We report the discovery of nontoxic fungicide fenarimol (1) as an inhibitor of Trypanosoma cruzi ( T. cruzi ), the causative agent of Chagas disease, and the results of structure-activity investigations leading to potent analogues with low nM IC(50)s in a T. cruzi whole cell in vitro assay. Lead compounds suppressed blood parasitemia to virtually undetectable levels after once daily oral dosing in mouse models of T. cruzi infection. Compounds are chemically tractable, allowing rapid optimization of target biological activity and drug characteristics. Chemical and biological studies undertaken in the development of the fenarimol series toward the goal of delivering a new drug candidate for Chagas disease are reported.

BackgroundThe trypanosome diversity of the Brush-tailed Bettong (Bettongia penicillata), known locally as the woylie, has been further investigated. At a species level, woylies are critically endangered and have declined by 90% since 1999. The predation of individuals made more vulnerable by disease is thought to be the primary cause of this decline, but remains to be proven.MethodsWoylies were sampled from three locations in southern Western Australia. Blood samples were collected and analysed using fluorescence in situ hybridization, conventional staining techniques and microscopy. Molecular techniques were also used to confirm morphological observations.ResultsThe trypanosomes in the blood of woylies were grouped into three morphologically distinct trypomastigote forms, encompassing two separate species. The larger of the two species, Trypanosoma copemani exhibited polymorphic trypomastigote forms, with morphological phenotypes being distinguishable, primarily by the distance between the kinetoplast and nucleus. The second trypanosome species was only 20% of the length of T. copemani and is believed to be one of the smallest recorded trypanosome species from mammals. No morphological polymorphism was identified for this genetically diverse second species. We described the trypomastigote morphology of this new, smaller species from the peripheral blood of the woylie and proposed the name T. vegrandis sp. nov. Temporal results indicate that during T. copemani Phenotype 1 infections, the blood forms remain numerous and are continuously detectable by molecular methodology. In contrast, the trypomastigote forms of T. copemani Phenotype 2 appear to decrease in prevalence in the blood to below molecular detectable levels.ConclusionsHere we report for the first time on the morphological diversity of trypanosomes infecting the woylie and provide the first visual evidence of a mixed infection of both T. vegrandis sp. nov and T. copemani. We also provide supporting evidence that over time, the intracellular T. copemani Phenotype 2 may become localised in the tissues of woylies as the infection progresses from the active acute to chronic phase. As evidence grows, further research will be necessary to investigate whether the morphologically diverse trypanosomes of woylies have impacted on the health of their hosts during recent population declines.

BackgroundIn natural aquatic environments biofilms are known to act as environmental reservoirs for Cryptosporidium parvum oocysts. However, the fate of these oocysts within biofilms has yet to be determined.MethodsThis study aimed to identify if biofilms have the ability to support the multiplication of Cryptosporidium by measuring the change in parasite number over time using quantitative polymerase chain reaction (qPCR) and detecting the possible extracellular developmental stages using a combination of confocal microscopy and immunolabelling techniques. Pseudomonas aeruginosa biofilm flow cell systems were established and C. parvum oocysts were constantly supplied over a six day period.ResultsA significant (P < 0.001) increase in Cryptosporidium was detected as the biofilm matured, with the total number of C. parvum multiplying 2–3 fold during this period. With this, various Cryptosporidium developmental stages (sporozoites, trophozoites, type I and II meronts) were identified from the biofilm.ConclusionThis is the first study demonstrating that biofilms not only serve as an environmental reservoir for oocysts, but are also capable of supporting the multiplication of Cryptosporidium over time in an aquatic environment.

BackgroundGiardia is now considered the most common enteric parasite in well cared for dogs and cats in developed countries. The ecology, epidemiology and clinical impact of infections with this parasite in such animals is still not fully understood due to variable results across different studies.MethodsFaecal samples were collected between 2009 and 2012 from privately owned cats and dogs in Germany presented to local veterinarians for a variety of reasons. Giardia positive samples were identified by microscopy and coproantigen methods. Total faecal DNA was extracted from Giardia positive samples and multilocus genotyping methods (18S rDNA, β-giardin, GDH) were applied. Relationships between host age, sex, and breed, season of presentation and the different species of Giardia detected were assessed.ResultsA total of 60 cat and 130 dog samples were identified as Giardia positive. Potentially zoonotic Giardia was identified in both animal species. Cats had a similarly high rate of infection with the G. duodenalis and G. cati. Cats less than 1 year were more likely to have G. duodenalis than cats older than 1 year. Pure breed cats demonstrated a greater proportion of zoonotic species than mixed breed cats. In samples from dogs, G. canis (C and D genotypes) were identified most commonly. Male dogs were more likely to have G. canis (genotype D) than female dogs. The 18S rDNA PCR protocol was the most successful followed by the β-giardin and GDH (amplifying from 92%, 42% and 13% of samples respectively).ConclusionsThe potentially zoonotic species G. duodenalis and G. enterica were found in cat and dog samples, with G. duodenalis found in greater numbers; however, this may be due to the detection techniques utilised. Cats appeared to show a relationship between G. duodenalis and G. cati with age and breed, which may be explained by different housing habitats for pure and mixed breed cats. The different success rates for the three loci utilised highlights the usefulness of the 18S locus as a screening tool, as well as the importance of using multiple loci for genotyping to fully determine the level of multiple infection of Giardia present.

BackgroundThe brush-tailed bettong or woylie (Bettongia penicillata) is on the brink of extinction. Its numbers have declined by 90% since 1999, with their current distribution occupying less than 1% of their former Australian range. Woylies are known to be infected with three different trypanosomes (Trypanosoma vegrandis, Trypanosoma copemani and Trypanosoma sp. H25) and two different strains of T. copemani that vary in virulence. However, the role that these haemoparasites have played during the recent decline of their host is unclear and is part of ongoing investigation.MethodsWoylies were sampled from five locations in southern Western Australia, including two neighbouring indigenous populations, two enclosed (fenced) populations and a captive colony. PCR was used to individually identify the three different trypanosomes from blood and tissues of the host, and to investigate the temporal and spatial dynamics of trypanosome infections.ResultsThe spatial pattern of trypanosome infection varied among the five study sites, with a greater proportion of woylies from the Perup indigenous population being infected with T. copemani than from the neighbouring Kingston indigenous population. For an established infection, T. copemani detection was temporally inconsistent. The more virulent strain of T. copemani appeared to regress at a faster rate than the less virulent strain, with the infection possibly transitioning from the acute to chronic phase. Interspecific competition may also exist between T. copemani and T. vegrandis, where an existing T. vegrandis infection may moderate the sequential establishment of the more virulent T. copemani.ConclusionIn this study, we provide a possible temporal connection implicating T. copemani as the disease agent linked with the recent decline of the Kingston indigenous woylie population within the Upper Warren region of Western Australia. The chronic association of trypanosomes with the internal organs of its host may be potentially pathogenic and adversely affect their long term fitness and coordination, making the woylie more susceptible to predation.