Preparation and diagnostic utility of a hemagglutination inhibition test antigen derived from the baculovirus-expressed hemagglutinin-neuraminidase protein gene of Newcastle disease virus

A recombinant hemagglutinin-neuraminidase (rHN) protein from Newcastle disease virus (NDV) with hemagglutination (HA) activity was expressed in Spodoptera frugiperda cells using a baculovirus expression system. The rHN protein extracted from infected cells was used as an antigen in a hemagglutination inhibition (HI) test for the detection and titration of NDV-specific antibodies present in chicken sera. The rHN antigen produced high HA titers of 2¹³ per 25 µL, which were similar to those of the NDV antigen produced using chicken eggs, and it remained stable without significant loss of the HA activity for at least 12 weeks at 4℃. The rHN-based HI assay specifically detected NDV antibodies, but not the sera of other avian pathogens, with a specificity and sensitivity of 100% and 98.0%, respectively, in known positive and negative chicken sera (n = 430). Compared with an NDV-based HI assay, the rHN-based HI assay had a relative sensitivity and specificity of 96.1% and 95.5%, respectively, when applied to field chicken sera. The HI titers of the rHN-based HI assay were highly correlated with those in an NDV-based HI assay (r = 0.927). Overall, these results indicate that rHN protein provides a useful alternative to NDV antigen in HI assays.     

The Detection of Specific Complement-Fixing Antibodies in Serum of White-Tailed Deer (Odocqileus Virginianus) with Theileria Infection

A complement-fixation (CF) antigen which has been prepared from Theileria infected erythrocytes is capable of reacting to specific serum antibodies of deer acutely infected with Theileria.

No sera from 17 deer known to be free of Theileria infection reacted positively to the CF test. Of 35 tests on sera from 12 infected deer having a parasitemia of 2% or less and no accompanying anemia, only 10 (29%) were positive, 2 (6%) were suspicious, and 23 (66%) were negative. Of 65 tests on 8 acutely infected deer, 49 (75%) were positive, 4 (6%) were suspicious and 12 (18%) were negative. Of the 8 deer in which acute theileriasis occurred all reacted to Theileria antigen at one time or another.

A significant correlation was found between CF titers and the degree of parasitemia in acute infections.

Rabbits were hyperimmunized using erythrocytes from either normal or Theileria infected deer. Reciprocal absorption of the hyperimmune sera with Theileria and normal erythrocytic antigens demonstrated the presence of antibodies specific for Theileria.

     

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.

     

Evaluation of different antigens in the complement-fixation test for diagnosis of Haemophilus pleuropneumoniae (parahaemolyticus) infections in swine

The identification of new serotypes of Haemophilus pleuropneumoniae (parahaemolyticus) and the frequency of pleural adhesions due to contagious pleuropneumonia in many fattening swine herds have prompted the study of the complement-fixation (CF) test as a diagnostic tool for use in swine. Whole cell antigens, mixed antigens, autoclaved antigens, and phenol-water-extracted antigens derived from different serotypes were prepared and tested with immunized-swine sera by the CF test. Mixed antigen consisting of whole cells from all known serotypes was the best screening antigen for routine use. This antigen gave positive titers with all sera in which a positive reaction against the separate serotype antigen was registered. The most highly serotype-specific reactions were obtained with antigens prepared by phenol-water extractions of whole cells. When whole-cell antigens were used in the CF test, antibodies to superficial serotype-specific and common species-specific antigens could be detected.     

The Effect of Unheated Normal Bovine Serum on The Complement Fixing Activity of Heat Inactivated Bovine Antiserum With Homologous Antigen: II. Aggregative Properties

To determine whether the greater fixation of complement observed in “modified” complement-fixation tests is related to an increased aggregation of the antigen-antibody complexes, parallel tests by the two methods have been made with two different particulate bacterial antigens and corresponding bovine antisera. At the end of the fixation period the mixtures were centrifuged, the supernatant fluids removed carefully, the sediments washed twice and re-suspended in a small volume of buffer. Smears of each sediment were examined by immunofluorescence microscopy using a fluorescein-labelled rabbit antibody for a globulin fraction of fresh guinea-pig serum containing first component of complement.

A greater degree of aggregation of the antigen-antibody complexes was observed in the sediments from tests with modified complement, that is complement supplemented with a diluted bovine serum globulin fraction prepared by dialysis. Aggregates from mixtures showing increased fixation of complement, as determined by titration of the residual hemolytic activity of the supernatant, appeared somewhat more brightly fluorescent.

Very faint or no fluorescence was evident in the stained washed sediments from mixtures of antigen and antibody without complement or from mixtures of antigen, heat-inactivated normal bovine serum and complement.

