F74 plasmids are major vectors of virulence genes in bovine NTEC2.

Necrotoxigenic Escherichia coli 2 (NTEC2) are defined as E. coli producing the toxin known as cytotoxic necrotizing factor 2 (CNF2), a potent toxin primarily found in bovine but also in humans. NTEC2 are mostly associated with bovine, and cnf2 is known to be carried by pVir-like plasmids. In this study, we looked for NTEC2 in a collection of E. coli collected between 2011 and 2018 in French bovine. Thirty-two isolates, collected from both sick (n = 19) and healthy (n = 13) animals, were identified and characterized using whole-genome sequencing. One F74 plasmid of this bacterial collection was long-read sequenced: its size was 138 121 bp and it carried the cnf2, F17cA-eG, cdtB, iutA, iucC and ompP virulence factors (VFs), but no resistance gene. A large variety of genetic backgrounds was observed, but all cnf2-carrying plasmids belonged to the IncF family, and most of them (78·1%) were of the F74 group. Similar F74 plasmids were also reported from bovine in the United Kingdom and the United States, as identified in the publically available databases. Consequently, these F74 plasmids, which are widely disseminated among E. coli from cattle in the French territory, are vectors of virulence determinants that largely went unnoticed until now.

Molecular Profiling of Shiga Toxin-Producing Escherichia coli and Enteropathogenic E. coli Strains Isolated from French Coastal Environments.

UNLABELLED: Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) strains may be responsible for food-borne infections in humans. Twenty-eight STEC and 75 EPEC strains previously isolated from French shellfish-harvesting areas and their watersheds and belonging to 68 distinguishable serotypes were characterized in this study. High-throughput real-time PCR was used to search for the presence of 75 E. coli virulence-associated gene targets, and genes encoding Shiga toxin (stx) and intimin (eae) were subtyped using PCR tests and DNA sequencing, respectively. The results showed a high level of diversity between strains, with 17 unique virulence gene profiles for STEC and 56 for EPEC. Seven STEC and 15 EPEC strains were found to display a large number or a particular combination of genetic markers of virulence and the presence of stx and/or eae variants, suggesting their potential pathogenicity for humans. Among these, an O26:H11 stx1a eae-ß1 strain was associated with a large number of virulence-associated genes (n = 47), including genes carried on the locus of enterocyte effacement (LEE) or other pathogenicity islands, such as OI-122, OI-71, OI-43/48, OI-50, OI-57, and the high-pathogenicity island (HPI). One O91:H21 STEC strain containing 4 stx variants (stx1a, stx2a, stx2c, and stx2d) was found to possess genes associated with pathogenicity islands OI-122, OI-43/48, and OI-15. Among EPEC strains harboring a large number of virulence genes (n, 34 to 50), eight belonged to serotype O26:H11, O103:H2, O103:H25, O145:H28, O157:H7, or O153:H2. IMPORTANCE: The species E. coli includes a wide variety of strains, some of which may be responsible for severe infections. This study, a molecular risk assessment study of E. coli strains isolated from the coastal environment, was conducted to evaluate the potential risk for shellfish consumers. This report describes the characterization of virulence gene profiles and stx/eae polymorphisms of E. coli isolates and clearly highlights the finding that the majority of strains isolated from coastal environment are potentially weakly pathogenic, while some are likely to be more pathogenic.

Fast detection of both O157 and non-O157 shiga-toxin producing Escherichia coli by real-time optical immunoassay.

UNLABELLED: Among bacterial pathogens involved in food-illnesses, seven serogroups (O26, O45, O103, O111, O121, O145 and O157) of Shiga-toxin producing Escherichia coli (STEC), are frequently identified. During such outbreak, and due to the perishable property of most foodstuff, the time laps for the identification of contaminated products and pathogens is thus critical to better circumvent their spread. Traditional detection methods using PCR or culture plating are time consuming and may present some limitations. In this study, we present a multiplexed immunoassay for the optical detection of most commonly enterohemorrhagic E. coli serogroups: O26, O45, O103, O111, O121, O145 and O157:H7 in a single device. The use of Surface Plasmon Resonance imaging not only enabled the label-free analysis of the samples but gave results in a real-time manner. A dedicated protocol was set up for the detection of both low contaminating bacterial concentrations of food samples (5 CFU per 25 g) and postenrichment aliquots. By combining one single device for the detection of O157 and non-O157 STEC in a label-free manner, this rapid approach may have an important economic and societal impact. SIGNIFICANCE AND IMPACT OF THE STUDY: This article presents a simple-to-operate immunoassay for the specific detection of Shiga-toxin producing Escherichia coli (STEC). This approach consists in the on-chip assay detection of viable cells on a specifically designed antibody microarray. By skipping any enrichment step and avoiding the use of labelling agent, this approach based on the Surface Plasmon Resonance imaging of the microarrays turns out to be much faster and more cost effective by comparison with standardized methods.

