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Brucella suis commonly infects swine but occasionally also other animal species and humans. Wild boars are the most important reservoir of B. suis biovar 2, continually infecting susceptible hosts through close contact. Nevertheless, the genetic diversity of B. suis in wildlife remains understudied. Here, we typed 17 Slovenian B. suis biovar 2 isolates obtained in 2017–2019 from wild boars (n = 16) and a hare (n = 1) using whole-genome sequencing (WGS). To assess the global phylogenetic diversity of B. suis, we compared them to 126 publicly available B. suis genomes. All Slovenian isolates fell within the biovar 2 lineage, confirming the previous multiplex PCR typing results. According to MLST-21, the wild boar isolates were of sequence types (STs) ST16 (n = 8) and ST153 (n = 8) the maximum genetic distance between isolates of the same ST was 28 wgMLST alleles. The ST153 isolates were restricted to the Slovenian-Croatian border and clustered together with the Croatian ST153 isolates from swine, indicating cross-border transmission of B. suis ST153 strain. The hare isolate was of ST40 and was genetically distant (≥ 489 alleles) from the wild boar isolates. The genome-wide phylogeny clearly separated different B. suis biovars. The present study is the first report on the population structure of B. suis in wildlife in Slovenia and shows that the Slovenian B. suis population is genetically heterogeneous. At the species level, B. suis biovars are clearly separated in the WGS-based phylogenetic tree and can therefore be reliably predicted using WGS.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) represents a concern in both human and veterinary medicine. The aim of this study was to investigate potential LA-MRSA transmission between animals and humans in rural settings. To this aim, a study was designed to include 14 farms in Slovenia, which were selected on the basis of a farmer (initial patient) with confirmed LA-MRSA infection and regular animal contacts. On all farms, the initial patients, their household members, animals and barn environment were analysed for the presence of LA-MRSA. In addition, the epidemiologically linked hospital-related LA-MRSA isolates were included to investigate possible nosocomial transmissions. On five farms, LA-MRSA was discovered both in animals and in humans. In total, 49 LA-MRSA isolates of different origins underwent whole-genome sequencing, antimicrobial susceptibility testing and spa typing. All 49 isolates belonged to the sequence type 398 (ST398), spa types t011 and t034, and harboured staphylococcal chromosomal cassette mec Vc. High levels of concordance between resistance phenotypes and genotypes were observed. No transmission pairs between animals and initial patients were discovered. However, several isolates originating from farm animals and other household members formed clusters with pairwise distances of ≤14 single nucleotide polymorphisms (SNPs), indicating recent transmission events. In addition, three closely related isolates (0 SNP) form hospitalized patients were observed, indicating a possible nosocomial transmission. Two hospital-related isolates harboured the immune evasion cluster genes, which are associated with adaptation to the human host; however, these two isolates differed in >30 SNPs from the remaining isolates. Characteristics of LA-MRSA from Slovenia reflect those observed previously in other European studies. Immune evasion cluster-positive LA-MRSA ST398 suggests its re-adaptation to the human host and calls for a closer monitoring of such emerging LA-MRSA lineages, in addition to monitoring and preventing the introduction of LA-MRSA from farms to hospitals where transmission is highly plausible.

Six Helicobacter -like isolates were recovered from 15 gastric mucosa samples of red foxes (Vulpes vulpes) shot by hunters in the surroundings of Ljubljana, Slovenia. Gram-negative, tightly coiled, intensely motile, 7–15 µm long and ≤1 µm wide bacteria grew on the biphasic blood agar plates. By using a genus-specific polymerase chain reaction (PCR), all isolates were confirmed as Helicobacter sp. and subsequently subjected to whole-genome sequencing (WGS). Five isolates showed a genome-wide average nucleotide identity (ANI) value of Helicobacter species and one isolate was classified as Helicobacter felis . In the five unidentified isolates, the 16S rRNA gene sequence similarity to the type strains of all Helicobacter species ranged from 98.6 to 98.9 %. Their taxonomic status was established using a polyphasic taxonomic approach comprising the core genome-based phylogeny, morphological and phenotypic characteristics, including an analysis of matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectra. Phylogeny revealed the existence of three novel and well-supported clusters, with Helicobacter bizzozeronii and Helicobacter baculiformis being the most closely related species. The isolates also differed from the previously described species in their MALDI-TOF profiles and some biochemical characteristics. In conclusion, the data presented herein indicate that the obtained isolates, excluding H. felis isolate, represent three novel Helicobacter species, for which the names Helicobacter labacensis sp. nov., Helicobacter mehlei sp. nov., and Helicobacter vulpis sp. nov. are proposed, with isolates L9T (=DSM 108823T=CRBIP 111719T), L15T (=DSM 108730T=CCUG 72910T) and L2T (=DSM 108727T=CCUG 72909T) as type strains, respectively.

