阅读说明：本技术 一种比较分析不同牛支原体菌株毒力的方法 (Method for comparatively analyzing virulence of different mycoplasma bovis strains ) 是由 郝华芳 陈胜利 储岳峰 颜新敏 蔡莉娟 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牛支原体菌株毒力比较分析方法。本发明的比较分析牛支原体菌株毒力的方法是将等活菌菌落浓度的经复苏传代的待比较分析的各待测牛支原体株、牛支原体标准株接种于鸡胚上,再将接种后的鸡胚置于恒温箱培养,定时检测鸡胚状态及死亡情况,统计接种各个菌株的卵黄囊接种鸡胚致死率、尿囊腔接种鸡胚致死率,结合死亡鸡胚尿囊液牛支原体分离鉴定情况,以此进行比较判定。本发明的方法具有操作简单、成本低、实验动物便宜且易得、无需复杂动物饲养管理条件、耗时短、重复性好等优点,极大减少动物试验工作量,避免了使用大动物牛所存在试验操作复杂、试验周期长、工作量大、成本昂贵、实验动物个体差异较大且要求高、重复性不理想等不足。(The invention discloses a comparative analysis method for virulence of different mycoplasma bovis strains. The method for comparatively analyzing the toxicity of the mycoplasma bovis strains comprises the steps of inoculating each mycoplasma bovis strain to be comparatively analyzed and a mycoplasma bovis standard strain which are subjected to resuscitative passage and are to be comparatively analyzed and have the same viable bacteria colony concentration on chick embryos, then placing the inoculated chick embryos in a constant temperature box for culture, regularly detecting the state and the death condition of the chick embryos, counting the lethality rate of the chick embryos inoculated with yolk sacs of each inoculated strain and the lethality rate of the chick embryos inoculated with allantoic cavities, and combining the separation and identification conditions of the mycoplasma bovis of the dead chick embryos to carry out comparison and judgment. The method has the advantages of simple operation, low cost, cheap and easily-obtained experimental animals, no need of complex animal feeding and management conditions, short time consumption, good repeatability and the like, greatly reduces the workload of animal experiments, and avoids the defects of complex operation, long experiment period, large workload, high cost, large individual difference and high requirement of experimental animals, unsatisfactory repeatability and the like when large animal cattle are used.)

1. A method for comparatively analyzing the virulence of mycoplasma bovis strains is characterized in that mycoplasma bovis standard strains to be comparatively analyzed, which are resuscitated and passaged and have similar viable bacteria colony concentrations, and each mycoplasma bovis strain to be detected are inoculated on chicken embryos, the inoculated chicken embryos are placed in a constant temperature box to be cultured, the states and death conditions of the chicken embryos are regularly detected, the mortality of the chicken embryos inoculated with each strain and the mortality of the chicken embryos inoculated in allantoic cavities are counted, and the strains to be detected are compared with the mycoplasma bovis standard strain PG45 or/and each mycoplasma bovis to be detected according to the separation and judgment conditions of the dead chicken embryos and the separation and identification conditions of the mycoplasma bovis.

2. The method of claim 1, wherein:

(1) SPF chick embryo preparation: preparing SPF (specific pathogen free) chick embryos which are 7-8 days old and have clear blood vessels and vigorous and healthy activities;

(2) preparing a mycoplasma bovis standard strain and a bacterial liquid of a to-be-detected strain: recovering and subculturing a mycoplasma bovis standard strain PG45 and a to-be-detected strain, culturing in a constant-temperature incubator at 37 ℃, centrifuging at 8000-12000 rpm for 15min when the mycoplasma bovis grows vigorously, and concentrating the bacterial solution by using normal saline until the bacterial solution concentration is 5.0-9.0 × 10⁹CFU/ml；

(3) Inoculation and observation of chick embryos: inoculating the prepared mycoplasma bovis bacterium liquid to be compared and analyzed to the chick embryos, inoculating each strain to SPF chick embryos according to 0.2 ml/egg yolk sac, after the test group is inoculated, incubating in an incubator, observing the state of the chick embryos every day, recording the death condition, performing autopsy on the dead chick embryos, and collecting allantoic liquid for pathogen separation and PCR identification;

(4) statistical analysis and result judgment: counting the death conditions of egg yolk sac inoculation and allantoic cavity inoculation of each strain, and the death number of chick embryos, calculating the lethality rate of egg yolk sac inoculation chick embryos and allantoic cavity inoculation chick embryos of each strain, combining the chick embryo dissection condition and the separation and identification condition of the death chick embryo allantoic fluid mycoplasma bovis, and comprehensively comparing, analyzing and judging the results.

