EPA-660/2-75-012
JUNE 1975
Environmental Protection Technology Series
Survival of Pathogens in Animal
Manure Disposal
National Environmental Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Corvallis, Oregon 97330
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development,
U.S. Environmental Protection Agency, have been grouped into
five series. These five broad categories were established to
facilitate further development and application of environmental
technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in
related fields. The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ENVIRONMENTAL PROTECTION
TECHNOLOGY STUDIES series. This series describes research
performed to develop and demonstrate instrumentation, equipment
and methodology to repair or prevent environmental degradation from
point and non-point sources of pollution. This work provides the
new or improved technology required for the control and treatment
of pollution sources to meet environmental quality standards.
EPA REVIEW NOTICE
This report has been reviewed by the Office of Research and
Development, EPA, and approved for publication. Approval does
not signify that the contents necessarily reflect the views and
policies of the Environmental Protection Agency, nor does mention
of trade names or commercial products constitute endorsement or
recommendation for use.
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EPA-660/2-75-012
JUNE 1975
SURVIVAL OF PATHOGENS
IN
ANIMAL MANURE DISPOSAL
By
Stanley L. Diesch
Philip R. Goodrich
Benjamin S. Pomeroy
Loren A. Will
University of Minnesota
St. Paul, Minnesota 55101
Grant #R802205
Program Element 1BB039
21ARS/02
Project Officer
S. C. Yin
Robert S. Kerr Environmental Research Laboratory
National Environmental Research Center
P.O. Box 1198
Ada, Oklahoma 74820
NATIONAL" ENVIRONMENTAL RESEARCH CENTER
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CORVALLIS, OREGON 97330
For sale by the Superintendent of Documents, U.S. Government
Printing Office, Washington, D.C. 20402
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ABSTRACT
A research project was conducted to measure and evaluate the public
health effects of pathogens in beef cattle manure found in the
extended aeration system of waste disposal.
Model oxidation ditches were used in laboratory studies. At simu-
lated summer and winter environmental conditions, determinations
were made of the viability and infectivity of leptospires in wean-
ling hamsters and salmonella in turkey poults. Salmonella was
transmitted by aerosols, but leptospires were not. In refeeding
contaminated feed and slurry contents, salmonella was transmitted
but leptospires were not. Leptospires isolated from the slurry of
the model ditch 17 days post seeding had lost measurable virulence.
Measurements of selected microbial aerosols were made in the vicin-
ity of a field ditch. Bacterial levels of 100-200 total colony-
forming units per liter of air were associated with the beef cattle
population in the housing unit and not with aerosols generated by
the oxidation ditch treatment system.
Studies were made on a model oxidation ditch simulating the field
ditch. The winter temperature conditions (2°-5° C) slowed the
degradation process considerably and high dissolved oxygen was
maintained.
This report was submitted by the University of Minnesota under the
partial sponsorship of the Environmental Protection Agency, Program
Element 1BB039 in fulfillment of Grant No. R802205. Work was
completed as of 9/30/73.
iii
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CONTENTS
Sections Page
I Conclusions 1
II Recommendations 3
III Introduction 4
IV Laboratory Model Pathogen Studies 6
Introduction 6
Materials and Methods 9
Phase I, Leptospiral Studies 9
Phase II, Salmonellae Studies 19
Results and Discussion 28
Summary 52
V Study of the Bioaerosol Production of a Field 54
Oxidation Ditch
Introduction 54
Materials and Methods 58
Results and Discussion 79
VI Model B Oxidation Ditch Studies 94
Introduction 94
Materials and Methods 94
Results and Discussion 96
VII References 119
VIII Appendix Irrigation Study 123
iv
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FIGURES
No. page
1 Schematic of Oxidation Ditch Model with Animal 8
Isolation Facility
la Model Oxidation Ditch with Animal Isolation Facility 11
2 Lundgren Electrostatic Aerosol Precipitator (LEAP) 11
Sampling the Air of Model A
3 Hamster Cage Inside the Animal Housing Unit Above 11
the Model A Oxidation Ditch
4 Sampling Sites of Model Oxidation Ditch - A 15
5 Oxidation Ditch at Rosemount 55
6 Rosemount Farmstead Buildings 56
7 Rosemount Farmstead Aerial Photo 57
8 All Glass Impinger (AGI) 59
9 Lundgren Electrostatic Aerosol Precipitator 60
10 Cylinder for Simultaneous AGI Sampling 64
11 Air Sampling Locations at Varying Distances from 74
Oxidation Ditch
12 Concurrent Sampling Device in Barn 78
13 Concurrent Sampling Device Outside of Barn 79
14 Chemical Oxygen Demand Vs. Time at 20° C for 3 RPMs 99
15 Chemical Oxygen Demand Vs. Time at 10° C for 3 RPMs 100
16 Chemical Oxygen Demand Vs. Time at 2° C for 3 RPMs 101
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TABLES
No. Page
1 Inoculation of MOD Manure Slurry With Leptospires 16
2 Detection of L. pomona MLS in Aerosols 17
3 Inoculation of MOD Manure Slurry With Salmonella 21
4 Determination of Minimum Infectious Dose (MID) of 23
Stock Salmonella typhimurium Inoculated Intra-
peritoneally (I/P)
5 Total Solids: Examples of Build-up 25
6 MOD Physical-Chemical Monitoring Summaries 30
7 Animal Exposure, Manure Slurry, and Aerosol Samp- 31
ling Results of Salmonella/Poult studies Utili-
zing Model Oxidation Ditch - A
8 Salmonella Animal Feeding and Recycled Feed Sample 32
Results
9 Feeding Salmonella typhimurium Contaminated Starter 33
Feed to Poults
10 Leptospira Serotype Pomona MLS: Infection via 34
Variable Routes of Inoculation
11 Infection of Hamsters by Various Routes of Inoculation 34
12 Leptospiral Infection of Hamsters by Various Routes 35
of Inoculation
13 Hamster Exposure to Microbioaerosol of Leptospira 36
Serotype Pomona MLS in an Isolation Chamber
VI
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TABLES (Cont.)
M_ Page
No. —*•*—
14 Infection of Turkey Poults by Various Routes of 37
Inoculation
15 Turkey Poult Exposure to Microbioaerosol of Salmonella 37
typhimurium in an Isolation Chamber
16 Salmonella Aerosol Experiments Conducted in an Iso- 38
lation Chamber
17 Initial Minimum Infectious Dose - Quantitation of 40
Leptospira pomona MLS Virulence
18 Minimal Infectious Dose Studies of March 24, 1972, 41
Leptospiral Ditch Isolates
19 Minimum Infectious Dose Study of Leptospira Serotype 42
pomona MLS Isolated from the Manure Slurry of Model
Oxidation Ditch A, on March 31, 1972. Inoculated
I/P into Hamsters
20 Minimum Infectious Dose Study of Leptospira Serotype 43
pomona MLS Stock Culture. Inoculated I/P into
Hamsters
21 Minimum Infectious Dose of Stock Leptospira Pomona MLS 44
22 Model Oxidation Ditch A - Sampling Turkey Poults. 46
Aerosol Data Taken When Turkeys Present and Absent
23 Comparison of Colony-Forming Units (CFU) Per Liter 47
of Air Sampled With LEAP. All Glass Impingers
(AGI) - Model Oxidation Ditch - (MOD-A) Field
Oxidation Ditch (FOD) at Summer Conditions
24 Results of AGI (All Glass Impinger) Sampling of Model 48
Oxidation Ditch A - Animal Housing Unit Microbio-
aerosol: Rotor Velocity Varied. November 16-17, 1972
vii
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TABLES (Cont.)
No. Page
25 Rosemount Field Oxidation Ditch: Microaerosol (AGI) 49
and Slurry Sampling for Leptospires
25a Model Oxidation Ditch: Animal Housing Unit Micro- 49
aerosol (LEAP) and Slurry Sampling for Leptospires
26 Animal Exposure, Manure Slurry, and Aerosol Sampling 51
Results of Leptospiral/Hamster Studies Utilizing
Model Oxidation Ditch - A
27 Leptospiral Animal Feeding and Recycled Feed Sample 52
Results
28 Comparison of AGI and Casella Counts of Colony- 62
Forming Units, Different Sampling Sites
29 Effect of AGI Collection Fluid on Observed Aerosol 62
Counts
30 Evaluation of Possible Contamination Sources 63
31 Simultaneous Duplicate Air Sampling with a Cylinder 65
32 Aerosol Fluctuations Associated with Time in the 67
West Barn
33 Aerosol Fluctuations Associated with Time in the 67
West Barn
34 Effects of Sampling for Various Lengths of Time in 68
the West Barn
35 Number of Bacteria in Rosemount Ditch 69
36 Preliminary Aerosol Data Taken November 17, 1971 to 73
May 18, 1972 When Cattle were Present
viii
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TABLES (Cont.)
No. Page
37 Preliminary Aerosol Data Taken February 15 to 75
February 24, 1972 When Cattle were Absent
38 Air Sampling at Varying Distances from Oxidation 75
Ditch on February 1, 1972
39 Aerosol Data Taken Weekly May 25, 1972 to June 13, 80
1973 When Cattle were Present - Rosemount
40 Aerosol Data Taken August 9, 1972 to September 13, 82
1972 and March 1, 1973 to March 5, 1973 - Cattle
Absent
41 Bioaerosol Data Taken on March 28, 1973 When Ditch 86
Had Only 6 cm Water with Rotor off
42 Bioaerosol Data Taken at Rotor Site when Rotor off 86
43 Influence of Meteorological Changes on Extramural 88
Upwind Counts
44 Bioaerosol Data Taken when West Barn Smelled of 89
Fuel Odor
45 Morris Sampling with AGI 91
46 Dairy Barn Sampling 93
47 Experiments Conducted on Model B Oxidation Ditch 95
48 Run Number 2, Rotor Speed 180 RPM, Temp. 20° C 102
49 Run Number 3, Rotor Speed 380 RPM, Temp. 20° C 104
50 Run Number 4, Rotor Speed 275 RPM, Temp. 20° C 106
51 Run Number 5, Rotor Speed 180 RPM, Temp. 2° C 108
ix
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TABLES (Cont.)
No
52 Run Number 6, Rotor Speed 275 RPM, Temp. 2° C 110
53 Run Number 7, Rotor Speed 380 RPM, Temp. 2° C 111
54 Run Number 8, Rotor Speed 275 RPM, Temp. 10° C 113
55 Run Number 9, Rotor Speed 380 RPM, Temp. 10° C 115
56 Run Number 10, Rotor Speed 180 RPM, Temp. 10° C 117
57 Aerosols From Spray Irrigation at Rosemount - 125
November 22, 1971
58 Aerosols From Spray Irrigation at Rosemount - 125
April 18, 1972
59 Aerosols From Pig Waste Spray Irrigation - 126
November 8, 1972
60 Aerosols From Spray Irrigation at Rosemount - 127
March 26, 1973
x
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ACKNOWLEDGMENTS
We greatly appreciate the guidance and support of Project Officer,
Mr. S. C. Yin, EPA, Ada, Oklahoma, during the last year of this
project, and during the first year, that of Dr. Mirdza Peterson,
formerly at EPA, Cincinnati, Ohio, but now of EPA, Chapel Hill,
North Carolina.
Excellent advice and counsel was received in the area of aerosol
sampling and aerosol microbiology from Dr. Velvl Greene, Professor
of Environmental Health, School of Public Health, University of
Minnesota. Dr. Frank Martin, Director and Associate Professor of
the Statistics Center, University of Minnesota, contributed in-
formation relative to research design and data interpretation.
We appreciate the efforts of Dr. Loren Will and the many others in
the Department of Microbiology and Public Health and in the Col-
lege of Veterinary Medicine for their interest and dedicated
efforts in research and cooperation in this multidisciplinary
research project.
We are grateful for the many contributions of Mrs. Genevieve
Trombley, Miss Sandra Spier, and for the work of veterinary
medical students James Geistfield and John Lillie and agricultural
engineering students Wayne Anderson and Francis Eikum. The co-
operation of Russell E. Larson and Richard 0. Hegg of the Agri-
cultural Research Service, U. S. Department of Agriculture, Agri-
cultural Engineering Division, is greatly appreciated because they
operated and maintained the field oxidation ditch at Rosemount
with concurrent beef housing studies.
Egon Strauman, Nicholas Horvath, Stephen Welsh and David Scholz in
the Agricultural Engineering Laboratory contributed many useful
ideas and maintained experimental equipment. In addition, they
conducted analyses when project personnel needed additional assis-
tance. We are grateful that so many persons contributed willingly
to move this project to completion.
xi
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SECTION I
CONCLUSIONS
Further refinement of simulation of the oxidation ditch field condi-
tions in the laboratory was accomplished in this two-year project
concerned with survival of pathogens in animal manures. A mechanism
was developed to simulate transmission of pathogenic microorganisms
from the laboratory model oxidation ditch to laboratory animals
housed above the ditch over the wheel-like rotor.
Improved methods of detection of leptospires in use and further re-
finement of the agar plate method were developed.
Quantitation studies indicated salmonella died (decimal reduction)
and leptospires lived or multiplied in the manure slurry environment
of the laboratory oxidation ditch.
In aerosol studies in the laboratory, leptospires were detected in
the air on one occasion and salmonella several times. Leptospires
were not transmitted to hamsters by recycled feed that was gathered
from leptospiral-contaminated manure slurry nor were the hamsters
infected via aerosols. Salmonella was transmitted to turkey poults
by feeding salmonella-contaminated feed and via aerosols.
There is some evidence that virulent leptospires exposed to an aer-
ated manure slurry environment in an oxidation ditch lose their
virulence and ability to infect or cause an antibody response in
weanling hamsters. Further work is needed.
Standard procedures for aerosol sampling at warm and cold environ-
mental temperatures were developed.
The air environment within a field beef-housing unit harbored a rich
bioaerosol of microorganisms. Counts of approximately 100 to 200
total colony-forming units per liter of air sampled were observed in
the year of air sampling. It was concluded that these high levels
were associated with the beef cattle population housed within the
oxidation ditch unit and not with the oxidation ditch treatment sys-
tem.
It was concluded that wet-floor environment in the buildings sup-
pressed aerosol formation. During cleaning periods with a dry
floor, unusually high, potentially hazardous, aerosol levels existed
within the barns.
Pathogens (leptospires and salmonella) were not isolated in the
1
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aerosol sampling of the field project. However, there was no evi-
dence that the animals were infected.
Total plate counts downwind for aerobic organisms were higher (6
versus 1.7 CFU/1) when the animals were present probably due to 'in-
creased wind turbulence. This conclusion was based on the informa-
tion that the coliform counts were still zero downwind when the
cattle were present. Also, the fecal streptococci were only sporad-
ically found and were, on the average, at the lower level of 0.04
CFU/1. None were detected at the 160-cm level. Most of the total
counts were probably aerobic soil microorganisms which had their
source in fields upwind from the sampling site and not from the oxi-
dation ditch.
In Model B laboratory model studies, the 20° C ditch temperature
promoted faster breakdown of the animal waste than the 10° C temper-
ature which, in turn, was more than the 2° C breakdown rate. Rotor
speed had an effect on breakdown only at the 20° C temperature, when
there was a high demand for oxygen by the microorganisms. In a
field oxidation ditch, the information gained in this research could
be used to significantly change the management of this system.
During cold weather, the rotor speed could be reduced with a signi-
ficant savings in energy and maintenance costs. The dissolved oxy-
gen levels can be maintained at a level high enough to promote aero-
bic degradation and minimize odor.
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SECTION II
RECOMMENDATIONS
There is evidence that salmonella and leptospires survive for long
periods of time in the manure slurry environment (this was based on
short-term, 5-day, seeding of virulent pathogens) and it has been
documented that infected cattle may shed leptospires in urine and
salmonella in feces for weeks to months, the slurry becomes an envi-
ronment to maintain viable pathogens. Therefore, we recommend that
manure effluent and sludge containing pathogens (leptospires and
salmonella) should be treated to kill the microorganisms.
There was evidence that leptospires lose their virulence and are
unable to infect when exposed to a manure environment. We recommend
further studies.
A high level of bacteria was found in aerosols of the field housing
unit during cleaning activity; face masks and protective clothing
are recommended.
Because the low temperature studies showed slow rates of degradation,
some field trials with slow rotor speeds should be attempted to see
if low odor levels are maintained. The limit of this recommendation
was tried during the winter of 1973-1974 at the Rosemount field
ditch. The rotor was shut off for the winter. Odors were not
offensive and considerable savings in energy and maintenance were
achieved.
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SECTION III
INTRODUCTION
Today, quality of environment and ecology are of vital concern to
every segment of society. In the U.S. the total volume of animal
waste is estimated at 1.5 million tons of which more than half is
produced by concentrated systems. Animal wastes are of concern in
the abatement of water, air, and soil pollution and the dissemina-
tion of infectious agents from animals to animals and man.
In nature, organisms which are pathogenic to man and animals may be
present in the excreta of domestic and wild animals. More than 150
diseases of animals are transmitted to man. Some of the most signi-
ficant bacterial zoonoses transmitted by animal wastes are salmonel-
loses, staphyloccal and streptococcal infections, tetanus, brucello-
sis, tuberculosis, leptospirosis and colibacillosis.
A literature survey concerned with the recovery of specific microor-
ganisms from urine and feces of inoculated infected animals indi-
cated that 14 microorganisms of specific disease entities were
recovered from the feces of infected cattle (anthrax, brucellosis,
foot and mouth, leptospirosis, psittacosis-ornithosis, Q fever,
rinderpest, tuberculosis, tularemia* and adenovirus, Coxsachie A
and Coxsachie B virus, enterovirus, and reovirus infections) and 7
microorganisms of disease from the urine of inoculated infected cat-
tle (brucellosis, foot and mouth, leptospirosis, Q fever, rinderpest,
tuberculosis and tularemia). The survey excluded most intestinal
diseases (1).
In an extensive literature survey of solid waste/disease relation-
ships, Hanks (2) states that the literature fails to supply data
which would permit a quantitative estimate of relationship between
solid waste and disease. However, he further states that circum-
stantial and epidemiologic information presented in reports does
support the definite relationship of disease and solid wastes. He
further states that in developed countries, incidence, prevalence,
and severity of human infection due to animal fecal wastes are low
from the standpoint of reported outbreaks, but suspicion is that the
amount of disease is actually much higher.
This research project was a continuation of a study made over a
three-year period concerned with the survival of pathogens in ani-
mal manure disposal. The major purposes of the initial three-year
study were: to measure survival of Leptospira pomona and Salmonella
typhimurium in beef cattle manure under specific, measured environ-
mental and physical conditions; to compare methods for measuring,
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detecting and preventing survival of pathogenic bacteria (lepto-
spires and salmonella) in animal manure; to simulate, produce and
maintain field environmental conditions in the laboratory; and to
establish criteria in the hydraulic and structural design of oxida-
tion channels, vertical aerators, other forms of extended aeration
devices, permissible loading rates of solid waste into aeration de-
vices, especially at warm and cold temperatures, and the effect on
survival of pathogens (3,4,5,6) (refer to Final Report, Survival of
Pathogens in Animal Manure Disposal, EP-00302, 1971).
This two-year research was designed to measure and evaluate the
public health effect of specific pathogens in beef cattle manure
found in the extended aeration system of waste disposal and if po-
tential pollution of the common environment of man and animals
occurs.
The purpose of this study was to determine and evaluate (a) the
public health hazards associated with potential pathogen transmis-
sion from the internal and external enviroment and from feed recy-
cled from animal manure disposal during aerobic treatment. Deter-
minations were made of the viability and infectivity of leptospires
and salmonella in aerosols caused by potential mechanical dissemi-
nation of these pathogens from manure of a model oxidation ditch.
Viability was measured in cultural media and infectivity in labora-
tory animals; (b) selected microbial aerosols generated during aero-
bic treatment of animal manures in an oxidation ditch under a beef
confinement housing unit. Environmental samplings of aerosols and
culturing of fecal-borne bacteria were made in proximity to the
field ditch; (c) relationships between temperature, loading rates
and degradation of manure in a model oxidation ditch were made
under controlled environment simulating the field ditch and further
utilized to develop design of the oxidation ditch. This research
was conducted in the laboratory and the field by the research team
from Veterinary Medicine, Agricultural Engineering, and Public
Health, University of Minnesota.
More specific information will be found at the beginning of each of
the following sections.
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SECTION IV
LABORATORY MODEL PATHOGEN STUDIES
INTRODUCTION
Two microorganisms selected for study were leptospires (shed in
urine) and salmonellae (shed in feces) of infected animals. In the
U.S. these pathogens are widespread and are of major public health
and economic importance to both man and animals.
In 1959, 22 states reported a total of 100 cases of human leptospi-
rosis. This figure indicated a 41% increase over the previous year.
In 43 of the 100 cases in which possible source of infection was
noted, 19 or 44% implicated water as the source (7). As many as
100,000,000 leptospires have been reported shed per ml of cattle
urine (8). Infected cattle shed the organisms for periods up to
several months. In the U.S., infections in cattle and swine are
common. The most common serotype infecting cattle is Leptospira
pomona. Leptospires survive for days to weeks outside the live
animal. In recent research, leptospires survived up to 138 days
in the manure of the field-simulated laboratory model of the oxida-
tion ditch.
As an epidemiologist in field study of leptospirosis, the principle
investigator has observed human cases of leptospirosis associated
with aerosol-borne transmission from the urine of infected cattle
in farmers, veterinarians, packinghouse workers, and hunters. The
likely problem of aerosol transmission of leptospirosis from animal
manures has not been defined. Infectivity had not been measured.
In the U.S. , salmonellosis is one of the major communicable dis-
ease problems. There are an estimated 2 million human cases per
year. In 1972 there were 22,151 isolations of salmonellae re-
ported from humans. Salmonella typhimurium was the most frequent-
ly isolated serotype from both human and nonhuman sources, includ-
ing cattle (9). A close correlation of the same serotype isolated
from human and animal cases was documented. From 1963-1967, S.
typhimurium was frequently isolated from individuals involved in
epidemics (10). Control of the ubiquitous salmonellae represents
a major challenge to the fields of public health, veterinary sci-
ence, agriculture, and the food industry. Salmonellosis causes
substantial losses to the livestock and poultry industry. In
recent years, enteritis in dairy and beef cattle caused by salmo-
nellae has increased. Salmonellae are discharged in cattle feces
for extended periods of time. In acute cases in calves, 10,000,000
organisms per gram of feces have been reported (11). The cost is
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estimated at $300,000,000 annually. Salmonellosis is a threat to
everyone as a food-borne disease.
Several researchers have demonstrated that salmonella occurs in the
upper respiratory tract before onset of gastrointestinal signs and
suggest that the transmission of the pathogen might be airborne
(12). In food-processing facilities, contaminated air supplies
leading to processing facilities have been found to be the direct
cause of the finished product contamination.
In experiments utilized to infect mice with mono-dispersed aerosols
of Salmonella typhimurium, the lethal dose was very much smaller
than required by ingestion and about equal to the intraperitoneal
lethal dose (13). In 1957, Moore reported that the conjunctival
route was far more effective than the oral route for producing sys-
temic salmonellae infection in guinea pigs (14).
Extreme growth temperature ranges for salmonellae are found between
7° C and 45° C, with a pH range of 4.1-9.0 and optimum pH of 6.5-
7.5 (12).
Gibson (15) in a review cites a number of references pertaining to
survival. Serotype typhimurium survived 120 days in water and 280
days in garden soil. In England, £. typhimurium survived for 12
weeks when seeded in cattle slurry (16). One project objective was
to study the potential transmission of disease-causing agents (path-
ogens) from cattle manure (solid waste) to animals by the aerosol
route. Potential public health effects of pathogens in the environ-
ment of animal production units which utilize the oxidation ditch
for treatment and disposal of manure wastes were studied. Pathogens
excreted by animals may become aerosolized and incorporated as re-
spired air breathed by animal and man alike. The first method of
approach was to utilize model situations and laboratory animals. A
1:10 scale model oxidation ditch with isolator animal housing unit
was used to simulate the Rosemount Pasveer field oxidation ditch and
animal production facility (Figure 1).
A most important consideration was the host-environment-agent (path-
ogen) interaction in the production of disease. If laboratory ani-
mals died or became ill during the study, it was incumbent to deter-
mine whether or not disease was produced by the pathogen under study
or if there was a cause-effect relationship. The experiments were
started with a pathogen of known virulence as determined by patho-
genicity in laboratory animal studies. A known dose not exposed to
the manure environment should have produced infection in host popu-
lations. Whether or not isolates from the manure were altered by
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00
AIR EXHAUST
FILTER
ISOLATOR / LAB
(air* tight)
HAMSTER CAGE
MODEL OXIDATION
DITCH "A"
ROTOR
Figure 1. Schematic of oxidation ditch model with animal isolation facility
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the environment to which they were exposed was an intriguing ques-
tion.
As described herein, effective control and measurement of the host,
the environment, and the agent in a simulated situation was accom-
plished.
