Livestock Drinking Water Microbiology and the Factors Influencing the Quality of Drinking Water Offered to Cattle

J. Dairy Sci. 84:1856–1862  American Dairy Science Association, 2001.

Livestock Drinking Water Microbiology and the Factors Influencing the Quality of Drinking Water Offered to Cattle J. T. LeJeune*, T. E. Besser*, N. L. Merrill†, D. H. Rice†, and D. D. Hancock† *Department of Veterinary Microbiology and Pathology and †Department of Veterinary Clinical Sciences, Field Disease Investigation Unit Washington State University, Pullman 99164

ABSTRACT The microbial quality of livestock drinking water was evaluated in 473 cattle water troughs located at 99 different cattle operations. The mean log10-transformed coliform and Escherichia coli concentrations per milliliter of trough water were 1.76 ± 1.25 (SD) and 0.98 ± 1.06 (SD), respectively. The degree of E. coli contamination was positively associated with the proximity of the water trough to the feedbunk, protection of the trough from direct sunlight, lower concentrations of protozoa in the water, and warmer weather. Salmonella sp. were isolated from 2/235 (0.8%) troughs and shigatoxigenic-E. coli O157 was recovered from 6/473 (1.3%) troughs. Four experimental microcosms simulating cattle water troughs were used to further evaluate the effects of protozoal populations on the survival of E. coli O157 in cattle water troughs. Escherichia coli O157 of bovine fecal origin proliferated in all microcosms. Reduction of protozoal populations by treatment with cycloheximide was associated with increased persistence of E. coli O157 concentrations in the microcosms. Water troughs are a major source of exposure of cattle to enteric bacteria, including a number of foodborne pathogens, and this degree of bacterial contamination appeared to be associated with potentially controllable factors. (Key words: water, cattle, microbiology, drinking) Abbreviation key: SMACCT = Sorbitol MaConkey Agar with cefixime and tellurite, MUG = methylumbelliferyl-beta-D-glucuronide, VRB-MUG = violet red bile MUG. INTRODUCTION An adequate supply of clean, fresh drinking water is widely considered essential for optimal cow health and maximum milk production (Church, 1991; Ens-

Received November 17, 2000. Accepted April 11, 2001. Corresponding author: D. Hancock; e-mail: [email protected].

minger et al., 1990). Physico-chemical properties of water suitable for livestock have been published but, despite the fact that waterborne transmission of pathogens among livestock has been long recognized, little information is actually available concerning the microbiological quality of water offered to cattle (Bitting, 1898; Hanninen et al., 1998; Reilly, 1981). Logically, livestock drinking water heavily contaminated with enteric bacteria could serve as a common source of exposure to potential pathogens to cattle that could result in infection of large numbers of animals during a relatively brief period. The extent to which water troughs serve as reservoirs for enteric microorganisms and the frequency that waterborne transmission of these pathogens occurs from water to cattle is not fully known. The purpose of the present study was to describe the microbiological quality of water commonly present on farms and examine some of the factors that might influence the microbial ecology within cattle water troughs and experimental microcosms. MATERIALS AND METHODS Survey Samples from 473 water troughs located on 98 dairy farms (n = 465) and in the holding area of one slaughter plant (n = 8) within the states of Washington, Oregon, and Idaho were collected between January and August 1996. Water and sediments were collected from the bottom of each trough in sterile plastic bags and transported to the laboratory on ice within 24 h. For each trough sampled the parameters recorded included 1) the trough construction material (metal, concrete, plastic, other); 2) whether or not the trough was exposed to direct sunlight; 3) whether or not the trough was enclosed (e.g., by a ball); 4) the winter heating practices; 5) interior or exterior location; 6) distance from the edge of the trough to the edge of the nearest feedbunk, and 7) time elapsed since the trough had been last cleaned (categories offered: <2 mo, 2 to 6 mo, 6 to 12 mo, and >12 mo).

