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

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

Angiotensin II Blood Serum Levels in Piglets, after Intra-Dermal or Intra-Muscular Vaccination against PRRSV

The Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) induces apoptosis in different organs. Angiotensin II (Ang II) is the main effector of the renin-angiotensin system and participates in apoptosis. Thus, this study aimed to investigate changes in piglet serum Ang II levels following intradermal (ID) and intramuscular (IM) vaccination with a commercial PRRS modified live virus (MLV) vaccine. The trial was conducted in a commercial pig farm, including 104 piglets which were randomly allocated to four groups: Group A—Porcilis PRRS ID, Group B—Porcilis PRRS IM, Group C—Diluvac ID and Group D—Diluvac IM. The study piglets were either vaccinated or injected at 2 weeks of age and they were tested by qRT-PCR for PRRSV and by ELISA for Ang II. The results indicated differences in viremia of tested piglets at 7 weeks of age, while piglets at 10 weeks of age were all found qRT-PCR positive for PRRSV. In addition, significant differences were noticed in Ang II in 7-week-old piglets. In conclusion, the present study provides evidence that ID vaccination induces less tissue damage, based on the lower measurements of Ang II in the serum of ID vaccinated piglets. © 2022 by the authors

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

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

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

Outbreaks of SARS-CoV-2 in naturally infected mink farms: Impact, transmission dynamics, genetic patterns, and environmental contamination

SARS-CoV-2 infection outbreaks in minks have serious implications associated with animal health and welfare, and public health. In two naturally infected mink farms (A and B) located in Greece, we investigated the outbreaks and assessed parameters associated with virus transmission, immunity, pathology, and environmental contamination. Symptoms ranged from anorexia and mild depression to respiratory signs of varying intensity. Although the farms were at different breeding stages, mortality was similarly high (8.4% and 10.0%). The viral strains belonged to lineages B.1.1.218 and B.1.1.305, possessing the mink-specific S-Y453F substitution. Lung histopathology identified necrosis of smooth muscle and connective tissue elements of vascular walls, and vasculitis as the main early key events of the acute SARS-CoV-2-induced broncho-interstitial pneumonia. Molecular investigation in two dead minks indicated a consistently higher (0.3–1.3 log10 RNA copies/g) viral load in organs of the male mink compared to the female. In farm A, the infected farmers were responsible for the significant initial infection of 229 out of 1,000 handled minks, suggesting a very efficient human-to-mink transmission. Subsequent infections across the sheds wherein animals were being housed occurred due to airborne transmission. Based on a R0 of 2.90 and a growth rate equal to 0.293, the generation time was estimated to be 3.6 days, indicative of the massive SARS-CoV-2 dispersal among minks. After the end of the outbreaks, a similar percentage of animals were immune in the two farms (93.0% and 93.3%), preventing further virus transmission whereas, viral RNA was detected in samples collected from shed surfaces and air. Consequently, strict biosecurity is imperative during the occurrence of clinical signs. Environmental viral load monitoring, in conjunction with NGS should be adopted in mink farm surveillance. The minimum proportion of minks that need to be immunized to avoid outbreaks in farms was calculated at 65.5%, which is important for future vaccination campaigns. Copyright: © 2021 Chaintoutis et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

West Nile virus state of the Art report of MALWEST project

During the last three years Greece is experiencing the emergence of West Nile virus (WNV) epidemics. Within this framework, an integrated surveillance and control programme (MALWEST project) with thirteen associate partners was launched aiming to investigate the disease and suggest appropriate interventions. One out of seven work packages of the project is dedicated to the State of the Art report for WNV. Three expert working groups on humans, animals and mosquitoes were established. Medical databases (PubMed, Scopus) were searched together with websites: e.g., WHO, CDC, ECDC. In total, 1,092 relevant articles were initially identified and 258 of them were finally included as references regarding the current knowledge about WNV, along with 36 additional sources (conference papers, reports, book chapters). The review is divided in three sections according to the fields of interest: (1) WNV in humans (epidemiology, molecular characteristics, transmission, diagnosis, treatment, prevention, surveillance); (2) WNV in animals (epidemiological and transmission characteristics concerning birds, horses, reptiles and other animal species) and (3) WNV in mosquitoes (control, surveillance). Finally, some examples of integrated surveillance programmes are presented. The introduction and establishment of the disease in Greece and other European countries further emphasizes the need for thorough research and broadening of our knowledge on this viral pathogen. © 2013 by the authors; licensee MDPI, Basel, Switzerland

Burden of infectious disease studies in Europe and the United Kingdom: a review of methodological design choices

This systematic literature review aimed to provide an overview of the characteristics and methods used in studies applying the disability-adjusted life years (DALY) concept for infectious diseases within European Union (EU)/European Economic Area (EEA)/European Free Trade Association (EFTA) countries and the United Kingdom. Electronic databases and grey literature were searched for articles reporting the assessment of DALY and its components. We considered studies in which researchers performed DALY calculations using primary epidemiological data input sources. We screened 3053 studies of which 2948 were excluded and 105 studies met our inclusion criteria. Of these studies, 22 were multi-country and 83 were single-country studies, of which 46 were from the Netherlands. Food- and water-borne diseases were the most frequently studied infectious diseases. Between 2015 and 2022, the number of burden of infectious disease studies was 1.6 times higher compared to that published between 2000 and 2014. Almost all studies (97%) estimated DALYs based on the incidence- and pathogen-based approach and without social weighting functions; however, there was less methodological consensus with regards to the disability weights and life tables that were applied. The number of burden of infectious disease studies undertaken across Europe has increased over time. Development and use of guidelines will promote performing burden of infectious disease studies and facilitate comparability of the results