馮永杰, 王英華, 楊玉榮

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綿羊新孢子蟲(chóng)病和弓形蟲(chóng)病血清流行病學(xué)調(diào)查及風(fēng)險(xiǎn)因素分析

馮永杰1, 王英華2, 楊玉榮1

目的調(diào)查河南、江蘇和浙江省綿羊的新孢子蟲(chóng)病和弓形蟲(chóng)病的感染狀況并進(jìn)行風(fēng)險(xiǎn)因素分析，為綿羊寄生蟲(chóng)病防控和羊肉肉品安全監(jiān)測(cè)提供基礎(chǔ)依據(jù)。方法采用間接免疫熒光(IFAT)和改良凝集實(shí)驗(yàn)法(MAT)分別檢測(cè)綿羊血清中的新孢子蟲(chóng)和弓形蟲(chóng)的抗體。統(tǒng)計(jì)分析風(fēng)險(xiǎn)因素的OR值及P值。結(jié)果綿羊新孢子蟲(chóng)的感染率為11.88%(62/522)，弓形蟲(chóng)抗體的陽(yáng)性率為17.81%(93/522)。地域和流產(chǎn)史是綿羊感染弓形蟲(chóng)的風(fēng)險(xiǎn)因素(P<0.05)，但與新孢子蟲(chóng)的感染不相關(guān)(P>0.05)；年齡和性別均與這兩種原蟲(chóng)的感染不相關(guān)(P>0.05)。結(jié)論河南、江蘇和浙江省綿羊新孢子蟲(chóng)病和弓形蟲(chóng)病的陽(yáng)性率均較高，羊肉肉品安全現(xiàn)狀堪憂。該地域綿羊的新孢子蟲(chóng)病流行情況首次報(bào)道。

綿羊；新孢子蟲(chóng)病；弓形蟲(chóng)??；風(fēng)險(xiǎn)因素；血清流行病學(xué)

Supported by the National Natural Science Foundation of China (No. 30800812) and the Scientific and Technological Project of Henan Province (No. B20140841)

弓形蟲(chóng)病和新孢子蟲(chóng)病可造成巨大的經(jīng)濟(jì)損失，常引起溫血?jiǎng)游锪鳟a(chǎn)、死胎和弱胎等生殖障礙，也包括反芻類(lèi)動(dòng)物[1-2]，新孢子蟲(chóng)感染是牛流產(chǎn)的最主要原因[2]。貓科動(dòng)物和犬科動(dòng)物分別為弓形蟲(chóng)(Toxoplasmagondii)和新孢子蟲(chóng)(Neosporacaninum)的終末宿主[1,3]，含有卵囊的貓和犬糞便污染的草料、草場(chǎng)和飲用水是反芻動(dòng)物感染的最主要途徑[4-5]。新孢子蟲(chóng)病可引起犬和牛反復(fù)流產(chǎn)[6-7]，患慢性新孢子蟲(chóng)病的?；蛉軌蛲ㄟ^(guò)胎盤(pán)傳染下一代[2,4]。已從草食動(dòng)物如牛、馬、鹿和綿羊體內(nèi)分離到活體新孢子蟲(chóng)蟲(chóng)株[2]。但是，國(guó)內(nèi)關(guān)于綿羊新孢子蟲(chóng)病的報(bào)道十分有限，新孢子蟲(chóng)病對(duì)綿羊等小反芻類(lèi)動(dòng)物造成的危害尚無(wú)法評(píng)估。弓形蟲(chóng)病是人畜共患的動(dòng)物源性寄生蟲(chóng)病，屬于我國(guó)二類(lèi)疫病。從羊肉、羊奶及其奶制品中成功分離到活體弓形蟲(chóng)蟲(chóng)株已被報(bào)道[8-9]。目前為止，從我國(guó)羊分離出新孢子蟲(chóng)的研究未見(jiàn)報(bào)道，僅分離到1株弓形蟲(chóng)[10]。因此，本研究針對(duì)河南、江蘇和浙江3省綿羊新孢子蟲(chóng)病和弓形蟲(chóng)病進(jìn)行流行病學(xué)調(diào)查，分析感染的風(fēng)險(xiǎn)因素，為羊的原蟲(chóng)寄生蟲(chóng)病的流行病學(xué)防控提供參考依據(jù)。

1　材料與方法

1.1材料2014年10月到2015年5月，從河南、江蘇和浙江3省(羊總飼養(yǎng)量約6 000萬(wàn)只)共采集農(nóng)村散養(yǎng)湖羊綿羊血液樣品522份(Epi Info7.1.5分析最低樣本量為384只)，分別來(lái)自河南67份，江蘇247份，浙江208份。采集頸靜脈血液，離心獲得血清，血清-20 ℃保存。并對(duì)羊進(jìn)行流行病學(xué)調(diào)查，詳細(xì)記錄地域、性別、年齡、流產(chǎn)史等信息。