General Materials and Methods Protocol Underlying Phase I (Lepto-
spiral/Hamster) and Phase II (Salmonella/Poult) Studies
Susceptible laboratory animals were housed in open-floored units
situated above the rotor of a 1:10 scale model oxidation ditch
(hood)to simulate beef cattle confinement facilities associated
with field Pasveer ditch operations. The MOD (model oxidation
ditch) manure slurry was inoculated (seeded) with the selected uri-
nary or fecal tract pathogen. Groups of laboratory animals were
exposed for short periods of time (1-4 weeks) to the potentially
pathogen-contaminated aerosol and surface. Small groups of labora-
tory animals were also fed recycled feed (RCF) or exposed to other
experimental conditions. If these situations were microbiologically
hazardous, the laboratory animals should monitor the extent by ex-
pressing disease, infection, or no response as determined serologi-
cally through antibody conversion or by isolation of the pathogen.
Four leptospiral and four salmonella experiments were conducted.
Two of each were at winter manure slurry temperatures (2° C) and the
remainder at summer manure slurry temperatures (20° C). These tem-
peratures had been determined from field data obtained in previous
research.
MATERIALS & METHODS
Phase I Leptospiral Studies
Oxidation Ditch, Model A (MOD-A) —
During the first three years (1968-1971) of this research grant en-
titled "The Survival of Pathogens in Beef Cattle Manure", a 1:10
scale model of the Pasveer field unit located at the University of
Minnesota's Rosemount Experiment Station was constructed. For these
studies the model (MOD-A) was overhauled and slight modifications
were made. Stainless steel rotor and rotor shield were added.
Since the original motor/gear burned out during a trial run, the
motor and speed-adjusting apparatus were replaced by newer, more
durable units. A thermal coil through which coolant circulated was
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attached to the center divider of the model ditch. Manure slurry
temperatures were regulated by altering the temperature of the cool-
ant circulating through this coil. The refrigeration/heating unit
was originally located beneath the MOD-A.
The Housing Unit —
A Fisher Isolator/Lab available in the Department of Veterinary Mi-
crobiology and Public Health was modified into an animal housing/
aerosol study chamber and located over the rotor of the MOD-A. The
airtight unit had a Cambridge absolute filter in the exhaust air-
stream for removal of pathogenic microbes. A stand for this isola-
tor was built so that it could be properly situated atop the model
oxidation ditch immediately above the rotor (Figures la and 2). A
hole was cut from the bottom of the isolator and either a cage ca-
pable of housing 20 hamsters or wire mesh flooring for poults was
placed within the isolator. Food and water were available at all
times in open troughs and waterers. Therefore, the food, water, and
environmental surfaces of animals were continuously exposed to aero-
sol contaminants if present in the ambient air (Figure 3).
Environment Control Room (ECR)^ —
A portion of a laboratory in the Department of Veterinary Microbiol-
ogy and Public Health was utilized to construct an environmental
control room consisting of interlocking insulated panels. The pre-
fabricated sections were purchased and installed by members of the
Department of Agricultural Engineering. This room assured uniform,
ambient, experimental temperature conditions. During 1972, instal-
lation of the environmental control room was completed. The ECR
unit enclosed the Model A oxidation ditch-animal housing unit com-
plex and the instrumentation for MOD-A slurry and environmental data
monitoring. Phase I and II experiments utilizing the MOD-A lab ani-
mal housing unit were conducted within the ECR with the exception of
the winter condition leptospiral studies.
Animal Model (Hamsters)—
A susceptible host to leptospiral infection is the weanling Syrian
hamster. Male, weanling (21-day-old, 20-40 grams) hamsters were
selected as subjects because of favorable colonizing and compatibil-
ity traits. Also, a continuous supply of the Syrian hamster was
assured.
During leptospiral experiments, some hamsters quartered in the ani-
mal housing unit above the MOD-A died. The cause was usually not
10
-------
Figure la. Model oxidation ditch
with animal isolation facility
Figure 2. Lundgren Electrostatic
Aerosol Precipitator (LEAP) sampling
the air Model A
Figure 30 Hamster cage inside the animal housing unit above the
Model A oxidation ditch
11
-------
ascertained since the remaining hamsters rapidly cannibalized their
dead or dying mate, usually leaving little or no material to cul-
ture. Such specimens salvaged were all found negative for Lepto-
spira sp., indicating that death resulted from battle rather than
disease (a "pecking order" is established and weakest hamsters are
killed). All such hamsters or hamster deaths were reported as neg-
ative results.
Agent, Pathogen —
On January 3, 1972, Leptospira serotype pomona MLS cultures were
obtained from Dr. Herman C. Ellinghausen, Jr., National Animal Dis-
ease Laboratory (NADL) at Ames, Iowa. Serotype re-confirmation was
made by two-way absorption test by the Bacteriology Section of the
Center for Disease Control, Atlanta, Georgia. These stock cultures
were maintained and used exclusively in Phase I research.
Agent; MOD-A Slurry Isolate —
During experiment first leptospiral, winter conditions, (L.I.W.),
isolation of leptospires from the previously seeded MOD-A manure
slurry was achieved with the filter-agar plate technique. These
isolates were serologically identified by the Center for Disease
Control as Leptospira serotype pomona MLS.
Minimum Infectious Dose (MID) Virulence Testing —
MID studies were conducted on stock Leptospira serotype pomona MLS
and MOD-A manure slurry isolate cultures. In each study, from 50
to 55 hamsters were used. Five hamsters per dilution/concentration
were injected intraperitoneally with 10-fold reductions from 20 x
10® to 2 x 10° or 2 x 101 leptospires and a control group injected
I/P with sterile diluent. The quantity of organisms was determined
nephelometrically and calibrated with a Roessler N20 standard.
Twenty-one days after inoculation, the hamsters were sacrificed,
blood was drawn, and kidneys were aseptically removed for culturing.
Serum agglutination testing for determination of antibody titer and
kidney culture isolation of inoculated hamsters were used to mea-
sure infection.
Media —
The basic leptospiral culture medium was Ellinghausen1s bovine serum
albumen (BSA) polysorbate 80 (17). Variations of this medium were:
(a) 1% of BSA diluent: a 200-ml stock bovine serum albumin in phos-
phate buffer, plus 800 ml of diluent containing phosphate buffers
12
-------
in water; (b) semisolid agar medium as 1% BSA medium with 2.5 g agar
for a 0.2% agar/1% BSA concentration and (c) agar plate medium as 1%
BSA with 12.5 g agar for a 1% agar/1% BSA concentration. The media
were prepared in the laboratory and incubated at 37.5° C, 29° C, and
room temperature for 24-hour periods and observed for contamination.
Media were stored at room temperature or in a 2° C refrigerator.
Manure and Water —
Beef cattle manure slurry (feces and urine mixture) was collected
from the field oxidation ditch at the University of Minnesota, Rose-
mount Experiment Station. Water was obtained from the Station's well
adjacent to the ditch facility. The water and manure were trans-
ported to the veterinary microbiology laboratory on the St. Paul Cam-
pus of the University where the manure was refrigerated at 0° to 1°
C until used. During the start-up of an experiment, these compo-
nents were mixed in the Model A oxidation ditch in such proportions
as to achieve a manure slurry in terms of microbial, chemical, and
physical processes, the model ditch was operated for one week prior
to inoculation with pathogens. Winter experiments were conducted
at 2° C manure temperature; summer studies at 20° C manure tempera-
ture. During this period, microbiological sampling was begun.
Monitoring the MOD Manure Slurry Physical Stare —
The parameters measured routinely in the Model A oxidation ditch
manure slurry were: pH, dissolved oxygen (D.O.), temperature, and
total solids. Sensing of the MOD-A manure slurry pH, temperature,
and dissolved oxygen (D.O.) was conducted by appropriate electrodes
and field-laboratory equipment. The electronically measured para-
meters were recorded by a Beckman 10" strip chart recorder at six-
hour intervals.
The ambient air temperatures and relative humidity were continuous-
ly recorded on a Serdex Hygrothermograph. Barometric pressure was
recorded daily by laboratory personnel from an aneroid barometer.
Vitamin B Analysis of the Manure Slurry —
Duplicate samples of the Model A oxidation ditch manure were sub-
mitted to WARF Institute, Madison, Wisconsin, for Thiamine HCL
(Vitamin B^) and Vitamin B^ determinations. These two vitamins
are essential for optimal leptospiral growth, according to Dr. H.
Ellinghausen.
13
-------
Seeding of the Model Oxidation Ditch-A Slurry —
During the initial stages of each leptospiral experiment, 37.3 bil-
lion log phase L. pomona MLS were inoculated into the manure slurry
of the Model A oxidation ditch. For each of the four experiments,
there were five daily seedings for a total of 1.9 x 1011 leptospires,
or 1.7 million/milliliter of manure slurry (Table 1). Thereafter,
no leptospires were seeded into this material. A modification of a
previously used procedure for preparation of the leptospiral inocu-
lum was made. Rather than resuspending the leptospires to a nephe-
lometer reading of 25 (Roessler Standard: N=20) which required a
large amount (373 ml) of diluent, the leptospiral organisms were
resuspended after centrifugation with the minimum amount of diluent
which would give a nephelometer reading. The number of organisms
was determined nephelometrically from this suspension, the total
quantity of which was generally about 75 ml, or 1/3 to 1/4 of the
amount previously utilized in seeding the MOD-A slurry. This pro-
cedure was considered more accurate because a direct nephelometer
reading was made on the inoculum, whereas we previously depended
upon proper dilution after nephelometric determination to quantify
the inoculum.
Sampling, MOD-A Manure Slurry —
Pipettes were used to aspirate manure slurry samples from MOD-A at
six sites, three effluent (top) and three sludge (bottom), (Figure
4). Two methods of culturing for leptospires from these slurry
samples were incorporated during the studies. Only the agar plate
technique was utilized throughout all four leptospiral experiments.
In the tube dilution technique, approximately one milliliter of ma-
nure was pipetted into a tube of 1% BSA diluent. Ten-fold serial
dilutions and transfer to Ellinghausen's BSA was made of this ma-
terial which was then incubated at 29° C and observed periodically
for 10 weeks by darkfield microscopy for the presence of leptospires.
This procedure was not efficient for isloation of leptospires from
highly contaminated material such as the manure used in these ex-
periments.
In the agar plate technique, six drops of manure slurry were placed
in the center of a Millipore, .22-n pore size, type GS filter which
was on the surface of BSA agar plate. The leptospires migrated
through this filter to establish growth in the agar medium. These
plates were incubated at 29° C and examined for evidence of cul-
tural detection of leptospiral survival and has allowed continuous
14
-------
®-SWABS IMMERSED 2.54cm. INTO THE SLURRY
O -SWABS IMMERSED IN THE SLURRY, ON THE BOTTOM
ANIMAL HOUSING UNIT
OO8*
r.
S^
L no**
i i
j
i i
i i
I ! i
- 1
i
i
1
1
•
— — «4
PHTOF
Figure 4. Sampling sites of model oxidation ditch-A
-------
Table 1. INOCULATION OF MOD MANURE SLURRY WITH LEPTOSPIRES
Experiment
Number
Date of Inoculation in
1972
L.I. Winter
L.I. Winter
L.3. Summer
L.3. Summer
March 7
March 8
March 9
March 10
March 13
May 30
May 31
June 1
June 2
June 3
July 26
July 27
July 28
July 31
August 1
October 10
October 11
October 12
October 13
October 14
leptospiral detection in the manure slurry until the termination of
each experiment. Sampling the manure of the model oxidation ditch
was conducted three times a week (Monday, Wednesday, Friday).
Aerosol of (MOD-A) laboratory animal housing unit —
A Lundgren Electostatic Aerosol Precipitator (LEAP) was selected for
sampling the ambient air of the model housing facility in preference
to the All Glass Impinger (AGI) which had been proposed. This choice
was reasonable since the LEAP samples up to 1,000 liters of air/min-
ute as opposed to 12.5 liters/minute by the AGI. Also, leptospires
were detected in samples collected with the LEAP but not by the AGI
in a pre-experiment study (Table 2). This experiment was conducted
in an isolator unit in our laboratory not associated with the
16
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Table 2. DETECTION OF L. POMONA MLS IN AEROSOLS
Method of Detection
Experiment Number
1234
1% BSA Liquid Media
1% BSA Agar Plates
Lundgren Electrostatic
Air Precipitator
Hamster (Serum agglu-
tination)
Kidney Isolation
Model A oxidation ditch. An attempt was made to saturate an en-
closed environment with an aerosol of L_. pomona MLS. Within the
isolator were located the following:
1. one nebulizer for production of homogeneous air dispersion
of L^. pomona MLS.
2. two AGI impingers for sampling the air.
3. five liquid BSA media in petri dishes.
4. five agar BSA plates.
5. five male weanling hamsters.
6. A LEAP mechanism capable of sampling the entire air con-
tent of the isolator was attached to the unit.
Furthermore, because detection was essentially by darkfield micro-
scopy, the larger volume of air and, thus, the concomitantly greater
bioload sampled by the LEAP provided greater sensitivity than the
AGI. The fluorescent antibody technique (FAT) was not utilized for
these studies. The FAT is a time-consuming detection method when
compared to the filter-agar plate technique which employs darkfield
microscopy successfully for detection of viable leptospires whereas
17
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the FAT does not indicate viability.
Utilizing the same arrangement as described above with the exception
of AGI and liquid medium plates, another study was conducted with
the L,. pomona MLS on March 21, 1973.
Effect of MOD-A Rotor Speed upon Aerosol Production —
On November 16 and 17, 1972, a series of aerosol samples were col-
lected from the MOD-housing unit with the Lundgren Electrostatic
Aerosol Precipitator (LEAP) and AGI. Each sample was collected at
various ditch rotor speeds designated as "slow" (normal operating
speed), "fast", and "fastest." The normal operating rotor speed
during all of the experiments was determined empirically as that ro-
tor speed which came closest to propelling the manure slurry at
45.72 cm/second which was not at the greatest rotor speed, but that
designated "normal" which was a relatively slow speed.
Refeeding Experiments —
In order to study the effects of recycling pathogen-contaminated ma-
nure to animals, refeeding experiments were conducted with hamsters.
Reclaimed solid wastes from the Rosemount pilot field oxidation ditch,
mainly corn, was utilized in our studies. Three hundred cubic centi-
meter volumes of recycled corn were wrapped in double-layer, 4-thick-
ness cheese cloth which was closed, tied, and pressed to eliminate
excess liquid. Thus prepared, the recycled feed (RCF) was frozen and
stored at 0° C for use as required. One sack of RCF was suspended in
the stream of the model oxidation ditch for successive one-week pe-
riods, after which time the leptospiral-contaminated content was fed
to a new group of five male weanling hamsters not housed in the unit.
Following consumption of the RCF (usually one day), these hamsters
were fed commercial hamster feed.
Various Routes of Inoculation Studies —
Since laboratory and domestic animals are exposed by various routes
while held in confinement facilities, testing of other than respira-
tory routes of infection was necessary. Therefore, experiments were
conducted utilizing hamsters. One droplet of a suspension of L_.
pomona MLS was inoculated topically onto nares, oral cavity, or con-
junctival surface. The suspensions were estimated to contain from
1 x 103 to 2 x 10^ L. pomona MLS per droplet.
Infection was measured by both the microscope agglutination (MA) test
of serum and by kidney culture techniques.
18
-------
Three variable route-of-inoculation studies utilizing L^ pomona MLS
were conducted on male weanling hamsters to determine either infec-
tiousness or the minimum dose required to cause infection. In one
experiment, pelleted rat chow (feed) was inoculated with pathogenic
leptospires and fed to susceptible hamsters.
PHASE II. Salmonellae Studies
During the period of November 15, 1972, to December 15, 1972, the
MOD-A and animal housing unit was overhauled and refitted to accom-
modate week-old turkey poults. A poultry feeder, water trough,
brooder heater, and light were installed within the housing unit.
The gear unit on the motor was rebuilt as was the ditch rotor and the
rotor mount. The cooling system was modified to exclude coolant cir-
culation through a diffuser in the environmental control room since
warm, ambient air conditions were required for this age poult as op-
posed to the cool temperatures preferred for hamsters. The system
continued to circulate coolant through the MOD-A to maintain and
regulate slurry temperature.
The change-over from hamsters to poults created several difficulties
which required time and experimentation to rectify. Young poults
were very susceptible to environmental stress; thus both morbidity
and mortality were required to obtain results and additional samples
were necessarily required because of the increased number of animals,
and more samples were taken from each bird. More labor and media
were required to follow a specimen through the salmonella culture
procedures than was the case for leptospiral detection and identifi-
cation.
Procedural Protocol —
Thirty-five poults, one-day-old, were purchased each week and grown
for approximately seven days to "week-old" poult stage. At one week
of age, this group was ideally to be separated into the following
categories:
Number Proposed:
Week-old Poults Use
20 Housed over MOD
7 Fed contaminated "starter" feed
5 Control
3 Expected "normal" mortality (10%)
19
-------
The actual numbers in the Salmonella poult experiments ranged as fol-
lows:
Number Utilized:
Week-old Poults Use
3-24 Housed over MOD
3-10 Fed contaminated starter
0-10 As controls
0-18 Mortality
Each salmonella experiment was conducted for a duration of about
five weeks: one initial week for stabilization of the MOD-A, five
days for seeding the manure slurry, and a final thirty days for ex-
periment run-time (Table 3).
One obstacle which was not overcome concerned the timing interval for
purchase of poults. Each week, 35 day-old poults were purchased from
the second largest turkey hatchery in Minnesota. This producer could
not assure an evenly spaced interval between poult hatches. The sup-
ply problem necessitated fluctuating poult ages and exposure duration
which potentially complicated statistical evaluation.
Poults —
Discussions were held to decide whether mice or poults should be the
laboratory animal model of choice for the salmonella studies. The
grant proposal was designed for poults but some practical considera-
tions, such as their size with increasing age, comparative suscepti-
bility to the agent, freedom from congenital or neonatal salmonello-
sis, and seasonal supply caused concern whether they would be suit-
able. Poults were selected.
A literature search disclosed that susceptible poults over 2 to 3
days of age would become infected with salmonellae, but changes of
fatal infection greatly diminished after this age. With this know-
ledge, the best sites for cultural isolation of salmonella from the
poults were determined. Routine sampling was conducted of the liver,
caecal junction, and oral cavity on all experimental poults. Once
the poults were dead or sacrificed, tongue, liver, or caecal tissue
was dissected from the poult, alcohol drenched, flamed, cut up, and
placed into Selenite Brilliant Green Sulfadiazene (SBGS) and followed
through as described.
Day-old poults were either purchased" from Moorhouse Turkey Hatchery,
Inc. or obtained from Dale Peterson Hatchery when Moorhouse could
20
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Table 3. INOCULATION OF MOD MANURE SLURRY WITH SALMONELLA
Experiment
Number
Date of Inoculation in
1973
S.I. Winter
S.2. Winter
S.3. Summer
S.4. Summer
Restart:
January 9
January 10
January 11
January 12
January 15
March 20
April 16
April 17
April 19
June 17
June 19
June 20
June 22
June 23
July 10
August 7
August 9
August 10
August 13
August 14
August 24
September 5
September 7
September 10
September 13
not supply the required number. Cloacal swabs were taken from all
35 week-old poults of that week's hatch before subjecting them to
experimental study.
Salmonella typhimurium stock cultures used in Phase II had been stored
on trypticase soy agar (T-Soy) slants and transferred at 3-6 month in-
tervals. Throughout the five years of study, this cultural line was
confirmed repeatedly both biochemically and serologically as being
21
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somatic antigen: Group B, 1, 4, 5, 12, and flagellar antigen: Phase
1 (i) and Phase 2 (1 complex).
Salmonella Sampling —
The general protocol for isolation of Salmonella has been: sample
-> Selenite Brilliant Green Sulfadiazene Broth (SBGS) and Brilliant
Green Bile Broth (BGB) 37° C/24 hrs -* Brilliant Green Sulfadiazole
(BGS) Plates and Xylose Lysine Desoxycholate (XLD) Plates 37& C/24
hrs -*• fish colonies to Triple Sugar Iron (TSI) agar slants 37°
C/24 hrs ->• biochemistry (dulcitol, salicine, urease, lysine decar-
boxylase -»• serotyping. Specimens or samples from other than the
manure slurry follow the same flow scheme except that only SBGS en-
richment and only one of the selective plates are utilized.
It is interesting to note that during the first three years of this
project, prior to incorporation of poults to the experiments, BGB
was the most successful enrichment medium. Thus far, less than one-
tenth of the Salmonella positive samples were grown in BGB. The
possibility that the medium was faulty was investigated, but also
consider that extraneous enteric microorganisms contributed by the
poults is competitive with and selected in preference to the sal-
monella in this medium.
Minimal Infectious Dose —
Four MID experiments were conducted to quantitate the virulence of
the S^. typhimurium which was being maintained on T-soy agar slants.
Thes¥ were transferred to GN (Hadjna) broth, incubated at 37° C for
various periods of time, and diluted. These culture dilutions were
plated on Brilliant Green Sulfadiazine (BGS) agar and a 1-cc aliquot
of each dilution was injected into test birds. Plate counts gave a
measurement of the quantities injected. The broth cultures were not
sonicated or shaken to break colony clumps into individual bacteria
(Table 4).
Manure and Water —
MOD-A manure slurry was that beef cattle manure slurry obtained from
the Rosemount FDD plus added well water from Rosemount for a proposed
initial total solids content of 5,000 to 10,000 mg/1 at the start of
each MOD-A study (Table 5 indicates the actual initial total solid
values). All manure and water were cultured for Salmonella or Lepto-
spira, depending on the experiment prior to use in the MOD-A.
22
-------
Table 4. DETERMINATION OF MINIMUM INFECTIOUS DOSE (MID) OF STOCK
SALMONELLA TYPHIMURIUM INOCULATED INTRA-PERITONEALLY (I/P)
Experiment No. 1 (1-2)
Group
Number
1
2
3
4
5
Control
Concentration in
Inoculum Administered
17
17
17
17
17
x 104
x 106
xlO10
xlO14
xlO1?
Initial #
of Poults
10
19
19
20
21
11
Number
Died
1
3
0
4
16
0
Number
Positive
7
14
18
20
21
0
All poults were cloacal swab culture negative for S_. typhimurium.
Experiment No. 2 (1-2)
Group No.
1
2
3
Control
No. of
Poults
6
9
4
8
Dose I/P
98 x 102
98 x 105
98 x 1011
0, Broth Only
Post -Mortem
No. Positive*
1 oral
8
4
0
Anti-Mortem
Positive (Oral)/
Population
1 Oral 1/6
Liver 0/6
Caecal 0/6
3 Oral 7/9
Liver 5/9
Caecal 6/9
4 Oral 4/4
Liver 3/4
Caecal 3/4
0 All 0/8
*0rgans
All poults were cloacal swab culture negative for S_. typhimurium.
1.0 cc inoculum I/P.
23
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Table 4. (continued) DETERMINATION OF MINIMUM INFECTIOUS DOSE (MID)
OF STOCK SALMONELLA TYPHIMURIUM INOCULATED
INTRA-PERITONEALLY (I/P)
Experiment No. 3 (1-3)
Group No.
1
No. of Poults Dose I/P No. Positive
10
80 x 1018
10 Oral 10/10
Liver 5/10
Caecel 9/10
8 80 x 1016 8 Oral 8/8
Liver 5/8
Caecel 6/8
All birds were positive orally antimortera. Cloacal swabs negative.
1 cc injected I/P.
Experiment No. 4 (1-4)
Group No. of
Number Poults
1
2
3
4
5
7
15
14
12
12
12
Control birds
Number Culture
S. typhimurium Sites
21
21
21
21
21
x
x
X
X
X
102
103
104
105
106
3
1
1
2
5
1
2
0
2
5
2
1
1
5
5
Death
0
1
0
0
0
- all negative
All cloacal swabs negative. 1 cc injected I/P.
Monitoring —
The MOD-A manure slurry was monitored for pH, D.O., and temperature
as described in Phase I.
24
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Table 5. TOTAL SOLIDS: EXAMPLES OF BUILD-UP
Date
Period
Prior to
and Includ-
ing 2
weeks of
L.l.W.
Control
Period to
and Includ-
ing 1 Week
of S.l.W.
1-14-72
1-28-72
3-3-72
3-27-72
12-7-72
1-3-73
1-17-73
Total Solids
(mg/1)
4,567
5,466
15,868
18,853
8,211
9,499
12,017
Seeding —
A loop of S_. typhimurium stock culture was removed from the T-Soy
slant to GN (Hadjna) broth which was incubated at 37° C for 8 to 24
hours. The broth culture was adjusted to 30 nephelometer units on
a Nephlo-colorimeter using a Roessler 20N standard as reference.
This material was then used for seeding by placing the required
amount of culture in 75 ml of buffered saline, then pouring into the
MOD-A manure slurry.
Sampling —
During sampling of the MOD-A for Salmonella early in the first Sal-
monella experiment, Calgiswab (tipped applicators) were taped to the
sides of the MOD-A channel walls with the tips of six swabs submerged
in the effluent (top-most 2.54 cm of manure slurry) and six in the
sludge portion (bottom-most 2.54 cm). Thus, there were two swabs at
each of six sites, three effluent and three sludge, as indicated in
Figure 4. These swabs dissolved in broth culture as the result of
chemical and microbial action. Thus, sampling began on the MOD-A
slurry at the prescribed positions with medicine droppers. The
dropper sample was 1-5 ml of manure slurry.
