Cow Fecal Samples Research Articles

Simultaneous Determination of Fourteen Veterinary Antibiotics in Animal Feces by Solid Phase Extraction and High Performance Liquid Chromatography

Abstract An efficient method for simultaneous determination of 14 veterinary antibiotics in animal feces by high-performance liquid chromatography was established under optimizing pretreatment and detection conditions. The results showed that 14 drugs could be separated thoroughly by 1% of acetic acid solution (pH 2.6) and acetonitrile. The detection limits for tetracyclines (TCs), sulfonamides (SAs), fluoroquinolones (FQs) and tylosin (TYL) were 35–90 μg kg −1 , 12–28 μg kg −1 , 9–17 μg kg −1 and 19 μg kg −1 , respectively (the signal-to-noise ratio was 3). After being extracted with a mixture of 50% methanol and 50% buffer solution, enriched and cleaned-up by hydrophi-liclipophilic balanced cartage (HLB), the corresponding recoveries were 58%–75%, 74%–93%, 74%–80% and 85% at a concentration of 1 mg kg −1 and were 66%–83%, 91%–101%, 80%–88% and 68% at a concentration of 10 mg kg −1 , respectively. The method was used to analyze the 14 antibiotics in samples of pig, chicken and dairy cow feces, which had been collected from concentrated animal feeding operations in Liaoning province. These drugs were observed in the range 0.75–22.34 mg kg −1 for TCs, 0.10–1.71 mg kg −1 for SAs, 0.38–4.46 mg kg −1 for FQs and 0.23–0.35 mg kg −1 for TYL.

Use of a culture-independent approach to characterize aerosolized bacteria near an open-freestall dairy operation

Animal manures are known to harbor a variety of zoonotic pathogens, which are suspected of being transported off-site as aerosols from confined feeding operations. In this study, aerosols were collected using a high-volume sampler downwind from a 10,000 cow open-freestall dairy and nearby fields being sprinkler irrigated with wastewater. DNA extracts were prepared from the aerosol samples, then a region of the16S ribosomal RNA gene was sequenced for bacterial identification and phylogenetic classification. At the dairy and irrigation sites, Proteobacteria (α-, β-, and γ-subdivisions) was the most abundant phylum, representing 78% and 69% of all sequences, respectively, while Actinobacteria, Bacteroidetes and Firmicutes represented only 10% or less of the sequences. Of the 191 clones sequenced from the dairy aerosol samples, 6 sequences were found to be homologous with uncultured bacteria from cow milk, rumen, and fecal samples. However, none of the sequence matches was affiliated with bacteria known to be pathogenic to otherwise healthy humans. Although our results do suggest a high diversity among the aerosolized bacteria, the sampling strategy employed in this study may not account for the variable nature of bioaerosol emissions.

Extreme Variability of Steroid Profiles in Cow Feces and Pig Slurries at the Regional Scale: Implications for the Use of Steroids to Specify Fecal Pollution Sources in Waters

Thirty-five samples of cow feces (cowpat and cow manure) and pig slurries subjected to different treatment processes and different storage times before land spreading were extracted and analyzed by gas chromatography-mass spectrometry to determine their fecal stanol profiles. The fresh pig slurry data presented here increase considerably the classical range of values obtained for steroid ratios, resulting in an overlap with the range for cow feces. These results lead to the inability to distinguish species source of feces on the basis of steroid ratios alone. The cause of these differences is not known, although it appears likely to be related to differences in the metabolism of animals in relation to their age and/or variations in diet, rather than to secondary mechanisms of steroid degradation during storage or/and treatment of the feces. Nevertheless, the specificity of steroids to serve as a tool to differentiate cow feces from pig slurries is restored by considering the fecal stanol profile, notably, the six most diagnostic stanol compounds, which are 5β-cholestan-3β-ol (coprostanol), 5β-cholestan-3α-ol (epicoprostanol), 24-methyl-5α-cholestan-3β-ol (campestanol), 24-ethyl-5α-cholestan-3β-ol (sitostanol), 24-ethyl-5β-cholestan-3β-ol (24-ethylcoprostanol), and 24-ethyl-5β-cholestan-3α-ol (24-ethylepicoprostanol). In this study, chemometric analysis of the fingerprint of these six stanols using principal components analysis (PCA) distinguished pig slurries from cow feces. The application of PCA to the stanol profiles, as developed in this study, could be a promising tool for identifying the animal source in fecal contamination of waters.

