RU2462263C2 - Use of bacterium clostridium perfringens type c for producing vaccine - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to veterinary science and biotechnology. The supernatant of the culture Clostridium perfringens type C is used for producing a vaccine for protection of mature pigs and piglets against the infection Clostridium perfringens type A.

EFFECT: use of the inventions leads to the considerable protection of piglets against the infection caused by Clostridium perfringens type A and improvement of breeding sow health.

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Description

The present invention relates to the use of the bacterium Clostridium perfringens type C for the manufacture of a vaccine.

Clostridia are gram-positive spore-forming anaerobic bacteria. Clostridia are widely known as pathogens in both domestic and wild animals. A review of the various clostridia and animal diseases that they cause is provided by Songer, J.G. at The Clostridia; Chapter 10: Clostridial Diseases of Animals, Molecular biology and Pathogenesis, ed. Rood, J.I. et al., Academic Press Ltd (1997) ISBN 0-12-595020-9.

Of the currently known species of clostridia, Clostridium perfringens is sometimes considered the most common bacterial pathogen, and of all types of clostridia, it is by far the most important cause of intestinal clostridial disease in domestic animals. The species Clostridium perfringens is divided into five types; types of AE, based on the major toxins that they produce.

Clostridium perfringens types A and C and Clostridium difficile are the main intestinal clostridial pig pathogens.

Clostridial intestinal infections in pigs are more specifically described by Songer, J.G. and Uzal, F.A. (J. Vet. Diagn. Invest 17: 528-536 (2005)).

Clostridium perfringens type C infects pigs, cattle, chickens, humans, sheep, dogs, and horses. Piglets are most vulnerable to C-type Clostridium perfringens infection. In 1-2 day old piglets, the infection results in a percentage of morbidity in the range of 30-50% and a mortality rate of 50-100% due to the disease. Older piglets (1-2 weeks) may have a longer infection. Pigs are a common source of infection.

A common approach for protecting piglets is to vaccinate pigs with Clostridium perfringens type C vaccine to induce antibodies that are passed on to their offspring through colostrum. This leads to a high level of protection in piglets at a time when they are most vulnerable to clostridial infection. Such vaccines are based on toxoids. They are usually detoxified supernatant vaccines in which no cells are present. Toxoid vaccines are described by ia Springer, S. and Selbitz, H.-J. (FEMS Immun. And Medical Microbiol. 24: 333-336 (1999)).

Clostridium perfringens type A infects lambs, goats, calves, chickens, pigs, horses, dogs, and humans. In mature pigs, Clostridium perfringens type A is a component of the normal intestinal flora. In this regard, it is currently assumed that the presence of Clostridium perfringens type A in the intestine is not adversely affected in mature pigs.

However, it can cause porcine necrotic enterocolitis in newborns and weaned pigs. Clostridium perfringens type A vaccines are rare. Currently, there is only one (conditionally licensed) commercially available vaccine.

One of the reasons for this may be the fact that, as noted above, type A Clostridium perfringens is a component of normal flora. In this regard, one would expect that it does not activate the immune system. As a consequence, one would expect that the production of antibodies against Clostridium perfringens type A would not be induced. If antibodies were induced, then Clostridium perfringens type A would not remain in the intestine, let alone be a component of the normal flora. As a further consequence, of course, one would expect that sows would not pass any protection to offspring through colostrum. For any reason, it is clear that Clostridium perfringens type A does not induce sufficient immune stimulation.

It has now surprisingly been found that Clostridium perfringens type C vaccines containing a detoxified culture supernatant are significantly capable of stimulating cross-immunity against Clostridium perfringens type A infection.

More unexpectedly, vaccination of sows with the supernatant of a Clostridium perfringens type C culture resulted in significant protection of their piglets against a Clostridium perfringens type A infection.

Even more unexpectedly, it was found that even the productive functions of sows improve after this vaccination. This is unexpected, since it was usually assumed, as noted above, that mature pigs, such as sows, are not adversely affected by type A Clostridium perfringens infection. This finding for the first time proves that the opposite is true.

