Comparison of culture-dependent and -independent bacterial detection results on nasal swabs in dogs with nasal discharge.

OBJECTIVES: The role of bacterial communities in the pathophysiology of canine nasal disease is still unclear. How and when to treat dogs with suspected secondary bacterial rhinitis and on which test to rely before making a decision to treat with antimicrobials has not been established. The objective is to compare the results of bacterial identification using agar-plate cultures and 16S rRNA gene amplicon sequencing in dogs with nasal discharge suspected to be of bacterial origin. MATERIALS AND METHODS: Twenty-nine client-owned dogs presented for investigation of nasal disease were included in the study. Paired swabs were collected from the same affected nasal cavity. One swab was streaked on 4 agar media (Columbia Blood Agar, MacConkey, Chapman and Edward's). The other swab was stored in a sterile cryotube at -80°. Extracted DNA underwent a polymerase chain reaction targeting the V1-V3 region of the 16S rRNA gene. RESULTS: At least one of the species detected by amplicon sequencing with a relative abundance of >10% was also identified by culture in 14 cases (48.3%), in association with marked predominance of one taxon (>80% relative abundance) in six of 14 cases. In 12 dogs (41.4%), the cultured isolates were rare or undetected components of the corresponding sequence libraries. A negative culture in the face of bacterial predominance (>50% relative abundance) of a potentially pathogenic bacteria detected by sequencing occurred in 17% (n=5) of cases; however, the use of other agar media may have decreased this percentage. CLINICAL SIGNIFICANCE: Standard culture does not reliably predict the bacterial profile detected by 16S rRNA gene amplicon sequencing.

Antimicrobial resistance of Escherichia coli isolated from freshwaters and hospital effluents in Belgium.

The purpose of this work was to evaluate the level of antimicrobial resistant Escherichia coli isolates in freshwaters and hospital effluents in Belgium. The samples were collected from 24 locations along the Ourthe, Vesdre, Amblève and Meuse rivers and in the wastewater effluents of several hospitals. The sampling stations in rivers were classified according to the dominant land covers of the rivers (rural, urban and forest areas). Two sampling campaigns were organized in May and October 2019 to highlight a possible seasonal effect. A total of 938 E. coli strains were isolated on Chromogenic Selective Tryptone Bile X-glucuronide (TBX) and TBX supplemented with amoxicillin (TBX+AMX) media. Disk diffusion assays were performed following the EUCAST's recommendations to assess the antimicrobial resistance against 12 antibiotics. A total of 32·7% of strains were at least resistant to one antibiotic and 24·6% were multiple antimicrobial resistant strains on TBX. The highest resistance rates were found for ampicillin (AMP), amoxicillin coupled with clavulanic acid (AMC) and sulfamethoxazole/trimethoprim (SXT). The lowest resistance rates were observed for meropenem (MEM) and ertapenem (ETP), which are last resort antibiotics. No significant difference was observed between both campaigns for the resistance rate to antibiotics.

Antimicrobial resistance and virulence characteristics in 3 collections of staphylococci from bovine milk samples.

