鲍消化道及其养殖水体异养菌的耐药性研究

采用纸片扩散法对分离自福建省皱纹盘鲍(Haliotis discus hannai)消化道及其养殖水体中的284株异养细菌(其中消化道菌131株,水体菌153株)进行药物敏感测试,以期了解其耐药性概况。结果显示,皱纹盘鲍消化道与养殖水体异养菌对多种抗菌药物的耐药率差异不明显,大部分菌株对青霉素、卡那霉素、庆大霉素、利福平产生耐药性,耐药率最高达83.95%,而对诺氟沙星、恩诺沙星、氯霉素耐药率较低,受试的水体异养菌对诺氟沙星耐药率为0;不同来源菌株的多重耐药(multiple antibiotic resistance,MAR)现象普遍,消化道及其养殖水体异养菌的多重耐药率均值分别为59.15%和51.31%。该研究同时发现相同分离源的菌株对青霉素、卡那霉素、庆大霉素和利福平的耐药率呈现一定的季节波动规律,消化道异养菌于9月耐药率最低,而水体异养菌耐药率在6月最低。结果表明,皱纹盘鲍消化道及水体异养菌的多重耐药率较高,耐药状况较严重。     

对虾育苗水体副溶血性弧菌耐药性分析

为了解广东、广西沿海2010-2012年对虾育苗水体中副溶血性弧菌的耐药性变化,为育苗期对虾病害预防提供依据.研究依据GB/T4789.7-2008对各地育苗水体样品进行菌株分离、鉴定,并用纸片法进行耐药性检测.结果显示,72株在TCBS培养基上显示绿色的弧菌中检出副溶血性弧菌30株,检出率为41.7％,分离菌株对青霉素、氨苄西林、苯唑青霉素、米诺环素、阿莫西林、四环素、阿洛西林显示高度耐药,对四环素的耐药率达97％.对虾育苗水体在TCBS培养基上显示绿色的弧菌主要以副溶血性弧菌为主,所有菌株耐药性比较强,应加强育苗期抗生素使用的管理.     

乌龟鲍曼不动杆菌的分离鉴定及药敏试验

自患病乌龟肝脏中分离出1株优势菌DL01221,对其进行形态学观察、生化试验、16SrRNA基因序列分析,同时采用Kriby-Bauer纸片扩散法进行药物敏感性试验。试验结果表明,该菌为革兰氏阴性球杆菌,将该菌株16SrRNA序列进行同源性和系统进化树分析,发现它与鲍曼不动杆菌处于同一群,同源性超过98%,结合菌株形态和生化指标,确定该菌株为鲍曼不动杆菌。致病性试验结果显示,其对乌龟具有较强的致病作用;耐药性结果显示其对头孢他啶、卡那霉素及氟罗沙星敏感,对其他多种抗生素耐药。     

副溶血弧菌质粒图谱及其抗药性研究

对112株副溶血弧菌提取弧菌质粒。质粒DNA样品经琼脂糖凝胶电泳检测,结果显示,在这112株弧菌中,有101株菌至少含有一条质粒,而另外11株菌中未检测到质粒,质粒的存在率高达90.18%。分析质粒电泳图谱发现,大部分副溶血弧菌仅携带一种质粒,占86.14%。30种抗生素的MIC测定结果显示,大部分菌株对青霉素类、氨基苷类、四环素类药物具有抗性,抗药率为87%～100%。另有些菌株对头孢菌素类、磺胺类抗菌药、氯霉素类等药物具有敏感性,其中以氯霉素最好,敏感率为84%。     

