92%
23
NR 024679.1
Mycetocola lacteus strain CM-10 16S ribosomal RNA, partial
sequence
841
841
100%
0.0
92%
24
NR 024678.1
Mycetocola saprophilus strain CM-01 16S ribosomal RNA, partial
sequence
841
841
100%
0.0
92%
25
NR 024677.1
Mycetocola tolaasinivorans strain CM-05 16S ribosomal RNA, partial
sequence
841
841
100%
0.0
92%
Isolate 16, Forward Primer, sequence
Battelle B-8
-------
ACTGANACACNGCCCANACTCCTACGGGAGGCAGNAGNGGNGAATATTGCNNAATGGGCGAAAGCCTGATGCANCNNCNCCGCGTGAGGNATGACNGCCTTCNGGTTG
TAAACNTNTTTTAGTAGGGAAGAANCGAAAGTGACGGTACCTGCAGAAAAAGCACCGGCTAACTACNTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTGTCCG
GAATTATTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCTGCTGTGAAATCCCGAGGCTCAACCTCGGGCTTGCAGTGGGTACGGGCAGACTANAGTGCGGTAGG
GGAGATTGGAATTCCTGGTGTAGCGGTGGAATGCGCAGATATCAGGAGGAACACCGATGGCGAAGGCAGATCTCTGGGCCGTAACTGACGCTGANNAGCGAAAGCGNG
GGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTANACGTTGNGCGCTAGATGTAGGGACCTTTCCACGGTTTCTGTGTNGTANCTNACNCATTAAGCGCCCC
GCCTGNNGAGTACGGCC
Isolate 19, Forward Primer
#
Accession
Description
Max
Score
Total
Score
Query
Coverage
E
value
Max
ident
1
NR 043997.1
Proteus mirabilis strain NCTC 11938 16S ribosomal RNA, partial
sequence
1170
1170
100%
0.0
99%
2
NR 025336.1
Proteus vulgaris strain DSM 30118 16S ribosomal RNA, partial
sequence
1122
1122
100%
0.0
98%
3
NR 043999.1
Proteus myxofaciens strain NCIMB 13273 16S ribosomal RNA, partial
sequence
1116
1116
100%
0.0
98%
4
NR 043998.1
Proteus penneri strain NCTC 12737 16S ribosomal RNA, partial
sequence
1079
1079
94%
0.0
99%
5
NR 043751.1
Morganella morganii strain DSM 14850 16S ribosomal RNA, partial
sequence
989
989
99%
0.0
95%
6
NR 043750.1
Morganella psychrotolerans strain U2/3 16S ribosomal RNA, partial
sequence
983
983
99%
0.0
94%
7
NR 042412.1
Providencia heimbachae strain : DSM 3591 16S ribosomal RNA,
complete sequence
974
974
100%
0.0
94%
8
NR 042415.1
Providencia vennicola strain : OP1 16S ribosomal RNA, complete
sequence
966
966
99%
0.0
94%
Battelle B-9
-------
Isolate 19, Forward Primer
#
Accession
Description
Max
Score
Total
Score
Query
Coverage
E
value
Max
ident
9
NR 024848.1
Providencia stuartii strain ATCC 29914 16S ribosomal RNA, partial
sequence
966
966
99%
0.0
94%
10
NR 042413.1
Providencia rettgeri strain : DSM 4542 16S ribosomal RNA, complete
sequence
961
961
99%
0.0
94%
11
NR 042411.1
Providencia rustigianii strain : DSM 4541 16S ribosomal RNA,
complete sequence
961
961
99%
0.0
94%
12
NR 028938.1
Morganella morganii strain Ml 1 16S ribosomal RNA, partial sequence
959
959
99%
0.0
94%
13
NR 041978.1
Pantoea agglomerans strain DSM 3493 16S ribosomal RNA, partial
sequence
937
937
98%
0.0
93%
14
NR 042327.1
Xenorhabdus ehlersii strain :DSM 16337 16S ribosomal RNA, partial
sequence
933
933
100%
0.0
93%
15
NR 042053.1
Providencia alcalifaciens DSM 30120 strain CIP8290T (ATCC9886T)
16S ribosomal RNA, complete sequence
933
933
99%
0.0
93%
16
NR 043648.1
Xenorhabdus hominickii strain KE01 16S ribosomal RNA, partial
sequence
931
931
100%
0.0
93%
17
NR 042822.1
Xenorhabdus beddingii strain DSM4764 16S ribosomal RNA, partial
sequence
931
931
99%
0.0
93%
18
NR 043645.1
Xenorhabdus mauleonii strain VC01 16S ribosomal RNA, partial
sequence
926
926
99%
0.0
93%
19
NR 042325.1
Xenorhabdus innexi strain :DSM 16336 16S ribosomal RNA, partial
sequence
926
926
100%
0.0
93%
20
NR 042811.1
Arsenophonus nasoniae strain ATCC 49151 16S ribosomal RNA,
partial sequence
922
922
100%
0.0
93%
21
NR 043643.1
Xenorhabdus griffiniae strain ID 10 16S ribosomal RNA, partial
sequence
922
922
100%
0.0
93%
22
NR 025875.1
Xenorhabdus poinarii strain G1 16S ribosomal RNA, partial sequence
922
922
100%
0.0
92%
23
NR 043634.1
Xenorhabdus stockiae strain TH01 16S ribosomal RNA, partial
sequence
920
920
99%
0.0
93%
24
NR 024644.1
Serratia rubidaea strain JCM1240 16S ribosomal RNA, partial
sequence
917
917
100%
0.0
92%
25
NR 043644.1
Xenorhabdus miraniensis strain Q1 16S ribosomal RNA, partial
sequence
915
915
99%
0.0
92%
Battelle B-10
-------
Isolate 19, Forward Primer, sequence
GCTTTCTTGCTGACGAGCGGCGGACGGGTGAGTAATGTATGGGGATCTGCCCGATAGAGGGGGATAACTACTGGAAACGGTGGCTAATACCGCATAATGTCTACGGACC
AAAGCAGGGGCTCTTCGGACCTTGCACTATCGGATGAACCCATATGGGATTAGCTAGTAGGTGGGGTAAAGGCTCACCTAGGCGACGATCTCTAGCTGGTCTGAGAGGA
TGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGA
AGAAGGCCTTAGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGTGATAAGGTTAATACCCTTRTCAATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCA
GCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTCAATTAAGTCAGATGTGAAAGCCCCGAGCTTAACTTGGG
AATTGCATCTGAAACTGGTTGGCTAGAGTCTTGTAGAGGGGGGTAGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAATACCGGTGGCG
Isolate 19, Reverse Primer
#
Accession
Description
Max
Score
Total
Score
Query
Coverage
E
value
Max
ident
1
NR 043997.1
Proteus mirabilis strain NCTC 11938 16S ribosomal RNA, partial
sequence
1242
1242
100%
0.0
99%
2
NR 043998.1
Proteus penneri strain NCTC 12737 16S ribosomal RNA, partial
sequence
1230
1230
100%
0.0
99%
3
NR 025336.1
Proteus vulgaris strain DSM 30118 16S ribosomal RNA, partial
sequence
1203
1203
100%
0.0
99%
4
NR 043648.1
Xenorhabdus hominickii strain KE01 16S ribosomal RNA, partial
sequence
1195
1195
100%
0.0
99%
5
NR 043999.1
Proteus myxofaciens strain NCIMB 13273 16S ribosomal RNA, partial
sequence
1182
1182
100%
0.0
98%
6
NR 043637.1
Xenorhabdus koppenhoeferi strain USNJ01 16S ribosomal RNA,
partial sequence
1175
1175
100%
0.0
98%
7
NR 042327.1
Xenorhabdus ehlersii strain :DSM 16337 16S ribosomal RNA, partial
sequence
1164
1164
100%
0.0
98%
Battelle B-ll
-------
Isolate 19, Reverse Primer
#
Accession
Description
Max
Score
Total
Score
Query
Coverage
E
value
Max
ident
8
NR 043646.1
Xenorhabdus kozodoii strain SaV 16S ribosomal RNA, partial
sequence
1162
1162
100%
0.0
98%
9
NR 042325.1
Xenorhabdus innexi strain :DSM 16336 16S ribosomal RNA, partial
sequence
1158
1158
100%
0.0
98%
10
NR 043634.1
Xenorhabdus stockiae strain TH01 16S ribosomal RNA, partial
sequence
1153
1153
100%
0.0
97%
11
NR 042326.1
Xenorhabdus budapestensis strain :DSM 16342 16S ribosomal RNA,
partial sequence
1147
1147
100%
0.0
97%
12
NR 027194.1
Xenorhabdus japonica strain SK-1T 16S ribosomal RNA, partial
sequence
1134
1134
100%
0.0
97%
13
NR 043642.1
Xenorhabdus doucetiae strain FRM16 16S ribosomal RNA, partial
sequence
1129
1129
100%
0.0
97%
14
NR 025875.1
Xenorhabdus poinarii strain G1 16S ribosomal RNA, partial sequence
1129
1129
100%
0.0
97%
15
NR 042822.1
Xenorhabdus beddingii strain DSM4764 16S ribosomal RNA, partial
sequence
1125
1125
100%
0.0
97%
16
NR 042820.1
Xenorhabdus bovienii strain DSM4766 16S ribosomal RNA, partial
sequence
1125
1125
100%
0.0
97%
17
NR 042328.1
Xenorhabdus szentinnaii strain :DSM 16338 16S ribosomal RNA,
partial sequence
1114
1114
100%
0.0
96%
18
NR 037074.1
Photorhabdus luminescens subsp. luminescens strain Hb 16S
ribosomal RNA, partial sequence
1114
1114
100%
0.0
96%
19
NR 025334.1
Obesumbacterium proteus strain 42 16S ribosomal RNA, partial
sequence
1114
1114
100%
0.0
96%
20
NR 042821.1
Xenorhabdus nematophila strain DSM3370 16S ribosomal RNA,
partial sequence
1112
1112
100%
0.0
96%
21
NR 026538.1
Dickeya paradisiaca strain LMG 2542 16S ribosomal RNA, partial
sequence
1110
1110
100%
0.0
96%
22
NR 042411.1
Providencia rustigianii strain : DSM 4541 16S ribosomal RNA,
complete sequence
1109
1109
100%
0.0
96%
23
NR 037112.1
Serratia proteamaculans strain 4364 16S ribosomal RNA, partial
sequence
1109
1109
100%
0.0
96%
24
NR 041972.1
Erwinia chrysanthemi strain DSM 4610 16S ribosomal RNA, partial
sequence
1109
1109
100%
0.0
96%
Battelle B-12
-------
Isolate 19, Reverse Primer
#
Accession
Description
Max
Score
Total
Score
Query
Coverage
E
value
Max
ident
25
NR 036851.1
Photorhabdus asymbiotica subsp. asymbiotica strain 3265-8 16S
ribosomal RNA, partial sequence
1109
1109
100%
0.0
96%
Isolate 19, Reverse Primer, sequence
TTCTTTTGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTAGCATTCTGATCTACGATTACTAGCGATTCCGACTTCATGGAG
TCGAGTTGCAGACTCCAATCCGGACTACGACAGACTTTATGAGTTCCGCTTGCTCTCGCGAGGTCGCTTCTCTTTGTATCTGCCATTGTAGCACGTGTGTAGCCCTACTC
GTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTATCACCGGCAGTCTCCTTTGAGTTCCCACCATTACGTGCTGGCAACAAAGGATAAGGGTTGCGC
TCGTTGCGGGACTTAACCCAACATTTCACAACACGAGCTGACGACAGCCATGCAGCACCTGTCTCAGCGTTCCCGAAGGCACTCCTCTATCTCTAAAGGATTCGCTGGAT
GTCAAGAGTAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCC
CAGGCGGTCGATTTAACGCGTTAGCTCCAGAAGCCACGGTTCAAGACCACAACCTCTAAATCGACATCGTTTACAGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTC
CCCACGCTTTCGCAC
Isolate 22, Forward Primer
#
Accession
Description
Max
Score
Total
Score
Query
Coverage
E
value
Max
ident
1
NR 043997.1
Proteus mirabilis strain NCTC 11938 16S ribosomal RNA, partial
sequence
1122
1122
100%
0.0
99%
2
NR 025336.1
Proteus vulgaris strain DSM 30118 16S ribosomal RNA, partial
sequence
1079
1079
100%
0.0
98%
3
NR 043999.1
Proteus myxofaciens strain NCIMB 13273 16S ribosomal RNA, partial
sequence
1068
1068
100%
0.0
98%
Battelle B-13
-------
Isolate 22, Forward Primer
#
Accession
Description
Max
Score
Total
Score
Query
Coverage
E
value
Max
ident
4
NR 043998.1
Proteus penneri strain NCTC 12737 16S ribosomal RNA, partial
sequence
1024
1024
93%
0.0
99%
5
NR 043751.1
Morganella morganii strain DSM 14850 16S ribosomal RNA, partial
sequence
941
941
100%
0.0
94%
6
NR 043750.1
Morganella psychrotolerans strain U2/3 16S ribosomal RNA, partial
sequence
941
941
100%
0.0
94%
7
NR 042412.1
Providencia heimbachae strain : DSM 3591 16S ribosomal RNA,
complete sequence
931
931
100%
0.0
94%
8
NR 042415.1
Providencia vennicola strain : OP1 16S ribosomal RNA, complete
sequence
922
922
100%
0.0
94%
9
NR 024848.1
Providencia stuartii strain ATCC 29914 16S ribosomal RNA, partial
sequence
922
922
100%
0.0
94%
10
NR 042413.1
Providencia rettgeri strain : DSM 4542 16S ribosomal RNA, complete
sequence
917
917
100%
0.0
94%
11
NR 042411.1
Providencia rustigianii strain : DSM 4541 16S ribosomal RNA,
complete sequence
917
917
100%
0.0
94%
12
NR 028938.1
Morganella morganii strain Ml 1 16S ribosomal RNA, partial sequence
911
911
100%
0.0
93%
13
NR 042053.1
Providencia alcalifaciens DSM 30120 strain CIP8290T (ATCC9886T)
16S ribosomal RNA, complete sequence
891
891
100%
0.0
93%
14
NR 041978.1
Pantoea agglomerans strain DSM 3493 16S ribosomal RNA, partial
sequence
891
891
100%
0.0
93%
15
NR 043648.1
Xenorhabdus hominickii strain KE01 16S ribosomal RNA, partial
sequence
885
885
100%
0.0
93%
16
NR 043645.1
Xenorhabdus mauleonii strain VC01 16S ribosomal RNA, partial
sequence
885
885
100%
0.0
93%
17
NR 042327.1
Xenorhabdus ehlersii strain :DSM 16337 16S ribosomal RNA, partial
sequence
885
885
99%
0.0
93%
18
NR 042811.1
Arsenophonus nasoniae strain ATCC 49151 16S ribosomal RNA,
partial sequence
880
880
100%
0.0
92%
19
NR 042822.1
Xenorhabdus beddingii strain DSM4764 16S ribosomal RNA, partial
sequence
880
880
100%
0.0
92%
20
NR 042325.1
Xenorhabdus innexi strain :DSM 16336 16S ribosomal RNA, partial
sequence
880
880
100%
0.0
92%
Battelle B-14
-------
Isolate 22, Forward Primer
#
Accession
Description
Max
Score
Total
Score
Query
Coverage
E
value
Max
ident
21
NR 024644.1
Serratia rubidaea strain JCM1240 16S ribosomal RNA, partial
sequence
880
880
100%
0.0
92%
22
NR 043643.1
Xenorhabdus griffiniae strain ID 10 16S ribosomal RNA, partial
sequence
874
874
100%
0.0
92%
23
NR 043642.1
Xenorhabdus doucetiae strain FRM16 16S ribosomal RNA, partial
sequence
874
874
100%
0.0
92%
24
NR 041974.1
Erwinia mallotivora strain DSM 4565 16S ribosomal RNA, partial
sequence
874
874
100%
0.0
92%
25
NR 026045.1
Pantoea ananatis strain 1846 16S ribosomal RNA, partial sequence
872
872
100%
0.0
92%
Isolate 22, Forward Primer, sequence
GCTTGCTTTCTTGCTGACGAGCGGCGGACGGGTGAGTAATGTATGGGGATCTGCCCGATAGAGGGGGATAACTACTGGAAACGGTGGCTAATACCGCATAATGTCTACG
GACCAAAGCAGGGGCTCTTCGGACCTTGCACTATCGGATGAACCCATATGGGATTAGCTAGTAGGTGGGGTAAAGGCTCACCTAGGCGACGATCTCTAGCTGGTCTGAG
AGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGT
ATGAAGAAGGCCTTAGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGTGATAAGGTTAATACCCTTATCAATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTG
CCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTCAATTAAGTCAGATGTGAAAGCCCCGAGCTTAACTTG
GGAATTGCATCTGAAACTGGTTGGCTAGAGTCTTGTAGAGGGGGGTAGAATTCCATGTGTAGCGGTGAAATG
Isolate 22, Reverse Primer
Battelle B-15
-------
#
Accession
Description
Max
Score
Total
Score
Query
Coverage
E
value
Max
ident
1
NR 043997.1
Proteus mirabilis strain NCTC 11938 16S ribosomal RNA, partial
sequence
1242
1242
100%
0.0
99%
2
NR 043998.1
Proteus penneri strain NCTC 12737 16S ribosomal RNA, partial
sequence
1230
1230
100%
0.0
99%
3
NR 025336.1
Proteus vulgaris strain DSM 30118 16S ribosomal RNA, partial
sequence
1203
1203
100%
0.0
99%
4
NR 043648.1
Xenorhabdus hominickii strain KE01 16S ribosomal RNA, partial
sequence
1195
1195
100%
0.0
99%
5
NR 043999.1
Proteus myxofaciens strain NCIMB 13273 16S ribosomal RNA, partial
sequence
1182
1182
100%
0.0
98%
6
NR 043637.1
Xenorhabdus koppenhoeferi strain USNJ01 16S ribosomal RNA,
partial sequence
1175
1175
100%
0.0
98%
7
NR 042327.1
Xenorhabdus ehlersii strain :DSM 16337 16S ribosomal RNA, partial
sequence
1164
1164
100%
0.0
98%
8
NR 043646.1
Xenorhabdus kozodoii strain SaV 16S ribosomal RNA, partial
sequence
1162
1162
100%
0.0
98%
9
NR 042325.1
Xenorhabdus innexi strain :DSM 16336 16S ribosomal RNA, partial
sequence
1158
1158
100%
0.0
98%
10
NR 043634.1
Xenorhabdus stockiae strain TH01 16S ribosomal RNA, partial
sequence
1153
1153
100%
0.0
97%
11
NR 042326.1
Xenorhabdus budapestensis strain :DSM 16342 16S ribosomal RNA,
partial sequence
1147
1147
100%
0.0
97%
12
NR 027194.1
Xenorhabdus japonica strain SK-1T 16S ribosomal RNA, partial
sequence
1134
1134
100%
0.0
97%
13
NR 043642.1
Xenorhabdus doucetiae strain FRM16 16S ribosomal RNA, partial
sequence
1129
1129
100%
0.0
97%
14
NR 025875.1
Xenorhabdus poinarii strain G1 16S ribosomal RNA, partial sequence
1129
1129
100%
0.0
97%
15
NR 042822.1
Xenorhabdus beddingii strain DSM4764 16S ribosomal RNA, partial
sequence
1125
1125
100%
0.0
97%
16
NR 042820.1
Xenorhabdus bovienii strain DSM4766 16S ribosomal RNA, partial
sequence
1125
1125
100%
0.0
97%
17
NR 042328.1
Xenorhabdus szentinnaii strain :DSM 16338 16S ribosomal RNA,
partial sequence
1114
1114
100%
0.0
96%
18
NR 037074.1
Photorhabdus luminescens subsp. luminescens strain Hb 16S
ribosomal RNA, partial sequence
1114
1114
100%
0.0
96%
Battelle B-16
-------
Isolate 22, Reverse Primer
#
Accession
Description
Max
Score
Total
Score
Query
Coverage
E
value
Max
ident
19
NR 025334.1
Obesumbacterium proteus strain 42 16S ribosomal RNA, partial
sequence
1114
1114
100%
0.0
96%
20
NR 042821.1
Xenorhabdus nematophila strain DSM3370 16S ribosomal RNA,
partial sequence
1112
1112
100%
0.0
96%
21
NR 026538.1
Dickeya paradisiaca strain LMG 2542 16S ribosomal RNA, partial
sequence
1110
1110
100%
0.0
96%
22
NR 042411.1
Providencia rustigianii strain : DSM 4541 16S ribosomal RNA,
complete sequence
1109
1109
100%
0.0
96%
23
NR 037112.1
Serratia proteamaculans strain 4364 16S ribosomal RNA, partial
sequence
1109
1109
100%
0.0
96%
24
NR 041972.1
Erwinia chrysanthemi strain DSM 4610 16S ribosomal RNA, partial
sequence
1109
1109
100%
0.0
96%
25
NR 036851.1
Photorhabdus asymbiotica subsp. asymbiotica strain 3265-8 16S
ribosomal RNA, partial sequence
1109
1109
100%
0.0
96%
Isolate 22, Reverse Primer, sequence
TTCTTTTGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTAGCATTCTGATCTACGATTACTAGCGATTCCGACTTCATGGAG
TCGAGTTGCAGACTCCAATCCGGACTACGACAGACTTTATGAGTTCCGCTTGCTCTCGCGAGGTCGCTTCTCTTTGTATCTGCCATTGTAGCACGTGTGTAGCCCTACTC
GTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTATCACCGGCAGTCTCCTTTGAGTTCCCACCATTACGTGCTGGCAACAAAGGATAAGGGTTGCGC
TCGTTGCGGGACTTAACCCAACATTTCACAACACGAGCTGACGACAGCCATGCAGCACCTGTCTCAGCGTTCCCGAAGGCACTCCTCTATCTCTAAAGGATTCGCTGGAT
GTCAAGAGTAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCC
CAGGCGGTCGATTTAACGCGTTAGCTCCAGAAGCCACGGTTCAAGACCACAACCTCTAAATCGACATCGTTTACAGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTC
CCCACGCTTTCGCAC
Battelle B-17
-------
Isolate 29, Forward Primer
#
Accession
Description
Max
Score
Total
Score
Query
Coverage
E
value
Max
ident
1
NR 043997.1
Proteus mirabilis strain NCTC 11938 16S ribosomal RNA, partial
sequence
