Comparative moleculo-immunological analysis of swamp- and riverine-type water buffaloes responses

Abstract

This moleculo-epidemiological and immunological study through cytokine response assessment was done to know the dynamics of cytokines in the initiation, persistence and association to physiological changes of a particular pathogen in water buffaloes. This is important to understand the magnitude and behavior of disease progression. Water buffalo blood samples gathered from different places in the Philippines revealed a 9.4%, 27.6%, 10.3% and 4.4% prevalence of bovine viral diarrhea virus (BVDV), bovine leukemia virus (BLV), Anaplasma marginale and Babesia bigemina infection, respectively. This was the first surveillance study of BVDV and BLV in the country. Furthermore, cytokine expression of these naturally infected animals was also quantified. BVDV-infected animals had up-regulated expressions of TNFα, IL-2 and IL-4; and down-regulated expressions of IFNγ and IL-12p40 while BLV positive animals had an up-regulated IL-4 and IL-6, and highly expressed IL-10 and IL-12p40 with unchanged IFNγ expression. Meanwhile, animals infected with A. marginale had all interleukins and IFNγ up-regulated with significant expression of IL-10 and IL-12p40 similar to the BLV positive animals. Since it was also observed that swamp-type buffaloes were more disease tolerant than riverine-type buffaloes based on the gathered infection rate of each examined pathogen, further assessment was done focusing on the two vital cytokines, IFNγ and TNFα. We quantified IFNγ and TNFα expressions in ConA-stimulated PBMC from both swamp and riverine buffaloes by real-time PCR. Cytokine expression from ConA-stimulated PBMC revealed that both IFNγ and TNFα were more highly expressed in swamp than in riverine buffalo. To further examine the probable cause of expression differences, the proximal promoter region of these two cytokines were sequenced for the presence of nucleotide polymorphism followed by luciferase assay to analyze the effect of these polymorphisms in gene transcription. A single nucleotide polymorphism was found in the IFNγ (−299) while eight polymorphisms in the TNFα promoter (−541, −553, −562, −596, −609, −655, −659, −688). Luciferase assay showed that both IFNγ promoter and TNFα promoter in swamp-type water buffalo had higher transcription activity compared to riverine-type water buffalo. These findings confirm that IFNγ and TNFα transcriptions in these animals were highly affected by the disparity in the cytokine promoter region. This suggests that disease tolerance or susceptibility of these buffaloes could be due to the differences in their relative cytokine transcription and may relate to pathogen–host specific pathogenesis.