JP2001342147A - Method for preventing johne's disease infection by m-cell uptake control and vaccine therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for preventing newborn animals, in particular calves from Johne's disease (ruminant paratuberculosis) infection. SOLUTION: This method for preventing Johne's disease infection comprises the following practice: Mycobacterium avium sp. paratuberculosis is heated into bacillus mort, which, in turn, is orally admininistered to newborn animals to induce the M-cell uptake control of the intestinal mucosa as a specific invasive route for Mycobacterium avium sp. paratuberculosis and thereby inhibit the subsequent live Mycobacterium avium sp. paratuberculosis invasion. Thus, by isolating calves from their dam immediately after birth and administering the calves with a vaccine as the above bacillus mort mixed with sterile colostrum, even in case not only dam's milk-derived Mycobacterium avium sp. paratuberculosis but also environment-derived Mycobacterium avium sp. paratuberculosis or the like is orally inoculated on the calves, the Mycobacterium avium sp. paratuberculosis can be prevented from invasion into the tissue (establishment of infection) of the calves by inhibiting the Mycobacterium avium sp. paratuberculosis from invasion via M-cells.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for preventing neonatal animal infection, particularly calf calf infection, and a vaccine for preventing infection with Johne's disease.

[0002]

2. Description of the Related Art Johne's disease is a chronic granulomatous diarrheal infectious disease caused by Mycobacterium tuberculosis, a kind of acid-fast bacterium. Regarding Johne's disease contamination in cattle herds in Japan, since 1980,
Both the number and location of the outbreaks are increasing. That is, there have been 100-200 outbreaks per year since the 1990s, and in 1999 over 800 cases.

[0003] Johne's disease is established when a calf is orally infected with Mycobacterium tuberculosis at an early stage after birth, but the process from infection to onset of Johne's disease is still largely unknown, and it is not possible at the individual level. Differences in the course of infection are unprecedented in other diseases. In the case of this disease, which can be said to be a super-chronic infectious disease, the disease is orally transmitted to a calf, and thereafter, the disease generally does not develop for an average of 3 to 6 years. In some cases, the disease does not develop for more than 10 years, and in extreme cases, it does not develop for life. In other words, cattle infected with S. tuberculosis include subclinically infected cattle and cattle that have developed the disease. There are carrier cows whose reaction and cellular immunity are difficult to detect.

[0004] Subclinically infected cattle (carrier cattle) in which these clinical symptoms and antibodies are not detected are subjected to immunological diagnosis (ELISA).
This is an infected cow that cannot be diagnosed under the law. Although the carrier cow has a specific antibody level below the diagnostic limit, it has lesions and irregular tooth removal, which is extremely problematic in epidemics. In particular, since the ELISA method is used worldwide as a standard for immunological diagnosis,
It can be said that the number of carrier cattle that cannot be detected by the ELISA method is relatively increasing as a result of the fact that the detection by the method A progresses and that the cows with low infection levels remain.

[0005] Irregular evacuation of this carrier cow is particularly important for the spread of infection, and isolating J. bacillus from feces is the only reliable diagnostic method possible at this time. However, diagnosis using this bacterial isolation also has a problem that several months must be spent for culturing the amount of bacteria required for the diagnosis.

[0006] In general, S. tuberculosis belongs to the atypical mycobacterium group III and is a auxotrophic bacterium that usually grows only on a medium supplemented with mycobactin. It is a very slow-growing bacterium that can take as long as 7-11 weeks for visible colonization, which slows the progress of diagnostics by bacterial isolation. Recently, a specific insertion sequence possessed by the DNA of Mycobacterium tuberculosis
In order to detect IS900, it has become possible to perform early identification by polymerase chain reaction (PCR) .However, the detection of M. paratuberculosis from feces is more sensitive than bacterial isolation due to the presence of a PCR inhibitor. May not be obtained.

