20
WATER PATHOGENS PRESENT IN THE WASTEWATER FLOW OF A LARGE DAIRY IN
THE SUMMER AND WINTER
C.W. Purdy, R.H. Raleigh, D.C. Straus, B.A Elmurodov
rom the Conservation and Production Research Laboratory, Renewable Energy, and Manure
Management Research Unit, Agricultural Research Service, United States Department of Agriculture,
P.O. Drawer 10, Bushland, Texas 79012 (Purdy); Veterinary Diagnostic Laboratory, Texas A&M
University, Amarillo Blvd West, Amarillo, Texas 79109 (Raleigh); Department of Microbiology and
Immunology, Texas Tech University Health Science Center, Lubbock, Texas 79430 (Straus); Uzbek
Veterinary Research Institute, Taylak, Samarkand, Republic of Uzbekistan 704453 (Elmurodov).
The authors thank Will Willis for technical assistance.
Abstract
Dairy wastewater is a valuable resource in semi-
arid regions that can be recycled, therefore it’s potential
content of epizootic pathogens, endotoxins and other hazardous substances is very timely and important. Bacterial
and fungal epizootic pathogens were studied in wastewater on a 4,500 cow dairy in the Southern High Plains, U.S.A..
The wastewater was collected in triplicate from 13 different collection points along the wastewater drainage system,
starting in the
milking
parlor and ending in a lagoon; and from an irrigation center pivot that applied the lagoon
wastewater mixed with fresh water to forage crops, later used as feed for dairy cows. Pathogens were found at all
collections points, except two points where chlorination was used. Eight
Salmonella
serovars were identified; most
wastewater samples contained
Escherichia coll
O157:H7; and
Listeria monocytogenes
was found in mean
concentrations from 1 x 10
4
to 1 x 10
5
CFU/ml. The mean concentrations of
Staphylococcus spp,
(1 x 10
4
to 1 x
10
7
CFU/ml) and
Enterococcus spp
(1 x 10
4
to 10 x10
5
CFU/ml) were also high. The mean concentration of mesophilic
fungi ranged from 1 x 10
2
to 1 x 10
5
CFU/ml. The center pivot wastewater contained fewer of each pathogen studied,
and no
Enterococcus spp
were identified from this site. One of the most biologically active substances found in the
lagoon wastewater was endotoxin which had a mean concentration of 37,712 EU/ml. These data show dairy
wastewater can be a significant source of epizootic pathogens and endotoxin.
Introduction
Water is very precious in a semi-arid region and dairy wastewater is used as a valuable resource. The 30
year average precipitation in this region was 470 mm/year, and 30 year normal temperature in degrees centigrade
was: maximum, 22, mean, 14, and minimum, 7. If this water contains dangerous pathogens there is the potential for
contaminating larger areas of crops and pasture land. This then becomes a reservoir that can infect mammals and
birds (Purdy, et al., 2001). Dairy wastewater that is used for dust control or for forage crops soon after irrigation,
become problematic as a potential source of enteric pathogens.
Modem dairies in the U.S.A, have an ingenious way of using water to carry away much of the solid waste
from the holding areas, milking parlors, and feed alleys located outside. The wastewater flows through ditches, and
settling basins, before entering waste holding ponds (lagoons). The lagoon wastewater is often pumped through pipes
to the irrigation center pivot irrigation systems, where it is usually blended with fresh well water prior to use.
The source of chlorinated water used to flush the fresh manure from the milking parlor floor was previously
used to cool the large milk reservoir tanks. This same source of water is frequently used in sprinklers directed up to
partially wash the cows udders of organic material while the cows wait in the holding area before they enter the
milking parlor. The water used to flush manure from the long feeding alleys is recycled lagoon water.
The objective of this study was to determine the concentration of bacteria, and fungi, and to identify specific
pathogen concentrations at 13 points along the flow of wastewater from the milking parlor to the lagoon and after
pumping to the center pivot used for irrigation. In addition, the endotoxin concentration was determined at four lagoon
sites.
Materials & Methods
Climate-
The 30 year average precipitation in the Southern High Plains was 120.4 mm in the winter and 349.5 mm
in the summer. The 30 year normal temperature in degrees centigrade for
21
the winter months (October through March) was: maximum, 15.1, mean, 7.5, and minimum, - 0.1; summer (April
through September) was: maximum, 28.8, mean, 21.0, and minimum, 13.2.
