KR101456160B1

KR101456160B1 - Ghost Salmonella and Vaccine for Preventing Gastrointestinal diseases containing the same

Info

Publication number: KR101456160B1
Application number: KR1020120143910A
Authority: KR
Inventors: 김삼웅; 김세원; 노정만; 이존화; 갈상완; 김태완
Original assignee: 경남과학기술대학교 산학협력단; 노정만
Priority date: 2012-12-11
Filing date: 2012-12-11
Publication date: 2014-11-04
Also published as: KR20140091083A

Abstract

The present invention relates to a ghost salmonella and a vaccine for preventing bacterial digestive diseases containing the ghost salmonella. More specifically, the present invention relates to a recombinant vector for inducing ghost salmonella, Salmonella transformed with the recombinant vector, The present invention relates to a method for producing ghost salmonella and a vaccine for preventing bacterial gastrointestinal diseases containing the ghost salmonella in an effective amount. The ghost vaccine delivery system according to the present invention is eco-friendly because it does not use an antibiotic mark. Unlike conventional vaccine production processes, it is not only manufactured to maintain natural antigens, but also can express antigens of other bacteria and can be secreted into an extracellular membrane capable of continuously maintaining immunogenicity. Therefore, Development of multivalent vaccine is possible. The antigen migrated to the outer membrane is protected by the micelle function of the outer membrane and can be maintained without being decomposed for a long time.

Description

[0001] Ghost Salmonella and Vaccine for Preventing Bacterial Digestive Disease Containing the same [0002] Ghost Salmonella and Vaccine for Preventing Gastrointestinal Diseases Containing the Same [

The present invention relates to a ghost salmonella and a vaccine for preventing bacterial digestive diseases containing the ghost salmonella. More specifically, the present invention relates to a recombinant vector for inducing ghost salmonella, Salmonella transformed with the recombinant vector, The present invention relates to a method for producing ghost salmonella and a vaccine for preventing bacterial gastrointestinal diseases containing the ghost salmonella in an effective amount.

Traditional vaccines against existing pathogenic bacteria are mainly killed vaccines or subunit vaccines. However, there is a disadvantage that the existing dead mold vaccine is unable to maintain the natural epiphope or deviate from the extracellular parts due to formalin treatment or heat treatment. Therefore, when the vaccine is treated to induce an immune response, a decrease in the immunity inducing ability often occurs. In order to overcome these drawbacks, recently, a toxin-resistant vaccine has been prepared by attenuating a strong toxic strain. However, the herbal medicine poisoning vaccine has the following disadvantages. First, it attenuates pathogenic strains, but it may cause disgust because it uses live bacteria. Second, it is possible to obtain a gene that has been lost for natural herb poisoning in a natural state, and to be converted to the original strong toxic strain. These side effects can lead to serious social and economic losses.

Conventional ghost vaccines have been developed with fish as the mainstream and have used antibiotic resistance genes as selective markers in vectors carrying ghost cassettes for the manufacture of ghost vaccines. The development of antibiotic resistant strains is currently a major social and cultural issue, so it is not desirable to use antibiotic resistance vectors in the manufacture of ghost vaccines and it is time to develop ghost cassettes that do not use antibiotic resistance markers.

Since antibiotic additions to all animal feeds have been banned by 2011, urgent measures against explosive increases in bacterial diseases are required. Until now, antibiotics have been added to the feed to prevent pig diseases, especially those caused by pathogenic bacteria, but they must now prevent pig disease without the use of antibiotics. Globally, the addition of antibiotics to livestock feeds is either decreasing or prohibiting the use of preventive vaccines to prevent disease. To this end, it is absolutely necessary to develop a preventive vaccine that has excellent defense effects by applying cutting-edge genetic engineering by biotechnology, which is a recent issue.

Most of the causes of mortality and growth decline in the mammalian period are due to digestive diseases caused by pathogenic Escherichia coli and salmonella. These pathogens first come into contact with the host animal through the gastrointestinal mucosa and cause disease through the mucosa. Escherichia coli induces diarrhea in the mucosa, and in the case of Salmonella, not only the mucosa but also the mucosal cells may enter the cell after adhering to the mucosal cells, which may lead to systemic infection. Because of these properties Salmonella is selected as an external antigen carrier and can induce mucosal immunity as well as mucosal immunity against variously transported antigens.

