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1.
The MET95 strain of a lentogenic Newcastle disease virus (NDV) isolated from a broiler in Japan, showed unique hemagglutination (HA) activity. The MET95 strain failed to show HA when examined by rapid glass plate method although they showed HA titer of 1:1,024 by micro-plate method. This unique HA was also observed after the MET95 strain was passaged ten times in chickens. The failure of HA by rapid glass plate method was not shown in any other NDVs examined.  相似文献   

2.
H D Stone 《Avian diseases》1985,29(3):721-728
Hemagglutination (HA) activity was recovered from the aqueous phases of commercially and experimentally prepared oil-emulsion (OE) Newcastle disease virus (NDV) vaccines using two methods: aqueous partition and freeze-thaw. Quantitation of the HA activity retrieved by the aqueous partition technique was directly related to the degree of protection conferred to chickens against a strain of velogenic viscerotropic (VV) NDV in nine of the ten vaccines analyzed. One of the ten vaccines yielded high levels of retrieved HA activity but induced low hemagglutination-inhibition (HI) antibody levels and low levels of protection. Therefore, the retrieval and quantitation of HA activity from OE NDV vaccines using the partition technique provides a useful adjunct to vaccine testing methods that doesn't require vaccination and challenge trials. The freeze-thaw method did not yield measurable HA titers for all vaccines with high efficacy, so its use should be restricted to only those vaccines for which it has been demonstrated to be suitable.  相似文献   

3.
Akabane virus was shown to lyse as well as to agglutinate pigeon erythrocytes. The hemolytic activity of the virus was markedly enhanced by repeated freeze-thawing, but its hemagglutinating activity was not affected. Hemolysis (HL) with the virus, like its hemagglutination, was affected by the NaCl concentration as well as by the pH of the diluent. HL was markedly affected by the incubation temperature, but hemagglutination (HA) was not; HL activity was highest at 37°C, somewhat lower at 25°C, very low at 4°C, and did not occur at 0°C. While pigeon erythrocytes were positive for both HL and HA, goose erythrocytes were positive for HA but negative for HL. Erythrocytes from cattle, sheep, rabbits, guinea pigs, mice and day-old chickens were tested for HA as well as for HL activity with negative results. A linear relationship was shown, in a wide range of the virus concentrations, between the percent HL and the virus concentration, as expressed on a logarithmic scale. Based on these findings we developed an assay method for Akabane virus hemolysin. Analysis by CsCl equilibrium density gradient centrifugation indicated the hemolytic as well as the hemagglutinating activity to be structurally associated with the virion. Scanning electron microscopy of pigeon erythrocytes undergoing HL with the virus revealed the appearance of a depressed area with a hole on the cell surface. The hemolytic activity of the virus was specifically inhibited by antisera to the virus and an HL-inhibition test was developed.  相似文献   

4.
The effects of formaldehyde, betapropiolactone (BPL), acetylethylenimine (AEI), and a deprived ionic environment on haemagglutinin (HA) and neuraminidase (NA) bound to PIV-3 were tested. Formalin elicited an increased NA activity and a significant decrease in the HA titres on three PIV-3 strains tested. The formalin-treated PIV-3 could be used in the neuraminidase inhibition (NI) test. The increased NA activity was due to an increased stability of the enzyme active sites of PIV-3. AEI and BPL did not affect the HA, but BPL caused an increased NA activity of a neuraminidase-weak strain.Following dialysis of PIV-3 against distilled water, an increased affinity of the virus-bound NA to the substrate sialolactose was found. The infectivity titres of PIV-3 were not altered by the dialysis, and virus preparations treated in this manner could be used in the NI-test. UV-treatment of PIV-3 resulted in a loss of infectivity and a loss of HA and NA activities.  相似文献   

