CZ2020249A3 - Non-pathogenic live strain of Escherichia coli CCM 8982 for oral vaccination of livestock - Google Patents

Abstract

The solution concerns a new non-pathogenic live strain of Escherichia colipro oral vaccination of livestock, which is intended for vaccination of freshly hatched poultry and newly weaned piglets and is stored in the Collection of Microorganisms in Brno, CZ, under the designation CCM 8982, and its use.

Description

Non-pathogenic live strain of Escherichia coli CCM 8982 for oral vaccination of livestock

Field of technology

The solution concerns a new non-pathogenic live strain of Escherichia coli (E. coli) CCM 8982 as a probiotic administered orally, especially in day-old chicks and weaned piglets after transport to the production hall, and which is based on rapid occupancy of the digestive tract, especially day-old chicks by E. coli CCM 8982, which by its presence performs the function of a probiotic in the digestive tract and thus protects it for about 14 days from colonization of the digestive tract by other bacteria dangerous to young.

State of the art

E. coli strains are one of the most common bacteria and are physiological only in the intestines in monogastres, such as poultry, as part of the intestinal microflora.

It is generally known that colonization of the intestinal tract in chickens by pathogens significantly reduces feed conversion and poultry health in general. In addition, infections in poultry halls, where there are usually one age in large numbers, spread very quickly. Pathogenic bacteria multiply in the digestive tract of infected poultry very quickly, and infectious birds spread the infection almost immediately using faeces throughout the farm. This causes great economic damage.

It can be clearly stated that probiotics can partially replace antibiotics in the fight against infectious diseases. Probiotics essentially prevent the colonization of an individual's intestine by another pathogen by modulating the mucosomal immune system, such as clostridia. They thrive in the mucus produced by inflammation, so controlling the formation of mucus-inflammation essentially controls clostridial infection. This is clearly demonstrated by Yanlong Jiang et al. , Pathog 11: 455-463, [PubMed: 15867585], who were able to control acute and chronic colitis in mice using a non-pathogenic strain of E. coli Nisslel917. Using the non-pathogenic strain E. coli Nissle 1917, they were able to eliminate Clostridium perfringens, a well-known and abundant gram-positive pathogen responsible for food spoilage and toxicity, necrotic enteritis, gangrene and in some cases the death of living organisms, in the poultry digestive tract. Non-pathogenic E. coli, Dis. 2014 April; 70 (3) and Kamada et al., Et al. Inflamm Bowel Dis. 2005; Nissle strain 1917, is a well-characterized probiotic E. coli strain that has a clear positive effect. In the study, the authors were able to show the positive effect of this E. Coli strain on growth, biofilm production and the inflammatory response of cytokines. The competitive effect between non-pathogenic E. coli, strain Nissle 1917, and Clostridium perfringens, type A strain CP4, in in vitro cultivation clearly showed that the presence of this strain inhibits CP4 growth, which further results in a reduction in atoxin gas production (α-toxin aNetB toxin) in the body.

Thus, in general, the use of a non-pathogenic strain of E. coli clearly inhibits the adhesion of pathogens and prevents their invasion into the digestive tract. The mechanism for doing so is not yet entirely clear, and it is widely accepted that probiotics eliminate pathogens by not giving them room to settle or access nutrients, and it is also known that they often surprisingly produce for pathogens. inhibitory substances such as lactic acid bacteria, other organic acidic substances, hydrogen peroxide and bactericidal substances.

The positive effect of the non-pathogenic E. coli strain EcN, and the strain (AIEC) (Boudeau, et al., 2003), has been shown, for example, to eliminate the occurrence of, for example, Salmonella (Schierack, et al., 2011), (Mital & Garg, 1995). , Bogovic-Matijasic, et al., 1998, Salminen, et al., 2010, Dobson, et al., 2012), where an adherent - invasive non-pathogenic strain of E. coli - AIEC which are targeted pathogens.

