SUMMARY OF MILK SA FUNDED RESEARCH ON LIVER FLUKE BETWEEN 2016 AND 2021.

Fasciola species are liver flukes that infect cattle and reduce the productivity by 15-30%. The infection can affect general health and feed intake that leads amongst others to morbidity, reduction in milk production and weight gains. One of the projects funded by Milk SA and done in the Tsitsikamma pasture-based dairy farms, is an investigation into the prevalence, seasonal occurrence and favourable/unfavourable environmental conditions of the intermediate snail host (Lymnaea truncatula) of the liver fluke parasite. The principle is that if one understands these factors, one can possibly control or manage the intermediate host more effectively, and as a result limit the development and infestation of the parasite which needs the snail to complete its life cycle.

The results have confirmed the farmers’ observation of dramatic differences in the importance of Fasciola prevalence between farms situated near, even bordering, each other. Mean totals of respectively zero and 42 of the intermediate hosts have been counted over three years per sampling site on two adjacent farms and even greater differences between adjacent sampling spots per farm. In fact, the number of L. truncatula present on one occasion in one of the most highly snail-infested sites was estimated to be greater than three million in total. This compared to mean totals of fewer than four L. truncatula recovered per sampling site in a survey of a large number of Belgian farms where Fasciola hepatica is highly prevalent. The consequence is that there is no doubt that this parasite would seriously threaten milk production on three of the four farms tested, should the liver fluke develop resistance to all of the available anthelmintics, as already of importance elsewhere, notably involving triclabendazole.

The prevalence and distribution of L. truncatula over seasons are clearly water and temperature dependent, but rain versus irrigation often have different outcomes as it affects the texture of muddy patches and therefore suitability of habitat of L. truncatula to thrive. The seasonal cycling of L. truncatula was shown to follow much the same pattern between the farms in the study, particularly as regards periods that are less conducive to the propagation of the snails. However, there was much variation between the farms, such as in time and number of peaks and variation in the time span of general peaking in numbers of snails recovered. This is to be expected, since open water is essential for the parasite to be able to infect the snail intermediate hosts, whereas the snail intermediate host can survive well in wet mud, in the absence of open water. Hence it is to be expected that the peaking of the parasite will not necessarily follow the peaking of L. truncatula very closely. Another factor is that the parasite has a depressive effect on the health of its snail intermediate host, which is not well adapted to the higher temperatures in midsummer. This implies that relatively high proportions of the infected snails would not survive long enough under such conditions to allow for completion of the development of the parasite in them. This is something that farmers can take note of.

Apart from the dependence on open water, the parasite eggs need to have been deposited in the vicinity of the snails long enough before the open water is present, for the eggs to have “matured” by the time they are required in the open water, so that the eggs are ready to hatch soon after they have become submerged in such water. These events need to be set in motion by periodic grazing of infected cattle on such marshy spots, in this way to allow the eggs to be disseminated to the spots and mature in depressions conducive to accumulation of open water. Once mature, the eggs can hatch soon after they have entered the water, hatch and successfully infect the snails, in this way to lead to infection of the cattle.  In other words, the young parasitic stage of Fasciola, the miracidium, needs to be fully developed to the level of being able to hatch soon after submersion, then swim in the water to find the snail intermediate host, penetrate, and develop to the next stage, the cercaria. Thereupon, once this next state has developed, it similarly requires open water to leave the snail, swim and encyst on herbage or other solid material in their surroundings to the metacercarial stage that is infective to their final hosts, for instance cattle and sheep. This information indicates that dairy farmers should manage their open water areas carefully.

An adjunct to this study was to test alternative identification methods. Traditional monitoring techniques such as physical identification and counting of individuals and species have shortcomings that include difficulties in correct identification of especially juvenile stages, dependence on weather conditions, shortage of expertise in taxonomy, the use of non-standardised sampling methods and the invasiveness of the field techniques. In contrast, environmental DNA (eDNA) methods are accurate, require less effort, are efficient and they offer good alternatives for monitoring the distribution of the species and larval stages. To test these possibilities, fresh water snails, mud and water samples were collected from different farm spots and laboratory cultures. DNA from both snail and immature stages of F. hepatica in both snails themselves and their environment (eDNA) was detected using PCR, followed by cloning and sequencing of amplifications.

PCR and sequence analysis confirmed the identity of the snail species. Fasciola hepatica DNA was detected in 9 of the 10 pre-selected L. truncatula snails (90%) after immature stages of the parasite had been detected in them, and in the three L. columella snails tested (100%). Snail DNA was detected in samples in which snail species had been cultured: 66.6% and 50% of water and mud samples respectively for L. truncatula and from two out of nine water samples (22%) for L. columella. Interestingly, the phylogenetic analysis showed the L. columella sequences grouped with those from South America. The results suggest that eDNA techniques may be useful in identification and quantification of Fasciola and its snail host, and may therefore support management decisions to disrupt the lifecycle of the parasite. The results of this investigation were published, the title of the paper being: Molecular detection of Fasciola hepatica and snail intermediate hosts and their environmental DNA, for sustainable management of fasciolosis.

The second project funded by Milk SA is based on biological control of fasciolosis (liver fluke infestation). This results because drug control is expensive and resistance to drugs on the market is now limiting this option. The objective is to develop a combination of botanical extracts and bio-control agents to control the different life stages of the liver flukes, and to control the intermediate snail hosts of the parasite. It was argued that the solution should be sustainable, as it is based on locally sourced botanical extracts to kill adult flukes and on locally sourced bio-control agents to kill the snails. Examples are fungi, bacteria and beneficial nematodes isolated from pastures and water bodies. The main points of the research focus were to:

1. Screen a range of non-toxic botanical extracts for activity against liver fluke adults and eggs in vitro and in vivo.
2. Isolate and screen a range of bio-control agents for the control of eggs, metacercariae, miracidia and cercariae, which are the stages that live in pastures even for brief periods before ingestion by snails or livestock.
3. Isolate and screen bio-control agents for the control of snail species that act as the intermediate host in the life cycle of Fasciola species.

In the inhibition work of the liver fluke, the efficacy of Morgana (Lilium spp), Moringa (Moringa oleifera), Funnel and Lemon grass (Cymbopogon spp) extracts were tested in vitro against fluke eggs using three dfferent solvents at 20 % concentration. Results showed that both Morgana and the Moringa plant extract was 100% effective. With ethanol extraction, both pineapple (Ananas comosus) and Moringa showed strong ovicidal effects against the eggs of the fluke. In the control of the snail host, Bacillus species, believed to have strong molluscicidal activity, were investigated. A total of 43 isolates were tested against the Lymnaea host. Screening trials revealed possible interference with the reproduction of the snails. Two isolates may have had an effect in the delay of reproduction and another isolate may have had an effect in the inhibition of reproduction. The results, however, were inconclusive but at the time further screening had to be abandoned since the post-doctoral student left to take up a position in Saudi-Arabia.

The Bacillus work has been picked up a year later. Due to difficulties in culturing and maintaining l. truncatula and L. columella in the laboratory, a population of aquatic snails (Physella acuta) was build up in tanks as a proxy for the fluke snail host. A total of 128 isolates that were identified as molluscicidal candidates from the initial 1193, are screened intensively. The results thus far have confirmed a general lack of direct activity against the aquatic snails, although two appear promising. In parallel, products of bacterial secondary metabolism are assessed for molluscicidal activity. This development may point to the right direction. The activity seems to be related to lipopeptide compounds which are bioactive surfactant molecules associated with the toxicity of Bacillus species.