     

猪细小病毒血凝抑制试验抗原、阳性血清与阴性血清的制备与鉴定

猪细小病毒（Porcine Parvovirus,PPV）血凝抑制试验抗原、阳性血清和阴性血清在猪细小病毒制品的效力检验中不可或缺,对猪细小病毒相关生物制品的质量控制至关重要。试验采用PPV 7909株病毒同步接种PK-15细胞制备血凝抑制试验抗原,用制备的抗原乳化后免疫豚鼠制备阳性血清,同时用未免疫的阴性豚鼠制备阴性血清。对抗原、阳性血清和阴性血清进行鉴定,结果表明,制备的PPV血凝抑制试验抗原HA效价达1:512;阳性血清HI效价达1:1024;阴性血清HI效价＜1:8,且特异性均良好。利用制备的血凝抑制试验抗原、阳性血清与不同PPV疫苗株的灭活抗原、阳性血清进行交叉反应试验,结果表明,制备的抗原与阳性血清具备良好的血清学交叉反应性,可用于PPV制品的统一评价。     

DNA免疫技术制备H10亚型禽流感病毒HA蛋白单因子血清的研究

为优化H10亚型禽流感病毒快速检测方法,按照鸡偏嗜性密码子将A/Jiangxi/IPB13/2013(H10N8)的血凝素(HA)基因序列优化后人工合成,克隆至真核表达载体pCAGGs。将重组质粒双酶切后测序证明该质粒构建成功。用200μg的表达载体免疫6周龄SPF鸡,分别在第一次免疫后的第30天和第60天用相同剂量实施第二次和第三次免疫,末次免疫后的第10天心脏采血,分离血清,制备单因子血清。间接免疫荧光试验和western blot试验证明HA蛋白成功表达,单因子血清制备成功。血凝抑制试验结果表明抗体效价≥128,单因子血清特异性强,与其他亚型禽流感病毒无交叉反应。该研究为H10亚型流感病毒快速鉴定和研究奠定了基础。     

A Study of Complement-fixation and Serum Agglutination Tests on Vibrio fetus Infected and Immunized Cattle

Complement-fixation (CF) and tube agglutination (TA) tests for demonstration of Vibrio fetus antibodies were conducted on the sera of three groups of ten heifers. One group was vaccinated subcutaneously with a commercial V. fetus var venerealis bacterin and challenged intra-utero, at the external os cervicus one month later; the second was infected only and the third vaccinated only.

The vaccinated cattle developed high CF serum titers, but no such increase was observed in animals infected only. A moderate increase in serum antibody titers was demonstrated by the TA test following either infection or vaccination; although titers observed were not higher than those observed in the sera of some apparently normal uninfected animals. The group receiving both vaccine and challenge was the only one in which significant serum antibody titers were demonstrable by the TA test. The sera of these animals also had significant titers in the CF tests.

The CF and TA tests detected serum antibodies produced by the parenteral inoculation of V. fetus antigen. These two tests were of limited value in detecting serum antibodies from animals with genital V. fetus var venerealis infection, although the formation of local antibodies was demonstrable by the vaginal mucus agglutination test.

     

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.

     

禽流感病毒H9亚型血凝抑制试验抗原国家参考品的研制

按照国家一级标准物质求,研制了一批禽流感病毒H9亚型血凝抑制试验抗原国家参考品,批号为200901。对其物理性状、无菌、效价、特异性、水分、真空度、均一性、稳定性等进行了测定,各项指标均符合求。经过8家单位对其协作标定,并经统计学分析,该批抗原血凝效价为1：（460±80）,最终定值为1：400。该禽流感病毒H9亚型血凝抑制试验抗原可以作为国家参考品。     

Serological Studies of Parasite Cattle: I. Complement-Fixing Activity of Serial Serum Samples

Complement-fixation (CF) tests with sonicated aqueous extracts of adult forms of five species of gastrointestinal nematodes as antigens have been made on serial biweekly bleedings from two groups of parasitized calves grazing on infected pastures during the 1963 or 1964 seasons. Three of these nematode species, Cooperia oncophora, Ostertagia ostertagi and Nematodirus helvetianus, were found in large numbers in most of these animals. The calves were negative serologically before being placed on pasture but within 2 to 4 weeks some had developed considerable CF activity with Cooperia and other nematode antigens. Ten calves died, however, before significant CF titres had been attained. Eight control calves in the 1963 or 1964 groups which were grazed on “clean” pastures, remained serologically negative during the summer and autumn months. Four of the six surviving exposed animals in the 1964 group showed a fall in CF activity during the winter months when they were stabled, whereas the six surviving controls developed low CF titres suggesting that they were becoming mildly parasitized. In the spring of 1965 CF titres began to rise in some of the previously exposed and control yearlings even before they were placed on infected pasture in June.     

