Detection of a virus related to betacoronaviruses in Italian greater horseshoe bats.

SUMMARYThe association between coronaviruses and bats is a worldwide phenomenon and bats belonging to genus Rhinolophus are the reservoir host for several coronaviruses, including a large number of viruses closely related genetically to severe acute respiratory syndrome-coronavirus (SARS-CoV). We carried out a survey in colonies of Italian bats (Rhinolophus ferrumequinum) for the presence of coronaviruses. Two of 52 R. ferrumequinum captured from different Italian areas tested positive by reverse transcription–PCR for a fragment of RNA-dependent RNA polymerase (RdRp) gene of viruses related to Coronavirus. Phylogenetic analysis revealed close correlations between one of the positive samples and SARS-related CoV belonging to the genus Betacoronavirus

Clinicopathological Findings in Cats Tested for Feline Immunodeficiency Virus (FIV) and Feline Leukaemia Virus (FeLV)

Abstract This retrospective study aimed to evaluate the clinicopathological changes in a population of cats tested for feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV), in an Italian Veterinary University Hospital, in the period between January 2002 and May 2016. During the period of 14 years, 1834 cats were tested, and of these 241/1834 (13.1%) were positive for FIV antibodies and 92/1834 (5%) cats were positive for FeLV antigen. These data confirm the presence of a high prevalence of these viruses on Italian territory. To the authors' knowledge, this study describes findings that have never been evaluated before, such as iron status in retrovirus-infected cats and urinalysis in FeLV-positive cats. In this study, FIV-positive cats were more likely to have higher serum protein concentration and lower albumin-globulin ratio than other groups of cats. Lower urine specific gravity and higher urine protein to creatinine ratio were also detected for FIV-positive cats when compared with negative and healthy cats. FeLV-positive cats were more likely to have cytopenia, decreased haemoglobin, haematocrit and RBC compared with other groups of cats. The data obtained underline the importance of considering retroviral infections in the presence of a broad spectrum of risk factors and laboratory anomalies

A Real-Time PCR Assay for Bat SARS-Like Coronavirus Detection and Its Application to Italian Greater Horseshoe Bat Faecal Sample Surveys

Bats are source of coronaviruses closely related to the severe acute respiratory syndrome (SARS) virus. Numerous studies have been carried out to identify new bat viruses related to SARS-coronavirus (bat-SARS-like CoVs) using a reverse-transcribed-polymerase chain reaction assay. However, a qualitative PCR could underestimate the prevalence of infection, affecting the epidemiological evaluation of bats in viral ecology. In this work an SYBR Green-real time PCR assay was developed for diagnosing infection with SARS-related coronaviruses from bat guano and was applied as screening tool in a survey carried out on 45 greater horseshoe bats (Rhinolophus ferrumequinum) sampled in Italy in 2009. The assay showed high sensitivity and reproducibility. Its application on bats screening resulted in a prevalence of 42%. This method could be suitable as screening tool in epidemiological surveys about the presence of bat-SARS-like CoVs, consequently to obtain a more realistic scenario of the viral prevalence in the population

Anaplasma phagocytophilum infection in thrombocytopenic dogs

Anaplasma and Ehrlichia spp. are tick-transmitted bacteria of clinical relevance in European dogs. The diagnosis of infection is often difficult due to the wide spectrum of disease caused by them. During infection, reduction in platelet count is considered the most common haematological abnormality, frequently representing the sole alteration in asymptomatic dogs. In this study, the presence of bacteria belonging to the genera Anaplasma and Ehrlichia was investigated in Northern Italy in blood samples from 159 thrombocytopenic dogs using a polymerase chain reaction (PCR) assay amplifying a portion of the heat shock gene (groEL). Obtained amplicons were sequenced and analysed. Two dogs were positive for A. phagocytophilum, while A. platys and E. canis were not detected. None of the PCR-positive dogs were diagnosed at the time of hospital admission, even in the presence of clinical signs and clinicopathological abnormalities potentially related to A. phagocytophilum infection. Nucleotide sequence analysis showed that the 2 detected strains belonged to the cluster Europe 1 and were different from each other. This study confirms the presence of A. phagocytophilum infections in dogs of Northern Italy, causing clinical signs and laboratory abnormalities that could not be properly diagnosed and treated

