MXPA98009690A - Process for the preparation of an actuobacillus pleuropneumoniae toxoid-immunogenic based on fractionated ultrafiltration for the prevention of contagious pleuropneumonia porc - Google Patents

Abstract

The present invention relates to a process for the production of a vaccine by means of a toxoid-immunogenic agent of actinobacillus pleuropneumoniae based on fractional ultrafiltration, for the prevention of contagious porcine pleuropneumonia, which consists in cultivating the reagent of each of the serotypes required, the result of the culture, is centrifuged obtaining a supernatant, this is done the immunological, which can be Apx I, Apx IIóel Apx III, each group being isolated, they will be packaged and stored vacun

Description

PROCESS FOR ELABORATING A TOXOID-IMMUNOGENOUS OF Actinobacil? Us pieuropneumoniae BASED ON FRACTIONATED ULTRAFILTRATION FOR THE PREVENTION OF PORTAIN CONTAGIOUS PLEUROPNEUMONIA.

FIELD OF THE INVENTION The present invention relates in general to a process for preparing a vaccine for the prevention of porcine Pleuropneumonia contagiosa.

BACKGROUND OF THE INVENTION I. DEFINITION OF THE DISEASE.

Contagious swine pleuropneumonia (PCP) is a devastating disease, produces high mortality and economic losses in susceptible pigs, due to a poor feed conversion in chronically infected animals. The disease can vary from a hyperacute to a chronic course, where the characteristic acute lesion is a necrotizing hemorrhagic pneumonia associated with fibropinous pleuritis and whereas the chronic lesion is distinguished by a consolidated, infarcted and encapsulated pulmonary tissue.

II. ETIOLOGICAL AGENT.

The bacterium Actinobacillus pieuropneumoniae is the agent responsible for PCP and is widely distributed in many countries. The etiological agent is considered today as Actinobacillus pieuropneumoniae, due to the studies carried out on the phenotypic bases and on the deoxyribonucleic acid (DNA) of these bacteria, caused that it was transferred from the species Haemophilus pieuropneumoniae to the species Actinobacillus pleuropneumoniae biovariety 1 if it is dependent on nicotinamide adenine dinucleotide (NAD) and the species Pasteurella haemolytica similar to the species Actinobacillus pieuropneumoniae biovar 2 but is dependent on NAD.

The bacterium is Gram negative and pleomorphic, usually observed as a short, encapsulated bacillus measuring 0.5 to 1.5 um long by 0.3 um wide, is a bacterium that lacks flagella and does not produce spores, however, it has cytoadherent fimbrias . It is an aerobic or anaerobic bacterium, a fundamental characteristic of this microorganism cs the depend on NAD, growth factor known as V, which has the following formula: C21, H27, N7, 014, P2; do not require hemin, known as growth factor X present in media enriched with blood.

For primary isolates it is important that e! NAD is given another microorganism in situ, by means of a stria on the suspected culture of Actinobacillus pleuropneumoniae, this phenomenon is known as satellitism.

The capsule is attached to the phospholipids of the cell membrane and serves as an anchor. The capsule is responsible for the specificity of the serotype, in the present ten serotypes designated with Arabic numerals from 1 to 10 are recognized, sc have designated two other serotypes on 1 1 and 12, but still the status of serotype 1 1 is not clear . The antibodies generated against the capsule only protect against death, but not against lung lesions and chronic infection. The virulence attributed to the capsule is variable between the different serotypes.

DETAILED DESCRIPTION OF THE INVENTION A. EXOTOXINS In the supernatants of Actinobacillus pieuropneumoniae, hemolytic activity has been detected and found. Hemolysin is very unstable, cyclic production (growth curve: late logarithmic phase and late stationary phase), is thermolabile, inactivated by formaldehyde and by protoelitic enzymes. This hemolysin is a protein that has a molecular weight of 104 to 1 10 Kd, induced by calcium and acidic (pH of 4.3), is immunogenic and immunologically protective. This hemolysin is identified as hemolysin or cytolysin 1 (Apx I: strongly hematological and cytotoxic) and is present in serotypes 1, 5, 9, 10 and 1 1. The hemolytic activity of this protein is responsible for the initial lesions of PCP, characterized by being hemorrhagic and necrotic.