禽流感病毒H5亚型血凝抑制试验抗原国家参考品的研制

按照国家一级标准物质要求和禽流感病毒（H5亚型）血凝抑制试验抗原制造及检验规程规定的抗原制备方法,研制了一批禽流感病毒（H5亚型）血凝抑制抗原国家参考品,用于检测禽流感病毒（H5亚型）制品生物效价和血凝抑制操作过程中的校验。该批抗原经过8家单位对其协作标定后对结果进行统计学分析,确定该批禽流感抗原的血凝效价为1：80。另对所研制的禽流感抗原均一性、稳定性、无菌检验、剩余水分测定、真空度测定等各项指标进行检测,结果均符合禽流感病毒（H5亚型）血凝抑制试验抗原制造及检验规程和国家一级标准物质要求。说明该禽流感病毒（H5亚型）血凝抑制抗原可以作为国家参考品。     

Observations on the preparation and stability of infectious bronchitis virus hemagglutination antigen from virus propagated in chicken embryos and chicken kidney cell cultures

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.     

Comparisons of the serologic response of chickens to Pasteurella multocida and its lipopolysaccharide

Chickens were inoculated with serotype 3 Pasteurella multocida cells or purified lipopolysaccharide (LPS), and their serologic responses to LPS and heat-stable antigens of 16 serotypes were compared. Chickens inoculated with cells or LPS had antibodies against LPS as determined by indirect hemagglutination tests; titers were highest 2-4 weeks after the initial inoculation. Sera from chickens inoculated with cells reacted with unheated and heated cell antigen in a tube-agglutination test. Sera from chickens inoculated with LPS reacted only with heated cell antigen in the tube-agglutination test. Nonspecific reactions with heat-stable antigens of other serotypes occurred in the gel-diffusion-precipitin test with sera from chickens inoculated with cells but not with sera from chickens inoculated with LPS. Antisera prepared against LPS could be used for serotyping field isolates of P. multocida.     

Complement-Fixing And Neutralizing Antibody Response To Bovine Viral Diarrhea And Hog Cholera Antigens

In calves inoculated with bovine viral diarrhea (BVD) viruses and soluble antigen, the complement-fixing (CF) antibodies appeared before serum-neutralizing (SN) antibodies and remained at high levels throughout the test period. A rapid rise in SN antibodies occurred after challenge with homologous virus with no apparent effect on CF antibody levels.

The CF antibody responses in calves infected with cytopathogenic NADL-MD and noncytopathogenic CG-1220 viruses were similar whereas SN antibody responses indicated strain specificity by reciprocal cross-neutralization tests.

The CF antibody levels in 5 hog cholera (HC) antisera were assayed using the soluble antigen of NADL-MD BVD virus. No demonstrable SN antibodies were present in four HC antisera tested against NADL-MD virus, but a significant titer was present in the commercially prepared antiserum.

Virus was reisolated from animals infected with BVD viruses by buffy coat culture technique during 3 weeks postinoculation, even when significant levels of CF and SN antibodies were present.

     

Subunit influenza vaccine candidate based on CD154 fused to HAH5 increases the antibody titers and cellular immune response in chickens

World Health Organization has a great concern about the spreading of avian influenza virus H5N1. To counteract its massive spread, poultry vaccination is highly recommended together with biosecurity measures. In our study, a recombinant vaccine candidate based on the fusion of extracellular segments of hemagglutinin (HA) H5 of avian influenza virus and chicken CD154 (HACD) is tested with the aim of enhancing humoral and cellular immune responses in chickens. Protein expression was carried out by transducing several mammalian cell lines with recombinant adenoviral vectors. HACD purification was assessed by three distinct purification protocols: immunoaffinity chromatography by elution at acidic pH or with a chaotropic agent and size exclusion chromatography. Humoral and cellular immune responses were measured using the hemagglutination inhibition assay and the semiquantitative real time PCR, respectively. The results showed that humoral response against HACD was significantly higher than the obtained with HA alone after booster (P<0.01, P<0.05). From HACD molecules purified by distinct protocols, only the obtained by size exclusion chromatography generated hemagglutinationin-inhibition activity. IFN-γ levels indicated that cellular immune response was significantly higher with HACD, in its pure or impure form, compared to its counterpart HA (P<0.01). These data demonstrate that HACD is able to significantly enhance humoral and cellular immune responses against HA antigen, which make this fusion protein a promising subunit vaccine candidate against H5N1 virus outbreaks.     