The spore-forming bacterium Paenibacillus larvae is the causative agent of American foulbrood (AFB), a devastating disease of honeybees (Apis mellifera). In the present study, we used whole-genome sequencing (WGS) to investigate an extensive outbreak of AFB in northwestern Slovenia in 2019. A total of 59 P . larvae isolates underwent WGS, of which 40 originated from a single beekeeping operation, to assess the diversity of P. larvae within the beekeeping operation, apiary and colony. By applying a case-specific single-linkage threshold of 34 allele differences (AD), whole-genome multilocus sequence typing (wgMLST) identified two outbreak clusters represented by ERIC II-ST11 clones. All isolates from a single beekeeping operation fell within cluster 1 and the median pairwise AD between them was 10 (range=1–22). The median pairwise AD for apiaries of the same beekeeping operation ranged from 8 to 11 (min.=1, max.=22). For colonies of the same apiary and honey samples from these colonies, the median pairwise AD ranged from 8 to 14 (min.=1, max.=20). The maximum within-cluster distance was 33 pairwise AD for cluster 1 and 44 for cluster 2 isolates. The minimum distance between the outbreak-related and non-related isolates was 37 AD, confirming the importance of associated epidemiological data for delineating outbreak clusters. The observed transmission events could be explained by the activities of honeybees and beekeepers. The present study provides insight into the genetic diversity of P. larvae at different levels and thus provides information for future AFB surveillance.

Salmonella enterica serovar Choleraesuis is a host-adapted serovar that causes serious infections in domestic pigs and wild boars. Here, we investigated an outbreak of salmonellosis in domestic pigs in Slovenia, 2018–2019. To assess the outbreak, 18 isolates from domestic pigs, wild boars, wild boar meat and a human patient underwent whole-genome sequencing (WGS). All isolates were of sequence type (ST) 145 and harbored no antimicrobial resistance genes or AMR-associated mutations. A single transmission cluster (≤ 6 alleles) of spatially (< 100 km) and temporally linked isolates was observed, comprising isolates of pig (n = 9), wild boar (n = 2) and human (n = 1) origin, and suggesting possible interspecies transmission. In all outbreak-related animal cases, septicemic salmonellosis was observed, accompanied in some cases by enteric symptoms. All pig isolates were linked to a single intensive breeding farm that distributed growers to small family farms. The same transport vehicles were used to distribute growers to family farms and also to transport livestock between neighboring countries. Both isolates that originated from the imported wild boar meat were genetically distant (≥ 122 alleles) from the outbreak cluster. The present results indicate the importance of screening domestic pigs and proper disinfection of transport vehicles to control the spread of S. Choleraesuis.

Paenibacillus larvae causes the American foulbrood (AFB), a highly contagious and devastating disease of honeybees. Whole-genome sequencing (WGS) has been increasingly used in bacterial pathogen typing, but rarely applied to study the epidemiology of P. larvae. To this end, we used 125 P. larvae genomes representative of a species-wide diversity to construct a stable whole-genome multilocus sequence typing (wgMLST) scheme consisting of 5745 loci. A total of 51 P. larvae isolates originating from AFB outbreaks in Slovenia were used to assess the epidemiological applicability of the developed wgMLST scheme. In addition, wgMLST was compared with the core-genome MLST (cgMLST) and whole-genome single nucleotide polymorphism (wgSNP) analyses. All three approaches successfully identified clusters of outbreak-associated strains, which were clearly separated from the epidemiologically unlinked isolates. High levels of backward comparability of WGS-based analyses with conventional typing methods (ERIC-PCR and MLST) were revealed; however, both conventional methods lacked sufficient discriminatory power to separate the outbreak clusters. The developed wgMLST scheme provides an improved understanding of the intra- and inter-outbreak genetic diversity of P. larvae and represents an important progress in unraveling the genomic epidemiology of this important honeybee pathogen.

