Partial Antigenic Characterization of Buffalopox Virus

The present study was undertaken to antigenically characterize the buffalopox virus (BPV). Six monoclonal antibodies (MAbs) against the BP4 strain of BPV have been produced and characterized. All six MAbs appeared to be specific to BPV, as none of them showed cross-reactivity with other poxviruses in antigen capture ELISA. Only two MAbs (20AB8 and 20CD11) bound significantly with different BPV isolates in antigen capture ELISA, whereas the remaining four MAbs bound weakly with the BPV. In Western blot analysis with purified BPV-BP4, the rabbit hyperimmune serum against purified BPV-BP4 reacted with 15 immunodominant polypeptides (100 kDa to 25 kDa), whereas two MAbs (21CB6, 21DB11) reacted with 42 kDa and 45 kDa polypeptides, respectively. However, three MAbs (20AB8, 20CD11, 21CB5) reacted with three degraded polypeptides (100 kDa, 40 kDa and 87 kDa) of BPV-BP4. In radioimmunoprecipitation assay (RIPA) with the rabbit hyperimmune serum to BPV-BP4, three virus specific polypeptides (69 kDa, 34 kDa, 32 kDa) were recognized in BPV-BP4, whereas two polypeptides (69 kDa, 34 kDa) were recognized in other BPV isolates (BPV-Bly, BPV-Vij96, BPV-Vij97). In virus neutralization test, none of the six MAbs tested showed any significant neutralizing ability to infection with different BPV isolates. However, the hyperimmune serum showed weak neutralizing ability to BPV infection.     

Characterization of monoclonal antibodies against avian reovirus S1133 protein sigmaA synthesized in Escherichia coli

Monoclonal antibodies (MAbs) were prepared against avian reovirus S1133 protein sigmaA (esigmaA) synthesized in Escherichia coli. MAbs were characterized and used to develop a diagnostic test. Ten MAbs were selected for competitive binding assay following coupling with horseradish peroxidase. The results indicated that these MAbs delineated two epitopes I and II of esigmaA. An immuno-dot binding assay was used to detect the effect of denaturation on antibody recognition of the epitopes. All MAbs bound to esigmaA in its native form. After denaturation by boiling in SDS and 2-mercaptoethanol, the binding of MAbs recognizing epitope I was fully abolished. However, the reactivity of MAbs recognizing epitope II was not affected. MAbs 31 and 32, recognizing epitopes I and II, respectively, were selected for the cross-reactivity to heterologous reovirus strains. The results suggest that the two epitopes are highly conserved among these virus strains. A MAb capture enzyme-linked immunosorbent assay (ELISA) procedure was developed using MAbs 32 and 31 to detect reovirus protein sigmaA in samples from tendon tissues of infected bird and chicken embryo fibroblast (CEF) cell cultures. Avian reovirus sigmaA antigens in tendon specimens were detected from the inoculated birds as early as 2 days post-inoculation (PI), approximated a peak at 7 days PI, and maintained this until 16 days PI, then decreased gradually. A clear difference in absorbance values between the tendon samples of the avian reovirus- and mock-infected birds is obtained. Positive results were also obtained from avian reovirus-infected CEF and from the tendon tissues of naturally infected broilers. These results indicated that the MAb capture ELISA is a useful methods for the detection of avian reovirus from chickens suspected to have avian reovirus infections.     

Foot-and-mouth disease virus C3 Resende subtype analysed by means of competition RIA using neutralizing monoclonal antibodies.

Foot-and-mouth disease virus (FMDV) was analysed using 30 monoclonal antibodies (MAbs) obtained from Balb/c mice immunized with FMDV C3 Resende (C3R) subtype 7 and 14 days before fusion No. 15 and 16 respectively. Fourteen MAbs were neutralizing and by means of competition radioimmuno assay it was possible to classify them into four groups. The first group consisted of MAbs specific for three sequential and three conformational epitopes. The second group consisted of MAbs specific for two conformational and for one sequential epitope. The third and the fourth groups consisted only of one MAb each, being specific for conformationally and one sequentially dependent epitope, respectively.     

Response of heifer mammary gland macrophages and neutrophils to interferon-gamma stimulation in vitro.

