Occurrences of avian encephalomyelitis virus in naturally infected chicks in Saudi Arabia's Eastern Province.

Background: A neurological infectious viral disease, avian encephalomyelitis was initially discovered in 2-week-old commercial chicks in 1930 and classified as a neurotropic viral disease. Aim: A neurological outbreak caused by avian encephalomyelitis virus (AEV) in young chicks was first reported in Al-Ahsa in the Kingdom of Saudi Arabia (KSA) in 2010. The aim of this article is to examine the AEV in KSA, Al-Ahsa Province. Methods: Gizzard, proventriculus, cerebrum, cerebellum, and medulla oblongata tissue samples were collected from infected chicks for histopathology test and molecular identification. Results: Infected chicks showed neurological signs particularly incoordination, mild head and neck tremors, stretching of legs, and lameness. The average morbidity and mortality rates were 35% and 10%, respectively. At necropsy, no obvious identifiable macroscopic lesions were found in the infected chicks. Nonsuppurative encephalomyelitis was found histopathologically in the central nervous system, mainly in the cerebral molecular layer. Microscopic lesions in the proventriculus showed masses of heavy numbers of small lymphocytes within the muscular layer. RT-PCR followed by sequence analysis revealed that The KSA strain (KJ939252) is intimately related to chicken European strains from Poland (KC912695) and the United Kingdom (AJ225173) with identity 99.6% than Chinese strains (AY225319, AY517471, and AY275539) with identity ranged between 94.6% and 95%. The phylogenetic tree analysis showed that the KSA strain is grouped in a similar clade with chicken European strains. Conclusion: The pattern of disease findings was typical of vertically transmitted AEV. The spread of AEV in Saudi Arabia is most likely due to the trade of birds and bird products with European countries.

Research Note: Pathogenetic characteristics of avian encephalomyelitis virus in Guangdong and Jiangxi Provinces, China.

In recent years, the infection rate of avian encephalomyelitis virus (AEV) infection in chickens has risen significantly, seriously endangering the development of the chicken industry. In order to study the current epidemiological status of AEV in China as well as the genetic and evolutionary patterns of the virus, we conducted a survey and genomic analysis of chicken AEV. The results showed that 46.26% (136/294) of the tissue samples tested (n = 294) were positive for AEV, with the highest positivity rate of 62.24% (61/98) among tissue samples from chickens aged 13 to 18 wk. The complete genomes of 2 representative AEV strains were determined, and the VP1 evolutionary tree results revealed that the 2 representative strains belonged to a novel AEV strain. Multiple alignment analysis showed that the ORF1 genes of the 2 representative strains differed by 82.3 to 99.9% at the amino acid level compared with the reference AEV strain, and the mutations at the key amino acid loci of VP2 and VP3 were the same as those in the chick embryo-adapted strain. The analysis makes up for the molecular epidemiological data and genetic variation of the 2 representative strains. The analysis makes up for the molecular epidemiological data and genetic variation of AEV and provides a basis for further understanding the spread of AEV in China.

Progress in research on the molecular biological detection techniques of avian encephalomyelitis.

Avian encephalomyelitis (AE) is a highly infectious disease caused by the avian encephalomyelitis virus (AEV), which primarily affects the central nervous system of 1- to 4-week-old chicks and causes significant economic losses in the worldwide poultry industry. Despite heavy dependency on vaccine immunization, AEV has persisted on farms for extended periods, which increases its virulence and makes quick and accurate detection crucial to preventing and controlling the disease. Classical diagnostic methods have been unable to meet the current requirements for rapid diagnosis of AE cases. To address this issue, this paper reviews the etiological and molecular biological detection techniques of AE, and it seeks to provide a reference for future research and to establish differential diagnostic techniques for AE epidemiological investigation, identification of epidemic strains, and early diagnosis of clinical cases. Through improving our understanding of AE, we can better combat the disease and protect the global poultry industry.

Detection of avian encephalomyelitis virus in chickens in Japan using RT-PCR.

A reverse transcription-polymerase chain reaction (RT-PCR) method was developed for broadly detecting the avian encephalomyelitis virus (AEV). The new primers were based on conserved sequences of the 5'-untranslated region of AEV, because the virus was not detected using previous reported RT-PCR. By applying this method to the chicken samples with suspected AEV infection in Japan, we successfully obtained PCR products of the predicted size from all samples, and we confirmed the presence of AEV via sequence analysis.

