Presented at the World Veterinary Poultry Association Congress, Budapest 1997

A COMMERCIAL SALMONELLA ENTERITIDIS ELISA TEST: RESULTS ARISING
FOR ITS USE IN MONITORING FOR INFECTION AND RESPONSE
TO AN INACTIVATED VACCINE


P.F. McMullin1, K.R.Gooderham, G.Hayes.

Poultry Health Services, Lakeside Veterinary Centre, Marsh Lane, Hemingford Grey, Huntingdon, Cambridgeshire, PE18 9EN, UK.

1 Present Address: Poultry Health Centre, Main Site Lane, Dalton, Thirsk, North Yorkshire, YO7 3JA UK.

Summary

Salmonella enteritidis (S.e.) infection in chickens has been a significant concern in recent years. A commercial Elisa (Guildhay) was used for routine monitoring of parent flocks of meat chickens. To demonstrate the effect of field challenge on S.e. serology we extracted the data for two contrasting companies. Company A had a history of repeated outbreaks of S.e. infections in broiler progeny. Company B had no isolates in routine hatchery monitoring and no evidence of the disease in the progeny. Sera were routinely collected at approximately 6 week intervals during production, alternating between 60 and 20 samples per air-space. All available data from unvaccinated flocks for a period of approximately 18 months were extracted. A total of 22120 assays representing 652 house/flock groups were included, with 10356 sera (317 groups) from company A, and 11764 (335 groups) from company B. Results were reported in serum-to-positive ratio bands in which band 0 was the negative range defined by the manufacturer, 1 the suspect range, and bands 1 to 12 progressively higher levels of positivity. Only sera reacting in band 6 (S:P 4.5 ) or higher were significantly more frequent in samples from the “problem” company. The great majority of samples in bands 6-12 occurred in a few flocks which underwent a distinct sero-conversion. Occasional strong reactions were obtained from the company with no bacteriological evidence of infection. Generally if only low-grade reactions were present their intensity was reduced when the flock was re-sampled after 2 weeks. Subsequent experience suggests that heat inactivation (56 C. for 30 minutes) causes most of the low-grade reactions in bacteriologically negative flocks to substantially weaken or disappear. To examine the response to vaccination, the data for 156 groups of sera from vaccinated birds were extracted. All birds were vaccinated with 2 doses of an inactivated product (Salenvac, Hoechst). A peak response was found at 22 weeks of age with a gradual decline in lay. This S.e. ELISA test was found to be a useful tool in monitoring programmes though the possibility of “false reactions” must be recognised.

Introduction

Serological testing for salmonellosis in poultry has a relatively long history. A tube agglutination test for Pullorum disease as described by Jones in 1913, a mere 14 years after the organism was first described (Rettger,1900). The subsequent introduction of a rapid plate serum agglutination test (Runnels et. al. 1927) and a stained antigen whole-blood test (Schaffer et.al. 1931) provided a practical basis on which to introduce eradication procedures.

The non-host-specific salmonellae, on the other hand, do not induce such a readily detectable antibody response. Micro-agglutination anti-globulin tests - (modified Coombes Test) were developed to improve test sensitivity (Williams and Whittemore, 1976). The sensitivity problem was solved by the development of the enzyme-linked-immuno- sorbent assay (Elisa) (Engvall & Perlman,1971) and its application to the measurement of antibody response to specific infections (Voller et alii, 1976). The development and application of experimental ELISA assays for Salmonella enteritidis has been described (Cooper et alii, 1989,Timoney et alii, 1990, Nicholas & Cullen, 1991). Two distinct types of test have been used. One uses a conventional indirect Elisa test with lipo-polysaccharide antigen absorbed on in the well, this captures antibody in field serum, and this, in turn is detected by the use of an anti-globulin/enzyme conjugate and substrate. In the alternative, competitive, method the field serum is used to prevent the attachment of an anti-gm flagellin antibody conjugate to the antigen bound to the plate. In this case positive reactions are seen as a reduction or lack of colour generation when the substrate is added. Salmonella enteritidis infection in both broiler parents and layer chickens has become a significant issue in recent years. In both classes of chicken there are public health implications, in addition infected broiler parents can produce progeny with clinical salmonellosis. Bacteriological screening of chicken parent flocks is required in the UK under the Poultry Breeding Flocks and Hatcheries Order (1993). In addition a substantial amount of serological screening has been carried out, mainly using commercial Elisa test kits.

Materials and Methods

All assay results summarised here are based on single tests of individual sera using a commercially available Elisa assay. The sera were obtained from conventional broiler parent chickens during routine screening procedures. Generally between 20 and 60 samples were examined per house or air-space. The test kit used (Guildhay “FLOCKSCREEN”TM S.e. Elisa kits) uses a conventional (non-competitive) assay and a lipo-polysaccharide antigen. Tests were carried out in accordance with the standard protocol. In-house control sera were used in addition to those provided by the kit manufacturer. Plates were repeated when the control sera did not come within the manufacturers recommended values. Raw data were read directly from an automatic plate reader (MR6000, Dynatech), and computed, stored and reported using a purpose-designed computer programme. Results are reported by indicating the number of sera in each group in each of 13 Serum-to-Positive Ratio Bands. Band 0 sera are negative, 1 suspect, and bands 2 through 12 are associated with increasingly intense reactions (Table 1). The bands for negative, suspect and positive interpretation were those recommended by the manufacturer when this work was carried out.