5.
D J King 《Avian diseases》1984,28(2):504-513
A total of 166 infectious bronchitis virus (IBV) hemagglutination (HA) antigen preparations were made during a 30-month period from allanto-amnionic fluid (AAF) from chicken embryos inoculated with 10 different IBV strains (Mass 41, Conn 46, H52, Florida 18288, Ark 99, JMK, T, Holte, EF, SE17). Antigens were prepared by inoculating 9- or 10-day-old embryos with 10(5.0) to 10(6.5) EID50 IBV, harvesting AAF after a 30-hour-postinoculation incubation, and phospholipase C (PLC) treatment of virus concentrated by pelleting from the AAF. Longer (48 hr) incubation times were tried, but production of H52 HA antigen was successful only from AAF harvested after 30 hours of incubation. AAF from JMK-infected embryos had lower infectivity titers and frequently yielded lower HA antigen titers than the other strains. The treatment of AAF with fluorocarbon did not enhance or diminish HA activity but did yield clearer antigens by removing extraneous material. Polyethylene glycol precipitation of virus was an acceptable alternative to pelleting virus at 39,000 X g. Inactivation of IBV with 0.1% betapropiolactone did not affect HA activity, whereas inactivation with 0.1% formalin caused a marked reduction in HA titer. Different buffer formulations including phosphate, tris, or HEPES were tried to optimize the conditions for PLC treatment of virus concentrate, but there were no apparent differences in the antigens prepared in the different buffers. The HA antigen preparations were stored and were stable at 4 C. Antigen titers of greater than or equal to 64 after storage for 20 months or longer were not uncommon. Addition of merthiolate as a preservative had no deleterious effect on HA activity. Antigen stability appeared to be enhanced by incorporating EDTA in buffer for virus pellet recovery and during enzyme treatment. Attempts to produce HA antigens from cell-culture-adapted virus propagated in chicken kidney cells were less satisfactory. An acceptable HA antigen was prepared from only two (Mass 41, SE17) of the seven strains that were tried. Virus propagation in chicken embryos is the better method of the two for IBV HA antigen production. Aside from the need to concentrate virus and treat the concentrate with PLC, there appeared to be considerable latitude in the procedures that can be used to make acceptable IBV HA antigens.  相似文献   

6.
K R Rhoades 《Avian diseases》1985,29(4):1170-1176
Hemagglutinating (HA) and nonhemagglutinating (NHA) variants were selected from each of two strains of Mycoplasma synoviae, WVU-1853 and Neb-3S. The HA titers of antigens prepared by 100-fold concentration of broth cultures were 1:2560 and 1:5120 for the HA variants and less than 1:5 for the NHA variants. Adsorption of erythrocytes to colonies of the variants was directly correlated with HA activity. The HA and NHA characteristics were stable in vitro, and there was no change in HA titers after repeated transfers in broth medium. Comparisons of pathogenicity indicated differences between strains but not between variants of each strain. Air-sac lesions resulting from exposure to variants of strain Neb-3S were marked, whereas those resulting from exposure to variants of WVU-1853 were slight. The HA titers of isolates recovered from turkey air sacs exposed to the Neb-3S variants varied considerably, suggesting in vivo instability of the HA characteristic.  相似文献   

7.
Characteristics of 2 encephalomyocarditis virus (EMCV) isolates (MN-25 and MN-30) recovered from aborted swine fetuses were examined along with 2 other swine isolates (NVSL-MDV and NVSL-PR) and a reference ATCC strain (VR-129). All 5 EMCV isolates were found to be serologically related by cross testing, using serum neutralization and fluorescent antibody assays. Hemagglutination (HA) properties of the 5 isolates were compared, using 5 diluents. The MN-25 and NVSL-MDV strains had HA activity with guinea pig RBC in all 5 diluents, whereas MN-30, NVSL-PR, and VR-129 had HA activity only in KCl-borate buffer. The HA ability with RBC of various animal species was examined, using KCl-borate diluent. All virus isolates had high HA titer (1:512 to 1:2,048) with guinea pig, rat, and horse RBC and lower HA titer (1:16 to 1:64) with sheep RBC. The MN-25 and NVSL-MDV isolates agglutinated dogs RBC, whereas MN-30, NVSL-PR, and VR-129 strains did not. Viral replication was evident in 8 of 10 cell lines tested, although infectivity titers of each virus varied by cell line used. Plaque-forming ability was similar for all 5 isolates, but plaque size was different by virus and cell culture used. Virus isolates were found to be stable after being heated at 56 C and subjected to a wide range of pH. A viral polypeptide pattern difference for all 5 isolates was not found by use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

