Ultracentrifugal Studies of Sera from Cattle Vaccinated or Naturally Infected with Brucella Abortus

In conformity with the findings of previous investigators, it was shown by density gradient ultracentrifugation that the antibodies in sera collected from calves shortly after vaccination with Brucella abortus, strain 19, were entirely or mainly rapidly-sedimenting. These macroglobulin (19S or IgM) antibodies showed complement-fixing as well as agglutinative activity with Br. abortus antigen. In later bleedings from the same vaccinated calves, antibodies with an intermediate sedimentation rate, (IgG), were present, as well as IgM. Sera from 15 of 22 non-vaccinated, relatively recent field cases of brucellosis appeared to contain only the IgG class of antibodies. In one herd, however, two cows with IgM only and five with both IgM and IgA were found; all seven of these cattle had been serologically negative before their introduction into this known infected herd a few months earlier. The agglutinative activity of sera from four cases of brucellosis of long standing and from eight cows, 4 to 13 years of age, that had been vaccinated as calves, was confined to the IgG fraction.     

Detection of pseudorabies virus antibodies: time of appearance by two serotests

This report describes a time-course comparison of detection of pseudorabies virus antibodies in experimentally infected swine by the virus-neutralization (VN) and indirect hemagglutination tests. Specific antibody titers were observed by the IHA test at 5 days after swine were inoculated, but not until 12 days by the VN test. The predominant immunoglobulin (Ig) class present in the serums of the swine at 5 and 7 days after inoculation was IgM, as determined by sulfhydryl reductions. The VN test lacked sensitivity to early Ig levels (IgM) in these experimentally infected swine, while the indirect hemagglutination test was highly sensitive to these same levels. On the basis of these results, it is possible that the VN test may read early infections as pseudorabies virus negative, due to the low presence of IgG in these samples.     

Immunologic responses of calves to aerosolized antigen: humoral response to ovalbumin

The humoral response of cattle to ovalbumin (OA), a nonenvironmental well-defined antigen, was studied. During 9 weeks of aerosolization, weekly serum and nasal secretion concentrations of immunoglobulin (Ig)G1, IgG2, IgM, IgA, and IgE were determined by enzyme-linked immunosorbent assay (ELISA) for OA specific antibody. Data from 3 calves given aerosol OA were compared and contrasted with data from 3 calves given aerosol saline solution and 1 calf given parenteral OA. The presence of cytotropic (skin sensitizing) antibody was evaluated during weeks 6 and 9 by direct skin testing with OA. A humoral response was induced in all 3 calves given aerosol OA. Serum IgG1 and IgG2 titers reached a maximum of 64,000 and 2,000, respectively, in calves given aerosol OA compared with 521,000 and 16,000, respectively, in the calf given parenteral OA. The ELISA did not detect an OA-specific IgM response. In contrast, all 3 calves given aerosol OA had serum IgA concentrations that increased to a peak by week 9. The mean IgA absorbance value for the 3 calves given aerosol OA was slightly greater than 5 times that of the calf given parenteral OA. Similarly, nasal secretions from calves given aerosolized OA had absorbance values that were 15-fold greater than that from the calf given parenteral OA. Calves given aerosol OA had antigen-specific IgE responses during weeks 6 to 8. The ELISA results were compared with results of passive cutaneous anaphylaxis tests. The presence of skin-sensitizing antibody was indicated by positive skin tests in the calves given aerosol OA and the calf given parenteral OA by week 9.     

Comparison of Latex Agglutination, Indirect Hemagglutination, and ELISA Techniques for the Detection of Toxoplasma gondii-specific Antibodies in the Serum of Cats

