Jonatas Campos de Almeida

Diagnosis and isolation of Toxoplasma gondii in horses from Brazilian slaughterhouses

This study aimed to investigate anti-Toxoplasma gondii antibodies and to isolate the parasite from the brains of horses processed at slaughterhouses in Brazil. We collected brain and blood samples from 398 horses of various ages, from six Brazilian states. Serum samples were evaluated by indirect fluorescent antibody test (IFAT cut-off titre ≥ 1:64), and brains were submitted to mouse bioassay. Among the 398 horses, positivity for T. gondii was identified in 46 (11.6%) by IFAT and in 14 (3.5%) by mouse bioassay. In 12 of those 14 bioassays, mice were positive only by IFAT (cut-off titre ≥ 1:16), T. gondii being isolated in the remaining two. Using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of 18S rDNA to differentiate among T. gondii, Neospora caninum, and Sarcocystis neurona, we found that two of the 14 brains were positive for T. gondii only. For genotyping of the two isolates and the PCR-positive brain, we performed PCR-RFLP based on 13 markers, and SAG2 all samples were Toxoplasma gondii type I. Collectively, IFAT of horse sera and mouse bioassay identified positivity in 60 (15%) of the samples. Our results show that some horses sent to slaughter in Brazil have been exposed to T. gondii.

Occurrence of Cryptosporidium spp. and Giardia spp. in a public water-treatment system, Paraná, Southern Brazil

The purpose of this study was to investigate the occurrence of Cryptosporidium spp. and Giardia spp. in a public water-treatment system. Samples of raw and treated water were collected and concentrated using the membrane filtration technique. Direct Immunofluorescence Test was performed on the samples. DNA extraction using a commercial kit was performed and the DNA extracted was submitted to a nested-PCR reaction (n-PCR) and sequencing. In the immunofluorescence, 2/24 (8.33%) samples of raw water were positive for Giardia spp.. In n-PCR and sequencing, 2/24 (8.33%) samples of raw water were positive for Giardia spp., and 2/24 (8.33%) samples were positive for Cryptosporidium spp.. The sequencing showed Cryptosporidium parvum and Giardia duodenalis DNA. In raw water, there was moderate correlation among turbidity, color and Cryptosporidium spp. and between turbidity and Giardia spp.. The presence of these protozoans in the water indicates the need for monitoring for water-treatment companies.

Cryptosporidium spp. and Giardia spp. in feces and water and the associated exposure factors on dairy farms

The aims of this study were to verify the prevalence of Cryptosporidium spp. and Giardia spp. in animal feces and drinking water on dairy farms and to identify a possible relation between the exposure factors and the presence of these parasites. Fecal samples from cattle and humans and water samples were collected on dairy farms in Paraná, Brazil. Analysis of (oo)cysts in the feces was performed by the modified Ziehl-Neelsen staining and centrifugal flotation in zinc sulfate. Test-positive samples were subjected to nested PCR amplification of the 18SSU ribosomal RNA gene for identification of Cryptosporidium and Giardia and of the gp60 gene for subtyping of Cryptosporidium. Microbiological analysis of water was carried out by the multiple-tube method and by means of a chromogenic substrate, and parasitological analysis was performed on 31 samples by direct immunofluorescence and nested PCR of the genes mentioned above. Identification of the species of Cryptosporidium was performed by sequencing and PCR with analysis of restriction fragment length polymorphisms. The prevalence of Giardia and Cryptosporidium was higher in calves than in adults. Among the samples of cattle feces, Cryptosporidium parvum was identified in 41 (64%), C. ryanae in eight (12.5%), C. bovis in four (6.3%), C. andersoni in five (7.8%), and a mixed infection in 20 samples (31.3%). These parasites were not identified in the samples of human feces. Thermotolerant coliform bacteria were identified in 25 samples of water (45.5%). Giardia duodenalis and C. parvum were identified in three water samples. The gp60 gene analysis of C. parvum isolates revealed the presence of two strains (IIaA20G1R1 and IIaA17G2R2) in the fecal samples and one (IIaA17G2R1) in the water samples. The presence of coliforms was associated with the water source, structure and degradation of springs, rain, and turbidity. The prevalence of protozoa was higher in calves up to six months of age. C. parvum and G. duodenalis were identified in the water of dairy farms, as were thermotolerant coliforms; these findings point to the need for guidance on handling of animals, preservation of water sources, and water treatment.

