CN104208104A - Method for constructing clinical mastitis model of two bacterial types in different udder regions of dairy cows - Google Patents
Method for constructing clinical mastitis model of two bacterial types in different udder regions of dairy cows Download PDFInfo
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Abstract
The invention provides a method for constructing a clinical mastitis model of two bacterial types in different udder regions of dairy cows. The method comprises the following steps: selecting a Chinese Holstein dairy cow as the experimental animal, wherein the selected dairy cow is healthy, does not have disease history, is normal in body condition, has excellent breast development and is at the middle stage of lactation for the first parity, SCC is less than100000 per mL, and results of milk bacterial culture in all udder regions are negative; respectively carrying out the following treatment on four udder regions of the dairy cow: injecting 1*10<7>CFU/mL of staphylococcus aureus bacterial suspension and 1*10<6> CFU/mL of streptococcus suspension into breasts of the dairy cow via ductus papillaris by using a breast clearing needle, injecting sterile PBS in equal amount for the control group, and not carrying out treatment on a blank group; and reversely pressing the breasts from the base of each nipple from bottom to top with hands in a way that the thumbs and the index fingers are connected into a circle so that the bacterial suspensions can uniformly diffuse to lactiferous ducts and do not accumulate in gland cisterns; and attacking with bacteria for 24 hours to make sure that the dairy cow mastitis model is successfully constructed. According to the method, the influence of individual difference on tests can be completely eliminated.
Description
Technical field
The invention belongs to bio-science field, particularly a kind of method utilizing the different newborn district of same milch cow to build two kinds of bacterial type clinic mastitis models.
Background technology
Bovine mastitis, also known as mastitis, is a kind of inflammatory reaction caused by Different types of etiopathogenises, is one of modal disease of milch cow, by the impact of the factors such as milch cow self genetic background, environmental condition, feeding and management condition.
Mastitis have had a strong impact on raw milk quality, the utilization periods of milch cow, milk cattle cultivating benefit, the nutritive value of milk product and the health of consumer.Bovine mastitis is that cattle industry production efficiency is low, the one of the main reasons that cost is high.The sickness rate of most countries bovine mastitis had been about 40% (comprising subclinical mastitis), and milk yield can reduce 15%, China due to technology immature, facility such as not to match at the reason.Every year because milch cow suffers from loss that mastitis cause up to billions of unit.Milk cattle infected mastitis, the toxin of a large amount of pathogenic microorganism and release thereof and residual antibiotic are contained in its Ruzhong, drink the phenomenons such as this kind of milk can cause the mankind to suffer from diarrhoea, vomits, fever, then have a strong impact on the health of the mankind.The cause of disease of mastitis has multiple, and wherein infected by microbes is the topmost reason causing bovine mastitis to occur, and the pathogen found reaches kind more than 150, and the case that staphylococcus aureus, streptococcus and escherichia coli cause is up to 90%.S. aureus L-forms and streptococcus are all amphimicrobian gram positive bacterias.S. aureus L-forms mainly causes the infection in mammal breast, and in milch cow, the intramammary infection of 25%-30% is all caused by S. aureus L-forms.What can cause mastitis in Streptococcus has streptococcus agalactiae, streptococcus dysgalactiae, streptococcus uberis, streptococcus pyogenes and streptococcus zooepidemicus.
Clinical mastitis symptom is obvious, and breast and milk all have abnormal symptom in various degree.The lighter's milk is thin, and with flocculus, grumeleuse, breast grade fever or pain; , sometimes there is pus in the thin out yellow of severe one milk, breast is red and swollen, matter hard, heating, pain, lactogenic reduces and secretion is smooth, and occur general reaction in various degree, as loss of appetite, body temperature rising, spirit depressing etc., sickness rate is about 21-23% simultaneously.Acute mastitis, serosity mastitis, Catarrhal mastitis, fibrin mastitis, mastitis purulenta, hemorrhagic mastitis, phlegmonous mastitis is divided into according to the difference of inflammation inflammatory process.But the foundation of clinic mastitis animal model is all a priority and difficulty problem, is subject to the impact of a lot of extraneous factor all the time.