The primary purpose of this study was to determine whether commercially available latex agglutination and indirect hemagglutination kits for the detection of Toxoplasma gondii-specific antibodies were capable of detecting T. gondii-specific immunoglobulin M (IgM) in the serum of cats. Serum samples from 35 cats containing either T. gondii-specific IgM, T. gondii-specific immunoglobulin G (IgG), or both were collected. Each serum sample was assayed using a latex agglutination kit, an indirect hemagglutination kit, an enzyme-linked immunosorbent assay (ELISA) for the detection of T. gondii-specific IgG, and an ELISA for the detection of T. gondii-specific IgM. When serum samples containing only T. gondii-specific IgM as determined by ELISA were assayed, the latex agglutination kit and the indirect hemagglutination kit detected antibodies in 33.3% and 13.3%, respectively. When T. gondii-specific IgG was present in a serum sample, the results from the latex agglutination kit, the indirect hemagglutination kit, and the IgG-ELISA were similar; however, there was a wide variation in titer magnitude results between the three assays. It was concluded that the latex agglutination kit and the indirect hemagglutination kit did not adequately detect T. gondii-specific IgM in feline serum.     

Induction of pulmonary antibodies to Pasteurella haemolytica following intraduodenal stimulation of the gut-associated lymphatic tissue in cattle.

The induction of pulmonary antibodies to a bacterial antigen following intraduodenal (D) stimulation of the gut-associated lymphatic tissue (GALT) was investigated. Six calves were divided into two groups of three calves each. The GALT-primed calves received an ID dose of live Pasteurella haemolytica A1 followed by a subcutaneous (SC) dose of killed P. haemolytica. The sham-primed calves received an ID dose of phosphate-buffered saline solution (PBSS) followed by a SC dose of killed bacteria. Serum and pulmonary lavage fluids were collected weekly from each calf and assayed for titers of leukotoxin neutralizing antibodies (LNA), as well as IgG and IgA (lavage fluids only) to P. haemolytica. The GALT-primed calves responded to the ID stimulation by bacteria with increased serum IgG. The sham-primed calves had no change in antibody titers following ID stimulation. The GALT-primed calves had increased serum IgG, lavage IgG and IgA and increased LNA titers in both lavage fluids and serum following the SC dose of killed bacteria. The sham-primed calves demonstrated only an increase in serum IgG following the SC inoculation. A challenge study to evaluate if antibodies induced by GALT stimulation could reduce pulmonary lesions was performed using six calves divided into two groups. One group received an ID dose of P. haemolytica followed two weeks later by a SC dose of killed P. haemolytica. The sham vaccinated calves received an ID dose of PBSS followed in two weeks by a SC dose of killed bacterin. Calves were challenged by an intrapulmonary dose of live P. haemolytica A1 eleven days after the SC inoculation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of calf age and Salmonella bacterin type on ability to produce immunoglobulins directed against Salmonella whole cells or lipopolysaccharide.

A commercially available Salmonella bacterin was administered to Holstein calves starting at 1 to 19 weeks of age. Serum samples were obtained before administering bacterin and at 2-week intervals thereafter. An ELISA with Salmonella dublin lipopolysaccharide (LPS) or S dublin whole cells as antigen, was used to measure specific IgG and IgM responses. Antibody responses to LPS were not detected from calves < 12 weeks old inoculated with killed bacterin. Immunoglobulin responses to whole-cell antigen were detected from all age groups of calves inoculated with the same killed Salmonella bacterin. Calves < 11 weeks old are able to produce immunoglobulins to some whole-cell antigens, but are unable to produce anti-LPS immunoglobulins when inoculated with killed Salmonella bacterin. This age-related response to killed Salmonella antigens may account, in part, for increased susceptibility to salmonellosis in calves < 12 weeks old. In comparison to the response for killed antigen, 8 calves given modified-live aromatic-dependent S dublin bacterin at 1 to 3 weeks of age had detectable anti-LPS immunoglobulins after immunization, although the response was not as rapid and was of a lesser magnitude than that of older calves given killed Salmonella bacterin.     

Evaluation of an antigenic fraction of Taenia hydatigena metacestode cyst fluid for immunodiagnosis of bovine cysticercosis

An antigenic fraction (ThFAS) isolated from Taenia hydatigena metacestode cyst fluid was used in an ELISA to detect antibodies to T saginata in experimentally and naturally infected cattle. In 10 calves given 1,000 to 100,000 T saginata eggs (20% to 60% viability), IgG and IgM antibodies were detected in all the calves by post-inoculation week 3. Immunoglobulin G antibody values remained increased until calves were slaughtered at post-inoculation weeks 13 to 26. Six naturally infected calves (determined by postmortem examination) were considered positive, using the ELISA. Shared antigens were demonstrated between ThFAS and T saginata and T crassiceps; there were no shared antigens between ThFAS and Haemonchus contortus or Fasciola hepatica. Specific lectin binding to ThFAS indicated the presence of glycoconjugates. Immunoblot analysis indicated that a low molecular weight polypeptide (10,000 Mr) bears the immunodiagnostic antigen.     