Mastitis is a prevalent disease in dairy cattle, and staphylococci are among the most common causative pathogens. Staphylococci can express resistance to a range of antimicrobials, of which methicillin resistance is of particular public health concern. Additionally, Staphylococcus aureus carries a variety of virulence factors, although less is understood about the virulence of non-aureus staphylococci (NAS). The aim of our study was to identify and characterize 3 collections of staphylococcal isolates from bovine milk samples regarding antimicrobial resistance, with emphasis on methicillin resistance, and their carriage of virulence genes typically displayed by Staph. aureus. A total of 272 staphylococcal isolates collected in Norway and Belgium in 2016 were included, distributed as follows: group 1, Norway, 100 isolates; group 2, Flanders, Belgium, 64 isolates; group 3, Wallonia, Belgium, 108 isolates. Species identification was performed by use of MALDI-TOF mass spectrometry. Phenotypic resistance was determined via disk diffusion, and PCR was used for detection of methicillin resistance genes, mecA and mecC, and virulence genes. Antimicrobial resistance was common in Staphylococcus epidermidis and Staphylococcus haemolyticus from all different groups, with resistance to trimethoprim-sulfonamide frequently occurring in Staph. epidermidis and Staph. haemolyticus as well as in Staph. aureus. Resistance to penicillin was most frequently observed in group 1. Ten Belgian isolates (1 from group 2, 9 from group 3) carried the methicillin resistance determinant mecA: 5 Staph. aureus from 2 different farms and 5 NAS from 3 different farms. Almost all Staph. aureus isolates were positive for at least 3 of the screened virulence genes, whereas, in total, only 8 NAS isolates harbored any of the same genes. Our study contributes to the continuous need for knowledge regarding staphylococci from food-producing animals as a basis for better understanding of occurrence of resistance and virulence traits in these bacteria.

Efficacy assessment of PEV2 phage on Galleria mellonella larvae infected with a Pseudomonas aeruginosa dog otitis isolate.

Pseudomonas (P.) aeruginosa is the most frequently isolated Gram-negative bacteria in dog otitis. Antimicrobial resistance is particularly prevalent in P. aeruginosa and phage therapy represents a promising alternative therapeutic strategy. The aim of this study was to assess the efficacy of the PEV2 phage against a clinical P. aeruginosa isolate from a canine otitis using a Galleria (G.) mellonella larvae model. The genomic DNA of PAV237 P. aeruginosa isolate was sequenced and analysed. In a first main experiment, the efficacy of PEV2 phage against PAV237 was assessed at different multiplicities of infection (MOI) (50,000, 5000, 500, 50) by analyzing the larvae survival rate during 4 days. In a second experiment, the bacterial and phage titer evolutions were assessed depending on two MOIs (50,000, 5000). No significant survival increase was observed with PEV2 therapy in the infected larvae groups. The generated Kaplan-Meier curves showed that the rate of alive larvae was significantly higher in the non-infected larvae compared to the infected-treated ones irrespective of phage MOIs. An increase of the phage titer was observed at 24 and 48 h post-inoculation (HPI) with both MOIs and the P. aeruginosa titers were lower with MOI 50,000 and 5000 compared to the infectivity control at 24 and 48 HPI. Even if an ineffectiveness of the PEV2 phage was observed on the larvae survival, PEV2 is active against P. aeruginosa in this model and PEV2 replication is correlated with a lower bacterial proliferation in the phage treated larvae.

Pathogenic potential of Escherichia coli O157 and O26 isolated from young Belgian dairy calves by recto-anal mucosal swab culturing.

AIMS: The purpose of this study was to investigate the occurrence of Escherichia coli O157 and O26 on Belgian dairy cattle farms, the presence of virulence genes in the confirmed isolates and the association of E. coli O26 presence with calf diarrhoea. METHODS AND RESULTS: In total, 233 recto-anal mucosal swabs (RAMS) were obtained from healthy and diarrheic dairy calves on three farms, each alternately visited three consecutive times. RAMS were analysed for presence of E. coli O157 and O26, and stx1, stx2 and eae virulence genes. Overall, 19% of RAMS tested positive for E. coli O157, while 31% tested positive for E. coli O26. The majority of isolates possessed both stx and eae, denoting a high pathogenic potential to humans. While both serogroups persisted at farm level, persistence within the same animal over time appeared to be relatively rare. Interestingly, E. coli O26 was already abundantly present at a younger age compared to E. coli O157. Calf diarrhoea could not be associated with presence of E. coli O26. CONCLUSIONS: Young dairy calves are important on-farm reservoirs of potentially pathogenic E. coli O157 and O26. A role of E. coli O26 in calf diarrhoea could not be confirmed. SIGNIFICANCE AND IMPACT OF THE STUDY: O157 and O26 are responsible for the majority of human STEC infections. Gaining more epidemiological information regarding their occurrence and persistence on cattle farms will contribute to a better understanding of STEC ecology and risk of human transmission.

