CN107893110A - A kind of screening technique of the feeding probiotics of anti-pig salmonella infection - Google Patents
A kind of screening technique of the feeding probiotics of anti-pig salmonella infection Download PDFInfo
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Abstract
The present invention discloses a kind of screening technique of the feeding probiotics of anti-pig salmonella infection, comprises the following steps：Salmonella infection morbidity piglet and the screening of disease-resistant piglet and its collection of intestinal contents sample；Salmonella infection morbidity piglet and the high-flux sequence of disease-resistant intestine of young pigs microorganism species；Salmonella infection morbidity piglet and disease-resistant intestine of young pigs microorganism metagenomics comparative analysis；And the screening and identification of the probiotics of anti-salmonella infection.The screening technique of the feeding probiotics of anti-pig salmonella infection proposed by the present invention, targetedly the probiotics with anti-salmonella infection function is filtered out from the huge microorganism species in disease-resistant piglet road, it is with strong points, avoid the blindness of screening, screening efficiency is improved, saves substantial amounts of human and material resources and time.
Description
Technical field
The present invention relates to additive for feed for piglets technical field, more particularly to a kind of feeding benefit of anti-pig salmonella infection
The screening technique of raw bacterium.
Background technology
Probiotics plays more and more important effect in terms of human and livestock health or some diseases of preventing and treating is ensured, particularly exists
China's antibiotic will disable comprehensively on the premise of, intestinal health and diarrhoea by be threaten China's animal husbandry development bottleneck problem.
Wherein, salmonellosis, also known as paratyphoid, it is various animals disease general name as caused by Salmonella bacteria, it is more in clinical aspect
Septicemia and enteritis are shown as, can also pregnant female is miscarried.Therefore, new Substitutes For Antibiotic is researched and developed, screening has
Prevent and treat the generation of animal salmonellosis, improve the feeding probiotics of the given efficacy such as animal anti-salmonella infection ability into
For the focus studied both at home and abroad.
Although current China has carried out the screening operation of substantial amounts of probiotics, and is widely used in production practices,
It is that these probiotics are mainly used for promoting digesting and assimilating for animal intestinal tract nutriment, promotes growth, and many probiotics are simultaneously
Non-sourcing in body animal, into host in after, it is difficult to be adapted to host, it is difficult to play its definite effect.It is and existing
Probiotics triage techniques mainly includes In Vitro Bacteriostasis method and attacking bacterium method in vivo, there is blindness, efficiency is low and specific aim not
By force and mostly external source bacterium the shortcomings of.
The content of the invention
The main object of the present invention is to propose a kind of screening technique of the feeding probiotics of anti-pig salmonella infection, it is intended to
The specific aim of the screening technique of the feeding probiotics of anti-pig salmonella infection is improved, improves screening efficiency.
To achieve the above object, the present invention proposes a kind of screening technique of the feeding probiotics of anti-pig salmonella infection,
Comprise the following steps：
Step S10, the morbidity piglet of salmonella infection and disease-resistant piglet, corresponding collection morbidity piglet and disease-resistant son are selected
The intestinal contents sample of pig；
Step S20, the intestinal microflora gene in the intestinal contents sample of extraction morbidity piglet and disease-resistant piglet
Group, and high-flux sequence is carried out to intestinal microflora；
Step S30, metagenomics comparative analysis is carried out to the sequencing data of high-flux sequence；
Step S40, according to the result of metagenomics comparative analysis, compare in morbidity piglet and disease-resistant intestine of young pigs flora
Difference flora, core flora and dominant microflora, and analyze it from the difference flora being only present in disease-resistant intestine of young pigs
In dominant bacteria and core bacterium, the purpose bacterial strain of probiotics to be screened is determined according to dominant bacteria and with the analysis result of core bacterium,
Then according to the bacterium colony characteristic of purpose bacterial strain, strain culturing is carried out using Selective agar medium, that is, filters out anti-pig salmonella sense
The probiotics of dye.
Preferably, step S10 includes：
On the pig farm of salmonella infection outburst, detected by clinicing symptom observation and pathology damage, and combine Salmonella
The qualitative PCR detection of bacterium, from the piglet group of qualitative PCR tests positive, is selected with typical clinical symptom and obvious pathology
The morbidity piglet of damage and the disease-resistant piglet without typical clinical symptom and obvious pathology damage；
Corresponding collection morbidity piglet and the intestinal contents sample of disease-resistant piglet, are saved backup in -80 DEG C；
Wherein, clinicing symptom observation includes piglet abdominal pain diarrhea, high heat dehydration, flatulence, nausea and vomiting and expiratory dyspnea, lost
There is the observation of the even dead typical clinical symptom of purple plague purpura under mass formed by blood stasis, basal part of the ear abdomen and front, and pathology damage detection includes piglet
It is mixed with excrement and does not digest cuing open for the pathology damage that food and a small amount of mucus, mesenterium purulence blood, lymph node oedema, intestinal villi contract
Inspection.
Preferably, step S20 includes：
The genome of microorganism species in the intestinal contents sample of morbidity piglet and disease-resistant piglet is extracted with kit, is obtained
Obtain the 16srRNA genes of different microorganisms in microorganism species；
Using 16srRNA universal primers amplification different microorganisms 16srRNA genes, and to amplified fragments carry out detection and
After concentration mensuration, recycle Tag primer to carry out second and expand, and concentration mensuration is carried out to the fragment of second of amplification, then
After the product dilution of second of amplification to same concentrations, mixed in equal amounts, to build sequencing library；
After the completion of building sequencing library, after the product of second of amplification is denatured with NaOH, sequencing kit is utilized
Gene sequencing is carried out with sequenator.
Preferably, step S30 includes：
Alpha diversity and beta diversity analysis are carried out to the sequencing data of high-flux sequence, to the enteric microorganism OTU of cluster
Carry out the species distribution of species annotation, analysis morbidity piglet and disease-resistant intestine of young pigs flora, and com-parison and analysis morbidity piglet and anti-
The core flora and difference flora of sick intestine of young pigs.
Preferably, step S40 includes：
According to the result of metagenomics comparative analysis, compare the difference bacterium in morbidity piglet and disease-resistant intestine of young pigs flora
Group, core flora and dominant microflora, and analyze advantage therein from the difference flora being only present in disease-resistant intestine of young pigs
Bacterium and core bacterium, the purpose bacterial strain of probiotics to be screened is determined according to the analysis result of dominant bacteria and core bacterium；
Disease-resistant intestine of young pigs flora is inoculated into broth bouillon and carries out Anaerobic culturel, then to the mesh in gut flora
Bacterial strain carry out the line culture of Selective agar medium solid plate, further according to the bacterium colony characteristic of purpose bacterial strain, select Selective agar medium
Positive bacterium colony on solid plate, which is inoculated into liquid selective medium, carries out Zengjing Granule, that is, obtains the anti-pig sramana screened
The probiotics of Salmonella infection；
Biochemical characteristic identification and 16s full genome sequencing identifications are carried out to the probiotics screened, identify screened benefit
Raw bacterium is Lactobacillus pentosus and clostridium butyricum.
