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An agar gel enzyme assay (AGEA) for simple detection of Salmonella enteritidis antibodies in chicken sera
203
DIAGN MICROBIOL INFECT DIS 1991;14:203-208
An Agar Gel Enzyme Assay (AGEA) for Simple Detection of Salmonella enteritidis Antibodies in Chicken Sera Chul Joong Kim and Kakambi V. Nagaraja
An agar gel enzyme assay (AGEA) was developed for the detection of antibodies to Salmonella enteritidis (SE). The assay was based on the ability of antibodies to diffuse through an agar gel and react with antigen coated on a polystyrene surface. The antigen-antibody reaction was then made visible by applying an enzyme-conjugated anti-immunoglobulin and the addition, subsequently, of a substrate-containing gel. The color change in circular zones was taken as the indication for the presence of antibodies. The present investigation reports identification of an antigen specific for SE and its use in the devel-
opment of a relatively simple AGEA procedure. The results of AGEA were compared with those of conventional microagglutination (MA) test and serum plate (SP) test. The percentage agreement between M A and AGEA in positive serum sample was found to be 94.4%, and in negative serum samples it was found to be 88.8%. The present results suggest that the AGEA could be a very useful screening test for the detection of SE antibodies because the assay is inexpensive, specific and simple to perform without much equipment, and give results within a 3-hr period.
INTRODUCTION
teritidis bacteria can be transmitted from infected laying hens directly into the interior of the eggs before the shells are formed. The infection in birds is best confirmed by the recovery of the organism. It usually requires 72-96 hr for the isolation and to be defined by its cultural and biochemical properties. Presently, the technique for isolating SE involves the culturing of doacal swabs and internal organs collected from suspected birds. The principal drawback with this method is that a single swabbing of each bird may not detect the presence of the SE organism due to the intermittent shedding of the organism by the bird. Repeated cloacal swabbing of all birds would certainly increase the chances of its isolation. However, continual swabbing of 100% of the birds in the field is a very timeconsuming and impractical method. An alternative method to culturing has been the detection of serum antibodies formed against the infecting SE organism. This detection remains theoretically more desirable for mass screening than culture of internal organs from birds on postmortem. However, none of the currently existing procedures for screening birds for Salmonella infection in poultry have been effective in detecting a reasonable percentage of birds infected with SE. The majority of tests rely on the agglutination of bacterial antigen and are consequently biased towards the detection
Salmonellosis due to Salmonella enteritidis (SE) has increased steadily starting in the late 1970s in the northeastern United States as a cause of gastroenteritis in humans. Chicken eggs that were contaminated with SE bacteria have caused some recent outbreaks of food-borne illness (St. Louis et al., 1988). The same authors have found that grade-A shell eggs were associated with the epidemic rise in SE infections. Unlike in the past, when Salmonella outbreaks were caused by cracked eggs soiled by chicken feces, the researchers have traced the recent outbreaks to uncracked eggs that had been washed and disinfected. In three other countries, Spain, France, and the United Kingdom, epidemic SE has been associated with shell eggs (Tauxe, 1988). It is believed that the eggs become contaminated during ovulation. Researchers strongly suspect that Salmonella enFrom the Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Minnesota, St. Paul, Minnesota, USA. Address reprint requests to Dr. K.V. Nagaraja, Room 301D, Veterinary Science Building, College of Veterinary Medicine, 1971 Commonwealth Avenue, St. Paul, MN 55108, USA. Received 18 April 1990; revised and accepted 11 September 1990.
© 1991 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010 0732-8893/91/$3.50
204
of IgM antibodies. The conventional tests, such as microagglutination (MA) test and serum plate agglutination (SPA) test, are not so sensitive and specific for SE (Fernando et al., 1988; Shivaprasad et al., 1988). It is also a practice in the field to use antigen made from cross-reacting Salmonella pullorum (SP) to test birds for infection with SE. This has not given consistent results in the detection of birds infected with SE. On a practical basis, detection of flock infections remains one of the most serious, unsolved problems in controlling salmonellosis in poultry. Increased interest in the control of SE in poultry has resulted in emphasis on the development of improved detection methods. In the past a radial immunodiffusion enzyme assay (RIDEA) has been examined for the detection of antibodies in serum samples (Johnson et al., 1984; Joo et al., 1984; Thawley et al., 1985; Gradil and Joo, 1988). However, to obtain the results, this assay requires incubation overnight. An enzyme-linked immunosorbent assay (ELISA) has been used for the detection of Salmonella infections (Lentsch, 1981). The ELISA procedure requires expensive equipment to conduct the test. The objective of this investigation was to identify an antigen specific for SE and to develop a relatively simple, yet highly sensitive and specific test. In this test, SE antibodies are b o u n d to antigen coated on a polystyrene plate after which the primary antigen-antibody reaction is visualized by the addition of an enzyme conjugate and substrate. Here the color intensity is proportional to the amount of antibody bound to the antigen.
