Antigenic relationships between the serotypes of Pasteurella haemolytica demonstrable by enzyme-linked immunosorbent assay (ELISA)

Mice and rabbits were immunised with sodium salicylate extracts (SSE) prepared from each of 12 serotypes of Pasteurella haemolytica, and the antisera to each were used in cross-indirect haemagglutination (IHA) tests and cross-enzyme-linked immunosorbent assays (ELISA) to study antigenic relationships between the serotypes. An indirect micro-ELISA demonstrated common antigenic relationships which were not apparent by IHA. Antisera from both species revealed considerable shared antigenicity between all the serotypes. Rabbit antisera presented clearer differences between the A biotypes on one hand and the T biotypes on the other, the T biotypes exhibiting much less cross-relatedness than that shown between the A serotypes.     

An enzyme-linked immunosorbent assay for detecting antibodies to Pasteurella multocida in chickens

An enzyme-linked immunosorbent assay (ELISA) was developed to detect the humoral antibody response in chickens receiving subcutaneous injections of the CU vaccine strain of Pasteurella multocida. Serum samples were collected twice weekly for 3 weeks, and chicken antibody responses were monitored using ELISA. The positive/negative ratio method of analysis was used to determine the antibody titer of vaccinated chickens. After a loge transformation of the ELISA titer, a linear relationship was confirmed between ELISA titer and positive/negative ratio. Regression analysis was used to construct a standard curve and derive an equation from this relationship. Using this equation, only one dilution was needed to determine the antibody titer of any unknown serum sample. The ELISA technique was used to monitor the mean antibody titer of vaccinated chickens over the 3-week period. A classic primary response curve occurred when titer was plotted against time.     

Rapid identification of Pasteurella multocida organisms responsible for haemorrhagic septicaemia using an enzyme-linked immunosorbent assay

Haemorrhagic septicaemia (HS) is caused by specific serotypes of Pasteurella multocida and is one of the major economic diseases of cattle and buffalo in South East Asia. Definitive diagnosis of the disease-causing organism with the available methods is labour intensive and not totally reliable, consequently, an ELISA system to identify P multocida organisms which cause HS was developed. One hundred and twenty-four P multocida isolates were tested, 58 were type strains and 66 were field isolates. Analysis of these strains indicated the assay had a specificity of 99 per cent and sensitivity of at least 86 per cent. The sensitivity could be an underestimate, as five isolates assumed to be false negative reactions may not all be HS-causing strains. The HS ELISA provides a rapid, simple, accurate and inexpensive diagnostic assay for identification of HS causing organisms but does not represent a new typing system for P multocida. This assay will also enable countries to assess the impact of HS more accurately.     

Comparison of enzyme-linked immunosorbent assay with dot immunobinding assay for detection of antibodies against Pasteurella multocida in turkeys

A dot immunobinding assay (DIA) was developed to detect antibodies against Pasteurella multocida in turkey serum. Five coating antigens, namely, whole-cell (WC) antigen, sonicated cell lysate (SCL), crude capsular extract (CE), formalin extract (FE), and heat-stable antigen (HSA), were compared by enzyme-linked immunosorbent assay (ELISA) and DIA using reference antisera against P. multocida organisms. WC and SCL antigens showed higher sensitivity, whereas FE and HSA antigens were more specific coating antigens in both assays. The specificity of DIA was greater than ELISA by comparing the P/N ratios of HSA against serum prepared from heterologous serotype of P. multocida. The DIA had also several distinct advantages over the ELISA, which included reduction of the manipulation time and more uniform binding of coating antigens onto the nitrocellulose membranes compared with binding of coating antigens to microtiter plates for ELISA.     

Comparison of vaccination protocols of broiler breeder hens for Pasteurella multocida utilizing enzyme-linked immunosorbent assay and virulent challenge

A commercial enzyme-linked immunosorbent assay (ELISA) to detect serological response to vaccination and virulent challenge with type 1 (X-73) Pasteurella multocida was used to determine the best vaccination protocol for broiler breeders against fowl cholera. Birds vaccinated twice, at 10 and 19 weeks of age, with the avirulent Clemson University (CU) strain both times, with a commercial bacterin first and the CU strain second, or with the CU strain first and bacterin second had the highest survival rates (98-100%) following challenge at 25 weeks. The two groups that received the CU strain and bacterin also produced the highest mean ELISA antibody titers (greater than 10,000). Birds vaccinated once, at 10 weeks, with the CU strain had the same survival rate as birds vaccinated twice with bacterin (90 and 91%). Under the conditions of this experiment, an ELISA titer greater than or equal to 1000 resulted in at least a 92% survival rate after virulent challenge (23% survival in nonvaccinates).     

