Mycoplasma gallisepticum virulent strain and application thereof

Abstract

The present application relates to a Mycoplasma gallisepticum isolated, identified and the establishment of animal infection model. Through the MG separation, identification and purification of the chicken flock with respiratory disease in Yantai, Shandong province, finally named as MG SD strain. Unlike the past air or continuous way of infection, using the MG pure culture to infect 28 and 56 day-old SPF chickens by tracheal inoculation, 80% of the SPF chickens appear pneumatocele 10 or 7 days after infection, and through 5 times continuous dilution of MG SD strain, 10 5.9 CCU 50 The dose of / chickens to 56 day-old SPF chickens by tracheal infection, 7 days after infection, the incidence of pneumatocele is still 80%, which shows that the strain has strong pathogenicity.

Description

[0001] SPECIFICATION TECHNICAL FIELD

[0002] The present application belongs to the field of biotechnology, and particularly relates to isolation and identification of Mycoplasma galliscepticum and establishment of an animal infection model. BACKGROUND

[0003] Mycoplasma galliscepticum MG-SD mainly causes chronic respiratory disease (CRD) in chickens and infectious sinusitis in turkeys, and the clinical symptoms include sneezing, coughing, nasal discharge, respiratory rale and air sac inflammation, and suborbital sinus swelling in turkeys. MG often causes mixed infection with other pathogens, such as infectious bronchitis virus, Newcastle disease virus and Escherichia coli, aggravates the symptoms of other respiratory diseases and promotes the spread of pathogens, resulting in more serious respiratory symptoms and economic losses.

[0004] At present, the inactivated vaccine CR strain and the live vaccine F-36 strain of Mycoplasma galliscepticum used in China are introduced from abroad, and there is still a big difference from the currently prevalent strains in China from the perspective of genetic typing and genetic evolution, and the vaccine culture titer (color change unit, CCU) is low. The CR virulent strain used for vaccine efficacy test is sprayed in the isolator, and the spraying operation is complex, the size of the sprayed droplets is strictly required, and fresh bacterial liquid needs to be sprayed. This method often causes failure of the challenge due to the non-compliance of the CCU of the fresh bacterial liquid with the relevant regulations, the poor size and uniformity of the droplets, etc., so that the vaccine cannot pass the quality test at one time. In addition, the vaccine efficacy is calculated by air sac score to determine whether the challenge is effective, and the result may still have the possibility of severe disease. Other scholars have proposed air sac challenge, multiple continuous injection and appropriate concentration of ammonia water stimulation assisted challenge, which all have problems of time-consuming, laborious, poor consistency, more resource occupation, etc. Therefore, it is urgent to establish a challenge model with simple operation, intuitive and reliable evaluation and stable challenge for the vaccine efficacy test of MG. SUMMARY

[0005] The present application aims to provide a Mycoplasma galliscepticum strain, and isolation and identification of the Mycoplasma galliscepticum strain and establishment of an animal infection model.

[0006] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0007] A strain of MG with stable genetic, high number of viable bacteria after culture, and specific genotype in China, named MG SD strain, was obtained by isolating, identifying and purifying MG from trachea and throat swabs of chicken infected with MG. The genotype of the strain is ST97. The strain has been preserved in China Center for Type Culture Collection on January 23, 2024, with the preservation number of CCTCC NO:V202408, and the address of China Center for Type Culture Collection is Wuhan, Wuhan University, China. The strain is classified and named as Mycoplasma galliscepticum MG-SD.

[0008] The construction method of the above-mentioned Mycoplasma galliscepticum comprises the following steps:

[0009] (1) Taking sick chickens suspected of being infected with MG from a chicken flock, sterilely collecting tracheal swabs and throat swabs, placing the sampling swabs in sterile mycoplasma culture medium, filtering through a 0.45 μm filter, and then culturing at 37°C for 18-20 hours. Then, inoculate fresh mycoplasma liquid culture medium at a volume ratio of 1:5, and then place it in a 37°C incubator. When the culture medium changes from rose red to orange or yellow, it is suspected that mycoplasma is growing and PCR detection is performed. Repeat the above steps to inoculate fresh liquid culture medium at a ratio of 1:10, and continuously pass it 3 times;

[0010] (2) Identifying the culture suspected of growing mycoplasma using MG-specific primers and MS-specific primers;

[0011] (3) Dilute the culture that has been passed 3 times blindly and identified as MG positive by 10 3 and 10 4 times, evenly spread the diluted bacterial solution on mycoplasma solid culture medium, and incubate at 37°C for 24-96 hours. Then, pick a single colony on mycoplasma liquid culture medium for culture. After the culture medium turns yellow, identify it by PCR. After the correct identification of MG culture, dilute and spread it on mycoplasma solid culture medium. After 3 consecutive cloning, the purified MG is named SD strain, and the culture is expanded in mycoplasma liquid culture medium. The culture is recorded as P1 generation.

