The Lethal Dose of Largemouth Bass Virus in Juvenile Largemouth Bass and the Comparative Susceptibility of Striped Bass

Abstract

Largemouth bass virus (LMBV) is an iridovirus that was isolated from wild adult largemouth bass Micropterus salmoides in the southeastern United States in 1994. Although originally isolated from moribund wild fish, its virulence to juvenile largemouth bass is uncertain. To help clarify this point, two LMBV titrations were made in juvenile largemouth bass. Titers of LMBV in fathead minnow cells were 10^4.8 and 10^5.8 tissue culture infectious doses—50% cytopathic endpoint (TCID50) per milliliter, respectively. Tenfold serial dilutions of LMBV employed in each cell culture titration, injected intraperitoneally (0.1 mL/fish) into largemouth bass produced calculated lethal dose—50% mortality endpoints (LD50s) of 282 (10^2.45) and 288 (10^2.46) infectious doses in two consecutive infectivity trials. Virus yield of assayed infected fish averaged 10^8.5 TCID50/g and 10^7.7 TCID50/g in viscera of moribund and dead fish in the two trials and 10^6.5 TCID50/g in surviving exposed fish 14 d after infection. In a second experiment, largemouth bass had 100% mortality 5 d after injection while virus immersed fish had a significantly (P ≤ 0.005) lower mortality of 17% at 14 d. Similarly treated juvenile striped bass Morone saxatilis suffered 63% mortality after injection and significantly (P ≤ 0.005) lower mortality of 10% after immersion. In a third study of 25 d, 100% of injected largemouth bass died by 5 d after injection, and all of them were virus-positive. Injected striped bass had a significantly (P ≤ 0.005) lower mortality of 24%; all three fish were virus-positive initially, two fish were virus-positive at 18 d, and none were positive at 25 d. Juvenile largemouth bass were highly susceptible to LMBV injection and striped bass were moderately susceptible, but both species were only mildly susceptible when exposed by immersion.     

Ultrastructure of the Spleen and Head Kidney of Striped Bass

Abstract

The spleen and head kidney of striped bass Morone saxatilis were examined by electron microscopy, including examination of ultrastructural localization of peroxidase. The spleen is composed of a loosely organized reticulum that supports the red and white pulp. The principal hematopoietic and mature blood cells observed were erythrocytes, erythroblasts, lymphocytes, macrophages, thrombocytes, and aggregates of pigmented macrophages. In addition, limited numbers of neutrophils, neutrophilic myelocytes (immature neutrophils), plasma cells, eosinophils and monocytes were identified. Reticular cells and fibers appeared to provide a structural framework for both cells and blood sinuses in the spleen. Like the spleen, the head kidney contained erythrocytes, macrophages, monocytes, lymphocytes, and other less frequently observed cell types. A distinct difference in the cytologic composition of the two organs, however, was in the greater number of neutrophilic myelocytes found in the head kidney, where these cells were often arranged in groups. Positive peroxidase reactions were observed for neutrophils, neutrophilic myelocytes, macrophage phagolysosomes, and pigmented macrophages in both the spleen and head kidney.     

Sinus Venosus Catheterization for Repeated Vascular Access in the Hybrid Striped Bass

Abstract

Placement of vascular cannulae for repeated venous blood sampling has proven to be a useful technique in many fish species. The anatomy and size of the hybrid striped bass (striped bass Morone saxatilis ♀ × M. chrysops ♂) makes this procedure challenging in this species. The sinus venosus was determined to be the best site for catheter placement based on size, accessibility, and ability to stabilize the catheter within it. Catheterization of the sinus venosus with a 20-gauge × 3.8-cm flexible Teflon catheter was unsuccessful because the catheter folded on itself, occluding the lumen. Catheterization with an 18-gauge × 3.8-cm hypodermic needle was also unsuccessful because the sinus wall occluded the needle lumen when negative pressure was applied. However, 18-gauge × 3.8-cm intravascular needles remained patent in free-swimming fish for 2 weeks without major complications. An external fixation device to prevent migration of the catheter out of the sinus venosus and laceration of the sinus wall during normal swimming movements improved the technique.     

Communications: Protective Immunity Induced by a Commercial Vibrio Vaccine in Hybrid Striped Bass

Abstract

Commercial Vibrio anguiliarum-V. ordalii bacterin was used to vaccinate hybrid striped bass (Morone saxatilis ♀ × M. chrysops ♂) to test the vaccine efficacy against vibriosis. Vaccination by direct immersion of fish in diluted Vibrio vaccine for 20 s resulted in increased protective immunity. The relative percent survival of hybrid striped bass challenged 35 d after vaccination was 66.7% for those challenged by 1-h immersion exposure to 7.03 × 10⁷ V. anguillarum cells/mL, 75.0% for those challenged by injection with 3.51 × 10⁵ cells/fish, and 86.7% for those challenged by injection with 3.51 × 10⁴ cells/fish.     

