A Sensitive Autoradlographic Procedure for Factor VIII-Related Antigen in Canine Plasma

A second-antibody autoradiographic technique was found to be suitable for visualization of precipitin arcs produced by the electroimmunoassay or crossed immunoelectrophoresis of factor VIII-related antigen in canine plasma. With the new autoradiographic procedure the electroimmunoassay was approximately ten times more sensitive than with the commonly used protein stain, Coomassie Blue R 250. Unlike earlier autoradiographic techniques, second-antibody autoradiography requires only commercially available reagents.     

Immunodiffusion reactions among porcine enteroviruses and other picornaviruses

Antisera raised in rabbits against the porcine enterovirus strains V13 and T80 produced two precipitin lines in immunodiffusion tests with the homologous crude antigens, and a single precipitin line with each of ten heterologous porcine enteroviruses which were tested, and with poliovirus type 1, coxsackievirus B4 and equine rhinovirus type 1, but not with a bovine rhinovirus. C and D antigens prepared from V13 virus by density gradient centrifugation produced single precipitin lines with V13 antiserum. A single precipitin line was also formed when the heated crude antigens of V13 and T80 viruses were reacted with the homologous antisera, indicating destruction of the D antigen, and these lines fused with those produced by the heterologous viruses. It was concluded that porcine enteroviruses contain C and D antigens, and that the C antigen is responsible for the serological cross-reactivity demonstrated among porcine enteroviruses and other picornaviruses by immunodiffusion.     

Diagnosis of infectious bronchitis (IB) by examination of tracheal mucus for IB-precipitating antigens

The use of tracheal exudates for detecting precipitating infectious bronchitis (IB) antigen by the agar-gel precipitin (AGP) technique is described. Of 47 experimentally infected tracheas, 24 gave positive precipitin lines between the second and 12th day postinfection; 9 out of 18 tracheas from birds from natural outbreaks of IB were also positive. Tests can be read within 18-24 hr. For best results, several antisera or an antiserum at various concentrations should be used.     

Cultural and histopathologic confirmation of canine blastomycosis diagnosed by an agar-gel immunodiffusion test

Sixteen sera from 17 dogs with blastomycosis produced a precipitin band identical with the diagnostically significant precipitin A band formed by a Blastomyces dermatitidis reference antiserum and a soluble B dermatitidis yeast-form antigen in the agar-gel immunodiffusion test (94% sensitivity). The other serum from a dog with histopathologic demonstration of B dermatitidis in pulmonic tissues produced an unrelated precipitin band. Sixteen of the 17 diagnoses made by the detection of precipitin A were confirmed by isolation and culture of B dermatitidis or by histopathologic demonstration of the pathogen. Three cases were confirmed by cultural isolation only, 10 by histopathologic demonstration only, and 3 by both. In three other dogs given amphotericin B, there were demonstrable changes in serum precipitin A reactions.     

泰氏锥虫抗原制备及实验室诊断研究

应用体外培养的泰氏锥虫制备可溶性抗原Ⅰ、抗原Ⅱ和代谢抗原.经测定,其蛋白含量每毫升分别为6.5mg、7.4mg和7.1mg.薄层等电聚焦电泳测定结果表明,抗原Ⅰ出现22条区带,抗原Ⅱ21条区带,代谢抗原28条区带,对照的伊氏锥虫琼脂免疫扩散抗原14条区带.经分析,泰氏锥虫抗原和伊氏锥虫抗原有4条区带在同一迁移率上.琼脂免疫扩散反应中,3种泰氏锥虫抗原均与相应免疫兔血清发生沉淀反应,抗原Ⅰ出现1条致密沉淀线,抗原Ⅱ和代谢抗原出现2～3条沉淀线,抗原效价为1:4～16.免疫电泳显示了类似的结果,抗原Ⅰ与免疫兔血清出现1条弧形沉淀线,抗原Ⅱ和代谢抗原与免疫兔血清出现了3条弧形沉淀线.间接血凝试验结果表明,泰氏锥虫自然感染牛血清效价为1:20～40,免疫兔血清为1:1280～5120.所制泰氏锥虫抗原对伊氏锥虫和媾疫锥虫血清也能很好地发生交叉反应,3份伊氏锥虫病马血清和3份伊氏锥虫人工感染兔血清血凝效价分别为1:10～40和1:8～1024;5份媾疫马血清有4份血凝效价为1:20～320.4份环形泰勒虫病牛血清均为阴性.     

