KR100430936B1 - Attenuated vaccine for bovine theileriosis - Google Patents

Abstract

The present invention is a bovine Tyreria disease stabilate vaccine using a Korean isolate, Theileria sergenti , Sungwhan stock, and a method for preparing the same, and the vaccine is administered to a cow. The present invention relates to a method for preventing Tyreria disease.

Description

Attenuated vaccine for bovine theileriosis

The present invention relates to an attenuated vaccine against bovine theileriosis. More specifically, the present invention is stabilized (stabilate) vaccine using a Korean isolate ( Tileria sergenti , Sungwhan stock) and its preparation method, and the vaccine is administered to cattle It relates to a method for preventing Tyreria disease.

Typhoid 's disease in cows is caused by infection of cattle via the tick ( Hamaphisalis longicornis ) of the family Piroplasmida and Theileriidae . It is a disease that causes swelling, dyspnea, anemia and jaundice. In Korea, mites, the mediators of this disease, are distributed throughout the country, with varying degrees. In particular, ticks are active in the spring, so there are many cases of grazing from about 1 month after grazing.

The main symptoms include jaundice and anemia, usually a fever around 41 ℃ after one week, but most of the fever is not found in a few days. After 10-14 days of infection, the protozoa begins to appear in red blood cells and peaks in about 1 month. As the protozoa proliferate, appetite declines, anemia progresses rapidly, and the thorn mucosa becomes pale. In the end, the body temperature drops below 38 ℃, collapses and cannot stand up. Such symptoms can be recovered after a few days with proper treatment at an early stage, but if the treatment is delayed or suddenly recovered, long-term anemia and jaundice will persist, and development will be reduced in rearing cattle.

Six to seven species of protozoa causing bovine Tyreria disease have been reported around the world, and the species present in Korea, Theileria sergenti , is classified as relatively weak in pathogenicity. However, once infected, most of the cows remain after the recovery because they are relieved when they are stressed, and this disease is endemic endemic disease in Korea. On the other hand, Korean cattle have some resistance to the disease, but cows introduced from abroad have no resistance to this disease, which can lead to anemia, developmental arrest, and severe death.

Currently in Korea, measures against this disease are limited to the prevention of infection through the treatment of ticks on pasture or the treatment of infected cows. In foreign countries, a method of immunizing immunity by separating the protozoan from the salivary glands of a disease-infected tick, inoculating the cow and inoculating it again and treating it is used. Tests to develop vaccines in Korea include protozoa isolated from blood and their metabolites. However, inoculation of protozoa and metabolites into cows may alleviate clinical symptoms somewhat, but not provide complete protection. In addition, these methods have the disadvantage that it is difficult to provide a sufficient amount of antigen when inoculated to many cattle.

Stabilate vaccines, meanwhile, are derived from an immunoassay against Theileria parva , which causes fever in Africa (InternationalLivestock Research Institute (ILRI), Kenya), and sporozoites infected with tick salivary glands ( After inoculating sporozoite with cows, treatment with chemotherapeutic agents such as long-acting oxytetracycline has been used to increase the effectiveness of the vaccine. In this case, the exact dose of sporozoite to be inoculated serves as an important factor in determining the success of the vaccine. It is also reported that the desired vaccine has been achieved without the use of therapeutic agents such as sustained release oxytetracycline. However, until now, such a stabilate vaccine was never produced using the Tyreria western titan strain.

As described above, the cattle ileary disease is a chronic wasting disease, causing enormous economic damage to farms, the need for a vaccine against this situation is increasing.

Therefore, the present inventors, while developing a new vaccine that can effectively prevent bovine Tyreria disease, during the ongoing research, stabilate vaccine derived from an immune test against Tyreria fava causing fever during Africa Focused on. The present inventors prepared a stabilate vaccine by separating the thyileria Seojeontai Sunghwan, the causative agent of domestic bovine Tyreria disease, from the tick salivary glands and attenuating it. In addition, it was confirmed that the vaccine can effectively prevent Tyreria disease by inducing a low level of infection in cattle to maintain immunity, and thus, the present invention has been completed.