At present, the research that direct application milch cow sets up mastitis model is less, generally replaces with lactogenic mice, lactogenic rabbit, to reduce costs, increases the motility of test.But the genetic background of the generation of mastitis, development and control and animal self has inseparable relation.Direct utilization milch cow can eliminate the impact of self genetic background on test undoubtedly as laboratory animal.
It is existing that to set up mastitis model method as follows:
1. the scorching model of mammary gland of mouse.Mainly through antibacterial culturing, then get 100 microlitres and inject bacterial infections mammary gland of mouse tissue, after merit bacterium 24h, observe the change of breast tissue external structure, cervical dislocation puts to death mice, takes out rapidly mammary gland tissue and does paraffin section, and a part is carried out HE dyeing and done pathological analysis.
2. rabbit mastitis model.Use the staphylococcus aureus be separated to from mastitis disease milk sample, manual-induced rabbit mastitis, set up rabbit mastitis model.And set up 104CFU, 106CFU and 108CFU 3 various dose groups and normal healthy controls group and normal saline group.Rabbit mammary gland is attacked bacterium 24h ear edge vein exploitating blood and is measured routine blood test.Observe attack bacterium rabbit clinical manifestation, Temperature changing, search for food, drinking-water situation, diagnose in conjunction with routine blood test examining report simultaneously, screen successful rabbit mastitis model.
The present invention starts with from the selection of laboratory animal, by the laboratory animal that a series of choose targets is suitable.Then attack the technology such as bacterium, sterile sampling by fixed point and set up same milch cow different two kinds, newborn districts bacterial type clinic mastitis model.
Summary of the invention
The object of this invention is to provide a kind of method building two kinds of bacterial type clinical mastitis models in the different newborn district of milch cow, directly application milch cow sets up mastitis model, to eliminate self genetic background of other method existence to the problem of the impact of test.
For achieving the above object, the present invention is by the following technical solutions:
Build a method for two kinds of bacterial type clinical mastitis models in the different newborn district of milch cow, it is characterized in that: comprise the following steps:
(1) choice criteria: choose health, without ill history, body condition is normal, breast development is good, be in for first parity lactogenic mid-term, SCC<10 ten thousand/mL, all newborn districts milk antibacterial culturing is negative china holstein cows as laboratory animal, tests after quarantining continuously 7 days;
(2) subregion attacks bacterium mode: be handled as follows respectively the 4 Ge Ru districts of above-mentioned milch cow: utilize lactogenesis pin respectively by 1 × 10
7the bacterial suspension of the staphylococcus aureus of CFU/mL and 1 × 10
6cFU/mL streptococcus suspension is injected in cow breast through papillary duct, and injected dose is 5mL, the sterilizing PBS of matched group injection equivalent, and blank group does not deal with;
(3) newborn district's pressing mode: thumb and forefinger are connected to circle, oppositely press from bottom to top from nipple base portion, make bacterium liquid evenly diffuse to lactiferous ducts, not in newborn pond accumulation, after attacking bacterium 24h, determine the success of mammitis of cow model construction.
Follow-up study can gather the mammary gland tissue of milch cow in sterile working immediately, puts into liquid nitrogen and takes back laboratory, proceed to-70 DEG C of refrigerators for subsequent use, omnidistance sterile working.
Attack the milk sample in each cow head Ge Ru of timing acquiring before and after bacterium district, carry out milk visual examination, BMT tests, and somatic number (SCC) measures, breast histological sections Physical examination; Milk bacteriological detection, judges whether single newborn district only infects inoculation bacterial type, and without cross infection, thus judge the success or not of milch cow two kinds of bacterial type clinic mastitis model constructions.
Following technical method is industry universal or national standard method, forms the art of this patent system together with the exclusive technology of this patent.
1) bacterial suspension makes and method of counting
Staphylococcus aureus (ATCC29213), streptococcus (C55912) comes from Yangzhou University's veterinary college.
Staphylococcus aureus (ATCC29213), is inoculated in broth bouillon, cultivates 24 h recoveries in 37 DEG C of electrothermostats.S. aureus L-forms after recovery is cultivated and gone down to posterity, and adjustment bacterial concentration also carries out bacterium counting." food microbiological examination of National Standard of the People's Republic of China's national food safety standard is checked by staphylococcus aureus " (GB4789.10-2010) that the separation of test staphylococcus aureus, cultivation, the operational approach such as qualification and count of bacteria and step are all promulgated in 2010 with reference to Ministry of Public Health carries out.
