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Background: Cryptosporidiosis is one of the most important parasitic diseases infecting a broad variety of animals and humans. In the present study, Nested PCR-RFLP-based assay was applied for genotyping of‏ ‏‏sheep cryptosporidiosis. The target of amplification was the 18S rRNA gene ‎‎used to identify Cryptosporidium species
‎Materials and Methods: In the first step, 1300 faecal samples were collected from sheep in Tehran province, then the samples were examined for the presence of ‎Cryptosporidium using modified acid fast staining. In the second step, DNA was extracted from the ‎positive samples. Next, 18S rRNA gene was amplified by ‎Nested-PCR in order to differentiate between the species. The PCR product was digested by Ssp1 restriction enzyme. ‎
Results: Twenty two positive ‎sheep samples were detected by modified acid fast method. The results were confirmed by molecular techniques. The 845 bp fragment of 18S rRNA was digested ‎by restriction enzymes. Twenty samples showed ‎a similar band on 2.5% agarose gel whereas 2 samples demonstrated different pattern. The sequences of two patterns indicated two species of C. andersoni and C. parvum.
Conclusion:  In spite of other studies results introducing C. parvum as the major agent of ‎cryptosporidiosis in sheep, in our study, C. andersoni was found to be dominant. 

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Aims: Toxoplasmosis is a cosmopolitan zoonotic disease caused by an obligate apicomplexan intracellular parasite known as Toxoplasma gondii (T. gondii). Recently, toxoplasmosis has been suggested as a risk factor for diabetes. Thus, the present study aimed to assess the association between T. gondii infection and two types of diabetes in Tehran, the capital of Iran.
Materials & Methods: In the current cross-sectional study, 98, 95, and 94 blood samples were collected from Type 1 and Type 2 diabetic and nondiabetic individuals, referring to Imam Sajad hospital from February to August 2018, respectively. Anti-T. gondii specific IgG and IgM antibodies were measured using enzyme-linked immunosorbent assay (ELISA). Moreover, a structured demographic questionnaire was completed for each person.
Results: IgG antibody was found to be positive in 16.32 (16 of 98) and 57.89% (55 of 95) of patients with diabetes Type 1 and Type 2 and 17.02% (16 of 94) of nondiabetic individuals as controls, respectively. However, the prevalence of positive IgM antibody in these groups was determined as 2.04 (2 of 98), 6.32 (6 of 95), and 17.02 % (16 of 94), respectively.
Conclusion: This finding revealed that toxoplasmosis could be considered as a possible risk factor for diabetes Type 2, while no statistically significant association was found between T. gondii infection and diabetes Type 1.  More research is required to be conducted in the future in order to better understand this association.

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Purpose: In the present work, clinical symptoms, serum immunoglobuline alteration and eosinophil count were investigated in the patients infected with Giardia lamblia. Materials and Methods: In this regard, 50 persons infected with Giardia and 50 healthy individuals were selected as the study and control groups respectively in Tehran. Sera of the groups were tested for IgG, IgE, IgA levels and their blood for the percentage of eosinophil. To measure IgA and IgG levels the SRID technique and to measure IgE level the ELISA technique were applied. Results: Results indicated that, 78%, 74%, 60%, 58% and 12% of the patients showed diarrhea, abdominal pain, weight loss, flatulence and visual disorder respectively, while no aforementioned symptoms were observed in non-infected individuals. In addition, the mean of eosinophil percentage as well as IgA, IgG and IgE levels in the infected group were found to be higher than that of the non infected group significantly (P<0.05). Meanwhile, no elevation was observed in the eosinophil percentage or immunoglobuline levels of the control group. Conclusion: In conclusion, Giardia lamblia can induce eosinophila and increase the level of IgE in the infected patients significantly.

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Purpose: The present study was conducted to evaluate the frequency of human hydatidosis in Ilam Province using Dot- ELISA techniques. Materials & Methods: Three thousands sera, collected from different cities of Ilam Province in the west of Iran were examined. The serum dilution of 1:400 was selected as the cut off and the results were confirmed by the ELISA technique. Results: Results indicated that, totally 37 individuals (1.2%) show positive sera. There were significant differences among various age groups of infected individuals. The most percentage of infection was found to be in the 20-30 age group. Among the sera tested, 0.56%, 1.57%, 10.77% were found to be positive in individuals living in cities, villages and migratory tribes respectively. Twenty two individuals of the infected people (1.47%) were female and 15 individuals (1%) were male. Most infected individuals were found to be in Mehran city (3.11%) and less infected individuals were found to be in Ilam city (0.79%). Conclusion: Most of the people living in migratory tribes were sheep keepers and were actually involved in animal production, wich can be regarded as a risk factor for their higher infection rate.

