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.

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.

PCR-based next-generation West Nile virus sequencing protocols.

The epidemiology of West Nile virus (WNV) is unpredictable and changing. Availability of whole genome sequences enables the detailed molecular epidemiology studies and the evaluation and design of diagnostic tools. In the present study we provide two PCR-based protocols which can be applied directly on biological samples from hosts infected by WNV strains belonging to lineage 1 or lineage 2. It was shown that the protocols worked successfully even on samples with relatively low viral load.

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.

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 105.6 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.

A highly sensitive semi-nested real-time PCR utilizing oligospermine-conjugated degenerate primers for the detection of diverse strains of small ruminant lentiviruses.

Small ruminant lentiviruses (SRLVs) are highly diverse retroviruses infecting sheep and goats. Although PCR-based testing is being utilized for diagnostics, its application is hampered by various factors. These include, among others, the exceptionally high genetic variability of SRLVs, as well as the low number of infected blood monocytes. For this reason, a highly sensitive and specific semi-nested real-time PCR for proviral DNA detection and quantification was developed. The method is innovative in that a) its design is based on selecting the preferred codon usage in the targeted conserved genomic regions and b) oligospermine-conjugated degenerate primers with increased Tm were utilized. Modifications permitted primer/template duplex formation in the cases of mismatches due to sporadic nucleotide polymorphisms in a number of variant SRLV strains and consequently, the detection of highly diverse SRLV strains. The potential loss of analytical sensitivity and specificity was counterbalanced by including a semi-nested step in combination with LNA probes. An in silico procedure for the evaluation of hybridization efficiency of the designed oligonucleotides to all known targeted variants was also implemented. The method presents a linear range of quantification over a 3-log10 range and a limit of detection of 3.9 proviral dsDNA copies per reaction. Its diagnostic performance was evaluated by testing field samples from seropositive and seronegative animals, followed by phylogenetic analysis of the strains detected. To further increase the diagnostic sensitivity, a DNA extraction protocol for blood leukocytes was developed and evaluated. A minimum of 500 ng input DNA is recommended for PCR-based detection of SRLV proviral DNA, given the low numbers of infected blood monocytes. The developed methodology may serve as a useful tool, which can be adjusted for the quantitative detection of viruses exhibiting high genetic variability.

Viability Quantitative PCR Utilizing Propidium Monoazide, Spheroplast Formation, and Campylobacter coli as a Bacterial Model.

A viability quantitative PCR (qPCR) utilizing propidium monoazide (PMA) is presented for rapid quantification of viable cells using the foodborne pathogen Campylobacter coli as a bacterial model. It includes optimized spheroplast formation via lysozyme and EDTA, induction of a mild osmotic shock for enhancing the selective penetration of PMA into dead cells, and exploitation of an internal sample process control (ISPC) involving cell inactivation to assess residual false-positive signals within each sample. Spheroplasting of bacteria in exponential phase did not permit PMA entrance into viable cells since a strong linear relationship was detected between simple qPCR and PMA-qPCR quantification, and no differences were observed regardless of whether spheroplasting was utilized. The PMA-qPCR signal suppression of dead cells was elevated using spheroplast formation. With regard to the ISPC, cell inactivation by hydrogen peroxide resulted in higher signal suppression during qPCR than heat inactivation did. Viability quantification of C. coli cells by optimized spheroplasting-PMA-qPCR with ISPC was successfully applied in an aging pure culture under aerobic conditions and artificially inoculated meat. The same method exhibited a high linear range of quantification (1.5 to 8.5 log10 viable cells ml-1), and results were highly correlated with culture-based enumeration. PMA-qPCR quantification of viable cells can be affected by their rigidity, age, culture media, and niches, but spheroplast formation along with osmotic shock and the use of a proper ISPC can address such variations. The developed methodology could detect cells in a viable-but-nonculturable state and might be utilized for the quantification of other Gram-negative bacteria.IMPORTANCE There is need for rapid and accurate methods to detect viable bacterial cells of foodborne pathogens. Conventional culture-based methods are time-consuming and unable to detect bacteria in a viable-but-nonculturable state. The high sensitivity and specificity of the quantitative PCR (qPCR) are negated by its inability to differentiate the DNAs from viable and dead cells. The combination of propidium monoazide (PMA), a DNA-intercalating dye, with qPCR assays is promising for detection of viable cells. Despite encouraging results, these assays still encounter various challenges, such as false-positive signals by dead cells and the lack of an internal control identifying these signals per sample. The significance of our research lies in enhancing the selective entrance of PMA into dead Campylobacter coli cells via spheroplasting and in developing an internal sample process control, thus delivering reliable results in pure cultures and meat samples, approaches that can be applicable to other Gram-negative pathogens.