Preferably, after the step s 40, in addition to：
Step S50, the probiotics screened is seeded in experiment piglet body, salmonella then is carried out to experiment piglet
Challenge test, diarrhea rate, the morbidity and mortality of statistical test piglet, and determination test intestine of young pigs 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-pig salmonella infection of probiotics；Wherein, the experiment piglet is the new cub immune without salmonella is carried out
Pig.
Preferably, step S50 includes：
Step S51, by 3 immune age in days piglets of no progress salmonella, manually powered milk substitute is fed, young as experiment
Pig, and it is randomly divided into I group, II group, III group and IV group；
Step S52, after the probiotics screened being carried out into vitro culture, to experiment piglet inoculation since 3 age in days mornings
Probiotics, 7 ages in days are grown to piglet is tested, wherein, I group of experiment piglet is inoculated with physiological saline, II group of experiment piglet inoculation penta
Sugared lactobacillus, III group of experiment piglet are inoculated with clostridium butyricum, and IV group of experiment piglet is inoculated with Lactobacillus pentosus and clostridium butyricum simultaneously；
Step S53, I group, II group, III group and IV group experiment piglet is carried out using salmonella in 7 ages in days evening attacking poison,
Attack continuous observation 48h after poison, the incidence of record experiment piglet, statistics diarrhea rate, morbidity and mortality；
Step S54, after attacking malicious 48h, taken a blood sample by vena cava anterior and gather the blood sample of experiment piglet and separate serum, test blood
Clear immune and inflammatory factor content；Meanwhile slaughter experiment piglet and its intestinal mucosa tissue sample being taken after blood sampling, determination test is young
Chitling road histology and morphology structure and immunocyte quantity and density, and intestinal mucosa functional protein expression quantity；According to diarrhoea
Rate, the statistical result of morbidity and mortality, sero-immunity and inflammatory factor content, intestinal tissue morphosis and immune thin
Born of the same parents' quantity and density, and intestinal mucosa functional protein expression quantity, to judge the ability of experiment piglet anti-salmonella infection.
Preferably, in step S52：
The dosage of experiment piglet from 3 ages in days to 7 ages in days inoculation probiotics is followed successively by 5 × 102cfu/100uL、5×103cfu/
100uL、5×104cfu/100uL、5×105cfu/100uL、5×106cfu/100uL；
The dosage of IV group of experiment piglet inoculation Lactobacillus pentosus is identical with the dosage for being inoculated with clostridium butyricum.
Preferably, in step S53：
The toxic agent amount of attacking that experiment piglet carries out attacking poison using salmonella is 2 × 108cfu。
Preferably, in step S54：
It is immunized in the serum and inflammatory factor includes interleukin-6, interleukin 8, interleukin 10, interference
Plain α, tumor necrosis factor-alpha and visible peristalsis visible intestinal peristalsis fatty acid binding protein；
The immunocyte includes goblet cell, lymphocyte and intrinsic confluent monolayer cells, the proinflammatory cytokines；
The intestinal mucosa functional protein includes heat shock protein 70, Caspase-3 albumen, Bax albumen, Occludin
Occludin and Claudin-1 and villin.
The screening technique of the feeding probiotics of anti-pig salmonella infection provided by the invention, by salmonella infection
Morbidity piglet and disease-resistant intestine of young pigs flora metagenomics big data analysis start with, targetedly from disease-resistant piglet road
The probiotics with anti-salmonella infection function is filtered out in huge microorganism species, the probiotics screened is from this
Body animal, the inadaptable and with strong points of probiotics and host is avoided, avoids the blindness of screening, improve screening effect
Rate, save substantial amounts of human and material resources and time.
Embodiment
, below will be in the embodiment of the present invention to make the purpose, technical scheme and advantage of the embodiment of the present invention clearer
Technical scheme be clearly and completely described.Unreceipted actual conditions person, builds according to normal condition or manufacturer in embodiment
The condition of view is carried out.Agents useful for same or the unreceipted production firm person of instrument, it is the conventional production that can be obtained by commercially available purchase
Product.
The present invention proposes a kind of screening technique of the feeding probiotics of anti-pig salmonella infection, provided by the invention anti-
In one embodiment of the screening technique of the feeding probiotics of pig salmonella infection, the feeding benefit of the anti-pig salmonella infection
The screening technique of raw bacterium comprises the following steps：
Step S10, the morbidity piglet of salmonella infection and disease-resistant piglet, corresponding collection morbidity piglet and disease-resistant son are selected
The intestinal contents sample of pig；
In the present embodiment, step S10 includes：
On the pig farm of salmonella infection outburst, detected by clinicing symptom observation and pathology damage, and combine Salmonella
Qualitative PCR (PCR, Polymerase Chain Reaction, the PCR) detection of bacterium, it is in from qualitative PCR detection
In positive piglet group, select morbidity piglet with typical clinical symptom and obvious pathology damage and without typical clinical symptom and
The disease-resistant piglet of obvious pathology damage；
Corresponding collection morbidity piglet and the intestinal contents sample of disease-resistant piglet, are saved backup in -80 DEG C；
Wherein, clinicing symptom observation includes piglet abdominal pain diarrhea, high heat dehydration, flatulence, nausea and vomiting and expiratory dyspnea, lost
There is the observation of the even dead typical clinical symptom of purple plague purpura under mass formed by blood stasis, basal part of the ear abdomen and front, and pathology damage detection includes piglet
It is mixed with excrement and does not digest cuing open for the pathology damage that food and a small amount of mucus, mesenterium purulence blood, lymph node oedema, intestinal villi contract
Inspection.
During the intestinal contents sample of morbidity piglet and disease-resistant piglet, more than 3 times Salmonellas are at least gathered
The different disease-resistant piglets and morbidity piglet on 3~5 different pig farms of bacterium infection eruption and prevalence, and pass through clinicing symptom observation, disease
The qualitative PCR detection of damage check and salmonella is managed, selects the morbidity with typical clinical symptom and obvious pathology damage
Piglet and each 20~30 of the disease-resistant piglet without typical clinical symptom and obvious pathology damage, the intestines of each piglet are gathered respectively
Road content (chyme or excrement) is used as sample.In this way, improving the preciseness of sample collection, avoid by gathered sample
The problem of causing final the selection result inaccuracy with contingency.