Virulence Genes in Expanded-Spectrum-Cephalosporin-Resistant and -Susceptible Escherichia coli Isolates from Treated and Untreated Chickens.

This study investigated antimicrobial resistance, screened for the presence of virulence genes involved in intestinal infections, and determined phylogenetic groups of Escherichia coli isolates from untreated poultry and poultry treated with ceftiofur, an expanded-spectrum cephalosporin. Results show that none of the 76 isolates appeared to be Shiga toxin-producing E. coli or enteropathogenic E. coli. All isolates were negative for the major virulence factors/toxins tested (ehxA, cdt, heat-stable enterotoxin [ST], and heat-labile enterotoxin [LT]). The few virulence genes harbored in isolates generally did not correlate with isolate antimicrobial resistance or treatment status. However, some of the virulence genes were significantly associated with certain phylogenetic groups.

Use of virulence determinants and seropathotypes to distinguish high- and low-risk Escherichia coli O157 and non-O157 isolates from Europe.

The presence of 10 virulence genes was examined using polymerase chain reaction (PCR) in 365 European O157 and non-O157 Escherichia coli isolates associated with verotoxin production. Strain-specific PCR data were analysed using hierarchical clustering. The resulting dendrogram clearly separated O157 from non-O157 strains. The former clustered typical high-risk seropathotype (SPT) A strains from all regions, including Sweden and Spain, which were homogenous by Cramer's V statistic, and strains with less typical O157 features mostly from Hungary. The non-O157 strains divided into a high-risk SPTB harbouring O26, O111 and O103 strains, a group pathogenic to pigs, and a group with few virulence genes other than for verotoxin. The data demonstrate SPT designation and selected PCR separated verotoxigenic E. coli of high and low risk to humans; although more virulence genes or pulsed-field gel electrophoresis will need to be included to separate high-risk strains further for epidemiological tracing.

Investigation of Clostridium botulinum in commercial poultry farms in France between 2011 and 2013.

Between 2011 and 2013, 17 poultry botulism outbreaks were investigated in France. All cases were associated with Clostridium botulinum type C-D. Presence of C. botulinum was studied in seven areas: poultry house, changing room, ventilation system, surroundings, animal reservoirs, water, and feed. Swabs, litter, soil, darkling beetles, rodents and wild bird droppings, feed and water samples were collected. The presence of C. botulinum type C-D in the environment of affected flocks was detected in 39.5% of the 185 samples analysed by real-time polymerase chain reaction. C. botulinum type C-D was reported in each area. Four areas were more frequently contaminated, being found positive in more than one-half of farms: darkling beetles (9/11), poultry house (14/17), water (13/16) and surroundings (11/16). After cleaning and disinfection, the ventilation system and/or the soil (in the houses and the surroundings) returned positive results in four out of eight poultry farms. Consequently, darkling beetles, the drinking water, the ventilation system and the soil in the surroundings and the houses were identified as the main critical contaminated areas to consider in poultry farms to prevent recurrence of botulism outbreaks.

Simultaneous enrichment and optical detection of low levels of stressed Escherichia coli O157:H7 in food matrices.

AIMS: Rapid detection of enterohaemorrhagic E. coli O157:H7 in large range of stress conditions occurring in food processing. METHODS AND RESULTS: Detection of E. coli O157:H7 in various food processing stress conditions using surface plasmon resonance imaging (SPRi) technique on an antibody microarray was evaluated. The direct detection method based on the culture/capture/measure (CCM) process consists of detecting bacteria during an enrichment step, which significantly decreases the overall assay duration. In optimized culture conditions, this method allows the specific detection of low CFU ml(-1) in <7 h. Detection of bacteria directly in contaminated food samples was also conducted. CONCLUSIONS: The CCM technique using an antibody microarray is a label-free immunoassay that allows rapid detection of E. coli O157:H7 in both food processing stress conditions and complex food matrices. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay is promising for detecting E. coli O157:H7 at different steps of food and drink processing and during storage. SPRi appears to be a suitable and powerful detection method for routine quality controls in food industry with important economic and societal impact.

