Iranian Journal of Parasitology
Volume:9 Issue: 4, Oct-Dec 2014

This study aimed to compare three staining methods including: Calcofluor white, Chromotrope and Quick Hot Gram chromotrope used in diagnosis of intestinal microsporidial spores. One hundred and seventy five stool specimens were collected from patients referred to Laboratory of Intestinal Protozoology at the School of Public Health, Tehran University of Medical Sciences during 2012-2013. All of specimens were evaluated by nested PCR. The formalin–fixed stool samples were prepared from each specimen and dried at room temperature for 10 min, followed by 10 min methanol fixation. All the collected stool samples were evaluated blindly by calcofluor white, Chromotrope and Quick Hot Gram chromotrope staining methods separately. Microsporidial spores were recognized using Chromotrope, Quick Hot Gram chromotrope and Calcofluor white, in16 of 18 (88.8%), 17 of 18 (94.4%) and 18 of 18(100%) samples that were positive by nested PCR respectively. Regarding 14 stool samples that were negative by nested PCR, 14 cases were negative by chromotrope and Quick hot Gram chromotrope and 13 samples were negative by Calcofluor white. One discordant sample interpreted as false positive. Calcofluor white staining had the best performance for the detection of intestinal Microsprora spores and can be used as initial screen test for the detection of intestinal Microspora spp.

Sensitive and glucantime (MA) resistance Leishmania tropica are referred to those isolates, which are responsive, or non-responsive to one or two full courses of treatment by MA systematically and/or intra-lesionally, respectively. In this study, we evaluated the antileishmanial activity of biogenic selenium nanoparti­cles (Se NPs) alone and in combination with MA against sensitive and glucantime-resistant L. tropica on in vitro model. The Se NPs were synthesized by employing the Bacillus sp. MSh-1. The antileishmanial effects of Se NPs alone and in combination with MA on pro­mastigote and amastigote stages of sensitive and glucantime-resistant L. tropica strains have been investigated using a colorimetric MTT assay and in a macrophage model. In addition hemolytic activity in type O+ human red blood cells and infectiv­ity rate of the promastigotes before and after treatment with the Se NPs was evaluated. In the promastigote stage, various concentrations of Se NPs significantly inhibited (P<0.05) the growth of promastigotes of both strains in a dose-dependent manner. Similarly, Se NPs especially in combination with MA significantly reduced the mean number of amastigotes of both strains in each macrophage. Se NPs showed no hemolytic effect on human RBCs at low concentrations. Moreover, infec­tion rate of macrophages by promastigotes significantly (P<0.05) was reduced when promastigotes pre-treated with Se NPs. The findings of this study suggest a first step in the search of Se NPs as a new antileishmanial agent. Further experiments are needed to investigate antileishmanial effects of biogenic Se NPs on L. tropica using a clinical setting.

The aim of this study was to detect the seroprevalence of human fascioliasis in Isfahan County, central Iran in 2013. Overall, 471 sera samples were collected from people recalled randomly to 20 health centers in the city of Isfahan and 10 related villages in 2014. Sera were examined using ELISA test. A questionnaire was filled out for each participant. Altogether eight cases (1.7%) were seropositive which had the OD absorb­ance in ELISA test more than the calculated cutoff of 0.36. All of them were female. One positive subject had a history of consuming Delar (Local dish) and three seropositive cases with history of eating Zeitoon-Parvadeh (Proceeded olive). Isfahan County might be considered as one area apt for fascioliasis. More studies in terms of veterinary investigation and verifying the risk factors are necessary.

