阅读说明：本技术 一种兔球虫病活疫苗及其制备方法和应用 (Rabbit coccidiosis live vaccine and preparation method and application thereof ) 是由 索勋 刘贤勇 索静霞 李超 顾小龙 汤新明 汪运舟 崔玉娟 陶鸽如 王丹 廖琴 于 2019-11-12 设计创作，主要内容包括：本发明涉及动物球虫病疫苗技术领域,具体涉及一种兔球虫病活疫苗及其制备方法和应用。本发明提供一种兔球虫病活疫苗组合物,其包含大型艾美耳球虫、肠艾美耳球虫和中型艾美耳球虫的早熟致弱虫株以及穿孔艾美耳球虫的早熟致弱虫株。本发明提供的兔球虫病活疫苗同时具有较高的免疫保护效力和安全性,能够有效防治兔球虫病,减少或杜绝抗球虫药物的使用,同时显著降低疫苗接种对于兔体重增重等生长和生产性能的不利影响,具有良好的经济效益和广阔的应用前景。本发明还提供球虫早熟致弱虫株的选育方法,该方法能够有效缩短早熟虫株的选育周期,提高球虫早熟致弱虫株的选育效率,获得的早熟虫株的遗传特性稳定、致病性显著降低、免疫原性良好。(The invention relates to the technical field of animal coccidiosis vaccines, in particular to a rabbit coccidiosis live vaccine and a preparation method and application thereof. The invention provides a rabbit coccidiosis live vaccine composition, which comprises Eimeria macrotype, Eimeria intestinalis and Eimeria intermedia precocious attenuated strains and Eimeria perforated precocious attenuated strains. The rabbit coccidiosis live vaccine provided by the invention has higher immune protection efficacy and safety, can effectively prevent and treat rabbit coccidiosis, reduces or eliminates the use of anti-coccidiosis drugs, and simultaneously obviously reduces the adverse effects of vaccination on growth and production performances of rabbits such as weight gain and the like, thereby having good economic benefits and wide application prospects. The invention also provides a breeding method of the coccidian prematurity attenuated strain, the breeding method can effectively shorten the breeding cycle of the coccidian prematurity attenuated strain, improve the breeding efficiency of the coccidian prematurity attenuated strain, and the obtained prematurity insect strain has stable genetic characteristic, obviously reduced pathogenicity and good immunogenicity.)

1. A rabbit coccidiosis live vaccine composition, which comprises at least 3 precocious attenuated strains of highly pathogenic rabbit eimeria and low pathogenic rabbit eimeria; the low-pathogenicity rabbit Eimeria tenella is a wild worm strain and/or a precocious attenuated worm strain.

2. The live rabbit coccidiosis vaccine composition of claim 1, wherein the oocyst numbers of the highly pathogenic rabbit eimeria praecox strain and the less pathogenic rabbit eimeria coccidiosis in the live rabbit coccidiosis vaccine composition are in a ratio of 1: (2-20).

3. The rabbit coccidiosis live vaccine composition of claim 1 or 2, wherein the low pathogenic rabbit eimeria comprises one or more of eimeria minutiae, eimeria perforata, eimeria vimentis;

preferably, the precocious attenuated strains of highly pathogenic rabbit eimeria comprise precocious attenuated strains of eimeria macrogola, eimeria intestinalis and eimeria intermedia; the low pathogenic E.rabbit comprises perforated E..

4. The rabbit coccidiosis live vaccine composition according to any one of claims 1 to 3, which comprises the following components: 100-3000 oocysts per head part of precocious attenuated strain of large Eimeria, 500-5000 oocysts per head part of precocious attenuated strain of intestinal Eimeria, 500-6000 oocysts per head part of precocious attenuated strain of medium Eimeria, and 1500-40000 oocysts per head part of wild Eimeria strain or precocious attenuated strain of perforated Eimeria.