[0012] Using P1 generation culture of SD strain at a dose of 10 7.0 CCU 50 / chicken, 28-day-old SPF chickens were challenged by tracheal and eye drop methods. After tracheal challenge, obvious tracheal pathological damage was observed, and 80% of the SPF chickens developed air sacculitis 10 days after challenge.

[0013] Using P1 generation culture of MG SD strain at a dose of 10 7.0 CCU 50 / chicken, 56-day-old SPF chickens were challenged by tracheal challenge. Seven days after challenge, the incidence of air sacculitis reached 80%.7.0 CCU 50 The incidence of air sacculitis is only 20% at 7 days after infection.

[0014] By 5 times of continuous dilution, 10 5.9 CCU 50 The incidence of air sacculitis is still 80% at 7 days after infection.

[0015] The advantages of the present application are that:

[0016] The present application successfully isolates a domestic unique genotype, strong virulence Mycoplasma galliscepticum MG-SD from Yantai, Shandong, and establishes a simple and easy-to-operate MG animal artificial infection model, which is necessary for further research of MG, development of effective prevention and control strategies, especially in the current antibiotic reduction action, more attention should be paid to the establishment of new, reliable and effective animal infection model for evaluating the effectiveness of drugs or prevention and control technology. Therefore, the Mycoplasma galliscepticum MG-SD strain isolated in the present application has high pathogenicity, and the Mycoplasma galliscepticum MG-SD strain is inoculated into 28 and 56-day-old SPF chickens by tracheal infection, which can cause severe air sacculitis, and has a high incidence of at least 80%, and the minimum infection dose is 10 5.9 CCU 50 / chicken, and the established infection model can be used as a strong virulent strain for MG vaccine potency test or effectiveness evaluation of other MG prevention and control schemes, and the MG SD strain has high titer and pathogenicity, and can also be used as a candidate strain for inactivated vaccine. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The CCU of MG is determined by using a 96-well plate; wherein the dilution degrees from left to right are 10 50 , 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 and negative control.

[0018] Figure 2The air sacculitis lesions of 28-day-old SPF chickens challenged with Mycoplasma galliscepticum MG-SD strain by tracheal infection.

[0019] Figure 3 The tracheal pathological damage of 28-day-old SPF chickens challenged with Mycoplasma galliscepticum MG-SD strain by tracheal infection. DETAILED DESCRIPTION

[0020] Mycoplasma galliscepticum MG-SD infected chicken flocks were subjected to MG pathogen isolation, purification and identification using mycoplasma liquid and solid culture media, and 28 and 56-day-old SPF chicken artificial infection models were established to determine the minimum challenge dose.

[0021] Example 1 Isolation, purification and identification of the strain

[0022] The sick chickens from a suspected MG infection flock in a large-scale chicken farm in Yantai, Shandong were taken, and the tracheal swabs and throat swabs were collected aseptically. The sampling swabs were placed in sterile mycoplasma culture medium, filtered through a 0.45 μm filter, and then placed in a 37°C incubator for 18-20 h. Fresh mycoplasma liquid medium was inoculated at a volume ratio of 1:5, and placed in a 37°C incubator. When the culture medium changed from rose red to orange or yellow, it was considered to be suspected mycoplasma growth and subjected to PCR detection. The above steps were repeated to inoculate fresh liquid medium at a ratio of 1:10, and the culture was continuously passaged for 3 times.

[0023] The test sample or culture was subjected to DNA extraction according to the instructions of the bacterial genomic DNA extraction kit, and the suspected mycoplasma growth culture was identified using Mycoplasma galliscepticum MG-SD specific primers MG-14F (5'-GAG CTA ATC TGT AAA CTT GGT C-3') / MG-13R (5'-GCT TCC TTG CGG TTA GCA AC) and MS specific primers MS-F (5'-GAG AAG CAA AAT AGT GAT ATC A-3') / MS-R (5'-CAG TCG TCT CCG AAG TTA ACA A-3') according to the national industry standard “Avian Mycoplasma PCR Detection Method” in 2015. The PCR product sizes were 185 bp and 207 bp, respectively.

