犬流感(H3N2亚型)血清学调查

为了解广东地区犬流感(CI)流行情况,以期为犬流感的预防提供参考依据,本研究采用ELSIA方法及血凝抑制试验(HI),对广东省内6地市的1440份犬血清样品进行抗体检测。结果显示,1440份犬血清中犬流感抗体平均阳性率为7.92%(ELISA)和6.25%(HI)。其中广州、深圳和惠州地区的犬流感抗体阳性率较高。1～3岁犬的犬流感抗体阳性率高于其他年龄段犬,雄性犬犬流感抗体阳性率高于雌性。结果表明,H3N2亚型犬流感病毒在广东局部地区流行。鉴于当前在部分地区CIV阳性率较高,结合CIV在犬群中迅速传播的流行特点,要避免CIV暴发流行及对人类的健康造成威胁,对于CIV的防制应结合当地实际情况,制定切实有效的防制措施。     

锦州地区犬流感的血清学调查

为了解辽宁省锦州地区犬流感的感染情况,于2010年9月至2011年5月收集了锦州地区10家宠物医院就诊犬、农家犬、流浪犬共158份血清样本,通过血凝抑制(HI)试验对犬流感进行了血清学调查。结果表明:158份血清样品中抗体阳性率为15.2%,其中H5抗体阳性率为3.2%,H3亚型抗体阳性率为10.8%,H9抗体阳性率为1.3%;调查结果同时表明,母犬犬流感血清抗体阳性率略高于公犬,流浪犬的HI抗体阳性率明显高于宠物犬和农家犬。     

遵义地区H3N2亚型犬流感病毒血清学调查

为了解遵义地区H3N2亚型犬流感病毒(Canine influenza virus,CIV)感染情况,本研究采用酶联免疫吸附试验对遵义市区及周边农村收集的320份血清样本进行了抗体检测。结果显示,遵义地区H3N2亚型CIV抗体整体阳性率为8.44%(27/320);农村田园犬抗体阳性率(9.24%)高于市区宠物犬(7.96%),但差异不具有显著统计学意义(P0.05);雄性犬的抗体阳性率(8.79%)高于雌性(7.97%),差异不显著,不具有统计学意义(P0.05);1～3岁的青年犬抗体阳性率最高(9.63%),其次为幼犬(8.62%),3岁以上犬最低(6.25%),差异不显著,不具有统计学意义(P0.05)。结果表明,遵义地区存在H3N2亚型犬流感病毒感染,相关部门应加强防范。     

犬流感病毒重组核蛋白抗体间接ELISA检测方法的建立

为建立H3N2亚型犬流感病毒(CIV)血清学检测方法,本研究利用H3N2亚型CIV重组核蛋白(r NP)作为检测抗原,通过优化反应条件,建立了检测CIV核蛋白血清抗体的间接ELISA方法。通过检测10份SPF犬血清样品确定阴阳性临界值为0.288。该方法检测犬瘟热病毒、犬细小病毒、犬副流感毒、犬腺病毒Ⅱ型、狂犬病病毒、犬弓形虫、犬弓首蛔虫、犬复孔绦虫的阳性血清均为阴性,具有良好的特异性,但与H1N1、H3N8和H9N2亚型CIV有交叉反应。该方法检测H3N2 CIV血清抗体的灵敏度为血凝抑制试验(HI)的3~12.5倍;而且其批内批间变异系数为1.85%~6.57%,重复性良好。通过对H3N2 CIV攻毒犬血清进行分析,表明该检测方法具有滞后性,检测到CIV抗体的时间晚于HI试验。利用本研究建立的方法和以H3N2 CIV为诊断抗原的HI检测方法对450份血清样品进行检测,结果显示该方法对血清样品具有初筛作用,两者阳性符合率为58.3%,阴性符合率为100%。本研究可以结合其它血清学方法为CIV流行病学调查进行快速、高效的检测。     

H3N2亚型犬流感病毒血凝及血凝抑制试验的研究

为优化H3N2亚型犬流感病毒(CIV)的血凝抑制(HI)试验方法,本研究应用不同种类红细胞进行CIV的血凝(HA)试验,应用不同种类红细胞和不同血清处理方法进行CIV的HI试验,评价其对HA和HI试验的影响。结果表明,H3N2亚型CIV对鸡和犬红细胞的凝集性最好,对小鼠、猪和牛红细胞的凝集性较差。HI试验应用鸡红细胞悬液效果最好,受体破坏酶(RDE)和高碘酸钾处理可以有效去除犬血清中非特异血凝抑制素,但高碘酸钾对血清特异性抗体有损耗。本研究筛选出H3N2亚型CIV HI试验的最佳方法,为犬流感的血清学诊断提供技术支持。     

