Expression and purification of P43 Toxoplasma gondii surface antigen

Toxoplasma gondii is an obligate intracellular protozoan parasite, capable of infecting all species of mammals including man. Congenital toxoplasmosis is more important during pregnancy for the first time. In this study we expressed and purified P43 Toxoplasma gondii tachyzoite and bradyzoite specific surface antigen. The recombinant pGEMEX-1 contained Toxoplasma P43 coding sequence was transformed into E. coli and mass cultured in LB medium contained 100 micro g/ml ampicillin at 37 [degree sign]C over night .The T7 promoter was induced by 1mM isopropyl-1-thio-beta-D-galactopyranoside [IPTG. Recombinant protein was purified by affinity chromatography and confirmed by gel diffusion dot blot and western blot,-using specific anti Toxoplasma antibodies. Recombinant plasmid was induced by IPTG and analyzed by SDS-PAGE. Recombinant protein was confirmed by Western-blot and dot blot using anti human Toxoplasma antibody. Recombinant Toxoplasma P43 was produced successfully

Comparison of three methods for diagnosis of cutaneous leishmaniasis

Leishmaniasis is one of the infectious parasitic diseases of highest incidence in the world. Cutaneous Leishmaniasis [CL] has long been reported in Shiraz, Southern Iran. There is a need to find a sensitive and specific method for treatment and control of the disease. We have compared the sensitivity of the conventional methods microscopy and cultivation of lesion scrapes against PCR amplification of parasite kinetoplast DNA from these samples. The samples [n=219] were obtained from the patients clinically suspected of CL. The smears were stained with Giemsa for microscopy and cultured in Novy-Nicolle-McNeal [NNN] blood agar for promastigote growth. For PCR, the dry smears were scraped off the slides and DNA was extracted. The positive rates from 219 specimens were 76.71%, 50.68%, and 93.61% for microscopy, cultivation, and PCR, respectively. The highest correlation was found between PCR and microscopy method [P= 0.014]. In PCR assay, 95.61%, 3.9%, and 0.49% of the samples were identified as Leishmania major, L.tropica, and dermatropic L.infantum, respectively. The PCR method appears to be the most sensitive for the diagnosis of CL and is valuable for identifying the other species of Leishmania with confusing dermatropic signs

prevalence of Toxoplasma infection among free-ranging chickens in Southern Iran using IFA and nested-PCR

As consumption of chicken meat may be as one of the sources of human infection, this study was undertaken to determine the prevalence of T. gondii in farm chickens[Gallu gallus domesticus] in Shiraz, southern Iran. Two hundred and thirty one blood samples were collected from farm chickens by a cluster random sampling method and tested for toxoplasmosis by indirect fluorescent antibody technique [IFAT]. The samples of the brain, heart, and liver of the chickens were tested by a Nested PCR method. The results were analyzed by SPSS software using Chi-Square test and a P value <0.05 was considered statically significant. Out of 58 seropositive chickens, 29 [1:16 in eight, 1:32 in 14, 1:64 in five and 1:128 in two birds] and out of seronegative chickens, three were enrolled in the study. The most infected tissue was liver [27 out of 29] and the lowest was the heart [16 out of 29] [alpha =0.05, P=0.002]. None of the seronegative chickens was positive in PCR method. Only 2 out of 8 cases with a titer of 1:16 [as cut off point] were negative in PCR method whereas the remained were positive. Based on cultural and food habits in our area, the meat and viscera of chicken may be important sources of infection in human when consuming semi-cooked meats. Considering the high prevalence of toxoplasmosis in chickens, standards in chicken breeding, education of environmental health personnel and standardization for preparation and handling techniques are required by Health and Veterinary organizations

In vitro infectivity of leishmania major isolated from patients with different clinical forms of cutaneous leishmaniasis and its association with parasite zymodems

The aim of this study was to characterize the Leishmania parasites isolated from cutaneous leishmaniasis [CL] patients in Pars Province in Iran and to compare the potential infectivity of the isolates in macrophage cell line. Moreover, attempt was made to find out the association between parasite infectivity and their zymodems. Twenty samples were taken from the skin lesion of CL patients. The samples were cultured in biphasic media followed by mass cultivation in RPMI medium. Each isolate was tested for the activity of the 5 enzymes including glucose phosphate isomerase [GPI], malate dehydrogenase [MDH], nucleoside hydrolase 1 and 2 [NH1 and NH2], and phosphoglucomutase [PGM]. The enzymatic profiles of the isolates were compared with WHO reference strains. Specific PCR [primers: LIN17 and LIN R4] and RAPD-PCR were used as complementary methods for characterization of the isolates. Isoenzyme electrophoresis showed that all of the isolates were L. major. PCR with LIN 17 and LIN R4 and RAPD-PCR with AB-07 primers further determined the isolates as L. major. Results of macrophage infectivity experiment, using J774 cell line, showed that the most virulent isolates were related to Z1 with 63% macrophage infectivity rate. A well correlation was found between the infectivity rate of the isolates and type of ulcer. Those isolates with high infectivity rate were involved in more severe, ulcerative or erythmatose lesions in CL patients. The most invasive isolates might be a good candidate for immunological studies and for vaccine development

seminested PCR based detection of Leishmania infantum infection in asymptomatic dogs in a new endemic focus of visceral leishmaniasis in Iran

