Serological response of chickens naturally infected with Salmonella typhimurium detected by ELISA.

Four laying flocks of chickens in Britain, each with a history of Salmonella typhimurium infection, were investigated serologically and bacteriologically. Blood samples were taken from identified birds from a single house on each site and sent to the Central Veterinary Laboratory, Weybridge for serological examination using enzyme-linked immunosorbent assays (ELISA) and rapid slide agglutination test (RST) using stained S. pullorum. The identified birds were taken to the local Veterinary Investigation Centre for bacteriological examination. On site A no salmonellae were recovered from birds in the house chosen for serological examination. Of these birds approximately 20% had antibodies to S. typhimurium in ELISA which used either a lipopolysaccharide (LPS) or heat-extract (HE) antigen from S. typhimurium. S. typhimurium was recovered from birds in one other of the four houses on the same site; these birds were not tested serologically. On site B, S. typhimurium was isolated from 8% of the birds examined. Of the total tested serologically, a third to half were seropositive by S. typhimurium ELISA using the LPS and HE antigen respectively. A small proportion of birds was seropositive by S. enteritidis ELISA and RST. No salmonellae were isolated from the other two sites although about 10% of birds tested on site C were seropositive in S. typhimurium ELISA. Cross-reactions were seen between S. typhimurium antigens in the ELISA and experimentally prepared antiserum to S. enteritidis. The S. enteritidis ELISA was generally more specific although cross-reacting antibodies were detected in sera from birds on sites A and B.     

Enzyme-linked immunosorbent assay for the detection of Salmonella enteritidis infection in chickens

An ELISA was developed and tested for its ability to detect antibodies against Salmonella enteritidis in chickens. Various features of the ELISA were evaluated and optimized. The outer membrane protein antigens selected by use of the protein immunoblotting method made the assay specific and sensitive. The assay was evaluated in chickens experimentally infected with S enteritidis. Blood samples collected at weekly intervals after experimental infection with S enteritidis were analyzed by ELISA. Results of the ELISA were compared with those of conventional serum plate and microagglutination tests. The ELISA was more sensitive and specific in the detection of S enteritidis infection than the other 2 conventional tests.     

Serological diagnosis of cysticercosis by an enzyme-linked immunosorbent assay in experimentally infected cattle.

Taenia saginata infections were established in four groups of calves by administering doses of 10, 10(2), 10(3) and 10(4) infective eggs respectively by gavage. A fifth group remained as uninfected controls. Sera were collected from all calves over a period of 210 days. The sera were examined by an enzyme-linked immunosorbent assay (ELISA) with a fraction of larval Taenia hydatigena cyst fluid as antigen for the presence of anti-T. saginata IgG antibodies. At slaughter, the tongue, masseter, diaphragm and cardiac muscles and liver were examined for cysticerci. The higher dose rates of T. saginata eggs were reflected in higher numbers of cysticerci found in the calves at necropsy. There was also a correlation between higher levels of antibodies produced as measured by the ELISA and the numbers of eggs given. Sero-conversion was first detected about 25 days postinfection in heavy infections and later in the lighter infections. Maximal levels of antibody occurred between 40 and 60 days postinfection, followed by a gradual decrease in levels of antibody. A secondary increase in antibody occurred between 160 and 200 days postinfection which might have been due to release of antigen after death of the cysticerci. The low level of circulating antibodies in light infections may result in false positive or false negative diagnoses depending upon the selection of the cut-off point.     

Prevalence of antithyroglobulin antibodies detected by enzyme-linked immunosorbent assay of canine serum

Antithyroglobulin antibody (ATA) values were higher in dogs with low total serum thyroxine (T4) and triiodothyronine (T3) values than in dogs with T3 and T4 values within reference value limits. In the population studied, Doberman Pinschers were predisposed to the development of ATA; there was no sex predilection for development of ATA. Antithyroglobulin antibodies and thyroid status were evaluated in 2 groups of healthy dogs (n = 30) and in 470 canine serum samples submitted for T3 and T4 value determination. Antithyroglobulin antibodies were evaluated by ELISA, and thyroid status was evaluated by measurement of total serum T3 and T4 by radioimmunoassay.     

Serodiagnosis of Salmonella dublin infection in Danish dairy herds using O-antigen based enzyme-linked immunosorbent assay.

Usefulness of two enzyme-linked immunosorbent assays (ELISA) for screening of dairy herds for antibodies to lipopolysaccharide (LPS) of Salmonella dublin (O:1,9,12) was investigated. Sera (3097) were collected from 40 dairy herds located in three areas of Denmark with different prevalence of salmonellosis: ten salmonellosis-free herds from the island of Samsø where there is no history of salmonellosis, ten salmonellosis-free herds from the island of Sealand where outbreaks are infrequent, and 20 salmonella infected herds from Jutland where salmonellosis is enzootic. The samples were analyzed for antibodies to S. dublin LPS using an indirect (O:9,12) and a blocking (O:9) ELISA. Using herd history of salmonellosis, herd location and clinical state of the herds as reference, the herd sensitivity and herd specificity of the tests were 100% and 100% in the indirect ELISA and 95% and 100% in the blocking ELISA, respectively. A significant correlation was found between the two tests (rs = 0.46, p < 0.001). However, the indirect ELISA detected more seropositive animals than the blocking ELISA (17% vs. 7%, respectively). In calves from Sealand, level of background reaction was significantly lower (p < 0.001) compared to the heifers and the cows. The percentages of seropositive calves in both tests were higher (p < 0.01) in comparison to cows (19 vs. 8 in indirect ELISA, and 14 vs. 6 in blocking ELISA, respectively). Results of the study indicated that it is possible to apply LPS ELISA in serological screening for salmonellosis.     

