Laboratory of Functional Helminthology, Institute of Parasitology, Biology Centre Czech Academy of Sciences, Branišovská 1160/31, 37005 České Budějovice, Czechia; Servicio de Análisis, Investigación y Gestión de Animales Silvestres (SAIGAS), Facultad de Veterinaria, Universidad Cardenal Herrera-CEU, CEU Universities, C/ Tirant lo Blanc, Alfara del Patriarca, 46115 Valencia, Spain.
Faculty of Science, University of South Bohemia, Branišovská 1760/31a, 37005, České Budějovice, Czechia; Institute of Entomology, Biology Centre Czech Academy of Sciences, Branišovská 1160/31, 37005, České Budějovice, Czechia.
Vet Parasitol. 2023 Nov;323:110050. doi: 10.1016/j.vetpar.2023.110050. Epub 2023 Oct 12.
The in vitro life cycle of zoonotic helminths is an essential tool for -omic translational studies focused on disease control and treatment. Anisakiosis is an emerging zoonosis contracted by the ingestion of raw or undercooked fish infected with the third stage larvae (L3) of two sibling species Anisakis simplex sensu stricto (s.s.) and Anisakis pegreffii, the latter being the predominant species in the Mediterranean basin. Recently, in vitro culture of A. pegreffii has been developed to enable fast and large-scale production of fertile adults. However, the conditions for larval development from hatching to infective L3 were not fulfilled to complete the cycle. Herein, we used a Drosophila medium supplemented with chicken serum and adjusted different osmolarities to maintain the culture of L3 hatched from eggs for up to 17 weeks. The highest survival rate was observed in the medium with the highest osmolarities, which also allowed the highest larval exsheathment rate. Key morphological features of embryogenesis and postembryogenesis studied by transmission electron microscopy revealed that the excretory gland cell is differentiated already up to 48 h post-hatching. Extracellular vesicles and cell-free mitochondria are discharged between the two cuticle sheets of the second stage larvae (L2). Contemporarly cultivated, two populations of adult A. simplex s.s. and A. pegreffii reached an average production of 29,914.05 (± 27,629.36) and 24,370.96 (± 12,564.86) eggs/day/female, respectively. The chromosome spreads of A. pegreffii obtained from mature gonads suggests a diploid karyotype formula of 2n = 18. The development of a reliable protocol for the in vitro culture of a polyxenous nematode such as Anisakis spp. will serve to screen for much needed novel drug targets, but also to study the intricated and unknown ecological and physiological traits of these trophically transmitted marine nematodes.
人畜共患寄生虫的体外生活史是进行疾病控制和治疗的 -omic 转化研究的重要工具。异尖线虫病是一种新兴的人畜共患病，由摄入受感染的生鱼或未煮熟的鱼而感染第三期幼虫（L3）引起，两种同缘种——严格意义上的单纯异尖线虫（s.s.）和佩氏异尖线虫（Anisakis pegreffii），后者在地中海盆地更为常见。最近，已经开发了佩氏异尖线虫的体外培养方法，以实现快速和大规模生产有生育力的成虫。然而，从孵化到感染性 L3 的幼虫发育条件尚未得到满足，无法完成整个周期。在此，我们使用含有鸡血清的果蝇培养基，并调整不同的渗透压来维持从卵中孵化出的 L3 的培养，最长可达 17 周。在渗透压最高的培养基中观察到最高的存活率，这也允许最高的幼虫蜕皮率。通过透射电子显微镜研究胚胎发生和胚胎发生后的关键形态特征表明，排泄腺细胞在孵化后 48 小时已经分化。细胞外囊泡和无细胞线粒体在第二阶段幼虫（L2）的两个角质层片之间排出。同时培养的两种单纯异尖线虫和佩氏异尖线虫成虫种群平均每天每只雌虫分别产生 29,914.05（±27,629.36）和 24,370.96（±12,564.86）个卵。从成熟性腺中获得的佩氏异尖线虫染色体扩散表明，其二倍体核型公式为 2n=18。建立可靠的多宿主线虫（如异尖线虫属）体外培养方法将有助于筛选急需的新型药物靶点，同时也有助于研究这些营养传播海洋线虫错综复杂和未知的生态和生理特征。
