[Prevalence of anti-toxocara antibodies in children with allergic manifestations referring to allergy clinics in Zanjan]

Background and Aim: Toxocariasis is a common parasitic zoonosis of humans, dogs and cats. Migration of Toxocara larva in the internal organs is accompanied by a variety of complications such as hepatomegaly, marked eosinophilia, pulmonary and ocular symptoms. In some cases, symptoms are nonspecific and the patients may only show allergy-like cutaneous and pulmonary manifestations. Considering the significant increase in the prevalence of allergic disorders, study of the possible etiologies of these disorders can lead to the early diagnosis and effective treatment of the disease. In this study prevalence of antitoxocara antibody and some hematological parameters were compared between allergic and non-allergic children

Materials and Methods: Our experimental group included 200 children under 15 years of age with skin and/or respiratory allergies referring to allergy clinics, and our control group consisted of 238 non-allergic children, matched to the experimental group in regard to age and sex. Specific anti-toxocara IgG titers were measured by TES/Ag-ELISA technique. Eosinophilia and leukocytosis were extracted from the CBC results. We used T-test and chisquare to compare the results between the two groups

Results: The prevalence of anti-toxocara IgG in the children with allergic manifestations was significantly higher [4.5%] than that in the healthy children [0.8%]. Children with allergic manifestations had a higher rate of eosinophilia [16.5%] compared to healthy children [0.4%], but there was no significant correlation between eosinophilia and toxocariasis serology

Conclusions: Regarding the higher prevalence of anti-toxocara antibodies in the children with skin and respiratory allergies, investigation of toxocariasis in the patients with allergic symptoms is recommended

Evaluation of Anti-Toxoplasma IgG, IgM, and IgA in Mothers with Spontaneous Abortion in Zanjan, Northwest Iran

Toxoplasma gondii is one of the major agents of infectious abortions and due to its worldwide distribution can threat healthy pregnant women who had no previous exposure to this parasite. The present study was designed to investigate the contribution of T. gondii to spontaneous abortions in Zanjan, Northwest of Iran, using ELISA method. Blood Samples were collected from 264 mothers referred to the provincial hospitals of Zanjan due to spontaneous abortion. The sera were isolated and subjected to evaluate the anti-Toxoplasma IgG, IgM and IgA antibodies. The results showed IgG positive (IgG+) in 99 cases (37.5%). A total of 68 women (25.8%) showed seroconversion with IgM or IgA or both IgM and IgA. They included: IgM+ in 21 (8.0%), IgA+ in 23 (8.7%) and both IgM+ and IgA+ in 24 (9.1%) subjects. In 23 cases, positive titers of IgM and IgG were accompanied. In general, the analysis of anti-Toxoplasma antibody patterns, showed that about 17% of the spontaneous abortions were associated with serological patterns of acute infection. According to these findings, a considerable proportion of spontaneous abortions can be attributed to T. gondii in the study area.

[Comparison and evaluation of Echinococcus granulosus protoscoleces excretory/secretory proteins in PBS complemented with glucose, DMEM and RPMI culture media]

Preparation of proper antigens is an important issue in serology of hydatidosis. Investigators have been able to obtain excretory/secretory antigens [E/S Ags] by short-term culture of protoscoleces in a couple of culture media. However, no data are available about production rate of such antigens in different culture media. The present study was carried out to evaluate the production of E/S Ags [proteins] in PBS complemented with glucose, DMEM and RPMI culture media. To obtain E/S proteins, protoscoleces of echinococcus were cultured in PBS complemented with 10% glucose, RPMI and DMEM for 72 hours. Proteins secreted in culture media were concentrated and assayed. To characterize different components, proteins were electrophoresed on SDSPAGE. Data were analyzed using One-way ANOVA and Tukey HSD tests. The mean concentration of E/S proteins in PBS medium in 24 hours of culture was significantly higher than DMEM and RPMI [P<0.05]. However, such a difference was not observed between E/S proteins in DMEM and RPMI media. E/S proteins obtained from PBS medium were separated into 12 major bands and the two other media into 14 major bands within a range of molecular masses of 16 to 67 kDa. PBS complemented with glucose is more appropriate than the two other media for E/S proteins production. The best time to obtain E/S proteins is the first 48 hours of culture

