CN107260796B

CN107260796B - Application of thyme compound plant essential oil in preventing and treating necrotic enteritis of chicken

Info

Publication number: CN107260796B
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: 2020-12-15
Anticipated expiration: 2037-06-08
Also published as: CN107260796A

Abstract

The invention discloses application of thyme compound plant essential oil in preventing and treating necrotic enteritis of chickens. The plant essential oil comprises thyme-nutmeg compound essential oil and thyme-basil compound essential oil. The applicant successfully constructs a chicken necrotic enteritis animal model in 1-2 years, and the model is fed by using wheat basal feed and added with fish meal at the later stage; the clostridium perfringens is adopted to singly attack the toxin, and typical lesions are obvious; can eliminate the confusion phenomenon between the previous coccidiosis concurrent model and the coccidiosis. The test period is short, and the repeatability is high. In addition to in vitro experiments, the applicant also utilizes the model to research the in vivo prevention and treatment effect of the plant essential oil, and really determines that the plant essential oil has the prevention and treatment effect on the necrotic enteritis of chickens.

Description

Application of thyme compound plant essential oil in preventing and treating necrotic enteritis of chicken

Technical Field

The invention belongs to the field of veterinary medicine, and particularly relates to application of thyme compound essential oil in prevention and treatment of necrotic enteritis in chickens.

Background

Necrotic enteritis in chickens was first discovered by Parish (1961) and is an acute digestive tract disease caused by clostridium perfringens, which is a ubiquitous disease of chickens. Causing losses of over 20 billion dollars per year to the poultry industry worldwide. In recent years, the ban of antibiotic growth promoters in feed in the european union has led to a large outbreak of necrotic enteritis in the chicken industry in european countries.

Necrotic enteritis usually occurs after 4 weeks of age, and occurs in poultry farming areas throughout the world (Long J R, 1973). Clinical symptoms are manifested as acute clinical symptoms and subclinical symptoms.

Acute clinical symptoms of necrotic enteritis: acute clinical symptoms are classically characterized by sudden increase in mortality in chickens, with no forewarning, and sometimes early signs of litter wetness. The disease is very rapid, and the death is within 1-2 h, and sometimes the death rate is increased to 50% (Riddell et al 1992).

Subclinical symptoms: subclinical symptoms have become more prevalent over the last few years, and this form of necrotic enteritis has no apparent clinical symptoms and does not increase mortality. However, chronic damage to the intestinal mucosa leads to malabsorption, a decrease in the rate of weight gain, an increase in the feed conversion ratio, and a huge loss of production (Elwinger et al 1992). In the case of subclinical infections, damage to the intestinal tract results in the arrival of bacteria in the bile ducts and portal veins, and a large number of clostridium perfringens colonize the liver, leading to cholangitis and hepatitis, with reddish or white lesions appearing on the liver (Onderka et al 1990).

Wherein, intestinal pathological changes include: macroscopic lesions usually appear in the small intestine, but other organs, such as the liver and kidney, also develop lesions. The duodenum, jejunum and ileum walls were found to be thinned and filled with gas by pathological dissection. The clinical symptom of necrotic enteritis is large area necrosis of small intestinal mucosa, and the surface is covered with yellowish brown pseudomembrane. Typical subclinical symptoms are ulcer with concave-convex shape on the surface of intestinal mucosa, yellowing of mucosa and attachment of cellulose-like substances on the surface. Necrotic enteritis exhibits a strong inflammatory response early in life, engorgement of the lamina propria, infiltration of a large number of inflammatory cells, primarily neutrophils. In the later stage of necrotic enteritis, diffuse necrosis of intestinal mucosa accounts for one third to one half of intestinal mucosa. A clear boundary between necrotic sites and other tissues occurs due to accumulation of neutrophils.

The necrotic enteritis inducing factors of the chickens comprise the following factors:

firstly, nutrition: the main risk factor for the development of necrotic enteritis is the formation of an environment in the intestine that favors the growth of clostridium perfringens. The nature of the ration is an important non-bacterial factor affecting the development of necrotic enteritis. The high level of indigestible water-soluble starch polysaccharides in the daily ration is more susceptible to necrotic enteritis, so barley, rye, oat and wheat are risk factors for necrotic enteritis, because the water-soluble starch polysaccharides increase the viscosity of chyme, prolong the time of passing through the intestinal tract and reduce the digestibility of nutrients (Craven et al 2000). High concentrations of proteins in the diet, such as fish meal, also increase the incidence of necrotic enteritis, as high protein feeds are not readily digestible in the intestinal tract, resulting in increased protein concentrations in the gastrointestinal tract, providing an abundant nutritional matrix for bacterial growth (Nauerby et al 2003). The size of the feed pellets also affects the incidence of necrotic enteritis, with larger feed pellets being more susceptible to necrotic enteritis than smaller ones (Engberg et al 2002).

II, coccidiosis: one of the most prominent causative factors of necrotic enteritis is coccidia, which damages the intestinal mucosa, resulting in exudation of protein plasma, and provides an abundant nutritional matrix for clostridium perfringens in the intestinal tract, facilitating its proliferation and toxin production (Williams et al 2005). Coccidiosis often precedes or coincides with necrotic enteritis. The test proves that the Eimeria and the Clostridium perfringens play a synergistic role in the necrotic enteritis lesion process. Al-sheikhly et Al found in the experiments with the test group co-infected with Clostridium perfringens and Eimeria that the lesions were more severe and mortality was higher compared to the group infected with Clostridium perfringens alone or with Eimeria alone.

Thirdly, pathogenic clostridium perfringens: the content of clostridium perfringens in intestinal tract of chicken suffering from necrotic enteritis reaches 10⁶~10⁸CFU/g, and the content of clostridium perfringens in the intestinal tract of healthy chickens is 0-10⁵CFU/g (Keyburn et al 2008). However, even if the large amount of perfringens is contained in the intestinal tractBacteria are also not sufficient to cause necrotic enteritis. The number of clostridium perfringens is therefore not directly related to necrotic enteritis, and not all clostridium perfringens are capable of inducing necrotic enteritis. The development of necrotic enteritis requires specific pathogenic conditions, and high numbers of clostridium perfringens may cause more serious lesions when all the pathogenic conditions are present, but provided that pathogenic strains are present.

