In vivo challenge studies on vaccinated chickens indicate a virus genotype mismatched vaccine still offers significant protection against NDV.

Although commercial vaccines against Newcastle Disease have been available for decades, outbreaks still occur in the face of vaccination Further vaccination may accelerate viral evolution resulting in a further reduction in vaccine efficacy. A key question is whether genotype-matched vaccines can confer better protection against contemporary type 1 Avian Paramyxoviruses. To assess this, an in vivo vaccine-challenge study was undertaken to assess protection afforded by 'genotype-matched' and commercial vaccine formulations. Groups of chickens were vaccinated twice (prime-boost) with an inactivated preparation of either La Sota Clone 30, AV632-chicken-Cyprus-13 (genotype VII.2), or mock vaccine, and later challenged with virulent AV632-chicken-Cyprus-13. Post vaccinal serological responses differed, although both vaccination/challenge groups showed similar levels of clinical protection compared to the unvaccinated group, where 100 % mortality was observed. Shedding was significantly reduced in the vaccinated groups compared to the unvaccinated group. Virus dissemination in the tissues of vaccinated birds was comparable, but onset of infection was delayed. Two mutations were observed in the HN gene of the heterologous vaccine group; H199N and I192M, the latter thought to be associated with increased fusogenic potential. These data demonstrate that existing vaccine formulations confer similar levels of clinical protection to contemporary strains and that the antigenic heterogeneity of circulating strains does not impact upon shedding profiles in immunised birds. In conclusion, the ability of virulent APMV-1 to cause disease in vaccinated flocks is unlikely to be the result of antigenic mismatch alone, and other factors likely contribute to vaccination failure and breakthrough.

Understanding the disease and economic impact of avirulent avian paramyxovirus type 1 (APMV-1) infection in Great Britain.

Newcastle disease (ND) is a notifiable disease affecting chickens and other avian species caused by virulent strains of Avian paramyxovirus type 1 (APMV-1). While outbreaks of ND can have devastating consequences, avirulent strains of APMV-1 generally cause subclinical infections or mild disease. However, viruses can cause different levels of disease in different species and virulence can evolve following cross-species transmission events. This report describes the detection of three cases of avirulent APMV-1 infection in Great Britain (GB). Case 1 emerged from the 'testing to exclude' scheme in chickens in Shropshire while cases 2 and 3 were made directly from notifiable avian disease investigations in chicken broilers in Herefordshire and on premises in Wiltshire containing ducks and mixed species, respectively). Class II/genotype I.1.1 APMV-1 from case 1 shared 99.94% identity to the Queensland V4 strain of APMV-1. Class II/genotype II APMV-1 was detected from case 2 while the class II/genotype I.2 virus from case 3 aligned closely with strains isolated from Anseriformes. Exclusion of ND through rapid detection of avirulent APMV-1 is important where clinical signs caused by avirulent or virulent APMV-1s could be ambiguous. Understanding the diversity of APMV-1s circulating in GB is critical to understanding disease threat from these adaptable viruses.

Game Birds Can Act as Intermediaries of Virulent Genotype VII Avian Orthoavulavirus-1 between Wild Birds and Domestic Poultry.

Newcastle Disease (ND), caused by virulent forms of Avian orthoavulavirus serotype-1 (AOAV-1) is an economically important avian disease worldwide. The past two incursions of ND into the United Kingdom occurred in game bird populations during 2005 and 2006. The nature of the game bird semi-feral rearing system, which can bring these birds into close contact with both wild birds and commercial or backyard poultry, has been hypothesized to act as a bridge between these two environments. As such, the risk that AOAV-1-infected game birds may pose to the UK poultry industry was investigated. Pheasants, partridges and chickens were experimentally infected with the virulent strain APMV-1/Chicken/Bulgaria/112/13, a genotype VII.2 virus associated with ND outbreaks in Eastern Europe. The study demonstrated that both chickens and pheasants are susceptible to infection with APMV-1/Chicken/Bulgaria/112/13, which results in high mortality and onward transmission. Partridges by contrast are susceptible to infection, but mortality was reduced, as was onward transmission. However, the data indicated that both pheasants and partridges may serve as intermediate hosts of AOAV-1 and may bridge the wild bird-domestic poultry interface enabling transmission into an economically damaging environment where morbidity and mortality may be high.

