FI96277C - Method for the preparation of a bacterial preparation useful for the prevention of Campylobacter infections in poultry - Google Patents

Description

5 96277

The present invention relates to a process for the preparation of a bacterial preparation for use in poultry, in particular for preventing the colonization of Campylobacter species.

10

Poultry is the main source of human campylobacter and salmonella infections. The role of Campylobacter ieiun and Campylobacter coli as a common cause of diseases of the human gastrointestinal tract is well documented. References-15 to King, E. O: "The laboratory recognition of Vibrio fetus and a closely related Vibrio isolated from cases of human vibrosis", Annals of the New York Academy of Sciences, 98: 700-711, 1962 proposed chickens as the primary human as a source of infections. Skirrow, M.B .: 20 "Campylobacter enteritis: a 'new' disease", Brit.Med.J.

2: 9-11 (1977) showed in some cases a link between human disease and contact with chickens carrying the organism on farms, butchers and home kitchens. This association was demonstrated later in 25 min in some epidemiological studies, e.g., Harris, N.V., Weiss, N.S. and Nolan, C.M .: "The role of poultry and meats in the etiology of Campylobacter lei coli enteritis." Am. J. of Public health 76: 407-411 (198-6). There have also been some reports of disease outbreaks in which an epidemiologically contaminated or suspected Campylobacter iosis intermediate has been raw, grilled or inadequately cooked chicken. WHO in April. According to Newsletter No 20 published in 1989, the number of campylobacter infections has exceeded the number of salmonella infections in recent years in some industrialized countries.

A study conducted by the Finnish Veterinary Department confirmed that the probable reason for the susceptibility of 96277 2 broilers to salmonella in the gut (growth and attachment) is the delayed development of normal intestinal bacterial growth. This delay is the result of mass-5 feeding methods in modern broiler farming. Such animals therefore have low resistance to exogenous bacteria entering the body. E. Nurmi and M. Rantala, Nature, 241: 210 to 211.1973 showed that normal adult gut contents given newly hatched chicks 10 increased their resistance to salmonella settling down. Based on this pioneering work, the theory of competitive foreclosure (hereinafter CE) was formulated. Bacterial preparations, in particular for the prevention of salmonella infections, have been described, for example, in EP 6696, EP 33854 and EP 154447. Soerjadi-Lien, Snoeyenbos, and Weinack, Avian Diseases 26: 520-524 20 (1982) and 28: 139-146 (1983) have reported that when a 10% stool suspension from specific pathogen-free (SPF) groups that were optimally -distributing microflora, was given to young chickens, their resistance to C ^. towards ieiun increased. This working group 25 further reported that the CE effect of the administered microflora decreased as the administered doses of Camplobacter species increased. According to other studies investigating the effect of CE on Campvlobacter species. no effect was observed when using either the CE-flora, which was obtained in 30 fecal or umpisuolieritteistä (Stern, Bailey and Blankenship, Avian Diseases 22: 330-334, 1988) and CE-flora, which was obtained from a healthy adult caecal flora of (Shanker, Lee & Sorrel, Epidemiology and Infection 100: 27-34, 1988).

The present invention provides a method of preparing a bacterial preparation useful for preventing the introduction of this human pathogenic bacterium into poultry, comprising bacteria derived from an adult bird, a microecological site where the pathogenic bacteria exist.

manufactured in accordance with the preparation 5 of the present invention comprises a bacteria flora, which is in particular derived from an adult bird's cecum flora of the mucous membranes and may be used to inhibit the human pathogenic bacteria to the bak 10, especially Campylobacter settling of the young birds. The results reported herein include a reduction in the growth of C. ieiu biotype 1 in broiler chickens. The bacterial preparation is also effective against other Campylobacter species and possibly also against other pathogenic bacteria.

The culture used in the invention is selected from the same microecological environment in which the Campylobacter tends to be present in the colon of broiler chickens. Very effective in cultures obtained by culturing the bacterial flora, which is derived from an adult bird's cecum wall mucosa, either anaerobic or microaerophilic conditions, preferably in mucin surface. It is also advantageous to select bacteria with good motility in mucin.

The crops used in the experiments were all derived from an older, laying hen.

30 CREATING CROPS

Bowel homogenate (culture) The entire caecum was homogenized and diluted with 0.1% peptone water in laboratory scale experiments and standard drinking water in farm scale experiments.

96277 4

The culture did not contain Salmonella or Campvlobacter species. The test method was as follows:

Salmonella: 1% buffered peptone-water (37 5 ° C / 20 h), Rappaport-Vassiliadis medium 41.5 ° C / 20 h) and tetrathionate medium (37 ° C / 20 h), Onöz and brilliant -green agar (37 ° C / 20 h),

Campylobacter: Brucella medium supplemented with 10 pyruvate, metabisulfite and ferric iron (FBP) (41.5 ° C / 20 h in a micro-aerophilic atmosphere) CCD agar (41.5 ° C / 44 h in a microaerophilic atmosphere).

