Introduction

Major histocompatibility complex (MHC) is a closely linked and highly polymorphic genetic region in the chromosome, composed of several gene loci. MHC-encoded molecules can recognize and present endogenous and exogenous antigens, followed by presenting the antigens to T cells, thereby leading to cellular and humoral immune responses (Millot 1978; Klein 1986). MHC polymorphism is closely related to resistance and susceptibility of many diseases, and MHC genes are considered as the primary candidates, currently rendering the phenomenon as one of the hotspots in the research of immunogenetics. In 1936, MHC genes were first shown in mice (Gorer 1937), and this have been followed in almost all the vertebrates. Although MHC and the encoded products vary across species, the gene structure and function are similar. In addition, the MHCs in mice and rats are named as H-2 complex and RT-1 complex, respectively. While the MHC of other species are named referring to the human leukocyte antigens (HLA). For instance, the MHC in sheep is named as ovine leukocyte antigens (OLA) (Mahdy et al. 1989; Hediger et al. 1991), which is localized on chromosome 20 and divided into three classes: I, II and III. The gene region of MHC class II in sheep is similar to that of humans and is subdivided into IIa and IIb. The IIa sub-region includes loci DRA, DRB1, DRB2, DRB3, DRB4, DQA1, DQA2, DQB1 and DQB2, and IIb includes DYA, DYB, DNA, DOB, DMA and DMB (Blattman et al. 1993; Escayg et al. 1996). The most polymorphic locus of MHC in sheep is DR (Andersson and Rask 1988), including the functional gene DRB1 and pseudogenes DRB2, DRB3 and DRB4 (Scott et al. 1991). Abundant polymorphism is observed on exon 2, and the adjacent intron 2 in DRB1. The β1 structural domain encoded by exon 2 constitutes the DR molecular peptide-binding region, which regulates the immune responses. It presents exogenous antigens to helper T-lymphocytes (Th, CD4+) to trigger the immune response, and hence, serves as a candidate gene for immune response and disease resistance (Brown et al. 1993; Palti et al. 2001). Currently, the reports on sheep MHC gene resistance/susceptibility primarily focus on class II, especially the polymorphism on exon 2 in the DRB1 gene (Schwaiger et al. 1995; Paterson et al. 1998; Sayers et al. 2005; Dukkipati et al. 2010). GenBank database has included >120 amino acid polymorphic sequences of DRB1 exon 2 encoding the β1 structural domain (Dutia et al. 1994; Jugo and Vicario 2001; Hemrrann et al. 2005). Also, a number of studies have reported the correlation between MHC in sheep breeds and disease (Ballingall and Riccardo 2010; Li et al. 2010, 2011a; Hui et al. 2012; Shen et al. 2014). However, none of the studies have yet addressed the correlation between sheep MHC-DRB1 gene polymorphism and mycoplasma pneumonia (MP) resistance.

MP, also known as mycoplasma pleuropneumonia, is a chronic respiratory tract infectious disease mainly caused by Mycoplasma ovipneumoniae (MO), manifested as cough, tachypnea, exercise intolerance, gasp, nasal discharge, progressive emaciation, and proliferative inflammation in pulmonary interstitium. The disease cannot be cured easily, and shows a high incidence among lambs. The sick lambs present weight loss, and the fatality rate is higher than that of the healthy adult sheep. The feed conversion ratio of the sick sheep is low, incurring a high medical cost, that in turn causes a huge economic burden to the sheep industry worldwide (Ayling and Nicholas 2007; Bottinelli et al. 2017). Xinjiang is a region with high incidence of the disease, and the loss accounts for >20% of all the diseases in sheep and goat breeding industry (Song et al. 2011). Occupants of Uyghurs ethnicity with large demand for mutton dominated this region. Since the availability of mutton in this area is low, several high-fecundity and meat sheep breeds has been introduced to improve the local breed in recent years. The epidemiological investigations of the new breeds have proved that high-fecundity Hu sheep is susceptible to MP (Li et al. 2005; Tao et al. 2006); especially, the infection rate of lamb is 40–60%, and the fatality rate is 10–20%. The Dorper sheep with superior meat quality is resistant to the disease; also, the hybrid generation is resistant to disease similar to the male parent (Li et al. 2005; Tao et al. 2006, 2007; Song et al. 2011). Interestingly, Dorper and Hu sheep are introduced from other places. However, after introduction into Xinjiang, these breeds showed a large differential resistance to MO, laying the foundation for the investigation of MO resistance in different sheep breeds.

