Prevalence of Toxoplasma gondii, Hepatitis E Virus, and Salmonella Antibodies in Meat Juice Samples from Pigs at Slaughter in Switzerland.

ABSTRACT: Toxoplasma gondii, hepatitis E virus (HEV), and Salmonella are zoonotic foodborne pathogens that may be transmitted to humans through the consumption of raw or undercooked pork. The aim of this study was to determine the seroprevalence of anti-T. gondii, anti-HEV, and anti-Salmonella antibodies from healthy pigs at slaughter in Switzerland. From August to September 2020, the diaphragm muscle of Swiss fattening pigs was collected in three Swiss abattoirs from 188 farms. Two randomly chosen pig carcasses per farm were selected. On the basis of the slaughter data, we noted the production system and the canton of origin, comparing indoor (n = 120) and free-range (n = 68) farming and regional allocation. The meat juice of these samples was analyzed for pathogen-specific antibodies by using commercial enzyme-linked immunosorbent assay kits. The seroprevalences were 1.3% for T. gondii, 71.8% for the HEV, and 5.3% for Salmonella, respectively. Comparing the origins, the results of many cantons were not meaningful due to the low number of samples. No regional accumulations were found for T. gondii and HEV. The results showed that 2.1% of the farms had least one T. gondii-seropositive animal, 80.3% had at least one HEV-seropositive animal, and 8.5% had at least one Salmonella-seropositive animal, respectively. The seropositivity of T. gondii was higher in free-range pigs than in indoor pigs, whereas anti-Salmonella antibodies were more common in pigs from indoor farming than in outdoor pigs. The seroprevalence of anti-HEV antibodies was similar in free-range and indoor farming pigs. Compared with studies from 2012, the seroprevalence of T. gondii has decreased, whereas the seroprevalence of the HEV has increased and is highly prevalent among fattening pigs in Switzerland. The low seroprevalence of Salmonella has remained stable in recent years.

Evaluation of Three Commercial Interferon-γ Assays in a Bovine Tuberculosis Free Population.

The interferon-γ assay has been used worldwide as an ancillary test for the diagnosis of bovine tuberculosis (bTB). This study aimed to describe, based on the bTB-free status in Switzerland, the difference of applying a more stringent cutoff point of 0.05 compared with 0.1 for bTB surveillance. Moreover, the effect of time between blood collection and stimulation, culture results, optical density values, and the influence of testing different breeds were evaluated. Blood samples from a total of 118 healthy cows older than 6 months were tested with three commercial interferon-gamma assays. To confirm the bTB-free status of the tested animals and to investigate potential cross-reactions with nontuberculous mycobacteria, pulmonary and abdominal lymph nodes in addition to ileal mucosa from each cattle were used for the detection of viable Mycobacteria spp. by specific culture. Significant differences regarding the proportion of false-positive results between the two Bovigam tests and between Bovigam 2G and ID Screen were found. Samples analyzed with Bovigam 2G were 2.5 [95% confidence interval (CI) 1.6-3.9] times more likely to yield a false-positive test result than samples analyzed with Bovigam TB. Similarly, the odds ratio (OR) for testing samples false-positive with ID Screen compared with Bovigam TB was 1.9 (95% CI 1.21-2.9). The OR for testing false-positive with ID Screen compared with Bovigam 2G was less to equally likely with an OR of 0.75 (95% CI 0.5-1.1). When using a cutoff of 0.05 instead of 0.1, the OR for a false-positive test result was 2.2 (95% CI 1.6-3.1). Samples tested after 6 h compared with a delayed stimulation time of 22-24 h were more likely to yield a false-positive test result with an OR of 3.9 (95% CI 2.7-5.6). In conclusion, applying a more stringent cutoff of 0.05 with the Bovigam 2G kit generates a questionable high number of false-positive results of one of three tested animals. Furthermore, specific breeds might show an increased risk to result false-positive in the Bovigam 2G and the ID Screen assays.

Correction: Wist et al. Phenotypic and Genotypic Traits of Vancomycin-Resistant Enterococci from Healthy Food-Producing Animals. Microorganisms 2020, 8, 261.

The authors wish to make the following correction to this paper [...].

Animal petting zoos as sources of Shiga toxin-producing Escherichia coli, Salmonella and extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae.

