Browse > Article
http://dx.doi.org/10.5713/ajas.2010.90323

PCR-SSCP of Serum Lysozyme Gene (Exon-III) in Riverine Buffalo and Its Association with Lysozyme Activity and Somatic Cell Count  

Sahoo, Nihar Ranjan (Molecular Genetics Laboratory, Division of Animal Genetics, Indian Veterinary Research Institute)
Kumar, Pushpendra (Division of Animal Genetics, IVRI)
Bhushan, Bharat (Division of Animal Genetics, IVRI)
Bhattacharya, T.K. (Project Directorate on Poultry, Hyderabad)
Sharma, Arjava (Division of Animal Genetics, IVRI)
Dayal, Sanker (Dept. of Animal Breeding and Genetics, Bihar Vety College)
Pankaj, Prabhat Kumar (Molecular Genetics Laboratory, Division of Animal Genetics, Indian Veterinary Research Institute)
Sahoo, Monalisa (Division of Pathology, IVRI)
Publication Information
Asian-Australasian Journal of Animal Sciences / v.23, no.8, 2010 , pp. 993-999 More about this Journal
Abstract
Serum lysozyme gene is one of the important genes influencing the immune system as its product can cause lysis of bacterial cell wall by cleaving the peptidoglycan layer. The present investigation on the serum lysozyme gene of Indian riverine buffalo was undertaken with the objectives to identify and characterize single nucleotide polymorphic patterns by PCR-SSCP method as well as to study the effect of different genotypes on serum lysozyme activity and somatic cell count. A total of 280 animals comprising four different famous bubaline breeds (Murrah, Mehsana, Surti and Bhadawari), spread over six different farms across the country were used for this study. A 276 bp (partial intron 2, complete exon 3 and partial intron 3) fragment of lysozyme gene was screened for polymorphism using the SSCP technique. Four genotypes namely AA, AB, BC and AC were observed, out of which BC genotype was found to be the most frequent. Among these three alleles, C allele (0.38) was most prevalent in these populations. Various SSCP allelic variants were cloned for sequencing and sequences were submitted to NCBI Genbank. From the alignment of the nucleotide sequences of various allelic variants, it was found that there were differences in 12 positions among the alleles, out of which maximum variation (at 8 places) was found in the intronic region. The allele A was closer to allele-C than allele-B. Allele B was phylogenetically equidistant from both of the other alleles. Mean lysozyme activity determined in serum samples of different animals of Murrah buffalo was $27.35{\pm}2.42\;{\mu}g$ per ml of serum, whereas the mean somatic cell count was $1.25{\pm}0.13{\times}10^5$ cells per ml of milk. The SSCP pattern-wise effects of various genotypes on lysozyme activity and SCC were analyzed. Although the mean values were apparently different in various genotypes, these differences were statistically non-significant. It can be concluded that the riverine buffaloes are sufficiently polymorphic with respect to serum lysozyme gene. The absence of AA genotype in Bhadawari breed of buffalo can be considered as a marker for breed characterization. The difference of four nucleotides in exon-3 indicates high selection pressure on the gene.
Keywords
Polymorphism; Lysozyme Gene; SSCP; Buffalo; Association; SCC; Sequencing;
Citations & Related Records
Times Cited By KSCI : 1  (Citation Analysis)
Times Cited By Web Of Science : 1  (Related Records In Web of Science)
Times Cited By SCOPUS : 0
연도 인용수 순위
1 Snedecor, G. W. and W. G. Cochran. 1994. Statistical methods.8th ed. Affiliated East-West Press, New Delhi. India. 1-86.
2 White, F. H., H. A. Mc Kenzie, D. C. Shaw and R. J. Pearee. 1988. Studies on partially purified bovine milk lysozyme. Biochem. Int. 16:521-528.
3 Orita, M., H. Iwahana and H. Kanazawa. 1989. Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc. Natl. Acad. Sci. USA. 86:2766-2770.   DOI
4 Osteras, O. 1999. Use of SCC as a guidance tool. Buskap. 2:54-55.
5 Paape, M. J., W. P. Wergin, A. J. Guidry and R. E. Pearson. 1979. Leukocytes- second line of defence against invading mastitis pathogens. J. Dairy Sci. 62:135-153.   DOI
6 Priyadarshini, S. and V. K. Kansal. 2002. Lysozyme activity in buffalo milk: Effect of lactation period, parity, mastitis, season in India, pH and milk processing heat treatment. Asian-Aust. J. Anim. Sci. 15:895-899.   DOI
7 Reiter, B. and A. J. Bramley. 1975. Defence mechanisms of the udder and their relevance to mastitis control. Pro. Seminar on Mastitis Control, IDF Bulletin Document. 85:210-222.
8 Sambrook, J. and D. W. Russell. 2001. Molecular cloning, a laboratory manual. 3rd Edition. Cold Spring Harbor Laboratory Press, NY, USA. 6.1-8.96.
9 Schalm, O. W., E. J. Carrol and N. C. Jain. 1971. Bovine mastitis. Lea and Fabiger, Philadelphia. 1-13.
10 Schalm, O. W., J. Lasmanis and E. J. Carrol. 1966. Significance of leukocyte infiltration into the milk in experimental Streptococcus agalactiae mastitis in cattle. Am. J. Vet. Res. 27:1537-1546.
11 Bassam, B. J., G. Caetano-Anolles and P. M. Gresshoff. 1991. Fast and sensitive silver staining of DNA in polyacrylamide gels. Anal. Biochem. 196:80-83.   DOI   ScienceOn
12 Seyfert, H. M. 1995. Bos taurus lysozyme (LZ) gene, complete cds./ http://www.ncbi.nlm.nih.gov ACCESSION U25810.
13 Seyfert, H. M., M. Henke, H. Interhal, U. Klussmann, D. Koczan, S. Natour, W. Pusch, B. Senft, U. F. Stenhoff, A. Tuckoricz and G. Hobom. 1996. Defining candidate genes for mastitis resistance in cattle; the role of lactoferrin and lysozyme. J. Anim. Breed. Genet. 113:169-176.
14 Sharma, A. K. 2002. Molecular characterization and immunological study in indigenous cattle. M.V.Sc Thesis submitted to IVRI Deemed University.
15 Lie, O. 1980. Genetic variation in the serum lysozyme activity in cattle. Acta. Vet. Scand. 21:448-450.
16 Di Luzio, N. R. 1979. Lysozyme activity: An index of macrophage functional status. In: Lysozymes in Biology and Pathology (Ed. J. T. Dingle, P. J. Jacques and J. H. Shaw). North Holland Publishing Company. Amsterdam. New York.Oxford:447-462.
17 Jolles, P. and J. Jolles. 1984. What's new in lysozyme research? Always a model system, today as yesterday. Mol. Cell Biochem. 63:165-189.
18 Kehrli, M. E. and D. E. Shuster. 1994. Factors affecting milk somatic cells and their role in health of bovine mammary gland. J. Dairy Sci. 77:619-627.   DOI