Clostridium difficile in young farm animals and slaughter animals in Belgium

Faecal carriage of Clostridium difficile in healthy animals has been reported recently, especially in piglets and calves. However there is limited data about carriage in animals just prior to slaughter in Europe. The main objective of this study was to determine the presence of C. difficile in pigs and cattle at the slaughterhouse. C. difficile was isolated in 6.9% of the cattle at the slaughterhouse. None of the pig slaughter samples were positive for C. difficile after an enrichment time of 72 h. For complementary data, a short study was conducted in piglets and calves at farms. C. difficile was more prevalent in piglets (78.3%) than in calves (22.2%) on the farms. Regarding the piglet samples, 27.8% of the positive samples were detected without enrichment of stools. The PCR ribotype 078 was predominant in farm animals. Samples isolated from slaughter cattle presented the widest range in PCR-ribotype variety, and the most prevalent PCR ribotype was 118a UCL. The results of this study confirm that C. difficile is present in slaughter animals in Belgium with a large percentage of toxigenic strains also commonly found in humans.     

Diversity and prevalence of Arcobacter spp. in broiler chickens

Ninety-nine strains of Arcobacter spp., isolated from 10 chicken carcasses purchased from a supermarket and 15 chicken carcasses collected from a poultry abattoir, were speciated using a variety of phenotypic identification methods. All were tested using API Campy test strips and the 16-test (Preston) identification scheme developed for campylobacters. Fifty strains were selected for examination using a more comprehensive probabilistic identification scheme, and the identity of representative strains confirmed by protein profiling using SDS-PAGE. All 25 carcasses yielded Arcobacter butzleri . Three supermarket and 10 abattoir carcasses also carried A. cryaerophilus , and two abattoir carcasses carried A. skirrowii . The API Campy scheme proved unsatisfactory for identifying these strains: only 20 of 99 strains were accurately identified, all of which were A. cryaerophilus , the only Arcobacter sp. included in the database. Moreover, 76 of 99 strains were misidentified. The 16-test scheme identified all the arcobacter strains as A. cryaerophilus, since neither A. butzleri nor A. skirrowii had been described when the scheme was developed. The computer-assisted probabilistic scheme succeeded in identifying all but one strain, the identity of which was clarified by the use of SDS-PAGE. To our knowledge this is the first time that arcobacters other than A. butzleri have been reported in poultry meat or any other food of animal origin. Their high prevalence in poultry products may be of significance to public health.     

Concentration and prevalence of Escherichia coli O157 in cattle feces at slaughter

The concentration and prevalence of Escherichia coli O157 in cattle feces at the time of slaughter was studied over a 9-week period from May to July 2002. Fecal samples (n = 589) were collected from the rectums of slaughtered cattle, and the animal-level prevalence rate was estimated to be 7.5% (95% confidence interval [CI], 5.4 to 9.6%) while the group prevalence was 40.4% (95% CI, 27.7 to 53.2%). Of the 44 infected animals detected, 9% were high shedders that contained E. coli O157 at concentrations of >10(4) CFU g(-1). These 9% represented >96% of the total E. coli O157 produced by all animals tested. All isolates possessed the vt(2) gene, 39 had the eaeA gene, and a further five had the vt(1) gene also. The presence of high-shedding animals at the abattoir increases the potential risk of meat contamination during the slaughtering process and stresses the need for correctly implemented hazard analysis and critical control point procedures.     

Ecology of Arcobacter species in chicken rearing and processing

AIMS: To investigate whether Arcobacter spp. colonize the poultry-rearing environment or whether they are contaminants acquired during transportation and/or from the processing plant. METHODS AND RESULTS: Samples were collected on poultry farms and in the processing plant during slaughter and dressing. Two cultural methods of detection were used. Isolates were identified to species level using a multiplex-polymerase chain reaction (m-PCR) method, either on the initial suspensions, or after enrichment, or on pure cultures of isolates. Of the 62 samples examined from poultry farms, arcobacters were found only outside the rearing sheds (in effluent sludge and stagnant water). Thirty-four samples were examined from the processing plant and 26 were positive for arcobacters. All the isolates were Arcobacter butzleri. Arcobacters were not found in any sample by direct plating nor by m-PCR on the initial suspensions, thus it was concluded that numbers were very low. CONCLUSIONS: Arcobacter spp. were not found in samples from the live birds and their immediate environment, but A. butzleri was found in effluent sludge and stagnant water outside the rearing sheds. However, A. butzleri is common in poultry abattoirs, and it appears that poultry carcasses are contaminated during processing. SIGNIFICANCE AND IMPACT OF THE STUDY: Arcobacters are not found inside poultry-rearing sheds, but are contaminants in the processing environment.     

