Evaluation of two monoclonal antibodies for serotyping Haemophilus paragallinarum

Two monoclonal antibodies (MAbs) were evaluated for their ability to serotype 108 isolates of Haemophilus paragallinarum. One MAb (E5C12D10) was raised against a Page serovar A strain and the other (F2E6) against a Page serovar C strain. In both dot blot and hemagglutination-inhibition tests, MAb E5C12D10 recognized the type strains of Page serovar A and Kume serovars A-1, A-2, A-3, and A-4. MAb F2E6 recognized the type strains of Page serovar C and Kume serovars C-1, C-2, and C-3. Neither antibody recognized the type strains of Page serovar B or Kume serovars B-1 and C-4. When evaluated with 97 field isolates in a dot blot test, the MAbs serotyped 81 isolates, which was better than agglutinin typing by the Page scheme (69 isolates serotyped). The field isolates that did not react with the MAbs were either Page serovar B/Kume serovar B-1 (three isolates), Page serovar C/Kume serovar C-4 (12 isolates), or nontypable by either the Page or Kume scheme (one isolate).     

Characterization and use of monoclonal antibodies to identify Haemophilus paragallinarum serovars

Two serovar-specific monoclonal antibodies (MAbs) to Haemophilus paragallinarum serovars A/1 and C/2 strains, respectively, were developed and characterized by hemagglutination-inhibition (HI) and dot-blotting tests using representative H. paragallinarum serovars A/1, B, and C/2 strains. In both the HI and dot-blotting tests, one MAb (E5C12D10), raised against strain 221, serovar A/1, reacted only with serovar A/1 strains, while the other MAb (F2E6), raised against strain S1 of serovar C/2, reacted with only serovar C/2 strains examined. In both tests, the two MAbs did not react with two serovar B strains. These results indicated that the two MAbs recognize serovar-specific hemagglutinating (HA) antigens of H. paragallinarum serovars A/1 and C/2 strains, respectively, and that a dot-blotting test using these MAbs is a practical alternative to the HI test for serotyping H. paragallinarum. Strains 0222 and Spross of serovar B, which did not react with these two MAbs, were found to possess serovar-specific HA antigen in cross-HI tests.     

Production and evaluation of a panel of monoclonal antibodies against Haemophilus paragallinarum

We report on the production and characterisation of monoclonal antibodies (MAbs) against Haemophilus paragallinarum, the causative agent of infectious coryza. A bank of 8 MAbs were produced by traditional techniques - four against the reference strain for Page serovar A (0083) and four against the reference strain for Page serovar C (Modesto). Seven of the eight MAbs were shown to be IgG(1) with one being nontypable. None of the MAbs had HI activity and none gave any detectable reaction when examined by Western blotting. None of the MAbs gave a positive reaction in the indirect ELISA with any of the eight type strains of Pasteurella species or sub-species. None of our 8 MAbs gave serovar specific reactions when used in an indirect ELISA format. There was a trend for the serovar A MAbs to give a higher titre with serovar A isolates/strains and a similar trend for the serovar C MAbs to give higher titres with the serovar C isolates/strains.     

Serological classification of Japanese isolates of Haemophilus paragallinarum using two serovar-specific monoclonal antibodies

A serological classification of 106 Japanese isolates of Haemophilus paragalinarum recovered from 1960 to 1984 was performed by dot-blotting and hemagglutination-inhibition (HI) tests using two serovar-specific monoclonal antibodies (MAbs), E5C12D10 and F2E6. By the dot-blotting test, 49 of the isolates were serovar A and 55 isolates were serovar C, and the two remaining isolates did not react with either MAb. These two nontypable strains had no hemagglutinating activity against chicken erythrocytes and were nonpathogenic to chickens. Although 49 serovar A isolates were serotyped by the HI test, only 23 of the 55 serovar C isolates could be serotyped. The remaining 32 isolates could not be serotyped because no or low hemagglutinating activity could be detected. Our results indicate that H. paragallinarum serovars A and C have both been present in Japan since 1960, with serovar A isolates being dominant before 1970 and serovar C isolates more prevalent than serovar A since 1970.     

Immunogenicity of Haemophilus paragallinarum serovar B strains.

Immunogenicity of three Haemophilus paragallinarum serovar B strains was investigated in cross-protection tests using monovalent vaccines prepared from the B strains, as well as one strain each of serovars A and C. A bivalent vaccine composed of the serovar A and C strains also was used. In the studies with the monovalent vaccines, the immunogenicity of serovar B strains was different from that of serovar A and C strains, although only partial serovar B-specific protection with the three strains was observed. Chickens vaccinated with the bivalent vaccine protected against challenge with one serovar B strain, as well as serovar A and C strains, but not against the other two serovar B strains.     

