Roberta Lemos Freire

Toxoplasma gondii infections in cats from Paraná, Brazil: Seroprevalence, tissue distribution, and biologic and genetic characterization of isolates

Cats are important in the epidemiology of Toxoplasma gondii because they are the only hosts that can excrete environmentally resistant oocysts. The prevalence of T. gondii was determined in 58 domestic cats from 51 homes from Santa Isabel do Ivai, Paraná State, Brazil where a water-associated outbreak of acute toxoplasmosis had occurred in humans. Antibodies to T. gondii were found with the modified agglutination test in 49 of 58 (84.4%) cats at a serum dilution of 1:20. Tissues (brain, heart, and skeletal muscle) of 54 of these cats were bioassayed in T. gondii–free, laboratory-reared cats; T. gondii oocysts were excreted by 33 cats that were fed feline tissues. Brains from these 54 cats were bioassayed in mice; T. gondii was isolated from 7. Skeletal muscles and hearts of 15 cats were also bioassayed in mice; T. gondii was isolated from skeletal muscles of 9 and hearts of 13. The results indicate that T. gondii localizes in muscle tissue more than the brains of cats. In total there were 37 T. gondii isolates from 54 cats. Most isolates of T. gondii were virulent for mice. Genotyping of the 37 isolates of T. gondii, using the SAG2 locus, revealed that 15 isolates were type I and 22 were type III. The absence of type II genotype in cats in this study is consistent with the previous studies on T. gondii isolates from Brazil and is noteworthy because most T. gondii isolates from the United States are type II. These findings support the view that Brazilian and North American T. gondii isolates are genetically distinct. This is the first report of genotyping of T. gondii isolates from the domestic cat.

Factors associated with seropositivity for anti-Toxoplasma gondii antibodies in pregnant women of Londrina, Paraná, Brazil

The aim of this study was to evaluate associations between seropositivity for IgG and IgM anti-Toxoplasma gondii antibodies and socio-economic and environmental variables in pregnant women of Londrina, state of Paraná, Brazil. We interviewed 492 pregnant women, each of whom answered an epidemiological questionnaire, and collected blood samples for measurement of IgG and IgM anti-T. gondii antibodies by chemiluminescence. A confirmatory diagnosis of acute infection was made by an IgG avidity test. Titres of specific IgG anti-T. gondii were obtained by IFAT. Seropositivity for IgG anti-T. gondii antibodies was observed in 242 women (49.2%) and, of these, six pregnant women (1.2%) showed seropositivity for IgM. Age group, level of education, per capita income, presence of a cat in the house and a habit of eating green vegetables were all factors associated with a greater chance of infection with T. gondii. This study showed that 250 (50.8%) pregnant women were susceptible to T. gondii and considered to be at high risk for toxoplasmosis during pregnancy. Based on the results obtained, is critical to establish a program of health surveillance for toxoplasmosis, in order to contribute to diagnosis and early treatment during the prenatal period. It is also necessary to introduce measures to prevent the Toxoplasma infection in seronegative pregnant women.

Toxoplasma gondii infection in pregnancy

Toxoplasmosis is caused by an intracellular protozoan, Toxoplasma gondii, which has a wide geographical distribution. The main infection routes are ingestion of cysts from raw or badly-cooked meat, ingestion of oocysts from substrates contaminated with the feces of infected felines and congenital transmission by tachyzoites. The congenital form results in a severe systemic disease, because if the mother is infected for the first time during gestation, she can present a temporary parasitemia that will infect the fetus. Many of the clinical symptoms are seen in congenitally-infected children, from a mild disease to serious signs, such as mental retardation. Early diagnosis during the pregnancy is highly desirable, allowing prompt intervention in cases of infection, through treatment of pregnant women, reducing the probability of fetal infection and consequent substantial damage to the fetus. Conventional tests for establishment of a fetal diagnosis of toxoplasmosis include options from serology to PCR. Prevention of human toxoplasmosis is based on care to avoid infection, understanding the disease and serological exams during gestation. Pregnant women should be tested serologically from three months gestation, until one month after childbirth. Inclusion of serology for congenital toxoplasmosis along with the basic Guthrie test for PKU is of fundamental importance for early diagnosis of infection and so that treatment is initiated, in order to avoid possible sequels in the infant.

