CN110819684B - Method for comparing virulence of mycoplasma capricolum goat pneumonia subspecies strain - Google Patents
Method for comparing virulence of mycoplasma capricolum goat pneumonia subspecies strain Download PDFInfo
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Abstract
The invention discloses a method for simply comparing the virulence of mycoplasma capricolum subsp pneumoniae strains. The method of the invention inoculates the resuscitated mycoplasma capricolum pneumonia subspecies of the goat with equal concentration on the chick embryo, then the inoculated chick embryo is placed in a constant temperature box for culturing, the chick embryo state and death condition are detected at regular time, and the chick embryo lethality rate of each strain is counted, so as to carry out comparison and judgment. The method has the advantages of simple operation, low cost, no need of complex animal feeding conditions, easy availability of experimental animals, short time consumption, good repeatability and the like, greatly reduces the workload of animal experiments, and avoids the defects of expensive price, large individual difference, high feeding requirement, complex experimental operation, long experimental period, large workload and the like of the experimental animal goats in the prior art.
Description
Technical Field
The invention relates to a method for comparing certain properties of different strains of a same kind of microorganism, in particular to a method for simply comparing the virulence of mycoplasma capricolum subsp pneumoniae strains.
Background
Contagious caprine pleuropneumonia (CCPP) is an animal epidemic that seriously harms the sheep industry, is currently prevalent mainly in Asia and Africa, and is an OIE official report epidemic. The pathogeny of the disease is mycoplasma capricolum pneumonia subspecies of goats, mainly infects goats and wild goats, and causes clinical symptoms such as fever, cough, dyspnea and the like. The research and the preparation of the mycoplasma capricolum subspecies pneumonia vaccine of the goat need to compare the virulence of different strains so as to obtain the result with the best immune effect. The virulence is also required to be measured in the research of the pathogenic mechanism and virulence gene of the mycoplasma capricolum subsp pneumonia of goats.
At present, the toxicity of mycoplasma capricolum pneumonia subspecies of goat is mainly determined by experimental animal goat, and the pathogenicity of the strain and the toxicity of the comparative strain are determined by the goat infection test. Strains with strong toxicity and immunogenicity are screened to be used as vaccine candidate strains. It is also often necessary to determine the virulence of mutant and wild strains in research on the excavation and identification of virulence genes of mycoplasma capricolum subsp pneumoniae. However, the experimental animal goat has the defects of high price, high feeding requirement, complex test operation, large workload and the like. Therefore, it is necessary to establish a simple method for comparing the virulence of mycoplasma capricolum and pneumonia subspecies of goat.
Disclosure of Invention
The invention aims to provide a method for simply and comparatively analyzing the virulence of different mycoplasma capricolum subsp pneumoniae strains.
A method for simply comparing the virulence of mycoplasma capricolum and pneumonia subspecies is characterized by that the resuscitated mycoplasma capricolum and pneumonia subspecies are inoculated on the chick embryo, then the inoculated chick embryo is placed in a constant temp. box to culture, the state and death condition of the chick embryo are regularly detected, and the chick embryo lethality rate of every strain is calculated, so that the comparison and judgement can be made.
The method can be used for comparing the virulence of the mycoplasma capricolum subsp pneumonia strain of the goat, and the specific method comprises the following steps:
(1) SPF chick embryo preparation: preparing healthy SPF (specific pathogen free) chick embryos of 7-8 days old, wherein the chick embryos are required to be good in development, clear in blood vessels and vigorous in activity, and discarding the clear eggs, dead embryos and weak embryos.
(2) Preparing bacterial liquid: resuscitating each mycoplasma capricolum subspecies pneumonia of goat to be comparatively analyzed, culturing in a constant-temperature incubator at 37 ℃, and waiting to cultureThe color of the base turns yellow (or the pH value is reduced to 6.8 or below), fresh bacteria liquid is centrifuged at 8000rpm for 15min, then the fresh bacteria liquid is resuspended by normal saline, and the titer of viable bacteria is adjusted to 10 7 CCU/ml。
(3) Inoculation and observation of chick embryos: the prepared mycoplasma capricolum goat pneumonia subspecies liquid is inoculated to the egg yolk sac of the chick embryo, and each strain is inoculated with 10 SPF chick embryos of 0.2 ml/egg. Inoculating 10 eggs of the negative control group of chick embryo yolk sac with 0.2ml of normal saline per egg; 10 chicken embryos of the blank control group are not treated; after inoculation, the incubator is continuously used for culturing until the chick embryos are 21 days old, the chick embryos are observed for 1 time every day, and the state and death condition of the chick embryos are observed and recorded.
