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1.
Pneumonic pasteurellosis is a common respiratory infection in cattle that has major economic and welfare implications world-wide and the incidence in the UK due to Pasteurella multocida, currently the same as that associated with Mannheimia haemolytica, is increasing. Whereas much is known regarding the pathogenesis of M. haemolytica infections little information is available on the pathogenic process of pasteurellosis initiated by P. multocida. In the present work calf systemic and innate immune responses to intratracheal challenge with formalin-killed P. multocida biotype A:3 and to subsequent experimental lung infection with live P. multocida were investigated. Eight-week-old calves were challenged intratracheally on day 0 with either 109 colony forming units (cfu) of formalin-killed P. multocida biotype A:3 in 300 ml saline (n=10) or 300 ml saline alone (n=10), followed, at day 21, by challenge with 109 cfu live P. multocida. Pathophysiological and lung phagocyte responses were assessed by clinical monitoring, sequential lung lavage and blood sampling. Results for samples obtained before, during and after challenge showed clinical and acute phase protein responses to both bacterial culture and saline control treatments, although higher responses were associated with bacterial challenge. Phagocytosis of P. multocida during 1 h incubation periods with lavaged cells in vitro was unaffected by exposure in vivo to killed P. multocida and there was evidence that P. multocida was able to survive intracellularly during this assay. There was no indication that lung exposure to formalin-killed P. multocida conferred protection against subsequent homologous live challenge.  相似文献   

2.
Several laboratory studies assessed the duration of immunity of a quadrivalent vaccine (Rispoval™4, Pfizer Animal Health) against bovine respiratory diseases (BRD) caused by bovine herpes-virus type-1 (BHV-1), parainfluenza type-3 virus (PI3V), bovine viral-diarrhoea virus type 1 (BVDV), or bovine respiratory syncytial virus (BRSV). Calves between 7 weeks and 6 months of age were allocated to treatment and then were injected with two doses of either the vaccine or the placebo 3 weeks apart. Six to 12 months after the second injection, animals were challenged with BHV-1 (n = 16), PI3V (n = 31), BVDV (n = 16), or BRSV (n = 20) and the course of viral infection was monitored by serological, haematological (in the BVDV study only), clinical, and virological means for ≥2 weeks. Infection induced mild clinical signs of respiratory disease and elevated rectal temperature in both vaccinated and control animals and was followed by a dramatic rise in neutralising antibodies in all treatment groups. Titres reached higher levels in vaccinated calves than in control calves after challenge with BHV-1, BVDV, or BRSV. On day 3 after PI3V challenge, virus shedding was reduced from 3.64 log10 TCID50 in control animals to 2.59 log10 TCID50 in vaccinated animals. On days 6 and 8 after BRSV challenge, there were fewer vaccinated animals (n = 2/10 and 0/10, respectively) shedding the virus than control animals (n = 8/10 and 3/10, respectively). Moreover, after challenge, the mean duration of virus shedding was reduced from 3.8 days in control animals to 1 day in vaccinated animals in the BVDV study and from 3.4 days in control animals to 1.2 days in vaccinated animals in the BRSV study. The duration of immunity of ≥6 months for PI3V, BHV-1 and BVDV, and 12 months for BRSV, after vaccination with Rispoval™4, was associated mainly with enhanced post-challenge antibody response to all four viruses and reduction of the amount or duration of virus shedding or both.  相似文献   