Another hemolysin with a molecular weight of 103 Kd has been found in serotypes 1 -2,3-4,5-6,7,8,9,1 1 and 12 and is identified as hemolysin or cytolysin 2 (Apx II: little hemolytic and moderately cytotoxic). Only serotype 10 produces Hemolysin I, while serotypes 7 and 12 produce Apx II. These hemolysins do not show antigenic differences between serotypes, although they are antigenically different among them.

The supernatant of the culture of Actinobacillus pleuropneaimoniae, has the toxic effect on adherent cells of lung washes (macrophages), as well as circulating monocytes. This exotoxin has a molecular weight of 120 Kd and is known as Cytolysin 3 (Apx HIr is not haemolytic but is strongly cytotoxic), however, it is also known as Pleurotoxin (PTX). The serotypes that are produced are the following 2,3,4,6 and 8.

Its production is inhibited by oxygen and cholesterol, it is thermolabile, it is not hemolytic and toxic to neutrophils (PMN), it is sensitive to proteases, it is an immunogenic protein that is neutralized by the sera of pigs convalescing to PCP.

B. APPROACH TO TECHNOLOGICAL DEVELOPMENT The technological development consisted of cultivating, preparing, purifying and concentrating the toxins (Apx I, Apx II and Apx III) of Actinobacillus pieuropneumoniae from serotypes 1, 3,5 and 7, to determine the hemolytic and cytolytic activity of each one. of the groups (A, B, and C). And to inactivate and prepare the toxoids (which will be known as PLEUROTOXOIDE A, B, and C. Finally the immunogens were validated in farms with contagious porcine pleuropneumonia problems.

C. MATERIALS AND METHODS. 1. MATERIALS. 1. 1 BACTERIAL STRAINS.

The following serotypes of Actínobacillus pleuroneumoniae are used to elaborate the immunogen and are divided into three groups: Group A; Actinobacíllus pieuropneumoniae serotypes 1, 5,9,10 and 1 1 for the production of the Apx I toxin, Group B: Actinobacillus pleuropneumoniae serotypes 2,3,6 and 8 for the production of the Apx III toxin.

Group C: Actinobacillus pleuropneumoniae serotypes: 1, 2,5,7,9,11 and 12, for the production of the Apx II toxin. 1. 2. CULTURE MEDIUM 1. 2.1 COMMERCIAL MEDIA.

The medium is prepared by adding 450 g of brain heart infusion broth (Bili, brain heart infusion: Difco Laboratories, Detroit, Mich or Bioxon de México, Oax.), In 12.2 L of distilled water (37 g / L), also You can use Tripticasa soy broth (Difco Laboratories, Detroit, Mich.), adding 450 g in 15.1 liters of distilled water (30 g L) and sterilizing at 121 ° C and 15 bs. of pressure for 15 minutes. 1. 2.2 SUPPLEMENTS.

At the time of culturing actinobacillus, add 10% (V / V) fresh yeast extract or from 0.01 to 0.025% (10 to 25 ug / ml) of nicotinamide adenine dinucleotide (NAD: Sigma Chemical Co., St Louis, Mo) or 0.1% (NAD: Eastman Kodak Co., Rochester, NY) - sterile It is also important to add 2.5 mM calcium chloride (Ca C12: Sigma Chemical Co., Ct. Louis, Mo. C 7902 ) for the cultivation of some serotypes: Group A and Ferric Pyrophosphate (fe4 (P207) 3: Sigma Chemical Co., St. Louis Mo. P 6526, for Group B and C serotypes, it is important to keep it in amber jars to protect the compound of light, and do not use the product if it changes from green to yellow or brown to a concentration of 0.25 g / liter for others, ferric nitrate is also used (fe (N03) 3.9H20: Sigma Chemical Co ., St. Louis Mo. F 8508) at a concentration of 0.020 g / liter of medium. 1. 2.3. PREPARATION OF YEAST EXTRACT OR NAD.

The fresh yeast extract is prepared in the following manner: 50 g are added. of fresh baker's yeast in 100 me. of 0.2 M KH2P04, heated between 80 and 85 ° C for 20 min., allowed to cool and clarified by filtration (Durapore TP 0.45 μm hydrophilic cartridge, Milillipore catalog number: CVHL 51 TP3), the pH is adjusted to 7.3 with a 1 N NaOH solution and sterilized by filtration (Durapore TPO.22 um hydrophilic cartridges, Millipore catalog number: CVGL 51 TP 3) to use these filters you need a 22-inch PS-1 Housing type cartridges, No Millipore catalog: YY16 022 14 with its accessories), The sterile yeast extract is packaged and stored at -20 ° C until use.

