嗜水气单胞菌胁迫下棘胸蛙脾脏的病理损伤

doi:10.3969/j.issn.1004-1524.20240499

浙江农业学报 ›› 2025, Vol. 37 ›› Issue (6): 1221-1232.DOI: 10.3969/j.issn.1004-1524.20240499

嗜水气单胞菌胁迫下棘胸蛙脾脏的病理损伤

任思宇(), 吴春艳^*(), 刘震坤, 杨延辉

重庆三峡职业学院动物科技学院,重庆 404155

收稿日期:2024-06-07 出版日期:2025-06-25 发布日期:2025-07-08
作者简介:任思宇(1987—),男,四川江油人,博士,副教授,研究方向为渔业资源与水生动物病害。E-mail:493319087@qq.com
通讯作者: *吴春艳,E-mail:281368189@qq.com
基金资助:
重庆市教委科研项目(KJQN202203514);万州区科技局博士直通车项目(wzstc20220120);重庆三峡职业学院创新团队(sxzy-2023td-006)

Pathological lesions in spleen of Chinese spiny frog (Quasipaa spinosa) after Aeromonas hydrophila infection

REN Siyu(), WU Chunyan^*(), LIU Zhenkun, YANG Yanhui

College of Animal Science and Technology, Chongqing Three Gorges Vocational College, Chongqing 404155, China

Received:2024-06-07 Online:2025-06-25 Published:2025-07-08

摘要/Abstract

摘要： 为探索败血症病原菌嗜水气单胞菌(Aeromonas hydrophila)胁迫下棘胸蛙(Quasipaa spinosa)脾脏的病理损伤,本研究采用浓度为1×10⁷ CFU·mL^-1的棘胸蛙源嗜水气单胞菌重悬液,以每尾0.2 mL的剂量腹腔注射感染体质量为(130±20)g的健康棘胸蛙,复制败血症模型,分别在2 h、4 h、8 h、12 h、16 h、24 h、32 h、40 h、48 h、60 h、72 h、96 h(4 d)、120 h(5 d)、144 h(6 d)、168 h(7 d)时观察脾的临床表现,并采样开展动态病理研究。结果显示,感染后的棘胸蛙精神萎靡,应激反应减弱,脾体指数逐步升高,在40 h~5 d期间与对照组差异极显著(P<0.01)。动态病理观察显示:随着病程发展,白髓淋巴细胞、网状细胞逐渐坏死减少,单个的黑色素巨噬细胞增加,被膜与被膜下的组织不同程度地变性坏死。在48~72 h期间,白髓内细胞成分严重坏死,网状细胞脱落肿大,白髓面积显著缩小至消失,血管壁及周围网状纤维断裂明显,黑色素巨噬细胞中心(melano-macrophage centres, MMCs)全部裂解消失。在嗜水气单胞菌引起的致死性败血症胁迫下,棘胸蛙脾组织崩解,细胞成分坏死,损害了机体免疫应答的结构基础。

关键词: 棘胸蛙, 嗜水气单胞菌, 致死性败血症, 病理损伤

Abstract:

In order to explore the pathological lesions in spleen of Chinese spiny frog (Quasipaa spinosa) after haemorrhagic septicemia infection, Aeromonas hydrophila isolated from diseased Chinese spiny frog, which was identified as the pathogenic bacterium, was used to replicate haemorrhagic septicemia in health individual with body weight of (130±20) g. The challenge concentration was 1×10⁷ CFU·mL^-1 and the injected dose was 0.2 mL per frog. The spleen was selected as representative of the internal organs for clinical symptoms and dynamic pathological observation was carried out at 2 h, 4 h, 8 h, 12 h, 16 h, 24 h, 32 h, 40 h, 48 h, 60 h, 72 h, 96 h(4 d), 120 h(5 d), 144 h(6 d), 168 h(7 d) after infection, respectively. It was shown that the main clinical signs included lethargy and depression with increasing of the spleen index which was significantly (P<0.01) different between the control group and the infection group from 40 h to 5 d. The observation of dynamic pathological showed necrosis and reduction of lymphocytes and reticular cells in white pulp district with an increase of single melano-macrophage, and varying degrees of degeneration and necrosis in tunica. Especially, the typical pathological damage included severe necrosis in white pulp and disappearance of melano-macrophage centres (MMCs) with reticular fibre decomposition from 48 h to 72 h. The research indicated that under the stress of lethal septicemia, the splenic structure of the frog was disintegrated, the cell components were severely necrotic, and the structural basis of the immune organ was destroyed.