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.     

The serologic response to Salmonella enteritidis and Salmonella typhimurium in experimentally infected chickens,followed by an indirect lipopolysaccharide enzyme-linked immunosorbent assay and bacteriologic examinations through a one-year period

Three groups of 100 individually marked salmonella-free chickens were followed for a period of 53 wk. The chickens were infected as day olds by crop instillation of 10(8) colony-forming units: one group with Salmonella enteritidis and a second group with Salmonella typhimurium. A third group was kept uninfected as controls. The groups were monitored bacteriologically by examination of cloacal swabs and organs and serologically by examination of serum and egg yolk by a lipopolysaccharide enzyme-linked immunosorbent assay throughout the period. Within the first week, 100% of birds in both infected groups were excreting salmonella bacteria in the feces. However, the number of fecal excretors declined rapidly with time, down to 6% in 16 wk for S. typhimurium and down to a similar level within the first 8 wk for S. enteritidis. For the latter, relapses with up to 40% positive birds were observed at the onset of egg production. For both S. typhimurium and S. enteritidis, positive bacteriologic cultures were obtained by sampling from internal organs at the end of the experiment, more than 1 yr from the time of infection. At the age of 6-7 wk, 50% of the chickens in the two infected groups showed a measurable serologic response in serum samples. The response persisted throughout the study in both serum and egg yolk samples. The inclusion of serologic methods is a valuable additional tool in the detection of salmonella in poultry, but serology should be used in conjunction with bacteriologic methods in surveillance programs, in particular to detect flocks in early stages of infection before a measurable serologic response has been raised.     

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.     

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.     

Serum antibody response to Mycoplasma hyopneumoniae measured by enzyme-linked immunosorbent assay after experimental and natural infection of pigs

Studies were conducted under experimental and field conditions to determine the effect of infection with M. hyopneumoniae on the immune response in serum as measured by ELISA. Following intratracheal challenge or contact exposure, serologically negative pigs derived from mycoplasma-free piggeries developed an immune response within 10 days. This response continued to rise for a further 50 days. In a field study in a commercial piggery, no animals (0/44) were observed to have M. hyopneumoniae antibodies at day 86 of life. However between day 86 and day 144, 97.7% (42/43) animals sero-converted. These results are discussed in terms of infection spread, particularly in the grower/finisher shed.     

Control panels of meat juice samples for a Salmonella enzyme-linked immunosorbent assay.

In the Danish pig production system, an indirect enzyme-linked immunosorbent assay (ELISA) for detection of antibodies in meat juice is used for Salmonella surveillance. Quality control (QC) of this ELISA was previously based on repeated testing of control serum samples. The purpose of the study reported here was to collect, characterize, and implement a panel of meat juice pools for supplemental internal QC. Muscle samples for extraction of meat juice were collected from slaughter pigs of 5 herds infected with Salmonella spp. and from 4 herds without Salmonella infection. A QC panel with 39 pools of meat juice, yielding ELISA optical density (OD) values covering the full range of expected OD values, was prepared and tested repeatedly to determine mean and SD OD values. Each pool was tested twice on each microtitration plate, and the results were used to determine limits for validity of future tests. This QC panel was included as an internal QC to be tested every month. Besides the QC panel, 2 panels containing 100 samples of meat juice with OD above the positive cut-off value and 100 samples with OD below that value were prepared for quarterly control of the diagnostic sensitivity (DSe) and the diagnostic specificity (DSp) of the ELISA. The inclusion of these panels in the QC system will provide information about drifts in DSe and DSp of the test. The procedures described here can be applied to other tests where meat juice samples are used for testing.     

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.     

Detection of antibodies to mouse thymic virus by enzyme-linked immunosorbent assay.

Enzyme-linked immunosorbent assay was used to detect serum antibodies to mouse thymic virus, a herpesvirus that causes thymic lesions and immunosuppression. Antibodies were detected in mice that had received single or multiple injections of the virus and were also found in mice housed in contact with the experimentally infected animals. By contrast, mice not exposed to mouse thymic virus or those inoculated with an uninfected thymus preparation remained seronegative. A serological survey of eight mouse colonies revealed one positive colony, confirmed by virus isolation. These results show that the test is sufficiently sensitive and specific to be used for routine screening of mice.     

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.     

Biotin-streptavidin enzyme-linked immunosorbent assay for the detection of antibodies to Campylobacter jejuni and C. coli in chickens.

An enzyme-linked immunosorbent assay (ELISA) was developed in a homologous system with bacterial ultrasonic-treated proteins as the antigen and antisera from chickens infected orally and subcutaneously with the strain Campylobacter jejuni serovar 6 (CJ 6). The cut-off level was determined using antisera from non-infected specific-pathogen-free chickens up to the age of 10 weeks. The suitability of the ELISA system was verified using antisera taken from chickens orally infected at the age of 4 weeks with CJ 1, 6, 28 or 36 or with Campylobacter coli serovar 28 (CC 28). The development of antibodies was monitored up to 6 weeks post-infection (p.i.). Sera from chickens infected with CJ 1, 6, 36 or CC 28 contained specific antibodies to Campylobacter, whereas in those infected with CJ 28 no specific antibodies were found. Distinct cross-reactions were observed between CJ 6, 28 and CC 28 antigens and their antisera 6 weeks p.i., while poor cross-reactions were found with antisera to CJ 1 and 28. Antibodies to strains of all heterologous serovars were successfully detected with an antigen pool comprised of CJ 1, 6 and 36 antigens. In 11 out of the 12 field sera obtained from 5- and 9-week-old broiler chickens suffering from campylobacteriosis, high specific antibody titres to Campylobacter jejuni were found.