Iranian Journal of Parasitology
Volume:7 Issue: 2, Apr-Jun 2012

Echinococcosis or hydatidosis is a chronic، zoonotic worldwide infection that occurs by the larval stages of taeniid cestodes of the genus Echinococcus. Iran is known as endemic region for this infection in the world. Vaccination has been considered as a good prevention method for this disease. Recombinant vaccines containing EG95 protein، against E. granulosus، has shown a high degree of protection against E. granulosus infection. In this study EG95 gene was extracted from Iranian isolates of E. granulosus and then cloned and expressed in expression vector. Protoscoleces were collected from sheep hydatid cysts. Then DNA and RNA were extracted from protoscoleces، and amplified by PCR and RT-PCR with specific primer. Afterward the purified RT-PCR products were successfully ligated into pTZ57R/T plasmid vector. The pcDNA3 plasmid was used as expression vector and Eg95 fragment sub cloned into this plasmid. The pcEG95 plasmid was digested by restriction enzymes to confirm cloning of this gene in pcDNA3 plasmid. In last step، the subcloned gene was expressed in CHO as eukaryotic cell. EG95 fragment successfully was subcloned in pcDNA3 and EG95 protein was expressed by eukaryotic cell. The recombinant EG95 protein was confirmed by SDS-PAGE and Western blot. Recombinant plasmid of pcEG95 was constructed successfully and express of recombinant EG95 protein was confirmed.

Plasmodium vivax is the predominant species causes of malaria with about 90% total annual reported malaria in Iran. This study conducted to determine the susceptibility of Plasmodium vivax isolates to chloroquine in Sistan and Balochistan Province، southeastern Iran. A total 270 subjects with symptomatic malaria and confirmed P. vivax infection completed the designed 28-day in vivo study. The thick and thin film blood smears were screened for malaria parasites by microscopy. The nested PCR was applied using the Plasmodium 18 subunit ribosomal ribonu­cleic (Ssr RNA) genes for detecting mixed infections and diagnosis of parasites in the samples with low parasite on days 0، 5، 6، 7، and 28. P. vivax was cleared in 15%، 50%، 95%، and 100% of patients on days 1، 2، 3، 4 respectively by microscopy assessment. Six patients were exhibited specific P. vivax band in nested PCR on day 5. No recurrence was observed on days 7، 14 and 28. Mean (±standard deviation) parasite clearance time was 2. 41 (±0. 8) days. P. vivax is still susceptible to chloroquine in Southeatern Iran. This finding is compati­ble with results of neighboring countries Pakistan and Afghanistan.

Fasciolosis is an important health and veterinary problem in Iran. The epidemiological pattern of disease has been changed markedly in recent years and there are regions that have potent capacity to be new focus of the disease. One of these areas is Yasuj district in southwest of Iran where animal fasciolosis has been quite common. The current study was conducted to determine the seroprevalence of human fasciolosis in this area and to reveal the epidemiological factors associated with the spreading of the disease in this region. One thousand blood samples were randomly collected from five villages in Yasuj district. ELISA, using Fasciola somatic antigen (SA), was carried out to detect anti Fasciola antibodies in the collected sera. Anti-Fasciola antibodies were detected in serum of 18(1.86%) individuals by ELISA. Out of 18 seropositive people, 9 (0.9) were female and 9 (0.9%) were male. Most of people (99.8%) had a history of consuming wild freshwater plants mainly Nasturtium microphyllum (local name Bakaloo) and/or Mentha logifolia (local name Pooneh). No significant correlation was found between seropositiv­ity to fasciolosis and sex, age, history of consumption of green leafy aquatic plants whe­reas correlation between seropositivity and abdominal pain was significant (P< 0.05). Results of this study showed that the seroprevalence rate of human fasciolosis in Yasuj district is relatively high and this area can be considered as a new emerging focus of the disease in Iran.

