草鱼、银鲫和青鱼捕捞后的应激反应

分别评价了捕捞对草食性(草鱼)、杂食性(银鲫)和肉食性(青鱼)鲤科鱼类血液指标(血浆史质醇、葡萄糖和乳酸浓度)、肝糖原含量和两种肝脏糖酵解酶(己糖激酶和丙酮酸激酶)活性的影响。结果显示:草鱼、银鲫和青鱼捕捞后血浆史质醇、葡萄糖和乳酸浓度均显著升高;草鱼和青鱼捕捞后2 h时肝糖原含量呈下降趋势,但银鲫捕捞前、后肝糖原含量未出现显著变化;捕捞前、后青鱼血糖浓度显著高于草鱼和银鲫。银鲫肝糖原含量显著高于草鱼和青鱼,其捕捞后血浆葡萄糖和乳酸浓度增加幅度较小,这意味着捕捞后银鲫应激反应强度相对较低。草鱼和银鲫捕捞后肝脏己糖激酶和丙酮酸激酶活性未发生显著变化,青鱼捕捞后2 h己糖激酶活性显著下降,这意味着捕捞应激后血糖升高未导致草鱼、银鲫和青鱼的肝脏糖酵解酶活性增强。     

热应激对大正三色锦鲤非特异性免疫指标及HSP70基因表达的影响

为探讨锦鲤的抗逆机理,在实验室条件下研究了持续热应激对大正三色锦鲤非特异性免疫指标及HSP70基因相对表达量的影响。分别于应激前、应激后2、6、10、14、18、22、26 h进行取样测定呼吸爆发、补体蛋白3(C3)、超氧化物歧化酶(SOD)、丙二醛(MDA)以及热休克蛋白70(HSP70)基因的相对表达量。结果发现,热应激后呼吸爆发降低,在应激后2、10、18～26h降低显著(P<0.05);热应激2 h后血清中C3含量略有上升,应激6～26 h过程中与应激前相比均下降,应激14～22 h显著地下降(P<0.05);热应激下血清中SOD有上升的趋势,但差异不显著(P>0.05);热应激下,血清中MDA浓度升高,应激10～18 h上升显著(P<0.05);应激后2、26 h HSP70基因的相对表达量与应激前相比显著性地上升了5.93倍、2倍(P<0.05),应激后6～22 h HSP70基因的相对表达量与应激前水平无显著性差异(P>0.05)。结果表明,热应激影响锦鲤非特性免疫指标,降低锦鲤非特异性免疫力;HSP70的表达受热应激调节,热应激下诱导合成的HSP70对锦鲤起到一定的应激保护作用。     

高碳水化合物日粮对异育银鲫血浆皮质醇含量和HSP70基因mRNA表达的影响

为了探讨高碳水化合物饲料是否会引起异育银鲫(Carassius auratus gibelio)的应激反应,选用体质量为(35.60±1.11)g的异育银168尾,随机分成2组,一组设为对照组(NS),投喂碳水化合物水平为35％的日粮;另一组设为高碳水化合物实验组(HS),投喂碳水化合物水平为50％的日粮,每组3个重复.在控温的循环水系统中饲养35 d和70 d后,测定异育银鲫血浆皮质醇激素含量及其肝、心脏、脾、肾中HSP70基因mRNA的表达量.结果显示,高碳水化合物实验组35 d时血浆皮质醇激素与对照组相比较显著升高(P＜0.05),肝和心脏HSP70基因mRNA表达较对照组明显增强(P＜0.05); 70 d时血浆皮质醇激素含量升高,且70 d时高碳水化合物组肝、心脏、脾和肾HSP70基因mRNA表达与对照组相比全部增强.各组饲养70 d后,饥饿24 h后重新投喂的0h、6h、12h、24 h、48 h,高碳水化合物实验组(HS)皮质醇含量在6h、48 h显著高于对照组(P＜0.05),在6h、12h、48 h肝HSP70基因mRNA表达与对照组相比均增强(P＜0.05),说明异育银鲫对50％碳水化合物含量的饲料的耐受性比较差,鱼体产生了应激反应.结果说明,当异育银鲫血清皮质醇激素含量升高时,其肝中HSP70基因mRNA的表达量却降低,反之亦然.本研究旨在为水生动物营养应激及营养免疫的相关研究提供新的思路,为开辟鱼类应激的营养调节提供科学依据.     

