Andrea Caprioli

Widespread Diffusion of Genotype 3 Hepatitis E Virus among Farming Swine in Northern Italy

Hepatitis E virus (HEV) causes acute hepatitis in humans, and infects several animal species, mostly asymptomatically. Swine and human HEV strains are genetically related suggesting both a zoonotic and a possible foodborne transmission. The prevalence of swine HEV was investigated in 274 randomly selected pigs from six different swine farms of Northern Italy, testing viral RNA in stools by nested reverse-transcription-polymerase chain reaction. HEV genome was detected in 115 stools (42%). All farms resulted positive for HEV, with a prevalence ranging between 12.8% and 72.5%. HEV-positive pigs were detected in all age groups and production stages tested, although infection was more prevalent in weaners than in the older fatteners (42.2% vs. 27.0%). Genetic characterization of swine strains identified was performed by sequencing and database alignment. Phylogenetic analysis on the nucleotide sequences from 16 positive PCR products indicated that all strains belonged to genotype 3. In particular, one group of seven Italian strains clustered close (91.6-96.2% identity) to human and swine European HEV strains.

Porcine Circovirus 2 Replication in Colostrum-deprived Piglets Following Experimental Infection and Immune Stimulation Using A Modified Live Vaccine against Porcine Respiratory and Reproductive Syndrome Virus

Porcine circovirus type 2 (PCV2) infection is now recognized as the major factor in the development of post‐weaning multisystemic wasting syndrome (PMWS). Although Koch’s postulates have been fulfilled for PCV2 and PMWS, the severe clinical expression of the disease observed in field cases has been difficult to reproduce experimentally. Some studies have demonstrated that immune stimulation associated with the use of some commercially available swine vaccines may trigger progression of PCV2 infection to disease and lesions characteristic of PMWS. Here we describe the effects on PCV2 infection in an experimental model following the use of a commercially available modified live vaccine to porcine respiratory and reproductive syndrome virus (PRRSV). Although none of the piglets infected with PCV2 developed clinical PMWS, the severity of microscopical lesions and the PCV2 antigen load associated with these lesions were higher in the PRRSV‐vaccinated piglets compared with those detected in the PCV2 only infected animals.

Is a diagnostic system based exclusively on agar gel immunodiffusion adequate for controlling the spread of equine infectious anaemia

To improve the efficiency of the National equine infectious anaemia (EIA) surveillance program in Italy, a three-tiered diagnostic system has been adopted. This procedure involves initial screening by ELISA (Tier 1) with test-positive samples confirmed by the agar gel immunodiffusion test (AGIDT) (Tier 2) and, in the case of ELISA positive/AGIDT negative results, final determination by immunoblot (IB) (Tier 3). During this evaluation, 74,880 samples, principally collected from two Regions of Central Italy (Latium and Abruzzo) were examined, with 44 identified as negative in AGIDT but positive in both ELISA and IB. As the majority of these reactions occurred in mules, an observational study was conducted in this hybrid equid species to investigate if there is a correlation between plasma-associated viral loads and serological reactivity, to test the hypothesis that false-negative or very weak positive AGIDT results are associated with elite control of EIA virus (EIAV) replication accompanied by reduced transmission risks. The study animals consisted of 5 mules with positive AGIDT readings, along with another 5 giving negative or very weak positive results in this test. All mules were seropositive in Elisa and IB. Samples were collected routinely during an initial 56-day observation period, prior to dexamethasone treatment lasting 10 days, to determine the effect of immune suppression (IS) on clinical, humoral and virological responses. All mules were monitored for a further 28 days from day 0 of IS. None of the animals experienced relevant clinical responses before IS and there were no significant changes in antibody levels in ELISA, IB or AGIDT. However, plasma-associated viral-RNA (vRNA) loads, as determined using TaqMan(®) based RT-PCR, showed unexpectedly high sample to sample variation in all mules, demonstrating host-mediated control of viral replication is not constant over time. Furthermore, there was no apparent correlation between vRNA loads and antibody reactivity in serological tests. Analysis of PCR products established all mules were infected with viruses possessing nucleotide sequence similarity, varying from 77 to 96%, to previously identified European EIAV strains. Following IS, all mules showed increases in plasma-associated vRNA loads, suggesting control of EIAV replication is mediated by immune responses in this hybrid species. However, only three mules showed anamnestic humoral responses to rises in viral loads, as defined by at least a four-fold increase in ELISA titre, while two remained AGIDT-negative. This study demonstrates that viral loads in equids with consistent ELISA/IB positive-AGIDT negative to very weak positive test results (Group N) can be equivalent to those that produce clearly positive results in all three serologic tests (Group P). Therefore, such animals do not pose inherently lower risks for the transmission of EIAV. Consequently, the exclusive use of the AGIDT, as prescribed by the World Organization of Animal Health (OIE) for diagnosis of EIA prior to the international movement of horses, can report as negative some EIAV-infected equids. These results dramatically underscore the necessity of combining the specificity of AGIDT with tests with higher sensitivity, such as the ELISA and the power of the IB to enhance the accuracy of EIA diagnosis.

