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Serosurveillance among animals, including pets, plays an important role in the current coronavirus disease 2019 (COVID‐19) pandemic, because severe acute respiratory coronavirus 2 (SARS‐CoV‐2) infections in animal populations could result in the establishment of new virus reservoirs. Serological assays that offer the required sensitivity and specificity are essential. In this study, we evaluated the diagnostic performance of three different commercially available immunoassays for the detection of SARS‐CoV‐2 antibodies in pets, namely two ELISA tests for the detection of antibodies against SARS‐CoV‐2 nucleocapsid [ID Screen SARS CoV‐2 double antigen multispecies (Double antigen) and ID Screen® SARS‐CoV‐2‐N IgG indirect ELISA (Indirect)] and one test for the detection of neutralizing antibodies against SARS‐CoV‐2 receptor‐binding‐domain [surrogate virus neutralization test (sVNT)]. The obtained results were compared with those of conventional virus neutralization test (VNT), which was regarded as reference method. A total of 191 serum samples were analysed. Thirteen (6.8%) samples showed VNT‐positive results. The overall sensitivity was higher for sVNT (100%) compared to nucleocapsid‐based ELISA assays (23% for Double antigen and 60% for Indirect). The specificity was 100% for Indirect ELISA and sVNT, when a higher cut‐off (>30%) was used compared to the one previously defined by the manufacturer (>20%), whereas the other test showed lower value (99%). The sVNT test showed the highest accuracy and agreement with VNT, with a perfect agreement when the higher cut‐off was applied. The agreement between each nucleocapsid‐based ELISA test and VNT was 96% for Indirect and 94% for Double antigen. Our findings showed that some commercially available serological tests may lead to a high rate of false‐negative results, highlighting the importance of assays validation for the detection of SARS‐CoV‐2 antibodies in domestic animals. [ABSTRACT FROM AUTHOR]

SARS‐CoV‐2 positive or seropositive owned cats have been reported worldwide. The detection of seropositive stray cats in the proximity of farms of infected minks, coupled with the demonstration of cat‐to‐cat transmission in experimental settings, raise the question whether stray cats may have an epidemiological role in the COVID‐19 pandemic and may act as sentinel for the circulation of SARS‐CoV‐2. The aim of this study was to evaluate the presence of SARS‐CoV‐2 RNA and anti‐SARS‐CoV‐2 antibodies in free roaming cats belonging to colonies located in an area highly affected by the COVID‐19 pandemic and to correlate the results with the positivity rate in people sharing the same area. Interdigital, cutaneous, oropharyngeal, nasal and rectal swabs, as well as blood samples, were collected from 99 cats living in colonies and admitted to our hospital for neutering. This caseload corresponds to the 24.2% of the feline population living in the 25 sampled colonies and to the 5.6% of all the free‐roaming registered cats. The presence of SARS‐CoV‐2 RNA in swabs was assessed using real time RT‐PCR. Anti‐SARS‐CoV‐2 serum antibodies were assessed using commercially available ELISA kits and confirmed by serum virus neutralization. In people, the SARS‐CoV‐2 positivity rate ranged from 3.0% to 5.1% (mean rate: 4.1%) and the seropositive rate from 12.1% to 16.3% (mean rate: 14.2%). Most of the colonies were in urban areas and resident cats had frequent contacts with external cats or people. A COVID‐19 positive caretaker was found, whereas all the cats were negative for SARS‐CoV‐2 RNA and seronegative. Although the negative results cannot exclude previous infections followed by decrease of antibodies, this study suggests that colony cats do not have an important epidemiological role in SARS‐CoV‐2 transmission dynamics. Further studies on larger caseloads are warranted, also in the light of the emerging new viral variants, on a One Health perspective. [ABSTRACT FROM AUTHOR]

Several emerging pestiviruses have been reported lately, some of which have proved to cause disease. Recently, a new ovine pestivirus (OVPV), isolated from aborted lambs, with high genetic identity to classical swine fever virus (CSFV), has proved to induce reproductive disorders in pregnant ewes. OVPV also generated strong serological and molecular cross-reaction with CSFV. To assess the capacity of OVPV to infect swine, twelve piglets were infected either by intranasal or intramuscular route. Daily clinical evaluation and weekly samplings were performed to determine pathogenicity, viral replication and excretion and induction of immune response. Five weeks later, two pigs from each group were euthanized and tissue samples were collected to study viral replication and distribution. OVPV generated only mild clinical signs in the piglets, including wasting and polyarthritis. The virus was able to replicate, as shown by the RNA levels found in sera and swabs and persisted in tonsil for at least 5 weeks. Viral replication activated the innate and adaptive immunity, evidenced by the induction of interferon-alpha levels early after infection and cross-neutralizing antibodies against CSFV, including humoural response against CSFV E2 and Erns glycoproteins. Close antigenic relation between OVPV and CSFV genotype 2.3 was detected. To determine the OVPV protection against CSFV, the OVPV-infected pigs were challenged with a highly virulent strain. Strong clinical, virological and immunological protection was generated in the OVPV-infected pigs, in direct contrast with the infection control group. Our findings show, for the first time, the OVPV capacity to infect swine, activate immunity, and the robust protection conferred against CSFV. In addition, their genetic and antigenic similarities, the close relationship between both viruses, suggest their possible coevolution as two branches stemming from a shared origin at the same time in two different hosts. [ABSTRACT FROM AUTHOR]

