False-Positive Anti-Toxoplasma Fluorescent-Antibody Tests in Patients with Antinuclear Antibodies

The indirect fluorescent-antibody (IFA) method for diagnosis of toxoplasmosis is widely used and is considered to be as specific as the Sabin-Feldman dye test. After observing a patient with systemic lupus erythematosus (SLE) who had a positive toxoplasma IFA test but a negative dye test, we studied sera with high titers of antinuclear antibodies from 16 SLE patients and from 2 with rheumatoid arthritis for Toxoplasma antibodies in the immunoglobulin G and M (IgG and IgM) IFA tests and the dye test. Results of these tests were compared with titers of antinuclear antibodies, precipitating antibodies to single-strand deoxyribonucleic acid (DNA), and binding antibodies by use of DNA labeled with (3)H-actinomycin D. Of 18 patients, 11 had IgG and 4 had IgM IFA Toxoplasma antibodies; only 2 had antibodies detectable in the dye test. The immunofluorescence patterns in the Toxoplasma IFA test were indistinguishable from those obtained in patients with toxoplasmosis without antinuclear antibodies. Absorption of SLE sera with DNA did not result in a decrease in Toxoplasma IFA titers. When SLE sera were absorbed with live T. gondii, a marked drop in IgG IFA titer was observed as well as a decrease in titers of antinuclear antibodies and (3)H-DNA binding. Treatment of Toxoplasma cells with deoxyribonuclease and ribonuclease did not decrease their fluorescence. These results suggest that T. gondii nuclear antigens can absorb antinuclear antibodies but do not have exposed substrates for deoxyribonuclease. Tests in which organisms containing "nuclear" antigens for IFA detection of antibodies to these organisms are used may result in "false-positives" with sera containing antinuclear antibodies.     

Experimental Toxoplasma gondii infection in striped skunk (Mephitis mephitis)

Twenty-three striped skunks (Mephitis mephitis) without demonstrable antibodies in 1:25 serum dilution in the modified agglutination test (MAT) were fed sporulated Toxoplasma gondii oocysts (9 skunks) or tissue cysts (10 skunks), and 4 skunks (controls) were not fed T. gondii. Skunks were bled before feeding T. gondii, 10 and 23- 25 days postinoculation (PI). All 9 seronegative skunks fed oocysts died of acute toxoplasmosis between 7 and 19 days PI; T. gondii tachyzoites were found in histological sections of many tissues. One of the 10 skunks fed tissue cysts and 1 of the 4 controls also died of acute toxoplasmosis days 19 and 20 PI; these animals probably became infected by ingestion of unexcysted oocysts passed in feces of skunks fed oocysts that were housed in the same room that skunks fed tissue cysts were housed. The remaining 9 skunks fed tissue cysts and the 3 controls developed only a mild illness and were killed in good health on days 23-25 PI. Antibodies to T. gondii were not found in 1:25 serum dilution of any of the 19 of 23 skunks that were alive on day 10 PI; 12 of 13 skunks had antibodies (MAT 1:80 or higher) on the day they were killed. Antibodies were not found in 1 skunk. Results indicate that skunks can develop IgG antibodies to T. gondii within 3 wk PI, and primary toxoplasmosis can be fatal in skunks.     

Experimental tissue cyst induced Toxoplasma gondii infections in dogs

Tissue cyst induced Toxoplasma gondii infections were examined in 2 beagle dogs orally inoculated with tissue cysts. Neither dog developed clinical signs of toxoplasmosis. Both dogs developed low antibody titers to T. gondii. The MAT and IFAT were superior to the LAT and IHT tests for detecting antibodies to T. gondii.     

Comparative study of oxidative stress and antioxidative markers in patients infected with Toxoplasma gondii

Recently, oxidative stress and antioxidative compounds have been described as potential biomarkers. However, there is no consensus on the most appropriate oxidative and antioxidative biomarkers for patients with Toxoplasma gondii. In the present study, we evaluated the levels of lipid, protein, DNA oxidative damage and antioxidants in samples from patients infected with T. gondii with and without ocular toxoplasmosis. The levels of MDA, TBARS, micronuclei, carbonyl, GSH, vitamin C and vitamin E were measured on samples from 8 patients positive for T. gondii antibodies with ocular toxoplasmosis (OT), 20 patients positive for T. gondii antibodies without ocular toxoplasmosis (non OT), and 12 healthy individuals negative for T. gondii antibodies. The levels of MDA, TBARS, carbonyl and micronuclei were significantly higher in non OT patients, while MDA and TBARS levels were lower in OT patients. In contrast, the antioxidative factors, GSH and vitamin E levels were significantly lower in non OT patients, while vitamin C was lower in non OT and OT patients. Additionally, non OT patients were indicated to be high producers of oxidative markers (TBARS, MDA, micronuclei and carbonyl), while control group was indicated to be high producer of antioxidative markers (GSH, vitamins C and E). However, OT patients were not found as high producers of oxidative nor antioxidative markers. Our results provide a starting point of possible markers to better understand the disease pathogenesis in patients infected with T. gondii. Additional studies are needed to clarify the potential contribution of oxidative and antioxidative markers in these patients population.     

