Detection of Salmonella Infection in Chickens by an Indirect Enzyme-Linked Immunosorbent Assay Based on Presence of PagC Antibodies in Sera

Abstract

The outcomes of infection of humans and animals with Salmonella range from a persistent asymptomatic carrier state to temporal mild gastroenteritis or severe systemic infection. A rapid and accurate diagnostic test would help formulate strategies for effective prevention of their infections in the animal population. Current sequencing data predict that the outer membrane protein, PagC, is present in all common Salmonella serovars with sequence similarities of more than 98%. PagC sequences in other bacterial species are less than 65% similarity at the amino acid level to those of Salmonella PagC. We hypothesized that PagC could be immunogenic and detection of antibodies to this protein could be an accurate indicator of Salmonella infection. The pagC gene from Salmonella enterica serovar Typhimurium CVCC542 was expressed in Escherichia coli. The purified recombinant PagC protein was immobilized in microtiter plate wells. Sera from SPF chickens infected with Salmonella or other non-Salmonella pathogens by injection were added and binding of PagC protein was detected by the horseradish peroxidase (HRP)-labeled goat anti-chicken antibody. Sera from Salmonella-infected chickens showed high specificity in contrast to the sera from chickens infected with other bacteria. When 87 Salmonella antibody-positive sera from Salmonella Pullorum orally infected SPF chicken and 93 negative sera from uninfected SPF chicken were tested, 98.3% agreement was detected. The rPagC enzyme-linked immunosorbent assay (ELISA) and agglutination had 80.6% agreement in detecting 252 clinical chicken sera samples. These results suggest that PagC antibody-based indirect ELISA can serve as a convenient and novel method for the diagnosis of Salmonella infection.

Introduction

S almonella is one of the most common causes of foodborne illnesses and is a worldwide public health concern (Majowicz et al., 2010). In poultry, this pathogen might spread through vertical and horizontal transmission, so it is not only a great threat to the poultry industry but also threatens the food safety. Eggs or egg products contaminated with Salmonella can lead to Salmonella disease outbreaks in humans (Uzzau et al., 2000; Pires et al., 2009). As of 2011, the incidence of foodborne illness caused by Shiga toxin–producing Escherichia coli (STEC) O157:H7, Campylobacter, and Listeria has fallen by at least 25% worldwide, but only by 10% for Salmonella-related illnesses (Osterholm, 2011). Efforts to reduce the infection of humans and animals with Salmonella can benefit from accurate and rapid diagnostic tests. A serological test such as an enzyme-linked immunosorbent assay (ELISA) could be an important tool, as such a test can be highly sensitive, convenient, and relatively cheap. While serological tests for detection of infection with typhoidal Salmonella are used routinely (Sharma et al., 2017; Tran Vu Thieu et al., 2017), similar tests for infection with nontyphoidal serovars are rarely used and are mostly based on detection of antibodies to lipopolysaccharides (LPS) or flagella proteins from specific Salmonella serovars (Kuhn et al., 2012). Therefore, a test that targets antibodies produced against a wider collection of Salmonella serovars would be desirable.

PagC is an outer membrane protein that belongs to the porin superfamily. This outer membrane channel protein shares a beta-barrel structure with 185 amino acid residues. PagC is positively regulated under magnesium deprivation by the PhoP-PhoQ two-component system (Alix et al., 2008). The specific function of PagC is unknown but is associated with reduced macrophage survival. The great antigenic specificity and immune reactivity raise the possibility that PagC could be a promising target for detecting Salmonella antibodies (Wang et al., 2003; Zhang et al., 2013).

The pagC gene without its signal peptide was cloned into the bacterial expression vector pET-28a and expressed in E. coli. An indirect ELISA was developed based on purified recombinant PagC and shown to have potential for sera-based detection of Salmonella infection of chickens. This method has the potential to contribute to rapid and accuracy diagnosis of Salmonellosis in chickens and to efforts to eradicate Salmonella.

Materials and Methods

Sequence analyses

A total of 48 amino acid sequences of PagC from different bacteria were downloaded from NCBI GenBank and MEGA6.0 was used to draw a phylogenetic tree by the maximum likelihood statistical method. MegAlign of DNAStar Lasergene 7.1 was used for amino acid sequence similarity analysis by the Clustal W method.

