Evidence that primary infection of Charollais sheep with Toxoplasma gondii may not prevent foetal infection and abortion in subsequent lambings.

A study carried out on a sheep farm examined whether Toxoplasma gondii foetal infection and associated abortion occur in successive lambings. We identified 29 ewes that gave birth to lambs on at least 2 successive years over our study period, 2000-2003. Tissue samples from the progeny of these ewes were analysed by PCR to determine infection status with T. gondii. T. gondii-infected lambs were born in 31% of successive pregnancies. T. gondii-positive lambs were aborted in successive pregnancies in 21% of lambings during study period, 2000-2003. The frequency of successive abortions within this flock over the period 1992-2003 was 18%. If a lamb was congenitally infected there was a high risk (69%) that the successive lamb from that ewe would also be congenitally infected. Similarly, if a lamb was aborted there was a high risk (55%) of abortion in the next lamb produced. These data suggest that life-long immunity to T. gondii infections may not always be acquired following an initial infection and raises the question as to whether the mechanisms of T. gondii transmission prior to and during ovine pregnancies are fully understood.

Does vertical transmission contribute to the prevalence of toxoplasmosis?

Toxoplasma gondii is a ubiquitous parasite with a widespread distribution both in terms of geographical and host range. Although the definitive host is the cat, it is also a major health hazard to domestic animals and humans. Three routes of transmission are recognised (infection from the cat, carnivory and congenital transmission). We aimed to assess the relative importance of congenital transmission, using sheep as a model system, due to the lack of carnivory. We report, using PCR as a diagnostic tool, that congenital transmission occurs with high frequency (69%). If transmission from oocysts was important in sheep, we would expect sheep reared under the same environmental conditions (i.e. a single farm) to have a random distribution of Toxoplasma infection. Using breeding records in conjunction with PCR, some families were found to have high Toxoplasma prevalence and abortion while others were free of Toxoplasma infection and abortion (P < 0.01). This supports the notion that Toxoplasma may be transmitted vertically. In humans, we conducted a similar study and showed that Toxoplasma was transmitted from mother to baby in 19.8% of cases. Vertical transmission in Toxoplasma may be more important than previously thought and this knowledge should be considered in any eradication strategies.

The prevalence of Neospora caninum and co-infection with Toxoplasma gondii by PCR analysis in naturally occurring mammal populations.

Neospora caninum and Toxoplasma gondii are closely related intracellular protozoan parasites associated with bovine and ovine abortion respectively. Little is known about the extent of Neospora/Toxoplasma co-infection in naturally infected populations of animals. Using nested PCR techniques, based on primers from the Nc5 region of N. caninum and SAG1 for T. gondii, the prevalence of N. caninum and its co-infection with T. gondii were investigated in populations of Mus domesticus, Rattus norvegicus and aborted lambs (Ovis aries). A low frequency of infection with N. caninum was detected in the Mus domesticus (3%) and Rattus norvegicus (4.4%) populations. A relatively high frequency of infection with N. caninum was detected in the brains of aborted lambs (18.9%). There was no significant relationship between N. caninum and T. gondii co-infection. Investigation of the tissue distribution of Neospora, in aborted lambs, showed that Neospora could not be detected in tissues other than brain and this was in contrast to Toxoplasma where the parasite could be frequently detected in a range of tissues.

Ecological impacts of the microsporidian parasite Pleistophora mulleri on its freshwater amphipod host Gammarus duebeni celticus.

The microsporidian parasite, Pleistophora mulleri, infects the abdominal muscle of the freshwater amphipod Gammarus duebeni celticus. We recently showed that P. mulleri infection was associated with G. d. celticus hosts being more vulnerable to predation by the invasive amphipod Gammarus pulex. Parasitized G. d. celticus also had a reduced ability to prey upon other co-occurring amphipods. We suggested the parasite may have pervasive influences on host ecology and behaviour. Here, we examine the association between P. mulleri parasitism and parameters influencing individual host fitness, behaviour and interspecific interactions. We also investigate the relationship between parasite prevalence and host population structure in the field. In our G. d. celticus study population, P. mulleri prevalence was strongly seasonal, ranging from 8.5% in summer to 44.9% in winter. The relative abundance of hosts with the heaviest parasite burden increased during summer, which coincided with high host mortality, suggesting that parasitism may regulate host abundance to some degree. Females were more likely to be parasitized than males and parasitized males were paired with smaller females than unparasitized males. Parasitism was associated with reduction in the host's activity level and reduced both its predation on the isopod Asellus aquaticus and aggression towards precopula pairs of the invasive G. pulex. We discuss the pervasive influence of this parasite on the ecology of its host.

