Astrid M. Tenter

Toxoplasma gondii: from animals to humans.

Toxoplasmosis is one of the more common parasitic zoonoses world-wide. Its causative agent, Toxoplasma gondii, is a facultatively heteroxenous, polyxenous protozoon that has developed several potential routes of transmission within and between different host species. If first contracted during pregnancy, T. gondii may be transmitted vertically by tachyzoites that are passed to the foetus via the placenta. Horizontal transmission of T. gondii may involve three life-cycle stages, i.e. ingesting infectious oocysts from the environment or ingesting tissue cysts or tachyzoites which are contained in meat or primary offal (viscera) of many different animals. Transmission may also occur via tachyzoites contained in blood products, tissue transplants, or unpasteurised milk. However, it is not known which of these routes is more important epidemiologically. In the past, the consumption of raw or undercooked meat, in particular of pigs and sheep, has been regarded as a major route of transmission to humans. However, recent studies showed that the prevalence of T. gondii in meat-producing animals decreased considerably over the past 20 years in areas with intensive farm management. For example, in several countries of the European Union prevalences of T. gondii in fattening pigs are now <1%. Considering these data it is unlikely that pork is still a major source of infection for humans in these countries. However, it is likely that the major routes of transmission are different in human populations with differences in culture and eating habits. In the Americas, recent outbreaks of acute toxoplasmosis in humans have been associated with oocyst contamination of the environment. Therefore, future epidemiological studies on T. gondii infections should consider the role of oocysts as potential sources of infection for humans, and methods to monitor these are currently being developed. This review presents recent epidemiological data on T. gondii, hypotheses on the major routes of transmission to humans in different populations, and preventive measures that may reduce the risk of contracting a primary infection during pregnancy.

Molecular and bioassay-based detection of Toxoplasma gondii oocyst uptake by mussels (Mytilus galloprovincialis)

Toxoplasma gondii is associated with morbidity and mortality in a variety of marine mammals, including fatal meningoencephalitis in the southern sea otter (Enhydra lutris nereis). The source(s) of T. gondii infection and routes of transmission in the marine environment are unknown. We hypothesise that filter-feeding marine bivalve shellfish serve as paratenic hosts by assimilation and concentration of infective T. gondii oocysts and their subsequent predation by southern sea otters is a source of infection for these animals. We developed a TaqMan PCR assay for detection of T. gondii ssrRNA and evaluated its usefulness for the detection of T. gondii in experimentally exposed mussels (Mytilus galloprovincialis) under laboratory conditions. Toxoplasma gondii-specific ssrRNA was detected in mussels as long as 21 days post-exposure to T. gondii oocysts. Parasite ssrRNA was most often detected in digestive gland homogenate (31 of 35, i.e. 89%) compared with haemolymph or gill homogenates. Parasite infectivity was confirmed using a mouse bioassay. Infections were detected in mice inoculated with any one of the mussel sample preparations (haemolymph, gill, or digestive gland), but only digestive gland samples remained bioassay-positive for at least 3 days post-exposure. For each time point, the total proportion of mice inoculated with each of the different tissues from T. gondii-exposed mussels was similar to the proportion of exposed mussels from the same treatment groups that were positive via TaqMan PCR. The TaqMan PCR assay described here is now being tested in field sampling of free-living invertebrate prey species from high-risk coastal locations where T. gondii infections are prevalent in southern sea otters.

Current research on Sarcocystis species of domestic animals

The genus Sarcocystis is composed of about 130 species of heteroxenous cyst-forming coccidia with differences in life cycle and pathogenicity. Pathogenic Sarcocystis spp. can cause disease in their intermediate hosts, in particular in ruminants. Research on Sarcocystis infections has been impeded by several facets of the parasites. Intermediate as well as definitive hosts can be parasitized by several different species with similarities in biology and morphology. Antigen preparations derived from pathogenic Sarcocystis spp. are highly cross-reactive with antibodies directed against non-pathogenic species. As a consequence, none of the currently available immunological tests is species-specific and can differentiate between pathogenic and non-pathogenic Sarcocystis spp. Over the last decade, new techniques in immunology, protein chemistry and molecular biology have facilitated more advanced studies on the molecular composition and molecular biology of Sarcocystis spp. in various laboratories. The development of species-specific monoclonal antibodies and analyses of the molecular composition of some life-cycle stages of Sarcocystis spp. of cattle and sheep showed that species-specific proteins and antigens exist in these species, although they are not highly abundant. In addition, comparisons of rRNA genes of different Sarcocystis spp. identified unique sequences in the rRNA of pathogenic Sarcocystis spp. that are suitable targets for species-specific identification. Thus, tools have become available that facilitate the development of methods for species-specific identification and differentiation of Sarcocystis spp. as well as the identification and study of molecules that are associated with pathogenicity of some of these parasites.