MATERIALS A N D METHODS Bacteria The strain of Salmonella enteritidis (SE) (no. 87.4390) provided from National Veterinary Services Laboratory (NVSL) (Ames, Ia), S. typhimurium (ST), and SP from our laboratory specimen bank were used. All three Salmonella serotypes used were of chicken origin. Biochemical tests and serotyping were performed to confirm that these cultures used were strains of SE, SP, and ST before use.
Antigen The procedure described by Filip et al. (1973) was used with few modifications. Briefly, tryptic soy agar (Difco Laboratories, Detroit, MI) in Roux flasks were inoculated with SE and incubated at 37~C for 24 hr. The bacterial ceils were collected in 10 mM Hepes buffer (Sigma Chemical Company, St. Louis, MO) (pH 7.4) and washed three times in saline. They
C.J. Kim and K.V. Nagaraja
were then broken by passing them through a French press (Wabash Metal Production Company, Wabash, IN) at 15,000 to 20,000 Ib/in 2. The broken cell suspension was centrifuged at 4000 g at 4°C for 20 min and the cell debris was discarded. The suspension was centrifuged at 100,000 g for 1 hr to get whole-membrane fraction. The gel-like pellet was dissolved in 2% Sarkosyly (Sigma) in 10 mM Hepes buffer and incubated overnight at 4°C. The suspension was again centrifuged at 100,000 g for 1 hr and the supernatant was discarded. The pellet, which contained mainly Sarkosyl insoluble outer membrane (OM) proteins, was dissolved in 10 mM TrisHC1 (pH 7.2) and dialyzed for 24 hr.
Antisera N e w Zealand white rabbits weighing 3 kg were used to produce antisera against SE, SP, and ST. Formalin-killed cultures containing 101° bacteria per milliliter were prepared from overnight stationary-phase 37°C Veal infusion broth (Difco) cultures and washed three times in saline. Equal volumes of formalinkilled suspension and mineral oil with arlacel-A (Sigma) were mixed well to obtain a homogenous oil emulsion suspension. Rabbits were hyperimmunized with bacteria in oil emulsion. Each rabbit received i ml of the suspension subcutaneously, five times at weekly intervals.
Western Blotting To identify proteins specific for SE, Western blotting was done using antiserum against SE, SP, and ST according to the procedure described by Talbot et al. (1984). The OM proteins of SE were separated on 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) by the Laemmli method (1970) with 4% staking gels for 15 hr at 4°C. Proteins separated in SDS-PAGE were electrophoretically transferred to a nitrocellulose filter using Transphor electrophoresis unit cell (Bethesda Research Laboratory, Gaithersburg, MD) with electroblotting buffer containing 25 mM Tris, 192 mM glycine, and 20% methanol, pH 8.3. The nitrocellulose filter strips were stained by Ponceau S red staining (Sigma) to check the transferred proteins and destained in distilled water. The strips were then immersed in Tris-buffered saline (TBS) containing 20 mM Tris, 500 mM NaCl, and 3% gelatin (Sigma), pH 7.5, for 1 hr at 37°C. The nitrocellulose filters were rinsed briefly in Tris-Tween buffer saline (TTBS) containing 0.05% Tween-20 in TBS buffer, pH 7.5. The resulting blots were incubated for 3 hr at 37°C either with serum containing rabbit anti-SE antibodies or rabbit anti-SP or -ST antibodies diluted in TTBS containing 1% gelatin. Un-
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AGEA for Antibody Detection
bound antibodies were removed by rinsing the blots in TFBS and b o u n d antibodies were detected by using conjugate labeled with horseradish peroxidase (HRP) (Bethesda Research Laboratory) and substrate 4 chlor-l-naphthol/(Sigma). Two protein bands from SE proteins over 40 kD molecular weight that did not react with antibodies to SP and ST were eluted from the polyacrylamide gel using Elutrap (Schleicher and Schuell, Keene, NH) equipment. The protein concentration of this solution was measured by Bradford (1976) protein assay and it was then lyophilized until used in the agar gel enzyme assay (AGEA).
Preparation of Antigen-Coated Petri Dishes Lyophilized SE protein antigen prepared earlier was reconstituted at 120 ng/ml in carbonate buffer (pH 9.5). Polystyrene Petri dishes (60 mm in diameter) were dispensed with carbonate buffer containing SE protein antigen. The volume dispensed was just enough to cover the entire bottom of the plate. The coating was allowed to proceed for 15 hr at 4°C, at which time the plates were washed three times with phosphate-buffered saline (PBS) containing 0.05% Tween-20 washing solution. To occupy unbound sites on the plates, we added 5 ml of 0.1% gelatin solution to each plate and allowed it to incubate for 1 hr at 37°C. The plates were washed three times with washing solution mentioned earlier. Five milliliters of 0.6% agarose containing 9% NaCI solution was added to each plate and allowed to harden. The SE antigen-coated plates were then stored at 4°C until used.
