An international inter-laboratory study to compare digital PCR with ISO standardized qPCR assays for the detection of norovirus GI and GII in oyster tissue.

An optimized digital RT-PCR (RT-dPCR) assay for the detection of human norovirus GI and GII RNA was compared with ISO 15216-conform quantitative real-time RT-PCR (RT-qPCR) assays in an interlaboratory study (ILS) among eight laboratories. A duplex GI/GII RT-dPCR assay, based on the ISO 15216-oligonucleotides, was used on a Bio-Rad QX200 platform by six laboratories. Adapted assays for Qiagen Qiacuity or ThermoFisher QuantStudio 3D were used by one laboratory each. The ILS comprised quantification of norovirus RNA in the absence of matrix and in oyster tissue samples. On average, results of the RT-dPCR assays were very similar to those obtained by RT-qPCR assays. The coefficient of variation (CV%) of norovirus GI results was, however, much lower for RT-dPCR than for RT-qPCR in intra-laboratory replicates (eight runs) and between the eight laboratories. The CV% of norovirus GII results was in the same range for both detection formats. Had in-house prepared dsDNA standards been used, the CV% of norovirus GII could have been in favor of the RT-dPCR assay. The ratio between RT-dPCR and RT-qPCR results varied per laboratory, despite using the distributed RT-qPCR dsDNA standards. The study indicates that the RT-dPCR assay is likely to increase uniformity of quantitative results between laboratories.

Prevalence of red panda amdoparvovirus infection in European zoos.

Red panda amdoparvovirus (RPAV) was first described in captive red pandas (Ailurus fulgens) at a zoo in the United States in 2018. Subsequently, the prevalence of infection in zoos in the United States was reported to be 50%; however, RPAV prevalence outside the United States remains unstudied. This study was conducted to investigate the prevalence of RPAV in 134 red pandas from zoos in Europe. Overall, RPAV was detected with PCR in 21 of 62 zoos (33.9%), and the virus prevalence among individuals was estimated to be 24.2% (95% confidence interval, 17.4%-32.0%). Remarkably, adult females tested positive for RPAV more frequently than adult males. Zoos where RPAV was detected reported a significantly higher occurrence of alopecia (and clinical signs in general), whereas other commonly reported problems (fecal disorders and dental disease) showed no difference. A repeated pooled sampling of two positive individuals further showed that RPAV excretion in feces is intermittent, with the viral DNA being only detected on 8 out of 14 sampling days. The intermittent nature of excretion implies that RPAV prevalence may be higher than the estimated value.

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.

Food Safety Knowledge among Professional Food Handlers in Slovenia: The Results of a Nation-Wide Survey.

The authors present and discuss the results of a nation-wide survey on food safety knowledge among professional food handlers in Slovenia. The data were collected via a telephone survey using a well-established questionnaire adapted to the Slovenian context. Altogether, 601 respondents from hotels, restaurants, catering, and confectionery units completed the questionnaire. To assess food safety knowledge among food handlers in both general and specific domains, three indexes (a General Knowledge Index, a Personal Knowledge Index, and a Temperature Knowledge Index) were created. Among them, the Temperature Knowledge Index revealed the largest gaps in knowledge. An insufficient transfer of food safety knowledge from managers and chefs to assistant chefs and kitchen assistants in establishments where more persons handle food was evident, while a course titled "Hygiene Minimum" of standardised training from the past still significantly contributes to food safety knowledge. The results suggest a need for improvement in the current system of food safety training courses for professional food handlers in Slovenia. The human factor in the food supply chain still has a significant role in ensuring food safety culture, and therefore must become a more important part of the food safety management system.

Identification of a Novel Papillomavirus Type (MfoiPV1) Associated with Acrochordon in a Stone Marten (Martes foina).

Papillomaviruses (PVs) are an extremely large group of viruses that cause skin and mucosal infections in humans and various domestic and wild animals. Nevertheless, there is limited knowledge about PVs in wildlife hosts, including mustelid species. This study describes a case in stone marten (Martes foina) with a clinical manifestation of skin tumor, which is rather atypical for infections with PVs. The result of the papillomavirus PCR performed on the skin tumor sample was positive, and the complete PV genome was determined in the studied sample using next-generation sequencing technology. The analysis of the PV genome revealed infection of the stone marten with a putative new PV type belonging to the Dyonupapillomavirus genus. The proposed new stone marten PV type was named MfoiPV1.

A molecular survey and phylogenetic analysis of porcine circovirus type 3 using oral fluid, faeces and serum.