Step S20, the intestinal microflora gene in the intestinal contents sample of extraction morbidity piglet and disease-resistant piglet
Group, and high-flux sequence is carried out to intestinal microflora；
In the present embodiment, step S20 includes：
With the kit (reagent dedicated for microbial genome in extraction chyme and excrement that Qiagen companies produce
Box) extraction morbidity piglet and disease-resistant piglet intestinal contents sample in microorganism species genome, obtain microorganism species
The 16srRNA genes of middle different microorganisms；
Using 16srRNA universal primers amplification different microorganisms 16srRNA genes, and to amplified fragments carry out detection and
After concentration mensuration, recycle Tag primer to carry out second and expand, and concentration mensuration is carried out to the fragment of second of amplification, then
After the product dilution of second of amplification to same concentrations, mixed in equal amounts, to build sequencing library；
After the completion of building sequencing library, after the product of second of amplification is denatured with NaOH, sequencing kit is utilized
(Qiagen companies) and sequenator (Miseq sequenators) carry out gene sequencing.
Step S30, metagenomics comparative analysis is carried out to the sequencing data of high-flux sequence；
In the present embodiment, step S30 includes：
Alpha diversity and beta diversity analysis are carried out to the sequencing data of high-flux sequence, to the enteric microorganism OTU of cluster
(activity classification unit, Operational Taxonomic Units, OUT) carries out species annotation, analysis morbidity piglet and disease-resistant
The species distribution of intestine of young pigs flora, and the core flora and difference flora of com-parison and analysis morbidity piglet and disease-resistant intestine of young pigs.
Using QIIME (Quantitative Insights Into Microbial Ecology) analysis platform, to hair
The gut flora of sick piglet and disease-resistant piglet carries out metagenomics comparative analysis, that is, compares its alpha diversity and beta diversity, right
The enteric microorganism OTU of cluster carries out the gut flora species distribution of species annotation, analysis morbidity piglet and disease-resistant piglet；With
LEFSe (LDA Effect Size) com-parison and analysis morbidity piglet and the core flora and difference flora of disease-resistant intestine of young pigs.
Step S40, according to the result of metagenomics comparative analysis, compare in morbidity piglet and disease-resistant intestine of young pigs flora
Difference flora, core flora and dominant microflora, and analyze it from the difference flora being only present in disease-resistant intestine of young pigs
In dominant bacteria and core bacterium, the purpose bacterial strain of probiotics to be screened is determined according to dominant bacteria and with the analysis result of core bacterium,
Then according to the bacterium colony characteristic of purpose bacterial strain, strain culturing is carried out using Selective agar medium, that is, filters out anti-pig salmonella sense
The probiotics of dye.
According to distributed number of the flora in intestinal microflora, morbidity piglet and disease-resistant intestine of young pigs can be analyzed
Difference flora, core flora and dominant microflora in microorganism species, and further analyze and be only present in disease-resistant intestine of young pigs
In difference flora in dominant bacteria and core bacterium, it should be noted that in the process, it is necessary first to guarantee finally filter out
Bacterial strain be the difference bacterium in difference bacterium in disease-resistant intestine of young pigs flora, and this difference bacterium is in all disease-resistant intestine of young pigs
(core bacterium i.e. as described herein) is distributed, while occupies higher percentage in all floras of disease-resistant intestine of young pigs
(dominant bacteria i.e. as described herein).Then according to the purpose that probiotics to be screened is confirmed to the analysis result of dominant bacteria and core bacterium
(in the embodiment that he provides in the present invention, the purpose bacterial strain to be screened determined after analysis is Lactobacillus pentosus and fourth to bacterial strain
Sour clostridium), finally carry out the processes such as selectivity culture, separation screening and identification to purpose bacterial strain.
In the present embodiment, step S40 includes：
According to the result of metagenomics comparative analysis, compare the difference bacterium in morbidity piglet and disease-resistant intestine of young pigs flora
Group, core flora and dominant microflora, and analyze advantage therein from the difference flora being only present in disease-resistant intestine of young pigs
Bacterium and core bacterium, the purpose bacterial strain of probiotics to be screened is determined according to the analysis result of dominant bacteria and core bacterium；
Disease-resistant intestine of young pigs flora is inoculated into broth bouillon and carries out Anaerobic culturel, then to the mesh in gut flora
Bacterial strain carry out the line culture of Selective agar medium solid plate, further according to the bacterium colony characteristic of purpose bacterial strain, select Selective agar medium
Positive bacterium colony on solid plate, which is inoculated into liquid selective medium, carries out Zengjing Granule, that is, obtains the anti-pig sramana screened
The probiotics of Salmonella infection；
Biochemical characteristic identification and 16s full genome sequencing identifications are carried out to the probiotics screened, identify screened benefit
Raw bacterium is Lactobacillus pentosus and clostridium butyricum.
Lactobacillus pentosus is a kind of probiotics, can adjust intestinal colony balance, suppress the life of intestinal toxic putrefactivebacteria
Long and toxin generation, promote digestion and the absorption function of intestines, while also there is the inhibitory action to pathogenic bacteria, it is pre- so as to play
Anti- or treatment bacterial diarrhea as caused by infection, and reduce infection risk caused by external pathogenic bacteria.Clostridium butyricum conduct
Feed addictive is applied in animal feed, can promote the propagation of animal intestinal tract profitable strain (Bifidobacterium, Bacillus acidi lactici)
And development, suppress growth, the breeding of harmful bacteria and spoilage organisms in enteron aisle, correct enteric flora disturbance, reduce the generation of enterotoxin,
And the drug resistance of animal bacteria can also be reduced, so as to play the positive role for ensureing animal health.In the present embodiment, pass through
The screening technique of the feeding probiotics of the anti-pig salmonella infection, the anti-pig salmonella infection screened it is prebiotic
Bacterium is Lactobacillus pentosus and clostridium butyricum, is the mushroom that prevention or therapeutic action can be played to intestine of young pigs disease, and have
The ability of Kang Shashi doors bacterium infection, meet expected screening purpose；Therefore, purpose bacterial strain is being put down using Selective agar medium solid
After plate line culture, it should select positive bacteria backwardness according to the bacterium colony characteristic of Lactobacillus pentosus and clostridium butyricum and be seeded to liquid choosing
Select and Zengjing Granule is carried out in culture medium.
