CN115364123A

CN115364123A - Brevibacillus laterosporus and application thereof in preventing and treating ruminant parasites

Info

Publication number: CN115364123A
Application number: CN202210783585.3A
Authority: CN
Inventors: 江国托; 单春乔; 刘艳; 王岩; 李娟�; 秦淑杰; 于洪敏; 翟红旭; 刘秋晨
Original assignee: Dalian Sanyi Animal Drug Co ltd; Jiangsu Sanyi Bioengineering Co ltd
Current assignee: Dalian Sanyi Animal Drug Co ltd; Jiangsu Sanyi Bioengineering Co ltd
Priority date: 2022-07-05
Filing date: 2022-07-05
Publication date: 2022-11-22
Anticipated expiration: 2042-07-05
Also published as: CN115364123B

Abstract

The invention belongs to the technical field of anti-parasite veterinary medicines, and particularly relates to Brevibacillus laterosporus and application thereof in preventing and treating ruminant parasites. In said use, the ruminant gut parasite is selected from the group consisting of nematodes, cestodes and coccidia, preferably the ruminant is a bovine or ovine. The brevibacillus laterosporus or the metabolite thereof has the function of broad-spectrum resistance to the ruminant alimentary canal parasite, can generate the effects of inhibiting development and killing ova and larvae, does not generate drug resistance, and does not have drug residue. The invention also finds that the combination of the brevibacillus laterosporus and other commonly used medicaments for preventing and treating the ruminant parasites can generate the synergistic anti-ruminant parasite effect, can obviously reduce the dosage of other commonly used medicaments for preventing and treating the ruminant parasites, and further reduces the toxic and side effects of the medicaments.

Description

Brevibacillus laterosporus and application thereof in preventing and treating ruminant parasites

Technical Field

The invention belongs to the technical field of anti-parasite veterinary medicines, and particularly relates to brevibacillus laterosporus and application thereof in preventing and treating ruminant parasites.

Background

Parasitic diseases are the most widespread, most varied and serious diseases in the world. It not only hinders the healthy development of the livestock breeding industry and causes great economic loss, but also threatens the health of human bodies and influences the safety of public health due to parasitic diseases suffered by human and livestock. Parasitic diseases of cattle and sheep of ruminants are common diseases in the process of breeding cattle and sheep, and serious threats are brought to the health of the cattle and sheep, so that the animal body is thinned and is malnourished, if the parasitic diseases cannot be treated in time, the cattle and the sheep can die, and great economic loss is caused to farmers.

At present, common ruminant anthelmintics mainly comprise organic phosphorus, ivermectin, doramectin, praziquantel and the like, and the dosage forms mainly comprise solution, pour-on preparation, injection and the like. If the organic phosphorus drugs are used for expelling the insects, serious pollution is brought to the environment if the organic phosphorus drugs are not properly treated after the medicated bath. The conventional dosage forms such as solution, pour-on and injection are easily limited by factors such as environment, grazing mode and the like when in use, are generally only suitable for large-scale ruminant animals in captive breeding mode, and have the problems of inconvenient administration and large consumption of manpower and material resources in the application process. And once the parasites cannot be completely killed in the medication process, the grazing cattle and sheep excreted excrement with the worm eggs and can pollute pasture, which increases the possibility of repeated infection and disease attack of ruminants.

The main gastrointestinal parasites of ruminants include nematodes, coccidia, cestodes and the like, and mixed infection is common (see, for example, liu Linke, etc., "investigation of sheep gastrointestinal parasite infection and identification of coccidia species in part of our country", livestock and veterinarians, vol.53, no. 4, p.117 to p.123 of 2021, so that development of veterinary drugs which can kill the above parasites simultaneously and have low toxicity and are environmentally friendly will be the future development direction.

Brevibacillus laterosporus (Brevibacillus laterosporus) is a rod-shaped, endospore-producing facultative anaerobic bacterium capable of producing its unique canoe-shaped parasporal body (CSPB) tightly attached to the spore side, thereby making its spore lateral. The brevibacillus laterosporus is a microbial resource which can resist bacteria and kill pests and has medical value. Early research mainly focuses on insecticidal activity, and most of the research is carried out abroad, and finished products of the biological insecticide are on the market. Brevibacillus laterosporus has various biological activities and exhibits a broad spectrum of antibacterial activities, particularly against bacteria and fungi. In addition, brevibacillus laterosporus is a probiotic bacterium of mammals and birds, and its specific antibiotic produced has been applied to the field of medicine.