注射葡萄糖对吉富罗非鱼血浆生化指标、胰岛素和糖酵解关键酶的影响

研究注射葡萄糖对吉富罗非鱼(Oreochromis miloticus)生化指标、胰岛素和糖酵解酶的影响。选取体质量约80 g的吉富罗非鱼150尾,随机分为2个实验组,对照组腹腔注射0.7%的无菌生理盐水,处理组按照30 mg/100 g(体质量)的剂量腹腔注射葡萄糖。结果表明:(1)吉富罗非鱼在注射葡萄糖后1 h血糖达到最高水平,而后显著下降(P<0.05),3 h后降低到正常水平。注射葡萄糖可显著提高胆固醇的含量(P<0.05),并显著降低谷草转氨酶水平(P<0.05),但对血浆蛋白、甘油三酯和谷丙转氨酶水平没有显著影响(P>0.05)。(2)肝糖原在葡萄糖注射后的6 h达到最高水平,而后显著下降(P<0.05),肌糖原没有发生变化。(3)血浆和肌肉中的胰岛素均在葡萄糖注射后的3 h达到最高水平,而后显著下降(P<0.05),而肝中的胰岛素水平没有发生变化。(4)丙酮酸激酶的活力在葡萄糖注射后6h达到最高水平(P<0.05),己糖激酶的活力在注射葡萄糖后没有发生变化。结果表明,注射葡萄糖可显著提高吉富罗非鱼血糖水平,且维持时间较长,胰岛素含量和丙酮酸激酶(PK)活力的提高均相对延迟,而己糖激酶活力的不足又限制了葡萄糖的酵解反应,从而引起血浆胆固醇升高,并伴有持续的血糖和肝糖原升高,从而产生营养胁迫的生理反应,加重鱼类对葡萄糖的代谢负担。     

氨氮胁迫对凡纳滨对虾肠道免疫相关指标的影响

为研究急性氨氮胁迫对凡纳滨对虾(Litopenaeus vannamei)肠道免疫功能的影响,将对虾暴露于氨氮浓度为20 mg·L^-1的海水中72 h,测定了不同时间点肠道中抗病原感染指标如酸性磷酸酶(ACP)、碱性磷酸酶(ALP)、溶菌酶(Lys)、酚氧化酶原(proPO)以及抗氧化功能指标如总抗氧化能力(T-AOC)、超氧化物歧化酶(SOD)、热休克蛋白70(HSP70)、热休克蛋白90(HSP90)等变化。结果显示,与对照组相比,氨氮胁迫后:1)ACP和ALP活性均于6 h显著升高(P<0.05),随后于48~72 h显著低于对照组(P<0.05);Lys活性于24 h显著升高至最大值(P<0.05),随后于72 h显著低于对照组(P<0.05)。2)T-AOC和SOD活性均于6 h和12 h显著高于对照组(P<0.05),随后于48 h和72 h显著降低(P<0.05)。3)HSP70基因表达水平于24 h显著升高至最大值,随后虽有降低,但仍显著高于对照组(P<0.05);HSP90和proPO基因表达水平均于12 h显著升高至最大值(P<0.05),随后于72 h降低至对照组水平(P>0.05)。研究表明,急性氨氮胁迫对凡纳滨对虾肠道免疫功能相关指标影响显著,对其肠道免疫防御系统有明显的损伤作用。     

热应激对大正三色锦鲤非特异性免疫指标及HSP70基因表达的影响

为探讨锦鲤的抗逆机理,在实验室条件下研究了持续热应激对大正三色锦鲤非特异性免疫指标及HSP70基因相对表达量的影响.分别于应激前、应激后2、6、10、14、18、22、26 h进行取样测定呼吸爆发、补体蛋白3(C3)、超氧化物歧化酶(SOD)、丙二醛(MDA)以及热休克蛋白70 (HSP70)基因的相对表达量.结果发现,热应激后呼吸爆发降低,在应激后2、10、18～26 h降低显著(P＜0.05);热应激2h后血清中C3含量略有上升,应激6～26 h过程中与应激前相比均下降,应激14 ～ 22 h显著地下降(P＜0.05);热应激下血清中SOD有上升的趋势,但差异不显著(P＞0.05);热应激下,血清中MDA浓度升高,应激10～18 h上升显著(P＜0.05);应激后2、26 hHSP70基因的相对表达量与应激前相比显著性地上升了5.93倍、2倍(P＜0.05),应激后6～22 hHSP70基因的相对表达量与应激前水平无显著性差异(P＞0.05).结果表明,热应激影响锦鲤非特性免疫指标,降低锦鲤非特异性免疫力;HSP70的表达受热应激调节,热应激下诱导合成的HSP70对锦鲤起到一定的应激保护作用.     