Accordingly, it is an object of the present invention to provide a bovine Tyreria disease stabilizer vaccine prepared from Tyreria west titanic patients.

Another object of the present invention is to provide a method for preparing the vaccine.

Still another object of the present invention is to provide a method for preventing bovine Tyreria disease using the vaccine.

1 is a flow chart of the production of Theileria sergenti stabilate;

2 is a photograph dissecting an infected tick to separate salivary glands;

3A and 3B are photographs confirming stabilate by staining salivary glands of infected ticks;

Figure 4 is a photograph confirming the stabilizers present in the salivary glands using indirect fluorescent antibody method;

FIG. 5 is a graph showing the change in infection rate (PPE%) after challenge with Tyreria in a cow vaccinated with Stabilate vaccine;

FIG. 6 is a graph showing the change in erythrocyte volume ratio (PCV%) after challenge with Tyreria in a cow vaccinated with Stabilate vaccine.

First, the present invention relates to an attenuated vaccine for bovine ilearia disease, which contains as an active ingredient stabilates isolated from the thyileria west gynecological strain.

In the present invention, the stabilate preferably comprises: (a) collecting the adult after vampire mite nymph to the calves infected with Tyreria; (b) harvested infected ticks were then wicked and sensitized to rabbits and harvested; (c) Isolated by aseptic sampling of saliva from collected ticks.

Second, the present invention

(a) vampire mite nymphs on calves infected with Tyreria and then recovered;

(b) harvested infected ticks were then wicked and sensitized to rabbits and harvested;

(c) aseptically collecting salivary glands from collected mites:

It relates to a method for producing the vaccine, including.

Third, the present invention relates to a method for preventing Tyreria disease, comprising administering the vaccine to a cow.

Hereinafter, the present invention will be described in detail.

The present invention relates to attenuated vaccines against ticks mediated by Theileria sergenti disease.

The method for producing a vaccine according to the present invention focuses on a method for preparing a vaccine that is used in the most pathogenic species present in Africa. In other words, the stabilate vaccine is derived from an immunoassay against Tyreria fava, which causes fever during Africa. Methods have been used to increase the effectiveness of the vaccine by zero treatment. In this case, the exact dose of sporozoite to be inoculated serves as an important factor in determining the success of the vaccine. However, it is also reported that the desired vaccine has been achieved without the use of therapeutic agents such as sustained release oxytetracycline.

To prepare the stabilate vaccine, the mite nymph was first attached to the ear of a calf with a infection rate of 10% or more of Tyreria, which was then sucked and dropped for about 6-7 days. After about one month, the molten adult was recovered, the intestine of the hatching adult was extracted, confirmed the infection, and then the infected tick was vampireized and sensitized to the rabbit. Three days later, mites vampires were forcibly collected, the mites were quickly dissected, and only fresh salivary glands were collected aseptically. The maturation of the stabilates was confirmed by salivary gland staining, and the stabilates were stored frozen in liquid nitrogen.

The target animal was divided into a control group and a vaccine group, and then challenged with a stabilizer vaccine prepared as described above. As a result, a statistically significant difference was obtained between the stabilate vaccine group and the control group over time in the blood infection rate (PPE%) and erythrocyte volume ratio (PCV%) between groups (p <0.05).

Vaccines according to the invention may be formulated in conventional formulations, preferably injectables, in the pharmaceutical art together with pharmaceutically acceptable carriers. The dose of the vaccine is usually 1 Unit based on cattle 6 months of age or older based on the active ingredient, but the dose may be increased or decreased depending on the age, weight, and health of the cattle to be administered. One unit is the amount of one inoculum of cows, and one ampule containing stabilate extracted from ten ticks.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited by them in any way.

