CN108165619A - The feeding prebiotic bacterial screening method of anti-chicken C.perfringens infection - Google Patents
The feeding prebiotic bacterial screening method of anti-chicken C.perfringens infection Download PDFInfo
- Publication number
- CN108165619A CN108165619A CN201711498189.1A CN201711498189A CN108165619A CN 108165619 A CN108165619 A CN 108165619A CN 201711498189 A CN201711498189 A CN 201711498189A CN 108165619 A CN108165619 A CN 108165619A
- Authority
- CN
- China
- Prior art keywords
- chicken
- probiotics
- perfringens
- broiler
- intestinal
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
-
- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
- C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- General Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention discloses a kind of feeding prebiotic bacterial screening method of anti-chicken C.perfringens infection, and this method includes：The screening of clinical C.perfringens infection morbidity chicken and disease-resistant chicken and the acquisition of enteron aisle chyme sample；C.perfringens infection morbidity chicken and the high-flux sequence of disease-resistant chicken intestinal microorganism species；C.perfringens infection morbidity chicken and disease-resistant chicken intestinal microorganism metagenomics comparative analysis；The screening and identification of the probiotics of anti-C.perfringens infection.The present invention is started with by the metagenomics big data analysis to morbidity broiler chicken and disease-resistant Intestine of Broiler flora, targetedly probiotics of the screening with the infection of anti-C.perfringens from disease-resistant Intestine of Broiler huge microorganism species, the probiotics screened derives from ontology animal, avoid the inadaptable of probiotics and host, it is and with strong points, the blindness of screening is avoided, saves a large amount of human and material resources and time.
Description
Technical field
The present invention relates to animal and veterinary technical field, more particularly to a kind of feeding benefit of anti-chicken C.perfringens infection
Raw bacterial screening method.
Background technology
C.perfringens is a kind of important humans and animals commensal gut bacterium and conditionity pathogenic bacteria, can be generated a variety of
Exotoxin, has people and many animals pathogenic, is especially of great significance to the outburst of chicken necrotizing enterocolitis.The disease is not
Breeding performonce fo animals and the price of deed can be only reduced, increases the death rate, causes huge economic loss, it can also be by chicken system
Product cause the infection of people, and significant threat is formed to human health.The generally use addition antibiotic control chicken necrosis in poultry husbandry
Property enteritis, with the continuous application of antibacterials, the antibody-resistant bacterium quantity in bacterium is also constantly increasing, at present, drug resistance
The problem of it is very serious, the difficulty that the infection of chicken C.perfringens is resisted using conventional medicine and conventional method is increasing,
As limitation of the antibiotics feed addictive in China uses, C.perfringens infection morbidity is in China's poultry husbandry
It can getting worse.A kind of feeding prebiotic bacterial screening method of anti-chicken C.perfringens infection is established, is conducive to filter out efficiently
Special probiotics, to prevention chicken C.perfringens infection be of great significance.
Probiotics plays increasingly important role in terms of human and livestock health or the certain diseases of prevention is ensured, particularly
Under the premise of China's antibiotic will disable comprehensively, intestinal health and diarrhea will be that the bottleneck of China's animal husbandry development is threatened to ask
Topic researches and develops new Substitutes For Antibiotic, and there is special efficacy to prevent animal diarrhea for screening, ensures animal intestinal tract health
Feeding probiotics has become the hot spot studied both at home and abroad.Although current China has carried out the screening operation of a large amount of probiotics,
And it is widely used in production practices, but these probiotics are mainly used for promoting the digestion of animal intestinal tract nutriment to inhale
Receive, promote growth, and many probiotics are not originating from ontology animal, into host in after, it is difficult it is difficult to adapted to host
To play its definite effect.
Traditional probiotics screening technique mainly includes In Vitro Bacteriostasis method and attacks bacterium method in vivo, and there is blindness, effects
Rate is low and specific aim is not strong and mostly external source bacterium the shortcomings of.
Invention content
The main object of the present invention is to provide a kind of feeding prebiotic bacterial screening method of anti-chicken C.perfringens infection,
It is intended to filter out efficient special probiotics, prevents chicken from infecting C.perfringens.
