Co-detection and isolation of Leishmania and Crithidia among naturally infected Tatera indica (Rodentia: Muridae) in Fars province, southern Iran

Objective: To explore the co-detection of natural infection of Trypanosomatidae parasites such as Leishmania and Crithidia in reservoir hosts of leishmaniasis. Methods: Rodent populations were monitored in two endemic foci of cutaneous leishmaniasis of Fars province, southern Iran from March to October 2016. Rodents were trapped alive in several parts of Shiraz and Kharameh cities. Afterwards, their organs were prepared for detection of Leishmania and Crithidia species by molecular, microscopic, and culture methods. Results: Totally, 115 rodents of five species; Tatera indica (T. indica) (85), Rattus rattus (12), Meriones libycus (9), Mus musculus (7), and Rattus norvegicus (2), were trapped alive and their tissue samples were examined using microscopic, cultivation, and molecular assays. Overall, 59 (51.3%) rodents were positive for Leishmania or Crithidia parasites. The highest rate (61.2%; 52/85) of Leishmania infection was related to the T. indica population. The cultivation, and molecular observations showed that two (2.4%; 2/85) of T. indica (foot-pad, and spleen samples) were positive to Crithidia. Conclusions: This is the first report of Crithidia infection in T. indica in Iran. Consequently, more epidemiological and ecological studies are needed to understand the role of Crithidia and Leishmania in T. indica.

Inter-and intraspecific variations of leishmania strains isolated from patients with cutaneous and visceral leishmaniases in pars province, south of Iran

Background: Cutaneous and visceral leishmaniases are present in Fars Province in the south of Iran. The current study aimed to evaluate the inter- and intragenic diversities of Leishmania species isolated from patients with leishmaniasis in Fars Province, using PCR-based analyses and DNA sequencing of the N-acetylglucosamine-1-phosphate transferase [nagf] gene

Methods: Clinical samples were taken from the skin lesions of 120 individuals with clinical suspicion of cutaneous leishmaniasis [CL] referred to the major health centers of Shiraz. Along with microscopic examination, a part of each sample was used for in vitro cultivation. DNA was extracted from the cultured parasites and the nagt gene was PCR-amplified. For RFLP analysis, the PCR product of the nagt gene was digested with the Accl restriction enzyme. Moreover, the PCR products of 23 isolates were sequenced and analyzed, using MEGA5

Results: From the 120 patients with clinical suspicion of CL, 110[91.7%] cases were found to be positive by direct microscopy while 77[64.1%] of the cultures were positive. Digestion of the PCR product with the Accl restriction enzyme detected L. major in 57 out of the 77 [74.1%] and Z. tropica, in 20 out of the 77 [25.9%] cases with CL. Phylogenetic analysis grouped the Leishmania isolates into 3 main clades, representing L. major, L. infantum, and L. rop/ca,encompassing 2, 2, and 2 haplotypes, respectively. Within the clades, the L. tropica intraspecies divergence was more pronounced in L. major

Conclusion: The findings of this study demonstrated that the causative agent of CL in Fars Province was mainly L. major and that there was considerable heterogeneity between the Leishmania species and also within the L. major isolates

Prevalence of Enteric Protozoan Oocysts with Special Reference to Sarcocystis cruzi among Fecal Samples of Diarrheic Immunodeficient Patients in Iran

The genus Sarcocystis is not usually considered as an important enteric pathogen in immune compromised patients. It might be expected that species for which humans are the final host (Sarcocystis hominis and Sarcocystis suihominis as well as possibly others) would be encountered increasingly often in immunodeficient persons. This study aimed to address how to detect and differentiate Sarcocystis oocysts and/or sporocysts from enteric protozoans in the diarrheal samples of immunodeficient patients in Shiraz, Iran. Diarrheal samples of 741 immunodeficient patients with recurrent persistent or chronic diarrhea were examined by microscopy and molecular biological analysis. Oocysts-positive samples were 68 Cryptosporidium spp., 9 Cystoisospora belli (syn. Isospora belli), 2 Cyclospora cayetanensis, and 15 microsporidia (Enterocytozoon bieneusi). Sarcocystis-like sporocysts found from a woman were identified as Sarcocystis cruzi through 18S rDNA amplification and phylogenetic analysis. To the best of our knowledge, this is the first report of S. cruzi from a human.

