Serological profile of anti-Toxoplasma gondii antibodies in apparently healthy dogs of the city of Botucatu, São Paulo state, Brazil

Toxoplasmosis is a zoonosis caused by Toxoplasma gondii, a coccidian protozoan of worldwide distribution. The seroprevalence in canine population can be an alternative for measuring T. gondii urban spreading. A total of 780 blood samples from dogs were collected, during the yearly anti-rabies campaign, carried out by the Department of Veterinary Hygiene and Public Health, School of Veterinary Medicine and Animal Husbandry (FMVZ), São Paulo State University, UNESP, together with the county health authorities, in August 1999. Using Indirect Fluorescent Antibody Test (IFAT) for detecting antibodies anti-T. gondii in the sera samples, we observed that 258 dogs (33.1%) were positive. The associations between the serological results and the epidemiological variables were studied. Statistically significant differences were not found regarding sex (32.2% male and 34.3% female reactors). Dogs without a defined breed showed seropositivity statistically higher than the pedigreed group. The occurrence of infection was considered higher with age.

Toxoplasma gondii: Comparison of a rhoptry-ELISA with IFAT and MAT for antibody detection in sera of experimentally infected pigs

Indirect ELISA and IFAT have been reported to be more sensitive and specific than agglutination tests. However, MAT is cheaper, easier than the others and does not need special equipment. The purpose of this study was to compare an enzyme linked immunosorbent assay using crude rhoptries of Toxoplasma gondii as coating wells (r-ELISA) with indirect fluorescence antibody test (IFAT) and modified agglutination test (MAT) to detect anti-T. gondii antibodies in sera of experimentally infected pigs. Ten mixed breed pigs between 6.5 and 7.5 weeks old were used. All pigs were negative for the presence of T. gondii antibodies by IFAT (titre < 16), r-ELISA (OD < 0.295) and MAT (titre < 16). Animals received 7 × 107 viable tachyzoites of the RH strain by intramuscular (IM) route at day 0. Serum samples were collected at days -6, 0, 7, 14, 21, 28, 35, 42, 50, and 57. IFAT detected anti-T. gondii antibodies earlier than r-ELISA and MAT. The average of antibody levels was higher at day 35 in IFAT (Log10 = 2.9) and in MAT (Log10 = 3.5), and at day 42 in r-ELISA (OD = 0.797). The antibody levels remained high through the 57th day after inoculation in MAT, and there was a decrease tendency in r-ELISA and IFAT. IFAT was used as gold standard and r-ELISA demonstrated a higher prevalence (73.3%), sensitivity (94.3%), negative predictive value (83.3%), and accuracy (95.6%) than MAT. Kappa agreements among tests were calculated, and the best results were shown by r-ELISA × IFAT (κ = 0.88, p < 0.001). Cross-reaction with Sarcocystis miescheriana was investigated in r-ELISA and OD mean was 0.163 ± 0.035 (n = 65). Additionally, none of the animals inoculated with Sarcocystis reacted positively in r-ELISA. Our results indicate that r-ELISA could be a good method for serological detection of T. gondii infection in pigs. © 2005 Elsevier Inc. All rights reserved.

Toxoplasma gondii: Detection by mouse bioassay, histopathology, and polymerase chain reaction in tissues from experimentally infected pigs

In the present study, we evaluated three techniques, mouse bioassay, histopathology, and polymerase chain reaction (PCR) to detect Toxoplasma gondii infection in tissues from experimentally infected pigs. Twelve mixed breed pigs, seronegative for T. gondii using an indirect immunofluorescent antibody test (IFAT), were used. Ten pigs were infected with 4 × 104 VEG strain oocysts, and two were maintained as uninfected controls. Animals were killed 60 days pos infection. Muscle (heart, tongue, diaphragm, and masseter) and brain samples were collected to investigate the presence of T. gondii tissue cysts by the different assay methods. For the bioassay, samples of brain (50 g) and pool of muscle samples (12.5 g of tongue, masseter, diaphragm, and heart) were used. PCR was performed using Tox4 and Tox5 primers which amplified a 529 bp fragment. The DNA extraction and PCR were performed three times, and all tissue samples were tested individually (brain, tongue, masseter, diaphragm, and heart). For histopathology, fragments of tissues were fixed in 10% of buffered formal saline and stained with HE. Histopathological results were all negative. PCR showed 25/150 (16.6%) positive samples, being 17/120 (14.1%) and 8/30 (26.6%) from muscle, and brain tissues, respectively. Tissue cysts of T. gondii were identified by mouse bioassay in 54/98 (55.1%) samples, being 31/48 (64.6%) from muscle samples, and 23/50 (46.0%) from brain samples. Toxoplasma gondii isolation in muscle samples by mouse bioassay was higher than in PCR (P < 0.01). Results indicate that DNA from pig tissues interfered with 529-bp-PCR sensitivity, and mouse bioassay was better than PCR in detecting T. gondii in tissues from pigs. © 2006 Elsevier Inc. All rights reserved.