Duplex real-time PCR detection of type III effector tccP and tccP2 genes in pathogenic Escherichia coli and prevalence in raw milk cheeses.

AIMS: To develop a duplex real-time PCR assay targeting enterohaemorrhagic Escherichia coli (EHEC) type III effector TccP/TccP2-encoding genes which are pivotal to EHEC-mediated actin cytoskeleton reorganization in human intestinal epithelial cells. METHODS AND RESULTS: The specificity of the assay was demonstrated with DNA from EHEC reference strains and non-E. coli bacterial species. The detection limit was determined as five tccP or tccP2 copies per reaction. The assay was then evaluated on a large collection of 526 E. coli strains of human, animal, food and environmental origins. The results showed that tccP was restricted to a limited number of serotypes (i.e. O5:H(-) , O55:H7, O125:H6 and O157:H7). The tccP2 gene was present in a higher number of serotypes including the five most frequent EHEC serotypes (i.e. O26:H11, O103:H2, O111:H8, O145:H28 and O157:H7), and a few other serotypes that caused human infections (i.e. O4:H(-) , O45:H2 and O55:H7). A minority of O26:H11 and O103:H2 strains however tested negative for tccP2, though it is not known whether the lack of tccP2 affected their pathogenic potential. Real-time PCR analysis of 400 raw milk cheeses revealed the presence of tccP and/or tccP2 genes in 19·75% of the cheese enrichment suspensions. CONCLUSIONS: A highly specific and sensitive duplex real-time PCR method was developed for rapid and simultaneous detection of tccP and tccP2. Unpasteurized dairy products may be contaminated with E. coli strains carrying tccP and/or tccP2. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed real-time PCR assay represents a valuable alternative to conventional PCR tests and should be useful for characterization of the virulome of pathogenic E. coli strains.

Evidence for the existence of a new genus Chlamydiifrater gen. nov. inside the family Chlamydiaceae with two new species isolated from flamingo (Phoenicopterus roseus): Chlamydiifrater phoenicopteri sp. nov. and Chlamydiifrater volucris sp. nov.

The family Chlamydiaceae currently comprises a single genus Chlamydia, with 11 validly published species and seven more taxa. It includes the human pathogens Chlamydia (C.) trachomatis, C. pneumoniae and C. psittaci, a zoonotic agent causing avian chlamydiosis and human psittacosis, as well as other proven or potential pathogens in ruminants, birds, snakes, reptiles and turtles. During routine testing of 15 apparently healthy captive flamingos in a zoo in 2011, an atypical strain of Chlamydiaceae was detected by real-time PCR of cloacal swab samples. Sequence analysis of the 16S rRNA gene revealed high similarity to the uncultured Chlamydiales bacterium clone 122, which previously had been found in gulls. As more samples were collected during annual campaigns of the flamingo ringing program in southern France from 2012 to 2015, Chlamydiaceae-specific DNA was detected by PCR in 30.9% of wild birds. From these samples, three strains were successfully grown in cell culture. Ultrastructural analysis, comparison of 16S and 23S rRNA gene sequences, whole-genome analysis based on de novo hybrid-assembled sequences of the new strains as well as subsequent calculation of taxonomic parameters revealed that the relatedness of the flamingo isolates to established members of the family Chlamydiaceae was sufficiently distant to indicate that the three strains belong to two distinct species within a new genus. Based on these data, we propose the introduction of Chlamydiifrater gen. nov., as a new genus, and Chlamydiifrater phoenicopteri sp. nov. and Chlamydiifrater volucris sp. nov., as two new species of the genus.

Simplex and multiplex real-time PCR assays for the detection of flagellar (H-antigen) fliC alleles and intimin (eae) variants associated with enterohaemorrhagic Escherichia coli (EHEC) serotypes O26:H11, O103:H2, O111:H8, O145:H28 and O157:H7.

