牛传染性鼻气管炎病毒单克隆抗体的制备及阻断ELISA方法的建立

为建立检测牛传染性鼻气管炎病毒(IBRV)血清抗体的阻断ELISA方法,本研究以经蔗糖密度梯度离心法纯化的IBRV作为免疫原制备1株单克隆抗体(MAb),命名为cp-1-1。经间接ELISA、IFA和western blot鉴定,该MAb与IBRV呈阳性反应,与牛病毒性腹泻病毒(BVDV)及牛副流感病毒3型(BPIV3)呈阴性反应,具有较强的特异性。质谱分析结果显示MAb cp-1-1识别的表位位于IBRV VP8蛋白。以纯化的IBRV作为包被抗原、MAb cp-1-1作为检测抗体,建立检测IBRV血清抗体的阻断ELISA方法。该检测方法的抗原包被量为0.89μg/孔,样品稀释度为12,检测抗体MAb量为1.3μg/孔,二抗稀释度为15000。利用50份IBRV抗体呈弱阳性的牛血清(中和抗体效价为14~116)作为标准参考血清,确定该检测方法的阻断率Cut Off值为52.06%,即阻断率高于52.06%时判为阳性,低于52.06%时判为阴性。阻断ELISA方法特异性试验显示仅IBRV阳性血清检测为阳性,而BVDV、BPIV3、牛腺病毒3型(BADV-3)和O型口蹄疫病毒(O-FMDV)阳性牛血清均检测为阴性,表明该方法具有较强的特异性;该方法可检测的最低中和抗体效价为14,与病毒中和试验的敏感性一致,表明该方法具有较高的敏感性;重复性试验显示该方法批内、批间变异系数均小于10%,显示较好的重复性。对130份现地牛血清检测结果显示,该方法与病毒中和试验的符合率为98.46%。用该方法对某牛场接种IBRV灭活疫苗的牛血清进行检测,抗体阳性率为99.51%(205/206)。另外,采用该方法对我国8个省(市、自治区)的801份牛血清进行检测,IBRV的抗体阳性率为41.6%(333/801)。本研究建立的阻断ELISA方法可以用于IBRV疫苗免疫监测和血清流行病学调查,为我国IBR的防控提供技术支持。     

Antibacterial activity of bovine lactoferrin hydrolysate against mastitis pathogens and its effect on superoxide production of bovine neutrophils

Antibacterial activity of bovine lactoferrin hydrolysates (LFH) on microorganisms isolated from bovine mastitis, and superoxide (O(2)(-)) production of bovine neutrophils were evaluated. Antibacterial effects of LFH were measured in vitro against Staphylococcus aureus, coagulase-negative staphylococci, Streptococci, Enterococci, Escherichia coli, Klebsiella pneumoniae, yeast-like fungi and Prototheca zopfii isolated from clinical cases of bovine mastitis. To compare susceptibilities against LFH, minimal inhibitory concentration (MIC) values were determined by a micro-plate assay method. Most organisms were sensitive to LFH. Prototheca zopfii was highly sensitive to LFH; the growth of the microorganism was inhibited completely even at 1 mug/ml. Staphylococcus aureus and Escherichia coli were resistant to LFH. The production of O(2)(-) by bovine neutrophils was used to evaluate the effect of LFH administration on functional activity. Increase in O(2)(-) production by bovine neutrophils occurred upon addition of LFH to neutrophils. These results demonstrate that LFH possesses antibacterial activity against pathogens that cause mastitis and activates neutrophil superoxide production.     

