Hussein Hala E, Johnson Wendell C, Taus Naomi S, Ueti Massaro W
Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA, USA.
Department of Biology, College of Arts and Sciences, Gonzaga University, Spokane, WA, USA.
Parasit Vectors. 2024 Feb 19;17(1):75. doi: 10.1186/s13071-024-06185-w.
Bovine babesiosis caused by Babesia bovis is one of the most important tick-borne diseases of cattle in tropical and subtropical regions. Babesia bovis parasites have a complex lifecycle, including development within the mammalian host and tick vector. In the tick midgut, extracellular Babesia parasites transform into gametes that fuse to form zygotes. To date, little is known about genes and proteins expressed by male gametes.
We developed a method to separate male gametes from in vitro induced B. bovis culture. Separation enabled the validation of sex-specific markers. Collected male gametocytes were observed by Giemsa-stained smear and live-cell fluorescence microscopy. Babesia male gametes were used to confirm sex-specific markers by quantitative real-time PCR. Some genes were found to be male gamete specific genes including pka, hap2, α-tubulin II and znfp2. However, α-tubulin I and ABC transporter, trap2-4 and ccp1-3 genes were found to be upregulated in culture depleted of male gametes (female-enriched). Live immunofluorescence analysis using polyclonal antibodies confirmed surface expression of HAP2 by male and TRAP2-4 by female gametes. These results revealed strong markers to distinguish between B. bovis male and female gametes.
Herein, we describe the identification of sex-specific molecular markers essential for B. bovis sexual reproduction. These tools will enhance our understanding of the biology of sexual stages and, consequently, the development of additional strategies to control bovine babesiosis.
由牛巴贝斯虫引起的牛巴贝斯虫病是热带和亚热带地区牛最重要的蜱传疾病之一。牛巴贝斯虫寄生虫具有复杂的生命周期，包括在哺乳动物宿主和蜱载体中的发育。在蜱的中肠中，细胞外的巴贝斯虫寄生虫转化为配子，配子融合形成合子。迄今为止，关于雄配子表达的基因和蛋白质知之甚少。
我们开发了一种从体外诱导的牛巴贝斯虫培养物中分离雄配子的方法。分离能够验证性别特异性标记。通过吉姆萨染色涂片和活细胞荧光显微镜观察收集的雄配子体。使用定量实时PCR，用牛巴贝斯虫雄配子来确认性别特异性标记。发现一些基因是雄配子特异性基因，包括蛋白激酶A、hap2、α-微管蛋白II和锌指蛋白2。然而，发现α-微管蛋白I和ABC转运蛋白、trap2 - 4和ccp1 - 3基因在缺乏雄配子（富含雌配子）的培养物中上调。使用多克隆抗体的活免疫荧光分析证实了雄配子表面表达HAP2，雌配子表面表达TRAP2 - 4。这些结果揭示了区分牛巴贝斯虫雄配子和雌配子的强标记。
在此，我们描述了鉴定牛巴贝斯虫有性繁殖所必需的性别特异性分子标记。这些工具将增进我们对有性阶段生物学的理解，从而有助于开发控制牛巴贝斯虫病的其他策略。
