MHC-DRB1/DQB1 Gene Polymorphism and Its Association with Resistance/Susceptibility to Cystic Echinococcosis in Chinese Merino Sheep
The aim of this study was to analyze the relationship between polymorphism of the MHC-DRB1/DQB1 gene and its resistance to Cystic Echinococcosis (C.E), as well as to screen out the molecular genetic marker of antiechinococcosis in Chinese Merino sheep. The MHCII-DRB1/DQB1 exon 2 was amplified by polymerase chain reaction (PCR) from DNA samples of healthy and hydatidosis sheep. PCR products were characterized by restriction fragment length polymorphism (RFLP) technique. Five restriction enzymes (Mval, HaeIII, SacI, SacII, and Hin1I) were employed to cut DRB1, while seven restriction enzymes (MroxI, ScaI, SacII, NciI, TaqI, Mval, and HaeIII) were employed to cut DQB1.Results showed that frequencies of patterns Mvalbb (), SacIab in DRB1 exon 2 (), and TaqIaa, HaeIIInn () in DQB1 exon 2 were significantly higher in the healthy group compared with the C.E individuals, which implied that there was a strong association between these genotypes and hydatidosis resistance or susceptibility. Chi-square test showed that individuals with the genic haplotype DRB1-SacIab/DRB1-Mvalbb/DQB1-TaqIaa/DQB1-HaeIIInn () were relatively resistant to C.E, while individuals with the genic haplotypes DRB1-Mvalbc/DQB1-Mvalyy/DQB1-TaqIab/DQB1-HaeIIImn () and DRB1-Mvalbb/DQB1-Mvalcc/DQB1-TaqIab/DQB1-HaeIIImn () were more susceptible to C.E. In addition, to confirm these results, a fielding experiment was performed with Chinese Merino sheep which were artificially infected with E.g. The result was in accordance with the results of the first study. In conclusion, MHC-DRB1/DQB1 exon 2 plays an important role as resistant to C.E in Chinese Merino sheep. In addition, the molecular genetic marker of antiechinococcosis (DRB1-SacIab/DRB1-Mvalbb/DQB1-TaqIaa/DQB1-HaeIIInn) was screened out in Chinese Merino sheep.
The major histocompatibility complex (MHC) gene of sheep is located on Chromosome 20 and is called Ovar . The MHC gene family includes two major subfamilies: class I and class II genes . Studies have shown the existence of class II loci that are homologous to HLA-DQB [3–6]. As in other vertebrate species, a high degree of polymorphism is found in the Ovar-DQB genes, with most of the polymorphic sites located in exon 2, which encodes the antigen-binding site . Due to its highly polymorphic character, a variety of studies have been applied in many fields. It has been well-reported that alleles of different MHC genes correlate with disease resistance in sheep ; furthermore, specific MHC alleles are associated with parasite resistance in sheep . Currently, relevant research on Ovar polymorphism and disease resistance or susceptibility mainly concentrates on Ovar-DRB1 [10–14] and Ovar-DQB [7, 15].
C.E is a cosmopolitan zoonotic parasitic disease caused by the larval stage (metacestode stage) of the tapeworm Echinococcus granulosus that cycles between canines, particularly dogs, as definitive hosts and various herbivores as intermediate hosts. In the intermediate hosts and humans, larvae develop into hydatid cysts in various organs, particularly the liver and lungs. C.E is associated with severe morbidity and disability, especially in pastoral areas in northwestern China, the prevalence of which not only results in a considerable decrease in livestock production, but also seriously affects the life quality of people. Chinese Merino sheep, well known as the character of well wool, is beneficial to local sheep husbandary; however it is relatively more susceptible to C.E. Therefore, this disease will result in low performance on Chinese Merino sheep.
