Histophilus somni Survives in Bovine Macrophages by Interfering with Phagosome-Lysosome Fusion but Requires IbpA for Optimal Serum Resistance.

Histophilus somni is capable of intracellular survival within professional phagocytic cells, but the mechanism of survival is not understood. The Fic motif within the direct repeat (DR1)/DR2 domains of the IbpA fibrillary network protein of H. somni is cytotoxic to epithelial and phagocytic cells, which may interfere with the bactericidal activity of these cells. To determine the contribution of IbpA and Fic to resistance to host defenses, H. somni strains and mutants that lacked all or a region of ibpA (including the DR1/DR2 regions) were tested for survival in bovine monocytic cells and for serum susceptibility. An H. somni mutant lacking IbpA, but not the DR1/DR2 region within ibpA, was more susceptible to killing by antiserum than the parent, indicating that the entire protein was associated with serum resistance. H. somni strains expressing IbpA replicated in bovine monocytes for at least 72 h and were toxic for these cells. Virulent strain 2336 mutants lacking the entire ibpA gene or both DR1 and DR2 were not toxic to the monocytes but still survived within the monocytes for at least 72 h. Monitoring of intracellular trafficking of H. somni with monoclonal antibodies to phagosomal markers indicated that the early phagosomal marker early endosome antigen 1 colocalized with all isolates tested, but only strains that could survive intracellularly did not colocalize with the late lysosomal marker lysosome-associated membrane protein 2 and prevented the acidification of phagosomes. These results indicated that virulent isolates of H. somni were capable of surviving within phagocytic cells through interference in phagosome-lysosome maturation. Therefore, H. somni may be considered a permissive intracellular pathogen.

Isolation and characterization of flagellar filaments from Bacillus cereus ATCC 14579.

Isolated flagellar filaments from the type strain of Bacillus cereus, ATCC 14579, were shown to consist of 34, 32 and 31 kDa proteins in similar proportions as judged by band intensities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequences of these three proteins of strain ATCC 14579 were identical with the deduced sequences of three flagellin genes BC1657, BC1658 and BC1659 in the whole genome sequence. Strain ATCC 14579 was classified into serotype T2 by a flagellar serotyping scheme for B. cereus strains that are untypeable into known flagellar serotypes H1 to H23. Flagellar filaments from a reference strain of serotype T2 contained two protein bands at 34 and 32 kDa, but a single protein band at 39 kDa was detected in flagellar filaments of a reference strain of serotype H1. Two murine monoclonal antibodies, 1A5 and 2A5, which recognize both the 34 and 32 kDa flagellins and a single flagellin of 32 kDa, respectively, were specifically reactive with B. cereus strains ATCC 14579 and serotype T2 in whole-cell ELISA and bacterial motility inhibition tests. In immunoelectron microscopy with monoclonal antibodies 1A5 and 2A5, colloidal gold spheres were shown to localize almost evenly over the entire part of flagellar filaments. Since strain ATCC 14579, and presumably strain serotype T2, are unusual among B. cereus strains in possessing multiple genes that encode flagellin subunits, a possible unique mechanism may contribute to assembly of multiple flagellin subunits into the filament over its entire length.

Characterization of discontinuous epitope of prion protein recognized by the monoclonal antibody T2.

The anti-prion protein (PrP) monoclonal antibody T2 has previously been prepared using PrP-knockout mice immunized with mouse recombinant PrP residues 121-231, however its interaction mechanism to PrP antigen has not been cleared. Here we identified and characterized the epitope of T2 antibody. The competitive ELISA with 20-mer synthetic peptides derived from PrP121-231 showed that T2 antibody had no affinity for these peptides. The analysis with deletion mutants of PrP revealed that 10 amino acids in the N terminus and 66 amino acids in the C terminus of PrP121-231 were necessary for reactivity with T2. Two far regions are necessary for complete affinity of the T2 antibody for PrP; either region alone is not sufficient to retain the affinity. The epitope recognized by T2 antibody is discontinuous and conformational. We examined the effect of disulfide bond and salt bridges. Alkylation of cysteine residues in C terminus of PrP121-231, which breaks a disulfide bond and disrupts the structure, had diminished the reactivity. Mutations induced in the PrP121-231 to break the disulfide bond or salt bridges, markedly had reduced the reactivity with T2 antibody. It suggests that T2 antibody recognized the structure maintained by the disulfide bond and salt bridges.

Virulence attributes of Histophilus somni with a deletion mutation in the ibpA gene.

