阅读说明：本技术 用于绵羊肺炎支原体毒力比较分析方法 (Comparative analysis method for virulence of mycoplasma ovipneumoniae ) 是由 郝华芳 储岳峰 陈胜利 颜新敏 袁婷 刘永生 于 2019-09-04 设计创作，主要内容包括：本发明公开一种不同绵羊支原体菌株毒力比较分析方法。本发明比较分析绵羊支原体菌株毒力的方法是将经复苏的各待比较分析的绵羊支原体分别接种于不同鸡胚上,再将接种后的鸡胚置于恒温箱内培养,定时检测鸡胚状态及死亡情况,统计各个菌株的鸡胚致死率和接种7d死亡率,结合死亡鸡胚尿囊液绵羊肺炎支原体的鉴定结果,以此进行比较判定。本发明的方法成本低、实验动物SPF鸡胚容易获得、操作简单方便、试验周期短、重复性好等优点,克服了现有技术存在的实验动物价格较为昂贵、对实验动物要求高,动物饲养繁琐、试验周期长、重复性较差等缺点。(The invention discloses a comparative analysis method for virulence of different mycoplasma ovis strains. The method for comparatively analyzing the mycoplasma ovis strain virulence comprises the steps of respectively inoculating each recovered mycoplasma ovis to be comparatively analyzed on different chick embryos, culturing the inoculated chick embryos in a constant temperature box, regularly detecting the state and death condition of the chick embryos, counting the chick embryo mortality and the inoculated 7d mortality of each strain, and carrying out comparison and judgment by combining the identification result of the mycoplasma ovis with the dead chick embryo allantoic fluid. The method has the advantages of low cost, easy acquisition of SPF chick embryos of experimental animals, simple and convenient operation, short test period, good repeatability and the like, and overcomes the defects of expensive experimental animals, high requirement on the experimental animals, complicated animal feeding, long test period, poor repeatability and the like in the prior art.)

1. A method for comparatively analyzing the virulence of mycoplasma ovis strains is characterized in that each recovered mycoplasma ovis to be comparatively analyzed is respectively inoculated on different chick embryos, the inoculated chick embryos are placed in a constant temperature box for culturing, the chick embryo states and death conditions are detected at regular time, the chick embryo mortality and the inoculated 7d mortality of each strain are counted, and the comparison and the judgment are carried out according to the identification results of the mycoplasma ovis with the dead chick embryo allantoic fluid.

2. The method of claim 1, wherein:

(1) SPF chick embryo preparation: preparing healthy SPF (specific pathogen free) chick embryos of 7-8 days old;

(2) preparing a bacterial solution to be detected of the mycoplasma ovipneumoniae: recovering the mycoplasma ovipneumoniae strain, inoculating the mycoplasma ovipneumoniae culture medium, culturing at the constant temperature of 37 ℃ for 1-2 days, subculturing, and allowing the culture medium to change colorWhen the yellow or pH value is not higher than 6.8, the normal saline adjusts the bacterial liquid concentration to make the bacterial liquid viable bacteria titer reach 10⁹CCU/ml。

(3) Inoculation and observation of chick embryos: inoculating a bacterial solution to be detected of mycoplasma ovipneumoniae to healthy SPF (specific pathogen free) chick embryos of 7-8 days old, inoculating yolk sacs, and inoculating 0.2ml of each strain; after inoculation, continuously culturing for 13-14 days in an incubator;

(4) statistical analysis and result judgment: and (4) counting the chick embryo mortality and the inoculation mortality of 7d of each strain, and performing comparative analysis and result judgment by combining the identification result of the dead chick embryo allantoic fluid mycoplasma ovipneumoniae.

3. Use of the method of claim 1 in the screening or preparation of a mycoplasma ovis vaccine.

4. Use of the method of claim 1 in the study of virulence of mycoplasma ovipneumoniae.

Technical Field

The invention relates to a method for comparing certain properties of microbial strains of the same species and different species, in particular to a method for comparing and analyzing the virulence of different mycoplasma ovis strains.

Background

Mycoplasma ovipneumoniae is a pathogen that infects sheep and goats and causes mainly respiratory diseases in sheep such as cough, asthma, rhinorrhea, etc., which are prevalent worldwide. Mycoplasma ovipneumoniae may be present in the normal nasal cavity of sheep. In recent years, reports of mycoplasma ovipneumoniae infection diseases in China are increasing, and healthy development of sheep raising industry is seriously harmed. Vaccination is an important means for preventing and controlling the disease, and is mainly an inactivated vaccine at present.