Nicotinamide adenine dinucleotide 0.01 to 0.25% (10 to 25 ug / ml.) (NAD: Sigma Chemical Co., St. Louis, Mo.) or 0.1% (NAD: Eastman Kodak Co., Rochester, NY) adjusts the pH to 7.3 with a 1 N NaOH solution and is sterilized by filtration (Durapore TP 0.22 um hydrophilic cartridges, Millipore Catalog No.: CVGL 51 TP3; to use these filters are used papercartuchos type PS-1 Housing 22 inches, catalog No. Millipore: YY16 022 14 with accessories). The sterile yeast extract is packaged and stored at -20 ° C until use. 1. 2.4 MAINTENANCE OF THE SCORES OF Actinobacillus pieuropneumoniae.

The different serotypes of Actinobacillus pieuropneumoniae for conservation and preparation of "seed" are grown on PPLO agar (Difco Laboratories, Detrit, Mich.), Supplemented with 10% yeast and 5% horse serum at a temperature of 37 ° C , in an atmosphere of 5% C02, for 18 to 24 hours. Subsequently, actinobacilli are harvested with sterile PBS, and 10% skim milk preservative previously sterilized in an autoclave is added to the preservation medium.

The bacteria harvested in this way with skim milk can be frozen at -20 ° C or freeze-dried and stored at 4 ° C. If 1% glycerol is added to skim milk, they can be frozen at -70 ° C but not lyophilized. Another alternative is to store the actinobacilli in the medium of suerc-inositol (horse serum and 5% mesoinositol) and freeze at -70 ° C. 1. 3 FILTRATION AND ULTRAFILTRATION EQUIPMENT 1. 3.1 FILTRATION EQUIPMENT A series of cartridge type filters are used (Durapore TP 0.45 um hydrophilic cartridges, Míllipore catalog: CVHL 51 TP3), to eliminate bacterial clumps and cartridge filters (hydrophilic cartridges that completely eliminate bacteria: To use these filters you need 22-inch housing type PS-1 Housing, catalog number of Miílípore, YYl or 022 14 with its accessories).

To achieve the necessary pressure it is important to have a Millipore XX80 000 00 peristaltic pump with head XX80 000 05 of 1.6 L / minute or to have tanks with inert gas (nitrogen) with pressure gauges and valves to regulate the pressure.

It also requires tubes for the heads of the peristaltic pump, Silicone (XX80 000 25); of Tygon (XX80 OOT 25.} or Norprene (XX80 OON 25). 1. 3.2 ULTRAFILTRATION EQUIPMENT An ultrafiltration system called Prostak-UF Pilot System is required to perform operations from 10 to 100 liters, one kilogram of output. This system has an expansion for membranes that can have an area of 0.93 m2 up to 4.65n m2.

This Prostak - UF Pilot system requires ultrafiltration membranes PTHK 100,000 NMWL (PT series) of polysulfone, which comes in modules with membranes that have an effective area of 0.18 m2 (with 2 stacks); 0.35 m2 (with 4 stak) or 0.9 m2 (with 10 stak) depending on the volume to ultrafill. 2. METHODOLOGY 2. 1 CULTIVATION OF THE CEPAS From the conserved "seed" strains, these are sown to have an inoculum of 100 ml in liquid medium, then a liter of commercial medium is prepared, with its yeast extract or commercial NAD, in addition to adding the calcium chloride and It is inoculated under completely aseptic conditions Actinobacillus pleroneumoniae serotype 1 or 7 (jugs of group A or C), in the same way Actinobacillus pleroneumoniae serotype 3 is inoculated (jug B) but it is added to ferric pyrosphate or ferric nitrate. For a period of 1 to 14 hours, incubate at 37 ° C, at the end of this time the contents of the flask are inoculated to the jugs A, B or C. or 20 liters of commercial media are prepared and the yeast extract is added or the commercial NAD is also added calcium chloride or ferric pyrosphate, depending on the serotype that you want to grow. To this volume one liter of the previously prepared inoculum is inoculated and allowed to incubate at 37 ° C for 12 hours. The incubation will depend on the serotype to be worked. In the case of Actinobacillus pleroneumoniae serotype 1 or 7, the incubation time will be from 24 to 26 hours and in the case of Actinobacillus pleroneumoniae serotype 3 it will be from 16 to 18 hours. 2. 2 SEPARATION OF TOXINS.

