PB-222 337
SURVIVAL OF PATHOGENS IN ANIMAL MANURE DISPOSAL
MINNESOTA UNIVERSITY
PREPARED FOR
ENVIRONMENTAL PROTECTION AGENCY
AUGUST 1973
DISTRIBUTED BY:
U1
National Technical Information Service
U. S. DEPAKTMENT OF COMMERCE
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BIBLIOGRAPHIC DATA
SHEET
1. Report No.
EPA-670/2-73-051
3. Recipient's Accession No.
PB-222 337
I. Title and Subtitle
SURVIVAL OF PATHOGENS IN ANIMAL MANURE DISPOSAL
5- Report Date
1973-issuing date
7. Author(s)
S. L. Diesch, B. S. Pomeroy, and E. R. Allred
8. Performing Organization Rept.
No.
9. Performing Organization Name and Address
University of Minnesota
St. Paul, Minnesota
10. Project/Task/Work Unit No.
11. Contract/Grant No.
EP-00302
12. Sponsoring Organization Name and Address
U.S. Environmental Protection Agency
National Environmental Research Center
Office of Research & Development
Cincinnati. Ohio 45268
13. Type of Report & Period
Covered
Final
14.
15. Supplementary Notes
16. Abstracts
A laboratory model (1:10 scale) of an operational field oxidation ditch
used in beef cattle production was utilized in survival and detection
stucjies of Lep tospira pomona and Salmonella ty phimurium. Minnesota
summer (20C) and winter (2C) temperatures, pH, and dissolved oxygen of
field.ditch manure slurry were simulated in laboratory model studies of
manure slurry, effluent, and sludge. Maximum leptospiral survival times
of 138 days (summer) and 18 days (winter) in the slurry were measured. Sal
monella survival of 47 days in slurry and 87 days in sludge (winter),
and;17 days in slurry (summer) were measured. Adequate laboratory
cultural detection and isolation techniques were developed to measure
survival. Findings from simulated studies in a second laboratory
modeliwere used to separate materials for recycling.
17. Key Words and Document Analysis. 17o. Descriptors
*Pathology, *Survival, *Animals
Models ,
Urinary
Sludge,
Oxidation, Beef cattle
Fertilizers, Wastes, *Waste disposal,
Leptospira . Salmonella typhimurium,
system, Feces , pH,
Isolation, Rotors
Dissolved gases, Simulation, Effluents
Reproduced by
NATIONAL TECHNICAL
INFORMATION SERVICE
US Department of Commerce
Springfield. VA. 22151
17b. Identif iers/Open-Ended Terms
Oxidation ditch, Leptospira pomona. Zoonotic disease pathogens,
Minnesota temperatures, Manure slurry, Suspension, *Solid waste
management, Resource, recovery
17e. COS/.TI Field/Group
18. Availability Statement
Release to public
19. Security Class (This
Report)
UNCLASSIFIED
20. Security Class (This
Page
UNCLASSIFIED
21. No. of Pages
22. Price /
FORM NTIS-35 '.REV, 3-72)
THIS FORM MAY BE REPRODUCED
14932-P72
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REVIEW NOTICE
The Solid Waste Research Laboratory of the
National Environmental Protection Agency has reviewed
this report and approved its publication. Approval
does not signify that the contents necessarily re-
flect the views and policies of this laboratory or
of the U.S. Environmental Protection Agency, nor
does mention of trade names or commercial products
constitute endorsement or recommendation for use.
The text of this report is reproduced by the
National Environmental Research Center - Cincinnati
in the form received from the Grantee; new prelimi-
nary pages have been supplied.
-ii-
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FOREWORD
Man and his environment must be protected from the
adverse effects of pesticides, radiation, noise and other
forms, of pollution, and the unwise management of solid
waste. Efforts to protect the environment require a
focus that recognizes the interplay between the com-
ponents of our physical environment--air, water, and
land. The National Environmental Research Centers
provide this multidisciplinary focus through programs
engaged in
• studies on the effects of environmental
contaminants on man and the biosphere, and
• a search for ways to prevent contamina-
tion and to recycle valuable resources.
In an attempt to solve one of the problems in-
volved in disposing of agricultural solid wastes, a
research project was conducted to study the survival
of pathogens in animal manure. Because leptospires
and salmonella are zoonotic disease pathogens that cause
significant problems in the United States, this report
will interest persons working not only in solid waste
management but in water pollution and public health.
A. W. Breidenbach, Ph.D.
Director.
National Environmental
Research Center, Cincinnati
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ABSTRACT
A three year research project entitled the Survival of
Pathogens in Animal Manure Disposal was conducted. A
laboratory model (1:10 scale ) of an operational field
oxidation ditch used in beef cattle production was uti-
lized in survival and detection studies of Leptospira
porno ntt and Salmonella typhimurium. In the United States
leptospires (urinary source)and salmonella (fecal source)
are zoonotic disease pathogens which cause significant
problems. Minnesota summer (20C) and winter (2C) tempera-
tures, pH and dissolved oxygen of field ditch manure
slurry were simulated in laboratory model studies in man-
ure slurry, effluent and sludge. Maximum leptospiral (sum-
mer) survival time of 138 days and 18 days (winter) in
the slurry were measured. Salmonella survival of 47 days
in slurry and 87 days in sludge (winter), and 17 days in
slurry (summer) were measured. Adequate laboratory
cultural detection and isolation techniques were developed
to measure survival.
Simulated studies in a second laboratory model were
conducted to define conditions to maintain maximum sus-
pension of solids. These findings were used to separate
materials for recycling. Maximum separation of solids
occurred when the rotor position was directly above the
collection sump.
Because of long term survival of pathogens in the model
oxidation ditch and previously documented periods of
shedding of weeks to months from infected cattle, a public
health problem is created. These findings suggest the
need for disinfection of effluents and sludge prior to
environmental application for the prevention of disease
in man and animals.
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CONTENTS
ABSTRACT iv
FIGURES vi
TABLES vii
CONCLUSIONS 1
RECOMMENDATIONS 5
CONTINUATION STUDY 7
INTRODUCTION. . 9
SURVIVAL AND DETECTION OF LEPTOSPIRES IN BEEF
CATTLE MANURE 27
APPENDIX A - LEPTOSPIROSIS 91
SURVIVAL AND DETECTION OF SALMONELLA IN BEEF
CATTLE MANURE 47
ENGINEERING STUDIES OF OXIDATION DITCH OPERATION 69
APPENDIX B - ENGINEERING STUDIES OF OXIDATION
DITCH OPERATION 115
ACKNOWLEDGEMENTS 81
REFERENCES 83
PROJECT PUBLICATIONS 89
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FIGURES
Figure 1 Plan view of the oxidation ditch and re-
search structures at Rosemount,Minnesota..18
Figure 2 Field oxidation ditch rotor 21
Figure 3 Cattle unit housing 18 steers above an
operational oxidation ditch 21
Figure 4 Laboratory model A oxidation ditch 22
Figure 5 Laboratory model A oxidation ditch with
monitoring probes and Selas candles 24
Figure 6 Model B oxidation ditch 26
Figure 7 Model B oxidation ditch, overhead view 26
Figure 2C Modified rotor as used in model B oxida-
tion ditch 70
Figure 7B Rotor locations and flow directions used
in solid placement tests 72
Figure 3B Tethered-ball meter used to measure low
velocities in model B oxidation ditch 73
Figure 4B Velocity meter as used to measure point
velocities in oxidation ditch (side view).73
Figure 5B Typical solids accumulation beneath rotor
of oxidation ditch 75
Figure 6B Solids accumulation in low velocity areas
in oxidation ditch (overhead view) 75
Figure 8B Effect of water depth on solid settlement
beneath rotor, with rotor at position A...76
Figure 9B Effect of water depth on solid settlement
beneath rotor, with rotor at position B...76
Figure 10B Effect of water depth on solid settlement
in sump, with rotor at position E 77
Figure 11B Effect of water depth on solid settlement
in sump, with rotor at position A 78
Figure 12B Effect of water depth on solid settlement
in sump, with rotor at position B 78
Figure 13-30 Velocity distribution tests, No. 1-18 125-
134
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TABLES
SURVIVAL AND DETECTION OF LEPTOSPIRES
Table IA Summary of survival and detection of
leptospires in a laboratory model
oxidation ditch at winter temperatures.... 35
Table IIA Survival and detection of leptospires in
an oxidation ditch at summer temperatures.37
Table IIIA Survival and detection of leptospires in
animal manure disposal. 40
Table IVA Summary of survival and detection of lepto-
spires in well and stream water 41
APPENDIX 91
Nephlos standard .92
Exp. 1LW-6LW Candle studies at winter temperature 97-99
Exp. 7LW-9W Effluent and sludge studies at winter
temperatures 100-101
Exp. 10LW Studies of seeded ditch at winter
temperatures 102
Exp. 1LS-5LS Candle studies at summer temperatures..103-105
Exp. 6LS-10LS Effluent and sludge studies at summer
temperatures 106-107
Exp. 11LS-12LS Studies of seeded ditch at summer
temperatures 108-114
SURVIVAL AND DETECTION OF SALMONELLA
Table 1 Calculated number of Salmonella typhimurium
seeded in manure 51
Table 2 Salmonella experiments. Broth and plate
media incorporated 57
Table 3 Survival and detection of Salmonella
typhimurium in a model oxidation
ditch or model effluent holding chambers..62
Table 4 Environmental conditions in a model oxida-
tion ditch and model effluent holding
chambers 63
Table 5 Detection of Salmonella typhimurium 65
ENGINEERING STUDIES OF OXIDATION DITCH OPERATION
Table 1 Solid-settlement tests 72
Table 2B-19B Velocity distribution studies
(Appendix) 116-124
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CONCLUSIONS
A laboratory model oxidation ditch of an operational field
oxidation ditch (Pasveer) was developed for studying pathogen
survival at simulated winter and summer environmental temper-
ature conditions. Methods were developed and utilized to
seed, measure survival and detect leptospires and salmonella
in an aerated manure environment of the laboratory model ditch
and settling chambers. Survival times of Leptospira pomona
and Salmonella typhimurium were measured under summer and winter
environmental conditions in beef cattle manure of the model
ditch in which temperature, pH and dissolved oxygen were monitored,
Leptospires were measured by artificial cultural techniques to
survive at Minnesota summer temperatures (20C) for up to 138
days, when seeded directly in the manure, up to 5 days in
effluent and 14 days in sludge of a model settling chamber.
At winter temperatures (2C), survival was measured for 18 days
when seeded directly in the manure, for 9 days in effluent and
11 days in sludge of the model settling chamber. Since shedding
of leptospires in urine of infected animals may occur from 3 to
6 months the problem is increased and these findings are signifi-
cant. Leptospirosis is widespread in animal populations. Based
on laboratory model ditch studies, the urine of infected animals
will result in long term sources and vehicles for transmission
of leptospires when manure is aerated in an oxidation ditch and
subsequently collected in settling chambers as effluent and
sludge, prior to discharge to the environment.
Salmonella were measured by cultural techniques, to survive
at summer temperatures for 17 days, when seeded in the manure
of the laboratory model oxidation ditch, 14 days in the efflu-
ent and sludge of a settling chamber. At winter temperatures,
survival was measured for 47 days in the manure of the oxida-
tion ditch, 87 days in the sludge and 66 days in effluent of
the model settling chamber. In the United States, salmonellosis
remains the major zoonotic problem. Animals shedding salmonella
in the feces may discharge the bacteria for weeks to months.
Based on laboratory model ditch studies the feces of infected
animals shedding will result in long term sources and vehicles
for transmission of salmonella when manure is aerated in an
oxidation ditch and subsequently collected in settling chambers
as effluent and sludge prior to discharge to the environment.
The research herein reported, has resulted in a better under-
standing of leptospirosis and salmonellosis problems as related
to the broad fields of agriculture, environment, pollution and
public health, as well as to the further knowledge of the
specific spirochaete and bacteria in terms of survival.
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The variation found in survival times of both leptospires and
salmonella may, in part, have resulted from a continuous im-
provement in laboratory detection and cultural methods of
isolating and purifying the pathogens for identification.
Despite qual itative measurement of the ability of these patho-
gens to survive, their subsequent virulence or the ability to
infect warm blooded animals and subsequently man was not deter-
mined. The public health effect was determined in that surviv-
al of a zoonotic pathogens creates a health problem.
From studies made at the Rosemount Oxidation Ditch it was found
that an appreciable portion of some feed rations pass through
beef animals and enter the oxidation ditch in undigested form.
Such residues are difficult to treat if allowed to remain in
the ditch. However, these residues have potential value to the
owner for re-feeding (re-cycling) purposes. Hazards of disease
transmission introduced when such residues are re-cycled for
feed must be defined. The engineering studies conducted in
laboratory Model B, provided some data upon which certain
design improvements were made in the field, as well as in the
laboratory models of the oxidation ditch. Since the principal
objective of the study was pathogen survival, major engineering
time and effort was directed toward the design, development
and maintaining the operation of the laboratory Model A.
Laboratory tests were run in Model B to define those conditions
necessary to maintain a condition of maximum suspension of
solids within an oxidation ditch. Difficulty was experienced
in efforts to design for those conditions necessary to keep
all solids in suspension since some particles, being of much
greater density than others, required abnormally high velocities
in order to remain in suspension. Attempts were made to define
conditions which would control, rather than prevent solids
settlement. Solids settlement data were used in an effort to
determine the feasibility of using the oxidation ditch as a means
of separating re-usable solid materials for recycling purposes.
Major factors affecting solid settlement were the location of
the rotor, rotor immersion depth, and liquid level in the ditch.
Regardless of these factors, solids accumulated directly beneath
the rotor. Maximum separation of solids from the liquid (87%)
occurred when the rotor was positioned directly above the
collection sump. Attempts were also made to eliminate reverse
flow conditions around each end of the ditch, at which points
excessive settlement of solids occurred. Although the temporary
insertion of warped sections at strategic locations within the
ditch showed some effect in reducing both sedimentation and re-
versed flow conditions, insufficient data and tests were made
to warrant conclusions at this time.
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A team effort approach for finding solutions to public
health and engineering problems, and those concerned with
veterinary medical and environmental problems is essential.
This project offered a direct approach to defining present
problems and to immediate control and prevention of disease
caused by pathogens transmitted by the manure vehicle of
domestic animals. The oxidation ditch is essentially a
closed system, or may be operated as a closed system. Based
on survival of leptospires and salmonella in the sludge and
effluents, disinfection appears to be needed prior to environ-
mental discharge. The expanding trend of livestock production
by automation and confinement practices presents a unique
opportunity for disease control of pathogens originating in
animal manures.
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RECOMMENDATIONS
Pathogenic microorganisms leptospires and salmonella are
capable of surviving in the beef cattle manure of a
laboratory model oxidation ditch at Minnesota summer (20C)
and winter (2C) environmental temperatures. Since leptospirosis
and salmonellosis are zoonotic diseases, they must be con-
sidered a public health problem and the feces and/or urine
of infected shedding cattle considered a source of these
pathogens.
Studies must be conducted to determine if the virulence of
these microorganisms continues to exist following survival
in the manure of an oxidation ditch.
Disinfection of the effluents and sludge of the extended
aeration ditch containing leptospires and salmonella is
needed, but to the best of our knowledge a reliable,
economical method of disinfection of huge volumes of animal
manures does not exist. Further research is essential.
Preceding page blank
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CONTINUATION STUDY
A continuation study of the Survival of Pathogens in Animal
Manure Disposal was approved for funding for two additional
years, (1971-1973). The following information briefly in-
dicates the research design, purpose and objectives.
This research was designed to measure and evaluate the public
health effect of pathogens, beef cattle manures, extended
aeration system of waste disposal and potential pollution
of the common environment of man and animals.
1. Determinations will be made of the viability and in-
fectivity of leptospires and salmonellae in aerosols
caused by potential mechanical dissemination of these
pathogens from manure of a model oxidation ditch.
Viability will be measured in artificial culture media
and infectivity in laboratory animals.
2. Determinations will be made of the viability and infec-
tivity of leptospires and salmonellae in the feed (corn)
recycled from the manure of the field oxidation ditch.
Viability will be measured in culture media and infec-
tivity in laboratory animals.
3. Measurements will be made of selected microbial aerosols
generated during aerobic treatment of animal manures in
an oxidation ditch under a beef confinement housing unit
Environmental samplings of aerosols and culturing of
fecal-borne bacteria will be made around the field ditch.
4. Relationships between temperature, loadi;ng rates and
degradation of manure in a model oxidation ditch will
be made under controlled environment simulating the
field ditch, and further utilized to develop design of
the oxidation ditch.
1. Objectives: The overall objectives are to determine
and evaluate:
a. The public health hazards associated with poten-
tial pathogen transmission from the internal
and external environment, and from feed recycled
from animal manure disposal during aerobic treat-
ment.
b. The public health hazards created by microbial
aerosols in the external environment, disseminated
from a field oxidation ditch during aerobic treat-
ment of animal manure.
Preceding page blank
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c. The relationship between temperature,
loading rates, degradation and the
effects of change in design in a model
oxidation ditch on manure.
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INTRODUCTION
Need for Study
Today, quality and ecology of environment is of concern
to every segment of society. Environmental pollution has in-
creased the responsibility and inquiry for all persons associ-
ated with animal and human health. On August 10, 1970,
President Nixon, in presenting the Report of the White House
Council on Environmental Quality, stated, "in dealing with the
environment, we must learn not to master nature but how to
master ourselves,our institutions, and our technology."
While much research has been conducted on handling and treat-
ment of human wastes, and determining the public health
significance; very little had been done to define similar prob-
lems involving animal wastes. As new methods of animal manure
disposal are developed, the public health significance of path-
ogenic organisms discharged in animal manures needs evaluation.
To keep pace with the changing livestock industry practice of
expanded confinement and concentration,new methods of manure
handling and disposal must be utilized. In 1966 it was reported
that 50% of the feedlot cattle fattened in the U.S. were located
in 300 feedlot areas (1).
In the United States, increasing centralization of live-
stock, milk, poultry and egg production has increased dis-
posal and recycling problems associated with more than 1.7
billion tons of animal wastes produced annually. Approximately
half of this amount is produced by concentrated systems. As
agri-business changes with expansions in animal and human
populations, consideration must be given to systematic manure
disposal and its public health effect.
Pathogens found in manure are potential contaminants of the
sludge when spread as fertilizer on land surface, and of the
effluent when discharged on land or into natural waterways;
both of these are sources of infection in the environment.
The demand for this study was intended to meet the inquiries
growing out of public awareness of the immense problem associated
with the effect of animal manures and wastes on environmental
quality. In 1965, an Environmental Pollution Panel (2) reported
that: "the problem of agricultural waste disposal has grown
to such dimensions that probably the major unsolved issue in
the confinement housing of livestock and poultry is the handling
and disposal of manure." The magnitude of the problem may be
visualized in simple terms by comparing the waste voided by
man and that by the animals he raises. For example, a cow
generates as much manure as 16.4 humans, one hog produces as
much manure as 1.9 people, and 7 chickens provide a disposal
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problem equivalent to that created by one person.
The total volume of animal wastes produced in the U.S. is
about 10 times the human population wastes, yet little con-
cern has been given to the former until recent times. In
the past, animals were largely produced in unconfined areas
where wastes were assimilated by the environment. The live-
stock industry has rapidly grown from small farm enterprises
into great agricultural industry, and the wastes have increased
in an unprecedented amount,—for example, the number of beef
cattle fattened in feedlots have doubled since 1950—to more
than 16,000,000.
The biochemical oxygen demand of wastes of a large mid-
western feedlot may be equated to one million people living
on 320 acres of land. If these animals are infected, a large
number of pathogenic agents may be shed into the environment.
Because of high cost of storing and handling animal manures
and their low nutrient value compared to commercial fertilizers,
such manures are not always economical for use as soil fertilizers,
Today, a large part of animal wastes are recycled to the land
as fertilizers by pastured animals depositing manure on the
ground. Other common methods are composting, direct spreading
on land, lagooning, or spraying.
In the past, economics of agricultural operation has largely
determined the management of livestock wastes. In the past
with unrestricted development and construction of feedlots,
minimal consideration was given to their public health effects
or to the resultant interaction of man and lower animals in
their common microbe-laden environment. Major changes in-
waste management methods would be costly for livestock pro-
ducers. Restrictions are being placed on agriculture. How-
ever, guidelines applicable to geographic conditions which
protect the livestock producer, the environment, and the health
and welfare of the public are essential. To protect public
health and reduce disease transmission, animal wastes are
being restricted from selected natural waters and land with
run-off potential, especially in areas of human population
density. Zoning should be considered.
In recent years, local, state and federal governmental agency
regulations have developed laws and guidelines, or are in the
process of developing laws to minimize the public health
hazards of livestock wastes and its subsequent environmental
effect. In 1971, the State of Minnesota developed and imple-
mented regulations for control of wastes from livestock feed-
lots, poultry lots, and other animal lots. In these regulations,
standards govern storage, transportation, and disposal of
animal wastes, and the registration and issuing of permits for
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construction and operation of animal waste disposal systems
for the protection of the -environment.
In order to comply with regulations, and because of economics,
new systematic approaches to livestock waste management are
being developed and utilized. As modern technology for
treating concentrated livestock waste is more commonly utilized,
the accompying health hazards to man and lower animals must
be evaluated.
Livestock wastes, which include dead animals, meat industry
wastes and animal manures, constitute a massive volume of
organic and inorganic materials that must be disposed of or
recycled.
The secondary treatment of livestock and other organic waste
materials involves two biologic processes - anaerobic and
aerobic. The anaerobic process involves the use of inorganic
compounds, other than oxygen, as the final electron acceptor.
Such compounds as used by anaerobic bacteria may be nitrates,
sulfates or carbonates. One of the principal advantages
of the anaerobic process is the high degree of stabilization
which is possible, with carbon dioxide and methane gas as the
primary end products. The major disadvantage of the anaerobic
systems, as applied to the treatment of livestock wastes, is
the high temperature required for optimum operation. The
process also requires considerable skill and attention to be
assured that proper mixing ratios, pH, and other conditions,
are maintained. While many industries and municipalities find
it practical to heat anaerobic digesters artifically, such a
practice is economically unfeasible for livestock operations.
Neither are livestock growers, in general, interested in
developing the skills required to operate a good anaerobic
digestion system.
The aerobic process,in contrast to anaerobic, utilizes molecular
oxygen as the final electron acceptor. Under many natural
conditions, such as in turbulent flowing streams, etc.,
sufficient oxygen is available to satisfy the needs. If the
supply and availability of natural oxygen is limited, however
mechanical means may be employed to provide additional oxygen.
Aerobic bacteria grow rapidly and degrade soluble organic
materials very effectively, provided adequate oxygen is
available. One principal advantage of the aerobic process
as applied to the treatment of livestock wastes is that
little or no odor is generated.
Supplemental oxygen may be provided to sustain an aerobic
condition in a variety of ways. In some instances shallow
ponds or lagoons (about 4 feet deep) may be used. If the
loading rate of the waste materials is too great, a deficiency
in naturally absorbed oxygen may occur with ponds or lagoons.
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Such conditions may require the use of special floating
aerators in order to maintain a sufficient supply of
oxygen.
