Mitochondrial genetic diversity and phylogenetic relationships of Echinococcus multilocularis in Europe.

The cestode Echinococcus multilocularis is the causative agent of alveolar echinococcosis, a fatal zoonotic parasitic disease of the northern hemisphere. Red foxes are the main reservoir hosts and, likely, the main drivers of the geographic spread of the disease in Europe. Knowledge of genetic relationships among E. multilocularis isolates at a European scale is key to understanding the dispersal characteristics of E. multilocularis. Hence, the present study aimed to describe the genetic diversity of E. multilocularis isolates obtained from different host species in 19 European countries. Based on the analysis of complete nucleotide sequences of the cob, atp6, nad2, nad1 and cox1 mitochondrial genes (4,968 bp), 43 haplotypes were inferred. Four haplotypes represented 62.56 % of the examined isolates (142/227), and one of these four haplotypes was found in each country investigated, except Svalbard, Norway. While the haplotypes from Svalbard were markedly different from all the others, mainland Europe appeared to be dominated by two main clusters, represented by most western, central and eastern European countries, and the Baltic countries and northeastern Poland, respectively. Moreover, one Asian-like haplotype was identified in Latvia and northeastern Poland. To better elucidate the presence of Asian genetic variants of E. multilocularis in Europe, and to obtain a more comprehensive Europe-wide coverage, further studies, including samples from endemic regions not investigated in the present study, especially some eastern European countries, are needed. Further, the present work proposes historical causes that may have contributed to shaping the current genetic variability of E. multilocularis in Europe.

First Molecular Detection of Neospora caninum in Feces of Grey Wolf (Canis lupus) and Golden Jackal (Canis aureus) Populations in Slovenia.

Neospora caninum is an obligate intracellular parasite that causes reproductive disorders and major economic losses in cattle, and induces neuromuscular disorders in canids. Exogenous infections are becoming increasingly important due to disease outbreaks. The sylvatic life cycle of N. caninum interferes with the domestic dog-ruminant life cycle, but understanding of it is scarce. The population of wild canids may play an important role in parasite dispersion. Feces from 42 grey wolves (Canis lupus) and 39 golden jackals (Canis aureus) were analyzed for the N. caninum Nc5 gene using a novel real-time PCR (qPCR) with a detection limit of 5 targets/µL in clinical samples. Three wolves (3/42; 7.1%) and one golden jackal (1/39; 2.6%) tested positive, which is the first detection of N. caninum in the population of grey wolves in Slovenia and the first detection of N. caninum DNA in the feces of a golden jackal. In addition to the grey wolf, we propose the golden jackal as a potential definitive host with hypothetical epidemiological importance for the sylvatic-domestic life cycle of N. caninum, due to its proximity to human habitats and its rapid expansion throughout Europe.

Wild Carnivore Survey of Echinococcus Species in Slovenia.

Wild carnivores are definitive hosts and potential reservoirs for the tapeworm Echinococcus sp. which can cause cystic and alveolar echinococcosis. Both are considered neglected and important food-borne pandemics. This study is the first to molecularly test Slovenian wild carnivores for Echinococcus species that can cause disease in humans. Fecal samples from 210 red foxes (Vulpes vulpes), 42 wolves (Canis lupus), 39 golden jackals (Canis aureus), 18 martens (Marten sp.), 2 Eurasian lynx (Lynx lynx), 2 European badger (Meles meles), and 1 Eurasian otter (Lutra lutra) were examined for Echinococcus granulosus sensu lato (EGsl: E. granulosus sensu stricto, E. canadensis) and E. multilocularis (EM) using real-time PCR. Red foxes (29.1%) and golden jackals (18%) were positive for EM. All animals examined were negative for EGsl. Univariate analysis showed no significant differences in EM prevalence with respect to animal species (red fox vs., golden jackal) (p = 0.22), age (p = 0.12), and sex (p = 0.18). Prevalence of EM was associated with the region (p < 0.001), with regions in central and southern Slovenia having higher EM prevalence and risk of infection. Due to the increase in population and expansion of habitat, the golden jackal may soon become as important definitive host for EM as the red fox.

