Seroprevalence of Toxoplasma gondii infection in domestic dogs in an area from northwest of Iran: a cross-sectional study using immunodominant surface antigen 1 (SAG1).

Toxoplasma gondii is an obligate intracellular protozoan parasite that infects humans and animals. T. gondii surface antigen 1 (SAG1) is an appropriate antigen with high specificity and sensitivity for the detection of T. gondii infection in humans and animal hosts. The aim of this study was to determine the seroprevalence of T. gondii infection using SAG1 antigen (P30) in ownership dogs in Meshkin-Shahr district in the northwestern Iran. The sera samples were collected from 171 domestic dogs and tested using indirect ELISA (SAG1 antigen). The data were analyzed using SPSS software version 13. From a total of 171 dogs, 82 (48 %) of them were sero-positive. No statistical significant difference was seen between T. gondii infection and gender (P = 0.995). The highest sero-prevalence of rate was observed in >5 years animals; but no statistical significant difference was seen between T. gondii infection and age (P = 0.589). Our findings indicate that Toxoplasma seropositivity rate is high in ownership dogs in northwest of Iran. This is probably due to high exposure to contaminated food, soil, or water sources with sporulated Toxoplasma oocysts.

Molecular analysis of isolates of the cestode Rodentolepis nana from the great gerbil, Rhombomys opimus.

Rodentolepis nana (syn. Hymenolepis nana) is a cyclophyllidean zoonotic enteric parasite with worldwide distribution. In humans, it is more prevalent in children, especially in temperate zones. A morphologically similar species of R. nana occurs in different rodents, including gerbils. In the present study molecular characterization of five isolates of R. nana from Rhombomys opimus in the Golestan Province (n= 2) and Razavi Khorasan Province (n= 3), both in north-eastern Iran, were analysed. After DNA extraction, the internal transcribed spacer 2 (ITS2) region of the R. nana isolates was amplified and sequenced, and genetic variation was analysed. Accordingly, two isolates from the Razavi Khorasan Province showed ITS2 signatures that differed from the isolates from the Golestan Province. The third isolate from Razavi Khorasan did not reveal these specific signatures, but exhibited sparse nucleotide polymorphisms in the ITS2 sequence. The ITS gene is conserved, and variation in this fragment could be an interesting subject for further inspection of existing variation in the genome of R. nana, among different domestic and wildlife host species and from different areas.

Correlation between clinical responses with the drugsusceptibility of parasites in Iranian cutaneous leishmaniasis caused by Leishmania major.

Reviews have shown increasing number of Iranian patients with cutaneous leishmaniasis (CL) who are unresponsive to pentavalent antimonial compounds such as meglumine antimoniate (Glucantime, MA). The present investigation aims to determine the correlation between clinical responses (healing, or non-healing) with susceptibility of Leishmania parasites to glucantime. Initially, in vitro susceptibility of Leishmania parasites was carried out on 93 isolates using macrophage models. Identification of these species was also performed by molecular methods including Nested-PCR and PCR-RFLP. The f indicated that total isolated were L. major. A significant association between the clinical outcome and the in vitro effective concentration 50% (EC50) values was observed. Leishmania derived from patients with non-healing lesions had EC50 values at least 3-fold higher than parasites isolated from lesions of healing patients. By molecular methods, patterns for both sensitive and resistant samples demonstrated restriction band which is related to L. major. The obtained findings in the present study demonstrated that MA-resistant L. major field isolates are now frequent in Iran. Such studies help to find strategies for rapidly diagnosing resistance in order to improve the clinical management of CL.

Interleukin 4 (IL-4) gene promoter polymorphisms in Rhombomys opimus, the main reservoir of zoonotic cutaneous leishmaniasis.

Great gerbils (Rhombomys opimus) are the most common gerbils in center to northeast of Iran as well as central Asia and serve as reservoirs for the zoonotic agents, including Leishmania major, the principal etiologic agent of zoonotic cutaneous leishmaniasis (ZCL). The outcome of L. major infection in gerbils is not uniform. Among several immune-related factors including cytokine genes, the polymorphism in interleukin 4 (IL-4) promoter gene showed a great impact on outcome and pathological symptoms of L. major infection at least in mouse model. In this study gerbils' IL-4 promoter gene polymorphism is assessed. Specific primers were designed to develop a PCR-based assay to amplify IL-4 promoter gene to possibly define IL-4 promoter gene polymorphism in great gerbil populations with a range of Leishmania infection and symptoms collected from different foci of the central, north and northeast regions of Iran. The results showed that the designed primers amplify 689bp of the promoter gene. Sequence analysis of the promoter gene revealed five polymorphic sites assembly six haplotypes among the gerbil populations. Further studies are needed to assess whether or not the five polymorphisms cause different outcome phenotypes following infection with L. major in great gerbils. The data might be used to characterize the immune responses of R. opimus against L. major infection.

