Molecular detection of Leishmania spp. Isolated from cutaneous lesions of patients referred to Herat regional hospital, Afghanistan

Cutaneous leishmaniasis is one of the main public health problems in Afghanistan, particularly in Herat. To identify Leishmania spp., molecular techniques were applied to samples from 64 cutaneous leishmaniasis patients referred to Herat regional hospital during 2013. Polymerase chain reaction [PCR]-restriction fragment length polymorphism [RFLP] analysis of the ribosomal RNA gene internal transcribed spacer-1 [ITS1] was used. Most of the patients demonstrated dry type single lesions on the head. The results of direct microscopy detection using Giemsastained skin scrapings were compared with that of ITS PCR-RFLP for the diagnosis of cutaneous leishmaniasis. Light microscopy examination showed 37/64 positive cases [58%]. PCR revealed 50 positive cases [78%], from which ITS PCR-RFLP identified 48 cases [96%] as L. tropica and 2 cases [4%] as L. major. Cutaneous leishmaniasis in Herat appears to be endemic and of the clinically dry type, caused mainly by L. tropica and occasionally by L. major

[Prevalence and severity of animal Fasciolosis in six provinces of Iran]

Fasciolosis is one of the most important parasitic disease common among both humans and livestock. Considering the health and economic importance of the disease, an understanding of the epidemiology of Fasciolosis is highly crucial. This study aimed to evaluate the prevalence and severity of Fasciola infection in animals from different geographical regions of Iran during 2009-10. In this descriptive study, 11100 livers taken from slaughtered sheep and cattle were carefully examined for Fasciola parasites at the six industrial slaughterhouses of East Azerbaijan, Khorasan-Razavi, Khuzestan, Fars, Mazandaran and Markazi provinces. All Fasciola parasites isolated from the livers of infected animals were transferred to the laboratory, and then the parasite species were identified and counted. Finally, the frequency distribution and the severity of infection were analyzed. In this study, 1.10% of the total sheep and cattle slaughtered in six industrial slaughterhouses were found positive for Fasciolosis. The severity of Fasciola in sheep and cattle livers was 7.77 +/- 0.42 and 15.24 +/- 1.78, respectively. Khorasan Razavi and Fars provinces had the highest [14.54 +/- 3.16] and lowest [7.75 +/- 0.79] severity of infection, respectively. Rresults of the study show a reduction in the prevalence and severity of Fasciolosis in sheep and cattle. But considering the importance of the disease and its endemicity, the preventive measures should be taken against the animal and human Fasciolosis in Iran

[ molecular beacon-based real time PCR assay for quantitative detection of Toxoplasma gondii in rat]

Application of quantitative real time PCR has evolved as a sensitive, specific, and rapid method for the detection of Toxoplasma gondii [T. gondii]. The present study aimed to evaluate the efficacy of real time PCR method, using B1 gene, for the diagnosis of toxoplasmosis in the experimentally infected rats. Parasites were cultured in peritoneal cavity of mice and then the DNA was extracted in tachyzoite stage. The B1 gene of T. gondii was amplified by PCR and detected by real time PCR method based on the molecular beacon probe. Finally, real time PCR was evaluated for the quantization of T. gondii in the blood of the experimentally infected rats. The B1 gene of T. gondii which was successfully amplified by PCR yielded an amplicon with an approximate length of 116 bp. Using this gene was evaluated highly appropriate for the quantization of T. gondii by real time PCR method. Application of real time PCR method is shown to be highly efficient in terms of sensitivity and rapidity for the detection of B1 gene as well as the quantization of T. gondii in blood of rat

[Evaluating the immunogenicity for plasmid encoding GRA5 antigen of Toxoplasma gondii in BALB/c mice]

Severe or lethal damages of toxoplasmosis clearly indicate the need for the development of a more effective vaccine. Immunization with recombinant plasmid encoding protective proteins is a promising vaccination technique. Therefore, this study aimed to evaluate the immunization with plasmid encoding GRA5 antigen of Toxoplasma gondii in BALB/c mice. In this experimental study, three groups of BALB/c mice [n=10 in each group] were selected using simple random sampling. GRA5 gene was cloned into pcDNA3 plasmid and purified by plasmid purification kits and then the product was injected [IM]. To determine the status of cellular and hurnoral immunity, the 11-4, IFN- gamma and IgG; IgG2a, IgG subtypes were evaluated respectively using the ELISA-based assay. The group immunized with pcGRA5 indicated a significant augmented response in humoral and cellular immunity [P

[Morphological and molecular characterization of dicrocoelium isolated from sheep in the north and center of Iran]

