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| Isolation, Identification and Pathogenicity Study of Lactococcus garvieae from Cultured Liza haematocheila |
| HONG Bao-Hua1, MA Rong-Rong1, YUAN Na1, ZHANG Ke-Xin2, LI Jin-Jin1, NI Si-Zhen1, ZHU Wei-Dong2, ZHANG Hong-Hu1, QIAN Dong1,* |
1 College of Marine Sciences, Ningbo University, Ningbo 315823, China;
2 Yuyao Fisheries Technology Extension Center, Yuyao 315400, China |
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Abstract In the summer of 2018, a large-scale persistent outbreak of Liza haematocheila occurred in a freshwater fishing farm in Ningbo, Zhejiang province, with the cumulative mortality rate more than 60%. The moribund fish swimming alone or spinning on the water surface with unresponsive, anorexic, swollen abdomen and exophthalmos, thickened opaque eyes. The dissected mullet showed ascites, enlarged liver, spleen, petechiae and local bleeding and other symptoms, reddish macular pseudomembrane was observed in the connection of the swim bladder and kidney in the abdomen with the inflated compartment. To study the pathogen and main pathogenesis of cultured L. haematocheila and other related species for further diseases scientific control, eight bacterial strains (named Lh-1~Lh-8) were isolated from the brain, liver, spleen and kidney of 3 diseased fish with typical symptoms by using nutrient agar (NA) and brain heart infusion agar (BHIA) medium. Round, smooth and greyish white colonies sized 1~4 mm were found on BHIA and confirmed as Gram-positive coccus with short chain. Lh-3, one of the isolates, was identified physiologically and biochemically as Lactococcus garvieae with 99% similarity by using VITEK-2-Compact. The 16S rDNA and gyrase B (gyrB) were used for molecular identification. Sequence homology alignment and construction of a phylogenetic tree showed that the similarity to Lactococcus garvieae reached 100% and 93.5%, respectively, and both showed Lactococcus garvieae. The symptoms similar to natural disease occurred in L. haematocheila 2 d after was injected intraperitoneally with Lh-3, with the LD50 of 5.86×104 CFU/ind. Lh-3 showed typical alpha hemolysis on blood agar plates, and no transparent circles appeared on skimmed milk plates. Institute of Cancer Research (ICR) mice (Mus musculus) were infected by intraperitoneal injection of Lh-3. The mice showed bristled, trembling and dispirited with reddish and swollen eyes after 12 h of challenging. Pathological changes such as congestion of liver, lung and abdominal wall were observed in infected mice, and the mortality within 24 h was 100% (1×109 CFU/ind). It could be concluded that the isolated Lactococcus garvieae was a causative pathogen to Lactococcus haematocheila with strong virulence, and potential zoonosis pathogen to mammals with certain pathogenicity. This study provides a reference for disease control of L. haematocheila and other aquaculture.
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Received: 03 January 2020
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Corresponding Authors:
*qiandong@nbu.edu.cn
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