Local meteorological conditions, dynamics of seroconversion to Toxoplasma gondii in cats (Felis catus) and oocyst burden in a rural environment.

The aim of this study was to analyse the spatio-temporal dynamics of Toxoplasma gondii infection in long-term monitoring of domestic cats (8-15 years) in three populations living in rural France. Overall seroprevalence was 52.7% (modified agglutination test > or =1:40). Incidence was 0.26-0.39 seroconversions/cat per year, and the estimated rate of soil contamination by T. gondii oocysts ranged between 31 and 3600 oocysts/m2 per year, depending on the population. Incidence risk in cats was related to mean precipitation, explaining both the spatial and temporal variability in risk: local conditions explained differences between the three study sites and incidence risk increased during rainy years. This study brings rare quantitative information on the level of contamination of the environment by T. gondii oocysts, and suggests that the spatio-temporal distribution of incidence risk in cats may reflect both the influence of rain on prey populations and infectivity of T. gondii oocysts.

Congenital toxoplasmosis in France in 2007: first results from a national surveillance system.

When immunocompetent people become infected with the parasite Toxoplasma gondii, the disease is generally asymptomatic. However, transplacental transmission of T. gondii may lead to severe congenital infection including in utero abortion, foetal death, or neurological or ocular damage of the foetus. France has had a national programme to prevent congenital toxoplasmosis since 1978. However, although estimated seroprevalence in pregnant women has fallen from 84% in the 1960s to 44% in 2003, no reliable data have been available on the annual number of cases of congenital toxoplasmosis or the severity of infection. In 2006, the French National Institute for Public Health Surveillance (Institut de Veille Sanitaire) and the National Reference Centre for Toxoplasmosis recommended that a national laboratory-based surveillance system be used for the surveillance of the disease. In 2007, 31 laboratories reported at least one congenital case through the surveillance system, giving a total of 272 cases. A total of 11 terminations of pregnancy were reported (six abortions and five foetal deaths). Of the live-born cases, 206 were asymptomatic, 28 were symptomatic and seven had a severe form of the disease. As there were 818,700 births in France and French overseas departments in 2007, the overall prevalence of congenital toxoplasmosis observed that year was 3.3 (95% confidence interval (CI): 2.9 to 3.7) per 10,000 live births and the incidence rate of the disease at birth was 2.9 (95% CI: 2.5 to 3.2) per 10,000 live births; the estimated incidence rate of symptomatic congenital toxoplasmosis was 0.34 (95% CI: 0.2 to 0.5) cases per 10,000 live births.

Multicenter comparative evaluation of five commercial methods for toxoplasma DNA extraction from amniotic fluid.

Over the past few years, a number of new nucleic acid extraction methods and extraction platforms using chemistry combined with magnetic or silica particles have been developed, in combination with instruments to facilitate the extraction procedure. The objective of the present study was to investigate the suitability of these automated methods for the isolation of Toxoplasma gondii DNA from amniotic fluid (AF). Therefore, three automated procedures were compared to two commercialized manual extraction methods. The MagNA Pure Compact (Roche), BioRobot EZ1 (Qiagen), and easyMAG (bioMérieux) automated procedures were compared to two manual DNA extraction kits, the QIAamp DNA minikit (Qiagen) and the High Pure PCR template preparation kit (Roche). Evaluation was carried out with two specific Toxoplasma PCRs (targeting the 529-bp repeat element), inhibitor search PCRs, and human beta-globin PCRs. The samples each consisted of 4 ml of AF with or without a calibrated Toxoplasma gondii RH strain suspension (0, 1, 2.5, 5, and 25 tachyzoites/ml). All PCR assays were laboratory-developed real-time PCR assays, using either TaqMan or fluorescent resonance energy transfer probes. A total of 1,178 PCRs were performed, including 978 Toxoplasma PCRs. The automated and manual methods were similar in sensitivity for DNA extraction from T. gondii at the highest concentration (25 Toxoplasma gondii cells/ml). However, our results showed that the DNA extraction procedures led to variable efficacy in isolating low concentrations of tachyzoites in AF samples (<5 Toxoplasma gondii cells/ml), a difference that might have repercussions since low parasite concentrations in AF exist and can lead to congenital toxoplasmosis.