Genetic characterization of Shigatoxigenic and enteropathogenic Escherichia coli O80:H2 from diarrhoeic and septicaemic calves and relatedness to human Shigatoxigenic E. coli O80:H2.

AIM: The purpose of this work was to identify and genetically characterize enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) O80:H2 from diarrhoeic and septicaemic calves in Belgium and to comparing them with human EHEC after whole genome sequencing. METHODS AND RESULTS: Ten EHEC and 21 EPEC O80 identified by PCR between 2009 and 2018 from faeces, intestinal content and a kidney of diarrhoeic or septicaemic calves were genome sequenced and compared to 19 human EHEC identified between 2008 and 2019. They all belonged to the O80:H2 serotype and ST301, harboured the eaeξ gene, and 23 of the 29 EHEC contained the stx2d gene. Phylogenetically, they were distributed in two major sub-lineages: one comprised a majority of bovine EPEC whereas the second one comprised a majority of stx2d bovine and human EHEC. CONCLUSIONS: Not only EPEC but also EHEC O80:H2 are present in diarrhoeic and septicaemic calves in Belgium and are genetically related to human EHEC. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings support the need to assess cattle as potential source of contamination of humans by EHEC O80:H2 and to understand the evolution of bovine and human EHEC and EPEC O80:H2.

Occurrence of 'gang of five' Shiga toxin-producing Escherichia coli serogroups on Belgian dairy cattle farms by overshoe sampling.

Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens responsible for global outbreaks. This study was conducted to investigate the occurrence of 'gang of five' STEC serogroups (O26, O103, O111, O145, O157) on Belgian dairy cattle farms by overshoe (OVS) sampling, and to evaluate the presence of virulence genes in the obtained isolates. A total of 88 OVS, collected from the pen beddings of 19 Belgian dairy cattle farms, were selectively enriched in mTSBn, followed by immunomagnetic separation and plating onto CT-SMAC for O157 STEC isolation, as well as in Brila broth, followed by a selective acid treatment and plating onto CHROMagarTM STEC and chromIDTM EHEC for non-O157 STEC isolation. Overall, 11 of 19 farms (58%) tested positive for presence of 'gang of five' STEC. O26 STEC was most frequently isolated from OVS (11/88; 12·5%), followed by O157 (10/88; 11·5%), O145 (3/88; 3·5%) and O103 (3/88; 3·5%). Additionally, 35% of the OVS collected from pens housing young cattle 1-24 months of age tested positive for 'gang of five' STEC, indicating that this age category is more likely to harbour STEC compared to new-born and adult cattle. Importantly, half of the obtained 'gang of five' STEC isolates (48%) possessed the eae and stx2 gene, suggesting a high pathogenic potential to humans.

In vitro and in vivo assessment of phage therapy against Staphylococcus aureus causing bovine mastitis.

OBJECTIVE: The aim of this study was to assess the efficacy of lytic bacteriophages on Staphylococcus aureus causing bovine mastitis, by in vitro and in vivo assays using Galleria mellonella and murine mastitis models. METHODS: Between May and December 2016, ten S. aureus (five methicillin-resistant and five methicillin-sensitive) isolates were isolated from milk samples of cattle with mastitis in Belgium and Norway. The isolates were assessed in vitro for their susceptibility to four lytic bacteriophages (Romulus, Remus, ISP and DSM105264) and subsequently in vivo in G. mellonella larvae and in murine mastitis model. RESULTS: Romulus, Remus and ISP showed a lytic activity against the S. aureus isolates in vitro. A larvae survival rate below 50% was observed at 4 days post-inoculation (DPI) in the groups infected with a methicillin-sensitive S. aureus isolate and treated with these three phages in vivo. An incomplete recovery of the mouse mastitis was observed at 48h post-inoculation (HPI) in the groups infected and treated with the ISP phage in vivo. CONCLUSIONS: The observations are much more pronounced statistically between the infected- phosphate buffered saline (PBS)-treated and infected-phage-treated groups in G. mellonella and the murine mastitis model demonstrating an effect of the phages against S. aureus associated with bovine mastitis.

