A Preliminary Study on Genetic and Serological Clustering of Pathogenic Pseudomonas plecoglossicida in Larimichthys crocea
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摘要:目的 内脏白点病是冬春季大黄鱼（Larimichthys crocea）最主要的细菌性疾病，变形假单胞菌（Pseudomonas plecoglossicida）是内脏白点病的主要病原。分析大黄鱼来源的变形假单胞菌病原聚类特征，为大黄鱼变形假单胞菌流行规律研究和科学防控提供参考。方法 克隆7株不同时空来源的大黄鱼变形假单胞菌16S rDNA、gyrB，测序并构建进化树；制备上述菌株的O-特异链血清（O抗原血清）与鞭毛蛋白血清（H抗原血清），进行菌体凝集试验。结果 16S rDNA和gyrB序列分析表明，所有的变形假单胞菌聚为一簇；O抗原和H抗原凝集结果表明，7株变形假单胞菌以及变形假单胞菌标准株均会发生凝集反应，凝集效价也无明显区别，与荧光假单胞菌（Pseudomonas fluoresenes）、溶藻弧菌（Vibrio alginolyticus）和哈维氏弧菌（Vibrio harveyi）不发生凝集反应。结论 本研究所用的7株不同时空来源的变形假单胞菌亲缘关系接近，可能具有共同的起源。Abstract:Objective Clustering of strains of Pseudomonas plecoglossicida, a major pathogen that causes the serious bacterial visceral white spot disease on Larimichthys crocea in winter and spring, was studied for disease control and epidemic prevention.Method The 16S rDNA and gyrB of 7 suspected P. plecoglossicida strains collected from different temporal and spatial sources were cloned and sequenced with evolutionary trees constructed. O and H antigen serums of these strains were prepared for agglutination tests.Result The 7 pathogens could be clustered into a same branch based on their sequences. Their O and H antigen sera agglutinated with those of the standard P. plecoglossicida reference without significant differences on titers, but not with that of P. fluoresenes, Vibrio alginolyticus, or V. harveyi.Conclusion It appeared that the 7 collected strains of P. plecoglossicida were genetically closely related and might have a common origin.
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图 1 基于16S rDNA序列构建的大黄鱼变形假单胞菌等菌株系统发育树
Figure 1. Phylogenetic tree of P. plecoglossicida strains based on 16S rDNA sequences
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图 2 变形假单胞菌gyrB PCR扩增结果
M：Marker；1、变形假单胞菌H17050402；2、变形假单胞菌H17052503；3、变形假单胞菌H17041302；4、变形假单胞菌H16042701；5、变形假单胞菌ATCC700383；6、变形假单胞菌H15122403；7、变形假单胞菌H16040803；8、变形假单胞菌H16041203；9、溶藻弧菌H21112601；10、哈维氏弧菌H21112606：11、空白。
Figure 2. PCR on gyrB of P. plecoglossicida
M: Marker; 1: P. plecoglossicida H17050402; 2: P. plecoglossicida H17052503; 3: P. plecoglossicida H17041302; 4: P. plecoglossicida H16042701; 5: P. plecoglossicida ATCC700383; 6: P. plecoglossicida H15122403; 7: P. plecoglossicida H16040803; 8: P. plecoglossicida H16041203; 9: V. alginolyticus H21112601; 10: V. harveyi H21112606; 11: blank.
