Serological Survey of Retrovirus and Coronavirus Infections, including SARS-CoV-2, in Rural Stray Cats in The Netherlands, 2020-2022.

Stray cats can host (zoonotic) viral pathogens and act as a source of infection for domestic cats or humans. In this cross-sectional (sero)prevalence study, sera from 580 stray cats living in 56 different cat groups in rural areas in The Netherlands were collected from October 2020 to July 2022. These were used to investigate the prevalence of the cat-specific feline leukemia virus (FeLV, n = 580), the seroprevalence of the cat-specific feline viruses feline immunodeficiency virus (FIV, n = 580) and feline coronavirus (FCoV, n = 407), and the zoonotic virus severe acute respiratory coronavirus-2 (SARS-CoV-2, n = 407) using enzyme-linked immunosorbent assays (ELISAs). ELISA-positive results were confirmed using Western blot (FIV) or pseudovirus neutralization test (SARS-CoV-2). The FIV seroprevalence was 5.0% (95% CI (Confidence Interval) 3.4-7.1) and ranged from 0-19.0% among groups. FIV-specific antibodies were more often detected in male cats, cats ≥ 3 years and cats with reported health problems. No FeLV-positive cats were found (95% CI 0.0-0.6). The FCoV seroprevalence was 33.7% (95% CI 29.1-38.5) and ranged from 4.7-85.7% among groups. FCoV-specific antibodies were more often detected in cats ≥ 3 years, cats with reported health problems and cats living in industrial areas or countryside residences compared to cats living at holiday parks or campsites. SARS-CoV-2 antibodies against the subunit 1 (S1) and receptor binding domain (RBD) protein were detected in 2.7% (95% CI 1.4-4.8) of stray cats, but sera were negative in the pseudovirus neutralization test and therefore were considered SARS-CoV-2 suspected. Our findings suggest that rural stray cats in The Netherlands can be a source of FIV and FCoV, indicating a potential risk for transmission to other cats, while the risk for FeLV is low. However, suspected SARS-CoV-2 infections in these cats were uncommon. We found no evidence of SARS-CoV-2 cat-to-cat spread in the studied stray cat groups and consider the likelihood of spillover to humans as low.

SARS-CoV-2 infection in dogs and cats is associated with contact to COVID-19-positive household members.

Several domestic and wild animal species are susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Reported (sero)prevalence in dogs and cats vary largely depending on the target population, test characteristics, geographical location and time period. This research assessed the prevalence of SARS-CoV-2-positive cats and dogs (PCR- and/or antibody positive) in two different populations. Dogs and cats living in a household with at least one confirmed COVID-19-positive person (household (HH) study; 156 dogs and 152 cats) and dogs and cats visiting a veterinary clinic (VC) (VC study; 183 dogs and 140 cats) were sampled and tested for presence of virus (PCR) and antibodies. Potential risk factors were evaluated and follow-up of PCR-positive animals was performed to determine the duration of virus shedding and to detect potential transmission between pets in the same HH. In the HH study, 18.8% (27 dogs, 31 cats) tested SARS-CoV-2 positive (PCR- and/or antibody positive), whereas in the VC study, SARS-CoV-2 prevalence was much lower (4.6%; six dogs, nine cats). SARS-CoV-2 prevalence amongst dogs and cats was significantly higher in the multi-person HHs with two or more COVID-19-positive persons compared with multi-person HHs with only one COVID-19-positive person. In both study populations, no associations could be identified between SARS-CoV-2 status of the animal and health status, age or sex. During follow-up of PCR-positive animals, no transmission to other pets in the HH was observed despite long-lasting virus shedding in cats (up to 35 days). SARS-CoV-2 infection in dogs and cats appeared to be clearly associated with reported COVID-19-positive status of the HH. Our study supports previous findings and suggests a very low risk of pet-to-human transmission within HHs, no severe clinical signs in pets and a negligible pet-to-pet transmission between HHs.

Serological Screening for Antibodies against SARS-CoV-2 in Dutch Shelter Cats.