25
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Microbioaerospl Sampling of the MOD-A Animal Housing Unit—
Sampling of the MOD-A poultry house air was conducted. Total bac-
terial count, fecal streptococci, coliform, and salmonella determi-
nations were made of the microbial profile as described in Section V,
Field Aerosol Studies. The Lundgren Electrostatic Aerosol Precipita-
tor (LEAP), sampling 1,000 liters of MOD-A poultry housing air per
minute, was utilized throughout these studies. The poultry housing
unit had an air volume of 11.1 cubic feet or 315.2 liters. Theoreti-
cally, the LEAP sampled the ambient poultry air approximately three
times every minute. The collecting fluid used was 0.1% peptone. The
samples were analyzed for total coliform, fecal streptococci, and
salmonella organisms.
The LEAP sampled airborne particles were deposited onto a thin,
moving film of liquid collecting medium. Air entered the sampler
through a nozzle that was calibrated for airflow rate up to 1,000
liters of air per minute. It then flowed over a rotating disc to
which is applied a high voltage. The air was then drawn radially
through this high potential, up to 20 kilowatts across the plate to
disc spacing, precipitating particles onto the collection disc. Col-
lecting liquid was supplied by a pump to the center of the collection
disc. Centrifugal force caused the liquid to flow in a thin, contin-
uous film outward across the disc to a pickup dish where the parti-
cles were washed. The sample liquid was pumped from the the pickup
dish to a container. From the LEAP samples taken at the MOD-A, 0.1
ml of properly diluted collecting fluid was placed on a plate count
agar (PCA) and incubated for 48 hours at 37° C. All colonies were
then counted.
Likewise, 0.1 ml of collecting fluid from the LEAP sample was placed
on Levine Eosin Methylen Blue Agar (L-EMB) and colonies that were
large, pigmented, gram negative reds, and fermented lactose with the
production of gas on TSI were counted.
Fecal streptococci determination was made by plating collecting
fluid on M-Enterococcus agar (M-ENT). Characteristic red colonies
produced on M-ENT were also tested for catalase negativity and the
production of a black color on Bile Eschelin Agar (BEA).
The presence or absence of salmonella was analyzed in samples taken
with the LEAP by placing 2 ml of collecting medium into enrichment
broth of Selenite Brilliant Green Sulfadiazene (BGS) and incubating
at 37° C for approximately 24-28 hours. From each broth solution
a loopful was streaked onto Brilliant Green Sulfadiazene (BGS) agar
and Xylose Lysine Desoxycholate (XLD) agar and incubated for 48 hours
26
-------
at 37° C. The characteristic reactions were classified as salmo-
nella. In addition, 0.1 ml of collecting fluid was placed directly
onto BGS and XLD agar to further check for the presence of salmonel-
la.
Aerosol experiments —
Initially, two experiments were conducted to test aerosol production
and microbioaerosol sampling ability. Later, five experiments were
conducted to ascertain quantitatively the infectivity of Salmonella
typhimurium aerosols. These experiments were conducted in an isola-
tion box identical to that converted for the MOD-A animal housing
unit complex. The salmonella broth cultures were nebulized with a
DeVilbiss Model 640 Clinical Glass Nebulizor. Th^ aerosol thus pro-
duced was sampled both by the LEAP connected to the isolator by a
galvanized metal 90° elbow and by open petri dishes (fall plates)
containing selective media (XLD or BGS) placed on the floor of the
isolator or on top of the cage within which the poults may or may
not have been placed.
During some of the aerosol experiments as well as other experiments
described below, the tongue-pharyngeal region was investigated as a
possible sampling site for the detection of salmonella infection.
Route of Inoculation —
A study was conducted with 54 poults to determine oral, ocular, and
nasal routes of susceptibility to S^. typhimurium infection. Culture
suspensions were placed by dropper onto these sites and streaked onto
BGS plates for quantitation.
Recycled Feed (RCF) —
Two types of studies were conducted to determine the effect of feed-
ing pathogen-contaminated RCF to poults. One series of experiments
was conducted as part of the four MOD-A experiments. Poult starter
feed wrapped in cheesecloth was submerged in the manure slurry of
the MOD-A for varying periods of time (one to several days), after
which it was cultured for salmonella and fed to week-old poults. The
design of this series was an attempt to simulate the feedxng to do-
mestic animals of manure slurry solids obtained from field oxidation
ditches. This feeding practice is currently occurring to some extent
at universities and in private animal industry throughout the U.S.
Initially during control experimentation, the slurry solids from the
Rosemount oxidation ditch were fed to the poults as had been done
with hamsters during Phase I research. The poults did not accept
27
-------
this material, however, and the "starter" ration was substituted
as an alternative to the recycled feed.
In another experiment, 120 one-week-old poults in groups of eight
were fed 2.86 kgs. of "starter" ration which was contaminated with
20 ml of various concentrations of _S. typhimurium. After the con-
taminated starter feed had been consumed, the poults were fed non-
contaminated starter. All poults were sacrificed and cultured one
week after being fed the salmonella-contaminated rations.
Oral dosing studies were also conducted. Eight week-old poults were
placed in each of two groups. Two poults from each group were sacri-
ficed on each of the first four days. Each group had been orally
inoculated with a different dose of £. typhimurium (154 x 10? and
154 x 102).
RESULTS AND DISCUSSION
The Phase I and II endeavor throughout the two-year period of Grant
No. R802205 was to study selected microbial pathogens and hosts in
association with an operational model oxidation ditch (MOD). The
conditions created for study were "practical worst care" situations,
that is, more severe conditions could have been established but
those created in the laboratory were similar to those expected in
the worst cases described in field situations. The MOD was a 1:10
scale model of a field unit. The winter (2° C) and summer (20° C)
MOD slurry temperatures were derived from data obtained from the
field unit. Likewise, the total solids (T.S.) parameter was deter-
mined from provious knowledge of oxidation ditch operation and func-
tion. Most important perhaps, was the determination of the quantity
of pathogen to be added to the MOD manure slurry which was based upon
known excretion rates. Calves may shed 10,000,000 salmonella per
gram of feces, and up to 100,000,000 leptospires per ml are excreted
in urine of infected, shedding animals.
A total of 1 x 10- Salmonella typhimurium were seeded at the begin-
ning of each salmonella experiment resulting in a concentration of
8.7 x 10-* organisms/ml of MOD manure slurry if a reduction of micro-
organisms did not occur. However, there was a decimal reduction
rate of ;S_. typhimurium in the slurry as evidenced by finite survival
duration which varied with MOD conditions (6). Furthermore, the
figures utilized are, at best, approximations which may flucuate by
an undetermined amount. The best estimation would be + 50% at the
time of nephelometer determination since the microorganisms are not
necessarily monodispersed in solution but, rather, may form aggre-
gates. The cultural measure used to calibrate the nephelometer may
28
-------
lack accuracy. It detects colony-forming units in addition to indi-
vidual organisms.
The same consideration was true also for Leptosp ir a serotype pomona
MLS seeding. There is a tendency toward clumping in cultural media.
After the initial seedings, 1.7 x 10 L_. pomona MLS were expected
(calculated) per milliliter of manure slurry. A decimal reduction
time existed for this pathogen in manure slurry, also based on sur-
vival time.
Cultural monitoring for pathogens, salmonella or leptospira, gave
the only indication of the presence of either. The critical factor
for determining the microbiological health hazard associated with
the MOD was the presence or absence of pathogen, and since the quan-
titation of seed numbers only verified that conditions were "worst
practical," it was necessary to reseed the slurry whenever the micro-
organisms could no longer be detected by methods utilized.
The manure slurry physical-chemical data summary for each experiment
is given in Table 6. The total solids increased significantly during
each experiment, as illustrated by examples in Table 5. The altera-
tion of the MOD manure slurry environment was obviously unavoidable,
but similar to real case situations. Survival of _S_. typhimurium
and/or _L. pomona MLS may be decreased as compared to previous re-
search conducted without the continuous introduction of extraneous
organic material. However, detection of survival may have been de-
creased also.
The detection of either L_. pomona MLS or J3. typhimurium was not nor
could it have been absolute. The methods for cultural detection of
microbiological pathogens must be altered with each situation until
a best method(s) is developed. Negative results regarding the manure
slurry, feed animals, and aerosol to which cultural detection methods
were applied in this research cannot necessarily be considered such
in the absence of positive results (Table 7). On at least one occa-
sion during each of the four experiments (5.I.W., week 3; S.2.W, week
6; S.3.S., weeks 4 and 5; S.4.S week 6), poults became infected with
^. typhimurium despite lack of cultural evidence of the pathogen's
presence in the RCF (Table 8). In fact, the probability of culturing
S. typhimurium from the starter feed was low. A study was conducted
to determine what concentration of S_. typhimurium in poult starter
feed was necessary for poult infection and/or cultural detection
(Table 9). Reasons for the negative results are conjectural: rapid
die-off of the microorganisms in the feed; absorption of the micro-
organism into the feed where it becomes isolated from the culture
media; or interaction of feed and media to either neutralize growth/
29
-------
Table 6. MOD PHYSICAL-CHEMICAL MONITORING SUMMARIES
Exp.
L.l.W.
2.W.
3.S.
4.S.
S.l.W.
2.W.
3.S.
4.S.
Total Solids
(mg/1)
5,466
5.655
4,688
13,723
9,499
10,754
8,277
19,287
6,560
D.O. (mg/1)
x; | range
0.5 1.4-01.4
4.4 0.0-06.8
2.6 0.0-10.2
1.1 0.0-06.0
4.4 0.0-24.0
2.5 0.0-09.8
Probe not
Functional
0.1 0.0-01.0
2nd Seeding
Temperature (°C)
x;
02.0
02.0
20.0
19.8
03.9
01.4
16.9
20.7
range
02.0-02.0
04.0-08.0
18.0-27.0
15.0-27.0
00.0-09.0
00.0-06.0
14.0-21.0
16.0-30.0
PH
x"; I range
6.5 6.3-6.7
7.2 6.4-7.6
6.3 4.5-7.7
6.4 5.0-6.9
7.2 6.0-8.1
7.2 5.8-7.7
7.7 5.1-8.4
7.2 5.8-8.0
reproduction or produce a toxic substance deleterious to growth and
detection.
In those instances where the RCF was culturally negative but poults
became infected, correlation was made with the MOD-A manure slurry
cultural results for _§_. typhimurium. If the slurry was positive for
jS_. typhimurium, it is assumed the presence of the pathogen was iden-
tified but transmission to the poult was not measured or may not
have occurred.
An alternative is to suspect that the poults themselves may have har-
bored j^. typhimurium and expressed infection when stressed during the
conditions of the studies. Throughout the course of the research
with poults occasionally salmonella would be detected on pre-commit-
ment to experiment by cloacal sample culture. These instances dis-
closed Arizonae spp. or non-S_. typhimurium salmonellae. Therefore,
whenever S_. typhimurium was detected in a poult, it had been intro-
duced to the poult by the experimental procedure through feeding,
exposure to aerosol, or inoculation by other routes.
Although this research was proposed to test the health hazard asso-
ciated with the potential aerosolization of pathogens from an oxida-
tion ditch (Pasveer) system, it was recognized that animals reared
in the area of such a facility would be exposed to more than simply
the respiratory route for infection. Microbioaerosols, if produced
by the operation of an oxidation ditch, potentially could settle on
30
-------
Table 7. ANIMAL EXPOSURE, MANURE SLURRY, AND AEROSOL SAMPLING RESULTS OF SALMONELLA/POULT
STUDIES UTILIZING MODEL OXIDATION DITCH-A
Experiment
Slurry
S.l.W.
0. Ditch
Check
Aerosol
Slurry
S.2.S.
0. Ditch
Check
Aerosol
Slurry
S.3.S.
0. Ditch
Check
Aerosol
Slurry
S.4.S.
0. Ditch
Check
Aerosol
Control
-
0/19
0/0
—
-
0/15
0/6
—
-
Poults
Not Avail
-
-
0/20
0/8
•*-
1
+
0/8
0/2
-
+
0/20
0/7
ND
+
7/15
0/7
-
+
0/18
0/5
~
2
+
0/11
0/3
—
+
0/20
NA
+
+
1/19
0/6
-
+
0/20
0/6
«_
3
+
1/3
0/1
-
+
0/20
0/4
—
+
1/13
0/3
+
+
0/19
0/5
™ ™
4
+
0/20
0/5
ND
+
0/19
0/5
-
+
0/15
1/10
-
+
0/24
0/5
Ml
5
-
0/20
0/8
ND
+
0/20
1/4
-
+
3/20
0/5
ND
+
0/17
0/3
«•»
6
+
0/19
0/6
-
+
5/21
0/5
-
+
0/20
0/6
ND
+
0/16
0/8
"
7
-
0/19
0/7
-
+
5/18
0/4
+
ND
ND
ND
ND
ND
ND
ND
ND
8
ND
ND
ND
ND
-
0/10
0/4
—
ND
ND
ND
ND
ND
ND
ND
ND
Total
1/119
0/41
20/163
1/39
12/102
1/37
0/134
0/40
-------
Table 8. SALMONELLA ANIMAL FEEDING AND RECYCLED FEED SAMPLE RESULTS
Experiment
S.l.W.
Feed
Check
RCF
S.2.W.
Feed
Check
RCF
S.3.S.
Feed
Check
RCF
S.4.S.
Feed
Check
RCF
Control
ND
0/9
ND
0/6
ND
Poults
NA
ND
0/8
1
ND
0/2
(AP)
0
0/7
0/7
(AP)
0
0/5
0/7
(AP)a
0
0/6
0/5
(AP)
2
0/7
0/3
+
NA
NA
(AP)
0
0/6
0/6
+
0/7
0/6
(AP)
3
2/4
0/1
(AP)
-
0/8
0/4
+
2/8
0/3
+
0/19
0/5
+
4
0/7
0/5
-
0/10
0/5
(AP)
"•
1/10
1/10
(AP^
—
0/6
0/5
-
5
0/6
0/8
-
0/8
1/4
+
1/10
0/5
(AP)
—
0/7
0/3
(AP)
6
0/8
0/6
(AP)
—
3/8
0/5
(AP)
""
0/9
0/6
—
4/8
0/8
(AP)
7
0/8
0/7
—
2/6
0/4
+
ND
ND
ND
ND
ND
ND
8
—
ND
ND
10/10
0/4
+
ND
ND
ND
ND
ND
ND
Total
2/40
15/57
4/48
4/53
CO
NJ
AP indicates aerosol positive for salmonella.
-------
Table 9. FEEDING SALMONELLA TYPHIMURIUM CONTAMINATED STARTER TO
POULTS
Experiment No. 1 (F-l)
Group
1
2
3
4
5
No. S.
25.5 x
25.5 x
25.5 x
25.5 x
25.5 x
typhlmurium
10°
10*
108
1012
1016
Results
Liver
0/8
0/8
0/8
0/8
0/8
Caecal
0/8
0/8
0/8
0/8
0/8
Cloacal
0/8
0/8
0/8
0/8
0/8
2.86 kgs of poultry starter plus 20 ml of a known concentration of
S. typhimurium were mixed in a plastic bag.
feed, water, and other exposed surfaces in the ambient environment;
that could contaminate wounds or ocular, oral, and/or nasal membranes,
Collateral research was conducted during the grant period to study
whether or not infection with L_. pomona MLS or S_. typhimurium could
be induced by pathogen contamination of feed, or eyes, nose, and/or
mouth and, if so, at what concentrations or dosage. Kidney culture
and/or serologic evidence of infection was induced in male weanling
Syrian hamsters by L_. pomona MLS when applied to eyes, nose, or
mouth but not in feed (Tables 10, 11, and 12). Efforts to infect
hamsters when exposed to virulent aerosols of leptospires failed
(Table 13) . Week-old poults became infected with S_. typhimurium
when applied to these three sites (Table 14) but not when fed to
the poults on feed (Table 9).
The higher dose rate was the only one to consistently induce infec-
tion in the poults. Comparison of these oral dosing rates with the
contaminated RCF dose rates and the route-of-inoculation rates indi-
cates that the RCF-S_. typhimurium dosages were above and below the
oral infectious dose in the oral dosing experiment. However, death
rate of the salmonella in this situation is not known and some fac-
tor may have played a role causing the nil infection rate.
33
-------
Table 10. LEPTOSPIRA SEROTYPE POMONA MLS: INFECTION VIA VARIABLE
ROUTES OF INOCULATION
Experiment
1
2
Totals
No. of
Leptospires
1.1 x 106
1.0 x 103
1.0 x 104
1.0 x 105
1.0 x 106
1.0 x 107
Control
Positive/Possible
Ocular
1/4
0/3
0/3
0/3
0/3
0/3
0/3
1/22
Nasal
3/3
0/3
0/3
1/3
2/3
3/3
0/3
0/21
Oral
1/3
0/3
0/3
0/3
0/3
0/3
0/3
1/21
Feed
ND
0/3
0/3
0/3
0/3
0/3
0/3
0/18
Table 11. INFECTION OF HAMSTERS BY VARIOUS ROUTES OF INOCULATION
Route
Nasal
Ocular
Oral
Control
No. of
Leptospirea
1.1 x 106
1.1 x 106
1.1 x 106
Saline
MA
1234
+ + + 0
+ - - -
+ - - 0
_ _ _ _
Cultural
1234
+ + + 0
+ _ _ _
+ - - 0
_ _ _
34
-------
Table 12. LEPTOSPIRAL INFECTION OF HAMSTERS BY VARIOUS ROUTES OF
INOCULATION
No. of
Leptospires
2 x 104
2 x 106
2 x 108
Control - BSA
Positive/Possible
Ocular
0/3
0/4
0/4
Nasal
0/4
0/3
0/4
0/5
Oral
0/3
0/4
0/4
Another set of experiments was conducted. Poults were inoculated
orally and I/P. The oral dose rate was 111 x 102 to 111 x 107; the
I/P rate was 130 x 103 to 130 x 1011.
In order to test the greater than "worst practical case" and deter-
mine a microbioaerosol dose for L_. pomona MLS and S^. typhimurium for
hamsters and poults, respectively, aerosol experiments were conducted
in an isolation chamber. These studies allowed the opportunity to
test the Lundgren Electrostatic Aerosol Precipitator (LEAP) and the
AGI aerosol samplers. Poults became infected when the ambient air
contained about 5-20 x 104 £. typhimurium/liter of air (Tables 15
and 16). Hamsters did not become infected when exposed to similar
aerosol concentrations of Ij. pomona MLS (Table 2).
Minimum infectious dose (MID) studies of both L_. pomona MLS and S^.
typhimurium provided a tool for quantitation of infectivity/or in-
fection potential or alteration thereof. Stock cultures of L pomona
MLS had a MID of 20 cells when injected intraperitoneally (I/P) into
male weanling hamsters (Table 17) . The S.. typhimurium MID was below
200 cells injected I/P for week-old poults (Table 4). Whenever a
new series of experiments was conducted, the virulence was tested or
had recently been determined. This was particularly important with
the L_. pomona MLS since quantitation was more precise for stored
cultures and virulence was a more labile characteristic than with
the S_, typhimurium.
L. pomona MLS (serotype confirmed by the Center for Disease Control
by cross agglutination) isolated from the MOD-A slurry 18 days post-
35
-------
Table 13. HAMSTER EXPOSURE TO MICROBIOAEROSOL OF LEPTOSPIRA SEROTYPE POMONA MLS IN AN ISOLATION
CHAMBER (five-minute exposure)
Inoculum
Organ Isms /ml
Control-PBS
Undiluted cul-
ture « 2 x 10 )
2 x 108
2 x 107
2 x 105
2 x 103
Amount
Nebulized (ml)
1.8
1.2
1.1
1.1
1.8
1.8
Agar Plates
0/5
0/5
0/5
0/5
0/5
0/5
LEAP
_
-
-
-
-
-
Results
Positive/Possible
0/5
0/10
0/10
0/9
0/8
0/7
Organisms/
liter of air
None
< 6.9 x 105
6.9 x 105
6.9 x 104
1.1 x 103
1.1 x 101
-------
Table 14. INFECTION OF TURKEY POULTS BY VARIOUS ROUTES OF INOCULA-
TION (week-old turkey poults, 0.1 cc inoculum used)
Group
1
2
3
4
5
6
7
No. of S_.
typhimurium
9.8 x 10~2
9.8 x 10°
9.8 x 104
9.8 x 108
9.8 x 1012
9.8 x 1016
Control
Positive/Possible
Ocular Oral Nasal
0/3
0/3
0/3
1/3
0/3
3/3
0/3
0/3
1/3
1/3
3/3
2/3
3/3
0/3
0/3
0/3
0/3
2/3
0/3
3/3
0/3
Table 15. TURKEY POULT EXPOSURE TO MICROBIOAEROSOL OF SALMONELLA
TYPHIMURIUM IN AN ISOLATION CHAMBER
Inoculum
(per ml)
Amount Nebulized
(ml)
Results
16
9.8 x 10
8.0 x
13.0 x 107
15.2 x 10C
18.7 x 10-
3.0
3.2
3.6
3.7
3.4
9.3 x 1014 Sal/1 of air
10/11 Poults
8.1 x 104 Sal/1 of air
1/9 Poults
14.9 x 105 Sal/1 of air
7/11 Poults
17.9 x 104 Sal/1 of air
0/12 Poults
20.0 x 107 Sal/1 of air
12/12 Poults
37
-------
Table 16. SALMONELLA AEROSOL EXPERIMENTS CONDUCTED IN AN ISOLATION
CHAMBER
Test
Results
(Sal/1)
Procedures (or Observations)
Exp. 1 (A-l)
No poults exposed, experiment con-
ducted to test production of and
sampling of a salmonella aerosol.
LEAP No. (5) and fall plate (3)
(open Petri dishes) samples were
negative for salmonella. Nebuli-
zer not functioning.
Exp. 2 (A-2)
No poults. Used a Devilbis 640
Nebulizer. Results: (4) LEAP and
(4) fall plates all positive for
salmonella but TNTC (too numerous
to count). Therefore, Nebulizer
functioning and aerosol sampled.
Exp. 3 (A-3)
Eleven poults plus 5 fall plates
exposed to a Salmonella typhimu-
rium aerosol 3.0 ml of broth cul-
ture (9.8 x 1016 IS. typhimurium/
ml). Five 1-minute LEAP samples
with 0.1 ml collecting fluid
plated.
LEAP (Lundgren
Electrostatic
Aerosol Pre-
cipitator)
9.3 x 10
14
5 LEAP plates - TNTC*
5 LEAP plates - TNTC
Poult tissue culture (positive/
possible)
Liver (4/11), Caecum (10/11),
Cloaca (0/11), Oral (9/11)
Exp. 4 (A-4)
8.1 x 10
Nebulized 3.2 ml of 8 x 106 £.
typhimurium ml broth culture. Ex-
posed 9 poults and 5 fall plates,
and took five 2-minute LEAP samples
No. of colonies/plate
LEAP 1, 0, 1, 1, and 2
Fall 2, 0, 0, 83 and 2
plates
Poults - 1 oral sample positive
38
-------
Table 16. (continued) SALMONELLA AEROSOL EXPERIMENTS CONDUCTED IN
AN ISOLATION CHAMBER
Test
Results
(Sal/1)
Procedures (or Observations)
Exp. 5 (A-5)
14.9 x 105
Nebulized 3.6 ml of a 130 x 106
S_. typhimurium 1-ml broth culture.
Exposed 11 poults 5 fall plates
and took five 2-minute LEAP sam-
ples.
No. of colonies/plate
LEAP 1, 4, 4, 1, 0
Fall 0, 0, 0, 0, 0
Plates
Poults - 7 positive; liver (4/11)
Caecum (4/11), oral (6/11;
best)
Exp. 6 (A-6)
17.9 x 10'
Nebulized 3.7 ml of a 152 x 103
S_. typhimurium/ml broth culture.
Poults were exposed and five 2-
minute samples were taken.
No. of colonies/plate
0, 0,
N.D.
0, 0, 0
LEAP
Fall
Plates
Poults (12) - All negative (cae-
cum, cloaca, liver and oral)
Exp. 7 (A-7)
2.0 x 10
8
Nebulized 3.4 ml of a 187 x 108
S. typhimurium/ml broth culture.
12 poults were exposed and five
2-minute LEAP samples were taken.
Three control birds not exposed.
No. of colonies/plat^
LEAP TNTC
Poults - 12 positive; Caecum
(10/12), Oral (10/12), Liver
(8/12)
*TNTC - Too numerous to count.
39
-------
Table 17. INITIAL MINIMUM INFECTIOUS DOSE - QUANTITATION OF
LEPTOSPIRA POMONA MLS VIRULENCE
Number of Lepto-
spires inoculated
0.2
2.0
20
200
2,000
20,000
200,000
2.0 x 106
20.0 x 106
20.0 x 107
0
0
Kidney culture
results (Positive/
No. inoculated)
0/5
0/5
1/5
1/5
3/5
1/5
2/5
5/5
2/5
3/5
0/5
0/5
Serum agglutination
results (Positive/
No. inoculated)
0/5
0/5
1/5
3/5
3/5
3/5
5/5
5/5
3/5
5/5
0/5
0/5
seeding had lost all virulence according to MID studies with the or-
ganism (Tables 17, 18, 19, and 20). The significance of this find-
ing cannot be understated. Does the virulence return with time or
is it environmentally switched off and on? Survival in wastes may
perpetuate in masked form a microbiological health hazard. A re-
check was made of the MID of the stock Leptospira pomona MLS cul-
ture (Table 21). Saprophytic members of the genus Leptospira are
commonly found in surface waters throughout the world. Environmen-
tal alteration of pathogenic leptospires may make them well-suited
for continued existence in soils or waters should they survive
waste disposal treatment conditions.
40
-------
Table 18. MINIMAL INFECTIOUS DOSE STUDIES OF MARCH 24, 1972,
LEPTOSPIRAL DITCH ISOLATES (studies in hamsters)
No. of Leptospires
Control
2x10°
2 x 101
2 x 102
2 x 103
2 x 104
2 x 105
2 x 106
2 x 107
2 x 108
Serum Antibody Titer
(Positive/ no . inoculated)
0/5
0/5
0/5
0/5
0/4
0/4
0/5
0/5
0/5
0/5
Kidney Culture
-
-
-
-
-
-
-
-
-
—
41
-------
Table 19. MINIMUM INFECTIOUS DOSE STUDY OF LEPTOSPIRA SEROTYPE POMONA MLS ISOLATED FROM THE
MANURE SLURRY OF MODEL OXIDATION DITCH A, ON MARCH 31, 1972 INOCULATED I/P INTO
HAMSTERS
Group Number
1
2
3
4
5
6
7
8
9
10
Concentration
(lepto/ml)
0 (control)
2 x 10°
2 x 101
2 x 102
2 x 103
2 x 104
2 x 105
2 x 106
2 x 107
2 x 108
Culture of Kidney
(positive/possible)
0/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
0/3
Serum Agglutination
(positive/possible)
0/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
0/2
Si
-------
Table 20. MINIMUM INFECTIOUS DOSE STUDY OF LEPTOSPIRA SEROTYPE POMONA MLS STOCK CULTURE?
INOCULATED I/P INTO HAMSTERSb
Group Number
1
2
3
4
5
6
7
8
9
10
Concentration
(lepto/ml)
0 (control)
2x10°
2 x 101
2 x 102
2 x 103
2 x 104
2 x 105
2 x 106
2 x 107
2 x 108
Culture of Kidney
(positive/possible)
0/4
2/4
4/5
5/5
1/5
0/5
0/5
0/5
0/5
0/3
Serum Agglutination
(positive/possible)
0/5
2/4
2/5c
4/5c
4/5
2/3
4/5
5/5
5/5
3/3
The stock L_. pomona MLS had been stored at toom temperature in closed cabinets. These stock
cultures were not subcultured since receipt in January, 1972.
3
Second MLS study.
Note failure of infected hamsters to seroconvert.
-------
Table 21. MINIMUM INFECTIOUS DOSE OF STOCK LEPTOSPIRA POMONA MLS
Group Number
1
2
3
4
5
6
7
8
9
10
Concentration
(lepto/ml)
(control)
2x10°
2 x 10l
2 x 102
2 x 103
2 x 104
2 x 105
2 x 106
2 x 107
2 x 108
Culture of Kidney
(positive/possible)
0/5
3/5
5/5
4/5
4/5
5/5
5/5
4/5
5/5
4/5
Serum Agglutination
(positive/possible)
0/4
2/5
1/1
2/2
1/1
0/2
0/5
1/3
2/4
2/3
Third MID study, March 21, 1973,
-------
Table 22 summarized the Model Oxidation Ditch A sampling. The
Rosemount sampling was simulated as shown by comparison of the bio-
aerosol counts. Again, the presence or absence of animals produces
the largest difference in counts. The ditch was seeded with Sal-
monella per ml of ditch liquid. As indicated in Table 22, only
minute quantities of air contain the indicator Samonella, and when
animals were absent, no Salmonella were detected. A surprisingly
large number of fecal streptococci were recovered compared to the
Rosemount sampling. The reason is unknown. The difference is not
due to the difference in sampler. Table 23 shows the results of a
comparative study using the LEAP and the AGI. The similarity should
be noted. Table 24 indicates greater microbioaerosol activity when
hamsters were present over the laboratory model ditch. Note lepto-
spires were isolated on all five samplings.
The University of Minnesota Rosemount Experiment Station field oxi-
dation ditch (FOD) manure slurry was discovered to be culturally
positive with an unidentified Leptospira in October, 1972. This was
a consequence of culturally monitoring this manure slurry-water mix-
ture for leptospires prior to seeding with L. pomona MLS in the
MOD-A for experiment L.4.5 (Tables 25 and 25a). The presence of
these "unknown" leptospires was not determined, however, until after
the pathogenic L. pomona MLS had been inoculated into the manure.
The long incubation time required for culture development prior to
examination by darkfield microscopy was longer than the control pe-
riod of one week. The isolate may have been a "saprophytic^ lepto-
spira since it did not agglutinate screening to six common patho-
gen" antisera (serotypes hardjo, autumnalis, pomona, canicola,
icterohemorrhagiae, erippotvphosa). A leptospiral antigen and hyper-
immune serum produced in rabbits is being evaluated by the Center
for Disease Control, Atlanta, Georgia, to confirm this possibility,
or that this is a less common pathogenic leptospira serotype.
The discovery of a leptospira in manure and/or water from the field
oxidation ditch (FOD) or well was interesting. Questions arose:
What was the source of the leptospira? Are there carriers/cases in
the herd? Can leptospires be detected in a field situation using
the technique and aerosol sampling technique?
In order to answer these questions to some extent, the aerosol sam-
ples from the Rosemount studies (see Section V), the manure slurry
of the MOD-A and the FOD, and the aerosol samples from the MOD-A
(Table 23) were cultured. The presence of the leptospiral contami-
nant probably did not affect the experiment other than in a positive
manner. For example, naturally occurring levels of leptospires can
be detected and isolated by the filter-agar plate procedure. Pre-
viously, studies with this technique were with laboratory-adapted
45
-------
Table 22. MODEL OXIDATION DITCH A - SAMPLING TURKEY POULTS AERO-
SOL DATA TAKEN WHEN TURKEYS PRESENT AND ABSENT
Viable Bacterial Aerosols (LEAP counts) on PCA (total, L-EMB (coli-
form), M-Ent (Fecal Streptococcus), and BGS enrichment (Salmonella),
37 C, 24 h4. Counts Given in Colony Forming Units/Liter (CFU/1)
Poults Present
Orga-
nism
Total
Coli-
form
F.
Strep.
Salmon.
No. of
Samples3
35
32
35
40
Total
CFU/1
585
2.0
142
.009
Range
CFU/1
0.7-173
.02-0.6
.02-26
.001-
.003
Average
CFU/1
17.00
0.90
04.00
4 x 10-5
Poults Absent
No. of
Samples3
12
10
11
13
Total
CFU/1
57.4
00.02
00.10
00.00
Range
CFU/1
0.40
0.00
0.2-.1
0.00
Average
CFU/1
1.4
0.2
0.009
2 x
ID'3
(9 Salmonella were detected
in 1.3 x 105 liters of air
samples.)
(No Salmonella were de-
tected in 4.3 x 10^
liters of air sampled.)
Each sampling represents 10,000 liters of air samples into approxi-
mately 25 ml of collecting fluid.
46
-------
Table 23. COMPARISON OF COLONY-FORMING UNITS (CFU) PER LITER OF AIR SAMPLED WITH LEAP3 ALL GLASS
IMPINGERS (AGI) - MODEL OXIDATION DITCH-A (MOD-A) FIELD OXIDATION DITCH (FOD) AT
SUMMER CONDITIONS
MOD-A Same Temp.
Date
Oct 30, 1972
Nov 1, 1972
Nov 7, 1972
Nov 12, 1972
Dec 21, 1972
(LEAP)
Dec 28, 1972
(LEAP)
Total
X
EMB!
0
0
0
0
0
0
0
0
M-Ent2
23
ND
17
6
ND
21
67
11
PCA3
146
6
503
33
40
39
767
128
FODb
Date
June 28, 1972
July 5, 1972
July 18, 1972
Aug 9, 1972
Aug 8, 1972
Sept 20, 1972
EMB1
0
0
0
0
1
3
4
1
M-Ent2
0
0
0
0
1
19
20
4
PCA3
149
37
82
6
176
109
559
96
aLEAP = Lundgren Electrostatic Aerosol Precipltator
^Selection of field data when external air temperature was between 15° C-30° C.
Barn Data. (Intramural)
Data Is East
l=Eosin Methylene Blue Agar; 2=M-Enterococcus Agar; 3=Plate Count Agar
-------
Table 24. RESULTS OF AGI (ALL GLASS IMPINGER) SAMPLING OF MODEL OXIDATION DITCH A - ANIMAL
HOUSING UNIT MICROBIOAEROSOL: ROTOR VELOCITY VARIED NOVEMBER 16-17, 1972
(Enumeration as Colony-forming Units/liter or CFU/1)
Hamsters Present
(Nov. 16, 1972)
Hamsters Absent
(Nov. 17, 1972)
Rotor Speed
Normal
Fast
Fastest
Normal
Fast
Fastest
PCA Aliquot No. 1
102,116
17, 13
23, 36
1, N.D.
2, 6
N.D.
PCAa Aliquot No. 2
97,130
2, 4
17,29
1, 1
5, 5
(splashing problem)
X
111
9
26
1
4
N.D.
Leptospires
Yes
Yes
Yes
Yes
Yes
N.D.
00
PCA = Plate Count Agar/Tryptone Glucose Yeast Extract Agar
-------
Table 25 . ROSEMOUNT FIELD OXIDATION DITCH: MICROAEROSOL (AGI)
AND SLURRY SAMPLING FOR LEPTOSPIRES
Sample
Air
Slurry
Date
12/20/72
12/27/72
I/ 2/73
1/10/73
12/20/72
2/20/73
3/26/73
Result (positive/possible)
Negative, (0/22)
Negative, (0/24)
Negative, (0/24)
Negative, (0/21)
Negative, (0/3)
Negative, (0/3)
Negative, (1/3)
These samples were aliquots obtained from the AGl's used
for routine microbioaerosol studies on these dates.
Positive result recognized in May, 1973.
Table 25a. MODEL OXIDATION DITCH: ANIMAL HOUSING UNIT MICROAEROSOL
(LEAP) AND SLURRY SAMPLING FOR LEPTOSPIRES
Sample
Air
MOD-Slurry
Date
12/18/72
12/20/72
12/22/72
12/28/72
12/29/72
I/ 3/73
I/ 4/73
I/ 9/73
1/11/73
1/12/73
12/18/72
12/20/72
12/27/72
12/29/72
I/ 3/72
I/ 4/72
I/ 8/72
1/10/72
1/12/72
Result (positive/possible)
Negative, (0/2)
Negative, (0/2)
Negative, (0/2)
Negative, (0/2)
Negative, (0/2)
Negative, (0/2)
Negative, (0/2)
Negative, (0/2)
Negative, (0/2)
Negative, (0/2)
Negative, (0/2)
Negative, (0/4)
Negative, (0/2)
Negative, (0/3)
Negative, (0/4)
Negative, (0/6)
Negative, (0/5)
Negative, (0/5)
Negative, (0/5)
plus 1 (?)
plus 1 (?)
plus 1 (?)
49
-------
pathogenic leptospires. Laboratory experience suggests that the
oxidation ditch may be a "cultural vat" for leptospires. The iso-
lation of leptospires from the FOD tends to confirm this contention.
Beef cattle associated with the FOD confinement feed operation were
also studied. On March 1, 1973, blood samples were obtained prior
to slaughter from 24 of the 36 beef cattle (Herefords) confinement-
housed above the FOD at Rosemount. The results were interesting.
All 24 sera were uniformly serologically negative by microscopic ag-
glutination test against Leptospira serotypes pomona, hardjo, grippo-
typhosa, canicola, icterohemorrhagiae, and autumnalis, but positive
at a 1:10 serum dilution against the "unknown" leptospiral ditch
isolate. A reaction at the 1:10 dilution is considered non-signifi-
cant, but the consistency of the agglutination for all 24 sera sug"
gests possible significance in view of the fact that other routine
sera tested from other state cattle herds submitted by veterinarians
for agglutination screening were completely negative to this "unknown"
leptospiral antigen.
Thirty-three of the 36 incoming replacement calves (Herefords) were
bled for serum one week later (3/8/73). The information concerning
the origin of these calves was not obtained, but they were kept at
another area of the Rosemount Experiment Station for about three
weeks prior to this time to be started on concentrate, feed, and
roughage. Their sero-agglutination reaction pattern may indicate
only vaccination or retained maternal antibodies, but also some
reactions to the leptospiral "unknown" at the 1:20 dilution.
These calves were bled again just prior to shipment for slaughter
in August, 1973.
Animal models exposed to the model oxidation ditch-confinement hous-
ing unit were not overwhelmed with L^ pomona MLS or S_. typhimurium
infection, nor did aerosol sampling of the ambient air with the LEAP
indicate high levels of pathogen contamination of the air (Tables 7,
8, 25, 25a, 26, and 27). Hamsters confined over an L^ pomona MLS-
contaminated slurry did not show evidence of leptospirosis. Poults
in 2 of 4 _S_. typhimurium experiments remained negative for signs of
salmonellosis but in S.2.W., 20 of 148 and in S.3.5., 12 of 102
poults became infected with _S. typhimurium. The overall low response
rate may have been due to local tissue immunity (with lack of sys-
temic immune involvement if the pathogens were present and contacted).
It was evident further that the MOD-A successfully simulated micro-
bioaerosol conditions present in a field oxidation ditch-cattle con-
finement unit (Table 23). The microbioaerosol contamination levels
in oxidation ditch housing are less than those associated with tra-
ditional dairy farm operations tested.
50
-------
Table 26. ANIMAL EXPOSURE, MANURE SLURRY, AND AEROSOL SAMPLING RE-
SULTS OF LEPTOSPIRAL/HAMSTER STUDIES UTILIZING MODEL
OXIDATION DITCH-A
Experiment
Slurry
L..1.W.
0. Ditch
Aerosol
Slurry
L.2.W.
0. Ditch
Aerosol
Slurry
L.3.S.
0. Ditch
Aerosol
Unknown
Slurry
L.4.S.
0. Ditch
Aerosol
Control
«
0/10
—
—
0/5
-
—
ND
ND
+
0/5
—
1
+
0/10
—
+
0/5
-
+
0/5
-
+
0/5
™
2
+
0/5
—
+
0/5
—
+
0/5
—
+
0/5
«.
3
+
0/5
—
+
0/5
—
+
0/5
—
+
0/5
"
4
+
0/4
—
+
0/5
—
+
0/5
—
+
0/5
"
5
+
0/5
—
+
0/5
—
+
0/5
—
+
0/5
6
+
0/5
—
+
0/5
—
+
0/5
—
+
0/19
+
7
+
0/10
—
ND
ND
ND
+
0/5
—
ND
ND
ND
8
+
0/1^
"-
ND
ND
ND
ND
ND
ND
ND
ND
ND
51
-------
Table 27. LEPTOSPIRAL ANIMAL FEEDING AND RECYCLED FEED SAMPLE
RESULTS
Experiment
Slurry
L.l.W.
Fed
RCF
Slurry
L.2.W.
Fed
RCF
Slurry
L.3.S.
Fed
RCF
Slurry
L.4.S.
Fed
RCF
Control
M
0/5
-
_
-
ND
_
0/5
—
+
ND
~
1
+
ND
—
+
0/5
—
+
0/5
—
+
ND
™
2
+
0/5
-
+
0/5
—
+
0/5
+
+
0/5
™
3
+
0/5
+
+
0/5
—
+
0/5
—
+
0/5
+
4
+
0/5
+
+
0/5
+
+
0/5
+
+
0/5
+
5
+
ND
-
+
ND
ND
+
0/5
+
+
0/5
+
6
+
ND
-
ND
ND
ND
4-
0/5
+
+
ND
—
7
+
0/5
+
ND
ND
ND
+
0/5
ND
+
ND
ND
8
+
o/
+
ND
ND
ND
ND
ND
ND
+
ND
ND
It is interesting to note that on the one date that leptospires
were detected in the MOD-A housing unit, testing was conducted for
microbioaerosol production at various rotor speeds. The "normal"
rotor speed was that derived from empirical experience as producing
the greatest rate of slurry flow. It apparently also produces the
greatest respiratory hazard. The other speeds are not practicable.
SUMMARY
Evaluation of laboratory data collected from research grant #R802205
suggests that the oxidation ditch aeration by rotor is probably not
a public health hazard in terms of aerosol dissemination of Ij. pornona
MLS and S_. typhimurium. However, if the contaminated manure slurry
is not disinfected in some manner, environmental health problems
52
-------
would probably result. Survival studies have demonstrated that
pathogenic leptospires OL. pomona) persist for 183 days in sterile
soil supersaturated with water and kept at 20° C (18); however, the
authors did not conduct virulence studies. Many cases of leptospi-
rosis associated with contaminated streams and recreational waters
are recorded. These cases, coupled with previous work describing
leptospiral viability for 138 days in the cattle manure slurry of a
model (Pasveer) oxidation ditch, suggest this problem is real. Lep-
tospires isolated from the manure slurry of the Model-A oxidation
ditch were serologically identical to those L_. pomona MLS inoculated
into the slurry. The oxidation ditch may serve as a "culture vat"
and reservoir for leptospires because detection of viable lepto-
spires for prolonged periods requires multiplication of the micro-
organisms. Previously, the finding of sufficiently high levels of
Thiamine and Vitamin 8^2 in the manure slurry suggested this possi-
bility since a source of nitrogen and oxygen is also provided.
Although the ditch isolates remained stable antigenically (as L_.
pomona MLS), the virulence (MID) studies demonstrate a decreased
virulence of the organism. The fact that virulence alters with en-
vironment is not surprising, but is significant to public health/
environmental health concern because the existence of a potential
pathogen in the uncontrolled environment could make it possible for
the attenuated pathogen to enter through many of the myriad routes
of disease transmission into another susceptible host, after which
the attenuated organism could revert to its original state, thereby
causing disease. In fact, survival in a non-host associated envi-
ronmental cycle is a new line of leptospiral disease transmission
not seriously considered previously. As improved methods of detec-
tion become available, the role of leptospires and salmonella in
public health will become more apparent. Failure of these organisms
to cause seroconversion after infection hinders these efforts, but
again, opens possible routes not before considered because of the
"masking" of infection not detectable with present diagnostic methods
used.
Our findings of an "unknown" pathogenic saprophytic leptospiral sero-
type in the Rosemount manure slurry and seroconversion of exposed
cattle serve to demonstrate exposure potential. The cattle either
brought the leptospires to the ditch or became infected from it.
In summary, it might be well to state that the oxidation ditch as a
means to an end (waste treatment) also serves as an instrument in
creating channels of disease transmission, but is not a cause. It
can serve as a dead end if a loss of virulence occurs or if a method
of disinfection is provided. Prevention of disease in animals so
housed is essential to the protection of the inhabitants of an envi-
ronment (both man and animal) from disease, whatever that disease
might be.
53
-------
SECTION V
STUDY OF THE BIOAEROSOL PRODUCTION OF
A FIELD OXIDATION DITCH
INTRODUCTION
Purpose
The purpose of this study was to monitor the bioaerosol production
from a rotor-aerated, operational field oxidation ditch (Pasveer) and
to assess the public health hazard in relation to bacteria produced by
this waste treatment facility.
Importance
The importance of this study can be determined by consideration of the
following:
1. Langmuir (19) presents a historic review of the literature
citing cases of airborne infection.
2. The droplet nuclei theory proposed by Wells (20) whereby a
particle originating from a liquid becomes small enough to
enter the lung.
3. The oxidation ditch is a treatment area with high concen-
trations of enteric bacteria, some of which may be patho-
genic for man and/or animals.
4. Animals housed on slats over the ditch and people working
in the area are exposed to airborne pathogens.
Description of the Oxidation Ditch Operation
The field oxidation ditch (Figure 5) is one of the facilities at the
University of Minnesota's Experiment Station located at Rosemount,
Minnesota. The oxidation ditch is located within the Livestock
Research Building on the Agricultural Engineering Farmstead (Figures
6 and 7). Two enclosed housing units have been constructed over the
oxidation channel. Within each unit, 18 beef cattle were fattened
on ordinary grain and concentrate typical of usual confinement pro-
duction units. Urinary and fecal wastes passing through the slotted
floors of the housing units combined to become the manure slurry of
the Pasveer aerobic treatment process. A rotor at the west end of the
oxidation channel served to propel and aerate the waste material. The
rotor and oxidation channel were covered by the housing unit and the
54
-------
• i
' -•
WEST OF
UNIT SITE
WEST VENT
f 1
15m NORTH OF
UNIT SITE
7.3152 m
BIO-DISC
SITE-*
WEST BARN SITE
Slotted Floor
iiiiiiiiiiiuiiiiiuiiiiiiiimiiiiii
OXIDATION DITCH
SIPHON
CHAMBER
— 15m-»-X
EAST OF
UNIT SITE
-EAST VENT
SITE
\
15m SOUTH OF
| UNIT SITE
X
Figure 5. Oxidation ditch at Rosemount
-------
LOCATION OF BUILDINGS
AT THE
ROSEMOUNT EXPERIMENTAL
STATION
North
TURKEY
ENVIRONMENT
BUILDING
NO. I
Q-WELL
"-^ HOUSE
TURKEY
ENVIRONMENT
BUILDING
NO. 2
-BOILER
HOUSE
.ELECTRIFICATION
RESEARCH
BUILDING
TRANSF.
HOUSE
OXIDATION DITCH
^LIVESTOCK
orr RESEARCH
BUILDING
SALMONELLA
RESEARCH
BUILDING
I CATTLE I
I PEN I
I I
FENCE
Figure 6. Rosemount farmstead buildings
-------
n
t
Figure 7. Rosemount farmstead aerial photo
-------
rotor enclosure. Two vents, one at each end of the ditch, allowed air
to exit from the ditch. Initially, vent fans drew the air from the
ditch and animal units. During the preliminary studies, a change was
made to positive pressure units which forced air continually into the
units. The air was forced down through the slats and exhausted through
the vents. The slat width space in the west unit was 3.81 cm and 3.18
cm in the east unit. There was a noticeable difference in exhaust air
speed between the two units* the west vent passing more air than the
east. This may have been due to the slat width. The normal operation
of the oxidation ditch was interrupted occasionally by mechanical
problems or experimental intervention. All such alterations are noted
in sampling reports.
Making Two Time Segments of the Research Effort Was Important
The initial period of preliminary studies was utilized to develop ex-
perimental procedures and gather baseline information for development
of standard protocol. A year-long study was then conducted with the
developed protocol during which time the field and laboratory situa-
tions were stabilized.
MATERIALS AND METHODS
In order to monitor the air for bioaerosols, a program of sampling
was developed.
Phase I, Preliminary Field Sampling Program
A preliminary program to develop field and laboratory procedures and
gather data and reference material occupied the first 10 months of
the 22-month study.
Field Work —
Sampler - Three different air samplers were evaluated: The All Glass
Impinger (AGI) (Figure 8), the Lundgren Electrostatic Aerosol Precipi-
tator (LEAP) (Figure 9), and the Casella Slit sampler. Although each
of these devices has its unique advantages and commendable qualities,
the AGI sampler was designated as the apparatus for routine monitoring
in the field. The LEAP and the Casella were too cumbersome for use
in the extramural and intramural environments in this study. Both
the LEAP and Casella sampled a wide range of particle sizes. Interest
was only in the 1 and 5 micron (y) size particles which can penetrate
to the depths of the pulmonary alveoli in the lung. The AGI was a
better sampler for this purpose because it theoretically samples parti-
cles in the size range of 1-10 microns. A simultaneous sampling with
58
-------
tn
\o
0 —
5-
-
4-
—
3-
2-
1-
•
-15
-14
LI3
~-\2
-10
— 9
- 8
~- 7
- 6
- 5
1 4
- 3
'- 2
- I
INCHES METRIC
Figure 8. All glass impinger (AGI)
-------
COLLECTION DISC
PICKUP DISH
PICKUP DISH MOTOR
DISC MOTOR
LIQUID
OUTPUT
LIQUID
INPUT
CERAMIC NOZZLE
HIGH VOLTAGE PLATE
CORONA NEEDLES
ASPIRATING PROBE
LIQUID SUPPLY LINE
AIR BLOWER
-PERISTALTIC PUMP
(TWO CHANNELS)
Figure 9. Lundgren electrostatic aerosol precipitator
-------
the AGI and the Casella (Table 28) was conducted. The AGI yielded 20
times the number of colonies per liter of air sampled than did the
Casella. This meant that approximately one particle contains 20 colo-
nies, indicating that particles sampled by the AGI harbor one or more
bacterial colony-forming units. A study of the efficiency of particle
collection done by Hay (21) also found that the impinger produced
highly variable results.
The AGI has several notable advantages. It is designed to simulate the
human respiratory system both with respect to sampling rate (12.5
liters per minute) and particle size retention (1-10 microns). Con-
sequently, it was ideal for a study of health hazards. It is relative-
ly inexpensive and permits the simultaneous sampling of multiple sites
with little extra effort as compared to the LEAP or Casella samplers.
Collecting fluid - A number of trials compared different sampling
fluids to be used in the AGI apparatus. These data are summarized in
Table 29. It is evident that collection fluids containing peptone
yielded higher counts. Ultimately a 0.1% peptone and 0.01% antifoam
fluid was adopted as the standard for use with the AGI protocol.
Determination of variables - The first six months of preliminary work
were used to determine field and laboratory variables that could
affect the aerosol analysis. Sources of variation in field data
studied were as follows:
1. Variability of duplicate samples.
2. Hourly variation.
3. Noticeable disturbances.
4. Sample storage.
5. Duration of sampling.
6. Ditch bacterial concentration.
7. Laboratory techniques.
Variables that could be controlled were sought. This was difficult
during this time due to continuous variation in the sampling procedure,
but some insights were gained. As the sampling protocol became stan-
dardized, so did the variables.
Contributing aerosol counts due to outside factors unrelated to the
ditch or animals need to be determined. The three-sided barn which
enclosed the housing units and the oxidation ditch also contained two
61
-------
Table 28. COMPARISON OF AGI AND CASELLA COUNTS OF
COLONY-FORMING UNITS, DIFFERENT SAMPLING SITES
(colony-forming units/liter)
Site
West Vent
East Vent
West Barn
North Vent
Downwind
X
Casella
18/liter
10/liter
12/llter
12/llter
I/liter
11/liter
AGI Counts
51/liter
150/liter
225 /liter
12/liter
4/liter
88/liter
Table 29. EFFECT OF AGI COLLECTION FLUID ON OBSERVED AEROSOL COUNTS
Mean viable airborne CFU/ liter
count 1.0% Peptone
0.85% 1.0% &
Location NaCl Peptone 0.1% Antifoam
West Vent 11 39 20
East Vent — — 17
West Barn 59 118 57
East Barn — — 68
Rotor 7 26
Field 0.5 0.5
0.1% Peptone
&
0.1% Antifoam
18
16
60
89
—
—
62
-------
other experimental units close by which possibly posed some influence
on the tests being conducted. For example, an exhaust fan from a
north barn (Figure 5) was vented into an area adjacent to the east
vent of the ditch. Further, exhaust from the biodisc room (Figure 5)
was vented into an area near the west ditch vent. Samples taken at
these sites showed that there was no detectable intermingling of air-
borne bacteria. The samples were taken one meter in front of the
vent, the 30-cm and 100-cm heights are lower. At a distance of another
600 cm, the counts were half those at 1 meter. Therefore, at the
east vent which is 15 meters away, there probably was no interference.
Likewise, interference from the biodisc at the west vent was not ob-
served. It was concluded that there were no contributing bacteria
from the north vent and biodisc being collected at the east and west
vent of the oxidation ditch (Table 30).
Table 30. EVALUATION OF POSSIBLE CONTAMINATION SOURCES
NORTH VENT
30-cm height 100-cm height 160-cm height
Number of samples
Average total CFU/1
33
7
39
5
32
19
BIO DISC LOCATION
Number of samples
Average total CFU/1
30-cm height
9
9
100-cm height
13
7
160-cm height
13
10
Variability of duplicate samples - The preliminary samples of air
showed that simultaneous duplicates differed by as much as 50%. An
understanding of this variation led to an investigation of possible
variables. A variation of air volume sampled was ruled out as a fac-
tor. The airflow measured through the AGl's (12.5 liters/minute) was
the same in all cases when checked using three different vacuum pumps.
To ensure that the duplicate AGl's were sampling a uniform aerosol, a
cylinder was devised which limited the air mass sampled by two AGl's
(Figure 10). The cylinder was 15 cm in diameter, 43 cm long, and
63
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Figure 10. Cylinder for simultaneous AGI sampling
-------
closed at one end except for two 1-cm openings. AGI nozzles sampling
through the apertures produced variable counts, indicating that the
bioairmass was not a homogenous mixture (Table 31). Higgins (22) and
May (21) also found with an impinger that bioaerosol counts fluctuated
a great deal. In Table 31 note that results from samplers 5 and 6,
and from 7 and 8 vary widely. The reason for this was the occasional
collection of large particles containing many bacteria. The impinging
breaks up the agglomeration to produce multiple colonies when plated.
In cases where duplicate results vary unrealistically, the result in-
dicating contamination has been deleted. In some cases, even mosquito
legs had been collected.
Table 31. SIMULTANEOUS DUPLICATE AIR SAMPLING WITH A CYLINDER
(colony-forming units/liter)
Location
Field
North of unit
South of unit
West barn
East barn
East vent
East of unit
Sampler
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Total plate determination
1
3
0.3
0.7
0.4
80
56
2659
26
43
37
16
86
97
8
4
6
3
65
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Hourly variation - Early samplings showed that sites where the great-
est variation in counts occurred were in the animal housing units.
In order to understand whether or not variation was due to some fac-
tor which could be controlled, such as the operator's presence or
movement, a series of hourly tests was taken in the west housing unit
(Table 32 and 33). It was observed (afternoon, Table 32) that many of
the cattle were lying down, as opposed to the morning when most of
the cattle were standing. The higher afternoon counts might then
have been contributed by incomplete venting of air due to the animals
blocking the air that would be passed through the slats. But, another
day of hourly sampling with careful notation of numbers of animals
lying vs. standing did not substantiate the hypothesis. The hourly
variations on Table 32 vary from 66 CFU/1 to 334 CFU/1 and those of
Table 33 from 18 CFU/1 to 66 CFU/1. The change in relative humidity
(R. H.) from hour to hour did not have any apparent influence on the
counts as is also seen on Table 33. The cause of the fluctuations
remained undetermined.
Noticeable disturbance - Often, drastic variations in counts could be
predicted from certain activities in the experimental area. High
bioaerosol counts were always observed when there were strong gusts
of wind, people working in the surrounding area, or feed bins being
filled. When such conditions existed, sampling was avoided if pos-
sible; however, when it was not possible, the results of the bioaerosol
counts have greater than usual variations. Such a case occurred
during sampling simultaneously at three heights at the vent exhaust
when a gust of wind occurred. The results were: at 30 cm, 247 CFU/1;
at 100 cm, 6 CFU/1; and at 160 cm, 1 CFU/1. When a noticeable distur-
bance as above occurred, the abnormal count was omitted (the 247).
Storage of samples - The storage time and temperature of the AGIs
during collection and bacteriological plating, another step in the
sampling procedure, was checked as a possible source for count vari-
ation since the collecting fluid could serve as a growth substrate.
The time period between collection and plating was kept at 2 to 3
hours. The temperature was maintained at around 5° C by keeping the
AGIs in an ice chest in the summer and in the winter in the chest
with no ice located in a 10° C room. On a hot day, duplicate samples
were compared by putting one of the duplicates in the cooler and the
other at ambient temperature of 27° C. The bioaerosol counts showed
no difference. Another check was done by comparing counts of samples
taken from an AGI immediately and those kept two hours. The two
counts were identical.
During preliminary sampling in the winter, the collection fluid froze
after 0.5 minute of sampling. Later, sampling times were prolonged
by addition of heat and storage of samplers in a heated area prior to
use.
66
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Table 32. AEROSOL FLUCTUATIONS ASSOCIATED WITH TIME
IN THE WEST BARN
(100 cm)
Time
9:30 a.m.
10:30
11:30
12:30 p.m.
1:30
2 -.20
Table 33.
Total CFU/1
Samples taken average
5
5
4
3
3
2
183
66
94
334
251
241
AEROSOL FLUCTUATIONS ASSOCIATED WITH
IN THE WEST BARN
(100 cm)
Range
69-305
47-96
60-123
240-515
223-269
208-274
TIME
RH
Time %
10:30 a.m. 87
11:30 78
12:30 p.m. 70
1:30 70
2:30 74
Temp. Number laying
OG (17 total)
16 3 laying
15 8 laying
14 9 laying
14 4 laying
15 0 laying
Samples
taken
2
2
2
2
2
Average
total CFU/1
66
53
18
24
45
67
-------
Duration of sampling - The length of sampling tine was an important
aspect to observe when analyzing causes for variation since, hypo-
thetically, some bacteria battered about in the agitated collecting
fluid during sampling may not survive.
A field experiment compared AGl's sampling the air of the west barn
for two times, 3 and 10 minutes. One set of AGls sampled the air
for 3 minutes concurrently and one, 10-minute AGI sample was begun.
After the first three-minute sample ceased, a second set (of 2)
three-minute samples was collected. The results are given in Table
34. The 10-minute sample was diluted 1:10 before plating. The re-
sults indicate that one 10-minute sample was an average of the two
consecutive 3-minute samples; however, the other 10-minute sample was
the same as the low counts. An additional test was done in the lab-
oratory with collecting fluid from two AGIs that had collected air
for five minutes. The total aerobic colonies per ml of fluid in the
AGI was determined in the normal way (see below) . The AGls then
sampled air from a clean area (set just inside a refrigerator with
the door open 2 cm) for 10 additional minutes. The colonies per ml
in both cases of the additional 10-minute sampling was nearly half
of the original counts. These data implied that to compare counts
from one area to another the sampling period should be the same.
This knowledge was incorporated into our final sampling procedure.
The air at every site associated with the field oxidation ditch was
sampled for five minutes.
Table 34. EFFECTS OF SAMPLING FOR VARIOUS LENGTHS OF TIME
IN THE WEST BARN
(colony-forming units/liter)
Time of sampling Average total
First set of 3 minute samples 271
Second set of 3 minute samples 94
Simultaneous 10 minute sample 187
First set of 3 minute samples 49
Second set of 3 minute samples 90
Simultaneous 10 minute sample 53
68
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Ditch bacterial concentration - A potential variable which may have in-
fluenced the production of a bioaerosol from the ditch was the concen-
tration of bacteria in the oxidation ditch. Higgins (22) and Darlow
(23) found that with increasing bacterial concentrations of liquid
from which droplet nuclei were formed the bioaerosol of such bacteria
also increased. On five different days, during different seasons,
when ditch samples were taken the bacterial counts varied little
(Table 35). Total aerobic counts per ml of ditch liquid averaged
50 x 10& and ranged from only 15 x 10& to 92 x 10&. Coliform organisms
per ml of ditch liquid averaged 8.9 x 10* and ranged from 2.7 x 10^
to 20 x 104. Fecal streptococci organisms per ml of ditch liquid
averaged 15 x 10* and ranged from 2 x 10* to 32 x 10*. The minor
change in concentration is not believed to enter in the fluctuation
of airborne counts.
Table 35. NUMBER OF BACTERIA IN ROSEMOUNT DITCH
Sample date
Sept. 27, 1972
Oct. 3, 1972
Feb. 20, 1973
Feb. 28, 1973
March 28, 1973
Average
Total
(CFU/ml)
18,800,000
15,000,000
47,000,000
77,000,000
92,000,000
50,000,000
Coliforms
(CFU/ml)
40,000
140 ,000
27,000
36,000
200,000
89,000
F. Streptococci
(CFU/ml)
20,000
170,000
240,000
20,000
320,000
150,000
Approach to the study - The obvious critical parameters influencing
the bioaerosol fluctuations during the sampling procedure have been
evaluated above. There are still fluctuations that have not been ex-
plained since to discover them was beyond the scope of this project.
The field study was used for evaluation of the health hazard of an
operational Pasveer oxidation ditch. It was concluded that the whole
cannot be subdivided into esoteric parts when dealing with problems,
especially of environmental concern.
69
-------
Laboratory - Once determined, standards of bacteriological culturing
and laboratory protocol did not play an important role in the count
variations. Laboratory personnel did not change, and all samples
were treated the same once the standard protocol was established.
Various methods or procedures were tested to determine the one most
routinely reproducible, efficient, and inexpensive to suit our re-
quirements. Each investigator of a new environment or new problem
must develop his own laboratory procedures.
1. Dilutions. Each AGI was etched to mark a volume of 30 ml.
The collecting fluid was added to this line. During sam-
ple collection some of the AGI fluid evaporated. In the
laboratory, the collecting fluid in the AGI was made up to
the 30-ml volume with sterile distilled water after sam-
pling so that the proper dilutions could be made. The
only alteration of this procedure was the dispensing method.
An Erlenmeyer wash bottle apparatus was finally devised to
dispense the liquid and was suitable for continuing use.
2. Plating. It was determined that plating of aliquots by
either a "pour plate" or membrane filter technique pro-
vided satisfactory and comparable results. However, since
both of these practices were time-consuming and cumber-
some for the processing of many samples in a reasonable
short peiod, a "surface spreading" technique which provided
counts sufficiently accurate for this work was used.
For total aerobic bioaerosol determination, Plate Count Agar
(PCA) (Difco) was used as recommended in Standard Methods.
Aliquots of AGI collecting fluid were surface spread on PCA.
When dry, the plates were inverted, incubated 24 hours (48
hours after preliminary work) at 27° C, then counted, and
total, colony-forming units per liter of air sampled was
calculated. All plating was done in a hood.
3. Characterizing Bacteria. In an attempt to characterize the
microbes in the aerosol, a variety of classical identifica-
tion techniques were employed on "typical" colonies picked
from enumeration plates (gram stain, hemolysis, catalase,
Salmonella enrichment, etc.) In the preliminary experiments,
Bacillus sp., Staphylocoecus sp., and Corynebacterium sp.
were the predominant types appearing in the aerosol. These
organisms are commonly found in the air but were not neces-
sarily associated with fecal excretion. Subsequent trials
to detect enteric microorganisms involved direct plating of
AGI fluid aliquots onto differential and selective media.
The enteric indicator organisms chosen to be studied were
70
-------
fecal streptococci (F. Strep.) and the coliform group (24,
25).
These organisms were chosen because there are well defined
steps leading to their identification and they are found in
the enteric tract. The two organisms behave quite different-
ly owing to the differences in gram stain, cell wall, and
motility. The differences offer possible explanations in
ability to become airborne and to survive airborne exposure
and detection.
It was found that coliform (gram negative, non-spore forming,
lactose fermenting) detection was better made on Levine's
Eosin Methylene Blue (L-EMB) agar. On Eosin Methyline Blue
(EMB) (less specific) numerous non-col±form colonies grew,
thus requiring further time and testing. Comparison of
the ability to detect actual coliform organisms on L-EMB
and EMB showed that the two media were essentially similar.
To further test the suspected colonies picked from L-EMB
(large and pigmented) , a gram stain was done and the colony
growth on Triple Sugar Iron (TSI) was checked for lactose
fermentation.
An enrichment procedure is generally done on samples (Stan-
dard^' Methods ). However, this was not feasible and the
procedure would have differed from that of identifying total
bioaerosol. A comparison was made, however, to see whether
or not enrichment increased the coliforms. From 60 AGI
samples the same aliquots were placed on L-EMB as in
Brilliant Green Bile broth (BGB) enrichment. These were
then streaked onto L-EMB. The results were the same, in-
dicating that the direct plating without enrichment was
adequate for our purposes.
The selective m-Enterococcus (m-ENT) agar was utilized for
identification of F. Strep. Therefore, another selective
agar, Bile Eschelin Agar (BES) (Difco) was used along with
the catalase test.
Preliminary Field Data —
During the six months of the preliminary studies, thirty-two sample
visits were made to the Rosemount site. More than 500 individual sam-
ples (totaling several thousand liters of air) were obtained at dif-
ferent locations inside and outside the animal housing units and oxi-
dation ditch, at different heights above grade, and different distances
from the ditch. This series of studies concerned itself with the
problem of determining the quantity of bacteria present in the ambient
71
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air of an operational oxidation ditch.
The data obtained from these samples were analyzed to develop final
laboratory and field protocol which would be the standard for a
one-year study of the field oxidation ditch bioaerosol status and
public health hazard. In other words, the critical sampling sites,
the mean aerosol counts associated with these sites, and the magni-
tude of fluctuation that could be anticipated with time and location
were identified. Characteristic data from the preliminary study are
summarized in Tables 36 and 37. These data are from a procedure that
was constantly changing; for example, samples were taken from various
heights, sampling durations varied, different amounts of collecting
fluid were cultured, different collecting solutions were used, and
ditch conditions were dissimilar. The ventilation system in the
housing units was altered. Until March, 1972, the housing units and
ditch had a negative pressure airflow since exhaust fans were utilized.
At that time, the exhaust fans were removed and intake fans placed in
the housing unit to create a positive pressure flow in the units.
The ditch was intermittently operational and the rotor cover was
sometimes removed. The ditch cover over the east end of the ditch was
occasionally removed (thus eliminating a point source for exhaust).
Generally, 3- to 15-minute duration air samples were made with the AGI,
and samples were taken at 30- to 100-cm heights. Duplicate samples
were taken at each site. In the laboratory, 0.5 to 5 ml of collecting
fluid was cultured by a pour plate method and incubated 24 hours at
27° C, colonies were counted, and calculations of "total colony-for-
ming units per liter of air sampled (CFU/1)" were made (Table 36).
The preliminary data (Tables 36, 37 & 38 and Figure 11) showed that
probably the most important single factor influencing the counts
during this time was the presence or absence of cattle in the confine-
ment housing units. Tables 36 and 37 summarize several weeks of com-
parable data during which the major independent variable was the
presence or absence of cattle in the housing units. Counts are con-
siderably less when cattle are absent and are in the same magnitude
of counts in periods of human activity.
From the preliminary data, standard sampling locations were decided
upon. Table 36 and Figure 6 show that samples at varying distances
from the ditch east, south, north, and the field (100 meters west of
unit) have the same order of total bacterial counts, indicating that
the ditch itself did not have any profound effect on airborne counts
more than a few meters away from the ditch, thus eliminating the need
to continue sampling these areas. Instead, a 15-meter upwind and
downwind sample was chosen for extramural sampling. This sampling
procedure would warn of extraneous aerosol contamination or indict the
ditch as a source.
72
-------
Table 36. PRELIMINARY AEROSOL DATA TAKEN NOVEMBER 17, 1971 TO MAY 18, 1972 WHEN
CATTLE WERE PRESENT
Viable Bacterial Aerosols at Different Sampling Sites (AGI Counts); on Plate Count Agar, 37^, 24 hr.
(colony forming units/liter)
West
vent*
East
vent
West
barn
East
barn
Rotor
Field
Ditch
15 m E.
of unit
15 m S.
of unit
15 m N.
of unit
Number
of
aamples
Range
CFU/1
18
18
32
29
20
19
12
10
2-399 1-126 8-1311 12-1208 11-192 0.05-10 3-158 0.1-2.8 0.12-7 fr.06-1.3
39 21 234 261 46 1 50 1.4 1.4 0.5
OJ
Average
CFU/1
* West Vent, east vent, west barn, and east barn counts are from samples taken at 30 and 100 cm from
the ground; each sample represents the average of duplicates taken at that site.
This Table Separates the Above Sites into Sampled Heights, 30 and 100 cm
West vent
East vent
West barn
East barn
Field
30 cm 100 cm 30 cm 100 cm 30 cm 100 cm 30 cm
100 cm 30 cm
100 cm
Number
of
samples
13
15
11
21
10
19
16
Range
CFU/1
1-113 5-399
1-126 19-29
26- 12- 22-
1311 6-1078 1208 1069
.05-2
.3-. 6
Average
CFU/1
20
89
21 24
323
188
326
230
0.4
-------
WIND 21 km/
TEMP.- 10° C
R.H. - 67%
TURKEY
BUILDING
I 1 BOILER
| I HOUSE
OXIDATION
C
DITCH
CATTLE I
PEN I
|
Figure 11. Air sampling, locations at varying distances from
oxidation ditch
74
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Table 37. PRELIMINARY AEROSOL DATA TAKEN FEBRUARY 15 TO FEBRUARY 24, 1972 WHEN CATTLE WERE ABSENT
Viable Bacterial Aerosols at Different Sampling Sites (AGI counts); on Plate Count Agar, 37°C, 24 hr.
(colony-forming units/liter)
Number of
samples
Range
Average
West
vent
7
1-11
6
East
vent
6
0.4-11
3
West
barn
12
0.2-13
it-
East
barn
11
0.3-29
8.3
Rotor
2
1-4
2
Field
7
0.5-6
2
Ditch
3
0.8-7
3.1
15 m E.
of unit
3
0.16-0.9
0.59
15 m S.
of unit
4
0.5-4
1.6
15 m N.
of unit
2
0.2-1.1
0.6
Ul
Table 38. AIR SAMPLING AT VARYING DISTANCES FROM OXIDATION DITCH ON FEBRUARY 1, 1972
(colony-forming units/liter)
Figure 11 Location
Location description
CFU/1 on PCA
1
2
3
4
5
100 m N.E.
1.8 m S.W.
12 m S.W.
24 m S.W.
1.8 m N.E.
0.22
0.36
0.53
0.36
0.11
-------
Table 36 indicates that there is a difference in counts due to varying
elevations above ground, thus necessitating sampling at more than one
elevation above grade-.
The ditch samples, which were taken 100 cm above the surface of the
ditch slurry (but beneath the ditch covering), showed that high counts
were also directly dependent upon the presence of animals. This sam-
pling was discontinued because the results would not indicate any
relative degree of health hazard since animal or human exposure at
that location is uncommon.
The need for consistency in sampling to assure statistical significance
was recognized. To meet this need, the sampling procedure was set
up to sample for the same length of time at each site and to make the
same dilutions in each case. This consistency need was also shown in
preliminary experiments. This is workable because in nearly all lo-
cations a countable number of total bacterial colonies appear. A
sampling cart was constructed to transport pumps, hoses, samplers and
electrical cords, greatly facilitating the sampling.
The early investigation of total aerobic bacteria present in the am-
bient air surrounding the ditch gave clues as to how to develop a
technical sampling procedure. The detection of total aerobic bacteria
did not indicate the amount of contamination from the ditch itself.
The aerosol investigation of enteric indicator organisms (fecal Strep.
and coliforms) was therefore added to the protocol.
Phase II, Finalized Sampling Program
Final Protocol —
At the end of May, 1972, the objectives specified in the preliminary
stages of the project were completed. We had demonstrated the feasi-
bility of an aerosol monitoring program and had learned enough about
the technology, the environment, and the bioaerosol itself to design
a sampling protocol whose results would be meaningful to both public
health and air researchers.
The protocol followed was:
1. Sampling visits were to be made once every 7 ± 3 days.
2. Sampling would be at each of the following 7 sites:
At the west vent on ditch housing
At the east vent on ditch housing
Inside the west housing =, unit
Inside the east housing unit
76
-------
Inside the rotor housing close to the turning rotor
Extramural site 15 meters upwind of the ditch
Extramural site 15 meters downwind of the ditch
3. At each site (except in the rotor housing) three samples
were taken concurrently at 3 heights above the ground:
30 cm (ground level), 100 cm (cattle respiratory level),
and 160 cm (human respiratory level). AGI nozzle openings
were positioned to face toward any wind movement. Con-
current sampling devices are shown in Figures 12 and 13.
4. The rotor housing site was sampled by duplicate AGIs
operating simultaneously at approximately 100 cm above
the ditch slurry surface.
5. Each sample consisted of 62.5 liters (i.e., a 5-minute
duration AGI sample) of air drawn into 30 ml of AGI
collecting fluid (0.1% peptone and 0.01% Dow antifoam).
6. AGI samples were transported to the laboratory in a refri-
gerated thermal chest.
7. In the laboratory, the volumes of the AGI were made up to
30 ml with sterile diluent (to replace any evaporation loss)
and 0.5>-ml aliquots of the collecting fluids were plated
in duplicate on prepared Plate Count Agar*, PCA (Tryptone-
Glucose-Yeast Extract), on Levine's-EMB (L-EMB), and on
m-Enterococcus* (m-Ent) agar. The aliquots were uniformly
distributed over the medium surface with sterile, bent
glass rods in a sterile hood. The plates were incubated
at 37° C for 48 hours. Counts were reported as Colony-
Forming Units per liter of air (CFU/1).
a. Total CFU/1, all colonies on PCA were counted for
computation.
b. Coliform CFU/1; large, pigmented colonies on L-EMB
that were gram negative rods and fermented lactose
with production of gas on Triple Sugar Iron* (TSI)
were counted for computation.
c. Fecal Streptococci CFU/1; red colonies on m-Ent that
were gram positive cocci, catalase negative, and that
turned Bile Eschelin Agar* (BSA) black were counted.
*Difco
77
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Figure 12. Concurrent sampling device in barn
78
-------
Figure 13. Concurrent sampling device outside of barn
Period of Sampling —
Using the standardized protocol described above, the routine monitoring
program was initiated on May 25, 1972. From that date to June 13, 1973,
47 sampling visits were made to Rosemount. A full year cycle of cli-
mate was included during the sampling period.
RESULTS AND DISCUSSION
Data
The data from the 47 sampling visits are summarized in Tables 39 and
40.
Height of Sample
The air sampling at different heights showed a definite pattern. For
example, at both east and west vents the 100-cm samplers gave, on the
average, higher counts. This was probably due to the fact that
79
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Table 39. AEROSOL DATA TAKEN WEEKLY MAY 25, 1972 TO JUNE 13, 1973 WHEN
CATTLE PEESENT - ROSEMOUNT
Viable Bacterial Aerosols at Different Sites (AGI Counts) on PCA
(Total), L-EMB (Coliform), and m-Ent (Fecal Streptococcus), 37°C,
48 hr. Counts Given in Colony-Forming Units/Liter (CFU/1)
Location
West
vent
East
vent
West
barn
East
barn
Height
Jam)
30
100
160
30
100
160
30
100
150
30
100
160
Organism
Total
Coliform
F. Strep.
Total
Coliform
F. Strep.
Total
Coliform
F. Strep.
Total
Coliform
F. Strep.
Total
Coliform
F. Strep.
Total
Coliform
F. Strep.
Total
Coliform
F. Strep.
Total
Coliform
F. Strep.
Total
Coliform
F. Strep.
Total
Coliform
F. Strep.
Total
Coliform
F. Stre£.
Total
Coliform
F. Strep.
Number
Of
samples
38
36
37
41
40
40
41
39
38
35
36
34
37
35
35
33
32
31
39
34
37
37
35
37
41
39
38
33
34
34
41
34
38
40
37
37
Total
CFU/1
657
0
1
2145
4
2
892
3
0
395
3
4
1856
4
10
648
0
2
5132
14
13
4411
8
25
4199
5
3
7991
10
31
8814
3
26
6418
5
47
Range
CFU/1
0-156
0-0
0-1
2-194
0-2
0-1
1-276
0-3
0-0
0-172
0-3
0-4
0-268
0-2
0-5
0-124
0-0
0-1
1-580
0-8
0-4
4-359
0-5
0-5
1-524
0-2
0-5
9-1428
0-10
0-14
4-837
0-3
0-5
9-794
0-3
0-33
Average
CFU/1
17
0
0.03
52
0.1
0.05
22
0.08
0
11
0.08
0.12
50
0.11
0.29
19
0
0.06
132
0.41
0.35
119
0.23
0.68
102
0.13
0.34
242
0.3
0.9
215
0.09
0.7
160
0.14
1.3
Average
CFU/1
3 heiehts
31
28
117
204
80
-------
Table 39 (continued). AEROSOL DATA TAKEN WEEKLY MAY 25, 1972 TO
JUNE 13, 1973 WHEN CATTLE PRESENT - ROSEMOUNT
Location
Rotor
Upwind
Downwind
Height
(cm)
30
100
160
30
100
160
Organism
Total
Coliform
F. Strep.
Total
Coliform
F. Strep.
Total
Coliform
F. Strep.
Total
Coliform
F. Strep.
Total
Coliform
F. Strep.
Total
Coliform
F. Strep.
Total
Coliform
F. Strep.
Number
Of
samples
40
38
37
40
37
37
39
38
37
38
35
36
39
37
37
39
38
38
38
35
35
Total Range
CFU/1 CFU/1
2188 2-191
6.5 0-5
5.5 0-2
261 0-120
0 0-00
0 0-00
370 0-260
0 0-0
0 0-0
181 0-29
0 0-0
0 0-0
178 0-40
0 0-0
1 0-1
359 0-116
0 0-0
1 0-1
137 0-41
0 0-0
0 0-n
Average
CFU/1
55
0.17
0.15
6.5
0
0
9.5
0
0
4.8
0
0
4.5
0
0.05
9.1
0
0.03
3.6
0
0
Average
CFU/1
3 heights
55
7
6
81
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Table 40. AEROSOL DATA TAKEN AUGUST 9, 1972 TO SEPTEMBER 13, 1972 AND
MARCH 1, 1973 TO MARCH 5, 1973 - CATTLE ABSENT
Location
West
vent
East
vent
West
barn
East
barn
Rotor
Height
(cm)
30
100
160
30
100
160
30
100
160
30
100
160
Organism
Total
Coliform
F. Strep.
Total
Coliform
F. Strep
Total
Coliform
F. Strep
Total
Coliform
F. Strep
Total
Coliform
F. Strep
Total
Coliform
F. Strep
Total
Coliform
F. Strep
Total
Coliform
F. Strep.
Total
Coliform
F. Strejr.
Total
Coliform
F. Strep.
Total
Coliform
F. Strep.
Total
Coliform
F. Strep.
Total
Coliform
F. Strep.
Number
of
samples
8
8
8
8
8
8
8
8
8
8
8
8
7
8
8
8
8
8
6
6
6
7
7
7
7
7
7
8
8
8
8
8
8
8
8
8
8
8
8
Total
CFU/lj
30
0
0
55
0
0
45
0
0
194
0
0
41
0
0
34
0
0
251
0
0
141
0
3
86
0
0
26
0
0
39
0
0
170
0
0
130
0
1
Range
CFU/1
2-8
0-0
0-0
2-18
0-0
0-0
0-16
0-0
0-0
0-182
0-0
0-0
0-12
0-0
0-0
0-10
0-0
0-0
12-131
0-0
0-0
10-32
0-0
0-2
6-25
0-0
0-0
0-7
0-0
0-0
0-14
0-0
0-0
0-148
0-0
0-0
8028
0-0
0-0.5
Average
CFU/1
3.8
0
0
6.9
0
0
5.6
0
0
24
0
0
5.9
0
0
4.2
0
0
42
0
0
20
0
0.42
12
0
0
3.2
0
0
4.9
0
0
21
0
0
16
0
0.12
Average
CFU/1
3 heights
5.4
12
24
10
16
82
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Table 40. AEROSOL DATA TAKEN AUGUST 9, 1972 TO SEPTEMBER 13, 1972 AND
MARCH 1, 1973 TO MARCH 5, 1973
Height Number
of
Location (cm) Organism samples
30
Upwind 100
160
30
Downwind 100
160
Total
Coliform
F. Strep.
Total
Coliform
F. Strep.
Total
Coliform
F. Strep.
Total
Coliform
F. Strep.
Total
Coliform
F. Strep.
Total
Coliform
F. Strep.
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
- CATTLE ABSENT
Total Range
CFU/1 CFU/1
6
0
0
19
0
0
13
0
0
14
0
0
16
0
0
10
0
0
0-6
0-0
0-0
0-9
0-0
0-0
0-6
0-0
0-0
0-6
0-0
0-0
0-6
0-0
0-0
0-2
0-0
0-0
Average
Average CFU/1
(CFU/1) 3 heights
0.75
0
0
2.4
0 1.6
0
1.6
0
0
1.8
0
0
2
0 1.7
0
1.2
0
0
the 100-cm height is most directly in line with the vent. The other
significant variation was in the two cattle housing units. In both
barns the highest bioaerosol concentrations were at the 30-cm height
and the lowest at the 160-cm height. This may mean that there is
some ground contamination that does not reach the higher heights. The
air is, however, being forced down through the slats and out due to
the positive pressure ventilation system. The extramural counts both
upwind and downwind showed the highest bioaerosol to be at the 100-cm
height. In essence, however, the counts do not vary considerably
from one height to another at any one site.
Housing Units
It is evident from the data that the air associated with the animal
housing units harbors a rich bioaerosol. Counts approximately 100
to 200 total CFU/liter are between one and two orders of magnitude
higher than those normally experienced by humans in non-agricultural
occupations. It is equally evident that these high counts can be
83
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attributed almost entirely to the active, crowded presence of the
livestock and not to the manure disposal system - the oxidation ditch
method of extended aerobic treatment. When the steers were removed
from the barn, within less than one hour, the bioload decreased to
levels approximating those of the extramural environment (Table 40),
even though the manure in the ditch was still being aerated by the
rotor. This phenomenon is remarkably similar to the one already pre-
sented in Table 37 of the preliminary data. In essence, it may be
said that the oxidation ditch contributes an insignificant number of
microbes to the air when compared to the air when compared to the
contribution of the living animals themselves.
Indicator Fecal Organisms
The numbers of indicator fecal bacteria enumerated in Tables 39 and
40 show that the high concentration is in the barns when animals are
present. This is a strong indication that aerobic fecal contamination
from the oxidation ditch, if present, is insignificant when compared
to the contribution from the animals themselves.
Wetness of Manure
A wet condition of manure wastes, whether in the ditch slurry or the
intramural surfaces of the housing units, inhibits aerosolization of
bacteria. In contrast, dry matter of varying size particles becomes
airborne by a slight amount of disturbance. For example, on one sam-
pling day during preliminary investigations, animals were not present
and therefore not wetting down the slats by urination. A worker had
been in both barns producing dust by scraping off the accumulated
residue on the slats. In both barns, counts were greater than 2,000
CFU/1, an unusually high level. On other days of the period when
animals were absent and no scraping occurred, the counts averaged less
than 10 total CFU/1.
Extramural Contamination
It was particularly gratifying to note that the extramural contamina-
tion levels, just a few dozen meters away from the barns, are con-
siderably lower than expected. This implies that the barn aerosols
do not persist for any significant distances or time periods extra-
mural ly .
Rotor Site
The fecal counts at the rotor site are most probably attributable to
the ditch liquid, since the rotor is enclosed and open only to the
ditch. This site has a microbioaerosol load which also fluctuates
84
-------
according to the presence or absence of animals. Table 41 indicates
that even with the rotor off and thus no splashing, the counts in this
area can be high. To further substantiate that the aerosol produc-
tion was from the animals rather than the ditch slurry, sampling was
done when the ditch contained only 6 cm of water and the rotor was
stopped. Table 42 indicates that the counts are similar to conditions
in Table 39 where the rotor is operating and the ditch slurry is
higher.
Particulate Nuclei Theory
It would seem however, that due to the documented particulate nuclei
theory (20) some of the ditch liquid would be aerosolized. Why then
were there so few viable aerosols from the ditch? The three possible
answers are: (1) bioaerosols created by the ditch are present but in
a much lower concentration than is contributed by the animals and
associated dust; (2) the physics of the manure slurry may inhibit the
biological droplet nuclei production from the ditch due to the con-
centration of bacteria in the ditch; and (3) there is rapid die off
of slurry resident microbes either in the slurry or in the air.
Comparison With Previously Reported Results
To evaluate the first possibility, other situations which produce drop-
let nuclei from a bacterial contaminate source were examined. Ladd
(26), when sampling near a sewage pre-aeration basin, utilizing the
Anderson-sieve sampler, was able to pick up tracer organisms that
were poured down the laboratory drain approximately 8 km from the sew-
age plant. He was able to detect the organism at a maximum concen-
tration of 0.008 CFU/1 5 hours after one pouring but none after the
eleventh hour. Sewage has a much lower solids content than existent
at all times in the oxidation ditch.
Randall (27) found in sampling air downwind from activated sludge units
with an Anderson sampler that, on the average, 17 CFU/1 of particle
size less than 5 microns were found downwind of the operation (6% of
which were Klebsiella).
Napolitano (28) studied concentrations of aerosols generated by activa-
ted sludge plants using EMB in an Anderson sampler. The highest count
obtained was 8 CFU/1 (no further selection was done). Further down-
wind at 15 and 30 meters, 0.18 CFU/1 was found.
Albrecht (29) found when sampling with both a Wells centrifuge and
midget impinger downwind from a trickling filter sewage treatment
plant that very few coliform organisms were recovered but growth on
nutrient agar showed recovery from 0.07 to 5 CFU/1 at the edge of the
85
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Table 41. BIOAEROSOL DATA TAKEN ON MARCH 28, 1973
WHEN DITCH HAD ONLY 6 cm WATER WITH ROTOR OFF
(colony-forming units/liter)
Location
West vent
East vent
West barn
East barn
Rotor
Upwind
Downwind
Average CFU/1 (total) over all 3 heights
40
50
80
144
92
2
0.3
Table 42. BIOAEROSOL DATA TAKEN AT ROTOR SITE WHEN ROTOR OFF
(colony-forming units/liter)
Date
Average total CFU/1
July 25, 1972
March 28, 1973
48
92
86
-------
trickling filter decreasing to 0.2 to 2.7 CFU/1 at a distance of 15
meters.
Adams (30) sampled the air with EMB in an Anderson sampler near a trick-
ling filter. Without classifying bacteria any further than counting
colonies on media, he found 19 CFU/1 at 15 meters downwind; 0.9 CFU/1
at 43 meters downwind and 0.003 CFU/1 at 1300 meters downwind.
In most of these instances, the bioaerosol load is similar (the extra-
mural counts of the oxidation ditch producing the lowest counts),
indicating that the production of bioaerosols of lung-penetrating size
from contaminated liquid sources is not as high as dry sources - the
Rosemount housing units for example. The following are also examples.
Cvjetanovic (31) monitored areas with a slit sampler and found total
bacteria in surgery rooms to be 0.5-2 CFU/1, hospital corridors 6
CFU/1, animal houses 7-70 CFU/1, and gyms 1-13 CFU/1.
Duguid (32) found, when sampling for total bacteria with a slit samp-
ler, that when a person is standing motionless in a room, 0.3 CFU/1
was produced; when there was slight activity, 4.3 CFU/1; vigorous ac-
tivity produced 28 CFU/1; and undressing and dressing produced 56
CFU/1.
Examining the physics of the situation, that is:
106 bacteria = 0.01 bacterium
1 sq. cm 1 sq. micron
it is doubtful that the concentration of ditch bacteria is high enough
to be incorporated into droplet nuclei. There are approximately 106
total bacteria in a ml of ditch liquid. At this level, the chances
of having a bacterial droplet nucleus 1 micron in size is 1 to 100.
Darlow (23), however, found that a tenfold reduction of total inoculum
did not reduce aerosol concentration proportionately. When sampling
with a slit sampler at seat level of a flushing toilet, he found
approximately 1 CFU/1 when the bacterial concentration in the toilet
was 5 x 105 cells/ml. He found that the number of bioaerosols produced
was reduced threefold when the toilet lid was open vs. closed. If these
results can be extrapolated to the ditch system, it seems that the ditch
(being a closed area except at the rotor site where sampling was done
100 cm above the surface) would produce less than 1 CFU/1 and, therefore,
is below the level of detection employed.
A literature review concerning the properties of the atmosphere which
affect the airborne survival of bacteria pointed out that unknown fac-
tors could be influencing the counts. For example, Webb (33, 34)
87
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studied the effects of chemical additives on airborne cells and found
that certain chemicals in the ditch could be influencing the survival.
Chemical composition also affects the evaporation rate of a droplet,
thus affecting the production of droplet nuclei (35).
Other studies showed that temperature and relative humidity (R.H.)
affected the airborne survival rate of bacteria (36, 37). The data
of this study was examined for patterns of counts which could be pre-
dicted due to meteorological variables, thereby modifying the tempera-
ture and R.H. if possible. The extramural atmosphere is, of course,
not subject to modification. However, no pattern could be identified.
Seasonal changes did not result in any change in counts. Table 43
illustrates the absence of seasonal meteorological influence upon
counts. Wright (38) also found no correlation between counts and
meteorological factors.
Table 43. INFLUENCE OF METEOROLOGICAL CHANGES ON EXTRAMURAL UPWIND
COUNTS
Extreme Meteorological Conditions Give Same Counts
Date Wind Temp RH%
Total CFU/1
Sky 30 em 100 cm 160 cm
12/20/72 11 km/hr (SE) -2 C 88 Overcast 0 0
5/16/73 30 km/hr (N) 9 C 48 Clear 0 0
0
0
Same Conditions Give Extreme Counts
Total CFU/1
Date
4/18/73
5/30/73
24
13
Wind
km/hr
km/hr
(SE)
(SW)
Temp
20 C
21 C
RH%
48
49
Sky
Clear
Clear
30 cm
0
9
100 cm
0
22
160
0
29
cm
Calculation of the average temperature and R.H. in the barns shows
that, for the west barn the average temperature was 19° C and R.H.
73%, while for the east barn it was 17° C, 75% R.H. This small
88
-------
difference then does not account for the higher concentration of bac-
teria in the east barn. This higher concentration is believed to be
due to the only known difference between barns, the slat operation,
allowing less air to pass through and, thus, less mass movement of
air containing aerosols from the housing unit.
Gaseous Odor Effects
One contributable alteration was the noticeable effect of a gaseous
odor upon counts. This odor of some sort of fuel in the west barn was
noticed on three days of sampling. Table 44 shows the reduced bio-
aerosol counts in the west barn when the odor was present, but the
counts in other areas were near average. The odor was determined to
be from preservative-treated lumber used to repair the inside of the
housing unit.
Table 44. BIOAEROSOL DATA TAKEN WHEN WEST BARN SHELLED OF FUEL ODOR
(average total CFU/1 over all 3 heights)
Location March 1, 1973 March 16. 1973 March 23. 1973 Average
West vent
East vent
West barn
East barn
Rotor
Upwind
Downwind
8
25
47
153
ND
2
8
7
4
7
553
ND
0
3
4
5
6
20
2
ND
ND
6
11
20
242
2
1
5.5
Health Hazard Evaluation
Essentially, the Rosemount study shows that the public health hazard
associated with the aerosols from the ditch is much less than that
associated with the animal housing operation.
89
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Comparison of Rosemount to Other Waste Treatment Facilities
Other University of Minnesota cattle waste treatment facilities were
in operation at Morris, Minnesota. These operations afforded the
opportunity to sample different systems to further evaluate microbio-
aerosol production in a beef animal facility.
At Morris, air samples were taken with the AGI inside three different
anima] housing units and at the exhaust air outlet venting pit area of
one of the enclosed slatted animal housing units. One of the housing
units sampled was roofed but open on one side; the animals were housed
on slats above a standing quiescent anaerobic pit (open-slat). Ano-
ther housing unit sampled was open on one side and the animals were
bedded with hay (open-bedded). The third unit was a warm, enclosed,
slatted floor beef unit with capacity for 70 animals. A quiescent
anaerobic pit collects the waste below the slats. The temperature,
R.H. and ventilation are similar to the Rosemount facility. The sam-
ples were taken at three heights consecutively (30, 100, 160 cm) ex-
cept at the vent where the sampling was directly in front of the vent.
The samples were analyzed for total, coliform, and fecal streptococci
organisms in the same manner as Rosemount.
Table 45 gives the bioaerosol counts of the Morris beef animal facility,
The results are remarkably similar to those taken at Rosemount. Ex-
tramural counts are zero CFU/1 and counts taken at the vent are 48
CFO/1. The slatted housing units, both open and closed, show bioaero-
sol counts decrease as the elevation of the sample increases. The
open bedded counts are believed to be lower than the other housing
units because the wind was blowing from the AGIs toward the animals
and hay. The comparison sampling of these other animal housing units
substantiates the counts of the Rosemount area. Note that the pit
holding tanks were not aerated, thus eliminating droplet nuclei for-
mation.
From the various sampling done, the expected microbioaerosol total and
indicator counts from the oxidation ditch manure disposal system and
the animal confinement areas of such systems have been revealed. How-
ever, the public health hazard can at this time only be assessed by
comparison with other waste management operations. For example, if it
takes a certain number of bacteria to infect, a comparison can be made
to the infection danger of other systems by evaluating the bioload of
fecal bioaerosols in those systems and indirectly assess the hazard.
For this reason and to compare a traditional system utilizing our pro-
tocol, lab, and personnel, a program of aerosol sampling at a St. Paul
Campus dairy barn was conducted from February 21, 1973, through June
11, 1973. The results of this investigation gave another parameter
with which to determine the relative public (occupational) health
90
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Table 45. MORRIS SAMPLING WITH AGI
(colony-forming units/liter of air sampled)
Location
Upwind
Open-slat
Open-bedded
Enclosed-slat
Exhaust-vent
Total
0
156
18
175
48
30 cm
Colif.
0
0
0
0
0
F. Strep.
0
3
0
2
0
Total
0
72
31
83
ND
100 cm
Colif.
0
0
0
0
ND
F. Strep.
0
2
0
3
ND
Total
ND
12
59
40
ND
160 cm
Colif .
ND
0
0
0
ND
F. Strep.
ND
0
0
0
ND
Sampling date: March 13, 1973
Extramural conditions: 13° C, 78% RH
Intramural conditions: 18° C, 55% RH
(enclosed-slat)
Partly cloudy sky
-------
hazard of an oxidation ditch to traditional methods of waste handling
uSSzed ifaniinal husbandry. This study continued on a weekly basis
to obtain microabtoaerosol data for comparison with the Rosemount in-
formation. The dairy barn manure disposal is by gutter cleaner, shovel,
wasSng when require!, and sweeping. Continuously operating fans ex-
haust air from the barn. Samples were taken in the center of the
alleyway between the two rows of producing cows. The samples were
analyzed for total plate count, coliform, and fecal Streptococci in
the same manner as at Rosemount.
Samples in the dairy bam were taken consecutively parting at the
l^est level (30 cm) and proceeding to the highest (160 cm) at a single
location, once in the morning and once in the afternoon, in °'deV;O
monitor variable conditions. In the morning, the animals were quiet,
in the afternoon the animals were eating and the floors were being
swept. Table 46 shows that the counts are considerably higher
during the afternoon activity. The counts show that the animals and
workers in the area are exposed to comparable numbers of total and
fecal bioaerosols to the Rosemount area. This is and has been a tra-
ditional system that has withstood any changes of xts environment due
to a public health necessity.
This comparative study and the previously given studies have given us
information with which to evaluate the oxidation ditch waste disposal
system to be non-hazardous to public health as far as the potential
contamination of microbioaerosols from the oxidation ditch.
92
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Table 46. DAIRY BARN SAMPLING
Aerosol Data Taken Weekly February 21, 1973 - June 11, 1973
When Cattle Present
Viable Bacterial Aerosols During Different Conditions (AGI Counts) on PCA (total),
L-EMB (coliform), and m-Ent (Fecal Streptococcus), 37°C, 24 hr.
Counts Given in Colony-Forming Units/Liter (CFU/1)
Height
above
floor
30 cm
100 cm
160 cm
Organism
Total
Coliform
F. Strep.
Total
Coliform
F. Strep.
Total
Co 11 form
F. Strep.
Quiet conditions
Number
of
samples
14
12
14
15
13
15
15
13
15
Range Average
CFU/1 CFU/1
38-266 112
0-0 0
0-3 0.43
22-222 92
0-0 0
Q-2 0.2
40-240 107
0-1 0.15
0-2 0.4
Sweeping and/or eating
Number
of Range Average
samples CFU/1 CFU/1
12 75-826 294
12 0-3 0.25
12 0-5 0.67
12 57-1168 370
12 0-0 0
12 0-3 0.67
12 78-820 261
12 0.-1 0.08
12 0-3 0.67
Compilation of
all 3 heights ,
both conditions
Ave rage
Total
CFU/1: 196
Average
Coliform
CFU/1: 0.08
Average
F. Strep.
CFU/1: 0.49
oo
-------
SECTION VI
MODEL B OXIDATION DITCH STUDIES
INTRODUCTION
The model oxidation ditch, MOD-B, was situated in the Agricultural En-
gineering Waste Management laboratory to conduct studies on the degra-
dation of waste. Field-scale oxidation ditches exposed to the constantly
changing environment have been studied extensively by Moore et. al.
(39) . More work was needed to study the degradation of beef manure
under more closely controlled conditions.
OBJECTIVE
The objective of the study was to determine relationships between tem-
perature and rotor speed and the breakdown of the beef waste under
controlled conditions simulating the field oxidation ditch at Rose-
mount.
MATERIALS AND METHODS
Model B Oxidation Ditch
The Model-B oxidation ditch was a modified version of the model used
by Diesch and Allred (40) during the initial Public Health Service
Grant Project. A steel, four-wheeled cart supported the l/10th scale
plexiglas model ditch on a steel frame. A variable speed reducer pro-
vided the power through a chain drive to the brush rotor in one end
of the ditch. To achieve the objective in the 2-year period instead
of the planned 3-year period, a third model ditch (MOD-C) was con-
structed and used during 1973.
Environmental Control Chamber^
To provide a controlled temperature environment and constant humidity
control, a control chamber was purchased and installed. Made up of
prefabricated sections of aluminum-clad styrofoam, the chamber was
easily constructed in the Agricultural Engineering Waste Management
Laboratory. The chamber is approximately 4 meters by 3 meters and 3
meters high. A water chiller unit was mounted on the roof and a dif-
fuser with a fan mounted inside the chamber. The temperature was con-
trolled by a thermostat which regulated a Jabsco pump that moved
water through a flexible hose from the water chiller to the diffuser.
Experimental Procedure
The Environmental control chamber was adjusted to one of the three
94
-------
temperatures for a specific test. Table 47 shows the various combina-
tions of rotor speed and temperature investigated. Next, the rotor
speed was adjusted and tested using a hand hold tachometer and a stop
watch. The ditch in the chamber was then filled with tap water to a
depth of 4.5 cm (1 3/4"). With the rotor operating, fresh beef cattle
feces were added until a level of approximately 0.25% total solids was
reached. After operating for several days to get a steady population
of microorganisms the tests were started. Fresh feces were added each
day during the work week. Wet chemical tests were performed twice a
week.
Table 47. EXPERIMENTS CONDUCTED ON MODEL B
OXIDATION DITCH
Dates
Shakedown run only
5-31-72 to 7-7-72
7-17-73 to 8-28-73
9-1-72 to 10-17-72
7-20-73 to 8-28-73
3-5-73 to 4-13-73
4-19-73 to 5-25-73
5-23-73 to 6-21-73
6-14-73 to 7-19-73
6-21-73 to 8-9-73
Run
#1
#2
#3
#4
#5
#6
#7
#8
#9
#10
Rotor Speed
(RPM)
180
380
275
180
275
380
275
380
180
Temperature
(°C)
20°
20°
20°
2°
2°
2°
10°
10°
10°
Laboratory Procedures^
The procedures described in Standard Methods for the Examination of
95
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Water and Waste Water, 13th Edition, 1971, were used to analyze COD,
solids, total nitrogen, and phosphate. Devarda's Alloy Method was
used in the determination of nitrate. Carbon and hydrogen were deter-
mined using a Coleman model 33 carbon hydrogen analyzer. Dissolved
oxygen was monitored using a Yellow Springs Instrument model 54
oxygen meter and pH was monitored with a Heath model EU-20-31 pH
recorder and electrometer.
RESULTS AND DISCUSSION
Data
The data from tests 2 through 10 on the model oxidation ditch B are
presented in tables 48 through 56 following this section.
Dissolved Oxygen Levels in the Ditch Slurry
One of the primary benefits derived from using an oxidation ditch is
that aerobic conditions are maintained as the animal waste is degraded.