Development and validation of a one-tube, nested real-time PCR method suitable for routine detection of Mycobacterium bovis in animal tissue.

AIMS: Development and validation of a real-time PCR test for high-throughput routine screening of animal tissue for Mycobacterium bovis and other Mycobacterium tuberculosis complex (MTBC) members. METHODS AND RESULTS: A preliminary study compared the results of a combination of five tissue preparation/DNA extraction methods and nine PCR assays on a panel of 92 cattle tissue samples of known M. bovis culture status (55 positive and 37 negative). The combination of DNA extraction and PCR was found to be important in achieving optimal detection of M. bovis. The optimal combination of a simple tissue preparation/DNA extraction method and a one-tube, nested real-time PCR to maximize the sensitivity of detection of an M. bovis-specific RD4 deletion and an IS1081 MTBC-specific target was selected for further evaluation. In total, tissue samples collected from 981 cattle and 366 non-bovine animals and submitted for routine TB culture were parallel tested with the selected method, as well as tissue samples obtained from 156 animals in certified TB-free cattle herds. CONCLUSION: For cattle, the optimized RD4-IS1081 PCR test exhibited a diagnostic sensitivity of 96% (95% CI: 94-97%) and specificity of 97% (95% CI: 95-98%) compared to culture. Specificity was 100% when testing the 156 samples from known TB-free cattle. For non-bovine species, the PCR had a diagnostic sensitivity of 93% (95% CI: 83-98%) and a specificity of 99% (95% CI: 97-100%).

Comparative pathogenesis of two genotype VI.2 avian paramyxovirus type-1 viruses (APMV-1) in pheasants, partridges and chickens.

Newcastle disease (ND) is caused by virulent forms of avian paramyxovirus-1 (APMV-1) and is an economically important disease of poultry world-wide. Pigeon paramyxovirus 1 (PPMV-1), a sub-group of APMV-1 is endemic in Columbiformes and can cause infections of poultry. An outbreak of ND in partridges in Scotland, UK, in 2006 (APMV-1/partridge/UK(Scotland)/7575/06) was identified as a class II, genotype VI.2.1.1.2.1, more commonly associated with PPMV-1. It has been hypothesized that game birds may be a route of transmission into commercial poultry settings due to the semi-feral rearing system, which potentially brings them into contact with both wild-birds and poultry species. Therefore, the pathogenesis and transmission of APMV-1/partridge/UK(Scotland)/7575/06 in game birds and chickens was investigated, and compared to a contemporary PPMV-1 isolate, PPMV-1/pigeon/UK/015874/15. Viral shedding and seroconversion profiles demonstrated that pheasants were susceptible to infection with APMV-1/partridge/UK(Scotland)/7575/06 with limited clinical signs observed although they were able to excrete and transmit virus. In contrast, partridges and pheasants showed limited infection with PPMV-1/pigeon/UK/015874/15, causing mild clinical disease. Chickens, however, were productively infected and were able to transmit virus in the absence of clinical signs. From the data, it can be deduced that whilst game birds may play a role in the transmission and epidemiology of genotype VI.2 APMV-1 viruses, the asymptomatic nature of circulation within these species precludes evaluation of natural infection by clinical surveillance. It therefore remains a possibility that genotype VI.2 APMV-1 infection in game birds has the potential for asymptomatic circulation and remains a potential threat to avian production systems.RESEARCH HIGHLIGHTS Demonstration of infection of game birds with Pigeon paramyxovirus-1 (PPMV-1).There are differing dynamics of infection between different game bird species.Differing dynamics of infection between different PPMV-1 isolates and genotypes in game birds and chickens.

Identification of Equine Arteritis Virus Immunodominant Epitopes Using a Peptide Microarray.

Using the commercially available PEPperCHIP® microarray platform, a peptide microarray was developed to identify immunodominant epitopes for the detection of antibodies against Equine arteritis virus (EAV). For this purpose, the whole EAV Bucyrus sequence was used to design a total of 1250 peptides that were synthesized and spotted onto a microarray slide. A panel of 28 serum samples representing a selection of EAV strains was tested using the microarray. Of the 1250 peptides, 97 peptides (7.76%) showed reactivity with the EAV-positive samples. No single peptide was detected by all the positive serum samples. Seven peptides repeatedly showed reactivity above the cut-off and were considered to have diagnostic potential. Five of these peptides were within the immunodominant GP5 protein and two were within the replicase polyprotein regions NSP2 and NSP10, located in ORF1. The diagnostic sensitivity of the seven peptides selected was low, ranging from 5% to 55%; however, the combined diagnostic sensitivity and specificity of the seven peptides was 90% and 100%, respectively. This data demonstrate that multiple peptide sequences would be required to design a comprehensive serological test to cover the diversity of the EAV strains and the individual immune responses of horses.

