Zoonotic helminths - why the challenge remains.

Helminth zoonoses remain a global problem to public health and the economy of many countries. Polymerase chain reaction-based techniques and sequencing have resolved many taxonomic issues and are now essential to understanding the epidemiology of helminth zoonotic infections and the ecology of the causative agents. This is clearly demonstrated from research on Echinococcus (echinococcosis) and Trichinella (trichinosis). Unfortunately, a variety of anthropogenic factors are worsening the problems caused by helminth zoonoses. These include cultural factors, urbanization and climate change. Wildlife plays an increasingly important role in the maintenance of many helminth zoonoses making surveillance and control increasingly difficult. The emergence or re-emergence of helminth zoonoses such as Ancylostoma ceylanicum, Toxocara, Dracunculus and Thelazia exacerbate an already discouraging scenario compounding the control of a group of long neglected diseases.

Kapsulotaenia tidswelli - an unusual cestode from the Australian goannas Varanus gouldii gouldii and V. giganteus.

Kapsulotaenia tidswelli is a proteocephalidean cestode that utilizes varanid lizards as definitive hosts. Fresh specimens of this cestode were observed with endogenous red pigmentation in the neck region that disappeared rapidly if specimens were not preserved in glutaraldehyde. The ultrastructural characteristics of the red pigment, which are described, suggest it is a carotenoid. Phylogenetic analysis confirmed a close relationship between K. tidswelli and other species of Kapsulotaenia for which sequence information is available. There is thus no reason to consider that the red pigmentation is because K. tidswelli is atypical, and it is proposed the carotenoids are likely to be associated with the diet of its varanid host.

Sensitivity testing of trypanosome detection by PCR from whole blood samples using manual and automated DNA extraction methods.

Automated extraction of DNA for testing of laboratory samples is an attractive alternative to labour-intensive manual methods when higher throughput is required. However, it is important to maintain the maximum detection sensitivity possible to reduce the occurrence of type II errors (false negatives; failure to detect the target when it is present), especially in the biomedical field, where PCR is used for diagnosis. We used blood infected with known concentrations of Trypanosoma copemani to test the impact of analysis techniques on trypanosome detection sensitivity by PCR. We compared combinations of a manual and an automated DNA extraction method and two different PCR primer sets to investigate the impact of each on detection levels. Both extraction techniques and specificity of primer sets had a significant impact on detection sensitivity. Samples extracted using the same DNA extraction technique performed substantially differently for each of the separate primer sets. Type I errors (false positives; detection of the target when it is not present), produced by contaminants, were avoided with both extraction methods. This study highlights the importance of testing laboratory techniques with known samples to optimise accuracy of test results.

Discovery of novel and potent benzhydryl-tropane trypanocides highly selective for Trypanosoma cruzi.

A benzhydryl tropinone oxime that is potently toxic to Trypanosoma cruzi has been previously identified. An SAR investigation determined that no part of the original compound was superfluous and all early SAR probes led to significant drops in activity. The only alteration that could be achieved without loss of activity was replacement of the aryl chloride substituent with chloro homologues. This led to the discovery of a trifluoromethyl-containing analogue with an EC(50) against T. cruzi of 30 nM and a cytotoxicity selectivity index of over 1000 relative to rat skeletal myoblast L-6 cells.

Morphological characterization of Cryptosporidium parvum life-cycle stages in an in vitro model system.

Cryptosporidium parvum is a zoonotic protozoan parasite that mainly affects the ileum of humans and livestock, with the potential to cause severe enteric disease. We describe the complete life cycle of C. parvum in an in vitro system. Infected cultures of the human ileocecal epithelial cell line (HCT-8) were observed over time using electron microscopy. Additional data are presented on the morphology, development and behavioural characteristics of the different life-cycle stages as well as determining their time of occurrence after inoculation. Numerous stages of C. parvum and their behaviour have been visualized and morphologically characterized for the first time using scanning electron microscopy. Further, parasite-host interactions and the effect of C. parvum on host cells were also visualized. An improved understanding of the parasite's biology, proliferation and interactions with host cells will aid in the development of treatments for the disease.

