Polarisation of Major Histocompatibility Complex II Host Genotype with Pathogenesis of European Brown Hare Syndrome Virus.

A study was conducted in order to determine the occurrence of European Brown Hare Syndrome virus (EBHSV) in Denmark and possible relation between disease pathogenesis and Major Histocompatibility Complex (MHC) host genotype. Liver samples were examined from 170 brown hares (hunted, found sick or dead), collected between 2004 and 2009. Macroscopical and histopathological findings consistent with EBHS were detected in 24 (14.1%) hares; 35 (20.6%) had liver lesions not typical of the syndrome, 50 (29.4%) had lesions in other tissues and 61 (35.9%) had no lesions. Sixty five (38.2%) of 170 samples were found to be EBHSV-positive (RT-PCR, VP60 gene). In order to investigate associations between viral pathogenesis and host genotype, variation within the exon 2 DQA gene of MHC was assessed. DQA exon 2 analysis revealed the occurrence of seven different alleles in Denmark. Consistent with other populations examined so far in Europe, observed heterozygosity of DQA (H o = 0.1180) was lower than expected (H e = 0.5835). The overall variation for both nucleotide and amino acid differences (2.9% and 14.9%, respectively) were lower in Denmark than those assessed in other European countries (8.3% and 16.9%, respectively). Within the peptide binding region codons the number of nonsynonymous substitutions (dN) was much higher than synonymous substitutions (dS), which would be expected for MHC alleles under balancing selection. Allele frequencies did not significantly differ between EBHSV-positive and -negative hares. However, allele Leeu-DQA*30 was detected in significantly higher (P = 0.000006) frequency among the positive hares found dead with severe histopathological lesions than among those found sick or apparently healthy. In contrast, the latter group was characterized by a higher frequency of the allele Leeu-DQA*14 as well as the proportion of heterozygous individuals (P = 0.000006 and P = 0.027). These data reveal a polarisation between EBHSV pathogenesis and MHC class II genotype within the European brown hare in Denmark

Development of a multiplex bead assay for simultaneous serodiagnosis of antibodies against Mycobacterium bovis, Brucella suis, and Trichinella spiralis in wild boar

This article belongs to the Special Issue Farm Animal and Wildlife Zoonotic Microorganisms.The aim of this study was to evaluate the diagnostic performance of a multiplex bead assay for the simultaneous detection of antibodies against Mycobacterium bovis, Brucella suis, and Trichinella spiralis. Sera from Eurasian wild boar of known serological status for TB (64 seropositive, 106 seronegative), Brucella (30 seropositive, 39 seronegative), and Trichinella (21 seropositive, 97 seronegative) were used for the development and evaluation of the assay. Magnetic beads coated with recombinant MPB83 antigen (TB), a whole-cell B. suis 1330 antigen, and an E/S T. spiralis antigen were used for the detection of specific antibodies using Bio-Rad Bio-Plex technology. The sensitivities (Se) and specificities (Sp) of the multiplex assay were, for M. bovis, 0.98 and 0.86; for B. suis, 1.00 and 0.97; and for T. spiralis, 0.90 and 0.99 (Se and Sp, respectively). The results show the diagnostic potential of this assay for the simultaneous detection of antibodies against M. bovis, B. suis, and T. spiralis in wild boar.We thankfully acknowledge the financial support of the European Union Seventh Framework Programme (2007–2013) under grant agreement no. 222633 (WildTech) titled “Novel Technologies for Surveillance of Emerging and Re-emerging Infections of Wildlife”.Peer reviewe

Εργαστηριακή διαγνωστική προσέγγιση σε λοιμώδη νοσήματα του σκύλου με ιδιαίτερη σημασία για την άγρια πανίδα και τη δημόσια υγεία