Cutaneous Leishmaniasis of the Eyelids: A Case Series with Molecular Identification and Literature Review

Cutaneous leishmaniasis (CL) is a protozoan disease which is endemic in Iran. It is transmitted by the Phlebotomus sand fly. The eyelid is rarely involved possibly because the movement of the lids impedes the sand fly from biting the skin in this region. Here, we report 6 rare cases of eyelid CL. The patients were diagnosed by skin scraping, culture, and PCR from the lesions. Skin scraping examination showed Leishmania spp. amastigotes in the cytoplasm of macrophages. Culture examination was positive for Leishmania spp. PCR was positive for Leishmania major and Leishmania tropica. The lesions were disguised as basal cell carcinoma, chalazion, hordeolum, and impetigo. The patients were treated with intramuscular meglumine antimoniate (20 mg/kg/day) for at least 3 weeks. They showed a dramatic response, and the lesions almost completely disappeared. We emphasized the importance of clinical and diagnostic features of lesions, characterized the phylogenetic relationship of isolated parasites, and reviewed the literature on ocular leishmaniasis.

Efficacy of some metronidazole derivatives against Giardia lamblia, in vivo study

Giardiasis is a protozoal infection of small intestine caused by giardia lamblia. The disease is usually asymptomatic though it can present as acute or chronic diarrhea. Giardiasis is a major cause of intestinal infection and Iran is an endemic area of the disease. Despite reports about drug resistance, long course treatment and various side effects, metronidazole is the drug of choice for giardiasis. In this study we investigated in vivo effects of five new derivatives [a-e] of metronidazole [MTZ] on the giardia lamblia trophozoite in infected mice. Giardia intestinalis cysts were isolated from a patient and purified by sucrose gradient method. Fifty Purified cysts were inoculated to mice and after development of infection, the new metronidazole derivatives were given to the mice and results were compared to metronidazole as positive control. Compounds a and b showed desirable antigiardiasis activity and could destroy the cyst and trophozoite of giardia lamblia in mice after both two and four days, but the activity of the other compounds appeared only after 4 days

Evaluation of Recombinant SAG1, SAG2, and SAG3 Antigens for Serodiagnosis of Toxoplasmosis

Serologic tests are widely accepted for diagnosing Toxoplasma gondii but purification and standardization of antigen needs to be improved. Recently, surface tachyzoite and bradyzoite antigens have become more attractive for this purpose. In this study, diagnostic usefulness of 3 recombinant antigens (SAG1, SAG2, and SAG3) were evaluated, and their efficacy was compared with the available commercial ELISA. The recombinant plasmids were transformed to JM109 strain of Escherichia coli, and the recombinants were expressed and purified. Recombinant SAG1, SAG2, and SAG3 antigens were evaluated using different groups of sera in an ELISA system, and the results were compared to those of a commercial IgG and IgM ELISA kit. The sensitivity and specificity of recombinant surface antigens for detection of anti-Toxoplasma IgG in comparison with commercially available ELISA were as follows: SAG1 (93.6% and 92.9%), SAG2 (100.0% and 89.4%), and SAG3 (95.4% and 91.2%), respectively. A high degree of agreement (96.9%) was observed between recombinant SAG2 and commercial ELISA in terms of detecting IgG anti-Toxoplasma antibodies. P22 had the best performance in detecting anti-Toxoplasma IgM in comparison with the other 2 recombinant antigens. Recombinant SAG1, SAG2, and SAG3 could all be used for diagnosis of IgG-specific antibodies against T. gondii.

In vitro and in vivo potential of RH strain of toxoplasma gondii [type I] in tissue cyst forming

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. 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. 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. 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

Molecular genotyping of toxoplasma gondii in human spontaneous aborted fetuses in Shiraz, southern Iran

Congenital toxoplasmosis is associated with variable complications including encephalitis, microcephaly, hydrocephaly, hepatitis, lymphadenopathy and even intrauterine death. Presence of Toxoplasma gondii in human placenta may induce congenital infection. The aim of this study was to determine the genotypes of Toxoplasma gondii infection in human spontaneous aborted fetuses in Shiraz, south of Iran. Five hundred and forty two paraffin-embedded blocks of aborted placenta were collected, from two university-affiliated hospitals in Shiraz. Occurrence of spontaneous abortion was confirmed by examine of the slides. After re-cutting of the blocks and dewaxing, semi-nested PCR assay was used to detect the fragments of T. gondii B1 gene in the samples. Also direct molecular genotyping was performed on positive samples with Restriction Fragment Length Polymorphism-PCR analysis on the SAG2 gene. Among the 542 tissue samples, the B1 gene was amplified from 78 [14.4%] of cases with the semi nested PCR and typed by RFLP. The genotype of Toxoplasma strains of 65 [out of 78] PCR-positive samples were evaluated and 54 out of 65 [83.1%] were found to be type II and 11 out of 65 [16.9%] were type I. Considering the high level of Toxoplasma infection in aborted fetuses in this study, Toxoplasma might largely contribute to spontaneous abortion in this area of Iran

