JP7550454B2 - Method for detecting Salmonella infection using BamA protein - Google Patents

Description

The present invention relates to a method for detecting Salmonella infection using BamA protein, as well as an antigen reagent and kit for use in the detection method.

Salmonella is a gram-negative bacillus that is subdivided into more than 2,600 serotypes. Salmonellosis (livestock and poultry) and poultry Salmonella infection are designated as notifiable and legally mandated infectious diseases, respectively, under the Livestock Infectious Diseases Prevention Law, and it is important to take measures to prevent these infections. When livestock or poultry are infected with Salmonella, the main symptoms are fever, loss of appetite, and loss of energy, and the disease can develop into acute septicemia and chronic diarrhea. However, due to the limitations of scientific methods, the occurrence of Salmonellosis in livestock and poultry both in Japan and overseas has not been completely suppressed. Furthermore, in farms where Salmonellosis has occurred, it can be difficult to purify the bacteria.

In an effort to purify the bacteria, monitoring is being carried out by isolating and culturing the bacteria from the host and measuring antibodies in serum. By periodically measuring antibodies in serum, it is possible to estimate the time of infection with Salmonella, making it possible to take appropriate infection control measures.

On the other hand, if salmonellosis occurs on a farm, a vaccine may be administered to prevent the spread of infection. The Salmonella vaccine currently administered to cows and chickens in Japan is a vaccine in which the bacteria is inactivated by treatment with formalin, and is a very effective means of preventing infection.

However, when anti-Salmonella antibodies in the serum of vaccinated individuals are measured, they show a positive reaction, just like those in individuals infected with Salmonella. In other words, vaccination results in a scientific flaw in that it becomes impossible to distinguish between individuals infected with Salmonella and those not infected. For this reason, there is a need for a test method that can distinguish between individuals infected with Salmonella and those vaccinated.

One known test method that can make such a distinction is, for example, a test method that uses proteins that make up the flagellar antigens of Salmonella bacteria (Patent Document 1).

International Publication No. 2004/055045

The present invention was made in light of these circumstances, and its purpose is to provide a new method that can detect individuals infected with Salmonella by distinguishing them from individuals who have been vaccinated with an inactivated vaccine. Another object of the present invention is to provide a reagent and kit for use in this detection.

The present inventors conducted extensive research to solve the above problems and found that BamA protein, a cell membrane protein of Salmonella bacteria, has a higher affinity for anti-Salmonella antibodies produced in individuals infected with Salmonella bacteria than for anti-Salmonella antibodies produced in individuals vaccinated with an inactivated vaccine, and that by using this protein as an antigen for detecting anti-Salmonella antibodies, it is possible to detect infection with Salmonella bacteria with high accuracy. On the other hand, when proteins secreted outside the cell by the type III secretion apparatus of Salmonella bacteria (SseG protein and SseF protein), cell membrane proteins (LptD protein), and LPS (lipopolysaccharide) were used as antigens, no significant difference was observed in the affinity for the above two anti-Salmonella antibodies. From the above, it was found that the specificity shown by BamA protein is not a phenomenon common to the molecules that constitute Salmonella bacteria.

Based on the above findings, the present invention relates to a method for detecting Salmonella infection using BamA protein, and a reagent and kit for use in said detection method, and more specifically provides the following:

(1) A method for detecting infection with Salmonella, comprising immunologically detecting anti-Salmonella antibodies in a sample using BamA protein or a functional fragment thereof as an antigen.

(2) An antigen reagent for immunologically detecting Salmonella infection, comprising a BamA protein or a functional fragment thereof.

(3) A kit for immunologically detecting Salmonella infection, comprising the antigen reagent described in (2).

The present invention makes it possible to easily and accurately detect infection with Salmonella bacteria using BamA protein. Salmonella bacteria are causative bacteria of zoonotic diseases that infect livestock and poultry, affecting their productivity and causing economic losses, as well as causing food poisoning in humans. By using the present invention, it becomes possible to efficiently prevent infection with Salmonella bacteria in animals such as livestock and poultry, and in humans. In particular, in chickens, the serum of vaccinated individuals may give false positives in pullorum rapid agglutination diagnostic liquid used to diagnose pullorum (poultry Salmonella infection), making it difficult to diagnose pullorum. The present invention can also be used as an adjunct to such existing diagnoses.