3.
Acidogenic diets were evaluated for their effects on lymphocyte proliferation in response to Staphylococcus aureus exotoxin B (SEB), and specific lymphocyte proliferation and serum-neutralizing antibody titers to four bovine respiratory viruses in vitro. Four Holstein steer calves, with an average weight of 213 +/- 42 kg, were fed a basal (control) diet consisting of 49% forage and 51% concentrate (DM basis), with 15% CP (on a DM basis). Three additional treatment diets were used: 1) the basal diet supplemented with 700 mL/d of butylene glycol (BG) to induce ketoacidosis by increasing blood beta-hydroxybutyate (BHBA); 2) the basal diet supplemented with 1.2 +/- 0.1 kg/d of anionic salts (AS; Soychor 16.7, West Central Soy, Ralston, IA) to induce a metabolic acidosis; and 3) the basal diet with all forage replaced by finely ground corn and soybean meal blended to provide 15% CP (HG), to induce lactic acidosis. The calves were fed each diet for 21 d in a 4 x 4 Latin square design. Blood samples were collected on d 18, 19, and 20 of each 21-d period and analyzed for pH; concentrations of BHBA; in vitro lymphocyte proliferation to SEB, bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV), parainfluenza-3 (PI-3), and bovine herpesvirus-1 (BHV-1); and titers of serum-neutralizing antibodies against the four viruses. Following treatment, the average pH of the serum samples was 7.38 for calves fed the control diet, 7.37 for the BG treatment, and 7.36 for the HG treatment, and was decreased (P < 0.05) to 7.33 for the AS treatment. All acidogenic diets decreased lymphocyte response to SEB (P < 0.05). The lymphocyte proliferative response, however, of each virus showed a different pattern of interaction with the three acidogenic diets tested. The AS diet was associated with increased lymphocyte proliferative response to BVDV and BRSV (P < 0.01) and increased serum neutralization titers to BHV-1 (P < 0.05). In calves fed the BHBA-inducing diet (BG), an increase in lymphocyte proliferation to BRSV was observed (P < 0.05). A similar relationship to blood BHBA concentration was not observed with the lymphocyte proliferation to BVDV, PI-3, or BHV-1. Titers of serum-neutralizing antibody against PI3 (P < 0.05) and BHV-1 (P < 0.01) were negatively correlated with blood pH, and titers of serum neutralizing antibodies to BHV-1 were negatively correlated to elevated circulating concentrations of BHBA (P < 0.05).  相似文献   

4.
Bovine respiratory disease complex is a very important health problem around the world. Present study describes serological distribution of bovine major respiratory viruses in non -vaccinated cattle population of Marmara region in north-western Turkey. Neutralising antibodies specific to bovine viral diarrhoea virus (BVDV), bovine herpesvirus 1 (BHV-1), bovine respiratory syncytial virus (BRSV), bovine parainfluenza virus 3 (PI-3), bovine adenovirus serotype 1 (BAV-1) and serotype 3 (BAV-3) were investigated. Among 584 serum samples collected from 39 establishments in 7 provinces, 41.4% were positive for BVDV, 17.1% for BHV-1, 73.0% for BRSV, 43.0% for PI-3, 89.5% for BAV-1 and 92.3% for BAV-3. There were significant differences observed between seroprevalence rates detected in neighbouring provinces. Serological prevalence of BVDV, BHV-1 and BRSV were extremely higher in large capacity dairy farms than of small capacity farms (p < 0.0001). This study demonstrates that herd capacity is a very important risk factor for respiratory viruses and, on the other hand bovine adenoviruses and BRSV are the common reason of respiratory diseases in the region.  相似文献   