Diversity of Campylobacter coli genotypes in the lower porcine gastrointestinal tract at time of slaughter

AIMS: To compare the genotypes of Campylobacter coli obtained from the rectal and ileal samples of pigs at the time of slaughter. METHODS AND RESULTS: Five animals were sampled following slaughter with ileal contents and anal swabs being taken post-evisceration. Swabs were directly plated onto charcoal cefoperazone desoxycholate agar (CCDA) while ileal contents were enriched in CCDA broth. Twenty isolates were picked from each site sampled and all 200 isolates were Camp. coli. Isolates were genotyped using random amplified polymorphic DNA (22 discrete types) and flaA (11 discrete types). Both methods found that 55% of the genotypes were unique to rectal samples. Only one animal yielded the same flaA type from ileal and rectal samples. CONCLUSIONS: Rectal sampling of pigs yielded a more diverse subset of Camp. coli genotypes than ileal contents, but failed to yield all of the genotypes carried by an individual animal. SIGNIFICANCE AND IMPACT OF THE STUDY: A small sample of pigs carried a very diverse population of Camp. coli genotypes; and sampling of a single site in the gut will recover only part of this population. Hence, any genotyping studies of Camp. coli in pigs must be interpreted with caution, and epidemiological studies could be confounded by the number of Camp. coli genotypes available.     

Antibiotic resistance and virulence of faecal enterococci isolated from food-producing animals in Tunisia

Antimicrobial agents exert a selection pressure not only on pathogenic, but also on commensal bacteria of the intestinal tract of humans and animals. The aim of this work was to determine the occurrence of different enterococcal species and to analyse the prevalence of antimicrobial resistance and the mechanisms implicated, as well as the genetic diversity in enterococci recovered from faecal samples of food-producing animals (poultry, beef and sheep) in Tunisia. Antimicrobial resistance and the mechanisms implicated were studied in 87 enterococci recovered from 96 faecal samples from animals of Tunisian farms. Enterococcus faecium was the most prevalent species detected (46 %), followed by E. hirae (33.5 %). High percentages of resistance to erythromycin and tetracycline were found among our isolates, and lower percentages to aminoglycosides and ciprofloxacin were identified. Most of the tetracycline-resistant isolates carried the tet(M) and/or tet(L) genes. The erm(B) gene was detected in all erythromycin-resistant isolates. The ant(6)-Ia, aph(3′)-Ia and aac(6′)-aph(2″) genes were detected in nine aminoglycoside-resistant isolates. Of our isolates, 11.5 % carried the gelE gene and exhibited gelatinase acitivity. The esp gene was detected in 10 % of our isolates and the hyl gene was not present in any isolate. The predominant species (E. faecium and E. hirae) showed a high genetic diversity by repetitive extragenic palindromic (REP)-PCR. Food animals might play a role in the spread through the food chain of enterococci with virulence and resistance traits to humans.     

Integron-containing bacteria in faeces of cattle from different production systems at slaughter

Aims:  To determine the prevalence and characteristics of integron-containing bacteria in faeces of cattle from grass-fed, lot-fed, or organically produced cattle.
Methods and Results:  Faecal samples from grass-fed ( n  =   125), lot-fed ( n  =   125) and organic ( n  =   135) cattle were tested for the presence of class 1 and class 2 integrons by using PCR and colony hybridisation. The prevalence of class 1 and class 2 integrase were higher in lot-fed cattle (71% and 62%) than grass-fed cattle (52% and 30%) which in turn were higher than organic cattle (25% and 11%). Isolation rates of integron-containing bacteria were reflective of PCR prevalence results.
Conclusions:  The antimicrobial resistance genes harboured by the integrons differed little across the three systems and were typically to antimicrobials that would rarely be used therapeutically or for growth promotion purposes. The differences in prevalence observed between the systems may be a function of the intensiveness of each system.
Significance and Impact of the Study:  Integron-containing bacteria may be present in all cattle production systems regardless of the amount of antimicrobial use and confirms that the prudent use of antimicrobials is required so that the development of integrons harbouring genes significant to human medicine is avoided.     