Animal petting zoos and farm fairs provide the opportunity for children and adults to interact with animals, but contact with animals carries a risk of exposure to zoonotic pathogens and antimicrobial-resistant bacteria. The aim of this study was to assess the occurrence of Shiga toxin-producing Escherichia coli (STEC), Salmonella, extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae and methicillin-resistant Staphylococcus aureus (MRSA) in animal faeces from six animal petting zoos and one farm fair in Switzerland. Furthermore, hygiene facilities on the venues were evaluated. Of 163 faecal samples, 75 contained stx1, stx2 or stx1/stx2 genes, indicating the presence of STEC. Samples included faeces from sika deer (100%), sheep (92%), goats (88%), mouflons (80%), camels (62%), llamas (50%), yaks (50%), pigs (29%) and donkeys (6%), whereas no stx genes were isolated from faeces of calves, guinea pigs, hens, ostriches, ponies, zebras or zebus. Salmonella enterica subsp. enterica serovar Stourbridge (S. Stourbridge) was detected in faecal samples from camels. A total of four ESBL-producing E. coli strains were isolated from faeces of goats, camels and pigs. PCR and sequencing identified the presence of blaCTX-M-15 in three and blaCTX-M-65 in one E. coli. Antimicrobial resistance profiling using the disk diffusion method revealed two multidrug-resistant (MDR) E. coli with resistance to ciprofloxacin, gentamicin and azithromycin, all of which are critically important drugs for human medicine. Multilocus sequence typing identified E. coli ST162, E. coli ST2179, extraintestinal high-risk E. coli ST410 and E. coli ST4553, which belongs to the emerging extraintestinal clonal complex (CC) 648. No MRSA was detected. On all animal petting venues, there were inadequacies with regard to access to hygiene information and handwashing hygiene facilities. This study provides data that underscore the importance of hygiene measures to minimize the risk of transmission of zoonotic pathogens and MDR, ESBL-producing E. coli to visitors of animal petting venues.

Seroprevalence of hepatitis E virus in dogs in Switzerland.

Hepatitis E virus (HEV) is the causative agent of an acute and in most cases self-limiting hepatitis. Of the four major HEV genotypes that infect humans, genotype 3 and 4 are zoonotic and have been identified in humans but predominantly in pigs and wild boar, which are considered the main reservoirs. However, the known host range of zoonotic HEV may be increasing to comprise additional species, including companion animals. Several studies have identified contact with dogs as a risk factor for HEV infection in humans, yet information on the occurrence of HEV in Swiss dogs is lacking. To examine a possible risk of exposure, this study was designed to assess the seroprevalence of HEV in 84 Swiss dogs. Serum and plasma samples collected from four veterinary clinics were screened for HEV-specific antibodies by HEV-antibody ELISA test kit. In addition, information of 22 dogs regarding the country of origin, the type of dog feed and any history of hunting was recorded. Samples from seropositive animals were also screened for the presence of HEV RNA by quantitative real-time RT-PCR (qRT-PCR). Overall, 38% (32 of 84) of the dogs tested seropositive for anti-HEV, indicating exposure to HEV. Among the 22 dogs for which information was available, HEV-specific antibodies were detected in three of five dogs that were born abroad, in one of two dogs that were fed a raw meat-based diet, and in one hunting dog. No viral RNA could be detected in any of the serum and plasma samples; thus, the genotype of the strains remained undetermined. This study provides further evidence for canine exposure and susceptibility to HEV and highlights the need to further assess the risks of HEV transmission to humans with contact to dogs.

Detection of Psychrophilic Clostridium spp. in Fecal Samples from Cattle of Different Ages Sampled at the Slaughterhouse Level.