Evolutionary dynamics of lineage 2 West Nile virus in Europe, 2004-2018: Phylogeny, selection pressure and phylogeography.

West Nile virus (WNV) is an arbovirus causing neuroinvasive disease to humans and equines. Since 2004, lineage 2 WNV strains have been identified in Europe and have been implicated in severe outbreaks, with that of 2018 exceeding the total number from the previous seven years. The aim of this study was to explore the evolutionary process that shapes the genetic diversity of lineage 2 WNV strains (belonging to the Central European/Hungarian subclade) and reconstruct the origin and transmission routes in Europe, and especially in the Balkans. For this purpose, a high number of whole genome sequences (WGSs) were analyzed, along with newly characterized sequences, including strains from the 2018 WNV transmission season in Greece. Maximum likelihood and Bayesian inference methods were used to perform the phylogenetic and phylodynamic analyses and phylogeographic reconstruction. The majority of the Central European/Hungarian lineage 2 strains are grouped in 2 phylogenetic subgroups (Central/South-West European and Balkan) with bush-like topology. Purifying selection shapes their evolution, however, strong evidence of positive selection was revealed in 7 non-structural protein codons of NS1, NS4B and NS5. Thirty-two amino-acid substitutions were fixed in different phylogenetic subgroups, indicating that random genetic drift is responsible for the majority of evolutionary changes. Virus migration, followed by subsequent local evolution is responsible for continuously evolving strains throughout Europe. In total, 10 virus transitions between discrete geographical locations, involving virus spread from Central Europe to other regions, were highly supported. Three novel, independent introductions from Hungary and Bulgaria were responsible for the 2018 re-emergence of WNV in Northern Greece, indicating that Hungary remains an important ecological niche for the virus and has a central role for the dissemination of novel strains in the Balkans. In Northern Greece, tMRCA estimations indicated that a 1-to 2-year period of silent enzootic transmission precedes spread to dead-end hosts. Reconstruction of WNV population dynamics, from WGS data, revealed epidemic patterns characterized by 3- to 5-year oscillations in Europe. Future studies are necessary to determine the possible driving factors for these fluctuations i.e. avian herd immunity and climatic conditions affecting mosquito and bird populations. Maintaining adequate epidemiological surveillance with emphasis on obtaining WGS data, in areas at risk, is crucial for understanding the epidemiology and transmission patterns of WNV. It can further support integrated programs for risk assessment of virus circulation dynamics, aiming to targeted prevention and response measures for veterinary and public health in Europe.

A PCR-based NGS protocol for whole genome sequencing of West Nile virus lineage 2 directly from biological specimens.

Lineage 2 West Nile virus (WNV) strains have been implicated in severe encephalitis outbreaks in humans and equines residing in Europe. WNV molecular characterization is important for the development of diagnostic assays, as well as for obtaining molecular information, which is necessary for epidemiological investigations of virus transmission in areas at risk. For whole genome sequencing of lineage 2 WNV strains, directly from biological specimens, a PCR-based NGS protocol was developed. The method was applied in WNV-positive specimens obtained from animal, human and mosquito hosts in Greece. The results of its application indicate that, even in cases of low virus titers, the developed PCR-based NGS approach is able to provide whole genome sequences of lineage 2 WNV strains.

Development of real-time PCR-based methods for the detection of enzootic nasal tumor virus 2 in goats.

Enzootic nasal adenocarcinoma (ENA) is a contagious neoplasm of sheep and goats, associated with the oncogenic retroviruses enzootic nasal tumor virus (ENTV) 1 and 2, respectively. It appears to be common in countries with substantial small ruminant-production. ENA diagnosis in goats is based on autopsy and histopathology, and there is no real-time PCR method available for ENTV-2 detection. Here, a novel one-tube real-time RT-PCR (RT-qPCR) method for the detection and quantification of ENTV-2 in nasal swabs is presented. The method targets the env gene/U3 region. For the design of ENTV-2-specific oligonucleotides, molecular characterization of seven Greek ENTV-2 strains was performed. Phylogenetic analysis revealed three distinct phylogenetic clades of ENTV-2 that correlate with the country of sample collection. Evaluation of the analytical performance of the RT-qPCR revealed an amplification efficiency of 92.8% and a linear range of quantification between 2 × 108 and 2 × 102 RNA transcripts. Analysis of nasal swabs from 23 histopathologically confirmed, naturally occurring ENA cases via RT-qPCR yielded positive results. Moreover, modification of the method for use in a real-time PCR (qPCR) assay enables detection of proviral DNA in tumor specimens. Both methods are highly specific and can be used for the confirmation of ENA-suspected cases. Future applications could include ante-mortem diagnosis, verification of the ENTV-2-free status in animal trade, disease surveillance, and control programs.

