Absorption of human serum antibodies against Toxoplasma gondii using Tachyzoites from the RH strain]

Absorption experiments to determine the number of T. gondii tachyzoites to extract the anti-Toxoplasma antibodies present in the human serum were carried out. The antibodies titer from each serum (n = 4) was determined by means of the Sabin-Feldman test (dye test). Ten millions of tachyzoites were sufficient to absorb the antibodies from sera presenting titer 1:16, 1:64 and 1:256. On the other side, the absorption of a 1:512 positive serum with 10 x 10(6) T. gondii decreased the titer to 1:64 and the dye test became negative when using 50 x 10(6) tachyzoites.     

Use of near-infrared raman spectroscopy to detect IgG and IgM antibodies against Toxoplasma gondii in serum samples of domestic cats.

Near-infrared Raman spectroscopy (NIRS) is a particularly promising technique that is being used in recent years formany biomedical applications. Optical spectroscopy has gained increasing prominence as a tool for quantitative analysis of biological samples, clinical diagnostic, concentration measurements of blood metabolites and therapeutic drugs, and analysis of the chemical composition of human tissues. Toxoplasmosis is an important zoonosis in public health, and domestic cats are the most important transmitters of the disease. This disease can be detected by several serological tests, which usually have a high cost and require a long time. The goal of this work was to investigate a new method to diagnosis Toxoplasma gondii infections using NIRS. In order to confirm antibody detection, 24 cat blood serum samples were analyzed by the Raman spectra, from which 23 presented positive serology to toxoplasmosis and one was a reference negative serum. Characteristic Raman peaks allowed differentiation between negative and positive sera, confirming the possibility of antibody detection by Raman spectroscopy. These results give the first evidence that this technique can be useful to quantify antibodies in cat sera.     

Prevalence of Toxoplasma gondii antibodies in dingoes

Serum samples from 62 dingoes (Canis familiaris dingo) trapped in five areas of southeastern New South Wales, Australia were tested for antibodies to Toxoplasma gondii. Six (10%) of the dingoes had direct agglutination test titers for T. gondii of greater than or equal to 1:64, and four of these animals had T. gondii-specific IgM, suggesting recent exposure.     

IgG antibodies to secretory component in normal human serum

While isolating free secretory component (FSC) by monoclonal antibody affinity chromatography, we demonstrated FSC-IgG complexes in human milk. We hypothesized that IgG antibody to secretory component (SC) might be transported into the milk from the serum. We therefore examined sera from 10 normal adults and 10 infants for IgG capable of binding to FSC in an enzyme-linked immunosorbent assay. Eight of 10 normal adult sera and nine of 10 infant sera demonstrated IgG binding to FSC with titers ranging from 1:54 to 1:4096. Quantitation of the IgG bound to FSC was hampered in adult sera by the binding of IgM and polymeric IgA to the FSC. Quantitation in five infant sera ranged from 0.5 to 6.4 micrograms/ml. A pepsin digest of an IgG fraction of serum demonstrated binding of the F(ab')2 fragments to the FSC. The specificity of the antibodies in human serum was evaluated by examining the binding to secretory IgA (sIgA) and FSC isolated from pooled human milk and polymeric IgA isolated from the ascitic fluid of a patient with an IgA myeloma. Eight of the 10 adults had antibody specific for FSC. Three of the eight, all female, also had antibody specific for sIgA. Two of the eight had antibody either to FSC and sIgA or to FSC plus an antibody that could bind to an epitope shared by sIgA and FSC. Competition experiments with monoclonal antibodies to human secretory component and sIgA were used to confirm and further define these specificities. The results of this study indicate that antibody to SC is common in normal adult and infant sera. The majority of antibodies seem to be directed against epitopes present on FSC but not on sIgA, which suggests sensitization to circulating or membrane-bound SC. The significance of these antibodies in normal human sera remains to be elucidated.     

Detection of antibodies in serum and cerebrospinal fluid to Toxoplasma gondii by indirect latex agglutination test and enzyme-linked immunosorbent assay.

