Cantos-Barreda Ana, Escribano Damián, Bernal Luis J, Cerón José J, Martínez-Subiela Silvia
Interdisciplinary Laboratory of Clinical Analysis, Interlab-UMU, Regional Campus of International Excellence "Campus Mare Nostrum", University of Murcia, Espinardo, Murcia, Spain.
Interdisciplinary Laboratory of Clinical Analysis, Interlab-UMU, Regional Campus of International Excellence "Campus Mare Nostrum", University of Murcia, Espinardo, Murcia, Spain.
Vet Parasitol. 2017 Aug 15;242:54-58. doi: 10.1016/j.vetpar.2017.05.017. Epub 2017 May 24.
Detection of serum anti-Leishmania antibodies by quantitative or qualitative techniques has been the most used method to diagnose Canine Leishmaniosis (CanL). Nevertheless, saliva may represent an alternative to blood because it is easy to collect, painless and non-invasive in comparison with serum. In this study, two time-resolved immunofluorometric assays (TR-IFMAs) for quantification of anti-Leishmania IgG2 and IgA antibodies in saliva were developed and validated and their ability to distinguish Leishmania-seronegative from seropositive dogs was evaluated. The analytical study was performed by evaluation of assay precision, sensitivity and accuracy. In addition, serum from 48 dogs (21 Leishmania-seropositive and 27 Leishmania-seronegative) were analyzed by TR-IFMAs. The assays were precise, with an intra- and inter-assay coefficients of variation lower than 11%, and showed high level of accuracy, as determined by linearity under dilution (R=0.99) and recovery tests (>88.60%). Anti-Leishmania IgG2 antibodies in saliva were significantly higher in the seropositive group compared with the seronegative (p<0.0001), whereas no significant differences for anti-Leishmania IgA antibodies between both groups were observed. Furthermore, TR-IFMA for quantification of anti-Leishmania IgG2 antibodies in saliva showed higher differences between seropositive and seronegative dogs than the commercial assay used in serum. In conclusion, TR-IFMAs developed may be used to quantify anti-Leishmania IgG2 and IgA antibodies in canine saliva with an adequate precision, analytical sensitivity and accuracy. Quantification of anti-Leishmania IgG2 antibodies in saliva could be potentially used to evaluate the humoral response in CanL. However, IgA in saliva seemed not to have diagnostic value for this disease. For future studies, it would be desirable to evaluate the ability of the IgG2 assay to detect dogs with subclinical disease or with low antibody titers in serum and also to study the antibodies behaviour in saliva during the treatment of CanL.
通过定量或定性技术检测血清抗利什曼原虫抗体一直是诊断犬利什曼病（CanL）最常用的方法。然而，唾液可能是血液的一种替代样本，因为与血清相比，唾液采集容易、无痛且非侵入性。在本研究中，开发并验证了两种用于定量唾液中抗利什曼原虫IgG2和IgA抗体的时间分辨免疫荧光分析（TR-IFMA）方法，并评估了它们区分利什曼原虫血清阴性和血清阳性犬的能力。通过评估分析方法的精密度、灵敏度和准确性进行分析研究。此外，用TR-IFMA分析了48只犬（21只利什曼原虫血清阳性和27只血清阴性）的血清。这些检测方法具有精密度，批内和批间变异系数低于11%，并且通过稀释线性（R = 0.99）和回收率测试（>88.60%）显示出高准确度。血清阳性组唾液中的抗利什曼原虫IgG2抗体显著高于血清阴性组（p<0.0001），而两组之间抗利什曼原虫IgA抗体未观察到显著差异。此外，用于定量唾液中抗利什曼原虫IgG2抗体的TR-IFMA在血清阳性和血清阴性犬之间显示出比血清中使用的商业检测方法更大的差异。总之，所开发的TR-IFMA可用于以足够的精密度、分析灵敏度和准确度定量犬唾液中的抗利什曼原虫IgG2和IgA抗体。唾液中抗利什曼原虫IgG2抗体的定量可能潜在地用于评估CanL中的体液反应。然而，唾液中的IgA似乎对该疾病没有诊断价值。对于未来的研究，期望评估IgG2检测方法检测亚临床疾病或血清中抗体滴度低的犬的能力，以及研究CanL治疗期间唾液中抗体的行为。
