Cordylobia anthropophaga Myiasis Mimicking Hyperproliferative Skin Disorder in Traveler Returning from Sub-Saharan Africa.

Myiasis is one of the most common skin diseases found in travelers returning from tropical and subtropical regions, where humans living in or visiting the African continent are most commonly infested by C. anthropophaga during the rainy season in regions with a warm climate. Here, we present a case of furuncular myiasis caused by C. anthropophaga in a Serbian patient returning from temporary work in Kenya, where the initial histology of skin lesion mimicked hyperproliferative skin disorder.

Is the Illegal Trade of Glass Eels (Anguilla anguilla) Increasing the Spread of Disease? A Case of EVEX.

The European eel (Anguilla anguilla) is a catadromous species that inhabits the rivers of the Adriatic watershed in Croatia. It is a critically endangered fish species, according to the IUCN, due to its declining abundance in European rivers caused by overfishing and trafficking and by diseases caused by nematodes, pathogenic bacteria and viruses. An illegal parcel of glass eels was confiscated at the Zagreb Airport and was intended to re-populate Croatian rivers. Barcoding was employed to determine species affiliation, and a thorough health check was carried out. This study reports the evaluation of gross lesions, histological findings, and EVEX virus isolation and identification. Since the confiscated glass eels were of unknown origin and given the serological and genetic similarities of EVA and EVEX, we designed primers and probes for almost whole genome sequencing to elucidate the origin of glass eels based on viral phylogeny. Bayesian phylogeny showed that the isolated strain had the most common ancestor with a Danish isolate and likely evolved from the French isolate of EVEX. These findings are discussed in light of the divergence of recently isolated strains and their possible contribution to the decrease of the abundance of the European eel in European waters.

The Consequences of Mitochondrial T10432C Mutation in Cika Cattle: A "Potential" Model for Leber's Hereditary Optic Neuropathy.

While mitogenome mutations leading to pathological manifestations are rare, more than 200 such mutations have been described in humans. In contrast, pathogenic mitogenome mutations are rare in domestic animals and have not been described at all in cattle. In the small local Slovenian cattle breed Cika, we identified (next-generation sequencing) two cows with the T10432C mitogenome mutation in the ND4L gene, which corresponds to the human T10663C mutation known to cause Leber's hereditary optic neuropathy (LHON). Pedigree analysis revealed that the cows in which the mutation was identified belong to two different maternal lineages with 217 individual cows born between 1997 and 2020. The identified mutation and its maternal inheritance were confirmed by Sanger sequencing across multiple generations, whereas no single analysis revealed evidence of heteroplasmy. A closer clinical examination of one cow with the T10432C mutation revealed exophthalmos, whereas histopathological examination revealed retinal ablations, subretinal oedema, and haemorrhage. The results of these analyses confirm the presence of mitochondrial mutation T10432C with homoplasmic maternal inheritance as well as clinical and histopathological signs similar to LHON in humans. Live animals with the mutation could be used as a suitable animal model that can improve our understanding of the pathogenesis of LHON and other mitochondriopathies.

Large-scale mitogenome sequencing reveals consecutive expansions of domestic taurine cattle and supports sporadic aurochs introgression.

The contribution of domestic cattle in human societies is enormous, making cattle, along with other essential benefits, the economically most important domestic animal in the world today. To expand existing knowledge on cattle domestication and mitogenome diversity, we performed a comprehensive complete mitogenome analysis of the species (802 sequences, 114 breeds). A large sample was collected in South-east Europe, an important agricultural gateway to Europe during Neolithization and a region rich in cattle biodiversity. We found 1725 polymorphic sites (810 singletons, 853 parsimony-informative sites and 57 indels), 701 unique haplotypes, a haplotype diversity of 0.9995 and a nucleotide diversity of 0.0015. In addition to the dominant T3 and several rare haplogroups (Q, T5, T4, T2 and T1), we have identified maternal line in Austrian Murbodner cattle that possess surviving aurochs' mitochondria haplotype P1 that diverged prior to the Neolithization process. This is convincing evidence for rare female-mediated adaptive introgression of wild aurochs into domesticated cattle in Europe. We revalidated the existing haplogroup classification and provided Bayesian phylogenetic inference with a more precise estimated divergence time than previously available. Occasionally, classification based on partial mitogenomes was not reliable; for example, some individuals with haplogroups P and T5 were not recognized based on D-loop information. Bayesian skyline plot estimates (median) show that the earliest population growth began before domestication in cattle with haplogroup T2, followed by Q (~10.0-9.5 kyBP), whereas cattle with T3 (~7.5 kyBP) and T1 (~3.0-2.5 kyBP) expanded later. Overall, our results support the existence of interactions between aurochs and cattle during domestication and dispersal of cattle in the past, contribute to the conservation of maternal cattle diversity and enable functional analyses of the surviving aurochs P1 mitogenome.

