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1.
检测禽流感病毒抗体的重组核蛋白间接ELISA方法的建立   总被引:9,自引:3,他引:9  
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。  相似文献   

2.
Transmissible viral proventriculitis (TVP) was experimentally reproduced in 2-wk-old specific-pathogen-free chickens and commercial broiler chickens by eyedrop inoculation of adenovirus-like virus (AdLV), isolate R1 1/3. No clinical signs and no weight gain depression were observed in chickens inoculated with AdLV (R11/3); however, gross and microscopic lesions characteristic of TVP were present in proventriculi of inoculated chickens. Proventriculi of AdLV (R11/3)-inoculated chickens were markedly enlarged, compared with sham-inoculated controls, by day 7 postinoculation (PI). Microscopic lesions in proventriculi of inoculated chickens were detected beginning on day 3 PI and consisted of degeneration and necrosis of glandular epithelium, ductal epithelial hyperplasia, replacement of glandular epithelium with ductal epithelium, and diffuse interstitial lymphoid infiltration; no microscopic lesions were observed in other tissues. AdLV (R11/3) antigens were detected in proventriculi by immunohistochemistry on days 3-10 PI in inoculated SPF chickens and days 3-21 PI in inoculated commercial broiler chickens; no viral antigens were detected in other tissues. AdLV (R11/3) was reisolated from proventriculi of inoculated SPF and commercial broiler chickens on days 5 and 7 PI. No virus, viral antigens, or lesions were detected in proventriculi collected from sham-inoculated chickens. These findings indicate an etiologic role for AdLV (R11/3) in TVP.  相似文献   

3.
Day-old specific-antibody-negative turkey poults were inoculated orally with cloned turkey reovirus isolate 81-68. Virus reisolations from 11 different tissues revealed widespread distribution at 3, 5, and 7 days postinoculation (PI). Virus was isolated from the intestines until 21 days PI. Virus was isolated from tendons until day 7 PI and again at day 28 PI. Reovirus serum-neutralization antibodies appeared as early as 7 days PI. All inoculated birds showed positive VN serum titers (greater than or equal to 1:20) by day 21 PI. No reovirus was isolated from control poults, and they remained antibody-negative during the entire experiment.  相似文献   

4.
The effects of fasting on the ability of swine serum to affect proliferation, protein synthesis and protein degradation in L6 myoblast cell culture bioassays were evaluated. Barrows (15 to 20 kg) were fitted with jugular catheters. Blood samples were collected at four evenly spaced intervals between 0800 and 1700 on collection days. Prefast blood samples were obtained on d 1 and 2 of the study, after which pigs were subjected to a 5-d fast. Fasted samples were obtained on the 1st, 3rd and 5th d of the fast (d 3, 5 and 7 of the study). Serum from each collection day was pooled and tested in the proliferation bioassay for each pig. Prefast and fasted serum pools were formed by pooling prefast days (1 and 2) or fasted days (3, 5 and 7), respectively, from all pigs in a study. These pools were tested in the proliferation and protein turnover bioassays as well as in a Somatomedin-C (SmC) radioimmunoassay. Serum from the fasted collection days showed a decrease in mitogenic activity compared with serum from the two prefast days (P less than .001). At high concentrations, sera obtained from fasted pigs inhibited muscle cell proliferation (P less than .001). Additionally, adding fasted serum to control swine serum (CSS) inhibited the mitogenic activity of CSS in a dose-dependent manner (P less than .025). Therefore, fasted sera showed a decreased ability to promote muscle cell proliferation and, in addition, appeared to contain a factor(s) that inhibits muscle cell proliferation. Fasted serum also caused a 21.6% increase in protein degradation compared with prefast serum.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