Species-level identification of trypanosomes infecting Australian wildlife by High-Resolution Melting - Real Time Quantitative Polymerase Chain Reaction (HRM-qPCR).

Conventional nested PCR and Sanger sequencing methods are currently the gold standards for detecting trypanosomes in wildlife. However, these techniques are time-consuming and can often overlook mixed infections. True trypanosome prevalence can thus be underrepresented. Here, we designed an 18S rDNA-based real-time quantitative PCR (qPCR) assay coupled with High-Resolution Melting Analysis (HRMA) to detect and discriminate three Trypanosoma species (T. copemani, T. noyesi, and T. vegrandis) commonly infecting Australian marsupials. A total of 68 genetically characterised samples from blood and tissue were used to validate the High-Resolution Melting - Real Time Quantitative Polymerase Chain Reaction (HRM-qPCR) assay. A further 87 marsupial samples consisting of blood, tissue and in vitro cultures derived from wildlife blood samples, were screened for the first time using this assay, and species identity confirmed using conventional PCR and Sanger sequencing. All three Trypanosoma species were successfully detected in pure cultures using the HRM-qPCR assay, and in samples containing mixed trypanosome infections. Of the 87 marsupial samples screened using the HRM-qPCR assay, 93.1% were positive for trypanosomes, and 8.0% contained more than one trypanosome species. In addition to the three targeted Trypanosoma species, this assay was also able to detect and identify other native and exotic trypanosomes. The turnaround time for this assay, from sample preparation to obtaining results, was less than 2 h, with a detection limit of 10 copies of the amplicon in a reaction for each of the targeted trypanosome species. This more rapid and sensitive diagnostic tool provides a high throughput platform for the detection, identification and quantification of trypanosome infections. It will also improve understanding of host diversity and parasite relationships and facilitate conservation management decisions.

Echinococcus, Giardia and Cryptosporidium: observational studies challenging accepted dogma.

The development of in vitro culture systems that allow the maintenance, and support the development of Echinococcus, Giardia and Cryptosporidium in the laboratory have had a significant impact on their biology and taxonomy and the epidemiology of infections they cause. This short retrospective review demonstrates how radical shifts in our understanding have occurred as a result of being able to grow these organisms in culture, and how molecular tools have helped in the interpretation of such research that often reflects the observations of earlier workers.

Vertical transmission of Toxoplasma gondii in Australian marsupials.

To date, little is known about the dynamics of vertical transmission of Toxoplasma gondii in Australian marsupials. Studies in mice demonstrate that vertical transmission of T. gondii is common and that chronically infected mice can transmit T. gondii to successive generations. In this study, PCR and immunohistochemistry were used to detect T. gondii in chronically infected marsupial dams and their offspring. T. gondii was detected in the unfurred pouch young of 2 out of 10 chronically infected western grey kangaroos (Macropus fuliginosus) and in the unfurred pouch young of a brush-tailed bettong (Bettongia penicillata). Results of the study suggest that vertical transmission of T. gondii can occur in chronically infected Australian marsupials.

The diversity, distribution and host-parasite associations of trypanosomes in Western Australian wildlife.

Little is known regarding the diversity, distribution or host-parasite associations of Trypanosoma spp. in Australian wildlife. Here we report on an investigation based on divergence of the 18S rRNA gene of trypanosomes isolated from a range of hosts and varied geographical locations. A total of 371 individuals representing 19 species of native animals from 14 different locations were screened. In total, 32 individuals from 9 different species tested positive for the parasite. Phylogenetic analysis revealed considerable parasite diversity with no clear geographical distribution and no evidence of host specificity. In general, it appears that Australian Trypanosoma spp. are widespread, with several genotypes appearing in multiple host species and in varied locations including both mainland areas and offshore islands. Some host species were found to be susceptible to multiple genotypes, but no individuals were infected with more than a single isolate.

Molecular epidemiology of Giardia and Cryptosporidium infections - What's new?

