Bovine Feces Research Articles

Evaluation of the host specificity of Bacteroides thetaiotaomicron alpha-1-6, mannanase gene as a sewage marker

The specificity of a short fragment functional gene marker (alpha-1-6, mannanase) in Bacteroides thetaiotaomicron was compared with two other Bacteroidales markers used for identifying sewage as the source of pollution in water. A total of 11 different animal species and 230 faecal samples were tested using quantitative polymerase chain reaction. The results showed that the alpha-1-6, mannanase marker had high specificity (0·97). Limited cross-reactivity was found in swine, gull and cat faeces with copy numbers close to detection limit. Positives were detected in all bovine and sheep faeces tested for the Human-Bac1 marker. The human-specific alpha-1-6, mannanase marker showed greater specificity (0·97) than the HF 183 Bacteroides PCR marker (0·71) and the Human-Bac1 (qPCR) marker. This is the first study that compares Bacteroides thetaiotaomicron alpha-1-6, mannanase marker with other markers. Our study showed that this marker has strong potential to be used as an indicator for sewage pollution in water.

Urine from Treated Cattle Drives Selection for Cephalosporin Resistant Escherichia coli in Soil

The U.S. Food and Drug Administration recently issued new rules for using ceftiofur in food animals in part because of an increasing prevalence of enteric bacteria that are resistant to 3rd-generation cephalosporins. Parenteral ceftiofur treatment, however, has limited effects on enteric bacteria so we tested the hypothesis that excreted ceftiofur metabolites exert significant selection pressure for ceftiofur-resistant Escherichia coli in soil. Test matrices were prepared by mixing soil with bovine feces and adding urine containing ceftiofur metabolites (CFM) (0 ppm, ∼50 ppm and ∼100 ppm). Matrices were incubated at 23°C or 4°C for variable periods of time after which residual CFM was quantified using a bioassay. Bla CMY-2 plasmid-bearing ceftiofur resistant (cefR) E. coli and one-month old calves were used to study the selection effects of CFM and transmission of cefR bacteria from the environment back to animals. Our studies showed that urinary CFM (∼13 ppm final concentration) is biologically degraded in soil within 2.7 days at 23°C, but persists up to 23.3 days at 4°C. Even short-term persistence in soil provides a >1 log10 advantage to resistant E. coli populations, resulting in significantly prolonged persistence of these bacteria in the soil (∼two months). We further show that resistant strains readily colonize calves by contact with contaminated bedding and without antibiotic selection pressure. Ceftiofur metabolites in urine amplify resistant E. coli populations and, if applicable to field conditions, this effect is far more compelling than reported selection in vivo after parenteral administration of ceftiofur. Because ceftiofur degradation is temperature dependent, these compounds may accumulate during colder months and this could further enhance selection as seasonal temperatures increase. If cost-effective engineered solutions can be developed to limit ex vivo selection, this may limit proliferation for ceftiofur resistant enteric bacteria while preserving the ability to use this important antibiotic in food animal production.

Molecular-based detection of the gastrointestinal pathogen Campylobacter ureolyticus in unpasteurized milk samples from two cattle farms in Ireland

Campylobacter jejuni and coli are collectively regarded as the most prevalent cause of bacterial foodborne illness worldwide. An emerging species, Campylobacter ureolyticus has recently been detected in patients with gastroenteritis, however, the source of this organism has, until now, remained unclear. Herein, we describe the molecular-based detection of this pathogen in bovine faeces (1/20) and unpasteurized milk (6/47) but not in poultry (chicken wings and caeca). This is, to the best of our knowledge, the first report of the presence of this potential gastrointestinal pathogen in an animal source, possibly suggesting a route for its transmission to humans.

Persistence ofEscherichia coliintroduced into streambed sediments with goose, deer and bovine animal waste

The focus of this work was to compare the survival of Escherichia coli introduced into streambed sediments from goose, deer and bovine faeces vs indigenous E. coli. The survival experiments were conducted in flow-through chambers for 32days using two sediments (mineral and organic) obtained from a first-order creek in Maryland. Bovine, goose and deer faeces were collected fresh and diluted or enriched so that added E. coli and indigenous populations were equivalent. Escherichia coli and total coliforms were enumerated using the Colilert-18 Quanti-Tray system. Patterns of E. coli survival and inactivation rates were virtually identical for indigenous strains in both mineral and organic sediments. The addition of E. coli strains from bovine, goose or deer faeces had relatively little impact on final E. coli concentrations, with the exception of deer-borne E. coli populations in the organic sediment. These results indicate that indigenous sediment-borne E. coli strains are generally, or more, persistent than those deposited into sediments, including wildlife. This is the first study on the survival of E. coli originating from wildlife faeces, in sediments, as opposed to bovine faeces or laboratory-cultured strains. As wildlife are likely to be the primary source of E. coli in most non agricultural watersheds, an understanding of the persistence of these strains is important to understanding microbial water quality.

