Tetrazolium-based cytotoxicity assay to determine anti-protozoal activity against the scuticociliate Anophryoides haemophila

Abstract

Anophryoides haemophila is a ciliated protozoan that causes 'bumper car' disease in American lobster Homarus americanus. Currently, there are no effective pharmaceuticals approved for use to treat the disease, which can be a significant source of mortality in lobster holding facilities. We have modified a tetrazoliurn-based cytotoxicity assay to determine the anti-protozoa1 activity of compounds towards A , haemophila. To obtain reproducible results, A. haemophila maintained in continuous culture were preferable to freshly isolated A. haemophila. It was also found that the ciliates must be replicating to produce reproducible results. Maximal anti-protozoa1 activity was evident after a 24 h Incubation period. Oxytetracycline was found to be relat~vely Inactive, while high concentrations (1 mM) of formaldehyde and sulphaquinoxaline were required to produce 50% cytotoxiciry. In contrast, 100 pM lasalocid produced 92 i 4 % and 100 p M pyrimethamine produced 84 k 6.5 % cytotoxiclty after 24 h. This objective assay provides a rapid means of screening conlpounds to identify those with potential In vjvo activity against Anophryoides haemophila.

In vivo efficacy of lasalocid and pyrimethamine treatments against Anophryoides haemophila, a ciliate pathogen of American lobster Homarus americanus

“…Furthermore, no pathology was observed, when the drug was administered to uninfected lobsters. The drug has been also shown effective during in vitro trials (Novotny et al, 1996, Cribb et al, 1999. As there is no effective treatment for the control of bumper car disease, on the basis of the results of the present study, we suggest that pyrimethamine is a good drug for the treatment of A. haemoplila infection in lobsters.…”

“…Επίσης, δεν παρα τηρήθηκαν παθολογικές αλλοιώσεις, όταν το φάρμακο δό θηκε σε μη μολυσμένα άτομα. Εξάλλου, το φάρμακο έχει αποδειχθεί ότι είναι αποτελεσματικό και σε in vitro δοκι μές (Novotny et al, 1996, Cribb et al, 1999.…”

“…Loss of motility, however, may not be an indication of ciliate death and the essay was subjective, that's why a more effective tetrazolium based cytotoxicity assay was developed for the purpose of an objective and rapid screening of compounds in vitro (Cribb et al, 1999). In this investigation, a series of compounds was assessed.…”

“…Η έλλειψη κινητι κότητας, πάντως, μπορεί να μην είναι σίγουρη ένδειξη θα νάτου του παρασίτου και έτσι η δοκιμή ήταν υποκειμενι κή. Για τους λόγους αυτούς, μία πιο αποτελεσματική μέ θοδος αναπτύχθηκε, η οποία βασιζόταν στην κυτταροτο-ξικότητα με βάση το tetrazolium και είχε ως σκοπό την α ντικειμενική και γρήγορη εκτίμηση των ουσιών in vitro (Cribb et al, 1999). Σε αυτή την έρευνα, μία σειρά από ου σίες δοκιμάστηκαν.…”

Pathogens, Parasites and Other Symbionts

“…Oxytetracycline and amprolium had little to no effect on the ciliate motility. Cribb et al (1999) developed an in vitro tetrazolium-blue formazan assay for testing the efficacy of various antibiotics against A. haemophila. They found that 100 M lasalocid or pyrimethamine was cytotoxic to the ciliate over 24 hours.…”