Seasonal dynamics of antibiotic-resistantEnterobacteriaceae in the gastrointestinal tract of domestic sheep

Considerable variation in counts of antibiotic-resistant enterobacteria in the ovine gastrointestinal tract was observed. The occurrence of ruminal and fecal isolates resistant to ampicillin (Ap), kanamycin (Km) and tetracycline (Tc) culminated in summer months, followed by rapid decline in subsequent months. Using PCR the tem1bla (Apr), aphA1 (Kmr) and tetB (Tcr) genes were found to be predominant. Under in vitro conditions all resistance genes were transferable into laboratory Escherichia coli strain with relatively high frequency (10(-3) transconjugants per recipient).     

Increased frequency of drug-resistant bacteria and fecal coliforms in an Indiana Creek adjacent to farmland amended with treated sludge

Many studies indicate the presence of human pathogens and drug-resistant bacteria in treated sewage sludge. Since one of the main methods of treated sewage disposal is by application to agricultural land, the presence of these organisms is of concern to human health. The goal of this study was to determine whether the frequency of drug-resistant and indicator bacteria in Sugar Creek, which is used for recreational purposes, was influenced by proximity to a farmland routinely amended with treated sludge (site E). Surface water from 3 sites along Sugar Creek (site E, 1 upstream site (site C) and 1 downstream site (site K)) were tested for the presence of ampicillin-resistant (Amp(R)) bacteria, fecal and total coliforms over a period of 40 d. Site E consistently had higher frequencies of Amp(R) bacteria and fecal coliforms compared with the other 2 sites. All of the tested Amp(R) isolates were resistant to at least 1 other antibiotic. However, no isolate was resistant to more than 4 classes of antimicrobials. These results suggest that surface runoff from the farmland is strongly correlated with higher incidence of Amp(R) and fecal coliforms at site E.     

Molecular epidemiology of antimicrobial-resistant commensal Escherichia coli strains in a cohort of newborn calves

Pulsed-field gel electrophoresis (PFGE) was used to investigate the dissemination and diversity of ampicillin-resistant (Amp(r)) and nalidixic acid-resistant (Nal(r)) commensal Escherichia coli strains in a cohort of 48 newborn calves. Calves were sampled weekly from birth for up to 21 weeks and a single resistant isolate selected from positive samples for genotyping and further phenotypic characterization. The Amp(r) population showed the greatest diversity, with a total of 56 different genotype patterns identified, of which 5 predominated, while the Nal(r) population appeared to be largely clonal, with over 97% of isolates belonging to just two different PFGE patterns. Distinct temporal trends were identified in the distribution of several Amp(r) genotypes across the cohort, with certain patterns predominating at different points in the study. Cumulative recognition of new Amp(r) genotypes within the cohort was biphasic, with a turning point coinciding with the housing of the cohort midway through the study, suggesting that colonizing strains were from an environmental source on the farm. Multiply resistant isolates dominated the collection, with >95% of isolates showing resistance to at least two additional antimicrobials. Carriage of resistance to streptomycin, sulfamethoxazole, and tetracycline was the most common combination, found across several different genotypes, suggesting the possible spread of a common resistance element across multiple strains. The proportion of Amp(r) isolates carrying sulfamethoxazole resistance increased significantly over the study period (P < 0.05), coinciding with a decline in the most common genotype pattern. These data indicate that calves were colonized by a succession of multiply resistant strains, with a probable environmental source, that disseminated through the cohort over time.     

Age-related decline in carriage of ampicillin-resistant Escherichia coli in young calves

The presence of ampicillin-resistant Escherichia coli (Amp(r) E. coli) in the fecal flora of calves was monitored on a monthly basis in seven cohorts of calves. Calves were rapidly colonized by Amp(r) E. coli, with peak prevalence in cohort calves observed in the 4 months after the calves were born. The prevalence of calves yielding Amp(r) E. coli in cohorts consistently declined to low levels with increasing age of the calves (P < 0.001).     

Use of antibiotic resistance analysis to identify nonpoint sources of fecal pollution.

