CN108251338B

CN108251338B - Mycoplasma hyorhinis virulent strain and application thereof

Info

Publication number: CN108251338B
Application number: CN201810198100.8A
Authority: CN
Inventors: 熊祺琰; 王佳; 邵国青; 冯志新; 华利忠; 韦艳娜; 甘源; 吴猛; 刘蓓蓓; 武昱孜
Original assignee: Jiangsu Academy of Agricultural Sciences
Current assignee: Jiangsu Academy of Agricultural Sciences
Priority date: 2018-03-12
Filing date: 2018-03-12
Publication date: 2021-01-29
Anticipated expiration: 2038-03-12
Also published as: CN108251338A

Abstract

The invention provides a mycoplasma hyorhinis virulent strain and application thereof, and relates to the technical field of microorganisms. The virulent strain of mycoplasma hyorhinis is mycoplasma hyorhinis (Mycoplasma hyorhinis)Mycoplasma hyorhinis) HEF16 strain with the preservation number of CCTCC NO: M2017462. The invention also provides application of the mycoplasma hyorhinis in establishing a mycoplasma hyorhinis challenge morbidity model, and by adopting pure culture of the virulent strain for challenge, various mycoplasma hyorhinis infection typical symptoms including multiple serositis and arthritis can be induced, the virulent strain is strong, the morbidity is fast, typical clinical manifestations can appear in 7 days, the morbidity situation is serious, the mycoplasma hyorhinis can be attacked in different background pigs, and the virulent strain can be used for establishing the mycoplasma hyorhinis morbidity model with the typical symptoms and is representative. The invention also provides the application of the mycoplasma hyorhinis in preparation of the inactivated vaccine, the strain has good immunogenicity, and the prepared inactivated vaccine has good virus attack protection efficacy.

Description

Mycoplasma hyorhinis virulent strain and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a mycoplasma hyorhinis virulent strain and application thereof.

Background

Mycoplasma hyorhinis (M.) (Mycoplasma hyorhinis) A member belonging to Mycoplasma genus (Mycoplasma) belonging to Mycoplasmataceae was originally isolated from the nasal cavity of infectious atrophic rhinitis pigs in 1953 by Carler and Mckay, and was designated Mycoplasma hyorhinis. Mycoplasma hyorhinis is a common pathogenic bacterium in clinical pig farms, is usually transmitted to piglets from sows or big pigs, and can be generally transmitted by upper respiratory infection through droplets or direct contact. Once a pig is infected, the mycoplasma spreads rapidly in the upper respiratory tract and can be isolated from the lungs and nasopharynx of infected pigs, and then can travel through the respiratory tract to the whole body. The mycoplasma hyorhinis can cause diseases such as porcine multiple serositis, arthritis, otitis, pneumonia and the like, wherein the multiple serositis and the arthritis are the most common diseases. The clinical infection rate can reach more than 60-70% in different countries and regions. Meanwhile, the mycoplasma hyorhinis can also form mixed infection with other pathogenic bacteria, so that the incidence and the severity of diseases are aggravated. In recent years, the research finds that the mycoplasma hyorhinis belongs to the pathogen of zoonosis, and the infection of the mycoplasma hyorhinis has obvious correlation with various human cancers. Furthermore, mycoplasma hyorhinis is also one of the most common mycoplasma causing culture contamination in cell culture.

At present, the control of the mycoplasma hyorhinis only depends on antibiotics, and no vaccine for marketization is available. The separation of virulent virus and the establishment of a pathogenesis model are the prerequisites for vaccine development, and the establishment of a pathogenesis model with typical symptoms and stability becomes an urgent need. However, the disclosed mycoplasma hyorhinis isolate is mainly pulmonary disease, does not meet the clinical typical morbidity characteristics of a pig farm, and is not representative; or the toxicity is not strong, the toxicity is attacked by adopting culture or cell tissue with higher titer, the disease onset is lighter, the disease onset time is later, or the disease onset can be realized by using colostrum-free pigs.