[0024] The culture that was passaged for 3 generations and was positive for Mycoplasma galliscepticum MG-SD by PCR was subjected to 10 3 and 10 4The diluted bacterial solution was evenly spread on the mycoplasma solid culture medium. After culturing at 37℃ for 24-96h, a single colony was picked and inoculated into the mycoplasma liquid culture medium for culture. After the culture medium turned yellow, it was identified by PCR. The correctly identified MG culture was diluted and spread on the mycoplasma solid culture medium. After cloning for three times, the purified Mycoplasma gallisceptum MG-SD was named SD strain, expanded in the mycoplasma liquid culture medium, and the culture was recorded as P1 generation and stored at -70℃ for future use.

[0025] After the MG strain storage solution stored at -70℃ was melted and balanced to room temperature, it was diluted 10-fold to 10 with mycoplasma liquid culture medium. -9 100 μL of diluted bacterial solution was added to 96-well plates, and 8 replicates were set up. The plates were placed in a 37°C incubator and cultured for 10 days. When the negative control did not change color (red), but the diluted bacterial solution changed color (turned yellow or orange), it was determined to be positive. CCU was calculated according to the Reed-Muench method. 50 After repeated determination, the number of colonies was 10 9.3 CCU 50 / ml( Figure 1 ).

[0026] Example 2 Culture Characteristics and Typing of Mycoplasma galliscepticum MG-SD Strain

[0027] The MG-SD strain of Mycoplasma galliscepticum requires nutrients such as nicotinamide adenine dinucleotide and porcine serum for growth, and its optimal culture temperature is 37°C. It grows well in different batches of mycoplasma liquid culture media. After culturing at 37°C for 2-5 days, the pH drops to 6.8-7.0 and the culture is harvested. The culture may appear slightly turbid. The viable count of cultures inoculated with different batches of culture media is greater than 10 8 CCU 50 / mL, up to 10 9.5 CCU 50 / mL. When the SD strain is inoculated with mycoplasma solid culture medium and cultured in a 37°C incubator for 2–5 days, “navel-shaped” or “fried egg-shaped” colonies may be seen.

[0028] The 7 housekeeping genes in the literature: atpG, plsC, mraW, ugpA, DUF3196, lgT, dppC and PCR method were used to amplify and sequence the housekeeping genes. The housekeeping gene sequencing results were uploaded to the PubMLST website (http: / / pubmlst.org / mgallisepticum / ), and the corresponding ST type was assigned, indicating that the SD strain was ST97, a genotype unique to China.

[0029] Example 3 Pathogenicity of Mycoplasma galliscepticum MG-SD strain to 28-day-old SPF chickens

[0030] Mycoplasma galliscepticum MG-SD strain was used to infect 28-day-old SPF chickens by tracheal or eye drop methods, and the differences in pathogenicity were compared, and a 28-day-old infection model was established. 45 28-day-old SPF chickens were randomly divided into 3 groups, 15 chickens in each group. The P1 generation culture of Mycoplasma galliscepticum MG-SD strain with CCU 50 determined was diluted with sterile medium to an appropriate concentration. The G1 group was injected with 0.2 mL of bacterial solution (containing 10 7 CCU 50 ) per trachea, the G2 group was attacked by eye drop with 0.1 mL of bacterial solution (containing 10 7 CCU 50 ) per eye, and the G3 group was not attacked as a negative control. After the attack, the clinical symptoms of the chickens were observed daily, mainly the respiratory symptoms, and the incidence was recorded. On the 10th, 20th and 30th day after the attack, 5 SPF chickens were randomly killed in each group to observe the clinical and pathological changes such as air sacculitis.

[0031] After the attack, no obvious respiratory symptoms were observed in the chickens of each group, and the main clinical manifestation after necropsy was air sacculitis, and no other symptoms such as foot pad swelling, sternal cyst, etc. On the 10th, 20th and 30th day after the attack, the air sacculitis incidence of the G1 group was 4 / 5 (80%), 3 / 5 (60%), and 4 / 5 (80%), respectively, and the air sacs were significantly thickened and had a large amount of yellow cheese-like material. No obvious air sacculitis was observed in the G2 and G3 groups. Figure 2 Histopathological observation of tracheal tissue showed that the tracheal mucosa layer was thickened and lymphocytes were obviously infiltrated on the 10th, 20th and 30th day after the tracheal attack, while eye drop attack only caused slight thickening of the tracheal mucosa layer in each of the 20 and 30 SPF chickens after the attack, and the control group did not have any tracheal pathological damage on the 10th, 20th and 30th day after the attack. Figure 3

[0032] ​Example 4 Establishment of artificial infection model of Mycoplasma galliscepticum MG-SD strain on 56-day-old SPF chickens

[0033] The CCU 50 of Mycoplasma galliscepticum MG-SD strain was determined, and the pathogenicity difference was compared between the strain and the CR strain, a virulent strain for efficacy test of MG vaccine specified in the current Chinese Veterinary Pharmacopoeia, by tracheal challenge infection of 56-day-old SPF chickens to establish a suitable challenge infection model.