Immunodiffusion and neutralization studies on porcine adenoviruses

Precipitating antigens were prepared from porcine kidney cell cultures infected with the four approved serotypes of porcine adenoviruses (PAV) and from human amnion cell cultures infected with serotype 5 human adenovirus. For extracellular precipitating antigens (EPA) concentration by ammonium sulphate precipitation from cell culture fluids was used. Cell-associated precipitating antigens (CAPA) were extracted from cell sediments by repeated freezing and thawing. All antigens reacted alike and formed a single coalescent precipitin line of identity when tested against sera collected from a sow infected in the field or from weaners intranasally infected with serotypes 3 or 4 of PAV. In order to determine the optimal time of harvesting cell culture materials for the preparation of precipitating antigens, the kinetics of production and release of infectious virus and precipitating activity of PAV serotype 3 in porcine kidney cell cultures were studied. Precipitating activity first appeared with CAPA 24 h p.i. and 12 h later with EPA. Infectivity titers did not correlate with precipitating activities of EPA or CAPA beyond that stage. At the time the infectivity of EPA was decreasing, its precipitating titer continued to increase. The peaks of precipitating activities of CAPA and EPA were demonstrated at 96 and 144 h p.i., respectively. Three 7-week-old weaners with serum neutralizing antibodies against the four serotypes of PAV, but without detectable precipitating antibodies, were inoculated intranasally with serotype 3 of PAV. Serum samples collected 1 week p.i. showed precipitating activities and steep increases in neutralizing antibody titers against the homologous serotype 3 and the heterologous serotypes 1, 2 and 4 of PAV. The serum neutralizing antibody titers remained nearly constant over a period of 18 weeks p.i. while the intensity of the precipitating reaction decreased. Intranasal infection of the pigs with serotype 4 PAV induced heterotypic anamnestic neutralizing antibody response as well as an increase of the precipitating antibodies.     

Type-I hypersensitivity as a component of eosinophilic myositis (muscular sarcocystosis) in cattle

Eight bovine hearts with lesions of eosinophilic myositis (EM) and 2 bovine hearts without EM lesions were collected at slaughter. Blood samples from these 10 hearts, and the heart of a newborn calf also were collected. Histologically, Sarcocystis cruzi was identified in the 8 hearts with EM lesions and the 2 hearts without EM lesions, but not in the heart of the newborn calf. Serum was harvested from the 10 blood samples and was used in homologous, modified, passive cutaneous anaphylaxis tests. Antigen was prepared from S cruzi bradyzoites isolated from the 2 hearts without EM lesions. Serum samples from the 8 cattle with EM lesions reacted positively to S cruzi antigen. When heat-inactivated IgE in serum (56 C for 4 hours) was used, all passive cutaneous anaphylaxis responses were considered negative. Using ELISA, serum IgE concentrations from the 10 cattle with and without EM lesions were 2.2 to 9 U/ml. As determined by radial immunodiffusion, IgM concentrations were 80 to 215 mg/dl. Immunoglobulin G concentrations were 420 to 2,050 mg/dl, but most were less than or equal to 1,700 mg/dl. Immunoglobulin A concentrations were 0 to 62 mg/dl; 1 steer with EM lesions had 0 mg/dl. Double-gel immunodiffusion confirmed the presence of Sarcocystis-specific precipitating antibodies. Sera from the 10 cattle with and without EM lesions formed at least 1 precipitin band.     

Infectious bronchitis agar-gel precipitin test--use of infected allantoic fluid as antigen

The use of infected allantoic fluid (AF) as precipitating infectious bronchitis (IB) antigen was investigated. The results show that unconcentrated AF harvested at the right time can be a very satisfactory precipitating IB antigen. With the majority of the virus strains used, unconcentrated AF, harvested 68 hours postinoculation (PI), showed more precipitating activity than that harvested at 20 or 120 hours PI. If further antigen concentration is required, dialysis with polyethyleneglycol MW 20,000, freeze-drying, and precipitation with polyethyleneglycol 6,000 are satisfactory methods of concentration.     

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.     