To achieve the above object, the feeding prebiotic bacterial screening method of anti-chicken C.perfringens infection proposed by the present invention,
Include the following steps：
Step 1：In the chicken house of C.perfringens infection outburst, pass through the observation of clinical symptoms and the inspection of pathology damage
It surveys, is detected with reference to the qualitative PCR of C.perfringens, select the morbidity chicken with typical clinical symptom and apparent pathology damage
With no typical clinical symptom and each 20-30 of disease-resistant chicken of apparent pathology damage only, the enteron aisle content of each broiler chicken is acquired respectively
Object；
Step 2：The 16srRNA genes of microorganism in each enteron aisle chyme are expanded using 16srRNA universal primers, and right
Amplified fragments are detected and concentration mensuration, and Tag primer is then recycled to carry out second and is expanded, and the segment to being expanded
It carries out concentration to measure, second of amplified production is diluted to identical concentration, mixed in equal amounts builds sequencing library, surveyed
Sequence；
Step 3：Using QIIME analysis platforms, two groups of chicken intestinal floras are analyzed, compare its alpha diversity (in group)
And beta diversity (between group), species annotation is carried out to the enteric microorganism OTU of cluster, analyzes two groups of chicken intestinal flora species point
Cloth；The difference floras of two groups of Intestine of Broiler, core flora and dominant microflora are compared in analysis, and from being only present in disease-resistant chicken intestinal
In difference flora in analyze dominant bacteria therein and core bacterium, determine to treat according to the analysis result of dominant bacteria and core bacterium
Screen the purpose bacterial strain of probiotics；
Step 4：Disease-resistant chicken intestinal flora is inoculated into meat soup, then Anaerobic culturel is put down with Selective agar medium solid again
Plate scribing line culture, according to the dominant bacteria and the bacterium colony characteristic of core bacterium determined in step 3, selects positive bacterium colony, is inoculated into respectively
Zengjing Granule in liquid selective medium；
Step 5：Carry out biochemical characteristic identification and the sequencing identification of 16s full genomes to the probiotics screened, and from wherein sieving
Have selected two probiotics.
It should be noted that core bacterium refers to what is be all distributed in all disease-resistant chicken intestinals or excrement, dominant bacteria refers to
Occupy the bacterium colony of the high ratio of comparison in difference flora.In general, the equal group of core and dominant microflora have overlapping.
Preferably, in the step 1, the clinical symptoms include：
Broiler fodder intake is reduced, body weight gains reduce, nutrient absorption is bad, diarrhea, serious enteron aisle are downright bad and the death rate
Increase the observation of waiting for death property cellulosic enteritis clinical symptoms, the broiler chicken group positive from the detection of C.perfringens qualitative PCR
In, select morbidity chicken and disease-resistant chicken
Preferably, the pathology damage includes enteric cavity expansion inflation, and intestinal wall thickens, and intestinal wall is congested, there is bleeding spot, intestines
Mucous membrane necrosis, in differ in size, different pseudomembrane sample necrosis region, muscle is pale seemingly to let off blood, liver and spleen enlargement,
In crineous.
Preferably, preservation under the conditions of the intestinal contents acquired in step 1 are placed in -80 DEG C.
Preferably, the step 3 further includes the type by determining the dominant bacteria and the core bacterium, obtains its bacterium
Fall characteristic.
Preferably, after step 5, it further includes：
The beneficial bacteria screened is seeded in the newborn Broiler Chicken of experiment, poison then is attacked to experiment broiler chicken C.perfringens
Experiment observes and records and attacks malicious broiler chicken incidence calculating morbidity and mortality, and measures experiment Intestine of Broiler tissue morphology
Structure and immunocyte, proinflammatory cytokines and mucous membrane functional protein be horizontal, according to diarrhea rate, morbidity and mortality
Statistical result, intestinal tissue morphosis and immunocyte, proinflammatory cytokines and mucous membrane functional protein are horizontal, to analyze
The effect of the anti-C.perfringens infection of probiotics screened.
Preferably, the step 5 further includes：
The 40 of the chicken house 1 age in days C.perfringens feminine gender broiler chicken that will be immunized purchased from no progress C.perfringens
Using conventional Diet, it is randomly divided into four groups, four groups are respectively probiotics A groups, probiotics B groups, probiotics A+B groups and right
According to group, every group of 10 broiler chicken；
After screened two groups of probiotics are carried out in vitro cultures, probiotic group broiler chicken is inoculated with since 1 age in days morning
The probiotics screened, until 7 ages in days；
The oral perfusion inoculation C. perfringens Type A CVCC-52 of every group of broiler chicken carries out attacking poison 8th age in days at night, attacks poison
Continuous observation 48h afterwards is observed and recorded and is attacked malicious broiler chicken incidence calculating morbidity and mortality；
After attacking malicious 48h, broiler chicken acquisition intestinal mucosa tissue sample is butchered, intestinal structure and immunocyte is measured, exempts from
Epidemic disease and inflammatory factor and mucous membrane functional protein level judge the effect of the anti-chicken C.perfringens infection of screened probiotics
Fruit.
Preferably, in the step 5,
Broiler chicken is tested from 1 age in days to 7 ages in days, first day dosage of inoculation is 5 × 102Then cfu/100uL is followed successively by 5 daily
×103cfu/100uL、5×104cfu/100uL、5×105cfu/100uL、5×106Cfu/100uL and 5 × 107cfu/
100uL。
Preferably, the toxic agent amount of attacking that experiment broiler chicken using C. perfringens Type A CVCC-52 attack poison is
109cfu/100uL。
Preferably, in steps of 5：
The immune and inflammatory factor includes interleukin-6, interleukin 8, interleukin 10, the interference of I type
Element, tumor necrosis factor-alpha and visible peristalsis visible intestinal peristalsis fatty acid binding protein.
