First-Time Detection of Porcine Reproductive and Respiratory Syndrome Virus and Porcine Circovirus 2 in an Albanian Farrow-to-Finish Herd

The purpose of this case report is to describe for the first time concurrent porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus 2 (PCV-2) infections in a commercial farrow-to-finish pig farm in Albania, as well as the phylogenetical analysis of isolated PRRSV strain. The present study reports on a farrow-to-finish commercial pig farm, located in South Albania. In a percentage of about 40% of weaners in each batch (60-70 piglets per batch), clinical signs, including fever, severe respiratory signs, wasting, jaundice, rough hairy coat, palpable inguinal lymphadenopathy, and high mortality rate, were performed. The clinical signs of sows included sporadic premature farrowings (22%), with increased number of stillbirth (3.3%) and weak piglets (4.1%) based on the record system of the farm. Blood samples were obtained from 8 sows (4 lactating and 4 dry-period sows), 25 piglets of 5 different batches (5 at 15-20 days, 5 at 40 days, 5 at 50 days, 5 of 60 days, and 5 of 70 days), and 5 finishers of 130-150 days of age. Moreover, tissue samples were collected from five weaners at 20-70 days of age. Histopathological examination of lung and lymph node sections revealed findings compatible with PRRSV and PCV-2 infection. Pigs between 15 and 130-140 days of age were positive for type 1 (European) PRRSV and pigs between 50 and 130-140 days of age were positive for PCV-2. Blood serum samples were tested by real-time polymerase chain reaction (PCR) for PCV-2 and one real-time reverse transcription-PCR-positive sample was selected for subsequent complete ORF5 (Gp5) gene sequencing. The results of this case report confirm the detection of PRRSV and PCV-2 concurrent infection in an Albanian farrow-to-finish pig farm. The full-length ORF5 sequence of the detected PRRSV strain (named "Mursi/AL/15") was successfully determined, revealing high nucleotide identity with other type 1 European isolates. © 2018, Mary Ann Liebert, Inc

Occurrence and molecular characterization of betanodaviruses in fish and invertebrates of the Greek territorial waters

Betanodaviruses are small ssRNA viruses that cause viral encephalopathy and retinopathy, a severe neuropathological infectious disease in marine fish species worldwide. In the present study, the occurrence of betanodaviruses was investigated in wild and cultured populations of fishes and invertebrates of the Greek territorial waters. Betanodaviruses were detected in 35 species belonging to 21 families and 12 orders. To our knowledge, 23 of those are reported for the first time in Greek waters, while 11 of them are reported for the first time globally. The positive samples were subjected to sequencing and phylogenetic analysis of partial segments of RNA1 and RNA2 genes. Almost all the viruses circulating in Greece fell within RGNNV genotype, while reassortant viruses were detected in three samples, namely two inter-RGNNV and one RGNNV/SJNNV. A novel unclassified Betanodavirus sequence was also identified. Most of the Greek sequence types have a restricted geographic distribution except for two RNA1 and one RNA2 sequence types that are widespread throughout the Mediterranean basin. The results of this study indicate the range of reservoirs/hosts of betanodaviruses and also their wide spread in the Greek territorial waters and reinforce the hypothesis that wild fish species transmit the virus to cultured ones and vice versa. © 2019 John Wiley & Sons Lt

A TaqMan probe-based multiplex real-time PCR method for the specific detection of wild type lumpy skin disease virus with beta-actin as internal amplification control.

&lt;p&gt;Lumpy skin disease (LSD) is a transboundary disease of economic importance affecting cattle and buffaloes. In South-Eastern Europe, immunization of cattle with homologous live attenuated vaccines for LSD control has prevented outbreaks since 2017, but has been associated with adverse reactions resembling disease symptoms. Thus, a diagnostic method suitable for disease surveillance in farms during vaccination campaigns with Neethling (Onderstepoort) and SIS type (Lumpyvax) live attenuated LSDV vaccines in Europe should be able to detect the wild type (WT) LSDV in animals with adverse reactions to the vaccines and samples with potentially high titers of the vaccine LSDV. To this end, a real-time PCR method targeting the EEV gene of LSDV was developed for the specific detection of WT strains, along with the use of beta-actin gene as an internal amplification control (IAC). Amplification efficiency of the WT virus target was 99.0% and 98.6%, in the presence and in the absence of high loads of vaccine LSDV, respectively. In the presence of 10 vaccine LSDV DNA copies, the limit of detection for WT LSDV was 12.6 DNA copies per reaction. The inter-assay CV was 0.04% for WT LSDV and 0.13% for beta-actin. The method can confirm diagnosis in suspect cases irrespective of the presence of the vaccine LSDV DNA by overcoming the masking effect of the WT LSDV. The simultaneous amplification of the beta-actin gene further assures the quality of diagnostic testing. The new method is a surveillance tool, complementing the DIVA real-time PCR during vaccination campaigns and can provide rapid insight on the targeted EEV gene in countries with novel and recombinant LSDV strains.&lt;/p&gt;</p

