# MHC-DRB1/DQB1 Gene Polymorphism and Its Association with Resistance/Susceptibility to Cystic Echinococcosis in Chinese Merino Sheep

Authors: Hong Shen; Guohua Han; Bin Jia; Song Jiang; Yingchun Du
Journal: Journal of Parasitology Research
 (2014)

---

## Abstract

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 (P
<
0.01), SacIab in DRB1 exon 2 (P
<
0.05), and TaqIaa, HaeIIInn (P
<
0.01) 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 (P
<
0.01) were relatively resistant to C.E, while individuals with the genic haplotypes DRB1-Mvalbc/DQB1-Mvalyy/DQB1-TaqIab/DQB1-HaeIIImn (P
<
0.01) and DRB1-Mvalbb/DQB1-Mvalcc/DQB1-TaqIab/DQB1-HaeIIImn (P
<
0.01) 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.


---

## Body

## 1. Introduction

The major histocompatibility complex (MHC) gene of sheep is located on Chromosome 20 and is called Ovar [1]. The MHC gene family includes two major subfamilies: class I and class II genes [2]. 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 [7]. 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 [8]; furthermore, specific MHC alleles are associated with parasite resistance in sheep [9]. 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′ [20]. 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 ofTaqpolymerase 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′) [20]. 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 ofDQB1 was amplified by primers FW: 5′-CCC CGC AGA GGA TTT CGT G-3′ and REV: 5′-ACC TCG CCG CTG CCA GGT-3′ [21]; 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 ofTaqpolymerase. 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.


### 2.3. RFLP

The cleavage map typing method and allele nomenclature referred to that of Konnai et al. [20]. Each 10 μL ofDRB1 PCR product was digested with 5 U ofSacI,Hin1I,HaeIII,MvaI, andSacII, respectively, in a total volume of 20 μL, including 2 μL 10× buffer. Each 10 μL ofDQB1 PCR product was digested with 5 U ofMroxI,ScaI,SacII,NciI,TaqI,MvaI, andHaeIII, 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 wereHaeIIImm,HaeIIInn,MvaIyy, andMvaIzz genotype, which are inconsistent with the previous reports [20]. 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 withE.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 withE.g. Eight of the sheep with the haplotype ofDRB1-SacIab/DRB1-MvaIbb/DQB1-TaqIaa/DQB1-HaeIIInn were taken as the test group, and the other eight sheep with the haplotypes ofDRB1-SacIab/DRB1-MvaIbc/DQB1-TaqIaa/DQB1-HaeIIInn,DRB1-SacIab/DRB1-MvaIbc/DQB1-TaqIaa/DQB1-HaeIIImn,orDRB1-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 byt-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 withE.g.


## 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′ [20]. 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 ofTaqpolymerase 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′) [20]. 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 ofDQB1 was amplified by primers FW: 5′-CCC CGC AGA GGA TTT CGT G-3′ and REV: 5′-ACC TCG CCG CTG CCA GGT-3′ [21]; 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 ofTaqpolymerase. 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.


## 2.3. RFLP

The cleavage map typing method and allele nomenclature referred to that of Konnai et al. [20]. Each 10 μL ofDRB1 PCR product was digested with 5 U ofSacI,Hin1I,HaeIII,MvaI, andSacII, respectively, in a total volume of 20 μL, including 2 μL 10× buffer. Each 10 μL ofDQB1 PCR product was digested with 5 U ofMroxI,ScaI,SacII,NciI,TaqI,MvaI, andHaeIII, 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 wereHaeIIImm,HaeIIInn,MvaIyy, andMvaIzz genotype, which are inconsistent with the previous reports [20]. 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 withE.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 withE.g. Eight of the sheep with the haplotype ofDRB1-SacIab/DRB1-MvaIbb/DQB1-TaqIaa/DQB1-HaeIIInn were taken as the test group, and the other eight sheep with the haplotypes ofDRB1-SacIab/DRB1-MvaIbc/DQB1-TaqIaa/DQB1-HaeIIInn,DRB1-SacIab/DRB1-MvaIbc/DQB1-TaqIaa/DQB1-HaeIIImn,orDRB1-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 byt-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 withE.g.


