Molecular investigation and genetic characterization of feline leukemia virus (FeLV) in cats referred to a veterinary teaching hospital in Northern Italy.

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.

Detection of Anaplasma spp. and Ehrlichia spp. in dogs from a veterinary teaching hospital in Italy: a retrospective study 2012-2020.

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.

No viable bacterial communities reside in the urinary bladder of cats with feline idiopathic cystitis.

Urinary microbial diversities have been reported in humans according to sex, age and clinical status, including painful bladder syndrome/interstitial cystitis (PBS/IC). To date, the role of the urinary microbiome in the pathogenesis of PBS/IC is debated. Feline idiopathic cystitis (FIC) is a chronic lower urinary tract disorder affecting cats with similarities to PBS/IC in women and represents an important problem in veterinary medicine as its aetiology is currently unknown. In this study, the presence of a bacterial community residing in the urinary bladder of cats with a diagnosis of FIC was investigated. Nineteen cats with clinical signs and history of FIC and without growing bacteria in standard urine culture were included and urine collected with ultrasound-guided cystocentesis. Bacterial community was investigated using a culture-dependent approach consisted of expanded quantitative urine culture techniques and a culture-independent approach consisted of 16S rRNA NGS. Several methodological practices were adopted to both avoid and detect any contamination or bias introduced by means of urine collection and processing which could be relevant due to the low microbial biomass environment of the bladder and urinary tract, including negative controls analysis. All the cats included showed no growing bacteria in the urine analysed. Although few reads were originated using 16S rRNA NGS, a comparable pattern was observed between urine samples and negative controls, and no taxa were confidently classified as non-contaminant. The results obtained suggest the absence of viable bacteria and of bacterial DNA of urinary origin in the urinary bladder of cats with FIC.

Concomitant Infections With Canine Parvovirus Type 2 and Intracellular Tick-Borne Pathogens in Two Puppy Dogs.

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.

Natural cases of polyarthritis associated with feline calicivirus infection in cats.

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 circovirus and Canine adenovirus type 1 and 2 in dogs with parvoviral enteritis.

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.

Integrated Use of Molecular Techniques to Detect and Genetically Characterise DNA Viruses in Italian Wolves (Canis lupus italicus).

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.

The detection of canine parvovirus type 2c of Asian origin in dogs in Romania evidenced its progressive worldwide diffusion.

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.

Natural distemper infection in stone martens (Martes foina): From infection to neutralizing antibodies.

We report an outbreak of canine distemper virus (CDV) among stone martens (Martes foina) in Italy. After being rescued in Northern Italy between April and June 2018, six subjects were kept in a wildlife and exotic animal rescue center in Bologna province. Subjects have been monitored for 15 months in captivity. Within this time-lapse, two subjects died, while among the remaining four, only one showed clinical symptoms referable to distemper. Surviving subjects have been regularly tested for CDV by means of reverse transcriptase-PCR from conjunctival and oropharyngeal swabs for eleven months. The identified viruses belonged to the Wildlife-Europe CDV genetic subgroup. Neutralizing antibodies were detected at the end of the eleven months, when all subjects tested reverse transcriptase-PCR negative. Our findings confirm the circulation of the Wildlife-Europe CDV genetic subgroup (Europe 1/South America 1 lineage) within the Italian wildlife, and improve knowledge on viral infection in stone martens.

Molecular detection of Anaplasma phagocytophilum in hair and spleen of cats revealed a possible underestimation of feline vector-borne pathogens.

Feline Vector-Borne Diseases show increased global prevalence and some Anaplasma and Ehrlichia species may pose a risk to human health. The diagnosis of Anaplasma and Ehrlichia species infection in cats is achieved by the combined use of different methods as cytologic examination evidencing intracytoplasmic morulae, serologic tests and molecular assays. The peripheral whole blood is considered the sample of choice for Anaplasma and Ehrlichia species DNA detection in cats, but false negative results are reported leading to underestimation of infection prevalence. In order to have a more accurate assessment of the spread of feline vector-borne pathogens, the presence of Anaplasma spp. and Ehrlichia spp. DNA in 37 owner and shelter cats subjected to necropsy were prospectively investigated by testing in end-point PCR spleen, bone marrow, blood clot and hair samples. The bacteria identified were genetically characterised. Three shelter cats tested positive for A. phagocytophilum DNA in spleen (one cat) or in hair samples (two cats). None of the cats tested positive in bone marrow and blood samples. From the results obtained, it can be assumed that the use of spleen or hair samples could allow a more reliable detection of A. phagocytophilum DNA in cats with blood tested negative. In the phylogeny constructed with a fragment of the heat shock (groEL) gene nucleotide sequences, all the identified A. phagocytophilum clustered with bacteria infecting a wide range of hosts, including humans, showing a potential zoonotic role.