Fourthly, bacteriocin: bacteriocins play an important role in the pathogenesis of necrotic enteritis. Through pulse field gel electrophoresis or amplified fragment length polymorphism analysis, healthy chicken flocks can separate A-type clostridium perfringens with different genotypes even in the same intestinal tract of the chicken. In contrast, only one subtype can be cloned in the intestine in cases of necrotic enteritis. After natural recovery or treatment, the bacteria in the chicken develop multiple genotypes again (Park et al, 2008). This single strain dominated phenomenon has been demonstrated in several studies, and is a major process in the development of necrotic enteritis. Isolates in necrotic enteritis secrete more growth factors inhibiting other clostridium perfringens than normal strains. This toxic protein produced by bacteria that inhibits the growth of other related strains is called bacteriocin. The production of bacteriocins inhibits the growth of other clostridium perfringens in the intestinal tract, so that the virulent strain replaces other strains.

Fifthly, NetB toxin: the NetB toxin was first found in clostridium perfringens type a, isolated from sick chickens with necrotic enteritis. Has similarity with amino acid sequences of various pore-forming toxins, and has 38 percent of homology with clostridium perfringens beta toxin and 31 percent of homology with alpha toxin produced by staphylococcus. The NetB toxin can make the liver cancer cells of the chicken round and dissolve, and pores with the diameter of about 1.6-1.8 nm are formed on cell membranes. The NetB toxin gene is located on a plasmid and is regulated by a VirsR two-component signal system. Clostridium perfringens knockout NetB toxin gene is unable to infect chickens or produce necrotic enteritis lesions in the intestine under test. However, the complementing mutant inserted with the NetB toxin gene is pathogenic as in the wild strain (Keyburn et al 2008). Thus, it can be seen that NetB toxin is a key factor in causing necrotic enteritis in chickens. Most of the strains isolated from necrotic enteritis cases carry the NetB toxin gene. In Canada, 95% of Clostridium perfringens from necrotic enteritis-affected chickens contain the NetB toxin gene, and only 35% of Clostridium perfringens isolated from healthy chickens are NetB toxin gene-positive (Chalmers et al 2008). The necrotizing enterocolitis isolate was 58% positive for the NetB toxin gene in the us isolate, and only 8.75% of the NetB toxin gene carried by the strain in the normal flora (Martin and Smyth 2009). Through a large number of broiler infection tests using clostridium perfringens type a with negative and positive NetB toxin genes, it is found that only clostridium perfringens with positive NetB toxin gene can produce necrotic enteritis lesions, and the lesion degree has a direct relationship with the production capacity of NetB toxin (Keyburn et al 2009).

With the development of globalization, the control of antibiotics in China must meet the international standard. Therefore, measures for treating necrotic enteritis in chicken are needed. Since necrotic enteritis is a complex, multifactorial disease, multiple aspects are considered to reproduce the disease.

The construction of the animal model of chicken necrotic enteritis has the following difficulties: such as screening of netB positive strains, identification of the pathogenic ability of different strains; before challenge, the bacteria need to be cultured in two different culture media for 30 hours continuously; searching experimental animal feed; and determining the concentration of the mixed material of the challenge bacteria liquid, and the like. Furthermore, the use of different scoring criteria results in different scoring systems not being able to perform comparative studies with each other. Because the establishment of an animal model of chicken necrotic enteritis is relatively complex, most studies are currently conducted only in vitro, not in animal models. The result of in vitro test alone often cannot truly reflect the control effect of the test drug on the necrotic enteritis of the chicken.

Disclosure of Invention

The invention aims to provide application of thyme compound essential oil in preparing a medicine for preventing and treating necrotic enteritis of chickens.

The technical scheme adopted by the invention is as follows:

the application of thyme compound essential oil in preparing medicine for preventing and treating necrotic enteritis in chicken is disclosed, wherein the thyme compound essential oil comprises thyme-nutmeg compound essential oil and thyme-basil compound essential oil.

Preferably, the thyme compound essential oil is thyme-nutmeg compound essential oil or thyme-basil compound essential oil.

Preferably, the volume ratio of the thyme essential oil to the nutmeg essential oil in the thyme-nutmeg compound essential oil is (1-3) to (3-1).

Preferably, the volume ratio of the thyme essential oil to the nutmeg essential oil in the thyme-nutmeg compound essential oil is 1: 1.

Preferably, the volume ratio of the thyme essential oil to the basil essential oil in the thyme-basil compound essential oil is (1-3) to (3-1).

Preferably, the volume ratio of the thyme essential oil to the basil essential oil in the thyme-basil compound essential oil is 1: 1.

In the research of the invention, various proportions of the thyme-nutmeg compound essential oil and the thyme-basil compound essential oil in the range of (1-3) to (3-1) are subjected to related experiments, and the following animal experiments are carried out with the proportion of 1:1 being good, simple and convenient, and the preferred volume ratio of 1: 1.

Preferably, the thyme compound essential oil is added into the daily ration of the chicken to prevent and treat necrotic enteritis of the chicken.

Preferably, the adding proportion of the thyme compound essential oil to the chicken daily feed is 80-150 mg/kg.

Preferably, the adding proportion of the thyme compound essential oil to the chicken daily feed is 100 mg/kg.

A feed additive for preventing necrotic enteritis in chicken is prepared from compound essential oil of herba Thymi or its acceptable food-grade adjuvants.

Preferably, the thyme compound essential oil comprises thyme-nutmeg compound essential oil and thyme-basil compound essential oil.

A feed for preventing and treating necrotic enteritis in chicken is prepared by adding thyme essential oil into chicken ration for preventing and treating necrotic enteritis; the thyme compound essential oil comprises compound essential oil mixed by thyme essential oil and nutmeg essential oil according to the volume ratio of (1-3) to (3-1), and compound essential oil mixed by thyme essential oil and basil compound essential oil according to the volume ratio of (1-3) to (3-1).

Preferably, the thyme compound essential oil is thyme-nutmeg compound essential oil or thyme-basil compound essential oil; the volume ratio of thyme essential oil to nutmeg essential oil in the thyme-nutmeg compound essential oil is 1: 1; the volume ratio of thyme essential oil to basil essential oil in the thyme-basil compound essential oil is 1: 1.