Serological relationship of a Japanese Babesia species and Babesia bigemina by the complement fixation and capillary-tube agglutination tests

The complement fixation (CF) test and the capillary-tube agglutination (CA) test were used to study the antigenic relationship between Babesia bigemina and the large Babesia species frequently infecting cattle in Japan. The CF antigen was prepared from parasitized erythrocytes by extraction with distilled water. The CA antigen was prepared from parasitized erythrocytes by mild sonification of mixtures of Babesia and erythrocyte stroma, following lysis of the erythrocytes with hypotonic saline solution. All the sera used were collected from experimentally-infected cattle. Cross reaction was demonstrated between the Japanese Babesia species and B. bigemina. There was, however, a difference of two dilutions in titer between homologous and heterologous antibody in the CF test, and a difference of more than three tubes in titer between both antibodies in the CA test. It was possible, therefore, to distinguish the Japanese Babesia species from B. bigemina by the CF and CA tests.     

Serological cross-reactivity of porcine reference antisera to Mycoplasma hyopneumoniae, M. flocculare, M. hyorhinis and M. hyosynoviae indicated by the enzyme-linked immunosorbent assay, complement fixation and indirect hemagglutination tests.

The enzyme-linked immunosorbent assay (ELISA) indicated significant cross-reactivity between the antigens of Mycoplasma hyopneumoniae ( HyoP ) and M. flocculare (Floc), another porcine mycoplasma of wide distribution but uncertain pathogenic significance, when porcine antisera of each specificity were tested against HyoP antigen. The titers of the anti-Floc sera ranged from threefold to 13-fold less than the titer of the anti- HyoP reference serum at different times after immunization. These values ranged from onefold less than to fourfold greater than the minimal positive titer of 80. The antisera to the other porcine mycoplasmal antigens [i.e. M. hyorhinis ( HyoR ) and M. hyosynoviae ( HyoS )] reacted less strongly to HyoP antigen but titers only slightly less than to slightly greater than the minimal positive titer were noted for some sera. Cross-reactivity was also detected by the complement fixation test, although the titers for this test were generally lower than for the ELISA, presumably reflecting lower sensitivity of the complement fixation test. Positive indirect hemagglutination titers to HyoP antigen were also observed for both anti-Floc sera obtained at one or more times during the immune response. With two exceptions (one anti- HyoR serum with a complement fixation titer of 16 and one anti- HyoR serum with an indirect hemagglutination titer of 10), none of the anti- HyoR or anti- HyoS sera had detectable indirect hemagglutination or complement fixation titers to HyoP antigen at any time after immunization. The levels of cross-reactivity detected by the complement fixation test and indirect hemagglutination and, especially, the ELISA would be of significance for the development of any practical sero-diagnostic test for mycoplasmal pneumonia of swine.     

National survey to determine uniformity of serological test results for avian mycoplasmosis

A national survey was conducted to address the concern over the uniformity of serological test results for avian mycoplasmosis. The National Veterinary Services Laboratories produced chicken and turkey mycoplasma serum-check test kits. Each kit had a total of 25 sera that consisted of negative and positive sera. Participating laboratories were requested to examine their kits using the serum plate agglutination test with the plate antigens currently being used and the hemagglutination inhibition test with the hemagglutinating antigens provided. A conclusion whether serum plate agglutination-positive sera were positive, negative, or suspicious was based on the hemagglutination inhibition test results. Results in each category were scored on the basis of 100 points. The average scores on the serum plate agglutination test, hemagglutination inhibition test, and conclusion were 95, 71, and 91, respectively, for the chicken serum test kit and 91, 71, and 89, respectively, for the turkey serum test kit. The results indicated a high degree of uniformity among laboratories in reporting serological test results for mycoplasmosis in chickens and turkeys.     

II. Detection of the Virus in Swine Tissues by Means of the Modified Direct Complement-Fixation Test

The modified direct complement-fixation test, supplemented with unheated normal calf serum, was used to demonstrate antibodies in sera of swine immunized to African swine fever virus. These antibodies did not react in the ordinary direct non-supplemented complement-fixation test.

African swine fever complement-fixing antigen in infected swine tissue is not denatured by extraction with fat solvents. Consequently, good antigens devoid of non-specific reactivity were obtained by extraction with a mixture of acetone and ether.

The virus was detected in infected swine tissue harvested one day after beginning of pyrexia. The modified direct complement-fixation test demonstrated cross-reactions between the six strains of virus studied.