ABSTRACT: Clostridium estertheticum and C. estertheticum-like spp. are obligate anaerobic psychrophiles causing "blown pack" spoilage of chilled vacuum-packed meat. The present study aimed at detecting and isolating these spoilage bacteria in fecal samples of cattle of different ages at the slaughterhouse level. One hundred two swab fecal samples were obtained and enriched anaerobically in prereduced peptone-yeast-glucose-starch (PYGS) medium for 3 weeks at 4°C and then screened for C. estertheticum and C. estertheticum-like spp. by using a 16S rRNA gene-based real-time PCR (RT-PCR) assay. The RT-PCR-positive samples were further enriched for 3 weeks in prereduced PYGS medium and then subjected to an ethanol (50%, v/v) and lysozyme (4 mg/mL) treatment. Isolation was carried out anaerobically on Columbia agar with 5% defibrinated sheep blood at 4°C for 3 weeks. Isolated strains were identified morphologically and by the 16S rRNA gene. Forty (39%) of 102 samples were RT-PCR positive. The frequency of positive samples was the following: 9 (45%) of 20 in calves (aged ≤160 days), 23 (43%) of 54 in young cattle (aged 161 to 1,000 days), and 8 (29%) of 28 in cows or bulls (aged >1,000 days). Six strains were isolated from 6 of 40 RT-PCR-positive samples. Of these, five were from the calves (n = 1) and young cattle (n = 4). The six isolates were identified as C. estertheticum (n = 1), Clostridium frigoriphilum (n = 1), and C. estertheticum-like spp. (n = 4). The present findings confirm that feces of cattle are an important source of psychrophilic Clostridium spp. The fecal carriage among livestock animals at slaughter is strongly correlated with the risk of carcass contamination. Therefore, the maintenance of slaughter hygiene is of central importance.

Draft Genome Sequence of Psychrobacter okhotskensis Strain 5179-1A, Isolated from a Raw Cured Ham Storage Crate.

We present the draft genome sequence of Psychrobacter okhotskensis strain 5179-1A, which was isolated from a raw cured ham storage crate. Its size and GC content are 3.4 Mb and 43.4%, respectively. The 16S rRNA sequences of strain 5179-1A and P. okhotskensis MD17T are 100% identical.

Phenotypic and Genotypic Traits of Vancomycin-Resistant Enterococci from Healthy Food-Producing Animals.

Food-producing animals may be a reservoir of vancomycin-resistant enterococci (VRE), potentially posing a threat to animal and public health. The aims of this study were to estimate the faecal carriage of VRE among healthy cattle (n = 362), pigs (n = 350), sheep (n = 218), and poultry (n = 102 flocks) in Switzerland, and to characterise phenotypic and genotypic traits of the isolates. VRE were isolated from caecum content of six bovine, and 12 porcine samples respectively, and from pooled faecal matter collected from 16 poultry flock samples. All isolates harboured vanA. Three different types of Tn1546-like elements carrying the vanA operon were identified. Conjugal transfer of vanA to human Enterococcus faecalis strain JH2-2 was observed for porcine isolates only. Resistance to tetracycline and erythromycin was frequent among the isolates. Our data show that VRE harbouring vanA are present in healthy food-producing animals. The vanA gene from porcine isolates was transferable to other enterococci and these isolates might play a role in the dissemination of VRE in the food production chain.

Microarray based genetic profiling of Staphylococcus aureus isolated from abattoir byproducts of pork origin.

Many parts of pork meat processing are currently not used for human consumption in Switzerland, although they are of great nutritional value. Therefore, data on the occurrence of pathogenic organisms on byproducts is extremely scarce and the prevalence and population structure of Staphylococcus aureus on meat processing sidestreams is unknown. Hence, abattoir byproducts of pork origin including ear, forefoot, heart, intestine, liver, rib bone, sternum, bladder, stomach, hind foot and tongue originating from six abattoirs were screened for S. aureus. The obtained isolates were investigated by spa typing and DNA microarray analysis to reveal their genomic profile and population structure. The prevalence of S. aureus was generally low with a mean of 8%. In total, 40 S. aureus strains were detected and assigned to 12 spa types (t015, t1491, t1778, t091, t337, t899, t2922, t7439, t1333, t208, t4049, t034) and seven clonal complexes (CC1, CC7, CC9, CC30, CC45, CC49, CC398). Detected enterotoxin genes included sea, seb, sec, seh, sel and egc encoded toxin genes seg, sei, sem, sen, seo, and seu. None of the isolates harbored genes conferring methicillin resistance, but blaZ/I/R genes causing penicillin resistance were frequently found. In addition, strains from CC398 exhibited tetM and tetK, conferring tetracycline resistance. Similarity calculations based on microarray profiles revealed no association of clonal complexes with particular body parts, but revealed a certain correspondence of clonal complex and originating abattoir.

Sequence Types and Antimicrobial Resistance Profiles of Streptococcus uberis Isolated From Bovine Mastitis.

