Bacterial preparation for treating the hatchery environment and use thereof

Abstract

The present invention relates to a bacterial preparation for the treatment of hatchery environment, which contains as an active ingredient a mixture of bacteria Chryseobacterium indologenes and Sphingobacterium multivorum. The invention further relates to the use of said preparation for the preventive improvement of the physiological condition of poultry, in particular for the prevention of infections of poultry by opportunistic pathogens selected from the group comprising E. coli, Clostridium sensu stricto. Enterococcus, Klebsiella, Enterobacter and Pseudomonas.

Description

[0001] Bacterial preparation for treating the hatchery environment and use thereof

[0002] Field of Art

[0003] The present invention provides a bacterial preparation for suppression of undesirable microbiota in hatcheries and subsequently improvement of the production parameters of poultry after placement on farms.

[0004] Background Art

[0005] Commercially produced chickens are hatched in hatcheries and are exposed to bacteria in the environment immediately after hatching. During hatching, waste consisting of eggshells and remnants of biological material from the eggs after the chickens hatch forms in the hatcheries. In the hatchery environment, the temperature is 37 °C and 80% relative humidity, which, in combination with the biological material from the remains of the shells, represent ideal conditions for bacterial growth. Chickens are then exposed to the bacteria growing in this material from the first moments of their lives. However, the microbial composition of this material is unknown, and consequences of chicken exposure to this microbiota on the health of the chickens after they are transported to farms is also unknown. In hatcheries, chicken production is highly concentrated, which on one hand increases the risk of improper development of the health of the chickens, but on the other hand represents an opportunity to positively influence the health of tens of thousands of chickens if appropriate intervention is taken. However, there is no information available on this matter and there is no microbial preparation that could be administered in hatcheries targeting the chickens indirectly by influencing the microbiota of the hatchery environment.

[0006] Disclosure of Invention

[0007] In the framework of the present invention, more than 50 samples of eggshell waste were studied, in which the composition of the bacterial microbiota was determined by sequencing part of the 16S rRNA gene. This composition was correlated with chicken mortalities after transport and placement on farms. Correlation analysis identified bacteria with an undesirable positive correlation with increased deaths of chickens in the first days of life as well as bacteria with a negative correlation to deaths, i.e. the more such bacteria were in the eggshell waste, the lower chicken mortalities were recorded on farms. We focused the cultivation on the latter bacteria. After cultivation on blood agar, suspect colonies were identified using mass spectrometry (MALDI Biotyper), the results of which were subsequently confirmed by sequencing part of the 16S rRNA gene and comparing the obtained sequences with the rRNA / ITS database in GenBank (https: / / blast.ncbi. nlm.nih.gov / Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LIN

[0008] K_LOC=blasthome). Finally, the entire genome sequence of the obtained isolates was determined.

[0009] Based on the above results, a preparation was developed to suppress undesirable microbiota in poultry hatcheries, which contains a mixture of bacteria Chryseobacterium indologenes and Sphingobacterium multivorum as the active ingredient. The bacterial preparation according to the invention has the following technical advantages over all similar products prepared so far:

[0010] - The contained bacteria (Chryseobacterium indologenes and Sphingobacterium multivorum) are commonly found in hatcheries, but are not found in intestinal microbiota of chickens, pigs, or humans.

[0011] - Both bacteria are among those that can be easily cultivated in vitro.

[0012] - Both bacteria can be cultivated in a medium free of any animal products.

[0013] - The bacteria from the product do not colonize the digestive or respiratory tract of poultry and thus do not affect health of the chickens.

[0014] - After application in the hatchery two days before the start of hatching, both bacteria remain in the environment until the chickens hatch, and compete with the growth of undesired bacteria in the environment, such as with E. coli. Clostridium sensu stricto, Enterococcus, Klebsiella, Enterobacter or Pseudomonas.

[0015] - In real flocks, application of the product reduces the percentage of confiscated animals at slaughter.

[0016] - The product can also be used in combination with probiotics colonizing chicken intestinal tract.

[0017] Definitions

[0018] BHI (brain heart infusion) is a brain heart bouillon used for growth of microorganisms.

[0019] ODeoonm is an abbreviation for the optical density of a sample measured at a wavelength of 600 nm. It is commonly used in microbiology to estimate the concentration of bacteria or other cells in a liquid culture.