Improving Current Knowledge on Seroprevalence and Genetic Characterization of Swine Influenza Virus in Croatian Pig Farms: A Retrospective Study.

In a total of 1536 blood serum samples analysed by ELISA, antibodies for IAV nucleoprotein (NP) were detected in 30.3%. Results from HI show that the most common subtype of swIAV in the Croatian pig population was H1N1 (44.6%), followed by H3N2 (42.7%) and H1N2 (26.3%). Antibodies to at least one subtype were detected in 62.19% of blood serum samples. Detection of swIAV antigen was performed by IHC and detected in 8 of 28 lung samples collected post-mortem. The matrix (M) gene was detected in nine of one hundred and forty-two lung tissue samples and in seven of twenty-nine nasopharyngeal swabs. Phylogenetic analysis of amplified HA and NA gene fragments in Croatian isolates suggests the presence of swIAV H1avN1av.

Classification of porcine reproductive and respiratory syndrome virus in Ontario using Bayesian phylogenetics and assessment of temporal trends.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important swine viruses globally, including in Ontario, Canada. Understanding the evolution and relation of the various PRRSV genotypes in Ontario can provide insight into the epidemiology of the virus. The objectives of this study were to i) describe the variability of PRRSV genotypes in Ontario swine herds, and ii) evaluate possible groupings based on PRRSV genomic data. Virus open reading frame 5 (ORF-5) sequences collected from 2010 to 2018 were obtained from the Animal Health Laboratory, University of Guelph and Bayesian phylogenetic models were created from these. The PRRSV population of Ontario was then categorized into 10 distinct clades. Model comparisons indicated that the model with a constant population assumption fit the data best, which suggests that the net change in the PRRS virus variation of the entire population over the last decade was low. Nonetheless, viruses grouped into individual clades showed temporal clustering during distinct time intervals of the entire study period (P < 0.01).

Le virus du syndrome reproducteur et respiratoire porcin (VSRRP) est l'un des virus porcins les plus importants au monde, y compris en Ontario, au Canada. Comprendre l'évolution et la relation des divers génotypes du VSRRP en Ontario peut donner un aperçu de l'épidémiologie du virus. Les objectifs de cette étude étaient de i) décrire la variabilité des génotypes du VSRRP dans les troupeaux de porcs de l'Ontario et ii) évaluer les regroupements possibles en fonction des données génomiques du VSRRP. Les séquences du cadre de lecture ouvert 5 du virus (ORF-5) recueillis de 2010 à 2018 ont été obtenues auprès du Laboratoire de santé animale de l'Université de Guelph et des modèles phylogénétiques bayésiens ont été créés à partir de ceux-ci. La population de VSRRP de l'Ontario a ensuite été classée en 10 clades distincts. Les comparaisons de modèles ont indiqué que le modèle avec une hypothèse de population constante correspondait le mieux aux données, ce qui suggère que le changement net de la variation du virus SRRP de l'ensemble de la population au cours de la dernière décennie était faible. Néanmoins, les virus regroupés en clades individuels ont montré un regroupement temporel à des intervalles de temps distincts de toute la période d'étude (P < 0,01).(Traduit par Docteur Serge Messier).

The impact of porcine reproductive and respiratory syndrome virus (PRRSV) genotypes, established on the basis of ORF-5 nucleotide sequences, on three production parameters in Ontario sow farms.

The porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped RNA virus, with high mutation rates and genetic variability; which is evident by the large number of discrete strains that co-circulate in swine populations. Veterinary practitioners frequently identify certain discrete PRRSV strains as having a higher clinical impact on production. However, with exception of a few strains, production impact is not well characterized for the majority of PRRSV variants. Predictive analytics, coupled with routine diagnostic sequencing of PRRSV, provide opportunities to study the clinical impact of discrete PRRSV strains on production. Thus, the primary objective of this research was to evaluate clinical impact of discrete PRRSV clades observed in Ontario sow farms. PRRS viruses were classified into discrete clades using Bayesian analysis of the nucleotide sequences of the ORF-5 region of the genome. Production data were gathered through veterinary clinics from herds participating in the ongoing PRRSV surveillance system. Data about pre-weaning mortality, sow mortality, and abortion rates were measured up to 8 weeks post initial PRRSV outbreak. Through conventional regression analysis, results support that clinical impact of the viruses varied among clades over time for abortion rate (p = 0.05) and pre-weaning mortality (p < 0.01). Using predictive modelling approaches based on grouped K-fold cross-validation, it was identified that PRRSV clade designations and other measured factors showed low predictive performance for abortion (R2 = 0.07), pre-weaning mortality (R2 = 0.09), and sow mortality (R2 = 0.04). Clade designation consistently showed moderate importance for abortion and pre-weaning mortality, with clade 2 viruses being identified, on average, as having higher impact. These results demonstrate that the prediction of clinical impact, through production parameters, based on phylogenetic classification of PRRS viruses is possible. However, very high impact outbreaks were difficult to predict across production parameters. More surveillance-derived data are required to continue to improve predictive performance of the models.

Evidence of CPV2c introgression into Croatia and novel insights into phylogeny and cell tropism.

Canine parvovirus type 2 (CPV2) emerged for the first time in 1978 and evolved into two antigenic variants CPV2a and CPV2b and the third new antigenic variant CPV2c reported in 2000 in Italy. During 2014 unexplained outbreaks of gastroenteritis were observed in kennels where an extensive vaccination program was ongoing and where vaccinated animals showed pathologic lesions consistent with typical parvovirosis. The aim of this study was to investigate whether CPV2 could have played a role in the emergence of these cases and to evaluate genetic or pathological specificities of the virus and the disease. Using PCR and phylogenetic analysis we showed that the CPV2c variant is circulating in Croatia and is in close relationships with isolates from North and South America. Histopathological lesions and cell tropism that are known for CPV2 we are reporting the identification of the virus in glial cells and ovaries. It seems that evolution of CPV and CPV2a-c and adaptation to dogs are two independent events. Croatian isolates had specific and some unique amino acid mutations under positive selection. The effect of the alterations on the immunoglobulin binding cannot be excluded.

Genetic diversity of PRRSV 1 in Central Eastern Europe in 1994-2014: origin and evolution of the virus in the region.

More than 20 years after the first outbreaks, the phylogenetic picture of PRRSV is still incomplete and full of gaps, especially in regards of PRRSV 1. Due to the exceptional diversity observed at the eastern borders of Europe and the low number of available sequences from Central Eastern European countries, the authors collected and analyzed both recent as well as already submitted sequences comparing them to a large backbone set of available ORF5 sequences representing the full spectrum of PRRSV 1 Subtype 1 diversity to conduct a systematic phylogenetic analysis and reclassification elucidating the diversity of the virus in these countries. Moreover, further analyses of the EUROSTAT data regarding the live pig movement trends revealed their influence of virus diversity and evolution. The results indicate that besides the effect of local, isolated divergent evolution and the use of modified live vaccines, the most important factor influencing a given country's virus diversity is the transboundary movement of live, infected animals.

Investigating porcine parvoviruses genogroup 2 infection using in situ polymerase chain reaction.