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Laboratory Analysis Enumeration of coliforms and Escherichia coli. The total coliform and E. coli concentrations in each sample were determined by a spread plate method. Tenfold serial dilutions of each sample were made in sterile deionized distilled water to 10−3. Each dilution and the stock samples (0.1 ml) were plated onto 150-mm violet red bile agar plates (Difco Laboratories; Detroit, MI) supplemented with 100 µg/L of 4per liter methylumbelliferyl-beta-D-glucuronide (MUG, Biosynth International, Inc. Naperville, IL) (VRB-MUG). After 18 to 24 h of incubation at 37.0°C, the number of lactose-positive colonies and proportion of lactose-positive colonies that were also MUG positive were recorded from dilution plates containing between 30 to 300 colonies. All lactose positive colonies were considered coliforms; those that were also fluorescent were considered E. coli. Three random colonies from each plate presumptively identified as E. coli were further tested for indole and oxidase production and typical reactions on triple sugar iron agar (Holt, 1994). Detection of E. coli O157 and Salmonella sp. A 30-ml aliquot of each sample was enriched with an equal volume of a 2× concentrate of tryptic soy broth (Difco Laboratories) containing 50 ng of cefixime/ml (Wyeth-Ayerst, Pearl River, NY) and 40 µg of vancomycin/ml (Sigma Chemical Company; St. Louis, MO). Enrichments were incubated overnight at 44.5°C rather than 37°C to increase the sensitivity of detection (LeJeune et al., 2001). Dilutions of the overnight enrichments were made to 10−3 in tryptic soy broth and 0.3 ml of the 10−2 and 10−3 dilutions were spread plated onto 150-mm Sorbitol MacConkey agar plates containing 2.5 µg of potassium tellurite/ml (Sigma Chemical Co.) and 50 ng of cefixime/ml (SMACCT) (Sanderson et al., 1995). After overnight incubation at 37.0°C, suspect E. coli O157 colonies were selected from the SMACCT plates and screened for typical biochemical reactions of E. coli O157, including the use of lactose and the absence of beta-glucuronidase activity (Ratnam et al., 1988). E. coli O157 suspect colonies were confirmed using a commercially available latex agglutination test for the O157 antigen (Oxoid, Basingstoke, Hampshire, UK). A second 30-ml aliquot from 235 of the samples (all samples collected during the summer and up to two samples per farm collected at other times of the year) was enriched overnight at 37.0°C in an equal volume of a 2× concentrate of selenite F broth (Difco). Enrichments were plated onto brilliant green sulfadiazine agar (Difco) and incubated overnight at 37.0°C. Salmonella suspect colonies were screened biochemically for

urease production and biochemical reactions typical of Salmonella enterica on triple sugar iron agar (Holt, 1994). Isolates were confirmed as Salmonella using Ogroup specific antisera. Protozoal and nematode enumeration. The number and size distribution of protozoa from each sample were determined in six microscope fields (400×) and then averaged. Size categories included < 50, 50 to 200, and > 200 µm. Samples were visually and microscopically examined for the presence of nematodes. Experimental Microcosms Experimental microcosms were established to determine the effects of protozoal grazing on the proliferation and survival of E. coli O157 in the sediments of recently contaminated cattle water troughs. Aliquots of bovine feces (5 g), freshly collected from a calf actively excreting a naladixic acid-resistant strain of E. coli O157 (WSU 2032), were inoculated into 12 sterile 250-ml polypropylene bottles each containing 90 ml of sterile deionized distilled water. Aliquots of sediment (5 g) collected from four in-use cattle water troughs were either left untreated (negative control), treated with 2 g of cycloheximide to inhibit endogenous protozoa, or sterilized by autoclaving (45 min, 121°C, 20 lb pressure) (positive control) before being added to each microcosm (Marino and Gannon, 1991). Microcosms were stored loosely capped in the dark without agitation at 20°C. Enumeration of E. coli O157. The concentration of E. coli O157 present in each microcosm was determined on d 0, 1, 3, 5, 7 and 14. Briefly, aggregates of bacteria present in the microcosms were separated using sonication (30 s) and allowed to settle for 10 min prior to withdrawing supernatant for enumeration (Davies et al., 1995). Serial tenfold dilutions of each supernatant were made in deionized distilled water and spread onto 150-mm MacConkey (Difco) agar plates containing 20 µg of naladixic acid/ml (United States Biochemical Corporation, Cleveland OH). Escherichia coli O157 suspects (lactose-positive colonies) were enumerated and up to 10 of these colonies from each plate confirmed as E. coli O157 based on the criteria described above. Statistical methods. All data analysis from the survey and the experimental microcosms were conducted using the SAS (Cary, NC) statistical software package. Crude analyses were conducted using the Wilcoxon rank sum tests (PROC NPAR1WAY function) and the multivariate models generated with the PROC GLM command. Journal of Dairy Science Vol. 84, No. 8, 2001