Dicroceliosis is a hepatic parasitic disease of clinical and financial significance for both human health and animal breeding. Considering the health and economic importance of the disease, this study aimed to determine the morphological and molecular characterization of 28S rDNA for Dicrocoelium isolated from sheep in the north and center of Iran during 2010-11. A total number of 200 trematodes were collected during an abattoir inspection from livers of naturally infected sheep in East Azerbaijan, Razavi Khorasan, Mazandaran and Tehran provinces in Iran. Adult worms were morphologically identified based on morphometric characterization and 60 specimens were characterized molecularly by sequencing. For molecular study, DNA was extracted and 28S rDNA region was amplified by PCR. Then, Tru1I fastdigest restriction enzyme and also RFLP technique were used to identify the parasite species. Finally, the PCR product was sequenced. A remarked morphological characteristic was that the orientation of testes in all isolates, were in tandem. Position the homological comparison of sequences showed that 28S rDNA in all isolates of Dicrocoelium had 963 bp and were similar to standard strain registrated in Genbank. RFLP pattern from D.dendriticum, which had 4 cut sites, produced 116, 145, 293 and 409 bp fragments. Although the morphological characterization in various provinces was significanly different, molecular identification showed that all specimens were identical [D.dendriticum] and there was not a significant difference between sequences of the collected parasites. Morphological and molecular assays show that Dicrocoelium dendriticum is the only species of Dicrocoelium among sheep in the north and center of Iran

Molecular cloning and expression of EG95 gene of Iranian isolates of Echinococcus granulosus

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

[Determination and cloning of the gene encoding EG95 protein in Iranian isolate of Echinococcus granulosus]

Echinococcus granulosus is a cestode parasite that causes cystic hydatid disease in humans worldwide. The gene encoding EG95 protein may be a good candidate to design a DNA vaccine to prevent the disease. Considering the importance of EG95 gene and the scarceness of research on it in Iran, this study was carried out to determine and clone the gene encoding EG95 from Iranian isolate of E. granulosus.At the first stage, protoscoleces was isolated from hydatid cyst fluid and then RNA was extracted from protoscoleces and after performing RT-PCR, the amplified cDNA samples were detected by gel electrophoresis. In next stage, the obtained gene was cloned in pTZ57R/T vector. Two methods were used for conformation of cloning: colony PCR amplification and digestion with the EcoRI and XhoI restriction enzymes. Finally, the cloned EG95 gene in pTZ57R/T vector was sequenced. Homological comparison of sequences showed that cDNA of EG95 in Iranian isolate of E. granulosus had 492 bp and was different from the standard strain of EG95 reported from New Zealand and Australia [X90928.1]. Moreover, cloning of EG95 gene in pTZ57R/T plasmid was confirmed by digestion of this plasmid with the restriction enzymes. The EG95 gene was cloned in pTZ57R/T plasmid successfully and this plasmid can be used to design a DNA vaccine in further studies

[Cloning and sequencing the plasmid encoding Toxoplasma gondii Microneme 3 protein]

Toxoplasma gondii, an obligatory intracellular protozoan parasite, causing toxoplasmosis in human and animal with worldwide spread. Microneme 3 [MIC3] protein, a 90 kDa parasite factor attaching to the host cells in the beginning of the invasion, is secreted in all stages of parasite development [e.g. sporozoite, tachyzoite and bradyzoite] and also is considered as a potent antigen. Therefore, besides the immunogenicity and the candidacy for vaccine design, the protein is used for diagnostic purposes, as well. The aim of the present study was to transfer MIC3 gene into plasmid vector [PTZ57R/T] for subcloning in eukaryotic and prokaryotic plasmids. Toxoplasmia genomic DNA extracted using phenol-chloroform method and MIC3 gene was then amplified by PCR with specific primers. Electrophoresis was performed by using agarose gel and PCR product was purified by T4 DNA ligase enzyme into a cloning vector. Finally, recombinant plasmid was transformed into E.coli [Top10 strain]. The extracted clone was verified with PCR, digestion enzymes and sequencing. The PCR product was seen as a 1052bp band in agarose gel [1%]. The recombinant plasmids was restricted by HindIII and EcoRV enzymes and two obtained 2886 and 1052bp bands showed that the MIC3 gene was cloned in PTZ57R/T plasmid. The results revealed that the cloning and transformation of MIC3 gene in pTZ57R/T was done successfully

Molecular identification and differentiation of fasciola isolates using PCR-RFLP method based on internal transcribed spacer [ITS1, 5.8S rDNA, ITS2]

