Research Article Immunoreactivity to Culture Filtrate Proteins of ...

Research Article

Advances in Animal and Veterinary Sciences

Immunoreactivity to Culture Filtrate Proteins of Mycobacterium avium Subspecies paratuberculosis in Naturally Infected Goat and Sheep Sera

Saurabh Gupta1, 2, Kundan Kumar Chaubey1, Shoor Vir Singh1*, Ashok Kumar Bhatia2, Naveen Kumar1, Anjana Goel2, Tarun Kumar Sachan1, Krishan Dutta Rawat1, Jagdip Singh Sohal3, Kuldeep Dhama4 Microbiology Laboratory, Animal Health Division, Central Institute for Research on Goats, Makhdoom, PO-Farah, Mathura, Uttar Pradesh, India; 2Department of Biotechnology, GLA University, Chaumuhan, Mathura, Uttar Pradesh, India; 3Amity Institute of Microbial Technology, Amity University Rajasthan, Jaipur, India; 4Division of Pathology, Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India. 1

Abstract | Mycobacterium avium subspecies paratuberculosis (MAP), the cause of granulomatous chronic enteritis in ruminants ( Johne’s disease) is under reported due to difficulties in diagnosing pre-clinical cases. Compromised specificity is a problem due to extensive sharing of antigens /epitopes among MAP and other mycobacterial strains. Culture filtrate (CF) proteins profile of native ‘Indian Bison Type’ strain of MAP was studied at different harvesting times (2-8 weeks of growth) in Middlebrook 7H9 medium supplemented with ADC, PANTA antibiotics and mycobactin J. Analysis of harvested CF proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the greater part of CF proteins had molecular masses ( USD 250 million economic losses has been reported in US Recent studies have focused on developing improved sealone (Ott et al., 1999). Though disease is endemic in In- rodiagnostics using species-specific multiple protein antidia and economic losses in dairy farm was estimated from gens (Singh et al., 2014c). Microfluidics and Lab-on-Chip reproductive disorders (Rs. 23400.0/cow/year), forced re- are some of the recent technologies that can predicate moval (Rs. 41,750.0/cow/year), reduced milk yield (Rs. development of laboratory-free diagnostic devices for

M

June 2015 | Volume 3 | Issue 6 | Page 347

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Advances in Animal and Veterinary Sciences mycobacterial infections (Wadhwa et al., 2012a; Li et al., Sodium Dodecyl Sulphate Polyacrylamide Gel 2011). Earlier reports have identified several antigens in- Electrophoresis (SDS- PAGE) ducing strong antibody responses but most of them have been found unsuitable for serodiagnosis because they are highly conserved within mycobacteria and hence cross-react with other mycobacterial pathogens. Recently, various MAP specific proteins/genes has been characterized, cloned, expressed and the protein was evaluated for their diagnostic potential (Cho et al., 2007). However, strain of MAP may also tune the sensitivity and specificity of test. Present study investigated CF protein profile of native Indian bison type (‘S 5’) MAP strain for the first time with reference to determine the antigenicity / reactivity of the CF proteins by immunoblotting with polyclonal antisera of goat and sheep infected with MAP and to develop diagnostic tests of higher sensitivity and specificity.

CF protein profile was analyzed by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and stained with Coomassie brilliant blue as per method given by Laemmli (1970).

Immunoblotting

The electrophoresed CF proteins were transferred on to PVDF-plus membrane (pore size 0.45 µm, Millipore) using Mini Trans-blot cell (Bio-Rad) for 1.5 hrs at 60 V, 100 mA as per Towbin et al. (1979) with some modifications. Membrane was blocked for 1 hr at room temperature in 5% skim milk (Himedia) containing TBS with 0.05% Tween-20 (TBST) and were probed with polyclonal primary antibody (goat serum) diluted 1:100 in 1X TBST for 2 hrs at room temperature. After incubation membrane was MATERIALS AND METHODS washed 3 times with 1X TBST. Reactivity was seen by incubating the PVDF-plus membrane with peroxidase-conjugated anti-goat IgG (Sigma, USA) in 1:2000 dilution for 1 Mycobacterial Strain Mycobacterium avium  subspecies  paratuberculosis (‘S 5’) hr at room temperature followed by washing 5 times (5 min strain was procured from the mycobacterial repository, each) with TBST. Visualization of immuno-reactive proCentral Institute for Research on Goats (CIRG), Makh- tein bands was done by diaminobenzidine (DAB) (Sigma). doom. The strain was maintained on Modified Herrold’s egg yolk medium with mycobactin J (HEYM) as per Singh Statistical Analysis et al. (1996) and was sub-cultured in Middlebrook 7H9 The correlation (R2) and Standard deviation of OD600 medium (as per the manufacturer, Becton Dickinson, BD) with time interval of growth measurements were analyzed supplemented with ADC (10% or 100ml/l), PANTA anti- using Graph Pad InStat. biotics and mycobactin J (2mg/l).

Growth Pattern

Growth of MAP in middlebrook 7H9 medium was monitored by taking absorbance (OD) of the culture at third day, then every week at 600nm for a period of 8 weeks (Figure 1). Growth curves were obtained by plotting the absorbance (OD) values versus incubation time in weeks. Turbidity in the medium was measured by Mcfarland standards (0.5, 1, 2, 3, 4, 5 and 6) to check the growth of the culture. To avoid clump formation the cultures were constantly shaken at 100 rpm during incubation.

Culture Filtrate (CF)

CF were  obtained by harvesting (4000×g, 20 min, 4°C) bacterial growth at different times points (2, 4, 6 and 8 weeks of incubation) and was filtered (using 0.22-μm pore size syringe filter, Millipore). CF were precipitated with saturated ammonium sulfate (Rankem) due to the high amount of albumin in the broth followed by extensive dialysis overnight against 10 mM phosphate-buffered saline (pH 7.4) until free of ammonium ions at 4°C. Dialysed CF were concentrated 10-fold using vacuum concentrator (SPD Speedvac, Thermo Savant). Concentration of CF proteins was quantified by Bradford protein assay kit (Genei) and was stored at -20°C.

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RESULTS

Growth Pattern

Mycobacterium avium  subspecies  paratuberculosis (‘S 5’) strain developed a granular growth with visible clumps suspended in the 7H9 medium up to 6 weeks, afterwards, clusters of floating cells were observed (Figure 1). The growth curve of MAP ‘S 5’ strain in 7H9 medium showed sharp bacterial growth phases delineation; however, after 3 weeks of incubation the culture was in log phase (early exponential) upto 8 weeks (mid to late exponential) in 7H9 medium (Figure 1). Total bacterial mass was with a maximum absorbance of 1.43 at 8 weeks. OD600 and time interval of growth measurements were highly correlated (R2 = 0.997, Figure 1). We determined in Indian Bison type ‘S 5’ strain of MAP that 1 McFarland unit was nearly equivalent to 0.26 OD600.

Immunoblot of Mycobacterium avium Subspecies paratuberculosis Secreted Proteins

The initial antigenic profiles were detected at 2, 4, 6 and 8 weeks of incubation in 7H9 medium. Analysis of harvested CF proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the greater part of CF proteins had molecular masses (