CN116267787A

CN116267787A - Chicken bursa synovialis mycoplasma infection model, and construction method and application thereof

Info

Publication number: CN116267787A
Application number: CN202310079060.6A
Authority: CN
Inventors: 张楠; 严秀; 宋姝缇; 姚兆锋; 贾艺鑫; 周明虎
Original assignee: Foshan University
Current assignee: Foshan University
Priority date: 2023-01-13
Filing date: 2023-01-13
Publication date: 2023-06-23

Abstract

The invention discloses a method for constructing a chicken bursa mycoplasma infection model, which comprises the following steps: and (3) taking the spotted-brown chickens as test animals, and injecting mycoplasma synoviae bacterial liquid into joints and foot pads of the spotted-brown chickens to infect the mycoplasma synoviae, so as to establish a chicken mycoplasma synoviae infection model. The method for constructing the chicken mycoplasma synoviae infection model provided by the invention can obtain the chicken mycoplasma synoviae infection model which has obvious disease symptoms, typical disease, high disease rate, low mortality rate, high MS bacteria-carrying capacity and stability.

Description

Chicken bursa synovialis mycoplasma infection model, and construction method and application thereof

Technical Field

The invention relates to the technical field of virus infection models, in particular to a chicken bursa mycoplasma synoviae infection model, a construction method and application thereof.

Background

In recent years, infection with Mycoplasma Synoviae (MS) has occurred in most intensive cultures. MS-infected chickens mainly cause synovitis, respiratory disease, and eggshell tip abnormalities (EAA). MS propagates vertically, which refers to propagation through eggs, and horizontally, which refers to propagation of contaminated feed, feathers, air, drinking water, etc. In addition to increasing embryonic mortality and the cost of treatment of broiler chickens, layer chickens and breeder groups, it also results in weight gain, feed conversion efficiency, egg production and hatchability. Previous studies reported that the genotype of MS clinical strains was mainly the K-type from the local chicken flock Vlha gene in china, and that the K-type resulted mainly in typical symptoms of chicken arthritis. At present, the mechanism of MS infection is not yet established, and thus antibiotics are particularly important for the treatment of MS.

There are significant differences in pharmacokinetics between healthy and diseased animals, particularly at the target site. Thus, the establishment of an MS infection model is a key to study the pharmacokinetics of tilmicosin in joints. Previous studies reported that 25 week old chickens challenged with MS through the footpad, and then pathological changes caused by MS were found by pathological sections after 10 days. Another study reported that infected chickens developed arthritis by intramuscular injection of MS to 20 day old SPF chickens after 12 days. However, these methods are time consuming and do not accurately quantify the amount of MS bacteria carried.

Tilmicosin is a semisynthetic macrolide antibiotic and has good antibacterial activity on gram-positive bacteria, partial gram-negative bacteria, mycoplasma, spirochetes and the like. Drug resistance is caused by the unreasonable use of antibacterial drugs. Tilmicosin is reported to have significantly reduced sensitivity to MS. The pharmacokinetic characteristics of the target site are of great significance in guiding clinical medication, so that the pharmacokinetic study of tilmicosin at the MS infected chicken target site is necessary to be carried out.

Disclosure of Invention

The invention aims to solve the technical problem of providing a method for constructing a chicken mycoplasma synoviae infection model, which can obtain the chicken mycoplasma synoviae infection model with obvious disease symptoms, typical lesions, high disease incidence, low mortality, high MS bacteria-carrying capacity and stability.

The invention also aims to solve the technical problems of providing a chicken bursa synovial mycoplasma infection model which has obvious symptoms, typical lesions, high morbidity, low mortality and high and stable MS bacteria carrying capacity.

In order to solve the technical problems, the invention provides a method for constructing a chicken bursa mycoplasma synoviae infection model, which comprises the following steps:

and (3) taking a chicken as a test animal, and injecting mycoplasma synoviae bacterial liquid into joints and foot pads of the test animal to enable the test animal to infect the mycoplasma synoviae, so as to establish a chicken mycoplasma synoviae infection model.

In one embodiment, the test animal is a spotted-brown chicken or an SPF chicken.

