Native Breed of Chicken Entertains Different B cell Target Antigens than Popular Breeds for Protection against Pathogenic E. coli

| To determine the B-cell target antigens of virulent E. coli that might be exploited to protect itself, serological responses were assessed in one of the best-adapted exotic breeds of backyard system (Rhode Island Red, RIR), one native breed (Haringhata Black, HB), and one commercial strain (broiler) upon experimental inoculation of virulent E. coli. Variation in detection of B cell target antigens of E. coli was observed in HB, RIR and broiler birds by Western blotting. It is concluded that B cell target antigens (78, 66, 43, 29, 5 kDa) are different in native birds against virulent E. coli that might be the driving factor of disease resistance as opposed to RIR and broiler birds where clinical symptoms were imminent.


ANTIGEN
The somatic soluble antigen of the E. coli isolate (SK3) was prepared using an ultrasonicator (Hielscher Ultrasonics GmBH, Germany).Ultra-sonication was done on ice at 150W with repeating duty cycles and 0.5 sec pulse pressure for two min with 30 sec interval (five times).The soluble sonicated extract was centrifuged at 10,000 g for 30 min at 4°C and the supernatant was collected as antigen (Choi et al, 1989).The somatic soluble antigen was kept at -20ºC for further use.

PROTEIN ESTIMATION
Protein concentration of somatic soluble antigen was estimated using commercially available protein estimation kit (Merck Biosciences, India).The absorbance was measured at 660 nm by UV-VIS Spectrophotometer (TechComp, Taiwan).

DETECTION OF IMMUNODOMINANT PEPTIDES BY WESTERN BLOTTING
NCP was kept in blocking buffer (5% skimmed milk powder in phosphate buffer solution, pH 7.4, PBS) for overnight at 4ºC.NCP was then washed with washing buffer (PBS-Tween 20).The NCP was incubated for two hr with pooled serum (n=3) from experimental birds (inoculated and control) of each variety collected at 14 DPI.The sera were diluted with blocking buffer (1:20).After washing, the NCP was incubated with rabbit anti-chicken horse radish peroxidase conjugate (Genei, India) for two hrs.The NCP was rinsed with substrate solution (H 2 0 2 and DAB tablet, Sigma-Aldrich, USA) for 2 min and dipped into distilled water to stop the reaction.Lastly, it was dried up and preserved.

PROTEIN CONTENT
The estimated protein of the soluble somatic antigen of the local E. coli isolate (SK-3) was 2.3 mg/ml.

DETECTION OF IMMUNODOMINANT PEPTIDES BY WESTERN BLOTTING
Western blot analysis of somatic soluble protein of the isolate (SK-3) was done with the experimental sera obtained from sensitized HB, RIR and broiler birds after inoculating the isolate.The polypeptide bands of crude protein of E. coli were found to be reactive at 158, 78, 66, 43, 29, 5 kDa against HB serum (Figure 2A).But in case of RIR serum, four immunoreactive bands were detected having molecular weights of 158, 138, 91 and 54 kDa (Figure 2B).In broiler birds, the crude protein showed immunoreactive polypeptide bands of 91, 54 and 26 kDa in Western blot (Figure 2C).

DISCUSSION
The present study was aimed to detect B-cell antigens in three breeds of poultry viz.Haringhata Black, a native breed of India, Rhode Island Red; a backyard breed and a commercial strain (broiler) upon experimental inoculation of pathogenic E. coli isolate.The study indicated the comparative role of serological activities in three different poultry breeds by which they could resist pathogenic E. coli.The E. coli strain (SK 3) was selected for experimental inoculation because it was isolated from a local diseased broiler.Further, the isolate possessed bla TEM gene which is one of the major ESBL genes produced by E. coli (bla TEM , bla SHV , bla CTX-M ) and the bacteria of reservoir poultry chiefly harbours bla TEM (Olsen et al., 2014).The broiler birds inoculated with pathogenic E. coli showed the clinical syndrome such as high fever, roughened feather and diarrhea within 9 days.In RIR bird, intensity of fever and diarrhea was less than the broiler birds.HB chickens didn't show any observable clinical sign and symptom.Probably the higher resistance capacity of the HB birds was responsible for the development of no clinical manifestation.
The polypeptide profile of the somatic antigen and immunodominant protein profile of the challenge bacterium was ascertained.Varied as well as similar B cell target antigens of E. coli isolate (SK-3) was observed in HB (158,78,66,43,29,5 kDa),RIR (158,138,91 and 54 kDa) and broiler birds (91, 54 and 26 kDa) by Western blot.This might be due to variation in species or strain of the birds used for the study.Previous report shows that the sonicated soluble protein of E. coli possessed bands of 77, 55, 52, 46, 42, 40, 37, 30, 26, 23, 19 and 12 kDa on SDS PAGE analysis (Malik et al., 1999) which is different from the present finding.Further, Mukherjee (2006) reported that 97, 58, 38 kDa bands of E. coli O44 were immunogenic as assessed by Western blot.Previously, on Western blot analysis two immune reactive bands were detected having molecular weight of 28 kDa and 39 kDa in O39 serotype of E. coli (Mitra, 2007).This variation might be due to different serotype used and the process of preparation of crude protein.Western blot was successfully used to determine serological responsiveness against pathogenic E. coli (Hopkins et al., 1995).Unique profile of B cell target antigens as observed in HB serum (78,66,43,29,5 kDa) should be given due importance as these might be the cause of protection against pathogenic E. coli through humoral immune responses.However, further studies are needed to establish this notion.
Thus the present investigation could detect varied humoral immune responses in the poultry breeds used in the study upon exposure to pathogenic E. coli by responding against different B cell antigens of E. coli.This type of response variation might be due to variation in their individual breed characteristics that encompass the disease resistance

Figure 1 :
Figure 1: Assessment of polypeptide profile of ESBL-producing E. coli O62 somatic antigen by SDS-PAGE followed by Commassie brilliant blue R-250 staining Lane S: Sample protein; Lane M: Standard molecular weight marker Figure 2: A) Western blot analysis of ESBL-producing E. coli O62 somatic antigen using Haringhata black antiserum; B) Western blot analysis of ESBL-producing E. coli O62 somatic antigen using RIR antiserum; C) Western blot analysis of ESBL-producing E. coli O62 somatic antigen using Broiler antiserum