Agar Gel Precipitation Test Research Articles

Quantitative counter-immunoelectrophoresis for estimation of antibodies to infectious bursal disease virus.

Quantitative counter-immunoelectrophoresis was standardized to detect antibodies to the avian infectious bursal disease virus. This technique correlated well with the conventional quantitative agar gel precipitation test in estimating antibodies to IBDV. The use of blood dried on filter paper as an alternative to serum is discussed. QCIE is simple, easy to perform and faster than QAGP.

Pathogenesis of highly virulent infectious bursal disease virus infection in intact and bursectomized chickens.

The pathogenesis of highly virulent infectious bursal disease (IBD) virus (IBDV) infection was studied using 6-week-old intact and 5-week-old bursectomized chickens inoculated with highly virulent strain 90-11 or reference strain I. Chickens inoculated with 10(0.7) EID50 of strain 90-11 showed neither clinical signs nor lesions during the 4-day observation period. In contrast, birds inoculated with 10(2.7) or 10(4.7) EID50 developed severe clinical IBD, as well as gross and histologic lesions, typical of IBD, and produced IBDV antigen demonstrable by immunostaining in the bursa of Fabricius (BF), thymus, spleen and bone marrow from day 2 post-inoculation (PI) onwards. The antigen was also detected by the agar-gel precipitation and latex microsphere agglutination tests in a bursal suspension of these birds from day 2 or day 3 PI on. Birds inoculated with 10(6.1) EID50 of strain I developed only slight clinical signs at day 4 PI. Their lesion- and antigen-scores in the BF were almost the same as those in virulent strain-infected chickens, but lesion- and antigen-scores in the other organs were negligible. Bursectomized chickens inoculated with strain 90-11 did not develop clinical IBD despite the presence of infection that was evidenced by histologic lesions in the thymus and spleen as well as IBDV antigen demonstrable by immunostaining in these organs.

A monoclonal antibody-based immunoperoxidase method for rapid detection of haemorrhagic enteritis virus of turkeys

An indirect immunoperoxidase ( ip) technique involving avidin-biotin peroxidase complex, using a monoclonal antibody was developed for the detection of haemorrhagic enteritis ( he) virus antigen in frozen and formalin-fixed paraffin-embedded tissue sections. This 1P procedure was compared with an indirect immunofluorescence antibody technique ( ifat) and an agar gel precipitation test ( agpt). Spleens from turkeys experimentally infected with HE virus were collected and examined for the presence of viral antigen. The IP staining procedure detected HE viral antigen as early as 48 hours after infection and continued to demonstrate the presence of viral antigen for up to 11 days after infection at which time the experiment was terminated. The antigen was detected from three to seven days and from two to nine days after infection by the agpt and ifat, respectively. The ifat and agpt had sensitivities of 74·19 and 48 per cent, respectively, compared with ip. Because of its high sensitivity and specificity, the ip technique could be useful for studying the pathogenesis and rapid laboratory detection of he virus.

Studies on thermostable antigens, production of species-specific antiadrenal sera and comparison of immunological techniques in meat speciation

Heat-stable antigens (BE forms: resistant to heat and ethanol precipitation) of adrenal and muscle tissues of cattle, buffalo, sheep, goat and pig were prepared for use in detection of adulteration in meats. The physico-chemical characteristics of these antigens revealed that the antigens of adrenals had only one component corresponding to ‘Troponin T’. Muscle antigens also contained a major troponin T component but were associated with low molecular weight fractions. Rabbit antiadrenal BE sera were developed and made species specific by immunoabsorption. The species-specific antisera were employed for identification of origin of fresh and cooked meats and their mixtures, using an immunodiffusion test—agar gel precipitation test (AGPT), counterimmunoelectrophoresis (CIEP), enzyme-linked immunosorbent assay (ELISA) and the unlabelled antibody peroxidase antiperoxidase (PAP) technique. The results indicated that absorbed antisera could successfully differentiate the fresh, cooked meats and the meat mixtures from the species under study. AGPT and CIEP were useful in identification of 5–10% addition, using water extracts of fresh meats and BE forms of cooked meats, whereas ELISA and PAP could detect adulteration down to the level of 1% when water extracts were used. Among the tests employed in the study, the PAP technique proved to be most sensitive. The antisera were also proved useful in identifying the species in canned meat products, milk, serum, plasma, semen, urine, organs, skin and spoilt flesh, employing AGPT and CIEP.