Contributions of immunoblotting, real-time PCR, and the Goldmann-Witmer coefficient to diagnosis of atypical toxoplasmic retinochoroiditis.

Ocular toxoplasmosis is a major cause of posterior uveitis worldwide. The diagnosis is based mainly on ophthalmological examination. Biological diagnosis is necessary in atypical cases, and this requires aqueous humor sampling by anterior chamber paracentesis. We evaluated real-time PCR targeting the Toxoplasma gondii 529-bp repeat element, the Goldmann-Witmer coefficient (GWC), and immunoblotting for the diagnosis of toxoplasmic retinochoroiditis in 54 patients with atypical uveitis. The results of these biological tests, applied to paired aqueous humor-serum samples, were compared to the clinical findings. Combining either PCR or the GWC with immunoblotting increased the sensitivity to 73% or 70%, respectively. Together, PCR and the GWC had 80% sensitivity. If feasible, sensitivity can be increased by combining the three methods (85% sensitivity). The interval between symptom onset and anterior chamber paracentesis strongly influenced the detection of specific intraocular antibody synthesis. The sensitivity of the GWC increased from 45% to 56% when sampling was performed 10 days after symptom onset, and that of immunoblotting increased from 53% to 72% when puncture was performed 30 days after symptom onset. PCR analysis of aqueous humor samples detected toxoplasmic DNA in 55% of patients. In contrast to the results of immunoblotting and the GWC, the results of PCR were not influenced by the interval between symptom onset and paracentesis. PCR was more informative than the GWC and immunoblotting for immunocompromised patients. Acute necrotizing retinal lesions were significantly larger in PCR-positive patients, with a mean of 3.5 optic disc diameters, than in PCR-negative patients, with a mean of 1.5 optic disc diameters.

Seroprevalence of Neospora caninum and Toxoplasma gondii antibodies in white-tailed deer (Odocoileus virginianus) from Iowa and Minnesota using four serologic tests.

The white-tailed deer (Odocoileus virginianus) is considered one of the most important wildlife reservoirs of Neospora caninum and Toxoplasma gondii in the US. Sera from white-tailed deer from Minnesota and Iowa were tested for antibodies to N. caninum by four serologic tests including the indirect fluorescent antibody (IFA) test (cut-off 1:25), Neospora caninum agglutination test (cut-off 1:25), an enzyme-linked immunoabsorbent assay, and Western blot (WB). Sera were also tested for antibodies to T. gondii using the modified agglutination test (cut-off 1:25). Of 62 adult deer from Minnesota antibodies to T. gondii were found in 20 (32.2%), N. caninum in 44 (71%), with dual infections in 18 deer. Of 170 (73 fawns, 9 yearlings, 88 adults) deer from Iowa, T. gondii antibodies were present in 91 (53.5%) with 37.0, 55.6 and 67.0% seropositivity in fawns, yearlings, and adults, respectively. Antibodies to N. caninum were found in 150 of 170 (88.2%) by any of the 3 tests (99 by Western blots, 135 by ELISA, 106 by IFA, and 118 by NAT). Dual infections with T. gondii and N. caninum were detected in 47 deer. Very high (84.9%) seropositivity of N. caninum in fawns suggests high rate of congenital transmission of the parasite. Seropositivity in each test at different titers is discussed.

[How can the cost of screening for toxoplasmosis during pregnancy be reduced?]. / Comment réduire le coût du dépistage de la toxoplasmose chez la femme enceinte?