New information generated since 2016 from the application of molecular tools to infections with Giardia and Cryptosporidium is critically summarised. In the context of molecular epidemiology, nomenclature, taxonomy, in vitro culture, detection, zoonoses, population genetics and pathogenicity, are covered. Whole genome sequencing has had the greatest impact in the last three years. Future advances will provide a much better understanding of the zoonotic potential of both parasites, their diversity and how this is linked to pathogenesis in different hosts.

African trypanosomiasis: sensitive and rapid detection of the sub-genus Trypanozoon by loop-mediated isothermal amplification (LAMP) of parasite DNA.

Control of human African trypanosomiasis (HAT) is dependent on accurate diagnosis and treatment of infected patients. However, sensitivities of tests in routine use are unsatisfactory, due to the characteristically low parasitaemias in naturally infected individuals. We have identified a conserved sequence in the repetitive insertion mobile element (RIME) of the sub-genus Trypanozoon and used it to design primers for a highly specific loop-mediated isothermal amplification (LAMP) test. The test was used to analyse Trypanozoon isolates and clinical samples from HAT patients. The RIME LAMP assay was performed at 62 degrees C using real-time PCR and a water bath. DNA amplification was detectable within 25min. All positive samples detected by gel electrophoresis or in real-time using SYTO-9 fluorescence dye could also be detected visually by addition of SYBR Green I to the product. The amplicon was unequivocally confirmed through restriction enzyme NdeI digestion, analysis of melt curves and sequencing. The analytical sensitivity of the RIME LAMP assay was equivalent to 0.001 trypanosomes/ml while that of classical PCR tests ranged from 0.1 to 1000 trypanosomes/ml. LAMP detected all 75 Trypanozoon isolates while TBR1 and two primers (specific for sub-genus Trypanozoon) showed a sensitivity of 86.9%. The SRA gene PCR detected 21 out of 40 Trypanosoma brucei rhodesiense isolates while Trypanosoma gambiense-specific glycoprotein primers (TgsGP) detected 11 out of 13 T. b. gambiense isolates. Using clinical samples, the LAMP test detected parasite DNA in 18 out of 20 samples which included using supernatant prepared from boiled blood, CSF and direct native serum. The sensitivity and reproducibility of the LAMP assay coupled with the ability to detect the results visually without the need for sophisticated equipment indicate that the technique has strong potential for detection of HAT in clinical settings. Since the LAMP test shows a high tolerance to different biological substances, determination of the appropriate protocols for processing the template to make it a user-friendly technique, prior to large scale evaluation, is needed.

Alpha 2 giardin is an assemblage A-specific protein of human infective Giardia duodenalis.

Of the 7 genetic assemblages of the parasite Giardia duodenalis only 2 (A and B) are known to cause infections in humans. These assemblages have been characterized in detail at the genomic level but few studies have examined differences in the proteins expressed. Employing one and two-dimensional PAGE we have identified an assemblage A-specific protein of human infective G. duodenalis; alpha 2 giardin. The protein difference was evident using both electrophoretic techniques. Alpha 2 giardin is known to be a structural protein and associates with the caudal flagella and the plasma membrane; however, its exact function is unknown. Although several proteins unique to assemblage B were also observed, we were unable to identify these proteins due to a lack of genomic data available for assemblage B isolates. Together, these proteins represent distinct phenotypic differences between the human infective assemblages of G. duodenalis and support the need to revise the taxonomy of this parasite.

Trypanosomes in a declining species of threatened Australian marsupial, the brush-tailed bettong Bettongia penicillata (Marsupialia: Potoroidae).

The brush-tailed bettong (Bettongia penicillata), or woylie, is a medium-sized macropod marsupial that has undergone a rapid and substantial decline throughout its home range in the Upper Warren region of Western Australia over a period of approximately 5 years. As part of an investigation into possible causes of the decline a morphologically distinct Trypanosoma sp. was discovered by light microscopy in the declining population but was absent in a stable population within the Karakamia Wildlife Sanctuary. Further investigations employing molecular methods targeting variations in the 18s rRNA gene determined that the trypanosome was novel and was also present within the Karakamia population albeit at a much lower overall prevalence and individual parasitaemia levels. Phylogenetic analysis suggests the novel Trypanosoma sp. to be closely related to other trypanosomes isolated from native Australian wildlife species. Although it appears unlikely that the parasite is solely responsible for the decline in woylie population size, it may (singularly or in conjunction with other infectious agents) predispose woylies to increased mortality.