The purpose of this study was to evaluate antileishmanial effects of ASA via NO pathway in Leishmania major infected Balb/c mice. Moreover، toxicity and pathological consequences of ASA administration were investigated. Balb/c mice were infected with L. major and ASA was inoculated orally after lesion appearance for its ability to modulate NO and to modify Leishmania infection in host، in order to evaluate the effects of NO production on size and lesion macroscopy، delay of lesion formation and proliferation of amasti­gotes inside macrophages. Liver، spleen، and lymph nodes were also studied as target organs to detect amastigotes. In addition، plasma was investigated for NO induction using Griess microassay. ASA increased NO production in plasma of both naïve and Leishmania test groups at the ultimate of the experimental period. A decline was observed in proliferation of amastigotes inside macrophages of test group when compared with control one. ASA reduced lesion size، inhibited Leishma­nia visceralisation in spleen، lymph node، and decreased hepato/splenomegaly in ASA treated animals. Some antileishmanial effects of ASA by NO-modulation were indicated during systemic leishmaniasis in mice. Despite slight effects on lesion size، ASA decreased parasite visceralization in target organs and declined their proliferation inside macrophages. Therefore، ASA may be indicated to inhibit systemic leishmaniasis via NO pathway in mice model.

Because of the strong immunologic responses of surface protein TaSp in Theileria annu­lata infected host، we tried to characterize this protein in a T. annulata isolate from Iran. The RNA prepared from T. annulata infected cells was used to produce SMART-DS-cDNA. The Double strand cDNA was then amplified with primers derived from TaSp mRNA se­quences. The PCR product was cloned in pTZ57R/T vector، sequenced and registered under acces­sion no. JQ003240 in GenBank. The sequence analysis showed 90%-94% nucleotide sequence identity and 68%-94% amino acid homology to the corresponding sequences of TaSp gene by T. annulata، T. sp. china I، T. sp. china and T. lestoquardi and three T. annulata reported from Iran respectively. Interestingly، the sequence analysis also showed small nucleotide sequence region near the 5` end in which the presented TaSp protein differed very strongly from the other known TaSp sequences. For the preparation of the recombi­nant protein، the cDNA was cloned in pQE-32 vector، the recombinant protein was pre­pared and assayed by Theileria infected bovine serum. The polymorphism in TaSp gene could be detected in intra- as well as inter species. The different characterized TaSp proteins had a common identic region، which may be helpful for develop­ment of broad band vaccine based on the recombinant proteins. The polymorphism in this gene، make this protein also interesting for the diagnostic purposes.

Infectivity of herbivores with Trichostrongylus nematodes is widespread in many countries، having a major economic impact on breeding، survivability، and productivity of domestic livestock. This study was carried out on Trichostrongylus species isolated from domestic livestock in order to develop an easy-to-perform method for species identification. Trichostrongylus isolates were collected from sheep، goat، cattle، and buffaloes in Khuzestan Province، southwest Iran. Primary species identification was carried out based on morphological characterization of male worms. PCR amplification of ITS2-rDNA region was performed on genomic DNA and the products were sequenced. Phylogenetic analysis of the nucleotide sequence data was conducted employing Bayesian Inference approach. Consequently، a restriction fragment length polymorphism (RFLP) profile was designed to differentiate Trichostrongylus species. A consensus sequence of 238 nucleotides was deposited in the GenBank for Iranian iso­lates of Trichostrongylus species including T. colubriformis، T. capricola، T. probolurus and T. vitrinus. The designated RFLP using restriction enzyme TasI could readily differentiate among species having differ­ent ITS2 sequence. The molecular analysis was in concordance with morphological findings. Phylogenetic analysis indicated a close relationship among the sequences obtained in this study and reference sequence of relevant species. ITS2-RFLP with TasI is recommended for molecu­lar differentiation of common Trichostrongylus species.

Geothermal waters could be suitable niches for thermophilic free living amoebae includ­ing Naegleria and Hartmannella. Ardebil Province، northwest Iran is popular for having many hot springs for recreational and health purposes activity. The present research is the first molecular based investigation regarding the presence of Naegleria and Hartmannella in the hot springs of Ardebil Province in Iran. Overall، 30 water samples were taken from waters of thermal hot springs in Ardebil Prov­ince، Iran during 2010-2011. All collected samples were transferred to Dept. of Parasitology and Mycol­ogy، Shahid Beheshti University of Medical Sciences، Tehran، Iran. Cultivation of concentrated wa­ter samples was performed using culture-enrichment method. Cloning of the target amoebae was ob­tained and morphological and molecular analysis was done using page key combined with two sets of primers، respectively. Sequence analysis and homology search was used for strains identification. Of 30 water samples، 8 (26. 7%) were positive for thermotolerant Vahlkampfiids and Hartman­nella based on morphological characteristics of vegetative form and double walled cysts. Clon­ing of the target amoebae were done successfully. Sequencing of the positive isolates revealed that the strains belonged to Naegleria (N. carteri and N. spp) and H. vermiformis. The result highlights a need for improved filtration and disinfection and periodic monitor­ing of recreational thermal waters in order to prevent disease related to free- living amoebae. This is the first comprehensive molecular study of thermophilic Naegleria and Hartmannella in hot springs of Iran.