Visceral Leishmaniasis [Kala-azar] is a serious health problem in some northern and south western parts of Iran. The incidence of kala-azar caused by Leishmania infantum has recently increased in Nourabad-Mamassani district of Fars Province, in the south of the country. This study was designed to determine the role of asymptomatic dogs as host reservoir of L. infantum in this new formed focus and detection of prevalence of infection near them. A total of 20 asymptomatic stray and sheep dogs were randomly sampled. The Buffy coat layer of their peripheral blood was used for DNA extraction and PCR. A species specific seminested PCR was used for DNA amplification using LINR4, LIN17 and LIN19 primers. These primers amplified variable area of the minicircle kDNA of Leishmania parasites. Of the 20 sampled dogs checked for leishmanial kDNA, six [30%] were found naturally infected. It is concluded that, dogs [Canis familiaris] even if asymptomatic, is considered as the domestic host reservoir of kala-azar in this endemic focus

[Phlebotomus [Paraphlebotomus] alexandri, the probable vector of visceral leishmaniasis [kalaazar] in southern Iran]

Visceral leishmaniasis [kala-azar], the most dangerous form of leishmaniasis, is endemic in some parts of Iran, e.g. Ardabil, Fars, East Azerbaijan, Bushehr and Qom provinces. In recent years, the incidence of VL has increased in the Nourabad-Mamassani district in Fars Province. This study was carried out to detect VL vectors and infection rates in this region over the 2003-2004 period. Sand flies were captured in the selected villages by means of sticky traps, aspirators and CDC miniature light traps. Heads and distal abdominal segments were used for species identification and other body parts were used for DNA extraction. We employed a semi-nested PCR technique to detect Leishmania, with specific kDNA primers [LIN R4 - LIN 17 - LIN19]. Some specimens were dissected for leptomonad infection. A total of 12688 sand flies were collected. Phlebotomus [Paraphlebotomus] alexandri was the second most prevalent species [17.34%]. The anthropophilic index of this species was 32.5%. Five cases [4.17%] of L. infantum infection were detected among the 120 P. alexandri examined by PCR method. We also observed two cases of leptomonad infection among the 112 dissected specimens. High prevalence rates and anthropophilic index of P. alexandri plus its natural infection with L. infantum provide enough evidence to implicate this species as the main vector species of VL in the region and the second proven kala-azar vector in Iran. Besides, the Mahoor-Milaty district of Noorabad-Mamassani was identified as a new endemic focus

Study on the genomic diversity of hymenolepis nana between rat and mouse isolates by RAPD-PCR

Hymenolepis nana is a common parasite of rodents as well as human intestine. This parasite has been reported from all over the world, including Iran. The infection rate has been reported up to 40% in some areas. The infection has various clinical manifestations. The parasite could establish severe hyperinfection in patients with immune deficiency. Regarding the rodents as hosts of the parasite, the infection may disseminate through these hosts to the nature. As H. nana is a zoonoses, phylogenic study of this parasite is of particular importance. Considering these criteria, the genomic diversity of 16 H. nana with the origin of Shiraz and Tehran were studied among the worms of mice and rats by RAPD-PCR. Genomic DNA extracted from individual worms by proteinase K method and three oligonucleotides primer [ABl-17, UBC-358, UBC-387] were used for RAPD-PCR. Similarity index were calculated by Nei and Li method. Data were analysed using UPGMA analysis and dendrograms were obtained by group average method with 100 bootstrapping analysis. The range of genomic similarity determined among specimens by ABl-17 primer was 48.3-90%, by UBC-358 primer 55-87% and by UBC-387 primer 53-97%. Regarding our data and genomic similarity indexes, various isolates were found in both specimens of rats and mice. However no differences were obtained between H. nana from rat or mouse isolates by these primers. The results showed that it is not possible to divide the isolates into two distinct groups based on their origin as Tehran and Shiraz

[First report of Kala-azar from Qeshm Island in Persian Gulf]

Visceral Leishmaniasis [VL] is a sever disease that is prevalent in Iran. We report a case of VL in a 3.5 year-old boy. Prolonged fever, chill, abdominal distention, and weight loss were important symptoms. Blood count showed pancytopenia and hypohemoglobinemia. Specific anti-leishmanial antibodies were detected by serological test [IFAT, DAT] but no Leishman body was observed in bone marrow. However, a 145 bp band of KDNA belong to L. infantum was detected by PCR method. Glucantime was administered and treatment was well tolerated. This is the first report of VL from Qeshm Island in Persian Gulf

