Isolation and genotyping of Toxoplasma gondii from free-ranging chickens from Argentina

The prevalence of Toxoplasma gondii in free-ranging chickens can be considered a good indicator of the prevalence of T. gondii oocysts in the environment because chickens feed from the ground. In the present study, prevalence of T. gondii in 29 free-range chickens (Gallus domesticus) from Argentina was investigated. Blood, heart, and brain from each chicken were examined for T. gondii infection. Antibodies to T. gondii, assayed with the modified agglutination test (MAT), were found in 19 of 29 (65.5%) chickens. Hearts and brains of seropositive (MAT > or = 1:5) chickens were bioassayed in mice. Toxoplasma gondii was isolated from 9 of 19 seropositive chickens. Genotyping of chicken isolates of T. gondii using the SAG2 locus indicated that 1 was type I, 1 was type II, and 7 were type III. This is the first report of isolation of T. gondii from chickens from Argentina.     

Prevalence and isolation of Toxoplasma gondii from white-tailed deer in Alabama

Nineteen white-tailed deer (Odocoileus virginianus) from 5 counties in Alabama were examined for infection with Toxoplasma gondii. Twenty-gram samples of heart tissue were bioassayed in mice, serum was examined for T. gondii antibodies using the direct agglutination test, and sections of heart muscle were examined histologically for tissue cysts. Toxoplasma gondii was isolated from 4 of 19 (21%) white-tailed deer hearts. Antibody titers of greater than or equal to 1:50 were found in sera from 7 of 16 (44%) white-tailed deer. Histological examinations of tissue sections from white-tailed deer hearts were negative for T. gondii.     

Prevalence of Toxoplasma gondii in slaughtered pigs in the state of Pernambuco, Brazil

The object of this study was to investigate Toxoplasma gondii antibodies and parasite DNA in pigs in the state of Pernambuco, Brazil. Blood samples were collected from 305 slaughtered pigs in 11 municipalities, and their sera were tested for T. gondii antibodies using the indirect fluorescent antibody test (IFAT, cutoff 1:64); 38 (12.5%) samples were positive. Attempts were made to detect T. gondii DNA in the heart tissue of seropositive pigs using the B 1 gene and PCR; 21 (55.2%) of the 38 hearts were positive. This is the first detection of T. gondii DNA in tissues of serologically positive swine in the State of Pernambuco, Brazil.     

Prevalence of Toxoplasma gondii infection in wild birds in Durango, Mexico

There is a lack of information concerning the prevalence of Toxoplasma gondii infection in wild birds in Mexico. In the present study, serum samples and tissues from 653 birds from Durango State, Mexico, were evaluated for T. gondii infection. Antibodies to T. gondii (modified agglutination test, titer 1∶25 or higher) were found in 17 (2.6%) of the 653 birds, including 1 of 2 curve-billed thrashers (Toxostoma curvirostre), 2 (1 Anas platyrhynchos, 1 Anas diazi) of 4 ducks, 1 of 2 eagles (Aquila sp.), 5 (27.8%) of 18 great-tailed grackles (Quiscalus mexicanus), 7 (1.3%) of 521 rock pigeons (Columba livia), and 1 (14.3%) of 7 quail (Coturnix coturnix). The seroprevalence of T. gondii infection in birds captured in a park outside the city zoo (11.6%, 8/69) was significantly higher than that found in birds from other regions (1.5%, 9/584, OR = 8.38; 95% CI: 2.82-24.77; P = 0.0001). Brains and hearts of 23 birds (17 seropositive, 6 seronegative) were bioassayed in mice for the isolation of T. gondii . Viable T. gondii was isolated from 1 of 7 seropositive pigeons. The DNA obtained from the T. gondii isolate from the pigeon was genotyped using the PCR-RFLP typing using 11 markers (B1, SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico) and revealed an atypical genotype. This is the first report of T. gondii infection in great-tailed grackles, the Mexican duck, and curved-billed thrashers and the first survey of wild birds in Mexico.     

Enzootic toxoplasmosis in sheep in north-central United States

Of 1,564 serum samples from adult ewes from 33 farms in Iowa, Minnesota, South Dakota, Kansas, and Nebraska where toxoplasmosis-induced ovine abortions had been diagnosed, 65.5% were found positive for Toxoplasma gondii antibodies using the modified agglutination test. Toxoplasma gondii antibody titers of ewes were: less than 64 (34.5%), 64 (14.9%), 256 (22.0%), 1,024 (14.5%), and greater than 4,096 (13.8%). Thus, 28.3% of sheep had high titers (greater than 1,024) indicating recently acquired T. gondii infection. On certain farms, up to 95% of ewes were seropositive. Prevalence of T. gondii antibodies increased with age of the ewe. Of 665 ewes, 53.6% of 1-yr-old ewes were seropositive (titers greater than 64) versus 75% of 5-yr-old ewes. Results indicate that T. gondii infection in sheep in the United States is widespread.     