451
451
100%
3e-
127
100%
2
NR 043999.1
Proteus myxofaciens strain NCIMB 13273 16S ribosomal RNA, partial
sequence
424
424
100%
6e-
119
98%
3
NR 025336.1
Proteus vulgaris strain DSM 30118 16S ribosomal RNA, partial
sequence
418
418
100%
3e-
117
98%
4
NR 042412.1
Providencia heimbachae strain : DSM 3591 16S ribosomal RNA,
complete sequence
381
381
100%
3e-
106
95%
5
NR 042415.1
Providencia vennicola strain : OP1 16S ribosomal RNA, complete
sequence
377
377
100%
4e-
105
95%
6
NR 042413.1
Providencia rettgeri strain : DSM 4542 16S ribosomal RNA, complete
sequence
372
372
100%
2e-
103
94%
7
NR 043750.1
Morganella psychrotolerans strain U2/3 16S ribosomal RNA, partial
sequence
368
368
100%
3e-
102
94%
8
NR 042411.1
Providencia rustigianii strain : DSM 4541 16S ribosomal RNA,
complete sequence
366
366
100%
le-
101
94%
9
NR 043751.1
Morganella morganii strain DSM 14850 16S ribosomal RNA, partial
sequence
357
357
100%
6e-99
93%
10
NR 024848.1
Providencia stuartii strain ATCC 29914 16S ribosomal RNA, partial
sequence
355
355
100%
2e-98
93%
11
NR 043998.1
Proteus penneri strain NCTC 12737 16S ribosomal RNA, partial
sequence
351
351
84%
3e-97
98%
12
NR 042053.1
Providencia alcalifaciens DSM 30120 strain CIP8290T (ATCC9886T)
16S ribosomal RNA, complete sequence
350
350
100%
le-96
93%
13
NR 028938.1
Morganella morganii strain Ml 1 16S ribosomal RNA, partial sequence
339
339
100%
2e-93
92%
14
NR 042811.1
Arsenophonus nasoniae strain ATCC 49151 16S ribosomal RNA,
partial sequence
329
329
100%
le-90
91%
15
NR 042822.1
Xenorhabdus beddingii strain DSM4764 16S ribosomal RNA, partial
sequence
324
324
100%
6e-89
91%
16
NR 042945.1
Xenorhabdus cabanillasii strain USTX62 16S ribosomal RNA, partial
sequence
322
322
95%
2e-88
91%
Battelle B-18
-------
Isolate 29, Forward Primer
#
Accession
Description
Max
Score
Total
Score
Query
Coverage
E
value
Max
ident
17
NR 025875.1
Xenorhabdus poinarii strain G1 16S ribosomal RNA, partial sequence
320
320
98%
8e-88
91%
18
NR 042325.1
Xenorhabdus innexi strain :DSM 16336 16S ribosomal RNA, partial
sequence
318
318
100%
3e-87
90%
19
NR 037110.1
Serratia odorifera strain PADG 1073 16S ribosomal RNA, partial
sequence
318
318
100%
3e-87
90%
20
NR 041975.1
Brenneria quercina strain DSM 4561 16S ribosomal RNA, partial
sequence
318
318
100%
3e-87
90%
21
NR 043643.1
Xenorhabdus griffiniae strain ID 10 16S ribosomal RNA, partial
sequence
313
313
100%
le-85
90%
22
NR 043634.1
Xenorhabdus stockiae strain TH01 16S ribosomal RNA, partial
sequence
313
313
100%
le-85
90%
23
NR 042327.1
Xenorhabdus ehlersii strain :DSM 16337 16S ribosomal RNA, partial
sequence
313
313
98%
le-85
90%
24
NR 024644.1
Serratia rubidaea strain JCM1240 16S ribosomal RNA, partial
sequence
313
313
100%
le-85
90%
25
NR 026050.1
Brenneria salicis strain ATCC 15712 16S ribosomal RNA, partial
sequence
311
311
100%
5e-85
90%
Isolate 29, Forward Primer, sequence
GCTTGCTTTCTTGCTGACGAGCGGCGGACGGGTGAGTAATGTATGGGGATCTGCCCGATAGAGGGGGATAACTACTGGAAACGGTGGCTAATACCGCATAATGTCTACG
GACCAAAGCAGGGGCTCTTCGGACCTTGCACTATCGGATGAACCCATATGGGATTAGCTAGTAGGTGGGGTAAAGGCTCACCTAGGCGACGATCTCTAGCTGGTCTGAG
AGG AT GAT CAGCCACACTGGGACT GA
Battelle B-19
-------
Isolate 29, Reverse Primer
#
Accession
Description
Max
Score
Total
Score
Query
Coverage
E
value
Max
ident
1
NR 043997.1
Proteus mirabilis strain NCTC 11938 16S ribosomal RNA, partial
sequence
1249
1249
100%
0.0
99%
2
NR 043998.1
Proteus penneri strain NCTC 12737 16S ribosomal RNA, partial
sequence
1238
1238
100%
0.0
99%
3
NR 025336.1
Proteus vulgaris strain DSM 30118 16S ribosomal RNA, partial
sequence
1210
1210
100%
0.0
99%
4
NR 043648.1
Xenorhabdus hominickii strain KE01 16S ribosomal RNA, partial
sequence
1203
1203
100%
0.0
99%
5
NR 043999.1
Proteus myxofaciens strain NCIMB 13273 16S ribosomal RNA, partial
sequence
1190
1190
100%
0.0
98%
6
NR 043637.1
Xenorhabdus koppenhoeferi strain USNJ01 16S ribosomal RNA,
partial sequence
1182
1182
100%
0.0
98%
7
NR 042327.1
Xenorhabdus ehlersii strain :DSM 16337 16S ribosomal RNA, partial
sequence
1171
1171
100%
0.0
98%
8
NR 043646.1
Xenorhabdus kozodoii strain SaV 16S ribosomal RNA, partial
sequence
1170
1170
100%
0.0
98%
9
NR 042325.1
Xenorhabdus innexi strain :DSM 16336 16S ribosomal RNA, partial
sequence
1166
1166
100%
0.0
98%
10
NR 043634.1
Xenorhabdus stockiae strain TH01 16S ribosomal RNA, partial
sequence
1160
1160
100%
0.0
98%
11
NR 042326.1
Xenorhabdus budapestensis strain :DSM 16342 16S ribosomal RNA,
partial sequence
1155
1155
100%
0.0
97%
12
NR 027194.1
Xenorhabdus japonica strain SK-1T 16S ribosomal RNA, partial
sequence
1142
1142
100%
0.0
97%
13
NR 043642.1
Xenorhabdus doucetiae strain FRM16 16S ribosomal RNA, partial
sequence
1136
1136
100%
0.0
97%
14
NR 025875.1
Xenorhabdus poinarii strain G1 16S ribosomal RNA, partial sequence
1136
1136
100%
0.0
97%
15
NR 042822.1
Xenorhabdus beddingii strain DSM4764 16S ribosomal RNA, partial
sequence
1133
1133
100%
0.0
97%
16
NR 042820.1
Xenorhabdus bovienii strain DSM4766 16S ribosomal RNA, partial
1133
1133
100%
0.0
97%
Battelle B-20
-------
Isolate 29, Reverse Primer
#
Accession
Description
Max
Score
Total
Score
Query
Coverage
E
value
Max
ident
sequence
17
NR 042328.1
Xenorhabdus szentinnaii strain :DSM 16338 16S ribosomal RNA,
partial sequence
1122
1122
100%
0.0
96%
18
NR 037074.1
Photorhabdus luminescens subsp. luminescens strain Hb 16S
ribosomal RNA, partial sequence
1122
1122
100%
0.0
96%
19
NR 025334.1
Obesumbacterium proteus strain 42 16S ribosomal RNA, partial
sequence
1122
1122
100%
0.0
96%
20
NR 042821.1
Xenorhabdus nematophila strain DSM3370 16S ribosomal RNA,
partial sequence
1120
1120
100%
0.0
96%
21
NR 026538.1
Dickeya paradisiaca strain LMG 2542 16S ribosomal RNA, partial
sequence
1118
1118
100%
0.0
96%
22
NR 042411.1
Providencia rustigianii strain : DSM 4541 16S ribosomal RNA,
complete sequence
1116
1116
100%
0.0
96%
23
NR 037112.1
Serratia proteamaculans strain 4364 16S ribosomal RNA, partial
sequence
1116
1116
100%
0.0
96%
24
NR 041972.1
Erwinia chrysanthemi strain DSM 4610 16S ribosomal RNA, partial
sequence
1116
1116
100%
0.0
96%
25
NR 041921.1
Dickeya dadantii strain CFBP 1269 16S ribosomal RNA, partial
sequence
1114
1114
100%
0.0
96%
Isolate 29, Reverse Primer, sequence
TTCTTTTGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTAGCATTCTGATCTACGATTACTAGCGATTCCGACTTCATGGAG
TCGAGTTGCAGACTCCAATCCGGACTACGACAGACTTTATGAGTTCCGCTTGCTCTCGCGAGGTCGCTTCTCTTTGTATCTGCCATTGTAGCACGTGTGTAGCCCTACTC
GTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTATCACCGGCAGTCTCCTTTGAGTTCCCACCATTACGTGCTGGCAACAAAGGATAAGGGTTGCGC
TCGTTGCGGGACTTAACCCAACATTTCACAACACGAGCTGACGACAGCCATGCAGCACCTGTCTCAGCGTTCCCGAAGGCACTCCTCTATCTCTAAAGGATTCGCTGGAT
GTCAAGAGTAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCC
CAGGCGGTCGATTTAACGCGTTAGCTCCAGAAGCCACGGTTCAAGACCACAACCTCTAAATCGACATCGTTTACAGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTC
CCCACGCTTTCGCACCTGA
Battelle B-21
-------
Battelle B-22
-------
APPENDIX C
WORK INSTRUCTIONS: DWI-01
-------
Work Instructions: DWI-01-02
Page 1 of 9
Key Words: Swab, DFU, Nucleic Acid, Microorganism
WORK INSTRUCTIONS FOR THE EXTRACTION OF
MICROORGANISMS, NUCLEIC ACIDS, AND PLGA MICROSPHERES
FROM ENVIRONMENTAL SAMPLES
Originated by: Date
Stacy Dean, Research Scientist
Applied Biology and Aerosol Technology
Reviewed by: Date
Jessica Wilcox, Research Associate
Applied Biology and Aerosol Technology
Approved by: Date
Chuck DeSanti, Manager
Applied Biology and Aerosol Technology
Battelle
Applied Biology and Aerosol Technology
505 King Avenue
Columbus, Ohio 43201
Battelle C-l
-------
Work Instructions: DWI-01-02
Page 2 of 9
Work Instructions for the Extraction of Microorganisms, Nucleic Acids, and
PLGA Microspheres from Environmental Samples
Scope
This Work Instruction (WI) describes the process that will be followed for the extraction of
environmental samples for nucleic acids, microorganisms, and PLGA microspheres. The method outlined
in this document pertains to project samples and may be altered if necessary for sample processing, but
deviations from the WI will be approved by the Principal Investigator (PI) or designee prior to beginning
work.