[ new endemic focus of visceral leishmaniosis in southeren Iran]

Visceral leishmaniosis [VL] is endemic in some parts of Iran. Mediterranean type of disease is present in Iran where its causative agent is Leishmania infantum and dogs are the main. reservoirs. Since many cases of the disease were reported from Noor-abad, in Fars province, we aimed to carry out an epidemiological survey on VL in human and animal reservoirs; [dogs] in Mahoor-Milaty district of Noor-Abad city at West North of Fars province. In this cross-sectional descriptive survey, E blood samples were randomly collected from all children

[Characterization of leishmania parasites isolated from kala- azar patients in kohgiloyeh and boyerahmad, using semi-nested PCR]

Visceral leishmaniasis [VL] is a disease commonly known as Kala-azar caused by protozoan parasites of the genus Leishmania including L. donovani, L. infantum and L. chagasi. VL is sporadic in manyareas of Iran and is endemic in a few provinces such as Fars, Azarbayjan, Bushehr, Ardabil and Qom. VL has been reported from some areas of Kohgiloyeh and Boyerahmad and this study aimed to characterize the causative agent of VL in this region. Bone marrow sample was obtained from 6 VL patients from children department in Imam Sajad hospital in Yasuj. DNA was extracted from the obtained samples and was checked by semi-nested PCR to determine the species of the parasite. To do that, a segment of minicircle kinetoplast DNA was amplified, using LINR4 and LIN17 primers. Products of PCR were evaluated by electrophoresis, using 1.5% agarose and stained with ethidium bromide. Parasitologically examination of bone marrow smears demonstrated amastigotes form of the parasite in the samples. For mass cultivation, isolated parasites were cultured in diphasic NNN followed by RPMI 1640 media. All the samples produced a 720 bp band in PCR assay. The isolates were compared with referent strains and it was revealed that all the isolates were L. infantum. Findings of this study demonstrated that the causative agent of VL in Kohgiloyeh and Boyerahmad was L. infantum. Further study is needed to explore other aspects of VL in this region

[Evaluation of hydatid cyst fluid as a substitute for fetal bovine serum [FBS] in culture of leishmania major]

Isolation and characterization of Leishmania parasites is a necessary strategy for control of Leishmaniasis. Free-cell culture media, rich with biologic fluids such as Fetal Bovine Serum [FBS] are among the best media for culturing of the parasite. In our country, FBS is very expensive and is not available everywhere. In this study, we evaluated Ovine Hydatid Cyst Fluid [HCF] as a substitute for FBS in liquid culture media of Leishmania major. Leishmania parasites were isolated from Balb/C mouse ulcer and characterized by PCR. Of six tubes by triplicate [totally 18 tubes] add to each tube 800,000 promastigotes of Leishmania major and then add 1 cc of media including, once BHI [Brain Heart Infusion broth], BHI plus 5% FBS, BHI plus 10% FBS, once Hydatid Cyst Fluid [HCF], BHI plus 5% HCF and BHI plus 10%HCF, respectively. After 24h, 72h, 144h, 192 hours the number of parasites in each tube counted and the means of them compared together. The result of this study showed that BHI plus HCF 10% medium could be use as a suitable substitute till 72 hours for Fetal Bovine Serum [FBS] in liquid Culture of Leishmania major parasites. In this study, we have introduced the Ovine Hydatid Cyst Fluid [HCF] as replacement for FBS in liquid Culture media

Characterization of leishmania parasites from archived geimsa-stained slides using nested polymerase chain reaction

In Iran, the clinical presentation of cutaneous leishmaniasis is mainly in the form of dry type [urban form] or wet type [rural form]. The microscopic finding of amastigotes in Giemsa-stained smears is the most practical laboratory test for the diagnosis of cutaneous leishmaniasis. However, determination of parasite species is not possible when using this method. Parasite characterization is made by various biochemical, immunological and molecular methods based on massive culture of the parasites. In this study nested PCR was used both for diagnosis as well as species identification. Giemsa-stained slides from forty-nine patients, that had been included in a drug resistance survey, were used in this study. From the available slides, forty-seven were diagnosed as having leishmaniasis using the nested PCR technique. Twenty of these were Leishmania tropica [L. tropica] and the remaining were Leishmania major [L. major]. Amastigotes were recovered from twenty-nine of these patients after standard treatment. This study revealed that clinically drug-resistant cases are more likely to be infected with L. tropica than with L. major, although this difference was not statistically significant. L. tropica was mostly present in facial lesions while L. major was mostly detected in hand and foot lesions. In patients with more than two lesions, L major was the predominant cause. L tropica was the cause of a more prolonged duration of disease. None of the above findings were, however, statistically significant. It can be concluded that nested PCR is a useful technique for studying the molecular epidemiology of leishmaniasis in the field