Cloning of pagC sequence

Salmonella enterica serovar Typhimurium CVCC542 strain was obtained from the China Veterinary Culture Collection Center. Genomic DNA was extracted using a standard phenol/chloroform method (Luk et al., 1993).

Forward (P1, 5′-CCAGAATTCGATACTAACGCCTTTTCC-3′) and reverse (P2, 5′-CAACTCGAGTCAGAAACGGTATCCAAC-3′) primers were used to amplify the pagC gene without signal peptide (NCBI Reference Sequence: NZ_CP007523.1) from Salmonella Typhimurium genomic DNA. The gel purified PCR product and the vector were double digested with EcoRI and XhoI and ligated to place pagC in-frame with the N-terminal 6 × histidine tag, generating the recombinant plasmid pET28a-6 × His-PagC (24–185)::Kan. Restriction enzyme digests and DNA sequencing were used to confirm correct insert orientation and reading frame.

Expression, purification, and Western blot assay of recombinant protein

Plasmid pET28a-6 × His-PagC (24–185)::Kan was transformed into E. coli BL21 (DE3)-competent cells. A single colony was inoculated into 5 mL of LB medium with 100 μg/mL of kanamycin (LB Kan) and incubated overnight at 37°C. The entire culture was added to 500 mL of LB Kan and allowed to grow with shaking at 180 rpm at 37°C until the midexponential growth phase. Expression of recombinant protein was induced by addition of isopropyl beta-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM and incubation for an additional 3 h. The bacteria were lysed through ultrasonication and centrifuged at 12,000 × g, 4°C for 20 min. The PagC protein was isolated in the form of inclusion bodies and 8 M urea was used to solubilize the peptides. Ni²⁻ agarose gel filtration was used to purify the recombinant peptides. Elution buffer was 200 mM imidazole in phosphate-buffered saline (PBS) with 8 M urea. The peptides were allowed to renature during dialysis against PBS.

Western blot analysis was performed to assess the immunoreactivity of PagC with rabbit anti-PagC serum prepared after three immunizations with PagC in 10-day intervals. In brief, the protein was transferred from the polyacrylamide gel onto a PVDF membrane by semidry electroblotting. The membrane was blocked with blocking buffer (5% skim milk powder in PBS +0.1% TWEEN-20) overnight at 4°C, followed by incubation with a mouse anti-PagC antibody. The membrane was washed and incubated with an horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody. After a final wash, the bands containing recombinant protein were visualized by adding the diaminobenzidine (DAB) chemiluminescent substrate.

Standardization of rPagC-ELISA

Indirect ELISA was performed as described previously (Sun et al., 2014). Briefly, 96-well ELISA plates were coated with 25, 50, 100, 200, 400, or 800 ng/well rPagC in 0.05 M carbonate buffer solution (CBS) (pH 9.6) and allowed to incubate at 4°C overnight. The blocking buffer contained 1% bovine serum albumin (BSA) (m/v), 2% BSA (m/v), 1% FBS (m/v), 5% skimmed milk (m/v), and 2% gelatin (v/v) in PBS +0.1% TWEEN-20 (v/v) (PBST). The chicken sera were diluted in PBST to 1:10, 1:20, 1:50, 1:100, and 1:200. HRP-conjugated sheep anti-chicken IgG secondary antibody was diluted in PBST to 1:1000, 1:2000, 1:3000, and 1:5000 (v/v). Finally, the substrate tetramethylbenzidine (TMB) and 2 M H₂SO₄ were added for color development and termination of the reaction, respectively. The plates were read at an optical density of 450 nm (OD₄₅₀) in TECAN Infinite^® M1000 Pro.

Thirty OD₄₅₀ values of Salmonella antisera presenting normal distribution (SPSS 20.0 was used in normality test) were used to calculate the mean OD₄₅₀ and standard deviation (SD). The cutoff value between antisera and control sera was calculated as the OD₄₅₀ of the 30 negative control sera plus 3 × SD of the mean. This calculation provides 99% confidence that all negative values fall within the defined range (Webster et al., 1997; Tiwari et al., 2013).