[0034] Fifty 56-day-old SPF chickens were divided into five groups, 10 chickens in each group. The P1 generation MG SD strain culture with determined CCU 50 was diluted with mycoplasma liquid medium, and 0.3 mL of MG SD strain bacterial solution (containing 10 7.0 CCU 50 ) was injected into the trachea of each chicken in G1, 0.3 mL of MG CR strain bacterial solution (10 9.0 CCU 50 ) was injected into the trachea of each chicken in G2, 0.3 mL of MG CR strain bacterial solution (10 8.0 CCU 50 ) was injected into the trachea of each chicken in G3, 0.3 mL of MG CR strain bacterial solution (10 7.0 CCU 50 ) was injected into the trachea of each chicken in G4, and G5 was not challenged as a negative control. After challenge, the clinical symptoms of chickens were observed daily, mainly respiratory symptoms, and the incidence was recorded. All chickens were killed on the 7th day after challenge, and clinical symptoms such as air sacculitis were observed.

[0035] No obvious respiratory symptoms were observed in chickens on the 7th day after challenge, and the main performance was air sacculitis. On the 7th day after challenge, the air sacculitis incidence rates of G1 and G2 were 8 / 10 and 10 / 10, respectively, the air sacs were significantly thickened, and there was a large amount of yellow cheese-like substance, the air sacculitis incidence rates of G3 and G4 were 8 / 10 and 2 / 10, respectively, and only a small amount of foam and a small amount of yellow cheese-like substance appeared in the air sacs. No air sacculitis lesions were observed in G5.

[0036] Example 5 Determination of minimum challenge dose

[0037] The CCU 50 of MG SD strain bacterial solution was determined, and the pathogenicity difference was compared between the strain and the CR strain, a virulent strain for efficacy test of MG vaccine specified in the current Chinese Veterinary Pharmacopoeia, by tracheal challenge infection of 56-day-old SPF chickens to establish a suitable challenge infection model.

[0038] Sixty 56-day-old SPF chickens were divided into six groups, 10 chickens in each group. The CCU 50The P1 generation MG SD strain culture was diluted 5 times in series using mycoplasma liquid culture medium. Each feather in the G1 group was injected into the trachea with 0.3 mL of Mycoplasma gallisepticum MG-SD bacterial solution (containing 10 8.0 CCU 50 ), and in group G2, 0.3 mL of Mycoplasma galliscepticum MG-SD solution (10 7.3 CCU 50 ), 0.3 mL of Mycoplasma galliscepticum MG-SD solution was injected into each feather trachea in group G3 (10 6.6 CCU 50 ), and in group G4, 0.3 mL of Mycoplasma galliscepticum MG-SD solution (10 5.9 CCU 50 ), and in group G5, 0.3 mL of Mycoplasma galliscepticum MG-SD solution (10 5.2 CCU 50 ), and Group G6 was not challenged and served as a negative control. After challenge, chickens were observed daily for clinical symptoms, primarily respiratory symptoms, and the incidence of disease was recorded. All chickens were sacrificed on day 7 after challenge, and clinical symptoms such as air sac inflammation were observed and air sac scoring was performed.

[0039] No obvious respiratory symptoms were observed in any group 7 days after challenge. Autopsies of the challenged groups primarily showed air sac inflammation. On day 7 after challenge, the air sac inflammation incidence in Group G1 was 10 / 10 (100%), with significant thickening of the thoracic and abdominal air sacs and the presence of a large amount of yellow, cheesy material. The highest average air sac score was 5.6. The air sac inflammation incidence in Groups G2, G3, and G4 was 8 / 10 (80%), with the air sac score gradually decreasing. Some birds had air sac inflammation in the thoracic or abdominal air sacs. The incidence of air sac inflammation in Group G5 was only 5 / 10 (50%), and no air sac inflammation lesions were observed in Group G6. Specific results are shown in Table 1.

[0040] Table 1 Air sac inflammation lesions after tracheal infection with different dilutions of MG SD strain

[0041]

[0042] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A Mycoplasma gallisepticum MG-SD strain characterized in that, The Mycoplasma gallisepticum MG-SD has been preserved in the China Center for Type Culture Collection on January 23, 2024, and the preservation number is CCTCC NO: V202408.

Images