Histophilus somni strain 2336 contains a large open reading frame of 12,285-bp length, ibpA, encoding the immunoglobulin binding protein (IbpA) which is associated with H. somni serum resistance. To elucidate other functions of the strain 2336 IbpA protein, an ibpA isogenic mutant, 2336.A1, was created by replacement of an 11.6-kb ibpA sequence with a kanamycin resistant gene cassette. Both the mutant strain 2336.A1 and the wild-type strain 2336 adhered at similar levels to bovine turbinate cells, bovine endometrial epithelial cells and bovine macrophage-like FBM-17 cells. However, a remarkable cytotoxic effect associated with disruption of actin filaments was observed in FBM-17 cells infected with strain 2336 but not with strain 2336.A1. Cytotoxicity was also noted with the wild type but not the mutant in assays with murine J774.1 macrophage cells and bovine primary monocytes. Inhibition of phagocytosis of microspheres was found in assays with murine J774.1 cells and bovine primary monocytes infected with strain 2336 but not with strain 2336.A1. These results indicate that H. somni IbpA protein inhibits phagocytic activity of macrophages and monocytes, probably by disruption of actin filament structure.

Lactoferrin induces cell surface retention of prion protein and inhibits prion accumulation.

Prion diseases are fatal neurodegenerative disorders, and the conformational conversion of normal cellular prion protein (PrP(C)) into its pathogenic, amyloidogenic isoform (PrP(Sc)) is the essential event in the pathogenesis of these diseases. Lactoferrin (LF) is a cationic iron-binding glycoprotein belonging to the transferrin (TF) family, which accumulates in the amyloid deposits in the brain in neurodegenerative disorders, such as Alzheimer's disease and Pick's disease. In the present study, we have examined the effects of LF on PrP(Sc) formation by using cell culture models. Bovine LF inhibited PrP(Sc) accumulation in scrapie-infected cells in a time- and dose-dependent manner, whereas TF was not inhibitory. Bioassays of LF-treated cells demonstrated prolonged incubation periods compared with non-treated cells indicating a reduction of prion infectivity. LF mediated the cell surface retention of PrP(C) by diminishing its internalization and was capable of interacting with PrP(C) in addition to PrP(Sc). Furthermore, LF partially inhibited the formation of protease-resistant PrP as determined by the protein misfolding cyclic amplification assay. Our results suggest that LF has multifunctional antiprion activities.

A monoclonal antibody (1D12) defines novel distribution patterns of prion protein (PrP) as granules in nucleus.

A monoclonal antibody (mAb) panel to bovine prion protein (PrP) was studied by immunoblotting and immunohistochemistry for scrapie and bovine spongiform encephalopathy. A mAb panel recognized both normal (PrP(C)) and abnormal (PrP(Sc)) isoforms of PrP in murine, ovine and bovine brain tissues. Interestingly, an anti-bovine PrP mAb, 1D12, prepared by immunizing PrP gene-knockout mice with a synthetic polypeptides corresponding to codons 153-166 of the bovine PrP gene showed novel patterns of reactivity for prion-uninfected neuronal cells. An enzyme-linked immunosorbent assay-mapping of the mAb epitopes resulted in a reaction of monoclonal 1D12 to YEDRY and M corresponding to amino acids 156-160 and 165 of bovine PrP. Several patterns of bovine PrP(C) distribution in PrP-deficient neuronal cells (HpL3-4) transfected with bovine PrP were observed after different fixation methods. Stained cell surface was observed after formalin fixation by immunofluorescent assay of 1D12 with confocal microscopy, whereas granules in nucleus were stained after acetone fixation. No reactivity in the nucleus was observed to HpL3-4, or HpL3-4mPrP cells expressing mouse PrP. This is the first paper that has reported the detection of the PrP(C) at both cell surface and nuclei of prion-uninfected cell line.

An advantageous method utilizing new homogenizing device BioMasher and a sensitive ELISA to detect bovine spongiform encephalopathy accurately in brain tissue.

A new screening method was developed to detect bovine spongiform encephalopathy (BSE). This method is advantageous because it has a simpler and safer protocol than commercial kits. A new device was developed for this method; it was named the BioMasher, to homogenize brain tissue by passing it through a porous rigid polypropylene filter. In this system, a purification step was eliminated in the sample preparation. Thus, the time needed for sample pretreatment is substantially shortened, and the risk of infection during sample processing is effectively reduced. Monoclonal antibodies to prion protein were created and used to construct a sensitive sandwich enzyme-linked immunosorbent assay system. The sensitivity of this assay kit using frozen BSE-positive brain is comparable or more sensitive than commercial kits. Moreover, the detection sensitivity for deteriorated samples, which were kept at 37 degrees C for 1 day, is 10- to 30-fold more sensitive than a commercial kit.

