血清4型禽腺病毒强毒株GY株的分离鉴定和致病性研究

从山东某疑似感染禽腺病毒的发病鸡群中采集病料并分离到一株禽腺病毒,克隆纯化后进行PCR检测和序列分析,鉴定该毒株为血清4型禽腺病毒,命名为GY株。用LMH细胞繁殖的GY株病毒滴度可以达到107.5 TCID₅₀/0.1mL,并建立了纯净稳定的种子批。动物致病性试验结果显示,攻毒鸡只8/10死亡,剖检均出现心包积液,肝脏肿大、出血或坏死,表明该毒株为强毒株。就攻毒途径、攻毒剂量等方面研究发现,GY株经胸部肌肉注射后发病率和发病时间相对颈部皮下注射途径更有优势,确定攻毒途径为肌肉注射;通过比较不同剂量(5×10^3.0~5×10^6.0 TCID50)GY株病毒攻击SPF鸡的致病性,将攻毒剂量定为5×10^5.0 TCID₅₀。研究表明,GY株可作为禽腺病毒4型攻毒用强毒株。     

猪伪狂犬病活疫苗(Bartha-K61株,传代细胞源)最小免疫剂量的研究

为了确定猪伪狂犬病活疫苗(Bartha-K61株,传代细胞源)的最小免疫剂量,本研究将3批猪伪狂犬病活疫苗(Bartha-K61株,传代细胞源)分别稀释成10TCID_(50)/mL、102.0TCID_(50)/mL、103.0TCID_(50)/mL,每批疫苗各个稀释度分别免疫仔猪1.0 mL/头,并设攻毒对照组和阴性对照组。免后10 d连同攻毒对照组用伪狂犬病病毒GD1株进行攻毒保护试验,阴性对照组不攻毒。结果表明,10TCID_(50)/头和102.0TCID_(50)/头的免疫剂量在免疫后10 d依然无法提供完全的免疫保护,保护率为20%～80%(1/5～4/5);103.0TCID_(50)/头的免疫剂量能够保护仔猪抵抗PRV强毒的攻击,保护率为100%(5/5);攻毒对照组发病率为100%(5/5),死亡率为80%(4/5);阴性对照组全部健活。由此确定猪伪狂犬病活疫苗(Bartha-K61株,传代细胞源)最小免疫剂量为103.0TCID_(50)/头。     

禽脑脊髓炎病毒VR株的致病性研究

为探索禽脑脊髓炎病毒强毒VR株对SPF鸡的致病性,开展了不同接种途径和不同日龄SPF鸡的致病力试验。不同接种途径致病力试验结果表明,AEV VR株口服后不能引起发病;刺种、肌肉注射途径接种随着日龄增大,发病率降低;脑内注射途径接种可引起试验鸡全部发病。最小致病量试验结果显示,VR株脑内注射攻毒的最小致病量为10~(2.0) EID_(50)。不同日龄SPF鸡的致病力试验结果表明,随着试验鸡日龄的增大,其致病力略有差异,日龄越小,发病率越高。结果表明,AEV VR株脑内攻毒70日龄内SPF鸡的发病率为80%及以上,攻毒剂量为10~(4.0)EID_(50)。     

猪口蹄疫O型灭活疫苗PD50效检攻毒剂量试验

采用PD50效检方法,对0304041-2批猪口蹄疫O型灭活疫苗(I型苗)进行不同攻毒剂量的PD50 检测.结果表明,分别用500 ID50 、1 000 ID50 、2 000 ID50 和4 000 ID50 的病毒进行攻毒,疫苗每头份分别含5.2、3.6、2.1和1.0个PD50 .该项试验的攻毒毒株ORMF8的ID50为10-7.0/2 mL,MID为10 -6.0 /2 mL.     

新城疫CHR株与La Sota株2种疫苗近期免疫效力比较试验

鸡新城疫CHR 株冻干疫苗中试产品3 批以及La Sota 株冻干疫苗( 符合质量要求) , 以5 种免疫途径: 肌注、滴鼻、喷雾、饮水、拌料分别接种11 日龄雏鸡。每批疫苗每种途径接种1 组, 20 只/ 组, 共20 组。雏鸡群免疫前血凝抑制抗体(HI) 效价为1∶2-13 , 于14 d 、28 d 后再测HI抗体水平, 并分别用10 000 鸡胚半数致死量(ELD50) 新城疫标准强毒北京株攻击。从而检测2 种疫苗用5 种免疫途径免疫后在不同时间的近期免疫效力。结果表明, 近期内CHR 株疫苗HI抗体呈上升趋势, 而La Sota 株疫苗HI抗体先升高后下降; CHR 株疫苗以饮水和拌料途径效果较好, 其它3 种途径效果相似。接种后14 d 攻毒, 2 种疫苗均能较好保护强毒攻击; 28 d 后, CHR 株保护率高于La Sota 株。     