AIMS: To develop real-time PCR assays targeting genes encoding the flagellar antigens (fliC) and intimin subtypes (eae) associated with the five most clinically important serotypes of enterohaemorrhagic Escherichia coli (EHEC), i.e. O26:H11, O103:H2, O111:H8, O145:H28 and O157:H7. METHODS AND RESULTS: Primers and probes specific to fliC(H2) , fliC(H7) , fliC(H8) , fliC(H11) , fliC(H28) , eae-ß1, eae-γ1, eae-ε and eae-θ were combined in simplex and multiplex 5'-nuclease PCR assays. The specificity of the assays was assessed on 201 bacterial strains and the sensitivity determined on serially diluted EHEC genomes. The developed PCR assays were found to be highly specific and detected as few as five EHEC genome equivalents per reaction. Furthermore, it was possible to detect the five major EHEC serotypes in cheese samples inoculated at concentration levels of ≤5CFU per 25g after overnight enrichment using the PCR assays. CONCLUSIONS: The PCR assays developed here were found to be sensitive and specific for the reliable detection of genes encoding the flagellar antigens and intimin variants belonging to the five most clinically relevant EHEC serotypes. SIGNIFICANCE AND IMPACT OF THE STUDY: Application of real-time PCR assays should improve the identification of foods contaminated by EHEC and facilitate the molecular typing of these organisms.

Evaluation of the 'GeneDisc' real-time PCR system for detection of enterohaemorrhagic Escherichia coli (EHEC) O26, O103, O111, O145 and O157 strains according to their virulence markers and their O- and H-antigen-associated genes.

AIMS: To evaluate the GeneDisc multiplex real-time PCR assay for detection of enterohaemorrhagic Escherichia coli (EHEC) O26, O103, O111, O145 and O157 strains. METHODS AND RESULTS: GeneDiscs for detection of genes encoding Shiga toxins (stx), intimins (eae), E. coli O157 (rfbE(O157)) and H7 (fliC(H7)) antigens as well as genes specific for EHEC O26 (wzx(O26)), O103 (wzx(O103)), O111 (wbd1(O111)), O145 (ihp1(O145)) and O157 (ihp1(O157)) were evaluated. The assay was run with native bacteria in 1 h in a GeneDisc Cycler. All genotypes of stx and eae, except stx(2f) and eae-rho, were identified. Escherichia coli strains belonging to O-groups O26, O103, O111, O157 as well as EHEC O145:[H28] strains were specifically detected with this assay. The ihp1(O157) gene was not found specific for EHEC O157. O-rough mutants of EHEC and non-motile EHEC O157 strains were reliably identified with the GeneDisc assay. Two to three colonies of EHEC strains were still detectable in a lawn of 50 000 apathogenic E. coli from agar plates. CONCLUSIONS: The GeneDisc assay is a specific and reliable assay for detection of major EHEC strains. It is robust enough to detect few EHEC colonies in mixed cultures of bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay is promising for its use in EHEC diagnostics and for EHEC monitoring with different kinds of samples.

Development of real-time PCR tests for detecting botulinum neurotoxins A, B, E, F producing Clostridium botulinum, Clostridium baratii and Clostridium butyricum.

AIMS: To develop real-time PCR assays for tracking and tracing clostridia responsible for human botulism. METHODS AND RESULTS: Real-time PCR assays based on the detection of the genes ntnh encoding the nontoxin-nonhaemagglutinin (NTNH) proteins or the most homologous regions of the botulinum neurotoxin (bont) genes have been developed together with four real-time PCR assays, each being specific of the genes bont/A, bont/B, bont/E, bont/F and enables a toxin type-specific identification. The specificity of the assays was demonstrated using a panel of botulinum toxin producing clostridia (29 strains), nonbotulinum toxin producing clostridia (21 strains) and various other bacterial strains. The toxin type-specific assays had a sensitivity of 100 fg-1000 fg of total DNA in the PCR tube (25-250 genome equivalents) which correspond to 10(3) to 10(4) cells ml(-1). After a 48 h enrichment in anaerobic conditions, these PCR assays allowed the detection of Clostridium botulinum type A in a naturally contaminated sample of 'foie gras' suspected in a C. botulinum outbreak. CONCLUSION: These PCR tests are specific and reliable for detection of heterogeneous BoNT producing clostridia responsible for human botulism. SIGNIFICANCE AND IMPACT OF THE STUDY: Adoption of these PCR assays is a step forward a reliable and rapid detection of these clostridia in food samples.

Screening of food raw materials for the presence of Shiga toxin-producing Escherichia coli O91:H21.