Key words: Quasipaa spinosa, Aeromonas hydrophila, haemorrhagic septicaemia, pathological lesions

中图分类号:

S948

任思宇, 吴春艳, 刘震坤, 杨延辉. 嗜水气单胞菌胁迫下棘胸蛙脾脏的病理损伤[J]. 浙江农业学报, 2025, 37(6): 1221-1232.

REN Siyu, WU Chunyan, LIU Zhenkun, YANG Yanhui. Pathological lesions in spleen of Chinese spiny frog (Quasipaa spinosa) after Aeromonas hydrophila infection[J]. Acta Agriculturae Zhejiangensis, 2025, 37(6): 1221-1232.

图/表 8

表1 人工感染后脾脏的大体动态病理变化

Table 1 Gross dynamic pathology of spleen after SZW-003 infection

采样时间 Sample time	颜色 Colour	被膜 Capsule	质地 Character	大小 Size
2~4 h	深红Deep red	有光泽Glossy	柔软Soft	正常Normal
8~16 h	暗红Dull red	被膜紧张、浑浊The capsule of spleen was tense and muddy	松软Soft	轻微肿大Slight swelling
24~40 h	发黑Nigrescence	被膜紧张、无光泽The capsule of spleen was tense without gloss	松散Loose	肿大Swelling
48~72 h	暗黑Dark	被膜紧张、无光泽The capsule of spleen was tense without gloss	松散Loose	显著肿大Significantly enlarged
4~7 d	暗红Dull red	被膜无光泽The capsule of spleen was not glossy	松软Soft	肿大但趋于正常Slight swelling

表2 人工感染后的脾体指数

Table 2 Spleen index after SZW-003 infection

t/h	脾体指数 Spleen index	t/h	脾体指数 Spleen index
0	0.108±0.019	40	0.320±0.103^**
2	0.120±0.011	48	0.293±0.054^**
4	0.121±0.024	60	0.321±0.114^**
8	0.108±0.036	72	0.311±0.083^**
12	0.199±0.037^*	96	0.231±0.073^**
16	0.194±0.050	120	0.208±0.043^**
24	0.212±0.039^*	144	0.168±0.036
32	0.177±0.035	168	0.190±0.082

图1 攻毒后白髓的动态病理变化(HE染色) a,正常脾脏,网状细胞(→)与淋巴细胞()结构清晰。b,攻毒后2~4 h,白髓内血窦充血明显(→);c,攻毒后8~16 h,网状细胞变性坏死(),嗜中性粒细胞浸润(),淋巴细胞染色浑浊不清(→);d,攻毒后24~40 h,淋巴细胞固缩(→),网状细胞变性坏死();e,攻毒后48~72 h,淋巴细胞()与网状细胞(→)严重坏死,可见大量细胞残体();f,攻毒后4~7 d,白髓充血明显(→),细胞较正常;g,对照组,脾脏网状细胞(→)与淋巴细胞()结构清晰。

Fig.1 Dynamic pathological changes of white pulp after SZW-003 infection (HE staining) a, Normal spleen showing the reticular cells (→)and lymphocytes()without pathological changes; b, 2-4 h after infection, hyperaemia was obvious in blood sinus of white pulp(→); c, 8-16 h after infection, necrosis of reticular cells(), and the pathological staining was not clear in lymphocytes(→)with neutrophilic granulocytes infiltration(); d, 24-40 h after infection, degeneration and necrosis of lymphocytes(→)and reticular cells(); e, 48-72 h after infection, serious necrosis of lymphocytes()and reticular cells(→)in white pulp with numerous debris(); f, 4-7 d after infection, the spleen showing hyperaemia with normal structure(→); g, Reticular cells (→)and lymphocytes()without pathological changes in the control group.

