Evidence of nuclear transport mechanisms in the protozoan parasite Giardia lamblia

Nuclear-cytoplasmic trafficking of proteins is a highly regulated process that modulates multiple biological processes in eukaryotic cells. In Giardia lamblia, shuttling has been described from the cytoplasm to nuclei of proteins during the biological cell cycle of the parasite. This suggests that a mechanism of nucleocytoplasmic transport is present and functional in G. lamblia. By means of computational biology analyses, we found that there are only two genes for nuclear transport in this parasite, named Importin α and Importin β. When these transporters were overexpressed, both localized close to the nuclear envelope, and no change was observed in trophozoite growth rate. However, during the encystation process, both transporters induced an increase in the number of cysts produced. Importazole and Ivermectin, two known specific inhibitors of importins, separately influenced the encysting process by inducing an arrest in the trophozoite stage that prevents the production of cysts. This effect was more noticeable when Ivermectin, an anti-parasitic drug, was used. Finally, we tested whether the enzyme arginine deiminase, which shuttles from the cytoplasm to the nuclei during encystation, was influenced by these transporters. We found that treatment with each of the inhibitors abrogates arginine deiminase nuclear translocation and favors perinuclear localization. This suggests that Importin α and Importin β are key transporters during the encystation process and are involved, at least, in the transport of arginine deiminase into the nuclei. Considering the effect produced by Ivermectin during growth and encystation, we postulate that this drug could be used to treat giardiasis.     

Giardia lamblia aurora kinase: a regulator of mitosis in a binucleate parasite

Giardia lamblia is a major cause of diarrhoeal disease worldwide. Since it has no known toxin, the ability of trophozoites to colonise the human small intestine is required for its pathogenesis. Mitosis in this protozoan parasite is a unique challenge because its two equivalent nuclei and complex cytoskeleton must be duplicated and segregated accurately. Giardial mitosis is a complex and rapid event that is poorly understood at the cellular and molecular levels. Higher eukaryotes have one to three members of the highly conserved Ser/Thr aurora kinase (AK) family that regulate key aspects of mitosis and cytokinesis. Giardia has a single AK orthologue (gAK) with 61% similarity to human AK A. In addition to the conserved active site residues, activation loop and destruction-box motifs characteristic of AKs, gAK contains a unique insert near the active site region. We epitope-tagged gAK at its C-terminus and expressed it under its own promoter. During interphase, gAK localises exclusively to the nuclei, but is not phosphorylated as shown by lack of staining with an antibody specific to phosphorylated AK A (pAK). In contrast, during mitosis pAK localises to the basal bodies/centrosomes and co-localises with tubulin to the spindle. During specific stages of mitosis, giardial pAK also localised dynamically to cytoskeletal structures unique to Giardia: the paraflagellar dense rods of the anterior flagella and the median body, whose functions are unknown, as well as to the parent attachment disc. Two AK inhibitors significantly decreased giardial growth and increased the numbers of cells arrested in cytokinesis. These inhibitors appeared to increase microtubule nucleation and cell-ploidy. Our data show that gAK is phosphorylated in mitosis and suggest that it plays an important role in the Giardia cell cycle. The pleiotropic localisation of AK suggests that it may co-ordinate the reorganisation and segregation of tubulin-containing structures in mitosis. We believe this is the first report of a signalling protein regulating cell division in Giardia.     

Babesia microti: Pathogenesis of parasite of human origin in the hamster

The pathogenesis of the disease in hamsters caused by the first human Babesia isolant, tentatively named Babesia microti, and the immunologic relationship of the organism to Babesia canis were studied. The patent phase of the disease was characterized by severe anemia and marked parasitemia which occurred between the 6th and 41st day following infection. An increase in total white cell count with a neutrophilia, eosinophilia, monocytosis, and lymphocytosis was observed during the patent phase. The patent phase was followed by development of a carrier state. This was demonstrated by relapse following splenectomy 113 days after infection. No statistically significant differences were observed between the serum profiles of infected and noninfected animals during the period monitored. A serologic relationship between B. microti and B. canis was revealed by the use of gel diffusion and indirect fluorescent antibody (IFA) tests. The IFA test was used to monitor serum antibody responses during the patent and carrier phases of the disease. Crossabsorption studies between B. canis and B. microti revealed that the two organisms possess common and specific antigens.     

