CN107260796A

CN107260796A - Application of the thyme compound plant essential oil in preventing and treating chicken necrotizing enterocolitis

Info

Publication number: CN107260796A
Application number: CN201710426928.XA
Authority: CN
Inventors: 彭新宇; 高彪; 袁明贵; 尹烨华; 徐志宏
Original assignee: Institute of Animal Health of Guangdong Academy of Agricultural Sciences
Current assignee: Institute of Animal Health of Guangdong Academy of Agricultural Sciences
Priority date: 2017-06-08
Filing date: 2017-06-08
Publication date: 2017-10-20
Anticipated expiration: 2037-06-08
Also published as: CN107260796B

Abstract

The invention discloses application of the thyme compound plant essential oil in preventing and treating chicken necrotizing enterocolitis.Plants essential oil includes thyme nutmeg compound essential oil, thyme sweet basil compound essential oil.Applicant successfully constructed chicken necrotizing enterocolitis animal model by the time of 12 years, and this model is fed using wheat basal diet, and added fish meal in the later stage；Poison is individually attacked using C.perfringens, typical cytopathic is obvious；The phenomenon that coccidia models of concurrency is obscured with global-worm illness before excluding.Test period is short, and repeatability is high.Except experiment in vitro, applicant also utilizes the vivo control effect of the scale-model investigation plants essential oil, really specify that above-mentioned plants essential oil has the effect of prevention and treatment for chicken necrotizing enterocolitis.

Description

Application of the thyme compound plant essential oil in preventing and treating chicken necrotizing enterocolitis

Technical field

The invention belongs to veterinary field, and in particular to thyme compound essential oil answering in preventing and treating chicken necrotizing enterocolitis With.

Background technology

Chicken necrotizing enterocolitis is that Parish (1961) has found first, is a kind of acute digestion caused by C.perfringens Tract disease, it is a kind of disease of chicken generally existing.The loss per year over 2000000000 dollars is caused to global aviculture. In recent years, European Union forbids adding antibiotic growth promoter in feed, result in poultry of the necrotic enteritis in European countries Largely broken out in industry.

Necrotic enteritis generally occur after 4 week old, have in global poultry farming area generation (Long J R, 1973).Clinical symptoms show as actual clinical symptom and inferior clinical symptom.

Necrotic enteritis actual clinical symptom：The classical feature of actual clinical symptom is exactly that chicken group's death rate increases suddenly, There is no tendency, the moist early symptom of bedding and padding sometimes occurs.Morbidity is very fast, dead in 1h~2h, sometimes dead Rate can increase to 50% (Riddell et al 1992).

Inferior clinical symptom：In past several years, more commonly, the necrotic enteritis of this form is not bright for inferior clinical symptom The aobvious clinical symptoms and death rate will not also increase.But, the chronic injury of intestinal mucosa can cause to digest and assimilate bad, weightening Rate declines, feed conversion rate increase, causes huge production loss (Elwinger et al 1992).In subclinical infection feelings Under condition, the damage of enteron aisle causes bacterium to reach bile duct and portal vein, and substantial amounts of C.perfringens parasitizes liver, result in courage Occur pale red or white focus (Onderka et al 1990) on Guan Yan and hepatitis, liver.

Wherein, enteron aisle lesion includes：Naked eyes lesions visible typically occurs in small intestine, but disease can also occur for other organs Become, such as liver and kidney.By pathological anatomy it can be found that duodenum, jejunum and ileum intestinal wall are thinning, and gassy.Necrosis Property enteritis clinical symptoms be small intestinal mucosa occur large area necrosis, surface is covered with yellowish-brown pseudomembrane.Typical subclinical disease Shape is that intestinal mucosal surface has concavo-convex ulcer, and mucous membrane turns yellow, and there is cellulose like material attachment on surface.Necrotic enteritis early stage Strong inflammatory reaction is shown, lamina propria is congested, substantial amounts of inflammatory cell infiltration, mainly neutrophil cell.Gangrenosum acne There is dispersivity necrosis in enteritis later stage, intestinal mucosa, and account for intestinal mucosa 1/3rd arrive half.Location of necrosis and its hetero-organization Between occur a clear lot line due to the accumulation of neutrophil cell.

Chicken necrotizing enterocolitis risk factor include it is following some：

First, nutrition：The major risk factors that necrotic enteritis occurs are exactly to form to be conducive to perfringens in enteron aisle The environment of clostridium growth.The property of daily ration is an important non-bacterial factor for influenceing necrotic enteritis to occur.In daily ration Necrotic enteritis is more susceptible to suffer from containing high-level heavy water soluble starch polysaccharide, therefore barley, rye, oat, wheat are bad The risk factors that dead property enteritis occurs, because water soluble starch polysaccharide can increase chyme viscosity, extend it when enteron aisle passes through Between, reduce nutritive digestibility (Craven et al 2000).High concentration protein in daily ration, such as fish meal, can also increase The incidence of disease of necrotic enteritis, because high protein feed is nondigestible in enteron aisle, causes the increase of intestines and stomach internal protein concentration, Abundant nutrient matrix (Nauerby et al 2003) is provided for the growth of bacterium.The size of feed granules can also influence bad The incidence of disease of dead property enteritis, feed granules it is big be more susceptible to suffer from necrotic enteritis (Engberg et al 2002) than small.

2nd, coccidia：One of topmost risk factor of necrotic enteritis is exactly coccidia, because coccidia causes to intestinal mucosa Damage, cause protein blood plasma to ooze out, provide abundant nutrient matrix for C.perfringens in enteron aisle, be conducive to it to increase Grow and produce (Williams et al 2005) with toxin.Global-worm illness is often to be broken out simultaneously prior to or with necrotic enteritis.Examination Checking is bright, and Eimeria and C.perfringens play synergy during necrotic enteritis lesion.Al-sheikhly etc. In test, ended with the test group and only infection C.perfringens or only infection of co-infection C.perfringens and Amy ear U.S. ear coccidia group compares discovery, and the former lesion is more serious and the death rate is higher.

3rd, pathogenic C.perfringens：C.perfringens content reaches in the enteron aisle of chicken with necrotic enteritis To 10⁶~10⁸CFU/g, and C.perfringens content is 0~10 in healthy chicken intestinal⁵CFU/g(Keyburn et al 2008).But be also not enough to cause necrotic enteritis even if containing substantial amounts of C.perfringens in enteron aisle.Therefore perfringens The quantity of clostridium does not have direct relation with necrotic enteritis, and not all C.perfringens can necrosis induced property Enteritis.The generation of necrotic enteritis needs specific pathogenic conditions, when lesion condition all in the presence of, the aerogenesis of high quantity Capsular clostridium may result in more serious lesion, but on condition that the presence of pathogenic bacterial strains.

