Epidemiological Investigation And Molecular Biological Diagnosis Of Histomoniasis

Histomonas meleagridis(H. meleagridis) is a flagellated protozoon which causes histomonosis of galliform birds. This disease is characterized by sulfur-colored droppings, necrotic foci in the liver, and thickening and ulceration of the caecal wall. It can be severe and may result in high mortality in turkey flocks. Recently due to public health concerns, all prophylactic and therapeutic drugs have been banned in the European Union. This has led to a reemerging of histomonosis with considerable economic losses, especially in free range birds. Recently, with the demonstration and promotion of domestic healthy breeding technologies such as large-scale ecological breeding and range breeding, histomonosis will be more and more serious, but few research related to this disease was reported. Therefore, it is of great significance to study this disease for the healthy development of poultry industry. In this study, epidemiological investigation on histomonosis in Jiangsu province and adjacent areas was first conducted. Further study on clearing the classification status and genetic evolution of H. meleagridis was also conducted. Then, sensitive, specific and practical diagnostic methods based on molecular biology were established, and clinical collected samples were tested. The main research contents are as following:1. Epidemiological investigation of histomoniasisDetailed classic epidemiological survey and analysis of69cases of histomoniasis in animal hospital of Yangzhou university were done from January2011to December2013. The survey results showed that, chickens at the age of31days to60days were the most susceptible, and the incidence of adult chickens was low. Concerning to the mortality rate,60.87%of cases were below10%. There was no obvious seasonality. The clinical diagnosis of this disease was easy according to the specific lesions of liver and cecum. The results of the survey provided a certain reference for clinical control of the disease.2. Cloning and phylogenetic analysis of the18S rRNA gene of H. meleagridisIn order to learn about the classification status and characteristics of H. meleagridis at molecular level,30sequences of18S rRNA gene of H. meleagridis were obtained by PCR, cloning and sequencing. The homology of18S rRNA gene was analyzed and the phylogenetic tree was constructed. Results indicated that the sequence similarity of18S rRNA gene was among the range of98.4%-100%, and the relationship was close. The18S rRNA of H. meleagridis was relatively conservative, which can be used as a molecular diagnostic target gene. At the same time, there are five obscure branches in the phylogenetic tree branch, indicating that there might be different genotypes, which needs further research.3. Cloning and phylogenetic analysis of the ITS region gene of H. meleagridisIn order to take the further study on whether there are different genotypes of H. meleagridis in this region, the phylogenetic analysis on ITS region of H. meleagridis in this area, others in Genebank and other relative parasites was carried out. Results indicated that the sequence similarity of ITS region gene was among the range of96.8%-99.8%, which formed five different phylogenetic branches. Different genotypes of H. meleagridis in this area may be existed. This study has laid a foundation for the further molecular epidemiology investigation and study of population genetics of H. meleagridis.4. Establishment and application of PCR assay for detection of H. meleagridis infectionA pair of specific primers were designed and synthesized according to18S rRNA gene of H. meleagridis. The PCR assay was optimized, the specificity and sensitivity were analyzed, and clinical samples of liver were detected. Results of sensitivity test indicated that the minimum detection limit of PCR was4ng/uL DNA of infected liver. The specificity test results showed no cross reaction with other common parasites in chickens. Among the14lesion-positive clinical samples of liver collected100%were positive detected by PCR assay, and the coincidence rate is100%compared with histopathological diagnosis results. In conclusion, the PCR assay is specific and sensitive, and should be useful for routine diagnosis of H. meleagridis in poultry.5. Loop-mediated isothermal amplification assay for detection of H. meleagridis infectionTo develop a rapid and sensitive method for specific detection of H. meleagridis, an assay based on loop-mediated isothermal amplification (LAMP) targeting the18S rRNA gene was established. The detection limit of the LAMP assay was10copies for standard plasmids containing18S rRNA gene fragment, which was superior to that of a classical PCR method. Specificity test revealed that there was no cross-reaction with other protozoa. This assay was evaluated for its diagnostic utility using field liver and caeca samples collected from suspected cases, the detection rate was100%and97.92%, respectively. These results indicate that the LAMP assay may be a useful tool for rapid detection and identification of H. meleagridis in poultry.