Marek's disease in chicken farms from Northwest Ethiopia: gross pathology, virus isolation, and molecular characterization.

Marek's disease virus (MDV) is a highly contagious, immunosuppressive, and oncogenic chicken pathogen causing marek's disease (MD). In this outbreak-based study, 70 dual-purpose chickens that originated from poultry farms in Northwest Ethiopia and suspected of MD were sampled for pathological and virological study from January 2020 to June 2020. Clinically, affected chickens showed inappetence, dyspnea, depression, shrunken combs, and paralysis of legs, wings, and neck, and death. Pathologically, single or multiple greyish white to yellow tumor-like nodular lesions of various size were appreciated in visceral organs. In addition, splenomegaly, hepatomegaly, renomegaly, and sciatic nerve enlargement were observed. Twenty-seven (27) pooled clinical samples i.e. 7 pooled spleen samples and 20 pooled feathers samples were aseptically collected. Confluent monolayer of Chicken Embryo Fibroblast cells was inoculated with a suspension of pathological samples. Of this, MDV-suggestive cytopathic effects were recorded in 5 (71.42%) and 17 (85%) pooled spleen and feather samples respectively. Molecular confirmation of pathogenic MDV was conducted using conventional PCR amplifying 318 bp of ICP4 gene of MDV-1, of which, 40.9% (9/22) tested positive. In addition, 5 PCR-positive samples from various farms were sequenced further confirming the identity of MDV. The ICP4 partial gene sequences were submitted to GenBank with the following accession numbers: OP485106, OP485107, OP485108, OP485109, and OP485110. Comparative phylogenetics showed, two of the isolates from the same site, Metema, seem to be clonal complexes forming distinct cluster. The other three isolates, two from Merawi and one from Debretabor, appear to represent distinct genotypes although the isolate from Debretabor is closer to the Metema clonal complex. On the other hand, the isolates from Merawi appeared genetically far related to the rest of the 3 isolates and clustered with Indian MDV strains included in the analysis. This study presented the first molecular evidence of MDV in chicken farms from Northwest Ethiopia. Biosecurity measures should strictly be implemented to hinder the spread of the virus. Nationwide studies on molecular characteristics of MDV isolates, their pathotypes, and estimation of the economic impact associated with the disease may help justify production and use of MD vaccines within the country.

Differential expression profile and in-silico functional analysis of long noncoding RNA and mRNA in duck embryo fibroblasts infected with duck plague virus.

BACKGROUND: Duck plague virus (DPV), belonging to herpesviruses, is a linear double-stranded DNA virus. There are many reports about the outbreak of the duck plague in a variety of countries, which caused huge economic losses. Recently, increasing reports revealed that multiple long non-coding RNAs (lncRNAs) can possess great potential in the regulation of host antiviral immune response. Furthermore, it remains to be determined which specific molecular mechanisms are responsible for the DPV-host interaction in host immunity. Here, lncRNAs and mRNAs in DPV infected duck embryonic fibroblast (DEF) cells were identified by high-throughput RNA-sequencing (RNA-seq). And we predicted target genes of differentially expressed genes (DEGs) and formed a complex regulatory network depending on in-silico analysis and prediction. RESULT: RNA-seq analysis results showed that 2921 lncRNAs were found at 30 h post-infection (hpi). In our study, 218 DE lncRNAs and 2840 DE mRNAs were obtained in DEF after DPV infection. Among these DEGs and target genes, some have been authenticated as immune-related molecules, such as a Macrophage mannose receptor (MR), Anas platyrhynchos toll-like receptor 2 (TLR2), leukocyte differentiation antigen, interleukin family, and their related regulatory factors. Furthermore, according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analysis, we found that the target genes may have important effects on biological development, biosynthesis, signal transduction, cell biological regulation, and cell process. Also, we obtained, the potential targeting relationship existing in DEF cells between host lncRNAs and DPV-encoded miRNAs by software. CONCLUSIONS: This study revealed not only expression changes, but also the possible biological regulatory relationship of lncRNAs and mRNAs in DPV infected DEF cells. Together, these data and analyses provide additional insight into the role of lncRNAs and mRNAs in the host's immune response to DPV infection.

