Brain tissue cysts in infected mice with RH-strain of Toxoplasma gondii and evaluation of BAG1 and SAG1 genes expression

Toxoplasma gondii is an obligate intracellular parasite that infects humans at high prevalence rates. The virulent RH strain of T. gondii is generally considered to have lost its cyst forming capacity. This study performed to obtain tissue cysts in mice infected with tachyzoites of RH strain treated with sulfadiazine [SDZ]. It provides the opportunity to analyze the conversion of tachyzoite to bradyzoite stage of the RH strain, followed by stage-specific gene-expression analyzing. Two groups of Swiss-Webster and BALB/c mice were infected subcutaneously with 10[4] tachyzoites of T. gondiiy RH strain and given SDZ [300 mg/l] with NaHCO3 [5 g[-1]] in drinking water from day 1 to day 14 post infection [p.i]. The infected mice were sacrificed on day 50 post infection. Their brains were removed and the numbers of tissue cysts were microscopically counted. Total RNA was extracted from brains and cDNA synthesis was carried out. Finally, RT-PCR [Reverse transcription PCR] was used to detect the expression of bradyzoite [BAG[1]] and tachyzoite [SAG[1]] specific genes during tachyzoite / bradyzoite stage conversion. Sixty five percent of all infected mice were survived. Cysts were detectable in mice brain [45%] on day 50 p.i. Also RT-PCR of the brain samples was positive for SAG1 and BAG1. It seems that conversion of tachyzoites to bradyzoites in brain of mice undergoing SDZ was not completed until 50 days after inoculation

Genotyping of hydatid cyst isolated from human and domestic animals in ilam province, western Iran Using PCR-RFLP

Hydatidosis or cystic hydatid disease is one of the most important diseases in human and animals. Identification of strains is important for improvement of control and prevention of disease. The aim of this study was to determine the strains isolated from human and domestic animals in Ilam Province, Iran, using PCR-RFLP method. Respectively, 30 and 4 animal and human hydatid cysts were collected from different slaughterhouses and hospitals of the province. Protoscolices were separated and their DNA genome was extracted by extraction kit. rDNA-ITS1 of each isolated samples was duplicated by BD1 [Forward] and 4s [Reverse] Primers. PCR products were studied by electrophoresis and then were digested using TaqI, HpaII, Rsal and Alul restriction enzymes. RFLP products were studied using electrophoresis on 1% agar gel. A fragment of l000bp was produced from amplification of rDNA-ITS1 of protoscolices using PCR method. After digestion of PCR product by Alul enzyme, 200bp and 800bp, by Rsal, 655bp and 345bp and by HpaII 700bp and 300bp sizes were obtained. TaqI enzyme had no change in fragment size and it remained l000bp. Considering the method. Ham strains was specified as E. granulosus sensu stricto [G1-G3]. Although sheep strain [G1] is dominated in human and different animal in Iran and the world, but more efforts should be done to clarify the true genotype of Ilam strains specified as E. granulosus sensu stricto [G1-G3]

Helminth parasites of Rhombomys opimus from golestan province, northeast Iran

The aim of the study was to determine the helminthic species occurring in great gerbil Rhombomys opimus collected from Maraveh Tappeh, Golestan Province, northeast Iran. During 2010-2011, a total of 77 R. opimus were captured from rural areas of Maraveh Tappeh, Golestan Province, using Sherman live traps and examined for infectivity with any larva or adult stages of helminthic parasites. Overall, 63 R. opimus [81.8%] were found infected with different helminthic species. The rate of infectivity with each species was as follows: Trichuris rhombomidis 31.2%, Trichuris muris 32.5%, Trichuris spp. 10.4%, Syphacia muris 2.6%, Dipetalonema viteae [Acanthocheilonema viteae] 37.7%, Skrjabinotaenia lobata 15.6%, Hymenolepis [=Rodentolepis] nanafraterna 5.2%, and Taenia endothoracicus larva 1.3%. R. opimus is host for several species of cestodes and nematodes in the study area. The high rate of infectivity with D. viteae indicates the susceptibility of these gerbils to this filarial nematode. Synchronous infections occurred up to four species of helminthes in one host

