GENETIC CHARACTERISTICS AND SPATIAL DISTRIBUTION OF DICROCOELIUM IN THE HIMALAYAN RANGES OF PAKISTAN

Abstract

Digenean lancet liver flukes of the family Dicrocoeliidae (Trematoda: Digenea) can infect the bile ducts of a variety of wild and domesticated mammals and humans around the globe. Three species of the genus Dicrocoelium, namely Dicrocoelium dendriticum, Dicrocoelium hospes, and Dicrocoelium chinensis, have been described as causes of dicrocoeliasis in domestic and wild ruminants. Among these, D. dendriticum is the most common and is distributed throughout Europe, Asia, North and South America, Australia, and North Africa. The main economic impact of dicrocoeliasis in livestock is the rejection of livers from slaughtered animals at meat inspection. However, in severe infections, affected animals may show clinical signs, including poor food intake, ill thrift, poor milk production, alteration in fecal consistency, photosensitization, and anemia. Pakistan is a semi-tropical country with ideal environmental conditions for the survival and reproduction of a wide range of parasitic organisms. Parasitic diseases are of critical importance and cause low productivity in livestock, but they are often overlooked by livestock owners due to the low mortality associated with parasitic infections. In comparison to other parts of Pakistan, the mean daily temperature ranges in the Himalayan ranges create an arid environment with only patchy coniferous tree cover, providing habitats that are mostly hostile to many snail species and potentially creating isolated habitats for Dicrocoelium spp. To improve livestock productivity, it is critical to investigate parasite hotspots, identify species, and understand parasite biology. The thesis is composed of various objectives and the first objective was to examine the morphometric variation and histopathological complications caused in the liver of sheep and goats infected with Dicrocoelium. For this purpose, one hundred and ninety adult Dicrocoelium parasitized sheep and goats from Khyber Pakhtunkhwa and Gilgit Baltistan, Pakistan, were analyzed morphometrically. To evaluate the degree of variation among the population, 26 characters were compared. The principal component analysis showed that the morphological traits of Dicrocoelium have no significant variations among the studied population. The histopathological analysis of the liver showed severe infiltration of the inflammatory cells, RBCs congestion, damaged hepatocytes, and sinusoids in the vicinities of the central vein and uterine eggs. The morphologically overlapping traits prevented accurate species identification; therefore, the present study confirmed the species identification of xxi Dicrocoeliid flukes by using molecular approaches, the molecular markers of rDNA and mitochondrial DNA were used. A phylogenetic comparison of published D. dendriticum ribosomal cistron DNA, cytochrome oxidase-1 (COX-1), and NADH dehydrogenase-1 (ND-1) mitochondrial DNA sequences with those from D. chinensis was performed to assess inter and intra species variation and reaffirm the use of species-specific single nucleotide polymorphism markers. The results of PCR and sequencing of 34 corresponding fragments of ribosomal DNA, and 14 and 3 corresponding fragments of mitochondrial DNA from the Chitral Valley flukes, reveal 10, 4, and 1 unique haplotypes, respectively. Additionally, these findings confirm the first-time molecular species identity of Pakistani lancet liver flukes as D. dendriticum and D. chinensis. Moreover, dicrocoeliasis is an important cause of production loss in ruminants due to the cost of liver condemnation at slaughter. Therefore, the objectives of the present study were to determine the prevalence of Dicrocoelium species infection and to predict the ecological niches and climatic variables that support dicrocoeliosis in the Himalayan ranges of Pakistan. Thirty-three (33) of 381 liver samples collected at slaughter and two hundred and thirty-eight (238) of 6060 blood samples from sheep and goat herds in the region were positive for Dicrocoelium. The results showed that the prevalence of dicrocoeliosis was higher in sheep than in goats and highest in females aged greater than three years. Therefore, an environmental risk map was created to predict active zones of transmission and showed the highest probability values in central parts of the Chitral district in the northwest of Pakistan. Further, the findings depict that the climatic variables of the mean monthly diurnal temperature range (Bio2), annual precipitation (Bio12), and normalized difference vegetation index (NDVI) were significantly (p<0.05) associated with the presence of Dicrocoelium infection. Similarly, charting antibody dynamics in Dicrocoelium-naive animals to determine the time of exposure of infection is needed to improve a strategic control program for liver fluke infection in sheep and goats in Pakistan. The current study also investigated the transmission patterns of Dicrocoelium in sheep farms (n=10) and goat (n=10) farms in Khyber Pakhtunkhwa and Gilgit Baltistan, Pakistan, between 2018 and 2019. 15-20 animals from each farm were screened for Dicrocoelium infection through ELISA and investigated their time of infection. The results point out that colostral transfer of Dicrocoelium antibodies from seropositive mothers was detected in sheep and goats up to xxii 16 weeks of age. In both sheep and goats, the estimated time of infection is different in various farms and years. However, the highest infection rate found in sheep farms was 41 in 2018 and 40 in 2019, while in goats the infection rate was 22 in 2018 and 18 in 2019. A minimum infection rate was reported in sheep farms-04, and 05 in 2018 and 2019 respectively. Furthermore, in goats’ the lowest infection rate was observed at farm-05 in 2018 and at farm-02, 03, 04, and 05 in 2019. However, the Dicrocoelium infection was found most prevalent in sheep and goats in September (n=84) and August (n=63) respectively. In conclusion, this study provides a preliminary illustration of a phylogenetic approach that could be developed to study the ecology, biological diversity, and epidemiology of Dicrocoeliid lancet flukes when they are identified in new settings. The findings of this study demonstrate the most suitable ecological niches and climatic variables influencing the risk of dicrocoeliasis in the Himalayan ranges of Pakistan. The methods and results could be used as a reference to inform the control of dicrocoeliasis in the region.