Hammondia hammondi harbors functional orthologs of the host-modulating effectors GRA15 and ROP16 but is distinguished from Toxoplasma gondii by a unique transcriptional profile.

Toxoplasma gondii and its nearest extant relative, Hammondia hammondi, are phenotypically distinct despite their remarkable similarity in gene content, synteny, and functionality. To begin to identify genetic differences that might drive distinct infection phenotypes of T. gondii and H. hammondi, in the present study we (i) determined whether two known host-interacting proteins, dense granule protein 15 (GRA15) and rhoptry protein 16 (ROP16), were functionally conserved in H. hammondi and (ii) performed the first comparative transcriptional analysis of H. hammondi and T. gondii sporulated oocysts. We found that GRA15 and ROP16 from H. hammondi (HhGRA15 and HhROP16) modulate the host NF-κB and STAT6 pathways, respectively, when expressed heterologously in T. gondii. We also found the transcriptomes of H. hammondi and T. gondii to be highly distinct. Consistent with the spontaneous conversion of H. hammondi tachyzoites into bradyzoites both in vitro and in vivo, H. hammondi high-abundance transcripts are enriched for genes that are of greater abundance in T. gondii bradyzoites. We also identified genes that are of high transcript abundance in H. hammondi but are poorly expressed in multiple T. gondii life stages, suggesting that these genes are uniquely expressed in H. hammondi. Taken together, these data confirm the functional conservation of known T. gondii virulence effectors in H. hammondi and point to transcriptional differences as a potential source of the phenotypic differences between these species.

Genotyping of samples from German patients with ocular, cerebral and systemic toxoplasmosis reveals a predominance of Toxoplasma gondii type II.

Toxoplasmosis is an important zoonosis transmitted from animals to humans world-wide. In order to determine Toxoplasma gondii genotypes in individuals living in Germany and to compare findings with those in animals, we analysed nine independent and unlinked genetic markers (nSAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico) by PCR-RFLP in 83 archived T. gondii-positive DNA samples from patients with ocular toxoplasmosis (n=35), toxoplasmic encephalitis (n=32), systemic toxoplasmosis after bone-marrow transplantation (n=15) and congenital toxoplasmosis (n=1). In 46 of these 83 samples the presence of T. gondii DNA was confirmed by conventional end-point PCR. Among these, 17 T. gondii-positive samples were typed at all nine loci. The majority (15/17, 88.2%) of these samples were of T. gondii type II (i.e., including both, the Apico type II and Apico type I variants). In addition, in one sample a T. gondii type II/type III allele combination and in another sample a T. gondii genotype displaying type III alleles at all markers was observed. In the remaining 11 samples, in which T. gondii could only be partially typed, exclusively type II (n=10) or type III (n=1) alleles were observed. Results of the present study suggest that the majority of patients in Germany are infected with type II T. gondii regardless of the clinical manifestation of toxoplasmosis. This finding is in accord with the predominance of type II T. gondii in oocysts isolated from cats and in tissues of other intermediate hosts in Germany.

Molecular identification of Sarcocystis spp. helped to define the origin of green pythons (Morelia viridis) confiscated in Germany.

Sarcocystis spp. represent apicomplexan parasites. They usually have a heteroxenous life cycle. Around 200 species have been described, affecting a wide range of animals worldwide, including reptiles. In recent years, large numbers of reptiles have been imported into Europe as pets and, as a consequence, animal welfare and species protection issues emerged. A sample of pooled feces from four confiscated green pythons (Morelia viridis) containing Sarcocystis spp. sporocysts was investigated. These snakes were imported for the pet trade and declared as being captive-bred. Full length 18S rRNA genes were amplified, cloned into plasmids and sequenced. Two different Sarcocystis spp. sequences were identified and registered as Sarcocystis sp. from M. viridis in GenBank. Both showed a 95-97% sequence identity with the 18S rRNA gene of Sarcocystis singaporensis. Phylogenetic analysis positioned these sequences together with other Sarcocystis spp. from snakes and rodents as definitive and intermediate hosts (IH), respectively. Sequence data and also the results of clinical and parasitological examinations suggest that the snakes were definitive hosts for Sarcocystis spp. that circulate in wild IH. Thus, it seems unlikely that the infected snakes had been legally bred. Our research shows that information on the infection of snakes with Sarcocystis spp. may be used to assess compliance with regulations on the trade with wildlife species.

Hammondia hammondi, an avirulent relative of Toxoplasma gondii, has functional orthologs of known T. gondii virulence genes.

Toxoplasma gondii is a ubiquitous protozoan parasite capable of infecting all warm-blooded animals, including humans. Its closest extant relative, Hammondia hammondi, has never been found to infect humans and, in contrast to T. gondii, is highly attenuated in mice. To better understand the genetic bases for these phenotypic differences, we sequenced the genome of a H. hammondi isolate (HhCatGer041) and found the genomic synteny between H. hammondi and T. gondii to be >95%. We used this genome to determine the H. hammondi primary sequence of two major T. gondii mouse virulence genes, TgROP5 and TgROP18. When we expressed these genes in T. gondii, we found that H. hammondi orthologs of TgROP5 and TgROP18 were functional. Similar to T. gondii, the HhROP5 locus is expanded, and two distinct HhROP5 paralogs increased the virulence of a T. gondii TgROP5 knockout strain. We also identified a 107 base pair promoter region, absent only in type III TgROP18, which is necessary for TgROP18 expression. This result indicates that the ROP18 promoter was active in the most recent common ancestor of these two species and that it was subsequently inactivated in progenitors of the type III lineage. Overall, these data suggest that the virulence differences between these species are not solely due to the functionality of these key virulence factors. This study provides evidence that other mechanisms, such as differences in gene expression or the lack of currently uncharacterized virulence factors, may underlie the phenotypic differences between these species.

