Effect of Temperature and Dissolved Oxygen Concentration on Edwardsiella ictaluri in Experimentally Infected Channel Catfish

Abstract

Channel catfish Ictalurus punctatus were experimentally infected with Edwardsiella ictaluri by immersion exposure. After clinical disease ran its course for 52 d, the surviving fish were exposed to one of the following environmental regimes in troughs: 25°C with aeration, 25°C with no aeration, or variable temperature (18–23°C) with no aeration. After 29 d of exposure to the environmental regimes, various organs and tissues of the fish were assayed to determine the effects of these conditions on E. ictaluri concentrations (colony-forming units/mL of tissue sample). The concentrations of this pathogen were significantly (P < 0.05) higher in all tissues (trunk kidney, liver, head kidney, blood, spleen, gallbladder, muscle, brain, and gonad) 52 d postinfection than 29 d after exposure to any of the environmental regimes (81 d postinfection). Fish exposed to a near-normal concentration of dissolved oxygen (6.4 mg/L) and a constant temperature of 25°C had E. ictaluri concentrations that were significantly (P < 0.01) lower than those offish exposed to a low oxygen concentration (2.6 or 1.8 mg/L) and either a constant or a variable temperature.     

Isolation and Partial Characterization of Extracellular Proteases Produced by Isolates of Flavobacterium columnare Derived from Channel Catfish

Abstract

Proteases of 23 isolates of Flavobacterium columnare derived primarily from channel catfish Ictalurus punctatus raised in the southeastern United States were isolated and partially characterized. The bacterial isolates were divided into two groups according to the apparent molecular masses of proteases after zymographic resolution by nonreducing, nondenaturing sodium dodccyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) with gelatin as the protease substrate. The 15 isolates in group 1 had two proteases with apparent molecular masses of 58 and 53.5 kilodaltons (kDa). Eight group-2 isolates produced three proteases with apparent molecular masses of 59.5, 48, and 44.5 kDa. Culture medium had an effect on the amount of protease produced by F. columnare LA 88–173. More protease was produced in a medium with low nutrients and salt (Ordal's medium) than in media with higher concentrations of nutrients or salts (TYES, Hsu-Shotts, modified Shieh's media). No differences were observed in the apparent molecular masses of the two proteases of F. columnare LA 88–173 produced in the various media or with different incubation times. Two proteases with apparent molecular masses of 58 and 53.5 kDa were seen as early as I d after inoculation, and these molecular masses did not change during the 7-d experiment. A sharp increase in protease production occurred during the first 24 h of incubation with minimal increase during the remaining 7 d of the experiment. All 23 isolates of F. columnare degraded the gelatin and casein incorporated into TYES agar medium but only 7 of the 23 isolates degraded elastin.     

斑点叉尾(鱼回)疑似疱疹病毒感染的病理形态学观察

对四川省一些养殖场斑点又尾鮰大规模死亡的病鱼组织进行了光镜和电镜观察．在其肾和肝组织的细胞中发现一种圆形或椭圆形病毒颗粒，有囊膜的病毒颗粒存在于胞浆中，直径150～200nm．无囊膜的病毒颗粒既存在于胞浆中，也存在于胞棱中，直径为80～110nm，同时在细胞核内可见无病毒核心的空衣壳，根据其形态特征初步确定为一种疱疹病毒。病鱼主要表现为鳍条基部和皮肤（特别是腹部和尾柄）充血、出血，腹部膨大，眼球突出．鳃发白．腹腔内充有淡黄色或淡红色的腹水，胃肠道扩张，其内充满大量淡黄色的粘液，肝、脾和肾肿大。病理组织学变化主要为全身组织器官广泛性水肿、出血、变性、坏死和炎症细胞浸润，特别是肾、肝、胃肠道、脾和脑的损伤较为严重。肾间质水肿，造血组织坏死，巨噬细胞和中性拉细胞浸润．肾小管上皮细胞空泡变性和坏死；肝水肿，狄氏间隙增宽．肝细胞空泡变性及坏死；胃肠道粘膜上皮变性，坏死．脱落，固有膜，粘膜下层水肿，淋巴细胞、巨噬细胞和中性枉细胞浸润；脾淤血．出血．淋巴细胞减少，大量巨噬细胞和中性粒细胞浸润；脑水肿，神经细胞肿胀．甚至坏死固缩。超微结构上．被病毒感染的细胞发生明显的痛变，线粒体肿胀，嵴断裂．溶解，粗面内质网扩张，核糖体颗粒脱落．细胞核体积增大，染色质浓缩．边集。     

