Concentration of Infectious Aquatic Rhabdoviruses from Freshwater and Seawater Using Ultrafiltration

Abstract

Infectious hematopoietic necrosis virus (IHNV), viral hemorrhagic septicemia virus, and spring viremia of carp virus were concentrated and detected from freshwater and seawater samples by using hollow-fiber ultrafiltration. Within 60 min, virus in a 50-L freshwater or saltwater sample was concentrated more than 70-fold, and virus retention efficiencies were consistently greater than 88%. Retention efficiency was highly dependent upon concentrations of column blocking and sample stabilization solutions. A large column with a surface area of 1.15 m² and a filtration capacity of 5–200 L exhibited optimal viral retention when blocked with 2% fetal bovine serum (FBS) and when the samples were supplemented with 0.1% FBS. Conversely, a small column with 100-fold less surface area and a filtering capacity of 0.5–2.0 L was optimized when blocked with 1% FBS and when the samples were supplemented with 0.1% FBS. The optimized ultrafiltration procedure was further validated with water from a tank that contained IHNV-exposed juvenile sockeye salmon Oncorhynchus nerka, resulting in an average virus retention efficiency of 91.6 ± 4.1% (mean ± SE). Virus quantification of concentrated samples demonstrated that IHNV shedding in sockeye salmon preceded mortality; shedding of the virus was observed to increase significantly as early as 7 d postchallenge and peaked at day 14, when virus levels reached 4.87 × 10³ plaque-forming units/mL. We conclude that ultrafiltration is a reliable and effective method for concentrating viable aquatic rhabdoviruses from large volumes of water and has application for the analysis of environmental water samples.

Received April 22, 2011; accepted August 4, 2011     

Formalin and footrot in sheep

Changes in formalin concentration were measured over time in uncontaminated solutions and solutions heavily contaminated by mud, faeces, wool or straw. Shallow plastic trays containing 1.11 of a 9% aqueous solution of formalin were exposed for 6 days (trial 1) or8 days (trial 2) to windy autumn weather. Evaporation of solution volumes was high (47–73%).

Concentration generally increased over time: by 13–31% in trial 1 and by 61–162% in trial 2. These figures represent actual increases in percentage formalin of up to 3.0% in trial 1 and up to 7.6% in trial 2. Exposure to mud, sheep faeces or wool did not affect the increase in concentration but there was no increase in the presence of straw.

As exposure to excessively concentrated formalin solutions produces hyperaemia, cracking of interdigital skin and lameness in sheep it is recommended that footbath solutions should be diluted to 2–5%, prepared on the day of use and discarded daily whether contaminated or not. Addition of formalin or water to existing solutions of unknown strength should be avoided.     

Purification of infectious bronchitis coronavirus by Sephacryl S-1000 gel chromatography

A procedure was developed to purify infectious bronchitis virus (IBV) by gel chromatography (GC) with a Sephacryl S-1000 column. Virus samples concentrated by centrifugation were applied to a Sephacryl S-1000 column and eluted by 0.02 M phosphate buffer (pH 7.2) containing 0.15 M NaCl. Virus particles were recovered mainly in the first peak. Purity of the samples was evaluated by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electron microscopy. Using electron microscopy, it was found that there were more spike-rich particles in the virus samples purified by GC than in those purified by sucrose density gradient centrifugation (SDGC). In addition, the hemagglutination unit [log10 (infectivity titer/hemagglutination titer)] of GC-purified virus samples was approximately 10 times lower than that of SDGC-purified virus samples. These results indicate that Sephacryl S-1000 gel chromatography is useful for purification of IBV.     