牛Ⅰ型干扰素受体α链与其配体结合活性的鉴定

为研究牛Ⅰ型干扰素受体α链（IFNAR1）与其配体结合的生物学性质，本研究采用RT-PCR方法从感染水疱性口炎病毒（vesicular stomatitis virus，VSV）的犊牛原代肾细胞中扩增得到IFNAR1基因，然后构建了表达BoIFNAR1胞外区多肽（BoIFNAR1-EC）的原核表达载体pET30a-BoIFNAR1-EC，在Rosetta^TM（DE3）pLysS宿主菌中表达出重组蛋白rBoIFNAR1-EC，并以纯化后的rBoIFNAR1-EC作为免疫原制备兔抗牛IFNAR1的多克隆抗体，随后鉴定BoIFNAR1与其配体的结合活性。结果表明，试验成功克隆得到1 685 bp的牛IFNAR1基因，编码560个氨基酸，由24个氨基酸组成的信号肽、414个氨基酸组成的胞外区、23个氨基酸组成的跨膜区及99个氨基酸组成的胞内区组成，与其他物种IFNAR1氨基酸序列同源性为63%~91%，在进化上具有高度保守性，其与羊的亲缘关系最近。随后构建了含牛IFNAR1胞外区基因的重组表达载体，并诱导表达了重组牛IFNAR1胞外区蛋白rBoIFNAR1-EC，其在大肠杆菌中几乎全部为可溶性表达，经纯化透析后作为免疫原制备了兔抗牛IFNAR1的特异性抗体，抗体效价高达1：204 800。配体结合试验表明，牛IFNAR1重组蛋白可与牛IFN-αA和IFN-ε结合，其多克隆抗体可阻断牛IFN-αA和IFN-ε与细胞表面的IFNAR1特异性结合，进而阻断牛IFN-αA和IFN-ε的抗病毒信号传导。     

Viral antibodies in bovine fetuses in Argentina

In order to establish the prevalence of viral infections of the bovine fetus in Argentina, a serological survey for antibodies against viral agents currently affecting cattle in this country was conducted. Antibodies against foot-and-mouth disease virus (FMDV), bovine herpesvirus-1 (BHV-1), bovine leukaemia virus (BLV), bovine rotavirus (BRV), bovine coronavirus (BCV), bovine viral diarrhoea virus (BVDV) and parainfluenza-3 (PI-3) were investigated in a total of 315 fetal serum samples. Conventional techniques were used: indirect immunofluorescence (FMDV, BHV-1, BVDv and BCV), radial immunodiffusion (BLV), ELISA (BRV) and haemagglutination inhibition (PI-3). Antibodies against BHV-1, BVDV and PI-3 were detected in samples from fetuses in the second and third trimester of gestation, with a prevalence of 1·21 per cent (two of 165), 2·03 per cent (four of 197) and 5·08 per cent (nine of 177), respectively. Either antibodies or non-antibody factors able to bind to BRV and Bcv antigens were detected with a prevalence of 2·44 per cent (five of 205) and 4·54 per cent (five of 110), respectively. In addition, 14·68 per cent of non-specific inhibitors of PI-3 mediated haemagglutination were found. No seropositives against FMDV and BLV were detected.     

Serological survey for antibodies against selected infectious agents among fallow deer (Dama dama) in central Italy

Sera were collected from 224 fallow deer (Dama dama) in reserves in central Italy. Samples were tested for antibodies against Chlamydia spp., Brucella spp. and Coxiella spp. with the complement fixation (CF) test. Indirect immunofluorescence was used to test for antibodies against Borrelia spp. and agglutination tests were conducted for Leptospira interrogans antibodies. Enzyme-linked immunosorbent assay (ELISA) tests were used to detect antibodies against bovine herpesvirus-1 (BHV-1) and bovine viral diarrhoea virus (BVDV). All samples were negative for antibodies against Brucella spp., L. interrogans, Coxiella spp. and BHV-1. Four samples (1.8%) had antibodies against Chlamydia spp., nine (4%) against Borrelia spp. and 10 (4.5%) against BVDV. These results indicate that fallow deer in central Italy have a low rate of exposure to pathogens typical of domestic livestock.     

Seroprevalence of OHV-2, BVDV, BHV-1, and BRSV in ranch-raised bison (Bison bison).

