The past, present and future of fluorescent protein tags in anaerobic protozoan parasites.

The world health organization currently recognizes diarrhoeal diseases as a significant cause of death in children globally. Protozoan parasites such as Giardia and Entamoeba that thrive in the oxygen-deprived environment of the human gut are common etiological agents of diarrhoea. In the urogenital tract of humans, the anaerobic protozoan parasite Trichomonas vaginalis is notorious as the most common non-viral, sexually transmitted pathogen. Even with high medical impact, our understanding of anaerobic parasite physiology is scarce and as a result, treatment choices are limited. Fluorescent proteins (FPs) are invaluable tools as genetically encoded protein tags for advancing knowledge of cellular function. These FP tags emit fluorescent colours and once attached to a protein of interest, allow tracking of parasite proteins in the dynamic cellular space. Application of green FPs-like FPs in anaerobic protozoans is hindered by their oxygen dependency. In this review, we examine aspects of anaerobic parasite biology that clash with physio-chemical properties of FPs and limit their use as live-parasite protein tags. We expose novel FPs, such as miniSOG that do not require oxygen for signal production. The potential use of novel FPs has the opportunity to leverage the anaerobe parasitologist toolkit to that of aerobe parasitologist.

Intercontinental distribution of a new trypanosome species from Australian endemic Regent Honeyeater (Anthochaera phrygia).

Establishing a health screening protocol is fundamental for successful captive breeding and release of wildlife. The aim of this study was to undertake a parasitological survey focusing on the presence of trypanosomes in a cohort of Regent Honeyeaters, Anthochaera phrygia, syn. Xanthomyza phrygia (Aves: Passeriformes) that are part of the breeding and reintroduction programme carried out in Australia. We describe a new blood parasite, Trypanosoma thomasbancrofti sp. n. (Kinetoplastida: Trypanosomatidae) with prevalence of 24·4% (20/81) in a captive population in 2015. The sequence of the small subunit rRNA gene (SSU rDNA) and kinetoplast ultrastructure of T. thomasbancrofti sp. n. are the key differentiating characteristics from other Trypanosoma spp. T. thomasbancrofti sp. n. is distinct from Trypanosoma cf. avium found in sympatric Noisy Miners (Manorina melanocephala). The SSU rDNA comparison suggests an intercontinental distribution of T. thomasbancrofti sp. n. and Culex mosquitoes as a suspected vector. Currently, no information exists on the effect of T. thomasbancrofti sp. n. on its hosts; however, all trypanosome-positive birds remain clinically healthy. This information is useful in establishing baseline health data and screening protocols, particularly prior to release to the wild.

Host origin determines pH tolerance of Tritrichomonas foetus isolates from the feline gastrointestinal and bovine urogenital tracts.

The ability for protozoan parasites to tolerate pH fluctuations within their niche is critical for the establishment of infection and require the parasite to be capable of adapting to a distinct pH range. We used two host adapted Tritrichomonas foetus isolates, capable of infecting either the digestive tract (pH 5.3-6.6) of feline hosts or the reproductive tract (pH 7.4-7.8) of bovine hosts to address their adaptability to changing pH. Using flow cytometry, we investigated the pH tolerance of the bovine and feline T. foetus isolates over a range of physiologically relevant pH in vitro. Following exposure to mild acid stress (pH 6), the bovine T. foetus isolates showed a significant decrease in cell viability and increased cytoplasmic granularity (p-value < 0.003, p-value < 0.0002) compared to pH 7 and 8 (p-value > 0.7). In contrast, the feline genotype displayed an enhanced capacity to maintain cell morphology and viability (p-value > 0.05). Microscopic assessment revealed that following exposure to a weak acidic stress (pH 6), the bovine T. foetus transformed into rounded parasites with extended cell volumes and displays a decrease in viability. The higher tolerance for acidic extracellular environment of the feline isolate compared to the bovine isolate suggests that pH could be a critical factor in regulating T. foetus infections and host-specificity.

Comparative transcriptomics reveals striking similarities between the bovine and feline isolates of Tritrichomonas foetus: consequences for in silico drug-target identification.