Concentration of infectious aquatic rhabdoviruses from freshwater and seawater using ultrafiltration

Infectious hematopoietic necrosis virus (IHNV), viral hemorrhagic septicemia virus, and spring viremia of carp virus were concentrated and detected from freshwater and seawater samples by using hollow-fiber ultrafiltration. Within 60 min, virus in a 50-L freshwater or saltwater sample was concentrated more than 70-fold, and virus retention efficiencies were consistently greater than 88%. Retention efficiency was highly dependent upon concentrations of column blocking and sample stabilization solutions. A large column with a surface area of 1.15 m2 and a filtration capacity of 5-200 L exhibited optimal viral retention when blocked with 2% fetal bovine serum (FBS) and when the samples were supplemented with 0.1% FBS. Conversely, a small column with 100-fold less surface area and a filtering capacity of 0.5-2.0 L was optimized when blocked with 1% FBS and when the samples were supplemented with 0.1% FBS. The optimized ultrafiltration procedure was further validated with water from a tank that contained IHNV-exposed juvenile sockeye salmon Oncorhynchus nerka, resulting in an average virus retention efficiency of 91.6 +/- 4.1% (mean +/- SE). Virus quantification of concentrated samples demonstrated that IHNV shedding in sockeye salmon preceded mortality; shedding of the virus was observed to increase significantly as early as 7 d postchallenge and peaked at day 14, when virus levels reached 4.87 x 10(3) plaque-forming units/mL. We conclude that ultrafiltration is a reliable and effective method for concentrating viable aquatic rhabdoviruses from large volumes of water and has application for the analysis of environmental water samples.     

Strain specific antibodies revealed by immunoabsorption studies with avian infectious bronchitis virus

Rabbit antisera prepared against the Massachusetts 41 (M41) strain of avian infectious bronchitis virus (IBV) and absorbed with chick embryo immunoabsorbent produced multiple precipitin lines in immunodouble-diffusion (IDD) tests with homologous or heterologous strains of virus. These precipitin lines were all removed by absorption with concentrated M41 virus preparations, but repeated absorption with concentrated, purified preparations of IBV strains: T, Holte, Connecticut, Beaudette or H120 failed to remove all precipitin lines produced to M41 virus, although all those to the heterologous viruses were removed. The remaining line(s) produced with M41 virus by sera absorbed with different heterologous viruses showed identity in IDD tests and was associated with the surface projections of the virus.     

苍耳提取物对萝卜蚜和粘虫作用方式及解毒酶活性影响的研究

采用室内生测法,研究了苍耳3种溶剂(丙酮、乙醇、氯仿)提取物对萝卜蚜和粘虫的作用方式及苍耳氯仿提取物对两种试虫的3种解毒酶活性的影响。结果表明,苍耳氯仿提取物对萝卜蚜有很强的忌避作用,当浓度为0.05 g/mL,处理4 h,忌避率达94.25%,48 h忌避率为84.53%;其次是触杀作用,校正死亡率最高为67.24%,LC₅₀为0.7420 g/mL;内吸作用极弱。对粘虫主要表现为拒食和生长发育抑制作用,拒食校正死亡率最高达98.27%,AFC₅₀为0.1985 g/mL;72 h体重增长率为-5.85%,与对照差异极显著;胃毒和触杀毒力较弱,LC₅₀分别为0.8997和1.3070 g/mL。苍耳氯仿提取物对萝卜蚜和粘虫的乙酰胆碱酯酶(AchE)有抑制作用,处理萝卜蚜24 h及处理粘虫4 h时,乙酰胆碱酯酶(AchE)抑制率最高,分别为37.55%和25.65%;对谷胱甘肽转移酶(GSTs)和多功能氧化酶(MFO)活力影响不明显。     

Journal of Aquatic Animal Health: Guide for Authors 2012

Abstract

A virus adsorption-elution (viradel) method for use with positively charged microporous filters was previously developed in our laboratory to concentrate waterborne infectious pancreatic necrosis virus (IPNV). In the present study, this method was evaluated for the detection of IPNV in water of laboratory aquaria containing IPNV-infected rainbow trout Onchorhynchus mykiss. Waterborne IPNV samples concentrated by the viradel method were compared with samples of sediment, fish tissue homogenate, and untreated water to determine how much the viradel method could improve rapidity and efficiency of virus detection. Results of three separate experiments indicated a significant, positive correlation between the virus titers in fish tissue and in the viradel method samples and between the sediment and untreated water samples.     

Separation of a Soluble Antigen and Infectious Particles of Bovine Viral Diarrhea Viruses and their Relationship to Hog Cholera

A soluble antigen present in infectious tissue culture fluids was separated from the infective virus particle by ultracentrifugation of two serologically related strains of bovine viral diarrhea viruses, NADL-MD and Oregon C24V.