Thus, in this study, the polymorphism of exon 2 in MHC-DRB1 among the three sheep groups was analysed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) genotyping. Also, the correlation between the genotype and resistance/susceptibility to MP was studied. The artificial MO-infection experiment was used to detect the disease resistance capability of sheep with candidate genotypes and explore the molecular mechanism, which provided a theoretical basis and reference for the application of anti-disease genotype of MHC class II gene in breeding to gain disease resistance.

Methods and methods

Animals and sample preparation

The blood samples were collected from 400 Hu sheep in Xinbao sheep farm, 560 Dorpers in Rifa sheep farm, and 255 F1 generations in Xinbao and Rifa sheep farm of Aksu, Xinjiang. A volume of 10 mL blood sample was withdrawn from each sheep, of which, 5 mL with anticoagulant was used to extract the genomic DNA, and the remaining was used to obtain serum at room temperature for further detection of the rate of MO.

PCR of MHC-DRB1 exon 2

The primer sequence was synthesized by Sangon Biotech (Shanghai) as described previously (Konnai et al. 2003). The 20 µL reaction system comprised of 1 μL of genome DNA at 150 ng/μL, 10 μL of 2× PCR mix, and 0.5 μL of upstream and downstream primers (100 μM) each. The amplification conditions were as follows: predenaturation for 5 min at 94°C; 35 cycles at 94°C for 30 s, 63°C for 30 s, 72°C for 30 s; 72°C for 7 min.

RFLP genotyping

ScaI, HaeIII, SacII, MvaI and BsaHI restriction endonucleases (table 1) were used to digest the products. The 20 µL reaction volume consisting of 5 μL of PCR product, 1 μL of 10× buffer, and 0.5 μL of 10 U/μL endonuclease was incubated at 37°C for 4 h or overnight. The cleaved products were detected using agarose gel electrophoresis and typed using standard restriction map provided by Konnai (Konnai et al. 2003).

Table 1 The genotypes of PCR-RFLP by restriction enzyme HaeIII in exon 2 of the MHC-DRB1 gene.
Full size table

MO indirect hemagglutination assay

The MO detection method previously described (Cheng et al. 2015) was used to detect the MO infection in sheep serum. Briefly, 96 V-shaped well plate was added with 25 μL of dilution buffer from the first to eight wells. In first well, 25 μL of the serum samples was added and mixed well. Twenty-five microliters of the mixture was add to the second well. The dilution continued until the eighth well. The plates were shaken for 2 min and then incubated at 37°C for 2 h without shaking. The test was considered positive when a layer of agglutinated erythrocytes was formed in wells at dilutions of 1:16 or higher, and positive and negative controls were included in each test. The positive rate of the infection was analysed in 100 samples.

MO experimental infection experiment in standard sheep

In total, 15 healthy with antibody negative, similar ages, body weights hybrid genotype F1 male sheep (similar type and weight) with HaeIII dd, MvaI cc and HaeIII ee were randomly selected from 255 F1 generations in Xinbao and Rifa sheep farm and divided into groups A, B and C, respectively (n = 5). One day before the experimental infection (−1 day), experimental infection (0 day) and on days 3, 7, 14, 21 and 27 after infection, two samples of fresh nonanticoagulant blood were collected at seven time points. At day 0, 5 mL of sheep MO was input into the trachea of the animal in the experimental groups as Y-98 standard strain culture medium (containing CFU = 106). The lung tissues infected with MO after death and 27 days after the experiment were collected for histopathological observation.