1.2IFAT和MAT檢測(cè)新孢子蟲(chóng)和弓形蟲(chóng)抗體綿羊血清的弓形蟲(chóng)抗體采用改良凝集法(modified agglutination test，MAT)檢測(cè)[1,11]。采用間接免疫熒光(indirect fluorescent antibody test，IFAT)方法檢測(cè)新孢子蟲(chóng)的抗體。每板均設(shè)置陽(yáng)性、陰性和空白對(duì)照，被檢血清抗體效價(jià)達(dá)到或超過(guò)1∶25者判定為弓形蟲(chóng)陽(yáng)性[1]，達(dá)到或超過(guò)1∶50判定為新孢子蟲(chóng)陽(yáng)性[12-13]，新孢子蟲(chóng)速殖子蟲(chóng)體表面呈現(xiàn)均一亮度的綠色為陽(yáng)性[14]。IFAT采用的二抗為異硫氰酸熒光素(FITC)標(biāo)記的兔抗綿羊IgG抗體(Abcam 公司，產(chǎn)品批號(hào)：ab150181)。經(jīng)過(guò)福爾馬林溶液固定純化的含有全抗原的弓形蟲(chóng)速殖子購(gòu)買(mǎi)于Kerafast 公司(產(chǎn)品批號(hào)：EH2002)。

1.3統(tǒng)計(jì)學(xué)分析數(shù)據(jù)采用軟件Graph Pad Prism 4.0 進(jìn)行統(tǒng)計(jì)分析。運(yùn)用卡方檢驗(yàn)或F檢驗(yàn)判定綿羊的性別、日齡、流產(chǎn)史和地域與其感染這兩種原蟲(chóng)之間存在的相關(guān)風(fēng)險(xiǎn)關(guān)系。當(dāng)P<0.05則認(rèn)為差異有統(tǒng)計(jì)學(xué)意義，即該因子是感染寄生蟲(chóng)的風(fēng)險(xiǎn)因子。

2　結(jié)　果

綿羊新孢子蟲(chóng)病的總感染率為11.88%(62/522，95% CI=14.76～21.34)，弓形蟲(chóng)病的陽(yáng)性率為17.81%(93/522，95% CI=9.36～14.95)。兩種原蟲(chóng)的混合感染率為3.45%(18/522, 95% CI=2.16～5.42)。

風(fēng)險(xiǎn)因素分析發(fā)現(xiàn)(表1，表2)，地理位置(OR=3.509)和流產(chǎn)史(OR=5.258)是綿羊感染弓形蟲(chóng)的風(fēng)險(xiǎn)因素(P<0.05)，但與感染新孢子蟲(chóng)病不相關(guān)(P>0.05)；年齡和性別因素均與綿羊感染這兩類(lèi)原蟲(chóng)不相關(guān)(P>0.05)。

表1綿羊新孢子蟲(chóng)病和弓形蟲(chóng)病的血清學(xué)流行病學(xué)

Tab.1Seroprevalence of T. gondii and N. caninum infection in sheep

CharacteristicsNo.ofsheeptestedNo.ofseropositivesheepatdifferenttiters1∶251∶501∶100Above1∶200Totalno.ofseropositivesheepPrevalence(%)95%CIT.gondii(MAT,cutoff:1∶25)Region河南67200245.971.91-14.80浙江2081301304421.1516.13-27.23江蘇247423364518.2213.88-23.53GenderFemale3971224567418.6415.10-22.78Male125700121915.209.87-22.61Abortionhistory(femalesheeponly,N=397)Yes92821273841.3031.78-51.52No305403293611.808.62-15.94Age(years)≤1374002616.227.27-31.52>14851524668717.9314.77-21.61Total5222012709317.8114.76-21.34續(xù)表(1)CharacteristicsNo.ofsheeptestedNo.ofseropositivesheepatdifferenttiters1∶251∶501∶100Above1∶200Totalno.ofseropositivesheepPrevalence(%)95%CIN.caninum(IFAT,cutoff:1∶50)Region河南67538--811.945.92-22.09浙江20818026--2612.508.62-17.74江蘇24724328--2811.347.92-15.94GenderFemale39738346--4611.598.78-15.13Male1259316--1612.807.93-19.88Abortionhistory(femalesheeponly,N=397)Yes929110--1010.875.83-19.04No30529236--3611.808.62-15.94Age(years)≤137312--25.410.57-18.63>148544560--6012.379.72-15.62Total52247662--6211.889.36-14.95