The plant essential oil can be well mixed with the chicken daily feed uniformly.

The invention has the beneficial effects that:

the applicant successfully constructed an animal model of chicken necrotic enteritis over a period of 1-2 years, which was fed with wheat basal feed and later supplemented with fish meal. The wheat contains non-starch polysaccharide, increases the viscosity of chyme, influences the digestive absorption of small intestine and provides conditions for the proliferation of clostridium perfringens. The fish meal added in the later period increases the protein content in the daily ration, can provide a large amount of amino acid for clostridium perfringens, and enables chickens to be more susceptible to necrotic enteritis. The clostridium perfringens is adopted to singly attack the toxin, and typical lesions are obvious; can eliminate the confusion phenomenon between the previous coccidiosis concurrent model and the coccidiosis. The model has short test period and high repeatability. In addition to in vitro experiments, the applicant also utilizes the model to research the in vivo prevention and treatment effect of the plant essential oil, and really determines the effect of the plant essential oil on preventing and treating the necrotic enteritis of the chicken.

Through a large number of researches, the thyme-nutmeg 1:1 mixed compound essential oil and the thyme-basil 1:1 mixed compound essential oil are preferably selected by the applicant, and the plant essential oil composition has a certain prevention and treatment effect on chicken necrotic enteritis.

The high-throughput sequencing technology is an advanced means for researching the microbial diversity at present, and the applicant utilizes the high-throughput sequencing technology to research the influence of the plant essential oil on the intestinal microorganisms of the necrotic enteritis of the chicken. The obtained test result has large data volume and high accuracy. Is superior to the traditional test method. The invention carries out high-throughput sequencing on intestinal flora of small intestines and cecal sections of 8 experimental groups. After quality control and splicing treatment, the number of the optimized sequence strips in each sample is more than 3000. As can be seen from the dilution curve, the amount and depth of sequencing is sufficient to cover most species in the sample for subsequent analysis. As can be seen from the 4 Alpha indices, namely, the ace index, the chao index, the shannon index and the simpson index, in the small intestine section, the abundance of species in the intestinal contents of each plant essential oil treatment group was reduced, but the diversity was increased, relative to the necrotic enteritis disease model group. The intestinal microflora is an important factor for maintaining the health of the intestinal tract, and a balanced microflora can effectively defend pathogens in the intestinal tract. The intestinal flora of the group treated with the plant essential oil varied to a different extent from that of the group artificially infected, and these changes may have a positive effect on the defense against diseases.

Drawings

FIG. 1 shows different changes of small intestine caused by necrotic enteritis in chickens; the method comprises the following steps: 1 is blank control, 2, 3, 4, 5, 6 and 7 are typical lesions of 1 point, 2 points, 3 points, 4 points, 5 points and 6 points respectively;

FIG. 2 is a genotype identification of Clostridium perfringens isolated from a lesion; wherein M is DL2000 Marker, and 1-20 are clostridium perfringens strains separated from focuses;

FIG. 3 shows the identification of the netB gene of Clostridium perfringens isolated from the lesions; wherein M is DL2000 Marker, and 1-20 are clostridium perfringens strains separated from focuses;

FIG. 4 shows the valid sequence length distribution statistics;

FIG. 5 is a dilutability curve;

FIG. 6 is a histogram of gated horizontal species distribution;

FIG. 7 is a histogram of the distribution of target horizontal species;

FIG. 8 is a histogram of family level species distribution;

FIG. 9 is a diversity matrix heatmap;

FIG. 10 is a multi-sample similarity dendrogram;

FIG. 11 is a PCoAPCoA scattergram based on unweighted unifrrac.

Detailed Description

Necrotic enteritis in chickens is an acute digestive tract disease caused by clostridium perfringens and is one of the most important intestinal tract diseases of chickens. Causing huge losses to the poultry industry worldwide. With the restriction of the use of antibiotic growth promoters at home and abroad, the incidence of the disease is increased. Finding a safe and effective novel medicament is a hot problem in the field of current cultivation.

The inventor establishes an artificial morbidity model of the necrotic enteritis of the chicken and lays a foundation for in vivo screening of the necrotic enteritis. The whole study includes part 7, part 1. isolation and identification of clostridium perfringens in chicken stool samples and intestinal tissues in necrotic enteritis cases; 2, carrying out genotype identification on the field isolate of the clostridium perfringens by a Multi-PCR method; 3. detecting a toxin gene (netB) of a field isolate NetB of clostridium perfringens; 4. detecting drug resistance of field isolate of clostridium perfringens; performing subtype analysis on the field isolate of the clostridium perfringens by ERIC-PCR; 6. establishing a necrotic enteritis model of the chicken; 7. the plant essential oil can be used for preventing and treating necrotic enteritis.

The experiment collects 550 parts of fecal samples and necrotic enteritis case intestinal tissues from 5 large-scale chicken farms in Qingyuan, Fushan, Yunfeng, Jiangmen, Guangzhou and other places in Guangdong province, adopts a selective culture medium TSC plate and a blood agar plate to carry out primary separation, and then carries out biochemical identification and buffalo milk characteristic identification on the separated strains. 47 strains of clostridium perfringens are primarily separated, the separation rate is 8.5 percent, and the separation rate is lower compared with the separation rate reported abroad.

And (3) designing primers aiming at four toxin genes of alpha, beta and iota, and establishing a Multi-PCR method. The primers used are as follows:

table 1: Muliti-PCR primer sequence

The PCR reaction system is 25 μ L: DNA template 2. mu.L, upstream and downstream primers 0.125. mu.L each, MaxMaster12.5. mu.L, nuclease free water 9.5. mu.L. Reaction procedure: 5min at 94 ℃; 1min at 94 ℃, 1min at 55 ℃, 1min at 72 ℃ and 30 cycles; 72 ℃ for 10 min; storing at 10 deg.C.

After the PCR reaction is finished, 8 mu L of PCR product is taken out, is subjected to electrophoresis in 1% agarose gel, is detected at 80V for 40min, is recorded and imaged by using a gel imaging system, and is stored in pictures.