图2 攻毒后红髓的动态病理变化(HE染色) a,正常脾脏的红髓;b,攻毒后2~4 h,红髓中心区血液充盈(→);c,攻毒后8~16 h,红髓与白髓之间可见明显的充血带(→);d,攻毒后24~40 h,网状细胞肿胀脱落呈吞噬细胞样(→),纤维基质肿胀断裂();e,攻毒后48~72 h,红髓结构严重破坏,网状细胞(→)与淋巴细胞()坏死严重;f,攻毒后4~7 d,网状细胞肿胀(),纤维基质水肿(→),结构较完整;g,对照组,脾脏红髓结构清晰。

Fig.2 Dynamic pathological changes of red pulp after SZW-003 infection (HE staining) a, Normal spleen showing the red pulp without pathological change; b, 2-4 h after infection, slight hyperaemia in blood sinus(→); c, 8-16 h after infection, serious hyperaemia between the white pulp and red pulp(→); d, 24-40 h after infection, the red pulp showing broken fiber()with cells descrete, and degeneration of reticular cells like phagocyte(→); e, 48-72 h after infection,the spleen showing severe pathological injury with necrosis of lymphocytes ()and reticular cells(→); f, 4-7 d after infection, the spleen showing swellen reticular cells()and fiber(→)with normal structure; g, Spleen in the control group showing red pulp without pathological change.

图3 攻毒后被膜的动态病理变化(HE染色) a,正常脾的被膜结构清晰;b,攻毒后2~4 h,脾脏未出现明显变化;c,攻毒后8~16 h,被膜结缔组织染色较深,并出现水肿(→);d,攻毒后24~40 h,被膜结缔组织水肿(),网状细胞脱落游离(→);e,攻毒后48~72 h,被膜显著变薄(→),被膜下纤维结构裂解();f,攻毒后4~7 d,被膜组织中结缔组织轻度增厚(→);g,对照组,脾脏被膜结构清晰。

Fig.3 Dynamic pathological changes of capsule after infection (HE staining) a, Normal spleen showing the capsule without pathological change; b, 2-4 h after infection, the spleen was normal; c, 8-16 h after infection,degeneration and swellen in capsule(→); d, 24-40 h after infection, the fiber structure was disorder with serious swellen in capsule(), and the reticular cell(→) presented degeneration and exfoliated under the capsule; e, 48-72 h after infection, the capsule(→) was significantly thinned with swellen and exfoliated reticular cells() under it; f, 4-7 d after infection, the capsule presented swellen(→); g, Spleen in the control group showing the capsule without pathological change.

图4 攻毒后脾内纤维的病理损伤 a,正常脾脏的被膜中弹性纤维连续(→),Tanake维多利亚蓝染色法;b,攻毒后48~72 h,被膜中弹性纤维减少(→),Tanake维多利亚蓝染色法;c,攻毒后48~72 h,静脉血管壁上的浅蓝色信号不连续(→),Tanake维多利亚蓝染色法;d,正常脾脏中网状纤维明显,排列连续(→),改良Gomori氨银法;e,攻毒后48~72 h,血管壁(→)及周围()的网状纤维排列杂乱,改良Gomori氨银法;f,攻毒后48~72 h,红髓中的网状纤维严重裂解,甚至消失(),改良Gomori氨银法;g,正常脾脏的红髓含有部分胶原纤维,蓝色信号较浅(→),Masson三色染色法;h,攻毒后48~72 h,红髓中的胶原纤维大面积地溶解断裂,Masson三色染色法;i,攻毒后4~7 d,红髓中的蓝色信号显著增强(→),Masson三色染色法;j,对照组,脾脏被膜中的弹性纤维连续(→),Tanake维多利亚蓝染色法;k,对照组,脾脏中的网状纤维明显,排列连续(→),Gomori氨银法;l,对照组,脾脏红髓中含有少量的胶原纤维(→),Masson三色染色法。