Streptococcus (C55912) is inoculated in 100 milliliters of Todd-Hewitt meat soups, puts 6h recovery in 37 DEG C of biochemical cultivation cases.Broth culture is diluted in aseptic mammal Ringer's mixture, obtains about 1 × 106 CFU/mL streptococcus suspension.The detection method of milk sample streptococcus intermedius is with reference to " inspection of National Standard of the People's Republic of China's microbiological test of food hygiene Hemolytic streptococcus " (GB/T 4789.11-2003).
2) Beijing mastitis test method(s) (BMT method)
BMT method is the method for somatic number in a kind of indirect detection milk, by Scalm and Noorlander, california mastitis test method (CMT) is proposed the earliest, the method is simple to operate, highly sensitive, low price, and can execute-in-place be carried out, be the diagnostic method that current various countries generally adopt, diagnostic criteria is in table 1.China have developed Beijing mastitis test method(s) (BMT), Shanghai mastitis test method(s) (SMT), Lanzhou mastitis test method(s) (Lanzhou Mastitis Test according to the principle of CMT, LMT), Hangzhou mastitis test method(s) (Hangzhou Mastitis Test, HMT) etc.
Table 1 Beijing bovine mastitis measures (BMT) standard
3) somatic cell from milk counting method
Milk somatic cell number refers to the total cellular score in every ml milk, and when milch cow generation mastitis, the somatic cell count in milk can significantly increase, and majority is leukocyte, accounts for 95% of bovine somatic cells number, and all the other are that mammary gland tissue damages the epithelial cell come off.Somatic number reflects the health status of milk quality and milch cow, and under normal circumstances, in milk, somatic number is generally at 20,000 ~ 200,000/mL.When the SCC number in milk meets or exceeds 500,000/mL, namely think that it is subclinical mastitis, now the infection rate of breast generally reaches 16%.A lot of enterprise is strict with somatic number when purchasing lactogenesis.This test utilizes the somatic number in Denmark Chemometec company SCC-100 type portable milk somatic cell fast detector direct-detection milk.
4) mammary gland tissue sampling, microsection manufacture and observational technique
After attacking bacterium 24h, sterile working gathers the mammary gland tissue → flesh tissue of milch cow to be fixed in 10% formalin → 48h after take out piece of tissue → be placed in flowing water rinse 12h → gradient alcohol dehydration → put into ethanol and dimethylbenzene equivalent mixed liquor 30min-1h process → dimethylbenzene transparent → waxdip and embedding → spend the night preserve (-20 DEG C) → cut into slices → open up sheet, scald sheet → dewaxing → dimethylbenzene I, after the rear Lignum Sappan uniformly dyeing core → washing of II → graded ethanol → washing, after hydrochloric acid differentiation → washing, PBS returns the rear eosin stains → washing → gradient alcohol dehydration → dimethylbenzene I of indigo plant → washing, II → gummy sealing → dry rear optical microphotograph Microscopic observation.
5) milk sample method of detecting bacterium after bacterium is attacked
Get the positive milk of about 5mL BMT in sterile test tube, get milk sample 1mL in serum broth, 37 DEG C of constant-temperature tables spend the night, each is increased the milk sample after bacterium to be inoculated on Sanguis Bovis seu Bubali agar culture medium and general graceful (37 DEG C of calorstats) cultivates 48 hours, judge antibacterial generic according to thalli morphology, haemolysis situation and dyeing property.
The invention has the beneficial effects as follows:
The feature that the present invention utilizes milch cow 4 Ge Ru district relatively to isolate, same milch cow is utilized to build the clinic mastitis model of two kinds of bacterial types, entirely eliminated the impact of individual variation on test, cause the pathogenesis of mastitis and the research of pathological change to have great significance to different bacterial type antibacterial.Be specially:
(1) the present invention can make full use of the different newborn district of same cow head and set up two kinds of bacterial type clinic mastitis models.Cause the factor of mammitis of cow a lot, the genetic background of self and immune state are important factors.The present invention is intended to set up two kinds of bacterial type clinic mastitis models with the difference breast district of same milch cow, for the research of comparative study different bacterium mastitis pathogenic process, pathogenesis and bovine mastitis resistance group, eliminate different genetic background and immune state impact, improve the accuracy of result of study.
