doi:10.5455/vetworld.2013.267-270 Characterization of immunogenic proteins of Cysticercus tenuicollis of goats

Aim: To identify immunodominant proteins of cystic fluid antigens and whole cyst lysate antigens of Cysticercus tenuicollis, the larval stage of the canine tapeworm Taenia hydatigena. Materials and Methods: Three numbers of cysts of C. tenuicollis were collected from the mesentry of small intestine of goats after slaughter. C. tenuicollis cysts of each sample were washed thoroughly with PBS (pH 7.4). Two types of antigens i.e. cystic fluid antigens and whole cyst lysate antigens were prepared from each sample. Polypeptide profiles of cystic fluid antigens and whole cyst lysate antigens preparations of C. tunicollis were analysed by Sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) containing 10% gel. C. tenuicollis proteins resolved by SDS-PAGE were electrophoretically transferred from the gel to a nitrocellulose membrane and probed by western blotting using natural immune sera obtained from infected goats. C. tenuicollis proteins that were resolved by SDS-PAGE and reactive by immune sera of goat was estimated by a Soft ware programme named KODAK 1D Image Analysis Software. Results: A total of eight major polypeptides of molecular weight (Mr) 149.4 kDa, 92.9 kDa, 74.2 kDa, 63.5 kDa, 36.2 kDa, 23.9 kDa, 15.7 kDa and 9.6 kDa were resolved by SDS-PAGE with minor variations. Out of these, three polypeptides of Mr 36.2 kDa, 23.9 kDa and 9.6 kDa were recognized as immunodominant polypeptides after western blotting. Both cystic fluid antigens and whole cyst lysate antigens resolved same Mr polypeptides by SDS-PAGE and identified same immunodominant polypeptides after western blotting. The immunodominant polipeptides of Mr 23.9 kDa and 9.6 kDa identified for the first time from both cystic fluid antigens and whole cyst lysate antigens prepared from Cysticercus tenuicollis. Conclusion: Eight polypeptides were resolved from cystic fluid antigens and whole cyst lysate antigens of Cysticercus tenuicollis by SDS-PAGe of which polypeptides of three polypeptides of Mr 36.2 kDa, 23.9 kDa and 9.6 kDa were recognized as immunodominant polypeptides after western blotting. These three immunodominant polypeptides could be explored for sero diagnosis of C. tenuicollis infection in goats.