Serum samples were collected at slaughter from 226 24-30-month-old American bison (Bison bison) bulls from Kansas, Minnesota, North Dakota, and Manitoba and assayed for antibodies to ovine herpesvirus type-2 (OHV-2), bovine viral diarrhea virus (BVDV), bovine herpesvirus type-1 (BHV-1), and bovine respiratory syncytial virus (BRSV). Antibodies were detected by serum neutralization for BVDV, BHV-1, and BRSV, while antibodies to OHV-2 were detected by competitive inhibition-ELISA (CI-ELISA). Detectable antibodies were found against all viruses: 10 of 226 (4.40%) against OHV-2, 125 of 226 (55.3%) against BVDV, 99 of 226 (43.8%) against BHV-1, and 208 of 226 (92.0%) against BRSV. Titers from 93.6% of the BVDV-positive animals, 79.8% of the BHV-1-positive animals, and 98.1% of the BRSV-positive animals were > or = 1.25. These data indicate that a low percentage of clinically normal bison are seropositive for OHV-2 while a high percentage of bison sampled are seropositive for BVDV, BHV-1, and BRSV.     

青海省牛弓形虫病的ELISA诊断及试剂盒建立

用弓形虫的重组蛋白SAG1作为ELISA诊断抗原,建立了ELISA诊断试剂盒,并对青海省牛群进行弓形虫病的血清学诊断。经对所收集的1856份牛血清进行弓形虫抗体检测,共检出阳性血清71份,阳性率约为3.83%。结果表明青海省牛群中存在弓形虫病的感染。     

Mapping of B-cell epitopes in the N-terminal repeated peptides of Anaplasma marginale major surface protein 1a and characterization of the humoral immune response of cattle immunized with recombinant and whole organism antigens

Major surface protein (MSP) 1a of the genus type species Anaplasma marginale (Rickettsiales: Anaplasmataceae) together with MSP1b forms the MSP1 complex. MSP1a has been shown to be involved in adhesion, infection and tick transmission of A. marginale, as well as to contribute to protective immunity in cattle. A differential antibody response to MSP1a and MSP1b was observed in cattle immunized with A. marginale derived from bovine erythrocytes (anti-MSP1a response) or cultured tick cells (anti-MSP1b response). In this study, we further characterized the MSP1a antibody response of cattle using several immunogens, including recombinant MSP1a (rMSP1a) protein, erythrocyte- or tick cell culture-derived A. marginale, or a combination of tick cell culture-derived A. marginale and rMSP1a. The MSP1a antibody response to all these immunogens was directed primarily against the N-terminal region of MSP1a that contains tandemly repeated peptides, whereas low antibody levels were detected against the C-terminal portion. Linear B-cell epitopes of MSP1a were mapped using synthetic peptides representing the entire sequence of the protein that were prepared by SPOT synthesis technology. Only two peptides in the N-terminal repeats were recognized by sera from immunized cattle. These peptides shared the sequence SSAGGQQQESS, which is likely to contain the linear B-cell epitope that was recognized by the pools of bovine sera. The average differential of antibody titers against MSP1a minus those against MSP1b correlated with lower percent reductions in PCV. A preferential antibody response to MSP1a was observed in cattle immunized with erythrocyte-derived, cell culture-derived plus rMSP1a or rMSP1a alone, and the percent reduction PCV was significantly lower in these cattle as compared with the other immunization groups. These results provide insight into the bovine antibody response against A. marginale and the role of MSP1a in protection of cattle against A. marginale infection.     

Serological survey of viral antibodies in llamas (Lama glama) in Argentina.

This study analysed sera from 390 llamas (Lama glama) from nine farms located in three different Argentine provinces: Buenos Aires, Cordoba and Jujuy. The samples were tested for antibodies against 8 virus known to infect cattle: bovine herpesvirus type 1 (BHV-1), bovine viral diarrhea virus (BVDV), bovine adenovirus (BAdV III), bovine enterovirus (BEV), bovine rotavirus (BRV), bluetongue virus (BTV), bovine leukaemia virus (BLV), and foot-and-mouth virus (FMDV) by conventional methods such as seroneutralization, immunoperoxidase staining, and agar gel immunodiffusion. The antibody prevalences detected in llamas were: BHV-1 in 0.77% (3/390), BVDV in 2.05% (8/390), BAdV III in 5.13% (20/390), BEV in 4.10% (16/390), BRV in 87.69% (342/390). No antibodies against BTV, BLV and VIAA (FMDV infection associated antigen) were detected.     

