Prevalence of Toxocara Spp. eggs in Public Parks in Tehran City,Iran

Background

The objective of the present research was to determine the frequency of Toxocara spp. eggs in soil samples of public parks, in the city of Tehran, Iran.

Methods

A total of 600 soil samples were taken from 120 parks between Aprils to November, 2008. Soil samples were collected from 5 distinct sites in the parks. The samples were washed with saline solution and the collected sediment from each park were equally divided and examined by floatation and Petri dish methods for Toxocara eggs.

Results

Ten percent were contaminated with Toxocara spp. eggs. The number of observed Toxocara eggs in each microscopic field was varied from 1-3. No significant differences were observed between floatation and Petri dish methods.

Conclusion

Our public parks showed a high risk of toxocariasis and the need for preventive studies.     

Aspicularis tetraptera Induced Hematological Parameters in Infected and Vaccinated Mice

Background

The present study deals with the effect of helminthic infection as Nematode parasite like Aspiculuris tetraptera on the haematological parameters of infected and vaccinated mice.

Methods

Totally 15 mice were used. Five mice were used for positive control, 5 mice used for negative control and 5 mice used for experiment. The hematological parameters were studied viz. RBC, Hb, and serum protein values.

Results

The mice carrying heavy infection showed decrease in the Hb, RBC, and serum protein but in the vaccinated mice, all studied parameters were become on normal range. The level of immune response was assessed based on above studied hematological parameters in infected and vaccinated mice with Aspiculuris tetraptera.

Conclusion

The increased value of RBC, Hb and Serum protein in infected and vaccinated mice compared to infected and non vaccinated suggested the involvement of blood parameters in immune response. This study also proves that somatic antigen of A. tetraptera was effective in imparting immunity in mice     

Rapid Detection of Toxoplasma gondii Antigen in Experimentally Infected Mice by Dot- ELISA

Background

Toxoplasmosis is a worldwide endemic disease. In congenitally infected infants and AIDS patients, toxoplasmosis causes high rates of morbidity and mortality. In these cases antibody detection is difficult; so detection of parasite or its components could be useful tool for early detection and following treatment of the infection.

Methods

Sixty-three BALB/c mice were injected intra-peritoneal with 5×10³ tachyzoites of Toxoplasma gondii RH strain, nine mice were sacrificed daily for 7 days. Fourteen mice were injected with phosphate buffer saline as control group. Dot–ELISA was performed for detection of T.gondii antigen in mice sera and capture – ELISA was done as golden standard assay too.

Results

Toxoplasma gondii antigen was detected from day 2 in mice sera; 22% of mice sera on day 2, 33% on day 3,77% on day 4 and 100% on day 5 till their death on day 7 had shown antigenemia by dot – ELISA, no positive result was detected in control mice by dot- ELISA.

Conclusion

Dot-ELISA is a sensitive method for diagnosis of T. gondii infection in the animal model; also, this technique is more rapid and easy to perform method in comparison with capture-ELISA.     

Immunoproteomic Analysis of the Excretory-Secretory Proteins from Spirometra mansoni Sparganum

Background

Sparganosis is caused by the invasion of Spirometra sparganum into various tissues/organs. Subcutaneous sparganosis can be diagnosed by biopsy, while visceral/cerebral sparganosis is not easy to be diagnosed. The diagnosis depends largely on the detection of specific anti-sparganum antibodies. The specificity of the ELISA could be increased by using S. mansoni sparganum excretory–secretory (ES) antigens, but it also had the cross-reactions with sera of patients with cysticercosis or paragonimiasis. The aim of this study was to identify early specific diagnostic antigens in S. mansoni sparganum ES proteins.

Methods

The sparganum ES proteins were analyzed by two-dimensional electrophoresis (2-DE) and Western blot probed with early sera from infected mice at 14 days post-infection. The immunoreactive protein spots were characterized by MALDI-TOF/ TOF-MS.