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

4. Use of the method of claim 1 for the study of the pathogenic mechanism or virulence of mycoplasma bovis.

Technical Field

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

Background

Mycoplasma (mycoplasma) is a prokaryotic cell type microorganism without a cell wall, with high polymorphism, capable of passing through a bacterial filter, widely present in the human or animal body, and partially pathogenic. Mycoplasma bovis (m.bovis) is an important pathogen that infects cows and cattle, mainly causing pneumonia, arthritis, and mastitis in calves. Mycoplasma bovis is an important pathogen of respiratory disease syndrome in cattle, and is also an important pathogen of cow mastitis. At present, mycoplasma bovis infection exists in the global range, causes huge economic loss for cattle raising industry, and seriously restricts the development of the cattle raising industry. Mycoplasma bovis is often isolated from lung of pneumonia calf, mammitis milk, arthritic joint fluid, and in addition, Mycoplasma bovis is isolated from part of upper respiratory tract of healthy cattle such as nasal cavity. The mycoplasma bovis exists widely in China and is popular in provinces and cities, and poses serious threat to the healthy sustainable development of the dairy cattle and cattle breeding industry in China

As for the virulence of mycoplasma bovis, studies have been made mainly on bovine bodies, and other animal models such as rabbits and mice have been tried. The mycoplasma bovis epidemic strains are numerous and have different genotypes, and the pathogenicity of the mycoplasma bovis is greatly different. Currently, the virulence determination of mycoplasma bovis mainly depends on the body of experimental animals, and the virulence differences of different mycoplasma bovis strains are distinguished through a bovine infection test. The selection of virulence strains is a key link in the research and development of inactivated vaccines, however, the experimental animals have the defects of high cattle cost, high feeding management requirements, high test operation difficulty and intensity, poor repeatability and the like. In the aspects of research on excavation and identification of virulence genes of mycoplasma bovis and research on pathogenic mechanisms, virulence genes or proteins can be screened and determined and functional research can be carried out by comparing the virulence of mutant strains (or subcultured strains) and wild strains. Therefore, establishing a method for simply analyzing the mycoplasma bovis virulence has important significance for the research, prevention and control of the mycoplasma bovis.

Disclosure of Invention

The invention provides a method for comparatively analyzing the virulence of different mycoplasma bovis strains.

The method for comparatively analyzing the toxicity of the mycoplasma bovis strains comprises the steps of inoculating each mycoplasma bovis strain to be comparatively analyzed and a mycoplasma bovis standard strain which are subjected to resuscitative passage and are to be comparatively analyzed and have the same viable bacteria colony concentration on chick embryos, then placing the inoculated chick embryos in a constant temperature box for culture, regularly detecting the state and the death condition of the chick embryos, counting the lethality rate of the chick embryos inoculated with yolk sacs of each inoculated strain and the lethality rate of the chick embryos inoculated with allantoic cavities, and combining the separation and identification conditions of the mycoplasma bovis of the dead chick embryos to carry out comparison and judgment.