Entamoeba moshkovskii and E. dispar are impossible to differenti­ate microscopically from the pathogenic species E. histolytica. Multiplex polymer­ase chain reaction (Multiplex PCR) is a widespread molecular biology technique for amplification of multiple targets in a single PCR experiment. For detection and differentiation of the three-microscopy indistinguish­able Entamoeba species in human, multiplex PCR assay using differ­ent DNA extraction methods was studied. A conserved forward primer was derived from the middle of the small-subunit rRNA gene, and reverse primers were designed from signature sequences specific to each of these three Entamoeba species. A 166-bp PCR product with E. histolytica DNA, a 580-bp product with E. moshkovskii DNA and a 752-bp product with E. dispar DNA were gener­ated in a single-round and multiplex PCR reaction. We recommend this PCR assay as an accurate, rapid, and effec­tive diagnostic method for the detection and discrimination of these three Enta­moeba species in both routine diagnosis of amoebiasis and epidemiological surveys.

Toxoplasma gondii, the obligate intracellular parasite is life threaten­ing in AIDS patients. Diagnosis of toxoplasmosis is based on serological methods especially increasing of IgM and IgG titers, but finding of parasite or its components (antigenemia) may be beneficial method in order to detection of acute toxoplasmosis in immunocompromised patients. Ninety-four serum samples from HIV positive patients were collected from Sanandaj, Kordistan west of Iran. These patients were lived in Sanandaj of whom 26 were prisoners infected with HIV virus in prison. Toxoplasma gondii antibodies were determined by IgG ELISA. T. gondii antigen was identified by cap­ture-ELISA. PCR was performed on samples with T. gondii antigenemia. CD4+ T cells counts had been determined by flowcytometry and were obtained from rec­ords of each patient. Among the examined HIV seropositive individuals, 19.1% (18/94) and 5.3% (5/94) were positive for Toxoplasma-IgG and antigenemia, respectively. Be­sides, one of the samples was positively detected by PCR method. Mean age of partici­pants was 37.9 ± 9.5 year. Prevalence of IgG antibody and antgenemia was higher in age group of 40-50 years old. The Mean of CD4+ T cells counts of partici­pants (total of HIV+ patients, IgG positive patients and patients with anti­genemia) was 699.2 ± 345.2, 655.1 ± 237.9 and 620.2 ± 215.1 respectively. Capture-ELISA and PCR could confirm the T. gondii acute infec­tion in HIV positive patients. For precise diagnosis of acute toxoplasmosis in HIV positive patient, performance of more studies based on more sensitive types of PCR is suggested.

Cryptosporidium parvum causes severe gastroenteritis in immunocompro­mised human and new borne animals. The organism can be transmit­ted through water. Since small number of C. parvum is infectious, the aim of the present study was to develop a chromatography method for the isolation of C. parvum oocyst in samples with limited number of oocysts. Antibody was prepared against whole antigen from C. parvum oocysts, the achieved Ab bound to the sepharose 4B and used for the isolation of oocysts. Antibody against P23 bound to the sepharose 4B, used also for the isolation of C. parvum oocyst. In comparison to these both methods, 2 traditional methods (Salt floatation and 55% sucrose floatation) were also performed. Both chromatography methods could bind oocysts with capacity depends on the column size. The isolated oocysts were free of bacteria. Our results showed that the traditional methods are useful for the isolation of oocysts from feces, in its smear stained with ziehl-nelsen, at least 3 oocyts are detectable in each microscopic field under 1000 X magnification. In contrast to the chromatography methods, the bacterial contamination was always observed in oocysts isolated with traditional methods. Immunochromatography could be used for the successful isolation of C. parvum oocysts from the samples containing limited number of oocysts.