Scheltinga (3) and other European investigators, have
utilized the Pasveer oxidation ditch as a means of introducing
supplemental oxygen into a waste treatment situation. This
method is presently in the developmental stage in the U.S.
for both municipal and animal manure waste disposal. In
1967 in the United States there were about 400 oxidation
ditches in operation; primary agricultural use was in swine
operations (4).
The basic operation of an oxidation ditch is similar to that
of an aerated pond or lagoon, except that with the former, the
liquid waste material is circulated by means of a horizontal-
shaft rotor. The purpose of the rotor is (a) to propel the
water at a velocity sufficient to keep most solids in suspension
and (b) to add oxygen to the waste material.
Recent investigations by Walker (5), Pasveer (6), Morris (7),
Irgens (8,9) Dale (10), (11), and Day (12) have shown that the
extended aeration process of aerobic digestion of animal
manures is practical and has specific advantages over anaerobic
digestion methods. The use of the oxidation channel for bio-
logic treatment of animal manures is expanding and appears
to be an effective and practical method.
Design criteria used for construction of animal oxidation
ditches, and other forms of the extended aeration method,
have been based largely on design and data obtained from
municipal treatment plants. Differences are noted when one
compares human waste to animal waste. Two of the more im-
portant differences are: human wastes as collected by the
water-carriage system are more diluted with water than are
manure wastes from feedlots, dairy or hog barns, or poultry
production units; animal wastes contain more slowly degradable
materials, such as grain hulls, cellulose and feed fibers.
New systematic approaches to waste disposal are being developed
in part in response to society's demand for bettering environ-
mental quality. Human disease problems associated with agri-
cultural occupations have been documented and there is growing
recognition and concern of man's contact with livestock wastes
through increased recreational and outdoor activities.
More than 150 zoonotic diseases are transmitted between
animals and man. Several hundred diseases are transmitted
from animal to animal. Many of the etiologic agents are shed
in animal wastes, or contaminate animal wastes where adequate
nutrients for survival and growth may be found. At times
agricultural and recreational use of lands may conflict j with
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combined usage of surface waters from the health point of
view.
Wedum and associates (13) reported on a survey of recovery
of specific microorganisms from urine and feces of
experimentally infected cattle and found that agents of 14
specific disease entities were recovered from the feces of
cattle infected with adenovirus, anthrax, brucellosis,
Coxsackie virus A, Coxsackie virus B, enterovlrus, foot and
mouth disease, leptospirosis, psittacosis - ornithosis, Q-fever,
reovirus, rinderpest, tuberculosis and tularemia; and 7 agents
from urine of experimentally infected cattle, (brucellosis,
foot and mouth, leptospirosis, Q-fever, rinderpest, tuberculosis,
and tularemia). This report excluded most intestinal diseases.
Epidemiologic investigations have associated pathogenic
microorganisms of animal waste origin with outbreaks of human
disease. Decker and Steele (14) report that human health
problems are created by bacterial zoonoses. These include
leptospirosis, salmonellosis, staphylococcal and streptococcal
infections, tetanus, brucellosis, tuberculosis and colibacillosis
and diseases by other classes of pathogenic agents which occured
following contact with wastes. Animal wastes also serve as
breeding grounds for many vectors essential for viral transmission.
In an extensive literature survey titled "Solid Waste/Disease
Relationships," Hanks (15) states that the literature
fails to supply data which would permit a quantitative estimate
of relationship between solid waste and disease. He further
states that circumstantial and epidemiologic information
presented in reports does support a definite relationship of
disease and solid waste —including animal waste. He further
states that in developed countries reported incidence of human
infections traceable to animal fecal wastes is low—but suspicion
is that the actual number of cases is probably much higher.
In implicating livestock wastes as vehicles of disease,
many variables affecting the host-agent-environment relation-
ship exist under field conditions.
A decision was made to study the pathogenic agents of two
disease entities that effect both animals and man. The
leptospiral and salmonella organisms were chosen because they
are shed, respectively, in urine and feces of infected animals.
It is anticipated that in the future many farmers in Minnesota
and other parts of the United States will construct
oxidation channel facilities similar to the field unit now
in operation at the Rosemount Agricultural Experiment Station
of the University of Minnesota. The following objectives
were developed.
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Major Objectives were on the:
1. Survival of Leptospira pomona and Salmonella typhimurium
in cattle manure disposal under specific, controlled en-
vironmental and physical conditions.
2. Comparision of methods for qualitatively determining the
survival of these two pathogens (L.pomona and _S. typhimurium)
in animal manure.
3. Simulation, production and maintenance of field environ-
mental conditions in laboratory model research units.
4. Establishment of criteria in the hydraulic and structural
design of oxidation channels, vertical aerators, other
forms of extended aeration devices; permissible loading
rates of solid waste into aeration devices, especially at
warm and cold temperatures, and the effect on survival of
pathogens.
The more specific aims were:
1. Determining and evaluating potential public health hazards
created by the extended aeration process, if survival of
Leptospira pomona and Salmonella typhimurium occur in
either the effluent or sludge of operational laboratory
. models.
2. Determining the effect of specific field environmental
conditions and chlorination upon the survival of L.
pomona and J3. typhimurium and the comparision of methods
for detection of pathogens in laboratory models.
3. Determining under laboratory-controlled conditions: the
performance of oxidation channel models when loaded with
beef animal manure.
Organization for Study
The overall project direction was provided by principal
investigator Dr. S.L. Diesch, Department of Microbiology and
Public Health, College of Veterinary Medicine. He was assisted
by co-principal investigator Dr. B.S. Pomeroy, Professor and
Head of the Department of Veterinary Microbiology and Public
Health.
For many years Dr. Pomeroy has been active in the prevention
and control of saImonellosis. During the final year of the
study, Dr. L. Will, Research Associate, Department of Veterinary
Microbiology and Public Health, contributed greatly to the
microbiologic aspects, the total research efforts and to the
preparation of this final project report.
14
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The microbiologic studies of the leptospires and
salmonella survival and detection were conducted in the labora-
tories of the Department of Veterinary Microbiology and Public
Health. Most of these studies were conducted in a laboratory
model designed and developed to simulate the field oxidation
ditch.
Two laboratory models were designed, constructed and
maintained by co-principal investigator Professor E.R. Allred
of the Department of Agricultural Engineering, University of
Minnesota. Professor Allred directed and conducted the engineer-
ing aspects of this research in the laboratories of the De-
partment of Agricultural Engineering. Total solids determin-
ations of the liquid manure from the field and laboratory ditch
model were conducted in the Agricultural Engineering laboratories
Mrs. Jenny Trombley and Mr. Egon Straumann were employed for
the total of the three year project and were invaluable
laboratory technical and scientific personnel. On a part-time
basis veterinary medical and engineering students were employed
throughout the study.
An essential part of this research project was the prior
development, construction and utilization of the field unit
oxidation ditch which had been operational at the Rosemount
Abricultural Experiment Station since 1967. Beef cattle were
housed on slatted floors and fattened over the ditch, thereby
setting-up a realistic field situation. Credit for the develop-
ment and maintenance of the field operational unit goes to
members of the Departments of Agricultural Engineering and
Animal Science. Financial support for the field project was
obtained from the University of Minnesota Agricultural Experi-
ment Station and the U.S. Department of Agriculture. Essential
environmental data to determine summer and winter conditions
were gathered from the field ditch unit. Samples of liquid
manure were collected from the field ditch unit and utilized
for survival studies in the laboratory model ditch of the De-
partment of Veterinary Microbiology and Public Health.
Nature and Scope of Study
Efforts were made to develop new methods and further refine
standard methods of pathogenic leptospira and salmonella detec-
tion. The measurements of maximum viability of these pathogens
were qualitative determinations.
Another important aspect of this study was the design,
development and utilization of laboratory models to simulate,
not duplicate, environmental field conditions. It was possible
to seed a laboratory model ditch with pathogenic bacteria.
Seeding a field ditch with pathogens would have been undesirable.
15
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We had initially proposed to collect and study effluents
discharged from the field operation ditch, however, during
the three years of study no effluents were discharged.
Effluents for research utilization were developed for patho-
gen survival and detection studies by allowing liquid manure
from the laboratory ditch model to separate by gravitation
into effluent and sludge.
Laboratory units were utilized as models to study the
survival of pathogens and the engineering aspects under simulated
conditions of the field units to determine the public health
significance. Studies were conducted to determine pathogen
survival at specific pH, dissolved oxygen, temperature and
total solids levels. From findings during the operation of
the field facility at Rosemount and from other studies, it
was apparent that because of differences, it was difficult to
employ the same design criteria for human wastes as for animal
wastes in the construction, operation of oxidation channels,
and the subsequent effect on pathogen survival. The most
difficult problems arose because of differences in: a) total
solids present in waste liquors; b) rate of settlement and
stablization of solids; and c) physical effects of large
solid particles on hydraulic flow patterns within an oxidation
ditch. There was evidence that the hydraulic efficiency of
the oxidation channel must be improved in order to avoid
settling-out of solids in certain areas, with subsequent
creation of odorous and anaerobic conditions. Laboratory trials
by Dale (8), and field ditch observations by Day (10) and
Allred (Co-principal investigator) indicate that screening of
undigested materials is desirable before the animal wastes
reach the ditch. Current investigators have generally found
the need for a holding basin (lagoon, pit, settling tank) to.
permit flocculation and settlement of solids. Studies to
determine the survival of pathogens and the effect on the
health of man and animals had not been evaluated. Research
was conducted to determine if the extended operation process
has a lethal effect, or no effect, or promotes viability
of pathogens. It was proposed that research could best and
most economically be conducted under laboratory controlled
conditions.
Future systems developed and utilized for animal manure
disposal must encompass consideration for all aspects of
public health as a means of prevention and control of
disease in both man and animals.
Methods of Procedure
Field Oxidation Ditch Facility
During recent years agricultural engineers at the University
16
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of Minnesota and the U.S. Department of Agriculture have con-
ducted experiments on treatment of beef cattle wastes in an
oxidation ditch. The function of the oxidation ditch is to
promote aerobic degradation of the animal manure. A liquid
medium is provided within the ditch to collect and treat the
animal waste as it enters. This treatment process varies,
depending on the objectives and the particular system involved.
One system provides partial treatment in the oxidation ditch
and relies on an external lagoon to provide additional treat-
ment until final disposal or utilization occurs. A batch
type oxidation ditch may be used to provide containment and
partial treatment until the waste can be removed and disposed
of on the land for final treatment.
The oxidation ditch provides several advantages to the beef
operator.
1. One of the primary concerns is odor control. A rotor
provides aeration and controls velocity of the flow.
In doing so, an aerobic condition is maintained and sub-
sequent degradation or breakdown of the waste is odor
free.
2. The second advantage offered is that of storage. Storage
reduces the possibility of runoff and pollution of surface
and groundwaters, since the operator may dispose or utilize
the animal waste on agricultural land when and where the
soil can accept the waste. If this is done sometime in
late spring through early fall, maximum utilization of the
nutrients and organic matter in the waste is obtained.
Spreading through the summer may also result in runoff
under certain conditions (in Minnesota).
3. The treatment received by the waste in oxidation ditches
reduces the pollutional strength of the material and in
so doing reduces the potential hazard which may occur from
land spreading. The levels of chemical and biochemical
oxygen demand, and solids can also be reduced in an
oxidation ditch.
4. The use of slattedfloors above an oxidation ditch also
results in a reduced labor input into the waste collection
treatment process.
The objective of the above research has been to explore
the use of the oxidation ditch for the handling of beef
cattle waste by providing a storage-treatment system with
maximum odor control and minimum labor requirements.
Figure 1 shows a floor plan of the pilot scale oxidation
ditch as it appears at the Rosemount Experiment Station,
Rosemount, Minnesota. It is a race-track type of oxidation
17
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24'-0"
ROTOR
OXIDATION DITCH
AGRICULTURAL ENGINEERING
UNIVERSITY OF MINNESOTA
ROSEMOUNT EXPERIMENT STATION
SIPHON
CHAMBER
Figure 1 Plan view of the oxidation ditch and research structures
at Rosemount, Minnesota.
18
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ditch which provides a continuous channel 172 feet long,
7 feet wide, and 4g feet deep. Poured-in-place concrete was
used to eliminate any losses to percolation. The ditch is
located within a rigid,framed, steel building enclosed on three
sides. Within this building are three beef-feeding, environmental
units constructed to evaluate the effects of the system on the
management and feeding of beef cattle. Two of these units are
located over the oxidation ditch and the waste generated passes
through the slots to the liquid below.
An initial group of 60 Holstein steers was placed in the field
unit on 11/4/67. Water was added to the ditch and the rotor
started in January, 1968. A second herd, of 45 Holstein steers
was placed over the unit on 6/6/68 and removed on 10/1/68.
The liquid ditch temperature ranged from 16 degrees to 20
degrees G. From this herd of cattle the liquid manure samples
were utilized in the laboratory of this research project.
After each herd was sold to slaughter most of the sludge was
removed and spread on the fields, and the remaining portion
used for seeding purposes in the next batch.
A third herd of 36 Hereford cattle was placed in the field
unit on 11/5/68. The liquid ditch manure temperature ranged
from 2.6 degrees to 0.2 degrees C. for November, December and
January. During this period, severe foaming of the ditch was
encountered which may predispose to aerosol transmission of
pathogenic organisms. The pH ranged from 8.2 - 8.3 during
this same period. The dissolved oxygen (D.O.) was 0.5 - 1 ppm
of the liquid manure and sludge were determined. This informa-
tion has been utilized in the laboratory model studies.
Tests of the treatment and storage of beef waste in the
oxidation ditch at Rosemount were begun in December, 1967.
Starting loading rates for four experiments have been 210, 138,
38, and 50 cubic feet of water per animal, respectively.
These experiments were conducted on a batch basis.
Continous measurements of dissolved oxygen, BOD, nitrogen,
and solids were made to define the functional operation of
the Rosemount Oxidation Ditch. No attempt was made, however,
to determine the extent of the survival of disease pathogens
in the Rosemount or other ditches. Construction and operation
of the Rosemount facility was financially supported by the
University of Minnesota Agricultural Experiment station and the
U.S. Department of Agriculture, (16, 17, 18, 19, 20, 21, 22).
The operational Rosemount Field unit of the oxidation ditch
was essential to this research project as it provided experi-
19
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mental parameters which were utilized in both laboratory models
(Figures 2,3). The data of environmental conditions from the
field oxidation ditch was utilized in the laboratory studies.
Development of Scale Laboratory Model Oxidation Ditches
Two laboratory models (1:10 scale) of the field oxidation
ditch at Rosemount were constructed. To simulate winter liquid
temperature conditions, as observed in the field ditch, it was
necessary to insulate the models by wrapping with two inch-
thick rigid styrofoam. In order to provide portability to the
models, the original cooling system was completely redesigned.
A three-quarter horsepower cooler-condenser unit, installed
directly beneath the ditch, served to cool the ethylene glycol
solution, which was pumped and circulated through the stainless
steel trough dividing wall within the ditch. An overflow of
coolant into the liquid manure caused termination of an experi-
ment on leptospiral survival and the loss of several weeks research
time. It was again necessary to redesign the cooling system into
a closed system of coils centered in the ditch. Since the models
were operated in indoor heated laboratories, the cooling system
operated much of the time. Provision for slight warming of
ditch liquids has also been provided and were used during sur-
vival studies, under summer (warm) conditions.
The final ditch design also included the addition of a
plexiglass cover. This cover provided the operating personnel
protection against aerosols which may contain pathogenic
microorganisms. It also provided greater uniform environmental
conditions at the liquid-atmosphere interface. Work in im-
proved engineering design criteria of the laboratory oxidation
ditches and its components were accomplished during the
construction phase of the laboratory models.
Utilization of .the Model A Oxidation Ditch
In the first model (hereafter referred to as Model A)
efforts were made to simulate environmental conditions exist-
ing at the Rosemount field oxidation ditch. (Figure 4). The
following seasonal data was collected in 1967-68 from the
operational field ditch over which 36 head of beef cattle were
housed: pH ranged from 6.8 - 8.4; winter ditch temperatures
ranged from 1.7 - 6.4C and summer from 13 - 25C. Total solids
ranged from 5,802 - 135,333 mg/liter. Since the field ditch
was tested experimentally for maximum loading capacity, no
effort was made to simulate the total solids levels of the field
20
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Figure 2. Field oxidation ditch, rotor. Roseraount
Experiment Station (University of Minnesota).
Figure 3. Cattle unit housing 18 steers above an operational
oxidation ditch. Rosemount Experiment Station,(University
of Minnesota).
NOT REPRODUCIBLE
2l
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NOT REPRODUCIBLE
Figure 4 Laboratory Model A, a 1:10 scale model of an operational field
oxidation ditch, contains beef cattle manure. Rotor is shown
on the left.
22
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ditch in Model A. Attempts were made to maintain the laboratory
model total solids at levels from 5,000 - 10,000 mg/liter.
This range of total solids had been reported as being maintained
in oxidation ditches used for. human sewage disposal. Attempts
were made to maintain the dissolved oxygen (D.O.) between 1
and 5 ppm in the liquid manure of the laboratory model.
Model A was filled with 113 liters of manure (liquid media)
from the field ditch. Following addition of manure the labora-
tory model oxidation ditch was allowed to function a minimum
of 1 week to stabilize the environmental conditions. At least
once a week, fresh samples of liquid manure were transported in
5 gallon jugs from the Rosemount field ditch to the laboratory.
The manure was refrigerated at 2C until added to the laboratory
model. Each lot was sampled and examined for presence of lepto-
spires by darkfield microscopy and fluorescent antibody technique.
Isolation attempts were made in artificial culture media. Lots
were examined for salmonellae by the fluorescent antibody
technique and cultural procedure. Only lots found negative
were added to the ditch. During initial experiments 2.2
Ibs. of liquid manure was added each day to the ditch. Due
to a build up of total solids (above 10,000 mg/liter) daily
additions of manure to the ditch were discontinued. Additions
were made intermittently to maintain the total solids at
10,000 mg/liter. It was occasionally necessary to add
unchlorinated well water from the Rosemount station well to
maintain desired operating range of total solids.
Portable effluent chambers,as settling chambers,were designed
and constructed by the agricultural engineers to hold 1,000 ml.
of liquid manure. The material used was plexiglass. Initially,
the environmental temperature was maintained by placing the
chamber in the liquid manure of the ditch (Figure 4). Subsequent
studies were conducted with the chamber removed from the laboratory
model ditch. During summer temperature studies, foaming of the
liquid manure in the laboratory ditch was a problem. This
foaming occurred during laboratory ditch start-ups. The foaming
was reduced by slowing the rotor speed and spraying the foam
with Dow-Corning Anti-foam A.* As the ditch stabilized, the
problem appeared to cease. During this same time period, foam-
ing was a problem in the field ditch operation, following re-
moval of total solids and re-stabilizing the ditch.
Temperature, pH and D.O. were monitored and recorded at 6
hour intervals by use of monitoring probes, (Figure 5). To
maintain uniform external environmental conditions, room
*Dow Corning Antifoam A Spray
Dow Corning Corporation
Greensboro, North Carolina
23
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NOT REPRODUCIBLE
•i
Figure 5 Laboratory model A oxidation ditch with monitoring probes shown
on left used to collect pH, D.O. and temperature data. Selas
candles shown on right were used to contain lep'tospires during
one phase of survival studies.
24
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temperature, barometric pressure and per cent relative
humidity were recorded daily.
Utilization of Model B Oxidation Ditch
The second oxidation ditch model, illustrated in Figures
6 and 7 and hereafter referred to as Model B, is a duplicate
of Model A except that a sump or storage pit was installed in
the former to facilitate the separation and removal of specific
solid materials. Two separate rotor designs were also construc-
ted and tested in Model B. Early tests were made using a
brush-type rotor but its use was discontinued when it became
obvious that insufficient water velocities were being generated,
Studies of the engineering aspects of oxidation ditch operation
were conducted in the Agricultural Engineering Waste Research
Laboratory, under the direction of Professors Evan R. Allred,
Co-principal Investigator, Phillip Goodrich, and James A.
Moore, all of the Department of Agriculture Engineering.
Investigations involving engineering design and operation
of the oxidation ditch focused upon the following major
objectives:
1. Determination of the effect of the location, speed and
immersion depth of the rotor upon solid settlement patterns.
2. To observe and measure the movement of solids for varying
stream-bed configurations and water depths.
3. Determination of the effect of rotor immersion and water
depths on velocity distribution patterns within the ditch.
4. Establishing criteria for design of field rotor units.
25
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Figure 6 Model B oxidation ditch in which velocity
distribution and solid sedimentation studies
were made.
NOT REPRODUCIBLE
Figure 7 Overhead view of Model B oxidation ditch.
26
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SURVIVAL AND DETECTION OF LEPTOSPIRES IN BEEF CATTLE MANURE
Introduction
Leptospires are pathogenic microorganisms which are mainly
shed in the urine of infected animals. The disease is wide-
spread in cattle, swine and many other animals. Large num-
bers of leptospires (100,000,000 per ml) in the urine have
been reported (23). Infected cattle are frequently shedders
of the organisms for periods up to several months. The agent
lives for extended periods of time in the environment and
is transmissible to both man and animals.
Leptospirosis has been referred to as the world's most wide-
spread, contemporary zoonosis. Although only 52 human cases
of leptospirosis were reported in the U.S. in 1970 (24) there
is evidence that many cases are not diagnosed and reported
(25,26). In the United States several outbreaks of human
leptospirosis have been associated with Ij. pomojia infection
from contact with water contaminated with urine from infected
cattle (25,27). In the U.S., serotype pornona is most commonly
reported in cattle. One outbreak described 40 human cases in
Iowa. In Iowa in 1963, Diesch and McCulloch (28) reported 15
cases following swimming in a farm creek and L,. porno na was
isolated from the swimming site. Forty cases were reported to
have occurred among packing house workers in Iowa (29). Sixty-
one human cases occurred in Washington following swimming in
irrigation ditch water contaminated by urine of infected
cattle pastured nearby (30).
According to Gillespie et. al. (31), Chang, Buckingham and
Taylor (32), and Ryu and Liu (33), leptospires may live in
water for several weeks. The overall public health effect
and impact of this disease has been well documented.
From 1951-1960 (34) the estimated average annual loss to the
1ivestock industry due to leptospirosis (dairy and milk)
was $12,189,000. The Leptospirosis Committee of the United
States Livestock Sanitary Association (35) stated that the
disease was not amenable to eradication and cited the need for
continuing prophylactic programs in the problem herds to pro-
vide protection against cyclic recurrence of infection.
Materials and Methods:
Development
The Leptospira pomona MLS strain originally isolated from
infected cattle, was selected as the serotype to be utilized
in the survival studies. In preliminary studies, intra-
peritoneal inoculation of leptospires into weanling hamsters
27
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caused death. Known cultural methods for maintaining and
growing adequate numbers and inoculating known numbers into
the laboratory model oxidation ditch were utilized, and
newer and improved methods were continuously evaluated.
Initially, there was a definite need for adequate laboratory
methods to measure survival of leptospires in animal manures
under specified field environmental conditions. Under standard
laboratory procedures it was difficult to obtain contaminant
free cultures. Developing satisfactory methods for selective
isolation of leptospires from massivly contaminated media, such
as beef cattle manure, posed a difficult problem.
