鳗鲡致病菌快速诊断试剂盒的研制及流行规律调查
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摘要
鳗鲡是中国重要的养殖经济鱼类,自70年代末以来,很快发展成为中国出口创汇最多的单项水产品之一。目前中国大陆的主要养殖品种有日本鳗鲡、欧洲鳗鲡和美洲鳗鲡。随着养殖集约化程度的提高,养殖环境日益恶化,鳗鲡病害成为困扰鳗鲡养殖业持续、健康发展的重要因素,尤其是细菌性病害,发病广、传播快、死亡率高,给鳗鲡养殖业者造成了巨大的经济损失。因此,快速的诊断疾病是有效防治的关键,但是目前对鳗鲡致病菌的快速诊断试剂盒的研究缺乏,对鳗鲡细菌病害的流行规律调查也甚少。鉴于此,本试验研制了15株主要鳗鲡致病菌免疫血清,并和实验室以往研制的20株主要鳗鲡致病菌免疫血清,制成35种快速诊断试剂盒,对鳗鲡主产区福建省不同养殖种类、不同季节、不同地区、不同规格的患病鳗鲡进行了检测,主要结果如下:
1.抗血清的制备。本试验采用静脉注射免疫法制备了15株鳗鲡致病菌的兔抗血清,并应用玻片凝集法对血清效价进行测定,结果显示:7株菌的抗血清效价达到2048,占46.7 % ;3株达到10~24,占20 %;3株达到512,占20 %;2株达到256,占13.3%。
2.抗血清交叉反应去除。采用玻片凝集法测定了每种免疫血清与其它34株鳗鲡致病菌(与血清对应的致病菌株除外)的交叉反应效价,并采用非特异性吸附沉淀法对存在交叉反应的血清进行了交叉去除,结果显示:具有明显交叉反应的血清有15种,占42.9 %;其中9种通过非特异性吸附沉淀法可完全去除,占存在交叉反应血清总数的60 %,且7种去除交叉后的血清效价没有下降,2种效价降低了1倍,去除交叉后的血清具有较高的效价和特异性;其余6种不能完全去除交叉反应的血清,在实际检测时可通过稀释血清使交叉反应不明显。
3.抗血清的检测灵敏度。采用玻片凝集法测定了35种血清的检测灵敏度,结果显示:检测灵敏度在1×10~8 cfu/mL以上的24种, 1×10~7 cfu/mL以上的9种, 1×10~6 cfu/mL以上的2种。
4.应用乳胶凝集实验方法检测鳗鲡致病菌的研究。为了提高试剂盒检测灵敏度,防止漏检,本试验在玻片凝集试验的基础上对乳胶凝集试验条件进行了探索。应用A蛋白亲和层析纯化后的抗体(S18和S31)致敏乳胶,并设计了不同偶联时间、抗体加入量、致敏时间和封闭时间的乳胶致敏效果对比试验,结果表明:碳化二亚胺(EDC)偶联3 h,加入纯化抗体量588.6μg/ml(S18); 568.7μg/ml(S31),25℃、200 r/min致敏10~ h,然后加入甘氨酸于25℃、200 r/min封闭30 min,得到的致敏乳胶抗体与对应抗原反应效价最高,达到512,抗原检测灵敏度最高,达到1×10~6 cfu/mL,且特异性强,重复性好,4℃保存4个月效价不变,室温保持1个月效价不变。乳胶凝集试验方法较直接玻片凝集法检测灵敏度高,但检测成本高于玻片凝集。
5.不同种类鳗鲡致病菌检测结果:3种养殖鳗鲡共检测到致病菌28株,共同的致病菌主要是嗜水气单胞菌,鳗弧菌主要感染欧洲鳗鲡和美洲鳗鲡。11株鳗鲡致病菌分别仅在一种鳗鲡中检出,其中欧洲鳗鲡、日本鳗鲡、美洲鳗鲡分别占5株、4株和2株,说明相当数量的致病菌具有种类特异性。