The taxonomy, phylogeny and transmission of Echinococcus.

The application of molecular tools to the characterisation of the aetiological agents of echinococcosis has revealed a series of largely host-adapted species and genotypes that are maintained in distinct cycles of transmission. They can be defined on both genetic and phenotypic characteristics which complement previous observations made by descriptive parasitologists many years ago. A revised taxonomy for species in the genus Echinococcus has been proposed and widely accepted, particularly with respect to forms maintained in transmission cycles involving sheep, horses and cattle. However, molecular epidemiological studies are required in a number of endemic areas in order to determine cycles of transmission responsible for maintaining the parasite. The taxonomic status of forms in cervids, pigs and camels has still to be resolved, and the status and epidemiological significance of newly described species in China requires further research.

The molecular characterisation of Giardia from dogs in southern Germany.

Faecal samples were obtained randomly from asymptomatic dogs visiting veterinary clinics (kept individually) and sanctuaries (kept in groups) in southern Germany. Of 60 Giardia positive samples, 55 samples successfully amplified at the 18S rDNA locus. In both dogs kept individually and dogs kept in groups, assemblage A was most prevalent (overall, n=33, 60%), followed by mixed infections with assemblages A&C (overall, n=15, 27.3%), while assemblages C&D alone were less often detected (overall, n=5, 9.1% and n=2, 3.6%). The occurrence of zoonotic genotypes was more prevalent in individual than in group dogs. Genotyping at the gdh locus confirmed the 18S results and clustered the assemblage A isolates into A-I. This is the first large-scale urban survey in Central Europe, which has not only confirmed the high prevalence of Giardia in asymptomatic domestic dogs, but has also shown that the zoonotic assemblage A occurs commonly in domestic dogs living in urban environments, and more frequently than the dog-specific assemblage. Although it was not possible to obtain samples from pet owners in this study, the results demonstrate that a significant proportion of dogs in urban areas of southern Germany harbour zoonotic Giardia, and should thus be considered a potential reservoir for infection in humans.

Genetic variability of Trypanosoma evansi isolates detected by inter-simple sequence repeat anchored-PCR and microsatellite.

Studies on genetic variability in Trypanosoma evansi have been limited by a lack of high-resolution techniques. In this study, we have investigated the use of inter-simple sequence repeats (ISSR) and microsatellites in revealing polymorphism among T. evansi isolates. Twelve ISSR primers and five microsatellite loci were used to generate polymorphic bands and alleles, respectively, to investigate the genetic variability among T. evansi isolates from Africa and Asia. Seven of the twelve ISSR primers showed variability between isolates with a total of 71 fragments of which 49(69%) were polymorphic. Microsatellite analysis revealed a total of 60 alleles. On average the ISSR markers revealed a higher genetic diversity (23%) than microsatellites (21.1%). The two techniques showed a strong agreement of r=0.95 for Dice and r=0.91 for Jaccard indices in estimating the genetic distances between isolates. The distance UPGMA tree revealed two major clusters of T. evansi which correlate with the minicircle classification of subtype A and B. The cophenetic correlation coefficient between Dice and Jaccard based matrices were r=0.79 for microsatellites and r=0.73 for ISSR indicating a strong agreement between dendrograms. The results suggest that both ISSR and microsatellites markers are useful in detecting genetic variability within T. evansi.

Biology and Systematics of Echinococcus.

The biology of Echinococcus, the causative agent of echinococcosis (hydatid disease) is reviewed with emphasis on the developmental biology of the adult and metacestode stages of the parasite. Major advances include determining the origin, structure and functional activities of the laminated layer and its relationship with the germinal layer; and the isolation, in vitro establishment and characterization of the multipotential germinal cells. Future challenges are to identify the mechanisms that provide Echinococcus with its unique developmental plasticity and the nature of activities at the parasite-host interface, particularly in the definitive host. The revised taxonomy of Echinococcus is presented and the solid nomenclature it provides will be essential in understanding the epidemiology of echinococcosis.

