Department of Preventive Veterinary Medicine, Laboratory of Animal Protozoology, Universidade Estadual de Londrina, Rodovia Celso Garcia Cid, PR 445 km 380, 86057-970, Londrina, PR, Brazil.
Parasitology and Zoology Laboratory, Universidade Estadual do Norte do Paraná - UENP, Jacarezinho, PR, Brazil.
Vet Parasitol. 2021 Aug;296:109515. doi: 10.1016/j.vetpar.2021.109515. Epub 2021 Jul 1.
The present study aimed to compare different indirect and direct diagnostic techniques to diagnose Toxoplasma gondii in free-range chickens. Samples of 386 chickens obtained from 24 Paraná properties were used for serological analysis by indirect fluorescent antibody test (IFAT), modified agglutination test (MAT), and enzyme-linked immunosorbent assay (ELISA). Animals positive by IFAT and/or MAT had their tissues submitted to the mouse bioassay, and those who were positive in this technique had their blood, tissues, and acidic pepsin tissue digestion submitted to PCR (conventional, nested, and quantitative-PCR (qPCR)). One hundred and nineteen chickens (30.8 %) were positive in at least one of the serological tests, being 102 (26.4 %) in the IFAT, 64 (16.6 %) in the MAT, and 62 (16.0 %) in the ELISA. The IFAT was used as a gold standard, and the MAT showed higher sensitivity (46.0 %) and specificity (94.0) compared to ELISA (43.5 % and 93.6 %, respectively). Ninety samples of eighteen chickens positive in the mouse bioassay were subjected to PCR, and according to molecular tests, the conventional PCR detected the T. gondii DNA in 30 % (27/90) of the samples, in 38.8 % (35/90) with nested-PCR and 40.0 % (36/90) with real-time. According to molecular analyzes, the sensitivity was higher in ITS1 nested-PCR (69.4 %) and specificity in conventional PCR-529bp (90.7 %), using the qPCR as the gold standard. MAT and ELISA had similarities in concordance analyzes. The IFAT was the serological technique with the highest agreement with the mouse bioassay, and serological tests in parallel showed to be a good screening option for the isolation of T. gondii in chick tissues. The PCR markers effectively detected the parasite DNA, and the heart was the tissue with the highest number of positives samples. The conventional PCR had sensitivity similar to nested-PCR and qPCR and could be a cheaper alternative to diagnose T. gondii infection in chicken tissues.
本研究旨在比较不同的间接和直接诊断技术，以诊断自由放养鸡中的刚地弓形虫。从巴拉那州的 24 个农场采集了 386 只鸡的样本，用于间接荧光抗体试验（IFAT）、改良凝集试验（MAT）和酶联免疫吸附试验（ELISA）的血清学分析。IFAT 和/或 MAT 阳性的动物将其组织进行小鼠生物检测，该技术阳性的动物将其血液、组织和酸性胃蛋白酶组织消化液进行 PCR（常规、巢式和定量-PCR（qPCR））检测。119 只鸡（30.8%）在至少一种血清学检测中呈阳性，IFAT 阳性 102 只（26.4%），MAT 阳性 64 只（16.6%），ELISA 阳性 62 只（16.0%）。IFAT 被用作金标准，与 ELISA（分别为 43.5%和 93.6%）相比，MAT 显示出更高的敏感性（46.0%）和特异性（94.0%）。18 只鸡中的 90 个生物检测阳性样本进行了 PCR，根据分子检测，常规 PCR 在 30%（27/90）的样本中检测到弓形虫 DNA，巢式 PCR 检测到 38.8%（35/90），实时 PCR 检测到 40.0%（36/90）。根据分子分析，ITS1 巢式-PCR 的敏感性更高（69.4%），常规 PCR-529bp 的特异性更高（90.7%），以 qPCR 作为金标准。MAT 和 ELISA 在一致性分析中有相似之处。IFAT 是与小鼠生物检测最一致的血清学技术，而平行的血清学检测显示是分离鸡组织中弓形虫的良好筛选方法。PCR 标志物有效地检测到寄生虫 DNA，心脏是阳性样本数量最多的组织。常规 PCR 的敏感性与巢式-PCR 和 qPCR 相似，可作为诊断鸡组织中弓形虫感染的更经济选择。