Echinococcosis is a zoonotic parasitic disease of humans and various herbivorous domestic animals transmitted by the contact with domestic and wild carnivores, mainly dogs and foxes. The aim of this study is the production, purifica­tion and evaluation immunogenicity of new construction of EG95 protein. The recombinant plasmid pET32-a+ used for Eg95 expression was con­structed with the EG95 gene of Echinococcus granulosus fused with the thioredoxin tag. This recombinant clone was over expressed in Escherichia coli BL-21 (DE-3). The expressed fusion protein was found almost entirely in the insoluble form (inclusion bodies) in cell lysate. The purification was performed under denaturing conditions in the presence of 8M urea by Ni–NTA column and dialysis. The purified recombinant proteins were confirmed with western blot analysis using polyclonal antiserum. To find out the immunogenicity of the purified protein, the BALB/c mice (10 mice/group) were immunized by injecting 20 µg rEG95 protein formulated in Freund’s and alum adjuvant. Immunization of mice with rEG95 using CFA/IFA and alum adjuvant generated high level of total antibody. In proliferation assay, the lymphocytes were able to mount a strong proliferative response with related production of IFN-ɣ, IL-12 and TNF-α but with low secretion of either IL-4 or IL-10. The humoral and cellu­lar immune responses against rEG95 suggested a mixed Th1/Th2 response with high intensity toward Th1. Our findings suggest that new construct of rEG95 formulated with CFA/IFA and alum adjuvant elicited strong cellular and humoral responses support­ing further development of this vaccine candidate.

Cystic echinococcosis (CE), a zoonotic parasitic infection caused by the metacestode (larvae) stage of dog tapeworm Echinococcus granulosus and recognized as a major economic and public health concern in the world. This study aimed to investigate the in vitro scolicidal effect of methanolic extract of Berberis vulgaris L. roots and its main compound, berberine against protoscole­ces of hydatid cysts. For this purpose, protoscoleces were aseptically aspirated from sheep livers having hydatid cysts. Various concentrations of the methanolic extract (0.25-2 mg/ml) and berberine (0.062- 0.5 mg/ml) were used for 5 to 30 min. Viability of protoscoleces was confirmed by eosin exclusive test. In the present study, all of the various concentrations of the B. vulgaris methanolic extract (0.25, 0.5, 1 and 2 mg/ml) and berberine (0.062, 0.125, 0.25 and 0.5 mg/ml) revealed significant (P<0.05) scolicidal effects against protoscoleces of E. granulosus in a dose-dependent manner. Both berberine and methanolic extract exhibited 100% inhibition against protoscoleces of E. granulosus at the concentration of 2.0 and 0.5 mg/ml after 10 min incubation respectively. According to the results, both B. vulgaris methanolic extract and berber­ine alone demonstrated high scolicidal activities against protoscoleces of hyda­tid cysts in low concentration and short exposure time on in vitro model. However, in vivo efficacy of B. vulgaris and berberine also requires to be evaluated using an ani­mal model with hydatid infection.

To determine the prevalence and intensity of helminths and their zoono­tic importance in small rodents inhabiting in the suburban areas of Hamadan City, Iran.The present survey was conducted on the helminth infections of two spe­cies of rodentsApodemussylvaticus(n=60) and Musmusculus(n=72) inthesuburban ar­eas of Hamadan City during 2010-2012. Rodents were collected and examined for helminth in the different organs. The nematodes were collected in 5% formalin solution and cleared in lactophenol, cestodes and trematodes collected from intes­tine fixed in AFA solution and stained byacetocarmine, cleared in xylol for identifica­tion.Helminths found in A. sylvaticus and M. musculus and their prevalence for the first time in suburban areas of Hamadan City were as follows; In A. sylvaticus: Cysticercusfascio­laris(3.33%),Syphaciafredrici(26.67%), S.stroma(8.33%),Anoplocephalidaesp. (1.67%), Skrjabinotaenialobata(5%), Plagiorchismuris(1.67%) and in M. musculus:Hymenolepis nana (16.67%), H.diminuta (5.55%), S. obvelata(30.56%), S.ohtarom (9.72%),Rodentole­piscrassa (1.39%), C.fasciolaris (1.39%). Among 11 species in two rodents 4 species includ­ing S. obvelata, H. nana, H.diminuta,and P. muris have zoonotic importance. Statisti­cally the relation between gender and their helminth infections was not signifi­cant in either M. musculus or A. sylvaticus (P>0.05).This study reports 11 species of helminths and on the other hand 3 species were identified for the first time in Iran and 5 species of them have potential health importance for public health and cat.