4th, bacteriocin：Bacteriocin plays an important roll in necrotic enteritis pathogenesis.Pass through PFGE Or amplified fragment length polymorphism, healthy chicken flock can isolate the A type C.perfringens of different genotype, even In the same enteron aisle of chicken.Opposite, a hypotype can only be cloned in enteron aisle in the case of necrotic enteritis.Naturally extensive After multiple or treatment, there are multiple genotype (Park et al, 2008) again in the bacterium in chicken body.This single bacterial strain is predominantly The phenomenon of position has been demonstrated that it is the main process of necrotic enteritis morbidity in multiple researchs.Separation in necrotic enteritis Strain can secrete the growth factor for more suppressing other C.perfringens than normal bacterial strain.It is this by bacteriogenic poisonous Can suppress other related strains growth protein referred to as bacteriocin.The generation of bacteriocin inhibits other perfringens shuttles Growth of the bacterium in enteron aisle so that surging strain instead of other bacterial strains.

5th, NetB toxin：NetB toxin finds the A type aerogenesis pods isolated in the diseased chicken with necrotic enteritis first In film clostridium.There is similitude with the amino acid sequence of a variety of pore-forming toxins, have with C. perfringens beta toxin homology 38%, the alpha toxin homology produced with staphylococcus has 31%.NetB toxin can make the liver cancer cells of chicken be rounded dissolving, 1.6~1.8nm or so hole is formed on cell membrane.NetB toxin genes are located on plasmid, by VirsR bi-component signals system The regulation and control of system.The C.perfringens of NetB toxin genes is knocked out, infected chicken is unable in test or can not be produced in enteron aisle Necrotic enteritis lesion.But, the supplement mutant after insertion NetB toxin genes has pathogenic as street strain (Keyburn et al2008).Therefore, it can be seen that NetB toxin is the key factor for causing chicken necrotizing enterocolitis.It is most of The bacterial strain isolated from necrotic enteritis case all carries NetB toxin genes.In Canada, from necrotic enteritis diseased chicken 95% all contains NetB toxin genes, the NetB toxin base from the C.perfringens that healthy chicken is isolated in C.perfringens Because the positive only accounts for 35% (Chalmers et al 2008).In U.S.'s separation strains, necrotic enteritis separation strains NetB toxin genes The positive is 58%, and bacterial strain carries NetB toxin genes and there was only 8.75% (Martin and Smyth 2009) in normal flora.It is logical Cross using the substantial amounts of broiler chicken infection experiment of A type C.perfringens that NetB toxin genes are negative and NetB toxin genes are positive, It was found that the only positive C.perfringens of NetB toxin genes could produce necrotic enteritis lesion, and lesion degree and NetB The generation ability of toxin has direct relation (Keyburn et al 2009).

With the development of globalization, control of the China to antibiotic will also integrate with international standard.So needing to carry out Tackle the measure of chicken necrotizing enterocolitis.Because necrotic enteritis is a kind of complicated, by the disease of multifactor impact, this is reappeared Disease will consider many aspects.

And there is following difficult point in the structure of chicken necrotizing enterocolitis animal model：Such as the screening of netB positive strains, different bacterium The identification of the pathogenic ability of strain；Attacking bacterium before poison needs to carry out the culture of continuous 30 hours in two kinds of different culture medias；It is real Test groping for animal feed；Attack determination of concentration of toadstool liquid spice, etc..In addition different scoring criterias is used, is caused Different points-scoring systems can not carry out mutual comparative studies.Because setting up for chicken necrotizing enterocolitis animal model is relative complex, So most research has only carried out testing in vitro at present, without carrying out the test in animal model.Only surveyed in vitro The result of examination tends not to real reaction for prevention effect of the reagent thing for chicken necrotizing enterocolitis.

The content of the invention

It is an object of the invention to provide application of the thyme compound plant essential oil in preventing and treating chicken necrotizing enterocolitis.

The technical solution used in the present invention is：

Application of the thyme compound essential oil in preventing and treating chicken necrotizing enterocolitis, thyme compound essential oil includes thyme-meat Cardamom compound essential oil, thyme-sweet basil compound essential oil.

It is preferred that, the volume ratio of thyme essential oil and nutmeg essential oil is (1-3) in thyme-nutmeg compound essential oil: (3-1)。

It is preferred that, the volume ratio of thyme essential oil and nutmeg essential oil is 1 in thyme-nutmeg compound essential oil:1.

It is preferred that, the volume ratio of thyme essential oil and Basil Essential is (1-3) in thyme-sweet basil compound essential oil:(3- 1)。

It is preferred that, the volume ratio of thyme essential oil and Basil Essential is 1 in thyme-sweet basil compound essential oil:1.

To thyme-nutmeg compound essential oil and thyme-sweet basil compound essential oil at (1-3) in present invention research:(3- 1) the various proportionings in the range of have done related experiment, find 1:1 effect is good, and simple and convenient, and all preferred volume ratios are 1:1 To carry out follow-up zoopery.

It is preferred that, thyme compound essential oil prevents and treats chicken necrotizing enterocolitis by being added in the daily ration of chicken.

It is preferred that, the ratio that thyme compound essential oil is added in daily grain of chicken is 80-150mg/kg.

It is preferred that, the ratio that thyme compound essential oil is added in daily grain of chicken is 100mg/kg.

A kind of feed addictive of prevention chicken necrotizing enterocolitis, additive is thyme compound essential oil or thyme compound essence Oily and its acceptable food grade accessories.

It is preferred that, thyme compound essential oil includes thyme-nutmeg compound essential oil, thyme-sweet basil compound essential oil.

It is a kind of prevent and treat chicken necrotizing enterocolitis method, be by thyme compound essential oil be added in the daily ration of chicken prevent and treat chicken it is bad Dead property enteritis；Thyme compound essential oil includes thyme essential oil and nutmeg essential oil according to volume ratio (1-3):(3-1) mixing Compound essential oil, thyme essential oil and sweet basil compound essential oil are according to volume ratio (1-3):The compound essential oil of (3-1) mixing.

Plants essential oil can be well mixed with daily grain of chicken well.