BACKGROUND: A program of systematic serology screening for toxoplasmosis during pregnancy has been running in France since 1978. The program involves monthly follow-ups for all non-immune pregnant women. Due to the steady decline in the seroprevalence of toxoplasmosis, the cost of the program is steadily increasing. Current screening is based on the detection of IgG and IgM isotypes. The aim of this work was to estimate the benefit of replacing combined dosage of two isotypes, by an alternative strategy that detects total anti-Toxoplasma immunoglobulins. METHODS: The rate of decreasing seroprevalence and the increasing burden on serological examinations was measured in a study population of pregnant women who were checked for toxoplasmosis by the parasitology laboratory of the Cochin Hospital, Paris. The increase in screening costs was estimated for the all-pregnant women and the expected benefits stemming from simply measuring total anti-Toxoplasma immunoglobulins compared to the double IgG-IgM assay were estimated. RESULTS: Between 1987 and 2008, the seroprevalence of toxoplasmosis measured at the Cochin hospital dropped from 70.8% to 48.6% with a 1.77% annual rate of decline. This downward trend is similar to that observed by the national perinatal surveys performed in 1995 and in 2003. As the number of non-immune women to follow-up each month is constantly increasing, the proportion of negative tests issued reached 87.6% in 2008. Extrapolating these results to the whole of France, we estimated that the number of required screening tests perform was increasing by 93,000 units per year with an additional associated cost of one million euros. Various alternative scenarios of antibody detection are proposed that could save between 40.2% and 48.4% of current screening costs. CONCLUSION: Replacement of combined dosage of IgG and IgM isotypes by determination of just total Ig would significantly reduce costs of toxoplasmosis screening for pregnant women, without effecting either the general strategy, or proven efficiency of the national program.

Transplacental toxoplasmosis in naturally-infected white-tailed deer: Isolation and genetic characterisation of Toxoplasma gondii from foetuses of different gestational ages.

Clinical toxoplasmosis is most severe in congenitally-infected hosts. In humans, transmission of Toxoplasma gondii from the mother to the foetus is considered to be most efficient during the last trimester of pregnancy but clinical congenital toxoplasmosis is more severe if transmission occurs during the first trimester. However, there are no data on the rate of congenital transmission of T. gondii with respect to gestational age in any host during natural infection. In the present study, attempts were made to isolate T. gondii by bioassay in mice inoculated with tissues from foetuses of 88 naturally-exposed white-tailed deer from Iowa and Minnesota. Viable T. gondii was isolated from foetuses of six of 61 deer in early pregnancy (45-85 days of gestation) from Iowa and foetuses of nine of 27 deer from Minnesota in mid-gestation (130-150 days) of a gestational period of 7 months. The 15 T. gondii isolates obtained from foetal deer were PCR-restriction fragment length polymorphism genotyped using polymorphisms at 10 nuclear markers including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and an apicoplast marker, Apico. Five genotypes were revealed, including the clonal Type II and III lineages, and three non-clonal genotypes. DNA sequencing analysis of representative isolates at loci SAG2, c22-8, L358 and PK1 revealed that the three non-clonal genotypes are closely related to the clonal Type I, II and III lineages. It is very likely that these non-clonal genotypes were derived from genetic crosses among the three clonal Type I, II and III lineages. The most common genotype was Type II, commonly found in humans in North America and Europe, suggesting the possible link of transmission from game animals to humans.

Seroprevalence of Neospora caninum and Toxoplasma gondii in black-tailed deer (Odocoileus hemionus columbianus) and mule deer (Odocoileus hemionus hemionus).

Deer are considered important intermediate hosts for the coccidian parasites, Toxoplasma gondii and Neospora caninum. Antibodies to N. caninum and T. gondii were determined in sera of 42 mule deer (Odocoileus hemionus hemionus) and 43 black-tailed deer (Odocoileus hemionus columbianus) from Washington state, USA, using direct agglutination test with specific antigens. A titer of 1:25 was considered diagnostic for both parasites. N. caninum antibodies were found in 7 of 42 mule deer and 8 of 43 black-tailed deer. T. gondii antibodies were found in 14 black-tailed deer but not in any of the mule deer. This is probably the first report of seroprevalence of N. caninum in these hosts.

Neospora caninum antibodies in wild carnivores from Spain.