Assessing the efficacy of a live vaccine against avian encephalomyelitis virus.

Avian encephalomyelitis virus (AEV) causes typical neurological symptoms in young chicks and a transient drop in egg production and hatchability in adult laying birds, resulting in huge economic losses in the poultry industry. An effective way to control and prevent this disease is vaccination of the flocks. Here, we assessed the efficacy of the live vaccine candidate strain GDt29 against avian encephalomyelitis virus. The GDt29 strain has low virulence, was confirmed safe, and showed no signs of pathogenicity. High titers of AEV-specific antibodies were detected in GDt29-vaccinated hens (S/P > 3.0) and their progeny (S/P > 2.0). Moreover, the eggs of GDt29-vaccinated hens with high levels of maternal antibodies were hatched successfully regardless of challenge with a heterologous AEV strain, and the GDt29 attenuated vaccine showed higher protective efficacy against AEV than the commercial vaccine. Furthermore, contact-exposed chicks bred with GDt29-vaccinated birds generated high titers against AE virus (S/P > 2.8). Collectively, our studies are proof of the principle that GDt29 might be an ideal vaccine candidate to prevent AEV infection, and they highlight the utility of using a live vaccine against AEV.

Dynamic distribution and tissue tropism of avian encephalomyelitis virus isolate XY/Q-1410 in experimentally infected Korean quail.

Avian encephalomyelitis (AE) is an important infectious poultry disease worldwide that is caused by avian encephalomyelitis virus (AEV). However, to date, the dynamic distribution of AEV in quails has not been well described. Quantitative real-time polymerase chain reaction (qPCR) and immunohistochemistry (IHC) assays were used to investigate the dynamic distribution and tissue tropism of AEV in experimentally infected Korean quail. AEV was detected in the cerebrum, cerebellum, proventriculus, intestine, liver, pancreas, spleen, bursa, lung and kidney as early as 3 days post-infection (dpi). The viral loads in the proventriculus, intestine, spleen and bursa were relatively higher than in other tissues. According to the qPCR results, AEV XY/Q-1410 infection lasted for at least 60 days in infected Korean quail. Immunohistochemistry-positive staining signals of AEV antigen were analysed by Image-Pro Plus software. A positive correlation between qPCR and IHC results was identified in most tissues. Our results provide an insight into the dynamic distribution of AEV in various tissues after infection. The distinct dynamic distribution of the viral genome in Korean quail in the early and late stages of infection suggests that AEV replication is affected by antibody levels and the maturity of the immune system of the host.

Serological evidence of avian encephalomyelitis virus and Pasteurella multocida infections in free-range indigenous chickens in Southern Mozambique.

A total of 398 serum samples from free-range indigenous chickens originating from four villages in Southern Mozambique were tested for the presence of avian encephalomyelitis virus (AEV) and Pasteurella multocida (PM) antibodies through commercial enzyme-linked immunosorbent assay (ELISA) kits. AEV and PM antibodies were detected in all villages surveyed. The proportion of positive samples was very high: 59.5% (95% confidence interval (CI) 51.7-67.7%) for AEV and 71.5% (95% CI 67.7-77.3%) for PM. Our findings revealed that these pathogens are widespread among free-range indigenous chickens in the studied villages and may represent a threat in the transmission of AEV and PM to wild, broiler or layer chickens in the region. Further research is warranted on epidemiology of circulating strains and impact of infection on the poultry industry.

Evolution of avian encephalomyelitis virus during embryo-adaptation.

Wild-type avian encephalomyelitis virus (AEV) causes neurological signs in young chicks but no disease in pullets after oral or intracutaneous infection. However, if the virus gets embryo-adapted by serial passaging in chicken embryos, it will cause AE after intracutaneous infection in chickens of all ages. Recently, several cases of AE in layer pullets occurring shortly after intracutaneous vaccination were described. The present investigation was initiated to determine if vaccines that had inadvertently been embryo-adapted were responsible for these outbreaks. Virus isolation was done from two vaccines and one field sample. One of the vaccines had been used in one of the flocks before the outbreak. After the first passage, regardless of the inoculum, no embryo was paralyzed, indicating that the vaccines and the field isolate were not embryo-adapted. After seven passages all three strains were fully embryo-adapted causing typical lesions in the embryos. Viral load as determined by RT-qPCR remained constant during the passages. Partial sequences of the VP2 gene of vaccines, the field sample and four other field isolates were nearly identical and highly similar to published sequences from all over the world; only sequences originating from non-vaccinated birds were clearly set apart. Analysis of whole genomes identified two single nucleotide polymorphisms (SNPs) that distinguished wild-type and embryo-adapted strains. Sanger sequencing brains and nerves of the five field isolates and of the first, third and fifth passages of the isolates showed that the mutations indicating embryo-adaptation were first observed in the fifth passage.