Seroprevalence and isolation of Toxoplasma gondii from sheep from São Paulo state, Brazil

Sheep are important in the epidemiology of Toxoplasma gondii infection but little is known of ovine toxoplasmosis in Brazil. Antibodies to T. gondii were assayed in sera of 495 sheep from 36 counties of S?o Paulo State, Brazil, using the modified agglutination test (MAT titer > or =1:25) and found in 120 (24.2%). Samples of brain, heart, and diaphragm of 82 seropositive sheep were pooled, digested in pepsin, and bioassayed in mice. Toxoplasma gondii was isolated from tissue homogenates of 16 sheep and the isolates were designated TgShBr1-16. Six of the 16 T. gondii isolates killed 100% of infected mice. Results indicate that asymptomatic sheep can harbor mouse-virulent T. gondii, and hence they can serve as a source of infection for humans.     

Seroprevalence of Toxoplasma gondii in pigs, sheep, goats, and cattle from Grenada and Carriacou, West Indies

Prevalence of Toxoplasma gondii infection in pregnant women in Grenada is considered high. Little is known of the epidemiology of T. gondii infection in Caribbean Islands. Serum samples of 750 food animals in Grenada and Carriacou were tested for antibodies to T. gondii by the modified agglutination test (MAT). Antibodies to T. gondii (MAT, 1∶25 or higher) were found in 23.1% of 247 pigs, 44.1% of 204 sheep, 42.8% of 180 goats, and 8.4% of 119 cattle. Seroprevalence increased with age, indicating postnatal acquisition of T. gondii. Antibody titers of 1∶200 or higher were present in 65 of 90 seropositive sheep, 61 of 77 seropositive goats, and 23 of 57 seropositive pigs. However, none of the cattle had a MAT titer of 1∶200, suggesting that bovines are a poor host for T. gondii. Results indicate that pigs, sheep, and goats could be important sources of T. gondii infection if their meat is consumed undercooked.     

High prevalence of toxoplasmosis in cats from Egypt: isolation of viable Toxoplasma gondii, tissue distribution, and isolate designation

Cats are important in the epidemiology of Toxoplasma gondii because they are the only hosts that excrete environmentally resistant oocysts in feces. In the present study, 158 feral cats from Giza, Egypt, were examined for T. gondii infection. Antibodies to T. gondii were found in 97.4% with the modified agglutination test. Viable T. gondii was isolated from tissues (brain, heart, tongue) of 115 of 137 cats by bioassay in mice. These isolates were designated TgCatEg 1-115; none of these isolates was virulent to out-bred Swiss Webster mice. Of the 112 seropositive cats whose tissues were bioassayed individually, T. gondii was isolated from the hearts of 83 (74.1%), tongues of 53 (47.3%), and brains of 36 (32.1%). Toxoplasma gondii oocysts were not detected in rectal contents of any of the 158 cats, probably related to high seropositivity (chronic infection) of cats surveyed. The high prevalence of T. gondii in feral cats in Egypt reported here indicates a high environmental contamination with oocysts.     

Toxoplasma gondii infections in red-tailed hawks inoculated orally with tissue cysts

The response to inoculation of Toxoplasma gondii tissue cysts was examined in 3 red-tailed hawks (Buteo jamaicensis). One hawk (hawk 1) was inoculated orally with 3.000 tissue cysts of the GT-1 isolate of T. gondii and 2 hawks (hawks 2 and 3) each were inoculated orally with 12,000 tissue cysts of a mixture of 8 isolates of T. gondii. None of the hawks developed clinical signs of toxoplasmosis. Serum antibodies were measured with the modified direct agglutination test using formalin-fixed tachyzoites. Hawk 1 had a titer of 1:40 prior to inoculation and did not have an increase in titer during the study. Hawks 2 and 3 had titers of 1:5 and 1:10, respectively, prior to inoculation, and both had increased titers (titers greater than or equal to 1:60) by 1 wk postinoculation and remained T. gondii antibody positive throughout the 10 wk of the study. Toxoplasma gondii was isolated from the heart and breast muscle of hawk 1. The biologic behavior of this T. gondii isolate was different from the 1 inoculated, and it probably represents a prior natural infection. Toxoplasma gondii was isolated from the brain, heart, breast muscle, and a mixture of gizzard and proventriculus from hawk 2 and from breast muscle of hawk 3. Toxoplasma gondii was not isolated from the eye, lung, liver, kidney, or spleen of any red-tailed hawk.     

