CS261584B1 - A method for obtaining Escheriohia coli bacteria clones equipped with a 987P protective antigen - Google Patents

Abstract

A method is provided that allows obtaining clones of Escherichia coli bacteria equipped with the protective antigen 987P, intended for the preparation of immunopreparations. This is achieved by inoculating liquid nutrient medium from stored production strains of E. coli and incubating it stationary at 37 °C until a blank is formed on the surface. Part of the formed blank is re-inoculated onto solid medium. Small colonies grown on this medium are examined by agglutination with serum 987P and in case of positive agglutination the rest of the colony is re-inoculated into liquid nutrient medium.

Description

The invention relates to a method for obtaining clones of Escherichia coli bacteria equipped with the protective antigen 987P for the preparation of immunopreparations, for the diagnosis, prevention and treatment of specific diarrhea of piglets.

Diarrheal diseases of newborn piglets in large-scale farms are still a serious economic problem. Most of these diseases have infectious etiology. In addition to epizootically-occurring transmissible gastroenteritis of pigs (TGE) viral gastroenteritis, enzootic enteric coliinfections and rotavirus infections are mainly involved in the complex of diarrhea in newborn piglets.

Enteral coli infections of piglets ‘are caused by enterotoxigenic types of Escherichia coli (ETEC), which have two essential virulence factors:

a) Produce enterotoxin either thermolabile - antigenic (LT), or thermostable - nonantigenic (ST), or both, which, by their action on the intestinal mucosa (by activation of adenyl- or puanylcyclase) disrupt the physiological ion transport, thereby increasing intestinal secretion at the expense of resorption; diarrhea occurs, often leading to dehydration and dying.

b) On their surface they are equipped with very fine fibrous structures (fimbria, pili) which are responsible for the adhesion of bacteria to the intestinal mucosa. These protein structures, called adhesins or colonization factors, allow their carriers to be colonized in the small intestine mucosa, prevent their removal by the intestinal peristalsis and thus allow the action of enlerotoxins secreted into the environment, i.e. directly on the mucosal surface.

Effective defense of newborn piglets against enteral coli infections is feasible by supplying specific antibodies against ETEC colonizing factors (adhesins) to the lumen of the intestine.

261 S84

This is possible either by sucking colostrum and milk from immunized mothers or by oral administration of specific sera.

In ETEC strains from piglets, three antigenically different types of colonization factors bearing the designation K88, K99 and 987P are registered.

The presence of colonization factors in ETEC cultures depends on the culture conditions, eg they are not produced at temperatures up to 20 ° C. Expression of the K99 antigen requires culture medium with a limited amount of nutrients and the presence of a number of trace elements (Coin Medium - Minimal Casein Hydrolysate Medium). Factor 9S7P production is subject to variation: P + # - * P- with a tendency to P-phase predominance, meaning that passage of cultures with the original presence of 987P results in the loss of this antigen.

Currently, a vaccine against coli infections of piglets K88, K99 is produced commercially, whose ETECs are used in three serotypes: O147'.K88ab, 0149: K88ac and 0101: K99. The strain with colonization factor 987P is not included in it. When the vaccine was introduced into production in 1983, the problems caused by the phenotypic phase variation in the production of factor 987P have not yet been overcome.

Some recent releases show the different position of factor 987P compared to factors K88 and K99. GAASTRA and DE GRAAF (1982) report a detailed genetic map and amino acid sequence of K88 and K99. However, for factor 987P these data are missing; the authors report only the diameter of the fimbriae (7nm), the molecular weight (18,900) and the isoelectric point (3.7). RE. ISAACSON, a worker who has been dealing with ETEC colonization factors and in particular 987P for many years, reports in 1986 a vaccine against enteral coli infections of newborn piglets containing isolated sawdust K88, K99 and 987? · K88 and K99 are derived from bacterial strains prepared by recombinant DNA technique. This technique is described in detail for producers of both types of fimbrios. Factor 987P is only briefly reported to be derived from the culture of the E. coli strain that produced it. A method for obtaining E. coli clones equipped with the 987P protective antigen is not disclosed.

This shows that there are some difficulties in producing factor 987P. These problems have been overcome by the process according to the invention, which consists in incubating the inoculum of the E. coli production strain in a stationary medium at 37 ° C in a liquid nutrient medium until a blank is formed on the head of 3 261 584 dine and after IS-hour incubation at 37 ° C, small colonies are searched for and examined by agglutination with anti-9871 serum, with the remainder of the positively agglutinating colony inoculated into the liquid nutrient medium »

Preferably, 2.5% Masopepton Broth containing 10g meat extract, 10g peptone and 5g sodium chloride v. 1 liter can be used to incubate the canned inoculum »

Part of the broth blank may be inoculated preferably after. blood agar, after which incubation at 37 ° C yields a mixture of large and small colonies. The remainder, tiny colonies, positively agglutinating with anti 937P serum, may preferably be inoculated into 2.5% masopepton broth.

