[ 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

[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

effect of vitamin D3 alone and mixed with IFN-gamma on tachyzoites of toxoplasma gondii [RH strain] proliferation and nitric oxide [NO] production in infected macrophages of BALB/C mice

Toxoplasma gondii is an obligatory interacelullar parasite that infects nucleated cells in its intermediate hosts. The aim of the present study was to determine the effect of vitamin D3 on the multiplication of T. gondii in peritoneal macrophage of Balb/c mice and nitric oxide production by macrophages. According to usage of vitamin D3 [one dose or seven doses] and INF gamma in vitro and in vivo, this study was divided into four experiments. In all experiments, the macrophages were collected from peritoneum and cultured in RPMI-1640. Then the supernatants were collected after 24 h and their nitric oxide was measure. After 96 h, the macrophages were collected and stained and the number of tachyzoites was measured. The first experiment [the mice were infected with tachyzoites and after 2 h, got one dose vita min D3 intraperitonealy] showed the best results. The mean of tachyzoites per macrophages was 2.37, and mean +/- SD of nitric oxide was 187.8 +/- 9. High-level production of nitric oxide may be related to the only one injection of vita min D3. The injection in long time might suppress the immune system

[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

effect of Alkanna tincturia and Peganum harmala extracts on leishmania major [MRHO/IR/75/ER] in vitro

Cutaneous leishmaniasis is an important health problem caused by Leishmania spp. As there is no vaccine, drug treatment is the only way to tackle leishmaniasis. In the present study, inhibitory and killing effects of Peganum harmala and Alkana tinctoria extracts on amastigotes and promastigotes forms of Leishmania were evaluated in-vitro. The seeds of Peganum harmala, Stems and roots of Alkanna tictoria were collected and crude extraction carried out. In this experimental study, Leishmania major promastigotes were cultured in RPMI-1640 with 10% FBS at 22-26°C, and infected macrophages with amastigotes were cultured in RPMI-1640 with 10% FBS at 37°C in 5% CO2. Then the extracts of each plant were added to cultivated parasites and incubated for 3 days. Promastigote and amastigote assay was carried out using counting assay based on growth inhibition. The results indicated that both extractions can inhibit the growth of promastigotes, and in concentrations of 40 micro g/ml of P. harmala, 200 micro g/ml of A. tincturia, and 20 micro g/ml of equal combination of P. hamala and A. tincturia are Inhibitory Concentration [IC50] for parasites growth. By adding these concentrations of the extracts to the infected macrophages in the culture, their effects were separately evaluated. The mean of amastigotes number in macrophages in the culture with P. harmala, A. ticturia, combination and control groups were 0.7, 0.7, 0.6, 2.3 amastigotes per macrophage, respectively. By this method, inhibition of intracellular and extracellular growth of L. major was demonstrated suggesting that, plant drugs with efficacy and safe products can be applied as new treatment for cutaneous leishmaniasis

[Study on variation of the sera folic acid, vitamin B12 and iron level in the 6-12 years old patients infected with Giardia lamblia in south Tehran]

Protozoa Giardia lambelia is caused to diarrhea in human and other mammals in worldwide. Giardia colonizes in duodenum and earliest jejunum. It can cover the intestinal surface and causes the stateorrhea, malabsorbtion syndrome and absorb disorder of vitamins A, E and D. Investigation the effect of Giardia on the vitamin B12, folic acid and iron of patients were the purpose of this study. A total of 30 children with giardiasis, aged between 6-12 years-old, selected from 3000 patients, who were admitted to the Emam Khomeiny hospital and diagnosed by stool examinations [direct and formalin-ether methods]. Blood of patients and control were collected. Amount of vitamin B12 and folic acid were evaluated with radioimmunoassay and iron was evaluated with Ferene method. According to the data, folic acid in the patient had no significant difference against control but vitamin B12 and iron in the patient with giardiosis was less than control and the differences were significant [P=0.01, P=0.04 respectively]. According to the results diagnosis of giardiosis in the early stage may prohibit intestinal damage and clinical symptoms due to vitamins and mineral elements shortage in the children

Seroepidemiological Survey of Hydatid cyst by ELISA in Kordestan Province

This study was conducted to evaluate the frequency of human hydatidosis in Kurdestan province by ELISA technique. In this study the sera of 1979 individuals were collected from different area [cities and villages] of Kordestan province. The serum dilution of 1/400 was selected for ELISA test. The results indicated that 1.12% of the individuals from Kordestan province showed positive sera. The results also showed that in Kordestan 0.9% and 1.42% of the people who live in the cities and villages had positive sera respectively. In this study 1.65% of female and 0.45% of male were positive. From the obtained result we found maximum number of infected people were in the range of 30-40 years [1.59%]. According to the results obtained from this study the highest percent of infection was found in the city of Ivandarre, the reason for this difference [1.69%] is due to the fact that most of the people who are involved in animal husbendary in the province live in this city

Natural infection of sand files Sergentomyia dentata in Ardebil to Lizard Leishmania

