Birds are hosts of many herpesviruses (HVs), in which the infection can go unnoticed or cause (lethal) disease. In contrast to HV in poultry, data on infection with and the presence and impact of HV in wild birds are limited. In birds, the full list of natural hosts of HV is unknown. Samples of organs and oropharyngeal and/or cloacal swabs of 1,212 live and dead free-living birds from 15 orders were examined for the presence of a fragment of the HV DNA polymerase gene. HV was detected in organ samples in eight out of 55 (14.5%) dead owls, in cloacal swabs in four out of 525 (0.7%) free-living songbirds caught during the autumn migration, in oropharyngeal and/or cloacal swabs in 34 out of 447 (7.5%) free-living birds admitted as wildlife casualties to the Clinic for Birds, Small Mammals, and Reptiles at the Faculty of Veterinary Medicine, and in oropharyngeal swabs in 16 out of 170 (9.4%) live free-living owls sampled on Mount Krim and the Jelovica Plateau. HVs were detected in individuals found dead as well as in live individuals with no clinical signs of illness. Furthermore, no productivity deviances (i.e., in clutch and brood size) were recorded in a breeding population of clinical healthy owls. Phylogenetic analysis of nucleotide sequences of the partial DNA polymerase gene of HVs showed that HVs detected in free-living birds belong to the subfamily Alphaherpesvirinae and that different HVs have been circulating in the population of free-living birds in Slovenia, which in most cases are different from known HV nucleotide sequences published in GenBank. Some HVs are specific to the host bird species, whereas in some cases HVs very similar to identical nucleotide sequences were found in different bird orders, meaning that HVs are not always restricted to bird host species. HV transmission could occur through bird predation or even superpredation. It was also found that small rodents are not a source of HV infections in owls, falcons, and birds of prey. The results of the research greatly expand the list of HV-susceptible host species of free-living birds and show that free-living birds with unknown infection status should be tested with a combination of oropharyngeal and cloacal swabs, which would maximize the probability of HV detection.
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