Significant familial differences in the frequency of abortion and Toxoplasma gondii infection within a flock of Charollais sheep.

A study was carried out to investigate the frequencies of abortion and congenital Toxoplasma gondii infection within 27 families (765 individuals) of a pedigree Charollais sheep flock maintained on a working farm in Worcestershire, UK, since 1992. Pedigree lambing records were analysed to establish the frequency of abortion for each family. The frequency of congenital infection was determined for each family by PCR analysis of tissue samples taken from newborn lambs. A total of 155 lambs were tested for congenital T. gondii infection, which were all born during the study period 2000-2003. Significant differences in the frequency of abortion between sheep families within this flock were observed with frequencies ranging between 0% and 48% (P < 0.01). Significantly different infection frequencies with T. gondii were also observed for different families and ranged between 0% and 100% (P<0.01). Although the actual cause of each abortion was not verified, a highly significant positive correlation was found to exist between the frequency of abortion and the frequency of T. gondii infection in the same families (P<0.01). The data presented here raise further questions regarding the significance of congenital transmission of T. gondii within sheep populations, the possible successive vertical transmission of T. gondii within families of sheep, and the potential role of inherited genetic susceptibility to abortion with respect to T. gondii infection. This work invites further study into the epidemiology of ovine toxoplasmosis and may have implications for sheep husbandry methods in the future.

High levels of congenital transmission of Toxoplasma gondii in longitudinal and cross-sectional studies on sheep farms provides evidence of vertical transmission in ovine hosts.

Recent research suggests that vertical transmission may play an important role in sustaining Toxoplasma gondii infection in some species. We report here that congenital transmission occurs at consistently high levels in pedigree Charollais and outbred sheep flocks sampled over a 3-year period. Overall rates of transmission per pregnancy determined by PCR based diagnosis, were consistent over time in a commercial sheep flock (69%) and in sympatric (60%) and allopatric (41%) populations of Charollais sheep. The result of this was that 53.7 % of lambs were acquiring an infection prior to birth: 46.4% of live lambs and 90.0% of dead lambs (in agreement with the association made between T. gondii and abortion). No significant differences were observed between lamb sexes. Although we cannot distinguish between congenital transmission occurring due to primary infection at pregnancy or reactivation of chronic infection during pregnancy, our observations of consistently high levels of congenital transmission over successive lambings favour the latter.

Ultrastructural and molecular characterization of Bacillidium vesiculoformis n. sp. (Microspora: Mrazekiidae) in the freshwater oligochaete Nais simplex (Oligochaeta: Naididae).

The development of a new species, Bacillidium vesiculoformis n. sp. (Microspora, Mrazekiidae), is described from the freshwater oligochaete Nais simplex (Oligochaeta, Naididae). Initial stages of parasite development consist of a monokaryotic merogony within a haemocyte of the intestinal blood sinus. The resulting hypertrophied haemocyte is attached to the chloragocytes of the sinus by fine cytoplasmic extensions with the sinus around the cell becoming greatly enlarged. The meronts within the haemocyte form diplokaryotic sporonts that undergo sporogenesis directly within the cytoplasm of the host cell. The infected cell becomes packed with spores and developmental stages, causing it dramatically to increase in size, eventually rupturing the oligochaete and cell. Sporogony appears to be disporoblastic. Released spores were observed to have an adhesive quality. Transmission studies conducted with mature spores failed to transmit the parasite horizontally although vertical transmission was observed. Phylogenetic analysis of the parasite demonstrated that B. vesiculoformis clustered with microsporidian parasites of bryozoa and two other microsporidians, Janacekia debaiseuxi and an unidentified Bacillidium sp.

Establishment of the new genus Paranosema based on the ultrastructure and molecular phylogeny of the type species Paranosema grylli Gen. Nov., Comb. Nov. (Sokolova, Selezniov, Dolgikh, Issi 1994), from the cricket Gryllus bimaculatus Deg.