Passive immunization protects guinea pigs from lethal toxoplasma infection

Abstract The cellular and humoral interactions that contribute to protective immunity in toxoplasmosis were studied by adoptive transfer of selective cell populations or immune serum and its fractions into normal syngeneic strain 2 guinea pigs. The results of this study with the RH strain of Toxoplasma gondii confirm and extend the findings of previous studies by showing that the passive transfer of parasite-sensitized T cells or of immune serum from previously infected donors protected recipient guinea pigs against lethal toxoplasmosis. An additional key finding was that similar levels of complete protection against lethal infection occurred in guinea pigs receiving partially purified anti- Toxoplasma immunoglobulins or immune cells that had been enriched for B cells prior to transfer. Cells residing in the spleen, lymph nodes and peritoneal cavity, but not the thymus, were equally effective in conferring immunity to challenged recipients. In addition, cell titration experiments revealed that guinea pigs could survive T. gondii infection by infusing them with as little as 2 × 10⁷ sensitized T cells or B cells. Unlike protection mediated by T cells, protection against lethal disease occurring in the B cell recipients was associated with the formation of Toxoplasma antibodies. These findings illustrate the major role of both humoral and cell-mediated immunity in affording protection against toxoplasmosis based on a guinea pig model of the human disease.     

Detection of Toxoplasma gondii DNA in milk of dairy cows from southern Brazil

Consumption of unpasteurized cow's milk may be a transmission route for some pathogenic microorganisms, but there is little information about the risk of Toxoplasma gondii infection. Blood and milk samples were collected in a paired and random fashion from 106 dairy cows and bulk-tank milk samples were also collected from each of the six farms, in southern Brazil. Serum anti-T.gondii antibodies (IgG) were detected by an indirect fluorescent antibody test (IFAT) with a cutoff point of 1:64. Nested PCR targeting the ITS1 was performed on milk samples to detect the Sarcocystidae family, confirmed to be T.gondii by Sanger sequencing. The occurrence of anti-T.gondii antibodies in the herds was 14.1%, (15/106) with seropositive cows in all herds. Antibody titers in positive samples ranged from 64 to 128. T.gondii DNA was detected in 2.8% (03/106) of the milk samples. The ITS1 sequences generated in this study were ON809793 - ON809794 and the sequencing revealed 98–100% identity with T. gondii DNA sequences deposited in GenBank. All cows PCR positive for T.gondii in milk were negative for IgG antibodies in serum, suggesting that naturally infected cows may shed T. gondii in milk in the acute phase of infection. The results of this study demonstrate that T. gondii DNA may be detected in raw cow's milk, so the potential risks of lactogenic infection should be considered. The presence of T. gondii DNA in milk does not confirm that the protozoa are viable and infective, and further investigations into the role of cow's milk in the epidemiology of toxoplasmosis are needed.     

High prevalence of aspartylglycosaminuria among school-age children in eastern Finland

Summary A high prevalence of the lysosomal storage disease aspartylglycosaminuria was found in a study of four birth cohorts of 12882 children in eastern Finland. Using school achievement tests and registers of mentally retarded individuals, 178 mentally retarded children were identified. Randomized urine samples from 151 of the 178 retarded children and from 101 healthy children were analyzed quantitatively for aspartylglucosamine by high-performance liquid chromatography. The results identified three affected individuals in the retarded group indicating an exceptionally high prevalence of aspartylglycosaminuria (1:3643) in the study population, consistent with a carrier frequency of 1:30. The 95% confidence limits for the prevalence are 1:4 352-1:16389. This is the highest prevalence described for any glycoproteinosis in any population and comparable to the incidence figures of the most common lysosomal storage diseases, Gaucher disease type I and Tay-Sachs disease among Ashkenazi Jews. In the study group, aspartylglycosaminuria was, after trisomy 21 (n = 19) and the fragile X syndrome (n = 6), the most common genetic cause for mental retardation.     