Passive Disease Surveillance of Alpine Chamois (Rupicapra r. rupicapra) in Slovenia between 2000 and 2020.

In this paper, we provide an overview of the causes of death of Alpine chamois (Rupicapra r. rupicapra) diagnosed in the national passive health surveillance of chamois in Slovenia. From 2000 to 2020, 284 free-ranging chamois provided by hunters were necropsied at the Veterinary Faculty, University of Ljubljana, Slovenia. Depending on the results of complete necropsy, histopathological, bacteriological, parasitological, and virological examinations, a descriptive data analysis was performed. The most common causes of death in chamois were infectious diseases (82.2%), followed by non-infectious diseases (11.8%). Of all the causes of death, parasitic infections accounted for 70.3%, trauma for 9.7%, and bacterial infections for 9.3% of all cases. Less common diseases were viral infections, neoplasms, winter starvation, and metabolic disorders.

The First Report of Elaphostrongylus cervi Infection in Two Imported Wapitis (Cervus canadensis) in Slovenia.

This study describes two female wapitis (Cervus canadensis) with neurological signs associated with an Elaphostrongylus cervi (E. cervi) infection. The original host of the nematode parasite is the Eurasian red deer (Cervus elaphus), although other cervids and small ruminants may also be affected. The two wapitis imported from Canada were kept in an enclosure with the Slovenian red deer herd. After developing debilitating neurological signs, the wapitis were euthanized and examined for possible causes. A histopathological examination of the brain of the first wapiti revealed severe diffuse perivascular meningoencephalitis with chronic vasculitis, and some cross-sections of nematodes were found in the leptomeninges. A necropsy of the second wapiti revealed severe pachymeningitis and leptomeningitis, where several adult nematode parasites were found. E. cervi was confirmed by molecular methods. The prevalence of E. cervi in the European red deer population is high, but no study has been conducted to assess its prevalence in Slovenia. This was the first confirmation of E. cervi in Slovenia and the first infection with this parasite described in Europe in a wapiti. Elaphostrongylus cervi should also be considered as a differential diagnosis in Europe for all ruminants grazing on pastures frequented by red deer and showing neurological clinical signs.

High Seroprevalence of Toxoplasma gondii in Slovenian Wild Boars (Sus scrofa).

Toxoplasma gondii is a zoonotic parasite of great public health concern. Wild boars could be considered an emerging source of toxoplasmosis in humans due to the popularity of venison and their increasing population. The aim of this study was to determine the seroprevalence of T. gondii in the Slovenian wild boar population and evaluate risk factors for human infection. Of 353 samples, 62% were positive for T. gondii using ELISA tests. This is the highest T. gondii seroprevalence reported to date in wild boar worldwide. The increase in prevalence with increasing age (p = 0.003) and weight (p = 0.002) were statistically significant, whereas gender was not (p = 0.781). Odds for being T. gondii-positive increased with age with the largest difference being between 2-3-year-old and 1-2-year-old animals (OR = 2.66, 95%CI: 1.03-6.85). Animals weighing 20-40 kg had a higher risk than animals weighing 0-20 kg (OR = 2.74, 95%CI: 1.21-6.20), whereas a further increase in the weight was not associated with increasing the odds. Due to the high Toxoplasma prevalence, the study concluded that the risk of exposure to T. gondii from handling raw or undercooked wild boar meat is high. Surveillance protocols should be established at the national level together with increased awareness within the hunting community.

Clostridioides difficile in bat guano.