[0007] Diagnosis of Johne's disease includes methods of detecting a host's cellular immune response to Y. tuberculosis in addition to bacterial isolation and antibody detection. An intradermal reaction, similar to the tuberculin reaction in tuberculosis infection, is used as the "yonin response", but a cellular immune response occurs only relatively early in the infection,
Due to the course of the specific cellular immune response in Mycobacterium tuberculosis infection, which subsequently declines and becomes undetectable, its application is limited relatively early after infection. Cellular immunity detection kits that utilize the phenomenon that T lymphocytes recognizing M. paratuberculosis antigens produce interferon gamma when they react and activate upon re-exposure to antigens have also been sold overseas. , Is currently discontinued.

Hitherto, diagnosis has been made by isolation of bacteria, detection of specific antibodies, detection of cell-mediated immunity, and histopathological diagnosis. Up to now, it has been difficult to adequately prevent and clean the disease. Thus, the mechanism of Johne's disease
The economic damage is extremely large and serious because there are many infected animals that are complicated and unclear, and cannot be diagnosed by bacterial isolation or immunological method. However, studies on pathology, bacteria, and immunology to prevent the spread of this disease are certainly progressing steadily, but at present, the presence or absence of Y. bacillus in feces is regularly checked. It can be said that the current state of diagnosis for Johne's disease is to examine or catch the spontaneous rise of antibodies in infected cattle and make an immunological diagnosis.

Regarding the process from infection to onset of Johne's disease, matters that have been clarified so far will be described below. Johne's disease does not cause bacterial proliferation or pathological damage to the host merely by the presence of the yeast in the intestinal lumen. Johnet teeth do not have the motility and cell invasiveness of Salmonella, Shigella, certain Escherichia coli, and Treponema, and it has long been unknown how to break through the intestinal mucosal barrier. Since the intestinal absorptive epithelial cells of newborn calves actively take up macromolecules such as colostrum antibodies by pinocytosis (drinking action), it is speculated that Y. bacilli swallowed with colostrum and milk will be taken up as well. It was later revealed that this was not the case (Chiodini RJ, Van Kruiningen HJ, Merkal R
S, Ruminant paratuberculosis (Johne's disease): th
e current status andfuture prospects.Cornell Vet.
74 (3): 218-62 (1984).

In an experiment using a calf ileum loop, the present inventors passively enter the epithelium due to the phagocytosis of extraordinary M cells that infect the intestinal Peyer's patch dome in the intestinal cavity. And is further taken up by intraepithelial and subepithelial macrophages (M
(φ) revealed by light microscopy and electron microscopy that it was further transported to the target part of the lymphatic device [Eikichi Hyakukei, J. Veterinary Society, 42: 229-237 (1992)]. In this experimental infection, M
Uptake of Mycobacterium tuberculosis by villus-absorbing epithelial cells other than cells, undifferentiated crypt epithelial cells, and embryonic cells was not observed. Furthermore, the present inventors have already shown that an antibody against M. paratuberculosis suggests the possibility of promoting the invasion of M. paratuberculosis into Peyer's patches. When a calf is infected with Y. bacillus, the bacteria excreted in the feces of the mother cow attach to the body surface and environment, such as the udder, and the calf is infected orally. 27% of the bacteria and 11.6% of the bacterium are detected in milk, so that in the infected area, bacilli are present not only in feces but also in milk and colostrum, and feces and milk are also important sources of infection. [Sweeney
R, Whitlock R, and Rosenberger AE, Journal of Clin
ical Microbiology 30: 166-171 (1992)].

The calf small intestinal mucosal lymph device accounts for 86% of the total small intestine tissue, one third of which is in the upper small intestine and the remaining 2 /
No. 3 is distributed in the ileum, and the dome epithelium of the ileal Peyer's patch consists of uniform constituent cells consisting of only M cells, while the dome of the lymphatic device distributed in the upper part of the small intestine is composed of M cells and normal absorptive epithelial cells. It is known that they are covered in a mixed state (Liebler E, Inaugural-Dessertation zur Er
largung des Grades eines Doctor Medicinae dir Tier
arzliche Hochshule Hannover (1985)]. Therefore,
The light microscopic / electron microscopic observations of the present inventors show that the invasion gate (M cells) of M. paratuberculosis is prepared more in the ileum, and the initial infection lesion of Johne's disease is located in the lower ileum. It can be said that it explains the reason why it is easily formed.