Sensitivity of anti-Toxoplasma antibody (IgG) test by enzyme-linked immunosorbent assay (ELISA) was evaluated in comparison with indirect latex agglutination (ILA) using 2,016 paired human samples of serum and cerebrospinal fluid (CSF). The samples were collected from neurologic patients in Korea with mass lesions in central nervous system (CNS) as revealed by imaging diagnosis (CT/MRI). When the sera were screened for anti-Toxoplasma antibody by ILA, 76 cases(3.8%) were positive (1:32 or higher titers). In the paired samples of CSF, no positive reactions were observed. When ELISA was performed using PBS extract of Percoll purified tachyzoites as antigen, cut-off absorbance was determined as 0.40 for serum and 0.27 for CSF tests. The antibody positive rates by ELISA were 7.0% in serum and 5.6% in CSF. Of them, 40 cases (2.0%) showed positive reactions in both serum and CSF. The antibody positive rates were higher in groups older than 40 years. The rates were higher in male (4.7% by ILA, 8.3% by ELISA) than in female (2.2% by ILA, 5.0% by ELISA). The rates in CSF showed no such sex difference. ELISA showed twice higher positive rates when serum was tested, and was sensitive enough to detect specific antibodies in CSF. Etiologic relations between positive antibody tests and CNS lesions remained unknown.     

Prevalence of antibodies to Toxoplasma gondii in dogs from northeastern Portugal

Prevalence of antibodies to Toxoplasma gondii was investigated in 673 domestic dogs from northeastern Portugal, using the modified agglutination test (MAT) with 1 : 20 as cutoff for seropositivity; antibodies were found in 256 dogs (38.0%). Differences between seroprevalence levels in males (36.7%) and females (41.8%) and between pure-breed (42.1%) and mixed-breed dogs (35.2%) were not statistically significant. Multiple logistic regression analysis identified age above 12 mo (odds ratio [OR] = 4.0), chance of eating birds or small mammals (OR = 4.0), housing exclusively outdoors (OR = 1.5), home-cooked meals (OR = 3.0), and eating raw meat or viscera (OR = 7.7) as risk factors for the canine T. gondii infection. Some control measures are suggested based on these findings.     

Toxoplasma gondii antibodies in free-living African mammals.

Twelve species of free-living African mammals from Kenya, Tanzania, Uganda and Zambia were tested for antibodies to Toxoplasma gondii using the indirect hemagglutination test. Of 157 animals sampled, 20 (13%) were seropositive. T. gondii antibodies were detected in Burchell's zebra, (Equus burchelli), hippopotamus (Hippopotamus amphibius), African elephant (Loxodonta africana), defassa waterbuck (Kobus defassa), lion (Panthera leo), and rock hyrax (Procavia capensis), The highest titers were found in elephants, two having titers of 1:4096 and one of 1:8192. These results are discussed in relation to the maintenance of T. gondii among African wildlife.     

Detection of Toxoplasma gondii Oocysts in Drinking Water

The world’s largest outbreak of waterborne toxoplasmosis occurred in a municipality in the western Canadian province of British Columbia. When drinking water emerged as a possible source of infection during the outbreak investigation, a laboratory method was needed to attempt detection of the parasite, Toxoplasma gondii. The method developed was based on the current U.S. Environmental Protection Agency method for detection of Cryptosporidium oocysts. Collection of large-volume drinking water samples and cartridge filter processing were unchanged, although identification of Toxoplasma oocysts in the filter retentate was carried out by using a previously described rodent model. Validation of the method developed was tested by using oocysts from a well-characterized Toxoplasma strain.     

