Comparative Analysis On The Susceptibility Of Three Chicken Lines To Marek's Disease Virus

Marek's disease(MD), one of the most common contagious lymphoproliferative diseases of domestic chickens, is caused by Marek's Disease Virus(MDV). Clinical signs of MD include bursal/thymic atrophy, rapid onset of monocytes that infiltrate peripheral nerves,visceral organs, muscle and skin. Semiparalysis or paralysis that caused by the aggregation of peripheral nerve tumor cells is also included.MDV replicates in chicken lymphocytes and establishes a latent infection within CD4+ T cells. The latently infected CD4+ T cells carry the virus to visceral organs, peripheral nerves, and feather follicle epithelium(FFE). FFE is the only anatomical site where infectious enveloped cell-free virus particles are produced and disseminated into the environment. White Leghorn chickens have been diversely selected for resistance or susceptibility(lines 63 and 72, respectively) to MDat Avian Disease and Oncology Laboratory(ADOL) according to the genetic resistance to MDV-induced tumors.After vaccination with the same MD vaccine, the chicken lines genetically resistant to MDV are more resistant to very virulent strains of MDV than the susceptible lines, expressing a low mortality rate.In this study, we established a model of line 63 and 72 to comparing the immunological responsescaused by MDV. Meanwhile, the model of line 15I5×71was established to further explore the immunological responses and mechanisms within the skin of MDV infected chickens.1. Comparative analysis of MDV-induced immunological responses in line 63 and 72One-day-old chicks from line 63 and 72 were randomly distributed into 2 groups of 25 birds each. Birds from one group of each line were inoculated intraperitoneally with 2000 pfu of r Md5 at 12 days post hatch. At 21 dpi, when the immunosuppression was clearly visible in thechickens, combs and spleens from each group were collected for immunohistochemistry,histopathology, Gram stain and Real-Time PCR analysis of select immune response genes to compare theimmunological responses of line 63 and 72 infected by MDV.Line 63 and 72 are two genetically diverse chicken lines with the same MHC haplotype(B2/B2). Line 63 is relatively resistant to MD, and exhibited some of the clinical signs relating to MD. In addition, the resistant line 63 develops necrosis of the combs and footpads.Chickens from line 72, on the other hand, are highly susceptible to MD infection and tumor development. Although line 72 never developed necrotic combs, paleness of tissue due to viral-induced anemia, stretched and overextended eyelids, high mortality rate due to transient paralysis and other complication were visible. Immunohistochemistry analysis revealed a massive infiltration of macrophages and lymphocytes into the combs of both lines. An increase number of infiltrated CD8+ T cells were observed only in necrotic combs of the resistant line 63, and no CD8+ T cells were detected in line 72. Histopathological analysis revealed erosion and thinning of epidermis layer within the infected tissues of the resistant line 63, lympho-plasmacytic infiltrates and heterophilic infiltration was detected within the connective tissues. Within the infected comb tissues of the susceptible line 72, moderate erosion of the epidermis and multifocal lympho-plasmacytic infiltrates was observed. Gram stain revealed the presence of Gram-positive micrococci. No such microorganism was observed in line 72. Real-time PCR-based gene expression analysis showed that there were minimal changes in the transcriptional activities of the other genes, except IL-8 and i NOS,within the combs of the infected resistant line 63. A substantial increase in the transcriptional activities of IL-6, IL-8, IL-12, i NOS, and IFN-? within the combs of the susceptible line 72 was observed. The RT-PCR analysis revealed that meq oncogene was expressed in the spleen tissues of all the infected birds of both the resistant and susceptible lines 63 and 72. The expression of meq was also detected in the comb tissue of the infected birds of line 72 but not the necrotic combs of line 63.There were different immunosuppression symptoms between MD-resistant line 63 and MD-susceptible line 72 after infected with MDV. Line 63 naturally developed necroticdermatitis that was exacerbated when infected with MDV. The results suggested that,selection for particular characteristics, such as resistance to MD, adversely affects the immunological competence of the line to other microbial pathogens or environmental stimuli.2. Immunological responses and mechanisms in the skin of MDV-infected chickens of line15I5×71One-day-old chicks of line 15I5×71, with the same susceptibility as line 72,were randomly distributed into 2 groups of 10 each. Birds from one group were inoculated intraperitoneally with 2000 pfu of r Md5 at 12 days post hatch. The second group served as un-inoculated negative control. Obvious symptoms of immunosuppression were observed at 26 dpi. Skin tissues were collected and analyzed by immunohistochemistry and Real-time PCR. This study investigated the immunological responses and mechanism of viral-induced immunosuppression and immune evasion in the FFE.Immunohistochemistry analysis revealed large number of virions produced in the FFE cells.There was also amassive infiltration of CD4+ T cells andmacrophages. However, the number of cytotoxic T lymphocytes(CTL) was limited. Real-Time PCR-based gene expression analysis showed a substantial increase in the transcriptional activities of most of the gene tested. In contrast, the transcriptional activities of the immune related genes(CD107a,ITGA6, TGF-62, IFN-6ased gene expression analys Although the overall gene expression pattern was suggestive of a Th1 type immune response,the replication of the cell-free enveloped virus particles in the FFE cells was uninhibited. It is speculated that the mechanism of MDV-induced immunosuppression appears to be through inhibition of CTL function due to down regulation of CD8 glycoprotein and/or blocking of CTL migration due to decrease expression of cell adhesion molecules. MDV-induced he absence of CTL or the lack of their biological functions resulted in the lack of an effective cellular immune response. Meanwhile,strong viral replication resulted in immunosuppression of the infected birds.