M cells do not take up intestinal bacteria present in the intestinal lumen indefinitely and indiscriminately, but it is said that they have a selective uptake mechanism. In Vibrio cholerae, only viable bacteria are derived from rabbits. It is said that it is taken up by intestinal M cells, but in the case of Bacillus subtilis, both live and dead bacteria are taken up, and the selection mechanism is not simple [Eikichi Hyakkei, Pathogenesis and prevention of Johne's disease Journal of the Japan Veterinary Medical Association, 42: 229-237 (1992)]. The intestine is a tissue that is required to take up many types of dietary antigens as nutrients, but there are no dangerous dietary antigens and pathogens that do not cause unnecessary or excessive immune reactions (such as food allergies). It has a mechanism for distinguishing bacterium antigens, including T
Lymphocytes are involved.

M cells, depending on macromolecules and types such as peroxidase, have been proven to take up bacteria. It is also known that Chlamydia sp and several kinds of viruses infect M cells, and the significance as a specific infection route has been elucidated as well as the biological defense function of antigen sampling and transport in the intestinal lumen. I have. However, to date, only a small portion has been revealed about the functions and roles of M cells [Ow
en RL, Uptake and transport of intestinal macromo
lecules and microorganisms by M cells in Peyer's p
atches--a personal and historical perspective.Semi
n Immunol 11: 157-63 (1999)]. The present inventors have already shown that if this function is elucidated, it will be possible to prevent infection in the form of inhibiting the uptake of M. paratuberculosis by M cells [Eikichi Hyakkei, pathogenesis of Johne's disease] And Japanese Journal of Veterinary Medicine 42: 229-237 (1992)].

[0014] By the way, during the incubation period in which the disease does not develop for an average of 3 to 6 years after the infection, M. paratuberculosis is present in the macrophages (Mφ) of the intestinal mucosal mesenteric lymph nodes in a quiescent state. The lesion of the experimentally infected calf tissue considered to be in an infected state is composed of a very small number of Mφ-type cells, shows only a very small number of bacteria, and often cannot prove the bacteria on the tissue section. In this incubation period, the trigger from the lesion to the onset period is that, in advance of diarrhea symptoms and a large amount of tooth removal (100 to 1 million per 1 g of stool), the proliferation of bacteria within the quiescent lesion, It is thought that the release of bacteria and fungal antigens out of the cells occurs accordingly.

As a result, Mφ aggregates and colonizes the lesion, and granuloma is formed and expanded, and at the same time, the humoral immune response is increased. Regarding the mechanism that enhances the growth of M. paratuberculosis latent in Mφ, the fact that M. paratuberculosis is mycobactin-dependent and has a very weak and special ability to acquire iron essential for growth and metabolism. The relationship between iron and iron-binding protein in tissue cells and bacterial growth is important because it depends on the presence or absence and iron ion concentration (Momotani E, Immunohistoc
chemical distribution ofyferritin, lactoferrin, and
transferrin ingranulomas of bovine paratuberculos
Infect Immun. 52: 623-627 (1986) .; Eiichi Hyakkei, Study on the Pathogenesis of Johne's Disease, Research Report on Livestock Hygiene Laboratory, Ministry of Agriculture, Forestry and Fisheries, 96: 275-280 (1991)].

The mechanism that enhances the growth of the latent Mycobacterium tuberculosis is presumed as follows. When acute infection or experimental inoculation of LPS or Mycobacterium tuberculosis cell wall results in hypoironemia, a non-specific host defense reaction to prevent sepsis, serum iron bound to transferrin (Tf) is then converted to lactoferrin ( Lf) intervenes and moves into Mφ, the iron concentration in Mφ increases, and the growth of M. paratuberculosis is promoted. It is an ironic phenomenon that when the host hid in the Mφ from the blood so that the iron which is indispensable for bacterial growth is not used by the bacterium, the fungus was waiting in the hiding place.