Molecular evolution of the vesicle coat component betaCOP in Toxoplasma gondii

Coatomer coated (COPI) vesicles play a pivotal role for multiple membrane trafficking steps throughout the eukaryotic cell. Our focus is on betaCOP, one of the most well known components of the COPI multi-protein complex. Amino acid differences in betaCOP may dictate functional divergence across species during the course of evolution, especially with regards to the evolutionary pressures on obligate intracellular parasites. A bioinformatic analysis of betaCOP amino acid sequences was conducted for 49 eukaryotic species. Cloning and sequence analysis of the Toxoplasma gondii betaCOP homologue revealed several amino acid insertions unique to T. gondii and one C-terminal insertion that is unique to apicomplexan parasites. These findings led us to investigate the possibility that betaCOP experienced functional divergence during the course of its evolution. Bayesian phylogenetic analysis revealed a tree consistent with pan eukaryote distribution and long-branch lengths were observed among the apicomplexans. Further analysis revealed that kinetoplast betaCOP underwent the most amount of change, leading to perhaps an overall change of function. In comparison, T. gondii exhibited subtle yet specific amino acid changes. The amino acid substitutions did not occur in the same places as other lineages, suggesting that TgbetaCOP has a role specific to the apicomplexans. Our work identifies 48 residues that are likely to be functionally important when comparing apicomplexan, kinetoplastid, and fungal betaCOP.     

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.     

Prevalence of antibodies to Toxoplasma gondii in wolverines from Nunavut, Canada

The prevalence of antibodies to Toxoplasma gondii was determined in blood and tissue exudates recovered from the spleens of 41 wolverines (Gulo gulo) collected in Nunavut, Canada, using a modified agglutination test (MAT). Antibodies to T. gondii were found in 17 (41.5%) of the 41 wolverines with MAT titers of 1:25 in 1, 1:50 in 4, 1:100 in 5, 1:200 in 6, and 1:400 in 1. This is the first report of antibodies to T. gondii in wolverines, and the results indicate that exposure is common.     

Detection and characterization of excretory/secretory proteins from Toxoplasma gondii by monoclonal antibodies

Excretory/secretory proteins (ESP) from Toxoplasma gondii were analyzed to define the function in the penetration process into host cells. Whole ESP obtained at 37 degrees C were composed of 15 bands with molecular mass of 110, 97, 86, 80, 70, 60, 54, 42, 40, 36, 30, 28, 26, 22, and 19 kDa. Five ESP of 86, 80, 42, 36, and 28 kDa were reacted with monoclonal antibodies (mAb), named as Tg386 (microneme), Tg485 (surface membrane), Tg786 (rhoptry), Tg378, and Tg556 (both dense granules), respectively. The ESP was released by a temperature-dependent/-independent manner and all at once whenever ready to pour out except Tg786. Each ESP was not exhausted within the parasite but the amount was limited. Tg786 was released continuously with increment, whereas Tg378 and Tg556 were ceased to release after 3 and 4 hr. Dense granular Tg378 and Tg556 were released spontaneously and constitutively before the entry into host cells also. The entry of T. gondii was inhibited by all the mAbs differentially. And the parasite deprived of ESP was inhibited to enter exponentially up to 90.1%. It is suggested that ESP play an essential function to provide appropriate environment for the entry of the parasite into host cells.     

Protease activity of 80 kDa protein secreted from the apicomplexan parasite Toxoplasma gondii

This study describes the characterization of 80 kDa protease showing gelationlytic property among three proteases in the excretory/secretory proteins (ESP) from Toxoplasma gondii. The protease activity was detected in the ESP but not in the somatic extract of RH tachyzoites. This protease was active only in the presence of calcium ion but not other divalent cationic ions such as Cu(2+), Zn(2+), Mg(2+), and Mn(2+), implying that Ca(2+) is critical factor for the activation of the protease. The 80 kDa protease was optimally active at pH 7.5. Its gelatinolytic activity was maximal at 37 degrees C, and significant level of enzyme activity of the protease remained after heat treatment at 56 degrees C for 30 min or 100 degrees C for 10 min. This thermostable enzyme was strongly inhibited by metal chelators, i.e., EDTA, EGTA, and 1,10- phenanthroline. Thus, the 80 kDa protease in the ESP secreted by T. gondii was classified as a calcium dependent neutral metalloprotease.     

