CN103349718A - 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 of Chinese medicine compound of preventing and treating the poultry Cryptosporidium spp and preparation method thereof and application.
Background technology
Cryptosporidiosis (cryptosporidiosis) is to be universal infecting both domestic animals and human protozoacide by what Cryptosporidium caused, is a kind of important paathogenic factor that causes man and animal acute diarrhea or chronic lethal diarrhoea.The main intestinal tract disease that is only second to rotavirus.Cryptosporidium extensively parasitizes mammal, birds, amphibian, Fish and people's digestive tract and other organ, and carries out a large amount of breedings at the parasitism place, causes the digestive and absorptive functions obstacle, causes animal diarrhoea and inapparent infection.Having now found that in the human case of 106 countries, is the important paathogenic factor of human body acute diarrhea or chronic lethal diarrhoea.Cryptosporidium spp comprises people's 260 many animals, studies show that, Cryptosporidium is mainly propagated by routes of transmission such as contact, drinking-water, food and the spittle, and the egg capsule of hidden spore worm environment to external world particularly (comprises O to chlorination
3Deng) very strong resistance is arranged so that traditional handicraft be difficult to its removal or kill (Feng Lihong. cryptosporidiosis progress [J]. preventive medicine journal of information, 2006 (1): 46-48.).Since this disease of reported first in 1976, there is the case of outburst in each continent in the whole world, and is wherein the most representative in the outburst of the U.S..Tracing to the source studies confirm that, has the Cryptosporidium behaviour Cryptosporidium of 2 great outbursts at least, and 1 time is Cryptosporidium parvum, but has the Cryptosporidium that repeatedly causes this disease outburst to derive from pig and cattle, companion animals and derive from poultry.These results have fully shown the threat of animal sources Cryptosporidium to human health.
Guangdong Province's located in subtropical zone, livestock and poultry cultivation density is large, and the sewage that produces for livestock and poultry cultivation at present, the processing of feces cause it that production, living environment and river water have been caused severe contamination owing to lacking general cost-effective disposal technology; In addition, area, Pearl River Delta population density is very big, it is reported, water source in Guangdong Province's is more single, mainly is surface water.In the city, the river course supplies water and accounts for 88%, and reservoir only accounts for 12%.Because the egg capsule of Cryptosporidium is environment to external world, particularly chlorination is had very strong resistance, traditional sterilization process is difficult to it is killed, in case the poultry polluted 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 derives from the C.felis of companion animals and 2 important Cryptosporidium worm kinds that C.canis causes the people to infect just (Dubey, 2009).
Although bibliographical information is arranged, some medicines have certain therapeutic effect to cryptosporidiosis, external present useful spiramycin, paromomycin treatment patient, also obtain curative effect with artificial high immune foremilk (hyperimmune bovine colostr μ m, HBC), Lac Bovis seu Bubali globulin, cattle transfer factor treatment.For example: (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, can use the Western medicine such as soybean trypsin inhibitor-sbti (soybean trypsin inhibitor) to carry out the control of Cryptosporidium.
Domestic Cryptosporidium spp with 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 medicines such as the ethanol extraction of Cortex Meliae, arecoline, Toosendanin, Semen Cucurbitae, artesunate, Fructus Quisqualis have preferably anthelminthic effect, and side effect is little.The medical material that cooperates simultaneously invigorating the stomach and promoting digestion, invigorating the spleen and stomach, invigorating QI and blood can greatly be alleviated the organ injury such as the intestines and stomach, dysfunction and the malnutrition that parasite causes and become thin.The anthelmintic Chinese medicine of using clinically at present adopts the multi-flavor medical material to be composited, existing plant antiscolic has again stomach invigorating and food calling composition (Liu Zonghu, Xia Weixin, to open beautiful. scale pig farm Chinese medicine and western medicine arrived [J] in conjunction with the epoch of anthelmintic. northern animal husbandry, 2008 (18) .).