Historical Aspects of Echinococcosis.

Echinococcosis is a zoonosis whose history dates back to antiquity. This article provides an overview on the general history of echinococcosis, including the elucidation of Echinococcus life cycles and the long controversy on the aetiology of the cystic and alveolar forms of echinococcosis (CE and AE), lasting about 100years since the middle of the 19th century. Furthermore, selected historical aspects of some fields of echinococcosis research are discussed and compared with our current knowledge, such as geographic distribution and epidemiology of CE (Echinococcus granulosus) and AE (Echinococcus multilocularis), clinical aspects and pathology, diagnosis in humans and animals, treatment (with focus on chemotherapy), control and basic research. A short paragraph is devoted to the neotropical forms of echinococcosis, caused by Echinococcus vogeli and Echinococcus oligarthrus. In this context the achievements of some ancestral pioneers of echinococcosis research are particularly highlighted and appreciated. Finally, the role of associations, international organizations (World Health Organization and others) and international working groups in echinococcosis research and control is briefly outlined. The retrospective reveals both the admirable achievements of our ancestors and the scientific progress of more recent times. But, it also shows the gaps in our knowledge, skills and resources that we need to control or even eradicate echinococcosis.

Host-Parasite Relationships and Life Histories of Trypanosomes in Australia.

Trypanosomes constitute a group of flagellate protozoan parasites responsible for a number of important, yet neglected, diseases in both humans and livestock. The most significantly studied include the causative agents of African sleeping sickness (Trypanosoma brucei) and Chagas disease (Trypanosoma cruzi) in humans. Much of our knowledge about trypanosome host-parasite relationships and life histories has come from these two human pathogens. Recent investigations into the diversity and life histories of wildlife trypanosomes in Australia highlight that there exists a great degree of biological and behavioural variation within and between trypanosomes. In addition, the genetic relationships between some Australian trypanosomes show that they are unexpectedly more closely related to species outside Australia than within it. These findings have led to a growing focus on the importance of understanding parasites occurring naturally in wildlife to (1) better document parasite biodiversity, (2) determine evolutionary relationships and degree of host specificity, (3) understand host-parasite interactions and the role of parasites in the natural ecosystem and (4) identify biosecurity issues of emerging disease in both wildlife and human populations. Here we review what is known about the diversity, life histories, host-parasite interactions and evolutionary relationships of trypanosomes in Australian wildlife. In this context, we focus upon the genetic proximity of key Australian species to the pathogenic T. cruzi and discuss similarities in their biology and behaviour that present a potential risk of human disease transmission by Australian vectors and wildlife.

Global Distribution of Alveolar and Cystic Echinococcosis.

Alveolar echinococcosis (AE) and cystic echinococcosis (CE) are severe helminthic zoonoses. Echinococcus multilocularis (causative agent of AE) is widely distributed in the northern hemisphere where it is typically maintained in a wild animal cycle including canids as definitive hosts and rodents as intermediate hosts. The species Echinococcus granulosus, Echinococcus ortleppi, Echinococcus canadensis and Echinococcus intermedius are the causative agents of CE with a worldwide distribution and a highly variable human disease burden in the different endemic areas depending upon human behavioural risk factors, the diversity and ecology of animal host assemblages and the genetic diversity within Echinococcus species which differ in their zoonotic potential and pathogenicity. Both AE and CE are regarded as neglected zoonoses, with a higher overall burden of disease for CE due to its global distribution and high regional prevalence, but a higher pathogenicity and case fatality rate for AE, especially in Asia. Over the past two decades, numerous studies have addressed the epidemiology and distribution of these Echinococcus species worldwide, resulting in better-defined boundaries of the endemic areas. This chapter presents the global distribution of Echinococcus species and human AE and CE in maps and summarizes the global data on host assemblages, transmission, prevalence in animal definitive hosts, incidence in people and molecular epidemiology.