Table 1 - Salmonella enteritidis Elisa Conversion table for Titre Groups and S/P ratios
Group S/P range Group S/P range
Neg. up to - 0.15 7 .50 -.549
Susp. .151-.249 8 .55 -.599
2 .25 -.299 9 .60 -.649
3 .30 -.349 10 .65 -.699
4 .35 -.399 11 .70 -.749
5 .40 -.449 12 .75- and above
6 .45 -.499    


Flock Monitoring: To demonstrate the effect of field challenge on S.e. serology we have extracted the data for two contrasting companies. Company A had a history of repeated outbreaks of S.e. infections in broiler progeny while company B had no isolates in routine hatchery monitoring and no evidence of a problem in the progeny. The data represent a period of approximately 18 months. Data from S.e. vaccinated flocks were excluded. A total of 22120 assays representing 652 house/flock groups were included, with 10356 sera (317 groups) from company A, and 11764 (335 groups) from company B.

Response to Vaccination: A second survey involved the extraction of data relating to flocks
vaccinated with an aluminium-hydroxide adjuvanted S.e. vaccine (SalenvacTM Hoechst). Flocks
were vaccinated at 10-11 weeks and again at 15-16 weeks. There was no evidence of
infection or transmission of S.e. in these flocks. The extraction yielded 156 groups of sera
from vaccinated stock. Some of these represented relatively small groups (4-6) birds
representing specific pens in vaccine field trials. Many of the data-points up to 22 weeks
however represent testing 20 sera per house in routine monitoring of vaccinal response.

Results

Infection Monitoring:
Table 2 shows the total % of samples received from each company and summarises the occurrence of suspect and positive reactions. The great majority of sera were negative. When viewed in this manner there was not a very marked difference in the prevalence of suspect and positive sera between the problem company and the non-problem company.

Table 2. Summary of sera tested - classification into negative, suspect and positive.

Figure 1 illustrates the distribution of the positive sera according to reporting band. There is now a clear difference evident between the two companies. However, low grade reactions in reporting bands 1 to 6 are poorly predictive of a history of-, or an ongoing-, S.e. problem. Many of these reactions became less intense when the same samples were repeat-tested. Generally if only low-grade reactions were present their intensity is reduced when the flock is re-sampled after 2 weeks. Subsequent experience has shown that heat inactivation of such sera (56 C for 30 minutes) reduces or eliminates many of these reactions. Reactions in bands higher than 6 were, on the other hand, very rare in company B, but were relatively common in company A, especially band 12. The great majority of samples in these bands occurred in a few flocks which underwent a distinct sero-conversion with a high percentage of birds becoming sero-positive.

Figure 1. % Prevalence of positive sera according to S/P reporting band.

Our standard serology reports also include a mean value for each group of sera. This is the simple arithmetic mean of the band values for each of the sera. Figures 3 and 4 below show the contrasting distribution of means for the two companies. Mean values above 2 occurred only in company A. These high mean values usually reflected a broad range of reactions with a substantial number of samples falling in the lower bands.

Figure 2, Company A : Mean S/P Bands according to flock age

Figure 3. Company B : Mean S/P bands according to flock age

Monitoring response to vaccination:
The mean S/P band for 156 groups of vaccinated birds is plotted in Figure 4. It must be kept in mind that about 30% of the groups shown in the figure refer to individual pens of vaccinated birds in controlled trials and these included only a small number of samples per group (generally 4-5 sera). The great majority of the groups of sera from vaccinated birds had a mean band in excess of 2. Lower values were more common at around 14 weeks of age. This may be due to too short an interval since the first application of vaccine. The vaccinal responses seen at 22 weeks include a large number of birds vaccinated under normal commercial conditions. Two regression lines have been added to the chart. One covers the period up to 23 weeks and represents the mean results during the period of response to vaccination. The second line is based on all data from 21 weeks to 55 weeks of age. This is intended to estimate the depletion of mean antibody response with age. It seems likely that the birds which have low levels of antibody or no detectable antibody response to vaccination will show sero-conversion when field challenge occurs with a virulent strain of S.e.. This remains to be investigated. The value of establishing these base-lines subsequently became evident when one company elected to try a modified vaccination programme.

Figure 4. Distribution of Mean S/P bands among groups of vaccinated chickens.

 

Early development work with the same vaccine in layer chickens had demonstrated satisfactory protection when birds are vaccinated at 1 day and 4 weeks of age. This company elected to vaccinate broiler parents and 3 and 8 weeks of age and it soon became evident that the humoral response as measured by this test was much reduced. Figure 5 above shows the mean S/P bands for flocks coming into lay over a two-year period. This figure was directly generated by our custom-designed serology reporting system. The early-vaccination policy was adopted for the flocks which came into lay between November 1996 and May 1996. Before and after this period mean S:P bands were 3 or above, during this period most were below 3.

Discussion

This commercial test is a sensitive measure of the antibody response to S.e. in broiler parent chickens. The significance of low-prevalence low-intensity positive reactions is questionable in that they occur in a significant number of birds with no microbiological evidence of infection. Perhaps its greatest value in unvaccinated birds is in the situation in which a hatchery isolate needs to be rapidly “connected” with a specific parent flock. Comparison of point-of-lay mean results in vaccinated birds with population data may form the basis for monitoring response to vaccination. The poor serological response to vaccination of young broiler parents was unexpected given previous work which showed that it is possible to protect layers very effectively with this sort of programme. It is however uncommon to apply inactivated vaccines in broiler parent chickens before 10 weeks of age. Physiological stresses associated with restricted feeding may reduce the response to such vaccines. Although the precise mechanism of protection is unknown a uniform and long-lasting response in circulating antibody is likely to be beneficial in S.e. control programmes.

References

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