Pathologic Characterization of Coinfection with Histomonas meleagridis, Marek's Disease Virus, and Subtype J Avian Leukosis Virus in Chickens.

Histomonas meleagridis is a trichomonad protozoan parasite that can cause an important poultry disease known as histomoniasis; Marek's disease virus (MDV) and subtype J avian leukosis virus (ALV-J) usually cause avian oncogenic diseases. Although these diseases have been reported in a single pathogen infection, information about their coinfection is scarce. This study reports a naturally occurring case of coinfection with H. meleagridis, MDV, and ALV-J in a local chicken flock at the age of 150 days. Necropsy revealed necrosis and swelling in the liver and spleen. Histologic analysis showed large areas of mild to severe necrosis of hepatocytes, with numerous intralesional trophozoites of H. meleagridis by H&E and periodic acid-Schiff staining; H&E staining showed pleomorphic and neoplastic lymphoid tumor cells in the liver and myeloid cells with eosinophilic cytoplasmic granules in the spleen. Coexpression of MDV and ALV-J antigens was detected in the liver by fluorescence multiplex immunohistochemistry staining. The 18S rRNA gene of H. meleagridis, meq gene of MDV, and gp85 gene of ALV-J were identified in mixed liver and spleen tissues by PCR and sequencing, respectively.

Reporte de caso­Caracterización patológica de la coinfección con Histomonas meleagridis, el virus de la enfermedad de Marek y el virus de la leucosis aviar subtipo J en pollos Histomonas meleagridis es un parásito protozoario tricomonial que puede causar una enfermedad avícola importante conocida como histomoniasis; El virus de la enfermedad de Marek (MDV) y el virus de la leucosis aviar subtipo J (ALV-J) suelen causar enfermedades oncogénicas aviares. Aunque estas enfermedades se han reportado como infecciones patógenas separadas, la información sobre coinfección es escasa. Este estudio reporta un caso natural de coinfección con H. meleagridis, el virus de la enfermedad de Marek y el virus de la leucosis aviar subtipo J en una parvada de pollos local a la edad de 150 días. La necropsia reveló necrosis e inflamación del hígado y el bazo. El análisis histológico mostró grandes áreas de necrosis de hepatocitos de leve a severa, con numerosos trofozoítos intralesionales de H. meleagridis por tinción de hematoxilina y eosina y por tinción de ácido periódico-Schiff. La tinción de hematoxilina y eosina mostró células linfoides neoplásicas y pleomórficas en el hígado y en el bazo presencia de células mieloides con gránulos citoplásmicos eosinofílicos. La coexpresión de antígenos del virus de Marek y de la leucosis aviar subtipo J se detectó en el hígado mediante tinción inmunohistoquímica de fluorescencia múltiple. El gene de ARNr 18S de H. meleagridis, el gene meq del virus de Marek y el gene gp85 del virus de la leucosis aviar subtipo J se identificaron en tejidos mixtos de hígado y bazo mediante PCR y secuenciación, respectivamente.

Genetic characterization of a Marek's disease virus strain isolated in Japan.

BACKGROUND: Marek's disease virus (MDV) causes malignant lymphomas in chickens (Marek's disease, MD). MD is currently controlled by vaccination; however, MDV strains have a tendency to develop increased virulence. Distinct diversity and point mutations are present in the Meq proteins, the oncoproteins of MDV, suggesting that changes in protein function induced by amino acid substitutions might affect MDV virulence. We previously reported that recent MDV isolates in Japan display distinct mutations in Meq proteins from those observed in traditional MDV isolates in Japan, but similar to those in MDV strains isolated from other countries. METHODS: To further investigate the genetic characteristics in Japanese field strains, we sequenced the whole genome of an MDV strain that was successfully isolated from a chicken with MD in Japan. A phylogenetic analysis of the meq gene was also performed. RESULTS: Phylogenetic analysis revealed that the Meq proteins in most of the Japanese isolates were similar to those of Chinese and European strains, and the genomic sequence of the Japanese strain was classified into the Eurasian cluster. Comparison of coding region sequences among the Japanese strain and MDV strains from other countries revealed that the genetic characteristics of the Japanese strain were similar to those of Chinese and European strains. CONCLUSIONS: The MDV strains distributed in Asian and European countries including Japan seem to be genetically closer to each other than to MDV strains from North America. These findings indicate that the genetic diversities of MDV strains that emerged may have been dependent on the different vaccination-based control approaches.