Ancient origin and genetic segregation of canine circovirus infecting arctic foxes (Vulpes lagopus) in Svalbard and red foxes (Vulpes vulpes) in Northern Norway.

Canine circovirus (CanineCV) is a relatively new viral species, belonging to the family Circoviridae, whose pathogenic role is still uncertain. Since its first description in one domestic dog in 2011 from the USA, several reports have been documenting its distribution worldwide. Recently, CanineCV was also detected in wild animals such as wolves, foxes and badgers. In order to investigate the presence and the genetic characteristics of CanineCV in foxes of Arctic and Sub-Arctic regions, the presence of CanineCV DNA in internal organs (liver and spleen) of 51 arctic foxes (Vulpes lagopus) from Svalbard archipelago and 59 red foxes (Vulpes vulpes) from Northern Norway, sampled from 1996 to 2001 and from 2014 to 2018, respectively, was screened by real-time PCR. CanineCV was detected in 11/51 arctic foxes and in 10/59 red foxes, backdating the circulation of the virus at least to 1996 in the arctic fox population. The complete genome of 14 identified CanineCV was sequenced and analysed showing an identity higher than 80.8% with the reference strains available to date. According to the species demarcation threshold of 80% genome-wide nucleotide sequence identity for members of the family Circoviridae provided by International Committee on Taxonomy of Viruses (ICTV), all the CanineCV belong to a single species. Phylogenetic analysis revealed that all the CanineCV were subdivided into five main clusters with one including only CanineCV identified in foxes. Furthermore, CanineCV identified in arctic foxes and red foxes formed two distinct lineages. From these data, we hypothesize that the viral transmission did not occur between the two species of foxes as a consequence of the lack of contact between the two hosts or that the virus acquired mutations in the time elapsed between the samplings.

Culture-Dependent and Sequencing Methods Revealed the Absence of a Bacterial Community Residing in the Urine of Healthy Cats.

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.

Serological, molecular and clinicopathological findings associated with Leishmania infantum infection in cats in Northern Italy.

OBJECTIVES: The aims of this study were to investigate the prevalence of Leishmania species infection in cats in Northern Italy and to evaluate the associations between infection and signalment and clinicopathological data. METHODS: The study was carried out in a veterinary university hospital from June to November 2017. Blood, urine, conjunctival swabs and hair were collected from all randomly selected cats. Leishmania species infection was evaluated using the indirect fluorescent antibody test (IFAT), setting a cut-off value of 1:80, and using real-time PCR on blood, conjunctival and hair samples. A complete blood count, serum chemistry profile, serum electrophoresis and urinalysis were also carried out. The cats were grouped on the basis of the results of the diagnostic criteria adopted in positive, negative and unconfirmed Leishmania cases. Non-parametric variables and continuous data were compared among the study groups using the χ2 test and the Mann-Whitney U-test, respectively. RESULTS: One hundred and fifty-two cats were included. Nineteen of the 152 (12.5%) cats were positive (18/152 [11.8%] showed an IFAT titre of ⩾1:80 and 1/152 [0.7%] was real-time PCR-positive from a hair sample); 106/152 (69.7%) cats were negative; and 27/152 (17.8%) cats were unconfirmed for Leishmania species. Total proteins, beta2-globulin and gamma-globulin were significantly increased in the positive Leishmania group compared with the negative group. CONCLUSIONS AND RELEVANCE: The results of the present study demonstrated the spread of Leishmania infantum infection in cats in Northern Italy. Hyperproteinaemia and hypergammaglobulinaemia appeared to be significant clinicopathological abnormalities in this population of cats with L infantum infection.

Genomic Characterization of Canine Circovirus Detected in Red Foxes (Vulpes vulpes) from Italy using a New Real-Time PCR Assay.

Data on canine circovirus circulation among red foxes (Vulpes vulpes) are limited. We report the detection of canine circovirus in a red fox from Italy. The virus was closely related to strains from dogs (Canis lupus familiaris) rather than those from foxes, suggesting a possible transmission between the two species.