Observation and scoring of pathological change in lung tissue

The clinical manifestations in lung tissues were observed, and the pathological change was scored based on the clinical symptoms and evaluation criteria proposed by Nicholas et al. (2002). Sheep were scored for mental state, respiratory symptoms, rogue, severity and duration of cough and arthritic lesions. Details of the clinical assessment are given in table 1 in electronic supplementary material at http://www.ias.ac.in/jgenet/.

Preparation of histopathological sections and pathological scoring

Lung tissue samples were immersed in 10% formaldehyde, dehydrated, cleared, embedded, consecutively sliced (thickness 5–7 μm), unfolded, dried, stained with haematoxylin-eosin, and observed under an optical microscope to describe the pathological characteristics. The pathological status of the experimental sheep was scored according to the MP histopathological standard in Golden Syrian Hamster (Cimolai et al. 1992). The histopathologist was blinded to the source of tissue and, the right and left lung sections from sheep were randomized within the entire set of slides to be examined. The pathology grading system consisted of a numerical score ranging from 0 to 26 (table 2 in electronic supplementary material). Each sagittal section was scored based on a cumulative total from five categories that incorporated an evaluation of: (i) number of bronchiolar and bronchial sites involved by the periluminal infiltrate, (ii) severity of periluminal infiltrate, (iii) severity of luminal exudate, (iv) frequency of perivascular infiltrate, and (v) severity of parenchymal pneumonia. The scores from each lung section were added and then divided by two to arrive at a score per sheep.

Detection of cytokines by ELISA

The sera were collected from experimental sheep before and after infection, and the change in the level of cytokines (TNFα, IFNγ, IL-1β, IL-2IL-4, IL-6, IL-10) was detected by ELISA kits. All the specific ELISA kits were purchased from BlueGene (Shanghai, China).

Statistical analysis

The correlation between the genotype frequency and MP was analysed by chi-square test. Hardy–Weinberg equilibrium analysis was also performed by chi-square test. Other data were analysed by one-way ANOVA using SPSS 17.0 software. The data were expressed as mean ± SD. P < 0.05 and P < 0.01 indicated significant difference.

Results

PCR-RFLP genotyping of MHC-DRB1 exon 2 gene

OLA-ERB1 and OLA-HL031 primers were used to amplify the exon 2 of the MHC-DRB1 gene in the three sheep groups that provided the products of 296 bp.

SacI, SacII, MvaI, BsaHI and HaeIII restriction endonucleases were used to cleave the amplified products of exon 2 in the MHC-DRB1 gene from 560 Dorper sheep, 400 Hu sheep and 255 D × H F1 hybrids. The polymorphic variations could be detected in the exon 2 of DRB1 gene in the three sheep groups using the specific restriction endonucleases mentioned above. The cleavage of SacI, SacII, MvaI and BsaHI was controlled by bialleles, representing three genotypes respectively (figure 1, a, c&d). By contrast, HaeIII restriction endonuclease exhibited a great difference among the three sheep groups (figure 1b), the PCR products showed three genotypes after MvaI, SacI, SacII and BsaHI endonuclease digestion, and the PCR product HaeIII endonuclease can produce up to 15 genotypes. The size of the digested fragments are shown in tables 1 and 2.

Figure 1
figure 1

PCR-RFLP genotyping of five endonucleases. (a)–(e) Represent the electrophoretic patterns of the second exon of MHC-DRB1 digested with SacI, HaeIII, BsaHI, SacII and MvaI, respectively. The lanes marked with the corresponding genotypes. M, pUC19 DNA marker.

