Serological Evaluation of EgAgB16 kDa, a Recombinant Antigen from Echinococcus granulosus for Diagnosis of Human Hydatidosis

Background: Regarding that accurate diagnosis of human hydatidosis still needs more investigations, the present study was conducted to clone, express, and evaluate the gene encoding AgB subunits (EgAgB16 kDa) from Echinococcus granulosus (Iranian G1 strain) and its evaluation by ELISA test.Methods: DNA was extracted from protoscoleces and was utilized by PCR for strain identification. Total RNA was prepared with RNeasy protect mini kit from E. granulosus (Iranian G1 strain) protoscoleces collected from naturally infected sheep with hydatid cyst. Recombinant AgB16 kDa was produced using pETDuet as vector and evaluated by ELISA method. A panel of sera including hydatid cyst-infected individu­als (n=72), healthy individual (n=48), toxoplasmosis (n=4), strongyloidosis (n=4), kala-azar (n=5) and tuberculosis (n=5) were examined using this recombinant antigen.Results: Recombinant protein was purified by affinity chromatography using His-Tag column. After purifica­tion, recombinant protein was confirmed by western blot analysis using His Tag monoclonal anti­body or hydatid positive human serum. The sensitivity, specificity; positive and negative predictive values were calculated as 93.5%, 95.6%, 96% and 92.9%, in that order. The cut-off point was detected 0.3 for rAgB16. Conclusion: While the produced recombinant AgB16 kDa showed promising results in diagnosing human hy­datidosis, but more investigations should be implemented to reach an accurate gold standard

Genotypic and Phenotypic Analysis of Fasciola Isolates

"nBackground: To identify the fasciolid species by morphometric and molecular methods in Zanjan, north­west of Iran. "nMethods: Adult Fasciola worms (n=584) were obtained from cattle and sheep in Zanjan slaughterhouse in 2007. Living flukes were washed, then worms' images were taken by 3CCD camera and finally apical zone of each worm was obtained. Morphometric values such as body length, body width, body area, body pe­rimeter and the distance between ventral sucker and posterior end of body were obtained using Auto­CAD image analysis software. Total gDNA was extracted from individual flukes by modified phenol-chloroform method. PCR amplification of ITS2 fragment was performed the isolated DNA samples and the amplicons were consequently subjected to RFLP assay and nucleotide sequencing to distinguish be­tween fasciolid species. "nResults: Mean of morphometric values in flukes from sheep was greater than those of cattle. Accordingly, the identified species included 31% F. hepatica-like, 7% F. gigantica-like and 62% intermediate forms. How­ever, ITS2 fragment of 535 amplified specimens, showed no variation at the species-specific nucleo­tide sites 230, 340 and 341. The amplified fragment composed of partial 5.8S sequence (62bp), the com­plete ITS2 sequence (361bp) and partial 28S sequence (34bp). The nucleotide contents of ITS2 region were 69 A, 116 T, 81 C and 95 G and the average G+C content was approximately 49%. Comparing of ITS2 sequences with the BLAST GenBank database, also confirmed that all specimens were F. hepatica. "nConclusion: A simple and rapid PCR-RFLP assay can be used for distinguishing between these species

Immunoregulatory Cytokine (TGF-ß And IL-10) Responses in Mice Inoculated With Protoscoleces and Major Hydatid Fluid Antigens of Cystic Echinococcosis

"nBackground: Our objectives were to investigate whether immunomodulatory cytokines, TGF-β and IL-10, are stimulated in response to cystic echinococcosis (CE) components in mice model, and whether major hydatid fluid antigens or live protoscoleces could equally contribute to such cytokines."nMethods: Protoscoleces were obtained by aseptic puncture of fertile sheep hydatid cysts. Hydatid fluid antigens (HFAgs) and Antigen B (AgB) were prepared by partial purification and electroelution, respectively. Of the 25 Balb/c mice assigned in four groups, the first group was inoculated ip with 2000 live protoscoleces; the second and the third groups were injected ip with 50 µg HFAgs and 50 µg AgB in 200 µl of PBS, respectively. Control group was only injected with PBS. The sera concentration of TGF-ß and IL-10 were determined by ELISA. Data were analyzed using One-Way ANOVA and Tukey-HSD tests to compare differences between means."nResults: The mean concentration of TGF-ß in those groups injected with protoscoleces, HFAgs and AgB were significantly higher than control group. However, in the case of IL-10 such differences were only detected in mice that were inoculated with protoscoleces (356±11 pg/ml) compared to control (207±9 pg/ml), HFAgs and AgB groups."nConclusion: TGF-ß and IL-10, two important immunomudulatory cytokines are induced by different molecules or components of CE, so that AgB could induce TGF-ß and components of protoscolex, other than AgB and Ag5, could induce IL-10