JMM Profile: Avian paramyxovirus type-1 and Newcastle disease: a highly infectious vaccine-preventable viral disease of poultry with low zoonotic potential.

Newcastle disease (ND) is a highly contagious disease of poultry caused by virulent avian paramyxovirus-1 (APMV-1) (previously termed avian avulavirus-1 and avian orthoavulavirus-1). APMV-1 is endemic in poultry in many developing countries, whilst outbreaks still occur in developed countries, affecting both commercial and backyard flocks. ND outbreaks can have substantial economic consequences due to high mortality rates and the imposition of trade restrictions. APMV-1 nucleic acid can be detected from swabs or tissues of suspected cases by PCR. Evidence of infection or vaccination may be demonstrated by the presence of specific antibodies against HN in serum samples. No anti-viral treatments exist, but vaccines are available, although there are currently concerns over their efficacy.

Development of an avian avulavirus 1 (AAvV-1) L-gene real-time RT-PCR assay using minor groove binding probes for application as a routine diagnostic tool.

Newcastle disease is a devastating disease of poultry caused by Newcastle disease virus (NDV), a virulent form of avian avulavirus 1 (AAvV-1). A rapid, sensitive and specific means for the detection of NDV is fundamental for the control of this notifiable transboundary virus. Although several real-time RT-PCR assays exist for the detection of AAvV-1, diagnostic sensitivity and specificities can be sub-optimal. In this study, we describe a modification to an existing AAvV-1 l-gene RT-PCR screening assay, where the original probe set was replaced with minor groove binding (MGB) probes, to create the MGB l-gene assay. The diagnostic sensitivity and specificity of this assay was evaluated against a broad panel of both Class I and Class II AAvV-1 viruses of diverse and representative lineages/genotypes in both clinical samples and amplified viruses, and compared with a number of previously published real-time RT-PCR screening assays for AAvV-1. The MGB l-gene assay outperformed all other assays in this assessment, with enhanced sensitivity and specificity, detecting isolates from a broad range of virus lineages/genotypes (including contemporaneously-circulating strains). The assay has also proved its value for screening original clinical samples for the presence of AAvV-1, thus providing an improved screening assay for routine detection of this notifiable disease agent.

The role of vaccination in risk mitigation and control of Newcastle disease in poultry.

Newcastle disease is regarded as one of the most important avian diseases throughout the world and continues to be a threat and economic burden to the poultry industry. With no effective treatment, poultry producers rely primarily on stringent biosecurity and vaccination regimens to control the spread of this devastating disease. This concise review provides an historical perspective of Newcastle disease vaccination and how fundamental research has paved the way for the development of instrumental techniques which are still in use today. Although vaccination programmes have reduced the impact of clinical disease, they have historically been ineffective in controlling the spread of virulent viruses and therefore do not always offer an adequate solution to the world's food security problems. However, the continued development of novel vaccine technology and improved biosecurity measures through education may offer a solution to help reduce the global threat of Newcastle disease on the poultry industry.

Development and evaluation of a multiplex enzyme-linked immunosorbent assay for the detection of antibodies to bovine respiratory diseases on the Meso Scale Discovery platform.

A multiplex enzyme-linked immunosorbent assay (ELISA) to detect antibodies to Bovine viral diarrhea virus (BVDV), Bovine herpesvirus 1 (BHV-1), Bovine parainfluenza virus 3 (BPV-3), and Bovine respiratory syncytial virus (BRSV) was developed on the commercially available Meso Scale Discovery (MSD) platform. The multiplex ELISA used the same antigens (relatively crude lysates of cultured virus) that are used in the current in-house separate ELISAs. Testing a panel of field samples demonstrated the multiplex assay could simultaneously detect the presence of several antibodies in a single assay and that there was good agreement between the multiplex ELISA and the in-house ELISAs. The use of a multiplex ELISA could offer significant benefits for routine serological testing through reduced staff time, reduced reagent cost, and streamlined laboratory operation.