A study was conducted to determine the reliability and repeatability of antibiotic resistance analysis as a method of identifying the sources of fecal pollution in surface water and groundwater. Four large sets of isolates of fecal streptococci (from 2,635 to 5,990 isolates per set) were obtained from 236 samples of human sewage and septage, cattle and poultry feces, and pristine waters. The patterns of resistance of the isolates to each of four concentrations of up to nine antibiotics were analyzed by discriminant analysis. When isolates were classified individually, the average rate of correct classification (ARCC) into four possible types (human, cattle, poultry, and wild) ranged from 64 to 78%. When the resistance patterns of all isolates from each sample were averaged and the resulting sample-level resistance patterns were classified, the ARCCs were much higher (96 to 100%). These data confirm that there are measurable and consistent differences in the antibiotic resistance patterns of fecal streptococci isolated from various sources of fecal pollution and that antibiotic resistance analysis can be used to classify and identify these sources.     

The prevalence of Salmonella spp. in bovine faecal,rumen and carcass samples at a commercial abattoir

AIMS: To determine the prevalence, serotype and antibiotic resistance profile of Salmonella isolates in cattle and on carcasses at a commercial Irish abattoir. METHODS AND RESULTS: Faecal, rumen and carcass samples were collected from a beef abattoir over a 12-month period and examined for the presence of Salmonella spp. Isolates were serotyped, phage typed (when serotype was found to be S. Typhimurium) and tested for susceptibility to a panel of antibiotics. Salmonella was isolated from 2% of faecal, 2% of rumen and 7.6% of carcass samples. Salmonella was most frequently isolated from samples taken during the period August to October. S. Dublin was isolated from 72% of positive samples. S. Agona and S. Typhimurium definitive type (DT)104 were each isolated from 14% of positive samples. All S. Typhimurium DT104 isolates were resistant to ampicillin, chloramphenicol, streptomycin, sulphafurazole and tetracycline (ACSSuT). On occasion, from a single animal, the same serotype was isolated from more than one sample (i.e. faeces and rumen; faeces and carcass; rumen and carcass; faeces, rumen and carcass). CONCLUSIONS: Salmonella is present in cattle at slaughter and on beef carcasses at an Irish abattoir, with a higher frequency of occurrence during the period August to October. Most isolates from the study are not commonly associated with human clinical infection, with the exception of S. Typhimurium DT104 (R-type ACSSuT). SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides epidemiological data that is necessary for the understanding of beef as a source of human Salmonella infection.     

Antimicrobial resistance in generic Escherichia coli isolates from wild small mammals living in swine farm, residential, landfill, and natural environments in southern Ontario, Canada

To assess the impacts of different types of human activity on the development of resistant bacteria in the feces of wild small mammals, we compared the prevalences and patterns of antimicrobial resistance and resistance genes in generic Escherichia coli and Salmonella enterica isolates from fecal samples collected from wild small mammals living in four environments: swine farms, residential areas, landfills, and natural habitats. Resistance to antimicrobials was observed in E. coli isolates from animals in all environments: 25/52 (48%) animals trapped at swine farms, 6/69 (9%) animals trapped in residential areas, 3/20 (15%) animals trapped at landfills, and 1/22 (5%) animals trapped in natural habitats. Animals trapped on farms were significantly more likely to carry E. coli isolates with resistance to tetracycline, ampicillin, sulfisoxazole, and streptomycin than animals trapped in residential areas. The resistance genes sul2, aadA, and tet(A) were significantly more likely to be detected in E. coli isolates from animals trapped on farms than from those trapped in residential areas. Three S. enterica serotypes (Give, Typhimurium, and Newport) were recovered from the feces of 4/302 (1%) wild small mammals. All Salmonella isolates were pansusceptible. Our results show that swine farm origin is significantly associated with the presence of resistant bacteria and resistance genes in wild small mammals in southern Ontario, Canada. However, resistant fecal bacteria were found in small mammals living in all environments studied, indicating that environmental exposure to antimicrobials, antimicrobial residues, resistant bacteria, or resistance genes is widespread.     

Escherichia coli K-12 mutants hyperproducing chromosomal beta-lactamase by gene repetitions.

