Study of Animal Mixing and the Dynamics of Hepatitis E Virus Infection on a Farrow-to-Finish Pig Farm.

In Europe, swine are a livestock reservoir for Hepatitis E virus genotype 3 (HEV-3). Consumption of food containing HEV-3 can cause zoonotic human infection, though risk is reduced by heat treatment. Implementing controls that limit infection in slaughter pigs may further reduce foodborne transmission risk but knowledge of infection dynamics on commercial farms is limited. This study addressed this knowledge gap and in particular investigated the influence of group mixing. Faeces were collected from grower (n = 212) and fattener (n = 262) pigs on a farrow-to-finish farm on four occasions. HEV RNA was detected on all occasions, and prevalence was higher in growers (85.8%) than fatteners (26.0%; p < 0.001). HEV-positive samples were also collected from the wider farm environment (n = 67; 64.7% prevalence), indicating potential sources for HEV re-circulation within the herd. Timing of infection in a cohort was also investigated. HEV was absent from all piglet faeces (n = 98) and first detected at weaner stage (25.7% prevalence), but only in groups weaned earlier or comprising pigs from many different litters. Farrowing sow faeces (n = 75) were HEV-negative but antibodies were detected in blood from two sows. Results suggest that multiple factors influence HEV infection dynamics on pig farms, and potential foci for further study into practical control solutions are highlighted.

Molecular and in vitro characterisation of hepatitis E virus from UK pigs.

Hepatitis E virus (HEV) infection is widespread in the global pig population. Although clinically inapparent in pigs, HEV infection is the cause of Hepatitis E in humans and transmission via the food chain has been established. Following a 2013 study that investigated prevalence of HEV infection in UK slaughter-age pigs samples indicating highest viral load were selected for further characterisation. High throughput sequencing was used to obtain the complete coding sequence from five samples. An in-frame insertion was observed within the HEV hypervariable region in two samples. To interrogate whether this mutation may be the cause of high-level viraemia and faecal shedding as observed in the sampled pigs virus isolation and culture was conducted. Based on viral growth kinetics there was no evidence that these insertions affected replication efficiency in vitro, suggesting as yet undetermined host factors may affect the course of infection and consequently the risk of foodborne transmission.

Characterisation of porcine circovirus type 2 in porcine circovirus disease cases in England and Wales.

Confirmed cases of porcine circovirus disease (PCVD) in Great Britain have shown a steady decline since the availability of porcine circovirus type 2 (PCV2) vaccines. However, PCVD is still occasionally diagnosed. The authors carried out a genotyping study to characterise PCV2 associated with confirmed PCVD cases in England and Wales from 2011 to January 2016 (n=65). A partial fragment of PCV2 genome encompassing ORF2 was amplified and sequenced from 45 cases of PCVD. The majority of sequences were genotype PCV2b but four sequences were PCV2d. The significance of the emergence of PCV2d in England and elsewhere in the world is not yet known, although it does appear to represent an ongoing global genotype shift.

Lack of genetic diversity in newly sequenced porcine circovirus type 1 strains isolated 20 years apart.

The complete genome sequences of a porcine circovirus type 1 (PCV1) strain isolated in 1990 and one isolated in 2011 were obtained and compared to the sequences of other available PCV1 isolates. Phylogenetic analyses revealed very low genetic diversity among these viruses, indicating an advanced state in the evolution of PCV1.

Ljungan virus and an adenovirus in Italian squirrel populations.

We report Ljungan virus infection in Eurasian red squirrels (Sciurus vulgaris) for the first time, and extend the known distribution of adenoviruses in both native red squirrels and alien gray squirrels (Sciurus carolinensis) to southern Europe.

Correction: Evidence for the Circulation of Equine Encephalosis Virus in Israel since 2001.

[This corrects the article on p. e70532 in vol. 8.].

Evidence for the circulation of equine encephalosis virus in Israel since 2001.

Equine encephalosis virus (EEV) distribution was thought to be limited to southern Africa until 2008 when we reported EEV in Israel. It was then assumed that the clinical presentation resembled the initial incursion in Israel. To investigate further we conducted a retrospective analysis of equine sera, which had been collected for diagnosis of other suspected diseases, via serum neutralisation test. The data demonstrated that EEV was circulating as early as 2001 with incidence ranging from 20-100% for time period 2001-2008. As the symptoms of EEV can be similar to other equine notifiable diseases this is a significant finding which highlights the need for vigilance and education to accurately diagnose new and emerging diseases.

Enteritis in an alpaca (Vicugna pacos) associated with a potentially novel adenovirus.

Adenovirus-associated enteritis was diagnosed by histopathology of small intestine in a 2-year-old alpaca (Vicugna pacos). Electron microscopy confirmed intracytoplasmic and intranuclear adenoviral particles within enterocytes. Nucleic acid was extracted from paraffin-embedded tissue sections, and a pan-adenovirus nested polymerase chain reaction (PCR) assay was employed to target a partial sequence of the polymerase gene. The PCR product (321 bp) was cloned and sequenced. Comparison of the nucleotide sequence against the National Center for Biotechnology Information (NCBI) nucleotide database demonstrated 68% identity with the isolates Canine adenovirus 1 and Bovine adenovirus 3. Comparison of the predicted amino acid sequence against the NCBI database demonstrated 75% identity with Bovine adenovirus 3. Phylogenetic analysis supported the relatively close relationship of this isolate to Bovine adenovirus 3, but the alpaca isolate was sufficiently distant to be considered a potentially novel adenovirus for this species.

Ontogeny of systemic cellular immunity in the neonatal pig: correlation with the development of post-weaning multisystemic wasting syndrome.