Zoonotic genotype of Giardia intestinalis detected in a ferret

Giardia intestinalis has been found in a variety of mammals, including humans, and consists of host-specific and zoonotic genotypes. There has been only 1 study of G. intestinalis infection in weasels, but the genotype of its isolate remains unclear. In this study, we report the isolation of Giardia in a ferret exhibited at a pet shop. The isolate was analyzed genetically to validate the possibility of zoonotic transmission. Giardia diagnostic fragments of the small subunit ribosomal RNA, beta-giardin, and glutamate dehydrogenase genes were amplified from the ferret isolate and sequenced to reveal the phylogenetic relationships between it and other Giardia species or genotypes of G. intestinalis reported previously. The results showed that the ferret isolate represented the genetic group A-I in assemblage A, which could be a causative agent of human giardiasis.     

Electrophoretic variation of aconitase and phosphoglucomutase detected in Microtus californicus.

Four electrophoretic variants of cytoplasmic aconitase (citrate (isocitrate) hydro-lyase, EC 4.2.1.3) were detected in a population of Microtus californicus when samples were screened by starch gel electrophoresis using Tris/citrate buffers at pH 7.0 and pH 8.7. Variation at what is presumed to be the phosphoglucomutase-3 locus (alpha-D-glucose-1,6-diphosphate:alpha-D-glucose-1-phosphate phosphotransferase, EC 2.7.5.1) was also detected with liver samples but was not detected in kidney samples or red blood cells lysates. This nongenetic variation is due to oxidation of free sulfhydryl groups.     

Changes in allozyme pattern of the protozoan parasite Giardia intestinalis.

The present study compares the allelic profiles of Giardia intestinalis grown in vivo and in vitro. Three clinical isolates of G. intestinalis were established in suckling mice and subsequently adapted to in vitro culture to test the null hypothesis that samples of the same clinical isolate grown in different culture conditions have identical allelic profiles. For each isolate, a mouse-derived and an axenically cultured sample were analysed electrophoretically at 11 enzyme loci. In each case, the axenically cultured sample of each isolate showed marked allelic differences from its corresponding in vivo sample. These data suggest that there may be either regulated expression of alternative genes encoding distinct isozymes (i.e. gene switching) or selection by different growth conditions of specific genotypes from a mixture present within the original clinical isolate. Although these hypotheses are not tested in this study, the data highlight the importance of confirming that allozymes (or isozymes) are stable genetic characters for the identification and characterization of protozoan taxa.     

Japanese Babesia microti cytologically detected in salivary glands of naturally infected tick Ixodes ovatus

Babesia microti protozoa were detected by light and electron microscopy in the salivary glands of field-collected Ixodes ovatus ticks; 6 of 85 adult ticks were demonstrated to be positive for B. microti DNA by polymerase chain reaction assays. In the salivary glands of unfed ticks, B. microti existed in the sporoblast stage in the granular acinus cells, and developed into the sporozoite stage during feeding on the host for 2 days. The present results indicated for the first time that I. ovatus can indeed carry B. microti and is not infected mechanically with the parasites by blood-sucking. This frequent infection of I. ovatus with B. microti demonstrates the significance of such a vector-pathogen relationship in Japan, and strongly suggests that I. ovatus is involved in the maintenance of B. microti in the fauna of Japanese rodents.     

Splenomegaly and Reticulocytosis Caused by Babesia microti Infections in Natural Populations of the Montane Vole, Microtus montanus

ABSTRACT. A survey for Babesia microti in rodents was conducted at six sites within Grand Teton National Park, Wyoming. Blood and spleen smears, hematocrits, and reticulocyte counts were made on all of the animals to evaluate parameters for the diagnosis of babesiosis. Ticks were removed for identification. Of 257 Microtus montanus , 103 were infected with B. microti. In addition, five of 12 Microtus pennsylvanicus and one of three Arvicola richardsoni were parasitized by B. microti. Peromyscus maniculatus (n = 40) were not infected. Concurrent infections by Hepatozoon sp., Trypanosoma sp., and the bacterium, Grahamella sp., were noted in blood smears from a number of M. montanus. Splenomegaly and reticulocytosis were significant parameters associated with babesiosis while decreased hematocrit was not. Ticks removed from the voles were identified as Ixodes eastoni and were the probable vectors of the B. microti.     