The beneficial effects of the invention are as follows：

Applicant successfully constructs chicken necrotizing enterocolitis animal model by 1-2 time, and this model uses wheat Basal feed is fed, and adds fish meal in the later stage.Wheat contains non-starch polysaccharide, adds chyme viscosity, have impact on Digesting and assimilating for small intestine, condition is provided for the propagation of C.perfringens.The fish meal that later stage adds, contains protein in daily ration Amount increase, can be supplied to the substantial amounts of amino acid of C.perfringens, chicken is more susceptible to suffer from necrotic enteritis.Using perfringens shuttle Bacterium individually attacks poison, and typical cytopathic is obvious；The phenomenon that coccidia models of concurrency is obscured with global-worm illness before excluding.This model test week Phase is short, and repeatability is high.Except experiment in vitro, applicant also utilizes the vivo control effect of the scale-model investigation plants essential oil, really It specify that plants essential oil has the effect of prevention and treatment for chicken necrotizing enterocolitis.

Applicant passes through numerous studies, preferably goes out thyme-nutmeg 1:1 mixing compound essential oil, thyme-sweet basil 1:1 Compound essential oil is mixed, above-mentioned plants essential oil combination all has certain prevention effect to chicken necrotizing enterocolitis.

High throughput sequencing technologies are the multifarious means of microorganisms more advanced at present, and applicant uses high flux Sequencing technologies study influence of the plants essential oil to chicken necrotizing enterocolitis enteric microorganism.Obtained result of the test data volume is big, accurate True property is high.Better than traditional test method.The present invention is carried out high by the gut flora of the small intestine to 8 experimental groups and caecum section Flux is sequenced.After Quality Control, splicing, the optimization band in each sample is more than 3000.Pass through dilution Curve understands that sequencing amount and sequencing depth cover most of species in sample enough, meet subsequent analysis.Referred to by 4 Alpha Number, i.e., ace indexes, chao indexes, shannon indexes and simpson indexes can be seen that in small intestinal segment, relative to gangrenosum acne The abundance of species reduces in enteritis model group, each plants essential oil treatment group intestinal contents, but diversity increase.Enteron aisle is micro- Biological flora is the key factor for maintaining intestinal health, the disease that the microorganism species balanced can be defendd effectively in enteron aisle Substance.There is different degrees of change relative to artificial challenge's group using the gut flora of plants essential oil treatment group, these changes Active influence may be produced to the defence of disease.

Brief description of the drawings

Fig. 1 is chicken necrotizing enterocolitis small intestine different lesions；Wherein note：1 is blank control, 2,3,4,5,6,7 corresponding points It is not typical 1 point, 2 points, 3 points, 4 points, 5 points, 6 points of lesions；

Fig. 2 is the C.perfringens genotype identification that focus is isolated；Wherein, M is DL2000Marker, and 1~20 is The C.perfringens bacterial strain that focus is isolated；

Fig. 3 is the C.perfringens netB identified for genes that focus is isolated；Wherein, M is DL2000Marker, 1~20 The C.perfringens bacterial strain isolated for focus；

Fig. 4 counts for ordered sequence distribution of lengths；

Fig. 5 is dilution linearity curve；

Fig. 6 is the horizontal species distribution histogram of door；

Fig. 7 is the horizontal species distribution histogram of mesh；

Fig. 8 is the horizontal species distribution histogram of section；

Fig. 9 is otherness matrix thermal map；

Figure 10 is Multi-example similarity dendrogram；

Figure 11 is the PCoAPCoA scatter diagrams based on unweighted unifrac.

Embodiment

Chicken necrotizing enterocolitis is a kind of acute disease of digestive tract caused by C.perfringens, is the most important enteron aisle disease of chicken One of disease.Aviculture to the whole world causes huge loss., should with the limitation used both at home and abroad antibiotic growth accelerator The incidence of disease of disease increased.Find the hot issue that a kind of safely, effectively pharmaceutical preparations are current cultivation fields.

Inventor establishes chicken necrotizing enterocolitis artificial onset's model, and is laid a good foundation for its internal screening.Entirely Research includes the separation identification of C.perfringens in the chicken manure sample of 7 part 1. and necrotic enteritis case intestinal tissue； 2.Multi-PCR methods carry out genotype identification to C.perfringens field separation strains；3. C.perfringens field is separated Strain NetB toxin genes (netB) detection；4. the Resistance detection of C.perfringens field separation strains；5.ERIC-PCR is to production Gas capsular clostridium field separation strains carry out Subtype；6. chicken necrotizing enterocolitis model is set up；7. plants essential oil is to gangrenosum acne intestines Scorching preventing and treating.

Test and acquire excrement from 5 feeding chicken in largely scale on the ground such as Qingyuan City of Guangdong Province, Foshan, Yunfu, Jiangmen, Guangzhou 550 parts of sample and necrotic enteritis case intestinal tissue, are carried out preliminary using selective medium TSC flat boards and blood agar plate Then separation strains have been carried out biochemical identification and the identification of iron content cow's milk characteristic by separation again.Initial gross separation goes out 47 plants of perfringens Clostridium, separation rate is 8.5%, relatively low relative to foreign countries' report separation rate.

Design of primers is carried out for tetra- kinds of toxin genes of α, β, ε and ι, Multi-PCR methods are set up.Use the following institute of primer Show：

Table 1：Muliti-PCR primer sequences

PCR reaction systems are 25 μ L reaction systems：The μ L of DNA profiling 2, each 0.125 μ L of upstream and downstream primer, MaxMaster12.5 μ L, the μ L of nuclease-free water 9.5.Response procedures：94℃5min；94 DEG C of 1min, 55 DEG C of 1min, 72 DEG C of 1min, 30 circulations；72 DEG C, 10min；10 DEG C of preservations.

After PCR reactions terminate, 8 μ L PCR primer is taken, in 1% agarose gel electrophoresis, 80V, 40min detections, using solidifying Glue imaging system is recorded as picture, and picture is preserved.

47 plants of C.perfringens are carried out with genotype identification using this method, it is left that 47 plants of separation strains amplify 400bp Right specific fragment, can be accredited as A types.Additionally by this method to Escherichia coli, bacillus subtilis, staphylococcus, Clostridium butyricum is expanded, and specific product does not occur, illustrates that this method has specificity.

The primer of NetB toxin genes is designed, it is as follows：

Sense primer：5’-GCTGGTGCTGGAATAAATGC-3’(SEQ ID NO：9)；

Anti-sense primer：5’-TCGCCATTGAGTAGTTTCCC-3’(SEQ ID NO：10).

