Introduction Our previous work demonstrated the suitability of buffalo polymorphonuclear (PMN) cells to act as a model in assessing the in vitro effects of aflatoxin B1 (1) and other membrane-acting agents on oxy- gen-dependent and partly independent microbicidal systems (2). In the latter case, there was evidence of in vitro protein and peptide synthesis in buffalo PMN cells exposed to lipopolysaccharide (LPS) from Escherichia coli. As a continuation of this pre- vious study, the results presented here report the biochemical and microbicidal characterisation of peptides such as β-defensins, from the PMN cell granules of healthy male buffaloes. These peptides were subsequently identified in mastitis-affected milk, by using matrix-assisted laser desorption ion- isation-time-of-flight (MALDI-TOF) mass spec- trometry. The induction of in vitro β-defensin gene expression in a bovine viral diarrhoea virus (BVDV)-infected bovine turbinate (BT) epithelial cell line, was evaluated by performing a reverse transcriptase-polymerase chain reaction (RT-PCR) with the cellular mRNA. The use of such pilot experiments involving the application of proteomics techniques such as MALDI-TOF and PCR, will permit the determina- tion of defensin biomarkers involved in the in vitro responses of host cells. This will potentially help in the identification of further refined biomarkers of cell injury, before any actual large-scale animal experimentation is undertaken for health manage- ment research. Materials and Methods β-Defensins in PMN cell granules Isolation of PMN cells and cytoplasmic granules Pooled peripheral blood was collected from four apparently healthy five- to six-year old male buf- faloes. PMN cells were isolated by cold hypotonic lysis of red blood cells, followed by differential centrifuga- tion (3). The procedure yielded 2 × 10 9 cells from 500ml of blood, with ≥ 90% purity and viability. The β-Defensin Antibiotic Peptides in the Innate Immunity of the Buffalo: In Vivo and In Vitro Studies Hemen Das, Naganath Swamy, Gyanaranjan Sahoo, Shahaj Uddin Ahmed and Tukaram More Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, India Summary — β-Defensin antimicrobial peptides are multifunctional biomolecules, which are a major com- ponent of the oxygen-independent microbicidal system of buffalo polymorphonuclear (PMN) cells. They have great potential for use as proteomic biomarkers of host cell responses in the presence of microbial agents. On purifying these peptides by RP-HPLC, four defensin peptides were revealed. The results from testing against Escherichia coli, Staphylococcus aureus, Streptococcus pyogenes, Candida albicans, Rinderpest Virus (RPV) and Newcastle Disease Virus (NDV), showed that the peptides possessed antimicro- bial and antiviral activities. Minimum inhibitory concentration (MIC) values varied according to the peptide amounts and the exposure time. Furthermore, an increase in the levels of these cationic antimicrobial pep- tides was apparent in milk obtained from natural cases of mastitis, as compared to the levels in normal milk. MALDI-TOF-based amino acid sequencing confirmed the expression of two β-defensins (LAP and BNBD-2) in mastitis milk. A comparison of peptide sequences revealed that buffalo LAP and BNBD-2 share 98.6% and 100% sequence identity, respectively, with those of cattle. In vitro, Bovine Viral Diarrhoea Virus (BVDV) infection was shown to induce the expression of the β-defensin gene, as evidenced by the PCR amplification of cDNA with specific primers. The determination of the enhanced expression of β-defensin peptides in mastitis milk and in PMN cells, can be considered as an advanced approach to the assessment of cellular and molecular responses to cell injury. It is hoped that in vitro studies on phagocytes such as PMN cells and other cell lines, will eventually replace the use of animals in elucidating the roles of these cytokines in response to microbe-derived cell damage. It will also be possible to use defensins as biomark- ers to correlate failure in host cell defence systems with peptide heterogeneity. Key words: antimicrobial, β-defensin, buffalo, BVDV, cationic, innate immunity, in vitro, in vivo, mastitis, PMN cell granules. Address for correspondence: T. More, Division of Biochemistry, Indian Veterinary Research Institute, Izatnagar 243122, Uttar Pradesh, India. E-mail: tmore_2001@yahoo.com ATLA 36, 429–440, 2008 429