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图 3 基于gyrB序列构建的大黄鱼变形假单胞菌等菌株系统发育树
Figure 3. Phylogenetic tree of P. plecoglossicida strains based on gyrB sequences
表 1 本研究用菌株及参考菌株
Table 1 Bacterial strains and reference used
菌株编号
Strains种属
Species来源地
Locations物种
Hosts分离部位
Tissues分离时间
DatesH15122403 变形假单胞菌
Pseudomonas plecoglossicida福建福鼎沙埕（沙埕港） 大黄鱼 脾 2015-12-24 H16040803 变形假单胞菌
Pseudomonas plecoglossicida福建霞浦东安（东吾洋） 大黄鱼 脾 2016-04-08 H16041203 变形假单胞菌
Pseudomonas plecoglossicida福建蕉城大湾（三都澳） 大黄鱼 脾 2016-04-12 H16042701 变形假单胞菌
Pseudomonas plecoglossicida福建蕉城黄湾（三都澳） 大黄鱼 肝 2016-04-27 H17041302 变形假单胞菌
Pseudomonas plecoglossicida福建蕉城泥土沃（三都澳） 大黄鱼 肾 2017-04-13 H17050402 变形假单胞菌
Pseudomonas plecoglossicida福建蕉城白秤潭（三都澳） 大黄鱼 肾 2017-05-04 H17052503 变形假单胞菌
Pseudomonas plecoglossicida福建蕉城白秤潭（三都澳） 大黄鱼 脾 2017-05-25 ATCC700383 变形假单胞菌
Pseudomonas plecoglossicida日本德岛县 香鱼 肾 1994-03 H13032003 荧光假单胞菌
Pseudomonas fluoresenes福建蕉城大湾（三都澳） 大黄鱼 脾 2013-03-20 H21112606 溶藻弧菌
Vibrio alginolyticus福建蕉城斗姆（官井洋） 大黄鱼 体表 2021-11-26 H21112601 哈维氏弧菌
Vibrio harveyi福建蕉城斗帽（官井洋） 大黄鱼 肝 2011-11-26 
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表 2 各菌株O抗原与抗O抗原血清的凝集反应结果
Table 2 Agglutination of O antigen and anti-O antigen sera
O抗原
O antigen抗O抗原血清凝集效价
Serum anti O antigen agglutination titerH15122403 H16040803 H16041203 H16042701 H17041302 H17050402 H17052503 H15122403 28 29 29 29 29 29 29 H16040803 28 29 28 28 28 28 28 H16041203 27 28 29 28 28 28 28 H16042701 28 29 29 28 27 29 28 H17041302 27 28 29 27 27 27 27 H17050402 27 28 29 26 27 29 28 H17052503 27 28 29 28 28 28 28 ATCC700383 27 28 28 29 28 28 29 H13032003 — — — — — — — H21112606 — — — — — — — H21112601 — — — — — — — 0.9%NaCl — — — — — — —
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表 3 各菌株H抗原与抗H血清的凝集反应结果
Table 3 Agglutination of H antigen and anti-H antigen sera
H抗原
H antigen抗H抗原血清凝集效价
Serum anti H antigen agglutination titerH15122403 H16040803 H16041203 H16042701 H17041302 H17050402 H17052503 H15122403 29 29 29 29 29 28 28 H16040803 29 29 29 29 29 28 28 H16041203 29 29 29 29 29 29 29 H16042701 29 29 29 29 29 29 28 H17041302 29 29 29 29 29 29 29 H17050402 29 29 28 28 29 29 29 H17052503 29 29 28 28 29 28 29 ATCC700383 29 28 29 29 29 28 29 H13032003 — — — — — — — H21112606 — — — — — — — H21112601 — — — — — — — 0.9%NaCl — — — — — — —
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表 4 其他临床分离菌株与7株变形假单胞菌抗血清的凝集结果
Table 4 Agglutination between individual strains and sera of 7 strains of P. plecoglossicida
抗原
Antigen抗O抗原血清/抗H抗原血清凝集效价
Serum anti O antigen/ Serum anti H antigen agglutination titer菌株
Strains时间
Dates来源
LocationsH15122403 H16040803 H16041203 H16042701 H17041302 H17050402 H17052503 H13032002 2013.03.20 宁德蕉城（三都澳） 28/29 28/29 28/28 29/29 27/27 26/26 27/28 H14060603 2014.06.06 宁德蕉城（三都澳） 29/28 29/28 29/27 29/28 29/29 29/28 29/28 H15012203 2015.01.22 宁德蕉城（三都澳） 29/29 29/28 29/29 29/29 28/28 29/28 29/29 H16031803 2016.03.18 宁德霞浦（东吾洋） 29/29 29/28 29/29 29/29 28/29 27/28 28/29 H16032302 2016.03.23 宁德福鼎（沙埕港） 29/28 28/29 27/29 28/29 29/29 28/29 28/28 H16032303 2016.03.23 宁德蕉城（三都澳） 29/28 29/28 29/27 28/29 29/28 28/29 29/29 H16042703 2016.04.27 宁德蕉城（三都澳） 29/28 29/29 29/29 29/29 29/28 29/28 28/29 H17030202 2017.03.02 宁德蕉城（三都澳） 29/29 29/29 29/28 28/29 29/29 28/29 28/28 H19040303 2019.04.03 宁德霞浦（东吾洋） 29/28 28/29 29/28 29/29 28/29 29/29 28/28 H20031102 2020.03.11 宁德蕉城（三都澳） 29/29 29/29 29/28 28/29 28/29 27/29 29/29 H22042903 2022.04.29 宁德蕉城（三都澳） 29/29 29/29 28/28 27/28 29/29 29/29 29/29 0.9%NaCl -/- -/- -/- -/- -/- -/- -/-
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