Isolation, identification and drug sensitivity of Pseudomonas veronii from diseased half smooth tongue-sole (Cynoglossus semilaevis Günther)
-
摘要: 为探究导致半滑舌鳎（Cynoglossus semilaevis）疾病发生的病原，从患病半滑舌鳎肝脏中分离纯化出一株细菌，命名为CLPV-114，结合细菌形态学观察、生化鉴定和16S rRNA序列分析进行病原鉴定，对其进行药敏试验和生长曲线测定，人工感染试验后测定半数致死剂量（LD50），并进行组织病理学观察。结果表明：菌株CLPV-114为革兰氏阴性杆菌，在系统发育树上与GenBank登录号分别为AF064460、AY144583的维罗纳假单胞菌（Pseudomonas veronii）亲缘关系最近，自然聚为一支；综合生理生化特征和16S rRNA基因序列分析鉴定其为维罗纳假单胞菌；药敏试验显示，CLPV-114对阿莫西林、氨苄西林和头孢哌酮等18种抗菌药物高度敏感，对呋喃妥因、阿奇霉素和四环素等11种抗菌药物中度敏感，对头孢克肟、磺胺异恶唑和甲氧嘧啶等4种抗菌药物耐药；人工感染试验显示，CLPV-114对半滑舌鳎具有致病性，人工感染后的患病鱼与自然患病鱼症状相似，CLPV-114菌株对半滑舌鳎的LD50为1.3×107 CFU/mL；组织病理学观察显示，CLPV-114可引起明显的组织学损伤，表现为肝细胞组织坏死，鳃血管中心充血，脾脏出现含铁血黄素沉积、出血，肠绒毛脱落。研究表明，本次从患病半滑舌鳎肝脏中分离的菌株CLPV-114为维罗纳假单胞菌，对半滑舌鳎具有较强的致病性，可引起不同组织出现明显病理变化，本研究结果可为半滑舌鳎维罗纳假单胞菌病的防控提供科学参考。Abstract: In order to investigate the pathogenesis of diseased half smooth tongue-sole (Cynoglossus semilaevis Günther), a bacterial strain was isolated from the liver of the diseased half smooth tongue-sole with body weight of 20 to 40 g, designed as CLPV-114, and identified by morphological observation, physiological, and biochemical features and the sequence analysis of 16S rRNA. The drug sensitivity, artificial infection, histopathological examination and determination of growth curve were examined in the strain CLPV-114. The median lethal dose was determined and histopathological observations were conducted in the artificial infection test. The results showed that the strain CLPV-114 was gram-negative bacterium, and closely related to Pseudomonas veronii (AF064460) and Pseudomonas veronii (AY144583), so identified as Pseudomonas veronii. The drug sensitivity test showed that CLPV-114 was highly sensitive to 18 kinds of drugs including amoxicillin, ampicillin, cefoperazone, moderately sensitive to 11 antibiotics such as nitrofurantoin, azithromycin, tetracycline, and resistant to antibiotics such as cefixime, sulfafurazole and sulfamethoxydiazine. Infection test showed that the clinical symptoms were similar to those of naturally diseased fish, with the LD50 of 1.3×107 CFU/mL in CLPV-114 against half smooth tongue-sole. Histopathological observations found that CLPV-114 strain caused obvious histological damage, such as hepatocellular tissue necrosis, vascular congestion of the central filament vessel of gill, haemorrhage and deposition of hemosiderin in the spleen, and the shedding of the intestinal villi. The finding indicates that the strain CLPV-114 isolated from the liver of diseased tongue sole is Pseudomonas veronii, which has strong pathogenicity to tongue sole and can cause significant pathological changes in different tissues, and provides an effective scientific reference for control of Pseudomonas veronii disease in half smooth tongue-sole.
-
Key words:
- Cynoglossus semilaevis /
- Pseudomonas veronii /
- 16S rRNA /
- pathogenicity /
- drug sensitivity
-
-
HAN Z R, SUN J F, LV A J, et al.Isolation, identification and characterization of Shewanella algae from reared tongue sole, Cynoglossus semilaevis Günther[J].Aquaculture, 2017, 468:356-362.
WANG Y Z, SUN J F, LV A J, et al.Histochemical distribution of four types of enzymes and mucous cells in the gastrointestinal tract of reared half-smooth tongue sole Cynoglossus semilaevis[J].Journal of Fish Biology, 2018, 92(1):3-16.
CHEN Z Q, YAO Z X, LIN M, et al.Study on pathogen of skin ulcer disease of half-smooth tongue sole(Cynoglossus semilaevis)[J].Journal of Fisheries of China, 2012, 36(5):764.
DU M, CHEN S L, LIU Y H, et al.MHC polymorphism and disease resistance to Vibrio anguillarum in 8 families of half-smooth tongue sole (Cynoglossus semilaevis)[J].BMC Genetics, 2011, 12:78.
