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.     

Cell-culture virus-neutralization test and enzyme-linked immunosorbent assay for evaluation of immunity in chickens against fowlpox

Two serological tests--the virus-neutralization (VN) test in chicken embryo fibroblasts (CEF) using a cell-culture-adapted virus, and the enzyme-linked immunosorbent assay (ELISA)--were used for evaluating the immune response in chickens against fowlpox virus. The VN test was conducted in 96-well tissue-culture plates using a fowlpox virus that was adapted to induce cytopathic effects (CPE) in CEF in 48 hr. The ELISA was carried out with an antigen prepared by precipitation of a cell-culture-propagated virus suspension with ammonium sulfate and concentration by centrifugation. A 0.1 M acetate buffer, pH 5, was used as the sensitizing solution for maximum specific binding of the antigen to the microplate plastic well. No antibodies were detected by the VN test in 228 serum samples taken from chickens at irregular intervals between 1 and 39 weeks of age, even though the birds were vaccinated against fowlpox at 13 weeks of age. However, in sera collected 4 weeks after a sample of laying hens was challenged with fowlpox virus, VN titers of 1/10 to 1/40 were detectable. On the other hand, significant antibody reactions were detected by the ELISA on sera from chickens during the growing period, following vaccination and challenge. Although no maternal antibodies were found at 1 week of age, a continuous increase in the mean ELISA titers to fowlpox was demonstrated during the entire experimental period. This study showed that the ELISA was considerably more sensitive and practical than the VN test.     

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.     

Antigenic properties of four serotypes of Pasteurella multocida determined by enzyme-linked immunosorbent assay

Broiler minibreeder hens were used to produce monovalent antisera to bacterins prepared from serotypes 1, 3, 4, and 3 X 4 cross (CU strain) of P. multocida and to a polyvalent fowl cholera bacterin containing serotypes 1, 3, and 4. Antiserum to the CU strain (live vaccine) was also produced. Monovalent enzyme-linked immunosorbent assay (ELISA) plate antigens were prepared by separately sonicating each of the strains. Polyvalent plate antigen (Poly 3) was prepared by combining, in equal amounts after sonication, antigens from serotypes 1, 3, and 4. Each antiserum was assayed against its homologous ELISA plate antigen and against all other heterologous plate antigens, including Poly 3. The strongest reactions, as indicated by the highest absorbance values, were observed in homologous ELISAs. The CU strain may be the best monovalent ELISA plate antigen for detecting antibodies formed in response to a commercial polyvalent bacterin and to vaccinations with the live CU strain. Overall, monovalent serotype 1 (strain X-73) antiserum did not react well with any other heterologous ELISA plate antigen, whereas monovalent antisera of serotypes 4 (strain P-1662) and 3 X 4 (CU strain) reacted equally strongly with monovalent serotype 4 ELISA plate antigen. Background binding of negative serum was significantly lower (P less than 0.05) when using CU plate antigen than when using any of the other plate antigens.     

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.     

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.     

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.     

Labeled avidin-biotin enzyme-linked immunosorbent assay for detecting antibody to infectious laryngotracheitis virus in chickens

A labeled avidin-biotin enzyme-linked immunosorbent assay (LAB-ELISA) for detecting antibody to infectious laryngotracheitis (ILT) virus in chicken sera was developed and compared with ordinary ELISA. Purified ILT virus, biotin-labeled anti-chicken IgG rabbit IgG conjugate, and horseradish-peroxidase-labeled avidin were used in the LAB-ELISA. When sera from farm chickens were tested by serum neutralization (SN) and two kinds of ELISA, the correlation rate between SN and LAB-ELISA was 50/50 (100%), and that between SN and ordinary ELISA was 39/50 (78%). In LAB-ELISA, all of the sera that were antibody-negative by SN had low absorbance (A) values (below 0.05), and the A values were closely correlated with the SN indexes. In ordinary ELISA, however, the sera antibody-negative by SN had various A values ranging from 0.06 to 0.32. LAB-ELISA had much lower nonspecific reactions than ordinary ELISA against sera from ILT-negative chickens, even when chickens were 30 weeks old. ILT antibody production after ILT vaccination could be detected by LAB-ELISA. A values peaked 5 weeks postinoculation and were maintained for 17 weeks.     