The method is used for carrying out genotype identification on 47 clostridium perfringens, and the 47 isolates are amplified to obtain specific fragments of about 400bp, which can be identified as type A. In addition, the method is used for amplifying escherichia coli, bacillus subtilis, staphylococcus and clostridium butyricum, no specific product is generated, and the method is proved to have specificity.

Primers for the NetB toxin gene were designed as follows:

an upstream primer: 5'-GCTGGTGCTGGAATAAATGC-3' (SEQ ID NO: 9);

a downstream primer: 5'-TCGCCATTGAGTAGTTTCCC-3' (SEQ ID NO: 10).

The PCR reaction system is 25 μ L: mu.L of template DNA, 0.5. mu.L of each primer, 12.5. mu.L of MaxMaster12, 9.5. mu.L of nuclease-free water. Reaction procedure: 94 ℃ for 2 min; at 94 ℃ for 45s, at 50 ℃ for 30s, at 72 ℃ for 45s, for 30 cycles; 10min at 72 ℃; storing at 10 deg.C.

The PCR method is used for detecting 47 strains of clostridium perfringens, and only 3 strains of clostridium perfringens are netB gene positive and are named as cpnetBB, cpnetBD and cpnetBF.

Drug susceptibility testing was performed on 47 isolates. The test uses 12 antibiotics and chemical synthesis drugs, wherein the drug resistance ratio to metronidazole and lincomycin is higher and is respectively 95.7 percent and 91.5 percent. The drug resistance ratio of the cefalexin to the terramycin to the doxycycline to the clindamycin to the erythromycin is 70-80%. Ampicillin resistance was the lowest, only 12.8%.

47 strains of Clostridium perfringens identified as type A by Multi-PCR were sub-typed using ERIC-PCR technology. The similarity is more than or equal to 0.60 as a demarcation point, 47 isolates can be divided into 5 main types, and the similarity is more than or equal to 0.70 as a demarcation point, 47 isolates can be divided into 8 subtypes.

In the model test, 1 day old healthy Ross 308 chicks were used and randomly divided into 6 groups of 10 chicks each. Free drinking water, and daily ration based on wheat. Wherein 1 group is blank control and the other groups are test groups. Except for the blank control group, the other groups adopt a bacterial liquid mixing mode to carry out toxicity attack. In 5 groups of experimental groups, 3 experimental groups respectively adopt 3 NetB gene positive strains to challenge the toxin, and in addition, 2 groups respectively adopt 2 NetB gene negative strains to challenge the toxin. The whole feeding process is divided into two stages: feeding wheat basal diet at 1-13 days old, and adding 50% of fish meal into basal feed at 14-23 days old. At 21-23 days old, 5 test groups were treated with bacterial liquid mixing for detoxification twice a day. The preparation method of the bacterial liquid used in the challenge test comprises the following steps: the test strains were first cultured anaerobically in bacon medium at 40 ℃ for 15h and then transferred to FT broth for further culture at 40 ℃ for 15 h. 24-day-old necropsy, small intestine disease was observed and scored using a 6-point system. As a result, no obvious lesions were found in the blank control group and in the other 2 strains challenge groups which were netB gene negative. Typical necrotic enteritis lesions appeared in all 3 strains challenge groups positive for the netB gene. The A-type clostridium perfringens with positive netB gene is separated from the focus of the sick chicken.

The antibacterial activity of the plant essential oil or the compound plant essential oil on clostridium perfringens is researched by adopting a paper diffusion method and a two-fold dilution method.

The present invention will be further described with reference to specific experiments, but is not limited thereto. Unless otherwise specified, the nutmeg essential oil, the thyme essential oil and the basil essential oil in the mixed compound essential oil formula are mixed according to the volume ratio. The essential oil product is referred to the national standard.

Experiment 1 establishment of necrotic enteritis model of chickens

1. Materials and methods

1.1 test strains

The bacterial strain used for experimental challenge is clostridium perfringens type A separated from necrotic enteritis cases, wherein NE01 and NE02 are netB gene negative bacterial strains, and cpnetBB, cpnetBD and cpnetBF are netB positive bacterial strains separated from regions of Borol, Dongguan and Foshan in Guangdong province.

1.2 test animals and design

The test was carried out using 1 day old Ross 308 commercial chicks from Guangdong Dongguan Zhengda Ji, Inc. The groups were randomized into 6 groups of 10 individuals. The test was divided into 6 groups, 1 group was blank control and not subjected to bacterial challenge, and the other 5 groups were subjected to bacterial liquid-feed challenge at 21 days of age, and the 5 groups of challenge bacteria were NE01, NE02, cpnetBB, cpnetBD and cpnetBF, respectively.

1.3 test daily ration

The basic ration and the Jiangchang steam fish meal are purchased from Jiangfeng practical company Limited in Guangzhou, and the composition and the nutrition level of the basic ration are shown in Table 2.

TABLE 2 basal diet composition and nutritional level

。

1.4 Breeding management

Cleaning the chicken coop 1 week before the test, sterilizing the coop and the ground by flame, fumigating and sterilizing by formaldehyde, and regularly sterilizing in the whole feeding process. Cage culture is adopted. The relative humidity is kept between 64% and 76%. The whole process keeps good ventilation, free food intake and free water drinking. Feeding basic ration for 1-13 days old, and adding fish meal into the basic ration for 14-23 days old to enable the content of the fish meal to reach 50%.

1.5 preparation of suspensions of challenge bacteria

The test groups of clostridium perfringens (including netB gene negative strains NE01 and NE02, netB positive strains cpnetBB, cpnetBD and cpnetBF) were revived, streaked onto TSC plates, and anaerobically cultured at 40 ℃ for 24 h. Black representative colonies on the plates were inoculated into meat culture media and anaerobically cultured at 40 ℃ for 15 h. The meat culture was inoculated into 400mL of FT broth and anaerobically cultured at 40 ℃ for 15 hours.

1.6 disease model establishment

Adding the FT culture into feed, and stirring uniformly, wherein the ratio of the bacterial liquid to the feed is (volume/weight; mL/g) 1: 1.5. feeding twice a day at 8:00 am and 4:00 pm for 3 d.

1.7 lesion Scoring

After 24 days, the patient was sacrificed by cervical dislocation, and lesions of duodenum and jejunum were observed by dissection, and scored for each group. The scoring criteria are shown in Table 3.

