Detection of Toxoplasma gondii in faeces of privately owned cats using two PCR assays targeting the B1 gene and the 529-bp repetitive element

Abstract

Toxoplasma gondii infection is a worldwide parasitic zoonosis with a high-health risk for humans. The key epidemiological role played by felids is related to oocyst shedding. The present study compared two amplification protocols for the molecular diagnosis of Toxoplasma infection in owned cats. A total of 78 owned cats referred to an Italian university-teaching hospital and exposed to various T. gondii-associated risk factors were sampled for blood and faeces. Faecal specimens were processed by flotation and tested using 2 copro-PCRs targeting the widely used B1 gene and the 529-bp repetitive element (RE). The sera were tested by the indirect immunofluorescent antibody test (IFAT) for the detection of immunoglobulins against T. gondii. Sixteen faeces (20.52%) tested positive for T. gondii DNA; 12 samples were positive only at B1-PCR, two at 529-bp RE-PCR and two at both genetic targets (overall agreement = 82.11%). The amplicons obtained were sequenced, and the Basic Local Alignment Search Tool analysis showed a high homology with the T. gondii strains available in reference databases. Two stool samples were microscopically positive for T. gondii-like oocysts and also tested positive by both B1 and 529-bp RE-PCRs. Thirty-three (42.3%) sera tested positive for antibodies; of which, seven were found to have T. gondii DNA-positive results using the B1 genetic target (overall agreement = 57.77%). The amplification sets targeting B1 and 529-bp RE showed substantially different yields. Further research is needed to better understand the significance and the sensitivities of using these multi-copy-targeted molecular methods from cat faeces before being used for routine diagnosis.

Prevalence and genotyping of Toxoplasma gondii in stray cats in Mashhad area, Iran.

“…This result may be due to consumption of meat contaminated with Toxoplasma cysts or due to the small number of Toxoplasma oocysts in fecal specimens which are difficult to determine by fecal examination. Similar to our results, a low prevalence of oocysts shedding in cats was determined at 1.2% in Iran [16], at 2.3% in Italy [17], at 0.3% in Japan [18], at 0.14 % in Germany [19], at 0.4% in Switzerland [20], at 4.7% in South Korea [21], at 0.9 in the USA [22], at 0.8% in Thailand [23], at 0.76 % in Finland [24] by microscopy and PCR methods. In contrast to serological studies, examining feces did not show any information about age and gender as risk factors, due to the low prevalence of oocyst shedding by the cats [13,25].…”

Prevalence and genotyping of Toxoplasma gondii in stray cats in Mashhad area, Iran.

“…This result may be due to consumption of meat contaminated with Toxoplasma cysts or due to the small number of Toxoplasma oocysts in fecal specimens which are difficult to determine by fecal examination. Similar to our results, a low prevalence of oocysts shedding in cats was determined at 1.2% in Iran [16], at 2.3% in Italy [17], at 0.3% in Japan [18], at 0.14 % in Germany [19], at 0.4% in Switzerland [20], at 4.7% in South Korea [21], at 0.9 in the USA [22], at 0.8% in Thailand [23], at 0.76 % in Finland [24] by microscopy and PCR methods. In contrast to serological studies, examining feces did not show any information about age and gender as risk factors, due to the low prevalence of oocyst shedding by the cats [13,25].…”

Optimization of flotation, DNA extraction and PCR methods for detection of Toxoplasma gondii oocysts in cat faeces

“…Numerous studies have been published analyzing effectiveness of the concentration method for the detection of parasitic protozoa in stool samples [18,19,20]. Several studies concerning the molecular investigation of parasitic protozoa in stool samples have also been published [21,22,23]. These studies were generally intended for specific parasites protozoa.…”

“…Also in the presented study, PCR targeting the B1 gene, having 35 repetition in T. gondii genome, was applied. In a recent study by Veronesi et al [23], in which as the targets in PCR the B1 gene and the 529-bp repetitive element (RE) were used, the lower sensitivity of the 529-bp RE-PCR assay in comparison to the B1-PCR was determined. In the study by Chemoh et al [11], a comparative analysis on the occurrence of T. gondii in cat faeces using PCR assays targeting the 529-bp RE and ITS-1 regions was performed, and showed a seven times higher detection by ITS-1 primers than the 529-bp RE set primers.…”