Preferably, two probiotics are bacillus subtilis and clostridium butyricum.
The present invention is started with by the metagenomics big data analysis to morbidity broiler chicken and disease-resistant Intestine of Broiler flora, is had
Pointedly probiotics of the screening with the infection of anti-C.perfringens from disease-resistant Intestine of Broiler huge microorganism species,
The probiotics screened derives from ontology animal, avoids the inadaptable and with strong points of probiotics and host, avoids sieve
The blindness of choosing saves a large amount of human and material resources and time.
Specific embodiment
Below in conjunction with the embodiment of the present invention, the technical solution in the embodiment of the present invention is clearly and completely retouched
It states, it is clear that described embodiment is only the part of the embodiment of the present invention, instead of all the embodiments.Based on this hair
Embodiment in bright, the every other reality that those of ordinary skill in the art are obtained without creative efforts
Example is applied, shall fall within the protection scope of the present invention.
The present invention is started with by the metagenomics big data analysis to morbidity broiler chicken and disease-resistant Intestine of Broiler flora, is had
Pointedly screening has the prebiotic of anti-chicken C.perfringens infection from disease-resistant Intestine of Broiler huge microorganism species
Bacterium, the main inventive step of the present invention are as follows：
Step 1：The screening of clinical C.perfringens infection morbidity chicken and disease-resistant chicken and the acquisition of enteron aisle chyme sample.
In the chicken house of C.perfringens infection outburst, by the observation of clinical symptoms and the detection of pathology damage, with reference to
The qualitative PCR detection of C.perfringens is reduced by broiler fodder intake, body weight gains reduce, nutrient absorption is bad, abdomen
It rushes down, serious enteron aisle is downright bad and the observation of death rate increase waiting for death property cellulosic enteritis clinical symptoms；Enteric cavity expansion inflation, intestines
Wall thickening, intestinal wall are congested, there is a bleeding spot, intestinal mucosa necrosis, in differ in size, different pseudomembrane sample necrosis region, muscle
It is pale seemingly to let off blood, liver and spleen enlargement, in the dissect of the pathology damages such as crineous, examined from C.perfringens qualitative PCR
It surveys in positive broiler chicken group, selects morbidity chicken with typical clinical symptom and apparent pathology damage and without typical clinical symptom
And each 20-30 of disease-resistant chicken of apparent pathology damage is only, acquires the intestinal contents of each broiler chicken respectively, it is standby in -80 DEG C of preservations
With.
Step 2：C.perfringens infection morbidity chicken and the high-flux sequence of disease-resistant chicken intestinal microorganism species.
The kit extraction dedicated for microbial genome in extraction chyme and excrement produced with Qiagen companies is each
Microbial genome in a Gut Segment, detection put forward the quality of genome, and its concentration is measured.
The 16srRNA genes of microorganism in each Gut Segment are expanded first with 16srRNA universal primers, and to expanding
Increase segment to be detected and concentration mensuration, Tag primer then recycled to carry out second and is expanded, and the segment to being expanded into
Row concentration measures, and second of amplified production is diluted to identical concentration, and mixed in equal amounts builds sequencing library.Library construction is good
Afterwards, after being denaturalized with NaOH, sequencing is carried out using the sequencing kit and Miseq sequenators of illumina companies.
Step 3：C.perfringens infection morbidity chicken and disease-resistant chicken intestinal microorganism metagenomics comparative analysis.
Using QIIME analysis platforms, two groups of chicken intestinal floras are analyzed, compare its a diversity and beta diversity, it is right
The enteric microorganism OTU of cluster carries out species annotation, analyzes two groups of chicken intestinal flora species distributions；Compare two with LEFSe analyses
The group core flora of Intestine of Broiler and difference flora.
Step 4：The screening and identification of the probiotics of anti-C.perfringens infection.
According to metagenomics analysis result, difference flora, core flora in more disease-resistant chicken and morbidity chicken intestinal flora
And dominant microflora, analysis are only present in the difference flora in disease-resistant chicken intestinal, then which is in these difference floras are analyzed
The dominant bacteria in disease-resistant chicken intestinal flora and core bacterium are appeared in, then selects different mirror for these dominant bacterias and core bacterium
Other culture medium.Chicken intestinal flora disease-resistant first is inoculated into meat soup, Anaerobic culturel, then uses Selective agar medium solid plate again
Scribing line culture according to the bacterium colony characteristic of expected screening probiotics, selects positive bacterium colony, is inoculated into liquid selective medium and increases
Bacterium is cultivated.