Therefore, in step s 40, the Selective agar medium flat board and liquid selective medium should be corresponded to use and be advantageous to
Lactobacillus pentosus and the culture medium of clostridium butyricum breeding, the Selective agar medium solid plate line culture, the training of Zengjing Granule
Foster method should be corresponded to using the cultural method for being advantageous to Lactobacillus pentosus and clostridium butyricum breeding；In addition, the broth bouillon
The culture medium being known to the skilled person, the Anaerobic culturel to enter by the way that well known to a person skilled in the art cultural method
OK, the biochemical characteristic identification and the identification of 16s full genomes are using well known to a person skilled in the art the side identified bacterial strain
Method is carried out.
The screening technique of the feeding probiotics of anti-pig salmonella infection provided by the invention, by salmonella infection
Morbidity piglet and disease-resistant intestine of young pigs flora metagenomics big data analysis start with, targetedly from disease-resistant piglet road
The probiotics with anti-salmonella infection is filtered out in huge microorganism species, the probiotics screened moves from body
Thing, the inadaptable and with strong points of probiotics and host is avoided, the blindness of screening is avoided, improves screening efficiency, is saved
Substantial amounts of human and material resources and time are saved.
In another embodiment of the screening technique of the feeding probiotics of anti-pig salmonella infection provided by the invention,
After step S40, in addition to：
Step S50, the probiotics screened is seeded in experiment piglet body, salmonella then is carried out to experiment piglet
Challenge test, diarrhea rate, the morbidity and mortality of statistical test piglet, and determination test intestine of young pigs 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-pig salmonella infection of probiotics；Wherein, the experiment piglet is the new cub immune without salmonella is carried out
Pig.
In the present embodiment, step S50 includes：
Step S51, by 3 immune age in days piglets of no progress salmonella, manually powered milk substitute is fed, young as experiment
Pig, and it is randomly divided into I group, II group, III group and IV group；
Step S52, after the probiotics screened being carried out into vitro culture, to experiment piglet inoculation since 3 age in days mornings
Probiotics, 7 ages in days are grown to piglet is tested, wherein, I group of experiment piglet is inoculated with physiological saline, II group of experiment piglet inoculation penta
Sugared lactobacillus, III group of experiment piglet are inoculated with clostridium butyricum, and IV group of experiment piglet is inoculated with Lactobacillus pentosus and clostridium butyricum simultaneously；
Step S53, I group, II group, III group and IV group experiment piglet is carried out using salmonella in 7 ages in days evening attacking poison,
Attack continuous observation 48h after poison, the incidence of record experiment piglet, statistics diarrhea rate, morbidity and mortality；
Step S54, after attacking malicious 48h, taken a blood sample by vena cava anterior and gather the blood sample of experiment piglet and separate serum, test blood
Clear immune and inflammatory factor content；Meanwhile slaughter experiment piglet and its intestinal mucosa tissue sample being taken after blood sampling, determination test is young
Chitling road histology and morphology structure and immunocyte quantity and density, and intestinal mucosa functional protein expression quantity；According to diarrhoea
Rate, the statistical result of morbidity and mortality, sero-immunity and inflammatory factor content, intestinal tissue morphosis and immune thin
Born of the same parents' quantity and density, and intestinal mucosa functional protein expression quantity, to judge the ability of experiment piglet anti-salmonella infection.
In step s 51, the experiment piglet is to be purchased from 3 ages in days without the immune pig farm of anti-salmonella is carried out
Salmonella feminine gender piglet, totally 40,4 groups are randomly divided into, every group of 10 piglets.Observe and record the mistake of experiment piglet incidence
Cheng Zhong, diarrhoea standard is separated into liquid, shapeless, liquid dung to test piglet excrement；It is depressed to test piglet pig spirit, food
It is intended to decline, body temperature rise, has difficulty in breathing, the symptom of diarrhoea stomachache, it is morbidity standard that purple plague purpura, which occurs, under the basal part of the ear, abdomen and front；With
Group records diarrhoea, morbidity head number and dead head number, calculation formula daily for unit：
Total diarrhoea head/(every group of piglet head number × experiment number of days) × 100% of every group of diarrhea rate (%)=in experimental period,
Total morbidity head/(every group of piglet head number × experiment number of days) × 100% of every group of the incidence of disease (%)=in experimental period,
The death rate (%)=every group of death head/every group of total head number × 100%.
Wherein, in step S52：The microbial strains that the extracorporeal culturing method of probiotics is known to the skilled person
Cultural method；Experiment piglet inoculation probiotics measures day by day incremental mode by 10 times and carried out, that is, tests piglet from 3 ages in days to 7 days
The dosage of age inoculation probiotics is followed successively by 5 × 102cfu/100uL、5×103cfu/100uL、5×104cfu/100uL、5×
105cfu/100uL、5×106cfu/100uL；For IV group of experiment piglet, it is inoculated with Lactobacillus pentosus and butyric acid shuttle simultaneously
During bacterium, Lactobacillus pentosus is identical with the dosage of inoculation of clostridium butyricum, i.e., two kinds of probiotics respectively account for half.
In step S53：The toxic agent amount of attacking that experiment piglet carries out attacking poison using salmonella is 2 × 108cfu。
In step S54：It is immunized in the serum and inflammatory factor includes interleukin-6 (IL-6), interleukin 8
(IL-8), interleukin 10 (IL-10), interferon-' alpha ' (IFN-α), tumor necrosis factor-alpha (TNF-α) and visible peristalsis visible intestinal peristalsis aliphatic acid knot
Hop protein (visible peristalsis visible intestinal peristalsis iFABP)；The immunocyte includes goblet cell, lymphocyte and intrinsic confluent monolayer cells, the immunoinflammatory
The factor；The intestinal mucosa functional protein includes heat shock protein 70 (HSF70), Caspase-3 albumen (Caspase-3), Bax
Albumen (Bax), Occludin Occludin and Claudin-1 and villin (Villin).
The probiotics infected the anti-pig Sharpe door bacterium screened by above-mentioned steps S10 to step S40 is inoculated into examination
Test in piglet, salmonella challenge test then is carried out to experiment piglet, diarrhea rate that statistical test piglet is attacked after poison, the incidence of disease
And the death rate, and determination test intestine of young pigs histology and morphology structure and immunocyte, proinflammatory cytokines and mucous membrane functional protein
Matter is horizontal, according to diarrhea rate, the statistical result of morbidity and mortality, intestinal tissue morphosis and immunocyte, immune inflammation
Sex factor and mucous membrane functional protein are horizontal, intuitively to analyze the effect of the anti-pig salmonella infection of screened probiotics,
Further to confirm the probiotics Lactobacillus pentosus and clostridium butyricum that are screened through above-mentioned steps S10 to step S40, there is anti-pig
The function of Sharpe door bacterium infection.
