Molecular study of free-ranging mule deer and white-tailed deer from British Columbia, Canada, for evidence of Anaplasma spp. and Ehrlichia spp.

Twenty-three free-ranging white-tailed deer (WTD; Odocoileus virginianus) and six mule deer (MD; Odocoileus hemionus) from south-central British Columbia, Canada, were tested for Anaplasma marginale by msp5 gene-specific PCR and Ehrlichia spp. by 16S rRNA or citrate synthase (gltA) gene-specific PCR, as well as by PCR with universal 16S rRNA primers detecting a wide range of bacteria. No deer tested positive for A. marginale. Amplification with universal 16S rRNA primers followed by sequencing of cloned fragments detected an Anaplasma sp. in one of 23 (4.3%) WTD and six of six (100%) MD and Bartonella sp. in four of 23 (17.4%) WTD. The Anaplasma sp. was genetically distinct from A. marginale and all other recognized members of the genus. Four of six (66.7%) MD and 0 of 23 (0%) WTD were Ehrlichia positive by PCR with primers for 16S rRNA and gltA genes. The sequences of gltA PCR fragments were identical to each other and to the respective region of the gltA gene of an Ehrlichia sp. which we detected previously in naturally infected cattle from the same area, suggesting the possibility of biological transmission of this rickettsia between cattle and wild cervids. Antibodies reactive with the MSP5 protein of A. marginale were detected using a competitive enzyme-linked immunosorbent assay in two of six (33.3%) MD, but not in WTD. The two seropositive MD were PCR positive for both the Anaplasma sp. and Ehrlichia sp. detected in this study, suggesting a reaction of antibodies against one or both of these rickettsias with the MSP5 antigen.

The schistosome in the mammalian host: understanding the mechanisms of adaptation.

In this review, we envisage the host environment, not as a hostile one, since the schistosome thrives there, but as one in which the relationship between the two organisms consists of constant communication, through signalling mechanisms involving sense organs, surface glycocalyx, surface membrane and internal organs of the parasite, with host fluids and cells. The surface and secretions of the schistosome egg have very different properties from those of other parasite stages, but adapted for the dispersal of the eggs and for the preservation of host liver function. We draw from studies of mammalian cells and other organisms to indicate how further work might be carried out on the signalling function of the surface glycocalyx, the raft structure of the surface and existence of pores in the surface membrane, the repair of the surface membrane, the role of the membrane structure in ion channel function (including recent work on the actin cytoskeleton and calcium channels) and the possible role of P-glycoproteins in the adaptation of the parasite to its environment. We are speculative in some areas, such as the suggestions that variability in surface properties of schistosomes may relate to the existence of membrane rafts and that parasite communities may exhibit quorum sensing. This speculative approach is adopted with the hope that future work on the whole organisms and their interactions will be encouraged.

Trypanosoma (Herpetosoma) kuseli sp. n. (Protozoa: Kinetoplastida) in Siberian flying squirrels (Pteromys volans).

All trypanosome species classified in the subgenus Herpetosoma in sciurid hosts have been recorded from ground and tree squirrels to date, but not from any flying squirrels. We describe in this paper a novel trypanosome species, Trypanosoma (Herpetosoma) kuseli sp. n., from Siberian flying squirrels (Pteromys volans) imported from China, and compare it with T. (H.) otospermophili in Richardson's ground squirrels (Spermophilus richardsonii) and Columbian ground squirrels (Spermophilus columbianus) from the USA. Due to a short free flagellum, the new species appeared stumpy compared with T. otospermophili (length of free flagellum 7.0 +/- 0.8 microm, total length 32.1 +/- 0.8 microm, n = 13 and length of free flagellum 15.5 +/- 1.6 microm, total length 35.9 +/- 1.0 microm, n = 13, respectively). Another conspicuous morphological feature of the new species was an anteriorly positioned kinetoplast, found approximately at the midpoint between the nucleus and the posterior end. These characters have not been recorded from any squirrel Herpetosoma trypanosome species. Comparison of the nucleotide sequences of the small and large subunit rRNA genes indicated that T. kuseli sp. n. was more homologous to T. otospermophili than murid Herpetosoma species, such as T. grosi, T. lewisi, T. musculi, T. microti and T. evotomys.

Detection of UV-induced thymine dimers in individual Cryptosporidium parvum and Cryptosporidium hominis oocysts by immunofluorescence microscopy.

