Low-cost molecular methods to characterise gastrointestinal nematode co-infections of goats in Africa.
Low-cost molecular methods to characterise gastrointestinal nematode co-infections of goats in Africa.
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DOI：
10.1186/s13071-023-05816-y
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发表时间：
2023-06-29
影响因子：
3.2
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中科院分区：
文献类型：
--
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兽医诊断有助于干预策略，跟踪人畜共患病，并指导牲畜的选择性育种计划。在反刍动物中，胃肠道线虫（GIN）寄生虫是造成生产损失的主要原因，但形态相似的物种限制了我们对特定的GIN合并感染如何在资源有限的环境中影响健康的理解。为了在物种水平上估计GINs和其他蠕虫的存在和相对丰度，我们试图开发一种低成本、低资源的分子工具包，应用于马拉维农村小农场的山羊。对马拉维利隆圭地区小农场的山羊进行了健康评分和粪便取样。感染强度通过粪便线虫卵计数估计，并将粪便亚样本干燥用于DNA分析。测试了两种DNA提取方法（低资源磁珠试剂盒和高资源自旋柱试剂盒），通过终点聚合酶链反应（PCR）、半定量PCR、定量PCR （qPCR）、高分辨率熔融曲线分析（HRMC）和“nemabiome”内部转录间隔物2 （ITS-2）扩增子测序筛选得到的DNA。尽管低资源磁珠法的DNA纯度和粪便污染物残留较差，但两种DNA分离方法都产生了类似的结果。无论感染强度如何，100%的样本均检测到GINs。大多数山羊存在GINs和球虫（艾美耳球虫属）的共感染，其中GIN种群以弯曲血蜱、色状毛圆线虫、轴状毛圆线虫和柱状食道口毛圆线虫为主。多重PCR和qPCR都能高度预测nemabiome扩增子测序获得的GIN物种比例；然而，HRMC在预测特定物种的存在方面不如PCR可靠。这些数据首次对非洲自然感染的小农山羊的GINs进行了“neneabiome”测序，并显示了个体动物之间共同感染GIN的可变性。通过半定量PCR方法检测到类似的粒度水平，这提供了物种组成的准确总结。因此，利用低成本的低资源DNA提取和PCR方法评估GIN合并感染是可能的，这可以在没有测序平台的地区增加分子资源的能力；也为廉价的分子GIN诊断打开了大门。鉴于牲畜和野生动物感染的多样性，这些方法有可能用于其他领域的疾病监测。在线版本包含补充材料，可在10.1186/s13071-023-05816-y获得。
Veterinary diagnostics aid intervention strategies, track zoonoses, and direct selective breeding programs in livestock. In ruminants, gastrointestinal nematode (GIN) parasites are a major cause of production losses, but morphologically similar species limit our understanding of how specific GIN co-infections impact health in resource-limited settings. To estimate the presence and relative abundance of GINs and other helminths at the species level, we sought to develop a low-cost and low-resource molecular toolkit applied to goats from rural Malawi smallholdings. Goats were subjected to health scoring and faecal sampling on smallholdings in Lilongwe district, Malawi. Infection intensities were estimated by faecal nematode egg counts with a faecal subsample desiccated for DNA analysis. Two DNA extraction methods were tested (low-resource magbead kit vs high-resource spin-column kit), with resulting DNA screened by endpoint polymerase chain reaction (PCR), semi-quantitative PCR, quantitative PCR (qPCR), high-resolution melt curve analysis (HRMC), and ‘nemabiome’ internal transcribed spacer 2 (ITS-2) amplicon sequencing. Both DNA isolation methods yielded comparable results despite poorer DNA purity and faecal contaminant carryover from the low-resource magbead method. GINs were detected in 100% of samples regardless of infection intensity. Co-infections with GINs and coccidia (Eimeria spp.) were present in most goats, with GIN populations dominated by Haemonchus contortus, Trichostrongylus colubriformis, Trichostrongylus axei, and Oesophagostomum columbianum. Both multiplex PCR and qPCR were highly predictive of GIN species proportions obtained using nemabiome amplicon sequencing; however, HRMC was less reliable than PCR in predicting the presence of particular species. These data represent the first ‘nemabiome’ sequencing of GINs from naturally infected smallholder goats in Africa and show the variable nature of GIN co-infections between individual animals. A similar level of granularity was detected by semi-quantitative PCR methods, which provided an accurate summary of species composition. Assessing GIN co-infections is therefore possible using cost-efficient low-resource DNA extraction and PCR approaches that can increase the capacity of molecular resources in areas where sequencing platforms are not available; and also open the door to affordable molecular GIN diagnostics. Given the diverse nature of infections in livestock and wildlife, these approaches have potential for disease surveillance in other areas. The online version contains supplementary material available at 10.1186/s13071-023-05816-y.
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影响因子：
3.7
作者：
Demeler J;Ramünke S;Wolken S;Ianiello D;Rinaldi L;Gahutu JB;Cringoli G;von Samson-Himmelstjerna G;Krücken J
通讯作者：
Krücken J
影响因子：
48
作者：
Callahan BJ;McMurdie PJ;Rosen MJ;Han AW;Johnson AJ;Holmes SP
通讯作者：
Holmes SP
影响因子：
2.6
作者：
Baker, RL;Mwamachi, DM;Thorpe, W
通讯作者：
Thorpe, W
影响因子：
1.8
作者：
Bath, G. F.;van Wyk, J. A.
通讯作者：
van Wyk, J. A.
影响因子：
2.6
作者：
Bisset, S. A.;Knight, J. S.;Bouchet, C. L. G.
通讯作者：
Bouchet, C. L. G.