Detection of IgM responses to bovine respiratory syncytial virus by indirect ELISA following experimental infection and reinfection of calves: abolition of false positive and false negative results by pre-treatment of sera with protein-G agarose.

The IgM responses in three panels of sera generated by infection and reinfection of calves with bovine respiratory syncytial virus (BRSV) were measured by indirect ELISA (I-ELISA). The effect of depleting serum IgG by pre-treatment with protein G agarose (PGA) was evaluated. Following primary infection a weak IgM response was detected in the untreated sera of 3 out of 4 calves with maternally derived antibody (MDA). Both the magnitude and duration of the specific IgM responses in these calves were increased by pre-treatment with PGA. In addition, the fourth infected calf tested gave a single positive IgM result following PGA treatment. Transient or persistent IgM responses which were abolished by pre-treatment of sera with PGA were detected in 4/8 calves following reinfection. These were considered to be false positive results, consistent with the influence of IgM rheumatoid factor (IgM-RF). One of these calves and two additional calves showed transient increases in IgM which were resistant to PGA treatment. These were considered to represent specific IgM responses to reinfection. The results indicate the ability of PGA treatment to eliminate both false positive and false negative results and emphasise the necessity for controlling the influence of IgM-RF in IgM-specific indirect ELISAs.     

Immune modulation by Ostertagia ostertagi and the effects of diet

IgG1 antibody responses to Ostertagia ostertagi third stage larvae (L3) and the third party antigen, keyhole limpet haemocyanin (KLH), and faecal egg counts were determined in calves infected with a single dose of O. ostertagi and in uninfected, pair-fed calves. The infected and uninfected calves were given diets either high (H) or low (L) in protein and energy. The diets were within the normal range of husbandry practice in the UK. IgG1 antibody responses to L3 antigen were significantly greater from 6 weeks post-infection in infected calves given the L diet than in infected calves given the H diet (P less than 0.05). The effects of diet and infection on anti-KLH IgG1 responses were independent of each other. IgG1 responses to KLH were decreased by infection and by the L diet compared with the H diet.     

Isotype-specific ELISAs for the detection of antibodies to bovine respiratory syncytial virus

Isotype-specific ELISAs for the detection of antibodies to bovine respiratory syncytial virus (BRSV) are described. BRSV-specific IgG1 and IgG2 were determined in indirect double antibody sandwich assays. For IgA and IgM antibody capture assays were used. The isotype specificity of the assays was confirmed by the observation that samples with a high titre of BRSV-specific antibodies of particular isotype were negative in the assays for the other isotypes and vice versa. Comparison of the results obtained in the ELISAs and in the virus neutralisation test showed that acute phase antibodies were more efficiently detected in the latter. It also showed that the presence of BRSV-specific IgA was not correlated with neutralising activity in vitro. The serum antibody response of BRSV-infected seronegative calves from the field consisted of a nearly simultaneous increase of IgM, IgA and IgG1-antibodies in the acute phase of the disease, while the IgG2-response followed at various intervals thereafter. In young animals with maternal antibodies a different pattern was found. There was no increase in IgG1 and IgG2, but six of eight animals showed a weak IgM response and two of these six calves also showed a weak and short lasting IgA response. Because maternal antibodies are insufficiently effective in protecting calves against BRSV, the presence of such antibodies at mucosal surfaces was investigated. Maternal immunity was found to be restricted to IgG1 antibodies in serum. This agrees with the failure of maternal antibodies to protect mucosal surfaces against BRSV infection.     

Isotype- and subclass-specific responses to infection and reinfection with parainfluenza-3 virus: comparison of the diagnostic potential of ELISAs detecting seroconversion and specific IgM and IgA.