美洲鳗鲡及其养殖水体分离耐药菌的多样性和耐药性分析

为了更有针对性地防控水产动物细菌性病害的发生和流行,本实验对美洲鳗鲡及其养殖水体耐药细菌的种属特征及耐药情况开展了相关研究。首先采集美洲鳗鲡不同部位(表皮、鳃、肠道)及其养殖水体的样品,经5种抗菌药物平板筛选耐药菌株,然后采用K-B纸片扩散法检测细菌对抗菌药物的耐药性,同时测定耐药菌株的16S rDNA序列,进而分析耐药菌的种属分布和多重耐药性。结果显示,经耐药平板筛选分离纯化得到108株细菌,分别属于气单胞菌属、柠檬酸杆菌属、不动杆菌属等20个属;其中,93.5%的菌株对3种(含)以上的抗菌药物具有耐药性,86.1%的菌株对3类(含)以上的药物具有抗性。对阿莫西林的耐药率高达90.7%,对四环素、利福平以及磺胺类和酰胺醇类药物类的耐药率为60%~80%,对头孢噻肟、新霉素以及喹诺酮类的耐药性弱(低于20%)。美洲鳗鲡肠道(0.40)、表皮(0.41)、鳃部(0.42)及水样(0.47)菌群的多重耐药指数显示各生态样品耐药程度较为严重,尤以水样为最。各菌属中,柠檬酸杆菌属(0.58)和克雷伯菌属(0.61)的多重耐药指数最高,而不动杆菌属(0.21)则相对较低。美洲鳗鲡及养殖水体普遍存在多重耐药菌株,对此应引起足够的重视;水产动物及养殖环境耐药细菌对某些水产用药如诺氟沙星、新霉素等耐药率低,可将其做为水产动物细菌性疾病治疗的首选药物。     

猪链球菌表型耐药观察

为掌握当前猪链球菌耐药性情况,实验对分离鉴定出的81株链球菌菌株选用12种抗生素进行药敏试验。试验结果表明,链球菌对各种常规药物敏感率普遍较低,大多数菌株对先锋V(85.1%)和利福平(81.4%)较敏感;其次是环丙沙星(66.6%)和恩诺沙星(62.6%);青霉素、红霉素、庆大霉素和阿莫西林的敏感率则在45%左右;对克林霉素、林可霉素、强力素和四环素等敏感率极低,而且同种药物对不同来源菌株的敏感性都不相同。和之前试验进行比较发现:链球菌耐药谱变化快,如先锋V、利福平其敏感率有所下降;部分抗生素阿莫西林、红霉素和青霉素其敏感率则有所上升。     

养殖大菱鲆(Scophthalmus maximus)腹水病的病原多样性及其耐药性分析

为了解引起养殖大菱鲆(Scophthalmus maximus)腹水病的病原多样性及其耐药性情况,针对2002–2010年由不同地区病样分离的27株细菌性病原进行了16S r DNA鉴定,并采用K-B法测定了27株细菌对22种抗生素的耐药性,分析了病原菌的耐药谱及耐药率变化。结果显示,大菱鲆腹水病病原菌主要有大菱鲆弧菌(Vibrio scophthalmi)、迟钝爱德华氏菌(Edwardsiella tarda)、鳗弧菌(Vibrio anguillarum)、哈维氏弧菌(Vibrio harveyi)、假交替单胞菌(Pseudoalteromonas espejiana)。山东青岛地区以大菱鲆弧菌为主,威海地区以迟钝爱德华氏菌为主,烟台地区菌株种类分布平均。5类细菌对青霉素类、头孢菌素类、大环内酯类、复方新诺明耐药率高于50%。只有1株迟钝爱德华氏菌对氟苯尼考产生了耐药,其余菌株对其均没有耐药性,且在长期使用中不易产生耐药性,证实氟苯尼考为当前防治腹水病的一种良好抗菌药物。27株病原菌的耐药谱数量为27个,每个菌株具备自己独特的耐药谱,74.1%的菌株对10种以上的抗菌药物产生了耐药性,均有多重耐药性。     

七彩神仙鱼肠炎病原菌的分离鉴定及药敏试验

为确定七彩神仙鱼(Symphysodon aequifasciatus)肠炎的致病菌并筛选敏感药物,利用传统培养法,从该鱼的肠道组织中分离出优势菌,通过生理生化试验及16S r DNA序列测序比对确定菌种信息,并使用药敏纸片法筛选致病菌的敏感药物。试验结果表明:优势菌株QC3为肠型点状气单胞菌(Aeromonas punctata),是七彩神仙鱼肠炎的致病菌株,此菌对氟苯尼考、强力霉素、庆大霉素、诺氟沙星高度敏感,对链霉素中度敏感,对土霉素、红霉素、青霉素、头孢唑林、氨苄西林表现出耐药性。根据研究结果,建议使用氟苯尼考、诺氟沙星、强力霉素、庆大霉素等药物对七彩神仙鱼肠炎病进行治疗。     

