CN103349718B - Livestock and poultry cryptozoite infection prevention and treatment compound traditional Chinese medicine, preparation method and application thereof - Google Patents
Livestock and poultry cryptozoite infection prevention and treatment compound traditional Chinese medicine, preparation method and application thereof Download PDFInfo
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Abstract
The present invention discloses a livestock and poultry cryptozoite infection prevention and treatment compound traditional Chinese medicine, a preparation method and an application thereof. The compound traditional Chinese medicine comprises the following raw materials, by weight: 10-20 parts of szechwan chinaberry fruit, 10-20 parts of betel nut, 5-15 parts of antifebrile dichroa root, 5-15 parts of pomegranata rind, and 5-15 parts of longleaf garden burnet root. According to the present invention, the szechwan chinaberry fruit, the betel nut, the antifebrile dichroa root and the pomegranata rind in the compound traditional Chinese medicine have an in vivo deworming effect, the longleaf garden burnet root has effects of blood cooling, bleeding stopping, heat clearing and detoxification, and has a corresponding immunity improvement effect, and livestock and poultry cryptozoite infection can be effectively prevented and treated.
Description
Technical field
The invention belongs to field of traditional Chinese, be specifically related to a kind ofly prevent and treat Chinese medicine compound of poultry Cryptosporidium spp and preparation method thereof and application.
Background technology
Cryptosporidiosis (cryptosporidiosis) be by Cryptosporidium cause in universal infecting both domestic animals and human protozoacide, be a kind of important paathogenic factor causing man and animal acute diarrhea or chronic lethal diarrhoea.Be only second to the primary bowel disease of rotavirus.Cryptosporidium extensively parasitizes mammal, birds, amphibian, the digestive tract of Fish and people and other organ, and carries out a large amount of breedings at parasitic place, causes digestive and absorptive functions obstacle, causes animal diarrhea and inapparent infection.Having now found that in 106 national human case, is the important paathogenic factor of human body acute diarrhea or chronic lethal diarrhoea.Cryptosporidium spp comprises 260 many animals of people, and research shows, Cryptosporidium is propagated mainly through routes of transmission such as contact, drinking-water, food and the spittle, and the egg capsule environment to external world of hidden spore worm, particularly (O is comprised to chlorination
3deng) have very strong resistance, make traditional handicraft be difficult to removed or killed (Feng Lihong. cryptosporidiosis progress [J]. preventive medicine journal of information, 2006 (1): 46-48.).Since this disease of reported first in 1976, in the whole world, there is the case of outburst in each continent, wherein representative in the outburst most of the U.S..Research of tracing to the source confirms, have the Cryptosporidium behaviour Cryptosporidium of 2 great outbursts at least, 1 time is Cryptosporidium parvum, but has and repeatedly cause the Cryptosporidium that this disease breaks out derive from pig and cattle, companion animals and derive from poultry.These results fully show the threat of animal sources Cryptosporidium to human health.
Guangdong Province is located in subtropical zone, and livestock and poultry cultivation density is large, and the sewage produced for livestock and poultry cultivation at present, the process of feces, owing to lacking general cost-effective disposal technology, cause it to cause severe contamination to production, living environment and river water; In addition, Pearl River Delta regional population's very dense, it is reported, water source in Guangdong Province's is more single, mainly surface water.In city, river course supplies water and accounts for 88%, and reservoir only accounts for 12%.Due to the egg capsule environment to external world of Cryptosporidium, particularly have very strong resistance to chlorination, traditional disinfection technique is difficult to be killed, once Livestock pollution water source, urban population faces the risk of great outburst.Again, because Pearl River Delta district economy is comparatively flourishing, the quantity of companion animals also sharply increases, and 2 that C.felis and C.canis deriving from companion animals causes people to infect just important Cryptosporidium worm kinds (Dubey, 2009).
Although there is bibliographical information, some medicines have certain therapeutic effect to cryptosporidiosis, external current useful spiramycin, paromomycin treatment patient, also curative effect is obtained with artificial high immune foremilk (hyperimmune bovine colostr μm, HBC), Lac Bovis seu Bubali globulin, cattle transfer factor treatment.Such as: (1) Okhuysen P C, Chappell C L, Kettner C, et al.(Cryptosporidium parvum metalloaminopeptidase inhibitors prevent in vitro excystation. [J] .Antimicrobial Agents and Chemotherapy, 1996, 40 (12): 2781-2784.), (2) Forney J R, Yang S, Du C, et al.Efficacy of serine protease inhibitors against Cryptosporidium parvum infection in a bovine fallopian tube epithelial cell culture system [J] .The Journal of parasitology, 1996:638-640.(3) Keithly J S, Zhu G, Upton S J, et al.Polyamine biosynthesis in Cryptosporidium parvum and its implications for chemotherapy [J] .Mol Biochem Parasitol, 1997, 88 (1-2): 35-42. mention in above-mentioned 3 parts of documents, the Western medicine such as soybean trypsin inhibitor-sbti (soybean trypsin inhibitor) can be used to carry out the control of Cryptosporidium.