In this regard, the first embodiment of the present invention relates to the use of the bacterium Clostridium perfringens type C for the production of a vaccine to protect mature pigs against infection with Clostridium perfringens type A.

A second embodiment of the present invention relates to the use of the bacterium Clostridium perfringens type C for the manufacture of a vaccine for the protection of sows against infection with Clostridium perfringens type A.

A third embodiment of the present invention relates to the use of the Clostridium perfringens type C bacterium for the production of a vaccine for sows to protect piglets against Clostridium perfringens type A infection.

A fourth embodiment of the present invention relates to the use of a bacterium Clostridium perfringens type C for the manufacture of a vaccine for the protection of piglets against infection with Clostridium perfringens type A.

As will be understood by one of skill in the art, the actual use of the Clostridium perfringens type C bacterium for vaccine production is a two-stage process: the Clostridium perfringens type C bacterium is used for growth in a culture medium to obtain a culture supernatant, which is the basis for the vaccine production in accordance with invention.

In this regard, another embodiment of the present invention relates to the use of a supernatant of a Clostridium perfringens type C culture for the manufacture of a vaccine for protecting mature pigs against Clostridium perfringens type A infection.

Another embodiment of the present invention relates to the use of a Clostridium perfringens type C culture supernatant for the manufacture of a vaccine for protecting sows against Clostridium perfringens type A infection.

Another embodiment of the present invention relates to the use of a supernatant of a Clostridium perfringens type C culture for the production of a vaccine for sows to protect piglets against a Clostridium perfringens infection A.

Also, one embodiment of the present invention relates to the use of a supernatant of a Clostridium perfringens type C culture for the manufacture of a vaccine for protecting piglets against Clostridium perfringens type A infection.

Within the type of toxin Clostridium perfringens type C, a separation can be made between strains that produce beta-2 toxin and strains that do not produce it. About 40% of all strains of Clostridium perfringens type C produce this toxin.

It has been found that the bacterium Clostridium perfringens type C, which produces beta-2 toxin, is even more suitable than the bacterium Clostridium perfringens type C, which does not produce beta-2 toxin. Such strains and their identification are described, in particular, by Fisher, DJ and others (Inf. And Immun. 74: 5200-5210 (2006)).

Thus, a preferred form of this embodiment relates to an application in which the bacterium Clostridium perfringens type C is used, which produces a beta-2 toxin.

Vaccines produced as described above are by no means the only vaccines administered to pigs in the process of commercial pig breeding. Clostridial vaccines are often given at about the same time as vaccines against other pathogens and swine viruses.

Thus, in another preferred embodiment, a vaccine produced as described in the present invention further comprises one or more antigens derived from pathogens or swine viruses, antibodies against these antigens or genetic information encoding such antigens.

Such organisms or viruses are preferably selected from the group of Pseudomonas rabies virus, PRRS virus, swine flu virus, swine parvovirus, infectious gastroenteritis virus, rotavirus, Escherichia coli, Erysipelothrix rhusiopathiae, Bordetella bronchiseptica, Salmonella typhimurium, Salmonellaociocleochemocidaera, Pulmonellaochemophidaidae, Salmonellaociochemophidaidae, Salmonellaociophidaidae, suis, Mycoplasma hyopneumoniae, Brachyspira hyodysenteriae and Actinobacillus pleuropneumoniae .

In this regard, a more preferred form of this embodiment relates to a vaccine produced as described in the present invention, which additionally contains one or more antigens derived from pathogenic organisms or viruses of pigs, where these organisms or viruses are selected from the group of rabies virus, PRRS virus, swine flu virus, swine parvovirus, infectious gastroenteritis virus, rotavirus, Escherichia coli, Erysipelothrix rhusiopathiae, Bordetella bronchiseptica, Salmonella typhimurium, Salmonella choleraesuis, Haemophilus parasuis, Pasteurella multococuma, uis, Mycoplasma hyopneumoniae, Brachyspira hyodysenteriae and Actinobacillus pleuropneumoniae .

