阅读说明：本技术 一种鸡源火鸡组织滴虫传代致弱株、其制备方法及用途 (Chicken source turkey tissue trichomonas passaged attenuated strain, preparation method and application thereof ) 是由 许金俊 孔令明 戎杰 陈乔光 王子静 禚振男 郭平 曲昌宝 刘丹丹 侯照峰 陶建 于 2019-10-11 设计创作，主要内容包括：本发明涉及生物技术领域一种鸡源火鸡组织滴虫传代致弱株、其制备方法及用途,提供的鸡源火鸡组织滴虫传代人工致弱株A162具有良好的安全性和免疫原性,其由分离自鸡群的虫株经传代致弱、冻存再经虫株复苏,获得的候选活疫苗成本低,使用方便,使用后无药物残留,不产生耐药性,不会对环境造成污染,免疫原性好,使用安全,能有效抵抗105个强毒虫体的感染。相比火鸡来源虫株所制备的疫苗,该疫苗对鸡群的组织滴虫病的预防更为科学和可靠,有望成为药物控制鸡群组织滴虫病的替代手段,从而有效克服该病防治目前面临的巨大瓶颈和困境。(The chicken source turkey tissue infusorium subculture artificial attenuated strain A162 has good safety and immunogenicity, and is prepared by carrying out successive attenuation on insect strains separated from chicken flocks, freezing and then reviving the insect strains. Compared with the vaccine prepared from the turkey source insect strain, the vaccine is more scientific and reliable in preventing the chicken histomoniasis, and is expected to become a substitute means for controlling the chicken histomoniasis by medicaments, so that huge bottlenecks and difficulties in preventing and treating the chicken histomoniasis are effectively overcome.)

1. A preparation method of a chicken source turkey tissue trichomonas passaged attenuated strain is characterized by comprising the following steps:

(1) and (3) weakening by passage: continuously carrying out passage to 162 generations of artificially attenuated turkey histomonas strains obtained by separating turkey histomonas from liver which is infected with histomoniasis and died of illness naturally in an in vitro culture process every 2-3 days;

(2) freezing and storing insect plants: mixing the culture solution, adding 1ml culture solution containing insect strain into each EP tube, and centrifuging at 3000r/min for 5 min; removing supernatant, and adding 1ml of M199 culture medium into each EP tube; uniformly mixing, transferring into freezing storage tubes, adding 100ul of dimethyl sulfoxide into each freezing storage tube, and uniformly mixing; screwing the cover; putting into a precooled gradient cooling box; immediately placing into an ultra-low temperature refrigerator at-80 deg.C; taking out after 12 hours; putting into a freezing storage box and putting into liquid nitrogen;

(3) recovering insect plants: quickly taking out the insect plants to be recovered from the liquid nitrogen, and slightly shaking the insect plants in a constant-temperature water bath kettle at 40 ℃ until the culture medium is melted; quickly transferring the culture medium in the freezing tube to a preheated 9ml culture medium for normal passage, and placing the culture medium in a constant-temperature incubator at 40 ℃ for anaerobic culture; observing whether the culture medium has the growth of the polypide after 36h, if so, carrying out next passage, if not, putting back the culture medium, observing after 20-30min, and selecting the sample with the growth of the polypide as a product.

2. The method for preparing the chicken-origin turkey tissue infusorian passaging attenuated strain according to claim 1, wherein when the strain is attenuated in the step (1), a cell culture bottle is cleaned and placed in an oven for drying; weighing 10mg of rice flour, filling into a culture bottle, wrapping the bottle mouth, and placing in an oven for drying and baking at 165 ℃ for 3-4h for sterilization; packaging with bottle cap, and conventional autoclaving for 30 min; adding 8ml of M199 and 1ml of horse serum into a prepared culture bottle in a super clean bench, covering a bottle cap, placing in a constant temperature incubator at 40 ℃, and preheating a culture medium for 30 min; sucking 1ml of turkey tissue infusorian body in a super clean bench, adding the preheated culture medium, covering a bottle cap, marking, placing in a dryer, sealing with vaseline, exhausting oxygen in the dryer by burning candle, making anaerobic environment, and culturing in a 40 deg.C constant temperature incubator; repeating the operation once every 2-3 days to obtain the process of passage breeding of the low virulent strain.

3. A chicken-derived turkey histomonas passaged attenuated strain characterized by being obtained according to the method of claim 2.

4. The use of the chicken-derived turkey histomonas campestris subcultured attenuated strain according to claim 3, wherein the strain comprises: the application of the vaccine as a vaccine for preventing and treating chicken trichomoniasis.

Technical Field

The invention relates to the technical field of biology, in particular to a chicken source turkey histomonas mellonus passage weakening strain for preventing chicken histomoniasis and a preparation method and application thereof.