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Non-pathogenic E. coli strains EcN have been demonstrated to be able to exclude several pathogenic bacteria from the settlement, such as:

Enterotoxigenic and enteropathogenic E. coli strains in biofilm formation (Hancock, et al., 2010). If non-pathogenic E. coli occupies a place in the gut of gram-negative bacteria, which are typically pathogenic, this will further allow the growth of gram-positive bacteria, such as Lactobacillus, and other same bacteria increase the synthesis of a wide range of beneficial metabolites that increase microvilli in the gut. efficiency of nutrient intake from feeding. A clear benefit is shown in the conversion of feed. Demand for non-pathogenic E. coli strains is still rising.

The essence of the invention

These shortcomings are eliminated by the non-pathogenic live strain Escherichia coli for oral vaccination of farm animals, which is intended for oral vaccination of pups and is stored in the Collection of Microorganisms in Brno, CZ, under the designation CCM 8982.

The non-pathogenic strain Escherichia coli CCM 8982 according to the invention is characterized in that it forms the basis for the production of a non-pathogenic vaccine for gastrointestinal protection in young farms, 1 ml of aqueous vaccine containing at least 2x10 ⁶ bacteria of this strain.

The non-pathogenic live strain of Escherichia coli CCM 8982 according to the invention is further characterized in that in aqueous form with a concentration of at least 2x10 ⁶ bacteria of this strain in 1 ml of water it is applied before or at the same time when freshly hatched poultry or weaned piglets.

The non-pathogenic live strain of Escherichia coli CCM 8982 according to the invention is also characterized in that it is applied in the form of an aqueous gel with a concentration of at least 2x10 ⁹ bacteria of this strain per ml in hatcheries on a belt.

The non-pathogenic live strain of Escherichia coli CCM 8982 according to the invention is also characterized in that it is intended for the protection of day-old chicks selected from the group of chickens, turkeys, pheasants, waterfowl or weaned piglets.

The non-pathogenic live strain of Escherichia coli CCM 8982 according to the invention is further characterized in that it is lyophilized with the addition of maltodextrin as a protective and is then distributed in closed plastic petri dishes and dissolved in water before use.

The non-pathogenic strain Escherichia coli CCM 8982 according to the invention as a probiotic preparation for pups was obtained by long-term selection on a blood agar plate from the hemolytic strain Escherichia coli O83: K24: H31 (from ColinfantNew Bom). This new strain of Escherichia coli CCM 8982 is characterized by the deletion of part of the hemolysin operon (GenBank: AM261284.1) in the region 8186-11440, i.e. 3254 base pairs. Due to the loss of hemolytic activity, the inventor had the entire operon sequenced and the results show that part of the hlyC gene, the entire hlyA gene and a small part behind the hlyA gene were deleted. In the new non-pathogenic strain Escherichia coli CCM 8982, the gene for hlyC posttranslational modification hlyA and the whole gene hlyA are excluded due to selection, therefore the newly isolated strain of this bacterium cannot be hemolytic and clearly different from Escherichia coli O83: K24: H31 (from ColinfantNew Bom ). The sequence of the entire hemolysin operon, including the indicated deleted portion, is shown in Figure 4.

The non-pathogenic live strain of Escherichia coli CCM 8982 according to the invention was isolated by repeated passage of cultures in which non-hemolytic colonies were isolated. The application of this non-pathogenic strain of Escherichia coli CCM 8982 according to the invention, which the inventor

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E.

The presence of Escherichia coli CCM 8982 according to the invention in the digestive tract of pups causes them to occupy a place in the gut of gram-negative bacteria, which are typically pathogenic, which in turn allows the growth of gram-positive bacteria such as Lactobacillus and other bacteria that increase broad scales of beneficial metabolites that increase microvilli in the gut and increase the efficiency of nutrient intake from feeding. There will be substantial protection of the young from unwanted disease, which is of favorable national economic importance.