PCR reaction systems are 25 μ L reaction systems：The μ L of template DNA 2, each 0.5 μ L, MaxMaster12.5 μ L of primer, nothing The μ L of nuclease water 9.5.Response procedures：94℃2min；94 DEG C of 45s, 50 DEG C of 30s, 72 DEG C of 45s, 30 circulations；72℃10min；10 DEG C preserve.

47 plants of C.perfringens are detected by PCR method, as a result only 3 plants C.perfringens are netB genes sun Property, sign as cpnetBB, cpnetBD and cpnetBF.

Drug sensitive test is carried out to 47 separation strains.Experiment has used 12 kinds of antibiotic and chemical synthetic drug, wherein to first nitre The resistance ratio of azoles and lincomycin is higher, and respectively 95.7% and 91.5%.To cefalexin, terramycin, Doxycycline, gram Woods mycin, erythromycin resistance ratio also between 70%~80%.Ampicillin-resistant ratio is minimum, and only 12.8%.

Hypotype is carried out to 47 plants using ERIC-PCR technologies by the Multi-PCR C.perfringens for being accredited as A types to divide Analysis.It is separation with similitude >=0.60,47 plants of separation strains can be divided into 5 principal modes, be separation, 47 with similitude >=0.70 Strain separation strains can be divided into 8 hypotypes.

In model test, using the baby chick of healthy Ross 308 of 1 age in days, 6 groups, every group 10 are randomly divided into.Free water, Daily ration based on feeding wheat.Wherein 1 group is blank control, and other groups are test group.Except blank control group, other groups are all Carry out attacking poison by the way of bacterium solution spice.3 experimental groups are respectively adopted 3 plants of NetB gene-positive strains and attacked in 5 groups of test groups Poison, attacks poison for 2 groups with the bacterial strain of 2 NetB gene negatives respectively in addition.Whole feeding process daily ration is divided into two stages：1~13 The daily ration on age in days feeding wheat basis, 14~23 ages in days add 50% fish meal in basal feed.21~23 ages in days are tried 5 Test a group progress bacterium solution spice and attack poison, twice daily.Bacterium solution compound method used in challenge test：Test strain is first in kitchen meat culture 40 DEG C of Anaerobic culturel 15h in base, are then transferred to 40 DEG C of Anaerobic culturel 15h of continuation in FT fluid nutrient mediums.24 age in days cut open inspections, see Examine small intestine lesion and scored, score and use 6 subsystems.As a result find, blank control group and other 2 netB genes Negative bacterial strain attacks poison group all without obvious lesion.The bacterial strain of 3 netB gene masculines attack poison group all occur in that it is typical bad Dead property enteritis lesion.The A type C.perfringens of netB gene masculines is isolated in the focus of morbidity chicken.

Using disk diffusion method and doubling dilution research plants essential oil or compound plant essential oil to C.perfringens Bacteriostatic activity.

With reference to specific experiment, the present invention is further illustrated, but is not limited thereto.Unless otherwise specified, originally Nutmeg essential oil, thyme essential oil, Basil Essential are entered according to volume ratio in mixing compound essential oil formula described in invention Row mixing.The essential oil product refers to national standard.

The necrotic enteritis model for testing 1 chicken is set up

1. materials and methods

1.1 test strain

It is the A type C.perfringens isolated of necrotic enteritis case that malicious bacterial strain uses therefor is attacked in experiment, wherein NE01 and NE02 is netB gene negative bacterial strains, and cpnetBB, cpnetBD and cpnetBF is are isolated from Guangdong Province's Boluo, Dongguan, Foshan Area's netB positive strains.

1.2 experimental animals and design

Experiment from 1 age in days the commercial baby chick of Ross 308, purchased from the just greatly healthy ground Co., Ltd of Dongguan, Guangdong.It is randomly divided into 6 Group, every group 10.Experiment is divided into 6 groups, and 1 group is that blank control attacks poison without bacterium, and other 5 groups carry out bacterium solution in 21 ages in days and mix Material attacks poison, and it is NE01, NE02, cpnetBB, cpnetBD and cpnetBF respectively that 5 groups, which are attacked malicious bacterium,.

1.3 experiment daily rations

The prosperous steam fish meal of basal diet and river is purchased from Guangzhou Jiang Feng Industrial Co., Ltd., basal diet composition and nutritive water It is flat to be shown in Table 2.

The basal diet of table 2 is constituted and trophic level

。

1.4 feeding management

First 1 week of experiment, clears up hen house, first with flame disinfection cage, ground, then is sterilized with formaldehyde fumigation, raises whole fixed Phase sterilizes.Using cage.Relative humidity is maintained at 64%~76%., whole process keeps good ventilation, free choice feeding and freedom Drinking-water.1~13 age in days feeds basal diet, and 14~23 ages in days add fish meal in basal diet, fish meal content is reached 50%.

1.5 attack the preparation of toadstool suspension

By the C.perfringens of test group (including netB gene negative bacterial strain NE01 and NE02, netB positive strain CpnetBB, cpnetBD and cpnetBF) recover, in streak inoculation to TSC flat boards, 40 DEG C of Anaerobic culturel 24h.Make even black on plate Color colonies typical is inoculated into cooked meat medium, 40 DEG C of Anaerobic culturel 15h.Then kitchen meat culture is inoculated into 400mL FT In fluid nutrient medium, 40 DEG C of Anaerobic culturel 15h.

1.6 disease models are set up

FT cultures are added in feed, mixed thoroughly, bacterium solution and feed ratio are (volume/weight；mL/g)1：1.5.Daily Twice, the morning 8:00, afternoon 4:00, continuously feed 3d.

1.7 lesion score

De- neck was put to death on 24th, cut open inspection observation duodenum, jejunoileum lesion, and the lesion to each group is scored respectively. Scoring criteria is shown in Table 3.

The necrotic enteritis lesion of table 3 scores

。

1.8 focuses separate C.perfringens genotype and netB genetic tests

With disposable swab sample is gathered from diseased region.After separation identification, C.perfringens un-mixing bases are carried out Because netB genetic tests are identified and carried out to type.

1.9 data processing

Data mean+SDRepresent, significance analysis is carried out with SPSS17.0.