The effect of high total ammonia concentration on the survival of channel catfish experimentally infected with Flavobacterium columnare

Ammonia concentrations in water can affect the severity of Flavobacterium columnare infections in fish. Two trials lasting 7 d each were conducted to determine the effect of a single immersion flush treatment of total ammonia nitrogen (TAN; 15 mg/L) on the survival of channel catfish Ictalurus punctatus infected with E columnare; the chemical was added while the water flowed continuously through the tanks. Both trials consisted of four treatments: (1) no ammonia exposure and no bacterial challenge (control), (2) ammonia exposure only, (3) bacterial challenge only, and (4) both ammonia exposure and bacterial challenge. Two hours after exposure to ammonia, the highest un-ionized ammonia level was 0.43 mg/L. The percent un-ionized ammonia is based on TAN, temperature, and pH. Caudal fins from three fish in each treatment were sampled at 24 h posttreatment to be analyzed by quantitative real-time polymerase chain reaction (qPCR). No significant difference in survival (mean +/- SE) was noted between the channel catfish in treatment 1 (95.2 +/- 1.2%) and those in treatment 2 (95.6 +/- 1.0%); however, survival in both treatments 1 and 2 differed significantly from that in treatments 3 (8.5 + 4.5%) and 4 (41.8 +/- 12.7%). Treatment 4 catfish had significantly higher survival than treatment 3 catfish. Quantitative PCR data showed that treatment 4 fish had significantly less F. columnare (7.6 x 10(5)) than did treatment 3 fish (1.2 x 10(7)), and treatment 2 fish (8.5 x 10(3)) had significantly less bacteria than did treatment 1 fish (6.9 x 10(4)), indicating that ammonia limited the F. columnare infection. The highest mean concentration of the bacteria (3.9 x 10(7)) was found on moribund fish. The ammonia concentrations tested did not negatively influence fish survival but interfered with the infection process. An in vitro assay was also conducted to evaluate the direct effects of ammonia on F columnare.     

Early Changes in Pigmented Macrophages in Head Kidney of Channel Catfish Infected with Aeromonas hydrophila

Abstract

Channel catfish Ictalurus punctatus with scarified skin were exposed to the bacterial pathogen Aeromonas hydrophila. Systemic infections developed in 80% of the exposed fish, and the remaining exposed fish had infections limited to the cutaneous lesion. Skin of control fish was injured in the same manner as for the exposed fish, but control fish were not exposed to A. hydrophila nor was A. hydrophila recovered from them. None of the fish died during the 3-d experiment, and gross lesions in head kidneys, including changes in the size or relative weight, were not found in infected fish. Macrophage aggregates in head kidney of systemically infected fish increased 68% in mean area, 28% in number, and 111% in total relative volume. Volume of lipofuscin, the predominate pigment found in channel catfish head kidney, in systemically infected fish was about twice that of control fish. Volume of hemosiderin was about four times as great and macrophages containing hemosiderin were about three times as numerous in systemically infected fish as in control fish. The amount of melanin in macrophages did not change in infected fish. Significant differences in macrophage aggregates and pigments were not found between controls and fish with only superficial infections. Microscopic lesions, other than changes in macrophage aggregates and pigments, were not found in the head kidney. Increases in macrophage aggregates, lipofuscin, and hemosiderin in head kidney of channel catfish were useful for quantification of injury caused by a systemic bacterial infection.     

猪流行性腹泻病毒实验感染猪排毒规律的研究

将健康猪12头,随机分为3组,每组4头,分别以肌肉接种、口服和滴鼻三种不同途径感染猪流行性腹泻病毒(PEDV)。用ELISA、电子显微镜技术对PEDV实验感染猪的排毒规律进行了研究。结果证明,PEDV感染猪除了可经粪便排毒外,还可经呼吸道分泌物向外排毒,即PEDV除了可在肠上皮细胞增殖外,还可在呼吸道内复制。实验感染猪一般于接种后2.5～5.2d尚未出现临床症状时开始排毒,持续排毒时间长达54～74 d。因此认为,不仅处于潜伏期的猪可以排毒,而且病愈后的猪也可以长期排毒,是潜在的危险传染源,在防疫上必须引起充分的重视。另外,也证明PEDV可经消化道、呼吸道和肌肉接种途径感染猪体。而此三种途径感染的猪,其临床表现存在着明显差异,即肌肉接种猪易感,日服猪次之,滴鼻猪不易感。     