广西黄颡鱼源嗜水气单胞菌及其致病性、耐药性调查

以常规方法分离细菌,API 20NE进行细菌鉴定,人工感染检测细菌的致病性,K-B纸片扩散法进行耐药性试验,对梧州市等3个不同养殖区域黄颡鱼源嗜水气单胞菌及其致病性、耐药性进行调查。结果显示,从广西梧州等3个市/县网箱养殖的患病黄颡鱼(Pseudobagrus fulvidrico)分离到17株嗜水气单胞菌,总检出率41.46%,永福县的检出率最高(76.92%),桂平市次之(26.67%),梧州市最低(23.08%)。除YF13无致病力外,其余16株菌株对健康黄颡鱼的平均致死率70.00%~100%。抑菌效果最好的药物有环丙沙星、左氟沙星和盐酸沙拉沙星3种,抑菌效果最差的是青霉素G和氨苄青霉素。高度敏感的药物梧州市有9种,永福县10种,桂平市12种;不敏感药物梧州市6种,永福县3种,桂平市2种。对3个养殖区域黄颡鱼源嗜水气单胞菌都高度敏感的药物有头孢他啶等8种,都不敏感的是青霉素G和氨苄青霉素2种。     

湛江水产品中大肠埃希氏菌耐药性检测与分析

采用K-B纸片扩散法,对从近江牡蛎、翡翠贻贝、菲律宾蛤仔、文蛤、华贵类栉孔扇贝等鲜活样品和罗非鱼片、熟虾等冷冻样品中分离的52株大肠埃希氏菌菌株进行青霉素类、喹诺酮类、单环内酰胺类和磺胺类等19种抗菌药物的药敏性分析,同时PCR扩增检测超广谱β-内酰胺酶(ESBLs)、质粒介导喹诺酮耐药(PMQR)基因,以检测湛江水产品中大肠埃希氏菌的耐药性及ESBLs、PMQR基因的携带情况。结果显示,52株大肠埃希氏菌菌株均对1种以上抗菌药物产生耐药性,其中对红霉素(90.4%)、复方新诺明(55.8%)、四环素(53.8%)、头孢噻肟(51.9%)的耐药率较高;76.9%的菌株表现出多重耐药,最多对9大类抗生素耐药;确证为产ESBLs菌株10株(19.2%),8株携带blaTEM基因,7株携带blaSHV基因,未检出blaCTX和blaOXA基因;检出PMQR菌株15株,检出率为28.8%,其中15株同时携带qnrA、aaac(6′)-Ib-c、qepA 3种耐药基因,9株携带qnrS基因,8株携带oqxB基因,1株携带qnrD基因,未检出qnrB、qnrC和oqxA耐药基因;2株大肠埃希氏菌同时携带ESBLs基因和PMQR基因。研究结果表明,湛江地区水产品中大肠埃希氏菌普遍耐药,且耐药谱广。菌株同时携带ESBLs基因和PMQR基因,有增加细菌耐药性的可能。     

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.     

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).     

Imported ornamental fish are colonized with antibiotic‐resistant bacteria

There has been growing concern about the overuse of antibiotics in the ornamental fish industry and its possible effect on the increasing drug resistance in both commensal and pathogenic organisms in these fish. The aim of this study was to carry out an assessment of the diversity of bacteria, including pathogens, in ornamental fish species imported into North America and to assess their antibiotic resistance. Kidney samples were collected from 32 freshwater ornamental fish of various species, which arrived to an importing facility in Portland, Oregon from Colombia, Singapore and Florida. Sixty‐four unique bacterial colonies were isolated and identified by PCR using bacterial 16S primers and DNA sequencing. Multiple isolates were identified as bacteria with potential to cause disease in both fish and humans. The antibiotic resistance profile of each isolate was performed for nine different antibiotics. Among them, cefotaxime (16% resistance among isolates) was the antibiotic associated with more activity, while the least active was tetracycline (77% resistant). Knowing information about the diversity of bacteria in imported ornamental fish, as well as the resistance profiles for the bacteria will be useful in more effectively treating clinical infected fish, and also potential zoonoses in the future.     