Aims An extensive source investigation was conducted on a dairy farm with neurolisteriosis and subclinical mastitis cases to identify infection source and potential transmission routes of Listeria monocytogenes. Methods and results A total of 36 L. monocytogenes isolates were obtained from animal clinical cases (neurolisteriosis and udder infection) and the farm environment (silage, faeces, water). Isolates were typed using pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing (WGS). Their virulence potential was assessed using the gentamicin protection assay and WGS-based identification of virulence genes. PFGE and WGS revealed a high genetic diversity of L. monocytogenes. An epidemiological link was confirmed for isolates from (i) several subclinical mastitis cases, (ii) silage and subclinical mastitis cases and (iii) different water sources. The neurolisteriosis isolate belonged to clonal complex (CC) 1, but infection source was not identified. A high occurrence (9/47 cows; 19·1%) of subclinical mastitis was observed with isolates belonging to CC2, CC4 and CC11. Conclusions The dairy farm environment was contaminated with diverse L. monocytogenes strains, including genotypes associated with human disease. Several isolates harboured genetic determinants associated with increased infectious potential in humans. Significance and impact of the study Results suggest that subclinical listerial mastitis should not be neglected as a potential source of milk contamination. The presence of hypervirulent CCs in subclinical mastitis cases calls for the implementation of improved mastitis detection.

The repeated occurrence of anthrax in grazing animals should be a reminder of a widespread presence of Bacillus anthracis spores in the environment. Its rapid diagnosis is critical to protect public health. Here, we report a case of anthrax in cattle that was investigated using conventional and molecular methods. In 2015, six cows suddenly died within three days and the number of dead animals increased to a total of 12 within two weeks. At necropsy, anthrax was suspected. Therefore, spleen tissue samples were collected (from 6/12 animals) and laboratory tests (microscopy, cultivation, and real-time PCR) performed. The results of tissue staining for microscopy and cultivation were in congruence, while B. anthracis real-time PCR outperformed both. Spleen tissues from all six animals were real-time PCR-positive, while B. anthracis was successfully cultivated and detected by microscopy from the spleen of only three animals. Additionally, the ear tissue from another (1/12) cow tested positive by real-time PCR, supporting the suitability of ear clippings for molecular confirmation of B. anthracis. Genotyping of the isolates using multiple-locus variable-number tandem repeat analysis (MLVA) revealed a common source of infection as all three typed isolates had an indistinguishable MLVA genotype, which has not been observed previously in Europe. The results indicate that molecular testing should be selected as the first-line tool for confirming anthrax outbreaks in animals to ensure timely protection of public health.

Staphylococcus pseudintermedius is a common cause of skin and soft tissue infections in dogs but can also cause infections in cats and humans. The frequency of methicillin-resistant S. pseudintermedius (MRSP) strains is increasing worldwide. Here, we obtained 43 MRSP isolates from dogs (n = 41), one cat (n = 1) and the small animal clinic environment (n = 1) in Slovenia from the period 2008-2018, which underwent whole-genome sequencing (WGS) and antimicrobial susceptibility testing. Five sequence types (STs) were identified, with ST71 (32/43) and ST551 (8/43) being the predominant. In Slovenia, ST551 was first detected in 2016, whereas a decrease in the frequency of ST71 was observed after 2015. All isolates were multidrug-resistant and most antimicrobial-resistant phenotypes could be linked to acquisition of the corresponding resistance genes or gene mutations. Core-genome multilocus sequence typing (cgMLST) revealed several potential MRSP transmission routes: (i) between two veterinary clinics by a single MRSP-positive dog, (ii) between the environment of a veterinary clinic and a dog, and (iii) between a canine and a feline patient through the contaminated environment of a veterinary clinic. Of the six dogs that were additionally sampled from 14 days to five months after the initial sampling, each harbored the same MRSP strain, suggesting a limited within-host diversity of MRSP in symptomatic dogs. The present results highlight the importance of MRSP-positive dogs in the spread of veterinary care-associated MRSP infections and call for the implementation of strict control measures to reduce MRSP contamination in veterinary clinic environments originating from animal-contact surfaces.

Paenibacillus larvae causes the American foulbrood (AFB), a highly contagious and devastating disease of honeybees. Whole-genome sequencing (WGS) has been increasingly used in bacterial pathogen typing, but rarely applied to study the epidemiology of P. larvae. To this end, we used 125 P. larvae genomes representative of a species-wide diversity to construct a stable whole-genome multilocus sequence typing (wgMLST) scheme consisting of 5745 loci. A total of 51 P. larvae isolates originating from AFB outbreaks in Slovenia were used to assess the epidemiological applicability of the developed wgMLST scheme. In addition, wgMLST was compared with the core-genome MLST (cgMLST) and whole-genome single nucleotide polymorphism (wgSNP) analyses. All three approaches successfully identified clusters of outbreak-associated strains, which were clearly separated from the epidemiologically unlinked isolates. High levels of backward comparability of WGS-based analyses with conventional typing methods (ERIC-PCR and MLST) were revealed; however, both conventional methods lacked sufficient discriminatory power to separate the outbreak clusters. The developed wgMLST scheme provides an improved understanding of the intra- and inter-outbreak genetic diversity of P. larvae and represents an important progress in unraveling the genomic epidemiology of this important honeybee pathogen.