2. chicken necrotizing enterocolitis model sets up result

2.1 clinical symptoms and enteron aisle lesion

Do not attack before poison that chicken is in good condition, start to attack after malicious 1d, in addition to blank group, slight abdomen occurs in remaining each group Rush down.Continuously attack after poison, 3 groups of netB gene-positive strains are attacked poison group symptom of diarrhea and aggravated, anorexia, row's red or dark brown coal Blood, intestinal submucosa tissue are mixed with tar-like excrement, some excrement.

After cut open inspection, blank group, intestinal mucosa is smooth, not damaged.

The bacterial strain of two plants of netB gene negatives attacks malicious group, and intestinal mucosa thickening phenomenon only occurs in appearance, and other are not obvious Lesions visible.

3 plants of netB positive strains attack malicious group, the different degrees of typical necrotic enteritis lesion of appearance, small intestinal mucosa surface There is concavo-convex ulcer, there is the attachment of fiber-like material on surface, and dispersivity necrosis occurs in serious intestinal mucosa.As a result Fig. 1 is seen.Fig. 1 is Chicken necrotizing enterocolitis small intestine different lesions；Wherein：1 is blank control, 2,3,4,5,6,7 it is corresponding be typical 1 point respectively, 2 Point, 3 points, 4 points, 5 points, 6 points of lesions.

2.2 small intestine lesion scores

Small intestine lesion score the results are shown in Table 4.

The small intestine lesion score result of table 4

Note：Shoulder note lowercase difference represents significant difference (P ＜ 0.05) in table, and capitalization difference represents that difference extremely shows Write (P ＜ 0.01), the identical expression difference of letter is not notable.

The result of table 4 is shown：Blank group does not have any lesion to occur.Two plants of bacterium of netB gene negatives are to cause intestinal wall Mucous membrane it is thickening, without other lesions visibles.Three groups of netB gene masculines all occur in that obvious necrotic enteritis lesion.Its Positive three groups of middle netB obtains split pole and is significantly higher than blank group and the negative groups of netB.Two groups of netB feminine gender group score differences are not notable.

2.3 focuses separate C.perfringens genotype and netB genetic test results

Genotype detection is carried out by Muliti-PCR methods, C.perfringens (Fig. 2 that all focuses are isolated is found And Fig. 3) 400bp or so fragment is amplified, it is determined as A type bacterium, netB genetic tests are also all the positive.

Fig. 2 is the C.perfringens genotype identification that focus is isolated；Wherein, M is DL2000Marker, and 1~20 is The C.perfringens bacterial strain that focus is isolated.

Fig. 3 is the C.perfringens netB identified for genes that focus is isolated；Wherein, M is DL2000Marker, 1~20 The C.perfringens bacterial strain isolated for focus.

The research of chicken necrotizing enterocolitis is concentrated in recent years and looks for the control sick method, and understands pathogenesis side Face.The basis that chicken necrotizing enterocolitis model is research is set up by artificial induction.In former research, concurrent frequently with coccidia Pattern induction experiment, but problem is that small intestinal coccidiosis and chicken necrotizing enterocolitis are easily obscured.Inventor in the present invention only with C.perfringens bacterium solution individually attacks malicious scheme, and effect is obvious.3 bacterial strain test groups of netB gene masculines all occur in that allusion quotation The necrotic enteritis lesion of type.And the C.perfringens bacterial strain isolated from focus is all the bacterial strain of A type netB gene masculines, Illustrate that 3 groups of netB positive bacterias for attacking poison can cause the necrotic enteritis of chicken.3 groups there is necrotic enteritis lesion experiment Group, lesion score is different, and lesion degree is variant, even if this explanation is all the C.perfringens of netB gene masculines, But still there is difference in the pathogenic of each bacterial strain.All there is not necrosis in separation strains and blank group negative 2 groups of netB Property enteritis lesion.This illustrates that netB genes are the major reasons of chicken necrotizing enterocolitis morbidity from the negative.

Wheat is used in experiment in basal feed, wheat contains non-starch polysaccharide, adds chyme viscosity, have impact on Digesting and assimilating for small intestine, condition is provided for the propagation of C.perfringens.The fish meal that later stage adds, contains protein in daily ration Amount increase, can be supplied to the substantial amounts of amino acid of C.perfringens, chicken is more susceptible to suffer from necrotic enteritis.

The initial success of this test model, is that further research is laid a good foundation, such as antibacterials, vaccine, virulence Factor evaluation etc..But due to reproduction of the different researchers to disease has different purposes, required disease severity Also it is different, thus using when also need to be adjusted correspondingly model.

Test the extracorporeal bacteria inhibitor test of 2 plants essential oils

It is all the main method for controlling necrotic enteritis all the time that antibiotic is added in daily ration, but as antibiotic promotees Growth stimulator is gradually disabled all over the world, and the research to new replacement therapy medicine turns into the task of top priority, and applicant preferably goes out Several essential oils are as a kind of safely and effectively Substitutes For Antibiotic, with important Development volue.

1. materials and methods

1.1 test strain

The 3 plants of netB positive strains isolated in pathological material of disease, i.e. cpnetBB, cpnetBD and cpnetBF.

1.2 plants essential oil

Nutmeg folk prescription essential oil, thyme folk prescription essential oil, sweet basil folk prescription essential oil, nutmeg and thyme essential oil 1:1 mixing Compound, nutmeg and sweet basil 1:1 mixing compound, thyme and sweet basil 1:1 mixing compound.Plants essential oil used is purchased from Guangzhou Certain commodity Co., Ltd.

The preparation of 1.3 filter papers

The filter paper that water imbibition is strong and homogeneous is selected, a diameter of 6mm disk is broken into puncher, the dry of cleaning is sub-packed in It is standby after 121 DEG C of autoclaving 15min in dry test tube.

1.4 bacterial strains are activated

Freeze 3 plants of bacterial strains are thawed, streak inoculation is on TSC flat boards, 40 DEG C of Anaerobic culturel 24h.Picking is typically black Color bacterium colony, is inoculated into 10mLFT fluid nutrient mediums, 40 DEG C of Anaerobic culturel 8h.Bacterium solution is diluted to according to 0.5 Maxwell opacity tube 10⁸CFU/mL。

1.5 disk diffusion method

100 μ L10 are drawn with liquid-transfering gun⁸On CFU/mL experiment bacterium solution to FT nutrient agars, uniformly applied with spreader Cloth 3 times and then smeared one week around plate edge, cover plate, put 3~5min of drying at room temperature.Each flat board is placed with the tweezer with sterilizing Sub careful gripping filter paper, is affixed on media surface, and light paper-pressing sheet, it is close to media surface, each scraps of paper center is apart More than 24mm, scraps of paper anomaly ware outer rim is more than 15mm.5 μ L plants essential oil stostes are drawn respectively to be added drop-wise on filter paper.By plate It is inverted, 40 DEG C of Anaerobic culturel 24h observe and measured the diameter of inhibition zone, and every group is done 3 repetitions, is averaged.

