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PubMed2004

Occurrence of Cystosporogenes sp. (Protozoa, Microsporidia) in a multi-species insect production facility and its elimination from a colony of the eastern spruce budworm, Choristoneura fumiferana (Clem.) (Lepidoptera: Tortricidae).

We have isolated a microsporidium from a laboratory colony of the eastern spruce budworm, Choristoneura fumiferana (Clem.) (Lepidoptera: Tortricidae). Light and electron microscopic investigations showed that gross pathology and ultrastructure of our isolate are similar to those described for Cystosporogenes legeri from the European grape vine moth, Lobesia botrana. Comparative phylogenetic analysis of the small subunit rDNA using maximum likelihood, maximum parsimony, and neighbour joining distance methods revealed perfect homology with the C. legeri sequence. The microsporidian was infectious to other Choristoneura species, as well as Malacosoma disstria, Lymantria dispar, and Lambdina fiscellaria. Incubation of infected egg masses at 41 degrees C for 20 min followed by 30 min in 33% formaldehyde did not reduce disease incidence in larval offspring. Exposure of one or two generations to fumagillin at 6000 ppm or higher eliminated infection in adult moths, but also reduced colony fitness. A clean colony was established by conducting individual matings and selecting disease-free offspring.

Animals↗
PubMed2005

Application of multiple DNA fingerprinting techniques to study the genetic relationships among three members of the subgenus Trypanozoon (Protozoa: Trypanosomatidae).

Three different DNA fingerprinting techniques, the mobile genetic element (MGE)-PCR, simple sequence repeat (SSR)-PCR and random amplified polymorphic DNA (RAPD)-PCR, were used to define a large set of genetic markers to study genetic similarity within and among Trypanosoma brucei, Trypanosoma equiperdum and Trypanosoma evansi strains (n=18) from China, Africa and South America and to investigate their genetic relationships. Using the three fingerprinting techniques, >890 bands (ranging in size from 0.2 to 2kb) were defined for all 18 strains of Trypanosoma. Within each of the strains, 39-59 bands were defined. The similarity coefficients between strains ranged from approximately 41 to 94%, with a mean of 65%. There was more genetic similarity among strains within T. evansi (mean of approximately 79%) compared with T. equiperdum ( approximately 65%) and T. brucei ( approximately 59%). The similarity coefficient data were used to construct the dendrogram, which revealed that (irrespective of species) the majority of strains from China and South America grouped together to the exclusion of those from Africa. The exceptions were a T. brucei strain from Africa and a T. equiperdum strain of unknown origin. Hence, employing data sets generated using the three different fingerprinting methods, it was not possible to unequivocally distinguish among T. brucei, T. evansi and T. equiperdum, although there was a tendency for T. evansi strains to group together to the exclusion of T. brucei. The findings provide support for the hypothesis that T. evansi originated from a mutated form of T. equiperdum and stimulate further investigations of the genetic make-up and evolution of members of the subgenus Trypanozoon.

Africa↗
PubMed2006

An opposite role is exerted by the acarian Myocoptes musculinus in the outcome of Toxoplasma gondii infection according to the route of the protozoa inoculation.

Infection with Toxoplasma gondii leads to a Th1 immune response. Alternatively, the acarian Myocoptes musculinus induces a disease in BALB/c mice that involves Th2 immune mechanisms. In this study, we investigated whether infestation by M. musculinus induces Th2 immune response in C57BL/6 mice and if this response influences the T. gondii-induced Th1 response when mice are inoculated by intraperitoneal or oral route. The animals were infected with M. musculinus and one month later with T. gondii ME-49 strain and the survival and immune response were monitored. The co-infected animals displayed higher mortality rate and the spleen cells showed a decreased IFN-gamma and elevated IL-4 and IL-5 production. These changes were associated with severe pneumonia and wasting condition. On the other hand, when mice were orally infected with 100 T. gondii cysts, co-infection prolonged the survival rates and ameliorated intestinal lesions in association with a significant drop in IFN-gamma levels in sera. These results indicate the interference of Th2 response induced by M. musculinus in a T. gondii-induced Th1 response. Altogether, these data demonstrate the profound interactions between the immune response induced against unrelated organisms T. gondii and M. musculinus, and suggest that this type of interactions may impact clinical disease.

Animals↗
PubMed2004

Improvement of recoveries for the determination of protozoa Cryptosporidium and Giardia in water using method 1623.

