那立冬,張世棟,董書(shū)偉,閆寶琪,楊洪早,桑夢(mèng)琪,賈 寧,王東升,嚴(yán)作廷
(1. 甘肅農(nóng)業(yè)大學(xué) 動(dòng)物醫(yī)學(xué)院,蘭州 730070;2. 中國(guó)農(nóng)業(yè)科學(xué)院 蘭州畜牧與獸藥研究所,蘭州 730050)
產(chǎn)后患子宮內(nèi)膜炎對(duì)奶牛血清和子宮黏液中免疫球蛋白質(zhì)量濃度的影響
那立冬1,2,張世棟2,董書(shū)偉2,閆寶琪2,楊洪早1,2,桑夢(mèng)琪2,賈 寧1,王東升1,2,嚴(yán)作廷1,2
(1. 甘肅農(nóng)業(yè)大學(xué) 動(dòng)物醫(yī)學(xué)院,蘭州 730070;2. 中國(guó)農(nóng)業(yè)科學(xué)院 蘭州畜牧與獸藥研究所,蘭州 730050)
為比較分娩后患子宮內(nèi)膜炎奶牛和健康奶牛血清及子宮黏液中IgA、IgM和IgG的變化,采用酶聯(lián)免疫吸附測(cè)定法分別對(duì)奶牛血清和子宮黏液中IgA、IgM和IgG的質(zhì)量濃度進(jìn)行定量檢測(cè)。結(jié)果表明,患病奶牛血清中的IgA和健康奶牛子宮黏液中的IgA持續(xù)降低,子宮黏液中IgA先降低后升高,健康奶牛血清中IgA變化不大;患病奶牛和健康奶牛子宮黏液和血清中IgG均先降低后升高;健康奶牛血清和子宮黏液中IgM先降低后升高,患病奶牛子宮黏液中IgM先升高后降低,后又升高,患病奶牛血清中IgM逐漸降低;患病奶牛血清中IgG始終高于健康奶牛的,說(shuō)明IgG在全身體液免疫中發(fā)揮主要作用;患病奶牛子宮黏液的IgA和IgM高于健康奶牛,說(shuō)明IgA和IgM在子宮局部免疫中發(fā)揮主要作用。
奶牛;子宮內(nèi)膜炎;免疫球蛋白;質(zhì)量濃度
子宮內(nèi)膜炎是導(dǎo)致奶牛不孕的主要生殖疾病[1-2],常引起奶牛子宮復(fù)舊延遲,卵泡發(fā)育和功能異常,繁殖力降低,屢配不孕,產(chǎn)奶量下降,致使治療費(fèi)用和淘汰率增加,經(jīng)濟(jì)效益降低,給奶牛養(yǎng)殖業(yè)造成嚴(yán)重的經(jīng)濟(jì)損失[3-5]。子宮內(nèi)膜炎發(fā)生的主要原因是由于分娩時(shí)子宮頸開(kāi)張和子宮內(nèi)環(huán)境的急劇變化,細(xì)菌等主要的病原微生物趁機(jī)進(jìn)入子宮,導(dǎo)致奶牛子宮細(xì)菌污染,尤其在產(chǎn)后1周內(nèi)奶牛子宮的污染率達(dá)90%~100%,產(chǎn)后7 d左右子宮細(xì)菌達(dá)到動(dòng)態(tài)平衡,即數(shù)量最多,產(chǎn)后10~14 d子宮內(nèi)持續(xù)存在多種條件性致病菌,正常奶牛產(chǎn)后2~4周內(nèi)大部分細(xì)菌被自發(fā)性排出,產(chǎn)后6周子宮污染物被徹底清除[6-8]。然而,至少20%的奶牛因?yàn)橐恍?qiáng)致病性細(xì)菌的持續(xù)存在,或因子宮的免疫防御體系遭到破壞,機(jī)體未能將細(xì)菌排出,會(huì)導(dǎo)致子宮感染引起子宮內(nèi)膜炎[9]。分娩后奶牛的免疫機(jī)能高低對(duì)子宮是否發(fā)生感染起著重要作用,特異性免疫球蛋白(immune globulin,Ig)在奶牛子宮復(fù)舊過(guò)程中發(fā)揮的作用極為關(guān)鍵[10]。Ig是體液免疫和局部黏膜免疫發(fā)揮重要作用的特異性免疫蛋白,普遍存在于動(dòng)物血液、淋巴液、組織液和外分泌液中,在奶牛產(chǎn)后對(duì)抗子宮感染中起著重要作用[11]。因此,比較產(chǎn)后患子宮內(nèi)膜炎奶牛和健康奶牛Ig質(zhì)量濃度變化規(guī)律可以反映產(chǎn)后子宮的狀態(tài)。近年來(lái),國(guó)內(nèi)外學(xué)者對(duì)分娩前后3周奶牛免疫狀態(tài)的研究較多[12-14],但對(duì)分娩3周后子宮復(fù)舊過(guò)程中奶牛免疫狀態(tài)的研究較少。本試驗(yàn)以分娩后21~42 d奶牛為研究對(duì)象,檢測(cè)血清和子宮黏液中IgA、IgG和IgM的質(zhì)量濃度,結(jié)合奶牛子宮狀態(tài)比較患子宮內(nèi)膜炎奶牛和健康奶牛Ig質(zhì)量濃度的變化趨勢(shì),為臨床診斷、治療和預(yù)防奶牛子宮內(nèi)膜炎提供參考。