Serological Survey of Viral Antibodies in Llamas (Lama glama) in Argentina

This study analysed sera from 390 llamas (Lama glama) from nine farms located in three different Argentine provinces: Buenos Aires, Cordoba and Jujuy. The samples were tested for antibodies against 8 virus known to infect cattle: bovine herpesvirus type 1 (BHV-1), bovine viral diarrhea virus (BVDV), bovine adenovirus (BAdV III), bovine enterovirus (BEV), bovine rotavirus (BRV), bluetongue virus (BTV), bovine leukaemia virus (BLV), and foot-and-mouth virus (FMDV) by conventional methods such as seroneutralization, immunoperoxidase staining, and agar gel immunodiffusion. The antibody prevalences detected in llamas were: BHV-1 in 0.77 % (3/390), BVDV in 2.05 % (8/390), BAdV III in 5.13 % (20/390), BEV in 4.10 % (16/390), BRV in 87.69 % (342/390). No antibodies against BTV, BLV and VIAA (FMDV infection associated antigen) were detected.     

Cytotoxic activity of Mannheimia haemolytica strains in relation to diversity of the leukotoxin structural gene lktA

The leukotoxin of Mannheimia haemolytica has a very high degree of amino acid diversity because the lktA gene has a complex mosaic structure that has been derived by horizontal DNA transfer and intragenic recombination. The objective of the present study was to determine the effect of this amino acid diversity on leukotoxin cytotoxicity against bovine and ovine cell types. This was done by comparing the luminol-dependent chemiluminescence response of bovine and ovine neutrophils after pre-incubation with the various leukotoxin types. The LktA1.1-type leukotoxin associated with bovine serotype A1 and A6 strains differs from the LktA1.2-type leukotoxin produced by ovine serotype A1 and A6 strains at a single amino acid position and has enhanced activity against bovine but reduced activity against ovine neutrophils. These findings, together with the exclusive association of the LktA1.1-type leukotoxin with bovine strains, suggest that this leukotoxin type has an adaptive advantage in the bovine host. Leukotoxins LktA6-LktA10 are associated with ovine strains and have complex mosaic structures and diverse amino acid sequences but similar levels of cytotoxic activity against bovine and ovine neutrophils, respectively. However, ovine neutrophils were more sensitive to the cytotoxic activities of these leukotoxins than were bovine neutrophils. LktA8- and LktA10-type leukotoxins are associated with serotype A2 and A7 strains that are responsible for the majority of ovine disease cases, but LktA6-, LktA7- and LktA9-type leukotoxins are associated with less common serotypes. These findings contribute to the growing body of evidence suggesting that factors other than leukotoxin cytotoxicity are responsible for the full expression of virulence in M. haemolytica. Overall, the extensive recombinational exchanges within the lktA gene of M. haemolytica have had little effect on leukotoxin function which is highly conserved.     

Evaluation of virucidal activity of three commercial disinfectants and formic acid using bovine enterovirus type 1 (ECBO virus), mammalian orthoreovirus type 1 and bovine adenovirus type 1

A modified version of the test method of the Comité Européen de Normalisation (CEN) was developed using formic acid and three commercial disinfectants to evaluate virucidal activity against three non-enveloped viruses, bovine enterovirus type 1 (ECBO virus), mammalian orthoreovirus type 1 and bovine adenovirus type 1 (BAV 1). Determination of the effects of temperature was carried out at 20 and 10 degrees C. All tests with protein load used bovine serum albumin (BSA) and yeast extract. The investigations were performed in suspension tests and in carrier tests using poplar wood virus carriers. The carrier tests showed that ECBO virus could be inactivated at 20 degrees C with 1% formic acid within a 60 min reaction time. For disinfection of ECBO virus at 10 degrees C within 60 min, a 2% concentration of formic acid was necessary. Formic acid was ineffective against reovirus and bovine adenovirus and cannot be recommended as a reference disinfectant. Inactivation of ECBO virus and adenovirus type 1 using a disinfectant containing aldehydes and alcohols could be achieved, but only at room temperature. The disinfection of reovirus type 1 at room temperature with this product was possible without a protein load. This disinfectant exhibited disinfection ability at 10 degrees C at a concentration of more than 2% or with a longer exposure time. A disinfectant containing aldehydes was effective at room temperature but its effect was reduced in the presence of organic matter. Inactivation at 10 degrees C was found only against adenovirus. The fourth disinfectant, which contained peroxiacetic acid, inactivated all test viruses at a concentration of 0.5% within 15 min independent of temperature and protein load.     

