罗非鱼无乳链球菌C5a肽酶(ScpB)的原核表达及其免疫原性

以尼罗罗非鱼(Oreochromis niloticus)为鱼体样本, 克隆了罗非鱼源无乳链球菌(Streptococcus agalactiae) scpB基因, 构建了原核表达质粒pET32a(+)-scpB, 转入大肠杆菌BL21(DE3)后, IPTG诱导表达, 表达的重组蛋白经镍离子金属螯合柱纯化及超滤管浓缩后, 进行SDS-PAGE和Western blot分析鉴定。纯化的重组蛋白以不同剂量(1 μg/g、3 μg/g、5 μg/g) 免疫尼罗罗非鱼后, 每周检测各实验组鱼体血清非特异性免疫指标[溶菌酶活性、超氧化物歧化酶(SOD)活力]及抗体水平变化情况, 并于免疫4周后以4倍LD₅₀的剂量对其进行人工攻毒。结果显示, 该重组蛋白在大肠杆菌中以包涵体的形式存在; 对尼罗罗非鱼的相对免疫保护率为69.66%~89.00%, 其中5 μg/g组的相对保护率最高; 受免鱼体血清中溶菌酶活性、SOD活力和抗体水平较对照组有极显著提高(P<0.01), 结果表明, 重组蛋白ScpB具有较强的免疫原性和保护作用。本研究旨在为GBS多肽疫苗的研制奠定基础。

     

溶藻弧菌感染后凡纳滨对虾鳃组织免疫相关基因的表达

分别给凡纳滨对虾(Litopenaeus vannamei)注射生理盐水、3×10⁶ CFU/mL(低浓度组)和9×10⁶ CFU/mL(高浓度组)溶藻弧菌(Vibrio alginolyticus)菌液, 采用荧光定量PCR技术, 检测不同处理后凡纳滨对虾鳃组织中Toll受体、IMD和溶菌酶基因表达量随时间的变化。结果表明, 处理42 h内, 注射生理盐水组对虾的Toll受体和IMD mRNA表达量无显著变化, 溶菌酶mRNA表达量在注射36 h后显著升高。急性感染溶藻弧菌后, 凡纳滨对虾鳃组织中Toll受体、IMD和溶菌酶mRNA的表达量峰值分别出现在感染后24、42和36 h; 溶藻弧菌的感染剂量不影响上述基因表达峰值的出现时间, 但显著影响上述基因的表达峰值(P<0.05), 各基因表达量峰值由大到小均依次为高浓度组、低浓度组、生理盐水组。急性感染初期, 对虾鳃组织中Toll受体mRNA表达量呈现显著下调, 而IMD和溶菌酶mRNA表达量在感染初期不存在显著下调现象。与感染前各基因的表达量相比, 高浓度溶藻弧菌感染组Toll受体mRNA表达量在2 h时显著下调, 低浓度溶藻弧菌感染组Toll受体mRNA表达量在3 h显著下调(P<0.05); 高浓度组IMD和溶菌酶mRNA表达量分别在36 h和12 h时开始有显著上调, 而低浓度组IMD和溶菌酶mRNA表达量则分别在42 h和24 h才有显著上调(P<0.05)。表明溶藻弧菌感染对凡纳滨对虾鳃组织中Toll受体、IMD和溶菌酶mRNA表达量有显著影响, 各基因的表达量与感染进程及溶藻弧菌剂量存在一定的相关性。

     

