Detection of small ruminant Lentivirus proviral DNA in red deer from Poland.

Small ruminant lentiviruses (SRLVs) are widespread and infect goats and sheep. Several reports also suggest that SRLVs can infect wild ruminants. The presence of specific antibodies against SRLVs has been identified in wild ruminants from Poland, but no studies have been conducted to detect proviral DNA of SRLVs in these animals. Therefore, the purpose of this study was to examine samples from Polish wild ruminants to determine whether these animals can serve as reservoirs of SRLVs under natural conditions. A total of 314 samples were tested from red deer (n = 255), roe deer (n = 52) and fallow deer (n = 7) using nested real-time PCR. DNA from positive real-time PCR samples was subsequently used to amplify a CA fragment (625 bp) of the gag gene, a 1.2 kb fragment of the pol gene and an LTR-gag fragment. Three samples (0.95%) were positive according to nested real-time PCR using primers and probe specific for CAEV (SRLV group B). All the samples were negative for the primers and probe specific for MVV (SRLV A group). Only SRLV LTR-gag sequences were obtained from two red deer. Phylogenetic analysis revealed that these sequences were more closely related to CAEV than to MVV. Our results revealed that deer can carry SRLV proviral sequences and therefore may play a role in the epidemiology of SRLVs. To our knowledge, this is the first study describing SRLV sequences from red deer.

First Molecular Characterization of Small Ruminant Lentiviruses Detected in Romania.

Small ruminant lentiviruses (SRLVs) are a group of retroviruses that cause multisystem chronic diseases in goats and sheep and lead to production losses in these animals, negatively affecting animal health and welfare. Although molecular characterization of SRLV field isolates has been performed in many countries, there is currently no information on SRLV genotypes circulating in sheep and goats in Romania. Therefore, the main objective of this study was to conduct a molecular and phylogenetic analysis of SRLVs from Romania and determine the degree of genetic relatedness of the obtained sequences to other known SRLV reference strains. A total of 81 sheep lung tissue samples and 41 sheep lung lymph node samples were tested using nested real-time PCR, and samples positive for real-time PCR were used to amplify an 800 bp gag-pol fragment and an overlapping 625 bp fragment of the gag gene. Pairwise DNA distance and phylogenetic analysis showed that the Romanian SRLV strains were closely related to the A2 and A3 strains based on gag-pol sequences and to the A3 and A17 subtypes based on gag sequences. No recombination events were found. Our results revealed that the Romanian sequences have similar epitope patterns to other existing subtypes, although E/K and R/K mutations in epitope 3 were found only in the Romanian sequences, which may have potential value in serological diagnosis. This study is the first report on the genetic characterization of SRLV strains circulating in Romania and provides new information on SRLV heterogeneity. Further detailed studies should be conducted to better understand the divergence of SRLV Romanian strains.

The genetic variability of small-ruminant lentiviruses and its impact on tropism, the development of diagnostic tests and vaccines and the effectiveness of control programmes.

Introduction: Maedi-visna virus and caprine arthritis encephalitis virus are two closely related lentiviruses which cause multisystemic, progressive and persistent infection in goats and sheep. Because these viruses frequently cross the species barrier, they are considered to be one genetic group called small-ruminant lentiviruses (SRLV). They have in vivo tropism mainly for monocytes and macrophages and organ tropism with unknown mechanisms. Typical clinical signs are pneumonia in sheep, arthritis in goats, and mastitis in both species. Infection with SRLV cannot currently be treated or prevented, and control programmes are the only approaches to avoiding its spread. These programmes rely mainly on annual serological testing and elimination of positive animals. However, the high genetic and antigenic variability of SRLV complicate their early and definitive diagnosis. The objective of this review is to summarise the current knowledge of SRLV genetic variation and its implications for tropism, the development of diagnostic tests and vaccines and the effectiveness of control and eradication programmes. Material and Methods: Subject literature was selected from the PubMed and the Google Scholar databases. Results: The high genetic diversity of SRLV affects the performance of diagnostic tools and therefore control programmes. For the early and definitive diagnosis of SRLV infection, a combination of serological and molecular tests is suggested. Testing by PCR can also be considered for sub-yearling animals. There are still significant gaps in our knowledge of the epidemiology, immunology and biology of SRLV and their impact on animal production and welfare. Conclusion: This information may aid selection of the most effective SRLV spread reduction measures.