## 3. Results

### 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).


### 3.2. PCR-RFLP

From restriction digestion ofDRB1 exon 2 PCR product, genotypes ofSacI,Hin1I,MvaI,SacII, andHaeIII (Table S2B) were observed, and some of genotypic restriction maps were in Figure 1. In addition, genotypes of restriction enzymesMroxI,ScaI,SacII,NciI,TaqI,MvaI, andHaeIII (Table S2B) forDQB1 PCR products were also observed, and some of their genotypic restriction maps were in Figure 2.(a) Part results of electrophoretic patterns of exon 2 of MHC-DRB1 digested withSacI in Chinese Merino sheep; M: pUC19 DNA marker. (b) Part results of electrophoretic patterns of exon 2 of MHC-DRB1 digested withSacII in Chinese Merino sheep; M: pUC19 DNA marker. (c) Part results of electrophoretic patterns of exon 2 of MHC-DRB1 digested withMvaI in Chinese Merino sheep; M: pUC19 DNA marker. (d) Part results of electrophoretic patterns of exon 2 of MHC-DRB1 digested withHaeIII in Chinese Merino sheep; M: pUC19 DNA marker.
(a)
(b)
(c)
(d)(a) Part results of electrophoretic patterns of exon 2 of MHC-DQB1 digested with TaqI in Chinese Merino sheep; M: pUC19 DNA marker. (b) Part results of electrophoretic patterns of exon 2 of MHC-DQB1. (c) Part results of electrophoretic patterns of exon 2 of MHC-DQB1 digested withHaeIII in Chinese Merino sheep; M: pUC19 DNA marker.
(a)
(b)
(c)