5. The rabbit coccidiosis live vaccine composition according to any one of claims 1 to 4, which comprises the following components: 500-3000 oocysts per head part of precocious attenuated strain of large Eimeria, 1000-5000 oocysts per head part of precocious attenuated strain of intestinal Eimeria, 1000-5000 oocysts per head part of precocious attenuated strain of medium Eimeria, and 3000-30000 oocysts per head part of wild Eimeria strain or precocious attenuated strain of perforated Eimeria.

6. The method for preparing the rabbit coccidiosis live vaccine composition of any one of claims 1 to 5, which comprises the breeding of a prematurity attenuated strain; the breeding of the early-maturing attenuated insect plants adopts a classical early-maturing pressure breeding method or a breeding method comprising the following steps: orally inoculating the rabbit to be attenuated coccidia strain, and orally inoculating the rabbit with the high-dose coccidia strain to be attenuated once or more for 12-72 hours after the first inoculation, and harvesting oocysts discharged earliest for passage amplification;

preferably, the first inoculation is followed by the second inoculation, and the time interval between the second inoculations is 12-60 hours; the high dose is 5-10 times of the immunization dose of the first inoculation.

7. The preparation method of claim 6, wherein the re-oral inoculation once or more times with the high-dose coccidia strain to be attenuated is re-inoculation twice within 12-72 hours and 24-84 hours after the first inoculation respectively;

preferably, the breeding method of the early-maturing attenuated insect strain comprises the following steps: the coccidia to be attenuated is added at 1.0X 10⁵～2.0×10⁵Inoculating dose of sporulated oocysts/rabbits to the rabbits orally, and inoculating the to-be-attenuated coccidian strains at 5.0 × 10 hours after the first inoculation⁵～1.0×10⁶The sporulated oocysts/rabbits were inoculated once more orally; then the first inoculation is carried out 24-84 hours later, the coccidia strain to be attenuated is treated at the ratio of 1.0 multiplied by 10⁶～2.0×10⁶The sporulated oocysts/rabbit were inoculated once orally at the inoculation dose and the earliest shed oocysts were harvested for passage expansion.

8. Use of the rabbit coccidiosis live vaccine composition of any one of claims 1-5 or the rabbit coccidiosis live vaccine composition prepared by the preparation method of claim 6 or 7 in preparation of a preparation for preventing or treating rabbit coccidiosis.

9. A breeding method of a premature and weak coccidia strain is characterized by comprising the following steps: orally inoculating the rabbit to be attenuated coccidia strain, and orally inoculating the rabbit with the high-dose coccidia strain to be attenuated once or more for 12-72 hours after the first inoculation, and harvesting oocysts discharged earliest for passage amplification;

preferably, the first inoculation is followed by the second inoculation, and the time interval between the second inoculations is 12-60 hours; the high dose is 5-10 times of the immunization dose of the first inoculation.

10. The breeding method of the coccidia precocious attenuated insect strain as claimed in claim 9, wherein the re-oral inoculation with high dose of the to-be attenuated coccidia strain is one or more times of re-inoculation for 12-72 hours and 24-84 hours after the first inoculation;

preferably, the breeding method comprises: the coccidia to be attenuated is added at 1.0X 10⁵～2.0×10⁵The sporulated oocyst/rabbit inoculation dose orally inoculates rabbits 12-7 after the first inoculationThe coccidia to be attenuated strain is treated at 5.0X 10 in 2 hours⁵～1.0×10⁶The sporulated oocysts/rabbits were inoculated once more orally; then the first inoculation is carried out 24-84 hours later, the coccidia strain to be attenuated is treated at the ratio of 1.0 multiplied by 10⁶～2.0×10⁶The sporulated oocysts/rabbit were inoculated once orally at the inoculation dose and the earliest shed oocysts were harvested for passage expansion.

Technical Field

The invention relates to the technical field of animal coccidiosis vaccines, in particular to a rabbit coccidiosis live vaccine and a preparation method and application thereof.

