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Characteristics of Toxoplasma gondii (strain GT-1) isolated from skeletal muscle of a goat are described. One oocyst, bradyzoite, or tachyzoite of GT-1 strain was lethal to mice. Toxoplasma tachyzoites were found in the peritoneal exudates and in the lungs 9 to 34 days after infection in mice inoculated intraperitoneally with tissues infected with strain GT-1.
Nine ponies were fed 100,000 infective Toxoplasma gondii oocysts and were given corticosteroid injections before and after feeding the T gondii oocysts. Titers to the Sabin-Feldman dye tests (1:2 to 1:16,384) developed within postinoculation days (PID) 7 to 21 and antibodies persisted to PID 133. Toxoplasma organisms were isolated from the tissues of 4 ponies (heart of 4, brain of 2, spinal cord of 3, diaphragm of 1, skeletal muscle of 1, liver of 1, kidneys of 1) killed between PID 36 and 63, but not from 5 ponies killed between PID 117 and 150. Seemingly, ponies are one of the more resistant species of animals to develop clinical toxoplasmosis.
Comparative investigations were carried out on the virulent and immunogenic properties of 5 cytogenic Toxoplasma strains (3 isolated from rabbits and 2--from fetuses of ewes that had miscarried). It was found that at shortening the interval of passage to 7--10 days and increasing the amount of the inoculate the virulence of the strains rose. Particularly virulent for albino mice proved the oocysts of T. gondii obtained after the feeding of kittens with cysts. The role was confirmed of Toxoplasma organisms in the etiology of abortions observed in experimentally infected guinea pigs and sheep. There were in the placenta and the brain of aborted fetuses characteristic histopathologic changes. Immunologic studies revealed that the isolated strains were immunologically identical.
Control of resistance to cyst burden following per-oral infection with Toxoplasma gondii has been mapped previously to a region of mouse chromosome 17 of approximately 140 kb. This region is contiguous with and contains the class I gene, Ld. Resistance to development of toxoplasmic encephalitis has also been reported to be controlled by genes in this region of H-2. TNF-alpha, D and L genes, as well as unidentified genes, are also in this region. The work described here was performed to identify definitively the gene(s) in this 140 kb region that confers resistance to cysts and encephalitis. The study demonstrates that relative resistance to T. gondii organisms and cyst burden in brain, and toxoplasmic encephalitis, 30 days following per-oral T. gondii infection is correlated absolutely with the presence of the Ld gene in inbred, recombinant, mutant and C3H.Ld transgenic mice. Mice with the Ld gene had lower cyst burdens and less encephalitis than those without the Ld gene. Specifically, 30 days after infection mice with the Ld gene had minimal perivascular inflammation and meningeal inflammation and very few Toxoplasma cysts or organisms in immunoperoxidase-stained preparations of their brains. Mice without the Ld gene had a similar pattern of inflammation, but in addition they had collections of inflammatory cells in the brain parenchyma. Free tachyzoites were found within these foci of inflammation and cysts were present in these areas as well as in contiguous areas without inflammatory cells. There were CD4+ and CD8+ T lymphocytes in the areas of inflammation and throughout the brain parenchyma. Mice that were resistant to cysts and encephalitis had little detectable brain cytokine mRNA expression, while mice that were susceptible had elevated levels of mRNA for a wide range of cytokines, consistent with their greater amounts of inflammation. The present work definitively demonstrates that a Ld-restricted response decreases the number of organisms and cysts within the brain and thereby limits toxoplasmic encephalitis and levels of interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha), interleukin-2 (IL-2), IL-6, IL-10, transforming growth factor-beta (TGF-beta), IL-1 alpha, IL1 beta and macrophage inhibiting protein (MIP) mRNA in the brain 30 days after per-oral infection.
Macrophages and monocytes have been shown to have an important role in the defence mechanism against Taxoplasma gondii infection. Antibodies in the presence of complement have been found capable of killing extracellular T. gondii. This study demonstrated that tachyzoites in the presence of antitoxoplasma antibodies with complement were detected in 39-58% of monocytes that had phagocytosed them and the mean number of Toxoplasma tachyzoites in this group was significantly low one hour post infection, while only 10-25% of monocytes phagocytosed Toxoplasma tachyzoites in absence of antibodies and complement with significant high number of replicated tachyzoites. This indicated that specific antibodies had a strong opsonizing action. Complement alone was weak in increasing the phagocytic activity.