At present, many studies focus on MHC-hydatid disease associations in human [16–19]. However, few reports have been published on the study of the Ovar association with C.E in sheep. In this study, efforts were made to investigate MHC-DRB1/DQB1 gene polymorphism and its association with resistance/susceptibility to C.E in Chinese Merino sheep, screening out the molecular genetic marker of antiechinococcosis.
2. Materials and Methods
2.1. Animal Sampling and Sample Preparation
We received blood samples from 204 2-year-old Chinese Merino sheep, donated from Mission 165, agricultural division 9, Xinjiang Production and Construction Corps. The C.E sheep and healthy sheep were distinguished by ovine hydatidosis ELISA kit (Shenzhen Combined Biotech Co., Ltd.). We chose 101 C.E sheep and 103 healthy controls. Samples of genomic DNA were obtained from whole blood and stored at −20°C until analysis. The major materials and reagents were obtained from Promega Company and Shanghai Sangon Biological Engineering Technology and Service Co., Ltd.
2.2. PCR Amplifications
The second exon of Ovar-DRB1 was amplified by nested PCR. The first round of PCR was performed with primers OLA-ERB1 (GC) 5′-CCG GAA TTC CCG TCT CTG CAG CAC ATT TCT T-3′ and HL031 5′-TTT AAA TTC GCG CTC ACCTCG CCG CT-3′ . 100 ng of genomic DNA was used as DNA template in a total volume of 20 μL PCR reaction which was composed of 1.5 mM MgCl2 and 120 μM dNTPs, to which 0.2 mM of each primer and 1.5 U of Taq polymerase were added. Reactions were performed in a thermocycler under the following conditions: one cycle of initial denaturation for 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 final extension at 72°C for 10 min. Three μL of first step PCR was used for the second step PCR by using primers OLA-ERB1(GC) and OLA-XRBI (5′-AGC TCG AGC GCT GCA CAG TGAAAC TC-3′) . The conditions were one cycle for 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 final extension at 72°C for 10 min. The second exon of DQB1 was amplified by primers FW: 5′-CCC CGC AGA GGA TTT CGT G-3′ and REV: 5′-ACC TCG CCG CTG CCA GGT-3′ ; 150 ng of Genomic DNA was amplified in a total volume of 112 50 μl, including 1.5 mM MgCl2, 100 μM dNTPs, 0.2 mM of each primer, and 2 U of Taq polymerase. Reactions were performed in a thermo cycler under the following conditions: one cycle of initial denaturation for 5 min at 94°C, followed by 33 cycles of 94°C for 30 s, 67°C for 30 s, and 72°C for 45 s, with final extension at 72°C for 10 min.
The cleavage map typing method and allele nomenclature referred to that of Konnai et al. . Each 10 μL of DRB1 PCR product was digested with 5 U of SacI, Hin1I, HaeIII, MvaI, and SacII, respectively, in a total volume of 20 μL, including 2 μL 10× buffer. Each 10 μL of DQB1 PCR product was digested with 5 U of MroxI, ScaI, SacII, NciI, TaqI, MvaI, and HaeIII, respectively. Samples were resolved by agarose gel electrophoresis at varying concentrations (Table S1) (see Supplementary Material available online at http://dx.doi.org/10.1155/2014/272601).
2.4. Cloning and Sequencing
According to the typing results of restriction digest, the samples 54 and 74 were selected for cloning and sequencing, because the samples were HaeIIImm, HaeIIInn, MvaIyy, and MvaIzz genotype, which are inconsistent with the previous reports . So the amplified PCR products of these samples were cloned into pGEM-T vector, the ligated plasmids was selected by blue-white colony screening, then masculine clone were sent to sequence.