1 is a graph showing the results of detecting anti-Salmonella antibodies in the serum of chickens infected with Salmonella bacteria and chickens vaccinated with an inactivated vaccine by ELISA using BamA protein as an antigen. 1 is a graph showing the results of detecting anti-Salmonella antibodies in the serum of chickens infected with Salmonella bacteria and chickens vaccinated with an inactivated vaccine by ELISA using SseF protein as the antigen. 1 is a graph showing the results of detecting anti-Salmonella antibodies in the serum of chickens infected with Salmonella bacteria and chickens vaccinated with an inactivated vaccine by ELISA using SseG protein as an antigen. 1 is a graph showing the results of detecting anti-Salmonella antibodies in the serum of chickens infected with Salmonella bacteria and chickens vaccinated with an inactivated vaccine by ELISA using the LptD protein as an antigen. 1 is a graph showing the results of detecting anti-Salmonella antibodies in the serum of chickens infected with Salmonella bacteria and chickens vaccinated with an inactivated vaccine by ELISA using LPS as an antigen.

The present invention provides a method for detecting infection with Salmonella spp., which comprises immunologically detecting anti-Salmonella antibodies in a sample using the BamA protein or a functional fragment thereof as an antigen.

The "Salmonella bacteria" to be detected as an infection in the detection method of the present invention include, but are not limited to, Salmonella bacteria of each serotype, such as Salmonella Typhimurium, Salmonella Choleraesuis, Salmonella Dublin, Salmonella Enteritidis, Salmonella Gallinarum, and Salmonella Pullorum, which belong to Salmonella enterica subsp. enterica.

In the present invention, the "sample" used to detect Salmonella infection is not particularly limited as long as it is a sample in which anti-Salmonella antibodies can be present. Generally, it is a blood sample such as serum, plasma, or whole blood collected from an individual that can be infected with Salmonella. The organism from which the sample is derived is not particularly limited as long as it is an organism that can be infected with Salmonella, and includes humans and other animals. The animal may be a livestock animal, a pet animal, an experimental animal, or an animal for other purposes. Examples of animals include, but are not limited to, chickens, ducks, geese, ducks, quails, pheasants, pigeons, turkeys, guinea fowl, cows, pigs, horses, sheep, goats, dogs, and cats.

These samples may be diluted or suspended in a diluent as necessary. Examples of the diluent include buffers such as phosphate buffer, Tris buffer, Good's buffer, borate buffer, acetate buffer, citrate buffer, glycine buffer, succinate buffer, and phthalate buffer. In addition, the samples of the present invention may be pretreated as necessary. Examples of pretreatment include freezing, concentration, fractionation, and purification, and addition of pH adjusters, stabilizers, preservatives, and antiseptics. The samples may be one of these treatments or a combination of two or more of them.

In the present invention, anti-Salmonella antibodies are detected in a sample as an indicator of Salmonella infection. An "anti-Salmonella antibody" is an antibody capable of specifically binding to the Salmonella bacteria, and more specifically, an antibody produced in the body of an individual from which the sample is derived. The isotype of the anti-Salmonella antibody may vary depending on the species of organism, and may be, for example, any of IgY, IgG, IgM, and IgA, or a combination of two or more of these. In addition, the isotype may be any subclass. The time of appearance and duration of anti-Salmonella antibodies in the body of an individual after infection with Salmonella may vary depending on the type of antibody. Therefore, the Salmonella infection detected in the present invention includes current and past infection with Salmonella bacteria.

The present invention is characterized in that the BamA protein or a functional fragment thereof is used as an antigen for detecting anti-Salmonella antibodies. The "BamA protein" is a bacterial membrane protein of Salmonella bacteria. A typical amino acid sequence of the BamA protein is shown in SEQ ID NO: 2, and a typical base sequence of the DNA encoding the protein is shown in SEQ ID NO: 1. However, it should be understood that in nature, the amino acid sequence of the protein may change due to genetic mutation. A "functional fragment" of the BamA protein may also be used as an antigen. Here, the functional fragment means a fragment of the BamA protein that has binding activity to anti-Salmonella antibodies. The binding activity of the BamA protein fragment to anti-Salmonella antibodies can be evaluated by the immunological method described below. The BamA protein or its functional fragment may be modified, such as by introducing a mutation, as long as the binding activity to anti-Salmonella antibodies is not impaired.