Virulence of the nine serovar reference strains of Haemophilus paragallinarum

The virulence of the reference strains of the nine currently recognized Kume serovars of Haemophilus paragallinarum was investigated. The capacity of the H. paragallinarum strains to cause the typical clinical signs of upper respiratory tract disease associated with infectious coryza in unvaccinated, nasal-challenged chickens was assessed. Differences in virulence were assessed by means of a standardized scoring system for clinical signs. All nine strains were pathogenic to chickens, producing typical clinical signs of infectious coryza. The highest clinical signs score was obtained for serovar C-1 (1.72), while the lowest clinical signs score was obtained for serovar C-4 (0.32). Our results indicate that virulence differences exist among the serovars of H. paragallinarum.     

B型副鸡嗜血杆菌的分离鉴定

从辽宁某公司鸡场疑似鸡传染性鼻炎的病鸡眶下窦分离到一株副鸡嗜血杆菌，用Page程序和Kume程序对其进行血清型鉴定，确认为B型副鸡嗜血杆菌，这是首次在我国分离到B型副鸡嗜血杆菌。     

Evaluation of a panel of monoclonal antibodies in the subtyping of Haemophilus paragallinarum.

A panel of four monoclonal antibodies (MAbs) was evaluated, using a hemagglutination-inhibition test, for its ability to subtype 76 isolates of Haemophilus paragallinarum. The results of the MAb reactions were compared with the results of both the Page and Kume serotyping schemes (the serovars of the Page scheme correspond to the serogroups of the Kume scheme). One MAb (E5C12D10) was raised against a Page serovar A strain and the remaining MAbs (F2E6, D6D8D5, and B3E6F9) against a Page serovar C strain. Six different reaction patterns were found among the 76 isolates of H. paragallinarum. There was total correlation between the MAb reaction pattern and the Page scheme, and thus the Kume scheme, to the serogroup level. All 19 Page serovar A (= Kume serogroup A) strains reacted only with MAb E5C12D10, whereas all five Page serovar B (= Kume serogroup B) strains failed to react with any of the MAbs. All 52 remaining strains were Page serovar C (= Kume serogroup C), and all failed to react with MAb E5C12D10 but showed varying reaction patterns with the three other MAbs. Although the MAbs recognized four subdivisions within Kume serogroup C, these subdivisions differed from the four Kume C serovars. This panel of MAbs can be used to assign isolates of H. paragallinarum to either Page serovars or Kume serogroups. Although the subdivisions recognized by the MAbs within the Page serovar C strains do not correspond to the Kume serovars, they may be useful in epidemiological applications.     

Production, characterization and protective effect of monoclonal antibodies to Haemophilus paragallinarum serotype A.

Monoclonal antibodies (mAbs) to Haemophilus paragallinarum serotype A were obtained by fusion of murine myeloma cells (P3-X63-Ag8-U1) and spleen cells from BALB/c mice immunized with whole cells of strain 221. Enzyme linked immunosorbent assay with whole cells was used to show that the monoclonal antibodies are specific for serotype A of H. paragallinarum. Four monoclonal antibodies indicated hemagglutination-inhibition (HAI) activity against serotype A; their titers were 10(4)-10(5). By western blotting, two of these monoclonal antibodies reacted with a protein of molecular weight 39,000. Chickens treated with mAbs possessing HAI activity survived without clinical signs of infection. No challenge strain was isolated from these chickens, indicating that four mAbs with HAI activity suppressed growth of the challenge strain in the nasal cavity, whereas mAbs without HAI activity showed no passive protective effect. These results demonstrated that HI antibodies contributed to protection, and strongly suggest that hemagglutinin (HA) antigen, especially the epitopes which were recognized by these mAbs are important for protective immunity in chickens.     

A monoclonal antibody-blocking enzyme-linked immunosorbent assay for the detection of serovar-specific antibodies to Haemophilus paragallinarum

Two monoclonal antibody-blocking enzyme-linked immunosorbent assays (B-ELISAs) were developed to detect serovar-specific antibodies to Haemophilus paragallinarum. One assay detected antibodies against serovar A and the other antibodies against serovar C. The assays were evaluated with sera derived from disease-free chickens as well as chickens experimentally immunized and/or challenged with H. paragallinarum strains 0083 (serovar A), Modesto (serovar C), or HP31 (serovar C). When tested with 440 negative sera (170 from a specific-pathogen-free and 30 from each of nine commercial layer flocks), both tests gave only a single false-positive reaction. The use of the B-ELISAs with the experimentally produced sera showed the assays to be serovar specific. With the exception of one serum, the serovar A B-ELISA detected antibodies only in the chickens vaccinated with 0083. Similarly, with the exception of one serum, the serovar C B-ELISA detected antibodies only in those chickens vaccinated with Modesto or those chickens challenged with HP31. Overall, the serovar A B-ELISA had a specificity of 99.7% and a sensitivity of 78.7%, whereas the serovar C B-ELISA had a specificity of 99.8% and a sensitivity of 64.7%.     