8.
Pseudorabies virus hemagglutinin was readily adsorbed on mouse erythrocytes at 4, 22, or 37 degrees C, but not on cattle erythrocytes. The adsorbed hemagglutinin could not be eluted from the cells by resuspending in phosphate-buffered saline (PBS), by incubating at 37 or 50 degrees C, or by incubating in the presence of neuraminidase. The receptor on mouse erythrocytes for the hemagglutinin was inactivated by trypsin, but not by neuraminidase, sodium deoxycholate (DOC), potassium periodate (KIO4), dithiothreitol (DTT), 2-mercaptoethanol (2-ME) and formalin. The hemagglutinin was inactivated by trypsin, alpha-amylase, pepsin, DOC, KIO4, and ethylendiamine-tetraacetic acid (EDTA), but not by papain, beta-glucosidase, phospholipase C, neuraminidase, DTT, 2-ME, Tween-80, ethylether, chloroform, trichloro-trifluoroethane, beta-propiolactone and formalin, suggesting that the hemagglutinin active component involved glycoproteins. The hemagglutinin was stable at 37 degrees C for lower temperatures but not at 60 degrees C or higher. The hemagglutinin activity was resistant to ultraviolet irradiation, while the infectivity was very susceptible. The hemagglutinin and the infectivity were readily sedimented by ultracentrifugation at 48,000 x g for 3 hr. In rate zonal centrifugation of the preparation on a sucrose density gradient, the hemagglutination (HA) activity showed a sharp peak at 1.22 g/ml coinciding with the peak of infectivity. The HA activity in the peak fraction seemed to be structually associated with virus particles. After fractionation of the virus by Nonidet P-40, the HA activity was found only in the fraction of the envelope material, indicating that the hemagglutinin is situated in the viral envelop.  相似文献   

9.
Hemagglutination (HA) activity of feline herpesvirus type 1 (FHV-1) propagated in feline lung cell culture and two established feline cell lines, CRFK and fcwf-4, was investigated. Intra- and extracellular crude samples obtained from those infected cell cultures did not show HA activity. However, when treated with tween 80-ether, HA activity appeared. There was no correlation between virus infectivity titers and the HA titers at various harvesting times, and besides, hemagglutinins were found in intracellular samples at the early stage of infection. By ultrasonic destruction of the infected fcwf-4 cells, high titer hemagglutinins were obtained. High titer hemagglutinins were also extracted successfully from infected fcwf-4 cell membranes by solubilization with any of the three detergents: Triton X-100, DOC, and CHAPS. The optimal concentrations of each detergent for solubilizing hemagglutinin were 0.05 (v/v)%, 0.5 (w/v)%, and 0.1-0.2 (w/v)%, respectively. The HA activities of both the ultrasonic-treated hemagglutinin and the detergent-soluble hemagglutinin from infected fcwf-4 cells were inhibited specifically by anti-FHV-1 sera. Therefore, either hemagglutinin could be used as HA antigen for the hemagglutination-inhibition test.  相似文献   

10.
A soybean lecithin‐based extender supplemented with hyaluronic acid (HA) was assayed for effectiveness to improve the quality of frozen–thawed ram semen. HA has not been tested yet in an extender containing soybean lecithin for freezing ram semen. Thus, the aim of this study was to analyse the effects of soybean lecithin at 1% or 1.5% along with HA at 0, 0.5 and 1 mg ml‐1 in a Tris‐based extender on the motion characteristics, membrane integrity (HOST), viability, GSH peroxidase (GSH‐PX) activity, lipid peroxidation and acrosomal status after freezing–thawing. Semen was collected from four Mehraban rams during the breeding season and frozen in the six lecithin×HA extenders. The extender containing 1.5% lecithin supplemented with no HA yielded higher total motility (52.5%±1.6), viability (55.8%±1.6) and membrane integrity (44.5%±1.7), but the effects of the lecithin concentration did not reach signification. Linearity‐related parameters, ALH, BCF, lipid peroxidation, GSH‐PX activity, morphology and acrosomal status were not affected by the extender composition. In general, adding HA significantly decreased sperm velocity (1 mg ml‐1 HA), total motility (only with 1.5% lecithin), viability (1 mg ml‐1 HA for 1% lecithin; both concentrations for 1.5% lecithin) and membrane integrity. In conclusion, adding HA to the freezing extender supplemented with soybean lecithin failed to improve quality‐related variables in ram semen. Increasing the lecithin content could have a positive effect, but further studies are needed.  相似文献   

11.
The erythrocytes of various species were tested in psittacine beak and feather disease (PBFD) virus haemagglutination (HA) and haemagglutination inhibition assays to determine which are suitable for use in these assays. HA activity was observed for erythrocytes of the salmon-crested cockatoo, the sulphur-crested cockatoo, the umbrella cockatoo, the goffin's cockatoo and the cockatiel, with differences amongst individuals within species, but not for erythrocytes of humans, the pig, the guinea pig, the chicken, the goose, the rose-ringed parakeet or the budgerigar. Anti-PBFD virus rabbit sera inhibited the virus-induced agglutination of erythrocytes, confirming the specificity of HA activity. This suggests that selection of suitable psittacine species as well as suitable individuals within a species is necessary when obtaining erythrocytes for the PBFD virus HA assay.  相似文献   