Serum samples from 251 wild carnivores from different regions of Spain were tested for antibodies to Neospora caninum by the commercial competitive screening enzyme linked immunosorbent assay (c-ELISA) and confirmed by Neospora agglutination test (NAT) and/or by indirect fluorescent antibody test (IFAT). Samples with antibodies detected by at least two serological tests were considered seropositive. Antibodies to N. caninum were found in 3.2% of 95 red foxes (Vulpes vulpes); in 21.4% of 28 wolves (Canis lupus); in 12.0% of 25 Iberian lynx (Lynx pardinus); in 16.7% of 6 European wildcats (Felis silvestris); in 6.4% of 31 Eurasian badgers (Meles meles); in 21.4% of 14 stone martens (Martes foina); in 66.7% of 3 pine martens (M. martes) and in 50% of 2 polecats (Mustela putorius). Antibodies to N. caninum in common genets (Genetta genetta) and Egyptian mongooses (Herpestes ichneumon) were only observed by c-ELISA but were not confirmed by IFAT and/or NAT. No antibodies were detected in 5 Eurasian otters (Lutra lutra) by any technique. Statistically significant differences were observed among species and among geographical areas. The highest seroprevalence of N. caninum infection was observed in the Cantabric Coastal region characterized by high humidity. To our knowledge, this is the first report of antibodies to N. caninum in free ranging wild carnivores, other than wild canids, in Europe. The existence of a possible sylvatic cycle could have important implications in both sylvatic and domestic cycles since they might influence the prevalence of infection in cattle farms in those areas.

Endemic toxoplasmosis in pigs on a farm in Maryland: isolation and genetic characterization of Toxoplasma gondii.

The prevalence of Toxoplasma gondii was investigated on a poorly managed pig farm in Maryland. Serum and tissue samples from 48 of the 100 pigs on the farm were available for T. gondii evaluation. Serological testing was performed using both ELISA and the modified agglutination test (MAT). Antibodies to T. gondii were detected by ELISA in 12 of 48 animals, while antibodies were detected in 34 of 48 pigs by MAT with titers of 1:10 in 1, 1:20 in 4, 1:40 in 7, 1:80 in 3, 1:160 in 8, 1:320 in 3, 1:640 in 4, and 1:1,280 in 4. Hearts of 16 pigs with MAT titers of 1:10 or higher were bioassayed for T. gondii in cats; 11 cats shed T. gondii oocysts. Hearts of 22 pigs were autolyzed and bioassayed only in mice; T. gondii was isolated from 3 of these 22 pigs. Genetic typing of the 14 T. gondii isolates using the SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico loci revealed 4 genotypes; 10 isolates belonged to type II lineage (genotypes 1 and 2), 3 belonged to genotype 3, and 1 belonged to genotype 4. Genotype 1 and 2 have type II alleles at all genetic loci, except the former has type II allele and the latter has a type I allele at locus Apico. Both genotypes 1 and 2 are considered to belong to the clonal type II lineages. Genotype 3 and 4 are nonclonal isolates. Results document high prevalence of T. gondii in pigs on a farm in Maryland.

Comparison of serological methods for the diagnosis of Neospora caninum infection in cattle.

The aims of this study were to evaluate the performance and agreement of various commercial and in-house Neospora caninum antibody assays used in dairy cattle in North America, and to investigate reproducibility of two assays performed in different laboratories. From 1998 to 2005, three enzyme linked immunosorbent assays (ELISAs, a competitive ELISA-VMRD Inc., an indirect ELISA-Biovet Inc., and another indirect ELISA-Herdchek IDEXX Corp.), two indirect fluorescent antibody tests (IFATs, VMRD Inc., and in-house USDA) and one N. caninum agglutination test (NAT, in-house USDA) were utilized to test 397 randomly selected dairy cattle serum samples from 34 herds in eastern Canada for antibodies to N. caninum. The manufacturers' recommended cut-off values were used to evaluate test performance and agreement between tests. One IFAT (VMRD Inc.) performed well (sensitivity and specificity: 0.97 and 0.97, respectively) using reference sera (n = 452), therefore, results from this IFAT on the 397 samples could subsequently be used as the reference standard to calculate test characteristics for the other assays. Only 11% of the 397 sera were found to be N. caninum-positive with the IFAT. Prevalence-adjusted bias-adjusted kappa (PABAK) ranged from 0.06 to 0.99. Positive agreement was moderate to very good (P(pos) = 0.25-0.96). Negative agreement was very good for all assays (P(neg) > 0.94) except NAT (P(neg) = 0.66). Sensitivity was > or =0.89 for all assays except the NAT, which had a significantly lower sensitivity (0.66). Specificity was high (>0.94) for all assays except for one indirect ELISA (specificity = 0.52). This indirect ELISA did not perform satisfactorily when used in 1998, but an improved version of the ELISA performed as one of the best assays in 2004. Reproducibility of the competitive ELISA was excellent, but the reproducibility of the indirect ELISA that was improved was low (concordance correlation coefficient = 0.90 and 0.36, respectively). The performance characteristics observed for most assays in this study make them useful for screening antibodies to N. caninum in cattle.