### 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 andHaeIIInn. In addition, two G-to-A point mutations at base positions 96 and 246 resulted in new alleles,MvaIy andMvaIz. 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 thatDRB1 genotypic frequencies ofMvaIbb (P
<
0.01),HaeIIIee, andSacIab (P
<
0.05) in negatives were higher than in C.E sheep, indicating a strong association between these genotypes and C.E resistance, while genotypes in terms ofSacIIab (P
<
0.05),HaeIIIdf (P
<
0.05),HaeIIIbd (P
<
0.01), andMvaIbc (P
<
0.01) inDRB1 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 ofTaqIaa andHaeIIInn (P
<
0.01),MvaIdz (P
<
0.05) in negatives were higher than in positives, while genotypes ofTaqIab andHaeIIImn (P
<
0.01),MvaIcz (P
<
0.05) in positives were higher than in negatives (Table 2). Therefore, we concluded thatDQB1 genotypes ofTaqIaa,HaeIIInn, andMvaIdz were resistant to C.E, while genotypes ofTaqIab,HaeIIImn, andMvaIcz were susceptible to C.E.Table 1
Genotypic frequencies ofDRB1 in Chinese Merino sheep with and without Cystic Echinococcosis.
Cystic Echinococcosis negative
Cystic Echinococcosis positive
Genotype
Number
Frequency
Genotype
Number
Frequency
SacI aa
42
0.3853
SacI aa
47
0.4700
SacI ab
58
0.5321*
SacI ab
38
0.3800
SacI bb
9
0.0826
SacI bb
15
0.1500
Hin1I aa
15
0.1376
Hin1I aa
14
0.1414
Hin1I ab
55
0.5046
Hin1I ab
43
0.4343
Hin1I bb
39
0.3578
Hin1I bb
42
0.4243
SacII aa
65
0.7471
SacII aa
50
0.6250
SacII ab
13
0.1494
SacII ab
22
0.2750*
SacII bb
9
0.1035
SacII bb
8
0.1000
MvaI aa
1
0.0115
MvaI aa
0
0
MvaI bb
68
0.7816**
MvaI bb
45
0.5556
MvaI cc
1
0.0115
MvaI cc
4
0.0494
MvaI dd
0
0
MvaI dd
0
0
MvaI ab
0
0
MvaI ab
0
0
MvaI ac
0
0
MvaI ac
1
0.0124
MvaI bc
17
0.1954
MvaI bc
31
0.3827**
HaeIII aa
17
0.1651
HaeIII aa
11
0.1100
HaeIII bb
9
0.0874
HaeIII bb
4
0.0400
HaeIII cc
5
0.0485
HaeIII cc
7
0.0700
HaeIII dd
0
0
HaeIII dd
1
0.0100
HaeIII ee
11
0.1068*
HaeIII ee
2
0.0200
HaeIII ff
11
0.1068
HaeIII ff
8
0.0800
HaeIII ab
1
0.0097
HaeIII ab
4
0.0400
HaeIII ac
12
0.1165
HaeIII ac
13
0.1300
HaeIII ae
1
0.0097
HaeIII ae
4
0.0400
HaeIII bd
1
0.0097
HaeIII bd
9
0.0900**
HaeIII be
2
0.0194
HaeIII be
5
0.0500
HaeIII cd
6
0.0583
HaeIII cd
0
0
HaeIII ce
16
0.1554
HaeIII ce
7
0.0700
HaeIII df
5
0.0485
HaeIII df
13
0.1300*
HaeIII ef
6
0.0583
HaeIII ef
12
0.1200
Note: the same genotypes ofDRB1 in Chinese Merino sheep with and without Cystic Echinococcosis, *
P
<
0.05, *
*
P
<
0.01.Table 2
Genotypic frequencies of DQB1 in Chinese Merino sheep with and without Cystic Echinococcosis.
Cystic Echinococcosis negative
Cystic Echinococcosis positive
Genotype
Number
Genotype
Number
Genotype
Number
MroxI aa
31
0.4493
MroxI aa
28
0.4375
MroxI ab
24
0.3478
MroxI ab
25
0.3906
MroxI aa
14
0.2029
MroxI aa
11
0.1719
ScaI aa
10
0.1176
ScaI aa
14
0.1972
ScaI ab
74
0.8706
ScaI ab
57
0.8028
ScaI bb
1
0.0118
ScaI bb
0
0
NciI xx
60
0.7058
NciI xx
66
0.7021
NciI gg
16
0.1882
NciI gg
20
0.2127
NciI xg
9
0.1058
NciI xg
8
0.0851
SacII aa
25
0.4464
SacII aa
37
0.5968
SacII bb
3
0.0536
SacII bb
0
0
SacII cc
5
0.0893
SacII cc
7
0.1129
SacII ab
10
0.1759
SacII ab
4
0.0645
SacII ac
12
0.2143
SacII ac
8
0.1290
SacII ad
1
0.0179
SacII ad
5
0.0806
SacII bd
0
0
SacII bd
1
0.0161
TaqI aa
85
0.8252**
TaqI aa
58
0.55743
TaqI bb
1
0.0097
TaqI bb
1
0.0099
TaqI ab
17
0.1650
TaqI ab
41
0.4059**
TaqI ac
0
0
TaqI ac
1
0.0099
MvaI aa
7
0.0737
MvaI aa
3
0.0361
MvaI bb
0
0
MvaI bb
1
0.0120
MvaI cc
13
0.1368
MvaI cc
18
0.2169
MvaI dd
12
0.1263
MvaI dd
4
0.0482
MvaI zz
22
0.2316
MvaI zz
16
0.1928
MvaI yy
15
0.1579
MvaI yy
14
0.1687
MvaI ad
1
0.0105
MvaI ad
0
0
MvaI az
3
0.0316
MvaI az
1
0.0120
MvaI bc
0
0
MvaI bc
1
0.0120
MvaI bd
0
0
MvaI bd
1
0.0120
MvaI bz
0
0
MvaI bz
1
0.0120
MvaI by
2
0.0211
MvaI by
0
0
MvaI cd
3
0.0316
MvaI cd
6
0.0723
MvaI cz
7
0.0737
MvaI cz
14
0.1687*
MvaI dz
9
0.0947*
MvaI dz
2
0.0241
MvaI dy
1
0.0105
MvaI dy
1
0.0120
HaeIII aa
3
0.0312
HaeIII aa
0
0
HaeIII mm
24
0.2500
HaeIII mm
32
0.3299
HaeIII nn
55
0.5729**
HaeIII nn
28
0.2887
HaeIII am
1
0.0104
HaeIII am
3
0.0309
HaeIII an
2
0.0208
HaeIII an
3
0.0309
HaeIII mn
11
0.1146
HaeIII mn
31
0.3196**
Note: the same genotypes ofDQB1 in Chinese Merino sheep with and without Cystic Echinococcosis, *
P
<
0.05, *
*
P
<
0.01.


