Detection and differentiation of Leishmania parasites in asymptomatic canine by High-Resolution Melting analysis of microsatellite fragment in ITS gene.

The high prevalence of Leishmania infection was reported in dogs as the main reservoir of CanL in many locations in the old world. Detection and firmly identification of Leishmania species in asymptomatic dogs by reliable method was considered and employed. Non-invasive and non-anesthetized blood sampling in asymptomatic dogs was conducted. Nested, conventional and real-time PCR with HRM technique was performed targeting ITS-rDNA gene. 88 asymptomatic dogs were sampled from three CanL endemic provinces of Iran in 2018-2019. 23 blood samples were Leishmania positive. L. major, L. tropica and L. infantum were accurately identified for the first time with HRM targeting ITS2-microsatellite. Three samples were mixed infections. CLC software TM predictions for microsatellite ITS-rDNA were 86.93 °C: L. major, 85.76 °C: L. tropica and 86.04 °C: L. infantum. Standard strains of Leishmania species were accurately separated with almost one to 2 °C deference (L. major: 86.61 °C, L. infantum: 85.41 °C, L. tropica: 84.82 °C). Each HRM curve represents one species in a sample for helping accurate identification of Leishmania species and even mixed infection when two curves are present. Detecting parasites at primary stages in asymptomatic cases is essential using Real-time HRM. As same as mammalian Leishmania in rodents which is present at early stages and non-pathogenesis, only L. major would exist and other Leishmania disappears. This can conclude also for L. major, L. infantum and L. tropica in dogs. The role of L. major existence in canine blood should be investigated more.

Genetic characterization of Echinococcus granulosus strains isolated from humans based on nad1 and cox1 gene analysis in Isfahan, central Iran.

Cystic echinococcosis (CE) is a medically important parasite-caused human disease. Humans may acquire the infection accidentally by ingestion of E. granulosus eggs. The parasite has a broad range of hosts and genotypes, which may affect its aetiological and biological characteristics. The present study aimed to determine the genetic characteristics of human isolates of E. granulosus in Isfahan, Iran. In this cross-sectional study, 50 surgically removed hydatid cysts were collected from hospitalized patients in Al-Zahra Hospital, Isfahan, Iran, over a period of 2 years (2015-2017). DNA was extracted from cyst material, and polymerase chain reactions (PCR) were performed targeting cox1 and nad1 genes. Amplicons were sequenced directly and the resulting sequences were aligned and analysed. Phylogenetic and genetic diversity analyses were also performed. Among the isolates, 43 (86%), 3 (6%) and 4 (8%) out of 50 were E. granulosus (G1), E. granulosus (G3) and E. intermedius (G6), respectively. In total, nine and eight haplotypes were identified by nad1 and cox1 gene analysis, respectively. The haplotype diversity index was higher by cox1 gene analysis (0.547) in G1 strains compared with nad1 (0.433). The G1 genotype was the most predominant isolate from human cases of CE, and the presence of G6 is indicative of an important role of camels in the development of human CE in Isfahan. This is the first report of the G3 genotype causing human CE in Isfahan. Moreover, cox1 gene analysis enables a higher resolution of the genetic diversity of the E. granulosus population compared with nad1 gene analysis.

Apoptosis of human lymphocytes after exposure to hydatid fluid.

BACKGROUND: Modulation of the immune response is an important strategy by which establishment and growth of hydatid cyst in the internal organs of human is warranted. Induction of apoptosis in the lymphocytes might be a considerable component. This study was designed to evaluate apoptotic impact of hydatid fluid (HF) on human lymphocytes. METHODS: Human lymphocytes were treated with hydatid fluid. After 6 hours of exposure, caspase-3 activity, the central enzyme of apoptosis cascade, was measured by fluorometric assay in the HF-treated lymphocytes and control cells. In addition, the expression of Bax (a pro-apoptotic protein) and Bcl-2 (an anti-apoptotic protein) mRNA was assessed by RT-PCR after 12 hours of exposure. RESULTS: Both the ratio of Bax/Bcl-2 mRNA expression and Caspase-3 activity were higher in the HF-treated lymphocytes relative to the control group. CONCLUSION: Apoptosis could be as a possible mechanism by which Echinococcus granulosus overwhelms host defenses.