2.5. Verification of Artificial Infection with E.g
To verify the validity and reliability of the above research results, sixteen 2-year-old Chinese Merino sheep, which were negative by hydatidosis ELISA kit detection, were chosen to conduct the experiment of artificial infection with E.g. Eight of the sheep with the haplotype of DRB1-SacIab/DRB1-MvaIbb/DQB1-TaqIaa/DQB1-HaeIIInn were taken as the test group, and the other eight sheep with the haplotypes of DRB1-SacIab/DRB1-MvaIbc/DQB1-TaqIaa/DQB1-HaeIIInn, DRB1-SacIab/DRB1-MvaIbc/DQB1-TaqIaa/DQB1-HaeIIImn,or DRB1-SacIab/DRB1-MvaIbb/DQB1-TaqIaa/DQB1-HaeIIImm, which were not associated with hydatidosis resistance or susceptibility, were taken as the control group. Each sheep was fed 10 adult cestodes with fertilized egg proglottis by mouth. These sixteen sheep were bred under the same conditions.
2.6. Statistical Analysis
Allelic and genotypic frequencies in C.E-negative and -positive Chinese Merino sheep were analyzed by -test to assess the relationship between different genotypes and C.E significance. The chi-square test was performed to analyze the relationship between the different haplotypes and C.E resistance. The C.E infection rates of the test and control groups were compared by Fisher’s exact test after artificial infection with E.g.
3.1. PCR Amplification
Ovar-DRB1 exon 2 was amplified by PCR with primers OLA-ERB1, OLA-HL031, and OLA-XRBI; one specific band of 296 bp was observed on 1.5% agarose (Figure S1B). Ovar-DQB1 exon 2 was amplified by PCR with primers FW and REV, and one specific band of 280 bp was observed on 2% agarose (Figure S1B).
From restriction digestion of DRB1 exon 2 PCR product, genotypes of SacI, Hin1I, MvaI, SacII, and HaeIII (Table S2B) were observed, and some of genotypic restriction maps were in Figure 1. In addition, genotypes of restriction enzymes MroxI, ScaI, SacII, NciI, TaqI, MvaI, and HaeIII (Table S2B) for DQB1 PCR products were also observed, and some of their genotypic restriction maps were in Figure 2.
3.3. Anlysis of Clonig and Sequncing
We verified the predicted RFLP profiles of Ovar-DRB1 alleles by sequencing cloned 184 amplified products, and all of the observed patterns of fragments matched exactly with those predicted from DNA sequences. Sequencing of Ovar-DQB1 exon 2 cloned amplified products revealed two single point mutations, T to G and A to G, at base positions 32 and 159, respectively, resulting in new alleles, HaeIIImm and HaeIIInn. In addition, two G-to-A point mutations at base positions 96 and 246 resulted in new alleles, MvaIy and MvaIz. Comparison of sequencing results to the original sequence of DQB1 exon 2 (GenBank, accession numbers: Z28523) are shown in Figure S3.
3.4. Analysis of the Relationship between Genotypes and C.E Resistance
Statistical comparisons of genotypic frequencies in C.E sheep and healthy controls revealed that DRB1 genotypic frequencies of MvaIbb (), HaeIIIee, and SacIab () in negatives were higher than in C.E sheep, indicating a strong association between these genotypes and C.E resistance, while genotypes in terms of SacIIab (), HaeIIIdf (), HaeIIIbd (), and MvaIbc () in DRB1 exon 2 occurred more often in C.E individuals when compared with the healthy group, which implied that there was a strong association between these genotypes and hydatidosis susceptibility (Table 1). DQB1 genotypic frequencies of TaqIaa and HaeIIInn (), MvaIdz () in negatives were higher than in positives, while genotypes of TaqIab and HaeIIImn (), MvaIcz () in positives were higher than in negatives (Table 2). Therefore, we concluded that DQB1 genotypes of TaqIaa, HaeIIInn, and MvaIdz were resistant to C.E, while genotypes of TaqIab, HaeIIImn, and MvaIcz were susceptible to C.E.