In this study, we used both ITS1 and ITS2 for molecular identification of Fasciola species. The region between 18S and 28S of ribosomal DNA was used in PCR-RFLP method for molecular identification of Fasciola species. Ninety tematodes of Fasciola were collected during abattoir inspection from livers of naturally infected sheep and cattle from Khorasan, East Azerbaijan, and Fars provinces in Iran. After DNA extraction, PCR was performed to amplify region ITS1, 5.8S rDNA, ITS2. To select a suitable restriction enzyme, we sequenced and analyzed the PCR products of F. hepatica and F. gigantica samples from sheep and cattle. Tsp509I fast digest restriction enzyme was selected for RFLP method that caused the separation specifically of Fasciola species. The fragment approximately 1000bp in all of the Fasciola samples was amplified and then digested with the Tsp409I restriction endonuclease. Seventy F. hepatica and 20 F. gigantica were identified of total 90 Fasciola isolates. The new PCR-RFLP assay using Tsp509I restriction enzyme provides a simple, practical, fast, low cost, and reliable method for identification and differentiation of Fasciola practical, fast, low cost, and reliable method for identification and differentiation of Fasciola isolates

Latent toxoplasmosis and human

Toxoplasmosis is one of the most common parasitic diseases worldwide. Although estimated that one third of the world's population are infected with Toxoplasma gondii, but the most common form of the disease is latent [asymptomatic]. On the other hand, recent findings indicated that latent toxoplasmosis is not only unsafe for human, but also may play various roles in the etiology of different mental disorders. This paper reviews new findings about importance of latent toxoplasmosis [except in immunocompromised patients] in alterations of behavioral parameters and also its role in the etiology of schizophrenia and depressive disorders, obsessive-compulsive disorder, Alzheimer's diseases and Parkinson's disease, epilepsy, headache and or migraine, mental retardation and intelligence quotients, suicide attempt, risk of traffic accidents, sex ratio and some possible mechanisms of T. gondii that could contribute in the etiology of these alterations

Morphometric study on male specimens of hyalomma anatolicum [acari: ixodidae] in west of Iran

Hyalomma anatolicum is the well-known hard tick, which is one of the most important livestock and human pathogens vector, wide range in host and distributed in all over the Hyalomma geographic fauna as well as in Iran. Taxonomy of the Hyalomma ssp. is debatable whereas their identification is a problematic work. The reasons for this claim is time consuming Delpy's researches in Iran also Schulze School, Feldman-Muhsam and the Russian tick workers. We would like to understand morphometric variation in the field collected H. anatolicum in Iran also validating some morphologic quantitative and qualitative characters. A total 247 field-collected tick specimens from different geographical regions in west of Iran includes Khuzestan and Lorestan Provinces were studied. The morphologic characters of the ticks were measured by the calibrated stereomicroscope armed scaled lens. The measurements were analyzed using SPSS for windows, version 16 on an IBM PC, so varied shapes of species in different geographic regions were drawn by the aid of a drawing tube connected to a light stereomicroscope. One way ANOVA test revealed significant differences among the quantitative parameters in five zones [P< 0. 00 1] also each zone to other zone by Post Hoc Tests e.g. LSD. No significant differences in the lateral grooves length/conscutum length ratio parameter were found. Morphometric variation in Hyalomma spp is poorly studied. The variation in range and quantity of the morphometric parameters of H.anatolicum underlies that the correct recognition and key construction for Hyalomma species dependes on a complement morphometric study on the other species

[Identification of Sarcocystis gigantea by PCR- RFLP]

Sarcocyst is one the most important protozoa belonged to apicomplexa. This parasite is prevalent among warm blooded animals throughout the world. In the present work, Sarcocystis gigantean was identified by amplification of 18SrRNA gene using PCR- RFLP. In this regard macroscopic cysts of sarcocystis were collected from esophagus and intra costal muscles of sheep in Shahriar slaughterhouse. Then genomic DNA of the parasites was extracted from specimens using phenol-chloroform method. 18S rRNA gene was amplified with specific primers. For RFLP two restricted enzymes of MspI and MobI were used and the patterns were analyzed accordingly. According to the resulted band, all the specimens were identified as Sarcocystis gigantean. This is the first report of molecular identification of Sacrocystis gigantean in Iran

[Primary research on gastro-intestinal cryptosporidium incidence rate in lambs and calves in amol city, Iran]

Cryptosporidium is an ubiqutous enteric protozoan parasite that infects a wide range of vertebrate hosts. In humans and many other mammals, cryptosporidium is recognized as a significant pathogen primarily as a cause of acute, severe diarrheal illness. At this investigation animal samples [stool] were collected from 708 heads of lambs [in the beginning of the birth to three months] and 713 heads of calves [in the beginning of the birth to six months] in spring, summer, autumn and winter seasons at amol city in 1374. the samples were examinated after staining using modified zihil - nelson technique. Results showed, 29 samples of lambs [4.09%] and 28 samples of calves [3.92%] were positive, also in winter season infestation rate was higher than the other seasons [4.65%]. whereas infestation rate in animals without clinical signs is high, so this subject is a important problems for public health

