Lu Xiangyi’s centrifuge is used for the study of pathogenic Vibrio harveyi.

Identification of Vibrio hollisae, the Pathogen of the Spine-tailed Goby, and Study on Its Specific Detection Method

Abstract: Between August and September 2009, mass mortality occurred among the Japanese mudskipper (Periophthalmus novemradiatus) cultured in mixed-species shrimp and crab ponds in Ganyu County, Jiangsu Province. The primary pathological features included hemorrhaging on the body surface and muscular ulcers. A dominant bacterium was isolated from deep-seated ulcerative tissues and livers of the Japanese mudskipper. Infection experiments demonstrated that this bacterium exhibited strong pathogenicity toward the Japanese mudskipper; when the bacterial concentration reached between 10^6 and 10^8 CFU/mL, it could cause the complete mortality of the Japanese mudskipper. The isolated bacterium was subjected to a series of biological tests, including morphological and physicochemical analyses. The homology of the 16S rRNA and gyrB gene sequences was also analyzed. Using PAUP4.0’s maximum parsimony method, phylogenetic trees based on these two genes were constructed. The results indicated that the pathogen responsible for the massive mortality of the Japanese mudskipper was Vibrio harveyi, a species belonging to the genus Vibrio. Furthermore, specific primer pairs targeting both the gyrB gene and the toxR gene were designed. These primers were then used for PCR amplification and sensitivity testing, followed by application to seafood samples, thereby establishing a rapid and highly sensitive PCR detection method for Vibrio harveyi. In the PCR amplification, the gyrB gene-specific primer pair A2B3 yielded an amplicon of 363 bp, with a detection limit as low as 0.0046875 ng/μL of Vibrio harveyi template DNA. For the toxR gene, the primer pair vh-tox-F/vh-tox-R generated an amplicon of 390 bp, with a detection limit as low as 0.75 ng/μL of Vibrio harveyi template DNA. The PCR detection technique based on the gyrB and toxR genes of Vibrio harveyi holds significant importance for epidemiological investigations of diseases caused by this bacterium.

The spear-tailed goby (Synechogobius hasta) is classified under the order Perciformes, family Gobiidae, and genus Synechogobius. It is commonly known as "fathead fish," "sea catfish," or "tumbaba fish." This warm-temperate, small-to-medium-sized, bottom-dwelling omnivorous fish exhibits exceptional tolerance to both temperature and salinity. The spear-tailed goby has an elongated body shape, with a sub-cylindrical anterior section and a laterally compressed tail. Its head is large and slightly flattened; its snout is moderately long and rounded; its mouth is large, with a slightly protruding lower jaw and small, densely packed teeth. The eyes are small, located on the upper side of the head, and the interorbital space is broad and flat. The body is covered with round scales, larger on the tail region. It has two dorsal fins that are separated from each other. The pelvic fin has evolved into a suction cup-like structure, enabling it to firmly attach itself to the seabed. The caudal fin is pointed and elongated. The fish’s body color is yellowish-brown with irregular, faint markings; its coloration varies depending on the habitat. The belly is lighter in color, turning pale yellow during sexual maturity, while the pelvic fin takes on a reddish hue. Typically, individuals measure 25–30 cm in length and weigh between 150–200 grams. The largest specimens can exceed 50 cm in length and weigh over 500 grams. As a large, warm-temperate goby species, it is found in muddy coastal areas along the Bohai Sea, East China Sea, and the coasts of Korea and Japan, as well as in estuarine regions. Its diet primarily consists of sandworms, white shrimp, mantis shrimp, and bigeye crabs, but it also consumes smaller gobies, juvenile herring, and other small fish larvae and juveniles.

1. In recent years, with the development of aquaculture, the number of farmers raising Sicyopterus japonicus has been steadily increasing. However, due to the combined effects of various factors—such as the overuse of drugs, gradual deterioration of seabed conditions, worsening aquatic ecological environments, and inadequate management practices—fish diseases have been breaking out repeatedly. Affected fish exhibit symptoms including yellowing and emaciation of the body, bending into an arc-shaped posture, ulcers of varying sizes on the body surface, purulent nodules in the intestinal tract, and visible lesions in many internal organs.2. The mortality rate caused by these fish diseases exceeds 70%, resulting in irreparable economic losses for aquaculture farmers. Between August and September 2009, large numbers of Sicyopterus japonicus raised in mixed culture ponds in Ganyu County, Lianyungang, Jiangsu Province, died suddenly. The primary pathological features were hemorrhaging on the body surface and muscular ulcers. We isolated a dominant bacterial strain from deeply ulcerated tissues and the liver of Sicyopterus japonicus, then conducted physiological, biochemical, and molecular identification on this strain. We analyzed the sequence homology of the 16S rRNA and gyrB genes and constructed a maximum parsimony (MP) phylogenetic tree based on these two genes using PAUP4.0. The results indicated that the pathogen responsible for the massive mortality of Sicyopterus japonicus was Vibrio harveyi Johnson and Shunk 1936, a species belonging to the genus Vibrio. Furthermore, we designed specific primers targeting the gyrB gene and the toxR gene of V. harveyi and performed PCR detection and sensitivity tests, thereby establishing a specific molecular biological method for detecting V. harveyi.

2 Materials and Reagents 2.1 Instruments and Equipment Instruments: Digital microscope, model DMD5-5, manufactured by Nikon Corporation of Japan; electrophoresis apparatus, model DYY-2C, manufactured by Beijing Liuyi Instrument Co., Ltd.; horizontal gel electrophoresis tank, model DYCP-31BN, manufactured by Beijing Liuyi Instrument Factory; conventional PCR amplifier, model TGradient, manufactured by Biometra GmbH of Germany; dual-person single-sided clean bench, model SW-CJ-2FD, manufactured by Suzhou Purification Microwave Oven, model P7021TP-6, manufactured by Galanz of China; vertical pressure steam sterilizer cylinder, model 01J203-04, manufactured by Shanghai Dongya Pressure Vessel Manufacturing Co., Ltd.; constant temperature shaker, model QYC 2102C, manufactured by Shanghai Fuma Experimental Equipment Co., Ltd.; electronic balance, model EL104, manufactured by METTLER TOLEDO Instruments (Shanghai) Co., Ltd.; refrigerated freezer, model BCD-206TDZA, manufactured by Qingdao Haier Co., Ltd.; biochemical incubator, model SPX-250B-Z, manufactured by Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory; desktop high-speed centrifuge, model TG16A-WS, manufactured by Shanghai Luxiang Centrifuge Instrument Co., Ltd.

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