Step 5：Biochemical characteristic identification and the sequencing identification of 16srRNA full genomes are carried out to the probiotics screened.By with
Upper experiment has tentatively filtered out two plants of probiotics A and B theoretically with the infection of anti-chicken C.perfringens.
The present invention is started with by the metagenomics big data analysis to morbidity broiler chicken and disease-resistant Intestine of Broiler flora, right
Intestinal microflora carries out high-flux sequence and metagenomics comparative analysis, targetedly from disease-resistant Intestine of Broiler Pang
Probiotics of the screening with the infection of anti-chicken C.perfringens in big microorganism species.On the one hand, the probiotics screened is
Specifically for the prevention of broiler chicken C.perfringens infection；On the other hand, the probiotics screened derives from ontology animal, keeps away
The inadaptable of probiotics and host is exempted from；Another further aspect avoids the blindness of screening, save a large amount of human and material resources and
Time.
Anti- C.perfringens infection Intestine of Broiler probiotic effect verification
It is the 1 of 38.94 ± 0.32g by the 40 of chicken house average weights being immunized purchased from no progress C.perfringens
Age in days C.perfringens feminine gender broiler chicken, using conventional Diet, is randomly divided into four groups, respectively：
Probiotics A groups, probiotics B groups, probiotics A+B groups and control group.
Every group of 10 broiler chicken；After two probiotics that in vitro culture is screened, incremental mode is measured day by day by 10 times, from 1
Age in days starts to be inoculated with probiotic group broiler chicken screened probiotics morning, until 7 ages in days, first day dosage of inoculation for 5 ×
102Then cfu/100uL is followed successively by 5 × 10 daily3cfu/100uL、 5×104cfu/100uL、5×105cfu/100uL、5
×106Cfu/100uL and 5 × 107cfu/100uL；Probiotics A groups are inoculated with probiotics A, probiotics B groups inoculation probiotics B, benefit
Raw bacterium A+B groups are inoculated with two kinds of probiotics of A and B, each probiotics respectively accounts for 1/2, and control group is inoculated with the physiological saline of same dose
(100uL).The oral perfusion inoculation C. perfringens Type A CVCC-52 of every group of broiler chicken carries out attacking poison 8th age in days at night, attacks poison
Dosage is：109cfu/100uL.Experimental period is 2 days, observes and records and attacks malicious broiler chicken incidence calculating incidence and death
Rate butchers broiler chicken acquisition intestinal mucosa tissue sample for 2 days after attacking poison, measures intestinal structure and immunocyte, be immunized and scorching
Sex factor and mucous membrane functional protein level judge the effect of the anti-chicken C.perfringens infection of screened probiotics.
Experiment effect
(1) incidence and mortality
It observes one week, is reduced with Feed consumption, body weight gains reduce, nutrient absorption is bad, diarrhea, serious intestines after attacking poison
The enteritis such as road necrosis symptom is morbidity standard, observes and records clinical symptoms and incidence, counts incidence and mortality.
Every group of number of always falling ill/(every group of broiler chicken number of elements × experiment number of days) × 100% of incidence (%)=in experimental period, extremely
Die rate (%)=every group of total death toll/every group of sum × 100%.
1. probiotics of table infects C.perfringens the influence of broiler chicken incidence of disease and the death rate
(2) intestinal structure and immunocyte
By thick about 5 μm of mucous membrane tissue, intestine colibacillosis is made after Hematoxylin-eosin dyes, using micro- sem observation,
And pass through pathology picture and text report analysis system and take figure and take pictures, measure control group, probiotics A groups, B groups and A+B groups experiment broiler chicken
The height of naps of small intestinal mucosa, Crypt depth, villus width and fuzzy surface product, immunocyte quantity and density, measure knot
Fruit is as shown in table 2.
Influence of 2 probiotics of table to C.perfringens infection Intestine of Broiler morphosis
Height of naps can directly reflect the function status of enteron aisle with Crypt depth, and intestinal villi atrophy can lead to maturation
Epithelial cell quantity is reduced, and ripe villus epithelial cells just have the function of to absorb nutrient, therefore ripe cell quantity is reduced
It can make nutritional ingredient that cannot fully absorb；Crypt depth reflects the production rate of villus epithelial cells, and crypts of small intestine shoals
Afterwards, illustrate that cell maturation rate rises, secreting function enhancing；The functional status of height of naps/Crypt depth reaction small intestine, ratio
Decline shows that mucous membrane is damaged digestibility decline.As shown in Table 2, it is high to be remarkably improved height of naps, villus by probiotics A and B
Ratio and the fuzzy surface product of degree/Crypt depth, reduce Crypt depth, illustrate that probiotics A and B can effectively reduce aerogenesis pod
Intestinal mucosa injury caused by the infection of film clostridium.