Host-specific 16S rRNA gene markers of Bacteroidales for source tracking of fecal pollution in the subtropical coastal seawater of Hong Kong

This study investigated the diversity of Bacteroidales communities in the feces of eight host species in Hong Kong (subtropical Asia), including human (in the form of sewage), cow, pig, horse, cat, dog, rabbit and rat. The analysis of terminal restriction fragment length polymorphism (TRFLP) in the 16S rRNA genes revealed significant differences in Bacteroidales communities among all host species, with the exception of dog and cat. Manual examination of TRFLP profiles resulted in six terminal restriction fragments (TRFs) that were potentially specific to the sewage (one TRF), cow (three TRFs) or pig (two TRFs) samples. All six TRFs were (1) present in 100% of the samples of the respective target host, (2) absent in other hosts or present only in low frequency and low intensity, and (3) verified for sizes using in silico digestion of DNA sequences in clone libraries. The six TRFs could reliably indicate the source of fecal contaminations in natural seawater amended with sewage, cow or pig fecal samples. In field tests conducted for two polluted and one unpolluted coastal site, the sewage-specific TRF was detected in all seawater samples of the sites known to be impacted by raw and treated sewage. However, only two of three cow-specific TRFs were detected for the two polluted sites, which also received fecal input from feral cows. No pig-specific TRF was detected, although one of the coastal sites was chronically polluted by pig farm run-offs. Nevertheless, the total absence of the six potentially host-specific TRFs in the seawater of an unpolluted site demonstrated the specificity of the TRFs as gene markers in indicating actual pollution.

Evaluation of new gyrB-based real-time PCR system for the detection of B. fragilis as an indicator of human-specific fecal contamination

A rapid and specific gyrB-based real-time PCR system has been developed for detecting Bacteroides fragilis as a human-specific marker of fecal contamination. Its specificity and sensitivity was evaluated by comparison with other 16S rRNA gene-based primers using closely related Bacteroides and Prevotella. Many studies have used 16S rRNA gene-based method targeting Bacteroides because this genus is relatively abundant in human feces and is useful for microbial source tracking. However, 16S rRNA gene-based primers are evolutionarily too conserved among taxa to discriminate between human-specific species of Bacteroides and other closely related genera, such as Prevotella. Recently, one of the housekeeping genes, gyrB, has been used as an alternative target in multilocus sequence analysis (MLSA) to provide greater phylogenetic resolution. In this study, a new B. fragilis-specific primer set (Bf904F/Bf958R) was designed by alignments of 322 gyrB genes and was compared with the performance of the 16S rRNA gene-based primers in the presence of B. fragilis, Bacteroides ovatus and Prevotella melaninogenica. Amplicons were sequenced and a phylogenetic tree was constructed to confirm the specificity of the primers to B. fragilis. The gyrB-based primers successfully discriminated B. fragilis from B. ovatus and P. melaninogenica. Real-time PCR results showed that the gyrB primer set had a comparable sensitivity in the detection of B. fragilis when compared with the 16S rRNA primer set. The host-specificity of our gyrB-based primer set was validated with human, pig, cow, and dog fecal samples. The gyrB primer system had superior human-specificity. The gyrB-based system can rapidly detect human-specific fecal source and can be used for improved source tracking of human contamination.