Severe, diffuse fibrinonecrotic pleuropneumonia in a cat affected by multiple viral infection

This communication describes the coinfection with feline panleukopenia virus (FPV), feline herpesvirus 1 (FeHV-1), feline calicivirus (FCV) and feline coronavirus (FCoV) in a 1 year\u2011old domestic cat living in a feline shelter. The cat was referred to veterinary hospital with clinical signs related to diffuse gastro-intestinal inflammation, it had developed a severe pneumopathy with fibrinous exudation in all body cavities and died 8 days after initial presentation. Pathological findings and biomolecular diagnostic test results were compatible with an initial FPV infection that, in consequence of the lymphoid depletion, has fostered coinfection or reactivation of chronic-latent infections with FeHV-1, FCV, and FCoV. In the reported case, the simultaneous presence of different viruses exacerbated the clinical status of the host, resulting in multiple organ damage and leading it to its death

Genetic complexity and multiple infections with more Parvovirus species in naturally infected cats

Parvoviruses of carnivores include three closely related autonomous parvoviruses: canine parvovirus (CPV), feline panleukopenia virus (FPV) and mink enteritis virus (MEV). These viruses cause a variety of serious diseases, especially in young patients, since they have a remarkable predilection for replication in rapidly dividing cells. FPV is not the only parvovirus species which infects cats; in addition to MEV, the new variants of canine parvovirus, CPV-2a, 2b and 2c have also penetrated the feline host-range, and they are able to infect and replicate in cats, causing diseases indistinguishable from feline panleukopenia. Furthermore, as cats are susceptible to both CPV-2 and FPV viruses, superinfection and co-infection with multiple parvovirus strains may occur, potentially facilitating recombination and high genetic heterogeneity. In the light of the importance of cats as a potential source of genetic diversity for parvoviruses and, since feline panleukopenia virus has re-emerged as a major cause of mortality in felines, the present study has explored the molecular characteristics of parvovirus strains circulating in cat populations. The most significant findings reported in this study were (a) the detection of mixed infection FPV/CPV with the presence of one parvovirus variant which is a true intermediate between FPV/CPV and (b) the quasispecies cloud size of one CPV sample variant 2c. In conclusion, this study provides new important results about the evolutionary dynamics of CPV infections in cats, showing that CPV has presumably started a new process of readaptation in feline hosts

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

Clinicopathological and Molecular Findings in a Case of Canine Anaplasma phagocytophilum

A documented case of canine granulocytic anaplasmosis coupled with the molecular characterization of the etiological agent is reported for the first time in Northern Italy. The patient showed nonspecific clinical signs such as fever and weight loss. The most relevant clinicopathological findings were thrombocytopenia, hypoalbuminemia, and normal azotemic proteinuria consistent with glomerular diseases. Blood smear examination revealed the presence of intracytoplasmatic inclusions in neutrophils associated with high positive serology for Anaplasma phagocytophilum. PCR analysis and sequencing of the amplicon confirm serological diagnosis of A. phagocytophilum. Phylogenetic analysis evidenced that the detected bacterial strain belongs to the A. phagocytophilum Europe 1 lineage. Data indicates that A. phagocytophilum circulates in natural environments of Emilia-Romagna region (Northern Italy) and its prevalence in dogs could be underestimated because the clinical signs are frequently nonspecific and a certain diagnosis requires the combination of clinicopathological and molecular assays. Pets living in this area should be regularly monitored and treated for ectoparasites to minimize health risks for humans and pets. Also, surveillance of A. phagocytophilum should be improved in Northern Italy and canine anaplasmosis should be considered in differential diagnosis of persistent proteinuria