But even to this day, there is no definite effectively medicine, vaccine for cryptosporidiosis emerges.Badly influence g and D and the human health of animal because of this disease, can cause the tremendous economic loss.For the most effective mode of prevention and control of cryptosporidiosis no more than clear and definite its epidemiology background, reduce infection chance (rich Chun-shan Mountain, Li Yu comes. the prevention and control of cryptosporidiosis [J]. herding and forage science, 2010 (Z2): 201-202.).Therefore, at present in the urgent need to studying a kind of medicine that can prevent and treat the 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 the poultry Cryptosporidium spp.
Another object of the present invention is to provide the preparation method of the Chinese medicine compound of above-mentioned control poultry Cryptosporidium spp.
A further object of the present invention is to provide the application of the Chinese medicine compound of above-mentioned control poultry Cryptosporidium spp.
For achieving the above object, the present invention adopts following technical scheme:
A kind of Chinese medicine compound of preventing and treating the poultry Cryptosporidium spp, described Chinese medicine compound is comprised 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 the Chinese medicine compound of the present invention, Semen Arecae, Radix Dichroae, Pericarpium Granati etc. have, Radix Sanguisorbae has the cooling blood for hemostasis antipyretic and antidote functions, and body is improved immunity also corresponding effect, can effectively prevent and treat Cryptosporidium.
The used raw material of Chinese medicine of the present invention all can be buied from the Chinese medicine shop, and its specification meets national medical standard.
Preferably, described Chinese medicine compound is comprised of the raw material of following weight portion: Fructus Toosendan 18 weight portions, Semen Arecae 12 weight portions, Radix Dichroae 18 weight portions, Pericarpium Granati 10 weight portions and Radix Sanguisorbae 10 weight portions.
The preparation method of the Chinese medicine compound of above-mentioned control poultry Cryptosporidium spp is pulverized Fructus Toosendan, Semen Arecae, Radix Dichroae, Pericarpium Granati and Radix Sanguisorbae, cross 120 mesh sieves, and mix homogeneously obtains described Chinese medicine compound.
The application of the Chinese medicine compound of above-mentioned control poultry Cryptosporidium spp in the preparation animal and fowl fodder, described Chinese medicine compound is added in the 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, the residual and resistant characterization of antibiotic-free, and control has significant curative effect to cryptosporidiosis.And the present invention fills a prescription and mainly considers with anthelmintic and two aspects of raising immunity of organism effect, uses trace to beat the powder technology medicine is effectively mixed, and with respect to existing anthelmintic Western medicine, has medicine irritability little, the advantage that trap is good.
Description of drawings
Fig. 1 is for infecting the feces figure of matched group chickling DPI12;
Fig. 2 detects figure for Cryptosporidium parvum oocysts suspended in the fabricius bursa that infects matched group chickling DPI8 amplifies 400 times;
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 that infects matched group and blank group chickling DPI1-15;
Fig. 5 is Western medicine group, the oocysts excreting regulation figure that infects matched group and blank group chickling DPI1-15;
Fig. 6 is the Electronic Speculum figure that 100 times and 400 times are amplified in the trachea section of 1 group of chickling DPI15 of compound recipe;
Fig. 7 is the Electronic Speculum figure that 100 times and 400 times are amplified in the trachea section of 2 groups of chickling DPI15 of compound recipe;
Fig. 8 is the Electronic Speculum figure that 100 times and 400 times are amplified in the trachea section of 3 groups of chickling DPI15 of compound recipe;
Fig. 9 is the Electronic Speculum figure that 100 times and 400 times are amplified in the trachea section of 4 groups of chickling DPI15 of compound recipe;
Figure 10 is the Electronic Speculum figure that 100 times and 400 times are amplified in the trachea section of 1 group of chickling DPI15 of Western medicine;
Figure 11 is the Electronic Speculum figure that 100 times and 400 times are amplified in the trachea section of 2 groups of chickling DPI15 of Western medicine;