Conventional and State-of-the-Art Detection Methods of Bovine Spongiform Encephalopathy (BSE).

Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative disease that belongs to a group of diseases known as transmissible spongiform encephalopathies (TSEs). It is believed that the infectious agent responsible for prion diseases is abnormally folded prion protein (PrPSc), which derives from a normal cellular protein (PrPC), which is a cell surface glycoprotein predominantly expressed in neurons. There are three different types of BSE, the classical BSE (C-type) strain and two atypical strains (H-type and L-type). BSE is primarily a disease of cattle; however, sheep and goats also can be infected with BSE strains and develop a disease clinically and pathogenically indistinguishable from scrapie. Therefore, TSE cases in cattle and small ruminants require discriminatory testing to determine whether the TSE is BSE or scrapie and to discriminate classical BSE from the atypical H- or L-type strains. Many methods have been developed for the detection of BSE and have been reported in numerous studies. Detection of BSE is mainly based on the identification of characteristic lesions or detection of the PrPSc in the brain, often by use of their partial proteinase K resistance properties. The objective of this paper was to summarize the currently available methods, highlight their diagnostic performance, and emphasize the advantages and drawbacks of the application of individual tests.

A large-scale study on the seroprevalence of small ruminant lentiviral infection in the Polish goat population.

A large-scale study was carried out in a Polish goat population in 2014-2022 to determine the herd-level (between-herd) and within-herd seroprevalence of small ruminant lentivirus (SRLV) infection. A total of 8354 adult goats (aged >1 year) from 165 herds located in various regions of Poland were serologically tested using a commercial ELISA. One hundred twenty eight herds were randomly selected while 37 were enrolled based on convenience non-random sampling. At least 1 seropositive result was obtained in 103 / 165 herds. For all these herds the probability that they were truly positive (herd-level positive predictive value) was calculated. It was ≥ 90% in 91 seropositive herds and 73% to < 90% in 12 herds in which only 1-4 goats were seropositive (22 goats in total). The seropositive goats in the latter herds were retested using a different commercial ELISA and 14 goats (9 males and 5 females) from 9 herds were confirmed to be seropositive (serial testing). The true herd-level seroprevalence was estimated at 61% (95% confidence interval [CI 95%]: 53%-68%). It differed significantly between herd size classes (p = 0.003): the highest prevalences were found in the medium (51 - 100 adult goats) and large herds (>100 adult goats) - 72% (CI 95%: 56-84%) and 86% (CI 95%: 67%-95%), respectively, while prevalences in very small (≤ 20 adult goats) and small herds (21 - 50 adult goats) were 46% (CI 95%: 34%-59%) and 57% (CI 95%: 43%-70%), respectively. The true herd-level seroprevalence differed significantly also between geographical regions of Poland (p = 0.003), with the highest values in the north-western and the lowest in the southern region of the country. The true within-herd seroprevalence estimated using a Bayesian approach ranged from 0.7% to 100% with the median (IQR) of 42% (17%-84%), and did not vary significantly between herd size classes (p = 0.393) or geographical regions of Poland (p = 0.570). Concluding, SRLV infection is widespread in the Polish goat population, the north-western region of Poland is most extensively infected, and herds counting > 50 adult goats are more often infected.

Genetic Diversity of the LTR Region of Polish SRLVs and Its Impact on the Transcriptional Activity of Viral Promoters.