Helminth Parasites of Rhombomys opimus from Golestan Province, Northeast Iran.

BACKGROUND: 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. METHODS: 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. RESULTS: 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) nana fraterna 5.2%, and Taenia endothoracicus larva 1.3%. CONCLUSION: 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.

BACKGROUND: The goal of this study was to know the identity of Leishmania species responsible of cutaneous leishmaniasis (CL) in Fars Province, southern Iran. METHODS: 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 lesions were examined both microscopically (after Giemsa staining) and molecularly (after PCR-RFLP). RESULTS: On the 44 examined patients, 39 exhibit Leishmania 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. CONCLUSIONS: Leishmania major is the most prevalent species in prospected area and L. tropica occurs in Shiraz and Ghir-Karzin counties.

Unusual presentation of disseminated cutaneous leishmaniasis due to Leishmania major: case reports of four Iranian patients.

We report four disseminated cutaneous leishmaniasis(DCL) cases referred to leishmaniasis laboratory at the School of Public Health, Tehran University of Medical Sciences with multiple nodular, ulcerative and crusted lesions extended on the face, trunk, and extremities. None of the patients had any complication and historical involvement in their immunological system conditions that suggest as the criteria for DCL. Direct smears of ulcers were positive for Leishmania parasite. The parasite was isolated from the active lesions and identified as Leishmania major (L. major) using PCR-RFLP assay and sequencing analysis.

Field evaluation of permethrin long-lasting insecticide treated nets (Olyset(®)) for malaria control in an endemic area, southeast of Iran.

Long lasting insecticide treated nets (LLINs) have been advocated as an effective tool for prevention and control of malaria. Olyset net was the first LLINs which became commercially available and obtained WHO approval. According to the national strategic plan on evaluation of Olyset net, a field trial was conducted to determine the efficacy of these nets against malaria vectors in an endemic area in the southeast of Iran. Fourteen villages with similar topographical and epidemiological situations were selected and randomly assigned to two clusters of the study: Olyset net and untreated net. Distribution of nets was carried out to cover 100% of the population in Olyset net and untreated net cluster. Anopheline mosquitoes were collected monthly using different WHO standard methods in both areas to determine their abundance, feeding pattern and resting behaviour. Human blood index was determined using ELISA test. Additionally, Olyset nets were evaluated for their biological activity using WHO cone bioassay test by susceptible colony of Anopheles stephensi (Beech strain) and then for insecticide residues by employing high performance thin layer chromatography. Malaria incidence was measured by passive and active case detection from all study population. In total 2115 adult anopheline mosquitoes were collected and identified using morphological characters. They comprised of seven species: Anopheles dthali (Liston), A. culicifacies (Giles), A. stephensi (Liston), A. superpictus (Grassi), A.fluviatilis (James), A. moghulensis (Christophers) and A. turkhudi (Liston). A. dthali, A. culicifacies and A. stephensi were most prevalent species in both areas. In the Olyset net study area, there was a significant reduction of 41.1%, 54.4%, 59.39% and 64.1% in the indoor-resting density of A. culicifacies, A. stephensi, A. dthali and A. superpictus, respectively, with an overall reduction of 39.3% in total mosquitoes in comparison with untreated net area. A significant reduction was also observed in human blood index of vector species in the Olyset net villages. Bioefficacy test results of Olyset nets showed that the median knockdown time was 1.48 and 3.25min, while the average mortality rate was 100% and 72.3%±7.07 in baseline and after 1 year of intervention, respectively. The average permethrin content reached to 68.31% (683.1mg/m(2)) of the initial insecticide dose of 937±21.69mg/m(2) (nearly 1000mg/m(2)) at the end of intervention. Malaria incidence was reduced by 96.6% and 64.8% in the village with Olyset nets and in the villages with untreated nets, respectively. During intervention period, there was a reduction of 93.2% in malaria incidence in Olyset net area as compared to the untreated area. This study indicated that Olyset nets have a major impact on malaria vectors and disease burden; therefore it could be recommended as an effective personal protection tool for malaria control in malarious areas.

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.

Comparison of microscopical examination and semi-nested multiplex polymerase chain reaction in diagnosis of Plasmodium falciparum and P. vivax.