Summary Results of the survey carried out in the territories of Tawny Owl Strix aluco in Ljubljana urban forests (Tivoli, Rožnik and Šišenski hrib Nature Park and Golovec Hill) were compared with the species territory density in non-urban forest of Mt. Krim. Surveys were performed with the point count method using playback in the springs of 2002 and 2016. The density established at Golovec Hill was 9.3 territories / 10 km2, while in Tivoli, Rožnik and Šišenski hrib Nature Park it reached 10.4 territories / 10 km2, which was higher than at Mt. Krim (4.1-5.8 territories / 10 km2), although densities between sites were not statistically different. Based on our surveys, the estimated population size of the urban Tawny Owl in Ljubljana would consist of 57 to 65 pairs. Our results suggest that the Tawny Owl can adapt well to the living conditions in the city urban forests and indicate the importance of the forest patches in urban areas.

Background Listeria monocytogenes is the causative agent of listeriosis, a serious disease affecting both animals and humans. Here, multilocus sequence typing (MLST) was used to characterize the genetic diversity of Listeria monocytogenes strains isolated from the natural environment and animal clinical cases in Europe. The prevalence of clonal complexes (CCs) obtained was compared according to (i) the origin of isolation – clinical cases vs. natural environment – and (ii) the clinical form of animal listeriosis – rhombencephalitis vs. abortion. To this aim, two datasets were constructed. The clinical dataset consisted of 350 animal clinical isolates originating from France and Slovenia and supplemented with isolates from Switzerland and Great Britain. The natural environment dataset consisted of 253 isolates from the natural environment originating from Slovenia and supplemented with isolates from nine other European countries. Results For the clinical cases, CC1, CC4-CC217 and CC412 were the most prevalent in rhombencephalitis and CC1, CC37 and CC4-CC217 in abortion. The hypervirulent CC1 and CC4-CC217 prevailed in both datasets. These results indicated that livestock is constantly exposed to hypervirulent CCs. CC1 was significantly associated with a clinical origin, whereas CC9, CC29 and CC14 were associated with the natural environment. CC1 was predominant among rhombencephalitis cases both in cattle and small ruminants, and its prevalence did not differ significantly between these two groups. A novel association of CC37 and CC6 with abortion cases was revealed. Conclusions Here, we show that CC1 and CC4-CC217 are prevalent in isolates of environmental and animal clinical origin, suggesting that ruminants are frequently exposed to hypervirulent CCs. The presence of CC4 in two mastitis cases calls for further attention due to direct threat to the consumer. We showed several associations between CCs and the origin of isolation or clinical form of listeriosis, e.g. CC37 and CC6 with abortion. This study improves our understanding of the population structure of L. monocytogenes isolates from the natural environment and animal clinical cases. Moreover, it provides a basis for future studies aiming to determine the underlying mechanisms of phenotypic traits of interest.

Campylobacteriosis is the most frequently reported bacterial food-borne illness in the European Union and contaminated broiler meat is considered the most important source of infection in humans. The aim of the present study was to evaluate real-time PCR (qPCR) and digital PCR (dPCR) for quantification of Campylobacter jejuni in 75 broiler neck-skin samples collected from a poultry slaughterhouse, and to compare them with the ISO 10272-2 standard plate count method. For qPCR standard curve, C. jejuni-negative neck-skin samples were spiked with C. jejuni suspension with a known number of bacterial cells. The observed CFU/g values by qPCR correlated greatly with the expected values and qPCR showed good performance with the reliable limit of detection (rLOD) and limit of quantification (LOQ) of three and 31 target copies per reaction, respectively. However, both rLOD (1219 CFU/g) and LOQ (12,523 CFU/g) were beyond the EFSA-proposed critical limit of 500–1,000 CFU/g of neck skin. Although C. jejuni cell counts were ≤1,000 CFU/g in only 7/75 samples by plate counting, they were ≤LOQ in 60/75 and ≤rLOD in 26/75 (≤1,000 CFU/g in 24/75) samples by qPCR. A strong and statistically significant correlation was observed between qPCR and dPCR. Both PCR-based methods correlated significantly with the plate count method; however, the correlation was moderate. Using the Bland–Altman analysis, an average agreement was noted between all three methods, although with a large standard deviation. A significant bias toward overestimation in dPCR was observed, probably due to the relatively high number of false positive calls. The linear dynamic range was comparable in both PCR-based methods; however, qPCR proved to be more suitable for routine use. In the future, the establishment of a reliable molecular quantification of C. jejuni in poultry samples showing a wide range of contamination levels down to the proposed critical limit is needed to enable time- and cost-effective surveillance throughout all stages in the food production chain. As both rLOD and LOQ were beyond this limit, a modification of the procedure is suggested to include less sample dilution prior to DNA extraction to enable PCR-based quantification of C. jejuni at the proposed microbiological criteria.