The BamA protein or a functional fragment thereof can be prepared as a recombinant protein or a natural protein by a method well known in the art. The recombinant protein can be prepared, for example, by inserting a DNA encoding the BamA protein (e.g., a DNA containing the base sequence set forth in SEQ ID NO: 1) or a DNA encoding a fragment of the protein into an appropriate expression vector, introducing the vector into an appropriate host cell, and purifying the protein from an extract or culture supernatant of the host cell. Examples of purification methods include, but are not limited to, ion exchange chromatography, reverse phase chromatography, and gel filtration. In addition, when the BamA protein or a fragment thereof is expressed in a host cell (e.g., E. coli, etc.) as a recombinant protein to which multiple histidines have been added, or as a fusion protein with a glutathione-S-transferase protein, the expressed recombinant protein can be affinity purified using a nickel column or a glutathione column, respectively. After purification of the fusion protein, regions other than the target protein or fragment can be removed by cleavage with thrombin or factor Xa as necessary. The natural protein can be prepared by methods known to those skilled in the art, for example, by contacting an extract of Salmonella with an affinity column to which an antibody against the BamA protein is bound.

The detection principle of the immunoassay used in the present invention is preferably the sandwich method, since it allows the construction of a highly sensitive detection system. In the sandwich method, anti-Salmonella antibodies are captured by a solid-phase capture molecule, which is then recognized by a detection molecule bound to a labeling substance, and after washing, detection is performed according to the type of labeling substance. For example, a plate such as a plastic plate, a fibrous material such as nitrocellulose, or particles such as magnetic particles or latex particles can be used as the solid phase.

In the sandwich method, BamA protein or a functional fragment thereof is used as an antigen for at least one of the molecules for capturing and detecting anti-Salmonella antibodies. There are no particular limitations on the other molecule as long as it can bind to the anti-Salmonella antibody, but for example, an antibody against the anti-Salmonella antibody (secondary antibody) or another protein that binds to the anti-Salmonella antibody can be used.

A labeling substance is usually bound to the detection molecule. The labeling substance is not particularly limited as long as it is a detectable substance, and examples thereof include enzymes such as horseradish peroxidase (HRP), alkaline phosphatase (ALP), and β-galactosidase (β-gal), fluorescent dyes such as fluorescein isothiocyanate (FITC) and rhodamine isothiocyanate (RITC), fluorescent proteins such as allophycocyanin (APC) and phycoerythrin (R-PE), radioisotopes such as ¹²⁵ I, gold particles, avidin, biotin, and latex. When an enzyme is used as a labeling substance, various detections can be performed depending on the substrate by adding a chromogenic substrate, a fluorescent substrate, or a chemiluminescent substrate as a substrate.

When quantifying anti-Salmonella antibodies in a sample, a sandwich method such as ELISA, in which microplate wells or beads are used as the solid phase, is preferred. The amount of anti-Salmonella antibodies from the obtained measured value can generally be determined by comparing it with the measured value from a standard sample. In this case, for example, the amount of anti-Salmonella antibodies in the sample can be determined by examining where the actual measured value is located on a standard curve created based on the measured values from the standard sample.

On the other hand, immunochromatography is suitable for detecting anti-Salmonella antibodies simply and quickly. One embodiment of an immunochromatographic device is described below. The device includes a detection zone in which capture molecules are immobilized in a line on a matrix made of a porous material such as a nitrocellulose membrane, and a labeling reagent zone carrying labeled detection molecules upstream of the detection zone. Usually, the labeling reagent zone is composed of a porous pad carrying labeled detection molecules. A developer tank storing a developer is provided at the upstream end of the matrix. In addition, usually, downstream of the detection zone, a development confirmation section in which molecules that capture the detection molecules are immobilized in a line to confirm the development of the labeled detection molecules, and downstream of that, a developer absorption zone in which a porous absorption pad for absorbing the developer is provided. Furthermore, when the label is an enzyme label, a substrate zone carrying a substrate for the labeling enzyme is provided upstream of the labeling reagent zone.