The mycoplasma ovipneumoniae genotypes are numerous, and the virulence has large difference. There were also differences in the degree of infection of cells by mycoplasma ovipneumoniae. At present, methods for comparing the virulence of mycoplasma ovipneumoniae mainly depend on animal experiments. The experimental animals are mainly sheep. However, the method has the defects of high requirement on experimental animals (healthy sheep or goats, negative mycoplasma pneumoniae and antibodies in sheep), long experimental period (generally 21-28 days or even longer), expensive price of experimental animals and sheep, high feeding requirement, complex experimental operation and the like. The establishment of a simple comparison method for the virulence of the mycoplasma ovipneumoniae is of great significance to both the virulence research of the mycoplasma ovipneumoniae and the screening of vaccine strains.

Disclosure of Invention

The invention provides a method for simply and comparatively analyzing the virulence of different mycoplasma ovis strains.

The method for simply and comparatively analyzing the mycoplasma ovis strain virulence comprises the steps of respectively inoculating various recovered mycoplasma ovis to be comparatively analyzed on different chick embryos, culturing the inoculated chick embryos in a constant temperature box, regularly detecting the state and death condition of the chick embryos, counting the chick embryo mortality and the inoculated 7d mortality of various strains, and carrying out comparison and judgment by combining the identification result of the mycoplasma ovis with the dead chick embryo allantoic fluid.

Preferably, the comparative method for analyzing the virulence of the mycoplasma ovis strain of the present invention is:

(1) SPF chick embryo preparation: healthy SPF (specific pathogen free) chick embryos of 7-8 days old are prepared, the chick embryos are vigorous and have clear blood vessels, and the chick embryos are cultured in an incubator (the temperature is 37.0-37.5 ℃, and the humidity is 50%).

(2) Preparing a bacterial solution to be detected of the mycoplasma ovipneumoniae: resuscitating the mycoplasma ovipneumoniae strain, inoculating the mycoplasma ovipneumoniae culture medium, culturing at the constant temperature of 37 ℃ for 1-2 days, subculturing, and adjusting the concentration of the bacterial liquid by using physiological saline when the color of the culture medium turns yellow or the pH value is not higher than 6.8 so that the viable bacterial titer of the bacterial liquid reaches 10⁹CCU/ml。

(3) Inoculation and observation of chick embryos: under the aseptic condition, inoculating a bacterial solution to be detected of mycoplasma ovipneumoniae to healthy SPF (specific pathogen free) chick embryos of 7-8 days old, inoculating yolk sacs, and inoculating each bacterial strain according to 0.2ml (per bacterial solution); and after inoculation, continuously culturing for 13-14 days in the incubator. In the specific operation, the bacterial liquid to be detected of the mycoplasma ovipneumoniae can be inoculated to healthy SPF (specific pathogen free) chick embryos of 7-8 days old under the aseptic condition, yolk sac inoculation is carried out, and each strain is inoculated with 10 SPF chick embryos of 0.2 ml/egg. And setting a negative control group. Inoculating 10 negative control groups of chick embryo yolk sac with 0.2 ml/piece of normal saline; 10 chicken embryos of the blank control group are not treated; after the test group is inoculated, the incubator continues to culture for 13-14 days.

(4) Statistical analysis and result judgment: the chick embryo mortality rate and the 7d inoculation mortality rate of each strain are counted. And performing comparative analysis and result judgment. The specific method is to observe and record the death condition of the chick embryos every day. Collecting dead chick embryos, placing the chick embryos in a refrigerator at the temperature of 2-8 ℃ for 6-24 hours, collecting allantoic fluid of the dead chick embryos, and carrying out mycoplasma ovipneumoniae separation and PCR identification; and (4) counting the death number of the chick embryos of each strain, calculating the death rate of the chick embryos, and meanwhile counting the death rate of 7d after inoculation. And comprehensively analyzing by combining the identification result of the dead chick embryo allantoic fluid mycoplasma ovipneumoniae, and comparing the virulence of different strains of mycoplasma ovipneumoniae.

The method can be applied to screening or preparation of the mycoplasma ovis vaccine, and can also be applied to virulence or pathogenic research of the mycoplasma ovipneumoniae.

Compared with the prior art, the method has the advantages of low cost, easy acquisition of SPF chick embryos of experimental animals, simple and convenient operation, short test period, good repeatability and the like, and overcomes the defects of expensive experimental animals, high requirement on the experimental animals, fussy animal feeding, long test period, poor repeatability and the like in the prior art. Therefore, the method can be used for screening virulent strains in the development of mycoplasma ovipneumoniae vaccines and can also be used for the virulence research of mycoplasma ovipneumoniae.