Isolation of Toxoplasma gondii from 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 previous serological surveys, >90% of bottlenose dolphins (Tursiops truncatus) from the coasts of Florida, South Carolina, and California had antibodies to T. gondii by the modified agglutination test (MAT). In the present study, attempts were made to isolate T. gondii from dead T. truncatus. During 2005, 2006, and 2007, serum or blood clot, and tissues (brain, heart, skeletal muscle) of 52 T. truncatus stranded on the coasts of South Carolina were tested for T. gondii. Antibodies to T. gondii (MAT 1:25 or higher) were found in 26 (53%) of 49 dolphins; serum was not available from 3 animals. Tissues (heart, muscle, and sometimes brain) of 32 dolphins (26 seropositive, 3 seronegative, and 3 without accompanying sera) were bioassayed for T. gondii in mice, or cats, or both. Tissues of the recipient mice were examined for T. gondii stages. Feces of recipient cats were examined for shedding of T. gondii oocysts, but none excreted oocysts. Toxoplasma gondii was isolated from hearts of the 3 dolphins (2 with MAT titers of 1:200, and 1 without accompanied serum) by bioassay in mice. Genotyping of these 3 T. gondii isolates (designated TgDoUs1-3) with the use of 10 PCR-RFLP markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico) revealed 2 genotypes. Two of the 3 isolates have Type II alleles at all loci and belong to the clonal Type II lineage. One isolate has a unique genotype. This is the first report of isolation of viable T. gondii from T. truncatus.     

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.     

Isolation and genotyping of Toxoplasma gondii from free-ranging chickens from Mexico

The prevalence of Toxoplasma gondii in free-ranging chickens is a good indicator of the presence of T. gondii oocysts in the environment because chickens feed from the soil. In the present study, prevalence of T. gondii in 208 free-range chickens (Gallus domesticus) from Mexico was investigated. Blood, heart, and brain from each animal were obtained to test for T. gondii infection. Antibodies to T. gondii, assayed with the modified agglutination test (1:10 or higher), were found in 13 (6.2%) chickens. Hearts and brains of 13 seropositive chickens were bioassayed in mice, and T. gondii was isolated from 6 chickens. All 6 isolates were avirulent for mice. Genotyping of chicken isolates of T. gondii using the SAG2 locus indicated that 5 were type III and 1 was type I. This is the first report of isolation of T. gondii from chickens from Mexico.     

Detection of Ocular Toxoplasma gondii Infection in Chronic Irregular Recurrent Uveitis by PCR

Toxoplasma gondii is a zoonotic parasite resulting in human infections and one of the infectious pathogens leading to uveitis and retinochoroiditis. The present study was performed to assess T. gondii infection in 20 ocular patients with chronic irregular recurrent uveitis (20 aqueous humor and 20 peripheral blood samples) using PCR. All samples were analyzed by nested PCR targeting a specific B1 gene of T. gondii. The PCR-positive rate was 25% (5/20), including 5% (1) in blood samples, 25% (5) in aqueous humor samples, and 5% (1) in both sample types. A molecular screening test for T. gondii infection in ocular patients with common clinical findings of an unclear retinal margin and an inflammatory membrane over the retina, as seen by fundus examination, may be helpful for early diagnosis and treatment.     

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.     

Triplex PCR using new primers for the detection of Toxoplasma gondii

Molecular methods are used increasingly for the detection of Toxoplasma gondii infection. This study developed a rapid, sensitive, and specific conventional triplex PCR for the detection of the B1 gene and ITS1 region of T. gondii using newly designed primers and an internal control based on the Vibrio cholerae HemM gene. The annealing temperature and concentrations of the primers, MgCl(2), and dNTPs were optimized. Two sets of primers (set 1 and 2) were tested, which contained different segments of the T. gondii B1 gene, 529 repeat region and ITS1 region. A series of sensitivity tests were performed using parasite DNA, whole parasites, and spiked human body fluids. Specificity tests were performed using DNA from common protozoa and bacteria. The newly developed assay based on set 2 primers was found to be specific and sensitive. The test was capable of detecting as little as 10 pg T. gondii DNA, 10(4) tachyzoites in spiked body fluids, and T. gondii DNA in the organ tissues of experimentally infected mice. The assay developed in this study will be useful for the laboratory detection of T. gondii infection.     

Evaluation of an indirect fluorescent antibody test (IFAT) for demonstration of antibodies to Toxoplasma gondii in the sea otter (Enhydra lutris)

An indirect fluorescent antibody test (IFAT) for detection of Toxoplasma gondii infection was validated using serum from 77 necropsied southern sea otters (Enhydra lutris nereis) whose T. gondii infection status was determined through immunohistochemistry and parasite isolation in cell culture. Twenty-eight otters (36%) were positive for T. gondii by immunohistochemistry or parasite isolation or both, whereas 49 (64%) were negative by both tests. At a cutoff of 1:320, combined values for IFAT sensitivity and specificity were maximized at 96.4 and 67.3%, respectively. The area under the receiver-operating characteristic curve for the IFAT was 0.84. A titer of 1:320 was used as cutoff when screening serum collected from live-sampled sea otters from California (n = 80), Washington (n = 21), and Alaska (n = 65) for T. gondii infection. Thirty-six percent (29 out of 80) of California sea otters (E. lutris nereis) and 38% (8 out of 21) of Washington sea otters (E. lutris kenyoni) were seropositive for T. gondii, compared with 0% (0 out of 65) of Alaskan sea otters (E. lutris kenyoni).     

