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

馮永杰, 王英華, 楊玉榮

?

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

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

綿羊；新孢子蟲??；弓形蟲?。伙L(fēng)險因素；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)

弓形蟲病和新孢子蟲病可造成巨大的經(jīng)濟損失，常引起溫血動物流產(chǎn)、死胎和弱胎等生殖障礙，也包括反芻類動物[1-2]，新孢子蟲感染是牛流產(chǎn)的最主要原因[2]。貓科動物和犬科動物分別為弓形蟲(Toxoplasmagondii)和新孢子蟲(Neosporacaninum)的終末宿主[1,3]，含有卵囊的貓和犬糞便污染的草料、草場和飲用水是反芻動物感染的最主要途徑[4-5]。新孢子蟲病可引起犬和牛反復(fù)流產(chǎn)[6-7]，患慢性新孢子蟲病的?；蛉軌蛲ㄟ^胎盤傳染下一代[2,4]。已從草食動物如牛、馬、鹿和綿羊體內(nèi)分離到活體新孢子蟲蟲株[2]。但是，國內(nèi)關(guān)于綿羊新孢子蟲病的報道十分有限，新孢子蟲病對綿羊等小反芻類動物造成的危害尚無法評估。弓形蟲病是人畜共患的動物源性寄生蟲病，屬于我國二類疫病。從羊肉、羊奶及其奶制品中成功分離到活體弓形蟲蟲株已被報道[8-9]。目前為止，從我國羊分離出新孢子蟲的研究未見報道，僅分離到1株弓形蟲[10]。因此，本研究針對河南、江蘇和浙江3省綿羊新孢子蟲病和弓形蟲病進行流行病學(xué)調(diào)查，分析感染的風(fēng)險因素，為羊的原蟲寄生蟲病的流行病學(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。并對羊進行流行病學(xué)調(diào)查，詳細記錄地域、性別、年齡、流產(chǎn)史等信息。

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

1.3統(tǒng)計學(xué)分析數(shù)據(jù)采用軟件Graph Pad Prism 4.0 進行統(tǒng)計分析。運用卡方檢驗或F檢驗判定綿羊的性別、日齡、流產(chǎn)史和地域與其感染這兩種原蟲之間存在的相關(guān)風(fēng)險關(guān)系。當P<0.05則認為差異有統(tǒng)計學(xué)意義，即該因子是感染寄生蟲的風(fēng)險因子。

2　結(jié)　果

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

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

表1綿羊新孢子蟲病和弓形蟲病的血清學(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、弓形蟲的風(fēng)險關(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)計學(xué)意義“*” indicates significant differences.

3　討　論

我國綿羊飼養(yǎng)量居世界第一，小反芻獸類的運輸和遷徙已成為寄生蟲病傳播和擴散的最主要原因，其中綿羊和山羊的貢獻顯著[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，增加了人類感染羊源性寄生蟲病的風(fēng)險。

新孢子蟲病可感染多種反芻類動物和非人靈長類[2,16]。新孢子蟲病已被證實是導(dǎo)致綿羊流產(chǎn)的原因之一[17]。我國關(guān)于新孢子蟲病的報道僅僅見于牛、狗、羊[18-20]，調(diào)查地域也十分有限，羊新孢子蟲病報道僅見青海省[20]。調(diào)查發(fā)現(xiàn)綿羊比山羊更加易感新孢子蟲病[21]。本次調(diào)查，河南、江蘇和浙江3省綿羊的新孢子蟲總感染率(11.88%)與青海省(10.33%)[20]的陽性率基本持平，3個省份各自的感染率均在11%左右，可見綿羊新孢子蟲的地域分布范圍較廣且分布水平相當。大于12月齡綿羊的新孢子蟲抗體的陽性率(12.37%)約是小于12月齡(5.41%)的2倍，但風(fēng)險因素分析發(fā)現(xiàn)年齡不是綿羊感染新孢子蟲病的風(fēng)險因子(P>0.05)。垂直傳播和食入新孢子蟲卵囊是反芻類動物感染新孢子蟲病的主要傳播途徑[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、牧場規(guī)模和衛(wèi)生條件均是綿羊感染新孢子蟲病的風(fēng)險因素[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病均不相關(guān)(P>0.05)。由此推測新孢子蟲可感染各個年齡階段的綿羊，綿羊感染新孢子蟲的主要原因是羊群周圍的環(huán)境，凈化羊群及草料儲存環(huán)境(如隔絕犬等)，阻斷新孢子蟲的傳播是防控該病的有效途徑。

弓形蟲病是人畜共患病，對畜牧生產(chǎn)和人類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省綿羊的弓形蟲陽性率(17.81%)處于我國已有報道的中等水平(2%～39%)。浙江和江蘇綿羊的弓形蟲陽性率均高于河南，江浙地區(qū)較為溫暖濕潤的氣候條件和豐富的物種資源更加有利于弓形蟲的生存和傳播。然而，危險因素分析發(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)，這說明河南和江蘇兩省的地域差異與弓形蟲的感染存在相關(guān)關(guān)系，但是存在差異的原因需要進一步探索。綿羊的流產(chǎn)史與弓形蟲陽性率之間相關(guān)性有統(tǒng)計學(xué)意義(P<0.01)，患弓形蟲病母羊流產(chǎn)率是健康母羊流產(chǎn)率的5.258倍。弓形蟲感染是母羊流產(chǎn)的主要原因之一，但弓形蟲是否可引起母羊反復(fù)流產(chǎn)，尚無確切證據(jù)，及時診斷和治療懷孕母羊弓形蟲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的風(fēng)險因素(P>0.05)，這與已報道的研究相吻合[22]，說明不同年齡和性別的綿羊均可受到弓形蟲的侵襲，綿羊弓形蟲病需要全面防控。因此，監(jiān)測綿羊新孢子蟲病和弓形蟲病的流行情況不僅能有效控制寄生蟲類疫病的擴散，而且為人畜等公共衛(wèi)生安全提供基礎(chǔ)保障。

(美國農(nóng)業(yè)部的J.P.Dubey教授贈予新孢子蟲和弓形蟲的陽性、陰性對照血清，中國農(nóng)業(yè)大學(xué)的劉群教授贈予包被新孢子蟲速殖子的96孔板，在此一并感謝。)

[1] Dubey JP. Toxoplasmosis of animals and humans[M]. 2nd ed. Boca Raton: CRC Press, Taylor & Francis Group, 2010: 1-313.

