Immunoinformatic analysis of immunogenic B- and T-cell epitopes of MIC4 protein to designing a vaccine candidate against Toxoplasma gondii through an in-silico approach

Purpose@#Toxoplasmosis, transmitted by Toxoplasma gondii, is a worldwide parasitic disease that affects approximately one-third of the world’s inhabitants. Today, there are no appropriate drugs to deter tissue cysts from developing in infected hosts. So, developing an effective vaccine would be valuable to avoid from toxoplasmosis. Considering the role of microneme antigens such as microneme protein 4 (MIC4) in T. gondii pathogenesis, it can be used as potential candidates for vaccine against T. gondii. @*Materials and Methods@#In this study several bioinformatics methods were used to assess the different aspects of MIC4 protein such as secondary and tertiary structure, physicochemical characteristics, the transmembrane domains, subcellular localization, B-cell, helper-T lymphocyte, cytotoxic-T lymphocyte epitopes, and other notable characteristic of this protein design a suitable vaccine against T. gondii. @*Results@#The studies revealed that MIC4 protein includes 59 potential post-translational modification sites without any transmembrane domains. Moreover, several probable epitopes of Band T-cells were detected for MIC4. The secondary structure comprised 55.69% random coil, 5.86% beta-turn, 19.31% extended strand, and 19.14% alpha helix. According to the Ramachandran plot results, 87.42% of the amino acid residues were located in the favored, 9.44% in allowed, and 3.14% in outlier regions. The protein allergenicity and antigenicity revealed that it was non-allergenic and antigenic. @*Conclusion@#This study gives vital basic on MIC4 protein for further research and also established an effective vaccine with different techniques against acute and chronic toxoplasmosis.

Antigenic properties of dense granule antigen 12 protein using bioinformatics tools in order to improve vaccine design against Toxoplasma gondii

Purpose@#Toxoplasma gondii is an opportunistic parasite infecting all warm-blooded animals including humans. The dense granule antigens (GRAs) play an important role in parasite survival and virulence and in forming the parasitophorous vacuole. Identification of protein characteristics increases our knowledge about them and leads to develop the vaccine and diagnostic studies. @*Materials and Methods@#This paper gave a comprehensive definition of the important aspects of GRA12 protein, including physico-chemical features, a transmembrane domain, subcellular position, secondary and tertiary structure, potential epitopes of B-cells and T-cells, and other important features of this protein using different and reliable bioinformatics methods to determine potential epitopes for designing of a high-efficient vaccine. @*Results@#The findings showed that GRA12 protein had 53 potential post-translational modification sites. Also, only one transmembrane domain was recognized for this protein. The secondary structure of GRA12 protein comprises 35.55% alpha-helix, 19.50% extended strand, and 44.95% random coil. Moreover, several potential B- and T-cell epitopes were identified for GRA12. Based on the results of the Ramachandran plot, 79.26% of amino acid residues were located in favored, 11.85% in allowed and 8.89% in outlier regions. Furthermore, the results of the antigenicity and allergenicity assessment noted that GRA12 is immunogenic and nonallergenic. @*Conclusion@#This research provided important basic and conceptual data on GRA12 to develop an effective vaccine against acute and chronic toxoplasmosis for further in vivo investigations. More studies are required on vaccine development using the GRA12 alone or combined with other antigens in the future.

Antigenic properties of dense granule antigen 12 protein using bioinformatics tools in order to improve vaccine design against Toxoplasma gondii