Figure 12 is the Electronic Speculum figure that 100 times and 400 times are amplified in the trachea section of 3 groups of chickling DPI15 of Western medicine;
Figure 13 is the Electronic Speculum figure that 100 times and 400 times are amplified in the trachea section of 4 groups of chickling DPI15 of Western medicine;
Figure 14 amplifies the Electronic Speculum figure of 100 times and 400 times for the trachea section of infecting matched group chickling DPI15;
Figure 15 is the Electronic Speculum figure that 100 times and 400 times are amplified in the trachea section of blank group chickling DPI15;
Figure 16 is the Electronic Speculum figure that 100 times and 400 times are amplified in the fabricius bursa section of 1 group of chickling DPI15 of compound recipe;
Figure 17 is the Electronic Speculum figure that 100 times and 400 times are amplified in the fabricius bursa section of 2 groups of chickling DPI15 of compound recipe;
Figure 18 is the Electronic Speculum figure that 100 times and 400 times are amplified in the fabricius bursa section of 3 groups of chickling DPI15 of compound recipe;
Figure 19 is the Electronic Speculum figure that 100 times and 400 times are amplified in the fabricius bursa section of 4 groups of chickling DPI15 of compound recipe;
Figure 20 is the Electronic Speculum figure that 100 times and 400 times are amplified in the fabricius bursa section of 1 group of chickling DPI15 of Western medicine;
Figure 21 is the Electronic Speculum figure that 100 times and 400 times are amplified in the fabricius bursa section of 2 groups of chickling DPI15 of Western medicine;
Figure 22 is the Electronic Speculum figure that 100 times and 400 times are amplified in the fabricius bursa section of 3 groups of chickling DPI15 of Western medicine;
Figure 23 is the Electronic Speculum figure that 100 times and 400 times are amplified in the fabricius bursa section of 4 groups of chickling DPI15 of Western medicine;
Figure 24 amplifies the Electronic Speculum figure of 100 times and 400 times for the fabricius bursa section of infecting matched group chickling DPI15;
Figure 25 is the Electronic Speculum figure that 100 times and 400 times are amplified in the fabricius bursa section of blank group chickling DPI15.
The specific embodiment
The present invention is described in further detail below in conjunction with embodiment and accompanying drawing, but embodiments of the present invention are not limited to this.
The key instrument of using in the embodiment of the 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 invention be purchased from academy of agricultural sciences, Guangdong Province herding institute, raw material of Chinese medicine is purchased from the large pharmacy of Wushan, Tianhe District, Guangzhou City Lu Likang hall.
With the Radix Bupleuri of 30 weight portions, the Pericarpium Citri Reticulatae of 18 weight portions, the Herba Artemisiae Annuae of 30 weight portions, the Radix Dichroae of 30 weight portions, the Radix Sophorae Flavescentis of 30 weight portions and the Herba Agrimoniae mixing of 20 weight portions, play the powder preparation by Chinese medicine trace powder beater, obtain Chinese medicine compound 1.
With the Herba Artemisiae Annuae of 30 weight portions, the Radix Dichroae of 30 weight portions, the Radix Sophorae Flavescentis of 30 weight portions, the Radix Stemonae of 18 weight portions, the Fructus Cnidii of 30 weight portions, the Radix Notoginseng mixing of 10 weight portions, play the powder preparation by Chinese medicine trace powder beater, obtain Chinese medicine compound 2.
18 Fructus Toosendans of weight portion, the Semen Arecae of 12 weight portions, the Radix Dichroae of 18 weight portions, the Pericarpium Granati of 10 weight portions and the Radix Sanguisorbae of 10 weight portions are pulverized, cross 120 mesh sieves by Chinese medicine trace powder beater, and mix homogeneously obtains Chinese medicine compound 3.
With the Herba Artemisiae Annuae of 30 weight portions, the Radix Dichroae of 30 weight portions, the Fructus Toosendan of 18 weight portions, the Pericarpium Granati of 10 weight portions, the Radix Sophorae Flavescentis of 30 weight portions, the Radix Bupleuri of 30 weight portions and the Herba Agrimoniae mixing of 20 weight portions, play the powder preparation by Chinese medicine trace powder beater, obtain Chinese medicine compound 4.
The dosage of soybean trypsin inhibitor-sbti (soybean trypsin inhibitor CAS:9035-81-8) being pressed 50ppm adds in the feedstuff, and mix homogeneously obtains feedstuff No. 5.
With Amastatin(amastatin CAS No.67655-94-1) to press in the dosage adding feedstuff of 75ppm, mix homogeneously obtains feedstuff No. 6.
