Characterization of Toxoplasma gondii from the feces of naturally infected cats

Feces of 1,000 cats from a humane shelter in Columbus, Ohio, were examined microscopically for oocysts of Toxoplasma gondii and by inoculation into mice. From the first 541 cats examined, oocysts of Toxoplasma were found in the feces of seven cats but in none of the remaining 459 cats. Results of the dye test in these seven cats showed titers of antibody of less than 1:2 in four cats, and of 1:8, 1:6, and 1:32 in the remaining three cats. The pathogenicity and infectivity of oocysts and cysts of all seven strains were compared in mice after oral and intraperitoneal inoculations. Oocysts and cysts were more pathogenic when administered by the oral route than by the intraperitoneal route. The cysts were less pathogenic than the oocysts. Excellent cross-immunity between six of these seven feline strains and the M-7741 strain was deomonstrated in cats by the fact that oocysts were not shed in feces of cats challenged with cysts of homologous or heterologous strains.     

Low seroprevalence of Toxoplasma gondii in feral pigs from a remote island lacking cats

Serum samples from 1,264 feral pigs from Ossabaw Island, Georgia were initially screened for antibodies to Toxoplasma gondii by the modified agglutination test (MAT) using whole-formalinized tachyzoites and mercaptoethanol. Seropositive samples were also tested by the Sabin-Feldman dye test, the latex agglutination test (LAT), and the indirect hemagglutination test (IHAT). Ossabaw Island is a remote, barrier island located southeast of Savannah, Georgia. Antibodies to T. gondii were found in 11 (0.9%) of 1,264 pigs. The antibody titers were 1:20 (1 pig), 1:80 (2 pigs), 1:160 (2 pigs), 1:320 (4 pigs), and 1:640 (2 pigs) by the MAT, and 1:8 (2 pigs), 1:16 (3 pigs), 1:32 (1 pig), 1:64 (2 pigs), 1:128 (1 pig), and > or = 1:256 (2 pigs) by the Sabin-Feldman dye test. By the LAT, 5 pigs had a titer of > or = 1:64 and by the IHAT all 11 pigs had a titer of < 1:64. Antibodies (MAT titer, > or = 1:25) were found in 31 (18.2%) of 170 feral pigs from mainland Georgia. This seroprevalence on the mainland was significantly higher (P < 0.0001) as compared on Ossabaw Island. The markedly low prevalence of T. gondii on Ossabaw Island was attributed to the virtual absence of cats on the Island; only 1 domestic cat was known to be present.     

Loss of stages after continuous passage of Toxoplasma gondii and Besnoitia jellisoni

Toxoplasma gondii, passed from mouse to mouse in the tachyzoite stage for 30-35 generations, developed cysts, which when fed to cats, failed to produce oocysts. Besnoitia jellisoni, passed similarly for 20 generations, lost the capacity to form cysts. These phenomena are explained by a loss of genomes or gene products during the rapid passage selecting for tachyzoites.     

Inhibition of Toxoplasma gondii replication in IFN-gamma-activated human intestinal epithelial cells

A therapeutic trial, involving 130 Schistosoma mansoni-infected children, with no previous history of antischistosomal treatment, was carried out to evaluate the efficacy of two different dose regimens of praziquantel. The study was carried out because low cure rates were described in this recently established (1990) S. mansoni focus in northern Senegal, following treatment with a standard dosage of 40 mg/kg. The subjects were randomly allocated into two groups: one group (1) received 40 mg/kg in one oral dose, the other group (2) was treated with two oral doses of 30 mg/kg at a 6-hr interval. Parasitologic examination and circulating anodic antigen (CAA) detection were performed before, 10 days, three, six, and 21 weeks after chemotherapy. No significant differences in cure rates were found between the two groups. Six weeks after treatment, 34% and 44% of the individuals were found to be stool negative in group 1 and group 2, respectively. However, only 10-15% became completely negative according to the serum CAA antigen assay. Mean egg counts were reduced by 99% in both groups. Antigen detection confirmed the parasitologic results. Fewer side effects were observed in the group treated with 2 x 30 mg/kg, which may be explained by split dosage administration. Our study shows that the low cure rates observed in this area could not be improved by using a higher dosage of praziquantel.     