Monoclonal antibodies to Hammondia hammondi allowing immunological differentiation from Toxoplasma gondii

Five murine monoclonal antibodies (MAbs) were developed against purified sporozoites of Hammondia hammondi. Despite a large antigenic similarity between the 2 closely related coccidia, H. hammondi and Toxoplasma gondii, these MAbs only reacted with H. hammondi. Three MAbs, ID3, 3F2, and 4C9-7, recognized antigens of 38 kDa localized in rhoptries (1D3), in rhoptries and in oocyst and cyst walls (3F2), and in rhoptries and the apical region (4C9-7). Another MAb, 4C9-10, reacted with a 27-kDa antigen in dense granules of sporozoites and tachyzoites, and MAB 11B3 labeled an antigen of >94 kDa located in the pellicular membrane of the 3 stages of the parasite. These MAbs could be used for a rapid discrimination of the 2 coccidia in epidemiological studies or for diagnostic purposes in tissues.     

Detection of Toxoplasma gondii parasitemia in experimentally inoculated cats

Toxoplasma gondii B1 gene polymerase chain reaction (PCR) amplification utilizing a flanking and nesting reaction was compared to mouse bioassay on feline whole blood samples collected before and after experimental inoculation with T. gondii. Samples were collected from 5 cats prior to inoculation with T. gondii and on days 3, 7, 10, 14, 21, 28, 35, 42, 49, 56, 63, 70, 84, 112, 140, 143, 147, 150, 154, 161, 168, 175, and 182 after inoculation. Cats were challenged with T. gondii orally on day 140. Bioassay was found to be less effective for detection of parasitemia than B1 gene PCR. Parasitemia was detected in all 5 cats by PCR multiple times after primary and challenge inoculation. Detection of T. gondii parasitemia by PCR utilizing the flanking reaction described here may be useful in predicting the oocyst shedding period in individual cats. As none of the cats developed signs of systemic illness, yet were chronically parasitemic, T. gondii whole-blood PCR is not helpful as a diagnostic test for clinical feline toxoplasmosis.     

Antibodies to rubellavirus, herpes simplex virus and Toxoplasma gondii in serial serum samples of pregnant and non-pregnant women.

By testing serial serum samples of 213 pregnant women for rubellavirus, of 196 for herpes simplex virus and of 134 for Toxoplasma gondii, it was found that during pregnancy there was a fall in the humoral antibody level. Presence and titre of antibodies were lower in sera of pregnant than of non-pregnant women. Alteration of the humoral antibody level during pregnancy may influence serological studies aimed at clarifying the role of infections in fetal malformations. Serial serum samples (4 samples from each pregnant woman involved) should be tested for obtaining reliable data regarding the frequency of infections during pregnancy.     

Prevalence of antibodies to Toxoplasma gondii in horses in Riyadh Province, Saudi Arabia

The aim of the present study was to determine the seroprevalence of Toxoplasma gondii in horses used for sporting purposes in the Province of Riyadh, Saudi Arabia. In total, 266 serum samples from clinically healthy horses were analyzed for anti- T. gondii antibodies using the Sabin-Feldman dye test. Antibodies to T. gondii were found in 84 (31.6%) horses, with specific titers of 1∶16 (78 with a prevalence of 29.3%), 1∶64 (4 with a prevalence of 1.5%), and 1∶256 (2 with a prevalence of 0.8%). The number of seropositive horses in Shaqra (43.7%) was considerably higher than in other regions, with the lowest occurring in Al-Majmaah (11.0%). However, the differences in seroprevalence between the 6 locations were not statistically significant (P > 0.05). In contrast, the overall seroprevalence in Riyadh province was higher than that reported in other countries. Horses are not a direct source of infection for human toxoplasmosis in this region. Usually human infection is facilitated via cats, which are the reservoir hosts for this parasite.     

Seroprevalence of antibodies to Toxoplasma gondii in the Pennsylvania bobcat (Lynx rufus rufus)

From 2000 to 2002 bobcat blood samples were collected, in association with the Pennsylvania Game Commission, during the recently reactivated bobcat hunting and trapping season. Sex, age, and county/township data were recorded for each animal. Blood was tested for antibodies to Toxoplasma gondii using the modified agglutination test. In the 2-yr study, 131 bobcat samples were collected in 14 Pennsylvania counties and 109 (83%) of these had antibodies to T. gondii (titer>or=25). A two-way Chi-Square test (95% confidence interval) yielded no significance differences in antibody prevalence between males (83%) and females (88%) or adults (83%) and juveniles (77%). All 14 counties had at least one bobcat with antibodies to T. gondii.