(3) immune and inflammatory factor
Using the method for enzyme-linked immunosorbent assay (ELISA), control group, probiotics A groups, B groups and the examination of A+B groups are measured
It tests serum of broilers and content with Correlative Inflammatory Factors is immunized, measurement result is as shown in table 3.
E. coli Infected Broiler is immunized 3 probiotics of table and the influence of inflammatory factor
The lymphokine that the T cell of interleukins (IL-6) activation and fibroblast generate, can make B cell precursor
As the cell for generating antibody；It is cooperateed with colony stimulating factor, the growth and differentiation of original bone marrow-derived cells can be promoted, enhanced
The cracking function of natural killer cells.Interleukin 8 (IL-8) is also known as the neutrophil cell factor, is diseases associated with inflammation
Important medium plays an important role in anti-infective, immune response adjusting and anti-tumor aspect.Interleukin 10 (IL-10) energy
Enough T cells for inhibiting activation generate cell factor, therefore be once known as cytokine synthesis inhibitor, particularly inhibit TH1 thin
Born of the same parents generate the cell factors such as IL-2, IFN-γ and lt, so as to inhibit cellullar immunologic response.IFN-α is known as interferon type Ⅰ, can be with
Inducing cell generates the transcription and translation that disease-resistant toxenzyme carrys out viral interference and achievees the effect that inhibit virus multiplication.Tumor necrosis factor
Son-α (TNF-α) be it is a kind of can direct killing tumour cell and to cell factor of the normal cell without overt toxicity.Visible peristalsis visible intestinal peristalsis fat
Fat acid binding protein (iFABP) plays an important role in the intake, transhipment and Metabolism regulation of long chain fatty acids.IFABP exists
Content in serum is very little, and iFABP can more early discharge, and membrane passage is larger in ischemic during intestine ischemia,
Molecule can be released into blood by cell membrane.As shown in Table 4, probiotics A and B can significantly reduce -1 β of serum IL, IL-6,
IL-8, IL-10, NF- κ B content significantly improve IFN-α, TNF-α, the content of iFABP, illustrate that probiotics A and B can be effective
The inflammation that C.perfringens infection causes is reduced, improves Organism immunoregulation ability.
Above-mentioned probiotics A and B be comform it is random select in multi-probiotics.It compares and finds by many experiments, it is withered
Careless bacillus and clostridium butyricum group have good resistance to C.perfringens infection, with reference to table 4.
4. bacillus subtilis group of table and clostridium butyricum group infect C.perfringens broiler chicken incidence of disease and the death rate
It influences
From table 4, it can be seen that the incidence of bacillus subtilis group and clostridium butyricum group is than probiotics A and benefit in table 1
Raw bacterium B is low, and the death rate is also significantly lower than probiotics A and probiotics B in table 1.In addition, bacillus subtilis and clostridium butyricum
Bacterial strain is combined, morbidity and mortality are well below the probiotics A and probiotics B in table 1, in addition to this, the combination strain
Corresponding morbidity and mortality will be low than bacillus subtilis group and clostridium butyricum group, illustrate bacillus subtilis group and
Clostridium butyricum group has effects that C.perfringens is resisted in collaboration.
The foregoing is merely the preferred embodiment of the present invention, are not intended to limit the scope of the invention, every at this
Under the inventive concept of invention, other phases of the equivalent transformation made using present specification or directly/be used in indirectly
The technical field of pass is included in the scope of patent protection of the present invention.
Claims (10)
1. a kind of feeding prebiotic bacterial screening method of anti-chicken C.perfringens infection, which is characterized in that include the following steps：
Step 1：In the chicken house of C.perfringens infection outburst, pass through the observation of clinical symptoms and the detection of pathology damage, knot
The qualitative PCR detection of C.perfringens is closed, selects morbidity chicken with typical clinical symptom and apparent pathology damage and without allusion quotation
Each 20-30 of disease-resistant chicken of type clinical symptoms and apparent pathology damage only, acquires the intestinal contents of each broiler chicken respectively；
Step 2：The 16srRNA genes of microorganism in each enteron aisle chyme are expanded using 16srRNA universal primers, and to expanding piece
Section is detected and concentration mensuration, and Tag primer is then recycled to carry out second and is expanded, and the segment to being expanded carry out it is dense
Degree measures, and second of amplified production is diluted to identical concentration, and mixed in equal amounts builds sequencing library, is sequenced；
Step 3：Using QIIME analysis platforms, two groups of chicken intestinal floras are analyzed, compare its alpha diversity and beta diversity,
Species annotation is carried out to the enteric microorganism OTU of cluster, analyzes two groups of chicken intestinal flora species distributions；Two groups of broiler chicken are compared in analysis
The difference flora of enteron aisle, core flora and dominant microflora, and analyzed from the difference flora being only present in disease-resistant chicken intestinal
Dominant bacteria therein and core bacterium determine the purpose bacterial strain of probiotics to be screened according to the analysis result of dominant bacteria and core bacterium；
Step 4：Disease-resistant chicken intestinal flora is inoculated into meat soup, Anaerobic culturel, then again with the choosing for prescreening probiotics
The scribing line culture of culture medium solid plate is selected, the bacterium colony characteristic of the probiotics of prescreening determined according to step 3 selects positive bacteria
It falls, is inoculated into Zengjing Granule in liquid selective medium respectively；
Step 5：Carry out biochemical characteristic identification and the sequencing identification of 16s full genomes to the probiotics screened, and from wherein filtering out
Two probiotics.