The pathogenesis of a North American H5N2 clade 2.3.4.4 group A highly pathogenic avian influenza virus in surf scoters (Melanitta perspicillata).

BACKGROUND: Aquatic waterfowl, particularly those in the order Anseriformes and Charadriiformes, are the ecological reservoir of avian influenza viruses (AIVs). Dabbling ducks play a recognized role in the maintenance and transmission of AIVs. Furthermore, the pathogenesis of highly pathogenic AIV (HPAIV) in dabbling ducks is well characterized. In contrast, the role of diving ducks in HPAIV maintenance and transmission remains unclear. In this study, the pathogenesis of a North American A/Goose/1/Guangdong/96-lineage clade 2.3.4.4 group A H5N2 HPAIV, A/Northern pintail/Washington/40964/2014, in diving sea ducks (surf scoters, Melanitta perspicillata) was characterized. RESULTS: Intrachoanal inoculation of surf scoters with A/Northern pintail/Washington/40964/2014 (H5N2) HPAIV induced mild transient clinical disease whilst concomitantly shedding high virus titers for up to 10 days post-inoculation (dpi), particularly from the oropharyngeal route. Virus shedding, albeit at low levels, continued to be detected up to 14 dpi. Two aged ducks that succumbed to HPAIV infection had pathological evidence for co-infection with duck enteritis virus, which was confirmed by molecular approaches. Abundant HPAIV antigen was observed in visceral and central nervous system organs and was associated with histopathological lesions. CONCLUSIONS: Collectively, surf scoters, are susceptible to HPAIV infection and excrete high titers of HPAIV from the respiratory and cloacal tracts whilst being asymptomatic. The susceptibility of diving sea ducks to H5 HPAIV highlights the need for additional research and surveillance to further understand the contribution of diving ducks to HPAIV ecology.

Complete genome sequence of an isolate of duck enteritis virus from China.

Here, we present the complete genomic sequence of duck enteritis virus (DEV) strain SD, isolated in China in 2012. The virus was virulent in experimentally infected 2-month-old ducks. The DEV SD genome is 160,945 base pairs (bp) in length. The viral genome sequence, when compared to that of strain DEV CSC, which was isolated in 1962, showed three discontinuous deletions of 101 bp, 48 bp and 417 bp within the inverted repeats. A comparison of the amino acid (aa) sequences of all ORFs of the CSC and SD isolates demonstrated an11-aa deletion, two single-aa deletions, and one single-aa deletion in LORF3, UL47, UL4, respectively. Moreover, 38 single aa variations were also detected in 24 different ORFs. These results will further advance our understanding of the genetic variations involved in evolution.

Identification of Marek's Disease Virus VP22 Tegument Protein Domains Essential for Virus Cell-to-Cell Spread, Nuclear Localization, Histone Association and Cell-Cycle Arrest.

VP22 is a major tegument protein of alphaherpesviruses encoded by the UL49 gene. Two properties of VP22 were discovered by studying Marek's disease virus (MDV), the Mardivirus prototype; it has a major role in virus cell-to-cell spread and in cell cycle modulation. This 249 AA-long protein contains three regions including a conserved central domain. To decipher the functional VP22 domains and their relationships, we generated three series of recombinant MDV genomes harboring a modified UL49 gene and assessed their effect on virus spread. Mutated VP22 were also tested for their ability to arrest the cell cycle, subcellular location and histones copurification after overexpression in cells. We demonstrated that the N-terminus of VP22 associated with its central domain is essential for virus spread and cell cycle modulation. Strikingly, we demonstrated that AAs 174-190 of MDV VP22 containing the end of a putative extended alpha-3 helix are essential for both functions and that AAs 159-162 located in the putative beta-strand of the central domain are mandatory for cell cycle modulation. Despite being non-essential, the 59 C-terminal AAs play a role in virus spread efficiency. Interestingly, a positive correlation was observed between cell cycle modulation and VP22 histones association, but none with MDV spread.

Use of real-time PCR to rule out Marek's disease in the diagnosis of peripheral neuropathy.