Serological evaluation of EgAgB16 kDa, a recombinant antigen from echinococcus granulosus for diagnosis of human hydatidosis

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. 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. 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 anti body 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. 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

[Identification of fasciola species by PCR-RFLP assay]

Background and Objective: fascioliasis is an important zoonotic disease that causes several health problems and economical losses in different parts of Iran including Zanjan. Fasciola hepatica and F. gigantica are recognized as causative agents of the disease. The differential diagnosis between these two species is very important for planning and control of infection. This study was designed to identify the Fasciola species by molecular methods in Zanjan [Iran]

Methods and Materials: a number of 535 adult Fasciola worms were collected from the natural infected livers of cattles and sheep in local slaughterhouse. Living flukes were washed extensively in PBS at 37 degreeC and then anterior half of adult worms were stored at -20 degreeC in 70% ethanol. Total genomic DNA was extracted from individual flukes by modified phenol-chloroform method. Nucleotide polymorphism of ITS2 fragment of rDNA was investigated using PCR-RFLP assay and sequencing technique

Results: the results of PCR-RFLP and comparison of ITS2 sequences with the BLAST GenBank database clarified that all specimens were F. hepatica. The obtained sequences are available in the GenBank, with accession numbers EU391412 to EU391424

Conclusion: the results of this study showed no evidence of F. gigantica infection in sheep and cattles in Zanjan as all of the isolates were found to be F. hepatica

Genotypic and phenotypic analysis of Fasciola isolates

To identify the fasciolid species by morphometric and molecular methods in Zanjan, northwest of Iran. 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. 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. A simple and rapid PCR-RFLP assay can be used for distinguishing between these species

Immunoregulatory cytokine [TGF-beta and IL-10] responses in mice inoculated with protoscoleces and major hydatid fluid antigens of cystic echinococcosis

Our objectives were to investigate whether immunomodulatory cytokines, TGF-beta 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. 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 micro g HFAgs and 50 micro g AgB in 200 micro l of PBS, respectively. Control group was only injected with PBS. The sera concentration of TGF-beta and IL-10 were determined by ELISA. Data were analyzed using One-Way ANOVA and Tukey-HSD tests to compare differences between means. The mean concentration of TGF-beta 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. TGF-beta and IL-10, two important immunomudulatory cytokines are induced by different molecules or components of CE, so that AgB could induce TGF-B and components of protoscolex, other than AgB and Ag5, could induce IL-10

Evaluation and comparison of antigen B-Elisa and antigen B-iimunoblotting in immunodiagnosis of cystic hydatid disease

Evaluation and comparison of the diagnostic value of ELISA using partial purified antigen B [AgB-ELISA] and antigen B subunits immunoblotting in immunodiagnosis of cystic hydatid disease [CHD]. Antigen B was obtained and partially purified from sheep hydatid cyst fluid. Serum samples were collected from surgically confirmed CHD patients, other parasitic disease, patients with malignancies and normal individuals. Sera were analyzed by ELISA using antigen B and immunoblotting based on antigen B subunits. School of Public Health serum blood bank, Tehran University of Medical Sciences, volunteers and selected CHD patients from different local hospitals. Subject: Serum samples were obtained from 64 surgically confirmed CHD patients, 55 from individuals infected with toxocariasis or fascioliasis, 50 from patients with malignancies and 73 from normal individuals. The sensitivity, specificity and cross-reaction of comparative tests evaluated. The sensitivity of AgB-ELISA was 89%, while that of 8/12-, 16-kDa immunoblot was 80%. Specificity of AgB-ELISA was 98% and that of 8/12-, 16-kDa immunoblot was 100%. The sensitivity and specificity of ELISA using crude hydatid fluid antigen [CHFAg-ELISA] were 94% and 83%, respectively. AgB-ELISA as well as 8/12-, 16-kDa immunoblot can be convenient in specific and confirmatory diagnosis of CHD