Lorenza Urbani

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.

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.

Canine adenovirus type 1 (CAdV-1) and type 2 (CAdV-2) are well known pathogens of domestic dogs but are little investigated in wild animals. The few available studies about CAdV-1 in wild animals show that it circulates in various species and that transmission of the virus in the interface between wildlife and domestic animals is a frequent event. Furthermore, wild animals are usually subject to asymptomatic infections, but cases of serious and fatal diseases have been documented, with possible effects on the conservation of the species. In contrast, CAdV-2 infection was reported only recently and sporadically in some wild animals, with few data regarding its pathogenic role in these species. However, the real prevalence of these viruses in wildlife is still uncertain due to the use of serological tests that are largely unable to distinguish antibodies against CAdV-1 and CAdV-2. This review, reporting all the data currently available on CAdV-1 and CAdV-2 infection in wild animals, highlights the importance of these pathogens for wildlife conservation and their role in the potential transmission of the infection to domestic dogs.

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.

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 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.

Anaplasma phagocytophilum, Anaplasma platys and Ehrlichia canis, responsible of diseases in dogs, are tick-borne pathogens with a proven or potential zoonotic role that have shown increasing prevalence worldwide. The aims of this retrospective study were to assess the frequency of Anaplasma spp. and Ehrlichia spp. exposure in dogs tested in a veterinary teaching hospital in Italy over a 9-year period, to compare the performance of the diagnostic tests used, to evaluate correlations with clinical data, and to genetically analyse the identified bacteria. During the study period, 1322 dogs tested by at least one of the rapid immunoenzymatic test, indirect immunofluorescent antibody test or end-point PCR assay for Anaplasmataceae detection were included. Dogs were tested if they had clinical signs or clinicopathological alteration or risk factors related to infection, and if they were potential blood-donor animals. Ninety-four of 1322 (7.1%) dogs tested positive for at least one pathogen: 53 (4.3%) for A. phagocytophilum, one (0.1%) for A. platys and 63 (4.6%) for E. canis. The number of dogs tested increased and the positivity rate progressively declined over the years. Comparison of tests showed a near-perfect agreement between serological tests and a poor agreement between PCR and indirect assays. A breed predisposition has been highlighted for A. phagocytophilum infection in hunting breed dogs and for E. canis infection in mixed breed dogs. Phylogeny confirmed potential zoonotic implications for A. phagocytophilum and showed no correlation of the identified bacteria with the geographical origin. Our study provides new insights into possible risk factors in dogs and evidenced discordant results between different tests, suggesting that a combination of serological and molecular assays is preferable for a correct diagnosis.

The limping syndrome is occasionally reported during acute feline calicivirus (FCV) infections or as consequence of vaccination. In this retrospective study, three clinical cases of lameness in household cats naturally infected by FCV were described and phylogeny of the virus were investigated by analysing the hypervariable E region of the ORF2 viral gene. Cats were diagnosed with polyarthritis and FCV RNA or antigens were detected in symptomatic joints. One cat, euthanized for ethical reasons, underwent a complete post-mortem examination and was subjected to histopathological and immunohistochemical investigations. No phylogenetic subgrouping were evident for the sequenced FCV. Histopathology of the euthanized cat revealed diffuse fibrinous synovitis and osteoarthritis eight months after the onset of lameness and the first detection of FCV RNA, supporting the hypothesis of a persistent infection. FCV was demonstrated by immunohistochemistry in synoviocytes and fibroblasts of the synovial membranes. This study provides new data on the occurrence of polyarthritis in FCV-infected cats, demonstrates by immunohistochemistry the presence of FCV in the synovial membranes of a cat with persistent polyarthritis and supports the absence of correlation between limping syndrome and phylogenetic subgrouping of viruses.

Canine parvovirus type 2 (CPV-2) is one of the most relevant pathogens associated with enteritis in dogs and is frequently reported in association with the detection of other pathogens in faeces. In this study the concomitant presence of Canine circovirus (CanineCV) and Canine adenovirus (CAdV) DNA in faecal or intestine samples of 95 dogs with parvovirus enteritis sampled in Italy (1995–2017) was investigated and the viruses identified were genetically characterised. Potential correlations with the antigenic variant of CPV-2 and with signalment data and outcome were evaluated. Twenty-eight of 95 (29.5%) CPV-2 infected dogs tested positive to other viruses: 7/28 were also positive to CanineCV, 1/28 to CAdV-1, 18/28 to CAdV-2, 1/28 to CanineCV and CAdV-2, and 1/28 to CAdV-1 and CAdV-2. The frequency of CAdV DNA detection and coinfections was significantly higher in purebred dogs compared to mixed breed ones (P = 0.002 and 0.009, respectively). The presence of coinfection was not associated with any other relevant data available, including CPV-2 variant and final outcome. The detection of CanineCV in a dog sampled in 2009 allowed to backdating its circulation in dogs. The eight CanineCV completely sequenced were phylogenetically related to the CanineCV identified in dogs, wolves and a badger from Europe, USA, Argentina and China. Nine CAdV were partially sequenced and phylogenetic analysis showed a separate branch for the oldest CAdV-2 identified (1995). From the results obtained in this study population, CanineCV and CAdV coinfections in dogs with parvoviral enteritis did not result in more severe disease.