AIMS: To provide information on the prevalence and detection, in foods, of Shiga toxin-producing Escherichia coli (STEC) O91:H21. METHODS AND RESULTS: Seven hundred fifteen minced beef meats and 205 raw milk samples were analysed by stx-specific PCR-ELISA. Samples positive for stx were subsequently tested for the presence of wzy-O91, fliC-H21 and the adhesin-encoding gene saa. For minced meat, 16 (2.2%) and 11 (1.5%) samples were found positive for (stx, wzy-O91, fliC-H21) and (stx, wzy-O91, fliC-H21, saa) combinations, respectively. For raw milk, seven (3.4%) samples were found positive for the (stx, wzy-O91, fliC-H21) combination but none of these contained saa. Two STEC O91:H21 saa-positive strains and three STEC O91 H21- and saa-negative strains were isolated by colony hybridization. CONCLUSIONS: A low prevalence of potentially pathogenic STEC O91:H21 in food products was found using a combination of PCR assays targeting stx, wzy-O91, fliC-H21 and saa. SIGNIFICANCE AND IMPACT OF THE STUDY: The PCR-based approach described here represents a valuable method for rapid screening of food samples contaminated by STEC O91:H21.

Chlamydia buteonis, a new Chlamydia species isolated from a red-shouldered hawk.

Chlamydiaceae are obligate intracellular bacterial pathogens for humans and animals. A recent study highlighted that a Chlamydiaceae intermediary between C. psittaci and C. abortus can infect hawks. Here, an isolate was obtained upon passage of cloacal and conjunctival sac material collected from a female hatch-year red-shouldered hawk (Buteo lineatus) in cultured cells. The diseased bird, one of 12 birds housed in a rehabilitation center, developed conjunctivitis and later died. Swabs from both sites tested positive for Chlamydia using the QuickVue Chlamydia test. The isolate, named RSHA, tested negative in qPCR assays specific for C. psittaci and C. abortus, respectively. Analysis of the 16S rRNA, 23S rRNA and whole genome sequences as well as MLST, ANIb and TETRA values reveal that C. psittaci and C. abortus are the closest relatives of RSHA. However, the overall results strongly suggest a phylogenetic intermediate position between these two species. Therefore, we propose the introduction of a new species designated Chlamydia buteonis with RSHAT as the type strain.

Development of a 5'-nuclease PCR assay for the identification of Escherichia coli strains expressing the flagellar antigen H21 and their detection in food after enrichment.

AIMS: To develop a real-time PCR assay targeting the Escherichia coli flagellar antigen H21 for identification and surveillance of clinically important Shiga toxin-producing E. coli (STEC) serotypes classified in seropathotype C. METHODS AND RESULTS: The fliC allele of STEC O91:H21 strain B2F1 was amplified and sequenced. The nucleotide sequence obtained was compared with fliC genes of E. coli O157:H21, O8:H21 and O113:H21 strains. A pair of oligonucleotide primers and a TaqMan minor groove binder probe specific for fliC-H21 were designed and used in a 5'-nuclease PCR assay. This method was evaluated using a panel of 138 diverse bacterial strains and was shown to be 100% specific for H21. PCR amplification of fliC-H21 from one cell per reaction mixture was possible, and an initial inoculum of 10 STEC H21 colony-forming units per 25 g of ground beef was detected after overnight enrichment. CONCLUSIONS: The PCR assay developed was found to be highly sensitive and specific for the identification and detection of E. coli H21 strains in ground beef. SIGNIFICANCE AND IMPACT OF THE STUDY: The real-time PCR assay targeting the H21 flagellar antigen described here offers a valuable method for the rapid detection and molecular typing of pathogenic STEC H21 strains in food.

Molecular characterization of O157:H7, O26:H11 and O103:H2 Shiga toxin-producing Escherichia coli isolated from dairy products.

Pathogenic Shiga toxin-producing E. coli (STEC) are recognized worldwide as environment and foodborne pathogens which can be transmitted by ingestion of ready-to-eat food such as raw milk-derived products. STEC show a prevalence rate in dairy products of 0.9%, yet comparably few outbreaks have been related to dairy products consumption. In this study, we used rt-qPCR to identify the virulence potential of O157, O26 and O103 STEC strains isolated from raw-milk dairy products by analyzing virulence-related gene frequencies and associations with O-island (OI) 44, OI-48, OI-50, OI-57, OI-71 and OI-122. Results showed that 100% of STEC strains investigated harbored genes associated with EHEC-related virulence profile patterns (eae and stx, with either espK, espV, ureD and/or Z2098). We also found similarities in virulence-related gene content between O157:H7 and O103:H2 dairy and non-dairy STEC strains, especially isolates from human cases. The O26:H11-serotype STEC strains investigated harbor the arcA-allele 2 gene associated with specific genetic markers. These profiles are associated with high-virulence seropathotype-A STEC. However, the low frequency of stx2 gene associated with absence of other virulence genes in dairy isolates of O26:H11 remains a promising avenue of investigation to estimate their real pathogenicity. All O26:H11 attaching-effacing E. coli (AEEC) strains carried CRISPRO26:H11SP_O26_E but not genetic markers espK, espV, ureD and/or Z2098 associated with the emerging potentially high-virulence "new French clone". These strains are potentially as "EHEC-like" strains because they may acquire (or have lost) stx gene. In this study, O157:H7, O103:H2 and O26:H11 STEC strains isolated from dairy products were assigned as potential pathogens. However, research now needs to investigate the impact of dairy product environment and dairy processing on the expression of their pathogenicity.