Specificity evaluation of rPagC-ELISA

SPF chickens were challenged with live Salmonella Pullorum, Salmonella Gallinarum, Salmonella Typhimurium, Salmonella Enteritidis, Salmonella Indiana, E. coli, Pasteurella multocida, Proteus mirabilis, and Staphylococcus gallinarum through wing muscles by injection (bacterial background is listed in Table 1). Sera were collected 45 days postchallenge with three times immunization interval of 15 days and tested using rPagC-ELISA to evaluate specificity.

Table 1.

Information of Bacteria in This Research

Bacterial strain	Source
Salmonella Pullorum 0079	Isolated from chicken by our laboratory
Salmonella Gallinarum YZ136	Isolated from chicken by Jiangsu Institute of Poultry Sciences
Salmonella Typhimurium 3692	Isolated from chicken by our laboratory
Salmonella Enteritidis 1205	Isolated from chicken by our laboratory
Salmonella Indiana YZ306	Isolated from chicken by Jiangsu Institute of Poultry Sciences
Escherichia coli O1 NJ023	Isolated from chicken by our laboratory
Pasteurella multocida type A CVCC1676	Purchased from CVCC, isolated from chicken
Proteus mirabilis YZ88	Isolated from chicken by Jiangsu Institute of Poultry Sciences
Staphylococcus gallinarum CVCC2234	Purchased from CVCC, isolated from chicken

CVCC, China Veterinary Culture Collection Center.

Repeatability and reproducibility evaluation of rPagC-ELISA

Twenty sera were selected randomly from clinical samples obtained from a Salmonella Pullorum-positive breeding chicken farm. These samples were used to evaluate the repeatability and reproducibility of rPagC-ELISA.

Wells of a 96-well microtiter plate were coated with PagC by incubation with 200 ng of the protein per well in 0.05 M CBS buffer at 4°C overnight. Four replicates of these samples were tested in the same assay to evaluate intra-assay variability (repeatability). Interassay variability was tested by carrying out the assays at three different times. The mean OD₄₅₀, SD, and coefficient of variation (CV) of each sera sample were calculated.

Evaluation of rPagC-ELISA with Salmonella Pullorum-positive and -negative sera

To evaluate rPagC-ELISA as a clinical tool, 87 reference antisera were collected from Salmonella Pullorum orally infected SPF chicken (1 × 10⁴ CFU/mL); these samples were tested by plate agglutination and antigen isolation for Salmonella Pullorum antibody and antigen. Ninety-three reference negative control sera were also isolated from SPF chickens free of Salmonella infection. The 180 reference positive and negative sera samples were tested using rPagC-ELISA and the agreement was determined.

Comparison of rPagC-ELISA and plate agglutination test for detection of sera of chicken against Salmonella

A total of 252 sera samples from breeding hens housed in a Salmonella-positive poultry farm in Jiangsu province were collected. The samples were tested with rPagC-ELISA and a plate agglutination kit (Pullorum disease agglutination detection kit) purchased from YEBIO in Qingdao. Agreement between rPagC-ELISA and the kit results was determined by counting the number of identical results and dividing it by the total number of samples.

Statistical analysis

The ELISA data were analyzed using the Student's t-test and p values <0.05 were considered statistically significant.

Results

Bioinformatics analysis of PagC

PagC is widely distributed in Salmonella enterica subsp. enterica. The phylogenetic tree showed that PagC of Salmonella had close genetic relationship (Supplementary Fig. S1A; Supplementary Data are available online at www.liebertpub.com/fpd). The amino acid of PagC in different bacteria was aligned and results are shown in Supplementary Figure S1B. Analysis of the amino acid sequence of PagC indicates that this protein is highly conserved in Salmonella. PagC has over 98% homology among most of different Salmonella enterica subsp. enterica serovars and less than 65% homology with those of other common non-Salmonella bacteria (Supplementary Fig. S1C). The close relationship of the PagC sequences from different Salmonella serovars and the distance of these sequences from those of other species suggest that PagC could be a promising antigen for detection of Salmonella infection.

Expression and Western blot assay of PagC protein

The 6 × His-PagC (24–185) was expressed from pET28a in the E. coli BL21 expression strain. Recombinant PagC protein had a molecular mass of about 23 kDa as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Supplementary Fig. S2A). Western blot with rabbit anti-PagC sera showed a single band with the same molecular weight as rPagC in Coomassie-stained SDS-PAGE (Supplementary Fig. S2B).