Purpose@#Toxoplasma gondii is an opportunistic parasite infecting all warm-blooded animals including humans. The dense granule antigens (GRAs) play an important role in parasite survival and virulence and in forming the parasitophorous vacuole. Identification of protein characteristics increases our knowledge about them and leads to develop the vaccine and diagnostic studies. @*Materials and Methods@#This paper gave a comprehensive definition of the important aspects of GRA12 protein, including physico-chemical features, a transmembrane domain, subcellular position, secondary and tertiary structure, potential epitopes of B-cells and T-cells, and other important features of this protein using different and reliable bioinformatics methods to determine potential epitopes for designing of a high-efficient vaccine. @*Results@#The findings showed that GRA12 protein had 53 potential post-translational modification sites. Also, only one transmembrane domain was recognized for this protein. The secondary structure of GRA12 protein comprises 35.55% alpha-helix, 19.50% extended strand, and 44.95% random coil. Moreover, several potential B- and T-cell epitopes were identified for GRA12. Based on the results of the Ramachandran plot, 79.26% of amino acid residues were located in favored, 11.85% in allowed and 8.89% in outlier regions. Furthermore, the results of the antigenicity and allergenicity assessment noted that GRA12 is immunogenic and nonallergenic. @*Conclusion@#This research provided important basic and conceptual data on GRA12 to develop an effective vaccine against acute and chronic toxoplasmosis for further in vivo investigations. More studies are required on vaccine development using the GRA12 alone or combined with other antigens in the future.

Evaluation of immune responses induced by GRA7 and ROP2 genes by DNA vaccine cocktails against acute toxoplasmosis in BALB/c mice

Background: The severe damages of toxoplasmosis clearly indicate the need for the development of a more effective vaccine. Immunization with plasmid DNA is a promising vaccination technique. Therefore, GRA7 plasmid was prepared to be used as a vaccine. The purpose of this study was evaluation of immunization with cocktail DNA vaccine including plasmids encoding Toxoplasma gondii ROP2 and GRA7 in BALB/c mice

Methods: In this study, 733 bp of GRA7 gene was cloned in pCDNA3.1 plasmid as an expression vector. The plasmids containing GRA7 and ROP2 genes were administered via IM according to immunized mice three times with a 3 week interval. For lymphocyte proliferation and cytokine assay, splenocytes of immunized mice were cultured for proliferation and cytokine assay. The other mice in each group were inoculated by the parasite and mortality of the mice was evaluated on a daily basis

Results: The cytokine assay results and lymphocyte proliferation response in cocktail DNA vaccines showed that IFN-gamma levels were significantly higher than controls [p<0.05], whereas IL-4 expression level in BALB/c mice immunized with cocktail was lower than that in control groups and these results are confirmed by MTT test. Predominance of the levels of IgG2a over IgG1 was observed in sera of the immunized mice. Furthermore, IgG2a values in cocktail DNA vaccines pcGRA7 were significantly higher than control group [p<0.01]. In contrast, IgG1 antibodies were similar between the two groups [p>0.5]. So, survival time in the immune groups was significantly prolonged in comparison to control ones [p<0.01]

Conclusion: The immunized mice by DNA vaccine produce higher titration of IFNgamma, indicated with Th1 response which is confirmed by high level of IgG2a. These data demonstrate that the cocktail GRA7/ROP2 is a potential vaccine candidate against toxoplasmosis

Molecular detection and genotypic characterization of toxoplasma gondii in paraffin-embedded fetoplacental tissues of women with recurrent spontaneous abortion

Background: Congenital toxoplasmosis is an important cause of spontaneous abortion worldwide. However, there is limited information on detection and genotypic characterization of Toxoplasma gondii [T. gondii] in women with recurrent spontaneous abortion [RSA]. The aim of this study is the molecular detection and genotypic characterization of T. gondii in formalin-fixed, paraffin-embedded fetoplacental tissues [FFPTs] of women with RSA that have referred to the Avicenna Research Institute in Tehran, Iran

Materials and Methods: This experimental research was undertaken on 210 FFPTs of women with RSA. The information of the patients was collected from the archives of Avicenna Research Institute in Tehran, Iran. After DNA extraction, the presence of T. gondii was examined by nested polymerase chain reaction targeting the GRA6 gene. Genotyping was performed on positive samples using polymerase chain reaction-restriction fragment length polymorphism [PCR-RFLP] that targeted the GRA6 and SAG3 genes. Sequencing was conducted on two GRA6 positive samples