Experimental Design Twenty 14-week-old white leghorn chickens were wing banded and housed in individual cages. All birds were determined to be SE free by rectal swab culture and serology using the currently available methods. They were infected with SE. Each bird was inoculated orally with 3.1 x 10 9 colony forming units (CFU)/ml of SE. Equal number of birds were kept as noninoculated controls. Serum samples from infected and noninfected birds were collected at weekly intervals and examined for the presence of antibodies to SE on AGEA, MA, and SP tests. Birds were examined for 16 weeks at which time the experiment was terminated.
Procedure for Screening Serum Samples for Salmonella enteritidis Antibodies Using the
AGEA Procedure Sera collected from birds infected and noninfected controls were individually absorbed onto the round
filter paper (6 mm in diameter) disks and these disks were placed on the gel of AGEA Petri dish. In each plate, reference filter paper disks impregnated with known sera negative and positive for SE antibodies were also included. Up to 14 disks were placed on each plate. The plates were allowed to incubate for 2 hr at room temperature to allow the serum from the filter paper disks to diffuse in the gel and to react with SE protein antigen. At the end of 2 hr, the gel was carefully peeled off and the plates were washed three times with washing solution and reacted with 3 M/plate of a 1/1000 dilution of peroxidase-conjugated goat anti-chicken IgG (Kirkegaard and Perry Laboratories, Gaithersburg, MD) for I hour at room temperature. The plates were then washed and were added with substrate [Sigma; 5 ml of a 1% agarose gel in PBS (pH 7.2) containing 0.1 ml of 0.3% hydrogen peroxide and 0.1 ml of 0.4% of 5-amino salicylic acid]. At the end of 30 min the reaction zones were read directly from the plate. A color change to dark brown was indicative of the presence of antibodies to SE and the absence of a color change was considered as a negative reaction or absence of antibodies to SE in the serum. Optimal conditions for AGEA on dilution and adsorption time for S. enteritidis antigen and peroxidase-conjugated goat antichicken IgG (Kirkegaard and Perry Laboratories) were predetermined by comparative testings. An optimal incubation time between antigen and antibody was evaluated with a different reaction time using positive and negative serum samples. Blood samples tested in AGEA system were also tested for the presence of antibodies to SE using conventional SP and MA tests currently used for salmonella, in general. The SP and MA test were conducted as described for SP and ST infections (Williams and Whittemore, 1971 and 1976).
Sensitivity and Specificity of AGEA Chicken serum samples from our laboratory specimen bank that were previously tested for the presence of SE antibodies on MA test were selected randomly. This included 157 samples tested positive and 89 samples tested negative for the presence of SE antibodies by MA test. These samples were tested on AGEA to evaluate its sensitivity and specificity compared with MA test results.
RESULTS Standardization of AGEA Optimum Period of Incubation Figure 1 shows the results of AGEA at 1-, 2-, and 3hr postincubation periods. After 2 hr of incubation, the reaction was easily visible and distinct for one
C.J. K i m a n d K.V. N a g a r a j a
206
FIGURE 1 Comparison of different incubation periods for AGEA: (left) 1 hr, (center) 2 hr, and (right) 3 hr.
to differentiate b e t w e e n positive a n d negative reaction; 1 hr of incubation a p p e a r e d not sufficient to read the results. Final color reaction w a s visible as early as 5 rain after the addition of agar containing substrate a n d h y d r o g e n peroxide.
Sensitivity of A G E A Figure 2 illustrates the sensitivity of AGEA. The letters N a n d S r e p r e s e n t s e r u m s a m p l e s f r o m k n o w n negative a n d specific p a t h o g e n - f r e e birds, respectively. There w e r e n o reactions (color zones) in the area w h e r e these disks (N, S) w e r e placed. Color d e v e l o p m e n t t o o k place only w h e r e the disks containing S. enteritidis positive s e r u m w e r e placed.
those of SP test a n d M A test (Table 1). The a n t i b o d y w a s first detected at 7 d a y s after infection in all three tests e x a m i n e d . At 6 w e e k s postinfection, five of 20 birds w e r e positive in SP test a n d M A test, w h e r e a s 18 of the s a m e 20 birds were f o u n d positive in AGEA. After 12 w e e k s of infection, n o n e of the birds w e r e positive in the SP test a n d a low n u m b e r of birds w e r e f o u n d positive in the M A test. H o w e v e r , the A G E A test w a s m u c h m o r e sensitive a n d able to detect a h i g h p e r c e n t a g e of e x p e r i m e n t a l l y infected birds u p to the e n d of the e x p e r i m e n t .
TABLE 1
Comparison of A G E A with M A and SP Tests The ability of A G E A to detect S. enteritidis antibodies in chicken s e r u m w a s tested a n d c o m p a r e d w i t h
FIGURE 2 Specificity of AGEA to detect antibodies to SE. Dark zones indicate the presence of antibodies to SE in the serum samples tested. N and S represent serum samples from known negative and specific pathogen-free birds, respectively. No color zone m the place of letters N and S represents the absence of antibodies to SE.