Seroprevalence of antibodies to Salmonella spp in semidomesticated reindeer in Norway,determined by enzyme-linked immunosorbent assay

An indirect ELISA was developed as a possible tool to detect the seroprevalence of antibodies to Salmonella spp in semidomesticated reindeer. To cover a broad spectrum of serogroups a lipopolysaccharide mix of S. typhimurium and S. choleraesuis was used as antigen in this pilot study. Sera from 31 culture-negative reindeer with no clinical or historical evidence of salmonellosis were used as negative serum control. After immunisation with an inactivated S. typhimurium vaccine, pooled sera from 6 reindeer were used as positive serum control as no serum from naturally infected animals was available. A seroprevalence of 0.6% in 2000 clinically healthy, slaughter-reindeer from Norway was determined by using this ELISA. No more information on Salmonella in reindeer in Norway is known to the authors. This is the first ELISA established for indirect detection of Salmonella in reindeer.     

Comparison of filter-paper-eluted whole blood with serum in fowl cholera serology using the enzyme-linked immunosorbent assay

Two methods for collecting blood for measuring antibody activity of Pasteurella multocida were compared. Whole blood was collected on filter-paper strips, dried for 48 hr at room temperature, and then stored in sealed plastic bags at 4 C. Blood was also collected in the usual manner with a needle and syringe, and serum was harvested and stored at -20 C until tested. Eluates of whole blood, obtained by overnight elution of two 4.8-mm discs in 200 microliters of buffered saline at 4 C, were compared with conventionally harvested serum for antibody activity by enzyme-linked immunosorbent assay (ELISA). Paired samples, taken from the same bird at the same time, showed no significant difference (P less than 0.05) in antibody activity as measured by absorbance when the disc-elution process itself was considered to be a 1:20 dilution. It was concluded that eluates of blood, derived from whole blood dried on filter-paper strips, may be used as an alternative to sera in ELISA for measuring P. multocida antibody activity.     

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.     

Serological response of chickens to infection with Salmonella gallinarum-S. pullorum detected by enzyme-linked immunosorbent assay.

The serological response to Salmonella pullorum and S. gallinarum infection in chickens was studied with an indirect enzyme-linked immunosorbent assay (ELISA). In broiler chickens, a more virulent strain of S. pullorum produced a significantly lower serum IgG titer than did a less virulent strain. In laying hens, the serum and egg-yolk IgG titers were very similar. In chickens infected with S. gallinarum, high IgG titers persisted for 30 weeks. In chickens reinfected with this strain, each reinfection was followed by transitory increases in IgG lasting no longer than 2 weeks. Serum samples from Brazil taken from a laying flock with evidence of fowl typhoid showed much higher antibody levels than did those from three uninfected flocks. Using lipopolysaccharide as the detecting antigen, infections caused by these salmonellae could be differentiated from those caused by other groups. Incorporation of the appropriate flagella antigen in the ELISA allowed differentiation between infections caused by S. pullorum and S. enteritidis.     

Diagnosis of bovine cryptosporidiosis by an enzyme-linked immunosorbent assay

This paper describes an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of cryptosporidiosis. A monoclonal antibody with a high affinity against an oocyst antigen was used to set up the test. The efficiency of this assay was compared with that of the flotation test; 275 calf faecal samples were examined by the two methods. There was 96% agreement between the two tests. For the 11 conflicting samples, the two tests were repeated and a modified Ziehl-Neelsen staining was performed on faecal smears. All these 11 samples contained few oocysts, but only five and six of them were shown to be positive by the ELISA and flotation tests, respectively. The degree of sensitivity of the ELISA and flotation tests is comparable; samples heavily or moderately contaminated with oocytes are detected by both methods. This ELISA is reliable and never gives rise to false positive results. Nevertheless, as with the flotation test, the occasional case containing very few oocysts will not always be detected by this test. If necessary, very accurate diagnosis can be made by a staining technique or by a direct immunofluorescent assay. In veterinary medicine, the ELISA seems to be a method of choice; it appears to be a fast and reliable technique which could be used as a routine test for the detection of Cryptosporidium oocysts. Nevertheless the degree of sensitivity must always be borne in mind. There is no need for a microscopic examination, which is an additional advantage.     