Introduction
from goats of India and abroad [4,8].High prevalence of infection (46.6%, 55.77% and 63.9%) have been The larval stage of the canine tapeworm Taenia reported in goats from Ethiopia and Benin, after hydatigena inside their intermediate host is known as abattoir study and adult goats were found more Cysticercus tenuicollis.The infection has been infected than kids [3,5,9].Cysticercus tenuicollis cysts reported to present in wild and domestic ruminants were found in 18.04% goats of Iran [10] and 10% goats throughout the world.The morphological features are of Iraq [11].used to identify the cyst but now-a-days molecular Almost similar percentage of infection (18.75%) tools are being used to define species of parasites in in goats has been reported from Maharashtra State, cases where the key morphologic features can not India [12].The prevalence of C. tenuicollis infection in detect species of parasites [1].The infection causes goats from Durg, Chattishgarh was reported as 21.01%condemnation of meat and thereby economic losses [2, [13].There is no effect of season on prevalence of this 3].It is generally found in the mesentries, omenta, infection [5].Significant level of calcium, sodium, cavities and also in the liver, lungs, kidneys and brain of potassium and considerable amount of Asparate infected animals [4,5].Aberrant location of C. aminotransferase (AST), Alkaline phosphatase (ALKP), tenuicollis inside the chorion-allantoic membrane of Lactate dehydrogenase (LDH) with low concentration goats has also been reported [6].C. tenuicollis of Alanine aminotransferase have been reported to be infection in liver causes hepatitis, burrowing canal, present in cystic fluid of C. tenuicollis of goats [14].granular degeneration, deposition of serofibrinous Examination of post mortem materials is the usual exudates and in lungs it causes pneumonitis [4,7].method of diagnosis.Detection of antibodies could be Besides this pathogenicity, outbreaks due to acute of interest as a tool for immunodiagnosis of the Cysticercus tenuicollis in goats have been reported infection.For this it is essential to know about the cystic fluid antigens, the fluid of each cyst was immunogenic proteins of C. tenuicollis.Hence, here an aspirated from the cyst with the help of a sterilized syringe and needle and collected directly in centrifuge attempt has been taken to identify the immunogenic tube.It was centrifuged at 15000xg for 15 minutes at proteins of C. tenuicollis using SDS-PAGE analysis and whole cyst Ags were estimated as 2.8 mg/ml and the appearance of almost similar molecular weight 3.2 mg/ml respectively.The antigens, thus prepared polypeptides for both Ags, might be due to their same o were stored in aliquots at -20 C till further use.composition.The slight difference may be due to the host variation.Although eight polypeptides were Source of immune sera: During slaughter, blood resolved after SDS-PAGE analysis, only three of them samples of the goats were collected in sterile condition have been found immunogenic in the present study, for and sera of the goats which were found positive for C. o both the Ags.This indicated that immunogenecity of tenuicollis infection were preserved at -20 C for further both Ags are same.Thus three immunodominant use at the time of immunoblot.
polypeptides of Mr 36.2 kDa, 23.9 kDa and 9.6 kDa SDS-PAGE and western blot analysis: For identifiwith minor variation were identified from both cystic cation of immunogenic proteins, polypeptide profiles fluid antigens and whole cyst lysate antigens.In a of cystic fluid antigens and whole cyst lysate antigen study, only one immuno reactive protein band of Mr 36 preparations of each C. tenuicollis were analysed by kDa has been reported from Turkey [20] and almost SDS-PAGE (Genei Bangalore, India) containing 10% similar immunodominant polypeptide of Mr 36.2 kDa gel under protein denaturing condition [18].Standard also identified in the present study.The additional twomolecular weight marker protein (Lonza, USA) was immunodominant polypeptides of Mr 23.9 kDa and 9.6 run simultaneously.Two separate gels for each sample kDa as identified in the present study might be due to were run in same conditions using the same antigen antigenic variation of the metacestode due to their samples prepared from C. tenuicollis.After completion strain variation and geographical distribution.These of running of the SDS-PAGE, one gel along with immunogenic antigens could be used for immunomolecular weight marker was shifted to stain with diagnosis of C. tenuicollis after purification using the Coomassie Brilliant Blue.Remaining one was washed technique either counter immunoelectrophoresis with transfer buffer and C. tenuicollis proteins resolved (CIEP) or agar gel precipitation test (AGPT) in the by SDS-PAGE were electrophoretically transferred field.The protein profiles of dialyzed cyst fluid, cyst (Genei, Bangalore) from the gel to a nitrocellulose membrane and scolex of C. tenuicollis resolved a total membrane as per the standard method [19] and probed 37 bands by SDS-PAGE, out of which 3 bands were by western blotting using natural immune sera observed in the cyst fluid, 22 in the cyst membrane and obtained from infected goats.The Mr for the C.
12 bands in the scolex of the cyst [15].They observed tenuicollis proteins that were resolved by SDS-PAGE cyst fluid proteins were least reactive, whereas and reactive by immune sera of goat was estimated by a membrane proteins elicited a consistent reaction with Soft ware programme named KODAK 1D Image the hyperimmune sera after western blot analysis.In a Analysis Software.
recent study from India, cystic membrane antigens of C. tenuicollis revealed 15 polypeptide bands of Mr 10

Results
to 131 kDa by SDS-PAGE analysis where naturally The polypeptide profiles of cystic fluid antigens infected goat serum identified two immunodominant and whole cyst lysate antigens as resolved by SDSbands of Mr 34 kDa and 14 kDa [16] and they PAGE have been shown in Fig- 1.A total of eight concluded that these two bands could be explored for polypeptides of Mr 149.4 kDa, 92.9 kDa, 74.2 kDa, sero diagnosis of T. hydatigena cysticercosis in goats.63.5 kDa, 36.2 kDa, 23.9 kDa, 15.7 kDa and 9.6 kDa

Conclusion
were resolved by SDS-PAGE from each samples with minor variations.Out of these three polypeptides of Mr Eight polypeptides were resolved from cystic 36.2 kDa, 23.9 kDa and 9.6 kDa were recognized as fluid antigens and whole cyst lysate antigens of immunodominant polypeptides after western blotting.
Cysticercus tenuicollis by SDS-PA.Ge of which All of them could be considered as major polypeptides.
polypeptides of three polypeptides of Mr 36.2 kDa, It could also be observed that both cystic fluid antigens 23.9 kDa and 9.6 kDa were recognized as immunoand whole cyst lysate antigens resolved same Mr dominant polypeptides after western blotting.These polypeptides.Fig- 2 represent the Western blot analysis three immunodominant polypeptides could be for identification of corresponding immuno-genic explored for sero diagnosis of C. tenuicollis infection proteins using immune sera.A total of three in goats.polypeptides of Mr 36.2 kDa, 23.9 kDa and 9.6 kDa