This article reports nine cases of neurological disease in brown layer pullets that occured in various European countries between 2015 and 2018. In all cases, the onset of neurological clinical signs was at 4-8 weeks of age and they lasted up to 22 weeks of age. Enlargement of peripheral nerves was the main lesion observed in all cases. Histopathological evaluation of nerves revealed oedema with moderate to severe infiltration of plasma cells. Marek's disease (MD) was ruled out by real-time PCR as none of the evaluated tissues had a high load of oncogenic MD virus (MDV) DNA, characteristics of MD. Based on the epidemiological data (layers with clinical signs starting at 5-8 weeks of age), gross lesions (peripheral nerve enlargement with a lack of tumours in other organs), histopathological lesions (oedema and infiltration of plasma cells), and no evidence of high load of MDV DNA, we concluded that those cases were due to peripheral neuropathy (PN). PN is an autoimmune disease easily misdiagnosed as MD, leading to a costly enforcement of the vaccination protocol. Additional vaccination against MD does not protect against PN and could worsen the clinical signs by over-stimulating the immune system. Differential diagnosis between PN and MD should always be considered in cases of neurological disease with enlargement of peripheral nerves as the only gross lesion. This case report shows for the first time how real-time PCR to detect oncogenic MDV is a very valuable tool in the differential diagnosis of PN and MD.

Development of reliable techniques for the differential diagnosis of avian tumour viruses by immunohistochemistry and polymerase chain reaction from formalin-fixed paraffin-embedded tissue sections.

A variety of techniques have been developed as diagnostic tools for the differential diagnosis of tumours produced by Marek's disease virus from those induced by avian leukosis virus and reticuloendotheliosis virus. However, most current techniques are unreliable when used in formalin-fixed paraffin-embedded (FFPE) tissues, which often is the only sample type available for definitive diagnosis. A collection of tumours was generated by the inoculation of different strains of Marek's disease virus, reticuloendotheliosis virus or avian leukosis virus singularly or in combination. FFPE tissue sections from tumour and non-tumour tissues were analysed by optimized immunohistochemistry (IHC) techniques and traditional as well as quantitative polymerase chain reaction (PCR) with newly designed primers ideal for DNA fragmented by fixation. IHC and PCR results were highly sensitive and specific in tissues from single-infected birds. Virus quantity was higher in tumours compared to non-tumour spleens from Marek's disease (MD) virus-infected birds. Thus, using FFPE sections alone may be sufficient for the diagnosis of MD by demonstration of high quantities of viral antigens or genome in tumour cells, along with the absence of other tumour viruses by traditional PCR, and if standard criteria are met based on clinical history and histology. IHC furthermore allowed detection of the specific cells that were infected with different viruses in tumours from birds that had been inoculated simultaneously with multiple viruses. Following validation with field samples, these new protocols can be applied for both diagnostic and research purposes to help accurately identify avian tumour viruses in routine FFPE tissue sections.

Characterizing the histopathology of natural co-infection with Marek's disease virus and subgroup J avian leucosis virus in egg-laying hens.

Marek's disease virus (MDV) and avian leucosis virus (ALV) are known to cause tumours in egg-laying hens. Here, we investigated the aetiology of tumours in a flock of egg-laying hens vaccinated against MDV. We carried out gross pathology and histopathological examinations of the diseased tissues, identified virus antigen and sequenced viral oncogenes to elucidate the cause of death in 21-22-week-old hens. At necropsy, diseased hens had distinctly swollen livers, spleens, and proventriculus, and white tumour nodules in the liver. The spleen and liver had been infiltrated by lymphoid tumour cells, while the proventriculus had been infiltrated by both lymphoid tumour cells and myeloblastic cells. Subtype J ALV (ALV-J) and MDV were widely distributed in the proventricular gland cells, and the lymphoid tumour cells in the liver and the spleen. In addition, positive ALV-J signals were also observed in parts of the reticular cells in the spleen. MDV and ALV-J antigens were observed in the same foci of the proventricular gland cells; however, the two antigens were not observed in the same foci from the spleen and liver. The amino acid sequence of the AN-1 (the representative liver tumour tissue that was positive for both ALV-J and MDV) Meq protein was highly similar to the very virulent MDV QD2014 from China. Compared to the ALV-J HPRS-103 reference strain, 10 amino acids (224-CTTEWNYYAY-233) were deleted from the gp85 protein of AN-1. We concluded that concurrent infection with MDV and ALV-J contributed to the tumorigenicity observed in the flock.