Epizootic haemorrhagic disease virus (EHDV) is a member of the Orbivirus genus in the Reoviridae family, and it is the etiological agent of an arthropod-transmitted disease that affects domestic and wild ruminants. Due to its significant economic impact, many attempts have been done in order to develop diagnostic immunoassays mainly based on the use of the viral protein 7 (VP7), that is, the immunodominant serogroup-specific antigen. In this work, a recombinant VP7 (recVP7) of EHDV serotype 2 was produced in a baculovirus system, and after purification using ion metal affinity chromatography, we obtained a high yield of recombinant protein characterized by a high degree of purity. We used the purified recVP7 as reagent to develop a competitive enzyme-linked immunoassay (c-ELISA), and we tested the presence of EHDV antibodies in 185 dromedary camel serum samples. The c-ELISA showed good performance parameters in recognising positive sera of naturally EHDV-infected dromedary camels; in particular, our developed test reached 85.7% of sensitivity, 98.1% of specificity, 93% of accuracy, and a high agreement value with results obtained by the commercial ELISA kit (Cohen's kappa value of 0.85) that we adopted as the reference method. This c-ELISA could be a useful screening test to monitor the virus spread in camels that are sentinel animals for endemic areas of disease. [ABSTRACT FROM AUTHOR]

Influenza D virus (IDV) was first reported in 2011 in swine in Oklahoma and consequently found in cattle, sheep, and goats across North America and Eurasia. Cattle have been proposed as the natural reservoir. In this study, we developed and validated a MAb‐based competitive ELISA for the detection of antibodies against IDV. Thirty‐one hybridomas specific to IDV were generated using Balb/C mice immunised with purified IDV/Swine/Italy/199724‐3/2015. The specificity of MAbs was determined by comparing their reactivity with the homologous and other influenza A viruses along with additional bovine and swine viruses. A solid‐phase competitive ELISA (IDV‐cELISA) was set up using the partially purified antigen coated to the plate and incubation of two serum dilutions (1/10 and 1/20) followed by addition of a peroxidase‐conjugated MAb as competitor, which had shown wide intratype cross‐reactivity and positivity in HI. To evaluate the diagnostic performances of IDV‐cELISA, we used 884 sera (414 negative and 470 positive) from different species. ROC analyses were performed to enable the selection of best cut‐off value and estimation of diagnostic specificity and sensitivity. The agreement between IDV‐cELISA and HI test was assessed by Cohen's kappa value (κ). The κ analysis showed an almost perfect agreement (κ = 0.93; 95%CI −0.899 to 0.961) between HI test and IDV‐cELISA. ROC analysis showed that IDV‐cELISA was accurate with an area under the curve (AUC) = 0.999, 95% CI 0.993–1.000). A cut‐off value of 65% was selected with Se and Sp values of 99.35 (95% CI 98.1–99.9) and 98.75 (95% CI 97.1–99.6). These results proved excellent diagnostic performances of IDV‐cELISA, which compared to HI presented major advantages, such as suitability for automation, low dependence on individual skills, spectrophotometric reading, and easy interpretation of the results. This assay can be exploited to detect anti‐IDV antibodies in different animal species. [ABSTRACT FROM AUTHOR]

Following the emergence of the A(H1N1)pdm09 in humans, this novel influenza virus was reverse transmitted from infected people to swine population worldwide. In this study we investigated the molecular evolution of A(H1N1)pdm09 virus identified in pigs reared in a single herd. Nasal swabs taken from pigs showing respiratory distress were tested for influenza type A and A(H1N1)pdm09 by real-time RT-PCR assays. Virus isolation from positive samples was attempted by inoculation of nasal swabs samples into specific pathogen free embryonated chicken eggs (ECE) and complete genome sequencing was performed on virus strains after replication on ECE or from original swab sample. The molecular analysis of hemagglutinin (HA) showed, in four of the swine influenza viruses under study, a unique significant amino acid change, represented by a two-amino acid insertion at the HA receptor binding site. Phylogenetic analysis of HA, neuraminidase, and concatenated internal genes revealed a very similar topology, with viruses under study forming a separate cluster, branching outside the A(H1N1)pdm09 isolates recognized until 2014. The emergence of this new cluster of A(H1N1)pdm09 in swine raises further concerns about whether A(H1N1)pdm09 with new molecular characteristics will become established in pigs and potentially transmitted to humans. [ABSTRACT FROM AUTHOR]