3.5. Verification of Artificial Infection with E.g
Analyzing the haplotype of resistant genotypes, it was found that the haplotype frequency of DRB1-SacIab/DRB1-MvaIbb/DQB1-TaqIaa/DQB1-HaeIIInn in C.E-negative sheep was higher than in C.E sheep (), indicating that this haplotype was the resistant haplotype of Chinese Merino sheep (Table 3). The result was verified by artificial infection hydatidosis. The haplotypes of DRB1-MvaIbc/DQB1-MvaIyy/DQB1-TaqIab/DQB1-HaeIIImn and DRB1-MvaIbb/DQB1-MvaIcc/DQB1-TaqIab/DQB1-HaeIIImn in positives were higher than in negatives (), which implied that these haplotypes were susceptible to C.E individuals.
Protoscoleces can develop into cysts within 20 days postinfection . The sixteen sheep that were artificially infected with E.g were slaughtered in the second month after E.g infection, and visual inspection of the liver and lung surfaces of each slaughtered animal was made for the detection of larval stages of cestodes . Results show that 3 sheep were infected with E.g in the test group, whereas 6 sheep were infected with E.g in the control group; therefore, the infection rate in the test group was significantly lower than that of the control group (). It is confirmed that the genic haplotype DRB1-SacIab/DRB1-MvaIbb/DQB1-TaqIaa/DQB1-HaeIIInn leads to C.E resistance in Chinese Merino sheep.
4. Discussion and Conclusion
The MHC gene is well known to be involved in the vertebrate immune system and encodes antigen recognition proteins used in the adaptive immune response. Polymorphism of this gene has become a hot topic in the past decades. A variety of studies, both overseas and domestic, have shown that MHC of sheep and goats introduces polybase mutation and affluent polymorphism. Amills et al. [24, 25]utilized the PCR-RFLP method to investigate polymorphism of DRB in goats, Konnai et al.  researched the polymorphism of DRB1 in some sheep, with results indicating that affluent polymorphism exists in the Ovis aries-DRB1 gene, and Dongxiao and Yuan  studied DRB3 polymorphism of Chinese local sheep and goats. In addition, Ovis aries-DQB1 gene investigations have been conducted abroad [21, 27], and Chinese scholars have studied MHC-DQB and DQA in human , swine , and cattle [30, 31]. However, there are still no domestic reports of Ovis aries-DQB1. In the present study, we used MroxI, ScaI, SacII, NciI, TaqI, HaeIII, and MvaI by PCR-RFLP to analyze DQB1 exon 2 and found the existence of 2, 2, 4, 2, 3, 3, and 6 alleles, as well as 3, 3, 7, 3, 4, 6, and 16 genotypes, respectively. The results of cloning and sequencing of the alleles, that is, HaeIIIm, HaeIIIn, MvaIy, and MvaIz, indicated that they are new alleles resulted from mutation in Chinese Merino sheep.
The extensive diversity at many MHC loci provides a valuable source of genetic markers for examining the complex relationships between host genotype and disease resistance or susceptibility . For example, Sayers et al.  suggested that the Ovar-DRB1 gene plays an important role in the enhanced resistance of Suffolk sheep to nematode infection. By comparing phenotypic frequencies of A.E patients with healthy controls, it has been speculated that HLA-DRB1*11 may have a certain resistance to A.E, but HLA-DQB1*02 would exacerbate the disease process . The potential immunogenetic predisposition for susceptibility and resistance to unilocular echinococcosis was investigated by HLA-DRB1 typing, and a statistically significant positive association was found between HLA-DR3 and HLA-DR11, and the occurrence of C.E. HLA-DR3 antigen was positively associated with the occurrence of isolated, multiple pulmonary cysts . Differences have been shown between HLA characteristics of A.E patients with different courses of E.m, notably the association of the HLA B8, DR3, and DQ2 haplotype with more severe forms of this granulomatous parasitic disease, which suggested that HLA characteristics of the host could influence immune-mediated mechanisms . This study found that the DRB1-SacIab/DRB1-MvaIbb/DQB1-TaqIaa/DQB1-HaeIIInn haplotype is echinococcosis resistant and selected the genetic markers of resistance to hydatidosis.
