Molecular epidemiology of canine parvovirus type 2 in Sicily, southern Italy: A geographical island, an epidemiological continuum.

Since it emerged as a major dog pathogen, canine parvovirus type 2 (CPV-2) has featured a remarkable genetic and phenotypic heterogeneity, whose biological, epidemiological, and clinical impact is still debated. The continuous monitoring of this pathogen is thus of pivotal importance. In the present study, the molecular epidemiology of CPV-2 in Sicily, southern Italy, has been updated by analysing 215 nearly complete sequences of the capsid protein VP2, obtained from rectal swabs/faeces or tissue samples collected between 2019 and 2022 from 346 dogs with suspected infectious gastrointestinal disease. The presence of the original CPV-2 type (4%) and CPV-2a (9%), CPV-2b (18%), or CPV-2c (69%) variants was documented. Over the years, we observed a decrease in the frequency of CPV-2a/-2b and a rapid increase of CPV-2c frequency, with a progressive replacement of the European lineage of CPV-2c by the Asian lineage. The observed scenario, besides confirming epidemiological relevance of CPV-2, highlights the occurrence of antigenic variant shifts over time, with a trend toward the replacement of CPV-2a, CPV-2b, and the European lineage of CPV-2c by the emerging Asian CPV-2c lineage. The comparison with other Italian and international sequences suggests the occurrence of viral exchange with other Italian regions and different countries, although the directionality of such viral flows could not be often established with confidence. In several instances, potential CPV-2 introductions led to epidemiological dead ends. However, major, long-lasting clades were also identified, supporting successful infection establishment, local spreading, and evolution. These results, besides demonstrating the need for implementing more effective control measures to prevent viral introductions and minimize circulation, stress the relevance of routine monitoring activities as the only tool to effectively understand CPV-2 epidemiology and evolution, and develop adequate countermeasures.

Biomolecular Analysis of Canine Distemper Virus Strains in Two Domestic Ferrets (Mustela putorius furo).

Canine distemper is a contagious and severe systemic viral disease that affects domestic and wild carnivores worldwide. In this study, two adult female ferrets (Mustela putorius furo) were evaluated for cutaneous lesions. Scab, fur, and swab samples from the external auditory canal, cutaneous lesions, and scrapings were analyzed. Canine distemper virus (CDV)-positive samples underwent RT-PCR/RFLP with the restriction enzyme PsiI, and the hemagglutinin gene sequence was obtained. According to the restriction enzyme and sequence analyses, the viral strains were typed as CDV field strains that are included within the Europe lineage and distinct from those including vaccinal CDV strains. The sequence analysis showed the highest nucleotide identity rates in older Europe lineage CDV strains collected from dogs and a fox in Europe. This study is the first to report on CDV infection in ferrets in southern Italy and contributes to the current knowledge about natural CDV infection in this species. In conclusion, vaccination remains crucial for preventing the disease and counteracting cross-species infection. Molecular biology techniques can enable the monitoring of susceptible wild animals by ensuring the active surveillance of CDV spread.

Persistence of DNA from canine parvovirus modified-live virus in canine tissues.

Canine parvovirus (CPV-2) modified-live virus vaccine strain can replicate in lymphoid tissues and intestinal mucosa after administration, being shed through canine faeces. Detection of vaccine strains has been reported in the bloodstream and faeces, potentially interfering with molecular diagnostic tests. The persistence of these strains in canine tissues has not yet been described. With this aim, canine tissues were tested during a molecular survey to screen for the presence of canine enteric viruses. Tissue samples from 165 dead dogs were tested by a conventional PCR assay. Positive samples and five commercial vaccines were subjected to sequence analysis. Vaccinal strains were detected and virus load was measured by using a set of real-time PCR assays using minor-groove binder (MGB) probes. Seventy-five dogs (45.4%) tested positive for CPV-2. Strains from 70 dogs were characterised as field variants. The presence of CPV sequences of vaccine origin was observed in the spleen, intestine, and mesenteric lymph nodes of five young dogs. Vaccinal strains were detected from 12 to 24 days after the last vaccine administration. Viral loads comprised between 6.3 × 102 and 9.95 × 104 DNA copies/10 µl of template. This study confirms that CPV vaccinal strains can be detected in canine tissues after vaccination, so post-mortem diagnosis of CPV infection needs further molecular analyses to assess the viral type (vaccine or field strains). The present study updates the current information on the persistence of CPV vaccine strains in canine tissues and their possible interference with molecular assays.