(4) Statistical analysis and result judgment: and (4) counting the chick embryo lethality rate of each strain, comparing and judging. The method can be applied to screening or preparing the mycoplasma capricolum subspecies pneumonia vaccine. The method can also be applied to the research of mining and identifying the virulent genes of the mycoplasma capricolum subspecies pneumonia of goats.
The beneficial effects of the invention are as follows: the method has the advantages of simple operation, low cost, no need of complex animal feeding conditions, easy availability of experimental animals, short time consumption, good repeatability and the like, greatly reduces the workload of animal experiments, and avoids the defects of expensive price, large individual difference, high feeding requirement, complex experiment operation, long experiment period, large workload and the like of the experimental animal goats in the prior art. Therefore, the method can be used for distinguishing the virulence of different mycoplasma capricolum subspecies pneumonia isolates, can also be used for screening strains in vaccine development, and can be used for mining and identifying and researching the virulence genes of the mycoplasma capricolum subspecies pneumonia of the goat.
Detailed Description
Example 1:
the invention is illustrated below with reference to examples.
The method for comparing the virulence of the mycoplasma capricolum subsp pneumoniae strain comprises the following steps:
(1) preparation of SPF chick embryo: a7-8 day-old healthy SPF chick embryo is prepared, and an SPF hatching egg is purchased from Shandong Hao Tai laboratory animal Breeding company Limited and is incubated in an incubator (the temperature is 37.0-37.5 ℃, and the humidity is 50%). And selecting the well-developed chick embryos with clear blood vessels and vigorous activity for inoculation, and discarding the eggs without sperm, dead embryos and weak embryos.
(2) Preparing mycoplasma capricolum and pneumonia subspecies liquid: resuscitating mycoplasma capricolum goat pneumonia subspecies F38 strain (England type bacterial culture Collection NCTC, number NCTC10192) and M1801 strain (preservation of Lanzhou veterinary institute of Chinese academy of agricultural sciences), respectively inoculating improved Thiaucort's culture medium (MTB culture medium), culturing in a constant temperature incubator at 37 deg.C until the color of the culture medium turns yellow (or pH value decreases to 6.8 or below), centrifuging at 8000rpm for 15min with fresh bacterial liquid, resuspending with physiological saline, and adjusting viable bacteria titer to 10 7 CCU/ml。
(3) Inoculating and observing chick embryos, and judging statistical results: inoculating prepared mycoplasma capricolum goat pneumonia subspecies bacterial liquid with equal concentration into chick embryo yolk sacs, inoculating 10 SPF chick embryos per strain with 0.2ml per strain, simultaneously setting a negative control group (inoculated with physiological saline, 0.2ml per strain and 10 strains) and a blank control group (10 strains without any treatment), after inoculation, continuously incubating in an incubator, observing the chick embryos every day, observing the survival state of the chick embryos, judging whether blood vessels are free or fuzzy, observing the death condition of the chick embryos, and recording. None of the negative control and blank control chick embryos died. Chick embryo lethality was counted and compared for each strain. Dead chick embryos are subjected to autopsy and observed, the dead chick embryos are mainly shown as hyperemia and hemorrhage, and no obvious lesion appears in the negative control group and the blank control group. Collecting allantoic fluid of dead chick embryos, inoculating improved Thiaucotrt's culture medium, and determining the growth condition of mycoplasma by observing the color change of the culture medium; performing PCR identification on the mycoplasma capricolum subspecies pneumonia of the goat on the allantoic fluid of the dead chick embryo.
The PCR identification method of mycoplasma capricolum and pneumonia subspecies of goat comprises the following steps:
upstream primer of SEQ ID No. 1: 5'-ATCATTTTTAATCCCTTCAAG-3' the flow of the air in the air conditioner,
downstream primer/5'-TACTATGAGTAATTATAATATATGCAA-3' of SEQ ID No. 2;
and (3) PCR system: PCR mix 12.5. mu. L, ddH 2 O8.5. mu.L, 1. mu.L of each of the forward primer and the reverse primer, and 2. mu.L of allantoic fluid DNA; and (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 5min, (95 ℃ 30s, 47 ℃ 30s, 72 ℃ 25s)35 cycles, 72 ℃ for 10min, 4 ℃ storage. The target fragment 316bp was amplified by PCR.
