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, β-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.

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.

Background: Highly pathogenic avian influenza (HPAI) H5Nx and human H1N1pdm2009 influenza viruses can infect cats. Infections in cats may result in viral adaptations or recombinant viruses, which may facilitate zoonotic transfer. Aim: We aimed to investigate the presence of HPAI H5 clade 2.3.4.4 and H1 influenza viruses and antibodies to these viruses in domestic and rural stray cats in the Netherlands and factors associated with exposure. Methods: Sera from stray and domestic cats, sampled 2020–2023, were analysed by ELISA and confirmed by hemagglutination inhibition assay (HAI) and pharyngeal swabs and lung tissue for influenza A virus by RT-qPCR. Results: In 701 stray cats, 83 (11.8%; 95% confidence interval (CI): 9.5–14.5) sera were positive for HPAI H5 and 65 findings were confirmed. In HAI, two sera were positive for both HPAI H5 and H1. In 871 domestic cats, four (0.46%; 95% CI: 0.13–1.2) sera were HPAI H5 positive and none were confirmed but 40 (4.6%; 95% CI: 3.3–6.2) sera were seropositive for H1 and 26 were confirmed. Stray cats living in nature reserves (odds ratio (OR) = 5.4; 95% CI: 1.5–20.1) and older cats (OR = 3.8; 95% CI: 2.7–7.1) were more likely to be HPAI H5 seropositive. No influenza A virus was detected in 230 cats. Conclusion: The higher HPAI H5 seroprevalence in stray cats compared with domestic cats suggests more frequent viral exposure, most likely due to foraging on wild birds. In contrast, exposure to H1 was more common in domestic cats compared with stray cats.

Dogs are susceptible to the currently circulating highly pathogenic avian influenza (HPAI) H5 and human H1N1_pdm2009 (pandemic H1N1) viruses, yet little is known about the extent to which dogs are exposed to both these viruses. Here we investigated HPAI H5 and human H1N1_pdm2009 virus exposure in domestic dogs–including dogs that participated in hunting-training–and investigated lifestyle factors associated with HPAI H5 virus exposure. We screened sera from 538 dogs, sampled between 2021 and 2023, for influenza A virus antibodies, using ELISA and hemagglutination inhibition assays (HAIs). We analyzed lung tissue and (naso)pharyngeal swabs for influenza A viruses using RT-qPCR. Seropositivity to HPAI H5 virus was more frequent (13.3 %) in hunting-training dogs than in companion dogs with unknown bird contact (3.7 %). In contrast, seropositivity to H1N1_pdm2009 was more frequent in companion dogs (7.1 %) than in hunting-training dogs (0.7 %). Based on owner questionnaires, seropositivity to HPAI H5 by ELISA in hunting-training dogs was significantly associated with recent bird contact in/near water (odds ratio 6.9). No influenza A viruses were detected in 207 necropsy dogs and 180 (hunting) dogs. Our findings suggest that dogs are frequently exposed to zoonotic influenza A viruses, and we recommend dog owners to avoid dog contact with sick/dead birds.

Objectives: Cats are susceptible to highly pathogenic avian influenza H5 clade 2.3.4.4 (HPAI H5) and human new pandemic H1N1 (H1N1pdm09) influenza A viruses. A simultaneous infection with multiple influenza A virus subtypes could potentially result in the generation of reassortant viruses with enhanced zoonotic potential. Previously, high seropositivity (11.8%) to HPAI H5 virus has been detected in rural stray cats in the Netherlands, presumably through contact with or feeding on infected birds. Seropositivity was much lower (0.46%) in concurrently sampled domestic cats with unknown outdoor access, while these were frequently (4.6%) seropositive to H1N1pdm09virus. As outdoor access is expected to increase the risk of HPAI H5 exposure, in this study we determined seropositivity to HPAI H5 as well as H1N1pdm09 in domestic cats with known outdoor access. Methods: Sera from 254 outdoor cats were collected in 2024, and screened for antibodies to HPAI H5 and H1N1pdm09 virus using in-house developed ELISAs and hemagglutination inhibition assays (HAI). Results: Antibodies to HPAI H5 virus were detected in seven cat sera (2.8%; 95% CI:1.1-5.6) by ELISA, but not by HAI. Antibodies to H1N1pdm09 were detected in 14 sera (5.5%; 95% CI: 3.1-9.1), of which seven were positive by HAI (2.8%; 95% CI: 1.1-5.6). Two sera reacted in ELISA to both HPAI H5 and H1N1pdm09 (0.79%, 95% CI: 0.1-2.8). Conclusions and relevance: Antibodies to both HPAI H5 and H1N1pdm09 viruses were detected in outdoor domestic cats, with a higher seroprevalence for H1N1pdm09. Seropositivity for HPAI H5 was higher than was previously detected in domestic cats with unknown outdoor access, but lower than in stray cats. This warrants further investigation into the potential role of outdoor domestic cats as mixing vessel and as source of (novel) zoonotic viruses.