Biologic and molecular characterization of Toxoplasma gondii isolates from pigs from Portugal.

Little is known of Toxoplasma gondii infections in animals in Portugal. In the present paper, we report the first isolation of viable T. gondii from pigs in Portugal. Antibodies to T. gondii were found in 52 (15.6%) of 333 pigs prior to slaughter using the modified agglutination test (MAT) at a serum dilution of 1:20. Attempts were made to isolate T. gondii from 37 seropositive pigs. Samples of brain and/or heart from each pig were digested in acid pepsin, and bioassayed into mice. Viable T. gondii was isolated from 15 pigs. Restriction fragment length polymorphism on products of SAG2 locus amplified by PCR and microsatellite analysis revealed that 11 isolates were Type II and four were Type III. The results indicate that phenotypically and genetically T. gondii are similar to isolates from pigs from the U.S.

Prevalence of Toxoplasma gondii in cats from Colombia, South America and genetic characterization of T. gondii isolates.

Cats are important in the epidemiology of Toxoplasma gondii infection because they are the only hosts that can excrete the environmentally-resistant oocysts. In the present study, prevalence of T. gondii was determined in serum, feces, and tissues of 170 unwanted cats from Colombia, South America. Antibodies to T. gondii were assayed by the modified agglutination test and found in 77 of 170 (45.2%) cats with titers of <1:5 in 93, 1:5 in eight, 1:10 in 17, 1:20 in 10, 1:40 in seven, 1:80 in four, 1:160 in eight, 1:320 in six, and 1:640 or higher in 17 cats. T. gondii oocysts were not found in feces of any cat as ascertained by bioassay in mice. Tissues (brain, heart, tongue) of 116 cats were bioassayed in mice or cats. T. gondii was isolated from tissues of 15 of the 42 cats with titers of 1:40 or higher and not from any of the 90 cats titers of 1:20 or lower. Of the 29 cats whose tissues were bioassayed individually, T. gondii was isolated from the tongues of nine, hearts of eight, and brains of five. Mice inoculated with tissues of 12 of 15 infected cats died of toxoplasmosis; with nine T. gondii isolates all infected mice died. Overall, 65 of 92 (70%) of T. gondii-infected mice died of toxoplasmosis. Genotyping of these 15 isolates using polymorphisms at the SAG1, SAG2, SAG3, BTUB, and GRA6 loci revealed that three isolates (TgCtCo1, 2, and 7) had Type I alleles and one isolate (TgCtCo8) had Type II allele at all five loci. Eleven isolates contained the combination of Type I and III alleles and were divided into three genotypes, with TgCtCo3,5,6,9,12,13 and 15 had alleles I, I, III, I and III, TgCtCo4,10,11 had alleles I, III, III, I and I, and TgCtCo14 had alleles I, III, III, III, and III, at loci SAG1, SAG2, SAG3, BTUB and GRA6, respectively. All infected mice from each group had identical genotype except one mouse infected with TgCtCo5 had a Type III allele at locus BTUB and a unique allele (u-1) at locus SAG1 indicating mixed infection for TgCtCo5, whereas the rest seven mice had a Type I alleles at both loci.

Seroprevalence of Toxoplasma gondii antibodies in humans from rural Western Amazon, Brazil.