Following infection with Clostridium perfringens, piglets often suffer from Escherichia coli infection . Escherichia coli is the cause of another severe intestinal disease in piglets. In this regard, piglets or pregnant sows are often vaccinated with Escherichia coli vaccines. E. coli vaccines are commonly used in the art and are commercially available. In this regard, a combination vaccine containing both the supernatant of the Clostridium perfringens type C culture and the components of the Escherichia coli vaccine is very desirable because it protects against Clostridium perfringens type C, Clostridium perfringens type A and Escherichia coli infections.

In this regard, the most preferred form of this embodiment relates to a vaccine produced as described in the present invention, which further comprises one or more antigens derived from the pathogenic organism of pig Escherichia coli .

The invention is illustrated, without limitation, in the following examples.

EXAMPLES

Obtaining antigen Clostridium perfringens type C

The standard strain Clostridium perfringens type C, strain 587 (also called CWC 1 / S, from the collection of Weybridge UK), was cultured in a fermenter until the onset of the stationary phase of growth. The culture was centrifuged and the pellet containing bacterial cells was separated. Formaldehyde was added to the supernatant to a final concentration of 0.5% v / v to inactivate toxins, followed by concentration by ultrafiltration and 0.2 μm filtration.

Vaccine preparation

The vaccine was prepared by mixing the Clostridium perfringens type C antigen and a tocopherol-based adjuvant. Each vaccine dose (2 ml) contained 20 μl of a 17-fold concentrated Clostridium perfringens type C culture antigen toxoid supernatant.

The vaccine was in accordance with the European Pharmacopoeia 0363 monograph ( Clostridium perfringens vaccine for veterinary use) regarding the safety and potency of type C vaccines.

Additionally, in this experiment, purified E. coli antigens (F4ab, F4ac, F5, and F6, fimbrial and heat labile toxin) were added to suppress possible E. coli infection. These components are known in the art, and one skilled in the art would know how to prepare a vaccine based on these components.

Alternatively, despite this, a qualified person could use a commercially available and ready-made E. Coli vaccine such as Porcilis Porcoli DF (available through Intervet Int. B.V., Wim de Korverstraat 35, Boxmeer, Netherlands).

The control vaccine contains the same components, with the exception of the Clostridium perfringens type C antigen, which was not present in the control group.

Sow protection

A Dutch pig farm that had a clinical outbreak of type A Clostridium perfringens was selected for a randomized, double-blind, field-effect study. It was found that the Clostridium perfringens type A isolate from the farm produces both an α-toxin and a β2-toxin.

Sows were vaccinated with IM during pregnancy approximately 6 and 2 weeks before farrowing, either with a vaccine containing only E. coli antigens (control group) or with a vaccine containing E. coli antigens and Clostridium perfringens type C antigen (test group). The vaccines were color coded (2x test vaccine, 1x control vaccine) to provide a blind method, and the distribution of pigs into groups was approximately 2: 1 (Table 1).

Table 1
Group sizes Group Number of sows Number of live-born piglets per sow Total number of piglets The control 41 11.6 453 Test 77 12.1 904

From the day of the first vaccination to the time of weaning piglets from sows (3-4 weeks after farrowing) sows were observed every 3 days for general health, feed intake and diarrhea. To facilitate statistical analysis, a clinical scoring system was used (table 2). Piglets born to vaccinated pigs were observed daily during the first week of life, once every three days from one week before weaning and weekly thereafter until the end of the nursery period. General health, feed intake, diarrhea, and mortality were recorded (table 2).