甲砜霉素在鸡肌肉中的残留消除规律

为研究甲砜霉素在鸡肌肉中的残留消除规律,鸡肌肉样品经丙酮、二氯甲烷提取,饱和正己烷脱脂,氮吹仪吹干浓缩后用流动相溶解,上高效液相色谱荧光检测器检测。测定鸡肌肉样品中甲砜霉素的检测限为1.5μg.kg-1(S/N=3)、定量限为5μg.kg-1(S/N=10)。各试验组鸡分别按体质量以10.0、20.0和50.0mg.(kg.d)-1剂量给药,每天1次,连续5d内服给药后,鸡肉中甲砜霉素残留量随给药剂量增大呈上升趋势,随休药期的延长而降低。休药第1天时,鸡肉中甲砜霉素残留量均达到峰值。休药前期甲砜霉素残留消除较快,后期消除缓慢。休药第5天时,鸡肉中甲砜霉素残留量均低于50μg.kg-1,休药第9天时,鸡肌肉中甲砜霉素残留量均低于检测限(1.5μg.kg-1);甲砜霉素在鸡肌肉中的残留量与给药剂量呈正相关。     

盐酸土霉素可溶性粉在肉鸡组织中的残留消除研究

在常规养殖环境中,对不同日龄肉鸡分别给予250 mg/L和333 mg/L 50%盐酸土霉素可溶性粉,集中饮水给药,每日1次,连续5 d。停药后4 h,1 d,3 d,5 d,6 d,7 d,8 d,9 d,10 d宰杀肉鸡,采集可食性组织(肌肉、肝脏和鸡胗)样品,肌肉、肝脏和鸡胗组织中的残留量采用UPLC-MS/MS测定。结果表明:土霉素主要残留在肝脏组织中;肌肉、肝脏和鸡胗组织的消除速率相似;停药5 d后,两个剂量组的肌肉、肝脏中土霉素残留量均低于最高残留限量(MRL)。休药期计算软件WTl.4结果显示,上述两种方法给药后,建议休药期分别为7、8 d。     

Nested Polymerase Chain Reaction Assay for Detection of Mycobacterium shottsii and M. pseudoshottsii in Striped Bass

Abstract

Wild striped bass Morone saxatilis in Chesapeake Bay are experiencing a high prevalence of mycobacteriosis, which produces granulomatous lesions of the skin and visceral organs. Culture-based studies have indicated that the newly described species Mycobacterium shottsii and M. pseudoshottsii are the dominant isolates from diseased fish. The classical fish pathogen M. marinum is also found, albeit at much lower frequencies. Both M. shottsii and M. pseudoshottsii are extremely slow-growing on standard selective media, and up to 12 months may be required for isolation and characterization. Epidemiological studies of mycobacteriosis in Chesapeake Bay would therefore benefit from rapid molecular assays with which to detect these species in fish. In this paper, we describe the development of polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) assays capable of detecting M. shottsii, M. pseudoshottsii, and, in most instances, coinfections thereof in striped bass tissues. In addition, PCR–RFLP assays were designed to detect M. marinum and other as-yet-undescribed Mycobacterium spp. present in Chesapeake Bay striped bass. Comparison of these molecular assays with culture-based techniques using splenic tissue from wild striped bass yielded generally concordant results and demonstrated the applicability of these techniques to the study of wild fish.     

蜜蜂采蜜期间链霉素和土霉素休药期的初步研究

以竞争酶联免疫分析手段,通过选择养蜂生产中常用的链霉素和土霉素作为试验代表药物,研究蜂蜜中药物残留消除情况,探讨药物的休药期,以找到合适的给药途径和休药期。试验结果显示,链霉素在给药后33 d,土霉素在给药后43 d,两者在蜂蜜中的残留量分别是5.67、7.77μg/kg,已经符合欧盟、日本及美国等进口方最严要求(≤10μg/kg)。     