Full size image
Table 2 The genotypes of PCR-RFLP by restriction enzymes in exon 2 of the MHC-DRB1 gene.
Full size table

Correlation between polymorphism of exon 2 in MHC-DRB1 and resistance/susceptibility of MP

To explore the correlation between MHC-DRB1 gene polymorphism and the trait of MP resistance or susceptibility, the genotypes from the MO-susceptible sheep population (Hu sheep) and MO-resistant populations (Dorper sheep and D × H F1 hybrids) were analysed. Table 3 summarizes the distribution of genotypes and frequencies. Hu sheep with a high risk of MP were used as control, while the others were experimental sheep groups. The results showed that genotype percentage of HaeIII ee and MvaI bb in the Hu sheep was higher than that of the experimental groups, while genotypes in terms of HaeIII dd and MvaI cc occurred more often in the Dorper sheep when compared with the control group (table 3). Further, significant frequency differences were observed in HaeIII ee, MvaI bb, HaeIII dd and MvaI cc genotypes between the control and experimental groups (P < 0.001). MvaI bb genotype was dominant with a frequency of 70% in the Hu sheep group, and the allele frequency of MvaI B was up to 80% (table 3 in electronic supplementary material). The frequencies of the other four dominant genotypes, SacI aa, SacII aa, BsaHI bb, and HaeIII ee, were >50%. Intriguingly, the trend of SacI aa, SacII aa, and BsaHI bb genotypes frequency in Dorper sheep was same as that of Hu sheep (table 4 in electronic supplementary material). However, MvaI and HaeIII restriction endonuclease cleavage sites differed markedly. The MvaI cc genotype frequency in Dorper was up to 60% but only 10% in the Hu sheep. The HaeIII dd genotype frequency in Dorper was up to 48% but was only 20% in the Hu sheep. Conversely, HaeIII ee genotype frequency in Hu sheep was up to 52.5% but was only 23% in the Dorper group.

Table 3 The frequencies of genotypes and alleles in MHC-DRB1 exon 2 of the three groups.
Full size table

To further confirm the correlation between MO-resistant/susceptible genotype and MP in sheep, a total of 100/1115 blood samples were randomly selected to detect the mycoplasma antibody level; of which, 21 were positive for MO. The ratios of HaeIII ee, MvaI bb, HaeIII dd and MvaI cc genotypes in the 21 blood samples were analysed, and the ratios of MO-resistant MvaI cc and HaeIII dd genotypes were lower than the theoretical genotype frequency. Moreover, the ratios of MO-susceptible HaeIII ee and MvaI bb genotypes were higher than the theoretical genotype frequency (figure 2). The frequency of the MO-resistant MvaI cc and HaeIII dd differed significantly from that of MO-susceptible HaeIII ee and MvaI bb genotypes (P < 0.05).

Figure 2
figure 2

Percentage of candidate susceptibility and resistant genotypes among 21 MO antibody positive animals. Gray colour, theoretical value; black colour, means actual positive; *P < 0.05.

Full size image

Comparison of experimental infection of MP and infection effect between MO-resistant and MO-susceptible genotypes

Isolation and diagnosis of mycoplasma after experimental infection

Smears were prepared from lung tissues samples obtained from deceased sheep that were stained and examined by microscopy. Further, after incubation at 37°C for two days, any pathogenic micro-organisms were not detected on the normal agar, blood agar and MacConkey agar plates, and the colour of the mycoplasma-specific culture medium was not altered. Subsequently, after incubation for seven days under 5% CO2, the colour of the medium changed from pink to yellow. The suspected mycoplasma culture was smeared on the cover glass and stained with Giemsa, which revealed spherical, ring, and multishaped mycoplasma by microscopic examination. The mycoplasma was seeded in a plate and incubated at 37°C in the presence of 5% CO2 for four days. The colony with typical mycoplasma morphology, as observed under the microscope (figure 3), indicated successful experimental infection.

Figure 3
figure 3

Morphology of MO (a) MO colony under a light microscope (40×); (b) MO colony with dienes staining under a light microscope (40×).

