Serological and virological survey of hepatitis E virus in wild boar populations in northwestern Italy: detection of HEV subtypes 3e and 3f.

Although rare in developed countries, most acquired human cases of hepatitis E virus (HEV) infection are associated with travel to developing countries where HEV is endemic. Increasingly, however, sporadic, non-travel-related HEV cases have been reported in developed countries. In Italy, only two studies to date have investigated the presence of HEV in wild boars. Here, we report a serological and virological survey of HEV in wild boar populations in northwestern Italy. During the hunting season, 594 serum and 320 liver samples were collected and screened for antibodies to HEV and HEV RNA. Overall, the seroprevalence was 4.9 %, and HEV RNA was detected in 12 liver samples (p = 3.7 %). No serum samples tested positive for HEV RNA. Phylogenetic analysis of the ORF2 region revealed that the isolates clustered within genotype 3, subtypes 3e and 3f, and were closely related to HEV strains previously detected in domestic pigs farmed in the same geographic area. Although the routes of viral transmission are still poorly understood, our data show that HEV genotypes 3e and 3f circulate in wild boars in northwestern Italy. Also, they provide evidence that autochthonous HEV infections in Italy could also be linked to wild boar populations, suggesting an increased risk for domestically acquired HEV infection in humans through wild animals. The HEV sequences determined in this study may be useful for comparing present and future human isolates to identify transmission events between wild boar, humans, and farmed pigs. Similarly to other more commonly known zoonotic agents, HEV should be included in national or regional disease surveillance programs for wild animals.

Association of a specific major histocompatibility complex class IIß single nucleotide polymorphism with resistance to lactococcosis in rainbow trout, Oncorhynchus mykiss (Walbaum).

Major histocompatibility complex (MHC) loci encode glycoproteins that bind to foreign peptides and initiate immune responses through their interaction with T cells. MHC class II molecules are heterodimers consisting of α and ß chains encoded by extremely variable genes; variation in exon 2 is responsible for the majority of observed polymorphisms, mostly concentrated in the codons specifying the peptide-binding region. Lactococcus garvieae is the causative agent of lactococcosis, a warm-water bacterial infection pathogenic for cultured freshwater and marine fish. It causes considerable economic losses, limiting the profitability and development of fish industries in general and the intensive production of rainbow trout, Oncorhynchus mykiss (Walbaum), in particular. The disease is currently controlled with vaccines and antibiotics; however, vaccines have short-term efficacy, and increasing concerns regarding antibiotic residues have called for alternative strategies. To explore the involvement of the MHC class II ß-1 domain as a candidate gene for resistance to lactococcosis, we exposed 400 rainbow trout to naturally contaminated water. One single nucleotide polymorphism (SNP) and one haplotype were associated with resistance (P < 0.01). These results are promising for using MHC class IIß as a molecular marker in breeding rainbow trout resistant to lactococcosis.

Evaluation of internal reference genes for quantitative expression analysis by real-time reverse transcription-PCR in somatic cells from goat milk.

Reverse transcription (RT) quantitative real-time PCR (qPCR) is the most accurate and easy-to-perform technique to measure the expression level of a selected gene of interest by quantifying mRNA transcripts. The use of reference genes is commonly accepted as the most reliable approach to normalize RT-qPCR data and reduce possible errors generated in the quantification of gene expression. The optimal number and choice of reference genes are experimentally validated for specific tissues or cell types and experimental designs. To date, data on qPCR normalization in goats are scarce and the most suitable reference genes in this species have been identified for only a limited number of tissues. The aim of this study was to determine an optimal combination of stably expressed reference genes in caprine milk somatic cells (MSC) from healthy and infected mammary glands. For the purpose, we performed RT-qPCR for 10 commonly used reference genes from various functional classes and then determined their expression level in MSC from goats intramammary challenged with Staphylococcus aureus and in MSC from healthy controls, with a view to select genes whose stability would be unaffected under infection conditions. The geNorm and NormFinder algorithms were used for validating the reference genes. Furthermore, to demonstrate the importance of normalization of gene expression with appropriate reference genes, we tested the effect of using a combination of the least stable genes for expression analysis evaluation. On the basis of our evaluation, we recommend the use of a panel of reference genes that should include G6PD, YWHAZ, and ACTB for caprine MSC gene expression profiling. The expression of the 2 genes of interest, pentraxin-related protein (PTX3) and secreted phosphoprotein 1 (SPP1), was evaluated by RT-qPCR in all samples collected pre- and postinfection, and the recommended reference genes were used to normalize the data. Our study provides a validated panel of optimal reference genes for the identification of genes differentially expressed by qRT-PCR in caprine MSC. Moreover, we provided a set of intron-spanning primer sequences that could be suitable for gene expression experiments using SYBR Green chemistry on other caprine tissues and cells.