With Pinacol(pinacol CAS No:76-09-5) to press in the dosage adding feedstuff of 100ppm, mix homogeneously obtains feedstuff No. 7.
With arginine (DL-a-(difluoromethyl) the arginine CAS No:69955-43-7 of DL-α-(Difluoromethyl)) dosage of pressing 50ppm adds in the feedstuff, and mix homogeneously obtains feedstuff No. 8.
Embodiment 9(effect embodiment):
Experiment material used in the present embodiment is as follows:
Object of study:
CSO(Cryptosporidi μ m Oocysts Cryptosporidium parvum oocysts suspended) by this laboratory sampling gained, be Columba livia source C.baily kind, this laboratory is preserved, use a cleaning level (no-special pathogen before using, Specific pathogen Free, SPF) chicken amplification goes down to posterity, and (the SPF chicken is purchased from the agricultural inventory company limited of the large China in Guangdong, report number: 20130216001, kind: the BWEL-SPF chicken).
For testing chickling: the Lingnan Yellow chicken, 1 age in days, the male and female dual-purpose is provided by academy of agricultural sciences, Guangdong Province herding institute zoopery center.Be 1 age in days during purchase, 2 ages in days begin to do experiment.
The normal birds of immunologic function, Cryptosporidium spp be self limiting often, and behind the chicken peroral infection hidden sub-worm's ovum capsule in 2 ages in week, the immunoreation of generation can be removed the polypide in the body, and almost completely can resist reinfect.In addition, chicken has age-dependent to the resistance of Cryptosporidium, above the average age for marriage chicken is less to the susceptibility of Cryptosporidium, shows as latent period and prolongs, and patent period shortens, symptom is lighter, feed conversion rate and weightening finish be substantially unaffected (Dai Yabin etc., fowl cryptosporidiosis, Chinese Poultry industry guide [J] also, 2002,19(24): 2-4).So this test and Selection experimental animal age is used just hatching 2 Japanese instar chicklings out, the interference effect of at first getting rid of other pathogen, other parasitic infection have been 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, 10 every group for the test chickling.According to " veterinary Chinese medicine, natural drug clinical trial technological guidance principle ", take the clinical medicine dose recommended as the basis, unified to wherein 9 groups artificially infect 1 * 10 for the test chickling
6The Cryptosporidium of an individual egg capsule/dosage (carry out the spice medicine feed during chickling 2 age in days, artificially infect Cryptosporidium baileyi during 3 age in days), remaining 1 group is that the blank group does not infect Cryptosporidium.Feeding environment potassium permanganate and ammonia fumigation, each organizes the isolated rearing of chickling difference in the chicken coop of flame burner sterilization.Get 8 groups and carry out Drug therapy the previous day freely drinking water to infect in 9 groups of infected group, remaining infected group is not carried out Drug therapy as infecting matched group.Concrete scheme is as shown in table 1.
The Drug therapy concrete grammar is: carry out free choice feeding with 1～No. 8 feedstuff of embodiment 1～8 preparation and feed, feed continuously until peak period of ovulation finishes.
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 is fed continuously until the peak period of ovulation end.
Table 1 experimental program table
(1) the average feed intake of feedstuff and each test chickling group average weight before and after the test:
The experiment administration divides into groups to weigh the previous day.Respectively at DPI0, DPI15 and DPI25 to the chickling of each test group by only weigh, record, calculate average weight and weightening finish; Measure DPI0 and the average feed intake of DPI15.The result is as shown in table 2:
Table 2DPI0, DPI15 respectively organizes the chickling average weight during DPI25 and each organizes average feed intake
" * " is the statistics symbol in the table 2: significant difference (P＜0.05), this test use SPSS software to carry out data analysis.Wherein n represents every group of number of elements that at every turn takes by weighing chicken.Natural law behind the DPI:Days post inoculated inoculation egg capsule, for example DPI15 for the inoculation egg capsule after 15 days; DPI25 for the inoculation egg capsule after 25 days.