We compared light microscopy examination and a semi-nested multiplex PCR (SnM-PCR) assay in endemic areas of the Islamic Republic of Iran. A total of 68 individuals with malaria-positive and suspected malaria symptoms were included in the study. Giemsa-stained thick blood films were examined under a light microscope for malaria parasites in 100 and 200 fields. DNA was extracted from blood samples and SnM-PCR based on the amplification of the small sub-unit ribosomal RNA (ssrRNA) gene sequences was applied. Microscopical examination showed that 48.5% (33.8% P. vivax and 14.7% P.falciparum) and 50% (35.3% P. vivax and 14.7% P. falciparum) of the samples were positive in 100 and 200 fields respectively. SnM-PCR showed the same results as the 200 field microscopy.

High specificity of semi-nested multiplex PCR using dried blood spots on DNA Banking Card in comparison with frozen liquid blood for detection of Plasmodium falciparum and Plasmodium vivax.

BACKGROUND: Venipuncture sampling in test tubes for detecting malaria parasites using PCR assays possesses a number of limitations such as reluctance of patients, some difficulties in transportation of blood samples and freezing them for long time. To overcome the mentioned limitations, some approaches have been employed by a number of authors. This study was proposed to compare between DNA Banking Card (DBC) filter papers containing dried finger-prick blood and venipunctured frozen liquid blood. METHODS: A total of 75 specimens was prepared from the equal enrolled individuals using three blood storage approaches; making Geimsa-stained thin and thick smears from each individual to determine the malaria-positive or -negative specimens, spotting two to three drops of finger-prick blood onto the DBC filter paper, and collecting a 2-ml venous blood sample into EDTA-contained test tube from each individual. A semi-nested Multiplex PCR technique with DNA extracted from the two latter sets of specimens was used for plasmodia diagnosis. RESULTS: DNA samples isolated from dried blood spotted on the DBC filter papers resulted in 32 (42.7%) positive and 43 (57.3%) negative cases comparable with the results outcome of frozen liquid blood with 35 (46.7%) positive and 40 (53.3%) negative cases. Statistical analysis revealed higher sensitivity for SnM-PCR using DNA from liquid blood with 100% vs. dried blood spotted on DBC with 97% but higher specificity for the DBC with 100% vs. liquid blood with 95.2%. CONCLUSIONS: Based on the results obtained from this study to overcome the problems of venipuncture frozen liquid blood sampling, replacement of a reliable filter paper for preserving finger-prick blood samples is a trustable and useful facilitator particularly in remote malaria-endemic areas.

Neospora caninum and Leishmania infantum Co-Infection in Domestic Dogs (Canis familiaris) in Meshkin-Shahr District, Northwestern Iran.

BACKGROUND: Mediterranean visceral leishmaniasis (MVL) is an infectious disease that affects both human and animals. Domestic dogs (Canis familiaris) are principal reservoir hosts of MVL caused by Leishmania infantum. Dogs are definitive hosts for Neospora caninum and a risk factor for infecting intermediate hosts. The immunosuppression caused by visceral leishmaniasis (VL) can promote the occurrence of co-infections with other agents such as neosporosis. This study aimed to determine the frequency of co-infection of the both protozoan parasites in the endemic areas of VL from Meshkin-Shahr District, north-west of Iran. METHODS: Altogether, 171 serum samples were collected from domestic dogs of Meshkin-Shahr District by multistage cluster sampling from October 2008 to August 2009. The collected serum samples were tested for the detection of simultaneous infection of L. infantum and N. caninum using direct agglutination test (DAT) and indirect ELISA, respectively. RESULTS: Of the 171 domestic dogs, 27 (15.8%) and 52 (30.4%) were showed antibodies against L. infantum and N. caninum, respectively. Simultaneous infections of N. caninum and L. infantum was found in 16 (9.4%) of the dogs. In VL-positive and VL-negative dogs, N. caninum infection was found in 59.3% and 25.0%, respectively. A statistically significant difference was found between VL-positive and VL-negative dogs with N. caninum infection (P= 0.001). CONCLUSION: These findings indicate that Meshkin-Shahr District in northwestern Iran is an active focus of canine visceral leishmaniasis (CVL). Neospora caninum and L. infantum co-infection is prevalent in the area and infection by L. infantum seems to enhance susceptibility to N. caninum infection in domestic dogs.

Epidemiological aspects of visceral leishmaniasis in baft district, kerman province, southeast of iran.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: It seems that visceral leishmaniasis is being endemic in southern villages of Baft district, southeast of Iran.

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.