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RESULTS Survey Bacteria. In the 472 farm water troughs assessed for bacterial numbers, total coliform and E. coli counts were 1.76 ± 1.25 and 0.98 ± 1.06 (mean ± SD log10 cfu/ ml), respectively. Escherichia coli O157 was isolated from six of 473 (1.3%) troughs. Salmonella sp. was isolated from two of 235 (0.8%) troughs. On rare occasions adequate samples were not available to conduct all analyses on all samples. Protozoa. Total protozoa counts in water from 469 water troughs were 1.6 ± 5.6 (mean ± SD per 400× microscopic field). Only 248 (52.9%) of the troughs had detectable protozoa, averaging 3.0 ± 7.5 per 400× field. Biotic associations. Escherichia coli counts were correlated to coliform counts (r = 0.75, P < 0.01) even though E. coli accounted for only a minority of total coliforms. Troughs from which E. coli O157 was isolated had higher log10 coliform counts/ml (2.82 vs. 1.75, P = 0.02), higher log10 E. coli/ml counts (1.76 vs. 0.97, P = 0.04), and greater protozoa counts (2.17 vs. 1.56, P = 0.04) compared with culture negative troughs. Escherichia coli and coliform counts were similar and not significantly different in the two troughs in which Salmonella was detected compared with other troughs. Protozoa counts were lower in the two Salmonella-positive troughs compared with the 237 Salmonella-negative troughs (0.90 vs. 2.2) but this difference was not significant. Nematodes were observed 28/471 (7%) of study troughs. Nematode-containing troughs had higher protozoa counts (2.39 vs. 1.51, P < 0.01). Escherichia coli and coliform counts were not associated with the presence of nematodes (P > 0.20). Effect of season. The effects of season on bacterial and protozoa parameters are shown in Table 1. Winter was considered January March; spring April–June; and fall July–September. E. coli and total coliform counts were higher in summer than in winter or spring (P < 0.05). Protozoa counts were lower in summer than in winter or spring (P < 0.05). Effect of trough cleaning. Of the 473 troughs sampled, data on time since last cleaning were obtained for 438. Forty-five percent of the troughs had not been cleaned in more than 12 mo, 17% between 12 to 6 mo previously, 14% between 2 and 6 mo previously, and 24% less than 2 mo previously. The effects of time since last cleaning on bacterial and protozoal parameters are shown in Table 2. Cleaning affected the total coliform count (P < 0.01), with troughs cleaned within the previous 2 mo having higher (P < 0.05) coliform counts compared with troughs cleaned more than 6 Journal of Dairy Science Vol. 84, No. 8, 2001

mo previously. However, there was no detectable effect of time since last cleaning on E. coli concentrations in the water (P = 0.41). No significant differences in E. coli O157 status were observed among the four times since last cleaning categories or when troughs cleaned within the previous 6 mo (1/166, 0.6%) were compared with less frequently cleaned troughs (5/272, 1.8%) (P = 0.26). Two Salmonella isolates were obtained, both from troughs that had not been cleaned in the previous 12 mo. Total protozoal counts were not associated with cleaning interval (P = 0.40). Effects of trough design. Usable responses regarding direct sunlight exposure were obtained for 468 troughs. Direct exposure to sunlight was associated with lower coliform (1.93 vs. 1.64 log10/ml; P = 0.02) and E. coli (0.85 vs. 1.15 log10/ml; P < 0.01) counts. Troughs exposed to direct sunlight also had more protozoa (2.15 vs. 0.91/400× field; P < 0.01) and higher percentages of small protozoa (60.1 vs. 40.2%, P < 0.01) and protozoa bearing chloroplasts (1.45 vs. 0.17/400× field; P < 0.01). Eschierichia coli O157 was isolated from 4/251 (1.6%) of troughs exposed to sunlight and in 2/217 (0.9%) of troughs not exposed to sunlight. Salmonella was isolated from two of 124 troughs shielded from direct exposure to sunlight and none of 108 troughs directly exposed to sunlight. Usable responses for construction material were obtained for 467 troughs; including 139 metal, 232 concrete, 88 plastic, and eight of other materials. When other factors were not controlled, metal troughs had lower coliform counts (1.53 log10 cfu/g) compared with other construction materials, (1.81, 2.0, and 2.6 log10 cfu/g, respectively, for concrete, plastic, and other; P < 0.01). Similarly, E. coli counts were lower for metal (0.71 log10 cfu/g) compared with other materials (1.1, 1.1, and 1.4 log10 cfu/g, respectively, for concrete, plastic, and other materials, P < 0.01). There were no significant differences in protozoa counts in troughs made of different materials. Usable responses were obtained regarding distance from the nearest feed bunk for 379 water troughs. These data were inadvertently not recorded for troughs sampled during the summer months. The square root of distance recorded was negatively correlated with log10 E. coli count (r = −0.14, P < 0.01) and log10 coliform counts (r = −0.24, P < 0.01). The distance to feedbunk was not associated with protozoa counts (r = 0.03, P = 0.55). When distance was dichotomized at 7.62 m, coliform counts were higher among water troughs near feed bunks (1.79 vs. 1.21 log10 cfu/g, P < 0.01). Similarly the E. coli counts were higher for water troughs near feed bunks (1.03 vs. 0.67 log10 cfu/g, P = 0.04).