Inhibition zone criterion：Bacteriostasis effect using antibacterial circle diameter be less than 10mm to be insensitive, 11~15mm it Between be sensitive, be medium sensitivity between 16~20, be extremely sensitive more than 20mm.

2 disk diffusion method results

The sensitiveness and suppression of folk prescription essential oil and compound essential oil to C.perfringens pathogenic bacteria are detected using disk diffusion method Bacterium acts on.It the results are shown in Table 5 and table 6.

Table 5：Antibacterial circle diameter (mm) of 3 kinds of folk prescription plants essential oils to three plants of pathogenic bacteria

Note：Inhibition zone criterion：Bacteriostasis effect is less than 10mm to be insensitive using antibacterial circle diameter, in 11~15mm Between be sensitive, be medium sensitivity between 16~20, be extremely sensitive more than 20mm.

3 kinds of folk prescription plants essential oils have stronger antibacterial work to 3 plants of C.perfringens pathogenic bacteria as can be seen from Table 5 With.Become apparent, reached extremely sensitive for netBC and netBD plants of effect.It is that moderate is quick for netBF plants of inhibitions Sense.

Table 6：Inhibitory action (mm) of the compound plant essential oil to three plants of pathogenic bacteria

It can be found that the fungistatic effect of 3 kinds of compound plant essential oils is also stronger from table 6.For netBC plants of compound plants The synergistic effect of essential oil is obvious.

Test minimal inhibitory concentration (MIC) experiment of 3 plants essential oils

1. materials and methods

The preparation of 1.1 plants essential oil test samples

Draw in 0.4mL plants essential oils to EP pipes, add Tween-80 0.1mL, be well mixed, add 0.5mL sterilized waters, Concentration is diluted to for 400 μ L/mL, is well mixed again.Milky white liquid, it is now with the current.

1.2 doubling dilution

Under aseptic condition, 10 test tubes are taken, are numbered, wherein No. 9 pipes are positive pipe, No. 10 Guan Weiwei negative tubes, remaining is Various concentrations pipe.With liquid-transfering gun to No. 1 pipe and No. 10 pipes plus 9mLFT culture mediums, remaining each pipe plus 5mLFT culture mediums.No. 1 is given again Pipe adds plants essential oil and supplies formula product, and piping and druming is uniform, draws 5mL and is put into No. 2 pipes, the like manage to No. 8.No. 10 pipes add plant Essential oil supplies formula product, and piping and druming is uniform, draws 5mL, discards.100 μ L bacteria suspensions are added to No. 1 pipe to No. 9 pipes.36 DEG C of ± 1 DEG C of anaerobism Cultivate 6~8h.Each μ L of liquid in pipe 100 are drawn respectively to FT nutrient agars, and coating is uniform.36 DEG C of ± 1 DEG C of anaerobism Culture 24 hours, observes result.The corresponding medicine highest diluted concentration of every asepsis growth flat board is this plants essential oil to production The minimal inhibitory concentration of gas capsular clostridium.Experiment is repeated 3 times.

2. Minimal inhibitory concentration result

It the results are shown in Table 7.

The plants essential oil of table 7 is to the MCI results (μ L/mL) to three plants of pathogenic bacteria

As shown in table 7, folk prescription and compound essential oil are respectively provided with certain inhibitory action to 3 plants of netB positive strains.For The MIC that six kinds of essential oils of netBC strains or essential oil are combined is 0.31 μ L/mL.MIC for netBD six kinds of essential oils of strain is 0.63 μ L/mL.For netBF plants of thymes, sweet basil and thyme+sweet basil effect preferably, MIC is 1.25 μ L/mL.

Test the animal experiment that 4 plants essential oils are used to prevent and treat chicken necrotizing enterocolitis

1. materials and methods

1.1 test strain

NetBF bacterial strains：For A type C.perfringens, netB gene masculines.

1.2 experimental animals and design

Experiment is randomly divided into 8 groups, every group 15 from the commercial baby chick 80 of Ross 308 of 1 age in days.1 group is blank Group, 1 group is to attack bacterium control group, and remaining 6 groups of group is that plants essential oil prevents and treats group, adds 100mg/ in feed after 17 ages in days respectively kg1:1 nutmeg folk prescription essential oil, thyme folk prescription essential oil, sweet basil folk prescription essential oil and compound plant essential oil.

1.3 experiment daily rations

Basal diet, fish meal is used such as experiment 1.

1.4 necrotic enteritis models are set up

Method according to used in experiment 1 carries out attacking poison to 7 test groups of remaining except blank control group.

1.5 small intestine lesion scores.

24 ages in days, randomly select 10 chickens, take off neck and put to death, cut open inspection observation duodenum, jejunum, ileum lesion, right respectively The lesion of each group is scored.Scoring criteria such as table 3.Data processing is as tested 1.

1.6 separation strain gene type netB identified for genes

With disposable swab sample is gathered from diseased region.After separation identification, aerogenesis is carried out according to the method for experiment 1 NetB genetic tests are identified and carried out to capsular clostridium isolated genes type.

1.7 high throughput sequencing technologies analyze the diversity of chicken intestinal flora

1.7.1 intestinal contents sample collection

In 24 age in days, each experimental group randomly selects 5 chickens, after analysing, in duodenum and caecum end two ends ligation Take out, ice chest is preserved.In superclean bench, enteron aisle is divided into two parts, small intestinal segment (duodenum, jejunum and ileum) and blind Intestinal segment.Intestinal contents are taken out under gnotobasis, and the intestinal contents of every group of 5 chickens are sufficiently mixed.- 80 DEG C of preservations.

1.7.2 bacteria total DNA is extracted

Bacteria total DNA is extracted in chicken intestinal content is taken out using QIAamp DNA Stool Mini Kit kits Carry, idiographic flow is with reference to kit specification.

1.7.3 Illumina miseq sequencing analysis

The enteric bacteria DNA of extraction is delivered into Wei Ji biotechnologies (Shanghai) Co., Ltd. and carries out high-flux sequence.