Background

Antibiotics and anticoccidial drugs have a long history as livestock and poultry feed additives, and are main means for guaranteeing the health of livestock and poultry in an intensive breeding mode. With the frequent occurrence of drug residues and drug-resistant strains (worms) and the threat to the environment and human health, the strategy of preventing and controlling animal epidemic diseases by using drugs gradually exposes the disadvantages. Outbreaks of coccidiosis in rabbits can cause massive rabbit death; chronic and subclinical coccidial infections lead to reduced animal productivity and reduced economic benefits. The continuous occurrence of coccidiosis is the main reason for the poor economic benefit and even loss of many rabbit farms. Therefore, under the dilemma that the use of anticoccidial drugs is limited and the drug resistance of coccidia frequently occurs, so that the prevention and control effects of the drugs are poor, the development of highly effective and safe rabbit coccidiosis vaccines is urgent.

The pathogeny of rabbit coccidiosis is 11 kinds of coccidiosis in Eimeria, which parasitizes on different parts of intestinal tract or liver, and the pathogenicity and prevalence rate of different kinds of coccidiosis are different, thus the harm to rabbit industry is different. Among them, Eimeria macrotype, Eimeria mesotype, Eimeria enterobacter and Eimeria punctata are the main species of infection and prevalence in rabbit farm, and the first three species have strong pathogenicity, cause severe diarrhea, and seriously affect productivity and economic benefits in rabbit industry. Therefore, the development of a rabbit coccidiosis vaccine must achieve two main goals: firstly, the pathogenic coccidian species can be well protected; secondly, the safety is high. The vaccine taking the live oocysts as the components can effectively resist the re-infection of the same coccidia and has great advantages in the prevention of coccidiosis of chickens and turkeys. Therefore, the live vaccine aiming at the epidemic coccidian species is developed for preventing the rabbit coccidiosis, the use of the anti-coccidian drug can be obviously reduced, the anti-coccidian drug is not used, and the food safety, the environment and the economic benefit are effectively improved, so that the application prospect is very wide.

Disclosure of Invention

In order to solve the problems in the prior art, the invention aims to provide a rabbit coccidiosis attenuated live vaccine with excellent safety and immune protection efficacy and a method for efficiently and quickly breeding a coccidiosis praecox attenuated strain.

In order to achieve the purpose, the technical scheme of the invention is as follows:

although the live oocysts of the rabbit coccidia are used as the immunogen of the vaccine, the immune protective efficacy of the vaccine is more favorably improved, but the risk of adverse effects on the growth and production of animals is higher, so that the technical problem in the development process of the rabbit coccidia vaccine is how to improve the safety of the live vaccine while ensuring the immune protective efficacy of the rabbit coccidia vaccine. In the research of rabbit coccidian vaccines, highly pathogenic species are mostly utilized for inoculation to obtain the immunity of corresponding species, and low-pathogenic or basically nonpathogenic rabbit coccidian is usually ignored. Furthermore, in order to improve the preparation efficiency of the vaccine, the invention develops a rapid breeding method of the early-maturing attenuated insect strain on the basis of the traditional early-maturing pressure breeding method, effectively shortens the passage and screening period of the early-maturing attenuated insect strain, and obtains the early-maturing attenuated insect strain with stable genetic characteristic, high safety and good immunogenicity.

Specifically, the technical scheme of the invention is as follows:

the invention provides a rabbit coccidiosis live vaccine composition, which comprises at least 3 precocious attenuated strains of highly pathogenic rabbit Eimeria and low pathogenic rabbit Eimeria; the low-pathogenicity rabbit coccidia is a wild insect strain and/or a prematurity attenuated insect strain.

Preferably, in the rabbit coccidiosis live vaccine composition, the ratio of the number of oocysts of the precocious attenuated strain of the highly pathogenic rabbit eimeria and the low pathogenic rabbit eimeria is 1: (2-20). Preferably 1: (5-20). The content proportion in the range is compounded for use, so that the safety and the immune protection efficacy of the vaccine can be better ensured at the same time.

Preferably, the low-pathogenicity rabbit coccidia adopts a precocious attenuated strain.

Preferably, the low pathogenic rabbit eimeria comprises one or more of eimeria minutissima, eimeria perforata, eimeria vimentis.