First report of Serotype-1 Marek's disease virus (MDV-1) with oncogenic form in backyard turkeys in Turkey: a molecular analysis study.

BACKGROUND: Marek's disease (MD) is a lymphoproliferative disease caused by Gallid alphaherpesvirus 2 (GaHV-2, MDV-1), which primarily affects chickens. However, the virus is also able to induce tumors and polyneuritis in turkeys, albeit less frequently than in chickens. RESULTS: This is the first study in Turkey reporting the molecular characterization of a MDV-1 strain detected in a flock of backyard turkeys exhibiting visceral lymphoma. Here, MEQ, vIL-8, pp38 and 132-bp tandem repeat regions, which are frequently preferred in the pathotyping of MDV-1, were examined. It was determined that the MEQ gene of MDV-1/TR-21/turkey strain obtained in the present study encoded 339 amino acids (1020 nt) and had four proline-rich repeat regions (PPPP). Based on the nucleotide sequence of the MEQ gene of the MDV-1/TR-21/turkey strain, a phylogenetic tree was created using the MEGA-X software with the Maximum Likelihood Method (in 1000 replicates). Our strain was highly identical (> 99.8) to the Italian/Ck/625/16, Polish (Polen5) and some Turkish (Layer-GaHV-2-02-TR-2017, Tr/MDV-1/19) MDV-1 strains. Also, nt and aa sequences of the MEQ gene of our strain were 99.1 and 99.41% identical to another Turkish strain (MDV/Tur/2019) originated from chickens. Sequence analysis of pp38 and vIL-8 genes also supported the above finding. The identity ratios of nucleotide and amino acid sequences of vIL-8 and pp38 genes of MDV-1/TR-21/turkey strain were 99.64-100% and 99.79-100%, respectively, when compared with those of the Polish strain. According to 132-bp tandem repeat PCR results, the MDV-1/TR-21/turkey strain had five copies. CONCLUSIONS: These results suggested that the MDV-1/TR-21/turkey strain obtained from backyard turkeys can be either very virulent or very virulent plus pathotype, though experimental inoculation is required for precise pathotyping.

Report of a new meq gene size: The first study on genetic characterisation of Marek's disease viruses circulating in Iranian commercial layer and backyard chicken.

1. In recent months, several outbreaks with clinical signs of MDV-1 were reported in Iranian parent and laying hen farms, in addition to backyard chickens. Several meq gene sequences from these outbreaks were amplified and molecularly characterised.2. The meq protein sequences revealed three different sizes, namely the standard 339 aa, a shorter form of 338 aa lacking a proline residue at position 191, and a very short (vs) size of 265 aa. Based on sequence and size, the 265 aa meq has never been reported from international research groups before. The protein has only one PPPP repeat motif suggesting it belongs to a highly virulent strain.3. The standard meq sequences showed 100% BLAST identity to the vv+ isolate Polen5. However, the 338 aa form clustered to the clade usually reported from North America.4. This is the first report on genetic analysis of MDV-1 from Iran, but further study is required to obtain a better picture of the diversity and prevalence of different MDV-1 strains circulating in the country's farms, backyard poultry and other bird species.

Molecular characterisation and phylogenetic analysis of Marek's disease virus in Turkish layer chickens.