Agglutinating antibodies to Toxoplasma gondii in sera from captive eastern barred bandicoots in Australia

Toxoplasmosis is considered a severe health risk for many marsupial species. The mainland Australian population of bandicoot is endangered. Therefore, a preliminary serosurvey was conducted to evaluate exposure to Toxoplasma gondii in 57 captive eastern barred bandicoot and to estimate the possible impact of Toxoplasma on recovering populations. Five (9%) bandicoot were classified as seropositive using a modified agglutination test. Nineteen additional bandicoot (33%) were classified as serosuspect using a direct agglutination test. No bandicoot showed signs of clinical disease. Seropositive titers were IgG associated, suggesting that infections were chronic and latent. Serostatus was not associated with either sex or being wild-caught, although each seropositive bandicoot was wild-caught. Seropositive animals ranged from 1.25- to 2.5-yr-old. Computer simulations using Vortex 5.1, based on the proportion of seropositive and seronegative bandicoot in this study, indicate that mortalities from T. gondii should have little impact upon captive populations. However, the potential impact of toxoplasmosis on recovery efforts for wild, mainland bandicoot populations is not clear.     

Seroprevalence of Toxoplasma gondii infection in chickens in Durango State, Mexico

Little is known concerning the seroprevalence of Toxoplasma gondii infection in chickens (Gallus domesticus) in Mexico. Antibodies to T. gondii were determined in 519 chickens in Durango, Mexico using the modified agglutination test (MAT). Two groups (A, B) of chickens were sampled. Group A chickens (n = 51) were raised in backyards in 7 municipalities in 3 geographical regions in Durango State. Group B chickens were raised in farms in the Mexican States of Sinaloa (n = 289) and Nayarit (n = 179) but slaughtered in 2 abattoirs in Durango City. Overall, antibodies to T. gondii were found in 36 (6.9%) of 519 chickens, with MAT titers of 1∶25 in 22, 1∶50 in 8, 1∶100 in 2, 1∶200 in 3, and 1∶400 in 1. Seroprevalence of T. gondii increased significantly with age and was significantly higher in Group A chickens than in Group B chickens. In Group A chickens, a 25.5% seroprevalence of T. gondii infection was found. Seropositive chickens were found in all 7 municipalities sampled. In Group B chickens, the seroprevalence of T. gondii infection was 4.9%. This is the first report of T. gondii infection in chickens in Durango State, Mexico.     

Isolation of Toxoplasma gondii from capybaras (Hydrochaeris hydrochaeris) from São Paulo State, Brazil

The capybara (Hydrochaeris hydrochaeris) is a large rodent used for human consumption in certain areas of South America. In the present study, viable Toxoplasma gondii was isolated for the first time from this host. Antibodies to T. gondii were assayed in the sera of 64 capybaras from 6 counties of S?o Paulo State, Brazil, using the modified agglutination test (MAT, > or =1:25 dilution) and the indirect fluorescent antibody test (IFAT, > or =1:16 dilution), and antibodies were found in 48 (75%) by MAT, and 49 (76.6%) by IFAT. Samples of brain, heart, and tongue of 40 seropositive capybaras were pooled, digested in pepsin, and bioassayed in mice. Toxoplasma gondii was isolated from tissue homogenates of 36 capybaras, and the isolates were designated TgCyBrl-36. Most isolates were lethal to mice; 17 of the 36 isolates killed 100% of infected mice, 11 isolates caused mortality in 25-90% of infected mice, and 8 isolates were nonpathogenic to mice. Results indicate that asymptomatic capybaras can harbor mouse-virulent T. gondii, and hence they can serve as a source of infection for humans.     

Isolation of Toxoplasma gondii from goats from Brazil

Goats are economically important in many countries, and little is known of caprine toxoplasmosis in Brazil. Antibodies to Toxoplasma gondii were assayed in the sera of 143 goats from 3 Brazilian states, using modified agglutination test (MAT titer > or = 1:25); 46 (32.2%) tested positive. Samples of brain, heart, diaphragm, and masseter of seropositive animals were pooled, digested in pepsin, and bioassayed in mice. Viable T. gondii specimens were isolated from tissue homogenates of 12 goats; the isolates were designated TgGtBr1-12. Ten of the 12 isolates killed 100% of infected mice, indicating that goats can harbor mouse-virulent T. gondii and, hence, can serve as a source of infection for humans.