Giardia and Cryptosporidium in inflowing water and harvested shellfish in a lagoon in Southern Italy

Giardia and Cryptosporidium spp. are important enteric protozoan pathogens for humans and animals, and have been found to contaminate water as well as edible shellfish all over the world. This is the first study to simultaneously investigate the presence of Giardia and Cryptosporidium in inflowing water and harvested shellfish in a geographically closed environment (Varano Lagoon, Southern Italy). Samples of treated wastewater were collected each month - at the outlet from the treatment plant, and downstream at the inlet into the lagoon - from the channels flowing into the Lagoon, together with specimens of Ruditapes decussatus and Mytilus galloprovincialis from shellfish-farms on the same lagoon. Giardia cysts were found by immunofluorescence (IF) microscopy in 16 out of 21 samples of treated wastewater and in 7 out of 21 samples from downstream water channels, and viable cysts were also detected by a beta-giardin RT-PCR. G. duodenalis Assemblages A and B were identified by small ribosomal subunit (18S-rDNA) and triosephosphate isomerase (tpi)-PCR, followed by sequencing. Cryptosporidium oocysts were found by IF in 5 out of 21 wastewater samples, and in 8 out of 21 samples from water channels. Molecular analysis identified the zoonotic species Cryptosporidium parvum by oocyst wall protein (COWP)-PCR and sequencing. Higher concentrations of Giardia cysts than Cryptosporidium oocysts were registered in almost all wastewater and water samples. IF and molecular testing of shellfish gave negative results for both protozoa. Wastewaters carrying Giardia and Cryptosporidium (oo)cysts are discharged into the Lagoon; however, the shellfish harvested in the same environment were found to be unaffected, thus suggesting that physical, ecological and climatic conditions may prevent contamination of harvested shellfish.     

Disease dynamics in cyclic populations of field voles (Microtus agrestis): cowpox virus and vole tuberculosis (Mycobacterium microti)

The possible role of pathogens in rodent population cycles has been largely neglected since Elton's 'epidemic hypothesis' of 1931. To revisit this question, 12 adjacent, cyclic but out-of-phase populations of field voles (Microtus agrestis) in North East England were studied and the initial results are presented here. The prevalences of antibodies to cowpox virus and of clinical signs of Mycobacterium microti infection (vole tuberculosis) showed delayed (not direct) density dependence (with a lag of three to six months). This did not result from changes in population structure, even though there were such changes associated with the different phases of the cycle. The prevalences rose as vole numbers rose, and peaked as numbers declined. The apparent lag in the numerical response of infection prevalence to changes in host abundance is consistent with the hypothesis that diseases, singly or in combination, play a hitherto underestimated role in the dynamics of cyclic populations.     

Prevalence of Chlamydophila felis by PCR among healthy pet cats in Italy

Conjunctival swabs were taken from 60 healthy pet cats and tested for Chlamydophila felis by PCR assays to amplify the ompA, omp2 and groEL genes. Chlamydial DNA was detected in 2 (3.3%) cats, one of which had been vaccinated against C. felis eight months before sample collection. The nucleotide and predicted amino acid sequences of three genes from two cats showed 100% identity with the same regions amplified from conjunctival swabs of cats in the same geographic area.     

Splenomegaly and reticulocytosis caused by Babesia microti infections in natural populations of the montane vole, Microtus montanus.

A survey for Babesia microti in rodents was conducted at six sites within Grand Teton National Park, Wyoming. Blood and spleen smears, hematocrits, and reticulocyte counts were made on all of the animals to evaluate parameters for the diagnosis of babesiosis. Ticks were removed for identification. Of 257 Microtus montanus, 103 were infected with B. microti. In addition, five of 12 Microtus pennsylvanicus and one of three Arvicola richardsoni were parasitized by B. microti. Peromyscus maniculatus (n = 40) were not infected. Concurrent infections by Hepatozoon sp., Trypanosoma sp., and the bacterium, Grahamella sp., were noted in blood smears from a number of M. montanus. Splenomegaly and reticulocytosis were significant parameters associated with babesiosis while decreased hematocrit was not. Ticks removed from the voles were identified as Ixodes eastoni and were the probable vectors of the B. microti.     