5.
以7日龄SPF雏鸡为试验动物,应用细胞培养和免疫酶技术,通过对外周血液免疫球蛋白含量、T和B淋巴细胞数量及其功能的检测,较全面系统地研究了鹅源H5N1亚型中强毒禽流感病毒(AIV)感染SPF雏鸡后,其外周血液上述指标的动态变化。结果发现,SPF雏鸡感染鹅源H5N1亚型AIV后,血清IgG和IgA含量在1~4d显著或极显著低于对照雏鸡(P<0.05或P<0.01),而IgM在病毒感染早期未见显著性差异,随后3种免疫球蛋白含量逐渐回升;血液T淋巴细胞数量显著低于对照雏鸡(P<0.05或P<0.01),而B淋巴细胞在1~5d明显降低(P<0.05或P<0.01);T、B淋巴细胞对ConA或PMA的增殖反应分别于1~8d、1~4d明显低于对照雏鸡(P<0.05或P<0.01)。上述结果表明,AIV感染SPF雏鸡外周血液无论是细胞免疫还是体液免疫功能均呈现一定抑制。  相似文献   

6.
将禽流感病毒血凝素 H9A基因克隆入插入载体 p FG11S中 ,通过酶切鉴定获得了正向转移载体 p FG11SHA;将其与禽痘病毒疫苗株 (w FPV)共转染鸡成纤维细胞 (CEF) ,通过蓝白斑筛选纯化得到重组病毒 r FPV- Ps- HA;以间接免疫荧光法证实 HA基因得到了表达。将该病毒经颈部皮下免疫 1日龄 SPF鸡 ,免疫后 15 d以 H9亚型禽流感病毒 F株翅静脉攻毒 ,攻毒后第 5天采集泄殖腔棉拭子样品进行病毒分离。将此重组病毒与以痘苗病毒 P7.5启动子表达相同基因的重组病毒 r FPV- P7.5 - HA作比较 ,结果表明 ,r FPV- Ps- HA相对于 r FPV- P7.5 - HA明显抑制了病毒的排出 ;攻毒后第 2、5、7、9、11天分别对 r FPV- Ps- HA、油乳剂灭活苗免疫鸡进行泄殖腔、气管排毒规律的检测 ,发现疫苗组均能很好地抑制排毒 ,攻毒对照组泄殖腔的排毒率明显高于气管排毒率  相似文献   

7.
Clinical signs of transmissible gastroenteritis were not observed in newborn pigs orally inoculated with the high-passaged vaccinal transmissible gastroenteritis virus (TO-163 strain). Vaccinal viral multiplication in digestive tract of newborn pigs fed colostrum before inoculation and kept at 21 to 22 C was diminished, but was not diminished in those fed colostrum and kept at 10 to 11 C. Other groups of newborn pigs inoculated with the attenuated vaccinal virus and kept at 18 to 22 C or at 31 to 34 C were challenge exposed with virulent intestinal virus on the 1st, 2nd, . . ., or 6th postinoculation (PI) days. In the groups kept at 18 to 22 C, 2 of 7 inoculated pigs challenge exposed with virulent virus on the 3rd PI day, 4 of 7 pigs exposed on the 4th PI day, and all of the pigs exposed on and after the 5th PI day survived the exposure. In the groups kept at 18 to 22 C, the attenuated vaccinal virus was distributed mainly in the respiratory organs and lymphatic tissues. On the contrary, in the groups kept at 31 to 34 C, all of the pigs died in 2 to 5 days after challenge exposure, and the attenuated vaccinal virus was scarcely detected in any of the pigs.  相似文献   

8.
In order to determine the actual prevalence of avian influenza viruses (AIV) in wild birds in Germany, extensive surveillance studies were carried out between March 2003 and January 2005. More than 3.000 samples of 79 different species of wild birds (migratory and resident birds) were taken and 1.151 established pools investigated. Samples came from 80 different regions of Germany. Forty AIV isolates representing 14 combinations of eight different hemagglutinin and eight neuraminidase subtypes, among them H5 and H7, were identified. All H5 and H7 isolates were found to be of low pathogenicity. The overall incidence of the investigated pools based on virus isolation was 3,5 % for AIV, with considerable variability noted among species, season and location. All AIV were isolated from birds sampled in autumn. Most of the AIV isolates came from the resting or wintering areas of mallards breeding far north. This study adds to the understanding of the ecology of influenza viruses in wild birds and empahsizes the constant need for surveillance in times of an ongoing and expanding epidemic of highly pathogenic AI.  相似文献   