虎血清中犬副流感病毒的抗体流行病学调查研究

为查明犬副流感病毒 (CPIV)对虎的感染情况 ,应用CPIV细胞培养物为血凝抑制 (HI)抗原 ,对上海、桂林、哈尔滨、宜昌、郑州等地区未曾使用过任何含CPIV疫苗免疫的 38只虎的血清进行HI抗体检测。结果表明 ,有 2 5份血清CPIV的HI抗体效价为 1∶4～ 1∶32 ,有 1 3份抗体效价 <1∶2 ,血清中CPIV的HI抗体阳性率达 65 79%。说明上述地区的虎可能存在着CPIV的隐性感染     

H3N2亚型犬流感病毒重组HA1蛋白间接ELISA检测方法的建立

为建立一种H3N2亚型犬流感病-毒(CIV)血清学检测方法,本研究利用CIV重组HA1蛋白作为检测抗原,建立H3N2亚型CIV抗体的间接ELISA检测方法,并优化反应条件.通过检测阴性血清样本30份确定其临界值为0.228.该方法与抗猪流感病毒H1N1、H1N2、H6N6、H9N2的阳性血清和犬瘟热病、犬细小病、犬副流感的阳性血清均无交叉反应.变异系数在1.01％～7.52％之间,具有较好的重复性.通过对150份临床样品进行检测并与血凝抑制试验检测结果比较,总符合率为98.6％.本研究为CIV流行病学调查提供了一种快速、方便、敏感的抗体检测方法.     

上海城区宠物犬犬流感的血清学调查

为了解上海城区宠物犬犬流感的感染情况,作者于2013年4月至2013年9月收集了上海城区20家宠物医院就诊犬共460份血清样本,通过ELISA方法检测对犬流感进行了血清学调查。结果表明:460份血清样品中抗体阳性率为14.57%,母犬犬流感血清抗体阳性率略高于公犬。     

上海浦东地区犬弓形虫病流行情况调查

目的了解上海浦东地区犬弓形虫病流行情况。方法对上海浦东地区城市犬血清样本150份、农村犬血清样本85份和养狗场犬血清8份用间接血凝试验进行了弓形体抗体检测。结果城市犬阳性率为5.3%(8/150),农村犬阳性率为5.9%(5/85),养狗场犬未检出阳性。结论农村犬和城市犬均存在HEV感染,农村犬感染比率略高于城市犬。     

H5亚型禽流感病毒感染犬、猫的流行病学调查

为探讨禽流感与禽类接触动物的关系,对来自东莞各养殖场所的698份犬血清和200份猫血清进行了H5亚型禽流感微量血凝(HA)与血凝抑制(HI)试验,另对与血清一一对应的898份犬、猫喉拭子进行H5亚型禽流感荧光RT-PCR检测试验。结果表明,698份犬血清H5抗体检出率为6.02%,抗体阳性率为0.29%;200份猫血清抗体检出率为2.5%,阳性率为0;898份犬、猫喉拭子中未检测到禽流感H5亚型病毒。     

部分猪场H1和H3亚型猪流感的血清学调查

为了解中国部分省市规模化猪场H1和H3亚型猪流感病毒的流行情况,采用血凝抑制试验对采集于广东、湖南、河南省12个市县28个规模化猪场的799份血清进行H1和H3亚型猪流感病毒的抗体检测。结果表明,H1亚型抗体阳性率在0~83.33%之间,猪抗体总阳性率为46.18%(369/799),猪场阳性率为89.29%(25/28)。H3亚型抗体阳性率在0~100.00%之间,猪抗体总阳性率为61.33%(490/799),猪场阳性率为85.71%(24/28)。广东、湖南和河南地区H1亚型抗体阳性率分别为48.91%、40.26%和50.67%,H3亚型抗体阳性率分别为58.55%、70.78%和78.67%。在被调查的上述3个地区的猪群中,H1和H3亚型猪流感病毒的感染较为普遍,其中H3亚型感染率高于H1亚型,且各地区猪流感病毒的流行情况存在地域性差异。     

Comparison of a commercial H1N1 enzyme-linked immunosorbent assay and hemagglutination inhibition test in detecting serum antibody against swine influenza viruses.