普安银鲫(Carassius auratus gibelio)卵黄囊期脂蛋白脂酶和肝脂酶基因的表达及葡萄糖、维生素C对其的影响

为了探讨普安银鲫(Carassius auratus gibelio)卵黄囊期脂蛋白脂酶(Lipoprotein lipase, LPL)和肝脂酶(Hepatic lipase, HL)的表达特点及葡萄糖和维生素C溶液分别浸泡对它们的影响,本研究采用荧光定量PCR技术检测LPL和HL基因在普安银鲫卵黄囊仔鱼发育中的表达情况及mRNA表达水平,并检测了葡萄糖、维生素C对这两种基因mRNA表达量的影响。结果显示,LPL和HL基因在普安银鲫内源营养期、混合营养期和外源营养期均有表达,且LPL和HL mRNA表达量呈上升变化。葡萄糖组LPL和HL mRNA表达量呈上升变化,维生素C组也呈上升变化。在内源营养期、混合营养期和外源营养期,葡萄糖能显著上调LPL和HL mRNA的表达量(P<0.05);在内源营养期、混合营养期和外源营养期,维生素C能显著上调LPL mRNA的表达量,而HL mRNA的表达量在混合营养期和外源营养期显著高于对照组(P<0.05)。研究表明,普安银鲫发育至混合营养期时,机体内脂质分解代谢增强。适宜水平的葡萄糖、维生素C能诱导LPL和HL mRNA的表达。     

Gln对中华鳖免疫应激后肝脏.脾脏及肠道HSP70表达的影响

本试验通过研究谷氨酰胺/脂多糖（Gln/LPS）对中华鳖肝脏、脾脏和肠道组织热应激蛋白HSP70表达的影响,考察Gln对中华鳖免疫应激的保护作用与其诱导组织HSP70表达的关系。试验选择体重接近的健康中华鳖24只,随机分为4组,分别腹腔注射0.7%Nacl、500 ugLPS/kgBW、100 mgGln/kgBW和500 ugLPS+100 mgGln/kgBW。采用免疫组化法检测中华鳖肝脏、肠道和脾脏中HSP70的表达,放射免疫法测定血浆皮质醇（COR）含量。结果表明：腹腔注射500 μgLPS/kgBW显著增强了中华鳖肝脏和脾脏组织HSP70的表达（P<0.05）,而补充100 mgGln/kg BW抑制了中华鳖肠道、肝脏和脾脏组织HSP70的表达。推测Gln对免疫应激中华鳖的保护作用与其诱导机体组织HSP70的表达无关。     

不同蛋白质和小麦淀粉水平对大黄鱼生长性能、糖酵解和糖异生关键酶活性的影响

本实验旨在研究不同蛋白质和小麦淀粉水平对大黄鱼生长性能、肝脏糖酵解和糖异生关键酶活性、血清指标、糖原含量及消化酶活性的影响。采用2×3双因素实验设计,共配制6组饲料,包含2个蛋白质水平(41%、46%)和3个小麦淀粉水平(10%、20%、30%),选取初重约为(14.84±0.16) g的大黄鱼900尾,随机分为6组(每组3个重复,每个重复50尾),进行8周的养殖实验。实验结果表明:饲料蛋白质和小麦淀粉水平的交互作用对大黄鱼增重率和特定生长率无显著影响(P>0.05),且在相同的饲料蛋白质水平下,饲料小麦淀粉水平对大黄鱼增重率和特定生长率无显著影响(P>0.05)。饲料蛋白质和小麦淀粉的交互作用对大黄鱼肝脏6-磷酸果糖激酶(phosphofructokinase,PFK)、丙酮酸激酶(pyruvate kinase,PK)、果糖-1,6-二磷酸酶(fructose-1,6-bisphosphatase,FBPase)活性有显著影响(P<0.05),但对葡萄糖激酶(glucokinase,GK)、葡萄糖-6-磷酸酶(glucose-6-phosphatase,G6Pase)、磷酸烯醇式丙酮酸羧激酶(phosphoenolpyruvate,PEPCK)活性无显著影响(P>0.05)。当饲料蛋白质水平为41%时,饲料小麦淀粉水平对葡萄糖激酶、6-磷酸果糖激酶和果糖-1,6-二磷酸酶活性有显著影响(P<0.05),且果糖-1,6-二磷酸酶活性随着饲料小麦淀粉水平的升高呈下降趋势,但对丙酮酸激酶、葡萄糖-6-磷酸酶、磷酸烯醇式丙酮酸羧激酶活性无显著影响(P>0.05);当饲料蛋白质水平为46%时,饲料小麦淀粉水平对葡萄糖激酶、6-磷酸果糖激酶、丙酮酸激酶、果糖-1,6-二磷酸酶活性有显著影响(P<0.05),且葡萄糖激酶、6-磷酸果糖激酶、丙酮酸激酶均随饲料小麦淀粉水平的升高呈上升趋势,但对葡萄糖-6-磷酸酶和磷酸烯醇式丙酮酸羧激酶活性无显著影响(P>0.05)。实验表明,大黄鱼在高蛋白质水平下能够有效调节糖酵解和糖异生关键酶活性,降低血糖含量及丙氨酸转氨酶、天冬氨酸转氨酶活性,并且未显著影响增重率;在低蛋白质水平,血糖含量和肝糖原含量随小麦淀粉水平的升高呈上升趋势,但增重率随着饲料小麦淀粉水平的升高呈上升趋势,表明在低蛋白质水平下高小麦淀粉水平对大黄鱼的生长有一定的改善作用。     

急性氨氮胁迫对黄颡鱼组织抗氧化酶活性及HSP70和HSP90基因mRNA表达水平的影响

为了探讨急性氨氮胁迫对黄颡鱼组织中抗氧化酶活性及HSP70和HSP90基因mRNA表达水平的影响,实验随机挑选了360尾黄颡鱼[初体质量(17.25±0.05)g],分别暴露于含有0(对照)、5.70(低浓度组)、28.50(中浓度组)和57.00(高浓度组)mg/L总氨氮浓度的水体中,进行96 h的急性胁迫实验。实验开始后,分别于0、12、24、48和96 h取样。结果显示,氨氮胁迫发生后,低、中浓度组实验鱼肝脏中超氧化物歧化酶(SOD)活性呈先升高后降低趋势,而高浓度组则持续降低;低、中、高浓度组实验鱼肝脏中丙二醛(MDA)含量在胁迫开始后显著升高;3 h时,高浓度组实验鱼肝脏中SOD活性达到最低,而MDA含量最高;24 h后,高浓度组实验鱼肝脏中过氧化氢酶(CAT)活性显著升高;低、中、高浓度组实验鱼肝脏中HSP70基因的mRNA表达量呈先降低后升高趋势,而鳃中HSP70基因表达量持续升高,但脑中HSP70基因在0 h后显著降低;氨氮胁迫3 h时,低、中、高浓度组实验鱼肝脏和脑中HSP70基因表达量显著低于对照组,而在鳃中正好相反;相比HSP70基因,高氨氮浓度组实验鱼肝脏和鳃中HSP90基因的mRNA表达量在24 h时达到最高。研究表明,不同浓度的氨氮胁迫会对黄颡鱼抗氧化酶活性造成不同程度的抑制,原因与丙二醛的积累量有关;相比HSP90基因,黄颡鱼HSP70基因的表达量在氨氮胁迫发生后迅速上调,这种生理调控机制提示HSP70在应对急性氨氮胁迫时发挥着更重要的作用。     

Association between the interannual variation in the oceanic environment and catch rates of bigeye tuna (Thunnus obesus) in the Atlantic Ocean