The U.S. Environmental Protection Agency has developed method 1623 for simultaneous detection of Cryptosporidium oocysts and Giardia cysts in water. Method 1623 includes four major steps: filtration, immunomagnetic separation (IMS), fluorescent antibody (FA) staining and microscopic examination. It was noted that the recovery levels following IMS-FA and FA staining were high, averaging more than 92.0% and 89.0% for C. parvum oocysts and G. lamblia cysts, respectively. In contrast, when the filtration step was incorporated, the recovery level of C. parvum oocysts declined significantly to 18.1% in seeded tap water, while a relatively high recovery level of 77.2% for G. lamblia cysts could still be achieved. Further study indicated that the recovery level of C. parvum oocysts could be enhanced significantly when an appropriate amount of silica particles was added to a water sample. The recovery level of C. parvum oocysts was affected by particle size and concentration. The optimal silica particle size was determined to be within the range of 5-40 microm, and the corresponding optimal silica concentration was 1.42 g for 10-l tap water. When both G. lamblia cysts and C. parvum oocysts were spiked into the tap water sample containing the optimum amount of silica particles, the average recovery levels of oocysts and cysts were 82.7% and 75.4%, respectively. The results obtained clearly suggested that addition of an appropriate amount of silica particles could improve the recovery level of C. parvum oocysts significantly and yet there was no noticeable deleterious effect on the recovery level of G. lamblia cysts. Further study indicated that the rotation time in the IMS procedure using the Dynal GC-Combo IMS kit (which was recommended in method 1623) was important for G. lamblia cyst detection. In contrast, the recovery level of C. parvum oocysts was not affected by the rotation time. Furthermore, it was found that the recovery levels of C. parvum oocysts using methods 1622 and 1623 were quite close although different IMS kits were used in the two methods.

Animals↗
PubMed2004

Molecular and phylogenetic characterization of syntaxin genes from parasitic protozoa.

Vesicular transport is an integral process in eukaryotic cells and the syntaxins, a member of the SNARE protein superfamily, are a critical piece of the vesicular transport machinery. We have obtained syntaxin homologues from diverse protozoan parasites (including Entamoeba, Giardia, Trichomonas and Trypanosoma), determined the paralogue affinity of the homologues by molecular phylogenetics and compared functionally critical amino acid sites identified in other syntaxins. Surprisingly, three sequences deviate at the signature glutamine residue position, conserved in all previously identified syntaxin homologues. It is known that, despite conserved structure and function of both the syntaxins and the proteins of the regulatory SM superfamily, the various syntaxin paralogues bind their respective SM partners at different regions of the syntaxin molecule. These sites of interactions have been identified down to the individual residues. The pattern of conservation at these residues, in our evolutionarily diverse sampling of syntaxin paralogues, is therefore used to gain further insight into the interaction of these proteins. Phylogenetic analysis confirms and extends previous conclusions that the syntaxin families are present in diverse eukaryotes and that the syntaxin sub-families diverged early in eukaryotic evolution. This result is expanded with the inclusion of new homologues for previously sampled taxa, newly sampled taxa, and newly sampled syntaxin sub-families. Because of their integral role in membrane trafficking, the syntaxin genes represent a valuable potential molecular marker for the experimental study of the endomembrane system of disease-causing protists.

Amino Acid Sequence↗
PubMed2005

A taxonomic re-appraisal of Sarcocystis nesbitti (Protozoa: Sarcocystidae) from the monkey Macaca fascicularis in Yunnan, PR China.

The first detection of Sarcocystis nesbitti Mandour, 1969 in the Chinese mainland is reported and the morphology of the sarcocyst is described in detail. The parasite was detected in the monkey, Macaca fascicularis, maintained on a monkey farm in Yunnan Province; the infection may have occurred via faecal contamination from local rats, mice and/or birds. S. nesbitti was characterized as follows: a macroscopic sarcocyst, length of the cyst up to 2 mm; cyst wall smooth, thin and no perpendicular protrusion is seen under the light microscope; border of cyst wall wavy, primary cyst wall thin (38-65 nm) and invaginated; ground substance about 0.5-0.76 microm thick with electron-dense granules and concentric spherical bodies. The cyst wall is described as type 1 by electron microscopy. It is suspected that S. nesbitti may utilize Macaca mulatta, M. fascicularis, Cercocebus atys, and Papio papionis, as well as human as intermediate hosts. The taxonomy of S. nesbitti is re-appraised in the light of a consideration of possible experimental artefacts and examination of the past literature. Evidence is presented that S. nesbitti may be one of the species infecting humans in South Asia and that the monkey may be a potential reservoir host.

Animals↗
PubMed2004

Parasitic protozoa: thiol-based redox metabolism.

In a thoughtful and stimulating article, Sylke Müller et al. describe the thiol-based redox metabolism of protozoan parasites and believe that the key enzymes involved in this metabolism have potential use as new drug targets. Additional information on this subject is provided here.

Animals↗
PubMed2004

Ultraviolet light inactivation of protozoa in drinking water: a Bayesian meta-analysis.