The aims of this study were to identify Blastocystis subtypes (STs) in a cohort of Turkish patients with various gastrointestinal symptoms using a novel Real Time PCR method developed recently for Blastocystis detection and assess the relation­ship between Blastocystis STs and patient symptoms. Totally, 617 stool samples of patients with gastrointestinal symptoms were examined with microscopy and inoculated in Jones medium. Blastocystis-positive sam­ples were further assessed to identify coinfections with other possible pathogens, includ­ing bacteria and viruses. Diagnostic efficacies of microscopy, culture and Real-Time PCR were compared. PCR products were sequenced to identify the subtypes of Blastocystis isolates. Totally 94 (15.24%) samples were positive for Blastocystis after all methods. Among these, 83 of 94 (88.3%) samples were identified with all methods, while 11 were positive only with Real Time PCR. Diarrhea and abdominal pain were the leading symp­toms in the patients. The only pathogenic agent identified in 76 of 94 (80.9%) patients was Blastocystis. Subtype 3 was the leading Blastocystis subtype (44.6%), while subtypes 6 and 7 were firstly isolated from symptomatic patients in our region. Comparison of three diagnostic methods indicated Real Time PCR as the most sensitive and specific method. Blastocystis was the only pathogenic agent among symptomatic patients, with subtype 3 being predominant. Patients with subtypes 6 and 7 need further assessments concerning the zoonotic potential of Blastocystis.

Giardia lamblia is one of the most common protozoal infections in human especially children. Metronidazol (MTZ) is the drug of choice for treatment of giardiasis; its chemical composition possesses major threats and is becoming less sensitive. This study aimed to search for natural extracts alternative to MTZ. In-vivo effects of dichloromethane extracts of ginger and cinnamon in doses of 10 and 20 mg/kg/day separately were studied on 30 experimentally in­fected albino rats divided into 6 groups (5 rats each). Plant extracts were started on the 6th day post infection for 7 successive days. The study was evaluated by fecal cyst and intestinal trophozoite counts, histopathology, scanning and transmission electron microscopic examinations of the small intestinal mucosa. Ginger and cinnamon caused reduction of fecal cyst and trophozoites counts. Histopathology, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) after exposure to each extract revealed evident improve­ment of intestinal mucosal damage produced by G. lamblia infection and direct structural injury to the trophozoites. However, these results were more obvi­ous after exposure to cinnamon extracts. We confirmed the potential therapeutic effects of ginger and cinna­mon extracts on G. lamblia infection in albino rats as a promising alternative ther­apy to the commonly used antigiardial drugs.

Giardia lamblia is one of the most prevalent intestinal flagellate protozoa that infects a wide range of vertebrate hosts causing severe intestinal disorder in children.This study was performed to determine subspecies of G.lamblia by the PCR-RFLP method, targeting the glutamate dehydrogenase(gdh)locus, in hospitalized children at Urmia Mutahhari Hospital, West Azerbaijan Province,Iran and determining the infection transformational storages in this area. Overall,720 stool specimens were collected from the hospitalized children, 34 samples were positive and Giardia cysts were detected under the microscope. Cysts were partially purified by the sucrose density gradient method and then washed with sterile distilled water to remove effectively the PCR inhibitors. Genomic DNA of G. lamblia isolates was extracted by freeze-thaw cycles followed by phenol/ chloro­form/isoamyl alcohol method. The single step PCR-RFLP assay was used to differenti­ate the assemblages between A and B, which were found in humans. In this method, 432 bp expected size was amplified, and then for detection of subspecies, specific restriction RsaI and BspLI enzymes were used. Totally 34 samples were positive in terms of Giardia cyst out of 720 examined samples microscopically, so the parasite spread rate is reported 4.72%. Analysis PCR-RFLP on these samples revealed that 28 samples (93.3%) have the genotype BIII and 2 samples (6.7%) belong to the subgroup BIV. PCR-RFLP is a proper analytical method for determining the genotype among parasite types, using the glutamate dehydrogenizes zone''s genes. Based on the results, an animal origin of infection cycle is suggested.

