Demonstration of an Antigenic Relationship Between Hog Cholera and Bovine Viral Diarrhea Viruses by Immunofluorescence

Specific staining of antigen within bovine embryo kidney tissue culture cells, infected with either Oregon C24V or NADL-MD bovine viral diarrhea virus, was accomplished using fluorescein-conjugated swine anti-hog cholera or bovine antiviral diarrhea globulin. Also specific staining of antigen within pig kidney tissue culture cells, infected with hog cholera virus, was accomplished using the same two types of conjugates. Specificity was confirmed by appropriate controls.     

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.

     

A Soluble Precipitating Antigen From Hog Cholera Virus Propagated in Tissue Culture: I. Preparation and Characterization of the Antigen

A precipitating hog cholera viral antigen was produced in a swine kidney tissue culture system. This antigen produced a single, sharp precipitin line with reference antiserum but no precipitin lines with nonimmune control sera. The precipitating antigen was present in the supernatant fluid after high-speed centrifugation, and was readily separated from infective virus by diethylaminoethyl cellulose (DEAE) chromatography. The antigen was found to be nondialyzable, stable to the action of chloroform and to storage at refrigeration temperature for at least 7 mo, but was inactivated at 56 C in 30 min.     

IV. Demonstration of the Viral Antigen by Means of Immunofluorescence

African swine fever immunofluorescent conjugates were prepared in swine and used successfully in the demonstration of viral antigen in frozen tissue sections and in inoculated tissue culture cells. Cross reactivity was observed with the six strains used in the inoculation of swine. The high antibody content of the serum of immune swine did not interfere with demonstration of the antigen in frozen tissue sections of certain of their organs. The localisation and extent of antigen varied with the stage of infection. The virus was demonstrated in spleen and other organs as early as after one day of pyrexia and until after death of the animal. A pool of hog cholera and African swine fever conjugates stained with dyes of different colours was used in the localisation of respective antigens in experimental mixed infection.     

Tween 80: a marker for differentiation of hog cholera and bovine viral diarrhea viruses.

Markers for differentiating hog cholera and bovine viral diarrhea viruses were studied using Tween 80, chloroform, trichlorotrifluoroethane and tri (n-butyl) phosphate. Attenuated A and virulent Ames strains of hog cholera virus were employed. Moreover, the NADL PK-15 cell culture adopted strain and low cell culture passaged Purdue strain of bovine viral diarrhea virus were used. These viruses were reacted with 2,500 micrograms/ml of Tween 80 for one hour at 37 degrees C. When attenuated A and virulent Ames strains of hog cholera virus with titers greater than 10(6) and 10(5) plaque forming units respectively, were reacted with Tween 80 the titer of each strains was reduced by approximately 10(4) plaque forming units of virus. When either strain of bovine viral diarrhea virus was reacted with Tween 80, virus was not detected.     

A Soluble Precipitating Antigen (HCA) from Hog Cholera Virus Propagated in Tissue Culture: II. Incidence of HCA-antibodies in Sera of Hog Cholera-Immune and Nonimmune Swine

Some conditions are presented under which swine develop antibodies for a soluble hog cholera viral antigen (HCA).     

The development of an international reference panel of monoclonal antibodies for the differentiation of hog cholera virus from other pestiviruses.

A panel of 30 monoclonal antibodies was defined and characterized with respect to the binding capacity in immunoperoxidase assay to different strains of pestivirus. Using the panel it was possible to identify specifically all strains and isolates of hog cholera virus, hog cholera vaccines derived from 'C' strains, and most strains of bovine viral diarrhoea/border disease (BVD/BD) viruses (including those isolated from pigs). A small proportion of BVD/BD isolates from pigs and ruminants reacted only with the monoclonals specific for pestivirus group antigen. It is recommended that monoclonal typing methods be introduced into official procedures for the diagnosis of hog cholera/classical swine fever.     