An entomological survey was carried out for Leishmania vector incrimination of sand flies in northwestern Iran. Among other specimens, 358 sand flies belong to the Sergentomyia Genus were tested for leptomonad infection using semi-nested PCR method as well as sequence analysis of ITS-rDNA fragment. Results of semi-nested PCR against kietoplast DNA showed reptile leptomonad infection in two specimens of S.dentata. The ITS2 sequence analysis of the specimens revealed 76% identity with those of Leishmania [sauroleishmania] adleri of Genbank. However, further studies need to clarify the species identity of the leptomonads. Interestingly, blood meal analysis of the sand flies determined an S.sintoni specimen with mammalian hemoglobin. This reptile related sauroleishmania parasites lacks the Lipophosphoglican [LQG] necessary for entrance to human phagocytes cells, and hence are not human pathogen. However, the GlycoInositoPhosphLipid[GIPL] molecules of this parasite reacts with sera of kala-azar patients and may cause false positive scores in sero-epidemiological surveys for kala-azar. Sauroleishmania can be transmitted to human infected bite of some Sergentomyia subgenera that show intermediate characteristics of Phlebtomus Genus. They are able to feed on human blood. This is the first report on presence of L. [sauroleishmania] adleri as well as ingestion of mammalian hemoglobin Sergentomyia sand flies in Iran

effect of betamethasone and IFN-gamma on replication of toxoplasma gondii [RH strain] and nitric oxide production in HeLa cell culture

Toxoplasmosis is a protozoal infection caused by Toxoplasma gondii. Toxoplasmosis produce severe damage in patients who are immunosuppresed. In those who are immunosupressed, latent infection can be reactivated resulting in acute disseminating disease. Betamethasone is a synthetic glycocorticoid, used as an anti-inflamatory and immunosuppressant in a wide variety of disorders. The aim of this study was evaluation of betamethasone as an immunosuppressor drug on infected cells by Toxoplasma gondii. In this study, at first HeLa cells were grown in 24 well culture plates in culture medium .When confluent monolayer was obtained, we compared 6 groups to evaluate the effect of betamethasone as a corticosteroid drug [two concentrations 4 and 40microg/ml] and the effect of IFN-gamma [100 IU/ml] on growth, replication and Nitric Oxide [NO] production. The results showed, that high number of plaques were seen in group with 40 microg/ml of betamethasone and the lowest number of plaques were seen in group with 100 IU of IFN-gamma. The difference between plaque number in control and groups treated with IFN-gamma and betamethasone was significant [P<0.05]. The groups with betamethasone or IFN-gamma without tachyzoites did not show any effect on cell structures. Replication rates in the wells treated with IFN-gamma were decreased significantly 72h post inoculation in comparison with control group [P< 0.05]. There was no significant difference among different groups in NO production. The results indicated that betamethasone increase the invasion of tachyzoites to host cells in vitro

In vitro effect of folic acid and cobalamin [vitamin B12] on adhesion and growth of Giardia lamblia

Giardia lamblia is one of the most common intestinal protozoan parasites infecting human in the world. The goal of this study was searching for in-vitro effect of folic acid and cobalamin on adhesion and growth of G. lamblia as two important mechanisms in the pathogenesis in TYI-S-33 medium. G. lamblia trophozoites were obtained by in- vitro excystation procedure. Three groups of Giardia trophozoites were analyzed: control group, G.lamblia was cultured in TYI-S-33 without any vitamin, 2nd group with 0.1 micro g/ml vitamin B12 or folic acid, and 3rd group with 0.5 micro g/ml of vitamin B12 or folic acid. All culture media tubes incubated at 37 °C. After 2 h of incubation, the adherence into borosilicate culture tubes, and after 24 h the growth of trophozoites were measured .The results showed that in vitamin B12 groups, the growth was increased significantly [P? 0.05] but the adherence decreased significantly [P

Clinical appearance, leishmanin test and elisa using monoclonal antibody in diagnosis of different forms of cutaneous leishmaniasis

Introduction: Cutaneous Leishmaniasis [CL] has 2 principal clinical forms in Iran: Anthroponotic and Zoonotic. These forms, previously called Dry and wet forms, are caused by Leishmania tropica and L. major respectively. Formerly, diagnosis of different forms was based on epidemiological status and clinical signs; but at present, definite diagnosis by advanced laboratory tests such as ELISA and Isoenzyme methods is possible. In order to investigate correlation between ELISA test, clinical appearance, and skin test, a study was undertaken in Emam Reza Hospital, Mashhad

Material and Methods: The study population was selected among the volunteers who had suspected skin lesions for C.L. Direct smear, culture and Leishmanin skin test was performed for 153 patients. ELISA using specific monoclonal antibodies [SMA] performed species determination

Results: The minimum and maximum ages of the patients were 19 months and 97 years old respectively. Most of the patients were Females [63.9%]. Among 72 patients whose cutaneous lesions were approved for Leishmaniasis, 91.6% had ulcers with dry appearance and 8.4% had appearance of wet form C.L. The etiologic agents isolated from the skin lesions of the latter patients were L.tropica [66.6%], L.major [28.8%] and unknown form [4.2%]. Among the patients who had skin lesions with wet appearance, the isolated agents were L.tropica [5.6%], L.major [1.4%] and unknown form [1.4%]. The sensitivity of Leishmanin Skin Test was higher in-patients infected by L. major

Conclusion: ELISA method using SMA is a sensitive and reliable test for differential diagnosis of Anthroponotic Cutaneous Leishmaniasis [ACL] and Zoonotic Cutaneous Leishmaniasis [ZCL]. Both ACL and ZCL are present in Mashhad. ACL is three times more prevalent than ZCL. Clinical appearance is not a valid factor for determination of species of Leishmania. The sensitivity of Leishmanin Skin Test is higher in wet form in relation to dry form. There may be other species of Leishmania causing cutaneous lesions in Mashhad