To investigate the effect of UV light on Cryptosporidium parvum and Cryptosporidium hominis oocysts in vitro, we exposed intact oocysts to 4-, 10-, 20-, and 40-mJ x cm-2 doses of UV irradiation. Thymine dimers were detected by immunofluorescence microscopy using a monoclonal antibody against cyclobutyl thymine dimers (anti-TDmAb). Dimer-specific fluorescence within sporozoite nuclei was confirmed by colocalization with the nuclear fluorogen 4',6'-diamidino-2-phenylindole (DAPI). Oocyst walls were visualized using either commercial fluorescein isothiocyanate-labeled anti-Cryptosporidium oocyst antibodies (FITC-CmAb) or Texas Red-labeled anti-Cryptosporidium oocyst antibodies (TR-CmAb). The use of FITC-CmAb interfered with TD detection at doses below 40 mJ x cm-2. With the combination of anti-TDmAb, TR-CmAb, and DAPI, dimer-specific fluorescence was detected in sporozoite nuclei within oocysts exposed to 10 to 40 mJ x cm-2 of UV light. Similar results were obtained with C. hominis. C. parvum oocysts exposed to 10 to 40 mJ x cm-2 of UV light failed to infect neonatal mice, confirming that results of our anti-TD immunofluorescence assay paralleled the outcomes of our neonatal mouse infectivity assay. These results suggest that our immunofluorescence assay is suitable for detecting DNA damage in C. parvum and C. hominis oocysts induced following exposure to UV light.

Cryptosporidium parvum sporozoites contain glutathione.

We used the fluorescent dye monochlorobimane (MCB) which binds glutathione (GSH) to localize between 2 and 6 distinctly labelled nuclear and cytoplasmic GSH foci in recently excreted and aged, intact Cryptosporidium parvum oocysts and sporozoites. Buthionine sulfoximine (BSO), a potent and specific inhibitor of GSH, was used to determine whether GSH is synthesized in BSO-treated C. parvum oocysts, by labelling treated oocysts with MCB. Both visual and electronic quantifications were performed. At 5 mM BSO, a significant inhibition of MCB fluorescence, reflecting reduced MCB uptake, was observed in GSH-depleted oocysts (mean +/- S.D. 35 +/- 3.7) compared with controls (3.3 +/- 1.2, P = 0). This clear reduction occurred only in viable oocysts. 1 mM BSO-treated oocysts exhibited weak or no MCB fluorescence, although they were viable (excluded propidium iodide, PI)), and intact and contained sporozoites by differential interference contrast microscopy (DIC). MCB was used in conjunction with PI to determine C. parvum oocyst viability. Oocysts labelled with MCB/PI or 4'6-diamidino-2-phenyl indole (DAPI)/PI produced comparable labelling patterns. Viable oocysts were labelled with MCB or DAPI whereas dead oocysts were labelled with PI only. The localization of GSH in viable, intact oocysts and excysted sporozoites and UV light-irradiated oocysts and sporozoites revealed no changes in MCB uptake at levels up to 40 mJ.cm(-2) irradiation. Although GSH can be detected following MCB localization in both the nucleus and cytoplasm of sporozoites, and can be specifically depleted by BSO treatment, MCB is unlikely to be useful as a surrogate for detecting UV damage in UV-treated Cryptosporidium oocysts.

The role of acidic organelles in the development of schistosomula of Schistosoma mansoni and their response to signalling molecules.

The cercariae of Schistosoma mansoni become transformed into schistosomula during host skin penetration. We have found that large acidophilic compartments are detected in schistosomula but not in cercariae or in any other stages of the parasite by use of the fluorescent dye LysoTracker, a dye specific for mammalian lysosomes. Some of these large acidic compartments incorporated monodansylcadaverine, a specific dye for autophagosomes. We have used potent inhibitors (wortmannin and 3-methyladenine) and a potent inducer (starvation) of autophagy to show that the pathway to the formation of the acidic compartments requires specific molecular signals from the environment and from the genome. Certain doses of ultraviolet light inhibited significantly the formation of the acidic compartments, which may indicate disruption of the lysosome/autophagosome pathway. We have also defined two proteins that are commonly associated with lysosomes and autophagosomes in mammalian cells, the microtubule-associated membrane protein (MAP-LC3) and lysosome-associated membrane protein (LAMP-1), in extracts of schistosomula. We suggest that the autophagy pathway could be developed in transformed schistosomula.