In the existing pigs, the vaccine for preventing pathogenic E. coli was prepared by inactivating pathogenic Escherichia coli by formalin treatment or heat, and then by inoculating the sows with the immune enhancer to induce the systemic immune response and to prevent the piglets. However, since formalin or heat treatment results in the elimination or alteration of extracellular proteins or other substances, which act as a major factor in the formation of immunity, the ability to induce an immune response is lowered and this phenomenon lowers the efficacy of the overall vaccine It causes. In addition, the immune response due to muscle inoculation sometimes fails to induce mucosal immunity and thus has a great difficulty in effectively preventing colicosis.

Because Salmonella is an invasive bacterium, live vaccines that can stimulate both mucosal immunity as well as mucosal immunity are needed. There is no vaccination against Salmonella spp. Using poisoned Salmonella in pigs domestically. Therefore, in order to effectively prevent these gastrointestinal disease-causing pathogens, an environmentally-friendly vaccine capable of inducing an effective mucosal immune response and cellular immunity is needed.

Korean Patent No. 10-0765357 US registered patent US 6,610,529

Under these technical backgrounds, the present inventors have made intensive efforts to complete the present invention.

It is an object of the present invention to provide a recombinant vector for inducing ghost salmonella.

Another object of the present invention is to provide Salmonella transformed with said recombinant vector.

It is still another object of the present invention to provide a method for producing ghost salmonella, which is obtained by culturing the salmonella under specific conditions.

It is still another object of the present invention to provide a vaccine for preventing bacterial digestive diseases containing an effective amount of the ghost salmonella.

According to one aspect of the present invention, a recombinant vector comprising the following genes can be provided.

1) lysis E gene;

2) a? PR promoter / cI857 repressor linked upstream of the lysis E gene;

3) the ParaBAD promoter and the araC gene, which are ligated downstream of the lysis E gene to produce the antisense RNA of the lysis E gene; And

4) asd (aspartate semialdehyde dehydrogenase) gene expressed independently of the above 1) to 3).

According to one embodiment, a recombinant vector may be provided which further comprises an MCS (multi cloning site) linked to a transmembrane domain (TMD) so as to label an external antigen on the outside of the cells transformed with the recombinant vector.

According to an aspect of the invention, asd deletion (asd ^-) transformed with the recombinant vector can be provided with a recombinant microorganism.

According to an aspect of the invention, transformed with the recombinant vector asd deletion (asd ^-) it may be provided with salmonella (Salmonella).

According to an aspect of the present invention, there can be provided a method for producing ghost salmonella comprising the following steps.

(a) culturing the Salmonella in a medium containing arabinose at 25 to 30 ° C;

(b) further culturing at a temperature of 40 to 43 DEG C in a medium containing no arabinose when the cell density (OD600) of the culture solution reaches 0.2 to 0.4; And

(c) obtaining the recombinant Salmonella typhimurium when the reduction in cell density of the culture solution is stopped.

According to one aspect of the present invention, a ghost salmonella produced by the above method and containing lysis E gene can be provided.

According to an aspect of the present invention, a vaccine for preventing bacterial digestive diseases containing an effective amount of the ghost salmonella can be provided.

The ghost vaccine delivery system according to the present invention is eco-friendly because it does not use an antibiotic mark. Unlike conventional vaccine production processes, it is not only manufactured to maintain natural antigens, but also can express antigens of other bacteria and can be secreted into an extracellular membrane capable of continuously maintaining immunogenicity. Therefore, Development of multivalent vaccine is possible. The antigen migrated to the outer membrane is protected by the micelle function of the outer membrane and can be maintained without being decomposed for a long time.