This thesis aimed to investigate specific canine infectious diseases of wildlife and public health significance by a laboratory diagnostic approach, combining laboratory techniques and environmental niche modelling, where it was considered appropriate. To this end, samples from diarrhoeic dogs were tested for the presence of Canine Parvovirus and Salmonella spp. Also, samples from European Brown hares were tested for the presence of Salmonella spp. only, as Canine Parvovirus strains prefer the carnivora as hosts. Canine Parvovirus is a pathogen of importance for dogs and other wild carnivore species while Salmonella spp. remain the second cause of human gastroenteritis cases in the EU; thus means that is of major Public Health importance.In the first chapter of this study, the international literature was critically reviewed, and health risks associated with human animal interactions were presented. More specifically, enteric pathogens of zoonotic risk which are transmitted by faeces of dogs and cats were referred in groups (Parasites/ Protozoa/ Bacteria/Viruses). Among them, Salmonellae, Campylobacter, Rotaviruses, Toxoplasma gondii, Echinococcus are of significant for many countries including Greece. The evaluation of the diagnostic accuracy of a commercial diagnostic tool for the detection of Canine Parvovirus is described in the second chapter. The rapid in-clinic test was compared with an established PCR method, which is considered as the most reliable diagnostic method. 78 faecal samples were collected from diarrhoeic dogs. Implementation of vaccination within a month prior to the onset of diarrhoea was reported for 12 of the sampled dogs. The rapid diagnostic test was performed in 23 of the faecal samples directly, while the rest were placed into a sterile swab and stored at −20 ◦C. The sensitivity of the rapid diagnostic test compared to PCR in the total number of samples, in samples from non-vaccinated dogs and in samples tested directly after collection were 22.22% (95% CI: 13.27–33.57%), 26.67% (95% CI: 16.08–39.66%) and 76.47% (95% CI: 50.10–93.04%) respectively, while the specificity of the test was 100% in any case. Based on these results, it can be argued that negative results do not exclude parvoenteritis, especially in dogs with early vaccination history, but a positive result almost certainly indicates CPV infection. The test should be conducted directly in order to expect improved sensitivity.In the third chapter, we aimed to investigate the presence of Canine Parvovirus in dogs with compatible clinical signs in Central Greece, to identify demographic and environmental factors associated with CPV infection, A total of 117 faecal samples were collected from diarrhoeic dogs and were tested for a fragment of VP2 capsid gene. Multivariate logistic regression, using R program, revealed that significant factors identified were age and utility of dogs (work dogs more probable to be positive versus pet-dogs). Geographical information system (GIS) together with the Ecological Niche Model (ENM) showed that the most important findings of the study were (i) Central plain of Thessaly were identified as high risk geographical areas, (ii) Livestock density (iii) land uses and specifically the category: discontinuous urban fabric and human population density are the most significant environmental parameters as risk factors according to the maximum entropy model. The last chapter concerns the detection of Salmonella spp., an important zoonotic pathogen in dogs and European Brown Hare, which is an important game species, in Thessaly. 50 samples (liver tissue) from hares hunted in Central Greece and 117 samples (faecal swabs) from dogs were examined for Salmonella using a validated PCR protocol (Malorny et al. 2003). Also, a spatial analysis using GIS was conducted to investigate possible geographical distribution and environmental risk factors, associated with Salmonella presence. 