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.     

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.     

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 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.     

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.     

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.     

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 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.     

The response of broiler breeder chickens to parenteral administration of avirulent Pasteurella multocida

Broiler breeder chickens were exposed to avirulent Pasteurella multocida at 14, 22, and 34 weeks of age either by stick wing 1 to 3 times or subcutaneously 3 times. Fowl pox vaccine was mixed with the first P. multocida exposure in some groups. Exposure did not impair egg production or hatch of fertile eggs. Challenge with pathogenic P. multocida serotype 1 at 68 weeks indicated that exposure to avirulent P. multocida 2 or 3 times provided better protection than 1 exposure. Mixing fowl pox vaccine with the avirulent P. multocida did not reduce immunity to fowl cholera or fowl pox.     

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.     

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.     

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.     

Comparison of dot immunobinding assay, enzyme-linked immunosorbent assay and immunodiffusion for serodiagnosis of paratuberculosis.

A rapid, simple and inexpensive dot immunobinding assay (DIA) was evaluated for the serodiagnosis of paratuberculosis in cattle. The assay was performed on nitrocellulose strips which were dotted with purified protoplasmic antigen of Mycobacterium paratuberculosis. After incubation with test serum samples, the bound antibodies were detected using an enzyme-amplified immunostaining procedure. The efficacy of DIA as a screening test for paratuberculosis was compared to that of an enzyme-linked immunosorbent assay (ELISA), a modified agar gel immunodiffusion (mAGID) test, and an AGID test using 329 serum samples from cattle which were examined for M. paratuberculosis infection by a sensitive fecal culture technique. The DIA and ELISA had comparable results and both of the enzyme immunoassays had higher sensitivity than tests based on AGID. The sensitivity of all four tests was influenced by the intensity of fecal bacterial shedding. Preabsorption of sera with Mycobacterium phlei increased the sensitivity of both enzyme immunoassays. the specificity but reduced the sensitivity of both enzyme immunoassays.     

Comparison of enzyme-linked immunosorbent assay and RT-PCR for the detection of porcine epidemic diarrhoea virus

Porcine epidemic diarrhoea (PED) is a contagious enteric disease of pigs caused by a coronavirus. A double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) based on the use of monoclonal antibodies was developed for the detection of porcine epidemic diarrhoea virus (PEDV). The DAS-ELISA was compared with RT-PCR in the examination of 506 specimens collected during 2006-2007 from pigs originating from different farms located in the Po valley. Both faecal samples obtained directly from the rectum of live animals showing clinical signs and intestinal samples collected from the caecum of deceased pigs were included in the study. The correlation between the two methods was higher when testing faecal samples (K = 0.97, 95% CI: 0.94-1.00) than testing intestinal samples (K = 0.62, 95% CI: 0.35-0.89). The use of ELISA technology provided an efficient and effective mean of evaluating the presence of coronavirus PED antigen in field samples and indicates that this procedure is a very useful tool in epidemiological studies.     

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.     

Comparison of hemagglutination and competitive enzyme-linked immunosorbent assay procedures for detecting canine parvovirus in feces

A competitive enzyme-linked immunosorbent assay (ELISA) for detection of canine parvovirus (CPV) antigen in fecal samples was developed. Fecal samples were tested by ELISA and a direct hemagglutination assay, and the results compared. The tests gave the same results in 83% and 88% of the fecal samples, depending on whether the samples were treated with chloroform. The discrepancies were due to the fact that each test detected virus in certain fecal samples that was not detected by the other. The use of a monoclonal anti-CPV conjugate resulted in a highly specific test. The performance of the ELISA as a competitive assay also increased the reproducibility and sensitivity over that which could be obtained from a classic sandwich-type procedure.     

Monoclonal-antibody-mediated enzyme-linked immunosorbent assay for detection of reticuloendotheliosis viruses

An enzyme-linked immunosorbent assay (ELISA) is described for the detection of reticuloendotheliosis viruses (REVs). The assay uses a mixture of monoclonal antibodies (MCAs) prepared against a 62-kilodalton REV envelope glycoprotein (gp62) to capture antigen, rabbit anti-REV serum as detection antibody, and peroxidase-conjugated anti-rabbit IgG as indicator antibody. The MCAs were reactive with REV strain T, chick syncytial virus, and duck infectious anemia virus but unreactive against Marek's disease and avian leukosis viruses. The ELISA was compared with complement fixation test and REV immunofluorescent assay of infected fibroblasts, plasmas, and egg albumen from infected chickens. The lower limit of gp62 detection was about 120 ng of REV protein. Limit dilution of infectious REV was detected after 7-8 days of cultivation of infected fibroblasts.     

Comparison of pepsin-digestion and enzyme-linked immunosorbent assay for the diagnosis of trichinosis in swine.