In use, a sample is added to the labeled reagent zone, and the developer pad is inserted into the developer tank by applying pressure to the pusher to move the protrusion, and the developer is supplied to the matrix through the developer pad. When the developer passes through the substrate zone, the substrate is dissolved in the developer, and the developer containing the substrate flows. When the developer passes through the labeled reagent zone, the labeled detection molecule and the sample are dissolved in the developer, and the developer containing the substrate, the labeled detection molecule, and the sample flows. If the sample contains anti-Salmonella antibodies, the anti-Salmonella antibodies and the labeled detection molecules bind to form a complex, and when this complex reaches the detection zone, the capture molecules on the detection zone bind to the anti-Salmonella antibodies in the complex. As a result, the labeled detection molecules are fixed to the detection zone via the anti-Salmonella antibodies. The anti-Salmonella antibodies are detected by measuring the label in the detection zone. If the sample does not contain anti-Salmonella antibodies, the labeled detection molecule will not be fixed to the detection zone and will move further downstream, and the label will not be detected in the detection zone. Note that the developer liquid confirmation section downstream of the detection zone has a solid phase molecule that captures the detection molecule, so the labeled detection molecule will be fixed to the developer liquid confirmation section. Therefore, if a label is detected in the developer liquid confirmation section, it means that the developer liquid has been developed correctly. The developer liquid is eventually absorbed by the absorbent pad downstream of that.

The detection method of the present invention makes it possible to distinguish between individuals infected with Salmonella bacteria and individuals vaccinated with inactivated Salmonella bacteria as a vaccine. Inactivated Salmonella bacteria to be used as a vaccine can be prepared, for example, by heat treatment of Salmonella bacteria, treatment with a chemical disinfectant, irradiation with radiation or ultraviolet light, or treatment with a mutagenic substance. As the inactivation treatment, heat treatment or treatment with formalin, a chemical disinfectant, is preferably used. The inactivated Salmonella bacteria may be a mixture of different Salmonella bacteria (for example, a mixture of Salmonella bacteria of different species or different serotypes).

The present invention also provides an antigen reagent for immunologically detecting infection with Salmonella genus bacteria, comprising the BamA protein or a functional fragment thereof. The antigen reagent may consist of only the antigen BamA protein or a functional fragment thereof, or may have a labeling substance bound thereto, or may be immobilized thereon. It may also be a composition containing these. Other components contained in the composition are not particularly limited, and may include, for example, sterilized water, physiological saline, a surfactant, a preservative, etc., and may be one type of these or a combination of two or more types thereof.

The present invention also provides a kit for immunologically detecting infection with Salmonella genus bacteria, comprising the antigen reagent. In addition to the antigen reagent, the kit may further comprise components that should be included in an immunological detection method. Examples of such components include, but are not limited to, standard samples (such as anti-Salmonella antibodies at various concentrations), control reagents, reaction buffers, diluents, and washing solutions. When the labeling substance is an enzyme, the kit may further comprise a substrate or a reaction stop solution necessary for detection and quantification of the labeling substance.

When the sandwich method is used for detection, and a solid phase to which BamA protein or a functional fragment thereof is bound is used as a molecule for capturing anti-Salmonella antibodies (one embodiment of the antigen reagent of the present invention), the kit of the present invention may further include a secondary antibody bound to a labeling substance as a molecule for detecting anti-Salmonella antibodies. On the other hand, when a labeled BamA protein or a functional fragment thereof is used as a molecule for detecting anti-Salmonella antibodies (one embodiment of the antigen reagent of the present invention), the kit of the present invention may further include a solid phase to which a secondary antibody or the like is bound as a molecule for capturing anti-Salmonella antibodies.

When immunochromatography is used for detection, the device may further include a zone carrying a capture molecule for anti-Salmonella antibodies and/or a labeled detection molecule. The device may include other components suitable for immunochromatography, such as a developer pad or an absorbent pad.

The kit of the present invention may further include instructions for use of the kit.

The present invention will be explained in more detail below based on examples and comparative examples.