Results

A total of approximately 149 proteins spots were detected with isoelectric point (pI) varying from 3 to 7.5 and molecular weight from 20 to 115 kDa and seven protein spots with molecular weight of 23-31 kDa were recognized by the infection sera. Three of seven spots were successfully identified and characterized as the same S. mansoni protein (cysteine protease), and the proteins of other 4 spots were not included in the databases.

Conclusion

The cysteine protease from S. mansoni ES proteins recognized by early infection sera might be the early diagnostic antigens for sparganosis.     

The Use of Recombinant 31 kDa Antigens of Trichinella spiralis for Serodiagnosis of Experimental Trichinellosis

Background:

We have previously reported that a 31 kDa protein was screened from the excretory-secretory (ES) proteins of Tichinella spiralis muscle larvae (ML) by immunoproteomics using early infection sera, and the gene encoding a 31 kDa protein from T. spiralis was cloned and expressed in an E. coli expression system. In this study, the recombinant 31 kDa antigens were used for detection of anti-Trichinella antibodies in serum of experimentally infected mice by ELISA.

Methods:

Anti-Trichinella IgG antibodies in sera of mice infected with Trichinella were assayed by ELISA with recombinant 31 kDa antigens, and its sensitivity and specificity were compared with ELISA with ES antigen.

Results:

The sensitivity and specificity of ELISA with recombinant antigens was 96.67% (29/30) and 96.87% (62/64), compared with 100% (30/30) and 98.44% (63/64) of ELISA with ES antigens was (P>0.05). In heavily, moderately and lightly infected mice (500, 300 and 100 larvae/mouse), anti-Trichinella antibodies were firstly detected by ELISA with recombinant antigens at 8, 12 and 14 dpi, respectively; then increased rapidly with a detection rate of 100% respectively at 28, 22 and 30 dpi. While the antibodies were firstly detected by ELISA with ES antigens at 10, 8 and 10 dpi, respectively, the antibody positive rate reached 100% at 14, 12 and 22 dpi, respectively.

Conclusion:

The recombinant 31 kDa antigens of T. spirali had a good sensitivity and specificity for detecting anti-Trichinella antibodies and might be the potential diagnostic antigen for trichinellosis.     

Detection and Identification of Toxoplasma gondii Type One Infection in Sheep Aborted Fetuses in Qazvin Province of Iran

Background

The aim of this study was to apply the nested-PCR and bioassay methods in detection and genotyping of Toxoplasma gondii infection in provided sheep aborted fetus samples from Qazvin Province of Iran.

Methods

Eighteen sheep aborted fetal samples were studied by nested-PCR-RFLP, histopathological observation and microbiological assay. Bioassay in mice was carried out by inoculating the brain samples intraperitoneally.

Results

The results demonstrated the frequency of 66% infected sheep aborted fetal samples with T. gondii type one. Although we could not isolate any parasite from inoculated mice even after three passages, but it was confirmed histopathologically formation of cyst like bodies in prepared mice brain sections.

Conclusion

The results of the performed nested-PCR and formation of brain cyst in inoculated mice exhibited that T. gondii type one infection might be considered as one of the major causative agents for abortion in ewes.     

Recombinant SAG1 Antigen to Detect Toxoplasma gondii Specific Immunoglobulin G in Human Sera by ELISA Test

Background

Although some serological tests for the detection of Toxoplasma gondii-specific immunoglobulin are commercially available, better diagnostic tools are needed. The aim of present study was to evaluate the usefulness of the recombinant Toxoplasma gondii SAG1 antigen for the recognition of toxoplasmosis by ELISA.

Methods

This study was conducted in Cellular and Molecular Biology Research Centers, Shahid Beheshti University, M.C., Tehran, Iran in 2008-2009. Surface antigen 1 (SAG1), a tachyzoite stage-specific protein, was subcloned into an expression vector and was subsequently transformed into BL21 (DE3) pLysS competent bacterial cells. After inducing expression of the recombinant antigen, the protein product was purified using Ni-affinity chromatography. The immunoreactivity of recombinant SAG1 (rSAG1) was analyzed by SDS-PAGE and western blotting. The reactivity of the rec-SAG1 protein was evaluated using an ELISA.