鸡传染性法氏囊病疫苗B87对SPF鸡289的免疫效果

30日龄SPF鸡经点鼻途径用鸡传染性法氏囊病病毒（1BDV）的中等毒力疫苗B87株免疫后14d，再以200个ELD50超强毒IBDV株GX8/99点鼻人工攻毒，所有鸡都没有发病死亡。而对照鸡有10/10发病和6/10死亡，并有典型的病理变化。这表明，对SPF鸡用中等毒力的IBDV疫苗免疫可诱发有效的免疫保护作用。并能完全抵抗超强毒IBDV的攻击。     

鸡传染性法氏囊病病毒地方流行毒株的免疫原性

从河北省一些发病鸡场分离到JD1～JD10共10株鸡传染性法氏囊病病毒(IBDV)毒株,用IBD标准阳性血清以琼扩试验进行了初步鉴定.并进行了IBDV分离物及其鸡胚适应毒免疫原对标准强毒IBDV-BC6/85株免疫保护试验,D78弱毒疫苗对IBDV各分离毒株的免疫保护试验以及分离毒株间交互免疫保护试验.结果表明,D78疫苗对JD2,JD5和JD10 IBDV分离株的保护率较低,分别为40%、50%和60%.分离毒株JD5、JD2及其鸡胚传代物E-JD2对强毒株的免疫保护率可达100%.交互免疫保护试验表明,JD2对其余各分离株的免疫保护指数达到80%以上,对标准强毒株和地方分离株均可产生有效免疫保护.     

鸡肾型传染性支气管炎病毒SF弱毒株的鉴定

针对IBV地方分离株SF株弱化毒进行鉴定,经特异性试验证实,SF株致弱毒能完全被本毒制备的血清所中和;血清交叉中和试验证明,SF株毒与Ark99株有高度的交叉保护;采用E1、E5代弱化毒接种SPF10日龄鸡胚,测定毒价EID50分别为10-6.88/0.1ml,10-6.68/0.1ml;同居感染试验未观察到健康鸡组织器官病变;连传SPF易感雏鸡5代无毒力返强;攻毒保护试验证实3000个EID50以上的免疫量使免疫雏鸡能耐受强毒攻击,达到完全保护;理化试验表明,该毒株对热、碱的耐受性较差,对乙醚、氯仿有机溶剂较为敏感,对酸有一定的耐受性;试验数据说明,SF株IBV已被成功弱化,抗原性好,毒力稳定、对雏鸡安全,不会产生毒力返强,并能产生较强的免疫力,是一株理想的弱毒苗株.     

禽腺病毒4型SD2015株对SPF鸡的致病性研究

2015年从临床发病肉鸡鸡群中分离到1株禽腺病毒,经PCR鉴定及同源性比对确定为血清Ⅰ群4型禽腺病毒,对其肝组织毒及适应SPF鸡胚与鸡胚肝细胞的细胞毒进行致病性研究。结果显示:肝组织毒毒力最强且传代稳定;肝组织毒经肌肉注射途径攻毒,试验鸡发病率及死亡率略高于皮下注射而远高于口服感染途径;随攻毒鸡日龄的增长,其对腺病毒的感染力下降;肝组织毒攻毒量为108.0TCID50/只,可引起攻毒鸡8/10以上死亡,10/10感染,感染鸡剖检有典型的心包积水、肝脏肿大、肾脏肿大等病变。本试验结果对Ⅰ群4型禽腺病毒灭活疫苗免疫效力评价具有重要意义。     

1株基因3型鸭甲肝病毒鸡胚化弱毒株的培育

为研发基因3型鸭甲肝病毒(Duckhepatitis virustype3,DHAV-3)的弱毒活疫苗,用SPF鸡胚对DHAV-3Y株进行了连续传代致弱,由此获得第80代鸡胚适应毒。致病性试验结果显示,第80代毒株对1日龄雏鸭已无致病性。免疫攻毒保护试验显示,雏鸭在1日龄时经肌肉注射途径免疫第80代鸡胚适应毒,能有效抵抗7日龄时的强毒感染,保护指数为100%。表明第80代鸡胚适应毒是一株具有良好免疫原性的鸡胚化弱毒疫苗候选株。     