### 3.5. Verification of Artificial Infection withE.g

Analyzing the haplotype of resistant genotypes, it was found that the haplotype frequency ofDRB1-SacIab/DRB1-MvaIbb/DQB1-TaqIaa/DQB1-HaeIIInn in C.E-negative sheep was higher than in C.E sheep (P
<
0.01), indicating that this haplotype was the resistant haplotype of Chinese Merino sheep (Table 3). The result was verified by artificial infection hydatidosis. The haplotypes ofDRB1-MvaIbc/DQB1-MvaIyy/DQB1-TaqIab/DQB1-HaeIIImn andDRB1-MvaIbb/DQB1-MvaIcc/DQB1-TaqIab/DQB1-HaeIIImn in positives were higher than in negatives (P
<
0.01), which implied that these haplotypes were susceptible to C.E individuals.Table 3
Assessment of the relationship between haplotype and Cystic Echinococcosis in Chinese Merino sheep.
Haplotype of MHC
Number of Cystic Echinococcosis positive cases
Number of Cystic Echinococcosis negative cases
χ
2
DRB1-SacIab/DRB1-MvaIbb/DQB1-TaqIaa/DQB1-HaeIIInn
19
1
17.5734**
DRB1-SacIab/DRB1-MvaIbc/DQB1-TaqIaa/DQB1-HaeIIInn
6
6
0.0012
DRB1-SacIab/DRB1-MvaIbc/DQB1-TaqIaa/DQB1-HaeIIImn
8
3
2.3000
DRB1-SacIab/DRB1-MvaIbb/DQB1-TaqIaa/DQB1-HaeIIImm
14
11
0.3460
DRB1-MvaIbc/DQB1-MvaIyy
/DQB1-TaqIab/DQB1-HaeIIImn
0
13
14.1600**
DRB1-MvaIbb/DQB1-MvaIcc/DQB1-TaqIab/DQB1-HaeIIImn
1
18
17.1439**
DRB1-MvaIbc/DQB1-MvaIbb/DQB1-TaqIab/DQB1-HaeIIInn
4
7
0.9280
DRB1-MvaIbc/DQB1-MvaIcc/DQB1-TaqIaa/DQB1-HaeIIImn
0
2
2.0600
DRB1-MvaIbb/DQB1-MvaIcz/DQB1-TaqIaa/DQB1-HaeIIImn
1
5
2.8292
Note:χ
2
>
χ
0.01
,
1
2
=
6.63, P
<
0.01. χ
2
>
χ
0.05,1
2
=
3.84, P
<
0.05. χ
2
<
χ
0.05,1
2
=
3.84, P
>
0.05.
*
P
<
0.05, *
*
P
<
0.01.Protoscoleces can develop into cysts within 20 days postinfection [22]. The sixteen sheep that were artificially infected withE.g were slaughtered in the second month afterE.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 [23]. Results show that 3 sheep were infected withE.g in the test group, whereas 6 sheep were infected withE.g in the control group; therefore, the infection rate in the test group was significantly lower than that of the control group (P
<
0.05). It is confirmed that the genic haplotypeDRB1-SacIab/DRB1-MvaIbb/DQB1-TaqIaa/DQB1-HaeIIInn leads to C.E resistance in Chinese Merino sheep.