15

Cultured colon homogenate (culture B) 1 ml of colon homogenate was used to inoculate 100 ml of Brucella medium supplemented with 1 g of Porcine Gastric 20 Mucin (SIGMA). Mucin was suspended in small portions in boiling water and neutralized before being added to other media. Cefoperazone (Cefobis, Pfizer) was added aseptically after autoclaving to a final concentration of 32 mg / l. Incubation was continued for 48 h at 41.5 ° C in a microaerophilic atmosphere.

Pure limakalvovilielmä the caecum (flora C)

A substantial portion of the immobile bacteria in culture B was discarded using semi-solid 10% mucin agar having the following composition

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5 96277

Semi-solid 10% musine agar:

Mucin 10.00 g 5 Brucella medium 0.50 g K2HPO4 0.25 g

Agaria 1.10 g

Water 100 ml 10 Mucin must be dissolved in boiling water before autoclaving. The pH is adjusted to 7.2 with 0.1 N NaOH.

Culture B was transferred to a 12 cm petri dish containing 33 ml of semi-solid 10% mucin agar.

After microaerophilic incubation (41.5 ° C / 48 h / oxygen 5%, CO 2 15% and nitrogen 80%), a small portion of the agar between 2 and 4 cm from the outside of the line was transferred to 10% isine-20 mucin medium. The composition of this medium was the same as that of mucin agar, except that it did not contain cefaprazone or agar. The procedure was repeated until no more immobile bacteria were detected on the medium.

25 Isolation of bacteria

Culture C was cultured on Brucella agar and in a microaerophilic atmosphere at 41.5 ° C for 48 to 96 hours. Mobile helical organisms were isolated.

Culture B was transferred to a 12 cm petri dish containing 33 ml of semi-solid 10% mucin agar.

After anaerobic incubation (41.5 ° C / 48 h), a small amount of 35 agar from 2 to 4 cm outside the line was transferred to 10% mucin medium II, which was also incubated in an anaerobic atmosphere. The procedure was continued until immobile bacteria were no longer detected on 96277 6 medium. After three repetitions, there were only monocotyledonous bacteria remaining on the medium, which were marked with Y.

5 CROP PROVISION

laboratory tests

In laboratory experiments, 60-72 newly hatched chickens 10 were divided into 12 groups. CE crops were given to groups of 2x4 birds with bird feed. The remaining 4 groups were used as controls and did not receive CE culture. The next day, C. ieiun biotype I Penner 15 or Penner 3/43/59 1000-3000 CFU was administered by the same method to all groups except two of the four control groups. Half of the birds were killed after a week and the rest after two weeks. A quantitative study of Campylobacter was performed on the colon of birds.

20 Field tests

In the field experiments, 1600-1700 chickens were divided into 16 groups, of which eight served as test groups, four as Campylobacter positive control groups, and four as control groups. CE cultures were mixed with the first drinking water of eight test groups. 4.16-5.80 log10 CFU of C. ieiun biotype 1 from Penner 15 was administered the following day to three infectious birds in each cage except in the cages of the four control groups. At each week-30 Sat, until the end of the growing season, three to five birds from each cage were killed and Campylobacter species were quantitatively examined from the cecum.

Brucella medium with FBT (ferrous sulphate, sodium metabisulphite and sodium pyruvate, all 0.5 g / l) and cefaperazone (32 mg / l) and carbon-Cefaperazone deoxycholate (CCD) agar was used for Campylobacter. 96277 in qualitative and quantitative studies of species. The media were incubated for 20 h and the agar for 44 h at 41.5 ° C in a microaerophilic atmosphere.

5 Results

Asettumistekijä is the arithmetic average of the CFU of individual birds in an amount per g of caecal content of the log 10 values for each test or control group.

10

According to the agreement, a Campylobacter-negative bird (in both quantitative and qualitative experiments) has a log10 value of 0.1 in the calculations.

15 Test 1

laboratory tests

Table 1 shows that microaerophilic incubation of culture B produced more effective competitive inhibition of Campylobacter species than anaerobic incubation of culture B.

Table 1. C.eiun biotype I thermostable serotype-25 pin 15 response dose 895 CFU / chicken.