Opsonic effect of monoclonal antibodies against Leptospira interrogans serovar copenhageni

The opsonic effect of monoclonal antibodies (McAbs) against Leptospira interrogans serovar copenhageni strain Shibaura was examined in vitro using radiolabeled organisms and mouse peritoneal exudate macrophages. Four IgG McAbs (all IgG3) and 2 IgM McAbs were used, all showed different reactivities and were bound to homologous lipopolysaccharide (LPS). IgG3 McAbs at the sub-agglutinating concentration were opsonic, but the degree of the opsonic effect varied among IgG3 McAbs. Opsonization with IgG3 McAbs at the concentration showing the same level of ELISA binding showed that the highest opsonic effect was exerted by Sw-6 antibody. The other IgG3 McAbs showed a similar but lower opsonic effect. IgM McAbs, which were not opsonic at the sub-agglutinating concentration even in the presence of complement, showed opsonic effect at the agglutinating concentration.     

Characterization of monoclonal antibodies against fowl poxvirus

Vaccines for the prevention of fowl pox in chickens and turkeys have been available for more than five decades. However, in recent years outbreaks have occurred in several previously vaccinated chicken flocks. Presumably, fowl poxviruses (FPVs) antigenically different from the attenuated vaccine strains are responsible for such occurrences. In support of this concept, we previously detected minor antigenic changes in field isolates based on comparative immunoblotting with polyclonal anti-FPV serum. Realizing the need for antibodies specific against the dominant antigens of FPV, monoclonal antibodies (MAbs) were produced by immunizing mice with either a field strain of FPV or a pigeon poxvirus, currently used for vaccination. Three hybridoma clones producing MAbs reacting with a specific FPV protein were selected from a total of 83 clones. In immunoblots, two of the MAbs, P1D9 and P2H10, recognized an antigen with an apparent molecular weight varying from 39 to 46 kD, depending on the FPV strain. The third MAb, P2D4, reacted with an approximately 80-kD protein, regardless of which FPV isolate was tested. Immunofluorescent staining with P1D9 and P2D4 revealed that these MAbs react with intracytoplasmic antigens in FPV-infected cells.     

Characterization of monoclonal antibodies directed against swine leukocytes

Hybridomas were produced from fusions of the SP2/0 mouse myeloma with splenic cells from: 1) an outbred Sprague Dawley rat immunized with swine peripheral blood mononuclear (PBM) cells; 2) a (CBA/NDub X BALB/c Dub) F1 mouse immunized with concanavalin A (Con A) activated swine PBM cells and 3) a (BALB/c Dub X C3H/He Dub) F1 mouse immunized with swine thymocytes. The resulting supernatants were screened by a microcytotoxicity assay for activity against swine PBM cells. Four hybridomas (MSA1, MSA2, MSA3 and MSA4) were selected, cloned and characterized by their cell reactivity and effect on mitogenic assays. MSA1 and MSA2 belong to the rat IgG2b subclass. MSA3 and MSA4 are of the mouse IgG2a subclass. These monoclonal antibodies reacted in the following manner: MSA1 with monocytes, granulocytes, red blood cells and bone marrow cells; MSA2 with subset of T cells; MSA3 with B cells and subsets of T cells and monocytes (class II molecule) and MSA4, a pan-T cell reagent (E-rosette receptor). The involvement of the various cell types reactive to the different monoclonal antibodies in the mitogenic response of swine PBM cells to Con A, phytohemagglutinin (PHA) or pokeweed mitogen (PWM) was investigated by cellular depletion with monoclonal antibody plus complement. Cellular depletion of PBM cells with the following monoclonal antibodies plus complement treatment resulted in: MSA1, almost total reduction in the mitogenic response to low doses of Con A or PWM; MSA2, partial reduction in the proliferative responses to any concentration of Con A, PHA or PWM; MSA3, partial reduction in proliferative responses to low concentrations of Con A or PWM and 4) MSA4, total elimination of any proliferative response to Con A, PHA or PWM.     