[0020] A master inoculum is a culture of microorganisms originating from a single batch, processed together in a single operation in such a way as to ensure homogeneity and stability and to avoid contamination.

[0021] Blood agar is a growth medium that is suitable for culture of microorganisms and allows the detection of various types of hemolysis in inoculated microorganisms. CFU is an abbreviation for the "Colony Forming Units" and expresses the number of viable microorganisms in a sample in units of CFU / g or CFU / ml.

[0022] The food-acceptable solvent comprises water, saline, or optionally a growth medium for bacteria.

[0023] An object of the present invention is a bacterial preparation for suppressing undesirable microbiota in poultry hatcheries, which contains as an active ingredient a mixture of Chryseobacterium indologenes and Sphingobacterium multivorum bacteria. Both of the above bacteria can be cultivated simultaneously, which has a positive impact on the prospective industrial production of said bacterial preparation. The bacterial preparation according to the invention is particularly suitable for administration by spraying in hatcheries 2 days before the start of chicken hatching, i.e. on the 19th day of embryonic development. The bacterial preparation was tested as safe for chickens after the spray application.

[0024] In one preferred embodiment, the bacterial preparation comprises a mixture of bacteria Chryseobacterium indologenes CCM 9369 and Sphingobacterium multivorum CCM 9368 as the active ingredient. Said bacterial strains are deposited in Czech Collection of Microorganisms, Department of Experimental Biology, Faculty of Science, Masaryk University, address: Kamenice 5 / budova A25, 625 00 Brno, Czech Republic. The date of deposit is 16.10.2023 and the depositors are: Ivan Rychlik, Martin Faldyna, Vyzkumny ustav veterinarniho lekafstvi, Hudcova 70, 621 00, Brno. In one embodiment, the bacterial preparation further comprises a water-based liquid carrier, for example a bacterial growth medium, in particular BHI. One milliliter of the bacterial preparation contains from 108to 1010CFU of each bacterium.

[0025] Further object of the present invention is a formulation for spray application comprising said bacterial preparation according to the present invention and at least one food-acceptable solvent. It was found that the bacteria from said preparation outcompete undesired bacteria, such as E. coli, Clostridium sensu slriclo. Enterococcus, Klebsiella, Enterobacter or Pseudomonas, from the hatchery environment, reduce their presence in hatcheries and therefore the exposure of newly hatched chickens to those bacteria. Although the bacteria mentioned are not considered as strictly pathogenic, exposure of newly hatched chickens without a developed immune system to these opportunistic pathogenic bacteria can lead to negative reactions associated with defects, which are then evaluated as reasons for confiscation at slaughter. The formulation for spray application for treating the hatchery environment therefore comprises the above described bacterial preparation and a water-based liquid carrier. The water-based liquid carrier can be water or saline or optionally a growth medium for bacteria, preferably consisting of soybean peptone, acid hydrolysate of soybean proteins and yeast extract, and bacterial fermentation products of this mixture. The bacterial formulation for spray application comprises at least 108CFU per milliliter of the liquid formulation of each of the two bacteria contained. The contained bacteria Chryseobacterium indologenes and Sphingobacterium multivorum in hatcheries compete in growth with other bacteria such as with E. coli, Clostridium sensu stricto, Enterococcus, Klebsiella, Enterobacter or Pseudomonas and ultimately reduce the exposure of hatched chickens to these undesired bacteria.

[0026] Description of Drawings

[0027] Figure 1. Microbiota composition in eggshell waste. Individual bar graphs show the microbiota composition or the abundance of selected species in individual eggshell waste samples. The most common bacterial species in eggshell waste include Chryseobacterium, E. coli, Clostridium sensu stricto, Enterococcus and Pseudomonas. A negative relationship was observed among Chryseobacterium, E. coli and Pseudomonas - if Chryseobacterium or Sphingobacterium were present in the samples, then E. coli and Pseudomonas were present at low abundance. This led to the hypothesis of active colonization of hatcheries by Chryseobacterium and Sphingobacterium, and limitation of the growth of undesired species.

