Molecular epidemiology of Blastocystis infections

Blastocystis is an enteric protist and one of the most frequently reported parasitic infections in humans and a variety of animal hosts worldwide. The genus Blastocystis consists of numerous genetically distinct groups, referred to as subtypes (STs). Some STs are highly host specific, while others display moderate or low host specificity. Therefore, the aims of this study were to determine the prevalence amongst various animal hosts (captive, free-ranging and wild), genetic diversity and zoonotic potential of Blastocystis. As polyparasitism is considered to be the norm in wildlife, the final aim of this study was to develop a molecular-based diagnostic method for the simultaneous detection of Blastocystis, Cryptosporidium sp. and Giardia duodenalis in Australian native fauna. These aims were achieved by sampling captive animals and their keepers from the Perth Zoo. Also, animal samples were obtained from other zoos in Australia and Europe. Samples from free-ranging and wild non-human primates (NHPs) and Australian native fauna were also included in this study. All samples were screened for Blastocystis using Polymerase Chain Reaction (PCR), followed by phylogenetic analyses to characterise these isolates in order to determine the genetic diversity and zoonotic potential of isolates within the Blastocystis genus. Blastocystis was detected in 13 species of animals from the Perth Zoo. It was also detected in NHPs from Belgian zoos. All wild and free-ranging NHP and Australian wildlife populations also harboured Blastocystis. This study describes the first reports of Blastocystis in the elephant, giraffe, Javan lutung, quokka, southern hairy nosed wombat and western grey kangaroo. Similarly, 12 Blastocystis STs, including six novel STs (STs 11 – 13 and 18 – 20), were identified in humans and animal hosts sampled as part of this study. Blastocystis STs 1, 2, 18 and 19 were identified in captive NHPs. However, STs 2, 8 and 20 were identified in wild NHPs. Australian native animals at the Perth Zoo harboured STs 1, 12 and 13, whereas free-ranging animals from Karakamia Sanctuary (KS) and wild animals from the Upper Warren Region (UWR) harboured STs 1 – 4 and 7. Captive elephants and giraffes from Australian and European zoos harboured STs 11 and 12, respectively. Higher prevalence of Blastocystis amongst zoo keepers and sequence homology of isolates from zoo keepers and animals at the Perth Zoo provide evidence of the zoonotic potential of Blastocystis. High prevalence amongst zoo keepers may be due to close contact between the animals and the zoo keepers, and other tasks carried out by the zoo keepers, such as cleaning of enclosures. Similarly, some Blastocystis isolates from Australian wildlife were also homologous to human isolates, and it seems that these hosts are natural hosts for the zoonotic ST 4. Other parasites, such as strongyle nematodes and coccidia were detected using microscopy. Various species of Australian wildlife are known to harbour these and other parasites, including zoonotic parasites, such as Cryptosporidium sp. and Giardia duodenalis. As polyparasitism is considered to be the norm in wildlife, a multiplex PCR (mPCR) was developed to detect Blastocystis, Cryptosporidium and Giardia simultaneously from Australian wildlife. This mPCR was evaluated against other diagnostic methods routinely used for the detection of these parasites, such as microscopy and nested PCRs. The multiplex PCR showed comparative and/or greater sensitivity and specificity to routinely utilised nested PCRs. The major advantages of the multiplex PCR are that it is less labour intensive and is cost effective in comparison to the nested PCRs used to amplify each parasite. In conclusion, the host range and genetic diversity of Blastocystis is much greater than previously anticipated. Some STs and/or subgroups of STs appear to be highly host specific, while others display moderate or low host specificity. Also, some STs which have a broad host range may be zoonotic. This study also provides further insight into polyparasitism amongst Australian wildlife.