A long terminal repeat (LTR) plays an indispensable role in small ruminant lentivirus (SRLV) gene expression. In this study, we present the LTR sequence of Polish SRLVs representing different subtypes, and analyzed their impact on SRLV promoter activity, as measured in transient transfection assays. Although certain nucleotide motifs (AML(vis), TATA box and the polyadenylation site (AATAAA)) were conserved across sequences, numerous mutations within the LTR sequences have been identified. Single nucleotide polymorphisms (SNPs) were detected in both regulatory (AP-1, AP-4, Stat and Gas) and non-regulatory sequences, and subtype-specific genetic diversity in the LTR region of Polish SRLVs was observed. In vitro assays demonstrated subtype-specific functional differences between the LTR regions of distinct SRLV subtypes. Our results revealed that the promoter activity of Polish strains was lower (1.64-10.8-fold) than that noted for the K1514 reference strain; however, the differences in most cases were not statistically significant. The lowest promoter activity was observed for strains representing subtype A5 (mean 69.067) while the highest promoter activity was observed for strain K1514 representing subtype A1 (mean 373.48). The mean LTR activities of strains representing subtypes A12, A17, A23, A18 and A24 were 91.22, 137.21, 178.41, 187.05 and 236.836, respectively. The results of the inter-subtype difference analysis showed that the promoter activity of strains belonging to subtype A5 was significantly lower than that for subtype A12 strains (1.32-fold; p < 0.00). The promoter activities of the A5 strain were 1.98-fold and 2.58-fold less active than that of the A17 and A23 strains, and the promoter activities of A12 strains were 1.955 and 1.5 times lower than the promoter activity of A23 and A17 strains, respectively. Furthermore, the promoter activity of A17 strains was 1.3 lower than the promoter activity of A23 strains. Our findings suggest that subtype-specific genetic diversity, mainly in the transcription factor's binding sites, has an impact on their transcriptional activity, producing a distinct activity pattern for the subtypes. This study provides new information that is important for better understanding the function of the SRLV LTR. However, further research including more strains and subtypes as well as other cell lines is needed to confirm these findings.

Characterisation of ORF3, M, N and E Gene Sequences of Porcine Epidemic Diarrhoea Virus from Domestic Pigs in Poland.

Introduction: Porcine epidemic diarrhoea virus (PEDV) is an enteric pathogen causing porcine epidemic diarrhoea and acute gastroenteritis in pigs of all ages. Previous analysis of the viral genome of PEDV in Poland was only based on the spike protein (S) gene sequences and no analysis of other genes has been performed. The aim of this study was to analyse the envelope (E), membrane (M) and nucleocapsid (N) protein and open reading frame 3 (ORF3) gene sequences. Material and Methods: Viral RNA from 18 Polish pig faecal samples that were quantitative reverse transcription PCR-positive for PEDV was analysed in four genomic regions (E, M, N and ORF3). Results: Phylogenetic analysis based on these regions' sequences revealed that Polish PEDV isolates were highly related and were clustered into group G2a across the four genes compared. Moreover, the Polish strains were located in distinct subclusters on the phylogenetic trees, which suggests the presence of at least three independently evolving PEDV genetic lines circulating in Poland. The occurrence of unique mutations in the sequences of Polish PEDV strains suggests that PEDV continues to undergo evolutionary processes, accumulating the mutations necessary for viral fitness in its natural hosts. The Polish PEDV strains differed genetically from the CV777 vaccine strain, suggesting the risk of relatively low vaccine efficacy if this strain is used. Conclusion: Our results promote a better understanding of the genetic diversity of PEDV field isolates in Poland and highlight the importance of molecular characterisation of PEDV field strains for the development of an effective vaccine against PEDV.

Current State of Molecular and Serological Methods for Detection of Porcine Epidemic Diarrhea Virus.

Porcine epidemic diarrhea virus (PEDV), a member of the Coronaviridae family, is the etiological agent of an acute and devastating enteric disease that causes moderate-to-high mortality in suckling piglets. The accurate and early detection of PEDV infection is essential for the prevention and control of the spread of the disease. Many molecular assays have been developed for the detection of PEDV, including reverse-transcription polymerase chain reaction (RT-PCR), real-time RT-PCR (qRT-PCR) and loop-mediated isothermal amplification assays. Additionally, several serological methods have been developed and are widely used for the detection of antibodies against PEDV. Some of them, such as the immunochromatography assay, can generate results very quickly and in field conditions. Molecular assays detect viral RNA in clinical samples rapidly, and with high sensitivity and specificity. Serological assays can determine prior immune exposure to PEDV, can be used to monitor the efficacy of vaccination strategies and may help to predict the duration of immunity in piglets. However, they are less sensitive than nucleic acid-based detection methods. Sanger and next-generation sequencing (NGS) allow the analysis of PEDV cDNA or RNA sequences, and thus, provide highly specific results. Furthermore, NGS based on nonspecific DNA cleavage in clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems promise major advances in the diagnosis of PEDV infection. The objective of this paper was to summarize the current serological and molecular PEDV assays, highlight their diagnostic performance and emphasize the advantages and drawbacks of the application of individual tests.