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Backgrounds: Trichomoniasis is one of the most common sexually transmitted infections in the world. The main aim of the present descriptive study was to determine the prevalence rate and clinical symptoms of trichomoniasis among women referring to the hospital in Mahshahr city in Khuzestan province, southwest of Iran.
Materials & Methods: Urine samples were collected from 2200 women referring to Imam Musa Kazim hospital in Mahshahr city. In addition, 500 Pap smear samples were used for early detection of Trichomonas vaginalis. At first, parasitological tests were performed to detect T. vaginalis in urine and dissolved Pap smear samples using microscopic examination. Finally, DNA extraction was performed on 34 parasites isolated from positive urine and Pap smear samples. Then the 18s rRNA gene of the parasite was amplified by PCR method. The PCR products of the 18s rRNA gene were finally sequenced.
Findings: The prevalence rate of this parasite was determined to be 1.54%. The highest prevalence rate of infection and clinical symptoms were observed in women aged 31-40 years. Totally, clinical symptoms were observed in 64.70% of infected women, including vaginal itching and irritation (64.70%) and abnormal discharge (26.47%).
Conclusion: The prevalence rate of Trichomonas infection was relatively low in women living in Mahshahr. In addition, about 35.29% of infected women were found to be clinically asymptomatic.

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Aims: Blastocystis is a common intestinal parasite among humans and various animals, including birds. The parasite has at least 28 known subtypes, of which nine subtypes have been reported in humans and livestock. The aim of this study was to determine the prevalence rate and common subtypes of Blastocystis hominis in pigeons and their owners in Tafresh city.
Materials & Methods: The present study was designed and conducted as a case control in Tafresh city (Markazi province) during 2020-2021. For this purpose, fecal samples were collected from pigeons (300 samples) and their owners (100 samples). Stool samples were studied by microscopic methods (direct and trichrome staining examinations). Then positive stool samples were examined by PCR method through amplification of 18 SrRNA gene and sequencing.
Findings: In direct stool examination, 39 (13%) out of 300 pigeon samples and 18 (18%) out of 100 human fecal samples were found to be positive for Blastocystis. In trichrome staining method, 18% of human samples and 15% of pigeon samples were positive, while in PCR test, only 2.5% of pigeon samples and 4.5% of human samples were Blastocystis positive. The alignment results showed that all Blastocystis strains isolated in this study (100%) were similar to subtype 3.
Conclusion: Due to the low prevalence rate of this parasite in pigeons in Tafresh city, their owners are less likely to be infected with this parasite. Therefore, the relative transmission risk of this parasite from pigeons to humans is low.

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Objectives: Toxoplasma gondii is a coccidian protozoon which forms tissue cyst in different organs of infected intermediate host. The present study was conducted to demonstrate the active form of parasite in different tissues of rat, which was experimentally infected with RH strain of Toxoplasma gondii using bioassay method inmice. Materials & Methods: In the experimental assay, 75 rats and 500 mice were used. The rats were infected experimentally with 40-50 thousands of tachyzoites intrapritoneally. Three rats were killed every 24 h then with up to three days 3 days interval for 60 days and their different organs were searched biologically for presence of the parasite. In this regard, the organs were squeezed with mortar in normal saline, diluted and following adding streptomycin and penicillin injected intrapritoneally in three outbreed mice each. Then the mice were followed up for 10 days. Results: Toxoplasma cyst was found in brain, liver and spleen of the rats within 5 to 9 post infection. The parasite was appeared in heart and muscles within 9-13 days after infection but not found in the kidney. The parasite was disappeared in spleen and liver following12 and 28 days of infection respectively but remain active in the brain, heart and muscles within 60 days of the study. Implication: Toxoplasma gondii could remain active in spleen and liver of infected rat for a short time. Meanwhile brain and muscles (heart and skeletal muscles) of the infected animal can keep the parasite for a long time. Kidney is not appropriate tissue for Toxoplasma gondii localization.

