Jenkins Mark C, Parker Carolyn, Campos Philip, Grimm Kenneth, Davies Cary, Tucker Matthew S, Heeder Carl, Quist Michael, Thompson Peter
Animal Parasitic Diseases Laboratory, Beltsville Agricultural Research Center, NE Area, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD 20705,
Animal Parasitic Diseases Laboratory, Beltsville Agricultural Research Center, NE Area, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD 20705.
Avian Dis. 2025 Jun;69(2):177-182. doi: 10.1637/aviandiseases-D-25-00006.
The purpose of this study was to evaluate a deep amplicon sequencing approach for estimating the relative abundances of different spp. oocysts in litter from commercial broiler farms that may or may not be experiencing necrotic enteritis (NE) infections. Oligonucleotide primers directed to the mitochondrial cytochrome oxidase I (COI) gene, a sequence that is conserved among all chicken spp., were first used to PCR amplify , , and oocyst DNA. COI amplification was applied to samples containing either a single species or an equal mixture of , , and oocysts. Amplicon sequencing and mapping to the relevant COI sequences in the GenBank database confirmed the expected ∼100% mapping to the appropriate sp. and in approximately equal percentages (∼33%) for mixtures of equal numbers of spp. oocysts. This approach was then applied to DNA derived from oocysts obtained at 0, 2, and 4 wk after chick placement (growout) from a total of 20 individual houses on six different commercial broiler farms. Of the seven spp. known to infect chickens, only five were consistently found in litter at each collection time point: , , , , and . The relative numbers of and non- () oocysts in all litter samples as estimated by COI deep amplicon sequencing showed a modest correlation with the respective or oocyst counts ( ∼ 0.30). The results revealed an interesting phenomenon that supports the role of in predisposing chickens to NE. In this study, the percentage of as estimated by deep amplicon sequencing at 0, 2, and 4 wk growout showed a strong positive correlation with NE incidence (0 wk, = 0.57; 2 wk, = 0.52; 4 wk, = 0.61). This study provides evidence for the usefulness of a deep amplicon sequencing approach to estimating the relative abundances of different oocysts infecting chickens because it allows reactions to take place in a single tube, thus avoiding the time-consuming, labor-intensive, species-specific internal transcribed spacer 1 (ITS1) PCR analyses. More importantly, it allows one to explore relationships between NE incidence and the abundance of minor species, which would have been missed by oocyst counting or ITS1 PCR because most species are not distinguishable by microscopy, and ITS1 PCR is not quantitative.
本研究的目的是评估一种深度扩增子测序方法，以估计商业肉鸡场垫料中不同种类球虫卵囊的相对丰度，这些肉鸡场可能正在经历或未经历坏死性肠炎（NE）感染。针对线粒体细胞色素氧化酶I（COI）基因的寡核苷酸引物首先用于PCR扩增柔嫩艾美耳球虫、堆型艾美耳球虫和巨型艾美耳球虫的卵囊DNA，该序列在所有鸡种中都是保守的。COI扩增应用于含有单个球虫种类或柔嫩艾美耳球虫、堆型艾美耳球虫和巨型艾美耳球虫卵囊等量混合物的样本。扩增子测序以及与GenBank数据库中相关COI序列的比对证实，预期约100%比对到合适的球虫种类，对于等量的不同球虫种类的混合物，比对率约为33%。然后将该方法应用于从六个不同商业肉鸡场的20个单独鸡舍中在雏鸡放置（育成期）后0、2和4周获得的球虫卵囊衍生的DNA。在已知感染鸡的七种艾美耳球虫中，在每个收集时间点的垫料中仅始终发现五种：柔嫩艾美耳球虫、堆型艾美耳球虫、巨型艾美耳球虫、毒害艾美耳球虫和布氏艾美耳球虫。通过COI深度扩增子测序估计的所有垫料样本中柔嫩艾美耳球虫和非柔嫩艾美耳球虫的相对数量与各自的柔嫩艾美耳球虫或非柔嫩艾美耳球虫卵囊计数显示出适度的相关性（r约为0.30）。结果揭示了一个有趣的现象，支持了柔嫩艾美耳球虫在使鸡易患坏死性肠炎方面的作用。在本研究中，通过深度扩增子测序在育成期0、2和4周估计的柔嫩艾美耳球虫百分比与坏死性肠炎发病率呈强正相关（第0周，r = 0.57；第2周，r = 0.52；第4周，r = 0.61）。本研究为深度扩增子测序方法在估计感染鸡的不同球虫卵囊相对丰度方面的有用性提供了证据，因为它允许在单个管中进行反应，从而避免了耗时、劳动密集的种特异性内部转录间隔区1（ITS1）PCR分析。更重要的是，它允许人们探索坏死性肠炎发病率与次要球虫种类丰度之间的关系，而这在卵囊计数或ITS1 PCR中会被遗漏，因为大多数球虫种类通过显微镜无法区分，并且ITS1 PCR不是定量的。
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