Cultivable microbiota of Lithobates catesbeianus and advances in the selection of lactic acid bacteria as biological control agents in raniculture

The cultivable microbiota of skin and cloaca of captive Lithobates catesbeianus includes microorganisms generally accepted as beneficial and potentially pathogenic bacteria. In order to select a group of potentially probiotic bacteria, 136 isolates were evaluated for their surface properties and production of antagonistic metabolites. Then, 11 lactic acid bacteria (LAB) strains were selected and identified as Lactobacillus plantarum, Lb. brevis, Pediococcus pentosaceus, Lactococcus lactis, L. garvieae and Enterococcus gallinarum. Studies of compatibility indicate that all the strains could be included in a multi-strain probiotic, with the exception of Ent. gallinarum CRL 1826 which inhibited LAB species through a bacteriocin-like metabolite. These results contribute to the design of a probiotic product to improve the sanitary status of bullfrogs in intensive culture systems, to avoid the use of antibiotics and thus to reduce production costs. It could also be an alternative to prevent infectious diseases during the ex situ breeding of amphibian species under threat of extinction.     

馆藏泽蛙标本壶菌病原实时PCR检测与系统发育分析

为研究、验证我国两栖类壶菌病的历史疫情,从时间和系统进化角度追溯壶菌的来源,该研究选取北京自然博物馆馆藏1982年采集于广东的泽蛙标本39只,利用Taqman-MGB荧光探针定量PCR技术进行壶菌检测,并对定量PCR产物克隆、测序,通过序列比对和系统发育分析判定其来源。最终得到定量PCR标准曲线:Y=-3.1X+32.65;相关系数R2=0.999 8;检测结果为阳性样本12只,检出率30.8%;同时系统发育分析表明,我国的壶菌存在一定程度的分化,一类与北美洲、南美洲、欧洲菌株呈现高度的亲缘关系;另一类则表现出与世界其他地区分布的壶菌有明显的不同,显示为独特类型。该研究把我国壶菌感染的最早记录推进到了20世纪80年代初期。     

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。     

鸡贫血病毒实验感染雏鸡胸腺与骨髓的超微结构观察

用鸡贫血病毒 Ｃｕｘ １ 毒株接种于 １ 日龄 Ｓ Ｐ Ｆ 雏鸡,在确认感染成功的基础上,对感染雏鸡胸腺和骨髓的超微结构进行了观察。电镜观察表明,感染鸡骨髓和胸腺细胞的形态变化具有较典型的凋亡特征,如细胞膜出泡、核染色质凝结边集、细胞核裂解、出现凋亡小体等,此外还见有可能由病毒粒子形成的环状物。本试验为证明鸡贫血病毒能引起细胞凋亡提供了依据。     

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.     

Detection of Anti-listeriolysin O and Listeria monocytogenes in Experimentally Infected Buffaloes (Bubalus bubalis)

The kinetics of antibody production against listeriolysin O (ALLO) and the recovery pattern of Listeria monocytogenes from bacteriological samples were studied following oral infection of buffalo calves with 3×10⁹ cells each of pathogenic L. monocytogenes. Antibodies to LLO appeared by 7–10 days post infection (PI), with a shallow peak between days 16 and 36 PI, when tested by indirect plate-ELISA. The titres of ALLO in all the animals then declined slowly but remained detectable up to day 70 PI. In dot-ELISA, ALLO could be detected by days 5 to 7 PI, and with higher titres than with the plate-ELISA. The pathogen was recovered at low rates as ALLO first appeared but was absent in the faecal, nasal and blood cultures as production of ALLO peaked.     

Immunofluorescent Studies of Bovine Para-Influenza 3 Virus I. Cell Cultures and Experimentally Infected Hamsters

Immunofluorescent techniques were used to follow the replication of the SF-4 strain of bovine para-influenza 3 (PI-3) virus in Madin-Darby Bovine Kidney (MDBK), secondary bovine kidney (BK), Embryonic Bovine Trachea (EBTr) cell cultures and experimentally infected hamsters. The virus replicated equally well in MDBK and secondary BK cell cultures but less successfully in EBTr cultures. Nuclear fluorescence was not observed in the cell systems studied.