Odor-free operating conditions are a result of this aerobic degrada-
tion. The dissolved oxygen levels indicate the amount of oxygen
available for the aerobic microorganisms. At no time during any of
the tests did the dissolved oxygen level even approach zero. In fact,
during the most severe test, at Summer temperature 20° C and slow
rotor speed (180 RPM), the D.O. reached 2.0 mg/1 on one occasion.
During cold-weather tests at 2° C, the D.O. was normally above 12
mg/1.
Evaporation From the Ditch
There was need to continually add water to the ditch to maintain the
desired level of slurry in the ditch. This was expected because the
aerobic decomposition products include water vapor. In the field
ditch, leaking waters and spilled water provided additional liquid
which was not added in the laboratory study. Also, it was observed
that the storage volume in field oxidation ditches did not build up
as fast as expected, due to evaporation. Thus, a slight benefit
was gained as less storage space was needed for extended detention
with the oxidation ditch when compared to anaerobic, liquid manure
tanks.
pH Values
The pH of the slurry was either neutral or slightly basic during all
of the tests. The aerobic process does not promote formation of acid
as is the case with the anaerobic process of degradation. Very little
fluctuation of pH occurred during the tests, from which it could be
96
-------
generalized that the system was well buffered also.
Total Solids Content
As would be expected, the total solids (TS) content of the slurry in-
creased as the individual test progressed. Because solids were being
added and the only solids removed were those in test samples, the TS
should continually increase. Also, because the solids added are not
all volatile (able to be biologically degraded) but partly ash, the
continual increase in TS was expected. Storage of the solids is
necessary and also affects the ability of the rotor in transferring
oxygen to the slurry. As a result, lower dissolved oxygen levels
were observed as the test would progress to higher solids content
levels. For example, in test number 2 on May 31, 1972, the TS was
9,232 mg/1 and the D.O. was 6.9 mg/1. In contrast, on July 7, 1972,
the TS was 12,562 mg/1 and the D.O. had dropped to 4.35 mg/1. The
tests did not continue as long as some which have been conducted in
the field oxidation ditch at Rosemount. The TS contents in the field
ditch have been allowed to reach at least 60,000 mg/1 on several
occasions. The dissolved oxygen has approached zero and the movement
of the ditch slurry slowed considerably. The model ditch reacted
within the scope of the experiment, as did the field ditch. Total
Solids is a prime indication of physical load being placed on the
moving rotor as the rotor lifts, aerates, and propels the slurry in
the raceway-shaped tank. When TS are elevated, more energy from the
rotor is expended moving the slurry and less is available to oxygenate
the slurry. The model ditch was able to maintain aerobic conditions
in the slurry at the loading levels imposed during these tests.
Nitrate in the Slurry
No conclusions could be derived from the nitrate tests performed.
The levels varied from no measurable amount to 175 mg/1.
Phosphate in the Slurry
Phosphate should be conserved in a closed system such as the oxidation
ditch. Phosphorus is not in volatile compounds normally associated
with bacterial degradation. The phosphate data have an increasing
trend in all tests with the dips probably related to sampling and
analytical errors. When the slurry is returned to the land at the
time the ditch contents are removed, the phosphate will again be in
the productive part of the phosphate cycle.
Total Volatile Solids
The total volatile solids (TVS) were affected by temperature. At the
97
-------
lower temperature, 2° C, the TVS were a larger fraction of the waste
because biological breakdown was progressing at a much slower rate.
Total Carbon
In each test, total carbon increased as more material was added. The
increase followed that of TVS as would be expected, since both are
indicative of organic matter in the slurry. Carbon levels were higher
in the test at colder temperatures, which indicates slower degradation
and buildup of organic matter.
Chemical Oxygen Demand
The chemical oxygen demand 'test (COD) is a measure of the waste
strength in terms of oxidizable matter. Figures 14, 15, and 16 show
COD vs. Time for the various BPMs at constant temperature on each
figure. At cold temperatures, the organic matter builds up at a
faster rate than at the 20° C temperature. At 20° C, KPM has no ob-
servable effect on breakdown rate as shown by the parallel trend of
COD buildup.
At 10° C an acclimatization effect with the COD buildup trend rising
sooner at 275 RPM occurred, then the 380 RPM, and, finally, the 180
RPM. This may be due to start-up problems and bacteria population
in the ditch. Overall breakdown is less as rotor speed is decreased.
At 2° C, the least amount of breakdown was occurring. Population
growth is slower at low temperatures so the breakdown is as expected.
Rotor speed does not show a noticeable effect because there is an
abundance of oxygen in the cold water to satisfy the aerobic micro-
organisms .
98
-------
Q
O
14,000
12,000
10,000
8,000-
6,000 -
4,000-
2,000 -
275 R.PM.
: TV
- X*
- a*/
//
1 1 1
10
20 30
i i i
40 50 60
TIME, DAYS
Figure 14. Chemical oxygen demand vs. time at 20° C for 3 RPMs
99
-------
18,000
16.000
14,000
12,000
10,000
Q
g 8,000
6,000
4,000
J 275 RPM.
180 RPM.
10
20 30 40
TIME, DAYS
50
Figure 15. "Chemical oxygen demand vs. time at 10° C for 3 RPMs
100
-------
18,000
16,000
14,000
\
E 12,000
0
<-> 10,000
8,000
6,000
4,000
/
/
/
^3
^^~ **
1
1
,'275 R.PM.
1
I
I
1
i
1
: ft
1 1
|i 380 R.PM.
i
• * •'<
• /" Y"'t!
~ / \ / ^
/q / \
y\ y \i80 RPM
L_ | I 1 -1 J
in ?0 3O 40 50 bO
TIME, DAYS
Figure 16. Chemical oxygen demand vs. time at 2° C for 3 RPMs
101
-------
Table 48. RUN NUMBER 2, ROTOR SPEED 180 RPM, TEMP. 20° C
Date
5-31-72
6-1-72
6-2-72
6-5-72
6-6-72
6-7-72
6-8-72
6-9-72
6-12-72
6-13-72
6-14-72
6-15-72
6-16-72
Waste
added
(g)
130.0
137.7
132.4
120.0
130.6
128.8
135.3
131.6
151.0
143.5
164.6
143.7
130.1
H20
added
(1)
8
4
4
ND
8
4
4
4
4
4
ND
4
4
Total
solids
(mg/1)
9,232
8,243
8,360
7,236
8,140
6,449
6,865
8,422
9.669
9,844
ND
10,255
11,618
Total
volatile
solids
(mg/1)
5,747
5,315
5,593
4,518
5,322
4,111
4,417
5,782
6,610
6,835
ND
7,149
8,372
PH
8.5
8.5
8.1
8.7
8.3
8.8
8.1
8.0
8.15
8.15
ND
8.05
8.25
Dis-
solved
oxygen
(mg/1)
6.9
7.1
4.6
7.4
6.6
4.0
3.9
8.3
6.7
5.0
ND
4.3
5.9
Chemical
oxygen
demand
(mg/1)
6,784
6,762 1
14,221
6,646
7,805
5,752
ND
7,770
7,126
7,918
7,170
9,042
7,548
PO,
(mg/1)
813
,056
ND
ND
590
437
237
553
581
ND
586
513
ND
NO 3
(mg/1)
175.0
ND
41.72
15.85
ND
55.0
8.24
ND
ND
ND
ND
3.66
1.93
Total
nitrogen
(mg/1)
384.3
377,4
140.0
350.3
374.6
271.6
259.5
336.5
138.2
274.0
ND
195.0
450.0
Car-
bon
U)
27.58
27.72
29 . 83
29.55
30.36
28.49
29.88
31.13
32.82
32.20
ND
32.78
35.30
Hydro-
gen
U)
4.32
4.39
4.54
4.51
4.67
4.45
4.56
4.88
4.87
5.15
ND
5.25
5.43
-------
Table 48 (continued). RUN NUMBER 2, ROTOR SPEED 180 RPM, TEMP'. 20° C
o
10
Date
6-19-72
6-20-72
6-21-72
6-22-72
6-23-72
6-26-72
6-27-72
6-29-72
6-30-72
7-3-72
7-5-72
7-6-72
7-7-72
Waste
added
(0
143.3
138.0
143.0
143.5
145.3
145.5
145.1
144.0
132.7
145.0
147.4
139.2
148.8
H-O Total
added solids
(1) (mg/1)
4
4
ND
4
4
ND
4
4
4
ND
ND
ND
10,063
11,072
10,993
11,124
10,499
10,870
ND
13,083
ND
12,957
ND
12,562
ND
Total Dis-
volatile solved
solids oxygen
(mg/1) pH (me/I)
6,950
8,228
7,897
8,180
7,469
7,727
ND
9,886
ND
9,850
ND
9,383
ND
8*05
8,4
8.5
8.51
8.50
8.50
8.35
8.5
8.4
ND
8.3
8.4
8.4
6.4
5.2
4.8
5.3
5.9
6.2
5.2
6.0
3.0
6.0
2.0
4.35
5.9
Chemical
oxygen
demand PO^ NOo
(mg/1) (mg/1) (mg/D
8,256
8,423
8,258
8,309
8,987
9,026
ND
9,853
ND
10,614
ND
10,736
ND
405
460
413
558
663
512
ND
633
ND
650
ND
695
ND
5.36
6.81
3.23
ND
5.51
5.76
ND
7.93
ND
6.49
ND
8.30
ND
Total Car-
nitrogen bon
(mg/1) (%)
446.0
502.0
525.0
482.0
506.0
336.0
ND
535.0
ND
547.0
ND
561.0
ND
32.89
34.18
33.17
33.80
32.46
33.60
ND
ND
ND
35.99
ND
36.09
ND
Hydro-
gen
(%)
5.13
5.37
5.33
5.24
5 . 18
4.99
ND
ND
ND
5.35
ND
5.34
ND
-------
Table 49. RUN NUMBER 3, ROTOR SPEED 380 RPM, TEMP. 20° C
Date
7-17-73
7-18-73
7-19-73
7-20-73
7-21-73
7-24-73
7-25-73
7-26-73
7-27-73
7-28-73
7-31-73
8-1-73
8-2-73
8-3-73
Waste H20
added added
(8) (1)
131.9
142.4
131.4
146.3
133.4
147.1
0
143.8
141.2
0
129.0
131.2
153.7
141.8
8
0
4
3
2
4
0
8
0
0
8
4
1
4
Total
solids
(ms/1)
3542
ND
ND
4534
ND
5040
ND
ND
ND
5496
ND
5277
ND
6677
Total
volatile
solids
(mg/1) pH
2623
ND
ND
3202
ND
3865
ND
ND
ND
4137
ND
3958
ND
5215
8.4
8.4
ND
8.3
8.35
8.45
ND
8.5
ND
ND
ND
6.45
6.3
6.2
Dis-
solved
oxygen
(mjj/1)
8.3
7.6
8.0
ND
7.3
7.7
ND
ND
ND
ND
ND
8.3
8.0
7.9
Chemical
oxygen
demand PO/
(mg/1) (mg/1)
3487
ND
ND
4016
ND
4658
ND
ND
ND
ND
ND
5538
ND
6246
185
ND
ND
185
ND
155
ND
ND
ND
235
ND
256
ND
219
Total
nitro-
NOo gen
(mgTl) (mg/1)
10.1
ND
ND
11.7
ND
11.62
ND
ND
ND
ND
ND
19.04
ND
ND
229
ND
ND
321.6
ND
297
ND
ND
ND
319.2
325.13
308.8
ND
401.2
Car-
bon
(%)
34.45
ND
ND
35.68
ND
37.62
ND
ND
ND
36.54
ND
35.95
ND
39.07
Hydro-
gen
(%)
5.50
ND
ND
5.63
ND
5.34
ND
ND
ND
5.16
ND
5.42
ND
5.61
-------
Table 49 (continued). RUN NUMBER 3, ROTOR SPEED 380 REM. TEMP. 20° C
Date
8-4-73
8-7-73
8-8-73
8-9-73
8-10-73
8-11-73
8-14-73
8-15-73
8-16-73
8-17-73
8-18-73
8-21-73
8-22-73
8-23-73
8-25-73
8-28-73
Waste
added
(8)
127.0
0
143.3
146.7
145.7
127.3
145.2
0
141.7
87.4
0
149.3
146,9
148.3
147.0
140.4
H?0 Total
added solids
(1) (mg/D
4
0
2
2
2
2
9
0
4
2
4
4
6
2
6
4
ND
5514
ND
ND
ND
ND
8659
ND
ND
9392
ND
9011
8751
ND
11060
10900
Total Dis-
volatlle solved
solids pH oxygen
(mg/1) (mg/1)
ND
4060
ND
ND
ND
ND
6646
ND
ND
7354
ND
6816
6572
ND
8773
8461
ND
6.4
8.50
8.55
8.42
8.5
6.4
ND
8.8
ND
ND
8.6
ND
ND
8.6
8.65
ND
ND
7.9
7.9
7.6
7.5
7.5
ND
7.0
7.3
ND
8.3
7.4
ND
7.0
7.8
Chemical
oxygen
demand P04
(mg/1) (mg/1) i
ND
5570
ND
ND
ND
ND
8472
ND
ND
11444
ND
ND
9273
ND
9841
10528
ND
136.3
ND
ND
ND
305.0
152.2
ND
ND
170
ND
152
168
ND
270
360
NO 3
(mg7D
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
12.2
ND
8.9
11.17
Total
nitro-
gen
(mg/D
ND
ND
ND
ND
ND
ND
466
ND
ND
452
ND
490.7
ND
ND
502
552
Car-
bon
(%)
ND
ND
ND
ND
ND
ND
36.20
ND
ND
38.81
ND
36.93
38.24
ND
38.75
38.65
Hydro-
gen
(%)
ND
ND
ND
ND
ND
ND
5.48
ND
ND
5.38
ND
4.96
5.18
ND
5.34
5.22
-------
Table 50. RUN NUMBER 4, ROTOR SPEED 275 RPM, TEMP. 20° C
Date
9-1-72
9-4-72
9-6-72
9-7-72
9-8-72
9-11-72
9-12-72
9-13-72
9-14-72
9-18-72
9-20-72
9-21-72
9-22-72
Waste
added
(8)
341
84.3
144.2
148.3
149.2
145
137.3
146.4
125.1
126.7
133.3
145.7
139.1
H20
added
(1)
39
10
10
2
4
6
4
2
2
6
4
2
4
Total
solids
Cmg/1)
ND
1714
ND
2491
ND
3639
ND
ND
5173
5518
ND
5955
ND
Total
Volatile
solids
(«g/l)
ND
ND
ND
1960
ND
2864
ND
ND
4219
4388
ND
4740
ND
Dis-
solved
pH oxygen
(«R/1)
ND ND
ND ND
ND ND
ND ND
8.45 7.4
8.50 7.5
8.50 8.5
8.5 7.8
ND ND
8.6 6.8
8.5 6.1
8.55 8.0
ND ND
Chemical
oxygen
demand
CmR/1)
ND
ND
ND
2588
ND
3488
ND
ND
4924
5687
ND
5194
ND
P04
(mg/1)
ND
ND
ND
ND
ND
ND
ND
ND
ND
185
ND
ND
367
NO 3
(mg/1)
ND
ND
ND
ND
SD
3.30
ND
ND
3.02
ND
ND
2.77
3.44
Total
Nitro-
gen
(mg/1)
ND
ND
ND
131.9
ND
194
ND
ND
264
313
ND
329
ND
Car-
bon
(%)
ND
ND
ND
41.20
ND
39.12
ND
ND
41.01
40.49
ND
41.34
ND
Hydro-
gen
(%)
ND
ND
ND
5.96
ND
5.96
ND
ND
5.89
5.93
ND
5.94
ND
-------
Table 50 (continued). RUN NUMBER 4, ROTOR SPEED 275 RPM. TEMP. 20° C
o
~»i
Date
9-27-72
9-28-72
9-29-72
10-2-72
10-3-72
10-5-72
10-6-72
10-10-72
10-11-72
10-12-72
10-13-72
10-16-72
10-17-72
Waste
added
140.8
129.7
156.6
175.6
170.8
200.3
149.2
262.6
205.0
196
180.8
170.5
180.3
H2°
added
(1)
8
4
4
8
4
4
2
8
0
4
4
4
4
Total
solids
(mg/1)
ND
6499
ND
7985
ND
8062
ND
8999
ND
10294
ND
11139
ND
Total
volatile
solids
(mg/1)
ND
5041
ND
ND
ND
6182
ND
6961
ND
8117
ND
8820
ND
PH
ND
ND
8.5
ND
ND
8.5
ND
ND
ND
8.5
ND
ND
8.55
Dis-
solved
oxygen
(mg/D
ND
ND
6.2
ND
ND
7.3
ND
ND
ND
6.4
ND
ND
6.8
Chemical
oxygen
demand
(mg/1)
ND
6904
ND
7555
ND
8734
ND
9603
ND
9940
ND
ND
11020
P04
(mg/1)
ND
ND
350
ND
ND
400
ND
511.9
ND
587.2
ND
ND
598.6
NO,
(mg/l)
ND
ND
6.44
ND
ND
5.68
ND
3.36
ND
ND
ND
3.86
ND
Total
nitro-
gen
(mg/1)
ND
304
ND
418
417
ND
ND
313
ND
504
ND
ND
ND
Car-
bon
ND
39.67
ND
37.08
ND
39.97
ND
39.81
ND
39.66
ND
39.11
ND
Hydro-
gen
ND
5.76
ND
5.80
ND
5.87
ND
5.69
ND
5.51
ND
5.29
ND
-------
Table 51. RUN NUMBER 5, ROTOR SPEED 180 RPM, TEMP. 2° C
o
00
Date
7-20-73
7-23-73
7-24-73
7-26-73
7-31-73
8-1-73
8-2-73
8-3-73
8-6-73
8-7-73
8-8-73
8-9-73
8-10-73
Waste
added
(«)
382.0
0
0
0
0
109.4
106.1
0
166.6
130.5
126.9
106.16
109.56
H20
added
<«
19
0
0
6
4
4
0
1
3
0
4
4
1
Total
solids
(mg/1)
ND
11230
ND
3470
4180
ND
ND
3450
3600
ND
ND
5817
ND
Total
volatile
so lids
(mg/1)
ND
9685
ND
3030
3793
ND
ND
3070
3040
ND
ND
5130
ND
Dis-
solved
pH oxygen
(mg/1)
ND ND
7.32 ND
ND ND
7.46 ND
7.74 15.5
ND ND
ND ND
7.88 12.3
8.05 12.0
ND ND
ND ND
7.46 11.9
ND ND
Chemical
oxygen
demand
(mg/1)
ND
19062
ND
12155
5388
ND
ND
6554
4630
ND
ND
4596
ND
(mg^L)
ND
1319
ND
608.5
19.2
ND
ND
649
131
ND
ND
212.5
ND
NO 3
(mg/1)
ND
NMA
ND
NMA
NMA
ND
ND
NMA
NMA
ND
ND
NMA
ND
Total
nitro-
gen
(mg/1)
ND
542
ND
168
213
ND
ND
246
235
ND
ND
343
ND
Car-
bon
(%)
ND
44.789
ND
45.755
ND
ND
ND
44.454
44.735
ND
ND
45.171
ND
Hydro-
gen
(%)
ND
6.260
ND
6.054
ND
ND
ND
6.630
6.139
ND
ND
6.104
ND
-------
o
VD
Date
8-13-73
8-15-73
8-16-73
8-20-73
8-24-73
8-27-73
8-28-73
Waste
added
(g)
121.9
124.6
109.6
137.5
109.3
0
0
H20
added
(1)
4
1
0
0
0
4
0
Total
solids
(mg/1)
5376
ND
5300
3870
ND
ND
9370
Total
volatile
solids
(mg/1)
4737
ND
4533
3410
ND
ND
8250
Dis-
solved
pH oxygen
(mg/1)
7.96 12.4
ND ND
7.43 13.0
7.89 12.0
ND ND
ND ND
7.23 13.5
Chemical
oxygen
demand
(mg/1)
5171
ND
4744
7126
ND
ND
4816
PC,
(mg/1)
259
ND
238
322
ND
ND
1646
NO 3
(mg/i:
NMA
ND
ND
NMA
ND
ND
NMA
Total
nitro-
gen
1 (mg/1)
287
ND
306
382
ND
ND
440
Car-
bon
(%)
ND
ND
44.401
41.230
ND
ND
43.204
Hydro-
gen
(%)
ND
ND
6.464
5.228
ND
ND
5.650
-------
Table 52. RUN NUMBER 6, ROTOR SPEED 275 RPM, TEMP. 2° C
Date
3-5-73
3-7-73
3-9-73
3-20-73
3-21-73
3-22-73
3-23-73
3-27-73
3-29-73
4-4-73
4-5-73
4-9-73
4-10-73
4-13-73
Waste
added
(g)
0
170.6
161,8
158.5
164.1
0
153.4
156,9
159.2
149.1
161.5
150
136
0
H20
added
(1)
0
2
2
15
2
0
2
2
2
3
3
3
3
0
Total
solids
(mg/1)
6303
7343
ND
6791
ND
ND
8032
7976
ND
10304
10339
ND
11313
ND
Total
volatile
solids
(mg/1)
5396
6316
ND
5790
ND
ND
6822
6703
ND
9156
8882
ND
9672
ND
PH
ND
ND
ND
8.3
ND
8.3
ND
8.2
8.3
ND
ND
ND
ND
ND
Dis-
solved
oxygen
(mg/1)
ND
ND
ND
12.0
ND
12.8
ND
11.9
ND
ND
ND
ND
ND
ND
Chemical
oxygen
demand
(mg/1)
ND
ND
ND
7236
ND
ND
ND
7968
16362
ND
16711
18945
ND
19585
POA
(mg/1) <
377
394
438
379
ND
39.48
ND
447.6
486
ND
557.3
ND
586.9
583.2
NO 3
top/I)
ND
ND
ND
NMA
ND
ND
ND
NMA
ND
ND
NMA
ND
ND
ND
Total
nitro-
gen
(mg/1)
ND
367
340
384
ND
350
ND
405
442.6
494.89
541
ND
635
ND
Car-
bon
(%)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Hydro-
gen
(«
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
-------
Table 53. RUN NUMBER 7, BDTOR SPEED 380 RPM, TEMP. 2° C
Date
4-19-73
4-24-73
4-26-73
4-27-73
5-1-73
5-2-73
5-3-73
5-4-73
5-7-73
5-9-73
5-10-73
5-14-73
5-15-73
Waste
added
(g)
1633
136.6
153.2
157.8
155.7
147.9
0
155.7
0
150.0
148.6
152.4
158.5
H20 Total
added solids
CD (mg/1)
60
0
3
3
3
9
0
3
0
3
3
3
3
ND
ND
ND
ND
ND
ND
6820
ND
8054
ND
8027
ND
9253
Total Dis-
volatile solved
solids pH oxygen
(mg/1) (mg/1)
ND
ND
ND
ND
ND
ND
6017
ND
7152
ND
7038
ND
8057
ND
ND
ND
ND
ND
ND
7.85
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Chemical
oxygen
demand PCU
(mg/1) (mg/1)
ND
ND
ND
ND
ND
ND
ND
6102
7738
ND
8478
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
258
ND
308
ND
422
Total
nitro-
N03 gen
(rag/l) (mg/1)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
387.3
ND
325.6
ND
452.0
ND
477.1
Car-
bon
(%)
ND
ND
ND
ND
ND
ND
40.345
ND
ND
ND
ND
ND
20.725
Hydro-
gen
(%)
ND
ND
ND
ND
ND
ND
6.236
ND
ND
ND
ND
ND
3.880
-------
Date
5-18-73
5-23-73
5-25-73
Waste
added
(g)
150.0
114.7
100
H20
added
4
4
32
Total
». solids
ND
9613
12240
Total
volatile
solids
(mg/ 1)
ND
8392
1283
Dis-
solved
pH oxygen
ND ND
8.20 NMA
7.97 ND
Chemical
oxygen
demand
(mg/1)
ND
5090
11885
PO,
(mg/1)
2873
263.
404.
*
NO 3
(mg/1)
ND
4 NMA
2 NMA
Total
nitro-
gen
(mg/1)
421.9
428.8
506.5
Car-
bon
24.039
41.52
37.937
Hydro-
gen
3.335
6.32
6.054
ho
-------
Table 54. RUN NUMBER 8 ROTOR SPEED 275 RPM, TEMP. 10° C
Date
5-23-73
5-25-73
5-29-73
5-30-73
5-31-73
6-1-73
6-4-73
6-5-73
6-6-73
6-7-73
6-8-73
6-11-73
6-13-73
Waste
added
(g)
0
100
0
113.6
106.4
140.1
157.3
150.1
160.6
149.7
154.4
124.4
117.8
HoO
added
(1)
0
4
8
4
0
2
4
1
0
2-
2
2
0
Total
solids
(rag/1)
5496
8147
ND
ND
ND
6398
5400
ND
ND
ND
6083
10820
ND
Total
volatile
solids
(mg/1)
4646
1061
ND
ND
ND
1042
899
ND
ND
ND
1160
1220
ND
Dis-
solved
pH oxygen
(mg/1)
7.41 ND
7.59 ND
8,14 ND
ND ND
ND ND
7.70 ND
8.13 ND
ND ND
ND ND
ND ND
7.99 ND
7.62 9.40
ND ND
Chemical
oxygen
demand
(mg/1)
4127
10372
5322
ND
ND
ND
6738
ND
ND
ND
7263
11600
ND
POA
(mg/1)
277.2
362
270.2
ND
ND
347
348
ND
ND
ND
464.2
428
ND
NO 3
(mg/1)
NMA
NMA
ND
ND
ND
NMA.