A survey for selected avian viral pathogens in backyard chicken farms in Finland.

Backyard poultry are regaining popularity in Europe and increased interest in the health and management of non-commercial farms has resulted. Furthermore, commercial poultry farm owners have become concerned about the risk represented by contagious avian diseases that nearby backyard poultry could transmit. Fifty-one voluntary backyard chicken farms were visited between October 2012 and January 2013. Blood samples and individual cloacal swabs were collected from 457 chickens. In 44 farms (86%), one or more of the tested chickens had antibodies against avian encephalomyelitis and chicken infectious anaemia viruses, 24 farms (47%) had chickens seropositive for infectious bronchitis virus, 10 farms (20%) had chickens seropositive for infectious bursal disease virus, six farms (12%) had chickens seropositive for infectious laryngotracheitis virus and two farms (5.4%) had chickens seropositive for avian influenza virus. No farms had chickens seropositive for Newcastle disease virus. Of the 51 farms, five (10%) had chickens positive for coronavirus reverse transcription polymerase chain reaction. A phylogenetic analysis showed that all backyard chicken coronaviruses collected were QX type infectious bronchitis viruses. All chickens tested for avian influenza and Newcastle disease viruses using real time reverse transcription polymerase chain reaction were negative. To our knowledge, there is no evidence to date to suggest that these diseases would have been transmitted between commercial and non-commercial flocks.

Health evaluation of African penguins (Spheniscus demersus) in southern Africa.

The African penguin (Spheniscus demersus) is an endangered seabird that breeds along the coast of Namibia and South Africa, and disease surveillance was identified as a priority for its conservation. Aiming for the establishment of baseline data on the presence of potential pathogens in this species, a comprehensive health assessment (blood smear examination, haematology, biochemistry and serology) was conducted on samples obtained from 578 African penguins at 11 breeding colonies and a rehabilitation centre. There were 68 penguins that were seropositive for at least one of seven pathogens tested: avian encephalomyelitis virus, avian infectious bronchitis virus, avian reovirus, infectious bursal disease virus, Newcastle disease virus, Mycoplasma gallisepticum and Mycoplasma synoviae. All samples were seronegative for avian influenza virus subtypes H5 and H7 and infectious laryngotracheitis virus. The apparent prevalence of Babesia sp. and Borrelia sp. in blood smears was consistent with previous studies. Babesia-infected individuals had a regenerative response of the erythrocytic lineage, an active inflammatory response and hepatic function impairment. These findings indicate that African penguins may be exposed to conservation-significant pathogens in the wild and encourage further studies aiming for the direct detection and/or isolation of these microorganisms.

Serological evidence of avian encephalomyelitis virus infection associated with vertical transmission in chicks.

Avian encephalomyelitis virus (AEV) can be transmitted both horizontally and vertically. In the present study, we report a typical case of AEV infection in broiler breeder chickens and their progeny identified by clinical survey of the disease, antibody detection, and reverse transcription (RT)-polymerase chain reaction (PCR) assay.

Serologic survey of wild turkeys (Meleagris gallopavo) and evidence of exposure to avian encephalomyelitis virus in Georgia and Florida, USA.

Wild Turkeys (Meleagris gallopavo) are susceptible to many of the same diseases as domestic turkeys. Before 2005, most Wild Turkeys in southern Georgia, US, had little or no exposure to commercial poultry operations. As part of a pathogen survey examining the effects of commercial poultry on Wild Turkeys, samples were collected from Wild Turkeys from March 2005 through May 2008. The turkeys were collected from 13 counties in southern Georgia and Madison County, Florida, and tested for antibodies to various pathogens of poultry. Three (13%) of the turkeys were positive for antibodies to Salmonella. Thirteen turkeys (54%) were positive for Newcastle disease virus antibodies, and 15 turkeys (63%) were positive for antibodies to reticuloendotheliosis virus. One turkey (4%) from Madison County was positive for avian encephalomyelitis virus antibodies.