Molecular characterization of the duck enteritis virus US10 protein.

BACKGROUND: There is little information regarding the duck enteritis virus (DEV) US10 gene and its molecular characterization. METHODS: Duck enteritis virus US10 was amplified and cloned into the recombinant vector pET32a(+). The recombinant US10 protein was expressed in Escherichia coli BL21 cells and used to immunize rabbits for the preparation of polyclonal antibodies. The harvested rabbit antiserum against DEV US10 was detected and analyzed by agar immunodiffusion. Using this antibody, western blotting and indirect immunofluorescence analysis were used to analyze the expression level and subcellular localization of US10 in infected cells at different time points. Quantitative reverse-transcription PCR (qRT-PCR) and pharmacological inhibition tests were used to ascertain the kinetic class of the US10 gene. A mass spectrometry-based strategy was used to identify US10 in purified DEV virions and quantify its abundance. RESULTS: The recombinant pET32a(+)/US10 protein was expressed as inclusion bodies, purified by gradient urea washing, and used to prepare specific antibodies. The results of qRT-PCR, western blotting, and pharmacological inhibition tests revealed that US10 is mainly transcribed in the late stage of viral replication. However, the presence of the DNA polymerase inhibitor ganciclovir and the protein synthesis inhibitor cycloheximide blocked transcription. Therefore, US10 is a γ2 (true late) gene. Indirect immunofluorescence analysis showed that US10 proteins were initially diffusely distributed throughout the cytoplasm, but with the passage of time, they gradually relocated to a perinuclear region. The US10 protein was detected in purified DEV virions by mass spectrometry, but was not detected by western blotting, indicating that DEV US10 is a minor virion protein. CONCLUSIONS: The DEV US10 gene is a γ2 gene and the US10 protein is localized in the perinuclear region. DEV US10 is a virion component.

A polymerase chain reaction assay for detection of virulent and attenuated strains of duck plague virus.

Sequence analysis of duck plague virus (DPV) revealed that there was a 528bp (B fragment) deletion within the UL2 gene of DPV attenuated vaccine strain in comparison with field virulent strains. The finding of gene deletion provides a potential differentiation test between DPV virulent strain and attenuated strain based on their UL2 gene sizes. Thus we developed a polymerase chain reaction (PCR) assay targeting to the DPV UL2 gene for simultaneous detection of DPV virulent strain and attenuated strain, 827bp for virulent strain and 299bp for attenuated strain. This newly developed PCR for DPV was highly sensitive and specific. It detected as low as 100fg of DNA on both DPV virulent and attenuated strains, no same size bands were amplified from other duck viruses including duck paramyxovirus, duck tembusu virus, duck circovirus, Muscovy duck parvovirus, duck hepatitis virus type I, avian influenza virus and gosling plague virus. Therefore, this PCR assay can be used for the rapid, sensitive and specific detection of DPV virulent and attenuated strains affecting ducks.

An attempt to develop a detection method specific for virulent duck plague virus strains based on the UL2 gene B fragment.

A comparison of the unique long region 2 (UL2) gene sequences between 10 virulent and 11 attenuated duck plague virus (DPV) strains (including all DPV UL2 gene sequences registered in GenBank) showed that the UL2 genes in the attenuated DPV strains had a 528 bp deletion in the B fragment. Primers were designed based on the B fragment sequence of the UL2 gene in an attempt to establish a fluorescence quantitative polymerase chain reaction (PCR) and a conventional PCR detection method that could specifically detect virulent DPV strains (i.e. positive detection for virulent DPV strains and negative detection for attenuated DPV strains). Additionally, PCR products were cloned for sequence analysis. These two methods detected five attenuated DPV strains in addition to the virulent DPV strains. Sequence analysis of the PCR products showed that the amplified products were the B fragments of the UL2 gene. These results indicated that detection methods specific for virulent DPV strains could not be established using primers designed based on the UL2 gene B fragment.

Preliminary study of the UL55 gene based on infectious Chinese virulent duck enteritis virus bacterial artificial chromosome clone.

