Isolation and Identification of the Pathogen Causing Skin Ulcer Disease in Cynoglossus semilaevis Günther and Its Sensitivity to Chinese Herbal Medicine

Guisheng GAO, Yanying ZHANG, Qiumei SHI*, Guangping GAO, Hongsheng HAN, Xiaoyuan HUANG, Baofeng JIAO, Yaoyao CHANG

1. Key Laboratory of Preventive Veterinary Medicine of Hebei Province/College of Animal Science and Technology, Hebei Normal University of Science & Technology, Qinhuangdao 066600, China;

2. Qinhuangdao Liangfeng Marine Ecological Science and Technology Co., Ltd., Qinhuangdao 066600, China

In recent years, coastal industrial aquaculture is rapidly developing,but the benefit of the aquaculture industry has been greatly affected by disease epidemics. In previous studies, common bacterial diseases and their epidemiological characteristics in a circulating water aquaculture plant of Cynoglossus semilaevis Günther in Qinhuangdao City were investigated.The results show that skin ulcer is the most common bacterial disease in cir culating water aquaculture of C.semilaevis Günther, which poses a serious threat to the reproductive survival rate.At present, few systematic studies have been reported on diseases of C. semilaevis Günther, but Little is available on occurrence and pathogenesis of these diseases. In this study, skin ulcer disease in aquaculture of C. semilaevis Günther was investigated systematically, aiming at providing basis for further prevention and control of skin ulcer disease.

Clinical Symptoms and Pathological Changes in Cynoglossus semilaevis Günther infected with Skin Ulcer Disease

In April 2015, skin ulcer disease occurs in a coastal industrial aquaculture plant of C. semilaevis Günther in Qinhuangdao City. C. semilaevis Günther infected with skin ulcer disease exhibited skin ulcers, fin base swelling, fin end ulcers, local gill filament edema with telangiectasia and congestion, liver and pancreas swelling, renal tissue degeneration,necrosis and ulceration, red blood cell deposition and hemoglobin precipitation in spleen,gallbladder enlargement with telangiectasia,and large amounts of catarrhal mucus in intestines (Fig.1,Fig.2).

Pathogen Isolation

Liver, pancreas, kidney tissues and ascites of C. semilaevis Günther infected with skin ulcer disease were collected, inoculated to a nutrient agar plate, and incubated at 28 ℃for 24 h.Morphological observation results show that round translucent, pale yellow or light brown bacterial colonies were grown with smooth and moist surface and neat edges (Fig.3). The colonies were inoculated to a blood agar plate and incubated at 28 ℃for 12-24 h.As shown in Fig.4,completely transparent zones of hemolysis formed on the plate.As shown in Fig.5,the pathogen was a Gram-negative bacterium with blunt round ends, and individual cells were slightly curved.

Physiological and Biochemical Identification of the Pathogen

Three predominant strains were isolated from liver, pancreas, kidney tissues and ascites of C. semilaevis Günther infected with skin ulcer disease to detect the physiological and biochemical characteristics (Table 1).According to the results, these strains were positive to lipase, arginine dihydrolase, D-trehalose, D-maltose and β-galactosidase but negative to D-glucose,L-arabitol,D-mannitol,malonate,D-arabinitol, L-aspartate arylamidase,β-glucosidase, ornithine decarboxylase, 5-ketogluconate and lysine decarboxylase, indicating that culture,physiological and biochemical characteristics of the isolated strains were consistent with Vibrio harveyi. Therefore, the pathogen causing skin ulcer disease in C. semilaevis Günther was identified as V.harveyi.

Recurrent Infection of C.semilaevis Günther with the Pathogen

A strain isolated from liver and pancreas tissues was incubated with LB broth and inoculated to five healthy C. semilaevis Günther. A C. semilaevis Günther was used as the control,and other four individuals were injected with 0.3 ml of bacterial liquid (containing 2 ×106colonies/ml). At 12 h post-infection, experimental fish swam slowly with lassitude, gradual skin ulceration and loss of appetite, which died successively within 72 h, but the control fish exhibited no abnormal performance. Autopsy results showed gill congestion, organ adhesion and liver congestion, with similar symptoms to naturally infected C. semilaevis Günther.Strains were isolated from dead C.semilaevis Günther for culture and identification. According to the results, the original injection bacterium exhibited consistent morphological and biochemical characteristics with V. harveyi, indicating that the original injection bacterium was the pathogen causing skin ulcer disease in C.semilaevis Günther.