Molecular Characterization of Small Ruminant Lentiviruses Isolated from Polish Goats with Arthritis.

Previous studies revealed that the small ruminant lentivirus (SRLV) population in Poland is highly heterogeneous. All SRLVs detected from Polish sheep and goats so far have belonged to subtypes B1, B2, A1, A5, A12, A13, A16, A17, A18, A23 and A24. However, all characterized strains originated from asymptomatic animals. This is the first study that characterizes the molecular properties of SRLVs isolated from different organs of six arthritic goats. Segments from three genomic regions (gag, LTR and env) were analyzed. In addition, we quantified the SRLV proviral load in the blood and different organs and examined its association with different degrees of histopathological lesions. All sequences obtained from the goats involved in this study were homogeneous, showing an average degree of variability of 4.8%, 3.7% and 8.8% for gag, LTR and env, respectively. Phylogenetic analysis revealed that the sequences from the analyzed goats were clustered within SRLVs group A and formed a new subtype within this group, tentatively named A27. The histopathological examination of the lung, mammary gland, synovial membranes of joints and brain of the analyzed goats revealed evidence of inflammatory processes associated with SRLV infection, which was confirmed by positive immunohistochemistry assays. No significant correlation was observed between histological features and alterations in the sequences from different tissues. No tissue-specific signature pattern was identified. It was shown that animals with a higher proviral load showed more lesion severity in various SRLV-affected tissues, indicating a positive association between these two parameters. Our results also revealed differences in the SRLV load between animals even though the sequences derived from all of the goats were closely related, suggesting that host factors may restrict and control viral replication. This study provides new information about SRLV variants isolated from arthritic goats; however, more studies, including the isolation and characterization of biological properties of these viruses, should be performed to evaluate their pathogenic potential.

Phylogenetic Analysis of Small Ruminant Lentiviruses Originating from Naturally Infected Sheep and Goats from Poland Based on the Long Terminal Repeat Sequences.

Introduction: Previous gag and env sequence studies placed Polish small ruminant lentiviruses (SRLVs) isolated from sheep and goats in subtypes B1, B2, A1, A5, A12, A13, A16-A18, A23, A24 and A27. This study extended the genetic/phylogenetic analysis of previously identified Polish SRLV strains by contributing long terminal repeat (LTR) sequences. Material and Methods: A total of 112 samples were analysed. Phylogenetic analyses were carried out on the LTR fragment using the neighbour-joining, maximum likelihood, and unweighted pair group method with arithmetic mean methods. Results: Polish caprine and ovine LTR sequences clustered within group A and grouped in at least 10 clusters (subtypes A1, A5, A12, A13, A16-A18, A23, A24 and A27). Most of the Polish strains (78%) belonged to the same subtype by the indication of the gag, env and LTR genomic regions. Discrepancies in affiliation depending on the particular sequence were observed in 24 (21%) strains, most of which came from mixed-species flocks where more than one SRLV genotype circulated. Sequences of the LTR reflected subtype-specific patterns. Several subtype-specific markers were identified, e.g. a unique substitution of T to A in the fifth position of the TATA box in A17, A27, A20 and B3. Conclusion: This study provides valuable insights into the genetic diversity of SRLV field strains in Poland, their phylogenetic relationships and their position in the recently established SRLV classification. Our results confirmed the existence of the ten subtypes listed and the readier emergence of new SRLV variants in mixed-species flocks.

Porcine Enteric Coronavirus Infections in Wild Boar in Poland - a Pilot Study.