C o m p a r i s o n of S e r u m Plate Test, Microagglutination Test, a n d A g a r Gel E n z y m e A s s a y for the Detection of Antibodies to SE
Weeks After Infection
SP Test a
MA b
AGEA c
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14d 15 16
0/20 10/20 15/20 20/20 18/20 12/20 5/20 6/20 7/20 3/20 5/20 3/20 0/20 0/20 0/20 0/19 0/19
0/20 16/20 19/20 20/20 20/20 15/20 5/20 7/20 7/20 11/20 5/20 5/20 6/20 3/20 0/19 3/19 3/19
0/20 19/20 19/20 20/20 20/20 20/20 18/20 19/20 19/20 19/20 19/20 19/20 19/20 18/20 17/19 16/19 16/19
Abbreviations as in text. aNumber positive for SE antibodies per number tested; 75% of the bacterial cells when agglutinated was taken as positive.
bNumber positive for SE antibodies per number tested; Titer 1:20 and higher was taken as positive. ~Number positive for SE antibodies per number tested; absorbance units at 0.32 and higher was taken as positive. dOne bird died.
AGEA for Antibody Detection
TABLE 2
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Percentage of Agreement Between Microagglutination Test and Agar Gel Enzyme Assay for the Detection of Antibodies to Salmonella enteritidis AGEA
MA Titer
Number of Serum Samples Examined
Positive
Negative
Percentage Correlation
Negative 1:20 1:40 1:80 1:160 >1:320
89 18 33 48 30 28
10 17 33 48 30 28
79 1 -----
88.8 94.4 100.0 100.0 100.0 100.0
Percentage Correlation
Table 2 shows the percentage agreement between MA test and AGEA in the detection of antibodies to SE in chicken sera. Among 89 serum samples tested on MA test and found negative for SE antibodies, 10 samples were found positive in AGEA (88.8% correlation). Among 18 samples found positive at 1:20 titer on MA test, only 17 of them were positive on AGEA (94.4% correlation). DISCUSSION The radial immunodiffusion enzyme assay has been described for quantitation of antibodies to different antigens (Elwing et al., 1980). In the present study, the procedure of testing serum samples was modified by using the filter paper disks. We soaked the round filter papers in serum samples, then placed them onto the gel, and let the antibodies in the filter paper diffuse through the low concentration of agarose gel containing high NaC1 concentration. This modification of the procedure reduced the period of antigen-antibody reaction to as low as 2 hr at room temperature. Therefore, the whole procedure to complete the assay takes only 3 hr. The antigen used in the AGEA was Sarkosyl insoluble outer membrane protein fraction of SE obtained after the disruption of cell at high pressure.
The highly sensitive and specific protein antigen was selected by Western immunoblotting technique. Western immunoblotting is a convenient, sensitive, and specific technique for the detection of antigens and antibodies. In the present study, we have applied this procedure to the analysis of OMP of SE. It was possible to adsorb this protein antigen easily onto a polystyrene surface. The serum plate and the MA tests are done with whole-cell antigens. They rely on the agglutination of bacteria and are consequently biased toward the detection of IgM antibodies. In AGEA, a protein antigen highly specific for SE was used. The results from AGEA showed high sensitivity in the detection of a low amount of antibodies to SE in serum samples as late as 16 weeks after infection (Table 1). This probably is due to the detection of IgG antibodies present at long postexposure intervals. In conclusion, described herein AGEA provides a high level of sensitivity and specificity and has the potential to become a valuable screening test. The AGEA results could be interpreted easily and fast. The assay is inexpensive, specific, and simple to perform without sophisticated equipment.
This work was supported by Southeast Poultry and Egg Association and Minnesota Agricultural Experiment Station.
REFERENCES
Bradford MM (1976) A rapid and sensitive method for the quantitafion of microgram quantifies of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-254. Elwing H, Lange S, Nygren H (1980) Diffusion-in-gel enzyme-linked immunosorbent assay (DIG-ELISA): optimal conditions for quantitation of antibodies. J Immunol Methods 39:247-256. Fernando WWD, Kradel DC, Clark CD (1988) Comparative serology studies on naturally and experimentally infected Salmonella enteritidis flocks. In Proceedings of the
60th Northeastern Conference on Avian Disease. Storrs, CT: University of Connecticut, p 5. Filip C, Fletcher G, Wulff JL, Earhart CF (1973) Solubilization of the cytoplasmic membrane of Escherichia coli by the ionic detergent sodium-lauryl sarcosinate. ] Bacteriol 115:717-722. Gradil C, Joo HS (1988) A radial immunodiffusion enzyme assay for detection of antibody to equine rhinopneumonitis virus (EHV-1) in horse serum. Vet Microbiol 17:315-322. Johnson EH, Smith BP, Reina-guerra M (1984) Diffusion
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in gel-enzyme linked immunosorbent assay (DIG-ELISA) to record the immunoglobulin response of calves vaccinated with salmonella. Vet Microbiol 10:71-86. Joo HS, Molitor TW, Leman AD (1984) Radial immunodiffusion enzyme assay for detection of antibodies to pseudorabies virus in swine serum. Am J Vet Res 45:20962098. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685. Lentsch RH, Batema RP, Wagner JE (1981) Detection of salmonella infections by polyvalent enzyme-linked immunosorbent assay. J Clin Microbiol 14:281-287. Shivaprasad HL, Timoney JF, Morales S, Baker RC (1988) Pathogenesis of Salmonella enteritidis in chickens: preliminary studies. In Proceedings of the 60th Northeastern Conference on Avian Disease. Storrs, CT: University of Connecticut, p 7. St. Louis ME, Morse DL, Potter ME, DeMelfi TM, Guzewich JJ, Tauxe RV, Blake PA (1988) The emergence of grade A eggs as a major source of Salmonella enteritidis