Escherichia coli K-12 ampicillin-resistant mutants hyperproducing chromosomal beta-lactamase arose spontaneously from strains carrying ampA1 ampC(+). Such mutants were found even in a recA background. Two Amp(r)-100 strains were analyzed genetically. The Amp(r)-100 resistance level of both strains could be transduced by direct selection for ampicillin resistance. Several classes of ampicillin-resistant transductants were found that differed from one another in the beta-lactamase activity and the ampicillin resistance mediated by an ampA1 ampC(+)-carrying strain. The data suggested that beta-lactamase hyperproduction was due to repetitions of the chromosomal amp genes. The size of the repeated region was calculated from cotransduction estimates, using the formula of Wu (Genetics 54:405-410, 1966), and was found to be about 1 min in one strain and 1.5 min in the other. Second-step Amp(r)-400 mutants were isolated from an Amp(r)-100 strain. The resistance of these mutants was apparently also due to repetitions, each mediating a resistance to about 10 mug/ml. Mutants of wild-type strains that were moderately resistant to ampicillin also gave rise to intermediate-resistance classes, suggesting repetitions of the wild-type amp alleles. F' factors hyperproducing chromosomal beta-lactamase by gene repetitions were constructed. They mediated levels of ampicillin resistance comparable to that of naturally occurring resistance plasmids. The expression of beta-lactamase hyperproduction was not affected by the presence of ampA and ampC alleles in trans and did not act in trans on the other alleles.     

Metagenomic analysis of virulence-associated and antibiotic resistance genes of microbes in rumen of Indian buffalo (Bubalus bubalis)

A major research goal in rumen microbial ecology is to understand the relationship between community composition and its function, particularly involved in fermentation process is of a potential interest. The buffalo rumen microbiota impacts human food safety as well as animal health. Although the bacteria of bovine rumen have been well characterized, techniques have been lacking to correlate total community structure with gene function. We applied 454 next generations sequencing technology to characterize general microbial diversity present in buffalo rumen metagenome and also identified the repertoire of microbial genes present, including genes associated with antibiotic resistance and bacterial virulence. Results suggest that over six percent (6.44%) of the sequences from our buffalo rumen pool sample could be categorized as virulence genes and genes associated with resistance to antibiotic and toxic compounds (RATC), which is a higher proportion of virulence genes reported from metagenome samples of chicken cecum (5.39%), cow rumen (4.43%) and Sargasso sea (2.95%). However, it was lower than the proportion found in cow milk (11.33%) cattle faeces (8.4%), Antarctic marine derived lake (8.45%), human fecal (7.7%) and farm soil (7.79%). The dynamic nature of metagenomic data, together with the large number of RATC classes observed in samples from widely different ecologies indicates that metagenomic data can be used to track potential targets and relative amounts of antibiotic resistance genes in individual animals. In addition, these data can be also used to generate antibiotic resistance gene profiles to facilitate an understanding of the ecology of the microbial communities in each habitat as well as the epidemiology of antibiotic resistant gene transport between and among habitats.     

Patterns of antimicrobial resistance observed in Escherichia coli isolates obtained from domestic- and wild-animal fecal samples, human septage, and surface water

A repeated cross-sectional study was conducted to determine the patterns of antimicrobial resistance in 1,286 Escherichia coli strains isolated from human septage, wildlife, domestic animals, farm environments, and surface water in the Red Cedar watershed in Michigan. Isolation and identification of E. coli were done by using enrichment media, selective media, and biochemical tests. Antimicrobial susceptibility testing by the disk diffusion method was conducted for neomycin, gentamicin, streptomycin, chloramphenicol, ofloxacin, trimethoprim-sulfamethoxazole, tetracycline, ampicillin, nalidixic acid, nitrofurantoin, cephalothin, and sulfisoxazole. Resistance to at least one antimicrobial agent was demonstrated in isolates from livestock, companion animals, human septage, wildlife, and surface water. In general, E. coli isolates from domestic species showed resistance to the largest number of antimicrobial agents compared to isolates from human septage, wildlife, and surface water. The agents to which resistance was demonstrated most frequently were tetracycline, cephalothin, sulfisoxazole, and streptomycin. There were similarities in the patterns of resistance in fecal samples and farm environment samples by animal, and the levels of cephalothin-resistant isolates were higher in farm environment samples than in fecal samples. Multidrug resistance was seen in a variety of sources, and the highest levels of multidrug-resistant E. coli were observed for swine fecal samples. The fact that water sample isolates were resistant only to cephalothin may suggest that the resistance patterns for farm environment samples may be more representative of the risk of contamination of surface waters with antimicrobial agent-resistant bacteria.     