This means that (i). The two types of iron chelates (liposoluble mycobactin and water-soluble exochelin) produced by acid-fast bacilli are compatible with the host iron-binding protein ｛ferritin (F).
t) demonstrating in vitro that it deprives Tf and Lf 鉄 of iron and supplies it to the fungus; (ii). Immunohistologically that these iron-binding proteins are localized in Johne's disease granuloma, (iii). It has been proved by a radiometric method that Mycobacterium tuberculosis grows using iron released from Tf and Lf in a low pH state where mycopactin does not exist. (Iv). Epithelioid cells constituting granuloma Is activated and differentiated Mφ, and it is known that the activated iron uptake of activated M is known to be significantly higher than that of Mφ (Eikichi Hyakukei, Pathogenesis mechanism of Johne's disease) Research, Ministry of Agriculture, Forestry and Fisheries Livestock Hygiene Laboratory
96: 275-280 (1991).

As described above, although the pathogenesis mechanism from the infection to the onset of Johne's disease has been considerably clarified, while Johne bacillus is present in the mesenteric lymph node Mφ in a quiescent state, Johne's disease occurs. Therapeutic methods for eliminating bacteria have not yet reached the level of practical use. In Japan, Johne's disease is designated as a domestic animal statutory infectious disease, so treatment with antibiotics is not performed. Although experimental reports have been reported, recurrence occurs after treatment has stopped, and antibiotic transfer into meat and milk is not practical (St-Jean G, Jernig
an AD, Treatment of Mycobacterium paratuberculosi
s infection in ruminants.Vet Clin North AmFood An
im Pract., 7: 793-804, (1991)). In addition, no definitive technology to prevent this infection has been developed so far, simply isolating newborn animals from the shedding cow and its contaminated environment,
It was only a matter of taking common preventive measures against infectious diseases, such as disinfecting the contaminated environment.

Conventionally, as a specific countermeasure against Johne's disease, there is a report that vaccination in which killed Johnella bacillus is killed subcutaneously with oil ashpand has been effective (van Schaik G, Kalis CH, Benedictus
G, Dijkhuizen AA, HuirneRB, Cost-benefit analysis
of vaccination against paratuberculosis in dairy
cattle.Vet Rec 139: 624-7 (1996), Molina JM, Anguia
no A, Ferrer O, Study on immune response of goats
vaccinated with a live strain of Mycobacterium pa
ratuberculosis.Comp Immunol Microbiol Infect Dis
19: 9-15 (1996)). However, although these methods enhance cellular immunity after infection, they have no protective effect on infection and only reduce the incidence. As a result, subclinical infected cows that continue to shed bacteria from the feces are generated, and there is a problem that not only the fundamental problem cannot be solved but also the contamination is expanded.

An oral vaccine against Johne's disease has been tried only once in the history of Johne's disease research, and a live bacterium of a non-pathogenic Johne's strain is orally administered to an adult sheep and subsequently infected with Johne bacillus. I have. It was reported that this oral vaccine did not provide protection against infection (Gilmou
r NL, Absense of immunogenicity of an oral vaccine
against Mycobacterium paratuberculosis in sheep,
J Comp Pathol 83: 437-445 (1973). In this experiment, a non-pathogenic, mycobactin-independent Yone strain was identified.31
The 6F strain was used, but subsequent reports have reported that the 316F strain is a different strain from M. paratuberculosis [Thor
el MF, Krichevsky M, Levy-Frebault VV, Numericalta
xonomy of mycobactin-dependent mycobacteria, emend
ed description of Mycobacterium avium, and descrip
tion of Mycobacterium avium subsp.
v., Mycobacterium avium subsp.paratuberculosis su
bsp.nov., and Mycobacterium avium subsp.silvatic
um subsp.nov.Int J Syst Bacteriol40: 254-60 (199
0); Ohene-Gyan KA, Haagsma J, Davies MJ, Hounsel
l EF, Novel glycolipids of Mycobacterium avium and
related M. paratuberculosis strains of relevance
to AIDS an Crohn's disease.Comp Immunol Microbio
lInfect Dis 18: 161-70 (1995)].

