An update on freshwater gastropods serving as intermediate hosts of amphistomes infecting livestock in South Africa

Improved surveillance of amphistome parasites and their intermediate hosts is crucial for comprehending their epidemiology and developing effective prevention and control measures, since amphistomosis is among the neglected parasitic diseases of ruminants, alongside schistosomiasis and fasciolosis. This study characterized the diversity of freshwater gastropods collected from six South African provinces and assessed their potential in the transmission of amphistomes infecting livestock. A total of 472 freshwater gastropods were collected from 6 provinces of South Africa between 2021 and 2023, and 11 snail species were identified based on shell morphology and confirmed using the mitochondrial cytochrome c oxidase I (COI) gene. Snails were screened for amphistome infection by cercarial shedding, followed by polymerase chain reaction (PCR) using the internal transcribed spacer 2 (ITS-2) region. Of these, 43 snails comprising Radix (R.) natalensis (n = 15), Physella (P.) acuta (n = 20), and Melanoides (M.) tuberculata (n = 8) shed cercariae. DNA was extracted from the shed cercariae and successfully amplified for amphistome DNA. However, only seven sequences obtained from cercariae shed by P. acuta (n = 5) and M. tuberculata (n = 2) were identified as amphistome spp. Cercariae from P. acuta were identified as Calicophoron microbothrium (n = 2) and Paramphistomidea spp. (n = 3), while the two cercarial samples from M. tuberculata were also identified as C. microbothrium. Whole tissue from the remaining 429 snails that did not shed cercariae was harvested and screened for amphistome DNA. Of these, 59 snails yielded an amplicon of the expected band size (380 bp) for amphistomes; however, the majority failed downstream quality control, while some amplified gastropod DNA. To the best of our knowledge, these findings provide the first confirmation of M. tuberculata as an intermediate host in the natural transmission of C. microbothrium in South Africa, as well as the first report of natural infection with C. microbothrium in P. acuta in sub-Saharan Africa. This study therefore represents a significant addition of this invasive snail species to the list of known intermediate hosts of amphistomes in Africa.