In this study, analysis of polymorphisms of MHC-DRB1/DQB1 by the PCR-RFLP method was performed, as well as screening of genetic markers of antiechinococcosis in Chinese Merino sheep. Artificial infection was used to verify the relationship between different haplotypes of polymorphic MHC gene loci and the resistance of echinococcosis, which would lay a theoretical foundation for sheep breeding of disease resistance in the future.
|MHC:||Major histocompatibility complex|
|OLA:||Ovine lymphocyte surface antigen|
|PCR:||Polymerase chain reaction|
|RFLP:||Restriction fragment length polymorphism|
Conflict of Interests
The authors declare that there is no conflict of interests regarding the publication of this paper.
Hong Shen and Guohua Han equally distributed to this paper.
We are grateful to the Mission 165, Ninth agricultural 264 ricultural division, Xinjiang Production and 265 Construction Corps for providing us with the experimental sheep. This study was supported 266 by the project of National Natural Science Foundation of China (Grant no. 30660124, Grant 267 no. 31260535, Grant no. 31060281) and Specialized Research Fund for the Doctoral Program of 268 Higher Education (Grant no. 20106518110003).
Fig S1: Electrophoretic patterns of PCR products of exon 2 of Ovar-DRB1/DQB1 in Chinese Merino sheep.
Fig S1: 10µl of DRB1 exon 2 PCR product was digested with 5U of Hin1I in Chinese Merino sheep, and then Samples were resolved by 1.5% agarose gel electrophoresis.
Fig S2: Each 10µl of DRB1 exon 2 PCR product was digested with 5U SacII, MroxI, ScaI, NciI respectively, The concentration of agarose gel electrophoresis is at2.5%.while concentration of agarose gel concentration of ScaI is 3%.
Fig S3: Comparison of MHC-DQB1 sequences of sheep.
TableS1: Conditions of restriction enzymes and examination for PCR products of the exon 2 of MHC-DRB1/ DQB1.
Table S2: The genotypes of PCR-RFLP in exon 2 of the Ovar-DRB1/gene.
R. Hediger, H. A. Ansari, and G. F. Stranzinger, “Chromosome banding and gene localizations support extensive conservation of chromosome structure between cattle and sheep,” Cytogenetics and Cell Genetics, vol. 57, no. 2-3, pp. 127–134, 1991.View at: Google Scholar
J. Klein, The Natural History of the Major Histocompatibility Complex, John Wiley and Sons, New York, NY, USA, 1986.
P. Chardon, M. Kirszenbaum, and P. R. Cullen, “Analysis of the sheep MHC using HLA class I, II, and C4 cDNA probes,” Immunogenetics, vol. 22, no. 4, pp. 349–358, 1985.View at: Google Scholar
P. C. Scott, J. F. Maddox, K. J. Gogolin-Ewens, and M. R. Brandon, “The nucleotide sequence and evolution of ovine MHC class II B genes: DQB and DRB,” Immunogenetics, vol. 34, no. 2, pp. 80–87, 1991.View at: Google Scholar
S. Paterson, K. Wilson, and J. M. Pemberton, “Major histocompatibility complex variation associated with juvenile survival and parasite resistance in a large unmanaged ungulate population (Ovis aries L.),” Proceedings of the National Academy of Sciences of the United States of America, vol. 95, no. 7, pp. 3714–3719, 1998.View at: Publisher Site | Google Scholar
F.-W. Schwaiger, D. Gostomski, M. J. Stear et al., “An ovine major histocompatibility complex DRB1 allele is associated with low faecal egg counts following natural, predominantly Ostertagia circumcincta infection,” International Journal for Parasitology, vol. 25, no. 7, pp. 815–822, 1995.View at: Publisher Site | Google Scholar