Bats are associated with the emergence of several mammalian diseases. Their sessional migration, and tendency to form large colonies in close proximity to human habitats enables effective intra- and inter-species transmission of pathogens. Clostridioides difficile is an important enteric pathogen in humans and animals; however, the source of its dissemination in the population is unknown. The purpose of this study was to determine the prevalence of C. difficile in bats, and to characterize C. difficile isolates. Feces (n = 93) was sampled from bats during their migration across Europe. Eighteen samples (19.4%) were positive for C. difficile; ribotypes 078, 056, and a new ribotype CDB3 were identified. Clostridioides difficile ribotypes 078 and 056 are associated with human and animal diseases. The C. difficile prevalence and ribotypes in this study do not necessarily identify bats as a significant source, but more likely as an indicator of C. difficile perpetuation in the environment.

Quantification of Clostridioides (Clostridium) difficile in feces of calves of different age and determination of predominant Clostridioides difficile ribotype 033 relatedness and transmission between family dairy farms using multilocus variable-number tandem-repeat analysis.

BACKGROUND: Community acquired Clostridioides (Clostridium) difficile infection (CA-CDI) is a significant health problem in human and veterinary medicine. Animals are often considered as potential reservoirs for CA-CDI. In Europe, family farming is the most predominant farming operation, with a complex interaction between animals and the community. Therefore, it is pertinent to evaluate transmission patterns of C. difficile on such prominent European farming model. Fecal samples from calves (n = 2442) were collected biweekly over a period of one year on 20 mid-size family dairy farms. Environmental samples (n = 475) were collected in a three month interval. Clostridioides difficile was detected using qPCR in 243 fecal samples (243/2442); positive samples were then quantified. Association between prevalence/load of C. difficile and age of the calves was estimated with logistic regression model. Most common C. difficile isolate from calves (n = 76) and the environment (n = 14) was C. difficile ribotype 033, which was further analyzed using multilocus variable-number tandem-repeat analysis (MLVA) to assess intra- and between-farm relatedness. RESULTS: Clostridioides difficile was detected in feces of calves less than 24 h old. Results showed a non-linear statistically significant decrease in shedding load of C. difficile with age (P < 0.0001). A nonlinear relationship was also established between the number of calves and the farm C. difficile prevalence, whereas the prevalence of C. difficile ribotype 033 increased linearly with the number of calves. MLVA revealed close intra-farm relatedness among C. difficile ribotypes 033. It also revealed that the between-farms close relatedness of C. difficile ribotypes 033 can be a direct result of farm to farm trade of calves. CONCLUSIONS: Implementation of better hygiene and management measures on farms may help decrease the risk of spreading CA-CDI between animals and the community. Trading calves older than 3 weeks would decrease the possibility C. difficile dissemination in the community because of lower prevalence and lower load of C. difficile in feces.

Risk factors associated with fecal shedding of Listeria monocytogenes by dairy cows and calves.

BACKGROUND: Listeria monocytogenes (LM) is an important foodborne pathogen affecting animals and humans. Listeriosis outbreaks in humans caused by consumption of unpasteurized dairy products are of serious concern. OBJECTIVE: To determine risk factors associated with fecal shedding of LM in family dairy farms. ANIMALS: Fecal samples were collected from cows and calves on 20 family dairy farms in 2-week intervals for a period of 1 year. METHODS: Longitudinal study. LM was detected using qPCR. Univariate mixed effect model and multivariate analyses were performed to associate risk factors (dietary change, breed, mastitis, other diseases, antibiotic treatment, other treatments, heat index, and meteorological season) with fecal shedding of LM. RESULTS: LM was isolated from all farms on at least 1 sampling day. The average yearly prevalence was 18.2% (98/540) and 8.4% (43/511) in cows and calves, respectively. Heat index (P = .05) and meteorological season (P = .04) affected fecal shedding of LM on a farm level. Meteorological season only influenced fecal shedding of LM in cows (P = .04), whereas heat index (P = .01) influenced fecal shedding of LM in calves. Spring season was identified as the major risk factor associated fecal shedding of LM on a farm level (P = .01) and in cows (P = .01). Dietary changes were associated with lower odds for fecal shedding of LM in calves (P < .01). CONCLUSIONS AND CLINICAL IMPORTANCE: Fecal shedding of LM is associated with environmental temperatures and the meteorological season. Farmers and veterinarians should use this information when implementing strategies to reduce risks for LM dissemination in animals and in the community.