副猪嗜血杆菌OMP5单克隆抗体的制备与鉴定

用副猪嗜血杆菌（HPS）血清5型灭活菌液免疫BALB/c小鼠,取其脾细胞与Sp2/0骨髓瘤细胞融合,以HPS外膜蛋白5（OMP5）为抗原,经间接ELISA法筛选阳性融合孔,并用有限稀释法对筛选的阳性孔进行亚克隆,获得2株分泌抗HPS OMP5单克隆抗体的杂交瘤细胞株1E2和8D9;间接ELISA法效价检测,1E2和8D9细胞培养上清抗体效价均为1∶2 560,纯化后腹水抗体效价分别为1∶204 800和1∶51 200;抗体型别鉴定,1E2和8D9分别分泌IgG2b、IgG2a型抗体;特异性检测表明,2株单抗均能特异识别OMP5。将2株杂交瘤细胞反复冻融3次复苏后仍能稳定分泌抗体。2株抗HPS OMP5单克隆抗体的成功制备,将为HPS感染快速检测方法的建立以及检测试剂的研制奠定基础。     

重组牛朊蛋白特异性抗体的制备及鉴定

以原核表达后经纯化的重组牛朊蛋白为免疫原,免疫prnp-/-基因敲除鼠。4次免疫后,利用淋巴细胞杂交瘤技术,取脾细胞和SP2/0骨髓瘤细胞进行细胞融合。间接ELISA方法筛选出阳性杂交瘤细胞,采用有限稀释法对阳性杂交瘤细胞进行3次克隆,用间接ELISA筛选出了稳定分泌针对牛重组朊蛋白特异性单克隆抗体的杂交瘤细胞株,命名为5C9D6。Western blotting鉴定结果表明,5C9D6均能特异性识别重组牛朊蛋白、健康牛、BALB/c脑组织匀浆中的PrPc,不识别prnp-/-基因敲除鼠脑组织匀浆液。本试验制备了可与牛、BALB/c鼠反应的单克隆抗体,同时也为牛海绵状脑病的研究及其诊断方法的建立奠定了基础。     

Outer-membrane proteins of Haemophilus paragallinarum

The outer-membrane protein (OMP) profiles of four isolates of Haemophilus paragallinarum (0083, 0222, Modesto, and HP31) were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. OMPs were isolated by sonic disruption followed by differential centrifugation and selective solubilization in Triton X-100. Although the isolates had similar profiles overall, two distinct OMP profile types, based on the variable molecular weight of a protein termed OMP C (39,000 or 38,000), were found. In addition, OMP C was found to be a heat-modifiable protein--being either absent or present in only minor amounts if the preparations were not heated at 100 C. Major and minor OMPs, some common to all four isolates, were recognized in immunoblots by an immune serum to isolate HP31.     

Further characterization of Haemophilus paragallinarum and Haemophilus avium

A total of 60 isolates of Haemophilus spp. from chickens, including four reference strains of H. paragallinarum and one of H. avium, were examined for their physiological and biochemical properties. The isolates could be placed into two groups. One group was identified as H. paragallinarum and consisted of 43 isolates including the four reference strains of H. paragallinarum. The other group was identified as H. avium and consisted of 17 isolates including the reference strain of H. avium. H. avium can be differentiated from H. paragallinarum by its possession of the enzymes catalase and alpha-glucosidase, capacity to grow in air, production of acid from galactose, and by the fact that its growth is not improved by the addition of chicken serum. In addition, the majority of H. avium isolates, unlike H. paragallinarum, possess a yellow pigment and produce acid from trehalose.     

Characterization of monoclonal antibodies against Actinobacillus pleuropneumoniae serotype 5.

Two monoclonal antibodies against Actinobacillus pleuropneumoniae serotype 5, designated as 5MAb-1 and 5MAb-6, were characterized. Enzyme-linked immunosorbent assay-inhibition tests with whole-cell antigens obtained from strains of serotype 1 through 12 of A pleuropneumoniae revealed that 5 MAb-1 bound to only serotype-5 strains. The epitope recognized by 5MAb-1 was a carbohydrate that was sensitive to periodate oxidation and resided on the structure of beta-1,6-linked D-galactose in an O-antigen polysaccharide of serotype-5 lipopolysaccharide. Analysis of these results revealed that the O-antigen polysaccharide of lipopolysaccharide was 1 of the antigenic determinants responsible for the serotype specificity of A pleuropneumoniae. On the other hand, 5MAb-6 reacted with strains of serotype 1 through 10 in varying degrees and its epitope was located on polypeptides sensitive to proteinase K. In an immunoblotting analysis, 5MAb-6 reacted with 2 polypeptide bands, with molecular weights of approximately 41,500 and 28,000, in the outer membrane protein-rich fraction obtained from strains of serotype 1 through 10. These results indicated that outer membrane proteins from several serotype strains of A pleuropneumoniae possessed common antigenic determinants.