5.
牛溶血性曼氏杆菌及牛荚膜A型多杀性巴氏杆菌是导致牛呼吸道疾病（bovine respiratory disease,BRD）的重要细菌性病原,每年给养牛业带来巨大的经济损失,目前对其疫苗研究仍显不足。本研究选用牛溶血性曼氏杆菌（Mannheimia haemolytica,Mh） Mh422株和牛荚膜A型多杀性巴氏杆菌（Pasteurella multocida,Pm） PmCQ2株作为疫苗菌株,分别制备了2种菌体浓度的Mh和Pm单价灭活菌苗及3种菌量配比（1∶1、2∶1和3∶1）的Mh-Pm二联灭活苗,以小鼠为模型,皮下多点免疫（0.2 mL）,加强免疫2次,免疫剂量均为首免的一半。首免后第7天及其后每隔5 d,小鼠尾静脉采血分离血清,ELISA方法检测抗体效价,三免后第20天,分别以Mh422或PmCQ2进行腹腔攻毒测定免疫保护效果。结果显示,所有小鼠接种疫苗均无不良反应,二免后第10天抗体达较高水平,三免后抗体水平持续升高,第15天到达高峰,其后25 d维持高水平,后缓慢下降。Mh单菌苗的2种免疫剂量对Mh422株攻毒的免疫保护率均为0,而Pm单菌苗的2种免疫剂量对PmCQ2株攻毒的免疫保护率全为100%;Mh和Pm间无交叉免疫保护作用;3种菌量配比的Mh-Pm二联疫苗对Mh422株和PmCQ2株攻毒的各自免疫保护率分别为53%～71%和100%。该研究结果表明,所制备的Mh422单菌苗对同型攻毒无免疫保护作用,在诱导机体抗体产生方面,Mh和Pm间无相互抑制作用,PmCQ2株具有促进Mh422株灭活疫苗对Mh422的免疫保护作用,这为牛溶血性曼氏杆菌和牛多杀性巴氏杆菌二联疫苗的进一步研究提供了理论基础。  相似文献   

6.
For many pathogens, adherence and/or invasion involve association with host extracellular matrix molecules, such as fibronectin (Fn). Pasteurella multocida was found to bind significantly to Fn and collagen type IX but not to laminin and collagen types IV and X. The binding of P. multocida to Fn was dose-dependent and was inhibited by heparin (Hep). Removal of polysaccharide capsule enhanced the binding capacity of the bacterium to Fn and inhibition by Hep. Protease treatment of bacteria decreased binding, implicating surface protein(s) as adhesive components. Investigation of the binding domain(s) of P. multocida on the Fn molecule revealed preferential binding to the N-terminal Hep-binding domain of Fn but not to the carboxyl-terminal Hep-binding domain. Furthermore, Fn, and anti-Fn antibodies inhibited P. multocida adherence to Madin-Darby bovine kidney cells, suggesting the involvement of Fn in the bacterium adherence to host cells. Ligand blotting, batch affinity purification and MALDI-TOF mass spectrometry implicated several proteins as putative adhesins of P. multocida in the Fn-mediated adherence. Taken together, the data suggest that P. multocida-Fn interaction may play a role in the bacterium adherence to host cells, and this may be mediated by bacterial surface proteins with preferential affinity for the Hep-1 binding domain of Fn.  相似文献   

7.
Six hundred and fifteen serum samples obtained from cows in five districts of Apure State, Venezuela, were tested by ELISA for antibodies to bovine virus diarrhoea virus (BVDV). The same samples were also ELISA-tested for antibodies to bovine herpesvirus type 1 (BHV-1) and bovine respiratory syncytial virus (BRSV). Additionally, the haemagglutination-inhibition (HI) test was used for detecting antibodies to parainfluenza virus type 3 (PIV-3). Overall, seroprevalence to BVDV was 36±7% (SE); seroprevalence varied by district (19–42%). BHV-1 seroprevalence was 67±4%; variation by district was similar to that of BVDV. However, the first 80 serum samples tested by BHV-1 ELISA all had a strong background reaction with the control antigen. Therefore, these sera were adsorbed to a homogenate of non-infected bovine kidney cell line (MDBK) and re-tested by ELISA. The non-specific reactivity was significantly reduced (p < 0.001 by Wilcoxon's signed-rank test). Compared to the virus-neutralisation (VN) test, the adsorbed BHV-1 ELISA showed 94% agreement and gave a κ value of 0.84, indicating that the adsorption did not interfere with test accuracy. Seroprevalence against BRSV was 85±3%, and showed differences across districts. Most of the cows (94±2%) were seropositive to PIV-3, and there were no significant differences among districts.  相似文献   