传染性造血器官坏死病毒-Sn1203株的基因型及糖蛋白的生物信息学分析

利用鲤(Cyprinus carpio)上皮细胞(epitheliaoma papulosum cyprini, EPC)培养传染性造血器官坏死病毒- Sn1203分离株(IHNV-Sn1203), 根据GenBank中IHNV G蛋白基因开放阅读框(open reading frame, ORF)的序列设计引物(GenBank序列编号AB288207), 采用RT-PCR的方法克隆得到IHNV-Sn1203株G蛋白全长ORF, 克隆至表达载体pET27b(+)中, 构建了pET27-G重组质粒, 并进行了测序分析。生物信息学分析结果显示, IHNV-Sn1203株G蛋白基因序列长度为1 527 bp, 与韩国株具有最高的核酸同源性(96.86%)和氨基酸同源性(97.05%)。该基因编码508个氨基酸残基, 推导分子量约为56.55 kD, 等电点为6.15; 氨基酸序列分析表明, G蛋白富含丝氨酸、苏氨酸和酪氨酸, 存在28个潜在的磷酸化位点; 存在4个潜在的N-糖基化位点和7个潜在的O-糖基化位点; G蛋白N端含有20个氨基酸的信号肽; 亲水性大于输水性; 位于483~508位氨基酸存在一跨膜区; 抗原表位预测显示抗原性良好; 系统进化树分析显示, IHNV-Sn1203株与日本株和韩国株聚为一簇, 都属于JRt基因型。

     

牙鲆有丝分裂雌核发育二倍体的制备

本研究采用紫外线灭活的真鲷(Pagrus major)精子激活牙鲆(Paralichthys olivaceus)卵子, 经过静水压机处理诱导有丝分裂雌核发育二倍体。优选施压起始时间、持续时间和压力大小3个方面参数, 获得最佳参数组合为: 起始时间60 min、持续时间6 min、施加压力650 kg/cm²。在此条件下的受精率最高为67.80%, 孵化率54.23%, 与其他处理组间达显著性差异(P<0.05)。用流式细胞仪检测未经静水压处理的胚胎, 其DNA含量约为普通二倍体的1/2, 即单倍体; 检测有丝分裂雌核发育胚胎, 其DNA含量与普通二倍体大体一致。结果表明, 应用该方法可成功诱导获得牙鲆有丝分裂雌核发育二倍体。

     

鲤 IGF-I 基因 2 个高度多态微卫星位点的鉴定及其与生长相关性 状的关联分析

??利用PCR结合测序方法, , 在鲤(Cyprinus carpio)类胰岛素生长因子-I (insulin-like growth factor-I, , IGF-I)基因内含子1和内含子2中各鉴定1个微卫星多态性位点, , 分别命名为intron1189和intron2310, , 这两个位点均以4碱基为重复单元。以黑龙江鲤(Cyprinus cario haematopterus, , YL)(n=263)、德国镜鲤选育系(Cyprinus carpio L. mirror, , JL)(n=229), 及荷包红鲤抗寒品系(Cyprinus carpio var. wuyuanensis, , HL)(n=255)为研究对象, , 评估了这两个位点不同基因型(频率>3%)与鲤4个生长时段(135 d 、325 d、335 d和445 d)生长表型的关联。结果显示, , 2个位点在3个鲤群体中均表现为高度多态(PIC>0.5)。intron1189位点多态性主要对YL的135 d、325 d体长和325 d、385 d体质量有显著影响(P<0.05), , 而intron2310位点多态性对JL各检测时段的体长和体质量均有显著影响(P<0.05)。对不同基因型个体的体长和体质量性状进行多重比较, , 结果显示, , 在intron1189位点, , 185/229基因型YL的135 d、325 d体长和325 d、385 d体质量最低, , 而205/221基因型YL的135 d、325 d体长和325 d体质量最高。在intron2310位点, , 290/350基因型JL在各检测时段的体长及135 d、325 d、385 d体质量最低, , 而318/350基因型JL的135 d、325 d、385 d体长和体质量均为最高。上述结果表明, , 鲤IGF-I基因内含子中的这两个高度多态微卫星位点潜在影响鲤的生长性能。

     

大鲵虹彩病毒流行株的分离及其主衣壳蛋白编码基因序列比较分析

大鲵虹彩病毒(giant salamander iridovirus, GSIV)是近年中国大陆新发现的引起人工养殖大鲵(Andrias davidianus)大规模死亡的病毒病原。为了揭示大鲵虹彩病毒流行株的基因型差异, 本研究对2010–2012年采集自全国不同大鲵养殖区域的患虹彩病毒病的大鲵样本进行了分子检测、病毒分离培养以及病毒主衣壳蛋白(major capsid protein, MCP)基因测序与比对分析。结果显示, 采自陕西、湖北、湖南、浙江、江西、福建等省的10个样本检测为阳性, 通过细胞培养获得10株病毒流行株。对该10株流行株MCP基因的测序与比对分析发现, 核苷酸序列相似性达到99.7%~100%, 其推测的氨基酸序列无明显差异, 证实中国大鲵虹彩病毒流行株属同一基因型。系统进化树分析结果表明, 所选大鲵虹彩病毒与蛙病毒分别聚为一枝, 但其亲缘关系较近。本研究结果旨为大鲵虹彩病毒病的疫苗研制及其免疫防控技术研究奠定基础。