(2) other animal (as mice, rabbit) mammary gland is utilized to set up mastitis model, in order to study formation and the pathogeny of cow mammary gland, this is a kind of round-about way, because the difference of mammary gland tissue autoimmune state and from mastitis milk isolated antibacterial different to other Animalsusceptibility, be difficult to obtain result of study accurately.The present invention can effectively avoid this kind of situation.
(3) cost of modeling is effectively reduced.The present invention sets up two kinds of bacterial type models on same cow head, reduces experiment ox head number, significantly reduces research cost.
Accompanying drawing explanation
Fig. 1 is the schematic diagram for examination milch cow SCC statistical result;
Fig. 2 is the schematic diagram of staphylococcus aureus general graceful culture medium;
Fig. 3 is streptococcus Sanguis Bovis seu Bubali agar culture medium schematic diagram;
Fig. 4 is the histiocytic schematic diagram of matched group cow mammary gland, and H.E dyes, 100 times;
Fig. 5 is the schematic diagram of staphylococcus aureus inductivity mastitis tissue paraffin section de;
Wherein: a: visible mammary gland interstitial edema, interval is broadening, HE 100 ×; B: mammary gland interstitial has inflammatory cell infiltration, interstitial hemorrhage HE 200 ×; C: have a large amount of exudate and a small amount of milk in alveolar lumen, epithelial cell come off because there is degeneration HE 400 ×.
Fig. 6 is the schematic diagram of streptococcus inductivity mastitis tissue paraffin section de;
Wherein: a: local field of view breast structure is smudgy, inflammatory cell disintegrate HE 100 in part alveolar lumen ×; B: the congestion of blood vessel of mammary gland interstitial is serious, in blood vessel a large amount of erythrocyte ooze out HE 200 ×; C: epithelial cell shedding in alveolar lumen, massive inflammatory cells infiltrated, cell connect closely HE 400 ×.
Detailed description of the invention
Below in conjunction with detailed description of the invention, the present invention will be further described.
The method building two kinds of bacterial type clinical mastitis models in the different newborn district of milch cow of the present invention comprises the following steps:
Step 1. choice experiment cattle
Select the healthy china holstein cows without ill history, body condition is normal, breast development is good, is in for first parity lactogenic mid-term, and SCC<10 ten thousand/mL, all newborn districts milk is negative through antibacterial culturing, tests after quarantining 7 days.
Step 2. strain
Staphylococcus aureus (ATCC29213), streptococcus (C55912) comes from Yangzhou University's veterinary college, is suffered from Lac Bovis seu Bubali juice being separated by mastitis.
Staphylococcus aureus (ATCC29213), is inoculated in broth bouillon, cultivates 24 h recoveries in 37 DEG C of electrothermostats.S. aureus L-forms after recovery is cultivated and gone down to posterity, and adjustment bacterial concentration also carries out bacterium counting." food microbiological examination of National Standard of the People's Republic of China's national food safety standard is checked by staphylococcus aureus " (GB4789.10-2010) that the separation of test staphylococcus aureus, cultivation, the operational approach such as qualification and count of bacteria and step are all promulgated in 2010 with reference to Ministry of Public Health carries out.
Streptococcus (C55912) is inoculated in 100mL Todd-Hewitt meat soup, puts 6h recovery in 37 DEG C of biochemical cultivation cases.Broth culture is diluted in aseptic mammal Ringer's mixture, obtains about 1 × 10
6cFU/mL streptococcus suspension.The detection method of milk sample streptococcus intermedius is with reference to " inspection of National Standard of the People's Republic of China's microbiological test of food hygiene Hemolytic streptococcus " (GB/T 4789.11-2003).
The foundation of step 3. mammitis of cow model
With reference to cow breast structure, utilize lactogenesis pin respectively by 1 × 10
7the bacterial suspension of the staphylococcus aureus of CFU/mL, 1 × 10
6cFU/mL streptococcus suspension and sterilizing PBS are injected in milch cow 3 Ge Ru district through papillary duct respectively, and injected dose is 5mL.A remaining Ge Ru district is blank group, does not deal with.