Technical scheme is described in further detail below according to specific embodiment, it will be appreciated that following real
Example is applied only to explain the present invention, is not intended to limit the present invention.
Embodiment 1
(1) pig farm of 5 salmonella infection outbursts is chosen, passes through piglet abdominal pain diarrhea, high heat dehydration, flatulence, nausea
There is the observation of the even dead typical clinical symptom of purple plague purpura under vomiting and expiratory dyspnea, septicemia, basal part of the ear abdomen and front, and
It is mixed with piglet excrement and does not digest food and a small amount of mucus, mesenterium purulence blood, lymph node oedema, the pathology damage of intestinal villi contracting
Cut open inspection, and combine salmonella qualitative PCR detect, from the piglet group of qualitative PCR tests positive, select with typical case
Clinical symptoms and the morbidity piglet of obvious pathology damage and the disease-resistant piglet each 25 without typical clinical symptom and obvious pathology damage
Head；Corresponding collection morbidity piglet and each Gut Segment sample of disease-resistant intestine of young pigs, are saved backup in -80 DEG C.
(2) kit dedicated for microbial genome in extraction chyme and excrement produced with Qiagen companies extracts
The genome of microorganism species in the enteron aisle Digesta samples of morbidity piglet and disease-resistant piglet, obtain different micro- lifes in microorganism species
The 16srRNA genes of thing；Using the 16srRNA genes of 16srRNA universal primers amplification different microorganisms, and amplified fragments are entered
After row detection and concentration mensuration, recycle Tag primer to carry out second and expand, and concentration is carried out to the fragment of second of amplification
Determine, after the product dilution for then expanding second to same concentrations, mixed in equal amounts, to build sequencing library；Structure is surveyed
After the completion of preface storehouse, after the product of second amplification is denatured with NaOH, using sequencing kit (Qiagen companies) and
Miseq sequenators carry out gene sequencing.
(3) alpha diversity is carried out to the sequencing data of step (2) and beta diversity is analyzed, to the enteric microorganism OTU of cluster
Carry out the species distribution of species annotation, analysis morbidity piglet and disease-resistant intestine of young pigs flora, and com-parison and analysis morbidity piglet and anti-
The core flora and difference flora of sick intestine of young pigs.
(4) according to the analysis result of step (3), compare morbidity piglet and difference flora in disease-resistant intestine of young pigs flora,
Core flora and dominant microflora, and analyze from the difference flora being only present in disease-resistant intestine of young pigs dominant bacteria therein and
Core bacterium, determine that the purpose bacterial strain of probiotics to be screened is Lactobacillus pentosus and fourth according to the analysis result of dominant bacteria and core bacterium
Sour clostridium；
Disease-resistant intestine of young pigs flora is inoculated into broth bouillon and carries out Anaerobic culturel, is then inoculated into gut flora
Line culture is carried out on the Selective agar medium solid plate of Lactobacillus pentosus and clostridium butyricum, further according to Lactobacillus pentosus and butyric acid
The bacterium colony characteristic of clostridium, selects the positive bacterium colony on Selective agar medium solid plate and is inoculated into liquid selective medium and increased
Bacterium is cultivated, that is, obtains the probiotics for the anti-pig salmonella infection screened；
Biochemical characteristic identification and 16s full genome sequencing identifications are carried out to the probiotics screened, identify screened benefit
Raw bacterium is Lactobacillus pentosus and clostridium butyricum.
Embodiment 2
(1) the salmonella feminine gender piglet that 40 are purchased from without 3 ages in days for carrying out the immune pig farm of salmonella, uses
Artificial powered milk substitute feeding, as experiment piglet, and is randomly divided into I group, II group, III group and IV group, every group of 10 piglets, wherein, I
Group is control group, and II group, III group and IV group is probiotic group；
(2) after the probiotics screened in embodiment 1 (including Lactobacillus pentosus and clostridium butyricum) being carried out into vitro culture,
By 10 times of amounts incremental mode day by day, to experiment piglet inoculation probiotics since 3 age in days mornings, 7 are grown to piglet is tested
Age in days, first day dosage of inoculation are 5 × 102Cfu/100uL, then follow-up daily dosage of inoculation be followed successively by 5 × 103cfu/
100uL、5×104cfu/100uL、5×105cfu/100uL、5×106cfu/100uL；Wherein, I group of experiment piglet inoculation physiology
Salt solution 100uL, II group of experiment piglet inoculation Lactobacillus pentosus 100uL, III group of experiment piglet inoculation clostridium butyricum 100uL, IV group
Experiment piglet is inoculated with Lactobacillus pentosus 50uL and clostridium butyricum 50uL simultaneously；
(3) I group, II group, III group and IV group experiment piglet is carried out using salmonella in 7 ages in days evening attacking poison, attacks poison
Dosage is 2 × 108cfu；Attack continuous observation 48h after poison, record experiment piglet body temperature (rectum), diarrhoea and incidence, with examination
Test piglet excrement and be separated into diarrhoea standard in liquid, shapeless, liquid dung；With test piglet pig spirit depressed, anorexia, body
Temperature rise, expiratory dyspnea, the symptom of diarrhoea stomachache, it is morbidity standard that purple plague purpura, which occurs, under the basal part of the ear, abdomen and front；It is every in units of group
Its record diarrhoea, morbidity head number and dead head number, and diarrhea rate, morbidity and mortality are counted, calculation formula is：Diarrhea rate
Every group of total diarrhoea head/(every group of piglet head number × experiment number of days) × 100%, the incidence of disease (%)=examination in (%)=experimental period
Every group of total morbidity head/(every group of piglet head number × experiment number of days) × 100% in the phase is tested,
The death rate (%)=every group of death head/every group of total head number × 100%.
The each group experiment body temperature of piglet, diarrhea rate, the statistical result of the incidence of disease and the death rate are as shown in table 1.
The diarrhea rate of 1 embodiment of table, 2 kinds of each group experiment piglets, morbidity and mortality statistical result
| I group | II group | III group | IV group | |
| Diarrhea rate (%) | 82.9a | 62.9b | 54.3b | 38.5c |
| The incidence of disease (%) | 87.1a | 64.3b | 60.0b | 42.9c |
| The death rate (%) | 70a | 20b | 20b | 10c |
| Body temperature (DEG C) | 42.3±0.6 | 40.1±0.3 | 39.8±0.2 | 39.4±0.3 |
From the statistical result of table 1, carried out using salmonella after attacking poison, probiotic group (II group, III group and IV group)
Experiment grice diarrhoea rate significantly reduces, caused by illustrating that Lactobacillus pentosus and clostridium butyricum can significantly reduce salmonella infection
Diarrhea rate, morbidity and mortality, while body temperature caused by can alleviating salmonella infection raises, and pentose breast is inoculated with simultaneously
It is better during bacillus clostridium butyricum.
