汕头牛田洋锯缘青蟹病害发生的环境因素及中草药防治研究
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摘要
锯缘青蟹(Scylla serrata,俗称青蟹)属肉食性、广盐、广温性海水经济甲壳动物,是我国东南沿海地区重要的海洋经济养殖蟹类,也是汕头市的传统特色优势水产养殖品种。自2004年秋冬以来,每年9~11月,汕头牛田洋养殖区的青蟹病害严重,经济损失较大。为了探讨青蟹病害的原因,寻找有效的病害治疗和防控方法,本课题组对此进行了较系统的研究。现已查明:副溶血弧菌、嗜水气单胞菌和寄生性腰鞭毛虫是牛田洋青蟹的主要病原。本论文主要从养殖水体理化因子(温度、盐度、溶解氧、pH、化学耗氧量、无机氮、无机磷)和生物因子(水体底泥中的异养菌与弧菌)等方面,探讨青蟹病害发生与养殖环境的关系;同时,开展青蟹细菌性病害(嗜水气单胞菌、副溶血弧菌)的中草药防治实验,试图寻求一种有效而安全的青蟹病害治疗方法。主要结果如下:
一.牛田洋青蟹病害爆发的环境因素
从牛田洋围垦区大顺公司的水产养殖基地选取4个100亩左右的青蟹养殖池塘作为采样、监测点,于2007年和2008年的4~12月,每月进行1~2次的定点环境监测、采样及病害调查,分析测定如下指标:水体的温度、盐度、溶解氧、pH,亚硝态氮(NO2--N)、硝态氮(NO3--N)、铵态氮(NH4+-N)及无机磷(PO43--P)的含量,化学耗氧量(COD),细菌数量(弧菌和异养菌);池塘底泥的细菌数量。结果发现:在07年和08年,养殖环境各因子与病害爆发都呈现出明显的季节性变化,且变化趋势基本一致。4~8月,青蟹健康状况良好,基本没有病害发生;自9月中下旬开始出现青蟹病害,10月份病害尤为严重,主要表现为黑鳃、甲壳黄斑、体内腹水等症状。两年内,各指标的变化范围:NH4+-N 76.63~235.63μg·L-1、NO2--N 6.15~27.75μg·L-1、NO3--N 22.33~174.83μg·L-1、PO43--P 8.27~44.81μg·L-1、水体异养菌27.25×105~157.93×105 cfu·L-1、水体弧菌9.75×103~116.75×103 cfu·L-1、底泥异养菌27.75×105~733.25×105 cfu·dw-1·g-1、底泥弧菌0.13×103~141.65×103 cfu·dw-1·g-1,它们的峰值均出现在病害高发的9~10月份;溶解氧和pH的变化范围分别是4.12~7.80 mg·L-1和6.21~8.22,它们的低值也出现在病害高发的9~10月份。结果表明:水体理化因子的恶化和条件致病微生物的大量增生是青蟹病害爆发的重要原因之一。
二.中草药防治青蟹细菌性病害的初步研究
通过药敏纸片法和倍比稀释法,分别测定了五倍子、连翘、贯众、地榆、大黄、黄芩、乌梅、黄连、马齿苋、石榴皮、益智仁、五味子等12味中草药对副溶血弧菌和嗜水气单胞菌的药物敏感性、最小抑菌浓度(MIC)和最小杀菌浓度(MBC)。结果显示,五倍子、乌梅和黄莲等对两菌株均具有明显的抑制作用,抑菌圈都大于19 mm,MIC均小于3.12 mg·mL-1。其中五倍子对两菌株的抑杀效果最明显,对副溶血弧菌和嗜水气单胞菌的抑菌圈分别达到37.00和42.29 mm,对两株菌的MIC和MBC分别为0.19和0.39 mg·mL-1。黄莲、石榴皮、马齿苋、大黄、五味子等对菌株有一定的抑杀作用,但贯众、石榴皮和益智仁的抑杀作用不明显。
接着,利用五倍子对青蟹副溶血弧菌病进行防治试验。实验设置4个组:阴性对照组(青蟹注射0.1 mL生理盐水,不药浴)、阳性感染组(注射0.1 mL 8×107 cfu·mL-1副溶血弧菌,不药浴)、五倍子治疗组(注射感染菌株后8h,用20 mg·L-1药液进行药浴3天)和五倍子对照组(注射生理盐水,药浴同治疗组),每组2个重复,每个重复6只青蟹。通过比较各组间青蟹的累积死亡率(CM),血淋巴中的细菌数量(TBC)及总血细胞数量(THC),血清中的酚氧化酶(PO)、溶菌酶(LSZ)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、酸性磷酸酶(ACP)及碱性磷酸酶(AKP)的活力,血清抗菌活力(ABA)、葡萄糖(Glu)及可溶性蛋白(Pro)的含量等免疫与代谢指标,以探究五倍子对感染副溶血弧菌青蟹的治疗效果,评估药浴对健康青蟹是否有副作用。结果显示,阳性感染组在染菌后第4天的CM即达100%,TBC显著增加,在感染二天后就达到105数量级,同时THC、PO、LSZ、ABA等指标较阴性对照组明显要低;五倍子治疗组7天的CM为25%,与阳性感染组相比,其TBC、THC、PO、LSZ、ABA等指标在治疗后第二天都有显著改善,到第七天时,各指标基本恢复到健康水平、与阴性对照组无显著差异;与阴性对照组相比,五倍子对照组青蟹除THC稍有降低外,其他指标均没有明显变化。结果表明,副溶血弧菌能引起青蟹较高的死亡率,注射107 cfu·mL-1菌液4天后即全部死亡;20 mg·L-1的五倍子药液能有效治疗青蟹由副溶血弧菌引起的疾病,该浓度药液对青蟹的免疫与代谢指标基本没有影响。
总之,本研究结果表明,养殖水体理化因子的恶化和条件致病菌的大量增生是青蟹病害爆发的重要原因之一,五倍子等中草药对于副溶血弧菌引起的青蟹病害具有良好的防治效果。研究成果可为青蟹病害防治提供科学依据,对于确保青蟹养殖业的健康发展具有重要的现实意义。