Serosurvey of anti-Toxoplasma gondii antibodies in sport horses from Paraiba state, Northeastern Brazil

Although the infection by Toxoplasma gondii in horses is usually asymptomatic, it may be characterized by the presence of tissue cysts and maintenance of detectable antibody titers. The aim of this study was to detect anti-T.gondii antibodies and to determine risk factors associated in sport horses from Paraíba State, northeastern Brazil. A total of 138 apparently healthy sport horses of different age and gender were sampled. Anti-Toxoplasma gondii antibodies were detected in 16/138 (11.6%; 95% CI: 6.77–18.14%) horses by indirect immunofluorescence antibody assay (IFA). A significant association was found between the water source and seropositivity to T. gondii (p<0.05). The present study highlights the importance of providing good quality water to the animals.

Toxoplasma gondii in backyard pigs: seroepidemiology and mouse bioassay

The aim of this study was to evaluate the occurrence of Toxoplasma gondii in backyard pigs destined for human consumption in Pernambuco state, Brazil. Blood and tissue samples (liver, heart, brain, lung and diaphragm) were collected from 224 pigs from legal slaughterhouses and tested for T. gondii infection. Antibodies to T. gondii were found in the sera of 37.9% (85/224) by using the immunofluorescence antibody test (cut-off–1:64). Tissue samples from seropositive pigs were bioassayed in mice. Tissue samples from seropositive pigs and from mice of the bioassay were submitted to histopathology, immunohistochemistry, polymerase chain reaction (PCR) and sequencing; 14.1% of pig tissue samples and 27.7% of bioassayed mouse samples were positive for T. gondii DNA, but all pig and mouse tissues were negative in histopathology analysis and immunochemistry. By using a risk assessment questionnaire, there was significant difference (p<0.001) in seroprevalence of 21.2% (reproducer) and 3.1% (finishing pig). These data serve as indicative of the sanitary conditions and risk of T. gondii infection for backyard pigs. Preventive measures must be implemented by health services to avoid toxoplasmosis human cases due to ingestion of pig meat.

Molecular and serological investigation of infectious diseases in captive and free-range crab-eating fox (Cerdocyon thous – Linnaeus, 1776) from northeastern Brazil

The aim of this study was to detect DNA and antibodies anti-Leishmania spp., Neospora caninum and Toxoplasma gondii in captive and free-range crab-eating fox (Cerdocyon thous) from northeastern Brazil. Twenty-five crab-eating foxes from different states of northeastern Brazil were sampled by this study. Blood samples were collected by cephalic or jugular vein punctures. The whole blood was submitted to PCR, and the sera samples to the serological analysis as follows: MAT for T. gondii, NAT for N. caninum, and ELISA for L. chagasi. The frequence of antibodies anti-T. gondii was 50% and 29.41% for free-range and captive wild canids, respectively. The frequence of antibodies anti-N. caninum observed by this study was 62.50% and 23.52% for free-range and captive wild canids, respectively. The frequence of antibodies anti-L. chagasi was 4.0% for captive wild canids. Co-infections cases were identified as follows: one captive wild canid seropositive for T. gondii and L. chagasi and two free-range animals seropositive for T. gondii and N. caninum. All PCR assays performed were negative for the pathogens analyzed. This study describes the presence of antibodies anti-T. gondii, N. caninum e L. chagasi in wild canids from northeastern Brazil and highlights the necessity of further studies on infectious diseases in free-range and captive wild canids.

Comparison of indirect fluorescent antibody test and the modified agglutination test for the detection of Toxoplasma gondii antibodies in stray dogs from Southern Brazil

The aim of the present study was to determine the prevalence of antibodies to Toxoplasma gondii by two serological techniques in sera of 364 stray dogs from Brazil by immunofluorescence antibody test (IFAT, cut off point 1:16) and to the modified agglutination test (MAT, cut-off points 1:25 and 1:50). A total of 175/364 (48.07%) sera were positive by IFAT, and 108/364 (29.67%) and 85/364 (23.35%) were positive by MAT with cutoff points 1:25 and 1:50, respectively were positive by MAT. Cohen’s Kappa Coefficient between IFAT and MAT was 0.81 (excellent) and 0.66 (substantial) with cutoff points 1:25 and 1:50, respectively. Using IFAT as gold standard, MAT sensitivity and specificity were 78% and 99% for 1:25 and 61% and 99% for 1:50, respectively. The results document of the usefulness of MAT for serological diagnosis because it does not require species-specific conjugate.