Comparison of quantitative PCR and MALDI-TOF mass spectrometry assays for identification of bacteria in milk samples from cows with subclinical mastitis.

AIMS: The objective of this study was to compare qualitatively and quantitatively the results of identification of the bacteria present in milk samples from cows with subclinical mastitis using multiplex qPCR assay and matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF MS® ) after bacteriological growth. METHODS AND RESULTS: A total of 182 samples were aseptically collected from 119 cows with high somatic cell counts (>2·105 SCC per ml) on 11 farms in Belgium in 2014. The mutiplex qPCR assay was carried out on 350 µl of milk with the PathoProof® Complete-16kit. Ten microlitre of milk was streaked on Columbia blood agar and three selective agar plates. Growing colonies were identified by MALDI-TOF MS. Of the 182 samples, 90 gave positive results with either or both tests for one or two bacterial species/genera. Total qualitative agreement of the bacteria identified was observed in 41 mono- or bi-bacterial samples (46%) and partial agreement in 19 bi-bacterial samples at both or either tests (21%). The results of both tests on those mono- and bi-bacterial samples were not significantly different (McNemar test; P = 0·395) with a fair agreement (Cohen's kappa test; k = 0·375; P = 0·055). Moreover, quantitative correlation between the qPCR intensity and the numbers of growing colonies was observed in half of the 60 samples with qualitative matching results. CONCLUSIONS: Both methods give identical qualitative and quantitative results with approximately a half and a quarter of the mono- and bi-bacterial samples respectively. Several reasons can explain the differences. The multiplex qPCR assay only targets the most important mammary gland pathogens and can detect DNA of bacteria both alive and dead. Conversely, bacteria only grow when alive and the MALDI-TOF MS databases do not include all bovine milk-associated bacterial species yet. SIGNIFICANCE AND IMPACT OF THE STUDY: This study further highlights the limitations and complementarity of the genetic and phenotypic tests for the identification of bacteria present in milk samples.

Characteristics of Shiga toxin producing- and enteropathogenic Escherichia coli of the emerging serotype O80:H2 isolated from humans and diarrhoeic calves in Belgium.

OBJECTIVES: Recently a highly virulent Escherichia coli O80:H2 pathotype carrying Shiga toxin genes, the intimin subtype eaeξ, and genes associated with the extraintestinal pathogenic E. coli (ExPEC) pS88 plasmid was described in France. In this study we examine the relatedness of Belgian E. coli O80:H2 isolated from humans and diarrhoeic calves as well their similarities with the French pathotype. METHODS: Eighteen Belgian E. coli O80:H2 strains (nine human Shiga toxin-producing E. coli (STEC) (2008-2016), two bovine STEC (1987) and seven bovine atypical enteropathogenic E. coli (aEPEC) (2009-2015)) were characterized with conventional PCR, disc diffusion susceptibility testing and whole genome sequencing. RESULTS: Only nine sporadic human STEC O80:H2 cases have been detected in Belgium. All patients were female, just two of them suffered from haemolytic uremic syndrome. All studied strains had the eaeξ subtype, belonged to the multi-locus sequence type ST-301, and carried virulence genes associated with the type III secretion system and effectors not encoded by the locus of enterocyte effacement (LEE). Multiple genes of the pS88 plasmid were detected in all but two strains (one human and one calf STEC). The Shiga toxin subtypes stx1a (n = 3; one human, two calf), stx2a (n = 2) and stx2d (n = 6) were detected. All strains were multidrug resistant, two were extended-spectrum ß-lactamase positive. Core genome MLST revealed that some human and calf E. coli differed by only 22 loci. CONCLUSIONS: The STEC/ExPEC O80:H2 pathotype was present in calves in Belgium as early as 1987, but human infections have been rare and mostly mild. The human STEC and bovine aEPEC cluster together and have the potential to be as virulent as the French isolates, as shown by their similar gene content.

