Clinical Evaluation of a Peptide‐ELISA based upon N‐terminal B‐cell Epitope of the VapA Protein for Diagnosis of Rhodococcus equi Pneumonia in Foals

A total of 227 field samples from naturally exposed foals aged between 3 weeks and 6 months were used in an evaluation of a peptide‐based enzyme‐linked immunosorbent assay (ELISA) for diagnosis of Rhodococcus equi infection. A biotinylated peptide derived from the virulence‐associated protein A (VapA) of R. equi, a horse pathogen, was synthesized and designated as PN11‐14. The peptide corresponds to the N‐terminal B‐cell epitope TSLNLQKDEPNGRASDTAGQ of the VapA protein. Based upon a serum immunoglobulin (Ig)G titre of 512 as a positive cut‐off value for the R. equi infection, the ELISA provided the overall sensitivity of 47.62%, specificity of 69.67% and an accuracy of 59.47% with a positive predictive value of 57.47% for true R. equi pneumonia. The assay was improved by detecting VapA‐specific IgGb antibodies against N‐terminal B‐cell epitope of the VapA protein rather than IgG antibodies. The VapA‐IgGb ELISA showed the overall sensitivity of 70.47%, specificity of 72.13% and accuracy of 71.36% with a positive predictive value of 68.52%. Diagnosis of R. equi disease in 6‐week‐old foals showed that the VapA‐IgGb ELISA provided an increasing trend (P = 0.0572) in sensitivity of 82.4% in comparison with the VapA‐IgG ELISA which showed the sensitivity of 58.8%. However, differences in specificity of both tests were statistically insignificant (P = 0.357) as analysed by the McNemar test. These results indicated that detection of VapA‐specific IgGb antibodies may be a better predictor of R. equi disease in foals.     

Isotype-specific antibody responses of cattle to Salmonella Dublin lipopolysaccharide and porin following Salmonella Dublin vaccination and acute and chronic infection.

Stimulation of different T-cell subsets during antigen presentation influences the antibody isotype response to an antigen. Salmonella infection and Salmonella bacterin vaccination are likely to stimulate different T-cell subtypes. The objective of this study was to determine whether there are differences in the isotype response of cattle to Salmonella antigens following Salmonella infection and Salmonella bacterin vaccination. Sera from Salmonella bacterin-vaccinated, experimentally infected, and chronically infected (carrier) adult cattle collected during previous studies was used to evaluate the IgG1, IgG2, and IgM isotype responses of cows to Salmonella serotype Dublin lipopolysaccharide (LPS) and porin. Following vaccination and experimental oral infection, IgG1 titers to LPS and porin rose more quickly and persisted longer than did IgG2 titers. In contrast to Salmonella infection, bacterin vaccination stimulated a weak response to Salmonella porin. Salmonella infection also induced a higher IgG2:IgG1 titer ratio to LPS than did bacterin vaccination. Chronic Salmonella infection induced the highest LPS and porin IgG2:IgG1 titer ratios and the highest correlation between LPS and porin titers. Response operating characteristic curves for each isotype-specific enzyme-linked immunosorbent assay (ELISA) were determined to evaluate the effect of isotype on the sensitivity and specificity of Salmonella ELISA serology for distinguishing sera of Salmonella carriers from those of vaccinated and acutely infected cows. IgG2 titers to LPS and porin provide a more specific indicator of chronic Salmonella infection status than do IgG1 titers to the same antigens with little to no loss in sensitivity.     

Acute Phase Protein Levels in Rabbits with Suspected Encephalitozoon cuniculi Infection

The objective of this study was to evaluate the application of acute phase protein assays for C-reactive protein (CRP), haptoglobin (HP), and serum amyloid A (SAA) in the diagnosis of Encephalitozoon cuniculi (ECUN) infection in pet rabbits. Serum samples from 48 pet rabbits were submitted from veterinary clinics within the United States. Participating veterinarians completed a questionnaire that was used to classify rabbits as either non-ECUN suspect (n = 19) or suspected of having ECUN infection (n = 29). A previously described enzyme-linked immunosorbent assay diagnostic test was used to detect immunoglobulin G (IgG) titers against ECUN. Samples were additionally tested for levels of CRP, HP, and SAA. A nearly 10-fold mean increase in CRP levels was observed in the ECUN-suspect group. This increase was significant (P < 0.05). There was no significant difference in HP or SAA levels between the clinical groups. These data support the use of CRP as an adjunct test in the diagnosis of ECUN infection in pet rabbits.     