Infection was limited to the epithelial lining of the upper respiratory tract, trachea and bronchi of experimentally infected hamsters. Virus was most easily identified in smears prepared from turbinate mucosa suggesting a potential diagnostic technique for use in identifying PI-3 infected animals.

     

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.     

2. Persistence of Equine Herpesviruses in Experimentally Infected Horses and the Experimental Induction of Abortion

An 8-month-old filly (No. 2) developed an acute vulvo-vaginitis and respiratory disease following inoculation of equine herpesvirus (EH virus) type 1 (EH 39 virus; equine rhinopneumonitis virus) into the vestibule of the vagina. The same virus produced acute respiratory disease but not balanoposthitis following intranasal, intravenous and intrapreputial inoculation of a 12-month-old colt (No. 3). A second 8-month-old filly (No. 1) developed a mild respiratory disease but not vulvo-vaginitis following intravestibular inoculation of EH 39 virus. EH viruses that were slowly cytopathic for equine foetal kidney cell cultures and serologically unrelated to the inoculated EH 39 virus were isolated from the buffy coat cells at 3 days and from the nasal cavity at 6 days after inoculation of horse No. 1. EH virus that was slowly cytopathic and serologically unrelated to EH 39 virus was isolated at 16 days from the vagina of the filly (No. 2) that developed acute vulvovaginitis and was frequently isolated from the nasal cavities of 2 of the 3 horses for 83 days and from the nasal cavity of the third horse for 57 days under conditions that precluded reinfection from other equidae except from each other. EH viruses were recovered from the 3 horses for a further 58 days under conditions where contact with other equidae may, although was not known to, have occurred between 83 and 141 days postinoculation. It was concluded that these viruses represented a single virus type that was present in the nasal cavity (designated EH 1–6 virus) perhaps also the blood stream of filly No. 1 at the time the 3 horses were purchased and that this virus was subsequently transmitted to the vagina of 1 and the nasal cavities of the other 2 horses. Accordingly a carrier state for EH 39 virus was not shown to occur. These findings are discussed in relation to the natural history of EH virus infections. Attempts to reactivate the EH viruses to cause clinical respiratory disease, by a series of injections of adrenalin and cortisone, were inconclusive. The 3 horses showed no clinical evidence of respiratory disease when they were reinfected intranasally with EH 39 virus 360 days (1 horse) and 412 days (2 horses) after the initial infection with this virus. Abortion was produced when EH 39 virus was inoculated directly into the allantoic or amniotic cavity of a pregnant mare although naturally occurring EH virus abortion remains unrecognised in Australia.     

实验性蛔虫感染鸡空肠粘膜和胸腺髓质肥大细胞的数量变化

对试验性蛔虫感染鸡的空肠粘膜和胸腺髓质肥大细胞的数量变化的研究表明 ,蛔虫感染鸡空肠粘膜肥大细胞较对照组有轻度增多 ,但无统计学显著差异性 (P>0 .0 5 )。然而 ,试验组鸡的胸腺髓质肥大细胞为 (136± 49)个 / m m2 ,较对照组的 (2 73± 5 6 )个 / mm2有极显著减少 (P<0 .0 0 1)。似乎试验感染鸡胸腺髓质肥大细胞的显著减少与空肠粘膜肥大细胞的相对增多之间存在某些相关性。     

Toxoplasmosis III. Studies Using the Complement-Fixation Test and Fluorescence-Inhibition Test with Sera of Experimentally Exposed Birds

The direct, modified direct and indirect complement-fixation tests and the fluorescence-inhibition test were investigated using sera from pigeons, chickens and turkeys which had been exposed to Toxoplasma gondii. The direct CF test was suitable for use with pigeon sera.

The indirect CF method effectively demonstrated antibodies in chicken and turkey sera. FI tests were less sensitive than the CF methods.