BACKGROUND: Lethal Duck Enteritis Virus (DEV) infection can cause high morbidity and mortality of many species of waterfowl within the order Anseriformes. However, little is known about the function of viral genes including the conserved UL55 gene among alpha herpes virus due to the obstacles in maintenance and manipulation of DEV genome in host cells. METHODS: In this paper, we constructed an infectious bacteria artificial chromosome (BAC) clone of the lethal clinical isolate duck enteritis virus Chinese virulent strain (DEV CHv) by inserting a transfer vector containing BAC mini-F sequence and selection marker EGFP into UL23 gene using homologous recombination. UL55 deletion and its revertant mutant were generated by two-step RED recombination in E. coli on basis of rescued recombinant virus. The function of UL55 gene in DEV replication and its effect on distribution of UL26.5 protein were carried out by growth characteristics and co-localization analysis. RESULTS: The complete genome of DEV CHv can be stably maintained in E. coli as a BAC clone and reconstituted again in DEF cells. The generated UL55 deletion mutant based on DEV CHv-BAC-G displayed similar growth curves, plaque morphology and virus titer of its parental virus in infected Duck Embryo Fibroblast (DEF) cells. Immunofluorescence assay indicated that the loss of UL55 gene do not affect the distribution of UL26.5 protein in intracellular. These data also suggest infectious BAC clone of DEV CHv will facilitate the gene function studies of DEV genome. CONCLUSIONS: We have successfully developed an infectious BAC clone of lethal clinical isolate DEV CHv for the first time. The generated UL55 gene mutant based on that demonstrated this platform would be a very useful tool for functional study of DEV genes. We found the least known DEV UL55 is dispensable for virus replication and UL26.5 distribution, and it could be a very promise candidate locus for developing bivalent vaccine. Experiment are now in progress for testifying the possibility of UL55 gene locus as an exogenous gene insertion site for developing DEV vectored vaccine.

A Chinese Variant Marek's Disease Virus Strain with Divergence between Virulence and Vaccine Resistance.

Marek's disease (MD) virus (MDV) has been evolving continuously, leading to increasing vaccination failure. Here, the MDV field strain BS/15 was isolated from a severely diseased Chinese chicken flock previously vaccinated with CVI988. To explore the causes of vaccination failure, specific-pathogen free (SPF) chickens vaccinated with CVI988 or 814 and unvaccinated controls were challenged with either BS/15 or the reference strain Md5. Both strains induced MD lesions in unvaccinated chickens with similar mortality rates of 85.7% and 80.0% during the experimental period, respectively. However, unvaccinated chickens inoculated with BS/15 exhibited a higher tumor development rate (64.3% vs. 40.0%), but prolonged survival and diminished immune defects compared to Md5-challenged counterparts. These results suggest that BS/15 and Md5 show a similar virulence but manifest with different pathogenic characteristics. Moreover, the protective indices of CVI988 and 814 were 33.3 and 66.7 for BS/15, and 92.9 and 100 for Md5, respectively, indicating that neither vaccine could provide efficient protection against BS/15. Taken together, these data suggest that MD vaccination failure is probably due to the existence of variant MDV strains with known virulence and unexpected vaccine resistance. Our findings should be helpful for understanding the pathogenicity and evolution of MDV strains prevalent in China.

Complete genome sequence and evolution analysis of a columbid herpesvirus type 1 from feral pigeon in China.

Here, we report the genome sequence of a feral pigeon alphaherpesvirus (columbid herpesvirus type 1, CoHV-1), strain HLJ, and compare it with other avian alphaherpesviruses. The CoHV-1 strain HLJ genome is 204,237 bp in length and encodes approximately 130 putative protein-coding genes. Phylogenetically, CoHV-1 complete genome resides in a monophyletic group with the falconid herpesvirus type 1 (FaHV-1) genome, distant from other alphaherpesviruses. Interestingly, the evolutionary analysis of partial genes of CoHV-1 isolated from different organisms and areas (currently accessible on GenBank) indicates that the CoHV-1 HLJ strain isolated from pigeon (Columba livia) is closely related to the strains isolated from peregrine falcon (Falco peregrinus) in Poland and owl (Bubo virginianus) in USA. These results may suggest possible transmission of the virus between different organisms and different geographic areas.

A novel alphaherpesvirus associated with fatal diseases in banded Penguins.