Optimization of parameters for rPagC-ELISA

The cutoff value for positive results was determined to be 0.328 through calculating the mean OD₄₅₀ values and SDs of 20 Salmonella-negative sera. The optimized work sheet of rPagC-ELISA is listed below (Table 2).

Table 2.

rPagC-Enzyme-Linked Immunosorbent Assay Work Sheet

Legend	Dilution	Buffer (pH)	Vol./well ( μL/well)	Inc. period	Temp. (°C)	Wash × time (min)
1st Coat	Antigen 2 μg/mL	0.05 M CBS	100	Over night	4	5 × 5
2nd Coat	Block	5% Skim milk	200	2 h	37	5 × 5
3rd Coat	Sera 1:50 dilution	5% Skim milk	100	1 h	37	5 × 5
4th Coat	Anti-chicken IgG secondary antibody 1:1000	5% Skim milk	100	1 h	37	5 × 5
Substrate	NA	TMB	100	10 min	37	NA
Stop	NA	2M H₂SO₄	50	10 min	24	NA
		Read at 450 nm
Positive cutoff value			0.328

CBS, carbonate buffer solution.

Specificity evaluation of rPagC-ELISA

The specificity evaluation demonstrated that all five Salmonella serovar sera samples provided readings above the cutoff value, which indicated that rPagC-ELISA could detect the Salmonella that frequently infects chickens in China. The OD values of sera positive for the other common chicken non-Salmonella bacterial pathogens were below the cutoff value, indicating that rPagC-ELISA reacted specifically with Salmonella (Supplementary Fig. S3).

Repeatability and reproducibility evaluation of rPagC-ELISA

The CV for the intra-assay comparison ranged from 2.73% to 5.10%, while the CV for the interassay comparison ranged from 2.04% to 7.41%. All the CVs were below 10%, which indicates that rPagC-ELISA has stable repeatability and reproducibility (Table 3) (Wang et al., 2016; Zhou et al., 2016).

Table 3.

Repeatability and Reproducibility of rPagC-Enzyme-Linked Immunosorbent Assay

	Intra-assay comparison (OD₄₅₀)				Intra-assay comparison (OD₄₅₀)
Sample no.	Mean	SD	CV%	Sample no.	Mean	SD	CV%
1	0.344	0.014	3.99	1	0.556	0.024	4.23
2	0.443	0.021	4.84	2	0.391	0.01	2.65
3	0.822	0.021	2.57	3	0.356	0.021	5.79
4	0.531	0.02	3.71	4	0.724	0.024	3.34
5	0.648	0.021	3.19	5	0.485	0.024	4.91
6	0.742	0.02	2.74	6	0.665	0.023	3.46
7	0.59	0.016	2.72	7	0.783	0.016	2.04
8	0.493	0.017	3.41	8	0.557	0.014	2.5
9	0.398	0.01	2.62	9	0.363	0.008	2.22
10	0.444	0.022	4.97	10	0.593	0.006	1.07
11	0.547	0.02	3.59	11	0.702	0.01	1.41
12	0.636	0.016	2.44	12	0.531	0.016	3
13	0.386	0.011	2.81	13	0.417	0.009	2.15
14	0.715	0.006	0.8	14	0.626	0.007	1.12
15	0.538	0.012	2.24	15	0.624	0.007	1.14
16	0.668	0.022	3.23	16	0.424	0.009	2.01
17	0.695	0.011	1.63	17	0.537	0.012	2.27
18	0.687	0.016	2.32	18	0.812	0.012	1.51
19	0.463	0.022	4.77	19	0.572	0.015	2.7
20	0.489	0.011	2.26	20	0.646	0.02	3.04

CV, coefficient of variation.

Results of rPagC-ELISA have great agreement with clinical control serum samples

The rPagC-ELISA correctly classified 84 of the 87 chickens infected with Salmonella Pullorum as positive, giving the method a sensitivity of 96.6%. All 93 control serum samples were negative by rPagC-ELISA, which translates to 100% specificity. The rPagC-ELISA results demonstrated 98.3% agreement with the produced positive and negative sera samples (Table 4).

Table 4.