Osmotic and Desiccation Tolerance in Escherichia coli O157:H7 Requires rpoS (σ38)

The contribution of RecA, Dps, and RpoS to survival of Escherichia coli O157:H7 during desiccation and osmotic stress was determined in Luria-Bertani broth with 12 % NaCl (LB-12) at 30 and 37 °C, on filter disks at 23 and 30 °C, and in sterile bovine feces at 30 °C. RecA did not significantly contribute to survival in any condition or temperature. The contribution of Dps to survival was only significant in LB-12 at 37 °C. RpoS was necessary for survival during desiccation and osmotic stress, and survival of the RpoS mutant was significantly less than the parent in all conditions and temperatures. The RpoS mutant survived up to 21 days in bovine feces, <4 days on filter disks, and >8 and <4 days in LB-12 at 30 and 37 °C, respectively. The parent, ΔrecA, dps, and dps/ΔrecA mutant strains survived >8 days in LB-12, >28 days on filter disks, and >28 days in bovine feces. Increased incubation temperatures were associated with decreased survival. E. coli O157:H7 can persist in desiccating and osmotically challenging environments, especially sterile feces, for an extended period time.

Two drying methods of bovine faeces for estimating n-alkane concentration, intake and digestibility: A comparison

An experiment was completed to evaluate effects of two drying methods applied to faecal and herbage samples on estimation of n-alkane concentration and the calculated dry matter (DM) intake, faecal output (FO) and apparent in vivo DM digestibility (DMD). Four Holstein–Friesian steers (182±12.1kg initial body weight) housed in individual pens were fed at 8:30 and 16:30h with ryegrass (Lolium perenne L.) hay for 18d. Refusals were removed and weighed every day at 8:30h before morning feeding. From days 9 to 18, the steers were dosed twice a day at feeding time, with cellucotton stoppers containing C32 (88.49mg/pellet) and C36 (87.12mg/pellet). During the last 5d of dosing, total collection of faeces was completed using harnesses. For the n-alkane analysis, faecal (n=64) and offered hay (n=4) samples were subdivided into two subsamples and prepared for two alternative drying methods: oven-drying at 60°C (OD) or freeze-drying (FD). The n-alkane profiles of the offered and refused hay oven-dried at 60°C did not differ. In offered hay subsamples, concentrations of n-alkanes were not affected by drying method. In faecal subsamples, drying method affected (P<0.05) concentrations of all n-alkanes, except for C23, C25 and C35, as well as the ratios C32:C31 and C32:C33. There were no differences between estimated and measured values when DM intake and apparent in vivo DMD were estimated based on the ratio C32:C33. In contrast, when the ratio C32:C31 was used, estimated values either differed (P<0.02) or tended to differ (P<00.09) from measured values for OD and FD subsamples, respectively. Estimates of FO from subsamples OD and FD did not differ from measured values. Faecal recovery increased with increasing carbon-chain length in both drying methods. Results show that oven-drying at 60°C could replace freeze-drying for sample dehydration to estimate DM intake and apparent in vivo DMD when ratio C32:C33 was used. However, due to the low number of replicates, further studies should be conducted before use of C32:C33 ratio in oven-dried samples can be recommended.