Effect of iodophor disinfection of non‐hardened Salmo trutta eggs on their bacterial and fungus load

This study investigated the efficiency of iodophor disinfection (135 ppm active iodine for 15–30 min) of non‐hardened Salmo trutta eggs against different groups of bacteria and against fungus. Egg samples were taken from non‐disinfected and from disinfected eggs, microorganisms were cultured on specific nutrient media and their mass was measured by turbidimetric methods. Bacteria and fungus mass of non‐hardened eggs could be reduced but not eliminated by iodophor disinfection with 135 ppm active iodine for 15 min. The extent of reduction was 47–65% (Experiment 1). The efficiency of disinfection increased with disinfection time as the reduction in bacteria and fungus mass was 40–55% after 15 min and 58–74% after 30 min (Experiment 2). Disinfection efficiency of iodophor solution diluted in water (reduction 49–57%) and of iodophor solution diluted in sodium chloride solution iso‐osmolar to the oocytes (reduction 52–61%) was similar (Experiment 3). The reduction in bacteria and fungus mass was persistent as it was 39–72% lower in embryos deriving from disinfected eggs than in embryos deriving from non‐disinfected ones (Experiment 4). In conclusion, the tested disinfection method is inadequate to eliminate pathogens completely but it could positively influence immune defence of eggs and embryos.     

Influence of the antibacterial herbs, Solanum trilobatum, Andrographis paniculata and Psoralea corylifolia on the survival, growth and bacterial load of Penaeus monodon post larvae

The indiscriminate use of antibiotics and chemicals in shrimp hatcheries has led to biomagnification and that in turn could lead to rejection of a whole consignment. The application of the bioencapsulation technique as a tool for curative treatment in shrimp larvae was investigated. Herbs having antibacterial properties such as Solanum trilobatum, Andrographis paniculata and Psoralea corylifolia (methanolic extracts) were bioencapsulated in Artemia and fed to Penaeus monodon post larvae PL 1–25. The post larvae were reared in a medium inoculated with pathogenic bacteria such as Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhi and Vibrio sp. Post larvae reared in the non-inoculated water and fed with non-enriched Artemia exhibited 90% survival, highest specific growth rate (12.43%) and reduced bacterial load. P. monodon reared in the bacterial inoculated water and fed with the non-enriched Artemia exhibited the lowest survival (10–30%), specific growth rate (8.42–9.1%) and increased bacterial load (2.86 × 10³ to 3.76 × 10⁵ cfu/g). The methanolic extracts of the herbs helped to increase survival and specific growth rate and reduced bacterial load in the P. monodon culture system. Among the three herbal extracts, P. corylifolia enriched Artemia fed post larvae showed the tendency to higher survival (>50%), growth rate (11.5 averaged) and low bacterial load (1.12 × 10⁵ cfu/g). This revised version was published online in August 2006 with corrections to the Cover Date.     

Effects of cortisol on hepatic carbohydrate metabolism and responsiveness to hormones in the sea raven,Hemitripterus americanus

The sea raven, Hemitripterus americanus, is a sit-and-wait, low metabolic rate, marine teleost. The objective of this study was to determine i) whether cortisol implantation (50 mg. kg^-1) for 7 days altered hepatocyte metabolism, and hepatocyte responsiveness to epinephrine, glucagon and insulin, and ii) whether 8 weeks of food-deprivation modified the above response. Cortisol implantation significantly increased hepatocyte total glucose production and oxidation from alanine compared to the sham group. There was no cortisol effect on glycogen breakdown, suggesting that the activation of other pathways, including gluconeogenesis, are required to account for the increased glucose production. Epinephrine-mediated (10^-5M) glycogen breakdown and insulin-mediated (10^-8M) total glucose production were enhanced in hepatocytes of cortisol implanted sea ravens, but there were no change in any glucagon (10^-7M) effects. The enhanced glycogen breakdown in the absence of similar increases in total glucose production with epinephrine indicates mobilization of carbohydrate reserves for endogenous use by the liver. Food-deprivation for 8 weeks significantly decreased condition factor, plasma cortisol concentration and liver glycogen content in the sea raven, but had no effect on plasma glucose concentration. Hepatocyte total glucose production and flux rates from alanine increased significantly with food-deprivation. Moreover, food-deprivation increased responsiveness of total hepatocyte glucose production to the actions of glucagon and insulin, but not to epinephrine; none of these effects were modified by cortisol implantation. Our results indicate that cortisol in the sea raven exerts both a direct and an indirect or permissive effect on hepatocyte metabolism by modifying hepatocyte responsiveness to epinephrine and insulin stimulation. Cortisol implantation did not modify the effects of glucagon or food-deprivation in this species.     