Disclosure of Invention

The invention aims to provide a mycoplasma hyorhinis virulent strain which can induce a plurality of mycoplasma hyorhinis including multiple serositis and arthritis to infect typical diseases, has strong toxicity, fast onset of disease, can show typical clinical manifestations within 7 days, has serious onset conditions, can be attacked on pigs with different backgrounds, can be used for establishing a mycoplasma hyorhinis pathogenesis model with the typical diseases, accords with clinical pathogenesis characteristics of a pig farm, and is representative.

The invention also aims to provide application of the mycoplasma hyorhinis virulent strain in establishing a mycoplasma hyorhinis challenge pathogenesis model, the virulent strain is attacked by adopting a pure culture of the virulent strain, the pathogenesis is fast, typical clinical manifestations can appear within 7 days, the pathogenesis is serious, the virulent strain can be attacked on pigs with different backgrounds, and various mycoplasma hyorhinis infection typical symptoms including multiple serositis and arthritis can be induced.

Still another object of the present invention is to provide a mycoplasma hyorhinis inactivated vaccine which provides good immunostimulation ability and protective efficacy against virus.

The purpose of the invention is realized by adopting the following technical scheme.

Virulent strain of swine rhinomycoplasma, and classified name is swine rhinomycoplasma (A)Mycoplasma hyorhinis) HEF16 strain, deposited in China center for type culture Collection with a preservation number of CCTCC NO: M2017462.

The specific sources of the strain are as follows: the pig herd in a certain pig farm of Anhui Hefei finds suspected mycoplasma hyorhinis infection symptoms such as joint swelling, lameness, depression, inappetence and the like, collects joint cavity liquid, inoculates a KM2 culture medium, is separated from the joint cavity liquid, and is identified as mycoplasma hyorhinis through 16 SrDNAPR sequencing. The pure culture of the strain is directly inoculated to a healthy negative pig, the pig has typical clinical symptoms within one week after virus attack, multiple serositis, arthritis and other related pathological changes can be observed by a caesarean section, mycoplasma hyorhinis is separated again from the pig, and the virus attack and re-separation strain is named as mycoplasma hyorhinis HEF16 strain.

The virulent strain has two advantages, namely, the virulent strain can induce various typical mycoplasma hyorhinis clinical symptoms including multiple serositis and arthritis and is more than other reported mycoplasma hyorhinis strains inducing pneumonia and otitisThe clinical morbidity characteristics of the pig farm are met, and the established morbidity model is more representative; secondly, the vaccine has strong toxicity, serious morbidity and early morbidity time, can be attacked in pigs with different backgrounds, can induce pathological changes in ordinary growing pigs, and is closer to clinic in toxicity attacking compared with some reports that CDCD (not eating colostrum during caesarean delivery) pigs are used. Simultaneously, only 10 pigs are needed for combating poison⁸A pure culture of CCU is immediate, and is more pure and requires lower titers than some reports using infected cell cultures. These characteristics are all favorable for the establishment and wide application of artificial morbidity models.

In the invention, the culture medium adopted by the mycoplasma hyorhinis virulent strain culture is KM2 culture medium, and the culture titer can reach 10 bacterial liquid per milliliter⁸-10¹¹CCU。

The invention provides application of the mycoplasma hyorhinis virulent strain in establishing a mycoplasma hyorhinis challenge morbidity model. The mycoplasma hyorhinis challenge pathogenesis model is used for vaccine efficacy test. In the preferred technical scheme, the virulent strain pure culture of the mycoplasma hyorhinis is adopted for virus attack. The concentration of the mycoplasma hyorhinis virulent strain pure culture is at least 10⁷CCU/ml。

In the invention, the virulent strain of mycoplasma hyorhinis is directly inoculated by using a strain culture, and can be inoculated by various ways, including nasal drip, abdominal cavity, intrapulmonary and intratracheal injection. Acute symptoms such as rough hair, fever, depression, inappetence, joint swelling, lameness or difficulty in walking, dyspnea, abdominal tenderness and the like can appear 3 to 7 days after inoculation, and even dying or death phenomenon can appear, after 14 days, the acute symptoms are generally relieved, but some pigs can continue to deteriorate or die.