BACKGROUND: Porcine parvovirus 2 (PPV2) was detected in swine serum without showing any relationship with disease. The emergence of the virus seemed to be a unique event until other genetically highly similar parvoviruses were identified in China and, later in 2012, the presence of the virus was also described in Europe. PPV2 is widely distributed in pig populations where it is suspected to be involved in respiratory conditions, based on its frequent detection in lung samples. In order to investigate the potential pathogenic involvement of PPV2, 60 dead pigs were examined from two farms. They were necropsied and tested for PPV2 and PCV2 (Porcine circovirus type 2) by PCR; by Brown and Brenn (B&B) staining for bacteria; by immunohistochemistry (IHC) to detect CD3, Swine leukocyte antigen class II DQ (SLAIIDQ), lysozyme, porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza (SIV), Mycoplasma hyopneumoniae (Mhyo); and by in situ hybridization (ISH) to detect ssDNA and dsDNA of PCV2. PPV2 positive samples were subjected to in situ polymerase chain reaction (IS-PCR) including double staining method to detect PPV2 and host cell markers. To calculate statistical difference we used GENMOD or LOGISTIC procedures in Statistical Analysis System (SAS®). RESULTS: We found that the PPV2 was localized mostly in lymphocytes in lungs, lymph nodes and liver. Neither CD3 antigen nor lysozyme was expressed by these infected cells. In contrast, low levels of SLAIIDQ were expressed by infected cells, suggesting that PPV2 may have a specific tropism for immature B lymphocytes and/or NK lymphocytes though possibly not T lymphocytes. CONCLUSION: The overall conclusion of this study indicates that PPV2 may contribute to the pathogenesis of pneumonia.

Revisiting the taxonomical classification of Porcine Circovirus type 2 (PCV2): still a real challenge.

BACKGROUND: PCV2 has emerged as one of the most devastating viral infections of swine farming, causing a relevant economic impact due to direct losses and control strategies expenses. Epidemiological and experimental studies have evidenced that genetic diversity is potentially affecting the virulence of PVC2. The growing number of PCV2 complete genomes and partial sequences available at GenBank questioned the accepted PCV2 classification. METHODS: Nine hundred seventy five PCV2 complete genomes and 1,270 ORF2 sequences available from GenBank were subjected to recombination, PASC and phylogenetic analyses and results were used for comparison with previous classification scheme. RESULTS: The outcome of these analyses favors the recognition of four genotypes on the basis of ORF2 sequences, namely PCV2a, PCV2b, PCV2c and PCV2d-mPCV2b. To deal with the difficulty of founding an unambiguous classification and accounting the impossibility to define a p-distance cut-off, a set of reference sequences that could be used in further phylogenetic studies for PCV2 genotyping was established. Being aware that extensive phylogenetic analyses are time-consuming and often impracticable during routine diagnostic activity, ORF2 nucleotide positions adequately conserved in the reference sequences were identified and reported to allow a quick genotype differentiation. CONCLUSIONS: Globally, the present work provides an updated scenario of PCV2 genotypes distribution and, based on the limits of the previous classification criteria, proposes new rapid and effective schemes for differentiating the four defined PCV2 genotypes.

Detection and characterisation of hepatitis E virus in naturally infected swine in Croatia.

Hepatitis E is a viral zoonotic disease infecting swine worldwide. Since pigs represent a likely animal reservoir for the hepatitis E virus, the epidemiology of naturally occurring hepatitis E was investigated in Croatian swine herds. Nearly all tested animals were seropositive for antibodies against the hepatitis E virus (55/60, 91.7%). Active infection was detected in all age groups by RT-PCR of viral RNA in serum (8/60, 13.3%) and bile samples (3/37, 8.1%), which was further confirmed by histopathological findings of characteristic lesions in the livers of the infected animals. Three new strains of hepatitis E virus were isolated from Croatian pig herds. Phylogenetic analysis using median-joining networks clustered those Croatian strains with isolates from various parts of the world, indicating their likely origin in international trade. Similarity to human isolates implies a zoonotic potential of Croatian strains, which raises a public health concern, especially in the light of the high prevalence of hepatitis E in the herds studied.

Emerging novel porcine parvoviruses in Europe: origin, evolution, phylodynamics and phylogeography.