1. There is no current data about the genotypes of Marek's disease virus (MDV) in Turkish poultry flocks; hence, this study was performed to analyse CVI988/Rispens, turkey herpesvirus (HVT) vaccine viruses and MDV field viruses as well as to perform phylogenetic analysis of MDV in Turkish layer chickens. 2. In 2017 and 2018, a total of 602 spleen samples from 49 layer flocks were collected from the Marmara, West Black Sea and Aegean regions. DNA was extracted from the spleen samples and the samples were analysed by real-time PCR probe assay to detect CVI988/Rispens and HVT vaccine viruses and MDV field strains. Samples found positive for MDV by real-time PCR were subjected to PCR using the Meq gene primers for phylogenetic analysis. 3. Amongst 49 flocks, virulent MDV was detected in nine flocks. CVI988/Rispens and HVT vaccine strains were detected in 47 flocks and HVT in all 49 flocks. Splenomegaly, hepatomegaly and tumours in the oviduct were observed in chickens of affected flocks. Virulent MDV was detected in 120 out of 602 spleen samples. Sequencing and phylogenetic analyses showed that MDVs detected in this study were closely related to MDV strains from Italy, Poland, Saudi Arabia, Iraq, India and China but showed diversity with MDV strains from Egypt and Hungary. Multiple sequence analysis of the Meq protein revealed several point mutations in deduced amino acid sequences. Interestingly, CVI988/Rispens vaccine virus from China (AF493555) showed mutations at position 66 (G66R) and 71 (S66A) along with two other vaccine strains from China (GU354326.1) and Russia (EU032468.1), in comparison with the other vaccine strain CVI988/Rispens (DQ534538). The molecular analyses of the Meq gene suggested that Turkish field strains of MDV are in the class of virulent or very virulent pathotypes. 4. The results have shown that MDV still affects poultry health, and the phylogenetic and amino acid variation data obtained will help in vaccination and control strategies.

Co-infection of vvMDV with multiple subgroups of avian leukosis viruses in indigenous chicken flocks in China.

BACKGROUND: In China, although the ALV eradication program and the MD vaccination strategy greatly reduce the disease burdens caused by the infection of ALV and MDV, the frequent emergence of novel ALV-K or vvMDV in the vaccinated chicken flock challenges the current control strategies for both diseases. RESULTS: In Guangdong Province, an indigenous chicken flock was infected with neoplastic disease. Hematoxylin-eosin staining of the tissues showed the typical characteristics of MDV and classical ALV infection. The PCR and sequencing data demonstrated that the identified MDV was clustered into a very virulent MDV strain endemic in domestic chickens in China. Moreover, subgroups ALV-A and ALV-K were efficiently recovered from two samples. The full genome sequence revealed that the ALV-K isolate was phylogenetically close to the ALV TW3593 isolate from Taiwan Province. CONCLUSIONS: A co-infection of vvMDV with multiple ALV subgroups emerged in a chicken flock with neoplastic disease in Guangdong Province. The co-infection with different subgroups of ALV with vvMDV in one chicken flock poses the risk for the emergence of novel ALVs and heavily burdens the control strategy for MDV.

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 and phylogenetic analysis of a virulent Marek's disease virus field strain in broiler chickens in Japan.

Marek's disease is a lymphoproliferative disease causing a serious threat in poultry production. Field strains of Marek's disease virus (MDVs) are continuously re-emerging, causing great economical losses to the poultry industry worldwide in spite of the intensive vaccination and restrictive management policy used. Histopathological and molecular characterizations of MDVs are essential for monitoring the changes of viruses and evaluating the effectiveness of existing vaccines. During 2016, 190 visceral tumour tissues representing 30 vaccinated chicken flocks from the Gifu prefecture, Japan, were analysed. A pathological examination revealed the presence of lymphoproliferative lesions in the visceral organs. Polymerase chain reaction screening of tissue specimens using specific primers for avian leucosis virus, reticuloendotheliosis virus, and MDV was positive only for MDV. The polymerase chain reaction products of meq, pp38, virus-induced IL-8 homology, and glycoprotein MDV genes were sequenced and used for homology, phylogenetic, and similarity level analysis with the published reference of MDVs in the database. The results revealed high similarity between the field isolates, vv and vv+ strains of MDV from the USA and China. Several point mutations in the nucleotide sequence of the field isolates and their deduced amino acid sequences were detected in those genes. The present molecular analyses indicated that nucleotide and amino acid changes could be valuable criteria for differentiation and determination of the pathogenicity and oncogenicity of MDVs according to the Avian Disease and Oncology Laboratory pathotyping in vivo studies. Furthermore, the results suggest that development of a new vaccine must be considered to overcome this devastating avian oncogenic viral disease.