LI W Y, ZUO Z H, ZHANG J J, et al.Isolation and identification of pathogens of ulcer disease in Cynoglossus semilaevis[J].Journal of Fisheries of China, 2020, 44(4):672-680.(in Chinese)
SWAIN P, BEHURA A, DASH S, et al.Serum antibody response of Indian major carp, Labeo rohita to three species of pathogenic bacteria:Aeromonas hydrophila, Edwardsiella tarda and Pseudomonas fluorescens[J].Veterinary Immunology and Immunopathology, 2007, 117(1/2):137-141.
NAM I H, CHANG Y S, HONG H B, et al.A novel catabolic activity of Pseudomonas veronii in biotransformation of pentachlorophenol[J].Applied Microbiology and Biotechnology, 2003, 62(2):284-290.
MOU X T, LI Y, LI Q J, et al.Analysis on microbial flora in three types of Pinus massoniana forests in Yunyang County of the Three Gorges Reservoir AreaⅡ.Microflora in woodland air, leaf and bark surface[J].Forest Research, 2010, 23(5):762-769.(in Chinese)
LIU B, LAN J L, LIN Y Z, et al.Characteristics of PLFA biomarkers for acephatemet resistant bacteria isolated from the soils[J].Asian Journal of Ecotoxicology, 2009, 4(5):734-744.(in Chinese)
HU X C, BIAN Y F, HU H X, et al.Isolation and identification of Pseudomonas veronii from gills of crucian carp(Carassius carassius)[J].Southwest China Journal of Agricultural Sciences, 2014, 27(1):439-441.(in Chinese)
CHENG Z W, SUN P F, JIANG Y F, et al.Kinetic analysis and bacterium metabolization of α-pinene by a novel identified Pseudomonas sp.strain[J].Journal of Environmental Sciences, 2012, 24(10):1806-1815.
QUEIPO-ORTUÑO M I, DE DIOS COLMENERO J, MACIAS M, et al.Preparation of bacterial DNA template by boiling and effect of immunoglobulin G as an inhibitor in real-time PCR for serum samples from patients with brucellosis[J].Clinical and Vaccine Immunology:CVI, 2008, 15(2):293-296.
DE LA CUESTA-ZULUAGA J J, SÁNCHEZ-JIMÉNEZ M M, MARTÍNEZ-GARRO J, et al.Identification of the virB operon genes encoding the type IV secretion system, in Colombian Brucella canis isolates[J].Veterinary Microbiology, 2013, 163(1/2):196-199.
BAUER A W, KIRBY W M M, SHERRIS J C, et al.Antibiotic susceptibility testing by a standardized single disk method[J].American Journal of Clinical Pathology, 1966, 45:493-496.
ROSSOLINI G M, MANTENGOLI E.Treatment and control of severe infections caused by multiresistant Pseudomonas aeruginosa[J].Clinical Microbiology and Infection:the Official Publication of the European Society of Clinical Microbiology and Infectious Diseases, 2005, 11(sup 4):17-32.
HIRAKAWA Y, SASAKI H, KAWAMOTO E, et al.Prevalence and analysis of Pseudomonas aeruginosa in chinchillas[J].BMC Veterinary Research, 2010, 6:52.
GAIKWADV J.Antibacterial targets in Pseudomonas aeruginosa[J].International Journal of Pharmaceutics, 2011, 2(3):159-164.
ELOMARI M, COROLER L, HOSTE B, et al.DNA relatedness among Pseudomonas strains isolated from natural mineral waters and proposal of Pseudomonas veronii sp.nov[J].International Journal of Systematic Bacteriology, 1996, 46(4):1138-1144.
ANZAI Y, KIM H, PARK J Y, et al.Phylogenetic affiliation of the pseudomonads based on 16S rRNA sequence[J].International Journal of Systematic and Evolutionary Microbiology, 2000, 50:1563-1589.
SANTOS Y, TORANZO A E, BARJA J L, et al.Virulence properties and enterotoxin production of Aeromonas strains isolated from fish[J].Infection and Immunity, 1988, 56(12):3285-3293.
AUGUSTIN J C, CARLIER V.Modelling the growth rate of Listeria monocytogenes with a multiplicative type model including interactions between environmental factors[J].International Journal of Food Microbiology, 2000, 56(1):53-70.
SALLAM K I.Antimicrobial and antioxidant effects of sodium acetate, sodium lactate, and sodium citrate in refrigerated sliced salmon[J].Food Control, 2007, 18(5):566-575.
KOKOU F, SASSON G, MIZRAHI I, et al.Antibiotic effect and microbiome persistence vary along the European seabass gut[J].Scientific Reports, 2020, 10:10003.
WU Y N, LIU P, LI Q, et al.Effects of a compound Chinese herbal medicine on immunity and disease resistance in sea cucumber Apostichopus japonicus[J].Journal of Dalian Ocean University, 2011, 26(4):338-343.(in Chinese)
计量
- 文章访问数: 270
- PDF下载数: 0
- 施引文献: 0
下载:
