Specificity of the indirect enzyme-linked immunosorbent assay for detection of pseudorabies virus antibodies in pigs exposed to bovine herpesvirus-1

Six 5-week-old pigs were inoculated intranasally (IN) with 10(7.6) TCID50 of bovine herpesvirus-1 (BHV-1). Three of the pigs also were inoculated IV with a similar dose of BHV-1. Clinical responses were not observed in these 6 pigs before oronasal challenge exposure with 10(7.8) TCID50 of virulent pseudorabies virus (PRV) at postinoculation day 42. Two pigs inoculated IN with BHV-1 and challenge exposed with PRV remained healthy, whereas the remaining 4 pigs developed severe clinical signs of pseudorabies and were moribund at postinoculation day 50 (8 days after challenge exposure). Anti-BHV-1 antibodies were demonstrable by ELISA in all 6 pigs and by serum neutralization (SN) in 5 pigs before challenge exposure with PRV. Anti-PRV antibody was not detected by ELISA or SN before challenge exposure to PRV. After challenge exposure to PRV, pigs with humoral antibody to BHV-1 responded anamnestically, and anti-PRV antibody activity was demonstrable by ELISA and SN in the 2 surviving pigs.     

Effects of a whey protein product and spray-dried animal plasma on growth performance of weanling pigs

Five experiments were conducted to evaluate the effects of a high-protein, whey protein product (WPP; 73% CP, 6.8% lysine, 12.8% fat, and 5% lactose) and spray-dried animal plasma (SDAP) on growth performance of weanling pigs. In all experiments, pigs were fed experimental diets from d 0 to 14 after weaning in a pelleted form and then a common diet in meal form for the remainder of the experiment. Dietary treatments were established by substituting WPP or SDAP for dried skim milk (Exp. 1) or soybean meal (Exp. 2, 3, 4, and 5) in the control diet. In Exp. 1, we maintained a constant level of lactose in all diets by adjusting the amount of added crystalline lactose. The amount of lactose in diets used in Exp. 2 through 5 varied slightly by the addition of WPP. In Exp. 1 and 2, 180 weanling pigs (initially 5.8 kg and 19 +/- 1 d of age or 5.5 kg and 17 +/- 1 d of age, respectively) were used. Treatment diets contained SDAP (2.5 and 5%) or WPP (2.7 and 5.4% in Exp.1, and 2.5 or 5.0% in Exp. 2). In Exp. 1, from d 0 to 7 after weaning, ADG and ADFI increased with increasing SDAP (linear, P < .01). No other treatment effects were observed during the d 0 to 14 period. In Exp. 2, from d 0 to 14 after weaning, ADG and G:F increased (linear, P < .04) with increasing SDAP or WWP. In Exp. 3, 305 weanling pigs (initially 4.1 kg and 12 +/- 1 d of age) were used. The control diet contained 2.5% SDAP. The experimental diets were similar to the control diet but contained an additional 2.5 or 5.0% SDAP or 2.5 or 5.0% WPP. From d 0 to 14 after weaning, ADG, ADFI, and G:F increased (quadratic, P < .05) with increasing SDAP up to 5.0%. Increasing WPP increased ADG (quadratic, P < .07) and ADFI (linear, P < .09). In Exp. 4 and 5, 329 and 756 weanling pigs (initially 4.1 kg and 12 +/- 1 d of age and 5.2 kg and 18 +/- 1 d of age, respectively) were fed diets in which WPP was substituted for 0, 25, 50, 75, and 100% (Exp. 4) or 0, 50, and 100% (Exp. 5) of the SDAP in the control diet. In Exp. 4 and 5, from d 0 to 14 after weaning, pigs fed a 1:1 blend of each protein source had better ADG (quadratic, P < .04) than those only fed SDAP. In conclusion, WPP can be used in combination with or as a total replacement for SDAP in diets for weanling pigs without reducing performance.     

