Reliable differentiation of a non-toxigenic tox gene-bearing Corynebacterium ulcerans variant frequently isolated from game animals using MALDI-TOF MS.

Corynebacterium (C.) ulcerans is a zoonotic member of the C. diphtheriae group and is known to cause abscesses in humans and several animal species. Toxigenic strains, expressing the tox gene encoding diphtheria toxin, are also able to cause diphtheria in humans. In recent years, a non-toxigenic but tox gene-bearing (NTTB) variant of C. ulcerans has been identified that was frequently isolated from clinically healthy as well as from diseased wildlife animals, especially wild boars (Sus scrofa scrofa) in Germany and Austria. The described clinical cases showed similar signs of disease and the isolated corynebacteria displayed common genetic features as well as similar spectroscopic characteristics, therefore being assigned to a so called wild boar cluster (WBC). This study describes the establishment and validation of a method using MALDI-TOF mass spectrometry for a reliable differentiation between various members of the C. diphtheriae group at species level as well as a reliable sub-level identification of C. ulcerans isolates of the WBC variant. For this study 93 C. ulcerans isolates from wildlife animals, 41 C. ulcerans isolates from other animals and humans, and 53 isolates from further representatives of the C. diphtheriae group, as well as 26 non-diphtheriae group Corynebacteria collected via the MALDI user platform from seven MALDI users were used. By assigning 86 C. ulcerans isolates to the WBC the extensive geographical distribution of this previously less noticed variant in two Central European countries could be shown.

Differences in antimicrobial resistance of commensal Escherichia coli isolated from caecal contents of organically and conventionally raised broilers in Austria, 2010-2014 and 2016.

The prevalence of antimicrobial resistance in commensal Escherichia coli isolated from organically raised broiler flocks was compared to the prevalence in isolates from conventional flocks. From 2010 to 2014, and in 2016, resistance trends and multidrug resistance in isolates from the caecal contents of flocks from both broiler production forms were analyzed. Samples were taken in four abattoirs accounting for at least 90% of the national slaughtered broiler population. In total, 962 commensal E. coli were obtained from organically raised broiler flocks (n = 142) and from conventionally raised broiler flocks (n = 820). The mean prevalence of commensal E. coli isolates, which were fully susceptible to the antimicrobials tested, was 43.3% in organically raised broiler flocks and thus significantly higher (P < 0.001) compared to 16.7% in conventionally operated flocks. During the study period, the proportion of fully susceptible isolates increased significantly in both broiler populations. Antimicrobial resistance rates were significantly lower in commensal E. coli isolated from organic compared to conventional production regarding ciprofloxacin (33.3% versus 69.1%), nalidixic acid (33.7% versus 67.4%), sulfamethoxazole (26.7% versus 39.9%), ampicillin (19.0% versus 33.8%) and trimethoprim (12.8% versus 24.9%). Regarding tetracycline, tigecycline and ceftazidime resistance rates were slightly but not significantly higher in isolates from organic flocks (27.6% versus 25.9%; 4.0% versus 1.4%; 2.0% versus 1.9%). This fact is surprising for tetracycline, as none of the investigated organic flocks had been treated with this antimicrobial during their lifetime. No resistances were found in isolates from both production forms against colistin and meropenem, and from organic flocks against azithromycin. The annual prevalence of resistance against ciprofloxacin and nalidixic acid decreased significantly in isolates from both broiler production forms. In isolates from organic flocks, it also decreased regarding ampicillin and sulfamethoxazole. Significant increasing trends were observed in the resistance prevalence against trimethoprim and borderline significantly for ampicillin in commensal E. coli from conventional flocks. Multidrug resistance was detected at a significantly higher prevalence in isolates from conventionally raised flocks (35.1%) compared to organic flocks (22.7%). Findings from this study clearly indicate the influences of organic compared to conventional broiler production practices on the prevalence of antimicrobial resistance in commensal E. coli from broiler flocks.

Petting zoos as sources of Shiga toxin-producing Escherichia coli (STEC) infections.

