B. China

Genome mapping of Clostridium perfringens strains with I-CeuI shows many virulence genes to be plasmid-borne

The intron-encoded endonuclease I-CeuI fromChlamydomonas eugametos was shown to cleave the circular chromosomes of allClostridium perfringens strains examined at single sites in the rRNA operons, thereby generating ten fragments suitable for the rapid mapping of virulence genes by pulsed-field gel electrophoresis (PFGE). This method easily distinguishes between plasmid and chromosomal localisations, as I-CeuI only cuts chromosomal DNA. Using this approach, the genes for three of the four typing toxins,β, ε, andı, in addition to the enterotoxin andλ-toxin genes, were shown to be plasmid-borne. In a minority of strains, associated with food poisoning, where the enterotoxin toxin gene was located on the chromosome, genes for two of the minor toxins,ϑ andμ, were missing.

Salmonella contamination of pigs and pork in an integrated pig production system.

This paper describes the monitoring of Salmonella in a closed pig production system in Belgium over a 2-year period. A sampling scheme including animal feeds and carcasses was designed to cover the entire chain of production from farrow to finishing pigs. Salmonella was detected by a method based on the use of semisolid Rappaport-Vassiliadis as a selective medium. The serotypes of the isolated strains were determined, and the antibiotic resistance of these strains to six antibiotics was also investigated. Feeds were found to be more contaminated than expected (10.2%, 34 of 332 samples). The percentage of positive fecal samples for pregnant sows (8.1%, 11 of 135 samples) was significantly higher than that for young and lactating sows (2.9%, 11 of 378 samples) (P<0.05). The percentage of positive samples for colon contents collected at the slaughterhouse (47.3%, 88 of 186 samples) was significantly higher than that for feces collected during the fattening stage (5.6%, 18 of 320 samples) (P<0.001). For carcass swab samples, the observed prevalence was 11.2% (17 of 152 samples). On farms, Salmonella recovery levels were higher for overshoe samples than for fecal samples, except for pregnant sows. Salmonella Typhimurium was the most frequently isolated serotype (32.2%, 55 of 171 samples), while Salmonella Brandenburg was predominant in the colon contents collected at the abattoir (21.4%, 18 of 84 samples). Feeds harbored a wide diversity of serotypes of minor epidemiological significance. Of 55 isolated strains of Salmonella Typhimurium, 11 (20%) were resistant to tetracycline, ampicillin, choramphenicol, streptomycin, trimethoprim, and nalidixic acid (R Type TeAmCSNa), while 12 (21.8%) were resistant to all of these antibiotics except nalidixic acid (R Type TeAmCS). The majority of Salmonella Typhimurium strains that exhibited resistance to more than four antimicrobial agents were characterized as Salmonella Typhimurium DT104 or as being closely related to Salmonella Typhimurium DT104 (7 of 12 isolates). In conclusion, our system of surveillance is effective in identifying most points of contamination in the production chain and will be useful in ongoing efforts to develop a Salmonella-free production system.

Hygiene indicator microorganisms for selected pathogens on beef, pork, and poultry meats in Belgium.

Several bacterial indicators are used to evaluate hygiene during the meat slaughtering process. The objectives of this study were to assess the Belgian baseline data on hygienic indicators and the relationship between the indicators and zoonotic agents to establish hygiene indicator criteria for cattle, pig, and chicken carcasses and meat. The study used the results from the official Belgian surveillance plan from 2000 to 2003, which included the monitoring of Escherichia coli counts (ECC), Enterobacteriaceae counts (EC), aerobic colony counts (ACC), and Pseudomonas counts (PC). The sampling method was the wet and dry swabbing technique for cattle and pig carcasses and neck skin excision for broiler and layer chicken carcasses. The 75th and 95th percentiles of ECC were -0.20 and 0.95 log CFU/cm2 for cattle carcasses, 1.20 and 2.32 log CFU/cm2 for pig carcasses, and 4.05 and 5.24 log CFU/g for chicken carcasses. The ACC were 2.1- to 4.5-log higher than the ECC for cattle, pigs, and chickens. For cattle and pig carcasses, a significant correlation between ECC, EC, and ACC was found. ECC for pork and beef samples and EC in pig carcasses were significantly higher in samples contaminated with Salmonella. In poultry samples, ECC were in general higher for samples containing Salmonella or Campylobacter. Thus, E. coli may be considered as a good indicator for enteric zoonotic agents such as Salmonella for beef, pork, and poultry samples and for Campylobacter in poultry samples.

