Indian J. Anim. Res., 50 (2) 2016:143-147 Print ISSN:0367-6722 / Online ISSN:0976-0555 AGRICULTURAL RESEARCH COMMUNICATION CENTRE www.arccjournals.com/www.ijaronline.in Molecular characterization of Ovar-DRB1 exon2 gene in Garole sheep resilient to gastrointestinal nematodes C. Paswan*, L.L.L. Prince, R. Kumar, C.P. Swarnkar, D. Singh and S. Kumar Central Sheep and Wool Research Institute, Avikanagar-304 501, India. Received: 07-02-2014 Accepted: 24-06-2014 DOI:10.18805/ijar.6710 ABSTRACT Ovine MHC DRB1 exon 2 (Ovar-DRB1.2) gene is arguably one of the critical genes, responsible for disease resistance against parasite in animals. This study was carried out for indepth analysis of polymorphism in MHC DRB1.2 gene and to explore the underlying molecular mechanisms for the development of disease resistance in Garole. PCR-RFLP study revealed that the gene is polymorphic in nature. The frequency of allele A for endonuclease SacI and Allele B for endonuclease Hin1I were significantly higher in Garole population. Key words: Ovar DRB1.2, Garole, Nematode Parasites, Disease Resistance, PCR-RFLP. INTRODUCTION Sheep is a vital source of meat and wool fiber particularly in Asia and Africa regions where it accounts for over 70% of world stocks (FAOSTAT, 2010).. However, it is assumed that the current levels of production will have to be increased to meet the future demands of increasing human population. Infection with gastrointestinal parasites mainly Haemonchus contortus is one of the most important constrains in sheep productivity enhancement. Out of total expenditure incurred on animal health management, 28% of expenditure is on parasite management ((IFAH Annual Report, 2009). Indiscriminate use of anthelmintics has resulted in the development of anthelmintic resistant parasites, persistence of drug residues in animal produce, environmental hazards and huge economic loss to the sheep producers (Waller, 1994; Windon 1996; Singh et al, 2002, Singh and Swarnkar 2008). New avenues of alternate control measures, including breeding strategies, are now being searched as components of sustainable parasite management systems (Sayers and Sweeney, 2005). To address this issue one of the strategies being used is either to develop a sheep breed which is resistant to parasite infection and to characterize and utilize those sheep breeds which are naturally resistant to nematode parasites. There is overwhelming evidence that genetic variation within and across the breeds of the sheep can be exploited to increase the resistance of sheep population to nematode parasites (Swarnkar et al.,2000; Subandriyo, 2002; Baker and Gray 2004; Yadav et al., 2006; Bishop and Morris, 2007; Mandal and Sharma 2008). India is home to many genetically diversified breeds of sheep. Garole is one sheep breed found in the low lying, swampy, hot and humid region of Sunderban delta spread across West Bengal of India and part of Bangladesh. In spite of grazing in knee length water and marshy land of Sundervan delta, this breed is resilient to parasite infection (Nimbkar et al., 2003). However, till now no work has been carried to understand the molecular mechanisms of resistance to nematode parasites in Garole sheep. Owing to immunological importance of MHC genes and their possible roles in disease resistance, this study was conducted to characterize second exon of Ovine MHC DRB1 (in this article abbreviated as Ovar-DRB1.2) in Garole sheep Ovar-DRB1.2 is a part of MHC which harbors clusters of immunological genes involved in immunological response against pathogens (Buitkamp et al., 1996). MATERIALS AND METHODS Blood samples were collected from hundred twenty (n=120) Garole sheep maintained at Central Sheep and Wool Research Institute (CSWRI), Avikanagar. Genomic DNA was extracted using phenol chloroform method (Sambrook et al., 2001). The quality of DNA was checked by spectrophotometer taking ratio of optical density (OD) value at 260 and 280 nm. Good quality DNA having OD ratio between 1.7 and 1.9 was used for genotyping. Nested PCR was used to amplify Ovar-DRB1.2 gene using primers OLA-ERB1 (5-3 CCGGAATTCCCGTCTC TGCAGCACATTTCTT), HL031 (5-3 TTTAAATTCG *Corresponding author s e-mail; drcdvet17@gmail.com. Address: Division of Animal Genetics and Breeding, CSWRI, Aviakanagar, Malpura Via; Jaipur, Rajasthan-304501.