A new approach for the diagnosis of hog cholera

Pestiviruses were isolated from seven cases of suspect hog cholera. Using peroxidase conjugates of monoclonal antibodies (Mabs) six isolates were identified as hog cholera viruses (HCV), while one isolate was of ruminant origin, possibly bovine viral diarrhea virus. In parallel attempts were made to develop an ELISA for the detection of HCV-specific antibodies in pig sera. The Mab HCTC26 coated to polystyrol plates efficiently captured the major viral glycoprotein gp53 from crude antigen suspensions prepared from infected cells. The immobilized gp53 served as diagnostic antigen. Five pigs experimentally infected with the HCV strain Glentorf were sequentially bled and the development of antibodies was monitored by neutralization tests and the ELISA. Results showed that both tests detected antibodies simultaneously after infection. Titres measured by ELISA were slightly higher than those registered by neutralization.     

Use of the agar diffusion precipitation test in the diagnosis of hog cholera

The application of the agar diffusion precipitation (ADP) test for diagnosing hog cholera was investigated. The test used as antigen, pancreatic tissue from 272 pigs that had been inoculated with hog cholera virus. The test was positive for 13.5% of the animals that were sick for 4 days or less, 40% of those sick for 5 days, and 77% of those sick for 6 days or more. The test was positive for 13.5% of all animals that had been vaccinated with crystal violet-glycerol hog cholera vaccine and had been sick for at least 6 days after challenge inoculation.     

Vaccination of Pigs Against Hog Cholera (Classical Swine Fever) With a Detergent Split Vaccine

Four pigs were inoculated subcutaneously with a detergent (triton X 100) split hog cholera virus in Freund’s incomplete adjuvant. Four other pigs were in the same way inoculated with a detergent split bovine viral diarrhoea virus, also in Freund’s incomplete adjuvant. In the experiment were used 3 control pigs. The vaccinations were repeated after 3 weeks. All pigs were challenged with highly virulent hog cholera virus (Tübingen) 12 weeks after primary inoculations. Signs of hog cholera were only noted in the control pigs.This introductory experiment was succeeded by a larger experiment with subcutaneous inoculations of: 10 pigs with detergent split hog cholera virus in Freund’s incomplete adjuvant, 10 pigs with detergent split hog cholera virus in a saponin (Quil A) solution, 10 pigs with detergent split bovine viral diarrhoea virus in Freund’s incomplete adjuvant, 10 pigs with detergent split bovine viral diarrhoea virus in the Quil A solution plus 5 control pigs. The vaccinations were repeated after 3 weeks, and finally all pigs were challenged 9 weeks later with the highly virulent hog cholera virus strain.With the exception of 1 animal which died accidentally, all animals survived in the groups inoculated with the Quil A vaccines and in the group inoculated with the detergent split hog cholera virus/oil adjuvant vaccine. In the group inoculated with the detergent split bovine viral diarrhoea virus/oil adjuvant vaccine, some of the pigs died of hog cholera.     

猪瘟兔化弱毒疫苗与我国近年猪瘟野毒的免疫保护相关性试验

２批猪瘟兔化毒睾丸细胞苗分别来自两家兽医生物药品厂，各分别免疫接种猪只，随后以５株猪瘟病毒野毒分别攻击，疫苗接种猪完全存活，对照猪完全死亡。此５株野毒分离自国内不同地区，并皆已经过细致的鉴定，以免疫荧光技术对猪扁桃体进行了活体检查，各对照猪材料上能在攻击后一周时出现病毒，而疫苗接种猪在整个观察期间未出现病毒，据此，显然可见猪瘟兔化毒细胞苗在预防国内猪瘟上极有效力     

Development and evaluation of an enzyme-labeled antibody test for the rapid detection of hog cholera antibodies.

A rapid enzyme-labeled antibody (ELA) microtechnique for the screening of swine for hog cholera antibodies was developed and evaluated with a blind study, using a 640-sample hog cholera serum bank. The total time to run a group of 22 samples was approximately 1 hour. The ELA test results correlated greater than 99% with hog cholera serum-neutralization test results on the same serums. Test results also indicated that the ELA test shares with the hog cholera serum-neutralization test the problem of cross reactions between the antibodies of hog cholera and bovine viral diarrhea.     