The environmental processes associated with variability in the catch rates of bigeye tuna in the Atlantic Ocean are largely unexplored. This study used generalized additive models (GAMs) fitted to Taiwanese longline fishery data from 1990 to 2009 and investigated the association between environmental variables and catch rates to identify the processes influencing bigeye tuna distribution in the Atlantic Ocean. The present findings reveal that the year (temporal factor), latitude and longitude (spatial factors), and major regular longline target species of albacore catches are significant for the standardization of bigeye tuna catch rates in the Atlantic Ocean. The standardized catch rates and distribution of bigeye tuna were found to be related to environmental and climatic variation. The model selection processes showed that the selected GAMs explained 70% of the cumulative deviance in the entire Atlantic Ocean. Regarding environmental factors, the depth of the 20 degree isotherm (D20) substantially contributed to the explained deviance; other important factors were sea surface temperature (SST) and sea surface height deviation (SSHD). The potential fishing grounds were observed with SSTs of 22–28°C, a D20 shallower than 150 m and negative SSHDs in the Atlantic Ocean. The higher predicted catch rates were increased in the positive northern tropical Atlantic and negative North Atlantic Oscillation events with a higher SST and shallow D20, suggesting that climatic oscillations affect the population abundance and distribution of bigeye tuna.     

Rare serotype occurrence and PFGE genotypic diversity of Streptococcus agalactiae isolated from tilapia in China

Previously, we reported 10 PEGE types of 85 tilapia Streptococcus agalactiae(GBS), which shifted from Streptococcus iniae in China, by using PEGE method. Presently, larger and more representative tilapia GBS were isolated, for the ?rst time in China, to characterize their serotypes and genetic diversities more precisely than had done before. 168 GBS strains were distributed in ?ve provinces of China, in which Guangdong, Guangxi and Hainan were the major ones, holding36.9%(62/168), 37.5%(63/168) and 19.6%(33/168), respectively. Serotypes, Ia, Ib and III, were observed in these strains and the most predominant one was Ia(95.2%), which mainly distributed in Guangdong, Guangxi and Hainan. Ia initially occurred in 2009, it shoot up to 32.1% in 2010,but decreased to 16.1% in 2011 before went up to 45.2% in 2012. Ib sporadically occurred during2007–2011, III onlyoccurred in 2012. 14 different PFGE types, including 4 new types(N, O,P and Q), were observed, in which B, D, F and G were the predominant types, holding 83.9%(141/168) of the total GBS strains. Ia corresponded to 11 PFGE types(A–H, N–P), in which type D predominated(51%). Ib represented 3 genotypes(I, J and Q) and III harbored only 2genotypes(N and F). Type N and Fsynchronously presented in Ia and III. In summary, the genetic diversity of tilapia GBS varied by serotypes and changed with geographical locations and years.Although Iastillpredominated, new rareserotypeIII alreadyoccurred in China.     

Evaluation of a Fast Method Based on the Presence of Two Restriction Sites in the Mitochondrial ND5 (mt ND5) Gene for the Identification of Scomber Species

The purpose of this work was to evaluate the suitability of a method based on the presence of two restriction sites (for Hae III and Hindf I) in the mitochondrial NADH dehydrogenase subunit 5 (mt ND5) gene to identify Scomber species. The evaluation was performed on 144 reference and market samples by sequencing of the entire 505-bp fragment of the mt ND5 gene and of a 464-bp fragment of the Kocher fragment of the cytochrome b gene (mt Cytb). Sequence analysis of any of the two fragments allows the identification of each of the four Scomber species, but S. japonicus and S. colias had the same restriction sites at the ND5 amplicon and would not have been differentiated by this analysis. Similarly, loss of the Hae III site in some S. scombrus individuals would have misidentified them as not being Scomber. All the market products were correctly labeled except one acquired in Spain labeled as originating in the Atlantic and containing S. japonicus.     

Growth performance,digestive enzyme activity and immune response of Japanese sea bass,Lateolabrax japonicus fed with fructooligosaccharide