Developing methods for containing leptospires in a specific area
of the model ditch was a major problem. This had to be resolved
by developing a suitable chamber to retain the leptospires
suspended in the manure medium. The chamber or container had
to contain an adequate number of leptospires for repeated
sampling and allow nutrient exchange and maintain comparable
environmental conditions as found in the laboratory ditch
manure. Several preliminary studies were conducted using
colloidal sacks, mylar sheeting, millipore filters and Rose
perfusion chambers, none of which were found to be suitable
for this study. The cellulose base filters were digested by
cellulose digesting organisms found in the beef cattle manures
and subsequently the leptospires escaped.
Methods were developed to wash and concentrate leptospires
for .inoculation into the laboratory oxidation ditch and the
isolation chamber. The organism is fragile and care must be
used in this procedure not to cause fragmentation or injury
to the leptospires.
The procedure for isolation of leptospires from the laboratory
model oxidation ditch and subsequent identification, to ascertain
survival times, was developed. These methods are described in
the text. In an attempt to develop new and more accurate
methods, alternative methods were constantly considered and
tested throughout the three year study. The solids and the
viscosity of the mixed liquor of the oxidation ditch posed
problems in attempted pre-filtering prior to isolation and
identification. Various methods of isolation by filtering,
centrifugation and gradient centrifugation, serial dilution and
agar plating techniques were tested as they were developed.
Growth of Leptospiral Inoculum
Leptospires for inoculation (seeding the laboratory model
ditch) were propagated in bovine serum albumin culture
medium (36). To inhibit contaminating bacteria, 5-fluorouracil
(100 meg/ml) was added to the media (37). Actively growing
28
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5-7 day cultures were centrifuged at 10,000 rpm for 5 minutes,
the supernate drawn off, and leptospires resuspended in phos-
phate buffered saline.
Numbers of concentrated leptospires were estimated by utiliza-
tion of the Coleman Model 9, Nephlo Colorimeter, and readings
from a standardized curve with Nephlos units against the num-
bers of leptospires per ml. of suspension. (Figure A, Appendix
A). The actual count of leptospires was determined with a
Petroff Hauser Counter.
Methods of Seeding for Survival Studies
1) Seeding in Selas Porcelain Candles Suspended in
Laboratory Model A Ditch.
Extensive research was conducted on the use of an isolet
chamber utilizing Gelman glass filters attached to the ends
of a circular pipe for containing leptospires. Because of
contaminant overgrowth within the chambers the use of the
isolet unit was discontinued. Following experimental evalua-
tion of many other methods, Selas porcelain candles, 0.3 micron
porosity, were utilized to contain leptospires and allow nutrient
exchange with the liquid manure environment. Three Selas candles
were used in each experiment indicated, and suspended in the man-
ure of Model A (Figure 5). The model oxidation ditch cover
shielded the open end of the candles from the external environ-
ment.
Candle A contained phosphate buffer solution and lepto-
spires (control).
Candle B contained sterile (autoclaved) manure and leptospires
(control).
Candle C contained manure and leptospires.
Three hundred million leptospires (3cc of 25 Nepholos BSA
culture) were placed in the material of each candle. During
each experiment conducted, the contents of the candles were
sampled daily and examined by Darkfield microscopy to detect
leptospires. The pH, D.O. and temperature were monitored from the
ditch environment.
2) Seeding in Liquid Manure of Laboratory Model A Ditch.
It was postulated that 36 beef cattle housed over the field
ditch may shed leptospires in the urine at a maximum rate of
100 million per ml. if infected. The leptospiral concentration
used for seeding amounted to a concentration of 1:1000 ratio,
and potential leptospires in urine (37.5 billion) were added daily
for 5 days.
3) Seeding in Effluent and Sludge of Model Settling Chamber
29
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The settling chamber was developed as a model of that designed
for use in the field oxidation ditch. Liquid manure (1,000 ml)
collected from the surface (top 1") of Model A was placed in
the settling chamber. In several hours, the liquid manure sep-
arated into distinct zones of effluent and sludge. Approx-
imately 1.2 billion leptospires were added to the chamber. The
environmental temperature of the settling chamber was maintained
at summer and winter conditions.
Maintenance of Study Environment in Laboratory Model -Oxidation
Ditch~"
1) Total Solids
Initially, attempts were made to add approximately 2.2 Ibs.
of liquid manure (collected from the field oxidation ditch)
each day to the ditch. This amount simulated the ratio of
defecation and urination of cattle housed on the slatted floor
above the field ditch. This daily addition was discontinued
and an intermittant addition of liquid manure was utilized to
maintain the total solids at 5,000 - 10,000 mgs/1. ;When total
solids were found higher than 10,000 mgs./l it was necessary to
add unchlorinated Rosemount well water to lower the total
solids. ,
2) pH _,,.•__
In most instances the pH range of the manure stabilized to
simulate the range of the field ditch (Appendix A). I)aily
additions of small amounts of manure lowered the alkaline pH.
3) Temperature
The ambient temperature was maintained in the range; of> 2 - 5C
(winter) by utilization of the refrigeration unit and
insulation of the laboratory model ditch. It was also necessary
to alter and stabilize the room temperature to effect maintain-
ing summer ambient temperature, 20C was more difficult to main-
tain. The situation was improved by installation of a window
air-conditioner to maintain a more stable room temperature.
4) Dissolved oxygen (D.O.)
During summer temperature studies, the D.O. of the liquid
manure was maintained at 5 ppm by regulating the speed of
the rotor. No difficulty was encountered in maintaining these
levels in summer studies.
i
During winter environmental studies (2 - 5C) it was nearly
impossible to maintain the D.O. at 5 ppm. When the rotor was
30
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stopped the D.O. reading was higher than 5 ppm. Addition of
small amounts of manure lowered the D.O. level reading.
Methods of Sampling for Leptospires
1) Selas Candles
Selas porcelain candles (No. 03 which are 0.3 porosity) were
utilized. Samples for detection and measuring'survival were
collected from the candles by use of a disposable pipette
with an attached bulb.
2) Laboratory Model Ditch Manure
Daily collection of manure samples were made by pipette from
the top, middle and bottom of the liquid manure in the labor-
atory model ditch. Two collection sites were (a) in front
rotor, where material was thoroughly mixed, and (b) other side
of divider, where settling of solids occurred.
3) Settling Chambers (Effluent and Sludge).
Samples were collected by pipettes at the top, middle and
bottom of the effluent settling chambers. The top and middle
zones were relatively clear and considered effluent. The
bottom was solids and considered sludge.
Methods of Detecting Leptospires
1) Fluorescent Antibody Studies (FA) to Detect Leptospires
After testing of several fluorescent antibody methods the
following procedure was developed to detect leptospires.
However, the FA technique does not measure survival.
For description of method used for fluorescent antibody
staining see Appendix A.
2) Darkfield microscopy.
All manure samples and inoculated culture media were examined
(645 x magnification) for presence or absence of leptospires.
Methods of Measuring Leptospiral Survival
(1) Tube Dilution Procedure for isolating leptospires
Tubes containing (8 ml.) liquid culture medium (Bovine serum
albumin) were inoculated daily with specimens from the manure
seeded with leptospires. A 10-fold serial dilution procedure
was utilized. The tubes were incubated for growth of lepto-
spires at 30C and examined microscopically at 7-12 day intervals
31
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for up to 6 weeks to determine leptospiral growth.
(2) Agar Plates for Isolating Leptospires
A modification of the Smibert's modification of Loesche and
Socransky's isolation agar plate technique was developed and
utilized. Johnson's Basal Medium with 1% agar ( 9 vols) and
rabbit serum(1 vol) was used. Small plastic (60 mm x 15 mm)
dispo plates contained the agar, and a .22 micron Millipore
filter (sterile) was placed on the agar. A sterile plastic
ring (1 in. diameter) was adhered to the filter with sterile
stop cock grease. A manure specimen from the inoculated candles
was placed within the ring. The plates were incubated at 30C.
The filter and ring were removed from the agar plate in 4-14
days. Initially, growth of leptospires were first read at
4-6 days. Plates were held for up to 6 weeks and examined
microscopically at weekly intervals for presence of leptospiral
growth. (For detail protocol see Appendix A).
Identification of Isolates
Following isolation in cultural medium leptospires were sub-
cultured and filtered to remove contaminants and preliminaryiy
identified by the microscopic agglutination (M.A.) tests
against specific pomona hyperimmune serum produced in rabbits.
Survival and Detection of Leptospires in the Laboratory Oxidation
Ditch at Winter Temperatures.'
The following survival studies were conducted to determine ,
the ability of leptospires to survive in the environmental
conditions of Model A summer and winter temperatures as occurs
in Northern climates. These studies were conducted with
leptospires seeded in Selas candles suspended in the manure of
the oxidation ditch in the laboratory, and seeded directly in
the manure of the ditch, and seeded in the sludge and effluent
of settling chambers.
The following experiments were conducted at winter environ-
mental temperatures:
1. Selas candles, 6 experiments No. 1LW, 2LW, 3LW, 4LW,
5LW, 6LW (Appendix A).
2. Settling chambers, 3 experiments No. 7LW, 8LW, 9LW
(Appendix A).
3. Direct seeding of Ditch, 1 experiment No. 10LW (Appendix A)
Survival and Detection of Leptospires in the Laboratory
Oxidation Ditcn Moaei at bummer Temperatures.
32
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The following experiments were conducted at summer environ-
mental temperatures.
1. Selas candles, 5 experiments No. 1LS, 2LS, 3LS, 4LS
5LS (Appendix A)
2. Settling chambers, 5 experiments No. 6LS, 7LS, 8LS,
9LS, 10LS (Appendix A)
3. Direct Seeding of Ditch, 2 experiments No. 11LS,
12LS (Appendix A)
Miscellaneous Studies
One study (1A) was conducted at a mean temperature of 12.6C
This temperature approximated spring or fall ambient temperatures,
A study was conducted to measure survival and detection of
leptospires seeded in unchlorinated well water collected from
a well at the Rosemount Agricultural Field Station and in a
natural stream water collected from a stream located in the
vicinity of the Rosemount oxidation ditch.
Results
All attempts to isolate or detect leptospires in manure from
field ditch samples (prior to adding to model ditch) were
negative.
Methods of Detecting Leptospires
1. Fluorescent antibody staining (FA) to detect leptospires.
A satisfactory method for the FA procedure was developed and
utilized. Leptospires were observed by FA in the manure for
the length of time in which survival was measured culturally
or for longer time periods.
2. Darkfield Microscopy
Leptospires were identified by darkfield microscopy of the
specimen examined. These findings were consistant with the
FA findings and many times of longer duration, than the sur-
vival which was measured culturally. Initially, motility of
leptospires was observed with a subsequent decrease until
non-motility was observed. Finally fragmentation and disinte-
gration were noted.
Methods of Measuring Survival
1. Tube dilution procedure for measuring leptospires.
33
-------
Serial dilutions of manure samples, 1/1,000, 1/10,000 and
1/100,000 in culture medium were found most satisfactory
for diluting contamination and measuring survival. Some
inoculated tubes, especially in lower (1/10 - 1/100) dilutions
were discarded prior to the 6 week reading as a result of
contamination over-growth. In later experiments only 1/1,000-
-1/100,000 dilutions were used, thus saving time, space and
culture medium.
2. Agar plates for isolating leptospires
Isolation of leptospires were made using this technique
during summer environmental temperatures, but attempts to
isolate at winter temperatures were negative.
Refer to Table I, Appendix A, for results and comparison of
data on the survival and detection of leptospires under winter
environmental conditions.
Experimental details of daily examinations under specified
environmental conditions are given for studies in Selas candles,
the settling chamber and direct seeding in the ditch in the
Tables (Appendix A). Note that daily readings, for example,
pH, D.O. and temperature are found in the tables. Those figures
are averages of 4 readings recorded each 24 hours during the
time of experiments.
Sampling for detection or measuring survival was conducted
once each day during the time in which.the experiment was
conducted.
Survival and Detection of Leptospires in a Model Oxidation
Ditch at Winter Temperatures
1. Leptospiral survival studies in Selas candles suspended in
the manure in ditch Model A.
Experiments 1LW, 2LW, 3LW, 4LW, 5LW, 6LW (Appendix A) are
summarized in Table lA. The maximum survival time measured was
11 days (Exp. 6LW). Survival was measured until
termination of the experiments. No survival was measured in
Exp. 3LW. The survival time of leptospires in Candle C in
Exp. 4LW, 5LW, and 6LW was considerably longer than control
candles A and B. With the exception of one isolation made in
(control) Exp. 1LW, the agar plate method of isolation was
found to be of no value at winter temperatures. Leptospires
were detected by darkfield microscopy and FA staining in nearly
all candles until termination of the experiments.
When the mean pH was 9 or higher, (compare Exp. 1LW, 2LW, and
3LW to Exp. 6LW), the measured leptospiral survival time was
34
-------
w
TABLE IA. SUMMARY OF SURVIVAL AND DETECTION OF LEPTOSPIRES IN A LABORATORY OXIDATION DITCH
AT WINTER TEMPERATURES.
In Selas Candles
Exp
No.
1 LW
2 LW
3 LW
4 LW
5 LW
6 LW
Days
Conducted
8
9
8
9
7
12
Mean
Temp
(C)
2.9
1.7
2.3
2.1
2.7
2.4
Mean
pH
9.2
9.0
9.0
8.4
8.4
8.2
Mean
D.O.
(ppm)
Survival
Tubes
13.4 ' I
13.4 1
12.7 0
7.3 8
6.4 7
5.4 11
and
Detection in Days
Plates
0
0
0
0
0
0
Darkfield
8
9
8
9
7
11
FA
8
9
8
9
7
11
In Settling Chambers
Exp
No.
7 LW
8 LW
9 LW
Exp
No.
Days
Conducted
9
7
12
Days
Conducted
Mean
Temp
(a
3.4
2.9
2.7
Mean
Temp
(C)
Mean
pH
8.7
8.3
8.2
Mean
pH
Mean
D.O.
(ppm)
Survival
Tubes
r* M* B*
3.5 9 9
4.0 7 7
6.8 11 11
Directly
Mean
D.O.
fppm")
8
7
11
in Ditch
Survival
Tubes
and
Detection in Days
Plates
T M B
0 0
0 0
0 0
and
0
0
0
Darkfield
T M B
979
111
11 11 11
FA
T M
9 9
7 7
11 11
B
9
7
11
Detection in Days
Plates
Darkfield
FA
10 LW
26
3.1
6.9
7.0
(22) 0
(18 days - post seeding)
25
24
*KEY
T = Top - effluent
M = Middle - effluent
B = Bottom - sludge
-------
shortened.
During winter environmental studies, great difficulty was
encountered in maintaining the D.O. manure environment below
5 ppm. Greater than 5 ppm D.O. was measured with the rotor
stopped for a period of time. Shorter survival times were
measured when D.O. was measured at more than 10 ppm.
2. Leptospiral survival in effluent and sludge of Model
Settling, chamber
Procedures used in these studies (Exp. 7LW, 8LW, and 9LW,
Appendix A) were summarized in Table IA and were identical to
those conducted during summer environmental studies except
that winter temperatures were maintained.
The maximum survival measured was 11 days or the duration of
the experiment conducted in effluent and sludge of Exp. 9LW.
3. Leptospiral survival studies in the Model A ditch
Approximately 37.5 billion leptospires were seeded directly
into the ditch liquid media daily for 5 days. Results are
found in Exp. 10LW, Appendix A and summarized in Table IA.
After the first day of seeding, leptospires survived for 22
days (18 days after 5th, final, day of seeding) in the sludge
and the liquid media of the ditch. No isolations were made
using the plate procedure. Leptospires were observed for
26 days by darkfield microscopy and 24 days by FA. The pH
mean was 6.9 and D.O., 7. These environmental conditions may
predispose the leptospires to longer survival.
The agar plate isolation method was found ineffective at
winter environmental temperatures. At this cold temperature
a lag time was observed in the growth phase in using the tube
method. While it was necessary to observe the cultue tubes
as long as 6 weeks, growth established in 4-7 days (summer) on
the agar plates. In both the darkfield and FA methods of
detection, leptospires were detected until the experiment was
terminated. The pH ranged from 8.2 to 8.7 and the D.O. was
about 5 ppm.
Survival and Detection of Leptospires in Model Oxidation
Ditch at Summer Temperatures.
Refer to Table IIA for results and comparison of data on the
survival and detection of leptospires under summer environ-
mental conditions. Table IIA summarizes the experiments. The
individual tables of experiments containing the daily observa-
tions made in Selas candles, direct seeding in the ditch, and
seeding in the effluent chambers are found in Appendix A.
36
-------
TABLE IIA
0)
SURVIVAL AND
TEMPERATURES.
DETECTION OF LEPTOSPIRES IN AN OXIDATION DITCH AT SUMMER
In Selas Candles
Exp
No.
1 LS
2 LS
3 LS
4 LS
5 LS
Days
Conducted
13
6
13
8
13
Mean
Temp
(C)
24.3
19.3
19.2
19.3
18.8
Mean
PH
8.4
8.4
8.6
7.9
7.0
Mean
D.O.
fppm)
Survival
Tubes
ND 0
1.1 0
2.8 2
4.0 1
5.0 6
and
Detection in Days
Plates
0
ND
1
1
1
Darkfield
13
5
11
5
13
FA
ND
3
13
1
12
In Settling Chambers
Exp
No.
6 LS
7 LS
8 LS
9 LS
10 LS
Exp
No.
11 LS
12 LS
Days
Conducted
8
4
5
6
7
Days
Conducted
24
Mean
Temp
CC)
20.0
19.2
18.8
20.0
18.8
Mean
Temp
fC}
18.8
20.1
Mean
pH
ND
8.7
9.0
ND
8.5
Mean
PH
7.9
6.4
Mean
D.O.
fppm)
Survival
Tubes
T M B
ND ND
7.7 220
8.9 554
ND 002
9.2 000
Directly in
Mean
D.O.
fppm)
and
Detection in Days
Plates
T
0
0
0
0
Ditch
Survival
Tubes
2.9 0
7.7 1
M
ND
0
0
0
0
and
B
0
0
0
0
DetectL
Plates
0
138
Daik field
T M B
883
444
553
621
732
on in Days
Darkfield
10
4
FA
T M
8 8
4 4
3 3
6 6
7 7
FA
11
0
B
8
4
1
1
7
KEY
ND = Not Done
T = Top - effluent
M = Middle - effluent
B = Bottom - sludge
-------
The studies conducted in the Selas candles are reported in
Table IIA and Experiments 1LS, 2LS, 3LS, 4LS, and 5LS
(Appendix A).
In these experiments leptospires survived in candle C for a
maximum of 6 days by the cultural method (Exp. 5LS), and no
survival in Exp. 1LS and 2LS. Comparing candle C with control
candles A and B, the leptospires survived longer in candle A,
containing leptospires and the phosphate buffer. In Exp. 5LS
the survival time measured was found identical in candles A,
B, and C.
Intact leptospires were detected by darkfield microscopic
examination and the fluorescent antibody staining for the
duration of the study (Table II, Exps. 1LS and 5LS). Isola-
tion procedures in some instances failed to measure viable
leptospires. It is significant that intact leptospires
existed in the manure environment for such long periods of
time. The darkfield examination of manure samples for lepto-
spires is a difficult procedure by which to evaluate viability.
Although motility may be observed, darkfield examination is not
a definitive measurement of survival. Leptospires were observed
as non-motile, but when inoculated into cultural medium, growth
and subsequent motility were observed. Due to the presence of
many kinds of microorganisms and artifacts the validity of
identifying non-motile leptospires is questionable.
Longer survival (6 days) was measured at neutral pH (Exp.
5LS) than at alkaline pH (Exp. 3LS), (2 days). The effect
of D.O. is noted when Exp. 2LS and Exp. 5LS are compared, with
no survival measured in Exp. 2LS with a D.O. of 1.1 ppm and
6 day survival measured in Exp. 5LS with a D.O. of 5.0 ppm.
Leptospiral experiments on leptospiral survival in effluent
and sludge of the model settling chamber.
The maximum time of survival measured in the effluent was 5
days and sludge 4 days (Exp. 8LS). Unfortunately, this exper-
iment was terminated on the 5th day. In Exp. 9LS and 10LS,
survival was not detected in the effluent in a 7 day period
but was detected in the sludge in the 3rd day (Exp. 9LS)
in most instances (Table IIA). Darkfield and FA techniques
detected intact leptospires for the duration of the experiment.
The survival of leptospires in Exp. 8LS effluent and sludge
existed under an extreme alkaline pH of 9.
A mean D.O. greater than 5 was measured in Exp. 7LS, 8LS,
and 10 LS. The higher D.O. may be explained by the presence
of H2S gas,, produced under anerobic conditions, resulting
in inaccurate D.O. determinations. Although the settling
chambers were not aerated, D.O. readings were recorded higher
than in Model A (aerated) experiments (Table IIA, Appendix),
reason, undetermined.
38
-------
(Table HA, Exp. 11LS) Surviving leptospires were not measured
in the mixed liquid manure or in the sludge. Being unable to
measure viable leptospires may have been due to the rapid death
of leptospires in the manure environment or failure of labora-
tory procedure to measure survival.
Leptospires were detected in the manure by darkfield microscopy
for 10 days and by FA techniques for 11 days. Based on pre-
vious survival experiments the D.O. level and pH appeared
adequate for survival, but no survival was measured by the
cultural technique.
Near the end of the project a repeat experiment of seeding
the laboratory model ditch (summer) was conducted. (Exp.
12LS, Appendix A). In this experiment only the rabbit serum
agar plate method of isolation was used. Isolation attempts
were conducted daily for 23 days and thereafter intermittently.
Manure (113 liters) was added to the model ditch only at the
beginning of the experiment.
Leptospires were cultured and isolated on the 82nd day post
seeding. Leptospires were isolated at all 6 sampling sites on
138 days postseeding by the plate culture technique. Morphological
identification was made by Darkfield microscopy. These isolates
were lost in sub-cultural techniques and definitive identifica-
tion was not made.
Miscellaneous Experiments Conducted on Leptospiral Survival
Temperature : Spring - Fall
One experiment (No. 1A) (Table IIIA) was conducted at a mean
temperature of 12.6C. This temperature is common in the fall
and spring seasons. Leptospires were found to survive for
5 days by the tube culture procedure and 7 days by the plate
culture technique. Leptospires were detected for the duration
of the experiment by the FA technique.
Leptospiral survival studies in Stream and Well Water from
Rosemount Ditch Area. (Table IVA)
Studies were conducted to determine the length of leptospiral
survival in Rosemount stream and well water. Three million
leptospires were placed in a Selas candle that was placed in
well water. In another experiment 1.5 x 10 leptospires were
placed directly into well and stream water held in containers.
Preliminary survival in water studies were conducted at summer
temperatures. Survival in well and stream water was measured to be
2 days. Leptospires were detected for longer periods of time
by darkfield and fluorescent antibody techniques. The tube
culture and plate agar techniques were comparable in measuring
39
-------
Table IIIA
SURVIVAL AND DETECTION OF LEPTOSPIRES IN ANIMAL MANURE DISPOSAL
Day
Manure
Added
(Ih)
Manure Environment
pH
Mean
Ditch
Temp .
cn
n.o.
Mean
(L>p.rO
Total
Solids
(PP-T)
Barom-
eter
Mean
(in.)
Experiment No, 1A Candle Studies at 11.5 - 13C.