检测猪伪狂犬病病毒gE抗体红细胞凝集试验的建立及应用

以纯化的抗人红细胞单链抗体(ScFv)—猪伪狂犬病病毒(PRV)gE蛋白双功能融合蛋白为抗原,建立了检测猪伪狂犬病毒gE抗体的红细胞凝集试验。利用方阵滴定试验筛选出最佳抗原工作浓度为55μg/mL,血清最佳稀释度为1∶20,作用时间15 min,与猪瘟(CSF)、猪细小病毒(PPV)、猪繁殖与呼吸综合征(PRRS)、猪乙型脑炎(JE)、猪布氏杆菌病(Brucellosis)阳性血清和PRV gE缺失疫苗接种的猪免疫血清均不出现红细胞凝集现象,与PRV标准阳性血清反应出现肉眼可见的凝集圈。与美国进口的PRV抗体检测gE-ELISA诊断试剂盒检测结果比较,50份猪血清的阴、阳性检出符合率均为100%。红细胞凝集试验检测方法具有操作简便、敏感性和特异性较高的特点,可用于PRV野毒感染的快速筛查。     

Comparison of wheat gluten and spray-dried animal plasma in diets for nursery pigs

Five experiments were conducted to determine the effects of different wheat gluten (WG) sources (Source 1 = enzymatically hydrolyzed, Source 2 = nonmodified ring-dried, Source 3 = spray-dried, and Source 4 = flash-dried) on growth performance of nursery pigs compared with soybean meal (SBM), spray-dried animal plasma (SDAP), or other specialty protein sources. In Exp. 1, pigs (n = 220, initially 6.1 +/- 2.5 kg) were fed a control diet containing (as-fed basis) 6% SDAP or WG Source 1 or 2. The WG and l-lysine*HCl replaced 50 or 100% of the SDAP. From d 0 to 21, increasing WG (either source) decreased ADG and ADFI (linear, P < 0.01), but improved (linear, P < 0.02) G:F. In Exp. 2, pigs (n = 252, initially 6.2 +/- 3.0 kg) were fed a negative control diet containing no SDAP or WG, diets containing (as-fed basis) 9% WG Source 1 or 5% SDAP, or combinations of WG and SDAP where WG and l-lysine*HCl replaced 25, 50, or 75% of SDAP. From d 0 to 14, pigs fed increasing WG had decreased ADG (linear, P < 0.05). In Exp. 3, pigs (n = 240, initially 7.0 +/- 2.5 kg) were fed a negative control diet, a diet containing (as-fed basis) either 3, 6, 9, or 12% WG Source 3, or a positive control diet containing 5% SDAP. The diets containing 9% WG and 5% SDAP had the same amount of SBM. From d 0 to 7, pigs fed 5% SDAP had greater (P < 0.04) ADG than pigs fed the diet containing 9% WG. From d 0 to 14, increasing WG had no effect on ADG, ADFI, or G:F. In Exp. 4, pigs (n = 200, initially 6.0 +/- 2.4 kg) were fed a negative control diet, the control diet with (as-fed basis) 4.5 or 9.0% WG Source 1, or the control diet with 2.5 or 5.0% SDAP. Diets containing WG and SDAP had similar SBM levels. From d 0 to 7 and 0 to 14, increasing SDAP tended to improve (linear, P < 0.06) ADG, but increasing WG had no effect. In Exp. 5, 170 barrows and gilts (initially 7.5 +/- 2.8 kg) were used to determine the effects of WG Source 1 and 4 compared with select Menhaden fish meal or spray-dried blood cells and a negative control diet (SBM) on the growth performance of nursery pigs from d 5 to 26 postweaning (d 0 to 21 of experiment). No differences were found in ADG or G:F, but pigs fed the diet containing (as-fed basis) 2.5% spray-dried blood cells had greater ADFI than pigs fed the negative control from d 0 to 21. Wheat gluten source had no effect on ADG, ADFI, or G:F. The results of these studies suggest that increasing WG in diets fed immediately after weaning did not improve growth performance relative to SBM or SDAP.     

