| Eel is one of the most important economic and cultivated fish species in China. The production of eels in the mainland of China takes up about 70% of total world production in recent years, the exported eel product earned has been the lead in the single aquatic product since 1970. Nowadays, the main species of cultivated eels in the mainland of China are Anguilla Anguilla , Anguilla Japonica and Anguilla rostrata. However, the intensively farmed eels are inevitable subjected to various pathogens such as parasites, bacteria and viruses. The diseases caused by pathogenic bacteria were often out-broken and widely spread during eel cultivation and sometimes caused large economic losses. Therefore, for preventing and controlling these bacterial diseases , it firstly need develop a rapid diagnosis of pathogenic bacteria . However, few kits for rapidly and accurately diagnosing pathogenic bacteria have been developed and knowledge on the epidemic of pathogenic bacteria of eels is scarce. In this study, 35 rapid diagnostic kits were prepared. And were used for investigating the epidemic rules of the pathogenic bacterial in cultivation. The main results showed as follows:1. The development of anti-sera. 15 immune sera were prepared using the method of intravenous injection with different strains of pathogenic bacteria isolated from eels. The titers of immune sera was measured by method of slide agglutination. The results showed that: seven of 15 sera is 2048,three is 10~24, and 512 respectively, and the rest two is 256.2. Purification of anti-sera. To some degree, fifteen immune sera of 35 sera showed cross-agglutination with other strains of pathogenic bacteria. The immune precipitation was used to remove the cross-agglutination fractions from those anti-sera. Nine cross agglutination sera could completely be dispelled by non-specific precipitation; The other 6 cross-agglutinated sera could not be completely removed but can be avoided by higher dilution of the sera. The titers of 7 sera after removed cross-agglutinate did not decline, but these of other two sera declined to half.3. Detection sensitivity of anti-sera. Detection sensitivities of 35 sera were detected by the method of slide agglutinate.The detection bacteria concentration of 24 sera were above 1×10~8 cfu/mL, and those of nine were above 1×10~7 cfu/mL, two were above 1×10~6 cfu/mL.4. Application of latex agglutination test to detect pathogenic bacteria of eels. In order to improve the detection sensitivity, this study adopted the latex agglutination methord and their better conditions of latex agglutination based on slide agglutinate was comparatively studied, the results showed that: Allergy-causing latex of high positive antigen reaction titer and high specificity was obtained with the condition: coupling EDC 3 h, joining the amount of purification antibody 588.6μg/ml and 568.7μg/ml for sera S18 and S31 sespectively, sentizing 10~ h on wave bed at a speed of 200 r/min, then add glycine closed 30 min on a wave bed of 200 r/min at 25℃. The detection titers of latex agglutination of S18 and S31 could detected bacteria concentration of 1×10~6 cfu/mL when the sera were diluted to 128 times. The retention time of the latex antibody could reached 4 months when in 4℃and one month in room temperature.The detection sensitivity of latex agglutinate is higher than direct slide agglutinate, but the cost is more than the latter.5. The detected pathogenic bacteria in different species of eels. 28 strains of pathogenic bacteria were detected ammong three species of eels. The main common pathogens are Aeromonas hydrophila of different serotypes. Vibrio anguillarum mainly infected Anguilla Anguilla and Anguilla rostrata. On the contrary, 11 strains of the 28 bacteria were only detected in one species of eel.6. The detected pathogenic bacteria during different seasons. 28 strains of pathogenic bacteria were detected from spring to autumn. eight strains of them were detected all the year around. A.hydrophila of different serotypes were the dominant pathogens of the year. However, 13 detected strains strains appeared in a single season, seven appeared only in spring, four only in summer and two only in autumn. It indicited that the infected pathogens were significantly different among three season. The main pathogens in spring were Klebsiella oxytoca, A.hydrophila (serotypeⅢ, B18) and Aermonas sobria; Those in summer were A.hydrophila (serotypeⅡ, B15) and V. anguillarum, and those in autumn was A. hydrophila (serotypeⅧ, B31).7. The detected pathogenic bacteria in different regions of Fujian province. 28 strains of pathogenic bacteria were detected in different regions of Fujian province. A. hydrophila was widely distributed and appeared simultaneously in the four regions; Yersinia ruckeri and Vibrio Hollis only been detected in the southern. Edwardsiella tarda and Enterobacter cloacae appeared in western fujian; 10~ strains were detected in two different regions; and the other nine only appeared in a single region.8. The detected pathogenic bacteria in different sizes of eels. 28 of pathogenic bacteria strains were detected. eight of them could be detected in all sizes of eel. The common main pathogens were different serotypes of A.hydrophila , V.anguillarum and Aeromonas veronii biovar sobria. two strains can be detected simultaneously in four sizes of eels, five strains can be detected in three sizes of eels, furthermore, five strains of the seven which were detected in a single size of eel only appeared in the sizes of eels less than 10~g body weight. They were Y. ruckeri , V. hollisae, Shewanella putrefaciens, one strain of nonfermentative bacteria and one strain of unidentified bacteria (B34). V. anguillarum mainly infected 10~-50g body weight of eels. In addition, Enterobacter dissolvens only been detected in the body weight of more than 200g of eels.This study showed that: High titer immune sera could be prepared by intravenous injection of eel pathogens to New Zealand rabbits. Most of the prepared sera existed cross-agglutinate but could been dispelled by non-specific precipitation. The diagnostic kits of immune sera developped in this study could be used and were specificity enough to rapidly detect the most major pathogenic bacteria of eels. The major pathogens of three species of eels were A.hydrophila of different serotypes. This study initially revealed that the major pathogens of cultured eels have specificity among different seasons, regions and specifications. |
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