First report of Salmonella 1,4,[5],12:i:- in free-ranging striped dolphins (Stenella coeruleoalba), Italy.

Between 2015 and the beginning of 2018 (January-March), 30 cetaceans were found stranded along the Ligurian Sea coast of Italy. Necropsies were performed in 22 cases and infectious diseases resulted the most common cause of death. Three striped dolphins, showed a severe coinfection involving the monophasic variant of Salmonella Typhimurium (Salmonella 1,4,[5],12:i:-). The isolates were characterized based on antimicrobial resistance, Multiple-Locus Variable-number tandem-repeat Analysis (MLVA) and whole-genome sequencing (WGS). All isolates demonstrated the same multidrug resistant genotype (ASSuT isolates), showed three different MLVA profiles, two of which closely related, and were identified as Sequence Type 34. Moreover, Single nucleotide polymorphisms (SNP) analysis confirmed strong correlations between two out of the three isolates. To our knowledge, S. 1,4,[5],12:i:-, one of the most common serovars in cases of human infection and food sources worldwide, has not previously been described in marine mammals, and reports of Salmonella-associated disease in free-ranging cetaceans are rare. These results highlight the role of cetaceans as sentinel species for zoonotic and terrestrial pathogens in the marine environment, suggest a potential risk for cetaceans and public health along the North Western Italian coastline and indicate cetaceans as a novel potential reservoir for one of the most widespread Salmonella serovars.

Genetic variability of the PRNP gene in goat breeds from Northern and Southern Italy.

AIMS: To determine the variability of the prion protein gene (PRNP) in goats from Northern and Southern Italy. METHODS AND RESULTS: Genomic DNA isolated from goat blood was polymerase chain reaction (PCR)-amplified for the coding region of the PRNP gene and then sequenced. In total, 13 polymorphic sites were identified: G37V, T110P, G127S, M137I, I142M, I142T, H143R, R154H, P168Q, T194P, R211Q, Q222K and S240P (substitutions I142T and T194P are novel) giving rise to 14 haplotypes. Clear frequency differences between Northern and Southern breeds were found and confirmed by genetic distance analysis. CONCLUSIONS: Differences in allele distribution were found between Northern and Southern goats, in particular regarding the M142 and K222 alleles, possibly associated to scrapie resistance; philogeographical analysis supported the idea that Northern and Southern breeds may be considered as separate clusters. SIGNIFICANCE AND IMPACT OF THE STUDY: In Italy only limited studies have been carried out on caprine PRNP genotype distribution; this study is important to fill this lack of information. Moreover the finding of significant differences among allele distributions in Northern and Southern goats, especially if involved in modulating resistance/susceptibility, need to be carefully considered for the feasibility of selection plans for resistance to scrapie.

Development and application of an enzyme-linked immunosorbent assay for detection of bovine viral diarrhea antibody based on Erns glycoprotein expressed in a baculovirus system.

The BVDV envelope glycoprotein E(rns)/gp48 and the C terminal 79 amino acids of the capsid protein coding region were expressed in a baculovirus system and antigenically characterized. Western blot assay was used to detect recombinant E(rns) (r-E(rns)) in infected insect cells using specific monoclonal antibodies. The r-E(rns) was then used in an indirect ELISA to detect BVDV specific antibodies in a panel of 540 well-characterized sera. Results of the r-E(rns) ELISA were compared to those obtained with a commercially available competitive ELISA targeting anti-NS2/3 antibodies. A good correlation was observed between the 2 ELISA (kappa = 0.916, 95% C.I.: 0.876, 0.956). Using the commercial NS2/3 ELISA as the reference test, the relative sensitivity of r-E(rns) ELISA was 97.5% (95% C.I.: 94.3%, 99.1%) and the relative specificity was 93.9% (95% C.I.: 89.4%, 96.9%), while relative specificity was 100% (95% C.I.: 97%, 100%) using true negative sera (derived from a negative herd). All but 1 antigen positive animals (n = 36) tested negative in the r-E(rns) ELISA; among them all 22 confirmed PI animals were negative by r-E(rns) ELISA. The ability of r-E(rns) ELISA to identify cattle immunized with inactivated vaccine was also demonstrated in a small group of cattle, compared to an NS2/3 antibody ELISA. Results suggest that r-E(rns) ELISA represents an alternative test for antibody generated by natural infection or BVDV vaccination.