口蹄疫疫苗效检模型动物测毒及口蹄疫种毒冻干试验

应用豚鼠作为口蹄疫疫苗效检模型动物检测口蹄疫病毒对模型动物的病原性.用体重400 g左右的豚鼠,将猪O型口蹄疫灭活疫苗效检攻毒毒株ORMF8经后肢蹠部皮内注射途径进行测毒.测毒结果表明,口蹄疫病毒可引起豚鼠出现典型发病,并产生明显病变,ORMF8种毒对豚鼠的毒价可达105.5ID50/0.2 mL.将乳鼠中和试验用种毒OMⅡ按一定比例加入5%蔗糖脱脂牛奶稳定剂进行冷冻真空干燥试验,3次冻干试验结果表明,种毒冻干后病毒含量有一定程度的下降,但下降程度不显著.     

The plasmid constructs producing shRNA corresponding to the conserved 3D polymerase of Foot and Mouth Disease virus protects guinea pigs against challenge virus

RNA interference (RNAi) has been used as an effective antiviral strategy for its specific silencing of viral gene expression in mammalian cells. In this study, shRNA targeting two regions of Foot and Mouth Disease Virus (FMDV) i.e. 3D and 5'UTR which are very essential in virus replication were evaluated. The constructs were made using h7K RNA polymerase III promoter. We investigated in vivo inhibitory effect of shRNA on FMDV replication in BHK-21 cells and guinea pigs. The results showed that transfection of 3D shRNA could reduce virus growth by three folds when cells were challenged with 10(2) TCID(50) of FMDV. Pretreated guinea pigs with 3DshRNA were protected 80% with 10(3) GPID(50) of FMDV. As a first report in guinea pigs which are recognized animal model for FMD vaccine potency testing, the study suggests that shRNA could be a viable therapeutic approach to control severity of FMD infection and spread.     

Foot-and-mouth disease - quantification and size distribution of airborne particles emitted by healthy and infected pigs

There is strong evidence to suggest that foot-and-mouth disease (FMD) can be transmitted by airborne virus up to many kilometres from a virus source. Atmospheric dispersion models are often used to predict where this disease might spread. This study investigated whether FMD virus (FMDV) aerosol has specific characteristics which need to be taken into consideration in these models. The characteristics and infectiousness of particles emitted by 12 pigs have been studied pre- and post-infection with O UKG 2001 FMDV. Aerosol generated by individual pigs was found log normally distributed in the range 0.015-20.0microm with concentrations between 1000 and 10000cm(-3) at the smallest size and <1cm(-3) above 10microm. No differences in either the total number of particles produced or their size distribution were detected between uninfected and infected pigs. However, a correlation between aerosol concentration and animal activity was found with a more active pig producing significantly greater concentrations than those that were less active. Viable virus was found up to a maximum of 6.3 log TCID(50)/24h/animal. The virus was distributed almost equally across the three size ranges; <3, 3-6 and >6microm. No correlation could be established between the production of virus and animal activity. In general the production of airborne virus closely followed the detection of viraemia in the blood and the presence of clinical symptoms. However, in one instance a pig excreted as much airborne virus as the other animals in the study, but with less virus detected in its blood. The results suggest that there is little merit in including a sophisticated virus release pattern based on physical activity periods or FMDV aerosol size spectrum, together with the appropriate dry deposition calculations, in models used to predict airborne spread of FMD. An estimate of the total daily virus production based on the clinical assessment of disease and virus strain is sufficient as input.     

水泡性口炎的的研究进展

水泡性口炎(Vesicular Stomatitis，VS)是由水泡性口炎病毒(Vesicular Stomatitisvirus，VSV)引起马、牛和猪的一种重要的传染病。临床表现为口腔粘膜、乳房和蹄部冠状带皮肤出现水泡和溃疡。VSV感染牛和猪时，在临床症状上极易与口蹄疫(FMD)、猪水泡病(SVD)、猪水泡疹(VES)混淆。且能够感染人。被国际兽疫局(OIE)列为A类传染病。因此，对VSV致病机理的研究有着重要的社会经济和公共卫生意义。本文从病原及其分子生物学特点、分布与生态学以及致病机理三个方面对水泡性口炎的研究进展进行综述。     