Isotype- and subclass-specific indirect enzyme-linked immunosorbent assays were developed to detect parainfluenza-3 virus-specific IgG1, IgG2, IgM, and IgA responses. Sera were treated with protein G-agarose prior to testing for specific IgM and IgA to eliminate the possibility of false-positive results due to IgM-rheumatoid factor and to remove interisotypic competition due to specific IgG. IgM and IgA absorbance values were expressed as a percentage of the absorbance values of positive reference sera included on each plate (S/P%), and respective positive/negative threshold values of 15.0% and 28.0% were determined. The mean interval between experimental infection of 3 calves and initial detection of specific IgG1 and IgG2 responses was 8.0 and 9.3 days respectively, rising rapidly to an initial plateau 13.7 and 11.0 days postinfection (dpi). Reinfection of these calves at 30 dpi resulted in further rapid increases, with higher plateau values reached 13.0 (IgG1) and 13.7 (IgG2) days later. The mean interval between infection and the first positive IgM and IgA responses was 6.7 and 12.3 days, respectively. IgM S/P% values peaked at 13.0 dpi, with all 3 calves showing a secondary anamnestic response to reinfection, peaking 4.7 days later. The IgA response to initial infection was weak, with only 2 calves showing an obvious peak response at 15.0 dpi. A strong anamnestic IgA response to reinfection occurred in 2 calves, with a peak response 9.5 days later. Apparent biphasic and triphasic IgM and IgA responses were evident in some calves. Acute and convalescent serum samples from 80 calves involved in 17 outbreaks of respiratory disease were tested for specific IgM and IgA. Positive IgM results were detected in 15 outbreaks, with 71 sera from 44 calves testing positive. Although IgA-positive results were detected in the same 15 outbreaks, only 42 sera from 31 calves were positive. In a previous study, seroconversion was detected in 21 of these calves from 10 outbreaks. Thus the diagnostic potential of the assays was in the order IgM > IgA > seroconversion. The correlations between IgM and IgA, IgM and seroconversion, and IgA and seroconversion results for each calf were 73.8%, 58.8% and 62.5%, respectively.     

Modulation of calf immune responses by Ostertagia ostertagi: the effect of diet during trickle infection.

The effects of Ostertagia ostertagi infection and diet on antibody responses to O. ostertagi third stage larval (L3) antigen and to an unrelated antigen, Keyhole Limpet Haemocyanin (KLH) were determined in calves experimentally infected with 3000 L3 on alternate days for 6 weeks. Calves were given one of two diets, and were either infected or not infected with O. ostertagi L3. The diets were either high (H) or low (L) in protein/energy and were within the range of normal husbandry practice in the UK. Both IgG1 and IgG2, but not IgA, responses to L3 antigen were increased in the L-diet compared with the H-diet. IgA responses to L3 antigen were not affected by dietary treatment. The effects of diet and infection on anti-KLH IgG1 were independent of each other; IgG1 anti-KLH responses were decreased by infection and by the L-diet compared with the H-diet. The data suggest that there is a strong interrelationship between diet and immunity during nematode infections.     

Systemic and pulmonary antibody responses of calves to Pasteurella haemolytica after intrapulmonary inoculation.

Systemic and pulmonary antibody responses of calves to Pasteurella haemolytica were evaluated by measuring immunoglobulin production in blood for 9 days and in pulmonary lavage fluid for 7 days after intrapulmonary inoculation. Clinical signs, pulmonary lesions, pulmonary and systemic inflammatory response, and amount of antigen in lavage fluid were used to evaluate the response of calves to challenge with P haemolytica. The pulmonary response consisted of production of IgG, IgE, and IgM antibodies to P haemolytica antigens and a 17- to 68-fold increase of cells in lavage fluid 8 hours after inoculation, with a gradual decrease toward normal. Antibodies of the IgM isotype to P haemolytica were demonstrated as early as 8 hours through 7 days after inoculation in 3 of 3 calves. Of the anti-P haemolytica isotypes, IgM was found in the highest concentration. In all of the inoculated calves, IgE was found 1 to 2 days after inoculation, and IgG was found in 2 of 3 inoculated calves from day 1 through 7 after inoculation. Detection of IgG correlated with smaller pulmonary lesions. Immunoglobulin A was not detected in lavage fluid. Serum was evaluated for IgG and IgM antibody response to P haemolytica. Specific IgM was detectable 5 days after inoculation, and IgG was detectable 7 days after inoculation. Pasteurella haemolytica antigens were not detected in serum or plasma. A transient increase in neutrophil count was found 8 hours after inoculation, with return to baseline values by 24 hours after inoculation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development and evaluation of an enzyme-linked immunosorbent assay for bovine antibody (IgG) to Pasteurella haemolytica