Caratterizzazione molecolare di ceppi di coronavirus felini ed analisi filogenetica comparativa

Obiettivo del presente progetto è quello di approfondire le conoscenze relative alle coronavirosi feline sia da un punto di vista virologico che da quello immunopatogenetico, per trarre informazioni utili sia per il controllo della diffusione dei coronavirus felini (FCoV) nelle popolazioni feline, sia per prevenire possibili zoonosi: da un lato, infatti, l'instabilità genetica dei CoV può essere responsabile di mutazioni o ricombinazioni responsabili di salti di specie, come accaduto nella recente epidemia di SARS; dall'altro, il sistema immunitario può determinare persistenza del virus nella popolazione favorendo così la comparsa di nuove varianti virali, potenzialmente patogene anche per l'uomo. Per raggiungere tale obiettivo si procederà al campionamento di sangue, feci e tessuti da: - gatti di allevamento, dove sono frequenti i portatori di FCoV e dove è possibile identificare in base al tipo di eliminazione fecale i gatti più o meno resistenti all'infezione - gatti clinicamente sani di proprietà FCoV-negativii - gatti con peritonite infettiva felina, la forma clinicamente manifesta di coronavirosi, che presentano varianti patogene di FCoV e/o stati immunitari in grado di determinare lo sviluppo della malattia I campioni di feci o tessuti verranno utilizzati per indagare i seguenti aspetti virologici: - identificazione, mediante sequenziamento ed analisi bioinformatica, delle caratteristiche genomiche dei ceppi di FCoVs identificati nei portatori sani e malati, comparandole con dati reperibili in letteratura e relativi ad altri coronavirus animali ed umani allo scopo di comprendere i meccanismi evolutivi e patogenetici dei FCoV. - Identifcazione delle quasispecie virali mediante analisi della variabilità di sequenza a carico dei geni S, 7b e 3c mediante SSCP (single strand conformational polymorphism) - Predizione degli epitopi immunostimolanti nella sequenza della nucleoproteina N dei FCoV attraverso analisi bioinformatiche e verifica in vitro di proteine ricombinanti prodotte sulla base delle sequenze identificate - Messa a punto di sistemi di isolamento in coltura cellulare dei ceppi di FCoVs "di campo" Sui campioni di sangue e tessuti verranno invece approfonditi i seguenti aspetti immunopatologici: - Identificazione di quadri clinico-patologici associati alle diverse forme di infezione da FCoV (peritonite infettiva felina o infezione asintomatica con eliminazione transitoria, persistente o intermittente) - Identificazione della risposta immunitaria innata (livelli e grado di sialilazione dell'alfa-1-glicoproteina acida) o specifica (quantificazione delle citochine prodotte da specifiche popolazioni cellulari) in gatti con diverse forme di infezione da FCoV. - Caratterizzazione della risposta citochinica in linee cellulari immunocompetenti feline prelevate da gatti FCoV negativi ma stimolate con ceppi virali dalle caratteristiche molecolari note (vedi sopra), con le proteine immunogene di FCoV prodotte nel corso del progetto, o con campioni biologici prelevati da gatti FCoV-infetti - Isolamento e sequenziamento di TLR3, TLR7 e TLR8 felini, probabilmente coinvolti in questo tipo di risposte ma dei quali non sono disponibili informazioni in letteratura: una volta caratterizzati i TLR, verrà valutato il loro eventuale polimorfismo genetico e quantificata la loro espressione in specifiche popolazioni cellulari di soggetti sani e sintomatici L'approccio sopra descritto consentirà di raccogliere una serie di dati sul virus e sulle risposte immunitarie in presenza di FCoV. Tali dati verranno integrati tra loro per valutare sia l'epidemiologia molecolare dei FCoV, sia l'influenza dei vari ceppi virali nel determinare le risposte immunitarie o, al contrario, l'esistenza di risposte immunitarie in grado di influenzare l'evoluzione delle quasispecie virali