| Litopenaeus vannamei is the main breed of shrimp in C hina.With the development of its farming,its output has been ranked first in the world in recent years and has made outstanding contributions to the development of agricultural economy in coastal areas and inland areas.However,the development of the industry is also accompanied by the occurrence of disease.In this paper,we focus on a new type of bacterial white spot disease(BWSD).During 2015 to 2016,the epidemiological investiga tion and pathogenicity of white spot occurred in Litopenaeus vannamei culture in Zhejiang,Hebei,Shandong,Jiangsu and Tianjin provinces were carried out.In the survey of more than 150 ponds in the north of Zhejiang province,it was found that the average detection rate of the symptoms was 84.7%.The samples from diseased shrimp with reference to GB / T 28630.2-2012 white spot syndrome(WSD)diagnostic protocols in nested PCR assay,were detected WSSV investigation.The results showed that the diseased shrimp on the crustacean was not infected with WSSV.And in the four main aquaculture modes of outdoor farming pond,outdoor higher-place pool,outdoor incubator and indoor circulating water pond are detected.Because of its occurrence from the impact of far ming patterns,leading to bacterial white spot disease can occur throughout the year.Under the naked eye,the white spots appearing on the carapace of the diseased shrimp are very similar to the white spot after infection with the white spot syndrome virus(WSSV),but the difference is that the shrimp with bacterial white spot is healthy and the normal growth is normal,active,and there is no obvious death phenomenon.However,the farmers are generally difficult to distinguish between bacterial white spot disease or viral white spot syndrome.In view of the past,the incidence of WSS can lead to a large number of shrimp deaths,resulting in serious economic losses,farmers usually take negative measures,and e ven emergency pond,end farming after finding the shrimp with white spots,resulting in unnecessary losses.In this paper,the pathophysiology of bacterial leukoplakia was studied.Under the optical microscope,the bacterial white spot was round,with obvious radiation ribs,extending from the white spot to the jagged edge;the white spot center is usually dark brown,and the shrimp carapace is eroded and pierced later.The white spots caused by WSSV infection were round,smooth edges,regular,round dense distribution of a large number of black spots,no obvious center.Pathology studies have shown that the white shrimp carapaces are destroyed and decomposed by bacteria,and the carapace are filled with a large number of tissue fragments and bacteria.The epithelial tissue and connective tissue are seve rely damaged,eroded and ruptured.Finally,the obvious structure is formed on the whole structure.High-power microscope observation found that the fracture of the epithelial tissue and connective tissue nucleus was basophilic deep staining,epithelial tissue and connective tissue associated with inflammation,and even necrosis.Under the electron microscope,the central area of white spot carapace was eroded by bacteria,perforation occurred,perforated around the carapace was flocculent,and a large number of rod-like bacteria adhesion,accumulation.At the same time,white spot carapace around the healthy shells also have a lot of rod-like bacteria attached.The results of the study laid the foundation for distinguishing between bacterial and viral white spots.6 dominant families with superiority of more than 80% were isolated from Zhejiang,Shandong(2015),Hebei,Tianjin,Jiangsu and Shandong(2016)with the head of the thoracic goat white spot of 6 groups of shrimp named ZJ201506,SD201508,HB201509,TJ201509,JS201510,SD201601.The results showed that the 6 dominant bacteria were Bacillus amyloliquefaciens or their late stage Bacillus velezensis,which were identified by Gram-pollination,Physiological and Biochemical Reaction.The results of susceptibility test were as follows: 6 strains of dominant bacteria were highly sensitive to ofloxacin,bacteria must be treated,cephalexin,gabsorlin,oxacillin and cefotaxime.For penicillin,rifampicin,azithromycin,acetyl spiramycin were insensitive.Subsequently,the healthy Litopenaeus vannamei was tested with the strain JS201506,setting the concentration in 5×102~5×106cfu/mL At the same time,the trauma experimental groups were set up.Traumatic treatment of the shrimp crust was carried out by using a scalpel.The trauma experimental group was set at 5×104cfu / mL and 5×105cfu / mL.The results showed that the actual detection of shrimp in the infected group at the concentration of 2×104 cfu / mL showed the same early white spot as that of the natural diseased shrimp at 27 days.Early white spots were appeared at 12 d and 10 d with the detection concentration 6.7×105cfu / mL and 9.1×105cfu / mL,and the late white spots were appeared at 29 d and 25 d with the detection concentration 6.7×105cfu / mL and 9.1×105cfu / mL.Traumatic experimental group of shrimp head on the chest armor appeared along the trauma of the growth of white spots,and white spots are more obvious.Above the prevalence of shrimp showed normal vitality,feeding status is good,did not appear obvious death.Thus,when the concentration of Bacillus amyloliquefaciens in water is higher than 104 cfu / mL,there is a risk of contamination of BWSD.A bacterial strain with a dominant degree of 87.1% was obtained by bacterial isolation of the diseased shrimp white spot in the e xperimental group 9.1×105cfu / mL.The dominant bacteria Gram stain was positive,rod-shaped,with spores.The results showed that the dominant strain was the same strain as the strain JS201506 by physiological and biochemical identification,drug sensitivity reaction and sequence analysis based on 16 s rDNA.The results of the bathing experiments on healthy Litopenaeus vannamei were conducted using the 3 kind of microecological preparations with Bacillus subtilis as the main constituent bacteria in group A to C.The microecological preparations were active by oxygen.And the amount of application in accordance with the instructions of the amount of 4-5 times.The results showed that typical cases of bacterial leukoplakia were observed in the A,B and C groups at 16 days,20 days and 18 days,respectively.While the test shrimp feeding status is good,no death phenomenon.Therefore,the breeding process should be a reasonable use of microecological preparations.In summary,Bacillus amyloliquefaciens or its late stage Bacillus velezensis were the main pathogens of bacterial white spots in shrimp of Zhejiang,Shandong,Hebei,Tianjin and Jiangsu provinces,and the occurrence of the disease is associated with an overdose of a microecological preparation of Bacillus as a major constituent bacterium.The above results for the correct distinction between the two different white spots pathogens caused by the virus and bacteria,as well as the development of shrimp healthy breeding and related diseases prevention and control has important guiding significance.At the same time,the conclusions of this paper also provide a theoretical basis for improving the ecological security awareness of aquaculture and the proper use of microecological preparations. |
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