某规模化猪场伪狂犬病的诊断

河南平顶山某猪场母猪出现较严重的流产和产死胎现象,且50日龄~70日龄仔猪出现神经症状,根据临床表现初步诊断为伪狂犬病。为排除猪繁殖与呼吸综合征和猪瘟,进行了实验室诊断。应用ELISA方法检测发病保育猪及母猪血清的伪狂犬病病毒野毒株gE抗体,并对发病仔猪病料进行了伪狂犬病病毒(PRV)、猪繁殖与呼吸综合征病毒(PRRSV)和猪瘟病毒(CSFV)的实时荧光定量PCR检测。结果显示,伪狂犬病病毒野毒抗体阳性,实时荧光定量PCR检测确定仔猪病料中PRV核酸阳性,PRRSV和CSFV核酸阴性。结合临床症状及实验室检测,确诊该猪场发生的是猪伪狂犬病。     

Influence of isoprinosine on lymphocyte function in virus-infected feeder pigs

Pseudorabies is a porcine herpesvirus of major importance in the swine industry. Isoprinosine is an immunomodulating drug that has been shown to be beneficial in treating herpesvirus infections. Twenty-four 7-week-old pigs were allotted within litters to 1 of 4 groups: control, isoprinosine (ISO), pseudorabies virus (PRV), or isoprinosine and pseudorabies virus (ISO-PRV). Isoprinosine was administered daily for 16 days to the ISO and ISO-PRV groups (75 mg/kg of body weight/day, PO). Immunity in pigs in the PRV and ISO-PRV groups was challenged with pseudorabies virus (10(5) TCID50 units) on day 4. Rectal temperatures and viral excretion were monitored daily; total and differential leukocyte counts, lymphocyte response to mitogens, and interleukin-2 production were monitored every 4 days. Pigs challenge-inoculated with pseudorabies virus became ill, with the ISO-PRV group most severely affected. Rectal temperatures were high (P less than 0.05) in virally challenged pigs on days 5 to 12 and 14 to 16; isoprinosine did not alter this effect. Pseudorabies virus-infected pigs had leukocytosis (P less than 0.05) on days 12 and 16, primarily caused by neutrophilia. Concanavalin A-stimulated lymphocyte proliferation was decreased (P less than 0.06) in both PRV and ISO-PRV groups on day 12, compared with control pigs, but only in the PRV group on day 16. Pokeweed mitogen-stimulated lymphocyte proliferation was decreased (P less than 0.02) in ISO-PRV pigs on day 8 of the experiment. Interleukin-2 concentrations, pooled over all sampling days, were decreased (P less than 0.03) in pseudorabies virus-infected pigs. Viral excretion was not altered by isoprinosine treatment. These data suggest that pseudorabies virus infection decreased lymphocyte proliferative responses and interleukin-2 production in pigs, and that isoprinosine did not mitigate these effects.     

Experimental dual infection of specific pathogen-free pigs with porcine reproductive and respiratory syndrome virus and pseudorabies virus

Twenty 6-week-old specific pathogen-free pigs were divided into four groups. On day 0 of the experiment, PRRSV-PRV (n = 6) and PRRSV (n = 4) groups were intranasally inoculated with porcine reproductive and respiratory syndrome virus (PRRSV) (10(5.6) TCID50). On day 7, the PRRSV-PRV and PRV (n = 6) groups were intranasally inoculated with pseudorabies virus (PRV) (10(3.6) TCID50). Control pigs (n = 4) were kept as uninoculated negative controls. Half of the pigs in each group were euthanized and necropsied on day 14 or 21. Clinical signs such as depression and anorexia were observed in the PRRSV-PRV and PRV groups after inoculation with PRV. Although febrile response was observed after virus inoculations, the duration of that response was prolonged in the PRRSV-PRV group compared with the other groups. The lungs in the PRRSV-PRV group failed to collapse and were mottled or diffusely tan and red, whereas the lungs of the pigs in the other groups were grossly normal. Histopathologically, interstitial pneumonia was present in all PRRSV-inoculated pigs, but the pneumonic lesions were more severe in the PRRSV-PRV group. Mean PRRSV titres of tonsil and lung in the PRRSV-PRV group were significantly (P < 0.05) higher than that in the PRRSV group on day 21. These results indicate that dual infection with PRRSV and PRV increased clinical signs and pneumonic lesions in pigs infected with both viruses, as compared to pigs infected with PRRSV or PRV only, at least in the present experimental conditions.     