The use of commercial immunoenzyme kits for the diagnosis of toxoplasmosis

The results of comparative study of sera obtained from donors and from several groups of patients with suspected toxoplasmosis are presented. The study has been carried out with the use of commercial enzyme immunoassay (EIA) kits: Toxoplasma gondii IgG EIA and Toxoplasma gondii IgM EIA manufactured by Labsystems (Finland), Sevatest ELISA IgG Toxo Micro I manufactured by Sevac (Czechoslovakia). Statistical processing of the results has confirmed the identity of these kits. The necessity of using evaluation criteria (the separation point, the scale for the interpretation of results) when working with the Sevac kits is emphasized. Comparative evaluation of antibody profiles in the sera under test suggests that the titer less than 1:1600 should be regarded as the separation point for these kits. IgM antibodies to T. gondii have been found only in 22% of patients with high titers of IgG antibodies.     

Natural toxoplasma gondii infections in European brown hares and mountain hares in Finland: proportional mortality rate, antibody prevalence, and genetic characterization

In material examined postmortem in Finland from May 2006 to April 2009, acute generalized toxoplasmosis was the immunohistochemically confirmed cause of death in 14 (8.1%) of 173 European brown hares (Lepus europaeus) and four (2.7%) of 148 mountain hares (Lepus timidus). Sera from 116 of the European brown hares and 99 of the mountain hares were screened with a commercial direct agglutination test for Toxoplasma gondii-specific IgG antibodies at a dilution of 1:40. All sera from cases of fatal toxoplasmosis had high titers of antibodies reactive to T. gondii. In contrast, none of 107 European brown hares and four (4%) of 96 mountain hares that died of other causes were antibody-positive. The proportional mortality rates and the T. gondii antibody prevalences among noncases differed significantly between the two host species (P<0.05). Direct genetic characterization of the causative agent was performed on DNA extracted from formalin-fixed, paraffin-embedded tissue of the hares with fatal toxoplasmosis. Based on the results with six microsatellite markers (B18, TUB2, TgM-A, W35, B17, and M33; all six in 15 cases and four in three cases), all the cases were caused by T. gondii genotype II; the size of the PCR product at the seventh marker (M48) varied (213-229 base pairs). The presence of T. gondii genotype II, which is endemic in Europe, is now confirmed in Finnish wildlife: Natural infections with T. gondii parasites belonging to this widespread genotype caused fatal generalized toxoplasmosis in the two species of wild hares.     

Seroprevalence of Toxoplasma gondii in wild toque macaques (Macaca sinica) at Polonnaruwa, Sri Lanka

From a natural population that inhabits the dry evergreen forest at Polonnaruwa, serum samples of 170 toque macaques were examined for antibodies to Toxoplasma gondii by the modified agglutination test. Of these, 21 (12%) were found with titers of 1:16 in 9, 1:32 in 9, 1:256 in 1, 1:1,024 in 1, and 1:4,096 in 1. There was no evidence of maternal transmission of antibodies or congenital toxoplasmosis. None of the infected macaques died within 1 yr after sampling. Toxoplasma gondii infection was closely linked to human environments where domestic cats were common. Macaques having frequent contact with human settlements showed a significantly greater (P < 0.0001) prevalence (19% infected) than macaques restricted to forest habitat, none of which was infected. Although infection with T. gondii has been noted in several species of Asian primates, this is the first report of T. gondii antibodies in toque macaques (Macaca sinica) that are endemic to the island of Sri Lanka.     

Age-dependent impairment of functional helper T cell responses to immunodominant epitopes of Toxoplasma gondii antigens in congenitally infected individuals

Human infection with Toxoplasma gondii is generally asymptomatic in immunocompetent adults while it causes significant morbidity in congenitally infected children. Cell mediated immunity plays the main role in host resistance to T. gondii infection and a Th1 cytokine profile is necessary for protection and control of infection. The present work focused on comparing the helper T cell response to the GRA1 antigen of the parasite between children with congenital toxoplasmosis and healthy adults with acquired infection. We demonstrated that in young children with congenital infection the specific T cell response to parasite antigens is impaired and that such hypo-responsiveness is restored during childhood. Also, we provided clear evidence that in individuals with congenital toxoplasmosis the acquisition of functional helper T cell responses is disease-unrelated and indistinguishable in terms of strength, epitope specificity, and cytokine profile from the corresponding responses in immunocompetent adults with asymptomatic acquired T. gondii infection.     