The phagocytic and killing abilities of heifer mammary gland macrophages (M phi) and neutrophils were evaluated after exposure to recombinant bovine interferon-gamma (rBoIFN-gamma) stimulation in vitro. Macrophages or neutrophils were cultured for 2 h with 0, 10(2), 10(4) and 10(5) units rBoIFN-gamma/mL. Phagocytosis assays were performed by incubation with Staphylococcus aureus at a leukocyte:bacteria ratio of 1:10. After 45 min, cells were stained with acridine-orange and phagocytic and killing abilities were determined. Although rBoIFN-gamma had no effect on M phi phagocytic activity, neutrophil phagocytic activity after incubation in 10(4) units rBoIFN-gamma (41.62%) was significantly higher than 0 (25.24%) or 10(2) units rBoIFN-gamma (24.73%). Neutrophil and M Phi killing abilities were not affected by any dose of rBoIFN-gamma. Results suggested that rBoIFN-gamma promoted neutrophil phagocytic activity, but did not affect neutrophil killing or overall M phi function in vitro.     

Physiological and morphological changes in bovine mammary glands following intramammary infusion of recombinant interferon-gamma.

Eleven lactating dairy cows were used to evaluate the response of bovine mammary glands to increasing doses of recombinant bovine interferon (rBoIFN)-gamma. Right front and rear quarters were intramammarily infused with eight different doses (10(2) U to 2 x 10(8) U/quarter) of rBoIFN-gamma; each dose was tested in at least two quarters. Left udder halves served as within animal controls in which quarters were injected with a saline placebo or were not infused at all. Milk secretion samples for compositional analysis were collected from each quarter prior to infusion and at 6, 24, 36 and 48 h following infusion. Animals were slaughtered immediately following the 48 h sampling period and mammary tissue was obtained for morphometric analyses. Milk composition was similar between control quarters and those quarters infused with up to 10(5) U of rBo-IFN-gamma during the entire sampling period. Quarters infused with 10(6) U and 10(7) U of rBoIFN-gamma had higher milk somatic cell counts (SCC) following treatment compared with preinfusion values. Changes in the composition of mammary secretion were most dramatic in quarters infused with greater than or equal to 10(8) U of rBoIFN-gamma as indicated by the significant increase in SCC and milk pH with a concomitant decrease in lactose concentration when compared with pre-infusion values or with control quarters. Morphometric analysis of tissue demonstrated an increase in stroma, a decrease in luminal area, and a marked increase in the number of infiltrating leukocytes in those quarters infused with the higher doses of rBoIFN-gamma.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development and characterization of monoclonal antibodies against FMD virus type Asia-1 and determination of antigenic variations in the field strains

Twelve mouse monoclonal antibodies (MAbs) were developed against an Indian vaccine strain of foot and mouth disease virus (FMDV) type Asia-1 WBN 117/85. The MAbs were tested for their ability to bind to whole virus particle, trypsin-treated 146S (TT-146S) virus particle, sub-viral (12S and disrupted virus) antigens by ELISA and to neutralize virus infectivity in cell culture. Extensive characterization of MAbs revealed the existence of three different groups based on the binding of non-overlapping epitopes. Eight type Asia-1 specific MAbs (RF7, RF8, RD10, RE11, RC11, RC10/O, RB11 and RC10/M), which formed group 1 (G1), were found to bind a neutralizing, trypsin-sensitive (TS) and conformational epitope. Two MAbs (WB8 and WC3) in group 2 (G2) were found to bind a non-neutralizing, trypsin-resistant, conformational and 12S-specific epitope, which was intertypically conserved in all the four serotypes of FMDV (O, A, C and Asia-1) prevalent in India. Two MAbs (KG10 and KF10), which formed group 3 (G3), were found to be against a non-neutralizing, TS and conformational epitope, common to types Asia-1 and A. Members of G1 were IgG2a isotype, while those of G2 and G3 were IgG1 and IgG2b isotypes, respectively. Antigenic analysis of 31 FMDV type Asia-1 field isolates and two vaccine strains, using a panel of type Asia-1-specific MAbs, revealed antigenic similarity of the virus isolates tested and non-existence of neutralization escape mutants. The developed MAbs have practical utility, especially in the manufacture of FMD vaccine, diagnosis and FMDV characterization.     

