Isolation, identification and histopathological study on oedema pathogen from cultured leech (Whitmania pigra)

doi:10.3969/j.issn.1004-1524.20221774

Acta Agriculturae Zhejiangensis ›› 2023, Vol. 35 ›› Issue (12): 2844-2853.DOI: 10.3969/j.issn.1004-1524.20221774

• Animal Science • Previous Articles Next Articles

Isolation, identification and histopathological study on oedema pathogen from cultured leech (Whitmania pigra)

TANG Yi¹(), YANG Qinglin¹, WANG Wei², YUAN Yuan², DING Shihua¹, SUN Hanchang³^,^*(), LYU Hao⁴

1. Key Laboratory of Freshwater Fish Reproduction and Development (Ministry of Education), College of Fisheries, Southwest University, Chongqing 400715, China
2. Chongqing Duoputai Pharmaceutical Co., Ltd., Chongqing 400800, China
3. Technology Innovation Center of Ecological Fishery Industrialization, College of Landscape and Life Science, Chongqing University of Arts and Science, Chongqing 402160, China
4. Chongqing Hechuan District Aquatic Technology Promotion Station, Chongqing 401520, China

Received:2022-12-13 Online:2023-12-25 Published:2023-12-27

Abstract

Abstract:

To identify the cause of the Whitmania pigra oedema bacteria disease outbreak and to provide new ideas for fishery medicine research, in the present study, the pathogenic bacterial strain (named CQ1808) was isolated from the body cavity fluid of the diseased leeches and bacterial examination, histopathology and antimicrobial drugs test were performed. The results demonstrated that CQ1808 strain was a Gram-negative short bacillus, and was naturally clustered with the 16S rDNA sequence of Citrobacter freundii by the BLAST search. The phylogenetic tree suggested that the strain was Citrobacter freundii. The antidrug profiles of the isolate showed that CQ1808 strain was highly sensitive to 21 antibiotics including aztreonam, ceftriaxone and cefoperazone; intermediately susceptible to 7 antibiotics including ceftazidime, cefazolin and kanamycin; resistant to 9 antibiotics including penicillin, cefuroxime and midecamycin. The symptoms of W. pigra artificially infected with CQ1808 strain were similar to naturally infected leeches. Histopathological analysis showed that the overall organizational structure of the naturally diseased leeches was abnormal and the most distinct pathological changes occurred in the crop. This study indicated that C. freundii was the causative agent of oedema in farmed leeches.

Key words: Whitmania pigra, oedema, Citrobacter freundii, pathogenicity

CLC Number:

S917.1

TANG Yi, YANG Qinglin, WANG Wei, YUAN Yuan, DING Shihua, SUN Hanchang, LYU Hao. Isolation, identification and histopathological study on oedema pathogen from cultured leech (Whitmania pigra)[J]. Acta Agriculturae Zhejiangensis, 2023, 35(12): 2844-2853.

Figures/Tables 8

References 36

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测定项目Test item	CQ1808	LMG 3246*	测定项目Test item	CQ1808	LMG 3246*
D-山梨醇D-Sorbitol	+	+	赖氨酸脱羧酶Lysine decarboxylase	-	-
鼠李糖Rhamnose	+	+	硝酸盐还原Nitrate reduction	+	+
甲基红试验Methyl red test	+	+	七叶苷Esculoside	-	-
吲哚Indole	-	-	蔗糖Sucrose	+	(-)
葡萄糖产酸Glucose acid	+	+	明胶液化Gelatin	-	-
葡萄糖产气Glucose gas	+	+	H₂S	+	+
氰化钾生长KCN growth	-	-	V.P.试验Voges-proskauer test	-	-
甘露糖Mannose	+	+	O-F试验O-F test	F	F
枸橼酸盐Citrate	+	+	肌醇Inositol	-	-
阿拉伯糖Arabinoso	+	+	麦芽糖Maltose	+	+
侧金盏花醇Adonitol	-	-	尿素Urea	-	-
木糖Xylose	+	+	果糖Fructose	+	+
水杨酸Salicylic acid	-	-	糊精Dextrin	-	-
氧化酶Oxidase	-	-	葡萄糖磷酸盐Glucose phosphate	-	-
精氨酸脱羧酶Arginine decarboxylase	+	+	半乳糖Galactose	+	+
苯丙氨酸脱羧酶Phenylalanine deaminase	-		D-纤维二糖D-Cellobiose	-	-
鸟氨酸脱羧酶Ornithine decarboxylase	-	-	水杨苷Salicin	+	+