(4) after attacking malicious 48h, taken a blood sample by vena cava anterior and gather the blood sample of experiment piglet and separate serum, test sera is exempted from
Epidemic disease and inflammatory factor content；Meanwhile slaughter experiment piglet and its intestinal mucosa tissue sample is taken after blood sampling, determination test piglet intestines
Road histology and morphology structure and immunocyte quantity and density, and intestinal mucosa functional protein expression quantity.Above-mentioned indices
Assay method and measurement result are as follows：
1. intestinal tissue morphosis and immunocyte quantity and density
Thick about 5 μm mucous membrane tissue is taken, intestine colibacillosis is made after Hematoxylin-eosin dyes, using micro- sem observation, and
Figure is taken by pathology picture and text report analysis system and taken pictures, determines the small intestinal mucosa of I group, II group, III group and IV group experiment piglet
Height of naps, Crypt depth, fine hair width and fuzzy surface product, immunocyte quantity and density, measurement result is respectively such as table
2nd, shown in table 3.
The measurement result of 2 embodiment of table, 2 kinds of each group experiment piglets morphosis
Height of naps can directly reflect the function status of enteron aisle with Crypt depth, and intestinal villi atrophy can cause the upper of maturation
Chrotoplast quantity is reduced, and ripe villus epithelial cells, which just have, absorbs nutrient function, therefore ripe cell quantity reduces meeting
Nutritional ingredient is set to fully absorb；Crypt depth reflects the production rate of villus epithelial cells, after crypts of small intestine shoals, says
Clear-cells maturing rate rises, secreting function enhancing；The functional status of height of naps/Crypt depth reaction small intestine, the petition of surrender under ratio
Bright mucous membrane is damaged digestibility and declined.
Understand that Lactobacillus pentosus and clostridium butyricum are remarkably improved suede with reference to the measurement result in above-mentioned analysis and table 2
Hair height, the ratio of height of naps/Crypt depth and fuzzy surface product, reduce Crypt depth, illustrate Lactobacillus pentosus and butyric acid
After clostridium acts on experiment piglet, intestinal mucosa injury caused by salmonella infection can be effectively reduced.
The measurement result of 3 embodiment of table, 2 kinds of each group experiment intestine of young pigs immunocyte quantity and density
| Project | I group | II group | III group | IV group |
| Jejunum | ||||
| Goblet cell quantity | 5.3±1.1b | 7.2±1.0b | 8.6±1.1a | 9.5±1.8a |
| Lymphocyte quantity | 26.7±3.7 | 28.4±3.9 | 29.6±4.2 | 28.0±4.3 |
| Lamina propria cell density | 6329±804c | 8044±921b | 9016±903b | 12358±839a |
| Lymphocyte density | 1419±323 | 1375±284 | 1348±298 | 1517±238 |
| Ileum | ||||
| Goblet cell quantity | 4.2±1.3b | 6.7±0.9a | 7.1±1.6a | 7.8±1.0a |
| Lymphocyte quantity | 26.6±2.3b | 31.4±4.2a | 30.8±3.9a | 31.5±2.9a |
| Lamina propria cell density | 8109±1081b | 8061±639b | 10692±1130a | 10989±974a |
| Lymphocyte density | 960±125 | 875±109 | 993±142 | 912±208 |
Goblet cell and the mucus of enterocyte secretion play lubrication protection to intestinal mucosa epithelium, are important are immunized
Relevant cell.Intestinal intraepithelial lymphocytes is distributed between enteron aisle simple columnar epithelium cell, on the basilar memebrane of mucosal epithelium, is had
Indispensable status, by scholar as one of standard for weighing intestinal immunological barrier performance.
Understand that Lactobacillus pentosus and clostridium butyricum are remarkably improved cup with reference to the measurement result in above-mentioned analysis and table 3
Shape cell and lymphocyte quantity and density, illustrate that Lactobacillus pentosus and clostridium butyricum are acted on after testing piglet, can improve
Its intestinal immunological barrier ability.
2. sero-immunity and inflammatory factor content
Using EUSA (ELISA) method, I group, II group, III group and IV group experiment piglet blood is determined
Sero-immunity and Correlative Inflammatory Factors content, measurement result in sample is as shown in table 4.
The measurement result of 4 embodiment of table, 2 kinds of each group experiment piglet sero-immunities and Correlative Inflammatory Factors content
| Project | I group | II group | III group | IV group |
| IL-6 | 103.6±12.8a | 78.3±9.6b | 73.9±8.3b | 79.1±7.2b |
| IL-8 | 181.4±21.6a | 153.4±14.9b | 138.9±10.5b | 152.6±11.9b |
| IL-10 | 89.5±8.7a | 66.5±7.9b | 58.7±6.7b | 61.2±7.3b |
| IFN-α | 101.6±12.9b | 148.9±22.7a | 154.9±12.8a | 161.8±13.3a |
| TNF-α | 97.8±8.5b | 128.9±11.6a | 137.6±14.5a | 129.8±13.1a |
| iFABP | 1.01±0.17b | 1.33±0.09a | 1.47±0.12a | 1.51±0.19a |
Interleukins (IL-6) be activation T cell and fibroblast caused by lymphokine, B cell precursor can be made
As the cell of antibody, and colony stimulating factor collaboration is produced, the growth and differentiation of original bone marrow-derived cells can be promoted, enhancing is certainly
The cracking function of Natural killer cell.Interleukin 8 (IL-8) is also known as the neutrophil cell factor, is the important of diseases associated with inflammation
Medium, played an important role in anti-infective, immune response regulation and anti-tumor aspect.Interleukin 10 (IL-10) can press down
The T cell of system activation produces cell factor, therefore is once referred to as CSIF, particularly suppresses the production of TH1 cells
The cell factors such as raw IL-2, IFN-γ and lt, so as to suppress cellullar immunologic response.IFN-α is referred to as interferon type Ⅰ, can induce
Cell produces the transcription and translation that disease-resistant toxenzyme carrys out viral interference and reaches the effect for suppressing virus multiplication.Tumor necrosis factor-alpha
(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 aliphatic acid
Associated proteins (visible peristalsis visible intestinal peristalsis iFABP) play 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 during intestine ischemia, and membrane passage is larger in ischemic, its
Molecule can be released into blood by cell membrane.