INTRODUCTION: Porcine epidemic diarrhoea virus (PEDV) of the Coronaviridae family causes significant economic losses in the pig industry worldwide. Wild boars contribute to the transmission of different viral, bacterial and parasitic infections to livestock animals and humans. However, their role in the maintenance and transmission of PEDV has not been established. MATERIAL AND METHODS: In this study, blood and faecal samples from 157 wild boars were collected from 14 provinces of Poland during the 2017-2018 hunting season. RNA was extracted from the faecal homogenate supernatant and subjected to quantitative RT-PCR (RT-qPCR), while clotted blood samples were used for detection of antibodies against PEDV by ELISA. RESULTS: Five blood samples (3.2%) were seropositive in ELISA, while none of the faecal samples were found positive using RT-qPCR assays. CONCLUSION: The results of this analysis indicate the need for additional studies incorporating a larger number of samples and preferably comparing different serological methods, to confirm whether wild boars in Poland act as PEDV reservoirs.

Quasispecies Composition of Small Ruminant Lentiviruses Found in Blood Leukocytes and Milk Epithelial Cells.

Small ruminant lentiviruses (SRLVs) exist as populations of closely related genetic variants, known as quasispecies, within an individual host. The privileged way of SRLVs transmission in goats is through the ingestion of colostrum and milk of infected does. Thus, characterization of SRLV variants transmitted through the milk, including milk epithelial cells (MEC), may provide useful information about the transmission and evolution of SRLVs. Therefore, the aim of this study was to detect SRLVs in peripheral blood leukocytes (PBLs) and milk epithelial cells of goats naturally infected with SRLVs and perform single nucleotide variations analysis to characterize the extent of genetic heterogeneity of detected SRLVs through comparison of their gag gene sequences. Blood and milk samples from 24 seropositive goats were tested in this study. The double immunolabeling against p28 and cytokeratin demonstrated that milk epithelial cells originated from naturally infected goats were infected by SRLVs. Moreover, PCR confirmed the presence of the integrated SRLVs proviral genome indicating that MECs may have a role as a reservoir of SRLVs and can transmit the virus through milk. The blood and MEC derived sequences from 7 goats were successfully sequenced using NGS and revealed that these sequences were genetically similar. The MEC and blood-derived sequences contained from 3 to 30 (mean, 10.8) and from 1 to 10 (mean, 5.4) unique SNVs, respectively. In five out of seven goats, SNVs occurred more frequent in MEC derived sequences. Non-synonymous SNVs were found in both, PBLs and MEC-derived sequences of analyzed goats and their total number differed between animals. The results of this study add to our understanding of SRLVs genomic variability. Our data provides evidence for the existence of SRLVs quasispecies and to our knowledge, this is the first study that showed quasispecies composition and minority variants of SRLVs present milk epithelial cells.

Molecular Characterization of Small Ruminant Lentiviruses in Polish Mixed Flocks Supports Evidence of Cross Species Transmission, Dual Infection, a Recombination Event, and Reveals the Existence of New Subtypes within Group A.

Small ruminant lentiviruses (SRLVs) are a group of highly divergent viruses responsible for global infection in sheep and goats. In a previous study we showed that SRLV strains found in mixed flocks in Poland belonged to subtype A13 and A18, but this study was restricted only to the few flocks from Malopolska region. The present work aimed at extending earlier findings with the analysis of SRLVs in mixed flocks including larger numbers of animals and flocks from different part of Poland. On the basis of gag and env sequences, Polish SRLVs were assigned to the subtypes B2, A5, A12, and A17. Furthermore, the existence of a new subtypes, tentatively designed as A23 and A24, were described for the first time. Subtypes A5 and A17 were only found in goats, subtype A24 has been detected only in sheep while subtypes A12, A23, and B2 have been found in both sheep and goats. Co-infection with strains belonging to different subtypes was evidenced in three sheep and two goats originating from two flocks. Furthermore, three putative recombination events were identified within gag and env SRLVs sequences derived from three sheep. Amino acid (aa) sequences of immunodominant epitopes in CA protein were well conserved while Major Homology Region (MHR) had more alteration showing unique mutations in sequences of subtypes A5 and A17. In contrast, aa sequences of surface glycoprotein exhibited higher variability confirming type-specific variation in the SU5 epitope. The number of potential N-linked glycosylation sites (PNGS) ranged from 3 to 6 in respective sequences and were located in different positions. The analysis of LTR sequences revealed that sequences corresponding to the TATA box, AP-4, AML-vis, and polyadenylation signal (poly A) were quite conserved, while considerable alteration was observed in AP-1 sites. Interestingly, our results revealed that all sequences belonging to subtype A17 had unique substitution T to A in the fifth position of TATA box and did not have a 11 nt deletion in the R region which was noted in other sequences from Poland. These data revealed a complex picture of SRLVs population with ovine and caprine strains belonging to group A and B. We present strong and multiple evidence of dually infected sheep and goats in mixed flocks and present evidence that these viruses can recombine in vivo.