table 2
Scoring system used for sows and piglets Clinical Score Behavior Appetite Feces consistency Feces Composition 0 Normal Normal Normal Normal one Depressive Slight loss of appetite Soft Mucus present 2 Lethargic Severe loss of appetite Liquid There is mucus with blood 3 Dying Does not suck Watery Intestinal bleeding is present

results

Table 3
Clinical observations of sows Score (N) 0 one 2 3 Average General behavior The control 687 99 51 twenty 0.30 Test 1623 33 0 0 0.02 Appetite The control 597 139 71 49 0.50 Test 1598 47 9 0 0.04 Feces consistency The control 385 250 153 68 0.89 Test 1564 67 17 7 0,07 Feces Composition The control 780 71 four 0 0.09 Test 1647 8 0 0 0.01

Table 3 shows the effect of vaccination with a vaccine containing Clostridium perfringens type C antigens compared with vaccination with a vaccine without Clostridium perfringens type C antigens.

As clearly follows from table 3, vaccination with a vaccine containing antigens of Clostridium perfringens type C, led to a significant improvement in the health status of sows.

Protecting offspring of vaccinated sows

Mortality in the litter of sows vaccinated with a vaccine containing Clostridium perfringens type C antigens was also compared with that in the litter of sows vaccinated with a vaccine without Clostridium perfringens type C antigens.

As clearly follows from table 4, the mortality in the litter of sows vaccinated with a vaccine containing Clostridium perfringens type C antigens was significantly lower than in the control group.

In addition, Table 5 shows that piglets from sows that were vaccinated with a vaccine containing Clostridium perfringens type C antigens had significantly lower clinical scores than the control group.

Table 4
Weekly mortality in the control and test litter Age (days) The control Test 0-6 29th 22 7-13 3 3 14-21 7 2 21-27 6 one 28-34 2 one 35-41 0 0 42-48 one one 49-55 one one 56-62 one 0 63-70 0 0 Total (%) 50 (11.04) 31 (3.43)

Table 5
Clinical observations of piglets Score (N) 0 one 2 3 Average General behavior The control 336 232 137 73 0.93 Test 1159 271 55 eleven 0.28 Appetite The control 426 166 128 58 0.77 Test 1336 125 31 four 0.13 Feces consistency The control 544 118 71 40 0.49 Test 1419 48 17 12 0.08 Feces Composition The control 636 104 31 6 0.24 Test 1414 70 10 one 0.06

Conclusion : the data demonstrate that vaccination of sows with a vaccine containing Clostridium perfringens type C antigens leads to a significant improvement in the health status of both vaccinated sows and their offspring under the threat of an outbreak of Clostridium perfringens type A.

Claims (8)

1. The use of Clostridium perfringens type C culture supernatant for the production of a vaccine for protecting mature pigs from Clostridium perfringens type A infection. 2. Use of a Clostridium perfringens type C culture supernatant for the production of a vaccine for protecting sows against Clostridium perfringens type A infection. 3. Use of Clostridium perfringens type C culture supernatant for vaccine production for sows to protect piglets from Clostridium perfringens type A infection. 4. Use of a Clostridium perfringens type C culture supernatant for vaccine production to protect piglets from Clostridium perfringens type A infection. 5. The use according to claims 1 to 4, characterized in that they use the bacterium Clostridium perfringens type C, which produces a beta-2 toxin. 6. The use according to claims 1 to 4, characterized in that for the production of the indicated vaccine, one or more antigens derived from pathogens or pig viruses, antibodies against these antigens, or genetic information encoding such antigens are additionally used. 7. The use according to claim 6, characterized in that said pathogen or virus is selected from the group of rabies virus, PRRS virus, swine flu virus, swine parvovirus, infectious gastroenteritis virus, rotavirus, Escherichia coli, Erysipelothrix rhusiopathiae, Bordetella bronchiseptica, Salmonella cholerarara , Salmonella typhimurium, Haemophilus parasuis, Pasteurella multocida, Streptococcus suis, Mycoplasma hyopneumoniae, Brachyspira hyodysenteriae and Actinobacillus pleuropneumoniae. 8. The use according to claim 6, characterized in that the pig pathogen is Escherichia coli.