TABLE 3 necrotic enteritis lesion score

。

1.8 Focus isolation Clostridium perfringens genotype and netB gene detection

Samples were collected from the lesion with a disposable swab. After separation and identification, carrying out clostridium perfringens separation genotype identification and carrying out netB gene detection.

1.9 data processing

Data are expressed as mean ± standard deviation (± s) and significance analysis was performed with SPSS 17.0.

2. Results of chicken necrotic enteritis model establishment

2.1 clinical symptoms and intestinal lesions

The chickens were in good condition before challenge, and after 1d of challenge, mild diarrhea appeared in all the other groups except the blank group. After continuous toxicity counteracting, 3 groups of netB gene positive strains counteract groups to aggravate diarrhea symptoms, reduce appetite, discharge red or black brown coal tar-like feces, and some feces are mixed with blood and intestinal mucosa tissues.

After the dissection, the intestinal mucosa was smooth and not damaged in the blank group.

Two netB gene negative strains attack the toxin group, only the phenomenon of intestinal mucosa thickening appears, and other obvious pathological changes do not appear.

Typical necrotic enteritis lesions with different degrees appear in 3 netB positive strain challenge groups, concave-convex ulcer is formed on the surface of small intestinal mucosa, fibrous substances are attached to the surface of small intestinal mucosa, and dispersive necrosis appears in severe intestinal mucosa. The results are shown in FIG. 1. FIG. 1 shows different changes of small intestine caused by necrotic enteritis in chickens; wherein: the number 1 is blank control, and the

numbers

2, 3, 4, 5, 6 and 7 are typical lesions of 1 point, 2 points, 3 points, 4 points, 5 points and 6 points, respectively.

2.2 Small bowel disease Scoring

The results of the small bowel disease scoring are shown in table 4.

TABLE 4 Small bowel disease scoring results

Note: in the table, the difference of the lower case letters indicates that the difference is significant (P < 0.05), the difference of the upper case letters indicates that the difference is very significant (P < 0.01), and the difference of the same letters indicates that the difference is not significant.

Table 4 the results show: the blank group did not show any lesions. The two strains negative to the netB gene only cause thickening of the intestinal wall mucosa and have no other visible lesions. Three groups positive for the netB gene showed marked necrotic enteritis lesions. The netB positive three groups scored very significantly higher than the blank and netB negative groups. The scores of the two netB negative groups were not significantly different.

2.3 detection results of focus-separated clostridium perfringens genotype and netB gene

After the Muliti-PCR method is used for genotype detection, the clostridium perfringens (figure 2 and figure 3) separated from all focuses is found to be amplified to a fragment of about 400bp, the bacteria are judged to be type A bacteria, and the netB gene detection is positive.

FIG. 2 is a genotype identification of Clostridium perfringens isolated from a lesion; wherein M is DL2000 Marker, and 1-20 are clostridium perfringens strains separated from focuses.

FIG. 3 shows the identification of the netB gene of Clostridium perfringens isolated from the lesions; wherein M is DL2000 Marker, and 1-20 are clostridium perfringens strains separated from focuses.

Recent studies on necrotic enteritis in chickens have focused on finding ways to control the disease and elucidating the pathogenesis. The establishment of a chicken necrotic enteritis model through artificial induction is the basis of research. In previous studies, concurrent pattern induction trials of coccidia were often used, but the problem was that small intestinal coccidiosis and necrotic enteritis in chickens were easily confused. The inventor only adopts the single toxin counteracting scheme of the clostridium perfringens bacterial liquid, and the effect is obvious. The experimental group of 3 strains positive for the netB gene showed typical necrotic enteritis lesions. And clostridium perfringens strains separated from the focus are all the strains with positive type A netB genes, which indicates that the offending 3 groups of netB positive bacteria can cause necrotic enteritis of the chickens. The 3 groups of experimental groups with necrotic enteritis lesions have different lesion scores and different lesion degrees, which shows that even though the strains are clostridium perfringens with positive netB genes, the pathogenicity of each strain is still different. Neither the 2 netB negative isolates nor the blank group showed necrotic enteritis lesions. This is a reverse indication that the netB gene is an important cause of the development of necrotic enteritis in chickens.

In the test, wheat is used as the basic feed, and the wheat contains non-starch polysaccharide, so that the chyme viscosity is increased, the digestive absorption of small intestine is influenced, and conditions are provided for the proliferation of clostridium perfringens. The fish meal added in the later period increases the protein content in the daily ration, can provide a large amount of amino acid for clostridium perfringens, and enables chickens to be more susceptible to necrotic enteritis.

The initial success of the test model lays a foundation for further research, such as antibacterial drugs, vaccines, virulence factor evaluation and the like. However, since different researchers have different purposes for the reproduction of diseases and the severity of diseases required is different, the model also needs to be adjusted accordingly when the method is applied.

Experiment 2 in vitro bacteriostatic test of plant essential oil

The addition of antibiotics in daily ration has been a main method for controlling necrotic enteritis, but as antibiotic growth promoters are gradually banned in various places in the world, the research on novel alternative treatment medicines becomes urgent, and the applicant preferably selects several essential oil formulas as a safe and effective antibiotic substitute and has important development value.

1. Materials and methods

1.1 test strains

3 netB positive strains, namely cpnetBB, cpnetBD and cpnetBF, isolated from the disease material.

1.2 plant essential oils

The essential oil is prepared from nutmeg single component essential oil, thyme single component essential oil, basil single component essential oil, nutmeg and thyme essential oil mixed compound at a ratio of 1:1, nutmeg and basil mixed compound at a ratio of 1:1, and thyme and basil mixed compound at a ratio of 1: 1. The plant essential oil is purchased from a daily product company, Inc. in Guangzhou city.

1.3 preparation of the Filter paper sheet

Selecting filter paper with strong water absorption and uniform texture, beating into round pieces with diameter of 6mm by using a puncher, subpackaging in clean dry test tubes, and autoclaving at 121 ℃ for 15min for later use.