规模化猪场繁殖障碍性疾病的血清学调查

运用ELISA、LAT和IHA对湖北省2006年度规模化猪场猪伪狂犬病(PR)、猪繁殖与呼吸综合征(PRRS)、猪乙型脑炎(JE)、猪细小病毒病(PP)、猪瘟(HC)、猪圆环病毒病(PCV-2)及弓形体病进行了血清学检测。分析调查结果发现PR、PRRS、JE、PP 、HC ５种疾病的免疫抗体平均阳性率分别为84.3%、78.3%、84.7%、58.0%、79.7%。非免PRRSV和PRV野毒的平均感染率分别为55.1%和17.7％,表明PRRSV感染情况比较严重。对58场次发病猪场的541份血清进行PRV、PRRSV、PCV-2混合感染情况的调查发现,规模化猪场中普遍存在二重或三重感染,从而使疾病更加复杂化,给防疫工作带来更大的困难。     

Evaluation in swine of a subunit vaccine against pseudorabies

A subunit vaccine against pseudorabies virus (PRV) was prepared by treating a mixture of pelleted virions and infected cells with the nonionic detergent Nonidet P-40 and emulsifying the extracted proteins incomplete Freund's adjuvant. Three 7-week-old pigs without antibodies against PRV were given 2 IM doses of this vaccine 3 weeks apart. Thirty days after the 2nd vaccination, 10(6) median tissue culture infective doses (TCID50) of a virulent strain of PRV were administered intranasally. Tonsillar and nasal swabs were collected daily between 2 and 10 days after challenge exposure. The pigs vaccinated with the subunit vaccine were not found to shed virulent PRV. Two groups of five 7-week-old pigs vaccinated with commercially available vaccines, either live-modified or inactivated virus, and subsequently exposed to 10(6) TCID50 of virulent PRV, shed virulent virus for up to 8 days. The subunit vaccine induced significantly higher virus-neutralizing antibody titers than either the live-modified or inactivated virus vaccine.     

Commercially produced spray-dried porcine plasma contains increased concentrations of porcine circovirus type 2 DNA but does not transmit porcine circovirus type 2 when fed to naive pigs

The porcine circovirus type 2 (PCV2) antibody and DNA status of porcine plasma products collected during the commercial spray-drying process were evaluated. Samples evaluated included 52 pooled liquid plasma (fresh) samples collected at 14 regional abattoirs before transport to 1 of 2 spray-drying facilities, 32 pooled liquid plasma (concentrated) samples collected after arrival at the spray-drying facilities at different stages before the spray-drying process, and 32 samples in powdered form (spray-dried) collected after spray drying. All 116 samples were positive for PCV2 antibody, with PCV2 ELISA sample-to-positive ratios ranging from 9.2 to 13.6 on a DM basis. Porcine circovirus type 2 DNA (4.5 to 7.9 log(10) PCV2 copies/mL, DM basis) was present in 82.7% (43/52) of the fresh plasma samples, 71.9% (23/32) of the concentrated plasma samples and 78.1% (25/32) of the spray-dried plasma samples, with a greater prevalence of PCV2b than PCV2a. To determine the infectivity of PCV2 DNA-positive commercial spray-dried plasma, nine 10-wk-old 68-kg PCV2-na?ve pigs were randomly assigned to 1 of 3 treatment groups and rooms: 1) a negative control (no plasma in the feed, not inoculated with PCV2); 2) a positive control (no plasma in the feed, inoculated with PCV2); and 3) plasma-fed pigs (4% porcine plasma in the feed for 42 d, not inoculated with PCV2). All positive control pigs became viremic by 7 d postinoculation and seroconverted by 42 d postinoculation, whereas pigs in the negative control group and in the spray-dried plasma group were PCV2 PCR negative and did not seroconvert to PCV2 for the duration of the study. The results indicate that PCV2 DNA and antibodies are commonly found in commercial spray-dried plasma. However, no evidence of infectivity of the PCV2 DNA was found in na?ve pigs when commercial spray-dried plasma was included in the diet under the conditions of this study.     