Despite their general low incidence, Shiga toxin-producing Escherichia (E.) coli (STEC) infections are considered an important public health issue due to the severity of illness that can develop, particularly in young children. We report on two Austrian petting zoos, one in Tyrol (2015) and one in Vorarlberg (2016), which were identified as highly likely infection sources of STEC infections. The petting zoo related cases involved a case of hemolytic uremic syndrome (HUS) due to STEC O157:HNM in 2015 and an outbreak of STEC O157:H7 infections affecting five young children and two adults in 2016. The HUS case accounted for 2.8% of the 36 STEC O157:HNM/H7 infections notified in Austria in 2015 (5,9% of 17 HUS cases). The seven cases described for 2016 accounted for 4.0% of the 177 human STEC infections documented for Austria in 2016, and for 19.4% of the 36 STEC O157:HNM/H7 infections notified that year. The evaluation of the STEC infections described here clearly underlines the potential of sequence-based typing methods to offer suitable resolutions for public health applications. Furthermore, we give a state-of-the-art mini-review on the risks of petting zoos concerning exposure to the zoonotic hazard STEC and on proper measures of risk-prevention.

Prevalence of most common human pathogenic Campylobacter spp. in dogs and cats in Styria, Austria.

The aim of this study was to determine the frequency of occurrence of most common human pathogenic Campylobacter species, Campylobacter jejuni (C. jejuni) and Campylobacter coli (C. coli), in dogs and cats in Styria, Austria. In the period from April 2010 to April 2012, 842 faecal samples from dogs and cats from Styria, Austria were examined for Campylobacter (C.) species (spp.). All samples were subjected to qualitative microbiological culture testing, and additionally, some of them have been studied using qualitative real-time PCR. In microbiological culture, 5.9% of all samples investigated were C. spp. positive. With 3.1% out of positive samples, C. jejuni was the most common type. Campylobacter upsaliensis (C. upsaliensis) was detected only in 0.5% of the samples. The remaining positive samples (2.4%) were classified as C. species (sp.). C. coli could not be found in any of the samples. A higher prevalence of C. jejuni was found in kittens with 14.3% and in diarrhoeic dogs (7.4%) and cats (23.8%). The real-time PCR revealed for dogs and cats together, 27% of C. jejuni-positive faecal and 8% positive faecal swap samples. The obtained C. jejuni strains underwent antibiotic resistance testing using three different tests (agar diffusion, MIC testing and E-test) with different numbers of antibiotics. From the antibiotics used in this study, several showed high test-dependent resistance rates (cephalexin, cefovecin, kanamycin, sulfamethoxazole/trimethoprim, penicillin G, ciprofloxacin, enrofloxacin, marbofloxacin, nalidixic acid). Overall, the prevalence of C. spp. in this study was very low compared to others, with the exception of C. jejuni in kittens and diarrhoeic animals. The results of the real-time PCR suggest that the rate of colonization of C. jejuni was actually higher than the results of the culture showed. As the resistance rates of C. jejuni isolates partly were very high, possible transmission of (multi-) resistant C. jejuni strains to humans especially from kittens and diarrhoeic animals must be expected.

Estimating sensitivity and specificity of a PCR for boot socks to detect Campylobacter in broiler primary production using Bayesian latent class analysis.

The present study compares three different assays for sample collection and detection of Campylobacter spp. in broiler flocks, based on (i) the collection of faecal samples from intestinal organs (caecum), (ii) individual faecal droppings collected from the bedding and (iii) faecal material collected by socks placed on the outside of a pair of boots (boot socks) and used for walking around in the flock. The two first methods are examined for Campylobacter using a culture method (ISO-10272-2:2006), while the boot socks are tested using PCR. The PCR-assay is a genus specific multiplex PCR with primers targeting 16S rDNA in Campylobacter and primers targeting Yersinia ruckerii. Sixty-seven broiler flocks from Austria and 83 broiler flocks from Denmark were included in this prospective study and 89 of these were found to be positive in at least one method (AT: 49 samples, DK: 40 samples) whereas 61 of these were negative in all assays. In Austria samples for the three assays were collected simultaneously, which facilitates a direct comparison of the diagnostic test performance. In Denmark, however, boot socks and faecal droppings were collected three days before slaughter while caecum samples were collected at slaughter. The results were evaluated in the absence of a gold standard using a Bayesian latent class model. Austrian results showed higher sensitivity for PCR detection in sock samples (0.98; Bayesian credible interval (BCI) [0.93-1]) than for culture of faecal droppings (0.86; BCI [0.76-0.91]) or caecal samples (0.92; BCI [0.85-0.97]). The potential impact of Campylobacter introduction within the final three days before slaughter was observed in Denmark, where four flocks were tested negative three days before slaughter, but were detected positive at the slaughterhouse. Therefore the model results for the PCR sensitivity (0.88; BCI [0.83-0.97]) and cultural ISO-method in faecal samples (0.84; BCI [0.76-0.92]) are lower than for caecal samples (0.93; BCI [0.85-0.98]). In our study, PCR detection on boot sock samples is more sensitive than conventional culture. In view of the advantage of rapid results before slaughter and low costs for sampling, especially in combination with existing Salmonella surveillance systems (just another pair of boot socks needed), this method-matrix combination could be a valuable surveillance tool in the broiler primary production.