Development of a SYBR Green real-time RT-PCR assay for the detection of avian encephalomyelitis virus.

Avian encephalomyelitis virus (AEV) causes epidemic diseases in poultry worldwide. A SYBR Green real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay was developed for the rapid detection and quantitation of AEV in this study. A pair of specific primers was designed in the highly conserved VP1 gene of this virus. When comparing this assay with conventional RT-PCR, the rRT-PCR assay was 100 times more sensitive and could detect levels as low as 10 standard DNA copies of the AEV SX strain. The specificity of this technique was evaluated in five other avian pathogens. The AEV RNA was detected as early as three days post-infection in chicken embryos. All 18 clinical chicken brains collected from an AEV outbreak in Northwestern China were detected to be positive (100%) using the rRT-PCR assay. However, only 5 of the 18 samples were positive (28%) using the conventional RT-PCR. The results were confirmed by virus isolation in chicken embryos. This high sensitivity, specificity, and simplicity of the SYBR Green rRT-PCR approach can be a more effective method than the conventional one for AEV diagnosis and surveillance.

Effect of envelope material on biosecurity during emergency bovine mortality composting.

The biosecurity of composting as an emergency disposal method for cattle mortalities caused by disease was evaluated by conducting full-scale field trials begun during three different seasons and using three different envelope materials. Process biosecurity was significantly affected by the envelope material used to construct the composting matrix. Internal temperatures met USEPA Class A time/temperature criteria for pathogen reduction in 89%, 67%, and 22%, respectively of seasonal test units constructed with corn silage, straw/manure, or ground cornstalks. In trials begun in the winter, survival times of vaccine strains of avian encephalomyelitis and Newcastle disease virus were noticeably shorter in silage test units than in the other two materials, but during summer/spring trials survival times in ground cornstalk and straw/manure test units were similar to those in test units constructed with silage.

Effects of blood sample mishandling on ELISA results for infectious bronchitis virus, avian encephalomyelitis virus and chicken anaemia virus.

This study determined the effect of sample mishandling on the performance of ELISAs for detection of antibodies against infectious bronchitis virus (IBV), avian encephalomyelitis virus (AEV) and chicken anaemia virus (CAV) in the serum of chickens. The effects of five different sample mishandling treatments were assessed: heat treatment, repetitive freezing and thawing and three levels of severity of haemolysis. These mishandling treatments simulated different conditions that might occur during routine blood collection, transport or storage in a clinical practice setting. Each mishandling treatment was experimentally applied under laboratory conditions and then samples were assayed for antibodies against IBV, AEV and CAV using commercial ELISA kits. Severe haemolysis had the most consistent detrimental effect on ELISA performance, producing results that were significantly different from the reference standard in all three ELISAs, although the direction of the effect varied (less positive for the IBV and CAV assays; more positive for the AEV assay). Moderate levels of haemolysis had a similar, but less consistent, effect to that of severe haemolysis, producing results that were significantly different from the reference standard only for the IBV (less positive) and AEV (more positive) ELISAs. Repetitive freeze-thawing also produced a significant effect on ELISA results for IBV (less positive) and AEV (more positive). The IBV ELISA appeared to be most susceptible to the effects of serum maltreatment. The findings from this study suggest that unpredictable variation in the results of ELISAs can occur due to different sample mishandling treatments.

Avian encephalomyelitis virus in reared pheasants: a case study.

An outbreak of neurological disease occurred in pheasant chicks on a game farm in 2007. The disease was first seen in the 10th hatching of chicks on the farm. Affected chicks showed trembling and incoordination from the time of hatching, and subsequently blindness and cataract formation was seen in some of the affected chicks at 3 weeks of age. The peak mortality and culling figure was 21.0% in the worst affected hatch, compared with a maximum of 11.7% in the first nine hatches. No further cases were evident by 7.5 weeks of age. Histopathological examination showed a moderate acute encephalomyelitis in some, but not all, of the chicks with neurological signs. The clinical presentation and histopathological findings were typical of vertically transmitted avian encephalomyelitis as seen in chickens, although avian encephalomyelitis virus could not be detected in inoculated embryonated chicken eggs. However, serological testing by enzyme-linked immunosorbent assay for antibodies to the virus was positive in four of five affected 3-week-old birds and in 23 out of 29 adult breeding birds, and reverse transcriptase-polymerase chain reaction testing of RNA extracted from brain and pancreas tissue of affected chicks yielded nucleotide sequences aligned 82% and 83% with three avian encephalomyelitis sequences in a sequence database. The evidence suggested that the neurological disease was attributable to infection with a strain of avian encephalomyelitis virus that appeared to have entered the flock at the start of the breeding season, and was possibly introduced by carrier pheasants brought on to the farm early in the season.