Molecular characterization of porcine epidemic diarrhoea virus (PEDV) in Poland reveals the presence of swine enteric coronavirus (SeCoV) sequence in S gene.

Porcine epidemic diarrhoea (PED) is a highly contagious enteric viral disease of pigs with a high morbidity and mortality rate, which ultimately results in huge economic losses in the pig production sector. The etiological agent of this disease is the porcine epidemic diarrhoea virus (PEDV) which is an enveloped, positive single-stranded RNA virus. The aim of this study was to perform molecular characterization of PEDV to identify the strains circulating in Poland. In this study, 662 faecal samples from 2015 to 2021 were tested with reverse transcription quantitative real-time PCR (RT-qPCR) and the results showed that 3.8% of the tested samples revealed a positive result for PEDV. A phylogenetic analysis of the complete genome and complete S gene sequences showed that Polish PEDV strains belonged to the G1b (S-INDEL) subgroup and were closely related to the European PEDV strains isolated from 2014 to 2019. Furthermore, RDP4 analysis revealed that the Polish PEDV strains harboured a recombinant fragment of ~400 nt in the 5' end of S gene with PEDV and swine enteric coronavirus (SeCoV) being the major and minor parents, respectively. Antigenic analysis showed that the aa sequences of neutralizing epitopes were conserved among the Polish PEDV strains. Only one strain, #0100/5P, had a unique substitution in the COE epitope. However, Polish PEDV strains showed several substitutions, especially in the COE antigen, as compared to the classical strain CV777. To the best of our knowledge, this is the first report concerning the molecular characterization of porcine epidemic diarrhoea virus strains, as well as the first phylogenetic analysis for PEDV in Poland.

Transcriptome Analysis for Genes Associated with Small Ruminant Lentiviruses Infection in Goats of Carpathian Breed.

Small ruminant lentiviruses (SRLV) are economically important viral pathogens of sheep and goats. SRLV infection may interfere in the innate and adaptive immunity of the host, and genes associated with resistance or susceptibility to infection with SRLV have not been fully recognized. The presence of animals with relatively high and low proviral load suggests that some host factors are involved in the control of virus replication. To better understand the role of the genes involved in the host response to SRLV infection, RNA sequencing (RNA-seq) method was used to compare whole gene expression profiles in goats carrying both a high (HPL) and low (LPL) proviral load of SRLV and uninfected animals. Data enabled the identification of 1130 significant differentially expressed genes (DEGs) between control and LPL groups: 411 between control and HPL groups and 1434 DEGs between HPL and LPL groups. DEGs detected between the control group and groups with a proviral load were found to be significantly enriched in several gene ontology (GO) terms, including an integral component of membrane, extracellular region, response to growth factor, inflammatory and innate immune response, transmembrane signaling receptor activity, myeloid differentiation primary response gene 88 (MyD88)-dependent toll-like receptor signaling pathway as well as regulation of cytokine secretion. Our results also demonstrated significant deregulation of selected pathways in response to viral infection. The presence of SRLV proviral load in blood resulted in the modification of gene expression belonging to the toll-like receptor signaling pathway, the tumor necrosis factor (TNF) signaling pathway, the cytokine-cytokine receptor interaction, the phagosome, the Ras signaling pathway, the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) (PI3K-Akt) signaling pathway and rheumatoid arthritis. It is worth mentioning that the most predominant in all pathways were genes represented by toll-like receptors, tubulins, growth factors as well as interferon gamma receptors. DEGs detected between LPL and HPL groups were found to have significantly enriched regulation of signaling receptor activity, the response to toxic substances, nicotinamide adenine dinucleotide (NADH) dehydrogenase complex assembly, cytokine production, vesicle, and vacuole organization. In turn, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway tool classified DEGs that enrich molecular processes such as B and T-cell receptor signaling pathways, natural killer cell-mediated cytotoxicity, Fc gamma R-mediated phagocytosis, toll-like receptor signaling pathways, TNF, mammalian target of rapamycin (mTOR) signaling and forkhead box O (Foxo) signaling pathways, etc. Our data indicate that changes in SRLV proviral load induced altered expression of genes related to different biological processes such as immune response, inflammation, cell locomotion, and cytokine production. These findings provide significant insights into defense mechanisms against SRLV infection. Furthermore, these data can be useful to develop strategies against SRLV infection by selection of animals with reduced SRLV proviral concentration that may lead to a reduction in the spread of the virus.