Dairy-The
dairy was located in the
Southern High Plains, where 4,500 head of cows were milked three times a day.
Sample collection for wastewater
and
dilution
technique-Three-two-liter wastewater samples were collected in
sterile plastic bottles from each of 13 points (milk parlor flush, holding area sprinklers, milk parlor effluent, beginning
feeding alley flush, end feeding alley flush, beginning sand ditch, end sand ditch, settling basin, North lagoon, South
lagoon, East lagoon, West lagoon, and center pivot). Each water sample was assayed for bacterial and fungal
concentration. Briefly, 10-fold serial dilutions (W
1
to 10'
7
) were prepared from each sample by inoculating 10 ml of
wastewater into glass bottles containing 90 ml of sterile water. Aliquots of 0.1 ml/per dilution were spread over the
surface of the Petri plates used in the dilution series for each medium used.
Culture medium
- Specialized media were used to determine the concentration of bacteria and fungi, and to identify
the pathogens (Brain-Heart-Infusion agar identified mesophilic and thermophilic bacteria[BHI], Difco; MacConkey
agar was used for identifing all coliforms [MAC], Difco; Enterococcosel agar for identifing
Enterococcus spp.
[EGA],
BBL;
Listeria
selective agar for identifing
Listeria monocytogenes.
[LSA], Oxoid; Bard Parker agar for identifing Gram-
positive cocci; inducted were
Staphylococcus aureus, S. epidermidis, S. saprophyticus, and Micrococcus spp
[BPA],Oxoid; Malt Extract agar for identifing thermal fungi [ME], Difco; Littman oxgall agar for identifing mesophilic
fungi [LOA], Difco. The fungi were further identified by colony formation, color and by spore identification (Crisan, EV,
1959; Barnett, HL., 1965; Larone, DH, 1995). The following inhibitory mediums (EC broth, Difco) were used for
differentiating and enumerating
Escherichia coli
O157:H7, and a selenite-brilliant green- sulfapyridine enrichment
broth medium (SBG plus Sulfa, Difco) was used in part for the isolation of
Salmonella spp.
Additional specific Difco media (blood agar base supported with 5% bovine blood [BA] was used to help in
the identification of hemolytic
Staphylococcus spp, Streptococcus spp, Corynebacterium spp,
and to determine that
Salmonella spp
were not hemolytic. In addition, Xylose-lysine-desoxycholate agar [XLD], Difco; Brilliant Green agar
[BG], Difco; Lysine Iron agar [LIA], Difco; and Triple Sugar Iron agar [TSI], Difco were used to differentiate
Salmonella
spp.
The
Salmonella spp
were serotyped by the National Veterinary Services Laboratory located at Ames, Iowa.
Culture incubation
temperatures-
Thermophilic bacteria (BHI medium) were incubated at 55°C, and thermophilic
fungi (LOA medium) were incubated at 50°C. Pathogens were grown at 37°C, and mesophilic bacteria and fungi were
incubated at 28°C.
Kinetic
Limulus
lysate
assay-Endotoxin was measured by use of the kinetic chromogenic quantitative
Limulus
ameobocyte lysate assay (Williams and Halsey, 1997). Methods for the assay are fully described by Purdy et al.
(2001).
Statistical analyses-Data
were analyzed by use of ANOVA, using the general linear models procedure
a
(SAS user’s
guide, 1988). Significant differences between wastewater assay positions were further evaluated by use of the
Bonferroni adjusted paired t-test. Differences were considered significant at P < 0.05.
Results
Eight
Salmonella
serotypes were identified in the winter and summer
(Table 1).
A total of 86
Salmonella
isolates
were identified, 6
Salmonella
were untypeable, and 4 were multiple
Salmonella
serotypes. Sixty
Salmonella
suspected isolates were not
Salmonella,
which gave a 63% recovery rate.
Salmonella agona
was only found in the
winter.
Salmonella spp
was found at 11 of the 13 collection sites, except in the milk barn flush water and the water
from the milk barn holding area floor sprinklers.
Endotoxin mean concentration for the lagoon wastewater differed significantly with the seasons (P > 0.007),
49,800 (std error 3006) EU/ml in the winter and 25,624 (3992) EU/ml in the summer.