Low prevalence of the 'gang of seven' and absence of the O80:H2 serotypes among Shigatoxigenic and enteropathogenic Escherichia coli (STEC and EPEC) in intestinal contents of healthy cattle at two slaughterhouses in Belgium in 2014.

AIMS: The purpose of this survey was to estimate the respective prevalence of the 'gang of seven' and 'non-gang of seven' serotypes of Shigatoxigenic and enteropathogenic Escherichia coli and to identify the O80:H2 serotype in 245 intestinal contents collected at two slaughterhouses in Belgium in 2014. METHODS AND RESULTS: After overnight enrichment growth, the 69 intestinal contents testing positive with PCR targeting the eae, stx1 and stx2 genes were inoculated onto four agar media. Of the 2542 colonies picked up, 677 from 59 samples were PCR confirmed. The most frequent virulotypes were eae+ in 47 (80%) samples, stx2+ in 20 (34%) samples and eae+ stx1+ in 16 (27%) samples. PCR-positive colonies belonged to different virulotypes in 36 samples. No colony was O80-positive, whereas two eae+ colonies from two samples were O26:H11, 50 eae+ stx1+ and eae+ from eight samples were O103:H2 and two eae+ stx1+ stx2+ colonies from one sample were O157:H7. CONCLUSIONS: The 'non-gang of seven' serotypes are more frequent than the 'gang of seven' serotypes and the O80:H2 serotype was not detected among Shigatoxigenic and enteropathogenic Escherichia coli in the intestines of cattle at these two slaughterhouses. SIGNIFICANCE AND IMPACT OF THE STUDY: Although the identification protocols of Shigatoxigenic Escherichia coli focus on the 'gang of seven' serotypes, several other serotypes can be present with possible importance in public health. Innovative selective identification procedures should be designed.

Identification of Shiga toxin-producing (STEC) and enteropathogenic (EPEC) Escherichia coli in diarrhoeic calves and comparative genomics of O5 bovine and human STEC.

Escherichia coli producing Shiga toxins (Stx) and the attaching-effacing (AE) lesion (AE-STEC) are responsible for (bloody) diarrhoea in humans and calves while the enteropathogenic E. coli (EPEC) producing the AE lesion only cause non-bloody diarrhoea in all mammals. The purpose of this study was (i) to identify the pathotypes of enterohaemolysin-producing E. coli isolated between 2009 and 2013 on EHLY agar from less than 2 month-old diarrhoeic calves with a triplex PCR targeting the stx1, stx2, eae virulence genes; (ii) to serotype the positive isolates with PCR targeting the genes coding for ten most frequent and pathogenic human and calf STEC O serogroups; and (iii) to compare the MLSTypes and virulotypes of calf and human O5 AE-STEC after Whole Genome Sequencing using two server databases (www.genomicepidemiology.org). Of 233 isolates, 206 were triplex PCR-positive: 119 AE-STEC (58%), 78 EPEC (38%) and 9 STEC (4%); and the stx1+eae+ AE-STEC (49.5%) were the most frequent. Of them, 120 isolates (84% of AE-STEC, 23% of EPEC, 22% of STEC) tested positive with one O serogroup PCR: 57 for O26 (47.5%), 36 for O111 (30%), 10 for O103 (8%) and 8 for O5 (7%) serogroups. The analysis of the draft sequences of 15 O5 AE-STEC could not identify any difference correlated to the host. As a conclusion, (i) the AE-STEC associated with diarrhoea in young calves still belong to the same serogroups as previously (O5, O26, O111) but the O103 serogroup may be emerging, (ii) the O5 AE-STEC from calves and humans are genetically similar.