Among the numerous highly pathogenic and less pathogenic rabbit coccidia, the present inventors have found that the perforated Eimeria is most effective in improving the safety of the large Eimeria, intestinal Eimeria and intermediate Eimeria component vaccines. Thus, in the rabbit coccidiosis live vaccine composition, the highly pathogenic prawned attenuated strains of rabbit eimeria coccidia preferably include prawned attenuated strains of eimeria macrolepia, eimeria intestinalis, and eimeria mellifera; the low pathogenic E.rabbit preferably comprises perforated E..

On the basis of meeting the content ratio of the components, in order to better ensure the safety and the immune protection efficacy of the vaccine at the same time, the rabbit coccidiosis live vaccine composition preferably comprises the following components: 100-3000 oocysts per head part of precocious attenuated strain of large Eimeria, 500-5000 oocysts per head part of precocious attenuated strain of intestinal Eimeria, 500-6000 oocysts per head part of precocious attenuated strain of medium Eimeria, and 1500-40000 oocysts per head part of wild Eimeria strain or precocious attenuated strain of perforated Eimeria.

More preferably, the rabbit coccidiosis live vaccine composition comprises the following components: 500-3000 oocysts per head part of precocious attenuated strain of large Eimeria, 1000-5000 oocysts per head part of precocious attenuated strain of intestinal Eimeria, 1000-5000 oocysts per head part of precocious attenuated strain of medium Eimeria, and 3000-30000 oocysts per head part of wild Eimeria strain or precocious attenuated strain of perforated Eimeria.

In order to expand the immune protection range of the vaccine, the rabbit coccidiosis live vaccine composition can comprise a plurality of high-pathogenic coccidia and low-pathogenic coccidia, thereby forming a multivalent coccidia vaccine.

As a preferred embodiment of the present invention, the present invention provides a tetravalent rabbit coccidia live vaccine composition consisting of a Prenylated strain of Eimeria macrotype, Eimeria intestinalis and Eimeria intermedia and a Periplaneta praecox strain of Eimeria punctata. The immunogen composition of the rabbit coccidiosis live vaccine composition is as follows: 1000-3000 oocysts per head part of precocious attenuated strain of large Eimeria, 1000-4000 oocysts per head part of precocious attenuated strain of intestinal Eimeria, 2000-5000 oocysts per head part of precocious attenuated strain of medium Eimeria, and 3000-30000 oocysts per head part of wild Eimeria strain or precocious attenuated strain of perforated Eimeria. Wherein, the ratio of the number of oocysts of the E.perforatum to the number of oocysts of E.macrogol, E.enterobacteriaceae and E.intermedia is preferably (5-20): 1.

the rabbit coccidiosis live vaccine has the following immunization modes: 20-50 day old rabbits were inoculated orally.

Further, the invention provides a preparation method of the rabbit coccidiosis live vaccine composition, which comprises the breeding of prematurity attenuated strains; the breeding of the early-maturing attenuated insect plants adopts a classical early-maturing pressure breeding method or a breeding method comprising the following steps: and (3) orally inoculating the rabbit with the to-be-attenuated coccidia strain, and orally inoculating the rabbit with the to-be-attenuated coccidia strain at a high dose once or more within 12-72 hours after inoculation, and harvesting oocysts discharged earliest for passage amplification.

Preferably, the first inoculation is followed by the second inoculation, and the time interval between the second inoculations is 12-60 hours; the high dose is 5-10 times of the immunization dose of the first inoculation.

Specifically, the high-dose coccidian strain to be attenuated is inoculated orally once or more times twice respectively at 12-72 hours and 24-84 hours after the first inoculation.

Preferably, the breeding method of the early-maturing attenuated insect strain comprises the following steps: the coccidia to be attenuated is added at 1.0X 10⁵～2.0×10⁵Inoculating dose of sporulated oocysts/rabbits to the rabbits orally, and inoculating the to-be-attenuated coccidian strains at 5.0 × 10 hours after the first inoculation⁵～1.0×10⁶The sporulated oocysts/rabbits were inoculated once more orally; then the first inoculation is carried out 24-84 hours later, the coccidia strain to be attenuated is treated at the ratio of 1.0 multiplied by 10⁶～2.0×10⁶The sporulated oocysts/rabbit were inoculated once orally at the inoculation dose and the earliest shed oocysts were harvested for passage expansion.