Fig.4 Dynamic pathological changes of fiber in spleen after infection a, Uninterrupted elastic fiber in capsule in normal spleen(→), Tanake Victoria blue staining; b, 48-72 h after infection, reduced elastic fiber in capsule(→),Tanake Victoria blue staining; c, 48-72 h after infection, discontinuous light blue signal on the venous vessel wall(→), Tanake Victoria blue staining; d, Obvious reticular fiber without interruption in normal spleen(→), modified Gomori silver ammonia method; e, 48-72 h after infection, disordered reticular fiber in the blood vessel wall(→)and around it(), modified Gomori silver ammonia method; f, 48-72 h after infectin, the reticular fiber in red pulp dissolved and disappeared(), modified Gomori silver ammonia method; g, Moderate collagen fibers in red pulp of normal spleen with weaker signal(→), Masson trichrome staining; h, 48-72 h after infection, the collagen fibers dissolved in red pulp, Masson trichrome staining; i, 4-7 d after infectin, significantly enhanced blue signal in the red pulp (→), Masson trichrome staining; j, Uninterrupted elastic fiber in capsule of spleen in the control group(→), Tanake victoria blue staining; k, Obvious and uninterruputed reticular fiber of spleen in the control group(→), modified Gomori silver ammonia method; l, Moderate collagen fibers in red pulp spleen with weaker signal in the control group(→), Masson trichrome staining.

图5 攻毒后黑色素巨噬细胞与黑色素巨噬细胞中心(MMCs)的动态病理变化 a,正常脾脏的MMCs结构致密,轮廓清晰(),HE染色;b,正常脾脏的MMCs普鲁士蓝染色较浅,间杂黄色信号(),普鲁士蓝染色;c,攻毒后8~16 h,黑色素巨噬细胞和小型MMCs的数量明显增加(),普鲁士蓝染色;d,攻毒后24~40 h,黑色素巨噬细胞和小型MMCs的数量明显增加(),普鲁士蓝染色;e,攻毒后24~40 h,MMCs()出现裂解,HE染色;f,攻毒后48~72 h,脾组织严重受损,MMCs全部裂解消失,HE染色;g,攻毒后4~7 d,MMCs经普鲁士蓝染色显示呈蓝色(),间杂浅黄色(→),小型的MMCs增加(▲),普鲁士蓝染色;h,对照组脾脏的MMCs结构致密,轮廓清晰(),HE染色;i,对照组脾脏的黑色素巨噬细胞数量较少(→),普鲁士蓝染色。

Fig.5 Dynamic pathological changes of melano-macrophages and melano-macrophage centres (MMCs) after infection a, MMCs in normal spleen were dense and clear in outline(),HE staining; b, MMCs in light blue()with a small amount of light yellow(→)in normal spleen, Perls staining; c, 8-16 h after infection, melano-macrophages and small MMCs increased obviously(), Perls staining; d, 24-40 h after infection, melano-macrophages and small MMCs increased obviously(), Perls staining; e, 24-40 h after infection, the MMCs presented histolysis(), HE staining; f, 48-72 h after infection, the spleen showed severe injury, the MMCs dissolved and disappeared, HE staining; g, 4-7 d after infection, the small MMCs increased(▲), and the MMCs presented mainly blue()with a small amount of light yellow(→), Perls staining; h, MMCs in spleen of the control group were dense and clear in outline(), HE staining; i, Fewer melano-macrophages in spleen of the control group(→), Perls staining.

图6 濒死蛙与幸存蛙脾脏的病理学变化(HE染色) a,濒死蛙淋巴细胞坏死、碎裂(→),网状细胞坏死变性();b,濒死蛙白髓结构坏死消失(▲),红髓显著充血,脾索断裂();c,幸存蛙红髓充血(),网状细胞肿胀(→),MMCs数量增加(▲);d,幸存蛙MMCs数量增加(▲),白髓结构正常,染色清晰。

Fig.6 Pathological changes of spleen in dying and survival Quasipaa spinosa (HE staining) a, Denegaration and necriosis of reticular cells()and lymphocytes(→)of dying Chinese spiny frog (Quasipaa spinosa); b, White pulp(▲)disappeared and red pulp presenting congestion with spleen cord rupture() of dying Chinese spiny frog; c, Red pulp showing hyperaemia() with swellen reticular cells(→), and MMCs increased(▲) in quantity in survival Chinese spiny frog; d, MMCs increased(▲) in quantity with normal white pulp of survival Chinese spiny frog.

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嗜水气单胞菌胁迫下棘胸蛙脾脏的病理损伤

Pathological lesions in spleen of Chinese spiny frog (Quasipaa spinosa) after Aeromonas hydrophila infection

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