Figure 1 is a schematic representation of the gene map of the ghost vector pMMP99-1 that retains the antibiotic mark.
Figures 2 to 4 illustrate the use of Ghost Vector 99-1 for the isolation of aspartate semialdehyde dehydrogenase (asd) gene from Escherichia coli (Fig. 2), Salmonella typhimurium (Salmonella typhimurium) (Fig. 3), and S. typhimurium (Fig. 4) lacking 3 genes of lon cpxR asd at 42 < 0 > C.
FIG. 5 shows results of in vitro challenge with oral salmonella after inoculation with oral and muscular with MMP13 [pMMP99-1].
Figure 6 is a schematic representation of a vector retaining a double-modulated ghost cassette comprising a non-antibiotic mark asd gene. A has a pBR replication initiation that maintains a high copy number, and B has a p15A replication initiation point that maintains an intermediate copy number.
FIG. 7 is a photograph of a Salmonella ghost cell taken by a transmission electron microscope.
Fig. 8 shows the number of viable cell counts according to the presence or absence of arabinose at 42 DEG C for MMP13 [pMMP184].
9 to 13 show total IgG (FIG. 9), IgG1 (FIG. 10), and IgG2a (FIG. 11) in the serum after immunization with MMP13 [pMMP184] , Fecal secretory IgA (Fig. 12) and vaginal secretory IgA (Fig. 13).

Hereinafter, the present invention will be described in more detail.

The ultimate goal of the present invention is to develop a multivalent vaccine using a genetic modification-based delivery system capable of effectively inducing mucosal immunity and cellular immunity and a vector having improved ability to secrete foreign antigens, To prevent disease.

1) lysis E gene;

2) a? PR promoter / cI857 repressor linked upstream of the lysis E gene;

In the preparation of the recombinant vector, a bacteriophage λPR promoter / cI repressor capable of inducing gene expression at a specific temperature condition was used as a regulatory region, and a lysis E of phiX174 phage to induce micropores in bacterial walls Genes were used.

A variety of origins can be used to control the copy number of the recombinant vector. According to one embodiment of the present invention, pBR ori (pMMP184) and p15A ori (pMMP187) were used, but other origins may be used depending on the purpose of use.

The vector of the present invention can produce a multivalent vaccine by further adding an external antigen in addition to the above essential elements.

In the following example, in addition to the lysis E gene regulatory system as described above, plasmid pMMP184 and pMMP187 were inserted into the P _R promoter with a portion corresponding to 8 TMD (transmembrane domain) region of E. coli ompA (outermembrane protein A) Respectively.

E. coli ompA consists of 1041 nts and 346 amino acids, of which 8 TMDs are retained in the cytoplasm where 1 to 540 nts of N-terminal (amino acids 1-180) there is a loop. The His ₆ epitope is attached to the ompA gene TMD in frame and the MCS (multi-cloning site) at the 3-terminal side of His ₆ is designed to clone the external antigen. His ₆ is readily detectable using a commercially available His-antibody. A more detailed schematic is shown in FIG.

According to an aspect of the invention, transformed asd deletion (asd ^-) with the recombinant vector, and Salmonella could be provided, according to one embodiment, the Salmonella can be a S. typhimurium (Salmonella typhimurium).

(a) culturing the Salmonella in a medium containing arabinose at 25 to 30 ° C;

(c) obtaining the Salmonella when the reduction of the cell density of the culture medium is stopped.

When the Salmonella is cultivated at a temperature of from 25 to 30 ° C in a culture medium containing arabinose at a temperature of from 180 to 220 rpm, the expression of lysis E gene can be strictly controlled through the simultaneous action of cI857 repressor and antisense. When the cultured cells reach stasis, they are transferred to a medium containing arabinose for expression of the lysis E gene and cultured at a temperature of 40 to 43 ° C to express the lysis E gene.

At this time, if the initial culture temperature condition is out of the above range, there is a problem that the efficiency of formation of a fine tunnel structure is deteriorated. If the temperature is elevated beyond the above range, the bacteria will grow over the temperature range of bacteria. The lysis E gene is induced by the temperature stimulation at 40 ~ 43 ° C to induce a fine tunnel structure of the cell wall, thereby ghosting the cytoplasm. After the temperature stimulation, the cell density was measured and collected when the decrease in cell density no longer occurred, followed by lyophilization and storage in the refrigerator.

Examples of bacterial digestive diseases include diarrhea, vomiting, and enteritis. And can be classified into Escherichia coli and Salmonella strains depending on the type of the causative bacteria.

Hereinafter, the present invention will be described in more detail with reference to Examples. It should be understood, however, that these examples are for illustrative purposes only and are not to be construed as limiting the scope of the present invention.