C.J. Kim a n d K.V. Nagaraja
infections: new implications for the control of salmoneUosis, lAMA 259:2103-2107. Talbot P], Knobler RL, Buchmeier MJ (1984) Western and dot immunoblotting analysis of viral antigens and antibodies: application to murine hepatitis virus. J Immunol Methods 73:177-188. Tauxe RV (1988) Proceeding of the National Public Meeting on Salmonella enteritidis. Washington, DC: Food Drug Administration and the US Department of Agriculture, pp 9-10. Thawley DG, Joo HS, Johnson ME, Solorzano RF (1985) Evaluation of the radial immunodiffusion enzyme assay for the detection of antibodies to pseudorabies virus. J Am Vet Med Assoc 186:1080-1083. Williams JE, Whittemore AD (1971) Serological diagnosis of pollorum disease with the microagglutination system. Appl Microbiol 21:394-399. Williams JE, Whittemore AD (1976) Comparison of six methods of detecting Salmonella typhimurium infection of chickens. Avian Dis 20:728-734.
DIAGN MICROBIOL INFECT DIS 1991;14:203-208
An Agar Gel Enzyme Assay (AGEA) for Simple Detection of Salmonella enteritidis Antibodies in Chicken Sera Chul Joong Kim and Kakambi V. Nagaraja
An agar gel enzyme assay (AGEA) was developed for the detection of antibodies to Salmonella enteritidis (SE). The assay was based on the ability of antibodies to diffuse through an agar gel and react with antigen coated on a polystyrene surface. The antigen-antibody reaction was then made visible by applying an enzyme-conjugated anti-immunoglobulin and the addition, subsequently, of a substrate-containing gel. The color change in circular zones was taken as the indication for the presence of antibodies. The present investigation reports identification of an antigen specific for SE and its use in the devel-
opment of a relatively simple AGEA procedure. The results of AGEA were compared with those of conventional microagglutination (MA) test and serum plate (SP) test. The percentage agreement between M A and AGEA in positive serum sample was found to be 94.4%, and in negative serum samples it was found to be 88.8%. The present results suggest that the AGEA could be a very useful screening test for the detection of SE antibodies because the assay is inexpensive, specific and simple to perform without much equipment, and give results within a 3-hr period.
INTRODUCTION
teritidis bacteria can be transmitted from infected laying hens directly into the interior of the eggs before the shells are formed. The infection in birds is best confirmed by the recovery of the organism. It usually requires 72-96 hr for the isolation and to be defined by its cultural and biochemical properties. Presently, the technique for isolating SE involves the culturing of doacal swabs and internal organs collected from suspected birds. The principal drawback with this method is that a single swabbing of each bird may not detect the presence of the SE organism due to the intermittent shedding of the organism by the bird. Repeated cloacal swabbing of all birds would certainly increase the chances of its isolation. However, continual swabbing of 100% of the birds in the field is a very timeconsuming and impractical method. An alternative method to culturing has been the detection of serum antibodies formed against the infecting SE organism. This detection remains theoretically more desirable for mass screening than culture of internal organs from birds on postmortem. However, none of the currently existing procedures for screening birds for Salmonella infection in poultry have been effective in detecting a reasonable percentage of birds infected with SE. The majority of tests rely on the agglutination of bacterial antigen and are consequently biased towards the detection
Salmonellosis due to Salmonella enteritidis (SE) has increased steadily starting in the late 1970s in the northeastern United States as a cause of gastroenteritis in humans. Chicken eggs that were contaminated with SE bacteria have caused some recent outbreaks of food-borne illness (St. Louis et al., 1988). The same authors have found that grade-A shell eggs were associated with the epidemic rise in SE infections. Unlike in the past, when Salmonella outbreaks were caused by cracked eggs soiled by chicken feces, the researchers have traced the recent outbreaks to uncracked eggs that had been washed and disinfected. In three other countries, Spain, France, and the United Kingdom, epidemic SE has been associated with shell eggs (Tauxe, 1988). It is believed that the eggs become contaminated during ovulation. Researchers strongly suspect that Salmonella enFrom the Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Minnesota, St. Paul, Minnesota, USA. Address reprint requests to Dr. K.V. Nagaraja, Room 301D, Veterinary Science Building, College of Veterinary Medicine, 1971 Commonwealth Avenue, St. Paul, MN 55108, USA. Received 18 April 1990; revised and accepted 11 September 1990.