The sensitivity of an indirect enzyme-linked immunosorbent assay (ELISA) for bovine IgG serum antibody to Pasteurella haemolytica was compared with that of an indirect hemagglutination (IHA) test. Pasteurella haemolytica serotypes were grown in a chemically defined cell culture medium, and soluble antigens released into the growth medium were used in the ELISA and IHA test. An ELISA with serotype-1 antigen consistently detected antibody in sera that were positive by IHA test (correlation, 99%). Sera reacting with serotype-1 ELISA antigens also reacted with ELISA antigens prepared from other serotypes. Although ELISA titers averaged 5 log2 units higher than IHA titers, plots of titers determined by the 2 methods were approximately linear. Titer increases detected in paired serum samples by either test were similar. The ELISA was more sensitive than was the IHA in detecting colostral IgG antibody in serum of newborn calves. The ELISA uses a simple, stable antigen preparation and detects antibody to P haemolytica serotypes that commonly infect cattle.     

Antibody dynamics in BRSV-infected Danish dairy herds as determined by isotype-specific immunoglobulins

Using specific ELISAs, antibody levels of four different isotypes to bovine respiratory syncytial virus (BRSV) were determined in calves, following experimental BRSV infection.Most calves experienced an increase in the specific IgM and IgG1 titres about 6-10 days after infection with BRSV. The IgM titre was transient showing positive titres for only 5-10 days, while specific IgG1 was present for a longer time. IgA was detected concomitantly with IgM but at a lower level. Production of IgG2 anti-BRSV antibodies was detected from 3 weeks after infection.In two closed herds, repeated blood samplings were performed on young stock to analyse maternal immunity. The passively transferred antibodies were mainly of the IgG1 isotype and the half-life of IgG1 to BRSV was estimated to be 26.6 days. One of the herds had an outbreak of enzootic pneumonia, diagnosed to be caused by BRSV. Furthermore, another herd with acute BRSV was followed by weekly blood samples in six calves; in both herds IgM and IgG1 was detected shortly after the appearance of clinical signs. Serum samples from 50 Danish dairy herds (453 samples) were tested for immunoglobulins of the isotypes IgG1, IgG2 and IgM. The presence of antibodies to BRSV was widespread and more than 54% of the samples had BRSV antibodies of both the IgG1 and IgG2 isotypes indicating a high herd prevalence to BRSV. Test samples from two herds out of 50 were free from all isotypes to BRSV.     

Escherichia coli Agglutinins in Cow Serum, Colostrum and the Nursing Calf

Immunochemical properties of Escherichia coli O antibodies present in bovine serum and colostrum were investigated. Dam and calf serum samples plus colostral whey samples were fractionated by gel filtration, and the 7S and 19S fractions isolated. Antibody activity against the O antigens of four recognized E. coli bovine pathogens was determined by the indirect hemagglutination test on the whole serum and colostral whey samples and the 7S and 19S fractions thereof. Mercaptoethanol reduction was used to chemically study the immunochemistry of the E. coli O antibodies.

The E. coli O antibodies in dam serum were entirely 19S macroglobulins and appeared to be IgM immunoglobulins. The antibodies in colostrum and calf serum were both 7S and 19S globulins. Reasons for believing these 7S antibodies may be IgG, and the 19S antibodies IgA, immunoglobulins are presented.