8.
In this study, viral pathogens associated with nine outbreaks of naturally occurring dairy calf pneumonia in Mashhad area of Khorasan Razavi province from September 2008 to May 2009 were assessed. Five diseased calves from each farm were chosen for examination. Acute and convalescent serum samples were taken from calves with signs of respiratory disease. Sera were analyzed for antibodies to bovine viral diarrhea virus (BVDV), bovine herpesvirus type 1 (BHV-1), bovine respiratory syncytial virus (BRSV), parainfluenza virus type 3 (PI-3V), and bovine adenovirus-3 (BAV-3) by indirect ELISA kits. Among 42 serum samples collected at sample 1, seroprevalence values for viruses BHV-1, BVDV, BRSV, PI-3V, and BAV-3 were 61.9% (26), 57.1% (24), 64.2% (27), 90% (38), and 61.9% (26), respectively. Seroconversion to BVDV, BRSV, PI-3V, and BAV-3 occurred in 11.9% (5), 16.6% (7), 26.1% (11), and 21.4% (9) of animals, and 52.3% (22) had generated antibodies against one or more viral infections at sample 2. In addition, no significant relationship between seroprevalence of BHV-1, BVDV, BRSV, PI-3V, and BAV-3 and dairy herd size was observed (P > 0.05). According to serological findings, BHV-1, BVDV, BRSV, PI-3V, and BAV-3 are common pathogens of the dairy calf pneumonia in dairy herds in Mashhad area of Khorasan Razavi province, Iran.  相似文献   

9.
hyaD为A型多杀性巴氏杆菌荚膜多糖合成相关基因,为探讨该基因对多杀性巴氏杆菌毒力及其免疫保护特性的影响,本研究利用同源重组方法,构建了牛源A型多杀性巴氏杆菌CQ2株（PmCQ2）的hyaD基因缺失株（ΔhyaD）。结果发现,与野生株相比,ΔhyaD的荚膜产生量及其感染后在脏器中的细菌定殖量均显著下降,其毒力显著降低。细胞试验发现,ΔhyaD更易黏附于巨噬细胞,被吞噬数量显著多于野生株,致使巨噬细胞相关炎性因子表达显著上调。hyaD基因的缺失,可调控与荚膜合成、LPS合成转运、铁转运等相关的基因表达显著下调,促使相关保护性抗原基因表达显著上调。以制备的PmCQ2株和ΔhyaD株灭活苗免疫小鼠（加强免疫1次）,免疫后第21天分别采用同源和异源多杀性巴氏杆菌攻毒,ΔhyaD株免疫小鼠肺组织感染后24 h无明显或轻微病理损伤,对牛源A型、B型和F型多杀性巴氏杆菌的免疫保护率分别为100%、100%和80%,对兔源、猪源和禽源A型多杀性巴氏杆菌的免疫保护率分别为90%、100%、100%;而野生株PmCQ2除对牛源A型多杀性巴氏杆菌的保护率在80%以上外,对牛源B型和F型及兔、猪、禽源A型多杀性巴氏杆菌均无明显交叉保护作用。研究结果表明,hyaD基因可通过调控荚膜产生及毒力相关因子表达影响菌株毒力;hyaD基因缺失可调控相关交叉保护性抗原表达,赋予菌株交叉免疫保护特性。该研究为多杀性巴氏杆菌通用型疫苗的研发提供了参考。  相似文献   

