Martin B. Slade

Patterns of Cryptosporidium Oocyst Shedding by Eastern Grey Kangaroos Inhabiting an Australian Watershed

ABSTRACT The occurrence of Cryptosporidium oocysts in feces from a population of wild eastern grey kangaroos inhabiting a protected watershed in Sydney, Australia, was investigated. Over a 2-year period, Cryptosporidium oocysts were detected in 239 of the 3,557 (6.7%) eastern grey kangaroo fecal samples tested by using a combined immunomagnetic separation and flow cytometric technique. The prevalence of Cryptosporidium in this host population was estimated to range from 0.32% to 28.5%, with peaks occurring during the autumn months. Oocyst shedding intensity ranged from below 20 oocysts/g feces to 2.0 × 106 oocysts/g feces, and shedding did not appear to be associated with diarrhea. Although morphologically similar to the human-infective Cryptosporidium hominis and the Cryptosporidium parvum “bovine” genotype oocysts, the oocysts isolated from kangaroo feces were identified as the Cryptosporidium “marsupial” genotype I or “marsupial” genotype II. Kangaroos are the predominant large mammal inhabiting Australian watersheds and are potentially a significant source of Cryptosporidium contamination of drinking water reservoirs. However, this host population was predominantly shedding the marsupial-derived genotypes, which to date have been identified only in marsupial host species.

An Immunoglobulin G1 Monoclonal Antibody Highly Specific to the Wall of Cryptosporidium Oocysts

ABSTRACT The detection of Cryptosporidium oocysts in drinking water is critically dependent on the quality of immunofluorescent reagents. Experiments were performed to develop a method for producing highly specific antibodies to Cryptosporidium oocysts that can be used for water testing. BALB/c mice were immunized with six different antigen preparations and monitored for immunoglobulin G (IgG) and IgM responses to the surface of Cryptosporidiumoocysts. One group of mice received purified oocyst walls, a second group received a soluble protein preparation extracted from the outside of the oocyst wall, and the third group received whole inactivated oocysts. Three additional groups were immunized with sequentially prepared oocyst extracts to provide for a comparison of the immune response. Mice injected with the soluble protein extract demonstrated an IgG response to oocysts surface that was not seen in the whole-oocyst group. Mice injected with whole oocysts showed an IgM response only, while mice injected with purified oocyst walls showed little increase in IgM or IgG levels. Of the additional reported preparations only one, BME (2-mercaptoethanol treated), produced a weak IgM response to the oocyst wall. A mouse from the soluble oocyst extract group yielding a high IgG response was utilized to produce a highly specific IgG1 monoclonal antibody (Cry104) specific to the oocyst surface. Comparative flow cytometric analysis indicated that Cry104 has a higher avidity and specificity to oocysts in water concentrates than other commercially available antibodies.

Cryptosporidium fayeri: diversity within the GP60 locus of isolates from different marsupial hosts

The highly polymorphic 60 kDa glycoprotein (GP60) of Cryptosporidium is an important tool for investigating the epidemiology of this parasite. Characterization of the GP60 gene has only been performed for 3 of the 20 known Cryptosporidium species, and has already enabled sub-typing and source tracking of species with human significance. We have characterised a fourth species, Cryptosporidium fayeri, at the GP60 locus using isolates (n=26) from different marsupial hosts to assess the diversity of GP60 within this species. The analysis demonstrated that C. fayeri isolates could be assigned to 6 subtypes which were associated with host species and locality. The intra-species diversity for the host-adapted C. fayeri was less than the diversity for human pathogenic species suggesting that the GP60 locus is under less selective pressure in these than host-adapted species.

A naturally occurring novel allele of Escherichia coli outer membrane protein A reduces sensitivity to bacteriophage.

ABSTRACT A novel Escherichia coli outer membrane protein A (OmpA) was discovered through a proteomic investigation of cell surface proteins. DNA polymorphisms were localized to regions encoding the proteins surface-exposed loops which are known phage receptor sites. Bacteriophage sensitivity testing indicated an association between bacteriophage resistance and isolates having the novel ompA allele.

Expression, purification and characterisation of secreted recombinant glycoprotein PsA in Dictyostelium discoideum

Dictyostelium discoideum is a newly developed eukaryotic expression system which is an alternative to tissue cultures for the production of recombinant proteins requiring eukaryotic folding and post-translational modifications. The homologous glycoprotein PsA (prespore specific antigen) is a glycosyl phosphatidylinositol (GPI) anchored membrane protein from D. discoideum. A truncated form of PsA has been expressed in D. discoideum and secreted into a peptone based broth at levels of 10 mg per 1 growth medium. A simple purification protocol for recombinant PsA (rPsA) involved three steps: the concentration of the culture supernatant by ammonium sulfate precipitation, Mono Q anion-exchange chromatography, followed by size exclusion chromatography on Superdex 75. 20 mg of rPsA was purified to 98% purity from 37.1 culture supernatant. Purified rPsA was characterised. The molecular mass of the purified rPsA is 15.6 kDa, which suggests that the molecule is secreted as a monomer and contains 12% (w/w) carbohydrate. The protein sequence of rPsA proved identical to that of the predicted DNA construct. Although the recombinant form of PsA is expressed at a different developmental stage from the native molecule, the same Thr residues that are O-glycosylated in the authentic molecule are glycosylated in the recombinant protein.