Comparison of various antigens and their ability to detect protective antibodies against Pasteurella multocida using enzyme-linked immunosorbent assay

Various antigenic extracts of the CU strain of Pasteurella multocida were prepared to determine their suitability as plate antigens for use in the enzyme-linked immunosorbent assay (ELISA) for the detection of fowl cholera antibodies. Antisera from two separate broiler breeder flocks with known fowl-cholera-vaccination histories were collected just before the birds were challenged with virulent strain X-73 P. multocida. A potassium thiocyanate (KSCN)-extracted antigen, a capsular (CAP) antigen, a lipopolysaccharide-protein antigen, and heat-stable, salt-soluble antigen were all suitable as ELISA plate-coating antigens. Filtered and unfiltered sonicates of the CU strain of P. multocida were also suitable ELISA plate antigens. The results suggested that different plate antigens were detecting different populations of antibodies formed in response to fowl cholera vaccinations. When antibody titers were correlated with survival after challenge, the KSCN and the CAP plate antigens placed more nonsurvivors into low-antibody-titer ranges and more survivors (protected birds) into the high-antibody-titer ranges than the other plate antigens.     

Comparison of enzyme-linked immunosorbent assay and indirect hemagglutination test for quantitating antibody responses in chickens against Pasteurella multocida

An indirect enzyme-linked immunosorbent assay (ELISA) was developed to measure humoral antibody responses of chickens against Pasteurella multocida. A standard indirect hemagglutination (IHA) test was used to compare serologic results with those of ELISA. The ELISA was also used following challenge with P. multocida to compare the efficacy of three commercial fowl cholera vaccination regimens. Although antibody titers measured by ELISA and IHA were highly correlated, ELISA was at least twice as sensitive as IHA. Antibody measured by ELISA and IHA also correlated significantly with protection against P. multocida challenge. No mortality occurred in any of the three vaccinated challenged groups. However, control unvaccinated chickens experimentally infected with P. multocida developed signs of acute pasteurellosis and died by the 10th day post-challenge. Impression smears made of hepatic tissue from all chickens were stained (Wright's stain), and typical bipolar rods characteristic of Pasteurella were identified in smears from unvaccinated challenged controls only.     

Serological cross-reactivity of avian adenovirus serotypes in an enzyme-linked immunosorbent assay

Fowl adenoviruses free of avian adenovirus-associated virus, representing 10 serotypes, were tested for cross-reactivity in an enzyme-linked immunosorbent assay (ELISA). All antigens and antisera prepared in chickens, along with uninfected control antigen and normal chicken serum, were reacted in a checkerboard pattern with ELISA. There was considerable cross-reactivity among all serotypes tested. Homologous reactions were generally, but not always, stronger than heterologous reactions. ELISA was about as sensitive as virus neutralization in detecting antibodies. The high sensitivity plus broad-spectrum reactivity should make ELISA a preferred test for the detection of adenovirus antibodies in poultry flocks.     

Development and evaluation of an enzyme-linked immunosorbent assay for bovine antibody to Pasteurella haemolytica: constructing an enzyme-linked immunosorbent assay titer

A 2-stage strategy was developed and evaluated for estimating serum antibody titer by use of ELISA and a series of dilutions. In stage 1, the linear response region and least-square estimate of the assay line slope were established from 9-point dilution assays. Provided that the reading was within the linear response region, this information was used in the stage-2 estimation of titer from a single absorbance reading. Operationally, 2 fixed dilutions were selected, one suitably low and one suitably high, to provide at least one reading within the linear region. The procedure should save considerable time when a large number of assays are to be performed. Stage 1 required approximately twenty 9-point assays, but all subsequent assays required only 2 fixed dilutions.     

Prevalence of trichinosis in Canadian swine determined serologically by enzyme-linked immunosorbent assay.

Fifteen thousand three hundred and eighteen porcine sera from all regions of Canada were examined for the presence of anti-Trichinella antibodies using the enzyme-linked immunosorbent assay with an excretory-secretory antigen. Four sera (0.026%) revealed the presence of anti-Trichinella antibodies, with titers (optical density readings) that fell in the low positive or high negative range on repeated examinations. One animal originated in British Columbia and three in Ontario. Serological examination of swine in the herds at time of traceback did not reveal further animals with anti-Trichinella antibodies.     