Patterns of Antimicrobial Resistance Observed in Escherichia coli Isolates Obtained from Domestic- and Wild-Animal Fecal Samples, Human Septage, and Surface Water

A repeated cross-sectional study was conducted to determine the patterns of antimicrobial resistance in 1,286 Escherichia coli strains isolated from human septage, wildlife, domestic animals, farm environments, and surface water in the Red Cedar watershed in Michigan. Isolation and identification of E. coli were done by using enrichment media, selective media, and biochemical tests. Antimicrobial susceptibility testing by the disk diffusion method was conducted for neomycin, gentamicin, streptomycin, chloramphenicol, ofloxacin, trimethoprim-sulfamethoxazole, tetracycline, ampicillin, nalidixic acid, nitrofurantoin, cephalothin, and sulfisoxazole. Resistance to at least one antimicrobial agent was demonstrated in isolates from livestock, companion animals, human septage, wildlife, and surface water. In general, E. coli isolates from domestic species showed resistance to the largest number of antimicrobial agents compared to isolates from human septage, wildlife, and surface water. The agents to which resistance was demonstrated most frequently were tetracycline, cephalothin, sulfisoxazole, and streptomycin. There were similarities in the patterns of resistance in fecal samples and farm environment samples by animal, and the levels of cephalothin-resistant isolates were higher in farm environment samples than in fecal samples. Multidrug resistance was seen in a variety of sources, and the highest levels of multidrug-resistant E. coli were observed for swine fecal samples. The fact that water sample isolates were resistant only to cephalothin may suggest that the resistance patterns for farm environment samples may be more representative of the risk of contamination of surface waters with antimicrobial agent-resistant bacteria.     

An investigation of the relationship between fecal and rumen bacterial concentrations in sheep

With no acceptable method for collecting fresh rumen fluid from zoo ruminants, it was proposed that fecal bacterial concentrations may be correlated with rumen bacteria. If so, fecal bacterial concentrations could be used to study both the effects of diet on rumen bacteria as well as rumen abnormalities. Total and cellulolytic bacterial concentrations were determined in whole rumen contents and feces of sheep using a most‐probable‐number (MPN) assay. In a Latin square design, four crossbred ewes were fed diets of 100% long or chopped orchardgrass hay (OH) and 60% ground or whole shelled corn plus 40% chopped OH. In a second trial, the sheep were fed a pelleted complete feed at varying levels of intake i.e., control at 2.0% of body weight and at 1.8, 1.6, and 1.2% of body weight. Higher total rumen bacterial concentrations (P<0.01) were found on the high concentrate diets as compared with the high forage diets. Grinding the corn also increased total bacterial concentrations (P<0.05). Fecal concentrations of total bacteria were higher (P<0.01) with the high concentrate diets. Chopping the forage decreased the concentration of fecal cellulolytic bacteria (P<0.05) but had no effect on their concentration in the rumen. An inverse linear relationship (P<0.01) was observed between total bacterial concentrations in the feces and diet intake. Although relationships were observed between the rumen and feces for total and cellulolytic bacterial concentrations, they were dependent on diet, particle size, and level of intake. Thus, fecal bacterial concentrations cannot be used to reliably predict rumen bacterial concentrations. Zoo Biol 27:100–108, 2008. © 2008 Wiley‐Liss, Inc.     

Antibiotic resistance of gram-negative enteric bacteria from pigs in three herds with different histories of antibiotic exposure

The antibiotic resistance patterns of gram-negative fecal bacteria from pigs in three herds with different histories of antibiotic exposure were examined. In general, smaller proportions of antibiotic-resistant or multiply resistant fecal isolates (P less than 0.05) were obtained from pigs in a herd not exposed to antimicrobial agents for 154 months than from pigs in a herd continuously exposed to antimicrobial agents at subtherapeutic doses or from pigs in a herd exposed only to therapeutic doses of antimicrobial agents. The proportions of antibiotic-resistant and multiply resistant strains were greater among isolates from pigs in the therapeutic herd than in the non-antibiotic-exposed herd (P less than 0.05). The proportion of antibiotic-resistant isolates in the non-lactose-fermenting population was greater than that in the lactose-fermenting population, regardless of herd. The results suggest that any form of antimicrobial exposure will increase the prevalence of antimicrobial resistance and multiple resistance of fecal bacteria.     

Antibiotic resistance of gram-negative enteric bacteria from pigs in three herds with different histories of antibiotic exposure.