The COVID-19 pandemic raised concerns that companion animals might be infected with, and could become a reservoir of, SARS-CoV-2. As cats are popular pets and susceptible to Coronavirus, we investigated the seroprevalence of SARS-CoV-2 antibodies in shelter cats housed in Dutch animal shelters during the COVID-19 pandemic. In this large-scale cross-sectional study, serum samples of shelter cats were collected during the second wave of human COVID-19 infections in The Netherlands. Seroprevalence was determined by using an indirect protein-based ELISA validated for cats, and a Virus Neutralization Test (VNT) as confirmation. To screen for feline SARS-CoV-2 shedding, oropharyngeal and rectal swabs of cats positive for ELISA and/or VNT were analyzed using PCR tests. In 28 Dutch animal shelters, 240 shelter cats were convenience sampled. Two of these cats (0.8%; CI 95%: 0.1-3.0%) were seropositive, as evidenced by the presence of SARS-CoV-2 neutralizing antibodies. The seropositive animals tested PCR negative for SARS-CoV-2. Based on the results of this study, it is unlikely that shelter cats could be a reservoir of SARS-CoV-2 or pose a (significant) risk to public health.

Serologic Screening of Severe Acute Respiratory Syndrome Coronavirus 2 Infection in Cats and Dogs during First Coronavirus Disease Wave, the Netherlands.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can infect many animal species, including minks, cats, and dogs. To gain insights into SARS-CoV-2 infections in cats and dogs, we developed and validated a set of serologic assays, including ELISA and virus neutralization. Evaluation of samples from animals before they acquired coronavirus disease and samples from cats roaming SARS-CoV-2-positive mink farms confirmed the suitability of these assays for specific antibody detection. Furthermore, our findings exclude SARS-CoV-2 nucleocapsid protein as an antigen for serologic screening of cat and dog samples. We analyzed 500 serum samples from domestic cats and dogs in the Netherlands during April-May 2020. We showed 0.4% of cats and 0.2% of dogs were seropositive. Although seroprevalence in cats and dogs that had unknown SARS-CoV-2 exposure was low during the first coronavirus disease wave, our data stress the need for development of continuous serosurveillance for SARS-CoV-2 in these 2 animal species.

Serological Screening of Influenza A Virus Antibodies in Cats and Dogs Indicates Frequent Infection with Different Subtypes.

Influenza A viruses (IAVs) infect humans and a variety of other animal species. Infections with some subtypes of IAV were also reported in domestic cats and dogs. In addition to animal health implications, close contact between companion animals and humans also poses a potential risk of zoonotic IAV infections. In this study, serum samples from different cat and dog cohorts were analyzed for IAV antibodies against seven IAV subtypes, using three distinctive IAV-specific assays differing in IAV subtype-specific discriminatory power and sensitivity. Enzyme-linked immunosorbent assays against the complete hemagglutinin (HA) ectodomain or the HA1 domain were used, as well as a novel nanoparticle-based, virus-free hemagglutination inhibition assay. Using these three assays, we found cat and dog sera from different cohorts to be positive for antibodies against one or more IAV subtypes and/or strains. Cat and dog serum samples collected after the 2009 pandemic H1N1 outbreak exhibit much higher seropositivity against H1 compared to samples from before 2009. Cat sera, furthermore, displayed higher reactivity for avian IAVs than dog sera. Our findings show the added value of using complementary serological assays, which are based on reactivity with different numbers of HA epitopes, to study IAV antibody responses and for improved serosurveillance of IAV infections. We conclude that infection of cats and dogs with both human and avian IAVs of different subtypes is prevalent. These observations highlight the role of cats and dogs in IAV ecology and indicate the potential of these companion animals to give rise to novel (reassorted) viruses with increased zoonotic potential.

Development and Validation of a S1 Protein-Based ELISA for the Specific Detection of Antibodies against Equine Coronavirus.

Equine coronavirus (ECoV) is considered to be involved in enteric diseases in foals. Recently, several outbreaks of ECoV infection have also been reported in adult horses from the USA, France and Japan. Epidemiological studies of ECoV infection are still limited, and the seroprevalence of ECoV infection in Europe is unknown. In this study, an indirect enzyme-linked immunosorbent assay (ELISA) method utilizing ECoV spike S1 protein was developed in two formats, and further validated by analyzing 27 paired serum samples (acute and convalescent sera) from horses involved in an ECoV outbreak and 1084 sera of horses with unknown ECoV exposure. Both formats showed high diagnostic accuracy compared to virus neutralization (VN) assay. Receiver-operating characteristic (ROC) analyses were performed to determine the best cut-off values for both ELISA formats, assuming a test specificity of 99%. Employing the developed ELISA method, we detected seroconversion in 70.4% of horses from an ECoV outbreak. Among the 1084 horse sera, seropositivity varied from 25.9% (young horses) to 82.8% (adult horses) in Dutch horse populations. Further, sera of Icelandic horses were included in this study and a significant number of sera (62%) were found to be positive. Overall, the results demonstrated that the ECoV S1-based ELISA has reliable diagnostic performance compared to the VN assay and is a useful assay to support seroconversion in horses involved with ECoV outbreaks and to estimate ECoV seroprevalence in populations of horses.