The aetiology of porcine post-weaning multisystemic wasting syndrome (PMWS) is poorly understood. Porcine circovirus type 2 (PCV-2) is an essential component of the experimental disease model for PMWS: however, evidence from experimental and field studies indicates that additional factors play a critical role in the aetiopathogenesis of PMWS. Current candidates include (1) immune stimulation (for example, via co-infection or vaccination), and (2) a novel infectious agent. A prospective, longitudinal case-control study was designed to investigate molecular triggers in leucocytes of neonatal piglets that may predispose to the development of PMWS. Blood samples were collected weekly from pigs (n=125) within five farms, from 1 week to 8 weeks of age: that is, before the appearance of clinical signs. Four colour flow cytometry was used to investigate changes in subsets of peripheral blood mononuclear cells, using monoclonal antibodies against the following cell associated markers; sIgG, CD3, MHCII dR, CD14, CD4a, CD8a, CD45RC, CD25, SWC3a, SWC8, CD163 and CD45. Sampling and laboratory analysis was supported by monitoring of clinical signs from 1 week to 20 weeks of age, or until disease supervened. At the conclusion of the study, 68 pigs (54%) were classified in Group 1 (no signs of clinical disease), 34 pigs (27%) in Group 2 (signs of clinical disease but not characteristic of PMWS), 17 pigs (14%) in Group 3 (suspect PMWS case) and 5 pigs (4%) in Group 4 (PMWS case). A single case of Porcine Dermatitis and Nephropathy syndrome (PDNS) was also diagnosed. Significant changes with age were demonstrated in clinically normal, neonatal pigs (Group 1), including an increase in B-cells and T-cells, and an increase in the proportion of total T-cells expressing MHCII. Within the T-cell subset, the proportion of CD8(+high) CD4(-) T-cells increased, in addition to the proportion of CD4(+) T-cells co-expressing CD8. Of the factors recorded, farm was found to have a highly significant effect on immune system development in the neonate. Comparison of Groups 1 and 4 cases identified significant differences between pigs which remained normal and those which subsequently developed PMWS. Pigs which went on to develop PMWS had a greater proportion of T-cells expressing MHCII in early life, higher mean intensity of expression of MHCII on T-cells, higher mean intensity of expression of MHCII on B cells and higher expression of CD25 on CD45RC(-) T-cells. These findings suggest that lymphocyte activation may be a key early event in the aetiology of PMWS.

Development of a TaqMan PCR assay with internal amplification control for the detection of African swine fever virus.

A closed-tube polymerase chain reaction (PCR) was developed to allow the rapid detection of African swine fever virus (ASFV) DNA. This assay targets the VP72 gene of ASFV and uses the 5'-nuclease assay (TaqMan) system to detect PCR amplicons, avoiding tube opening and potential cross-contamination of post-PCR products. An artificial mimic was engineered with the TaqMan probe site replaced by a larger irrelevant DNA fragment allowing discrimination from ASFV by using two-colour TaqMan probe reporters. When added to the samples, successful amplification of this mimic demonstrated the absence of substances inhibitory to PCR, thereby validating negative results. Assay sensitivity was confirmed by obtaining positive signals with a representative selection of ASFV isolates. Many of the clinical and post-mortem features of ASF resemble those of classical swine fever (CSF) and porcine dermatitis and nephropathy syndrome (PDNS). Therefore, fast and reliable detection of ASFV is essential not only for the implementation of control measures to prevent the spread of ASF, but also in the differential diagnosis from CSF and PDNS. This assay should prove to be a valuable tool in the laboratory diagnosis of ASF and will complement existing molecular methods to provide rapid differential diagnosis in cases of suspected swine fever.

Evaluation of automated RT-PCR to accelerate the laboratory diagnosis of foot-and-mouth disease virus.

Automated fluorogenic (5' nuclease probe-based) reverse transcription polymerase chain reaction (RT-PCR) procedures were evaluated for the diagnosis of foot-and-mouth disease (FMD) using suspensions of vesicular epithelium, heparinised or clotted blood, milk and oesophageal-pharyngeal fluid ('probang') samples from the United Kingdom (UK) 2001 epidemic and on sera from animals experimentally infected with the outbreak serotype O FMD virus strain. A MagNA Pure LC was initially programmed to automate the nucleic acid extraction and RT procedures with the PCR amplification carried out manually by fluorogenic assay in a GeneAmp 5700 Sequence Detection System. This allowed 32 samples to be tested by one person in a typical working day or 64 samples by two people within 10-12 h. The PCR amplification was later automated and a protocol developed for one person to complete a single test incorporating 96 RT-PCR results within 2 working days or for two people to do the same thing in around 12 h. The RT-PCR results were directly compared with those obtained by the routine diagnostic tests of ELISA and virus isolation in cell culture. The results on blood, probang and milk samples were in broad agreement between the three procedures but specific RT-PCR protocols for such material have to be fully optimised as perhaps have the positive-negative acceptance criteria. However, the automated RT-PCR achieved definitive diagnostic results (positive or negative) on supernatant fluids from first passage inoculated cell cultures and its sensitivity was greater than ELISA on suspensions of vesicular epithelium (ES) and at least equivalent to that of virus isolation in cell culture. The combined tests of ELISA, virus isolation in cell culture and RT-PCR might, therefore, only be required for confirmation of a first outbreak of FMD in a previously FMD-free country. Should a prolonged outbreak subsequently occur, then either ELISA plus RT-PCR or else RT-PCR alone could be used as the laboratory diagnostic tool(s). Either approach would eliminate the requirement for sample passage in cell culture and considerably advance the issue of laboratory diagnostic test results.