Avendaño Catalina, Ramo Ana, Vergara-Castiblanco Claudia, Monteagudo Luis V, Sánchez-Acedo Caridad, Quílez Joaquín
Faculty of Animal Sciences, Universidad de Ciencias Aplicadas y Ambientales, U.D.C.A, Calle 222 # 55 - 37, Bogotá, Colombia.
Department of Animal Pathology, Faculty of Veterinary Sciences, University of Zaragoza, Miguel Servet 177, 50013, Zaragoza, Spain.
Acta Trop. 2019 Apr;192:151-157. doi: 10.1016/j.actatropica.2019.02.005. Epub 2019 Feb 7.
The intra-species genetic diversity of Cryptosporidium parvum in dairy cattle farms in the central area of Colombia was investigated using a multilocus fragment typing approach with nine variable-number tandem-repeat (VNTR) loci and the gp60 gene. Genomic DNA of 70 C. parvum isolates from pre-weaned calves in 32 farms was analysed. Most markers showed two (ML1, MSB, CP47, and MSC6-7) or three alleles (5B12, Cgd2_3850, and Cgd6_5400), although they exhibited a major allele accounting for more than 69% of specimens, which explains their low discriminatory index. The TP14 microsatellite was monomorphic while a total of six alleles were found at the ML2 microsatellite. The two novel allelic variants (219bp, 245bp) exhibited by more than 36% of specimens at the latter locus were a remarkable finding. The 10-markers typing tool provided a Hunter-Gaston discriminatory value of 0.940 (95% CI, 0.918 - 0.961) and differentiated 22 multilocus subtypes (MLTs). Nevertheless, the combination of the three most informative markers (ML2, gp60, and Cgd2_3850) differentiated 68% of MLTs and hardly impaired the discriminatory index. The fact that many MLTs (13/22) were distinctive for individual farms provides evidence for the endemic nature of the infection and the major role played by transmission within farms. The eBURST algorithm suggested a low degree of genetic divergence. All but three MLTs were clustered in a clonal complex with a star-like topology typical of clonal expansion, however linkage analysis did not find evidence of linkage disequilibrium. Bayesian analysis also identified a genetic structure with K = 3 being the best estimation of ancestral clusters, although a large proportion of isolates (35%) could not be allocated to a single population, which indicates their mixed origin. The results confirm the genetic distinctiveness of C. parvum in cattle farms in this geographical area. This is the first multilocus analysis on the intra-specific variability of Cryptosporidium from calves in South America.
采用包含9个可变数目串联重复序列（VNTR）位点和gp60基因的多位点片段分型方法，对哥伦比亚中部地区奶牛场中的微小隐孢子虫种内遗传多样性进行了研究。分析了来自32个农场断奶前犊牛的70株微小隐孢子虫分离株的基因组DNA。大多数标记显示出两个（ML1、MSB、CP47和MSC6 - 7）或三个等位基因（5B12、Cgd2_3850和Cgd6_5400），尽管它们都有一个占标本69%以上的主要等位基因，这解释了它们较低的鉴别指数。TP14微卫星是单态的，而在ML2微卫星上共发现了6个等位基因。在后者位点上超过36%的标本表现出的两个新的等位基因变体（219bp、245bp）是一个显著的发现。这种10标记分型工具的Hunter - Gaston鉴别值为0.940（95%置信区间，0.918 - 0.961），并区分出22种多位点亚型（MLT）。然而，三个信息量最大的标记（ML2、gp60和Cgd2_3850）的组合区分出了68%的MLT，并且几乎没有损害鉴别指数。许多MLT（13/22）在各个农场中具有独特性这一事实为感染的地方性本质以及农场内传播所起的主要作用提供了证据。eBURST算法表明遗传分化程度较低。除了三个MLT外，所有MLT都聚集在一个具有典型克隆扩张的星状拓扑结构的克隆复合体中，然而连锁分析未发现连锁不平衡的证据。贝叶斯分析也确定了一个遗传结构，其中K = 3是对祖先簇的最佳估计，尽管很大一部分分离株（35%）无法分配到单个种群中，这表明它们具有混合起源。结果证实了该地理区域奶牛场中微小隐孢子虫的遗传独特性。这是南美洲首次对犊牛隐孢子虫种内变异性进行的多位点分析。