The collection of step 4. milk sample
Sterile working gathers milk sample, adopt milk sample before attacking bacterium one day twice (morning 10 point, afternoon 5 point) attack bacterium after every 6h adopt a milk sample.First to four Ge Ru district disinfections (clean breast by 0.1% bromo geramine after warm water cleaning, then use the iodophor solution dipping nipple of 1%), sterile gloves is then brought to carry out hand milking.Each newborn district first squeezes and decaptitates 2-3 milk, and then respectively gets milk sample 50mL, label by sterile working's method to each newborn room of milch cow, is sent to experimental determination somatic number (SCC) immediately, and residue milk sample is freezing for subsequent use.
Step 5. organizes sample collection
After attacking bacterium 24h, determine the success of mammitis of cow model construction, sterile working gathers the mammary gland tissue of milch cow, puts into liquid nitrogen at once and takes back laboratory, proceed to-70 DEG C of refrigerators subsequently for subsequent use, omnidistance sterile working.
The foundation of step 6. staphylococcus aureus type mastitis model
1) after attacking bacterium, milch cow clinical manifestation changes
With 6h after staphylococcus aureus artificial challenge cow mammary gland, measure Gong Junru district Ruzhong somatic number, occur obviously raising.After attacking bacterium, 12h milch cow starts to occur mastitis clinical symptoms, and cow breast occurs slight red and swollen, and body temperature raises, and pressing breast milch cow is with mild pain sense.After attacking bacterium, 24h symptom is aggravated, and cow breast is hardening, and redness, distending pain are serious, and milk occurs abnormal, and have foster newborn, the newborn appearance of blood of grumeleuse, floccule, water, a cattle spirit depressing, appetite is absolutely useless, can be judged to be that clinical mastitis makes disease success.
2) milch cow BMT detection case after bacterium is attacked
Before staphylococcus aureus artificial challenge cow mammary gland, all newborn district BMT detect and are all negative.Attack 6h after bacterium, all there is weak positive reaction in experimental group breast district, presents light green, have floccule to occur during inclination in dish, and matched group and blank group are still negative.Attack 24h after bacterium, all there is strong positive reaction in experimental group, presents green or bottle green in dish, is g., jelly-like during shake, has a circle translucency jelly around.Before matched group and blank group BMT detect, 18h detects and is negative, and occurs mild infection afterwards, but the order of severity can not show a candle to test group.
3) milch cow Ruzhong SCC situation of change after bacterium is attacked
After staphylococcus aureus artificial challenge cow mammary gland, in the milk of infected area there is obvious ascendant trend in somatic number (SCC), reaches the highest when 24h, and obvious rising does not appear in matched group and blank Zu Ru district SCC.
4) milk bacteriological detection result
Before infection, the Ge Ru district of milch cow that selects of this experiment is not all by infection of staphylococcus aureus.After artificial challenge's cow mammary gland tissue after 12h, 24h, the milk sample gathering milch cow Ge Ru district respectively carries out bacteriological detection, and after the but graceful culture medium culturing of Pu, visible infected group has the growth of staphylococcus aureus, without other miscellaneous bacterias; Matched group and blank group are without staphylococcus aureus growth.Result illustrates, the staphylococcus aureus through artificial challenge has affected the lactogenic function of mammary gland, but does not infect other newborn districts.
5) bacterium rear milk area pathology section examination is attacked
Certified milk district mammary gland tissue observation by light microscope: breast structure is complete, interstitial is thin and evenly, NIP and hypertrophy phenomenon, in interstitial, blood vessel is obvious, visible a small amount of lymphocyte.Mammary gland alveolus cavity shape is different, arrangement is tight, and the visible a small amount of milk of intracavity, occurs without other cells, monolayer galactophore epithelial cell marshalling, connect closely, without obvious pathological change.