Results: T. gondii DNA was detected in 3.8% [8/210] of the samples. Genotyping showed that all positive samples belonged to type III of the T. gondii genotype. Sequencing two genomic DNAs of the GRA6 gene revealed 99% similarity with each other and 99-100% similarity with T. gondii sequences deposited in GenBank. There were six patients with histories of more than three abortions; one patient had a healthy girl and another patient had two previous abortions. Abortions occurred in the first trimester of pregnancy in seven patients and in the second trimester of pregnancy in one patient

Conclusion: The results of this study have indicated that genotype III is the predominant type of T. gondii in women with RSA in Tehran, Iran. Also, our findings suggest that toxoplasmosis may play a role in the pathogenesis of RSA. However, further studies are needed to elucidate a clear relationship between T. gondii infection and RSA

DNA vaccination with a plasmid encoding LACK-TSA fusion against Leishmania major infection in BALB/c mice

Vaccination would be the most important strategy for the prevention and elimination of leishmaniasis.The aim of the present study was to compare the immune responses induced following DNA vaccinationwith LACK (Leishmania analogue of the receptor kinase C), TSA (Thiol-specific-antioxidant) genesalone or LACK-TSA fusion against cutaneous leishmaniasis (CL). Cellular and humoral immuneresponses were evaluated before and after challenge with Leishmania major (L. major). In addition,the mean lesion size was also measured from 3th week post-infection. All immunized mice showed apartial immunity characterized by higher interferon (IFN)-γ and Immunoglobulin G (IgG2a) levelscompared to control groups (p<0.05). IFN-γ/ Interleukin (IL)-4 and IgG2a/IgG1 ratios demonstratedthe highest IFN-γ and IgG2a levels in the group receiving LACK–TSA fusion. Mean lesion sizesreduced significantly in all immunized mice compared with control groups at 7th week post-infection(p<0.05). In addition, there was a significant reduction in mean lesion size of LACK-TSA andTSA groups than LACK group after challenge (p<0.05). In the present study, DNA immunizationpromoted Th1 immune response and confirmed the previous observations on immunogenicity ofLACK and TSA antigens against CL. Furthermore, this study demonstrated that a bivalent vaccinecan induce stronger immune responses and protection against infectious challenge with L. major.

[Evaluation of the effect of achillea biebersteinii afan essential oil on leishmania major promastigote and amastigote growth under in vitro conditions]

Objective: Cutaneous leishmaniasis is an endemic disease in certain areas of Iran. The use of pentavalent antimony compounds as first line treatment has been reported, however they are associated with limitations and adverse events. Hence, an attempt to find a new, effective compound has been under consideration. This study examines the effect of Achillea biebersteinii afan as, a native plant in Iran, against Leishmania major promastigote and amastigote growth under in vitro conditions

Methods: This experimental study was performed at Tarbiat Modares University in 1392.We extracted the essential oil of the Achillea biebersteinii afan plant by steam distillation and analyzed it by gas chromatography mass spectrograph. Then, we evaluated the effect of different concentrations [10%, 15%, 25% and 50%] of the oil on the growth of the promastigotes stage of Leishmania and infected macrophages that contained amastigotes under in vitro conditions. Effectiveness of the oil on promastigotes and amastigotes was assessed by direct count and the MTT assay. In all tests, each of the wells that contained culture media and parasites without drug were considered the control group. Data analyses were conducted with ANOVA

Result: The MTT results indicated significant differences among the number of parasites in the control and case groups treated with 10%, 15%, 25%, and 50% of the oil within 24, 48 and 72 hours after culture. The concentration of 50% of the oil killed 66% of the macrophages that contained amastigotes after 72 hours

Conclusion: Achillea biebersteinii afan oil was effective in killing Leishmania major promastigotes and infected macrophages that contained amastigotes. We have proposed to study the extract in vivo for treatment of cutaneous leishmaniasis lesions