Immunosuppression in sheep induced by cyclophosphamide, bluetongue virus and their combination: Effect on clinical reaction and viremia.

The main purpose of this work was to establish an experimental model for immunosuppression in sheep, and evaluate its possible effects on bluetongue viremia. Animals were allocated in 4 groups: Cy (cyclophosphamide), BT (bluetongue), CyBT (combined Cy and BT) and Co (control), and underwent clinical evaluations, virological testing, peripheral blood immunophenotyping and determination of antiviral humoral immune responses. Intravenous administration of cyclophosphamide (37.5 mg/kg body weight) resulted in immunosuppresion induction, as significant drops were observed in blood leukocytes and lymphocyte subset counts (CD2+, CD4+, CD8+, CD19+), lasting 3-10 days after its administration. Reduction in B-cell (CD19+) counts was more pronounced than in T-/NK-cell (CD2+) counts (92% and 59%, respectively). BTV-9 inoculation resulted in pronounced lymphocytopenia observed from day 1 post-inoculation. Their combined administration resulted in a more intense immunosuppressive effect, as indicated by the greater reduction in lymphocyte, granulocyte, CD4+ and CD8+ cell counts. In group CyBT, earlier initiation of fever by one day (day 6 p.i.) compared to group BT (day 7 p.i.), and delay in antibody responses by one day was observed, compared to group BT. Neutralizing antibodies in both groups (BT, CyBT) were detectable from day 10 p.i., but no significant titer differences were observed. Infectious virus titers were detected from day 4 p.i. in group BT and from day 3 in group CyBT. Statistical significances in virus titers were also observed (greatest mean titer difference: 1.4 log10 CEID50/ml RBCs at day 5 p.i., P < 0.001), indicating possible impact of immunosuppression on virus transmission and epidemiology of bluetongue.

Epidemiological characteristics and clinicopathological features of bluetongue in sheep and cattle, during the 2014 BTV serotype 4 incursion in Greece.

During 2014, an outbreak of Bluetongue virus (BTV) infections attributed to serotype 4 occurred in Greece and spread to south-eastern Europe. In the present article, the clinical and epidemiological data of 15 sheep flocks and 5 dairy cattle herds affected in Greece are described. In sheep, the most frequent clinical signs observed were fever, hyporexia, and edema of the face. A number of clinically affected sheep had chronic laminitis resulting in chronic lameness. Confirmation of suspect clinical cases was performed using BTV-specific real-time RT-PCR, and serotype 4-specific RT-PCR. The average morbidity of bluetongue in the sheep flocks was estimated to be 15.3 % (95 % C.I. 6.8-23.8 %) and the average mortality and case fatality were 4.5 % (95 % C.I. 1.5-7.6 %) and 32.0 % (95 % C.I. 18.1-42.9 %), respectively. The BTV seroprevalence and the ratio of clinical manifestations-to-infections determined in seven of these flocks, were on average 36.5 % (95 % C.I. 15.7-57.3 %) and 24.6 % (95 % C.I. 12.8-36.3 %). BTV ratio of clinical manifestations-to-infections was higher in the imported western European sheep breeds examined compared to the local ones. In dairy cattle, the average herd prevalence of viremia was 48.8 % (95 % C.I. 15.3-82.4 %) and none had signs associated with bluetongue. The results of this study indicate that the 2014 Greek BTV-4 has significant impact on the health status and the viability of sheep in affected flocks but does not cause clinical signs in cattle, despite the high prevalence of viremia.

Evidence of Schmallenberg virus circulation in ruminants in Greece.

During March 2013, we investigated the presence and the levels of Schmallenberg virus (SBV) circulation in three dairy cow herds and three sheep flocks in Central Macedonia, Greece. In two cow herds, a high number of abortions had been observed during the winter. Six bulk-tank milk samples and 147 individual sera were screened for SBV-specific antibodies by ELISA. Positive reactions were obtained from 5 out of 6 bulk-tank milk samples, 58 out of 90 sera from the 3 cow herds, and 2 sera from 2 of the 3 sheep flocks. Twenty-two ELISA-positive sera were tested by serum neutralization test (SNT). SNT confirmed the presence of neutralizing antibodies against SBV in all samples tested, with titers ranging between 1:32 and ≥1:256. No neutralizing antibodies against Akabane virus (AKAV) or Shamonda virus (SHAV) were detected, indicating that neutralizing antibodies against SBV do not cross react with AKAV or SHAV in SNT. ELISA testing of bulk-tank milk samples proved to be convenient and reliable. None of the tested sera was found positive for SBV by real-time RT-PCR, indicating that the sampling was conducted past the viremia stage. This is the first report of SBV circulation in Greece.