Influence of recombinant bovine interferon gamma and dexamethasone on pneumonia attributable to Haemophilus somnus in calves

The influence of recombinant bovine interferon gamma (rBoIFN-gamma) treatment on resistance of clinically normal and dexamethasone-treated calves to Haemophilus somnus infection was evaluated. Four groups of 6 calves each were treated with saline solution (controls), dexamethasone (0.04 mg/kg of body weight/for 3 days), rBoIFN-gamma (2 micrograms/kg for 2 days), or dexamethasone and rBoIFN-gamma (aforementioned dosages). All treatments were started 24 hours before intrabronchial challenge exposure with 5 x 10(9) colony-forming units of H somnus. Rectal temperature and WBC count were monitored daily. Two of the dexamethasone-treated calves died of pneumonia 4 days after challenge exposure and were necropsied. All other calves were euthanatized and necropsied 7 days after challenge exposure. All calves had pneumonia of variable intensity. Dexamethasone-treated calves had increased volume of pneumonic lung (P less than 0.05) and increased severity of pneumonia, compared with control calves. Recombinant bovine interferon gamma treatment resulted in reduction in pneumonic lung volume and severity of pneumonia in dexamethasone-treated calves (P less than 0.05), although it did not influence severity of pneumonia in nondexamethasone-treated calves.     

Antigenic relationships of foot-and-mouth disease virus serotype Asia-1 isolates demonstrated by monoclonal antibodies.

A panel (26) of monoclonal antibodies (MAbs) was elicited against three distinct isolates of foot-and-mouth disease virus (FMDV) serotype Asia-1. Each MAb was characterized according to the location of its epitope: Class I, restricted to the intact virion (140S); Class II, restricted to 140S and the virion protein subunit (12Sps); Class III, available on 140S, 12Sps and virus protein 1; Class IV, restricted to 12Sps. In addition, the MAbs were further categorized by isotype, neutralization of viral infectivity, capacity to bind in radioimmunoassay and precipitation in the Ouchterlony reaction. Neutralization of FMDV infectivity by a MAb of the IgA isotype is reported for the first time. A minimum of seven distinct neutralization epitopes were described on FMDV Asia-1. Some of the neutralizing MAbs bound FMDVs in addition to those that they neutralized. The MAbs defined epitopes common to FMDV serotypes Asia-1, A, O1 and C but neutralizing capacity was restricted to serotype Asia-1. Class IV MAbs defined epitopes highly conserved throughout the FMDV serotypes. Identification of FMDV neutralization epitopes makes possible the direct selection of optimal FMDV strains for vaccine fabrication. In addition, these data are crucial to the design of future synthetic vaccines.     

具有HI活性的H1亚型流感病毒特异性单克隆抗体的制备与应用

为了制备具有HI活性的HI亚型流感病毒特异性单克隆抗体(MAb),本研究以H1N1亚型猪流感病毒(SIV)株A/Swine/Guangdong/718/01(H1N1)为免疫原,免疫BALB/c小鼠,经常规细胞融合后,血凝抑制(HI)方法进行检测,融合细胞经稀释克隆纯化后,获得11株能稳定分泌抗血凝素特异性HI MAb的杂交瘤细胞株。鉴定表明,所获MAb与其他具有血凝活性的病毒以及其他14个HA亚型的流感病毒均不具有HI交叉反应,表明这11株MAb均具有良好的流感病毒亚型特异性。其中A6F、2BBF和2BB与其他H1亚型流感病毒分离株的HI试验证实我国不同地区分离株之间的抗原性存在一定差异。11株MAb对H1SIV抗原的HI试验结果显示其HI效价有明显差异。叠加实验表明这些MAb分别识别HA抗原的不同表位。间接免疫荧光试验表明,2BBF、8HB、1DH、7FC和2BB均可与2009年流行H1N1病毒A/California/04/2009HA抗原发生特异性反应。这些MAb特异性的研制为H1亚型流感病毒的疫情病原学快速诊断以及病毒抗原性变异的相关研究提供了物质基础。     

Recombinant bovine interferon-gamma, but not interferon-alpha, potentiates bovine neutrophil oxidative responses in vitro