Investigation of Marek's disease virus from chickens in central Ethiopia.

Marek's disease (MD) is a lymphoproliferative and neuropathic disease of domestic chickens and less commonly, turkeys and quails, caused by a highly contagious, cell-associated, oncogenic herpesvirus. In Ethiopia, MD is believed to be introduced with importation of exotic and crossbred to improve the poultry production and has been reported to be a potential threat to the poultry sector both in backyard and commercial farming systems. This study was aimed at isolation and molecular analysis of MD virus isolates circulating in chicken population in the central part of Ethiopia where commercial farms are populated. From September 2013 to January 2014, clinical and post-mortem examination were conducted on diseased chickens suspected of MD virus infection. Representative spleen and feather follicle samples were collected following sterile procedure, and infectious virus isolation was performed using primary chicken fibroblast cell culture. Cell culture inoculated with suspension of pathological samples developed characteristic MD virus cytopathic effect of rounding of the cells and small plaques. Further analysis of the virus was conducted by conventional PCR amplifying the ICP4 gene fragment from eleven tissue samples using MD virus specific primers. PCR products were further sequenced and analyzed. Nucleotide sequence similarity search of the local isolates resulted a high degree of sequence similarity with Gallid Herpes virus type 2 strain (Marek's disease virus type 1, JN034558). To our knowledge, the present study is the first report conducted on virus isolation and molecular characterization of MD virus isolates circulated in Ethiopia. Eleven ICP4-like gene fragment (318 bp) sequences generated in the present study were uploaded in the public database (KU842366-76). Further research on virus isolation, genetic characterization, and infection dynamics is recommended targeting chickens of all age groups reared in different agro-ecological zones under different production system.

Molecular and pathogenicity characterization of Gallid herpesvirus 2 newly isolated in China from 2009 to 2013.

During the course of our continuous surveillance of Gallid herpesvirus 2 (GaHV-2), 44 isolates were obtained from GaHV-2-positive chickens of different flocks in China from 2009 to 2013. The meq gene, considered as a major GaHV-2 oncogene, was sequenced and was found to contain an open reading frame of 1020 nucleotides encoding a 339 amino acid (aa) polypeptide in all isolates. Compared with the GaHV-2 GA strain, the meq genes in 15.9 % (7/44) of the isolates analyzed in this study contained an aa substitution mutation at position 88 (A to T) of which is the first report. The main characteristics of Chinese GaHV-2 isolates meq genes included the substitutions K77E, D80Y, V115A, T139A, P176R, and P217A, and the aa substitution frequency at positions 139 and 176 showed an increase. To test the pathogenicity of the isolates, a pathogenicity study and a vaccination-challenge test were performed on three selected isolates (ZY/1203, WC/1203, and WC/1110) and reference strain GA. The results showed that the three isolates induced gross Marek's disease (MD) lesions in 95.0-100 % cases, which was a higher rate than that obtained for strain GA (82.4 %). Three isolates induced mortality in 10-21.1 % of specific-pathogen-free chickens, which was similar to results with strain GA (23.5 %). The commercially available CVI988 vaccine induced lower protective indices (PIs) against ZY/1203 (82.4) and WC/1110 (83.3) as compared to those against WC/1203 (100) and GA (100). These results showed an evolving trend in the meq genes of the isolates; three isolates exhibited higher morbidity as compared to the reference strain and the vaccine induced lower PIs against two isolates as compared to that against the reference strain.

Disease eradication on large industrial farms.