Evaluation of a gel diffusion precipitin test for porcine parvovirus

The use of a gel diffusion precipitin (GDP) test for the detection of porcine parvovirus (PPV) infection in pigs is described. The close correlation between gel diffusion precipitin and haemagglutination inhibiting (HI) antibody titres indicates that, with careful standardisation, a high level of sensitivity can be achieved with the GDP test and that it is a simple and relatively inexpensive alternative to the more commonly used HI test. Experimental infection of 2 groups of pigs showed that GDP and HI antibody responses were closely correlated and that GDP antibodies to PPV persisted for at least 41 weeks after infection. In a commercial herd study, serological evidence of declining passive immunity and subsequent acquisition of active immunity was demonstrated by measuring the GDP and HI antibody titres in sequential serum samples of pigs from a known PPV endemic farm. The GDP test described was shown to be less sensitive than haemagglutination (HA) in the detection of viral antigen but was, nevertheless, considered useful as a simple screening test for the amounts of antigen usually present in PPV infected mummified foetuses.     

Antigen and Antibody in Aleutian Disease in Mink II. The Reaction of Antibody with the Aleutian Disease Agent Using Immunodiffusion and Immunoelectroosmophoresis

Aleutian disease viral (ADV) antigen was prepared by fluorocarbon extraction of spleen, liver, and lymph nodes from mink experimentally infected ten days previously. Using a potent ADV antigen, antibody was detected by immunodiffusion (ID) and immunoelectroosmophoresis (IEOP). Utilizing these precipitin tests, antibody was detected in all the mink sera tested as early as seven days after experimental infection. Titer of antibody increased throughout the infection period. Titers of more than 100 were reached by 15 days post infection, titers of 1,000 at one month, and titers of more than 5,000 to 10,000 were achieved at two months post infection and thereafter. The immunodiffusion test gave similar or slightly lower titers than those detected by the IEOP.

The IEOP test promises to be a most useful technique for the diagnosis of aleutian disease because it is simple, rapid and specific and is capable of detecting infection early in the course of the disease. It is suggested that this test should be utilized especially for the screening of animals purchased or imported as breeding stock onto ranches.

     

Immunoglobulin response to bluetongue virus soluble antigen in subcutaneous chambers.

Group-specific antibodies were produced by inoculation of bluetongue virus soluble antigen into polyethylene chambers implanted subcutaneously in 8 rabbits and 2 sheep. For comparison, 5 rabbits and 1 sheep were inoculated intramuscularly with the soluble antigen in Freund's complete adjuvant. Antibodies present in the serum and chamber fluids were detected by the agar gel precipitin or serum-neutralization tests, qualitatively examined by immunoelectrophoresis and immunofluorescence, and quantitated by electroimmunodiffusion.     

Comparison of inoculation regimes for the experimental production of swine erysipelas arthritis: II Serological findings in a gel diffusion precipitin test and enzyme-linked immunosorbent assay

The serological response of pigs to Erysipelothrix rhusiopathiae inoculation was monitored by a gel diffusion precipitin test (GDPT) using a crude, serotype-specific, autoclaved antigen and an enzyme-linked immunosorbent assay (ELISA) using a heat-extracted, alcohol precipitated and molecular seived antigen previously shown to react with serum from pigs infected with serotypes 1 or 2. All pigs receiving 3 or 5 weekly intravenous inoculations of either a highly virulent (VRS 229) or a lowly virulent isolate (VRS 252) produced GDPT-reactive antibody within 3 weeks, but only 44% were still reactive at 8 to 9.5 weeks. The ELISA response was significantly higher in pigs inoculated with the highly virulent strain, and was similar in pigs receiving 3 or 5 doses of either strain. In a dose-response trial, after 3 doses of VRS 229, GDPT reactivity occurred earlier and was stronger in pigs given higher doses of E. rhusiopathiae, but the response peaked 3 to 5 weeks after the start of challenge and was short lived. GDPT reactivity correlated with dose, but not with the severity of arthritis. The ELISA demonstrated specific IgG antibody was present by 2 weeks, and persisted to at least 11 weeks. The ELISA reactivity was significantly higher in pigs with arthritis than in pigs that received low doses and were not arthritic. Within groups of pigs with arthritis a significant, dose dependent, linear ELISA response developed but did not correlate with the presence or degree of arthritis at slaughter. Non-arthritic pigs had similar low ELISA responses to uninoculated controls.     