In this study we have addressed the in vitro effects of recombinant bovine interferon-gamma (rBoIFN-gamma) and interferon-alpha (rBoIFN-alpha 1) on oxidative functions of bovine neutrophils. Treatment with rBoIFN-gamma, but not rBoIFN-alpha 1, enhanced the luminol-dependent chemiluminescence (LDCL) response of bovine neutrophils to both opsonized zymosan particles and phorbol myristate acetate. Pre-incubation of neutrophils for 2 h at 39 degrees C with rBoIFN-gamma resulted in a 40% increase in both LDCL and release of hydrogen peroxide by neutrophils stimulated with opsonized zymosan. This enhancement was observed at doses ranging from 0.2 to 2000 units of rBoIFN-gamma per ml. In contrast to the results observed in the LDCL and hydrogen peroxide assays, preincubation of neutrophils with rBoIFN-gamma had no effect on the levels of superoxide anion released in response to opsonized zymosan. Pre-incubation with rBoIFN-gamma increased phorbol myristate acetate (PMA)-stimulated LDCL by 30%, although it had no effect on either superoxide anion or hydrogen peroxide release in response to PMA stimulation. Neither recombinant interferon directly elicited an oxidative burst from neutrophils in the absence of zymosan or PMA stimulation.     

Antigenic characterization of Anaplasma marginale isolates from different regions of Brazil

Antigenic characterization of A. marginale isolates has contributed to identifying the presence of common and restricts epitopes of major surface proteins (MSPs). The data may improve vaccine development to protect against A. marginale isolates from different regions. Brazilian A. marginale isolates were characterized antigenically by Western blot with monoclonal antibodies (MAbs) against MSPs and rabbit anti-MSP-4 from Florida strain. Six A. marginale isolates from MS, MG (AUFV1), SP, PR-L1, PR-HV, RS and Florida strain were tested with ANA22B1 to MSP-1a, AMR36A6 to MSP-1b, ANAF19E2 to MSP-2, AMG75C1 and AMG76B2 to MSP-3 and ANAF16C1 to MSP-5. ANA22B1 recognized MSP-1a epitope in all A. marginale isolates, and reacted with polypeptides of different size ranging 46-105kDa. MSP2 was not detected in MS and SP isolates by ANAF19E2, and only PR-L1 and MG (AUFV1) isolates reacted with MAbs which recognize MSP3 epitope. MSP4 and MSP5 were detected in all A. marginale isolates analyzed. The results revealed conservation of MSP-1a and MSP-5 epitopes among all Brazilian isolates, and showed antigenic variability to MSP-1b, MSP-2 and MSP-3 proteins, agreeing with recent data about the genetic diversity found in the polimorphic multigene family responsible for these proteins.     

Generation of monoclonal antibodies to porcine interleukin 6 (PIL-6) using the recombinant PIL-6 expressed in Escherichia coli

Porcine interleukin-6 (PIL-6) protein without signal peptide was expressed as a glutathione S-transferase (GST) fusion protein in Escherichia coli. The fusion protein was expressed in an insoluble fraction, however, it was solubilized by refolding procedure using urea. From the solubilized protein, the recombinant PIL-6 (rPIL-6) was purified by a batch method using glutathione sepharose 4B and PreScission protease cleavage. By the B3B1 hybridoma cell proliferation assay, biological activity of the purified rPIL-6 was confirmed. Three monoclonal antibodies (MAbs) named 2B-1, 5A-8 and 4C-3 were generated by using the rPIL-6 as an immunogen. Immunoglobulin isotypes of the MAbs were IgG2a (4C-3) and IgG2b (2B-1 and 5A-8). For the epitope analysis, additive enzyme-linked immunosorbent assay and immunoblot analysis using deletion mutants of PIL-6 were performed. These experiments revealed that the two MAbs (2B-1 and 5A-8) recognize an overlapped epitope and the other (4C-3) recognizes a distinct epitope, and all epitopes reside in the region of aa26-64 of PIL-6.     