The invention also claims the application of the mycoplasma hyorhinis virulent strain in the preparation of the mycoplasma hyorhinis inactivated vaccine and the inactivated vaccine taking the inactivated mycoplasma hyorhinis virulent strain as an active component. The inactivated vaccine also comprises an adjuvant. The adjuvant is oil emulsion adjuvant or water-based adjuvant. In a preferred technical scheme, the adjuvant is white oil.

In the invention, the mycoplasma hyorhinis virulent strain is used for preparing inactivated vaccinesIn the process, the inactivated culture is directly used without concentration, the process is simple, and the cost is low. When immunizing, the inoculation dose is 10⁸-10⁹Each head of CCU can be immunized once or twice according to different adjuvants and animal species, and the interval period of the two immunizations is 2-3 weeks generally.

The mycoplasma hyorhinis virulent strain can induce various mycoplasma hyorhinis including multiple serositis and arthritis to infect typical diseases, has strong toxicity and rapid onset of disease, can show typical clinical manifestations within 7 days, has serious onset conditions, can be attacked on pigs with different backgrounds, can be used for establishing a mycoplasma hyorhinis pathogenesis model with the typical diseases, accords with clinical pathogenesis characteristics of a pig farm, and is representative. The inactivated vaccine prepared by the mycoplasma hyorhinis virulent strain can provide good immune stimulation capability and virus attack protection efficacy.

Drawings

FIG. 1 shows the colony morphology of Mycoplasma hyorhinis strain HEF16 on solid plates.

FIG. 2 is a scanning electron micrograph of Mycoplasma hyorhinis strain HEF 16.

FIG. 3 shows the status of the sick pig after challenge with Mycoplasma hyorhinis strain HEF 16.

FIG. 4 shows the arthritic symptoms after challenge with Mycoplasma hyorhinis HEF16 strain. (A) Joint swelling; (B) effusion in joint cavity.

FIG. 5 shows the symptoms of multiple serositis after challenge with Mycoplasma hyorhinis HEF16 strain. (a) pleurisy; (B) pericarditis; (C) peritonitis.

Detailed Description

The KM2 culture medium is prepared by the following method: collecting 5000ml of eagle's solution (containing MEM48.47g, L-glutamine 1.5 g), 24.00g NaCl, 6.00g KCl, 8.695g Na₂HPO₄、0.60gKH₂PO₄、0.30gCaCl₂、0.30gMgCl₂50.00g of hydrolyzed milk protein, 50.00g of Yeast Extract, 2000ml of healthy pig serum, 100-200 ten thousand IU penicillin potassium salt, 125ml of 1 percent thallium acetate solution and 16.67ml of 0.4 percent phenol red aqueous solution are mixed uniformly, then 1N NaOH solution is used for correcting the pH value to 7.4-7.6, and redistilled water is used for fixing the volume to 10000 ml.

Example 1: isolation, culture and identification of Mycoplasma hyorhinis strain HEF16

A certain pig farm swinery of Anhui Hefei discovers suspected mycoplasma hyorhinis infection symptoms such as joint swelling, lameness, depression, inappetence and the like, collects joint cavity liquid, inoculates a KM2 culture medium, separates a mycoplasma strain from the joint cavity liquid, and carries out 16SrDNAPCR sequencing. The sequence is shown as SEQ ID NO. 1, and through homology comparison, the homology with the mycoplasma hyorhinis sequence is up to 99 percent, and the mycoplasma hyorhinis is identified.