[
1
2
3
4
5
6
7
2.2
2.2
2.2
2.2
2.2
2.2
2.2
8 !
9 1
10 !
11
12
13
14
2.2
2.2
2.2
8.5
8.5
8.4
8.5
8.3
8.5
8.5
8.6
8.8
8.9
8.8
8.7
8.8
2.2 8.8
1
15 . 2.2 ; 8.8
16 i 2.2 8.7
17
18
19
2.2 1 8.7
2.2 8.9
2.2 8.9
i :
. 1 !
13.8
12.5
13.5
13.0
12.8
12.1
13.0
12.8
13.2
12.3
13.0
12.0
11.9
13.4
12.1
12.0
11.9
11.8
12.9
i i
( • i
Mean
i 8.7
12.6
!
i
ND
ND
ND
i
1
Survival and Detection Measurenu:;. L~s
Tubes
Candle
ABC
ND ND ND
P P P
P P P
P P P
P - P
TP - TP
_ _ _
P - P
_ _ _
— _ _
_ _ _
_ _ _
— _ _.
_
— _ _
_ _ _
M _ _
« _ _
_ _ _
Darkfield
Candle
A B C
++ ++ ++
++ ++ ++
+ ++ +
++ ++ ++
ND ND ND
++ + ++
++ - ++
+ + +
++ - ++
+ + +
+ + +
+ - +
+ + +
+ + +
+ + +
+ + +
+ + +
+ - +
+ - -
T = tube
P = plate
PA
Candle
A B C
ND ND ND
+ + -f
+ + +
+ + +
+ + +
•4- + +
+ - +
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
+ + -f
++ = motility
f
.p-
o
-------
TABLE IVA SUMMARY OF SURVIVAL AND DETECTION OF LEPTOSPIRES IN WELL WATER AND STREAM WATER.
Exp
No.
Days Mean
Conducted Temp
fC)
7A
Selas 5 19.9
Candles
in well
water
8A
Selas 9 10.4
Candle
in stream
water
1.0A 9 ditch
18.6
Be a Kup
19.0
Mean
pH
Mean
D.O.
fppm)
8.6 2.7
7.9. 4.0
ditch ditch
7.9 5.7
Bea Kup Bea Kup
8.3 5.8
Survival and Detection in Days
Tubes Plates Darkfield FA
2 2 5 • 5
2 25 1
A&B A&B A&B A & B
22 84
KEY
A = Well
B = Stream
-------
survival in water.
Additional Observations
1. The laboratory Model A which contained 113 liters of
manure was housed in the departmental laboratory and found
to be nearly odorless when aerated to maintain a D.O. of 5
ppm.
2, During the year, predominantely during summer environ-
mental temperature studies, foaming was found to be a major
problem which occurred especially when the ditch was being
stabilized. During this time foaming also occurred in the
field ditch. After the rotor was changed from a brush to a
paddle type, foaming decreased and was no longer a problem.
3. At summer environmental ditch temperatures, active degrada-
tion of manure occurred in the laboratory model.
4. In February, 1970, four feeder cattle (approximately 1100
pounds average weight) were found dead over the Rosemount
oxidation ditch. Following necropsy examination, a tentative
diagnosis of idiopathic toxicosis was made since prior to the
death the rotor had failed to function for hours and death
followed start-up. Consideration of possible emission of
toxic gases from the manure located in the oxidation ditch was made,
Discussion '•'....•'
The laboratory model of the field oxidation ditch developed
was found to be an adequate environment for leptospiral
survival and detection studies of leptospires under simulated
winter and summer temperature conditions. It should be emphasized
that these conditions simulate, but did not duplicate, the
environmental conditions (pH, D.O., temperature, and total
solids) that existed in the operational field ditch unit. When
the D.O. was maintained at 5 ppm or greater in front of the rotor,
the manure of the model ditch was essentially odorless in the
laboratory. Under winter conditions difficulty was encountered
in maintaining the D.O. at 5 ppm in front of the rotor. The
rotor speed was reduced to minimize the aeration. Daily add-
itions of small amounts of manure caused a lowered pH of ditch
manure.
For measurement of survival the results indicated that the
tube dilution isolation technique was found to be the most
adequate under winter conditions. In addition to problems of
contamination, the modified agar plate technique was found '
unsatisfactory for isolation of leptospires, for at cold
temperatures (1.7 - 6.4C), there may have been a lag in the
leptospires ability to penetrate the filter to establish growth
42
-------
in the agar medium. Using this method, no isolations were ob-
served during winter studies.*
In the leptospiral survival studies under simulated summer
environmental conditions, the beef cattle manure in the labora-
tory model system of the Rosemount field oxidation ditch was
adequately utilized. In the laboratory methods of isolation,
the tube dilution procedure was utilized for culturing and
growth of leptospires from the manure. However, overgrowth
of contaminants was a problem and may have resulted in failure
to measure maximal survival time of the leptospires. The modified
agar plate method was utilized in filtering out contaminants and
isolating leptospires. Findings in Experiment 12LS (Table IIA)
indicate that the changes in procedure resulted in a longer
term and more accurate determination of survival time.
For detection, the fluorescent antibody procedure developed
was adequate for identifying the presence of leptospires in the
manure for a period equal to or greater than the cultural
isolation. Controls were essential due to a 1 irge amount of
extraneous materials and possibility of reading false positives.
Darkfield examination was found adequate for detection and
measuring motility of leptospires. This procedure requires a
skilled observer to differentiate leptospires from artifacts
and other numerous microorganisms. Darkfield is generally
unreliable when the leptospires become non-motile. This examina-
tion requires a trained, skilled observer. Neither of the above
methods definitively measure survival.
The survival time (Table IA) of 18 days in the ditch was the
maximum time measured (Exp. 10LW). A mean pH of 6.9 was observed.
In comparison, survival studies in Selas candles (1LW, 2LW)
with the pH of 9.0 - 9.2, the survival time was measured to be
one day. The leptospires survived for a longer time in the
aerated ditch than in the settling chamber (sludge and effluent).
In the settling chamber the survival time was measured for a
maximum of 11 days in both effluent and sludge. Unfortunately,
these tests were not continued for a longer time period.
In Table IIA measurements indicated that leptospires survived
for at least 6 days in the manure in Selas candles, for 138 days
(82 day definitively) in the manure of the oxidation ditch model ,
and for 5 days in effluent and sludge of a settling chamber in
*In later studies of leptospiral survival in beef cattle
manure at winter temperatures, the plate agar technique,
following additional modifications was very effectively used
for isolation.
43
-------
experiments conducted at summer environmental temperature.
Leptospires survived only 3 days in well and stream water.
Although Leptospira pornona had been isolated for about 18
days in winter studies utilizing the tube dilution procedure,
developments in cultural isolation techniques utilizing the
agar plate technique allowed for isolation of _L. porno na for
approximately 138 days under summer conditions. These isola-
t ions were relatively routine regarding ease of isolation and
the number of organisms present indicates that the leptospires
not only survived but may have also muliplied in the model
oxidation ditch manure environment. Additional research is
needed to definitively quantitate and document leptospiral
multiplication•
These findings indicate a greater survival time of leptospires
in aerated liquid manure of the model oxidation ditch than in
effluents or sludge, indicating that aeration provides a more
suitable environment for survival. Further studies are needed.
Little research has been conducted on the survival of patho-
genic leptospires in animal manures. One report indicated
that L. icterohaemorrhagiae could not survive in human feces
for morethan 24 hours and that polluted water, sewage, and
soil will not keep icterohaemorrhagiae alive for more than
3 days (41).
Reports indicate that pH less than 5.0 or greater than 8.5
is detrimental to leptospires. The optimum appears to be
7.2 - 7.4 (42).
Other researchers have indicated that leptospires survive in
the environment for varying lengths of time. Chang, Buckingham
and Taylor (32) found that leptospires survived in river water
8-9 days at 5 - 6C, 5 -6 days at 25 - 27C, and 3-4 days
at 31 - 32C. Indications were that lower temperatures were
more conducive to survival. It was also indicated that presence
of other microorganisms probably was detrimental to leptospires.
In domestic sewage,survival of leptospires was only 12 - 14
hours but rose to 2 - 3 days when aerated. Survival was 7-8
days when sewage was diluted with tap water to 1% of its strength,
Survival was measured for 6-7 days in 10% sewage in tap
water at 5 - 6C (pH 7.1 - 7.2).
Ryu and Liu (33), in laboratory viability studies with
leptospires, attempted to define a survival pattern in rice-
planted paddy water, found a decreasing survival time at high
temperatures (40 - 43C, viable 3-6 hours)as compared to
low temperatures (0 - 30C, viable 7-14 days).
In environmental studies Okazaki and Ringen (43) reported
44
-------
that temperatures below 7 to IOC or above 34 to 36C, and pH
below 6.0 or above 8.4 were detrimental to survival of sero-
type pornona. Seroytpe pomona survived for less than seven
days under simulated natural conditions, and longer in stag-
nant pools, than in moving water. Addition of small amounts
of fresh water to stagnant pools increased the organisms
survival time. In soil studies leptospires survived for 30
.minutes in air-dried soil, 3-5 days in damp soil and 183
days in soil super-saturated with water.
In studies of four leptospiral strains in Malaya, Smith and
Turner (44) reported that leptospires survived longer in alkaline
than in acid buffered distilled water, and significant differences
between the four serotypes were found in resistance to pH.
Survival at pH under 7.0 ranged from 10-117 days and at pH
over 7.0 from 21-152 days.
The aerated beef cattle manure, effluents, and sludge appeared
suitable for survival of leptospires. Infected cattle may shed
leptospires for several months in the urine. Up to 100,
000,000 leptospires per ml. of urine have been reported (31).
The finding of viable leptospires in the aerated ditch for more
than 138 days in the summer and for 18 days in the winter is
significant. A critical need exists to define whether or not
the surviving leptospires have maintained virulence to infect
animal hosts. The oxidation ditch containing beef cattle manure
constitutes an adequate environment for the survival of
pathogenic leptospires of significantly long duration. Such
contaminated manure hauled to the land, or effluent may be
discharged into natural waters, thus constituting a potential
health hazard.
45
-------
SURVIVAL AND DETECTION OF SALMONELLA IN BEEF CATTLE MANURE.
Introduction
Epidemiologic surveillance indicates that we have not con-
trolled animal-associated salmonella. In the United States
there are an estimated 2 million human cases annually,. (45)
There is increasing concernfcr the salmonellosis problem in
both man and animals. In 1970 there were 24,216 human cases
of salmonellosis (excluding typhoid fever) reported in the
United States with 335 in Minnesota. Salmonella organisms
have been found in surface waters contaminated by animal
manure. In 1966, a large waterborne outbreak at Riverside,
California resulted from contamination of the water supply
by j>almonella typhimurium.
This serotype is widely found in domestic animals. There is
speculation that the water may have been contaminated by
seepage from distant cattle feedlots (14). From June, 1964-
October, 1965, an investigation of water pollution along
the Upper Mississippi River and its major tributaries was
conducted in Minnesota and Wisconsin. Pathogenic bacteria
and viruses were isolated from stream and waste samples.
Fourteen species of Salmonella were found in sewage effluent
at a sewage treatment plant. Several species of Salmonella
were found in the river at locations six and ten miles
downstream (46). Salmonellae have been isolated in influents
and a considerably lesser amount in unchlorinated effluents
of the Municipal Sewerage Plant at Glenwood, Minnesota, which
utilizes the oxidation channel method. Chlorinated effluents
were not examined for Salmonella (47).
The single-host species of typhoid and paratyphoid organisms
are being encountered less frequently as the etiologic
agent of human suffering. However, the ubiquitous, multi-
host salmonellae are being isolated from warm and cold-
blooded animals, and as a contaminant from food, feeds, and
numerous other materials. The infections they cause have
been reported more often, especially in persons very young,
very old, or debilitated. Wherever ecologic research and
investigation probe into our environment, salmonellae are
found.
Although 1.300 distinct serotypes of unadapted salmonellae
exist, 96% of the cultural isolates from animals and man
belong to only 55 serotypes. Salmonella typhimurium is the
salmonella most common to infect man and domestic animals.
Although 95% of Americans live and associate essentially in
urban areas, man and animals do share a common environment
Preceding page blank
47
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for work and recreation. There is a tremendous interaction
between host, pathogenic agent, and environment. It is
difficult to comprehend this interplay because many factors
in the disease transmission remain unknown.
How will salmonellosis be controlled? Generally speaking,
immunization or prevention of exposure are the two methods used
to protect against infection. And yet, vaccination against
salmonellosis appears inefficient and ineffective (48, 49,
50, 51) and drug resistance studies indicate that salmonellae
cannot be eliminated with chemotherapeutic agents (45 , 48) .
For the most part, even the minimum infective dose is not
known as there are so many types and strains of salmonella.
Therefore, the main thrust in efforts to control salmonellosis
must be to decrease the exposure potential within our society.
But, as populations grow, animals, as well as people, both
of which carry salmonella, are often found living in crowded
conditions.
Economic pressure has forced producers to cloister live-
stock in large numbers into more and more congested housing
which often does not satisfy basic sanitation requirements.
Cittle may harbor and excrete salmonellae in feces, and
subsequently pulverise this waste which becomes airborne as
dust laden with the microorganisms and spread to other areas.
Or, the salmonella-laden manure accumulates until such times
when it is washed by land run-off into streams, rivers,
lakes, and on through the water ways to pollute harbors and
estuaries. Wild animals, fish, mollusks, and other lower
animal forms living in and near this environment may become
infected (52, 53). Furthermore, this is the common environ-
ment in which we grow food and seek recreation.
Cattle afflicted with salmonellosis may shed 10 million
microorganisms per gram of feces (54).
The principle of the oxidation ditch is to maintain manure
as suspended solids of biological value in water of sufficient
oxygen content to maintain an aerobic condition allowing for
microbial degradation. This is accomplished within an end-
to-end channel through which the manure is propelled by a
rotor which serves to aerate the material. (Figure 1). The
aerobic process is nearly odorless. These two factors thus
facilitate handling and reuse.
After a period of time, the waste from an oxidation ditch
field unit can be disposed of in a number of ways. It may
be mechanically pumped from the ditch into a tank truck and
then spread onto land. Or, the effluent may.be discharged
into an irrigation channel or stream. Studies are being
conducted to ascertain the feasibility of recycling cattle
48
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and poultry manure wastes as feed for domestic animals (55,
56) and at a profit, too.
i
British investigators recently reported survival of Salmonella
typhimurium, ^. dublin, Staphylococcus aureus, Escherichia
coli, and Brucella abortus in cattle manure slurries for
12 weeks (57).
Methods
In order to evaluate the potential health effects of salmonella
in cattle manure, research was conducted in a laboratory
model oxidation ditch and effluent holding chambers simulating
field environmental conditions at summer and winter temperatures,
The objectives of the research herein reported were: to
measure salmonella survival time, to develop and improve
bacteriologic methods of measurement of detection and survival
of pathogens in beef cattle manure.
All field samples of manure were examined for Salmonellae
prior to adding to the model. During experiments, DO, pH,
and temperature data were monitored at 6-hour intervals.
The 2C manure temperatures were adequately maintained by
the cooling system as originally conceived. This cooling
system consisted of condenser, fan, cooling coil, and
channel trough, antifreeze, holding vat, and thermostat.
However, on or about April 27, 1970, it was discovered that
the refrigerant in the open channel coolant trough was leak-
ing into the model ditch manure, thus killing the microorgan-
isms. The trough was then replaced with refrigerator coils/
tubes which made the cooling/coolant system a closed system.
This redesign prevented further known leakage and did not
affect the salmonella experiments.
The 20C (summer) manure temperatures were difficult to main-
tain. When the ambient air\temperature was near 20C the ditch
manure would acclimate with it. The warm-to-hot Minnesota
summer temperatures which frequently range as high as 32C
and above outdoors, cause equally as high or higher indoor
temperatures in the lab, thus precluding maintenance of the
20C ambient temperature without the aid of a room air con-
ditioner for the lab. It had been hoped that the cooling
system on the model A unit would be able to maintain 20C,
but its control range would allow only 17-18 degrees as a
controlable maximum temperature. The problem encountered
was that the air conditioning unit, though of adequate
capacity for the room-size, did not function well enough.
Occasionally it would overload the lab circuit and shut off.
Periods of high relative humidity tended to cause the air
conditioning unit to ice up, especially if the daytime
temperatures were extremely hot and the nightime temperatures
IOC cooler. As a result, the summer oxidation ditch A
manure temperatures were more fluctuating than the winter
temperatures, even though on average both winter and summer
49
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manure temperatures were close to 2C and 20C as specified.
Styrofoam insulation placed about the outside periphery
of the 2C ditch did aid to maintain proper temperatures
during June, July, and August. Ice would build-up on the
refrigerating coils of the model oxidation ditch.
Through prolonged continuous use, the bushing of the paddle
wheel assembly, which was constructed of ordinary steel
would wear to a point whereby the functional capacity of ,
the paddle was not adequate. The bushings were replaced
with lubricant-impregnated brass which provided a longer
wearing potential than the original bushings.
The Effluent Chambers/Flask (Experiment 27B)
Studies of Salmonella typhimurium survival in effluent
was considered necessary in light of two facts. First,
the Pasveer oxidation channel can be operated on a batch
load-unload basis or as a continuous skim-off system
whereby effluent is continuously being discharged from the
ditch. Second, consideration of the material itself,
which is a suspension of solids in liquid, indicated a bi-
phasic system whereby both phases, effluent and sludge,
required testing.
Effluent chambers were designed to contain 1 liter of
aerated ditch waste were utilized in experiments 27B, 29B
and 29C, and experiment SOB. The waste was allowed to settle
and the effluent (top) and sludge (bottom) were sampled to
detect Salmonella typhimurium. Monitoring of the pH, D.O.,
and temperature was also achieved. In experiment 29B and 29C
the chambers were placed in the channel of the oxidation
ditch to acclimatise with it. The chambers of experiments
27B and SOB were separate from the ditch. The sampling
procedure was the same as for sampling the model oxidation
ditch A. Experiment 27B was in a flask.
Contamination Studies Involving the Effluent Chambers
Contamination Testing.
During experiment #29 wherein the effluent chambers of
#29B and #29C were in the stream of the model A oxidation
channel, it became apparent to us that contamination of
ditch manure or effluent chamber material may have occured.
Two probable sources of cross-contamination between ditch
and chambers were: aerosol transmission, and/or the contamin-
ated instrument probes for the D.O., temperature, and pH
determinations even though they were rinsed during the
interval between samplings. Procedures were established to
determine the probability of such cross-contamination.
Washings from the probes were cultured for salmonella, and
50
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TABLE 1. CALCULATED NUMBER OF
SALMONELLA TYPHIMURIUM SEEDED
INTO MANURE.
Experiment No. Day No . S a Imo ne 1 1 a
26 A 0 33 x 106
1 33 x 106
2 33 x 10J?
3 33 x 10b
4 33 x 10b
27 A 0 33 x lOJj
1 33 x 10b
2 33 x 10°
3 33 x 10°;
4 33 x 10
27 B 0 3.0 x 10'
28 A 0 33 x
1 33 x
2 33 x 10
3 33 x 10?.
4 33 x 10°
29 A 0 33 x lof
1 33 x 10°
2 33 x 10b
3 33 x 10°
4 33 x 10b7
29 B 0 3.0 x 10'
1 3.0 x 10
29 C 0 1/113 of 2 day
seeded ditch
manure
30 A 0 33 x 106
1 33 x 10C
2 33 x
3 33 x 10
4 33 x 10b
30 B 0 1/113 of 5 day
seeded ditch
manure
51
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S-S (Salmonella-Shigella) and BGS (Brilliant Green Sulfadiazine)
plates were exposed for 24 hours at three locations in the
model ditch, that is, on each of the two effluent chambers,
and atop the rotor housing.
All results from the washings and the exposed plates were
negative. It was decided, however, to isolate the effluent
chambers from the model A oxidation ditch in further studies
by separating them. Thus, during experiment #30, which was
conducted at 2C, the effluent chambers were kept stable at
2C in refrigerators, and not monitored for pH, D,O. and
temperature once seeding of either ditch or chambers had
occured.
During salmonella experiments #26-30 additional manure was
introduced into the oxidation ditch on two occassions. The
physical data recorded from the model oxidation ditch in
experiments #26, #27, and #28 indicated little or no
change in the parameters after the addition of the manure to
the original slurry. Therefore, it was decided to not add
extraneous manure after the original charging load in subse-
quent experiments to eliminate as much as possible any
undetermined error. Experiment #29 was conducted for at least
15 days, and experiment #30, for 28 days longer than previous
experiments.
The three parameters, D.O., pH, temperature, were consistantly ..
comparable one to the other. Thus, it was felt that the
addition of manure increments after the initial charge did
not contribute considerably to the physical make-up of the
waste, and neither added nor subtracted from the quality of
the micro-biological climate of the material. Although
salmonella organisms were detected in the manure for
periods longer than those in the manure experiment #29 and
#30, the longer survival times were considered to be the
2C degree winter ditch temperature, as well as the improved
cultural detection. The samples were not only processed in
a manner different from original cultures, but also more
samples per sampling site were analyzed routinely, that is,
more data were recorded.
Maintenance of Conditions
Model Oxidation Ditch
The oxidation ditch model containing salmonella-free well
water and manure from Rosemount field unit was stabilized
with regard to pH, temperature, dissolved oxygen, total
solids, and mechanical operation initially before being
seeded with Salmonella typhimurium stock culture. On
two occasions, Salmonella was detected in the manure and
52
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these lots were discarded. Throughout each experiment,
efforts were made to maintain the pH between 6.5 and 8.0
the total solids between 5,000 and 10,000 mgs/liter,
the dissolved oxygen at 5 ppm., and the temperature near
20C for summer conditions, and 2C for winter conditions.
These conditions were achieved by regulation of temperature
alone for the most part.
Effluent Holding Chambers/Flasks
The monitoring of pH, D.O., and temperature in the chambers,
incorporated the same probes used in monitoring the
oxidation ditch.
Temperature was the only parameter maintained in the effluent
holding chambers/flasks. Accurate monitoring and recording
of pH and dissolved oxygen was accomplished in experiments
#29B and #29C, and nearly identical conditions are assumed
for experiment #30B.
Seeding of Salmonella
Stock cultures of biochemically and serologically pure
Salmonella typhimurium grown on tryptic soy agar were inoculated
into biphasic veal infusion broth agar (1-2.5%) for cultiva-
tion. These cultures were again screened for J3. typhimurium
and utilized for direct seeding into either the oxidation
ditch or the effluent holding chambers of experiments
#27B (Flask) and #29B.
Calculations on the basis of reference (54) indicated that
5-6 billion organisms daily were required for seeding the
model ditch to simulate the Rosemount operational oxidation
ditch at maximum j5. typhimurium shedding from the 36 beef
cattle there. However, such a high estimate of salmonella
load was based on the assumption that the herd consisted of
"scouring" calves with severe,acute diarrhea. In practice
fewer Salmonella typhimurium would be expected. The model
oxidation ditch A was inoculated daily with approximately
33 million Salmonella typhimurium organisms (Table 1). The
number of J3. typhimurium in veal infusion broth-agar was
estimated by counting on a Petroff-Hauser grid the number of
organisms per one milliliter of 30 nephlos Salmonella in
VIB on a nephlometer. The inoculum
was then diluted with 100 mis of sterile veal infusion broth
for mixing with the oxidation ditch manure. During each
experiment the laboratory oxidation ditch was seeded on the
five initial consecutive days with the calculated number of
Salmonella typhimurium.