Mediation analysis to estimate direct and indirect milk losses associated with bacterial load in bovine subclinical mammary infections.

Milk losses associated with mastitis can be attributed to either effects of pathogens per se (i.e. direct losses) or to effects of the immune response triggered by the presence of mammary pathogens (i.e. indirect losses). Test-day milk somatic cell counts (SCC) and number of bacterial colony forming units (CFU) found in milk samples are putative measures of the level of immune response and of the bacterial load, respectively. Mediation models, in which one independent variable affects a second variable which, in turn, affects a third one, are conceivable models to estimate direct and indirect losses. Here, we evaluated the feasibility of a mediation model in which test-day SCC and milk were regressed toward bacterial CFU measured at three selected sampling dates, 1 week apart. We applied this method on cows free of clinical signs and with records on up to 3 test-days before and after the date of the first bacteriological samples. Most bacteriological cultures were negative (52.38%), others contained either staphylococci (23.08%), streptococci (9.16%), mixed bacteria (8.79%) or were contaminated (6.59%). Only losses mediated by an increase in SCC were significantly different from null. In cows with three consecutive bacteriological positive results, we estimated a decreased milk yield of 0.28 kg per day for each unit increase in log2-transformed CFU that elicited one unit increase in log2-transformed SCC. In cows with one or two bacteriological positive results, indirect milk loss was not significantly different from null although test-day milk decreased by 0.74 kg per day for each unit increase of log2-transformed SCC. These results highlight the importance of milk losses that are mediated by an increase in SCC during mammary infection and the feasibility of decomposing total milk loss into its direct and indirect components.

Genotypic and phenotypic characterization of methicillin-resistant Staphylococcus aureus (MRSA) isolated from milk of bovine mastitis.

The aim of this study was to evaluate the presence of methicillin-resistant Staphylococcus aureus (MRSA) among a (S. aureus) collection (n = 430) isolated from milk of cows suffering from mastitis in Belgium and to compare their genotypic as well as phenotypic characteristics. Pulsed field gel electrophoresis (PFGE) and PCR-based typing techniques (MLST, spa, SCCmec, and agr typing) have been applied and supplemented by capsule serotyping, biofilm production quantification and antimicrobial susceptibility testing. Nineteen MRSA were isolated. Seven distinct ApaI PFGE patterns were observed. All isolates, except one, were identified as ST398 strains. Three spa types (t011, t567 and t108) and two SCCmec types (IV and V) were identified. All isolates belonged to agr type I and capsule type 5 and were Panton-Valentine leukocidin (PVL) negative. All isolates produced biofilm in TSBglc , whereas the majority did not in milk serum. Twelve resistance patterns were observed, with almost two-thirds of the isolates being resistant to at least six antibiotics, including penicillin and tetracycline. Our study confirms that the emerging ST398 LA-MRSA clone has attained Belgian cattle. With regard to genotypic and phenotypic typing, the 19 MRSA isolated in this study form a homogenous group and do not differ much from one another, neither from what has been previously described.

Pyrosequencing of epizootic rabbit enteropathy inocula and rabbit caecal samples.

The aetiological agent of epizootic rabbit enteropathy (ERE) is still unknown although a bacterial infection seems the most likely hypothesis. In this study, amplification of the V5 and V6 regions of 16SrDNA from four virulent and two non-virulent caecal samples was performed using a pyrosequencing platform. The virulent samples did not group in the same cluster. The bacterial flora identified was both different and richer than the cultivable bacterial flora. These findings highlight the need for biomolecular techniques to identify the aetiological agent of ERE.

Prevalence of Salmonella serotypes in male mule ducks in Belgium.