From the data of table 2, can see, C.baileyi(Cryptosporidium baileyi, C.baileyi, Cryptosporidium baileyi) infection makes the feed intake of chickling and body weight all present downward trend, 4 groups of compound recipe groups are compared the weightening finish that has to a certain degree with the infection matched group, the average weight that compound recipe is 1 group is the highest, but also all is lower than the blank group.On the feed intake, C.baileyi(Cryptosporidium baileyi, C.baileyi, Cryptosporidium baileyi) infect matched group and be starkly lower than each compound recipe group, 4 groups of 1 group of compound recipe and compound recipes are near the blank group.1～4 group of DPI15 compound recipe, 1～2 group in Western medicine and blank group diversity be (P＜0.05) significantly; 4 groups of DPI25 compound recipes, 1 group in Western medicine, infection matched group and blank group diversity be (P＜0.05) significantly.1～4 group of feedstuff that is added with Chinese medicine compound that is embodiment 1～4 prepares of compound recipe can improve infection C.baileyi(Cryptosporidium baileyi to a certain extent, C.baileyi, Cryptosporidium baileyi) function of chickling body impels feed intake and body weight all to increase to some extent.Relatively comprehensive, 3 groups of effects of compound recipe are best in the Chinese drug-treated group, the claimed compound recipe group of namely the present invention.
(2) infect rear feces egg capsule counting:
Collect feces every day behind the Chickens Infected, and be stored in 4 ℃ of refrigerators, checks within 24h and counting.Check egg capsule with Sheater's sugar flotation technique.If it is positive that check result shows Cryptosporidium, then take by weighing 5g feces with the dilution of 30mL tap water, filter through 60 orders and 100 order copper sieve, 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 centrifugal egg capsule precipitation mixing, with the blood cell counting plate counting, five large grids of average computation are averaged, and calculate egg sac number.
Through observing, infect the matched group clinical symptoms: DPI9-DPI14 is C.baileyi(Cryptosporidium baileyi, C.baileyi, Cryptosporidium baileyi) peak period shows respiratory symptom, and is indivedual for coughing and rale, the mental status is slightly poor, does not like.Each infected group finds in the feces blood sample feces is arranged during DPI12-DPI15, and indivedual chickling have the proctoptosis phenomenon, infects matched group and infects rear the 12nd day feces as shown in Figure 1.Each infected group test chickling respiratory symptom alleviates during DPI15, cuts open the visible annulus trachealis mucosa of each infected group chickling of inspection hyporrhea, toughness secretions; Fabricius bursa enlargement, hemorrhage, in more viscous secretion is arranged.
Each infected group chickling is got annulus trachealis mucosa secretions and fabricius bursa secretions, through the floating microscopy of saturated sucrose solution, the visible a large amount of Cryptosporidium parvum oocysts suspendeds of fabricius bursa, the annulus trachealis egg sac number is relatively few, and accompanying drawing 2 shines for infecting 400 times of microscopies of the 8th day fabricius bursa of matched group.
Continuous Observation is respectively organized chickling at row's egg sac number of the 1st to 15 day, and the result is shown in accompanying drawing 3, accompanying drawing 4 and accompanying drawing 5.Can see from accompanying drawing 3～5, be 4d the incubation period of Cryptosporidium baileyi, and the peak period concentrates on 9-12d.1～4 group of compound recipe is compared with infecting matched group, all decreases to some degree on row's egg sac number, and particularly compound recipe is 3 groups, and trend significantly decreases on the egg sac number.Wherein mechanism infers it is the interior anthelmintic action of body that has owing to the Fructus Toosendan in 3 groups of Chinese medicine compound of compound recipe, Semen Arecae, Radix Dichroae, Pericarpium Granati etc., and Radix Sanguisorbae has the cooling blood for hemostasis antipyretic and antidote functions, and body is improved immunity also corresponding effect.Fabricius bursa is the central immune organ of birds, can produce bone-marrow-derived lymphocyte, finishes specific immunne response thereby produce specific antibody.And fabricius bursa is C.baileyi(Cryptosporidium baileyi, C.baileyi, Cryptosporidium baileyi) one of main parasitic organ, behind the Chickens Infected, can cause immunosuppressant and defective, destroy on the one hand the immune system of body, cause the chickling immunity of organisms to descend, the secondary infection that easily excites other pathogen to cause, on the other hand, C.baileyi(Cryptosporidium baileyi, C.baileyi, Cryptosporidium baileyi) effect of itself makes chickling show a series of clinical symptoms.The effect of Chinese medicine compound of the present invention (being 3 groups of compound recipes) is this two aspects of anthelmintic in enhancing human body immunity power and the body mainly, and synthesis result plays a role.