Staphylococcus aureus infects rear mammary gland tissue pathology sections observation: its mammary gland of S. aureus L-forms induction type breast district has obvious pathological changes, local field of view visible mammary gland interstitial is with obvious inflammatory edema and hypertrophy phenomenon, interstitial is broadening, slight bleeding and have a large amount of neutrophil and lymphocytic infiltration.Alveolar lumen shrinkage deformation, galactophore epithelial cell connects tight, and intercellular substance is obvious, and part epithelial cell comes off because of generation granular degeneration and steatosis.A small amount of cotton-shaped milk and a large amount of secretions is had, the inflammatory cells such as the galactophore epithelial cell mainly come off and polymorphonuclear neutrophisls, macrophage, lymphocyte in alveolar lumen.
7. streptococcus processed group mastitis model is set up
1) clinical symptoms change
Cow mammary gland checks milch cow after infecting streptococcus comprehensively.There is ANOMALOUS VARIATIONS significantly, lymphonodi supramammarici swelling in breast, the milk in 24h postoperative infection breast district, quality is hardening, erythrosis, touches breast milch cow pain obvious, a cattle loss of appetite with hands; Have floccule in milk, breast amount reduces sharply.Be diagnosed as clinic mastitis.There is not exception in matched group and blank Zu Ru district.
2) procuratorial work of milk outward appearance and BMT detect
When giving milk cow milking, can find that milch cow has the behavior of refusing to milk; There is floccule even curdling block in Ruzhong; Infect in streptococcic newborn district, the thin person of milk sample, color and luster jaundice, thickness, indivedual in the thick shape of pus, and matched group is normal with blank group milk.BMT testing result: the milk BMT in Ge Ru district detects result before artificial challenge and is feminine gender, and after artificial challenge, streptococcus Zu Ru district milk should be positive or strong positive, milk all forms gel, to in centralized during revolution, not easily scatter, after stopping stirring, grumeleuse adheres to and tries to get to the heart of a matter; The milk in matched group and blank Zu Ru district is without significant change, and flowing is smooth, does not occur grumeleuse.Illustrate that the somatic number in streptococcus Zu Ru district milk sharply increases.
3) somatic cell from milk number (SCC) measures
After artificial challenge streptococcus, in infected newborn district milk, somatic number rises rapidly.To attack before bacterium at 100,000/below mL, attack more than 1,000,000/mL after bacterium 12h, matched group and blank group are all at 200,000/below mL; Attack more than 2,000,000/mL after bacterium 24h, matched group and blank group are all at 300,000/below mL.
4) milk bacteriological detection result
Before infection, the Ge Ru district of milch cow that selects of this experiment is not all by streptococcal infection.After artificial challenge's cow mammary gland tissue after 12h, 24h, the milk sample gathering milch cow Ge Ru district respectively carries out bacteriological detection, and after menses slat chain conveyor, visible infected group has streptococcic growth, without other miscellaneous bacterias; Matched group and blank group are without streptococcus growth.Result illustrates, the streptococcus through artificial challenge has affected the lactogenic function of mammary gland, but does not infect other newborn districts.
5) bacterium rear milk area pathology section examination is attacked.
Streptococcus infects rear mammary gland tissue pathology sections observation: streptococcus infects thin vessels and the telangiectasis of rear mammary gland tissue interstitial, congested serious, a large amount of erythrocyte oozes out in conduit, in interstitial, polymorph neutrophile leucocytes, lymphocyte and a small amount of macrophage invade profit, blood capillary and the expansion of mammary gland interstitial thin vessels, congested serious, connective tissue tails off.The exudates such as a large amount of macrophage, lymphocyte, leukocyte and the epithelial cell that comes off are full of in alveolar lumen.The disorderly avalanche of acinus cavity configuration in local field of view, smudgy, in part alveolar lumen there is the phenomenon of downright bad disintegrate with the epithelial cell come off in inflammatory cell.