Detection by ELISA of the humoral immune response in rabbits naturally infected with Trichophyton mentagrophytes

An indirect enzyme linked immunosorbent assay (ELISA) was developed and its diagnostic potential evaluated for rabbits infected by Trichophyton mentagrophytes. Within-run and between-run coefficient of variance varied from 2.3 to 7.7% and from 5.9 to 8.5%, respectively, indicating satisfactory reproducibility of the ELISA. There was no significant cross-reaction with antigens of Microsporum canis, Malassezia pachydermatis and Aspergillus fumigatus. The level of specific IgG to Trichophyton mentagrophytes was measured in sera of 25 11-week-old and 12 younger infected rabbits. There was no significant difference in the IgG level between 12 5-week-old infected rabbits and controls (p = 0.38). The antibody response was higher in 12 7-week-old rabbits compared with controls (p = 0.001). The IgG level in 25 11-week-old rabbits differed from the controls very significantly (p < 0.0001). Increased specific IgG in 11-week-old rabbits exhibited 96% sensitivity and 94% specificity. Predictive values of a positive and a negative test were 96 and 94%, respectively. Western immunoblotting associated three protein bands (21.5, 31, 44 kDa) with Trichophyton mentagrophytes infection.     

Clinical evaluation of a peptide-ELISA based upon N-terminal B-cell epitope of the VapA protein for diagnosis of Rhodococcus equi pneumonia in foals

A total of 227 field samples from naturally exposed foals aged between 3 weeks and 6 months were used in an evaluation of a peptide-based enzyme-linked immunosorbent assay (ELISA) for diagnosis of Rhodococcus equi infection. A biotinylated peptide derived from the virulence-associated protein A (VapA) of R. equi, a horse pathogen, was synthesized and designated as PN11-14. The peptide corresponds to the N-terminal B-cell epitope TSLNLQKDEPNGRASDTAGQ of the VapA protein. Based upon a serum immunoglobulin (Ig)G titre of 512 as a positive cut-off value for the R. equi infection, the ELISA provided the overall sensitivity of 47.62%, specificity of 69.67% and an accuracy of 59.47% with a positive predictive value of 57.47% for true R. equi pneumonia. The assay was improved by detecting VapA-specific IgGb antibodies against N-terminal B-cell epitope of the VapA protein rather than IgG antibodies. The VapA-IgGb ELISA showed the overall sensitivity of 70.47%, specificity of 72.13% and accuracy of 71.36% with a positive predictive value of 68.52%. Diagnosis of R. equi disease in 6-week-old foals showed that the VapA-IgGb ELISA provided an increasing trend (P=0.0572) in sensitivity of 82.4% in comparison with the VapA-IgG ELISA which showed the sensitivity of 58.8%. However, differences in specificity of both tests were statistically insignificant (P=0.357) as analysed by the McNemar test. These results indicated that detection of VapA-specific IgGb antibodies may be a better predictor of R. equi disease in foals.     

Development and validation of an ELISA for the detection of leptospire-specific antibodies in rodents

An enzyme-linked immunosorbent assay (ELISA) for the detection of IgG antibodies in rodents was developed and validated with the microscopic agglutination test (MAT) and leptospiral cultures. Sonicated antigen from cultures of serovars tarassovi and pyrogenes was used. As conjugate, a combination of anti-rat and anti-hamster IgG labeled with peroxidase was used. The optimal cut-off point was determined by plotting the sensitivity and specificity for various cut-off point values by means of receiver operating characteristic (ROC) curve. Concordance between ELISA and each of the MAT titers was measured by kappa (kappa). Proportions of positive results were compared by means of McNemar's test. Total 214 rodents were trapped, but only 117 could be processed by the three techniques (culture, ELISA, MAT; 1:20, 1:40, 1:50) and used for statistical analysis. Although, MAT titers in rodents infected with the serogroup Ballum tended to be lower than those infected with the serogroup Icterohaemorrhagiae, all (20/20) were ELISA-positive and almost all (19/20) were MAT-positive.The percentage of positive results obtained by ELISA, 47.0% exceeded significantly the 40.2% obtained by MAT (1:50). Difference between ELISA and MAT (1:40) was not significant and no differences were observed between ELISA and MAT (1:20). Agreement, specificity, sensitivity and the consequent area under the ROC curve between ELISA and MAT were higher as MAT cut-off points were lowered, being optimal at 1:20. The fact that differences between ELISA and MAT were significant at 1:50, but not at 1:40 or 1:20, supports the suggestion that lower MAT titers should be considered positive in rodents. The ELISA developed to detect leptospire-specific antibodies had optimal sensitivity and specificity in relation to MAT and it is concluded that it may constitute a very useful indicator for epidemiological purposes of past or present leptospiral infection in rodents.     