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LIVESTOCK DRINKING WATER MICROBIOLOGY Table 1. Bacterial and protozoal parameters in water troughs classified by season. Season Parameter

Winter

Spring

Summer

Mean coliforms (log10/ml) SEM Number tested Mean Escherichia coli (log10/ml) SEM Number tested

1.60a 0.10 176 0.83a 0.08 176

Mean protozoan/400× field SEM Number tested

2.59a 0.55 173

Percent positive for E. coli O157 Number tested Percent positive for Salmonella Number tested Percent with nematodes Number examined

1.1%a 176 1.3%a 78 9.1%a 175

1.72a 0.08 224 0.99a 0.07 225 1.20a 0.33 225 1.3%a

2.30b 0.13 72 1.29b 0.12 72 0.25b 0.09 71 1.4%a

225 0.0%a 92

72 1.5%a 65

5.8%a 225

7.0%a 71

Different superscript within a given row indicates a statistical significant difference (P < 0.05).

a,b

Usable responses were obtained regarding the use of electric trough heaters for 468 troughs of which 172 were sampled during winter months (27 heated and 145 not heated). Total coliforms, E. coli concentrations, and protozoa counts were similar and not significantly associated with the use of electric trough heaters. Considerable confounding was present among the cleaning and trough design variables. For example, metal troughs were more likely to be exposed to sunlight than other trough types. A general linear model was used in an effort to evaluate the effects of the variables after accounting for confounding. The independent variables used were cleaning frequency (dichotomized at 2 mo), construction material, exposure

to sunlight, being completely enclosed, and distance to nearest feed bunk (dichotomized at 7.62 m). A season × electric heater interaction was forced into the model on theoretical grounds, and no other interactions appeared to be significant. Only 349 troughs had usable responses for all variables; all of these were sampled in winter and spring. Three separate models were run, one each with coliforms, E. coli, and protozoal counts as dependent variables. The results are shown in Table 3; only variables associated with one or more dependent variable (P < 0.1) are included. Trough construction material was associated with total coliform counts (P = 0.04), with metal troughs having lower counts than either concrete or plastic troughs. Exposure to

Table 2. Bacterial and protozoal parameters in water troughs classified by cleaning interval. Time since trough last cleaned* Parameter

< 2 mo

2– 6 mo

6–12 mo

> 12 mo

Mean coliforms (log10/ml) SEM Number tested Mean Escherichia coli (log10/ml) SEM Number tested Mean protozoa/400× field SEM Number tested Percent positive for E. coli O157 Number tested Percent positive for Salmonella Number tested

2.18a 0.12 104 1.18a 0.12 104 0.96a 0.50 104 1.0% 104 0.0% 54

1.81b 0.17 62 0.99a 0.13 62 2.52b 0.96 62 0.0% 62 0.0% 35

1.53b 0.14 73 0.93a 0.11 73 1.45b 0.70 73 2.7% 73 0.0% 29

1.64b 0.08 199 0.92a 0.07 199 1.76b 0.39 198 1.5% 199 1.9% 104

Percent with nematodes Number examined

0.9%a 104

11.3%b 62

5.5%ab 73

10.6%b 198

Different superscript within a given row indicates a statistical significant different (P < 0.05).