The effluent holding chambers were inoculated either by
53
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PETROFF-HAUSER COUNTING CHAMBER
FOR DETERMINATION OF BACTERIA *
A MODIFICATION.
The Salmonella typhimurium were motile and viable
when used with this method, that is, not stained.
Salmonella
1. The number of bacteria in 20 squares of the chamber were
counted, and a mean calculated.
2. The squares on the grid of the counting chamber aire
l/20mm by l/20mm and the chamber is l/50mm deep.
3. Therefore, by multiplying the mean of 20 squares by
20 million (20x20x50x1000) the number of bacteria were
calculated.
Salmonella: Veal Infusion Broth culture suspensions of
30 nephelos were utilized.
*Simmons and Gientzkow: Laboratory Methods of U.S. Army,
1944, p. 404.
54
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directly seeding 3.0 x 10 J5. typhimurium into the chamber
containing 1,000 ml of dilute manure,(experiments 27B and
29B) or indirectly by placing 1,000 ml of seeded oxidation
ditch manure into the effluent chambers (experiments 29C
and SOB).
Sampling
Six sampling sites were selected at positions about the
periphery of the model oxidation ditch A as indicated in
Figure 7 , below. Sites 1,2,3 were
areas 1 inch below the surface of the manure, whereas,
sites 4,5.6 were located at the same positions as 1,2,3
but between the floor of the model to 1 inch above the
floor. The Moore (58) technique of sampling was standard
protocol wherein two sterile cotton swabs were taped into
position at each site and remained in place until replaced
in 24-72 hours with fresh sterile cotton swabs. Personel
handling contaminated material, swabs, and the like wore
plastic gloves.
Modifications of the Moore procedure were tried also. In
experiment #26A sterile cotton-tipped swabs were dipped
momentarily into the ditch manure, then placed in enrichment
media. In experiment #28 1 ml samples were taken for inocu-
lation into enrichment media. Sampling was done daily for
1-2 weeks, then every three days for the remainder of each
experiment. All samples were placed in enrichment broth
prior to selective media for microbial isolation.
55
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Methods of Measuring Salmonella Survival
Cultural Procedure
The general flow of the cultural schema was: sample to selec-
tive enrichment for 24 hours at 37.5C followed in turn by
selective plating for isolation and differentiation on TSI
and biochemistry. The biochemical results were verified by
serological testing (Figure 6).
Original cultural methods included enrichment in tetrathionate-
brilliant green medium and brilliant green (BG)-sulfadizaine
medium with an incubation period of 6 to 48 hours followed
by plating on Eosin-Methylene Blue (EMB), MacConkey, and
Salmonella-Shigella (SS) agars. Subsequent work also utilized
GN (Hajna) broth, Brilliant Green-Bile broth and Selenite-
BG-sulfadiazine agar plates for isolation (see Table 2).
Differentiation was accomplished biochemically and
serologically. The biochemistry utilised triple sugar iron
(TSI), urea, decarboxylase base, lysine decarboxylase broth,
salicin, dulcitol, and KCN broth base. H-broth and motilit.y
test media were utilized when necessary to enhance proper
functioning of the serological procedure. Salmonella A
and poly-B somatic antisera, and flagellar Hj_ and H-^ complex
antisera were utilized for serotyping the isolates obtained
through enrichment and biochemistry. All were commercial
products.*
Serologic Procedures
TSI cultures were utilized for the serological confirmation
of isolates. Difco commercial preparations were utilised
in this procedure. Somatic poly A and B antisera, and later,
only the B antisera, were used in the slide agglutination test
to identify the isolation as to Ohnehauch heat stable antigen,
Group B. Most isolates obtained through TSI and the bio-
chemical reactions were positive at this stage with only a
few rough forms being encountered. Flagella H antigen
analysis was accomplished by the tube test of Edwards and
Bruner (59) incorporating H complex (phase 2) and H.
(phase 1) flagellar antisera. -1
After completion of each experiment, 5 TSI cultures of
salmonella isolates which had been serologically determined
in our lab as being S. typhimurium were randomly selected
and carried to Mrs. L. York in the Department of Veterinary
*Difco, Detroit, Michigan.
56
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TABLE 2. SALMONELLA EXPERIMENTS: BROTH AND PLATE MEDIA INCORPORATED
BROTHS: Tetrathionate Brilliant Green Selenite Brilliant Gram
Brilliant Green Sulfadiazine Brilliant Green Green- Negative
Sulfadiazine Bile (Haj na)
Experiment
Number
26 X
27
28
29
30
PLATES: MacConkeys
Experiment
Number
26 X
27
28
29
30
X
X X X
X X
X X
X X X
EMB Brilliant Green Salmonella
Sulfadiazine Shigella
X X X
X X
X X
X X
X X
-------
Microbiology typing lab for analysis. Mrs. York tested these
isolates by the Spicer-Edwards procedure. All cultures
examined were identified as _S. typhimurium by Mrs. York.
This use of allied laboratory facilities allowed not only
for a check on our results but also upon the validity of
our procedure.
Fluorescent Antibody Studies
The concept of being able to distinguish one genera of
microorganisms from among the many others in waste, or for
that matter in any environment has led the interested
scientist to attempt many different approaches to attain
prompt recognition of the microorganism. Among the methods
available, the fluorescent antibody technique (F.A.T.)
seemed to be quite promising.
The theory of fluorescent antibody action indicated a
selective, sensitive method by which Salmonella typhimurium
could be "spotted" in the midst of the myriad other microor-
ganisms present in manure waste. It was for this reason that
the FAT was considered for use in our project.
However, despite attempting various modifications of the
methods of Groeffert and Hicks (60) and the National Animal
Disease Lab. (61) we feel that F.A.T. as applied in our lab
was not as satisfactory as the cultural methods utilised in
this study. Recently a procedure (Harrington, 61) has come
to our attention. This procedure , if employed, could not
only improve the feasibility of F.A. for our work, but also
make the cultural aspects more rapid as well. It was not
applied as we have not dealt with salmonella since learning
of the technique.
Difficulties with fluorescent antibody technique:
A. Non-fluorescence or repression of fluorescence.
B. Lack of readily available conjugate which we could
use with confidence.
C. No one good procedure whose modifications worked
well.
The direct FA technique incorporating commercially** avail-
able rabbit antiserum to Salmonella O (antigens 4,5,12) and
Salmonella H. and H^ complex were utilized. Positive and
**Sylvana, Millburn, New Jersey.
58
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negative controls were tested. Conjugates were undiluted
and diluted 1:4 and 1:9. These were prepared in the same
manner as were the leptospiral conjugates.
Chlorination Studies
It was found from the survival studies involving both
Salmonella typhimurium and Leptospira jpomonj* that these
microorganisms doindeed live on in the extended aeration
processed livestock waste (manure) for a period of time.
Although the salmonella experience a decimal reduction
rate, which was not determined, and do become undetectable
in time, the leptospires were continually detected for as
long as 4 months once isolation procedures were derived,
indicating multiplication rather than mere survival. Further
quantitative studies are needed to confirm this belief.
The implication, of course, is that, regardless of whether
death occurs after a period of time or multiplication
succeeds, any leakage of non-disinfected manure from the
treatment/holding facilities where pathogens are present
is a potential health hazard, either immediate or remote,
in terms of disease transmission from a reservoir (port :
of exit).
Therefore, chlorination of pathogen contaminated wastes
was attempted on a laboratory scale using wastes from the
model A oxidation ditch. Titration of residual chlorine was
used to indicate the amount of chlorine involved in the
treatment of the waste, and cultural isolation was the
criteria used to gauge the effectiveness of chlorination
as a disinfecting method for manure wastes.
In actual fact, the slurry from the Pasveer oxidation ditch
is a suspension of solids in a water vehicle and the solids
portion is allowed to settle out, thus separating "sludge"
waste from "effluent" waste. The two are generally handled
as a single medium for spreading onto land, but more and
more the two are being allowed to separate in holding ponds
or the like, with the effluent portion being discharged
into surface waters and only the sludge being hauled to
the land as fertilizer. This surely facilitates handling
because of the decreased bulk and time consumption. Patho-
gens exist in both portions of the waste and both must be
considered potential hazards. However, often it is only
the effluent which is chlorinated.
In chlorination studies, only effluent was chlorinated
after it had separated from the suspended solids. Even
so, the titration of residual chlorine could only be
accomplished amperometrically as other methods depended upon
59
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color change which could not be visualized/detected in the
turbid, green effluent. The residual chlorine determina-
tions were performed with some difficulty. A Wallace and
Tiernan Amperometric Titrator model #WIA790-l-2 was used
in such titrations. The Spinning electrode agitates the
sample while measuring current flow. The effluent being
rich in organic matter would foam and spill out over the
sample container making the quantitation of residual chlorine
inaccurate.
Furthermore, after a period of time the electrode portion of
the titrator cell would deteriorate to the point of leaking
electrolyte solution and allowing the ingression of effluent
into the cell chamber.
The procedure developed is listed on the following page.
More work is to be conducted on the chlorination of patho-
gen contaminated manure. Thus far we can report that a
contact time of approximately 60 minutes will be necessary
to destroy pathogens in the oxidation ditch effluent.
Although chlorination of effluents may be accomplished, it
doesn't appear that this method is practical due to the
high organic load and probable NH_ content.
o
Results
Salmonella typhimurium survived for 17 days post-seeding in
the model oxidation ditch at summer temperatures (20C) and
for 47 days under winter conditions (2C) . Further, studies
in model settling chambers holding 1,000 ml of model oxidation
ditch effluent indicate that ^. typhimurium can be detected
for even greater periods of time than at similar temperatures
in the oxidation ditch. The longest survival time was 87
days, experiment 5b, effluent holding chamber sludge (Table 3),
The data indicates that survival is of greatest duration in
the sludge portion of the settling chambers. Information
derived from the investigation of a stream gave similar
results in that salmonella recovery rates for stream bottom
sediments were-greater than those from surface water. (62)
All isolates were biochemically and serologically identi-
fied as Salmonella typhimurium.
The ambient environmental conditions monitored were main-
tained within the preselected norms as determined from the
field ditch (Table 4).
Based upon results obtained, the three sampling methods:
60
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Chlorination Procedure*
1. Beef cattle manure having been processed in an oxidation
ditch (extended aeration process) is allowed to separate
by stasis (gravity) into sludge and effluent portions.
2. Calculations indicated that to study chlorination of the
model A oxidation ditch effluent, 8.8 x 10 Salmonella
typhimurium are required to proportionately simulate-the
concentration of pathogens (calculations of total pathogen
content of the model A oxidation ditch, see appendix, p. 32),
Salmonella is added to 195 ml of effluent which is contained
in the vessel of an amperometric titrator.**
3. Ca (OCl2)2 at 47 mS Per liter is added to this vessel.
4. Samples are incubated at room temperature for varying
lengths of time (15, 30, 45, and 60 minutes).
5. The agitator is started.
6. 5 ml of phenylarsene oxide is added to the chlorinated
effluent.
7. 4 ml of pH 4.0 buffer solution is then also added to the
material.
8. 1 ml of potassium iodide solution is added.
9. A 0.0282N iodine solution is added from a burette to
titrate the residual chlorine. The end-point is then
detected on the micro-Ammeter of the titrator which is
registered as a deflection of the needle of the meter.
* from: Wallace and Tiernan, Book #WIA 790-1-2, "Determin-
ation of residual chlorine in waste-water."
** Wallace and Tiernan, model series A-790012.
61
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TABLE 3
SURVIVAL AND DETECTION OF SALMONELLA TYPHIMURIUM IN
A MODEL OXIDATION DITCH OR MODEL EFFLUENT HOLDING CHAMBERS
Experiment No.
Bacteriological Isolation
F.A.
Ditch;
26a
27a
28a
29a
30a
Chamber;
27b
29b
29c
30b
Days
14
14
17
40
47
14
15-effluent
18-sludge
21-effluent
52-sludge
66-effluent
87-sludge
Days
10
4
48
10
10
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CO
TABLE 4
ENVIRONMENTAL CONDITIONS IN A MODEL OXIDATION DITCH AND
MODEL EFFLUENT HOLDING CHAMBERS
Exp. Days
No. Conducted
Ditch:
26a
27a
28a
29a
30a
Chamber:
27b
29 b
29c
30b
38 ;
28
38
53
66
18
46
54
103
Total Solids (mg/L)
x ; range
6799; 6154-7256
5747; 5317-6927
7575; 4183-9058
5509; 3069-7258
6363; 6100-7258
(1>
(1)
(1)
(1)
PH
x ; range
6.7;6.1-8.5
7. 4; 6. 8-8. 2
7.6;6.4-8.2
8.5;8.1-8.7
7.8;6.9-8.4
7.4; NA
T 8.0;7.6-8.3
B 7. 4; 7. 6-8. 3
T 8.0; 7. 6-8. 3
B 7. 7; 7. 3-8. 4
T 8.3*
7.8;
B 8.2*
Temp (C) D.O. (ppm)
x ; range x ; range
19. 4;16. 0-21.0
20.2;17.0-24.0
20.6; 4.0-22.5
1.3; 0.0-3.0
2.1; 1.0-4.0
20.2; NA
2.9;2.0-5.5
2.6;1.5-6.5
2.9;2.0-7.5
2.6;0.0-7.0
2.0*
2.0;
2.5*
3. 8; 0.4-7. 4
1.8; 0.0-3. 6
3. 8;0. 0-10.6
12. 7; 9. 2-22. .0
14. 0;5. 0-20.0
ND
4.3; 0.1-9.2
3.4; 0.1-8.7
3.8; 0.7-11.0
0.7; 0.1-4.5
3.8*
13.0;
1.2*
T = top ; B = bottom
NA ; not available
ND ; not done
(1); waste material separates into effluent and sludge portions which were not monitored
for T.S.
* = not monitored continuously
-------
temporary swab, prolonged swab, and increment removal,
were comparable in isolation efficiency.
The greatest success for measuring survival thus far has
been achieved utilizing BG-Bile and Selenite-BG-sulfadiazine
as the enrichment phase, and SS and Selenite-BG-sulfadizaine
the plating phase for isolation. (Table 5)
Original fluorescent antibody efforts were not as successful
as anticipated. Attempts to retrieve positive flourescing
J>. typhimurium from the enrichment phase media were found
more successful than sampling directly from the oxidation
ditch manure.
During the progression of experiment 2, wherein the hold-
ing chambers were maintained in the stream of the oxidation
channel, it was considered that cross-contamination from
chamber-to-ditch or, conversely, from ditch-to-chamber may
occur either by aerosol transmission, or from the set of
monitor electrodes as only one set of instrumentation was
available for monitoring both experimental subunits.
Therefore, it was elected to not measure D.O., T.S., pH and
temperature in experiment 28b (settling chamber) and to house
the chamber within a refrigerator with the temperature held
constant at 2C. The behavior of the parameters could be
implied from the extensive data obtained in experiments
29b and 29c, and experiment 30b would be a control for these
previous two studies. Experiment 30a (ditch) terminated
prior to 30b (chamber) and one reading was made of the
settling chamber manure. From the data it can be determined
that chamber SOB had behaved as the chamber of experiments
29b and 29c.
SaImonella typhimurium was not detected after disinfection
of the contaminated effluent with chlorine at a residual of
1.0 to 5.0 ppm. and a contact time of at least 60 minutes.
Discussion
In an attempt to simulate field conditions, salmonella re-
search was conducted in a laboratory utilizing a model of
an oxidation channel. The procedures are laboratory protocol
and attempts were made to simulate, but never exactly dupli-
cate, field conditions. This fact was exemplified in that
manure was added to the model at intermittant intervals.
During field conditions continous urination and defecation
of beef cattle occured in the housing unit through slatted
floors located immediately over the ditch. In field practice
the extended aeration process of treatment is altered by
the operator to meet individual needs. Most producers
appreciate the nearly odorless operation and discard the
64
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TABLE 5. DETECTION OF SALMONELLA
TYPHIMURIUM
PLATES
No. Days
Experiment No. BGS SS
29A 40 28
29B 19 19
29C 52 48
30A 51 32
30B 91 80
Note on broths: in 10 of 12 columns of data
for the above experiments, the Brilliant-
Green Bile broth was the media of initial
enrichment
65
-------
manure in batches as the ditch holding capacity is reached,
regardless of loading rate. Thus, dissolved oxygen, B.O.D.,
pH,and total suspended solids may fluctuate drastically
between emptying times. Others operate their channels for
longer periods of time by allowing a lower loading rate by
housing fewer cattle over the ditch and continually siphon
off the effluent. Some units are improperly designed or
misused. Because of these and the many other situations
involving animal waste treatment, there continues to be
excessive pollution of the environment with little control
of contained and subsequently disposed zoonotic pathogens,
including Salmonella.
As the livestock production industry becomes more familiar
with this new method of trfflting and disposing of solid
waste, and better guidelines develop to aid the operator,
problems facing the producer today will be resolved. Systems
will be adopted which will make the operation of an oxida-
tion ditch less difficult. However, the aerobic process
mil remain and the waste will continue to contain pathogens
which will survive the manure treatment, perhaps even grow
and multiply within it.
Within the conduct of these experiments on the survival of
Salmonella typbimurium the MPN (Most Probable Number) was
not calculated. Nevertheless, since multiple tube methods
of detection were used, the data validly indicated that
^>. typhimurium did not multiply in the manure of the model
ditch, but rather declined in number with time. This state-
ment is made on the basis of two criteria:
1. The proportionate number of samples positive deminished
with time, and
2. They became more difficult to isolate with time, i.e.,
longer incubation times were required, and fewer colonies
were found per plate. Although the most probable number
was not done at any time, we are quite certain that the
salmonella did not multiply, but rather followed a decimal
reduction time which was shorter at summer temperatures
(20C) than at winter temperatures (2C).
This finding is in accord with Rankin, et. al. (57) who did
MPN and obtained similar results from animal waste slurry
studies.
The current studies with Salmonella typhimurium utilized as
a model organism revealed that this enteric pathogen survived
in oxidation ditch conditions for 47 days, and in the field,
could feasibly contaminate the environment once released
66
-------
into it. Thus, the interaction within the world of health
would be altered and the effects far-ranging. The spread of
drug-resistant pathogens and the ensueing transfer of this
resistance which has developed from the treatment of live-
stock or the use of medicated feed (63, 64) are good examples
of the type of present day and future problems with which
we must cope. If this situation cannot be confined to the
environment of the housing unit itself, the public health
effects are sure to be felt throughout the entire livestock
industry and its related environment. The world of health
is an all-encompassing one in which we find man and his
domestic animals co-existing with other living beings.
Appropriate health practice must not be jeoprodized by
contamination of our environment.
Perhaps one approach to controlling the salmonellae problem
in animal manures is to chlorinate, or otherwise treat and
disinfect the waste emanating from confinement housing units.
However, with present methods, disinfection is not practical.
Preliminary information from experiments conducted in the
laboratory indicates that chlorination of oxidation ditch
effluent destroys J5. typhimurium. Due to the high organic
content and the cost, disinfection of sludge, or manure
itself, to eliminate pathogens would be extremely difficult.
If chlorination or disinfection of feedlot, poultry lot,
and other animal lot wastes were possible, it would be a
meaningful measure of disease prevention and control.
Additional research is required before guidelines can be
established. Livestock do produce enormous amounts of
manure waste, and the trend toward confinement housing and
concentration of livestock makes this industry a tremendously
important potential polluter if new methods and systems are
not developed for handling and treatment of wastes. The
increasing size of livestock production units in many
instances creates public health problems of equal or
greater magnitude than do municipal sewage plants which do
not always destroy salmonellae (65,66).
67
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ENGINEERING STUDIES OF OXIDATION DITCH OPERATION
One of the major problems with oxidation ditch operation
is the inability to keep all solids in suspension. Mass
accumulation of solids in any portion of the ditch pro-
mo.tes anaerobic conditions because of insufficient oxygen
within the body mass. As stated earlier, the principal
objective of the Model B studies was to determine and evaluate
those factors affecting solid settlement patterns within the
ditch. When it is impossible to eliminate the settlement of
solids it may be desirable (or necessary) to remove them
from the ditch for disposal by other means.
Solid Settlement Studies: Initial tests with the Model B
oxidation ditch (Figures 6 and 7, see Introduction) were run
using beef manure from the Rosemount field oxidation ditch as
the solids material. It was found, however, that use of such
wastes created extremely murky conditions making it difficult
to observe and photograph the movement and settlement of
the solids. A preliminary testing program was also made for
proper selection of rotor design (Figure 2C).
For the above reasons a search of various organic and in-
organic materials was made in an attempt to find a more
suitable material to serve as the solids medium. Following
extensive testing, small plastic spheres were found to be most
suitable. Such spheres are commercially available in various
diameters and specific gravities. The spheres used were
chemically inert, of two diameters (1/8" and 3/32"), and of
four specific gravities (0.91, 1.14, 1.17, and 1.30). Each
type of sphere was color-coded, according to its particular
size and specific gravity, thus permitting ready identification.
Specific gravities were chosen so as to represent a range
known to exist in typical beef animal manure wastes.
Unexpected problems of water quality were also encountered.
Data obtained from certain early runs proved invalid because
of a gradual incrustative condition which developed along
the bottom and walls of the channel. Such incrustation, which
occurred as a result of using tap water containing some 10
grains per gallon of hardness forming minerals, resulted in
unpredictable roughness conditions within the channel. De-
mineralized water was then used in an effort to overcome the
incrustation problem arid was found to be satisfactory. Because
of the relatively large amounts of air entrained in the deminer-
alized water, however, small bubbles formed on the bottom of
the channel and on a large percentage of the plastic spheres,
causing them to move in a retarded and unatural fashion. The
latter difficulty was overcome by allowing the water to stand
overnight, thus de-aerating itself before a test was made.
Preceding page blank
69
-------
I
Fig.2C Modified Rotor as Used in Model B
Oxidation Ditch
NOT REPRODUCIBLE
70
-------
Each solid-settlement test was run for a period of 15 min-
utes. Before the commencement of any given test the plastic '
spheres were dry-weighed and distributed evenly on the bed of
the model ditch. The rotor, which had previously been positioned
at one of three locations (A, B or E, as shown on page 72)
was started. The rotor speed for all tests reported was 150
revolutions per minute. During the test period (15 minutes)
the solids were forced to move within the ditch in a fashion
as dictated by the particular test conditions, as enumerated
in a fashion as dictated by the particular test conditions,
as enumerated in Table 1 . Following each test period the rotor
was stopped and the percentage of solids found in the storage
sump and beneath the rotor was determined.
Velocity Distribution Studies: Fluid flow in open channels
is seldom uniform in nature. This nonuniformity is especially
evident where curvilinear conditions are imposed, as at the
ends of the typical oxidation ditch. Under such conditions,
velocities vary widely both horizontally across the channel
width as well as with water depth. The ability of a stream to
transport solids depends to a large extent upon its velocity.
Regions of a channel where low velocities exist, therefore,
have less ability to suspend and carry solids than do regions
of higher velocity. Depending upon the size and density of
the solid, a critical velocity exists below which settlement
of the solid will occur. Since in most oxidation ditch opera-
tions it is important to keep the finer particles in suspension
at all times, the velocity must always equal or exceed the
critical velocity for such solids.