Detection of Hepatitis E Virus (HEV) in Italian pigs displaying different pathological lesions

In this study we investigated the HEV prevalence in Italian pigs displaying different pathological lesions, possible risk factors related to the infection, and the possible relations occurring between HEV and other concomitant pig pathogens. Genetic characterization of some of the identified strains was also performed. Detection of HEV RNA was accomplished using a nested reverse-transcription polymerase chain reaction on bile samples from 137 pigs of 2-4months of age submitted for diagnostic purposes. Forty-one of the 137 examined pigs (29.9%) tested positive for HEV RNA. Animals of 80-120days of age showed a higher prevalence of HEV infection (46.9% against 20% of younger animals). No statistically significant correlations between HEV positivity and the presence of other pathological conditions detected at necropsy, or concomitant coinfections with PCV2 and/or PRRSV were detected. All identified strains belonged to genotype 3, and were similar to other HEV subtypes 3e, 3f, 3c circulating in Europe.

Should the domestic buffalo (Bubalus bubalis) be considered in the epidemiology of Bovine Herpesvirus 1 infection

Only limited information is available on the epidemiology and pathogenesis of Bovine Herpesvirus 1 (BoHV-1) in domestic buffalos. In this study, a virulent BoHV-1 field strain isolated from cattle was inoculated into buffaloes to evaluate their susceptibility to the virus and to investigate the establishment of viral latency through clinical, virological and serological investigations. Latency was also studied by attempting viral reactivation using pharmacological induction. Six of seven male, 5 months old buffaloes were intranasally inoculated with BoHV-1; the other animal was kept as negative control. The animals were clinically monitored during the post-infection (P.I.) and the post-pharmacological induction (P.P.) periods. During these periods, nasal and rectal swabs, and blood samples, with and without anticoagulant, were collected at 2-3 day intervals. On culling the animals, 206 days P.I., their trigeminal ganglia and tonsils were collected. No clinical signs referable to BoHV-1 were observed throughout the experimental period. However, seropositivity was detected in all infected animals within day 20 P.I., using BoHV-1 glycoprotein E and glycoprotein B competitive ELISAs (IDEXX) and virus neutralisation test. In real-time PCR (RT-PCR), five of these animals were positive, at least once, for nasal or rectal swabs, during the P.I. period. The sixth infected animal was found positive only in the trigeminal ganglia after culling. Ganglia were also positive for two other animals. Virus isolation in permissive cell-lines was successful for a part of the RT-PCR positive samples. The detected viruses were confirmed by genetic analysis as identical to the inoculated strain. No evidence of infection was observed in the negative control. This study represents the first experimental transmission of BoHV-1 in buffaloes, confirming their susceptibility to infection and their possible role as host/reservoirs of BoHV-1.