[ killing effect of silver nanoparticles and direct electric current induction on leishmania major promastigotes in vitro]

Objective: Worldwide, Leishmania major is one of the major causes of cutaneous leishmaniasis, including Iran. In the present study we investigate the effect of a direct electricity current in combination with silver nanoparticle on the killing of Leishmania major in vitro

Methods: We evaluated the effects of different concentrations of silver nanoparticles against Leishmania major promastigotes in vitro, then the half maximal inhibitory concentration [IC50] of the nanoparticles was determined. In the second step, the killing effect of silver nanoparticles alone or in combination with 3mA of direct electric current was assessed in promastigote cultures for 10 minutes. Next, we evaluated the survival rate of treated promastigotes with the MTT assay

Results: The parasite count showed that the various concentrations of silver nanoparticles significantly decreased the numbers of live promastigotes over time compared with the control group after 24, 48 and 72 hours of culture. The IC50 of the nanoparticles was 39.8 microg/ml after 48 hours of cultivation. Promastigote mortality occurred in 33.5% with the use of silver nanoparticles alone at concentrations of 160 microg/ml and 100% when combined with 3 mA direct current electricity after 10 minutes

Conclusion: Silver nanoparticles alone did not completely kill Leishmania major promastigotes. However, the combined use of both direct current electricity and silver nanoparticles had a significant synergistic effect on promastigote mortality

[Identification of trichomonas in the lung sputum of patients with asthma and chronic pulmonary disease admitted to masih daneshvari hospital by PCR technique]

Trichomonas species usually reside in the mouth and occasionally in the respiratory tract. These species can be found in the lungs of humans. Although the pathogenicity of the parasite in the respiratory system has not been proven, it is more prevalent in people who lack good oral health or suffer from asthma or chronic pulmonary diseases. In the present descriptive study, we have identified Trichomonas by direct microscopic observation of stained smears and by PCR on the lung sputum of patients with asthma and chronic lung diseases that include lung cancer, bronchiectasis, COPD, and malignant pulmonary disease who were admitted to Masih Daneshvari Hospital, Tehran, Iran. For direct examination of 133 sputum samples, we stained the smears with giemsa. In addition a total of 60 samples were used for DNA extraction by an extraction kit [Cinnagen]. Nested-PCR was used for amplifying the ITS1 target gene of the parasite. Finally the DNA sequence of the gene was determined. According to the results, in direct examination of the sputum smears there were only 4 positive cases identified, whereas 22 [36.66%] of the samples were identified as Trichomonasby nested PCR. According to gender, 33.33% of the female samples and 38.46% of the male samples were found to be positive. Considering the high prevalence of this parasite in the study group, chronic pulmonary disease and asthmatic patients may be more susceptible to Trichomonas

Evaluation of immunogenicity of novel isoform of EG95 [EG95-5G1] from echinococcus granulosus in BALB/C mice

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 micro g 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-gamma, IL-12 and TNF-alpha 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

[ identification of human cryptosporidium species in Tehran by PCR/RFLP]

Cryptosporidiosis is one of the most important parasitic infections in Iran which causes diarrhea in humans and animals. The identification of the Cryptosporidium species among humans is necessary. This study aims to identify species of Cryptosporidium isolated from patients that referred to three hospitals in Tehran based on the 18s rRNA gene by nested PCR-RFLP assay. In the first step of the present descriptive cross-sectional study, 1128 human fecal samples were collected from patients that referred to three hospitals [Ali Asgar, Mofid and Imam Khomeini] in Tehran. The samples were examined for Cryptosporidium by modified acid fast staining. In the second step, DNA of the positive samples were extracted, then gene of 18s rRNA was amplified by nested PCR in order to differentiate between species. The PCR products were subsequently digested by Vsp1 restriction enzyme and their sequences determined. The modified acid fast method detected 12 [1.06%] positive samples which was confirmed by a molecular technique. The 845bp fragment of 18s rRNA was digested by restriction enzymes. There were 10 samples identified as Cryptosporidium parvum that showed similar patterns on 2.5% agarose gel; 2 other samples were identified as Cryptosporidium homonis and Cryptosporidium andersoni based on the different patterns and sequence results. Although Cryptosporidium parvum is introduced as the major agent for cryptosporidiosis in humans, Cryptosporidium hominis and Cryptosporidium andersoni may also infect humans

