Cytologic detection of Toxoplasma gondii in the cerebrospinal fluid of a dog and in vitro isolation of a unique mouse-virulent recombinant strain.

Parasites resembling Neospora caninum or Toxoplasma gondii were detected by cytologic examination of cerebrospinal fluid (CSF) from a dog with neurologic disease. The dog became severely ill and was euthanized. Canine tissue homogenates were used for direct parasite isolation in cell culture, bioassay in 2 mouse lineages, and PCR. T. gondii was isolated in monkey kidney cells, and species identity was confirmed by PCR. Inoculated parasites were highly virulent for mice, which developed clinical signs and were euthanized immediately. PCR-RFLP for T. gondii using the cultured isolate (TgDgBA22) was conducted with 12 genetic markers, and a unique recombinant strain was identified. Detection of T. gondii by CSF cytology, although described in humans, had not been reported previously in dogs, to our knowledge, and was crucial for the diagnosis of toxoplasmosis in the examined dog.

Genetic Diversity of Toxoplasma gondii Isolates From Free-Range Chickens In Bahia, Brazil.

The genotyping of 25 isolates of Toxoplasma gondii from free-range chickens in the state of Bahia, Brazil, was performed by PCR-restriction fragment length polymorphism using 11 genetic markers: SAG1, 5'+3'SAG2, alt.SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, Apico, and CS3. The analysis revealed 8 genotypes, 3 of which had not been previously reported. Four genotypes were represented by single isolates, whereas the other genotypes were represented by 2 or more isolates. Five isolates showed mixed infections, and 2 of them were identical. None of the clonal types I, II, or III were found, but 2 isolates corresponded to the Brazilian clonal lineage BrIII. There was a single allele for the c22-8 marker. The CS3 marker demonstrated efficiency in the evaluation of virulence in mice. This study reaffirms the diverse genetic variability of T. gondii in Brazil.

Comparison of methods for detection of Toxoplasma gondii in tissues of naturally exposed pigs.

This study aimed to compare the sensitivity of five diagnostic methods commonly used for the detection of Toxoplasma gondii in tissues of naturally infected pigs. We purchased 20 heads of pigs in butcher shops in the city of Ilhéus, Bahia. Brain and tongue fragments were taken from each animal for the performance of PCR against T. gondii. The rest of these two tissues were processed and inoculated into three mice. These rodents were observed for 42 days and euthanized. We prepared slides with brain and lungs of each mouse for the visualization of T. gondii. From the tissues of mice, we carried out polymerase chain reaction (PCR), histopathology, and immunohistochemistry in an attempt to identify the parasite. The PCR direct from the tissue of pigs showed 10% (2/20) of positive samples, all from the brain. PCR in tissue from mice found that 55% (11/20) of pigs were positive: 55% (11/20) and 45% (9/20) for brains and tongues, respectively. Mice were inoculated with material obtained from the samples and examined by various methods for resulting Toxoplasma infection (bioassay). Cyst detection in bioassay mice identified 25% (5/20) and immunohistochemistry 30% (6/20) of the samples pigs as positive for T. gondii. Histopathology of mice tissue could not detect parasite; only suggestive pathological changes such as inflammation with foci of necrosis were seen. The results indicated PCR of mice tissue as the most sensitive among those tested.