Result

Sensitivity and specificity of the generated recombinant-ELISA (rec-ELISA) compared to a commercially available ELISA (com-ELISA) were 88.4% and 88%, respectively.

Conclusion

Recombinant SAG1 produced in E. coli is a promising antigen that can be used in diagnostic assays for the detection of specific antibodies against T. gondii.     

Validation of PCR Assay for Identification of Sarcoptes scabiei var. hominis

Background

Infestation of the skin by the “itch mite” Sarcoptes scabiei var. hominis results in a contagious skin infection in humans called “scabies”. By resolving morphology issues, the present study was designed to be acquainted with itch mite by molecular markers.

Methods

The mite samples were collected from scabies patients by visiting government hospitals of twin City, Pakistan. For successful molecular detection approach, preparation of Sarcoptes mite DNA by commercial DNA extraction kit method. Furthermore, two primers i.e. Sarms 15 F/R and 16S D1/D2 were used to amplify target sequence by using PCR. The amplified products were then separated by agarose gel, electrophoresis and analyzed after staining and visualizing in UV transilluminator.

Results

Analysis of PCR product showed one specific band of 178 bp with primer Sarms 15 F/R, while, with primer 16S D1/D2 bands of 460 bp and 600 bp were observed on 2% agarose gel. The appearance of different band of 600 bp revealed that it might be due to heteroplasmy state present in the Pakistani Sarcoptes mites population.

Conclusion

Current study adds validity to the claim that PCR is more accurate, specific and sensitive in the detection of the ectoparasites even in smallest amount.     

Larva Migration and Eosinophilia in Mice Experimentally Infected With Gnathostoma spinigerum

Background

Gnathostoma spinigerum causes larva migran in human which is endemic in Southeast Asia. Information regarding larva migration is limited. In this study, we investigated the parasite migration by recovery of worms from the whole body of mouse after oral infection with advanced third stage larvae (AL3). The percentage of blood eosinophils was examined in parallel.

Methods

Mice were orally infected with AL3 and histological study of organs was investigated in order to study the migration of AL3, along with blood eosinophilia.

Results

At 1 hr post infection (PI), the larvae remained in the stomach, thereafter at 3, 5, 7, 10 and 24 hr PI; they were recovered from various organs including liver, mesentery, esophagus, diaphragm, lung, heart and dorsal fat. At day 15 PI, they were mostly found in muscles (76.47%). The average worm recovery (5 months) was 78.03%. The worms were found in the liver at every time point. Larva encystment was detected. There was a significant difference in blood eosinophils between the 8 larvae- (average 9.33% + 6.25%) and the 15 larvae-infected groups (average 22.66% + 11.03%). Surprisingly, the blood eosinophils (average 19.00% + 2.92%) were not higher in the higher infective dose- group (25 larvae).

Conclusion

Liver was involved by G. spinigerum throughout the study. We detected larva encystment which had never been reported in human gnathostomiasis. The highest percentage of eosinophil occurred during the invasive stage.     

Urinary Schistosomiasis in Urban and Semi-Urban Communities in South-Eastern Nigeria

Background

In view of the massive rural-to-urban migration in Nigeria, investigations on transmission of urinary schistosomiasis were carried out in urban and semi-urban communities in Nike Lake area of Enugu State, Nigeria.

Methods

Urine samples of school children were tested for micro-haematuria using reagent strips followed by microscopic examination for Schistosoma haematobium eggs. Water contact sites were also identified and sampled for snails.

Results

The overall prevalence of S. haematobium eggs in school children was 4.64%. The mean intensity of infection was 1.14 ± 0.41 eggs/10ml urine. Males had insignificantly higher prevalence and intensity of S. haematobium infection than females. The youngest age group (4-7 years) had no infection. The prevalence of micro-haematuria (6.2%) was higher than that of microscopy, and this correlated positively with prevalence (r = 0.65, P < 0.01) and intensity (r = 0.50, P < 0.01) of the infection. Potential intermediate host of human shistosome collected were: Bulinus globosus, B. senegalensis and Biomphalaria pfeifferi. However, only B. globosus shed cercariae of S. haematobium, with a snail infection rate of 0.73%. Transmission was in the dry season coinciding with the drying of wells.