The mud crab Scylla serrata is a predacious, eurythermal and euryhaline crustacean. It is of important economic values and mainly cultured along the coast of southeastern China, and is also the traditional and characteristic aquaculture breed of Shantou. Since the autumn of 2004, S. serrata cultured in Niutianyang of Shantou have been suffered from serious diseases between September and November, which resulted in high mortality and great economic loss. For understanding fully the cause of the diseases and finding out a method to cure and control the diseases, we carried out a systematic investigation. Up to now, we have known that Vibrio parahaemolyticus, Aeromonas hydrophila and parasitic dinoflagellate of the genus Hematodinium are the main pathogens of diseases. This thesis made investigations on water physico-chemical parameters and biological factors so as to study the relationship between environmental factors and the outbreak of diseases; besides, cure-experiments with Chinese herbs against the Vibrio parahaemolyticus, Aeromonas hydrophila were carried out, so as to find a secure and efficient treatment against crab diseases. The main results are as following:
1. The environmental factors to the outbreak of S. serrata disease
During 2007 and 2008, four ponds were chosen as the sampling and monitoring sites from the mud crab culture area of Dashun Company in Niutianyang, and once or twice investigations were performed in each month from April to November. It was found that: both the outbreak of disease and the environmental factors displayed obvious seasonal change and their variation tendency were almost the same in both years. The crabs were in good status between April and August, some of them became diseased in September, and the death climax appeared in October, such as the yellow spot, black gill, yellow water and black ulcer of carapace diseases. During the two years, the data ranges of the investigated physico-chemical parameters and biological factors are: NH4+-N 76.63~235.63μg·L-1, NO2--N 6.15~27.75μg·L-1, NO3--N 22.33~174.83μg·L-1, PO43--P 8.27~44.81μg·L-1, water heterotrophic bacteria 27.25×105~157.93×105 cfu·L-1, water vibrio bacteria 9.75×103~116.75×103 cfu·L-1, sediment heterotrophic bacteria 27.75×105~733.25×105 cfu·dw-1·g-1, sediment vibrio bacteria 0.13×103~141.65×103 cfu·dw-1·g-1. The above data peaked in September or October, which are also the outbreak period of diseases. The range of dissolved oxygen and pH are respectively 4.12~7.80 mg·L-1 and 6.21~8.22, and the lowest value also appeared in September and October. These results suggested that both the deterioration of environmental factors and the proliferation of conditional pathogens are the important reasons for the outbreak of S. serrata diseases.
