Claudio Caruso

Extended Genetic Diversity of Bovine Viral Diarrhea Virus and Frequency of Genotypes and Subtypes in Cattle in Italy between 1995 and 2013

Genetic typing of bovine viral diarrhea virus (BVDV) has distinguished BVDV-1 and BVDV-2 species and an emerging putative third species (HoBi-like virus), recently detected in southern Italy, signaling the occurrence of natural infection in Europe. Recognizing the need to update the data on BVDV genetic variability in Italy for mounting local and European alerts, a wide collection of 5′ UTR sequences (n = 371) was selected to identify the frequency of genotypes and subtypes at the herd level. BVDV-1 had the highest frequency, followed by sporadic BVDV-2. No novel HoBi-like viruses were identified. Four distribution patterns of BVDV-1 subtypes were observed: highly prevalent subtypes with a wide temporal-spatial distribution (1b and 1e), low prevalent subtypes with a widespread geographic distribution (1a, 1d, 1g, 1h, and 1k) or a restricted geographic distribution (1f), and sporadic subtypes detected only in single herds (1c, 1j, and 1l). BVDV-1c, k, and l are reported for the first time in Italy. A unique genetic variant was detected in the majority of herds, but cocirculation of genetic variants was also observed. Northern Italy ranked first for BVDV introduction, prevalence, and dispersion. Nevertheless, the presence of sporadic variants in other restricted areas suggests the risk of different routes of BVDV introduction.

A new genotype of border disease virus with implications for molecular diagnostics

Border disease virus (BDV) is a (+) single-stranded RNA pestivirus affecting mainly sheep and goats worldwide. Genetic typing of BDV has led to the identification of at least seven major genotypes. This study reports the detection of a BDV strain from a goat in northwestern Italy during routine investigations. Sequence analysis revealed mutations in the 5’-UTR of the virus with implications for BDV molecular diagnostics. Moreover, subsequent phylogenetic analysis based on the combined 5’-UTR and Npro/partial C genes, showed divergence from known BDV genotypes, revealing the detection of a novel pestivirus group, for which we propose the name BDV genotype 8.

Potential Pathogenetic Role of Bovine Herpesvirus 4 in Two Dairy Cows with Dermatitis-Pyrexia-Hemorrhagic Syndrome

ABSTRACT Dermatitis, pyrexia, and hemorrhagic syndrome (DPHS) is a rare bovine syndrome of unclear etiology. We describe two DPHS cases, the first to occur in Italy, with clinicopathological findings suggesting a potential pathogenetic role of bovine herpesvirus-4 (BoHV-4).

Evidence of circulation of the novel border disease virus genotype 8 in chamois

Evidence of association between the novel putative border disease virus genotype 8 (BDV-8) and fatal disease in an Alpine chamois (Rupicapra rupicapra) is reported. Diagnostically, we also demonstrated, as already previously reported, the failure of BDV-specific primers (PDB1 and PDB2) to detect BDV-8.

Surveillance of Infectious Bovine Rhinotracheitis in marker-vaccinated dairy herds: Application of a recombinant gE ELISA on bulk milk samples

Infectious Bovine Rhinotracheitis (IBR) occurs worldwide, requiring significant resources for eradication programs or surveillance purposes. The status of infection is usually detected by serological methods using the virus neutralization test (VNT) or enzyme-linked immunosorbent assay (ELISA) on individual sera. The gE DIVA (Differentiating Infected from Vaccinated Animals) vaccines approach, adopted in order to reduce the virus circulation and prevent clinical signs, have tightened the range of available methods for the serological diagnosis. Different gE blocking ELISA could be performed to detect specific antibodies in sera of infected or whole virus-vaccinated animals but with less sensitivity if applied to bulk milk samples, especially in marker-vaccinated herds. A new rec-gE ELISA was recently developed in Italy and applied with good performances on blood serum samples. The present paper focuses on the application of a rapid protocol for purification/concentration of immunoglobulin G (IgG) from bulk milk and on the use of the new rec-gE indirect ELISA. The study involved three different partners and 225 herds (12,800 lactating cows) with different official IBR diagnostic statuses. The diagnostic specificity of the method was demonstrated closed to 100% while the diagnostic sensitivity was strictly related to the herd-seroprevalence. Considering 2.5% as the limit of detection of within-herd seropositivity prevalence, the diagnostic sensitivity showed by the proposed method was equal to 100%. A single reactivation of a whole strain vaccine in an old cow was detected inside a group of 67 lactating cows, showing the field applicability of the method.

First report of malignant catarrhal fever in a captive pudu (Pudu puda).

A male, six-year-old pudu (Pudu puda) from an Italian zoo was submitted for postmortem examination after sudden death. Necroscopy revealed non-suppurative bronchopneumonia and degeneration of the liver and haemorrhagic lesions of the thymus, pericardium and spleen. Microscopically, multifocal perivascular mononuclear cell infiltrates were observed in the kidneys, lungs, spleen, and the portal triads of the liver. Histological examination of the brain showed meningitis, vasculitis and perivascular cuffs of mononuclear inflammatory cells. A region of the DNA polymerase gene of malignant catarrhal fever viruses was amplified by real-time PCR and nested PCR. PCR products from the tissue samples were sequenced and analysed. The sequences showed 99% similarity with a portion of the caprine herpesvirus 2 DNA polymerase gene. This is the first report of malignant catarrhal fever in a captive pudu.