144 INDIAN JOURNAL OF ANIMAL RESEARCH CGCTCACCTCGCCGCT) and OLA-XRB1 (5-3 AGCTC GAGC GCTGCACAGTGAAACTC) (van Eijak et al., 1992). First round of PCR was set in 20 l PCR mix, using: 10 x buffer, 90-100 ng DNA template, 100 M each dntps, 1.5 mm MgCl 2, 1U of Taq polymerase (Fermentas) and 10 pm of each ERB1 and HLO31 primers., Thirty cycles of PCR was performed using initial denaturation at 95 0 C for five minutes, denaturation at 94 0 C for 30 sec, annealing at 50 0 C for 30 sec, extension at 72 0 C for 1 min and final extension at 72 0 C for 10min in thermocycler (M J research Inc USA). Product of first round PCR was used as template for second round keeping concentration of PCR componenets and temperature profiles same as that of first round of PCR, except the annealing temperature at 60 0 C. 8 l PCR products were digested overnight with 5U of SacI and Hin1I restriction enzymes (RE) at 37 0 C. The RE product was run on 4% ultra fine agarose (Sigma) gel containing ethedium bromide at 80 volts for 3 hours and pattern of RE digestion was documented under gel doc system (UVP), 50 bp DNA marker or Lambda DNA HindIII EcoRI double digest was used for estimation of the fragments sizes. Data was analyzed using Chi square method. RESULTS AND DISCUSSION PCR amplification of Ovar-DRB1.2 gene generated a fragment of 296 bp size which is similar to that of bovine size (Fig.1). Primers used in this study were basically designed for BoLA (van Eijak et al., 1992). This shows that MHC genes are highly conserved across different species including human (Gao et al. 2010). Major histocompatibility complex (MHC) class II molecules are glycoproteins composed of two non-covalently linked and chains. MHC class II molecules selectively bind different sets of peptides derived from extracellular antigens and present them to helper T cells. When helper T cells recognize the foreign antigen associated with the MHC class II molecule, they release B cell growth factor and interleukins which induce the clonal expansion of B lymphocytes and cluster differentiation (CD8+) T cells respectively (Tizard, 1987). Exon 2 of DRB1 was selected as it encodes for 1 domain of MHCII molecules, 1 forms the part of peptide binding regions (PBR) and they are likely to be related to functionality such as disease resistance (Brown et al., 1993). Earlier study reported that the intensity of gastrointestinal nematode infection was affected by the level of inheritance of Garole blood in its crossbred progenies with other Indian sheep breeds. It was found that higher the percentage of Garole blood in Garole cross, better is the resistance to gastrointestinal nematode infection (Nimbkar et al., 2003; Singh et al., 2011). Similar attempts were made to study the genetic variation in nematode resistance within and across several breeds of sheep across the world (Mugambi et al., 1997; Gruner et al., 2003; Wanyangu et al., 1997; Baker et al., 2003). Selection of sheep breed for nematode resistance based on Fecal Egg Count (FEC) has given encouraging results in Australia (Windon, 1996). However, no such attempts were made to unravel the molecular mechanisms conferring resistance to GI parasites infestations in Garole. This is the first effort to study genetic variability of MHCII genes in Garole. PCR amplicons of MHC DRB1.2 gene were subjected to restriction digestion using SacI and Hin1I endonucleases to study structural variation in DRB1.2 gene. Both of the endonuclease produced three RFLP bands of different molecular weights, implicating three different genotypes (Fig.2 & 3). Three genotypes observed for SacI endonuclease were AA (296 bp), AB (229, 97 bp) and BB (296, 229, 97 bp) (Table 1). Similarlly for HinII were AA (296 bp), BB (178, 118 bp) and AB (296, 178, 118) (Table1). This study revealed that MHC DRB1.2 is highly polymorphic in Garole. Several studies related to polymorphism of MHC Fig 2: RE pattern of Ovar-DRB1 by Sacl M:100bp ladder Fig 1: PCR Amplification of Ovar-DRB1 M:100bp ladder. Fig 3: RE pattern of Ovar-DRB1 by Hinll M:100bp ladder