10 hares out of 50 and 8 out of 117 dogs were found to be Salmonella positive. GIS analysis using Max Entropy Montel showed that risk for the exposure to Salmonella in hares are driven by environmental parameters; precipitation seasonality, precipitation of the coldest quarter and livestock density being the most significant. Respectively, for dogs being positive to Salmonella and its potential distribution, the significant environmental parameters were: maximum temperature of the warmest month, mean temperature of warmest quarter of the year, livestock density and normalized difference vegetation index (NDVI) for August. Livestock density is an environmental parameter which is shared between dogs and hares positive to Salmonella, indicating that there is a probable spill-over among farm animals, dogs and wildlife. This study, which is the first of this kind from Greece, suggests that the role of hares and dogs in Salmonella shedding and transmission deserves further elucidation.This thesis provides evaluation of existing diagnostic methods for parvoenteritis, which is present in the canine population of the Greek mainland. It also explores the association of CPV presence in diarrhoeic dogs with certain demographic and environmental factors. Apart from that, the Salmonella presence in diarrhoeic dogs and hares is reported and associated with environmental factors which differ between the two animal species, suggesting that in this case the different habits of the hosts are depicted. The continuous surveillance of canine populations in combination with targeted wildlife species could provide information on the pathogens (especially zoonotic) circulating posing risk for wildlife, domestic animals and humans.Ο στόχος της παρούσας διατριβής ήταν η μελέτη ορισμένων λοιμωδών νοσημάτων του σκύλου, ιδιαίτερης σημασίας για την άγρια πανίδα και τη Δημόσια Υγεία μέσω διαγνωστικής εργαστηριακής προσέγγισης, συνδυάζοντας εργαστηριακές τεχνικές και οικολογικού μοντέλου θέσης, όπου αυτό κρίθηκε απαραίτητο. Για το σκοπό αυτό, δείγματα από σκύλους που παρουσίαζαν διάρροια εξετάστηκαν για την εύρεση του ιού Canine Parvovirus και σαλμονέλλας. Επίσης, δείγματα από ευρωπαϊκούς καφετί λαγούς εξετάστηκαν για την παρουσία σαλμονέλλας, εφόσον τα στελέχη Canine Parvovirus προτιμούν ως ξενιστές σαρκοφάγα είδη. Ο ιός Canine Parvovirus επιλέχθηκε ως παθογόνος παράγοντας με ιδιαιτέρη σημασία για τους σκύλους και άλλα άγρια είδη σαρκοφάγων ενώ η Σαλμονέλλα παραμένει η δεύτερη συχνότερη αιτία γαστρεντερίτιδας σε ανθρώπους στην Ευρωπαϊκή Ένωση, δηλαδή αποτελεί παθογόνο παράγοντα με μεγάλη σημασία για τη Δημόσια Υγεία. Στο πρώτο κεφάλαιο της μελέτης, έγινε κριτική ανασκόπηση της διεθνούς βιβλιογραφίας και παρουσιάστηκαν κίνδυνοι για την ανθρώπινη υγεία που προκύπτουν από την αλληλεπίδραση ανθρώπων και ζώων. Πιο συγκεκριμένα, ζωονοτικοί παθογόνοι παράγοντες που εντοπίζονται στον εντερικό σωλήνα και μπορούν να μεταδοθούν μέσω των κοπράνων παρουσιάστηκαν σε κατηγοριοποίηση (Παράσιτα/ Πρωτόζωα/ Βακτήρια/ Ιοί). Μεταξύ αυτών, στελέχη Salmonellae, Campylobacter, ροταϊοί, Toxoplasma gondii και ο εχινόκοκκος αποτελούν πρόβλημα σε πολλές χώρες, συμπεριλαμβανομένης της Ελλάδας. Η εκτίμηση της διαγνωστικής ακρίβειας μιας εμπορικής διαγνωστικής μεθόδου για την ανίχνευση του Canine Parvovirus περιγράφεται στο δεύτερο κεφάλαιο. Γίνεται σύγκριση της ταχείας εμπορικής δοκιμής με συγκεκριμένη PCR μέθοδο, που θεωρείται η πιο αξιόπιστη μέθοδος για την ανίχνευση του ιού. 78 δείγματα κοπράνων συλλέχθηκαν από σκύλους με διάρροια. Σε 12 σκύλους αναφέρθηκε εμβολιασμός σε διάστημα ενός μήνα πριν την έναρξη των συμπτωμάτων. Η ταχεία δοκιμή έγινε σε 23 δείγματα άμεσα, ενώ τα υπόλοιπα αποθηκεύτηκαν στους -20◦C. Η ευαισθησία της εμπορικής μεθόδου σε σχέση με