6.不同季节鳗鲡致病菌检测结果:3个季节共检测到28株致病菌,8株全年流行,嗜水气单胞菌是全年最主要的致病菌,但在不同季节检测到的具有血清型差异。13株致病菌在单一季节出现,包括春季7株,夏季4株,秋季2株,显示了较强的季节特异性。春季主要致病菌是酸克雷伯氏菌、嗜水气单胞菌(血清Ⅲ型,B18)和温和气单胞菌;夏季主要嗜水气单胞菌(血清Ⅱ,B15)和鳗弧菌;秋季以嗜水气单胞菌(血清Ⅷ型,B31)为主。
7.不同地区鳗鲡致病菌检测结果:检测到28株鳗鲡致病菌,其中嗜水气单胞菌(不同血清型)分布范围广,在4个地区均出现,鲁氏耶尔森菌和霍利斯弧菌只在闽南出现,迟钝爱德华氏菌和溶解阴沟肠杆菌流行于闽西;10~株在2个不同地方同时检出;9株致病菌菌仅在单一地区出现,说明部分鳗鲡致病菌具有地域差异。
8.不同规格鳗鲡致病菌检测结果:从5个规格共检测到28株鳗鲡致病菌,其中8株在所有规格都能检出,共同的主要致病菌为不同血清型的嗜水气单胞菌、鳗弧菌和威隆气单胞菌温和生物变种;7株在单一规格检测到的致病菌中有5株出现在10~g以下的鳗鲡,它们是鲁氏耶尔森菌、霍利斯弧菌、腐败希瓦菌、1株非发酵菌和1株未鉴定结果的菌(B34),鳗弧菌主要危害10~-50g的鳗鲡,50g以上的鳗鲡以感染嗜水气单胞菌和豚鼠气单胞菌为主,溶解阴沟肠杆菌唯一在200g以上的鳗鲡中检出。说明一些致病菌具有种类差异。
本研究结果表明:静脉注射鳗鲡致病菌免疫新西兰大白兔可以制备高效价的免疫血清,而非特异性吸附法能够对大多数存在交叉反应的血清进行交叉去除,去除交叉后的免疫血清诊断试剂盒特异性高,检测快速、操作简便,可以在现场对鳗鲡致病菌进行准确检测。不同血清型的嗜水气单胞菌是三种主要养殖鳗鲡的主要致病菌,本研究结果初步揭示了养殖鳗鲡的主要致病菌具有一定的种类、季节、地区和规格的特异性。
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.
1.抗血清的制备。本试验采用静脉注射免疫法制备了15株鳗鲡致病菌的兔抗血清,并应用玻片凝集法对血清效价进行测定,结果显示:7株菌的抗血清效价达到2048,占46.7 % ;3株达到10~24,占20 %;3株达到512,占20 %;2株达到256,占13.3%。
2.抗血清交叉反应去除。采用玻片凝集法测定了每种免疫血清与其它34株鳗鲡致病菌(与血清对应的致病菌株除外)的交叉反应效价,并采用非特异性吸附沉淀法对存在交叉反应的血清进行了交叉去除,结果显示:具有明显交叉反应的血清有15种,占42.9 %;其中9种通过非特异性吸附沉淀法可完全去除,占存在交叉反应血清总数的60 %,且7种去除交叉后的血清效价没有下降,2种效价降低了1倍,去除交叉后的血清具有较高的效价和特异性;其余6种不能完全去除交叉反应的血清,在实际检测时可通过稀释血清使交叉反应不明显。
3.抗血清的检测灵敏度。采用玻片凝集法测定了35种血清的检测灵敏度,结果显示:检测灵敏度在1×10~8 cfu/mL以上的24种, 1×10~7 cfu/mL以上的9种, 1×10~6 cfu/mL以上的2种。