氟苯尼考及其代谢物氟苯尼考胺在鸡肌肉中的残留消除规律

本试验旨在研究氟苯尼考及其代谢物氟苯尼考胺在鸡肌肉中的残留消除规律。鸡肌肉样品经丙酮、二氯甲烷提取,饱和正己烷脱脂,氮吹仪吹干浓缩后,以乙腈-磷酸二氢钠溶液(0.01mol.L-1,含0.005mol.L-1十二烷基硫酸钠和0.1%三乙胺)(体积比35∶65)为流动相,流速为1.0mL.min-1,在激发波长225nm、发射波长285nm处用高效液相色谱荧光检测器检测。结果:测得鸡肌肉中氟苯尼考、氟苯尼考胺的检测限分别为1.5和0.5μg.kg-1(S/N=3)、定量限分别为5和2μg.kg-1(S/N=10)。各试验组京海黄鸡分别按体质量以25.0、50.0和100.0mg.kg-1.d-1剂量给药,每天1次,连续5d内服给药后,休药第1天时,鸡肌肉中氟苯尼考及其代谢物氟苯尼考胺的残留量均达到峰值,且残留量随给药剂量的增大而增大,随休药期的延长而降低。休药第7天时,鸡肌肉中氟苯尼考及其代谢产物氟苯尼考胺的总残留量均低于MRLs(100μg.kg-1);休药第11天时,鸡肌肉中氟苯尼考及其代谢产物氟苯尼考胺的总残留量均低于检测限;氟苯尼考、氟苯尼考胺在鸡肌肉中的残留量及其总残留量均与给药剂量呈正相关。     

猪肉中氯霉素残留量测定能力验证分析

为了解我国食品检测实验室的氯霉素残留检测能力,CNAS于2012年委托中国兽医药品监察所组织实施了猪肉中氯霉素残留量测定的能力验证工作.全国21个省、市、自治区的66家实验室参加了本次能力验证,采用的测试方法主要是液相色谱-串联质谱法和气相色谱-质谱法.结果显示:参加实验室满意结果率为80.3％,可疑结果率为9.09％,不满意结果率为10.6％,说明参加能力验证的绝大多数实验室可以准确检测猪肉中氯霉素残留情况.     

鸡蛋和鸡肉中尼卡巴嗪残留检测高效液相色谱 串联质谱法研究

建立了鸡蛋和鸡肉中尼卡巴嗪残留标示物4,4'-二硝基碳酰替苯胺(DNC)检测的高效液相色谱-串联质谱方法(HPLC-MS/MS).液相色谱条件为色谱柱为XBridge C18柱(150 mm×2.1 mm,5 μm),流动相为甲醇 0.1 mol/L的乙酸铵水溶液(75 25,V/V),柱温30 ℃,流速0.2 mL/min,进样量20 μL.质谱条件为电喷雾离子源(ESI-),多反应监测(MRM)方式进行采集;DNC-D8同位素内标法定量.结果表明,DNC在10～500 ng/mL浓度范围内呈现良好的线性关系,R2为0.999 7;鸡蛋和鸡肉中DNC残留检测方法检测限均为0.5 ng/g,定量限均为1 ng/g;鸡蛋中从2、5和10 ng/g三个添加浓度检测结果可以看出,方法平均回收率为95.9%～102.2%,批内RSD为0.9%～4.4%,批间RSD为0.3%～2.1%.鸡肉组织从100、200和300 ng/g三个添加浓度检测结果可以看出,方法平均回收率为94.0%～103.3%,批内RSD为2.0%～6.3%,批间RSD为3.1%～5.2%.     

紫外分光光度法和高效液相色谱法测定盐酸土霉素可溶性粉的含量

采用紫外分光光度法和高效液相色谱法测定盐酸土霉素可溶性粉中土霉素的含量。紫外分光光度法试验表明,用0.01 mol/L盐酸溶液作参比液,以268 nm为测定波长,土霉素在4~32μg/mL范围内吸光度与浓度呈良好的线性关系,相关系数r=0.9999,平均回收率(n=5)为101.04%,RSD=1.02%。高效液相色谱法试验表明,以0.05 mol/L草酸铵溶液-二甲基甲酰胺-0.2 mol/L磷酸氢二铵溶液(75∶20∶5)为流动相,以280 nm为检测波长,土霉素在0.02~0.14mg/mL范围内浓度与峰面积线性关系良好,相关系数r=0.9999,平均回收率(n=5)为100.52%,RSD=0.25%。紫外分光光度法、高效液相色谱法与抗生素微生物检定法比较,结果基本一致。     

Differences between Plasma and Serum Samples for the Evaluation of Blood Chemistry Values in Rainbow Trout,Channel Catfish,Hybrid Tilapias,and Hybrid Striped Bass