Hepatitis E Virus: A Cross-Sectional Serological and Virological Study in Pigs and Humans at Zoonotic Risk within a High-Density Pig Farming Area.

An increase in autochthonous hepatitis E virus (HEV) infections has been recorded in Italy suspected to be zoonotically transmitted from pigs; this study was carried out to determinate the seroprevalence and risk factors associated with hepatitis HEV exposition, both in swine and humans working in pig farms, located within a high-density pig farming area in Piedmont region, north-western Italy. The presence of viral RNA in human and swine samples was also evaluated, and phylogenetic analysis was performed on HEV-positive samples. Forty-two swine farms were sampled; 142 workers were enrolled in the study and classified into two groups: (i) 69 workers with occupational contact with swine (including veterinarians and farmers) recruited in the 42 sampled farms; (ii) 73 without occupational contact with swine. Forty-one of 42 (97%) swine farms resulted positive to enzyme-linked immunosorbent assay test for HEV antibodies (Abs). Overall seroprevalence in swine was 50% (441/879), with seropositivity rate higher in sows (333/469, 71%). HEV RNA in stool samples was detected in animals from 13 of 42 tested farms (31%), and a higher positivity resulted in weaners (40/246, 16.3%). Phylogenetic analysis classified all HEV isolates within genotype 3 (subtypes 3f, 3e, 3c). All humans were negative for HEV viral genome in blood. Five of 142 sera were positive for IgG anti-HEV with an overall prevalence of 3.52% with no statistically significant differences in prevalence rates between workers at zoonotic risk and the control group (5.7% versus 1.3%). In contrast, a significant difference (OR 10.1) was observed within the subgroup including subjects exposed for short periods (veterinarians) compared with those who worked for long periods (farmers) suggesting a correlation between the time of exposure and the likelihood of HEV infection. Reporting HEV infection is not mandatory in Italy, but a constant epidemiological surveillance should be ensured to clarify the epidemiology of this disease.

Hepatitis E Virus: First Description in a Pet House Rabbit. A New Transmission Route for Human?

In this work, we identified for the first time hepatitis E virus (HEV) in a pet house rabbit, an adult 7 years old female of domestic rabbit (Oryctolagus cuniculus). Importantly, the resulting phylogenetic tree showed that the HEV strain identified in the pet house rabbit was closely related to a human HEV sequence; this finding reawakens concerns regarding the zoonotic risk represented by HEV in animals and expands to house rabbit the spectrum of potential source of infection for humans. Potential for domestic transmission of HEV to humans should be taken into account.

Detection of Invasive Borrelia burgdorferi Strains in North-Eastern Piedmont, Italy.

Following reports of human cases of Lyme borreliosis from the Ossola Valley, a mountainous area of Piemonte, north-western Italy, the abundance and altitudinal distribution of ticks, and infection of these vectors with Borrelia burgdorferi sensu lato were evaluated. A total of 1662 host-seeking Ixodes ricinus were collected by dragging from April to September 2011 at locations between 400 and 1450 m above sea level. Additional 104 I. ricinus were collected from 35 hunted wild animals (4 chamois, 8 roe deer, 23 red deer). Tick density, expressed as the number of ticks per 100 m(2), resulted highly variable among different areas, ranging from 0 to 105 larvae and from 0 to 22 nymphs. A sample of 352 ticks (327 from dragging and 25 from wild animals) was screened by a PCR assay targeting a fragment of the 16S rRNA gene of B. burgdorferi s.l. Positive samples were confirmed with a PCR assay specific for the 5S-23S rRNA intergenic spacer region and sequenced. Four genospecies were found: B. afzelii (prevalence 4.0%), B. lusitaniae (4.0%), B. garinii (1.5%) and B. valaisiana (0.3%). Phylogenetic analysis based on the ospC gene showed that most of the Borrelia strains from pathogenic genospecies had the potential for human infection and for invasion of secondary body sites.

Detection of border disease virus (BDV) genotype 3 in Italian goat herds.