An ELISA for detection of Toxoplasma gondii-specific IgA in feline serum was developed. A group of cats (n = 7) was inoculated orally with T gondii bradyzoites. Toxoplasma gondii-specific serum IgM, IgG, and IgA responses were followed sequentially by use of the ELISA for 34 weeks. Serum IgA was detected later than IgM or IgG, and was detected in most cats on week 34 after inoculation. None of the cats was seropositive for IgA during the oocyst-shedding period. A group of client-owned cats with suspected clinical toxoplasmosis and a group of healthy cats were tested for T gondii-specific IgA in serum. A trend toward association of T gondii-specific IgA in serum of cats with ocular disease was observed.
The most important cyst-forming coccidian parasites in human and veterinary medicine belong the genera of Toxoplasma, Neospora and Sarcocystis. Toxoplasma gondii shows its clinical relevance in congenital infections and opportunistic infections in immunodeficient patients. In veterinary medicine the parasite is predominantly the cause of important economic loss in livestock production. Neospora causes diseases resembling toxoplasmosis; neosporosis is one of the most important causes of bovine abortion in the US. Neospora caninum leads to myositis and paralysis in dogs. The potential implication of Neospora in toxoplasmosis-like diseases in humans is not yet known. Sarcocystis is usually a relatively harmless intestinal parasite in humans. Recent data from tropical areas suggest that man can also become an intermediate host for certain Sarcocystis species, which potentially represents a source of opportunistic infection and disease in areas with increasing HIV prevalence. In veterinary medicine, Sarcocystis causes muscle diseases and also abortion or myeloencephalitis with lethal outcome in certain animal species. Molecular-epidemiological investigations have resulted in a new understanding of biological and population-genetic mechanisms relevant to the disease. Recently developed molecular techniques, such as transfection in protozoan parasites, are presently used not only to elucidate molecular-pathogenetic events in the course of disease, but also to prepare potential new immuno-therapeutic tools for future vaccination against infection or disease.
Cats that have shed Toxoplasma gondii oocysts are considered to be immune to reshedding of oocysts. To investigate if this immunity persists in cats for 6 yr, 12 4-6-mo-old cats without T. gondii antibodies were inoculated orally with tissue cysts of the ME-49 strain (6 cats) and the TS-2 strain (6 cats) of T. gondii. All of them shed > or = 20 million oocysts between 4 and 13 days after feeding tissue cysts. Two cats became ill between 11 and 13 days after primary infection; 1 died on the 13th day, and the other had to be killed on the 11th day because of generalized acute toxoplasmosis. Toxoplasma gondii oocysts were not found on the hair of 10 cats examined 7 days after cats had shed millions of oocysts. On day 39 after primary infection, 5 cats (2 infected with the ME-49 strain and 3 infected with the TS-2 strain) were challenged orally with tissue cysts of the ME-49 strain. None of the challenged cats shed oocysts. One cat died due to causes unrelated to toxoplasmosis. Seventy-seven months after primary infection, the remaining 9 cats were challenged orally with tissue cysts of the P89 strain of T. gondii. Four of these 9 cats re-shed T. gondii oocysts; 3 of them had been challenged also at 39 days after primary infection. Two control cats housed together with chronically infected cats for 6 yr remained seronegative for T. gondii; both of these shed oocysts after challenge with the P89 strain.
The distribution of anti-toxoplasma antibodies among the aborigines in Malaysia and its association with other soil transmitted infections and eosinophilia were studied. A total of 415 serum samples were collected and tested by IFA test. Overall prevalence was 10.6%, lower than previously reported. The antibody titers showed a unimodal distribution peaking at 1:8 dilution. There was a higher proportion of high antibody titer (> 1:128) in the adult compared to the children with no significant difference in prevalence rate by sex. The pattern of infection does not differ from other soil transmitted infections and there was no association between raised Toxoplasma antibodies with eosinophilia.
