CZ20131048A3 - Use of Salmonella enterica ssp. enterica serovar Typhimurium antigens for serological differentiation of infected and vaccinated pigs - Google Patents

Abstract

The solution describes a method for serologically distinguishing Salmonella enterica ssp. Enterica serovar Typhimurium-infected pigs from vaccines based on inactivated Salmonella enterica ssp. Enterica serovar Typhimurium vaccine, in which an ELISA or westernblot level of antibodies against at least one native or recombinant a protein selected from the group SseB, Gene ID: 1252916; SipB, Gene ID: 1254408 and SipD, Gene ID: 1254406 and at least one native or recombinant protein selected from the group of FliC, Gene ID: 1253480 and YfgL, Gene ID: 1254042. The infected animals are identified as having a higher level than animals vaccinated antibodies to SseB or SipB or SipD, while having lower levels of antibodies to FliC or YfgL, whereas animals vaccinated are identified as having higher levels of antibodies to FliC or YfgL than animals infected with lower levels of antibodies to SseB or SipB or SipD. The serological distinction between animals infected with animals vaccinated is necessary for the application of curative procedures aimed at reducing the percentage of Salmonella positive slaughter pigs. Another reason for the need to distinguish animals infected from animals vaccinated is the elimination of the false positivity of vaccinated pigs in estimating the prevalence of Salmonella ssp. Pigs by using established serological methods in some Western European countries.

Description

The invention relates to serological diagnostics and immunoprophylaxis of salmonella infections in pigs. Serological differentiation of infected and vaccinated animals is necessary for the application of recovery procedures aimed at reducing the percentage of Salmonella positive pigs for slaughter. Another reason for the need to differentiate between infected and vaccinated animals is to exclude false positivity of vaccinated pigs when estimating the infestation of Salmonella ssp pigs by means of established serological methods in some Western European countries. The invention allows serological differentiation of pigs infected with Salmonella enterica ssp. Enterica serovar Typhimurium from pigs vaccinated with an inactivated culture of Salmonella enterica ssp. Enterica serovar Typhimurium.

BACKGROUND OF THE INVENTION

The most common agents of swine salmonella infections in the Czech Republic and most of the European Union are the non-atypical Salmonella enterica ssp. Enterica serovars, especially the Typhimurium and Derby serovars, which occur in approximately 65% of cases of Salmonella cultivation. The other 25% of Salmonella culture capture cases in pigs are Enteritidis and Agona serovars (EFS A 2008). One method of controlling the occurrence of non-pyrophic Salmonella infections in pigs may be to vaccinate pigs against the infectious agents, but the only vaccine so far authorized (in Germany and Poland) is based on a live vaccine strain and has been little used so far. The inventors experimentally verified the efficacy of vaccinating sows with an inactivated culture of Salmonella enterica ssp. Enterica serovar Typhimurium to reduce salmonella levels in organs of suckling piglets (Matiašovic et al. 2013) and confirmed the efficacy of pig vaccination with inactivated Salmonella enterica ssp. Enterica serovar Typhimurium. previously at herd level (Roesler et al. 2006).

For the successful introduction of any vaccine against Salmonella enterica ssp. Enterica serotypes, it is necessary to distinguish animals vaccinated from animals infected. Established serological methods for the detection of antibodies against non-phytoid serovar Salmonella enterica ssp.

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_ν j 4 <'Ϊ d t * t <*' * enterica pigs are based on detection of antibodies to O-antigens of serotypes. After natural infection of pigs with non-phytoidal serovars of Salmonella enterica ssp. Enterica and after vaccination of pigs with any vaccine containing Salmonella enterica ssp. Enterica O-antigens, IgG antibodies against these antigens are produced. Thus, prior serological methods have identified pigs vaccinated as well as infected pigs.

The possibility of serologically distinguishing infected pigs from vaccinated pigs is the development of vaccines based on vaccine strains lacking O-antigens (Leyman et al. 2011). Targeted or accidental genetic modifications of vaccine strains complicate vaccine development, and the possible use of genetically modified strains makes vaccine registration difficult.

Live attenuated vaccine registered in Germany and Poland (IDT Biologica GmbH, Germany) is further developed to differentiate post-infectious and post-vaccine antibodies. The vaccine induces the production of antibodies against O-antigens. The authors chose a deletion mutant method where the vaccine strain lacks the ompD protein and therefore does not form it upon vaccination of the antibody, as opposed to a wild strain that stimulates the pig's immune system to produce antibodies against the ompD protein. Thus, the differentiation of vaccinated animals from infected animals is based on serological detection of antibodies against the ompD protein. The problem of a modified live vaccine is that it is less protective against the original vaccine strain (Selke et al. 2007).