Example 1: Mites and Bovine Infections of Tyreria

The protozoan used in this example was a stellar adult carcinoma, and used for 17 generations of parasites using the Korean cattle calf extracted from the veterinary quarantine. This protozoan is manufactured and preserved by the National Veterinary Research and Quarantine Service (Freezing) containing isopropyl alcohol by mixing with a medium consisting of 10% DMSO (DIMETHYL SULFOXIDE), 10% serum and 80% medium (a (alpha) -MEM). A person who intends to conduct a test or research after standing in a container at -80 ° C for one day and storing it in liquid nitrogen can apply for the sample to the Quarantine Service Division (TEL: 031-467-1953). Those skilled in the art can easily obtain the microorganism. The ticks ( Haemaphysalis longicornis ) fed the rabbit's blood were recaptured, and the test tube, which maintains humidity above 60%, was kept at room temperature to induce spawning and hatching to obtain hatching larvae. The larva was further blood-sucked into rabbits for 6 to 7 days to bleed and drop out. The fallen larvae were kept in the same in vitro to induce molting to obtain nymphs after about one month. 50 ml of dexamethasone (dexamethason, 1mg / ml) was treated for 1 week in spleen-extracted calves infected with Tyreria, and the infection rate of red blood cells of protozoa was more than 10%. Thereafter, the hatching nymph was attached to the calves' ears, and the animals were sucked and dropped for about 6 to 7 days. Dropped ticks were recovered and stored in vitro under the same conditions. The vampires that have been vampires escape and become adults, and the adults are used to produce stabilate vaccine by separating the salivary glands.

Example 2: Mass Production of Stabilates

After about one month, the infested adult intestine was extracted and confirmed by artificial infection using PCR (polymerase chain reaction) technique. In order to perform PCR, DNA was isolated from salivary glands using phenol extraction. The separated DNA was purified using 100% ethanol and then used for PCR. PCR reaction conditions were induced 5 minutes at 94 ℃, 30 seconds at 57 ℃, 30 seconds at 72 ℃ 30 times at 30 ℃, 94 ℃ 30 times and gave a reaction time of 3 minutes at 72 ℃. The PCR sample was electrophoresed in 2% agarose gel and then the size of the PCR product appeared on the gel. Primers used for PCR are as follows. Thp-2: 5'-CCAAGTTCACCCCAACTGTCG-3 ', Thp-5: 5': GCACTGTTCATGGCGTGCAAA-3 '.

Thereafter, infected ticks were confirmed to be infected with rabbits about 100 times, and then vampled and sensitized. Three days later, the mites vampires were forcibly removed and the mites were quickly dissected. The state is shown in FIG. Thereafter, only fresh salivary glands were collected aseptically. In order to collect aseptically, the dissection of ticks was carried out with a lamp on a test table sterilized with alcohol, and the tools and saline used for dissection were sterilized. The maturation of the stabilates was confirmed by salivary gland staining, and the stabilates were stored frozen in liquid nitrogen.

The production flow chart of such stabilizer is shown in FIG. In addition, stabilates in the tick salivary glands could be confirmed using gimja (giemsa) staining and fluorescent antibody assay (see FIGS. 3 and 4). In order to prevent laver dying, first spread the salivary glands separated from the tick so that they do not overlap on the slide and dry them. The dried salivary glands were fixed with methanol for 10 minutes and then stained with 10% gimsa dye for 1 hour. After the dyeing was washed with running water and observed under a microscope. In order to identify the protozoa present in the salivary glands, a fluorescent antibody test was also performed using an antibody that specifically reacts with the protozoa. After drying, the immobilized salivary glands reacted with protozoal specific antibodies for 1 hour at 37 ° C. and washed, and again reacted with fluorescent secondary antibodies. After the reaction was washed and observed using a fluorescence microscope. Salivary glands whose stabilates were identified by laver and fluorescent staining were stored in liquid nitrogen.