2. the feeding prebiotic bacterial screening method of anti-chicken C.perfringens infection as described in claim 1, which is characterized in that institute
It states in step 1：
The clinical symptoms include the reduction of broiler fodder intake, body weight gains reduce, nutrient absorption is bad, diarrhea, serious enteron aisle
Necrosis and the death rate increase, dead property cellulosic enteritis.
3. the feeding prebiotic bacterial screening method of anti-chicken C.perfringens infection as claimed in claim 1 or 2, feature exist
In the pathology damage includes enteric cavity expansion inflation, and intestinal wall thickens, and intestinal wall is congested, there is bleeding spot, intestinal mucosa necrosis, in big
Small not grade, different pseudomembrane sample necrosis region, muscle is pale seemingly to let off blood, liver and spleen enlargement, in crineous.
4. the feeding prebiotic bacterial screening method of anti-chicken C.perfringens infection as described in claim 1, which is characterized in that
Preservation under the conditions of the intestinal contents acquired in step 1 are placed in -80 DEG C.
5. the feeding prebiotic bacterial screening method of anti-chicken C.perfringens infection as described in claim 1, which is characterized in that
After step 5, further include：
The beneficial bacteria screened is seeded in the newborn Broiler Chicken of experiment, then to testing broiler chicken C.perfringens challenge test,
Observe and record and attack malicious broiler chicken incidence and calculate morbidity and mortality, and measure experiment Intestine of Broiler histology and morphology structure and
Immunocyte, proinflammatory cytokines and mucous membrane functional protein are horizontal, according to diarrhea rate, the statistics knot of morbidity and mortality
Fruit, intestinal tissue morphosis and immunocyte, proinflammatory cytokines and mucous membrane functional protein are horizontal, to analyze what is screened
The effect of the anti-C.perfringens infection of probiotics.
6. the feeding prebiotic bacterial screening method of anti-chicken C.perfringens infection as claimed in claim 5, which is characterized in that institute
Step 5 is stated to further include：
By the 40 of chicken house 1 age in days C.perfringens feminine gender broiler chicken being immunized purchased from no progress C.perfringens using normal
Diet is advised, is randomly divided into four groups, four groups are respectively probiotics A groups, probiotics B groups, probiotics A+B groups and control group, often
10 broiler chicken of group；
After screened two groups of probiotics are carried out in vitro cultures, the inoculation of probiotic group broiler chicken is sieved since 1 age in days morning
The probiotics of choosing, until 7 ages in days；
The oral perfusion inoculation C. perfringens Type A CVCC-52 of every group of broiler chicken carries out attacking poison 8th age in days at night, is held after attacking poison
Continuous observation 48h is observed and recorded and is attacked malicious broiler chicken incidence calculating morbidity and mortality；
After attacking malicious 48h, butcher broiler chicken acquisition intestinal mucosa tissue sample, measure intestinal structure and immunocyte, be immunized and
Inflammatory factor and mucous membrane functional protein level judge the effect of the anti-chicken C.perfringens infection of screened probiotics.
7. the feeding prebiotic bacterial screening method of anti-chicken C.perfringens infection as claimed in claim 6, which is characterized in that
In the step 5,
Broiler chicken is tested from 1 age in days to 7 ages in days, first day dosage of inoculation is 5 × 102Cfu/100uL, then it is followed successively by 5 daily ×
103cfu/100uL、5×104cfu/100uL、5×105cfu/100uL、5×106Cfu/100uL and 5 × 107cfu/100uL。
8. the feeding prebiotic bacterial screening method of anti-chicken C.perfringens infection as claimed in claim 6, which is characterized in that examination
It is 10 that broiler chicken, which is tested, using the C. perfringens Type A CVCC-52 toxic agent amounts of attacking for carrying out attacking poison9cfu/100uL。
9. the feeding prebiotic bacterial screening method of anti-chicken C.perfringens infection as claimed in claim 6, which is characterized in that
In step 5：
The immune and inflammatory factor includes interleukin-6, interleukin 8, interleukin 10, interferon type Ⅰ, tumour
Necrosis factor-alpha and visible peristalsis visible intestinal peristalsis fatty acid binding protein.
