Thesis defense Mirjam Duijvestijn

The Netherlands’ dense human and livestock populations, diverse wildlife, and water‑rich landscape creates favourable conditions for the emergence and spread of zoonotic pathogens, such as respiratory coronaviruses (CoVs) and influenza A viruses (IAVs). Dogs and cats, which live in close contact with humans and may interact with wildlife, could facilitate cross‑species transmission of these viruses, and may even serve as bridges for virus adaptation to humans. In this thesis the role of dogs and cats in the ecology of animal‑ and human‑origin CoVs and IAVs in the Netherlands was assessed, focusing on their potential capacity to act as virus hosts, sources, or reservoirs.

Virus exposure in cohorts of dogs and cats that differed in lifestyle and habitat was evaluated using both molecular (qPCR) and serological methods (ELISAs, pseudovirus neutralization and hemagglutination inhibition assays), complemented by risk factor analyses based on host, habitat, and lifestyle data. The inclusion of antibody detection was essential to avoid underestimation of exposure, as virus presence in active infections is expected to be short lived. In addition, to explore tissue-susceptibility to avian‑ and human IAVs, lectin histochemistry was used to explore the potential distribution of α2‑3‑ and α2‑6‑linked sialic acid receptors in canine and feline nasal, tracheal, and conjunctival epithelia.

Among rural stray cats, severe acute respiratory coronavirus-2 exposure was limited and provided no evidence for establishment of a feline reservoir. Across multiple dog and cat cohorts with differing habitats and lifestyles, approximately five percent had antibodies against human H1N1pdm2009 virus, consistent with frequent exposure through infected owners or caretakers. Seropositivity to highly pathogenic avian influenza H5 (HPAI H5) virus in dogs and cats varied markedly by habitat and activity: it was highest in hunting‑training dogs—especially those with recent bird contact in or near water—and in stray cats that lived in nature reserves, with lower but notable seropositivity in companion dogs and outdoor cats. Spatial and temporal clustering of HPAI H5‑seropositive stray cats suggested common‑source exposure and raised the possibility of cat‑to‑cat transmission. Despite frequent serological evidence of exposure, active IAV infection was not detected in diseased or deceased dogs and cats, underscoring challenges of short detection windows, mild or asymptomatic infections, and suboptimal sampling methods.

Co‑seropositivity to H1N1pdm2009 and HPAI H5 viruses in dogs and cats indicated that sequential or concurrent infections may occur, creating opportunities for reassortment. The possibility of overlapping presence of avian‑ and human‑type sialic acids in canine and feline respiratory tissues supports their potential role as mixing vessels for IAV evolution. Overall, the findings emphasize that habitat and lifestyle strongly shape pathogen exposure in dogs and cats and that structured, One Health‑oriented surveillance in dogs and cats—including systematic antibody testing, targeted sampling of risk groups, and biobanking of surplus veterinary samples—can improve early detection of zoonotic threats and strengthen pandemic preparedness.