Bovine mastitis is one of the most common diseases among dairy cows and causes high economic losses in dairy industries worldwide. Streptococcus uberis is one of the most frequently identified pathogens causing the disease. In this study, 153 S. uberis strains isolated from mastitis milk samples were analyzed for their genetic diversity using multi locus sequence typing (MLST). Moreover, antibiotic susceptibility testing was performed using a microdilution assay and 11 antimicrobial agents including penicillin, which is the first line agent for treatment of bovine mastitis in Switzerland. MLST was successful for 152 (99.3%) of the strains. Overall, 103 different sequence types (STs) were determined, including 91 novel STs. S. uberis belonging to clonal complex (CC) 5 represented 47 (30.7%) of the mastitis cases. Two (1.3%) of the strains belonged to CC86 and one (0.7%) to CC143. The population structure identified in this work suggests that environmental transmission is the predominant route of infection in herds in Switzerland. Antimicrobial susceptibility testing determined a resistance rate of 11.8% for pirlimycin and elevated MIC90-values for marbofloxacin as well as for erythromycin. This study highlights the importance of genetic characterization of S. uberis and the need for veterinary breakpoints for surveillance of antimicrobial resistance in S. uberis.

Molecular types, virulence profiles and antimicrobial resistance of Escherichia coli causing bovine mastitis.

BACKGROUND: Escherichia coli is an important aetiological agent of bovine mastitis worldwide. METHODS: In this study, 82 E. coli from bovine mastitis milk samples from 49 farms were analysed for their genetic diversity using phylogenetic grouping and multilocus sequence typing. The isolates were examined by PCR for a selection of virulence factors (VFs). Antimicrobial susceptibility profiles were assessed using the disk diffusion method. RESULTS: The most prevalent phylogroups were group B1 (41.5 per cent of the isolates) and group A (30.5 per cent). A variety of 35 different sequence types (STs) were identified, including ST1125 (11 per cent), ST58 (9.8 per cent), ST10 (8.5 per cent) and ST88 (7.3 per cent). Aggregate VF scores (the number of unique VFs detected for each isolate) ranged from 1 to 3 for 63.4 per cent of the isolates and were at least 4 for 12.2 per cent. For 24.4 per cent of the isolates, the score was 0. The three most frequent VFs were traT, fyuA and iutA. The majority (72 per cent) of the isolates harboured traT. The majority (68.3 per cent) of the isolates were fully susceptible to all antimicrobials tested, with 22 per cent resistant to ampicillin and 14.6 per cent to tetracycline. Resistance rates were low for gentamicin (3.7 per cent), amoxicillin/clavulanic acid (2.4 per cent) and ceftiofur (1.2 per cent), respectively. CONCLUSION: Among the study's sample population, E. coli strains were genotypically diverse, even in cows from the same farm, although some STs occurred more frequently than others. Susceptibility to clinically relevant compounds remained high.

Serotypes and virulence profiles of Shiga toxin-producing Escherichia coli strains isolated during 2017 from human infections in Switzerland.

Since 2015, the Swiss Federal Office of Public Health registered an increase of notifications of STEC, probably due to the adoption of culture independent stx screening tests in diagnostic laboratories. This study aimed to identify the serotypes and virulence genes of 120 STEC isolated from human clinical stx positive specimens during 2017 in order to estimate any changes in serotype distribution and toxin profiles of STEC compared to the time span 2010-2014. Culturing of STEC from stool samples was achieved using the streak plate technique on MacConkey agar. We performed O and H serotyping by PCR and by micro array. Virulence genes were identified and subtyped using molecular methods, including stx1 and stx2 subtypes, and the intimin encoding gene, eae. STEC were recovered from 27.5% of the stx positive samples. STEC O157:H7 accounted for 7.5% of all isolates, and STEC O80:H2, O91:H10/H14/H21, O103:H2/H11, and O26:H11 accounted for 36.9% of the non-O157 strains. Forty-five isolates with stx1 variants, 47 with stx2 variants and 28 isolates with both stx1 and stx2 variants were identified. Forty (33.3% of all isolates) carried the subtypes associated with high pathogenic potential, stx2a, stx2c, or stx2d. The eae gene for intimin was detected in 54 strains (45% of all strains). Compared to 2010-2014, our data show that the proportion of the so called "top five" serogroups, STEC O26, O111, O103, and O157 declined from 53.7% to 28.3% in 2017. The proportion of isolates with stx2a, stx2c, or stx2d decreased from 50.5% to 33.3%. We also observed an increase of STEC harbouring the low pathogenic subtypes stx2b and stx2e from 12.6% to 29.2%, and of eae negative STEC from 29.5% in 2010-2014 to 55% in 2017. Simultaneously, there was a sharp increase of the patients' median age from 24 years to 46.5 years. Clinical manifestations in the patients included abdominal pain without diarrhea (22.3%), diarrhea (77.7%), and the haemolytic-uremic syndrome (HUS) (7.4%). Our data show that a greater number and a wider range of STEC serotypes are detected by culture-independent testing, with implications for public health services.