Cutaneous leishmaniasis is a neglected parasitic disease، which imposes massive human distress and financial costs to the endemic countries. Better understanding of host immune response to the parasite leads to helpful strategies for disease control. Interleukin (IL) -10 and transforming growth factor (TGF) -β are important immune regulatory cytokines، which appear to develop non-healing forms of leishmaniasis. However، there is little information about the function of IL-10 and TGF-β in old world cutaneous leismaniasis. The aim of this study was to analyze the role of IL-10 and TGF-β in human cutaneous leishmaniasis due to Leishmania major infection. Biopsies were obtained from lesions of twenty proven cases of L. major induced cutaneous leishmaniasis. IL-10 and TGF-β positive cells were detected by immunofluorescence staining of frozen sections and compared between two groups of patients with early and late lesions. The mean percentage of IL-10 positive cells were significantly (P= 0. 035) higher in late lesions (0. 51±0. 24) than early ones (0. 15±0. 07). Similar results were obtained for TGF-β with mean percentages of 0. 16±0. 05 and 0. 53±0. 28 in early and late lesions respectively (P= 0. 008). IL-10 and TGF-β are present in lesions of L. major induced cutaneous leishmaniasis and contribute to the pathogenesis of long lasting disease forms.

The present study deals with the effect of helminthic infection as Nematode parasite like Aspiculuris tetraptera on the haematological parameters of infected and vaccinated mice. Totally 15 mice were used. Five mice were used for positive control، 5 mice used for nega­tive control and 5 mice used for experiment. The hematological parameters were studied viz. RBC، Hb، and serum protein values. The mice carrying heavy infection showed decrease in the Hb، RBC، and serum protein but in the vaccinated mice، all studied parameters were become on normal range. The level of immune response was assessed based on above studied hematological parameters in infected and vaccinated mice with Aspiculuris tetraptera. The increased value of RBC، Hb and Serum protein in infected and vaccinated mice compared to infected and non vaccinated suggested the involvement of blood parameters in immune response. This study also proves that somatic antigen of A. tetraptera was effective in imparting immunity in mice

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.

Bovine theileriosis results from infection with obligate intracellular protozoa of the genus Theileria. The phylogenetic relationships between two isolates of Theileria annulata، and 36 Theile­ria spp.، as well as 6 outgroup including Babesia spp. and coccidian protozoa were analyzed using the 18S rRNA gene sequence. The target DNA segment was amplified by PCR. The PCR product was used for direct sequencing. The length of the 18S rRNA gene of all Theileria spp. involved in this study was around 1،400 bp. A phylogenetic tree was inferred based on the 18S rRNA gene sequence of the Iran and Iraq isolates، and other species of Theileria available in GenBank. In the constructed tree، Theileria annu­lata (Iran vaccine strain) was closely related to other T. annulata from Europe، Asia، as well as T. lestoquardi، T. parva and T. taurotragi all in one clade. Phylogenetic analyses based on small subunit ribosomal RNA gene suggested that the percent identity of the sequence of Iran vaccine strain was completely the same as Iraq sequence (100% identical)، but the similarity of Iran vaccine strain with other T. annulata reported from China، Spain and Italy determined the 97. 9 to 99. 9% identity.

The aim of this study was to determine the Toxoplasma antibodies in pregnant women in Zanjan، by ELISA method. Blood samples were taken from 500 pregnant women referred to the health centers of Zanjan City، North West Iran، IgM and IgG titers were primarily evaluated. The collected data were analyzed with SPSS 11. 5 using Chi-Square test. Anti Toxoplasma IgM and IgG were positive in 1. 4% and 37. 2% respectively. Seropositive subjects were more frequently seen in those with >30 years old compared to younger women (<20 years old). No significant relationship was found between the seroprevalence of T. gondii infection and level of education، residence area، history of abortion and gestational age. The rate of IgM positive was low; however، a large number of the studied population were IgG positive، indicative of having a latent infection due to the past exposure to Toxoplasma para­site in this region.