Comparison of the enzyme linked immunosorbent assay, the haemagglutination inhibition test and the agar gel precipitation test for the detection of antibodies against infectious bronchitis and Newcastle disease in commercial broilers

Serological results as obtained by the agar gel precipitation (AGP) test, haemagglutination inhibition (HI) test (M41, D274, D1466) and an enzyme linked immunosorbent assay (ELISA) from commercial broiler flocks, which had suffered from a clinical infectious bronchitis (IB) infection in the fattening period, were compared with serological findings of control broiler flocks. In addition the Hi-test and ELISA for Newcastle disease (ND) were compared. The AGP results differ significantly between groups. The test had a low sensitivity. The results of the HI-IBV tests were similar for both groups and no difference was seen between flocks with or without clinical IB problems. The IBV ELISA showed a highly significant difference between the two groups. In contrast to the low and not significant correlation coefficients (r) between the IBV HI tests and the ELISA, the value of r between the NDV HI and ELISA was high. The results indicate an ELISA as the preferred test for the diagnosis of IB in broilers.

Prevalence of subclinical infectious bursal disease and its significance in India.

From a total of 32 poultry flocks, 1,176 serum samples were screened by the agar gel precipitation test and 314 (26.7%) were positive for antibodies to infectious bursal disease virus (IBDV). Prevalence of infection on the farms ranged from 8.88 to 53.84 per cent. The prevalence was highest (61.82%) in chickens between 7 and 11 weeks old and lowest (3.92%) in those above 22 weeks of age. In commercial broilers and layers 51.61 and 17.78% respectively were seropositive reactors. The high prevalence of subclinical IBD and its economic significance are discussed.

Pathogenicity and preliminary antigenic characterization of six infectious bursal disease virus strains isolated in France from acute outbreaks.

Six isolates originating from acute outbreaks of infectious bursal disease recently reported in broiler and pullet flocks in France were studied with respect to their pathogenicity and their antigenic relatedness to the Faragher 52/70 reference strain. Although the mortality experimentally induced in susceptible chickens by the field strains was sometimes four times higher than that which followed the inoculation of the reference strain (16 to 48% versus 12%), neither mortality nor morbidity were observed in chickens previously vaccinated with a commercial live vaccine and then challenged under the same conditions. Agar gel precipitation tests demonstrated the existence of common antigens in the different strains, and high cross-neutralization indices measured in embryonated specific pathogen free eggs showed them all to belong to serotype I. These data are discussed with reference to previous European and North-American studies on the antigenic status of infectious bursal disease virus.

Détection des anticorps anti-adénovirus dans le sérum de chevaux Polo au Nigeria

Serum samples obtained from 107 Polo horses showing clinical signs of viral respiratory disease were tested for precipitating antibodies to adenovirus by agar gel precipitation test and counter-immunoelectrophoresis method. The results obtained demonstrate serological evidence of adenovirus infection in Polo horses in Nigeria. The counter-immunoelectrophoresis method was observed to be about 3 times more sensitive than the agar gel precipitation test with 19.3 vs 64.5%. It could thus be used to screen a large number of serum samples within a short period.

Dot immunobinding assay for the detection of bluetongue virus antibodies in sheep experimentally inoculated with bluetongue virus type 1

Dot immunobinding assay (DIA) was evaluated for the detection of bluetongue virus (BTV) antibodies in sheep experimentally inoculated with BTV 1. Serum samples collected on 14, 21, 28, 43 and 60 days post infection (dpi) were positive for precipitating antibodies by the agar gel precipitation test (AGPT) while antibodies could be detected as early as 7 dpi by DIA and ELISA. Virus neutralizing antibodies were detected first at 14 dpi. The sensitivity of the four tests was compared on the same serum samples collected at different intervals. The results indicated that DIA was more sensitive than AGPT and the serum neutralization test and as sensitive as ELISA. Thus due to sensitivity simplicity and economy, DIA could replace AGPT for diagnosis and serological survey for BTV infection in animals.

Comparative studies on pathogenical, virological and serological properties of Marek's disease virus isolated from Japanese quail and chicken

The QM-1 strain of Marek's disease virus (MDV) isolated from Japanese quail (JQ) induced higher mortality (96%) with MD lesions in quail than the JM strain of MDV isolated from chickens (C) (21%). In contrast both viruses induced MD in 90% of chickens. Pathological changes produced in quail inoculated with JQ-MDV or C-MDV were similar. However, the incidence of tumour lesions in the duodenum of quail inoculated with JQ-MDV was significantly higher. Neither virus induced tumour lesions in the duodenum of chickens. Appearance of viraemia in quail inoculated with MDV was delayed and its level was low as compared with chicken cases. MDV antigens in feather tips appeared in quail inoculated with JQ-MDV but not with C-MDV. No MDV was recovered from the MDV-inoculated quail by direct kidney cell cultures, although MDV was recovered by subpassage of the cultures into chicken embryo fibroblasts. No antibodies were detected in the affected quail by agar gel precipitation test.