However, at present, there is no report that Brevibacillus laterosporus is used for preventing and controlling ruminant digestive tract parasites, such as nematodes, cestodes and the like.

Disclosure of Invention

In order to solve the problem of the prior art that a broad-spectrum antiparasitic medicament for preventing and treating the parasites in the alimentary canal of the ruminants is insufficient, the invention provides brevibacillus laterosporus and application thereof in preventing and treating the parasites in the ruminants.

Specifically, the invention is realized by the following technical schemes:

in a first aspect, the present invention provides the use of Brevibacillus laterosporus (Brevibacillus laterosporus) or a metabolite thereof for the manufacture of a medicament for the control of ruminant parasites.

Alternatively, in the above use, the ruminant parasite is a ruminant gut parasite.

Alternatively, in the above use, the ruminant gut parasite is selected from one or more of: nematodes, cestodes or coccidia.

Preferably, the Brevibacillus laterosporus has a broad spectrum of action against ruminant gut parasites, which are nematodes, cestodes and coccidia.

Alternatively, in the above use, the ruminant is selected from a cow, a sheep, an alpaca, an antelope, a camel or a deer.

Alternatively, in the above use, the ruminant is selected from cattle or sheep.

Alternatively, in the above use, the classification of brevibacillus laterosporus is named: brevibacillus laterosporus CB-42 (Brevibacillus laterosporus CB-42) with the preservation number of CCTCC NO: m2022755, the preservation time is 2022, 5 and 30 days, and the preservation places are: china Center for Type Culture Collection (CCTCC), the collection unit address: wuhan university, wuhan, china.

Alternatively, in the above use, the metabolite of brevibacillus laterosporus is a protease.

Alternatively, in the above use, the metabolite of brevibacillus laterosporus is alkaline protease BLG4.

The alkaline protease BLG4 is a protease with the molecular weight of 30kDa purified from Brevibacillus laterosporus G4, and the alkaline protease BLG4 can exfoliate the outer layers of the cuticles of nematodes, cestodes and coccidia, and the inclusion is exuded, so that the insect body is dead.

Alternatively, in the above use, the medicament further comprises other medicaments for controlling ruminant parasites.

Alternatively, in the above use, the other medicament for controlling ruminant parasites is selected from one or more of: ivermectin, abamectin, doramectin, albendazole, fenbendazole, nitrochlorophenol, or triclabendazole.

Preferably, the medicament comprises or consists of: brevibacillus laterosporus and ivermectin.

Preferably, the medicament comprises or consists of: brevibacillus laterosporus and albendazole.

Preferably, the medicament comprises or consists of: brevibacillus laterosporus and triclabendazole.

More preferably, in the medicament, the ratio of the Brevibacillus laterosporus and other medicaments for controlling the ruminant parasites is 10.

Still more preferably, the ratio of brevibacillus laterosporus to other ruminant parasite control drugs in the drug is 10, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1 by weight.

Further preferably, the ratio of the brevibacillus laterosporus to the other ruminant parasite control drugs in the drugs is 10, 9:1, 8:1, 7:1, 6:1, 5:1 or 4:1, calculated in weight ratio.

In the medicament, the combination of the brevibacillus laterosporus and other medicaments for preventing and treating the ruminant parasites can generate a synergistic anti-ruminant parasite effect, so that the dosage of other medicaments for preventing and treating the ruminant parasites can be obviously reduced.

In a second aspect, the present invention provides a Brevibacillus laterosporus (Brevibacillus laterosporus) having an animal parasite control effect, the Brevibacillus laterosporus being classified and named as: brevibacillus laterosporus CB-42 (Brevibacillus laterosporus CB-42) with the preservation number of CCTCC NO: m2022755, the preservation time is 2022, 5 and 30 days, and the preservation places are: china Center for Type Culture Collection (CCTCC), preservation Unit Address: wuhan, wuhan university.