We decided to determine whether Toxoplasma gondii-specific IgM or IgG is produced locally or is deposited in the aqueous humor of T gondii-naive cats after primary or secondary inoculation with T gondii. Cats were orally inoculated with T gondii tissue cysts during weeks 0 and 36. Aqueous humor and serum T gondii-specific IgM and IgG were measured, using ELISA, during weeks 0, 2, 4, 6, 8, 12, 16, 20, 26, 34, 38, 40, 42, 44, 48, 56, 62, 66, and 72 after primary oral inoculation. Total immunoglobulin-based Goldmann-Witmer coefficients were calculated to verify intraocular antibody production. Toxoplasma gondii-specific IgM was not detected in the aqueous humor of any cat. Data indicate that cats have transient local production of T gondii-specific IgG in the aqueous humor after primary and secondary oral inoculations with T gondii tissue cysts. The intraocular immune response to systemic T gondii infection may signal ocular recruitment of antigen-specific lymphocytes that function independently from the general humoral response to T gondii infection. Cautious interpretation of results that suggest intraocular production of T gondii-specific IgG in cats with uveitis is warranted.
During the hunting season of 1993, hearts from 28 black bears (Ursus americanus) from Pennsylvania were examined for Toxoplasma gondii infection. Serum samples from heart blood were examined for T. gondii antibodies by the use of the modified agglutination test (MAT), the Sabin-Feldman dye test (DT), the latex agglutination test (LAT), and the indirect hemagglutination test (IHA). One-hundred grams of myocardium from each bear were bioassayed in mice. Approximately 500 g of myocardium from 11 bears were fed to 11 cats, and feces from those cats were examined for T. gondii oocysts. Toxoplasma gondii MAT antibodies (> or = 1:40) were found in 22 bears in titers of 1:40 (4 bears), 1:80 (3 bears), 1:160 (7 bears), 1:320 (4 bears), and > or = 1:2,000 (4 bears). Antibodies to T. gondii by DT (> or = 1:10), LAT (> or = 1:32), and IHA (> or = 1:64) tests were found in 21, 9, and 6 bears, respectively. All 6 bears that did not have T. gondii antibodies in MAT were also negative in DT, IHA, and LAT. Viable T. gondii was isolated from 3 bears by bioassays in mice and from an additional 7 bears by bioassays in cats. All 10 bears that had viable T. gondii had MAT and DT antibodies but only 6 and 5 of them had antibodies by LAT and IHA, respectively.
The obligate intracellular parasite Toxoplasma gondii produces a nucleoside triphosphate hydrolase (NTPase) (nucleoside-triphosphatase, EC 3.6.1.15) activable by dithiol-containing compounds. We have isolated the genomic DNA for the NTPase from the RH strain of Toxoplasma and determined the nucleotide sequence of three tandemly arranged open reading frames termed NTP1, NTP2, and NTP3. We have also isolated and sequenced cDNAs for NTP1 and NTP3; no cDNA for NTP2 was obtained. The two cDNA clones encode proteins that are more than 97% identical at the amino acid level but significantly differ within two small domains, indicating the presence of NTPase isoforms. Both possess N-terminal signal sequences and two regions with partial homology to certain known ATP binding motifs: the glycine-rich loop common to many ATP binding proteins and the beta-phosphate binding domain found in the hexokinase-actin-hsp70 family. Antiserum against a NTP1-fusion protein immunoprecipitated NTPase activity from extracellular parasites that was increased in activity by treatment with dithiothreitol, confirming the identity of the cloned genes. By immunofluorescence, the NTPase is located in vesicular structures within the parasite, and in infected cells it is secreted into the vacuolar space and becomes partially associated with the parasitophorous vacuolar membrane. Since the vacuolar membrane is freely permeable to small molecules of < 1300 Da, host cell ATP may serve as a substrate for the NTPase and supply the energy for parasite-directed processes in the vacuolar space.