Geographical distribution of Leishmania species of human cutaneous leishmaniasis in Fars province, southern Iran

The goal of this study was to know the identity of Leishmania species responsible of cutaneous leishmaniasis [CL] in Pars Province, southern Iran. Five counties of Shiraz, Firouz Abad, Ghir-Karzin, Farashband and Larestan were prospected. Forty-four patients exhibiting cutaneous lesions were selected. Samples collected on skin le sions were examined both microscopically [after Giemsa staining] and molecularly [after PCR-RFLP]. On the 44 examined patients, 39 exhibit Leishmama sp. by microscopical examination, all confirmed by PCR. For five patients with negative microscopical examination, PCR was positive for three of them. Among these 42 positive samples, 3 [7%] were infected by L tropica and 39 [93%] by L. major. Leishmania major is the most prevalent species in prospected area and L. tropica occurs in Shiraz and Ghir-Karzin counties

Seroepidemiologcal investigation of visceral leishmaniasis in stray and owned dogs in alborz province, central Iran using direct agglutination test

The aim of present study was to determine the seroprevalence of canine visceral leishmaniasis [CVL] among stray and owned dogs in Kouhsar district of Alborz Province, central Iran. The study was performed from March 2011 to July 2011 using Direct Agglutination Test [DAT]. Three hundred and thirty seven dogs including 257 stary and 80 owned dogs were selected by random sampling. The agreement between serological data and sex, age, life style of dogs and clinical signs were assessed by Chi-square. DAT showed that from 337 serum samples collected from owned and stray dogs, 12sera [3.6%] were positive. The seroprevalance was 10% [8/80] among owned dogs and 1.6% [4/257] among stray dogs. A significant difference in seroplevalance was seen between owned and stray dogs [P = 0.01]. The highest seroprevalence rate [14%] was observed among the ownership dogs of 5 years old and above. Statistical analysis revealed significant relation between seroprelvalence and age [P= 0.02]. There was no statistically significant relation between male [6.3%] and female [2.2%] seroprevalence [P= 0.085]. This survey indicates the importance and necessity of serologic screening of visceral leishmaniasis in human and dogs in Kouhsar district

Gene regulation of pteridine reductase 1 in leishmania promasti-gotes and amastigotes using a full-length antisense construct

Pteridine metabolic pathway is unusual features of Leishmania, which is necessary for the growth of parasite. Leishmania has evolved a complex and versatile pteridine salvage network which has the ability of scavenging a wide area of the conjugated and unconjugated pteridines especially folate and biopterin. In this study, we focus on the inhibition of ptr1 gene expression. L. major ptr1 gene was cloned into pcDNA3 and digested using KpnI and BamHI. The gene was subcloned so that antisense will transcribe and called pcDNA-rPTR. Leishmania major was cultured and late logarithmic-phase promastigotes were harvested. The promastigotes were divided into two groups. One group was transfected with 50 micro g of pcDNA-rPTR, whereas the other group was transfected with pcDNA3. Transfected cells were cultured and plated onto semi-solid media. Mouse pritonean macrophages were transfected using pcDNA-rPTR-tansfected promastigotes. Western blotting was performed on mouse transfected pritonean macrophages and extracts from transfected promastigotes of L. major using a L. major ptr1 antibody raised in rabbits. The PTR1 protein was not expressed in pcDNA-rPTR- tansfected promastigotes and mouse macrophage transfected with pcDNA-rPTR- tansfected promastigotes. This approach might be used to study the pteridine salvage pathway in Leishmania or to assess the possibility of using gene expression inhibition in the treatment of leishmaniasis.

