Validation of a real-time PCR assay for the detection of African swine fever virus in fresh pork meat juice.

African swine fever virus (ASFV) is the etiological agent of African swine fever (ASF), a disease with detrimental effects on the health, welfare, and production of domestic and wild pigs. The ASF laboratory confirmation is based on the analysis of blood, serum and organ samples. However, testing these samples could not be always convenient, economically feasible or possible. This study describes the validation process of a PCR-based assay targeting a portion of p72 gene, used for the molecular detection of ASFV, from meat juice samples obtained from pigs succumbed to ASFV. More specifically, we investigated the capability of a real-time PCR assay to detect ASFV DNA in meat juices obtained from the diaphragmatic muscle along with the correspondent spleens of 55 ASFV-positive pigs and wild boars sampled from confirmed outbreaks in Romania and from 73 ASFV-negative and regularly slaughtered healthy pigs collected in the Abruzzo region (Italy). The test was able to detect viral DNA in both types of samples, with lower Ct values in spleens (mean=21.11, median=20.61) than meat juices (mean=23.08, median=22.40). However, distributions of Ct values were strongly correlated each other (R2= 0.83, P<0.001). Considering the distribution of the observed Ct values in the 55 positive meat juice samples, a 1:10 dilution would be able to detect 90 % of positive samples, whereas a 1:100 dilution would reduce the detectability to 78 % of more contaminated samples. As meat juice could be obtained easily from muscles and considering the potential use of this test on pooled samples, it could represent a tool to aid the investigation of ASFV spread.

First Reported Circulation of Equine Influenza H3N8 Florida Clade 1 Virus in Horses in Italy.

BACKGROUND: Equine influenza (EI) is a highly contagious viral disease of equids characterized by pyrexia and respiratory signs. Like other influenza A viruses, antigenic drift or shift could lead to a vaccine-induced immunity breakdown if vaccine strains are not updated. The aim of this study was to genetically characterize EIV strains circulating in Italy, detected in PCR-positive samples collected from suspected cases, especially in the absence of formal active surveillance. METHODS: Between February and April 2019, blood samples and nasal swabs collected from each of the 20 symptomatic horses from North and Central Italy were submitted to the National Reference Centre for Equine Diseases in Italy to confirm preliminary analysis performed by other laboratories. RESULTS: None of the sera analysed using haemagglutination inhibition and single radial haemolysis presented a predominant serological reactivity pattern for any antigen employed. All nasal swabs were positive with IAV RRT-PCR. Only one strain, isolated in an embryonated chicken egg from a sample collected from a horse of a stable located in Brescia, Lombardy, was identified as H3N8 Florida lineage clade 1 (FC1). In the constructed phylogenetic trees, this strain is located within the FC1, together with the virus isolated in France in 2018 (MK501761). CONCLUSIONS: This study reports the first detection of H3N8 FC1 in Italy, highlighting the importance of monitoring circulating EIV strains to verify the vaccine composition appropriateness for maximum efficacy.

One health system supporting surveillance during COVID-19 epidemic in Abruzzo region, southern Italy.

The Istituti Zooprofilattici Sperimentali (IZSs) are public health institutes dealing with the aetiology and pathogenesis of infectious diseases of domestic and wild animals. During Coronavirus Disease 2019 epidemic, the Italian Ministry of Health appointed the IZSs to carry out diagnostic tests for the detection of SARS-CoV-2 in human samples. In particular, the IZS of Abruzzo and Molise (IZS-Teramo) was involved in the diagnosis of SARS-CoV-2 through testing nasopharyngeal swabs by Real Time RT-PCR. Activities and infrastructures were reorganised to the new priorities, in a "One Health" framework, based on interdisciplinary, laboratory promptness, accreditation of the test for the detection of the RNA of SARS-CoV-2 in human samples, and management of confidentiality of sensitive data. The laboratory information system - SILAB - was implemented with a One Health module for managing data of human origin, with tools for the automatic registration of information improving the quality of the data. Moreover, the "National Reference Centre for Whole Genome Sequencing of microbial pathogens - database and bioinformatics analysis" - GENPAT - formally established at the IZS-Teramo, developed bioinformatics workflows and IT dashboard with ad hoc surveillance tools to support the metagenomics-based SARS-CoV-2 surveillance, providing molecular sequencing analysis to quickly intercept the variants circulating in the area. This manuscript describes the One Health system developed by adapting and integrating both SILAB and GENPAT tools for supporting surveillance during COVID-19 epidemic in the Abruzzo region, southern Italy. The developed dashboard permits the health authorities to observe the SARS-CoV-2 spread in the region, and by combining spatio-temporal information with metagenomics provides early evidence for the identification of emerging space-time clusters of variants at the municipality level. The implementation of the One Health module was designed to be easily modelled and adapted for the management of other diseases and future hypothetical events of pandemic nature.

Brucella abortus Strain RB51 Administered to Prepubescent Water Buffaloes, from Vaccination to Lactation: Kinetics of Antibody Response and Vaccine Safety.