Recently a commercial enzyme-linked immunosorbent assay (ELISA) kit for detecting antibody against H1N1 swine influenza virus (SIV) has been made available to diagnosticians and veterinary practitioners. Because the hemagglutination inhibition (HI) test has been considered the standard test for SIV serology, diagnostic performance of the new ELISA was evaluated using positive (n = 60) and negative (n = 188) serum samples from young pigs with known status of SIV infection and compared with that of the HI test. Both ELISA and HI test identified all negative animals correctly. None of the serum samples (n = 64) from pigs inoculated with H3N2 SIV was positive by ELISA for SIV antibody. The H1N1 SIV antibody detectable by ELISA appears to develop more slowly in comparison with antibody detectable by HI test. Although antibody was detected by HI test in all inoculated animals (n = 20) by day 7 postinoculation (PI), antibody was detected by ELISA in 0%, 75%, and 100% of the inoculated animals on days 7, 14, and 28 PI, respectively. Discrepancy in test results between the 2 serologic tests appeared to be because of differences in antibody isotypes detected by each test. Enzyme-linked immunosorbent assay mainly detected IgG antibody, whereas the HI test detects IgM antibody very efficiently as well as IgG antibody. Collectively, the commercial ELISA is highly specific for antibody to H1N1 SIV but may not identify positive animals at the early stage of infection as effectively as the HI test, particularly when SIV is introduced to a na?ve swine population.     

Evidence of pandemic H1N1 influenza exposure in dogs and cats,Thailand: A serological survey

Influenza A virus causes respiratory disease in both humans and animals. In this study, a survey of influenza A antibodies in domestic dogs and cats was conducted in 47 animal shelters in 19 provinces of Thailand from September 2011 to September 2014. One thousand and eleven serum samples were collected from 932 dogs and 79 cats. Serum samples were tested for influenza A antibodies using a multi‐species competitive NP‐ELISA and haemagglutination inhibition (HI) assay. The NP‐ELISA results showed that 0.97% (9/932) of dogs were positive, but all cat samples were negative. The HI test against pandemic H1N1, human H3N2 and canine H3N2 showed that 0.64% (6/932) and 1.20% (1/79) of dogs and cats were positive, respectively. It is noted that all six serum samples (5 dogs and 1 cat) had antibodies against pandemic H1N1. In summary, a serological survey revealed the evidence of pandemic H1N1 influenza exposure in both dogs and cats in the shelters in Thailand.     

Serological evidence of avian influenza virus and canine influenza virus infections among stray cats in live poultry markets,China

From January 2010 to January 2012, we collected sera samples from 700 stray cats living in close proximity to poultry farms or poultry markets in 4 provinces in China. A number of cats had evidence of avian and canine influenza virus infection: avian H9N2 [24 by HI ≥1:20 and 16 by microneutralization (MN) assay ≥1:80]; avian H5N1 (9 by HI ≥1:20 and 3 by MN assay ≥1:80) and canine H3N2 (32 by HI ≥1:20 and 18 by MN ≥1:80). Bivariate analyses revealed that cats sampled near live poultry markets and cats with influenza-like-illness were at increased risk of having elevated antibody titers by HI against avian H9N2, avian H5N1, or canine H3N2 viruses. Hence, cats may play a very important role in the ecology of novel influenza viruses and periodic epidemiological surveillance for novel influenza infections among stray cats could serve as an early warning system for human threats.     

The antibody responses to swine influenza virus (SIV) recombinant matrix 1 (rM1), matrix 2 (M2), and hemagglutinin (HA) proteins in pigs with different SIV exposure

The influenza invariant matrix 2 (M2) protein is a potential subunit vaccine candidate to induce protective immunity against broader strains of influenza A viruses (IAV). Antibodies to M2 protein have not been well characterized in IAV natural hosts. To characterize M2-specific antibodies in pigs, an ELISA to the extracellular region of the M2 (M2e) protein was developed. Sera from pigs experimentally infected with three different swine influenza virus (SIV) subtypes, immunized with an SIV inactivated vaccine, or positive for SIV maternally derived antibodies (MDA) in the absence of SIV infection were tested in assay. Confirmation of antibody titer status of pigs, was determined using a hemagglutination-inhibition (HI) test and the presence of antibodies to matrix 1 (M1) protein was measured by a recombinant M1 (rM1)-based ELISA. The antibody titers to the HA and M2e proteins but not to the rM1 were directly correlated to the dose of virus used to infect the pigs and the level of antibodies detected by the HI assay varied according to SIV subtype. Pigs experimentally infected with SIV produced low levels of M2e antibodies compared to antibodies detected by the HI and rM1 assays. Vaccination alone followed by infection did not increase the levels of M2e antibodies in contrast to HA and rM1 antibodies. Pigs with MDA had different levels of HA antibodies and were positive to M2e antibodies, but results were not correlated to HA antibodies levels and inconsistently present.     

