Protein profile of Chlamydophila abortus isolates from Kerala , India

Chlamydiae are of microbiological interest because of their mode of interaction with eukaryotic host cells and their specialized life cycle with unique features of parasitism. Reports regarding prevalence of infections of Chlamydophila abortus, the causative organism for chlamydial abortions in livestock, was the basis of the study. Two isolates, one each from cattle and goat abortion along with a reference isolate, were used for characterization with Sodium Dodecyl Sulphate-Poly Acrylamide Gel Electrophoresis (SDS-PAGE). Elementary bodies infected Mc Coy cells, harvested from bottle cultures were disrupted by Teflon coated magnetic pellet. Urografin-76 diluted with Tris-Potassium hydrochloride was used for purification of Elementary bodies of Chlamydophila abortus organism. On protein estimation of Elementary bodies by Biuret method, all the three isolates revealed protein concentration between 500-1000 mg/100ml, which were sufficient for electrophoresis. Ten percent of resolving gel and five percent of stacking gel of polyacrylamide in which 10g of processed isolate samples along with standard protein marker and Mc Coy cell protein (control) were electrophoresed. Using Alpha Imager Gel Documentation System, the protein bands were analyzed. Twelve bands each for local bovine isolate and reference isolate were noticed while only 10 bands were there in the caprine isolate. Additional bands of 148 kDa and 135 kDa were present in bovine isolate, compared to the reference isolate, while 152 kDa and 137 kDa bands were unique for caprine isolate.


Materials and Methods
Chlamydial organism causes economically Chlamydial organisms isolated from bovine and important diseases such as pneumonitis, polyarthritis, caprine abortion samples were the materials for the enteritis, encephalomyelitis, conjunctivitis and placentitis study.The primary isolation was done by inoculating the suspected samples in chick embryo through Yolk leading to abortion in domestic animals (Storz, 1971).sac route.The identity of the local samples with the Research work carried out in India had proved the reference isolate was confirmed by Agar Gel Precipiprevalence of chlamydial agents in the northern states tation Test and sensitivity for sodium sulphadiazine of India (Batta et al., 1996 andKatoch, 1997).In (Mani et al., 2006).Kerala, Chlamydial organism had been isolated from The samples were inoculated in Mc Coy cell pneumonic lungs in goats, aborted foetus and semen culture for the purpose of purification of elementary samples from bovines (Francis, 1988 and Mani et al., bodies (EBs) of the organism.Chlamydial cell culture 2006).Based on latest classification two genera harvests of 100 ml were disrupted using Teflon coated named Chlamydia and Chlamydophila comes under o family Chlamydiaceae.The organisms that cause magnetic pellet for 5 minutes at 4 C to remove cell abortion in ruminants have been characterized as debris.The supernatant was layered on to 30% (v/v) Chlamydophila abortus.The present study was to urografin-76 diluted with Tris-Potassium hydrocompare and understand the protein profile of two chloride (T-KCl).Centrifuged at 50000 x g for 45 local isolates of Chlamydophila abortus based on a minutes and pellet was re-suspended in one milliliter reference isolate from Department of Veterinary of T-KCl.It was layered on to 30 to 60 percent (v/v) Microbiology, Veterinary College, Palampur, urografin-76 gradient in T-KCl and then centrifuged at Himachal Pradesh.50000 x g for two hours.The band seen at the middle of

Results and Discussion
the 40 and 50 percent gradient was collected, diluted with three millilitres of T-KCl and centrifuged again at Those isolates confirmed with sodium sulpha 50000 x g for 45 minutes.The pellet was resuspended diazine treatment and Agar gel Precipitation Test, were in 0.5ml of T-KCl.This was taken as purified EBs of used for studying the protein profile.All the three Chlamydophila abortus isolates.Fifty milliltres of isolates revealed protein concentration between 500uninfected Mc Coy cell harvest was disrupted with a 1000 mg/100ml, which were sufficient for electroteflon coated magnetic pellet.This was sonicated at phoresis.After electrophoresis the polypeptide bands o 40% duty cycle for 5 minutes at 4 C in Branson were clearly visible on staining with coomassie sonifier 450 using microtip.This was used as the cell brilliant blue.control.The protein estimation of EBs was done by Control well showed six bands, of which the Biuret method, using Protein Estimation Kit.
band at 36 kDa was present in all the three isolates and Sodium dodecyl sulphate -poly acrylamide gel so was not taken in to account.Thus a total of 12 electrophoresis (SDS-PAGE) was used for characteriprotein bands were observed in the reference and zation of protein fractions of different Chlamydophila bovine isolates.Of the 12 bands observed most of abortus isolates, following the procedures described them were common.Additional bands of molecular by Laemmli (1970).Sample containing about 10g of weight 148 kDa and 135 kDa were present in reference purified elementary body was mixed with equal isolate, while 152 kDa and 137 kDa were unique for o volume of sample buffer, heated in a water bath at 90 C local bovine sample.Ten bands only were noticed in o for one minute, cooled and stored at 4 C.The cell the local caprine sample and were lacking bands at protein was also prepared as above.
ranges of 152 kDa, 148 kDa, 137 kDa, 135 kDa, 32 The discontinuous system of polyacrylamide gel kDa, 18 kDa, 15 kDa and 7 kDa.Bands of 155 kDa, 19 electrophoresis was employed (Markey et al., 1993).kDa, 12.2 kDa and 6.4 kDa were present only in One millimetre thick spacer was used for separating caprine sample.the glass plates.A 10% and 5% of gels were casted as In the present study only 12 polypeptide bands resolving and stacking gel respectively.Wells of the were prominent in both the bovine isolates while 10 bands were present for caprine isolate.Eventhough the gel were loaded with 20µl of each of the above prepared reference and bovine isolates gave same number of samples.The standard protein marker (3 kDa to 97.4 bands, the bands at molecular weights 148 kDa and kDa) was from Genei Bangalore.A voltage of 150 V was used for the electrophoresis.The gel was stained 135 kDa were unique for the reference isolate.152 with coomassie brilliant blue by standard procedure.kDa and 137 kDa were seen only in local bovine isolate.In local caprine isolate 155 kDa, 19 kDa, 12.2 Using Alpha Imager Gel Documentation System, the kDa and 6.4 kDa bands were unique.Six bands of protein bands were analyzed and photographed.
A -Coomassie brilliant blue stained gel after SDS-PAGE B -Representation of protein bands on each well analysed by Alpha Imager Gel Documentation System; 1-Marker, 2-Reference strain, 3-Bovine isolate, 4-caprine isolate, 5-Mc Coy cell protein.