vitro study on cytotoxic effects of zno nanoparticles on promastigote and amastigote forms of leishmanta major

Although pentavalent antimony compounds are used as antileish-manial drugs but they are associated with limitations and several adverse complications. Therefore, always effort to find a new and effective treatment is desired. In this study, the effect of ZnO nanoparticles with mean particle size of 20 nanometers [nm] on Leishmania major promastigotes and amastigotes was evaluated. Viability percentage of promastigotes after adding different concentrations of ZnO nanoparticles [30, 60, 90 and 120 microg/ml] to the parasite culture was evaluated by MTT assay. In the flow cytometry study, Annexin V-FITC Apoptosis detection Kit was used to study the induced apoptosis and necrotic effects. IC50 after 24 hours of incubation was 37.8 microg/ml. ZnO nanoparticles exert cytotoxic effects on promastigotes of L major through the induction of apoptosis. A concentration of 120 microg/ml of ZnO nanoparticles induced 93.76% apoptosis in L major after 72 hours. ZnO NPs can induce apoptosis in L major by dose and time-depended manner in vitro condition

Evaluation of protective effect of IL-22 and IL-12 on cutaneous leishmaniasis in BALB/c mice

OBJECTIVE@#To investigate the protective effect of IL-22 and IL-12 on cutaneous leishmaniasisin BALB/c mice.@*METHODS@#The protective effect of IL-22 and IL-12 on cutaneous leishmanias in BALB/c mice was evaluated by measurement of IL-4, INF-γ, total IgG, IgG1 and IgG2a after challenge with Leishamania major. Clinical evaluations were performed by measurement of lesion diameter, and survival rate of the mice.@*RESULTS@#In week 27 post infection, the mortality rates for control groups were 100%. While the survival rates for the IL-12, IL-12 + IL-22, and IL-22(5 ng/g) groups were 100%. The size of lesions decreased in the presence IL-22 (5 ng/g) of mice weight, which was statistically significant in comparison with other groups (P<0.05). Mean of total IgG, IgG1 and IgG2a for IL-22 (5 ng/g) group was more than other groups. In IL-22 group (5 ng/g), INF-γ production was significantly higher than other groups and IL-4 was significantly lower than other groups.@*CONCLUSIONS@#The results obtained indicate the effectiveness of IL-22 and its effect on IL-12 in protection of cutaneous leishmaniasis.

Evaluation of protective effect of IL-22 and IL-12 on cutaneous leishmaniasis in BALB/c mice

Objective: To investigate the protective effect of IL-22 and IL-12 on cutaneous leishmaniasisin BALB/c mice. Methods: The protective effect of IL-22 and IL-12 on cutaneous leishmanias in BALB/c mice was evaluated by measurement of IL-4, INF-γ, total IgG, IgG1 and IgG2a after challenge with Leishamania major. Clinical evaluations were performed by measurement of lesion diameter, and survival rate of the mice. Results: In week 27 post infection, the mortality rates for control groups were 100%. While the survival rates for the IL-12, IL-12 + IL-22, and IL-22(5 ng/g) groups were 100%. The size of lesions decreased in the presence IL-22 (5 ng/g) of mice weight, which was statistically significant in comparison with other groups (. P<0.05). Mean of total IgG, IgG1 and IgG2a for IL-22 (5 ng/g) group was more than other groups. In IL-22 group (5 ng/g), INF-γ production was significantly higher than other groups and IL-4 was significantly lower than other groups. Conclusions: The results obtained indicate the effectiveness of IL-22 and its effect on IL-12 in protection of cutaneous leishmaniasis.