9.
The strain BA-1 of caprine herpesvirus-1 (CpHV-1), isolated from latently infected goats, was inoculated intranasally into three five-year-old goats. The animals developed fever and leukopenia. The signs began on post-inoculation day (PID) 4 and lasted 7 days. In one goat herpes-like lesions appeared on the vulvar area on PID 7. Virus was consistently recovered from the nasal and the vaginal swabbings obtained from the three goats. Virus was never recovered from the ocular and rectal swabbings nor from any buffy coat samples. However, the buffy coats were positive for viral DNA detected by polymerase chain reaction (PCR). All isolates from the experimental goats were identical in their restriction patterns to the original BA-1 and were similar to the reference E/CH strains of CpHV-1.  相似文献   

10.
Two experiments were conducted to evaluate the effect of marble spleen disease virus (MSDV) infection on the immune response of pheasants. In the first, 15 ring-necked pheasants were inoculated orally with cell-culture-propagated MSDV and 15 received saline (controls). On days 7, 21, and 35 postinoculation (PI), all birds received sheep erythrocytes intravenously. Hemagglutination titers to sheep erythrocytes were determined for serum samples collected weekly for 6 weeks. The virus-inoculated group had significantly (P less than 0.05) lower hemagglutination titers than the control group. In the second experiment, 30 pheasants were allotted into two groups as above. Whole blood was collected from each bird weekly for 5 weeks. The blood was cultured in microtiter plates with or without optimum concentrations of concanavalin A. Five of 10 MSDV-inoculated pheasants had significantly depressed T lymphocyte transformation on either day 7 or day 14 PI. Overall, the depression of T lymphocyte transformation was transient and mild.  相似文献   

11.
Five-week-old specific-pathogen-free chickens inoculated intravenously with a waterfowl-origin type A influenza virus (A/mallard/Ohio/184/86) had swollen and mottled kidneys on days 3, 5, and 7 postinoculation (PI) and multiple raised nodules on days 5, 10, and 20 PI. Histologically, the kidneys had multifocal heterophilic tubulointerstitial nephritis with epithelial necrosis on day 3 PI, lymphoplasmacytic tubulointerstitial nephritis on day 5 PI, and fibrosing interstitial nephritis with cortical lobular collapse, atrophic tubules, glomerular aggregates, and interstitial lymphoid follicles and aggregates on days 7, 10, and 20 PI. Heterophilic intratubular medullary-cone nephritis was present in dead or moribund chickens on days 3 and 5 PI. Furthermore, the presence of mild multifocal heterophilic tubulointerstitial nephritis on day 20 PI suggests that a waterfowl-origin strain of type A influenza virus of low pathogenicity has the potential to produce acute and chronic active nephritis in the chicken and that the kidney is a potential site for influenza viral persistence. The acute, subacute, and chronic histopathologic renal lesions of this influenza virus in chickens are similar to lesions reported for some nephropathogenic infectious bronchitis viruses and avian nephritis picornavirus.  相似文献   