10. the feeding prebiotic bacterial screening method of anti-chicken C.perfringens infection as described in claim 1, which is characterized in that
Two probiotics are bacillus subtilis and clostridium butyricum.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711498189.1A CN108165619A (en) | 2017-12-29 | 2017-12-29 | The feeding prebiotic bacterial screening method of anti-chicken C.perfringens infection |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711498189.1A CN108165619A (en) | 2017-12-29 | 2017-12-29 | The feeding prebiotic bacterial screening method of anti-chicken C.perfringens infection |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108165619A true CN108165619A (en) | 2018-06-15 |
Family
ID=62516865
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711498189.1A Pending CN108165619A (en) | 2017-12-29 | 2017-12-29 | The feeding prebiotic bacterial screening method of anti-chicken C.perfringens infection |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108165619A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110607262A (en) * | 2019-09-25 | 2019-12-24 | 君维安(武汉)生命科技有限公司 | Probiotic composition for intervening inflammatory enteritis and screening method and application thereof |
| CN110839761A (en) * | 2019-12-03 | 2020-02-28 | 广东容大生物股份有限公司 | Research and development method of broiler chicken composite probiotic preparation |
| CN113308411A (en) * | 2021-07-09 | 2021-08-27 | 广东海纳川生物科技股份有限公司 | Culture method and application of bacillus subtilis |
| CN113881607A (en) * | 2021-11-17 | 2022-01-04 | 宜兴市天石饲料有限公司 | Research and development method for preventing and treating avian clostridium perfringens |
| CN115094008A (en) * | 2022-07-21 | 2022-09-23 | 宜兴市天石饲料有限公司 | Research and development method of biological antibacterial preparation for preventing and treating avian clostridium perfringens |
| CN117757891A (en) * | 2024-02-22 | 2024-03-26 | 潍坊华卓生物科技有限公司 | Reverse screening method and application of functional probiotics for preventing H9 subtype avian influenza virus |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103243041A (en) * | 2012-07-27 | 2013-08-14 | 四川农业大学 | Bacillus licheniformis H2 and application thereof |
| CN105039223A (en) * | 2015-08-14 | 2015-11-11 | 山东宝来利来生物工程股份有限公司 | Bacillus subtilis with effect of inhibiting clostridium perfringens and application of bacillus subtilis |
| CN105105115A (en) * | 2015-08-21 | 2015-12-02 | 暨南大学 | Probiotics compound for crowds with blood types of A, B and O and method and application of probiotics compound |
| CN106479924A (en) * | 2016-10-31 | 2017-03-08 | 湖北绿雪生物科技有限公司 | The preparation of a kind of Clostridium butyricum and clostridium butyricum active bacteria preparation and application |
| CN107446838A (en) * | 2016-05-31 | 2017-12-08 | 赢创德固赛有限公司 | Bacillus subtilis strain with probiotic active |
-
2017
- 2017-12-29 CN CN201711498189.1A patent/CN108165619A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103243041A (en) * | 2012-07-27 | 2013-08-14 | 四川农业大学 | Bacillus licheniformis H2 and application thereof |
| CN105039223A (en) * | 2015-08-14 | 2015-11-11 | 山东宝来利来生物工程股份有限公司 | Bacillus subtilis with effect of inhibiting clostridium perfringens and application of bacillus subtilis |
| CN105105115A (en) * | 2015-08-21 | 2015-12-02 | 暨南大学 | Probiotics compound for crowds with blood types of A, B and O and method and application of probiotics compound |
| CN107446838A (en) * | 2016-05-31 | 2017-12-08 | 赢创德固赛有限公司 | Bacillus subtilis strain with probiotic active |
| CN106479924A (en) * | 2016-10-31 | 2017-03-08 | 湖北绿雪生物科技有限公司 | The preparation of a kind of Clostridium butyricum and clostridium butyricum active bacteria preparation and application |
Non-Patent Citations (6)
| Title |
|---|
| DRAGANA STANLEY ET AL.: "Changes in the caecal microflora of chickens following Clostridium perfringens challenge to induce necrotic enteritis", 《VETERINARY MICROBIOLOGY》 * |
| MARCIO C. COSTA ET AL.: "Comparison of the Fecal Microbiota of Healthy Horses and Horses with Colitis by High Throughput Sequencing of the V3-V5 Region of the 16S rRNA Gene", 《PLOS ONE》 * |
| YANNI FENG ET AL.: "Identification of changes in the composition of ileal bacterial microbiota of broiler chickens infected with Clostridium perfringens", 《VETERINARY MICROBIOLOGY》 * |
| 杨天龙 等: "宏基因组技术在鸡肠道微生物中应用的研究进展", 《中国畜牧兽医》 * |
| 贾丽楠 等: "丁酸梭菌对肉仔鸡肠道致病菌抑菌作用研究", 《中国家禽》 * |
| 阳艳林 等: "不同益生菌对产气荚膜梭菌抑制效果的比较", 《中国畜牧杂志》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110607262A (en) * | 2019-09-25 | 2019-12-24 | 君维安(武汉)生命科技有限公司 | Probiotic composition for intervening inflammatory enteritis and screening method and application thereof |