The Cryptosporidium spp of domestic garlicin treatment people cryptosporidiosis, Radix Angelicae Sinensis decoction for tonifying blood and Polysaccharides on Mice thereof has certain effect.Herbal mixture proves in clinical observation in modern age and pharmacological research: the medicine such as ethanol extraction, arecoline, Toosendanin, Semen Cucurbitae, artesunate, Fructus Quisqualis of Cortex Meliae has good anthelminthic effect, and side effect is little.Coordinate the medical material of invigorating the stomach and promoting digestion, invigorating the spleen and stomach, invigorating QI and blood simultaneously, greatly can alleviate organ injury, dysfunction and the malnutrition such as the intestines and stomach that parasite causes and become thin.The anthelmintic Chinese medicine applied clinically at present adopts multi-flavor medical material to be composited, existing plant antiscolic, has again stomach invigorating and food calling composition (Liu Zonghu, Xia Weixin, Zhang Yan. the epoch of scale pig farm Integrated TCM anthelmintic arrive [J]. northern animal husbandry, 2008 (18) .).
But even to this day, definite effectively medicine, vaccine be there is no for cryptosporidiosis to emerge.Because this disease badly influences the g and D of animal and the health of the mankind, tremendous economic can be caused to lose.For the most effective mode of prevention and control of cryptosporidiosis no more than its epidemiology background clear and definite, reduce infection chance (rich Chun-shan Mountain, Li Yu comes. the prevention and control [J] of cryptosporidiosis. herding and forage science, 2010 (Z2): 201-202.).Therefore, at present in the urgent need to studying a kind of medicine can preventing and treating poultry Cryptosporidium spp.
Summary of the invention
For solving the shortcoming and defect part of prior art, primary and foremost purpose of the present invention is to provide a kind of Chinese medicine compound of preventing and treating poultry Cryptosporidium spp.
Another object of the present invention is to the preparation method of the Chinese medicine compound that above-mentioned control poultry Cryptosporidium spp is provided.
Another object of the present invention is the application of the Chinese medicine compound providing above-mentioned control poultry Cryptosporidium spp.
For achieving the above object, the present invention adopts following technical scheme:
Prevent and treat a Chinese medicine compound for poultry Cryptosporidium spp, described Chinese medicine compound is made up of the raw material of following weight portion: Fructus Toosendan 10-20 weight portion, Semen Arecae 10-20 weight portion, Radix Dichroae 5-15 weight portion, Pericarpium Granati 5-15 weight portion and Radix Sanguisorbae 5-15 weight portion.
Anthelmintic action in the body that Fructus Toosendan in Chinese medicine compound of the present invention, Semen Arecae, Radix Dichroae, Pericarpium Granati etc. have, Radix Sanguisorbae has cooling blood for hemostasis antipyretic and antidote functions, and improving immunity to body also has corresponding effect, effectively can prevent and treat Cryptosporidium.
The present invention's raw material of Chinese medicine used all can be buied from Chinese medicine shop, and its specification meets national medical standards.
Preferably, described Chinese medicine compound is made up of the raw material of following weight portion: Fructus Toosendan 18 weight portion, Semen Arecae 12 weight portion, Radix Dichroae 18 weight portion, Pericarpium Granati 10 weight portion and Radix Sanguisorbae 10 weight portion.
The preparation method of the Chinese medicine compound of above-mentioned control poultry Cryptosporidium spp, Fructus Toosendan, Semen Arecae, Radix Dichroae, Pericarpium Granati and Radix Sanguisorbae pulverized, cross 120 mesh sieves, mix homogeneously obtains described Chinese medicine compound.
The Chinese medicine compound of above-mentioned control poultry Cryptosporidium spp is preparing the application in animal and fowl fodder, and described Chinese medicine compound is added in animal and fowl fodder, and addition is 0.5% of animal and fowl fodder quality.
Compared with prior art, the present invention has the following advantages and beneficial effect:
The present invention is full Chinese medicine formula, and antibiotic-free remains and resistant characterization, has significant curative effect to cryptosporidiosis control.And formula of the present invention mainly considers with anthelmintic and raising immunity of organism effect two aspects, uses trace to beat powder technology and makes medicine effectively mix, relative to existing anthelmintic Western medicine, have medicine irritability little, the advantage that trap is good.
