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 toT. gondii.

本研究旨在调查刚地弓形虫（Toxoplasma gondii）抗体，并从巴西屠宰场屠宰马匹的脑组织中分离该寄生虫。我们采集了来自巴西6个州的398头不同年龄段马匹的脑组织与血液样本。采用间接荧光抗体试验（indirect fluorescent antibody test, IFAT，临界滴度≥1:64）检测血清样本，脑组织样本则开展小鼠生物试验。在398头受试马匹中，经IFAT检测出46头（11.6%）刚地弓形虫抗体阳性，经小鼠生物试验检出14头（3.5%）阳性。在14例生物试验阳性样本中，12例仅通过IFAT检测呈阳性（临界滴度≥1:16），剩余2例成功分离得到刚地弓形虫。通过针对18S rDNA的聚合酶链反应-限制性片段长度多态性（polymerase chain reaction-restriction fragment length polymorphism, PCR-RFLP）分析，以区分刚地弓形虫、新孢子虫（Neospora caninum）与神经元肉孢子虫（Sarcocystis neurona），我们发现14份脑组织样本中仅2份为刚地弓形虫阳性。为对这2株分离株及PCR阳性脑组织进行基因分型，我们基于13个标记位点开展PCR-RFLP分析，且经SAG2基因检测，所有样本均为刚地弓形虫I型毒株。综上，对马匹血清的IFAT检测与小鼠生物试验共检出60份（15%）样本呈阳性。本研究结果表明，巴西送往屠宰场的部分马匹曾暴露于刚地弓形虫。