Development and application of an ELISA for detecting antibodies to Salmonella enteritidis in chicken flocks.

Enzyme-linked immunosorbent assays (ELISAs) were developed for the detection of IgG antibody to Salmonella enteritidis in poultry flocks. A lipopolysaccharide (LPS) and heat-extracted (HE) antigen for use in the ELISA were evaluated together with the rapid slide test (RST), microagglutination test (MT) and the microantiglobulin (MAG) test. In experimentally infected specific pathogen free chickens, good correlation was seen between all tests although, generally, the MT and MAG detected antibody earlier and titres peaked earlier than the ELISAs. The LPS antigen detected antibody earlier than the HE antigen but the latter gave higher titres in the later stages of infection. Cross reactions were seen between S enteritidis and S typhimurium in the ELISAs although homologous reactions were always much higher. Antisera to S montevideo or S senftenberg gave weak positive reactions in both S enteritidis ELISAs. Serological and bacteriological examinations of representative samples from two commercial chicken flocks were carried out. In flock A the HE-ELISA and MAG test detected antibody in nearly all birds. The LPS-ELISA detected antibody in over 60 per cent of birds, while the MT and RST detected few seropositive birds. The whole blood test using the stained S pullorum antigen on the farm detected antibody in just under 25 per cent of the birds. S enteritidis was isolated from the organs of 25 per cent of the birds. All birds in flock B were seronegative by all tests; no salmonellae were isolated from the organs of these birds.     

Detection of antibody to Salmonella enteritidis and S typhimurium in the yolk of hens' eggs

Enzyme-linked immunosorbent assays (ELISAs) have been developed to detect IgG antibodies to Salmonella enteritidis and S typhimurium in the yolk of hens' eggs. Better discrimination and more consistent results were obtained between eggs from experimentally infected and uninfected hens by using saline-dilution of yolk rather than chloroform extraction. Threshold absorbance values were determined in three salmonella-free flocks, and on the basis of these results ELISA optical density values greater than 0.25 were considered to be positive for antibodies to salmonella. Four flocks with a history of salmonella infection were examined; three contained birds which were seropositive for S enteritidis by ELISA and from which S enteritidis was isolated, and a large proportion of eggs from these birds contained antibody to S enteritidis. Eggs from the fourth flock had no detectable antibody, although serum antibody was detected in some birds. No salmonellae were isolated from the yolks of the eggs from any of the four flocks.     

A disc ELISA for the detection of salmonella group D antibodies in poultry

An ELISA using lipopolysaccharide antigens prepared from Salmonella gallinarum and S enteritidis was developed for the serological diagnosis of fowl typhoid and S enteritidis infection in poultry. There was good agreement between the results of the ELISA and conventional serological tests when samples from naturally infected birds and S enteritidis immunised birds were tested. Some cross reactions were observed when serum samples from S typhimurium infected birds were tested by ELISA. Subsequently a disc ELISA, using filter paper discs, was developed to facilitate sampling and testing of poultry. There was good correlation between the results of the disc and serum ELISAs and the test is recommended for the field testing of birds.     

Salmonellosis in breeds of pigs with latent infections and in salmonellosis foci

The occurrence of salmonellae in pigs in Slovakia is described for the period from 1971 to 1980. On the whole, 1430 strains (11 serological types) of salmonellae were isolated in stocks with latent infections. The proportions of the serological types were as follows: S. agona 0.69%, S. anatum 0.14%, S. arizona 0.07%, S. bareilly 0.14%, S. decatur 0.07%, S. enteritidis 1.12%, S. give 0.28%, S. heidelberg 0.07%, S. choleraesuis 93.71%, S. panama 0.07% and S. typhimurium 2.45%. In 1315 salmonellosis foci 1333 strains (six serological types) of salmonellae were isolated. The proportions of the serological types were as follows: S. agona 0.37%, S. anatum 0.07%, S. bareilly 0.22%, S. enteritidis 1.20%, S. choleraesuis 90.59% and S. typhimurium 5.30%. The annual pattern of the occurrence of the most frequent serological types is described.     