Understand that Lactobacillus pentosus and clostridium butyricum can significantly reduce serum with reference to the measurement result in above-mentioned analysis and table 4
IL-6, IL-8 and IL-10 content, IFN-α, TNF-α and iFABP content are significantly improved, illustrate Lactobacillus pentosus and butyric acid shuttle
After bacterium acts on experiment piglet, the inflammation of salmonella infection initiation can be effectively reduced, improves Organism immunoregulation ability.
3. intestinal mucosa functional protein expression quantity
Using immunoblotting (Western blot), the enteron aisle of I group, II group, III group and IV group experiment piglet of measure
Mucous membrane albumen relative expression quantity, measurement result are as shown in table 5.
The measurement result of 5 embodiment of table, 2 kinds of each group experiment intestine of young pigs mucous membrane functional protein expression quantity
| Project | I group | II group | III group | IV group |
| HSP70 | 2.35±0.31a | 1.04±0.29b | 0.97±0.28b | 1.06±0.17b |
| Caspase-3 | 1.83±0.19a | 1.29±0.35b | 1.36±0.31b | 1.27±0.30b |
| Bax | 1.29±0.18a | 0.83±0.03b | 1.04±0.16ab | 1.05±0.08ab |
| Occludin | 0.82±0.23b | 1.26±0.31a | 1.33±0.28a | 1.28±0.29a |
| Claudin-1 | 1.31±0.39b | 1.83±0.32a | 1.89±0.24a | 1.71±0.38a |
| Villin | 1.54±0.06a | 1.09±0.08b | 0.97±0.14b | 0.88±0.09b |
Heat shock protein 70 (HSP70) performs most basic physiological function in cell, such as protein folding, stretching, extension, turns
Fortune, the formation of oligomer and depolymerization etc., existence and the function of cell are maintained, when cell is by various harmful stress,
HSP70 can be discharged into the inflammation and immune response of extracellular regulation body in the form of the multiple biological activities factor, stress but work as
When excessive, to cytoprotection with regard to very little, body still can sustain damage HSP70.Bax is the water homologous with BCL-2
Dissolubility GAP-associated protein GAP, it is most important rush apoptogene member in Bcl-2 families, Bax can form heterologous with Bcl-2 protein bindings
Dimer, suppress Bcl-2 function, promote Apoptosis.Caspase is one group of cysteine proteinase family, its family into
Member plays an important role in apoptosis process.Villin villin is actin Fibronectin, is a kind of important thin
Born of the same parents' scaffolding protein, being played a significant role in brush border epithelial cell atomization, it is the mark of the ripe propagation of intestinal cell,
It is also important tumor markers simultaneously.Close connection is a kind of principal mode connected between epithelial cell, has and safeguards epithelium
The difference of cell both sides material and the critical function for keeping cell polarity, transmembrane protein and peripheral protein interaction form
Tight junction protein plastidome, for Occludin Occludin and Claudin-1 as a member in system, major function is to participate in dimension
Hold close-connected barrier function.
Understood with reference to the measurement result in above-mentioned analysis and table 5, compared with control group (I group), two kinds of probiotics pentose breasts
Bacillus and clostridium butyricum significantly reduce Jejunal mucosa HSP70, Caspase-3 and Bax expressing quantity, improve
Viliin, Occludin, Claudin-1 expressing quantity.Illustrate two kinds of probiotic actions of Lactobacillus pentosus and clostridium butyricum
After piglet is tested, the integrality of its intestinal structure is maintained, alleviates the damage of protection of intestinal mucosal barrier cells caused by ablactation stress
Wound and apoptosis.
In summary, the screening technique of the feeding probiotics of anti-pig salmonella infection provided by the invention, by sand
Door Salmonella infection morbidity piglet and disease-resistant intestine of young pigs flora metagenomics big data analyze start with, targetedly from
The probiotics with the infection of Kang Shashi doors bacterium is filtered out in the huge microorganism species in disease-resistant piglet road, at least with following excellent
Point：(1) probiotics screened is specific to the preventing and treating of piglet salmonella infection；(2) probiotics screened derives from
Body animal, the inadaptable of probiotics and host is avoided, (3) are with strong points, avoid the blindness of screening, improve screening
Efficiency, save substantial amounts of human and material resources and time.Meanwhile the probiotics screened can be effectively reduced by salmonella infection
The generation of caused piglet diseases, intestine of young pigs health is ensured, the bottleneck problem for solving puzzlement pig industry.
The preferred embodiments of the present invention are these are only, are not intended to limit the scope of the invention, for this area
For technical staff, the present invention can have various modifications and variations.It is all in the spirit and principles in the present invention etc, that is made is any
Modification, equivalent substitution, improvement etc., it all should be included within the scope of the present invention.
Claims (10)
1. a kind of screening technique of the feeding probiotics of anti-pig salmonella infection, it is characterised in that comprise the following steps：
Step S10, the morbidity piglet of salmonella infection and disease-resistant piglet are selected, corresponding collection morbidity piglet and disease-resistant piglet
Intestinal contents sample；
Step S20, the intestinal microflora genome in the intestinal contents sample of extraction morbidity piglet and disease-resistant piglet, and
High-flux sequence is carried out to intestinal microflora；
Step S30, metagenomics comparative analysis is carried out to the sequencing data of high-flux sequence；
Step S40, according to the result of metagenomics comparative analysis, the difference in morbidity piglet and disease-resistant intestine of young pigs flora is compared
Different flora, core flora and dominant microflora, and analyze from the difference flora being only present in disease-resistant intestine of young pigs therein
Dominant bacteria and core bacterium, the purpose bacterial strain of probiotics to be screened is determined according to dominant bacteria and with the analysis result of core bacterium, then
According to the bacterium colony characteristic of purpose bacterial strain, strain culturing is carried out using Selective agar medium, that is, filters out anti-pig salmonella infection
Probiotics.
2. the screening technique of the feeding probiotics of anti-pig salmonella infection as claimed in claim 1, it is characterised in that step
S10 includes：
On the pig farm of salmonella infection outburst, detected by clinicing symptom observation and pathology damage, and combine salmonella
Qualitative PCR detects, and from the piglet group of qualitative PCR tests positive, selects with typical clinical symptom and obvious pathology damage
The morbidity piglet and disease-resistant piglet without typical clinical symptom and obvious pathology damage；
Corresponding collection morbidity piglet and the intestinal contents sample of disease-resistant piglet, are saved backup in -80 DEG C；
Wherein, clinicing symptom observation includes piglet abdominal pain diarrhea, high heat dehydration, flatulence, nausea and vomiting and expiratory dyspnea, sepsis
There is the observation of the even dead typical clinical symptom of purple plague purpura under disease, basal part of the ear abdomen and front, and pathology damage detection includes piglet excrement
Just it is mixed with and does not digest cuing open for the pathology damage that food and a small amount of mucus, mesenterium purulence blood, lymph node oedema, intestinal villi contract
Inspection.