Prevalence and molecular typing of attaching and effacing Escherichia coli among calf populations in Belgium.

Abstract Attaching and effacing Escherichia coli are involved in diarrhea in 2 to 8-week old calves. The virulence factors of these bacteria include: (i) the secretion of proteins (i.e. EspB) involved in microvilli effacement, (ii) the production of the intimin, a 94kDa outer membrane protein encoded by the eaeA gene and involved in the intimate attachment of bacteria to epithelial cell and (iii) the production of verotoxins: VT1 and/or VT2. We investigated the presence and the pathotype of these strains in several calf populations by colony hybridization or by genetic amplification. Using the colony hybridization method we showed first that only 5% of calves who died from diarrhea presented EaeA+ E. coli strains and secondly that 19% of healthy calves showed an asymptomatic carriage. However, using colony hybridization and genetic amplification, we identified EaeA+ strains in 91% of calves living in farms with recurrent diarrhea problems. In 66% of the calves, there was a correlation between the presence of AEEC and diarrhea. At the pathotype level, most of the EaeA+ isolates were negative for VT probes. In VT+ bacteria, the majority were VT1+. The number of VT positive bacteria was significantly higher in calves who died from diarrhea than in healthy or sick calves. This underlined the aggravating role of verotoxins in the disease. Moreover, only 25% of the bovine AEEC were positive with the EaeB probe. Surprisingly, the proportion of EaeB+ strains was significantly higher in healthy calves than in other populations.

Belgian surveillance plans to assess changes in Salmonella prevalence in meat at different production stages

From 1997 to 1999, the prevalence of Salmonella was assessed at different stages through the pork, poultry, and beef meat production chains. Different dilutions of the initial sample suspension were analyzed to provide a semiquantitative evaluation of Salmonella contamination and to determine the most representative dilution necessary to detect a reduction in prevalence. An average of 300 samples for each type of meat were analyzed. According to Fishers exact test, the dilution to be used to detect a reduction in prevalence was chosen based on an initial prevalence of 20 to 26%. Based on this introductory study, a new sampling plan representative of the nationwide Belgian meat production process was used from 2000 through to 2003. This study confirmed the consistently high rate and level of contamination of poultry meat: broiler and layer carcasses were the most contaminated samples followed by broiler fillets and poultry meat preparations. A constant and significant decrease in Salmonella prevalence was observed for pork carcasses, trimmings, and minced meat and for beef minced meat. Less than 3% of beef carcasses and trimming samples were positive for Salmonella. The Belgian plan, as utilized from 2000 to 2003, was suitable for monitoring of zoonoses because the sampling plan was representative of nationwide production processes, covered all periods of the year, and was executed by trained samplers and the analyses were carried out by recognized laboratories using an identical analytical method.

Heterogeneity of the eae genes in attaching/effacing Escherichia coli from cattle: comparison with human strains.

Enteropathogenic (EPEC) and enterohaemorrhagic (EHEC) Escherichia coli isolated from cattle were studied by DNA colony hybridization to subtype their intimin-encoding (eae) gene with probes derived from the variable parts of the eae alpha gene of the human EPEC strain E2348/69, the eae gamma gene of the human O157:H7 EHEC strain ATCC43888, and the eae beta gene of the bovine O26:H- EHEC strain 193, whose eae gene was first cloned and sequenced during this work. The EPEC and EHEC had been isolated from diarrhoeic calves (143 EPEC and 48 EHEC) and from healthy animals at the slaughterhouse (10 EPEC and 34 EHEC). The 191 bovine EPEC and EHEC isolated from diseased calves were positive with the Eae beta probe (55 and 27% respectively) and with the Eae gamma probe (9 and 73% respectively), whereas 52 EPEC (36%) were negative with the Eae alpha, Eae beta, and Eae gamma probes. The results were different for the 44 bovine EPEC and EHEC isolated from healthy cattle at slaughterhouses: most tested positive with the Eae gamma probe (80 and 82% respectively) and the remaining (20 and 18% respectively) with the Eae beta probe. Nine O26 human EHEC tested positive with the Eae beta probe and seven O111 with the Eae gamma probe. The bovine and human EPEC and EHEC belonging to these two serogroups gave identical results: the 18 bovine and human O26 isolates tested positive with the Eae beta probe, whereas the 13 O111 isolates were positive with the Eae gamma probe. In contrast, the isolates belonging to other serogroups (O5, O15, O18, O20, and O118) gave more variable results. The eae beta and eae gamma, but not the eae alpha, variants were thus distributed amongst bovine EPEC and EHEC. The eae beta variant seemed to be more frequently associated with the presence of clinical signs in calves, but one third of EPEC from diarrhoeic calves carried an eae gene variant other than the alpha, beta, or gamma variants. In addition, the use of these gene probes did not enable differentiation between bovine and human EHEC belonging to the same O serogroup.