Genotypic analysis of Toxoplasma gondii isolates from pigs

To determine the prevalence of the 3 primary clonal lineages of Toxoplasma gondii (strain types I, II, and III) in a potential food source of infection for humans, we analyzed 43 isolates of T. gondii that had been collected from pigs at an abattoir in Iowa. Parasites were harvested as in vitro-grown tachyzoites, and their genotypes were determined at the SAG1 and SAG2 loci. On the basis of the allele identified at the SAG2 locus, isolates were grouped into 1 of the 3 primary lineages. Type II strains were by far the most prevalent, accounting for 83.7% of the isolates. The type III genotype was identified in only 16.3% of the isolates. These prevalences differ significantly from a previous sampling of isolates from animals but are similar to the frequencies with which they occur in human disease cases. Similar to the previously characterized strain P89, strains P62 and P105 appeared to have recombinant genotypes. The type I genotype was not identified in the isolates from pigs although these strains have previously been shown to account for approximately 10-25% of toxoplasmosis cases in humans.     

Isolation of developmentally regulated genes from Toxoplasma gondii by a gene trap with the positive and negative selectable marker hypoxanthine-xanthine-guanine phosphoribosyltransferase

For more than 60 years, evolutionary cytogeneticists have been using naturally occurring chromosomal inversions to infer phylogenetic histories, especially in insects with polytene chromosomes. The validity of this method is predicated on the assumption that inversions arise only once in the history of a lineage, so that sharing a particular inversion implies shared common ancestry. This assumption of monophyly has been generally validated by independent data. We present the first clear evidence that naturally occurring inversions, identical at the level of light microscopic examination of polytene chromosomes, may not always be monophyletic. The evidence comes from DNA sequence analyses of regions within or very near the breakpoints of an inversion called the 2La that is found in the Anopheles gambiae complex. Two species, A. merus and A. arabiensis, which are fixed for the "same" inversion, do not cluster with each other in a phylogenetic analysis of the DNA sequences within the 2La. Rather, A. merus 2La is most closely related to strains of A. gambiae homozygous for the 2L+. A. gambiae and A. merus are sister taxa, the immediate ancestor was evidently homozygous 2L+, and A. merus became fixed for an inversion cytologically identical to that in A. arabiensis. A. gambiae is polymorphic for 2La/2L+, and the 2La in this species is nearly identical at the DNA level to that in A. arabiensis, consistent with the growing evidence that introgression has or is occurring between these two most important vectors of malaria in the world. The parallel evolution of the "same" inversion may be promoted by the presence of selectively important genes within the breakpoints.     

Nucleotide sequence of ToxPK1 gene from Toxoplasma gondii

We report here for the first time a complete nucleotide sequence (6.8 kb) of a protein kinase gene (ToxPK1) from the obligate intracellular protozoan parasite of man, Toxoplasma gondii. This gene comprising putatively of 9 exons and 8 introns forms the Toxoplasma gene with the largest number and size of introns reported so far. The predicted protein with 508 amino acids contains the 15 invariant residues as well as the characteristic motifs specific to protein serine/threonine kinases. Homology-based computational comparisons suggested that TOXPK1 belongs to or closely resembles the SNF1 subfamily of protein-serine/threonine kinases. Based on the functions of SNF1 homologs in other organisms and our RT-PCR results, it is likely that TOXPK1 may be transiently expressed to up-regulate glycogen biosynthesis during the development of tachyzoites into bradyzoites.     

Toxoplasma gondii isolates of free-ranging chickens from Rio de Janeiro, Brazil: mouse mortality, genotype, and oocyst shedding by cats

Most isolates of Toxoplasma gondii can be grouped into 3 genetic lineages. In the present study, 67 isolates of T. gondii were obtained by bioassay in mice inoculated with brains and hearts of 96 asymptomatic chickens from an area highly endemic to human infection in Rio de Janeiro, Brazil. Of the 48 isolates genotyped using the SAG2 locus, 34 (70%) were of type I and 13 (27%) were of type III. No isolate of type II was recovered. Isolates from 1 chicken contained a type I and type III mixed infection, indicating natural multiparasite infection in the same animal. Cats fed mice infected with 11 type I strains shed 19-535 million oocysts in their feces, indicating that type I isolates can circulate in the environment.     

