The Establishment Of SPF Chicken Lines With Unique MHC Haplotypes And Their Resistance To MD

The BWEL-SPF chicken lines kept by the HVRI of CAAS have a relatively homogenous genetic background following a closed breeding program over 17 generations, and are the only Chinese SPF chicken genetic resource with independent intellectual property rights. Chicken major histocompatibility complex (MHC) molecules present peptides to T cells to initiate immune response and are the major genetic control over host resistance to a variety of avian diseases, including the Marek's disease (MD), leukemia, Rous sarcoma disease as well as Salmonellosis. Therefore it would be very important to explore the possibility of establishment of BWEL-SPF chicken lines with unique MHC haplotypes to be used for the challenge experiments to investigate particularly the pathogenic mechanisms of and control measures to the MD.A total of 498 BWEL15-SPF chickens were first genotyped for a microsatellite DNA marker LEI0258 mapped within MHC-B region and 17 genotypes including four homozygous ones were detected. Based on these results, two homozygous lines, named as BW1 (263/263) and BW3 (543/543), and two heterozygous lines, BW4 (206/298, 206/362, 206/543) and BW5 (206/298, 263/298), were established as F0 generation. Three hundred and thirty-seven individuals of F1 generation from the four lines were further genotyped for four microsatellites of LEI0258, MCW0371, MCW0321 and MHC-D, all located within the MHC-B region. Twenty-six genotypes including seven homozygous haplotypes were found. Subsequently, six lines with different and homozygous MHC haplotypes were restructured as follows: G1 (206/206-202/202-217/217-317/317), G2 (263/263-203/203-217/217-310/310), G3 (263/263-206/206-217/217-307/307), G5 (298/298-209/209-217/217-313/313), G6 (362/362-205/205- 217/217-310/310) and G7 (543/543-205/205-217/217-317/317).In order to examine the genetic resistance of BWEL-SPF chicken lines with unique MHC haplotypes to the MD, 39 chicks of G3 line and 30 chicks of G7 line of F2 generation were infected with very virulent MDV Md5 strain. The chicks were first inoculated with turkey herpes virus (HVT) by a dosage of 3000 pfu per individual at their age of three days and then inoculated intraperitoneally by the Md5 at a dosage of 500 pfu per individual at their age of 10 days. Feathers were collected every week post challenge of the Md5 from all individuals for 10 weeks for DNA extraction. The virus copies per million plume pulp cells (viral load) of each individual were quantified using a real-time fluorescent PCR with both the unique meq gene of very virulent MDV strain and the chicken ovotransferrin gene (ovo) used as targets. The clinical symptoms of the infected chicks were monitored daily and all chicks survived over the infection were killed at end of the challenge experiments. One chick died in the course of infection and one chick each carrying either a low or high viral load and killed by the end of experiments were autopsied to check their clinical symptom and histological and pathological changes (HE staining).The results showed that the average viral load of the G3 line peaked with a log value of 5.94 at the 4th week post infection, followed by a slow decline till to a log value of 5.15, very close to the initial level of viral load of 5.08, at the 6th week post infection. This viral load was then maintained until the end of the experiment at a log value of 5.52. For the G7 line, the average viral load peaked with a log value of 5.67 for the first time also at the 4th week post infection, followed by a slight decrease with no significant difference (P > 0.05) from that of the G3 line by the 6th week post infection. From the 7th week post infection, the viral load began to rise until the end of the experiment at a log value of 6.82, which was significantly (P < 0.01) higher than that of the G3 line. The viral load values of all 10 batches of samples in the G3 line ranged between 3.38 and 7.98, of which the percentage of values higher than 6.00 was 21.14%. However, the viral load values in the G7 line was between 3.61 and 7.72 and the percentage was 39.69%. The death rates of the G3 and G7lines were 5.56% and 28.57%, respectively. All these data indicated that the G3 line had a stronger genetic resistance to the very virulent MDV strain than the G7 line. Furthermore, all the dead chicks over the challenge had a relatively high viral load, e.g. the log value > 6.00, and also severe clinical symptoms. The clinical symptoms, histological and pathological changes of the killed chicks with low or high viral load by the end of experiment revealed a positive correlation of the high viral loads with the severe pathological damages over the infection.In conclusion, this study provided clear evidence of usefulness of the establishment of different BWEL-SPF chicken lines with unique MHC haplotypes in understanding the mechanism of genetic resistance to avian diseases.