2. The preliminary study on therapy with Chinese herb to bacterial disease of S. serrata
The inhibitory effects of twelve Chinese herbs on the V. parahaemolyticus and A. hydrophila, which are the main pathogens of S. serrata diseases, were respectively tested via the in vitro antimicrobial susceptibility disk method and doubling dilution method. The results showed that Rhus chinensis, Prumus mume and Coptis chinensis displayed highest effect on both of the pathogens, the inhibitory plaque diameter was wider than 19 mm, and the minimal inhibitory concentration was lower than3.12 mg·mL-1. Coptis chinensis, Pomegranate, Portulaca oleraceal, Pheum officinale, Fructus schisandrae chinensis showed some effect on the pathogens, while the other herbs did not bacteria-inhibit or bactericide effectively, such as, Dryoptersis crassirhizoma, Pomegranate, Alpinia oxyphylla.
To confirm the inhibitory effect of R. chinensis on V. parahaemolyticus, a therapy experiment was conducted. The experiment included four groups, i.e., negative control group (S. serrata injected with 0.1 mL saline, non-medicated bath), positive infection group (injected with 0.1 mL 8×107 cfu·mL-1 V. parahaemolyticus, non-medicated bath), R. chinensis-healing group (injected with 0.1 mL 8×107 cfu·mL-1 V. parahaemolyticus and 8h later,medicated bath in 20 mg·L-1 R. chinensis for three days) and R. chinensis control group (injected with 0.1 mL saline,medicated bath as the R. chinensis-healing group). Duplicate in each group and each with 12 crabs. By comparing the cumulative mortality (CM), total bacteria counts (TBC), total haemocytes counts (THC) in haemolyph, and other immuno-metabolism indexes (Phenoloxidase PO, Lysozyme LSZ, et al.) among groups, the therapy effect of R. chinensis to V. parahaemolyticus-infected disease and the adverse effect of R. chinensis on normal crabs were evaluated. It was found that: during the experiment period, the positive infection group showed high mortality, all crabs died after 4 days, and noticeable changes in TBC (up to 105 cfu·mL-1 2 days after infection) and the immuno-metabolism indexes (THC, PO, LSZ, et al.) were observed. In R. chinensis-healing group, low CM (25% after 7 days) was observed, and the indexes of TBC, THC, and PO showed significant improvement comparing to positive group, 2days after medicated bath; On the seventh day, every indexes get back to normal on the whole, without noticeable change to control group. Compared with the negative control group and the R. chinensis control group, we can know that 20mg·L-1 R. chinensis medicated bath played no adverse function on normal crab, except causing some decreasing in total haemocyte counts. Those results indicated that R. chinensis at 20mg·L-1 medicated bath could effectively cure the S. serrata disease caused by V. parahaemolyticus and cause little adverse effects on normal crab at such a dose.
In summary, the results demonstrate that both the deterioration of environmental factors and the proliferation of conditional pathogens are the important reasons for the outbreak of S. serrata diseases, and R. chinensis has a good therapy effect on S. serrata diseases caused by V. parahaemolyticus. The results of the study could provide scientific evidence for preventing mud crab diseases, and play a great practical importance in the healthy and continuous development of the crab industry.
一.牛田洋青蟹病害爆发的环境因素
从牛田洋围垦区大顺公司的水产养殖基地选取4个100亩左右的青蟹养殖池塘作为采样、监测点,于2007年和2008年的4~12月,每月进行1~2次的定点环境监测、采样及病害调查,分析测定如下指标:水体的温度、盐度、溶解氧、pH,亚硝态氮(NO2--N)、硝态氮(NO3--N)、铵态氮(NH4+-N)及无机磷(PO43--P)的含量,化学耗氧量(COD),细菌数量(弧菌和异养菌);池塘底泥的细菌数量。结果发现:在07年和08年,养殖环境各因子与病害爆发都呈现出明显的季节性变化,且变化趋势基本一致。4~8月,青蟹健康状况良好,基本没有病害发生;自9月中下旬开始出现青蟹病害,10月份病害尤为严重,主要表现为黑鳃、甲壳黄斑、体内腹水等症状。两年内,各指标的变化范围:NH4+-N 76.63~235.63μg·L-1、NO2--N 6.15~27.75μg·L-1、NO3--N 22.33~174.83μg·L-1、PO43--P 8.27~44.81μg·L-1、水体异养菌27.25×105~157.93×105 cfu·L-1、水体弧菌9.75×103~116.75×103 cfu·L-1、底泥异养菌27.75×105~733.25×105 cfu·dw-1·g-1、底泥弧菌0.13×103~141.65×103 cfu·dw-1·g-1,它们的峰值均出现在病害高发的9~10月份;溶解氧和pH的变化范围分别是4.12~7.80 mg·L-1和6.21~8.22,它们的低值也出现在病害高发的9~10月份。结果表明:水体理化因子的恶化和条件致病微生物的大量增生是青蟹病害爆发的重要原因之一。