Incidence of salmonellae in domestic fowl in Slovakia from 1971 to 1980

In flocks with latent infections, 2724 strains of salmonellae (19 serotypes) were isolated from the birds. The following species were represented as follows: S. gallinarum-pullorum (65.08%), S. typhimurium (9.10%), S. bareilly (6.90%), S. enteritidis (2.38%) and S. agona (1.98%). In the foci of salmonelloses 893 strains of salmonellae (19 serotypes) were isolated. The highest representation was found in the S. gallinarum-pullorum (54.20%), S. typhimurium (24.53%), S. bareilly (7.72%), S. choleraesuis (2.29%), S. enteritidis (1.68%), S. infantis (1.54%) and S. anatum (1.23%). Post-mortem examination resulted in recording 15 887 strains of salmonellae (41 serotypes). The following were represented by the largest proportions: S. gallinarum-pullorum (26.62%), S. typhimurium (25.20%), S. bareilly (18.93%), S. infantis (7.20%), S. enteritidis (4.62%), S. agona (3.51%), S. choleraesuis (3.17%), S. anatum (2.19%), S. lille (1.54%) and S. bredeney (1.16%).     

Trials with an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of subclinical genital infections in rams caused by Histophilus ovis and Actinobacillus seminis

The performance of an enzyme-linked immunosorbent assay (ELISA) was evaluated in the serological diagnosis of subclinical genital infection in 6 naturally infected ram flocks and 2 experimentally infected ram hoggets. The test employs lipopolysaccharide (LPS) antigen prepared by autoclaving from Actinobacillus seminis and Histophilus ovis. A total of 193 sheep (118 unmated virgin rams and 75 mature breeding rams) were examined clinically, serologically (by ELISA) and bacteriologically (semen bacteriology) at the same time. Serum samples from all animals were also tested by an ELISA employing LPS antigen prepared from Brucella ovis in the same way. Shedding of A. seminis and H. ovis did not show close correlation with serological positivity (Table 1), as only 9 (15.0%) out of the 60 A. seminis shedders were ELISA seropositive at the same time. As regards H. ovis only 10 (19.2%) out of the 52 H. ovis shedders were ELISA seropositive at the same time. The results indicate that, when used alone, the ELISA employing LPS antigen is unsuitable for diagnosing subclinical genital infection caused by H. ovis and A. seminis in rams. The authors discussed shortly the employing fields of this ELISA test in the diagnostic work.     

Serologic detection of experimental Salmonella enteritidis infections in laying hens by fluorescence polarization and enzyme immunoassay

Detection of infected poultry flocks is essential for controlling eggborne transmission of Salmonella enteritidis to humans. The present study evaluated the detection of antibodies in the sera of experimentally infected chickens by a fluorescence polarization assay with a tracer prepared from the O-polysaccharide of S. enteritidis and an enzyme-linked immunosorbent assay (ELISA) with an S. enteritidis flagellin antigen. In two trials, groups of specific-pathogen-free laying hens were infected orally with either 10(6) or 10(8) colony-forming units (CFU) of S. enteritidis (phage type 13a) or with 10(8) CFU of Salmonella typhimurium. Serum samples were collected before inoculation and at five subsequent weekly intervals. Both assays successfully detected the majority of hens infected with S. enteritidis at either dose level, but they also identified a substantial number of hens infected with S. typhimurium as seropositive. The fluorescence polarization test detected S. enteritidis infection significantly more often and cross-reacted with sera from hens infected with S. typhimurium significantly less often than the ELISA. The fluorescence polarization assay also offered advantages in terms of speed and methodologic simplicity.     

Serological and bacteriological investigations of chickens from flocks naturally infected with Salmonella enteritidis

Groups of 10 birds were obtained from four flocks which had shown evidence of natural salmonella infection. S enteritidis had been isolated from three flocks and S typhimurium from the fourth. Each bird was housed in a separate cage and blood samples and cloacal swabs were taken weekly to follow the course of natural infection. After four weeks the birds were killed and examined post mortem. The isolation of Salmonella species could not be related to the serological results. In individual birds the rapid slide test and tube agglutination test could not be relied upon to detect infection; the microantiglobulin test and the enzyme-linked immunosorbent assay (ELISA) were more sensitive than the other tests and detected some infected birds that were negative by the rapid slide and tube agglutination tests, and also showed high titres in some birds from which Salmonella species could not be isolated post mortem. Sera obtained from two flocks which had a history of natural S enteritidis infection were evaluated by all the tests; evidence of infection was found with the microantiglobulin and ELISA tests but not with the other tests.     

