The relationship between MHC-DRB1 gene second exon polymorphism and hydatidosis resistance of Chinese merino (Sinkiang Junken type), Kazakh and Duolang sheep

The present study aimed at detecting the association of ovine major histocompatibility complex class II (Ovar II) DRB1 gene second exon and susceptibility or resistance to hydatidosis in three sheep breeds of Sinkiang. The MHC-DRB1 second exon was amplified by polymerase chain reaction (PCR) from DNA samples of healthy sheep and sheep with hydatidosis. PCR products were characterized by the restriction fragment length polymorphism (RFLP) technique. Five restriction enzymes, MvaI, HaeIII, SacI, SacII, Hin1I, were used, yielding 14 alleles and 31 restriction patterns. Frequencies of patterns MvaIbc, Hin1Iab, SacIIab, HaeIIIde, HaeIIIdf, HaeIIIdd (P < 0.01) in Kazakh sheep, SacIab (P < 0.05) in Duolang sheep, and HaeIIIab, HaeIIIce, HaeIIIde, HaeIIIee (P < 0.01) in Chinese Merino (Sinkiang Junken type) sheep, were significantly higher in healthy sheep compared with infected sheep. These results indicated a strong association between these patterns and hydatidosis resistance. In contrast, the frequencies of MvaIbb, SacIIaa, Hin1Ibb, HaeIIIef (P < 0.01) and HaeIIIab (P < 0.05) in Kazakh sheep, SacIbb, HaeIIIae, Hin1Iab (P < 0.05), HaeIIIaa, HaeIIIbe, HaeIIIef (P < 0.01) in Duolang sheep, SacIIaa (P < 0.05) and HaeIIIbd, Hin1Ibb, HaeIIIcf, HaeIIIef (P < 0.01) in Chinese Merino sheep (Sinkiang Junken type) were significantly lower in healthy sheep compared with infected sheep. This indicated a strong association between these patterns and hydatidosis susceptibility. In addition, sheep with the pattern of HaeIIIef demonstrated a high hydatidosis susceptibility (P < 0.01) in all three breeds, while sheep with the pattern HaeIIIde demonstrated significant hydatidosis resistance (P < 0.01) in Kazakh and Chinese Merino sheep (Sinkiang Junken type). These results suggest that the Ovar-DRB1 gene plays a role in resistance to hydatidosis infection in the three sheep breeds.


INTRODUCTION
H ydatidosis (Echinococcus granulosus) is recognized as one of the world major zoonoses, and is found all over the world (Rausch, 1995;Andersen et al., 1997;Dalimi et al., 2002;Eckert & Deplazes, 2004;Jenkins et al., 2005). Sinkiang Autonomous Region of China is a prevalent area of hydatidosis. In sheep, the overall prevalence rate for hydatidosis cysts is 38.89 % to 61.25 % (Li et al., 2005). Kazakh sheep and Duolang sheep, the local sheep of Sinkiang, are bred for meat and fat. Chinese Merino sheep (Sinkiang Junken type) produce excellent wool. In Sinkiang, hydatidosis in farm animals causes considerable economic problems due to the loss of meat and edible liver, as well as the value of the fleece from infected sheep. Therefore it also affects the life quality of herdsmen. Original contribution Parasite, 2011, 18, 163-169 In recent years, many animal breeding studies have focused on MHC genes as candidate genes for disease resistance and susceptibility. The MHC is a multigene family that controls immunological self/non-self recognition. They include genes for cell surface glycoproteins that present peptides of foreign and self proteins to T cells, thereby controlling both cell-and antibody-mediated immune responses (Klein, 1986). A striking characteristic of MHC genes is their extreme polymorphism. Diversity driven by pathogens implies a strong association between MHC alleles and patterns of resistance to specific autoimmune or infectious diseases. Such a link was first shown for chickens, in which the B21 haplotype (MHC class IIB) confers the strongest resistance to the herpes virus responsible for Marek's disease (Briles et al., 1977;Longenecker & Gallatin, 1978). Equally well known is the role of the chicken class I MHC in providing resistance to the Rous sarcoma virus (Schierman & Collins, 1987;Kaufman & Venugopal, 1998). The polymorphism of Ovar-DRB1 plays an important role in resistance to nematode infection in the Suffolk breed (Sayers et al., 2005). MHC Class II Ovar-DRB1 was chosen as the immune response gene in this study because it is highly polymorphic, transcribed, and there are over 100 different DRB1 alleles reported in Genbank based upon either restriction fragment length polymorphisms (RFLP) or the deduced amino acid sequence for the β1 domain encoded by exon 2 (Dutia et al., 1994;Schwaigger et al., 1996;Jugo & Vicario, 2001;Konnai et al., 2003;Herrmann et al., 2005). Others have shown specific MHC-DRB1 alleles associate with resistance and/or less severe clinical signs in human hydatidosis (Gottstein et al., 1996;Li et al., 2003). Konnai et al. (2003) detected the Ovar-DRB1 exon 2 polymorphisms of Suffolk sheep by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). In the present study, the polymorphism of the class II Ovar-DRB1 exon 2 was detected by PCR-RFLP analysis in three sheep breeds. We characterized the relationship between the Ovar-DRB1 exon 2 polymorphism and hydatidosis resistance, and screened the genotypes associated with hydatidosis resistance and susceptibility in each sheep breed. Results of the present study may play an important role in developing new sheep breeds that are resistant to hydatidosis.