Preferably, the animal is selected from ruminants, poultry, livestock, waterfowl or aquatic animals and the parasite is selected from one or more of: nematodes, cestodes or coccidia.

Further preferably, the ruminant is selected from the group consisting of cattle, sheep, alpaca, antelope, camel and deer.

More preferably, the ruminant is selected from cattle or sheep.

Compared with the prior art, the invention has the following beneficial effects:

(1) The brevibacillus laterosporus or the metabolite thereof has the function of broad-spectrum resistance to the ruminant alimentary canal parasite, can generate the effects of inhibiting development and killing ova and larvae, does not generate drug resistance, and does not have drug residue.

(2) The invention also finds that the combination of the brevibacillus laterosporus and other commonly used medicaments for preventing and treating the ruminant parasites can generate a synergistic effect of resisting the ruminant parasites, and can obviously reduce the dosage of other commonly used medicaments for preventing and treating the ruminant parasites so as to reduce the toxic and side effects of the medicaments.

Drawings

FIG. 1 is a photograph of an experimental site and an experimental animal of a Shengda farm in Zhangzhou prefecture, zhang kou, hebei province.

FIG. 2 is a photograph of an experimental site and an experimental animal of a Shengda farm in Zhangzhou prefecture, zhang kou, hebei province.

FIG. 3 is a photograph showing the procedure of the detection by the McMaxim's egg counting method.

FIG. 4 is a photograph showing the procedure of the detection by the Macmester egg counting method.

Detailed Description

The invention is further illustrated with reference to specific examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the scope of the invention.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or instruments used are conventional products which are not known to manufacturers and are available from normal sources.

The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples are all commercially available products unless otherwise specified.

Example (b): bacillus laterosporus insecticidal test report

1 materials and methods

1.1 Main Equipment

Refrigerator, microscope, electric heating furnace, electronic balance, beaker, plate, penicillin bottle, rubber head dropper, glass slide, glass rod, sealing bag, disposable gloves, etc.

1.2 Primary reagents

Bacillus laterosporus CCTCC M2022755 CB-42, albendazole powder and saturated saline solution (the preparation of the saturated saline solution is that 400g of salt is dissolved in 1000mL of boiling water to prepare the saturated saline solution, and the saturated saline solution is cooled for standby application).

1.3 test animals and sites

Test animals: sheep flocks (80-90 kg) were selected for testing without anthelmintic, free-range, grazing as the primary feeding means, as shown in figures 1 and 2. 100 sheep with positive parasites are finally confirmed by detection, and only a part of sheep are randomly selected for insecticidal test.

Test site: shengda farm in Zhanjiakou city of Hebei province

1.4 fecal sample collection method

Fresh fecal samples, each about 20g, were taken through the rectum in the grazing season, and then the feces were packed into clean sealed bags with air squeezed out as much as possible. Marking and collecting the information of the excrement, the sex, the age, the weight and the like, numbering, placing in an incubator, posting to a laboratory, placing in a refrigerator at 4 ℃, and completing the detection within two days.

1.5 fecal ova examination

And (3) inspecting the sheep cestode eggs, the nematode eggs, the coccidian eggs and the protozoan oocysts by using a saturated saline floating method to determine the condition of the sheep infected with the parasites. The specific method comprises the following steps:

(1) placing 2g of the excrement in a plate, lightly crushing the excrement by using a glass rod, then adding 10 times of saturated saline water, and stirring and mixing by using the glass rod;

(2) filtering the liquid dung into a small beaker by using gauze;

(3) pouring the filtered liquid dung into a clean penicillin bottle until the liquid dung in the pipe is approximately horizontal to the pipe opening, and then dripping the liquid dung into the penicillin bottle by using a rubber-head dropper until the liquid dung is dripped to the liquid level and slightly protrudes out of the bottle opening without overflowing. Standing for 30min;

(4) dipping the liquid level of the standing liquid dung by a cover glass, placing the liquid dung on a glass slide, and performing microscopic examination.