Evaluation of rPagC-Enzyme-Linked Immunosorbent Assay with Control Sera of Salmonella Pullorum

	rPagC-ELISA results
Control sera	Positive	Negative	Total	% Agreement
Positive	84	3	87	NA
Negative	0	93	93	NA
Total	84	96	180	98.30

ELISA, enzyme-linked immunosorbent assay.

Performance of rPagC-ELISA and plate agglutination test

As shown in Table 5, 71 and 132 sera samples tested positive and negative, respectively, with both methods. However, discrepant results were obtained for 12 sera that were positive by the plate agglutination test but negative by rPagC-ELISA. Thirty-seven sera were found to be positive by rPagC-ELISA but negative by the plate agglutination test (Table 5). Therefore, these two detection methods were in agreement for 80.6% of the tested samples.

Table 5.

Summary of Plate Agglutination Test and rPagC-Enzyme-Linked Immunosorbent Assay Results

	rPagC-ELISA results
Plate agglutination test results	Positive	Negative	Total	% Agreement
Positive	71	12	83	NA
Negative	37	132	169	NA
Total	108	144	252	80.60

Discussion

Bacterial culture is the gold standard for Salmonella diagnosis owing to the accuracy of the testing method, but its complexity and time requirement limit its application in clinical application (Funk et al., 2005). In the present study, an indirect ELISA method was developed based on the detection of PagC protein antibody to identify Salmonella infections of chicken. PagC protein has high homology in Salmonella enterica subsp. enterica, it is a major constituent of Salmonella membrane vesicles and actived by acid conditions, which means this protein has high expression level in macrophage phagosomes (Kitagawa et al., 2010). When Salmonella is ingested by macrophages, PagC is likely to be presented to lymphocytes to induce production of PagC-specific antibody.

Prior studies have noted PagC has good antigenic specificity and immunoreactivity (Wang et al., 2003; Zhang et al., 2013). The results with sera from five Salmonella-infected chickens and four chickens infected with non-Salmonella (E. coli, P. multocida, P. mirabilis and S. gallinarum) subjected to the rPagC-ELISA method support the utility of PagC antibody detection for identification of Salmonella infections. Many serological test detections of nontyphoidal Salmonella have been developed, but generally without a focus on Salmonella-infected chickens (Kuhn et al., 2015). The only official standard serological Salmonella detection method for chicken is the agglutination test in China. It is widely used for poultry typhoid and pullorum disease testing since it is convenient and cheap. However, the interpretations of agglutination results are somewhat subjective and prone to error. The use of rPagC-ELISA to detect the positive and negative sera of chicken demonstrated an agreement of 98.3% between the detection results and the truth of these tested control serum samples. In contrast, the agreement between the results of the agglutination test and these tested control serum samples only reached to 80% (data not shown). These results suggest that the PagC ELISA method is better able to detect Salmonella infection.

Although the rPagC-ELISA is high in efficiency and sensitivity, it still requires further optimization. For example, the OD readings obtained for sera from chickens infected with non-Salmonella pathogens were close to those of the cutoff value, suggesting a certain level of cross-reactivity. It will be necessary to test more sera from chickens not infected with Salmonella, but infected with other disease agents to further assess cross-reactivity. It might be possible to utilize particular PagC epitopes to decrease cross-reactivity. The rPagC-ELISA results from more clinical chicken sera samples will also be helpful in determining a more appropriate cutoff value to reduce the interference of cross-reactivity.

In conclusion, this study developed an indirect ELISA method for Salmonella infection diagnosis in chicken based on PagC antibody detection. This method has high accuracy and specificity. It can serve as a convenient and novel method for the diagnosis of Salmonella infection and contribute to the eradication of salmonellosis in chicken.

Footnotes

Acknowledgments

This work was supported by the National Key Research and Development Program (2016YFD0501607); Special Fund for Agro-scientific Research in the Public Interest (201403054); the National Natural Science Foundation of China (31772746, 31302093, 31272581); the Natural Science Foundation of Jiangsu Province (BK20130676); the PhD Program of the Foundation of the Ministry of Education of China (20130097120024); the National Transgenic Major Program (2014ZX0800946B); the Key Project of Independent Innovation of the Fundamental Research Fund for the Central Universities of Nanjing Agricultural University (KYZ201630); the Jiangsu Province Science and Technology Support Program (BE2013433); and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Disclosure Statement

No competing financial interests exist.

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