翘嘴鲌源维氏气单胞菌的分离鉴定及组织病理观察

Isolation, Identification and Histopathological Observation of Aeromonas veronii from Culter alburnus

为确定患病翘嘴鲌(Culter alburnus)的病原,本研究从患病鱼肝脏中分离到一株优势菌株CQ200825,对该菌进行形态学观察、生理生化鉴定、16S rRNA和gyrB基因序列分析、人工感染实验、毒力基因检测、组织病理观察以及药敏试验。结果显示,菌株CQ200825为短杆状的革兰氏阴性菌,通过16S rRNA和gyrB基因序列比对、系统进化树的构建和生理生化特性,确认为维氏气单胞菌(Aeromonas veronii)。人工感染实验组与自然发病鱼都表现为鳍条基部出血、肝脾肿大、有腹水等症状,同时从人工感染濒死鱼体内分离到与菌株CQ200825理化及分子特性一致的优势菌株,表明CQ200825为患病翘嘴鲌的病原菌,并计算出菌株CQ200825对翘嘴鲌的半数致死量为2.7×10^(6)CFU/mL。毒力基因检测结果显示,菌株CQ200825携带外膜蛋白(ompA)、溶血素(hlyA)、热稳定性肠毒素(ast)和细胞毒性肠毒素(act)4种毒力基因。组织病理观察发现,患病翘嘴鲌的肝、脾、肾和肠均有不同程度的病变,即肝细胞肿胀、坏死,血管中血细胞凝集,脾脏红白髓界限不清、胞核有轻微肿胀,肾小管上皮细胞发生颗粒变性,肠绒毛大部分坏死脱落。药敏试验结果显示,菌株CQ200825对多西环素等17种抗菌药物高度敏感,对复方新诺明等4种抗菌药物中度敏感,对阿莫西林等12种抗菌药物表现为耐药。本研究可为翘嘴鲌维氏气单胞菌病的诊断与防治提供参考依据。

Culter alburnus is a common freshwater aquaculture fish in China. It is popular because of its tender meat, rich nutrition, and high-protein and low-fat contents. With the breakthrough in compound feed and artificial breeding technology, the scale of C. alburnus cultivation is increasing, and it is cultivated in more than 10 provinces and many cities in China, including Anhui, Hubei, Jiangsu, Zhejiang,and Chongqing. However, with the increase in culturing scale and density of C. alburnus, the water quality is continuously deteriorating, leading to decreased immunity and spread of diseases, especially various bacterial, fungal and parasitic diseases, among which bacterial diseases are the most serious,causing huge economic losses. In August 2020, C. alburnus died in a farm, and the cumulative death rate was 20% in Rongchang District, Chongqing. The main clinical symptoms was loose scales, bleeding at the base of the fin, a large amount of ascites in the abdominal cavity, and hepatosplenomegaly. In order to identify the pathogen responsible for the death of C. alburnus, the pathogenic mechanism was investigated and appropriate drugs to treat the disease were screened. A dominant strain named CQ200825 was isolated from the liver of the diseased fishes. An artificial infection experiment was performed to verify pathogenicity of the strain CQ200825. This was combined with the morphological, physiological,and biochemical analysis of the bacteria for identification;16S rRNA sequence and gyrB sequence analysis was also done. Several analyses were performed for accurate identification of the species,virulence gene detection, histopathological observation, and to probe its virulence factor and tissue pathological changes caused after infection. Finally, effective drugs were screened by drug susceptibility test. The results showed that naturally occurring fishes swam slowly, had decreased appetite, or did not feed at all. Their scales were partially detached and the eyeballs were protruding. An obvious hyperemia and hemorrhage around the orbit, gill cover, and base of the fin was also observed. Autopsy revealed a large amount of ascites in the abdominal cavity and the liver and spleen were swollen. The bacterium grew at 28℃ for 24 h on BHI medium, and its colony diameter was 1.0~1.5 mm, which had smooth surface and rounded edges. Strain CQ200825 was gram negative, and its size was about 0.5~1.0×1.2~2.4 μm;the strain was short rod-shaped, and most single permutation. The physiological and biochemical characterization of the strain CQ200825 showed that it could produce indole but not H2S. The M.R test and V-P test results were positive. The test results for the production of lysine decarboxylase and oxidase were positive, whereas those for arginine hydrolase, phenylamine deaminase, ornithine decarboxylase,and urease were negative. The strain could utilize D-mannitol, D-glucose, and cellobiose, but could not utilize arabinose, salicin, sorbitol, and inositol. The evolutionary tree constructed by 16S rRNA and gyr B gene sequences of the strain showed that it clustered with Aeromonas veronii, and the similarity was 99.60% and 99.63%, respectively. Based on morphology and physiological and biochemical properties,the strain CQ200825 was identified as A. veronii. Both the artificially infected and the naturally pathogenetic fishes showed symptoms of hemorrhage at the base of the fin, hepatosplenomegaly, and ascites. Meanwhile, a dominant strain was isolated from the artificially infected dying fishes, which was the same as the strain CQ200825 based on its physicochemical and molecular properties, indicating that the strain CQ200825 was the pathogen of the diseased C. alburnus. The LD50 value of strain CQ200825 against C. alburnus was 2.7×10^(6) CFU/mL. Four virulence genes were found in the strain CQ200825,including the outer membrane protein(ompA), haemolysin(hlyA), cytotonic heat-stable enterotoxin(ast),and cytotoxic enterotoxin(act), and sequencing analysis revealed that the lengths of these genes were 903 bp,592 bp, 504 bp, and 249 bp, respectively. Pathological analysis revealed that the liver, spleen, kidney, and intestine of the diseased C. alburnus had different degrees of pathological changes. Liver cells of the diseased fish were swollen and necrotic, and hemocytes agglutinated in blood vessels;splenic tissue was necrotic and the cells were abnormal, the boundary of red and white pulp was unclear, and the nucleus had slight swelling;the epithelial cells of renal tubules showed granulosa degeneration and glomerular atrophy;and the intestinal structure was severely damaged, serous membrane was removed, most of the intestinal villus were necrotic and exfoliated, and goblet cells epithelial cells were necrotic. The results of the drug susceptibility test revealed that the strain CQ200825 was highly sensitive to 17 antibacterial drugs,namely doxycycline, enrofloxacin, florfenicol, ceftazidime, kanamycin, cefoperazone, streptomycin,ciprofloxacin, and pipemidic acid;moderately sensitive to four antibacterial drugs, including erythromycin, cotrimoxazole, sulfisoxazole, and neomycin;and resistant to 12 antibacterial drugs namely penicillin, amoxicillin, gentamicin, midecamycin, kitasamycin, carbenicillin, cefazolin, and lincolmensin.This study can provide reference for the diagnosis and prevention of A. veronii infection in C. alburnus.