Antibodies to Toxoplasma gondii were assayed in sera of 266 humans from 71 farms located at Rondônia State, Western Amazon, Brazil, by the modified agglutination test (MAT) and the indirect immunofluorescent antibody test (IFAT). Antibodies were found in 195 humans (73.3%), with MAT titers of 1:25 in 11, 1:50 in 11, 1:100 in 16, 1:200 in 27, 1:400 in 38, 1:800 in 37, 1:1,600 in 22, and 1:3,200 or higher in 33. From the 71 farms visited, 69 had seropositive humans. Prevalence of anti-T. gondii antibodies increased with age of the people (P < 0.05), and no difference was observed in the occurrence by gender (P > 0.05). A sanitary questionnaire was applied in each farm, and statistical association between the serologic status and several variables were analyzed. Home-grown vegetable consumption and origin of drinking water (well or river) were the independent variables that displayed significant association (P = 0.002 and 0.02, respectively). Higher values of occurrence were found in people with consumption of home-grown vegetables (76.1%) and people that drink well water (75.4%) compared with people that did not consume this type of food (61.9%) and drink river water (55.2%). By IFAT (> or = 1:16), 194 of 266 (73%) humans were seropositive and there was a good correlation between MAT and IFAT.

Characterization of Toxoplasma gondii isolates in free-range chickens from Portugal.

The prevalence of Toxoplasma gondii in free-ranging chickens is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. The prevalence of T. gondii in 225 free-range chickens (Gallus domesticus) from Portugal was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT) and found in 61 chickens with titers of 1:5 in 8, 1:10 in 6, 1:20 in 3, 1:40 in 23, 1:80 in 5, 1:160 in 4, 1:320 in 8, and 1:640 or higher in 4. Hearts, leg muscles, and brains of 15 seropositive (MAT 1:10 or higher) chickens were bioassayed individually in mice. Tissue from 38 chickens with titers of 1:5 or less were pooled and fed to a T. gondii-free cat. Feces of the cat were examined for oocysts, but none was found. Toxoplasma gondii was isolated from 16 of 19 chickens with MAT titers of 1:10 or higher. Genotyping of 12 of these 16 isolates with polymorphisms at the SAG2 locus indicated that 4 were type III, and 8 were type II. None of the isolates was lethal for mice. Phenotypically, T. gondii isolates from chickens from Portugal were different from those of T. gondii isolates from chickens from Brazil.

Prevalence of antibodies to Neospora caninum in wild animals.

Antibodies to Neospora caninum were determined in several species of wild animals in the United States by the Neospora agglutination test (NAT). Antibodies (NAT 1:40 or higher) were found in 5 of 249 bison (Bison bison), 5 of 160 caribou (Rangifer tarandus), 4 of 162 moose (Alces alces), 4 of 122 wolves (Canis lupus), and 1 of 224 musk ox (Ovibos moschatus) but not in 197 black bears (Ursus americanus). To our knowledge, this is the first report of antibodies to N. caninum in bison and caribou. The total absence of N. caninum antibodies in black bears indicates that bears are not a host for N. caninum and that there is no cross-reactivity between the NAT and the modified agglutination test (MAT) for Toxoplasma gondii, because more than 80% of black bears in eastern United States have MAT antibodies at a 1:25 serum solution.

A comparison of several serologic tests to detect antibodies to Toxoplasma gondii in naturally exposed bottlenose dolphins (Tursiops truncatus).

Toxoplasma gondii infection in marine mammals is intriguing and indicative of contamination of the ocean environment and coastal waters with oocysts. In a previous study, 138 of 141 (97.8%) bottlenose dolphins (Tursiops truncatus) from the coasts of Florida and California had antibodies to T. gondii by the modified agglutination test (MAT). Although the MAT has been found to be highly sensitive and specific for T. gondii antibodies from several species of terrestrial animals, it has not yet been validated for T. gondii infections in marine mammals. Furthermore, T. gondii has yet not been isolated from dolphins. In the present study, sera from 146 (60 from the 2004 samples and 86 from the 2003 samples) T. truncatus from the coastal areas of South Carolina and Florida were tested for antibodies to T. gondii. Sera from 2004 were tested by the MAT, the indirect fluorescent antibody test (IFAT), the Sabin-Feldman dye test (DT), an indirect hemagglutination test (IHAT), an enzyme-linked immunosorbent assay (ELISA), and Western blot. All 60 dolphins were seropositive, with MAT titers of 1:20 in 3, 1:40 in 19, 1:80 in 29, 1:160 in 2, 1:1,280 in 3, 1:2,560 in 2, and 1:5,120 or higher in 2, and these results were confirmed in another laboratory. The DT titers of these dolphins were <1:10 in 53, 1:800 in 3, 1:1,600 in 2, and 1:3,200 in 2. The IHAT titers were <1:64 in 52, 1:128 in 1, 1:512 in 2, and 1:2,048 in 5. The IFAT titers were <1:20 in 3, 1:20 in 11, 1:40 in 36, 1:80 in 2, 1:160 in 1, and 1:320 or higher in 7. All 7 DT-positive dolphins had high MAT titers, but 2 were negative by the IHAT. Western blot results closely followed MAT results; ELISA results matched MAT results, which were 1:40 or higher. In sera from the 2003 samples, MAT antibodies were found in 86 of 86 dolphins with titers of 1:25 in 29, 1:50 in 23, 1:100 in 27, 1:200 in 3, 1:1,600 in 1, and 1:3,200 in 3; these sera were not tested by other means. Overall, MAT antibodies were found in all 146 dolphin sera tested. Because marine mammals are considered sentinel animals indicative of contamination of the coastal and marine waters by T. gondii oocysts, serologically positive infections need to be validated by the detection of T. gondii organisms in the tissues of seropositive animals.

