石斑鱼养殖病原混合感染研究与分析

Study and analysis of pathogen co-infection in grouper culture

摘要:

目的 了解养殖石斑鱼常见疾病病原混合感染特性及规律，以指导石斑鱼的病害防控。方法 采用PCR方法对从广西地区 (北海、钦州、防城港市)石斑鱼养殖场采集的发病石斑鱼展开病毒性病原及细菌性病原的流行病原调查，并将发病鱼中的病原通过细胞特异性方法分离病毒性病原，利用平板 (LB和TCBS)划线分离法分离细菌性病原。然后将分离的病原感染石斑鱼细胞以确定病原的感染性。结果 发病的石斑鱼存在虹彩病毒 (Singapore grouper iridovirus, SGIV)、神经坏死病毒 (nervous necrosis virus, NNV)、哈维氏弧菌、创伤弧菌、溶藻弧菌、副溶血弧菌、嗜水气单胞菌及美人鱼发光杆菌单一感染及混合感染现象，且细菌性疾病中还存在细菌的种间与种内混合感染。此外，石斑鱼病毒性疾病和细菌性疾病在夏季和秋季病原的检出率均高于春季与冬季，其中秋季病原检出率最高。与此同时，从发病鱼体内分离的病毒性病原与细菌性病原均可感染石斑鱼单一细胞。结论 在发病石斑鱼中，无论是病毒性疾病或是细菌性疾病，多以二重感染为主，细菌多重感染也是常见类型，且混合感染类型较为复杂。结果可为石斑鱼病原混合感染致病机理研究提供方向，并为石斑鱼养殖的疾病防控提供理论依据。

Abstract:

Co-infection is a common phenomenon in nature, and large-scale morbidity in livestock farms is caused by multiple pathogenic factors, with pathogen co-infection being a major contributor. Co-infection complicates clinical diagnosis and treatment. However, most studies focus solely on the pathogenic characteristics and mechanisms of single pathogens, and many prevention and control methods and drug treatment strategies are also limited to a single pathogens. Consequently, these methods are often ineffective against co-infections involving multiple pathogens. In fact, co-infection can cause more severe symptoms and damage to the host compared to single-pathogen infections. Moreover, interactions between co-infected pathogens can be synergistic, antagonistic, or non-interfering, and they may alter the pathogenic mechanisms of the original single pathogens. Therefore, developing rapid, sensitive, and efficient detection methods for multiple pathogens and exploring the pathogenesis of co-infection are of great significance for the prevention and treatment of co-infection. Pathogen co-infection has been extensively studied in livestock and poultry, including the detection technology and pathogenic mechanisms of virus-virus, virus-bacteria, and bacteria-bacteria co-infections. However, co-infection in aquaculture remains underexplored, with few studies reported. Grouper is a highly valued marine fish, known for its delicate and nutritious meat and significant economic importance. With the expansion of aquaculture and increaing industrialization and urbanization, the deterioration of the offshore grouper aquaculture environment has intensified, leading to more severe disease outbreaks in grouper populations. To understand the characteristics and patterns of common pathogen infections in cultured grouper, this study used PCR to investigate viral and bacterial pathogens in diseased grouper collected from farms in Guangxi (Beihai, Qinzhou, Fangchenggang City) for the first time. Pathogens were isolated using cell-specific isolation methods for viral pathogens and plate (LB and TCBS) streak separation for bacterial pathogens. The infectivity of the isolated pathogens was then confirmed using grouper cell lines. Results showed that Singapore grouper iridovirus (SGIV), nervous necrosis virus (NNV), Vibrio harveyi, V. vulnificus, V. alginolyticus, V. parahaemolyticus, A. eromonas hydrophila and Photobacterium damselaesingle were detected grouper singly or in combination. V. harveyi, V. vulnificus, V. alginolyticus and V. parahaemolyticus were the primary bacterial pathogens infecting cultured grouper in Guangxi, and interspecies and intraspecies co-infections observed. The detection frequency of V. harveyi was the highest, indicating that it is the main causative agent of Vibrio disease in cultured grouper in Guangxi. Detection rates of viral and bacterial pathogens veried significantly by season. The detection rate of NNV was 13% in spring (March to May), higher than that of SGIV. In summer (June to August), SGIV had a detection rate of 19%, higher than that of NNV. The highest detection rates for SGIV and NNV were observed in autumn (September to November) at 58% and 40%, respectively, while the lowest rates were in winter (December to February) at 4% and 6%, respectively. Autumn also had the highest detection rate for bacterial pathogens, with V. harveyi, V. vulnificus, V. alginolyticus, V. parahaemolyticus, A. hydrophila and P. mermaid detected at rates of 32%, 6%, 9%, 15%, 6%, and 15%, respectively. V. harveyi was detected in diseased grouper across all seasons, while V. alginolyticus had high detection rate in summer, autumn and winter; V. vulnificus was mainly detected in spring and autumn; and V. parahaemolyticus and P. mermaid were detected in summer and autumn. In summary, diseases in cultured grouper in Guangxi are characterized by co-infections involving multiple pathogens, with complex and diverse co-infection patterns. Our findings provided a direction for exploring the pathogenic mechanisms of grouper pathogen co-infection, offering a theoretical basis for epidemic prevention and control, and shedding new light on aquatic pathogen co-infection research.