家蚕体内细菌感染模型建立及不同菌株的协同致病特征分析

Establishment of a bacterial infection model in silkworm(Bom⁃byx mori)and synergistic pathogenicity characteristics of different bacterial strains

【目的】建立家蚕体内细菌感染模型,揭示不同细菌协同感染家蚕的致病力,明确云南蚕区家蚕细菌病的发病原因及其规律,为指导家蚕生产过程中细菌病的防治提供科学依据。【方法】从家蚕龄期、感染浓度和感染方法3个因素出发构建家蚕体内细菌感染模型,然后选用从云南地区自然发病蚕体中分离获得的5株肠道致病菌[Mammaliicoccus sciuri(葡萄球菌属)、粘质沙雷氏菌(Serratia marcescens)、弗氏柠檬酸杆菌(Citrobacter freundii)、Klebsiella variicola(克雷伯菌属)和Pseudomonas soli(假单胞菌属)],开展5种单菌感染及17种组合协同感染试验。【结果】通过建立家蚕体内细菌感染模型发现,家蚕群体添食细菌比单头添食的存活率低;细菌感染浓度选择时建议二龄期家蚕感染选用5.0 OD/mL、四龄期和五龄期家蚕感染选用10.0 OD/mL。选取的5株细菌对家蚕均具有致病力,整体上表现为随着细菌感染时间的延长,家蚕存活率逐渐降低;小蚕期感染细菌对家蚕的影响更明显,至五龄期有大量家蚕发病死亡,与养蚕过程中的细菌病发生规律相同。无论是小龄期感染还是大龄期感染,细菌协同感染的家蚕存活率总体上低于单菌感染,尤其是粘质沙雷氏菌参与的协同感染,至上蔟期的家蚕存活率均较低。此外,无论是二龄期感染还是四龄期感染,夏季家蚕的存活率总体上均高于春季家蚕,说明云南蚕区夏季家蚕的抗病力优于春季家蚕。【结论】5株分离自云南地区自然发病蚕体的病原菌对家蚕产生明显致病性,其致病性与感染龄期、感染浓度、不同细菌组合及感染季节等均有关,总体上具体表现为小蚕期感染较大蚕期感染对家蚕的影响更明显,细菌协同感染比单菌感染的危害更严重,且夏季家蚕感染后的存活率高于春季家蚕。

【Objective】This study aimed to establish a bacterial infection model in silkworm(Bombyx mori)to investi gate the synergistic pathogenicity of different bacteria,to clarify the pathogenesis and epidemiological characteristics of bacterial diseases of silkworm in Yunnan sericulture area,providing scientific guidance for the prevention and control of bacterial diseases during silkworm rearing.【Method】A bacterial infection model in B.mori was constructed based on three factors:instar,infection concentration and infection method.Five strains of intestinal pathogens[Mammaliicoccus sciuri(Staphylococcus sp.),Serratia marcescens,Citrobacter freundii,Klebsiella variicola(Klebsiella sp.),and Pseu domonas soli(Pseudomonas sp.)]were isolated from naturally infected silkworm in Yunnan.The pathogenicity of single infections with each strain and 17 synergistic infection combinations were conducted.【Result】The established bacterial in fection model in silkworm showed that silkworm larvae infected through population feeding of bacteria had a lower sur vival rate than those infected individually.An infection concentration of 5.0 OD/mL was recommended for second instar larvae,and 10.0 OD/mL for fourth and fifth instar larvae.All of the five selected bacterial strains were pathogenic to silk worm.The survival rate of infected silkworm generally decreased with the prolongation of infection time.Notably,infec tion at early instars had more obvious impact on silkworm leading to a large number of death of the fifth instar silkworm,which was consistent with the occurrence pattern of bacterial diseases in sericulture.The survival rate of silkworm with synergistic infection was generally lower than that of single-strain bacterial infections in both early and late instar infec tions,especially those synergistic infections involving S.marcescens,which resulted in lower survival rates until the spin ning stage.Additionally,the survival rate of summer rearing silkworm was generally higher than that of spring rearing silkworm in both second and fourth instar infections,indicating that the disease resistance of summer rearing silkworm in Yunnan sericulture areas was stronger than that of spring rearing silkworm.【Conclusion】The five strains of pathogenic bacteria isolated from naturally infected silkworm in Yunnan show obvious pathogenicity to silkworm,and their pathoge nicity is related to the infection instar,infection concentration,different bacterial combinations,and infection season.Overall,the specific performance is that the infection at early instars has a more significant impact on silkworm than that at late instars;synergistic infection is more severe than the single-strain bacterial infection;the survival rate of summer rearing silkworm after infection is higher than that of spring rearing silkworm.