The vaccine based on an inactivated culture of Salmonella enterica ssp. Enterica serovar Typhimurium (Matiašovic et al. 2013) is being developed into commercial form by teams of both applicants. The vaccine induces the production of antibodies against O-antigens. Another vaccine based on an inactivated culture of Salmonella enterica ssp. Enterica serovar Typhimurium or an inactivated culture of other Salmonella enterica ssp. Enterica serovars is not commercially available.

It is known that after infection of humans or animals with a live strain of Salmonella enterica ssp. Enterica, antibodies to various antigens of this pathogen, including most of the proteins of the invention, are produced (SseB - Lee et al. 2012, SipB Durur et al. 2013, SipD - Desin et al 2010, FliC - Bergman et al 2005). The present invention allows comparisons of antibody levels against certain Salmonella enterica ssp. Enterica serovar Typhimurium serums to differentiate pigs infected with Salmonella enterica ssp.

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Literature for the prior art

1. The EFSA Joumal, 2008, 135: 1-111.

2. Matiasovic, J., Kudlackova, H., Babickova, K., Stepanova, H., Volf, J., Rychlik, I., Babak, V., Faldyna, M. Impact of matemally-derived antibodies against Salmonella enterica Serovar Typhimurium suckling piglets. Veterinary Joumal. 2013, 196: 114-115.

3. Roesler, U., Heller, P., Waldmann, KH, Truyen, U., Hensel, A. Immunization of sows in an integrated pig-breeding herd using a homologous inactivated Salmonella vaccine decreases the prevalence of Salmonella typhimurium infection in the offspring. Joumal of Veterinary Medicine B, 2006, 53, 224-228.

4. Leyman, B., Boyen, E., Van Parys, A., Verbrugghe, E., Haesebrouck, E., Pasmans, F. Salmonella Typhimurium LPS mutations for use in vaccines allowing differentiation of infected and vaccinated pigs. Vaccine, 2011, 29: 3679-3685.

5. Selke, M., Meens, J., Springer, S., Frank, R., Gerlach, G.F. Immunization of pigs to prevent disease in humans: construction and protective efficacy of Salmonella enterica serovar Typhimurium live negative marker vaccine. Infection and Immunity, 2007, 75: 2476-2483.

6. Lee, SJ, Liang, L., Juarez, S., Nanton, MR, Gondwe, EN, Msefula, CL, Kayala, MA, Necchi, E, Heath, JN, Hart, P., Tsolis, RM, Heyderman RS, MacLennan, CA, Felgner, PL, Davies, DH, McSorley, SJ Identification of a common immune signature in murine and human systemic Salmonellosis. Proc Natl Acad Sci USA, 2012, 109: 4998-5003.

7. Durand, D., Ochoa, TJ, Bellomo, SM, Contreras, CA, Bustamante, VH, Ruiz, J., Cleary, TG Detection of secretory immunoglobulin A in human colostrum and mucosal response to proteins of type III secretion system of Salmonella, Shigella, and enteropathogenic Escherichia coli. Pediatrician Infect Dis J, 2013, 32: 1122-1126.

8. Desin, T. S., Mickael, C. S., Lam, P. K., Potter, A. A., Koster, W. Protection of epithelial cells from Salmonella enterica serovar Enteritidis invasion by antibodies against the SPI-1 type III secretion system. Can J Microbiol., 2010, 56: 522-526.

9. Bergman, M.A., Cummings, L.A., Alaniz, R.C., Mayeda, L., Fellner, I., Cookson, B.T. CD4 + -T-cell responses generated during murine Salmonella enterica serovar Typhimurium infection are directed towards multiple epitopes within the natural antigen of FliC. Infection and Immunity, 2005, 73: 7226-7235.

SUMMARY OF THE INVENTION

To date, there is no known way to distinguish animals infected with Salmonella enterica ssp. Enterica serovar Typhimurium from pigs vaccinated with a vaccine based on an inactivated culture of a wild strain of Salmonella enterica ssp. Enterica serovar Typhimurium. SUMMARY OF THE INVENTION The present invention is based on the finding that after vaccination of pigs with a vaccine based on an inactivated culture of Salmonella enterica ssp. Enterica serovar Typhimurium, antibodies against Salmonella enterica ssp. Enterica serovar Typhimurium are produced qualitatively in a different way than after infection. from animals vaccinated.