The mycoplasma separated is inoculated to a healthy negative pig through a nasal drip, abdominal cavity or trachea way, the pig has typical clinical symptoms within one week after challenge, multiple serositis and arthritis related pathological changes can be observed through a caesarean section, the mycoplasma hyorhinis is separated from the mycoplasma hyorhinis again, and the PCR sequencing result of the 16SrDNA is the same as that of SEQ ID NO. 1. The reisolated strain was then subjected to 6-gene MLST typing (Trueb B et al, Veterinary Microbiology, 2016, 191: 9-14), which was identical to the ST type of the challenge strain. The results prove that the strain is a virulent strain of mycoplasma hyorhinis. The challenge reisolated strain was named as Mycoplasma hyorhinis HEF16 strain and used as a seed virus.

The mycoplasma hyorhinis HEF16 strain can form a typical 'fried egg' type colony when cultured on a solid culture medium (figure 1), and the growth titer in a liquid culture medium is 10⁸-10¹¹CCU/ml. The scanning electron microscope shows that the spherical. A suspected secretory vesicle structure with a diameter of about 50nm was occasionally observed around the bacterial cells (FIG. 2).

The culture medium adopted by the mycoplasma hyorhinis HEF16 strain is KM2, the mycoplasma hyorhinis HEF16 strain is cultured for 1-3 days at 37 ℃, and the culture titer can reach 10 bacterial liquid per milliliter⁸-10¹¹CCU。

Strain preservation is carried out on the mycoplasma hyorhinis HEF16 strain, and the preservation information is as follows:

and (3) classification and naming: mycoplasma hyorhinis strain HEF16

Mycoplasma hyorhinisStrain HEF 16;

the preservation unit: china center for type culture Collection;

address: wuhan, Wuhan university; the preservation date is as follows: 31/8/2017;

the preservation number is: CCTCC NO: M2017462.

Example 2: challenge morbidity test 1 for mycoplasma hyorhinis strain HEF16

Animals: the three-way hybrid pig which is delivered aseptically and does not eat colostrum is raised to 3 months old, and is detected to be healthy as a challenge animal. The group with the toxin being attacked is 5, and the group with the toxin being attacked is 5.

Toxic materials: the mycoplasma hyorhinis HEF16 strain is cultured in KM2 culture medium at 37 deg.C for 1-3 days for resuscitation, and is used after 2-3 generations.

Counteracting toxic substances: taking a fresh culture of the mycoplasma hyorhinis HEF16 strain, and adjusting the titer to 10⁷CCU/mL, 5mL each, 10 mL total, by nasal and intrapulmonary injection⁸Each head of the CCU.

Disease occurrence judgment standard: clinical symptoms are observed after toxin attack, pathological change conditions are observed after 21-28 days of autopsy, and the disease can be judged to be the onset of disease according to any one of the following two items: (1) the swine rhinomycoplasma infection causes death. (2) The mycoplasma hyorhinis has obvious clinical symptoms and autopsy lesions, namely rough hair, mild fever, depression, inappetence and joint clinical symptoms after infection, wherein the symptoms last for at least 3 days, and the joint clinical symptoms refer to lameness or joint swelling; multiple serositis (pericarditis, pleuritis, peritonitis) or arthritic lesions can be seen by caesarean section.

Clinical and anatomical observations: the HEF16 virus challenge group has all diseases, the control group has no abnormal clinical manifestations, and no pathological changes are found by autopsy. The pathogenesis of the HEF16 strain challenge group (see figures 3-5) is as follows: the pigs subjected to the toxicity attacking test have rough hair, mild fever, depression, inappetence, difficulty in walking and joint swelling 2-3 days after infection, and the clinical symptoms are more than 3 days. The test pigs are obviously thinned after 14 days of infection, 2 pigs are killed in advance in an endangered state, the symptoms of the rest pigs are stable after 14 days, the disease course is not obviously aggravated, and the autopsy is carried out for 21 days. The autopsy result shows that joint swelling and joint fluid increase are mainly seen, and a part of pigs are accompanied with yellow suppurative foci; the pericardium is thickened and adhered, the thoracic cavity is adhered with cellulosic exudation, part of the abdominal cavity of the pig is adhered, and the surfaces of the liver, the spleen and the intestinal tract have milky cellulosic exudation, namely, the multiple serositis and the arthritis lesion are simultaneously generated.