Toxoplasma gondii sexual cross in a single naturally infected feline host: generation of highly mouse-virulent and avirulent clones, genotypically different from clonal types I, II and III.

Tachyzoite clones obtained from a single Toxoplasma gondii oocyst field sample were genotyped and characterized regarding mouse virulence. PCR-RFLP genotyping of tachyzoites initially isolated from interferon-γ-knockout (GKO) mice, BALB/c mice and VERO cell culture using the nine independent, unlinked genetic markers nSAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico revealed mixed T. gondii infections showing combinations of type II and type III alleles at different loci. Forty-five individual clones were obtained from all mixed T. gondii tachyzoite cell cultures by limiting dilution. Sixteen T. gondii clones showed type III alleles at all loci and 29 clones displayed a combination of type II and type III alleles at different loci. Five clone groups were identified in total, four of which include T. gondii clones that showed a non-canonical allele pattern and have never been described in natural infections before. All tested clones, except two, were highly virulent in BALB/c mice. The isolation of different non-canonical T. gondii clones originating from an oocyst sample of a single naturally infected cat demonstrate that sexual recombination as well as re-assortment of chromosomes via a sexual cross of T. gondii occur under natural conditions and result in the emergence of clones with increased virulence in mice.

Comparison of different commercial DNA extraction kits to detect Toxoplasma gondii oocysts in cat faeces.

The cat is the definitive host of Toxoplasma gondii and plays an important role in the transmission of this and other coccidian parasites, e.g. Hammondia hammondi, a protozoon closely related and morphologically similar to T. gondii. A number of techniques to detect T. gondii nucleic acids in feline faeces are described and several extraction kits for isolating pathogen DNA from faeces or soil are commercially available. To compare the performance of such kits with regard to isolating oocyst DNA, a feline sample that had tested negative for coccidian parasites including T. gondii and H. hammondi was spiked with 10(4), 10(3), 10(2), 50 and 10 H. hammondi oocysts. Several ready-to-use stool or soil kits and an in-house method were then used to extract parasite DNA from these spiked faecal samples. Of six kits tested, two were found suitable for the detection of H. hammondi oocysts DNA by the polymerase chain reaction (PCR) in faecal samples with a detection limit of 250 oocysts per 1 g of faecal sample. These two kits revealed a similar, even slightly lower detection limit (50 oocysts per 1 g of sample) when tested with faecal samples spiked with T gondii oocysts.

Isolation and genotyping of Toxoplasma gondii causing fatal systemic toxoplasmosis in an immunocompetent 10-year-old cat.

A 10-year-old male, neutered domestic shorthair cat was presented with fever, anorexia, vomiting, and diarrhea. Serologic testing for Feline immunodeficiency virus and Feline leukemia virus were negative. Fine-needle aspirates of mesenteric lymph nodes revealed the presence of banana-shaped apicomplexan parasites. The cat died after 4 days of hospitalization. Postmortem polymerase chain reaction (PCR) analysis confirmed the presence of Toxoplasma gondii in all examined organs. Parasites were ex vivo isolated in outbred mice and subsequently transferred into cell culture. Genotyping, using genetic markers for SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico for PCR-restriction fragment length polymorphism, revealed infection with type II T. gondii displaying type II alleles at all loci except Apico, which exhibited a type I allele. This is the most frequently identified genotype among cats acting as definitive hosts in central Europe, but to the authors' knowledge, it has never been associated with systemic toxoplasmosis in an adult, immunocompetent cat.

Studies on the CPA cysteine peptidase in the Leishmania infantum genome strain JPCM5.

BACKGROUND: Visceral leishmaniasis caused by members of the Leishmania donovani complex is often fatal in the absence of treatment. Research has been hampered by the lack of good laboratory models and tools for genetic manipulation. In this study, we have characterised a L. infantum line (JPCM5) that was isolated from a naturally infected dog and then cloned. We found that JPCM5 has attributes that make it an excellent laboratory model; different stages of the parasite life cycle can be studied in vitro, it is accessible to genetic manipulation and it has retained its virulence. Furthermore, the L. infantum JPCM5 genome has now been fully sequenced. RESULTS: We have further focused our studies on LiCPA, the L. infantum homologue to L. mexicana cysteine peptidase CPA. LiCPA was found to share a high percentage of amino acid identity with CPA proteins of other Leishmania species. Two independent LiCPA-deficient promastigote clones (DeltaLicpa) were generated and their phenotype characterised. In contrast to L. mexicana CPA-deficient mutants, both clones of DeltaLicpa were found to have significantly reduced virulence in vitro and in vivo. Re-expression of just one LiCPA allele (giving DeltaLicpa::CPA) was sufficient to complement the reduced infectivity of both DeltaLicpa mutants for human macrophages, which confirms the importance of LiCPA for L. infantum virulence. In contrast, in vivo experiments did not show any virulence recovery of the re-expressor clone DeltaLicpaC1::CPA compared with the CPA-deficient mutant DeltaLicpaC1. CONCLUSION: The data suggest that CPA is not essential for replication of L. infantum promastigotes, but is important for the host-parasite interaction. Further studies will be necessary to elucidate the precise roles that LiCPA plays and why the re-expression of LiCPA in the DeltaLicpa mutants complemented the gene deletion phenotype only in in vitro and not in in vivo infection of hamsters.