Seroprevalence and phylogenetic analysis of Toxoplasma gondii from domestic cats, captive wild felids, free-range wild felids and rats in certain regions of Thailand

Toxoplasmosis is an important zoonotic disease caused by Toxoplasma gondii, an obligated zoonotic apicomplexan parasite. The infection varies according to geographical areas. This work aimed to study the seroprevalence and genotype of T. gondii infection in domestic, captive and free-range wild felids, and in their small mammal prey, rats (Rattus spp). Two hundred and ninety three sera, received from the 4 individual animal groups in Thailand, were tested using the indirect latex agglutination test (ILAT) for specific antibody detection. The nested-PCR for glycerol-3-phosphate (B1) and bradyzoite surface antigen (SAG4) gene detection was used to detect seropositive animals and PCR product was submitted for DNA sequencing. Out of the 293 sera, ILAT showed 11.68% positive results. T. gondii were found 3.48% seropositive in the domestic cats (n=86), 18.84% seropositive in the captive wild felids (n=138), 14.28% seropositive in the free-range wild felids (n=7), and 6.67% seropositive in the murine prey (n=60). Tissues from the seropositive animals such as liver, heart, brain and skeletal muscle were collected, and then DNA was extracted to perform nested-PCR and sequence analysis. By the nested-PCR, the brain and muscle tissues received from 3 black rats and a clouded leopard (1.37%) were found positive for T. gondii. SAG4 and B1 might serve as novel genetic markers for population genetic studies of T. gondii isolates. Based on the ML phylogenetic tree analysis of SAG4 and B1 coding sequences, T. gondii found in 3 murine prey and a clouded leopard was close to T. gondii RH type I strain with approximately 99-100% similarity. This is the first report on the relation of T. gondii infection with strain identification in domestic cats, captive and free-range felids, and murine in Thailand. Better understanding of the genetic diversity will lead to better management, prevention and treatment of this disease in the valuable species of wild felids

ELISA based on a recombinant Paragonimus heterotremus protein for serodiagnosis of human paragonimiasis in Thailand

Abstract Background Paragonimus heterotremus is the main causative agent of paragonimiasis in Thailand. In Western blot diagnostic assays for paragonimiasis, the 35 kDa band present in crude P. heterotremus somatic extracts represents one of the known diagnostic bands. This study aimed to use a P. heterotremus cDNA library to create a recombinant version of this antigen for use in immunodiagnosis of paragonimiasis. Methods To accomplish this aim a cDNA expression library was constructed from adult worm mRNA and immuno-screened using antibodies from mice that had been immunized with the 35 kDa antigen. Screening resulted in the identification of an immunoreactive protein encoded by clone CE3, which contained an inserted sequence composed of 1292 base pairs. This clone was selected for use in the construction of a recombinant P. heterotremus protein because of its similarity to proactivator polypeptide. For recombinant protein expression, the CE3 gene sequence was inserted into the plasmid vector pRset and the resulting product had the expected molecular weight of 35 kDa. An IgG-ELISA based on the CE3 recombinant protein was evaluated by using sera from healthy individuals, from patients with paragonimiasis and other parasitic infections. This ELISA was performed by using human sera diluted at 1:2000, an optimized antigen concentration of 1 μg/ml, and anti-human IgG diluted at 1:4000. Results The cut-off optical density value was set as the mean + 2 standard deviations (0.54), which resulted in the test having a sensitivity of 88.89% and a specificity of 95.51%. The recombinant antigen could react with antibodies from P. heterotremus, P. pseudoheterotremus and P. westermani infections. Cross-reactivity occurred with a few cases of Blastocystis hominis infection (2/3), Bancroftian filariasis (1/10), opisthorchiasis (3/10), strongyloidiasis (4/10) and neurocysticercosis (1/11). Conclusions Given the high test sensitivity and specificity, reflected in the low level of heterologous infection cross-reactivity (11/215 serum samples), observed in the IgG-ELISA, this 35 kDa antigen may be useful for the detection of paragonimiasis