García-Lunar P, Moré G, Campero L, Ortega-Mora L M, Álvarez-García G
SALUVET, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid, Ciudad Universitaria s/n, 28040-Madrid, Spain.
Laboratorio de Inmunoparasitología, Facultad de Ciencias Veterinarias, Universidad Nacional de La Plata, Calle 60 y 118, 1900 La Plata, Argentina; Comisión Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina.
Vet Parasitol. 2015 Nov 30;214(1-2):49-54. doi: 10.1016/j.vetpar.2015.09.011. Epub 2015 Sep 8.
Bovine besnoitiosis control remains a challenge because the disease continues to spread and control relies solely on accurate diagnosis coupled to management measures. However, recent studies have reported that routinely used ELISAs may raise a high number of false-positive results. Herein, cross-reactions between Besnoitia besnoiti antigens and anti-Neospora caninum and/or anti-Sarcocystis spp.-specific antibodies were studied in an in house ELISA since N. caninum and Sarcocystis spp. are closely related parasites, and both infections are highly prevalent in cattle worldwide. The serum panel was composed of the following categories: sera from B. besnoiti-seronegative (n=75) and -seropositive cattle (n=66), B. besnoiti-based-ELISA false-positive reactors (n=96) together with N. caninum (n=36) and Sarcocystis spp. (n=42) -seropositive reference cattle sera. B. besnoiti tachyzoite based western blot (WB) results classified animals as seropositive or seronegative. Sera were analyzed for the detection of anti-N. caninum by WB and ELISA and anti-Sarcocystis spp.-specific antibodies by WB and IFAT. Those samples recognizing a Sarcocystis spp. 18-20 kDa antigenic region and N. caninum 17-18 kDa immunodominant antigen were considered to be Sarcocystis spp. and N. caninum seropositive, respectively. The category of B. besnoiti based-ELISA false-positive reactors showed the highest number of sera with specific anti-Sarcocystis spp. and anti-N. caninum antibodies (74%; 71/96), followed by the N. caninum-seropositive cattle category (52.8%; 19/36). In contrast, few B. besnoiti-seronegative and -seropositive cattle showed antibodies against Sarcocystis spp. and N. caninum (10.7%; 8/75 and 1.5%; 1/66), respectively). This study revealed that B. besnoiti false-positive ELISA results were associated not only with the presence of anti-N. caninum and anti-Sarcocystis spp. antibodies (χ(2): 78.36; p<0.0001; OR: 34.6; CI: 14-88) but also with high antibody levels against them using ELISA and IFAT tests, respectively (p<0.05; t-test). These results may explain why only some animals seropositive to Sarcocystis spp. and/or N. caninum are Besnoitia false-positive reactors. Therefore, sera meeting these requirements should be included in future validations of serological tests for bovine besnoitiosis.
牛贝诺孢子虫病的防控仍然是一项挑战，因为该病仍在蔓延，且防控完全依赖于准确诊断并辅以管理措施。然而，最近的研究报告称，常规使用的酶联免疫吸附测定（ELISA）可能会产生大量假阳性结果。由于犬新孢子虫和肉孢子虫属是密切相关的寄生虫，且这两种感染在全球范围内的牛群中都非常普遍，因此本文在一种内部ELISA中研究了贝诺孢子虫抗原与抗犬新孢子虫和/或抗肉孢子虫属特异性抗体之间的交叉反应。血清样本分为以下几类：贝诺孢子虫血清阴性（n = 75）和血清阳性牛（n = 66）的血清、基于贝诺孢子虫的ELISA假阳性反应动物（n = 96）以及犬新孢子虫（n = 36）和肉孢子虫属（n = 42）血清阳性的参考牛血清。基于贝诺孢子虫速殖子的蛋白质印迹法（WB）结果将动物分类为血清阳性或血清阴性。通过WB和ELISA分析血清以检测抗犬新孢子虫抗体，并通过WB和间接荧光抗体试验（IFAT）检测抗肉孢子虫属特异性抗体。那些识别肉孢子虫属18 - 20 kDa抗原区域和犬新孢子虫17 - 18 kDa免疫显性抗原的样本分别被认为是肉孢子虫属和犬新孢子虫血清阳性。基于贝诺孢子虫的ELISA假阳性反应动物类别中，具有特异性抗肉孢子虫属和抗犬新孢子虫抗体的血清数量最多（74%；71/96），其次是犬新孢子虫血清阳性牛类别（52.8%；19/36）。相比之下，很少有贝诺孢子虫血清阴性和血清阳性的牛显示出抗肉孢子虫属和抗犬新孢子虫的抗体（分别为10.7%；8/75和1.5%；1/66）。本研究表明，贝诺孢子虫ELISA假阳性结果不仅与抗犬新孢子虫和抗肉孢子虫属抗体的存在有关（χ(2)：78.36；p < 0.0001；OR：34.6；CI：14 - 88），而且分别与使用ELISA和IFAT检测时针对它们的高抗体水平有关（p < 0.05；t检验）。这些结果可能解释了为什么只有一些对肉孢子虫属和/或犬新孢子虫血清阳性的动物是贝诺孢子虫假阳性反应动物。因此，满足这些要求的血清应纳入未来牛贝诺孢子虫病血清学检测的验证中。