Characterization of monoclonal antibodies directed against the bovine herpesvirus-1 glycoprotein E and use for the differentiation between vaccinated and infected animals

A panel of seven monoclonal antibodies (MAbs) directed against the bovine herpesvirus-1 (BHV-1) glycoprotein E (gE) was obtained. For that purpose, mice were either tolerized to BHV-1 gE-negative virus and then immunized with wild type BHV-1 or immunized with plasmid DNA expressing the gE and gI glycoproteins. The MAbs were characterized by their reactivity with the gE protein or the gE/gI complex and by competition experiments. Results showed that the MAbs were directed against three antigenic domains, two located on the gE glycoprotein and one on the gE/gI complex. Blocking experiments were performed with sera from experimentally vaccinated and infected cattle. A competition was observed between gE-positive bovine sera and six of the seven MAbs. The bovine sera thus recognized two of the three antigenic sites. Field sera were then tested in blocking enzyme-linked immunosorbent assay using one horseradish peroxidase-conjugated MAb. A specificity of 98.2% and a sensitivity of 98.2% compared to the commercially available test were observed.     

Evidence of bovine immunoglobulin G1 (IgG1) protease activity in partially purified culture supernate of Pasteurella haemolytica A1.

In the bovine respiratory tract, IgG1 is a major secretory immunoglobulin (Ig), and both IgG1 and IgG2 are believed to be important in defense against pneumonic pasteurellosis (shipping fever) in calves. Here we provide evidence for hydrolysis of IgG1 in the presence of partially purified culture supernate (ppCS) from the respiratory pathogen Pasteurella haemolytica A1. Bovine IgG1 was hydrolysed sequentially into three distinct bands (approximately 39, 12, and 7 kDa respectively). Furthermore, partial hydrolysis of bovine IgG2 was observed, but neither bovine IgA nor IgM were affected by incubation with ppCS. These findings suggest that the production of an IgG1-specific protease by P. haemolytica A1 may be a virulence mechanism contributing to the pathogenesis of bovine pneumonic pasteurellosis.     

Evaluation of the protective effect of bovine lactoferrin against lipopolysaccharides in a bovine mammary epithelial cell line

Lactoferrin (Lf) is a non-haem iron-binding glycoprotein with a molecular weight of about 80 kDa, synthesized by glandular epithelial cells and stored in the secondary granules of neutrophils. The physiological significance of Lf is related to non-specific immune defence against pathogens, immunomodulatory activity, iron homeostasis, antioxidant properties and regulation of cell growth. Lf is a bioactive component of the mammary secretions and its modulatory and defensive functions do affect the newborn and the mammary gland as well. In this work a bovine mammary epithelial cell line (BME-UV1) was used as an in vitro model of the bovine mammary epithelium to examine the protective role of exogenous bovine Lf (bLf) against the cytotoxic damage induced by bacterial lipopolysaccharides (LPS) and the endogenous bLf mRNA expression after LPS exposure. In the in vitro model used, exogenous bLf exerts a protective effect against endotoxin cytotoxicity, which could be mediated by the LPS-neutralizing capability of bLf. In addition, in BME-UV1 cells the response to LPS exposure does not involve bLf mRNA expression, suggesting that this cell line lack of functional LPS-responsive elements.     