Full size image

Observation and scoring of clinical manifestation

Any significant clinical manifestations were not observed in the three groups after seven days post-MO infection. However, after 16 days, two sheep in group C presented clinical manifestations, such as anorexia, diarrhea, emaciation, light cough, serous nasal or purulent nasal, astasia, and severe symptoms such as dyspnea and recumbency. The two sheep perished on days 18 and 25, respectively. Table 4 describes the clinical manifestation sum scores of the three groups, and the score of clinical manifestation in the group C was significantly higher than that in the groups A and B (P < 0.05); however, no significant difference was found between groups A and B (P > 0.05) (table 4).

Table 4 Score of clinical symptoms in the three groups of sheep.
Full size table

Pathological observation and score of lung tissue

The changes in the histopathological sections of the lung tissues from the sheep deceased due to sickness and slaughter were observed and scored. As shown in figure 4, the lung was infected in different degrees in the three groups. The lesion in the MO-susceptible group C showed maximal pleural effusion. The MO infection altered the serous fibrous pleural pneumonia, massive pleural effusion, pulmonary pleural fibrosis, pink consolidation in different degrees on the lung surface, haemorrhagic spot, interstitial broadening, and marble-like section in the pneumonic patch due to dry and interstitial edema (figure 4). The degree of injury, lung colour, adhesion between lung tissues and thoracic cavity, and pleural effusion were used as indicators to score the lung tissues in the MO-infected sheep. The pathological change score of group C was significantly higher than that in groups A and B (P < 0.01). However, no significant difference was observed between groups A and B (P > 0.05) (table 5).

Figure 4
figure 4

Pathological changes in the lung of sheep infected with MO. (a)–(c) Represents the pathological changes in the infected lung in a subset of samples in resistance groups (a&b). (d)–(f) Represents pathological changes in the infected lung in a subset of samples in the susceptibility group (c). Arrows indicate the marble-like surface changes.

Full size image
Table 5 Scores of pathological changes in the lung tissue in the three groups.
Full size table

The pathological changes in the experimental groups were further scored based on the slices. The average values in groups A and B were 9.2 and 10.2, respectively. The pathological lesion was light, representing the broadening between the alveolar and pulmonary interstitium. A small amount of lymphocytic infiltration was observed in the mesothelium, while a small amount of inflammatory cell infiltration was observed in the lumen. The alveolar wall was expanded by the capillary blood, and the red blood cells were dissolved in individual vessels (figure 5). The average value in group C was 21.8, which is significantly higher than that of groups A and B (P < 0.01) (table 6), thereby indicating a severe pathological change. This phenomenon indicated that the lesion was wide with a large area, the pulmonary interstitium was significantly broadened, a large amount of lymphocytic infiltration was observed in the septum, the bronchial epithelial structure was damaged or fell off, and significant number of lymphocytes exuded in the lumen of the bronchiole accompanied by congestion in the blood capillary of pulmonary interstitium and venule near the bronchus. Compared to the infected sheep alveoli, the alveolar score in the normal group was <1 without any significant pathological change.

Figure 5
figure 5

Pathological morphology of sheep infected with MO. N, normal lung without infection; A, B, and C, MO-infected lung of A, B, and C groups, respectively. N, normal alveolar (↘) and alveolar interstitial ( ); A1, alveolar (↘) and widened alveolar interstitial ( ), alveolar wall with capillary blood is highly dilated and filled with red blood cells ( ); A2, multiple lymphocytes ( ) infiltrate on the side of the small bronchi ( ); B1, the structure of the lung tissue is controversial. Inflammatory cells (lymphocyte infiltration) are observed in the respiratory bronchus ( ). C1, the structure of the lungs disappeared, a large number of lymphocytes infiltrated around the small bronchioles ( ) in the lungs, lymphocytes infiltrated in the small bronchi, and necrotic foci were observed. C2, alveolar (↘) is reduced, alveolar wall capillary blood is highly dilated ( ), and erythrocytosis is increased; C3, multiple lymphocytes infiltrating on the side of the respiratory bronchioles ( ), and red blood cells are dissolved in individual blood vessels.