Serum levels of zinc, copper, vitamin B12, folate and immunoglobulins in individuals with giardiasis

Giardia lamblia is one of the most important intestinal parasites. The aim of this study was to measure serum levels of IgA, IgE, zinc, copper, vitamin B12 and folate in individuals with giardiasis in comparison to normal subjects. The study was carried out among 49 Giardia positive and 39 age and sex matched healthy volunteers. Examination of stool samples was done by direct wet smear and formol-ether concentration method. Serum samples were obtained for further laboratory examination. IgA levels were measured by Single Radial Immune Diffusion [SRID]. IgE levels were measured by ELISA kit. Zinc and copper levels was measured by Ziestchem Diagnostics Kit and colorimetric endpoint-method respectively. Vitamin B12 and folate levels were measured by DRG Diagnostics Kit and Enzyme Immunoassay method respectively. All data were analyzed using SPSS version 17. There was a statistically significant difference in IgA, IgE, copper and zinc levels between positive and negative groups [P<0.05]. There was no significant difference between vitamin B12 and folate levels between the two groups. Mean values of Giardia positive and negative groups for IgA were 309.26 and 216.89 mg/dl, IgE 167.34 and 35.49 IU/ml, copper 309.74 and 253.61 micro g/dl and zinc 69.41 and 144.75 micro g/dl respectively. The results showed levels of IgA may correlate more closely with giardiasis than IgE. Regarding trace elements, giardiasis elevated serum copper levels, while it decreased serum zinc. Finally, there was no significant difference in serum levels of vitamin B12 and folic acid between the two groups

Seroprevalence of canine visceral leishmaniasis in asymptomatic dogs in Iran

Clinically infected dogs have been identified as the main reservoir hosts of visceral leishmaniasis [VL] caused by Leishmania infantum. Recently asymptomatic infected dogs were regarded to be as important as clinically ill dogs. This study was aimed to determine the seroprevalence of L. infantum infection among asymptomatic dogs in the southwest and central parts of Iran and to investigate possible risk factors associated with this infection. 548 serum samples were collected from dogs in three Iranian provinces and subjected to direct agglutination test [DAT] in dilutions of 1:80 to 1:20480. Fifty three [9.67%] of the dogs had detectable anti-L. infantum antibodies at dilutions of >/= 1:80. Living status of the dogs [household or free roaming] was a potential risk factor for the infection; seroprevalence was significantly higher in free roaming dogs [P<0.001]. Dogs of more than 2-year-old had a significantly higher infection rate in comparison with younger dogs [P<0.001]. No significant statistical differences were seen between seroprevalences of the male and female dogs. The results of this study show relatively high sero-prevalence of L. infantum infection in evaluated regions and higher seroprevalence in old and free roaming dogs, which shows the importance of environmental contamination and access of the dogs to the other reservoir hosts

Production and evaluation of Toxoplasma gondii recombinant surface antigen 1 [SAG1] for serodiagnosis of acute and chronic toxoplasma infection in human sera

The assays currently available for the detection of specific anti-Toxoplasma antibodies may vary in their abilities to detect serum immunoglobulins, due to the Lack of a purified standardized antigen. The aim of this study was evaluation the recombinant Toxoplasma gondii SAG1 antigen for the serodiagnosis of acute and chronic toxoplasmosis. This study describes an ELISA using recombinant SAG1 for detection of IgM and IgG antibodies against Toxoplasma gondii in human sera. Genomic DNA of T. gondii [RH Strain] was isolated and PCR reaction was performed. Recovered DNA was cloned into PTZ57R cloning vector. The recombinant plasmid was detected by restriction analysis. The SAG1 gene was subcloned in the pET- 28a expression vector. Protein production was then induced with 1 mM isopropyl-D - thiogalactopyranoside [IPTG]. A total of 204 sera were tested using a commercial IgG and IgM ELISA kit [Trinity, USA] as gold standard prior to testing them with the recombinant antigen. Tested sera were divided into the following groups:[a] The 74 T. gondii IgG positive [b] 70 T.gondii IgM positive [c] 60 sera who had no serological evidence of toxoplasmosis as negative sera.To determine the specificity of the test, we used other parasitic diseases including echinococusis [N=5], malaria [N=14], leishmaniasis [N=7],fasciolasis [N=4], sterengyloidiasis [N=1]. Sensitivity and specificity of the generated recombinant IgG ELISA in comparison with commercial ELISA [Com ELISA] were 93% and 95%, and the sensitivity and specificity of the generated recombinant IgM ELISA were 87% and 95% respectively. The results acquired here show that this antigen is useful for diagnostic purposes and could be replaced by lysed, whole cell antigens for diagnosis of chronic toxoplasmosis