a,b

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LEJEUNE ET AL. Table 3. Least square means (se) of coliformns, Escherichia coli and protozoa derived from generalized linear model with management factors and season as independent variables. Effect

N

log10 E. coli /ml

log10 coliforms/ml

log10 Protozoa/400× field

Construction material Metal Concrete Plastic Other

116 179 79 5

P* = 0.65 0.84 0.88 0.93

P = 0.04 1.58 (0.17)a 1.74 (0.19)b 2.12 (0.19)b 2.22 (0.61)ab

P = 0.43 0.19 (0.05) 0.26 (0.05) 0.29 (0.05) 0.18 (0.17)

P < 0.01 169 210

P = 0.08 1.03 (0.20) 0.62 (0.19)

P = 0.02 2.04 (0.22) 1.79 (0.22)

0.17 (0.06) 0.27 (0.06)

P = 0.09 342 37

P = 0.38 1.00 (0.16) 0.65 (0.25)

P = 0.80 2.02 (0.19) 1.81 (0.29)

0.21 (0.05) 0.23 (0.08)

P = 0.19 75 274

P < 0.01 0.91 (0.21) 0.74 (0.17)

P = 0.04 2.21 (0.25) 1.62 (0.20)

0.17 (0.07) 0.26 (0.06)

P = 0.02 268 111

P < 0.01 0.98 (0.19) 0.68 (0.20)

P = 0.20 2.23 (0.22) 1.60 (0.23)

0.19 (0.06) 0.25 (0.06)

Exposed to sunlight No Yes Enclosed No Yes Time since cleaned <2 mo >2 mo Distance to feedbunk <7.62 m >7.62 m

0.47 (0.15) (0.17) (0.17) (0.53)

a,b Different superscript for a given effect indicates a statistical significant difference (P < 0.05). Only those independent variables significantly (P < 0.10) associated with at least one dependent variable are shown. *Statistical significance of variable in model.

sunlight was associated with lower E. coli (P < 0.01) counts and higher protozoa counts (P = 0.02). Enclosed troughs had lower E. coli counts than open troughs (P = 0.09). Troughs that were cleaned less than 2 mo before sample collection had higher coliform (P < 0.01) counts and lower protozoa counts (P < 0.05) than troughs cleaned less frequently, although the E. coli counts did not differ by time since last cleaned (P = 0.19). Troughs less than 7.62 m from the nearest feed bunk had higher coliform (P < 0.01) and E. coli (P < 0.02) counts than those located further away.

DISCUSSION The results of this study demonstrate that drinking water offered to cattle is often of poor microbiological quality. The association between the water quality parameters and the ecological factors measured suggest that many of the same factors that influence the survival and proliferation of bacteria in natural aquatic ecosystems have parallels in cattle water troughs. Cattle water troughs have previously been compared to natural aquatic ecosystems, but the quantitative ef-

Experimental Microcosms The effects of cycloheximide and autoclaving on E. coli O157 counts in experimental microcosms are depicted in Figure 1. During the first 72 h, E. coli O157 increased in numbers 100 to 1000-fold. Subsequently, E. coli O157 concentrations declined differently depending upon treatment (P = 0.05). Concentrations were highest in the sterilized microcosms through the duration of the experiment (P < 0.05 vs. both other treatments). Escherichia coli O157 concentrations in cycloheximide treated microcosms were lower than those in the autoclaved group but higher than the untreated control group. Journal of Dairy Science Vol. 84, No. 8, 2001

Figure 1. Proliferation and persistance of Escherichia coli O157 in microcosms simulating cattle water troughs autoclaved (x), left untreated (䊉), or treated with cycloheximide (䊏) to inhibit protozoa.