表2地理位置，性別，年齡和流產(chǎn)史與綿羊感染新孢子蟲(chóng)、弓形蟲(chóng)的風(fēng)險(xiǎn)關(guān)系

Tab.2Odds ratio of geographical origin, gender, abortion history and age of sheep as risk factors for T. gondii and N. caninum antibody

FactorCategoryOR95%CIPT.gondiiRegion河南---江蘇3.5091.2140-10.14000.0131*浙江1.2040.7573-1.91500.4771GenderFemale---Male1.2780.7375-2.21500.4233AbortionhistoryNo---Yes5.2583.0590-0.03800.0001*Age(years)≤1--->11.1290.4571-2.79101.0000N.caninumRegion江蘇---河南1.0610.4593-2.44900.8320浙江1.0540.4524-2.45301.0000GenderFemale---Male1.1200.6097-2.05800.7515AbortionhistoryYes---No1.0970.5219-2.30701.0000Age(years)≤1--->12.4710.5791-10.54000.2929

比值比：Odds ratio(OR); “*”差異有統(tǒng)計(jì)學(xué)意義“*” indicates significant differences.

3　討　論

我國(guó)綿羊飼養(yǎng)量居世界第一，小反芻獸類(lèi)的運(yùn)輸和遷徙已成為寄生蟲(chóng)病傳播和擴(kuò)散的最主要原因，其中綿羊和山羊的貢獻(xiàn)顯著[15]。涮羊肉，羊肉燒烤和手抓羊肉等烹飪習(xí)慣不能充分熟化羊肉，增加了人類(lèi)感染羊源性寄生蟲(chóng)病的風(fēng)險(xiǎn)。

新孢子蟲(chóng)病可感染多種反芻類(lèi)動(dòng)物和非人靈長(zhǎng)類(lèi)[2,16]。新孢子蟲(chóng)病已被證實(shí)是導(dǎo)致綿羊流產(chǎn)的原因之一[17]。我國(guó)關(guān)于新孢子蟲(chóng)病的報(bào)道僅僅見(jiàn)于牛、狗、羊[18-20]，調(diào)查地域也十分有限，羊新孢子蟲(chóng)病報(bào)道僅見(jiàn)青海省[20]。調(diào)查發(fā)現(xiàn)綿羊比山羊更加易感新孢子蟲(chóng)病[21]。本次調(diào)查，河南、江蘇和浙江3省綿羊的新孢子蟲(chóng)總感染率(11.88%)與青海省(10.33%)[20]的陽(yáng)性率基本持平，3個(gè)省份各自的感染率均在11%左右，可見(jiàn)綿羊新孢子蟲(chóng)的地域分布范圍較廣且分布水平相當(dāng)。大于12月齡綿羊的新孢子蟲(chóng)抗體的陽(yáng)性率(12.37%)約是小于12月齡(5.41%)的2倍，但風(fēng)險(xiǎn)因素分析發(fā)現(xiàn)年齡不是綿羊感染新孢子蟲(chóng)病的風(fēng)險(xiǎn)因子(P>0.05)。垂直傳播和食入新孢子蟲(chóng)卵囊是反芻類(lèi)動(dòng)物感染新孢子蟲(chóng)病的主要傳播途徑[2]。然而，小于12月齡的綿羊獲得新孢子蟲(chóng)抗體的具體原因有待研究。青海省的調(diào)查發(fā)現(xiàn)，狗的存在、放牧方式、牧場(chǎng)規(guī)模和衛(wèi)生條件均是綿羊感染新孢子蟲(chóng)病的風(fēng)險(xiǎn)因素[20]。本次調(diào)查分析，地理位置，母羊流產(chǎn)史和性別與綿羊新孢子蟲(chóng)病均不相關(guān)(P>0.05)。由此推測(cè)新孢子蟲(chóng)可感染各個(gè)年齡階段的綿羊，綿羊感染新孢子蟲(chóng)的主要原因是羊群周?chē)沫h(huán)境，凈化羊群及草料儲(chǔ)存環(huán)境(如隔絕犬等)，阻斷新孢子蟲(chóng)的傳播是防控該病的有效途徑。

