Phylogenetic analysis of porcine reproductive and respiratory syndrome virus isolates from Northern Ireland.

To investigate the genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV) in Northern Ireland, the ORF5 gene from nine field isolates was sequenced and phylogenetically analysed. The results revealed relatively high diversity amongst isolates, with 87.6-92.2% identity between farms at the nucleotide level and 84.1-93.5% identity at the protein level. Phylogenetic analysis confirmed that all nine isolates belonged to the European (type 1) genotype and formed a cluster within the subtype 1 subgroup. This study provides the first report on PRRSV isolate diversity in Northern Ireland.

Detection of a novel gammaherpesvirus (genus Rhadinovirus) in wild muntjac deer in Northern Ireland.

This study represents the initial part of an investigation into the potential for non-native, wild, free-living muntjac deer (Muntiacus reevesi) to carry viruses that could be a threat to livestock. A degenerate PCR assay was used to screen a range of tissues from muntjac deer culled in Northern Ireland for the presence of herpesviral nucleic acids. This was followed by sequencing of PCR amplicons and phylogenetic analysis. We report the detection of a novel gammaherpesvirus most closely related to a type 2 ruminant rhadinovirus from mule deer. It remains to be determined if this new virus is pathogenic to deer or presents a risk to food security through the susceptibility of domestic livestock.

Molecular detection of kobuviruses in livestock in Northern Ireland and the Republic of Ireland.

Kobuviruses have been detected in a wide range of mammals including cats, dogs, pigs, cattle, goats, sheep and bats. Kobuviruses have been detected in symptomatic and asymptomatic animals; however, the clinical significance of infection in animals is still unclear. To date, there is no information regarding kobuvirus prevalence in livestock in Ireland. This study reports the first detection of kobuviruses in pigs, bovines and ovines using quantitative PCR. In this study, mesenteric lymph node was collected from cattle (n = 57), pigs (n = 53) and sheep (n = 50) from farms in Northern Ireland and the Republic of Ireland, from animals which had been submitted by private veterinary practitioners from 2009 to 2011 for routine post mortem and clinico-pathological examination. Kobuviruses were detected in 14 cows (24.5%), 5 pigs (9.4%) and 1 sheep (2%). Phylogenetic analysis of Irish kobuviruses from cattle and pigs revealed that the isolates clustered according to their host species. Interestingly, the sheep kobuvirus clustered with bovine kobuviruses detected in this study and other published kobuvirus strains. The data presented in this study contributes to the understanding of the epidemiology of these viruses in animals and to the genetic diversity that these viruses possess.

Bioreduction of sheep carcasses effectively contains and reduces pathogen levels under operational and simulated breakdown conditions.

Options for the storage and disposal of animal carcasses are extremely limited in the EU after the introduction of the EU Animal By-products Regulations (ABPR; EC/1774/2002), leading to animosity within the livestock sector and the call for alternative methods to be validated. Novel storage technologies such as bioreduction may be approved under the ABPR provided that they can be shown to prevent pathogen proliferation. We studied the survival of Enterococcus faecalis, Salmonella spp., E. coli O157 and porcine parvovirus in bioreduction vessels containing sheep carcasses for approximately 4 months. The vessels were operated under two different scenarios: (A) where the water within was aerated and heated to 40 °C, and (B) with no aeration or heating, to simulate vessel failure. Microbial analysis verified that pathogens were contained within the bioreduction vessel and indeed reduced in numbers with time under both scenarios. This study shows that bioreduction can provide an effective and safe on-farm storage system for livestock carcasses prior to ultimate disposal. The findings support a review of the current regulatory framework so that bioreduction is considered for approval for industry use within the EU.

Development of a novel real-time PCR-based strategy for simple and rapid molecular pathotyping of Newcastle disease virus.

A novel real-time PCR strategy was applied to simultaneously detect and to discriminate low-pathogenic lentogenic and virulent meso/velogenic Newcastle disease virus (NDV). The pathotyping is achieved by a three-step semi-nested PCR. A pre-amplification of the cleavage site (CS) region of the F gene is followed by a two-level duplex real-time PCR directly targeting the CS, combining detection and pathotyping in a single tube. A wide range of NDV isolates spanning all genotypes were successfully detected and pathotyped. Clinical samples from outbreaks in Sweden in 2010 that were positive by the novel PCR method were also successfully pathotyped. The method is time-saving, reduces labour and costs and provides opportunities for rapid diagnosis at remote locations and in the field. Since the same strategy was also recently applied to avian influenza virus pathotyping, it shows promise of finding broad utility in diagnostics of infectious diseases caused by different RNA viruses in various hosts.