Heavy Metal Levels in Milk and Serum of Dairy Cows from Different Farms Located near an Industrial Area.

Background: Heavy metals are toxic, non-biodegradable substances able to enter the food chain of cows and then transfer to their milk. This study investigated the relationship between the heavy metal concentrations in serum and milk and the environmental pollutants exposure at two different farms in Ragusa, Italy. Methods: To evaluate the concentrations of aluminum (Al), chromium (Cr), iron (Fe), copper (Cu), zinc (Zn), arsenic (As), cadmium (Cd), and lead (Pb), milk and serum samples were collected from 40 Friesian dairy cows from farm 1 at about 3.7 km from an industrial area (group 1) and 40 Friesian dairy cows from farm 2 at about 400 mt from a greenhouse and 6.0 km from a chemical fertilizer factory (group 2). Results: The concentrations of heavy metals measured in serum and milk showed no statistically significant differences between group 1 and group 2. No significant correlation between heavy metals in serum and milk in group 1 was observed. A positive correlation between Zn concentrations measured in milk and serum samples was observed in group 2 (r = 0.35, p = 0.03). Conclusions: The determination of heavy metals in serum and milk can be an important tool to detect the exposure to environmental pollutants and in monitoring the hygienic state of the environment in which milk is produced.

Identification and Molecular Characterization of a Divergent Asian-like Canine Parvovirus Type 2b (CPV-2b) Strain in Southern Italy.

Canine parvovirus type 2 (CPV-2) is an infectious agent relevant to domestic and wild carnivorans. Recent studies documented the introduction and spread of CPV-2c strains of Asian origin in the Italian canine population. We investigated tissue samples from a puppy collected during necropsy for the presence of viral enteropathogens and all samples tested positive only for CPV-2. The full coding sequence of a CPV-2b (VP2 426Asp) strain was obtained. This virus was related to CPV-2c strains of Asian origin and unrelated to European CPV-2b strains. The sequence had genetic signatures typical of Asian strains (NS1: 60Val, 545Val, 630Pro; VP2: 5Gly, 267Tyr, 324Ile) and mutations rarely reported in Asian CPV-2b strains (NS1: 588N; VP2: 370Arg). Phylogenetic analyses placed this strain in well-supported clades, including Asian CPV-2c-like strains, but always as a basal, single-sequence long branch. No recombination was observed for this strain, and we speculate that it could have originated from an Asian CPV-2c-like strain that acquired the 426Asp mutation. This study reports the first evidence of an Asian-like CPV-2b strain in Italy, confirming the occurrence of continuous changes in the global CPV-2 spread. Since positive convergent mutations complicate data interpretation, a combination of phylogenetic and mutation pattern analyses is crucial in studying the origin and evolution of CPV-2 strains.

Immune and Inflammatory Response in Horse Vaccinated Against Equine Herpesviruses 1 (EHV-1) and 4 (EHV-4) Assessed by Serum Protein Electrophoretic Pattern and Leukocyte Population.

Protection against infectious diseases can be obtained with vaccines generating immunogenic response through a combination of humoral and cellular immunity. In this study haematological and serum protein electrophoretic profiles of horses vaccinated against herpesvirus 1 (EHV-1) and 4 (EHV-4) were evaluated. Blood samples were collected from 16 horses before (T0), after 24h, 48h, 72h, 1st week, 2nd week and 3rd week (T1I, T2I, T3I, T7I, T14I and T21I) from the first EHV vaccine-dose administration as well as before (TPREII), and after 24h, 48h, 72h, 1st week, 2nd week, 3rd week and 4th week (T1II, T2II, T3II, T7II, T14II, T21II and T28II) from the EHV vaccine-booster. Total leukocyte values increased at T1I, T1II, T3II and T28II compared to T0 (P < .01). Higher lymphocytes and lower neutrophils values were found after first vaccine-dose and vaccine-booster administration compared to the T0 (P < .01). Monocytes showed higher values at T14II than T0 (P < .01). Higher serum values of total proteins, α1-, α-2-, ß1-, ß2- and γ-globulins were found in horses after first vaccine-dose and vaccine-booster administration (P < .01). Gathered results suggest that horses vaccinated against EHV1 and EHV-4 exhibited a dynamic change of WBC, lymphocytes, neutrophils and monocytes. Moreover, the analysis of serum electrophoresis pattern suggested that EHV vaccination induced the development of inflammation and antibody response in vaccinated horses as highlighted by the increase of α-, ß- and γ-globulin fractions. These changes probably reflect the systemic immunological adaptation of animals to EHV vaccine.