[Implementation and results of the EU-wide baseline studies on the prevalence of Salmonella spp. in slaughter and breeding pigs in Austria]. / Umsetzung und Ergebnisse der EU-weiten Grundlagenerhebung zur Prävalenz von Salmonella spp. in Schlacht- und Zucht- schweinen in Osterreich.

The Member States of the European Union are following a common strategy on the control of Salmonella and other foodborne pathogens (Anonym, 2003). Within that framework baseline studies on the most relevant animal populations have been carried out. This paper describes the implementation and the results of the baseline studies on Salmonella spp. in slaughter and breeding pigs in Austria. A total of 647 slaughter pigs were sampled in 28 slaughterhouses between October 2006 and September 2007. Samples were taken from the ileocaecal lymphnodes to detect infection in pigs and from the surface of the carcasses to detect contamination. Out of the 617 datasets included in the final analysis, Salmonella prevalences of 2% in lymphnodes and 1.1% on the carcass surface were observed. S. Derby, S. Enteritidis and S. Typhimurium were the three most frequently identified serovars. In an additional study, a total of 252 holdings with breeding pigs has been sampled between January and December combined multiplier herds. Respectively prevalences of 5, 8, 5, 3 and 9.1% were obtained, with S. Typhimurium being the most frequently isolated serovar. Overall, compared to neighbouring Member States a rather low prevalence of Salmonella spp. in pigs was documented for Austria, in particular in slaughter pigs.The serovar distribution seemed to be similar throughout the pig populations, some also being represented in Austrian human isolates. Contamination of feed seems to play a minor role considering the overall low prevalence, but nevertheless has to be taken into account in any future control or monitoring strategy for Salmonella spp. in pigs.

[Implementation and results of the EU-wide baseline study on the prevalence of Salmonella spp. in flocks of turkeys in Austria]. / Umsetzung und Ergebnisse der EU-weiten Grundlagenstudie zur Prävalenz von Salmonella spp. in Putenherden in Osterreich.

The member states of the European Union (EU) are following a common strategy on the control of Salmonella and other foodborne zoonotic pathogens (Anonym, 2003). In order to establish the prevalence of Salmonella (S.) spp. in turkeys, a baseline survey was organised in between October 2006 and September 2007. In Austria a total of 202 turkey flocks in 104 holdings was included. By doing so, all holdings with fattening turkeys were covered by the sampling scheme, whereas breeding turkey flocks did not exist in the survey period. A prevalence of Salmonella spp. of 17.3% in flocks or 25% of holdings was obtained. A total of nine different serovars were identified, S. Hadar being the most frequently isolated with eleven flocks infected representing 31.4% of all. S. Montevideo and S. Saintpaul were both found in eight flocks, and S. Senftenberg in six flocks. Two flocks were colonized with S. Newport, while one flock each had S. Typhimurium, S. Derby, S. Blockley and a monophasic strain of Salmonella group B. Of the two serovars of highest relevance for human health, S. Typhimurium was detected in only 0.5% of flocks or 1% of holdings tested and S. Enteritidis was not detected at all. Taking into account the high prevalence of the various Salmonella serovars however control measures to prohibit horizontal transmission and entry of Salmonella spp. from the turkey reservoir into the food chain in Austria seem justified.

A 2010 Austrian Salmonella enteritidis PT4 outbreak associated with a laying hen holding previously involved in an S. enteritidis PT4 cluster: pitfalls of regulatory responses in risk management.

We report on an outbreak caused by Salmonella enteritidis phage type 4 (PT4) among 143 participants at a soccer camp in Austria in August 2010. The outbreak affected 34 persons, including 24 epidemiologically related cases and 10 laboratory-confirmed cases. Food-specific cohort analyses revealed spaetzle (homemade noodles) (relative risks (RR): 2.68; 95% CI: 1.13-6.45), hamburger (RR: 2.70; 95% CI: 1.13-6.45) and potato salad (RR: 2.91; 95% CI: 1.69-5.02) as the most biologically plausible infection sources. Eggs used as ingredients were considered to be the vehicle of infection for the outbreak strain. The sole egg producer supplying the hotel that housed the soccer camp participants with table eggs operated two flocks. One flock had been epidemiologically and microbiologically related to a previous S. enteritidis PT4 outbreak affecting the same Austrian province in the four months preceding the August outbreak. We hypothesize that eggs from this flock, already condemned for industrial use only, were falsely declared table eggs and sold among eggs from the non-banned flock causing the subsequent outbreak. In Austria, the illegal distribution of eggs designated for industrial use (i.e., false declaration of these eggs as table eggs) has been previously documented. Our findings underscore the potential of proper epidemiological outbreak investigation to identify the pitfalls of regulatory responses in risk management.