检测禽流感病毒抗体的重组核蛋白间接ELISA方法的建立

以大肠杆菌系统表达的H9N2亚型禽流感病毒（AIV）核蛋白（NP）为抗原，建立了禽流感间接酶联免疫吸附试验抗体检测技术（NP—ELISA）。对263份待检血清（包括临床收集的243份血清和20份H9N2亚型AIV免疫鸡阳性血清）进行检测，NP—ELISA与琼脂免疫扩散试验（AGP）的总符合率为83．3％，与血凝抑制试验（HI）的总符合率为92％。特异性试验表明，NP—ELISA方法可以检测H5、H7和H9亚型AIV特异性抗体，检测为阳性的血清样品能够被阳性鸡胚尿囊液阻断。敏感性试验证实，NP—ELISA最早可以检测鸡感染后7d的血清样品，并于感染后10d确定100％血清阳性，而AGP检测直到首免后21～28d才出现部分血清阳性，HI检测直到10～14d才出现部分血清阳性，并且NP-ELISA要比HI敏感4～40倍。试验证明，NP—ELISA是检测AIV血清型特异性抗体的一种特异、敏感、快速、经济的血清学检测技术。     

Seroprevalence of subtype H3 influenza A virus in South Korean cats

To investigate the potential transmission of subtype H3 influenza virus to cats, a serological survey was carried out in South Korea. Serum samples (n = 1027) were obtained from 809 pet cats and 218 domesticated cats living in urban colonies (D-cats) from 2008 to 2010, and tested using an influenza anti-nucleoprotein (NP)-specific enzyme-linked immunosorbent assay (ELISA) and the haemagglutination inhibition (HI) test, which was recommended by the World Organization for Animal Health. Anti-influenza virus antibodies were detected in 3.12% and 2.43% of cat sera tested using the NP-specific ELISA and HI test, respectively. Anti-H3 antibodies were also identified when the HI assay was used for influenza virus serotyping. These data may indicate the sporadic transmission of subtype H3 influenza virus from other infected species to cats in South Korea.     

Development of Indirect Enzyme-linked Immunosorbent Assay with Nucleoprotein as Antigen for Detection and Quantification of Antibodies against Avian Influenza Virus

Avian influenza (AI) is a serious infectious disease caused by avian influenza virus (AIV) belonging to type A Orthomyxovirus. In the present study, we developed an indirect enzyme-linked immunosorbent assay (ELISA) employing E. coli-expressed full-length nucleoprotein (NP) of H9N2 avian influenza virus for the detection and quantification of antibodies against AIV nucleoprotein. The NP-ELISA was compared with the AI agar gel propagation (AGP) test, haemagglutination inhibition (HI) test, and IDEXX-FlockChek ELISA using 263 sera. The NP-ELISA was significantly more sensitive than the AGP and HI tests, and showed 96.2% agreement ratio with IDEXX-FlockChek ELISA. With results obtained using the NP-ELISA, an ELISA titre (ET) prediction equation, with which the ELISA titres of a flock or individual chickens can be determined, was derived from a positive/negative (P/N) ratio standard curve. The NP-ELISA enables an alternative rapid serological diagnosis and is suitable for influenza A antibody screening, especially in species that harbour several influenza subtypes.     

Influenza surveillance in birds in Italian wetlands (1992-1998): is there a host restricted circulation of influenza viruses in sympatric ducks and coots?

We report the results of a 6-year serological and virological monitoring performed in ducks and coots in Italy, in order to assess the degree of influenza A virus circulation in these birds during wintering. A total of 1039 sera collected from 1992 to 1998 was screened by a double antibody sandwich blocking ELISA (NP-ELISA): seroprevalence of antibodies to influenza A viruses was significantly higher in ducks compared to coots (52.2% vs. 7.1%, respectively). The hemagglutination-inhibition (HI) assay, performed on NP-ELISA positive sera, showed that 16.9% of these duck sera and 33.3% of these coot sera had antibodies to at least one influenza virus HA subtype: ducks showed HI antibodies against most of the HA subtypes, except for the H3, H4, H7, and H12; coots were seropositive to the H3 and H10 subtypes, only. From 1993 to 1998, 22 virus strains were obtained from 802 cloacal swabs, with an overall virus isolation frequency of 2.7%. Viruses belonging to the H1N1 subtype were by far the most commonly circulating strains (18/22) and were isolated mainly from ducks (17/18). The remaining viruses were representative of the H10N8, H5N2 and H3N8 subtypes. Our data indicate some differences between influenza A virus circulation in sympatric ducks and coots and a significant antigenic diversity between some reference strains and viruses recently isolated in Italy.     

三种检测禽流感抗体方法的比较

应用胶体金免疫层析法(GICA)、血凝抑制试验(HI)和琼脂扩散试验(AGID)三种方法同时检测所采集的342份鸡血清样品,并对三者的检测结果进行比较。结果发现,HI试验的阳性检出率最高(78.65%),其次为GICA(69.88%)和AGID试验(59.36%);GICA与HI的符合率为89.47%,与AGID的符合率为84.21%,HI与AGID的符合率为80.70%。结果表明,GICA比AGID试验更为敏感,且与HI试验有较好的符合率,可用于禽流感的血清学监测。