Accompanying drawing explanation
Fig. 1 is the feces figure infecting matched group chickling DPI12;
Fig. 2 is that in the fabricius bursa of infection matched group chickling DPI8, Cryptosporidium parvum oocysts suspended amplifies 400 times of detection figure;
Fig. 3 is the oocysts excreting regulation figure of each experimental group chickling DPI1-15;
Fig. 4 is compound recipe group, the oocysts excreting regulation figure infecting matched group and blank group chickling DPI1-15;
Fig. 5 is Western medicine group, the oocysts excreting regulation figure infecting matched group and blank group chickling DPI1-15;
Fig. 6 is the trachea section amplification 100 times of compound recipe 1 group of chickling DPI15 and the Electronic Speculum figure of 400 times;
Fig. 7 is the trachea section amplification 100 times of compound recipe 2 groups of chickling DPI15 and the Electronic Speculum figure of 400 times;
Fig. 8 is the trachea section amplification 100 times of compound recipe 3 groups of chickling DPI15 and the Electronic Speculum figure of 400 times;
Fig. 9 is the trachea section amplification 100 times of compound recipe 4 groups of chickling DPI15 and the Electronic Speculum figure of 400 times;
Figure 10 is the trachea section amplification 100 times of Western medicine 1 group of chickling DPI15 and the Electronic Speculum figure of 400 times;
Figure 11 is the trachea section amplification 100 times of Western medicine 2 groups of chickling DPI15 and the Electronic Speculum figure of 400 times;
Figure 12 is the trachea section amplification 100 times of Western medicine 3 groups of chickling DPI15 and the Electronic Speculum figure of 400 times;
Figure 13 is the trachea section amplification 100 times of Western medicine 4 groups of chickling DPI15 and the Electronic Speculum figure of 400 times;
Figure 14 infects the trachea section amplification 100 times of matched group chickling DPI15 and the Electronic Speculum figure of 400 times;
Figure 15 is the trachea section amplification 100 times of blank group chickling DPI15 and the Electronic Speculum figure of 400 times;
Figure 16 is the fabricius bursa section amplification 100 times of compound recipe 1 group of chickling DPI15 and the Electronic Speculum figure of 400 times;
Figure 17 is the fabricius bursa section amplification 100 times of compound recipe 2 groups of chickling DPI15 and the Electronic Speculum figure of 400 times;
Figure 18 is the fabricius bursa section amplification 100 times of compound recipe 3 groups of chickling DPI15 and the Electronic Speculum figure of 400 times;
Figure 19 is the fabricius bursa section amplification 100 times of compound recipe 4 groups of chickling DPI15 and the Electronic Speculum figure of 400 times;
Figure 20 is the fabricius bursa section amplification 100 times of Western medicine 1 group of chickling DPI15 and the Electronic Speculum figure of 400 times;
Figure 21 is the fabricius bursa section amplification 100 times of Western medicine 2 groups of chickling DPI15 and the Electronic Speculum figure of 400 times;
Figure 22 is the fabricius bursa section amplification 100 times of Western medicine 3 groups of chickling DPI15 and the Electronic Speculum figure of 400 times;
Figure 23 is the fabricius bursa section amplification 100 times of Western medicine 4 groups of chickling DPI15 and the Electronic Speculum figure of 400 times;
Figure 24 infects the fabricius bursa section amplification 100 times of matched group chickling DPI15 and the Electronic Speculum figure of 400 times;
Figure 25 is the fabricius bursa section amplification 100 times of blank group chickling DPI15 and the Electronic Speculum figure of 400 times.
Detailed description of the invention
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
The key instrument used in the embodiment of the present invention has: dehydrator, Chinese medicine trace powder beater, blood cell counting plate (Hawksley BS.748), desk type high speed frozen centrifugation (German Eppendorf 5804R), ultramicroscope (Leica 512661), paraffin slicing machine.
Feedstuff described in the embodiment of the present invention be purchased from herding institute of academy of agricultural sciences of Guangdong Province, raw material of Chinese medicine is purchased from the large pharmacy of Wushan, Tianhe District, Guangzhou City Lu Likang hall.
Embodiment 1
By the mixing of the Herba Agrimoniae of the Radix Dichroae of the Herba Artemisiae Annuae of the Pericarpium Citri Reticulatae of the Radix Bupleuri of 30 weight portions, 18 weight portions, 30 weight portions, 30 weight portions, the Radix Sophorae Flavescentis of 30 weight portions and 20 weight portions, play powder preparation by Chinese medicine trace powder beater, obtain Chinese medicine compound 1.
Chinese medicine compound 1 is added in feedstuff, mix homogeneously, obtain No. 1 feedstuff; Wherein the addition of Chinese medicine compound 1 is 0.5% of feedstuff quality.
Embodiment 2
The Radix Notoginseng of the Fructus Cnidii of the Radix Stemonae of the Radix Sophorae Flavescentis of the Radix Dichroae of the Herba Artemisiae Annuae of 30 weight portions, 30 weight portions, 30 weight portions, 18 weight portions, 30 weight portions, 10 weight portions is mixed, plays powder preparation by Chinese medicine trace powder beater, obtain Chinese medicine compound 2.
Chinese medicine compound 2 is added in feedstuff, mix homogeneously, obtain No. 2 feedstuffs; Wherein the addition of Chinese medicine compound 2 is 0.5% of feedstuff quality.