(3) Immune Organs Index:
The Chickens Infected Cryptosporidium baileyi, in DPI0, DPI15 and DPI25, every group of stochastical sampling, getting its fabricius bursa and spleen divides another name its quality, calculate fabricius bursa 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 the fabricius bursa index is as shown in table 4:
Table 3DPI0, DPI15 respectively organizes the chickling index and spleen index during DPI25
" * " is the statistics symbol in the table 2: significant difference (P＜0.05), this test use SPSS software to carry out data analysis.Wherein n represents the quantity of every group of chicken of choosing.Natural law behind the DPI:Days post inoculated inoculation egg capsule, for example DPI15 for the inoculation egg capsule after 15 days; DPI25 for the inoculation egg capsule after 25 days.
Table 4DPI0, DPI15 respectively organizes chickling fabricius bursa index during DPI25
" * " is the statistics symbol in the table 2: significant difference (P＜0.05), this test use SPSS software to carry out data analysis.Wherein n represents the quantity of every group of chicken of choosing.Natural law behind the DPI:Days post inoculated inoculation egg capsule, for example DPI15 for the inoculation egg capsule after 15 days; DPI25 for the inoculation egg capsule after 25 days.
Data from table 3 and table 4 can find out, 1～4 group of spleen and fabricius bursa to the Cryptosporidium parvum oocysts suspended chickling of compound recipe is grown the effect that all has in various degree.1～4 group of compound recipe in preinfection to the chickling Development of The Spleen without obvious facilitation, 3 groups of compound recipes among the index and spleen index DPI25, infect matched group and blank group diversity remarkable (P＜0.05); 4 groups of compound recipes and blank group diversity remarkable (P＜0.05) among the fabricius bursa index D PI25.1 group of spleen value of 2 groups of compound recipes and Western medicine is the most approaching with the blank group.1～4 group of compound recipe is grown the facilitation that all has in various degree in preinfection to the chickling fabricius bursa, because fabricius bursa is the main parasitic organ of Cryptosporidium baileyi, during DPI25,1～4 group of chickling fabricius bursa of compound recipe index is compared with the blank group, all shows the fabricius bursa index and reduces.But compare with infecting matched group, except 2 groups of compound recipes, each dosage group all has the growth that promotes fabricius bursa.
Immune Organs Index is the important indicator of Immune Organs of Body growth promoter degree, immune level (the Yue Daoyou that can reflect to a certain extent body, 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, behind the Cryptosporidium baileyi infection chickling, the atrophy of chickling fabricius bursa is diminished, and the enlargement of spleen pathological proliferation by the spice feeding of 1～4 group of compound recipe, has contrast with the infection matched group, and improvement is to a certain degree all arranged.