人工感染绵羊肺腺瘤病毒内蒙株羔羊的临床病理学研究

试验旨在探讨试验性感染绵羊肺腺瘤病毒内蒙株（jaagsiekte sheep retrovirus,JSRV-NM）对羊的淋巴细胞亚群、淋巴细胞凋亡、血气指标的影响。用含JSRV-NM株的病毒悬液1 mL气管内接种1日龄羔羊,对人工感染羊进行了临床症状观察、血液学检查、病理生理学的系统病理学研究。在不同时段用流式细胞仪检测外周血中淋巴细胞及其亚群的动态变化及淋巴细胞凋亡情况。同时,对血液做血气分析和血清酶的检测。人工感染羔羊均出现了一些轻微的绵羊肺腺瘤病（sheep pulmonary adenomatosis,SPA）临床症状。SPA攻毒组感染JSRV-NM株后前60 d,外周血中CD4+、CD8+ T细胞保持较高水平;到感染后90 d呈现下降趋势。对淋巴细胞凋亡检测的结果显示,在感染JSRV-NM株前60 d,凋亡细胞都保持较高水平;而感染第90天后,晚期细胞凋亡显著低于对照组。血气分析结果显示,阴离子隙（AnGap）、细胞外液碱剩余（BE(ecf)）、氯离子浓度（Cl-）、氧分压p（O2）、实际碳酸氢根（HCO3act）、标准碳酸氢根（HCO3std）、二氧化碳总量（TCO2）、氧饱和度（O2SAT）、氧含量（O2CT） 9项在不同时期与对照组存在显著差异。接毒羔羊尚未发生SPA的典型症状,但试验羔羊的病情已经出现,处于SPA疾病的早期表现;病羊在感染JSRV-NM病毒株后,在一定时期内,血液中CD4+ T淋巴细胞和CD8+ T淋巴细胞的变化是SPA病羊在疾病过程中细胞免疫和淋巴细胞受损的重要表现之一;外周血淋巴细胞会呈现不同程度的凋亡,JSRV-NM株诱导机体免疫细胞凋亡是一种重要的临床表现;本试验结果初步证明,血气分析方法在临床诊断绵羊肺腺瘤病时有一定的参考价值。     

头孢噻呋体外抗菌活性及其对实验性感染鸡大肠杆菌病的疗效

采用肉汤微量稀释法测定头孢噻呋对大肠杆菌、沙门菌、溶血性巴氏杆菌、金黄色葡萄球菌、链球菌等病原菌的最小抑菌浓度,采用棋盘稀释法测定头孢噻呋与氧氟沙星、甲氧苄啶(TMP)的联合抑菌浓度指数.结果表明,头孢噻呋、氧氟沙星对5种病原菌都具有很强的抗菌活性;头孢噻呋与氧氟沙星联合使用时,对大肠杆菌、沙门菌、链球菌可产生协同作用,对溶血性巴氏杆菌和金黄色葡萄球菌表现为无关作用;头孢噻呋与TMP联合用药时,对上述5种病原菌均具有协同作用.对实验性感染鸡大肠杆菌病的药效学试验结果表明,头孢噻呋高(100mg/L)、中(50mg/L)剂量对感染鸡的有效率明显高于对照药物庆大霉素(P<0.05),极显著高于感染对照组(P<0.01);头孢噻呋高剂量组(100mg/L)感染鸡的增重率与健康对照组相比差异不显著(P>0.05).建议临床应用头孢噻呋治疗鸡大肠杆菌病时,可采用饮水给药,剂量为100mg/L.     

Closantel缓释剂驱除山羊实验感染肝片吸虫的效果

选用 5只实验感染肝片吸虫山羊进行 Closantel(氯氰碘柳胺 )缓释剂驱虫试验。试验用山羊每只一次性口服肝片吸虫囊蚴 15 0个 ,感染后第 15周随机将山羊分成对照组 (n=2 )和驱虫组 (n=3) ,驱虫组每只羊口服 2丸 Closantel缓释剂 (含 15 .5 % Closantel,每丸 7.6 g) ,每周定时采集颈静脉血和进行粪便检查 ,每月称重 ,试验结束时剖检测量肝重和计数肝内虫体数 ,以驱虫前后山羊的红细胞、白细胞、嗜酸性粒细胞、血清抗体和一氧化氮的变化、体重变化、剖检时肝重 /体重之比、虫卵的变化以及虫体减少率作为衡量驱虫效果的指标。结果表明 ,投药后嗜酸性粒细胞在第 1、2、3周与对照组相比有显著下降 ,其他各项指标与对照组相比虽有一定差异但不显著。虫体减少率为 4 1.2 2 %。提示Closantel缓释剂对驱除实验感染肝片吸虫有一定效果 ,在虫体感染前投药会起到预防作用。     