Susceptibility and Resistance to Canine Leishmaniose is Associated to Polymorphisms of the Canine TNF-α Gene

The prevalence of canine leishmaniosis (CanL) infection in an enzootic area is considerably higher than the overall prevalence of the disease, suggesting a role of host genetics related to the outcome of the disease. It is accepted that one determining factor for the outcome of CanL is the type of the triggered immune response, which seems to be genetically determined. TNF-α is a cytokine which plays a crucial role during the immune response against Leishmania parasites. In the present study a case-control study with 20 resistant and 20 susceptible dogs was performed. The distribution of breeds was equal in both groups. By Sanger method the nucleotide sequence upstream the Open Reading Frame of the canine TNF-α gene was determined and four polymorphisms were identified (−40 C/A, −1134 T/G, −1150 T/C κα −1243 C/G). Statistical analysis showed that the polymorphism TNF-α −40 C/A is correlated with susceptibility to CanL, while the polymorphism TNF-α −1243 C/G is correlated with resistance to CanL. Further statistical analysis, regarding the possible correlation of gender as well as clinical manifestations of the disease with the above-mentioned polymorphisms of the TNF-α gene, showed no significant findings. Further analysis of the above polymorphisms, as well as identification of more polymorphisms in candidate genes, is required to provide a better understanding of the complex underlying immune response in CanL

Investigation of Fas (APO-1)-Related Apoptosis in Piglets Intradermally or Intramuscularly Vaccinated with a Commercial PRRSV MLV

Porcine reproductive and respiratory syndrome virus (PRRSV) induces apoptosis through the activation of death receptors, including cell-surface Fas receptor. The aim of this study was to investigate the impact of intradermal (ID) and intramuscular (IM) vaccination with a commercial PRRSV-modified live vaccine in piglets on Fas-related apoptosis. The study included 104 suckling piglets from a commercial farrow-to-finish pig farm, suffering from positive unstable PRRSV status. Animals were assigned in four groups: group A - Porcilis PRRS ID-vaccinated pigs, group B - Porcilis PRRS IM-vaccinated pigs, group C - Diluvac ID adjuvant-administered pigs, and group D - Diluvac IM adjuvant-administered pigs. Vaccines were administered at 2 weeks of age. Blood samples were collected from the same pigs at 4, 7, and 10 weeks of age. Sera were examined by quantitative real-time reverse transcription-PCR (qRT-PCR) for PRRSV and by ELISA for soluble Fas (sFas). At 4 weeks of age, all groups were negative qRT-PCR for PRRSV; at 7 weeks only group A was negative; and at 10 weeks all groups were positive. sFas was significantly increased in groups C (4 vs. 7, 4 vs. 10, and 7 vs. 10 weeks) and D (7 vs. 10 weeks). Significant differences among groups were noticed only at 10 weeks (A vs. C, A vs. D, B vs. C, B vs. D). A significant positive and moderate correlation between PRRSV viral load and Fas level was observed. In unvaccinated piglets, increased serum sFas levels reveal apoptotic suppression compared with vaccinated piglets. In the latter, vaccine-derived antibodies limit the infection and may attribute to the reduced Fas expression, suggesting a weak induction of lymphocyte-mediated cytotoxicity. Copyright © 2022 Mary Ann Liebert, Inc

Colonic mucosal and serum expression of microRNAs in canine large intestinal inflammatory bowel disease