The aim of this study was to determine intestinal and liver hel­minth infections in Rattus rodents in Tehran Iran. Overall, 306 traps were put in 39 different regions in Tehran from 2009 to 2010. Rodents, including R. rattus and R. norvegicus were caught by live-traps. They become unconscious and the spinal cords were cut, afterwards the body was dissected and the stomach, small intestine, large intestine, liver, and cecum were studied separately. The dominant type and the prevalence rate of parasites in the rodents were determined based on the infected parts of their body. After recognition of the helminthes’ types, among the 120 total number of rodents, 39 belonged to males, while among the infected rodents, 57(47.5%) were female and 18(15%) were male. The prevalence of infection in Tehran was 62.5%. Seventy cases (58.33%) of helminth infections were observed in R. rattus and 5 cases (4.16%) were observed in R. norvegicus. The maximum prevalence (15.5%) was seen in the center and east part of Tehran, while the minimum (9.16%) was in the north part of the city. The helminthes types and the corresponding percent­ages were Hymenolepis nana fraterna (35.8%), Heterakis spumosa (17.5%), Hymenole­pis diminuta (7.5%) and Capillaria annulosa (1.6%). The dominant rodent was Rattus rattus and among the identified helminthes, Hymenolepis diminuta and Hymenole­pis nana fraterna are zoonotic ones. The information presented here improves our understanding of the major parasitic infections that rodents harbor and can transmit to human and ani­mal populations in Iran. To prevent infectivity of human, the hazard of the identi­fied zoonotic species needs to be contemplated.

Malaria is a major international public health problem. Drug-resistant parasites have made treatment and control of malaria more difficult. Therefore, safe, afforda­ble and effective new drugs are urgently needed. Traditional medicine is an important source for new drugs. Determining the ancient medicinal books was the first step of this study for finding malaria or disease that has symptoms like ma­laria. Then the plants that used to treat “Ghebbe Khalesseh fever” were listed. Fi­nally, recent antimalarial researches were explored. About 31 plants were identified. Information from these resources is valuable for the selection of plants for antiplasmo­dial screening programs.

Heartworm (Dirofilaria immitis) is mosquito-borne filarial nematode capable of causing serious cardiopulmonary disease in canines and felines, and pulmo­nary dirofilariasis in man. This research was conducted with the objectives of determining the incidence and assessing possible risk factors of canine heartworm in the southeast of Iran. From October 2012 to September 2013, blood samples from 87 dogs from Zabol area in Sistan and Baluchestan and 33 dogs from Bam area in Kerman Province were examined for detection of Dirofilaria immitis using modified knott test and serology. Out of 120 dogs, 29 (24.2%; 95%CI: 16.6-31.8%) were positive, serologi­cally. The overall seroprevalence of D. immitis in dog in Zabol and Bam was 27.5% (95% CI: 24.7-32.5%) and 15.15% (95% CI: 12.3-20.7%), respectively. 28.8% of stray dogs and 20.6% of housed dogs in the study areas were seropositive. Seropreva­lence of D. immitis was not significantly different between stray and housed dogs (P=0.295). Investigation of seasonal dynamic of infection with D. im­mitis in stray and housed dog showed that the proportion of infected dog in spring and summer was greater than colder season (autumn and winter) which was not significant. The prevalence of infection with D. immitis in >5 years old stray dogs (53.8%) was greater than other age categories while in housed dogs infection rate was greater in 3-5 years old (27.3%). It is important to point out the increased incidence of canine heatrworm in Iran. In order to stop the spread of canine heartworm, preventive measures must be taken now.