Forage enrichment with copper and zinc in beef grazing systems in Argentina

The presence of trace elements in water and soils, their absorption by plants, and their transfer to the food chain, is a phenomenon that has been studied in different regions around the world. In Argentina, few studies exist regarding trace element content in soils, and the studies on forages are furthermore scarce. The interest to quantify the levels of trace elements in cattle forage lies in the intrinsic connection between the quality of forage and the nutritional (i.e. Cu and Zn) aspects of cattle health and products for human consumption. Feed lots use feed, which incorporates core minerals whose basic components are Fe, Se, I, Mn, Co, Cu, and Zn. The excess of these trace elements are eliminated through the feces into the environment, therefore, the use of manure from these systems, as a source of organic fertilizer, could reduce environmental contamination, and supplement soils, in areas which are naturally deficient in these micronutrients, for forage growth intended for livestock consumption. Soils from different areas in Argentina were characterized, mainly for their Cu and Zn content. Soil–plant transfer of these trace elements to natural forage was analyzed for agricultural soils with higher content of micronutrients. On the other hand, pot experiments were performed for the enrichment of the forage species, Lolium perenne (ryegrass), with Cu and Zn through fertilization with bovine feces spiked with known concentrations of these trace elements. Soils from copper‐deficient areas used for livestock breeding were used in these experiments. Differences in Cu and Zn concentrations were observed between the fertilized and non-fertilized reactors, while there were no differences found in the Cu and Zn concentrations considering the method of fertilization (homogenization or superficial). These results show that manure from feed lots may be used for forage enrichment with micronutrients.

Complete Genome Sequence of the Strong Mutator Salmonella enterica subsp. enterica Serotype Heidelberg Strain B182

In bacteria, normal mutation frequencies are mostly around 10(-10) per base pair. However, there exists natural isolates, called "mutators," that exhibit permanent mutation occurrences up to 1,000-fold greater than usual. As mutations play essential roles, particularly in the evolution of antibiotic resistance, bacteria showing elevated mutation rates could have an important responsibility in the emergence of antibiotic resistance, especially in the clinical background. In this announcement, we report the first complete genome sequence of the Salmonella enterica subsp. enterica serotype Heidelberg B182 mutator strain, isolated from bovine feces (France), which consists of a 4,750,465-bp circular chromosome (cB182_4750; GC, 52.2%) and one circular plasmid of 37,581 bp (pB182_37; GC, 42.8%).

Prevalence and Serotypes ofListeria monocytogenesContamination in Chinese Beef Processing Plants

The aim of this work was to study the epidemiology of Listeria spp., particularly Listeria monocytogenes, and to identify the serotypes present in contaminated samples from beef processing plants in China. A total of 439 samples were obtained from bovine feces, hides, and carcasses at three commercial processing plants. A standard protocol (ISO 11290-1) was followed to detect Listeria spp. and L. monocytogenes, and multiplex polymerase chain reaction was used to identify the various L. monocytogenes serotypes. The overall prevalences of Listeria spp. and L. monocytogenes were 65.6% and 26.4%, respectively, and the contamination was highest in the hide samples. The identified L. monocytogenes serotypes were 1/2c and 1/2a. The results of the current study indicate that Listeria spp. contamination is common in Chinese beef processing plants; specific measures should be taken to prevent and/or treat L. monocytogenes contamination of feces and hides in beef slaughter plants. Furthermore, because Listeria spp. contamination was found to be prevalent, it should, therefore, be studied further. The prevention of cases of sporadic listeriosis in China should also be addressed.

Origin of fecal contamination in waters from contrasted areas: Stanols as Microbial Source Tracking markers

Improving the microbiological quality of coastal and river waters relies on the development of reliable markers that are capable of determining sources of fecal pollution. Recently, a principal component analysis (PCA) method based on six stanol compounds (i.e. 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)) was shown to be suitable for distinguishing between porcine and bovine feces. In this study, we tested if this PCA method, using the above six stanols, could be used as a tool in “Microbial Source Tracking (MST)” methods in water from areas of intensive agriculture where diffuse fecal contamination is often marked by the co-existence of human and animal sources. In particular, well-defined and stable clusters were found in PCA score plots clustering samples of “pure” human, bovine and porcine feces along with runoff and diluted waters in which the source of contamination is known. A good consistency was also observed between the source assignments made by the 6-stanol-based PCA method and the microbial markers for river waters contaminated by fecal matter of unknown origin. More generally, the tests conducted in this study argue for the addition of the PCA method based on six stanols in the MST toolbox to help identify fecal contamination sources. The data presented in this study show that this addition would improve the determination of fecal contamination sources when the contamination levels are low to moderate.