First Draft Genome for a Burkholderia mallei Isolate Originating from a Glanderous Mule from Brazil.

Burkholderia mallei is the etiological agent of glanders. Here, we present the draft genome sequence of Burkholderia mallei strain 16-2438_BM#8 that was isolated from a mule found dead in Pernambuco, northeast Brazil. It is the first available genomic sequence from a strain isolated on the American continent.

Shiga toxin-producing Escherichia coli strains isolated from dairy products - Genetic diversity and virulence gene profiles.

Shiga toxin-producing Escherichia coli (STEC) are widely recognized as pathogens causing food borne disease. Here we evaluate the genetic diversity of 197 strains, mainly STEC, from serotypes O157:H7, O26:H11, O103:H2, O111:H8 and O145:28 and compared strains recovered in dairy products against strains from human, meat and environment cases. For this purpose, we characterized a set of reference-collection STEC isolates from dairy products by PFGE DNA fingerprinting and a subset of these by virulence-gene profiling. PFGE profiles of restricted STEC total DNA showed high genomic variability (0.9976 on Simpson's discriminatory index), enabling all dairy isolates to be differentiated. High-throughput real-time PCR screening of STEC virulence genes were applied on the O157:H7 and O26:H11 STEC isolates from dairy products and human cases. The virulence gene profiles of dairy and human STEC strains were similar. Nevertheless, frequency-wise, stx1 was more prevalent among dairy O26:H11 isolates than in human cases ones (87% vs. 44%) while stx2 was more prevalent among O26:H11 human isolates (23% vs. 81%). For O157:H7 isolates, stx1 (0% vs. 39%), nleF (40% vs 94%) and Z6065 (40% vs 100%) were more prevalent among human than dairy strains. Our data point to differences between human and dairy strains but these differences were not sufficient to associate PFGE and virulence gene profiles to a putative lower pathogenicity of dairy strains based on their lower incidence in disease. Further comparison of whole-genome expression and virulence gene profiles should be investigated in cheese and intestinal tract samples.

Occurrence of C. botulinum in healthy cattle and their environment following poultry botulism outbreaks in mixed farms.

Ten cattle farms located in an area with a recent history of poultry botulism outbreaks were investigated to evaluate the occurrence of toxigenic C. botulinum in healthy cattle. Environmental samples in the 10 cattle farms and bovine fecal contents in farms with a confirmed environmental contamination were collected. Detection of C. botulinum toxin genes C, D, C/D, D/C and E was performed using real-time PCR. 4.9% (7/143) of the environmental samples collected in the 10 investigated cattle farms were positive for C. botulinum type C/D. Theses samples (boot-swabs in stalls and on pasture and water of a stream) were collected in 3 different farms. One cow dung sample and 3 out of 64 fecal contents samples collected in a single farm were also positive for C. botulinum type C/D. This study demonstrates that cattle are probably indirectly contaminated via poultry botulism in the area and that they can be intermittent carrier of C. botulinum type C/D after poultry botulism outbreaks in mixed farms.

Investigation of animal botulism outbreaks by PCR and standard methods.

A double PCR procedure is proposed for identification of Clostridium botulinum C and D. This method consists of a first PCR amplification with a degenerate primer pair able to amplify a 340 bp common DNA fragment from botulinum neurotoxin (BoNT) C1 and D genes, followed by two subsequent PCR amplifications with two primer pairs specific for BoNT/C1 and D respectively (198 bp DNA fragment). This method was found to be specific for C. botulinum C and D, amongst 81 strains of C. botulinum and 21 different species of other Clostridium and bacteria tested. The detection limit ranged from 10 to 10(3) bacteria in the reaction volume according to the C. botulinum C and D strains. In 160 naturally contaminated animal and food samples submitted to a 48 h enrichment culture, the double PCR showed an 89.4% correlation rate with the standard mouse bioassay. A clear distinction between botulism type C and D was obtained. The double PCR provides a reliable alternative for detection and identification of C. botulinum C and D in clinical and food samples.