10.
试验旨在探索羊源D型多杀性巴氏杆菌入侵宿主脾脏组织中引发的免疫应答途径。首先利用羊源D型多杀性巴氏杆菌(HN01菌株)感染小鼠,建立羊源D型多杀性巴氏杆菌感染的动物模型;之后利用转录组测序技术获得感染小鼠与正常小鼠的脾脏转录组数据,并使用COG、KOG、eggNOG、GO、KEGG数据库对测序结果中的差异表达基因(differentially expressed genes,DEGs)进行功能注释与分析,同时对于显著富集到关键免疫通路的差异表达基因使用STRING软件和KEGG mapper进行蛋白互作分析,筛选出核心通路中起关键作用的基因;最后选取关键的10个基因进行实时荧光定量RT-PCR验证。转录组分析结果显示,与正常组相比,感染组中筛选出3 380个差异表达基因(P<0.01,log2|FoldChange|≥0.5),其中1 691个基因上调,1 689个下调。基因功能富集分析结果表明,感染组脾脏中的差异表达基因主要发挥信号转导的功能,其主要参与的生物途径包括细胞因子与细胞因子受体互作通路、趋化因子信号通路、HIF-1信号通路、TNF信号通路。蛋白互作分析筛选出约28个核心差异表达基因,结合实时荧光定量RT-PCR验证后,其中9个基因的表达结果与测序一致,分别是C3、Cd4、Cxcl13、Lck、Gnai1、Grap2、IL-6、Cxcr6及Serping1基因。本研究初步证明了脾脏在抵抗多杀性巴氏杆菌入侵中参与了一系列免疫应答反应,为进一步研究羊源D型多杀性巴氏杆菌与宿主之间相互作用的分子机制奠定理论基础。  相似文献   

11.
Strains of Mycoplasma ovipneumoniae and Pasteurella haemolytica isolated from sheep affected with chronic pneumonia were inoculated by endobronchial route to conventionally-reared and SPF (Specific Pathogen-Free) lambs. Changes resembling those of the naturally-occurring disease were produced in most lambs given the organisms in combination and in some given M. ovipneumoniae alone. Similar but less extensive changes were seen in SPF lambs and fewer animals were affected. Different strains of M. ovipneumoniae did not affect the extent of changes produced in SPF lambs. M. ovipneumoniae became established in the lungs of both types of sheep; P. haemolytica did so less readily.

It was concluded that chronic pneumonia may be reproduced in conventional animals by combined inoculation of M. ovipneumoniae and P. haemolytica. Age and status of immunity to mycoplasmas may account for the different responses of conventional and SPF lambs.  相似文献   


12.
Sixteen litters of seven pigs from each of nine Danish farrow-to-finish herds were followed to investigate the serological patterns caused by natural infection with Mycoplasma hyopneumoniae, Pasteurella multocida toxin and Actinobacillus pleuropneumoniae serotypes 2, 5–7, 12. In seven of the herds, pigs were followed as two separate cohorts started 4 weeks apart, and in two herds only one cohort was followed.

A total of 999 pigs were included in the study. The pigs were blood sampled at weaning and subsequently every fourth week until slaughter. All pigs were examined for antibodies against M. hyopneumoniae (enzyme-linked immunosorbent assay), P. multocida toxin (enzyme-linked immunosorbent assay) and A. pleuropneumoniae serotypes 2, 5–7, 12 (complement-fixation tests). The most-common pattern (28%) of seroconversion was that of pigs first seroconverting to A. pleuropneumoniae serotype 2, followed by seroconversion to M. hyopneumoniae. Each herd had a dominant serotype of A. pleuropneumoniae to which most pigs seroconverted. Seroconversion to the respiratory pathogens occurred mainly in the growing-to-finishing units (8–24 weeks). The risk of seroconversion to the P. multocida toxin was very low (<20%) and occurred late.

None, four and seven herds tested seropositive to PRRS and to swine influenza virus subtypes H3N2 and H1N1, respectively, when testing 10 pigs per herd (selected randomly among the study pigs) at the age of 20 weeks.  相似文献   