This part of the study was aimed at the problem of velocity
distribution in an oxidation ditch, and to determine those
regions within the ditch having velocities sufficiently low to
cause solid settlement. To measure low velocities, as they
occur in such a hydraulic model, it became necessary to
construct and calibrate a suitable velocity measuring insturment
or device. After thorough investigation it was decided that
a "tethered sphere" meter* was most suitable. Figures 3B
and 4B show the basic construction of such a meter, which i
consists principally of a wax ( or plastic) sphere suspended
by a fine fiber (in this case a single strand.of dental floss).
When placed in a stream, the sphere is deflected from a vertical
position in'response to the magnitude of the velocity. In
addition to its ability to measure low velocities, such a meter
is capable of measuring point velocities in areas having
*0riginal research on tethered sphere meters made by Dr. Henize
Stefan and Frank R. Schiebe, and reported in Technical Paper No.
32, Series A, of the St. Anthony Falls Hydraulic Laboratory,
University of Minnesota, Minneapolis, Minnesota.
71
-------
Figure 7 i<
Rotor Locations and Flow Directions Used in Solid
Settlement Tests.
Table 1. Solid-Settlement Tests
Rotor
Location
B
B
A
A
E
E
A
A
B
B
E
E
A
A
B
B
E
E
Water
Depth
2"
2"
2"
2"
2"
2"
1 I/?."
1 1/2"
1 1/2"
1 1/2"
1 1/2"
1 1/2"
1"
1"
1"
1"
1"
1"
% Solids
in
Sump*
29.3
19.5
36.1
18.9
31.5
39.9
35. A
14.2
45.4
24.0
52.6
56.2
35.6
37.9
69.8
49.8
87.1
72.5
% Solids
Under
Rotor*
51.5
38.6
38.8
45.1
___ —
37.0
39.2
49.5
45.1
j
48.9
39.2
22.1
21.2
__ __.
Rotor
Vane
in
out
in
out
in
out
in
out
in
out
in
out
in
out
in
out
in
out
* Values shcvn represent average value of two or more runs for each
condition.
72
-------
Figure 3B. Tethered-ball meter used to measure low
velocities in Model B oxidation ditch.
NOT REPRODUCIBLE
4R
Fibure Velocity meter as used to measure point
velocities in oxidation ditch (side view),
73
-------
severe space limitations.
Results
On the basis of the observations made in this study it is
very difficult to prevent solids settlement and accumulation
immediately beneath the rotor. As indicated earlier, such
accumulations are undesirable since anaerobic conditions are
likely to develop with subsequent odor problems. An inspection
of the data shown in Table 1, together with Figures 5B and 6B
shows that regardless of water depth in the ditch, solids did
accumulate beneath the rotor. As to be expected, these data
indicate that maximum separation of solids from the liquid
occurs when the rotor is placed in a position directly above
the collection sump. For water depths of 1", with rotor
positioned above the sump, (Figure 7B) shows from 72 to 87
percent of the solids were collected in the sump.
In an effort to reduce the accumulation of solids beneath
the rotor, a vane, shaped in the form of a Venturi throat-
section, was designed and installed in the rotor vicinity of
the ditch. Data shown in Figures 8B through 10B indicate that
such a vane had an insignificant effect on the removal of solids
from beneath the rotor. Whether vanes of these configurations
might have a significant effect upon the accumulation of solids
in other areas of the ditch was not determined.
The use of the throat-shaped vane in the vicinity of the
rotor had significant effect upon percentage of solids collected
in the sump, when the rotor was located at the A and B positions,
(see Figures 11B and 12B). Again, however, the presence of
the vane showed no effect with the rotor at the E position, as
shown in Figure 10B.
Tables 2B through 19B (Appendix B) show velocity values as
measured at 24 locations in the ditch. All velocities were
measured at the one-inch depth. As noted, velocity distribution
measurements were taken under varying conditions of water
depth, rotor immersion depth, and rotor location. These
velocity data were then plotted on scaled outlines of the
ditch, Figures 13B through SOB (Appendix B) from which the
velocity at any given point were determined. Shaded areas
indicate those points or regions where reverse velocities
occurred.
Inspection of the tabulated data, and of Figures 13B through
SOB (Appendix B) shows that reverse velocity conditions
occurred in many runs. The presence or absence of reversed
flow conditions in a given area is of critical importance
since surrounding such areas velocities must be at or near
zero. Such low velocity conditions cause deposition of solids,
74
-------
>. -*.
^§
Fipire 5B. Typical solids accumulation beneath rotor
of oxidation ditch.
NOT REPRODUCIBLE
Figure 6B. Solids accumulation in low velocity areas in
oxidation ditch (overhead view).
75
-------
80
ROTOR at (A)
Svoi
CO
q 30
„!
0 50
co
-------
ROTOR at (E)
WATER
DEPTH
Figure 10B. Effect of Water Depth on Solid Settlement in Sump,
with Rotor at Position E.
77
-------
ROTOR ~ at (A)
60
(.0
z: 40,
IX-
30
q
"f ?Q
a '~^
^o 10
G *
0
Figure 11B-
%
WITH
WITHOUT L
0
2"
WATER
DEPTH
Effect of Water Depth on Solid Settlement in Sump,
with Rotor at Position A.
ROTOR at (B)
WTHOUT VANES
2"
WATER
DEPTH
Figure 12B- Hffect of Water Depth on Solid Settlement in Sump
with Rotor at Position B.
-------
which as stated previously, may create partial clogging of the
ditch, anaerobic decomposition and other undesirable problems.
Observations of the shape and extent of the settled solids in
low velocity regions provides some insight as to the corrections
necessary for modifying the contour confirmation of the stream-
bed.
79
-------
ACKNOWLEDGEMENTS
The project staff is indebted to many individuals for assis-
tance, cooperation and information during the research period.
Among those who have been particularly helpful are:
Dr. W.T.S. Thorp, Dean, College of Veterinary Medicine,
University of Minnesota
Dr. H.O. Halvorson, Professor, Department of Biochemistry,
College of Biological Sciences, University of Minnesota
Dr. H.C. Ellinghausen, Jr., National Animal Disease Laboratory,
USDA, Ames, Iowa
Dr. R.C. Johnson, Associate Professor of Microbiology, Health
Sciences Center, University of Minnesota
Dr. P.R. Goodrich, Assistant Professor, Department of Agricultural
Engineering, College of Agriculture, University of Minnesota
J.A. Moore, Instructor, Department of Agricultural Engineering,
College of Agriculture, University of Minnesota
R.E. Larson, Associate Professor (USDA), Department of
Agricultural Engineering, College of Agriculture, University
of Minnesota
Dr. L.L. Boyd, Professor and Head, Department of Agricultural
Engineering, College of Agriculture, University of
Minnesota
R.O. Hegg, Instructor (USDA) Department of Agricultural
Engineering, College of Agriculture, University of Minnesota
We are especially greateful to Dr. Mirdza Peterson, Project
Officer, to Louis W. Lefke and Daniel J. Keller of the
Division of Solids Wastes, Environmental Protection Agency,
Cincinnati, Ohio, for their excellent support of this re-
search project.
Preceding page blank
81
-------
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62. Hendricks, C.W., Increased Recovery Rate of Salmonellae
from Stream Bottom Sediments Versus Surface Waters,
App. Microb., 21, No. 2, Feb., 1971, 379-380.
63. Loken, K.I., Wagner, W. and Henke, C., Transmissible
Drug Resistance in Enterobacteriaceae Isolated from
Calves Given Antibiotics, Am. J. Vet. Res., 32, No. 8,
Aug., 1971, 1207-1212.
64. Ewing, W.H., Excerpts from "An Evaluation of the
Salmonella Problem," USDHEW, PHS, HS and MHA, National
Communicable Disease Center, Atlanta, Georgia, June 30,
1969.
65. Kampelmacher, E.H. and van Nodrle Jansen, Lucretia M.,
Salmonella—Its Presence in and Removal from a Waste-
water System, J.W.P.C.F., December, 1970.
66. Kabler, P., Removal of Pathogenic Microorganisms by
Sewage Treatment Processes, Sewage and Solid Wastes
Journal, 31, 1959, 1373.
88
-------
PROJECT PUBLICATIONS
1. Diesch, S.L.: Disease Transmission of Water Borne
Organisms of Animal Origin, in Agricultural Practices
and Water Quality. ed by Willrich and Smith. The
Iowa State University Press. Ames, Iowa. 1970, 265-
285, (English)
2. Diesch, S.L.: Transmission De Enfermedades Por Organis-
mos Hidrico's De Origen Animal. Boletin De La Oficina
Sanitaria Pan americana LXIX No. 4 /Oct.) 1970, 314-
330 (Spanish)
3. Diesch, S.L., Pomeroy, B.S. and Allred, E.R., Survival
and Detection of Leptospires in Aerated Beef Cattle
Manure in Livestock Waste Management and Pollution
Abatement. The Proceedings of the International Live-
stock Symposium on Livestock Wastes, Pub. by Am. Soc.
of Agric. Eng., St. Joseph, Michigan. 1971, 263-266.
4. Diesch, S.L., Survival of Leptospires in Cattle Manure.
Scientific Proceedings of the 108th Annual Meeting of
the AVMA. 159. Dec. 1, 1971, 1513-1517.
5. Will, L.A., Diesch, S.L. and Pomeroy, B.S., Survival
of Salmonella Typhimurium in Animal Manure Disposal in
a Model Oxidation Ditch. Presented-section of Veterinary
Public Health, A.P.H.A., Minneapolis, Minn., Oct. 12,
1971. In Press A.P.M.A. Journal, 1972.
6. Diesch, S.L. : Microbiologic Contaminants of Livestock
Wastes As A Public Health Problem. Proc. of the 1971
United States Animal Health Association Mtg., 1971,
321-324.
89
-------
LEPTOSPIROSIS
APPENDIX-A
Preceding page blank
91
-------
110
100 !
Q
DC
c/
u:
u-1
o
pi
c/j
O
UJ
CL,
u
90 ;
80
70 :
60
50 ,
40 :
30
10 j
..o
0 20 40 60 80 100 120 140 160 180 200 220 240 2bO 280 300
NUMBER OF LEPTOSPIRES PER MTLL1LITER x 1 MILLION
-------
Fluorescent Antibody Methods Used in Leptospire Detection
A Zeiss FA microscope was used in the fluorescent antibody
procedure, equipped with light source HBO Osram 200 W/4
(high intensity micro-scope illuminator), darkfield condensor,
heat absorbing filter KG I, and exciter filters BG 38, and
BG 12. Leptospira pomona fluorescein conjugate (Sylvan Co.)
was diluted, 1 part with 9 parts PBS (phosphate buffered
saline) and incubated with 0.1 gm bovine liver powder for 30
min. in 37C water bath with inversion every 5-10 min. The
conjugate was then centrifuged at 3,000 rpm for 20 min. and the
supernate drawn off and stored at -5C in 0.5 ml quantities.
The following controls were used during FA studies.
Slide 1. Known antigen L. pomona incubated with known
L. pomona conjligate~
Slide 2. Known antigen L. pomona incubated with specific
antiserum, rinsed briefly with PBS, and incubated
with specific conjugate.
Slide 3. Known antigen L. pomona incubated with anti-
brucella conjugate.
Slide 4. Sterile PBS and incubated with specific conjugate,
The following FA procedure was used to detect leptospires in
manure.
Slides were pre-soaked in a 6% Tween 80. One drop of feces
was spread over a 15mm^ area and allowed to air dry, or dry
with circulating heated air. The material was then fixed in
acetone 5 minutes and dryed at 37C 5 minutes, followed by
rinses in 3% Tween 80 in PBS 10 minutes and PBS 3 minutes.
Excess moisture was removed and conjugate applied for 30
minutes at 37C in a moist chamber. The slides were rinsed
in PBS and distilled water and dried. Cover slips were then
mounted with elvanol or Difco mounting fluid and observed.
93
-------
Agar Plate Technique for Isolating Leptospires
In order to substantiate that leptospires survive in animal
manures the organisms must be isolated culturally. The
abundance and broad spectrum of microorganisms in animal
manure appeared to be an overwhelming demand for this re-
search project entitled the Survival of Pathogens in Animal
Manure Disposal. In 1957 Cox and Larson (38) described the
first successful growth of leptospirae as isolated colonies,
and many workers have continued to improve techniques or de-
vise new methods in a quest for rapid, efficient, and pure
isolation of the organisms.
The retrieval of serotype pomona from seeded beef cattle
manure was essential to measure survival and to fulfill one
objective of this research project.
Smibert (39) isolated leptospires by taking advantage of the
selective penetration through Millipore filters, and Rittenberg,
et. a 1. (40) illustrated the feasibility of Swinny filtering
cultTTre broth to isolate leptospires.
After much experimentation with previously described tech-
niques, including the tube dilution schema, and many attempts
to culturally isolate leptospirae which could be observed with
darkfield and Fluorescent Antibody microscropy, a method was
developed which achieved the results hoped for.
Other bacterial growth was selectively restricted or inhibited
pharmacologically with use of 5-fluorouracil (37). A dilution
procedure was also used to advantage to isolate leptospirae
from contaminated cultures.
By taking advantage of these previously mentioned techniques,
we were able to develop a procedure with which we are able
to isolate leptospires in pure culture from animal manure
held at 20C.
MateriaIs
1. Rabbit serum agar plates with 0.22 Millipore filters
arranged as according to Smibert.
2. Liquid media: Bovine Serum Albumin according to Johnson (36).
3. Semi-solid: Bovine Serum Albumin-Agar according to Johnson(36)
4. Millipore Swinny hypodermic adapter fitted with millipore
HA 0.45 m filter.
94
-------
Method
1. Pipette approximately 5 ml seeded manure sample.
2. Place 3-5 drops of the material onto the center portion of
the Millipore-curtain ring arrangement on the rabbit serum
agar plates.
3. Incubate at 29C for 4 to 14 days (7 preferred) with the
filters and ring in place. This prevents surface over-
growth by fungi which are present in the manure and the
environment. It is important to maintain an ambient
humidity of 80-90% to prevent drying of the plates.
4. After the 4-14 day period of incubation the filter and
ring are removed.
5. A plug of plate media suspected of harboring leptospiral
growth is crushed between a coverslip and glass slide and
examined for leptospires by darkfield microscopy.
6. If leptospires are observed a plug of the agar media is
removed with a loop and placed within the barrel of a
3 cc disposable syringe and the plunger replaced.
7. Approximately 1 cc of liquid medium may be aspirated
into this syringe.
8. The material was forced through a 23 gauge needle into
the barrel of a 5 cc glass syringe fitted with a Millipore
Swinny hypodermic adapter containing a 0.45 filter.
9. The glass plunger was replaced and liquid medium was
aspirated through the Swinny to make approximately 3-5
cc of material in the glass syringe.
10. This material was forced through the Swinny filter into
a tube containing liquid medium.
11. A side-by-side series of 6 liquid and 6 semi-solid media
was arranged in a rack and serial 10 fold dilutions and
cultures made.
12. Incubate at 29C and examine according to media. Generally,
we would darkfield examine the liquid cultures at 3-7
days and the semi-solid cultures at 5-14 days.
-------
Laboratory Procedure Used to Determine Total Solids (TS)
Three (or more) porcelain evaporating dishes (50 to 100
ml. capacity) were placed for drying in a 103'- 105'C
oven for at least 20 minutes. A measured quantity or sample
of the liquid waste was poured into each evaporating dish
and total weight determined. The evaporating dishes (with
waste liquid) were placed on a steam table, where they re-
mained until all visible water was evaporated. The dish
and remaining solids residue were then transferred to 103'
105'C oven for overnight drying period. Dishes with dry
residue then removed from oven, cooled in desiccator, and
weighted.
Calculation:
mg residue X 1,000
mg/1 residue on evaporation = ml sample
Laboratory Procedure Used to Determine Total Volatile Solids (TVS)
Dry residue samples obtained in (TS) procedure above ignited
in electric furnace at 600'C to constant weight, usually re-
quiring one hour. Loss on ignition reported as mg/1 total
volatile solids and the residue as mg/1 fixed solids. Dishes
allowed to cool briefly in air then cooled in desiccator.
Although most TVS values exceeded 1,000 mg/1, calculations
were reported to four significant figures.
mg/1 = mg (TS) residue - mg fixed solids
ml sample X 1
96
-------
SURVIVAL AND DETECTION OF LE?TOSPIRES IN ANIMAL MANURE DISPOSAL
<£>
Day
Exp
1
2
3
M
5
6
7
8
9
lean
Exp
1
2
3
M
5
6
7
8
9
10
lean
Manure
Added
(Ib)
eriment N
eriment N
Ma nLir e Environment
PH
Mean.
o. 1 LW
9.2
9.2
9.2
9.3
9.3
9.3
9.3
9.2
9.2
9.2
o. 2 LW
9.2
9.2
9.1
8.9
8.9
8.9
8.9
8.9
8.9
8.9
9.0
Ditch
Temp.
Mean
f.ci
Candle
3.5
M.O
3.5
3.0
3.0
3.0
2.5
2.0
1.5
2.0
Candle
2.0
1.0
1.0
1.7
2.0
2.0
2.0
1.0
3.5
M.O
1.7
n.o.
Mean
(ppm)
Studies <
10.1
13.6
1M.2
1H. 2
13.9
13.5
13.5
13.6
13.8
13. H
studies £
13.2
13.0
13.2
13.5
13.7
13.7
13.3
12.3
12.0
12.0
13. M
Total
Solids
(PPm}.
at Winter
12,239
s,mn
Jt Winter
7,960
7,731
Barom-
eter
Mean
(in.)
Temperati
30.30
30.33
30.27
ND
ND
ND
ND
30.26
30.25
30.29
Tempera ti
29.81
30. 1M
29.96
30.08
ND
ND
30.42
29.75
30.23
30.06
30.06
Survival and Detection Measurements
Tubes
Candle
ABC
ires
ND ND ND
TP - T
_
_ _ _
_
_
_
_
_
ires
ND ND ND
T - T
_
_
_
_
_
_
_
_ • _
Darkf ield
Candle
ABC
ND ND ND
++ + +
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
++ ++ ++
+ + +
++ + +
++ + +
. + '+ +
+ + +
+ + +
+ + +
+ + +
+ + +
PA
Candle
A B C
ND-ND ND
+ + +
+ + +
+ - +
+ ? ?
+ ? ?
+ ? +
+ + +
+ + +
ND ND ND
+ + +
+ + +
ND ND ND
ND ND ND
ND ND ND
ND ND ND
ND ND ND
ND ND ND
+ + +
-------
SURVIVAL AND DETECTION OF LEPTOSPIRES IN ANIMAL MANURE DISPOSAL
Day
Exp
1
2
3
4
5
6
7
8
9
lean
Manure
Added
(Ib)
criment t*
Experiment t>
1
2
3
4
5
6
7
8
9
10
ean
Manure Environment
oil
Mean
o. 3 Lfo
9.0
9.0
9.0
9.0
9.0
9.0
9.0
9.0
9.0
9.0
o. M LW
8.4
8.4
8.5
8.5
8.5
8.5
8.M
8.5
8.3
8.3
8.4
Ditch
Temp .
Mean
fC)
Candle
2.0
2.0
2.5
2.0
2.7
2.0
2.0
2.0
3.0
2.3
Candle
2.0 .
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.5
2.0
2.0
n.o.
Mean
(ppm)
Total
Solids
(PPtf)
studies at Winter
12.5
12. M
12.9
13.0
12.7
12.5
12.0
13.0
13. M
12.7
Studies ;
5.9
8.4
8.1
7.3
7.2
7.0
7.8
7.9
7.0
6.8
7.3
6,748
6,596
6,268
3t Winter
9,511
9,913
9,643
Barom-
eter
Mean
(in.)
Temperati
30.22
30.20
30.06
29.96
30.05
29.87
30.00
30.17
30.12
30.07
Survival cine! Detection Measurements
Tubes
Candle
ABC
ires
ND ND ND
_
_ _ _
_
_
_
_
_
-
Temperatures
30.14
29.98
30.08
29.99
29.06
29.31
29.32
29.94
29.94
29.47
29.72
ND ND ND
_
_
- - -
_
_
_
_
- - T
_
Darkfield
Candle
ABC
ND ND ND
++ ++ ++
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
ND ND ND
++ ++ ++
++ ++ +
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
+ + 4-
PA
Candle
ABC
ND ND ND
+ + +
ND ND ND
ND ND ND
ND ND ND
ND ND ND
ND ND ND
ND ND ND
+ + +
ND ND ND
+ + +
ND ND ND
ND ND- ND
ND ND ND
ND ND ND
ND ND ND
. ND ND ND
ND ND ND
4- + +
00
-------
SURVIVAL AND DETECTION OF LEPTOSPIRES IN ANIMAL MANURE DISPOSAL
<£>
Day
Exp
1
2
3
4
5
6
7
8
•lean
Exp
1
2
3
4
5
6
7
8
9
10
11
12
lean
Manure
Added
(Ib)
eriment Is
2.2
2.2
2.2
2.2
2.2
2.2
eriment ts
2.2
2.2
.. 2.2
2.2
2.2
2.2
2.2
Manure Fnvironinen t
PH
Mean
o. 5 LV
8.2
8.3
8.5
8.2
8.2
8.5
8.5
8.4
8.4
o. 6 LV\
8.4
8.3
8.2
8.1
8.2
8.3
8.2
8.2
8.2
8.2
8.1
8.2
8.2
Ditch
Temp .
Mean
ccn
Cnndle
2.3
3.0
2.0
2.0
3.0
3.0
3.1
3.3
2.7
Candle
3.0
14. 0
2.8
2.8
2.3
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.14
P.O.
Mean
(pprn)
Studies
6.7
6.7
6.9
7.3
6.0
5.9
6.0
5.9
6.4
Studies
5.4
5.7
5.8
5.6
5.4
5.6
5.3
5.4
5.1
5.3
5.14
5.4
5.43
Total
Solids
. (PPr-i;
at Winter
9,480
8,596
8,425
at Winter
8,126
8,M3M
9,045
Barom-
eter
Mean ' ..
(in.)
Temperati
29.67
29.48
29.07
29.58
29.71
29.37
29.60
29.75
29.53
Temperat
29.95
29.83
29.84
29:92
29.93
29.87
ND
29.80
29.45
29.48
29.85
29.84
Survival and Detection Measurements
Tubes
Candle
ABC
ares
ND ND ND
T - T
- - T
- T
- - T
- - T
- - T
- - T
ares
ND ND ND
- - T
T - T
T - T
_
- - T
_
- - T
- - T
- - T
- - T
- - T
Darkfield
Candle
A B C
ND ND ND
+ + +
+ + +
++ + +
+ + +
+ + +
+ + +
+ + +
ND ND ND
++ ++ ++
+ + +
++ + - +
++ + +
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
PA
Candle
A B C
ND ND ND
+ + +
ND ND ND
ND ND ND
ND ND ND
ND ND ND
ND ND ND
+ + +
ND ND ND
+ + +
+ + +
ND ND ND
• ND ND ND
ND ND ND
ND ND ND
ND ND ND
ND ND ND
ND ND ND
ND ND ND
+ + +
-------
SURVIVAL AND DETECTION OF LEPTOSPIRES IN ANIMAL MANURE DISPOSAL
Day
Exp
1
2
3
i)
5
6
7
8
9
10
"lean
Exp
1
2
3
M
5
6
7
8
•lean
Manure
Added
(Ib)
eriment Is
Manure Environment
Pi!