© 1991 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010 0732-8893/91/$3.50
204
of IgM antibodies. The conventional tests, such as microagglutination (MA) test and serum plate agglutination (SPA) test, are not so sensitive and specific for SE (Fernando et al., 1988; Shivaprasad et al., 1988). It is also a practice in the field to use antigen made from cross-reacting Salmonella pullorum (SP) to test birds for infection with SE. This has not given consistent results in the detection of birds infected with SE. On a practical basis, detection of flock infections remains one of the most serious, unsolved problems in controlling salmonellosis in poultry. Increased interest in the control of SE in poultry has resulted in emphasis on the development of improved detection methods. In the past a radial immunodiffusion enzyme assay (RIDEA) has been examined for the detection of antibodies in serum samples (Johnson et al., 1984; Joo et al., 1984; Thawley et al., 1985; Gradil and Joo, 1988). However, to obtain the results, this assay requires incubation overnight. An enzyme-linked immunosorbent assay (ELISA) has been used for the detection of Salmonella infections (Lentsch, 1981). The ELISA procedure requires expensive equipment to conduct the test. The objective of this investigation was to identify an antigen specific for SE and to develop a relatively simple, yet highly sensitive and specific test. In this test, SE antibodies are b o u n d to antigen coated on a polystyrene plate after which the primary antigen-antibody reaction is visualized by the addition of an enzyme conjugate and substrate. Here the color intensity is proportional to the amount of antibody bound to the antigen.
MATERIALS A N D METHODS Bacteria The strain of Salmonella enteritidis (SE) (no. 87.4390) provided from National Veterinary Services Laboratory (NVSL) (Ames, Ia), S. typhimurium (ST), and SP from our laboratory specimen bank were used. All three Salmonella serotypes used were of chicken origin. Biochemical tests and serotyping were performed to confirm that these cultures used were strains of SE, SP, and ST before use.
Antigen The procedure described by Filip et al. (1973) was used with few modifications. Briefly, tryptic soy agar (Difco Laboratories, Detroit, MI) in Roux flasks were inoculated with SE and incubated at 37~C for 24 hr. The bacterial ceils were collected in 10 mM Hepes buffer (Sigma Chemical Company, St. Louis, MO) (pH 7.4) and washed three times in saline. They
C.J. Kim and K.V. Nagaraja
were then broken by passing them through a French press (Wabash Metal Production Company, Wabash, IN) at 15,000 to 20,000 Ib/in 2. The broken cell suspension was centrifuged at 4000 g at 4°C for 20 min and the cell debris was discarded. The suspension was centrifuged at 100,000 g for 1 hr to get whole-membrane fraction. The gel-like pellet was dissolved in 2% Sarkosyly (Sigma) in 10 mM Hepes buffer and incubated overnight at 4°C. The suspension was again centrifuged at 100,000 g for 1 hr and the supernatant was discarded. The pellet, which contained mainly Sarkosyl insoluble outer membrane (OM) proteins, was dissolved in 10 mM TrisHC1 (pH 7.2) and dialyzed for 24 hr.
Antisera N e w Zealand white rabbits weighing 3 kg were used to produce antisera against SE, SP, and ST. Formalin-killed cultures containing 101° bacteria per milliliter were prepared from overnight stationary-phase 37°C Veal infusion broth (Difco) cultures and washed three times in saline. Equal volumes of formalinkilled suspension and mineral oil with arlacel-A (Sigma) were mixed well to obtain a homogenous oil emulsion suspension. Rabbits were hyperimmunized with bacteria in oil emulsion. Each rabbit received i ml of the suspension subcutaneously, five times at weekly intervals.
Western Blotting To identify proteins specific for SE, Western blotting was done using antiserum against SE, SP, and ST according to the procedure described by Talbot et al. (1984). The OM proteins of SE were separated on 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) by the Laemmli method (1970) with 4% staking gels for 15 hr at 4°C. Proteins separated in SDS-PAGE were electrophoretically transferred to a nitrocellulose filter using Transphor electrophoresis unit cell (Bethesda Research Laboratory, Gaithersburg, MD) with electroblotting buffer containing 25 mM Tris, 192 mM glycine, and 20% methanol, pH 8.3. The nitrocellulose filter strips were stained by Ponceau S red staining (Sigma) to check the transferred proteins and destained in distilled water. The strips were then immersed in Tris-buffered saline (TBS) containing 20 mM Tris, 500 mM NaCl, and 3% gelatin (Sigma), pH 7.5, for 1 hr at 37°C. The nitrocellulose filters were rinsed briefly in Tris-Tween buffer saline (TTBS) containing 0.05% Tween-20 in TBS buffer, pH 7.5. The resulting blots were incubated for 3 hr at 37°C either with serum containing rabbit anti-SE antibodies or rabbit anti-SP or -ST antibodies diluted in TTBS containing 1% gelatin. Un-
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AGEA for Antibody Detection
bound antibodies were removed by rinsing the blots in TFBS and b o u n d antibodies were detected by using conjugate labeled with horseradish peroxidase (HRP) (Bethesda Research Laboratory) and substrate 4 chlor-l-naphthol/(Sigma). Two protein bands from SE proteins over 40 kD molecular weight that did not react with antibodies to SP and ST were eluted from the polyacrylamide gel using Elutrap (Schleicher and Schuell, Keene, NH) equipment. The protein concentration of this solution was measured by Bradford (1976) protein assay and it was then lyophilized until used in the agar gel enzyme assay (AGEA).