C.baileyi(Cryptosporidium baileyi, C.baileyi, Cryptosporidium baileyi) infect after the percentage ratio of different times chickling spleen percentage of liveweight change (table 3) and show: respectively organize chickling index and spleen index no difference of science of statistics (P〉0.05) before the infection, after infecting 15, the percentage ratio of 25d infection experiment group spleen percentage of liveweight is higher than the blank group in various degree, infect matched group and be higher than other each test group.Wherein, 3 groups of compound recipes, infection matched group and blank group diversity remarkable (P＜0.05) among the DPI25.As seen, infect the growth that has as if stimulated spleen in early days, allow spleen to a certain degree hypertrophy occur, 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 infecting, in the C.baileyi invasive procedure, the chickling spleen is to C.baileyi(Cryptosporidium baileyi, C.baileyi, Cryptosporidium baileyi) the immunohistology reaction and the C.baileyi(Cryptosporidium baileyi that infect, C.baileyi, Cryptosporidium baileyi) Immunohistological lesion that infects the chickling spleen occurs together.Infection is early stage to have stimulated the growth of chickling spleen when causing certain Immunohistological lesion, caused immunohistology reaction to a certain degree, causes the weightening finish of infected group spleen.In addition, respectively organize chickling index and spleen index no difference of science of statistics (P〉0.05) before the infection, 15d after infecting, the percentage ratio of 25d infection experiment group fabricius bursa percentage of liveweight is lower than the blank group, except 2 groups of DPI25 compound recipes, infect matched group fabricius bursa index and all be lower than other each experimental grouies, 4 groups of DPI25 compound recipes and blank group are diversity significantly (p〉0.05), as seen 1～25d C.baileyi(Cryptosporidium baileyi after infecting, C.baileyi, Cryptosporidium baileyi) normal development of fabricius bursa produced obvious inhibitory action, this report bright with swimming loyalty is runed counter to, identical (the Yue Daoyou of conclusion with Yue Daoyou research, 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:
Test oocysts excreting regulation according to infecting chickling, drawing in the time of the DPI15 left and right sides is ovulation capsule peak latter stage, and this moment, Cryptosporidium baileyi was maximum to the infringement of immune organ, therefore the annulus trachealis when getting DPI15 and fabricius bursa tissue are made tissue slice, and the observation pathological change.
The concrete steps of microsection manufacture are as follows:
1. draw materials and fixing: get fresh fabricius bursa tissue, annulus trachealis tissue, 10% formalin is fixed;
2. flushing: the piece of tissue after fixing must be used flowing water flush away fixative before dewatering, and the fixing tissue of formaldehyde fixative is generally washed more than 6 hours, also can wash and spend the night;
3. dehydration: the dehydration of alcohol of transparent and waxdip variable concentrations commonly used, used alcohol concentration and process are as follows: one night of 70%()---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. embedding: embedding is exactly that the good piece of tissue of infiltrating is embedded in the impregnant (paraffin), and carries out the numbering labelling;
5. section: after the wax stone molding, cut into slices;
6. open up sheet and bonding die: slice thickness 4 μ m, 42 ℃ warm water extends piece, and fishes for wax disk(-sc) after wax disk(-sc) fully flattens by the time, puts the oven dry of 45 ℃ of baking sheet machines, then can dye;
7. dyeing and sealing: HE(haematoxylin-Yihong is adopted in dyeing) dyeing, process is as follows:
Get the section of drying, be put in and dewax about 10min in the dye vat that fills dimethylbenzene, the section of having taken off behind the wax can be dyeed, concrete dyeing course and time are 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) distillation washing 1～2min, (7) haematoxylin dye liquor 10～20min, (8) common washing 1min, (9) the hydrochloride alcohol differentiation several seconds, (10) tap water flushing 30min, (11) 70% ethanol 2～5min, (12) 80% ethanol 2～5min, (13) 95% ethanol 2～5min, (14) Yihong dyeing 20s, (15) anhydrous alcohol I 3min, (16) anhydrous alcohol II 3min, (17) anhydrous alcohol adds equivalent dimethylbenzene 2min, (18) dimethylbenzene number minute is till transparent, and (19) take out section in dimethylbenzene, be placed in the lattice plate, drip a small amount of natural gum in tissue central authorities, (20) get a coverslip, gently with one side of coverslip near microscope slide, then put down rapidly coverslip, natural gum has been proofreaied and correct the coverslip direction around namely being expanded to rapidly.Be placed in the calorstat section of sealing dry.
At last, the loose colour in the section that drying is good is cleaned, and is stained with label at the left end of slide, indicates title, colouring method, fixing means and the date of tissue slice.
Observed result such as accompanying drawing 6 to shown in the accompanying drawing 25.25 can see from accompanying drawing 6 to accompanying drawing, and except the blank group, other each infection experiment group fabricius bursa and annulus trachealis are all lost in various degree, infect the matched group performance serious.