Neutralizing antibodies against the two viruses were absent in four hog cholera antisera, but present in significant titer in the commercially prepared antiserum. Precipitin tests utilizing the agar double diffusion technique formed a single line of identity between the concentrated soluble antigen of both viruses and NADL-MD and hog cholera antisera. No lines were observed using concentrated virus pellet and noninfected BEK cell antigens or control SPF calf and swine sera.

     

Development of an indirect ELISA for detection of antibody to wobbly possum disease virus in archival sera of Australian brushtail possums (Trichosurus vulpecula) in New Zealand

AIMS: To develop an indirect ELISA based on recombinant nucleocapsid (rN) protein of wobbly possum disease (WPD) virus for investigation of the presence of WPD virus in Australian brushtail possums (Trichosurus vulpecula) in New Zealand.

METHODS: Pre- and post-infection sera (n=15 and 16, respectively) obtained from a previous experimental challenge study were used for ELISA development. Sera were characterised as positive or negative for antibody to WPD virus based on western-blot using WPD virus rN protein as antigen. An additional 215 archival serum samples, collected between 2000–2016 from five different regions of New Zealand, were also tested using the ELISA. Bayesian modelling of corrected optical density at 450?nm (OD₄₅₀) results from the ELISA was used to obtain estimates of receiver operating characteristic (ROC) curves to establish cut-off values for the ELISA, and to estimate the prevalence of antibody to WPD virus.

RESULTS: Western blot analysis showed 5/14 (36%) pre-infection sera and 11/11 (100%) post-infection sera from experimentally infected possums were positive for antibodies to WPD virus. Bayesian estimates of the ROC curves established cut-off values of OD₄₅₀≥0.41 for samples positive, and OD₄₅₀<0.28 for samples negative for antibody to WPD virus, for sera diluted 1:100 for the ELISA. Based on the model, the estimated proportion of samples with antibodies to WPD virus was 0.30 (95% probability interval=0.196–0.418). Of the 230 archival serum samples tested using the ELISA, 48 (20.9%) were positive for antibody to WPD virus, 155 (67.4%) were negative and 27 (11.7%) equivocal, using the established cut-off values. The proportion of samples positive for WPD virus antibody differed between geographical regions (p<0.001).

CONCLUSION: The results suggested that WPD virus or a related virus has circulated among possums in New Zealand with differences in the proportion of antibody-positive samples from different geographical regions. Antibodies to WPD virus did not seem to protect possums from disease following experimental infection, as one third of possums from the previous challenge study showed evidence of pre-existing antibody at the time of challenge. These results provide further support for existence of different pathotypes of WPD virus, but the exact determinants of protection against WPD and epidemiology of infection in various regions of New Zealand remain to be established.

CLINICAL RELEVANCE: Availability of the indirect ELISA for detection of WPD virus antibody will facilitate prospective epidemiological investigation of WPD virus circulation in wild possum populations in New Zealand.     

猪瘟病毒持续感染对母猪繁殖性能及仔猪猪瘟疫苗免疫效力的影响

利用来源于同一猪场的2头猪瘟病毒(HCV)持续感染的带毒母猪及所产35头仔猪(包括13头死胎)和6头阴性对照猪,观察母猪的胎儿发育成活状况、仔猪HCV带毒率及HCV垂直传播对仔猪猪瘟兔化弱毒疫苗(HCLV)免疫效力的干扰作用,同时进行水平传播试验和观察HCV持续感染对母猪繁殖功能的影响。结果表明：HCV持续感染对其中1头母猪的胎儿发育和成活有明显影响,而对另1头母猪的胎儿发育没有明显影响;HCV持续感染母猪可经过胎盘垂直传播病毒给仔猪,传播率达45％～86％;吃初乳和接种HCLV不能阻止带毒仔猪的死亡,9头带毒仔猪在45d内死亡4头;免疫HCLV不能使带毒仔猪产生免疫保护力。5头猪在强毒攻击后死亡4头;HCV垂直传播的带毒猪可发生水平传播,并引起3／4感染猪死亡;HCV持续感染可引起母猪生殖系统病理变化。导致繁殖障碍。     

Prevalence of antibodies to equine viruses in the Netherlands

Summary

The prevalence of antibodies to various viruses was investigated in a series of serum samples collected from horses in the Netherlands between 1963 and 1966 and from 1972 onwards. Neutralizing antibodies to equine rhinopneumonitis virus, equine arteritis virus and to equine rhinovirus types 1 and 2 were detected in respectively 76%, 14%, 66% and 59% of the equine serum samples tested.