Identification of risk factors influencing Clostridium difficile prevalence in middle-size dairy farms.

Farm animals have been suggested to play an important role in the epidemiology of Clostridium difficile infection (CDI) in the community. The purpose of this study was to evaluate risk factors associated with C. difficile dissemination in family dairy farms, which are the most common farming model in the European Union. Environmental samples and fecal samples from cows and calves were collected repeatedly over a 1 year period on 20 mid-size family dairy farms. Clostridium difficile was detected in cattle feces on all farms using qPCR. The average prevalence between farms was 10% (0-44.4%) and 35.7% (3.7-66.7%) in cows and calves, respectively. Bacterial culture yielded 103 C. difficile isolates from cattle and 61 from the environment. Most C. difficile isolates were PCR-ribotype 033. A univariate mixed effect model analysis of risk factors associated dietary changes with increasing C. difficile prevalence in cows (P = 0.0004); and dietary changes (P = 0.004), breeding Simmental cattle (P = 0.001), mastitis (P = 0.003) and antibiotic treatment (P = 0.003) in calves. Multivariate analysis of risk factors found that dietary changes in cows (P = 0.0001) and calves (P = 0.002) increase C. difficile prevalence; mastitis was identified as a risk factor in calves (P = 0.001). This study shows that C. difficile is common on dairy farms and that shedding is more influenced by farm management than environmental factors. Based on molecular typing of C. difficile isolates, it could also be concluded that family dairy farms are currently not contributing to increased CDI incidence.

Prevalence and molecular characterization of Clostridium difficile isolated from European Barn Swallows (Hirundo rustica) during migration.

BACKGROUND: Clostridium difficile is an important bacterial pathogen of humans and a variety of animal species. Birds, especially migratory passerine species, can play a role in the spread of many pathogens, including Clostridium difficile. Barn Swallows (Hirundo rustica) nest in close proximity to human habitats and their biology is closely associated with cattle farming. Therefore, we hypothesized that Barn Swallows can be the reservoir of Clostridium difficile. RESULTS: Barn Swallows (n = 175) were captured on their autumn migration across Europe to sub-Saharan Africa. Droppings were collected from juvenile (n = 152) and adult birds (n = 23). Overall prevalence of Clostridium difficile was 4% (7/175); 4.6% (7/152) in juvenile birds and 0/23 in adults. Clostridium difficile ribotypes 078, 002 and 014 were identified, which are commonly found in farm animals and humans. Three new Clostridium difficile ribotypes were also identified: SB3, SB159 and SB166, one of which was toxigenic, harbouring genes for toxins A and B. CONCLUSIONS: Results of this study indicate that Barn Swallows might play a role in national and international dissemination of Clostridium difficile and could serve as a source for human and animal infection. Clostridium difficile ribotype 078 was identified, which has been reported as an emerging cause of community-associated Clostridium difficile infection in humans. Based on this and other studies, however, it is more likely that Barn Swallows have a more indicative than perpetuating role in Clostridium difficile epidemiology.

An improved qPCR protocol for rapid detection and quantification of Clostridium difficile in cattle feces.