Quantitative diversity of 67 kda protective antigen among serovar 2 strains of Erysipelothrix rhusiopathiae and its implication in protective immune response

Mouse monoclonal antibodies (MAbs), raised against the NaOH-extracted antigen of Erysipelothrix rhusiopathiae strain Kyoto (serovar 2), recognized two different epitopes on a single protein of molecular weight 67 kDa. The MAbs were classified as protective or non-protective against strain Fujisawa (serovar 1). In immunoblotting analysis using the MAbs, fifteen wild strains were shown to contain different amounts of 67 kDa protective antigen. Each formalin-killed whole cell vaccine (bacterin) prepared from the fifteen wild strains conferred different levels of protection against strain Fujisawa in mice. Bacterins prepared from wild strains with larger amounts of 67 kDa protective antigen tended to give high levels of antigen-specific antibody and better protection to mice. These results indicate that the amount of 67 kDa protective antigen which influences the induction of protective immune responses may vary substantially among the strains of E. rhusiopathiae (serovar 2).     

Monoclonal antibodies preventing invasion of Neospora caninum tachyzoites into host cells

Twelve monoclonal antibodies (MAbs) against Neospora caninum tachyzoites were produced to specify the antigens related to the invasion of tachyzoites into host cells. In the assay to evaluate the inhibition activity, all these MAbs prevented the cultured Vero cells from the invading by the tachyzoites. These MAbs recognized approximately a 73 kDa antigen in Western blot analysis. Immunofluorescence assay and immune electron microscopy revealed that this 73 kDa antigen is a part of the surface antigens of N. caninum tachyzoite, and that the tachyzoite antigen identified plays an important role for invasion of host cells.     

Production of monoclonal antibodies against serum immunoglobulins of black rockfish (Sebastes schlegeli Higendorf)

The present study was undertaken to produce monoclonal antibodies (MAbs) against immunoglobulin (Ig) purified from black rockfish (Sebastes schlegeli Higendorf) serum using protein A, mannan binding protein, and goat IgG affinity columns. These three different ligands were found to possess high affinity for black rockfish serum Ig. All of the Igs purified eluted at only 0.46 M NaCl concentration in anion exchange column chromatography and consisted of two bands at 70 kDa and 25 kDa in SDS-PAGE; they also had similar antigenicity for MAbs to Ig heavy chain in immunoblot assays. Therefore, black rockfish Ig is believed to exist as a single isotype within serum. The MAbs produced against Ig heavy chain reacted specifically with spots distributed over the pI range from 4.8 to 5.6 with a molecular weight of 70 kDa on two dimensional gel electrophoresis immunoblot profiles.     

Modulation of bovine mammary neutrophil function during the periparturient period following in vitro exposure to recombinant bovine interferon gamma

Effects of recombinant bovine interferon (rBoIFN) gamma on mammary gland neutrophil activity during the periparturient period were studied. Bovine mammary gland neutrophils were isolated and incubated in mammary gland secretions obtained from Holstein-Friesian cattle during the last 2 weeks of gestation. Cell functions were evaluated following treatment with 10 U, 100 U, and 1000 U of rBoIFN-gamma. Bacterial phagocytosis, bactericidal activity and chemiluminescence were significantly lower for neutrophils incubated in mammary gland secretions when compared with control neutrophils incubated in Hank's balanced salt solution. Treatment of mammary neutrophils with rBoIFN-gamma reversed the suppressive effects of mammary secretions resulting in higher chemiluminescent activity and significantly more bacterial phagocytosis and bactericidal activity when compared with untreated controls. Results from these preliminary in vitro data suggest that rBoIFN-gamma therapy may modulate mammary gland neutrophil functions in vivo and possibly facilitate the rapid clearance of mastitis-causing pathogens mammary glands during the periparturient period.     

A characterization of monoclonal antibodies prepared against Pasteurella haemolytica serotype 1 surface antigens.

The production and characterization of monoclonal antibodies against Pasteurella haemolytica serotype 1 is described. Ten monoclonal antibodies were produced and divided, on the basis of their properties, into six different groups. One produced bacteria agglutination only of P. haemolytica serotype 1. Three antibodies bound with P. haemolytica serotypes 1, 5-8 and 12 and the antigen was identified in immunoblots as lipopolysaccharide. Two antibodies bound P. haemolytica serotypes 1, 2, 5-8 and 12 and P. multocida serotypes 1-7, 9, 12, 15 and 16, recognizing an epitope present on a 29 kDa outer membrane protein. One antibody bound all P. haemolytica and P. multocida serotypes. The antigen was a hexosamine less than 30 kDa which contained a formalin sensitive epitope. One antibody bound only to P. haemolytica serotype 1 and the antigen was identified as a 66 kDa outer membrane protein. Two antibodies bound P. haemolytica serotypes 1, 2, 5-9 and 12 and the antigen, while not identified, was localized on the outer membrane. This study identified antigens which contribute to the cross-reactions among P. haemolytica and P. multocida serotypes and the antibodies may be useful in investigating the pathogenesis of pneumonic pasteurellosis.     