Aujeszky's Disease in Red Fox (Vulpes vulpes): Phylogenetic Analysis Unravels an Unexpected Epidemiologic Link

Abstract We describe Aujeszkys disease in a female of red fox (Vulpes vulpes). Although wild boar (Sus scrofa) would be the expected source of infection, phylogenetic analysis suggested a domestic rather than a wild source of virus, underscoring the importance of biosecurity measures in pig farms to prevent contact with wild animals.

Efficient isolation on Vero.DogSLAMtag cells and full genome characterization of Dolphin Morbillivirus (DMV) by next generation sequencing

The Dolphin Morbillivirus (DMV) genome from the first Mediterranean epidemic (1990-’92) is the only cetacean Morbillivirus that has been completely sequenced. Here, we report the first application of next generation sequencing (NGS) to morbillivirus infection of aquatic mammals. A viral isolate, representative of the 2006-’08 Mediterranean epidemic (DMV_IZSPLV_2008), efficiently grew on Vero.DogSLAMtag cells and was submitted to whole genome characterization by NGS. The final genome length was 15,673 nucleotides, covering 99.82% of the DMV reference genome. Comparison of DMV_IZSPLV_2008 and 1990-’92 DMV strain sequences revealed 157 nucleotide mutations and 47 amino acid changes. The sequence similarity was 98.7% at the full genome level. Whole-genome phylogeny suggested that the DMV strain circulating during the 2006-’08 epidemics emerged from the 1990-’92 DMV strain. Viral isolation is considered the “gold standard” for morbillivirus diagnostics but efficient propagation of infectious virus is difficult to achieve. The successful cell replication of this strain allowed performing NGS directly from the viral RNA, without prior PCR amplification. We therefore provide to the scientific community a second DMV genome, representative of another major outbreak. Interestingly, genome comparison revealed that the neglected L gene encompasses 74% of the genetic diversity and might serve as “hypervariable” target for strain characterization.

A comparative study on subacute toxicity of arsenic trioxide and dimethylarsinic acid on antioxidants and antioxidant-related enzymes in crandell rees feline kidney (CRFK), human hepatocellular carcinoma (PCL/PRF/5) and epithelioma papulosum cyprini (EPC) cell lines

ABSTRACT Arsenic (As) is a global contaminant of terrestrial and aquatic environments posing concern for environmental and human health. The effects of subacute concentrations of arsenic trioxide (AsIII) and dimethylarsinic acid (DMAV) were examined using Crandell Rees feline kidney (CRFK), human hepatocellular carcinoma (PLC/PRF/5), and epithelioma papulosum cyprini (EPC). Whole monolayer with suffering cells (confluence 100%, pyknosis and refractive cells; value scale = 2) led to identification of subacute As concentrations for the three cell lines. The selected AsIII concentrations were 1.33 µM for CRFK and 33.37 µM for PLC/PRF/5 and EPC, at 48 hr time point. The selected DMAV concentrations were 0.67 mM for PLC/PRF/5, 1.33 mM for CRFK, and 2.67 mM for EPC for 48 hr. Unlike the AsIII test, the three cell lines did not exhibit marked susceptibility to DMAV-mediated toxicity. Several oxidative stress biomarker levels, directly or indirectly associated with reactive oxygen species (ROS) elimination including superoxide dismutase, catalase, glutathione peroxidases, glutathione reductase, glutathione S-transferase, glyoxalase I, glyoxalase II, and total glutathione, were determined in the three cell lines at 24 and 48 hr. Antioxidant responses in metal-treated cells were significantly altered compared to controls, suggesting a perturbation of redox state. The weakening of antioxidant pathway in either healthy or tumoral cells was greater using AsIII than DMAV. Differences in level of several oxidative stress biomarkers suggest that the oxidative stress mechanism induced by AsIII is distinctly different from DMAV. Multifaceted mechanisms of action underlying ROS generation in tumor and nontumor cells versus AsIII and DMAV exposure are thus involved. Since As-mediated toxicity is quite complex, more data regarding both oxidant-enhancement and oxidant-lowering strategies may be useful to improve knowledge regarding the influence of As on human and animal cells.

Microbial agents in macroscopically healthy mammary gland tissues of small ruminants

Background Health of mammary glands is fundamental for milk and dairy products hygiene and quality, with huge impacts on consumers welfare. Methods This study aims to investigate the microbial agents (bacteria, fungi and lentiviruses) isolated from 89 macroscopically healthy udders of regularly slaughtered small ruminants (41 sheep, 48 goats), also correlating their presence with the histological findings. Multinomial logistic regression was applied to evaluate the association between lesions and positivity for different microbial isolates, animal age and bacteria. Results Twenty-five samples were microbiologically negative; 138 different bacteria were isolated in 64 positive udders. Coagulase-negative staphylococci were the most prevalent bacteria isolated (46.42%), followed by environmental opportunists (34.76%), others (10.14%) and pathogens (8.68%). Most mammary glands showed coinfections (75%). Lentiviruses were detected in 39.3% of samples. Histologically, chronic non-suppurative mastitis was observed in 45/89 glands, followed by chronic mixed mastitis (12/89) and acute suppurative mastitis (4/89). Only 28 udders were normal. Histological lesions were significantly associated with the animal species and lentiviruses and coagulase-negative staphylococci infections. Goats had significantly higher risk to show chronic mixed mastitis compared to sheep. Goats showed a significantly lower risk (OR = 0.26; 95% CI [0.06–0.71]) of being infected by environmental opportunists compared to sheep, but higher risk (OR = 10.87; 95% CI [3.69–37.77]) of being infected with lentiviruses. Discussion The results of the present study suggest that macroscopically healthy glands of small ruminants could act as a reservoir of microbial agents for susceptible animals, representing a potential risk factor for the widespread of acute or chronic infection in the flock.