Purpose
The purpose of this document is to provide detailed instructions for the extraction of samples for further
analysis of viable microorganisms, nucleic acids, and PLGA microspheres.
References
Material Safety Data Sheets (MSDS), as appropriate
Biosafety Manual, Recommended Practices for Biosafety Level 2 Agents, Building 20 Biosafety Manual,
(current version)
Definitions
BSC—Biological Safety Cabinet
BSL2—Biological Safety Level 2
hsDNA—Herring sperm DNA
SDS—Sodium Dodecyl Sulfate
Battelle C-2
-------
Work Instructions: DWI-01-02
Page 3 of 9
Procedures
Special Concerns:
All samples will be handled using aseptic technique and all manipulations will occur in a decontaminated
BSC following the procedures outlined in the Building 20 Biosafety Manual for BSL-2 microorganisms.
General:
Staff members working on this effort will review this WI and program specific information not included
in this WI before operations occur.
Staff members will note the locations of nearest emergency equipment, including the nearest exits,
eyewash, safety shower, and fire extinguishers.
Staff members will don safety glasses and lab coats upon entering the lab. At a minimum, one pair of
latex gloves or nitrile gloves will be worn for all procedures which involve handling of biological
materials or chemicals. Two pairs of gloves, at least one pair nitrile, will be worn while handling reactant
materials and decontamination solutions and while working in the BSC, including during the cleanup
process.
Waste Disposal—strict adherence to the "Discharge to Drain" permits is required. If a Discharge to Drain
permit is not in place, chemicals and media must be collected for proper disposal.
Equipment Needed:
Nitrile or latex gloves or equivalent
Disposable pipettes: 10 mL, 25 mL, sterile
Pipet Aid
Biohazard bags, various sizes
Steriflip-GP, 0.22 |a,m (Millipore Cat. No. SCGP00525)
Refrigerator, 4 +3 ° C
Variable volume pipettes (such as Pipetman P-2, P-20, P-200, and P-1000)
Pipette tips for variable volume pipettes, aerosol resistant, sterile
Vortex
Incubator, 65 +2 ° C
Battelle C-3
-------
Work Instructions: DWI-01-02
Page 4 of 9
Biological safety cabinet, Class II
Timer
Vacuum
Materials Required:
DNA erase or equivalent
Isopropyl alcohol
10-15% bleach
IX phosphate buffered saline (PBS), pH 7.2
20% SDS
Nuclease-free water, sterile
DNA typing-grade hsDNA
250 mL bottles, sterile
2 mL polypropylene cryovial tubes
50 mL conical tubes
Procedure:
Thoroughly decontaminate the biosafety cabinet and all processing areas (bench tops, incubators, etc.)
before use according to the three-step ABAT decontamination regimen. Decontaminate all samples,
reagents, and other materials passed into or out of the BSC.
Collect pre-swabs according to DWI-02-01
Working in a BSC, aseptically (using sterile forceps, if needed) transfer each original sample, or portion
of original sample, into a 250 mL bottle. Decontaminate gloves between samples, change gloves if they
come into contact with any portion of the original samples.
Obtain a negative matrix control (one for each matrix type in the sample set) and record on Form B.
Battelle C-4
-------
Work Instructions: DWI-01-02
Page 5 of 9
Obtain a positive matrix control (one for each matrix type in the sample set) that has been previously
spiked with B. atrophaeus DNA and PLGA beads and record on Form B.
Add sterile IX PBS to pre-wet each sample according to the following table:
Matrix
Pre-Wet
Extraction
Volume (mL)
Volume (mL)
Gauze (-58 cm2)
5
10
Membrane Filter (<100 cm2)
2
10
Grease (1 g)
5
10
Crax (1 g)
5
10
Vortex each sample for 30 sec. to mix (record on Form A)
Add additional IX PBS (extraction volume) and vortex each sample for 30 sec. to mix (record on Form
B).
Allow samples to sit at room temperature for 15 min, vortex for 30 seconds, incubate at room temperature
for another 15 min, and then vortex again for 30 seconds. Document the incubation and vortex times on
Form A.
Remove a 1 mL aliquot from each sample into separate, sterile, 15 mL conical tubes. Microbiological
extracts may be stored at 4 °C for 24 h prior to analysis, if necessary.
Plate each sub-sample onto BHIA for microbial analysis (200 |jL/plate).
Streak B. atrophaeus onto BHIA (3 or 4 phase streak) for comparison.
Record the volume remaining in the bottle on Form B.
Battelle C-5
-------
Work Instructions: DWI-01-02
Page 6 of 9
Add 20% SDS to the remaining volume, to achieve a final concentration of 0.1% v/v.
The amount required should be calculated by converting the sample volume to microliters, then dividing
by 200. For example, to calculate the amount required for a 12 mL sample:
12 mL x 1000|a,L/lmL = 12000 jiL /200 = 60 jiL 20% SDS
Add hsDNA to each sample 1 |jL/mL and record on Form B.
Vortex each sample for 30 sec. to mix (record on Form A).
Incubate at 65 +2 ° C for 15 min, vortex for 30 seconds; incubate an additional 15 min at 65 +2 ° C, and
then vortex again for 30 seconds. Document the incubation and vortex times on Form A.
Transfer the extracted volume to a 50 mL conical tube (use a pipet to squeeze as much liquid out of the
matrix as possible).
Filter the extract with a Steriflip filter to remove large particles.
The filtered extract will proceed to the isopropanol precipitation procedure ABAT-V-012, and will
ultimately be analyzed on the 7900HT.
Decontaminate the BSC and all processing areas.
Maintenance Procedures:
Maintenance will be performed in accordance with the individual equipment manuals or SOPs. There is
no specific maintenance required for this procedure.
Emergency/First Aid Procedures:
Battelle C-6
-------
Work Instructions: DWI-01-02
Page 7 of 9
In the event of an emergency, staff will turn off equipment as possible, evacuate the laboratory, and notify
Battelle security if necessary.
First Aid/Self-Aid Procedures
If physical injuries occur, first aid or self-aid will be administered and Health Services will be called (4-
4444 or 911) on internal Battelle phones.
Quality Control
Training and documentation of competency of personnel as being proficient in the use of this procedure is
required.
All verifications, data, and data manipulations will be documented/recorded and available upon request
by the Program Manager or Principal Investigator to facilitate review.
Forms and Attachments
Documentation of Extraction Steps
Documentation of Volumes for Extraction
Battelle C-7
-------
Work Instructions: DWI-01-02
Page 8 of 9
Form A. Documentation of Extraction Steps
Project
DYNAMAC
Sample Set
Date
1. Remove samples from storage Room/Temperature
Record sample matrices on Form B, column A and transfer to 250 mL Corning storage bottle.
Pre-wet samples with extraction buffer, record volume of buffer added on Form B, column B. ~ Vortex briefly
(~30 sec.).
Add extraction buffer to samples and record volume of buffer added on Form B, column C. ~ Vortex briefly (~30
sec.).
Incubate the samples at 25 + 3°C for 15+3 min. Start time: End time: ~ Vortex
briefly.
Incubate the samples 25 + 3°C for 15 + 3 min. Start time: End time: ~ Vortex briefly.
Remove microbiological extract for analysis and transfer to 15 mL conical. Record on Form B, column E.
Record volume remaining for nucleic acid analysis on Form B, column F.
Add SDS to a final concentration of 0.1% v/v, record volume added on Form B, column G.
SDS Lot # Expiration Date:
Add hsDNA (1 |_iL hsDNA/mL sample), record volume added on Form B, column H.
hSDNA Lot # Expiration Date:
Vortex briefly. Incubate the extracts at 65 + 3°C for 15 + 3 min. Thermometer # Cal. Due
Actual Temperature: Start time: End time: ~ Vortex briefly.
Incubate the extracts at 65 + 3°C for 15 + 3 min. Thermometer #
Actual Temperature: Start time: End time: ~ Vortex briefly.
Transfer extracts to 50 mL conical tubes and clarify by filtering through Steiiflip filter units.
Measure volumes recovered and place into pre-labeled OakRidge tube; note on Form B, column I. Proceed to
Alcohol Precipitation using ABAT-V-012.
Battelle C-8
-------
Work Instructions: DWI-01-02
Page 9 of 9
Performed by
Reviewer:
Date:
Date:
Battelle C-9
-------
Form B. Documentation of Volumes for Extraction
Project
DYNAMAC
Set Number
Date
Sample #
A
B
C
D
E
F
G
H
I
Sample
matrix
IX
PBS
Pre-wet
Vol.
Added
(mL)
IX
PBS
Extract
ion Vol.
(mL)
Total
Volume
IX
PBS
Added
(mL)
B+C)
Volume
Extract
Remove
d for
Micro
Analysi
s
(mL)
Volume
Remain
ing for
Nucleic
acid
Analysi
s
(mL)
(D-E)
Volume
hsDNA
Added
OiL)
Volume
SDS
Added
(HL)
Volume
recover
ed from
Sterifli
P
Wash
(mL)
Record sample notes on additional pages if necessary.
Performed by: Date:
Reviewer: Date:
Form C. Microbiological Analysis
-------
Work Instructions: DWI-01-02
Page 11 of 9
Project
DYNAMAC
Set Number
Date
Sample #
Colony Morphology (Describe: size, color, edge, shape, roughness)
1
2
3
6
5
B. atrophaeus
Control
2mm,
orange,
entire.
N/A
N/A
N/A
N/A
Example description: B. atrophaeus should be ~2 mm, orange, entire, raised, and smooth.
Performed by: Date:
Reviewer: Date:
Battelle C-11
-------
APPENDIX C. PHOTOLOG OF PLANT ACTIVITIES
C-1
-------
DARLING
Subject: Darling International, Inc. Plant
Site: Darling International, Inc.
Photograph No.: 1
Direction: Northeast
Daniell
Date: 10/19/2010
Photographer: Neil
Subject: DATS collected samples from the processing building on the rendering facility.
Site: Darling International, Inc.
Photograph No.: 2 Date: 10/19/2010
Direction: Northeast Photographer: Neil Daniell
C-2
-------
Subject: Receiving floor of Darling International, Inc. Plant. Pit is in the background.
Site: Darling International, Inc.
Photograph No.: 3 Date: 10/19/2010
Direction: East Photographer: Mike
Marshall
Subject: Swab samples 1a and 1b taken from the southeast corner of the
receiving floor.
Site: Darling International, Inc.
Photograph No.: 4 Date: 10/19/2010
Direction: West and down Photographer: Mike
Marshall
C-3
-------
D
Subject: Swab amples 2a and 2b taken from the northwest corner of the receiving floor.
Site: Darling International, Inc.
Photograph No.: 5 Date: 10/19/2010
Direction: Northwest and down Photographer: Mike
Marshall
Subject: DATS collected swab amples 3a and 3b from the central vertical wall of the
tipping floor pit.
Site: Darling International, Inc.
Photograph No.: 6 Date: 10/19/2010
Direction: West Photographer: Mike
Marshall
C-4
-------
C-5
-------
Subject: Swab amples 4a and 4b taken from the northwest side (back vertical wall) of
the pit.
Site: Darling International, Inc.
Photograph No.: 7 Date: 10/19/2010
Direction: East Photographer: Mike
Marshall
Subject: Waste water collection sump. Swab amples 5a and 5b were collected from
the foreground area (south side).
Site: Darling International, inc.