Settlement factor 1 week 2 weeks 30 Comparison 1 5.13 7.74

Comparison 2 5.96 8.35

Culture B microaerophilic culture Group 1 <2.00 <2.00 35 Group 2 <2.00 <2.00

Group 3 <2.00 <2.00

Group 4 2.34 <2.00 96277 8

Culture B anaerobic culture Group 1 <2.00 3.24

Group 2 4.91 <2.00

Group 3 3.89 <2.00 5 Group 4 3.70 7.04

Test 2

Culture B was purified on a mobility and atmospheric basis to microaerophilic culture C and anaerobic bacterial culture Y as described above. Anaerobic bacteria Y alone did not elicit a CE effect. The combination of these cultures (1: 1) brought back the CE effect, as shown in Groups 3 and 4, Table 2.

15

laboratory tests

Table 2. C. ieiun biotype I thermostable serotype 15 response dose 245 CFU / chicken 20 setting factor 1 week 2 weeks

Check 1 7.78 7.58

Check 2 8.33 8.74 25

Crop Y alone

Group 1 1.90 6.70

Group 2 6.55 8.06 30 Crop Y + crop C (1: 1)

Group 3 1.80 <2.00

Group 4 3.22 <2.00

Field experiments 16 x 100 broiler chickens were reared in a normal rearing unit for six weeks. Eight groups

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35,996,277 were used for the competitive inhibition experiment. Four groups served as negative comparisons not shown in the figure. The test groups were given two species of ‘defined intestinal growth’ in their first drinking water.

5 The dilution of the crops was 1/30. Each bird received an estimated 0.25 ml of treatment fluid.

Four test groups were given mucin-adapted anaerobic motile bacteria (culture Y). The remaining four groups received a combined culture (1: 1) containing culture Y and culture C, a mucin-adapted microaerophilic culture as described above.

36 broiler chickens were infected with Campylobacter ieiun 15 biotype 1, Penner serotype 15 at one day of age. The dose was log10 4.16 CFU. Three of these infectious individuals were divided into each group.

For each group were examined quantitatively the content of the three bird's cecum campylobacteria 20 relative to the CCD agareilla week intervals. Asettumistekijä was calculated from the arithmetic mean of the log 10 values of the individual birds CFU per gram of the amount of cecal contents in each group. The negative control remained qualitatively negative for Campylobacter species until slaughter.

The results are shown in Figure 1. It can be seen that mucin-adapted anaerobic motile bacteria (culture Y) did not protect the colon from the settlement of Campylobacter species, although the bacterial count of Campylobacter species was slightly reduced (<1 log10 units). The combination of culture Y and culture C (1: 1) delayed the onset of establishment of Campylobacter species by two weeks. 3 vii-35 h after infection, the bacterial count of Campylobacter species was still 4 log10 units lower than in the control groups.

Experiment 3 96277 10

The diluted homogenate of the caecum (flora A) or CE-kutusta was compared to an unspecified anaerobic atmosphere for 5 air-umpisuolikasvuston increased, the CE-effect.

The numbers of birds and groups tested as well as the number of CE crops administered per bird were the same as in the previous experiment. Four test groups received culture A and four groups of unspecified colon growth 10 grown in an anaerobic atmosphere. This crop is called 427 / C. Eighteen broiler chickens were infected with Campylobacter ieiun biotype 1, Penner serotype 15, at 18 days of age. The dose was log10 5.80 CFU. Three of these infectious agents were assigned to each group.

Figure 2 shows that flora A (the diluted homogenate of the caecum) showed the more efficient CE effect.

20 Culture A apparently settled in the caecum of chickens and prevented C. ieiun from settling in two groups and reduced the degree of colonization in two groups (by four log, 0-unit). Culture 427 / C delayed the onset of infection by 1-2 weeks, but it is likely that after two weeks, the amount of Campylobacter in the colon of chickens would be at the same level as in the colon of control groups (fully established in Campylobacter species).

ii: a & .i i l i e ·. . ·

Claims (4)

96277 1. A method for the preparation of bacterial preparations on the basis of a prophylactic infection of a Campylobacter infection, 25 kännetecknat därav. 1. Process for prevention of Campylobacter infections in poultry useful in the preparation of the bacterial preparation 5, characterized in that the culturing of bacteria derived from an adult bird cecal mucosa, and is isolated from the bacteria. 2. A method according to claim 1, comprising 30 to 30, for producing a bacterial or microaerophilic or anaerobic atmosphere. A method according to claim 1, characterized in that the bacteria are cultured in a microaerophilic or anaerobic atmosphere to produce a preparation. 3. A patent according to claim 1 or 2, which comprises a bacterial head or mucinmedium. 35 Method according to Claim 1 or 2, characterized in that the bacteria are cultured on a mucin medium. Method according to one of Claims 1 to 3, characterized in that helical motile bacteria are isolated. 20 4. An embodiment of the invention according to claims 1-3, which comprises an isolated spiral-shaped bacterium with a rope.