Serological Screening for Coronavirus Infections in Cats.

Coronaviruses (CoVs) are widespread among mammals and birds and known for their potential for cross-species transmission. In cats, infections with feline coronaviruses (FCoVs) are common. Several non-feline coronaviruses have been reported to infect feline cells as well as cats after experimental infection, supported by their ability to engage the feline receptor ortholog for cell entry. However, whether cats might become naturally infected with CoVs of other species is unknown. We analyzed coronavirus infections in cats by serological monitoring. In total 137 cat serum samples and 25 FCoV type 1 or type 2-specific antisera were screened for the presence of antibodies against the S1 receptor binding subunit of the CoV spike protein, which is immunogenic and possesses low amino acid sequence identity among coronavirus species. Seventy-eight sera were positive for antibodies that recognized one or more coronavirus S1s whereas 1 serum exclusively reacted with human coronavirus 229E (HCoV-229E) and two sera exclusively reacted with porcine delta coronavirus (PDCoV). We observed antigenic cross-reactivity between S1s of type 1 and type 2 FCoVs, and between FCoV type 1 and porcine epidemic diarrhea virus (PEDV). Domain mapping of antibody epitopes indicated the presence of conserved epitope(s) particularly in the CD domains of S1. The cross-reactivity of FCoV type 1 and PEDV was also observed at the level of virus neutralization. To conclude, we provide the first evidence of antigenic cross-reactivity among S1 proteins of coronaviruses, which should be considered in the development of serological diagnoses. In addition, the potential role of cats in cross-species transmission of coronaviruses cannot be excluded.

Enteropathogen infections in canine puppies: (Co-)occurrence, clinical relevance and risk factors.

Laboratory confirmation of the causative agent(s) of diarrhoea in puppies may allow for appropriate treatment. The presence of potential pathogens however, does not prove a causal relationship with diarrhoea. The aim of this study was to identify specific enteropathogens in ≤12 month old puppies with and without acute diarrhoea and to assess their associations with clinical signs, putative risk factors and pathogen co-occurrence. Faecal samples from puppies with (n=113) and without (n=56) acute diarrhoea were collected and screened for Canine Parvovirus (CPV), Canine Coronavirus (CCoV), Salmonella spp., Campylobacter spp., Clostridium perfringens, Clostridium difficile, ß-hemolytic Eschericha coli (hEC), Giardia spp., Toxocara spp., Cystoisospora spp., and Cyniclomyces guttulatus. One or more pathogens were detected in 86.5% of diarrhoeic puppies and in 77.8% of asymptomatic puppies. Significant positive associations were found between CPV and CCoV, CPV and Cystoisospora spp., Toxocara spp. and hEC, Giardia spp. and C. guttulatus. Only CPV and CCoV were significantly associated with diarrhoea, hEC with a subset of puppies that had diarrhoea and severe clinical signs. CPV was more prevalent in puppies under 3 months of age. Puppies from high-volume dog breeders were significantly at increased risk for CPV (OR 4.20), CCoV (OR 4.50) and Cystoisospora spp. (OR 3.60). CCoV occurred significantly more often in winter (OR 3.35), and CPV in winter (OR 3.78) and spring (OR 4.72) as compared to summer. We conclude that routine screening for CPV, CCoV and hEC is recommended in puppies with acute diarrhoea, especially if they are under 3 months of age and originate from high-volume dog breeders. Routine screening for other pathogens may lead to less conclusive results.

Integration of immunodeficiency virus in oocytes via intracytoplasmic injection: possible but extremely unlikely.