Response of early-weaned pigs to spray-dried porcine or animal plasma-based diets supplemented with egg-yolk antibodies against enterotoxigenic Escherichia colil

Two experiments involving 168 10-d-old weaned pigs were conducted to compare growth-promoting properties of dietary spray-dried animal plasma (SDAP), spray-dried porcine plasma (SDPP), and chicken egg-yolk antibodies (EYA) or egg-yolk powder (EYP, contains no specific antibodies) from d 0 to 14 postweaning. In Exp. 1, 96 pigs (3.2 +/- 0.2 kg BW) were used to test the hypothesis that the superior performance of piglets fed SDPP-based diets was partly due to the presence of specific antibodies against enterotoxigenic Escherichia coli (ETEC), which could be replaced with EYA. Four experimental diets in a completely randomized design and arranged in a 2 x 2 factorial (SDPP without or with autoclaving [AuSDPP] and without [EYP] or with supplementation of EYA) were used. Autoclaving SDPP at 121degrees C for 15 min completely destroyed anti-K88/F18 antibodies. Overall feed intake and gain:feed ratio were similar (P > 0.05) among treatments and averaged 122.7 g/d and 0.688, respectively. However, pigs fed AuSDPP+EYP diets had poorer (P < 0.001) ADG compared with those fed SDPP+EYP or SDPP+EYA from 0 to 14 d. Scours were four times higher (P < 0.05) for treatment AuSDPP+EYP compared with all other treatments. Plasma urea nitrogen concentration was higher (P < 0.05) in AuSDPP+EYP- and AuSDPP+EYA-fed pigs. Also twice the number of piglets fed AuSDPP+EYP appeared unhealthy compared with piglets on treatment AuSDPP+EYA. In Exp. 2, 72 10-d-old weaned pigs (3.5 kg BW) were used to compare the effect of EYA supplementation and oral challenge of ETEC strain F18 on performance and visceral organ weights. The experimental diets consisted of SDAP+EYP, SDAP+EYA, SDPP+EYP, and SDPP+EYA. From d 0 to 7, and the entire experimental period, dietary treatment did not influence (P > 0.05) growth rate and feed consumption. Plasma urea N concentration was higher (P < 0.05) in piglets fed the SDAP+EYP diet before and after the oral challenge. Gain:feed ratio, organ weights, villi heights, and crypt depths were not affected (P > 0.05) by dietary treatments. The results indicate that SDPP contains specific anti-ETEC antibodies, which is one of the factors responsible for its superior growth-enhancing effects. Spray-dried animal plasma, SDPP and EYA have similar growth promoting effect in early-weaned pigs.     

Antibody response of pigs to inactivated monovalent and bivalent vaccines for porcine parvovirus and pseudorabies virus

Groups of pigs vaccinated with an inactivated bivalent vaccine containing porcine parvovirus (PPV) and pseudorabies virus (PRV) developed geometric mean titers (GMT) of humoral antibody for each of the viruses as high or slightly higher than those of other groups of pigs that were vaccinated with inactivated monovalent vaccines containing one or the other of the same viruses. An increase in GMT after challenge exposure of vaccinated pigs to live virus indicated that vaccination did not prevent virus replication. However, an indication that replication was less extensive in vaccinated pigs was provided by the following. Although neither vaccinated nor nonvaccinated (control) pigs had clinical signs after exposure to the live PPV, the effect of vaccination was evident by the fact that GMT were higher in nonvaccinated pigs after exposure than they were in vaccinated pigs. Conversely, all pigs exposed to live PRV had clinical signs, but these signs varied between mild-to-moderate and transient for vaccinated pigs to severe and fatal for nonvaccinated pigs.     

Study of the potential involvement of pseudorabies virus in swine respiratory disease.

In order to investigate the potential involvement of pseudorabies virus (PRV) in swine respiratory disease, nine week old pigs were intranasally inoculated with the PRV strain 4892. Two doses of infection were used: 10(4.5) median tissue culture infectious doses (TCID50)/pig and 10(3.5) TCID50/pig, with ten pigs per group. In the group of pigs inoculated with 10(4.5) TCID50, seven out of ten pigs died within six days after inoculation. The mortality rate in the group of pigs inoculated with the lower dose was only two out of ten and, there were several pigs in this group that showed signs of respiratory distress besides some mild nervous signs. Pseudorabies virus was isolated from various tissues collected postmortem, including alveolar macrophages. Virus localization in tissues was also detected by in situ hybridization. The histopathological examination of the respiratory tract tissues revealed a pathological process that was progressing from mild pneumonia to severe suppurative bronchopneumonia. The isolation of virus from alveolar macrophages provides support to the hypothesis that replication of PRV during the course of infection produces an impairment of the defense mechanisms in the respiratory tract.     