The antibiotic resistance patterns of gram-negative fecal bacteria from pigs in three herds with different histories of antibiotic exposure were examined. In general, smaller proportions of antibiotic-resistant or multiply resistant fecal isolates (P less than 0.05) were obtained from pigs in a herd not exposed to antimicrobial agents for 154 months than from pigs in a herd continuously exposed to antimicrobial agents at subtherapeutic doses or from pigs in a herd exposed only to therapeutic doses of antimicrobial agents. The proportions of antibiotic-resistant and multiply resistant strains were greater among isolates from pigs in the therapeutic herd than in the non-antibiotic-exposed herd (P less than 0.05). The proportion of antibiotic-resistant isolates in the non-lactose-fermenting population was greater than that in the lactose-fermenting population, regardless of herd. The results suggest that any form of antimicrobial exposure will increase the prevalence of antimicrobial resistance and multiple resistance of fecal bacteria.     

Serotypes,Virulence Factors,and Antimicrobial Susceptibilities of Vaginal and Fecal Isolates of Escherichia coli from Giant Pandas

Although Escherichia coli typically colonizes the intestinal tract and vagina of giant pandas, it has caused enteric and systemic disease in giant pandas and greatly impacts the health and survival of this endangered species. In order to understand the distribution and characteristics of E. coli from giant pandas, 67 fecal and 30 vaginal E. coli isolates from 21 giant pandas were characterized for O serogroups, phylogenetic groups, antimicrobial susceptibilities, and pulsed-field gel electrophoresis (PFGE) profiles. In addition, these isolates were tested for the presence of extraintestinal pathogenic E. coli (ExPEC) and diarrheagenic E. coli (DEC) by multiplex PCR detection of specific virulence genes. The most prevalent serogroups for all E. coli isolates were O88, O18, O167, O4, and O158. ExPEC isolates were detected mostly in vaginal samples, and DEC isolates were detected only in fecal samples. Phylogenetic group B1 predominated in fecal isolates, while groups B2 and D were frequently detected in vaginal isolates. Resistance to trimethoprim-sulfamethoxazole was most frequently observed, followed by resistance to nalidixic acid and tetracycline. All except five isolates were typeable by using XbaI and were categorized into 74 PFGE patterns. Our findings indicate that panda E. coli isolates exhibited antimicrobial resistance, and potentially pathogenic E. coli isolates were present in giant pandas. In addition, these E. coli isolates were genetically diverse. This study may provide helpful information for developing strategies in the future to control E. coli infections of giant pandas.     

临床分离的变形杆菌中整合子分型研究

目的了解临床分离变形杆菌中1、2类整合子的流行现状及其耐药性。方法PCR扩增146株变形杆菌中1、2类整合酶基因,M-H肉汤稀释法检测146株变形杆菌的药敏情况。结果1类整合子检出率为36.9%(54/146),2类整合子检出率为38.4%(56/146),同时携带1、2类整合子的检出率为18.5%(27/146)。结论1、2类整合子在变形杆菌感染临床分离株中所占比例已相当高,1、2类整合子与变形杆菌的多重耐药具有相关性。     

Determining sources of fecal pollution in a rural Virginia watershed with antibiotic resistance patterns in fecal streptococci

Nonpoint sources of pollution that contribute fecal bacteria to surface waters have proven difficult to identify. Knowledge of pollution sources could aid in restoration of the water quality, reduce the amounts of nutrients leaving watersheds, and reduce the danger of infectious disease resulting from exposure to contaminated waters. Patterns of antibiotic resistance in fecal streptococci were analyzed by discriminant and cluster analysis and used to identify sources of fecal pollution in a rural Virginia watershed. A database consisting of patterns from 7,058 fecal streptococcus isolates was first established from known human, livestock, and wildlife sources in Montgomery County, Va. Correct fecal streptococcus source identification averaged 87% for the entire database and ranged from 84% for deer isolates to 93% for human isolates. To field test the method and the database, a watershed improvement project (Page Brook) in Clarke County, Va., was initiated in 1996. Comparison of 892 known-source isolates from that watershed against the database resulted in an average correct classification rate of 88%. Combining all animal isolates increased correct classification rates to > or = 95% for separations between animal and human sources. Stream samples from three collection sites were highly contaminated, and fecal streptococci from these sites were classified as being predominantly from cattle (>78% of isolates), with small proportions from waterfowl, deer, and unidentified sources ( approximately 7% each). Based on these results, cattle access to the stream was restricted by installation of fencing and in-pasture watering stations. Fecal coliforms were reduced at the three sites by an average of 94%, from prefencing average populations of 15,900 per 100 ml to postfencing average populations of 960 per 100 ml. After fencing, <45% of fecal streptococcus isolates were classified as being from cattle. These results demonstrate that antibiotic resistance profiles in fecal streptococci can be used to reliably determine sources of fecal pollution, and water quality improvements can occur when efforts to address the identified sources are made.     

