Epidemiological Survey And Genotyping Classification Of Leishmania

Kala-azar (commonly known as visceral leishmaniasis) is a chronic endemic disease systemically caused by Leishmania donovani and Communicated by sandfly. It is seriously harmful to human health as a parasitic zoonotic Disease.And it's a world popular disease which is highly endemic in China,North India and East Mediterranean regions.The disease is is one major target disease of World Health Organization.During recent six years,in the six provinces(Xinjiang, Gansu, Inner Mongolia, Shaanxi, Shanxi and Sichuan)of Western China, it takes on sporadic state. There are about 400 new occurring cases every year.In Xinjiang, Gansu and Sichuan, the number of incidence accounts for over 90% percent of the new number of the infected,and it has the trend of spreading to non-endemic districts. Especially after the earthquake in 2008, quantities of dogs as source of infection wandered around. The number of patients is 9 cases in 2008,and 6 patients appeared during the first half year of 2009.During the same year, an outbreak of kala-azar appeared in jiashi county of kashi city.It caused a lot of health problems for the residents.so it is significant to grasp asymptomatic infection in dogs in epidemic area and the situation of current infection of Leishnmnia donovani.In our country, Kala-azar can be classified in to three types of diseases which are plain, desert and hill foci.There are significant epidemiological differences among the three types and it's indistinguishable from pathogenic Morphology.The importance of revealing difference between strains is self-evident on prevention of Kala-azar. In our study,we chose the kDNA and specific ITS-1 fragments from rDNA as Gene targets.Two pairs of primers RV1,RV2 and K13A,K13B were designed and synthesized according to conserved region of kDNA.PCR was used that amplified different kinds of Leishmania ribosomal repeats and they were successfully amplified. It proves that the two pairs of primers can be used on identification of leishmania.75 blood samples were collected from patients,their families and neighbors in Kashgar city of Xinjiang province.And 106 blood samples were picked from dogs in heishui county of sichuan province. Each blood sample was collected in types of anti-coagulation and non-anticoagulant. The results of PCR and ELISA revealed the positive detection rates of easy feeling crowd in xinjiang were 69.33% and dogs infection in sichuan is 19.05%. This shows a higher Leishmania infection rate still exists in Kashgar city in Xinjiang and Heishui county in Sichuan. Meanwhile, the questionnaire was designed for the objects in Kashgar we chosed and a survey was conducted by the statistical software SPSS Logistic regression analysis. It showed that peoples being bitten outdoor and whose neighbors or family members are kala-azar patients may be predisposed to kala-azar.Family members or neighbors who are disease are main transmission factors.Kala-azar in Kashgar of Xinjiang taken on significant family aggregation phenomenon.Sequences of ITS-1 of Shandong isolates,Sichuan isolates,Xinjiang isolates and Jiashi isolates of the three L.d isolates from plain, desert and hill foci of China were amplified by PCR and to sequence them. DNA sequence of the three L.d isolateswere got.And it shows that different degrees of variation exist among ITS-1 of Shandong, Sichuan, Xinjiang and Jiashi isolates by Sequence Comparison. The most frequently gene mutation of ITS-1 is Jiashi isolates whose Sequence polymorphism is the most significant. Phylogenetic tree of leishmania was constructed in the research.It shows that there are significant difference in homology of three epidemic area. The evolutionary distance between the desert isolates and plain isolates is far. So is the distance between desert isolates and hill isolates. It's a little closer between plain isolates and hill isolates. Homology beween desert isolates and hill isolates is less than that beween desert isolates and plain isolates. The results indicate that the disease can spread through the sand flies from wild animal hosts directly to person. The results indicate that the disease can spread through the sand flies from wild animal hosts directly to person. This is related to the patient directly accessing into the wilderness.In conclusion,firstly in this study, we chose Leishmania kinetoplast DNA (Kinetoplast DNA) as target genes for PCR, confirming that the two pairs of primers RV1,RV2 and K13A,K13B can be used for identification of leishmania. Dog blood samples of Heishui county in Sichuan and blood samples of easy feeling crowd in Kashgar in Xinjiang were treated by PCR for detection of the infection.The results indicated that the status of kala-azar in Heishui county of Sichuan province and Kashgar city of Xinjiang province is still severe. Secondly, the result of analysis to dangerous factors of the kala-azar indicates that Kala-azar in Kashgar of Xinjiang taken on significant family aggregation phenomenon. It is good for us to keep abreast of infection status of kala-azar,and to provide epidemiological data for the prevention and treatment of kala-azar. It further illustrates the differences between desert, hills and plain isolates of leishmania from genetic level. It explained the difference on kala-azar's treatment from clinical symptoms,drug treatment,epidemiology and other aspects of this disease.It also further reveals the relationship between different isolates,and provides scientific basis for the prevention and treatment of different types of kala-azar.