Toxoplasma gondii genotyping in a dog co-infected with distemper virus and ehrlichiosis rickettsia

This paper reports a toxoplasmosis, erhlichiosis and distemper co-infection in a dog with an exuberant neuropathological clinical picture. Primary involvement was discussed based on information collected in the analysis of the clinical case, such as neurological impairment, epidemiological data, poor immunoprophylactic scheme of the dog affected and the role of these diseases on immunosuppression. Canine distemper and ehrlichiosis were diagnosed based on epidemiologic data, clinical signs, hematological and cytological evaluation. Toxoplasma gondii was isolated and genetically characterized as Type I using restriction analysis (RFLP) with SAG-2 genes. Immunosuppression features of both dogs and human beings are discussed, as well as implications on animal and public health. This is the first report on toxoplasmosis, ehrlichiosis and distemper co-infection in a dog in Brazil, associated with genotyping determination of the T. gondii strain involved.

Presença de anticorpos anti-Neospora caninum e Toxoplasma gondii em cães com leishmaniose visceral da região de Araçatuba, São Paulo, Brasil

Dogs that had positive and negative sera to Leishmania chagasi from the region of Araçatuba, São Paulo, Brazil, were evaluated for the presence of anti-Neospora caninum and anti-Toxoplasma gondii antibodies as potential co-infecting agents. Blood samples were collected from 204 dogs and out of them 98 were carriers of leishmaniosis. Sera were tested for the presence of anti-L. chagasi antibodies by ELISA, and anti-T. gondii and anti-N. caninum by an indirect fluorescent antibody test (IFAT). Age, gender, and association between the presences of anti-L. chagasi antibodies and seroprevalence to N. caninum and T. gondii were analyzed by chi-square test. Out of the 204 sera investigated, 36 (17.6%) were positive for N. caninum (IFAT=50) and 75 (36.8%) to T. gondii (IFAT=16) with titers that varied from 50 to 6400 for N. caninum, and from 16 to 16384 for T. gondii. The copresence of anti-L. chagasi, N. caninum and T. gondii antibodies was observed in 17 (8.3%) dogs. Antibodies to N. caninum were observed in four (3.8%) out of 106 dogs that were negative for L. chagasi, and in 32 (32.6%) out of the 98 dogs that were positive for L. chagasi. Anti-T. gondii antibodies were found in 40 (41.0%) and in 35 (33.0%) of the 98 positive dogs and in 106 negative dogs for L. chagasi, respectively. An association between the presence of antibodies against L. chagasi and a positive response to N. caninum (p<0.001) was observed. The gender and age of the dogs did not show an association between the presence of antibodies and any of the agents studied (p>0.05), with the exception of age and presence of anti-L. chagasi antibodies, in which only a slight association was observed (p=0.038). Within this interaction, a higher number of dogs, older than four years, were positive for this agent when compared to other age groups.