Experimental Toxoplasma gondii infection in grey seals (Halichoerus grypus)

Laboratory-reared animals were used to assess the susceptibility of seals (Halichoerus grypus) to Toxoplasma gondii infection. Four seals were each orally inoculated with 100 or 10,000 oocysts of T. gondii (VEG strain), and another 4 seals served as negative controls. Occasionally, mild behavioral changes were observed in all inoculated seals but not in control animals. A modified agglutination test revealed the presence of antibodies to T. gondii in sera collected from inoculated seals and mice inoculated as controls. No evidence of the parasite was found on an extensive histological examination of seal tissues, and immunohistochemical staining of tissue sections from inoculated seals revealed a single tissue cyst in only 1 seal. Control mice inoculated with 10 oocysts from the same inoculum given to seals became serologically and histologically positive for T. gondii. Cats that were fed brain or muscle tissue collected from inoculated seals passed T. gondii oocysts in feces. This study demonstrates that T. gondii oocysts can establish viable infection in seals and supports the hypothesis that toxoplasmosis in marine mammals can be acquired from oocysts in surface water runoff and sewer discharge.     

Prevalence of antibodies to Toxoplasma gondii in ostriches (Struthio camelus)

Serum samples from 973 ostriches (Struthio camelus) in Canada were examined for antibodies to Toxoplasma gondii by the modified agglutination test incorporating mercaptoethanol and formalin-fixed whole tachyzoites. Twenty-eight (2.9%) of the 973 birds were found to be seropositive for antibodies to T. gondii at titers of 1:25 in 15 birds, 1:50 in 12 birds, and 1:500 in 1 bird. This is the first record of T. gondii exposure in ostriches, and it supports the hypothesis that all avian species are susceptible to Toxoplasma infection. Nevertheless, the results of this study suggest that the risk of acquiring toxoplasmosis from ostriches as a food source is low.     

Lacrimal secretory IgA in active posterior uveitis induced by Toxoplasma gondii

It is quite difficult to diagnose active toxoplasmosis in patients with ocular toxoplasmosis. Active posterior uveitis presumably due to Toxoplasma gondii infection (APUPT) is seldom produced during a prime-infection; hence most patients do not show high IgM antibodies. High levels of IgA have been described in active toxoplasmosis. The purpose of this study was to investigate possible association between APUPT and the specific anti-parasite sIgA in tears. The study was carried out as case-control. Tears of 25 clinically confirmed APUPT patients and 50 healthy control subjects were analyzed. All were IgG seropositive. Specific sIgA was determined by ELISA assay using T. gondii RH strain crude extract. Anti-T. gondii sIgA was found in 84% of the cases and in 22% of the control subjects. The intensity of the reaction was higher in APUPT cases (P = 0.007). There was strong association between APUPT patients and lacrimal sIgA (odds-ratio 18.61, P = 0.0001). ELISA test sensitivity was 84% and specificity 78%. Our data suggest that anti-T.gondii secretory IgA found in tears may become an important marker for active ocular toxoplasmosis.     

Two viable Toxoplasma gondii isolates from red-necked wallaby (Macropus rufogriseus) and red kangaroo (M. rufus)

Wallabies and kangaroos are susceptible to Toxoplasma gondii. However, little information concerning T. gondii infection in captive macropods is available. Three dead macropods collected from a zoo exhibited no clinical symptoms associated with toxoplasmosis. Heart fluids were tested for T. gondii antibodies using a modified agglutination test. T. gondii DNA samples derived from macropod tissues were tested by Polymerase Chain Reaction. Viable T. gondii were isolated from myocardium of macropods via mouse bioassay. Tissues (brain, lungs, or mesenteric lymph nodes) from T. gondii-positive mice were seeded into Vero cell culture flasks. The virulence of the isolated T. gondii strains was evaluated in Swiss mice. The DNA from T. gondii tachyzoites obtained from cell cultures was characterized by 10 PCR-RFLP markers and the virulence genes, ROP18 and ROP5. T. gondii antibodies were identified in two of the three macropods (Macropod^#5 and ^#7). T. gondii DNA was obtained from the heart and lungs of Macropod^#7. Two viable T. gondii strains were isolated from the myocardium of Macropus rufogriseus (Macropod^#5) and M. rufus (Macropod^#7) via mouse bioassay and designated as TgRooCHn2 and TgRooCHn3, respectively. TgRooCHn2 was ToxoDB genotype^#3, and TgRooCHn3 was ToxoDB genotyp^#2. Both 10⁴ TgRooCHn2 and TgRooCHn3 tachyzoites had intermediate virulence in mice. M. rufogriseus (Macropod^#5) and M. rufus (Macropod^#7) may have been in the initial stages of toxoplasmosis, due to a recent T. gondii infection with oocysts. This study is the first to document the T. gondii ToxoDB^#3 isolate in macropods. T. gondii infection in captive macropods indicates the urgent need to control the transmission of this parasite in the environment, food and water of zoo animals.     