BACKGROUND: Few, if any, protozoan parasites are reported to exhibit extreme organ tropism like the flagellate Tritrichomonas foetus. In cattle, T. foetus infects the reproductive system causing abortion, whereas the infection in cats results in chronic large bowel diarrhoea. In the absence of a T. foetus genome, we utilized a de novo approach to assemble the transcriptome of the bovine and feline genotype to identify host-specific adaptations and virulence factors specific to each genotype. Furthermore, a subset of orthologs was used to characterize putative druggable targets and expose complications of in silico drug target mining in species with indefinite host-ranges. RESULTS: Illumina RNA-seq reads were assembled into two representative bovine and feline transcriptomes containing 42,363 and 36,559 contigs, respectively. Coding and non-coding regions of the genome libraries revealed striking similarities, with 24,620 shared homolog pairs reduced down to 7,547 coding orthologs between the two genotypes. The transcriptomes were near identical in functional category distribution; with no indication of selective pressure acting on orthologs despite differences in parasite origins/host. Orthologs formed a large proportion of highly expressed transcripts in both genotypes (bovine genotype: 76%, feline genotype: 56%). Mining the libraries for protease virulence factors revealed the cysteine proteases (CP) to be the most common. In total, 483 and 445 bovine and feline T. foetus transcripts were identified as putative proteases based on MEROPS database, with 9 hits to putative protease inhibitors. In bovine T. foetus, CP8 is the preferentially transcribed CP while in the feline genotype, transcription of CP7 showed higher abundance. In silico druggability analysis of the two genotypes revealed that when host sequences are taken into account, drug targets are genotype-specific. CONCLUSION: Gene discovery analysis based on RNA-seq data analysis revealed prominent similarities between the bovine and feline T. foetus, suggesting recent adaptation to their respective host/niche. T. foetus represents a unique case of a mammalian protozoan expanding its parasitic grasp across distantly related host lineages. Consequences of the host-range for in silico drug targeting are exposed here, demonstrating that targets of the parasite in one host are not necessarily ideal for the same parasite in another host.

Afternoon shedding of a new species of Isospora (Apicomplexa) in the endangered Regent Honeyeater (Xanthomyza phrygia).

The Regent Honeyeater (Xanthomyza phyrigia) is an endangered Australian bird species. Breeding populations have been established at Australian zoos in support of re-introduction programmes. This species is the host of a new species of Isospora (Apicomplexa). Oocysts are spherical, 25·8 (22·5-28·75) by 23·8 (20-26·25) µm with a colourless to pale yellow smooth wall undergoing rapid exogenous sporulation, 90% sporulated oocysts in 8 h at 20°C. Each oocyst contains 1 polar granule. Sporocysts are ovoid, 18·67 (17-19) by 9·49 (9-10) µm with a flat Stieda body and spherical substieda body devoid of a hyaline body. The asexual stages and sexual phase is within the enterocytes of the duodenum and jejunum. Faeces collected in the morning (AM, n=84) and in the afternoon (PM, n=90) revealed significant diurnal periodicity in oocyst shedding; 21% (18 of 84) of the AM were positive with the mean of 499 oocysts.g-1 compared to the PM with 91% (82 of 90) bird faeces positive with the mean of 129 723 oocysts.g-1. Therefore, parasite checks for these birds should be carried out in the afternoon to obtain an accurate result. The ecological significance of the high parasite burden in captive birds requires further investigation and comparison to the wild counterparts.

High prevalence of Tritrichomonas foetus 'bovine genotype' in faecal samples from domestic pigs at a farm where bovine trichomonosis has not been reported for over 30 years.

Bovine venereal trichomonosis caused by the flagellate Tritrichomonas foetus is a notifiable disease in Australia. While, T. foetus is pathogenic in both cattle and cats, it has long been established that the same T. foetus colonises the stomach, caecum and nasal cavity of pigs without apparent clinical significance. Multi-locus genotyping grouped the non-pathogenic porcine T. foetus with the pathogenic 'bovine genotype', rather than with the 'feline genotype' T. foetus. Bovine trichomonosis is now uncommon due to wide-spread use of artificial insemination, however, whether T. foetus remains prevalent in pigs where bovine trichomonosis has been eradicated remains unknown. We surveyed faecal samples from pigs farmed in close proximity with T. foetus-negative cattle. The Modified Diamond's Medium assay used were 77.4% (24/31) positive for trichomonads and 64.50% (20/31) were T. foetus-positive based on real-time PCR and conventional PCR. An axenic reference strain of T. foetus, designated PIG30/1 was established. In addition, a novel trichomonad ITS rDNA, PIG12, closely related to sequences from Trichomitus spp is reported. Multi-locus genotyping at nine loci matched PIG30/1 to the 'bovine genotype' T. foetus. In conclusion, cross-species transmission of T. foetus between pigs and cows from environmental exposure of T. foetus-contaminated pig faeces is unlikely. Domestic T. foetus-positive pigs possess a negligible risk of a successful T. foetus transmission event to cattle.