Photograph No.: 8 Date: 10/19/2010
Direction: North Photographer: Neil Daniell
C-6
-------
Subject: DATS collected samples 5a and 5b from the plant floor on the south side of
the raw pit sump.
Site: Darling International, Inc.
Photograph No.: 9 Date: 10/19/2010
Direction: North and down Photographer: Mike
Marshall
Subject: Wastewater samples 13a and 13b collected from inside the sump during the
surrogate selection phase. Inset is a close-up of the collection process.
Site: Darling International, inc.
Photograph No.: 10 Date: 10/19/2010
Direction: North and down Photographer: Mike
Marshall
C-7
-------
C-8
-------
Subject: Incline auger leading from the pit to the cooking process.
Site: Darling International, Inc.
Photograph No.: 11 Date: 10/19/2010
Direction: East Photographer: Mike
Marshall
Subject: DATS collected samples 6a and 6b from the side wall of the incline auger
Site: Darling International, Inc.
Photograph No.: 12 Date: 10/19/2010
Direction: South Photographer: Mike
Marshall
C-9
-------
Subject: Large grinder used in the rendering process
Site: Darling International, Inc.
Photograph No.: 13
Direction: Northeast
Daniell
Date: 10/19/2010
Photographer: Neil
Subject: DATS collected samples 7a and 7b from the large grinder used in the
rendering process.
Site: Darling International, Inc.
Photograph No.: 14 Date: 10/19/2010
Direction: East and down Photographer: Mike
Marshall
C-10
-------
Subject: Stairwell between the tallow tanks (left) and the cooker (right).
Site: Darling International, inc.
Photograph No.: 15 Date: 10/19/2010
Direction: East Photographer: Mike
Marshall
Subject: Swab samples 8a and 8b were collected from the stairwell between the tallow
tanks and the cooker.
Site: Darling International, Inc.
Photograph No.: 16 Date: 10/19/2010
C-11
-------
Direction: East and down
Marshall
Photographer: Mike
Subject: The load out area used by the Darling International plant.
Site: Darling International, Inc.
Photograph No.: 17 Date: 10/19/2010
Direction: South Photographer: Mike
Marshall
Subject: DATS collected swab samples 9a and 9b from an undisturbed locale in the
rear of the load out area.
C-12
-------
Site: Darling International, Inc.
Photograph No.: 18 Date: 10/19/2010
Direction: South and down Photographer: Mike
Marshall
Subject: The crax grinder used by the Darling International plant. Note heavy dust
presences.
Site: Darling International, Inc.
Photograph No.: 19 Date: 10/19/2010
Direction: Northwest Photographer: Mike
Marshall
C-13
-------
i, ,1 ¦
Subject: Swab samples 10a and 10b were collected from the base (southeast corner)
of the crax grinder.
Site: Darling International, Inc.
Photograph No.: 20 Date: 10/19/2010
Direction: Northwest and down Photographer: Mike
Marshall
Subject: Crax grinder storage used by the Darling International plant.
Site: Darling International, Inc.
Photograph No.: 21 Date: 10/19/2010
Direction: Northwest Photographer: Mike
C-14
-------
Marshall
Subject: DATS collected samples 11a and 11b from an undisturbed area of the crax
grinder storage bins.
Site: Darling International, Inc.
Photograph No.: 22 Date: 10/19/2010
Direction: West and down Photographer: Mike
Marshall
Subject: Truck receiving bay used by the Darling International plant. Swab samples
12a and 12b were collected from the background area near the drain.
C-15
-------
Site: Darling International, Inc.
Photograph No.: 23
Direction: Southwest
Marshall
Date: 10/19/2010
Photographer: Mike
Subject: Samples 12a and 12b were collected the southwest side the truck receiving
bay.
Site: Darling International, inc.
Photograph No.: 24 Date: 10/19/2010
Direction: Southwest and Down Photographer: Mike
Marshall
Subject: Darling International plant employees cleaning the tipping floor for the
C-16
-------
rendering study.
Site: Darling International, inc.
Photograph No.: 25
Direction: Southeast
Date: 10/16/2011
Photographer: Neil Daniell
Subject: DATS personnel performaing background air sampling inside Darling
International plant.
Site: Darling International, Inc.
Photograph No.: 26 Date: 10/19/2011
Direction: South Photographer: Neil Daniell
Subject: Air sampling pump collecting a sample from inside Darling International plant
C-17
-------
during the rendering study.
Site: Darling International, inc.
Photograph No.: 27
Direction: South
Date: 10/19/2011
Photographer: Neil Daniell
Subject: Background air sampling outside of the Darling International plant
Site: Darling International, Inc.
Photograph No.: 28 Date: 10/19/2011
Direction: Northeast Photographer: Neil Daniell
Subject: DATS collecting a background wipe sample on the tipping floor during the final
study.
Site: Darling International, Inc.
Photograph No.: 29 Date: 10/19/2011
Direction: East Photographer: Leroy
C-18
-------
Mickelsen
V
V
v
Subject: DATS collecting a background wipe sample on the wall of the auger during the 1
final study.
Site: Darling International, Inc.
Photograph No.:
30 Date: 10/19/2011
Direction: South
Photographer: Leroy
Mickelsen
-------
Subject: EPA personnel inoculating an incoming load of carcasses.
Site: Darling International, Inc.
Photograph No.: 31 Date: 10/20/2011
Direction: South Photographer: Melissa
Ivancevich
Subject: Air sampling outside of the Darling International plant during processing of
inoculated loads.
Site: Darling International, inc.
Photograph No.: 32 Date: 10/20/2011
Direction: East Photographer: Melissa
Ivancevich
C-20
-------
Subject: Inoculated load of carcasses being deposited onto the tipping floor.
Site: Darling International, inc.
Photograph No.: 33 Date: 10/20/2011
Direction: South Photographer: Melissa
Ivancevich
Subject: DATS personnel collecting a wipe sampling from the tipping floor during
processing of inoculated carcasses.
Site: Darling International, Inc.
Photograph No.: 34 Date: 10/20/2011
Direction: East Photographer: Melissa
Ivancevich
C-21
-------
Subject: DATS wipe sampling wall of auger during processing of inoculated loads.
Site: Darling International, Inc.
Photograph No.: 35 Date: 10/20/2011
Direction: East Photographer: Melissa
Ivancevich
Subject: Cleaning of a truck using a bleach solution after it has dumped its inoculated
load.
Site: Darling International, Inc.
Photograph No.: 36 Date: 10/20/2011
Direction: South Photographer: Melissa
Ivancevich
C-22
-------
Subject: Post-inoculation air sampling inside of plant.
Site: Darling International, Inc.
Photograph No.: 37 Date: 10/21/2011
Direction: East Photographer: Leroy
Mickelsen
Subject: DATS post-inoculation wipe sampling of inside wall of auger.
Site: Darling International, Inc.
Photograph No.: 38 Date: 10/21/2011
Direction: South Photographer: Leroy
Mickelsen
C-23
-------
Subject: DATS collecting a sample of the tippling floor during the post-inoculation
phase.
Site: Darling International, inc.
Photograph No.: 39 Date: 10/21/2011
Direction: Southeast Photographer: Leroy
Mickelsen
Subject: DATS collecting a sample from the wall of the tipping floor pit during the post-
inoculation phase. Note that plant personnel frequently push material against this wall
using a front loader and deposit it into the pit.
Site: Darling International, Inc.
Photograph No.: 40 Date: 10/21/2011
Direction: Southeast Photographer: Leroy
Mickelsen
C-24
-------
Appendix D - Sample Chain of Custody Sheets
-------
CDA USE PA Contract Laboratory Program
t P3r\ Generic Cha in of Custody
Reference Case
Client No: 1
5 DG No: 1—
Dale Shipped: 1 0/22*2011
Carrier Name: Fee Ex
Airbill:
Shipped to: Battelle
505 King Ave
Columlbus OH 43201
<513) 362-2600
Chain of Custody Record
Sarrpfer
Slgiature:
For Lab Use Only
Lad Contract No:
RellnqulBhed By |DateTime)
Race toed By (Date f Time)
1 Anne Busher 10/22
¦¦ 10/22/1:
¦ Unit Price:
2
Transfer To:
3
Lab Contract No:
4
Unit Price:
Station Name
Sample No
Composite
Grab Matrix
Sample Comments
Sample Sample
Sample Date Start Time End Time Concentration
Analysis Analysis
Location Name Preservative Name Name 2 Analysis Name 3
Enumeration
PLGA
Bacterial ID-PCR
Darlir
g Inte
na
onal
IRP-AIR -10-19-11-ABC-B1
G
Ambient Air
START -10:32; STOP -12:32; FLOW -1.058 Liter Per Minute ;
Cooker Room near scrubber intake.
10/19/2011
10:32
12:32
L
Cooker Room
near scrubber
intake
Ice Only
Darlir
g Inte
na
onal
IRP-AIR -10-19-11-ABC-B2
G
Ambient Air
START -10:39; STOP -12:39; FLOW -1.078 Liter Per Minute ;
Window Btw Sump & Tipping Floor
10/19/2011
10:39
12:39
L
Window Btw
Sump & Tipping
Floor
Ice Only
Darlir
g Inte
na
onal
IRP-AIR -10-19-11-ABC-B3
G
Ambient Air
START -10:44; STOP -12:55; FLOW -1.013 Liter Per Minute ;
DAF Tank Area
10/19/2011
10:44
12:55
L
DAF Tank Area
Ice Only
x
x
x
Darlir
g Inte
na
'onal
IRP-AIR -10-19-11-ABC-B4
G
Ambient Air
START -10:47; STOP -13:04; FLOW -1.018 Liter Per Minute ;
Soap Stock Receiving Tanks
10/19/2011
10:47
13:04
L
Soap Stock
Receiving Tanks
Ice Only
Darlir
g Inte
na
onal
IRP-AIR -10-19-11-ABC-B5
G
Ambient Air
START -10:52; STOP -12:57; FLOW -1.037 Liter Per Minute ;
East of innoculating area
10/19/2011
10:52
12:57
L
East of
innoculating area
Ice Only
Darlir
g Inte
na
onal
IRP-AIR -10-19-11-ABC-B6
G
Ambient Air
START -10:56; STOP -12:58; FLOW -1.060 Liter Per Minute ;
West of Innoculating Area
10/19/2011
10:56
12:58
L
West of
Innoculating Area
Ice Only
Darlir
g Inte
na
onal
IRP-AIR -10-19-11-ABC-B7
G
Ambient Air
START-11:03 STOP -13:08; FLOW -1.012 Liters Per Minute;
Front Parking Lot
10/19/2011
11:03
13:08
L
Front Parking Lot
Ice Only
x
x
x
Darlir
g Inte
na
'onal
IRP-AIR -10-19-11-ABC-B8
G
Field QC
Field Blank
10/19/2011
Field Blank
14:10
L
Field Blank
Ice Only
X
X
X
Darlir
g Inte
na
onal
1RP-AIR-10-20-ll-ABC-001
G
Ambient Air
START - 8:18; STOP -12:18; FLOW -1.052 Liter Per Minute ;
Cooker Room near scrubber intake.
