P. J. O'Donoghue

Molecular and phylogenetic characterisation of Cryptosporidium from birds.

Avian isolates of Cryptosporidium species from different geographic locations were sequenced at two loci, the 18S rRNA gene and the heat shock gene (HSP-70). Phylogenetic analysis of the sequence data provided support for the existence of a new avian species of Cryptosporidium infecting finches and a second species infecting a black duck. The identity of Cryptosporidium baileyi and Cryptosporidium meleagridis as valid species was confirmed. Also, C. baileyi was identified in a number of isolates from the brown quail extending the host range of this species.

Drug resistance in the sexually transmitted protozoan Trichomonas vaginalis

ABSTRACTTrichomoniasis is the most common, sexually transmitted infection. It is caused by the flagellated protozoan parasite Trichomonas vaginalis. Symptoms include vaginitis and infections have been associated with preterm delivery, low birth weight and increased infant mortality, as well as predisposing to HIV/AIDS and cervical cancer. Trichomoniasis has the highest prevalence and incidence of any sexually transmitted infection. The 5-nitroimidazole drugs, of which metronidazole is the most prescribed, are the only approved, effective drugs to treat trichomoniasis. Resistance against metronidazole is frequently reported and cross-resistance among the family of 5-nitroimidazole drugs is common, leaving no alternative for treatment, with some cases remaining unresolved. The mechanism of metronidazole resistance in T. vaginalis from treatment failures is not well understood, unlike resistance which is developed in the laboratory under increasing metronidazole pressure. In the latter situation, hydrogenosomal function which is involved in activation of the prodrug, metronidazole, is down-regulated. Reversion to sensitivity is incomplete after removal of drug pressure in the highly resistant parasites while clinically resistant strains, so far analysed, maintain their resistance levels in the absence of drug pressure. Although anaerobic resistance has been regarded as a laboratory induced phenomenon, it clearly has been demonstrated in clinical isolates. Pursuit of both approaches will allow dissection of the underlying mechanisms. Many alternative drugs and treatments have been tested in vivo in cases of refractory trichomoniasis, as well as in vitro with some successes including the broad spectrum anti-parasitic drug nitazoxanide. Drug resistance incidence in T. vaginalis appears to be on the increase and improved surveillance of treatment failures is urged.

Ultrastructure of the attachment of Cryptosporidium sporozoites to tissue culture cells

The attachment of Cryptosporidium sporozoites to Madin-Darby canine kidney (MDCK) cells was examined using transmission electron microscopy. As the anterior end of the sporozoite came into close proximity to the MDCK cell, the host cell membrane evaginated around the sporozoite, forming a parasitophorous vacuole. A dense band formed below the host cell membrane at the site nearest to the conoid. Variably electron-dense material was apparently released from the conoid and a large membrane-bound vacuole was formed in the anterior end of the sporozoite, displacing the typical anterior electron-dense organelles (rhoptries and micronemes). The outer membrane of the sporozoite pellicle then fused with the host cell membrane immediately adjacent to the conoid. The membrane surrounding the anterior vacuole was also fused with the common host-parasite membrane, forming Y-shaped membrane junctions where each limb was a unit membrane. A direct link was thereby established between the anterior vacuole of the sporozoite and the host cell cytoplasm. The anterior vacuole membrane separating the sporozoite and the host cell cytoplasm was the precursor of the feeder organelle.

Transcriptional changes in the hookworm, Ancylostoma caninum, during the transition from a free-living to a parasitic larva.

Background Third-stage larvae (L3) of the canine hookworm, Ancylostoma caninum, undergo arrested development preceding transmission to a host. Many of the mRNAs up-regulated at this stage are likely to encode proteins that facilitate the transition from a free-living to a parasitic larva. The initial phase of mammalian host invasion by A. caninum L3 (herein termed “activation”) can be mimicked in vitro by culturing L3 in serum-containing medium. Methodology/Principal Findings The mRNAs differentially transcribed between activated and non-activated L3 were identified by suppression subtractive hybridisation (SSH). The analysis of these mRNAs on a custom oligonucleotide microarray printed with the SSH expressed sequence tags (ESTs) and publicly available A. caninum ESTs (non-subtracted) yielded 602 differentially expressed mRNAs, of which the most highly represented sequences encoded members of the pathogenesis-related protein (PRP) superfamily and proteases. Comparison of these A. caninum mRNAs with those of Caenorhabditis elegans larvae exiting from developmental (dauer) arrest demonstrated unexpectedly large differences in gene ontology profiles. C. elegans dauer exiting L3 up-regulated expression of mostly intracellular molecules involved in growth and development. Such mRNAs are virtually absent from activated hookworm larvae, and instead are over-represented by mRNAs encoding extracellular proteins with putative roles in host-parasite interactions. Conclusions/Significance Although this should not invalidate C. elegans dauer exit as a model for hookworm activation, it highlights the limitations of this free-living nematode as a model organism for the transition of nematode larvae from a free-living to a parasitic state.