To elucidate the spatiotemporal phylodynamics, dispersion and evolutionary processes underlying the emergence of novel porcine parvovirus 2 (PPV2), PPV3 and PPV4 species, we analysed all available complete capsid genes, together with ours, obtained in Europe. Bayesian phylogeography indicates that Romania (PPV2 and PPV4) and Croatia (PPV3) are the most likely ancestral areas from which PPVs have subsequently spread to other European countries and regions. The timescale of our reconstruction supported a relatively recent history of the currently circulating novel PPV species (1920s to 1980s) in the domestic or sylvatic host. While PPV2 strains exhibited a large genetic exchange characterized by significant recombination and gene flow between distinct regions and hosts, PPV3 and PPV4 showed a diversification reflected by the accumulation of geographically structured polymorphisms. The RNA-like evolutionary rates detected inter- and intrahost recombination and the positive selection sites provided evidence that the PPV2-4 capsid gene plays a prominent role in host adaptation.

Phylogeny, spatio-temporal phylodynamics and evolutionary scenario of Torque teno sus virus 1 (TTSuV1) and 2 (TTSuV2) in wild boars: fast dispersal and high genetic diversity.

Torque teno sus viruses (TTSuV1-2), members of the newly established family Anelloviridae are considered non-pathogenic emerging viral agents of Suidae species. However, the genetic diversity, phylogeny and evolutionary processes responsible for the emergence and spread of TTSuVs in wild boars remain poorly understood. Here we implemented phylogenetic and evolutionary analyses to address key questions on the genetic diversity, evolutionary scenario and spatio-temporal dynamics of emerging TTSuVs in wild boars of the Western region (Transylvania) of Romania. High levels of genetic diversity of wild boar origin TTSuV1-2 have been found as well as a new TTSuV1 genotype and several new subtypes. Phylogenies suggest that several wild boar viral strains in both TTSuV species are likely to have emerged from a well-defined ancestor approximately 40 (TTSuV1) and 18 (TTSuV2) years ago and showed independent evolutionary trajectories. Bayesian phylogeography showed an intense flow of viral strains throughout the Transylvanian counties possibly related to wild host migrations, facilitating the rapid spread of TTSuVs. The intra-genotype and inter- and intrahost level recombination, intense spatio-temporal viral flow and the positively selected sites found in the ORF2 genes should be considered important driving forces shaping TTSuVs evolution. The first reported rates of nucleotide substitution for porcine anelloviruses, estimated to be 5.29-5.51 × 10(-4)subs site(-1)year(-1), are in line with those measured previously for mammalian ssDNA viruses and RNA viruses. These high evolutionary rates of TTSuVs, independent of recombination, are reflections of adaptive evolution, an important factor in the emergence of novel viral variants which may explain their ability to emerge in Suidae hosts.

Accumulation of the beta(2)-adrenergic agonist clenbuterol in mouse dark hair.

The aim of the present study was to evaluate the suitability of dark hair as a matrix for determination of the beta(2)-adrenergic agonist clenbuterol residues using previously validated enzyme-linked immunosorbent assay (ELISA) as a screening method for its quantitative determination. The experimental group of mice (n = 60) were treated with two different anabolic dosages of clenbuterol for 15 days, whereas the control group of animals (n = 30) was left completely untreated. Hair samples were collected on days 0, 5, 10, and 15 of treatment. Validation of the ELISA analytical procedure showed good recovery (mean recovery 74%) with an acceptable intra-assay variation in individual measurements for all hair samples to which 5, 10, and 50 ng/g clenbuterol were added (CV < 10%). Low blank levels of clenbuterol (2.4 +/- 0.6 ng/g) were measured in hair of untreated mice, whereas significantly higher clenbuterol concentrations rising proportionally with the time of treatment were recorded in hair of mice treated with lower (6.5 mg/kg body mass) and higher (12.5 mg/kg body mass) dose of clenbuterol. The peak hair concentration of clenbuterol measured on the last day of treatment (day 15) was 1553.9 +/- 140.1 ng/g and 6248.3 +/- 589.4 ng/g in the lower and higher dose group, respectively. Study results clearly indicated dark hair as a pigmented tissue to have a high accumulation potential for clenbuterol residues, thus being the target matrix of choice for detection of clenbuterol abuse as an anabolic in meat production.