Characteristics of foot and mouth disease virus in Taiwan

Since March 1997 two strains of foot and mouth disease (FMD) virus have found their way into Taiwan, causing severe outbreaks in pigs and in Chinese yellow cattle. Outbreaks occurred in March 1997 were caused by a pig-adapted virus strain (O/Taiwan/97) which did not infect other species of cloven-hoofed animals by natural route. The epidemic spread over the whole region of Taiwan within two months and the aftermath was 6,147 pig farms infected and 3,850,746 pigs destroyed. In June 1999, the second strain of FMD virus (O/Taiwan/99) was isolated from the Chinese yellow cattle in the Kinmen Prefecture and in the western part of Taiwan. By the end of 1999, Chinese yellow cattle were the only species infected and those infected cattle did not develop pathological lesions. Seroconversions of serum neutralization antibody and on non-structural protein (NSP) antibodies were the best indicators for infection in non-vaccinated herds. The infected animals, however, excreted infectious levels of virus to infect new hosts. Based on the detection of the specific antibody to FMD virus, and virus isolation from oesophageal-pharyngeal (OP) fluid samples, ten herds of Chinese yellow cattle located in Kinmen and Taiwan were declared to have been infected. During the period of January to March 2000, however, five outbreaks caused by FMD virus similar to the O/Taiwan/99 virus occurred in four prefectures of Taiwan. The infected species included goats, Chinese yellow cattle and dairy cattle. Those outbreaks have caused high mortality in goat kids under two weeks old and also developed typical clinical signs of infection in dairy cattle.     

Cedivac-FMD can be used according to a marker vaccine principle

In this study, we investigated whether Cedivac-FMD, an emergency vaccine against foot-and-mouth disease (FMD), is suitable for use conjointly with a screening program intended to confirm freedom from disease in vaccinated herds based on evidence of virus replication in vaccinates. Different sets of sera were tested using the Ceditest FMDV-NS ELISA for the detection of antibodies against non-structural proteins (NSPs) of FMD virus. During a vaccine safety study, serum samples were collected from 10 calves, 10 lambs and 10 piglets following administration of a double dose and a repeat dose of high payload trivalent Cedivac-FMD vaccine. All serum samples collected both 2 weeks following the administration of a double dose as well as those collected 2 weeks after the single dose booster (given 2 weeks after the double dose) were negative in the Ceditest FMDV-NS ELISA. In a series of vaccine potency experiments, serum samples were collected from 70 vaccinated cattle prior to and following exposure to infectious, homologous FMD virus. When testing cattle sera collected 4 weeks after vaccination with a regular dose of monovalent >6 PD(50) vaccines, 1 of 70 animals tested positive in the NSP antibody ELISA. After infection with FMD virus, antibodies to NSP were detected in 59 of 70 vaccinated cattle and 27 of 28 non-vaccinated control animals within 7 days. Cedivac-FMD vaccines do not induce NSP antibodies in cattle, pigs or sheep following administration of a double dose or a repeat dose. FMD-exposed animals can be detected in a vaccinated group within 7-14 days. Because Cedivac-FMD does not induce NSP antibodies, the principle of 'marker vaccine' applies.     

Foot-and-mouth disease: a review of intranasal infection of cattle, sheep and pigs

In an outbreak of foot-and-mouth disease (FMD) it is important to identify animals at risk from airborne virus. Investigations have been carried out over the years to determine the dose required to infect cattle, sheep and pigs by the intranasal route. This paper reviews the results of investigations for animals which have been infected by instillation or spraying a virus suspension into the nostrils or by exposure to affected animals through a mask or by indirect contact. The lowest doses were found by use of a mask. With virus from affected pigs given through a mask, doses of 18 infectious units (IU) in cattle and 8 IU in sheep were found to cause infection and give rise to lesions. Overall, cattle required the least amount of virus followed by sheep. Pigs required a dose of 22 IU to cause infection and a dose of 125 IU to give rise to lesions. In many experiments pigs failed to become infected. With all three species the dose varied with the individual animal and the virus strain. For modelling previous outbreaks and in real time, a dose of 8 IU or 10 and 50% infectious doses (ID50) could be used where cattle and sheep were involved. Experience in the field, combined with the results from experiments involving natural infection, indicate that pigs are not readily infected by the intranasal route. However, for modelling purposes a dose of about 25 IU should be used with care. Investigations are needed to determine doses for virus strains currently in circulation around the world. In addition, the nature of the aerosol droplets needs to be analysed to determine how the respective amounts of infective and non-infective virus particles, host components and, in later emissions, the presence of antibody affect the survival in air and ability to infect the respiratory tract. Further work is also required to correlate laboratory and field findings through incorporation of the doses into modelling the virus concentration downwind in order that those responsible for controlling FMD are provided with the best available assessment of airborne spread. Finally, the doses found for infection by the intranasal route could be applied to other methods of spread where virus is inhaled to assess risk.     