The observed incidence of serum samples positive to equine adenovirus in the complement fixation test was 39%. Precipitating antibodies to equine infectious anaemia virus were detected only in serum samples from two horses imported from abroad. Haemagglutination inhibiting antibodies to Myxovirus influenzae A / equi‐1, M. Influenzae A / equi‐2, and Reovirus types 1, 2, and 3 were present in respectively 82%, 50%, 10%, 33% and 3.6% of the serum samples tested.

The most frequently observed incidence of antibodies to the various equine respiratory viruses occurred in the groups of horses having repeatedly contact with other horses.     

重组M蛋白-乳胶凝集试验检测PRRS病毒血清抗体的研究

利用纯化的 PRRS病毒重组 M蛋白致敏乳胶制成乳胶抗原 ,成功地建立了一种检测 PRRS病毒血清抗体的乳胶凝集试验 (L AT)诊断方法。用制备的乳胶 M抗原分别检测猪瘟、猪伪狂犬病、猪细小病毒病、猪弓形体病、猪衣原体病、猪乙型脑炎阳性血清 ,结果均为阴性 ,无交叉反应 ,说明建立的 L AT方法具有良好的特异性。用建立的乳胶凝集试验方法与国外IDEXX公司 PRRS病毒抗体检测试剂盒同时对 76份猪血清样本进行检测 ,结果表明建立的 L AT方法的特异性和敏感性均为 95 % ,两种方法的总符合率为 87% ,检出率基本一致。研究结果表明 L AT方法具有操作简便、快速、敏感性高、特异性强、价格低廉且可用于现场检测等优点 ,是一种适合基层兽医单位用于 PRRS病毒血清抗体检测的新方法     

Serological Studies of African Horse-Sickness Virus with Emphasis on Neutralization Test in Tissue Culture

In place of mice, monkey kidney stable (MS) cell cultures were used successfully in serologic studies of African horse-sickness virus.

The maintenance medium containing 2% serum was chosen as the virus diluent. Maximum neutralization occurred after 1-hour incubation at 37 C., and maintained the same titer during an additional 4-hour incubation period. No significant difference was observed between neutralization titers titrated using the same antiserum mixed with two different passage levels of virus.

Rabbit and guinea pig antiserums prepared using virus grown in MS cell cultures had antibody titer as high as those prepared in the same manner using mouse brain suspension.

African horse-sickness virus strains isolated in Asia were serologically identified using a standard neutralization technique in tissue culture. All the strains were closely related to each other and all had antigenic similarity to Type 6 virus (strain 114).

     

Effect of menhaden fish oil supplementation and lipopolysaccharide exposure on nursery pigs. II. Effects on the immune axis when fed simple or complex diets containing no spray-dried plasma

A trial using 64 weanling pigs (TR4×PIC C22) was conducted to determine the effects of menhaden fish oil supplementation and diet complexity on performance and immune response of nursery pigs. Pigs (17 days and 6.27±1.16 kg) were weaned into a segregated early wean facility and given free access to a complex diet for 7 days post-weaning. At day 0 (day 7 post-weaning), pigs were blocked by weight and allotted to 64 pens. Treatments (Trt) were arranged as a 2×2×2 factorial arrangement. Main effects included diet (complex versus simple), oil (menhaden fish (MFO) versus corn (CO)), and immunogen (saline versus lipopolysaccharide (LPS)). Experimental diets contained 6% oil (6% CO or 5% MFO+1% CO) and were fed for 14 days. On day 12, i.v. injections of either LPS (150 μg/kg) or saline were given, followed by blood collection at 30 min intervals for 6 h. After the immune challenge (day 14), pigs were placed onto a common corn-soybean meal fortified diet and growth performance was evaluated until termination of the study (day 28). Pigs were weighed and feed intakes recorded at 7, 14, and 28 days. Prior to immune challenge (day 12), there were differences in BW for pigs fed complex versus simple diets (P<0.01; 13.1 and 12.1 kg, respectively) and pigs fed CO versus MFO diets (P<0.05; 12.9 and 12.3 kg, respectively). During the challenge period, for pigs treated with LPS there was a Time×Immunogen×Oil effect (P<0.001) for serum cortisol with MFO fed pigs having lower serum cortisol as compared to CO fed pigs. Also, during the challenge period, for pigs treated with LPS there was a Time×Diet×Immunogen×Oil effect (P<0.001) for serum tumor necrosis factor- (TNF-) with pigs fed complex diets supplemented with CO having higher serum TNF- as compared with pigs fed complex diets supplemented with MFO. At days 14 and 28, LPS-treated pigs had lower BW than saline injected controls (P<0.001 and 0.01, respectively). In addition, pigs fed simplified diets continued to have lower BW after challenge compared to pigs fed a complex diet. Interestingly, there were no differences (P>0.10) in BW after challenge in pigs fed MFO. This study suggests that MFO supplementation alters the immune response during LPS challenge and that simplified diets may compromise nursery performance.     