Detection of Outer Membrane Protein Gene (OMP)of the Pathogen

Genomic DNA of V. harveyi was extracted in accordance with the introductions of genomic DNA extraction kit(Fig.6). The results showed that genomic DNA of three V. harveyi strains was successfully extracted.Using genomic DNA of V. harveyi as a tem-plate, 933 bp specific band were amplified by PCR, which was consistent with the expected size (Fig.7),indicating that outer membrane protein gene(OMP) of V. harveyi was successfully amplified and detected.

The amplified OMP gene of V. harveyi was sequenced and submitted to GenBank. By using BLAST software, four reference strains published in GenBank were obtained for comparison and analysis of nucleotide homology. The results showed that OMP gene sequences of three V.harveyi strains shared 98.7%-100% homologies with reference strains. To be specific, OMP gene sequence of representative strain 3 shared 100% homology with reference strain JQ946883; OMP gene sequences of three representative strains shared 98.8%-99.4% homologies with each other(Fig.8).

Table 1 Biochemical identification results

Sensitivity Test of the Pathogen to Chinese herbal medicine

As shown in Fig.9 and Table 2,among 12 single herbs used in this study,Galla Chinensis,Fructus Mume,Fructus Hippophae and Lignum Sappan exerted strong antibacterial effects against V.harveyi,among which Galla Chinensis had the highest antibacterial activity; Pericarpium Granati exerted no significant antibacterial effect against V. harveyi; Pericarpium Citri Reticulatae, Rhizoma Acori Graminei,Herba Houttuyniae, Herba Portulacae,Herba Andrographis,Eucalyptus globulus Labill. and Herba Menthae Heplocalycis had little effect on V.harveyi.

According to the results of sensitivity test of the pathogen to 12 single herbs, Galla Chinensis, Fructus Mume, Fructus Hippophae and Lignum Sappan, which exerted the strongest antibacterial effects, were prepared into prescription 1 (Galla Chinensis + Fructus Mume), prescription 2 (Galla Chinensis + Fructus Hip-pophae)and prescription 3(Galla Chinensis + Lignum Sappan) for sensitivity test. The results showed that prescription 1 (Galla Chinensis+Fructus Mume) exhibited the strongest antibacterial effect.

Table 2 Antibacterial circle diameter mm

Discussions

In the current aquaculture production, bacterial diseases are still the most important diseases endangering cultured fishes[1-4].According to the disease records from Key Laboratory of Preventive Veterinary Medicine of Hebei Province and existing researches,bacterial diseases of C. semilaevis Günther are mainly caused by Vibrio,and most pathogens belong to Gramnegative bacteria, such as V. harveyi,V.splendidus and V.anguillarum.Vibrio strains causing other flatfish diseases can also cause bacterial diseases in C. semilaevis Günther to some extent. In this study, V. harveyi is the main pathogen causing skin ulcer disease in C. semilaevis Günther.Statistics show that V. harveyi is the most important pathogen that causes diseases of maricultural animals[5],which can induce ulceration symptoms in Scophthalmus maximus[6], Fugu obscurus[7], Pseudosciaena crocea and other maricultural species, cause degeneration and necrosis of internal organs of diseased fishes[8-9], lead to physiological metabolic disorders of diseased fishes[10]and reduce their immunity and resistance to diseases,thereby causing the death of diseased individuals. In the present study, the pathogen causing skin ulcer disease in C. semilaevis Günther was identified as V.harveyi;all the three representative strains harbored OMP gene. According to the results of sensitivity test of the pathogen to Chinese herbal medicine, V. harveyi was strongly pathogenic to C. semilaevis Günther.Galla Chinensis, Fructus Mume,Fructus Hippophae and Lignum Sappan exerted strong antibacterial effects against V. harveyi; prescription 1(Galla Chinensis + Fructus Mume) exhibited the strongest antibacterial effect.

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