The invention also provides application of the rabbit coccidiosis live vaccine composition or the rabbit coccidiosis live vaccine composition obtained by the preparation method in preparation of a preparation for preventing or treating rabbit coccidiosis.

The invention provides a rabbit coccidiosis live vaccine which comprises the rabbit coccidiosis live vaccine composition.

Preferably, the rabbit coccidiosis live vaccine further comprises an adjuvant and an auxiliary material which are allowed in the field of animal vaccines.

On the other hand, the invention provides a breeding method of a premature and weak coccidia strain of the coccidia, which comprises the following steps: and (3) orally inoculating the rabbit to be attenuated coccidian strain, and orally inoculating the rabbit again with the high-dose coccidian strain to be attenuated for one time or more 12-72 hours after the first inoculation, and harvesting oocysts discharged earliest for passage amplification.

Preferably, the first inoculation is followed by the second inoculation, and the time interval between the second inoculations is 12-60 hours; the high dose is 5-10 times of the immunization dose of the first inoculation. More preferably, the time interval between the first inoculation and the second inoculation is 12-48 hours, and the time interval between the first inoculation and the second inoculation is 12-24 hours.

The rapid weakening breeding method of the rabbit coccidia is different from the most commonly adopted classical early maturing pressure breeding method at present, the cycle of the early maturing weakening insect strain obtained by increasing one or more times of high dose inoculation and collecting oocysts discharged at the earliest stage for passage and amplification is shortened by 2-5 generations compared with that of the classical early maturing pressure breeding method, and the obtained early maturing insect strain is stable in genetic characteristic, remarkably reduced in pathogenicity and kept good in immunogenicity.

Preferably, the oral inoculation once or more times of the high-dose coccidia strain to be attenuated is twice the inoculation 12-72 hours and 24-84 hours after the first inoculation respectively. More preferably, the secondary inoculation is performed twice at 12 to 48 hours and 24 to 72 hours after the primary inoculation, respectively.

More preferably, the breeding method comprises: the coccidia to be attenuated is added at 1.0X 10⁵～2.0×10⁵Inoculating dose of sporulated oocysts/rabbits to the rabbits orally, and inoculating the to-be-attenuated coccidian strains at 5.0 × 10 hours after the first inoculation⁵～1.0×10⁶The sporulated oocysts/rabbits were inoculated once more orally; then the first inoculation is carried out 24-84 hours later, the coccidia strain to be attenuated is treated at the ratio of 1.0 multiplied by 10⁶～2.0×10⁶The sporulated oocysts/rabbit were inoculated once orally at the inoculation dose and the earliest shed oocysts were harvested for passage expansion.

The invention further provides application of the breeding method of the coccidian presenile attenuated strain in breeding of the coccidian attenuated strain or preparation of a coccidiosis vaccine.

The invention also provides a coccidiosis live vaccine which comprises the coccidiosis prematurity attenuated strain obtained by breeding by using the breeding method of the coccidiosis prematurity attenuated strain.

The invention has the beneficial effects that:

the rabbit coccidiosis live vaccine provided by the invention has higher immune protection effect and safety, can effectively prevent and treat rabbit coccidiosis, has long immune duration, can effectively reduce or eliminate the use of anti-coccidiosis drugs, reduces the breeding cost, obviously reduces the adverse effects of vaccination on growth and production performance of rabbits such as weight gain and the like, improves the breeding benefit, is easy to produce, has no pollution to animal products and environment, and has good economic benefit and wide application prospect.

The breeding method of the coccidian prematurity attenuated strain provided by the invention can effectively shorten the breeding cycle of the coccidian prematurity attenuated strain, effectively improve the breeding efficiency of the coccidian prematurity attenuated strain, and the obtained prematurity insect strain has stable genetic property, obviously reduces pathogenicity, and simultaneously keeps good immunogenicity, thereby providing an efficient method for breeding the coccidian prematurity attenuated strain and preparing a coccidian vaccine.

Detailed Description

Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.