1.1 試驗(yàn)動(dòng)物及分組
試驗(yàn)選用的中國(guó)荷斯坦奶牛由甘肅省某奶牛養(yǎng)殖場(chǎng)提供。選擇年齡在3~8歲、分娩時(shí)間相近、無(wú)其他疾病的產(chǎn)后奶牛,在產(chǎn)后21 d通過(guò)直腸檢查和陰道分泌物性狀鑒定,篩選出健康奶牛和臨床型子宮內(nèi)膜炎奶牛(患病牛)各9頭,試驗(yàn)用奶牛采用牛場(chǎng)標(biāo)準(zhǔn)飼養(yǎng)管理程序進(jìn)行飼喂。
1.2 樣品采集和處理
于產(chǎn)后21、28、35和42 d采集每頭奶牛的血液和子宮黏液。通過(guò)頸靜脈采集血液約10 mL,分離血清,分裝,低溫保存帶回實(shí)驗(yàn)室,-80 ℃保存,待用;通過(guò)子宮頸按摩方法將子宮黏液收集至試管,約50 mL。低溫保存帶回實(shí)驗(yàn)室,-80 ℃保存至少24 h,待黏液凍結(jié)后將凍結(jié)的黏液組織迅速取出約0.4 g,轉(zhuǎn)移至組織研磨器中,按照1∶4(質(zhì)量∶體積)的比例加入生理鹽水,充分研磨后,3 000 r/min離心10 min,吸取上清液,分裝,-80 ℃保存,待用。
1.3 主要試劑及器材
牛IgA ELISA定量檢測(cè)試劑盒(貨號(hào)140321)、牛IgG ELISA定量檢測(cè)試劑盒(貨號(hào)140711)和牛IgM ELISA定量檢測(cè)試劑盒(貨號(hào)140605),Bethyl 公司產(chǎn)品;酶標(biāo)板恒溫振蕩器(Abson;micbio-Ⅱ);酶標(biāo)儀(Thermo;MK3);臺(tái)式離心機(jī)(Scilogex;DMO412)等。
1.4 免疫球蛋白質(zhì)量濃度測(cè)定
采用酶聯(lián)免疫吸附雙抗體夾心法定量檢測(cè)奶牛血清和子宮黏液中IgA、IgM和IgG的質(zhì)量濃度。試驗(yàn)操作嚴(yán)格按照試劑盒操作說(shuō)明進(jìn)行。
1.5 數(shù)據(jù)分析
數(shù)據(jù)以“平均值±標(biāo)準(zhǔn)差”表示,采用SPSS 19.0和GraphPad prism 5.0軟件進(jìn)行統(tǒng)計(jì)分析。組間數(shù)據(jù)比較采用t檢驗(yàn)。
2.1 產(chǎn)后奶牛血清和子宮黏液中IgA的質(zhì)量濃度變化
由圖1和圖2可知,健康奶牛血清中IgA質(zhì)量濃度從產(chǎn)后21 d(0.183 g/L)開(kāi)始下降,在28 d(0.116 g/L)后略有升高,42 d降至最低(0.107 g/L)。患病奶牛血清中IgA質(zhì)量濃度在21 d最高(0.300 g/L),35 d降至最低(0.124 g/L)(圖1);健康奶牛子宮黏液中IgA質(zhì)量濃度在產(chǎn)后21 d 最高(0.419 g/L),21~42 d逐漸降低,42 d最低(0.096 g/L)?;疾∧膛W訉m黏液中IgA質(zhì)量濃度在21 d(0.692 g/L)開(kāi)始降低,35 d最低(0.297 g/L),而在42 d迅速升高(1.311 g/L)(圖2);患病奶牛血清在21 d極顯著高于健康奶牛(P<0.01),在28 d顯著高于健康奶牛(P<0.05)。奶牛子宮黏液在21 d和28 d患病奶牛顯著高于健康奶牛(P<0.05),42 d患病奶牛極顯著高于健康奶牛(P<0.01);患病奶牛血清中IgA質(zhì)量濃度持續(xù)降低,健康奶牛血清中IgA質(zhì)量濃度變化不大。患病奶牛子宮黏液中IgA質(zhì)量濃度先降低后升高,健康奶牛子宮黏液中IgA質(zhì)量濃度持續(xù)降低。
產(chǎn)后時(shí)間相同的患病奶牛與健康奶牛相比,*表示差異顯著(P<0.05),**表示差異極顯著(P<0.01)。下圖同。Compared endometritis cows having same postpartum days with healthy cows,* means significant difference atP<0.05,** means significant difference at theP<0.01.The same as below.