The results show that obvious clinical and caesarean change can appear after the strain HEF16 is detoxified, the pig has quick disease occurrence, clinical manifestations can appear within 7 days, the caesarean lesion is typical, and the disease occurrence is serious. The above experimental results show that the strain HEF16 is a virulent strain.

Example 3: challenge morbidity test 2 for mycoplasma hyorhinis strain HEF16

Animals: the two-element hybrid pig which is delivered aseptically and does not eat colostrum is raised to 2 months old, and is detected to be healthy as a challenge animal. The group with the toxin being attacked is 5, and the group with the toxin being attacked is 5.

Counteracting toxic substances: a fresh culture of Mycoplasma hyorhinis HEF16 strain (the culture method is the same as that in example 2) was taken, and the titer was adjusted to 10⁷CCU/mL, 5mL each, 10 mL total, intratracheal and intraperitoneal routes⁸Each head of the CCU.

Disease occurrence judgment standard: as above.

Clinical and anatomical observations: after 3 days of toxin attack, the body temperature begins to rise, poor appetite, joint swelling, lameness, joint palpation pain, rough hair and depression begin to appear on the 4 th day, and the clinical symptoms last for more than 3 days. In the second week after the challenge, the test pigs in the challenge group had difficulty in moving, emaciation and 3 deaths; after the third week of toxin attack, the appetite of the toxin attack group is moderately recovered, and the acute symptoms are relieved. 21 days after the toxin attack, the multiple serositis and arthritis typical pathological changes appear as follows: the pleural cavity is seriously adhered and contains a small amount of pleural effusion, the pericardium is thickened, pericardial effusion exists, a large amount of cellulose on the surface of the heart seeps out, and a large amount of effusion and a suppurative focus exist in the joint cavity. The mycoplasma hyorhinis in the focus effusion or the tissue is PCR positive, and the strain is successfully separated. Therefore, the toxic group had all the diseases. The control group had no abnormal clinical manifestations and no pathological changes by autopsy. As can be seen from the results, after the strain HEF16 is detoxified, the pig has a rapid onset of disease, and the typical clinical manifestations and the severe cases of the disease can appear within 7 days. The above experimental results show that the strain HEF16 is a virulent strain.

Example 4: challenge morbidity test 3 for mycoplasma hyorhinis strain HEF16

Animals: 4-month-old healthy Bama pigs 10 pigs were randomly grouped, and 5 pigs in the challenge group and 5 pigs in the control group.

Counteracting toxic substances: a fresh culture of Mycoplasma hyorhinis HEF16 strain (the culture method is the same as that in example 2) was taken, and the titer was 10⁷CCU/mL, 5mL each, 10 mL total, by nasal and intraperitoneal routes⁸Each head of the CCU.

Disease occurrence judgment standard: as above.

Clinical and anatomical observations: pigs in the toxin counteracting group begin to have clinical manifestations of rough hair, mild fever, mental depression, appetite reduction, mild lameness, reluctance to walk, joint swelling, poor mental status and the like 3 days after toxin counteracting, and last for more than 3 days. After 28 days of toxin attack, the lung and the chest are obviously adhered, the lung lobes are adhered, the pericardium is slightly thickened, a large amount of effusion and a suppurative focus exist in the joint cavity, namely, multiple serositis (pericarditis, pleuritis and peritonitis) and arthritis lesion appear simultaneously; the joint swab and the nose swab detect the positive of the mycoplasma hyorhinis, the strain is successfully separated, and the serum antibody turns positive. Therefore, the toxic group had all the diseases. The control group had no abnormal clinical manifestations and no pathological changes by autopsy.