Toxoplasma gondii in semen of experimentally infected swine

Eight reproductive boars were divided into three groups and inoculated with Toxoplasma gondii [GI (n=3) 1.5×104 oocysts strain P; GII (n=3) 1.0×106 tachyzoites strain RH; and GIII (n=2) non-inoculated control]. Clinical, hematological, parasitemia and serological tests and studies of the parasite in the semen through bioassay and PCR, and in reproductive organs (Bioassay and immunohistochemical analyses) were conducted to evaluate the toxoplasmic infection. Blood and semen were collected on day -2, -1, 1, 3, 5, 7, 9, 11, 14 and weekly up to 84 days post-inoculation (DPI). No clinical or hematimetric alteration was observed in the boars. Parasitemia was detected in one boar inoculated with oocysts at the 7th DPI and in another boar infected with tachyzoites (GII) at the 3rd and 49 th DPI. Serological tests revealed antibodies against T. gondii in animals inoculated with oocysts or tachyzoites at the 7th DPI with dilutions of 1:256 and 1:64, which reached peaks of 1:4096 at day 11 and 9, respectively. The bioassays revealed the presence of the parasite in semen samples of a boar inoculated with oocysts (GI) at 3, 49 and 56 DPI and from two boars infected with tachyzoites (GII), one animal at 5 and two animals at 49 days DPI. Mice inoculated with semen from the control group (GIII) remained serologically negative. PCR analysis showed T. gondii DNA in the semen of Boar 1 and Boar 3 inoculated with tachyzoites and oocysts, respectively. The immunohistochemical tests showed T. gondii in the reproductive organs of Boar 1 and Boar 2, inoculated with tachyzoites and oocysts, respectively. These findings suggest the possible occurrence of venereal transmission of T. gondii in swine.

Cytomegalovirus and Toxoplasma gondii seroprevalence in a Brazilian liver transplant waiting list

Cytomegalovirus (CMV) disease is a major cause of morbidity and mortality in solid organ transplantation. Disseminated toxoplasmosis after liver transplantation is a rare but fatal event. Serologic screening of the donor and the recipient is essential to prophylactic management, early diagnosis and therapeutic strategies to minimize the consequences of these infections. The aim of the present study was to determine the seroprevalence of CMV and Toxoplasma gondii (TG) in a Brazilian liver transplant waiting list (LTWL). Serological data were collected from 44 candidates on the LTWL between May 2003 and November 2004. Serological investigation of antibodies IgM and IgG against CMV (anti-CMV) and TG (anti-T. gondii) was performed using fluorometry commercial kits. IgG anti-CMV was positive in 37 patients (94.9%) out of 39 available results. There were not IgM anti-CMV positive results. Out of 36 analyzed patients, 22 (61.1%) presented positive IgG anti-T. gondii and none had positive IgM anti-T. gondii. The high CMV seroprevalence among our LTWL reinforces the need for appropriate protocols to avoid related complications, like reactivation and superinfection by CMV. Environmental and drug prophylactic strategies against primary infection and reactivation, as well as early diagnosis and treatment of toxoplasmosis complications, are essential for the good outcome of transplant patients.

Utilização do método de aglutina ção direta e da reação de imunofluorescência indireta na detecção de anticorpos para Toxoplasma gondii em cavalos naturalmente infectados

Infection by Toxoplasma gondii in equines is usually not apparent, it being characterized by presence of antibody titers and tissue cysts. This study was aimed at verifying the presence of anti-Toxoplasma antibodies in equine serum by modified agglutination test (MAT) and reaction to indirect immunofluorescent antibody test (IFAT). 1984 samples of serum were examined, by MAT, using whole formalin fixed tachyzoites of T.gondii as antigen. The samples reacting in the MAT test, and 150 other negative samples, chosen at random, were also tested by IFAT, utilizing anti-equine IgG. The association among the test results was verified by the McNemar test. 138 samples were positive in the MAT test, with 60 (46.38%) presenting reaction at a dilution of 1:64; 52 (37.7%) at 1:256; 19 (13.8%) at 1:1024; five (3.6%) at 1:4096; and two (1.45%) at 1:16384. Of 132 positive MAT samples, 14 were negative in the IFAT test, but the statistical analysis indicated general agreement in results of the tests. The results obtained showed agreement among the tests utilized, and the possibility of participation of equines in the transmission of toxoplasmosis to carnivorous animals, and also to humans.