Accordingly, the present invention is based on the use of two groups of Salmonella enterica ssp., Enterica serovar Typhimurium proteins as antigens for the detection of antibodies in serum, colostrum and pig meat juice. These two groups of proteins differentially induce antibody production after infection of Salmonella enterica ssp. Enterica serovar Typhimurium and after vaccination of pigs with a vaccine based on inactivated Salmonella enterica ssp. Enterica serovar Typhimurium.

Against SseB proteins, Gene ID: 1252916; SipB, Gene ID: 1254408, high levels of antibodies are produced after natural pig infection, whereas after vaccination of pigs with an inactivated culture of Salmonella enterica ssp. Enterica serovar Typhimurium, antibodies against these proteins do not develop at all or only very little. This makes it possible to detect animals infected with Salmonella enterica ssp. Enterica serovar Typhimurium.

Against FliC proteins, Gene ID: 1253480; YfgL, Gene ID: 1254042 there is little or no antibody production after natural pig infection, while high levels of antibodies are generated against these proteins after vaccination of pigs with an inactivated culture of Salmonella enterica ssp. Enterica serovar Typhimurium. This makes it possible to detect animals vaccinated with a vaccine based on an inactivated culture of Salmonella enterica ssp., Enterica serovar Typhimurium.

Evaluation of the levels of antibodies against proteins JESG, SipB, SipD against proteins and FliC _x YfgL and thus enables to distinguish infected animals from animals vaccinated with inactivated vaccine based on culture of Salmonella enterica ssp. Enterica serovar Typhimurium. High levels of antibodies against SseB or SipB or SipD, as well as low levels of antibodies against FliC or YfgL, identify animals infected with Salmonella enterica ssp. Enterica with Typhimurium or other Salmonella enterica ssp. Enterica, depending on the antigenic affinity of the proteins. High levels of antibodies against FliC or YfgL, as well as low levels of antibodies against SseB or SipB or SipD, identify animals vaccinated with a vaccine based on an inactivated culture of Salmonella enterica ssp.

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4. * *, <f f τ t 6 * * * culture - Salmonella-entericassp ·. enterica serovar Typhimurium. High levels of antibodies against both groups of proteins are identified by both infected and vaccinated animals. Low or no antibody levels against both groups of proteins identify animals that have either not been infected or vaccinated or animals that have not developed antibodies after infection or vaccination.

Examples

Serological differentiation of animals infected from animals vaccinated using the antigens of the invention is possible, for example, by an ELISA assay or a westemblot.

ELISA test

When using the isolated SseB protein (GeneID: 1252916) as an antigen in an ELISA assay, the measured absorbance in a serum sample of pigs infected with Salmonella enterica ssp. Enterica serovar Typhimurium is at least 2x higher than in a serum sample of pigs vaccinated with Salmonella enterica ssp. enterica serovar Typhimurium and than in a pooled serum sample of control uninfected pigs.

Using the isolated FliC protein (GeneID: 1253480) as an antigen in an ELISA assay, the measured absorbance in a serum sample of pigs vaccinated with an inactivated culture of Salmonella enterica ssp. Enterica is at least 3 times higher than in a serum sample of pigs infected with Salmonella enterica ssp. enterica serovar Typhimurium and than in a pooled serum sample of control uninfected pigs.

The above ELISA tests are prepared by coating the wells of one microtiter plate with recombinant SseB protein and the other microtiter plate with recombinant FliC protein, both at a working dilution of 1 pg / ml in carbonate-bicarbonate buffer (0 ^ 05 M, pH 9.6) in a volume of 100 μΐ per well (incubation 16 hours, 4 ° C). Subsequently, the plates are washed three times with PBS containing 0.05% Tween 20 (PBS-T, pH 7.2). The pig serum or plasma samples to be examined are diluted 100-fold in PBS-T with 0.5% casein hydrolyzate and added to the wells of the microtiter plate at a volume of 100 μΐ. After incubation (1 h, 37 ° C, humid chamber), plates are washed three times with PBS-T solution and 100 µL of horseradish peroxidase-labeled conjugate (Bethyl Laboratories, Montgomery, USA) is added to the wells. working dilution. After incubation (60 min, 37 ° C, humid chamber), the plates are washed again three times and 100 µl of TMB Complete substrate solution (Test-line, Brno, Czech Republic) is added to the wells. The enzyme reaction stops after 10 minutes with 50 μΐ 2M sulfuric acid. The resulting absorbance is read using a multichannel spectrophotometer at 450 nm, in our case Synergy H1 (Biotek, USA).