Embodiment 3
Pulverized by Chinese medicine trace powder beater, crossed 120 mesh sieves by the Radix Sanguisorbae of the Radix Dichroae of the Semen Arecae of 18 Fructus Toosendans of weight portion, 12 weight portions, 18 weight portions, the Pericarpium Granati of 10 weight portions and 10 weight portions, mix homogeneously, obtains Chinese medicine compound 3.
Chinese medicine compound 3 is added in feedstuff, mix homogeneously, obtain No. 3 feedstuffs; Wherein the addition of Chinese medicine compound 3 is 0.5% of feedstuff quality.
Embodiment 4
The Herba Agrimoniae of the Radix Sophorae Flavescentis of the Pericarpium Granati of the Fructus Toosendan of the Radix Dichroae of the Herba Artemisiae Annuae of 30 weight portions, 30 weight portions, 18 weight portions, 10 weight portions, 30 weight portions, the Radix Bupleuri of 30 weight portions and 20 weight portions is mixed, play powder preparation by Chinese medicine trace powder beater, obtain Chinese medicine compound 4.
Chinese medicine compound 4 is added in feedstuff, mix homogeneously, obtain No. 4 feedstuffs; Wherein the addition of Chinese medicine compound 4 is 0.5% of feedstuff quality.
Embodiment 5
Soybean trypsin inhibitor-sbti (soybean trypsin inhibitor CAS:9035-81-8) is added in feedstuff by the dosage of 50ppm, mix homogeneously, obtain No. 5 feedstuffs.
Embodiment 6
By Amastatin(amastatin CAS No.67655-94-1) add in feedstuff by the dosage of 75ppm, mix homogeneously, obtains No. 6 feedstuffs.
Embodiment 7
By Pinacol(pinacol CAS No:76-09-5) add in feedstuff by the dosage of 100ppm, mix homogeneously, obtains No. 7 feedstuffs.
Embodiment 8
By DL-α-(Difluoromethyl) arginine (DL-a-(difluoromethyl) arginine CAS No:69955-43-7) add in feedstuff by the dosage of 50ppm, mix homogeneously, obtains No. 8 feedstuffs.
Embodiment 9(effect example):
Experiment material used in the present embodiment is as follows:
Object of study:
CSO(Cryptosporidi μm of Oocysts Cryptosporidium parvum oocysts suspended) by this lab samples gained, for Columba livia source C.baily kind, this laboratory is preserved, use front cleaning grade (no-special pathogen, Specific pathogen Free, SPF) chicken amplification go down to posterity, (SPF chicken is purchased from Guangdong great Hua Nong inventory company limited, report number: 20130216001, kind: BWEL-SPF chicken).
For test chickling: Lingnan Yellow chicken, 1 age in days, male and female dual-purpose, is provided by herding institute of academy of agricultural sciences of Guangdong Province animal experimental center.Be 1 age in days during purchase, 2 ages in days start to test.
The normal birds of immunologic function, Cryptosporidium spp self limiting often, after the hidden sub-worm's ovum capsule of chicken peroral infection in 2 week age, the polypide in body can be removed by the immunoreation of generation, and almost can resist subinfection more completely.In addition, the resistance of chicken to Cryptosporidium has age-dependent, the susceptibility of above the average age for marriage chicken to Cryptosporidium is less, and show as latent period and extend, patent period shortens, symptom is lighter, feed conversion rate and weightening finish also substantially unaffected (Dai Yabin etc., fowl cryptosporidiosis, Chinese Poultry industry guide [J], 2002,19(24): 2-4).So this test and Selection experimental animal age use just hatching 2 Japanese instar chicklings out, first the interference effect of other pathogen is got rid of, other parasitic infection are prevented, secondly, the immune system of chickling is immature, infect C.baileyi(Cryptosporidium baileyi, C.baileyi, Cryptosporidium baileyi) effect relatively good a lot.
The test method that the present embodiment adopts is: will be divided into 10 groups for test chickling, and often organize 10.According to " veterinary Chinese medicine, natural drug clinical trial technological guidance principle ", based on the clinical medicine dose recommended, unified to wherein 9 groups carry out artificially infecting 1 × 10 for test chickling
6the Cryptosporidium (carry out spice medicine feed during chickling 2 age in days, artificially infect Cryptosporidium baileyi during 3 age in days) of an individual egg capsule/dosage, remaining 1 group is that blank group does not infect Cryptosporidium.Feeding environment potassium permanganate and ammonia fumigation, the isolated rearing of each group chickling difference is in the chicken coop of sterilizing through flame burner.In 9 groups of infected group, get 8 groups carry out Drug therapy the previous day in infection of freely drinking water, remaining infected group does not carry out Drug therapy as infection matched group.Concrete scheme is as shown in table 1.