The Use of an Experimental Vaccine in Gestating Beef Cows to Reduce the Shedding of Escherichia coli O157:H7 in the Newborn Calf

Beef cows in the last trimester of pregnancy were used to determine if vaccinating against Escherichia coli O157:H7 would increase antibody titers in the serum and also result in the transfer of these antibodies to the neonatal calf. Seventy-one cows were vaccinated 30 d before parturition with an experimental vaccine and then commingled with 66 nonvaccinated cows. Cow fecal and venous blood samples were collected at trial initiation and again about 14 d after parturition. Calf feces and serum were collected at about 14 d after parturition and 60 d later. The serum was analyzed for antibody titers to E. coli O157:H7, and the prevalence of E. coli O157:H7 in feces was determined by the Barkocy-Gallagher procedure. Initial cow antibody titers to E. coli O157:H7 were not different (P = 0.50) between treatments, but by parturition, the antibody titers for E.coli O157:H7 in vaccinated cows were 11 times higher (P < 0.001) than for control cows (917 vs. 83). The serum titers for calves suckling vaccinated cows were higher (P < 0.001) than control calves (1,485 vs.135) at about 14 d after calving. By 60 d, titer levels were still higher (P < 0.001) for calves suckling vaccinated cows. Initial fecal E. coli O157:H7 concentrations for cows were negative for both treatments and remained low. There were no differences in fecal E. coli O157:H7 at 60 d postpartum among calves; less than 5% of calves were shedding. Results suggest that vaccinating gestating cows for E. coli O157:H7 resulted in elevated antibody titers cows, and these antibodies transferred to the calf.

Potential of Antimicrobial Agents to Inhibit Fungi to Aid Isolation of Campylobacter

In preliminary studies conducted to isolate thermophilic Campylobacter spp. from cull dairy cow fecal samples, growth of fungal strains on Abeyta-Hunt-Bark agar and charcoal cefoperazone desoxycholate agar (CCDA) interfered with isolation of target bacteria. Concentrations of antifungal substances in the agar media were not effective in suppressing growth of antibiotic resistant fungi. The objective of the current study was to determine the efficacy of supplemental antimicrobial agents to inhibit growth of fungi of fecal origin on Campylobacter isolation media where microbial growth is overwhelmed by fungi. The inhibitory effect of rifampicin and seven antimycotic agents--cycloheximide, 5-fluorouracil, 5-fluorocytosine, sodium propionate, potassium sorbate, nystatin, and amphotericin B--was evaluated. Antimicrobial agents were added singly or combined at concentrations ranging from 12.3 to 227 mg/L. Incubation was conducted at 42 degrees C for 24-48 h in gas jars (CO2+H2). The most effective antibiotic combinations that inhibited fungal growth without affecting Campylobacter growth were CCDA selective supplement SR155E with amphotericin B (59.5 or 114 mg/L) or a mixture of rifampicin, 5-fluorocytosine, and nystatin (119 or 227 mg/L each). Results of the present study indicate that addition of supplemental antifungal agents in the presence of antibiotics such as rifampicin may enhance suppression of fungal overgrowth on Campylobacter isolation agar media.

Competitive Metagenomic DNA Hybridization Identifies Host-Specific Microbial Genetic Markers in Cow Fecal Samples

Several PCR methods have recently been developed to identify fecal contamination in surface waters. In all cases, researchers have relied on one gene or one microorganism for selection of host-specific markers. Here we describe the application of a genome fragment enrichment (GFE) method to identify host-specific genetic markers from fecal microbial community DNA. As a proof of concept, bovine fecal DNA was challenged against a porcine fecal DNA background to select for bovine-specific DNA sequences. Bioinformatic analyses of 380 bovine enriched metagenomic sequences indicated a preponderance of Bacteroidales-like regions predicted to encode membrane-associated and secreted proteins. Oligonucleotide primers capable of annealing to select Bacteroidales-like bovine GFE sequences exhibited extremely high specificity (>99%) in PCR assays with total fecal DNAs from 279 different animal sources. These primers also demonstrated a broad distribution of corresponding genetic markers (81% positive) among 148 different bovine sources. These data demonstrate that direct metagenomic DNA analysis by the competitive solution hybridization approach described is an efficient method for identifying potentially useful fecal genetic markers and for characterizing differences between environmental microbial communities.