Characterization of monoclonal antibodies against the second-generation schizonts of Leucocytozoon caulleryi

Monoclonal antibodies (MAbs), R1 and M5, were established against the second-generation schizont of Leucocytozoon caulleryi (L. caulleryi). Both antibodies reacted to membrane and internal structure proteins of the second-generation schizont by immunofluorescence microscopy. Molecular weight of the second-generation schizont (2GS) antigen was analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. At least 40 protein bands were detected in 2GS antigen by SDS-PAGE under reduced condition and ranged from 10 to 270 kDa. MAb R1 reacted to polypeptides of 150-268 kDa in 2GS antigen, whereas MAb M5 did with that of 66 kDa. Injection with a protein of 2GS antigen fractionated by affinity chromatography using MAbs R1 and M5 protected chickens against challenge with sporozoites of L. caulleryi. These results suggest that MAbs, R1 and M5, recognize 2GS antigen of L caulleryi.     

Quantitation of bovine immunoglobulin isotypes and allotypes using monoclonal antibodies.

A panel of 10 monoclonal antibodies specific for bovine immunoglobulins M, A, G1, G2 and light chains were produced and enzyme-linked immunosorbent assays developed to measure Ig levels in body fluids and culture supernatants using this panel of MAbs. An inhibition ELISA was accurate and sensitive for MAbs of high affinity, detecting levels as low as 10 ng ml-1 of IgM using a high-affinity MAb, IL-A50 (dissociation constant = 1.3 X 10(-11) M). For MAbs of lower affinity (KD of less than 0.25 X 10(-9) M) a sandwich ELISA was more sensitive, detecting 0.1-1.0 microgram ml-1 Ig, provided a conjugate of an anti-light chain MAb was used. Using these ELISA techniques, four pairs of MAbs specific for bovine IgM, IgA, IgG1 and IgG2 respectively, were screened on sera from over 100 cattle of different breeds to determine whether any detected a polymorphic epitope. MAbs IL-A30, IL-A60, IL-A66, IL-A71, IL-A72, IL-A73 and IL-A74 were shown to recognise monomorphic determinants on their respective heavy chains. In contrast, the epitope recognised on the mu-heavy chain by MAb IL-A50, which had previously been shown to be polymorphic, was found to be allelic and inherited under the control of a single gene, probably Cu.     

Identification of B-cell epitopes in the NSP1 protein of porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) was divided into North American and European genotypes. NSP1 was an important non-structural protein of PRRSV, which was auto-cleaved from the replicase polyprotein into NSP1α and NSP1β subunits and played an important role in the immune suppression. In this study, six monoclonal antibodies (MAbs) against the recombinant PRRSV NSP1, expressed in Escherichia coli system, were screened out and identified. Western blot and IFA results indicated that 4 out of 6 MAbs recognized the recombinant NSP1α and 2 MAbs recognized NSP1β. Epitope mapping results indicated that MAb 4H2 recognized the linear epitopes E(54)EPLRW(59) in NSP1α, MAbs (2G5, 3E11 and 4D4) recognized the epitopes H(157)VLTNLP(163) in NSP1α, and MAbs 3C7 and 1H7 reacted with the epitopes 185aa to 232aa in NSP1β. Protein sequence alignment of NSP1 indicated E(54)EPLRW(59) was conserved in all North American PRRSV strains, whereas European type strains has variable amino acids in this region. The epitope H(157)VLTNLP(163) was relatively conserved among all PRRSV strains, except for a L162→S162 change in European type strains. The epitope 185-232aa was variable among North American PRRSV strains. These results may facilitate future investigations into the function of NSP1 of PRRSV and diagnostic methods for PRRSV infection.