1. Newly developed diagnostic kit for toxoplasma infection, Platelia Toxo IgG, IgM and IgA were compared with IFA IgG and IgM, revealing high co-positivity between the results obtained by the two methods. 2. The serum containing antinuclear antibody, rheumatoid factor and so on, which are often regarded as cause of false positive results, showed no nonspecific reaction completely. 3. Platelia Toxo IgM and IgA were applied for the serum obtained from the patients who were laboratory infected and/or acquired toxoplasma infection, most of the specimen revealing IgM and IgA antibody at the acute stage of the infection.
Blood inoculation in mice showed that Toxoplasma organisms circulate in blood after 1 h of oocyst infection. Parasites were detected up to 15 days later and then disappeared from the bloodstream concomitantly with cyst formation in the brain, probably due to antibody presence. Immunosuppression caused by cortisone acetate treatment induced Toxoplasma bloodstream invasion in chronically infected mice and hamsters, causing death in some. Natural dissemination is discussed in relation with congenital toxoplasmosis. Induced immunosuppressive effect is compared with that produced by natural diseases such as Hodgkin, lymphoma, AIDS and others.
The obligate intracellular parasite Toxoplasma gondii resides within a specialized, nonfusogenic vacuole termed the parasitophorous vacuole. We are exploring the hypothesis that parasite proteins contained in dense core granules and secreted into the vacuole after cell invasion contribute to the modification of the parasitophorous vacuole membrane (PVM). The dense granule protein GRA3 associates preferentially with the PVM. Here we show that GRA3 liberated from dense granules is a soluble protein. Following secretion, one-third or more of GRA3 in the parasitophorous vacuole becomes stably associated with the vacuole membrane as judged by sucrose flotation gradients and Triton X-114 phase partitioning experiments. GRA3 cannot be removed from the membrane by extraction with sodium carbonate at pH 11.0, nor can it be labeled in infected cells with [1-14C]acetate. In sucrose velocity sedimentation gradients, GRA3 from vacuolar membranes solubilized in Triton X-100 sediments as an oligomer. Membrane-associated GRA3 cross-links to itself, migrating as a ladder of bands in SDS-polyacrylamide gel electrophoresis. No other parasite or cell proteins co-precipitate with cross-linked GRA3. We conclude that a portion of GRA3 secreted from the parasite as a soluble protein directly inserts into the vacuole membrane in an oligomeric form. This is the first report in Toxoplasma or related parasites of a protein which inserts into the vacuole membrane for some purpose other than to lyse that membrane.
A total of one hundred and fifty Saudi blood donors were examined for Toxoplasma gondii antibodies (IgG-IgM) by three serological techniques, 50 normal cross matched controls were subjected to the same examination. The number of positive donors for IgG by Indirect Haemagglutination test (IHA) was 36 (24%), by Enzyme Linked Immunosorbent Assay (ELISA) was 70 (46.7%), and by Immunofluorescent test (IFAT) 74 (49.3%). As to IgM the ELISA and IFAT showed 3 positive cases among the blood donors (2%). Rheumatoid factor and antinuclear antibodies were done for positive cases, and showed no significant cross reaction, IgG antibodies were significantly high in donors than in controls. The three tests were more or less dependable. However regarding simplicity, specificity, accuracy and time consumption factor IFAT was recommended for Toxoplasma IgG for all blood donors to avoid the risk of in infection.
A specific 32P labelled DNA probe of the cloned DNA fragment (1.1kb) was used in Southern hybridization to determine seven tachyzoites of toxoplasma isolated from dead fetus of malformation during the period of 1986-1989. The results show that seven parasite strains (SH) have identical bands in the same position (2.0kb) compared with RH, Zs2, CN strains, and this provides reliable evidence for identification of toxoplasma.
The proteins and antigens of nine strains of Toxoplasma gondii were analyzed by SDS-PAGE and immunoblotting. These strains including RH strain, Zs-2 strain, CN strain and SH1, SH2, SH3, SH4, SH5, SH8 strains, were obtained from other laboratories or isolated from deformity fetus in our laboratory. SDS-PAGE analysis revealed that these strains were very close in the major bands of proteins and differences were observed only in part. There were common components in antigens of all strains using IgG antibody prepared from high-titre rabbit antisera raised against RH strain of Toxoplasma.