Hog Cholera: I. Investigation of the Agar Double-Diffusion Precipitation Test for The Detection of the Virus in Swine Tissue

The agar double-diffusion precipitation test was investigated to detect hog cholera virus in splenic and pancreatic tissues of swine. Special attention was given to the conditions influencing the sensitivity and specificity of the test. These studies emphasize the strict techniques and methods required in the test in order to detect specific antigen — antibody reaction. Absorption studies performed on serum fractions prepared by DEAE cellulose chromatography and studied electrophoretically, indicated the specific reaction was given by fractions, migrating in the gamma-globulin region and was absorbed by infected but not by normal spleens. The sensitivity of the test is dependent to a great extent on the successful liberation of the viral antigen from the tissue.     

Differentiation of hog cholera and bovine virus diarrhoea viruses in pigs using monoclonal antibodies

Monoclonal antibodies against hog cholera and bovine viral diarrhoea viruses were assayed on organ tissue sections of experimentally infected animals. The animals had been infected simultaneously with both viruses. The antibodies were tested using an indirect immunofluorescence test and an indirect enzyme immunoassay with a biotin/streptavidin/peroxidase detection system. A polyclonal hyperimmune serum was used as a control in direct immunofluorescence tests. Both techniques based on monoclonal antibodies were more sensitive and more specific than the conventional test, the enzyme immunoassay being more sensitive than the immunofluorescence test. Small amounts of BVD viral antigen were demonstrable with monoclonal antibodies in most organ tissues.     

实验室人工感染诱发猪瘟野毒垂直传播

利用2头人工感染中等毒力猪瘟野毒耐过猪进行配种,诱发了猪瘟野毒垂直传播.带毒母猪于带毒后171 d产下9头仔猪,其中3头为死胎,6头为木乃伊.直接免疫荧光抗体试验和RT-PCR检测,9头均为阳性.测序结果表明,3头测序仔猪中,2头仔猪所分离病毒E2基因主要抗原编码区序列与公猪所接种病毒的一致;另1头仔猪所分离病毒E2基因主要抗原编码区序列与公猪所接种病毒的同源性高于与母猪所接种病毒的同源性.母猪在与公猪配种前后,其所分离病毒E2基因主要抗原编码区序列发生了变化,配种后与公猪所分离病毒的一致,说明猪瘟病毒在猪体内的繁殖存在一定的优势选择现象.     

Hog cholera diagnostic techniques.

Clinical signs and lesions can sometimes provide the basis for a presumptive diagnosis of hog cholera (HC). However, an accurate diagnosis requires laboratory testing. The usual procedure for the detection of viral antigen is the examination of cryostat sections stained with fluorescein-conjugated HC antiserum. A more definitive technique is isolation of the virus in PK-15 cell cultures and identification of the viral antigen in cells using an HC fluorescent antibody conjugate. As bovine viral diarrhea (BVD) virus will cross-react with HC virus, isolation must be confirmed by the comparison of BVD and HC staining or, preferably, by the use of monoclonal antibodies that can differentiate between HC and BVD viruses. Hog cholera surveillance must rely on serology. The fluorescent antibody virus neutralization (FAVN) test is the classical technique, and HC and BVD antibody can usually be differentiated if HC-positive serum samples are tested against both viruses. Recently the enzyme-linked immunosorbent assay (ELISA) and peroxidase-labeled antibody tests have become the commonly used techniques.     

Natural infection of pigs with bovine viral diarrhea virus and its differential diagnosis from hog cholera.

Natural infection of pigs with bovine viral diarrhea virus (BVDV) through contact with infected cattle has caused problems in diagnosing hog cholera (HC). Low cross-reacting serum antibody titers against HC caused by BVDV infection were found in clinically normal pigs as well as those suspected of having HC. Bovine viral diarrhea virus was isolated from specimen tissues and initially identified as HC virus (HCV), using the fluorescent antibody cell culture technique. Additional cell cultures, as well as pig and calf trials, were necessary to identify it as BVDV. The isolate caused clinical signs of illness in the calves, whereas the pigs remained healthy. Bovine viral diarrhea virus may be detected in tissue sections or isolated in cell cultures and confirmed as HCV, using the HC fluorescent antibody conjugate. Laboratories performing the neutralization test for HC should use discretion when interpreting HC titers unless BVD titers are determined on the same serums.     