Escherichia coli
O157:H7 was recovered from all EC enriched water samples, except those collected from
the center pivot in the winter. The water from the origin of milk barn flush
22
and from the floor sprinklers were considered sterile, however occasional microbe contaminate were encountered
due to the collection process.
The most numerous mesophylic fungal pathogens identified from the wastewater collection sites
were
Mucor spp,
and
Aspergillus spp,
and the most numerous thermophilic fungi were
Mucor pusillus
and
Aspergillus fumigatus.
The overall mean concentrations of bacteria and fungi found in the dairy wastewater collection
areas are reported as colony forming units (CFU)/ml for selective media
(Table 2).
There were significant
differences (P < 0.045) between the summer and winter and among the collection sites (P < 0.004) for
mesophilic bacterial concentrations. There were no significant differences between mean concentration of
thermophilic bacteria between seasons or among collection sites. There were significant differences
between seasons and collection sites for the mean concentration of mesophilic fungi
(Table 2).
The mean
concentrations of theromphilic fungi were determined only in the winter. At that time the mean
concentrations were significantly different among collection sites (P < 0.0001).
Discussion
Dairy wastewater appears to be a significant source of enteric pathogens,
Salmonella spp,
and
Escherichia coli
O157:H7. In addition numerous Gram-positive pathogens such as S.
aureus,
Streptococcus spp,
and
Corynebacterium spp
were isolated from the wastewater.
Listeria monocytogenes
was recovered most frequently. The endotoxin concentration of the dairy wastewater was high 37,712
(5121) EU/ml, but lower than that found in feedyard playas (8,321 ng/ml or 83,210 EU/ml) (Purdy, CW, et
al., 2001).
Thermal fungi and thermal bacteria in wastewater were 100 times less frequent than mesophilic
fungi and mesophilic bacteria at each collection site. (Data not shown). It is interesting that one to two log
differences were seen at different sides of the lagoon using the selective media. This indicates that the
lagoon microflora are not thoroughly mixed in the lagoon even under continuous multiple aerators.
The variety of bacterial pathogens and high levels of endotoxin should be considered before using
this water for aerial spraying to abate dust. This practice would certainly increase environmental
contamination, and increase the aerosol risk to animals and humans. The use of this untreated wastewater
in the irrigation of plants that will be fed as forage without sustaining some drying (curing) period should be
discouraged.
The spread of epizootic pathogens by dairy wastewater to other animals should be avoided. For
example, in this study, eight
Salmonella
serovars were identified and many more serovars were probably
present. We recommend that dairy wastewater used to settle dust be chlorinated to kill the pathogens
present. It should be realized that the less organic material that the wastewater contains the more efficient
chlorination will be. Additional animal research is needed to determine if the aerosolized endotoxin level
induced by high pressure application of wastewater to settle dust is sufficient to lower milk production and
induce fever in exposed livestock.
It appears that the wildlife most vulnerable to these wastewater pathogens would be migratory
water fowl, which can gain access to the lagoon, regardless of fencing.
References
Barnett, H.L. 1965. Illustrated Genera of Imprefect Fungi, 2
nd
Ed., Burgess Publishing Co., Minneapolis,
Minnesota.
Crisan, E.V. August 1959. The isolation and identification of Thermophilic Fungi. A Master of Science
Thesis Submitted to the Faculty of Purdue University, Department, of Microbiology, Purdue Press,
Larone, D.H. 1995. Medically important fungi: A guide to identification, 3
rd
Ed., ASM Press, Washington, D
C.
Purdy, C.W., Straus, D C., Parker, D.B., and Williams, B.P. 2001. Water quality in cattle feedyards playas
in winter and summer. American Journal of Veterinary Research. 62:1402- 1407.
SAS
Users’ Guide:Version 6.03 Edition.
1988. Cary, NC: SAS Institute Inc.
23
Williams P.B. and Halsey, J.E. 1997. Endotoxin as a factor in adverse reactions to latex gloves.
Annals of Allergy, Asthma, and Immunology 79:303-310. ______________________________
Table 1.
Salmonella
serovars identified from dairy wastewater and the number of -each serovar
isolated.
Salmonella
serovars
Number isolated
agona
12
give
6
minnesota
14
montevideo
6
muenster
1
newport
10
typhimurium
36
Worthington
1
Untypeable Salmonella
4
Multiple Salmonella
serotypes
6
Table 2. Mean microbial
colorfy.
forming units (CFU)/ml based on the dairy wastewater collection site and the
selective media on which the wastewater dilutions were grown.