Example 3 Protective Experiments of Stabilate Vaccine

In order to investigate the protective ability of the Stabilate vaccine prepared in Example 2, four out of six three to four month-old calves were placed in the control group (# 5 and # 6) with two in the vaccine group (# 1 to # 4). All the calves used in the experiment were spleened. After dissolving the stabilate vaccine stored in liquid nitrogen rapidly in a 37 ° C. water bath, and washing it three times with sterile PBS (Phosphate Buffered Saline), the vaccine group contains two units of stabilate vaccine per day. Two times, subcutaneous injections were made near the superficial cervical lymph nodes. The control group was inoculated with the same amount of sterile PBS.

Tyreria with 8.6% infection shortly after vaccination and 30 and 32 days after vaccination Blood was collected from infected cows, washed three times with PBS, and challenged two times with 50 ml of red blood cells per calf. At weekly intervals, changes in infection concentration (PPE%, Parasite Per Erythrocyte) and erythrocyte volume ratio (PCV%, Packed Cell Volume) were observed up to 84 days post-inoculation to investigate the protective ability of the stabilate vaccine.

The blood infection rate (PPE%) of main hematopoietic insects is shown in FIG. 5. As shown in FIG. 5, the vaccination group showed an infection rate of 2.0-4.7% only after 7 days of inoculation, and then gradually decreased, and the infection rate was lowered to 0.1-0.2% two days before challenge. Slightly increased after challenge, the infection rate was 0.5-0.8% at 30 days after challenge (63 days after vaccination). On the other hand, the control group showed a continuous increase in the infection rate after challenge vaccination, showing an infection rate of 5.8 to 7.2% at 40 days after the challenge (70 days after the start of the experiment) and 16.5 to 20.3% at 84 days after the experiment. Showed an infection rate. On the other hand, the vaccination group showed a very slight increase in the infection rate (0.5-0.8%) seen at 63 days after vaccination, and 0.3-2.2% infection at 84 days after the start of the experiment.

The change in the individual red blood cell volume ratio (PCV%) of the main hematopoiesis is shown in FIG. 6. As shown in FIG. 6, the vaccinated group showed a slightly reduced volume ratio of 30-37% after 7 days of vaccination. Afterwards, it gradually decreased, and recovered 2 to 34% before challenge. After inoculation, it was slightly decreased, but returned to normal at 34-36% at 30 days after inoculation (63 days after vaccination). After that, most of the volume ratios were shown until the end of the experiment (84 days after the start of the experiment). In comparison, the control group showed a continuous decrease in the volume of red blood cells after challenge, with infection rates of 25-27% at 40 days (70 days after the start of the challenge) and 22 at 84 days after the experiment. Infection rate was ˜24%.

As described above, as a result of comparing and analyzing the change in blood infection rate and erythrocyte volume ratio over time between the groups, there was a statistically significant difference between the stabilate vaccine group and the control group (p <0.05).

As described above, since the stabilized vaccine prepared according to the present invention was recognized to have a statistically significant protective ability, the vaccine of the present invention can be effectively used for the prevention of bovine Tyreria disease.

Claims (5)

An attenuated vaccine against bovine ilearia disease , which contains stabilate isolated from Theileria sergenti , Sungwhan stock as an active ingredient. The method of claim 1, wherein the stabilate is (a) blood vaccination and dropping the tick nymph to the cilia infected with Tyreria to recover the adult; (b) harvested infected ticks were then wicked and sensitized to rabbits and harvested; (c) A vaccine isolated by aseptic harvesting of salivary glands from collected ticks. The vaccine of claim 1 or 2 formulated as an injection. (a) vampire mite nymphs on calves infected with Tyreria and then recovered; (b) harvested infected ticks were then wicked and sensitized to rabbits and harvested; (c) aseptically collecting salivary glands from collected mites: Including, the method of producing a vaccine according to claim 1. A method of preventing bovine tyreria disease, comprising administering a vaccine according to claim 1 to a bovine.