In one embodiment, the spotted-brown chicken is 4 weeks old, 6 weeks old, 8 weeks old, 10 weeks old, or 12 weeks old.

In one embodiment, the period of challenge by injecting the mycoplasma synoviae fluid into the joints and foot pads of the test animal is 1 time/1 day, 2 times/3 days, 2 times/5 days, or 2 times/7 days.

In one embodiment, the mycoplasma synoviae bacterial fluid is injected into the joints and foot pads of the test animal at a dose of 0.5mL, 1mL, 2mL, 4mL, or 5mL per challenge.

In one embodiment, the concentration of the mycoplasma synoviae bacterial fluid is 4×10 ⁸ CFU/mL-6×10 ⁸ CFU/mL。

In one embodiment, the isolated culture of mycoplasma synoviae is prepared by the following method:

inoculating the freeze-dried powder of a standard mycoplasma synoviae strain onto a liquid culture medium under the aseptic condition, then growing in a constant-temperature incubator at 37 ℃, representing the mycoplasma synoviae to a logarithmic growth phase when the culture medium changes from red to orange, diluting a bacterial liquid and transferring the bacterial liquid onto a solid culture medium, and then culturing in a carbon dioxide incubator at 37 ℃ for 5-7 days to obtain colonies of the mycoplasma synoviae.

In one embodiment, the process of injecting the mycoplasma synoviae bacterial fluid into the joints and the foot pads of the test animal further comprises the step of placing the test animal in a cold-hot alternating environment for stress.

In order to solve the technical problems, the invention also provides a mycoplasma synoviae infection model constructed by the method for constructing the mycoplasma synoviae infection model.

Correspondingly, the chicken bursa mycoplasma infection model is applied to research on pharmacokinetics of tilmicosin.

The implementation of the invention has the following beneficial effects:

the chicken mycoplasma synoviae infection model constructed by the method provided by the invention has the characteristics of obvious disease symptoms, typical pathological changes, high disease rate, low mortality and high and stable MS bacteria-carrying capacity, and lays an important foundation for developing the research on prevention and treatment of chicken mycoplasma synoviae infection.

Meanwhile, the chicken bursa mycoplasma infection model is applied to research on pharmacokinetics of tilmicosin in joints, and theoretical support can be provided for solving the problem that the sensitivity of tilmicosin to MS is obviously reduced.

Drawings

FIG. 1 shows MS bacterial load in joints of SPF chickens and spotted-brown chickens after challenge in example 1 of the present invention;

FIG. 2 shows colony morphology (4-fold) of MS under a microscope in example 1 of the present invention;

FIG. 3 is an electropherogram of the PCR product of example 1 of the present invention. Wherein, 1 is a control group; 2 is standard strain WVU18534;4 is an isolate.

Detailed Description

The present invention will be described in further detail below in order to make the objects, technical solutions and advantages of the present invention more apparent.

Unless otherwise indicated or contradicted, terms or phrases used herein have the following meanings:

in the present invention, the use of "a combination thereof", "any combination thereof", and the like includes all suitable combinations of any two or more of the listed items.

In the present invention, "preferred" is merely to describe embodiments or examples that are more effective, and it should be understood that they are not intended to limit the scope of the present invention.

In the invention, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.

In the present invention, the numerical range is referred to, and both ends of the numerical range are included unless otherwise specified.

The invention provides a method for constructing a chicken bursa mycoplasma infection model, which comprises the following steps: and (3) taking a chicken as a test animal, and injecting mycoplasma synoviae bacterial liquid into joints and foot pads of the test animal to enable the test animal to infect the mycoplasma synoviae, so as to establish a chicken mycoplasma synoviae infection model.

Specifically, in one embodiment, the experimental animal is a spotted-brown chicken and an SPF chicken, and the joint and the foot pad of the experimental animal are injected with a mycoplasma synoviae bacterial liquid. Moreover, the spotted-brown chickens are common broiler breeds in China and are closer to clinical application. In addition, the joint volume of the spotted-brown chicken is larger, and the probe is positioned accurately. Preferably, the test animal is a spotted-brown chicken, and the spotted-brown chicken is selected as the test animal, so that the incidence rate is highest, and the average MS bacterial load of the infected chicken is also highest.