Extracts of Sida cordifolia contain polysaccharides possessing immunomodulatory activity and rosmarinic acid compounds with antibacterial activity.

BACKGROUND: The overuse of antibiotics has led to increased antimicrobial resistance, but plant-derived biological response modifiers represent a potential alternative to these drugs. This investigation examined the immunomodulatory and antibacterial activities of Sida cordifolia (used in ethnomedicinal systems to treat infectious disease). METHODS: Successive extractions were performed from the roots of these plants in hexane, chloroform, methanol and water. Immunomodulatory activity was determined in a series of experiments measuring the responses of splenocytes, macrophages and an in vivo model of innate immunity (Galleria mellonella). Antibacterial activity was assessed by determining minimum inhibitory/bactericidal concentrations (MIC/MBCs) for various Gram-positive and Gram-negative bacterial strains. RESULTS: Immunomodulatory activity was confined to the aqueous extract, and further fractionation and biochemical analysis yielded a highly potent polysaccharide-enriched fraction (SCAF5). SCAF5 is a complex mixture of different polysaccharides with multiple immunomodulatory effects including immune cell proliferation, antibody secretion, phagocytosis, nitric oxide production, and increased expression of pro-inflammatory cytokines. Furthermore, Galleria mellonella pre-treated with SCAF5 produced more haemocytes and were more resistant (P < 0.001) to infection with methicillin-resistant Staphylococcus aureus (MRSA) with a 98% reduction in bacterial load in pre-treated larvae compared to the negative control. The antibacterial activity of Sida cordifolia was confined to the methanolic fraction. Extensive fractionation identified two compounds, rosmarinic acid and its 4-O-ß-d-glucoside derivative, which had potent activity against Gram-positive antibiotic-resistant bacteria, including MRSA. CONCLUSIONS: Sida cordifolia counters bacterial infections through a dual mechanism, and immunomodulatory polysaccharides from this plant should be isolated and characterised to realise their potential as anti-infective agents. Such properties could be developed as an antibiotic alternative (1) in the clinic and (2) alternative growth promoter for the agri-food industry.

The Growth Performance, Nutrient Digestibility, Gut Bacteria and Bone Strength of Broilers Offered Alternative, Sustainable Diets Varying in Nutrient Specification and Phytase Dose.

This study assessed the use of locally sourced sustainable feed ingredients, rapeseed meal (RSM) and maize dried distiller grains with solubles (DDGS) in diets over traditional ingredients on the growth performance, bone strength and nutrient digestibility of broilers. This work also investigated the effects of supplementing exogenous phytase in two doses (500 vs. 1500 FTU/kg). Using male Ross 308 chicks (n = 320) assigned to receive one of four experimental diets: (1) Positive control diet 1 (PC1), a wheat, soya-based diet + 500 FTU/kg phytase. (2) Positive control diet 2, RSM/DDGS diet + 500 FTU/kg phytase (PC2). (3) Negative control (NC) reduced nutrient RSM/DDGS diet, no phytase. (4) The NC diet plus 1500 FTU/kg phytase (NC+). PC1 birds displayed higher feed intake and body weight gain consistently throughout the trial (p < 0.001) as well as increased body weight by 28 d and 42 d (p < 0.001). Whole-body dual emission X-ray absorptiometry (DXA) analysis revealed PC1 birds also had higher bone mineral density (BMD), bone mineral content (BMC), total bone mass, total lean mass and total fat mass than birds offered other treatments (p < 0.01). Diet had no significant effect on bone strength. Phytase superdosing improved the digestibility of dry matter (DM), neutral detergent fibre (NDF), gross energy (GE), calcium (Ca), potassium (K) and magnesium (Mg) compared to birds in other treatment groups. The phytase superdose also improved performance in comparison to birds offered the NC diet. Phytase superdosing increased the IP6 and IP5 degradation and increased the ileal inositol concentration of the birds. N excretion was lower for birds offered the traditional wheat−soya diet and highest for those offered the high-specification RSM/DDGS diet with a commercial dose of phytase. The addition of a phytase superdose to the negative control diet (NC+) reduced P excretion of birds by 15% compared to birds offered NC.

Evaluation of Mycoplasma hyopneumoniae bacterins for porcine torque teno virus DNAs.