1. Containing an antibiotic mark ghost Production of cassettes

The ghost cassette received from Professor Kim Ki-hong (Department of Fish Pathology, Pukyong National University) for vaccination using salmonella ghost in livestock was pHCE :: GAPDH SDM37 which was additionally mutated in cI857 and was named pMMP98-1. More specifically, the plasmid pMMP98-1 was mutated to cI857 via site-directed mutagenesis (SDM). That is, the sequence of OR2 for cI binding is 5'-TAACACCGCG C GTGTTG-3 ', in which C corresponding to the 11th (underlined region) is changed to T. This mutation increases the binding of cI857, and it is possible to inhibit the expression of the lysis E gene up to 37 ° C., which is a relatively high temperature at a temperature of 30 ° C. or higher. In addition, the cI857 :: P _R and lysis E genes are fused at the transcription level. To analyze the ghosting, only the ghost cassette was amplified by PCR in a T-vector (promega), and inserted into it, and named pMMP99-1. This is illustrated schematically in FIG.

When the E. coli cells carrying the plasmid pMMP99-1 were cultured at 37 ° C to A600 0.35 and then moved to 42 ° C, the OD value was increased to about 0.15 until about 1 hour, and then 0.25 (Fig. 2). Ghost screen initially ^{10 9 CFU (colony forming unit)} / ml which was the living cell is 42 ℃ movement 210 ⁴ decrease in CFU / ml in less than hour, and then with 10 ⁴ CFU / ml to the culture for 3 hours, down to up, 4 From the time point of view, it can be seen that it is maintained at about 10 ⁴ CFU / ml. The non-ghosting mutation frequency was very low and remained stable below about 10 ⁴ CFU / ml. This result is consistent with the previously reported literature.

pMMP99-1 was transformed into E. coli MMP13 and CK110 in order to observe the ghost frequency in asd-deficient strains. Since these strains are DAP (diaminopimellic acid) auxotrophic strains, they supplemented them with medium. As shown in FIG. 3, in the case of MMP13, the increase and decrease of the OD value were found to occur gently, unlike the case of the common E. coli strain. In addition, it took 48 hours for the ghost to reach 10 ³ CFU / ml, which was clearly different from the general aspect. In particular, in the case of CK110, the ghost frequency is very high, about ¹⁰⁶ CFU / ml after 48 hours.

MMP13 is an asd-deficient strain, and CK110 is a lon cpxR asdA16-deficient strain. Asd - deficient strains were observed to be delayed by 1.5 to 2 times compared to wild - type strains. This growth delay is presumed to be caused by the failure of the strain to become a complete state even if DAP is provided from the outside, and it is presumed that such a phenomenon causes a non-specific phenomenon in ghosting.

In CK110, cpxR deficiency induces abnormal expression of fimbriae, and lon deficiency leads to abnormal cell division and induction of capsule formation and various abnormalities. The abnormal physiological characteristics of this strain are considered to cause the strain to be exposed to highly stress inducing conditions. The cassette of lysis E gene does not completely block the expression of lysis E gene due to temperature, induces the expression of leaky lysis E protein in a small amount, and induces the death of many strains at an early stage . This phenomenon can be inferred to be due to abnormal transfection efficiency and mutation of the lysis E gene in the transformed strain. This condition is presumed to provide a potent environment for mutating CK110, and as a result, it is estimated that ghosting does not progress to below 10 ⁶ CFU / ml as shown in Fig.

2. Wild Salmonella Challenge infection

In order to observe whether ghost vaccine is effective against virulent Salmonella, the following experiment was conducted. First, the ghost vaccine was administered once or more by mouth or muscle to BALB / c mice. At 2 weeks after the inoculation, S. typhimurium χ 3339 was diluted to 2 × 10 ⁷ CFU / ml and then inoculated 20 μl each. Mice were observed twice daily for 30 days after oral challenge with challenge.

As a result, as shown in Fig. 5, mice in the control group died from 4-5 days after administration of salmonella and died after 80 days after 20 days. The protective effects of wild salmonella on the number of administration and route of administration were as follows.

A single oral dose of the vaccine showed a 25% protection effect, while the oral effect increased to 58.4% when the oral dose was increased by two. In the case of administration of intramuscular ghost vaccine, a protection effect of 50% in one administration and 37.5% in two administrations was observed. The Salmonella ghost vaccine has a protective effect of around 50% depending on the number of administrations and Lute.