A novel avian alphaherpesvirus, preliminarily designated sphenicid alphaherpesvirus 1 (SpAHV-1), has been independently isolated from juvenile Humboldt and African penguins (Spheniscus humboldti and Spheniscus demersus) kept in German zoos suffering from diphtheroid oropharyngitis/laryngotracheitis and necrotizing enteritis (collectively designated as penguin-diphtheria-like disease). High-throughput sequencing was used to determine the complete genome sequences of the first two SpAHV-1 isolates. SpAHV-1 comprises a class D genome with a length of about 164 kbp, a G+C content of 45.6 mol% and encodes 86 predicted ORFs. Taxonomic association of SpAHV-1 to the genus Mardivirus was supported by gene content clustering and phylogenetic analysis of herpesvirus core genes. The presented results imply that SpAHV-1 could be the primary causative agent of penguin-diphtheria-like fatal diseases in banded penguins. These results may serve as a basis for the development of diagnostic tools in order to investigate similar cases of penguin diphtheria in wild and captive penguins.

Co-occurrence of Mycoplasma Species and Pigeon Herpesvirus-1 Infection in Racing Pigeons ( Columba livia).

Oropharyngeal swab samples were collected from 438 live racing pigeons ( Columba livia), with and without signs of respiratory disease, that were housed in 220 lofts in 3 provinces in the western part of the Netherlands. Polymerase chain reaction (PCR) was used to identify Mycoplasma species and pigeon herpesvirus-1 (PHV-1) from the samples. In 8.6% of the pigeon lofts tested, signs of respiratory disease were present in pigeons at sampling, and in 30.9% of the sampled pigeon lofts, respiratory signs were observed in pigeons during the 6-month period immediately before sampling. A total of 39.8% of tested pigeons (54.5% of tested lofts) were positive for Mycoplasma species, and 30.6% of tested pigeons (48.6% of tested lofts) were positive for PHV-1. In 15.8% of the tested pigeons (26.8% of tested pigeon lofts), coinfection by Mycoplasma species and PHV-1 was identified. The number of pigeon lofts having pigeons coinfected by Mycoplasma species and PHV-1 was higher than that where only one of the infections was identified. Neither the presence of Mycoplasma species, PHV-1, nor the co-occurrence of both infections was significantly associated with signs of respiratory disease.

Field studies of the detection, persistence and spread of the Rispens CVI988 vaccine virus and the extent of co-infection with Marek's disease virus.

OBJECTIVE: To use specific real-time qPCR to determine (1) the vaccination success of Rispens CVI988 vaccine in feathers and dust; (2) persistence of Rispens infection in vaccinated layer chickens; (3) extent of co-infection with wild-type Marek's disease virus (MDV) in vaccinated layers; and (4) presence of Rispens virus in unvaccinated broiler flocks. METHODS: Feather, dust and serum samples were collected from birds aged 3 days to 91 weeks from three layer farms. qPCR was used to detect MDV and Rispens in DNA extracted from dust and feathers. Previously tested MDV-positive dust samples from 100 broiler flocks were tested for the presence of Rispens using qPCR, while serum samples were used to detect anti-MDV antibody using ELISA. RESULTS: Overall, 66% and 93% of feather and dust samples, respectively, from Rispens-vaccinated layers were Rispens-positive. Viral load in these samples varied between farms during early life, reaching readily detectable levels at 2-3 weeks of age. Vaccinated chickens maintained a high Rispens load in feathers and dust and high MDV antibody levels until 91 weeks of age. MDV infection was detected in 6.7% of feather samples from vaccinated chickens. Rispens virus was detected in 7% of samples from unvaccinated broiler flocks. CONCLUSION: Vaccine take can be measured effectively by Rispens-specific qPCR of feathers or dust from approximately 3 weeks post vaccination. Infection with Rispens is persistent, with lifelong shedding and serological response. The detectable infection rate of vaccinated chickens with MDV is low and there is preliminary evidence of escape of Rispens virus to unvaccinated flocks.

Outbreak of Marek's disease in a vaccinated broiler breeding flock during its peak egg-laying period in China.