First neuroinvasive human case of West Nile Disease in Southern Italy: Results of the 'One Health' approach.

BACKGROUND: West Nile Disease (WND) is a zoonotic mosquito-borne infection involving viral pathogens, human and animal hosts, vectors and environment. Cooperation among medical, veterinary and entomological fields has been promoted by the Italian Public Health Authorities, and an integrated West Nile Virus (WNV) Surveillance Plan has been in force in Italy since 2016 to prevent the transmission risk of WND to humans through an early detection of viral circulation by animal and entomological surveillance. This managing model is unique in Europe. OBJECTIVES: This survey aimed at presenting the 'One Health' approach applied in 2016 to the first autochthonous human case of West Nile Neuroinvasive Disease (WNND) in Sicily (Southern Italy). METHODS: Serological (anti-WNV IgM and IgG ELISA, anti-WNV neutralizing antibodies) and molecular tests were conducted on blood, liquor and urine of a 38-year-old man with encephalitis and meningitis. Overall, 2704 adult culicides from 160 mosquito catches were morphologically identified. Female mosquitoes were analysed in pools for WNV RNA detection. Serological (anti-WNV IgM and IgG ELISA) and molecular analyses for WNV were carried out in 11 horses, 271 chickens and two dogs sampled in farms around the man's residence. RESULTS AND CONCLUSIONS: WNND was confirmed by serological analysis on patient's liquor and serum. Collected mosquito species included Culex pipiens (93.56%, CI95% 92.64%-94.49%), Aedes albopictus (5.25%, CI95% 4.41%-6.09%), Culex hortensis (0.59%, CI95% 0.30%-0.88%), Culiseta longiareolata (0.55%, CI95% 0.27%-0.83%) and Anopheles maculipennis s.l. (0.04%, CI95% -0.04% to 0.11%). Mosquito pools were negative for WNV RNA. Two dogs (100%) and two horses (18.18%, CI95% -4.61 to 40.97%) resulted positive for anti-WNV specific antibodies. The 'One Health' approach allowed to report the first human neuroinvasive WND in Sicily and to confirm the local circulation of WNV in animals of the same area where the clinical case occurred, defining the autochthonous origin of the infection.

Detection and Molecular Characterization of Two Gammaherpesviruses from Pantesco Breed Donkeys during an Outbreak of Mild Respiratory Disease.

Equid and asinine gammaherpesviruses (GHVs; genus Percavirus) are members of the Herpesviridae family. Though GHVs have been reported in horse populations, less studies are available on gammaherpesviral infections in donkeys. This study reports the co-infection with two GHVs in Pantesco breed donkeys, an endangered Italian donkey breed. Samples (n = 124) were collected on a breeding farm in Southern Italy from 40 donkeys, some of which were healthy or presented erosive tongue lesions and/or mild respiratory signs. Samples were analysed by using a set of nested PCRs targeting the DNA polymerase, glycoprotein B, and DNA-packaging protein genes, and sequence and phylogenetic analyses were performed. Twenty-nine donkeys (72.5%) tested positive, and the presence of Equid gammaherpesvirus 7 and asinine herpesvirus 5 was evidenced. In 11 animals, we found evidence for co-infection with viruses from the two species. Virions with herpesvirus-like morphology were observed by electron microscopic examination, and viruses were successfully isolated in RK-13-KY cell monolayers. The histological evaluation of tongue lesions revealed moderate lympho-granulocytic infiltrates and rare eosinophilic inclusions. The detection of GHVs in this endangered asinine breed suggests the need long-life monitoring within conservation programs and reinforces the need for further investigations of GHV's pathogenetic role in asinine species.