Full size image
Table 6 Pathological scores of the lungs in the three groups of sheep.
Full size table

Altered expression level of serum cytokines before and after artificial MO infection

To explore the immune response of MO-resistant and MO-susceptible genotypes before and after MO-infection, the cytokines response in groups A (HaeIII dd genotype), B (MvaI cc genotype) and C (HaeIII ee genotype) was detected. The expression of TNF-α, IFN-γ, IL-1β, and IL-2 increased initially followed by a decrease. The peak times of cytokine secretion in groups A and B were earlier than that of group C. The expression levels of TNF-α, IFN-γ, and IL-1β in group C were higher than those of groups A and B, differing significantly after 14 days and markedly at some point of time. The expression level of IL-2 in group C was lower than that of groups A and B, reaching a significant difference at day seven postinfection and an extremely significant difference at days 14 and 21. At day 27, the trend returned to baseline. The humoral cytokines IL-4, IL-6 and IL-10 before and after MO infection showed an increasing trend, followed by a decrease, and the amplitude of change was significant with a shorter peak time as compared to the cellular cytokines (TNF-α, IFN-γ, IL-1β). In addition, the expression levels of IL-4 and IL-6 in groups A and B were lower than those of group C, and the difference became significant at day three after experimental infection. The content of IL-10 in group C was high, which was reduced subsequently as compared to that of groups A and B (figure 6).

Figure 6
figure 6

The expression level of serum cytokines. (a), TNF-α; (b), IFN-γ; (c), IL-1β; (d), IL-2; (e), IL-4; (f), IL-6; (g), IL-10. A & B, resistance groups; C, control susceptibility group.

Full size image

Discussion

MHC plays a key role in recognition of adaptive immunity and antigen presentation. The high polymorphism is conducive to the expansion of presentation range of antigen in the animal population and the survival and continuation of human and animals, which has been widely proved in mammals (Ammer et al. 1992; Ujvari and Belov 2011; La Gruta et al. 2018; Trier et al. 2018). Some mutations are associated with the diseases of human and animals (Daly and Day 2001; Shiina et al. 2004; Liu et al. 2004; Li et al. 2011a, b). In this study, we found that the frequency of HaeIII ee and MvaI bb genotypes in Hu sheep was significantly higher than the Dorper with strong MO resistance. On the other hand, the HaeIII dd and MvaI cc genotypes were commonly observed in Dorper, suggesting that the HaeIII ee and MvaI bb are susceptible genotypes of MP, while HaeIII dd and Mval cc were the resistant genotypes. Next, all the typed samples were randomly sampling inspected to detect the positive rate of MO antibody. These results proved that the frequency of HaeIII dd and MvaI cc was lower than the infection rate of susceptible genotypes, further indicating that HaeIII dd and MvaI cc could resist the MO infection. The MHC gene polymorphism is correlated with the resistance/susceptibility of the disease, which is currently the research hotspot in the field of molecular breeding for disease resistance in livestock. Hitherto, the susceptibility of a large amount of bacteria, virus and parasite infectious diseases has been found to be correlated with MHC, e.g. Herpes virus, Rous sarcoma virus (Briles et al. 1977; Longenecker and Gallatin 1978; Schierman and Collins 1987; Kaufman and Venugopal 1998; Mejdell et al. 1994) in chicken, mastitis, leukaemia, intestinal parasite and tick in cows (Aarestrup et al. 1995; Sharif et al. 1998), Pasteurella multocida, diarrhea in piglet, and hereditary malignant skin melanoma and colibacillosis in pigs (Tissot et al. 1989; Renard et al. 2001). Further, the sheep carrying DRB1 with allele 70–71 encoding Arg–Lys resisted the infection of bovine leucosis virus (BLV) and inhibited the tumour development (Nagaoka et al. 1999). In 2003, Konnai et al. (2003) found that the sheep with different exon 2 genotypes differed markedly in the expression of immune response factors after BLV infection, which was proposed as the molecular mechanism underlying BLV resistance in sheep. Sayers et al. (2005) found that MHC-DRB1 genotype exerted genetic resistance in Suffolk and Poll Dorset that was linked with nematode infection. In addition, the sheep MHC polymorphism was closely related to the development and incidence of echinococcosis (Gottstein and Bettens 1994; Eiermann et al. 1998; Godot et al. 2000; Li et al. 2011b; Shen et al. 2014). To the best of our knowledge, the present study, for the first time, reported the strong correlation between MHC-DRB1 gene polymorphism and sheep MP.