Seroprevalence of human hydatidosis using ELISA method in Qom Province, Central Iran

The objective of this study was to determine the prevalence of cystic echinococcosis [CE] in Qom Province, central Iran using ELISA test. Overall, 1564 serum samples [800 males and 764 females] were collected from selected subjects by randomized cluster sampling in 2011-2012. Sera were analyzed by ELISA test using AgB. Before sampling, a questionnaire was filled out for each case. Data were analyzed using Chi-square test and multivariate logistic regression for risk factors analysis. Seropositivity was 1.6% [25 cases]. Males [2.2%] showed significantly more positivity than females [0.9%] [P= 0.03]. There was no significant association between CE seropositivity and age group, occupation, and region. Age group of 30-60 years encompassed the highest rate of positivity. The seropositivity of CE was 2.1% and 1.2% for urban and rural cases respectively. Binary logistic regression showed that males were 2.5 times at higher risk for infection than females. Although seroprevalence of CE is relatively low in Qom Province, yet due to the importance of the disease, all preventive measures should be taken into consideration

Comparative proteomics study on meglumine antimoniate sensitive and resistant Leishmania tropica isolated from Iranian anthroponotic cutaneous leishmaniasis patients

In order to define the protein expressional changes related to the process of meglumine antimoniate resistance in anthroponotic cutaneous leishmaniasis [CL], we performed a comparative proteomics analysis on sensitive and resistant strains of Leishmania tropica isolated from Iranian CL patients. Cell proteins were analysed with 2-dimensional electrophoresis and differentially expressed proteins were identified by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry. Image analysis of the matched maps identified 7 proteins that were either over- or down-expressed: activated protein kinase c receptor [LACK], alpha tubulin [X2], prostaglandin f2-alpha synthase, protein disulfide isomerase, vesicular transport protein and a hypothetical protein. The study shows the usefulness of proteomics in identifying proteins that may express differences between sensitive and resistant L. tropica isolates

Prevalence of trichomonas vaginalis infection in Hamadan city, Western Iran

Infection with Trichomonas vaginalis is one of the most common sexually transmitted diseases [STDs] in humans. The prevalence of infection in Iran has been reported between 2 to 8%, depending on deferent socio-cultural conditions. This study aimed to determine the prevalence of T. vaginalis in women referred to gynecologic clinics in Hamadan city, West of Iran. This descriptive cross-sectional study was conducted on 750 women who referred to Gynecologic clinics in Hamadan from November 2010 to July 2011. Vaginal samples were obtained from them and examined by wet mount and culture methods for the detection of T. vaginalis. Sixteen out of 750 vaginal swab specimens [2.1%] were culture positive for T. vaginalis and 13 of these positive specimens [1.7%] were wet mount positive. Only 12 of 42 patients who were clinically diagnosed as having T. vaginalis infection, confirmed by culture method. Five hundred and fifty of the participants women [73.3%] had at least one of signs and symptoms of trichomoniasis. No statistical correlation was observed between clinical manifestations and parasitological results [p>0.05]. This study showed low prevalence of T. vaginalis infection in the study population. Since clinical signs of trichomonal vaginitis are the same of other STDs, a confirmatory laboratory diagnosis is necessary. Wet smear as well as culture are sensitive for detection of T. vaginalis

Inhibition of leishmania major PTR1 gene expression by antisense in escherichia coli