测定项目Test item	CQ1808	LMG 3246*	测定项目Test item	CQ1808	LMG 3246*
D-山梨醇D-Sorbitol	+	+	赖氨酸脱羧酶Lysine decarboxylase	-	-
鼠李糖Rhamnose	+	+	硝酸盐还原Nitrate reduction	+	+
甲基红试验Methyl red test	+	+	七叶苷Esculoside	-	-
吲哚Indole	-	-	蔗糖Sucrose	+	(-)
葡萄糖产酸Glucose acid	+	+	明胶液化Gelatin	-	-
葡萄糖产气Glucose gas	+	+	H₂S	+	+
氰化钾生长KCN growth	-	-	V.P.试验Voges-proskauer test	-	-
甘露糖Mannose	+	+	O-F试验O-F test	F	F
枸橼酸盐Citrate	+	+	肌醇Inositol	-	-
阿拉伯糖Arabinoso	+	+	麦芽糖Maltose	+	+
侧金盏花醇Adonitol	-	-	尿素Urea	-	-
木糖Xylose	+	+	果糖Fructose	+	+
水杨酸Salicylic acid	-	-	糊精Dextrin	-	-
氧化酶Oxidase	-	-	葡萄糖磷酸盐Glucose phosphate	-	-
精氨酸脱羧酶Arginine decarboxylase	+	+	半乳糖Galactose	+	+
苯丙氨酸脱羧酶Phenylalanine deaminase	-		D-纤维二糖D-Cellobiose	-	-
鸟氨酸脱羧酶Ornithine decarboxylase	-	-	水杨苷Salicin	+	+

药物 Drug	抑菌圈直径判定标准 The judgment standard of inhibition zone diameter/mm			药物含量 Drug content/ (μg·disc^-1)	抑菌圈直径 Inhibition zone diameter/mm	药物敏感性 Drug sensitivity
药物 Drug	R	I	S	药物含量 Drug content/ (μg·disc^-1)	抑菌圈直径 Inhibition zone diameter/mm	药物敏感性 Drug sensitivity
青霉素Penicillin	≤19	>19~<28	≥28	10	0	R
氨苄西林Ampicillin	≤13	>13~<17	≥17	10	0	R
氨曲南Aztreonam	≤15	>15~<22	≥22	30	38.5	S
头孢曲松Ceftriaxone	≤13	>13~<21	≥21	30	29.3	S
头孢他啶Ceftazidime	≤14	>14~<18	≥18	30	17.4	I
头孢哌酮Cefoperazone	≤15	>15~<21	≥21	75	32.5	S
头孢噻肟Cefotaxime	≤14	>15~<23	≥23	30	35.5	S
头孢呋辛Cefuroxime	≤13	>13~<19	≥19	30	0	R
头孢拉定Cefradine	≤14	>14~<18	≥18	30	20.7	S
头孢唑啉Cefazolin	≤14	>14~<18	≥18	30	16.7	I
头孢克洛Cefaclor	≤14	>14~<18	≥18	30	28.9	S
头孢氨苄Cephalexin	≤14	>14~<18	≥18	30	22.3	S
卡那霉素Kanamycin	≤13	>13~<18	≥18	30	13.5	I
丁胺卡那霉Amikacin	≤14	>14~<17	≥17	30	15.2	I
庆大霉素Gentamycin	≤12	>12~<15	≥15	10	15.0	S
新霉素Neomycin	≤12	>12~<17	≥17	30	13.5	I
链霉素Streptomycin	≤11	>11~<15	≥15	10	22.2	S
大观霉Spectinomycin	≤14	>14~<18	≥18	100	15.8	I
罗红霉素Erythmmycin	≤13	>13~<23	≥23	15	0	R
麦迪霉素Midecamycin	≤13	>13~<18	≥18	30	0	R
克林霉素Clindamycin	≤14	>14~<21	≥21	2	0	R
氯霉素Chloramphenicol	≤12	>12~<18	≥18	30	22.5	S
氟苯尼考Florfenicol	≤12	>12~<18	≥18	30	21.7	S
四环素Tetracycline	≤14	>14~<19	≥19	30	20.0	S
米诺环素Minocycline	≤14	>14~<19	≥19	30	31.9	S
多西环素Doxycycline	≤10	>10~<14	≥14	30	25.8	S
多西环素Deoxycycline	≤12	>12~<16	≥16	30	21.5	S
复方新诺明Sulfamethoxazole	≤10	>10~<16	≥16	20	31.2	S
磺胺异恶唑Sulfafurazole	≤12	>12~<17	≥17	300	30.3	S
氧氟沙星Ofloxacin	≤12	>12~<16	≥16	5	22.3	S
左氧氟沙星Levofloxacin	≤13	>13~<17	≥17	5	15.3	I
诺氟沙星Norfloxacin	≤12	>12~<17	≥17	10	11.2	R
环丙沙星Ciprofloxacin	≤15	>15~<21	≥21	5	14.2	R
恩诺沙星Enrofloxacin	≤14	>15~<18	≥18	10	18.7	S
呋喃妥因Nitrofurantoin	≤14	>15~<17	≥17	300	23.5	S
呋喃唑酮Furazolidone	≤14	>15~<17	≥17	100	19.7	S
利福平Rifampicin	≤16	>15~<20	≥20	5	0	R