Integrated visceral leishmaniasis surveillance system in primary care for children in Meshkin-Shahr district, north-western Islamic Republic of Iran.

In 2001 a visceral leishmaniasis (VL) surveillance system was set up for children aged < or = 12 years in the primary health system in Meshkin-Shahr district of Ardebil province, north-western Islamic Republic of Iran. All cases with clinical signs and symptoms of VL and positive by the direct agglutination test were referred for physical examination and treatment. The mean annual incidence of VL decreased significantly from 1.88 before (1985-2000) to 0.77 per 1000 child population after the intervention (2001-07). In a control area with no surveillance, it increased from 0.11 to 0.23 per 1000. Early detection of VL using practical serological tests and timely treatment of cases could decrease the mortality and morbidity rates of VL in endemic areas.

Isolation and characterization of pathogenic Leishmania turanica from Nesokia indica (Rodentia, Muridae) by PCR-RFLP and ITS1 sequencing in Iran.

Zoonotic cutaneous leishmaniasis, primarily an infection of desert rodents, is an important endemic disease in some parts of Iran. In spite of reporting Leishmania infection in Nesokia indica microscopically, its role remains unclear in Iran. We report the first natural infection by Leishmania turanica in a wild-caught N. indica in the west part of Iran, near the border with Iraq. The evidence is in the form of PCR-RFLP testing based on internal transcribed spacer 1 (ITS1) rDNA with HaeIII enzyme and ITS1 sequence analysis. This isolate was highly pathogenic in BALB/c mice and golden hamsters and caused lethal systemic infection in the former.

Molecular data on vectors and reservoir hosts of zoonotic cutaneous leishmaniasis in central Iran.

Due to the increasing number of positive cases of cutaneous leishmaniasis with occurrence of new foci, a study was carried out to investigate on vectors and reservoirs of the disease in the Shahrood district, central Iran during 2005-2006. Sandflies and rodents were collected using sticky papers and Sherman live traps respectively More than 1700 sandflies were collected and identified, mainly Phlebotomus papatasi species. RAPD-PCR analysis of sandflies showed that 10% of P. papatasi and 4.2% of P. caucasicus were naturally infected with Leishmania major. Two species of rodents, potential reservoirs, Rhombomys opimus (92.5%) and Nesokia indica (7.5%) were trapped in the district. Microscopy identification from rodents confirmed that 91.9% of the Rhombomys opimus were positive to amastigotes. Species identification of isolated parasites revealed Leishmania major DNA in the infected Rhombomys using RAPD-PCR technique. This epidemiological data highlight the importance of the disease in the region and could help people involved in control programs.

PCR based on identification of vectors of zoonotic cutaneous leishmaniasis in Shahrood District, central of Iran.

A study was made in rural region of Shahrood city, Semnan province in the central of Iran during 2005 to investigate of vectors of zoonotic cutaneous leishmaniasis. Sticky traps and an aspirator were used for collecting of sandflies. Three species of Phlebotomus papatasi, P. caucasicus and Sergentomyia sintoni were collected and identified and the first species was dominant (53%). Nested PCR method were employed for identifying of isolated parasites of dissected female of sandflies. Among the dissected sand flies 3 out of 24 (12.5%) Phlebotomus papatasi and 2 out of 48 (4.2%) Phlebotomus caucasicus were found naturally infected with promastigotes. Species-specific amplification ofGiemsa staind promastigote slides revealed specific PCR production of Leishmania major DNA in the infected P. papatasi and P. caucasicus sand flies. Having high prevalence and infection rate provide enough evidence to incriminate of P. papatasi as the main and proven vector of cutaneous leishmaniasis to human in the region and the species of P. caucasicus play the second role for maintenance of disease between rodents.

Characterization of Leishmania infection in rodents from endemic areas of the Islamic Republic of Iran.

Between 1991-2000, Leishmania species were isolated and characterized by isoenzyme and molecular analysis from rodents caught in various parts of the Islamic Republic of Iran. In areas endemic for cutaneous leishmaniasis, parasites were observed by direct microscopy in smears from 18.6% of 566 specimens. L. major was isolated from 4 species: Rhombomys opimus, Meriones libycus, Tatera indica and Mer. hurrianae. L. turanica was isolated from R. opimus for the first time in this country. In endemic areas of visceral leishmaniasis, parasites were observed in liver and spleen from 13.7% of 504 rodents. Two species were positive on culture; promastigotes isolated from Mer. persicus were characterized as L. donovani zymodeme LON50 and from Mesocricetus auratus as L. infantum LON49.