弓形蟲(chóng)病是人畜共患病，對(duì)畜牧生產(chǎn)和人類(lèi)的健康已造成顯著的危害。本次調(diào)查發(fā)現(xiàn)，河南、江蘇和浙江3省綿羊的弓形蟲(chóng)陽(yáng)性率(17.81%)處于我國(guó)已有報(bào)道的中等水平(2%～39%)。浙江和江蘇綿羊的弓形蟲(chóng)陽(yáng)性率均高于河南，江浙地區(qū)較為溫暖濕潤(rùn)的氣候條件和豐富的物種資源更加有利于弓形蟲(chóng)的生存和傳播。然而，危險(xiǎn)因素分析發(fā)現(xiàn)，僅江蘇地區(qū)與河南的感染率存在顯著差異(P<0.05)，這說(shuō)明河南和江蘇兩省的地域差異與弓形蟲(chóng)的感染存在相關(guān)關(guān)系，但是存在差異的原因需要進(jìn)一步探索。綿羊的流產(chǎn)史與弓形蟲(chóng)陽(yáng)性率之間相關(guān)性有統(tǒng)計(jì)學(xué)意義(P<0.01)，患弓形蟲(chóng)病母羊流產(chǎn)率是健康母羊流產(chǎn)率的5.258倍。弓形蟲(chóng)感染是母羊流產(chǎn)的主要原因之一，但弓形蟲(chóng)是否可引起母羊反復(fù)流產(chǎn)，尚無(wú)確切證據(jù)，及時(shí)診斷和治療懷孕母羊弓形蟲(chóng)病是提高羊繁殖率的有效方法。年齡和性別不是綿羊感染弓形蟲(chóng)的風(fēng)險(xiǎn)因素(P>0.05)，這與已報(bào)道的研究相吻合[22]，說(shuō)明不同年齡和性別的綿羊均可受到弓形蟲(chóng)的侵襲，綿羊弓形蟲(chóng)病需要全面防控。因此，監(jiān)測(cè)綿羊新孢子蟲(chóng)病和弓形蟲(chóng)病的流行情況不僅能有效控制寄生蟲(chóng)類(lèi)疫病的擴(kuò)散，而且為人畜等公共衛(wèi)生安全提供基礎(chǔ)保障。

(美國(guó)農(nóng)業(yè)部的J.P.Dubey教授贈(zèng)予新孢子蟲(chóng)和弓形蟲(chóng)的陽(yáng)性、陰性對(duì)照血清，中國(guó)農(nóng)業(yè)大學(xué)的劉群教授贈(zèng)予包被新孢子蟲(chóng)速殖子的96孔板，在此一并感謝。)

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Seroprevalence and risk factors of sheep in neosporosis and toxoplasmosis

FENG Yong-jie1, WANG Ying-hua2, YANG Yu-rong1

(1.CollegeofAnimalScienceandVeterinaryMedicine,HenanAgriculturalUniversity,Zhengzhou450000,China;2.CenterforAnimalDiseaseControlandPreventionofHenanProvince,Zhengzhou450000,China)

The aim of this research was to investigate the neosporosis and toxoplasmosis for sheep in Henan, Jiangsu and Zhejiang provinces and analyze the risk factors of infection, and provide a foundation of prevention and control in this two parasitosis for sheep and monitoring in mutton safety. The indirect fluorescent antibody test(IFAT) and modified agglutination test(MAT) were used to detect the antibodies ofNeosporacaninumandToxoplasmagondii. Odds ratio(OR) andPvalue were analyzed to distinguish the correlativity of risk factors. The results showed that the seropositive ofNeosporacaninumwas 11.88%(62/522), and the infection rate of toxoplasmosis was 17.81%(93/522). Abortion history and region of sheep were the infection risk factors of toxoplasmosis(P<0.05), but not for neosporosis(P>0.05). Meanwhile, gender and age had no relationship of infection in sheep for this two protozoons(P>0.05). In conclusion, a high positive infection percentage for neosporosis and toxoplasmosis in sheep in Henan, Jiangsu and Zhejiang provinces. Improving and reinforcing the prevention and control measures were urgent, and safety of sheep meat should be focused. This was the first report of the infection of neosporosis in sheep from central and eastern China.

sheep; neosporosis; toxoplasmosis; risk factors; seroprevalence

Yang Yu-rong, Email: yangyu7712@sina.com

楊玉榮，Email: yangyu7712@sina.com

1.河南農(nóng)業(yè)大學(xué)牧醫(yī)工程學(xué)院，鄭州450000；2.河南省動(dòng)物疾病預(yù)防控制中心，鄭州450000

R531.8

A

1002-2694(2016)07-0613-05

2016-03-29；

2016-06-16

DOI：10.3969/j.issn.1002-2694.2016.07.005

國(guó)家自然科學(xué)基金 (No. 30800812)；河南省科技攻關(guān)項(xiàng)目(No.B20140841)