ETEC cultures with colonization factor 9871 are characterized in that they contain a mixture of bacterial cells equipped with factor 9S7P (P + phase) and bacterial cells without this factor (P-) phase ®

In their normal passage on nutrient media, the P- phase prevails and the P + phase disappears. However, only the P + phase is a protective antigen. The P + phase is characterized by a hydrophobic character, so that in stationary cultivation in a liquid medium, a minor part of it concentrates on the surface of the medium and forms a fine blank. This phenomenon is rarely addressed in bacteriological laboratories. In this case, however, this is a crucial point. This blank represents an important phase in obtaining a clone of a bacterial strain with a predominance of cells equipped with a colonization factor of 987P. The culture in the liquid medium with the formed blank should be handled with great care, as even with the smallest impact on the culture tube, the blank drops into the culture »

Carefully inoculate from the culture surface blank into additional liquid soils and solid soils, eg Masopepton agar (I, TA) or blood agar (KA) on Petri dishes »Different colonies grow on solid soils on solid soils. white, containing only the P- phase, smaller colonies, white, containing a mixture of both phases, and greyish smaller colonies, which contain a P + phase predominance.

Example 1

Two tubes of Masopepton broth, Petri dish with blood agar (KA) and Petri dish with End-soil were inoculated from the stock culture on Dorset's broth. After incubation for 1 hour (37 ° C), the broth cultures in the tubes are carefully removed from the thermostat and inspected for blank formation on the surface. If the blank was not formed, the cultures were returned to the thermostat (37 ° C) and inspected for a further 2 days until the blank was formed at the broth level. Initially, i

- 4 261 584 blank very fine whitish-gray, with progressive incubation growing. Carefully vaccinate from the formed blank to the next KA. After 18 hours incubation of the inoculum on KA (37 ° C), growth, consisting of larger and small colonies, is checked. A small aliquot is removed from the small greyish colony on KA and examined by slide agglutination with anti-987P serum. directly to immunize animals to obtain anti 987P serum. • After inoculation, both Dorset broth and broth are incubated for 18 hours at 37 ° C. The grown culture Dorset broth (stock culture) is stored at 4 ° C and serves as a can. The broth-grown culture is intended as a vaccine inoculum.

Example 2

In a fermenter filled with sterile caseinhylorolysate broth after heating to 37 ° C, the broth grown according to Example 1 is transferred and cultured submerged at 37 ° C to a concentration of 2 < 9 > For grown culture, a purity test is performed by inoculation on KA and End broth and checked for the presence of protective antigen by agglutination with anti-987P serum. * The culture is inactivated by the addition of 0.5% formalin. After inactivation, sterile aluminum hydroxide gel as adjuvant is added in an amount of 2 liters per 8 liters of bacterial culture and mixed well.

The vaccine is intended to immunize pregnant sows that protect suckling piglets from infection by antibodies contained in the mammary gland secretions.

Claims (4)

SUBJECT OF THE INVENTION 261 584 Method for preparing 987P antigen-producing Escherichia coli clones, characterized in that the canned inoculum of an E. coli production strain is incubated stationary at 37 ° C in a liquid nutrient medium until a blank is formed at the surface, part of the blank is then seeded to a solid and after incubation at 37 ° C for 18 hours, small colonies are screened and examined by agglutination with anti 9S7P serum, with the remainder of the positive agglutinating colony inoculated into the liquid nutrient medium. 2. A process for preparing Escherichia coli clones according to claim 1, characterized in that 2.5% Masopepton broth containing 10g meat extract, 10g peptone and 5g sodium chloride per liter is used to incubate the inoculum from the production strain. 3. A process for the preparation of Escherichia coli clones according to claims 1 and 2, characterized in that a portion of the blank is inoculated after blood agar and incubated at 37 DEG C. to obtain a mixture of large and small colonies. 4. Method for the preparation of Escherichia coli clones (I p After The composition is characterized in that the rest of the small colony positive not agglutinating with anti 987P serum is inoculated into a 2.58 m sopepton broth containing 10 g of meat extract, 10 g pe. pton and 5g of sodium chloride per liter.