12.
Postoperative abdominal adhesions are known to present clinical challenges to the surgeon. Adhesion formation is a balance modulated by the fibrinolytic system. The key components involved are the tissue plasminogen activators (tPAs) and plasminogen activator inhibitors (PAI-1 and PAI-2). Sodium hyaluronate (HA) has been shown to reduce the incidence and severity of adhesions in horses. The objectives of this study were to measure tPA and PAI-1 activity in equine peritoneum and evaluate the effect of 0.4% HA solution on local tPA and PAI-1 activity. An exploratory laparotomy was performed and local serosal trauma was induced by using an established abrasion model. Our study involved two groups: in the first group (n = 6) 0.4% HA was used in all intestinal manipulations, whereas in the second group (n = 6) sterile saline was used. Parietal peritoneum, jejunal seromuscular biopsies at abraded sites (AJ) and nonabraded sites, and peritoneal fluid samples were taken at time 0- and at 30-minute intervals up to 120 minutes. Peritoneum tPA activity was significantly decreased at 60 and 90 minutes. Interestingly, AJ contained significantly higher tPA activity than nonabraded sites at 30-, 60-, 90-, and 120-minute intervals in control horses. The increase in tPA activity with AJ in treated (HA) horses was significantly attenuated as compared with the control (saline). Detectable levels of PAI-1 activity could not be identified in our samples. The results of our study indicate that exploratory celiotomy in horses is associated with a significant decrease in peritoneal tPA activity, and HA significantly decreases the fibrinolytic response of the jejunum to surgical trauma. Further characterization of these responses will hopefully lead to new pharmacologic strategies for adhesion prevention.  相似文献   

13.
经RT-PCR扩增了禽流感病毒A/PFV／Restock／1／34（H7N1）1．7kbHA基因的cDNA，将其克隆到pMD18-T中并测序。在去除编码HA信号肽的核苷酸序列后，亚克隆到杆状病毒转移载体pBlueBacHis，筛选到重组质粒命名为pBlueBacHisH7HA，测序正确后与线性化的杆状病毒DNA（Bac-N-Blue^TMDNA）共转染sf9昆虫细胞，挑取蓝色蚀斑，经三轮蚀斑纯化，获得数株重组杆状病毒rpBlueHisH7HA。提取重组病毒DNA，经PCR证明目的基因片段已插入到杆状病毒基因组中，血凝实验、SDS-PAGE和Western blot实验结果表明HA基因在重组杆状病毒感染的HFive细胞中获得了表达。  相似文献   

14.
利用RT-PCR方法扩增猪流感病毒A/Swine/Henan/11/2005(H1N1)血凝素(HA)基因,克隆于pMD18-T载体进行测序。以pMD18-HA为模板、利用带酶切位点的引物再次扩增HA基因的开放阅读框(ORF),克隆到表达载体pET32a中,经双酶切、测序及PCR鉴定得到阳性重组表达质粒pET-HA,将质粒转化到表达宿主菌BL21(DE3)中,经终浓度为1 mmol/L的IPTG诱导,SDS-PAGE结果显示,HA蛋白获得了高效表达,经Western-blot检测证实表达产物具有良好的免疫学活性,在间接ELISA中的初步应用表明具有良好的抗原反应性。本研究为以重组HA蛋白为抗原建立H1亚型猪流感抗体的ELISA检测方法奠定了基础。  相似文献   

15.
ＩＢＶ毒株Ｈ１２０、Ｈ５２、ＭＡ５对ＮＤＶ－ＬａＳｏｔａ的干扰实验证明ＩＢＶ特异性干扰ＮＤＶ增殖。１）采取不同顺序的同胚接种法：ＩＢＶ接种之后再接种ＮＤＶ;或ＮＤＶ接种之后再接种ＩＢＶ,及ＩＢＶ,ＮＤＶ同时接种,ＩＢＶ均干扰ＮＤＶ的增殖。２）ＮＤＶ接种３６小时之内,干扰现象最为明显,Ｈ１２０,Ｈ５２的干扰能力稍强于ＭＡ５。３）ＮＤＶ血清中和实验结果显示,不同顺序同胚接种ＮＤＶ、ＩＢＶ时,ＮＤＶ－ＬａＳｏｔａ不影响ＩＢＶ的增殖能力。同胚增殖ＩＢＶ,ＮＤＶ的关键是控制ＮＤＶ、ＩＢＶ的接毒量及选择合适的收毒时间。  相似文献   

