Seroprevalence of infectious pathogens of zoonotic and veterinary importance in wild ruminants from Slovenia.

Introduction: Wildlife represents an increasingly important source of pathogens of medical and veterinary importance. Surveillance in wildlife offers an insight on current epidemiological status of selected pathogens and help to prevent spillovers to humans and livestock. Material and methods: Our study included 312 wild ruminants belonging to five species: Roe deer (n = 134), red deer (n = 113), Alpine chamois (n = 53), European mouflon (n = 10) and Alpine ibex (n = 2). Seven pathogens that may have profound effect on human/livestock health and economic viability of the farms were tested using serological methods. Results: Antibodies against Toxoplasma gondii, Neospora caninum, Coxiella burnetii, Brucella spp., Chlamydophila abortus, Mycobacterium avium subsp. paratuberculosis (MAP) and Mycobacterium bovis were detected in 34.62% (108/312), 0.96% (3/312), 2.24% (7/312), 0, 0.96% (3/312), 0, 0.64% (2/312) of animals tested, respectively. Because of low prevalences, risk factors were assessed only for T. gondii. Sex (female>male) and species (roe deer>red deer, roe deer>Alpine chamois) were significantly associated with the T. gondii positive outcome, while age was not. Discussion: Adult males had the lowest T. gondii prevalence which offers future research opportunities. The lower seroprevalence of most investigated pathogens suggests game meat, if properly cooked, as being relatively safe for human consumption. This is the first study investigating the seroprevalence and associated risk factors of selected pathogens in wild ruminants in Slovenia.

Bovine Tuberculosis in Wild Boar (Sus scrofa) in Slovenia.

Mycobacteria of the Mycobacterium tuberculosis complex (MTC) are capable of infecting a wide variety of animals. Wild boar (Sus scrofa) has been recognized as an important wildlife reservoir for bovine tuberculosis. We screened wild boar in Slovenia for the presence of (1) Mycobacterium bovis in tissues and (2) antibodies to M. bovis in blood samples. In 2016 and 2017, 1284 tissue samples from 676 wild boar were subjected to cultivation. In 2018 and 2019, blood samples from 132 wild boar were examined using an ELISA kit. None of the MTC species were isolated from the tissue samples, and no antibodies to M. bovis were detected in the blood samples. Several nontuberculous mycobacteria (NTM), identified by 16S rRNA sequencing and/or matrix-assisted laser desorption ionization time-of-flight mass spectrometry, were found in the tissues of 9.8% of the wild boar: Mycobacterium nonchromogenicum, Mycobacterium peregrinum/Mycobacterium septicum, Mycobacterium avium, Mycobacterium engbaekii, Mycobacterium arupense, Mycobacterium algericum, Mycobacterium bohemicum, Mycobacterium confluentis, Mycobacterium flavescens, Mycobacterium fortuitum, Mycobacterium thermoresistibile, and Mycobacterium vaccae. Species-level identification was not possible for 21.2% of the isolates. At the time of the study, wild boar in Slovenia were not at risk from bTB; the significance of the presence of NTM in wild boar remains to be clarified and evaluated from a One Health perspective.

Phylogeography of Brucella suis biovar 2 with focus on Slovenian wildlife.

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.

Canine Leishmaniasis Prevalence in the Slovenian Dog Population.

INTRODUCTION: Leishmaniasis is a life-threatening zoonosis of which dogs are the major reservoir and sandflies are the vectors. Until now, the prevalence of canine leishmaniasis (CanL) in the Slovenian dog population was unknown. MATERIAL AND METHODS: Epidemiological data, eye swabs and blood samples were taken from 465 dogs born in Slovenia and older than one year. Commercial ELISA kits and real-time PCR were used. For ELISA-positive samples, an immunofluorescence antibody test (IFAT) was performed. Descriptive statistics were used to characterise the samples. The one-sample nonparametric chi-square test was used to test whether the categories of a variable were equally distributed. RESULTS: A 59.9% proportion of the recruited dogs had travelled to endemic regions and 62.1% of them had not been protected by insect repellents. Skin symptoms that might be CanL-related were described in 109 of the dogs' histories (23.4%), inappetence and/or weight loss in 25 (5.4%), and anaemia, intermittent fever, and/or lymphadenopathy in 19 (4.1%). At the time of recruitment, all dogs were asymptomatic. All samples were PCR negative, nine (1.9%) were ELISA positive, but none were IFAT positive. Five of the nine ELISA-positive dogs were non-travellers. CONCLUSION: We conclude that the seroprevalence of canine leishmaniasis of 1.9 % in the autochthonous Slovenian dog population may pose a risk of endemic spread of the disease.

Infectious Abortions in Small Ruminants: Challenges for Diagnosis and Public Health.

In this study, we report an abortion outbreak in a ruminant herd consisting of goats, sheep, and cows, with scenarios in two consecutive years. In early 2017, abortions occurred in ∼70% of goats and 66% tested positive for Coxiella burnetii (C. burnetii) and 40% of goats were positive for Chlamydophila abortus (C. abortus). In February 2018, the same herd reported an abortion rate of 75%, with 55% positive for C. burnetii, 36% for C. abortus, and 22% for Toxoplasma gondii. Six goat milk samples were positive for C. burnetii by molecular analysis. Three family members were positive for C. burnetii. C. burnetii could be considered as the main cause of abortions in the first and second year. Animals that undergo an infection and abortion are prone to secondary infections. Vaccination or other rapid interventions should be initiated to protect animals and humans.