Simplified preparation of a specific S. enteritidis antigen for ELISA and other immunological techniques

This study was conducted to prepare a specific S. enteritidis antigen (FG-Antigen) for the serological detection of S. enteritidis infections in chicken flocks. This antigen (FG-Antigen) consistent mainly of the flagellar fraction H:g and partly of the fimbrial fraction SEF14 from a S. enteritidis-phage type 4 strain. The initial steps followed in the preparation of this antigen were conducted based on a previously described procedure, which involved the application of heat at 60 degrees C. The purification process (filtration and concentration) enabled the exclusion of the cross-reaction causing LPS antigens from the preparation and allowed the retention of S. enteritidis-specific antigens composed of fimbria and H:g fractions. As a result, no cross-reaction with S. typhimurium nor with S. gallinarum was exhibited by the prepared FG-antigen. To characterize and determine its specificity, the following laboratory tests were conducted: indirect ELISA, immunoblotting and a SEF14 agglutination test. In these examinations, rabbit and chicken reference sera as well as chicken field sera and absorbed hyperimmune sera against H:g-carrying serovars were used.     

Salmonella typhimurium infection in domesticated fowl in a children's zoo.

Salmonella typhimurium infection occurred in a children's zoo where 11 fowl and 85 mammals were kept. Initially, the guinea pigs were infected and transmitted the infection to the fowl and rabbits. These mammals responded to medication and cleared of the infection; however, the birds were judged to contain excreters despite four regimens of treatment with antibiotics. Cloacal swabs were taken from all the birds. One turkey was positive for Salmonella and was destroyed. Pooled fecal samples from the birds were again positive. All the birds were tested serologically, and two birds, a goose and a turkey, were positive with Salmonella pullorum-gallinarum antigen, which was assumed to be a cross reaction with S. typhimurium antigen. The two birds were destroyed and the goose yielded Salmonella. The infection was finally eradicated, and the serologic examination was considered to be the most useful procedure for detection of the excreters.     

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 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.     

Sources of salmonellae in an uninfected commercially-processed broiler flock.

Cultural monitoring was used to study the incidence and sources of salmonellae in a 4160 bird broiler flock during the growing period, transport and processing in a commercial plant. No salmonellae were isolated from any of 132 litter samples of 189 chickens cultured during the seven-week growing period, even though nest litter samples from four of the eight parent flocks yielded salmonellae and Salmonella worthington was isolated from the meat meal component of the grower ration. On arrival at the plant, 2/23 birds sampled carried S. infantis on their feathers, although intestinal cultures failed to yield salmonellae. Three of 18 processed carcasses samples yielded salmonellae (S. infantis, S. heidelberg, S. typhimurium var copenhagen). The most likely source of these salmonellae was the plastic transport crates, since 15/107 sampled before the birds were loaded yielded salmonellae (S. infantis, S. typhimurium). The crate washer at the plant did not reduce the incidence of Salmonella-contaminated crates, since 16/116 sampled after washing yielded salmonellae (S. infantis, S. typhimurium, S. heidelberg, S. schwarzengrund, S. albany).     

Serological detection of experimental Salmonella enteritidis infections in laying hens

The antibody response of laying hens to experimental Salmonella enteritidis infection was evaluated in microagglutination, tube agglutination, and rapid whole-blood plate agglutination assays. Hens of three different ages were infected by either oral inoculation or horizontal contact transmission. Blood was collected at weekly intervals, and the presence of specific antibodies was assessed by reaction with antigens prepared from strains of S. enteritidis and S. pullorum. The sensitivity of detection of infected hens did not vary significantly between the assays, as all three tests effectively identified most exposed hens as seropositive. Within each test, however, variation was observed in the detection sensitivity when different antigens were used. The microagglutination titers of serum samples were determined by serial dilution. Antibody titers peaked at 1 to 2 weeks postinoculation and declined steadily, although most birds were still identified as seropositive at 10 weeks postinoculation. The mean microtest titers obtained with S. enteritidis antigens were higher than with an S. pullorum antigen, indicating greater test sensitivity. However, use of the S. pullorum antigen resulted in fewer false positives when sera from uninfected control hens were tested. The titers of contact-exposed hens peaked later and at lower values than did those of inoculated hens, but these two groups of hens had similar antibody titers after the third week postinoculation.     