Consensus sequence of translational initiation sites from Toxoplasma gondii genes

Clinical analysis and the set of laboratory studies, performed in 25 patients one, two, three or six years after surviving acute period of trichinellosis, documented complaints in 22 patients (88.0%) in the form of muscle complaints (68.2%), cardiovascular complaints (45.4%), generalized weakness (40.9%) and fatigability (31.8%). No significant alterations were demonstrated in electrocardiographic records. In 71.4% examined patients lactic dehydrogenase activity was augmented. Presence of IgG antibodies against the E/S antigen of Trichinella sp. was disclosed in 24 (96%) patients, including 22 patients (88.0%), in whom high titres of the antibodies were found. Morphological studies on muscle tissue (performed in 5 patients) disclosed alterations typical of trichinellosis in 4 patients and presence of Trichinella larvae, calcified to a significant extent, in 2 patients. The long term persistence of IgG class antibodies against Trichinella antigen in patients who survived acute period of trichinellosis a few years earlier points to a chronic antigenic stimulation, probably reflecting progressive destruction of Trichinella larvae in muscle tissue. This may also be expressed in complaints reported by the patients. The problem requires further observations and clinical studies.     

Seroprevalence of Bartonella henselae and Toxoplasma gondii infections among pet cats in Kanagawa and Saitama Prefectures

Seroprevalence of Bartonella henselae and Toxoplasma gondii was investigated among 471 pet cats obtained from seven private animal hospitals in Kanagawa and Saitama Prefectures during the period from May 1994 to June 1995. 'Furthermore, 67 randomly selected from the 471 serum samples were examined for the feline immunodeficiency virus (FIV) antibody and feline leukemia virus (FeLV) antigen. The antibody to B. henselae was examined by an indirect immunofluorescent antibody test. T. gondii, FIV and FeLV infections in cats were detected with respective commercial kits. Of the cat serum samples tested, 43 (9.1%) were found to be seropositive for B. henselae and 41 (8.7%) for T. gondii. The B. henselae-positive rate (12.9%) of male cats was significantly higher than that (5.2%) of female cats. On the other hand, T. gondii-positive rate was 9.1% in male and 8.7% in female cats and there was no significant difference in the positivity between sexes. The positive rate in each hospital varied from 0 to 19.5% for B. henselae and 4.9 to 18.8% for T. gondii. The ages of B. henselae- and T. gondii-positive cats were distributed from < 1-year-old to 14-year-old and the seropositivity increased with age of cats. Of the 67 cat serum samples, 16 and 6 cases were positive for FIV and FeLV, respectively. There was no relationship between these viral and B. henselae infections in cats.     

Meningoencephalitis caused by Toxoplasma gondii diagnosed by isolation from cerebrospinal fluid in an HIV-positive patient

Toxoplasmosis of the central nervous system is a frequent opportunistic infection in AIDS patients, usually presenting as a mass brain lesion detected by computerized axial tomography scanning or magnetic resonance imaging. A case of diffuse meningoencephalitis with no radiological evidence of brain lesions is described. Diagnosis was made by culturing cerebrospinal fluid (CSF) on THP1 cells where tachyzoites of Toxoplasma gondii were demonstrated after 8 days of incubation by both direct observation and immunofluorescence. CSF examination with culture should be considered in AIDS patients with neurological signs and symptoms but without radiological evidence of cerebral lesions.     

The role of the cytoskeleton in host cell invasion by Toxoplasma gondii

The protozoan parasite Toxoplasma gondii provides a model system for studying invasion by intracellular parasites belonging to the phylum Apicomplexa. Taking advantage of the versatility of T. gondii for genetic and cell biological studies, we have shown that parasite motility and cell invasion are powered by an actin-myosin based motor in the parasite. Unlike bacterial cell uptake, parasite invasion does not involve significant alterations in the host cell cytoskeleton. Instead, invasion is an active process of penetration into the host cell by the parasite. The force for cell penetration is provided by a unique form of substrate-dependent motility termed gliding. Gliding motility is characterized by the rearward capping of surface membrane proteins that propels the parasite forward in a helical spiral. Both actin and myosin are localized beneath the plasma membrane in the parasite where they presumably combine to produce the force necessary for motility. During cell invasion, the rearward capping of cell surface receptors envelopes the parasite in a unique vacuole derived from the host cell plasma membrane. This system offers insights into force generation and motility in a simple organism that is also an important human pathogen.     