Patents relating to immunity involving M cells include, for example, stimulating IgA production sites such as host Peyer's patch cells, promoting IgA production activity, increasing the amount of IgA production, and preventing infection and allergic reactions. For preventing oral infections by orally administering protoplasts or cytoplasmic membranes of Bifidobacterium (Japanese Patent Application Laid-Open No. 4-34253).
No. 3), liposomes, emulsions, and the like in order to efficiently transport drugs to the Peyer's patch in the digestive tract after oral administration.
Alternatively, a technique of including a specific substance such as phosphatidylserine, a mannose derivative, or a mannan derivative in a membrane component of a lipid-spreading structure for oral administration such as a water-soluble micelle (Japanese Patent Laid-Open No. 5-17344). In order to efficiently transfer an active substance, a technique for orally administering a preparation containing a liposome containing phosphatidylcholine, cholesterol and phosphatidylinositol as a lipid component and a physiologically active substance encapsulated in the liposome (Japanese Patent No. 2814307) No.) etc.
The fact that specific antibodies are completely ineffective at protecting Johne's disease from infection does not particularly suggest prevention or treatment of Johne's disease.

Further, although the causal relationship has not been clarified, the possibility of the involvement of S. tuberculosis as a cause of human Crohn's disease (an intractable disease specified by the Ministry of Health and Welfare) has been increasing in recent years. From the standpoint of the government, drastic measures against Johne's disease are urgently needed, and there is a need for early Johne's disease prevention methods and means for cleaning (Coll)
ins MT, Mycobacterium paratuberculosis: a potentia
l food-borne pathogen? J Dairy Sci 80: 3445-8 (199
7); Engstrand L, Mycobacterium paratuberculosis a
nd Crohn's disease.Scand J Infect Dis Suppl 98:27
-9, 1995)).

[0023]

SUMMARY OF THE INVENTION The present invention has been made based on the background concerning Johne's disease described above. That is, an object of the present invention is to provide a method for preventing Johne's disease infection in newborn animals, particularly calves, and a vaccine for preventing Johne's disease infection. This will allow the growth and production of calves without Johne's disease, establish a herd free of Johne-infected calves, and proactively address measures against Johne's disease, a problem of economic depletion worldwide. To provide a solution.

[0024]

Means for Solving the Problems The present inventors have found that Peyer's patch M cells in the intestinal tissue of neonatal calves are the only entry gates for Bacillus subtilis, and these cells excessively take up antigen information in the intestinal tract. Although it has been clarified earlier that it has no mechanism, after extensive studies, it has been found that the uptake control mechanism can be artificially induced, that is, Peyer's patch of newborn animals, especially newborn calves M cells in the dome epithelium
Induced by the oral administration of artificially killed bacteria, the property that does not sustain the uptake of Mycobacterium tuberculosis (suppression of phagocytosis)
By suppressing the passive invasion (invasion by uptake) from M cells, which are the specific invasion gates of live Y. fungi,
The inventors have found that calf can be prevented from infection with Johne's disease, and have completed the present invention. The oral vaccine of the present invention is a completely new method in that the colostrum is administered with killed Mycobacterium tuberculosis immediately after birth, and the Gilmoure (1973)
It is a vaccine based on a completely different concept and mechanism from that of the report.

That is, the present invention resides in (1) a method for preventing infection of cattle by inducing the control of M cell uptake. And (2) Mycobacteriu (Mycobacteriu)
m avium subspecies paratuberculosis) was killed by heating, and the killed S. tuberculosis was orally administered to a newborn animal to induce the uptake control of M cells in the intestinal mucosa, which is a specific invasion route of S. tuberculosis. The present invention relates to a method for preventing Johne's disease infection, which comprises inhibiting the invasion of living Mycobacterium tuberculosis. And (3), Mycobacteriu (Mycobacteriu)
mavium subspecies paratuberculosis) is killed by heating, and the killed S. tuberculosis is orally administered to neonatal calves. And (4) a method for preventing Johne's disease infection by orally administering killed S. tuberculosis along with clean colostrum. And (5), Mycobacteriu (Mycobacteriu)
mavium subspecies paratuberculosis). Also, (6) a vaccine for preventing infection with Johne's disease, comprising killed Mycobacterium tuberculosis in normal colostrum.