Listeria monocytogenes is the causative agent of listeriosis, a serious disease affecting both humans and animals. While listeriosis outbreaks in humans are commonly investigated in detail, routine typing of L. monocytogenes is generally not performed in animal outbreaks. Here, seven presumable listeriosis outbreaks in small ruminants were retrospectively identified based on the pulsed-field gel electrophoresis (PFGE) profiles. Outbreaks were further characterised using three different analytical approaches based on the whole-genome sequencing (WGS) data: core-genome multilocus sequence typing (cgMLST), whole-genome MLST (wgMLST) and whole-genome single nucleotide polymorphism (wgSNP) typing. A monoclonal pattern of all seven outbreaks was identified using all three approaches, indicating common-source outbreaks. The outbreak strains belonged to sequence types (STs) 1 (n = 3), ST18 (n = 1), ST21 (n = 2) and ST184 (n = 1). Two epidemiologically linked ST1 outbreaks with indistinguishable PFGE profiles showed a polyphyletic nature and differed in >78 SNPs; thus, they were classified as separate outbreaks according to WGS. In ST184, the outbreak strain was also found in faeces of apparently healthy ruminants, silage and water collected from the trough, which were the most likely source(s) of infection. The outbreak-associated isolates differed in 0–7 cgMLST alleles, 0–12 wgMLST alleles and 1–13 SNPs. The minimum genetic diversity between outbreak-associated isolates and epidemiologically unrelated isolates of the same ST was low in all analysed cases, approaching the maximum diversity within the outbreak cluster. The results suggest that a fixed threshold to define the outbreak cluster should only be considered as a guide and highlight the role of epidemiological data for outbreak confirmation. The identified cgMLST clusters may be further investigated by wgMLST and/or wgSNP typing to increase confidence during investigations of outbreaks caused by highly clonal L. monocytogenes groups. This study gives an overview of the inter- and intra-outbreak genetic diversity of L. monocytogenes strains involved in animal outbreaks, hence improving their investigation.

Wildlife species are an important reservoir of mycobacterial infections that may jeopardise efforts to control and eradicate bovine tuberculosis (bTB), caused by Mycobacterium bovis. Slovenia is officially free of bTB, but no data on the presence of mycobacteria in wild animals has been reported. In this study, samples of liver and lymph nodes were examined from 306 apparently healthy free-range wild animals of 13 species in Slovenia belonging to the families Cervidae, Suidae, Canidae, Mustelidae and Bovidae. Mycobacteria were isolated from 36/306 (11.8%) animals (red deer, roe deer, fallow deer, wild boar and jackal) and identified by PCR, commercial diagnostic kits and sequencing. Non-tuberculous mycobacteria identified in five species were Mycobacterium peregrinum, M. avium subsp. hominissuis, M. intracellulare, M. confluentis, M. fortuitum, M. terrae, M. avium subsp. avium, M. celatum, M. engbaekii, M. neoaurum, M. nonchromogenicum and M. vaccae.

Among 1280 cultured and wild adult Mediterranean mussels, Mytilus galloprovincialis, collected over a 1-year surveillance period from the Slovene Adriatic Sea, 0.3% were histologically positive for the presence of Marteilia spp. The infection was concentrated in winter. Employing the molecular methods of PCR, cloning, DNA restriction and sequencing, only Marteilia refringens type M was detected in all the infected mussels. Although all life-cycle stages of M. refringens severely infected digestive glands, only sporadic disruption of epithelial cells of digestive tubules and focal destruction of digestive tubules were observed in the infected mussels. This was the first detection of M. refringens in M. galloprovincialis from the Slovene Adriatic Sea with the lowest prevalence reported to date. In addition, our results highlight the need for sequencing to complement the established PCR-RFLP analysis for correct parasite typing.

Brucella spp. that cause marine brucellosis are becoming more important, as the disease appears to be more widespread than originally thought. Here, we report a whole and annotated genome sequence of Brucella ceti CRO350, a sequence type 27 strain isolated from a bottlenose dolphin carcass found in the Croatian part of the northern Adriatic Sea.

Bovine viral diarrhea virus (BVDV) subgenotype 1e was isolated for the first time in Slovenia in 2006. Here, we report the complete genome sequence of BVDV-1e, strain SLO/2407/2006. The published genome will increase our understanding of the molecular characteristics of the BVDV-1e strains circulating in Europe.