16.
Seventy-two isolates of Haemophilus paragallinarum were serotyped according to the Page scheme, using a new hemagglutination-inhibition (HI) test. The results were compared with the plate agglutination method conventionally used in the Page scheme. The HI test used washed cells of H. paragallinarum, glutaraldehyde-fixed chicken erythrocytes, and rabbit antisera originally produced for the agglutination method. For 49 of the isolates, there was complete correlation between the results of the HI serotyping test and the previously performed agglutination test--23 were serovar A, two were serovar B, and 24 were serovar C. The other 23 isolates were nontypable by the agglutination test, but 21 of them could be serotyped by the HI method--six as serovar A, two as serovar B, and 13 as serovar C. Nine isolates required treatment of the bacterial cells with hyaluronidase for the expression of hemagglutination (HA) activity. Two isolates did not have HA activity despite hyaluronidase treatment and so could not be serotyped by the HI test.  相似文献   

17.
利用RT-PCR扩增猪流感病毒A/Swine/Zhejiang/7/2004(H9N2)(SW/ZhJ7/04)HA基因,测序后将其克隆到真核表达载体pCAGGS上,经酶切分析、PCR鉴定和测序验证,得到重组表达质粒pCAGGS-HA,之后将其转染293T细胞,48h后收集转染细胞样品,进行SDS-PAGE分析,结果显示HA蛋白获得了表达。经Western blot检测,结果表明pCAGGS-HA能够在体外瞬时表达具有免疫学活性的HA蛋白,通过免疫小鼠,对其免疫效果进行了初步研究。  相似文献   

18.
血凝素是副鸡嗜血杆菌（Haemophilus paragallinarum，Hpg）的主要抗原成分之一，鸡只对Hpg的免疫力与血凝素抗体滴度成正相关。根据GenBank上已发表的B型Hpg Dalian株的血凝素基因序列（AY622378），设计合成了1对特异扩增Hpg血凝素基因的引物。以大连分离株Hpg中提取的细菌DNA为模板，利用PCR扩增出了1038bp的血凝素基因，将其克隆到pET-32a载体上，构建了pET-HA原核表达质粒并在BL21。大肠杆菌中过量表达。血凝试验显示纯化的重组蛋白具有血凝活性；Western试验证明该重组蛋白可以被B型Hpg特异性鸡血清所识别。本研究在国内首次对Hpg的血凝素基因进行克隆表达，并分析了重组蛋白的血凝活性。  相似文献   

19.
提高鸡传染性支气管炎病毒血凝抗原滴度途径及抗原灭活   总被引：4，自引：0，他引：4  
将７株传染性支气管炎病毒（ＩＢＶ）标准株（Ｍ４１、Ｈ１２０、Ｈｏｌｔｅ、Ｇｒａｙ、Ｃｏｎｎｅｃｔｉｃｕｔ、Ｉｏｗａ６０９和Ｔ株）和６株分离株（ＮＩＢＶ、ＧＩＢＶ、Ｍ、ＳＨ、Ｊ和Ｈ株）分别接种于鸡胚，收获尿囊液，经浓缩后，用魏氏梭菌培养液处理，制备血凝抗原。其中，Ｈ１２０株血凝滴度最高，Ｔ、Ｍ、Ｊ和Ｈ株无血凝性。应用含有不同滴度ＩＢＶ母源抗体的鸡胚增殖病毒制备抗原，效价与用ＳＰＦ鸡胚增殖病毒制备的抗原效价一致。尿囊液经反复冻融后再制备抗原会使血凝价降低。抗原分别用甲醛、高碘酸钠、硼氢化钾和ＳＤＳ灭活，其中甲醛灭活效果最理想。抗原对氯仿敏感，对乙醚稳定。适宜浓度的Ｎａ＋、Ｍｇ２＋可显著提高抗原的血凝性  相似文献   

20.
The interference phenomenon of infectious bronchitis virus (IBV) with growth of Newcastle disease virus (NDV) in embryonating chicken eggs (ECE) was used as a diagnostic method. Fifteen field isolates obtained from presumptively infectious-bronchitis-affected chickens were analyzed by the IBV-NDV interference test. Eight isolates were capable of interfering with the growth of the La Sota strain of NDV, as measured by hemagglutination (HA) activity when IBV was inoculated 10 hr before NDV into ECE. The interference was considered specific for IBV, because it could be eliminated by adding homologous anti-IBV serum. The sensibility of this method could be demonstrated, because in some cases low-passage levels of IBV isolates showing HA interference ability were not capable of producing lesions in ECE. Furthermore, serologically negative IBV samples did not interfere with NDV growth. From these results, the IBV-NDV interference test appears to be a potential diagnostic alternative for identifying IBV field isolates.  相似文献   

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