Evaluation of an indirect enzyme-linked immunosorbent assay (ELISA) using recombinant toxin for detection of antibodies against Pasteurella multocida toxin

To facilitate the control of progressive atrophic rhinitis (PAR) of swine caused by toxigenic Pasteurella multocida, an enzyme-linked immunosorbent assay (ELISA) and a serum neutralization test (NT) have recently been developed to detect antibodies against the P. multocida dermonecrotic toxin (PmDNT). However, the NT is a cumbersome and time-consuming technique. To overcome these drawbacks, we developed an indirect ELISA, using recombinant PmDNT expressed in Escherichia coli, for the detection of antibodies to PmDNT in serum samples from pigs. The practical usefulness of this ELISA was compared with the NT using serum samples obtained from experimentally infected and naturally infected pigs. In the pigs experimentally inoculated with vaccine including PmDNT toxoid, the ELISA and neutralization antibodies were detected at almost the same time, and a good correlation was demonstrated between both tests (P<0.01, R(2)=0.807). Therefore, the ELISA can be used to evaluate the immune reaction of pigs after vaccination with P. multocida toxoid. In a survey conducted on a field herd with a history of clinical AR, the seropositivity by ELISA in pigs of age 4.5-6 months was increased even though the NT was negative, and the correlation was low between the results obtained with the two tests (P<0.01, R(2)=0.38). Therefore, the results indicated that this ELISA might be a useful alternative to the NT currently used to detect the antibody to PmDNT after vaccination or infection with P. multocida.     

Lipopolysaccharides of the Heddleston serotypes of Pasteurella multocida

Lipopolysaccharides (LPS) were extracted from 13 of the 16 Heddleston serotypes of Pasteurella multocida by phenol-chloroform-petroleum ether (PCP). Serotypes 3, 9, and 13 were extracted only by phenol-water (PW). After extraction of LPS of serotype 9 by PW, an additional LPS was isolated by PCP. All LPS contained glucose, 2-keto-3-deoxyoctonate, and heptose. Two isomers of heptose, D-glycero-D-mannoheptose and L-glycero-D-mannoheptose, were found in serotypes 2 and 5. Antisera made against purified LPS of serotypes 2 and 5 reacted with both heat-stable antigens and LPS from serotypes 2 and 5 in the gel-diffusion precipitin test. Antisera against serotype 2 LPS protected turkeys against challenge with capsulated serotype 5, indicating that a structural relationship exists between LPS of strains that cause hemorrhagic septicemia and fowl cholera. Rhamnose was a component of serotype 9 LPS, and galactose was found in all LPS, except for serotype 11. The LPS of serotype 13 contained an isomer of heptose that has not been identified. The LPS had buoyant densities in CsCl of 1.40 +/- 0.0148 g/ml, and all hemagglutinated chicken and turkey, but not sheep or horse, RBC.     

Fowl cholera immunity in broiler breeder chickens determined by the enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies in broiler breeders vaccinated (wing web) with the CU fowl cholera vaccine. Birds were bled weekly from 9 to 26 weeks, every other week from 26 to 40 weeks, and every 4 weeks from 40 to 56 weeks of age. Overall mean ELISA antibody titers (9 to 56 weeks) reported as log10 values and survivability of the vaccinates after virulent challenge were as follows: unvaccinated--5.75, 48%; birds vaccinated once at 8 weeks--5.91, 78%; birds vaccinated twice at 8 and 14 weeks--6.11, 100%; birds vaccinated thrice at 8, 14, and 20 weeks--6.23, 100%; birds vaccinated twice at 8 and 20 weeks--6.12, 100%; and birds vaccinated twice at 8 and 20 weeks (plus fowl pox at 8 weeks)--6.08, 95%. Survivability in the vaccinates after virulent challenge with strain X-73 Pasteurella multocida was 100% in birds with ELISA antibody titers (log10) greater than 6.02. Under the conditions of this experiment, birds vaccinated two or three times between 8 and 20 weeks proved to be sufficiently immune at 56 weeks of age to withstand a virulent fowl cholera challenge. Birds not vaccinated or vaccinated only once at 8 weeks were not sufficiently immunized to withstand virulent challenge.     