1.4 Strain activation

The frozen 3 strains were thawed, streaked and inoculated on TSC plates, and anaerobically cultured at 40 ℃ for 24 h. A typical black colony was picked, inoculated into 10mLFT liquid medium, and anaerobically cultured at 40 ℃ for 8 hours. Diluting the bacterial liquid to 10 degrees according to 0.5 McLeod's turbidimetric tube⁸CFU/mL。

1.5 paper diffusion method

By moving liquidsThe gun sucks 100 muL 10⁸And (3) putting the CFU/mL test bacterial liquid on an FT nutrient agar culture medium, uniformly coating for 3 times by using a coater, then coating for a circle around the edge of the plate, covering the plate, and drying at room temperature for 3-5 min. And carefully clamping filter paper sheets by using sterilized tweezers on each flat plate, sticking the filter paper sheets on the surface of the culture medium, slightly pressing the paper sheets to ensure that the filter paper sheets are tightly stuck to the surface of the culture medium, wherein the distance between the centers of the paper sheets is more than 24mm, and the distance between the paper sheets and the outer edge of the flat plate is more than 15 mm. Respectively sucking 5 mu L of plant essential oil stock solution and dripping the plant essential oil stock solution on a filter paper sheet. The plates were inverted, anaerobically cultured at 40 ℃ for 24h, the diameters of the zones of inhibition were observed and measured, 3 replicates per group were performed, and the average was taken.

The judgment standard of the inhibition zone: the bacteriostasis effect is insensitive when the diameter of the bacteriostasis ring is less than 10mm, sensitive between 11 mm and 15mm, moderate sensitive between 16 mm and 20mm and high sensitive when the diameter is more than 20 mm.

2 paper diffusion method results

And (3) detecting the sensitivity and the bacteriostatic action of the single essential oil and the compound essential oil on pathogenic bacteria of clostridium perfringens by using a paper diffusion method. The results are shown in tables 5 and 6.

Table 5: bacteriostatic circle diameter (mm) of 3 single-component plant essential oil on three pathogenic bacteria

Note: the judgment standard of the inhibition zone: the bacteriostasis effect is insensitive when the diameter of the bacteriostasis ring is less than 10mm, sensitive between 11 mm and 15mm, moderate sensitive between 16 mm and 20mm and high sensitive when the diameter is more than 20 mm.

From table 5, it can be seen that 3 kinds of single-component plant essential oils have strong bacteriostatic effects on 3 kinds of pathogenic bacteria of clostridium perfringens. The effect is more obvious for netBB and netBD strains, and high sensitivity is achieved. The inhibitory effect was moderately sensitive to the netBF strain.

Table 6: inhibition of three pathogenic bacteria by compound plant essential oil (mm)

From table 6, it can be found that the bacteriostatic effect of the 3 kinds of compound plant essential oils is also stronger. Has obvious synergistic effect on the netBB compound plant essential oil.

Experiment 3 Minimal Inhibitory Concentration (MIC) test of plant essential oils

1. Materials and methods

1.1 preparation of plant essential oil test sample

Sucking 0.4mL plant essential oil into an EP tube, adding Tween-800.1 mL, mixing well, adding 0.5mL sterile water, diluting to a concentration of 400 μ L/mL, and mixing well again. Milk white liquid, it is used as it is.

1.2 two-fold dilution method

Under the aseptic condition, 10 test tubes are taken and numbered, wherein the number 9 tube is a positive tube, the number 10 tube is a negative tube, and the rest are tubes with different concentrations. 9mLFT medium was added to tubes 1 and 10 using a pipette gun, and 5mLFT medium was added to each of the remaining tubes. Adding plant essential oil into the No. 1 tube for providing products, blowing and beating uniformly, sucking 5mL of the product, putting the product into the No. 2 tube, and repeating the steps until the product reaches the No. 8 tube. Adding plant essential oil into No. 10 tube, blowing and beating uniformly, sucking 5mL, and discarding. Add 100. mu.L of bacterial suspension to tubes No. 1 to 9. Anaerobic culture is carried out for 6-8 h at 36 +/-1 ℃. And respectively sucking 100 mu L of liquid in each tube to the FT nutrient agar medium, and uniformly coating. Anaerobic culture is carried out for 24 hours at 36 +/-1 ℃, and the result is observed. The highest dilution concentration of the corresponding medicine of the sterile growth plate is the minimum inhibitory concentration of the plant essential oil to clostridium perfringens. The experiment was repeated 3 times.

2. Test result of minimum inhibitory concentration

The results are shown in Table 7.

TABLE 7 MCI results (μ L/mL) of plant essential oils against three pathogens

As shown in table 7, both single and compound essential oils had certain inhibitory effects on 3 netB positive strains. The MICs for the six essential oils or combination of essential oils for the netBB strain were all 0.31 μ L/mL. MICs for six essential oils of netBD strain were all 0.63. mu.L/mL. The best effect was obtained for the netBF strains thyme, basil and thyme + basil, with an MIC of 1.25. mu.L/mL.

Experiment 4 animal experiment of plant essential oil for preventing and treating necrotic enteritis in chicken

1. Materials and methods

1.1 test strains

netBF strain: is clostridium perfringens type A, and has positive netB gene.

1.2 test animals and design

80 Ross 308 commercial chicks of 1 day old were selected for testing and randomly divided into 8 groups of 10 chicks each. 1 group is blank group, 1 group is attack bacteria control group, and the other 6 groups are plant essential oil control group, and 100mg/kg1:1 single nutmeg essential oil, thyme single essential oil, basil single essential oil and compound plant essential oil are added into feed after 17 days old respectively.

1.3 test daily ration

Basal diet, fish meal were used as in experiment 1.

1.4 necrotic enteritis model establishment

The remaining 7 test groups except the blank control group were challenged according to the method used in experiment 1.

1.5 Small bowel disease scoring.

At 24 days of age, 10 chickens were randomly selected, sacrificed by taking off the neck, examined by dissection for lesions in duodenum, jejunum and ileum, and scored for lesions in each group. The scoring criteria are shown in table 3. Data were processed as in experiment 1.

1.6 genotype netB gene identification of isolates

Samples were collected from the lesion with a disposable swab. After separation and identification, the clostridium perfringens separation genotype identification and the netB gene detection are carried out according to the method of experiment 1.