Prevalence of Salmonella spp. in Austrian broiler flocks in the context of the EU-wide baseline survey 2005-2006.

In Austria an EU-wide baseline survey on the prevalence of Salmonella spp. in broilers organized by the EU commission was conducted from October 2005 to September 2006. The aim of this study was to produce comparable data on the prevalence of Salmonella in broiler flocks and holdings for all member states and for the EU-Commission to set EU-wide targets for the control of Salmonella in the broiler populations. A randomised sampling plan was designed according to EU-commission parameters (p = 50%; CI = 95%, a = 5%). Sampling was carried out regularly throughout the whole year. On every farm one flock was sampled with five pairs of boot swabs and analysed in the lab according to appendix D of ISO 6579 (2002). In Austria, 363 flocks on farms consisting of at least 5000 broilers each were tested. 28 flocks (7.7%) showed infections with Salmonella spp., eight flocks (2.2%) had either S. Enteritidis (six flocks) or S. Typhimurium (two flocks). In detail, S. Enteritidis (1.7%), S. Typhimurium (0.6%), S. Montevideo (4.1%), S. Infantis 0.6%, S. Senftenberg, S. Tennessee and S. Virchow (0.3% each) have been found. Data indicated that the risk of vertical transmission of Salmonella spp. to broiler flocks has almost been kept at bay; however, the risk of horizontal transmission still needs attention. Contamination of feeding stuff, possible persistence, spreading between barns of a farm as well as introduction of Salmonella spp. through individuals or materials are important factors for future control strategies.

Clostridium difficile: a new zoonotic agent?

Clostridium difficile is mainly considered a nosocomial pathogen associated with diarrhea and pseudomembranous colitis in hospitalized patients. Austrian hospitals reported 2761 cases of C. difficile infection (including 277 lethal outcomes) in 2007, compared with 777 cases (including 54 lethal outcomes) in 2003. The occurrence of community-acquired C. difficile infection is also increasingly reported. Recent studies have shown the occurrence of C. difficile in food and animals. The aim of the present study was to determine the occurrence of C. difficile in food and animals in Austria. Between March and July 2008, gut or fecal samples from 67 cows, 61 pigs and 59 broiler chickens were collected at Austrian abattoirs. Between February and April 2008 meat samples (51 beef [25 ground], 27 pork [17 ground] and 6 samples of chicken meat) were purchased at retail outlets. Of the 187 samples tested, eight yielded C. difficile: in cows 3/67 samples (4.5%) were positive, in pigs 2/61 (3.3%), in broiler chickens 3/59 (5%). Six of the eight isolates yielded toxigenic C. difficile (toxins A and B): 2/67 (3%) cow samples, 2/61 (3.3%) pig samples, 2/59 (3.4%) chicken samples. Genes for the binary toxin were detected in one of the two pig isolates, a PCR ribotype 126 strain. None of the 84 meat samples yielded C. difficile. The results of this Austrian study suggest that animal reservoirs are possible sources, via food, of human C. difficile infection.

A foodborne outbreak of Salmonella Enteritidis phage type 6 in Austria, 2008.

In April 2008, a general foodborne outbreak of Salmonella Enteritidis phage type 6 affected nine persons (two households in Salzburg and one household in Tyrol; eight microbiologically confirmed cases and one possible case). Epidemiological investigation revealed that all cases had eaten lunch together at a farm. Homemade bread dumpling loaf, prepared with eggs from the farm, was the most likely vehicle for the pathogen. Six eggs were bacteriologically tested and yielded the outbreak strain on the egg shells. The farm's egg laying flock consisted of 12 hens, which were identified as the source of infection by isolating the outbreak strain from samples of hen fecal material and dust. Molecular subtyping using multiple loci variable number of tandem repeats analysis (MLVA) revealed a pattern unique to isolates from the outbreak, different from nine epidemiologically unrelated isolates tested for comparison. The laying hens were culled and no further cases of salmonellosis were associated with the farm. This example demonstrates the considerable potential of outbreak investigation to elucidate the transmission of infection along the food chain and to provide information essential for implementation of targeted measures for prevention of foodborne illness.