Drug therapy concrete grammar is: 1 ~ No. 8 feedstuff prepared with embodiment 1 ~ 8 carries out free choice feeding and feeds, and feeds continuously until peak period of ovulation terminates.
Wherein blank group and infect matched group and carry out free choice feeding with the feedstuff of not dosing and feed and freely drink water, feeds continuously until peak period of ovulation terminates.
Table 1 experimental program table
(1) the average feed intake of feedstuff and each test chickling group average weight before and after test:
Experimental administration carries out grouping and weigh the previous day.Respectively at DPI0, DPI15 and DPI25 to the chickling of each test group by only weighing, record, calculate average weight and weightening finish; Measure the average feed intake of DPI0 and DPI15.Result is as shown in table 2:
Chickling average weight and each group of average feed intake is respectively organized when table 2DPI0, DPI15, DPI25
In table 2, " * " is statistics symbol: significant difference (P<0.05), and this test uses SPSS software to carry out data analysis.Wherein n represents the number of elements often organized and take chicken at every turn.DPI:Days post inoculated inoculates natural law after egg capsule, and such as DPI15 is after inoculation egg capsule 15 days; DPI25 for inoculation egg capsule after 25 days.
Can see from the data of table 2, C.baileyi(Cryptosporidium baileyi, C.baileyi, Cryptosporidium baileyi) infection make the feed intake of chickling and body weight all present downward trend, 4 groups of compound recipe groups with infect the weightening finish had compared with matched group to a certain degree, the average weight that compound recipe is 1 group is the highest, but also all lower than blank group.In feed intake, C.baileyi(Cryptosporidium baileyi, C.baileyi, Cryptosporidium baileyi) infect matched group and be starkly lower than each compound recipe group, compound recipe 1 group and compound recipe 4 groups are closest to blank group.DPI15 compound recipe 1 ~ 4 group, 1 ~ 2 group, Western medicine and blank group diversity are significantly (P<0.05); DPI25 compound recipe 4 groups, 1 group, Western medicine, infection matched group and blank group diversity are significantly (P<0.05).The feedstuff being added with Chinese medicine compound prepared by compound recipe 1 ~ 4 group i.e. embodiment 1 ~ 4 can improve infection C.baileyi(Cryptosporidium baileyi to a certain extent, C.baileyi, Cryptosporidium baileyi) function of chickling body, impel feed intake and body weight to increase all to some extent.Integrated comparative, in Chinese drug-treated group, compound recipe 3 groups of effects are best, the claimed compound recipe group of namely the present invention.
(2) feces egg capsule counting after infecting:
After Chickens Infected, every day collects feces, and is stored in 4 DEG C of refrigerators, checks and counting within 24h.Egg capsule is checked with Sheater's sugar flotation technique.If check result shows that Cryptosporidium is positive, then take the dilution of 5g feces 30mL tap water, through 60 orders and the sieved filter of 100 order copper, the centrifugal 10min of filtrate 3000rpm, abandons supernatant, and precipitation adds the saturated sucrose solution of 15mL and stirs evenly, the centrifugal 10min of 3000rpm.Get saturated sucrose solution and the mixing of centrifugal egg capsule precipitation, with blood cell counting plate counting, average computation five block plaid are averaged, and calculate egg sac number.
Through observing, infect matched group clinical symptoms: DPI9-DPI14 is C.baileyi(Cryptosporidium baileyi, C.baileyi, Cryptosporidium baileyi) peak period, show respiratory symptom, indivedual is cough and rale, the mental status is slightly poor, does not like.During DPI12-DPI15, each infected group finds there is blood sample feces in feces, and indivedual chickling has proctoptosis phenomenon, infects matched group and infects rear 12nd day feces as shown in Figure 1.During DPI15, each infected group test chickling respiratory symptom alleviates, and cuts open each infected group chickling of inspection visible annulus trachealis mucosa hyporrhea, toughness secretions; Fabricius bursa enlargement, hemorrhage, inside there is more viscous secretion.
Get annulus trachealis Mucosal secretions and fabricius bursa secretions to each infected group chickling, through the floating microscopy of saturated sucrose solution, the visible a large amount of Cryptosporidium parvum oocysts suspended of fabricius bursa, annulus trachealis egg sac number is relatively few, and accompanying drawing 2 shines for infecting matched group the 8th day fabricius bursa 400 times of microscopies.