From virus to vaccine: developments using the simple eukaryote, Dictyostelium discoideum

Mass vaccination compaigns against viral diseases, both human and anim al, depend on the availability of cheap viral antigens. The eukaryote Dictyostelium discoideum has simple growth requirements and rapid growth rates and forms stable cell lines. These features, together with the possibility of secreting recombinant (glyco)proteins into a defined buffer, make the D. discoideum expression system an attractive option for producing economical recombinant subunit vaccines.

Purification of a membrane glycoprotein with an inositol-containing phospholipid anchor from Dictyostelium discoideum

Large-scale purification of a Dictyostelium discoideum cell surface glycoprotein, which is anchored in the membrane via a glycosylphosphatidylinositol (GPI) moiety, is described. The purification protocol involved four steps: separation of crude cell membranes by low-speed centrifugation, delipidization of these membranes using acetone, extraction of the membrane proteins using the detergent Octyl beta-D-thioglucopyranoside (OTP), and purification of a specific membrane protein by monoclonal antibody immunoaffinity chromatography. The protein purified, PsA (prespore-specific antigen), is a developmentally regulated membrane glycoprotein found on a subset of cells from the cellular slime mould, D. discoideum. The protocol provides an efficient, economical, and technically simple way to purify GPI proteins in sufficient quantities for structural and functional studies. PsA was recovered at a yield of about 60%; with a purity of 97%, the extraction of 1 x 10(10) cells (1.1 g dry weight) yielded about 0.5 mg PsA glycoprotein. Techniques are described for growing kilogram quantities of D. discoideum cells in stainless steel trays at little cost. D. discoideum has considerable potential as a novel expression system for the production of foreign membrane-associated proteins. The purification strategy provides a means of purifying other GPI proteins, including those produced by protein engineering techniques.

Dictyostelium discoideum Mitochondrial DNA Encodes a NADH:Ubiquinone Oxidoreductase Subunit Which Is Nuclear Encoded in Other Eukaryotes

Complex I, a key component of the mitochondrial electron transport system, is thought to have evolved from at least two separate enzyme systems prior to the evolution of mitochondria from a bacterial endosymbiont, but the genes for one of the enzyme systems are thought to have subsequently been transferred to the nuclear DNA. We demonstrated that the cellular slime mold Dictyostelium discoideum retains the ancestral characteristic of having mitochondria encoding at least one gene (80-kDa subunit) that is nuclear encoded in other eukaryotes. This is consistent with the cellular slime molds of the family Dictyosteliaceae having diverged from other eukaryotes at an early stage prior to the loss of the mitochondrial gene in the lineage giving rise to plants and animals. The D. discoideum mitochondrially encoded 80-kDa subunit of complex I exhibits a twofold-higher mutation rate compared with the homologous chromosomal gene in other eukaryotes, making it the most divergent eukaryotic form of this protein.

Recombinant glycoprotein production in the slime mould Dictyostelium discoideum

Dictyostelium discoideum is a well known amoeboid organism, with unicellular and multicellular life-cycle stages, that is used for studying cell and developmental biology. With advances in gene-disruption technology and transformation of this organism, many homologous proteins have been expressed either to complement defective proteins or to study basic cell biology. Now, D. discoideum is being used to express heterologous proteins that are difficult to study in other systems, and its unique cell biology is being exploited to facilitate a wide range of protein modifications. In the past year, substantial progress has been made in expressing correctly folded forms of malarial circumsporozoite antigen and rotavirus surface glycoprotein VP7. Exciting developments have also been made in expressing human muscarinic receptors.

Cryptosporidium in Eastern Grey Kangaroos Macroqus Giganteus

Publisher Summary This chapter illustrates the first epidemiological investigation of Cryptosporidium in a wild marsupial population. The molecular epidemiology of Cryptosporidium in marsupial hosts is not well comprehended. . Molecular characterization of Cryptosporidium in marsupials consists of the identification of the Cryptosporidium parvum “marsupial” genotype in a koala, a red kangaroo, and in captive yellow-footed rock wallabies. The eastern grey kangaroo is one of Australias largest and most abundant species of kangaroo. With its distribution confined to Eastern Australia, the population density in New South Wales alone during the year 2000 was estimated to be 3.7 million. The presence of eastern grey kangaroos in water catchment areas and their likely contribution of fecal contamination to riparian zones highlight the importance of understanding the epidemiology of Cryptosporidium in this host. This study demonstrates that Cryptosporidium is present within this eastern grey kangaroo population all year and that prevalence varies with season, peaking during late summer to early autumn. The numbers of oocysts shed ranges from 10 to 2 X 107 oocysts/g feces with the majority of animals shedding < 1000 oocysts/g feces.