1.7 high throughput sequencing technology for analyzing diversity of chicken intestinal flora

1.7.1 intestinal content sample Collection

At 24 days of age, 5 chickens were randomly selected from each experimental group, dissected, ligated to the duodenum and cecum ends, and then harvested and stored in ice boxes. In the clean bench, the intestine is divided into two sections, the small intestine section (duodenum, jejunum and ileum) and the cecal section. The intestinal contents were removed under sterile conditions and the intestinal contents of 5 chickens per group were thoroughly mixed. Storing at-80 deg.C.

1.7.2 Total DNA extraction of bacteria

The extraction of total DNA of bacteria in intestinal contents of chicken is carried out by adopting a QIAamp DNA pool Mini Kit, and the specific flow refers to the Kit specification.

1.7.3 Illumina misseq sequencing analysis

The extracted intestinal bacterial DNA was sent to microorganism-based biotechnology (Shanghai) Co., Ltd for high-throughput sequencing.

1.7.4 bioinformatics analysis

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

2 results of animal experiments

2.1 Small bowel disease scoring results, see Table 8.

Table 8: small bowel disease scoring

As seen from table 8: compared with the toxicity attacking group, the thyme single-component essential oil treatment group has no significant difference, and other groups have significant differences. The results show that the treatment groups of the single nutmeg essential oil, the single basil essential oil, the 1:1 mixed compound of the nutmeg essential oil and the thyme essential oil, the 1:1 mixed compound of the nutmeg essential oil and the basil essential oil and the 1:1 mixed compound of the thyme and the basil essential oil can obviously reduce the damage of clostridium perfringens to the intestinal tract. Thyme single treatment group had no effect. From the severity of pathological changes, the thyme single-component plant essential oil has the best effect, and compared with other groups, only 1 chicken with 4-6 grades of pathological changes are in the nutmeg single-component plant essential oil group. Compared with the single formula of the nutmeg and basil compound plant essential oil, the number of chickens with lesions of 4-6 is reduced.

In conclusion, in animal experiments, the single nutmeg essential oil, the single basil essential oil, the 1:1 mixed compound of nutmeg and thyme essential oil, the 1:1 mixed compound of nutmeg and basil and the 1:1 mixed compound of thyme and basil all have certain prevention and treatment effects on necrotic enteritis. However, according to the scoring results, more lesions appeared in the treated groups, probably due to the fact that the plant essential oils, although having bacteriostatic effect, did not inactivate the toxins. In the experimental animal model, the strain solution after continuous culture is used for mixing and counteracting toxic substances, and the culture solution contains a large amount of toxins, so that the intestinal mucosa is damaged. In addition, the amount of the plant essential oil and the adding time may affect the control effect. In practical application, the feeding time of the plant essential oil can be increased, so that the proliferation of clostridium perfringens can be controlled in the early stage, the bacterial quantity and the toxin content are greatly reduced, and the occurrence of diseases is reduced.

2.2 focal isolation of Clostridium perfringens genotype and netB Gene detection results

The clostridium perfringens separated from all focuses by genotype detection is amplified to obtain fragments of about 400bp, and the clostridium perfringens is judged to be type A bacteria, and the netB gene detection is positive.

2.3 Effect of different plant essential oils on the intestinal flora of chickens

2.3.1 sequence number results

The results are shown in tables 9 and 10, and FIG. 4.

TABLE 9 effective sequence statistics

Note: in the table, G1, G2, G3, G4, G5, G6, G7, and G8 represent a blank group, an offensive control group, a nutmeg group, a thyme group, a basil group, a nutmeg + thyme 1:1 mixed complex group, a nutmeg + basil 1:1 mixed complex group, and a thyme + basil 1:1 mixed complex group, respectively. A represents a small intestine section and B represents a cecal section. Unless otherwise specified, all the meanings are as defined herein.

TABLE 10 optimized sequence statistics

The results in tables 9 and 10 and fig. 4 show that the effective sequences of the high-throughput sequencing are about 50000, the length of the effective sequences is mostly about 450bp, and after quality control and splicing treatment, the band sequence obtained by each sample is over 30000.

Complex microbial flora exists in intestinal tracts of chickens, and the composition of the microbial flora plays an important role in aspects of host health, production performance, development, nutrient digestion and absorption and the like. The research on the structural composition and change of the intestinal flora is of great significance. The high-throughput sequencing technology is an advanced means for researching the microbial diversity at present, and the experiment researches the influence of the plant essential oil on the intestinal microorganisms of the necrotic enteritis of the chicken by using the high-throughput sequencing technology. The obtained test result has large data volume and high accuracy. Is superior to the traditional test method.

This experiment was performed by high throughput sequencing of the intestinal flora of the small intestine and the cecal section of 8 experimental groups. After quality control and splicing treatment, the number of the optimized sequence strips in each sample is more than 3000. As can be seen from the dilution curve, the amount and depth of sequencing is sufficient to cover most species in the sample for subsequent analysis.

2.3.2 analysis of abundance and diversity of intestinal flora

The dilution curve of the bacterial population in the sample is shown in FIG. 5. As can be seen from FIG. 5, the slope gradually decreases with the increase of the number of sequencing sequences, and tends to plateau. Indicating that the sequencing depth is sufficient to cover most species in the sample for subsequent analysis.

The number of OTUs, ace index, chao index, shannon index and simpson index for each sample are shown in Table 11.

TABLE 11 sample diversity results

As can be seen from Table 11, more OTUs can be detected in all samples, and the number of OTUs is distributed between 192 and 274. This indicates that the abundance of each group of species is high in the intestinal contents sampled this time, but the species composition difference between different groups is large. As can be seen by the 4 Alpha indices, the abundance of species in the intestinal contents of each treatment group was reduced, but the diversity was increased, in the small intestine relative to the necrotic enteritis disease model group. The nutmeg + thyme group was the most diverse. In the caecum, the myristica fragrans + basil group flora is most abundant and diverse relative to the necrotic enteritis disease model group.

2.3.3 structural analysis of intestinal flora

Each sample was analyzed at 3 levels from phylum, order and family, and histograms of relative abundance of species at different levels were plotted, as shown in fig. 6, 7 and 8. And a community heatmap is drawn.