新肝宝对鲶爱德华氏菌感染黄颡鱼的保护作用研究

为了考察新肝宝对鲶爱德华氏菌感染黄颡鱼的保护作用,采用不同粉碎粒度的新肝宝拌饲投喂7 d,随后用鲶爱德华氏菌腹腔注射感染,观察死亡率、酶学指标及组织病理变化。结果显示,新肝宝组死亡率(48.57%和44.28%)低于阴性对照组(54.29%),溶菌酶及超氧化物歧化酶活性显著高于对照组(P<0.05),肝脏、肾脏、脾脏损伤情况均小于阴性对照组,其中600目作用效果优于200目。结果表明,新肝宝能够提高黄颡鱼非特异性免疫力,增强黄颡鱼对鲶爱德华氏菌的抵抗能力,在一定程度上减轻鲶爱德华氏菌感染导致的肝、脾、肾损伤,其中600目作用效果优于200目。     

柔嫩艾美尔球虫实验感染雏鸡体液免疫机制研究

本文通过人工复制的鸡球虫病鸡,利用免疫组化技术等方法,检测了柔嫩艾美耳球虫(E.tenella)初次感染雏鸡后,盲肠局部和免疫器官中IgG生成细胞数的动态变化。结果表明:攻毒后雏鸡盲肠粘膜、脾脏、法氏囊、盲肠扁桃体中的IgG生成细胞分别于第3、3、3、2d开始增殖,在第12、12、16、9d达到峰值,随后即开始下降,盲肠扁桃体中的IgG生成细胞数在第22d仍高于对照组,且差异显著(P<0.05)。     

Closantel缓释剂对实验感染肝片吸虫山羊机体自由基影响的研究

选用5只实验感染肝片吸虫山羊研究Closantel缓释剂对机体抗氧化功能的影响，实验用山羊每只一次性口服肝片吸虫囊蚴150个，感染后第15周随机将山羊分成对照组（n=2）和驱虫组（n=3），驱虫组每羊口服两颗Closantel缓释剂，每周定时采集颈静脉血液，检测血清抗氧化酶的变化，观察服用Closantel缓释剂对其的影响。结果表明投药后山羊血清中GSH－Px、CAT均有不同程度的升高，SOD变化不显著，而脂质过氧化产物MDA含量则下降，提示Closantel的缓释剂型对实验感染肝片吸虫山羊机体的抗氧化产生了影响，对机体有一定保护作用。     

Effect of Vitamin E on the Immune Response of Channel Catfish to Edwardsiella ictaluri

Abstract

Three-month-old fingerling channel catfish Ictalurus punctatus were fed purified diets supplemented with ∝-tocopherol acetate to provide 0, 60, and 2,500 mg vitamin E/kg for 180 d. A 30-s immersion bath and an oral booster were used to deliver a bacterin of formalin-killed Edwardsiella ictaluri to half of the fish from each dietary treatment. Resistance of red blood cells to peroxidation was used as an index of antioxidant status. The susceptibility of red blood cells to oxidative hemolysis decreased with increasing levels of dietary vitamin E. Vaccinated and nonvaccinated fish were evaluated for agglutinating antibody titers and macrophage activity. Humoral antibody titers in response to E. ictaluri were significantly (P ≤ 0.05) higher in vaccinated fish than in nonvaccinated fish; however, no such differences in agglutinating antibody titers were detected among any of the dietary treatment groups. Both vaccination and vitamin E significantly enhanced the ability of macrophages to phagocytize virulent E. ictaluri. Results of this study indicate that elevated levels of dietary vitamin E may affect the ability of channel catfish to respond immunologically to bacterial pathogens.     

SPF鸡人工感染鸡败血霉形体后呼吸系统的病理变化

10日龄SPF鸡气囊接种鸡败血霉形体（MG）R株，3－5天后出现的典型的MG感染症状，呼吸系统发生一系列典型的病理组织学变化。感染鸡和对照鸡的气管经固定、脱水、临界点干燥、镀金后置于扫描电镜下观察，感染鸡气管粘膜明显水肿，部分纤毛脱落或全部脱落。电镜下可见感染鸡的气管粘膜水肿增厚近2倍，粘膜下层单核细胞积聚，其间有数量不等的淋巴细胞聚集，粘液细胞显著减少或消失；气囊粘膜水肿增厚；肺组织中有淋巴细胞增生形成的结节，对照鸡呼吸系统的组织切片无变化。     

The Effect of Storage on Ammonia Concentration in Canine Packed Red Blood Cells.