If we take a critical look at what the occupation of the digestive tract of fresh pups by the non-pathogenic live strain Escherichia coli CCM 8982 according to the invention allows, then the following key factors can be highlighted:

• Protection of young from early infection with pathogenic bacteria • Support for the formation of natural non-pathogenic microflora in the intestines • Protection and saving of bedding

These three main factors of using the non-pathogenic strain Escherichia coli CCM 8982 according to the invention as a new probiotic for pups are extremely important for production in prospective poultry and pigs without antibiotics, as they eliminate the main cause of antibiotics in day-old pups, especially poultry or weaned piglets. Another indisputable benefit of the new strain is the very good quality of the bedding, which does not need to be changed.

Clarification of drawings

In the accompanying drawings, Figure 1 shows a graph 1 which compares the weights of chicks at standard fattening times.

Separate colonies of non-hemolytic Escherichia coli CCM 8982 are clearly visible in the photographs in Figure 2.

The photographs in Figure 3 show the differences between the new isolated non-hemolytic strain Escherichia coli CCM 8982 - (already a new preparation in the vaccine) and the hemolytic strain E. coli Zhly +, which is the commonly used Colinfant New Bom.

In FIG. 4 is a schematic representation of the sequence of the entire hemolysin operon, including the indicated deleted portion, in the novel non-pathogenic strain of Escherichia coli CCM 8982 according to the invention.

The non-pathogenic strain Escherichia coli CCM 8982 according to the invention was prepared by the inventor in the laboratory of Hena, s.r.o., Dolní Bořek, CZ, and successfully tested in practice on a private farm near Hradec Králové, CZ.

The following non-pathogenic strains of Escherichia coli CCM 8982 according to the invention only illustrate but do not limit them.

Examples of embodiments of the invention

Example 1

Isolation of non-hemolytic non-pathogenic strain Escherichia coli CCM 8982.

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The non-pathogenic live Escherichia coli strain CCM 8982 for oral vaccination of livestock according to the invention was isolated by the inventor from Colinfant New Bom (Dyntec). One dose of Colinfant contains Escherichia coli Zhyla - criodesiccata (O83: K24: H31) in the number of 0.8x10 ⁸ to 1.6x10 ⁸ bacteria.

The non-pathogenic strain Escherichia coli CCM 8982 according to the invention as a probiotic preparation for pups was obtained by selection on an agar plate from Escherichia coli Žhly + (from Colinfant New Bom), which was seeded on a 5% blood LB plate (solid LB medium), and when after about 25 passages, a non-hemolytic non-pathogenic strain of Escherichia coli CCM 8982 was obtained. In Fig. 2 and Fig. 3, separate colonies of non-hemolytic Escherichia coli CCM 8982 are clearly visible. The non-hemolytic colony was transferred to a new LB plate with 5% blood. Next, the bacteria were propagated in shaken culture in liquid LB medium at 37 ° C, the bacteria were separated from the medium by centrifugation and washed in PBS. Bacterial suspensions with a concentration of 2xl0 ⁹ bacteria / ml (equivalent to OD = 2.5 at 600 nm) was then administered orally to poultry chicks at 50 μΐ directly into the beak. When applied to drinkers in the form of an aqueous vaccine, a concentration of 2x10 ⁶ bacteria per 1 ml was used.

Experiment 1

Application of Escherichia coli CCM 8982 for mutual comparison with Escherichia coli Žhly + (Colinfant) and poultry control group. Oral administration of the preparations in both groups on the first day after birth in a dose of 0.05 ml (concentration 2x10 ⁹ / ml). The control group was without application, in each group there were 20 one-day-old broilers of the ROSS 308 type. The fattening took place according to standard conditions for 35 days. At each weighing, faeces were collected to determine the presence of applied non-pathogenic Escherichia coli CCM 8982.

Results:

Group 1, where the new isolated Escherichia coli CCM 8982 was applied, and the control group clearly had the highest weight and the two groups did not differ from each other. In the group where Colinfant was applied, a lower weight of chickens was recorded. No deaths were reported in group 1 and control groups, and 1 deaths in the Colinfant group. The percentage of mortality in such a small population did not make sense to evaluate, and therefore it was tried to test this product in a larger number of turkeys, ie the breeding of the most delicate species of poultry, where the breeder thought there would be a clear positive consequence of early gastrointestinal occupancy Escherichia coli CCM 8982 .