     

The Genetics of Mortality and Survival of Broiler Chicks Infected as Embryos by Subgroup A Rous Sarcoma Virus

A study using two high-performance broiler lines, a synthetic male line (SML) and a synthetic dam line (SDL), was undertaken to investigate the pattern of mortality due to induced liver tumours (LT) and the immune response to subgroup A virus inoculated via the chorioallantoic membrane (CAM) route. The distribution patterns of the four possible phenotypes were similar in both sire and dam lines. The occurrence of conversely associated phenotypes was about 30% in both the lines. The percentages of CAM(+) LT(–) and CAM(–) LT(+) were 14.26% and 14.46% in the dam line and 20.0% and 9.57% in the male line. The LT mortality was 30–50% in the birds with low pock counts, whereas it was 80–93% in the birds with high pock counts. The group specific antigen shedding status did not influence death due to LT. In birds in the high pock count group, 98% of deaths due to LT were completed by the sixth week, whereas in those in the low pock count group, death due to LT was spread over 24 weeks. The SDL birds survived better than SML birds in the high pock count groups. In both lines, about 20% of deaths occurred owing to non-specific causes. The average survival time after hatching before death from LT was 26 days, whereas that for non-specific death was 81 days.     

Bovine Viral Diarrhea Virus in Swine: Characteristics of Virus Recovered from Naturally and Experimentally Infected Swine

A noncytopathogenic field strain of bovine viral diarrhea virus (BVDV) was isolated from an Iowa farm brood sow and from her hysterectomy-derived, colostrum-deprived (HDCD) piglets. This field isolant was fully virulent for a neonatal calf. The NADL strain of BVDV was passaged through a series of HDCD piglets with no resultant loss of virulence for neonatal calves. Most of the BVD viral isolants recovered from pigs had been changed from a cytopathogenic biotype to a noncytopathogenic biotype. Circumstantial evidence points to swine as “carrier” hosts of BVDV.     

Humoral Antibody Responses of Swine Infected Experimentally with Group E Streptococcus II. Antistreptokinase and Antistreptodornase Responses

Swine infected experimentally with group E Streptococcus (GES) produced significant antibody titers against streptokinase (streptococcal fibrinolysin, SK) and streptodornase (streptococcal deoxyribonuclease, SD). The antibodies directed against SK (antistreptokinase, ASK) appeared two to nine weeks postexposure and persisted for the duration of the experiment (nearly six months). The ASK inhibited SK produced by GES antigenic types III, IV, and V, by GES devoid of type specific antigen, and by a group P Streptococcus. Selected strains of GES serotypes I and II and group U Streptococcus did not produce detectable SK.

The antibodies directed against SD (antistreptodornase, ASD) appeared two to three weeks postexposure, reached a peak about six weeks postexposure, and persisted at high levels for nearly six months (the duration of the experiment). The ASD inhibited SD produced by all known antigenic types of GES, by GES devoid of type specific antigen, and by strains of groups P and U Streptococcus. The antibodies failed to inhibit SD produced by group C Streptococcus.

The potential utilization of ASK and ASD titers as serological means of identifying swine infected with GES (carrier swine) is discussed.

     

Capsid Protein Genetic Analysis and Viral Spread to the Spinal Cord in Cats Experimentally Infected with Feline Calicivirus (FCV)

We investigated primitively the molecular basis of the neural spread of a feline calcivirus isolate (FCV-S) from the spinal cord of a cat that died after manifesting excitation. Experimental infections of cats with three clones from parent virus isolate FCV-S, isolated based on plaque size, were performed, and virus recovery from the spinal cord and the nucleotide and predicted amino acid sequences of the viral capsid protein region (ORF2) were compared. In the experimental infection with the one-time cloned virus (C1L1) isolated from a large plaque, the C1L1 was recovered from the spinal cord. In contrast, seven-times cloned C6L7 (from large plaque) and five-times cloned C5S2 (isolated from small plaque) were not recovered from the spinal cord. Genetic analysis of the capsid protein gene of the three viral clones revealed that four bases were different and two amino acids were different at positions 34 (Val in C6L7 and Ala in C1L1 and C5S2) and 46 (Leu in C6L7 and Pro in C1L1 and C5S2) between C6L7 (with large plaque) and C5S2 (with small plaque). The amino acid at position 434 of C1L1 was different from those of C6L7 and C5S2 (Gly in C1L1, D (Asp) in C6L7 and C5S2). From these results, the plaque size seemed not to be related to the spread of virus to the spinal cord. Clone C1L1, which spread to the spinal cord, had a difference of one amino acid from the other two clones, which may be related to the ability to spread to the spinal cord.     