二.中草药防治青蟹细菌性病害的初步研究
通过药敏纸片法和倍比稀释法,分别测定了五倍子、连翘、贯众、地榆、大黄、黄芩、乌梅、黄连、马齿苋、石榴皮、益智仁、五味子等12味中草药对副溶血弧菌和嗜水气单胞菌的药物敏感性、最小抑菌浓度(MIC)和最小杀菌浓度(MBC)。结果显示,五倍子、乌梅和黄莲等对两菌株均具有明显的抑制作用,抑菌圈都大于19 mm,MIC均小于3.12 mg·mL-1。其中五倍子对两菌株的抑杀效果最明显,对副溶血弧菌和嗜水气单胞菌的抑菌圈分别达到37.00和42.29 mm,对两株菌的MIC和MBC分别为0.19和0.39 mg·mL-1。黄莲、石榴皮、马齿苋、大黄、五味子等对菌株有一定的抑杀作用,但贯众、石榴皮和益智仁的抑杀作用不明显。
接着,利用五倍子对青蟹副溶血弧菌病进行防治试验。实验设置4个组:阴性对照组(青蟹注射0.1 mL生理盐水,不药浴)、阳性感染组(注射0.1 mL 8×107 cfu·mL-1副溶血弧菌,不药浴)、五倍子治疗组(注射感染菌株后8h,用20 mg·L-1药液进行药浴3天)和五倍子对照组(注射生理盐水,药浴同治疗组),每组2个重复,每个重复6只青蟹。通过比较各组间青蟹的累积死亡率(CM),血淋巴中的细菌数量(TBC)及总血细胞数量(THC),血清中的酚氧化酶(PO)、溶菌酶(LSZ)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、酸性磷酸酶(ACP)及碱性磷酸酶(AKP)的活力,血清抗菌活力(ABA)、葡萄糖(Glu)及可溶性蛋白(Pro)的含量等免疫与代谢指标,以探究五倍子对感染副溶血弧菌青蟹的治疗效果,评估药浴对健康青蟹是否有副作用。结果显示,阳性感染组在染菌后第4天的CM即达100%,TBC显著增加,在感染二天后就达到105数量级,同时THC、PO、LSZ、ABA等指标较阴性对照组明显要低;五倍子治疗组7天的CM为25%,与阳性感染组相比,其TBC、THC、PO、LSZ、ABA等指标在治疗后第二天都有显著改善,到第七天时,各指标基本恢复到健康水平、与阴性对照组无显著差异;与阴性对照组相比,五倍子对照组青蟹除THC稍有降低外,其他指标均没有明显变化。结果表明,副溶血弧菌能引起青蟹较高的死亡率,注射107 cfu·mL-1菌液4天后即全部死亡;20 mg·L-1的五倍子药液能有效治疗青蟹由副溶血弧菌引起的疾病,该浓度药液对青蟹的免疫与代谢指标基本没有影响。
总之,本研究结果表明,养殖水体理化因子的恶化和条件致病菌的大量增生是青蟹病害爆发的重要原因之一,五倍子等中草药对于副溶血弧菌引起的青蟹病害具有良好的防治效果。研究成果可为青蟹病害防治提供科学依据,对于确保青蟹养殖业的健康发展具有重要的现实意义。
The mud crab Scylla serrata is a predacious, eurythermal and euryhaline crustacean. It is of important economic values and mainly cultured along the coast of southeastern China, and is also the traditional and characteristic aquaculture breed of Shantou. Since the autumn of 2004, S. serrata cultured in Niutianyang of Shantou have been suffered from serious diseases between September and November, which resulted in high mortality and great economic loss. For understanding fully the cause of the diseases and finding out a method to cure and control the diseases, we carried out a systematic investigation. Up to now, we have known that Vibrio parahaemolyticus, Aeromonas hydrophila and parasitic dinoflagellate of the genus Hematodinium are the main pathogens of diseases. This thesis made investigations on water physico-chemical parameters and biological factors so as to study the relationship between environmental factors and the outbreak of diseases; besides, cure-experiments with Chinese herbs against the Vibrio parahaemolyticus, Aeromonas hydrophila were carried out, so as to find a secure and efficient treatment against crab diseases. The main results are as following:
1. The environmental factors to the outbreak of S. serrata disease
During 2007 and 2008, four ponds were chosen as the sampling and monitoring sites from the mud crab culture area of Dashun Company in Niutianyang, and once or twice investigations were performed in each month from April to November. It was found that: both the outbreak of disease and the environmental factors displayed obvious seasonal change and their variation tendency were almost the same in both years. The crabs were in good status between April and August, some of them became diseased in September, and the death climax appeared in October, such as the yellow spot, black gill, yellow water and black ulcer of carapace diseases. During the two years, the data ranges of the investigated physico-chemical parameters and biological factors are: NH4+-N 76.63~235.63μg·L-1, NO2--N 6.15~27.75μg·L-1, NO3--N 22.33~174.83μg·L-1, PO43--P 8.27~44.81μg·L-1, water heterotrophic bacteria 27.25×105~157.93×105 cfu·L-1, water vibrio bacteria 9.75×103~116.75×103 cfu·L-1, sediment heterotrophic bacteria 27.75×105~733.25×105 cfu·dw-1·g-1, sediment vibrio bacteria 0.13×103~141.65×103 cfu·dw-1·g-1. The above data peaked in September or October, which are also the outbreak period of diseases. The range of dissolved oxygen and pH are respectively 4.12~7.80 mg·L-1 and 6.21~8.22, and the lowest value also appeared in September and October. These results suggested that both the deterioration of environmental factors and the proliferation of conditional pathogens are the important reasons for the outbreak of S. serrata diseases.