圖1 奶牛血清中IgA質(zhì)量濃度
Fig.1 Mass concentration of IgA in cows serum
圖2 奶牛子宮黏液中IgA質(zhì)量濃度Fig.2 Mass concentration of IgA in cows uterus mucus
2.2 產(chǎn)后奶牛血清和子宮黏液中IgG的質(zhì)量濃度變化
由圖3和圖4可知,健康奶牛血清中IgG質(zhì)量濃度在產(chǎn)后21 d最高(26.929 g/L),35 d降至21.786 g/L,到42 d升高至25.280 g/L?;疾∧膛Q逯蠭gG質(zhì)量濃度在21 d最高(37.990 g/L),35 d降至最低(27.181 g/L),42 d小幅度升高(27.731 g/L)(圖3);健康奶牛子宮黏液中IgG在產(chǎn)后21 d為9.793 g/L,28 d(6.919 g/L)小幅度降低,28~42 d先迅速升高后緩慢升至最高(34.309 g/L)?;疾∧膛W訉m黏液中IgG質(zhì)量濃度在21~28 d緩慢升至19.249 g/L,在35 d降至最低(9.960 g/L),42 d快速升至最高(31.074 g/L)(圖4);患病奶牛血清IgG質(zhì)量濃度在21 d顯著高于健康奶牛(P<0.05),28 d極顯著高于健康奶牛(P<0.01)?;疾∧膛W訉m黏液IgG質(zhì)量濃度21 d顯著高于健康奶牛(P<0.05),28 d患病奶牛極顯著高于健康奶牛(P<0.01),在35 d健康奶牛極顯著高于患病奶牛(P<0.01)。健康和患病奶牛血清中IgG質(zhì)量濃度先降低后升高,且患病奶牛血清中IgG質(zhì)量濃度始終高于健康奶牛。健康和患病奶牛子宮黏液中IgG質(zhì)量濃度在21~42 d先降低后升高。
圖3 奶牛血清中IgG質(zhì)量濃度Fig.3 Mass concentration of IgG in cows serum
圖4 奶牛子宮黏液中IgG質(zhì)量濃度Fig.4 Mass concentration of IgG in cows uterus mucus
2.3 產(chǎn)后奶牛血清和子宮黏液中IgM的質(zhì)量濃度變化
由圖5和圖6可知,健康奶牛血清中IgM質(zhì)量濃度在產(chǎn)后21 d最高(3.543 g/L),35 d降至最低(2.540 g/L),42 d升高至2.817 g/L?;疾∧膛Q逯蠭gM質(zhì)量濃度在21 d最高(3.374 g/L),在42 d降至最低(2.591 g/L)(圖5);健康奶牛子宮黏液中IgM質(zhì)量濃度在產(chǎn)后21 d為0.442 g/L,28 d降至最低(0.044 g/L),在35 d升高至0.201 g/L,在42 d降低至0.043 g/L。患病奶牛子宮黏液中 IgM 質(zhì)量濃度在21 d為1.164g/L,28d升至最高(1.650g/L),35d降低至1.294 g/L,42 d質(zhì)量濃度升高至1.363 g/L(圖6);21~42 d患病奶牛子宮黏液中IgM質(zhì)量濃度極顯著高于健康奶牛(P<0.01);患病奶牛血清中IgM質(zhì)量濃度逐漸降低,健康奶牛血清中IgM質(zhì)量濃度先降低后升高?;疾∧膛W訉m黏液中IgM質(zhì)量濃度先升高后降低,后又升高,健康奶牛子宮黏液中IgM質(zhì)量濃度先降低后升高。
圖5 奶牛血清中IgM質(zhì)量濃度Fig.5 Mass concentration of IgM in cows serum
圖6 奶牛子宮黏液中IgM質(zhì)量濃度Fig.6 Mass concentration of IgM in cows uterus mucus
產(chǎn)后奶牛子宮感染導(dǎo)致子宮內(nèi)膜炎發(fā)生與機(jī)體免疫狀態(tài)有著密切聯(lián)系,Ig作為體液免疫的重要成分,在子宮局部黏膜中發(fā)揮重要作用,由病原微生物感染引起子宮炎癥時(shí),機(jī)體產(chǎn)生一系列的免疫答應(yīng),引起血液中Ig質(zhì)量濃度迅速升高,部分Ig會(huì)被轉(zhuǎn)移至子宮,同時(shí)子宮局部黏膜也會(huì)產(chǎn)生Ig,這些Ig一方面會(huì)直接作用于病原微生物,并在激活的補(bǔ)體系統(tǒng)的協(xié)助下使其溶解,同時(shí)也會(huì)通過(guò)對(duì)中性粒細(xì)胞調(diào)控使其發(fā)揮吞噬作用,所以Ig質(zhì)量濃度變化可有效反映子宮的免疫狀態(tài)[15-16]。本試驗(yàn)測(cè)定產(chǎn)后21~42 d健康奶牛與患子宮內(nèi)膜炎奶牛血清和子宮黏液中IgA、IgG和IgM的質(zhì)量濃度,并對(duì)健康奶牛和患子宮內(nèi)膜炎奶牛的這些指標(biāo)進(jìn)行比較,分析產(chǎn)后患子宮內(nèi)膜炎對(duì)奶牛血清和子宮黏液中Ig質(zhì)量濃度的影響。