Comparison of the environmental survival characteristics of Salmonella Dublin and Salmonella Typhimurium

To examine possible correlations in bovine Salmonella isolates between environmental survival and serovar-associated epidemiological patterns, bovine field isolates of Salmonella serovars Typhimurium and Dublin (two each) were inoculated into bovine faeces slurry and tested monthly by culture for survival during a six-month period of storage at a variable ambient temperature in a disused animal transporter. Low moisture conditions, where the slurry was dried onto wooden dowels, increased detectable survival of a low-level inoculum by up to five months, compared with wet slurry. A more modest increase of survival time was seen with storage of wet slurry under refrigeration at 4°C. Under both dry and wet conditions, the concentration of culturable Salmonella Typhimurium declined at a slower rate than did that of Salmonella Dublin. Salmonella that was naturally contaminating bovine faeces from farms with Salmonella Typhimurium did not show superior survival times compared with Salmonella Typhimurium that had been artificially inoculated into samples. The differing survival characteristics of the two serovars that was observed in environmental faeces may complement their different modes of infection in cattle. Salmonella Dublin, being a bovine host-adapted strain that establishes chronic infection in some animals, may have less need to survive for a prolonged period outside of its host than does Salmonella Typhimurium.

Efficacy of Octenidine Hydrochloride for Reducing Escherichia coli O157:H7, Salmonella spp., and Listeria monocytogenes on Cattle Hides

The efficacy of octenidine hydrochloride (OH; 0.025, 0.15, and 0.25%) for inactivating Escherichia coli O157:H7, Salmonella spp., and Listeria monocytogenes on cattle hides was investigated at 23°C in the presence and absence of bovine feces. All tested concentrations of OH were effective in decreasing more than 5.0 log CFU of bacteria/cm(2) in 5 min (P < 0.01). The results suggest that OH could be used to decontaminate cattle hides; however, further studies under commercial settings are necessary to validate these results.

Seropathotypes, Phylogroups, Stx Subtypes, and Intimin Types of Wildlife-Carried, Shiga Toxin-Producing Escherichia coli Strains with the Same Characteristics as Human-Pathogenic Isolates

The objectives of this study were to investigate the presence of Shiga toxin-producing Escherichia coli (STEC) strains in wildlife that have spread in Europe, living near human settlements; to analyze their epidemiological role in maintenance and transmission to domestic livestock; and to assess the potential health risk of wildlife-carried strains. STEC strains were recovered from 53% of roe deer, 8.4% of wild boars, and 1.9% of foxes sampled in the northwest of Spain (Galicia). Of the 40 serotypes identified, 21 were classified as seropathotypes associated with human disease, accounting for 81.5% of the wildlife-carried STEC strains, including the enterohemorrhagic serotypes O157:H7-D-eae-γ1, O26:[H11]-B1-eae-β1, O121:H19-B1-eae-ε1, and O145:[H28]-D-eae-γ1. None of the wildlife-carried strains belonged to the highly pathogenic serotype O104:H4-B1 from the recent Germany outbreak. Forty percent of wildlife-carried STEC strains shared serotypes, phylogroups, intimin types, and Stx profiles with isolates from human patients from the same geographic area. Furthermore, wildlife-carried strains belonging to serotypes O5:HNM-A, O26:[H11]-B1, O76:H19-B1, O145:[H28]-D, O146:H21-B1, and O157:H7-D showed pulsed-field gel electrophoresis (PFGE) profiles with >85% similarity to human-pathogenic STEC strains. We also found a high level of similarity among STEC strains of serotypes O5:HNM-A, O26:[H11]-B1, and O145:HNM-D of bovine (feces and beef) and wildlife origins. Interestingly, O146:H21-B1, the second most frequently detected serotype in this study, is commonly associated with human diarrhea and isolated from beef and vegetables sold in Galicia. Importantly, at least 3 STEC isolates from foxes (O5:HNM-A-eae-β1, O98:[H21]-B1-eae-ζ1, and O146:[H21]-B1) showed characteristics similar to those of human STEC strains. In conclusion, roe deer, wild boar, and fox in Galicia are confirmed to be carriers of STEC strains potentially pathogenic for humans and seem to play an important role in the maintenance of STEC.