3. Double adjustment Cost Construction of cassettes

If intracellular expression occurs in the unregulated state of the lysis E gene, the cell into which the gene is inserted will die or mutate. When the lysis E gene is expressed at a proper time while preventing such a phenomenon, it is considered that ghosting can be induced well.

Under such technical background, cI857 and antisense RNA were introduced to strictly control the expression of the lysis E gene. cI857 was introduced to allow regulation by temperature. Antisense RNA was induced to be induced by the ara system. This concept is designed to inhibit the expression of the lysis E gene by expression of antisense when arabinose is present, as opposed to the conditional lethal system by araC P _araBAD :: asd.

The framework of the pMMP184 plasmid containing the tightly regulated lysis E gene is derived from pYA3342 and contains pBR ori, MCS and asd The gene is derived from pYA3342 (Fig. 6A). In order to lower the copy number of plasmid, pMMP187 was constructed by pYA3332 which is a p15A ori origin (Fig. 6B).

The ghost cassette consists of the cI857-P _R :: E gene, and cI857 acts as a temperature-dependent inhibitor of the P _R promoter. E gene fused with cI857-P _R to be at the translation level. This is most likely to be regulated by cI857, as regulation may be different in some cases if linked at the transcription level. In order to analyze the function of the ara system inducing the production of antisense RNA depending on the temperature-dependent control and the presence of arabinose, the MMP13 strain transformed with pMMP184 was cultured to a certain point (the cell density (OD600) reached 0.4) After that, the temperature was raised to 42 캜 and cultivation was carried out in the presence and absence of arabinose. As a result, it was observed that the ghost formation was completed within 36 hours when the temperature was raised and arabinose was absent (FIGS. 7 and 8). It shows that the newly constructed system is making ghost construction well.

In addition to the ghosting function of the plasmids pMMP184 and pMMP187, 8 TMD (transmembrane domain) regions of E. coli ompA (outermembrane protein A) were cloned into the P _R promoter for use as an external antigen delivery system. E. coli ompA was composed of 1041 nts and 346 amino acids, of which 8 TMDs were retained and 1 to 540 nts N-terminal (amino acids 1-180) region was cloned. The N-terminal and C-terminal of this gene are present in the cytoplasm and there are four loops outside the cell. The His ₆ epitope is attached to the ompA gene TMD in frame and the MCS (multi-cloning site) at the 3-terminal side of His ₆ is designed to clone the external antigen. His ₆ is readily detectable using a commercially available His-antibody. A more detailed schematic diagram is shown in Fig.

The constructed pMMP184 and 187 consist of an environmentally friendly vector system that does not use the antibiotic mark. In order to use as a marker for plasmid selection, asd (aspartate semialdehyde dehydrogenase) is deleted in Salmonella strains to construct auxotrophs and the conditional autotroph system ( asd gene on plasmid) .

4. ELISA ( Enzyme - linked immunosorbent assay ) To induce animal immunity

Serum, vaginal washing samples, and fecal samples were collected at 2, 4, 6, 8, and 10 weeks before inoculation and after inoculation. Serum was collected from the orbital vein and centrifuged at 4,000g for 5 minutes. Serum was separated from the supernatant and stored at -20 ° C. The fecal secretion sample was weighed, suspended in PBS to 100 mg / ml, centrifuged for 10 minutes, and the supernatant was separated and stored at -20 ° C.

Salmonella was detected in serum, fecal secretion samples, vaginal secretion samples (2, 4, 6, 8, 10 weeks) ELISA was performed to measure antibodies specific for LPS. In the sample wells, salmonella LPS was added at a concentration of 200ng / well, and goat anti-mouse IgG / IgG1 / IgG2a or rabbit anti-mouse IgA was added at 200ng / well And then coated at 4 ° C overnight. The coated plates were washed 3 times with PBS and then treated with blocking buffer (1% skim milk in PBS).