Background

Histomoniasis (Histomonosis), also known as cecal hepatitis (Infectious enterohepatitis), is a protozoal disease of fowls of the order galliformes caused by Histomonas meleagris (h.meleagris), and is mainly characterized by liver necrosis, cecal enlargement and thiopard-like feces; the disease birds will have blood circulation disorder in the later period of disease, resulting in purple heads, which is also called Blackhead disease (Blackhead). The disease mainly attacks turkeys, the morbidity and mortality can reach 100 percent, the disease is distributed worldwide, the disease is very serious and common in major countries and regions for raising turkeys in Europe, America and the like, and the disease has great harm to turkey breeding industry. Although the feeding amount of turkeys is not large in China, the prevalence of histomoniasis in poultry flocks, particularly in chicken flocks, is very serious, according to the statistical analysis of poultry cases registered by the outpatient department of Yangzhou university animal hospital between 1 month 2012 and 12 months 2013, the histomoniasis cases account for 13.3% of the total number of the poultry cases to be diagnosed and are positioned at the first outpatient cases of the mass outbreak in the animal hospital; after statistical analysis of 69 cases of histomonas case data which are recorded in detail, local hens are most susceptible in poultry, the number of cases accounts for 88.4% of the total number of cases, local variety three-yellow chickens in Jiangsu area account for 8.7% of the total number of cases, and white feather broilers, caged laying hens and special poultry are few; the disease mainly occurs to poultry which are kept and housed, accounts for 98.6 percent of the total cases, and the disease incidence proportion of the poultry which are kept on the net and raised in cages is lower; in the sick chicken flocks, the morbidity is between 1.6 and 100 percent, the average is 27.4 percent, and the 60.9 percent case mortality is less than 10 percent, and can reach more than 40 percent at most. Although the susceptibility is slightly inferior to that of turkeys, due to the wide existence of the polypide and various transmission media in the external environment and the demonstration application and popularization of the domestic poultry large-scale ecological captive breeding and stocking and other health breeding technologies in recent years, the prevalence of histomoniasis in poultry flocks, particularly chicken flocks, is expected to be more serious, and the damage is also expected to be more serious.

For many years, chemical drugs have been the main means of preventing and treating histomoniasis and other parasitic diseases, and have played a great role. Many chemical drugs have been used for the prevention and treatment of this disease, among which more drugs are quintavalent arsenicals of nitrophenylarsonic acid and nitroimidazoles of dinucleomidazole. The drug resistance of the insects after the drugs are widely applied is not obvious at present, but the drugs still have potential toxicity and carcinogenicity and remain in meat and eggs to cause certain harm to human bodies. Currently, the european union has completely banned all chemical drugs with curative effect on histomoniasis (CEC, 1995, 2002), the united states has banned the use of other chemical drugs besides nitrobenzarsonic acid, and the 193 rd publication of the department of agriculture in china has also listed nitroimidazoles in the list of veterinary drugs and other compounds banned for food animals, which is also the main reason for the current epidemic of the disease. In more than ten years, the use of natural plant extracts to replace chemical drugs has become a hotspot of the research on the prevention and treatment of histomoniasis. However, although a few plant extracts have been shown to have some inhibitory effect on the in vitro reproduction of insect bodies, the results reported to date show that plant extracts have little effect on natural or artificially infected histomoniasis. In conclusion, the control of histomoniasis by using chemical drugs and plant extracts is not an ideal method and means, and the prevention and treatment of histomoniasis is in great trouble at present, so that a safe, efficient and convenient new strategy and a new way for prevention and treatment are urgently needed to be developed.

With the public health concern degree and the continuous expansion of the demand of people on green food, the traditional chemical drug control method for animal parasitic diseases is seriously challenged, the problems of drug residue and drug pollution become important factors for limiting the use and development of drugs, and the development of vaccines becomes the main research direction and development trend of the current parasitic disease control. Parasites are more complex eukaryotes than bacteria and viruses, have a complex life history, most parasites cannot be cultured away from a host under artificial conditions, and the complex, easy variation, camouflage and surface antigen shedding and renewal of parasite antigens often make it difficult for the host to generate protective immune response. Thus, parasite vaccines are not as easily successful as against bacteria and viruses. Due to the unique immunological characteristics of the parasite, the attenuated live vaccine which can imitate natural infection and stimulate the body to generate effective immune response and is safe and reliable has absolute advantages compared with other types of vaccines, is the main attack direction of the current parasite vaccine development, and has reduced toxicity through special animal passage or in vitro culture, thus being the main development trend of the current attenuated live vaccine research.

In the case of turkey histomonas, birds are infected by eating the eggs of the nematode, various transmission media or cloaca that carry the eggs, which are in contact with the excreta of the sick bird. After entering the cecum tissue, the polypide is propagated by adopting a binary fission method and enters the liver to be propagated through the portal blood flow, so that the liver and the cecum tissue are damaged, and the inflammation and necrosis of the liver and the cecum are caused. It has been demonstrated that protective immunity elicited by a worm after infection is similar to that of Eimeria gallinarum, and that cellular immunity is the main and humoral immunity is very small. The cultured inactivated insect antigen is adopted to immunize turkeys, or high-dose IgG is directly injected into the turkeys, which can not provide effective immune protection for the turkeys although the turkeys can produce high IgG level in serum. Therefore, the development of a safe attenuated live vaccine capable of stimulating cellular immunity is the only way to immunoprophylaxis the disease. There are many methods of parasite attenuation in vitro, and reduction of virulence by passage through specific animals or continuous culture in vitro is the most commonly used method of attenuation. In recent years, foreign scholars Liebhart, Sulejmanovic, Nguyen and the like continuously culture turkey tissue infusorium from turkeys in vitro or continuously passage turkey in vivo, the toxicity is obviously reduced, the turkey and turkey are safe and have no side effect, the infection of virulent strains can be effectively resisted after immunization, and the practical feasibility of preventing the tissue trichomoniasis by using attenuated live vaccines is preliminarily proved.