Evaluation of IFA, MAT, ELISAs and immunoblotting for the detection of anti-Toxoplasma gondii antibodies in paired serum and aqueous humour samples from experimentally infected pigs

The study evaluated the efficiency of diagnostic laboratory methods to detect anti-Toxoplasma gondii antibodies in paired serum and aqueous humour samples from experimentally infected pigs. 18-mixed breed pigs were used during the experiment; these were divided into two groups, G1 (infected group, n = 10) and G2 (uninfected group, n = 8). Infection was performed with 4 × 10 4 VEG strain oocysts at day 0 by the oral route in G1 animals. All pigs were euthanized at day 60, when retina, aqueous humour, and blood samples were collected. Anti-T. gondii antibody levels were assessed in serum (s) and aqueous humour (ah) by indirect immunofluorescence assay (IFA), modified agglutination test (MAT), m-ELISA (using crude membranes from T. gondii tachyzoites as antigen) and r-ELISA (using rhoptries from T. gondii tachyzoites as antigen). Polymerase chain reactions (PCR) of samples from the retina were performed by using Tox4 and Tox5 primers. Antibody titers of G1 animals ranged from 128 to 1024 and from 16 to 256 in serum and aqueous humour, respectively. There were differences in the correlation coefficients between IFA(s) × IFA (ah) (r = 0.62, P = 0.05), MAT(s) × MAT (ah) (r = 0.97, P < 0.0001); however, there was no significant difference between r-ELISA(s) × r-ELISA (ah) (r = 0.14, P = 0.7). Antibodies present in serum and aqueous humour recognized similar antigens. Samples of retina were positive by PCR in 30% (3/10) of infected pigs. G2 animals remained without antibody levels and were PCR negative throughout the experiment. These results suggest that the use of a combination of tests and immunoblotting for paired aqueous humour and serum samples could improve the sensitivity and specificity for the diagnosis of ocular toxoplasmosis. © 2007 Elsevier Ltd. All rights reserved.

Semen variables of sheep (Ovis aries) experimentally infected with Toxoplasma gondii

The influence of Toxoplasma gondii on semen variables and sperm morphology of sheep was evaluated in eight reproductive males distributed into three experimental groups: GI, three sheep inoculated with 2.0 × 105 of P strain oocytes; GII, three sheep infected with 1.0 × 106 of RH strain tachyzoites and; GIII two control sheep. Clinical (rectal temperature, cardiac and respiratory frequencies), parasite and serology exams (IIF) were realized. Sperm variables (volume, motility, vigor and concentration) and semen morphology for each sheep were also evaluated. Thus, semen and blood collections were assessed on post-inoculation days (PIDs)-1,3,5,7,11,14 and weekly thereafter up to PID 70. Clinical alterations were observed (hypothermia and anorexia) in infected sheep from groups GI and GII. Parasitic outbreaks were detected in five sheep. All the infected sheep produced antibodies against T. gondii from PID 5 onwards, reaching a peak of 4096 and 8192 for group GI and GII sheep, respectively. Differences (P < 0.05) were observed regarding the ejaculate volume between the inoculated groups (oocytes and tachyzoites) and control. Even though experimental toxoplasmic infection resulted in clinical symptomology in the inoculated sheep, the minimal alterations in sperm pathologies could not be directly attributed to T. gondii. © 2008 Elsevier B.V. All rights reserved.

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 ≥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. © 2009 American Society of Parasitologists.

Serological survey of Toxoplasma gondii in captive Neotropical felids from Southern Brazil

Toxoplasma gondii is the causative intracellular protozoan of toxoplasmosis in human being and animals. Members of the Felidae family are considered the single definitive host for the infection; both wild and domestic cats are able to excrete oocysts in the environment. Wild cats maintained in captivity may serve as source of infection for other clinically susceptible animals in the same environment. The aim of this study was to determine the frequency of T. gondii IgG antibodies in 57 neotropical felids (1 Leopardus geoffroyi; 3 Puma yagouaroundi; 17 Leopardus wiedii; 22 Leopardus tigrinus; and 14 Leopardus pardalis) kept at the Bela Vista Biological Sanctuary, Itaipu Binacional, Southern Brazil, by the modified agglutination test (MAT) using titer 16 as cut-off point. Seropositivity was observed in 38/57 (66.67%; 95% CI 53.66-77.51%) samples, with higher frequency in ocelots (71.43%). Wild-caught felids were three times more likely to be infected when compared to zoo-born animals (P≤ 0.05) and age of wild-caught animals (P= 0.6892; 95% CI. = 0.7528-1.66) was not significant as a risk factor for the infection, the same occurring with zoo-born animals (P= 0.05; 95% CI. = 0.6267-24.052). These results suggest that, despite efforts to control T. gondii infection in zoo facilities, such as individual pens, hygiene monitoring, veterinary care and pre-frozen meat offered as food, non-domestic felids kept in captivity, particularly the wild-caught specimens, may be invariably exposed to infection due to other environmental sources. © 2010 Elsevier B.V.