Continuous Observation respectively organizes chickling at row's egg sac number of the 1 to 15 day, and result is as shown in accompanying drawing 3, accompanying drawing 4 and accompanying drawing 5.Can see from accompanying drawing 3 ~ 5, the incubation period of Cryptosporidium baileyi is 4d, and peak period concentrates on 9-12d.Compound recipe 1 ~ 4 group is compared with infection matched group, and all decreases to some degree, particularly compound recipe 3 groups on row's egg sac number, egg sac number significantly decreases trend.Wherein mechanism infers it is anthelmintic action in the body that has due to the Fructus Toosendan, Semen Arecae, Radix Dichroae, Pericarpium Granati etc. in compound recipe 3 groups of Chinese medicine compound, and Radix Sanguisorbae has cooling blood for hemostasis antipyretic and antidote functions, and improving immunity to body also has corresponding effect.Fabricius bursa is the central immune organ of birds, can produce bone-marrow-derived lymphocyte, thus generation specific antibody completes specific immunne response.And fabricius bursa is C.baileyi(Cryptosporidium baileyi, C.baileyi, Cryptosporidium baileyi) one of main parasitic organ, after Chickens Infected, immunosuppressant and defect can be caused, destroy the immune system of body on the one hand, chickling immunity of organisms is caused to decline, easily excite the secondary infection that other pathogen cause, on the other hand, C.baileyi(Cryptosporidium baileyi, C.baileyi, Cryptosporidium baileyi) effect itself, make chickling show a series of clinical symptoms.The effect of Chinese medicine compound of the present invention (i.e. compound recipe 3 groups) is mainly started with from anthelmintic these two aspects in enhancing human body immunity power and body, and synthesis result plays a role.
(3) Immune Organs Index:
Chickens Infected Cryptosporidium baileyi, in DPI0, DPI15 and DPI25, often organize stochastical sampling, getting its fabricius bursa and spleen divides another name its quality, calculate bursal index ［ fabricius bursa quality (mg)/body weight (g) ］ and index and spleen index ［ spleen weight (mg)/body weight (g) ］, average.Index and spleen index result is as shown in table 3, and bursal index is as shown in table 4:
Chickling index and spleen index is respectively organized when table 3DPI0, DPI15, DPI25
In table 2, " * " is statistics symbol: significant difference (P<0.05), and this test uses SPSS software to carry out data analysis.Wherein n represents the quantity of the chicken that every group is chosen.DPI:Days post inoculated inoculates natural law after egg capsule, and such as DPI15 is after inoculation egg capsule 15 days; DPI25 for inoculation egg capsule after 25 days.
Chickling bursal index is respectively organized when table 4DPI0, DPI15, DPI25
In table 2, " * " is statistics symbol: significant difference (P<0.05), and this test uses SPSS software to carry out data analysis.Wherein n represents the quantity of the chicken that every group is chosen.DPI:Days post inoculated inoculates natural law after egg capsule, and such as DPI15 is after inoculation egg capsule 15 days; DPI25 for inoculation egg capsule after 25 days.
Can find out from the data table 3 and table 4, compound recipe 1 ~ 4 group of spleen to Cryptosporidium parvum oocysts suspended chickling and fabricius bursa grow the effect all had in various degree.Compound recipe 1 ~ 4 group before infection the phase to chickling Development of The Spleen without obvious facilitation, compound recipe 3 groups in index and spleen index DPI25, infect matched group and blank group diversity remarkable (P<0.05); In bursal index DPI25, compound recipe 4 groups and blank group diversity are significantly (P<0.05).Compound recipe 2 groups and Western medicine 1 group of spleen value follow blank group closest.Phase grows the facilitation all had in various degree to compound recipe 1 ~ 4 group to chickling fabricius bursa before infection, due to the main parasitic organ that fabricius bursa is Cryptosporidium baileyi, during DPI25, compound recipe 1 ~ 4 group of chickling bursal index, compared with blank group, all shows bursal index and reduces.But compared with infection matched group, except compound recipe 2 groups, each dosage group all has the growth promoting fabricius bursa.
Immune Organs Index is the important indicator of Immune Organs of Body growth promoter degree, immune level (the Yue Daoyou of body can be reflected to a certain extent, Zhao Jinfeng, Li Jiacheng, Deng. Chong Zuji alpha interferon is to the therapeutic effect [J] of the tentative Cryptosporidium baileyi disease of chickling. Agricultural University Of He'nan's journal, 2011,45 (1): 102-106.).Result of the test shows, and Cryptosporidium baileyi makes the atrophy of chickling fabricius bursa diminish, the enlargement of spleen pathological proliferation after infecting chickling, by the spice feeding of compound recipe 1 ~ 4 group, has contrast, all have improvement to a certain degree with infection matched group.