ANIMALS SAMPLING AND SAMPLE PREPARATION
B lood samples of Chinese Merino sheep (Sinkiang Junken type; 604 healthy animals and 425 animals with hydatidosis) were donated by agricultural construction division 9 in Sinkiang. Blood samples of Duolang sheep (122 healthy sheep and 70 sheep with hydatidosis) were donated by agricultural construction division 3. Blood samples of Kazakh sheep (400 healthy sheep and 302 sheep with hydatidosis) were donated by agricultural construction division 4. The sheep with hydatidosis were distinguished from healthy sheep by a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Shenzhen Combined Biotech Co., Ltd, Shanghai, China). Genomic DNA were obtained from whole blood by phenol-chloroform method (Liu et al., 1997), and stored in a -20 °C freezer until analysis.

DESIGN OF OVAR-DRB1 EXON 2-SPECIFIC PRIMERS AND PCR AMPLIFICATION
The second exon of Ovar-DRB1 was amplified by PCR in two rounds. The first round of PCR was performed with primers OLA-ERB1 (5'-CCG GAA TTC CCG TCT CTG CAG CAC ATTTCT T-3') and HL031 (5'-TTT AAA TTC GCG CTC ACCTCG CCG CT-3') (adopted from Konnai et al., 2003). We subjected 100 ng of genomic DNA to PCR amplification in a total volume of 20 µl, containing 1.5 mM MgCl 2 , 120 µM dNTP, 0.2 mM each primer, and 1.5 U of Taq polymerase (TIANGEN Biological Engineering Technology And Service Company, Beijing, China). Reactions were performed in a thermocycler (Bio RAD, Germany) under the following conditions: a single cycle of 5 min at 94 °C, followed by 15 cycles of 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 60 s, with a final extension at 72 °C for 10 min. We used 3 µl of the resulting mixture and primers OLA-ERB1and OLA-XRBI (5'-AGC TCG AGC GCT GCA CAG TGAAAC TC-3') (adopted from Konnai et al., 2003) for the second round of PCR. The cycling conditions for the second round were: a single cycle of 5 min at 94 °C, followed by 30 cycles of 94 °C for 30 s, 63 °C for 30 s, and 72 °C for 60 s with a final extension at 72 °C for 10 min.

POLYMORPHISM DETECTION BY RFLP
PCR products (10 µl) from the second round were digested for 4 h at 37 °C with 5 U of MvaI, HaeIII, SacI, SacII, or Hin1I (Shanghai Sangon Biological Engineering Technology And Service Co., Ltd., Shanghai, China) in a total volume of 20 µl. The products of enzyme digestion were analyzed by a 2.5 % or 3 % agarose gel electrophoresis.