1.7.4 bioinformatic analysis

Including sequencing data processing, OUT analyses, species information analysis, Alpha diversity analysis and Beta diversity point Analysis.

2 animal test results

2.1 small intestine lesion score results, are shown in Table 8.

Table 8：Small intestine lesion score

Found out by table 8：Compared with attacking malicious group, thyme folk prescription essential oil treatment group does not have significant difference, and other units are equal There is significant difference.As a result nutmeg folk prescription essential oil, sweet basil folk prescription essential oil, nutmeg and thyme essential oil 1 are shown:1 mixing is multiple Side, nutmeg and sweet basil 1:1 mixing compound, thyme and sweet basil 1:1 mixing compound treatment group can significantly mitigate perfringens Damage of the clostridium to enteron aisle.Thyme folk prescription treatment group does not have effect.From the point of view of lesion severity, thyme folk prescription plant essence Preferably, relative to other groups, lesion has 1 to oily effect in 4~6 points of chicken.Nutmeg+sweet basil compound plant essential oil is relative When its folk prescription, lesion has also declined in 4~6 points of chicken.

In summary, nutmeg folk prescription essential oil, sweet basil folk prescription essential oil, nutmeg and thyme essential oil 1 in animal experiment:1 Mix compound, nutmeg and sweet basil 1:1 mixing compound, thyme and sweet basil 1:1 mixing compound all has one to necrotic enteritis Fixed prevention effect.But according to result of scoring from the point of view of, still there is more lesion to occur in treatment group, though this be probably due to Right plants essential oil has fungistatic effect, but can not inactivate toxin.In current experimental animal model, use continuous Bacterium solution spice after culture, which is attacked, contains substantial amounts of toxin in poison, nutrient solution, cause the damage of intestinal mucosa.In addition, plants essential oil Consumption, its prevention effect may can all be influenceed by adding length etc. of time.Plants essential oil feeding can be increased in practical application Time, so can beforehand control C.perfringens propagation so that bacterial number and content of toxins are all substantially reduced, from And reduce the generation of disease.

2.2 focuses separate C.perfringens genotype and netB genetic test results

The C.perfringens isolated by all focuses of genotype detection amplifies 400bp or so fragment, sentences It is set to A type bacterium, netB genetic tests are also all the positive.

Influence of the 2.3 different plants essential oils to chicken intestinal flora

2.3.1 sequence number result

It the results are shown in Table 9 and table 10, and Fig. 4.

The ordered sequence of table 9 is counted

Note：G1, G2, G3, G4, G5, G6, G7, G8 represent blank group, attacked in malicious control group, nutmeg group, hundred respectively in table Fragrant group, sweet basil group, nutmeg+thyme 1:1 mixing compound group, nutmeg+sweet basil 1:1 mixing compound group, thyme+sweet basil 1: 1 mixing compound group.A represents small intestinal segment, and B represents caecum section.In the present invention unless otherwise specified, this implication is represented.

The optimization of table 10 is counted

Table 9 and table 10 and Fig. 4 results show that the ordered sequence of high-flux sequence is in 50000 or so, ordered sequence Length is mostly in 450bp or so, and after Quality Control and splicing, the band sequence obtained by each sample is at 30000 More than.

There are the microorganism species of complexity in the enteron aisle of chicken, the compositions of these floras to the health of host, production performance, Development and nutrient digestion all play an important role in terms of absorbing.Studying its intestinal microflora composition and change has important Meaning.High throughput sequencing technologies are the multifarious means of microorganisms more advanced at present, and high flux is used in this experiment Sequencing technologies study influence of the plants essential oil to chicken necrotizing enterocolitis enteric microorganism.Obtained result of the test data volume is big, accurate True property is high.Better than traditional test method.

This experiment carries out high-flux sequence by the gut flora of the small intestine to 8 experimental groups and caecum section.By matter After control, splicing, the optimization band in each sample is more than 3000.Pass through dilution curve, sequencing amount Most of species in sample are covered enough with sequencing depth, meet subsequent analysis.

2.3.2 the abundance and diversity analysis of gut flora

Dilution linearity curve such as Fig. 5 of flora in sample, as shown in Figure 5, with the increase of sequencing sequence number, its slope is gradually Reduce, tend to plateau.Illustrate that sequencing depth covers most of species in sample enough, meet subsequent analysis.

The OTU numbers of each sample, ace indexes, chao indexes, shannon indexes and simpson indexes are as shown in table 11.

The sample diversity result of table 11

As shown in Table 11, more OTU is can detect that in all samples, OTU distributed number is between 192~274. This shows in the intestinal contents of this sub-sampling that each group species abundance is very high, but the difference that species are constituted between different group compared with Greatly.It is can be seen that by 4 Alpha indexes in small intestine, relative to necrotic enteritis disease model group, each treatment group's enteron aisle The richness of species reduces in content, but diversity increase.Wherein nutmeg+thyme group diversity highest.In caecum, Relative to necrotic enteritis disease model group, nutmeg+sweet basil group flora richness highest, diversity are best.

2.3.3 intestinal microflora is analyzed

Finishing analysis are carried out from door, mesh, 3 levels of section to each sample, the species relative abundance post on varying level is drawn Shape figure, such as Fig. 6, Fig. 7 and Fig. 8.And draw group's thermal map.

From fig. 6, it can be seen that in door level, in the different intestinal segments of different samples, Firmicutes (Frimicutes) (Firmicutes) it is main bacterium door.In small intestine, necrotic enteritis model group denaturation bacterium door (Proteobacteria) Account for 18.9%.But in plants essential oil treatment group, sweet basil, nutmeg+thyme, nutmeg+sweet basil and thyme+sweet basil group In, square line bacterium door (Actinobacteria) ratio increase, respectively 32.5%, 14.6%, 45.3%, 23.3%.Wherein hundred In in perfume (or spice)+sweet basil group Bacteroidetes (Bacterodetes) (Bacterodetes) ratio be 23.3%, with other each group differences It is larger.In caecum, Bacteroidetes (Bacterodetes) (Bacterodetes) is the door of each group content more than second, but phase For attacking malicious group, each plants essential oil prevents and treats the increase of Bacteroidetes in group (Bacterodetes) (Bacterodetes) ratio.