BACKGROUND: Outbreaks of Marek's disease (MD), caused by Marek's disease virus (MDV), primarily occur in 10-12-week-old hens. CASE PRESENTATION: We report a case of MD in a breeding flock of 24-30-week-old vaccinated broilers in China. The clinical signs in the affected chickens appeared at 24 weeks, and the incidence of tumours peaked at 30 weeks. The morbidity and mortality of the hens were 5 % and 80 %, respectively. Hematoxylin-eosin staining of the tissues showed the typical characteristics of MD. MDV infection was confirmed in the hens with an agar gel diffusion precipitation assay for the MD antigen in the feather follicle epithelium. An MDV strain, designated AH1410, was isolated from the blood lymphocytes. Sequence analyses of the pp38, meq, and gB genes revealed that strain AH1410 had molecular features consistent with a virulent, previously identified MDV. CONCLUSION: Our data provide evidence that not only is MDV becoming more virulent, but that the period of its onset in chickens is expanding. These findings provide the basis the molecular surveillance and further study of virulent MDV mutants and control strategies for MD in China.

In vivo characterisation of two Australian isolates of Marek's disease virus including pathology, viral load and neuropathotyping based on clinical signs.

OBJECTIVE: To evaluate the pathogenicity of Australian Marek's disease virus (MDV) isolate MPF23 (1985) against the reference strain MPF57 based on pathology, viral load and neuropathotyping on the basis of clinical signs. PROCEDURE: Two MDV challenge isolates (MPF57 or MPF23) were administered to unvaccinated specific-pathogen free (SPF) layer chicks on day 5 after hatch at three challenge doses (500, 2000 or 8000 plaque-forming units (pfu)/chick). Mortality, body weight, immune organ weights, MDV load in peripheral blood lymphocytes (PBL) and clinical signs were measured to 56 days post challenge (dpc). RESULTS: MPF23 was the more pathogenic of the two viruses, inducing higher mortality (81% vs 62%) and incidence of MD lesions (100% vs 76%). MPF23 induced earlier, more sustained and more severe neurological signs in the period 26-56 dpc. However, there were few differences during the 0-23 dpc used in the neuropathotyping classification under test. The observed pattern during this earlier period classified both viruses as neuropathotype B, consistent with a very virulent pathotype. MDV load in PBL at 7 and 44 dpc did not differ between virus isolates, but the load at 7 dpc was significantly and negatively associated with time to euthanasia or death. CONCLUSION: MPF23 appears to be as, or more, virulent than the MDV strains isolated over the subsequent two decades. The neuropathotyping system developed in the USA did not clearly differentiate between the two isolates under test; however, extension of the period of assessment of clinical signs beyond 26 dpc did reveal clear differences.

Establishment of six homozygous MHC-B haplotype populations associated with susceptibility to Marek's disease in Chinese specific pathogen-free BWEL chickens.

The highly polymorphic chicken major histocompatibility complex (MHC) is associated with different levels of immunologic responses to certain avian pathogens. MHC-B haplotype chickens are an important genetic resource for studying the genetic determination of pathogen resistance and susceptibility. The BWEL chicken population is the only specific pathogen-free (SPF) chickens bred and developed by the State Center of Poultry Genetic Resources of Laboratory Animals in China. In this study, we successfully established six homozygous MHC-B haplotype populations from the BWEL chickens using microsatellite marker technology, named as BW/G(1, 2, 3, 5, 6, 7) lines, and their molecular genotypes were matched to six serologically defined MHC-B haplotypes, B13, B15, B2, B5, B21 and B19, respectively. The sequences of BF genes exons 2 and 3 from four successive generations (F1-F4) of the BW/G(n) lines were completely consistent with those of serologically defined MHC-B haplotypes. Subsequently, six BW/G(n) line specific allo-antisera were prepared by immunization with red blood cells (RBCs) and hemagglutination tests results showed the BW/G(n) SPF chickens could be serologically differentiated. Additionally, susceptibility to Marek's disease (MD) in the BW/G3 (B2 haplotype) and BW/G7 (B19 haplotype) lines were determined by comparing mortality, macroscopic and histopathological lesions, and viral loads in feather pulp. The BW/G7 line showed greater genetic susceptibility to the very virulent MD virus (MDV) strain than the BW/G3 line. The establishment of MHC-B haplotype chicken populations associated with susceptibility to MD will be helpful for studying host immune responses and further developing the more effective vaccines in the context of MHC specificities, and they are also very useful for an understanding of MHC genes architecture and function.