The properties of acidic compartments in developing schistosomula of Schistosoma mansoni.

A variety of fluorescent probes have been used to study the acidic compartments in cercariae and schistosomula of Schistosoma mansoni. Freshly transformed schistosomula treated with the LysoTracker Red dye specific for lysosomes showed large acid-containing compartments (0.5-10 microm in size). The uptake of the dye is an energy-dependent process that depends on the metabolic activity of schistosomula. The compartments were quantified individually with respect to area, quantity of fluorescence and the total number/schistosomulum. Under normal conditions these compartments were not found in untreated cercariae, but appeared in cercariae slightly damaged by poly-L-lysine. The formation of these compartments seemed to be related to the development of cercariae into schistosomula as the number of compartments and uptake of fluorescence increased with time after transformation. Also, the method of transformation as well as the in vitro incubation of the parasite affected the percentage area of compartments/schistosomulum. Acid phosphatase enzyme activity was assessed using an endogenous phosphatase probe. Living and fixed schistosomula displayed the presence of enzyme activity in compartments of the same size and distribution as the acid-rich compartments. This was confirmed by histochemical staining showing deposition of enzyme-generated lead at the sites of phosphatase activity. We suggest that the development of acidic compartments is important during the transformation process or as a consequence of damage.

A study of the effect of surface damage on the uptake of Texas Red-BSA by schistosomula of Schistosoma mansoni.

In this paper we describe the effect of poly-L-lysines of different molecular weight on the schistosomula. In the control sample, the schistosomula of Schistosoma mansoni take up fluorescent Texas Red conjugated to bovine serum albumin (TxR-BSA) into the gut. Following slight damage by 24.0 kDa poly-L-lysine, a high proportion of schistosomula take up fluorescent TxR-BSA into the excretory system. Subsequently, the dye diffused into the bodies of the schistosomula. We suspected that this diffusion involved the process of endocytosis so we investigated this with the use of endocytosis inhibitor, Latrunculin A. Addition of the endocytosis inhibitor Latrunculin A following poly-L-lysine treatment inhibited gut uptake of TxR-BSA as well as the diffusion of excretory-ingested TxR-BSA molecules.

A study of some characteristics of individual clones of Schistosoma mansoni with emphasis on the biological and metabolic activities.

The variability within schistosome populations was explored using mixed populations of cercariae from multimiracidial snail infections and individual clones of Schistosoma mansoni cercariae obtained from monomiracidial snail infections. We investigated the heterogeneity between different clones of S. mansoni with respect to infectivity and metabolism. One difference between clones of cercariae was found in the recovery of adult worms from Balb/C mice. Recovery of adult worms was greater after infections with a mixed population than with a clonal population. To investigate some biochemical features of individuals in clones or mixed populations, the uptake of [35S]methionine into individual parasites and their membrane proteins was measured. Isoelectric focusing of a soluble membrane fraction: the frozen-thawed supernatant extracted from individual clones, showed the presence of proteins of isoelectric point between 7.2 and 8.2 in all clones. These proteins were less labelled with [35S]methionine in the clones than in the mixed population. It was concluded that basic proteins are synthesized by all clones and in the mixed population but at different rates. Differences in the rate of incorporation of [35S]methionine into the surface membranes of schistosomula and adult worms derived from individual clones are reported. In addition, a direct correlation between the percentage of recovery of adult worms from mice infected with individual clones of S. mansoni and the rate of incorporation of [35S]methionine into schistosomula of these particular clones was observed. It is suggested that the high rate of metabolism shown by an individual clone may account for the enhanced survival of the cercariae derived from that clone during penetration of the skin and migration through the vertebrate host. In order to examine individuals in a population of schistosomula, from a clone or mixed population, the lysosome-specific fluorescent probe LysoTracker DND-99 was used to label the parasites and quantitative fluorescent measurements were made on individual parasites. There were significant differences between clones and a mixed population. Furthermore, the variation between individuals from a mixed population was greater than from that in any clone, just as was found in the infectivity studies. Freshly transformed schistosomula of individual clones labelled with the LysoTracker DND-99 showed less variations in the quantitative uptake of the dye within a single clone when compared to the mixed population. We conclude that for any biochemical and biological parameter, a population of cercariae consists of individuals showing a wide range of values, with a much greater range in a mixed population. This variability is likely to have great relevance for infectivity of the final host and the efficacy of drugs and the immune system.