OBJECTIVE: To determine whether commercial Mycoplasma hyopneumoniae bacterins sold for use in swine contain porcine torque teno virus (TTV). SAMPLE POPULATION: 22 commercially available M hyopneumoniae bacterins. PROCEDURES: Direct and nested PCR assays for genogroup-specific TTV DNAs were performed on serials of M hyopneumoniae bacterins by use of published and custom-designed primer pairs at 3 laboratories in North America and Europe. RESULTS: Of the 22 bacterins tested by use of direct and nested PCR assays, 7 of 9 from the United States, 2 of 5 from Canada, and 4 of 8 from Europe contained genogroup 1- and genogroup 2-TTV DNAs. In some bacterins, the TTV DNAs were readily detected by use of direct PCR assays. CONCLUSIONS AND CLINICAL RELEVANCE: Analysis of these data indicated that many of the commercially available M hyopneumoniae bacterins were contaminated with TTV DNA. It is possible that some of these bacterins could inadvertently transmit porcine TTV infection to TTV-naïve swine.

Molecular beacon real-time PCR detection of swine viruses.

Rapid and reliable detection of viral pathogens is critical for the management of the diseases threatening the economic competitiveness of the swine farming industry worldwide. Molecular beacon assays are one type of real-time polymerase chain reaction (PCR) technology capable of fast, specific, sensitive, and reliable viral detection. In this paper, the development of molecular beacon assays as novel tools for the rapid detection of Aujeszky's disease virus, African swine fever virus, porcine circovirus type 2 and porcine parvovirus is described. The assays are capable of rapidly detecting 2 x 10(1) copies of target and are linear between 2 x 10(9) and 2 x 10(2) copies. They can detect virus specifically in clinical samples such as whole blood, serum and tissue. In comparison to conventional PCR they are either as sensitive or more sensitive. As such these molecular beacon assays represent a powerful tool for the detection of these viruses in swine.

The development of a real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay using TaqMan technology for the pan detection of bluetongue virus (BTV).

Bluetongue virus (BTV) is an infectious, non-contagious viral disease of domestic and wild ruminants that is transmitted by adult females of certain Culicoides species. Since 2006, several serotypes including BTV-1, 2, 4, 6, 8, 9 and 16, have spread from the Mediterranean basin into Northern Europe for the first time. BTV-8 in particular, caused a major epidemic in northern Europe. As a result, it is evident that most European countries are at risk of BTV infection. The objective of this study was to develop and validate a real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) assay based on TaqMan technology for the detection of representative strains of all BTV serotypes. Primers and probes were based on genome segment 10 of the virus, the NS3 gene. The assay was tested for sensitivity, and specificity. The analytical sensitivity of the rRT-PCR assay was 200 copies of RNA per reaction. The assay did not amplify the closely related orbivirus epizootic hemorrhagic disease virus (EHDV) but successfully detected all BTV reference strains including clinical samples from animals experimentally infected with BTV-8. This real time RT-PCR assay offers a sensitive, specific and rapid alternative assay for the pan detection of BTV that could be used as part of a panel of diagnostic assays for the detection of all serotypes of BTV.

Reproduction of post-weaning multi-systemic wasting syndrome in an animal disease model as a tool for vaccine testing under controlled conditions.

Snatch farrowed, colostrum deprived piglets were inoculated with different combinations of porcine circovirus 2, porcine parvovirus and Erysipelothrix rhusiopathiae candidate vaccines. 10 piglets were mock-vaccinated. Following virus challenge with a combined porcine circovirus 2/porcine parvovirus inoculum, all animals were monitored and samples taken for serology, immunohistochemistry and qPCR. At 24 dpc all non-vaccinated animals remaining were exhibiting signs of post-weaning multi-systemic wasting syndrome which was confirmed by laboratory analysis. Details of the study, analysis of samples and performance of the candidate vaccines are described.

Adaptation of an Invader assay for the detection of African swine fever virus DNA.

A closed tube isothermal Invader assay (Third Wave Technologies Inc., Madison, Wisconsin, USA) was adapted for the detection of African swine fever virus (ASFV) DNA. Several ASFV Invader assays were designed successfully and tested on a real-time PCR instrument (iCycler, BioRad). The assay exhibiting the lowest signal/noise ratio (VP73 ASFV Invader Assay) was analysed further using serial 10-fold dilutions of Lisbon 60 ASFV viral genome. The assay sensitivity was determined to be in the order of 2500 copies of ASFV DNA and showed a dynamic range of 4 logs, from 2.5x10(6) to 2500 copies. The high specificity of the test was demonstrated by the lack of cross-reactivity to the clinically similar but heterologous virus, classical swine fever virus. The sensitivity of the Invader assay is sufficient for the testing of acutely infected viremic animals in which the viral load will be high. The robustness and ease of use of the ASFV Invader assay, combined with the possibility to run and read the assay using simple and relatively inexpensive equipment, makes it suitable for laboratories lacking containment facilities and/or real-time PCR instrumentation or on a regional basis for on-site diagnosis close to putative sites of ASFV outbreaks.