Many modern farms exhibit all-in-all-out dynamics in which entire cohorts of livestock are removed from a farm before a new cohort is introduced. This industrialization has enabled diseases to spread rapidly within farms. Here we look at one such example, Marek's disease. Marek's disease is an economically important disease of poultry. The disease is transmitted indirectly, enabling the spread of disease between cohorts of chickens who have never come into physical contact. We develop a model which allows us to track the transmission of disease within a barn and between subsequent cohorts of chickens occupying the barn. It is described by a system of impulsive differential equations. We determine the conditions that lead to disease eradication. For a given level of transmission we find that disease eradication is possible if the cohort length is short enough and/or the cohort size is small enough. Marek's disease can also be eradicated from a farm if the cleaning effort between cohorts is large enough. Importantly complete cleaning is not required for eradication and the threshold cleaning effort needed declines as both cohort duration and size decrease.

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.

First survey of the occurrence of duck enteritis virus (DEV) in free-ranging Polish water birds.

Duck plague (DP) caused by anatid herpesvirus 1, also called duck enteritis virus (DEV), presents one of the most important concerns in mass waterfowl production. Apart from geese and ducks, free-ranging water birds are the most notorious infection carriers. The epidemiology of DEV in Western Europe remains unknown. Therefore, it was reasonable to conduct a study on its occurrence using modern but simple real-time loop-mediated isothermal amplification (LAMP). Analysis of 132 field isolates showed the presence of DEV in 96 birds (72.7 %), and it was found predominantly in wild ducks (Anas platyrhynchos) and mute swans (Cygnus olor). This virus was also found in graylag geese (Anser anser), tundra bean geese (Anser fabalis), and grey herons (Ardea cinerea). The results were recorded as green colour of positive samples, fluorescence under ultraviolet light, and florescent curves in a real-time PCR system. This study indicates the high prevalence of DEV among free-ranging water birds in Poland and the possible transmission to other birds settling in the water environment. This is the first report of DEV detection among free-ranging water birds in Poland.

Molecular evolution of Marek's disease virus (MDV) field strains in a 40-year time period.

Marek's disease (MD) presents a serious threat in poultry production. The disease has been limited for over 40 yr by protective vaccination. The widely applied vaccination against MD is also one of the factors causing evolutionary pressure onto field Marek's disease virus (MDV) virulent strains. Molecular evolution of MDV genes involved in oncogenesis may increase the pathogenicity of MDV virulent strains. The goal of the presented study was to sum up the molecular evolution of MDV field strains isolated in the last 40 yr in Poland. In total, 85 field MDV strains collected between 1974 and 2012 were propagated in chicken embryo fibroblasts. After DNA extraction, three sets of primers were designed for PCR complementary to the MDV076 (RLORF7) region encoding the meq oncogene as well to the MDV077 (23 kDa protein binding alpha-enolase) and MDV077.5 (RLORF6) genes. The obtained 85 MDV076, 60 MDV077, and 58 MDV077.5 cloned fragments were sequenced and aligned with the sequences of reference MDV strains showing different pathogenicity levels. The retrieved nucleotide (nt) and deduced amino acid sequences of RLORF7, 23 kDa protein, and LORF6 of Polish field strains showed several mutations and substitutions homologous to those observed in reference strains with a determined pathogenicity. The observed changes indicated the continuous evolution of field MDV strains. The RLORF7 nt sequence of analyzed MDV isolates showed similarity to virulent and very virulent MDV reference strains. The obtained 23 kDa and LORF6 nt sequences provided more important data and were more similar to mildly pathogenic strains than to virulent and very virulent MDV. The specific nt motifs in all three genes may indicate an increase of MDV virulence and were found in strains starting from 2006. According to the obtained results, the strains isolated in 2012 are similar to the very virulent plus MDV group. The study showed that RLORF7, 23 kDa protein, and RLORF6 fragments harbor sequence motifs that may have some association with MDV pathogenicity level. However, the exact role of the investigated regions in pathogenicity should be further examined by knock-out MDV strains. Also, the true MDV pathotype may only be determined by traditional in vivo experiments.