A. Materials and Methods (1) Purification of Antigens The Salmonella -derived molecules used as antigens are as follows:

(a) Example BamA protein (804 amino acids, molecular weight 90422, bacterial membrane protein) (SEQ ID NOs: 1 and 2)

(b) Comparative Example (i) SseG protein (230 amino acids, molecular weight 24237, protein secreted outside the bacterial cell by the type III secretion apparatus) (SEQ ID NOs: 3 and 4)
(ii) SseF protein (261 amino acids, molecular weight 26595, protein secreted outside the bacterial cell by the type III secretion apparatus) (SEQ ID NOs: 5 and 6)
(iii) LptD protein (786 amino acids, molecular weight 89540, bacterial membrane protein) (SEQ ID NOs: 7 and 8)
(iv) LPS (Lipopolysaccharide)

(2) Cloning of antigen genes
PCR was performed using the DNA of Salmonella enterica serovar Typhimurium x3306 strain as a template and various primers listed in Table 1.

The PCR products were then cloned using the TOPO cloning method, and each of the Salmonella proteins listed in (1) was then highly expressed in E. coli. pET151/D-TOPO was used as the plasmid for protein expression. A His tag (6x) was added to the N-terminus of the protein expressed using this plasmid.

(3) Purification of antigen The Salmonella bacteria were pre-cultured (static culture) overnight in 5 mL of LB medium (containing ampicillin). Then, the bacteria were shake-cultured in 100 mL of LB medium until the absorbance (600 nm) reached 0.3, and isopropyl-β-D-thiogalactopyranoside was added and cultured for another 6 hours. After the culture, the bacteria were collected and disrupted by ultrasonic treatment. The disrupted bacteria were then dissolved in Urea lysis buffer, and various proteins were captured and purified using a nickel column, and eluted with imidazole elution buffer.

LPS was purified from Salmonella bacteria that had been statically cultured overnight at 37°C in 5 ml of LB medium using an LPS extraction kit (iNtRON).

(4) Chicken Serum Chickens (White Leghorn) were inoculated twice with Salmonella enterica serovar Typhimurium or an inactivated vaccine (heat-inactivated dead bacteria) at 1 and 3 weeks after birth, and serum was collected two weeks later.

In the Salmonella-infected group, Salmonella enterica serovar Typhimurium aroA mutant strain (1×10 ⁷ CFU/ml/200 μl PBS) was inoculated intravenously twice.

The vaccinated group was inoculated twice intravenously with Salmonella enterica serovar Typhimurium aroA mutant strain (1 x ¹⁰⁷ CFU/ml/200 µl PBS) that had been inactivated by heat treatment at 100°C for 5 minutes.

(5) ELISA method Antigen (recombinant Salmonella protein) adjusted to 0.5 μg/ml was reacted at 37° C. for 1 hour to immobilize the antigen, and then blocked at room temperature for 1 hour using Blocking One (Nacalai Tesque). Next, serum from the Salmonella-infected group and the vaccinated group were reacted with the immobilized proteins at 37° C. for 1 hour, and a secondary antibody (HRP-labeled rabbit anti-chicken IgY antibody) was reacted at 37° C. for 1 hour. Next, TMB (3,3',5,5'-tetramethylbenzidine) was added as a substrate to develop the color of the HRP label, and chicken IgY was quantified using an absorbance plate reader (450 nm).

B. Results (1) ELISA using recombinant BamA protein as an antigen in chickens ELISA was performed on chicken serum using BamA protein, a bacterial membrane protein, as an antigen. As a result, the antibody titer in serum was higher in the Salmonella spp.-infected group than in the vaccinated group (Fig. 1A). On the other hand, when SseG protein, SseF protein, LptD protein, a membrane protein secreted from the type III secretion system, and LPS derived from Salmonella enterica serovar Typhimurium x3306 were used as antigens, no significant difference in serum antibody titer was observed between the Salmonella spp.-infected group and the vaccinated group (Fig. 1B-E). From the above results, it was found that BamA protein is an antigen that can distinguish vaccinated individuals from Salmonella spp.-infected individuals.

According to the present invention, it is possible to identify individuals infected with Salmonella by distinguishing them from individuals who have been vaccinated with an inactivated vaccine. The present invention is particularly applicable in the fields of agriculture and medicine.

SEQ ID NOs: 9 to 16
<223> Primer sequence

Claims (3)

A method for detecting infection with Salmonella, comprising immunologically detecting anti-Salmonella antibodies in a sample using BamA protein or a functional fragment thereof as an antigen. An antigen reagent for immunologically detecting Salmonella infection, comprising BamA protein or a functional fragment thereof. A kit for immunologically detecting Salmonella infection, comprising the antigen reagent according to claim 2.