An anti-human mu chain monoclonal antibody: use for detection of IgM antibodies to Toxoplasma gondii by reverse immunosorbent assay.

A precipitating anti-human mu chain monoclonal antibody (designated Tibi 82 McAb) was produced by the cell fusion technique. This McAb (isotype: IgG1 kappa) reacted by radioimmunoassay with all 10 human IgM proteins tested. In contrast, no reactivity was observed with IgG, IgA, IgE, lambda and kappa chains. 19 S IgM proteins were precipitated by Tibi 82 McAb using the Ouchterlony method under standard conditions. Hence specificity of this McAb for the C mu 2 domain was characterized by inhibition of precipitin reactions using human IgM fragments. Despite its narrow specificity for the C mu 2 domain, such a McAb could be used for IgM capture in the detection of specific IgM to Toxoplasma gondii employing the IgM immunosorbent agglutination assay (IgM-ISAGA). Tibi 82 McAb was compared with 3 anti-human IgM polyclonal reagents in the routine analysis of 117 sera. With 2 of them, a correlation coefficient of 0.976 was obtained and Tibi 82 McAb was more sensitive than the third polyclonal reagent tested. The IgM-ISAGA technique was shown to be reproducible using Tibi 82 McAb and similar anti-human mu chain McAbs could permit the wider development of reverse immunosorbent methods for the detection of specific IgM in various infectious diseases.

Seroprevalence of antibodies to Neospora caninum and Toxoplasma gondii in water buffaloes (Bubalus bubalis) from Egypt.

Sera from 75 water buffaloes from Egypt were examined using a direct agglutination test incorporating mercaptoethanol for antibodies to Neospora caninum and Toxoplasma gondii. Antibodies to N. caninum were found in 51 (68%) of 75 buffaloes in titres of 1:20 (six buffaloes), 1:40 (15 buffaloes), 1:160 (one buffalo), 1:320 (one buffalo) and > or = 1:640 (28 buffaloes), using N. caninum formalin-preserved whole tachyzoites as antigen. Antibodies to T. gondii were not found in a 1:100 dilution of serum of any of the 75 buffaloes, using T. gondii as antigen, indicating specificity in the detection of antibodies to N. caninum. This is the first report of N. caninum prevalence in water buffaloes, which are economically very important domestic animals in developing countries.

High prevalence of antibodies to Neospora caninum in white-tailed deer (Odocoileus virginianus).

Serum samples of 400 white-tailed deer (Odocoileus virginianus) from 16 preserves in northeastern Illinois were tested for Neospora caninum antibodies in the N. caninum agglutination test using mouse-derived N. caninum tachyzoites and mercaptoethanol. Antibodies were found in 162 deer with titres of 1:40 (47 deer), 1:80 (32 deer), 1:160 (17 deer), 1:200 (eight deer), 1:400 (19 deer), 1:800 (17 deer) and > or = 1:1600 (22 deer). There were no significant differences in prevalence between age or sex of the deer. The high prevalence of N. caninum infection in deer is consistent with a sylvatic cycle of N. caninum.