Marek's Disease in a Flock of Japanese Quails (Coturnix japonica) in Germany.

A case of increased mortality was investigated at a German farm housing Japanese quails (Coturnix japonica) for egg production. Different age groups were kept in aviaries in one barn. The quail chicks had diarrhea and the adults were emaciated, some also with diarrhea and conjunctivitis. Postmortem examination showed gross tumorlike lesions in 7 of 15 adult quails examined, with ocular lesions in 2 of them. On histopathological examination, infiltrates of monomorphic round cells were found in liver, spleen, lung, and proventriculus. In the eyes of two quails, similar cells were infiltrating the choroid layer, limbus, and adjacent parts of the cornea. Malignant lymphoma was diagnosed. Immunohistochemical examination identified tumor cells as T cells, and Mardivirus gallidalpha 2-specific PCR was positive for five quails with gross lesions. Additionally, Proteus mirabilis and Klebsiella pneumoniae were detected in the chicks and Clostridium spp. and coccidia in the adults. Marek's EcoQ protein (Meq) gene from Mardivirus gallidalpha 2 was sequenced and analyzed, confirming Marek's disease. The results of our examinations demonstrate that the ocular lesions were caused by Marek's disease and that the Meq gene from Mardivirus gallidalpha 2 was detected in the flock of quails. As a control strategy for Marek's disease in quails, an all-in/all-out system was introduced. Additional laying quails acquired from a breeder at 35 days of life were vaccinated at stabling with a combined turkey herpesvirus (HVT)-Rispens vaccine and 14 days later with a Rispens vaccine. Subsequently, the losses and laying rates returned to normal.

Reporte de caso- Enfermedad de Marek en una parvada de codornices japonesas (Coturnix japonica) en Alemania. Se investigó un caso de aumento de la mortalidad en una granja alemana que alberga codornices japonesas (Coturnix japonica) para la producción de huevo. Se alojaron diferentes grupos de edad en una caseta. Los polluelos de codorniz tenían diarrea y los adultos presentaron emaciación, algunos también con diarrea y conjuntivitis. El examen post mortem mostró lesiones tumorales macroscópicas en 7 de 15 codornices adultas examinadas, con lesiones oculares en dos de ellas. En el examen histopatológico se encontraron infiltrados de células redondas monomórficas en el hígado, el bazo, los pulmones y el proventrículo. En los ojos de dos codornices, células similares infiltraron la capa coroidea, el limbo y las partes adyacentes de la córnea. Se diagnóstico linfoma maligno. El examen inmunohistoquímico identificó células tumorales como células T, y el método de PCR específico para Mardivirus gallidalpha 2 fue positivo con cinco codornices que presentaban lesiones macroscópicas. Además, se detectó Proteus mirabilis y Klebsiella pneumoniae en los polluelos y Clostridium spp. y coccidias en las aves adultas. Se secuenció y analizó el gene de la proteína EcoQ (Meq) de Mardivirus gallidalpha 2, lo que confirmó la enfermedad de Marek. Los resultados de estos exámenes demuestran que las lesiones oculares fueron causadas por la enfermedad de Marek y que el gene Meq del Mardivirus gallidalpha 2 fue detectado en la parvada de codornices. Como estrategia de control de la enfermedad de Marek en codornices, se introdujo un sistema todo dentro/todo fuera. Codornices de postura adicionales adquiridas de un criador a los 35 días de vida fueron vacunadas cuando se alojaron en la caseta con una vacuna combinada contra el herpesvirus del pavo (HVT) y Rispens y 14 días después con una vacuna Rispens. Posteriormente, las pérdidas y los índices de postura volvieron a la normalidad.

Molecular characterization of Marek's Disease virus reveals reticuloendotheliosis virus-long terminal repeat integration in the genome of the field isolates in Egypt.

