Paz Gustavo F, Rugani Jeronimo M N, Marcelino Andreza P, Gontijo Célia M F
Grupo de Estudos em Leishmanioses, Instituto René Rachou, Fundação Oswaldo Cruz, 30190-002, Belo Horizonte, MG, Brazil.
Grupo de Estudos em Leishmanioses, Instituto René Rachou, Fundação Oswaldo Cruz, 30190-002, Belo Horizonte, MG, Brazil.
Acta Trop. 2018 Jun;182:198-201. doi: 10.1016/j.actatropica.2018.03.018. Epub 2018 Mar 12.
The aim of this study was to evaluate the relationship between naturally occurring Leishmania spp. infections in dogs (Canis familiaris) and the practical implications of the use of serological and molecular methods to confirm diagnoses. The study population consisted of 96 domestic dogs in southeastern Brazil. Serum samples were tested for the presence of anti-Leishmania immunoglobulin G (IgG) antibodies using four commercial canine visceral leishmaniasis kits. Dogs confirmed positive by immunofluorescence antibody test (IFAT) were culled and samples from mesenteric lymph nodes, spleen border, bone marrow and ear skin were taken and submitted to DNA extraction. PCR reactions were performed using primers that amplify a 300-350 bp fragment of the Leishmania ribosomal internal transcribed spacer 1 (ITS1) region. The ITS1 amplified products were analyzed by PCR-RFLP using Hae III restriction endonuclease. To confirm the Leishmania species detected by PCR, each purified sample was sequenced in duplicate. Of the 96 serum samples submitted to serological assays, 8 (8.3%) tested positive for Leishmania by IFAT, 4 (4.1%) by ELISA, 2 (2.1%) by rK39 RDT and 7 (7.3%) by DPP. Four of these infected dogs (50%) were found to be infected only by Leishmania braziliensis or Leishmania amazonensis, and their serum samples tested positive by IFAT and DPP. These findings demonstrate for the first time that cross-reactivity of L. braziliensis and L. amazonensis infection in dogs can be found using the DPP serum test. This is the first record of Leishmania (Leishmania) amazonensis confirmed by a specific molecular marker in dogs (Canis familiaris) from Belo Horizonte, Brazil.
本研究的目的是评估犬（家犬）自然感染利什曼原虫属与使用血清学和分子方法确诊的实际意义之间的关系。研究对象为巴西东南部的96只家犬。使用四种商用犬内脏利什曼病检测试剂盒检测血清样本中抗利什曼原虫免疫球蛋白G（IgG）抗体的存在情况。通过免疫荧光抗体试验（IFAT）确诊为阳性的犬被扑杀，并采集肠系膜淋巴结、脾脏边缘、骨髓和耳部皮肤样本进行DNA提取。使用扩增利什曼原虫核糖体内部转录间隔区1（ITS1）区域300 - 350 bp片段的引物进行PCR反应。使用Hae III限制性内切酶通过PCR - RFLP分析ITS1扩增产物。为了确认通过PCR检测到的利什曼原虫种类，每个纯化样本进行两次重复测序。在接受血清学检测的96份血清样本中，8份（8.3%）通过IFAT检测为利什曼原虫阳性，4份（4.1%）通过ELISA检测为阳性，2份（2.1%）通过rK39快速诊断检测（RDT）为阳性，7份（7.3%）通过双重免疫扩散试验（DPP）为阳性。其中4只感染犬（50%）被发现仅感染巴西利什曼原虫或亚马逊利什曼原虫，其血清样本通过IFAT和DPP检测为阳性。这些发现首次证明，使用DPP血清检测可发现犬感染巴西利什曼原虫和亚马逊利什曼原虫时的交叉反应性。这是巴西贝洛奥里藏特地区通过特定分子标记在犬（家犬）中确诊亚马逊利什曼原虫（利什曼原虫属）的首次记录。