Effects of prior coinfection with different Salmonella serovars on the progression of a Salmonella enterica serovar enteritidis infection in hens undergoing induced molt

Four trials were conducted to evaluate whether prior infection with Salmonella enterica serovar typhimurium (S. typhimurium) or Salmonella enterica serovar muenchen (S. muenchen) would modify the severity or the transmission of Salmonella enterica serovar enteritidis (S. enteritidis) challenge in hens undergoing molt via feed withdrawal. Hens were separated into two groups where one group received a prior S. typhimurium or S. muenchen infection, whereas the other group remained untreated until S. enteritidis challenge. In trials 1 and 2, one group of hens was infected with S. typhimurium 5 days prior to feed withdrawal. Both groups of hens were then challenged with S. enteritidis on day 4 post feed withdrawal. In trials 3 and 4, one group of hens received S. typhimurium or S. muenchen, respectively, 1 day after feed was withdrawn. Transmission of S. enteritidis was evaluated by challenging the center hen in rows of 11 hens per row with S. enteritidis at 4 days post feed withdrawal and following the progression of the S. enteritidis down the row of hens over time. In trials 1 and 2, where hens received S. typhimurium 5 days prior to feed withdrawal, shedding of the S. enteritidis challenge was significantly reduced in hens on day 10 postchallenge in trial 1 and on days 3 and 10 postchallenge in trial 2 compared with the hens subjected only to the molt procedure. Significantly fewer S. enteritidis were recovered in livers and spleens at day 9 postchallenge in trial 2 from hens receiving the prior S. typhimurium infection. In trial 3, where hens received S. typhimurium 1 day after feed withdrawal, S. enteritidis transmission was significantly reduced in these hens on days 3, 10, and 24 postchallenge. In trial 4, similar in methodology to trial 3 except that, rather than S. typhimurium, hens received S. muenchen, a Salmonella organism totally lacking any antigen cross-reactive with S. enteritidis, S. enteritidis transmission was significantly reduced on days 3, 10, 17, and 24 postchallenge, suggesting that factors other than specific immunity were involved in the observed resistance to S. enteritidis infection. These results indicate that prior infection of a flock with a non-S. enteritidis paratyphoid Salmonella can reduce S. enteritidis problems that may occur during a molt.     

Flock infection and transport as sources of salmonellae in broiler chickens and carcasses.

Cultural monitoring was used to determine the incidence and sources of salmonellae in a 4160-bird broiler flock raised on litter in 32 pens. Twenty-five of the pens remained apparently free of salmonellae during the 49-day growing period. Salmonella johannesburg, first detected in the meat meal component of the starter ration, was recovered from the litter of seven pens and from the intestines of dead or culled chicks from two pens. Salmonella alachua was also recovered from two of these pens. Culture of swabs collected from the plastic crates used to transport this flock for processing showed that 97/112 (86.6%) were contaminated with salmonellae (15 serovars) before the birds were loaded. The crate washer at the plant did not remove salmonellae from these crates: 97/132 (73.5%) crates sampled after washing yielded salmonellae. Eleven serovar were recovered, including S. johannesburg and S. alachua introduced by the infected flock. Twelve of 31 chickens (38.7%) collected when the birds were unloaded at the processing plant were intestinal carriers of S. johannesburg and/or S. alachua and 29 (93.5%) were external carriers. Salmonella johannesburg, S. alachua and four other serovars were isolated from the feathers of these birds. Eleven of 25 (44%) carcasses tested from this flock yielded salmonellae. Salmonella johannesburg or S. alachua, first isolated from the infected flock, were recovered from five carcasses and S. haardt and S. Typhimurium, first isolated from the transport crates were recovered from six carcasses.     