BACKGROUND: Porcine circovirus type 3 is the most recently discovered porcine circovirus, and an emerging pathogen. In this study the status of its presence on some Slovenian farms is reported. The effectiveness of the vaccine against porcine circovirus type 2 was assessed against porcine circovirus type 3. Group samples of oral fluid, faeces and individual serum samples were taken from six different pig categories and tested for presence of viral DNA, using both real time and conventional PCR. Positive samples were subjected to direct Sanger sequencing. Nucleotide sequences were analyzed and compared to GenBank PCV3 sequences. RESULTS: Positive samples were sent for genome sequencing, which confirmed the presence of virus in all different pig categories on five farms. A high to moderate correlation of strong statistical significance was found between individual serum samples, oral fluid and faeces. Slovenian PCV3 was found to be distributed in a way similar to that of other countries. Slovenian PCV3 nt sequences are highly related, sharing more than 99.5% nt identity. On one farm a commercially available vaccine against porcine circovirus type 2 was used on 3-week-old pigs. It did not affect the presence of porcine circovirus type 3 in oral fluid or sera of any of the seven age groups of pigs, each with two control groups. CONCLUSIONS: The results constitute the first discovery of the virus in Slovenia. Genome sequencing has revealed a high degree of similarity between Slovenian and GenBank isolates.

Molecular detection of porcine reproductive and respiratory syndrome virus, porcine circovirus 2 and hepatitis E virus in oral fluid compared to their detection in faeces and serum.

BACKGROUND: Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), Porcine Circovirus Type 2 (PCV2) and Hepatitis E virus (HEV) are common and economically important viral disease causative agents detected in pig oral fluid (OF), faeces and serum at some infection stages. The purpose of this study was to detect PRRSV, PCV2 and HEV on six pig farms to determine which of the three sample types, OF, faeces or serum is appropriate for the diagnosis of these viruses in different pig categories. The following pig categories were included: 5 weeks-old (w/o), 7 w/o, 9 w/o, 11 w/o weaners, fatteners and breeding sows. Pursuant to the preliminary detection of each pathogen at the selected farms, OF samples, faeces, serum pools and 10 individual sera were examined, using PCR, for each age category. If any of the viruses were found in pools of faeces and OF, then faeces and OF from positive farms were tested separately for each pig category. The viral nucleic acids were detected using RT-PCR, PCR and real-time RT-PCR, for PRRSV, PCV2 and HEV respectively. RESULTS: PRRSV and HEV were detected on one farm and PCV2 on three others, positive results being more often obtained from the OF than from the faeces of the same animals. Ten individual serum samples from pigs from the same group of animals were also tested. The viruses were detected in almost all individual sera and OF in the same pig category with some exceptions: PRRSV was detected in the OF of fatteners but was absent in their sera; on Farm 2, PCV2 was detected in sera of 11 w/o pigs and fatteners but absent in group samples of their OF and, vice versa, in case of 9 w/o animals; HEV was detected in the OF of the youngest, 5 w/o weaners and absent in sera of the same age group. CONCLUSIONS: The primary finding of the study is that OF is a welfare-friendly, non-invasive and highly efficient matrix for pathogen detection, thus evidencing the usefulness of pig OF as a matrix in which each of the three viruses considered can be detected with the highest probability.

Clostridioides difficile in national food surveillance, Slovenia, 2015 to 2017.

BackgroundClostridioides difficile is an important human and animal intestinal pathogen. Because of increasing indications of an association between C. difficile and food, in 2015, the Administration of the Republic of Slovenia for Food Safety, Veterinary Sector and Plant Protection (UVHVVR) included C. difficile in its national food surveillance.AimWe aim to report the results and experience with a nationwide and long-term testing of food for C. difficile as a part of a regular national food surveillance programme.MethodsRetail minced meat and meat preparations (beef, pork and poultry) were sampled within a three-year period, 2015 to 2017. Selected raw retail vegetables, leaf salads and root vegetables, and ready-to-eat salads were only sampled during 2016 and 2017. Seafood was only sampled in 2017.ResultsAltogether, 434 samples were tested, with 12 of 336 (3.6%) meat samples and 6 of 98 (6.1%) raw vegetables contaminated with C. difficile. Twelve of 18 recovered food isolates were toxigenic (toxinotypes 0, III, V, XII). The isolates belonged to 13 different PCR ribotypes, 001 being most common (5 isolates). Several food types with an increased potential of being contaminated with C. difficile were detected by surveillance.ConclusionThe three-year C. difficile testing within the national food surveillance revealed a low proportion of C. difficile-contaminated food and high genotype variability. Because the risk of C. difficile infection associated with C. difficile-contaminated food is unknown, no measures were recommended in the case of positive results.