2. The preliminary study on therapy with Chinese herb to bacterial disease of S. serrata
The inhibitory effects of twelve Chinese herbs on the V. parahaemolyticus and A. hydrophila, which are the main pathogens of S. serrata diseases, were respectively tested via the in vitro antimicrobial susceptibility disk method and doubling dilution method. The results showed that Rhus chinensis, Prumus mume and Coptis chinensis displayed highest effect on both of the pathogens, the inhibitory plaque diameter was wider than 19 mm, and the minimal inhibitory concentration was lower than3.12 mg·mL-1. Coptis chinensis, Pomegranate, Portulaca oleraceal, Pheum officinale, Fructus schisandrae chinensis showed some effect on the pathogens, while the other herbs did not bacteria-inhibit or bactericide effectively, such as, Dryoptersis crassirhizoma, Pomegranate, Alpinia oxyphylla.
To confirm the inhibitory effect of R. chinensis on V. parahaemolyticus, a therapy experiment was conducted. The experiment included four groups, i.e., negative control group (S. serrata injected with 0.1 mL saline, non-medicated bath), positive infection group (injected with 0.1 mL 8×107 cfu·mL-1 V. parahaemolyticus, non-medicated bath), R. chinensis-healing group (injected with 0.1 mL 8×107 cfu·mL-1 V. parahaemolyticus and 8h later,medicated bath in 20 mg·L-1 R. chinensis for three days) and R. chinensis control group (injected with 0.1 mL saline,medicated bath as the R. chinensis-healing group). Duplicate in each group and each with 12 crabs. By comparing the cumulative mortality (CM), total bacteria counts (TBC), total haemocytes counts (THC) in haemolyph, and other immuno-metabolism indexes (Phenoloxidase PO, Lysozyme LSZ, et al.) among groups, the therapy effect of R. chinensis to V. parahaemolyticus-infected disease and the adverse effect of R. chinensis on normal crabs were evaluated. It was found that: during the experiment period, the positive infection group showed high mortality, all crabs died after 4 days, and noticeable changes in TBC (up to 105 cfu·mL-1 2 days after infection) and the immuno-metabolism indexes (THC, PO, LSZ, et al.) were observed. In R. chinensis-healing group, low CM (25% after 7 days) was observed, and the indexes of TBC, THC, and PO showed significant improvement comparing to positive group, 2days after medicated bath; On the seventh day, every indexes get back to normal on the whole, without noticeable change to control group. Compared with the negative control group and the R. chinensis control group, we can know that 20mg·L-1 R. chinensis medicated bath played no adverse function on normal crab, except causing some decreasing in total haemocyte counts. Those results indicated that R. chinensis at 20mg·L-1 medicated bath could effectively cure the S. serrata disease caused by V. parahaemolyticus and cause little adverse effects on normal crab at such a dose.
In summary, the results demonstrate that both the deterioration of environmental factors and the proliferation of conditional pathogens are the important reasons for the outbreak of S. serrata diseases, and R. chinensis has a good therapy effect on S. serrata diseases caused by V. parahaemolyticus. The results of the study could provide scientific evidence for preventing mud crab diseases, and play a great practical importance in the healthy and continuous development of the crab industry.
引文
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