The Hu sheep with a high risk of MP differed in the breed, size and genetic characteristics as compared to Dorper with low risk. However, the F1 generation with a low incidence of MP shared genetic characteristics with their parents, such that the genetic background was relatively consistent. Thus, the F1 generation was the optimal group for investigating the susceptibility of MP and resistance gene. HaeIII dd and MvaI cc genotypes with resistance and HaeIII ee genotype with susceptibility in the F1 generation were selected for artificial MO-infection. The clinical manifestations and histopathological scores indicated that MO anti-infection of HaeIII dd and MvaI cc genotypes was significantly stronger than that of HaeIII ee. The resistant group did not show any severe symptoms of MP, such as severe cough, asthma, high temperature after MO infection, rather only light cough and exudation of a small amount of sticky secretions from the nasal cavity were observed. Subsequently, in the MP-susceptible group, severe infection symptoms were noted over a period, and two sheep deceased during the infection period. The score of pnaeumonia degree in the MO-susceptible group was significantly higher than that in the high-resistance group. Cimolai used MP to infect golden Syrian hamster to establish a pnaeumonia model and a histopathological scoring system of MP (Cimolai et al. 1992). Zhang et al. (2012) used the scoring system for evaluating the histopathology of sheep MP. This evaluation system could objectively evaluate the grade of pnaeumonia severity. In addition, the pulmonary inflammatory response and pathological change in the lung were included in the evaluation indices, and the final score objectively evaluated the severity of pnaeumonia. Thus, this scoring system was used in the present study, and the obtained histopathological scores of resistant/susceptible sheep were consistent with the pathological scores, which was in line with the previous studies (Turner 1996; Zhang et al. 2012).

Reportedly (Gottstein and Hemphill 1997; Rigano et al. 2003), mycoplasma infection can produce various cellular immune factors that bind to receptors on the cell surface, mediating and regulating the immune function and inflammatory response. Some studies demonstrated that these cell immune factors had complicated correlations among each other. For example, these proteins interacted with each other and influenced the formation of various immune globulins, complements and acute phase reactive protein, thereby building a complex network system (Torcal et al. 1996; Zhang et al. 2003). In the current study, the expression levels of serum TNFα, IFNγ, IL-1β, IL-2, IL-4, IL-6 and IL-10 in the sheep infected with MO were initially increased and then decreased, suggesting that various inflammatory cytokines participated in the occurrence and development of MP, resulting in immune function disorder. After infection, the cytokines TNFα, IFNγ, IL-4, IL-6, and IL-1β, which initiate the inflammatory response or promote an inflammatory response in the susceptible group were rapidly activated, and the expression was significantly higher in the middle and late stages of infection as compared to that of the resistant groups. Further, the levels of IL-2 and IL-10, which promote the immune response of the body and inhibit the inflammatory response were significantly lower than the expression in the resistant group at seven days postinfection. The expression of Th1 cytokine in the HaeIII ee-susceptible sheep was significantly lower than that of the HaeIII dd and MvaI cc-resistant groups. However, the expression of IL-4 of Th2 cytokine was significantly increased as compared to that of the HaeIII dd and MvaI cc resistant group. The high expression of IL-2 plays a role against the mycoplasma infection. The interaction of the IL-4 cytokine with eosinophils stimulated the secretion of inflammatory mediators and aggravated the local inflammatory response. These data were consistent with the pathological changes in the sheep infected with MP in the MO-susceptible/resistance groups. Taken together, it can be concluded that HaeIII dd and MvaI cc were MP-susceptible genotypes, and HaeIII ee was MO-resistant genotype.