Morphological and molecular characterization of amphistomes from wild ruminants and their snail intermediate hosts in Matebeleland region of Zimbabwe.

Abstract

Amphistomosis is a tropically neglected disease that affects both wild and domestic ruminants. The disease is not well understood as to date most amphistome species have no known intermediate hosts. Wild ruminants harbor several amphistome species which they share with domestic ruminants, however, very few studies have focused on amphistomes of these wild ruminants. The lack of molecular information on amphistomes of wild ruminants and their intermediate host snails prompted the need to do a study on the identity of amphistomes species of wild ruminants in Zimbabwe. The main objectives of the study were to (a) conduct a systematic review of amphistomes found in wild ruminants across Africa (b) to conduct molecular and morphological characterization of amphistomes present in the wildlife ruminants, and (c) to detect the presence of DNA of these amphistomes in the intermediate host snails collected from wildlife conservancies and game parks in Matebeleland region of Zimbabwe. The study sites for all this work were areas Matebeleland, located in the southwestern region of Zimbabwe. A systematic search of literature on the amphistomes of wild ruminants in published between 1900 and 2022 in Africa showed that there are a total of 38 amphistome species belonging to the genera Calicophoron (C.), Gastrothylax (G.), Carmyerius (Car.), Cotylophoron (Cot.), Leiperocotyle (L.), Bilatorchis (Bi.), Paramphistomum (P.), Stephanopharynx (S.) and Gigantocotyle (Gi.) identified and recorded in wild ruminants. It was observed that most studies on amphistomes of wild ruminants were conducted and documented before the year 2000, which not only show the paucity of data in this area but also highlighted the less attention given to these group of parasites by researchers. Studies done post 2000 mostly shifted and focused on amphistomes and other helminth parasites of domestic animals such as schistosomiasis including zoonotic diseases such as fascioliasis which are more common and have a higher impact on public health in Africa. This is understandable as domestic animals are seen as an investment and of economic importance to most countries in Africa. As a result, current amphistome prevalence estimates in Africa may not completely reflect the diversity of host species or geographical areas. Results also revealed that only one study has been done to date on molecular characterization of amphistomes of wild ruminants and future research gaps were identified. A total of 313 hunter-killed wild ruminants from game parks and wildlife conservancies from Hwange, Nyamandlovu and Beitbridge, were randomly selected and assessed for amphistome infection. The wild ruminant species were identified as Syncerus caffer (African buffalo), Aepyceros melampus (impala), Redunca arundinum (reedbuck), Kobus ellipsiprymnus (waterbuck), Hippotragus niger (sable), Tragelaphus strepsiceros (kudu), Connochaetes taurinus taurinus (blue wildebeest), Sylvicapra grimmia (common duiker), Tragelaphus sylvaticus (bushbuck) and Damaliscus lunatus (tsessebe). From these, amphistome infections were found in African buffalo, sable, waterbuck, kudu, eedbuck and blue wildebeest. The overall prevalence of infection was 10.22% (32/313), with the highest infection rate recorded in 63 % (7/11) in waterbuck. The highest prevalence of amphistomes per locality was in Hwange at 35% (7/20) whilst the lowest prevalence was observed in Nyamandlovu at 5.62% (15/267). Morphology revealed eight amphistome species which included C. raja, C. microbothrium, C. phillerouxi, C. clavula, L. gretillati, G. crumenifer, Gi. symmeri and Orthocoelium (O.) dicranocoelium. Molecular analysis using ITS-2 region confirmed identity of some of the isolates with a percentage identity of above 97%. This is a first record of Gastrothylax crumenifer, Leiperocotyle gretillati and Orthocoelium dicranocoelium in Zimbabwe. This was followed by a study on detection of amphistome DNA and identification of snail intermediate hosts of amphistomes from the following six areas in the Matebeleland region of Zimbabwe; Matopos, Inyathi, Beitbridge, Nyamandlovu, Ntabazinduna and Esigodini. A total of 487 freshwater snails were collected from 9 of 19 surveyed water points (from the six mentioned areas) where wild ruminants had been spotted or frequently drink and were morphologically identified as Biomphalaria (Bio.) pfeifferi, Bulinus (B.) tropicus, B. truncatus, B. globosus, Lymnaea (Radix) natalensis, Physa (Phy.) acuta, Bellamya spp. and Melanoides (M.) tuberculata. Of these snails, species identifcication was confirmed for Bio. pfeifferi, Bul. tropicus, Bul. truncatus, Bul. globosus, L. (R.) natalensis, based on the Cox1 gene. Bulinus tropicus and Phy. acuta were collected in abundance. Amphistome DNA was detected in 11.9 % (58/487) snails, with the highest infection rate being detected in B. globosus (44.4 %). Amphistome DNA from M. tuberculata was successfully sequenced and identified as C. microbothrium. An additional band was detected in M. tuberculata, B. tropicus and B. trancatus which showed a 96.42% similarity to Paragonimus spp. sequence in the GenBank. Although mapping was done in the review paper, it shows that there still is a lack of current information on amphistomes of wild ruminants. More work therefore needs to be done on wild ruminants as they may be potential reservoirs of amphistomes. In conclusion, this study revealed how wild ruminants harbor diverse amphistome species, some of which have not been documented before in Zimbabwe. This may be attributed to either movement of wild animals across barriers or establishment of new and invasive snails in different parts of the country. Furthermore, the detection of Calicophoron microbothrium DNA in snails such as Melanoides tuberculata shows that there could be many more snail intermediate hosts of amphistomes. Future work should focus on screening more snails for amphistome infection.