Detection of hepatitis E virus in Italian pig herds

HEPATITIS E is a public health concern in many developing countries, where it is primarily transmitted by the faecal-oral route through contaminated water and food (Emerson and Purcell 2003). The disease is caused by a small, non-enveloped single-stranded RNA virus classified as the separate genus Hepevirus. Although hepatitis E disease occurs only sporadically in countries with good health care systems, the seroprevalence in healthy individuals can be high (Emerson and Purcell 2003). Porcine hepatitis E virus (HEV) is not pathogenic to general pig populations, but there is evidence that the virus may be a zoonotic agent and that animal reservoirs may exist. Experimental interspecies transmission of HEV between non-human primates and pigs has been demonstrated (Meng and others 1998), and seroepidemiological studies have shown that pig handlers are at higher risk of HEV infection than the general population (Meng and others 2002). In Japan, studies have supported the possibility of zoonotic transmission, as consumption of undercooked pig organs or meat and, in one case, of deer meat, was closely linked to cases of hepatitis E in human beings (Tei and others 2003, Yazaki and others 2003). The first porcine strain of HEV was characterised in the USA in 1997 (Meng and others 1997). Since then, several other porcine strains have been described worldwide. In the past few years, sporadic cases of autochthonous hepatitis E in human beings have been reported in several European countries, including Italy (Zanetti and others 1999). In many of these cases, the infecting HEV strain showed a high degree of homology with porcine strains of HEV detected in the same country (van der Poel and others 2001, Clemente-Casares and others 2003, Banks and others 2004a). In recent years in Europe, HEV in pig herds has been reported only in Spain (Clemente-Casares and others 2003), the UK (Banks and others 2004b) and the Netherlands (van der Poel and others 2001). This short communication describes the detection, by a nested reverse transcriptase-PCR (nested RT-PCR), of HEV in two Italian pig farms, and the phylogenetic analysis of the viral strains. Thirty-four faecal and 22 serum samples were collected from five different farrow-to-finish farms located in northcentral Italy. Samples were collected from healthy pigs between two and five months of age. Faecal samples represented pools of faeces from animals of the same age group. Total RNA was extracted from 140 μl of faecal suspension or serum using a QIAmp viral RNA kit (Qiagen), according to the manufacturer’s instructions. Template cDNA was reverse transcribed using random hexamers, according to standard protocols. A 145 base pair (bp) fragment of the open reading frame 2 of HEV was amplified from the prepared cDNA by nested PCR, as described by Erker and others (1999). A faecal suspension from a human patient with hepatitis E was used as a positive control. Nested RT-PCR products were visualised on 2 per cent agarose gel, and bands of the correct size were excised and purified with the QIAquick Gel Extraction Kit (Qiagen). Nucleotide sequencing was performed using the ABI PRISM BigDye Terminator kit, version 2·0 (Applied Biosystems). Sequences were assembled with SEQMAN (DNASTAR), and alignment was performed using the ClustalX algorithm. The HEV genome was detected in two faecal pools (5·9 per cent) collected at two different farms, but all the serum samples were negative. The positive faecal pools were obtained from groups of pigs aged 4·5 and 2·5 months, respectively. Phylogenetic analysis of the two viral sequences (113 bp), termed HEVBO/01 and HEVPI/01, was performed by the neighbour-joining method using PHYLIP 3·6. Bootstrap confidence values (500 replicates) were calculated by using the Seqboot and Consense programs. A phylogenetic tree (Fig 1) was created with the Treeview software using an avian HEV isolate as out group. Phylogenetic analysis of the viral sequences showed that the two Italian strains, HEVBO/01 and HEVPI/01, belonged to genotype 3, as did other porcine and human HEV strains indigenous to Europe. However, they differed significantly from each other, being only 84 per cent identical (18 nucleotide changes). The two Italian strains clustered with strains from countries where HEV is considered non-endemic. In particular, HEVPI/01 was related (with 90 per cent identity) to a human strain (AY540113) detected in a sporadic case of acute autochthonous hepatitis E in Spain (Buti and others 2004). In conclusion, this report represents the first description of HEV in Italian pig herds, and confirms the presence of the virus in apparently healthy pigs. These findings are important, because of the potential risk of transmission of porcine HEV to human beings, either by contact with infected pigs or by ingestion of contaminated undercooked meat. However, further studies are needed to address the true zoonotic potential of HEV in pigs. Studies are in progress to evaluate the prevalence of the infection in Italian pigs. The study of a high number of viral strains will be necessary to assess intraspecies and interspecies HEV homologies and to understand whether zoonotic transmission of HEV may occur in Italy.