C.baileyi(Cryptosporidium baileyi, C.baileyi, Cryptosporidium baileyi) infect after percentage ratio change (table 3) of different times chickling spleen percentage of liveweight show: each group chickling index and spleen index no difference of science of statistics (P>0.05) before infecting, after infecting 15, the percentage ratio of 25d infection experiment group spleen percentage of liveweight in various degree higher than blank group, infect matched group higher than other each test group.Wherein, compound recipe 3 groups, infection matched group and blank group diversity remarkable (P<0.05) in DPI25.Visible, infect the growth seeming have stimulated spleen in early days, spleen is allowed to occur hypertrophy to a certain degree, this result of the test bright with swimming loyalty is to identical (You Zhongming, Jiang Jinshu. Cryptosporidium baileyi infects the morphological observation [J] of immune organs of chicken treated. Scientia Agricultura Sinica, 1998,31 (4) .).The same with the relation of anti-infectious immunity as infection, in C.baileyi invasive procedure, chickling spleen is to C.baileyi(Cryptosporidium baileyi, C.baileyi, Cryptosporidium baileyi) infect immunohistology reaction with C.baileyi(Cryptosporidium baileyi, C.baileyi, Cryptosporidium baileyi) infect the Immunohistological lesion of chickling spleen is occurred together.Infect in early days while causing certain Immunohistological lesion, have stimulated the growth of chickling spleen, cause immunohistology reaction to a certain degree, cause infected group spleen to increase weight.In addition, each group chickling index and spleen index no difference of science of statistics (P>0.05) before infecting, 15d after infecting, the percentage ratio of 25d infection experiment group fabricius bursa percentage of liveweight is lower than blank group, except DPI25 compound recipe 2 groups, infect matched group bursal index all lower than other each experimental grouies, DPI25 compound recipe 4 groups and blank group are diversity remarkable (p>0.05), 1 ~ 25d C.baileyi(Cryptosporidium baileyi after visible infection, C.baileyi, Cryptosporidium baileyi) obvious inhibitory action is created to the normal development of fabricius bursa, this report loyal bright with trip is runed counter to, (Yue Daoyou identical with the conclusion that Yue Daoyou studies, Zhao Jinfeng, Li Jiacheng, Deng. Chong Zuji alpha interferon is to the therapeutic effect [J] of the tentative Cryptosporidium baileyi disease of chickling. Agricultural University Of He'nan's journal, 2011, 45 (1): 102-106.).
(4) section pathological change:
According to infection chickling test oocysts excreting regulation, draw when about DPI15 to be ovulation capsule peak latter stage, the now infringement of Cryptosporidium baileyi to immune organ is maximum, therefore annulus trachealis when getting DPI15 and fabricius bursa tissue make tissue slice, observation pathological change.
The concrete steps of microsection manufacture are as follows:
1. draw materials and fix: get fresh fabricius bursa tissue, annulus trachealis tissue, 10% formalin is fixed;
2. rinse: the piece of tissue after fixing must wash away fixative with flowing water before dewatering, the tissue that formaldehyde fixative is fixed generally washes more than 6 hours, also can wash and spend the night;
3. dewater: transparent and waxdip commonly uses the dehydration of alcohol of variable concentrations, alcohol concentration used and process as follows: 70%(mono-night)---80%(2 hour)---90%(1 hour)---95%(1 hour)---100% (I) (30 minutes)---100% (II) (15 minutes)---dimethylbenzene (15 minutes)---dimethylbenzene (10 minutes)---paraffin (2 ~ 2.5 hours)
4. embed: the piece of tissue of having infiltrated is embedded in impregnant (paraffin) by embedding exactly, and carries out numbering labelling;
5. cut into slices: cut into slices after wax stone molding;
6. open up sheet and bonding die: slice thickness 4 μm, the warm water of 42 DEG C extends piece, and wax disk(-sc) fishes for wax disk(-sc) after fully flattening by the time, puts 45 DEG C and dries the oven dry of sheet machine, then can dye;
7. dyeing and sealing: dyeing adopts HE(Hematoxylin-eosin) dyeing, process is as follows:
Get the section of drying, be put in the interior dewaxing of dye vat about the 10min filling dimethylbenzene, take off the section after wax can dye, concrete dyeing course and the time as follows: (1) 100% ethanol 2 ~ 5min, (2) 95% ethanol 2 ~ 5min, (3) 90% ethanol 2 ~ 5min, (4) 80% ethanol 2 ~ 5min, (5) 70% ethanol 2 ~ 5min, (6) washing 1 ~ 2min is distilled, (7) haematoxylin dye liquor 10 ~ 20min, (8) common washing 1min, (9) the hydrochloride alcohol differentiation several seconds, (10) tap water 30min, (11) 70% ethanol 2 ~ 5min, (12) 80% ethanol 2 ~ 5min, (13) 95% ethanol 2 ~ 5min, (14) eosin stains 20s, (15) anhydrous alcohol I 3min, (16) anhydrous alcohol II 3min, (17) anhydrous alcohol adds equivalent dimethylbenzene 2min, (18) dimethylbenzene number is minute to transparent, (19) in dimethylbenzene, section is taken out, be placed in lattice plate, drip a small amount of natural gum in tissue central authorities, (20) coverslip is got, gently with coverslip while close to microscope slide, then coverslip is put down rapidly, namely natural gum be expanded to surrounding rapidly, correct coverslip direction.The section of sealing is placed in calorstat dry.
Finally, the loose colour in dried section is cleaned, and be stained with label at the left end of slide, indicate the title of tissue slice, colouring method, fixing means and date.