NMA
ND
ND
ND
NMA
NMA
ND
Total
nitro-
gen
(mg/1)
342.5
436.2
322.2
ND
ND
474
363.3
ND
ND
ND
384.3
477
ND
Car-
bon
(%)
37.943
ND
38.32
ND
ND
ND
40.423
ND
ND
ND
40.874
38.293
ND
Hydro-
gen
(%)
6.055
ND
5.93
ND
ND
ND
5.763
ND
ND
ND
5.836
5.264
ND
-------
I-1
£
Date
6-14-73
6-18-73
6-19-73
6-20-73
6-21-73
Waste
added
_ JJZ>
ND KID
7.30 $.00
Chemical
oxygen
demand
WD
ND
15643
W>
ND
17740
PO/
(mg/D
ND
536.4
ND
ND
533
NQ3
Cmg/1)
ND
NMA
ND
ND
NMA
Total
nitro-
gen
Cms/1)
ND
695
ND
ND
370
Car-
bon
GO
ND
43.990
ND
ND
42.502
Hydro-
gen
-------
Table 55. RUN NUMBER 9, ROTOR SPEED 380 RPM, TEMP. 10° C
Date
6-14-73
6-18-73
6-19-73
6-20-73
6-21-73
6-22-73
6-25-73
6-26-73
6-27-73
6-28-73
6-29-73
7-2-73
7-3-73
Waste HoO
added added
(g) CD
1713
126.6
135
119.5
118.8
141.2
66.11
123.4
134.1
131.3
62.42
0
64.01
8
0
3
0
3
4
2.75
2
0
3
6
0
Total
solids
(mg/1)
9427
0
0
8640
0
9580
0
0
0
12290
11740
0
Total Dls-
vo la tile solved
solids pH oxygen
(mg/1) (mg/1)
1107
ND
ND
920
ND
993
ND
ND
ND
11105
10500
ND
ND
7.17
ND
ND
7.60
ND
7.47
ND
ND
ND
7.58
7.53
ND
ND
8.50
ND
ND
8.9
ND
9.40
ND
ND
ND
7.60
9.20
ND
Chemical
oxygen
demand PO/
(mg/1) (mg/1)
ND
8057
ND
ND
8416
ND
11191
ND
ND
ND
11684
12303
ND
ND
386.4
ND
ND
388.5
ND
358
ND
ND
ND
416
523
ND
Total
nitro- Car-
NOo gen bon
(mg/1) (mg/1) (%)
ND
NMA
ND
ND
NMA
ND
NMA
ND
ND
ND
NMA
NMA
ND
ND
372.4
ND
ND
598
ND
405
ND
ND
ND
423
395
ND
ND
45.139
ND
ND
46.814
ND
46.586
ND
ND
ND
44.936
44.498
ND
Hydro-
gen
(%)
ND
4.221
ND
ND
6.198
ND
6.539
ND
ND
ND
5.433
6.165
ND
-------
Table
Date
7-4-73
7-5-73
7-6-73
7-9-73
7-10-73
7-11-73
7-12-73
7-13-73
7-16-73
7-17-73
7-19-73
55 (continued). RUN NUMBER 9, ROTOR SPEED 380 RPM
Waste H2<) Total
added added solids
(K) (1) (mg/1)
63.1
112.2
110.2
112.7
130.9
95.47
103.1
113
113.6
123.1
0
6
3
0
4
3
3
4
4
3
6
3
0
9390
0
6700
0
0
6790
0
11140
ND
10430
Total
volatile
solids pH
(mg/1)
ND
8190
ND
5180
ND
ND
5420
ND
9670
ND
ND
ND
7.61
ND
7.85
ND
ND
8.24
ND
7.73
7.73
7.60
Dis-
solved
oxygen
(mg/1)
ND
9.20
ND
8.5
ND
ND
12.80
ND
5.9
5.9
9.0
Chemical
oxygen
demand P04
(mg/1) (rag/l)
ND
8819
ND
7207
ND
ND
9134
ND
ND
ND
13635
ND
458
ND
408.7
ND
ND
435.6
ND
516
ND
238.1
, TEMP
NOo
(mg/1)
ND
NMA
ND
NMA.
ND
ND
NMA
ND
NMA
NMA
NMA
. 10° C
Total
nitro-
gen
(mg/1)
ND
329
ND
306.6
ND
ND
400
ND
505
ND
510
Car-
bon
(%)
ND
44.802
ND
ND
ND
ND
40.993
ND
44.456
ND
44.685
Hydro-
gen
(%)
ND
5.981
ND
ND
ND
ND
4.957
ND
5.772
ND
4.816
-------
Table 56. RUN NUMBER 10, ROTOR SPEED 180 RPM, TEMP. 10° C
Date
6-21-73
6-25-73
6-26-73
6-27-73
6-28-73
6-29-73
7-2-73
7-3-73
7-4-73
7-5-73
7-6-73
7-9-73
7-10-73
Waste
added
(g)
1074.39
67.21
117.0
131.7
141.6
57.79
0
73.56
0
128.4
118.5
128.5
125.9
H20 Total
added solids
(1) (mg/D
4
0
0
0
3
4
0
3
3
0
4
10
ND
9580
ND
ND
ND
7620
8960
ND
ND
9400
ND
5100
ND
Total Dis-
volatile solved
solids pH oxygen
(mg/1) (mg/1)
ND
993
ND
ND
ND
6830
8050
ND
ND
8420
ND
4090
ND
ND
7.19
ND
ND
ND
7.31
7.30
ND
ND
7.26
ND
7.69
ND
ND
6.70
ND
ND
ND
6.30
7.80
ND
ND
6.80
ND
5.5
ND
Chemical
oxygen
demand POA
(mg/1) (mg/1)
ND
9406
ND
ND
ND
10707
9117
ND
ND
7692
ND
6606
ND
ND
213
ND
ND
ND
332
390
ND
ND
367.5
ND
405.5
ND
Total
nitro-
NOo gen
(mg/1) (mg/1)
ND
NMA
ND
ND
ND
NMA
NMA.
ND
ND
ND
ND
NMA
ND
ND
238
ND
ND
ND
317
383
ND
ND
329
ND
264.6
ND
Car-
bon
(%)
ND
41.280
ND
ND
ND
44.655
44.838
ND
ND
43.451
ND
ND
ND
Hydro-
gen
(%)
ND
6.804
ND
ND
ND
5.763
5.601
ND
ND
5.281
ND
ND
ND
-------
Table 56 (continued). RUN NUMBER 10, ROTOR SPEED 180 RPM. TEMP. 10° C
H
00
Date
7-11-73
7-12-73
7-13-73
7-16-73
7-17-73
7-19-73
7-20-73
7-23-73
7-24-73
7-26-73
7-31-73
8-3-73
8-6-73
8-9-73
Waste
added
(8)
112.5
114.7
116
126.6
111.2
0
120.4
0
121.7
115.6
0
0
0
0
H20
added
(1)
1
0
4
3
3
2
ND
3
4
6
7
0
4
0
Total
solids
CmR/l)
ND
6990
ND
9300
ND
9920
9920
ND
ND
10440
9600
9880
9710
8747
Total
volatile
solids
(mg/1)
ND
5736
ND
8147
ND
ND
ND
ND
ND
9090
8266
8630
8350
7467
PH
ND
7.87
ND
7.45
ND
7.43
ND
ND
ND
7.40
7.41
7.99
7.88
7.48
Dis-
solved
oxygen
(ing/1)
ND
8.70
ND
9.9
ND
5.6
ND
ND
7.4
11.1
ND
8.30
5.70
4.5
Chemical
oxygen
demand
(mg/1)
ND
7813
ND
9949
ND
9817
ND
ND
ND
12170
10596
10910
10109
14639
PO,
(mg/1)
ND
449
ND
482
ND
ND
ND
ND
ND
158.5
318
248
482
463.2
N03
(mg/1)
ND
NMA
ND
NMA
ND
NMA
ND
ND
ND
NMA
NMA
NMA
NMA
NMA
Total
nitro-
gen
(mg/1)
ND
442
ND
478
ND
502
ND
ND
ND
546
540
533
492
260
Car-
bon
(%)
ND
ND
ND
41.606
ND
45.089
ND
ND
ND
43.214
39.929
43.531
43.386
42.287
Hydro-
gen
(%)
ND
ND
ND
4.919
ND
6.150
ND
ND
ND
5.137
3.989
5.679
5.167
5.107
-------
SECTION VII
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ing Progress Monograph Series No. 2. American Institute of
Chemical Engineers. 1954. 122 p.
36. Kethley, T. W., E. L. Fincher, and W. B. Cown. The effect of
sampling method upon the apparent response of airborne bacteria
to temperature and relative humidity. Journal of Infectious
Diseases 100:(1) 97-102. 1957.
37. Brown, A. D. The survival of airborne microorganisms, effects
of temperature. Aust. Journal of Biol. Sci. 7: 444. 1954.
38. Wright, T. J., V. W. Greene and H. J. Paulus. Viable micro-
organisms in an urban atmosphere. AFCA Journal 19; 337. 1969.
39. Moore, J. A., R. E. Larson, R. 0. Hegg, and E. R. Allred. Beef
confinement systems - oxidation ditch. Trans, of the ASAE 16;
168-171. 1973.
40. Diesch, S. L., B. S. Pomeroy, and E. R. Allred. Survival of
pathogens in animal manure disposal. Final report on grant
EP-00302. U. S. Environmental Protection Agency. 1971. 189 p.
41. Merz, R. C. Third report on the study of waste water reclamation
and utilization. California State Water Pollution Control Board.
Publication No. 18. Sacramento, California. 1957. 102 p.
42. National Biocentric, Inc. Evaluation of potential environmental
health problems associated with virus transmission from spray
irrigation of waste water treatment plant effluent. Report sub-
mitted to Sanitary Sewer Board, Alexandria Lake Area Sanitary
District, Alexandria, Minnesota. National Biocentric, Inc.
2233 Hamline Avenue N., St. Paul, Minnesota. June 1, 1972. 66 p.
43. Riley, R. L., C. C. Mills, W. Nyka, N. Weinstock, P. B. Storey,
L. V. Sultan, M. C. Riley, and W. F. Wells. Aerial dissemination
of pulmonary tuberculosis. American Journal Hygiene 70: 185.
1959.
44. Riley, R. L., and L. O'Grady. Airborne Infection. Macmillan
Publishing Co., Inc. 1961. 180 p.
122
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SECTION VIII
APPENDIX IRRIGATION STUDY
INTRODUCTION
The operation of spray-irrigation ditch material plays an important
role in treatment of cattle waste in an oxidation ditch. This is
necessary because cattle manure solids build up in the ditch until
the ditch can only function as a holding tank. At this point, ideally
sometime before, the ditch material must be removed, and spray-irri-
gation pumping is one such method. The mechanical ejection of ditch
manure into the air by a sprinkler-irrigating pump provided the
opportunity to examine the production of airborne bacteria. Aerosol
monitoring indicated the relative degree of public health hazard
associated with this method of handling waste from an oxidation ditch.
For this study, samples of air were taken during irrigation in order
to monitor the aerosolized microbes produced in such an operation.
RELATED STUDIES
Previous aerosol studies of operations similar to spray-irrigation
have been done (22, 27, 28, 29, 30, 41). The similarity between these
studies and ours was the production of a visible spray of sewage
from trickling filters, aeration tanks, laboratory-generated sprays,
and to other sprinklers. The differences, however, warranted
carrying out our own study. For example, no other study dealt with
cattle waste alone without the other additives of the sewage system
such as plastics, paper, disinfectants, etc. The droplet size pro-
duced by the different operations varies due to pressure, spray
nozzle size (19), and bubble size (22), which adds to the different
conditions. In addition, we felt it was important to compare the
bioaerosol production of the spray-irrigation to our central study
of monitoring the bioaerosol from the oxidation ditch where the same
system of sampling was employed; for example, the same samplers, lo-
cation, procedure, and operator.
RESULTS AND DISCUSSION
The four studies during spray-irrigation are presented in Tables 57,
58, 59, 60. Each sampling time has been analyzed separately because
daily and even hourly varying conditions prevented overall generali-
zations without first analyzing data under constant conditions.
From Table 57 it can be seen that at the edge of the spray the air-
borne bacterial counts were lower than at distances farther downwind.
The high counts at 82 meters downwind may have been due to an un-
noticed wind change, an unknown spreading pattern which had a high
concentration of bacteria at this distance and height, or an unknown
123
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factor. The counts of the sample taken after spraying were lower
than all the counts except those taken at the edge of spray.
Data from Table 58, however, show no pattern of zonal concentration;
but the total counts during spraying were higher, although not sig-
nificantly (during - average of 3, before - 1, upwind - 0.4), than
the counts before spraying and the counts upwind.
Table 58 shows that there was no difference in numbers of airborne
bacteria between the 100- and 30-cm above-ground samples at any dis-
tance. There was no zonal concentration apparent, but comparing two
15 meter downwind samples it is found that the counts downwind in-
crease when the wind increases. No indicator organisms (coliform or
F. streptococci) were detected from AGI samples but were found on all
exposed plates during irrigation—implying that particles from the
ditch were airborne but not detectable in sizes that would penetrate
the lung (1-5 microns). The total count, however, has increased from
0 before spraying to 30 CFU/1 during spraying, showing that some
small particles are airborne. The after-spray counts show that the
small and large fallout particles are quickly removed from or diluted
out of the air.
The ditch liquid samples show the enteric indicator organisms to be
approximately 300 times less than the concentration of the total
number of organisms present. This same concentration proportion is
borne out in the AGI samples of the air, but the open plates show
proportionately higher concentrations of enteric organisms recovered.
One explanation of this may be that since open plates collect par-
ticles that are falling, therefore large particles, each colony
probably has many bacteria, thus making the actual number of bacteria
present much more than counted. Also, since there are 300 times the
number of total bacteria than enteric bacteria in the sprayed liquid,
the chances of having many enteric organisms per falling particle
are small. So the number of enteric colonies on the plates approximates
the actual number. Unlike other air samples taken near the animal
housing unit where F. streptococci outnumber coliform, the fallout
plates show similar numbers of coliform and F. streptococci present.
A speculated reason for this is that in spite of the fact that coliform
organisms do not survive long when airborne (22), it is possible that
they are protected by a coating of ditch material when they are in
the fallout droplets, thus accounting for the coliform counts approach-
ing those of the fecal streptococci.
The few samples on Table 59 show that when the large fallout particles
are present in large numbers (overgrowth) the smaller, lung-penetrating
sizes are not necessarily present (1 CFU/1).
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Table 57. AEROSOLS FROM SPRAY IRRIGATION AT ROSEMOUNT
NOVEMBER 22, 1971
Time
During spray
During spray
During spray
During spray
After spray
Temperature :
Wind:
Sky:
Sampler:
Meters Downwind
0
61
82
113
82
-3° C
8-16 km/hr
overcast
AGI
Total CFU/1
0.4
15
120
25
4
Table 58. AEROSOLS FROM SPRAY IRRIGATION AT ROSEMOUNT
APRIL 18, 1972
Time
Before spray
During spray
During spray
During spray
During spray
Meters Downwind
0
37
52
67
98
Total CFU/1
1
3
4
2
5
During spray
Meters Upwind
37
0.4
Temperature:
Wind:
Sky:
Sampler:
8° C
16 km/hr
overcast 79% RH
AGI
125
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Table 59. AEROSOLS FROM PIG WASTE SPRAY IRRIGATION -
NOVEMBER 8, 1972
Meters AGI: CFU/1 Plates-CFU/5 min. expos.
downwind Total Coll F-Strep PGA L-EMB m-ENT
Before
spray
During
spray
33
15
0
0
0
64
0
ovgr ovgr ovgr
Temperature:
Wind:
Sky:
Sampler:
10° C
11 km/hr
overcast
AGI, plus exposed plates at ground level
The data collected by others add additional light on the subject and
support to the data collected in our experiment.
Albrecht (29) found when sampling with both a Wells centrifuge and
midget impinger downwind from a trickling filter sewage treatment
plant that very few coliform organisms were recovered, but growth on
nutrient agar showed recovery from 0.07 to 5 CFU/1 at the edge of the
trickling filter, decreasing to 0.2 to 2.7 CFU/1 at a distance of
15 meters. He noticed an increase in counts downwind with increasing
wind velocity.
Randall (27) found in sampling air downwind from activated sludge units
with an Anderson sampler that on the average 17 CFU/1 of particle size
less than 5 microns were found downwind of the operation (6% of which
were Klebsiella). He noted that as the wind increased so did counts
and also there was a rough correlation between higher counts at
higher R.H. He concluded by saying there existed a definite possibili-
ty of airborne infection from activated sludge units.
Adams (30) sampled the air with EMB in an Anderson sampler near a
trickling filter. Without classifying bacteria any further than
126
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Table 60. AEROSOLS FROM SPRAY IRRIGATION AT ROSEMOUNT
MARCH 26, 1973
Agar plates (ground)
AGI; CFU/1 CFU/5 min. exposure
Down 30 cm 100 cm
wind F- F-
(m) Total Coll Strep Total Coli Strep PCA L-EMB m-ENT
Before
spray 0100 400 ovgr* 0 0
During
spray 15 2 0 0 6 0 0 348 28 27
During
spray 15** 23 00 30 00 >2000 98 188
1 5
During
spray 33 23 00 26 00 221
After
spray 50 0 0 0 000 ovgr* 0 0
Up-
wind
During
spray 33 200 000 77 0 1
Bacterial counts of ditch liquid collected during irrigation:
92 x 10^ total aerobic bacteria per ml ditch.
20 x 10^ Coliform bacteria per ml ditch
32 x 104 F. Streptococci per ml ditch
* ovgr denotes overgrowth with a spreading organism.
** during this sampling the wind speed was greater than the other 15
meter downwind sample.
Temperature: 11° C
Wind: 8 km/hr
Sky: Clear, 57% RH
Sampler: Samples with AGI were taken simultaneously at two heights,
30 and 100 cm, plus exposed plates on ground.
127
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counting colonies on media, he found 19 CFU/1 at 15 meters downwind,
0.9 CFU/1 at 42 meters downwind, and 0.003 CFU/1 at 1280 meters
downwind. He observed greater recoveries with high wind velocity,
high R.H., and darkness.
Napolitano (28) studied concentrations of aerosols generated by acti-
vated sludge plants using EMB in an Anderson sampler. The report
says that sampling counts were highest at the aeration source where a
noticeable spray was produced. Here 8 CFU/1 were obtained on EMB
(no further selection was done; however, it was stated that Aerobacter
aerogenes was predominate). At 15 and 30 meters downwind 0.18 CFU/1
was found.
Merz (41) found when sampling with an impinger downwind from a sprink-
ler discharging settled sewage onto a golf course that he detected
coliforms in the air only at distances where spray could be felt.
He concluded that there was no hazard except that of direct contact
with unevaporated droplets.
Higgins (22) found in a laboratory study of generated bioaerosols by
aeration and the production of bubbles that the production of viable
bacterial aerosols by bursting bubbles is highly dependent upon species.
E. coli had a very low aerosolization rate. In addition, he found that
the aerosol production depended on composition of aeration liquid,
wind velocity, and concentration of cells. He could not make any
statement on the effects of air and water temperature or R.H.
Ladd (26) found when sampling near a sewage pre-aeration treatment
with the Anderson-sieve sampler he was able to pick up a tracer or-
ganism, Bacillus subtilis var. gobgii, that was poured down the labora-
tory drain (approximately 8 km from the sewage plant) . He was able to
detect the organism at a maximum concentration of 0.008 CFU/1 (15
organisms per 60 ft3) 5 hours after the pouring but detected none after
the eleventh hour. A concentrated suspension solution of 4.5 liters
was poured into the drain - approximately 106 (our estimate) organisms
per ml. He also found 8 times as many bacteria were emitted by the
pre_aeration tank as measured by upwind controls. He concluded that
harmful bacteria were being emitted from the treatment plants and
could be harmful to operators and others in the area.
CONCLUSION
The assessment of the public health hazard of spray-irrigating liquid
from an oxidation ditch involved many factors, many of which are un-
known. But two likely ways exist in looking at the problem. One
method is by examining the greatest bioaerosol load produced by any
such operation, fitting into the equation the known infectious doses
128
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of bacteria potentially inhabiting the aerosol and survival curves of
airborne bacteria, also including the expected contaminated air a person
breathes (15,000 liters per day) (44), then extrapolating to find the
probability of infection. A second method would be to compare disease
rates produced from an operation of similar bioaerosol loads; for
example, animal housing units or, in the case of sewage, comparison
with a hospital.
in dealing with cattle waste an evaluation by the first method is
difficult because most studies on, for example, airborne survival have
been done on human airborne disease,not cattle. What is known in this
area, however, is that a dose as low as one tubercle bacillus can
cause tuberculosis, whereas 10^ pneumococci are needed to infect a
mouse by airborne route (44). In addition, very little is known of
the respiratory infectious dose of enteric bacteria which are the
potential hazards being looked at in irrigating an oxidation ditch.
This lack of information with which to analyze the hazard leads to
the alternate analysis of comparing similar situations.
In none of the bioaerosol studies with the AGI during irrigation were
any enteric organisms present (less than 1 CFU/1), and total counts
increased from 1 CFU/1 before spraying to a maximum of 120 CFU/1 and
an average of 20 CFU/1 during and downwind of spray. Other cited
researchers present this same number when sampling downwind in the
vicinity of sewage spray. The counts of samples taken seconds after
spraying stopped are the same as before spraying samples, indicating
that there is no buildup of bacteria. The previously reported sampling
of animal housing units frequently have enteric F. Strep.; also, the
average Rosemount animal housing unit total count is 161. In addition,
the samples of a dairy barn show averages of 196 total aerobic bac-
teria and 0.5 F. Strep. Therefore, the relative hazard of spraying
compared to confinement housing is small as far as cattle health and
people working in the housing areas are concerned. As far as the res-
piratory hazard of workers in the area there is no denial of the
increase in bioaerosol from the ditch during spraying in the immediate
area, but this hazard is present for a short while. Therefore, it is
recommended that, during spraying, workers in the area wear protective
masks. A more possible danger is through contact with large droplets
of spray which are visible during the operation. Diseases can arise
in the nasal mucosa, tonsils, or respiratory mucosa of the upper
respiratory tract through inhalation of large particles (37). Again,
a mask in the areas during spraying is recommended.
129
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
REPORT NO.
EPA-660/2-75-012
3. RECIPIENT'S ACCESSION»NO.
4. TiTLE AND SUBTITLE
Survival of Pathogens in Animal Manure Disposal
5. REPORT DATE
6. PERFORMING ORGANIZATION CODE
Stanley L. Diesch, Philip R. Goodrich* Benja-
min S. Pomeroy, Loren A. Will
nf Agrii-nl turql Engi'nppri'ng
9. PERFORMING ORG \NIZATION NAME^AND ADDRESS
College of Veterinary Medicine
University of Minnesota
St. Paul, Minnesota 55108
8. PERFORMING ORGANIZATION REPORT NO.
10. PROGRAM ELEMENT NO.
1BB039
11. CONTRACT/GRANT NO.
R 802205
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Protection Agency
Robert S. Kerr Environmental Research Laboratory
P.O. Box 1198
Ada, Oklahoma 74820
13. TYPE OF REPORT AND PERIOD COVERED
Final 8/15/71
14. SPONSORING AGENCY CODE
15. SUPPLEMENTARY NOTES
16. ABSTRACT
A research project was conducted to measure and evaluate the public health effects
of pathogens in beef cattle manure found in the extended aeration system of waste
disposal.
Model oxidation ditches were used in laboratory studies. At simulated summer and win-
ter environmental conditions determinations were made of the viability and infecti-
vity of leptospires in weanling hamsters and salmonella in turkey poults. Salmonella
was transmitted by aerosols, but leptospires were not. In refeeding contaminated
slurry contents salmonella was transmitted but leptospires not. Leptospires isolated
from the slurry of the model ditch 17 days post seeding had lost measurable virulence.
Measurements of selected microbial aerosols were made in the vicinity of a field ditch.
Bacterial levels of 100-200 total colony-forming units per liter of air were associ-
ated with the beef cattle population in the housing unit and not with aerosols gen-
erated by the oxidation ditch treatment system.
Studies were made on a model oxidation ditch simulating the field ditch. The winter
temperature conditons (2° - 5° C) slowed the degradation process considerably and
high dissolved oxygen was maintained.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
Agricultural Wastes
Biological aerosols
Veterinary Medicine
Public Health
Salmonella typhimurium
Environmental sanitation
Dispersions
Zoonoses
Leptospira
Leptopirosis
Beef Cattle
Hamsters, Turkeys
Oxidation ditch
Agricultural Engineering
Pasveer ditch
06/06
Waste treatment Seasonal variations
13. DISTRIBUTION STATEMENT
Release Unlimited
19. SECURITY CLASS (ThisReport)
Unclassified
136
20. SECURITY CLASS (Thispage)
Unclassified
22. PRICE
EPA Form 2220-1 (9-73)
ft U. S. GOVERNMENT PRINTING OFFICE: I975-698-779 /I69 REGION !0
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