The highly contagious, immunosuppressive, and cancer-causing Marek's disease virus (MDV) infects chickens. The financial costs of Marek's disease (MD) are significant for the chicken industry. In this study, a total of 180 samples from chicken farms suspected to be MDV-infected were collected. The chickens were sampled during the period between the months of October 2016 and February 2018 at Dakahlia and Damietta Governorates, Egypt. A total of 36 pooled samples were created. The prepared samples were inoculated into embryonated chicken eggs (ECEs). Indirect fluorescent antibody technique (IFAT) and ICP4 gene-based polymerase chain reaction (PCR) were used for MDV identification. For the genetic characterization of the identified virus, The ICP4 gene sequence was identified and compared with the sequences available from various regions of the world. Furthermore, the genomes of all detected MDVs were screened for the long terminal repeat (LTR) region of reticuloendotheliosis (REV) in their genomes. The results showed that 31 out of 36 pooled samples (86.1%) inoculated into ECEs displayed the characteristic pock lesions. By using IFAT and PCR to identify MDV in ECEs, positive results were found in 27 samples (75%). The Egyptian virus is thought to be genetically closely related to MDVs circulating in Ethiopia, China, and India. REV-LTR was amplified from 6 out of 27 field isolates genomes (22.2 %) while MDV vaccine strains were free from REV-LTR insertion. The integrated REV-LTRs depicted a close genetic relationship with those integrated in fowl poxvirus (FWPV) circulating in Egypt as well as those integrated in FWPVs and MDVs from China, USA, South Africa, and Australia. To the best of our knowledge, this investigation represents the first identification and characterization of REV-LTR insertions in Egyptian MDV field isolates. Given the findings above, additional research in the future seems crucial to determine how the REV-LTR insertions affect MDV pathogenesis, virulence, and insufficient vaccination protection.

Molecular characterization of the meq oncogene of Marek's disease virus in vaccinated Brazilian poultry farms reveals selective pressure on prevalent strains.

Marek's disease virus (MDV) has become an increasingly virulent pathogen in the poultry industry despite vaccination efforts to control it. Brazil has experienced a significant rise of Marek's disease (MD) outbreaks in recent years. Our study aimed to analyze the complete meq gene sequences to understand the molecular epidemiological basis of MD outbreaks in Brazilian vaccinated layer farms. We detected a high incidence rate of visceral MD (67.74%) and multiple circulating MDV strains. The most prevalent and geographically widespread genotype presented several clinical and molecular characteristics of a highly virulent strain and evolving under positive selective pressure. Phylogenetic and phylogeographic analysis revealed a closer relationship with strains from the USA and Japan. This study sheds light on the circulation of MDV strains capable of infecting vaccinated birds. We emphasize the urgency of adopting preventive measures to manage MDV outbreaks threatening the poultry farming industry.

Isolation and analysis of a very virulent Marek's disease virus strain in China.

BACKGROUND: A severe MD was broken out at a farm in Shandong, China, despite FC126 vaccination of the chickens at 1-day-old. The mortality of the flocks reached up to 38.3%. The infected chickens were found to have MD pathological changes, including enlargement of spleens, livers and kidneys, and tumors occured on organs later. Samples were collected from the chickens for diagnosis. METHODS: The collected samples were inoculated into primary duck embryo fibroblast (DEF) cells, and the MDV strain named SD2012-1 was isolated. In order to identify the isolate, amplification by PCR and sequencing of oncogenic Meq and vIL-8 gene were processed, the obtained sequences were compared with the sequences of reference strains, and SD2012-1 was used to challenge immunized SPF chickens. RESULTS: A very virulent MDV isolate strain, SD2012-1, was isolated from a chicken flock in Shandong Province, China, the isolate had the characteristics of very virulent MDV-1, nucleotide and deduced amino acid sequence comparisons of Meq and vIL-8 gene of SD2012-1 with those of reference strains showed SD2012-1 had high homology with MDV strains isolated from China, SD2012-1 could break through the protection provided by HVT vaccine and HVT + SB-1 vaccine immunization and caused the mortality of SPF chickens over 60%. The immune failure occured at the farm could be due to the improper selection of vaccines. SD2012-1 produced death later and the gross postmortem lesions of chickens died early and later were different. CONCLUSIONS: MDV strain SD2012-1 isolated from Shandong Province, China was found to have the characteristics of very virulent MDV-1, which could break through the protection provided by HVT vaccine and HVT + SB-1 vaccine, the virus seemed to have a long latent period, and cause different gross postmortem lesions of chickens between chickens died early and later. A better immunization way should be chosen to prevent infection of this MDV strain in field.