Observed result is as shown in accompanying drawing 6 to accompanying drawing 25.Can see from accompanying drawing 6 to accompanying drawing 25, except blank group, other each infection experiment group fabricius bursa and annulus trachealis are lost all in various degree, infect matched group performance serious.
Infecting matched group annulus trachealis ring mucosa comes off seriously, and the epithelium generation atrophy of mucosal surface, microvillus does not almost have, only remaining very thin one deck, and has inflammatory cell infiltration phenomenon.The inner mucosa of blank group annulus trachealis ring is complete, and thickness is even, and the microvillus marshalling of mucosal surface, without inflammatory cell infiltration.Compound recipe 1 ~ 4 group all has pathological change in various degree, seriously compound recipe 2 groups and compound recipe 4 groups, and mucosal surface partial exfoliation, presents irregular wavy.Compound recipe 3 groups and compound recipe 4 groups of annulus trachealis surface mucosa are relative complete, and microvillus substantially also exists, and compound recipe 1 group of annulus trachealis uneven distribution is even, and surface mucosa is Old City Wall shape, and compound recipe 4 groups is also incomplete.Western medicine 1 ~ 2 group of pathological change is serious, and coming off appears in mucous layer, presents Old City Wall shape.Western medicine 3 ~ 4 groups of relative performance's is not obvious.
Can see from fabricius bursa slice map, the relative Western medicine of compound recipe 1 ~ 4 group 1 ~ 4 group of effect will be got well.Infect matched group fabricius bursa section sample can see, 400 times of lower fabricius bursa surface mucosa adhere to many Cryptosporidiums, and topical epithelial is thickening because of hyperplasia, and another part epithelium then atrophy occurs; Thus epithelium is uneven, and in irregular wave wave-like, and then develops into protruding and the recessed Old City Wall shape replaced; The visible macrophage of epithelium.Interstitial connective tissues hypertrophy, part connective tissue enters folliculus, and folliculus is split; The congestion of blood vessel is hemorrhage, and hyaline thrombus appears in part blood vessel.
In sum, treatment by Chinese herbs effect is better than Western medicine relatively, infers that the drug resistance with Western medicine uses relevant, and the use of Western medicine can increase the weight of the injury of immune organ, make pathological manifestations more obvious, compound recipe 3 groups and Western medicine 4 groups of pathological changes the faintest, it is optimum that therapeutic effect has been reacted in side.
The present embodiment is evaluated 4 kinds of herbal mixtures by multiple test index, wherein chickling body weight change and feed intake, fabricius bursa and trachea detection Cryptosporidium baileyi egg sac number, tissue slice pathological change three index expression compound recipes 3 groups of effects are better than other compound recipe groups, Immune Organs Index achievement data analysis performance is not remarkable, but has certain treatment trend.
The present embodiment by four kinds of Chinese medicine compound and four kinds of Western medicine respectively to C.baileyi(Cryptosporidium baileyi, C.baileyi, Cryptosporidium baileyi) infection model carry out therapeutic test, grope its treatment active drug, respectively from chickling different times feed intake and gain in weight, clinical symptoms observation of symptoms and oocysts excreting regulation, immune indexes, the aspects such as pathological section are observed, comprehensive discovery, each group of medicine all has certain effect, wherein compound recipe 3 groups i.e. Chinese medicine compound general performance of the present invention is optimum, row's egg sac number and feed intake gain in weight all take an evident turn for the better, Western medicine group Chinese medicine and western medicine 4 groups of general performances are optimum.
Western medicine group 4 kinds of medicines all possess some special knowledge in the test of the enzymatic activity of Cryptosporidium and Cell culture invitro test, all show certain therapeutic effect, this test is got these 4 kinds of Western medicine and is carried out the research of body cylinder therapeutic effect, on the one hand return zoopery to evaluate medication effect, be also on the one hand to herbal mixture effect comparative evaluation.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (3)
1. prevent and treat a Chinese medicine for poultry Cryptosporidium spp, it is characterized in that, described Chinese medicine is made up of the raw material of following weight portion: Fructus Toosendan 18 weight portion, Semen Arecae 12 weight portion, Radix Dichroae 18 weight portion, Pericarpium Granati 10 weight portion and Radix Sanguisorbae 10 weight portion.
2. the preparation method of the Chinese medicine of control poultry Cryptosporidium spp according to claim 1, is characterized in that, described preparation method is by Fructus Toosendan, Semen Arecae, Radix Dichroae, Pericarpium Granati and Radix Sanguisorbae pulverizing, crosses 120 mesh sieves, and mix homogeneously obtains described Chinese medicine.
3. the Chinese medicine of control poultry Cryptosporidium spp according to claim 1 is preparing the application in animal and fowl fodder, and it is characterized in that, described Chinese medicine adds in animal and fowl fodder, and addition is 0.5% of animal and fowl fodder quality.
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