As can be seen from the results, after the strain HEF16 is detoxified, the pig has a rapid onset of disease, and the typical clinical manifestations and the severe cases of the disease can appear within 7 days. From the challenge experiments of examples 2-4, it can be found that the HEF16 strain can be attacked in pigs with different backgrounds after challenge, and the HEF16 strain is a virulent strain.

Example 5: immunity protection test of porcine mycoplasma rhinotracheale HEF16 strain inactivated vaccine

Preparing a vaccine: taking a fresh culture of the mycoplasma hyorhinis HEF16 strain, and adjusting the thallus concentration to 10⁹Adding formaldehyde into CCU/ml for inactivation treatment to obtain inactivated antigen solution. Adopting a conventional method to mix the inactivated antigen solution and the white oil according to the volume ratio of 1: 3, mixing and emulsifying to obtain the mycoplasma hyorhinis HEF16 strain inactivated vaccine.

Animals: 30 healthy ternary hybrid pigs of 7-21 days old are randomly divided into 3 groups: the G1 group is a healthy control group and is not immune; the G2 group is a vaccine immunization group, 2 mL of the inactivated vaccine of the mycoplasma hyorhinis HEF16 strain is injected into each head muscle, the immunization is performed once after 2 weeks, and the immunization dosage method is the same as the first time; g3 group for counteracting toxic pathogenControl group, no immunization. G1 group animals did not attack virus, G2 group animals attacked virus one and a half month after the first immunization, and 5ml of each fresh culture of Mycoplasma hyorhinis (10 ml) was inoculated by nasal drip and intraperitoneal injection⁸CCU per head), time, method and dose of challenge in group G3 were the same as in group G2.

Evaluation of vaccine protection: counting the number of pathogenic animals after attacking according to pathogenic standards, and calculating the attacking protection rate according to the following formula: the protection rate of offensive toxin = (number of offensive animal-number of diseased animal)/number of offensive animal x 100%.

As a result: animals in the control group which were attacked after the attack of the toxin showed acute clinical symptoms, all pigs suffered from the disease, and all pigs had typical pathological changes of multiple serositis and arthritis by the caesarean section, of which 3 were dead; and only 2 animals in the immune group have disease symptoms, no animal is dead, and the protection rate reaches 80 percent. See in particular the table below.

Group of	Total number of animals	Number of onset of disease	Number of dead heads	Survival rate (%)	Protective Rate (%)
						G1	10	0	0	100	-
G2	10	2	0	100%	80%
						G3	10	10	3	70%	-

SEQUENCE LISTING

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Claims

1. Virulent strain of swine rhinomycoplasma, and classified name is swine rhinomycoplasma (A)Mycoplasma hyorhinis) HEF16 strain with the preservation number of CCTCC NO: M2017462.

2. The use of a virulent strain of mycoplasma hyorhinis as claimed in claim 1 for establishing a model of mycoplasma hyorhinis attack pathogenesis.

3. The use according to claim 2, wherein said model of mycoplasma hyorhinis challenge is used in a vaccine efficacy test.

4. Use according to claim 2 or 3, characterized in that: the virulent strain of mycoplasma hyorhinis of claim 1 is used for challenge.

5. The use according to claim 4, wherein: the concentration of the mycoplasma hyorhinis virulent strain pure culture is at least 10⁷CCU/ml。

6. The use of a virulent strain of mycoplasma hyorhinis as claimed in claim 1 in the preparation of an inactivated vaccine against mycoplasma hyorhinis.

7. An inactivated vaccine of Mycoplasma hyorhinis, wherein the active ingredient is the inactivated virulent strain of Mycoplasma hyorhinis of claim 1.

8. The inactivated vaccine for Mycoplasma hyorhinis according to claim 7, further comprising an adjuvant.

9. The inactivated vaccine for mycoplasma hyorhinis according to claim 8, wherein the adjuvant is an oil emulsion adjuvant or an aqueous adjuvant.

10. The inactivated vaccine for mycoplasma hyorhinis according to claim 9, wherein the adjuvant is white oil.