Protozoa related to Trypanosome family including Leishmania, synthesize enzymes to escape from drug therapy. One of them is PTR1 that its enzymatic activity is similar to dihydrofolate reductase [DHFR]. Dihydrofolate reductase - thymidylate synthase has a major role in DNA synthesis, if it is inhibited, the result would be the death of parasite. Since PTR1 activity is similar to DHFR, causes the decrease of inhibition effect of drug. The aim of this study was inhibition of Iranian L. major PTR1 expression with mRNA antisense in prokaryotic system as an approach to appear of the drugs therapeutic effects more. PTR1 gene was ligated to pACYCDuet-1 and pcDNA3 plasmids as sense and antisense plasmids, respectively. Simultaneously transfer of sense and antisense plasmids was done in E. coli strain M15. SDS-PAGE and western blot analysis were carried out to analyze the expression. Sense and antisense plasmids were prepared and confirmed by restriction analysis and PCR then simultaneously transfer of them was done. SDS-PAGE and western blot analysis showed PTR1 gene was inhibited by mRNA antisense in bacterial cells. Expression of PTR1 gene in sense plasmid was inhibited successfully by antisense plasmid

Epidemiological aspects of visceral leishmaniasis in Baft district, Kerman province, southeast of Iran

Visceral leishmaniasis [kala-azar] is an endemic disease in some areas of Iran. A cross-sectional study was conducted for sero-epidemiological survey of visceral leishmaniasis [VL] in Baft district from Kerman Province, southeast of Iran. Blood samples were collected from children up to 12 years old and 10% of adult population from Baft villages with a multi-stage randomized cluster sampling. In addition, blood samples were collected from 30 domestic dogs from the same areas. All the collected blood samples were tested by direct agglutination test [DAT] for the detection of anti-Leishmania antibodies in both human and dog using the cut-off value of >/= 1:3200 and >/= 1:320, respectively. Parasitological, molecular, and pathological were performed on infected dogs. Chi-square and Fisher exact tests were used to compare sero-prevalence values. From 1476 collected human serum samples, 23 [1.55%] showed anti-Leishmania antibodies at titers of 1:800 and 1:1600 whereas 14 [0.95%] showed anti-Leishmania infantum antibodies at titers of >/= 1:3200. No statistically significant difference was found between male [1.18%] and female [0.69%] sero-prevalence [P=0.330]. Children of 5-8 years showed the highest sero-prevalence rate [3.22%]. Seven out of 30 domestic dogs [23%] showed anti-Leishmania antibodies at titers >/= 1:320. Leishmania infantum was identified in five infected dogs by nested -PCR assay. It seems that visceral leishmaniasis is being endemic in southern villages of Baft district, southeast of Iran

Rapid detection of Toxoplasma gondii antigen in experimentally infected mice by dot- ELISA

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. Sixty-three BALB/c mice were injected intra-peritoneal with 5x10[3] 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. 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. 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

problem of mixing up of leishmania isolates in the laboratory: suggestion of ITS1 gene sequencing for verification of species

Leishmaniasis is endemic in Iran. Different species of Leishmania [L.] parasites are causative agents of this disease. Correct identification of Leishmania species is important for clinical studies, prevention, and control of the diseases. Mix up of Leishmania isolates is possible in the laboratory, so there is need for verification of species for isolates of uncertain identity. Different methods may be used for this purpose including isoenzyme electrophoresis and molecular methods. The isoenzyme electrophoresis, due to its drawbacks, is feasible only in specialized laboratories while molecular methods may be more feasible. The aim of this research was to study the application of the internal transcribed spacer 1 [ITS1] sequencing method, in comparison to isoenzyme electrophoresis method, for verification of Leishmania species. Six Leishmania isolates were received from different research institutions in Iran. The species of these isolates were known by donating institution according to their isoenzyme profile. The species of these isolates were re-identified in Pasteur Institute of Iran by PCR amplification of ITS1 followed by sequencing and comparison of these sequences with Leishmania sequences in GenBank. Isoenzyme electrophoresis confirmed the confirmation of the results of ITS1. ITS1 sequence showed that some isolates were mixed up or contaminated with Crithidia. Isoenzyme electrophoresis confirmed the results of ITS1 sequences. ITS1 Sequencing is relatively more feasible than the traditional isoenzyme electrophoresis method and is suggested for verification of Leishmania species

