CN103484414A

CN103484414A - Mycopasma hyopneumoniae strain

Info

Publication number: CN103484414A
Application number: CN201310498958.3A
Authority: CN
Inventors: 赵凯; 刘洪斌; 张凤强; 毛春玲
Original assignee: Heilongjiang University
Current assignee: Heilongjiang University
Priority date: 2013-10-22
Filing date: 2013-10-22
Publication date: 2014-01-01

Abstract

The invention discloses a mycoplasma hyopneumoniae strain which is preserved in the Ordinary Microorganism Center of the Chinese Microorganism Culture Collection and Management Committee which is located in #3, No.1 Yard, Beichen Road, Chaoyang District, Beijing, the preservation number is CGMCC No.8096, and the preservation date is August 15th, 2013. The name of the strain is mycoplasma hyopneumoniae HDZK-Mhp57. According to the mycoplasma hyopneumoniae HDZK-Mhp57, the viable bacteria titer of a prepared acellular culture medium is as high as 1012CCU/mL, the immunogenicity is good, the pneumonia lesion is reduced by more than 90%, the generation cost of an animal biological product is greatly lowered, the stain can be prepared into preventing animal biological products or veterinary medicines, and a foundation for researching and preparing mycoplasma hyopneumoniae inactivated vaccines, genetic engineering vaccines and mycoplasma hyopneumoniae diagnosis kits is laid.

Description

One strain mycoplasma hyopneumoniae bacterial strain

Technical field

The present invention relates to a strain mycoplasma hyopneumoniae bacterial strain.

Background technology

Porcine mycoplasmal pneumonia (Mycoplasmal pneumonia of swine, MPS), claim again swine enzootic pneumonia or epidemic swine pneumonia, MPS is a kind of chronic, contact, non-lethal respiratory system disease, domestic sickness rate reaches more than 80%, this disease can affect swinery production performance and weightening finish and the price of deed, but other transmissible disease of secondary infection again is one of transmissible disease of pig industry priority control.The pathogenic agent of porcine mycoplasmal pneumonia is mycoplasma hyopneumoniae (Mycopasma hyopneumoniae, Mhp), can with health pig, directly contact by sick pig or by aerosol transmission, general propagation distance can reach 4.7km, current research finds that its propagation distance can be as far as 9.2km, therefore, the extremely difficult prevention and control of Mhp.Pig infect after Mhp there will be that expiratory dyspnea, body are become thin, immunosuppression etc.

Mycoplasma hyopneumoniae is moccasin guiding principle, Mycoplasmas, Mycoplasmataceae, Mycoplasma, mycoplasma hyopneumoniae kind.The acellular wall of Mhp, have the height polymorphism, differs in size, and in liquid culture and lung contact, often is spherical, diameter 0.2-0.5 μ m, and, by the coccoid ring-type that is concatenated into, diameter 0.8-20 μ m, occasionally have crescent, long-chain shape and thread.The mycoplasma hyopneumoniae vitro culture requires harsh to growth conditions, nutritional requirement is high and the speed of growth is slow, is subject to the impact of other mycoplasmas (as mycoplasma hyorhinis) of pig in sepn process, causes mycoplasma hyopneumoniae separation screening difficulty larger.

Summary of the invention

The object of the invention is to provide a strain mycoplasma hyopneumoniae bacterial strain.

One strain mycoplasma hyopneumoniae bacterial strain, in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, deposit number is CGMCC No.8096, preservation date is on August 15th, 2013, the preservation address is No. 3, No. 1, Beichen Lu, Chaoyang District, Beijing City institute, its called after mycoplasma hyopneumoniae (Mycopasma hyopneumoniae) HDZK-Mhp57.

Mycoplasma hyopneumoniae (Mycopasma hyopneumoniae) HDZK-Mhp57, its colonial morphology is that typical mycoplasma is decocted pocket egg type bacterium colony; Physiological and biochemical property: the glucose response positive, seminose reacting positive, arginine reaction negative, urease reaction negative, antiserum(antisera) suppress the growth experiment positive, the fluorescence antibody experiment is positive.

The molecular biology identification result of mycoplasma hyopneumoniae (Mycopasma hyopneumoniae) HDZK-Mhp57: the homology of the mycoplasma hyopneumoniae HN0613 of the close kind of announcing in its 16S rRNA sequence and NCBI is 98%, is a new mycoplasma bacterial strain.

The present invention's one strain mycoplasma hyopneumoniae bacterial strain, in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, deposit number is CGMCC No.8096, preservation date is on August 15th, 2013, the preservation address is No. 3, No. 1, Beichen Lu, Chaoyang District, Beijing City institute, its called after mycoplasma hyopneumoniae (Mycopasma hyopneumoniae) HDZK-Mhp57.

Mycoplasma hyopneumoniae in the present invention (Mycopasma hyopneumoniae) HDZK-Mhp57, in the acellular substratum of preparation, the viable bacteria titre is up to 1012CCU/mL; Immunogenicity is good, and pneumonia pathology decrement can reach more than 90%, can greatly reduce the manufacturing cost of veterinary biologics.Mycoplasma hyopneumoniae (Mycopasma hyopneumoniae) HDZK-Mhp57 can be used for being prepared into veterinary biologics or veterinary drug for prevention, for development porcine mycoplasmal pneumonia inactivated vaccine, recombinant vaccine and mycoplasma hyopneumoniae diagnostic kit are laid a good foundation.

