Characterization of leukocyte subsets in buffalo (Bubalus bubalis) with cross-reactive monoclonal antibodies specific for bovine MHC class I and class II molecules and leukocyte differentiation molecules

Highlights

• Monoclonal antibodies were identified that recognize orthologues of LDMs and MHC I and II molecules in buffalo (Bubalus bubalis).

• Phenotypic analysis of buffalo lymphocyte subsets showed that all WC1⁺ γδ T cells express CD8 with a small subset co-expressing CD2.

• The extensive set of cross reactive mAbs will allow for comparison of the similarities and differences in the immune response to pathogens affecting the health of both cattle and buffalo.

Abstract

Although buffaloes (Bubalus bubalis) are a major component of the livestock industry worldwide, limited progress has been made in the study of the mechanisms regulating the immune response to pathogens and parasites affecting their health and productivity. This has been, in part, attributable to the limited availability of reagents to study immune responses in buffalo. As reported here, a set of cross-reactive monoclonal antibodies (mAbs), developed against bovine, ovine and caprine leukocyte differentiation molecules (LDM) and major histocompatibility complex (MHC) molecules, were identified and used to compare expression of LDM in Italian and Egyptian buffalo. The results show most of the epitopes identified with the mAbs are conserved on LDM and MHC I and II molecules in both lineages of buffalo. Comparison of the composition of lymphocyte subsets between buffalo and cattle revealed they are similar except for expression of CD2 and CD8 on workshop cluster one (WC1) positive γδ T cells. In cattle, CD8 is expressed on a subset of CD2+/WC1- γδ T cells that are present in low frequency in blood of young and old animals, whereas, CD8-/CD2-/WC1+ γδ T cells are present in high frequency in young animals, decreasing with age. In the buffalo, CD2 is expressed on a subset of WC1+ γδ T cells and CD8 is expressed on all WC1+ γδ T cells. The availability of this extensive set of mAbs provides opportunities to study the immunopathogenesis of pathogens and parasites affecting the health of buffalo.

Introduction

Buffaloes are an essential component of the livestock industry in many countries including Italy and Egypt. They are used as draught animals and also as a major source of milk and meat products (Borghese, 2005, Council, 1981, Wikipedia, 2014). Although considerable progress has been made in defining the genome of buffalo and in improving methods for selective breeding to enhance performance (Michelizzi et al., 2010), progress has been constrained on management of animal health. This is attributable to the limited information available on the mechanisms regulating the immune response to pathogens and parasites (Borghese, 2005, Council, 1981). Infectious diseases of water buffalo are currently managed similarly to those of cattle, on the assumption that the immune responses are similar. Buffalo are susceptible to many of the diseases affecting cattle, and many of the vaccines and chemotherapeutic regimens developed for use in cattle have proven effective in buffalo (Council, 1981, Tomar and Tripathi, 1987). However, there are also clear differences in the response to some pathogens, emphasizing the need to modify the bovine-based strategies of management to meet the needs unique to buffalo (Council, 1981). To achieve this objective, it will be necessary to identify and characterize the immune mechanisms that account for differences in resistance and disease susceptibility observed between cattle and buffalo. Before this can be accomplished, however, the immune system of buffalo needs to be characterized.

Until now, very few studies have been conducted to develop or identify the mAb reagents needed to characterize the immune system of buffalo. This is attributable to the lack of resources and limited number of investigators available to develop mAb reagents needed for research in buffalo. To circumvent these limitations, we have taken advantage of the phylogenetic conservation of the structure and function of orthologous molecules in different species. Investigators that participated in the Third International Workshop on Leukocyte Antigens of Cattle and Sheep screened the mAbs submitted to the workshop for cross reactivity with buffalo LDM (Naessens and Hopkins, 1996, Vilmos et al., 1996). Multiple mAbs were identified, some of which identified epitopes conserved on orthologues in multiple species including buffalo. Of the 159 mAbs examined, 110 reacted with molecules expressed on buffalo leukocytes, indicating it might be possible to use this strategy to identify most of the mAbs needed for studies in buffalo. Sixty five of the mAbs used in the study were submitted by our laboratory. These were included in a subsequent study of over 200 mAbs examined specifically for cross reactivity with LDM in buffalo (Davis et al., 2001). MAbs were identified that were specific for MHC I and MHC II molecules and some specific for orthologues of LDM (numeric name designation for each molecule, cluster of differentiation molecules, CD) characterized in humans. Additional mAbs were identified that recognized epitopes conserved on sIgM, λ light chain, granulocytes, the δ and γ chains of the γδ T cell receptor, the workshop cluster 1 (WC1) molecules, B cells and molecules with upregulated expression on concanavalin A (conA) activated lymphocytes (Davis et al., 2001). Some mAbs were duplicates of mAbs that recognized the same or different epitopes on the same CD molecule. Initial data were obtained on the frequency of leukocyte subsets (Davis et al., 2001). Only a few animals were available for the initial studies, limiting our ability to accurately compare the phenotype and frequency of subsets in animals of different ages. The objectives of the present study were twofold 1) verify cross-reactivity of mAbs identified in the previous studies and validate activity of recently characterized mAbs for cross reactivity with orthologues of buffalo CD molecules and 2) compare and extend information on the composition and frequency of leukocyte subsets in buffalo at different ages.