Identification of a sporozoite-specific antigen from Toxoplasma gondii

Reduction of risk for human and food animal infection with Toxoplasma gondii is hampered by the lack of epidemiological data documenting the predominant routes of infection (oocyst vs. tissue cyst consumption) in horizontally transmitted toxoplasmosis. Existing serological assays can determine previous exposure to the parasite, but not the route of infection. We have used difference gel electrophoresis, in combination with tandem mass spectroscopy and Western blot, to identify a sporozoite-specific protein (T. gondii embryogenesis-related protein [TgERP]), which elicited antibody and differentiated oocyst- versus tissue cyst-induced infection in pigs and mice. The recombinant protein was selected from a cDNA library constructed from T. gondii sporozoites; this protein was used in Western blots and probed with sera from T. gondii -infected humans. Serum antibody to TgERP was detected in humans within 6-8 mo of initial oocyst-acquired infection. Of 163 individuals in the acute stage of infection (anti- T. gondii IgM detected in sera, or < 30 in the IgG avidity test), 103 (63.2%) had detectable antibodies that reacted with TgERP. Of 176 individuals with unknown infection route and in the chronic stage of infection (no anti- T. gondii IgM detected in sera, or > 30 in the IgG avidity test), antibody to TgERP was detected in 31 (17.6%). None of the 132 uninfected individuals tested had detectable antibody to TgERP. These data suggest that TgERP may be useful in detecting exposure to sporozoites in early T. gondii infection and implicates oocysts as the agent of infection.     

Pathology of experimental toxoplasmosis in eastern barred bandicoots in Tasmania

Wild-caught eastern barred bandicoots (Perameles gunnii) initially seronegative to Toxoplasma gondii, were inoculated orally with approximately 100 T. gondii oocysts. The bandicoots were maintained in indoor pens under laboratory conditions and observed daily. Serial blood samples were tested for agglutinating antibodies to T. gondii. Inoculated bandicoots died 15 and 17 days post infection. A rise in Direct Agglutination Test (DAT) titres was detected at the time of death (1:256, 1:64 respectively). Clinical observations, serological changes, gross findings at necropsy, and histopathological changes were consistent with acute toxoplasmosis. The findings indicate that eastern barred bandicoots are likely to die from primary T. gondii infection, often even before detectable antibodies are produced, reinforcing the significance of toxoplasmosis as a potential contributor to the reduction in numbers of wild populations of eastern barred bandicoots.     

Prevalence of antibody to Toxoplasma gondii in the moose (Alces alces americana Clinton) of Nova Scotia, Canada

The prevalence of antibodies to Toxoplasma gondii was investigated in the sera of 125 moose taken by hunters in 5 countries of Nova Scotia. Nineteen of these sera (15%) were positive by the indirect passive hemagglutination test, with titers above 1:64. This study adds further evidence to the prevalence of antibodies to T. gondii in the wildlife, extending this evidence to eastern Canada. The possibility that humans may acquire toxoplasmosis by ingesting undercooked infected meat from game animals is supported by the results of this investigation; the implications of these findings to public health are obvious.     

Prevalence of antibodies to Toxoplasma gondii in raccoons (Procyon lotor) from an urban area of Northern Virginia

Raccoons (Procyon lotor) are intermediate hosts for Toxoplasma gondii, and clinical toxoplasmosis in raccoons has been reported. A 2-yr serological survey was conducted to determine the prevalence of antibodies to T. gondii in raccoons collected from Fairfax County, Virginia, a suburban/urban area outside Washington, D.C. Serum samples from 256 raccoons were examined for T. gondii antibodies at a 1:50 dilution using the modified direct agglutination test. Results indicated that 216 (84.4%) of the raccoons had been exposed to T. gondii. Our results indicate that raccoons in this area of Virginia are frequently exposed to T. gondii. Domestic cats were common in the study area and may have served as a source of oocysts for raccoons and the food items of raccoons.     

The frequency of X-linked mental retardation

600 mentally retarded children, 339 boys and 261 girls aged 5-9 years were examined. Boys to girls ratio (B:G) was found in the total group to be 1.3. Among 257 children with mental retardation of confirmed genetic origin B:G = 1.4, in 129 children with confirmed exogenous defects B:G = 1.04. The significant prevalence of boys over girls was characteristic of children with monogenic forms. The frequency of X-linked mental retardation in the total group, in all mentally retarded boys and in boys with genetic forms was 12.5 +/- 1.3%, 22.1 +/- 2.2% and 28.5 +/- 2.8%, respectively. The frequency of X-linked mental retardation was higher in boys with genetic forms of imbecility.