Electrophoretic and immunoblot analysis of Cryptosporidium oocysts

Cryptosporidium oocysts were recovered by density gradient centrifugation from diarrhoeal faeces of four human patients and one goat kid. Goat‐derived oocysts were further treated with excystation medium and the excysted oocyst walls purified by isopycnic ultracentrifugation. Soluble extracts from intact oocysts and the oocyst wall preparation were analysed by SDS‐PAGE. Fifty‐one polypeptide bands were detected in intact oocyst preparations: 48 were in the range 14 000–200 000 molecular weight (MW), two bands were less than 14 000 MW and one band was above 200 000 MW. Twenty‐one bands were detected in the oocyst wall preparation, all within the range 14 000–200 000 MW. Immunoblot analysis of Cryptosporidium polypeptides using acute or convalescent human and goat sera revealed a large number of reactive bands. Varying degrees of heterogeneity were observed within and between the two serum groups. Nine of the 10 human sera and all of the goat kid sera reacted with a 23 000 MW and 32 000 MW antigen. A 15 500 MW antigen was also detected by all the goat and four of the 10 human sera. Both serum groups reacted with various antigens above 40 000 MW. Surface labelling of three human isolates of Cryptosporidium oocysts with 125I was performed using the Bolton and Hunter reagent. The solubilized preparations were separated by SDS‐PAGE on 12% and 18% slab gels and autoradiographed. Common bands were seen at 15 500, 32 000, 47 500, 79 000 and 96 000 MW. Some variation in the molecular weight of polypeptides labelled with 125I was observed among the three isolates. These studies indicate that the 32 000 MW antigen appears to be a suitable candidate for the preparation of an antibody probe because it is common to many isolates and is located on the exterior of the oocyst wall.

Phylogenetic relationships of Trypanosoma chelodina and Trypanosoma binneyi from Australian tortoises and platypuses inferred from small subunit rRNA analyses

Trypanosome infections are often difficult to detect by conventional microscopy and their pleomorphy often confounds differential diagnosis. Molecular techniques are now being used to diagnose infections and to determine phylogenetic relationships between species. Complete small subunit rRNA gene sequences were determined for isolates of Trypanosoma chelodina from the Brisbane River tortoise (Emydura signata), the saw-shelled tortoise (Elseya latisternum), and the eastern snake-necked tortoise (Chelodina longicollis) from southeast Queensland, Australia. Partial sequence data were also obtained for T. binneyi from a platypus (Ornithorhynchus anatinus) from Tasmania. Phylogenetic relationships between T. chelodina, T. binneyi and other species were examined by maximum parsimony and likelihood methods. The Australian tortoise and platypus trypanosomes did not exhibit any close phylogenetic relationships with those of mammals, reptiles or amphibians, but were closely related to each other, and to fish trypanosomes. This contra-indicates their co-evolution with their vertebrate hosts but does not exclude co-evolution with different groups of invertebrate vectors, notably insects and leeches.

Tick paralysis in Australia caused by Ixodes holocyclus Neumann

Abstract Ticks are obligate haematophagous ectoparasites of various animals, including humans, and are abundant in temperate and tropical zones around the world. They are the most important vectors for the pathogens causing disease in livestock and second only to mosquitoes as vectors of pathogens causing human disease. Ticks are formidable arachnids, capable of not only transmitting the pathogens involved in some infectious diseases but also of inducing allergies and causing toxicoses and paralysis, with possible fatal outcomes for the host. This review focuses on tick paralysis, the role of the Australian paralysis tick Ixodes holocyclus, and the role of toxin molecules from this species in causing paralysis in the host.

Immune and pathophysiological responses to different strains of Giardia duodenalis in neonatal mice

Numerous studies have demonstrated various strain differences between Giardia isolates, but little is known about the immunology and pathogenesis of infections. This study aimed to compare host responses to strains of Giardi duodenalis differing in levels of virulence and pathogenicity and, by doing so, elucidate the mechanisms via which pathogenic strains establish infections. Marked differences were found in the infection dynamics, histopathological responses and serum antibody responses of neonatal mice infected with either G. duodenalis strain BRIS/83/HEPU/106 (isolated from a human) or BRIS/95/HEPU/2041 (isolated from a sulphur-crested cockatoo, Cacatua galerita). Infections with the bird strain were more intense (6.7-times greater) and persisted longer (by 14days) than infections with the human strain. The bird strain was more pathogenic and caused greater pathophysiological alteration to the gut mucosa, including increased villous atrophy, hyperplasia of goblet cells and vacuolated epithelial cells. Mice infected with the bird strain produced less serum anti-Giardia IgA and IgM, but more total (non-specific) serum IgA than those infected with the human strain of Giardia. This suggests that avian G. duodenalis strains are infective for mammalian hosts and may contribute to zoonotic infections. Furthermore, infection of mice with BRIS/95/HEPU/2041 serves as a good experimental model to provide further insight into the mechanisms via which G. duodenalis causes disease.

Cryptosporidium Infections in Birds and Mammals and Attempted Cross-Transmission Studies

Infections by Cryptosporidium were detected in association with clinical disease in 11 humans (Homo sapiens), 19 calves (Bos taurus), nine common quail (Coturnix coturnix), six mallard ducks (Anas platyrhynchos), five ring-necked pheasant (Phasianus colchicus) and a single budgerigar (Melopsittacus undulatus). Infections in mammals were accompanied by transient diarrhoea and anorexia, whereas infected birds exhibited clinical signs of respiratory distress. Repeated cross-transmission studies revealed apparent strain differences or differences in the host specificity of several mammalian and avian isolates for homologous vertebrate classes only. Oocysts from humans and calves were infective to mice, pigs or lambs, but not to chickens, whereas oocysts from quail and pheasant were infective to chickens, but not to mice.

Coccidia in sheep in South Australia

Faecal samples from 136 sheep from four different locations in South Australia were examined to determine the types and numbers of Eimeria spp. present. Coccidian oocysts were detected in 80% of the sheep and 11 different species of Eimeria were identified. The species detected (and their prevalence) were E. crandallis/E. weybridgensis (76%), E. ovina (55%), E. ovinoidalis (54%), E. granulosa (49%), E. parva/E. pallida (44%), E. intricata (37%), E. ahsata (31%), E. faurei (24%), and E. punctata (1%). No major differences were observed in the patterns of infection between the four locations examined. Faecal samples were also collected each month for a year from 48 lambs at two of the locations and oocyst counts were found to decrease markedly in all lambs after 6 months of age but to persist at low levels until the end of sampling at 17 months of age.