## 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).


## 3.2. PCR-RFLP

From restriction digestion ofDRB1 exon 2 PCR product, genotypes ofSacI,Hin1I,MvaI,SacII, andHaeIII (Table S2B) were observed, and some of genotypic restriction maps were in Figure 1. In addition, genotypes of restriction enzymesMroxI,ScaI,SacII,NciI,TaqI,MvaI, andHaeIII (Table S2B) forDQB1 PCR products were also observed, and some of their genotypic restriction maps were in Figure 2.(a) Part results of electrophoretic patterns of exon 2 of MHC-DRB1 digested withSacI in Chinese Merino sheep; M: pUC19 DNA marker. (b) Part results of electrophoretic patterns of exon 2 of MHC-DRB1 digested withSacII in Chinese Merino sheep; M: pUC19 DNA marker. (c) Part results of electrophoretic patterns of exon 2 of MHC-DRB1 digested withMvaI in Chinese Merino sheep; M: pUC19 DNA marker. (d) Part results of electrophoretic patterns of exon 2 of MHC-DRB1 digested withHaeIII in Chinese Merino sheep; M: pUC19 DNA marker.
(a)
(b)
(c)
(d)(a) Part results of electrophoretic patterns of exon 2 of MHC-DQB1 digested with TaqI in Chinese Merino sheep; M: pUC19 DNA marker. (b) Part results of electrophoretic patterns of exon 2 of MHC-DQB1. (c) Part results of electrophoretic patterns of exon 2 of MHC-DQB1 digested withHaeIII in Chinese Merino sheep; M: pUC19 DNA marker.
(a)
(b)
(c)