Single Nucleotide Polymorphisms in Genes Encoding Toll-Like Receptors 7 and 8 and Their Association with Proviral Load of SRLVs in Goats of Polish Carpathian Breed.

Toll-like receptors (TLRs) 7 and 8 are important in single-stranded viral RNA recognition, so genetic variation of these genes may play a role in SRLVs infection and disease progression. Present study aimed to identify SNPs in genes encoding TLR7 and TLR8 in goats of Carpathian breed and analyze their association with the SRLVs provirus concentration as index of disease progression. A total of 14 SNPs were detected, 6 SNPs in the TLR7 gene locus and 8 SNPs in the TLR8 gene. Nine of the 14 identified polymorphisms, 4 in the TLR7 gene and 5 in TLR8 gene, were significantly associated with the SRLVs proviral concentration. These SNPs were located in 3'UTR, 5'UTR and intron sequences as well as in the coding sequences, but they led to silent changes. Homozygous genotypes of three TLR7 SNPs (synonymous variant 1:50703293, 3'UTR variant 1:50701297 and 5'UTR variant 1:50718645) were observed in goats with lower provirus copy number as well as in seronegative animals. The results obtained in this study suggest that SNPs of TLR7/TLR8 genes may induce differential innate immune response towards SRLVs affecting proviral concentration and thereby disease pathogenesis and progression. These findings support a role for genetic variations of TLR7 and TLR8 in SRLVs infection and warrants further studies on the effect of TLR7/TLR8 polymorphisms on SRLVs infection in different populations.

Mutations of p53 Gene in Canine Sweat Gland Carcinomas Probably Associated with UV Radiation.

INTRODUCTION: Apocrine sweat gland carcinomas (ASGCs) are rare malignant skin tumours in dogs and humans. The literature published so far focuses mostly on the clinico-epidemiological aspect of these tumours, but little is known about their pathogenesis. In this study we aimed to determine whether the p53 gene is involved in the carcinogenesis of the apocrine sweat gland in dogs and whether ultraviolet radiation (UV) is related to it. MATERIAL AND METHODS: Forty canine ASGCs were submitted to laser capture microdissection to isolate neoplastic cells, from which DNA was subsequently extracted. PCR amplification and sequencing of p53 exons 2-8 was then performed, followed by computer analysis of the obtained sequences. RESULTS: Sixteen mutations within the p53 gene were found in 13 tumours. The mutations involved C → T, T → C, G → A, and CC → TT transitions, C → G transversion and adenine deletion, which are gene alteration types known to be related to UV radiation in the process of skin carcinogenesis in humans. Six of the thirteen tumour cases displayed the C → T transitions in the same location in exon 4 and three of the thirteen cases displayed T → C in the same location in exon 5. CONCLUSION: The results of the present study indicate both the participation of the p53 gene and the influence of UV radiation in the formation of ASGCs in dogs.

Molecular Characterization of Small Ruminant Lentiviruses of Subtype A5 Detected in Naturally Infected but Clinically Healthy Goats of Carpathian Breed.