The ultrastructure of the microsporidian parasite Nosema grylli, which parasitizes primarily fat body cells and haemocytes of the cricket Gryllus bimaculatus (Orthoptera, Gryllidae) is described. All observed stages (meront, meront/sporont transitional stage ("second meront"), sporont, sporoblast, and spore) are found in direct contact with the host cell cytoplasm. Nuclei are diplokaryotic during almost all stages of the life cycle, but a brief stage with one nucleus containing an abundance of electron-dense material is observed during a "second merogony." Sporogony is disporous. Mature spores are ovocylindrical in shape and measure 4.5+/-0.16micromx2.2+/-0.07 microm (n=10) on fresh smears and 3.3+/-0.06 micromx1.4+/-0.07 microm (n=10) on ultrathin sections. Spores contain 15-18 coils of an isofilar polar filament arranged in one or two layers. Comparative phylogenetic analysis using rDNA shows N. grylli to be closely related to another orthopteran microsporidian, Nosema locustae, and to Nosema whitei from the confused flour beetle, Tribolium confusum. Antonospora scoticae, a parasite of the communal bee Andrena scotica, is a sister taxon to these three Nosema species. The sequence divergence and morphological traits clearly separate this group of "Nosema" parasites from the "true" Nosema clade containing Nosema bombycis. We therefore propose to change the generic name of N. grylli and its close relative N. locustae to Paranosema n. comb. We leave N. whitei in former status until more data on fine morphology of the species are obtained.

High levels of congenital transmission of Toxoplasma gondii in a commercial sheep flock.

Our current understanding of congenital transmission of Toxoplasma gondii from ewe to lamb dictates that infection frequently results in abortion and the death of the developing foetus, that the birth of live infected lambs occurs rarely and that the cat is the predominant source of infection in ewes. Using direct polymerase chain reaction detection of T. gondii, we report high levels of congenital transmission occurring in a commercially managed sheep flock. We sampled foetal-derived placental tissue and tissues from aborted lambs and showed that congenital transmission was detected in these tissues from 61% of all pregnancies. Where pregnancies resulted in the death of one or more lambs, T. gondii was detected in the lamb tissue for all but one of 18 (94%) pregnancies. Of the successful pregnancies resulting in the birth of live lambs we were able to detect T. gondii in foetal-derived placental tissue from 37 of 70 (42%) pregnancies. These results show that congenital transmission is occurring in a high percentage of lambings including normal healthy lambings, at this farm, suggesting that this route of transmission from generation to generation may be much more significant than that reported previously. These results may have implications for sheep husbandry and future epidemiological studies of T. gondii.

MGE-PCR: a novel approach to the analysis of Toxoplasma gondii strain differentiation using mobile genetic elements.

The position of mobile genetic elements (MGE) within eukaryotic genomes is often highly variable and we have exploited this phenomenon to develop a novel approach to strain differentiation in Toxoplasma gondii. Two PCR based strategies were designed in which specific primers were used to amplify T. gondii MGE's revealing information on element size and positional variation. The first PCR strategy involved the use of a standard two primer PCR while the second strategy used a single specific primer in a step-up PCR protocol. This approach was applied to T. gondii reference strains which were either acute virulent or avirulent to mice. The use of a standard two primer PCR reaction revealed the presence of a virulence related marker in which all avirulent strains possessed an additional 688 bp band. The single primer PCR strategy demonstrated that all virulent strains had identical banding patterns suggesting invariance within this group of strains. However, all avirulent strains had different banding patterns indicating the presence of a number of individual lineages within this group. The applicability and sensitivity of MGE-PCR in epidemiological studies was demonstrated by direct amplification of T. gondii from sheep tissue samples. All sheep isolates, tested in this way, gave identical banding patterns suggesting the presence of an endemic Toxoplasma strain on this farm.

Transovarial transmission in the microsporidia.

The microsporidia are an ancient and diverse group of protists which have many unusual characteristics. These include prokaryotic-like 70s ribosomes, enclosed nuclear division, a lack of mitochondria and complex life cycles which frequently involve vertical transmission. This use of vertical transmission is unparalleled by other protists and is seen only among bacterial endosymbionts and sex ratio distorters and in host cell organelles. Transovarially transmitted microsporidia can have unusual and profound effects on host population sex ratios. We here consider the mechanisms of transovarial transmission and its implications for parasite evolution. We review parasite/host relationships and the evolution of virulence under transovarial transmission and consider the implications of these parasites for host ecology and evolution.

Ultrastructural characterisation and molecular taxonomic identification of Nosema granulosis n. sp., a transovarially transmitted feminising (TTF) microsporidium.