药物 Drug	抑菌圈直径判定标准 The judgment standard of inhibition zone diameter/mm			药物含量 Drug content/ (μg·disc^-1)	抑菌圈直径 Inhibition zone diameter/mm	药物敏感性 Drug sensitivity
药物 Drug	R	I	S	药物含量 Drug content/ (μg·disc^-1)	抑菌圈直径 Inhibition zone diameter/mm	药物敏感性 Drug sensitivity
青霉素Penicillin	≤19	>19~<28	≥28	10	0	R
氨苄西林Ampicillin	≤13	>13~<17	≥17	10	0	R
氨曲南Aztreonam	≤15	>15~<22	≥22	30	38.5	S
头孢曲松Ceftriaxone	≤13	>13~<21	≥21	30	29.3	S
头孢他啶Ceftazidime	≤14	>14~<18	≥18	30	17.4	I
头孢哌酮Cefoperazone	≤15	>15~<21	≥21	75	32.5	S
头孢噻肟Cefotaxime	≤14	>15~<23	≥23	30	35.5	S
头孢呋辛Cefuroxime	≤13	>13~<19	≥19	30	0	R
头孢拉定Cefradine	≤14	>14~<18	≥18	30	20.7	S
头孢唑啉Cefazolin	≤14	>14~<18	≥18	30	16.7	I
头孢克洛Cefaclor	≤14	>14~<18	≥18	30	28.9	S
头孢氨苄Cephalexin	≤14	>14~<18	≥18	30	22.3	S
卡那霉素Kanamycin	≤13	>13~<18	≥18	30	13.5	I
丁胺卡那霉Amikacin	≤14	>14~<17	≥17	30	15.2	I
庆大霉素Gentamycin	≤12	>12~<15	≥15	10	15.0	S
新霉素Neomycin	≤12	>12~<17	≥17	30	13.5	I
链霉素Streptomycin	≤11	>11~<15	≥15	10	22.2	S
大观霉Spectinomycin	≤14	>14~<18	≥18	100	15.8	I
罗红霉素Erythmmycin	≤13	>13~<23	≥23	15	0	R
麦迪霉素Midecamycin	≤13	>13~<18	≥18	30	0	R
克林霉素Clindamycin	≤14	>14~<21	≥21	2	0	R
氯霉素Chloramphenicol	≤12	>12~<18	≥18	30	22.5	S
氟苯尼考Florfenicol	≤12	>12~<18	≥18	30	21.7	S
四环素Tetracycline	≤14	>14~<19	≥19	30	20.0	S
米诺环素Minocycline	≤14	>14~<19	≥19	30	31.9	S
多西环素Doxycycline	≤10	>10~<14	≥14	30	25.8	S
多西环素Deoxycycline	≤12	>12~<16	≥16	30	21.5	S
复方新诺明Sulfamethoxazole	≤10	>10~<16	≥16	20	31.2	S
磺胺异恶唑Sulfafurazole	≤12	>12~<17	≥17	300	30.3	S
氧氟沙星Ofloxacin	≤12	>12~<16	≥16	5	22.3	S
左氧氟沙星Levofloxacin	≤13	>13~<17	≥17	5	15.3	I
诺氟沙星Norfloxacin	≤12	>12~<17	≥17	10	11.2	R
环丙沙星Ciprofloxacin	≤15	>15~<21	≥21	5	14.2	R
恩诺沙星Enrofloxacin	≤14	>15~<18	≥18	10	18.7	S
呋喃妥因Nitrofurantoin	≤14	>15~<17	≥17	300	23.5	S
呋喃唑酮Furazolidone	≤14	>15~<17	≥17	100	19.7	S
利福平Rifampicin	≤16	>15~<20	≥20	5	0	R

Isolation, identification and histopathological study on oedema pathogen from cultured leech (Whitmania pigra)

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