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

Isoenzyme electrophoresis of 30 isolates of Giardia from humans and felines

Thirty isolates of Giardia duodenalis from humans and felines were compared by isoenzyme electrophoresis. Using 10 enzyme systems, 13 different zymodemes were distinguished. The majority of zymodemes could be divided into two groups: one group comprising human and feline isolates with worldwide geographic distribution; the other group containing human isolates restricted to Western Australia. A number of isolates showed multiple-banded patterns and the genetic significance of these findings is discussed. The marked heterogeneity of G. duodenalis demonstrated in this study is considered in relation to the epidemiology of giardiasis. The findings are consistent with felines serving as a reservoir of infection to humans

Genotypic characterisation of Giardia from domestic dogs in the USA

The first large-scale urban survey of Giardia infections in dogs was undertaken in the USA. It involved several locations in the Western United States with Giardia isolates from microscopy-positive samples characterised by multi-locus PCR and sequencing. A high prevalence of Giardia was confirmed in asymptomatic domestic dogs, and for the first time, provides evidence that zoonotic assemblages/subgroups of Giardia occur frequently in domestic dogs living in urban environments, and more frequently than the dog specific assemblages

Echinococcus multilocularis coproantigen detection by enzyme-linked immunosorbent assay in fox, dog, and cat populations

A sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of Echinococcus multilocularis coproantigens (EM-ELISA) was developed with polyclonal rabbit (solid phase) and chicken egg (catching) antibodies that were directed against E. multilocularis coproantigens and somatic worm antigens, respectively. In experimentally infected dogs and cats, coproantigens were first detectable 6-17 days postinfection (PI) in samples of 8 dogs (worm burdens at necropsy: 6,330-43,200) and from 11 days PI onward in samples of 5 cats infected with 20-6,833 worms. After anthelmintic treatment of 4 dogs and 5 cats at day 20 PI, coproantigen excretion disappeared within 3-5 days. The sensitivity of the ELISA was 83.6% in 55 foxes infected with 4-60,000 E. multilocularis, but reached 93.3% in the 45 foxes harboring more than 20 worms. The EM-ELISA was used in surveys of 'normal' dog and cat populations in Switzerland. Among 660 dogs and 263 cats, 5 dogs and 2 cats exhibited a positive reaction. In 2 of these dogs (0.30%) and 1 cat (0.38%), intestinal E. multilocularis infections were confirmed by necropsy, polymerase chain reaction PCR, or both. The specificities of the ELISA in these groups were found to be 99.5% and 99.6%, respectively, if positive ELISA results that could not be confirmed by other methods were classified as 'false positive' reactions

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

Cryptosporidium — What is it?

Cryptosporidium is a ubiquitous enteric protozoan pathogen of vertebrates, and although recognised as a cause of disease in humans and domestic animals for over 50 years, fundamental questions concerning its biology and ecology have only recently been resolved. Overwhelming data now confirm that, like its close relatives, Cryptosporidium is a facultatively epicellular apicomplexan that is able to multiply in a host cell-free environment. These data must be considered in the context of the phylogenetic reclassification of Cryptosporidium from a coccidian to a gregarine. Together, they dictate an urgent need to reconsider the biology and behaviour of Cryptosporidium, and perhaps help to explain the parasite's incredible genetic diversity, distribution and host range. Improved imaging technologies have complemented phylogenetic studies in demonstrating the parasite's affinities with gregarine protozoa and have further supported its extracellular developmental capability and potential role as an environmental pathogen. These advances in our understanding of Cryptosporidium as a protozoan pathogen are examined with emphasis on how they may influence control strategies in the future

Is Toxoplasma gondii a threat to the conservation of free-ranging Australian marsupial populations?