Taenia multiceps is a cestode parasite with its larval stage (metacestode), Coenurus cerebralis, mainly encysts in the central nervous system of sheep and other livestock causing cerebralis coenurosis. Since treatment of coenu­rosis with chemotherapy showed little effect and surgical removal of cysts is not advisable in field conditions, vaccination is useful to control coenurosis. Previous study indicated that immunization with T. multiceps metacestode antigens could induce protection in sheep against coenurosis, so the aim of this study was to iden­tify T. multiceps metacestode antigens in order to find potential vaccine develop­ment candidates for further study. The protein extracts from the larval T. multiceps were analyzed by two-dimensional electrophoresis (2-DE) and characterized by mass spectrometry. A total of 150 protein spots were detected with isoelectric point (pI) value from 4.97 to 9.65 and molecular weight from 14 to 98 kDa. Twenty-two pro­tein identities were determined by mass spectrometry and 15 unique proteins were obtained. Functional annotation revealed that some of these proteins are involved in catalytic activity, binding, metabolic, cellular process and stress response. Among these molecules are antioxidant proteins (peroxiredoxin and glutathione-S-transfer­ase), glycolytic enzymes (malate dehydrogenase and enolase), proteins with chaper­one activity (heat shock protein 70 and small heat shock protein), and structural proteins (actin, actin modulator protein and paramyosin). The identification of T. multiceps metacestode protein will provide valuable information to elucidate their specific roles in the parasitism and screen new targets for vaccine development.

Leishmania Homologue of receptors for Activated C Ki­nase (LACK) antigen is a 36-kDa protein, which provokes a very early im­mune response against Leishmania infection. There are several reports on the expression of LACK through different life-cycle stages of genus Leishmania, but only a few of them have focused on L.tropica. The present study provides details of the cloning, DNA sequenc­ing and gene expression of LACK in this parasite species. First, several local isolates of Leishmania parasites were typed in our laboratory using PCR technique to verify of Leishmania parasite species. After that, LACK gene was amplified and cloned into a vector for sequencing. Fi­nally, the expression of this molecule in logarithmic and stationary growth phase promastigotes, as well as in amastigotes, was evaluated by Reverse Transcription-PCR (RT-PCR) technique. The typing result confirmed that all our local isolates belong to L.tropica. LACK gene sequence was determined and high similarity was observed with the sequences of other Leishmania species. Furthermore, the expression of LACK gene in both promastigotes and amastigotes forms was confirmed. Overall, the data set the stage for future studies of the proper­ties and immune role of LACK gene products.

Leishmaniainfantum is the most frequent cause of visceral leishmaniasis and L. tropica has been rarely linked to the disease in Iran.In this study, bone marrow aspi­rates were collected from 10 child patients, suspected with visceral leishmania­sis referred to the Pediatric Wards of Kerman Medical Hospitals, Kerman, Iran during 2002–2011. Leishmania species were identified by using nested PCR in all slides. The PCR samples from nine patients indicated L. infantum as principal causa­tive agent of visceral leishmaniasis and one L.tropica as a minor species.

Cerebral coenurosis is the intermediary larval stage of Taenia multiceps, which affects intermediate hosts, particularly sheep and goats. In this report, gross and microscopic features of three scarce natural coenurosis cases, a one-year-old ram and two lambs of 7 month old from a flock are explained. At nec­ropsy, numerous small cysts measuring 5 to 10 mm in diameter were ob­served on both cerebrum and cerebellum surfaces, likewise multiple deep parts of which. In histopathological examination of the neural tissue, severe tissue destruction, a distinct layer of Gitter cells formation around the cysts, neuronophagia, gliosis and perivascular infiltration of lymphocytes were observed. In this early stage of parasite life cycle, larval migration and destruction of tissue, also aggregation of glial cells around the cysts cause a loose connection between cysts and neural tissue.

Ophionyssus natricis is a purely blood sucking parasite of snakes and of worldwide distribution. Infected snakes often exhibit lethargy, pruritus, crusting dermatitis, and behavioral changes. Ophionyssus natricis can also attack humans, causing popular vesiculo-bullous eruption of the skin. A 29 years old man working in zoo, Sari, Mazandaran, Iran, presented itchy papullar eruption of the skin. He had noticed small insects fixed on his skin and large numbers of these same insects on a python and its cage in the zoo. Regarding totheir morphological characteristics they were diagnosed as O. natricis (Geravis, 1844), a snake mite. It is the first report of O. natricis from Iran.