Identification of live toxoplasma gondii by the nasba method in rat

Toxoplasmosis is a worldwide disease, for which different detection methods have been used. The nucleic acid sequence-based amplification [NASBA] method is shown to be highly efficient for diagnosis of live microorganisms. The present research evaluates the molecular isothermal method of NASBA to identify live Toxoplasma gondii [T. gondii] in rat. Tachyzoites of T. gondii were inoculated in the peritoneal cavities of mice [Mus musculus] and their RNA was extracted. The NASBA method was then used to amplify the tachyzoite B1 rRNA gene. Next, we examined blood samples from 30 experimentally infected case and control rats [Rattus norvegicus] by NASBA. Finally, the resultant band was investigated on an agarose gel. The B1 genes extracted from both the tachyzoites and blood samples were successfully amplified by the NASBA method. This amplified gene yielded an amplicon of approximately 116 bp on gel agarose. NASBA is highly efficient for the identification of live T. gondii. This method can be applied for early diagnosis of active toxoplasmosis in both newborns and immunocompromised individuals

In vitro study of the effect of artimisinin on promastigotes and amastigotes of leishmania major

Cutaneous leishmaniasis is an endemic infectious disease considered to be a crucial health problem in many countries, including Iran. As such, there is a need for new medications with few side effects. In the present research we have studied the effect of artimisinin on Leishmania major [L. major] and cell death in vitro. A specific number of promastigotes of L. major were grown in the presence of different concentration of artimisinin to achieve IC[50] of the drug. The MTT method was applied to evaluate the cytotoxic effect of the artiminisinin on L. major. Various densities of this drug were applied to study the induction of apoptosis by flow cytometry on L. major promastigotes. We calculated the IC[580] of artimisinin to be 25 microg/ml by promastigote assay. Promastigotes were incubated at 72 hours incubation with various doses of artimisinin [10, 25, 50 and 100 microg/ml]. The dose 100 ?g/ml showed the most apoptosis [68.16%] by Annexin-V FITC. Whereas the 10 microg/ml dose had the least apoptosis [12.78%]. There was no change in the control group. According to MTT, the toxic effect of artiminisinin on L. major promastigotes increased with increasing drug concentration. This study revealed that artimisinin has a little toxic effect on macrophages. According to the flow cytometry and MTT results, artimisinin can be suggested as an appropriate drug for in vivo antileishmanial study

[Effect of Toxoplasma gondii infection on spermatogenesis of male rats]

Evaluation of the effects of Toxoplasma gondii infection on spermatogenesis in male rats. The RH strain of T. gondii tachyzoites were injected interaperitoneally in an infected group of 35 rats, while 21 rats were used as controls. Each ten days from 10-70 days of postinfection [PI], 5 rats from infected group and 3 rats from control group were scarified. The percentage of body weight to testis weight ratio [BTR] as well as sperm parameters and fructose levels in seminal vesicles and coagulating glands [SVCG] were investigated. An IgG ELISA kit was designed for serologic diagnosis of infection in the rats. All rats injected with T. gondii tachyzoites were infected from 10-70 PI. Sperm motility from 10-70 PI, sperm viability from 10-60 PI and sperm concentration from 20-60 PI were significantly decreased in the infected group [P<0.05] ; sperm abnormality was significantly increased in the infected group on days 30, 40 and 50 PI [P<0.05]. BTR in the infected group was not significantly changed compared to control group [P>0.05]. Fructose level in SVCG in the infected group was significantly decreased on days 10-50 PI [P<0.05] compared to control. According to the results, toxoplasmosis can cause impermanent impairment on the spermatogenesis in the male rats

Alteration in some serum enzyme levels of rats during experimental infection with Toxoplasma gondii

