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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.

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.

Background Canine parvovirus (CPV) is one of the most important pathogens of dogs. Despite vaccination, CPV infections are still ubiquitous in dogs, and the three antigenic variants 2a, 2b and 2c are variously distributed in the canine population worldwide. To date, no information is available on CPV variants circulating in some European countries. The aim of this study was to genetically characterise the CPV detected in ten dogs with clinical signs of acute gastroenteritis in Romania. The presence of Carnivore protoparvovirus 1 DNA was investigated in faecal samples using an end-point PCR targeting the complete VP2 gene and positive amplicons were sequenced and analysed. Results All ten dogs with acute gastroenteritis tested positive to Carnivore protoparvovirus 1 DNA in faecal samples. The identified viruses belonged to CPV-2c type, showed identical sequences of the VP2 gene and were characterised by distinctive amino acid residues in the deduced VP2 protein: 5-glicine (5Gly), 267-tirosine (267Tyr), 324-isoleucine (324Ile) and 370-arginine (370Arg). These distinctive amino acid residues have already been reported in CPV-2c widespread in Asia and occasionally detected in Italy and Nigeria. Conclusions Since CPV-2c with VP2 amino acid residues 5Gly, 267Tyr, 324Ile and 370Arg were never reported before 2013, it can be assumed that this virus is progressively expanding its spread in the world dog population. This study adds new data about the presence of this new virus in Europe and underline worrying questions about its potential impact on the health of the canine population.

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.

The Protoparvovirus carnivoran 1 viral species includes relevant pathogens as feline panleukopenia virus (FPV) and canine parvovirus type 2 (CPV-2), which are mainly responsible for immunosuppression and gastroenteritis in domestic and wild carnivores. Differently to FPV, CPV is frequently subjected to mutations. To date, the original antigenic type CPV-2 is mainly used in vaccine production, while field strains have been progressively replaced by the CPV antigenic variants 2a, 2b, and 2c. In recent years, additional distinctive mutations have been identified in different CPV antigenic variants classified as “Asian-like.” The variability of these viruses can impact on the reliability of molecular diagnostic tests potentially leading to false-negative results or delays in diagnosis. To improve diagnostic accuracy and efficiency, innovative molecular techniques such as High-Resolution Melting (HRM) analysis have been developed. These methods reduce execution time, facilitate diagnosis, and enable the differentiation of species or variants without the need for sequencing. In this study, two real-time PCR (qPCR) assays with HRM analysis were developed to complement existing tools for the detection and genetic differentiation of circulating FPV and CPV. Specifically, a test capable of differentiating FPV, original CPV-2 and CPV-2 antigenic variants, and a test for the identification of Asian-like CPV strains were validated. Furthermore, the FPV and CPV-2 identified in 33 dogs and cats diagnosed with parvoviral infection in a veterinary teaching hospital in Northern Italy between 2017 and 2023 were genetically characterized by sequencing. Based on specific VP2 amino acid residues, 33.3% viruses were FPV, 6.1% were original CPV-2, 6.1% were CPV-2a, 21.2% were CPV-2b and 33.3% were CPV-2c. FPV were detected only in cats and showed high amino acid similarity, confirming its evolutionary stasis. The 45.4% of CPV identified in this study, carried amino acid residues resembling those of Asian-like viruses, suggesting an origin linked to an initial importation and subsequent local diffusion. In contrast, the other CPV-2a, 2b, and 2c viruses exhibited greater genetic heterogeneity and their autochthonous origin was supposed. The two qPCR-HRM assays successfully detected and classified all the FPV and CPV tested, highlighting their reliability and usefulness for both diagnostic and epidemiological purposes.

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.

In this report the concomitant infection with canine parvovirus type 2 (CPV-2), Hepatozoon canis and Ehrlichia canis in two puppy dogs from Southern Italy is described. Dogs were referred to a veterinary university hospital for the acute onset of lethargy and gastrointestinal signs. A complete clinical and clinicopathological evaluation was carried out and the multiple infection was confirmed by microscopic detection of inclusion bodies in peripheral blood smear, rapid immunoenzymatic tests, indirect fluorescent antibody tests, and molecular assays. Sequence analysis revealed that the CPV-2 identified belonged to the 2c variant and had amino acid residues in the predicted VP2 protein typical of “Asian-like” strains widespread in Asia and occasionally reported in Romania, Nigeria and Italy, particularly in the region of Sicily. Numerous monocytes were infected by both H. canis gamonts and E. canis morulae, suggesting that this co-infection is not accidental and that E. canis preferably infects those cells parasitized by H. canis . The clinical presentation of these animals was severe but supportive cares associated with early etiological therapy allowed a good prognosis. Movement of puppies from geographic areas where vector-borne pathogens are endemic must be carefully evaluated and core vaccinations and ectoparasite prevention treatments must be rigorously adopted.

In this study, internal organs (tongue, intestine, and spleen) of 23 free-ranging Italian wolves (Canis lupus italicus) found dead between 2017 and 2019 were tested for Carnivore protoparvovirus 1, Canine adenovirus (CAdV), and Canine circovirus (CanineCV) using real-time PCR assays. Genetic characterisation of the identified viruses was carried out by amplification, sequencing, and analysis of the complete viral genome or informative viral genes. All the wolves tested positive for at least one of the DNA viruses screened, and 11/23 were coinfected. Carnivore protoparvoviruses were the most frequently detected viruses (21/23), followed by CanineCV (11/23) and CAdV (4/23). From the analysis of the partial VP2 gene of 13 carnivore protoparvoviruses, 12 were canine parvovirus type 2b, closely related to the strains detected in dogs and wild carnivores from Italy, and one was a feline panleukopenia-like virus. Of the four CAdV identified, two were CAdV-1 and two were CAdV-2. The complete genome of seven CanineCVs was sequenced and related to the CanineCV identified in dogs, wolves, and foxes worldwide. Close correlations emerged between the viruses identified in wolves and those circulating in domestic dogs. Further studies are needed to investigate if these pathogens may be potentially cross-transmitted between the two species.

A growing number of studies suggest that the lower urinary tract of humans and dogs can harbor a urinary microbiota. Nevertheless, a certain concern has developed that the microbiota reported could be due to unaccounted contamination, especially in low-biomass samples. The aim of this study was to investigate the bacterial community which populates the urine of healthy cats using two approaches: a culture-dependent approach which consisted of the expanded quantitative urine culture (EQUC) techniques capable of identifying live bacteria not growing in standard urine cultures, and a culture-independent approach which consisted of 16S ribosomal RNA next generation sequencing (16S rRNA NGS) capable of identifying bacterial DNA and exploring microbial diversity with high resolution. To avoid confounding factors of possible bacterial contamination, the urine was sampled using ultrasound-guided cystocentesis, and several sample controls and negative controls were analyzed. The urine sampled from the 10 cats included in the study showed no bacterial growth in the EQUC procedure. Although several reads were successfully originated using 16S rRNA NGS, a comparable pattern was observed between urine samples and the negative control, and no taxa were statistically accepted as non-contaminant. Taken together, the results obtained allowed stating that no viable bacteria were present in the urine of healthy cats without lower urinary tract disease and urinary tract infections, and that the bacterial DNA detected was of contaminant origin.