Current status of Marek's disease in the United States and worldwide based on a questionnaire survey.

A questionnaire was widely distributed in 2011 to estimate the global prevalence of Marek's disease (MD) and gain a better understanding of current control strategies and future concerns. A total of 112 questionnaires were returned representing 116 countries from sources including national branch secretaries of the World Veterinary Poultry Association, vaccine, breeder, and production companies, as well as MD researchers from various backgrounds. Each country listed on a questionnaire was recorded as an individual entry, and on average there were 2.0 entries per country (median 1; range 1-13). All flock types were listed as having increased MD incidence during the last 10 yr in close to 50% of countries by at least one respondent, with the majority of these countries located within French-speaking Africa, Eastern Europe, East Asia, and South America. Only 18 countries (16%) indicated increasing MD incidence was likely due to higher virulent strains, while the presence of other immunosuppressive diseases was a more common explanation. Increased use of CVI988/Rispens was cited as the most likely reason for decreasing MD incidence in 49 countries (42%). In the United States, MD incidence has continued to decrease during the last 10 yr, reaching a record low in 2007 (0.0008%) as measured by leukosis condemnation rates in broilers at slaughter. However, a recent increase of leukosis condemnations in North Carolina and Pennsylvania needs to be closely monitored.

Prevalence of Marek's disease virus in different chicken populations in Iraq and indicative virulence based on sequence variation in the ecoRI-q (meq) gene.

A cross-sectional survey was conducted in six provinces in southern Iraq to determine the point prevalence of Marek's disease virus (MDV) in different chicken populations followed by sequencing the meq gene for phylogenetic analysis and virulence-associated polymorphisms. A total of 109 samples from unvaccinated flocks were analyzed comprising 52 dust and 30 spleen samples from commercial broiler farms and 27 spleens from local layer chickens purchased in the town markets. The overall prevalence of MDV was 49.5% with no significant differences between provinces (P = 0.08) or sample types (P = 0.89). Prevalence ranged from 36.8% in Karbala and Nasiriyah to 65% in Amarah. The percentages of positive samples were 59.1%, 46.7%, and 48.1% in broiler dust, broiler spleen, and layer spleen, respectively. The overall mean (+/- SEM) Log10 MDV viral copy number per milligram of dust or spleen as determined by quantitative PCR was 1.78 +/- 0.19, with no significant differences between provinces (P = 0.10) or sample types (P = 0.38). In positive samples only, the overall mean was 3.43 +/- 0.18. Sequencing of the meq gene from samples that showed high levels of MDV target in qPCR testing was attempted. Nine samples were sequenced. These sequences were compared with meq sequences of MDVs of different pathotype. All the Iraqi MDVs had a short meq gene of 897 base pairs because of the deletion of 123 bp relative to the reference strain Md5. The Iraqi meq sequences also contained single-nucleotide polymorphisms, resulting in differences in the amino acid sequence. All of the nine Iraqi meq genes encoded two repeats of four-proline sequences. The published negative association between four-proline repeat number and MDV virulence suggests that the Iraqi MDVs are likely to be highly virulent, but this needs to be confirmed by in vivo testing. Taken together, these results indicate that MDV is common in unvaccinated commercial and village chickens in southern Iraq, that there is limited meq gene sequence variation, that all sequenced samples had a short meq with two four-proline repeats, and that this is consistent with a high level of virulence.