13.
The prevalence of bovine viral diarrhea virus (BVDV) infections was determined in a group of stocker calves suffering from acute respiratory disease. The calves were assembled after purchase from Tennessee auctions and transported to western Texas. Of the 120 calves, 105 (87.5%) were treated for respiratory disease. Sixteen calves died during the study (13.3%). The calves received a modified live virus BHV-1 vaccine on day 0 of the study. During the study, approximately 5 wk in duration, sera from the cattle, collected at weekly intervals, were tested for BVDV by cell culture. Sera were also tested for neutralizing antibodies to BVDV types 1 and 2, bovine herpesvirus-1 (BHV-1), parainfluenza-3 virus (PI-3V), and bovine respiratory syncytial virus (BRSV). The lungs from the 16 calves that died during the study were collected and examined by histopathology, and lung homogenates were inoculated onto cell cultures for virus isolation. There were no calves persistently infected with BVDV detected in the study, as no animals were viremic on day 0, nor were any animals viremic at the 2 subsequent serum collections. There were, however, 4 animals with BVDV type 1 noncytopathic (NCP) strains in the sera from subsequent collections. Viruses were isolated from 9 lungs: 7 with PI-3V, 1 with NCP BVDV type 1, and 1 with both BVHV-1 and BVDV. The predominant bacterial species isolated from these lungs was Pasteurella haemolytica serotype 1. There was serologic evidence of infection with BVDV types 1 and 2, PI-3V, and BRSV, as noted by seroconversion (> or = 4-fold rise in antibody titer) in day 0 to day 34 samples collected from the 104 survivors: 40/104 (38.5%) to BVDV type 1; 29/104 (27.9%) to BVDV type 2; 71/104 (68.3%) to PI-3V; and 81/104 (77.9%) to BRSV. In several cases, the BVDV type 2 antibody titers may have been due to crossreacting BVDV type 1 antibodies; however, in 7 calves the BVDV type 2 antibodies were higher, indicating BVDV type 2 infection. At the outset of the study, the 120 calves were at risk (susceptible to viral infections) on day 0 because they were seronegative to the viruses: 98/120 (81.7%), < 1:4 to BVDV type 1; 104/120 (86.7%) < 1:4 to BVDV type 2; 86/120 (71.7%) < 1:4 to PI-3V; 87/120 (72.5%) < 1:4 to BRSV; and 111/120 (92.5%) < 1:10 to BHV-1. The results of this study indicate that BVDV types 1 and 2 are involved in acute respiratory disease of calves with pneumonic pasteurellosis. The BVDV may be detected by virus isolation from sera and/or lung tissues and by serology. The BVDV infections occurred in conjunction with infections by other viruses associated with respiratory disease, namely, PI-3V and BRSV. These other viruses may occur singly or in combination with each other. Also, the study indicates that purchased calves may be highly susceptible, after weaning, to infections by BHV-1, BVDV types 1 and 2, PI-3V, and BRSV early in the marketing channel.  相似文献   

14.
为构建表达牛呼吸道合胞体病毒(BRSV)G蛋白基因的牛疱疹病毒Ⅰ型(BHV-1)重组病毒,本研究将人工合成的BRSV全长G蛋白基因编码序列插入到巨细胞病毒(CMV)启动子之下构建TK基因缺失转移载体。利用磷酸钙-DNA沉淀法将该转移载体与亲本病毒BHV-1/TK-/LacZ+的基因组DNA共转染牛鼻甲细胞后收获增殖的病毒。通过反向蚀斑筛选,得到重组病毒BHV-1/TK-/G+。PCR检测结果证实G蛋白基因已经插入到了亲本病毒BHV-1/TK-/LacZ+的基因组中,间接免疫荧光试验和western blot证实BHV-1/TK-/G+中的G蛋白基因在感染的细胞中获得了表达。本研究为研制BRSV及其他重要牛传染病的BHV-1病毒活载体疫苗奠定了基础。  相似文献   