As can be seen from Figure 7, in mesh level clostridium mesh (Clostridiules) and lactobacillus mesh (Lactobacillales) it is the main mesh in each sample enteron aisle.In small intestine, plants essential oil is prevented and treated in group, sweet basil and Pork and beans In cool+sweet basil group, relative to attack malicious group, clostridium mesh (Clostridiules) proportion is reduced, and attacks clostridium mesh in malicious group (Clostridiules) ratio is 55.3%, and 57 ratios are respectively 20.1% and 16.0%, and the bud in nutmeg+sweet basil group The increase of spore bacillus mesh (Bacillales) content, is 15.0%.Bacteroid mesh (Bcateroidales) in thyme+sweet basil group Content is higher than other groups, accounts for 23.3%.In caecum, bacteroid mesh (Bcateroidales) turns into the third main mesh, each group Bacterial community changes little.

As it can be observed in the picture that in section's level, in small intestine, attacking in poison group Bacterial community, main section is Lactobacillaceae (Lactobacillaceae), enterobacteriaceae (Enterobacteriaceae), Ruminococcus section (Ruminococcaceae) and Hair spiral Cordycepps (Lachnospiraceae) proportion is 19.5%, 18.3%, 5.1% and 3.6%.Relative to attacking malicious group, In each plants essential oil treatment group, Bacterial community changes in sweet basil, nutmeg+thyme, nutmeg+sweet basil and thyme+sweet basil group Become larger, in sweet basil group, Corynebacteriaceae (Corynebacteriacea) accounts for 31.4%；In nutmeg+sweet basil group, cud ball Cordycepps (Ruminococcaceae) accounts for 19.8%, and Corynebacteriaceae (Corynebacteriacea) accounts for 13.3%, hair spirillum Section (Lachnospiraceae) accounts for 11.8%；Ratio highest section is bar-like Bacteriaceae in nutmeg+sweet basil group (Corynebacteriacea) account for 27.3%, and other group is visibly different is, staphylococcaceae (Staphylococcaceae) increase with Brevibacteriaceae (Brevibacteriacea) content, ratio is respectively 13.0% He 10.2%；In thyme+sweet basil group, reason grinds the increase of Cordycepps (Rikenellaceae) content, accounts for 23.3%.In caecum, various masters It is Lactobacillaceae (Lactobacillaceae), hair spiral Cordycepps (Lachnospiraceae) and Ruminococcus section to want flora (Ruminococcaceae), each group difference is little.The relative abundance level of each species, can pass through face in thermal map representative sample The gradient and similarity of color come reflect sample in all kinds of levels group constitute similitude and otherness.

The change of flora is also had opposite impacts on to the feed conversion rate of chicken in enteron aisle, and plants essential oil is shown from side Effect to host's production performance.Intestinal microflora is the key factor for maintaining intestinal health, the microorganism of a balance The pathogen that flora can be defendd effectively in enteron aisle.The gut flora of present invention experiment Zhong Ge treatment groups is organized all relative to poison is attacked There is different degrees of change, these changes may produce active influence to the defence of disease.

2.3.4 Beta diversity analysis

It is utilized respectively otherness matrix thermal map (Fig. 9), Multi-example similarity dendrogram (Figure 10) and PCoA scatter diagrams (figure 11) similarity between each sample, in matrix thermal map, color representative sample distance value, the redder representative sample phase of color are represented Higher like spending, color is more blue then to represent that similitude is lower；In PCoA scatter diagrams, sample composition is more similar, its distance in figure It is about near.The intestinal bacteria structure of each group it can be seen from Fig. 9, Figure 10 and Figure 11, sweet basil group and nutmeg+sweet basil group and sky White group structure of community is similar, and to attack malicious control group difference larger.Other Ji Ge treatment groups are inoculated with closely with malicious control group similitude is attacked. In caecum, the intestinal microflora of each group is much like.

Each group caecum intestinal microflora is much like.This explanation occurs mainly in small intestinal segment due to necrotic enteritis lesion, The poison of attacking of C.perfringens does not produce obvious influence to caecum section.Act on, planted to caecum additionally, due to metabolic adsorption etc. The content of thing essential oil reduces, and effect is also accordingly reduced.

Above-described embodiment is preferably embodiment, but embodiments of the present invention are not by above-described embodiment of the invention Limitation, other any Spirit Essences without departing from the present invention and the change made under principle, modification, replacement, combine, simplification, Equivalent substitute mode is should be, is included within protection scope of the present invention.

SEQUENCE LISTING

<110>Institute of Animal Health,Guangdong Academy Of Agricultural Sciences

<120>Application of the thyme compound plant essential oil in preventing and treating chicken necrotizing enterocolitis

<130>

<160> 10

<170> PatentIn version 3.5

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Claims

1. application of the thyme compound essential oil in preventing and treating chicken necrotizing enterocolitis, it is characterised in that：Thyme compound essential oil includes Thyme-nutmeg compound essential oil, thyme-sweet basil compound essential oil.

2. application according to claim 1, it is characterised in that：In thyme-nutmeg compound essential oil thyme essential oil and The volume ratio of nutmeg essential oil is (1-3):(3-1).

3. application according to claim 1, it is characterised in that：Thyme essential oil and sieve in thyme-sweet basil compound essential oil The volume ratio for strangling essential oil is (1-3):(3-1).

4. the application according to claim any one of 1-3, it is characterised in that：Thyme compound essential oil is by being added to chicken Chicken necrotizing enterocolitis is prevented and treated in daily ration.

5. a kind of feed addictive of prevention chicken necrotizing enterocolitis, it is characterised in that additive is thyme compound essential oil or hundred In fragrant compound essential oil and its acceptable food grade accessories.

6. feed addictive according to claim 5, it is characterised in that：Thyme compound essential oil includes thyme-Pork and beans Cool compound essential oil, thyme-sweet basil compound essential oil.

7. feed addictive according to claim 6, it is characterised in that：Thyme in thyme-nutmeg compound essential oil The volume ratio of essential oil and nutmeg essential oil is (1-3):(3-1).

8. feed addictive according to claim 6, it is characterised in that：Thyme essence in thyme-sweet basil compound essential oil The volume ratio of oil and Basil Essential is (1-3):(3-1).

9. a kind of method for preventing and treating chicken necrotizing enterocolitis, it is characterised in that：It is the daily ration that thyme compound essential oil is added to chicken Middle preventing and treating chicken necrotizing enterocolitis；Thyme compound essential oil includes thyme essential oil and nutmeg essential oil according to volume ratio (1-3): The compound essential oil, thyme essential oil and sweet basil compound essential oil of (3-1) mixing are according to volume ratio (1-3):The compound essence of (3-1) mixing Oil.