Enzyme-linked immunosorbent assay with a Salmonella enteritidis antigen for differentiating infected from vaccinated poultry

The specificity and sensitivity of indirect ELISA, based on the use of four different antigenic extracts obtained from a clinical isolate of Salmonella enteritidis, were compared with those obtained with the gm-flagellin based ELISA (IDEXX). A total of 116 serum samples from salmonellae free, naturally infected and vaccinated hens were studied. The results showed that the indirect ELISA, based on lipopolysaccharide (LPS), O-polysaccharide (PS) or membrane sediment (SD) antigens, enable the identification of a greater number of infected birds and discriminated field antibody responses from vaccinal ones better than the commercial IDEXX test. The indirect ELISA that used a O-polysaccharide rich fraction (PS) proved to be the most specific and sensitive test, suggesting that this indirect ELISA could be used to confirm IDEXX results, especially when the differentiation between vaccinated and infected poultry is required.     

Antibody response to experimental Salmonella typhimurium infection in chickens measured by ELISA

An indirect ELISA has been developed to detect Salmonella typhimurium antibodies in chicken sera, using whole bacterial cell protein, flagellar protein or lipopolysaccharide as antigens. In experimental infections high concentrations of S typhimurium-specific IgG persisted after the faecal excretion of S typhimurium had ceased, whereas the specific IgM response was transitory. Some uninfected chickens placed in contact with experimentally infected birds developed high IgG titres in the absence of detectable faecal excretion. Other S typhimurium strains, which varied in their invasive abilities, also induced high titres of IgG. The ELISA allowed chickens infected experimentally with S typhimurium to be differentiated from chickens infected with 10 other serotypes, including S enteritidis. The use of whole blood in place of serum in the ELISA reduced the titres slightly. The storage of serum dried on to filter paper strips for four weeks produced little change in ELISA antibody titre, and the treatment of such strips with phenol or chloroform vapour had little or no effect on the antibody titre.     

Characterisation of monoclonal antibodies against a fimbrial structure of Salmonella enteritidis and certain other serogroup D salmonellae and their application as serotyping reagents

A panel of 13 monoclonal antibodies from different hybridomas was produced against a novel salmonella fimbrial antigen expressed predominantly by Salmonella enteritidis strains. The specificity of the monoclonal antibodies to this antigen (SEF14) was confirmed by enzyme-linked immunosorbent assay (ELISA) using purified SEF14, immune electron microscopy and, with 11 monoclonal antibodies, the identification of a repeating protein subunit (14,300kDa) on the antigen. Blocking-ELISA with the monoclonal antibodies identified epitopes in at least three, non-overlapping clusters which appeared evenly distributed on SEF14 in immune electron microscopy. The use of the monoclonal antibodies in direct-binding ELISA on a range of salmonella serotypes suggested that the epitopes on SEF14 are highly conserved and were expressed by all the S enteritidis strains examined; some strains of S dublin and the only strain of S moscow available were the only other serotypes that expressed SEF14. A latex agglutination reagent based on a monoclonal antibody was developed and used to test for SEF14 on 280 strains (representing 120 serotypes in 24 serogroups of salmonellae) that had been grown on Sensitest agar for 18 hours at 37 degrees C. All S enteritidis strains (64) and most S dublin strains (28 of 33) produced SEF14 as did the two strains representing S blegdam and S moscow. SEF14 was not detected in any other strains of serotypes from serogroup D or from any other serogroup examined.     

Dynamics of Salmonella contamination in a commercial quail operation.

Control of carcass contamination requires knowledge of the source and dynamics of spread of Salmonella in commercial poultry production. We examined Salmonella contamination at a U.S. commercial quail operation. Pulsed-field gel electrophoresis (PFGE) was used to type isolates in order to trace Salmonella throughout this production environment. During a 6-mo survey, Salmonella serotypes hadar, typhimurium, typhimurium variant Copenhagen, and paratyphi were encountered within this poultry operation. Ninety-four percent of the Salmonella isolated from breeder and production houses and from carcass rinses belonged to Salmonella serotypes typhimurium variant Copenhagen and hadar. There were six distinct S. typhimurium variant Copenhagen genetic types, as identified by PFGE, present within this particular poultry operation. Seventy-nine percent of S. typhimurium variant Copenhagen identified from the environment of the breeder and production houses produced the same PFGE pattern. Thirty-eight percent of S. typhimurium Copenhagen isolated from carcass rinses and the breeder house shared the same PFGE DNA pattern. This study demonstrates the transmission of salmonellae throughout this production environment, from the breeders to their progeny and to the birds ultimately processed for human consumption.