την PCR ήταν 22.22% (95% CI: 13.27–33.57%) όσον αφορά το σύνολο των δειγμάτων, στα δείγματα από μη εμβολιασμένους σκύλους 26.67% (95% CI: 16.08–39.66%) και στα δείγματα που εξετάστηκαν αμέσως μετά τη συλλογή τους 76.47% (95% CI: 50.10–93.04%). Η ειδικότητα της δοκιμής ήταν 100%. Σύμφωνα με τα προαναφερθέντα αποτελέσματα, εξάγεται το συμπέρασμα ότι τα αρνητικά αποτελέσματα της ταχείας δοκιμής δεν αποκλείουν την ύπαρξη του ιού, αλλά το θετικό αποτέλεσμα σχεδόν σίγουρα υποδηλώνει μόλυνση από CPV. Η ταχεία δοκιμή είναι προτιμότερο να διεξάγεται, αμέσως μετά τη συλλογή των δειγμάτων.Στο τρίτο κεφάλαιο, στόχος ήταν η ανίχνευση της παρουσίας του ιού Canine Parvovirus σε σκύλους με συμβατά κλινικά συμπτώματα στην Κεντρική Ελλάδα καθώς και η διαπίστωση ύπαρξης δημογραφικών και περιβαλλοντικών παραγόντων που ενδέχεται παίζουν κάποιο ρόλο στην εμφάνιση του. Συνολικά εξετάστηκαν 117 δείγματα κοπράνων σκύλων έναντι ενός τμήματος του γονιδίου VP2. Η πολυπαραγοντική λογιστική παλινδρόμηση, με τη χρήση του προγράμματος R, έδειξε ότι οι παράγοντες που σχετίζονται με την εμφάνιση του ιού είναι η ηλικία και η χρήση του σκύλου (οι σκύλοι εργασίας είναι πιο πιθανό να μολυνθούνε από ότι οι σκύλοι αμιγώς συντροφιάς). Τα ευρήματα του Γεωγραφικού Πληροφοριακού Συστήματος μαζί με το οικολογικό μοντέλο θέσης ήταν τα εξής: (i) η πυκνότητα του κτηνοτροφικού κεφαλαίου και (ii) οι χρήσεις γης (συγκεκριμένα ο διακεκομμένος αστικός ιστός) και η πυκνότητα ανθρώπινου πληθυσμού αναγνωρίζονται ως οι πιο σημαντικοί περιβαλλοντικοί παράγοντες για την εμφάνιση του ιού. Το τελευταίο κεφάλαιο αφορά την ανίχνευση Salmonella spp., ενός σημαντικού ζωονοτικού παθογόνου, σε σκύλους και ευρωπαϊκούς καφετί λαγούς, ενός διαδεδομένου είδους θηράματος. 50 δείγματα από θηρευμένους λαγούς και 117 δείγματα σκύλων από την Κεντρική Ελλάδα εξετάστηκαν με αξιολογημένη μέθοδο PCR. Επιπλέον, έγινε χωρική ανάλυση με τη χρήση ΓΠΣ για τη διερεύνηση της πιθανής γεωγραφικής κατανομής και των σημαντικών περιβαλλοντικών παραγόντων για την παρουσία του βακτηρίου. Βρέθηκαν θετικοί 10 από τους λαγούς και 8 από τα 117 δείγματα σκύλων. Η ανάλυση ΓΠΣ μαζί με το μοντελοποίηση ειδών έδειξε τα εξής: για τους λαγούς σημαντικοί περιβαλλοντικοί παράγοντες ήταν η εποχικότητα της βροχόπτωσης, η βροχόπτωση κατά το πιο ψυχρό τρίμηνο του έτους και η πυκνότητα του κτηνοτροφικού κεφαλαίου. Αντιστοίχως, για σκύλους θετικούς στη σαλμονέλλα σημαντικοί βρέθηκαν: η μέγιστη θερμοκρασία του πιο θερμού μήνα, η μέση θερμοκρασία του πιο θερμού μήνα, η πυκνότητα του κτηνοτροφικού κεφαλαίου και ο δείκτης βλάστησης για τον Αύγουστο. Η πυκνότητα του κτηνοτροφικού κεφαλαίου είναι ένας κοινός παράγοντας που σχετίζεται με την παρουσία σαλμονέλλας και στα δύο ζωικά είδη, υποδεικνύοντας ότι ενδεχομένως υπάρχει μετάδοση του παθογόνου μεταξύ των παραγωγικών ζώων, των σκύλων και της άγριας πανίδας. Αυτή η μελέτη, που έγινε για πρώτη φορά στην Ελλάδα υποδηλώνει ότι ο ρόλος των λαγών και τον σκύλων στην απέκκριση και μετάδοση των Salmonellae χρειάζεται περαιτέρω διερεύνηση. Σε αυτή τη διδακτορική διατριβή, έγινε εκτίμηση μιας εμπορικά διαθέσιμης διαγνωστικής μεθόδου για την παρβοεντερίτιδα, η οποία είναι ακόμα παρούσα στην ηπειρωτική Ελλάδα. Εξερευνά επίσης τη σχέση μεταξύ της παρουσίας του ιου CPV με συγκεκριμένους δημογραφικούς και περιβαλλοντικούς παράγοντες. Επίσης, η παρουσία της σαλμονέλλας ανιχνεύεται σε σκύλους με διάρροια και λαγούς, ενώ συνδέεται με περιβαλλοντικούς παράγοντες, οι οποίοι διαφοροποιούνται μεταξύ των δύο ζωικών ειδών, υποδεικνύοντας ότι σε αυτή την περίπτωση αντανακλώνται οι συνήθειες των ξενιστών. Η συνεχής επιτήρηση των πληθυσμών σκύλων σε συνδυασμό με στοχευμένους πληθυσμούς ειδών άγριας πανίδας είναι δυνατό να δώσουν πληροφορίες για παθογόνους παράγοντες (ειδικά ζωονοτικούς) που κυκλοφορούν αποτελώντας κίνδυνο για την άγρια πανίδα, τα κατοικίδια ζώα και τους ανθρώπους