Preparation of Antigen-Coated Petri Dishes Lyophilized SE protein antigen prepared earlier was reconstituted at 120 ng/ml in carbonate buffer (pH 9.5). Polystyrene Petri dishes (60 mm in diameter) were dispensed with carbonate buffer containing SE protein antigen. The volume dispensed was just enough to cover the entire bottom of the plate. The coating was allowed to proceed for 15 hr at 4°C, at which time the plates were washed three times with phosphate-buffered saline (PBS) containing 0.05% Tween-20 washing solution. To occupy unbound sites on the plates, we added 5 ml of 0.1% gelatin solution to each plate and allowed it to incubate for 1 hr at 37°C. The plates were washed three times with washing solution mentioned earlier. Five milliliters of 0.6% agarose containing 9% NaCI solution was added to each plate and allowed to harden. The SE antigen-coated plates were then stored at 4°C until used.
Experimental Design Twenty 14-week-old white leghorn chickens were wing banded and housed in individual cages. All birds were determined to be SE free by rectal swab culture and serology using the currently available methods. They were infected with SE. Each bird was inoculated orally with 3.1 x 10 9 colony forming units (CFU)/ml of SE. Equal number of birds were kept as noninoculated controls. Serum samples from infected and noninfected birds were collected at weekly intervals and examined for the presence of antibodies to SE on AGEA, MA, and SP tests. Birds were examined for 16 weeks at which time the experiment was terminated.
Procedure for Screening Serum Samples for Salmonella enteritidis Antibodies Using the
AGEA Procedure Sera collected from birds infected and noninfected controls were individually absorbed onto the round
filter paper (6 mm in diameter) disks and these disks were placed on the gel of AGEA Petri dish. In each plate, reference filter paper disks impregnated with known sera negative and positive for SE antibodies were also included. Up to 14 disks were placed on each plate. The plates were allowed to incubate for 2 hr at room temperature to allow the serum from the filter paper disks to diffuse in the gel and to react with SE protein antigen. At the end of 2 hr, the gel was carefully peeled off and the plates were washed three times with washing solution and reacted with 3 M/plate of a 1/1000 dilution of peroxidase-conjugated goat anti-chicken IgG (Kirkegaard and Perry Laboratories, Gaithersburg, MD) for I hour at room temperature. The plates were then washed and were added with substrate [Sigma; 5 ml of a 1% agarose gel in PBS (pH 7.2) containing 0.1 ml of 0.3% hydrogen peroxide and 0.1 ml of 0.4% of 5-amino salicylic acid]. At the end of 30 min the reaction zones were read directly from the plate. A color change to dark brown was indicative of the presence of antibodies to SE and the absence of a color change was considered as a negative reaction or absence of antibodies to SE in the serum. Optimal conditions for AGEA on dilution and adsorption time for S. enteritidis antigen and peroxidase-conjugated goat antichicken IgG (Kirkegaard and Perry Laboratories) were predetermined by comparative testings. An optimal incubation time between antigen and antibody was evaluated with a different reaction time using positive and negative serum samples. Blood samples tested in AGEA system were also tested for the presence of antibodies to SE using conventional SP and MA tests currently used for salmonella, in general. The SP and MA test were conducted as described for SP and ST infections (Williams and Whittemore, 1971 and 1976).
Sensitivity and Specificity of AGEA Chicken serum samples from our laboratory specimen bank that were previously tested for the presence of SE antibodies on MA test were selected randomly. This included 157 samples tested positive and 89 samples tested negative for the presence of SE antibodies by MA test. These samples were tested on AGEA to evaluate its sensitivity and specificity compared with MA test results.
RESULTS Standardization of AGEA Optimum Period of Incubation Figure 1 shows the results of AGEA at 1-, 2-, and 3hr postincubation periods. After 2 hr of incubation, the reaction was easily visible and distinct for one
C.J. K i m a n d K.V. N a g a r a j a
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FIGURE 1 Comparison of different incubation periods for AGEA: (left) 1 hr, (center) 2 hr, and (right) 3 hr.
to differentiate b e t w e e n positive a n d negative reaction; 1 hr of incubation a p p e a r e d not sufficient to read the results. Final color reaction w a s visible as early as 5 rain after the addition of agar containing substrate a n d h y d r o g e n peroxide.
