Dubert Javier, Nelson David R, Spinard Edward J, Kessner Linda, Gomez-Chiarri Marta, da Costa Fiz, Prado Susana, Barja Juan L
Departamento de Microbiología y Parasitología, CIBUS-Facultad de Biología y Instituto de Acuicultura, Universidad de Santiago de Compostela, Santiago de Compostela 15782, Spain.
Department of Cell and Molecular Biology, University of Rhode Island, Kingston, RI 02881, United States.
J Invertebr Pathol. 2016 Jan;133:27-33. doi: 10.1016/j.jip.2015.11.008. Epub 2015 Nov 25.
Vibriosis represents the main bottleneck for the larval production process in shellfish aquaculture. While the signs of this disease in bivalve larvae are well known, the infection process by pathogenic Vibrio spp. during episodes of vibriosis has not been elucidated. To investigate the infection process in bivalves, the pathogens of larvae as V. tubiashii subsp. europaensis, V. neptunius and V. bivalvicida were tagged with green fluorescent protein (GFP). Larvae of Manila clam (Ruditapes philippinarum) were inoculated with the GFP-labeled pathogens in different infection assays and monitored by microscopy. Manila clam larvae infected by distinct GFP-tagged Vibrio spp. in different challenges showed the same progression in the infection process, defining three infection stages. GFP-tagged Vibrio spp. were filtered by the larvae through the vellum and entered in the digestive system through the esophagus and stomach and colonized the digestive gland and particularly the intestine, where they proliferated during the first 2h of contact (Stage I), suggesting a chemotactic response. Then, GFP-tagged Vibrio spp. expanded rapidly to the surrounding organs in the body cavity from the dorsal to ventral region (Stage II; 6-8h), colonizing the larvae completely at the peak of infection (Stage III) (14-24h). Results demonstrated for the first time that the vibriosis is asymptomatic in Manila clam larvae during the early infection stages. Thus, the early colonization and the rapid proliferation of Vibrio pathogens within the body cavity supported the sudden and fatal effect of the vibriosis, since the larvae exhibited the first signs of disease when the infection process is advanced. As a first step in the elucidation of the potential mechanisms of bacterial pathogenesis in bivalve larvae the enzymatic activities of the extracellular products released from the wild type V. neptunius, V. tubiashii subsp. europaensis and V. bivalvicida were determined and their cytotoxicity was demonstrated in fish and homeothermic cell lines for the first time. That activity was lost after heat treatment.
弧菌病是贝类养殖幼体生产过程中的主要瓶颈。虽然双壳贝类幼体中这种疾病的症状广为人知，但致病性弧菌属在弧菌病爆发期间的感染过程尚未阐明。为了研究双壳贝类的感染过程，将幼虫病原体如塔氏弧菌欧洲亚种、海弧菌和双壳弧菌用绿色荧光蛋白（GFP）标记。在不同的感染试验中，用GFP标记的病原体接种菲律宾蛤仔的幼体，并通过显微镜进行监测。在不同挑战中被不同GFP标记弧菌属感染的菲律宾蛤仔幼体在感染过程中表现出相同的进展，确定了三个感染阶段。GFP标记的弧菌属被幼体通过鳃过滤，经食道和胃进入消化系统，并在消化腺尤其是肠道中定殖，它们在接触的最初2小时内在肠道中增殖（第一阶段），表明存在趋化反应。然后，GFP标记的弧菌属从背侧到腹侧区域迅速扩展到体腔中的周围器官（第二阶段；6 - 8小时），在感染高峰期（第三阶段）（14 - 24小时）完全定殖于幼体。结果首次证明，在早期感染阶段，弧菌病在菲律宾蛤仔幼体中是无症状的。因此，弧菌病原体在体腔内的早期定殖和快速增殖支持了弧菌病的突然和致命影响，因为幼体在感染过程进展时才表现出疾病的最初症状。作为阐明双壳贝类幼体中细菌致病潜在机制的第一步，首次测定了野生型海弧菌、塔氏弧菌欧洲亚种和双壳弧菌释放的细胞外产物的酶活性，并在鱼类和恒温细胞系中证明了它们的细胞毒性。热处理后该活性丧失。