[0028] Figure 2. A correlation between bacteria in eggshell waste and chicken mortalities after placement on farms. The correlation between the abundance of individual bacteria in eggshell waste and mortalities on the 7th, 14th, 21stand 28thday of fattening. If Chryseobacterium indologenes and Sphingobacterium multivorum dominated in eggshell waste, lower chicken mortalities were recorded in flocks. On the contrary, if the genera Klebsiella, Pseudomonas, Enterococcus and Enterobacter were present in this material at a higher abundance, increased mortalities were recorded in flocks.

[0029] Figure 3. Effect of treatment using the bacterial preparation on the percentage of confiscated animals at slaughter. Treatment with the preparation significantly reduced the percentage of confiscated animals at slaughter.

[0030] Examples Example 1: Identification and isolation of suitable bacterial species specific to the microbiota in eggshell waste

[0031] In designing the bacterial preparation according to the present invention, we identified bacteria in eggshell waste by DNA isolation, PCR through a part of the 16S rRNA gene and subsequent sequencing of the resulting PCR products. The correlation analysis between microbial composition and chicken mortality after transport to farms led to the identification of bacterial species that correlated with low chicken mortality on farms (Fig. 1 and 2). We therefore focused the subsequent culture of those species. Said cultivation was carried out from eggshell waste samples collected in chicken hatcheries, diluted in sterile saline with pH 7.5 at 25 °C. Individual dilutions were plated on blood agar (commercially available e.g. from Biovendor a.s.) and incubation was carried out aerobically for 72 hours at 37 °C. From the grown colonies, an aliquot was resuspended in 1.0 ml of BHI (commercially available e.g. from Sigma- Aldrich) with 10% glycerol and frozen at -70 °C for further use. DNA was isolated from another aliquot and used for complete bacterial DNA sequencing and subsequent species identification. With the knowledge of the whole genome sequence, genes specific to each of the listed bacteria were identified and targeted by SybrGreen real-time PCR for detection and quantification of target bacteria in any type of sample using the following primers:

[0032] Individual purified bacterial strains were also identified based on the 16S rRNA gene sequence.

[0033] For the bacteria used in other tests, this is as follows:

[0034] Chryseobacterium indologenes CCM 9369

[0035] CATACAATGGAGAGTTTGATCCTGGCTCAGGATGAACGCTAGCGGGAGGCCTAACA

[0036] CATGCAAGCCGAGCGGTAGAGATCTTTCGGGATCTTGAGAGCGGCGTACGGGTGCG

[0037] GAACACGTGTGCAACCTGCCTTTATCTGGGGGATAGCCTTTCGAAAGGAAGATTAAT

[0038] ACCCCATAATATGTTGGATGGCATCATTCGACATTGAAAACTCCGGTGGATAGAGAT

[0039] GGGCACGCGCAAGATTAGATAGTTGGTGAGGTAACGGCTCACCAAGTCTGCGATCT

[0040] TTAGGGGGCCTGAGAGGGTGATCCCCCACACTGGTACTGAGACACGGACCAGACTC

[0041] CTACGGGAGGCAGCAGTGAGGAATATTGGACAATGGGTGAGAGCCTGATCCAGCCA

[0042] TCCCGCGTGAAGGACGACGGCCCTATGGGTTGTAAACTTCTTTTGTATAGGGATAAA

[0043] CCTACTCTCGTGAGAGTAGCTGAAGGTACTATACGAATAAGCACCGGCTAACTCCGT GCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTATCCGGATTTATTGGGTTTA AAGGGTCCGTAGGCGGATCTGTAAGTCAGTGGTGAAATCTCACAGCTTAACTGTGA AACTGCCATTGATACTGCAGGTCTTGAGTGTTGTTGAAGTAGCTGGAATAAGTAGTG TAGCGGTGAAATGCATAGATATTACTTAGAACACCAATTGCGAAGGCAGGTTACTA AGCAACAACTGACGCTGATGGACGAAAGCGTGGGGAGCGAACAGGATTAGATACC CTGGTAGTCCACGCCGTAAACGATGCTAACTCGTTTTTGGGCTTTCGGGTTCAGAGA

[0044] CTAAGCGAAAGTGATAAGTTAGCCACCTGGGGAGTACGAACGCAAGTTTGAAACTC AAAGGAATTGACGGGGGCCCGCACAAGCGGTGGATTATGTGGTTTAATTCGATGAT ACGCGAGGAACCTTACCAAGGCTTAAATGGGAAATGACAGGTTTAGAAATAGACTT TTCTTCGGACATTTTTCAAGGTGCTGCATGGTTGTCGTCAGCTCGTGCCGTGAGGTGT TAGGTTAAGTCCTGCAACGAGCGCAACCCCTGTCACTAGTTGCCATCATTAAGTTGG