Mean
.
!o. 7 LV
8.8
i 8.7
eriment Is
8.7
8.7
8.7
8.7
8.7
8.5
8.6
8.7
o. 8 LV\
8.0
3.5
8.5
8.M
8.3
8.M
8.2
8.2
8.3
Ditch
Temp.
Mean
fCI
Effluer
M.3
5.0
3.3
3.3
3.5
2.8
3.2
2.8
2.8
3.M
1 Effluen
3.0
3.5
2.5
2.0
2.6
3.5
3.0
3.0
2.9
D.O.
Mean
(ppm)
t and SI
M.2
3.5
3.5
2.6
3.6
3.5
3.6
3.5
3.5
3.5
t and SI
7.3
2.7
2.5
2.6
5.5
5.6
5.M
5.3
M.6
Total
Solids
(PPm)
'
udge Stud
udge Stud
Barom-
eter
Mean
(in.):
ies at Wi
ies at Wi
Survival and Detection Measurements
Tubes
Candle
ABC
iter Tempera-
ND ND ND
_
_
_
_
_
_
T - -
T - T
T T -
nter Tempera1
ND ND ND
_
- T -
- T -
- - T
T T. -
T - -
T T T
Darkf ield
Candle
ABC
rures
ND ND ND
+ + +
+ + +
— 4* —
_
+ + -
+ + +
+ + -
+
+ - +
tures
ND ND ND
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
PA
Candle
A B C
ND ND ND
+ + +
ND ND ND
ND ND ND
ND ND ND
ND ND ND
ND ND ND
ND ND ND
ND ND ND
+ + +
ND ND ND
+ + +
ND ND ND
ND ND ND
ND ND ND
ND ND ND
ND ND ND
.+ + +
o
o
-------
SURVIVAL AND DETECTION OF LEPTOSPIRES IN ANIMAL MANURE DISPOSAL
Day
Exp
1
2
3
M
5
6
7
8
9
10
11
12
•lean
Manure
'Added
(Ib)
•eriment N
MG r.ure Environment
Pi!
Mean
o. 9 LV
8.3
8.2
8.2
8.1
8.M
8.M
8.M
8.3
8.2
8.0
7.9
ND
8.2
Ditch
Temp .
Mean
cn
Effluen
3.3
3.0
2.8
2.9
3.0
2.M
2.6
2.6
2.5
2.5
2.5
ND
2.7
n.o.
Mean
(pprn)
t and SI
7.1
6.8
6.1
6.5
7.8
6.5
7.0
7.0
6.9
6.8
6.5
ND
6.8
Total
Solids
(pprn)
jdge Stud
Barom-
eter
Mean
(in.)
ies at Wi
Survival and Detection Measurements
Tubes
Candle
ABC
nter Temperal
+ + +
+ +
+ +
+ + +
+ — —
+ — —
- + +
+ + -
+ + +
+ + +
+ + +
Darkfield
Candle
ABC
rures
4- + +
+ + +
+ + +
+ + +
+ + +
+ + +
+ '+ +
+ + +
+ + -
+ + +
+ + +
PA
Candle
A B C
+ + +
_
ND ND ND
ND ND ND
ND ND ND
ND ND ND
ND ND ND
ND ND ND
ND ND ND
ND ND ND
+ + +
-------
SURVIVAL AND DETECTION OF IJiPTOSPIRES IN ANIMAL MANURE DISPOSAL
Day
Manure
Added
(Ib)
Experiment Is
1
2
3
4
5
6
7
8
2.2
2.2
2.2
9 i 2.2
10 j
11 t
12
13
1M
15
16
17
18
4.3
4.4
19 j
20
21
22
23
24
25
26
27
Mean
Manure Environment
pi!
Mean
Ditch •
Tc'ir.o. '
"Mean
• rev
D.O.
Me: an
(ppm)
Total
Solids
(ppr-0
Barom-
eter
Mean
(in.)
'o. 10 LW Studies of Seeded Ditch 'at Winter
7.6
7.5
7.3
7.1
6.9
6.9
7.0
7.2
6.8
6.7
6.7
6.7
6.7
6.7
6.7
6.6
6.8
6.8
7.0
6.9
6.8
6.7
6.6
6.6
6.6
6.6
6.6
6.9
2.0
2.0
3.0
3.3
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.3
M.O
M.O
M.O
3.0
3.0
3.0
M.O
M.O
3.0
3.0
3.3
3.1
5.7
5.5
5.9
5.9
6.6
6.8
6.1
8.M
8.7
8.8
8.9
8.7
8.6
8.6
8.2
8.14
9.1
9.1
7.8
6.7
6.9
7.M
7.9
8.2
8.3
8.6
9.2
9,621
9,653
9,625
10,700
10 ,'542
10,752
7.0 '
Survival and Detection Measurerr.cn ts
Tubes -
X Y
1 Temperature
29.76 ND ND
29. 6M
29.80
29.81
29.69
29.68
29.93
29.97
30.07
29.92
29.82
30.03
30.19
30.08
30.13
30.05
29.74
29.75
29.62
29.60
29.60
29.72
29.67
29.94
29.95
29.70
29.86
29. 8M
+ +
+
+
+ +
+ +
+ +
+ +
+
+ +
_ _
-
+
-
-
+
-
- -
-
ND ND
ND ND
+
+ +
- -
_
ND ND
— —
Darkfield
X Y
s
ND ND
+ ++
+ ++
+ +
+
++ +
++ +
++
++
-
+
_
+
'
+
+
++
+
+
-
_ _
+
+
+
+ +
+ +
_ _
FA
X Y
ND ND
-
_ _
ND ND
ND ND
-
ND ND
ND ND
ND ND
-
_ _
+
ND ND
+
ND ND
ND ND
ND ND
ND ND
ND ND
+
+ +
+
+ +
+ +
+ +
- -
_ _
++=motility
-------
SURVIVAL AND DETECTION OF LEPTOSPIRES IN ANIMAL MANURE DISPOSAL
o
CO
Day
Manure
Added
(Ib)
Experiment
f
1
2
3
M
5
6
7
8
9
10
11
12
13
m
Mean
Ex
1
2
3
M
5
6
7
Mean
2.2
2.2
2.2
2.2
2.2
2.2
2.2
2.2
2.2
2.2
2.2
2.2
2.2
2.2
Jeriment
2.2
•
Manure Environment
pH
Mean
Ditch
Temp .
Mean
ra
n.o.
Mean
(ppm)
.
Total
Solids
(ppm)
Barom-
eter
Mean
(in.)
Survival and Detection Measurements
Tubes
Candle
A B . C
flo. 1 LS Candle. Studies , at Summer Temperatures
8.0
8.2
8.3
8.5
8.5
8.5
8.5
8.5
8.M
8.3
8.3
8.H
8.5
8.3
8.36
to. 2 L
8.5
8.5
8.M
8.5
8.M
8.5
8.3.
8. MM
25.5
25.8
25.3
2M.1
2M.6
2M.O
22.8
23.2
23.5
2M.3
2M.5
2M.M
23.7
2M.7
2M.29
3 Candle
19.6
20.2
18.7
18.7
19.0
19.0
20.0
19.31
ND
Studies
0.5
0.6
0.5
O.M
ND
3.M
1.1
1.08
6,230
at Summer
20,906
14,MM6
12,709
6,268
ND
Temperat
ND
29.32
ND
29.99
ND
ND
ND
29.66
_
_ _ _
_
_
_
_
- - -
_
_ _ _
_
_ _ _
_
_
_
Tares
ND ND ND
_
_
_
_
_
_ _ _
Darkfield
Candle
ABC
++ ++ ++
++ ++ ++
++ + ++
+ - +
+ - +
+ - +
+ - +
+ - +
+ - +
+ - +
+ - +
+ - +
+ - +
+ - +
++ +4- ++
++ + +
+ + +
+ + +
+ + +
+
_ _ _
PA
Candle
A B C
ND ND ND
ND ND ND
ND ND ND
ND ND ND
ND ND ND
ND ND ND
ND ND ND
ND ND ND
ND ND ND
ND ND ND
ND ND ND
ND ND ND
ND ND ND
ND ND ND
+ + +
+ + +
+ + +
+ + +
ND ND ND
ND ND ND
ND ND ND
-------
SURVIVAL AND DETECTION OF LEPTOSPIEES IN ANIMAL MANURE DISPOSAL
Day
Ex
1
2
3
L|
r
6
7
8
9
10
11
12
13
14
ean
1 Mnnure Environme it
Manure ' FDitdi
n.o.
, ' pit | Temp. Mean
r, '. ' ! Mean i Moan
(" ) i f'p-,
i i
Total
Solids
(ppin) i fpp.r)
l-iarom-
Survival and Delrection Measurements
Tubes
ett-ir Candle
Mean
! -(in.)
ABC
1
i i
periment No. 3 LS Candle Studies at Summer Temperatures
I
2.2
2.2
2.2
2.2
2.2
2.2
2.2
8.4
8.4
8.5
8.7
8.8
8.9
8.7
8.6
2.2 8.6
2.2 | 8.6
2.2
2.2
2.2
2.2
8.5
8.6
8.6
18.8
18.6
18.8
19.0
19.2
19.0
18.0
18.5
19.6
19.6
20.4
21.1
19.6
8.6 18.9
8.61
i
i
i
i
i
19.22
i
4.4 1 5,009
2.8
2.4
3.0
4,749
2.0
3.0
2.5
3.3
3.7
3.9
0.4
ND
ND
ND
2.84
5,106
6,092
29.95
29.10
30.19
29.70
ND
ND
30.20
29.50
29.57.
ND
ND
ND
ND
ND
29.77
i
TP*TP*TP*
XP*TP*TP*
+ + +
+
+ - -
_ _ _
_
ND ND ND
ND ND ND
ND ND ND
ND ND ND
ND ND ND
ND ND ND
ND ND ND
*Measuremen
3 hours du
day.
++ = Motili
T = Tube
P = Plate
Dark fie Id
Candle
ABC
++ ++ ++
++ ++ ++
++ ++ ++
++ ++ ++
++ ++ +
ND ND ND
+ + +
+ + +
+ + +
++ + +
+ + +
+ + +
+ +
t of survival
ring first an
ty
PA
Candle
ABC
+ + +
+ + +
ND ND ND
ND ND ND
ND ND ND
ND ND ND
ND ND ND
ND ND ND
ND ND ND
ND ND ND
ND ND ND
ND ND ND
ND ND ND
+ - +
every
I second
-------
SURVIVAL AND DETECTION OF LEPTOSPIRES IN ANIMAL MANURE DISPOSAL
o
01
Day
1 Manux^ Environment
Manure
A - - A PH
Aciaed
t ~\ i~~\
(lb>
Mean .
Ditch
Temp .
Mean
fCI
D.O.
Mean.
(ppm)
•
i
Total
Solids
(PPrc)
Barom-
eter
Mean
(in.)
Survival and Detection Measurements
Tubns
Candle
ABC
Experiment No. 5 LS Candle Studies at Summer Temperatures
1 2.2
2
3
"
5
6
7
8
9
10
11
12
13
14
Mean
7.0
7.1
7.0
6.9
6.9
2.2 7.0
2.2 1 7.0
2.2
2.2
2.2
2.2
2.2
Experiment
1 i 2.2
2
3
4
5
6
7
2.2
2.2
2.2
8 I
9
6.9
6.9
6.9
6.9
7.1
7.1
7.0
7.0
^o. 8A
8.2
7.9
8.0
8.4
7.9
7.8
7.8
7.7
7.7
19.4
18.6
19.0
18.9
19.0
19.0
18.5
18.9
18.3
18.3
19.0
19.0
18.8
18.8
18.8
4.3
3.0
5.7
6.4
8,183
6.5
7.7
6.M
3.3
4.4
2.1
1.7
4.0
6.4
7.1
4.9
Candle Studies a
20.0
19.3
18.7
19.7
19.3
18.9
19.6
19.8
19.5
4.9
4.0
6.6
5.9
1.8
3.7
3.M
2.0
3.9
8,263
9,336
t Summer
5,761
6,012
6,053
30.14
30. Ml
30.02
ND
ND
29.80
29.62
30.19
29.75 .
29.84
ND
ND
29.91
29.39
ND ND ND
TP TP TP
P T -
T T T
P - -
TP T T
TP T T
_
_ _ _
_
_
_
_
29.91 |
Temperatures
30.10
29.91
ND
30.22
30.22
30.18
30.18
ND
ND
ND ND ND
TP TP TP
P - -
TP - -
_
_
_
- -. -
\ -
Darkfield
Candle
A B C •
++ ++ ++
++ ++ ++
++ ++ ++
++ + +
++ + +
++ ++ +
++ ++ +
+ + +
+ + +
++ + +
+ + +
+ + +
+ + +
+ + +
++ ++ ++
++ ++ ++
++ + +
++ + +
++ + +
++ + +
+ + -
+ +
+ + -
PA
Candle
ABC
ND ND ND
+ + +
ND ND ND
ND ND ND
ND ND ND
ND ND ND
ND ND ND
ND ND ND
ND ND ND
ND ND ND
ND ND ND
+ + +
+ + +
+ + -
+ + +
+ + +
ND ND ND
ND ND ND
ND ND ND
ND ND ND
ND ND ND
ND ND ND
+ + -
Mean 7.88 . 19.37 3.96 30.14
-------
SURVIVAL AND DETECTION OF IJiPTOSPIRES IN ANIMAL MANURE DISPOSAL
Day
Exj;
v,
i\annre
Addod-
(Ib)
t
pH
Mean
>eriment No. 6 [,J
j
1
2
ND
i
3
'1
5
6
7
8 i
9
Experiment No. 7 L£
1
2
3
_
M !
5
Mean j
9.1
9.0
8.7
8.5
8.3
8.71
Experiment No. 8 LS
l
1
2
3
M
5
6
Mean
8.5
9.2
9.2
9.0
8.9
8.6
9.0
Ditch
Tcnip.
Me np.
rci
D.O.
Mean
(ppm)
•
Totr:l j Barom-
Solids
(ppr.O
otor
Mean
(in.)
; 1 '
Effluent and Sludge Studies at Sun
ND
Tempera
Effluen
19.0
19.0
19.5
19.5
19.0
19.2
Effluen
18.0
19.0
19.0
19.0
19.0
19.0
ND
ture at
20°C
t and Sludge Stud:
10.0
M.2
8.M
8.1
8.0
7.7
t and Sli
8.3
9.1
9.6
9.2
8.6
8.5
18.8 \ 8.9
I
idge Studi
Survival and Detection Moasureir.nnirs
Tubes
T MB
«ner Temperat
i
es at SUIT
ND
es at Sum
ND
i
ND
mer Temperat
ND ND ND
_
T T -
_ _ _
_
imer Temperat
_
_
_
_ _ _
T T T
T T -
Darkfield
T M B
ures
++ ++ ++
+ ++ ++
+ ++ ++
++ ++ ++
++ + -
+ ++
++.++ -
+ ++
- -f -
ures
+ •+ +
+ -i- +
+ + +
+ + +
+ + +
ares
+ ++ ++
+ + +
+ + +
+ + +
+ + -
+
PA
T M B
+ +. +
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
+ + • +
+ + +
ND ND ND
+ + +
+ + +
+ . + +
+ + +
+ + +
+ + +
+ + -
+ + -
_ _ _
_ _ _
-------
SURVIVAL AND Dl-TCCTJON OF LEPTOSPIRES IN ANIMM, MANURE DISPOSAL
i Si;r\n'vol and DtrL-seti on
Day
Ex;
1
2
3
5
7
Manure
Added
(Ib)
p- • ' - ' • —1
pl!
Mean
K.'riment No. 9 [,£
Experiment b
1
2
3
M.
5
6
7
8
Mean
.
!
Jo. 10 r.
8.9
8.5
8.M
8.M
8.M
8.M
8.M
8.M
8.5
!>! tch
Tetr.p .
Mean
rci
0 . 0 .
Me. -in
(nprn)
Effluent and Sli
No Exte
,S Efflue
19 . 5
19. S
19.0
19.0
18.5
18.0
17.5
19.5
18.8
Effluen-
rnal Dati
nt and S
8.5
8.3
9.7
9.0
9.8
10.0
10.2
8.5
9.2
Solids
(pprr.)
jdge Stud:
: at 20°
i Made
.Lid go Stuc
ctcr
Mean
(in.)
.es at Sun
lies at SL
ND
Tubes
T M D .
imer Tc-mi'orat
_
- - T
_
_
immer Tempera
ND ND ND
_
_
_
_
- — —
Darkfield
T M B
ures
++ ++ +
++ + +
+ - I
; : :
tures
+ + +
+ + +
+ + +
+ + -
+
+ " "
PA
T M B
+ + +
+ + I
+ * :
ND ND ND
+ + +
ND ND ND
ND ND ND
ND ND ND
ND ND ND
ND ND ND
-------
SURVIVAL AND DETECTION OF Ua'TOSPIRES IN AXIMAL MANURE DISPOSAL
o
00
Day
Manure
Added
(Ib) -
Manure' F.nvlronrv.on t
PH
Mean
Ditch-
Temp-.
Mean
iC)
D.O. ..
Me cm
(ppm)
Tornl
Solids
(ppnO
Barom-
eter
Mean
(in.)
Survival and Detection Measurei;.r:n^s
Tubes
X Y
Darkfield
X Y
Experiment No. 11 LS Studies of Seeded Ditch 'at Summer Temperaturels
1
2
' 3
4
5
6
7
2.2
9 I 2.2
10 2.2
I
11 ! 2.2
12
13
14
2.2
15
16 1
17
18
19
20
21
22
23
24
25
lean
2.2
8.2
8.2
8.2
8.2
8.1
8.4
8.4
8.3
8.3
8.2
8.2
8.2
8.1
8.2
m
ND
ND
ND
ND
7.8
7.6
ND
7.1
6.8
6.7
7.9
18.7
19. M
18. M
17.0
14.0
17.3
17.9
18.3
18.5
17.6
17.0
17.3
18.0
18.0
ND
ND
ND
ND
ND
18.2
18.3
ND
18.8
19.3
19.3
18.0
M.I
4,902 j 29.74
3.4
3.2
2.6
3.2
3.0
3.4
2.5
2.0
1.9
l.M
ND
ND
7.7
ND
ND
ND
ND
ND
3.8
1.2
ND
2.0
2.2
2.3
2.9
4,978
4,798
5,817
5,685
5,977
6,733
29.72
29.66
ND
29.70
29.78
29.87
29.70
29.83
29.73
29.61
29.72
29.72
29.84
ND
ND
ND
ND
ND
29.92
ND
ND
ND
29.73
29.67
29.75
ND ND
ND ND
-
-
-
_ _
-
-
-
_ ' _
-
-
-
-
-
-'
-
-
-
-
-
-
-
-
-
X = Efflue
++ =
ND ND
ND ND
-
ND ND
++
++ +
+ +
+ ++
+ +
+ +
+
-
-
.-
-
-
-
-
-
ND ND
-
-
-
- -
-
it; Y = Slud'j
motility
PA
X Y
ND ND
ND ND
ND ND
ND ND
+ +
+ +
+ +
+ +
+ +
+ +
-
+ +
ND ND
-
-
-
-
-
-
-
-
-
ND ND
ND ND
ND ND
e
-------
SURVIVAL AND DETECTION OF LEPTOSPIRES IN ANIMAL MANURE DISPOSAL
Manure Environment
T"1 3 \ /
LJ Q y
Ex
0
1
2
3
4
5
G
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
PH
Mean
perimLM)
5.9
6.0
6.0
6.3
6.3
6.4
6.4
6.4
6.4
6.4
6.4
6.5
6 . 7
7.0
6.9
6.7
6.3
6 . 4
6.4
6.3
6.3
6.4
6.4
6.4
Ditch
Temp .
Mean
(C)
t No. 12
21
20
21
19
21
22
23
20
19
17
22
23
24
21
19
19
20
19
19
20
17
20
20
19
D.O.
Mean
(ppm)
I,S Stud
4.5
2.9
1.7
1.0
1.3
8.9
18.0
17.0
14.0
11.0
5.0
4.0
3.0
o.5
1M.2
5.2
1.0
1.2
2.0
1.7
2.4
2.2
2.2
2.1
"
Total
Solids
(ppm)
ic.-s of So
8218
MD
ND
ND
ND
857S
MD
ND
8373
ND
ND
ND
7396
ND
ND
7334
ND
ND
ND
7226
MD
MD
ND
ND
Barom-
eter
Mean
(in.)
fded Ditc
29.50
29.80
29. 85
29.81
29.78
29.78
29.78
29.98
29.76
29.75
29.61
ND
29.74
29.75
29.73
29.75
29.98
29.71
29.26
29.15
29.44
30.00
31.50
30.08
Survival (Rabbit Serum Agar)
Position" X
Top
i at Si
ND
+
-
+
+
-
-
-
-
-
-
ND
-
-
- •
_
_
-
-
-
+
+
+
+
Middle
jiiimer Ternc
ND
+
_
+
+
+
+
-
-
_
+
ND
-
-
_
-
-
-
-
-
_
4.
+
-
Bottom
eratures
ND
-
-
ND
ND
-
_
-
-
_
+
ND
-
-
_
_
-
-NG
-NG
-
+
+
+
+
Position Y
Top
ND
+
ND
+
-
-
+
+
+
+
-
ND
-
_
_
_
-
_
-
_
+
+
^
+
Middle
MD
-
_
_
_
_
_
-
_
_
_
MD
_
_
_
_
_
_
-
_
_
+
. +
Bottom
ND
-
_
+
+
_
_
+
+
_
_
ND
_
_
_
_
_
_
-
_
+
+
+
o
CD
-------
No. 12 LS
SURVIVAL AND DETECTION OF LEFTOSPIRES IN ANIMAL MANURE DISPOSAL
Manure Environment
Day
24
25
26
27-
40
Ml
42-
46
47 '
48
49
50
51"
52
53.
54
55
56
57
58
61
62
63
64
65
PH
Mean
6.4
6.5
O.G
ND
ND
ND
ND
ND
ND '
ND
7:8
7.8
7.8
ND
ND
ND
ND
ND
ND
ND
ND
7.7
7.8
7.9
7.9
Ditch
Temp .
Mean
(C)
19
18
20
ND
ND
ND
ND
ND
ND
ND
16
17
23
ND
ND
ND
ND
ND
ND
ND
ND
20
24
19
18
D.O.
Mean
(ppm)
1.6
1.6
0.8
ND
ND
ND
ND
ND
ND
ND
4.4
4.0
4.8
ND
ND
ND
ND
ND
ND
ND
ND
4.8
5.0
5.0
5.3
Total
Solids
(ppm)
ND
ND
ND
ND
ND
ND
ND
ND
5713
5198
ND
ND
ND
ND
ND
4424
ND
ND
3990
ND
ND
6237
ND
5160
ND
Barom-
eter
Mean
(in.)