The incidence and clinical symptoms of different day-old chickens vary, and in one embodiment, the spotted-brown chickens are 4 weeks old, 6 weeks old, 8 weeks old, 10 weeks old or 12 weeks old, and the spotted-brown chickens are injected with mycoplasma synoviae bacterial fluid at the joints and foot pads. Preferably, the spotted-brown chicken is a spotted-brown chicken of 8 weeks of age. The spotted-brown chickens of 8 weeks of age are selected, the incidence rate is high, and the infection symptoms are obvious.

The incidence and average bacterial load of the spotted-brown chickens in different challenge cycles are different, and in one embodiment, the challenge cycle of injecting the mycoplasma synoviae bacterial liquid into the joints and foot pads of the spotted-brown chickens is 1 time/1 day, 2 times/3 days, 2 times/5 days or 2 times/7 days. 1/1 day here means 1 day of injection, and 1 time of injection is used in the same day; 2 times/1 day refers to 1 day of injection, 2 times of injection on the same day; 2 times/3 days refers to 3 days of injection, 2 times per day; 2 times/5 days means 5 days of injection, 2 times per day; 2 times/7 days refers to 7 days of injection, 2 times per day. Preferably, the period of toxicity attack of the injection of the mycoplasma synoviae bacterial liquid into the joints and the foot pads of the spotted-brown chickens is 2 times/5 days. The continuous 5 days are selected as the toxin attacking period, the incidence rate of the spotted-brown chickens is highest, and the average MS bacterial load of infected chickens is also highest.

In addition, the inventor finds that the toxicity attacking dose and the bacteria carrying amount of the infected chicken are not in a proportional relation, in one embodiment, when the toxicity attacking dose of the mycoplasma synoviae bacterial liquid injected into the joints and the foot pads of the spotted-brown chickens is 0.5mL, 1mL, 2mL, 4mL or 5mL, a higher infection rate can be obtained, and when the toxicity attacking dose is 2mL, the average bacteria carrying amount of the infected chickens is the highest.

In one embodiment, the isolated culture of mycoplasma synoviae is prepared by the following method: inoculating the freeze-dried powder of a standard mycoplasma synoviae strain onto a liquid culture medium under the aseptic condition, then growing in a constant-temperature incubator at 37 ℃, representing the mycoplasma synoviae to a logarithmic growth phase when the culture medium changes from red to orange, diluting a bacterial liquid and transferring the bacterial liquid onto a solid culture medium, and then culturing in a carbon dioxide incubator at 37 ℃ for 5-7 days to obtain colonies of the mycoplasma synoviae. After the colony of the mycoplasma synoviae is obtained, the mycoplasma synoviae bacterial liquid is prepared, preferably, the concentration of the mycoplasma synoviae bacterial liquid is 4 multiplied by 10 ⁸ CFU/mL-6×10 ⁸ CFU/mL。

In addition, the inventor finds that setting stress has a certain influence on the morbidity, average MS bacterial load and mortality of chickens in the infection process. In one embodiment, the process of injecting the mycoplasma synoviae bacterial fluid into the joints and the foot pads of the test animal further comprises the step of placing the test animal in a cold-hot alternating environment for stress. The average MS-bearing capacity of infected chickens stressed in a cold-hot alternating environment is higher, but the mortality rate is higher.

Correspondingly, the invention also provides a chicken mycoplasma synoviae infection model constructed by the method for constructing the chicken mycoplasma synoviae infection model. The chicken mycoplasma synoviae infection model has the characteristics of obvious disease symptoms, typical disease, high disease rate, low death rate, high and stable MS bacteria carrying capacity, and lays an important foundation for developing chicken mycoplasma synoviae infection prevention and treatment research.

