Morphological and molecular characterization of Fasciola hepatica and Fasciola gigantica phenotypes from co-endemic localities in Mpumalanga and KwaZulu-Natal provinces of South Africa

Fasciolosis is a food- and water-borne disease caused by digenean trematode species, Fasciola hepatica and F. gigantica. They are widely distributed and infect a wide range of definitive hosts, causing enormous economic loss due to reduced productivity in domestic ruminants. The two species have been previously reported to be co-endemic in KwaZulu-Natal and Mpumalanga provinces of South Africa. Hybridization between the two species has been reported elsewhere. Despite the overlap of the two species in two provinces, there has been no attempt to determine the presence of the intermediate forms or hybrids. Therefore, this study aimed at morphological and molecular characterization of Fasciola spp. collected from cattle slaughtered at abattoirs located in the two provinces of South Africa, where two species are endemic. A total of 71 fluke specimens were collected cattle from abattoirs in Enhlazeni and Nelspruit in Mpumalanga province and Pietermaritzburg in KwaZulu-Natal province of South Africa, and Zimbabwe (Bulawayo abattoir). Fasciola gigantica specimen collected from Zimbabwe where it has been confirmed as the only species occurring and this was used as control in the morphological and molecular assessment of the collected specimens. Of the 71 specimens collected, 37 were classified as F. hepatica, 12 as F. gigantica and 22 as Fasciola spp using morphological characters. Of these species, 11 of 37F. hepatica and 6 of 22 Fasciola sp were found to be aspermic or having very scanty sperm. Fifteen flukes which were spermatic were all identified morphologically as F. gigantica whilst 5 flukes which were aspermic were identified morphologically as F. hepatica. Molecular analysis of the same 15 spermatic specimens confirmed the presence of F. hepatica (n=9) and F. gigantica (n=6) using the CO1 marker and as F. hepatica (n=4), F. gigantica (n=7) and Fasciola sp. (n=1) for the same specimens using the ITS-1/5.8S/ITS-2 marker. The remaining 4 aspermic flukes (one did not resolve) were all identified morphologically as F. hepatica and molecular analysis confirmed them as F. hepatica (n=4) by both CO1 and ITS-1/5.8S/ITS-2. Phylogenetic analysis based on both CO1 and ITS-1/5.8S/ITS-2 showed that F. hepatica species formed a moderately supported monophyletic clade with F. gigantica. Their ancestral history was further confirmed by haplotype network, which formed novel haplotypes that corresponded with the structure of the phylogenetic tree. Results from this study showed that morphological characters alone have limitations in identifying F. hepatica and F. gigantica in areas where the two species occur, although both methods confirmed the presence of F. gigantica occurring in Zimbabwe, F. hepatica in KwaZulu-Natal, and both species occurring in Mpumalanga province. Therefore, the use of morphological techniques, complemented by molecular techniques are recommended, especially in endemic areas where the two species are co-endemic.