Evidence of paratuberculosis in Ohio's white-tailed deer, as determined by an enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) was developed and used to detect antibodies to Mycobacterium paratuberculosis in serum samples obtained in December of 1983 from 954 hunter-killed white-tailed deer (Odocoileus virginianus) in 13 Ohio counties. Positive or negative status was determined by calculating a signal-to-noise ratio, a ratio between the optical density of the test serum and negative reference sera; a ratio of greater than or equal to 3.0 was considered positive. Twenty-four samples (2.5%) were found to be assay positive, using this method. A statistically significant difference among age groups was found, with those less than or equal to 6 months of age having a lower proportion of positives. Differences by sex were not observed. To determine the validity of the ELISA in deer, serum samples from 46 fallow (Dama dama) and axis deer (Axis axis) harvested from a known infected population were tested by ELISA and agar-gel immunodiffusion. The agar-gel immunodiffusion test showed evidence of exposure of the deer to M paratuberculosis or a related antigen. The ELISA closely approximated the prevalence of paratuberculosis infection as previously determined by fecal culture in this population. As a result of these tests, it was concluded that free-ranging Ohio deer have been infected with M paratuberculosis or exposed to a closely related antigen.     

Fowl cholera immunity induced by various vaccines in broiler minibreeder chickens determined by enzyme-linked immunosorbent assay

Broiler minibreeder hens were vaccinated for protection against fowl cholera at 12 and 21 weeks of age using several vaccination schemes, which included a live Pasteurella multocida (CU strain) vaccine, two commercial polyvalent fowl cholera oil-based bacterins, and two experimentally prepared polyvalent oil-based bacterins. Some treatment groups received only live or killed vaccines, whereas others received a live vaccine at 12 weeks followed by a killed product at 21 weeks. At 42 weeks of age, all birds that received the live CU vaccine twice or once followed by a bacterin survived challenge. Birds that received killed vaccines only were significantly less protected but still showed a respectable survival rate of 86%. All unvaccinated controls died within 72 hr after challenge. At 72 weeks of age, overall protection was lower than that at 42 weeks, regardless of vaccination treatment. Antibody titers were usually higher in birds that received bacterins than in those receiving live vaccines, yet overall protection was still greater in those birds that received the live cholera vaccine twice.     

Specific identification of Pasteurella multocida serogroup-A isolates by PCR assay

A polymerase chain reaction (PCR) assay targeting the hyaC-hyaD gene was developed and used to identify strains of Pasteurella multocida belonging to serogroup-A. A set of serogroup-specific-PCR primers amplified a 564 bp product from genomic DNA prepared from bacterial cells or directly from bacterial colonies. This method detected as low as 10 ng of bacterial DNA and had a specificity of 100% for P. multocida serogroup-A. A nested PCR method yielded a single 374 bp product. All fifty isolates were also shown to be identical by restriction fragment length polymorphism (RFLP) analysis of the PCR products after digestion with BglII.     

Age-related changes in porcine corticosteroid-binding globulin (pCBG) as determined by an enzyme-linked immunosorbent assay

The objectives of this study were to develop an assay for the direct measure of porcine corticosteroid-binding globulin (pCBG) and to confirm age-related changes in plasma pCBG concentration. Isolation and purification of pCBG from plasma was performed by affinity chromatography and HPLC–DEAE anion exchange techniques. Analysis by SDS–PAGE revealed two polypeptides (54 and 59 kDa) having similar amino acid homology (>50%) to previously reported sequences of seven mammalian species for the first 33 amino acids. Porcine CBG (20 ng/well) was immobilized to microtiter plates and standards or samples added along with rabbit antiserum developed against the purified pCBG. Goat anti-rabbit IgG-alkaline phosphatase conjugate was added followed by p-NPP substrate. The resultant color development was read at 405 nm. Intra- and interassay coefficients of variation (n=26) of a pooled sample were 10 and 15%, respectively. Age-related changes (P<0.001) in plasma pCBG concentration (n=203) from day 3 through 168 of age confirmed that, in the pig, changes seen in the percent distribution of cortisol among protein bound and free forms around day 28 of age are associated with an increase in CBG concentration.     

Serodiagnosis of canine leptospirosis by solid-phase enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) to detect antibodies to Leptospira interrogans serotype canicola in dogs was developed and evaluated. Comparison of the ELISA with the microscopic agglutination test (MAT) showed that, during the first two weeks after an experimental infection with serotype canicola, the ELISA detected antibody at higher dilutions than the MAT. After the second week post-infection both tests detected antibody at almost equal titres (r = 0.89). The outer envelope (OE) antigen of serotypes icterohaemorrhagiae, copenhageni and canicola was fairly serotype-specific, whereas the pellet (P) antigen showed more cross-reactivity. Both OE and P antigen of Leptospira biflexa strain Patoc I could be used as cross-reacting antigen in the ELISA. Compared to the MAT, the ELISA has some technical advantages. It is suggested that the ELISA would be useful as a screening test.