Cloning and sequencing of Leishmania major thiol-specific-antioxidant antigen [TSA] gene

Leishmaniasis is caused by parasitic protozoa of the genus Leishmania which, in the infected host are obligate intracellular parasite. TSA is the immuno-dominant antigen of Leishmania major which is considered as the most promising molecule for a recombinant or DNA vaccine against leishmaniasis. Genomic DNA of TSA protein was extracted and amplificated as a template. Then the PCR product was cloned into pTZ57R/T vector. Finally, the recombinant plasmid was extracted from transformed Escherichia coli [TG1 strain] and sequenced. MRHO/IR/75/ER [an Iranian strain] of L. major and TSA gene [Accession number LmjF15.1080] were used. Sequence analysis of cloned TSA gene into pTZ57R/T vector showed high homology of 90% with LmjF15.1080 [TSA gene] and strain "LV39" [Accession no. AF069386] and strain "Friedlin" [Accession no.AF044679]. We cloned TSA gene of L. major successfully. Recombinant plasmid was confirmed. It is ready to express recombinant protein for further studies

alteration of liver function indices in rat infected with Toxoplasma gondii [RH strain]

Toxoplasmosis is a parasitic disease caused by Toxoplasma gondii. The migration of the parasite in blood as well as some organs like liver may cause some changes in physiological and biochemical indices in infected individuals. These may change the level of some indices like urea, total bilirubin, total protein, and albumin. The present investigation was conducted to study alteration of some liver functional indices of rats which were experimentally infected with Toxoplasma gondii. 80 non-infected rats and 60 non-infected mice were selected. Rats were divided into two groups of 60 test and 20 control rats. The test group was infected intraperitoneally with 5xl0[4] tachyzoites. Every 3 days for 60 days, three rats from test group and one rat from control group were bled. Standard techniques were used for urea, bilirubin, total protein and albumin tests. In addition, the livers of infected rats were searched biologically for presence of the parasite using intrapritoneal injection in mice method. The results indicated that, Toxoplasma cyst was present in the liver of infected rats within 6 to 27 days post infection. The parasite disappeared in liver after 28 days of infection. Biochemical results indicated that, urea from 6[th] to 60[th] day, total bilirubin from 6[th] to 27[th] day, albumin and total protein from 6[th] to 12[th] day post infection were increased but decreased to normal values afterward. Generally, temporary alteration of some biochemical indices during experimental infection of rat with toxoplasmosis may occur. The alteration mainly is due to the parasite migration to various tissues of the animal and it shifts to the normal condition following cyst formation in brain or muscles

study on giardiasis in pregnancy

In this study 404 pregnant women in Islamshahr, a district southwest of Tehran, were examined for giardiasis. Results showed 7.92% of them to be positive. Abdominal pain, nausea and diarrhea were observed as the most common symptoms in infected mothers. Results also indicated that hematological parameters and weight gain of infected pregnant women did not differ significantly from those without any parasitic infection. Also, birth weight, length and head circumference of the neonates of these two groups of mothers did not show any significant difference

[ study on three cestode species of subfamily dipylidinae from stray cats of tehran]

A total of 102 stray cats were collected from different regions of Tehran. After autopsy, the small intestine of the animals were searched for the presence of helminths. Three species of Dipylidinae, Diplopylidium nolleri, Joyeuxiella pasqualei and Dipylidium caninum were recovered. The infection rate of Diplopylidium nolleri, Joyeuxiella pasqualei and Dipylidium caninum were 37.25%, 3.92% and 4.90%, respectively. In addition, these species were described, compared and drawn by camera lucida. Tins is the first report of Diplopylidium nolleri and Joyeuxiella pasqualei from in Iran

[ study of helminth parasites of Jackals in the northern parts of Iran]

A total of 100 Jackals were collected from different regions of Mazandaran and Guilan Provinces, in the north of Iran. After autopsy, different organs were examined for the presence of the helminthes. From a total of 25 isolated species 12 species had zoonotic importance which ten species including alata, Phagiccla sinoecum, Echinochasmus [E] schwartzi, Euparyphium sp. Plagiorchis sp. Spirometera erinacei, Spirometrahoughtoni, Strongyloides stercoralis, Taenia pisiformis, and Physalopterea sp., are reported for the first time in Iran. The highest prevalence of infection in Jackals belonged to hookwoums and following, Mesocestoides lineatus 35%, 23% and 22% prevalence respectively