The serum was diluted 1: 100 with PBS, the vaginal secretion sample and the fecal secretion sample were diluted 1: 2, and then 100 μl was added to each well. The mixture was reacted at 37 ° C for 2 to 4 hours. For peroxidase-conjugated goat anti-mouse IgG, IgG1, IgG2a HRP and IgA levels in serum, peroxidase-conjugated goat anti-mouse IgA HRP was added at a ratio of 1: 2,000 Diluted to a multiplication factor, and 100 μl was dispensed into each well, followed by reaction at 37 ° C for 2 hours. After the PBS solution was completely removed, the reaction was carried out for 10 to 90 minutes in a solution containing the reaction substrate 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS). After the reaction was stopped with 0.1% SDS, the expression level was observed with an ELISA reader at a wavelength of 405 nm.

As a result, as shown in Figs. 9, 10 and 11, it was confirmed that the expression of IgG, IgG1, and IgG2a in the serum significantly increased from the fourth week after administration of ghost vaccine as compared with the control. In addition, as shown in Fig. 12, the expression of sIgA in fecal secretion samples tends to increase slightly overall, but it is not judged to be reliable because the values of 0 and 2 are high.

Expression of sIgA in the vaginal secretion sample showed an increase in expression at 4 weeks and a tendency to gradually decrease after reaching the maximum value (Fig. 13). The inability to detect IgA in the feces is presumed to be due to the rapid degradation of IgA by many proteases in the feces.

When Salmonella Typhimurium, a state-of-the-art genetic engineering technique of synthetic biology, is orally infected with a delivery system, it is possible that not only Salmonella itself but also pathogenic Escherichia coli antigens, due to the characteristics of Salmonella, It can induce not only immune but also cellular immunity.

Existing inoculum vaccines are inactivated by formalin treatment or heat, but the ghost vaccine delivery system according to the present invention is eco-friendly because it does not use an antibiotic mark. Unlike conventional vaccine production processes, it is not only manufactured to maintain natural antigens, but also can express antigens of other bacteria and can be secreted into an extracellular membrane capable of continuously maintaining immunogenicity. Therefore, Development of multivalent vaccine is possible. The antigen migrated to the outer membrane is protected by the micelle function of the outer membrane and can be maintained without being decomposed for a long time.

In addition, since the ghost microbial vaccine delivery system can induce mucosal immunity by oral infection and induce humoral immune response by muscle inoculation, when the live microbial system and the germ system are all used, It is possible to develop a multivalent vaccine that can more effectively prevent pathogenic coliform and salmonellosis. In addition to the delivery system, the efficacy of the multivalent vaccine is determined by the high expression of the foreign antigen and its ability to induce the cell membrane or extracellular space.

The polyvalent vaccine developed as described above can be manufactured in the form of a powder by lyophilization immediately after culturing, which makes it easy to manufacture and store. The ghost vaccine is simple and economical to manufacture because it can increase production as needed with culture and ghost induction technology. In addition, the development of multivalent vaccines with excellent defense effects and the practical use in the field can enhance the export potential to foreign countries in addition to the effect of imported vaccines.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims

A recombinant vector comprising the following genes.
1) lysis E gene;
2) a? PR promoter / cI857 repressor linked upstream of the lysis E gene;
3) the ParaBAD promoter and the araC gene, which are ligated downstream of the lysis E gene to produce the antisense RNA of the lysis E gene; And
4) asd (aspartate semialdehyde dehydrogenase) gene expressed independently of the above 1) to 3).

2. The recombinant vector according to claim 1, further comprising a MCS (multi cloning site) linked to a TMD (transmembrane domain) so as to mark an external antigen on the outside of the cell transformed with the recombinant vector.

Asd deletion (asd ^- ) microorganism transformed with the recombinant vector of claim 1 or 2.

Claim 1 or claim 2 of the asd deletion transformed with a recombinant vector (asd ^-) Salmonella (Salmonella).

A method for producing ghost salmonella comprising the steps of:
(a) culturing the salmonella of claim 4 in a culture medium containing arabinose at 25 to 30 ° C;
(b) further culturing at a temperature of 40 to 43 DEG C in a medium containing no arabinose when the cell density (OD600) of the culture solution reaches 0.2 to 0.4; And
(c) obtaining the Salmonella when the reduction of the cell density of the culture medium is stopped.

6. A ghost salmonella, produced by the method of claim 5, containing the lysis E gene.

A vaccine for the prevention of bacterial digestive diseases comprising an effective amount of the ghost salmonella of claim 6.