Detailed Description

The following description is given in conjunction with embodiments of the present invention.

A comparative analysis method for the virulence of mycoplasma ovipneumoniae comprises the following steps:

1. preparing a healthy SPF chick embryo (an SPF hatching egg is purchased from Shandong Hao Tai laboratory animal Breeding company, Ltd.) of 7 days old, hatching in a hatching box at the temperature of 37.0-37.5 ℃ and the humidity of 50%, irradiating the egg before inoculation, and discarding the egg without sperm, the dead embryo and the weak embryo.

2. Preparing a mycoplasma ovipneumoniae bacterial solution: recovering mycoplasma ovipneumoniae Y98 strain (purchased from China veterinary microbial culture collection center CVCC, number CVCC384) and MoGH3-3 strain (separated and stored by Lanzhou veterinary research institute of China agricultural academy of sciences), respectively inoculating mycoplasma ovipneumoniae culture medium (improved Thiaubert's culture medium), culturing at constant temperature of 37 ℃ for 1-2 days, subculturing, centrifuging at 8000rpm for 15min when the color of the culture medium turns yellow (or the pH value is not higher than 6.8), regulating the concentration of bacterial liquid by using normal saline, and enabling the titer of the viable bacterial liquid to reach 10⁹CCU/ml。

3. Inoculation and observation of chick embryos: inoculating the prepared mycoplasma ovipneumoniae bacterial liquid into chick embryo yolk sacs, inoculating 10 SPF chick embryos per strain with each strain with 0.2ml per cell, simultaneously setting a negative control group (inoculated with physiological saline with 0.2ml per cell and 10 cells) and a blank control group (10 cells without any treatment), and continuously culturing in an incubator for 13-14 days after inoculation.

4. Observation and statistical analysis: chick embryo death was observed and recorded daily. And (3) collecting dead chick embryos, placing the chick embryos in a refrigerator at the temperature of 2-8 ℃ for 6-24 hours, collecting chick embryo allantoic fluid for later use, and performing mycoplasma ovipneumoniae separation and identification. None of the negative control and blank control chick embryos died; and (4) counting the death number of the chick embryos of each strain, calculating the mortality of the chick embryos, and meanwhile counting the mortality of 7d after inoculation.

The method for separating and identifying the mycoplasma ovipneumoniae comprises the following steps: collecting allantoic fluid of dead chick embryos, inoculating improved Thiaucotrt's culture medium, and carrying out separation and identification on mycoplasma ovipneumoniae. Extracting chick embryo allantoic fluid bacterial DNA and carrying out PCR analysis.

SEQ ID No.1 upstream primer 5' -TGAACGGAATATGTTAGCTT-3,

downstream primer 5'-GACTTCATCCTGCACTCTGT-3' of SEQ ID No. 2;

PCRmix 12.5μL、ddH₂o8.5. mu.L, forward primer 1.0. mu.L, and reverse primer 1.0. mu. L, DNA 2.0.0. mu.L. The PCR procedure was as follows: at 95 deg.C for 5min, for 35 cycles (95 deg.C for 30s, 55 deg.C for 30s, 72 deg.C for 30s), at 72 deg.C for 10min, and at 4 deg.C. The target fragment of PCR was 361 bp.

The results are shown in Table 1:

TABLE 1

As can be seen, the death time of the mycoplasma ovipneumoniae Y98 strain and the MoGH3-3 strain infected chick embryos is 1-10 days after inoculation; at 7d of inoculation, the mycoplasma ovipneumoniae Y98 strain died only 10% (1/10), while the mycoplasma ovipneumoniae MoGH3-3 strain died 50% (5/10); the death rate of the mycoplasma ovipneumoniae MoGH3-3 strain inoculated with the 7d strain is 50%, and the death rate of the mycoplasma ovipneumoniae Y98 strain inoculated with the 7d strain is 10%; the chicken embryo fatality rate of the mycoplasma ovipneumoniae Y98 strain is 40%, and the chicken embryo fatality rate of the MoGH3-3 strain is 50%; therefore, the mortality rate of chicken embryo inoculation 7d and chicken embryo mortality rate of the MoGH3-3 strain are higher than those of the Y98 strain. The allantoic fluid of dead chick embryos contains mycoplasma ovipneumoniae pathogen. From the results of the chick embryos, the mycoplasma ovipneumoniae MoGH3-3 strain has higher virulence than the Y98 strain, which is consistent with strong and weak virulence to sheep. The method has the advantages of low cost, easy acquisition of experimental animals, simple operation, short test period, good repeatability, small workload and the like.

It should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

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