Conclusion

The results revealed that urinary schistosomiasis is prevalent, and that B. globosus and not B. truncatus as previously reported is the main intermediate host of urinary schistosomiasis in this part of Enugu State.     

Immunogenicity of MHC Class I Peptides Derived from Leishmania mexicana Gp63 in HLA-A2.1 Transgenic (HHDII) and BALB/C Mouse Models

Background

Leishmania is an intracellular parasite infecting humans and many wild and domestic animals. Recent studies have suggested an important role for cytotoxic T cells against Leishmania. Peptide-based vaccines targeting short sequences derived from known immunogenic proteins have been shown to elicit cellular immune responses against disparate pathogens.

Methods

We predicted four HLA-A2 peptides derived from L. mexican/major gp63 and tested these in HHD II mice, as well as four peptides for mouse MHC class I from the same proteins tested in BALB/ mice.

Results

The results revealed immunogenicity for three of the four peptides predicted for HLA-A2. Immunisation with these peptides, along with IFA, induced CTL responses detected by standard 4-hour cytotoxicity assay and significantly upregulated the production of IFN-γ. When HHDII mice were injected IM with L. mexicana gp63 cDNA and splenocytes were restimulated with blasts loaded with the immunogenic peptides, two of the peptides were able to induce significant level of IFN-γ detected by ELISA. None of the peptides predicted for Balb/c mouse MHC class I elicited CTL activity or significantly upregulated the IFN-γ.

Conclusion

The results may help in developing a peptide-based vaccine, which can be applied alone or in combination with drugs against Leishmania.     

Genetic Variation and Selection of Domain I of the Plasmodium vivax Apical Membrane Antigen-1(AMA-1) Gene in Clinical Isolates from Iran

Background

Apical Membrane antigen 1 (AMA-1) is positioned on the surface of merozoite and it may play a role in attack to red blood cells. The main aim of present study was to determine the genetic variation, as well as, to detect of selection at domain I of AMA-1 gene Plasmodium vivax isolates in Iran.

Methods

Blood samples were collected from 58 patients positive for P. vivax, mono infection and the domain I of AMA-1 gene was amplified by nested PCR and then sequenced.

Results

A total 33 different haplotypes were identified among 58 Iranian sequences. The 23 new haplotypes were determined in this study that was not reported previously in other regions of the world. There were totally observed 36 point mutations at the nucleotide level in the analyzed sequences. Sequences analyses indicated 25 amino acid changes at 20 positions in which 5 sites demonstrated thrimorphic polymorphism and the others were dimorphic in the domain I of the Iranian PvAMA-1 isolates.

Conclusion

Our findings indicated relatively high level of allelic diversity at the domain I of PvAMA-1 among P.vivax isolates of Iran. Since, PvAMA-1 is considering as vaccine candidate antigen, these data provide valuable information for the development of a PvAMA-1 based malaria vaccine.     

Inhibition of Murine Systemic Leishmaniasis by Acetyl Salicylic Acid via Nitric Oxide Immunomodulation

Background

The purpose of this study was to evaluate antileishmanial effects of ASA via NO pathway in Leishmania major infected Balb/c mice. Moreover, toxicity and pathological consequences of ASA administration were investigated.

Methods

Balb/c mice were infected with L. major and ASA was inoculated orally after lesion appearance for its ability to modulate NO and to modify Leishmania infection in host, in order to evaluate the effects of NO production on size and lesion macroscopy, delay of lesion formation and proliferation of amastigotes inside macrophages. Liver, spleen, and lymph nodes were also studied as target organs to detect amastigotes. In addition, plasma was investigated for NO induction using Griess microassay.

Results

ASA increased NO production in plasma of both naïve and Leishmania test groups at the ultimate of the experimental period. A decline was observed in proliferation of amastigotes inside macrophages of test group when compared with control one. ASA reduced lesion size, inhibited Leishmania visceralisation in spleen, lymph node, and decreased hepato/splenomegaly in ASA treated animals.