The results are shown in Table 1:
as can be seen, the death time of the chicken embryo of the mycoplasma capricolum subsp pneumoniae F38 strain is within 7d, and the mortality rate of the chicken embryo is 30% (3/10); and all the chick embryos inoculated with the M1801 strain die after 4d of inoculation, and the mortality rate of the chick embryos is 100% (10/10). The chicken embryo lethality rate of the mycoplasma capricolum pneumonia subspecies M1801 strain is obviously higher than that of the F38 strain. Inoculating allantoic fluid of dead chick embryos into an improved Thiaucotrt's culture medium, wherein the color of the allantoic fluid turns yellow; the allantoic fluid of the dead chick embryo is subjected to PCR identification, and mycoplasma capricolum subspecies pneumonia pathogens of goats can be detected. The result shows that mycoplasma capricolum pneumonia subspecies can infect lethal chick embryo; from the chicken embryo lethal result, the toxicity of the M1801 strain of the mycoplasma capricolum pneumonia of the goat is stronger than that of the F38 strain, which is consistent with the observed toxicity of the goat of the experimental animal. The method has the advantages of simple operation, low cost, no need of complex animal feeding conditions, easy obtainment of experimental animals, short time consumption, good repeatability, small workload and the like.
TABLE 1
Finally, 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
<110> Lanzhou veterinary research institute of Chinese academy of agricultural sciences
<120> method for comparing virulence of mycoplasma capricolum subspecies pneumonia strain
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> Artificial sequence upstream Primer (PR)
<400> 1
atcattttta atcccttcaa g 21
<210> 2
<211> 27
<212> DNA
<213> Artificial sequence downstream Primer (PF)
<400> 2
tactatgagt aattataata tatgcaa 27
Claims (3)
1. A method for comparing the virulence of mycoplasma capricolum and goat pneumonia subspecies is characterized in that the mycoplasma capricolum and goat pneumonia subspecies which are revived in equal concentration and are to be compared and analyzed are inoculated on a chick embryo, the inoculated chick embryo is placed in a constant temperature box for culturing, the chick embryo state and death condition are detected at regular time, the chick embryo mortality of each strain is counted, and comparison and judgment are carried out according to the chick embryo lethality;
the specific method comprises the following steps:
(1) preparation of SPF chick embryo: preparing healthy SPF (specific pathogen free) chick embryos of 7-8 days old, wherein the chick embryos are required to be good in development, clear in blood vessels and vigorous in activity, and clear eggs, dead embryos and weak embryos are removed;
(2) preparing bacterial liquid: resuscitating each mycoplasma capricolum pneumonia subspecies strain to be compared and analyzed, culturing in a constant temperature incubator at 37 deg.C, centrifuging at 8000rpm for fresh bacteria liquid when the color of the culture medium turns yellow or the pH value is reduced to below 6.8, resuspending with normal saline, and adjusting viable bacteria titer to 10 7 CCU/ml；
(3) Inoculation and observation of chick embryos: inoculating the prepared mycoplasma capricolum goat pneumonia subspecies liquid into the egg yolk sac of the chick, inoculating each strain into SPF chick according to 0.2 ml/piece, continuously culturing the chick in an incubator until the chick is 21 days old, observing the chick for 1 time every day, and observing and recording the state and death condition of the chick;
(4) statistical analysis and result judgment: and (4) counting the chick embryo lethality rate of each strain, comparing and judging.
2. Use of the method of claim 1 in the screening or preparation of a mycoplasma capricolum subsp pneumoniae vaccine strain.
3. Use of the method of claim 1 in research on the excavation and identification of virulence genes of mycoplasma capricolum subsp pneumoniae.
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| CN106798919A (en) * | 2017-01-23 | 2017-06-06 | 六安市绿丰牧业科技有限公司 | A kind of goat immune method |
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| CN106798919A (en) * | 2017-01-23 | 2017-06-06 | 六安市绿丰牧业科技有限公司 | A kind of goat immune method |
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