The Properties and Classification of Bovine Viral Diarrhea Virus

Examination of the C24V (Oregon) and MAC A (Ontario) strains of bovine viral diarrhea viruses have shown them to be ribonucleic acid containing viruses, with essential lipid and having compound helical symmetry with the diameter of the helix being in the neighbourhood of 180 A. Because of these properties it is suggested that the virus should be considered a member of the Myxovirus group. Hog cholera virus is related to bovine viral diarrhea virus by means of a “soluble” antigen, and also possesses essential lipid. It is therefore suggested that hog cholera virus represents still another veterinary myxovirus.     

从疑似猪瘟病料中检出牛病毒性腹泻病毒

根据牛病毒性腹泻病毒（ＢＶＤＶ）ＮＡＤＬ株和猪瘟病毒（ＨＣＶ）Ａｌｆｏｒｔ株的核苷酸序列，设计合成了１对ＢＶＤＶ引物和３对ＨＣＶ引物。以从吉林、长春、哲盟３个地区经临床及病理学诊断为猪瘟的病料中提取的ＲＮＡ为模板，采用反转录－聚合酶链反应（ＲＴ－ＰＣＲ），分别以ＢＶＤＶ和ＨＣＶ引物进行扩增。结果，用ＢＶＤＶ引物从哲盟地区的疑似猪瘟病料中扩增出大小约４００ｂｐ的片段，而用３对ＨＣＶ引物在不同的条件下均未从该病料中扩增出相应大小的ＨＣＶ基因片段。此外，用ＨＣＶ的ＰＥ０／ＰＥ４引物从吉林、长春的病料中扩增出了ＨＣＶ的基因片段，但用ＢＶＤＶ引物从这两个地区的病料中均未扩增出ＢＶＤＶ的基因片段。从哲盟疑似猪瘟病料中扩增出的片段经克隆、序列测定及计算机分析证实，该片段的核苷酸序列和氨基酸序列与ＢＶＤＶ的同源性明显高于与ＨＣＶ的同源性。将哲盟病料接种于ＭＤＢＫ传代细胞，出现典型而规律的ＢＶＤＶ样细胞病变。由此证明，从哲盟疑似猪瘟病料中检测出的是ＢＶＤＶ而不是ＨＣＶ     

Detection of antibodies against hog cholera virus and bovine viral diarrhea virus in porcine serum. A comparative examination using CF, PLA and NPLA assays

The antibodies in serum samples from an outbreak of low-virulent hog cholera in Spielbach, West Germany, 1966, as well as serum samples from pigs inoculated with hog cholera (HG) virus and bovine viral diarrhea (BVD) virus, respectively, were examined by means of 3 different methods:

1. A modified direct complement fixation (GF) test,
2. A peroxidase-linked antibody (PLA) assay based on microplates with fixed, viral-antigen containing cells,
3. A neutralization assay carried out in microplates using the “chessboard” principle and read by means of the peroxidaselinked antibody (NPLA) assay.

A good correlation was found in their ability to detect the antibodies. Generally neutralizing antibodies could be found 2 weeks after inoculation. By CF and PLA antibodies could be detected at the same time or up to 2 weeks later. All sera were tested by the 3 methods against both HG viral antigen and BVD viral antigen. HC-antibodies could not be distinguished from BVD-antibodies by CF but to a certain degree by PLA. BVD-antibodies could to a certain degree be distinguished from HG-antibodies by CF but not by PLA. This means that CF and PLA together provide a good possibility for differentiation between the two types of antibodies. NPLA could to a high degree of reliability distinguish between HG-antibodies and BVD-antibodies.