Clostridium difficile (CD) can cause a significant and transmissible disease in animals and humans, with poorly understood epidemiology. Animals have been suggested as a possible source of infection and environment contamination. It is necessary that a precise and rapid diagnostic tool is available for the detection of CD from clinical and/or environmental samples. A quantitative real-time PCR (qPCR) protocol for CD detection defined by Penders et al. (FEMS Microbiol Lett, 243, 2005, 141-147) was modified. The modified protocol, supported by a novel extraction method, was tested on CD-spiked cattle feces and clinical fecal samples from calves. Quantification was performed targeting CD 16S rRNA gene. Three different commonly used TaqMan universal PCR master mixes were also compared. Results indicate that the modified protocol is very sensitive with an LOD of 7.72 CD cells per g CD-spiked feces. The protocol is capable of precise quantification with an LOQ of 77.2 CD cells per g CD-spiked feces, R(2) between 0.9957 and 0.9968, isolation efficiency from 87.89% to 90.96%, and an interassay CV ranging from 3.71% to 9.57%. The qPCR protocol for the detection and quantification of CD from animal feces investigated and described in this article using MIQE guidelines has the lowest detection and quantification limits published to date. Therefore, it can be implemented for precise epidemiological investigations of CD infections in animals and humans.

Evaluation of combined high-efficiency DNA extraction and real-time PCR for detection of Mycobacterium avium subsp. paratuberculosis in subclinically infected dairy cattle: comparison with faecal culture, milk real-time PCR and milk ELISA.

BACKGROUND: Johne's disease is caused by Mycobacterium avium subsp. paratuberculosis (Map) and it is one of the most important diseases in cattle worldwide. Several laboratory tests for Map detection are available; however, these are limited by inadequate sensitivity and specificity when used in subclinically infected populations. To identify Map shedders in subclinically infected cattle, we used a new, high-yield method for DNA-extraction from Map in faeces combined with quantitative real-time PCR (qPCR) for amplification of the insertion sequence IS900 of Map (HYDEqPCR). Evaluation of HYDEqPCR was carried out in comparison with faecal culture, milk qPCR, and milk enzyme-linked immunosorbent assay (ELISA), on 141 faecal and 91 milk samples, from 141 subclinically infected dairy cattle. RESULTS: The qPCR proved to be highly sensitive, with a detection limit of 2 IS900 DNA copies/µl in 67 % of the reactions. It also showed 100 % specificity, as determined from 50 Map and non-Map strains, and by the sequencing of qPCR amplicons. The detection limit of HYDEqPCR was 90 Map/g Map-spiked faeces, which corresponds to 2.4 colony forming units/g Map-spiked faeces, with an estimated efficiency of 85 % (±21 %). When tested on the field samples, HYDEqPCR showed 89 % of the samples as positive for Map, whereas faecal culture, milk qPCR, and milk ELISA detected 19 %, 36 % and 1 %, respectively. Fisher's exact tests only show statistical significance (p ≤0.05) for the correlation between HYDEqPCR and faecal culture. The agreement between HYDEqPCR and milk qPCR and milk ELISA was poor, slight, and non-significant. CONCLUSIONS: This study highlights the advantages of HYDEqPCR for detection of Map in subclinically infected populations, in comparison with faecal culture, milk qPCR and milk ELISA. HYDEqPCR can detect low-level Map shedders that go undetected using these other methods, which will thus underestimate the proportions of Map-shedders in herds. Identification of these shedding animals is extremely important for prevention of the spread of Map infection in an animal population. Due to the relatively high sensitivity and specificity of HYDEqPCR, it can be applied to test for Map at the herd or individual level, regardless of animal age or production stage. HYDEqPCR will allow early detection and control of Map in any population at risk.

Zero prevalence of Clostridium difficile in wild passerine birds in Europe.

Clostridium difficile is an important bacterial pathogen of humans and a variety of animal species, where it can cause significant medical problems. The major public health concern is the possibility of inapparent animal reservoirs of C. difficile and shedding of bacteria to noninfected individuals or populations, as well as being a source of food contamination. Migrating birds can be a key epizootiological factor for transmission and distribution of pathogens over a wide geographic range. Therefore, the purpose of this study was to investigate whether migrating passerine birds can be a source of spread of C. difficile along their migration routes. Cloacal samples were taken from 465 passerine birds during their migration south over the Alps. Selective enrichment was used for detection of C. difficile. Clostridium difficile was not isolated from any of the samples, which indicates that migrating passerine birds are unlikely to serve as a reservoir and a carrier of C. difficile.