Equine piroplasmosis is a tick-borne disease caused by Babesia and Theileria species. Despite its presence in Europe, no laboratory testing is required for animal movement, even though some countries remain free of this disease. Differentiating between species and genotypes is crucial to determine the most effective treatment, as dosage, active compounds, and duration vary. However, diagnosis is often challenging due to genetic variability and the limited sensitivity of molecular methods. The aims of this study were to compare the performances of different molecular diagnostic tests to identify the most effective assay for piroplasm DNA detection and to genetically characterize the piroplasms identified in horses in Northern Italy from 2016 to 2022. Among 63 horses tested, 24 (38.1%) were positive in at least one of the tests used. Four horses tested positive for Babesia caballi with identical nucleotide sequences, while 22 horses tested positive for genetically different Theileria species, including Theileria equi, Theileria haneyi-like species, and Theileria sp. Africa. Two horses were coinfected by Babesia caballi and Theileria haneyi-like species. The best diagnostic approach to avoid false negative results was a combination of different assays. Further studies will be necessary to better assess the prevalence and genetic diversity of piroplasms involved in equine piroplasmosis.

Feline leukemia virus (FeLV) is responsible for feline leukemia syndrome in domestic cats. The prevention and control of disease caused by FeLV are primarily based on vaccination and identification and isolation of infected subjects. Antigen diagnostic methods, which are the most widely used in clinical practices, can be associated to molecular tests to characterize the FeLV detected. In this study, a quantitative SYBR Green Real-Time PCR (qPCR) assay was used to detect FeLV proviral DNA in blood samples from antigen positive cats referred to a veterinary teaching hospital in Northern Italy in 2018–2021. To genetically characterize the identified viruses, a portion of the viral envelope (env) gene was amplified using six different end-point PCRs and sequenced. Twenty-two of 26 (84.6%) cats included in the study tested positive by qPCR assay. This suggests a high performance of the qPCR adopted but further studies are required to investigate the cause of discordant results between the antigen test and qPCR in four cats. From env gene analysis, 15/22 qPCR-positive cats were infected by FeLV subtype A and 5/15 shown coinfection with subtype B.

Animals, including wildlife, are part of One-Health concept since many infectious diseases can affect both humans and animals. In this study, 126 red foxes (Vulpes vulpes) from Northern Italy in 2022–2023 were tested by molecular assays for Protoparvovirus carnivoran 1 (PPVC-1), Canine adenovirus type 1 and 2 (CAdV-1 and CAdV-2), Circovirus canine (CanineCV), Canine distemper virus (CDV), and Leptospira spp. A total of 39 of 126 (30.9%) red foxes were infected with at least one pathogen and five of these were coinfected: 20/126 (15.9%) red foxes tested positive for PPVC-1, 3/126 (2.4%) for CAdV, 20/126 (15.9%) for CanineCV, and 2/126 (1.6%) for Leptospira spp. DNA. No foxes tested positive for CDV RNA. The pathogens identified were genetically analysed. New findings were reported such as a fox with multiple feline panleukopenia virus (FPV) and canine parvovirus type 2b (CPV-2b) infection associated with quasispecies dynamics, typical genetic characteristics of the identified CanineCV, and the first detection in red foxes of Leptospira ST198 related to L. interrogans serogroup Australis. Further studies are necessary to investigate the transmission between domestic animals and wildlife and to understand the role of red foxes in the maintenance of these pathogens not only in the wild but also in urban and peri-urban environments.

Feline calicivirus (FCV) is widespread in multi-cat environments and typically causes acute upper respiratory tract disease (URTD). FCV also causes outbreaks of virulent systemic disease (VSD), mainly in adults, with multiple organ involvement. In this study, an FCV-VSD infection was described in a less-one-month-old Maine Coon kitten originating from a cattery where an outbreak of FCV-URTD had previously been reported. After spontaneous death, post-mortem examination as well as histopathological, immunohistochemical, bacteriological and virological investigations were carried out. Pathological findings were consistent with severe pneumonia and cutaneous oedema of the footpads. No concomitant bacterial infection was detected. FCV RNA was detected in several organs and the highest amount of viral RNA was observed in the lung sample, in which the presence of the FCV antigen was confirmed by immunohistochemistry. With the same immunohistochemical technique, the IBA-1 antibody detected sparse alveolar macrophages, the main viral target cell and pulmonary replication site. The nucleotide sequences of the viral ORF2 gene amplified from all positive tissues were identical with each other and phylogeny confirms that highly virulent FCV strains are not distinguishable from FCV-URTD phenotypes. Our findings reinforce the hypothesis that VSD outbreaks can occur even in small populations, due to the high genetic variability of FCV.

Feline immunodeficiency virus (FIV) is responsible for immunodeficiency syndrome in cats. Several viral subtypes have been identified, each with a variable geographical distribution. To date, the subtype B is known to be the genotype spread in Italy. In this study, the genetic diversity of FIV in northern Italy was assessed by detecting proviral DNA in the blood samples of 50 cats determined to be positive through an anti-FIV antibodies test. These cats were tested using six different PCR assays, and the identified viruses were sequenced and analyzed. Forty-eight cats were confirmed positive, and several FIV subtypes were characterized. As expected, the subtype B was the most commonly observed, and the subtype A was reported for the first time in Italy. Moreover, a new taxon possibly representing an additional FIV subtype was detected, and one virus belonging to subtype B potentially had a recombinant origin. The genetic variability between the FIV viruses that emerged in this study may lead to the potential diagnostic failure of single molecular tests. Therefore, a new diagnostic strategy, which adopts different molecular tests and sequencing, is recommended to monitor the evolution and spread of FIV.

Protoparvovirus carnivoran 1 (PPVC-1), Canine adenovirus type 1 and 2 (CAdV-1 and CAdV-2), and Canine circovirus (CanineCV) are highly diffusive viruses that affect domestic and wild carnivores worldwide, yet limited data are available on their circulation in Eastern European countries. In this retrospective study, the presence of these DNA viruses was investigated using molecular assays on fecal samples from 89 companion animals (56 dogs and 33 cats) collected in Romania between 2019 and 2021. The pathogens identified were analyzed genetically. Overall, 36/56 (64.3%) dogs and 5/33 (15.2%) cats tested positive for PPVC-1, 1/56 (1.8%) dogs for CAdV-1 and CAdV-2, and 15/56 (26.8%) dogs for CanineCV. In total, 40/56 (71.4%) dogs were positive for at least one of the screened pathogens. Novel findings in dogs included the frequent detection of canine parvovirus type 2c of Asian origin (Asian-like CPV-2c) and the first genetic data on CAdV-1 and CanineCV circulating in Romania. This study provides new insights into the epidemiology of DNA viruses in dogs and cats from Romania and highlights the need for ongoing monitoring of circulating pathogens to safeguard animal health, prevent outbreaks and limit potential transboundary spread.