15.
通过对多杀性巴氏杆菌(Pasteurella multocida)CS株全基因组的毒力基因分析,以期从基因组角度了解P. multocida CS的致病性机理。常规方法分离细菌,并进行毒力鉴定。基于二代测序平台对P. multocida CS进行测序,应用比较基因组学方法将其与GenBank中具全基因组来源于不同地域和宿主的P. multocida基因组进行比较分析。结果显示：猪肺疫病料中分离细菌滴鼻感染小鼠,测得其LD50为5×102 CFU·mL-1,显示较强毒性。将测序后的P.multocida CS株全基因组信息提交至NCBI,获得登录号SUB11119617。通过对P.multocida CS全基因组序列的分析表明,P.multocida CS株全基因组大小为2 599 048 bp,G+C含量为39.932%,编码CDs为2 580个,占整个基因组长度的69.6%,编码CDs的平均长度为952 bp。此外还有53个tRNA基因和3个rRNA基因。有87.95% 的编码CDs可进行COGs功能分类,29个为功能未知基因。利用RaAXML的最大释然法(maximum likelihood,ML)进行系统进化分析发现,P. multocida CS株与1个猪源性、2个牛源血清A型毒株,和1个猪源性D型毒株有较近的亲缘关系。P. multocida CS株含254个毒力相关基因,分为4大类11小类。其中铁摄取系统、黏附系统、分泌系统和双组分调控系统相关的多个基因在不同的毒株(包括血清型)的分布存在多态性。通过对基因出现频率的分析发现,铁摄取系统相关的tbpA和铁复合物外膜受体蛋白基因(iron complex outer membrane receptor protein gene,irp)以及黏附的相关的ppdD和ompA基因在血清A型出现的频率较高,这是否与不同菌株的毒力差异相关有待进一步证实。P. multocida CS的分泌系统由Ⅰ型(hly基因)、Tat型、Sec-SRP型3种类型组成。其中,hlyD基因在不同的血清型的分布频率存在多态性。P. multocida CS株的双组分信号转导系统(TCSs)由16个调节相关基因,12个感应相关基因和2个有hybrid相关基因。这些基因在不同血清型分布的多态性与不同菌株毒力的关系有待进一步研究。综上所述,组成铁摄取系统、黏附系统、分泌系统和双组分调控系统的基因在不同的血清以及同一血清型内出现频率存在多态性,它们与P. multocida菌株毒力强弱的关系有待进一步研究。  相似文献   

16.
为建立适用于羊源多杀性巴氏杆菌的重组酶聚合酶扩增（RPA）诊断方法,本研究依据前期测序鉴定的羊源多杀性巴氏杆菌KMT1基因序列构建pET-28a （＋）-KMT1质粒标准品。设计基于RPA技术的特异性引物及RPA荧光探针,建立实时荧光RPA方法的最适反应体系。采用10倍梯度连续稀释的质粒标准品检测该RPA方法的敏感性并绘制相关性曲线;以10种不同菌株的基因组DNA为模板验证方法的特异性;用感染多杀性巴氏杆菌的山羊及小鼠组织样品对方法的可靠性进行验证。结果显示,本试验建立的实时荧光RPA方法最适反应温度为39 ℃,最佳引物为KMT1-Fe1,灵敏度达100拷贝/μL,检测下限为10拷贝/μL。与大肠杆菌、金黄色葡萄球菌、副伤寒沙门氏菌、副溶血弧菌、绿脓杆菌、产酸克雷伯菌、布鲁氏菌S2株和鲍曼不动杆菌均无交叉反应。对13个组织样本进行检测,阳性率为76.9%,与实时荧光定量PCR检测结果的符合率达92.3%。综上所述,本研究建立的羊源多杀性巴氏杆菌实时荧光RPA方法具有特异性强、灵敏度高、可靠、快速便捷等特点,适用于多杀性巴氏杆菌的临床分子诊断。  相似文献   

17.
In 961 calves up to an age of 6 months which were sent to the animal health center in Oldenburg between March 1987 and March 1990 for necropsy the results of determination of different viruses were calculated: BVD-, rota-, corona-, parainfluenza-3- (PI-3)-, bovine herpes-1 (BHV-1)- and bovine respiratory syncytial virus (BRSV). In 122 and 104 randomly collected health calves of 22 farms antibodies against BRSV and bovine adeno virus-types 5, 7 and 8 were determined. 50.1% of the necropsied calves were one and two weeks old. In this group in 40.2% rotavirus and in 19.0% coronavirus could be isolated. All over the calves the frequencies of isolated viruses were 13.3% for BVDV, 4.6% for BRSV, 3.2% for BHV-1, and 2.1% for PI-3. The percentages of positive findings for rota- and coronavirus increased up to 7 days after birth, and thereafter both decreased. The frequencies of BVDV and BRSV were higher in older groups. The frequency of PI-3 was low and remained constantly. Infections with rota-, corona- and with both viruses were accompanied by BVDV in 11.3, 5.3 and 14.3%, respectively. Against bovine adenoviruses and BRSV in the first 8 weeks and after 14 weeks of life in more than 70% of the calves antibodies were detected.  相似文献   