Diversity of serotypes of Campylobacter jejuni and Campylobacter coli isolated in laboratory animals

One hundred nineteen isolates of Campylobacter jejuni and Campylobacter coli from nine laboratory animal species were serotyped using antisera to 20 Penner serotypes commonly isolated from cases of human enteric infections. Although C. jejuni and C. coli were isolated from laboratory animals with diarrhea, the majority were cultured from asymptomatic animals (81%). Seven of twenty-two isolates from animals with diarrhea were serotype 4 (32%) and three were serotype 1 (14%). Sixty-one of the 119 isolates (51%) were typeable using the 20 Penner antisera indicating that many of the isolates obtained from 29 nonhuman primates (five species), 20 ferrets, 7 hamsters, 15 cats and 48 dogs are serotypes commonly associated with human enteritis. Among typeable strains, 13 different serotypes were identified. Two particular serotypes, 4 and 19 were isolated from several species of animals and comprised 24% of the isolates studied. Since asymptomatic laboratory animals of several different species harbor serotypes of C. jejuni and C. coli that are potentially pathogenic to man, appropriate precautions should be instituted to minimize exposure of personnel to the organisms in laboratory animal feces. If suspected cases of zoonotic-related enteric campylobacteriosis involving laboratory animals do occur, serotyping of isolates would be a useful epidemiologic marker in studying the outbreak.     

Campylobacter jejuni/coli in commercially reared beagles: prevalence and serotypes

A 5 year longitudinal study involving 187 commercially reared beagles from three suppliers was undertaken to determine prevalence and serotypes of Campylobacter jejuni and C. coli. Campylobacter jejuni or C. coli was isolated from the feces in 62 of 177 asymptomatic beagles and 8 of 10 dogs with diarrhea for an overall prevalence of 37%. A total of 36 isolates were serotyped on the basis of thermostable antigens with 20 antisera prepared against frequently occurring serotypes isolated from humans with campylobacter associated enteritis (15 C. jejuni, 5 C. coli serotypes). Of these isolates, 17 (47%) serotyped with antisera to 7 C. jejuni serotypes frequently isolated in human cases of enteric campylobacteriosis (serotypes 1, 4, 10, 16, 18, 19, 37). One C. coli reacted to antisera 24, 34, 37, one strain of C. coli to antisera type 37, and another C. coli to antisera type 34. All three C. coli belonged to serotypes frequently encountered in diarrheic human patients.     

Incidence and ecology of Campylobacter jejuni and coli in animals

Since its initial emergence in the 1970s, Campylobacter has become one of the most common causative agents of bacterial foodborne illness. Campylobacter species readily colonize the gastrointestinal tracts of domestic, feral and wild animals and while they rarely cause clinical disease in food animals, they can produce severe acute gastroenteritis in humans. Prevalence of Campylobacter in food animals can exceed 80% thus challenging processors to employ post-harvest pathogen reduction strategies. Reduction of pathogens before arrival to the abattoir is also of interest because the implementation of pre-harvest interventions may compliment existing post-harvest control techniques to further diminish possible retail sources of infection. Such multiple hurdle approaches that simultaneously utilize pre- and post-harvest control techniques are expected to be the most effective approach for decreasing human illness associated with foodborne pathogens.     

Metronidazole resistance and microaerophily in Campylobacter species

Metronidazole is active against most anaerobic organisms and is also used in the treatment of the microaerophilic bacterium Helicobacter pylori. Resistance to metronidazole is uncommon in most anaerobic organisms, but it is increasingly prevalent in H. pylori. Previously we have suggested that metronidazole resistance in H. pylori is inherent in the microaerophilic nature of the organism and therefore would be present in other microaerophiles such as Campylobacter. Short periods of anaerobiosis caused metronidazole-resistant (Mtr^R) strains of Campylobacter spp. to become sensitive to metronidazole. Under microaerophilic conditions, cultures of the Mtr^R mutant Campylobacter coli R1 at bacterial cell densities of greater than 10⁸ cfu/ml lost viability, whereas no loss in viability was observed in cultures at cell densities of less than 10⁸. The Mtr^S C. coli strain lost viability at all cell densities. Comparisons of NAD(P)H oxidase activity between MtrS and Mtr^R strains indicated that the Mtr^S C. coli strain contained fourfold higher NADH oxidase activity and twofold higher NADPH oxidase activity than did the Mtr^R Campylobacter strains. These results show that Mtr^R Campylobacter spp. display resistance characteristics similar to those of H. pylori, suggesting that the resistance mechanism is a phenomenon of the microaerophilic nature of these bacteria. Received: 9 March 1998 / Accepted: 17 June 1998     

Bovine renal lipofuscinosis: Prevalence, genetics and impact on milk production and weight at slaughter in Danish cattle

Background

Bovine renal lipofuscinosis (BRL) is an incidental finding in cattle at slaughter. Condemnation of the kidneys as unfit for human consumption was until recently considered the only implication of BRL. Recent studies have indicated a negative influence on the health of affected animals. The present study investigated the prevalence, genetics and effect of BRL on milk yield and weight at slaughter.