Calcium transport and catabolism of adenosine triphosphate in the protozoan parasite Giardia lamblia

1. Calcium uptake by washed trophozoites of Giardia lamblia was dependent on inorganic orthophosphate and stimulated by glucose. Uptake was both rapid and substantial: 224 +/- 73 nmoles Ca2+/mg protein/min. 2. Known inhibitors of Ca2+ uptake in mammalian cells also impeded Ca2+ influx into G. lamblia. 3. The inhibitor studies indicated that Ca2+ transport in G. lamblia was an active process. Energy for such a process could be provided by the action of ATPases. 4. Two types of ATPases were found in the parasite; one, a membrane-associated enzyme activated by Ca2+; the other, a soluble, cytosolic enzyme activated by Mg2+. 5. These enzymes differed not only in their intracellular distribution and divalent cation requirements, but also in their sensitivity to calmodulin antagonists. The particulate enzyme was sensitive to these inhibitors whereas the soluble ATPase was not. 6. Our data indicate that Ca2+ transport in G. lamblia is mediated by a membrane-bound, calmodulin-regulated, Ca2+-ATPase.     

A comparison of Giardia microti and Spironucleus muris cysts in the vole: an immunocytochemical, light, and electron microscopic study

We have shown that cysts of the genus Spironucleus share many common morphological features with Giardia cysts including: 2-4 nuclei, flagellar axonemes, a distinct cyst wall, and they even display the same immunostaining as Giardia cysts when labeled with antibodies specific for Giardia cyst wall. A direct comparison of Spironucleus muris and Giardia microti cysts have revealed that cysts of S. muris are significantly smaller than cysts of G. miroti. At the ultrastructural level, the cyst walls are similar in fibrillar appearance, but the width of the S. muris cyst wall is significantly less than that of G. microti. The cysts of S. muris also differ from G. microti in that they contain a striated rootlet fiber, flagellar sheath, and numerous glycogen rosettes. Characteristic features of Giardia include the adhesive disc and median body. Although the cysts of Spironucleus and Giardia are similar in appearance, these unique morphological features can be used to distinguish between the 2 protozoa and should be employed in the detection of Giardia cysts in water samples.     

Sarcocystis cernae: A parasite increasing the risk of predation of its intermediate host,Microtus arvalis

Summary 1)The transmission dynamics of the protozoan parasite Sarcocystis carnae (erná and Louková 1976) (Apicomplexa, Eimeroidea, Sarcocystidae) in natural populations were studied in the Lauwersmeerpolder in the northern Netherlands. This parasite needs two hosts to complete its life cycle; the common vole (Microtus arvalis) as its intermediate host and the kestrel (Falco tinnunculus) which preys on the vole, as its final host. 2) Seasonal variation in prevalence of infection in snap-trapped common voles was determined in two years, 1984 and 1985. It was found to be lowest in November (6% of the voles infected) and it increased gradually to a peak in May (33%). 3) Data collected in three successive kestrel breeding seasons (1983–85) revealed that voles in the kestrel summer diet are infected twice as frequently as those in snap-trap samples, 21% and 9% respectively. This difference (P<0.05, X²-test) suggests that the parasite influences its intermediate host behaviour in such a way that it enhances the probability of parasite transmission to the final host.     

Evidence of a continuous endoplasmic reticulum in the protozoan parasite Entamoeba histolytica

Entamoeba histolytica, the cause of amebiasis, is believed to have no continuous endoplasmic reticulum (ER), with ER functions occurring in vesicles. Here, using an ER-targeted green fluorescent protein fusion protein and fluorescence loss in photobleaching, we have unambiguously demonstrated the presence of a continuous ER compartment in living E. histolytica trophozoites.     

Evidence of plasticity in the reproduction of a trematode parasite: the effect of host removal

The parasitic trematode Proctoeces lintoni requires 3 hosts (intertidal mussels, keyhole limpets, and clingfish) to complete its life cycle. The densities and size structure of host communities are modified by selective human harvesting. This study examined clutch and egg size of P. lintoni in 3 adjacent sites in rocky intertidal areas of central Chile presenting differences in the levels of human disturbance (i.e., from a fully protected marine reserve to free open-access areas). We found significant differences in parasite fecundity among sites. An increase in number of eggs was observed inside protected marine areas compared with open-access areas, suggesting a plastic response of the parasite reproductive strategies to the host community modification. These results show that host removal by humans in coastal ecosystems can strongly influence parasite life history traits.