Comparative Biochemical Profile and Analysis of Genetic Varitions in Echinococcus Granulosus and its Control with the Selected Medicinal Plants

Abstract

Cystic Echinococcosis is a serious zoonotic disease caused by Echinococcus granulosus species complex. The current study is the first attempt to determine the level of infection in domestic livestock and to explore the CE-related knowledge and awareness among livestock farmers in different districts of Khyber Pakhtunkhwa, province of Pakistan. A total of 1297 animals were examined for hydatid cysts including 538 cows, 428 buffaloes, 208 sheep, and 123 goats, at different slaughter houses in different districts of Khyber Pakhtunkhwa in 2 years from September 2015 to September 2017. For epidemiological investigations, prevalence in association with various factors (climate, age, and gender), organ specificity, types of cysts (fertile, sterile, or calcified), and viability of cysts parameters was recorded. Basing on the results obtained, areas with high prevalence were selected for further follow-ups and administration of questionnaires to the farmers and dog owners, to provide baseline data about this parasitic disease and to identify potential areas of emergence with correspondence animal and of public health significance. The finding of this study revealed the presence of CE in livestock of KP, Pakistan. The prevalence of hydatid cysts was the highest in buffaloes (15.88%) followed by cows (15.79%), sheep (15.38%), and goats (3.25%). Our investigation revealed close relationship between prevalence and animal age and gender in different months of the year. These findings also showed the highest prevalence of hydatid cysts in liver (63.49%), followed by lungs (23.80%) and mesentery (2.64%). Fertile and viable cysts were observed in all animal species except goats. The highest percentage of fertile and viable cysts was reported from the liver and lungs of sheep. For evaluation of risk factors, a total of 384 respondents were investigated. The results of current study revealed that 97.9% of farmers are not familiar with CE and transmission of this infection from dogs to human and livestock. The present study shows that CE will continue to be of medical and veterinary importance in Pakistan. Many molecular techniques like DNA sequence and PCR based methods are been used largely to distinguish genotypes (G1-G10) of E. granulosus, providing new approach in the prevention and control program of hydatid cysts. The genotypic diversity of Echinococcus species in livestock of province Khyber Pukhtunkhwa, Pakistan is poorly explored. Therefore, this study was conducted to identify the genotypes of E. granulosus in domestic animals by using the mitochondrial cox1 gene sequence. For this purpose a total of 30 hydatid cysts samples were collected from different organs of naturally infected domesticated animals (Cows, buffaloes, sheep and goats). DNA was extracted from the protoscolices of individual fertile cysts and used for polymerase chain reaction. PCR amplified products (17) were subjected to Sanger sequencing by Genetic Analyzer 3700 ABI. The collected data was analyzed for homology using NCBI Genotyping Tool Program and Bio Edit. Codon Code Aligner Software was used to edit/clean the sequence. Phylogenetic analysis were further performed with the Molecular Evolution Genetic Analysis (MEGA) software package (version 7.0.1.4), for the confirmation of subtype determinations. The sequencing of the amplified product revealed the presence of Echinococcus granulosus senso stricto (G1-3) genotype as a dominant genotypes in all collected samples. Further studies covering a large number of isolates might be necessary to investigate if there are other genotypes in the hydatid cyst population in this region of Pakistan. There are several protoscolicidal agents for the inactivation of hydatid cysts protoscolices during surgery but many of them are associated with adverse side effects. This study was aimed to evaluate the antiprotoscolices effect of ethanol extracts of selected medicinal plants commonly found in the study area including Buxus Wallichiana, Morus nigra, Berberis vulgaris, Callistemon citrinus, Euphorbia hirta, Carthamus lanatus and Euphorbia heliscopia against Echinococcus granulosus in in-vitro and in-vivo model. Protoscolices were aseptically aspirated from naturally infected livestock and viability of protoscolices was confirmed by 0.1% eosin red stain method. Protoscolices were exposed to three different concentrations of alcoholic extracts (10 to 50mg/ml) for 10 to 60 min. Phytochemical analysis was performed to identify bioactive compounds in the alcoholic extracts of all the selected medicinal plants. The highest efficacy was shown by B. vulgaris (100.00%) followed by B. wallichiana (86.97%) and E. heliscopia (78.19%) respectively, after exposure of 60 minutes at 50mg/ml concentration, that lead to the significant reduction in the viability of protoscolices. In addition alkaloids, flavonoids, phenols, tannins and saponnins were identified qualitatively and weighted quantitatively, that might help in the identification of bioactive compounds involved in selective action on the tegument layer of protoscolices. The in-vivo study showed decrease in cysts number, decrease in diameter treated with B.vulgaris-Oral, B.vulgaris-Intraperitoneal, B.Wallichiana-Oral, B.Wallichiana-Intraperitoneal, and increase in reduction percentage of secondary hydatid cysts in the groups under investigation. Difference in the biochemical profile of hydatid cyst fluid was observed after in-vitro assay. These findings suggest that these three plants can be a promising source of potent antiprotoscolices effect. However, the mechanism by which plant extracts killed protoscolices and also their safety for living cells are unclear and needed to be more investigated.