12.
为了研究一株从鹭中分离到的禽流感病毒(AIV)A/Heron/Guangdong/C1/2013(H5N6)对鸭和小鼠的致病力,本研究通过对鸭和小鼠滴鼻点眼和鸡的颈静脉注射进行攻毒试验,观察其致病力和组织病理学等变化,对其生物学特性进行初步研究。结果显示,该毒株的鸡胚半数感染量(EID50)为10-8.16/0.1 mL,静脉接种致病指数(IVPI)为2.76。对鸭的半数致死量(LD50)为10-4.0/0.2 mL,对小鼠的LD50为10-4.67/0.05 mL。以106 EID50/只滴鼻点眼感染鸭,主要表现为食欲下降、精神萎靡、肿头流泪等症状,大多数鸭在感染后4~7 d死亡,感染后第7 天肝脏、肺脏、肾脏仍在排毒,解剖可见心包积液、肺脏淤血、肾脏肿大等症状,病理切片可见心脏、肝脏、脾脏、肾脏炎性细胞浸润,脑细胞核固缩等病变。以5×105 EID50/只滴鼻感染小鼠,主要表现为食欲下降、精神萎靡、被毛粗乱、聚堆等症状,大部分小鼠在感染后5~7 d死亡,第7天时只有肺脏仍在排毒,各脏器解剖学病变不明显,病理切片可见心脏、肾脏、肺脏炎性细胞浸润,脑细胞核固缩等病变。研究结果表明,该H5N6亚型AIV毒株对鸭和小鼠具有很强的致病力,IVPI大于1.2,为高致病性AIV,本研究为H5N6亚型AIV研究和防控提供了理论基础。  相似文献   

13.
Five antigen capture immunoassay test kits, Directigen Flu A (Becton Dickinson), QuickVue Influenza test kit (Quidel), FLU OIA (ThermoBiostar), Zstat Flu (ZymeTx, Inc.) and NOW FLU A Test (Binax) were used to detect avian influenza virus (AIV) in clinical specimens as per manufacturers' protocols. Each kit was shown to be specific for AIV propagated in embryonating chicken eggs (ECE); other respiratory viruses of poultry tested gave negative results. The Directigen Flu A kit proved to be 10-fold more sensitive than the other kits, capable of detecting 10(4.7) mean embryo lethal dose (ELD50)/ml in allantoic fluid; this is more sensitive than the hemagglutination test using chicken erythrocytes. None of the kits proved to be sufficiently sensitive to reliably detect AIV in oropharyngeal and cloacal swabs collected from chickens experimentally infected with AIV subtype H6N2. In two different experiments, individual swabs and pools of five or six swabs were tested. By virus isolation, 39 individual oropharyngeal swabs tested positive for AIV, but Directigen and Flu OIA only detected 2/39 and NOW FLU A 1/39. Zstat and QuickVue did not detect any. Five individual cloacal swabs positive by virus isolation were negative with all five kits. In a second experiment using pools of five swabs, 26 swab pools were positive by virus isolation and 5/26 were positive by Directigen, the only kit to provide any positive results. Five cloacal swab pools were also positive by virus isolation and 1/5 was positive by Directigen; all other test kits were negative. All of these experiments were performed using the H6N2 subtype of AIV. The results are disappointing, as the kits have proven to be insensitive for detecting AIV when compared with the gold standard, virus isolation. This limits their use in diagnostic field investigations. Individual or groups of chickens could be assumed to be positive for AIV if positive by any of the kits, but a negative result with any of the kits would not prove that birds were AIV free.  相似文献   

14.
Fourteen calves were inoculated intranasally (i.n.) with the viral isolates as follows: 5 with 85/BH 16TV, 1 with 85/BH 17TV, 1 with 85/BH 18TV, 2 with 85/BH 231TN and 5 with 85/BH 232TN. Strain 85/BH 16TV was the only one which caused overt respiratory-like disease in all inoculated calves. Onset of the disease was observed after 7-8 days of incubation and was characterized by fever, depression, nasal discharge and coughing. Virus was isolated from the nasal swabbings of calves obtained from post-infection day (PID) 2-10. The other viral strains did not cause any sign of disease although virus was isolated regularly from the nasal swabbings of the inoculated calves. Virus was recovered from central nervous system tissues of calves that were infected with 85/BH 16TV or 85/BH 232TN strains and were killed on PID 4 or 8. Virus was also isolated from other tissues, such as lymph node, nasal mucosa (PID 8), or lung (PID 4). It was speculated that the nervous system could be one of the target areas of the virus of the naturally occurring infection by BHV-4. This might indicate a possible role of the nervous system (site of latency?) in the pathogenesis of BHV-4 as is the case in certain herpesviral infections of man and the lower animals.  相似文献   