P. M. Outteridge, L. Andersson, P. G. C. Douch et al., “The PCR typing of MHC-DRB genes in the sheep using primers for an intronic microsatellite: application to nematode parasite resistance,” Immunology and Cell Biology, vol. 74, no. 4, pp. 330–336, 1996.View at: Google Scholar
L. M. Herrmann-Hoesing, S. N. White, M. R. Mousel, G. S. Lewis, and D. P. Knowles, “Ovine progressive pneumonia provirus levels associate with breed and Ovar-DRB1,” Immunogenetics, vol. 60, no. 12, pp. 749–758, 2008.View at: Google Scholar
M. A. Hohenhaus and P. M. Outteridge, “The immunogenetics of resistance to Trichostrongylus colubriformis and Haemonchus contortus parasites in sheep,” British Veterinary Journal, vol. 151, no. 2, pp. 119–140, 1995.View at: Google Scholar
M. E. Azab, S. A. Bishara, R. M. R. Ramzy, N. M. Oteifa, L. M. El-Hoseiny, and M. A. Ahmed, “The evaluation of HLA-DRB1 antigens as susceptibility markers for unilocular cystic echinococcosis in Egyptian patients,” Parasitology Research, vol. 92, no. 6, pp. 473–477, 2004.View at: Publisher Site | Google Scholar
D.-A. Vuitton, G. Mantion, B. Bartholomot, P. Giraudoux, and S. Bresson-Hadni, “Parasite-host relationships and treatment,” Bulletin de l'Academie Nationale de Medecine, vol. 192, no. 6, pp. 1103–1117, 2008.View at: Google Scholar
V. Godot, S. Harraga, I. Beurton et al., “Resistance/susceptibility to Echinococcus multilocularis infection and cytokine profile in humans. II. Influence of the HLA B8, DR3, DQ2 haplotype,” Clinical and Experimental Immunology, vol. 121, no. 3, pp. 491–498, 2000.View at: Publisher Site | Google Scholar
S. Konnai, Y. Nagaoka, S. Takesima, M. Onuma, and Y. Aida, “DNA typing for ovine MHC DRB1 using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP),” Journal of Dairy Science, vol. 86, no. 10, pp. 3362–3365, 2003.View at: Google Scholar
P. Feichtlbauer-Huber, M. J. Stear, R. Fries, and J. Buitkamp, “Reference-strand-mediated conformation analysis of MHC alleles: a new method for high-resolution typing of the Ovar-DQB genes,” Immunogenetics, vol. 51, no. 1, pp. 65–68, 2000.View at: Google Scholar
H. Liu, G. F. Su, S. H. Wei et al., “Association of human leucocyte antigen-DRB and -DQB alleles with Eales' disease,” Chinese Journal of Ocular Fundus Diseases, vol. 22, no. 2, pp. 90–93, 2006.View at: Google Scholar
M.-Y. Fang, X.-X. Hu, N. Li, and C.-X. Wu, “Polymorphism analysis of the exon 2 of SLA-DQB gene in Xiao Meishan, Zhong Meishan and Yorkshire pigs with PCR-RFLP,” Acta Genetica Sinica, vol. 29, no. 8, pp. 685–687, 2002.View at: Google Scholar
K. Wang, D. X. Sun, R. H. Xu et al., “Polymorphism in upstream regulatory region of BoLA-DQB genes,” Acta VEterinaria Et Zootechnica Sinica, vol. 36, no. 6, pp. 521–525, 2005.View at: Google Scholar
S. X. Gao, S. Z. Xu, J. Q. Li et al., “Analysis of polymorphisms of BoLA-DQA and DRB3 exon2 and the relationship between the gene and mastitis in dairy cattle,” Acta VEterinaria Et Zootechnica Sinica, vol. 37, no. 4, pp. 317–320, 2006.View at: Google Scholar
T. H. Eiermann, F. Bettens, P. Tiberghien et al., “HLA and alveolar echinococcosis,” Tissue Antigens, vol. 52, no. 2, pp. 124–129, 1998.View at: Google Scholar