一例猪圆环病毒2型和大肠杆菌、溶血葡萄球菌混合感染的诊断

山东德州某猪场发生猪高热、呼吸系统疾病甚至死亡的疫情。采集病料提取病变组织总DNA或RNA进行猪繁殖与呼吸综合征病毒、猪圆环病毒2型、猪细小病毒、猪伪狂犬病病毒、猪瘟病毒的PCR或RT-PCR检测。PCR扩增出353 bp的猪圆环病毒2型特异性条带。同时进行细菌分离培养、生化鉴定等试验,诊断为猪圆环病毒2型和大肠杆菌、溶血葡萄球菌混合感染。     

Effects of pelleting and pellet conditioning temperatures on weanling pig performance

We conducted two experiments to study the effects of pelleting and pellet conditioning temperature on weanling pig performance. In Exp. 1, 252 weanling pigs (PIC, L326 x C22) averaging 6.0 +/- 1.3 kg and 21 +/- 3 d of age were used to evaluate six corn-soybean meal-based diets containing 15% dried whey and formulated to contain 1.4% lysine. Treatments consisted of a control diet without spray-dried animal protein (SDAP) fed in meal form, a diet with 5% SDAP fed in meal form, and four diets with 5% SDAP that were conditioned at 60, 66, 71, or 77 degrees C for 10 s prior to pelleting. Pellets had a 3.97-mm diameter. The experimental diets were fed from d 0 to 14 after weaning, and all pigs were fed a common diet in meal form from d 14 to 28 after weaning. From d 0 to 7 after weaning, pigs fed diets containing SDAP had greater ADG, gain/feed (P < 0.001), and ADFI (P < 0.05) than pigs fed the control diet. No differences (P > 0.10) were observed between pigs fed the pelleted diets and those fed the SDAP diet in meal form. Conditioning temperature had no effect (P > 0.10) on weanling pig performance from d 0 to 14, and the diet fed from d 0 to 14 had no effect on overall performance (d 0 to 28). In Exp. 2, 252 weanling pigs (6.3 +/- 1.5 kg and 22 +/- 4 d of age) were used to evaluate diets with same composition as in Exp. 1, but treatments consisted of diets with or without SDAP conditioned at 60 degrees C before pelleting, and four diets containing 5% SDAP that were conditioned at 68, 77, 85, and 93 degrees C before pelleting. As in Exp. 1, conditioning lasted 10 s, pellets were 3.97 in mm diameter, and experimental diets were fed for the first 14 d of the 28-d experiment. From d 0 to 7, pigs fed the SDAP diet conditioned at 60 degrees C had greater ADFI (P < 0.05) and tended (P = 0.12) to have greater ADG than pigs fed the diet without SDAP and conditioned at 60 degrees C. From d 0 to 7, ADG (quadratic effect, P < 0.03) and ADFI (linear effect, P < 0.002) decreased as conditioning temperature increased, with the largest decrease observed above 77 degrees C. From d 0 to 14 and 0 to 28, ADG was not affected (P > 0.10) by pellet conditioning temperature or SDAP fed from d 0 to 14. The results of these studies suggest that conditioning diets containing 5% SDAP at temperatures above 77 degrees C decreases weanling pig growth performance.     