Apparition du syndrome d’amaigrissement et de retard de croissance sur des poulets de chair au Soudan

The first outbreak of reovirus infection in broiler chickens was observed in the Sudan in the largest poultry industry of the country. The disease was characterized clinically by growth retardation, lameness, poor feathering, shank depigmentation and occasionally retraction of the head. Grossly there was enlargement of the proventriculus, atrophy of the pancreas and bone abnormalities. Histopathologic changes included hepatitis, nephritis, myocarditis, pericarditis, catarrhal enteritis, pancreatic necrosis, encephalomalacia and ricketic changes. Details were described. Reovirus was isolated from affected birds and reisolated from experimentally infected chicks. Infection was confirmed by immunoelectrophoresis and agar gel precipitation tests.

Detection of Marek's disease virus antigen in chickens by a novel immunoassay

An immunoassay was developed to detect Marek's disease virus (MDV) antigen on the tips of feathers obtained from MDV-infected chickens. MDV in follicular debris on the feather tip was demonstrated by use of a specific monoclonal antibody. The principle of an indirect ELISA was employed and the feather tip was used as the solid phase. Presence of MDV was reflected by a dark brown precipitate on the feather tip which could be observed by naked eye. This test system proved to be more sensitive than the agargel precipitation (AGP) test as all feather tips of MDV-infected animals gave a positive reaction in the feather tip-ELISA whereas about a half yielded a detectable precipitate in the AGP. Advantages of this feather tip-ELISA and applications are discussed.

The Production of Peste des Petits Ruminants Hyperimmune Sera in Rabbits and their Application in Virus Diagnosis

Hyperimmune sera were produced by serial inoculation of rabbits with Vero cell-adapted, sucrose gradient-purified Nigerian peste des petits ruminants virus (PPRV) isolate. Two antisera produced, neutralized the homologous PPRV but not the heterologous rinderpest Kabette "O" virus. The antisera gave strong precipitin lines with purified PPRV antigens and were used to detect PPRV and rinderpest virus antigens from ante-mortem secretions and post-mortem tissue homogenates from PPR and rinderpest virus infected goats and cattle by the agar gel precipitation tests (AGPT). The hyperimmune sera gave good titration curves with both purified Nigerian goat and the United Arab Emirate wildlife PPRV isolates in the indirect enzyme linked immunosorbent assay (ELISA). Results of indirect ELISA showed that although there were some cross reactions with the rinderpest, canine-distemper and measles viruses, at 1:100 dilution, the antisera would give a positive signal with only the homologous PPR virus.

Use of milk samples and monoclonal antibodies directed against BLV-p24 to identify cattle infected with Bovine leukemia virus (BLV)

An indirect double-antibody sandwich (IDAS) enzyme-linked immunosorbent assay (ELISA) using milk samples was developed to identify cows infected with bovine leukemia virus (BLV). Two monoclonal antibodies (McAbs) were used. One, which was directed against BLV core protein p24, was used to coat ELISA plates; the other was used to prepare a horseradish peroxidase (HRP) conjugate directed against bovine immunoglobulin. The IDAS-ELISA detected antibodies directed against BLV-p24 in 97% of the milk samples collected from known seropositive cows identified by the agar gel precipitation test (AGTP). Even when milk samples were diluted 1:50, 93% of the seropositive cows were identified. Only 0.43% of the 4000 milk samples collected in The Netherlands reacted nonspecifically. Nonspecific binding disappeared, however, when these samples were diluted 50 times in BLV-negative milk. In a comparative evaluation of BLV test-kits in various European laboratories, our IDAS-ELISA using McAb directed against p24 was one of the most sensitive.

The role of the reticuloendothelial system in the immunopathology of Marek's disease

The role of macrophages in immunity against Marek's disease (MD) was studied. Chickens of one group were subjected to depletion of macrophages using repeated doses of Francil amorphous silica and those of another group were subjected to activation of macrophages using repeated doses of brewer's thioglycollate broth. Chickens of a third group were vaccinated with herpesvirus of turkeys FC 126 vaccine followed by depletion of macrophages. Chickens of these three groups, as well as groups of healthy unvaccinated and healthy vaccinated chickens, were challenged with virulent MD virus. A sixth group of healthy uninfected chickens was kept as a control. The results, based on clinical signs, gross and histopathological studies and agar gel precipitation test (AGPT) for antibodies, indicated that activation of macrophages enhanced immunity against MD and depletion of macrophages had the opposite effect. The protective effect of vaccination against MD was also lowered by depletion of macrophages. The results of AGPT indicated retardation of MD virus replication by macrophage activation and the reverse on depletion.