Experiment 2

In experimental group A), 2,200 one-day-old turkeys were stopped for deep litter. The application of Escherichia coli CCM 8982 with a final concentration of 2x10 ⁶ was carried out in the hat drinkers at the time of placing the imported one-day-old turkeys in the hall.

The control group was group B), which was in the same hall and in which Escherichia coli CCM 8982 was not applied, and it was the same number of 2200 turkeys.

The following turkey deaths were recorded here in 14 days:

Group A, Escherichia coli CCM 8982 20 turkeys = 0.7%

Group B, control 41 turkeys = 1.86%

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Experiment 3

In experimental group A), 1400 one-day-old turkeys were stopped for deep litter. The application of Escherichia coli CCM 8982 with a concentration of 2x10 ⁶ was carried out in the hat drinkers at the time of placing the imported one-day-old turkeys in the hall. The control group was group B), which was in the same hall and in which Escherichia coli CCM 8982 was not applied, and there were 800 turkeys.

During the 14 days there was a total mortality of turkeys as follows:

Group A, Escherichia coli CCM 8982 15 turkeys = 0.93%

Group B, control 37 turkeys = 4.6%

Extremely important results of these practical experiments on a private farm near Hradec Králové, CZ, were the fact that the groups where the new non-pathogenic strain Escherichia coli CCM 8982 according to the invention was used did not have to change or modify the litter. There was a very clear consequence of the application of the new strain - the turkeys did not have diarrhea at all, so there was no wetting of the litter, as was the case in the control groups, where the diarrhea was reflected in the number of deaths in all three trials. The results also show that this non-hemolytic strain of Escherichia coli CCM 8982 is able to remain in the gut of poultry for at least 14 days after its application, which in turn protects the individual from pathogenic bacteria and results in a significant reduction in deaths and elimination of diarrhea. .

According to the breeders, the turkeys in which the Escherichia coli strain CCM 8982 according to the invention was applied did not have diarrhea during the first 14 days of fattening and the mortality of the turkeys was significantly reduced. The breeders also highly appreciated the unpretentiousness of poultry farming, as the litter was not covered in groups where the Escherichia coli strain CCM 8982 according to the invention was used.

Experiment 4

In experimental group A), 500 one-day-old turkeys were stopped for deep litter. The application of Escherichia coli CCM 8982 with a concentration of 2x10 ⁶ was carried out in the hat drinkers at the time of placing the imported one-day-old turkeys in the hall. In experimental group B), 500 one-day-old turkeys were stopped for deep litter. Escherichia coli CCM 8982 was in this case pre-lyophilized and before application this mixture was dissolved in drinking water and in the same amount as in group A was then applied to the hat drinkers at the time of placing the imported one-day-old turkeys in the shed. It was also a control group, which was in the same hall and in which Escherichia coli CCM 8982 was not applied, and there were 1000 turkeys.

During the 14 days there was a total mortality of turkeys as follows:

Group A, Escherichia coli CCM 8982 5 turkeys = 1%

Group B, lyophilized. Escherichia coli CCM 8982 6 turkeys = 1.2%

Group C, control group 45 turkeys = 4.5%

Experiment 5

Oral administration of products in both groups on the first day after birth (concentration in drinking water 2x10 ³ / ml).

In experimental group A), 700 one-day-old turkeys with a final concentration of Escherichia coli CCM 8982, about 2x10 ³ / ml were stopped on deep litter. The application of the product was carried out in hat drinkers at the time of placing the imported one - day - old turkeys in

-5CZ 2020 - 249 A3 hall. The control group was group B), which was in the same hall and in which Escherichia coli CCM 8982 was not applied, and there were 700 turkeys.

During the 14 days there was a total mortality of turkeys as follows:

Group A, Escherichia coli CCM 8982 Group B, control of turkeys = 3.57% turkeys = 4.14%

The litter in the experimental group was similar to the control.