First Complete Genome of Lake Sinai Virus Lineage 3 and Genetic Diversity of Lake Sinai Virus Strains From Honey Bees and Bumble Bees.

The complete genome of Lake Sinai virus 3 (LSV3) was sequenced by the Ion Torrent next-generation sequencing (NGS) technology from an archive sample of honey bees collected in 2010. This strain M92/2010 is the first complete genome sequence of LSV lineage 3. From October 2016 to December 2017, 56 honey bee samples from 32 different locations and 41 bumble bee samples from five different locations were collected. These samples were tested using a specific reverse transcriptase-polymerase chain reaction (RT-PCR) method; 75.92% of honey bee samples and 17.07% of bumble bee samples were LSV-positive with the RT-PCR method. Phylogenetic comparison of 557-base pair-long RNA-dependent RNA polymerase (RdRp) genome region of selected 23 positive samples of honey bees and three positive bumble bee samples identified three different LSV lineages: LSV1, LSV2, and LSV3. The LSV3 lineage was confirmed for the first time in Slovenia in 2010, and the same strain was later detected in several locations within the country. The LSV strains detected in bumble bees are from 98.6 to 99.4% identical to LSV strains detected among honey bees in the same territory.

A molecular survey, whole genome sequencing and phylogenetic analysis of astroviruses from roe deer.

BACKGROUND: Although astroviruses (AstV) have been detected in a variety of host species, there are only limited records of their occurrence in deer. One of the most important game species in Europe, due to its meat and antlers, is roe deer. Infected game animals can pose a threat to the health of other animals and of humans, so more attention needs to be focused on understanding the diversity of viruses in wildlife. The complete genome and organization of the roe deer AstV genome have not so far been described. RESULTS: In our study, 111 game animals were screened for the presence of AstV. While no AstVs were detected in red deer, wild boar, chamois and mouflon, AstV RNA was present in three samples of roe deer. They were further subjected to whole genome sequencing with next generation sequencing. In this study, two AstV genomes were assembled; one in sample D5-14 and one in sample D12-14, while, in sample D45-14, no AstV sequences were identified. The complete coding sequences of the AstV SLO/D5-14 strain genome and of the almost complete genome of the AstV SLO/D12-14 strain were determined. They showed a typical Mamastrovirus organization. Phylogenetic analyses and amino acid pairwise distance analysis revealed that Slovenian roe deer AstV strains are closely related to each other and, also, related to other deer, bovine, water buffalo, yak, Sichuan takin, dromedary, porcine and porcupine AstV strains - thus forming a highly supported group of currently unassigned sequences. CONCLUSIONS: Our findings suggest the existence of a new Mamastrovirus genogroup might be constituted while this aforementioned group is distantly related to Mamastrovirus genogroups I and II. In this study, additional data supporting a novel taxonomic classification are presented.

Genome sequence of an aichivirus detected in a common pipistrelle bat (Pipistrellus pipistrellus).

The family Picornaviridae includes important human and animal pathogens that are associated with a wide range of diseases and, in some cases, have zoonotic potential. During epidemiological surveillance of bats, we identified, by next-generation sequencing (NGS) techniques, the presence of picornavirus RNA in a common pipistrelle bat (Pipistrellus pipistrellus). By coupling NGS, primer-walking strategies, and sequence-independent protocols to obtain the sequences of the 5' and 3' termini, we reconstructed the genome sequence of picornavirus strain ITA/2017/189/18-155. The genome of the bat picornavirus is 8.2 kb in length and encodes a polyprotein of 2462 amino acids. A comparison of polyprotein sequences revealed that this virus is distantly related (65.1% and 70.9% sequence identity at the nucleotide and amino acid level, respectively) to a bat aichivirus identified in 2010. Phylogenetic analysis showed that this picornavirus clustered closely with members of the genus Kobuvirus, which also includes human and animal aichiviruses. The identification of aichiviruses in several animal hosts is providing hints that will lead to an understanding of their origin and evolutionary patterns.

Effect of Composting Under Semipermeable Film on the Sewage Sludge Virome.