First Case of Human Anisakiosis in Greece: Acute Invasive Infection Mimicking Peritoneal Malignancy.

Consumption of raw and mildly processed seafood, in the context of modern Western world eating trends, is recognized as a major driver for human fish-borne infections. However, these zoonoses and their unfamiliar risks remain neglected and underappreciated among European diagnosticians. In contemporary Europe anisakidosis is one of the most important fish-borne zoonoses. It is caused by ingesting the third-stage infective larvae of the nematode parasites that belong to the family Anisakidae. The case described herein, is an intestinal and ectopic form of anisakiosis (Anisakis spp.), causing symptoms of subacute abdomen and masquerading as an intraperitoneal malignancy. It is the first anisakidosis case reported in Greece, affecting a young patient who had been repeatedly exposed to the parasite by consuming homemade raw fish. Right hemicolectomy, omentectomy and excision of a descending colon nodule were uneventfully performed. The pathology report confirmed granulomatous tissue with eosinophilic infiltration and parasites that were morphologically and molecularly identified as Anisakis spp. Although challenging, acquiring an accurate diagnosis of anisakidosis can prevent unnecessary surgery, as the infection typically is self-resolving, and if treatment is deemed necessary, it can be limited to antiparasitic medication. However, in rare cases, extra-gastrointestinal migration of larvae can cause severe damage with practically unknown risks, posing a diagnostic and therapeutic dilemma. In such a clinical case scenario, surgical exploration can decisively contribute to a definitive diagnosis and early identification of intraabdominal complications necessitating surgical intervention.

First case of macrocyclic lactone-resistant Dirofilaria immitis in Europe - Cause for concern.

Heartworm disease caused by the nematode Dirofilaria immitis is one of the most important parasitoses of dogs. The treatment of the infection is long, complicated, risky and expensive. Conversely, prevention is easy, safe, and effective and it is achieved by the administration of macrocyclic lactones (MLs). In recent years, D. immitis strains resistant to MLs have been described in Southern USA, raising concerns for possible emergence, or spreading in other areas of the world. The present study describes the first case of ML-resistant D. immitis in a dog in Europe. The dog arrived in Rome, Italy, from USA in 2023. Less than 6 months after its arrival in Italy, the dog tested positive for D. immitis circulating antigen and microfilariae, despite it having received monthly the ML milbemycin oxime (plus an isoxazoline) after arrival. The microfilariae suppression test suggested a resistant strain. Microfilariae DNA was examined by droplet digital PCR-based duplex assays targeting four marker positions at single nucleotide polymorphisms (SNP1, SNP2, SNP3, SNP7) which differentiate resistant from susceptible isolates. The genetic analysis showed that microfilariae had a ML-resistant genotype at SNP1 and SNP7 positions, compatible with a resistant strain. It is unlikely that the dog acquired the infection after its arrival in Europe, while it is biologically and epidemiologically plausible that the dog was already infected when imported from USA to Europe. The present report highlights the realistic risk of ML-resistant D. immitis strains being imported and possibly transmitted in Europe and other areas of the world. Monitoring dogs travelling from one area to another, especially if they originate from regions where ML-resistance is well-documented, is imperative. Scientists, practitioners, and pet owners should be aware of the risk and remain vigilant against ML-resistance, in order to monitor and reduce the spreading of resistant D. immitis.

Comparison of disk diffusion and broth microdilution methods for antimicrobial susceptibility testing of Campylobacter isolates of meat origin.

The aim of this study was to compare the disk diffusion (DD) and the broth microdilution (BMD) methods in determining the antimicrobial susceptibility of 36 Campylobacter isolates of meat-origin to six antibacterial drugs (erythromycin, ciprofloxacin, tetracycline, streptomycin, gentamicin and nalidixic acid). All the available zone diameter and minimum inhibitory concentration (MIC) breakpoints of C. jejuni and C. coli as recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) were utilized. In addition, the zone diameter breakpoints of Enterobacterales for nalidixic acid, gentamicin, and streptomycin, as recommended by the Clinical and Laboratory Standards Institute (CLSI), were applied. All Campylobacter isolates were categorised as susceptible to erythromycin and gentamicin by both methods indicating completely concordant classification results. The overall highest 'Very major error' (VME) and 'Major error' (ME) rates were detected for nalidixic acid (13.3%) and tetracycline (26.3%), respectively, whereas a 'Minor error' (mE) rate was detected only for ciprofloxacin (60.1%). However, the Cohen's kappa statistic indicated a substantial concordance between the DD and BMD classification results for tetracycline and streptomycin, and almost perfect agreement for nalidixic acid, with corresponding categorical agreement rates of over 86% and approximately up to 92%. The correlation between the complementary inhibition zones and MIC breakpoints was strong and statistically highly significant (p < 0.001) for ciprofloxacin, tetracycline, streptomycin, and nalidixic acid.