[0045] GGACTCTAGTGAGACTGCCTACGCAAGTAGAGAGGAAGGTGGGGATGACGTCAAAT CATCACGGCCCTTACGCCTTGGGCCACACACGTAATACAATGGCCGGTACAGAGGG CAGCTACACAGCGATGTGATGCAAATCTCGAAAGCCGGTCTCAGTTCGGATTGGAG TCTGCAACTCGACTCTATGAAGCTGGAATCGCTAGTAATCGCGCATCAGCCATGGCG CGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCAAGCCATGGAAGTCTGGG

[0046] GTACCTGAAGTCGGTGACCGTAACAGGAGCTGCCTAGGGTAAAACAGGTAACTAGG GCTAAGTCGTAACAAGGTAGCCGTACCGGAAGGTGCGGCTGGAACATCTCATTTTA G (SEQ ID NO. 5)

[0047] Sphingobacterium multivorum CCM 9368

[0048] ATGGAGAGTTTGATCCTGGCTCAGGATGAACGCTAGCGGCAGGCCTAATACATGCA AGTCGGACGGGATCCATCGGAGAGCTTGCTCGAAGATGGTGAGAGTGGCGCACGGG TGCGTAACGCGTGAGCAACCTACCTCTATCAGGGGGATAGCCTCTCGAAAGAGAGA TTAACACCGCATAACATCAACAGTTCGCATGTTCGGTTGATTAAATATTTATAGGAT AGAGATGGGCTCGCGTGACATTAGCTAGTTGGTAGGGTAACGGCCTACCAAGGCGA

[0049] CGATGTCTAGGGGCTCTGAGAGGAGAATCCCCCACACTGGTACTGAGACACGGACC AGACTCCTACGGGAGGCAGCAGTAAGGAATATTGGTCAATGGGCGGAAGCCTGAAC CAGCCATGCCGCGTGCAGGATGACTGCCCTATGGGTTGTAAACTGCTTTTGTCCAGG AATAAACCTAAATACGTGTATTTAGCTGAATGTACTGGAAGAATAAGGATCGGCTA ACTCCGTGCCAGCAGCCGCGGTAATACGGAGGATCCGAGCGTTATCCGGATTTATTG

[0050] GGTTTAAAGGGTGCGTAGGCGGCCTATTAAGTCAGGGGTGAAATACGGTGGCTCAA CCATCGCAGTGCCTTTGATACTGATGGGCTTGAATCCATTTGAAGTGGGCGGAATAA GACAAGTAGCGGTGAAATGCATAGATATGTCTTAGAACTCCGATTGCGAAGGCAGC TCACTAAGCTGGTATTGACGCTGATGCACGAAAGCGTGGGGATCGAACAGGATTAG ATACCCTGGTAGTCCACGCCCTAAACGATGATAACTCGATGTTGGCGATAGACAGCC

[0051] AGCGTCCCAGCGAAAGCGTTAAGTTATCCACCTGGGGAGTACGCCCGCAAGGGTGA AACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGAGGAGCATGTGGTTTAATTCG ATGATACGCGAGGAACCTTACCCGGGCTTGAAAGTTAGTGAAGAGTGCAGAGACGC ACTCGTCCTTCGGGACACGAAACTAGGTGCTGCATGGCTGTCGTCAGCTCGTGCCGT GAGGTGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATGTTTAGTTGCCAGCATG

[0052] TCATGGTGGGGACTCTAAACAGACTGCCTGTGCAAACAGAGAGGAAGGTGGGGACG ACGTCAAGTCATCATGGCCCTTACGTCCGGGGCTACACACGTGCTACAATGGATGGT ACAGCGGGCAGCTACATAGCAATATGATGCTAATCTCTAAAAGCCATTCACAGTTCG GATTGGGGTCTGCAACTCGACCCCATGAAGTTGGATTCGCTAGTAATCGCGTATCAG CAATGACGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCAAGCCATGA

[0053] AAGTTGGGGGTACCTAAAGCATGTTACCGCAAGGAGCGTGTTAGGGTAAAACCGAT AATTGGGGCTAAGTCGTAACAAGGTAGCCGTACCGGAAGGTGCGGCTGGAATACCT CCTTT (SEQ ID NO. 6) Example 2: Manufacturing of the bacterial preparation

[0054] Pure cultures of both of the above isolates (Chryseobacterium indologenes and Sphingobacterium multivorum) are stored in BHI medium (commercially available e.g. from Sigma-Aldrich) with 10% glycerol at -70 °C. When in culture, individual cultures are plated on blood agar (commercially available e.g. from Biovendor a.s.) and incubated for 36 hours at 37 °C. Then, individual cultures are separately resuspended in 5 ml of BHI medium with 10% glycerol to ODeoonm = 1, which corresponds to the concentration of IxlO9CFU / ml. Subsequently, five milliliter volumes of both cultures are mixed and distributed into one milliliter master inocula. In the case of preparing a probiotic mixture, 1 ml of the master inoculum is used to inoculate 3 liters of growth medium and is incubated aerobically in a medium without animal proteins (soybean peptone (enzymatic digestate), acid hydrolysate of soy protein and yeast extract) at 37 °C for 36 hours. The resulting fermentate concentration reaches IxlO8CFU / ml. After cultivation, the fermentate of Ch. indologenes and S. multivorum is centrifuged at 5000g at 20 °C for 20 min. After removal of the supernatant, the pellet is resuspended in 30 ml of BHI medium with 10% glycerol and frozen at -70 °C. The resulting preparation thus contains IxlO10CFU / ml and one dose of the preparation with a volume of 30 ml is sufficient to treat one hatchery with 30,000 eggs.

[0055] Example 3: Effect of bacterial treatment on the percentage of confiscated animals at slaughter. On the nineteenth day of embryonic development, the bacterial preparation containing at least 1010CFU / ml was diluted 100 times in sterile saline and sprayed into the hatchery environment. A total of 13 flocks treated with the bacterial preparation (a total of 487,700 chickens) were compared with 5 control flocks (207,800 chickens). The treatment with the bacterial preparation significantly reduced the percentage of confiscated animals at slaughter (Fig. 3).

[0056] Conclusions

[0057] The bacteria Chryseobacterium indologenes and Sphingobacterium multivorum from the bacterial preparation according to the present invention colonize the hatchery environment, where they compete with undesirable microbiota. The preparation is preferably applied by spraying in the hatchery on the 19thday of embryonic development. The preparation does not show any negative effects, no deaths of poultry were recorded after its administration and the bacteria from the preparation do not colonize poultry. Therefore, the preparation according to the present invention can preferably be used as a preparation for improving the overall health of poultry after hatching, indirectly through the protection of the environment in hatcheries. The resulting effect is manifested in a lower percentage of confiscated poultry at slaughter after the end of fattening.

Claims

CLAIMS1. A preparation for treating the hatchery environment, characterized in that it comprises a mixture of Chryseobacterium indologenes and Sphingobacterium multivorum bacteria as the active ingredient.

2. The preparation according to claim 1, characterized in that it comprises a mixture of Chryseobacterium indologenes CCM 9369 and Sphingobacterium multivorum CCM 9368 bacteria as the active ingredient.

3. The preparation according to claim 1 or 2, characterized in that it further comprises a waterbased liquid carrier, preferably a growth medium for bacteria.

4. The preparation according to claim 1, 2 or 3, characterized in that it comprises from 108to IO10CFU of each bacteria in one milliliter of said preparation.

5. A formulation for spray application, characterized in that it comprises the preparation according to any one of the preceding claims 1 to 4, and further comprises at least one water-based liquid carrier, preferably selected from the group comprising water or saline, wherein the content of each bacteria in one milliliter of the formulation is at least 107CFU.

6. The formulation according to claim 5, characterized in that it comprises from 108to 109CFU of each bacteria in one milliliter of the formulation.

7. The preparation according to any one of the preceding claims 1 to 4 or the formulation according to claim 5 or 6 for use for preventive improvement of physiological condition of poultry.

8. The preparation according to any one of claim 1 to 2 or the formulation according to claim 5 or 6 for use in preventing infections of poultry by opportunistic pathogens selected from the group comprising E. coli. Clostridium sensu stricto, Enterococcus, Klebsiella, Enter obacter and Pseudomonas.

Images