Two dimensional electrophoresis evaluation of milk from bovine with Johne’s disease

Introduction Johne’s disease is a chronic granulomatous enteropathy affecting all ruminants. The etiologic agent is Mycobacterium avium subsp. paratuberculosis (MAP), a slow-growing, facultative intracellular mycobacteria. There is also a concern that MAP might be a causative agent of some cases of inflammatory bowel disease in humans, especially Crohn’s disease (1). However, the ‘cause and effect’ relationship remains controversial due in part to the difficulties in detecting MAP in human tissues using in vitro culture. Food products including pasteurized bovine milk have been suggested as potential sources of human infection. In the present study a two dimensional electrophoresis approach has been used to analyse milk from bovines with paratuberculosis in order to characterize two dimensional profile of milk proteins come from infected bovines. This approach could be able to study putative markers for more sensitivity and specific diagnosis of paratuberculosis. Methods Milk samples were collected from healthy and infected bovine and prepared for 2-DE by dilution in IEF rehydration solution containing 7M urea, 2M thiourea, 4% CHAPS, 1% DTT, 2.0% v/v Ampholine (2). IEF was subsequently performed on IPG strip with pH range 4-8 and ultra narrow pH gradient 4-5.5 using a step-voltage programme from 100 to 8000 V for a total focusing time of approximately 100 kVh. Proteins were detected by coomassie G-250 and Pro-Q diamond (to detect phosphorylations) and gel images acquired using a dedicated scanner Pharos FX (Biorad). Conclusions Quantitative analysis performed on coomassie-stained gels have shown major differences in the area with pH 4.5. Our results have shown that caseins are most abundant in milk from infected bovines that in controls milk. This is unexpected result and further investigations are working on. References: 1) Grant I.R.et al., J Appl Microbiol. 2005;98(6):1282-93. 2) Roncada P. et al., Acta Paediatr. 2005 Dec;94(12):1708-13.