Free-living amoeba of the genus Acanthamoeba are ubiquitous protozoa involved in opportunistic and non-opportunistic infection in humans, such as granulomatous amoebic encephalitis and amoebic keratitis. Both infections have challenging characteristics such as the formation of the resistant cysts in infected tissues, hampering the treatment and most usual diagnosis depending on time-consuming and/or low sensitivity techniques. The use of monoclonal antibodies presents itself as an opportunity for the development of more effective alternative diagnostic methods, as well as an important and useful tool in the search for new therapeutic targets. This study investigated the possibility of using a previously produced monoclonal antibody (mAb3), as a diagnostic tool for the detection of Acanthamoeba trophozoites by direct and indirect flow cytometry and immunofluorescence. Immunoprecipitation assay and mass spectrometry allowed the isolation of the antibody's target and suggested it is a transporter part of the CPA (cation: proton antiporter) superfamily. In vitro tests indicate an important role of this target in Acanthamoeba's encystment physiology. Our results support the importance of studying the role of CPA2 transporters in the context of acanthamoebiasis, as this may be a way to identify new therapeutic candidates.

The trematode Parorchis acanthus is widely distributed and exhibits a broad host spectrum, with larval stages occurring in marine gastropods and adult flukes parasitizing on various seabird species. Unlike the majority of digeneans, its cercariae do not invade a second intermediate host but encyst on a substrate in the open. In a series of laboratory experiments, we investigated factors influencing cercarial emergence and settlement of P. acanthus. Emergence of cercariae from the gastropod intermediate host Nucella lapillus increased with increasing temperature, whereas cercarial encystment success decreased. Numbers of emerged cercariae were highest in the presence of light and water. However, although in much lower numbers, cercariae were also released from N. lapillus and successfully encysted under relatively dry conditions. Cercariae were found to settle predominantly on filtering blue mussels Mytilus edulis, suggesting that cercarial concentration on the valves is largely facilitated by the bivalve's inhalation current. After being inhaled, cercariae actively emerged from the mussels using their suckers and subsequently encysted on the shells, preferably along the rim of the valves. Barnacle epibionts Semibalanus balanoides were observed to impair cercarial settlement on mussels, by disturbing or preying on cercariae. Whilst settlement success of cercariae on M. edulis increased with increasing mussel density, intensity of encysted metacercariae decreased. Furthermore, cercariae settled more efficiently on small than on large mussels. Our findings suggest that the transmission of P. acanthus cercariae is regulated by a range of abiotic and biotic factors, with filtering activity of mussels playing a key role by enhancing accumulation of cercariae on a substrate suitable for transmission to the bird final host.

The toxicity of cadmium, zinc and cadmium/zinc mixtures at concentrations ranging from 1000 to 50000μg/l were investigated against cercariae and metacercariae of Parorchis acanthus obtained from the dog whelk Nucella lapillus. Cercarial encystment at concentrations of 25000μg/l or higher was significantly impaired by all test metals; however, at lower concentrations only zinc demonstrated toxicity. Mixtures of cadmium and zinc had a synergistic effect compared with single metal toxicity but only at 50000μg/l. Excystment in vitro was only significantly affected by cercariae exposed to cadmium/zinc mixtures whilst encysting. Twenty-four h exposures of fully formed cysts had no effect on excystment in vitro. Effects on in vitro excystment rates over a 2h period demonstrated widespread effects for cercariae-exposed P. acanthus. No effects were evident on excystment rates of cyst-exposed parasites.