Antimicrobial Resistance and Class 1 Integrons in Pathogenic Escherichia coli from Dairy Farms

The goal of this study was to assess the prevalence of antimicrobial resistance and class 1 integrons, including integron-associated genes, in 24 Escherichia coli isolates from dairy farms. Escherichia coli isolates (n = 14) from dairy cows with mastitis (ECDM), Shiga toxin-producing (STEC) O157:H7 from cull dairy cow fecal samples (n = 9) and bulk tank milk (n = 1) were evaluated for sensitivity to 19 antimicrobial agents used commonly in human and/or veterinary medicine. Multiplex PCR was used to determine presence of genes associated with class 1 integrons (intI1, qacEDelta1, and sulI1). Class 1 integrons were found only in eight of 10 isolates (one STEC O157:H7 and seven ECDM) that demonstrated antimicrobial resistance, and seven of these were resistant to two or more antimicrobial agents. Eight of 10 STEC O157:H7 and six of 14 ECDM were susceptible to all commonly used antibiotics. Five ECDM demonstrated multiple resistances to four or more antibiotics. Most of the 24 isolates examined exhibited resistance against sulfamethoxazole, followed by streptomycin and tetracycline. STEC O157:H7 strains had less prevalence of antibiotic resistance and integron carriage than ECDM. The multiplex PCR method developed for detection of intI1, qacEDelta1, and sulI1 can be used routinely for monitoring presence of these genes. Class 1 integrons were found in eight of 10 E. coli strains that demonstrated antimicrobial resistance; seven of these were resistant to two or more antibiotics. It appears that integrons played a role in the incidence of antimicrobial resistance of the strains used in this study.

Molecular Characterization of Salmonella spp. Isolated from Bulk Tank Milk and Cull Dairy Cow Fecal Samples

The consumption of meat from cull dairy cows and of raw milk has been associated with foodborne salmonellosis. This survey was conducted to establish the prevalence of Salmonella in cull dairy cow fecal samples and bulk tank milk and to determine the proportion of Salmonella-positive dairy farms (n = 30) in east Tennessee. Food and Drug Administration bacteriological analytical protocols were generally used for Salmonella isolation. Primary enrichment was performed with lactose broth, and secondary enrichment was conducted with tetrathionate broth. Eosin methylene blue, hektoen enteric, xylose lysine desoxycholate, bismuth sulfite, and brilliant green (BG) were used as isolation agars. BG agars supplemented with individual antibiotics and/or sulfur compounds were also evaluated. Six of 268 (2.24%) bulk tank milk samples and 9 of 415 (2.17%) fecal samples from 7 of 30 (25.3%) dairy farms were Salmonella-positive. Most isolates (11 of 15) were obtained between September and December. Salmonella isolates were further characterized using polyvalent somatic O Salmonella antiserum, o-nitrophenyl-β-d-galactopyranoside (ONPG), and Analytical Profile Index (API) 20E strips for Enterobacteriaceae. Serological evaluation of presumptive positive Salmonella isolates resulted in substantial numbers of false positives (41.2%). ONPG and API 20E tests enabled further biochemical distinction of the majority of Salmonella spp. from Salmonella Arizonae and closely related members of Enterobacteriaceae like Citrobacter youngae. Pulsed-field gel electrophoresis of SpeI-digested Salmonella DNA was used to subtype isolates. The isolates grouped into four clusters. The baseline information generated in this survey is being used to develop preharvest pathogen reduction programs on selected farms.