Seroepidemiology and occupational and environmental variables for leptospirosis, brucellosis and toxoplasmosis in slaughterhouse workers in the Paraná State, Brazil

Leptospirosis, brucellosis and toxoplasmosis are widely-distributed zoonosis, being the man an accidental participant of their epidemiological chains. The aim of this paper was to make a seroepidemiological report and identify occupational and environmental variables related to these illnesses in 150 workers in a slaughterhouse in the Northern region of Paraná. For the diagnosis of leptospirosis a microscopical seroagglutination test was applied; for brucellosis, the tamponated acidified antigen test and the 2-mercaptoetanol tests were used, and for toxoplasmosis the indirect immunofluorescence reaction test. For each employee an epidemiological survey was filled, which investigated occupational and environmental variables which could be associated with these infections. Positive results for leptospirosis were found in 4.00% of the samples, for brucellosis in 0.66% of samples and toxoplasmosis in 70.00%. From the three diseases researched, only the results for leptospirosis suggest occupational infection.

Occurrence of antibodies to Neospora caninum and Toxoplasma gondii in dairy cattle from the northern region of the Paraná State, Brazil

Three-hundred and eighty-five serum samples were taken from dairy cows on 90 farms in 12 counties from the northern region of the Parana State, Brazil. The samples were analyzed by IFAT for the detection of anti-Neospora caninum and anti-Toxoplasma gondii IgG antibodies. Forty-five (12%) samples were seropositive to N. caninum, while 102 (26%) samples were seropositive to T. gondii. Only four animals were seropositive to both coccidia. No significant difference was observed between the N. caninum serology and any of the variables studied, such as dairy cattle management, milk production, reproductive problems, feeding, and presence of dogs, cats and rodents. These data suggest that neosporosis is present among dairy cattle in the studied geographic region and the simultaneous detection of serum positive animals to both types of coccidian parasite demonstrates the independent occurrence of these coccidia in dairy cows.

Evaluation of experimental Toxoplasma gondii (Nicolle and Manceaux, 1909) infection in pigs by bioassay in mice and polymerase chain reaction

The aim of the present experiment was to standardize a nested polymerase chain reaction (nPCR) for the detection of Toxoplasma gondii in tissues of experimentally infected pigs and to compare the performance of nPCR with the standard isolation technique, the bioassay in mice. Comparison between the two methods was done testing eight 4 month-old pigs orally inoculated with 5 x 104 oocysts of Toxoplasma gondii (AS-28 strain) and three non-infected pigs at the same age, kept as control. All animals were euthanatized 47 days after infection and samples of brain, heart, tongue and retina were collected from each animal for analysis by nPCR and bioassay in mice. By using the bioassay, Toxoplasma gondii was detected in 4 infected pigs, being two in the retina, one in the heart and one in the tongue. Toxoplasma gondii DNA was detected in five of the inoculated pigs, being: three in the tongue, two in the brain and heart and one in retina. The detection threshold of the nPCR on mouse brain suspension artificially infected with the RH strain of Toxoplasma gondii was 10 tachyzoites/ml. Although both techniques were unable to detect the parasite in all infected pigs, nPCR showed better performance as it was accomplished in a shorter period of time. When used concurrently, both techniques detected the agent in seven infected animals. The only way to increase sensitivity of either method is to increase the amount of tissue to be examined.

Experimental infection by Toxoplasma gondii using contaminated semen containing different doses of tachyzoites in sheep

Toxoplasmosis is a parasitic disease caused by Toxoplasma gondii that affects reproductive performance in small ruminants. Although the T. gondii life cycle is well understood since 1960s, several aspects related to its infection remain unclear. In the present study we hypothesized that sheep inseminated with T. gondii-contaminated semen would develop toxoplasmosis. In order to test that hypothesis, 41 sheep were experimentally infected with semen spiked with the organism. Females were divided in three groups (G1-G3): (a) females in G1 group were inseminated with semen containing 6.5 x 10(4) tachyzoites; (b) females in G2 group with semen containing 4 x 10(7) tachyzoites; and (c) females in G3 group with tachyzoite-free semen (control group). To confirm T. gondii infection via semen, serological tests were performed using indirect immunofluorescence reaction and the detection of parasite DNA in the blood stream using the nested PCR test. While in G1 group only 5/15 (33.3%) of the females presented seroconversion, all sheep in G2 15/15 (100%) seroconverted. The nested PCR test showed that 14/15 (93.3%) of the females in the G1 and 14/15 (93.3%) in the G2 group were positive for T. gondii while in the G3 group all samples were negative. In addition, ultra-sound test evidenced that in sheep presented embryonic reabsorption in animals from the infected groups. In conclusion, insemination using fresh semen experimentally contaminated with different infectant doses of T. gondii tachyzoites was able to infect sheep, leading to the possibility of toxoplasmosis transmission via semen.