4.应用乳胶凝集实验方法检测鳗鲡致病菌的研究。为了提高试剂盒检测灵敏度,防止漏检,本试验在玻片凝集试验的基础上对乳胶凝集试验条件进行了探索。应用A蛋白亲和层析纯化后的抗体(S18和S31)致敏乳胶,并设计了不同偶联时间、抗体加入量、致敏时间和封闭时间的乳胶致敏效果对比试验,结果表明:碳化二亚胺(EDC)偶联3 h,加入纯化抗体量588.6μg/ml(S18); 568.7μg/ml(S31),25℃、200 r/min致敏10~ h,然后加入甘氨酸于25℃、200 r/min封闭30 min,得到的致敏乳胶抗体与对应抗原反应效价最高,达到512,抗原检测灵敏度最高,达到1×10~6 cfu/mL,且特异性强,重复性好,4℃保存4个月效价不变,室温保持1个月效价不变。乳胶凝集试验方法较直接玻片凝集法检测灵敏度高,但检测成本高于玻片凝集。
5.不同种类鳗鲡致病菌检测结果:3种养殖鳗鲡共检测到致病菌28株,共同的致病菌主要是嗜水气单胞菌,鳗弧菌主要感染欧洲鳗鲡和美洲鳗鲡。11株鳗鲡致病菌分别仅在一种鳗鲡中检出,其中欧洲鳗鲡、日本鳗鲡、美洲鳗鲡分别占5株、4株和2株,说明相当数量的致病菌具有种类特异性。
6.不同季节鳗鲡致病菌检测结果:3个季节共检测到28株致病菌,8株全年流行,嗜水气单胞菌是全年最主要的致病菌,但在不同季节检测到的具有血清型差异。13株致病菌在单一季节出现,包括春季7株,夏季4株,秋季2株,显示了较强的季节特异性。春季主要致病菌是酸克雷伯氏菌、嗜水气单胞菌(血清Ⅲ型,B18)和温和气单胞菌;夏季主要嗜水气单胞菌(血清Ⅱ,B15)和鳗弧菌;秋季以嗜水气单胞菌(血清Ⅷ型,B31)为主。
7.不同地区鳗鲡致病菌检测结果:检测到28株鳗鲡致病菌,其中嗜水气单胞菌(不同血清型)分布范围广,在4个地区均出现,鲁氏耶尔森菌和霍利斯弧菌只在闽南出现,迟钝爱德华氏菌和溶解阴沟肠杆菌流行于闽西;10~株在2个不同地方同时检出;9株致病菌菌仅在单一地区出现,说明部分鳗鲡致病菌具有地域差异。
8.不同规格鳗鲡致病菌检测结果:从5个规格共检测到28株鳗鲡致病菌,其中8株在所有规格都能检出,共同的主要致病菌为不同血清型的嗜水气单胞菌、鳗弧菌和威隆气单胞菌温和生物变种;7株在单一规格检测到的致病菌中有5株出现在10~g以下的鳗鲡,它们是鲁氏耶尔森菌、霍利斯弧菌、腐败希瓦菌、1株非发酵菌和1株未鉴定结果的菌(B34),鳗弧菌主要危害10~-50g的鳗鲡,50g以上的鳗鲡以感染嗜水气单胞菌和豚鼠气单胞菌为主,溶解阴沟肠杆菌唯一在200g以上的鳗鲡中检出。说明一些致病菌具有种类差异。
本研究结果表明:静脉注射鳗鲡致病菌免疫新西兰大白兔可以制备高效价的免疫血清,而非特异性吸附法能够对大多数存在交叉反应的血清进行交叉去除,去除交叉后的免疫血清诊断试剂盒特异性高,检测快速、操作简便,可以在现场对鳗鲡致病菌进行准确检测。不同血清型的嗜水气单胞菌是三种主要养殖鳗鲡的主要致病菌,本研究结果初步揭示了养殖鳗鲡的主要致病菌具有一定的种类、季节、地区和规格的特异性。
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.
引文
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