The addition of compost from sewage sludge to soils represents a sustainable option from an environmental and economic point of view, which involves the valorisation of these wastes. However, before their use as a soil amendment, compost has to reach the quality levels according to the normative, including microbial parameters. Viruses are not included in this regulation and they can produce agricultural problems and human diseases if the compost is not well sanitised. In this study, we carried out the analysis of the viral populations during a composting process with sewage sludge at an industrial scale, using semipermeable cover technology. Viral community was characterised by the presence of plant viruses and bacteriophages of enteric bacteria. The phytopathogen viruses were the group with the highest relative abundance in the sewage sludge sample and at 70 days of the composting process. The diversity of bacterial viruses and their specificity, with respect to the more abundant bacterial taxa throughout the process, highlights the importance of the interrelations between viral and bacterial communities in the control of pathogenic communities. These results suggest the possibility of using them as a tool to predict the effectiveness of the process.

High detection rate and high genetic diversity of genogroup I Picobirnaviruses from roe deer.

Picobirnaviruses (PBVs) have been characterized as opportunistic enteric pathogens detected in various domestic, zoo and wild animals, suggesting a wide host range of these viruses. It is thus important to monitor wild animals for the presence of various human and animal pathogens in order to identify a potential reservoir of infectious diseases. In this study, the first phylogenetic analysis of PBV from roe deer (Capreolus capreolus) was performed with a total of 70 investigated samples of feces from roe deer collected in 2014 and 2015 during a survey throughout Slovenia. A high detection rate of PBVs was observed with newly designed specific primers, 42 samples out of 70 (60%) being positive. Phylogenetic analysis of the partial RdRp gene showed that roe deer PBV sequences were distributed over the whole phylogenetic tree and were distributed between 7 highly supported groups and 12 separate branches within the PBV genogroup I. The animal PBV strain most closely related to roe deer PBV strains was the Rhesus macaque PBV/BGD/PbV-55 strain, with 89.1% nucleotide identity to that of PBV SLO/D80-14. Overall nucleotide sequence identity between PBV strains obtained from roe deer ranged from 60.4 to 100%, confirming the high genetic diversity with no subtypes related to host species or geographic location in general. This first phylogenetic survey of roe deer PBVs provides further knowledge concerning the putative host range and confirms the high genetic diversity of these PBVs.

Genetic Diversity of Deformed Wing Virus From Apis mellifera carnica (Hymenoptera: Apidae) and Varroa Mite (Mesostigmata: Varroidae).

Deformed wing virus (DWV) is one of the most widespread viruses that infect honey bee colonies. The route of infection is directly through contaminated food, feces, and air, or indirectly through the varroa mite, which acts as a vector. Positive DWV samples were obtained from Carniolan honey bee (Apis mellifera carnica) colonies and of varroa mites from the whole territory of Slovenia during a survey between 2007 and 2014. Nucleotide sequences of 471 nucleotides for the L protein gene and 573 nucleotides for the helicase gene were compared. High genetic diversity was observed among these Slovenian Carniolan honey bee DWV field samples, as well as with almost all the strains previously found in other countries in Europe. Phylogenetic analyses in two regions of the viral genome show that several of the DWV strains obtained from honey bees and varroa are genetically very closely related, confirming the important role of varroa in the transmission of DWV. Identification of closely related sequences also confirmed that the same strains of DWV have been successfully transmitted between various honey bee colonies and apiaries. It has also been established that simultaneous infection, in one apiary, of honey bees with two or more different strains of DWV is quite frequent. This is phylogenetic study that compares honey bee and varroa DWV strains from Carniolan honeybees.

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 novel reassortant mammalian orthoreoviruses from bats in Slovenia.

BACKGROUND: Recently, mammalian orthoreoviruses (MRVs) were detected for the first time in European bats, and the closely related strain SI-MRV01 was isolated from a child with severe diarrhoea in Slovenia. Genetically similar strains have also been reported from other mammals, which reveals their wide host distribution. The aim of this study was to retrospectively investigate the occurrence and genetic diversity of MRVs in bats in Slovenia, from samples obtained throughout the country in 2008 to 2010, and in 2012 and to investigate the occurrence of the novel SI-MRV01 MRV variant in Slovenian bats. RESULTS: The detection of MRVs in bat guano was based on broad-range RT-PCR and specific bat MRV real-time RT-PCR. Subsequently, MRV isolates were obtained from cell culture propagation, with detailed molecular characterisation through whole-genome sequencing. Overall, bat MRVs were detected in 1.9% to 3.8% of bats in 2008, 2009 and 2012. However, in 2010 the prevalence was 33.0%, which defined an outbreak of the single SI-MRV01 strain. Here, we report on the identification of five MRV isolates of different serotypes that are designated as SI-MRV02, SI-MRV03, SI-MRV04, SI-MRV05 and SI-MRV06. There is high genetic variability between these characterised isolates, with evident genome reassortment seen across their genome segments. CONCLUSIONS: In conclusion, we have confirmed the presence of the SI-MRV01 strain in a Slovenian bat population. Moreover, according to genetic characterisation of S1 genome segment, all three MRV serotypes were present in the bat population. In this study, five independent MRV isolates were obtained and detailed whole genome analysis revealed high diversity between them. This study generates new information about the epidemiology and molecular characteristics of emerging bat MRV variants, and provides important molecular data for further studies of their pathogenesis and evolution.