1.6 Mexmaster eggs.

And (3) counting the eggs of the sheep by using a Macmaster method for the fecal sample of the sheep which is positive in egg detection: as shown in fig. 3 and 4, 2g of feces were put into a beaker and ground, and 58mL of saturated saline was added thereto and mixed by shaking; the liquid manure was then filtered into another beaker; stirring the filtered liquid dung, sucking a small amount of the liquid dung by a rubber head suction pipe while uniformly mixing, dripping the liquid dung into a Mcmaster counting chamber, placing the Mcmaster counting chamber on a microscope, and standing for 3min. And (3) after standing, carefully observing by using a microscope low-power microscope, counting all eggs in the two counting chambers, and multiplying the average value by 200 to obtain the number of eggs (eggs per gram, EPG) in each gram of excrement.

Per gram of fecal ova = average number of ova in two graduated rooms × 200.

1.7 Bacillus laterosporus insecticidal test protocol

In the insect expelling test, 48 positive sheep infected by parasites determined in the earlier stage are selected and randomly and evenly divided into four groups of 12, wherein the group A is a blank control group, the group B is a single-use group of brevibacillus laterosporus, the group C is a single-use group of albendazole, and the group D is a group of brevibacillus laterosporus and albendazole. Firstly, counting the fecal ova of four groups of sheep, and recording the EPG of each group of sheep before expelling insects; 2mL of normal saline (containing 9mg of bacillus laterosporus and suspended by normal saline) is administered to each sheep in the group A, 2mL of normal saline (containing 9mg of bacillus laterosporus and suspended by normal saline) is administered to each sheep in the group B, 2mL of normal saline (containing 1mg of albendazole powder and suspended by normal saline) is administered to each sheep in the group D according to the instruction, and 2mL of normal saline (containing 9mg of bacillus laterosporus and 1mg of albendazole powder and suspended by normal saline) is administered to each sheep in the group D. Then taking feces at the fourth day after administration and counting the EPG value; and simultaneously carrying out second administration, carrying out second fecal examination on the seventh day after the first administration, and recording the EPG value after insect expelling.

Sampling and detecting: about 20g of feces were collected before drenching, on the 4 th day and on the 7 th day after drenching, as described in section 1.4. Observing the result by using a microscope, and counting the eggs.

1.8 determination of insect-repellent Effect

After expelling parasites, the clinical conditions of the sheep in each group are respectively observed and recorded, and the number of eggs of the sheep in each group, the parasite rate of the sheep, the egg reduction rate and the parasite infection reduction rate between an experimental group and a control group are counted, wherein the specific formula is as follows:

the Macmarster method:

(1) Number of eggs per gram of feces (EPG) = [ (n 1+ n 2)/(2 × 0.15) ] × 60 ÷ 2

Wherein (n 1+ n 2)/2 is the average number of worm eggs in each counting chamber, 0.15 is the effective volume of each counting chamber is 0.15mL,60 is the total volume of the liquid dung is 60mL, and 2 is the gram number of the used excrement is 2g.

(2) Sheep parasite rate (%) = number of positives/total number of samples 100

(3) Egg reduction rate = (control group egg average value-experimental group average value)/control group egg average value

(4) Reduction rate of parasite infection = (number of positive control group-number of positive experimental group)/number of positive control group

2 results and analysis

2.1 insecticidal effect of each administration group on sheep for 4 days

The results are shown in Table 1, and after 4 days of administration, the egg positive rates and the average egg values of the group B Brevibacillus laterosporus single-use group, the group C albendazole single-use group and the group D Brevibacillus laterosporus + albendazole group were all reduced to different degrees compared with the group A blank control group. The change range of the B group and the D group is obvious, and the animals in the C group are possibly administered with low dosage although the albendazole which is a common broad-spectrum antiparasitic drug is administered, so that the antiparasitic effect is not obvious after the animals are administered for 4 days, and the positive rate of eggs and the average value of eggs are respectively 92% and 685. However, unexpectedly, as shown in group D, when low doses of albendazole were used in combination with brevibacillus laterosporus, the egg positivity and average egg values were significantly reduced compared to groups B and D alone, with 67% and 268, respectively.