Serological distinction of bovine Salmonella carriers from vaccinated and acutely infected cows.

Identification of Salmonella carriers using lipopolysaccharide (LPS) ELISA serology in a Salmonella-infected herd requires distinction of chronically infected cattle from convalescent and vaccinated cows. Cows responding to Salmonella infection and vaccination produce titers to Salmonella LPS that overlap with the lower titers of some Salmonella carriers. The objective of this study was to determine if the LPS antigen specificity of the bovine humoral immune response to Salmonella LPS antigens differs following vaccination and acute and chronic Salmonella infection. The study focused on the nondiscriminatory area of Salmonella ELISA serology, specifically, peak-titered sera from Salmonella bacterin-vaccinated and experimentally infected cows and low-titered sera from Salmonella carriers. The LPS serogroup specificity of the IgG1 and IgG2 response following acute and chronic Salmonella serotype Dublin infection and Salmonella bacterin vaccination was evaluated using 5 Salmonella serogroup (B, D, E1, C3, and C1) LPS ELISA assays. IgG, titers of carriers, vaccinated, and acutely infected cows were predominantly O antigen specific. Similarly, the IgG2 titers of acutely infected cows were also O antigen specific. In contrast, Salmonella carriers produced an IgG2 response to each of the heterologous LPS antigens (B, E1, C3, and C1) examined. The results of this study indicate that the bovine IgG1 isotype response to Salmonella LPS is serogroup specific. Conversely, production of IgG2 antibodies to core Salmonella LPS antigens shared across Salmonella serogroups is a feature of chronic Salmonella infections.     

Comparison of cellular and humoral immunoassays for the assessment of summer eczema in horses

The objective of this study was to compare and analyze three common diagnostic methods for summer eczema (SE) in horses, an allergic dermatitis caused by bites of Culicoides spp. Nine horses with a medical history of SE and nine control animals were intradermally challenged with whole body extracts (WBE) and the saliva of a native (C. nubeculosus) and exotic (C. sonorensis) Culicoides species. Blood and serum samples of the horses were examined for basophil reactivity by a histamine release test (HRT) and for Culicoides-specific serum immunoglobulin E (IgE) and G (IgG) by enzyme-linked immunosorbent assay (ELISA). The results of intradermal testing (IDT) at 30min (immediate reactivity) and 4h (late-phase reactivity) post challenge with most insect preparations revealed significant differences between horses with and without SE. Overall, the HRT showed the most accurate results with a sensitivity of 1.00 for all Culicoides preparations and specificities of 0.78 (WBE) and 1.00 (saliva). By contrast, delayed reactions of the IDT (24h), and levels of Culicoides-specific IgE and IgG in the native serum showed little or no distinction between allergic and non-allergic horses. However, the use of purified serum IgE and IgG indicated the possibility for elevated titers of insect-specific serum immunoglobulins in horses with SE. The IDT and HRT did not reveal obvious differences in onset and intensity of positive reactions for the native verses exotic Culicoides species, whereas the ELISA showed slightly higher numbers of positive reactions for serum IgG with the indigenous species. Saliva, as compared to WBE, was found to have improved sensitivity and/or specificity for the HRT and for the late-phase immune reactions as measured by the IDT. Overall, the results indicate that allergy tests utilizing effector cells (mast cells, basophils) are more accurate in diagnosing SE in horses than serological analysis by ELISA.     

Development and evaluation of an enzyme-linked immunosorbent assay based on recombinant TgSRS2 for serodiagnosis of Toxoplasma gondii infection in cats

An enzyme-linked immunosorbent assay (ELISA) based on recombinant SAG1-related sequence 2 of Toxoplasma gondii (rTgSRS2) was developed to detect toxoplasmosis in cats. The specificity and sensitivity of rTgSRS2 ELISA were confirmed using a series of serum samples from T. gondii-experimentally infected mice. A total of 76 field samples from cats were examined by the developed ELISA. The rTgSRS2 ELISA showed a good diagnostic performance characterized by high concordance (88.16) and kappa value (0.76) with latex agglutination test (LAT). The sensitivity and specificity of the test were 92.68% and 82.86%, respectively. These results suggest that the ELISA based on rTgSRS2 could be a useful tool for serodiagnosis of T. gondii infection in cats.     