Soroepidemiologia da toxoplasmose em gestantes a partir da implantação do Programa de Vigilância da Toxoplasmose Adquirida e Congênita em municípios da região oeste do Paraná

PURPOSE: To evaluate the susceptibility to toxoplasmosis in pregnant women in the public health service from two cities in the western region of Parana, Brazil. METHODS: Four thousand twenty-two pregnant women were evaluated for anti-Toxoplasma gondii IgG and IgM by ELISA and MEIA. Seronegative pregnant women repeated the serology in the second and third trimester of pregnancy. Neonatal screening of 27 newborns was also performed in one of the cities to detect IgM anti- Toxoplasma gondii by fluorometry. All pregnant women answered an epidemiological questionnaire to analyze the factors associated with the risk of infection by Toxoplasma gondii. For statistical analysis, the presence of IgG anti-Toxoplasma gondii was considered as the dependent variable and the variables contained in the epidemiological questionnaire as the independent ones. RESULTS: The prevalence of anti-Toxoplasma gondii IgG in pregnant women was 59.8 and 60.6%. In one of the cities, the variables associated with the presence of IgG antibodies were low educational level and more than one pregnancy. There was no association with other factors studied such as consumption of raw or undercooked meat, consumption of raw vegetables, consumption of colonial salami, handling soil or sand, the presence of a home vegetable garden and cats in the household. In the other city there was no statistical association with the variables studied. No case of acute infection and no seroconversion were confirmed in either city. None of the infants evaluated were positive for toxoplasmosis. CONCLUSION: Toxoplasmosis is common in pregnant women attended by the public health service in the region studied and 40% of them are susceptible to the infection. These data reinforce the need to keep the screening program in these cities.

Fatores de risco associados a soropositividade para leptospirose em matrizes suínas

Leptospirosis is a widespread zoonosis of great social and economic importance for swine production sistem. Two hundred and ninety-eight sows from farms in the north of Parana state were studied to individualize management practices or environmental condition that are related to risk of leptospirosis infection. All the animals were submitted to the microscopic seroaglutination test and an epidemiological questionnaire was filled out for each one to investigate variables that could be associated with this infection. The results were submitted to the c2 test, followed by multivariate analysis. Antibody titers of >100 were detected in 132 sows (44.3%) most frequently against icterohaemorrhagiae serovar (98.5%). The final model of multivariate analysis showed the risk factors to be flooded areas near the installation (OR=1.73; 1.04<IC95%<2.80), canal type water source (OR=1.58; 0.98<IC95%<2.55) and absence of cleaning of the water reservoirs (OR=2.25; 0.79<IC95%<6.43).

Detection of Toxoplasma gondii by PCR and mouse bioassay in commercial cuts of pork from experimentally infected pigs

The distribution of T. gondii in commercial cuts of pork (ham, tenderloin, spareribs and arm picnic) by PCR and bioassay from experimentally infected pigs, was evaluated. Eighteen mixed breed pigs were divided into two groups (G). The G1 animals (n=10) were infected with 4 x104 oocysts of the T. gondii VEG strain and the G2 animals (n=8) were used as control. Pigs of both groups were slaughtered at 59th day after infection, and meat samples were collected for bioassay and PCR. All animals from G1 were positive by at least one or both tests, and all control animals were negative. T. gondii was identified in pork by mouse bioassay and PCR in 27/40 (67.5%) and in 9/40 (22.5%) of the evaluated samples, respectively. There were no statistical differences in the distribution of tissue cysts from commercial cuts of pork by bioassay (P>0.05). However, statistical differences were observed when mouse bioassay and PCR were compared (P<0.01).