     

环境因子对鼠尾藻生殖托生长及光合特性的影响

鼠尾藻(Sargassum thunbergii)样本采自青岛太平角, 以光照、温度、盐度及营养盐水平4项环境因子分别设置4组单因素实验, 研究其对鼠尾藻生殖托的生长、表观光合作用、暗呼吸作用以及色素积累的影响。结果显示: (1)光照度为8 000 lx、温度为20℃和盐度为20的条件下生殖托的比生长速率(SGR)较大, 表观光合作用较强。光照12 000 lx抑制生殖托的光合作用与生长, 15℃下比生长速率比20℃小50.64%(P<0.05), 盐度从31下降到28, 比生长速率与表观光合速率显著增大。(2)色素含量与光照度、温度呈显著的负相关性, 叶绿素a和类胡萝卜素含量受环境因子影响较大, 而叶绿素c相对稳定。(3)氮磷营养盐的浓度配比对生殖托的生长、光合作用影响显著, 适宜的氮磷比(N/P)范围为5∶1~1∶1, 而50∶1显著抑制生殖托的生长。(4)相比氮元素而言, 磷浓度变化对生殖托光合作用影响更大, 说明磷元素对于生殖托的构建至关重要。(5)氮磷比对生殖托色素含量影响不显著, 而富氮、富磷培养液有利于色素的积累。结果表明, 上述4项环境因子对鼠尾藻生殖托的生长及光合特性均有显著影响, 适宜的培养条件为光照度8 000 lx、温度20℃、盐度20、氮磷浓度比(N/P)在5∶1~1∶1。本研究旨在为鼠尾藻人工育苗技术的优化提供科学依据。

     

低盐度和不同硫酸铜浓度对银鲳鳃离子调节酶和肝抗氧化功能的影响

开展银鲳(Pampus argenteus)试养海域的盐度易随气候降低, 且受到一定程度Cu等重金属污染, 研究低盐条件下硫酸铜对银鲳的影响十分必要。本实验首先进行银鲳幼鱼低盐度适应, 将盐度以4的幅度从24逐步降低至12。稳定后进行硫酸铜胁迫, 在盐度12下CuSO₄·5H₂O浓度梯度设为0、0.1、0.3、0.5 mg·L^–1, 盐度24下CuSO₄·5H₂O浓度梯度设为0、0.5 mg·L^–1, 胁迫持续144 h。通过检测两种鳃离子调节酶: Na⁺/K⁺-ATP酶(NKA)和V-H⁺-ATP酶(VHA), 以及3种肝抗氧化活性物质－还原型谷胱甘肽(GSH)、超氧化物歧化酶(SOD)和过氧化氢酶(CAT), 探究低盐条件下铜离子对银鲳幼鱼上述指标的影响。结果显示, 盐度逐步降低后, NKA与VHA活力呈上升后下降的变化, 之后NKA活力随硫酸铜浓度增加而减弱; VHA活力加入硫酸铜后都出现显著下降, 其中CuSO₄·5H₂O 0.3 mg·L^–1与0.5 mg·L^–1组在72 h时下降更为明显; 盐度24硫酸铜组NKA和VHA活力都在24 h时增强而后减弱。GSH含量和SOD活力在盐度降低时出现跃升, CAT活力则呈波动变化, 加入硫酸铜后, 0.3 mg·L^–1与0.5 mg·L^–1组GSH含量持续下降后显著上升而后回落; 各硫酸铜组SOD活力都出现增强后回落的变化; 0.3 mg·L^–1与0.5 mg·L^–1组CAT活力在72 h有显著增强。本实验表明, 铜离子对NKA与VHA有抑制作用, 盐度降低时其作用更强; GSH、SOD和CAT的变化能反映低盐度和铜离子对银鲳的伤害程度。银鲳对水体铜离子有一定的抗性, 但在盐度降低等环境变化时, 应当密切注意水体中铜等重金属浓度。