## 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 andHaeIIInn. In addition, two G-to-A point mutations at base positions 96 and 246 resulted in new alleles,MvaIy andMvaIz. 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 thatDRB1 genotypic frequencies ofMvaIbb (P
<
0.01),HaeIIIee, andSacIab (P
<
0.05) in negatives were higher than in C.E sheep, indicating a strong association between these genotypes and C.E resistance, while genotypes in terms ofSacIIab (P
<
0.05),HaeIIIdf (P
<
0.05),HaeIIIbd (P
<
0.01), andMvaIbc (P
<
0.01) inDRB1 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 ofTaqIaa andHaeIIInn (P
<
0.01),MvaIdz (P
<
0.05) in negatives were higher than in positives, while genotypes ofTaqIab andHaeIIImn (P
<
0.01),MvaIcz (P
<
0.05) in positives were higher than in negatives (Table 2). Therefore, we concluded thatDQB1 genotypes ofTaqIaa,HaeIIInn, andMvaIdz were resistant to C.E, while genotypes ofTaqIab,HaeIIImn, andMvaIcz were susceptible to C.E.Table 1
Genotypic frequencies ofDRB1 in Chinese Merino sheep with and without Cystic Echinococcosis.
Cystic Echinococcosis negative
Cystic Echinococcosis positive
Genotype
Number
Frequency
Genotype
Number
Frequency
SacI aa
42
0.3853
SacI aa
47
0.4700
SacI ab
58
0.5321*
SacI ab
38
0.3800
SacI bb
9
0.0826
SacI bb
15
0.1500
Hin1I aa
15
0.1376
Hin1I aa
14
0.1414
Hin1I ab
55
0.5046
Hin1I ab
43
0.4343
Hin1I bb
39
0.3578
Hin1I bb
42
0.4243
SacII aa
65
0.7471
SacII aa
50
0.6250
SacII ab
13
0.1494
SacII ab
22
0.2750*
SacII bb
9
0.1035
SacII bb
8
0.1000
MvaI aa
1
0.0115
MvaI aa
0
0
MvaI bb
68
0.7816**
MvaI bb
45
0.5556
MvaI cc
1
0.0115
MvaI cc
4
0.0494
MvaI dd
0
0
MvaI dd
0
0
MvaI ab
0
0
MvaI ab
0
0
MvaI ac
0
0
MvaI ac
1
0.0124
MvaI bc
17
0.1954
MvaI bc
31
0.3827**
HaeIII aa
17
0.1651
HaeIII aa
11
0.1100
HaeIII bb
9
0.0874
HaeIII bb
4
0.0400
HaeIII cc
5
0.0485
HaeIII cc
7
0.0700
HaeIII dd
0
0
HaeIII dd
1
0.0100
HaeIII ee
11
0.1068*
HaeIII ee
2
0.0200
HaeIII ff
11
0.1068
HaeIII ff
8
0.0800
HaeIII ab
1
0.0097
HaeIII ab
4
0.0400
HaeIII ac
12
0.1165
HaeIII ac
13
0.1300
HaeIII ae
1
0.0097
HaeIII ae
4
0.0400
HaeIII bd
1
0.0097
HaeIII bd
9
0.0900**
HaeIII be
2
0.0194
HaeIII be
5
0.0500
HaeIII cd
6
0.0583
HaeIII cd
0
0
HaeIII ce
16
0.1554
HaeIII ce
7
0.0700
HaeIII df
5
0.0485
HaeIII df
13
0.1300*
HaeIII ef
6
0.0583
HaeIII ef
12
0.1200
Note: the same genotypes ofDRB1 in Chinese Merino sheep with and without Cystic Echinococcosis, *
P
<
0.05, *
*
P
<
0.01.Table 2
Genotypic frequencies of DQB1 in Chinese Merino sheep with and without Cystic Echinococcosis.
Cystic Echinococcosis negative
Cystic Echinococcosis positive
Genotype
Number
Genotype
Number
Genotype
Number
MroxI aa
31
0.4493
MroxI aa
28
0.4375
MroxI ab
24
0.3478
MroxI ab
25
0.3906
MroxI aa
14
0.2029
MroxI aa
11
0.1719
ScaI aa
10
0.1176
ScaI aa
14
0.1972
ScaI ab
74
0.8706
ScaI ab
57
0.8028
ScaI bb
1
0.0118
ScaI bb
0
0
NciI xx
60
0.7058
NciI xx
66
0.7021
NciI gg
16
0.1882
NciI gg
20
0.2127
NciI xg
9
0.1058
NciI xg
8
0.0851
SacII aa
25
0.4464
SacII aa
37
0.5968
SacII bb
3
0.0536
SacII bb
0
0
SacII cc
5
0.0893
SacII cc
7
0.1129
SacII ab
10
0.1759
SacII ab
4
0.0645
SacII ac
12
0.2143
SacII ac
8
0.1290
SacII ad
1
0.0179
SacII ad
5
0.0806
SacII bd
0
0
SacII bd
1
0.0161
TaqI aa
85
0.8252**
TaqI aa
58
0.55743
TaqI bb
1
0.0097
TaqI bb
1
0.0099
TaqI ab
17
0.1650
TaqI ab
41
0.4059**
TaqI ac
0
0
TaqI ac
1
0.0099
MvaI aa
7
0.0737
MvaI aa
3
0.0361
MvaI bb
0
0
MvaI bb
1
0.0120
MvaI cc
13
0.1368
MvaI cc
18
0.2169
MvaI dd
12
0.1263
MvaI dd
4
0.0482
MvaI zz
22
0.2316
MvaI zz
16
0.1928
MvaI yy
15
0.1579
MvaI yy
14
0.1687
MvaI ad
1
0.0105
MvaI ad
0
0
MvaI az
3
0.0316
MvaI az
1
0.0120
MvaI bc
0
0
MvaI bc
1
0.0120
MvaI bd
0
0
MvaI bd
1
0.0120
MvaI bz
0
0
MvaI bz
1
0.0120
MvaI by
2
0.0211
MvaI by
0
0
MvaI cd
3
0.0316
MvaI cd
6
0.0723
MvaI cz
7
0.0737
MvaI cz
14
0.1687*
MvaI dz
9
0.0947*
MvaI dz
2
0.0241
MvaI dy
1
0.0105
MvaI dy
1
0.0120
HaeIII aa
3
0.0312
HaeIII aa
0
0
HaeIII mm
24
0.2500
HaeIII mm
32
0.3299
HaeIII nn
55
0.5729**
HaeIII nn
28
0.2887
HaeIII am
1
0.0104
HaeIII am
3
0.0309
HaeIII an
2
0.0208
HaeIII an
3
0.0309
HaeIII mn
11
0.1146
HaeIII mn
31
0.3196**
Note: the same genotypes ofDQB1 in Chinese Merino sheep with and without Cystic Echinococcosis, *
P
<
0.05, *
*
P
<
0.01.