ND
29.75
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
29.74
ND
ND
ND
ND
ND
ND
ND
ND
29.74
29.76
29.82
30.00
Survival (Rabbit Serum Asar)
Position X
Top
ND
ND
ND
ND
+
ND
ND
ND
+
+
+
+
ND
ND
_
ND
ND
ND
ND
ND
+
ND
+
ND
Middle
ND
ND
ND
ND
+
ND
ND
ND
+
+
+
•(-
ND
ND
+
ND
ND
ND
ND
ND
+
ND
+
ND
Bottom
ND
ND
ND
ND
+
.ND
ND
ND
+
+
+
+
ND
ND
+
ND
ND
ND
ND
ND
+
ND
+
ND
Position Y
Top
ND
ND
ND
ND
+
ND
ND
ND
+
+
+
+
ND
ND
+
ND
ND
ND
ND
ND
+
ND
+
ND
Middle
ND
ND
ND
ND
+
Contarnir
ND
ND
ND
+
+
+
+
ND
ND
+
ND
ND
ND
ND
ND
+
ND
+
ND
Bottom
ND
ND
ND
ND
+
a ted
ND
ND
ND
+
+
+
+
ND
ND
+
ND
ND
ND
ND
ND
+
ND
+
ND
-------
Mo. 12 LS
SURVIVAL AND DETECTION OF LEPTOSPIRES IN ANIMAL MANURE DISPOSAL
Manure Environment
Day
66
67
68
69
70
71
72
73
7M
75 .
76
77
78
79.
80
.81
82
83
84
85
86
87
88
89
PH
Mean
7.9
M.9
7.9
7.9
8.0
ND
ND
ND
ND
8.0
8.0
8.0
8.0
8.0
8.2
8.2
8.0
8.0
8.0
8.1
-8.0
8.0
7.9
8.0
Ditch
Temp.
Mean
(C)
18
18
18
17
20
ND
ND
ND
ND
23
23
21
2M
20
20
23
22
22
21
21
20
20
22
22
D.O.
Mean
(ppm)
5.1
5.0
M.5
M.M
M.M
ND
ND
ND
ND
5.1
6.0
6.3
6.6
6.0
5.6
6.0
6.0
6.0
5.8
5.8
5.9
6.2
7.0
6.7
Total
Solids
(ppm)
ND
ND
5556
ND
ND
MM03
ND
ND
ND
4229
ND
ND
8109
ND
ND
ND
8162
ND
ND
ND
ND
ND
ND
ND
Barom-
eter
Mean
(in.)
ND
29.70
ND
29.68
29.67
ND
ND
ND
ND
29.80
ND
ND
ND
29.77
ND
ND
ND
29.78
29.75
29.77
29.78
29.69
29.83
29.92
Survival fRabbit Serum Ajrar")
Position X
Top
ND
ND
+
ND
-
ND
+
ND
ND
ND
+
ND
ND
ND
ND
ND
ND
ND
ND
Middle
ND
ND
+
ND
_
ND
+
ND
ND
ND
+
ND
ND
ND
ND
ND
ND
ND
ND
Bottom
ND
ND
+
ND
ND
+
ND
ND
ND
+
ND
ND
ND
ND
ND
ND
ND
ND
Position Y
Top
ND
ND
+
ND
+
ND
+
ND
ND
read
too
soon
ND
+
ND
_
ND
ND
+
ND
+
ND
ND
ND
ND
Middle
ND
ND
+
ND
ND
+
ND
ND
ND
+
ND
+
ND
ND
ND
ND
+
ND
ND
ND
Bottom
ND
ND
+
ND
ND
+ •
ND
ND
ND
+
ND
ND
ND.
ND
ND
ND
ND
ND
-------
No. 12 LS
SURVIVAL AND DETECTION OF LEPTOSPIRES IN ANIMAL MANURE DISPOSAL
Manure Environment
Day
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
11M
115
116
. PH
Mean
8.0
8.0
8.0
8.0
8.0
8.0
8.0
8.2
8.0
8.0
8.0
7.9
8.0
8.1
8.0
7.9
7.9
8.0
7.9
7.9
7.8
7.8
7.8
7.8
7.9
8.0
7.9
Ditch
Temp.
Mean
(C)
22
22
22
23
24
25
25
19
19
18
19
18
19
20
19
18
18
19
18
18
19
18
18
18
19
18
18
D.O.
Mean
(ppm)
6.2
6.8
7.0
7.2
6.5
6.2
5.8
5.1
5.1
6.3
6.1
5.9
6.9
6.8
6.6
6.3
7.4
8.0
8.0
7.3
7.0
6.5
6.9
9.0
8.5
8.6
7.3
Total
Solids
(ppm)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND .
ND
ND
ND
ND
ND
ND
ND
ND
ND
Barom-
eter
Mean
(in.)
29.92
30.00
30.00
30. 04
ND
ND
29.65
ND
27.86
29.63
29.61
ND
ND
ND
29.92
29.76
29.78
29.78
ND
29.81
29.75
29.75
29.76
29.76
29.90
ND
ND
Survival (Rabbit Serum Asar)
Position X
Top
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Middle
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Bottom
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Position Y
Top
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Middle
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
N
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Bottom
ND "
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
-------
No. 12 LS
SURVIVAL AND DETECTION OF LEPTOSPIKES IN ANIMAL MANURE DISPOSAL
Manure Environment
Day
117
118
119
120
121
122
123
124
125
1 PR
O.C VJ
127
128
129
130
131
132
133
134
135
136
137
138
139
PH
Mean
7.8
8.0
8.1
8.0
8.0
8.0
8.2
8.0
7.9
7.9
8.0
8.0
8.0
7.9
8.0
8.0
8.0
8.0
8.0
8.0
8.0
8.0
Ditch
Temp.
Mean
(C)
18
18
18
19
18
17
17
17
17
17
16
17
17
18
17
16
18
18
18
18
19
20
D.O.
Mean
(ppm)
9.1
11.0
8.8
10.2
10.5
11.0
10.6
9.3
10.8
10.0
11.0
10.0
14.0
10.7
14.0
14.0
11.8
11.0
11.0
li.o
11.0
12.0
Total
Solids
(ppm)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Barom-
eter
Mean
(in.)
29.93
29.94
29.88
29.76
29.52
29.91
29.61
29. 6S
29.78
.PFM MOT I
.LlZtlN INwl I
29.78
29.76
29.80
29.75
ND
29.75
29.76
29.26
ND
ND
ND
29.79
29.92
Survival ( Rabbit Serum Agar")
Position X
Top
ND
ND
ND
ND
ND
ND
ND
ND
ND
OR K T MP
UIxJXXlNv
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Middle
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Bottom
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Position Y
Top
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Middle
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Bottom
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND -..
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
CO
-------
No. 12 LS
SURVIVAL AND DETECTION OF LEPTOSPIRES IN ANIMAL MANURE DISPOSAL
Manu.ro Environment
Day
140
141
142
143
144
145
Means
PH
Mean
8.0
7.8
7.8
8.0
7.6
Ditch
Temp .
Mean
(C)
•
20
20
21
21
19.6
D.O.
Mean
(PPm)
12.0
12.0
12.0
12.0
7.1
Total
Solids
(PPm)
ND
ND
ND
ND
FNH
6367
Barom-
eter
Mean
(in.)
30.18
-STOPPED-
30.19
ND
ND
OF EXPER
30.15
•
Survival (Rabbit Serum Agar")
Position X
Top
ND
+
ND
ND
TMFMT-.
Middle
ND
+
ND
ND
* ISOlc
Bottom
ND
+
ND
ND
ted + D.F
Position Y
Top
ND
+
ND
ND
•
Middle
ND
+
ND
ND
Bottom
ND
+*
ND
ND
-------
ENGINEERING STUDIES OF OXIDATION DITCH OPERATION
Appendix - B
115
-------
Velocity Distribution Studies
Table 2^ Table 3J3
Test Number 1 Test Number 2
Rotor Immersion Depth 1/2" Rotor Immersion Depth 1/2"
Water Depth_2^ Water Depth 2"
Rotor Position A Rotor Position B
Lateral Position
Station I II
III
Lateral Position
Station I II
III
Velocity*
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
.04
.07
.07
.10
.10
0
.01
0
.16
.14
.30
.27
.07
.07
0
0
0
0
-.01
0
.04
.30
.19
.25
.12
.14
.19
.21
.25
.39
.42
.39
.25
.19
19
.14
.10
.19
.16
.14
.16
.14
.16
.30
.19
.14
.14
.14
.07
.12
.16
.04
.14
0
.04
.27
.14
.14
.30
.04
.19
.16
.16
.25
.30
.30
.36
.25
.07
.01
.01
.07
Velocity*
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
0
0
0
0
.01
.01
.01
.21
.25
.30
.2
0
.16
0
.04
.07
.07
.07
.12
.19
.21
.36
.04
0
.10
.12
.12
.10
.12
.07
.12
.14
.16
.16
.27
.30
.16
.30
.19
.16
.12
.12
.10
.12
.10
.14
.27
.36
.30
.27
.21
.21
.16
.16
.12
.10
0
.01
.10
.25
.10
0
.04
.10
.10
.07
.07
.04
0
0
.04
.30
*Velocities measured in units of feet per second.
116
-------
Velocity Distribution Studies (con't)
Table 4£ Table 5B^
Test Number 3_ Test Number 4_
Rotor Immersion Depth 1/2" Rotor Immersion Depth
Water Depth 2J^ Water Depth 2_^
Rotor Position E Rotor Position A
Lateral. Position
Station I II
III
Velocity*
1
2
3
4
5
6
7
8
9
10
11
12 .
13
14
15
16
17
18
19
20
21
22
23
24
-.01
.33
.33
.21
.14
.10
.16
.39
.36
.42
.21
0
0
0
.01
.07
.04
.07
.10
.36
.30
.36
.16
.48
.27
.33
.25
.27
.21
.21
.16
.19
.21
.39
.39
.19
.25
.27
.21
.25
.25
.16
.25
.13
.16
.30
0
.01
-.09
.12
.14
.19
.12
.10
.04
.04
.19
.46
.39
.42
.36
.36
.33
.25
.19
.07
.04
.04
.12
.33
Lateral Position
Station I II
III
Velocity*
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
0
.48
.27
.25
.27
.21
.25
.52
.48
.48
.45
0
0
0
.01
.01
.04
.07
.07
.42
.48
.45
.21
.52
.52
.36
.33
.30
.27
.27
.30
.27
.33
.42
.45
.30
.27
.21
.27
.25
.27
.27
.27
.30
.27
.39
.52
-.01
.19
.16
.14
.16
.19
.14
.10
.12
.27
.48
.48
.48
.30
.39
.27
',27
.27
.16
.12
.07
.25
* Velocities measured in units of feet per second
117
-------
Velocity Distribution Studies (con't)
Table ]3B Table TB
Test Number 5 Test Number 6
Rotor Immersion Depth 1"
Water Depth 2^_
Rotor Position JB
Lateral Position
Station
I
II
III
Velocity*
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
.01
-.07
.01
.01
.16
.07
.12
.48
.42
.48
.33
.33
0
.46
.46
.48
.33
.33
.36
.48
.48
.48
.21
0
.25
.25
.25
.30
.30
.27
.25
.42
.33
.36
.36
.52
.52
.48
.48
.48
.42
.42
.39
.36
.39
.36
.46
.48
.42
.42
.39
.42
.36
.33
.30
.14
.10
.07
.25
.52
-.33
-.16
-.21
-.16
.30
.30
.33
.19
.19
.12
.27
.48
Rotor Immersion Depth 1"
Water Depth 2^
Rotor Position E
Lateral Position
Station
I
II
III
Velocity*
1
2
3
4
5
6
7
8
9
10
11
12
13
m
15
16
17
18
19
20
21
22
23
24
.21
.21
.19
.33
.39
.42
0
.16
.46
.52
.48
.46
.12
.12
.01
0
0
.07
.10
0
.30
.42
.42
.39
.27
.27
.33
.36
.39
.42
.46
.46
.39
.33
.30
.33
.33
.33
.30
.27
.36
.30
.36
.42
.39
.21
.19
.27
.14
.12
.14
.07
0
-.10
-.16
.42
.21
.07
.01
.10
.25
.25
.33
.36
.36
.48
.48
.48
.25
.10
.04
.12
*Velocities measured in units of feet per second.
118
-------
Velocity Distribution Studies (con't)
Table 8B_ Table 9_B
Test Number 7 Test Number 8
Rotor Immersion Depth 1^"
Water Depth 2^
Rotor Position A
Lateral Position
Station
I
II
III
Velocity*
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
.36
.36
.30
.30
.33
.30
.27
.25
.42
,M6
.142
.16
.12
.33
.04
0
.27
.07
.12
.36
.48
.46
.M2
.36
.36
.36
.36
.36
.39
.M6
.36
.36
.30
.36
.36
.36
.39
.36
.M2
.39
.42
.48
.42
.30
.30
.36
.36
.36
.30
.30
.25
.25
.07
.30
.14
.04
.25
.39
.42
.42
.42
.48
.48
.48
.48
.30
.12
.04
.25
Rotor Immersion Depth
Water Depth 2^
Rotor Position B
Station
Lateral Position
II
III
Velocity*
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
0
0
0
.01
.10
.14
.12
.42
.36
.48
.07
.25
.46
.39
.42
.36
.33
.33
.45
.46
.48
.07
.01
.33
.36
.33
.33
.36
.33
.36
.36
.33
.36
.42
.45
.46
.39
.36
.36
.36
.36
.36
.30
.36
.42
.39
.48
.48
.42
.42
.42
.39
.42
.19
.07
.14
.27
0
0
.04
.19
.21
.16
.27
.12
.07
.12
.27
.48
*Velocities measured in units of feet per second.
119
-------
Velocity Distribution Studies fcon't)
Table 10B Table 11B
Test Number 9.
Rotor Immersion Depth
Water Depth 2^_
Rotor Position
Lateral Position
Station
I
II
Ill
Velocity*
1
2
3
M
5
6
7
8
9
10
11
12
13
1M
15
16
17
18
19
20
21
22
23
2M
.1M
.21
.21
.30
.33
.27
.25
.M2
.42
.42
.12
.01
-.12
0
.07
.21
.25
.27
.30
.42
.42
.36
.33
.M2
.33
.36
.33
.30
.30
.25
.25
.27
.42
.42
.27
.33
.36
.33
.36
.33
.36
.27
.36
.42
.16
.21
.27
.30
.30
.30
.25
.19
.12
.10
.25
.42
.48
.42
.36
.36
.36
.30
.36
.07
.12
.30
Test Number 10
Rotor Immersion Depth
Water Depth 3_^_
Rotor Position A
Lateral Position
Station
I
II
III
Velocity*
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
.12
.16
.16
.14
.10
.16
0
-.07
.12
.36
.25
.25
.14
0
.04
.04
0
-0.1
-0.1
0
.12
.30
.25
.30
.19
.19
.16
.19
.27
.30
.46
.30
.19
.14
.19
.19
.16
.19
.16
.14
.14
.16
.27
.25
.14
.14
.14
.14
.12
.10
.10
.04
-.07
0
.10
.04
.04
.10
.14
.14
.25
.27
.33
.27
.42
.30
.14
.07
.07
.07
*Velocities measured in units of feet per second.
120
-------
Velocity Distribution Studies (con't)
Table 12B Table 13B
Test Number 11
Rotor Immersion Depth ^"
Water Depth 3^
Rotor Position £
Lateral Position
Station
I
II
III
Velocity*
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
2M
0
0
. 0
0
.01
.10
.07
.36
.M2
.M2
.12
.12
.12
.10
.21
.21
.14
.19
.14
.14
.36
0
0
.12
.14
.14
.19
.21
.16
.21
.25
.19
.21
.33
.48
.39
.33
.25
.21
.16
.16
.16
.14
.16
.30
.25
.36
.36
.36
.30
.19
.19
.21
.12
.07
.12
.21
-.07
-.07
.12
.07
.10
.16
.14
.10
.10
.07
.12
.27
Test Number 12
Rotor Immersion Depth ^"
Water Depth 3^
Rotor Position E^
Lateral Position
Station
I
II
Ill
Velocity*
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
-.07
.12
.21
.14
.21
.16
.14
.36
.36
.42
.01
0
-.01
0
.01
.10
.12
.10
.14
.30
.36
.36
.10
__-
.48
.39
.30
.25
.25
.25
.19
.19
.14
.25
.36
.33
.21
.19
.25
.21
.21
.25
.25
.25
.19
.19
.30
0
-.07
.01
.16
.16
.19
.19
.07
.04
.10
.21
.33
.36
.36
.33
.27
.19
.25
.21
.14
.10
.10
.16
*Velocities measured in units of feet per second.
121
-------
Velocity Distribution Studies (cpn't)
Table 1MB Table 15B
Test Number 13
Rotor Immersion Depth 1"
Water Depth ^_
Rotor Position A
Lateral Position
Station
I
II
III
Velocity*
1
2
3
i)
S
6
7
8
9
10
11
12
13
1M
15
16
17
18
19
20
21
22
23
24
.25
.27
.27
.33
.33
. .30
.36
-.07
.48
.42
.42
.14
.16
.19
.12
0
0
-.07
0
.25
.48
.48
.46
.33
.30
.33
.30
.36
.39
.48
.42
.27
.25
.30
.36
.33
.33
.30
.33
.21
.25
.46
.33
.21
.25
.30
.25
.16
.25
.16
.12
-.16
0
.30
.16
.12
.12
.27
.27
.36
.39
.46
.46
.48
.46
.30
.14
.10
.07
Test Number 14
Rotor Immersion Depth 1"
Water Depth 3_^
Rotor Position B.
Lateral Position
Station
I
II
III
Velocity*
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
.07
.12
.07
.12
.16
.16
.21
.42
.48
.48
.33
— -
.10
.30
.30
.21
.19
.19
.21
.42
.42
.48
.01
.01
.19
.30
.27
.30
.33
.30
.33
.33
.30
.33
.42
.48
.48
.36
.33
.33
.27
.30
.30
.25
.30
.42
.27
.46
.42
.42
.42
.39
.33
.36
.16
.12
.16
.39
-.16
-.07
.07
.14
.30
.22
.22
.16
.04
.14
.22
.42
*Velocities measured in units of feet per second.
122
-------
Velocity Distribution Studies (con't)
Table 16B Table 17B
Test Number 15
Rotor Immersion Depth 1"
Water Depth 3^
Rotor Position £
Lateral Position
Station
I
II
Ill
Velocity*
1
2
3
4
5
6
7
8
9
10
11
12
13
m
15
16
17
18
19
20
21
22
23
24
-.12
-.01
.16
.25
.19
.27
.25
.42
.48
.48
.07
.04
-.07
.16
0
.0
.21.
.19
.16
.14
.42
.48
.36
.48
.39
.39
.30
.27
.33
.30
.30
.25
.30
.48
.39
.10
.10
.30
.36
.33
.27
.33
.33
.25
.30
.48
.07
.21
.30
.33
.30
.27
.27
.14
.07
.16
.30
.42
.48
.46
.39
.36
.30
.33
.27
42
.07
.12
.21
Test Number 16
Rotor Immersion Depth
Water'Depth 3_^
Rotor Position A
Station
Lateral Position
II
III
Velocity*
1
2
3
4
5
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
.33
.36
.39
.42
.46
.46
.46
.39
.48
.48
.48
.33
.25
.9
.16
.07
-.07
-.16
.07
.16
.48
.48
.48
.36
.33
.33
.33 j
.33
.30
.48
.42
.36
.30
.33
.33
.36
.27
.33
.36
.21
.27
.46
.42
.25
.21
.30
.21
.21
.14
.16
.07
.12
.01
.33
.21
.16
.16
.33
.36
.27
.39
.48
.50
.48
.48
.33
.14
.07
.07
*Velocities measured in units of feet per second.
123
-------
Velocity Distribution Studies (con't)
Table 18B Table 19B
Test Number 17
Rotor Immersion Depth lV"
Water Depth 3^
Rotor Position 13
Lateral Position
Station
I
II
III
Velocity*
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
0
0
.DM
.12
.14
.21
.30
.48
.48
.48
.48
.48
.50
.46
.42
.42
.42
.36
.48
.48
.42
.12
0
.16
.25
.30
.36
.30
.36
.36
.36
.33
.36
.48
.50
.33
.36
.36
.30
.36
.33
.33
.27
.27
.42
.33
.48
.48
.42
.39
.42
.42
.30
.14
.10
.19
.36
.12
.12
,07
.14
.14
.14
.25
.12
.12
.16
.33
.42
Test Number 18
Rotor Immersion Depth
Water Depth 3_^
Rotor Position E
Station
Lateral Position
II
III
Velocity*
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
.21
.30
.36
.36
.33
.36
.36
.48
.48
.42
.16
.04
-.16
.07
.12
.16
.27
.19
.30
.48
.42
.42
.36
.50
.39
.27
.33
33
.36
.36
.30
.30
.36
.42
.30
.30
.27
.36
.39
.33
.36
.36
.36
.27
.33
.42
0
.07
.07
.16
.19
.27
.27
.21
.12
.19
.33
.42
.48
.42
.42
.48
.30
.33
.27
.19
.07
.14
.25
*Velocities measured in units of feet per second.
124
-------
to
Vi
Figure 13
Velocity Distribution Teat No. 1
Water Depth - 2"
Rotor Immersion Depth - 1/2"
Figure 14
Velocity Distribution Test No. 2
Water Depth - 2"
Rotor Immersion Depth - 1/2"
-------
Figure 15
Velocity Distribution Test No. 3
Water Depth - 2"
Rotor Immersion Depth - 1/2"
ON
Figure 16
Velocity Distribution Test No. 4
Water Depth - 2"
Rotor Immersion Depth - 1"
-------
to
Figure 17
Velocity Distribution Test No. 5
Jfeter Depth -2"
RDtor Imaerslon B»pth - 1"
Figure 18
Velocity Distribution Teat Wo. 6
Water Depth - 2"
Rotor Immersion Depth - 1"
-------
oo
Figure 19
Velocity Distribution Test No. 7
Water Depth - 2"
Rotor Immersion Depth - 1 1/2"
Figure 20
Velocity Distribution Test No. 8
Water Depth - 2"
Rotor Immersion Depth - 1 1/2"
-------
(0
<£>
Figure 21
Velocity Distribution Teat No. 9
Water Depth - 2"
Rotor Immersion Depth - 1 1/2"
-------
Figure 22
Velocity Distribution Test No. 10
Water Depth - 3"
Rotor Immersion Depth - 1/2"
Figure 23
Velocity Distribution Test No. 11
Water Depth - 3"
Rotor Iismersion Depth - 1/2"
-------
CO
Figure 24
Velocity Distribution Test No. 12
Water Depth - 3"
Rotor Immersion Depth - 1/2"
Figure 25
Velocity Distribution Test. No. 13
Water Depth - 3"
Rotor Immersion Depth - 1"
-------
Figure 26
Velocity Distribution Teat. No. 14
Water Depth - 3"
Rotor Immersion Depth - 1"
Figure 27
Velocity Distribution Test No. 15
Water Depth - 3"
Rotor Immersion Depth - 1"
-------
-co
CO
Figure 28
Velocity Distribution Test No. 16
Water Depth - 3"
Rotor Immersion Depth - 1 1/2"
.Figure 29
Velocity Distribution Test No. 17
Water Depth - 3"
Rotor Immersion Depth - 11/2"
-------
Figure 30
Velocity Distribution Test No. 18
Water Depth - 3"
Bator Immersion Depth - 11/2"
------- |