     

Persistence of antibodies and anamnestic response in calves vaccinated with inactivated infectious bovine rhinotracheitis virus and parainfluenza-3 virus vaccines

Persistence of antibodies in calves vaccinated with 2 types of inactivated infectious bovine rhinotracheitis (IBR) virus and parainfluenza-3 (PI-3) virus vaccines were determined. Calves seronegative for IBR and PI-3 viruses were inoculated with 2 doses of inactivated IBR virus-PI-3 virus vaccines administered 2 weeks apart. Blood samples were obtained from the calves for serum at 2 weeks, 6 months, and 1 year after vaccination. The serums were tested by serum-neutralization tests. Antibody response to the vaccines persisted on a declining scale for 1 year. The anamnestic responses to the vaccines were determined by inoculating the same calves with a booster dose of vaccine 1 year after the original 2 doses were given. Blood samples were obtained from the calves for serum 2 weeks later. The serums were tested by serum-neutralization tests. The single booster dose of vaccine elicited an anamnestic response to both IBR and PI-3 viruses.     

Estimation of immunity in the developing calf: cellular and humoral responses to keyhole limpet haemocyanin

The efficacy of keyhole limpet haemocyanin (KLH) as a test antigen was determined in calves. Humoral and cellular (in vivo and in vitro) responses were compared. Calves were immunized with KLH at either 3 weeks or up to 5 months of age and immune responses were subsequently tested. Class and subclass antibody responses were detected by ELISA, lymphocyte blastogenesis was measured using a whole blood culture technique (LTT) and skin sensitivity responses were measured as an increase in skin thickness following intradermal injection. In young calves, skin test responses were maximal at 24 h and were found to correlate with IgG1 and IgG2 responses (P less than 0.01), with IgA (P less than 0.05) but not with IgM or LTT. Histological examination of skin swellings found a sequence of cellular events, with polymorphonuclear cells dominating until 48 h after intradermal injection, when mononuclear cells became involved. However, in older calves, skin test responses correlated not only with IgG1 and IgA responses but also with lymphocyte transformation (P less than 0.05). These findings suggest that, while immune responses to KLH may be a useful indicator of immune competence in calves, interpretation should be made with caution particularly in young calves.     

Total and antigen-specific serum immunoglobulin isotype concentrations in hyperimmunized cattle that have undergone plasmapheresis

The effects of prolonged plasmapheresis of cattle on total and antigen-specific immunoglobulin production were evaluated. Five adult cows were hyperimmunized by repeated IV administration of live, logarithmic-phase Pasteurella haemolytica A1 organisms. Three of the cows underwent plasmapheresis daily for 3 weeks. From 2 cows, serum was only obtained periodically. Anti-P haemolytica antibody was assayed by indirect hemagglutination and a kinetic-augmented, antigen-capture ELISA for capsular polysaccharide and lipopolysaccharide/outer membrane protein antigens. Total serum immunoglobulin concentration was determined for IgM, IgG1, and IgG2 by primary radial immunodiffusion. Anti-P haemolytica A1 activity increased rapidly after immunization. After beginning plasmapheresis, the antigen-specific antibody activities remained nearly constant. In general, antilipopolysaccharide/outer membrane protein activity (in terms of concentration) was higher than anti-capsular polysaccharide activity and was not affected as much by the plasmapheresis. Total serum Ig concentration decreased transiently by a small amount after beginning plasmapheresis.     

Serum antibody response to soluble extract of the third-larval stage of Ostertagia ostertagi in cattle

Serum IgG, IgM, and IgA antibody responses against L₃ antigens of Ostertagia ostertagi were monitored by enzyme-linked immunosorbent assay (ELISA) after one, two or multiple sequential inoculations of this nematode in calves. Following the first infection, antibody levels did not change. After a second inoculation, IgG increased significantly (P < 0.05) after 2 months. IgG was not significantly increased 1 month after challenge inoculation. IgM and IgA antibody levels did not change following the first or second inoculations of L₃. IgG antibody levels rose only slightly following multiple sequential inoculations with infectious L₃.

Results indicate that calves with ostertagiasis have very weak serum antibody responses to L₃, and these appear to be of little value in detection of the infection in these animals.