Phylogenetic analysis based on full-length large subunit ribosomal RNA gene sequence comparison reveals that Neospora caninum is more closely related to Hammondia heydorni than to Toxoplasma gondii.

Since its first description in the late 1980s, Neospora caninum has been recognised as a prominent tissue cyst-forming parasite due to its ability to induce congenital disease and abortion in animals, especially cattle. It is found worldwide and is a cause of significant economic losses for the livestock industry. However, its place within the family Sarcocystidae, like that of several other taxa, remains unresolved. Neospora caninum shares several morphological and life cycle characters with Hammondia heydorni, although it is most commonly thought of as being a close relative of Toxoplasma gondii. This study presents information regarding the phylogenetic relationship of N. caninum to species currently classified into the genus Hammondia, as well as to two strains (RH and ME49) of T. gondii based on the full-length large subunit ribosomal RNA gene. Phylogenetic analyses using two alignment strategies and three different tree-building methods showed that the two species in the genus Hammondia are paraphyletic. Neospora caninum was shown to form a monophyletic clade with H. heydorni instead of T. gondii, which in turn was shown to be most closely related to H. hammondi. The finding that N. caninum and H. heydorni are closely related phylogenetically may aid the elucidation of currently unknown aspects of their biology and epidemiology, and suggests that H. heydorni should be considered in the differential diagnosis of N. caninum from other apicomplexan parasites.

Phylogeny of the tissue cyst-forming coccidia.

Publisher Summary This chapter reviews the currently used classifications of tissue cyst-forming coccidia and discusses the recent phylogenetic studies based on molecular data that have provided new insights into the phylogeny and genetic relatedness of some of these parasites. In particular, the genetic relationships of species placed into the two oldest genera of tissue cyst-forming coccidia (Sarcocystis and Toxoplasma) are discussed. The chapter presents the strategies to infer phylogenetic relationships or genetic relatedness of tissue cyst-forming coccidia from molecular data. The chapter also discusses the distinct differences in the biology, life cycle, and morphology between the heteroxenous tissue cyst-forming coccidia and homoxenous coccidia. Over the last decade, phenotypic characters have gradually been replaced by more objective and more phylogenetically valid molecular characters for the inference of phylogenetic relationships of a broad range of protozoa.

Phylogenetic analysis of coccidia based on 18S rDNA sequence comparison indicates that Isospora is most closely related to Toxoplasma and Neospora.

The phylogenetic relationships and taxonomic affinities of coccidia with isosporan‐type oocysts have been unclear as overlapping characters, recently discovered life cycle features, and even recently discovered taxa. continue to be incorporated into biological classifications of the group. We determined the full or partial 18S ribosomal RNA gene sequences of three mammalian Isospora spp., Isospora felis, Isospora ohioensis and Isospora suis, and a Sarcocystis sp. of a rattlesnake, and used these sequences for a phylogenetic analysis of the genus Isospora and the cyst‐forming coccidia. Various alveolate 18S rDNA sequences were aligned and analyzed using maximum parsimony to obtain a phylogenetic hypothesis for the group. The three Isospora spp. were found to be most closely related to Toxoplasma gondii and Neospora caninum. This clade in turn formed the sister group to the Sarcocystis spp. included in the analysis. The results confirm that the genus Isospora does not belong to the family Eimeriidae, but should be classified together with the cyst‐forming coccidia in the family Sarcocystidae. Furthermore, there appear to be two lineages within the Sarcocystidae. One lineage comprises Isospora and the Toxoplasma/Neospora clade which share the characters of having a proliferative phase of development preceding gamogony in the definitive host and an exogenous phase of sporogony. The other lineage comprises the Sarcocystis spp. which have no proliferative phase in the definitive host and an endogenous phase of sporogony.