Furthermore, the chicken bursa mycoplasma infection model is applied to the research on the pharmacokinetics of tilmicosin, especially the pharmacokinetics in joints of infected chicken. Tilmicosin is an effective drug for clinically treating MS infection, but tilmicosin drug resistance occurs frequently. Pharmacokinetic studies of MS-infected chickens at joint sites are critical for reducing tilmicosin resistance, but no related study exists at present. The invention utilizes the established chicken bursa mycoplasma infection model and combines the microdialysis technology to research the pharmacokinetics of tilmicosin in joints, thereby providing a theoretical basis for clinical application of tilmicosin for treating MS.

The invention is further illustrated by the following examples:

example 1

Construction method of chicken bursa synovial mycoplasma infection model

Material method

Material

MS standard strain (MSWVU-1853) was purchased from China veterinary drug administration. Tilmicosin phosphate was purchased from Hebei Tianxiang biopharmaceutical Co., ltd (tilmicosin content 74.82%, specification 1kg, lot number: A202003005). Tilmicosin standard was purchased from the chinese veterinary drug institute (content 98.60%). Acetonitrile, methanol and formic acid and the remaining analytical grade reagents were purchased from Fisher Scientific company. Ammonium acetate was purchased from Jiangsu Tianan chemical Co., ltd. MS antibody kit was purchased from Shenzhen Lvshiyuan Biotechnology Co., ltd.

Experimental animal

SPF chickens of 8 weeks old are purchased from Xin Dahua nong fowl eggs limited company, and the ephedra slow feather chickens of 8 weeks old are purchased from fowl breeding factories in south China sea area of Buddha, guangdong, transported to experimental animal centers of Buddha animal college for feeding, and fed with complete feed and clean drinking water for free feeding.

Culture of mycoplasma synoviae

An appropriate amount of lyophilized powder (MSWVU-1853) was inoculated onto a liquid medium under aseptic conditions, and MS was grown in an incubator at 37 ℃. When the medium turns from red to orange, it is diluted and dropped on a solid medium, cultured in a carbon dioxide incubator at 37℃for 5 to 7 days, and the area of the number of MSs between 30 and 300 is counted by a microscope, and the concentration of MS is calculated.

Establishment of synovial cyst mycoplasma infection model

The MS infection model is established by adopting six aspects of different varieties, different toxin attacking ways, different day ages, different doses, different toxin attacking periods and whether stress exists. MS antibody detection of spotted-brown chickens is negative.

(1) Comparison of different varieties

40 SPF chickens (8 weeks old, male and female halves, body weight 300-400 g) were randomly divided into 2 groups (20 in each group) and the infected groups were 5X 10 ⁸ The CFU/mL MS detoxify the joint-bound footpad, 2mL each time, 2 times a day, 5 days in succession, and the control group detoxify with normal saline. The group number of spotted-brown chickens (8 weeks old, male and female halves, 500-600 g) is the same as that of SPF chickens.

(2) Comparison of different toxin-attacking modes

120 spotted-brown chickens were randomly divided into 6 groups (20 per group), 5 of which were infected groups and 1 of which were control groups. For the infected group 5X 10 ⁸ The CFU/mL MS is subjected to toxicity elimination through the trachea, the foot pad, the joint, the nose drops and the joint combined foot pad, 2mL each time, 2 times a day, and 5 days continuously, and the control group uses physiological saline.

(3) Comparison of different age of offensive day

120 spotted-brown chickens were randomly divided into 6 groups (20 per group), 5 of which were infected groups and 1 of which were control groups. The infections include 4 weeks of age, 6 weeks of age, 8 weeks of age, 10 weeks of age and 12 weeks of age, respectively.For the infected group 5X 10 ⁸ CFU/mL MS detoxify the joint-bound footpad, 2mL each time, 2 times daily for 5 days. Saline was administered to the control group.

(4) Comparison of different toxin counteracting doses

120 spotted-brown chickens were randomly divided into 6 groups (20 per group), 5 of which were infected groups and 1 of which were control groups. The infected group was 5X 10 ⁸ The CFU/mL MS attacks the toxin on the joint-binding footpad 2 times a day for 5 consecutive days, with toxin attacks in amounts of 0.5mL, 1mL, 2mL, 4mL and 5mL, respectively. Saline was administered to the control group.