Objective: To determine the effect of storage on ammonia concentration in canine packed red blood cell (pRBC) units.
Design: In vitro and in vivo study.
Setting: University Veterinary Teaching Hospital.
Interventions: Ammonia concentration was measured in 7 units of canine pRBC prepared in citrate-phosphate-dextrose (CPD) and Adsol^a on Days 1 and 35 of storage. Ammonia was measured in 4 additional units of canine pRBC on Days 0, 7, 14, 21, 28, and 35. Plasma ammonia was also determined in 5 anemic dogs receiving pRBC.
Measurements and Main Results: Ammonia concentration increased from 73 ± 15 mmol/L (mean ± SD) on Day 1 to 800 ± 275 mmpl/L on Day (p<0.001). When measured every 7 days in 4 units of canine pRBC, ammonia concentration increased from 23 ± 8 mmol/L on Day 0 to 179 ± 13 mmol/L (Day 7), 276 ± 56 mmol/L (Day 14). 383 ± 47 mmol/L (Day21), 466 ± 30 mmol/L (Day 28), and 562 ± 27 mmol/L (Day 35) (p<0.05 for all comparisons). In a preliminary study, plasma ammonia concentration measured in blood samples from 5 anemic dogs without primary liver disease immediately before and after transfusion with 5–10 ml/kg of stored pRBC remained in the normal reference range.
Conclusions: The ammonia concentration in stored canine pRBC increased markedly with time. In this preliminary study, ammonia concentrations in dogs without primary liver disease did not increase above the reference range after transfusion with pRBC.     

The association of Flavobacterium columnare strains of high and low virulence with gill tissue of black mollies (Poecilia sphenops).

The ability of a high virulence strain (AJS 1) and a low virulence strain (AJS 4) of Flavobacterium columnare (Flexibacter columnaris) to attach to the gills of black mollies (Poecilia sphenops) was investigated. For that purpose, two groups of 25 black mollies each were immersed in a bath containing 10(6) CFU/ml of F. columnare AJS 1 or AJS 4. At regular intervals from 1 to 12 h after the contact infection, fish were sacrificed and gills, skin, spleen and heart were sampled for bacteriology. Samples of the gills were taken for immunohistochemical and electron microscopic examination. Bacteriological examination proved that the number of gill-associated F. columnare was higher for AJS 1 than for AJS 4. Strain AJS 1 was isolated from the heart and spleen of 6 and 1 of the 16 examined animals, respectively. Strain AJS 4 was not isolated from the internal organs of any fish. When examined immunohistochemically, strain AJS 1 was found closely associated with gill epithelium whereas this was not the case for strain AJS 4. The adherence of bacteria to the gill tissue challenged with the virulent strain AJS 1 was also clearly demonstrated using scanning and transmission electron microscopy. These results indicate that adhesion of F. columnare to the gill tissue constitutes an important step in pathogenesis.     

企鹅新城疫强毒人工感染北京雏鸭的研究

以分离鉴定的企鹅新城疫强毒（ＰＮＤＶ）经静脉注射、肌肉注射和洋鼻点眼等途径分别人工感染７日龄和１４日龄的北京雏鸭,结果只有静脉注射组雏鸭发病死亡,其致死率分别为１００％和５０％,且自死亡雏鸭脑组织中均可重新分离到ＰＮＤＶ。而肌肉注射组雏鸭和滴鼻点眼组雏鸭于接毒后１４天内均未表现任何异常,但可诱导产生较高效价的特异性抗体,然而抗体效价在短时间内迅速下降,且春脑和脾脏中均未分离到ＰＮＣＶ。试验结果表明北京鸭对ＰＮＤＶ的易感性不强,即使经静脉注射静致死率也随着日龄的增大而降低;而经肌肉注射或滴鼻点眼途径均未能使北京雏鸭发病,且其脑和脾脏的带毒时间不超过１４天。     

实验性鸡马立克氏病羽髓病变研究

通过光镜1电镜等方法对鸡马立克氏病羽髓组织进行了形态学研究。结果显示，羽髓远端坏死，羽髓中有多量肿瘤细胞浸润，肿瘤细胞呈弥漫性或局部性浸润，电镜观察，肿瘤细胞核异型性明显，核膜凹陷，核内出现假核内包含物，在一些羽髓中，巨噬细胞吞噬肿瘤细胞较多见，而且观察到一些肿瘤细胞发生调亡，结果表明，鸡马立克氏病羽髓病变十分明显，羽髓的形态学变化可用于鸡马立克氏病的诊断。