Development of a non-radioactive digoxigenin cDNA probe for the detection of avian encephalomyelitis virus.

A non-radioactive digoxigenin cDNA probe for detection of avian encephalomyelitis virus (AEV) was developed. The cDNA probe hybridized specifically with a fragment of VP1 gene of AEV genome was found to be sensitive with as little as 10 pg target DNA fragment in a sensitivity test. Infected chicken embryos were strongly labelled by testing the probe against a range of AEV strains, and no non-specific reaction was observed in non-infected chicken embryos as well as five other avian pathogenic virus-infected samples used as negative controls. Furthermore, the cDNA probe was capable of detecting AEV from chicken embryo brain at 3 days post-inoculation as compared with an immunofluorescence assay, which required up to 5 days of incubation in the embryos. In clinical application, five out of 16 clinical brain samples that were negative by the immunofluorescence assay were positive for AEV by the cDNA probe. Both the sensitivity and specificity of the developed cDNA probe indicated that it is a highly promising and reliable diagnostic tool for the detection of AEV infections.

The VP1 protein of avian encephalomyelitis virus is a major host-protective immunogen that serves as diagnostic potential.

Avian encephalomyelitis virus (AEV) is an important pathogen of poultry and is classified as a member of Picornaviridae. To investigate the protective immunity induced by AEV structural proteins, recombinant VP1, VP0, and VP3 proteins were expressed in a baculovirus system. The result of in vivo protection assays shows that the VP1 protein is a major host-protective immunogen against AEV challenge and demonstrates further that the antibody raised against VP1 protein could neutralize more effectively AEV infection than antibody against VP3 or VP0 protein in a virus neutralization test. These purified recombinant proteins were subsequently evaluated as enzyme-linked immunosorbent assay (ELISA) antigens for detection of AEV infection. A total number of 50 positive sera and 30 negative sera were tested for ELISA validation. Results obtained by testing 193 sera from chickens suspected of being infected AEV further showed that the diagnostic sensitivities of the VP1, VP3, and VP0 protein-based ELISAs were 98.1, 80.6, and 51.9%, and their specificities were 100, 87.9, and 81.8%, respectively. Both sensitivity and specificity of the VP1 protein-based ELISA were comparable with a commercially available test, indicating that the VP1 protein has a highly promising and reliable diagnostic potential, and thus is a suitable antigen for ELISA detection of AEV antibodies in chickens.

The picornavirus avian encephalomyelitis virus possesses a hepatitis C virus-like internal ribosome entry site element.

Avian encephalomyelitis virus (AEV) is a picornavirus that causes disease in poultry worldwide, and flocks must be vaccinated for protection. AEV is currently classified within the hepatovirus genus, since its proteins are most closely related to those of hepatitis A virus (HAV). We now provide evidence that the 494-nucleotide-long 5' untranslated region of the AEV genome contains an internal ribosome entry site (IRES) element that functions efficiently in vitro and in mammalian cells. Unlike the HAV IRES, the AEV IRES is relatively short and functions in the presence of cleaved eIF4G and it is also resistant to an inhibitor of eIF4A. These properties are reminiscent of the recently discovered class of IRES elements within certain other picornaviruses, such as porcine teschovirus 1 (PTV-1). Like the PTV-1 IRES, the AEV IRES shows significant similarity to the hepatitis C virus (HCV) IRES in sequence, function, and predicted secondary structure. Furthermore, mutational analysis of the predicted pseudoknot structure at the 3' end of the AEV IRES lends support to the secondary structure we present. AEV is therefore another example of a picornavirus harboring an HCV-like IRES element within its genome, and thus, its classification within the hepatovirus genus may need to be reassessed in light of these findings.