Salmonella species infections of male mule ducks were studied for 32 months in 100 flocks on nine duck farms in Belgium. The prevalence of Salmonella species infections changed significantly over time (P<0.001) with infection rates of 50, 13.4, 6.7, 2.6 and 2.9 per cent, respectively, at the time of arrival on the farm, at three, six and nine weeks of age, and when the ducks left the breeding unit to enter the force-feeding rooms (at 11 or 12 weeks of age). During the study period, 95 strains of Salmonella were isolated, belonging to 11 serotypes. S Indiana (42.1 per cent) and S Regent (36.8 per cent) were the two most common serotypes, whereas S Typhimurium and S Enteritidis were found only once (1.1 per cent). All isolated strains were resistant to at least two antimicrobials, but resistance to more than five antimicrobials was observed in 21.6 per cent of the strains.

Typing of O26 enterohaemorrhagic and enteropathogenic Escherichia coli isolated from humans and cattle with IS621 multiplex PCR-based fingerprinting.

AIMS: This study evaluated a typing method of O26:H11 enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC) based on the variation in genomic location and copy numbers of IS621. METHODS AND RESULTS: Two multiplex PCRs, targeting either the left (5') or right (3') IS/chromosome junction of 12 IS621 insertion sites and one PCR specific of another truncated copy, were developed. Thirty-eight amplification profiles were observed amongst a collection of 69 human and bovine O26:H11 EHEC and EPEC. Seventy-one per cent of the 45 EHEC and EPEC with identical IS621 fingerprints within groups of two, three or four isolates had >85% pulsed field gel electrophoresis (PFGE) profile similarity, including four groups of epidemiologically related EHEC or EPEC, while most of the groups had <85% similarity between each others. Epidemiologically related EHEC from each of three independent outbreaks in Japan and Belgium also exhibited identical IS621 fingerprints and PFGE profiles. CONCLUSIONS: The IS621 fingerprinting and the PFGE are complementary typing assays of EHEC and EPEC; though, the former is less discriminatory. SIGNIFICANCE AND IMPACT OF THE STUDY: The IS621 printing method represents a rapid (24 h) first-line surveillance and typing assay, to compare and trace back O26:H11 EHEC and EPEC during surveys in farms, multiple human cases and outbreaks.

Epizootic rabbit enteropathy: comparison of PCR-based RAPD fingerprints from virulent and non-virulent samples.

Epizootic rabbit enteropathy is a gastrointestinal disorder of unknown aetiology of farmed rabbits characterised by inanition and mortality. Genomic analyses of virulent and non-virulent samples of inocula were performed using random amplification of polymorphic DNA (RAPD). Differences in bacterial DNA composition were found between inocula, but specific sequences were not linked with field cases of epizootic rabbit enteropathy.

Enteropathogenic (EPEC), enterohaemorragic (EHEC) and verotoxigenic (VTEC) Escherichia coli in wild cervids.

AIMS: The aim of this study was to investigate the presence of enteropathogenic (EPEC), enterohaemorragic (EHEC) and verotoxigenic (VTEC) Escherichia coli strains in free-ranging wild ruminants in Belgium and to characterize the positive isolates (serogroups and virulence-associated factor-encoding genes). METHODS AND RESULTS: Escherichia coli strains isolated from faeces of wild cervids were characterized by PCR targeting genes coding for the main virulence properties of EPEC, EHEC and VTEC strains. The prevalence rate of these pathogenic strains in faecal samples obtained from the wild ruminants was found to be 15%. No pathogenic isolate was found to belong to the O157, O26, O111, O103 or O145 serogroups. Moreover, a new gene, eibH, showing 88% identity with eibG was detected in VTEC strains. CONCLUSIONS: The results reveal that wild ruminants could be considered as a potential source of VTEC and EPEC infection for humans and possibly also for domestic ruminants. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study suggests the potential risk of transmission of VTEC, EHEC and EPEC strains from wild ruminants to humans via the consumption of venison and to domestic ruminants because of sharing of the same pasture. Indeed, many serogroups other than O157 EHEC have also been shown to be responsible for outbreaks in humans in several countries, and studies focusing solely on O157:H7 EHEC tend to underestimate this risk of transmission.