Sensitivity of A G E A Figure 2 illustrates the sensitivity of AGEA. The letters N a n d S r e p r e s e n t s e r u m s a m p l e s f r o m k n o w n negative a n d specific p a t h o g e n - f r e e birds, respectively. There w e r e n o reactions (color zones) in the area w h e r e these disks (N, S) w e r e placed. Color d e v e l o p m e n t t o o k place only w h e r e the disks containing S. enteritidis positive s e r u m w e r e placed.
those of SP test a n d M A test (Table 1). The a n t i b o d y w a s first detected at 7 d a y s after infection in all three tests e x a m i n e d . At 6 w e e k s postinfection, five of 20 birds w e r e positive in SP test a n d M A test, w h e r e a s 18 of the s a m e 20 birds were f o u n d positive in AGEA. After 12 w e e k s of infection, n o n e of the birds w e r e positive in the SP test a n d a low n u m b e r of birds w e r e f o u n d positive in the M A test. H o w e v e r , the A G E A test w a s m u c h m o r e sensitive a n d able to detect a h i g h p e r c e n t a g e of e x p e r i m e n t a l l y infected birds u p to the e n d of the e x p e r i m e n t .
TABLE 1
Comparison of A G E A with M A and SP Tests The ability of A G E A to detect S. enteritidis antibodies in chicken s e r u m w a s tested a n d c o m p a r e d w i t h
FIGURE 2 Specificity of AGEA to detect antibodies to SE. Dark zones indicate the presence of antibodies to SE in the serum samples tested. N and S represent serum samples from known negative and specific pathogen-free birds, respectively. No color zone m the place of letters N and S represents the absence of antibodies to SE.
C o m p a r i s o n of S e r u m Plate Test, Microagglutination Test, a n d A g a r Gel E n z y m e A s s a y for the Detection of Antibodies to SE
Weeks After Infection
SP Test a
MA b
AGEA c
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14d 15 16
0/20 10/20 15/20 20/20 18/20 12/20 5/20 6/20 7/20 3/20 5/20 3/20 0/20 0/20 0/20 0/19 0/19
0/20 16/20 19/20 20/20 20/20 15/20 5/20 7/20 7/20 11/20 5/20 5/20 6/20 3/20 0/19 3/19 3/19
0/20 19/20 19/20 20/20 20/20 20/20 18/20 19/20 19/20 19/20 19/20 19/20 19/20 18/20 17/19 16/19 16/19
Abbreviations as in text. aNumber positive for SE antibodies per number tested; 75% of the bacterial cells when agglutinated was taken as positive.
bNumber positive for SE antibodies per number tested; Titer 1:20 and higher was taken as positive. ~Number positive for SE antibodies per number tested; absorbance units at 0.32 and higher was taken as positive. dOne bird died.
AGEA for Antibody Detection
TABLE 2
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Percentage of Agreement Between Microagglutination Test and Agar Gel Enzyme Assay for the Detection of Antibodies to Salmonella enteritidis AGEA
MA Titer
Number of Serum Samples Examined
Positive
Negative
Percentage Correlation
Negative 1:20 1:40 1:80 1:160 >1:320
89 18 33 48 30 28
10 17 33 48 30 28
79 1 -----
88.8 94.4 100.0 100.0 100.0 100.0
Percentage Correlation
Table 2 shows the percentage agreement between MA test and AGEA in the detection of antibodies to SE in chicken sera. Among 89 serum samples tested on MA test and found negative for SE antibodies, 10 samples were found positive in AGEA (88.8% correlation). Among 18 samples found positive at 1:20 titer on MA test, only 17 of them were positive on AGEA (94.4% correlation). DISCUSSION The radial immunodiffusion enzyme assay has been described for quantitation of antibodies to different antigens (Elwing et al., 1980). In the present study, the procedure of testing serum samples was modified by using the filter paper disks. We soaked the round filter papers in serum samples, then placed them onto the gel, and let the antibodies in the filter paper diffuse through the low concentration of agarose gel containing high NaC1 concentration. This modification of the procedure reduced the period of antigen-antibody reaction to as low as 2 hr at room temperature. Therefore, the whole procedure to complete the assay takes only 3 hr. The antigen used in the AGEA was Sarkosyl insoluble outer membrane protein fraction of SE obtained after the disruption of cell at high pressure.
The highly sensitive and specific protein antigen was selected by Western immunoblotting technique. Western immunoblotting is a convenient, sensitive, and specific technique for the detection of antigens and antibodies. In the present study, we have applied this procedure to the analysis of OMP of SE. It was possible to adsorb this protein antigen easily onto a polystyrene surface. The serum plate and the MA tests are done with whole-cell antigens. They rely on the agglutination of bacteria and are consequently biased toward the detection of IgM antibodies. In AGEA, a protein antigen highly specific for SE was used. The results from AGEA showed high sensitivity in the detection of a low amount of antibodies to SE in serum samples as late as 16 weeks after infection (Table 1). This probably is due to the detection of IgG antibodies present at long postexposure intervals. In conclusion, described herein AGEA provides a high level of sensitivity and specificity and has the potential to become a valuable screening test. The AGEA results could be interpreted easily and fast. The assay is inexpensive, specific, and simple to perform without sophisticated equipment.
This work was supported by Southeast Poultry and Egg Association and Minnesota Agricultural Experiment Station.
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