Detection and characterisation of novel bocavirus (genus Bocaparvovirus) and gastroenteritis viruses from asymptomatic pigs in Ireland.

BACKGROUND: Livestock animals have been the assumed source of several human epidemics in recent years, for example, influenza H1N1, rotavirus G8/G9, and MERS-CoV. Surveillance of novel viruses in animals is essential to evaluate the risk to human and animal health and to determine any economic impact, for example, failure to thrive. There is a paucity of data regarding detection and characterisation of gastroenteritis viruses, particularly novel viruses, in porcines in Ireland. Recently, a number of small novel porcine DNA viruses have emerged globally, for example, torque teno sus virus, porcine bocavirus, and parvoviruses 2 & 4, and little is known about the biology and potential pathogenicity of these viruses. Bocaparvovirus is a genetically distinct group of viruses which has been recently detected in humans and animals. METHODS: In this study, the presence of gastroenteritis viruses (rotavirus A, porcine circovirus, adenovirus, and porcine bocavirus) was investigated in a selection of archived faecal samples from asymptomatic piglets from a commercial farm in Ireland. A total of 104 specimens were pooled and screened using conventional molecular techniques (PCR and RT-PCR), a subset of specimens (n=44) were then examined individually. Viral diversity was then investigated using statistical and phylogenetic techniques. RESULTS: Initial screening showed a high prevalence of PBoV in this farm, with the formation of three distinct groups in phylogenetic analysis. Other viruses were also investigated in this study with the first report of PCV, PAdV and lineage I G5 RVA in Ireland. Some specimens contained >1 virus, with statistical analysis indicating a strong correlation for mixed infections of PBoV and PAdV on this farm. CONCLUSION: Investigating the diversity of circulating enteric viruses on Irish porcine farms is important to improve the prophylactic tools available and to facilitate the early detection of changes in circulating viruses.

The development of an accelerated reverse-transcription loop mediated isothermal amplification for the serotype specific detection of bluetongue virus 8 in clinical samples.

In 2006 bluetongue virus serotype 8 (BTV 8) was identified for the first time in the Netherlands causing a major epidemic in sheep and cattle that quickly spread to neighbouring Belgium, Germany and beyond to France and the UK. This resulted in severe animal health and welfare problems as well as substantial economic losses to the agrifood industries of these countries. Given that the early diagnosis of BTV infection 'in-the-field' is extremely useful to its subsequent management and control, this study was established to design a novel, sensitive and rapid nucleic acid diagnostic test for the serotype-specific detection of BTV 8, which could be used without the use of advanced laboratory support and equipment. Primers for the detection of BTV 8 were based on genome segment 2 of the virus, the VP2 gene. The assay was assessed using a full panel of BTV reference strains and clinical samples. Positive amplification was observed using a fluorescent detection reagent. The sensitivity of the RT-LAMP assay was 102 copies of RNA. The assay did not amplify the closely related orbivirus EHDV. This novel RT-LAMP offers a sensitive, specific and rapid method of detecting BTV 8. The approach is inexpensive and easy to use and could potentially be used in a 'pen-side' setting 'in the field' or by smaller less well-equipped laboratories in developing countries.

Detection of a porcine boca-like virus in combination with porcine circovirus type 2 genotypes and Torque teno sus virus in pigs from postweaning multisystemic wasting syndrome (PMWS)-affected and non-PMWS-affected farms in archival samples from Great Britain.

In this study we detail the detection and genetic analysis of a novel porcine boca-like virus (PBo-likeV) in archival sera and tissue samples from pigs from farms in Great Britain. We also investigate the distribution of porcine circovirus type 2 (PCV2) genotypes and Torque teno sus virus (TTSuV) genogroups 1 and 2 in combination with this novel PBo-likeV. PBo-likeV was detected in over 70% of all tissues investigated. Over 24% of all tissues recovered from PMWS-affected animals had all viruses present and 25% of tissues recovered from non-PMWS-affected pigs were positive for all 4 viruses.