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Objectives: Malaria is the most important tropical disease in terms of morbidity and mortality. The diagnosis of malaria can be conveniently subdivided into clinical, parasitological, biochemical, serological and molecular biological detection. The objective of the present work was to compare two techniques, blood smear and PCR-RFLP, for detection of Plasmodium species. Materials & Methods: Totally, 46 positive blood samples of malaria were examined by these two methods. In parasitological detection, direct observation of Plasmodium in Geimsa-stained thick and thin blood smears were carried out. In polymerase chain reaction (PCR) method, the target DNA was a segment of 18S rRNA gene. In RFLP technique three enzymes, Hinf I, Hae III and Tsp45 I, were used. Results: The results indicated that, by direct observation of thin smear, 35 and 11cases were identified as Plasmodium vivax and Plasmodium falciparum respectively. But molecular analysis showed that 2 of 11 cases of Plasmodium falciparum were Plasmodium vivax whereas 3 of 35 cases of Plasmodium vivax were Plasmodium falciparum. Conclusion: In diagnosis of human Plasmodium species, the PCR-RFLP technique was found more appropriate and sensitive than blood smear technique.

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Objective: An entomological survey was carried out for Leishmania vector incrimination of sand flies in northwestern Iran. Materials and Methods: 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 anlalysis of ITS-rDNA fragment. Results: 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. Conclusion: This reptile related sauroleishmania parasites lacks the Lipophosphoglican (LPG) necessary for entrance to human phagocytes cells, and hence are not human pathogen. However, the GlycoInositoPhosphoLipid (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 by infected bite of some Sergentomyia subgenera that show intermediate

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Objective: This study was conducted to evaluate the frequency of human hydatidosis in Kurdestan province by ELISA technique. Materials and Methods: 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. Results: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%). Conclusion: According to the results obtained from this study the highest percent of infection was found in the city of Ivandarre, the reasone for this defference (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.

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Objective: Toxoplasmosis may cause significant damage to the developing fetus and is a life-threatening opportunistic infection in immunocompromised persons. Molecular methods are known to be more sensitive and more specific than serological assays for diagnosis of toxoplasmosis. Application of quantitative PCR has evolved sensitive, specific, and rapid method for the detection of RH strain of Toxoplasma gondii DNA. Materials and Methods: In the present study, quantitative PCR-ELISA (Polymerase Chain Reaction-enzyme linked immunosorbent assay) was used for quantization of Toxoplasma gondii in the blood of 15 rats (Rattus norvegicus) infected experimentally with the parasite. In this regard Polymerase PCR-ELISA was developed for rapid detection of Toxoplasma gondii. Specific primers targeting RE gene were selected and used for the amplification of toxoplasmic DNA. DIG-labeled (digoxigenin-labeled) amplicons were hybridized with a specific biotinylated oligonucleotide probe in solution phase and subsequently transferred to streptavidin coated plates. The captured DNA-DNA hybrids were colorimetrically detected by the addition of anti-digoxigenin antibody peroxidase conjugate and substrate. Results: DNA of Toxoplasma gondii were efficiently detected within 4 hours and no interference was encountered in the amplification and detection of the parasite. Conclusion: Efficiency of the PCR-ELISA system was evaluated we found with several advantages in terms of sensitivity, rapidity and simplicity in this system.

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Objective: Bacterial meningitis is a dangerous and sometimes fatal infection that affects the central nervous system. Because some antibiotics can prevent some types of these Bacteria and supress them from spreading and infecting, therefore it is important to know what type of virus or bacterium is causing meningitis. Haemophilus influenzae and Neisseria meningitides are the two main pathogens causinig acute bacterial meningitis. Different methods are used for the detection of H. influenzae and N. meningitidis but they are of low sensitivity, taking long time and difficult to perform. Therefore, complementary methods are necessary for more sensitive detection of these agents. Materials and Methods: In this study, a multiplex polymerase chain reaction (mPCR) assay was developed for detection of H. influenzae and N. meningitidis. These strains were confirmed by biochemical methods. Two specific primer pairs were designed for lic-1 and opa genes of H. influenzae and N. meningitidis respectively. Results: DNA amplification product fragments were 150 bp and 320 bp for H. influenzae and N. meningitidis, respectively. Streptococcus pneumoniae used as a negative control and did not yield a PCR product. Conclusion: The results of this study indicated that PCR is a useful complementary diagnostic technique, especially when Gram stain, culture, or antigenic detection is negative or inconclusive.