(5) Comparison of different attack cycles

The 120 spotted-brown chickens were randomly divided into 6 groups (20 in each group), wherein 5 groups are infected groups, 1 group is control group, and 5×10 infected groups are adopted ⁸ The CFU/mL MS attacks the toxin on the joint-combined foot pad, and the toxin attack period is only 1 time/1 day, 2 times/3 days, 2 times/5 days and 2 times/7 days respectively, wherein the disposable toxin attack group adopts 5mL large-dose injection of the joint-combined foot pad, and the rest attack the toxin 2 times a day, 2mL each time and 2 times a day. Saline was administered to the control group.

(6) Comparison of whether stress

60 spotted-brown chickens 8 weeks old were randomly divided into 3 groups, 2 of which were infected groups and 1 of which were healthy groups. The infectious components are stressed and non-stressed. Wherein, the stress group is placed in a cold-hot alternating environment to carry out stress, and the non-stress group does not carry out corresponding operation. For the infected group 5X 10 ⁸ CFU/mL MS detoxify the joint-bound footpad, 2mL each time, 2 times daily for 5 days. The control group was given normal saline and was not stressed.

Identification of model of synovial mycoplasma infection

The success of the establishment of the infection model is determined by methods such as clinical symptoms, pathological anatomy, pathogen separation identification (micro-morphological identification and PCR identification), detection of a bursa of mycoplasma synoviae antibody kit and the like. The PCR identification method is as follows: genotyping was performed using a single copy conserved region at the 5' end of the variable lipoprotein hemagglutinin vlhA gene using previously reported PCR primers (Wetzel et al 2010): 5' GGCCATTGCTCTCTCTGTTAT-3 ' and 5'-AGTAACCGATCCGCTTAATGC-3'. The theoretical amplification length calculated using the model standard strain was 370bp. Primer synthesis and DNA sequencing of amplified samples was performed by Sangon Biotech (Shanghai, china).

Results

(1) Comparison of different varieties

Table 1 shows the infection of Mahuang chickens and SPF chickens of the same age at the same challenge conditions. The results showed that the incidence of both breeds was 95%. The bacterial load of MS after detoxification is shown in figure 1. After 5 days of continuous detoxification, the bacteria load in the SPF chickens is in an ascending trend, and the spotted-brown chickens are in a stable level. Therefore, the pheasant is more suitable for establishing an MS acute infection model, because the bacterial load of MS in the pheasant is in a stable trend, and the bacterial load in the pheasant is higher.

Table 1 shows the infection of different breeds of chickens

(2) Comparison of different toxicity attack pathways

Table 2 shows the clinical symptoms, morbidity and mortality of chickens infected by different challenge routes. The toxicity attack of the trachea, nose drops, foot pads, joints and joints combined with the foot pads increases morbidity and clinical symptoms. The morbidity and mortality of the joint-bound foot pad were highest, 95% and 15%, respectively. The chicken infected with severe MS may die in the late stage of challenge. Therefore, joint bonding of the foot pad is the best way of counteracting toxic substances. The control group had no MS infection.

Table 2 shows the infection conditions of different toxin-attacking modes

(3) Comparison of different age of offensive day

The incidence and clinical symptoms of chickens of different ages are shown in Table 3. The incidence and clinical symptoms gradually increase over 4-8 weeks. The incidence rate of the chicken groups of 8-12 weeks of age is over 95 percent, and the clinical symptoms are similar. The control group had no symptoms.

Table 3 shows the infection of Mahuang chickens of different ages

(4) Comparison of different toxin counteracting doses

The average MS load for the different challenge doses is shown in table 4. The result shows that the toxicity attacking dose is not in direct proportion to the bacteria carrying amount of infected chickens, and when the toxicity attacking dose is 2mL, the average bacteria carrying amount is the highest and is 3.6X10 ⁶ CFU/mL. The control group had no MS infection.