通過(guò)酶聯(lián)免疫吸附試驗(yàn)方法檢測(cè)Ig質(zhì)量濃度,具有操作簡(jiǎn)單、靈敏度高的特點(diǎn),且能定量反映Ig質(zhì)量濃度變化。Ig變化的時(shí)間早于臨床表現(xiàn)、病理變化,是判定子宮內(nèi)膜炎發(fā)生的重要指標(biāo)??梢赃\(yùn)用此方法對(duì)奶牛子宮內(nèi)膜炎癥的變化進(jìn)行動(dòng)態(tài)監(jiān)測(cè),Ig變化幅度較大時(shí),意味著可能發(fā)生子宮感染,應(yīng)引起高度重視。但此檢測(cè)方法應(yīng)用也存在問(wèn)題,血清中的Ig是全身性抗原感染的綜合表現(xiàn),不能定位局部。
有研究表明,產(chǎn)后健康奶牛血清中Ig以IgG和IgM為主,IgA質(zhì)量濃度很低[17-19],與本研究結(jié)果一致。奶牛子宮黏液中Ig以IgG和IgA為主,IgM質(zhì)量濃度則很低[20]。但本研究對(duì)患病奶牛Ig質(zhì)量濃度檢測(cè)發(fā)現(xiàn),患病奶牛子宮黏液中IgM的質(zhì)量濃度高于IgA,且極顯著高于健康奶牛,可能是由于子宮復(fù)舊過(guò)程健康奶牛子宮中主要以IgG和IgA免疫為主,但發(fā)生子宮感染則會(huì)誘導(dǎo)IgM大量產(chǎn)生并參與抗感染作用。本研究還發(fā)現(xiàn),患病奶牛血清中IgM質(zhì)量濃度與健康奶牛并無(wú)差異,可能原因是子宮感染后血液中增加的IgM轉(zhuǎn)移至子宮,引起子宮黏液IgM的升高,增強(qiáng)子宮的抗感染作用。
產(chǎn)后奶牛子宮遭受到病原微生物污染時(shí),大部分奶牛在分娩后2~4周通過(guò)自身的免疫調(diào)節(jié)和子宮復(fù)舊能清除病原微生物,從而恢復(fù)子宮的正常功能[8,18]。本研究發(fā)現(xiàn),患病奶牛血清和子宮黏液中IgA和IgG質(zhì)量濃度在21~28 d均高于健康奶牛,并在28~35 d逐漸降低,在35 d除子宮黏液中IgG遠(yuǎn)低于健康奶牛外,其他均與健康奶牛無(wú)顯著差異?;甲訉m內(nèi)膜炎奶牛Ig質(zhì)量濃度普遍高于健康奶牛這種變化規(guī)律與其他學(xué)者報(bào)道一致[6,12]。但在35 d健康子宮黏液中IgG的質(zhì)量濃度突然升至相對(duì)較高水平,在42 d患病奶牛子宮黏液中IgA和IgG的質(zhì)量濃度也突然的升至較高水平,這種急速的變化可能與產(chǎn)后子宮復(fù)舊程度有關(guān)。
試驗(yàn)結(jié)果表明,患病奶牛血清中IgG質(zhì)量濃度始終高于健康奶牛,說(shuō)明IgG在全身體液免疫中發(fā)揮主要作用;患病奶牛子宮黏液中IgA和IgM高于健康奶牛,說(shuō)明IgA和IgM在子宮局部免疫中發(fā)揮主要作用。
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(責(zé)任編輯:顧玉蘭 Responsible editor:GU Yulan)
Effects of Dairy Cows with Endometritis on Immunoglobulin Mass Concentration in Serum and Uterus Mucus of Postpartum
NA Lidong1,2,ZHANG Shidong2,DONG Shuwei2,YAN Baoqi2,YANG Hongzao1,2,SANG Mengqi2,JIA Ning1,WANG Dongsheng1,2and YAN Zuoting1,2
(1. College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China; 2.Lanzhou Institute of Animal Husbandry and Pharmaceutical Sciences,Chinese Academy of Agricultural Sciences,Lanzhou 730050,China )
In order to compare healthy cows with the mass concentration variation of IgA,IgM and IgG in serum and uterus mucus in postpartum dairy cows sufering from endometritis.The mass concentration of IgA,IgM and IgG in uterus mucus and serum were detected by the method of enzyme-linked immunosorbent assay. The results showed that the mass concentration of IgA reduced continuously both serum of endometritis cows and uterus mucus of healthy cows. The IgA mass concentration in uterus mucus decreased initially and then increased in cows