| CN110839761A (en) * | 2019-12-03 | 2020-02-28 | 广东容大生物股份有限公司 | Research and development method of broiler chicken composite probiotic preparation |
| CN113308411A (en) * | 2021-07-09 | 2021-08-27 | 广东海纳川生物科技股份有限公司 | Culture method and application of bacillus subtilis |
| CN113881607A (en) * | 2021-11-17 | 2022-01-04 | 宜兴市天石饲料有限公司 | Research and development method for preventing and treating avian clostridium perfringens |
| CN115094008A (en) * | 2022-07-21 | 2022-09-23 | 宜兴市天石饲料有限公司 | Research and development method of biological antibacterial preparation for preventing and treating avian clostridium perfringens |
| CN117757891A (en) * | 2024-02-22 | 2024-03-26 | 潍坊华卓生物科技有限公司 | Reverse screening method and application of functional probiotics for preventing H9 subtype avian influenza virus |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Sumon et al. | Growth performance, digestive enzyme activity and immune response of Macrobrachium rosenbergii fed with probiotic Clostridium butyricum incorporated diets | |
| CN108165619A (en) | The feeding prebiotic bacterial screening method of anti-chicken C.perfringens infection | |
| Hossain et al. | Probiotics as potential alternative biocontrol agents in the agriculture and food industries: A review | |
| Yu et al. | Improved extraction of PCR-quality community DNA from digesta and fecal samples | |
| Pirarat et al. | Modulation of intestinal morphology and immunity in nile tilapia (Oreochromis niloticus) by Lactobacillus rhamnosus GG | |
| Bandyopadhyay et al. | Effect of a probiotic bacterium Bacillus circulans PB7 in the formulated diets: on growth, nutritional quality and immunity of Catla catla (Ham.) | |
| Zommiti et al. | Probiotics—live biotherapeutics: a story of success, limitations, and future prospects—not only for humans | |
| Jahan et al. | Probiotic yeast enhances growth performance of rohu (Labeo rohita) through upgrading hematology, and intestinal microbiota and morphology | |
| Amin et al. | In vitro screening of lactic acid bacteria isolated from gastrointestinal tract of Atlantic Salmon (Salmo salar) as probiont candidates | |
| Coble et al. | Distinct lines of chickens express different splenic cytokine profiles in response to Salmonella Enteritidis challenge | |
| Liu et al. | Effects of dietary Lactobacillus plantarum and AHL lactonase on the control of Aeromonas hydrophila infection in tilapia | |
| EP1715755A1 (en) | Use of live bacteria for growth promotion in animals | |
| Hossain et al. | Multi-species probiotics improve growth, intestinal microbiota and morphology of Indian major carp mrigal Cirrhinus cirrhosus | |
| Klakegg et al. | Enhanced growth and decreased mortality in Atlantic salmon (Salmo salar) after probiotic bath | |
| Amenyogbe et al. | Probiotic potential of indigenous (Bacillus sp. RCS1, Pantoea agglomerans RCS2, and Bacillus cereus strain RCS3) isolated from cobia fish (Rachycentron canadum) and their antagonistic effects on the growth of pathogenic Vibrio alginolyticus, Vibrio harveyi, Streptococcus iniae, and Streptococcus agalactiae | |
| Liu et al. | Tributyrin administration improves intestinal development and health in pre-weaned dairy calves fed milk replacer | |
| Śmiałek et al. | The use of probiotics in the reduction of Campylobacter spp. prevalence in poultry | |
| Ahiwe et al. | Dietary hydrolysed yeast cell wall extract is comparable to antibiotics in the control of subclinical necrotic enteritis in broiler chickens | |
| CN107937504A (en) | The screening technique of the feeding probiotics of anti-swine escherichia coli infection | |
| Luo et al. | Early-life nutrition interventions improved growth performance and intestinal health via the gut microbiota in piglets | |
| Dobrowolski et al. | Structural changes in the small intestine of female turkeys receiving a probiotic preparation are dose and region dependent | |
| Hosseindoust et al. | Dietary Bacillus subtilis B2A strain in laying hens challenged with Salmonella gallinarum: effects on egg production, egg quality, blood haptoglobin and targeted intestinal Salmonella shedding | |
| CN110226568A (en) | A kind of application in terms of regulating and controlling aquatic animal intestinal health of method and fly maggot improving fly maggot antibacterial peptide fungistatic effect | |
| LC et al. | Use of lactic acid bacteria from Nile tilapia Oreochromis niloticus as probiotics for sustainable production and improvement in fish welfare | |
| Song et al. | Effects of Lactobacillus plantarum on growth traits, slaughter performance, serum markers and intestinal bacterial community of Daheng broilers |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180615 |