Nematophagous fungi in fresh feces of cattle in the Mata region of Minas Gerais state, Brazil

The capacity to survive gut passage is one of the desirable characteristics for nematophagous fungi to be considered potential biological control agents of gastrointestinal nematodes of livestock. From April 1995 to April 1996, a pool of 10 cow fecal samples and 10 individual samples of feces from heifers, which were raised under partial (cows) or total (heifers) confinement in the Mata Region of Minas Gerais State, Brazil, were examined monthly for the presence of nematophagous fungi. A total of 10 isolates was found in the survey. Eight isolates were recovered from the pooled samples of cow feces and two from the individual samples of heifers. Fungi were present in the cow feces during the dry months of August (two isolates of Arthrobotrys oligospora and one Monacrosporium eudermatum) and September (one isolate of Harposporium lilliputanum and one of M. gephyropagum). Fungi were also recovered at the beginning and middle of the rainy season: one isolate of A. musiformis in October, and one isolate of M. gampsosporum and one unidentified fungus which produced septate hyphae and adhesive buds in December. In the individual samples collected from heifers, fungi were present only in the months of September (end of dry season) and March (end of rainy season). One isolate each of H. lilliputanum and A. oligospora were found, respectively. Additional studies to further characterize these isolates should be encouraged.

Clostridium perfringens as a point source indicator in non-point polluted streams

Clostridium perfringens spores were evaluated as an indicator of point source pollution in small streams whose bacteriological water quality was influenced by both point and non-point sources. Samples were collected from small streams (the Rio San Jose, New Mexico and Worm Creek, Idaho/Utah); the Grants, New Mexico; Preston, Idaho; Hyrum, Utah; and San Jose/Santa Clara, California; sewage treatment plant effluents; and the Franklin, Idaho; Logan, Utah; and Wellsville, Utah: facultative sewage lagoons. In both of the small streams, C. perfringens spores could be detected, in decreasing concentrations, for more than 10 km downstream from municipal wastewater treatment plant effluents. Coliform and fecal streptococci concentrations varied widely along the streams in apparent response to non-point sources. Samples of cow, horse and sheep feces; farmlot runoff; oxbow lakes that receive fecal material from animal feeding facilities; and from animal grazing area soil were low in C. perfringens spores, but contained high concentrations of other fecal indictor bacteria. C. perfringens spores appear to be a sensitive indicator for microorganisms entering streams with municipal wastewater, even when agricultural non-point sources of fecal indicator bacteria are important in the receiving stream. Samples of effluents taken from sewage treatment plants with sand filtration processes, and from facultative sewage lagoons, indicate that C. perfringens spores concentrations may be low in these effluents.

Evaluation of culture media for the isolation of Salmonellae from feces.

A study conducted on 300 fecal samples from a cow and a pig, each artificially contaminated with approximately four Salmonella organisms revealed that, of the three enrichment broths used in conjunction with the three selective media, the maximum number of isolations were obtained with Brilliant Green MacConkey broth (BGMB), followed by those obtained with tetrathionate (TTB), and the least with selenite broth. The combination of BGMB with Brilliant Green-neutral red-lactose agar (BGNRLA), and TTB with desoxycholate citrate agar (DCA) gave an equal number of isolations. Of the three selective media used in conjunction with the three enrichment broths, the maximum number of recoveries were obtained on BGNRLA, followed by those on DCA, and least number of isolations on bismuth sulfite agar (BSA). The combination of selenite F broth-BSA appeared to be somewhat inhibitory for the growth of Salmonella organisms. Of the two selective media combinations, the DCA-BGNRLA combination yielded the highest number of isolations. The use of all three selective media gave still better results. Selenite and tetrathionate broths were found unsuitable for isolating Salmonella choleraesuis from feces. BGMB, containing 100 mug of Brilliant Green per ml, proved to be a useful enrichment medium for the isolation of this organism from cow and pig fecal samples, each inoculated with 16 organisms. Observations recorded seemed to suggest that contaminant bacteria perhaps outgrow and mask S. choleraesuis. An incubation period of 24 to 30 hr was found optimum for the three enrichment broths. A longer period was detrimental in the case of TTB but not with selenite broth and BGMB.