10/20/2011
8:18
12:18
M
Cooker Room
near scrubber
intake
Ice Only
Darlir
g Inte
na
'onal
1RP-AIR-10-20-11-ABC-002
G
Ambient Air
START -10:00; STOP -14:00; FLOW -1.050 Liter Per Minute ;
Window Btw Sump & Tipping Floor
10/20/2011
10:00
14:00
M
Window Btw
Sump & Tipping
Floor
Ice Only
Darlir
g Inte
na
onal
IRP-AIR-10-20-ll-ABC-003
G
Ambient Air
START -10:40; STOP -14:40; FLOW -1.005 Liter Per Minute ;
DAF Tank Area
10/20/2011
10:40
14:40
M
DAF Tank Area
Ice Only
x
x
x
Darlir
g Inte
na
'onal
IRP-AIR-10-20-ll-ABC-004
G
Ambient Air
START -14:33; STOP -18:33; FLOW -1.024 Liter Per Minute ;
Soap Stock Receiving Tanks
10/20/2011
14:33
18:33
M
Soap Stock
Receiving Tanks
Ice Only
Darlir
g Inte
na
onal
IRP-AIR-10-20-ll-ABC-005
G
Ambient Air
START - 8:13; STOP -12:16; FLOW -1.011 Liter Per Minute ;
East of innoculanting area
10/20/2011
8:13
12:16
M
East of
innoculanting
area
Ice Only
Darlir
g Inte
na
onal
IRP-AIR-10-20-ll-ABC-006
G
Ambient Air
START - 8:15; STOP -12:15; FLOW - 0.9941 Liter Per Minute ;
West of Innoculating Area
10/20/2011
8:15
12:15
M
West of
Innoculating Area
Ice Only
Darlir
g Inte
na
onal
IRP-AIR-10-20-ll-ABC-007
G
Ambient Air
START- 8:34 STOP -12:34; FLOW -1.014 Liters Per Minute;
Front Parking Lot
10/20/2011
8:34
12:34
M
Front Parking Lot
Ice Only
x
x
x
Darlir
g Inte
na
'onal
IRP-AIR-10-20-ll-ABC-008
G
Field QC
Field Blank
10/20/2011
Field Blank
14:00
L
Field Blank
Ice Only
x
x
x
Darling International IRP-AIR-10-21-11-ABC-0010
START: 10:18 STOP: 14:18; FLOW RATE: 1.011; East side of
innoculation area
10/21/2011 10:18 14:18 M
East side of
innoculation area Ice Only
OTfeana* Iw Cm
id! |ipj 9; Lv is* UHtfli f«l ItfLHJ* dltf! f QC.
-'JiJUUH.h -j L- r. 7-1 *1
ijpor Ricaqpt:
l rain or uusioay seai wumoec:
Af4ty*>4 K4y:
C4«ie*rita,t(i6ri: j. - UWr4 M - l^p i^w/r h - ngn
- C. - G
Cuil&tfy #*au IntiS? __
(h pnr-fil It*#? _
ENLfM ¦ F.yOfttttn ¦ PLWA, FC-* ¦ i|>?CR
TR Number: 7-083090244-102211-0001
PR pfiftviriM pratf nnnaiy n*uKf RAfiiiAH'A fiv jwailm in irv rauirt* it Iwuitt :in.i ryhr*l rrv*f a
Senc Copy za: Sample Wi-ageR-enl Ofice 1iC ?".• iin'eitraf D'„ Chanlilly, VA. 2D151-1E1S Plwne 7C3va 1 3^203; Fa* 7D3VS1
LABORATORY COPY
Hvi1.HI page 1 of 6
-------
CDA USE PA Contract Laboratory Program
t P3r\ Generic Cha in of Custody
Reference Case
Client No: 1
5 DG No: 1—
Dale Shipped: 10/22*2011
Carrier Name: Fee Ex
Airbill:
Shipped to: Battelle
505 King Ave
Columbus OH 43201
<513) 362-2600
Chain of Custody Record
Sarrpfer
Slgiature:
For Lab Use Only
Lad Contract No:
RellnqulBhed By (Data / Him)
Race toed By (Date f Time)
1
Unit Price:
2
Transfer To:
3
Lad Contract No:
4
Unit Phce:
Station Name
Sample No
Composite
Grab Matrix
Sample Comments
Sample Sample
Sample Date Start Time End Time Concentration Location Name Preservative
Analysis
Name
Analysis
Name 2 Analysis Name 3
10/20/2011
Wipe Sample Wall of Auger
Wall of Auger
ce Only
10/20/2011
Field QC
ce Only
10/21/2011
Wipe Sample PD 2 - GRINDER WALL; LEFTSIDE
ce Only
10/21/2011
Wipe Sample PD 4 - Between floor drains; in front of electrical panel
ce Only
10/21/2011
Wipe Sample PD 6 - top of steps; large grinder
ce Only
10/21/2011
Wipe Sample PD 8 - Three feet south of western tallow tank
ce Only
10/21/2011
Wipe Sample PD 10 - Floor in Front of Cooker Control Panel
ce Only
10/21/2011
Wipe Sample PD 12 - Floor approximately 1.5 feet from control panel
ce Only
10/21/2011
Wipe Sample PD 14 - 2 feet from small crax grinder control panel
ce Only
10/21/2011
Wipe Sample PD 16 - Center of doorway near M3 Tallow Tank
ce Only
Wipe Sample PD 13 - Front of Crax Loadout area
10/21/2011
ce Only
Wipe Sample PD 15 - 3 feet wall near M2 tallow tank; in walkway
10/21/2011
ce Only
Wipe Sample PD 9 - Office Door; Cooker Room
10/21/2011
ce Only
Wipe Sample PD 7 - end of railroad tracks
10/21/2011
ce Only
Wipe Sample PD 1 - CENTER LEFT OF TIPPING FLOOR NEAR DOOR
10/21/2011
ce Only
Wipe Sample PD 11 - Railroad Tracks next to floor drain; ~ Tank D6
10/21/2011
ce Only
Wipe Sample PD 5 - Between floor drains; in front of electrical panel
10/21/2011
ce Only
Wipe Sample PD 3-AUGER COVER
10/21/2011
ce Only
Wipe Sample Tipping Floor; 12 feet from pit wal
10/20/2011
Tipping Floor; 12
ce Only
Wipe Sample Tipping Floor; 12 Feet from Pit Wall
10/20/2011
Tipping Floor; 12
ce Only
OTfeana* Iw Cm
COTI54«*?S
id! Iifrj 9; Lv is* UHtfli f«l IdLnj* dltf! f QC.
-'JUI .IUH.H -j L- r. 7-1 »|
?vr|jvfilro
ijpor
l rain or uuscny sea numoar
Af4ty*>4 K4y:
C«n«*nt-vflon: L - UMr* m - LMt MHrWi. N - !-tjn
Cuil&tfy #*au IntiS? __
f h prrint _
ENLm ¦ FwOfHCtn ¦ PLWA, FC-* ¦ i|>?CR
TR Number: 7-083090244-102211-0001
PR pfiftviriM pratf nnnaiy n*uKf RAfiiiAH'A fiv jwailm in irv rauirt* it Iwuitt an.i vHp.iI rrv*f a
Senc Copy za: Sample Hi-iaen-ent Ofice 1 iC ?".• iin'eitraf D'„ Chanlilly, VA. 2D151-1B1G Plwne 7C3va 1 3^203; Fa* 7D3VS16^2
LABORATORY COPY
Hvi1.HI page 1 of 6
-------
CDA USE PA Contract Laboratory Program
t P3r\ Generic Cha in of Custody
Reference Case
Client No: 1
5 DG No: 1—
Dale Shipped: 10/22*2011
Carrier Name: Fee Ex
Airbill:
Shipped to: Battelle
505 King Ave
Columbus OH 43201
<513) 362-2600
Chain of Custody Record
Sarrpfer
Slgiature:
For Lab Use Only
Lad Contract No:
RellnqulBhed By |DateTime)
Race toed By (Date f Time)
1
Unit Price:
2
Transfer To:
3
Lad Contract No:
4
Unit Phce:
Station Name
Sample No
Composite
Grab Matrix
Sample Comments
Sample Sample
Sample Date Start Time End Time Concentration
Location Name Preservative
Analysis
Name
Analysis
Name 2 Analysis Name 3
Sump & Tippir
10/21/2011
ce Only
Soap Stock
10/21/2011
ce Only
START -10:47; STOP -14:47; FLOW -1.008 Liter Per Minute ;
Ambient Air Front Parking Lot
10/21/2011
10/21/2011
Front Parking Lot
ce Only
ce Only
START: 10:15 STOP: 14:15; FLOW RATE: 1.017; West side of
10/21/2011
ce Only
Tipping Floor; 12
feet from pit
10/19/2011
Wipe Sample Tipping Floor; 12 feet from pit
ce Only
The claw; tippir
10/19/2011
WipeSample Theclaw; tipping room
ce Only
10/19/2011
Field QC
ce Only
10/20/2011
Field QC
ce Only
10/20/2011
Wipe Sample Wall of Auger
Wall of Auger
ce Only
10/20/2011
Wipe Sample Wall of Auger
Wall of Auger
ce Only
START -10:43; STOP -14:43; FLOW -1.048 Liter Per Minute ;
Ambient Air Window Btw Sump & Tipping Floor
START -10:33; STOP -14:33; FLOW -1.006 Liter Per Minute ;
10/21/2011
ce Only
Wipe Sample Wall of Auger
10/20/2011
Wall of Auger
ce Only
Wipe Sample Wall of Auger; LeftSide
10/19/2011
Wall of Auger;
ce Only
Wipe Sample Tipping Floor; 12 feet from pit
10/20/2011
Tipping Floor; 12
feet from pit
ce Only
Field QC
Postive Control; Innoculant Water
10/20/2011
ce Only
START -10:30; STOP -14:30; FLOW -1.015 Liter Per Minute ;
Ambient Air Soap Stock Receiving Tanks
START -10:37; STOP -14:37; FLOW -1.009 Liter Per Minute ;
10/21/2011
ce Only
Wipe Sample Tipping Floor; 12 Feet from Pit Wall
10/20/2011
Tipping Floor; 12
ce Only
Wipe Sample Drain; middle of floor; 12 feet from new wal
10/19/2011
Drain; middle of
floor; 12 feet
ce Only
START -10:22; STOP -14:22; FLOW -1.042 Liter Per Minute ;
10/21/2011
ce Only
Wall of Auger
OTfeana* Iw Cm
COTI54«*?S
id! Iifrj 9; Lv is* UHtfli f«l ItfLHJ* dltf! f QC.
