Molecular cloning, sequencing and structural studies of granulocyte-macrophage colony-stimulating factor (GM-CSF) from Indian water buffalo (Bubalus bubalis).

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that is essential for growth and development of progenitors of granulocytes and monocytes/macrophages. In this study, we report molecular cloning, sequencing and characterization of GM-CSF from Indian water buffalo, Bubalus bubalis. In addition, we performed sequence and structural analysis for buffalo GM-CSF. Buffalo GM-CSF has been compared with 17 mammalian GM-CSFs using multiple sequence alignment and phylogenetic tree. Three-dimensional model for buffalo GM-CSF and human receptor complex was built using homology modelling to study cross-reactivity between two species. Detailed analysis was performed to study GM-CSF interface and various interactions at the interface.

Comparative in vitro toll-like receptor ligand induced cytokine profiles of Toda and Murrah buffaloes-Identification of tumour necrosis factor alpha promoter polymorphism.

The objective of this study was to assess cytokine production upon activation of pattern recognition receptors responsible for sensing bacterial and viral pathogen associated molecular patterns in two genetically diverse buffalo breeds, Toda and Murrah. A very limited molecular-epidemiological analysis showed a higher prevalence of Anaplasma and Theileria in Murrah than Toda buffaloes. Toda buffalo peripheral blood mononuclear cells (PBMC) produced significantly higher levels of IFN γ and/or TNF α mRNAs in response to peptidoglycan, poly I:C, lipopolysaccharide, imiquimod and CpG. Flagellin stimulation did not result in any significant differences in the expression levels of the cytokines tested between these breeds. The levels of ligand induced IFN γ and TNF α mRNA and proteins also correlated except when induced with CpG. The proximal promoter region of TNF α across these two breeds were also sequenced to detect SNPs and promoter assay performed to determine their role in altering the transcriptional activity. Two polymorphisms were identified at -737 (T/A) and -1092 (G/T) positions in Toda buffalo TNF α promoter and promoter assay revealed higher transcription activity in Toda buffalos than in Murrah. This suggests that disease tolerance of these buffalo breeds could be due to the differences in their cytokine transcription levels in response to the respective PAMPs that may be at least in part determined by polymorphisms in the cytokine promoter regions.

Detection of egg drop syndrome virus antigen or genome by enzyme-linked immunosorbent assay or polymerase chain reaction.

Mouse monoclonal antibodies (mAbs) were produced against an Indian isolate of egg drop syndrome (EDS) virus and characterized. Four hybridoma clones were secreting mAbs that bound to a 100 kDa protein, presumably the hexon protein. These mAbs were found to cross-react with two other Indian isolates of EDS virus and to the reference UK 127 strain. Three of these mAbs were mapped to the same epitope compared with the other mAb (F8), which bound to a different epitope. An antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) was developed using the F8 mAbs as capture antibody and polyclonal chicken serum against EDS virus as detection antibody. A polymerase chain reaction (PCR) was used to detect the EDS viral genome. Following experimental infection of oestrogen-treated chickens with EDS virus, cloacal swabs, oviduct, uterus and spleen were collected at different days post-infection and used in both AC-ELISA and PCR, directly and after a single passage in embryonated duck eggs. The sensitivity and specificity of antigen detection by AC-ELISA or PCR was 95% and 98%, respectively. For diagnosis of EDS viral infections, PCR is recommended due to its ease and the lack of requirement of prepared reagents such as mAbs or conjugates. We recommend that PCR be performed directly on boiled tissue homogenates. Any negative samples may be passaged in embryonated duck eggs and the allantoic fluids tested by PCR before a conclusive negative diagnosis is given.

Protectotypic differentiation of avian infectious bronchitis viruses using an in vitro challenge model.

Two vaccine and three virulent strains of infectious bronchitis virus (IBV) were used to infect day-old specific-pathogen-free chickens. Precocious development of oviducts was induced in young female chicks by oestrogen injections. Tracheal and oviduct organ cultures prepared from immunised chickens were challenged in vitro with homologous and heterologous viruses to assess tracheal and oviduct cross-protection. Tracheal cross-protection was seen between serologically related and unrelated viruses. Protection at at the level of the oviducts was lower compared to that seen at the tracheal level. However, the yields of the homologous challenge viruses from immunised oviducts were significantly reduced as compared to yields from normal oviducts. After challenge, tracheal and oviduct ciliary scores were used to group the five strains of IBV into protectotypes. The five viruses could be grouped into three protectotypes and four serotypes. The virulent IBV strains were found to have caused complete stasis of oviduct cilia when examined at three weeks post-infection. Oestrogen treatment of chicks did not significantly alter the lymphocyte proliferation responses to concanavalin A or pokeweed mitogen. The applicability of this model for the study of protection afforded to the oviducts, in terms of protection against virus-induced damage, rather than drop in egg production, is discussed.