Simulation and quantification of enrichment and retention processes in the southern Benguela upwelling ecosystem

Important environmental processes for the survival and recruitment of early life stages of pelagic fishes have been synthesized through Bakun's fundamental triad as enrichment, concentration and retention processes (A. Bakun, 1996, Patterns in the Ocean. Ocean Processes and Marine Population Dynamics. San Diego, CA, USA: University of California Sea Grant). This conceptual framework states that from favourable spawning habitats, eggs and larvae would be transported to and/or retained in places where food originating from enrichment areas would be concentrated. We propose a method for quantifying two of the triad processes, enrichment and retention, based on the Lagrangian tracking of particles transported within water velocity fields generated by a three‐dimensional hydrodynamic model. We apply this method to the southern Benguela upwelling ecosystem, constructing putative maps of enrichment and retention. We comment on these maps regarding main features of the circulation in the region, and investigate seasonal variability of the processes. We finally discuss the results in relation to available knowledge on the reproductive strategies of two pelagic clupeoid species abundant in the southern Benguela, anchovy (Engraulis encrasicolus) and sardine (Sardinops sagax). Our approach is intended to be sufficiently generic so as to allow its application to other upwelling systems.     

Effects of capture,loading density and transport stress on the mortality,physiological responses,bacterial density and growth of rohu <Emphasis Type="Italic">Labeo</Emphasis><Emphasis Type="Italic"> rohita</Emphasis> fingerlings

This study evaluated the effects of three levels of loading density (200, 300 and 400 g L⁻¹) and four levels of duration (1, 3, 6 and 9 h) on the mortality, plasma cortisol and chloride levels and growth of rohu Labeo rohita fingerlings in a transport simulation. While total immediate mortality was low (4–12%), delayed mortality was high (27–49%). Initial capture and handling were found to constitute the most stressful phase as indicated by elevated plasma cortisol. The levels of cortisol continued to rise at 6 and 9 h after transport, with the highest level observed at 9 h after transport. The level of plasma chloride fell after transport across all density levels and treatment durations. Specific growth rate (SGR, %/day) and total bacterial density (cfu/g) were not different between and within treatments, and between treatments and the controls. This study demonstrates that transport has a profound effect on the stress responses of rohu fingerlings. Although the level of immediate mortality was low, the higher level of delayed mortality has serious implications in terms of production inefficiency for grow-out farmers.     

The effects of introduced tilapias on native biodiversity

• 1. The common name ‘tilapia’ refers to a group of tropical freshwater fish in the family Cichlidae (Oreochromis, Tilapia, and Sarotherodon spp.) that are indigenous to Africa and the southwestern Middle East. Since the 1930s, tilapias have been intentionally dispersed worldwide for the biological control of aquatic weeds and insects, as baitfish for certain capture fisheries, for aquaria, and as a food fish. They have most recently been promoted as an important source of protein that could provide food security for developing countries without the environmental problems associated with terrestrial agriculture. In addition, market demand for tilapia in developed countries such as the United States is growing rapidly.
• 2. Tilapias are well‐suited to aquaculture because they are highly prolific and tolerant to a range of environmental conditions. They have come to be known as the ‘aquatic chicken’ because of their potential as an affordable, high‐yield source of protein that can be easily raised in a range of environments — from subsistence or ‘backyard’ units to intensive fish hatcheries. In some countries, particularly in Asia, nearly all of the introduced tilapias produced are consumed domestically; tilapias have contributed to basic food security for such societies.
• 3. This review indicates that tilapia species are highly invasive and exist under feral conditions in every nation in which they have been cultured or introduced. Thus, the authors have concluded that, despite potential or observed benefits to human society, tilapia aquaculture and open‐water introductions cannot continue unchecked without further exacerbating damage to native fish species and biodiversity. Recommendations include restricting tilapia culture to carefully managed, contained ponds, although exclusion is preferred when it is feasible. Research into culture of indigenous species is also recommended.

Copyright © 2005 John Wiley & Sons, Ltd.