Leishmania major in Tatera indica in Estahban, Southern Iran: microscopy, culture, isoenzyme and PCR

This study identifies the infected rodent hosts to Leishmania major in Estahban town, southern Iran during 2004-2005. The rodents were caught alive from April 2004 to April 2005 in Estahban town, south of Iran and examined for any skin lesion. An impression was provided from the tissues of feet, tail, ears and any patent lesion, stained with Giemsa and studied microscopically for the presence of amastigotes. All samplings were cultured at 25°C in rabbit blood agar and considered negative if no promastigotes were visible during a two months period. The parasites from any positive culture were cryopreserved in liquid nitrogen pending their identification in PCR and isoenzyme electrophoresis. The femoral bones were histologically and ultrastrucrurally studied. Among 13 captured rodents, 8 were Tatera indica [5 male and 3 female Indian gerbils] and 5 were Rattus rattus [3 males and 2 females]. Just one female T. indica was smear-positive for amastigotes in Mohmmad Abad village. This rodent was also found culture positive for leishmanial infection which was confirmed by PCR and enzyme electrophoresis. At histological and ultrastructural levels, many clusters of amastigotes were noticed in the foamy macrophages of the femoral bone bone marrow. T. indica was found for the first time in the area and can be one of the rodents to be a potential reservoir host of L. major. It was also shown that femoral bone marrow was the tissue of choice to confirm the presence of macrophages containing the amastigote form of the parasite

[Application of superior enzymatic systems for characterization of causative agents of visceral leishmaniasis [kala-azar] in Iran using isoenzyme electrophoresis]

Visceral leishmaniasis [Kala-azar] is one of the parasitic zoonotic diseases which is caused by Leishmania donovani complex parasites. Thus, the aim of this study is the application of different enzymatic systems for discrimination species and strains visceral leishmaniasis agent. In this experimental study, reference strains of Leishmania infatum, Leishmania major, Leishmania tropica in addition, the leishmania parasites isolated from bone marrow of subjects and internal organs of dogs infected to visceral leishmaiasis [VL] were inoculated on RPMI + FBS 10% medium for mass cultivation. Then, using electrophoresis on polyacrilamid gel, six enzymatic systems including GPI, PGM, MDH, G6PD, 6PGD and NH2 were examined in order that identification of species and strains of the isolates and thus finding the appropriate enzymatic systems for discrimination of these compared with reference strains. Isoenzymatic profile of six enzymatic systems mentioned above for these isolates were compared with reference strains and also relative migration were calculated. Finally, the results were showed that only five enzymatic systems, except 6PGD, had discriminated ability of different species. In the present study, GPI and G6PD enzymes had the most heterogeneity while NH2 enzyme had the most homogeneity. Moreover, PGM, GPI and MDH were highly active enzymes

Zoonotic cutaneous leishmaniasis in Shiraz, Southern Iran: a molecular, isoenzyme and morphologic approach

Cutaneous leishmaniasis [CL] with diverse clinical manifestations is prevalent and remains a major public health problem in Iran and its incidence has been doubled over the last decade. The present study is about the potential role of rodents in the epidemiology of CL in Kharameh district in Shiraz, Southern Iran. From April 2004 to April 2005, a total of sixteen rodents were collected in live traps from the endemic area of CL in Kharameh district in Shiraz. Evans medium was used for culture. Specific polymerase chain reaction and isoenzyme electrophoresis methods were performed to characterize the parasite. The rodent species were Tatera indica. Three samples from Tatera indica were found positive [2 males and 1 female in Kafdehak and Sejel-Abad villages] for L. major. Macrophages in the bone marrow of femoral bone were infected with the amastigote form of the parasite. It seems that T. indica is the reservoir host for CL in Kharameh [a district in Shiraz, Southern Iran]. It was shown that the bone marrow of the rodents is the tissue of choice for light and ultrastructural studies of L. major