Infect matched group annulus trachealis ring mucosa and come off seriously, the epithelium generation atrophy on mucosa top layer, microvillus does not almost have, only remaining very thin one deck, and the inflammatory cell infiltration phenomenon is arranged.The inner mucosa of blank group annulus trachealis ring is complete, even thickness, and the microvillus marshalling on mucosa top layer is without inflammatory cell infiltration.1～4 group of pathological change that all has in various degree of compound recipe, 4 groups of serious is 2 groups of compound recipes and compound recipes, the mucosa surface part comes off, and presents irregular wavy.4 groups of annulus trachealises surfaces of 3 groups of compound recipes and compound recipe mucosa is relative complete, and microvillus substantially also exists, and 1 group of annulus trachealis uneven distribution of compound recipe is even, and surperficial mucosa is the Old City Wall shape, and 4 groups of compound recipes also are incomplete.1～2 group of pathological change of Western medicine is serious, and coming off appears in mucous layer, presents the Old City Wall shape.3～4 groups of relatively show not obvious of Western medicine.
Can see from the fabricius bursa slice map, 1～4 group of effect of 1～4 group of relative Western medicine of compound recipe will be got well.Infecting matched group fabricius bursa section sample can see, 400 times of lower fabricius bursa surface mucosas are adhering to many Cryptosporidiums, and local epithelium is because of the hyperplasia thickening, and atrophy then occurs another part epithelium; Thereby epithelium is uneven, and is irregular wave shape, and then develops into projection and the recessed Old City Wall shape that replaces; The visible macrophage in epithelium below.Between the matter connective tissue proliferation, the part connective tissue enters folliculus, and folliculus is cut apart; The congestion of blood vessel is hemorrhage, and hyaline thrombus appears in the part blood vessel.
In sum, the 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, 4 groups of pathological changes of 3 groups of compound recipes and Western medicine are the faintest, and it is optimum that therapeutic effect has been reacted in the side.
The present embodiment is by a plurality of test indexes 4 kinds of herbal mixtures to be estimated, wherein chickling body weight change and feed intake, fabricius bursa and trachea detect the Cryptosporidium baileyi egg sac number, 3 groups of effects of three index expression compound recipes of tissue slice pathological change are better than other compound recipe groups, Immune Organs Index achievement data analysis performance is not remarkable, but certain treatment trend is arranged.
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 carries 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, the comprehensive discovery, each is organized medicine and all has certain effect, wherein 3 groups of compound recipes are that Chinese medicine compound general performance of the present invention is optimum, all take an evident turn for the better on row's egg sac number and the feed intake gain in weight, 4 groups of general performances of Western medicine group Chinese medicine and western medicine are optimum.
4 kinds of medicines of Western medicine group all possess some special knowledge in the test of the enzymatic activity of Cryptosporidium and cell in vitro culture experiment, 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 be to return zoopery to estimate medication effect, also be on the one hand to the herbal mixture effect the comparative evaluation.
Above-described embodiment is the better embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (4)
1. Chinese medicine compound of preventing and treating the poultry Cryptosporidium spp, it is characterized in that, described Chinese medicine compound is comprised 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.
2. Chinese medicine compound according to claim 1 is characterized in that, described Chinese medicine compound is comprised of the raw material of following weight portion: Fructus Toosendan 18 weight portions, Semen Arecae 12 weight portions, Radix Dichroae 18 weight portions, Pericarpium Granati 10 weight portions and Radix Sanguisorbae 10 weight portions.
3. the preparation methoies of the Chinese medicine compound of claim 1 or 2 described control poultry Cryptosporidium spps, it is characterized in that, described preparation method is that Fructus Toosendan, Semen Arecae, Radix Dichroae, Pericarpium Granati and Radix Sanguisorbae are pulverized, cross 120 mesh sieves, and mix homogeneously obtains described Chinese medicine compound.
4. the application of Chinese medicine compound in the preparation animal and fowl fodder of claim 1 or 2 described control poultry Cryptosporidium spps is characterized in that, described Chinese medicine compound is added in the animal and fowl fodder, and addition is 0.5% of animal and fowl fodder quality.
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Effective date of registration: 20151208 Address after: 527223 Guangdong city of Luoding province Sheng Jiang Zhen Sheng Jiang Cun cattle ridge Patentee after: LUODING YUANSHI LAYING HEN CULTIVATION CO.,LTD. Address before: 510642 Tianhe District, Guangdong, No. five road, No. 483, Patentee before: South China Agricultural University |
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