Methods

BRL status of slaughter cattle was recorded at four abattoirs during a 2-year-period. Data regarding breed, age, genetic descent, milk yield and weight at slaughter were extracted from the Danish Cattle Database. The prevalence of BRL was estimated stratified by breed and age-group. Furthermore, total milk yield, milk yield in last full lactation and weight at slaughter were compared for BRL-affected and non-affected Danish Holsteins and Danish Red cattle.

Results

433,759 bovines were slaughtered and 787 of these had BRL. BRL was mainly diagnosed in Danish Red, Danish Holstein and crossbreds. The age of BRL affected animals varied from 11 months to 13 years, but BRL was rarely diagnosed in cattle less than 2 years of age. The total lifelong energy corrected milk (ECM) yields were 3,136 and 4,083 kg higher for BRL affected Danish Red and Danish Holsteins, respectively. However, the median life span of affected animals was 4.9 months longer, and age-corrected total milk yield was 1,284 kg lower for BRL affected Danish Red cows. These cows produced 318 kg ECM less in their last full lactation. Weight at slaughter was not affected by BRL status. The cases occurred in patterns consistent with autosomal recessive inheritance and several family clusters of BRL were found. Analysis of segregation ratios demonstrated the expected ratio for Danish Red cattle, but not for Danish Holsteins.

Conclusion

The study confirmed that BRL is a common finding in Danish Holsteins and Danish Red cattle at slaughter. The disorder is associated with increased total milk yield due to a longer production life. However, a reduced milk yield was detected in the end of the production life in Danish Red. The study supports that BRL is inherited autosomal recessively in the Danish Red breed and Danish Holsteins, but with incomplete penetrance of the genotype in Danish Holsteins.     

Occurrence,Diversity, and Host Association of Intestinal Campylobacter,Arcobacter, and Helicobacter in Reptiles

Campylobacter, Arcobacter, and Helicobacter species have been isolated from many vertebrate hosts, including birds, mammals, and reptiles. Multiple studies have focused on the prevalence of these Epsilonproteobacteria genera in avian and mammalian species. However, little focus has been given to the presence within reptiles, and their potential zoonotic and pathogenic roles. In this study, occurrence, diversity, and host association of intestinal Epsilonproteobacteria were determined for a large variety of reptiles. From 2011 to 2013, 444 cloacal swabs and fecal samples originating from 417 predominantly captive-held reptiles were screened for Epsilonproteobacteria. Campylobacter, Arcobacter, and Helicobacter genus specific PCRs were performed directly on all samples. All samples were also cultured on selective media and screened for the presence of Epsilonproteobacteria. Using a tiered approach of AFLP, atpA, and 16S rRNA sequencing, 432 Epsilonproteobacteria isolates were characterized at the species level. Based on PCR, Campylobacter, Arcobacter, and Helicobacter were detected in 69.3% of the reptiles; 82.5% of the chelonians, 63.8% of the lizards, and 58.0% of the snakes were positive for one or more of these genera. Epsilonproteobacteria were isolated from 22.1% of the reptiles and were isolated most frequently from chelonians (37.0%), followed by lizards (19.6%) and snakes (3.0%). The most commonly isolated taxa were Arcobacter butzleri, Arcobacter skirrowii, reptile-associated Campylobacter fetus subsp. testudinum, and a putative novel Campylobacter taxon. Furthermore, a clade of seven related putative novel Helicobacter taxa was isolated from lizards and chelonians. This study shows that reptiles carry various intestinal Epsilonproteobacteria taxa, including several putative novel taxa.     

Specific detection of Arcobacter and Campylobacter strains in water and sewage by PCR and fluorescent in situ hybridization

The aim of this study was to evaluate PCR and fluorescent in situ hybridization (FISH) techniques for detecting Arcobacter and Campylobacter strains in river water and wastewater samples. Both 16S and 23S rRNA sequence data were used to design specific primers and oligonucleotide probes for PCR and FISH analyses, respectively. In order to assess the suitability of the methods, the assays were performed on naturally and artificially contaminated samples and compared with the isolation of cells on selective media. The detection range of PCR and FISH assays varied between 1 cell/ml (after enrichment) to 10(3) cells/ml (without enrichment). According to our results, both rRNA-based techniques have the potential to be used as quick and sensitive methods for detection of campylobacters in environmental samples.