The accompanying drawing explanation

Fig. 1 is fluorescent antibody test in embodiment figure as a result.

Embodiment

Embodiment one: present embodiment one strain mycoplasma hyopneumoniae bacterial strain, in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, deposit number is CGMCC No.8096, preservation date is on August 15th, 2013, the preservation address is No. 3, No. 1, Beichen Lu, Chaoyang District, Beijing City institute, its called after mycoplasma hyopneumoniae (Mycopasma hyopneumoniae) HDZK-Mhp57.

A strain mycoplasma hyopneumoniae bacterial strain in present embodiment, its sepn process is as follows:

One, get mycoplasma pneumonia pathology lungs, in super clean bench, with aseptic operation scissors clip site of pathological change edge tissues, put into aseptic plate, pathology lungs tissue is cut into small pieces, inoculation Friis meat soup, 37 ℃ are cultured to the nutrient solution variable color;

Two,, after the nutrient solution variable color, get culture that 0.5mL filters through 0.45 μ m filter and join in the Friis cultured solution of broth that 1.5mL added penicillin, 37 ℃ of cultivations;

Three, go down to posterity week about once, first two weeks will add 50% mycoplasma hyorhinis specific serum in nutrient solution, reaches the 9th week culture is diluted to coated plate, and screening singly falls, and preserves bacterial strain after triplicate.

Identify according to the following steps:

One, microscopy

Friis plating culture, 37 ℃ of cultivations; Picking list bacterium colony smear, carry out respectively gramstaining and Wright's staining, microscopy;

Result:

Pathological tissues is inoculated in the Friis nutrient solution, nutrient solution variable color after 6d; Filter after inoculation nutrient solution after 5-6d and become yellow; After being inoculated into dull and stereotyped 37 ℃ of cultivation 6-7d, the typical mycoplasma of appearance is decocted pocket egg type bacterium colony.

Gramstaining is not found bacterium, and Wright Stain is found the mycoplasma thalline.

Two, biochemical test

Urease test: picking bacterial strain from the Friis flat board, be inoculated in urea medium, 37 ℃ of cultivations, observe after 24h, continues to be cultured to four as negative and do final judgement.

The breakdown of glucose test: picking bacterial strain from the Friis flat board, be inoculated in the liquid nutrient medium containing glucose and cultivate, observe colour-change.

The arginine hydrolysis experiment: picking bacterial strain from the Friis flat board, be inoculated in the liquid nutrient medium containing arginine and phenol red indicator and cultivate, observe colour-change.

The seminose decomposition run: picking bacterial strain from the Friis flat board, be inoculated in the liquid nutrient medium containing seminose and cultivate, observe colour-change.

Result:

The glucose response positive, seminose reacting positive, arginine reaction negative, urease reaction negative.

Three, growth inhibition test

Friis plating culture, be adsorbed in undiluted antiserum(antisera) the filter paper of sterilizing, has the filter paper of serum to be attached at planar surface suction, and 37 ℃ to be cultured to bacterium colony visible; Simultaneously, the filter paper of processing with normal serum is done contrast.

Result:

Have around sero-fast filter paper and formed the inhibition zone that diameter is about 200mm in suction, and the filter paper that normal serum is processed is without inhibition zone.

Four, fluorescent antibody test

Get the nutrient solution smear, the fixing 10min of acetone after seasoning; Afterwards, with PBS gently rinsing for several times, drip the mycoplasma hyopneumoniae fluorescence antibody, put into wet 37 ℃ of senses of box and make 30min, PBS rinses, when half-dried at the fluorescence microscopy Microscopic observation.

Result: as shown in Figure 1, the fluorescent antibody test result is positive.

The molecular biology identification result: the homology of the mycoplasma hyopneumoniae HN0613 of the close kind of announcing in its 16S rRNA sequence and NCBI is 98%, be a new mycoplasma bacterial strain, called after mycoplasma hyopneumoniae (Mycopasma hyopneumoniae) HDZK-Mhp57.

Mycoplasma hyopneumoniae (Mycopasma hyopneumoniae) HDZK-Mhp57, in the acellular substratum of preparation, the viable bacteria titre is up to 10 ¹²cCU/mL; Immunogenicity is good, and pneumonia pathology decrement can reach more than 90%, can greatly reduce the manufacturing cost of veterinary biologics.Mycoplasma hyopneumoniae (Mycopasma hyopneumoniae) HDZK-Mhp57 can be used for being prepared into veterinary biologics or veterinary drug for prevention.

Claims

1. a strain mycoplasma hyopneumoniae bacterial strain, it is characterized in that it is in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, deposit number is CGMCC No.8096, preservation date is on August 15th, 2013, the preservation address is No. 3, No. 1, Beichen Lu, Chaoyang District, Beijing City institute, its called after mycoplasma hyopneumoniae (Mycopasma hyopneumoniae) HDZK-Mhp57.