Detecção de anticorpos anti-Toxoplasma gondii em cavalos carroceiros da região metropolitana de Curitiba, Paraná, Brasil

Toxoplasma gondii, the agent for toxoplasmosis, has worldwide distribution. Horses normally play a secondary role in its life cycle, but movement around urban areas, feeding on grass and the increasing use of carthorses for gathering recyclable material in some urban areas of Brazil may increase their exposure to T. gondii infection. The aim of the present study was to investigate the frequency of anti-T. gondii antibodies in carthorses in the metropolitan region of Curitiba, PR. IgG antibodies against T. gondii were detected using the indirect fluorescence antibody test (IFAT) (titers ≥ 64). Seventeen (17.0%) of the 100 horses sampled were seropositive. There were no statistical differences in relation to sex (p = 0.28) or age (p = 0.15). Our findings suggest that carthorses are exposed to T. gondii infections and that no associations with age or sex exist.

Ocorrência de anticorpos contra Leishmania spp., Neospora caninum e Toxoplasma gondii em soros de cães atendidos no Hospital Veterinário da Universidade Estadual de Londrina-Pr

The aim of this study was to detect the presence of IgG antibodies anti-Leishmania spp., Toxoplasma gondii and Neospora caninum in dogs from a Veterinary Hospital from Universidade Estadual de Londrina. Blood samples from 112 animals were obtained by jugular venipuncture to obtain sera. The samples were tested by indirect immunofluorescence to detect antibodies anti-Leishmania spp., anti-N. caninum and anti-T. gondii. Thirteen (11.61%), 25 (22.32%), and 57 (50.89%) samples were positive for Leishmania spp., N. caninum, and T. gondii, respectively. The co-presence of anti-Leishmania spp. and N. caninum was observed in 6 (5.36%), anti-Leishmania spp. and anti-T. gondii in 8 (14.7%), and anti-N. caninum and anti-T. gondii in 18 (16.07%) samples. The co-presence of anti-Leishmania spp., anti-N. caninum and anti-T. gondii was observed in 5 (4.46%) dogs. There was a higher prevalence of Leishmania in Toxoplasma and Neospora positive animals, however, these results were not statistically significant (range p = 0.052 p = 0.06). The dogs have an important role in the epidemiological cycle of these diseases, which are important in animal and public health. The northern state of Paraná is an endemic area for human cutaneous leishmaniasis, therefore, studies should be conducted to uncover the real role of dogs as reservoirs of Leishmania to humans in the state.

Falhas reprodutivas associadas com a presença de do vírus da artrite-encefalite caprina, toxoplasma gondii e neospora caninum em caprinos no estado de São Paulo, Brasil

This study aimed at assessing the occurrence of antibodies against the caprine arthritis-encephalitis virus (CAEV), Toxoplasma gondii and Neospora caninum, as well as the associations between the presence of antibodies and the occurrence of reproductive failures in goats. Serum samples were collected from 923 goats of both sexes, over 3 months of age, from 17 dairy farms located in different municipalities of São Paulo State, Brazil. Infections by T. gondii, N. caninum and CAEV were evaluated by indirect methods of diagnosis based on indirect fluorescence antibody test (IFAT), Neospora agglutination test (NAT), and agar gel immunodiffusion (AGID), respectively. A survey was conducted on the farms to obtain information about reproduction dates (abortions, stillbirths and births of weak and premature kids) and zoosanitary management. Antibodies against CAEV, T. gondii and N. caninum was found in 37.81%, 23.62% and 17.23% respectively. There was no significant association between the presence of anti-CAEV antibodies and CAEV/T. gondii or CAEV/N. caninum co-infection, suggesting that CAEV does not predispose goats to infection by these agents. However, when CAEV/T. gondii (p<0.01) or CAEV/N. caninum (p<0.001) co-infection was present, the occurrence of reproductive failures was significantly higher what could indicate that CAEV-induced immunosuppression may predispose goats to develop the clinical symptoms of toxoplasmosis and neosporosis increasing the risks of the reproductive failures.