Westemblot

When using isolated SipB protein (Gene ID: 1254408) or SipD (Gene ID: 1254406) as an antigen in a westemblot test, the intensity of the strip after incubation with a serum sample of pigs infected with Salmonella enterica ssp. a serum sample of pigs vaccinated with an inactivated culture of Salmonella enterica ssp. enterica serovar Typhimurium and than with a pooled serum sample of control uninfected pigs.

When using isolated YfgL protein (Gene ID: 1254042) as an antigen in a westemblot test, the intensity of the strip after incubation with a serum sample of pigs vaccinated with an inactivated culture of Salmonella enterica ssp. Enterica serovar Typhimurium is at least 2 times higher than after incubation with a pig serum sample. enterica serovar Typhimurium and than with a pooled serum sample of control uninfected pigs.

The above westemblots were prepared by dividing the antigen (recombinant SipB or YfgL protein) on a 7 cm acrylamide 10 SDS-PAGE gel using a MiniPROTEAN® II Electrophoresis Cell (Bio-Rad, USA) (20 min at a constant voltage of 80jV) The gel proteins are transferred (75 min at a constant current of 340 mA) to a PVDF membrane (Amersham Hybond-P, GE) using a semi-dry westemblot apparatus (Omni-Bio, Czech Republic). The PVDF membrane is blocked in 0.5% casein hydrolyzate in PBS containing 0.05% Tween 20 (PBS-T, pH 7.2) for 16 hours at 4 ° C. The membrane is then incubated for 1 hour with being examined sample of pig sera in lOOnásobném t '* · from ⁴ - * dilutions in the same solution - PBS-T / casein hydrolyzate with shaking at room temperature. Thereafter, the membrane was washed five times with PBS-T aL | and incubated at room temperature with a conjugate of labeled horseradish peroxidase (Goat anti-pig IgG conjugate, Bethyl Laboratories, Montgomery, USA, 100x diluted in PBS-T / casein hydrolyzate) and washed again 5 times (PBS-T). Chromogenic substrate diaminobenzidine (13 mg in 40 ml PBS, after addition of 40 μΐ hydrogen peroxide) is used for antigen visualization. The reaction is stopped by addition of excess water. The color intensity of the strips is measured, for example, on a GelLogic 2200 PRO (Carestream Health, USA). The color intensity of the strips can also be evaluated visually.

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Evaluation

High levels of antibodies against SseB or SipB or SipD, as well as low levels of antibodies against FliC or YfgL, identify animals infected with Salmonella enterica ssp. Enterica serovar Typhimurium or other Salmonella enterica ssp. Enterica serovar, depending on the antigenic affinity of said proteins. High levels of antibodies against FliC or YfgL, as well as low levels of antibodies against SseB or SipB or SipD, identify animals vaccinated with an inactivated Salmonella enterica ssp. Enterica serovar Typhimurium culture vaccine. High levels of antibodies against both groups of proteins are identified by both infected and vaccinated animals. Low or no antibody levels against both groups of proteins identify animals that have either not been infected or vaccinated or animals that have not developed antibodies after infection or vaccination.

Industrial applicability

Creation of detection systems (eg ELISA, westemblot) allowing serological differentiation of pigs infected with Salmonella enterica ssp. Enterica serovar Typhimurium from pigs vaccinated with a vaccine based on an inactivated culture of Salmonella enterica ssp. Enterica serovar Typhimurium.

On behalf ofHH-spol. Bioveta, a.s.

Name: Ing. Libor Bittner

Function: Chairman of the Board of Directors

----------- J menem of the Research Institute of Veterinary Medicine, v.v. i.,

Name: RNDr. Miroslav Machala Function: Person in charge of management

Name: MVDr. Vladimir Vrzal, CSc. Function: Member of the Board of Directors

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Claims (1)

Method for serologically distinguishing pigs infected with Salmonella enterica ssp. Enterica serovar Typhimurium from pigs vaccinated with a vaccine based on an inactivated culture of Salmonella enterica ssp. Enterica serovar Typhimurium, characterized in that the serum sample is determined by ELISA or westernblot against at least one native or recombinant protein selected from the group of SseB, Gene ID: 1252916; SipB, Gene ID: 1254408 and SipD, Gene ID: 1254406 and at least one native or recombinant protein selected from the group FliC, Gene ID: 1253480 and YfgL, Gene ID: 1254042, wherein the infected animals are identified as having higher vaccinated animals levels of antibodies against SseB or SipB or SipD while having a lower level of antibodies against FliC or YfgL, whereas animals vaccinated are identified as having a higher level of antibodies against FliC or YfgL, while having a lower level of antibodies against the protein SseB or SipB or SipD.