Epidemiology of taeniosis/cysticercosis in Europe, a systematic review: Western Europe.

BACKGROUND: Taenia solium and Taenia saginata are zoonotic parasites of public health importance. Data on their occurrence in humans and animals in western Europe are incomplete and fragmented. In this study, we aimed to update the current knowledge on the epidemiology of these parasites in this region. METHODS: We conducted a systematic review of scientific and grey literature published from 1990 to 2015 on the epidemiology of T. saginata and T. solium in humans and animals. Additionally, data about disease occurrence were actively sought by contacting local experts in the different countries. RESULTS: Taeniosis cases were found in twelve out of eighteen countries in western Europe. No cases were identified in Iceland, Ireland, Luxembourg, Norway, Sweden and Switzerland. For Denmark, Netherlands, Portugal, Slovenia, Spain and the UK, annual taeniosis cases were reported and the number of detected cases per year ranged between 1 and 114. Detected prevalences ranged from 0.05 to 0.27%, whereas estimated prevalences ranged from 0.02 to 0.67%. Most taeniosis cases were reported as Taenia spp. or T. saginata, although T. solium was reported in Denmark, France, Italy, Spain, Slovenia, Portugal and the UK. Human cysticercosis cases were reported in all western European countries except for Iceland, with the highest number originating from Portugal and Spain. Most human cysticercosis cases were suspected to have acquired the infection outside western Europe. Cases of T. solium in pigs were found in Austria and Portugal, but only the two cases from Portugal were confirmed with molecular methods. Germany, Spain and Slovenia reported porcine cysticercosis, but made no Taenia species distinction. Bovine cysticercosis was detected in all countries except for Iceland, with a prevalence based on meat inspection of 0.0002-7.82%. CONCLUSIONS: Detection and reporting of taeniosis in western Europe should be improved. The existence of T. solium tapeworm carriers, of suspected autochthonous cases of human cysticercosis and the lack of confirmation of porcine cysticercosis cases deserve further attention. Suspected cases of T. solium in pigs should be confirmed by molecular methods. Both taeniosis and human cysticercosis should be notifiable and surveillance in animals should be improved.

Antimicrobial susceptibility of animal and human isolates of Clostridium difficile by broth microdilution.

A total of 188 human (n = 92) and animal (n = 96) isolates of Clostridium difficile of different PCR ribotypes were screened for susceptibility to 30 antimicrobials using broth microdilution. When comparing the prevalence of antimicrobial resistance, the isolates of animal origin were significantly more often resistant to oxacillin, gentamicin and trimethoprim/sulfamethoxazole (P<0.01). The most significant difference between the animal and human populations (P = 0.0006) was found in the level of imipenem resistance, with a prevalence of 53.3 % in isolates of human origin and 28.1 % in isolates of animal origin. Overall, the results show similar MICs for the majority of tested antimicrobials for isolates from human and animal sources, which were collected from the same geographical region and in the same time interval. This supports the hypothesis that C. difficile could be transmissible between human and animal hosts. Resistant isolates have been found in all animal species tested, including food and companion animals, and also among non-toxigenic isolates. The isolates of the most prevalent PCR ribotype 014/020 had low resistance rates for moxifloxacin, erythromycin, rifampicin and daptomycin, but a high resistance rate for imipenem. Multiresistant strains were found in animals and humans, belonging to PCR ribotypes 012, 017, 027, 045, 046, 078 and 150, and also to non-toxigenic strains of PCR ribotypes 010 and SLO 080.

The First Report of Mycobacterium celatum Isolation from Domestic Pig (Sus scrofa domestica) and Roe Deer (Capreolus capreolus) and an Overview of Human Infections in Slovenia.

Mycobacterium celatum, a slowly growing potentially pathogenic mycobacterium first described in humans, is regarded as an uncommon cause of human infection, though capable of inducing invasive disease in immunocompromised hosts. According to some reports, a serious disease due to M. celatum may also occur in individuals with no apparent immunodeficiency. In animals, an M. celatum-related disease has been described in three cases only: twice in a domestic ferret (Mustela putorius furo) and once in a white-tailed trogon (Trogon viridis). In this paper, we report the first detection of M. celatum in a domestic pig (Sus scrofa domestica) and roe deer (Capreolus capreolus). A nation-wide overview of human M. celatum infections recorded in Slovenia between 2000 and 2010 is also given. Pulmonary disease due to M. celatum was recognized in one patient with a history of a preexisting lung disease.

IS1245 RFLP-based genotyping study of Mycobacterium avium subsp. hominissuis isolates from pigs and humans.

Mycobacterium avium subsp. hominissuis, ubiquitous environmental mycobacterium, is an opportunistic pathogen that is regularly isolated from pigs and humans in Slovenia. Genetic diversity of 114 isolates from pigs (n = 57) and humans (n = 57), identified by means of bacteriology, DNA-RNA hybridization techniques, IS901 PCR and IS1245 PCR, was investigated in this study, using IS1245 restriction fragment length polymorphism (RFLP) analysis. Identical IS1245 RFLP profiles were found in isolates from pigs, isolates from humans and isolates from both origins. The proportion of clustered isolates varied as it depended on the similarity level (100% and 75%) chosen for the cluster limits. Using IS1245 RFLP, it was possible to detect monoclonal, polyclonal and recent infections and to monitor the genetic variability of the strains from individual patients. Our findings indicate the environment as the source of infection for both pigs and humans. The questions about the possibility of intra- and inter-species transmission of infection remain to be answered.