Risk and Environmental Factors Associated with the Presence of Canine Parvovirus Type 2 in Diarrheic Dogs from Thessaly, Central Greece

Canine parvovirus type 2 (CPV-2) primarily infects dogs, which are the main host reservoir, causing severe gastrointestinal disease associated with immunosuppression. The present study was conducted in Thessaly, Greece and aimed to identify risk and environmental factors associated with CPV-2 infection in diarrheic dogs. Fecal samples were collected from 116 dogs presenting diarrhea and were tested by polymerase chain reaction (PCR) for the presence of CPV-2 DNA. Supplementary data regarding clinical symptoms, individual features, management factors and medical history were also gathered for each animal during clinical evaluation. Sixty-eight diarrheic dogs were found to be positive for the virus DNA in their feces. Statistical analysis revealed that CPV-2 DNA was less likely to be detected in senior dogs, while working dogs, namely hounds and shepherds, had higher odds to be positive for the virus. Livestock density and land uses, specifically the categories of discontinuous urban fabric and of human population density, were identified as significant environmental parameters associated with CPV-2 infection by using Geographical Information System (GIS) together with the Ecological Niche Model (ENM). This is the first description of the environmental variables associated with the presence of CPV-2 DNA in dogs’ feces in Greece

Diagnostic performance of a rapid in-clinic test for the detection of Canine Parvovirus under different storage conditions and vaccination status

Canine parvovirus (CPV) is one of the most common causes of acute haemorrhagic enteritis in young dogs, while clinical diagnosis is often indecisive. The aim of our study was to evaluate the diagnostic accuracy of an in-clinic rapid test in the detection of CPV infection in dogs. To this end, we compared the Rapid Diagnostic Kit of Canine Parvovirus, Coronavirus and Rotavirus antigen (Quicking (R)) to PCR, which is considered as the most reliable diagnostic method. A total of 78 duplicated faecal samples were collected from diarrhoeic dogs. Vaccination history within a month prior to the onset of diarrhoea was reported for 12 of the sampled dogs. The rapid diagnostic test was performed in 23 of the faecal samples directly, while the rest were placed into a sterile cotton tipped swab suitable for collection and transportation of viruses (Sigma Sigma-VCM (R)) and stored at -20 degrees C The sensitivity of the Quicking rapid diagnostic test compared to PCR in the total number of samples, in samples from non-vaccinated dogs and in samples tested directly after collection were 22.22% (95% CI: 13.27-33.57%), 26.67% (95% CI: 16.08-39.66%) and 76.47% (95% CI: 50.10-93.04%) respectively, while the specificity of the test was 100% in any case. In conclusion, negative results do not exclude parvoenteritis from the differential diagnosis, especially in dogs with early vaccination history, but a positive result almost certainly indicates CPV infection. An improved sensitivity may be expected when the test is performed immediately. (C) 2015 Elsevier B.V. All rights reserved

Epidemiological analysis of African swine fever in the European Union (September 2019 to August 2020).