Table 4 shows the infection of spotted-brown chickens with different amounts of challenge agent

(5) Comparison of different attack cycles

The incidence and average MS load of infected chickens in different challenge cycles are shown in table 5. The incidence of MS and average MS bacteria load increase in the period of 1/1 day, 2/1, 3 and 5 days, while the incidence and average MS bacteria load decrease in the period of 7 days. In the continuous 5-day period, the incidence rate is highest and is 100%, and the average MS bacterial load of infected chicken is highest and is 3.7X10 ⁶ CFU/mL. The control group had no MS infection.

Table 5 shows the infection of spotted-brown chickens with different toxin-attacking cycles

(6) Comparison of stress and absence

The morbidity, average MS load and mortality in the stressed groups are shown in table 6. The incidence of the stressed group was 100% and the incidence of the non-stressed group was 90%. There was no significant difference in average MS load between stressed and non-stressed groups (P > 0.05). The mortality rate was 10% in the non-stressed group and 35% in the stressed group. The control group had no MS infection.

Table 6 shows the infection of the stressed spotted-brown chickens

Identification of infection models

MS-infected chickens exhibit mental depression, difficulty standing, wasting, lameness, joint swelling. The joint cavity of MS-infected chickens was dissected and yellow gummy mucus was found. The serum antibody of the infected group is positive, and the serum antibody of the healthy group is negative. MS isolated from the joints showed typical mycoplasma morphology of the poached eggs under a microscope, and the results are shown in figure 2. The electropherogram of the vlhA gene is shown in FIG. 3. MS amplified a specific PCR band of 370bp.

Finally, by injecting 2mL of joint-bound footpad into 8-week-old spotted-brown chickens, 2 times daily for 5 days as the optimal infection regimen, the average bacterial load was 3.70X10% ⁶ CFU/mL, MS infection model was successfully established.

Example 2

Application of chicken bursa synovial mycoplasma infection model

Method

Pharmacokinetics of tilmicosin in infected chickens

The infected group of spotted-brown chickens (n=6) were fixed on a chicken cage, the joints were locally anesthetized, and then the microdialysis probe was passed through the joint cavity. Prior to dosing, blank plasma samples and dialysate samples were collected in the infected group. The infected group took 15mg/kg b.w tilmicosin orally. Plasma samples were collected after 5, 15, 30min, 1, 2, 4, 8, 12, 24, 48, 72 hours. Collecting joint dialysate sample and blood plasma sample at 0.5-1.5h, 1.5-2.5h, 2.5-3.5h, 3.5-4.5h, 4.5-5.5h, 5.5-6.5h, 6.5-7.5h, 7.5-8.5h, 8.5-9.5h, and 9.5-10 h. The dialysate samples were stored at-20 ℃ and tested over two weeks.

Determination of tilmicosin concentration in plasma and dialysate

By triple quadrupole high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS, dayThe shimadzu corporation) determines the concentration of tilmicosin in the plasma sample and the dialysate sample. Analyte pass-through Shim-pack GIST-HPC ₁₈ The column (50X 21mm,3 μm, shimadzu (China), co., ltd.) was separated and the mobile phase consisted of water and 0.1% formic acid (A solvent) and acetonitrile (B solvent). The elution procedure is 0-1 min15% B, 1.5-4 min 65% B, 4-4.5 min 95% B, 4.5-5.5 min 95% B, 5.5-10 min15% B, and the flow rate is 0.3mL/min. The sample volume was 5. Mu.L.

Plasma samples were pretreated and 1% acetic acid-acetonitrile was added. The solution was centrifuged, the supernatant removed, and the extraction was repeated, the supernatants combined and evaporated. The residue was suspended and filtered through a 0.22mm membrane filter prior to HPLC-MS/MS analysis. Without any pretreatment of the dialysate. The concentration of the standard curve was 5, 10, 20, 50, 100, 200, 500ng/mL. The detection limit is defined as the signal-to-noise ratio (S/N) not less than 3, and the quantification Limit (LOQ) is defined as the signal-to-noise ratio (S/N) not less than 10. The recovery and coefficient of variation within and between batches were determined.

Pharmacokinetic analysis

Results of tilmicosin concentration in plasma and dialysate were analyzed by non-compartmental model using winnonlin6.1 software and PK parameters were obtained: maximum concentration (C) _max ) Peak time of arrival (T) _max ) Half-life of elimination (t) _1/2 ) Area under the drug-time curve (AUC) and Mean Residence Time (MRT).