Potency of microbial inocula from bovine faeces and rumen fluid for in vitro digestion of different tropical forage substrates

AbstractIn vitro ruminal fermentation techniques rely on the availability of fistulated ruminants for rumen fluid (RF), a major constraint for resource‐poor institutions. An alternative would be to use faecal microbes. This study was therefore designed to compare the potency of fresh bovine faeces against RF as sources of microbial inocula for fermenting six contrasting tropical forages using the Tilley and Terry (Journal of British Grassland Society, 18, 104–111) technique. Faecal inocula were prepared at concentrations of 250 g (Faec250), 300 g (Faec300), 350 g (Faec350), 400 g (Faec400) and 450 g (Faec450) fresh cow faeces per litre bicarbonate buffer. In vitro organic matter digestibility (IVOMD) increased with increasing concentration of faeces at 12‐, 24‐ and 48‐h incubation intervals. All faecal inocula except Faec450 had lower potency when compared with RF at all incubation intervals. Digestibility of forage substrates with Faec450 (533 ± 23 g kg−1 DM IVOMD) was comparable to RF (566 ± 5 g kg−1 DM IVOMD) after 48‐h incubation. Faec450 showed greater potency on more fibrous and low‐protein substrates than RF. The coefficient of determination of the regression models for predicting 12‐, 24‐ and 48‐h RF IVOMD from Faec450 were 0·914, 0·75 and 0·756 respectively. It was concluded that inocula prepared from 450 g cow faeces demonstrated great potential as a substitute and predictor for RF in 48‐h IVOMD assays of tropical forages.

Molecular characterization ofEscherichia coliO157:H7 strains isolated from different sources and geographic regions

Escherichia (E.) coli serotype O157:H7 is a globally distributed human enteropathogen and is comprised of microorganisms with closely related genotypes. The main reservoir for this group is bovine bowels, and infection mainly occurs after ingestion of contaminated water and food. Virulence genetic markers of 28 O157:H7 strains were investigated and multilocus enzyme electrophoresis (MLEE) was used to evaluate the clonal structure. O157:H7 strains from several countries were isolated from food, human and bovine feces. According to MLEE, O157:H7 strains clustered into two main clonal groups designated A and B. Subcluster A1 included 82% of the O157:H7 strains exhibiting identical MLEE pattern. Most enterohemorrhagic E. coli (EHEC) O157:H7 strains from Brazil and Argentina were in the same MLEE subgroup. Bovine and food strains carried virulence genes associated with EHEC pathogenicity in humans.

High diversity and differential persistence of fecal Bacteroidales population spiked into freshwater microcosm

Bacteroidales markers are promising indicators of fecal pollution and are now widely used in microbial source tracking (MST) studies. However, a thorough understanding of the persistence of Bacteroidales population after being released into environmental waters is lacking. We investigated the persistence of two host specific markers (HF183 and CF193) and temporal change of Bacteroidales population over 14 days in freshwater microcosms seeded with human or bovine feces. The concentrations of HF183/CF193 and Escherichia coli were determined using quantitative polymerase chain reaction (qPCR) and standard cultivation method, respectively. Shifts in the Bacteroidales population structure were fingerprinted using PCR-denaturing gradient gel electrophoresis (DGGE) and subsequent sequencing analysis targeting both 16S rDNA and rRNA-transcribed cDNA. Both HF183 and CF193 decayed significantly faster than E. coli but the decay curves fit poorly with first-order model. High diversity of Bacteroidales population was observed for both microcosms, and persistence of different species in the population varied. Sequence analysis indicated that most of the bovine Bacteroidales populations in our study are unexplored. DGGE and decay curve indicated that RNA decayed faster than DNA, further supporting the use of rRNA as indicator of metabolically active Bacteroidales population. Evaluations with more realistic scenarios are warranted prior to extending the results of this study to real field settings.

Genetic diversity and population structure of Escherichia coli from neighboring small-scale dairy farms

The genetic diversity and population structure of Escherichia coli isolates from small-scale dairy farms were used to assess the ability of E. coli to spread within the farm environment and between neighboring farms. A total of 164 E. coli isolates were obtained from bovine feces, bedding, cow teats and milk from 6 small-scale dairy farms. Ward's clustering grouped the isolates into 54 different random amplified polymorphic DNA (RAPD) types at 95% similarity, regardless of either the sample type or the farm of isolation. This suggests that RAPD types are shared between bovine feces, bedding, cow teats, and milk. In addition, transmission of RAPD types between the studied farms was suggested by the Ward grouping pattern of the isolates, Nei's and AMOVA population analyses, and genetic landscape shape analysis. For the first time, the latter analytical tool was used to assess the ability of E. coli to disseminate between small-scale dairy farms within the same producing region. Although a number of dispersal mechanisms could exist between farms, the genetic landscape shape analysis associated the flow of E. coli RAPD types with the movement of forage and milking staff between farms. This study will aid in planning disease prevention strategies and optimizing husbandry practices.