Immunoreactivity of fractionated antigens obtained from autoclaved extracts of an arthritogenic isolate of Erysipelothrix rhusiopathiae

The immunoreactive antigens in heat-extracted (autoclaved) preparations of an arthritogenic strain of Erysipelothrix rhusiopathiae (isolate VRS 229, serotype 1a) have been identified by gel diffusion precipitin (GDP) tests and a novel application of the enzyme linked immunosorbent assay (ELISA) procedure. Antigens precipitated by ethanol treatment of autoclaved extracts of this strain were resolved into 4 major peaks (A,B,C and D) after gel permeation chromatography on Sephacryl S200. Peak A was confirmed as a protein peak (Lowry positive) which was excluded from the gel. This peak was identified to be ELISA-reactive when assayed with serum from pigs infected with other isolates corresponding to serotypes 1a, 1b and 2. However, it did not form precipitin lines in GDP tests. Peak B was Lowry-positive and also contained carbohydrates. It was not as reactive in ELISA tests but rapidly formed precipitin lines with serum from pigs infected with the homologous isolate, but only erratically with serums from pigs infected with other serotype 1a and 1b isolates, and not with serotype 2 isolates. Peaks C and D were high in carbohydrate and phosphate content respectively but were both non-reactive in GDP tests and only slightly so by ELISA. Since serotypes 1 and 2 are the most predominant among isolates from infected pigs it is likely that the commonly recognised A antigen is a useful ELISA reagent for the diagnosis of E. rhusiopathiae infection; B antigen on the other hand, would probably be of limited diagnostic value.     

Equine antibody to bovine serum induced by several equine vaccines as a source of extraneous precipitin lines in the agar gel immunodiffusion test for equine infectious anemia.

Precipitin lines not associated with equine infectious anemia (EIA) were observed in routine agar gel immunodiffusion (AGID) testing for the infection. The serums which produced these lines were obtained from horses which had been given multiple vaccinations with commercially available cell culture-origin equine virus vaccines as part of a comprehensive herd health program. The lines formed against cell culture-derived, but not spleen-derived EIA viral antigens. Investigation revealed that bovine serum proteins in the vaccines induced precipitating antibodies which reacted with bovine serum proteins in cell culture-derived antigens. A vaccination trial, utilizing 4 commercially available vaccines in various combinations, indicated that as few as 2 vaccinations could induce AGID-detectable antibodies to bovine serum proteins in individual ponies. These antibodies were very transitory, usually lasting no longer than a week. Some horses, however, which had been given 4 vaccinations developed similar antibodies which persisted 3 months beyond the last vaccination. The extraneous precipitin lines produced by these antibodies in the AGID test for EIA were readily distinguished from true EIA-associated reactions and did not result in false-positive interpretations of the test. However, heavy percipitin lines due to strong antibovine serum activity did mask weakly positive EIA reactions.     

Counterimmunoelectrophoresis (immunoelectroosmosis) and serum electrophoretic pattern in serologic diagnosis of canine blastomycosis

Counterimmunoelectrophoresis (CIEP) with blastomyces and histoplasma antigens was used in a serologic study of 181 dogs clinically suspected of having blastomycosis and of 8 dogs with confirmed blastomycosis or histoplasmosis. Thirteen of the 181 dogs, positive by CIEP, were euthanatized, and the diagnosis was confirmed by cultivation and/or microscopic detection of Blastomyces dermatitidis. Additional CIEP-positive dogs were confirmed by staining of aspirates collected in vivo. Radiographic support for the diagnosis was reported in 4 other dogs in which histoplasmosis was excluded by a negative CIEP with histoplasma antigen. The precipitating antibody may disappear during the course of the disease, as it did in 1 dog treated with amphotericin B, but not cured. This dog reverted from CIEP-positive to CIEP-negative within 17 months of treatment (with a weak reaction after 10 months of treatment). The CIEP-detectable antibody was present only in 1 dog without a confirmation by histopathologic findings or cultivation among 24 well-documented cases and 181 total tested sera. The CIEP was more sensitive and specific than was the gel-diffusion precipitin test, eliminated the problems of anticomplementarity that often affected the results of complement-fixation tests with canine sera, and served well in detecting dogs with blastomycosis. Electrophoretic pattern of sera from CIEP-positive dogs with blastomycosis showed a decrease in albumin and an increase in alpha 2- and often in beta- and gamma-globulins, with a substantial decrease of the albumin/globulin ratio.     