Volume 50 Issue 2 (2016) 145 region have reported that the genes located in MHC regions constitute the most polymorphic loci known in vertebrates (Hedrick 1994; Hughes and Hughes 1995). Similar kinds of results were shown in different sheep breeds (Ballingal et al., 1992; Konnai, et al., 2003; Ballingal et al., 2008; Table 1: RE (SacI and Hin1I) Fragment Size of DRB1 2 Gene in Garole Restriction Enzymes (RE) SacI Hin1I Fragment sizes (bp) 296, 229, 67 296, 178, 118 Table 2: Genotypic and allelic Frequency of Ovar-DRB1 2 Gene in Garole Restriction Number of Genotypic frequencies Allelic frequencies chi-square test Enzyme Animals AA AB BB A B SacI 122 0. 80 0.18 0.02 0. 89 0.11 P<0.01 Hin1I 122 0.19 0.34 0.47 0.36 0.64 P<0.01 Schwaiger et al., 1995). The variability of the MHCmolecules is correlated with the diversity of the T-lymphocyte receptors which in turn determines the disease and parasite resistance of an organism (Hedrick et al. 2001; Hedrick et al. 2001; Paterson et al. 1998; Hedrick, 1994; Langefors et al. 2001). On the other hand Crawford et al. (1997) could not associate the role of MHC genes with incidence of gastrointestinal parasites infestation in sheep. Genotypic frequency of AA, AB and BB was 0.88, 0.18 and 0.02, respectvely for ScaI endoclease. Similarly, the genotypic frequency of AA, AB and BB for HinII were 0.19, 0.34 and 0.47, respectively. Genotypic frequency of AA (0.80) was significantly higher for SacI endonuclease (Table2). However, the genotypic frequency of BB (0.47) was significantly higher in case of Hin1I endonulease (Table2). One of the characteristic features of the MHC genes is their co-expression of the genes which is well supported by this study too (Dukkipati et al. 2006). This study revealed that Garole population was skewed towards homozygosity for DRB1.2 gene, it may be attributed to the fact that this population has become closed nuclear breeding unit as no animals have been introduced from outside since 1997. This is in contrast to the hypothesis of heterozygous advantage or symmetrical over-dominance, which states that more the heterozygous population better is the immune response hence the disease resistance (Doherty and Zinkernagel, 1975). Allelic frequency of A (0.89) and B (0.64) (Table2) were higher for SacI and Hin1I, respectively. In spite of being more homozygote population under study had very low incidence of nematode parasite infestation, indicating the prospects of selecting theses animals having alleles A (SacI) and B (Hin1I) for parasite resistance in future. CONCLUSION This study revealed that Ovar-DRB1.2 is polymorphic in Garole sheep. The frequency of allele A for SacI and Allele B for Hin1I were significantly higher as compared to other alleles. Genomic variability in Ovar- DRB1.2 gene may be responsible for conferring resistance in Garole against GI nematode. There is need to validate the association between these genotypes and fecal egg counts in Garole and its cross so that these genotypes can be used for establishing disease resistance flock in future. ACKNOWLEDGEMENT Authors are thankful to the Director, Central Sheep and Wool Research Institute, Avikanagr, Malpura, for funding and providing necessary facilities to carry out this research work. REFERENCES Baker, R. L., Gibson, J. P., Irqi, F. A., Menge, D. M., Mugambi, J. M., Hanotte, O., Nagda, S., Wakelin, D., Behnke, J. M., (2003). Exploring the genetic control of resistance to gastrointestinal helminth infections in sheep and mice. Proceedings of the Association for Advancement in Animal Breeding and Genetics, 15: pp 183 190 7 11 July 2003 Melbourne, Victoria, Australia. Baker, R. L. and Gray, G. D. ( 2004). Worm Control for Small Ruminants in Tropical Asia. pp 63 95. (Eds ) Sani, R A, Gray, G D, Baker, R L. ACIAR Monograph 113, Canberra, Australia. Ballingal, K. T., Fardoe, K., Mckeever, D. J. (2008). Genomic organisation and allelic diversity within coding and noncoding regions of the Ovar-DRB1 locus. Immunogenetics 60 (2): 95 103. Ballingall K. T., Wright H., Redmond J., Dutia B. M., Hopkins J., Lang J., Deverson E. V., Howard J. C., Puri N., and Haig D. (1992). Expression and characterization of ovine major histocompatibility complex class II (OLA-DR) genes. Anim. Genet. 23:347 359. Bishop, S. C., Morris, C. A. (2007). Genetics of disease resistance in sheep and goats Small Ruminant Res. 70: 48 59.
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