Seroprevalence of Toxoplasma gondii infection in dogs in Tehran, Iran

Toxoplasma gondii infects a wide range of animals; felines are definitive hosts and other animals including the dogs are intermediate hosts. The aim of this study was to determine the seroprevalence of T. gondii infection in dogs in Tehran, capital of Iran and to investigate possible associated risk factors. Three hundreds ninety six serum samples were collected during 2007-8 from the dogs. Collected samples were tested using an indirect fluorescent antibody test [IFAT] in dilutions of 1:16 and more. All procedures were carried out in Shahrekord University, Iran. All the data were analyzed using SPSS software, qui square test with confidence interval of 0.95. From evaluated samples, 89 [22.47%] were positive in titers of at least 1:16. further evaluations in other dilutions showed positive results in dilutions of maximum 1:16, 1:32, 1:64, 1:128 and 1:256 in 38, 29, 15, 2 and 5 dogs respectively. Investigation of the role of risk factors showed no sex predisposition while infection rate was significantly higher in dogs older than one year old. Living places were of significant importance; infection rate was significantly higher in stray or guard dogs in compare with household dogs [P<0.05]. Relatively high seroprevalence of T. gondii infection in dogs in Tehran shows high environmental contamination. It is recommended that the dogs with suspected clinical signs be tested for T. gondii infection

[Establishment and integration of a visceral leishmaniasis surveillance system in the primary health care system and its evaluation, Meshkin-Shahr District, Iran]

Visceral leishmaniasis is a systemic parasitic disease with a high fatality rate in under-5-year-old children. The disease is endemic in some parts of Iran, particularly in the north-west region. In 2001 a visceral leishmaniasis [VL] surveillance system was established for children aged>/=12 years in the primary health system in Meshkin-Shahr District, Ardebil Province, situated in the north-west of Islamic Republic of Iran. All cases with clinical signs and symptoms of VL and confirmed positive by the direct agglutination test [DAT] were referred for physical examination and treatment. The mean annual incidence of VL decreased significantly from 1.88 per 1000 children before [1985-2000], to 0.77 per 1000 child population after [2001-07], the intervention. In the control area with no surveillance, it increased from 0.11 to 0.23 per 1000. Early detection of VL using serological tests and timely treatment of cases can decrease the mortality and morbidity rates of VL in endemic areas

Toxoplasma infection in schizophrenia patients: a comparative study with control group

Schizophrenia is a serious, chronic, and often debilitating neuropsychiatric disorder. Its causes are still poorly understood. Besides genetic and non-genetic [environmental] factors are thought to be important as the cause of the structural and functional deficits that characterize schizophrenia. This study aimed to compare Toxoplasma gondii infection between schizophrenia patients and non-schizophrenia individuals as control group. A case-control study was designed in Tehran, Iran during 2009-2010. Sixty-two patients with schizophrenia and 62 non-schizophrenia volunteers were selected. To ascertain a possible relationship between T. gondii infection and schizophrenia, anti-Toxoplasma IgG antibodies were detected by indirect-ELISA. Data were statistically analyzed by chi- square at a confidence level of 99%. The sero-positivity rate among patients with schizophrenia [67.7%] was significantly higher than control group [37.1] [P <0. 01]. A significant correlation between Toxoplasma infection and schizophrenia might be expected

Disseminated leishmaniasis caused by Leishmania tropica in a puppy from Karaj, central Iran

A 5-month old puppy with muco-cutaneous lesions in the chin, around lips and eyes was examined physically and microscopically for leishmaniasis. Muco-cutaneous lesions containing a large number of amastigotes of Leishmania spp. were observed. Amastigotes were also detected in liver and spleen of the puppy. The animal was positive with Dipstick rK39 kit and high level of anti-Leishmania antibodies was detected by direct agglutination test [DAT]. DNA, Using PCR-RFLP technique extracted from cultured Leishmania promastigotes and L. tropica was identified. This is the first report of concurrent mucosal and visceral involvement of L. tropica in a puppy from Iran