Collection
site
Mean
CFU/ml BHI
Mean
CFU/ml LOA
Mean CFU/ml
MAC
Mean
CFU/ml BPA
Mean
CFU/ml LSA
Mean
CFU/ml ECA
Milk Parlor
Effluent
1.35E+08
(8.96E+07)
1.62E+05
(9.10E+04)
1.65E+05
(6.92E+04)
4.08E+06
(1.82E+06)
5.97E+05
(1.60E+05)
6.37E+05
(4.73E+05)
Alley Flush
Top
4.28E+07
(1.03E+07)
8.10E+05
(3.33E+05)
4.26E+04
(1.25E+04)
2.55E+06
(9.35E+05)
1.34E+05
(5.55E+04)
5.23E+04
(7.80E+03)
Alley Flush
Bottom
2.66E+07 (9.51
E+06)
4.30E+05
(6.63E+04)
9.25E+04
(1.79E+04)
1.12E+07 (6.41
E+06)
1.19E+05
(8.73E+04)
7.26E+04
(3.09E+04)
Sand Ditch
Top
5.53E+07
(1.94E+07)
2.06E+05
(9.30E+04)
2.38E+05
(7.12E+04)
6.33E+08
(2.45E+06)
2.34E+05
(7.14E+04)
9.38E+04
(1.45E+04)
Sand Ditch
Bottom
3.33E+07
(4.32E+06)
3.80E+05
(1.71E+05)
1.51E+05
(5.10E+04)
6.69E+06
(2.47E+06)
2.26E+05 (6.31
E+04)
1.07E+05
(3.22E+04)
Collection
site
Mean
CFU/ml BHI
1
Mean CFU/ml
LOA
2
Mean CFU/ml
MAC
3
Mean CFU/ml
BPA
4
Mean CFU/ml
LSA
5
Mean CFU/ml
ECA
6
Settling Basin
7.95E+07 (2.51
E+07)
3.76E+05
(1.74E+05)
3.61E+04
(1.38E+04)
2.04E+07
(1.09E+07)
2.14E+05
(5.57E+04)
1.26E+05
(5.62E+04)
Lagoon North
6.23E+06
(2.40E+06)
1.00E+05
(8.16E+04)
3.62E+04
(1.54E+04)
2.88E+06 (1.41
E+06)
1.70E+05
(1.23E+04)
1.84E+04
(7.52E+03)
Lagoon
South
5.62E+06
(2.05E+06)
7.17E+02
(3.00E+02)
6.05E+04 (2.91
E+04)
8.47E+05
(2.92E+05)
1.33E+05
(1.52E+04)
7.97E+03
(3.56E+03)
Lagoon East
1.84E+07
(7.38E+06)
5.02E+04
(5.00E+04)
1.83E+05
(9.34E+04)
3.86E+06
(1.78E+06)
1.77E+05
(2.90E+04)
1.78E+04
(7.23E+03)
Lagoon West
1.19E+06
(3.05E+05)
4.37E+04
(2.29E+04)
7.06E+04
(2.17E+04)
5.80E+05
(7.29E+04)
1.26E+05
(1.82E+04)
6.72E+03
(1.97E+03)
Collection
site
Mean
CFU/ml BHI
Mean
CFU/ml LOA
Mean CFU/ml
MAC
Mean
CFU/ml BPA
Mean
CFU/ml LSA
Mean
CFU/ml ECA
Center Pivot
3.68E+05
(3.39E+05)
2.54E+03
(1.83E+03)
6.33E+02
'
(3.31
E+02)
4.57E+02
(2.38E+02)
1.67E+00
(1.67E+00)
0.00E+00
(0.00E+00)
1. Brain heart infusion agar is a rich all purpose bacterial medium; 2. Littman oxgall agar specific for fungal
isolation; 3. MacConkey agar specific for Gram-negative bacteria and followed EC enrichment broth which limited
the growth to
Escherichia coli
and
E. coli
O157:H7; 4. Bard Parker selective media for
Staphylococcus spp
and
Micrococcus spp;
5.
Listeria
selective agar for
Listeria monocytogenes;
Enterococcosel agar selective medium for
Enterococcus spp.