Embodiment
The foundation of No. 9133 (milch cow 1), No. 9130 (milch cow 2), No. 8107 (milch cow 3) china holstein cowses two kinds of bacterial type clinic mastitis models
Select healthy in cattle farm, Yangzhou suburb, without ill history, body condition is normal, breast development is good, be in for first parity lactogenic mid-term, SCC<10 ten thousand/mL, all newborn districts milk antibacterial culturing is 3 negative china holstein cowses, cattle number is respectively: 9133, 9130, 8107, carry out attacking bacterium (table 2) with said method, carry out milk visual examination afterwards, BMT detects (table 3), Somatic Cell Count (adopts SCC-100 type milk somatic cell fast detector because this test SCC measures, therefore the highest test result that can only show 2,000,000/mL.When SCC is higher than 2,000,000/mL, by milch cow occur clinical symptoms and BMT detect a cattle situation is judged) (Fig. 1), milk bacteriological detection (Fig. 2, Fig. 3), mammary gland tissue pathology sections observation (Fig. 4, Fig. 5, Fig. 6), all prove that this cattle infects clinic mastitis, wherein milk bacteriological detection shows, right front (A2) breast Simple infection staphylococcus aureus of this milch cow 1, right back (A3) breast Simple infection streptococcus, left front (A1), left back (A4) breast are all without bacteriological infection; Right back (B3) breast Simple infection staphylococcus aureus of milch cow 2, left back (B4) breast Simple infection streptococcus, left front (B1), right front (B2) breast are all without bacteriological infection; Left back (C4) breast Simple infection staphylococcus aureus of milch cow 3, right back (C4) breast Simple infection streptococcus, left front (C1), right front (C2) breast are all without bacteriological infection.Result shows, 3 cow head staphylococcus aureuses, streptococcus infect different newborn districts clinical mastitis model respectively and are all successfully established.
Table 2 subregion attacks bacterium example
Note: Bos primigenius number: 9133(milch cow 1); 9130(milch cow 2); 8107(milch cow 3)
A1---C4: organize sample number after corresponding newborn area code and mammary gland tissue sampling
Table 3 is for test milch cow BMT testing result
Note: "-" represents negative, and "+" represents the weak positive, " ++ " represents positive, and " +++ " represents strong positive.
Claims (5)
1. build a method for two kinds of bacterial type clinical mastitis models in the different newborn district of milch cow, it is characterized in that: comprise the following steps:
(1) choice criteria: choose health, without ill history, body condition is normal, breast development is good, be in for first parity lactogenic mid-term, SCC<10 ten thousand/mL, all newborn districts milk antibacterial culturing is negative china holstein cows as laboratory animal;
(2) subregion attacks bacterium mode: be handled as follows respectively the 4 Ge Ru districts of above-mentioned milch cow: utilize lactogenesis pin respectively by 1 × 10
7the bacterial suspension of the staphylococcus aureus of CFU/mL and 1 × 10
6cFU/mL streptococcus suspension is injected in cow breast through papillary duct, the sterilizing PBS of matched group injection equivalent, and blank group does not deal with;
(3) newborn district's pressing mode: thumb and forefinger are connected to circle, oppositely press from bottom to top from nipple base portion, make bacterium liquid evenly diffuse to lactiferous ducts, not in newborn pond accumulation, after attacking bacterium 24h, determine the success of mammitis of cow model construction.
2. build the method for two kinds of bacterial type clinical mastitis models as claimed in claim 1 in the different newborn district of milch cow, it is characterized in that: in step (1), the china holstein cows chosen is tested after quarantining continuously 7 days.
3. build the method for two kinds of bacterial type clinical mastitis models as claimed in claim 1 in the different newborn district of milch cow, it is characterized in that: in step (2), injected dose is 5mL.
4. the method for two kinds of bacterial type clinical mastitis models is built as claimed in claim 1 in the different newborn district of milch cow, it is characterized in that: follow-up study can gather the mammary gland tissue of milch cow in sterile working immediately, put into liquid nitrogen and take back laboratory, proceed to-70 DEG C of refrigerators for subsequent use, omnidistance sterile working.
5. the method for two kinds of bacterial type clinical mastitis models is built as claimed in claim 1 in the different newborn district of milch cow, it is characterized in that: the milk sample of attacking each cow head Ge Ru of timing acquiring before and after bacterium district, carry out milk visual examination, BMT tests, somatic number (SCC) measures, breast histological sections Physical examination; Milk bacteriological detection, judges whether single newborn district only infects inoculation bacterial type, and without cross infection, thus judge the success or not of milch cow two kinds of bacterial type clinic mastitis model constructions.
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| CN111387133B (en) * | 2020-02-25 | 2021-04-16 | 中国农业大学 | Method for establishing model for inhibiting mouse mastitis by using Klebsiella pneumoniae bacteriophage |
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