Hydatidosis is one of the major zoonotic diseases that cause considerable public health problems in Iran. The present study was designed to investigate pediatric hydatidosis in pa­tients referred to the Children Medical Center Hospital in Tehran، Iran during 2005-2010. Data were collected from the records of 17 patients referred to the center with hydatidosis. Data included demographic data; laboratory results، type، and site of cysts، clinical manifestations، and treatment. Nine patients were boys (52. 9 %) and eight (47. 1 %) were girls. Most patients referred from central areas of Iran (58. 8%). Seven patients had cysts in their lungs (41. 2%) and three cases (17. 6 %) in liver. Six cases (35. 3 %) had simultaneous lung and liver cysts، 3 patients (17. 6%) had brain cysts (alone or in combination with other organs involvement) and 2 patients (11. 7%) showed multi-organ involvement. All patients were treated by albendazole and underwent surgery، recurrence was seen in 4 (23. 5 %) of the cases and one patient died due to rupture of the cyst and anaphylactic shock. Multi-organ involvement seems to be on the rise in children، this has led to the necessity for physicians to be more aware of clinical features، search، and rule out other organs for involvement diagnosis once a cyst is detected in one organ.

Hadjelia truncata is a nematode that causes lesions in the gizzard lining of pigeons، which may even lead to death. The aim of this study was to introduce Alphitobius diaperinus as a new intermediate host for Hadjelia truncata. H. truncata infection was identified in a pigeon flock in Ahvaz City، Khuzestan Province، Iran by performing fecal examination and autopsy. Adult and larval stages of beetles were collected from the litter of pigeon houses، and identified morphologically. The beetle larvae were cultured in a medium، containing feces of the infected pigeons. Nematode larval stages from naturally and experimentally (culturally) infected adult beetles were fed to two groups of pigeons. The collected beetles were identified as Alphitobius diaperinus. Average length and width of the adult beetles were 6. 31 mm and 2. 88 mm respectively. Infection rates of naturally and experimentally infected beetles with larval stages of the nematode were 66. 2% and 45. 1% respectively. The adult nematodes collected from gizzards of experimentally infected pigeons were identified as H. truncata. Nematode infection rates in pigeons after feeding the infective larvae collected from naturally and experimentally infected beetles were 44. 7% and 32. 5 % respectively. A. diaperinus can serve as a natural intermediate host for H. truncata.

Cutaneous leishmaniasis is one of the most important parasitic diseases، which are endemic in different parts of Iran. Leishmania major and L. tropica are the primary causative agents of this disease. The aim of the present study was to detect the multiple forms of L. major in lung. Ppromastigotes of L. major at stationary phase were injected to BALB/c mice. After 60 days، the different forms of Leishmania parasites were checked in lung tissue. Promastigote and amastigote forms of Leishmania parasites were detected.

The heartworm disease is an infectious disease of dogs with Dirofilaria immitis combined with cardiovascular and circulatory abnormalities. The heartworm disease can become a serious health risk when associated with a severe infection. In this study، a male، 8 year-old dog that died suddenly was necropsied and all tissues were examined grossly. Major organs including heart، lungs، liver، spleen، kidneys، brain، eyes، and testis were fixed in 10% neutral formalin، embedded in paraffin، sectioned at 5-µm thickness، stained with hematoxylin and eosin، and examined with a light microscope. For each examined organ، paraf­fin-embedded tissues were cut and placed in eppendorf tubes for genomic DNA extraction. PCR was performed using two sets of primers for amplification of a 302 bp ITS-2 gene fragment and a 203 bp cytochrome oxidase subunit 1 (CO1) gene fragment of D. immitis. During the necropsy examination، 46 adult D. immitis were found in the portal vein، right ventricle، and atrium of the heart and pulmonary trunk. Microscopically، microfilarias were found throughout the vessels of different organs including lungs، kidneys، liver، heart، brain، and spleen. All tissues examined by PCR were positive for D. immitis ITS-2 and CO1. PCR technique now represents an effective method for identification of D. immitis from formalin-fixed samples.