Staining of Parasitic Helminths by Extracts of Allium cepa, Juglans regia, and Rubia tinctorum: An Approach to Herbal Dyes

Background: Although carmine, as a synthetic dye, is the major substance for staining helminths, it may impose some adverse effects on human health. In the present study, we evaluated the aqueous extracts of onion (Allium cepa) skin, walnut (Juglans regia) husk, and madder (Rubia tinctorum) roots as potential herbal dyes for staining parasitic helminths. Methods: Aqueous solutions (5%, 10% and 20%, w/v) of each herbal prepared from dried and powdered husk of walnut, skins of red onion, and madder roots in distilled water. Parasitic helminths including Fasciola spp., Dicrocoelium denderiticum, Echinococcus gronulosus protoscolices, Moniezia spp., and Haemonchus contortus were stained by different concentrations of herbal dyes according to carmine staining method. The structural clarity and quality of stained internal organs of the helminths such as suckers, intestine, and reproductive systems were scored by semi-quantitative evaluation in comparison with carmine stained samples. Results: The optimum concentrations of extracts for helminths staining were 10% (w/v) of A. cepa and J. regia, and 20% (w/v) of R. tinctorum with final scores of 3.1, 3 and 2.8, respectively. In general, A. cepa and J. regia extracts showed higher quality in staining Platyhelminthes, while R. tinctorum extract presented relatively higher quality in staining Nematoda. Conclusion: Considering proper quality of A. cepa, J. regia and R. tinctorum extracts in staining the helminths, they may be safe, eco-friendly, and inexpensive alternatives to carmine dye

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

Molecular detection of Taenia spp. in dogs' feces in Zanjan Province, Northwest of Iran

Aim: Echinococcus and Taenia spp. are important but neglected zoonotic helminths of dogs. Dogs as the most relevant definitive hosts harbor several species of Taenia and Echinococcus simultaneously in their gastrointestinal lumen which are morphologically indistinguishable. In this study, we used a multiplex polymerase chain reaction (PCR) method to identify Taeniid infections which seem to be highly distributed in the study region. Materials and Methods: A total of 450 dog fecal samples were collected from eight different areas of Zanjan province, northwest of Iran, and examined using a flotation method followed by multiplex PCR for detection and identification of parasites' eggs. Results: Gastrointestinal parasites were found in 86 out of 450 fecal samples (19.1%) by microscopic examination. Taeniid eggs were observed in 5.6% of samples, containing 0.45%, 3.8%, and 1.3% Echinococcus granulosus, Taenia spp., and mix infection of both E. granulosus and Taenia spp., respectively. Echinococcus multilocularis was absent in the samples. Conclusion: A relatively low rate of E. granulosus (1.8%) was observed in this study. However, risks of this parasite should not be overlooked, and control programs need to be extended for this species and other Taeniid spp. In particular, dogs are recommended to be dewormed more frequently

PCR-based Diagnosis of Toxoplasma Parasite in Ocular Infections Having Clinical Indications of Toxoplasmosis

Background: The diagnosis of ocular toxoplasmosis is mainly based on clinical features. However, ocular fluid testing by PCR may be very helpful for approval or rejection of this etiology. In this study, we utilized a nested-PCR technique, targeting the B1 partial sequence to analyze the aqueous and vitreous samples for evaluating the presence of the Toxoplasma DNA. Methods: Fifty aqueous or vitreous humor samples were obtained from patients with clinical features of ocular toxoplasmosis admitted to ophthalmology hospitals and clinics in Iran, within 2014. The samples were subsequently subjected to DNA extraction and purification. For nested amplification of the Toxoplasma B1 gene, two primer pairs were used. The outer and inner primers are expected to produce a 193 bp and a 96 bp fragments, respectively. Results: The first-round PCR resulted in the detection of T. gondii in 58% of samples by amplification of the expected 193bp DNA fragment. The nested-PCR using the inner primers, detected 15 additional samples from those with negative amplicons in the first round PCR (overall positivity of 88%). In addition, vitreous samples showed relatively more positive cases than aqueous humor in detection of the infection. Conclusion: The nested-PCR protocol using the B1 gene, with the high detection power, could be a useful complimentary method to clinical diagnose of ocular toxoplasmosis