Studies On The Pathogen Of Skin-ulcer Disease In Juvenile Hybrid Groupers Epinephelus Fuscoguttatus(♀)×E.Lanceolatus(♂)

The hybrid grouper, Epinephelus fuscoguttatus(♀) × E. lanceolatus(♂), is a newly bred seawater cultivated fish species with high value. Owning to its delicious meat, rapid growth, strong disease-resisting capability, the commercially high price, this kind of hybrid grouper has become one of the most extensive and important maricultured fish in China. The yield has been rapidly increased. Due to the industry grow too fast and lack the measures for disease control, the disease of hybrid grouper is serious and cause a large number of economic loss. During the last two years, a ‘skin ulcer disease’ with high mortality occurred in cultured hybrid grouper E. fuscoguttatus(♀) × E. lanceolatus(♂) in a grouper hatchery. Based on the field investigation of the disease, pathogens analysis and artificial challenge experiment, we confirmed the pathogen infected the hybrid grouper, E. fuscoguttatus(♀) × E. lanceolatus(♂),was Vibrio harveyi ML01.The epidemics status of the ‘skin ulcer disease’ of juvenile hybrid grouper was investigated in 2014 and 2015. This disease usually occurs in summer(from June to September) with water temperature of 25 ~ 29 °C. The body length of the diseased fish was generally 1 ~ 6 cm. The juveniles with the body length of about 4 cm were most vulnerable to disease. If this disease was not treated timely, the cumulative mortality could reach as high as 90%. During the early stage of this disease, fish exhibited anorexia, unresponsive, poor vitality, and lying on the bottom of water tank. With the disease became worsening, a number of symptoms occurred, including scales shedding, epidermis bleeding, skin and mouth jaw ulcer. At the most serious time, their caudal peduncle ulcerated up to muscle.Through screening the pathogens, we eliminated the parasite- and virus-infection, and isolated one bacterial strain from the moribund fish suffered from severe skin ulceration. Bacterial challenge tests were performed with the isolate on the health juvenile hybrid grouper through exposure by immersion, immersion following dermal abrasion and intraperitoneal injection, respectively. Within 14 days after infection, the isolate named ML01 could cause mass mortality in affected juveniles through immersion following dermal abrasion and intraperitoneal injection, and the diseased juveniles displayed obvious symptoms of skin ulcer. The LD50 of ML01 was 1.10×105 CFU by intraperitoneal injection. These results showed that the isolate ML01 was the pathogen of the skin ulcer in juvenile hybrid grouper. The strain ML01 was then identified as Vibrio harveyi by bacterial morphological observation, API system identification, 16 S rDNA sequence and multilocus sequence analysis of recA, rpoA, and pyrH genes. This study suggested that the ML01, which was identified as V. harveyi, was the causal agent of ‘skin ulcer disease’ of juvenile hybrid grouper E. fuscoguttatus(♀) × E. lanceolatus(♂). Susceptibility to antibiotics tests showed that strain ML01 was sensitive to cefatriaxone, doxycycline and minocycline.In recent years, Vibrio harveyi has become an important pathogen for mariculture animals. It caused serious diseases and mortalities of many aquaculture animals and induced huge economic loss worldwide. The isolation and identification of virulence factors of V. harveyi become research hotspot recently. The V. harveyi strain ML01(CGMCC No. 11720) was isolated from diseased juvenile hybrid grouper Epinephelus fuscoguttatus(♀) × Epinephelus lanceolatus(♂) suffering from severe skin ulcer. In this research, the virulence factors including extracellular products(ECPs) and secretory proteins of strain ML01 were extracted and purified by cellophane plate and gel filtration techniques. Furthermore, these virulence factors were characterized and identified through toxicity tests, mass spectrometry MALDI-TOF/TOF, and molecular cloning methods. The results showed that ECPs of strain ML01 have the activity of gelatinase, amylase, lipase and caseinase, but have not the activity of urease. The hemolysis to sheep red blood cells of ECPs was negative. The toxicity tests showed that ECPs of strain ML01 was lethal to zebrafish and the LD50 value was 19.55 μg protein/g body mass. Three major secretory proteins(P42, P36 and P31) corresponding to the molecular weight of 42 KDa, 36 KDa and 31 KDa in SDS-PAGE were purified from strain ML01. These proteins were identified as membrane proteins of OmpU, OmpN and a hypothetical protein of V. harveyi by mass spectrometry MALDI-TOF/TOF. Based on homology cloning techniques, the genes of proteins P42, P36 and P31 were amplified from the genome of strain ML01 and sequenced. The sequence alignment results showed that the open reading frames(ORFs) similarities between strain ML01 and V. harveyi ATCC 33843 were 97.08%(P42 gene), 100%(P36 gene) and 99.67%(P31 gene), while the similarities of peptide sequences were 99.71%(P46), 100%(P36) and 99.93%(P31), respectively. This research is of significance to further analysis on the pathogenic mechanism and the development of subunit vaccines of V. harveyi strain ML01.