Immune response of pregnant cows to bovine rotavirus immunization

Fifteen pregnant Holstein cows were freely assigned to 3 experimental groups (5 cows in each group). Cows in group I were inoculated IM and intramammarily (IMm) with Ohio Agricultural Research and Development Center (OARDC) tissue culture-propagated modified-live Nebraska calf diarrhea bovine rotavirus with added adjuvant (OARDC vaccine-immunized cows). Group II cows were given IM injections of a commercial modified-live rotavirus-coronavirus vaccine (commercial vaccine-immunized cows), and the remaining 5 cows were noninoculated controls (group III). Rotavirus antibody in colostrum and milk was mainly associated with immunoglobulin (Ig)G1, and less so with IgG2, IgA, and IgM, as analyzed by the enzyme-linked immunosorbent assay (ELISA), using monospecific anti-bovine IgG1, IgG2, IgM, and IgA sera. In serum, the rotavirus antibody was distributed almost equally between IgG1 and IgG2. The same relationships appeared in both immunized and nonvaccinated cows. All OARDC vaccine-injected cows had virus-neutralization (VN) and ELISA IgG1 rotavirus antibody titers in serum and mammary secretions at significantly increased levels (at least 100-fold; P less than 0.05) compared with the titers in groups II (commercial vaccine-immunized cows) and III (controls). Serum, colostrum, and milk antibody titers from these latter 2 groups did not differ statistically. The ELISA IgG2, IgA, and IgM rotavirus antibody titers also were significantly greater in mammary secretions from OARDC vaccine-immunized cows than in groups II and III cows. There was a high correlation between ELISA IgG1 and VN rotavirus antibody titers for all samples tested (r = 0.97, P less than 0.001), but ELISA IgG1 antibody titers were consistently higher than VN titers. The ELISA IgG1 and VN antibody titers of milk samples collected from cows 30 days after parturition were higher from the OARDC vaccine-immunized cows (ELISA IgG1, geometric mean titer (GMT) = 3,511; VN GMT = 1,689) than were titers from the group II cows (ELISA IgG1 GMT = 39; VN GMT = 33) or group III cows (ELISA IgG1 GMT = 21; VN GMT = 19). These results indicate that IM plus IMm immunization of pregnant cows, using modified-live bovine rotavirus with added adjuvant, may significantly enhance serum, colostrum, and milk rotavirus antibody titers, whereas IM vaccinal inoculation of pregnant cows with a commercial modified-live rotavirus-coronavirus vaccine may not.     

Comparison of a clinic-based ELISA test kit with the immunofluorescence test for the assay of Ehrlichia canis antibodies in dogs.

The "gold standard" for the detection of antibodies to Ehrlichia canis, the cause of canine monocytic ehrlichiosis (CME), is the indirect immunofluorescence antibody (IFA) test. The IFA test however is generally available only in selected laboratories and requires extensive equipment and trained personnel. A double-blind study was conducted to compare the ability of an in-clinic standardized enzyme-linked immunosorbent assay (ELISA) test kit to measure E. canis IgG antibodies in dogs compared with the standard IFA technique. A good correlation was found between the 2 techniques (r2 = 0.8793; P < 0.0001). Evidence for the sensitivity of the ELISA technique for the early detection of E. canis IgG antibodies was demonstrated by comparing the appearance of E. canis antibody titers by the IFA and ELISA techniques after artificial infection of 2 sets of dogs. In both experimental infections, both tests were equally sensitive for the early detection of IgG antibodies against E. canis, and the results correlated well with the appearance of fever and clinical signs. Proposed application of the in-clinic ELISA test is to aid in the diagnosis of CME.     