von Willebrand factor in lysates of washed canine platelets

Canine and human platelets (washed 4 times in a solution containing EDTA, prostaglandin E1, and theophylline to prevent release of alpha-granule constituents) were lysed by being frozen and thawed in the presence of detergent. Radioelectroimmunoassay for von Willebrand factor (vWf) in 5 human platelet lysates produced precipitin rockets, shaped like those produced from vWf in plasma from healthy human beings, and indicated that the mean von Willebrand factor antigen (vWf:Ag) content in platelets from healthy human being was 526 +/- 87 human U/10(12) platelets. Radioelectroimmunoassay for vWf in platelet lysates from 17 healthy dogs with normal plasma. vWf:Ag concentration produced precipitin rockets that looked different from those produced from canine plasma and indicated vWf:Ag content of 59 +/- 35 canine U/10(12) platelets. Inclusion of protease inhibitors in the lysing solution did not normalize the appearance of the precipitin rockets or substantially alter the measured platelet content of vWf:Ag. The array of vWf multimers revealed by sodium dodecyl sulfate-agarose gel electrophoresis of canine platelet lysates had a distinct appearance that differed from that of vWf in canine or human plasma and platelets; the intensity of the canine platelet vWf multimer bands was skewed, with relatively greater density in the lower molecular weight region and faint or undetectable multimer bands in the higher molecular weight region. Electrophoretograms with visible multimers in the high molecular weight region had vWf components that had higher molecular weight than did any vWf components in canine plasma. Radioelectroimmunoassay for fibronectin in these same canine platelet lysates indicated that the fibronectin content in platelets was 2.89 +/- 1.10 mg/10(12) platelets.(ABSTRACT TRUNCATED AT 250 WORDS)     

A trivalent antigen for the detection of type I avian adenovirus precipitin

A double immunodiffusion antigen prepared from cell-culture-propagated CELO virus was not capable of detecting precipitin directed against all of the type I avian adenovirus (fowl adenoviruses) isolates tested. However, an antigen pool containing CELO-4, B-3, and IBH-2 (Tipton) fowl adenovirus isolates detected precipitin directed against representative isolates of 10 type I serotypes. Additionally, this antigen pool markedly improved detection of adenovirus field exposure.     

Evaluation of the agar-gel immunodiffusion test for the detection of precipitating antibodies against progressive pneumonia virus of sheep.

Various cell cultures were evaluated for their ability to support progressive pneumonia virus infection in vitro. Ovine trachea cells supported progressive pneumonia virus infection for an extended time,were extremely durable and could be passaged up until 30 passages. Progressive pneumonia virus infected ovine trachea cells were then used for the production of antigen for agar-gel immunodiffusion. A method for concentrating antigen, diafiltration, was compared to dialysis against polyethylene glycol. Using diafiltration, the concentrated virus was easily quantitated, less viscous (and therefore easier to apply) and only produced one precipitation line. Agar-gel immunodiffusion was used to survey 401 animals from two sheep flocks. One flock (96 sheep) was free of progressive pneumonia while the other flock had 111 of 305 total animals positive for precipitating antibodies. The incidence of precipitating antibodies in sheep ranged from 23% for yearling ewes to 80% in ewes seven years old.     

鸭疫里默氏菌6型、12型与16型之间的交叉反应

鸭疫里默氏菌6型和12型仅在玻片凝集试验中存在较弱的交叉反应，这一交叉反应可经血清吸收试验得到消除；12型和16型在玻片凝集试验和试管凝集试验中均表现较强的双向交叉反应，但在琼扩试验中表现为较弱的单向交叉反应；6型和16型之间没有交叉反应。12型和16型之间的交叉反应和交叉沉淀反应均可经血清吸收后得以消除，但经过16型参考菌株吸收过的12型抗血清，还与6型菌株存在交叉凝集反应，只有同时用6型和16型菌株吸收，12型抗血清才具有血清型特异性。血清吸收对6型和12型抗血清的同型凝集反应能力和沉淀反应能力没有影响，但对16型抗血清的同源凝集能力和沉淀反应能力均有较大的影响，经、2型参考菌株吸收后，16型抗血清与同型抗原之间的凝集效价下降2个滴度，而且不再形成清晰的沉淀线。结果表明，6型和12型之间、12型和16型之间均存在ab-bc的抗原关系。