Comparative RNA-seq analysis of the Tritrichomonas foetus PIG30/1 isolate from pigs reveals close association with Tritrichomonas foetus BP-4 isolate 'bovine genotype'.

Tritrichomonas foetus was described as a commensal of the stomach, caecum and nasal cavity of pigs before it was recognised as the cause of reproductive tract disease of cattle. T. foetus also causes chronic large bowel diarrhoea in domestic cats. Multi-locus genotyping and comparative transcriptome analysis has previously revealed that T. foetus isolated from cat and cattle hosts are genetically distinct, referred to as the 'feline genotype' and 'bovine genotype', respectively. Conversely, multi-locus genotyping has grouped porcine T. foetus with the 'bovine genotype'. To compare the extent of the similarity between porcine T. foetus and cattle 'bovine genotype' isolates, RNA-sequencing (RNA-seq) was used to produce the first cell-wide transcriptome library of porcine T. foetus PIG30/1. Comparative transcriptome analysis of the PIG30/1 with the published bovine (BP-4) and feline (G10/1) transcriptomes revealed that the porcine T. foetus shares a 4.7 fold greater number of orthologous genes with the bovine T. foetus than with the feline T. foetus. Comparing transcription of the virulence factors, cysteine proteases (CP) between the three isolates, the porcine T. foetus was found to preferentially transcribe CP8 like the 'bovine genotype' T. foetus, compared to thehigh transcription of CP7 seen for 'feline genotype' T. foetus. At the cell-wide transcriptome level, the porcine T. foetus isolate (PIG30/1) groups closer with the 'bovine genotype' T. foetus rather than the 'feline genotype' T. foetus.

Quantitative PCR detection of Theileria equi using laboratory workflows to detect asymptomatic persistently infected horses.

Equine piroplasmosis is the most important tick-borne disease of horses. Regulations on movement of horses into disease-free countries are in place to preserve international trade. Introduction of infectious disease, such as equine piroplasmosis, into non-endemic countries remains a substantial risk owing to the wide-spread distribution of vectors. Identification and restriction of movement of Theileria equi persistently infected horses is an integral part of control strategies, because persistently infected horses with low parasitaemia are an important reservoir. We used real-time PCR for diagnosis of T. equi DNA in clinically healthy horses in an equine piroplasmosis endemic area. The sensitivity was assessed using a synthetic plasmid DNA and a laboratory workflow was developed to maximise detection of persistently infected horses. The detection limit was 10 rDNA copies of the plasmid DNA. Assuming that each red blood cell contains a single T. equi genome the detection limit corresponded to 2.5 T. equi/µl of total blood and parasitaemia as low as 2-3.8 × 10(-5)%. A laboratory workflow was developed and assessed on samples from Saudi Arabia. The laboratory workflow focused on samples returning no or single positive result in duplicate PCR. In total, we obtained 42% (59/141; 95% confidence interval: 33.85-50.15) T. equi positive samples, 26% (37/141) negative for T. equi samples. The remaining 45 samples were judged as suspect with no definitive diagnosis made. The Saudi Arabia's T. equi small subunit ribosomal DNA (SSU rDNA) sequencing (n=16) demonstrated A clade (n=15) as the dominant T. equi clade. Clade B was sequenced in a single case. We present an approach for diagnostic workflow to detect T. equi in clinically healthy but persistently infected horses. Results from Saudi Arabia confirm that T. equi is widespread in the Middle East region. High proportion of horses with low parasitaemia calls for caution with results based on a single blood sample. Understating of the fluctuation of the parasitaema in persistently infected horses in endemic areas is needed to establish the required sample numbers for reliable detection of T. equi.

Identification of Chromera velia by fluorescence in situ hybridization.

Chromera velia is evolutionarily the closest free-living and photosynthetic organism to the medically important obligatory parasitic apicomplexans that cause diseases including malaria and toxoplasmosis. In this study, a novel oligonucleotide probe targeting C. velia's small subunit ribosomal RNA was designed. To enable usage of this probe as a detection tool, a fluorescence in situ hybridization (FISH) protocol was optimized. The results obtained showed that when used in combination, the C. velia CV1 probe and optimized FISH protocol enabled efficient detection of C. velia in culture. This new technique will allow a better understanding of the ecological role of C. velia within the coral microhabitat.