Pan-serotypic detection of foot-and-mouth disease virus using a minor groove binder probe reverse transcription polymerase chain reaction assay.

A novel assay for the pan-serotypic detection of foot-and-mouth disease virus (FMDV) was designed using a 5' conjugated minor groove binder (MGB) probe real-time RT-PCR system. This assay targets the 3D region of the FMDV genome and is capable of detecting 20 copies of a transcribed RNA standard. The linear range of the test was eight logs from 2 × 10¹ to 2 × 108 copies and amplification time was approximately 2 h. Using a panel of 83 RNA samples from representative FMDV isolates, the diagnostic sensitivity of this test was shown to be equivalent to a TaqMan real-time RT-PCR that targets the 5' untranslated region of FMDV. Furthermore, the assay does not detect viruses causing similar clinical diseases in pigs such as swine vesicular disease virus and vesicular stomatitis virus, nor does it detect marine caliciviruses causing vesicular exanthema. The development of this assay provides a useful tool for the differential diagnosis of FMD, potentially for use in statutory or emergency testing programmes, or for detection of FMDV RNA in research applications.

Isolation in cell cultures and initial characterisation of two novel bocavirus species from swine in Northern Ireland.

We report the isolation in cell cultures of two novel bocavirus species in pigs from farms in Northern Ireland with clinical postweaning multisystemic wasting syndrome (PMWS). We have designated the isolates as porcine bocavirus-3 (PBoV3) and porcine bocavirus-4 (PBoV4). To date 5082 and 4125 bps of PBoV3 and PBoV4 have been sequenced, respectively. PBoV3 and PBoV4 show nucleotide homology to other known bocaviruses in swine and other organisms. Open reading frame (ORF) analysis has shown that these viruses have a third small ORF, equivalent to the NP1 ORF that distinguishes the bocaviruses from other parvoviruses. A panel of porcine field sera was screened by indirect immunofluorescence against both viruses. Of the 369 samples analysed, 32 (8.7%) and 35 (9.5%) sera were seropositive for PBoV3 and PBoV4 respectively, thus providing serological evidence of the exposure of swine in the field to bocavirus-like viruses. To date, the clinico-pathological significance of these novel swine bocaviruses, as primary pathogens or as immunosuppresive triggers for other infectious agents, is undetermined.

Sensitive detection of African swine fever virus using real-time PCR with a 5' conjugated minor groove binder probe.

The design of a 5' conjugated minor groove binder (MGB) probe real-time PCR assay is described for the rapid, sensitive and specific detection of African swine fever virus (ASFV) DNA. The assay is designed against the 9GL region and is capable of detecting 20 copies of a DNA standard. It does not detect any of the other common swine DNA viruses tested in this study. The assay can detect ASFV DNA in a range of clinical samples. Sensitivity was equivalent to the Office International des Epizooties (OIE) recommended TaqMan assay. In addition the assay was found to have a detection limit 10-fold more sensitive than the conventional PCR recommended by the OIE. Linear range was ten logs from 2x10(1) to 2x10(10). The assay is rapid with an amplification time just over 2h. The development of this assay provides a useful tool for the specific diagnosis of ASF in statutory or emergency testing programs or for the detection of ASFV DNA in research applications.

Detection of a novel porcine boca-like virus in the background of porcine circovirus type 2 induced postweaning multisystemic wasting syndrome.

Porcine circovirus type 2 (PCV-2) has been found to be the causative agent of postweaning multisystemic wasting syndrome (PMWS). However, PCV-2 is a ubiquitous virus in the swine population and a majority of pigs infected with PCV-2 do not develop the disease. Different factors such as age, maintenance, the genetics of PCV-2, other pathogens, etc. have been suggested to contribute to the development of PMWS. However, so far no proven connection between any of these factors and the disease development has been found. In this study we explored the possible presence of other so far unknown DNA containing infectious agents in lymph nodes collected from Swedish pigs with confirmed PMWS through random amplification and high-throughput sequencing. Although the vast majority of the amplified genetic sequences belonged to PCV-2, we also found genome sequences of Torque Teno virus (TTV) and of a novel parvovirus. The detection of TTV was expected since like PCV-2, TTV has been found to have high prevalence in pigs around the world. We were able to amplify a longer region of the parvovirus genome, consisting of the entire NP1 and partial VP1/2. By comparative analysis of the nucleotide sequences and phylogenetic studies we propose that this is a novel porcine parvovirus, with genetic relationship to bocaviruses.