Campylobacteriosis is the most frequently reported bacterial food-borne illness in the European Union and contaminated broiler meat is considered the most important source of infection in humans. The aim of the present study was to evaluate real-time PCR (qPCR) and digital PCR (dPCR) for quantification of Campylobacter jejuni in 75 broiler neck-skin samples collected from a poultry slaughterhouse, and to compare them with the ISO 10272-2 standard plate count method. For qPCR standard curve, C. jejuni-negative neck-skin samples were spiked with C. jejuni suspension with a known number of bacterial cells. The observed CFU/g values by qPCR correlated greatly with the expected values and qPCR showed good performance with the reliable limit of detection (rLOD) and limit of quantification (LOQ) of three and 31 target copies per reaction, respectively. However, both rLOD (1219 CFU/g) and LOQ (12,523 CFU/g) were beyond the EFSA-proposed critical limit of 500–1,000 CFU/g of neck skin. Although C. jejuni cell counts were ≤1,000 CFU/g in only 7/75 samples by plate counting, they were ≤LOQ in 60/75 and ≤rLOD in 26/75 (≤1,000 CFU/g in 24/75) samples by qPCR. A strong and statistically significant correlation was observed between qPCR and dPCR. Both PCR-based methods correlated significantly with the plate count method; however, the correlation was moderate. Using the Bland–Altman analysis, an average agreement was noted between all three methods, although with a large standard deviation. A significant bias toward overestimation in dPCR was observed, probably due to the relatively high number of false positive calls. The linear dynamic range was comparable in both PCR-based methods; however, qPCR proved to be more suitable for routine use. In the future, the establishment of a reliable molecular quantification of C. jejuni in poultry samples showing a wide range of contamination levels down to the proposed critical limit is needed to enable time- and cost-effective surveillance throughout all stages in the food production chain. As both rLOD and LOQ were beyond this limit, a modification of the procedure is suggested to include less sample dilution prior to DNA extraction to enable PCR-based quantification of C. jejuni at the proposed microbiological criteria.

Background Bacterial intercellular communication, called quorum sensing, takes place via the production and collective response to signal molecules. In Gram-negative bacteria, like Pseudomonas aeruginosa, these signaling molecules are N-acylhomoserine lactones (AHLs). P. aeruginosa is a common cause of inflammation of the ear canal (otitis externa) in dogs. It employs quorum sensing to coordinate the expression of host tissue-damaging factors, which are largely responsible for its virulence. The treatment of P. aeruginosa-associated otitis is challenging due to a high intrinsic resistance of P. aeruginosa to several antibiotics. Attenuation of quorum sensing signals to inhibit bacterial virulence is a novel strategy for the treatment of resistant bacterial pathogens, including P. aeruginosa. Therefore, it is important to recognize and define quorum sensing signal molecules in clinical samples. To date, there are no reports on determination of AHLs in the veterinary clinical samples. The purpose of this study was to validate an analytical procedure for determination of the concentration of AHLs in the ear rinses from dogs with P. aeruginosa-associated otitis externa. Samples were obtained with rinsing the ear canals with physiological saline solution. For validation, samples from healthy dogs were spiked with none or different known amounts of the selected AHLs. With the validated procedure, AHLs were analyzed in the samples taken in weekly intervals from two dogs, receiving a standard treatment for P. aeruginosa-associated otitis externa. Results Validation proved that the procedure enables quantification of AHLs in non-clinical and clinical samples. In addition, a time dependent reduction of AHL concentration was detected for the treated dogs. Conclusions Our results indicate that liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is superior in detecting AHLs compared to other chromatographic techniques. This is the first report on determination of AHLs in the clinical samples of veterinary importance. The analytical procedure described in this paper is capable of supporting novel antimicrobial strategies, which target quorum sensing.

Fish are commonly infected with non-tuberculous mycobacteria (NTM), which should be regarded as potential pathogens when handling aquarium fish and equipment. This study examined 107 aquarium fish from pet shops. Cultivation of the fish samples using different selective media was conducted for identification of NTM. Isolates were identified using the GenoType Mycobacterium common mycobacteria and additional species assays, sequencing of the 16S rRNA and rpoB genes, and real-time PCR assay for identification of Mycobacterium (M.) marinum. Among the investigated fish, 79.4% (85/107) were positive for mycobacteria, with 8.2% (7 of 85) having two mycobacterial species present. Among the positive fish, the common pathogens M. marinum, Mycobacterium fortuitum (M. fortuitum group) and Mycobacterium chelonae were identified in approx. 90% of fish and other NTM species in 10%, including Mycobacterium peregrinum/septicum, Mycobacterium gordonae, Mycobacterium arupense, Mycobacterium kansasii, Mycobacterium ulcerans and Mycobacterium setense. The well-known human pathogen M. marinum was present in 10.6% of the positive fish (9 of 85). The species of mycobacteria identified in the study are not only recognized as aquarium fish pathogens, but can also cause pathology in humans. Microbiological and clinical communities should therefore be sensitized to the role of NTM in infections associated with exposure to aquarium fish.