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

(1) SPF chick embryo preparation: preparing SPF (specific pathogen free) chick embryos which are 7-8 days old and have clear blood vessels and vigorous and healthy activities;

(2) preparing a mycoplasma bovis standard strain and a bacterial liquid of a to-be-detected strain: recovering and subculturing a mycoplasma bovis standard strain PG45 and a to-be-detected strain, culturing in a constant-temperature incubator at 37 ℃, centrifuging at 8000-12000 rpm for 15min when the mycoplasma bovis grows vigorously, and concentrating the bacterial solution by using normal saline until the bacterial solution concentration is 5.0-9.0 × 10⁹CFU/ml；

(3) Inoculation and observation of chick embryos: inoculating the prepared bacterial liquid of each mycoplasma bovis to be detected and mycoplasma bovis standard strain PG45 to the chick embryo, inoculating each strain to SPF chick embryo according to 0.2 ml/egg yolk sac, inoculating each strain to 10 chick embryos, after the test group is inoculated, incubating in an incubator, observing the state of the chick embryos every day, recording the death condition, performing dissecting inspection on the dead chick embryos, and collecting allantoic liquid for pathogen separation and PCR identification;

(4) statistical analysis and result judgment: counting the death conditions of the yolk sac inoculated and the allantoic cavity inoculated chick embryos and the death number of the chick embryos of each strain, calculating the mortality rate of the yolk sac inoculated chick embryos and the allantoic cavity inoculated chick embryos of each strain, combining the chick embryo dissection condition and the separation and identification condition of the death chick embryo allantoic fluid mycoplasma bovis, comparing the strain to be detected with the mycoplasma bovis standard strain PG45 or/and each mycoplasma bovis to be detected, and comprehensively comparing, analyzing and judging the result.

The method for comparatively analyzing the virulence of the mycoplasma bovis strain can be applied to screening or preparation of mycoplasma bovis vaccines.

The method is applied to research on pathogenic mechanism or virulence of mycoplasma bovis.

The invention has the beneficial effects that: the method has the advantages of simple operation, low cost, cheap and easily-obtained experimental animals, no need of complex animal feeding and management conditions, short time consumption, good repeatability and the like, greatly reduces the workload of animal experiments, and avoids the defects of complex operation, long experiment period, large workload, high cost, large individual difference and high requirement of experimental animals, unsatisfactory repeatability and the like when large animal cattle are used. Therefore, the method is suitable for simply analyzing the virulence of different mycoplasma bovis strains, can also be used for strain screening in vaccine development and application in research of excavation and identification of virulence genes of mycoplasma bovis.

Detailed Description

The invention is implemented as follows:

(1) SPF chick embryo preparation: preparing healthy SPF (specific pathogen free) chick embryos (with clear blood vessels and vigorous activity) of 7-8 days old, incubating the chick embryos in an incubator (the temperature is 37.0-37.5 ℃ and the humidity is 50%), illuminating the eggs before inoculation, and removing the eggs without sperm, the dead embryos and the weak embryos.

(2) Preparing a mycoplasma bovis standard strain and a bacterial liquid of a to-be-detected strain: respectively inoculating a standard mycoplasma bovis PG45 strain (American culture Collection ATCC, number ATCC 25523) and a mycoplasma bovis 08M strain to be detected (isolated and stored by Lanzhou veterinary institute of Chinese academy of agricultural sciences), respectively inoculating improved Thiaucorn's culture medium, culturing in a constant-temperature incubator at 37 ℃, centrifuging at 8000rpm for 15min when the mycoplasma bovis grows vigorously (when the color of the culture medium turns yellow or the pH value is reduced to 6.8-7.0), and concentrating the bacterial solution to a bacterial solution concentration of about 7.0 × 10 by using physiological saline⁹CFU/ml。

(3) Inoculating and observing chick embryos, and analyzing data: inoculating the prepared mycoplasma bovis bacterium liquid to chick embryos, inoculating 10 SPF chick embryos to each strain yolk sac, and inoculating 0.2 ml/egg of 10 SPF chick embryos to each strain allantoic cavity. Meanwhile, a negative control group is arranged, and the same way is used for inoculating physiological saline 0.2 ml/piece for 20 pieces in total; 10 chicken embryos of the blank control group are not treated; after inoculation, the incubator is used for incubation, chick embryos are observed for 1 time every day, the state of the chick embryos is observed, whether blood vessels are free or not or whether gaps are fuzzy or not is judged, and the death condition of the chick embryos is observed and recorded. None of the negative control and blank control chick embryos died; and (4) performing autopsy on dead chick embryos, and collecting allantoic fluid for pathogen detection (pathogen separation and PCR identification). Counting the death number of egg yolk sac inoculated and allantoic cavity inoculated chick embryos of each strain, and calculating the lethality rate of the egg yolk sac inoculated chick embryos and the allantoic cavity inoculated chick embryos of each strain.