[0026]

DETAILED DESCRIPTION OF THE INVENTION The present invention is based on the idea that Peyer's patch M cells in the intestinal tissue of neonatal calves are the only invasion gates of Mycobacterium tuberculosis, and these M cells have a mechanism that does not excessively take up antigen information in the intestinal tract. However, by applying the fact that the uptake mechanism is artificially induced, that is, when heat-killed bacteria are orally administered to neonatal calves, M cells inhibit the subsequent invasion of living M. tuberculosis by applying the fact that M. It is for prevention (see FIG. 1). Specifically, a certain amount of heat-killed Bacillus paratuberculosis is mixed with aseptic freeze-dried colostrum of cattle as a preparation, diluted as appropriate with warm water, and delivered to newborn calves isolated from the mother after calving. It is orally administered for a day, and specifically controls uptake of M. tuberculosis by M cells after administration as appropriate. At this time, by giving the vaccine together with aseptic colostrum, it is possible to enhance the immune resistance of calves and prevent infection of Yeast bacteria via mother's milk.

[0027]

EXAMPLES The production of the vaccine of the present invention and its application are as follows:
It is performed through the following steps (1) to (5). (1) Mycobacterium avium subspecies par
culture of M. avium subspecies paratuberculosis (ATCC 10698)
2mg / L (Allied Laboratories, Inc.)
INC, Fayette, Mo, USA) for 10 days in a Middlebrook 7H9 liquid medium (DIFCO, USA). Use a cell culture flask (800 ml capacity, Sumilon MS-20800) for culture.
Perform the culture at a depth of 1-2 cm at 37 ° C. 10 ⁸ CFU / ml bacteria are obtained from 10 ⁵ cfu / ml Mycobacterium tuberculosis by culturing for 10 days. The culture solution is dispensed into 50 ml TPX centrifuge tubes (Sumilon MS-57150), and
Centrifuge at 00G to obtain a bacterial pellet. 0.01% of this
Add 0.01M sterile PBS, resuspend and centrifuge. (2) Preparation of killed bacteria for oral administration Heat at 72 ° C for 20 minutes, cool and freeze or freeze-dry and store. (3) Mix with aseptic colostrum Aseptic colostrum or dried colostrum (mother milk manufactured by Nippon Nosan Kogyo Co., Ltd.) is mixed in hot water heated to 45 ° C. to a concentration of 300 g / 900 ml. Add and adjust so that it becomes × 1010 / 900ml colostrum. (4) Oral administration to newborn calves after parturition Calves are isolated from their mothers immediately after parturition and placed in a clean environment, and within 30 minutes after parturition, 900 ml of colostrum containing killed Yone bacteria is delivered in a stomach tube. / 30 to 40kg / head, given. 12
Administer the same amount within hours. The next day, the colostrum concentration was adjusted to (3)
The same amount is administered twice a day, reduced to half. (5) After the treatment of (4), isolate and raise with sterile dried colostrum or artificial milk.

[0028]

[Experiment 1] Confirmation of uptake of Mycobacterium tuberculosis and suppression of continuous uptake of M. parasites by calf M cells Lay it down, make multiple ileal loops by double ligation at the distal end of the ileum by surgical laparotomy, inject a viable or dead bacterial suspension of Mycobacterium tuberculosis, and 15, 15, 60 minutes after inoculation, under general anesthesia. The intestinal tissue was taken out, embedded in formalin-fixed paraffin, a 4 μm tissue specimen was prepared, stained with hematoxylin-eosin and Thiel-Nelsen, and observed with an optical microscope. Further, the same experiment was carried out using eight colostrum-uninfected calves, and the intestinal tissue was removed again under general anesthesia 6 to 20 hours after the injection of the viable or dead bacterial suspension of the Y. fungus, formalin-fixed paraffin. Embedding was performed, a 4 μm tissue specimen was prepared, stained with hematoxylin-eosin and Thiel-Nelsen, and observed with an optical microscope.