The COVID-19 Pandemic Enhanced Virology Research in Greece.

The emergence of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presented an unprecedented public health threat, being the cause of one of the most devastating pandemics in history [...].

Evaluation of Intradermal PRRSV MLV Vaccination of Suckling Piglets on Health and Performance Parameters under Field Conditions.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes respiratory disease in weaning and growing pigs. A vaccination against PRRSV is one of the most important control measures. This trial aimed to evaluate the effect of the intradermal (ID) administration of a PRRSV-1 modified live virus (MLV) vaccine in comparison to the intramuscular (IM) administration on the piglets' health and performance. A total of 187 suckling piglets of a PRRSV-positive commercial farrow-to-finish farm were assigned to four groups: group A­PRRSV ID, group B­PRRSV IM, group C­control ID, and group D­control IM. At 2 weeks of age, all the study piglets were either vaccinated with a PRRSV-1 MLV vaccine or injected with the vaccine adjuvant (controls). The collected blood serum samples were tested by ELISA and qRT-PCR. The side effects, body weight (BW), average daily gain (ADG), mortality rate, and lung and pleurisy lesions scores (LLS, PLS) were also recorded. The ELISA results indicated that the vaccination induced an important seroconversion at 4 and 7 weeks. Significant differences in the qRT-PCR results were noticed only at 10 weeks in group A vs. group C (p < 0.01) and group B vs. group C (p < 0.05). High viral loads, as evidenced by the qRT-PCR Ct values, were noticed in animals of both non-vaccinated groups at 7, 10, and 13 weeks. An ID vaccination has a positive impact on the BW at the piglets' slaughter, while both an ID and IM vaccination had a positive impact on the ADG. The mortality rate was lower in vaccinated groups at the finishing stage. The LLS and PLS were significantly lower in the vaccinated groups. In conclusion, our study demonstrated that the ID vaccination of suckling piglets with a PRRSV-1 MLV vaccine has a positive effect on the piglets' health and performance, including an improved BW and a lower LLS and PLS index at their slaughter, as well as a decreased mortality rate at the growing/finishing stage.

Limited cross-species transmission and absence of mutations associated with SARS-CoV-2 adaptation in cats: A case study of infection in a small household setting.

In the present study, the course of SARS-CoV-2 natural infection in two asymptomatic cats, which were negative for immunosuppressive retroviral infections, is investigated. The source of the virus for the cats was their COVID-19-affected owner, with whom they were in continuous proximity in a small household setting. The owner's signs included fatigue, sneezing, anosmia and loss of taste, and diagnosis was confirmed 4 days after symptom onset. Oropharyngeal and faecal swabs were collected from the cats, to investigate the course of SARS-CoV-2 RNA concentrations, as well as the directionality of the chain of virus transmission. Both infected cats were real-time RT-PCR-positive on various time-points. Pharyngeal shedding of at least 6 days was observed in them, with high SARS-CoV-2 titres (> 7 Log10 copies/swab) on the first sampling time-point, that is, 7 days after the onset of owner's clinical signs. In one cat, after the initial decline, slightly increasing virus titres were measured 3 to 6 days after the first real-time RT-PCR-positive swab. Serological testing of this cat revealed absence of seroconversion. The course of viral RNA concentrations in the faecal swabs of the other cat was similar to that in its pharynx. The detected SARS-CoV-2 strains, from both infected cats and their owner, underwent whole-genome sequencing, revealing the absence of emergence of cross-species adaptive mutations in cats. The results support the notion that human SARS-CoV-2 strains are relatively well-adapted to cats. It is still unclear whether asymptomatic animals could play a role in COVID-19 epidemiology, in case of interaction with naïve animals and/or people. Our findings highlight difficulties in SARS-CoV-2 transmission to cats, as neither the two infected cats nor their owner was able to transmit the virus to a third cat living in the same small flat, despite their very close contact during the days corresponding to high virus shedding.

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.

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.