Epidemiology and pathogenesis of fasciolosis in eastern Nepal

Following a general introduction, the thesis is divided into three parts, covering epidemiology, pathogenesis and molecular biology. Each part contains relevant literature review, materials and methods, results and a discussion for that particular part. The thesis ends with a general discussion which, includes an estimate of the financial loss resulting from fasciolosis in Nepal but concentrates on the major conclusions reached from this study and the major recommendations.In part one, following a review of literature on the factors affecting epidemiology of fasciolosis, a 19 months field survey on the epidemiology of fasciolosis in eastern Nepal is described. Four Lymnaea spp., namely L. auricularia race rufescens, L. auricularia sensu stricto, L. viridis and L. luteola were identified. L. auricularia race rufescens was the predominant species. The main snail habitats consisted of springs or stream fed rice-fields, irrigation channels, ponds and road-side pools. The monsoon rains and rice cultivation practices contributed to the creation and expansion of the snail habitats. The snail population density was high during the dry period and declined with the onset of the monsoon. Snail egg masses and young snails were observed throughout the year. In the hills, mature Fasciola spp. infections were found in snails from May to February, while in the Terai, infected snails were found throughout the year.In the hills, the highest prevalence of fasciolosis was recorded in buffaloes (57.9%) followed by cattle (44.8%), goats (22.4%) and sheep (18.2%). The relatively high prevalence of fasciolosis observed in stall-fed buffaloes was due to feeding metacercariae contaminated, rice-straw and grass, cut and carried from rice-fields. In the Terai, the highest prevalence was found in cattle (51.4%) followed by buffaloes (41.3%) and goats (13.3%). Slaughter place surveys revealed that F. gigantica is the predominant species, although infection with F. hepatica and an intermediate form were also found. The mean fluke burden in infected buffaloes was 203.2 ± 17.9, in infected goats this was 23.7 ± 3.3. Fluke burdens increases with the age of the animals. The seasonal pattern of the infections in the intermediate and definitive hosts indicated that the bulk of the infection was derived from fluke eggs deposited on the pasture during March-May and again in October and November.A control programme for fasciolosis based on these findings requires that all animals, including goats and sheep, be treated with appropriate anthelmintics, such as oxyclosanide or triclabendazole, in February and again in late August.In part two, the literature on the pathogenesis of fasciolosis with special reference to its effects on productivity of ruminants is reviewed. Descriptions of the 5 experimental studies on the pathogenesis of fasciolosis carried out in Edinburgh and in Nepal follow. A pilot comparative study in sheep, conducted in Edinburgh indicated that F. gigantica was more pathogenic than F. hepatica. A second pilot experiment was conducted in Nepal using 11 to 14 months old 9 Baruwal sheep. With a mean burden of only 5 F. gigantica, the mean weekly liveweight gain of the infected sheep over 35 weeks was 22.0% less than that of the uninfected controls. The pathogenesis in goats was investigated using 15 to 18 months old 18 Nepalese hill goats. Burdens of more than 1.3 flukes/kg of initial liveweight produced clinical chronic fasciolosis. A burden of 3.6 flukes/kg caused death of a goat at about 33 weeks after infection. There was a significant reduction in weight gain in the infected goats.Two experiments were carried out to study the pathogenesis of fasciolosis in 12 to 18 months old 32 Nepalese hill buffaloes. During the 35 weeks of post infection monitoring, no clinical signs were observed in the buffalo calves which harboured 1.3 flukes/kg resulted in clinical fasciolosis. A chronic disease occurred in those 6 buffalo calves which had burdens between 1.3 and 4.9 flukes/kg. Two of these became moribund around 26 weeks after infection while the remaining 4 survived until the end of the experiment. The presence of 7.4 to 9.1 flukes/kg caused subacute fasciolosis resulting in the death of buffalo calves between 14 and 18 weeks after infection. In both experiments, the liveweight gain of the infected buffaloes was affected; a longer infection period with smaller fluke burden has a proportionally greater effect on the depression of liveweight gain than a shorter infection period with larger fluke burden. The mean dressing percentage was reduced by about 12.4% in the infected buffaloes.Although, eosinophils and the increased serum GLDH and GGT levels appeared to be the sensitive indicators of Fasciola spp. infections, these were not related to the intensity of infections. Infiltration of eosinophils in periportal areas and bile ducts lamina propria was markedly evident in sheep and goats, but not in buffaloes. The inferior eosinophils together with the absence of liver tissue infiltration by eosinophils in infected buffaloes may well be related to the fact that buffaloes have poorer ability to develop resistance to infection of F. gigantica even than sheep and goats. A prominent feature in subacute infections in sheep and buffaloes was aberrant flukes in various organs and accompanying haemorrhagic areas.In part three, the available literature on the advantages and disadvantages of the various methods available for speciation and differentiation of Fasciola spp. is reviewed. This is followed by a description of the molecular biology work which was undertaken and which resulted in the development of species-specific (MHFh and MHFg) and cross-reactive (MHFxl and MHFx2) DNA piobes for the identification of Fasciola spp. None of the probes reacted with any of the control or host DNA's tested. If used in conjunction these DNA probes can clearly differentiate between F. hepatica and F. gigantica. As such they present useful tools for species identification