Lessons learned from a Salmonella enteritidis phage type 4 outbreak in Austria, 2005.

An outbreak of gastroenteritis due to Salmonella Enteritidis phage type 4 occurred in people who attended a traditional hunting festivity in a small village in western Austria 6 through 11 November 2005. Of approximately 250 attendees, 227 had consumed dishes offered at the festival, and of these consumers 35 persons fulfilled the outbreak case definition (attack rate of 15.4%). Spätzle (traditional pastalike side dish) was most likely the contaminated part of the incriminated main course (relative risk of 18.9, 95% confidence interval of 4.6 to 76.7; P < 0.001). Thirteen eggs that remained from the preparation of the spätzle were negative for Salmonella when tested individually without shell disinfection, as were 1200 eggs collected at the egg production plant and examined with shell disinfection. The back-traced egg production farm had been initially certified as Salmonella free by a voluntary quality control program. However, an intensified environmental investigation of the incriminated egg production farm performed in the first quarter of 2006 and based on an appropriate method of sampling revealed Salmonella Enteritidis phage type 4 in 4 of 13 flocks. Although a combination of epidemiological and microbiological investigations allowed elucidation of the mode of spread, no restrictions were placed on the incriminated flocks of laying hens. These flocks were kept in production until they were stalled out due to age in August 2006. In June 2006, a cluster of 23 cases of Salmonella Enteritidis phage type 6 infection was again associated with this egg production farm. Evidence provided by epidemiological analyses is often disregarded by decision makers. However, negative results from microbiological testing of food involved in an outbreak are often weighted as strong evidence against a causal association between that food and the outbreak.

[Screening for antibodies against zoonotic agents among employees of the Zoological Garden of Vienna, Schönbrunn, Austria]. / Untersuchungen auf Antikörper gegen Zoonoseerreger bei Angestellten des Wiener Tiergartens Schönbrunn.

The aim of this study was to investigate the prevalence of antibodies against zoonotic agents in employees of the zoological garden of Vienna, Schönbrunn, Austria. Sixty out of 120 employees participated in the study. In 97% of them antibodies to at least one zoonotic agent were identified. Only two participants were free of antibodies to the zoonotic agents tested. The following seroprevalences (in brackets) were obtained: Viral zoonotic (and potentially zoonotic) agents: Influenzavirus A/H1N1 (58%), Influenzavirus A/H3N2 (85%), Lymphocytic choriomeningitis virus (13%), Encephalomyocarditis virus (5%), Orthopox- (Cowpox-) virus and Hantavirus type Puumala (3%). Hantavirus type Hantaan and Borna disease virus (all negative). Bacterial zoonotic agents: Bartonella henselae (65 %), Borrelia burgdorferi (10%), Leptospira interrogans serovar copenhageni and serovar icterohaemorrhagiae as well as Chlamydophila psittoci (2% each). Brucella spp., Coxiella bumetii, and Francisella tularensis (all negative). Parasitic zoonotic agents: Toxoplasma gondii (53%), Toxocara spp. (21%), Capillaria hepatica (2%), Fasciola hepatica, Schistosoma mansoni, E. multilocularis, and E. granulosus (all negative). The remarkably high seroprevalence to the causative agent of cat scratch disease, Bartonella henselae, is probably due to the private contact of the employees to cats. Regarding viral zoonotic agents it has to be mentioned that Influenzavirus vaccination and/or human-to-human transmission of especially A/H3N2 Influenzaviruses probably attributed significantly to the very high seroprevalence to both Influenzavirus types A/H1N1 and A/H3N2. When investigating parasitic zoonotic agents, high prevalence rates were found against Toxoplasma gondii and Toxocara spp., however, it was not possible to establish a causal link between seropositivity and the professional activity in the zoo. Interestingly, in the case of antibodies to T. gondii, the typical correlation with age was not found in this study, while in the case of the Toxocara spp. positive subjects a correlation was identified with both age and duration of employment in the zoo. Regarding the later two zoonotic parasites, employees of the zoological garden showed significantly higher seroprevalences than the average Austrian population. Antibodies to Capillaria hepatica, a hepatic-parasite in rodents which is diagnosed in humans rarely, were identified in one employee and another one showed a questionable positive result. Further investigations did not exhibit clinical infestations with the parasite in these two individuals so far.