In January 2011, cases of abortion, stillbirth and weak live kids were reported in two goat herds in northern Italy. Samples from 18 kids found dead, 12 fetuses, and two stillborn kids were analyzed for pestivirus antigen using an ELISA kit and a border disease virus (BDV)-specific RT-PCR. Positive results were obtained in six kids and one fetus. Phylogenetic analysis based on 225 bp of the 5'UTR fragment of the BDV genome from positive samples showed that the goats were infected with BDV genotype 3. Serum and blood samples collected from all animals in both herds were analyzed using competitive ELISA to detect p80 antibodies and RT-PCR to detect viraemia. Pestivirus antibodies were detected in 61/67 goats in herd A and in 38/169 in herd B. A persistently infected (PI) goat was found in herd A. The PI animal was submitted to the laboratory for BDV diagnosis with Ag-ELISA, viral isolation, and nested RT-PCR on tissue samples from the spleen, kidney, brain, liver, lung, ileocaecal valve, mesenteric lymph nodes, and skin. All of the tests were positive for BDV in each of the tissues analyzed. The BDV sequence of the PI was identical to BDV sequences found in other positive animals. This is the first description of a BDV PI goat and the first evidence of BDV genotype 3 circulation in Italy. The study raises questions about the real impact this virus has on breeding goats.

Molecular characterization of Pseudomonas fluorescens isolates involved in the Italian "blue mozzarella" event.

Between June and September 2010, widespread Italian consumer reports of unusual blue spoilage on fresh dairy products were publicized, resulting in the so-called blue mozzarella event. An inordinately high number of samples from mozzarella and whey cheese products of Italian and German production subsequently tested positive for Pseudomonas fluorescens. The aim of this study was to verify whether a selected P. fluorescens strain was responsible for this apparently unusual event. Molecular characterization of 181 isolated P. fluorescens strains was conducted using a newly optimized pulsed-field gel electrophoresis protocol. Although a high number of pulsotypes was found (132), only four pulsotypes were associated with more than one production plant, and only one German isolate had the same pulsotype as was detected in two Italian plants. This is the only evidence of possible cross-contamination among cheeses from the two countries. The overall results did not support the spread of contamination from German to Italian plants or the presence of one environmental strain that spread in both countries.

Co-existence of classical scrapie and Nor98 in a sheep from an Italian outbreak.

Nor98 is an atypical scrapie strain characterized by a molecular pattern and brain distribution of the pathological prion protein (PrP(Sc)) different from classical scrapie. In Italy, 69 atypical cases have been identified so far and all were characterized as Nor98 strain. In this paper we report an unusual case in a sheep which showed immunohistochemical and molecular features of PrP(Sc) different from the other atypical cases. The sheep was from an outbreak where the index and the other four cases were affected by classical scrapie. Histopathological, immunohistochemical and Western blot analyses on the brain of the unusual case revealed the simultaneous presence of pathological features characteristic of Nor98 and classical scrapie. Interestingly, the prevalent disease phenotype in the brainstem was classical scrapie-like, while in the cerebral cortex and cerebellum the Nor98 phenotype was dominant. The sub-mandibular lymph node was positive and showed a PrP(Sc) molecular pattern referable to classical scrapie. The PrP genotype was AL(141)RQ/AF(141)RQ. Taken together, the occurrence of classical scrapie in the outbreak, the PrP genotype, the involvement of different cellular targets in the brain and the pathological and molecular PrP(Sc) features observed suggest that this unusual case may result from the co-existence of Nor98 and classical scrapie.

Nocardia otitidiscaviarum pneumonia in an Alpine chamois (Rupicapra rupicapra rupicapra).

Nocardia otitidiscaviarum was cultured from the lung of an Alpine chamois (Rupicapra rupicapra rupicapra) with suppurative bronchopneumonia. This is the first report of both nocardiosis and Nocardia otitidiscaviarum in this wild ungulate species.

A case of scrapie in a sheep carrying the lysine-171 allele of the prion protein gene.

Susceptibility to scrapie in sheep depends on the host PrP genotype. No data about the linkage of the rare ARK allele to differential scrapie susceptibility are currently available. Several tissues isolated from sheep from an Italian scrapie outbreak and carrying the ARK allele were examined for the presence of the pathological prion protein. A weak positivity was detected only by Western blot in the brainstem of one ARK/ARH sheep. This result shows that the ARK allele does not confer full resistance against scrapie and that the allele needs to be studied further before it can be considered for breeding purposes.

Identification of prion protein gene polymorphisms in goats from Italian scrapie outbreaks.

Susceptibility to scrapie in sheep is influenced by polymorphisms of the prion protein (PrP) gene, whereas no strong association between genetics and scrapie has yet been determined in goats due to the limited number of studies on these animals. In this case-control study on 177 goats from six Italian scrapie outbreaks, the association between PrP alleles and the occurrence of scrapie was studied. Three silent mutations and 11 PrP polymorphisms were identified, of which two polymorphisms (L133Q and M137I) and one silent mutation (T202T) have not been reported previously. Twelve alleles were determined by cloning. Statistical analysis suggested a possible protective role against scrapie for the glutamine to lysine mutation at codon 222.