However, the existing turkey histomonas mellifera used for attenuated vaccine development is separated from turkey groups, not from chicken groups, and the susceptibility of different varieties of birds to the turkey histomonas mellifera and the pathogenicity and immunogenicity of insect strains from different varieties have certain differences, so that the insect strains separated from the chicken groups are necessary to prepare the attenuated vaccine.

Disclosure of Invention

The invention aims to provide a turkey histomonas mellonus attenuated strain separated from sick chicken flocks, and a preparation method and application thereof, and aims to solve the problems that a turkey histomonas mellonus field strain has strong pathogenicity and is not suitable for being directly used for preparing vaccines and the existing turkey strain is not matched with the chicken flocks.

The purpose of the invention is realized as follows: a preparation method of a chicken source turkey tissue trichomonas passaged attenuated strain comprises the following steps:

(1) and (3) weakening by passage: continuously carrying out passage to 162 generations of artificially attenuated turkey histomonas strains obtained by separating turkey histomonas from liver which is infected with histomoniasis and died of illness naturally in an in vitro culture process every 2-3 days;

(2) freezing and storing insect plants: mixing the culture solution, adding 1ml culture solution containing insect strain into each EP tube, and centrifuging at 3000r/min for 5 min; removing supernatant, and adding 1ml of M199 culture medium into each EP tube; uniformly mixing, transferring into freezing storage tubes, adding 100ul of dimethyl sulfoxide into each freezing storage tube, and uniformly mixing; screwing the cover; putting into a precooled gradient cooling box; immediately placing into an ultra-low temperature refrigerator at-80 deg.C; taking out after 12 hours; putting into a freezing storage box and putting into liquid nitrogen;

(3) recovering insect plants: quickly taking out the insect plants to be recovered from the liquid nitrogen, and slightly shaking the insect plants in a constant-temperature water bath kettle at 40 ℃ until the culture medium is melted; quickly transferring the culture medium in the freezing tube to a preheated 9ml culture medium for normal passage, and placing the culture medium in a constant-temperature incubator at 40 ℃ for anaerobic culture; observing whether the culture medium has the growth of the polypide after 36h, if so, carrying out next passage, if not, putting back the culture medium, observing after 20-30min, and selecting the sample with the growth of the polypide as a product.

When the passage is weakened in the step (1), cleaning a cell culture bottle, and drying the cell culture bottle in an oven; weighing 10mg of rice flour, filling into a culture bottle, wrapping the bottle mouth, and placing in an oven for drying and baking at 165 ℃ for 3-4h for sterilization; packaging with bottle cap, and conventional autoclaving for 30 min; adding 8ml of M199 and 1ml of horse serum into a prepared culture bottle in a super clean bench, covering a bottle cap, placing in a constant temperature incubator at 40 ℃, and preheating a culture medium for 30 min; sucking 1ml of turkey tissue infusorian body in a super clean bench, adding the preheated culture medium, covering a bottle cap, marking, placing in a dryer, sealing with vaseline, exhausting oxygen in the dryer by burning candle, making anaerobic environment, and culturing in a 40 deg.C constant temperature incubator; repeating the operation once every 2-3 days to obtain the process of passage breeding of the low virulent strain.

The tissue infusorian attenuated strain of the chicken-origin turkey has the application as a vaccine for preventing and treating chicken group trichomoniasis.

The chicken source turkey tissue infusorian passage artificial weakening strain A162 provided by the invention has good safety and immunogenicity, is a passage weakening candidate live vaccine prepared from the infusorian strain separated from chicken flocks, has the advantages of low cost, convenient use, no drug residue after use, no drug resistance, no environmental pollution, good immunogenicity, safe use and effective resistance to 10⁵Infection of a virulent insect. Compared with the vaccine prepared from the turkey source insect strain, the vaccine is more scientific and reliable in preventing the chicken histomoniasis, and is expected to become a substitute means for controlling the chicken histomoniasis by medicaments, so that huge bottlenecks and difficulties in preventing and treating the chicken histomoniasis are effectively overcome.

Drawings

FIG. 1 is a flow chart of the present invention.

FIG. 2 is an enlarged view of the prepared chicken-derived turkey histomonas mellons attenuated live vaccine (magnification × 400).

FIG. 3 shows the group A of attenuated immunogobies (left cecum right liver).

FIG. 4 shows the group B of the non-immunized aggressor control group (left cecum right liver).

FIG. 5 shows group C of the non-immunized non-attacked control group.

Detailed Description