A Preliminary Study ofSalmonella, VerocytotoxigenicEscherichia coli/Escherichia coliO157 andCampylobacteron Four Mixed Farms

The aims of this study were to investigate the incidence of Salmonella, verocytotoxigenic Escherichia coli (VTEC)/Escherichia coli O157 and Campylobacter on four mixed farms and to characterize the isolates in terms of a range of virulence factors. Eighty-nine composite (five different samples from the same animal species combined) faecal [cattle (24), pigs (14), sheep (4), poultry (4), horses (7), deer (4), dogs (9), rodents (2) and wild birds (20)] samples, 16 composite soil samples plus 35 individual water samples were screened using culture-based, immunomagnetic separation and molecular methods. Salmonella was detected in bovine faeces, cattle and poultry house water. Salmonella serotypes/phage types included Dublin, Kiel and Typhimurium DT193, and most isolates were spvC, invA and rck positive. The pefA and rck genes were found exclusively in the non-Typhimurium strains, while Salmonella Dublin and Salmonella Kiel strains carried Salmonella genomic island I marker(s). VTEC/E. coli O157 were found in deer and dog faeces only. The E. coli O157 isolate was an enteroinvasive E. coli, while the VTEC isolate was untypable but carried the vt1, eaeA, hlyA, tir and eptD genes. This article reports the first confirmed carriage of E. coli O157 in Irish deer. Campylobacter species were not detected over the course of this study. It was concluded that [1] Salmonella, VTEC and Campylobacter have low (<5%) prevalence or are absent on the farms in this study; [2] water was an important source of bacterial pathogens; [3] both dogs and deer may act as a source of pathogenic E. coli and [4] key virulence and resistance determinants are widespread in farm Salmonella strains. This study highlights the need to control water as a source of pathogens and suggests that the domestic pets and deer should be considered in any farm risk assessment.

Identification of host‐specific Bacteroidales 16S rDNA sequences from human sewage and ruminant feces

The need to identify the source of fecal contamination of water has led to the development of various fecal source identification methods, a field known as microbial source tracking (MST). One promising method of MST focuses on fecal members of the order Bacteroidales, some of which exhibit a high degree of host-specificity. In order to identify host-specific Bacteroidales genetic markers, a ∼1060 bp section of Bacteroidales 16S rDNA was amplified from human sewage (n = 6), and bovine (n = 6) and ovine fecal (n = 5) samples and used for the generation of three clone libraries. Phylogenetic analysis of sequences from the three clone libraries revealed that the Bacteroidales species found in both human sewage and bovine and ovine feces were a highly diverse group of organisms, many of which were not represented by previously characterised 16S rDNA. Ovine and bovine feces appear to host similar populations of Bacteroidales species and these species were more diverse and less closely related to cultivated species than the Bacteroidales population found in human sewage. Species of Bacteroidales from the ruminant and human feces formed isolated clusters containing putatively host-specific sequences. These sequences were subsequently exploited for the design of host-specific primers which were used in MST studies.

Filling One More Gap: Experimental Evidence of Horizontal Transmission of Vaccinia Virus Between Bovines and Rodents

Vaccinia virus (VACV) has been associated with several exanthematic outbreaks in bovine, human, and equine species in Brazilian rural areas. Little is known about VACV reservoirs, although it is believed that rodents could be associated with VACV outbreaks. With the goal of filling one more gap in the VACV ecological puzzle, the present work aimed at mimicking a potential transmission route of VACV between cows and rodents, both known as natural VACV hosts. Balb/c mice were exposed to feces of experimentally VACV infected cows for 20 days, and samples from these mice were examined by using molecular and serological tests. VACV DNA was detected in feces and blood samples after several days of exposure; infectious VACV particles were also detected in the feces. The presence of anti-VACV neutralizing antibodies in murine sera further suggested horizontal transmission. If the transmission model described here can be applied to natural environments, exposure to bovine feces could be considered a risk factor for the spread of VACV; consequently, the traditional use of bovine manure as a fertilizer in agricultural activities may be promoting the infection of rodents.