Conclusions

Some antileishmanial effects of ASA by NO-modulation were indicated during systemic leishmaniasis in mice. Despite slight effects on lesion size, ASA decreased parasite visceralization in target organs and declined their proliferation inside macrophages. Therefore, ASA may be indicated to inhibit systemic leishmaniasis via NO pathway in mice model.     

Development of 116 kDa Fraction for Detecting Experimental Toxoplasma gondii Infections in Mice

Background

Serological diagnosis of Toxoplasma gondii infection using crude antigens may not be more accurate. To increase the diagnostic potency of antigens, isolation of their immunogenic fractions could be useful. The current research adopted to obtain an affinity isolated fraction from RH strain using CNBr Sepharose 4B column coupled with infected mice sera helping in detection of IgM and IgG of toxoplasmosis due to RH strain and other strains.

Methods

The isolated fraction was characterized by SDS-PAGE. Moreover, the diagnostic potency of the fraction was assessed by indirect ELISA in mice experimentally infected with RH strain and two other local strains; one of sheep origin and the other of human origin.

Results

The fraction was found to be consisted of a single band of 116 kDa compared with 17 bands ranged from 116 to 16 kDa associated with crude extract. The fraction proved potent diagnostic potentials of acute and chronic mice toxoplasmosis. Where it was detected both IgM and IgG antibodies as early as two days and as late as 2 months post experimental infection with any of the three strains. The level of detected IgM and IgG by RH fraction was higher in mice infected with RH strain than with local strains except IgM due to sheep strain parasite.

Conclusions

The 116 kDa fraction of T. gondii tachyzoites can be considered as a candidate in improving of serodiagnosisof Toxoplasma infections.     

Immunotherapy Using Autoclaved L. major Antigens and M. vaccae with Meglumine Antimoniate,for the Treatment of Experimental Canine Visceral Leishmaniasis

Background

To evaluate immunotherapy against canine visceral leishmaniasis, Leishmania major antigen and heat-killed Mycobacterium vaccae (SRL172) were used as stimulators of immune defense mechanisms and the results were compared with standard chemotherapy meglumine antimoniate.

Methods

Nineteen mongrel dogs aging 1-3 years old were used in this experiment. Infection was carried out in 15 out of 19 dogs using L. infantum, isolated from a naturally infected poly-symptomatic dog.

Results

All the cases showed positive serologic results by direct agglutination test during 30-60 days following inoculation. In the first group, which was under chemotherapy (Glucantime^R), one of the members showed recurrence of the disease despite rapid effect of the therapeutic protocol. Immunotherapy using SRL172 caused complete cleaning of the parasite in group 2, but the speed was less than Glucantime. Immunotherapy using L. major antigen combined with M. vaccae in group 3 and combine administration of immunotherapy and chemotherapy in group 4 both were with relapsing of one case in each group. Group 5 and 6 were consisted of positive and negative control dogs, respectively.

Conclusion

Immunotherapy seems to be an adjuvant in treatment of canine leishmaniasis but it needs more investigation for final confirmation.     

Expression and Purification of P43 Toxoplasma gondii Surface Antigen

Background

Toxoplasma gondii is an obligate intracellular protozoan parasite, capable of infecting all species of mammals including man. Congenital toxoplasmosis is more important during pregnancy for the first time. In this study we expressed and purified P43 Toxoplasma gondii tachyzoite and bradyzoite specific surface antigen.

Methods

The recombinant pGEMEX-1 contained Toxoplasma P43 coding sequence was transformed into E. coli and mass cultured in LB medium contained 100 μg/ml ampicillin at 37°C over night. The T7 promoter was induced by 1mM isopropyl-1-thio-ß-D-galactopyranoside (IPTG. Recombinant protein was purified by affinity chromatography and confirmed by gel diffusion dot blot and western blot,-using specific anti Toxoplasma antibodies.

Results

Recombinant plasmid was induced by IPTG and analyzed by SDS-PAGE. Recombinant protein was confirmed by Western-blot and dot blot using anti human Toxoplasma antibody.