STATISTICAL ANALYSIS
Hardy-Weinberg equilibrium of Ovar-DRB1 genotypes was analyzed by χ 2 test. The distribution of genotypic frequency in healthy sheep and sheep with hydatidosis within a breed was analyzed by χ 2 test. SPSS version 13.0 was used for statistical analysis. Original contribution Parasite, 2011, 18, 163-169

Restriction enzymes
The genotypes of each restriction enzyme

PCR-RFLP
Restriction enzyme analysis with SacI, Hin1I, MvaI, SacII and HaeIII produced restriction patterns and allele frequencies in accordance with that reported by Konnai et al. (2003). Restriction patterns are shown in Table I (SacI,Hin1I,MvaI,SacII) and Table II (HaeIII); fragments the genotypic restriction map is shown in Figs 2-6; and a diagram of this exonic region, the cleavage sites is shown in Fig. 7. The Ovar-DRB1 exon 2 of Chinese Merino, Duolang and Kazakh sheep was analyzed by PCR-RFLP using restriction enzymes SacII (two alleles, three genotypes), MvaI (two alleles, three genotypes), SacI (two alleles, three genotypes), Hin1I (two alleles, three genotypes), and HaeIII (six restriction profiles, 19 patterns). Polymorphisms were detected at base pairs 229,225,208,210,178,173,159,87.

DISCUSSION AND CONCLUSION
A t present, both domestic and international studies have indicated that MHC genes show extensive polymorphism in humans, mice, cattle (Blattman et al., 1993;Xu et al., 1993), sheep, goats (Amills & Francino, 1995, 1996Yang et al., 2006 ( The same genotypes in healthy group and hydatidosis group of one breed sheep, * P < 0.05, ** P < 0.01.  Sun et al., 2004), and chickens (Xu et al., 2005). Using PCR-RFLP, Yang et al. (2006) and Sun et al. (2004) investigated the MHC-DRB3 polymorphism in sheep and goats. Konnai et al. (2003) determined the Ovar-DRB1 exon 2 polymorphisms of 52 Suffolk sheep by PCR-RFLP with restriction enzymes SacI (two alleles), SacII (two alleles), Hin1I (two alleles), and HaeIII (six alleles), which was consistent with our results. Peng et al. (2007) determined Ovar-DRB1 exon 2 polymorphisms of 211 Chinese Merino sheep (Sinkiang Junken type) by PCR-RFLP with restriction enzymes SacI (two alleles, three genotypes) and Hin1I (two alleles, three genotypes), which was also consistent with our results. However, six alleles and 15 patterns were found using the restriction enzyme HaeIII in their study, while six alleles and 19 patterns were found using this enzyme in the present study. This difference may be due to different numbers and species of sheep. Last but not the least, the genotypes of HaeIII bc and bf weren't appearance in the population which we chosen. Perhaps they will be found in a larger population.
Ovar-DRB1 is a principal member of MHC class II DRB in sheep (Deverson et al., 1991;Scott et al., 1991;Ballingall et al., 1992). It is often used as a genetic marker in disease association studies (reviewed by Dukkipati et al., 2006). Azab et al. (2004) demonstrated that people who carry human leukocyte antigen (HLA)-DR3 and HLA-DR11 were at high risk for cystic echinococcosis (CE), and those with HLA-DR3 were more susceptible to complications. Shcherbakov & Monje- Barredo (1989) showed that people with HLA-B5 and B18 of HLA I antigen were at high risk for CE, while those with HLA-B14 and B27 had resistance to CE. In addition, Schwaiger et al. (1995) and Sayers et al., (2005) found that MHC-DRB1 was related to nematode resistance. Taken together, these results show that MHC polymorphisms are closely associated with parasite resistance or susceptibility. In the present study, the Ovar-DRB1 exon 2 polymorphisms in three breeds of sheep were also shown to be associated with hydatidosis resistance and susceptibility (  et al., 2006). The Chinese Merino sheep (Sinkiang Junken type) is a cross between a Merino ram from Australia and Sinkiang Junken sheep. The difference among the three breeds in HaeIII patterns of the Ovar-DRB1 second exon may be due to different breeding histories.
In the present research, we screened restriction patterns associated with hydatidosis resistance and susceptibility in three sheep breeds by PCR-RFLP. Additional research is needed to determine whether these patterns could serve as genetic markers for hydatidosis.