Stimulation of human T lymphocytes obtained from Toxoplasma gondii-seronegative persons by proteins derived from T. gondii

Toxoplasma gondii antigens are superantigens in mice. To investigate a superantigen effect in humans, lymphocytes from T. gondii-seronegative subjects were studied for proliferation to T. gondii antigens (TA). Marked cellular proliferation, predominantly of CD4+ lymphocytes, was apparent. TA elicited expansions of Vbeta-bearing lymphocytes in all subjects, but different Vbeta-bearing lymphocytes were expanded in different subjects in both CD4+ and CD8+ subpopulations. Cord blood cells also proliferated to TA. Previously fixed antigen-presenting cells were unable to present TA. Thus, T. gondii appears to produce a molecule(s) that induces polyclonal activation of human T cells and requires antigen processing to mediate this effect. That T. gondii does not appear to behave as a superantigen in humans is important in understanding the pathogenesis of T. gondii infection in immunocompromised hosts and in the design of anti-T. gondii vaccines.     

Toxoplasma gondii: an ultrastructural study of host-cell invasion by the bradyzoite stage

The invasion of Toxoplasma gondii tachyzoites and bradyzoites was followed in bovine kidney cells via electron microscopy. The process of invasion differed between bradyzoites and tachyzoites. In the early stages of entry there was evidence of localised formation of membrane projections in the host cell adjacent to the parasite. Parasite reorientation and rhoptry release appeared to be necessary for invasion; however, the tight junction could not be clearly discerned and there was no evidence of constriction or of any membrane shedding from the parasite. The resulting parasitophorous vacuole was smaller than the tachyzoite vacuole and parasites were frequently found to lie immediately under the host cell membrane. The vacuole was rapidly adapted by the release and formation of an intra-phagosomal membrane network, while the parasitophorous vacuole formed a relationship with host-cell endoplasmic reticulum.     

Determination of genotypes of Toxoplasma gondii strains isolated from patients with toxoplasmosis

To determine the genotypes of Toxoplasma gondii strains associated with human toxoplasmosis, we developed a sensitive approach for typing parasites grown from clinical samples by short-term in vitro culture. A newly described nested PCR assay was capable of amplifying genomic DNA from as few as five parasites in the presence of host tissues. Typing was based on DNA polymorphisms at the SAG2 locus, encoding tachyzoite surface antigen p22. Restriction fragment length polymorphisms in PCR-amplified SAG2 products were used to classify strains into one of the three major lineages of T. gondii. This approach was successfully used to determine the genotypes of 68 of 72 samples that had been previously isolated from patients with congenital, cerebral, and disseminated toxoplasmosis. Type II strains of T. gondii were found in a majority of the samples, accounting for 55 (81%) of the 68 toxoplasmosis cases. In contrast, type I and III strains were found in only 7 (10%) and 6 (9%) of the 68 cases, respectively. The results of this study support the previous finding that type II strains are most often associated with human toxoplasmosis. Nested PCR analysis at the SAG2 locus provides rapid assignment of T. gondii to a specific genotype that should be useful in analyzing a variety of clinical samples.     

Prevalence of Neospora caninum and Toxoplasma gondii antibodies in sera from camels from Egypt

Sera from camels from Egypt were examined by the direct agglutination tests incorporating mercaptoethanol for antibodies to Neospora caninum and Toxoplasma gondii. Antibodies to N. caninum were found in 6 of 161 camels in titers of 1:40 (2 camels) and 1:80, 1:160, 1:640, and 1:1280 in 1 camel each, using N. caninum formalin preserved whole tachyzoites as antigen. Antibodies to T. gondii were found in 17.4% of 166 camels in titers of 1:25 (3 camels), 1:50 (18 camels). and > 1:500 (8 camels) using T. gondii tachyzoites. All 6 camels with N. caninum antibodies had no T. gondii antibodies in 1:4 dilution of serum, indicating specificity of the reaction. This is the first report of N. caninum prevalence in Egypt.