Evaluation of a 3A-truncated foot-and-mouth disease virus in pigs for its potential as a marker vaccine

Foot-and-mouth disease (FMD) is a highly contagious and economically devastating disease of cloven-hoofed animals in the world. The disease can be effectively controlled by vaccination of susceptible animals with the conventional inactivated vaccine. However, one major concern of the inactivated FMD virus (FMDV) vaccine is that it does not allow serological discrimination between infected and vaccinated animals, and therefore interferes with serologic surveillance and the epidemiology of disease. A marker vaccine has proven to be of great value in disease eradication and control programs. In this study, we constructed a marker FMDV containing a deletion of residues 93 to 143 in the nonstructural protein 3A using a recently developed FMDV infectious cDNA clone. The marker virus, r-HN/3A_93–143, had similar growth kinetics as the wild type virus in culture cell and caused a symptomatic infection in pigs. Pigs immunized with chemically inactivated marker vaccine were fully protected from the wild type virus challenge, and the potency of this marker vaccine was 10 PD₅₀ (50% pig protective dose) per dose, indicating it could be an efficacious vaccine against FMDV. In addition, we developed a blocking ELISA targeted to the deleted epitope that could clearly differentiate animals infected with the marker virus from those infected with the wild type virus. These results indicate that a marker FMDV vaccine can be potentially developed by deleting an immunodominant epitope in NSP 3A.     

Comparison of sensitivity and specificity in three commercial foot-and-mouth disease virus non-structural protein ELISA kits with swine sera in Taiwan

Three commercialized ELISA kits for the detection of antibodies to the non-structural proteins (NSPs) of FMD virus were compared, using sera from uninfected, vaccinated, challenged and naturally infected pigs. The kinetics of the antibody response to NSPs was compared on sequential serum samples in swine from challenge studies and outbreaks. The results showed that ELISA A (UBI) and ELISA B (CEDI) had better sensitivity than that of the 3ABC recombinant protein-based ELISA C (Chekit). The peak for detection of antibodies to NSPs in ELISA C was significantly delayed in sera from natural infection and challenged swine as compared to the ELISA A and B. The sensitivity of the three ELISAs gradually declined during the 6-month post-infection as antibodies to NSP decline. ELISA kits A and B detected NSP antibody in 50% of challenged pigs by the 9-10th-day and 7-8th-day post-challenge, respectively. ELISA B and C had better specificity than ELISA A on sequential serum samples obtained from swine immunized with a type O FMD vaccine commercially available in Taiwan. Antibody to NSPs before vaccination was not detected in swine not exposed to FMD virus, however, antibody to NSPs was found in sera of some pigs after vaccination. All assays had significantly lower specificity when testing sera from repeatedly vaccinated sows and finishers in 1997 that were tested after the 1997 FMD outbreak. However, when testing sera from repeatedly vaccinated sows or finishers in 2003-2004, the specificity for ELISAs A, B and C were significantly better than those in 1997. This effect was less marked for ELISA A. The ELISA B was the best test in terms of the highest sensitivity and specificity and the lowest reactivity with residual NSP in vaccinates.     

Procedures for preventing the transmission of foot-and-mouth disease virus to pigs and sheep by personnel in contact with infected pigs

The most effective method of containing an outbreak of foot-and-mouth disease (FMD) is by the culling of livestock. However, qualified people must diagnose the disease before the culling can begin, and they must avoid susceptible animals after having been in contact with infected premises, to prevent them from transmitting the virus. To test the effectiveness of biosecurity procedures in preventing the transmission of FMD virus (O/UK/35/2001) investigators contacted and sampled pigs inoculated with FMD virus for approximately 45 minutes and then contacted and sampled sentinel pigs and sheep after either using no biosecurity procedures, or washing their hands and donning clean outerwear, or showering and donning clean outerwear. The virus was detected in the nasal secretions of one investigator immediately after the postmortem investigation of the inoculated pigs but was not detected in samples collected between approximately 12 and 84 hours later. After the contaminated personnel had showered and changed into clean outerwear they did not transmit the strain of FMD virus to susceptible pigs and sheep.