Mycoplasma bovis PCR identification (uvrC gene):

upstream primer of SEQ ID No. 1: 5'-TTACGCAAGAGAATGCTTCA-3' the flow of the air in the air conditioner,

downstream primer of SEQ ID No. 2: 5'-TAGGAAAGCACCCTATTGAT-3', respectively;

and (3) PCR system: PCR mix 12.5. mu.L, forward primer 1. mu.L, reverse primer 1. mu. L, DNA 2. mu.L, ddH2O 8.5.5. mu.L, 25. mu.l system; and (3) PCR reaction conditions: at 95 deg.C for 5min, for 35 cycles (95 deg.C for 30s, 52 deg.C for 30s, 72 deg.C for 1min40s), at 72 deg.C for 5min, and at 4 deg.C. The target fragment was amplified by PCR 1626 bp.

The yolk sac inoculation result is shown in table 1, the death time of the chicken embryo of the mycoplasma bovis standard PG45 strain is 1-10 d, the mortality of the chicken embryo is 80% (8/10), and the mortality of the inoculated 7d is 70%; and the death time of the mycoplasma bovis 08M strain chick embryo is 1-12 d, the mortality rate of the chick embryo is 70% (7/10), and the mortality rate of the chick embryo after inoculation for 7d is 50%. The chicken embryo fatality rate (80%) of the standard strain PG45 of Mycoplasma bovis inoculated with yolk sac is higher than that of the 08M strain of Mycoplasma bovis (70%).

The result of allantoic cavity inoculation is shown in Table 2, the death time of the chick embryo of the mycoplasma bovis standard strain PG45 is 1-14 days, and the death rate of the chick embryo is 70% (7/10); and the death time of the mycoplasma bovis 08M chick embryo is 1-7 days, and the chick embryo mortality rate is 30% (3/10). As can be seen, the allantoic cavity inoculation, the chicken embryo fatality rate (70%) of the mycoplasma bovis standard strain PG45 is significantly higher than that (30%) of the mycoplasma bovis 08M strain.

Inoculating allantoic fluid of dead chick embryos into an improved Thiaucotrt's culture medium, wherein the color of the allantoic fluid turns yellow; and (4) performing PCR identification on the allantoic fluid of the dead chick embryos, wherein the PCR is positive.

The death conditions of the chicken embryos of the to-be-detected mycoplasma bovis strain and the standard strain PG45 are statistically analyzed, and the results of the mortality rate of the chicken embryos inoculated by the yolk sac and the mortality rate of the chicken embryos inoculated by the allantoic cavities are integrated, so that the mycoplasma bovis standard strain PG45 and the mycoplasma bovis 08M can kill the chicken embryos, and allantoic fluid of the dead chicken embryos can be separated into mycoplasma bovis pathogens; the lethality of the chicken embryo inoculated by the yolk sac of the mycoplasma bovis standard strain PG45 and the lethality of the chicken embryo inoculated by the allantoic cavity are both higher than that of the mycoplasma bovis 08M strain. The result shows that the toxicity of the mycoplasma bovis standard strain PG45 is stronger than that of the 08M strain, the 08M strain has stronger toxicity, which is consistent with the literature report that the mycoplasma bovis standard strain PG45 has stronger pathogenicity to calves and is stronger than that of a plurality of mycoplasma bovis domestic isolates, and the mycoplasma bovis domestic isolate 08M strain can also cause the calves pathogenic result. The method for determining the virulence of the mycoplasma bovis has the advantages of low cost, simple and convenient operation, cheap and easily obtained experimental animals, good repeatability and the like.

TABLE 1

TABLE 2

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.

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