After the incubation period, the contents of the jugs (A, B or C) are filtered as follows: a) First, a pre-filtration with cartridges (Durapore TP 0.45 um hydrophilic cartridge, Catalog No. Mil pore: CVHL 51 TP3), for the elimination of bacterial clumps and with a filtration (Durapore TP 0.22 um hydrophilic cartridges, catalog no. Millipore: CVGL 51 TP3) to completely eliminate bacteria. Subsequently, the pressure is inverted and the bacterial biomass is recovered. b) With the Prostak-UF Pilot System and the PTHK 100,000 NMWL (PT series) ulystrone membranes of polysufone, which comes in modules, the ultrafiltration is carried out in a cut-off of 100.00 Kd molecular weight, with the filtrate of each garrafón, in each operation try not to mix the membranes of a serotype with those of any other. The filtrate is stored on one side to determine the presence of endotoxins and on the other the retentate to determine the protein content, trying to store small amounts, before inactivating with formaldehyde, to titrate the hemolysins and cytotoxins, as well as the endotoxin. 2. 3 TOXOID PREPARATION.

The retentate is rehydrated with sterile PBS at the same original volume and "inactivated" with formaldehyde at a final concentration of 0.1 to 0.4% at a temperature of 4 ° C for 24 hours. 2. 4 PREPARATION OF THE IMMUNOGEN Depending on the concentration of proteins, the adjuvant, aluminum hydroxide, is added to the total content of the jug in the appropriate amount. 2. 5 STORAGE AND PACKAGING OF THE PRODUCT. The toxoid is packaged in bottles that store 25, 50 and 100 doses of 2.0 ml each, and should be stored between two and eight degrees Celsius until the time of use and should not be frozen, it will only be necessary to store the bottles with the immunogen in the refrigerator. 2. 6 QUALITY AND SPECIFICATIONS OF RAW MATERIALS. 1. All strains of bacteria used should be of reference and from the American Type Culture Collection, ATCC. 2. All chemical reagents used must have a purity of 99%. 3. To dissolve all raw materials used for the manufacture of the reagent, deionized water should be used. 4. All culture media must guarantee the growth of the strains of Actinobacillus pieuropneumoniae. 1. PREPARATION OF THE REAGENT OR "PNEUMOTEST" The bacterial biomass obtained is rehydrated with PBS and inactivated with formaldehyde or with phenolated physiological saline solution, and the reagent is prepared according to the rapid technology to diagnose pig respiratory diseases.

Claims (1)

CLAIMS Having described the present invention, this is considered a novelty, for which the contents of the following clauses are claimed.