-'JUI .IUH.H -j L- r. 7-1 j(
?vr|jvfilro
ijpor
l rain or uuscny sea n umoec:
Af4ty*>4 K4y:
C«n«*nt-vflon: l - UMr* m - LMt MHrwi. N - !-»gpn
Cuil&tfy #*4i Intac*"? __
f h prrint _
ENLfM ¦ FwOfHCtn ¦ PLWA, FC-* ¦ BHHrill i|>?CR
TR Number: 7-083090244-102211-0001
PR pfiftviriM pratf nnnaiy n*uKf RAfiiiAH'A fiv jwailm in irv rauirt* it Iwuitt an.i vHp.iI rrv*f a
Senc Copy za: Sample Hi-iaen-ent Ofice 1 iC ?".• iin'eitraf D'„ Chanlilly, VA. 2D151-1B1G Plwne 7C3va 1 3^203; Fa* 7D3VS16^2
LABORATORY COPY
Hvi1.HI page 1 of 6
-------
pDA USE PA Contract Laboratory Program
t P3r\ Generic Cha in of Custody
Reference Case
Client No: 1
5 DG No: 1—
Dale Shipped: 10/22*2011
Carrier Name: Fee Ex
Airbill:
Shipped to: Battelle
505 King Ave
Columbus OH 43201
<513) 362-2600
Chain of Custody Record
Sarrpfer
Slgiature:
For Lab Use Only
Lad Contract No:
RellnqulBhed By |DateTime)
Received By (Date (Time)
1
Unit Price:
2
Transfer To:
3
Lad Contract No:
4
Unit Phce:
Station Name
Sample No
Composite
Grab
Matrix
Sample Comments
Sample Date
Sample
Start Time
Sample
End Time
Concentration Location Name
Preservative
Analysis
Name
Analysis
Name 2
Analysis Name 3
Enumeration
PLGA
Bacterial ID-PCR
Darl
ng International
IRP-WIPE-10-21-11-ABC-0027
G
Wipe Sample
PD 17 - Walkway; 4 feet from stairs near SS5 Tank
10/21/2011
NA
16:39
PD 17
Ice Only
x
x
x
Darl
ng International
IRP-WIPE-10-2 l-ll-ABC-0028
G
Wipe Sample
PD 18 -12 feet from control panel; center of flexing & tanks
room
10/21/2011
NA
16:47
PD 18
Ice Only
x
x
x
Darl
ng International
IRP-WIPE-10-2 l-ll-ABC-0029
G
Wipe Sample
PD 19 - 6 inches from drain near maint. roll up door
10/21/2011
NA
16:54
PD 19
Ice Only
x
x
x
Darl
ng International
IRP-WIPE-10-2 l-ll-ABC-0030
G
Wipe Sample
PD 20- Center of Ramp; 5 feet from door
10/21/2011
NA
17:01
PD 20
Ice Only
x
x
x
Darl
ng International
IRP-WIPE-10-2 l-ll-ABC-0031
G
Wipe Sample
PD 21-12 feet from wall; near floor traing {new wall near the
sump)
10/21/2011
NA
17:16
PD 21
Ice Only
x
x
x
Darl
ng International
IRP-WIPE-10-21-11-ABC-0032
G
Wipe Sample
PD 22- Center of Bay Door; 5 feet from door
10/21/2011
NA
17:22
PD 22
Ice Only
x
x
x
Darl
ng International
IRP-WIPE-10-2 l-ll-ABC-0033
G
Wipe Sample
Wall of Auger; Stage 2 Process Sampling
10/21/2011
NA
10:05
Wall of Auger;
Stage 2 Process
Sampling
Ice Only
Darl
ng International
IRP-WIPE-10-2 l-ll-ABC-0034
G
Wipe Sample
Tipping Floor; 12 feet from pit
10/21/2011
NA
10:18
Tipping Floor; 12
feet from pit
Ice Only
x
x
x
Darl
ng International
IRP-WIPE-10-2 l-ll-ABC-0035
G
Wipe Sample
Wall of Auger; Stage 2 Process Sampling
10/21/2011
NA
12:02
Wall of Auger;
Stage 2 Process
Sampling
Ice Only
Darl
ng International
IRP-WIPE-10-2 l-ll-ABC-0036
G
Wipe Sample
Tipping Floor; 12 feet from pit
10/21/2011
NA
12:12
Tipping Floor; 12
feet from pit
Ice Only
x
x
x
Darl
ng International
IRP-WIPE-10-21-11-ABC-0037
G
Wipe Sample
Wall of Auger; Stage 2 Process Sampling
10/21/2011
NA
14:01
Wall of Auger;
Stage 2 Process
Sampling
Ice Only
Darling International IRP-WIPE-10-21-11-ABC-0038
Wipe Sample Tipping Floor; 12 feet from pit
10/21/2011 NA
Tipping Floor; 12
feet from pit Ice Only
Wall of Auger;
Stage 2 Process
Darlin
I International
IRP-WIPE-10-2 l-ll-ABC-0039
G
Wipe Sample
Wall of Auger; Stage 2 Process Sampling
10/21/2011
NA
16:00 L
Sampling
Ice Only
X
X
X
Darlin
I International
IRP-WIPE-10-2 l-ll-ABC-0040
G
Wipe Sample
Tipping Floor; 12 feet from pit
10/21/2011
NA
16:08 L
Tipping Floor; 12
feet from pit
Ice Only
x
x
x
Darlin
I International
IRP-WIPE-10-2 l-ll-ABC-0041
G
Field QC
Field Blank B4; inside warehouse
10/21/2011
NA
11:08 L
B4
Ice Only
X
X
X
Darlin
I International
IRP-WIPE-10-2 l-ll-ABC-0042
G
Field QC
Field Blank B8; inside warehouse
10/21/2011
NA
11:50 L
B8
Ice Only
X
X
X
Darlin
I International
IRP-WIPE-10-2 l-ll-ABC-0043
G
Field QC
Field Blank B12; inside warehouse
10/21/2011
NA
15:00 L
B12
Ice Only
X
X
X
Darlin
I International
IRP-WIPE-10-2 l-ll-ABC-0044
G
Field QC
Field Blank B16; inside warehouse
10/21/2011
NA
16:32 L
B16
Ice Only
X
X
X
Darlin
I International
IRP-WIPE-10-2 l-ll-ABC-0045
G
Field QC
Field Blank B20; inside warehouse
10/21/2011
NA
17:07 L
B20
Ice Only
X
X
X
Darlin
I International
IRP-WIPE-10-2 l-ll-ABC-0046
G
Field QC
Field Blank BA1; inside warehouse
10/21/2011
NA
14:06 L
BA1
Ice Only
X
X
X
Darlin
I International
IRP-WIPE-10-21-11-ABC-0047
G
Field QC
Field Blank BA2; inside warehouse
10/21/2011
NA
15:53 L
BA2
Ice Only
X
X
X
Darlin
I International
IRP-WIPE-10-2 l-ll-ABC-0048
G
Field QC
Field Blank BTF1; inside warehouse
10/21/2011
NA
14:20 L
BTF1
Ice Only
X
X
X
Darlin
I International
IRP-WIPE-10-2 l-ll-ABC-0049
G
Field QC
Field Blank BTF2; inside warehouse
10/21/2011
NA
16:19 L
BTF2
Ice Only
X
X
X
OTfeana* Iw Cm
COTI54«*?S
id! |ipj 9; Lv is* UHtfli f«l ItfLHJ* dltf! f QC.
-'JiJI IUH.h -j L- r. 7-1 fcr ik--31 *1
ijpor
l rain or uuscny sea n jrna&r:
Af4ty*>4 K4y:
C-cncvnb-rtlen: m - LHriMOilt. H - !-tjn
- C. - 4
Cuil&tfy #*4i IrlrtaH^
f h prrint _
ENLfM ¦ FwOfHCtn ¦ PLWA, FC-* ¦ BHHrill iE^PCR
TR Number: 7-083090244-102211-0001
PR priflwiriM pratf nnnaiy n*uKf RAfiiiAH'A fiv jwailm ^ irv ruuirbi *' it Iwuitt :in.i vHp.iI rrv*f a
Senc Copy za: Sample Hi-iaen-ent Ofice 1iC ?".• iin'eitraf D'„ Chanlilly, VA. 2D151-1E1S Plwne 7C3va 13^203; Fa* 7D3VS1
LABORATORY COPY
Hvi1.HI page 1 of 6
-------
CDA USE PA Contract Laboratory Program
t P3r\ Generic Cha in of Custody
Reference Case
Client No: 1
5 DG No: 1—
Dale Shipped: 10/22*2011
Carrier Name: Fee Ex
Airbill:
Shipped to: Battelle
505 King Ave
Columbus OH 43201
<513) 362-2600
Chain of Custody Record
Sarrpfer
Slgiature:
For Lab Use Only
Lad Contract No:
RellnqulBhed By |DateTime)
Race toed By (Date f Time)
1
Unit Price:
2
Transfer To:
3
Lad Contract No:
4
Unit Phce:
Station Name
Sample No
Composite
Grab Matrix
Sample Comments
Sample Sample
Sample Date Start Time End Time
Analysis Analysis
Concentration Location Name Preservative Name Name 2 Analysis Name 3
10/24/2011 NA
Wipe Sample Next to Yellow Pole
ce Only
10/24/2011 NA
Wipe Sample In center of room on metal grate
ce Only
10/24/2011 NA
Wipe Sample Outside wall of grinder on south side of tipping floor
ce Only
10/24/2011 NA
Wipe Sample Near door and below chute to pit
ce Only
10/24/2011 NA
Wipe Sample Pathway at top of stairs of roto strainer behind skimmer tanks
ce Only
10/24/2011 NA
Wipe Sample Top of stairs of large grinder in auger room
ce Only
10/24/2011 NA
Wipe Sample center of floor, 3 feet in from drair
ce Only
10/24/2011 NA
Wipe Sample End of railroad tracks
ce Only
10/24/2011 NA
Wipe Sample Middle of room in front of D2 tank
ce Only
10/24/2011 NA
Wipe Sample In front of bay door
ce Only
Wipe Sample In front of office door/emergency shower
10/24/2011 NA
ce Only
10/24/2011 NA
Wipe Sample End of cooker, in front of bay door
ce Only
Wipe Sample Field Blank; inside warehouse
10/24/2011 NA
ce Only
Wipe Sample End of cooker, to the right of the control panel
10/24/2011 NA
ce Only
Wipe Sample Middle of railroad tracks, in front of Tank D6, near drair
10/24/2011 NA
ce Only
Wipe Sample Field Blank; inside warehouse
10/24/2011 NA
ce Only
Wipe Sample 1 foot in from doorway to auger room
10/24/2011 NA
ce Only
Wipe Sample Field Blank; inside warehouse
10/24/2011 NA
ce Only
Wipe Sample In front of doorway, 1.5 feet in from drair
10/24/2011 NA
ce Only
Wipe Sample Middle of floor in center of room
10/24/2011 NA
ce Only
Wipe Sample Lid
10/24/2011 NA
ce Only
Field QC
Field Blank @ PC 5; inside warehouse
10/24/2011 NA
ce Only
Wipe Sample Or
on north side of tipping floor
10/24/2011 NA
ce Only
Wipe Sample In front of Bay Door on south side
10/24/2011 NA
ce Only
OTfeana* Iw Cm
COTI54«*?S
id! Iifrj 9; Lv is* UHtfli f«l ItfLHJ* dltf! f QC.