Porcine epidemic diarrhea virus (PEDV) was detected for the first time in Slovenia in January 2015. The complete genome sequence of PEDV strain SLO/JH-11/2015, obtained from a fecal sample of a fattening pig with diarrhea in September 2015, is closely related to recently detected European strains.

The objective of the present work was to establish effective and rapid diagnostic methods for the detection of Bacillus anthracis, a highly virulent zoonotic pathogen, in the air, soil and animal (or human) tissue samples. Liquid culture of B. anthracis was aerosolized and four air sampling procedures were employed. Detection of B. anthracis in the air samples was successful with RCS High Flow sampler (culturebased detection) and when sampling through the air filter (molecular detection using SmartHelix Complex Samples DNA Extraction Kit). Liquid B. anthracis culture was also employed for spiking the homogenised bovine lymphatic gland tissue and soil samples. DNA extraction was performed using three different commercial kits for each sample type. High Pure PCR Template Preparation Kit was the most effective for DNA extraction from animal tissue samples. Detection in the soil was successful when PowerSoil DNA Isolation Kit was used. Our results indicate that B. anthracis can be monitored in different matrices by rapid molecular methods when appropriate sampling and DNA extraction procedures are employed prior to PCR assay. The selected rapid protocols can be implemented in specialized veterinary or human diagnostic laboratories with moderate costs.

During a comparison of 16S rDNA PCR-denaturant gradient gel electrophoresis (DGGE) profiles of methanogenic archaea from rumen fluid, rabbit caecum and pig feces, a unique band common to all rabbit caecum samples was observed. DGGE profiling also showed that the methanogen community from the New Zealand White adult rabbits is different and less complex than the methanogen communities from the rumen and pig feces. Small subunit ribosomal gene sequences of methanogenic archaea were subsequently retrieved from the constructed rabbit caecum 16S rDNA gene library. Results of the phylogenetic analysis indicated that rabbit caecum is inhabited by members of the genus Methanobrevibacter and is possibly one-species dominated, because all the retrieved sequences exhibited similarity values of 99% or higher. This species may well be a novel species of the genus Methanobrevibacter. It belongs to a distinct phylogenetic group containing Methanobrevibacter woesei, Methanobrevibacter thaueri and Methanobrevibacter gottschalkii strains isolated from animal feces, and Methanobrevibacter smithii from the predominating methanogen population of the human large bowel.

Since listeriosis, caused by Listeria monocytogenes, is one of the important concerns of public health in Europe related to foodborne zoonoses, an efficient protocol for isolate typing is necessary when performing epidemiological studies. Three standardized PFGE protocols available for L. monocytogenes were briefly reviewed. Since observing a poor-quality of ApaI pulsotypes in our laboratory, enzymes from three different manufacturers were compared. The obtained pulsotypes showed that restriction digestion with ApaI from New England BioLabs should be complemented with a subsequent overnight incubation of PFGE plugs in TE buffer for better performance.

Six slaughter batches deriving from six typical industrial broiler flocks were examined for the presence, quantity and genetic characteristics of contaminating Campylobacter jejuni (C. jejuni) during various stages of slaughtering and carcass processing. To assess the contamination dynamics of the carcasses, the analyses were always conducted on neck-skin samples from the same pre-selected and carefully marked carcasses in each batch. The skin samples were taken sequentially at three successive slaughter-line locations in the evisceration room, after three-day refrigeration and after three-day freezing procedure. Caecal samples from the same animals were also tested, as well as samples from the slaughterhouse environment before and after slaughtering. The samples were analysed by the ISO10272 isolation method; campylobacters from neck-skin samples were also quantified. Isolates were species-identified and genotyped by pulsed-field gel electrophoresis (PFGE). On average, the highest C. jejuni skin contamination was detected at the first sampling point (post-plucking), suggesting that the majority of Campylobacter contamination actually occurs before the entrance to the eviscerating room, probably during the preceding plucking stage. In two out of five positive batches, an additional increase in contamination was recorded after the evisceration step. An evident trend of increasing contamination level was detected when successive batches were compared at each of two initial sampling sites in the evisceration room, indicating an accumulation of contaminating C. jejuni at some point before the evisceration room. Three-day refrigeration and three-day freezing caused a 4.5- and 142-fold drop in mean C. jejuni CFU counts, respectively. All pre-slaughtering samples from the slaughterhouse environment were negative and all post-slaughtering samples, except water from the scalding tank, were positive. Pulsotypes were limited: altogether five different types were detected, typically one type per batch. The PFGE results from the slaughterhouse environment isolates indicate that cross-contamination is possible (multiple pulsotypes detected in e.g. eviscerating machine). Nevertheless, this was not confirmed in carcasses: analyses of neck-skin isolates suggest that carcasses are contaminated by their own caecal/farm/flock pulsotype.

