A comparative study on the impact of two artemisinin derivatives, artemether and artesunate, on the female reproductive system of Fasciola hepatica

Highlights

• The artemisinins caused severe disruption and breakdown of the vitelline follicles.

• The ovary, Mehlis’ gland and ootype remained unaffected by drug action.

• The vitellaria changes lead to a drop in egg production.

• Eggs produced by the triclabendazole-resistant flukes were abnormal and lacked sperm.

Abstract

An in vivo study in the laboratory rat model has been carried out to monitor changes to the female reproductive system in adult Fasciola hepatica following treatment with the artemisinins, artemether and artesunate. Rats infected with the triclabendazole (TCBZ)-resistant Sligo isolate were dosed orally with artemether at a concentration of 200 mg/kg and flukes recovered at 24, 48 and 72 h post-treatment (pt). Rats infected with the TCBZ-resistant Oberon isolate were dosed orally with artesunate at a concentration of 200 mg/kg and flukes recovered 24, 48, 72 and 96 h pt. The flukes were processed for histological and transmission electron microscope (TEM) examination of the uterus, Mehlis’ gland, ovary and vitellaria.

After treatment with artemether, egg production had become abnormal by 72 h pt, with free vitelline cells and masses of shell protein material within the uterus; spermatozoa were absent. The Mehlis’ gland and ovary retained a normal morphology over the 3-day period. A change in the cell population in the vitelline follicles was seen at 48 h pt, with a decline in the number of immature cells. This became more marked by 72 h and the follicles became progressively vacuolated over the 3-day period. At the TEM level, there were changes in the immature vitelline cells at 24 h pt, as evidenced by a decrease in shell protein production and the presence of lipid droplets and abnormal mitochondria. Spaces in the follicles separated the cells from each other. The changes became progressively more severe with time, so that, by 72 h pt, the follicles were very disrupted, containing cells in the advanced stages of apoptotic breakdown. In extreme cases, the follicles were scarcely recognisable and had become filled with cellular debris. Fine structural changes to the vitelline cells induced by artesunate treatment were similar to those described for artemether, but generally occurred more quickly and were greater; this was particularly true of the swelling of the ger cisternae.

Overall, the results have shown that artemisinin treatment has a severe impact on egg production by TCBZ-resistant flukes, an effect that is mediated by disruption of the vitelline cells.

Introduction

Triclabendazole (TCBZ) remains the most effective flukicide for the treatment of fasciolosis in ruminants because of its high activity against the most clinically damaging immature, migratory stage of infection. However, the over-dependence on TCBZ has led to the almost inevitable development of drug-resistant fluke populations. Since the first report of TCBZ resistance (in Australia, by Overend and Bowen, 1995), further well-documented reports have identified resistance in Australia (Walker et al., 2004, Brockwell et al., 2014), in South America (Olaechea et al., 2011, Ortiz et al., 2013) and in a number of European countries, including The Netherlands, UK and Ireland (e.g. Coles et al., 2000, Gaasenbeek et al., 2001, Flanagan, 2010, Mooney et al., 2009, Gordon et al., 2012, Hanna et al., 2015). Taken on its own, the existence of TCBZ resistance in diverse areas of the World is a matter of considerable concern and represents a challenge to management strategies in the future. Other drugs are available commercially, but their activity is confined to the adult stage and so they do not share the same range or spectrum of activity as TCBZ. Moreover, the position of TCBZ as market leader is being compromised by farmers responding to anecdotal reports of resistance by switching to alternative (and less effective or potentially inappropriate) drugs, even when this may not be warranted. However, there is another area of concern, namely, the recent and likely continued future increase in the incidence and spread of the disease (discussed by Fairweather, 2011b). These two issues (of resistance and altered epidemiology) should serve as drivers in the search for new drugs. One group of potential new compounds is the artemisinins.

The artemisinins are important antimalarial agents and anti-schistosomal compounds (e.g. Woodrow et al., 2005, Utzinger et al., 2007, Cui and Su, 2009, Ding et al., 2011, Keiser and Utzinger, 2012). More recently, they have received a considerable amount of attention as potential fasciolicides. A number of semi-synthetic and fully-synthetic artemisinin derivatives have been tested against Fasciola hepatica, and activity demonstrated under in vitro conditions and in rodent (rat) infections, even against a TCBZ-resistant isolate of F. hepatica (Keiser et al., 2006a, Keiser et al., 2006b, Keiser et al., 2007, Duthaler et al., 2010, Duthaler et al., 2012, Zhao et al., 2010, Kirchhofer et al., 2011, Kirchhofer et al., 2012, Wang et al., 2011). This activity has not been fully or consistently translated to infections in larger ruminants such as sheep (Keiser et al., 2008, Keiser et al., 2010a, Keiser et al., 2010b, Meister et al., 2013). Nevertheless, it is important for the future design and testing of new artemisinin derivatives that there is a better understanding of their action against fluke. There have been few morphological studies and they have concentrated on external surface changes to flukes following treatment (Keiser and Morson, 2008a, Keiser and Morson, 2008b, Halferty et al., 2009, O’Neill et al., 2009, O’Neill et al., 2015, Kirchhofer et al., 2011, Duthaler et al., 2012).

The aim of the present investigation was to assess the impact of two semi-synthetic derivatives, artemether and artesunate, on the reproductive capacity of the fluke, by observing changes in those components of the female reproductive system that are involved in egg production, namely, the ovary, vitellaria, Mehlis’ gland and uterus. The main focus of the paper will be on the vitelline cells, which provide shell protein and energy reserves for egg formation. They have a very high level of secretory activity and rapid turnover, are very sensitive to drug action and previously have been good models for studies on drug action against F. hepatica (Fairweather et al., 1988, Skuce and Fairweather, 1990, Stitt and Fairweather, 1996, Colhoun et al., 1998, Halferty et al., 2009, Toner et al., 2011, McConville et al., 2012). Additional histological observations on changes induced by artemether in other female tissues will provide a more complete picture of drug action and the downstream impact on egg production. Furthermore, the results may help to explain the faecal egg count reductions recorded post-treatment with these two artemisinins (Keiser et al., 2008, Keiser et al., 2010a). Two well-defined TCBZ-resistant fluke isolates were used in the study.