15.
In vitro studies with organ (oviduct and trachea) and chicken embryo kidney cell cultures were attempted to assess the pathogenicity of locally isolated infectious bronchitis virus (IBV-P:120) initially isolated from the oviduct of young chicks. In oviduct cultures infected with IBV, ciliary movements decreased as early as 24 hours postinoculation (PI), and on the 6th day ciliary movements ceased completely. Cytopathic changes were also noticed. Immunofluorescent antigen was detected from 1 to 6 days PI, the maximum being on the 3rd day. The characteristic microscopic changes in the oviduct explants were reduced by 24 hours PI and had completely ceased on the 5th day. Cytopathic effect and immunofluorescent antigen were present from 1 to 8 days PI, being maximum on the 5th day. Histological changes marked by loss of cilia, rounding of the epithelial cells, degeneration, and sloughing were detected from 2 to 8 days PI. Low-embryo-passaged (EP-7) IBV did not produce cytopathic effect on the chicken embryo kidney cell cultures. On the contrary, high-embryo-passaged (EP-14) virus produced cytopathic effect at the third tissue-culture-passage level.  相似文献   

16.
为探究参虎败毒颗粒抗猪繁殖与呼吸综合征病毒(PRRSV)的作用,将20头PRRSV阴性仔猪随机平均分为4组,即提前药物处理感染组(A组)、感染同时药物处理组(B组)、无药物处理感染组(C组)和无药物处理不感染组(D组).A和B组分别于攻毒前3d和攻毒时在基础日粮中添加参虎败毒颗粒(每头10g·d-1),连续饲喂至攻毒后...  相似文献   

17.
Between 1993 and 2000, gallinaceous birds, waterfowl, and environmental specimens from the live bird markets (LBMs) of the northeastern United States and non-LBM premises were tested for the presence of avian influenza virus (AIV), pathogenic properties of AIV subtypes, especially of hemagglutinin (H) subtypes H5 and H7, and a possible association between LBM and non-LBM infections. Ten H subtypes of AIV were isolated from the LBM specimens: H1, H2, H3, H4, H5, H6, H7, H9, H10, and H11. During this period, the 10 subtypes also were isolated from birds in non-LBM premises. In the LBMs, subtypes H2, H3, H4, H6, H7, and H11 were present for 5-8 yr despite efforts to clean and disinfect the premises. The H5 or H7 subtypes present during the same year in both LBMs and non-LBMs within a state or in contiguous states were (subtype/year): H5N2/1993, 1999, and H7N2/1994-99. The AIV subtypes including the H5 and H7 that were evaluated for pathogenicity in chickens were low pathogenic. The deduced amino acid sequence at the H cleavage site of H5 and H7 subtypes was consistent with those of low pathogenic AIV. Although the H5N2 and H7N2 subtypes remained low pathogenic, they did undergo mutations and acquired an additional basic amino acid at the H cleavage site; however, the minimum number of basic amino acids in correct sequence (B-X-B-R, where B = basic amino acid, X = need not be basic amino acid, and R = arginine) required for high pathogenicity was lacking. A low pathogenic H5 or H7 subtype may become highly pathogenic by acquiring additional basic amino acids at the H cleavage site. The LBMs have been and will likely continue to be a source of AIV for commercial poultry.  相似文献   