A novel microsporidian parasite is described, which infects the crustacean host Gammarus duebeni. The parasite was transovarially transmitted and feminised host offspring. The life cycle was monomorphic with three stages. Meronts were found in host embryos, juveniles, and in the gonadal tissue of adults. Sporoblasts and spores were restricted to the gonad. Sporogony was disporoblastic giving rise to paired sporoblasts, which then differentiated to form spores. Spores were not found in regular groupings and there was no interfacial envelope. Spores were approximately 3.78 x 1.22 microns and had a thin exospore wall, a short polar filament, and an unusual granular polaroplast. All life cycle stages were diplokaryotic. A region from the parasite small subunit ribosomal RNA gene was amplified and sequenced. Phylogenetic analysis based on these data places the parasite within the genus Nosema. We have named the species Nosema granulosis based on the structure of the polaroplast.

Segregation of a microsporidian parasite during host cell mitosis.

We investigated the segregation of an intracellular microsporidian parasite during host cell division. A time-course experiment was carried out to examine the distribution of parasites relative to host chromosomal DNA via light and electron microscopy. Fluorescent light microscopy and EM studies showed that the parasite lay in the perinuclear zone of the host cell during interphase and segregated to daughter cells at mitosis. At metaphase, the parasite was frequently closely associated with host microtubules and mitochondria. Electron-dense bridges were observed between the parasites and the host microtubules and also between host mitochondria and microtubules. The study suggests that both the parasite and the host cell organelles segregate in association with spindle microtubules.

Within-host transmission strategies of transovarial, feminizing parasites of Gammarus duebeni.

The amphipod Gammarus duebeni harbours several species of vertically transmitted microsporidian parasites. G. Duebeni were collected from 3 localities in the UK. Animals from Budle Bay, Northumberland, were infected with Octosporea effeminans, and those from Millport, Isle of Cumbrae and Fenham Flats, Northumberland were infected with microsporidia of the genus Nosema. We derived expected distributions of parasites per host embryonic cell by modelling parasite transmission as a multitype, Galton-Watson branching process. Parasite prevalence (proportion of females infected) was significantly heterogeneous among localities. Parasite burden in zygotes was much higher for females infected with Nosema than in animals infected with O. effeminans. There was no significant difference between localities in the number of Nosema in the zygotes. Comparison of models and data from 64-cell host embryos showed that the distributions of parasites per cell were consistent with the hypothesis that sorting of parasites into daughter cells is biased for at least 1 cell lineage. Host embryos infected with O. effeminans could expect to contain a growing number of parasites in each cell generation within such biased cell lineages; similar estimates for Nosema predict a decline in the number of parasites per cell within a biased lineage. We discuss the possibility that the 2 species of parasite may be employing different strategies in order to ensure transmission to the next host generation.

Cellular distribution of a feminizing microsporidian parasite: a strategy for transovarial transmission.

The cellular distribution of a vertically transmitted, feminizing microsporidian was followed in its host Gammarus duebeni. In adult females the parasite was restricted to gonadal tissue, in particular primary and secondary follicle cells. Spores were diplokaryotic with a thin spore wall and a short polar filament, characteristics typical of 'early' spores involved in autoinfection. The diplokaryotic life-cycle, absence of spore groupings and of a pansporoblast membrane typify the genus Nosema. However, the unusual globular polaroplast of the spore and restriction of this stage to host ovarian tissue have not previously been described in Nosema. Sporogony occurred only in follicle cells adjacent to developing oocytes and was in synchrony with the process of vitellogenesis. Oocytes were infected after formation of intracellular connections with follicle cells but harboured only vegetative stages of the parasite. Parasites were associated with the perinuclear cytoplasm and, in developing embryos, segregated to daughter cells along the axis of the spindle. In juvenile animals there was no evidence of pathology linked with feminization and the parasite was found at low density in cells under the cuticle. The parasite is highly adapted to transovarial transmission with an efficient mechanism of oocyte infection and no evidence of pathology.

The implantable neurocybernetic prosthesis system.

The neurocybernetic prosthesis system (NCP) is an implantable, multiprogrammable pulse generator that delivers constant current electrical signals to the vagus nerve for the purpose of reducing the frequency and severity of epileptic seizures. The signals are delivered on a predetermined schedule, or may be initiated by the patient with an external magnet. The device is implanted in a subcutaneous pocket in the chest just below the clavicle, similar to pacemaker placement. The stimulation signal is transmitted from the prosthesis to the vagus nerve through a lead connected to an electrode which is a multi-turn silicone helix, with a platinum band on the inner turn of one helix. The prosthesis can be programmed with any IBM- compatible personal computer using NCP software and a programming wand.