## 3.5. Verification of Artificial Infection withE.g

Analyzing the haplotype of resistant genotypes, it was found that the haplotype frequency ofDRB1-SacIab/DRB1-MvaIbb/DQB1-TaqIaa/DQB1-HaeIIInn in C.E-negative sheep was higher than in C.E sheep (P
<
0.01), indicating that this haplotype was the resistant haplotype of Chinese Merino sheep (Table 3). The result was verified by artificial infection hydatidosis. The haplotypes ofDRB1-MvaIbc/DQB1-MvaIyy/DQB1-TaqIab/DQB1-HaeIIImn andDRB1-MvaIbb/DQB1-MvaIcc/DQB1-TaqIab/DQB1-HaeIIImn in positives were higher than in negatives (P
<
0.01), which implied that these haplotypes were susceptible to C.E individuals.Table 3
Assessment of the relationship between haplotype and Cystic Echinococcosis in Chinese Merino sheep.
Haplotype of MHC
Number of Cystic Echinococcosis positive cases
Number of Cystic Echinococcosis negative cases
χ
2
DRB1-SacIab/DRB1-MvaIbb/DQB1-TaqIaa/DQB1-HaeIIInn
19
1
17.5734**
DRB1-SacIab/DRB1-MvaIbc/DQB1-TaqIaa/DQB1-HaeIIInn
6
6
0.0012
DRB1-SacIab/DRB1-MvaIbc/DQB1-TaqIaa/DQB1-HaeIIImn
8
3
2.3000
DRB1-SacIab/DRB1-MvaIbb/DQB1-TaqIaa/DQB1-HaeIIImm
14
11
0.3460
DRB1-MvaIbc/DQB1-MvaIyy
/DQB1-TaqIab/DQB1-HaeIIImn
0
13
14.1600**
DRB1-MvaIbb/DQB1-MvaIcc/DQB1-TaqIab/DQB1-HaeIIImn
1
18
17.1439**
DRB1-MvaIbc/DQB1-MvaIbb/DQB1-TaqIab/DQB1-HaeIIInn
4
7
0.9280
DRB1-MvaIbc/DQB1-MvaIcc/DQB1-TaqIaa/DQB1-HaeIIImn
0
2
2.0600
DRB1-MvaIbb/DQB1-MvaIcz/DQB1-TaqIaa/DQB1-HaeIIImn
1
5
2.8292
Note:χ
2
>
χ
0.01
,
1
2
=
6.63, P
<
0.01. χ
2
>
χ
0.05,1
2
=
3.84, P
<
0.05. χ
2
<
χ
0.05,1
2
=
3.84, P
>
0.05.
*
P
<
0.05, *
*
P
<
0.01.Protoscoleces can develop into cysts within 20 days postinfection [22]. The sixteen sheep that were artificially infected withE.g were slaughtered in the second month afterE.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 [23]. Results show that 3 sheep were infected withE.g in the test group, whereas 6 sheep were infected withE.g in the control group; therefore, the infection rate in the test group was significantly lower than that of the control group (P
<
0.05). It is confirmed that the genic haplotypeDRB1-SacIab/DRB1-MvaIbb/DQB1-TaqIaa/DQB1-HaeIIInn leads to C.E resistance in Chinese Merino sheep.


## 4. Discussion and Conclusion

TheMHC 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 ofDRB in goats, Konnai et al. [20] researched the polymorphism ofDRB1 in some sheep, with results indicating that affluent polymorphism exists in the Ovis aries-DRB1 gene, and Dongxiao and Yuan [26] studiedDRB3 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 andDQA in human [28], swine [29], and cattle [30, 31]. However, there are still no domestic reports of Ovis aries-DQB1. In the present study, we usedMroxI,ScaI,SacII,NciI,TaqI,HaeIII, andMvaI by PCR-RFLP to analyzeDQB1 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, andMvaIz, 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 [10]. For example, Sayers et al. [11] 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 [32]. 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 [16]. Differences have been shown between HLA characteristics of A.E patients with different courses ofE.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 [19]. This study found that theDRB1-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.



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*Source: 272601-2014-03-24.xml*