Detection of Neospora caninum in dog organs using real time PCR systems

Neospora caninum is a parasite responsible for paresis in dogs. The dog can harbour enkysted parasites in several organs. The detection of N. caninum was performed using 3 different real time PCR systems all amplifying the NC5 DNA region. One system was based on Sybrgreen, one on Plexor technology and the last on Taqman probe. Comparison of the three methods indicated that the detection limit was 1 equivalent genome on pure DNA but that this detection limit increased in the presence of foreign DNA using the Sybrgreen and Plexor systems. Therefore, the Taqman system was chosen to detect N. caninum in liver and spleen of naturally infected dogs. The overall prevalence was 32.2%. Comparison between PCR results and serological results using IFAT showed that among the 28 PCR positive dogs only 9 were seropositive and that 8 seropositive dogs were PCR negative. Therefore serology can underestimate the real carriage in dogs. However, PCR methods must be improved in terms of sensitivity and inhibition problems.

Discrimination between Bifidobacterium species from human and animal origin by PCR-restriction fragment length polymorphism.

Bifidobacteria are normal intestinal flora in humans and animals. The genus Bifidobacterium includes 31 species of significant host specificity. Taking into account their properties, we proposed to use bifidobacteria as fecal contamination indicators. PCR-restriction fragment length polymorphism on the 16S rDNA gene was used to distinguish the different Bifidobacterium species. Sixty-four strains belonging to 13 different species were differentiated from animal or human origin using one or two restriction enzymes. Moreover, the primers used were specifics of the Bifidobacterium genus. Therefore, this method made it possible to determine both the presence of bifidobacteria in a sample and its origin of contamination.

Clinical signs, reproduction of attaching/effacing lesions, and enterocyte invasion after oral inoculation of an O118 enterohaemorrhagic Escherichia coli in neonatal calves.

Abstract Attaching and effacing (AE) lesions are produced among others by enteropathogenic Escherichia coli and enterohaemorrhagic E. coli (EHEC), which differs from the former by the production of cytotoxins active on various cell cultures, the verocytotoxins, or shigacytotoxins. EHEC are associated with diarrhoea and dysentery in humans and in ruminants, mainly calves from two to eight weeks of age. Clinical signs and/or lesions have been reproduced experimentally with EHEC strains belonging to serotypes O5:K4/Nm, O26:K-:H11, O111:Nm, and O157:H7 which are isolated from cattle and/or humans. The purpose of this work was to develop an experimental model of infection in newborn calves with a bovine EHEC strain isolated from a calf which of died of diarrhoea, and belonging to the O118:H16 serotype, which is also common to both cattle and humans. The bovine O118:H16 EHEC strain was able to colonize the gut of three newborn calves, and to induce diarrhoea twenty-four hours after challenge and to produce AE lesions in the small and/or large intestines. AE lesions were detected microscopically and ultrastructurally in the small intestine of one calf and in the whole intestinal track of two calves. Internalization of bacteria and also of pedestal-bacteria complex inside of the enterocyte was observed in two of the three calves. The significance of this stage is unknown but may be related to the invasion of the calf by the bacteria. The challenge strain was isolated from the mesenteric lymph nodes of the same two calves but not from other organs or from heart blood. No blood was observed in the faeces of any of the three calves, nor were any lesions in the internal organs, which may have been related to the production of a verotoxin whose role is still unknown in cattle.

Prevalence of enterohaemorrhagic Escherichia coli from serotype O157 and other attaching and effacing Escherichia coli on bovine carcasses in Algeria

Aims:  Bovine meat is the principal source of human contamination of attaching and effacing Escherichia coli, including enterohaemorrhagic E. coli O157. The aim was to study the prevalence of these strains on bovine carcasses in Algeria.