A methicillin-resistant Staphylococcus aureus (MRSA) Sequence Type 8, spa type t11469 causing infection and colonizing horses in Italy

A Methicillin-resistantStaphylococcus aureus(MRSA) was isolated in Italy from a pathological sample of a mare presenting chronic purulent sinusitis and that had undergone frontal-sinus surgery three months before. Humans, horses, dogs and environmental samples were subsequently collected at the mares stable and at the Veterinary Hospital, where the mare was operated/hospitalized, and screened for the presence of MRSA that was detected from other horses and from the environment at both sites. All the MRSA isolates belonged to clonal complex (CC)8, ST8-t11469-SCCmec-IVa, and showed similar phenotypic and genetic multidrug resistance patterns and macrorestriction-pulsed-field gel electrophoresis profiles. The only MRSA detected from humans was a CC1, ST1-t127-SCCmec-IVa. This paper represents the first report of a clinical MRSA infection in a horse in Italy. This study also supports the opinion that improper use of antibiotics and hospitalization/surgery can represent risk factors for MRSA colonization/infection in horses, and that the environment is among important sources for exposure.

A New Multilocus Sequence Typing Scheme and Its Application for the Characterization of Photobacterium damselae subsp. damselae Associated with Mortality in Cetaceans

Photobacterium damselae subsp. damselae (PDD) is a known pathogen of fish, humans and marine mammals. In this study, a Multilocus Sequence Typing (MLST) scheme based on six housekeeping genes (glp, gyrB, metG, pnt, pyrC, and toxR) was developed to better understand the PDD population structure and used to type 73 PDD isolates from cetaceans, mainly striped dolphins (Stenella coeruleoalba) involved in mortality episodes, and from a few marine chelonians. Five reference ATCC strains were also included in the study. Typing allowed the discrimination of groups of PDD strains isolated from different host species, at different times and from different geographic areas, suggesting that a clonal PDD group may have spread in the Tyrrhenian sea at the time of an Unusual Mortality Event (UME) among cetaceans, mainly striped dolphins, occurred in early 2013 along the Italian western coasts.

Evolution of equine infectious anaemia in naturally infected mules with different serological reactivity patterns prior and after immune suppression.

Information on equine infectious anaemia (EIA) in mules, including those with an equivocal reaction in agar gel immunodiffusion test (AGIDT), is scarce. For this, a study was conducted to evaluate the clinical, viral loads and pathological findings of two groups of naturally infected asymptomatic mules, respectively with a negative/equivocal and positive AGIDT reactivity, which were subjected to pharmacological immune suppression (IS). A non-infected control was included in the study that remained negative during the observation period. Throughout the whole study, even repeated episodes of recrudescence of EIA were observed in 9 infected mules, independently from their AGIDT reactivity. These events were generally characterised by mild, transient alterations, typical of the EIA acute form represented by hyperthermia and thrombocytopenia, in concomitance with viral RNA (vRNA) peaks that were higher in the Post-IS period, reaching values similar to those of horses during the clinical acute phase of EIA. Total tissue viral nucleic acid loads were greatest in animals with the major vRNA activity and in particular in those with negative/equivocal AGIDT reactivity. vRNA replication levels were around 10-1000 times lower than those reported in horses, with the animals still presenting typical alterations of EIA reactivation. Macroscopic lesions were absent in all the infected animals while histological alterations were characterised by lymphomonocyte infiltrates and moderate hemosiderosis in the cytoplasm of macrophages. On the basis of the above results, even mules with an equivocal/negative AGIDT reaction may act as EIAV reservoirs. Moreover, such animals could escape detection due to the low AGIDT sensitivity and therefore contribute to the maintenance and spread of the infection.

Author Correction: Abundance and diversity of the faecal resistome in slaughter pigs and broilers in nine European countries

In the version of this Article originally published, the surname of author Oksana Lukjancenko was spelt incorrectly as ‘Lukjacenko’. This has now been corrected.