The experiment clearly showed the need for a higher and recommended dose per 1 ml of drinking water: at least 2x10 ⁶ bacteria of this new strain.

Experiment 6

In experimental group A), 600 one-day-old pheasants of both sexes were stopped for deep litter. The application of Escherichia coli CCM 8982 according to the invention with a concentration of 2x10 ⁶ was carried out in hat drinkers at the time of placing the pheasants in the hall. Control group B) was in the same number in the same hall from the same hatching, but was not treated with Escherichia coli CCM 8982. In both groups, the antibiotic normally prescribed by a veterinarian during pheasant breeding was not used. The experiments took place in part on a farm in Maršovice and Mezn, CZ. It was clearly shown that in group A), where the new non-pathogenic strain Escherichia coli CCM 8982 according to the invention was used, the litter was in very good condition, animals without diarrhea and no antibiotics were needed and this fact is very important as it is in line with EU policy on the use of antibiotics in poultry. In total, this experiment was repeated twice with a different number of animals with the same result.

During 14 days there was a total death of pheasants as follows:

Group A, Escherichia coli CCM 8982 up to 1%

Group B, control of 30 pheasants = 5%

Example 2

In the experimental group A), stopped with 100 pieces of freshly weaned piglets with an average weight of 27 kg at the age of about one month and were administered Escherichia coli CCM 8982 according to the invention at a concentration of 2xl0 ^9th The application of Escherichia coli CCM 8982 was via a medicator in drinking water. The delivered dose was dissolved in 5 liters of water and added to the drinking schedule at a concentration of 2% by weight. The dose was drunk in 4 to 6 hours. There were 1 death within 14 days. After 14 days of transport, the average weight of all piglets in this group was 40 kg. Above-average gains were achieved during the first 14 days of fattening. No antibiotics or other treatments were used in this group.

In control group B, which also consisted of 100 freshly weaned piglets of the same age, the average weight was 37 kg and 4 deaths.

Industrial applicability

The new non-pathogenic strain Escherichia coli CCM 8982 for oral vaccination of livestock significantly reduces the mortality of freshly hatched pups and weaned piglets, as it protects flocks of poultry and herds of piglets from early infection by pathogenic bacteria and promotes the formation of natural non-pathogenic microflora in the intestines. At the same time, there is no need to change and get bedding, which is now in short supply due to the dry weather.

Claims (6)

PATENT CLAIMS 1. Non-pathogenic live strain of Escherichia coli for oral vaccination of livestock, which is intended for vaccination of freshly hatched poultry and newly weaned piglets and is stored in the Collection of Microorganisms in Brno, CZ, under the designation CCM 8982. Use of a non-pathogenic live strain of Escherichia coli CCM 8982 according to claim 1 for the production of a non-pathogenic vaccine for the protection of the digestive tract in young farms, wherein 1 ml of aqueous vaccine contains at least 2x10 ⁶ bacteria of this strain. Use of a non-pathogenic live strain of Escherichia coli CCM 8982 according to claim 1 for the production of an aqueous solution with a concentration of at least 2x10 ⁶ bacteria of this strain in 1 ml of water for application before or at the same time when placing freshly hatched chicks in breeding premises. Use of a non-pathogenic live strain of Escherichia coli CCM 8982 according to claim 1 for the production of an aqueous gel with a concentration of at least 2x10 ⁹ / ml of bacteria of this strain for application in hatcheries on the belt by spraying pups in transport crates. Use of a non-pathogenic live strain of Escherichia coli CCM 8982 according to claim 1 for the protection of day-old chicks selected from the group of chickens, turkeys, pheasants, poultry chicks and weaned piglets. Use of a non-pathogenic live strain of Escherichia coli CCM 8982 according to claim 1 for the preparation of a solution, wherein said strain is lyophilized with maltodextrin as binder and then distributed in closed petri dishes and diluted in water and applied either by gel spraying in hatcheries before use. and maternity wards, or directly into drinking fountains.