Brucella RB51 is a live modified vaccine. Its use in water buffalo has been proposed using a vaccination protocol different to that used for cattle, but knowledge of the long-term effects of RB51 vaccination in this species remains incomplete. The aim of the study was to evaluate the safety and kinetics of antibody responses in water buffaloes vaccinated according to the protocol described for the bovine species in the WOAH Manual, modified with the use of a triple dose. Water buffaloes were vaccinated with the vaccine RB51. A booster vaccination was administered at 12 months of age. When turning 23-25 months old, female animals were induced to pregnancy. RB51-specific antibodies were detected and quantified using a CFT based on the RB51 antigen. Vaccinated animals showed a positive serological reaction following each vaccine injection, but titers and the duration of the antibody differed among animals. For 36 weeks after booster vaccination, the comparison of CFT values between vaccinated and control groups remained constantly significant. Afterwards, antibody titers decreased. No relevant changes in antibody response were recorded during pregnancy or lactation. In conclusion, results indicated that the vaccination schedule applied is safe and allows for vaccinated and unvaccinated controls to be discriminated between for up to 8 months after booster vaccination.

Campylobacter and risk factors associated with dog ownership: a retrospective study in household and in shelter dogs.

Campylobacteriosis has been the most frequently reported zoonotic disease in humans in Europe. The scientific literature has reported that the role of dogs may be relevant. The objectives of this work are to improve the knowledge about Campylobacter spp. carriage, infection and antimicrobial resistance in household and shelter dogs in Italy, and to assess risk factors at the dog/human interface. During the 2015­2016 period, rectal swabs were collected from 431 household vet­visiting dogs and 173 dogs housed in shelters. A total of 3 veterinary clinics, located in three Italian regions (Abruzzo, Molise and Tuscany) and 10 shelters, five in Abruzzo and five in Molise, were included in the study. Relevant risk factors for the transmission of Campylobacter spp. from dogs to humans were assessed by means of a questionnaire administered to owners of household dogs. For Campylobacter spp. isolation, selective cultivation methods were used, followed by confirmation and species identification with the PCR method. Phenotypic antibiotic resistance profiles assayed using antimicrobial susceptibility testing were combined. Campylobacter spp. were isolated from 9 household dogs (2.1% CI 1.1% ­ 3.9%) and from 13 shelter dogs (7.5 % CI 4.5% ­ 12.4%). In household dogs C. jejuni was the most represented species (0.9%). In shelter dogs, the most common species was C. jejuni (5.2%). Campylobacter spp. isolates were resistant to ciprofloxacin (22.73%), nalidixic acid (22.73%), tetracyclines (27.27%), streptomycin (9.09%) and erythromycin (4.55%). The main C. jejuni Clonal Complex identified in dogs were CC21, CC45, CC206, CC403, CC42 and CC658. The risk of contracting Campylobacteriosis from dogs remains a concrete reality. This risk is increased in the presence of common habits, as shown by the data from the questionnaire. Prevalence control of Campylobacter spp. in household and shelter dogs would be important in order to reduce the transmission to humans.

Myocardial Injury Complicated by Systolic Dysfunction in a COVID-19-Positive Dog.

A six-year-old Cavalier King Charles spaniel was referred with a two-month history of severe exercise intolerance and syncope. Clinical signs had developed during a local wave of coronavirus disease (COVID-19) two weeks after its family members had manifested symptoms of this viral disease and their positivity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was confirmed. Cardiologic assessment documented myocardial injury complicated by systolic dysfunction. An extensive diagnostic work-up allowed us to rule out common causes of myocardial compromise, both infective and not. Accordingly, serological and molecular tests aimed at diagnosing SARS-CoV-2 infection were subsequently performed, especially in light of the dog's peculiar history. Results of such tests, interpreted in the light of previous findings and current knowledge from human medicine, supported a presumptive diagnosis of COVID-19-associated myocardial injury, a clinical entity hitherto poorly described in this species.

Neutralization of SARS-CoV-2 Variants by Serum from BNT162b2 Vaccine Recipients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evolved rapidly, leading to viral lineages characterized by multiple mutations in the spike protein, which could potentially confer to the virus the ability to avoid the vaccine-induced immune response, making the vaccines less effective or ineffective. Here, we initially evaluated the neutralization capabilities in vitro by serum neutralization (SN) of six serum samples collected from recipients of the BNT162b2 vaccine against 11 SARS-CoV-2 isolates belonging to the major SARS-CoV-2 lineages that had been circulating in Italy. Then, we considered 30 additional serum samples by SN assay against the dominant B.1.617.2 (Delta) variant. A B.1 lineage isolate was used as a reference. In the first analysis, significant differences when compared with the reference strain (p > 0.05) were not evidenced; instead, when the panel of 30 sera was tested against the B.1.617.2 (Delta) variant, a significant (p = 0.0015) 2.38-fold reduction in neutralizing titres compared with the reference after the first vaccine dose was demonstrated. After the second vaccine dose, the reduction was not significant (p = 0.1835). This study highlights that the BNT162b2 vaccine stimulates a humoral response able to neutralize all tested SARS-CoV-2 variants, thus suggesting a prominent role in mitigating the impact of the SARS-CoV-2 pandemic in real-world conditions. Long-term follow-up is currently ongoing.