Studies on Trånsmissible Gastroenteritis of Swine: III. The Effect of Selective Inhibitors of Viral Replicåtion on a Cytopathogenic Virus from Transmissible Gastroenteritis

The effect on the plaque production of the Purdue strain of cytopathogenic virus from transmissible gastroenteritis of swine by 5-bromo-2'-deoxyuridine (BUDR), 5-iodo-2'-deoxyuridine (IUDR), actinomycin-D, puromycin, and amantadine-HCI (Symmetral) has been studied.

Amantadine-HCI reduced the plaque-forming units of virus per ml by approximately 98%. Puromycin prevented almost all virus reproduction while actinomycin-D caused approximately a 22% reduction. Both IUDR and BUDR produced approximately a 20% increase in plaque-forming units of virus per ml.

Swine testis cells stained with acridine orange early in the course of infection contained brick-red particles in the cytoplasm, indicative of a ribonucleic acid (RNA) type virus.

     

Immunofluorescence Study of Wild Rabies Virus and Antibody

A concentrate of wild rabies antibody was prepared from hyperimmune serums of three dogs refractory to wild rabies. The animals resisted repeated intramuscular injections of large doses of wild rabies virus in emulsions of whole brain, in emulsions of submaxillary salivary glands, and in emulsified mixtures of brain and submaxillary glands taken from naturally rabid dogs.

The antibody was conjugated with fluorochrome and then absorbed by a procedure that gave “cell-free” working solutions of fluorescent antibody. The procedure entailed parallel absorption steps with minced pathological canine submaxillary glands from (1) naturally rabid dogs (these glands contained specific, undegraded, natural antigens of live wild rabies virus plus nonspecific substances and antigens) and (2) nonrabid dogs from a rabies endemic region (these glands contained nonspecific substances and antigens).

Extracts from submaxillary glands of the three naturally rabid dogs and one nonrabid dog were stained with a cell-free solution of the fluorescent antibody. The glands of the rabid dogs contained fluorescent aggregates of intense green spherical and filamentous particles. When nonfluorescent canine hyperimmune serum was incubated with rabies-containing submaxillary extract, the rabies antigens were quenched. When nonfluorescent equine fixed virus antiserum was incubated with such extracts, the aggregates still retained bright fluorescence.

     

Physicochemical Characterization of a Neonatal Calf Diarrhea Virus

Physicochemical characteristics of two isolates of a neonatal calf diarrhea virus were investigated. Neither isolate was sensitive to ether or chloroform, both were stable at pH 3.0, were relatively heat resistant, but were thermolabile when heated to 50°C for one hour in the presence of 1.0 M MgCl₂. Multiplication of virus was not inhibited by concentrations of 5-iodo-2'-deoxyuridine (IDUR) up to 500 µg/ml, which indicated that the nucleic acid was ribonucleic acid (RNA). Also, multiplication was not inhibited by concentrations of actinomycin D (AD) up to 0.5 µg/ml. Thermal denaturation studies demonstrated that the nucleic acid had a high melting temperature.