     

NaCl盐度和NaHCO3碱度对鲤、鲫和大鳞鲃的精子活力及其受精率的影响

研究了不同NaCl盐度(0、1、2、3、4、5、6、7、8、9、10、11、12)和NaHCO₃碱度(0 mmol/L、10.00 mmol/L、15.85 mmol/L、25.12 mmol/L、39.81 mmol/L、63.10 mmol/L)对松浦镜鲤(Cynipus carpio)、方正鲫(Carassius auratus gibelio)和大鳞鲃(Barbus capito)精子活力及其受精率的影响。结果表明: 1) 在盐度为4时, 方正鲫和大鳞鲃精子的激烈运动时间、快速运动时间和寿命最长, 方正鲫分别为(90.11±9.03) s、(126.34±13.90) s和(154.27±11.36) s; 大鳞鲃分别为(48.91±1.43) s、(62.19±4.28) s和(90.68±4.46) s。在盐度为5时松浦镜鲤精子的激烈运动时间、快速运动时间和寿命最长, 分别为(72.44±9.42) s、(102.16±8.82) s和(206.99±6.65) s。2) 当盐度达到8以上时, 3种鱼的精子激活将受到抑制; 盐度大于10时, 方正鲫和大鳞鲃精子死亡; 盐度大于11时, 松浦镜鲤精子死亡。3) 在碱度为15.83 mmol/L时, 3种鱼的精子激烈运动时间、快速运动时间和寿命均最长, 且显著高于其他碱度条件下的精子活力(P<0.05)。4) 在盐度为1时, 方正鲫和大鳞鲃受精率达到最高分别为63.0%和68.0%, 在盐度为3时, 松浦镜鲤受精率达到最高为72.3%, 当盐度大于3时, 受精率开始呈明显下降趋势。碱度为10.00 mmol/L时, 3种鱼的受精率均最高, 分别为75.4%、54.0%和66.0%。本实验旨在通过测定不同NaCl盐度、NaHCO₃碱度条件下3种鱼类精子的活力和受精率所受的影响, 为北方地区碳酸型盐碱水域的渔业开发利用提供基础资料。 

     

克隆牙鲆和普通牙鲆血液生理生化指标的比较

以中国水产科学研究院北戴河中心实验站养殖的双单倍体(DH)牙鲆(Paralichthys olivaceus)作为亲鱼, 采用冷休克方法抑制第二极体排出, 建立牙鲆克隆家系。以普通牙鲆的一个家系作为对照组, 在同样饲养条件下进行培育。实验组和对照组均为2⁺ 龄鱼, 克隆家系随机抽取5尾, 体质量(1 398±88) g; 普通群体随机抽取10尾, 体质量(1 749±125) g。分别采集血液进行血液生理和生化指标的比较分析。结果表明: 1) 克隆牙鲆的红细胞数量(RBC)、血红蛋白数量(HGB)、平均血红蛋白量(MCH)、红细胞压积(HCT)极显著低于普通牙鲆(P<0.01), 而在其他血液生理指标上二者没有显著性差异(P>0.05); 2) 克隆牙鲆在白球比(ALB/GLB)、谷草转氨酶/谷丙转氨酶(AST/ALT)比值、谷草转氨酶(AST) 3个指标上显著高于普通牙鲆(P<0.05), 其中AST差异达到极显著水平(P<0.01), 而碱性磷酸酶(ALP)、总胆固醇(CHO)、尿素氮(BUN) 3项指标, 克隆牙鲆极显著低于普通牙鲆(P<0.01)。克隆牙鲆血液生理指标的平均变异系数为5.50%, 普通牙鲆的则为9.21%, 其中克隆牙鲆红细胞平均体积(MCV)的变异系数仅为1.52%。克隆牙鲆血液生化指标的变异系数平均为10.56%, 普通牙鲆的则为27.23%, 达到克隆牙鲆的2.58倍。结果证实, 克隆牙鲆个体间的一致性较好, 作为指标生物, 效果优于普通牙鲆等海产鱼类。

     

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).