peer reviewedAn update on the African swine fever (ASF) situation in the 10 affected Member States (MS) in the EU and in two neighbouring countries from the 1 September 2019 until the 31 August 2020 is provided. The dynamics of the proportions of PCR- and ELISA-positive samples since the first ASF detection in the country were provided and seasonal patterns were investigated. The impact of the ASF epidemic on the annual numbers of hunted wild boar in each affected MS was investigated. To evaluate differences in the extent of spread of ASF in the wild boar populations, the number of notifications that could be classified as secondary cases to a single source was calculated for each affected MS and compared for the earliest and latest year of the epidemic in the country. To evaluate possible risk factors for the occurrence of ASFV in wild boar or domestic pigs, a literature review was performed. Risk factors for the occurrence of ASF in wild boar in Romanian hunting grounds in 2019 were identified with a generalised linear model. The probability to find at least one PCR-confirmed ASF case in wild boar in a hunting ground in Romania was driven by environmental factors, wild boar abundance and the density of backyard pigs in the hunting ground area, while hunting-related variables were not retained in the final model. Finally, measures implemented in white zones (ASF-free zones that are geographically adjacent to an area where ASF is present in wild boar) to prevent further spread of ASF were analysed with a spatially, explicit stochastic individual-based model. To be effective, the wild boar population in the white zone would need to be drastically reduced before ASF arrives at the zone and it must be wide enough. To achieve the necessary pre-emptive culling targets of wild boar in the white zone, at the start of the establishment, the white zone should be placed sufficiently far from the affected area, considering the speed of the natural spread of the disease. This spread is faster in denser wild boar populations. After a focal ASF introduction, the white zone is always close to the infection hence pre-emptive culling measures in the white zone must be completed in short term, i.e. in a few months

Epidemiological analyses of African swine fever in the European Union: (September 2020 to August 2021).

peer reviewedThis report provides a descriptive analysis of the African swine fever (ASF) Genotype II epidemic in the affected Member States in the EU and two neighbouring countries for the period from 1 September 2020 to 31 August 2021. ASF continued to spread in wild boar in the EU, it entered Germany in September 2020, while Belgium became free from ASF in October 2020. No ASF outbreaks in domestic pigs nor cases in wild boar have been reported in Greece since February 2020. In the Baltic States, overall, there has been a declining trend in proportions of polymerase chain reaction (PCR)-positive samples from wild boar carcasses in the last few years. In the other countries, the proportions of PCR-positive wild boar carcasses remained high, indicating continuing spread of the disease. A systematic literature review revealed that the risk factors most frequently significantly associated with ASF in domestic pigs were pig density, low levels of biosecurity and socio-economic factors. For wild boar, most significant risk factors were related to habitat, socio-economic factors and wild boar management. The effectiveness of different control options in the so-named white zones, areas where wild boar densities have been drastically reduced to avoid further spread of ASF after a new introduction, was assessed with a stochastic model. Important findings were that establishing a white zone is much more challenging when the area of ASF incursion is adjacent to an area where limited control measures are in place. Very stringent wild boar population reduction measures in the white zone are key to success. The white zone needs to be far enough away from the affected core area so that the population can be reduced in time before the disease arrives and the timing of this will depend on the wild boar density and the required population reduction target in the white zone. Finally, establishing a proactive white zone along the demarcation line of an affected area requires higher culling efforts, but has a higher chance of success to stop the spread of the disease than establishing reactive white zones after the disease has already entered in the area

UPGMA phylogenetic tree of the eight <i>Lepus europaeus</i> DQA exon 2 alleles.

<p>UPGMA phylogenetic tree resulting from the analysis of the eight <i>Lepus europaeus</i> DQA exon 2 alleles identified in this study, together with sequences assessed in the study of Koutsogiannouli et al. 2009 available from GenBank. Numbers indicate the percentage bootstrap support (10000 replicates). The evolutionary distances were computed using the maximum composite likelihood method and are in the units of the number of base substitutions per site. The populations in which each allele was present are given abbreviated in parentheses. (The neighbour-joining tree produced similar results with comparable bootstrap values).</p

Groups of Danish brown hares individuals according to the presence of EBHSV and the variation in exon 2 DQA gene.

<p>Numbers (N) and percentages of Danish brown hares assessed for the four groups defined in this study: EBHSV negative (EBHSV⁻), EBHSV positive (EBHSV⁺), susceptible (EBHSV⁺ Dead), resistant (EBHSV⁺ not Dead).</p