Isolation, Identification, and Pathogenicity of Two Field Strains of Infectious Bursal Disease Virus

Using a sentinel bird approach, two field isolates of infectious bursal disease virus (IBDV) were isolated from broiler farms in two major broiler-producing areas of the state of Georgia. These farms had a history of subclinical IBD associated with respiratory problems and poor performance. Isolates designated as U-28 and 3212 were isolated using specific-pathogen-free chicken embryos and chicken embryo bursal cells. These isolates were identified by means of agar gel precipitation and virus-neutralization tests, direct immunofluorescence, histopathology, and electron microscopy. Isolates U-28 and 3212 appear to differ in antigenicity and pathogenicity from previously known serotype I IBDV isolates. In evaluating the extent of bursal damage caused by these field isolates, an association was found between the bursa of Fabricius/body weight index, histopathology scoring of atrophy, and morphometric analysis of the total follicle area.

Further characterisation of a variant type of turkey herpesvirus which releases large quantities of cell‐free viruses into culture medium

A variant type of turkey herpesvirus (HVT/VT) which releases large quantities of cell-free virus into its culture medium was compared with the prototype of the FC 126 strain of HVT (HVT/WT) as to the biological and serological characteristics. The lack of replication-defective HVT/VT in chickens was considered to be due to the inability of the virus to infect and replicate in lymphocytes and not to temperature-sensitive defects at 41 degrees C. No obvious antigenic differences were found between HVT/VT and HVT/WT in agar gel precipitation and neutralisation tests using four specific antisera prepared in chickens hyperimmunised with cells infected with HVT/WT or HVT/VT and cultural supernatants of cells infected with HVT/WT or HVT/VT. Group-specific (gs) A and gs B antigens were released into the culture medium from cells infected with HVT/VT.

Deteccion Del Antigeno Y Anticuerpos De Viruela Caprina Mediate La Prueba De Contrainmunoelectroforesis

The counterimmunoelectrophoresis (CIE) test was standardised for the detection of goat pox antigen and antibody using inactivated antigens. The chloroform inactivated and live antigens were equally sensitive for detection of goat pox precipitins. The precipitinogens of goat pox virus (GPV) were found to be soluble in nature. The CIE test was quick as well as more sensitive than the agar gel precipitation test for detection of GPV antibody/antigen. The CIE employing inactivated antigen has been used for the first time in the detection of GPV antibodies/antigens.

Counter-immunoelectrophoresis for the detection of Brucella antigen and antibodies in the diagnosis of brucellosis in buffaloes.

Counter-immunoelectrophoresis was employed for the detection of Brucella antigen in stomach contents of aborted buffalo fetuses and antibody in aborted as well as apparently healthy in contact buffaloes. Five of 16 aborted cases were serologically positive for brucellosis but isolation of Brucella abortus was successful in only two cases. By counter-immunoelectrophoresis, Brucella antigen was detected in the fetal stomach contents of four serologically positive cases. Of the 68 serum samples from in contact healthy buffaloes, 10 were positive with counter-immunoelectrophoresis: more than were detected by tube agglutination, Rose Bengal plate agglutination, complement fixation and agar gel precipitation test.

Serological monitoring of eastern wild turkeys for antibodies to Mycoplasma spp. and avian influenza viruses.

From 1981 through 1986, plasma or serum samples were obtained from 322 wild turkeys (Meleagris gallopavo) from Georgia (n = 111), Kentucky (n = 21), Louisiana (n = 22), North Carolina (n = 118), Tennessee (n = 19), Missouri (n = 24) and Iowa (n = 7). These samples were tested for antibodies to Mycoplasma gallisepticum (MG) and in most instances, M. synoviae (MS), M. meleagridis (MM), and avian influenza (AI) virus. All 322 turkeys were seronegative for MG by the rapid plate agglutination (RPA) test. All of a subsample (n = 147) also were negative (titer less than or equal to 1:40) for MG by the hemagglutination inhibition (HI) test. Five of 253 turkeys (2%) were seropositive (+4 reaction) for MS by the RPA test; however, HI tests for MS on these five turkeys were negative as were attempts to isolate MS from trachea and homogenized lung tissue. Three of 253 turkeys (1%) were seropositive (+1 to +3 reactions) for MM by the RPA test. None of 210 turkeys had antibodies to AI by the agar gel precipitation test. These data suggest that populations of native eastern wild turkeys are not important in the epizootiology of MG, MS, MM, or AI.