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Objective: 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. Materials and Methods: 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. Results: 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. Conclusion: 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.

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Objective: 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. Materials and Methods: 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. Results: The LACK gene (Accession no LmjF28.2740) of MRHO/IR/75/ER & 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). Conclusion: 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.

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Objective: Toxoplasmosis, caused by an intracellular protozoan parasite, and the Toxoplasma gondii, is widespread throughout the world. In recent years, significant progress has been made in the identification of vaccine candidates which could induce a protective response. GRA7, an excretory 29 kDa Toxoplasma gondii a dense granular antigen released by infected host cells. In tachyzoite-infected cells, p29 accumulates within the parasitophorous vacuole and co-localizes with its delimiting membrane. Materials and Methods: In the present work, first genomic DNA of Toxoplasma gondii was extracted and used for amplifying of GRA7 gene as a template. Then PCR product was extracted from agarose gel and cloned into TOPO vector. The plasmid containing GRA7 gene was extracted from the transformed bacteria (TOP10 strain) and sequenced. Results: Sequence analysis of GRA7 gene cloned into TOPO vector showed only one base difference when composed with the gene bank sequence for RH strain was only one base. Conclusion: The results indicated that this clone is suitable for subcloning in Prokaryotic and Eukaryotic plasmid.

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Objective: 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. Materials and Methods: 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. Results: During the study, the following changes were observed: AST in the first 8 hours, from the 32th hour till the 40th hour and from the 48th hour till the 56th hour; ALT in the first 8 hours and from the 48th hour till the 56th hour; ALK/P from the 24th hour till the 40th hour and from the 48th hour till the 64th hour; CPK and LDH from the 24th hour till the 40th hour and from the 48th hour till 56th hour; ACP from the 16th hour till the 48th hour. But afterward, whole offer mentioned enzyme shifted to normal levels. Conclusion: Alteration in serum enzyme levels of rats during infection with Toxoplasma gondii found is not permanent.

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Objective: Toxoplasma gondii is an obligate intracellular protozoan that causes Toxoplasmosis in human and animal. In recent years, significant progress has been made in the identification of vaccine candidates which can induce protective responses. In this study we used complete Rhoptry protein 2 gene of Toxoplasma gondii as a single DNA vaccine and evaluated its immune responses in comparison with control groups. Materials and Methods: BALB/c mice were immunized intramuscularly with three weaks time interval with pcROP2 (as case group) and pc-DNA3 and PBS (as control groups). After immunization, we evaluated the immune response using cytokine and antibody measurements. Results: The results of cytokine (IFN-γ, IL-4) assays showed that mice immunized with pcROP2, elicited stronger Th1-type cellular immune responses than those immunized with empty plasmid, or PBS (high level of IFN-γ and low-level of IL-4).Also Anti-T. gondii IgG titres (OD) increased markedly in the pcROP2 group, which was significantly higher than those of control groups (P<0.05). When challenged with the highly virulent Toxoplasma gondii RH strain, mice immunized with pcROP2 had siginificantly higher survival rates compared to control groups (P<0.05). Conclusion: This study showed that pc-ROP2 as a single DNA vaccine is effective to prime enhanced and balanced cellular and humeral immunity responses, and relatively improved mice survival time against toxoplasmosis.

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Objective: Toxoplasmosis can lead to severe pathological effects in both infected humans and animals. The various DNA vaccines against Toxoplasma compose of single or cocktail antigens have been investigated but they have partial protective against disease. In this study, we used pcROP1 as a DNA vaccine and aluminium phosphate and aluminium hydroxide to compare their efficacy as mineral adjuvants. Materials and Methods: BALB/c mice immunized with pcROP1 alone or with co-administration of Alpo4 or Alum and the effectiveness of these two adjuvants were compared using lymphocyte proliferation assay, cytokine and antibody assay and survival time. Results: The group co-administered alum elicited stronger humoral and Th1-type cellular immune responses than the group co-administered Alpo4, while immune response in group administered with pcROP1 alone is higher than them. When challenged with Toxoplasma gondii RH strain, mice immunized with or without alum had significantly higher survival rates, whereas there was no notable enhancement of survival rate in Alpo4 group (P≤0.05). Conclusions: Our result suggest that pcROP1 plus alum and aluminium phosphate not strongly potentiate the efficacy of this DNA.