Garcia João Luis, Gennari Solange Maria, Machado Rosângela Zacarias, Navarro Italmar Teodorico
Microbiology and Immunology laboratory, Departamento de Biologia, Universidade Estadual do Centro Oeste-UNICENTRO, R. Simeão Camargo Varela de Sá, 03, Bairro Cascavel, 85040-080 Guarapuava, PR, Brazil.
Exp Parasitol. 2006 Aug;113(4):267-71. doi: 10.1016/j.exppara.2006.02.001. Epub 2006 Mar 20.
In the present study, we evaluated three techniques, mouse bioassay, histopathology, and polymerase chain reaction (PCR) to detect Toxoplasma gondii infection in tissues from experimentally infected pigs. Twelve mixed breed pigs, seronegative for T. gondii using an indirect immunofluorescent antibody test (IFAT), were used. Ten pigs were infected with 4 x 10(4) VEG strain oocysts, and two were maintained as uninfected controls. Animals were killed 60 days pos infection. Muscle (heart, tongue, diaphragm, and masseter) and brain samples were collected to investigate the presence of T. gondii tissue cysts by the different assay methods. For the bioassay, samples of brain (50 g) and pool of muscle samples (12.5 g of tongue, masseter, diaphragm, and heart) were used. PCR was performed using Tox4 and Tox5 primers which amplified a 529 bp fragment. The DNA extraction and PCR were performed three times, and all tissue samples were tested individually (brain, tongue, masseter, diaphragm, and heart). For histopathology, fragments of tissues were fixed in 10% of buffered formal saline and stained with HE. Histopathological results were all negative. PCR showed 25/150 (16.6%) positive samples, being 17/120 (14.1%) and 8/30 (26.6%) from muscle, and brain tissues, respectively. Tissue cysts of T. gondii were identified by mouse bioassay in 54/98 (55.1%) samples, being 31/48 (64.6%) from muscle samples, and 23/50 (46.0%) from brain samples. Toxoplasma gondii isolation in muscle samples by mouse bioassay was higher than in PCR (P<0.01). Results indicate that DNA from pig tissues interfered with 529-bp-PCR sensitivity, and mouse bioassay was better than PCR in detecting T. gondii in tissues from pigs.
在本研究中，我们评估了三种技术，即小鼠生物测定法、组织病理学和聚合酶链反应（PCR），以检测实验感染猪组织中的刚地弓形虫感染。使用了12头通过间接免疫荧光抗体试验（IFAT）检测为刚地弓形虫血清阴性的杂种猪。10头猪感染了4×10⁴个VEG株卵囊，2头作为未感染对照。感染后60天处死动物。采集肌肉（心脏、舌头、膈肌和咬肌）和脑样本，通过不同的检测方法调查刚地弓形虫组织包囊的存在情况。对于生物测定法，使用脑样本（50克）和肌肉样本池（12.5克舌头、咬肌、膈肌和心脏）。使用Tox4和Tox5引物进行PCR，扩增出一个529bp的片段。DNA提取和PCR进行了三次，所有组织样本均单独检测（脑、舌头、咬肌、膈肌和心脏）。对于组织病理学，组织片段用10%的缓冲甲醛固定并用苏木精-伊红染色。组织病理学结果均为阴性。PCR显示25/150（16.6%）的样本呈阳性，分别有17/120（14.1%）和8/30（26.6%）来自肌肉和脑组织。通过小鼠生物测定法在54/98（55.1%）的样本中鉴定出刚地弓形虫组织包囊，其中31/48（64.6%）来自肌肉样本，23/50（46.0%）来自脑样本。通过小鼠生物测定法在肌肉样本中分离出刚地弓形虫的比例高于PCR（P<0.01）。结果表明，猪组织中的DNA干扰了529bp-PCR的敏感性，在检测猪组织中的刚地弓形虫方面，小鼠生物测定法优于PCR。