伪狂犬病病毒弱毒株LY株的分离鉴定

从辽阳某猪场的10日龄仔猪中分离到1株病毒,经纯化后测得其毒价为107.29TCID50/mL.细胞中和试验表明,该病毒能被猪伪狂犬病病毒标准阳性血清所中和.电镜下可见到典型的疱疹病毒粒子,具有囊膜及外周纤突.所分离的病毒对氯仿、胰蛋白酶、乙醚敏感,在pH5.0～9.0下稳定,56℃ 30 min可以灭活.应用特异性引物,通过PCR能扩增出伪狂犬病病毒1 240 bp的gD基因.分离病毒对3日龄乳鼠有一定的致病力,但对家兔、3～5日龄仔猪及妊娠母猪都有很高的安全性.用不同剂量的病毒培养液肌肉注射于3～5日龄仔猪,14 d后用105.7TCID50伪狂犬病病毒强毒攻击,所有试验仔猪均可得到有效保护.用分离毒免疫母猪,其后代可获高滴度的母源抗体,15日龄的仔猪能抵抗105.7TCID50强毒的攻击.试验的结果初步说明,所分离的病毒为伪狂犬病病毒(命名为PRV LY株),并可能是一株弱毒株,而且具有很好的免疫保护作用.     

膜分离法纯化浓缩猪伪狂犬病疫苗毒的效果

本研究使用不同孔径的陶瓷(有机)膜过滤器,对不合格的猪伪狂犬病毒细胞收获液(病毒含量≤104TCID50/mL)滤除杂蛋白、超滤浓缩、除菌处理得到纯化浓缩的猪伪狂犬病疫苗病毒液;然后对纯化浓缩的猪伪狂犬病疫苗病毒液分别进行杂蛋白去除率检验与无菌检验、病毒含量测定、安全检验、效力检验;将检验合格的纯化浓缩的猪伪狂犬病疫苗病毒液添加保护剂冻干,并对纯化浓缩的猪伪狂犬病冻干活疫苗进行以上各项检验,以及进行免疫猪体内抗体消长变化的检测。结果表明:纯化的猪伪狂犬病疫苗杂蛋白去除率平均达到68.3%以上,病毒含量≥105TCID50/mL,效力检验合格;免疫猪体内抗猪伪狂犬病毒抗体增长幅度比同时期未纯化的常规疫苗显著,其中免疫至84 d时中和抗体效价平均高达40.35稀释倍数左右,比常规疫苗中和抗体效价平均高出14.22稀释倍数。此项研究为畜禽疫苗的纯化提供一定的参考。     

应用PCR技术检测伪狂犬病病毒

根据伪狂犬病病毒(PRV)的gE基因序列,设计并合成了一对引物,以闽A株DNA为模板,建立了检测PRV的PCR方法。该方法能从猪细小病毒闽A株和FB株中扩增出一条长度为1 808 bp的片段,对Bartha株、gE-株检测为阴性;而以猪圆环病毒、猪繁殖与呼吸综合征病毒、猪伪狂犬病病毒、猪瘟病毒和正常细胞的核酸为模板的均为阴性;敏感性试验表明,该体系可检测到10pg的猪细小病毒基因组DNA。表明该方法适用于检测猪伪狂犬病病毒野毒株或非gE基因缺失弱毒株。     

猪五种繁殖障碍性疫病病原体多重PCR的建立及初步应用

为了建立能够同时检测猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪伪狂犬病病毒(PRV)、猪圆环病毒2型(PCV2)和猪细小病毒(PPV)的多重PCR,并用于猪场感染情况的动态监控以及临床诊断,根据GenBank中已发表的5种病毒的基因序列,针对CSFV的E2、PRRSV的Nsp2、PRV的gB、PCV2的ORF2和PPV的VP2基因,分别设计了特异性引物。在建立的单项PCR基础上,通过优化反应条件,建立了能同时检测5种病毒的多重PCR,并具有较高的灵敏性和良好的特异性。采用建立的多重PCR对127份疑似病猪的扁桃体活体组织进行检测,检出了5种病毒的存在,其中感染2种及以上病毒的样品比例为38.6%(49/127),表明该方法是一种快速、灵敏、高效的病原学检测手段。     

猪细小病毒、伪狂犬病病毒和猪圆环病毒2型多重PCR检测方法的建立及应用

根据猪细小病毒(PPV)、伪狂犬病病毒(PRV)和猪圆环病毒2型(PCV-2)的基因序列,分别选取各自的保守区段设计引物,通过反应条件的优化,建立了检测PPV、PRV和PCV-2的多重PCR方法.用建立的方法对采自陕西省部分猪场的286份病料及血样进行检测,从对临床健康猪全血样品中PPV、PRV和PCV-2的检测结果看...