In this experiment, a feeding trial was performed to determine the effects of fructooligosaccharide (FOS) on growth performance, digestive enzyme activity and immune response of Japanese sea bass, Lateolabrax japonicus juveniles (initial weight 38.3 ± 0.5 g), and the fish were examined following feeding with six levels of FOS (0, 0.5, 1, 2, 4 and 6 g/kg) for 28 days. Significant enhancement of weight gain (WG) and specific growth rate (SGR) was found in fish fed 1 g/kg FOS incorporated diets (p < .05), while the feed conversion ratio (FCR) in the 1, 2 g/kg FOS groups reduced significantly compared with the control (p < .05). Besides, the crude lipid in the 4, 6 g/kg FOS groups increased significantly compared with the control (p < .05). On the other hand, the erepsin and lipase activities significantly elevated in intestine of fish fed 2 g/kg FOS (p < .05) and the lysozyme activity in serum of fish fed 2 g/kg FOS were significantly higher than that in the control (p < .05). Moreover, the alkaline phosphatase activities in serum of fish fed 0.5, 1, 2 g/kg FOS were significantly higher than in control (p < .05). Regression analysis showed that the relationships between dietary FOS levels and either SGR, FCR, erepsin or lysozyme activities were best expressed by regression equations, and the optimal inclusion levels are 1.37, 1.80, 3.06, 3.11, 1.93 and 1.80 g/kg for SGR, FCR, erepsin, lipase, lysozyme and total superoxide dismutase activities, respectively. Overall, this study revealed that FOS incorporated diets could beneficial for L. japonicus culture in terms of increasing the growth, digestion and immune activities. Under the present experimental condition, the optimal supplementary level of FOS in the diet of L. japonicus is 1–3 g/kg.     

Growth hormone (GH) and reproduction: a review

Interaction between growth and reproduction occurs in many vertebrates and is particularly obvious at certain stages of the life cycle in fish. Endocrine interactions between the gonadotropic axis and the somatotropic axis are described, the potential role of GH being emphasised. A comparative analysis of these phenomena in mammals, amphibians and fish, suggests a specific role of GH in the physiology of puberty, gametogenesis and fertility. It also shows the original contribution made by studies on the fish model in this field of investigations.     

Purification and partial characterization of GtHs (cfLH and cfFSH) from Indian walking catfish (Clarias batrachus) (L.) and development of a homologous ELISA for cfLH

Two gonadotropins (GtH; Qa and Qb) were purified by gel filtration and ion exchange chromatography from the pituitaries of Indian walking catfish (Clarias batrachus). The presence of GtH during purification was assessed by in vitro oocyte maturation and in vivo steroidogenic activity, and their identities were determined by elution profiles, molecular weight, biological activities and yield. The molecular weights of Qa and Qb were 37 and 42 kDa, respectively, and composed of distinct subunits (Qa: 20 and 14 kDa and Qb: 26 and 18 kDa). Polyclonal antibodies raised against Qa immunostained Qa, Qb and pituitary GtH cells. A competitive Qa‐ELISA was developed whose sensitivity was 6.25 ng mL^?1 (1.25 ng well^?1) with intra‐ (3.5%) and inter‐ (12.4%) assay coefficients of variation. Displacement curves parallel to the standard were obtained with plasma and pituitary extracts of catfish, Qb and carp GtHII. The assay was validated by measuring the plasma Qa levels after LHRH treatment and in relation to ovarian growth in the female catfish during different reproductive phases. Based on the results, Qa and Qb corresponded to fish LH and FSH respectively. The findings will increase the knowledge of the mechanisms controlling fish reproduction and identification of sensitive phases in fish in captivity for hormonal manipulation.     

Controlled infection of Poecilia reticulata Peters (guppy) with Tetrahymena by immersion and intraperitoneal injection

Tetrahymena is a protozoan parasite, which infects guppy, Poecilia reticulata Peters, and causes substantial economical losses in commercial farms worldwide. Studies of guppy infected by Tetrahymena require standardized infection protocols. The LD50 for Tetrahymena infection of guppies by intraperitoneal (IP) injection was calibrated, and the level obtained was 946 parasites per fish. Guppy infection with Tetrahymena by immersion, imitating the natural route of infection via the integument, was studied under normal or stress conditions. Exposure to cold and netting (CNI) and to cold only (CI) followed by immersion exposure to 10 000 Tetrahymena per mL resulted in 22.5% and 19.2% mortality, respectively, as compared to 14.2% and 10% in groups that were netted only (NI) or non‐stressed (I). Histopathology revealed that immersion infection resulted in a systemic infection. Lysozyme levels, measured 3 weeks after infection, were significantly higher in the CNI group (288 μg per mg protein) compared with CI‐, NI‐ and I‐treated groups (94.5, 64 and 62.3 μg mg^?1, respectively). There was no evident parasite immobilization activity in body homogenates, suggesting no development of acquired immunity. Re‐infection by IP injection revealed no increase in protection in any of the treatment groups, mortality range of 56.3–75%, higher than in the non‐exposed control (40.6% mortality).