Conclusion

Recombinant Toxoplasma P43 was produced successfully.     

Plasmodium berghei ANKA Infection in ICR Mice as a Model of Cerebral Malaria

Background

Animal models with various combination of host-parasite have long been employed to study malaria pathogenesis. Here, we describe the combination of Plasmodium berghei ANKA infection in inbred ICR mice as a model of cerebral malaria (CM).

Methods

Infection in mice was initiated by intraperitoneal injection of 2 x 10⁷ (0.2ml) parasitized red blood cells (PRBCs).

Results

This model can produce a severe degree of infection presented by the high degree of parasitaemia followed by death 6-7 days post infection. Severe anemia, splenomegaly, hepatomegaly and discolourations of major organs were observed. Histopathological findings revealed several important features mimicking human CM including, microvascular sequestration of PRBCs in major organs, particularly in the brain, hypertrophy and hyperplasia of the kupffer cells in the liver, pulmonary edema and hyaline membrane formation in the lungs and haemorrhages in the kidney''s medulla and cortex. Proinflammatory cytokines TNFα, IFNγ, IL-1, IL-6 and IL-18, and anti-inflammatory cytokine IL-10 were all found to be elevated in the plasma of infected mice.

Conclusion

This model can reproduce many of the important features of CM and therefore can be used as a tool to advance our understanding of the disease pathogenesis.     

Strongyloides stercoralis: The Most Prevalent Parasitic Cause of Eosinophilia in Gilan Province,Northern Iran

Background

Eosinophilia occurs in a wide variety of situations such as parasitic infections, allergic disorders, and malignancies. Most cases of eosinophilia of parasitic origin, especially those with a tissue migration life cycles consists of human infections by helminth parasites. The aim of present study was to determine the parasitic causes of eosinophilia in patients in a major endemic area of human fascioliasis in Gilan Province, northern part of Iran.

Methods

One hundred and fifty patients presenting with an elevated eosinophilia attending infectious disease clinics with or without clinical symptoms, were examined. After clinical history evaluation and physical examination, coprological examinations were performed using the formalin-ether and the Kato-Katz techniques for detection of Fasciola sp. and intestinal parasites.

Results

Forty two percent of patients were infected with S. stercoralis, nine (6%) were found to be infected with Fasciola sp. while only a single patient (0.7%) were infected by Ttrichostrongylus sp.

Conclusion

Local clinicians in Gilan may consider eosinophilia as a suggestive indication for diagnosis of human fascioliasis, especially when microscopic stool and/or serological tests are negative. Based on the results, local physicians should consider S. stercoralis as the potential causes of eosinophilia in patients with elevated eosinophilia.     

In Vitro and In Vivo Potential of RH Strain of Toxoplasma gondii (Type I) in Tissue Cyst Forming

Background

Based on recent studies, there are controversial reports on the capacity of tissue cyst forming of Toxoplasma gondii RH strain. In this study, the capacity was evaluated by in vivo and in vitro experiments.

Methods

RH strain was subcutaneously inoculated to ten Wistar rats. After one month, their blood, brain, tongue and diaphragm were collected and evaluated by MAT, PCR, pathological and bioassay methods. The parasite was cultivated in the cell monolayer. To change to bradyzoite, the media pH was altered to 6.8. Biological aspect of the bradyzoites was evaluated by incubation in acidic pepsin and it''s inoculation in ten BALB/c mice.

Results

All rats showed antibodies to Toxoplasma at titers ≥1:320 but no DNA and tissue cyst were detected in the tissues. Following intraperitoneal inoculation of rats’ brain homogenate into BALB/c mice, no infection was established in none of the animals. During presence of cell culture, in acid media for a 3-5 days period, cyst-like structures were noticed when they were stained with PAS. The visible bradyzoites in the cysts that were incubated in acid pepsin medium were not able to kill any mice.

Conclusion

This study confirmed that Iranian RH strain has lost the potential of tissue cyst forming in rats and bradyzoites cultivated in cell culture lost their resistance to acidic condition, so this strain can be a candidate for future vaccine researches.