Statistical analysis of data

Tilmicosin concentration data were processed with IB MSPSS statistical software and PK parameters were calculated using winnonlin version 6.1.0 software, and metering data were expressed as (mean±sd).

Results

HPLC-MS/MS analysis

The linearity of tilmicosin in plasma and dialysate is good (R ² >0.999). Tilmicosin has a limit of detection (LOD) of 2ng/mL and a limit of quantification (LOQ) of 4ng/mL. The variation Coefficient (CV) of tilmicosin in the blood plasma is less than 13%, and the recovery rate is 80-88%.

Pharmacokinetics of tilmicosin in plasma and dialysate

Determination of temozolomide in MS-infected chickens after oral administration at a dose of 15mg/kgPlasma and joint dialysate concentrations of combretastatin. Pharmacokinetic parameters of plasma and dialysate in infected chickens are shown in table 7. Results show t _1/2 No significant difference from MRT (P>0.05). However, AUC and C of plasma and dialysate _max There was a significant difference, the AUC of tilmicosin in plasma was 2.1 times (P<0.01 Tilmicosin C in blood plasma _max Is 1.4 times (P)<0.05). Tilmicosin has a distribution coefficient of 0.51 (AUC _{Dialysate liquid} /AUC _{Plasma of blood} ＝0.51)。

Table 7 shows the comparison of the principal pharmacokinetic parameters in infected chicken blood and dialysate samples (n=6)

The results show that the concentration of tilmicosin at joints of MS-infected chickens is far lower than the plasma concentration, and the penetration of tilmicosin at joints to plasma is about 50%. This may lead to the generation of drug resistant strains. Therefore, the recommended dose should be appropriately increased when guiding clinical medication for the treatment of MS infection.

While the foregoing is directed to the preferred embodiments of the present invention, it will be appreciated by those skilled in the art that changes and modifications may be made without departing from the principles of the invention, such changes and modifications are also intended to be within the scope of the invention.

Claims

1. The method for constructing the chicken bursa mycoplasma infection model is characterized by comprising the following steps of: and (3) taking a chicken as a test animal, and injecting mycoplasma synoviae bacterial liquid into joints and foot pads of the test animal to enable the test animal to infect the mycoplasma synoviae, so as to establish a chicken mycoplasma synoviae infection model.

2. The method for constructing a model of mycoplasma synoviae infection according to claim 1, wherein the test animal is spotted-brown chicken or SPF chicken.

3. The method of claim 2, wherein the spotted-brown chicken is 4 weeks old, 6 weeks old, 8 weeks old, 10 weeks old or 12 weeks old.

4. The method according to claim 1, wherein the period of challenge of the mycoplasma synoviae bacterial liquid injected into the joints and foot pads of the test animal is 1 time/1 day, 2 times/3 days, 2 times/5 days or 2 times/7 days.

5. The method for constructing a model of mycoplasma synoviae infection according to claim 1, wherein the injection of mycoplasma synoviae bacterial liquid into the joints and foot pads of the test animal is performed at a dose of 0.5mL, 1mL, 2mL, 4mL or 5mL each time.

6. The method for constructing a model of mycoplasma synoviae infection according to claim 1, wherein the concentration of the mycoplasma synoviae bacterial liquid is 4 x 10 ⁸ CFU/mL-6×10 ⁸ CFU/mL。

7. The method for constructing a chicken mycoplasma synoviae infection model according to claim 1, wherein the separation culture of mycoplasma synoviae is prepared by the following method:

8. The method for constructing a model of mycoplasma synoviae infection according to claim 1, wherein said injecting a mycoplasma synoviae bacterial liquid into the joints and foot pads of said test animal further comprises subjecting said test animal to stress in a cold and hot alternating environment.

9. A model of mycoplasma synoviae infection constructed by the method of constructing a model of mycoplasma synoviae infection as defined in any one of claims 1 to 8.

10. Use of a model of mycoplasma synoviae infection as defined in claim 8 in the study of the pharmacokinetics of tilmicosin.