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fects of specific farm management practices and environmental conditions on the microbiological quality of the water have not been reported (Locke and Collins, 1996). The presence of bacteria in natural aquatic ecosystems is dependent upon the rate of contamination and the equilibrium that establishes between bacterial proliferation in that environment and the rate of its elimination. Bacterial contaminants in cattle water troughs may arise from multiple sources. Cattle may contaminate the troughs with cud or fecal material. Extraneous matter including dust, feed, or bedding may also enter the trough. In some instances, depending upon the source, water may contain high levels of bacterial contamination before it even enters the trough. In this study, bacterial contamination was higher in troughs that were closest to the feedbunk. Proximity of the troughs to the feedbunk may have permitted a greater amount of feed to enter the trough, thus increasing the level of contamination as well as providing a nutrient-rich substrate for bacterial growth and survival at the bottom of the trough. It is not uncommon for bacteria to concentrate up to 1000 times higher in sediments than in the overlying water column (Ashbolt et al., 1993). In addition to the nutrient content of the water, several other factors may influence the survival rate of bacteria in water, including the exposure to direct sunlight and temperature and competition with other microorganisms (Barcina, 1995). The lower E. coli densities in the troughs exposed to direct sunlight observed in this study is consistent with the reported deleterious effects of visible light on E. coli survival in other aquatic systems (Barcina et al., 1989). The observed seasonal fluctuations in E. coli counts in water parallel the seasonal trend in total bacterial counts reported in a longitudinal study of troughs on a single farm (Van der Veer and Van der Veer, 1992). Bacteria in aquatic systems are more likely to proliferate as the water temperature increases, especially above 15°C (LeChevallier et al., 1996). The seasonal increase of E. coli and the proliferation of E. coli O157 during the first 3 d of the experiment in all four of the experimental microcosms derived from different troughs provides further evidence that the sediments of cattle water troughs are environments conducive to extensive bacterial proliferation, even at moderate environmental (20°C) temperatures. The seasonal peaks in E. coli counts in livestock drinking water quality may have relevance to food safety: Troughs with higher generic E. coli counts were also the ones most likely to test positive for E. coli O157. The reported increases in infection of cattle with E. coli O157 during summer months may result, in part, from increased concentra-

tions of this agent in contaminated water troughs (Hancock et al., 1994). Although the more recently cleaned troughs had higher coliforms counts, the cleaning interval had no significant effect on the E. coli counts. Moreover, it is notable that both E. coli O157 and Salmonella tended to be isolated more frequently in the less recently cleaned troughs. The ability of E. coli and Salmonella sp. to survive in other aquatic environments suggests that, once introduced, these bacteria may persist and possibly proliferate as endogenous flora within the troughs, whereas recently cleaned troughs would be less likely to harbor these particular strains of bacteria until they are recontaminated from an outside source (Burton et al., 1987; Marino and Gannon, 1991). Competition with and predation by other microorganisms is considered to be one of the most important factors influencing the elimination of bacteria from natural aquatic systems (Gonzalez et al., 1992; Mallory et al., 1983; Marino and Gannon, 1991). The role of protozoa in influencing the degree of bacterial contamination of cattle water troughs in this study was supported in two distinct ways: 1) the inverse relationship between E. coli counts and protozoal counts. 2) The higher concentrations of E. coli persisting in the experimental microcosms following cycloheximide treatment, a potent inhibitor of protozoa. When considered together, these factors strongly suggest an important role for protozoa in the ecology of E. coli cattle water troughs. The extent of bacterial contamination observed in the drinking water offered to cattle demonstrates that the animals’ daily exposure to E. coli from this source alone can be substantial. Multiple factors that influence the survival and persistence of bacteria in natural aquatic systems also appear to have an effect on the complex ecosystems present in cattle water troughs. Additional research is required to quantify the risks associated with microbial contamination of livestock drinking water. REFERENCES Ashbolt, N., G. Grohmannand, and C. Kueh. 1993. Significance of specific bacterial pathogens in the assessment of polluted receiving water of Sydney. Water Sci. Technol. 27:449–452. Barcina, I. 1995. Survival strategies of enteric bacteria in aquatic systems. Microbiologia 11:389–392. Barcina, I., J. M. Gonzalez, J. Iriberri, and L. Egea. 1989. Effect of visible light on progressive dormancy of Escherichia coli cells during the survival process in natural fresh water. Appl. Environ. Microbiol. 55:246–251. Bitting, A. W. 1898. The relation of water supply to animal diseases. Bull. 70 Purdue University Agriculture Experiment Station IX:42–51. Burton, G. A., Jr., D. Gunnison, and G. R. Lanza. 1987. Survival of pathogenic bacteria in various freshwater sediments. Appl. Environ. Microbiol. 53:633–638. Journal of Dairy Science Vol. 84, No. 8, 2001

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