Further, the synergy index q value is determined according to a formula for calculating the "golden average" known in the art (see, for example, the "addition in concomitant medication", the chinese pharmacology bulletin, 12 months 1980, 1 (2) 70-76), and the q value is obtained by the following formula: q = PA + B/(PA + PB-PA x PB). Wherein PA, PB and PA + B are the reduction rate of eggs or parasite infection of group B, group C and group D, respectively. q is less than 1, which indicates that the two medicines generate antagonism after being used together; q > 1 indicates that the two drugs produce synergistic effect after being combined together, and q =1 indicates that the two drugs produce additive effect after being combined together. Calculated q values for group D were 1.39 and 1.61 for the rate of egg reduction or parasite infestation reduction, respectively. Therefore, 4 days after the drug is taken, the combination of the brevibacillus laterosporus and the albendazole can effectively expel parasitic ova of nematodes, tapeworms and coccidia in the sheep of 1-2.5 years old, and the combination of the brevibacillus laterosporus and the albendazole generates a synergistic effect.

TABLE 1 Positive rate of eggs and average value of eggs

2.2 insecticidal effect of each administration group on sheep for 7 days

The results are shown in Table 2, and the results were consistent with the trend of the results after 4 days of administration, and after 7 days of administration, the egg positivity and the average value of the eggs of the group B Brevibacillus laterosporus single-use group, the group C albendazole single-use group and the group D Brevibacillus laterosporus + albendazole group were all reduced to different degrees compared with the group A blank control group. The change range of the animals in the group B and the group D is obvious, and the animals in the group C are possibly low in dosage although the common broad-spectrum antiparasitic drug albendazole is given, so that the antiparasitic effect of the animals in the group C is still not strong after 7 days of administration, and the positive rate of eggs and the average value of eggs are respectively 75% and 425. However, unexpectedly, as shown in group D, when low doses of albendazole were used in combination with brevibacillus laterosporus, the egg positivity and average egg values were significantly reduced compared to groups B and D alone, with 8% and 36, respectively.

And, according to the calculation formula as described above, the q values of group D were calculated to be 1.34 and 1.21 with respect to the reduction rate of eggs or the reduction rate of parasite infection, respectively. Therefore, after the medicine is taken for 7 days, the combination of the brevibacillus laterosporus and the albendazole can effectively expel parasitic ova of nematodes, tapeworms and coccidia in sheep aged 1-2.5 years, and the combination of the brevibacillus laterosporus and the albendazole generates a synergistic effect.

TABLE 2 Positive rate of eggs and average egg value

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims

1. Use of Brevibacillus laterosporus (Brevibacillus laterosporus) or its metabolite in preparing medicine for preventing and treating ruminant parasites.

2. Use according to claim 1, characterized in that: the ruminant parasite is a ruminant gut parasite.

3. Use according to claim 1 or claim 2, characterized in that: the ruminant gut parasite is selected from one or more of the following: nematodes, cestodes or coccidia.

4. Use according to any one of claims 1 to 3, characterized in that: the ruminant is selected from cattle, sheep, alpaca, antelope, camel or deer.

5. Use according to claim 1, characterized in that: the classification name of the Brevibacillus laterosporus is Brevibacillus laterosporus CB-42 (Brevibacillus laterosporus CB-42), the collection number is CCTCC NO: m2022755, preservation time 2022, 5 months and 30 days, and preservation place: china Center for Type Culture Collection (CCTCC), preservation Unit Address: wuhan, wuhan university.

6. Use according to claim 1, characterized in that: the metabolite of the brevibacillus laterosporus is protease.

7. Use according to claim 6, characterized in that: the metabolite of the brevibacillus laterosporus is alkaline protease BLG4.

8. Use according to any one of claims 1 to 6, characterized in that: the medicament also comprises other medicaments for preventing and treating the ruminant parasites.

9. Use according to claim 8, characterized in that: the other medicament for preventing and treating the ruminant parasites is selected from one or more of the following medicaments: ivermectin, abamectin, doramectin, albendazole, fenbendazole, nitrochlorophenol, or triclabendazole.

10. Brevibacillus laterosporus (Brevibacillus laterosporus) having an effect of controlling parasites on animals, characterized in that: the classification of Brevibacillus laterosporus is named as: brevibacillus laterosporus CB-42 (Brevibacillus laterosporus CB-42) with the preservation number of CCTCC NO: m2022755, the preservation time is 2022, 5 and 30 days, and the preservation places are: china Center for Type Culture Collection (CCTCC), preservation Unit Address: wuhan, wuhan university.