18.
To identify antigens which may be important for stimulating immunity to pneumonic pasteurellosis, a bovine antiserum to whole P. haemolytica was used to screen a recombinant lambda gt11/P. haemolytica expression library. One of the recombinant bacteriophage clones identified with the bovine antiserum, SW20C, expressed a fusion protein which was also recognized by rabbit antiserum to partially purified P. haemolytica culture supernatant and was found to be immunogenic in guinea pigs. The guinea pig antibody recognized a 100 kDa protein in P. haemolytica cell lysates. Sequence analysis of the cloned DNA from SW20C identified a fragment of 1443 bp with a small open reading frame that was contiguous with the lacZ sequence. The 153 bp P. haemolytica-specific open reading frame encoded a polypeptide of approximately 6kDa. Homology searches of Genbank and the EMBL data bases revealed no homology of this open reading frame with any other bacterial sequences including P. haemolytica leukotoxin and Ssa1. Evaluation of sera from calves that were scored either susceptible or resistant to experimental pneumonic pasteurellosis demonstrated a significant (P < 0.001) correlation between the intensity of the antibody response to the SW20C antigen and resistance to disease.  相似文献   

19.
Sera from healthy goats were collected during October 1979 through October 1980. These sera were tested for bovine herpesvirus-1 (BHV-1), bovine viral diarrhea virus (BVDV), parainfluenza-3 (PI-3) virus, bovine adenoviruses (BAV) -3 and -7, and goat respiratory syncytial virus (GRSV) antibodies by microtitration virus-neutralization test. The number of herds with seropositive goats for each virus were: 5/38 (13.2%) for BHV-1; 9/38 (23.7%) for BVDV; 8/38 (21.1%) for PI-3 virus; 1/38 (2.6%) for BAV-3; 15/38 (39.5%) for BAV-7; and 26/34 (76.5%) for GRSV. Seropositive rates for each virus for the individual goats tested were: 6/502 (1.2%) for BHV-1; 9/498 (1.8%) for BVDV; 49/458 (10.75) for PI-3 virus; 1/487 (0.025) for BAV-3; 40/448 (8.9%) for BAV-7; and 166/332 (50.0%) for GRSV.  相似文献   

20.
The objective of this study was to verify whether a mixed infection in calves with bovine viral diarrhea virus (BVDV) and other bovine viruses, such as bovid herpesvirus-4 (BHV-4), parainfluenza-3 (PI-3) and infectious bovine rhinotracheitis (IBR) virus, would influence the pathogenesis of the BVDV infection sufficiently to result in the typical form of mucosal disease being produced.

Accordingly, two experiments were undertaken. In one experiment calves were first infected with BVDV and subsequently with BHV-4 and IBR virus, respectively. The second experiment consisted in a simultaneous infection of calves with BVDV and PI-3 virus or BVDV and IBR virus.

From the first experiment it seems that BVDV infection can be reactivated in calves by BHV-4 and IBR virus. Evidence of this is that BVDV, at least the cytopathic (CP) strain, was recovered from calves following superinfection. Moreover, following such superinfection the calves showed signs which could most likely be ascribed to the pathogenetic activity of BVDV. Superinfection, especially by IBR virus, created a more severe clinical response in calves that were initially infected with CP BVDV, than in those previously given the non-cytopathic (NCP) biotype of the virus. Simultaneous infection with PI-3 virus did not seem to modify to any significant extent the pathogenesis of the experimentally induced BVDV infection whereas a severe clinical response was observed in calves when simultaneous infection was made with BVDV and IBR virus.  相似文献   


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