The entry of Toxoplasma gondii into the bloodstream and other tissues [such as liver, muscle, cardiac muscle, ...] of intermediate hosts and its multiplication in nucleated cells may cause changes in plasma levels of various enzymes due to tissue damage. In present study the serum levels of AST, ALT, ALK/P, CPK, LDH, and ACP in rats infected experimentally with Toxoplasma gondii have been investigated. Totally, 116 uninfected rats were divided into 87 as case group and 29 as control group. The case group was infected intraperitoneally with 50000 tachyzoites. Blood specimens were taken from cases and its control once every 8 hours in the first three days and then once every three days for a period of 60 days and serum levels were measured for the mentioned enzymes. During the study, the following changes were observed: AST in the first 8 hours, from the 32[th] hour till the 40[th] hour and from the 48[th] hour till the 56[th] hour; ALT in the first 8 hours and from the 48th hour till the 56[th] hour; ALK/P from the 24[th] hour till the 40[th] hour and from the 48[th] hour till the 64[th] hour; CPK and LDH from the 24[th] hour till the 40[th] hour and from the 48[th] hour till 56[th] hour; ACP from the 16[th] hour till the 48[th] hour. But afterward, whole offer mentioned enzyme shifted to normal levels. Alteration in serum enzyme levels of rats during infection with Toxoplasma gondii found is not permanent

[Cloning and sequencing of L. major leishmania homologue of receptors for activated C kinase [LACK] gene]

Leishmaniasis is caused by parasitic protozoa of the genus Leishmania which in the infected host is obligate intracellular parasite. LACK gene is conserved among related Leishmania species. LACK is the immuno-dominant antigen of L.major which is considered as the most promising molecule for a recombinant or DNA vaccine against leishmaniasis. In this study the genomic DNA of an Iranian standard strain of Leishmania major [MRHO/IR/75/ER] was ertracted and the LACK gene was amplilified by PCR. Then the PCR product was cloned into pTZ57R/T cloning vector, The PT-LACK recombinant plasmid was extracted from transformed E.coli bacteria [TG1 strain] and sequenced. The LACK gene [Accession no LmjF28.2740] of MRHO/IR/75/ER and L. major was amplified using PCR method. LACK gene was cloned into pTZ57R/T coloning vector. Sequence analysis of the cloned LACK gene showed high homology 89% with LmjF28.2740 [LACK gene]. The LACK gene of L.major was cloned in pTZ57R/T vector successfully. Recombinant plasmid was confirmed and could be used for the production of recomiomort antigens is DNA vaccines, for further studies

[ study on single nucleotide polymorphisms in Plasmodium fakiparum chloroquine resistance during two years in Chabahar]

Plasmodium falciparum chloroquine resistance is a major problem in malaria endemic areas. Single nucleotide polymorphisms in pfcrt and pfmdr1 genes are known to be associated with chloroquine resistance in some parts of the world. The major goal of the present study was to detect the five single nucleotide polymorphisms in pfmdr1 gene and one single nucleotide polymorphisms in pfcrt gene. Total of 26 blood samples were collected from falciparum malaria infectious person with chloroquine failure in Chabahar, a harbor located in Sistan baluchestan during 2 years. Detection of single nucleotide polymorphisms were carried out by Real-Time PCR using Light CyclerTM hybridization probe assay. Our data showed that the pfmdr1 N86Y mutation was detected in 6[23%] samples. Although this mutation was not observed in the first year but in the second year it was substancial. In addition the pfcrt K76T mutation was detected in 11 samples [42.3%] of CVMNT haplotype, 7 samples [26.9%] of CVIET haplotype, 5 samples [19.2%] of SVMNT haplotype and 2 samples [7.6%] of SVIET haplotype. The mutations considerably have increased during 2 years. Our results showed single nucleotide polymorphisms in pfmdr1 and pfcrt genes. This could be considered as chloroquine resistance markers for malaria control in Chabahar