Experimentally Induced Whirling Disease I. Dose Response of Fry and Adults of Rainbow Trout Exposed to the Triactinomyxon Stage of Myxobolus cerebralis

Abstract

The intensity and prevalence of whirling disease was tested by exposure of 2-monthold fry and 1-, 2-, and 3.5-year-old adults of rainbow trout Oncorhynchus mykiss to a known number of laboratory-produced Myxobolus cerebralis at the actinosporean triactinomyxon stage. Fry exposed to graded concentrations of infectivity (triactinomyxons) for 3 h were individually examined for spores of Myxobolus cerebralis 5 and 6 months later. Exposure of fish to the lowest doses, 1 and 10 triactinomyxons per fish, did not result in detectable myxosporean spores. Fish that became lightly infected by a dose of 100 triactinomyxons per fish experienced a decrease in the incidence of infection between 5 and 6 months after exposure. A linear relationship was found between the numbers of recovered myxosporean spores and doses of 100–10,000 triactinomyxons per fish, and the spore burden appeared to plateau at doses of 10,000–100,000 triactinomyxons per fish. Adult fish continuously exposed to the highest dose of triactinomyxons for 3.5 months were infected and asymptomatic, however, the severity of myxosporean infection decreased with increased age of fish. This information may help in controlling whirling disease in salmonids.     

Usefulness of Polymerase Chain Reaction in Early Detection and Tissue Tropism of Fowl Adenovirus in Experimentally Infected Chicken

Veterinary Research Communications -     

啤酒酵母粉替代部分鱼粉对牛蛙生长、抗氧化能力以及肝脏和肠道组织形态学的影响

本试验旨在研究啤酒酵母粉替代部分鱼粉对牛蛙生长性能、体组成、肝脏抗氧化指标、血清生化指标、肠道消化酶活性以及肝脏和肠道组织形态学的影响。选取初始体重为(34.15±1.10) g的牛蛙135只,随机分为3组,每组3个重复,每个重复15只。以含20%鱼粉的饲料为对照饲料,用啤酒酵母粉分别替代对照饲料中25%(Y25%)和50%的鱼粉(Y50%),配制3种等氮等脂的试验饲料,进行为期8周的生长试验。结果显示:用啤酒酵母粉替代25%或50%的鱼粉后牛蛙的各生长性能指标均没有受到显著影响(P>0.05)。2个替代组蛙体水分含量显著高于对照组(P<0.05),蛙体粗灰分含量显著低于对照组(P<0.05),而蛙体粗蛋白质和粗脂肪含量则与对照组无显著差异(P>0.05)。对照组和Y25%组干物质和粗蛋白质的表观消化率均显著高于Y50%组(P<0.05)。粗脂肪的表观消化率随着啤酒酵母粉替代水平的升高而显著降低(P<0.05),而磷的表观消化率则随着啤酒酵母粉替代水平的升高而显著升高(P<0.05)。啤酒酵母粉替代25%或50%的鱼粉对牛蛙血清中谷草转氨酶(AST)、谷丙转氨酶(ALT)的活性均无显著影响(P>0.05),Y50%组血清碱性磷酸酶(ALP)、溶菌酶(LZM)活性显著高于对照组(P<0.05)。Y25%和Y50%组肝脏中总抗氧化能力(T-AOC)显著高于对照组(P <0.05)。肝脏中总超氧化物歧化酶(T-SOD)与过氧化氢酶(CAT)活性以及还原型谷胱甘肽(GSH)含量均随啤酒酵母粉替代水平的升高而显著升高(P<0.05)。2个替代组肠道蛋白酶、脂肪酶活性均显著低于对照组(P<0.05),而肠道淀粉酶活性则与之相反。组织学观察发现各组肝脏和肠道组织结构未见明显异常,但2个替代组肝细胞脂肪沉积较对照组减少。肝脏中胰岛素样生长因子-Ⅰ(IGF-Ⅰ)基因的相对表达量各组间没有显著差异(P> 0.05),但2个替代组肝脏中热休克蛋白70(HSP70)基因的相对表达量显著高于对照组(P<0.05)。综上,啤酒酵母粉是牛蛙饲料中优质的蛋白质源,其替代鱼粉水平不超过50%时可维持牛蛙正常的生长性能,并有利于维护肝脏健康。