Detection of Aspergillus-specific antibodies by agar gel double immunodiffusion and IgG ELISA in feline upper respiratory tract aspergillosis

Feline upper respiratory tract aspergillosis (URTA) is an emerging infectious disease. The aims of this study were: (1) to assess the diagnostic value of detection of Aspergillus-specific antibodies using an agar gel double immunodiffusion (AGID) assay and an indirect immunoglobulin G (IgG) ELISA; and (2) to determine if an aspergillin derived from mycelia of Aspergillus fumigatus, Aspergillus niger and Aspergillus flavus can be used to detect serum antibodies against cryptic Aspergillus spp. in Aspergillus section Fumigati. Sera from cats with URTA (group 1: n = 21) and two control groups (group 2: cats with other upper respiratory tract diseases, n = 25; group 3: healthy cats and cats with non-respiratory, non-fungal illness, n = 84) were tested. Isolates from cats with URTA comprised A. fumigatus (n = 5), A. flavus (n = 1) and four cryptic species: Aspergillus felis (n = 12), Aspergillus thermomutatus (Neosartorya pseudofischeri, n = 1), Aspergillus lentulus (n = 1) and Aspergillus udagawae (n = 1).Brachycephalic purebred cats were significantly more likely to develop URTA than other breeds (P = 0.013). The sensitivity (Se) of the AGID was 43% and the specificity (Sp) was 100%. At a cut-off value of 6 ELISA units/mL, the Se of the IgG ELISA was 95.2% and the Sp was 92% and 92.9% for groups 2 and 3 cats, respectively. Aspergillus-specific antibodies against all four cryptic species were detected in one or both assays. Assay Se was not associated with species identity. Detection of Aspergillus-specific antibodies by IgG ELISA has high Se and Sp for diagnosis of feline URTA.     

RELATIONSHIP BETWEEN RADIOGRAPHIC HILAR LYMPHADENOPATHY AND SEROLOGIC TITERS FOR COCCIDIOIDES SP. IN DOGS IN AN ENDEMIC REGION

Hilar lymphadenopathy is a common radiographic finding in coccidioides infections. Serologic studies are used most often for the diagnosis of coccidioidomycosis in endemic areas, with IgG titers ≥1:8 considered positive for infection and lower IgG titers of <1:8 considered indicative of exposure and not necessarily related to organism presence. The objective of this study was to determine the relationship of hilar lymphadenopathy to coccidioidomycosis titers for dogs in an endemic area. A positive association between these parameters would allow treatment to be initiated before obtaining titer results. Thoracic radiographs of 131 dogs from an endemic area were reviewed for evidence of hilar lymphadenopathy. These results were compared with serology results. There was a significant association between hilar lymphadenopathy and a positive serology result (P<0.001). With hilar lymphadenopathy as a predictor of a positive titer result, sensitivity was 28.0%, specificity was 91.5%, the positive predictive value was 43.8%; and the negative predictive value was 84.4%. There was no association between the titer result and gender, age, or weight. The radiographic finding of hilar lymphadenopathy appears to be a useful indicator of coccidioidomycosis infection in an endemic population of dogs supporting the treatment of patients for coccidioidomycosis when hilar lymphadenopathy is present and before obtaining serology results.     

Antigen requirements and specificity of enzyme-linked immunosorbent assay for detection of canine IgG against canine distemper viral antigens

An enzyme-linked immunosorbent assay (ELISA) was developed for detection of specific immunoglobulin G (IgG) against canine distemper virus (CDV) antigens. Sucrose gradient separation of viral and cellular proteins was required to produce coating antigens for the ELISA. The specificity of the ELISA was demonstrated by blocking CDV-positive canine sera with CDV-specific antisera produced in goats and rabbits and adsorption of positive sera with CDV antigens. A comparison of the ELISA with the serum-neutralization technique for the detection of CDV antibodies was conducted. Anti-CDV IgG was detected in conventional dogs as early as 6 days after inoculation with a commercial vaccine to CDV. Paired sera from the immunized dogs were evaluated by both techniques and a statistically (P less than 0.01) significant agreement between the ELISA and the serum-neutralization technique was shown (r = 0.6121, n = 75).     

Bayesian estimation of diagnostic accuracy of a new bead-based antibody detection test to reveal Toxoplasma gondii infections in pig populations

The success of a Toxoplasma gondii surveillance program in European pig production systems depends partly on the quality of the test to detect infection in the population. The test accuracy of a recently developed serological bead-based assay (BBA) was investigated earlier using sera from experimentally infected animals. In this study, the accuracy of the BBA was determined by the use of sera from animals from two field subpopulations. As no T. gondii infection information of these animals was available, test accuracy was determined through a Bayesian approach allowing for conditional dependency between BBA and an ELISA test. The priors for prevalence were based on available information from literature, whereas for specificity vague non-informative priors were used. Priors for sensitivity were based either on available information or specified as non-informative. Posterior estimates for BBA sensitivity and specificity were (mode) 0.855 (Bayesian 95% credibility interval (bCI) 0.702–0.960) and 0.913 (bCI 0.893–0.931), respectively. Comparing the results of BBA and ELISA, sensitivity was higher for the BBA while specificity was higher for ELISA. Alternative priors for the sensitivity affected posterior estimates for sensitivity of both BBA and ELISA, but not for specificity. Because the difference in prevalence between the two subpopulations is small, and the number of infected animals is small as well, the precision of the posterior estimates for sensitivity may be less accurate in comparison to the estimates for specificity. The estimated value for specificity of BBA is at least optimally defined for testing pigs from conventional and organic Dutch farms.     