Population structure and virulence gene profiles of Streptococcus agalactiae collected from different hosts worldwide.

Streptococcus agalactiae is a leading cause of morbidity and mortality among neonates and causes severe infections in pregnant women and nonpregnant predisposed adults, in addition to various animal species worldwide. Still, information on the population structure of S. agalactiae and the geographical distribution of different clones is limited. Further data are urgently needed to identify particularly successful clones and obtain insights into possible routes of transmission within one host species and across species borders. We aimed to determine the population structure and virulence gene profiles of S. agalactiae strains from a diverse set of sources and geographical origins. To this end, 373 S. agalactiae isolates obtained from humans and animals from five different continents were typed by DNA microarray profiling. A total of 242 different S. agalactiae strains were identified and further analyzed. Particularly successful clonal lineages, hybridization patterns, and strains were identified that were spread across different continents and/or were present in more than one host species. In particular, several strains were detected in both humans and cattle, and several canine strains were also detected in samples from human, bovine, and porcine hosts. The findings of our study suggest that although S. agalactiae is well adapted to various hosts including humans, cattle, dogs, rodents, and fish, interspecies transmission is possible and occurs between humans and cows, dogs, and rabbits. The virulence and resistance gene profiles presented enable new insights into interspecies transmission and make a crucial contribution to the identification of suitable targets for therapeutic agents and vaccines.

Complete Genome Sequence of Anoxybacillus flavithermus Strain 52-1A Isolated from a Heat-Processed Powdered Milk Concentrate.

The thermophilic spore-forming bacterium Anoxybacillus flavithermus is responsible for powdered milk product spoilage, and its presence in dairy processing environments is a concern. Here, the complete genome sequence of the A. flavithermus strain 52-1A isolated from a heat-processed powdered milk product concentrate in Switzerland is presented.

Extended-spectrum-ß-lactamase-producing Enterobacteriaceae isolated from vegetables imported from the Dominican Republic, India, Thailand, and Vietnam.

To examine to what extent fresh vegetables imported into Switzerland represent carriers of extended-spectrum-ß-lactamase (ESBL)-producing Enterobacteriaceae, 169 samples of different types of fresh vegetables imported into Switzerland from the Dominican Republic, India, Thailand, and Vietnam were analyzed. Overall, 25.4% of the vegetable samples yielded one or more ESBL-producing Enterobacteriaceae, 78.3% of which were multidrug resistant. Sixty isolates were obtained: Escherichia coli, 26; Klebsiella pneumoniae, 26; Enterobacter cloacae, 6; Enterobacter aerogenes, 1; and Cronobacter sakazakii, 1. We found 29 isolates producing CTX-M-15, 8 producing CTX-M-14, 7 producing CTX-M-55, 3 producing CTX-M-65, 1 each producing CTX-M-1, CTX-M-3, CTX-M-27, and CTX-M-63, 5 producing SHV-2, 3 producing SHV-12, and 1 producing SHV-2a. Four of the E. coli isolates belonged to epidemiologically important clones: CTX-M-15-producing B2:ST131 (1 isolate), D:ST405 (1 isolate), and D:ST38 (2 isolates). One of the D:ST38 isolates belonged to the extraintestinal enteroaggregative E. coli (EAEC) D:ST38 lineage. Two of the K. pneumoniae isolates belonged to the epidemic clones sequence type 15 (ST15) and ST147. The occurrence of antibiotic-resistant pathogenic and commensal Enterobacteriaceae in imported agricultural foodstuffs constitutes a source of ESBL genes and a concern for food safety.