Phylogenetic relationships of the genus Frenkelia: a review of its history and new knowledge gained from comparison of large subunit ribosomal ribonucleic acid gene sequences

The different genera currently classified into the family Sarcocystidae include parasites which are of significant medical, veterinary and economic importance. The genus Sarcocystis is the largest within the family Sarcocystidae and consists of species which infect a broad range of animals including mammals, birds and reptiles. Frenkelia, another genus within this family, consists of parasites that use rodents as intermediate hosts and birds of prey as definitive hosts. Both genera follow an almost identical pattern of life cycle, and their life cycle stages are morphologically very similar. However, the relationship between the two genera remains unresolved because previous analyses of phenotypic characters and of small subunit ribosomal ribonucleic acid gene sequences have questioned the validity of the genus Frenkelia or the monophyly of the genus Sarcocystis if Frenkelia was recognised as a valid genus. We therefore subjected the large subunit ribosomal ribonucleic acid gene sequences of representative taxa in these genera to phylogenetic analyses to ascertain a definitive relationship between the two genera. The full length large subunit ribosomal ribonucleic acid gene sequences obtained were aligned using Clustal W and Dedicated Comparative Sequence Editor secondary structure alignments. The Dedicated Comparative Sequence Editor alignment was then split into two data sets, one including helical regions, and one including non-helical regions, in order to determine the more informative sites. Subsequently, all four alignment data sets were subjected to different tree-building algorithms. All of the analyses produced trees supporting the paraphyly of the genus Sarcocystis if Frenkelia was recognised as a valid genus and, thus, call for a revision of the current definition of these genera. However, an alternative, more parsimonious and more appropriate solution to the Sarcocystis/Frenkelia controversy is to synonymise the genus Frenkelia with the genus Sarcocystis.

Phylogenetic relationships of Sarcocystis species from sheep, goats, cattle and mice based on ribosomal RNA sequences

Partial sequences of the small subunit ribosomal RNA of four species of Sarcocystis were obtained by reverse transcription of total cellular RNA. The semi-conserved regions of these four species were aligned with homologous sequences of two other Sarcocystis species and a range of other eukaryotes including Toxoplasma, Eimeria and Cryptosporidium. Parsimony analysis of the aligned sequences showed that Sarcocystis and Toxoplasma had a more recent common ancestor with Eimeria than with Cryptosporidium. The six Sarcocystis spp. did not cluster together in this analysis; two monophyletic groups were observed, one formed by the two Sarcocystis spp. with felids as definitive hosts, and another by the four Sarcocystis spp. with canids as definitive hosts. These two clades were segregated by Toxoplasma. An analysis of nucleotide divergence suggests that the difference between the two groups of Sarcocystis spp. is similar to that between invertebrates and vertebrates. The results obtained here question the validity of a separation of the genus Sarcocystis from Toxoplasma and refute classifications that place these two genera into two different subfamilies of the Sarcocystidae.

Development and application of different methods for the detection of Toxoplasma gondii in water.

ABSTRACT Two methods, centrifugation and flocculation, were evaluated to determine their efficiencies of recovery of Toxoplasma gondii oocysts from contaminated water samples. Demineralized and tap water replicates were inoculated with high numbers of sporulated or unsporulated T. gondii oocysts (1 × 105 and 1 × 104 oocysts). The strain, age, and concentration of the seeded oocysts were recorded. Oocysts were recovered either by centrifugation of the contaminated samples at various g values or by flocculation with two coagulants, Fe2(SO4)3 and Al2(SO4)3. The recovery rates were determined with the final pellets by phase-contrast microscopy. Sporulated oocysts were recovered more effectively by flocculation with Al2(SO4)3 (96.5% ± 21.7%) than by flocculation with Fe2(SO4)3 (93.1% ± 8.1%) or by centrifugation at 2,073 × g (82.5% ± 6.8%). For the unsporulated oocysts, flocculation with Fe2(SO4)3 was more successful (100.3% ± 26.9%) than flocculation with Al2(SO4)3 (90.4% ± 19.1%) or centrifugation at 2,565 × g (97.2% ± 12.5%). The infectivity of the sporulated oocysts recovered by centrifugation was confirmed by seroconversion of all inoculated mice 77 days postinfection. These data suggest that sporulated Toxoplasma oocysts purified by methods commonly used for waterborne pathogens retain their infectivity after mechanical treatment and are able to induce infections in mammals. This is the first step in developing a systematic approach for the detection of Toxoplasma oocysts in water.

Identification of Synapomorphic Characteris in the Genus Sarcocystis Based on 18S rDNA Sequence Comparison

ABSTRACT. In order to further investigate synapomorphic characters in the genus Sarcocystis, the small subunit ribosomal RNA gene sequences of Sarcocystis capracanis and Sarcocystis moulei were determined and used to infer the phylogenetic position of these two organisms within the cyst‐forming coccidia. Phylogenies derived using distance, maximum parsimony and maximum likelihood methods demonstrated that S. capracanis groups with Sarcoystis tenella and Sarcocystis arieticanis as a clade that shares the characteristic of using canids as their definitive host. S. moulei was shown to group with Sarcocystis gigantea and Sarcocystis fusiformis as a clade that shares the characteristic of using felids as their definitive host.