Comparison of parasitological and serological diagnosis of trichinosis in swine was carried out on 36 pigs given 15,400 infective larvae each by gavage. Circulating eosinophil levels were determined and sera were examined by enzyme-linked immunosorbent assay for anti-Trichinella antibodies. Two pigs were killed per day from days 15 to 29 postinfection. Muscle was examined by pepsin-digestion and comparable tissue was fed to a rat. Eosinophil counts increased at about day 6 and reached peak levels about day 25 postinfection and returned to approximate preinfection levels about two months postinfection in those pigs still in the study. Infective larvae were recovered from all pigs killed at greater than or equal to 18 days postinfection. Using the criterion of 5 x mean optical density readings of negative sera as positive, seroconversion occurred between days 19 and 26 postinfection. Use of a lower criterion of 3 x mean optical density readings of negative sera resulted in only three of 30 pigs killed greater than or equal to 18 days postinfection seroconverting less than or equal to 18 days postinfection, when infective larvae were first recovered in the musculature. In pigs, even in those heavily infected, there is a lag between the period that trichinae in musculature become infective and development of antibodies as detected by enzyme-linked immunosorbent assay which results in false negative reactions in many animals. This study demonstrated that the enzyme-linked immunosorbent assay using an excretory-secretory antigen should not be used to certify pork or pork products free of infective Trichinella larvae or safe for human consumption.     

A competitive enzyme-linked immunosorbent assay for determination of chloramphenicol

Chloramphenicol is a broad-spectrum antibiotic shown to have specific activity against a wide variety of organisms that are causative agents of several disease conditions in domestic animals. Chloramphenicol has been banned for use in food-producing animals for its serious adverse toxic effects in humans. Due to the harmful effects of chloramphenicol residues livestock products should be free of any traces of these residues. Several analytical methods are available for chloramphenicol analysis but sensitive methods are required in order to ensure that no traces of chloramphenicol residues are present in edible animal products. In order to prevent the illegal use of chloramphenicol, regulatory control of its residues in food of animal origin is essential. A competitive enzyme-linked immunosorbent assay for chloramphenicol has been locally developed and optimized for the detection of chloramphenicol in sheep serum. In the assay, chloramphenicol in the test samples and that in chloramphenicol-horseradish peroxidase conjugate compete for antibodies raised against the drug in camels and immobilized on a microtitre plate. Tetramethylbenzidine-hydrogen peroxide (TMB/H2O2) is used as chromogen-substrate system. The assay has a detection limit of 0.1 ng/mL of serum with a high specificity for chloramphenicol. Cross-reactivity with florfenicol, thiamphenicol, penicillin, tetracyclines and sulfamethazine was not observed. The assay was able to detect chloramphenicol concentrations in normal sheep serum for at least 1 week after intramuscular injection with the drug at a dose of 25 mg/kg body weight (b.w.). The assay can be used as a screening tool for chloramphenicol use in animals.     

The response of broiler breeder hens to dietary lysine and methionine.

1. Broiler breeder hens were used in an experiment lasting 10 weeks (29 to 38 weeks of age) to measure the responses to dietary lysine and methionine, the main objective being to determine whether the coefficients of response to these amino acids were the same for broiler breeders and for laying pullets. 2. The hens were offered 150 g/d of one of 20 dietary treatments, 10 being lysine-limiting and 10 being methionine-limiting. The diets were mixed by diluting one of two concentrate (summit) mixes with a protein-free dilution mixture. The lysine-limiting summit diet was designed to supply approximately 1300 mg lysine/bird d, while the other supplied 520 mg methionine/bird d, when fed at 150 g/bird d. 3. Birds on the 5 lowest concentrations of both lysine and methionine did not consume the allotted amount of food, the amount decreasing, in a curvilinear fashion, to approximately 105 g/bird d. 4. The minimum egg weight recorded was 0.8 of the maximum, whereas the rate of lay of birds fed on the diets with the lowest amino acid concentrations was 0.2 of the maximum. 5. Using the Reading Model, the coefficients of response were calculated to be (for lysine) 16.88 E and 11.2 W, and for methionine, 7.03 E and 1.52 W, where E = egg output, g/bird d, and W = body mass, kg/bird. An average, individual, broiler breeder of 3 kg, producing 45 g of egg output per day, would need 793 mg of lysine and 321 mg of methionine daily. This intake of methionine is similar to that estimated by means of coefficients used for laying pullets, but the lysine requirement would be underestimated by 0.18 if the coefficients for laying pullets were used. 6. The coefficients for maintenance for both lysine and methionine, determined in this experiment, are considerably lower than values published previously, whilst the coefficients for egg output are, in both cases, higher. The resultant flock response curves therefore differed significantly from those in which the coefficients of response for for laying pullets were used.(ABSTRACT TRUNCATED AT 400 WORDS)