[2] Dubey JP, Schares G. Neosporosis in animals-the last five years[J]. Vet Parasitol, 2011, 180(1/2): 90-108.

[3] Donahoe SL, Lindsay SA, Krockenberger M, et al. A review of neosporosis and pathologic findings ofNeosporacaninuminfection in wildlife[J]. Int J Parasitol Parasites Wildl, 2015, 4(2): 216-238.

[4] Dubey JP, Lindsay DS. Neosporosis, toxoplasmosis, and sarcocystosis in ruminants[J]. Vet Clin North Am Food Anim Pract, 2006, 22(3): 645-671.

[5] Panadero R, Painceira A, Lopez C, et al. Seroprevalence ofToxoplasmagondiiandNeosporacaninumin wild and domestic ruminants sharing pastures in Galicia (Northwest Spain)[J]. Res Vet Sci, 2010, 88(1): 111-115.

[6] Pabon M, Lopez-Gatius F, Garcia-Ispierto I, et al. ChronicNeosporacaninuminfection and repeat abortion in dairy cows: a 3-year study[J]. Vet Parasitol, 2007, 147(1/2): 40-46.

[7] Dubey JP, Koestner A, Piper RC. Repeated transplacental transmission ofNeosporacaninumin dogs[J]. J Am Vet Med Assoc, 1990, 197(7): 857-860.

[8] Dubey JP, Casey SJ, Zajac AM, et al. Isolation and genetic characterization ofToxoplasmagondiifrom alpaca(Vicugnapacos) and sheep (Ovis aries)[J]. Trop Anim Health Prod, 2014, 46(8): 1503-1507.

[9] Dubey JP, Verma SK, Ferreira LR, et al. Detection and survival ofToxoplasmagondiiin milk and cheese from experimentally infected goats[J]. J Food Prot, 2014, 77(10): 1747-1753.

[10] Zhou P, Zhang H, Lin RQ, et al. Genetic characterization ofToxoplasmagondiiisolates from China[J]. Parasitol Int, 2009, 58(2): 193-195.

[11] Dubey JP, Desmonts G. Serological responses of equids fedToxoplasmagondiioocysts[J]. Equine Vet J, 1987, 19(4): 337-339.

[12] Soares HS, Ahid SM, Bezerra AC, et al. Prevalence of anti-Toxoplasmagondiiand anti-Neosporacaninumantibodies in sheep from Mossoro, Rio Grande do Norte, Brazil[J]. Vet Parasitol, 2009, 160(3-4): 211-214.

[13] Helmick B, Otter A, McGarry J, et al. Serological investigation of aborted sheep and pigs for infection byNeosporacaninum[J]. Res Vet Sci, 2002, 73(2): 187-189.

[14] Martins J, Kwok OC, Dubey JP. Seroprevalence ofNeosporacaninumin free-range chickens (Gallus Domesticcus) from the Americas[J]. Vet Parasitol, 2011, 182(2/4): 349-351.

[15] Vasileiou NG, Fthenakis GC, Papadopoulos E. Dissemination of parasites by animal movements in small rumi nant farms[J]. Vet Parasitol, 2015, 213(1/2): 56-60.

[16] Barr BC, Conrad PA, Sverlow KW, et al. Experimental fetal and transplacentalNeosporainfection in the nonhuman primate[J]. Lab Invest, 1994, 71(2): 236-242.

[17] Gonzalez-Warleta M, Castro-Hermida JA, Regidor-Cerrillo J, et al.Neosporacaninuminfection as a cause of reproductive failure in a sheep flock[J]. Vet Res, 2014, 45: 88.

[18] Yao L, Yang N, Liu Q, et al. Detection ofNeosporacaninumin aborted bovine fetuses and dam blood samples by nested PCR and ELISA and seroprevalence in Beijing and Tianjin, China[J]. Parasitology, 2009, 136(11):1251-1256.

[19] Yang YR, Zhang QF, Kong YG, et al. Low prevalence ofNeosporacaninumandToxoplasmagondiiantibodies in dogs in Jilin, Henan and Anhui provinces of the People’s Republic of China[J]. BMC Vet Res, 2014, 10: 295.

[20] Liu ZK, Li JY, Pan H. Seroprevalence and risk factors ofToxoplasmagondiiandNeosporacaninuminfections in small ruminants in China[J]. Prev Vet Med, 2015, 118(4): 488-492.

[21] Diakoua A, Papadopoulos E, Panousis N, et al.ToxoplasmagondiiandNeosporacaninumseroprevalence in dairy sheep and goats mixed stock farming[J]. Vet Parasitol, 2013, 198(3/4): 387-390.

[22] Bartova E, Machacova T, Sedlak K, et al. Seroprevalence of antibodies ofNeosporaspp. andToxoplasmagondiiin horses from southern Italy[J]. Folia Parasitol (Praha), 2015, 62: 043.

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

國家自然科學(xué)基金 (No. 30800812)；河南省科技攻關(guān)項目(No.B20140841)