Slovenia has no history of health problems related to proliferative kidney disease (PKD) either in farmed or in wild fish. However, due to the past molecular evidence for the presence of Tetracapsuloides bryosalmonae DNA in tissues of some fish from open waters, a survey was conducted on wild salmonids that were primarily sampled for other purposes. In winter 2010-2011, specimens from a total of 244 rainbow trout, Oncorhynchus mykiss (Walbaum), and brown trout, Salmo trutta L., from 30 bodies of fresh water were examined for T. bryosalmonae using a PCR method. The sampled fish showed no clinical signs or gross pathological lesions characteristic of PKD. Nineteen (7.8%) fish from seven (23.3%) fresh waters were positive for T. bryosalmonae. The identity of PCR amplicons was confirmed by sequencing. With one exception, all the positive fish were found in waters from the regions where the average yearly temperatures and the environmental pollution are higher. This preliminary countrywide survey provided the first insight into the situation regarding T. bryosalmonae infection of wild salmonids in Slovenia.

Denaturant gradient gel electrophoresis (DGGE) enables insight into the diversity of the studied microbial communities on the basis of separation of PCR amplification products according to their nucleotide sequence composition. However, the success of the method is accompanied by the inherent appearance of various sequence artifacts that bias the impression of community structure by generating additional bands representing no virtual microbes. PCR-DGGE artifacts require optimization of the method when aiming at the phylogenetic identification of the selected DGGE bands. The aim of our study was to develop a procedure which will increase the reliability of the identification. Samples of rumen fluid were used for the optimization since they contain a complex microbial community that supports the generation of artifactual bands. An optimized procedure following band excision and elution of microbial DNA is proposed including nuclease treatment, selection of DNA polymerase with proofreading activity, and cloning prior to sequencing and identification analysis.

Epidemiological studies on Mycobacterium avium are requisite for revealing infection sources and disease transmission. They are based upon genotyping methods like RFLP and MIRU–VNTR. In our study, MIRU–VNTR typing was applied to 121 previously RFLP typed M. avium field isolates to compare the discriminatory power of both methods. The applicability of MIRU–VNTR typing was studied for isolates from a limited geographic area, namely 41 M. avium subsp. avium and 80 M. avium subsp. hominissuis isolates. Among the former, exhibiting 12 IS 901 RFLP types, five MIRU–VNTR types were found with discriminatory index ( DI ) of 0.716. Among the latter, exhibiting 56 IS 1245 RFLP types, 18 MIRU–VNTR types were found with DI of 0.866. Concomitant use of both methods increased DI to 0.981 and 0.995, respectively. MIRU–VNTR typing employing the selected markers provided discernible discrimination among M. avium subsp. hominissuis isolates, but more discriminative markers are needed for M. avium subsp. avium isolates.

Bacteria of the genus Salmonella cause a global health problem related to contaminated foodstuffs and faecally-polluted water supplies. The aim of our work was to determine the efficiency and rapidness of Salmonella detection in spiked tap water samples by comparison of different methods for DNA extraction. Water samples were spiked with different Salmonella loads. Four DNA extraction methods were employedČ foodproof Sample Preparation Kit I (Biotecon Diagnostics) and QIAamp DNA Stool Mini Kit (Qiagen) for DNA extraction after an overnight enrichment step, and Adiapure Water DNA Extraction and Purification Kit (Adiagene) and SmartHelix Complex Samples DNA Extraction Kit (developed recently in our laboratory) for direct DNA extraction. The extracted DNA was subjected to PCR amplification using Salmonella genus-specific primer pair. The SmartHelix extraction kit proved tobe more efficient than the Adiapure Water kit for direct detection of Salmonella DNA in water. The overnight enrichment step improved the detection of Salmonella spp. in samples spiked with low bacterial load, however it extended the analysis time. Results suggest that the newly developed SmartHelix extraction kit should be regarded as a considerable choice when rapid detection of pathogens in water samples is aimed for to prevent disease outbreaks and support safe food assurance.