Small ruminant lentiviruses (SRLVs) are widespread in sheep and goats in Poland, and several subtypes were identified and molecularly characterized up to date. This is the first study that characterizes the molecular properties of A5 strains of SRLV detected in naturally infected, but clinically healthy, Carpathian goats. Segments from three genomic regions (gag, env, and LTR) were analyzed. Genetic distance, pairwise comparison, and phylogenetic analysis revealed that Polish SRLV A5 sequences are closely related to the Swiss and German A5 sequences suggesting a common origin. The epidemiological linkage was identified particularly between the small ruminants of Germany and Poland. Amino acid sequences of immunodominant regions in CA protein were well-conserved within analyzed strains; however, they showed some remarkable changes like substitution (D) to (E), at position 90 in Major Homology Region (MHR) and (T) to (S), at position 141 in epitope 3. In contrast, aa sequences of surface glycoprotein exhibited the highest variability confirming type-specific variation in SU5 epitope. Two deletions in the U3 region of A5 strains were noted: One (8 nt) located near the 5' end of the U3 region and the other (29 nt) located in the central region of U3. Additionally, all A5 strains had specific deletion (10 nt) in the R region. Furthermore, we did not find a correlation between copies of the CAAAT motif and clinical manifestation in infected animals. These data showed some remarkable features in the viral genome of A5 strains, which may be related to the attenuated phenotype in vivo, characterized by the lack of any clinical signs in infected goats. Certainly, more studies are required to support the hypothesis that these A5 viruses are of low pathogenicity for goats. We want to focus our future studies on the analysis of the whole genomes of these isolates and their biological properties, as well as on clinicopathological studies of goats infected by A5 SRLV, aiming to clarify the pathogenic potential of these viruses.

Seroprevalence of small ruminant lentivirus (SRLV) infection in wild cervids in Poland.

Small ruminant lentiviruses (SRLV) are widespread amongst domesticated sheep and goats worldwide. Infection of wild ruminants in close contact with affected domesticated small ruminants has been proposed as an actor in SRLV epidemiology, but studies are limited. The aim of this study was to estimate the apparent (AP) and estimated prevalence (EP) of exposure to SRLV infection in wild ruminants from Poland. Samples originating from 198 free-living cervids comprising 142 European red deer and 56 roe deer were serologically tested using a multi-epitope recombinant antigen ELISA representing subtypes A1, A13, B1, and B2 of SRLV and a commercial ELISA test. The estimated prevalence of SRLV infection was estimated using the Bayesian approach with models that adjusted for the misclassification of animals because of a small population and lack of sampling method, the imperfect performance of the ELISAs and because sera of different species were tested. The calculated estimated prevalence ranged from 5.3 % (95 % CI 0.3, 12.5) to 24.6 % (95 % CI 3.3, 38.5) for the ELISA with multi-epitope antigens while estimated prevalence using the commercial ELISA was 2.5 % (95 % CI 0.2, 6.6). These results may suggest the existence of a new SRLV reservoir in Poland and highlight the importance of surveilling and controlling SRLV infection in domestic and wild ruminants sharing pasture areas.

Compartmentalization of Subtype A17 of Small Ruminant Lentiviruses between Blood and Colostrum in Infected Goats Is Not Exclusively Associated to the env Gene.

The compartmentalization of small ruminant lentiviruses (SRLVs) subtype A17 was analyzed in colostrum and peripheral blood leukocyte cells of three naturally infected goats. This study aimed to analyze heterogeneity of the SRLV env (V4V5) gene, which encodes neutralizing epitopes of SU glycoprotein, the gag gene encoding capsid protein (CA), and LTR, a noncoding region, responsible for determination of cell tropism. Compartmentalization was assessed using six established tree or distance-based methods, including permutation test to determine statistical significance. We found statistical evidence of compartmentalization between blood and colostrum in all infected goats although phylogenetic evidence of such compartmentalization was not obvious. Our study demonstrated that compartmentalization is not exclusively specific to the env gene, as we revealed that gag and LTR sequences are also compartmentalized between blood and colostrum. The work also confirms the combined use of different methods as essential for reliable determination of intrahost viral compartmentalization. Identifying and characterizing distinct viral subpopulations and the genetic evolution of SRLV in specific anatomical sites enhances our overall understanding of SRLV pathogenesis, immune control, and particularly virus transmission.