suffering from endometritis,There was no great change in the mass concentration of IgA in the serum of healthy cows ; IgG levels of the uterus mucus and serum in endometritis cows and healthy cows decreased firstly and then increased; IgM mass concentration in serum and uterus mucus of healthy cows decreased firstly and then increased,IgM mass concentration in uterus mucus of cows with endometritis increased firstly and then decreased,the IgM levels in serum of endometritis cows gradually reduced. These indicated that IgG in serum of cows suffering from endometritis was always higher than that of healthy cows,so it is appoved that IgG played a major role in systemic humoral immunity. The mass concentration of IgA and IgM in uterus mucus of postpartum dairy cows with endometritis was higher than that of healthy cows,this results indicated that IgA and IgM played the key role in local immunity of the uterus.
Dairy cows;Endometritis;Immuneglobulin;Mass concentration
NA Lidong,male,master student.Research area:diagnosis and prevention of cow disease. E-mail:17709317797@189.cn
YAN Zuoting,male,research fellow.Research area:R&D of new traditional Chinese veterinary medicine,diagnosis and prevention of cow disease. E-mail:yanzuoting@caas.cn
日期:2017-03-30
2016-07-19
2016-09-30
中央級(jí)公益性科研院所基本科研業(yè)務(wù)費(fèi)專(zhuān)項(xiàng)(1610322015003);“十二五”國(guó)家科技支撐計(jì)劃(2012BAD12B03);中國(guó)農(nóng)業(yè)科學(xué)院農(nóng)業(yè)科技創(chuàng)新工程(CAAS-ASTIP-2014-LIHPS-03)。
那立冬,男,碩士研究生,研究方向?yàn)槟膛<膊≡\斷與防治。E-mail:17709317797@189.cn
嚴(yán)作廷,男,研究員,研究方向?yàn)橹蝎F藥研發(fā)、奶牛疾病診斷與防治。E-mail:yanzuoting@caas.cn 王東升,男,副研究員,研究方向?yàn)橹兴幩幚砼c奶牛繁殖疾病。E-mail:lzmyswds@126.com
S857.23;S858.23
A
1004-1389(2017)04-0503-06
WANG Dongsheng,male,associate research fellow.Research area:pharmacology of Chinese medicine and dairy cow reproduction disease. E-mail:lzmyswds@126.com
網(wǎng)絡(luò)出版地址:http://kns.cnki.net/kcms/detail/61.1220.S.20170330.1508.006.html
Received 2016-07-19 Returned 2016-09-30
Foundation item Basal Scientific Research Funds for Central Public-Interest Scientific Institutions(No.1610322015003); National Science and Technology Support Program in “12th Five-year” Plan(No.2012BAD12B03);Agricultural Science and Technology Innovation Program (No.CAAS-ASTIP-2014-LIHPS-03).