Work-related increases in titer of Campylobacter jejuni antibody among workers at a chicken processing plant in Miyazaki prefecture,Japan, independent of individual ingestion of edible raw chicken meat

Workers in poultry abattoirs may be frequently exposed to Campylobacter jejuni, which is a leading cause of bacterial food poisoning in Japan. The present study was conducted to measure the titers of IgG and IgA antibodies against C. jejuni among 104 female workers in a chicken processing plant in Miyazaki prefecture, Japan. Information regarding habitual ingestion of raw chicken meat and potential occupational risk factors was collected using a questionnaire. Acid extracts of four C. jejuni strains representing the genotypes most dominant in Miyazaki were used as antigens. The levels of both immunoglobulins measured by ELISA were not correlated with ingestion of edible raw chicken meat, the amount consumed in one sitting, or its frequency. Although age was correlated with antibody levels, the length of employment was not. Furthermore, the IgG and IgA levels in workers at the evisceration step were significantly higher than those at other locations in the plant. To identify the bacterial proteins recognized by the workers’ IgG and IgA antibodies, Western blotting followed by LC/MS was conducted. Flagellin was identified as the common protein recognized in the sera of workers for whom ELISA demonstrated both the highest and lowest antibody levels. We concluded that the titers of IgG and IgA against C. jejuni in workers at the processing plant had been increased by occupational exposure to Campylobacter, regardless of raw chicken meat ingestion.     

Aspects of the humoral immune response to Staphylococcus intermedius in dogs with superficial pyoderma,deep pyoderma and anal furunculosis

Bacterial infection (pyoderma) of the canine skin is largely caused by Staphylococcus intermedius and may be a superficial or deep infection. Pyoderma may be a primary, idiopathic disease or secondary to a range of other dermatological disorders. In this study, the serum concentrations of IgG, IgA, antistaphylococcal IgG and antistaphylococcal IgA were measured by ELISA in normal dogs (n = 22), dogs with idiopathic deep pyoderma (n = 22), atopic dermatitis and superficial pyoderma (n = 24), atopic dermatitis without pyoderma (n = 25), flea bite dermatitis with superficial pyoderma (n = 8), pustular demodicosis (n = 8) and German shepherd dogs with anal furunculosis (n = 28). The serum IgG was significantly increased in dogs with atopy and superficial pyoderma (p < 0.001), and lower than normal in dogs with idiopathic deep pyoderma (p < 0.015). The concentration of serum IgA was significantly lower than normal in dogs with atopy uncomplicated by pyoderma (p < 0.015). The concentration of antistaphylococcal IgG in all clinical sera was significantly elevated (p < 0.001) when compared to normal dogs but concentrations of antistaphylococcal IgA were no greater than in normal dogs. Western blotting analysis for determination of the specificity of serum IgG antistaphylococcal antibody revealed that there were nine major epitopes. Discriminant analysis demonstrated that particular combinations of these epitopes were recognised more frequently by sera from dogs in different clinical groups.     

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.     

Blocking enzyme-linked immunosorbent assay for detection of antibodies against Actinobacillus pleuropneumoniae serotype 6 in pig serum

A blocking enzyme-linked immunosorbent assay (ELISA) detecting antibodies against Actinobacillus pleuropneumoniae (Ap) serotype 6 was developed. The blocking ELISA was based on the inhibition of a polyclonal antibody raised against Ap serotype 6. Purified lipopolysaccharide from Ap serotype 6 was used as antigen. The blocking ELISA was tested against sera from pigs experimentally infected with the 12 serotypes of Ap biotype 1. Cross-reaction with serotypes 3 and 8 but not with other serotypes was observed. The sensitivity and specificity of the test on a herd level were evaluated with sera from herds naturally infected with serotypes 2, 6, 8 or 12 and with sera from herds free of infection with any Ap serotype. The blocking ELISA showed a high herd sensitivity (1.00 (0.79-1.00)) and specificity (0.97 (0.93-0.99)).