As can be seen from fig. 6, at the phylum level, Firmicutes (Firmicutes) are the most predominant phyla among the different sections of intestine of the different samples. In the small intestine, the necrotic enteritis model group degenerative bacteria (Proteobacteria) accounted for 18.9%. However, in the plant essential oil treatment groups, the rates of phyla quadratica (actinobacilla) were increased at 32.5%, 14.6%, 45.3%, and 23.3% in the basil, nutmeg + thyme, nutmeg + basil, and thyme + basil groups, respectively. Wherein the ratio of bacteroidetes (bacteroides) in thyme + basil group is 23.3%, which is different from other groups. In the caecum, bacteroidetes (bacteroides) is the second most abundant phylum in each group, but the bacteroides (bacteroides) ratio in each plant essential oil control group is increased relative to the challenge group.

As can be seen from fig. 7, the order clostridiales (clostridiales) and Lactobacillales (Lactobacillales) were the main orders in the intestine of each sample as seen at the eye level. In the small intestine, in the plant essential oil control group, the proportion of clostridiales (clostridium) in basil and nutmeg + basil groups is reduced relative to the challenge group, the proportion of clostridiales (clostridium) in the challenge group is 55.3%, the proportion of clostridiales (clostridium) in the challenge group is 20.1% and 16.0%, respectively, and the content of Bacillales (Bacillales) in nutmeg + basil group is increased to 15.0%. The content of Bacteroides (Bcateroidales) in the thyme + Ocimum group was 23.3% higher than that in the other groups. In the caecum, Bacteroides (Bcateroidales) is the third main objective, and the group structures are not changed much.

As can be seen from fig. 8, at the family level, in the small intestine, the proportion of the main families in the structure of the offending group flora, namely, the families of lactobacillus (lactobacillus), Enterobacteriaceae (Enterobacteriaceae), Ruminococcaceae (Ruminococcaceae) and heliciaceae (Lachnospiraceae), was 19.5%, 18.3%, 5.1% and 3.6%. Compared with the toxicity counteracting group, in each plant essential oil treatment group, the structures of the flora in the basil, nutmeg + thyme, nutmeg + basil and thyme + basil groups are greatly changed, and in the basil group, corynebacteriaceae (corynebacteriaceae) accounts for 31.4 percent; in the myristica fragrans + basil group, the Ruminococcaceae (Ruminococcaceae) accounted for 19.8%, the corynebacteriaceae (corynebacteriaceae) accounted for 13.3%, the Lachnospiraceae (Lachnospiraceae) accounted for 11.8%; the family with the highest proportion in the nutmeg + basil group is corynebacteriaceae (corynebacteriaceae) accounting for 27.3%, which is significantly different from the other groups in that the contents of Staphylococcaceae (staphyloccaceae) and brevibacteriaceae (brevibacteriaceae) are increased by 13.0% and 10.2%, respectively; in the thyme + basil group, the content of Rikenellaceae (rikenella ceae) was increased, accounting for 23.3%. In the caecum, various major bacterial groups are Lactobacillaceae (Lactobacillus), Lachnospiraceae (Lachnospiraceae) and Ruminococcaceae (Ruminococcaceae), and the groups are not very different. The heat map represents the relative abundance levels of each species in the sample, and the similarity and difference of the community composition of the sample at each level can be reflected by the gradient and similarity of the color.

The change of the flora in the intestinal tract also has different influences on the feed conversion rate of the chickens, and the effect of the plant essential oil on the production performance of the host is laterally shown. The intestinal microflora is an important factor for maintaining the health of the intestinal tract, and a balanced microflora can effectively defend pathogens in the intestinal tract. The intestinal flora of the treatment groups in the test of the invention varied to a different extent compared to the challenge group, and these changes may have a positive effect on the defense against the disease.

2.3.4 Beta diversity analysis

Respectively representing the similarity between samples by using a difference matrix heat map (figure 9), a multi-sample similarity tree map (figure 10) and a PCoA scatter map (figure 11), wherein in the matrix heat map, the color represents a sample distance value, the redder the color represents the sample similarity, and the bluer the color represents the similarity; in the PCoA scattergram, the more similar the sample composition, the closer they are in the graph. As can be seen from fig. 9, fig. 10, and fig. 11, the small intestine bacterial structures of the respective groups, the basil group and the nutmeg + basil group, were similar to the blank colony structure, and were greatly different from the challenge control group. The other treatment groups and the challenge control group are similarly inoculated. In the cecum, the intestinal flora structures of the groups are very similar.

The cecal intestinal flora structures of the groups are very similar. This indicates that the challenge of clostridium perfringens does not have a significant effect on the cecal segment, since necrotic enteritis lesions occur mainly in the small intestine segment. In addition, due to metabolism and absorption, the content of caecum plant essential oil is reduced, and the effect is correspondingly reduced.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

SEQUENCE LISTING

<110> institute of animal health of academy of agricultural sciences of Guangdong province

Application of thyme compound plant essential oil in prevention and treatment of necrotic enteritis in chicken

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Claims

1. The application of the thyme compound essential oil in preparing the medicine for preventing and treating necrotic enteritis of chickens is characterized in that: the thyme compound essential oil is thyme-nutmeg compound essential oil or thyme-basil compound essential oil;

wherein the volume ratio of thyme essential oil to nutmeg essential oil in the thyme-nutmeg compound essential oil is 1: 1;

the volume ratio of thyme essential oil to basil essential oil in the thyme-basil compound essential oil is 1: 1.

2. Use according to claim 1, characterized in that: the thyme compound essential oil is added into the daily ration of the chicken to prevent and treat necrotic enteritis of the chicken.

3. A feed additive for preventing necrotic enteritis in chicken is characterized in that the additive is thyme compound essential oil or thyme compound essential oil and acceptable food-grade auxiliary materials thereof;

wherein the thyme compound essential oil is thyme-nutmeg compound essential oil or thyme-basil compound essential oil;

the volume ratio of thyme essential oil to nutmeg essential oil in the thyme-nutmeg compound essential oil is 1: 1;

4. The feed for preventing and treating necrotic enteritis of chickens is characterized by comprising the following components in parts by weight: adding thyme compound essential oil into the daily ration of chicken to prevent and treat necrotic enteritis of the chicken;

the volume ratio of thyme essential oil to basil essential oil in the thyme-basil compound essential oil is 1: 1;

the adding proportion of the thyme compound essential oil to the chicken daily ration is 80-150 mg/kg.