Development and Validation of a New Protocol for Detecting and Recovering Clostridium difficile from Meat Samples.

There is no recommended protocol for detecting and isolating Clostridium difficile present in food samples. Here, we have evaluated the recovery of C. difficile in meat samples after incubating them in various enrichment broths. The media were as follows: cycloserine-cefoxitin fructose broth supplemented with taurocholic acid, d-cycloserine, cefoxitin, and lysozyme; cycloserine-cefoxitin mannitol broth with taurocholate and lysozyme; and cycloserine-cefoxitin fructose broth supplemented with taurocholic acid, C. difficile moxalactam norfloxacin selective supplement, and lysozyme. Samples were inoculated with various strains and quantities of C. difficile and then enriched in the different broths for 1, 4, and 7 days. C. difficile was isolated on agar plates and detected with quantitative real-time PCR (qPCR). The procedure using enrichment in cycloserine-cefoxitin fructose broth supplemented with taurocholic acid, d-cycloserine, cefoxitin, and lysozyme and incubation for 4 days for qPCR detection and 7 days for isolation (plating on C. difficile agar base with added C. difficile selective supplement and 7% [v/v] defibrinated horse blood after alcoholic shock and centrifugation) was validated. Samples of different kinds of meat and meat preparation were contaminated and used for validation of the chosen protocol. The sensitivity of detection with qPCR was 100%, and the sensitivity of the isolation method was 96%.

Complete Genome Sequence of Roe Deer Picobirnavirus Strain PBV/roe_deer/SLO/D38-14/2014.

Picobirnaviruses (PBVs) have been detected in feces from various animal species and humans. Here, we report the complete genome sequence of the PBV/roe_deer/SLO/D38-14/2014 strain, which is the first PBV detected in roe deer, providing additional knowledge about the high diversity and host range of PBVs.

Whole genome sequence and a phylogenetic analysis of the G8P[14] group A rotavirus strain from roe deer.

BACKGROUND: Group A rotaviruses (RVA) are associated with acute gastroenteritis in children and in young domestic and wild animals. A RVA strain was detected from a roe deer for the first time during a survey of game animals in Slovenia in 2014. A further RVA strain (SLO/D110-15) was detected from a roe deer during 2015. The aim of this study was to provide a full genetic profile of the detected RVA strain from roe deer and to obtain additional information about zoonotic transmitted strains and potential reassortments between human rotavirus strains and zoonotic transmitted rotavirus strains. The next generation sequencing (NGS) analysis on Ion Torrent was performed and the whole genome sequence has been determined together with a phylogenetic analysis. RESULTS: The whole genome sequence of SLO/D110-15 was obtained by NGS analyses on an IonTorrent platform. According to the genetic profile, the strain SLO/D110-15 clusters with the DS-1-like group and expresses the G8-P[14]-I2-R2-C2-M2-A3-N2-T6-E2-H3 genome constellation. Phylogenetic analysis shows that this roe deer G8P[14] strain is most closely related to RVA strains found in sheep, cattle and humans. A human RVA strain with the same genotype profile was detected in 2009 in Slovenia. CONCLUSIONS: The G8P[14] genotype has been found, for the first time, in deer, a newly described host from the order Artiodactyla for this RVA genotype. The finding of a rotavirus with the same genome segment constellation in humans indicates the possible zoonotic potential of this virus strain.

Detection and Whole-Genome Analysis of a Zoonotic G8P[14] Rotavirus Strain Isolated from a Child with Diarrhea.

The group A rotavirus strain SI-2987/09 was detected in a child with severe diarrhea. The whole-genome analysis revealed the G8-P[14]-I2-R2-C2-M2-A3-N2-T6-E2-H3 genome constellation, which reflects the zoonotic transmission of the strain most probably from the ungulate group. This was also confirmed by the high nucleotide identity to those animal rotavirus strains.