Abstract

Blood chemistry analytes are determined for fish from either serum or plasma samples. Serum and plasma are similar in that they both represent the fluid component of blood; however, plasma contains clotting factors that are not present in serum. This study was conducted to determine whether the type of sample—plasma or serum—had an effect on measured blood analytes in rainbow trout Oncorhynchus mykiss, channel catfish Ictalurus punctatus, hybrid tilapia Oreochromis spp., and hybrid striped bass (striped bass Morone saxatilis × white bass M. chrysops). Paired plasma and serum samples were analyzed for the following standard biochemical analytes: total protein, albumin, globulin, creatinine, total bilirubin, alkaline phosphatase, aspartate aminotransferase, sodium, potassium, chloride, calcium magnesium, phosphorus, glucose, and cholesterol. For all four taxa, values for potassium were lower in the serum and magnesium and phosphorus values were higher in the serum. Glucose values were lower in the serum from rainbow trout, hybrid striped bass, and channel catfish; whereas cholesterol values were higher in the serum of rainbow trout, channel catfish, and hybrid tilapias. The differences observed between serum and plasma were distinct from changes occurring with hemolysis and, therefore, do not represent release of erythrocyte constituents. The differences most likely represent metabolic utilization of blood constituents while the blood was clotting. This work indicates that plasma should be used preferentially to serum for biochemical analysis because analyte levels determined from serum may not accurately reflect those found in circulating blood.     

金霉素和代谢物差向金霉素在肉鸡肌肉和肝脏中的残留分布

为了研究金霉素(CTC)、差向金霉素(ECTC)在肉鸡肌肉、肝脏中的残留分布情况,样品经提取液提取后过Oasis HLB SPE小柱净化,采用电喷雾正离子模式(ESI+)和多反应监测模式(MRM),用高效液相色谱-串联质谱仪检测;试验组鸡按体重以50 mg/(kg·d)剂量内服CTC,连续5 d,每天给药1次。结果表明:该方法测定肉鸡肌肉和肝脏中CTC检测限和定量限分别为5.0、10.0μg/kg。ECTC检测限和定量限分别为20.0、30.0μg/kg。CTC和ECTC在肉鸡肌肉中平均回收率分别在73.0%~95.20%和81.80%~101.60%,相对标准偏差(RSD)分别在8.85%~12.18%和2.05%~9.29%;在肉鸡肝脏中平均回收率分别在67.06%~90.80%和78.64%~106.98%,RSD分别在2.90%~8.88%和4.72%~9.48%;肉鸡肌肉和肝脏中CTC、代谢物ECTC的残留量分别在给药后第1、5 d时达到最高。肉鸡肝脏中ECTC的残留量约占总残留量的1/2。停药后CTC及其代谢物ECTC代谢缓慢,在停药第6 d时,仍能检测到CTC及其代谢物ECTC,但其残留量均低于最高残留限量(MRLs)。从保障食品安全方面考虑,建议我国将CTC和ECTC均作为金霉素的残留标示物进行监测。     

长效土霉素注射液在猪体内的生物等效性研究

研究自制长效土霉素注射液与进口长效土霉素注射液(得米先注射液)在猪体内单剂量注射的药代动力学和生物等效性,为临床应用提供参考和依据.采用双处理、双周期随即交叉试验设计.12头健康猪分为两组,分别先后单剂量肌肉注射自制长效土霉素注射液与得米先注射液,用高效液相色谱法测定血浆中土霉素的浓度,3P97软件计算药代动力学参数.自制长效土霉素注射液与参比制剂的主要药动学参数如下:AUC分别为(145.41 ±3.59)、(159.28 ±4.47) μg·mL-1·h,T1/2β分别为(84.776±2.21)、(72.740±1.95)h,Cmax分别为(3.519±1.66)、(3.924±1.34)μg/mL,Tmax分别为(1.848±0.54)、(1.760±0.41)h.采用3P97药代动力学软件分析比较AUC和Cmx90％置信区间,自制长效土霉素注射液的AUC、Cmax的90％置信区间分别为94.7％～105.2％、85.0％ ～114.6％,分别落在参比制剂的80％～125％、70％ ～143％范围内,两组参数差异不显著(P＞0.05),结果表明二者具有生物等效性.     

土霉素在鸡体内的生理房室模型研究

为预测土霉素在鸡体内的药动学特点,建立了土霉素在鸡体内的血流限速生理房室模型。结果显示,土霉素在鸡体内的药动学参数:Tmax(达峰时间)为2.22h,Cmax(峰浓度)为0.62μg/mL,AUC(药时曲线下面积)为7.61(μg/mL)×h,Ka(吸收速率常数)为1.21h-1,Ke(消除速率常数)为0.10h-1,T1/2Ka(吸收半衰期)为0.57h,T1/2Ke(消除半衰期)为6.73h,V(表观分布容积)为6.38L/kg,CL(血浆清除率)为0.66L/h·kg。其结果表明,土霉素在鸡体内的药动学特点为:吸收迅速,分布广泛,消除缓慢。因此,运用生理房室模型可以预测药物的药动学参数。