[Experimental Results] No invasion of Mycobacterium tuberculosis was observed in the ileal Peyer's patch at 15, 30, and 60 minutes after inoculation. However, various numbers of M. paratuberculosis were observed in the intestinal lumen of the ileum tissue 6-20 hours after inoculation. In the ileal tissue, a limited number of viable and dead bacteria was observed in the Peyer's patch in the ileal tissue, but was scarcely observed before and after that. Despite the presence of a large number of Mycobacterium tuberculosis in the intestinal lumen, the number of bacteria taken into Peyer's patches is generally very small, and in some cases, no bacteria are found. From these experiments, it was shown that uptake of live and dead bacteria of M. paratuberculosis into the mucous membrane was established by uptake of M cells, but continuous uptake of M. paratuberculosis did not occur.
It has been clarified that there is at least a control mechanism for the uptake of M cells into living and dead bacteria of Mycobacterium tuberculosis. This experiment showed that if a sufficient amount of killed bacteria was orally administered immediately after parturition, the uptake of live bacteria could be inhibited after the uptake of killed S. tuberculosis by certain M cells (see FIG. 2).

[0030]

[Experiment 2] [Experimental method] Mice cause oral infection with M. paratuberculosis. Based on the data obtained in the above calf experiments, mice were administered live mice of M. paratuberculosis once to continuously,
Pathological proof of Mycobacterium tuberculosis in Peyer's patches at different elapsed times was performed. 24 × 14 weeks old C57BL / 6 mice, females, 14 weeks old, were proliferated with 2 × 10 7 cf / 0.5 ml of live Yeast strain (ATCC 10698 strain) in PBS and orally inoculated only once with a gastric tube. While 16
Similarly, the same mice were orally inoculated with the killed bacteria. At 0,4,5,6,7,21,22,23 hours after inoculation, 3 mice were inoculated with killed mice, and 2 mice were killed under over anesthesia with ether. All intestinal tracts at the site where the plate was present were collected and fixed in 20% formalin. Paraffin-embedded tissue specimens were stained with acid-fast bacilli to observe the localization of Mycobacterium tuberculosis.

[Experimental Results] Viable bacteria in the Peyer's patch were observed in each case at 4 hours and 22 hours, and a few dead bacteria were observed at 7, 21, and 22 hours. Various numbers of mycobacteria were found in the intestinal lumen. From this experiment, it was clarified that when administered orally to mice, both live and dead Staphylococcus paratuberculosis were taken up in the Peyer's patch of mice, but the number of bacteria was limited (see FIG. 3).

[0032]

[Experiment 3] [Experimental method] It was examined whether or not the uptake into the Peyer's patch occurs continuously when the M. tuberculosis orally administered to the mouse is always present in the intestinal tract, using killed M. tuberculosis. Experimental method: The same mice as in Experiment 2 were inoculated three times at 1 hour intervals with a gastric tube with killed S. tuberculosis suspension. Thereafter, at 0, 2, 3, 4, 5, 6, and 7 hours, the intestinal tract site where the Peyer's patch was present was collected, and the localization of S. tuberculosis was observed histopathologically.

[Experimental Results] Only one B. fungus was observed in one case at 5 hours, and no B. fungi in the tissue was observed in the other cases. Various numbers of mycobacteria have been demonstrated in the intestinal lumen. From this experiment, it was confirmed that even in a state in which the fungus is always present in the intestinal lumen, the fungus is not always observed in the Peyer's patch present in the same intestinal tract site. This finding implies that even if a large number of M. tuberculosis is present adjacent to the Peyer's patch, the M cells of the Peyer's patch do not continuously take up this (see FIG. 4).