1. Process to elaborate a toxinoid-immunogen of actinobacillus piéuropneumoniae based on fractional ultrafiltration for the prevention of contagious porcine pleuropneumonia, the serotypes of actinobacillus pleuropneumonie are cultured a) in agar, b) yeast extract c) and horse serum at 37 ° C already in an atmosphere of C02, during a period of 18 to 24 hours; d) the actinobacilli are harvested in susupension of sterile phosphates, e) skim mílk is added; the harvested bacteria are frozen at -20 ° C, being freeze-dried and stored at 4 ° C; if glycerol is added to the skim milk they can be frozen at -70 ° C but not frozen; í) preparing a liter of commercial medium with its extract, add CaCl, actinobacillus pleopneumoniae serotype 1 or 7 is inoculated; In the same way serotype 3 will be made, adding ferric pyrophosphate or ferric nitrate, the process is carried out in a period of 12 hours at 37 ° C and agitation of 30 to 12 hours; at the end of this, the content is inoculated; later, g) they are prepared from 10 to 20 It and the yeast extract is added, calcium chloride or ferric pyrophosphate is added, depending on the serotype one wishes to grow; to this, a liter of the previously prepared inoculum is inoculated for a period of time of 12 to 15 hours at 37 ° C; the incubation will depend on the serotype to be worked, for serotype 1 or 7 the incubation time is 24 to 26 hours and for serotype 3 it goes from 16 to 18 hours; h) the mixture is filtered to eliminate bacterial lumps, by means of a pump or tanks with inert gas can be maintained with pressure gauges and valves to regulate the pressure; i) an ultrafiltration is then carried out at volumes ranging from 10 to 100 It; the strains are then cultured, j) filtered, k) the toxoid preparation is rehydrated and inactivated with formaldehyde, at a temperature of 4 ° C in 24 hours; 1) depending on the concentration of proteins aluminum hydroxide is added, finally it is packed in bottles of 25, 50 and 100 doses of 2.0 ml cadauna and stored between 2 and 8o C. Process for preparing a toxoid-immunogen of actinobacillus pieuropneumoniae based on fractional ufiltration for the prevention of porcine contagious pleuropneumonia, in accordance with clause 1, characterized in that the supernatants of actinobacillus pieuropneumoniae have hemolytic activity, hemolycin is a protein , which is induced by calcium and acid, is immunogenic and immunological protective. Process for preparing a toxoid-immunogen of actinobacillus pieuropneumoniae based on fractional ultrafiltration for the prevention of porcine contagious pleuropneumonia, in accordance with clause 1, characterized in that a hemoiicin or cytokine with a molecular weight of 104 to 1 10 Kd is present in serotypes 1,5,9,10 and 11, which have been attributed the properties of being hemorrhagic and nacrotic, which is called Apx I. Process for preparing a toxoid-immunogen of actinobacillus pieuropneumoniae based on fractional ultrafiltration for the prevention of porcine contagious pyeuropneumonia, in accordance with clause 1, characterized in that another hemolysin of molecular weight 103 Kd, is present in the serotypes of 1,2 , 3,4,5,6,7,8,9,1 1 and 12, which produces the Apx II cytosine. Process for preparing a toxoid-immunogen of actinobacillus pieuropneumoniae based on fractional ultrafiltration for the prevention of porcine contagious pleuropneumonia, in accordance with clause 5, characterized in that cytokine 3 or Apx III, is not homozygous but is strongly cytotoxic. Serotypes that produce it are: 2,3,4,6 and 8. Process for preparing a toxoid-immunogen of actinobacillus pieuropneumoniae based on fractional ultrafiltration for the prevention of porcine contagious pleuropneumonia, in accordance with clause 1, characterized in that the supernatant of the culture actinobacillus pieuropneumoniae, whose toxic effect on adherent cells of the pulmonary lavage, as well as in circulating monocytes, this exotoxin has a molecular weight of 120 Kd, known as the cytokine 3. Process for preparing a toxin-immunogen of actinobacillus pieuropneumoniae based on fractional ultrafiltration for the prevention of porcine contagious pneumoniae, in accordance with clause 1, characterized in that filters are used to eliminate bacterial clumps and for the total elimination of bacteria. cartridge and to reach the necessary pressure is resorted to a peristaltic pump. Process for preparing a toxoid-immunogen of actinobacillus pieuropneumoniae based on fractional ultrafiltration for the prevention of porcine contagious pleuropneumonia, in accordance with clause 1, characterized in that an ultrafiltration system is required. Process to prepare a toxoid-immunogen of actinobaciííus pleuropneumopiae based on fractional ultrafiltration for the prevention of porcine contagious pleuropneumonia, in accordance with clause 1, characterized in that immunogens are prepared from the culture supernatant result of the strains. Process to prepare a toxoid-immunogen of actinobacillus pieuropneumoniae based on fractional ultrafiltration for the prevention of porcine contagious pleuropneumonia, in accordance with clause 1, characterized in that for the case of actinobaciílus pieuropneumoniae of the serotype 1 or 7 class the time of incubation will be from 24 to 26 hours. Process for preparing a toxoid-immunogen of actinobacillus pieuropneumoniae based on fractional ultrafiltration for the prevention of porcine contagious pleuropneumonia, in accordance with clause 1, characterized in that for the case of actinobacillus pieuropneumoniae of the class of serotype 3 the incubation time will be from 4 to 6 pm Process for preparing a toxoid-immunogen of actinobacillus pieuropneumoniae based on fractional ultrafiltration for the prevention of porcine contagious pleuropneumonia, in accordance with clause I, characterized by the preparation of the immunological and depending on the concentration of proteins, is added a adyubant which is aluminum hydroxide. Process to elaborate a toxoid-immunogen of actinobacillus pieuropneumoniae based on ultrafiltration, fractionated for the prevention of porcine contagious pleuropneumonia, in accordance with clause 1, characterized in that toxoid 3 is packaged in flasks that store 25.50 and 100 doses of 2.0 ml . and it is stored at a temperature that goes from 2 ° C to 8 ° C, until the moment of its use, afterwards it will only be kept in refrigeration.