■丝虫线虫通常在脊椎动物宿主的血流中产生幼虫期(微丝虫),微丝虫存在于血液或皮下组织中。丝虫线虫引起人类疾病,比如河盲症和象皮病,被广泛研究。然而,在鸟类中,它们被忽视,因为它们是非致病性的。在泰国,在野生鸟类和家鸡中可以发现微丝虫病。最近,向兽医诊断实验室提交的血液样本数量的增加可能会增加微丝虫的数量。因此,有关丝虫物种的知识和可靠的检测方法很重要。因此,本研究旨在探讨血沉棕黄层涂片和基于聚合酶链反应(PCR)的方法检测家鸡微丝菌的效果。此外,使用细胞色素c氧化酶亚基1(COX1)基因的序列鉴定寄生虫。
■重新分析了来自先前研究的Giemsa染色的血沉棕黄层涂片。这些可用的血沉棕黄层涂片是由在泰国南部散养的55只家鸡制成的。从鸡血中提取的57个冷冻基因组DNA用于检测Onchocercidae线虫中COX1基因的存在。用于扩增COX1基因的OnchoCOI_R2-OnchoCOI_R2片段的巢式PCR方案应用于先前的报道。分析了COX1的序列以鉴定Onchocercidae线虫,以及它们是单一感染还是混合感染。我们构建了贝叶斯系统遗传学来鉴定寄生虫并评估鸟类和其他脊椎动物宿主中丝虫线虫之间的关系。
■来自15个样本的棕黄层涂片显示微丝菌。在这15个样本中,只有8例COX1巢式PCR扩增呈阳性.另外两个血沉棕黄层阴性样品的巢式PCR也是阳性的。对这11个巢式PCR阳性样品进行测序显示,几乎所有样品都是Onchocercidae线虫。贝叶斯系统发育分析表明,鸡the。与其他禽类丝虫线虫分组。然而,所有鸡Onchocercidaespp。在测序色谱图中显示了一个双峰,表明混合丝虫感染(物种或单倍型)。因此,国家生物技术信息中心没有保藏鸡Onchocercidae序列,GenBank。
■Giemsa染色的血沉棕黄层涂片是在常规兽医诊断实验室中检测鸡肉微丝菌的可靠方法。开发新的基于PCR的方法是必要的。该方法可以提供更高的检测灵敏度和特异性。此外,PCR方法使我们能够获得线虫的遗传特征,这有助于我们最大限度地了解线虫。进一步调查,如丝虫线虫对鸡及其潜在载体的致病性,是必需的。
UNASSIGNED: Filarial nematode typically produces a larval stage (microfilariae) in the bloodstream of vertebrate hosts, where microfilariae reside in the blood or subcutaneous tissues. Filarial nematodes cause human diseases, such as river blindness and elephantiasis, which are widely studied. However, in avian species, they are overlooked because they are nonpathogenic. In Thailand,
microfilaria can be found in wild birds and domestic chickens. Recently, an increase in the number of blood samples submitted to veterinary diagnostic laboratories may have increased the number of microfilariae. Therefore, knowledge about filarial species and reliable detection methods are important. Therefore, this study aimed to investigate the efficacy of buffy coat smear and polymerase chain reaction (PCR)-based methods for the detection of
microfilaria in domestic chickens. In addition, parasites were identified using the sequence of the cytochrome c oxidase subunit 1 (COX1) gene.
UNASSIGNED: Giemsa-stained buffy coat smears from a previous study were reanalyzed. These available buffy coat smears were prepared from 55 domestic chickens raised as backyard free-ranging in Southern Thailand. Fifty-seven frozen genomic DNA extracted from chicken blood were used to detect the presence of the COX1 gene in Onchocercidae nematodes. The nested PCR protocol for amplification of the OnchoCOI_R2-OnchoCOI_R2 fragment of the COX1 gene was applied from a previous report. Sequences of COX1 were analyzed to identify Onchocercidae nematodes and if they were single or mixed infections. We constructed Bayesian phylogenetics to identify parasites and assessment of the relationship between filarial nematodes in avian species and other vertebrate hosts.
UNASSIGNED: Buffy coat smears from 15 samples revealed
microfilaria. Of these 15 samples, only eight were positive for COX1 nested-PCR amplification. The other two buffy coat-negative samples were also positive for nested-PCR. Sequencing of these 11 nested PCR-positive samples revealed that almost all of them were Onchocercidae nematodes. Bayesian phylogenetic analysis showed that chicken Onchocercidae spp. were grouped with other avian filarial nematodes. However, all chickens Onchocercidae spp. showed a double peak in the sequencing chromatogram, indicating mixed filarial infection (species or haplotypes). Therefore, no chicken Onchocercidae sequence was deposited on National Center for Biotechnology Information, GenBank.
UNASSIGNED: Giemsa-stained buffy coat smear was a reliable method for the detection of chicken
microfilaria in routine veterinary diagnostic laboratories. Development of a new PCR-based method is necessary. This method may provide greater sensitivity and specificity of detection. In addition, the PCR method allowed us to access the genetic characteristics of nematodes, which helped us maximize our knowledge of nematodes. Further investigations, such as the pathogenicity of filarial nematodes in chickens and their potential vectors, are required.