To assess the dose of UV light needed to achieve specified levels of Giardia spp. cysts and Cryptosporidium spp. oocysts inactivation in drinking water, a Bayesian meta-analysis is used to analyze experimental data from several studies. Of the 20 studies identified by an extensive data collection effort, 14 (five reported experiments on Giardia and nine on Cryptosporidium) were selected for analysis based on a set of criteria. A substantial amount of the log inactivation data are reported as greater than a given inactivation level (i.e., censored data). The Bayesian hierarchical modeling approach used in this study not only properly addresses the common concerns in a meta-analysis but also provides a robust method for incorporating censored data. Different statistical models will result in different estimates of the UV doses needed to achieve a specific inactivation level. The Bayesian approach allows us to present the uncertainty in terms of risk, which is better suited for supporting US EPA in developing regulations.

Animals↗
PubMed2006

Removal of viruses, parasitic protozoa and microbial indicators in conventional and membrane processes in a wastewater pilot plant.

The aim of this study was to investigate variations in the occurrence and removal of enterovirus and norovirus genomes, Giardia cysts, Cryptosporidium oocysts and the most commonly used faecal indicators in a Swedish wastewater pilot plant. Paired samples were taken from the inlet and outlet of each treatment line: tertiary filtration, membrane bioreactor (MBR) and upflow anaerobic sludge blankets (UASB). (Oo)cysts and indicators were enumerated using standard methods and viruses using RT-PCR. Giardia cysts and enteroviruses were constantly detected, mean numbers 10(3.11) cysts and 10(4.0) PCR units L(-1), respectively. Oocysts were found in 5/19 samples, mean number 5 L(-1). Noroviruses were found in 6/7 influent samples, with an average titre of 10(3.28)L(-1), during winter, but only in 2/15 in the rest of the year (mean 200 L(-1)). MBR treatment removed indicators more efficiently than did the other two lines, with 5log removal of E. coli. Human virus genome removal did not differ between the MBR and tertiary treatment line. Microorganism removal in UASB was significantly lower for all the organisms studied. E. coli, enterococci and Cl. perfringens removal was correlated (p<0.05) with enterovirus genome removal, with R-values around 0.4. However, values for removal of indicators were more strongly correlated to each other. Removal of viruses based on enumeration using RT-PCR probably underestimates infectious virion removal.

Animals↗
PubMed2001

Arginine kinase of the flagellate protozoa Trypanosoma cruzi. Regulation of its expression and catalytic activity.

In epimastigotes of Trypanosoma cruzi, the etiological agent of Chagas' disease, arginine kinase activity increased continuously during the exponential phase of growth. A correlation between growth rate, enzyme-specific activity and enzyme protein was observed. Arginine kinase-specific activity, expressed as a function of enzyme protein, remains roughly constant up to 18 days of culture. In the whole range of the culture time mRNA levels showed minor changes indicating that the enzyme activity is post-transcriptionally regulated. Arginine kinase could be proposed as a modulator of energetic reserves under starvation stress condition.

Animals↗
PubMed2001

Spermidine is essential for normal proliferation of trypanosomatid protozoa.

Trypanosomatid parasites containing a metabolically unstable ornithine decarboxylase (ODC) are naturally resistant to high levels of alpha-difluoromethylornithine (DFMO) because this ODC inhibitor, though causing a drastic reduction of intracellular putrescine, elicits only a moderate decrease of the spermidine endogenous pool. In this study we have used a combination of DFMO with cyclohexylamine (CHA; bis-cyclohexylammonium sulfate), an inhibitor of spermidine synthase, to reach a more complete depletion of spermidine. Under these conditions we have observed the arrest of proliferation not only in trypanosomatids with stable ODC but also in parasites with an enzyme of high turnover rate. In all cases the reinitiation of proliferation occurred only after the addition of exogenous spermidine, and neither putrescine nor spermine were able to induce the same effect.

Animals↗
PubMed2002

Magic-angle spinning (31)P NMR spectroscopy of condensed phosphates in parasitic protozoa: visualizing the invisible.

We report the results of a solid-state (31)P nuclear magnetic resonance (NMR) spectroscopic investigation of the acidocalcisome organelles from Trypanosoma brucei (bloodstream form), Trypanosoma cruzi and Leishmania major (insect forms). The spectra are characterized by a broad envelope of spinning sidebands having isotropic chemical shifts at approximately 0, -7 and -21 ppm. These resonances are assigned to orthophosphate, terminal (alpha) phosphates of polyphosphates and bridging (beta) phosphates of polyphosphates, respectively. The average polyphosphate chain length is approximately 3.3 phosphates. Similar results were obtained with whole L. major promastigotes. (31)P NMR spectra of living L. major promastigotes recorded under conventional solution NMR conditions had spectral intensities reduced with respect to solution-state NMR spectra of acid extracts, consistent with the invisibility of the solid-state phosphates. These results show that all three parasites contain large stores of condensed phosphates which can be visualized by using magic-angle spinning NMR techniques.

Animals↗