Circulation of diverse protoparvoviruses in wild carnivores, Italy.

Protoparvovirus is a monophyletic viral genus that includes the species Carnivore protoparvovirus-1 infecting domestic and wild carnivores. In this paper, the results of an epidemiological survey for Carnivore protoparvovirus-1 in wild carnivores in Italy are reported. Overall, 34 (11.4%) out of 297 tested animals were positive for Carnivore protoparvovirus-1, but the frequency of detection was much higher in intestine (54%) than in spleen samples (2.8%), thus suggesting that the intestine is the best sample to collect from wild animals for parvovirus detection. Feline panleukopenia virus (FPV) was detected in red foxes (Vulpes vulpes) (2.8%, 7/252) and Eurasian badgers (Meles meles) (10%, 1/10), whilst canine parvovirus (CPV) was found in wolves (54.3%, 19/35), Eurasian badgers (60%, 6/10) and one beech marten (Martes foina) (100%, 1/1), with more than one parvovirus type detected in some animals. Protoparvoviral DNA sequences from this study were found to be related to CPV/FPV strains detected in Asia and Europe, displaying some amino acid changes in the main capsid protein VP2 in comparison with other parvovirus strains from wildlife. In particular, the two most common mutations were Ile418Thr and Ala371Gly, which were observed in 6/12 (50%) and 5/12 (41.7%) of the CPV sequences from this study. Continuous surveillance for parvoviruses in wild carnivores and genetic analysis of the detected strains may help obtain new insight into the role of these animals in the evolution and epidemiology of carnivore parvoviruses.

Epidemiology, pathological aspects and genome heterogeneity of feline morbillivirus in Italy.

Feline morbillivirus (FeMV) is an emerging morbillivirus first described in cats less than a decade ago. FeMV has been associated with chronic kidney disease of cats characterized by tubulointerstitial nephritis (TIN), although this aspect is still controversial and not demonstrated with certainty. To investigate FeMV prevalence and genomic characteristics, an epidemiological survey was conducted in a total number of 127 household cats originating from two Italian regions, Abruzzi and Emilia-Romagna. A total number of 69 cats originating from three feline colonies were also enrolled for the study. Correlation with TIN was investigated by employing a total number of 35 carcasses. Prevalence of FeMV RNA was higher in urine samples collected from cats of colonies (P = 31.8%, CI 95% 22.1-43.6) compared to household cats (P = 8.66%, CI 95% 4.9-14.9) and in young and middle-aged cats while prevalence of FeMV Abs was higher in old cats. Sequences obtained straight from infected biological samples, either partial or complete, cluster into two clades within FeMV genotype 1, distantly related to FeMV genotype 2. Immunohistochemistry analysis of kidney sections of FeMV RNA positive cats revealed immunoreactivity within epithelial cells of renal tubuli and inflammatory cells. However, statistically significant association between FeMV and renal damages, including TIN, was not demonstrated (p= 0.0695, Fisher exact test). By virus histochemistry performed with FeMV-negative feline tissues and a FeMV isolate, tropism for different cellular types such as inflammatory cells residing in blood vessels of kidney and brain, airway epithelial cells, alveolar macrophages and to a lesser extent, the central nervous system, was demonstrated. Additional studies are warranted in order to establish viral tropism and immune response during the early phases of infection and to disentangle the role of FeMV in co-infection processes.

Campylobacter and antimicrobial resistance in dogs and humans: "One health" in practice.

Increasing antimicrobial resistance in both medicine and agriculture is recognised as a major emerging public health concern. Since 2005, campylobacteriosis has been the most zoonotic disease reported in humans in the European Union. Human infections due to Campylobacter spp. primarily comes from food. However, the human-animal interface is a potential space for the bidirectional movement of zoonotic agents, including antimicrobial resistant strains. Dogs have been identified as carriers of the Campylobacter species and their role as a source of infection for humans has been demonstrated. Furthermore, dogs may play an important role as a reservoir of resistant bacteria or resistance genes. Human beings may also be a reservoir of Campylobacter spp. for their pets. This review analyses the current literature related to the risk of Campylobacter antimicrobial resistance at the dog-human interface.

A real-time RT-PCR assay for molecular identification and quantitation of feline morbillivirus RNA from biological specimens.

The aim of this study was to develop a real-time RT-PCR to detect and quantitate feline morbillivirus (FeMV) RNA in biological samples. Primers and probe were targeted on a conserved region of FeMV P/V/C gene. To validate the assay with field samples, a total number of specimens of cats have been recruited including 264 urine and blood samples and compared with a generic RT-PCR targeting the L protein encoding gene of morbilliviruses. In addition, 385 tissue samples from 35 carcasses of cats have been also employed. RNA titres were low in all tested samples. Results also indicated the absence of cross-reaction with related morbilliviruses and existing pathogens of cats. In tissues with low levels of FeMV RNA, the presence of viral antigen was also evidenced by immunohistochemistry targeting the N viral protein. This newly described assay allows for a rapid, accurate and reliable quantitative detection of FeMV RNA that can be applied for diagnostics and research studies.