18.
Two experiments were undertaken to evaluate whether porcine reproductive and respiratory syndrome (PRRS) virus was able to cross the placenta and infect midgestation fetuses following intranasal inoculation of sows and whether PRRS virus directly infected fetuses following in utero inoculation. In experiment 1, eight sows between 45 and 50 days of gestation were intranasally inoculated with PRRS virus (ATCC VR-2332), and four control sows were inoculated with uninfected cell culture lysate. Virus inoculated sows were viremic on postinoculation (PI) days 1, 3, 5, 7 and 9, shed virus in their feces and nasal secretions, and became leukopenic. Sixty-nine of 71 fetuses from principal sows euthanized on PI day 7, 14 or 21 were alive at necropsy and no virus was isolated from any of the fetuses. Two principal sows that farrowed 65 and 67 days PI delivered 25 live piglets and three stillborn fetuses. The PRRS virus was isolated from two live piglets in one litter. In experiment 2, laparotomies were performed on five sows between 40 and 45 days of gestation and fetuses were inoculated in utero with either PRRS virus alone, PRRS virus plus a swine serum containing PRRS antibodies, or uninfected cell culture lysate. Three sows were euthanized on PI day 4 and two sows on PI day 11. Viral replication occurred in fetuses inoculated with virus alone and was enhanced in fetuses inoculated with virus plus antibody. No virus was isolated from control fetuses.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

19.
To investigate the pathogenicity of A/Hero/Guangdong/C1/2013(H5N6), an AIV strain isolated from Heron on ducks and mice, pathogenicity of this new virus strain and changing of histopathology as well as a preliminary study on its biological characteristics were studied by virus challenges test via intranasal and eye-drop and in chicken via jugular vein injection.Results showed that the EID50 of this strain of virus was 10-8.16/0.1 mL in embryonated chicken eggs and the intravenous inoculation of pathogenic index (IVPI) was 2.76.In ducks and mice, the 50% lethal doses (LD50) of it were determined to be 10-4.0/0.2 mL and 10-4.67/0.05 mL, respectively.Symptoms of infection included loss of appetite, depression, swollen head and tears after being infected with 106 EID50 per duck by intranasal and eye-drop administration.Most of ducks died 4 to 7 days post-infection, liver, lung and kidney still eliminated toxicants at day 7 post-infection.Anatomy showed symptoms of pericardial effusion, pulmonary congestion and kidney enlargement, while pathological section showed pathological change like karyopycnosis and inflammatory cell infiltration in heart, liver, kidney and spleen.Mice developed symptoms of infection like loss of appetite, depression, shaggy coat and ruffled coat after being infected with 5×105 EID50 per mouse by intranasal and eye-drop administration.Most of the infected mice died 5 to 7 days post-infection and only liver still eliminated toxicants at day 7 post-infection.Although organ anatomy showed no obvious pathological changes, pathological section showed pathological changes like karyopycnosis and inflammatory cell infiltration in heart, kidney and lung.Our research demonstrated that this H5N6 subtype AIV had a strong pathogenicity and could be defined as a highly pathogenic AIV strain as its IVPI was greater than 1.2.Our work laid a theory foundation for study, prevention and control of H5N6 subtype AIV.  相似文献   

20.
为研究鹅源H5N1亚型禽流感病毒(AIV)人工感染雏鸡免疫器官细胞凋亡的动态变化,本研究将50只1日龄SPF雏鸡随机分为两组。试验组雏鸡于7日龄,分别经鼻、眼、口同时感染105TCID50的鹅源H5N1亚型AIV,分别于感染后3d、4d、5d、7d和14d迫杀,采取胸腺、法氏囊和脾脏,应用TUNEL染色法和透射电镜技术观察其细胞凋亡的动态变化情况。结果显示,试验组雏鸡的胸腺和脾脏凋亡细胞数量在感染后3d~7d极显著高于对照组(p<0.01);法氏囊凋亡细胞数量在感染后3d~4d比对照组明显增加(p<0.05或p<0.01)。组织器官超微结构检测可见凋亡细胞核染色质固缩并凝结成块,聚集在核膜周围,呈新月状或环状;细胞质浓缩。表明鹅源H5N1AIV能够诱导感染雏鸡免疫器官发生细胞凋亡。  相似文献   

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