[0034]

[Experiment 4] The effect of the M-cell vaccine was confirmed using a ileal loop system of a goat that develops infection with Johne's disease as in cattle. [Experimental method] Three Shiba goats were quarantined immediately after birth, and until day 2, bovine colostrum dry milk (M-cell vaccine) mixed with killed Mycobacterium tuberculosis was administered. After that, return to the mother's goat, raise it while feeding with mother's milk and normal artificial milk, and under general anesthesia on the 20th day after birth, form a loop at the end of the ileum as described above, ) 10 ⁹ cfu / ml of live bacteria was inoculated into the loop. At 6 hours and 20 hours after the inoculation, the loop was surgically excised again under general anesthesia, and histopathologically observed for localization of S. tuberculosis. As a control to which this vaccine was not administered, observation was performed at 20 hours using a normal-bred goat of the same age.

[Experimental Results] A few mycobacteria were observed in the macrophages in the Peyer's patch dome tissue of the ileal loop of the non-vaccinated sheep goat. However, no bacilli were observed in Peyer's patch epithelium and dome tissues of vaccinated sheep goats. In each case, a remarkably large number of Mycobacterium tuberculosis was observed in the intestinal lumen of the ileal loop. From this experiment, it was found that in Shibayagi inoculated with killed S. tuberculosis with colostrum immediately after birth, M. cell uptake of S. tuberculosis by M cells even in the inoculation of S. tuberculosis into the ileal loop enriched in M cells, which is an invasion route of S. tuberculosis Was suppressed. This result clearly shows that the vaccine can be applied to prevent the infection of Johne's disease in other ruminants such as cattle (see FIG. 5).

[0036]

EFFECTS OF THE INVENTION Immediately after parturition, calves are isolated from mother cows, and the killed vaccine mixed with aseptic colostrum is given to the bacterium, so that not only yeasts derived from breast milk but also yeasts derived from the environment can be obtained. Even if is orally inoculated, the invasion of M. paratuberculosis into tissues (establishment of infection) can be prevented by inhibiting the invasion of M. paratuberculosis from M cells. As a result, by the preventive method using the vaccine of the present invention, even in an area that has already been contaminated with Johne's disease, grow and produce calves without Johne's disease, and establish a clean cow herd that is free of Johne's disease infection. It is possible.

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a diagram of a Peyer's patch and a dome portion of a small intestine, which is an invasion gate of Mycobacterium tuberculosis, and a diagram showing a mechanism of suppressing M cell uptake by killed Mycobacterium tuberculosis.

FIG. 2 is a diagram showing the results of Experiment 1.

FIG. 3 is a diagram showing the results of Experiment 2.

FIG. 4 is a diagram showing the results of Experiment 3.

FIG. 5 is a diagram showing the results of Experiment 4.

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[Procedure amendment]

[Submission date] June 1, 2000 (2006.1)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Name of invention

[Correction method] Change

[Correction contents]

Title: Method for preventing Johne's disease infection by controlling M cell uptake and vaccine thereof

Claims (6)

[Claims] 1. A method for preventing bovine infection by inducing the control of M cell uptake. 2. Mycobacterium avium subspe
cies paratuberculosis) was killed by heating, and the killed S. paratuberculosis was orally administered to a newborn animal to induce the uptake of M cells in the intestinal mucosa, which is a specific invasion pathway of S. tuberculosis, and then lived. A method for preventing Johne's disease infection, which comprises inhibiting the invasion of Mycobacterium tuberculosis. 3. A Mycobacterium avium subspe
cies paratuberculosis) by heating, and orally administering to the neonatal calf the Johnes bacillus that has been killed, to prevent Johne's disease infection in cattle. 4. The method for preventing Johne's disease infection according to claim 2 or 3, wherein killed S. tuberculosis is orally administered together with clean colostrum. 5. A Mycobacterium avium subspe
cies paratuberculosis), a vaccine for preventing Johne's disease infection for newborn calves, comprising a heat-killed bacterium of Cies paratuberculosis). 6. A vaccine for preventing infection with Johne's disease, which comprises killed Mycobacterium tuberculosis in normal colostrum.