-'JUI .IUH.H -j L- r. 7-1 j(
?vr|jvfilro
ijpor
l rain or uuscny sea n umoec:
Af4ty*>4 K4y:
C«n«*nt-vflon: l - UMr* m - LMt MHrwi. N - !-»gpn
Cuil&tfy #*4i Intac*"? __
f h prrint _
ENLfM ¦ FwOfHCtn ¦ PLWA, FC-* ¦ BHHrill iE^PCR
TR Number: 7-083090244-102211-0001
PR pfiftviriM pratf nnnaiy n*uKf RAfiiiAH'A fiv jwailm in irv rauirt* it Iwuitt an.i vHp.iI rrv*f a
Senc Copy za: Sample Hi-iaen-ent Ofice 1 iC ?".• iin'eitraf D'„ Chanlilly, VA. 2D151-1B1G Plwne 7C3va 13^203; Fa* 7D3VS16^2
LABORATORY COPY
Hvi1.HI page 1 of 6
-------
CDA USE PA Contract Laboratory Program
t P3r\ Generic Cha in of Custody
Reference Case
Client No: 1
5 DG No: 1—
Dale Shipped: 10/22*2011
Carrier Name: Fee Ex
Airbill:
Shipped to: Battelle
505 King Ave
Columbus OH 43201
<513) 362-2600
Chain of Custody Record
Sarrpfer
Slgiature:
For Lab Use Only
Lad Contract No:
RellnqulBhed By (Data / Him)
Race toed By (Date f Time)
1
Unit Price:
2
Transfer To:
3
Lad Contract No:
4
Unit Phce:
Station Name
Sample No
Composite
Grab Matrix
Sample Comments
Sample Sample
Sample Date Start Time End Time
Concentration Location Name Preservative
Analysis
Name
Analysis
Name 2 Analysis Name 3
10/24/2011 NA
Wipe Sample Back of crax load out area
ce Only
10/24/2011 NA
WIpeSample 1 foot f ro m cont ro I pa ne I
ce Only
10/24/2011 NA
Wipe Sample In front of doorway; 20 feet ir
ce Only
Wipe Sample In front of T3 wastewater / doorway to cooker room
10/24/2011 NA
ce Only
10/24/2011 NA
Wipe Sample In front of fire extinguisher; about 4 ft from wal
ce Only
10/24/2011 NA
Wipe Sample center of floor, 5 feet from stairs
ce Only
10/24/2011 NA
Wipe Sample Beside F4 tank
ce Only
10/24/2011 NA
Wipe Sample behind supm room wall next to control panel
ce Only
10/24/2011 NA
Wipe Sample 12 feet from wall; end of grate
ce Only
10/24/2011 NA
Wipe Sample 10 feet from maintenance shed
ce Only
10/24/2011 NA
WIpeSample Rear of bay
ce Only
Wipe Sample south of fleshing tank
10/24/2011 NA
ce Only
Wipe Sample Center of ramp
10/24/2011 NA
ce Only
Wipe Sample 2 feet in front of control panel
10/24/2011 NA
ce Only
Wipe Sample Field blank
10/24/2011 NA
ce Only
Wipe Sample Field blank
10/24/2011 NA
ce Only
Wipe Sample
10/24/2011 NA
ce Only
Wipe Sample Behind DAF tank, about 6 feet from wal
10/24/2011 NA
ce Only
Wipe Sample In front of Ml Tank; 6 feet out
10/24/2011 NA
ce Only
Wipe Sample 12 feet; front of fleshing tank
10/24/2011 NA
ce Only
Wipe Sample Field Blank; inside warehouse
10/24/2011 NA
ce Only
Wipe Sample In front of Raw SoapstockTank 2, about 4 feet away
10/24/2011 NA
ce Only
OTfeana* Iw Cm
COTI54«*?S
id! Iifrj 9; Lv is* UHtfli f«l ItfLHJ* dltf! f QC.
-'JUI .IUH.H -j L- r. 7-1 fcr j(
?vr|jvfilro
ijpor
l rain or uuscny sea n umoec:
Af4ty*>4 K4y:
C«n«*nt-vflon: l - UMr* m - LMt MHrwi. N - !-»gpn
Cw«tc4y #*4i Intac*"? __
f h prrint _
TR Number: 7-083090244-102211-0001
PR pfiftviriM pratf nnnaiy n*uKf RAfiiiAH'A fiv jwailm in irv rauirt* it Iwuitt an.i vHp.iI rrv*f a
Senc Copy za: Sample Hi-iaen-ent Ofice 1 iC ?".• iin'eitraf D'„ Chanlilly, VA. 2D151-1B1G Plwne 7C3va 13^203; Fa* 7D3VS16^2
LABORATORY COPY
Hvi1.HI page 1 of 6
-------
CDA USE PA Contract Laboratory Program
t P3r\ Generic Cha in of Custody
Reference Case
Client No: 1
5 DG No: 1—
Dale Shipped: 1 0/22*2011
Carrier Name: Fee Ex
Airbill:
Shipped to: Battelle
505 King Ave
Columlbus OH 43201
<513) 362-2600
Chain of Custody Record
Sarrpfer
Slgiature:
For Lab Use Only
Lad Contract No:
RellnqulBhed By |DateTime)
Rtecetved By (Date f Time)
1
Unit Price:
2
Transfer To:
3
Lab Contract No:
4
Unit Price:
Station Name
Sample No
Composite
Grab Matrix
Sample Comments
Sample Sample
Sample Date Start Time End Time Concentration Location Name Preservative
Analysis
Name
Analysis
Name 2 Analysis Name 3
Enumeration
PLGA
Bacterial ID-PCR
Darling
Inte
na
'onal
IRP-WIPE-10-24-11-ABC-097
G
Wipe Sample
Field Blank
10/24/2011 NA
9:19 L
Field Blank
Ice 0
ly
x
x
x
Darling
Inte
na
'onal
IRP-WIPE-10-24-11-ABC-098
G
Wipe Sample
Wall of Auger
10/24/2011 NA
8:52 L
PC Grinder Study
Auger
Ice 0
iv
x
x
x
Darling
Inte
na
onal
IRP-WIPE-10-24-11-ABC-099
G
Wipe Sample
12 feet from wall
10/24/2011 NA
8:41 L
PC Grinder Study
Tipping Floor
Ice 0
iv
Darling
Inte
na
'onal
IRP-FPG-10-24-11-ABC-001
G
Grease
Sample
from spigot off take
10/24/2011 NA
13:54 L
GreaseTank
Ice 0
iv
x
x
x
Darling
Inte
na
onal
IRP-F PC-10-24-ll-ABC-001
G
Crax Sample
directly from auger screw as it filled truck
10/24/2011 NA
13:59 L
Crax Load out
Area
Ice Only
x
x
x
Darling
Inte
na
onal
1RP-AIR-10-24-ll-ABC-018
G
Ambient Air
START - 8:59; STOP -14:59; FLOW -1.040 Liter Per Minute ;
Window Btw Sump & Tipping Floor
10/24/2011 NA
14:59 L
Station 2
Ice Only
x
x
x
Darling
Inte
na
'onal
1R P-AIR-10-24-ll-ABC-019
G
Ambient Air
START - 8:42; STOP - 9:45; FLOW -1.003 Liter Per Minute ;
Station 5 - South of Building; East coner
10/24/2011 NA
9:45 L
Station 5
Ice 0
iv
x
x
x
Darling
Inte
na
'onal
IRP-AIR-10-24-ll-ABC-020
G
Ambient Air
START - 8:51; STOP -14:51; FLOW -1.010 Liter Per Minute ;
Station 7 - Front Parking Lot
10/24/2011 NA
14:51 L
Station 7
Ice 0
iv
x
x
x
Darling
Inte
na
'onal
IR P-AIR-10-24-ll-ABC-021
G
Ambient Air
START - 8:40; STOP -14:40; FLOW -1.012 Liter Per Minute ;
Station 6 -West of Innoculating Area
10/24/2011 NA
14:40 L
Station 6
Ice Only
x
x
x
Darling
Inte
na
onal
IRP-AIR-10-24-ll-ABC-022
G
Ambient Air
START - 9:03; STOP -15:03; FLOW -1.010 Liter Per Minute ;
Station 4 -Wall near soap stock tanks
10/24/2011 NA
15:03 L
Station 4
Ice 0
iv
x
x
x
Darling
Inte
na
'onal
IR P-AIR-10-24-ll-ABC-023
G
Ambient Air
START - 8:55; STOP -14:55; FLOW -1.023 Liter Per Minute ;
Station 8 -Near Grinder
10/24/2011 NA
14:55 L
Station 8
Ice Only
x
x
x
Darling
Inte
na
onal
IRP-AIR-10-24-ll-ABC-024
G
Ambient Air
Field Blank
10/24/2011 NA
15:47 L
Field Blank
Ice Only
X
X
X
Darling
Inte
na
'onal
1RP-AIR-10-24-11-ABC-025
G
Ambient Air
Field Blank
10/24/2011 NA
15:48 L
Field Blank
Ice 0
iv
X
X
X
Darling International IRP-AIR-10-24-11-ABC-027
START - 8:48; STOP -11:45; FLOW -1.006 Liter Per Minute ;
Ambient Air Station 3 - On rotary skimmer in DAF Room
10/24/2011 NA
OTfeana* Iw Cm
COTI54«*?S
id! |ipj 9; Lv is* UHtfli f«l ItfLHJ* dltf! f QC.
¦-'JiJI .lUI TjL- r. 1 5 fcr J k-' "si J!
ijpor
l rain or uuscny seai numoer
Af4ty*>4 K4y:
C-cncvnb-rtlen: m - LHriMOilt. H - !-»gpn
- C. - G
Cuil&tfy #*au IntiS? __
f h prrint _
ENLfM ¦ FwOfHCtn ¦ PLWA, FC-* ¦ BHHrill iE^PCR
TR Number: 7-083090244-102211-0001
PR pfiftviriM pratf nnnaiy n*uKf RAfiiiAH'A fiv jwailm in irv rauirtu it Iwuitt :in.i ryhr*l rrv*f a
Senc Copy za: Sample Wi-ageR-enl Ofice 1 iC ?".• iin'eitraf D'„ Chanlilly, VA. 2D151-1B1G Plwne 7C3va 13^203; Fa* 7D3VS1
LABORATORY COPY
Hvi1.HI page 1 of 6
-------
APPENDIX E
Formulation of Fluid D
-------
VAkibou,
18 July 2013
Neil L),*wit-li
Pyiumac Cotp
80'> stto.jrnside Dr
McUonough GA 30252
Neil:
Per your question regarding the formulation for the Fluid D (Lot # FD2841) used for
your study wth Barillas dtfopiiaens. I am providing the specifics noted below. Also
attached is n copy fiom USP 29 with the instructions for preparation of Fluid D
f-'ysentialSy I luid O is f luid A {dilution peptone) with added surfactants II ts used
widely m microbiological tabs to keep otganisms suspended in solution during dilution
piucosses etc, ns it has no deleterious ettecf on their viabiiily.
Specifics fo> I of I- 02341;
Reagents.
0.1% (by weight) Difco Proteose Peptone #3 (Product # 211693; lot # G086506)
0.1% ihv weajhl' Fisher Tween 80 {lot# 952224)
Water; Triton Distiiied Water, Lot 05-04-12,
Procedure;
1 wo uiams of each of the two reagents were added to 2 liters of distilled water and
stirred until completely dissolved The pH was adjusted to 7 18 with dilute NaOH (initial
pH was 5 BRl The? solution was then sterilized by membrane filtration.
Please contact me if additional information is needed.
Regards,
•i\
Joseph P Daimasso
President
I •- < l ¦ 5 K< Apex, NC 27602 34 \ tt Ph '
suftiji^yaKibu
'-4930 Fax;
-------
SEPA
United States
Environmental Protection
Agency
PRESORTED STANDARD
POSTAGE & FEES PAID
EPA
PERMIT NO. G-35
Office of Research and Development (8101R)
Washington, DC 20460
Official Business
Penalty for Private Use
$300
------- |