Background: Canine inflammatory bowel disease (IBD) is a group of chronic gastrointestinal (GI) disorders of still largely unknown etiology. Canine IBD diagnosis is time-consuming and costly as other diseases with similar signs should be initially excluded. In human IBD microRNA (miR) expression changes have been reported in GI mucosa and blood. Thus, there is a possibility that miRs may provide insight into disease pathogenesis, diagnosis and even treatment of canine IBD. The aim of this study was to determine the colonic mucosal and serum relative expression of a miRs panel in dogs with large intestinal IBD and healthy control dogs. Results: Compared to healthy control dogs, dogs with large intestinal IBD showed significantly increased relative expression of miR-16, miR-21, miR-122 and miR-147 in the colonic mucosa and serum, while the relative expression of miR-185, miR-192 and miR-223 was significantly decreased. Relative expression of miR-146a was significantly increased only in the serum of dogs with large intestinal IBD. Furthermore, serum miR-192 and miR-223 relative expression correlated to disease activity and endoscopic score, respectively. Conclusion: Our data suggest the existence of dysregulated miRs expression patterns in canine IBD and support the potential future use of serum miRs as useful noninvasive biomarkers. © 2020 The Author(s)

SARS-CoV-2 wastewater monitoring using a novel PCR-based method rapidly captured the Delta-to-Omicron ΒΑ.1 transition patterns in the absence of conventional surveillance evidence

Conventional SARS-CoV-2 surveillance based on genotyping of clinical samples is characterized by challenges related to the available sequencing capacity, population sampling methodologies, and is time, labor, and resource-demanding. Wastewater-based variant surveillance constitutes a valuable supplementary practice, since it does not require extensive sampling, and provides information on virus prevalence in a timely and cost-effective manner. Consequently, we developed a sensitive real-time RT-PCR-based approach that exclusively amplifies and quantifies SARS-CoV-2 genomic regions carrying the S:Δ69/70 deletion, indicative of the Omicron BA.1 variant, in wastewater. The method was incorporated in the analysis of composite daily samples taken from the main Wastewater Treatment Plant of Thessaloniki, Greece, from 1 December 2021. The applicability of the methodology is dependent on the epidemiological situation. During Omicron BA.1 global emergence, Thessaloniki was experiencing a massive epidemic wave attributed solely to the Delta variant, according to genomic surveillance data. Since Delta does not possess the S:Δ69/70, the emergence of Omicron BA.1 could be monitored via the described methodology. Omicron BA.1 was detected in sewage samples on 19 December 2021 and a rapid increase of its viral load was observed in the following 10-day period, with an estimated early doubling time of 1.86 days. The proportion of the total SARS-CoV-2 load attributed to BA.1 reached 91.09 % on 7 January, revealing a fast Delta-to-Omicron transition pattern. The detection of Omicron BA.1 subclade in wastewater preceded the outburst of reported (presumable) Omicron cases in the city by approximately 7 days. The proposed wastewater surveillance approach based on selective PCR amplification of a genomic region carrying a deletion signature enabled rapid, real-time data acquisition on Omicron BA.1 prevalence and dynamics during the slow remission of the Delta wave. Timely provision of these results to State authorities readily influences the decision-making process for targeted public health interventions, including control measures, awareness, and preparedness. © 2022 Elsevier B.V

Development and validation of a TaqMan probe-based real-time PCR method for the differentiation of wild type lumpy skin disease virus from vaccine virus strains.

&lt;p&gt;Lumpy skin disease (LSD) is a transboundary viral disease of cattle with severe economic impact. Immunization of cattle with homologous live attenuated vaccines poses a number of diagnostic problems, as it has been associated with adverse reactions resembling disease symptoms. The latter hampers clinical diagnosis and poses challenges in virus identification. To this end, a duplex quantitative real-time PCR method targeting the GPCR gene was developed and validated, for the concurrent detection and differentiation of wild type and vaccine Lumpy skin disease virus (LSDV) strains. The method was evaluated in three laboratories. The evaluation included a panel of 38 poxvirus isolates/strains and the analytical characteristics of the method were determined. Amplification efficiencies were 91.3% and 90.7%, for wild type and vaccine LSDV, respectively; the limit of detection was 8 DNA copies for both targets and the inter-assay CV was 0.30% for wild type and 0.73% for vaccine LSDV. The diagnostic performance was assessed using 163 LSDV-positive samples, including field specimens and samples from experimentally vaccinated/infected animals. The method is able to confirm diagnosis in suspect cases, it differentiates infected from vaccinated animals (DIVA) and can be regarded as an important tool for effective LSD surveillance and eradication during vaccination campaigns.&lt;/p&gt;</p