problem of mixing up of leishmania isolates in the laboratory: suggestion of ITS1 gene sequencing for verification of species

Leishmaniasis is endemic in Iran. Different species of Leishmania [L.] parasites are causative agents of this disease. Correct identification of Leishmania species is important for clinical studies, prevention, and control of the diseases. Mix up of Leishmania isolates is possible in the laboratory, so there is need for verification of species for isolates of uncertain identity. Different methods may be used for this purpose including isoenzyme electrophoresis and molecular methods. The isoenzyme electrophoresis, due to its drawbacks, is feasible only in specialized laboratories while molecular methods may be more feasible. The aim of this research was to study the application of the internal transcribed spacer 1 [ITS1] sequencing method, in comparison to isoenzyme electrophoresis method, for verification of Leishmania species. Six Leishmania isolates were received from different research institutions in Iran. The species of these isolates were known by donating institution according to their isoenzyme profile. The species of these isolates were re-identified in Pasteur Institute of Iran by PCR amplification of ITS1 followed by sequencing and comparison of these sequences with Leishmania sequences in GenBank. Isoenzyme electrophoresis confirmed the confirmation of the results of ITS1. ITS1 sequence showed that some isolates were mixed up or contaminated with Crithidia. Isoenzyme electrophoresis confirmed the results of ITS1 sequences. ITS1 Sequencing is relatively more feasible than the traditional isoenzyme electrophoresis method and is suggested for verification of Leishmania species

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

[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