OBJECTIVE: To determine if human oocytes can be infected with HIV-1 via intracytoplasmic injection and to determine the infection threshold. DESIGN: Twenty-eight donated immature and unfertilized human oocytes from HIV-negative women were injected with 4 × 10(4) HIV-1 virions and 13 oocytes were used as uninjected controls. To determine the infection threshold, 543 cat oocytes were injected with 4 × 10(4), 4 × 10(2), or 40 copies of feline immunodeficiency virus (FIV) and 376 oocytes were used as controls. SETTING: Academic hospital. PATIENT(S)/ANIMAL(S): Donated immature human oocytes and mature cat oocytes. INTERVENTION(S): Injection with HIV-1 or FIV. MAIN OUTCOME MEASURE(S): Viral integration as measured by fluorescent in situ hybridization with HIV-1-specific probes or by nested FIV polymerase chain reaction. RESULT(S): We detected viral integration in three of 28 (11%) human oocytes injected with 4 × 10(4) copies of HIV-1. When injected with high dose FIV (4 × 10(4) copies) 16%-49% of cat oocytes showed viral integration. This decreased to 2%-7% and 0.6%-1.8% when an intermediate (4 × 10(2) copies) or low (40 copies) dose was injected, respectively. CONCLUSION(S): Human and cat oocytes can be infected with HIV-1 and FIV respectively, when injected with high amounts of virus. The probability of viral integration is extremely low when small amounts of virus particles are injected. Taking into account the small volume injected during intracytoplasmic injection, the chances of viral integration are 0.00002%.

Three-color flow cytometry detection of virus-specific CD4+ and CD8+ T cells in the cat.

We describe a three-color flow cytometry assay for the detection of virus-specific CD4+ and CD8+ T cells in the cat. The assay is based upon detection of intracellular TNFalpha using the cross-reactive mAb 6401.1111, raised against the human cytokine. Allophycocyanin-conjugated mAb 6401.1111 specifically stained feline TNFalpha-producing murine cells and also Staphylococcus aureus Enterotoxin B-stimulated feline T cells, thus providing formal evidence for cross-reactivity. By using the anti-TNFalpha mAb in combination with PE- and FITC-conjugated mAbs against feline CD4 and CD8, respectively, antiviral CD4+ and CD8+ T cells could be identified in the peripheral blood and in the spleens of feline infectious peritonitis virus-infected cats. Moreover, feline calicivirus (FCV)-specific CD4+ T cells were detected in the spleens of FCV-vaccinated cats. As antigen-presenting cells (APCs), we used immortalized autologous fibroblast cell lines, PBMC or splenocytes. A straightforward protocol, in which splenocyte preparations served both as APCs and effector cells, consistently yielded best results. The assay will permit further studies of the cellular immune responses in cats during natural and experimental viral infections. It will contribute to vaccine development against feline viruses by facilitating the identification of T cell antigens and epitopes, and by allowing the quantitative detection of virus-specific T cells after vaccination. Furthermore, the assay will add to the value of those systems in which viral infections of the cat serve as models for human disease.

Envelope gene sequences encoding variable regions 3 and 4 are involved in macrophage tropism of feline immunodeficiency virus.

The envelope is of cardinal importance for the entry of feline immunodeficiency virus (FIV) into its host cells, which consist of cells of the immune system including macrophages. To characterize the envelope glycoprotein determinants involved in macrophage tropism, chimeric infectious molecular clones were constructed containing envelope gene sequences from isolates that had been propagated in peripheral blood mononuclear cells (PBMC). The progeny virus was examined for growth in PBMC and bone marrow-derived macrophages and viruses with different replication kinetics in macrophages were selected. Envelope-chimeric viruses revealed that nucleotide sequences encoding variable regions 3 and 4 of the surface glycoprotein, SU, are involved in macrophage tropism of FIV. To assess the biological importance of this finding, the phenotypes of envelope proteins of viruses derived from bone marrow, brain, lymph node and PBMC of an experimentally FIV-infected, healthy cat were examined. Since selection during propagation had to be avoided, provirus envelope gene sequences were amplified directly and cloned into an infectious molecular clone of FIV strain Petaluma. The viruses obtained were examined for their replication properties. Of 15 clones tested, 13 clones replicated both in PBMC and macrophages, two (brain-derived clones) replicated in PBMC only and none replicated in Crandell feline kidney cells or astrocytes. These results indicate that dual tropism for PBMC and macrophages is a common feature of FIV variants present in vivo.