Resistance to lipid solvents, stability at an acid pH, relatively high thermostability, type of nucleic acid, plus previous reports from this laboratory on general morphology and cytopathogenicity suggest that the virus may belong to the diplornavirus (reovirus) group. However, thermolability in the presence of 1.0 M MgCl₂ is not consistent with characteristics of this group.

     

Fruit availability,frugivore satiation and seed removal in 2 primate‐dispersed tree species

During a mast‐fruiting event we investigated spatial variability in fruit availability, consumption, and seed removal at two sympatric tree species, Manilkara bidentata and M. huberi (Sapotaceae) at Nouragues Natural Reserve, French Guiana. We addressed the question of how Manilkara density and fruits at the community level might be major causes of variability in feeding assemblages between tree species. We thus explored how the frugivore assemblages differed between forest patches with contrasting relative Manilkara density and fruiting context. During the daytime, Alouatta seniculus was more often observed in M. huberi crowns at Petit Plateau (PP) with the greatest density of Manilkara spp. and the lowest fruit diversity and availability, whereas Cebus apella and Saguinus midas were more often observed in M. bidentata crowns at both Grand Plateau (GP), with a lowest density of M. bidentata and overall greater fruit supply, and PP. Overall, nearly 53% and 15% of the M. bidentata seed crop at GP and PP, respectively, and about 47% of the M. huberi seed crop were removed, otherwise either spit out or defecated beneath trees, or dropped in fruits. Small‐bodied primates concentrated fallen seeds beneath parent trees while large‐bodied primate species removed and dispersed more seeds away from parents. However, among the latter, satiated A. seniculus wasted seeds under conspecific trees at PP. Variations in feeding assemblages, seed removal rates and fates possibly reflected interactions with extra‐generic fruit species at the community level, according to feeding choice, habitat preferences and ranging patterns of primate species.     

Physical, Chemical and Biological Characteristics of Two Bovine Enterovirus Plaque Variants

Large plaque (4LP) and small plaque (4SP) variants were derived from a parent bovine virus strain by serial plaque passage. Both 4LP and 4SP were resistant to chloroform and stabilized at 50°C for one hour by 1.0 M magnesium chloride. Both 4LP and 4SP had buoyant densities in cesium chloride of 1.36 gm/ml. Antigenically, 4LP and 4SP were reciprocally cross neutralizable.

The nucleic acid of 4LP was shown to be ribonucleic acid (RNA) by resistance of its infectivity to deoxynuclease (DNase) but not ribonuclease (RNase) and by increased incorporation of [³H]-uridine into cytoplasmic RNA in cells of virus infected cultures. In growth characteristics, both 4LP and 4SP had maximum adsorption times of 75 to 90 minutes but 4LP had more rapid replication and release rates and yielded nearly twice as many infectious units per cell as 4SP. The differences in growth properties correlated directly with the differential in plaque diameter which was 40-50%.

     

Egg production in relation to the results of a long term serological survey of 73 flocks of fowl

Summary

Seventy‐three flocks of fowl were tested at regular intervals for the presence of precipitins to fowl adenovirus (AV) and infectious bronchitis virus (IBV), haem‐agglutinating inhibiting antibodies to BC14 virus, and of agglutinins to Mycoplasma gallisepticum (M.g.) and Mycoplasma synoviae (M.s.). In all the eight flocks affected with Egg Drop Syndrome (EDS ‘76), egg production problems were associated with increasing numbers of BCI4 virus reactors and AV reactors. In flocks showing production problems other than EDS ‘76 without any apparent cause, the average percentage of AV reactors increased significantly after the rearing period; this was not true of IBV reactors. BC14 reactors were either absent or present only once, in small numbers and with low titres, during the test period. The average percentage of AV reactors did not increase after the rearing period either in normally producing flocks or in flocks with production problems for which other diseases or dietary errors plausibly accounted for these problems. All this suggests a pathogenic role of AV in production problems. One can conclude from the high percentage of reactors in all groups of flocks that sub‐clinical IBV infections are common.

The percentage of IBV reactors during the laying period of flocks with EDS ‘76 was significantly higher than that of normally producing flocks. It is therefore suggested that subclinical IBV infection could be among the factors causing stress, acting as a trigger for EDS ‘76.

All M.g.‐infected flocks showed production problems; M.s. infections could not be related to egg production disturbances.