3. the screening technique of the feeding probiotics of anti-pig salmonella infection as claimed in claim 1, it is characterised in that step
S20 includes：
The genome of microorganism species in the intestinal contents sample of morbidity piglet and disease-resistant piglet is extracted with kit, is obtained micro-
The 16srRNA genes of different microorganisms in biological flora；
Using the 16srRNA genes of 16srRNA universal primers amplification different microorganisms, and amplified fragments are detected and concentration
After measure, recycle Tag primer to carry out second and expand, and concentration mensuration is carried out to the fragment of second of amplification, then by the
The product dilution of secondary amplification is to after same concentrations, mixed in equal amounts, to build sequencing library；
After the completion of building sequencing library, after the product of second of amplification is denatured with NaOH, sequencing kit and survey are utilized
Sequence instrument carries out gene sequencing.
4. the screening technique of the feeding probiotics of anti-pig salmonella infection as claimed in claim 1, it is characterised in that step
S30 includes：
Alpha diversity and beta diversity analysis are carried out to the sequencing data of high-flux sequence, the enteric microorganism OTU of cluster is carried out
Species are annotated, the species distribution of analysis morbidity piglet and disease-resistant intestine of young pigs flora, and com-parison and analysis morbidity piglet and disease-resistant son
The core flora and difference flora in chitling road.
5. the screening technique of the feeding probiotics of anti-pig salmonella infection as claimed in claim 1, it is characterised in that step
S40 includes：
According to the result of metagenomics comparative analysis, compare morbidity piglet and difference flora in disease-resistant intestine of young pigs flora,
Core flora and dominant microflora, and analyze from the difference flora being only present in disease-resistant intestine of young pigs dominant bacteria therein and
Core bacterium, the purpose bacterial strain of probiotics to be screened is determined according to the analysis result of dominant bacteria and core bacterium；
Disease-resistant intestine of young pigs flora is inoculated into broth bouillon and carries out Anaerobic culturel, then to the purpose bacterium in gut flora
Strain carries out the line culture of Selective agar medium solid plate, further according to the bacterium colony characteristic of purpose bacterial strain, selects Selective agar medium solid
Positive bacterium colony on flat board, which is inoculated into liquid selective medium, carries out Zengjing Granule, that is, obtains the anti-pig salmonella screened
The probiotics of infection；
Biochemical characteristic identification and 16s full genome sequencing identifications are carried out to the probiotics screened, identify screened probiotics
For Lactobacillus pentosus and clostridium butyricum.
6. the screening technique of the feeding probiotics of anti-pig salmonella infection as claimed in claim 5, it is characterised in that in step
After rapid S40, in addition to：
Step S50, the probiotics screened is seeded in experiment piglet body, then carrying out salmonella to experiment piglet attacks poison
Experiment, diarrhea rate, the morbidity and mortality of statistical test piglet, and determination test intestine of young pigs histology and morphology structure and immune
Cell, proinflammatory cytokines and mucous membrane functional protein are horizontal, according to diarrhea rate, the statistical result of morbidity and mortality, intestines
Road histology and morphology structure and immunocyte, proinflammatory cytokines and mucous membrane functional protein are horizontal, come analyze screened it is prebiotic
The effect of the anti-pig salmonella infection of bacterium；Wherein, the experiment piglet is the newborn piglet immune without salmonella is carried out.
7. the screening technique of the feeding probiotics of anti-pig salmonella infection as claimed in claim 6, it is characterised in that step
S50 includes：
Step S51, by 3 immune age in days piglets of no progress salmonella, manually powered milk substitute is fed, as experiment piglet, and
It is randomly divided into I group, II group, III group and IV group；
Step S52, it is prebiotic to experiment piglet inoculation since 3 age in days mornings after the probiotics screened being carried out into vitro culture
Bacterium, 7 ages in days are grown to piglet is tested, wherein, I group of experiment piglet is inoculated with physiological saline, II group of experiment piglet inoculation pentose breast
Bacillus, III group of experiment piglet are inoculated with clostridium butyricum, and IV group of experiment piglet is inoculated with Lactobacillus pentosus and clostridium butyricum simultaneously；
Step S53, I group, II group, III group and IV group experiment piglet is carried out using salmonella in 7 ages in days evening attacking poison, attacks poison
Continuous observation 48h afterwards, the incidence of record experiment piglet, statistics diarrhea rate, morbidity and mortality；
Step S54, after attacking malicious 48h, taken a blood sample by vena cava anterior and gather the blood sample of experiment piglet and separate serum, test sera is exempted from
Epidemic disease and inflammatory factor content；Meanwhile slaughter experiment piglet and its intestinal mucosa tissue sample is taken after blood sampling, determination test piglet intestines
Road histology and morphology structure and immunocyte quantity and density, and intestinal mucosa functional protein expression quantity；According to diarrhea rate, hair
The statistical result of sick rate and the death rate, sero-immunity and inflammatory factor content, intestinal tissue morphosis and immunocyte quantity
And density, and intestinal mucosa functional protein expression quantity, to judge the ability of experiment piglet anti-salmonella infection.
8. the screening technique of the feeding probiotics of anti-pig salmonella infection as claimed in claim 7, it is characterised in that in step
In rapid S52：
The dosage of experiment piglet from 3 ages in days to 7 ages in days inoculation probiotics is followed successively by 5 × 102cfu/100uL、5×103cfu/
100uL、5×104cfu/100uL、5×105cfu/100uL、5×106cfu/100uL；
The dosage of IV group of experiment piglet inoculation Lactobacillus pentosus is identical with the dosage for being inoculated with clostridium butyricum.
9. the screening technique of the feeding probiotics of anti-pig salmonella infection as claimed in claim 7, it is characterised in that in step
In rapid S53：
The toxic agent amount of attacking that experiment piglet carries out attacking poison using salmonella is 2 × 108cfu。
10. the screening technique of the feeding probiotics of anti-pig salmonella infection as claimed in claim 7, it is characterised in that
In step S54：
In the serum be immunized and inflammatory factor include interleukin-6, interleukin 8, interleukin 10, interferon-' alpha ',
Tumor necrosis factor-alpha and visible peristalsis visible intestinal peristalsis fatty acid binding protein；
The immunocyte includes goblet cell, lymphocyte and intrinsic confluent monolayer cells, the proinflammatory cytokines；
The intestinal mucosa functional protein includes heat shock protein 70, Caspase-3 albumen, Bax albumen, Occludin
Occludin and Claudin-1 and villin.
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Application publication date: 20180410 |