| In China, Pacific abalone, Haliotis discus hannai distribute naturally along thecoast of Liaodong and Shandong Peninsulas and is one of the most importantmariculture shellfishes. At present, the farmed area of Pacific abalone has extended tothe southern party of China such as Fujian and Guangdong provinces. Artificialculture that its range is wide turns toward the intensive direction of high-density. Inrecent years, although the germplasm and cultivation technique both has improvedgreatly, management of aquaculture environment and disease prevention are stillrelatively backward. Therefore there are little of effective measures to prevent theoutbreak of diseases in the process of breeding. In2011and2012, an incidence ofmassive mortality of Pacific abalone broke out in Fujian and Guangdong provinces.We collected the moribund2-month-old juvenile abalones in early December,2011from Nanhong Seafood Breeding Co., Ltd in Shantou of Guangdong province andisolated2pathogens which were named as bb3and bb4, respectively. In order tounderstand the pathogenic mechanism of two strains for this disease in the SouthChina sea,we conducted the following experiment: the morphological observation andidentification of strains, the optimum growth temperature range and antibioticsusceptibility test of strains, the virulence test for one-year-old juvenile abalones, theinfluence of ambient environment on the virulence of strains, the immune response ofthe host, the results were summarized as follows:1The morphological observation and identification of strainsStrains bb3and bb4were purified by streaking and re-streaking on LB solidmedium, strains bb3and bb4showed a pale yellow, the round colony, smooth surface,the central uplift, neat edge. Under the same culture conditions, the volume of bb4was smaller than the volume of bb3. Gram-staining and scanning electron microscopywere used to observe the bacterial morphology, the result showed that strains bb3andbb4were both Gram-negative bacteria and bacilliform, and bb4had a flagellum.The16S rRNA sequence analysis showed that bb3had the highest homology to the16S rRNA of Vibrio alginolyticus with a similarity value of99%, bb4had thehighest homology to the16S rRNA of V. harveyi with a similarity value of100%. TheMLSA of five housekeeping genes (topA, ftsZ, gapA, mreB, gyrB) showed that bb3and bb4had the closest relationship with V. alginolyticus and V. harveyi, the resultwas consistant with16S rRNA sequence analysis, so strains bb3and bb4were definedas V. alginolyticus and V. harveyi, respectively.2The optimum growth temperature range and antibiotic susceptibility test ofstrainsWe counted for bacterial suspension by the plate dilution method, the result werebb3:1OD=12.4×10~8cfu/ml, bb4:1OD=10×10~8cfu/ml. In order to understand theinfluence of temperature for the growth of strains, the experiment set the temperaturegradient:20,24,28,32,34,36℃, the growth rate of two vibrio strains increasedfirstly and then decreased with the increase of temperature, the most appropriatetemperature for the growth was32℃. On the other hand, antibiotic susceptibility testindicated that the both strains were highly sensitive to six and four of total20antibiotics for bb3and bb4, respectively. Especially, both strains were found sensitivein common to ceftriaxone and amikacin, indicating certain drug-resistance of thestrains.3The pathogenic effect of strains bb3and bb4Strains bb3and bb4were diluted into the suspension liquid of differentconcentrations. Abalones (3.12±0.14cm in shell length,4.01±0.54g in whole wetweight) were injected with the bacterial suspension of different concentrations in theexperiment group while with PBS in the control group. Each abalone wasmuscle-injected with100μl corresponding bacterial suspension or PBS. Afterwards,abalones were put into the24℃water environment and death of the abalones wasrecorded daily for2weeks. The result showed that:(1) abalones began to die at18h,the peak of death appeared at the third day;(2) The two-week cumulative mortality ofabalones in the control group was2.22%;(3) The two-week cumulative mortalities ofabalones injected with100μl5×10~4,5×10~5,5×10~6,5×10~7,5×10~8cfu/ml bb3were15.56,31.33,58.00,85.56,100.00%, respectively, LD50of bb3was1.94×10~6cfu/ml; (4) The two-week cumulative mortalities of abalones injected with100μl5×10~4,5×10~5,5×10~6,5×10~7cfu/ml bb4were38.67,70.00,95.56,98.89%, respectively, LD50of bb4was1.11×10~5cfu/ml. These results demonstrated that the mortality of abaloneswas positive to the concentrations of strain, the virulence of bb4was higher than thevirulence of bb3.4The influence of ambient environment on the virulence of strains bb3and bb4In order to understand the influence of ambient environment on the virulence ofstrains bb3and bb4, firstly, abalones were infected with the bacterial suspension ofbb3(2.5×10~7cfu/ml), bb4(2×10~7cfu/ml) or PBS, then abalones were placed into theambient environment of the different temperatures (12,16,18,20,24,28℃) or thedifferent salinities (24,26,28,30‰). The two-week cumulative mortalities ofabalones would elevate with the increase of the water temperature, the two-weekcumulative mortalities of the three groups were the highest at24‰. The mortalities ofabalones infected with strains bb3and bb4were23.33%,73.33%at24‰and8.89%,62.22%at30‰. The cumulative mortalities of abalones infected with strain bb3under24‰and30‰had significant differences.5The immune-related material changed when abalones were infected by strainbb4The relative expression level of c-type lysozyme gene in the mantle and thecontent of hemocyanin in the blood both ocurred the significant change after abaloneswere infected with strain bb4. By RT-PCR analysis, after abalones were infected withstrain bb4, the relative expression level of Lys in the mantle increased firstly and thenrestored to the initial level, the relative expression level of Lys was the highest (6.24times of the blank group) at6h which was significant differences when comparedwith the relative expression level at0h. On the other hand, the relative content ofhemocyanin was measured by the absorbance values under346nm. It turned out thatthe content of hemocyanin showed a downward trend and dropped to the lowest pointat48h after abalones were infected with strain bb4. The content of hemocyanin at48hwas reduced by28.4%than the control group. The content of hemocyanin began to recover after48h. |
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