It has often been asserted that Australian marsupial species are particularly susceptible to Toxoplasma gondii infection and to clinical toxoplasmosis following infection. This implicates T. gondii as a potential threat to marsupial population viability, and contrasts to what is known of T. gondii in populations of several other host species. We reviewed the literature, and found a lack of scientifically robust evidence addressing the occurrence of T. gondii infection in free-ranging populations of Australian marsupial species, and the impacts of the infection on population health. Key limitations included a lack of studies in free-ranging marsupial populations, study findings susceptible to substantial chance influences, and selection, misclassification and confounding biases. The lack of scientifically robust data available on this topic indicates that assertions that free-ranging populations of Australian marsupials are particularly susceptible to T. gondii infection and to toxoplasmosis are premature. The threat of T. gondii to the viability of free-ranging marsupial populations should therefore be regarded, at this stage, as a hypothesis

Loop-mediated isothermal amplification (LAMP) method for rapid detection of Trypanosoma brucei rhodesiense

Loop-mediated isothermal amplification (LAMP) of DNA is a novel technique that rapidly amplifies target DNA under isothermal conditions. In the present study, a LAMP test was designed from the serum resistance-associated (SRA) gene of Trypanosoma brucei rhodesiense, the cause of the acute form of African sleeping sickness, and used to detect parasite DNA from processed and heat-treated infected blood samples. The SRA gene is specific to T. b. rhodesiense and has been shown to confer resistance to lysis by normal human serum. The assay was performed at 62°C for 1 h, using six primers that recognised eight targets. The template was varying concentrations of trypanosome DNA and supernatant from heat-treated infected blood samples. The resulting amplicons were detected using SYTO-9 fluorescence dye in a real-time thermocycler, visual observation after the addition of SYBR Green I, and gel electrophoresis. DNA amplification was detected within 35 min. The SRA LAMP test had an unequivocal detection limit of one pg of purified DNA (equivalent to 10 trypanosomes/ml) and 0.1 pg (1 trypanosome/ml) using heat-treated buffy coat, while the detection limit for conventional SRA PCR was ∼1,000 trypanosomes/ml. The expected LAMP amplicon was confirmed through restriction enzyme RsaI digestion, identical melt curves, and sequence analysis. The reproducibility of the SRA LAMP assay using water bath and heat-processed template, and the ease in results readout show great potential for the diagnosis of T. b. rhodesiense in endemic regions

Factors influencing the development and carbohydrate metabolism of Echinococcus granulosus in dogs

Echinococcus granulosus adult worms, 35 days postinfection, were measured for dispersion in the intestines of 10 dogs, a range of morphological characters, and the excreted end products of carbohydrate catabolism following 4 hr incubation in vitro. Most worms were found in the proximal sections of the small intestine, but the pattern of dispersion differed between dogs. Worm development varied both between dogs and between different regions of the small intestine of individual dogs. Overall there was a high level of variability with no simple patterns. Worm metabolism was related to worm development and, also independently, to local population density within the intestine. Larger, more mature worms produced less lactate and, at higher densities. worms tended to produce more acetate and succinate (pathways with a higher energy yield than lactate) and less ethanol. Thus, both more developed worms and high population density are associated with a shift from cytosolic to mitochondrial metabolism. The variation between worm populations along the small intestine along with the observed variation between worm populations from sibling dogs infected with genetically identical parasites suggests that the local host environment has a significant effect on parasite development

Phylogenetic analysis of Cryptosporidium isolates from captive reptiles using 18S rDNA sequence data and random amplified polymorphic DNA analys

Sequence alignment of a polymerase chain reaction-amplified 713-base pair region of the Cryptosporidium 18S rDNA gene was carried out on 15 captive reptile isolates from different geographic locations and compared to both Cryptosporidium parvum and Cryptosporidium muris isolates. Random amplified polymorphic DNA (RAPD) analysis was also performed on a smaller number of these samples. The data generated by both techniques were significantly correlated (P < 0.002), providing additional evidence to support the clonal population structure hypothesis for Cryptosporidium. Phylogenetic analysis of both 18S sequence information and RAPD analysis grouped the majority of reptile isolates together into 1 main group attributed to Cryptosporidium serpentis, which was genetically distinct but closely related to C. muris. A second genotype exhibited by 1 reptile isolate (S6) appeared to be intermediate between C. serpentis and C. muris but grouped most closely with C. muris, as it exhibited 99.15% similarity with C. muris and only 97.13% similarity with C. serpentis. The third genotype identified in 2 reptile isolates was a previously characterized 'mouse' genotype that grouped closely with bovine and human C. parvum isolates