北京地区规模化奶牛场三种病毒性腹泻病的血清学调查

为了解近年北京地区奶牛腹泻性疾病的流行情况,采用酶联免疫吸附试验(ELISA)对北京地区密云、怀柔和昌平3个区县的未免疫接种牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)、牛冠状病毒(Bovine coronavirus,BCV)和牛轮状病毒(Bovine rotavirus,BRV)疫苗的31个规模化奶牛场的1 650份血清样品进行了BVDV、BCV、BRV感染抗体检测。结果显示,BVDV抗体平均阳性率为48.2%,BCV抗体平均阳性率为57.2%,BRV抗体平均阳性率为52.2%,BVDV、BCV及BRV感染在密云、怀柔和昌平3个区县的牛群中普遍存在,需进一步加强奶牛腹泻性疾病的综合防控。     

Monoclonal antibody against bovine alpha1-acid glycoprotein

Monoclonal antibodies (Mabs) against bovine alpha1-acid glycoprotein (alpha1AGP) were prepared from mouse hybridoma cell line. Bovine alpha1AGP as antigen was purified by using ion exchange column chromatography and the yield from 500 ml serum was about 10 mg. Immunoglobulin isotypes of 3 Mabs obtained were IgM and light chain types were kappa. The Mabs reacted with bovine alpha1AGP on immunoblot analysis, but not with alpha1AGP digested with N-glycosidase, suggesting that an epitope recognized by these Mabs may be associated with a glycan side chain of bovine alpha1AGP.     

Demonstration of antibodies against bovine leukemia virus (BLV) by blocking ELISA using bovine polyclonal anti-BLV immunoglobulin.

A blocking enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against bovine leukemia virus (BLV) is described. The test is based on the biotin-streptavidin system using unlabelled polyclonal bovine IgG against BLV as catching antibody and biotinylated bovine anti-BLV IgG as detecting antibody. The sensitivity was found to be 50-100 times higher than the agar gel immunodiffusion test, with a specificity of practically 100%. The blocking ELISA proved to be suitable for detection of antibodies against BLV in serum and milk. In 34 paired milk/serum samples, the average ratio of BLV antibody titres was 1:26. So far, more than 700,000 sera have been screened by blocking ELISA for BLV antibodies in the course of the Danish surveillance programme for BLV infection.     

Non-specific natural cytotoxic factor released from bovine peripheral blood lymphocytes.

Natural cytotoxicity against bovine leukemia cells (PC-3 cells) was found in bovine peripheral blood lymphocytes (PBL), and in non-adherent cells but not in adherent cells to nylon-wool column. Natural cytotoxic cells (NCC), which have natural cytotoxic activity, are found in T cell-rich fraction. When NCC were cocultured with PC-3 cells, natural cytotoxic factor (NCF) was released rapidly from NCC, and dose-response curve for NCF was almost linear induction. Cytotoxicity against PC-3 cells by NCC or NCF was increased with an increment of incubation period. Cytotoxicity against K562 cells, CL-1 cells, M1 cells or EL-4 cells by NCF was almost the same level as that against PC-3 cells, but that against those cell lines by NCC was not found. NCF activity in culture fluid from NCC cocultured with K562 cells or CL-1 cells was lower than that from NCC cocultured with PC-3 cells.     

Stability through the gastrointestinal tractus of newborns and tolerance of bovine immunoglobulines with a high agglutination titer against pathogenens E. Coli given per os (author's transl)]

The aim of this study was to investigate the stability of bovine immunoglobuline and maintenance of their efficacity given per os to newborns, and the tolerance to this preparation. This preparation is designed for prevention and/or treatment of infant gastroenteritis by passive immunity.It contains bovine immunoglobulines IgG (75% of total), IgM and IgA with a high titer against frequent serotypes of E. coli. It was given per os to 10 newborns, 8 being aged 1 to 5 months, and 2 aged 18 months. Samples of stool were collected before (controls) and after treatment (tests). Bovine IgG were estimated in the stools by standard immunodiffusion test, specific titer against coli antigens by agglutination technic. Blood formula was checked throughout.Tolerance to the preparation was good. In the stools of 9 of the 10 newborns, bovine immunoglobulines were found at higher concentrations in tests than in controls, both in term of quantity (several mg IgG per gram of stool) and in term of specificity against E. coli serotypes. The stools of one child only (aged 18 months) was negative for bovine IgG and specific immunoglobulines.These results demonstrate that bovine immunoglobulines of this preparation given per os are stable through the gastrointestinal tractus and that their specificity is maintained, in particular in children aged less than 5 months.