Considerations when using discriminant function analysis of antimicrobial resistance profiles to identify sources of fecal contamination of surface water in Michigan

The goals of this study were to (i) identify issues that affect the ability of discriminant function analysis (DA) of antimicrobial resistance profiles to differentiate sources of fecal contamination, (ii) test the accuracy of DA from a known-source library of fecal Escherichia coli isolates with isolates from environmental samples, and (iii) apply this DA to classify E. coli from surface water. A repeated cross-sectional study was used to collect fecal and environmental samples from Michigan livestock, wild geese, and surface water for bacterial isolation, identification, and antimicrobial susceptibility testing using disk diffusion for 12 agents chosen for their importance in treating E. coli infections or for their use as animal feed additives. Nonparametric DA was used to classify E. coli by source species individually and by groups according to antimicrobial exposure. A modified backwards model-building approach was applied to create the best decision rules for isolate differentiation with the smallest number of antimicrobial agents. Decision rules were generated from fecal isolates and applied to environmental isolates to determine the effectiveness of DA for identifying sources of contamination. Principal component analysis was applied to describe differences in resistance patterns between species groups. The average rate of correct classification by DA was improved by reducing the numbers of species classifications and antimicrobial agents. DA was able to correctly classify environmental isolates when fewer than four classifications were used. Water sample isolates were classified by livestock type. An evaluation of the performance of DA must take into consideration relative contributions of random chance and the true discriminatory power of the decision rules.     

Isolation and identification of fecal bacteria from adult swine.

An examination of the fecal microflora of adult swine was made with regard to the efficiency of several roll tube media in enumeration and recovery of anaerobes, the effects of medium constituents on recovery, and the isolation and identification of the predominant kinds of bacteria. Total number of organisms by microscopic bacterial counts varied among fecal samples from 4.48 X 10(10) to 7.40 X 10(10) bacteria/g (wet weight). Comparison of different nonselective roll tube media indicated that about 30% of the fecal bacteria could be recovered with a rumen fluid (40%, vol/vol) medium (M98-5). Recoveries of 21 and 15%, respectively, were obtained with M10 and rumen fluid-glucose-cellobiose agar (RGCA) media. Rumen fluid, Trypticase, sugars, and CO2 gas phase were important components required for maximum recovery with this medium. Similar high recoveries of anaerobes were also obtained with M98-5 containing swine cecal extract of place in rumen fluid or M10 plus swine cecal extract. Significantly lower recoveries were observed with RCGA, media supplemented with swine fecal extracts, reinforced clostridial medium, brain heart infusion agar, and prereduced blood agar. Ninety percent of the bacteria isolated from roll tube media were gram positive and consisted of facultatively anaerobic streptococci, Eubacterium sp., Clostridium sp., and Propionibacterium acnes. The remainder of the flora (8%) included several other species of anaerobes and Escherichia coli. Rumen fluid (or volatile fatty acids), Trypticase, and yeast extract additions to basal media stimulated the growth of anaerobic strains. Variation in the relative proportions of the predominant fecal microflora was observed. This work indicates that satisfactory enumeration, isolation and cultivation of the predominant microflora in swine feces can be obtained when strict anaerobic culture methods and a rumen fluid medium are used.     

Isozyme and morphological characteristics of the anaerobic fungus Piromyces mae isolated from the duodenum, rumen and faeces of sheep

Abstract Isolates of anaerobic fungi obtained from the rumen, duodenum and faeces of sheep were identified as Piromyces mae based on their morphological characteristics observed using light microscopy. There was no significant morphological variation among the isolates of P. mae from the rumen, duodenum and faeces. Isozymes of 12 isolates of P. mae (one each from the rumen, duodenum and faeces from 4 different sheep) were analysed by PAGE. A total of 12 isozymes were studied and 5 isozyme loci were successfully typed. They were malic enzyme, malate dehydrogenase, shikimate dehydrogenase, α-esterase and β-esterase. All the isolates of P. mae regardless of whether they were from the rumen, duodenum or faeces or from different animals produced very similar isozyme banding patterns for each of the enzyme systems. The similar isozyme profiles of the isolates indicate that they are of the same species although they exist in different regions of the alimentary tract.