An enzyme-linked immunosorbent assay for the detection of antibodies to Mycoplasma gallisepticum in experimentally infected chickens

An indirect enzyme-linked immunosorbent assay (ELISA) was developed and tested for its ability to detect humoral response to Mycoplasma gallisepticum in chickens. Two antigens were used in the solid phase of the assay. Antigen 1 was a membrane-derived sodium dodecyl sulfate (SDS)-solubilized preparation; Antigen 2 was prepared in the same manner as Antigen 1 but was passed through an immunoadsorbent column containing rabbit anti-medium antibodies. Test conditions were optimized for incubation times and temperatures. Antigen, serum, and enzyme conjugate concentrations were standardized, and reproducibility was determined. A baseline value, representing a positive or negative result, was established independently for both antigens. The assay was then used to detect anti-M. gallisepticum antibodies in experimentally infected chickens. Serum samples collected at 0, 2, 5, 7, 10, 14, 21, 28, and 35 days postinfection (PI) were analyzed by serum plate agglutination (SPA), hemagglutination-inhibition (HI), and ELISA with both Antigens 1 and 2. ELISA was found to be less sensitive but more specific than SPA and more sensitive than HI. The ELISA was more sensitive with Antigen 1 than with Antigen 2. The former assay correctly identified 79% of the serum samples positive for M. gallisepticum by 7 days PI and 100% of the positive birds by 35 days PI. When the absorbance values for each group of birds were averaged, the ELISA successfully identified the M. gallisepticum-infected birds as uniformly positive 7 through 35 days PI and correctly identified all other groups negative for M. gallisepticum through 35 days PI.     

An enzyme-linked immunosorbent assay for detection of antibodies to avian adenovirus and avian adenovirus-associated virus in chickens

An enzyme-linked immunosorbent assay system (ELISA) was adapted for the detection of antibodies to avian adenovirus (AV) and avian adenovirus-associated virus (A-AV). Both before and after exposure, sera from chickens undergoing natural and experimental infections were assayed by ELISA, virus neutralization (VN), and immunodiffusion (ID) for antibody to both CELO virus and A-AV. The ELISA system was found to be comparable to VN for determining antibody concentrations to CELO virus and A-AV. In many cases, ELISA was found to be more sensitive than ID.     

Detection of Corynebacterium equi-specific antibody in horses by enzyme-linked immunosorbent assay

An enzyme-linked immunosorbant assay was developed to measure naturally occurring Corynebacterium equi specific antibody in horse serum. Antibody against C equi was demonstrated in normal adults and was passively transferred to foals. Adult levels of specific antibody were reached by 5 to 6 months of age in healthy foals. Decreased early antibody levels were demonstrated in a limited number of foals with confirmed C equi infection.     

A study of breeder vaccination programs and problems in the broiler progeny in Saskatchewan utilizing enzyme-linked immunosorbent assay

A survey of antibodies against infectious bursal disease virus (IBDV), infectious bronchitis virus (IBV), Newcastle disease virus (NDV), and reovirus (RV) was conducted in broiler-breeder flocks and selected progeny broiler flocks utilizing the enzyme-linked immunosorbent assay. Marked differences in antibody titers between different breeder flocks were related to differences in vaccination programs. Poor performance in some progeny broiler flocks was related to low antibody titers against IBDV in the source breeder flocks. Progeny broiler flocks in which there was a high incidence of condemnations for airsacculitis had elevated antibody titers against IBV. A few progeny broiler flocks that experienced high mortality due to gangrenous dermatitis had no antibody titers against IBDV at processing. Antibody titers against RV were very variable and could not be related to any production problems.