Evaluation of Culture Media for the Isolation of Salmonellae from Feces

A study conducted on 300 fecal samples from a cow and a pig, each artificially contaminated with approximately four Salmonella organisms revealed that, of the three enrichment broths used in conjunction with the three selective media, the maximum number of isolations were obtained with Brilliant Green MacConkey broth (BGMB), followed by those obtained with tetrathionate (TTB), and the least with selenite broth. The combination of BGMB with Brilliant Green-neutral red-lactose agar (BGNRLA), and TTB with desoxycholate citrate agar (DCA) gave an equal number of isolations. Of the three selective media used in conjunction with the three enrichment broths, the maximum number of recoveries were obtained on BGNRLA, followed by those on DCA, and least number of isolations on bismuth sulfite agar (BSA). The combination of selenite F broth-BSA appeared to be somewhat inhibitory for the growth of Salmonella organisms. Of the two selective media combinations, the DCA-BGNRLA combination yielded the highest number of isolations. The use of all three selective media gave still better results. Selenite and tetrathionate broths were found unsuitable for isolating Salmonella choleraesuis from feces. BGMB, containing 100 mug of Brilliant Green per ml, proved to be a useful enrichment medium for the isolation of this organism from cow and pig fecal samples, each inoculated with 16 organisms. Observations recorded seemed to suggest that contaminant bacteria perhaps outgrow and mask S. choleraesuis. An incubation period of 24 to 30 hr was found optimum for the three enrichment broths. A longer period was detrimental in the case of TTB but not with selenite broth and BGMB.

A Critical Study of Energy Determination in Fresh and Dried Cow Feces

Summary An accurate method of determining energy in fresh feces is described in detail. The energy content of eight samples of cow feces was determined in freeze-dried, oven-dried, and fresh feces. Moisture was determined by freeze drying, by the toluene distillation method, and by oven drying at 80° C. The three methods are compared and discussed. The results of the duplicate energy determinations in the feces dried by two different methods, and the triplicate determinations in the fresh feces calculated on a comparable basis by means of the three different moisture determinations, are compared and evaluated and their errors discussed. The difference between the energy content of freeze-dried, oven-dried, and fresh feces calculated to a comparable basis by means of the most reliable water determination used in this study (freeze drying) was less than 2%.

A Comparison of Nitrogen and Energy Determinations on Fresh and Oven-Air Dried Cattle Feces

Summary Samples of fresh cow feces, obtained from 12 different animals, were subjected to analysis for total moisture (dry matter), nitrogen, and gross energy. These analyses were made both on the fresh and on the oven–air dried materials. Total moisture of the fresh feces was determined in three ways, and gross energy of the fresh feces was determined with the aid of two different primers, 95% ethyl alcohol and benzoic acid. Gross energy of the dried feces was determined without the use of any primer. The samples of fresh feces were dried in a forced-air oven for a period of 22 hr. at a temperature of 65° C. Small but consistent losses of nitrogen, average of 5.15%, occurred upon drying, as well as the liberation of 727mg. of carbon dioxide per 100g. of fresh feces. The average levels of the determinations of gross energy were 66.31, 71.17, and 71.24 Calories per 100g. of fresh feces for the ethyl alcohol, benzoic acid, and oven–air dry methods, respectively. The composite standard errors between duplicates for the energy determinations, expressed as Calories per 100g. of fresh feces, were 3.70, 2.17, and 0.22 for the ethyl alcohol, benzoic acid, and oven–air dry methods, respectively. Under the conditions of drying fresh cow feces employed in this experiment, only small losses of nitrogen occurred, and there was no loss of gross energy.