A Predominant Clonal Thromboembolic Meningoencephalitis Group of Histophilus somni Assigned by Major Outer Membrane Protein Gene Sequencing and Pulsed-Field Gel Electrophoresis.

Histophilus somni, a member of the family Pasteurellaceae, causes a variety of diseases, including thromboembolic meningoencephalitis (TEME) and respiratory diseases, which result in considerable economic losses to the cattle and sheep industries. In this study, 132 chronologically diverse isolates from cattle in Japan and 68 isolates from other countries comprising 49 from cattle and 19 from sheep were characterized using major outer membrane protein (MOMP) gene sequence and pulsed-field gel electrophoresis (PFGE) analyses. The H. somni isolates formed nine MOMP genetic clades (clade Ia, Ib, and II-VIII) and 10 PFGE clusters (HS1-HS10). Except for two (1.0%), all isolates fell into one of the nine MOMP genetic clades, while 62 (31.0%) isolates belonged to no PFGE cluster. MOMP genetic clade Ia and PFGE cluster HS1 were the major groups, and all HS1 isolates possessed the clade Ia MOMP gene. Isolates from TEME cases were significantly associated with these major groups (chi-square test, p < 0.0001), as 88.2% of the TEME isolates belonged to MOMP genetic clade Ia and PFGE cluster HS1, which formed the most predominant clonal group. After an inactivated vaccine using an HS1 strain with the clade Ia MOMP gene was introduced in Japan in late 1989, the number of TEME cases and isolates assigned into the clonal group decreased simultaneously. However, the proportions of clade Ia and cluster HS1 isolates from TEME cases remained high after 1990. These results suggest a close association of TEME with PFGE cluster HS1 and MOMP genetic clade Ia, and imply the presence of factors or characteristics commonly possessed by those strains that contribute to the development of TEME.

Detection of bovine mastitis pathogens by loop-mediated isothermal amplification and an electrochemical DNA chip.

Bovine mastitis causes significant economic losses in the dairy industry. Effective prevention of bovine mastitis requires an understanding of the infection status of a pathogenic microorganism in a herd that has not yet shown clinical signs of mastitis and appropriate treatment specific for the pathogenic microorganism. However, bacterial identification by culture has drawbacks in that the sensitivity may be low and the procedure can be complex. In this study, we developed a genetic detection method to identify mastitis pathogens using a simple and highly sensitive electrochemical DNA chip which can specifically detect bacterial DNA in milk specimens. First, we selected microorganisms belonging to 12 families and/or genera associated with mastitis for which testing should be performed. Next, we optimized the conditions for amplifying microorganism DNA by loop-mediated isothermal amplification (LAMP) using 32 primers and the use of a DNA chip capable of measuring all pathogens simultaneously. Sample detection could be completed in just a few hours using this method. Comparison of the results obtained with our DNA chip method and those obtained by bacterial culture verified that when the culture method was set to 100%, the total positive concordance rate of the DNA chip was 85.0% and the total negative concordance rate was 86.9%. Furthermore, the proposed method allows both rapid and highly sensitive detection of mastitis pathogens. We believe that this method will contribute to the development of an effective mastitis control program.

Molecular characterization of the major outer membrane protein of Haemophilus somnus.

The major outer membrane protein (MOMP) of Haemophilus somnus shows antigenic and molecular mass diversity that forms the basis of a preliminary grouping system for H. somnus strains. In this study, the gene encoding MOMP of H. somnus strain 8025 was cloned in three overlapping fragments by PCR techniques, and then sequenced. The gene consists of a 1164-bp open reading frame encoding a deduced 380-amino acid protein with a 19-amino acid signal sequence, giving a mature protein with a calculated molecular mass of 39,913 Da. Significant homology was found between MOMP and porin protein sequences of bacteria in Pasteurellaceae species. When expressed in Escherichia coli, the protein from the MOMP gene directed by the T7 promoter was identical in size (approximately 40 kDa) to native MOMP and reacted with MOMP-specific antibodies. Comparisons of the MOMP gene sequences from six unrelated strains of H. somnus to that of strain 8025 revealed that the genes of three MOMP type 1 strains were highly conserved with that of strain 8025 in length and sequence. However, two MOMP type 3c strains and one MOMP type 3a strain differed markedly from the MOMP of strain 8025 in their 3'-terminal halves. Their deduced MOMP amino acid sequences differed in sequence (3c, 80.5 and 82.7% identity; 3a, 62.4% identity) and in length (3c, 384 and 376; 3a, 316), indicating that the molecular differences are the basis of antigenicity and molecular mass differences of H. somnus MOMP. In the predicted MOMP secondary structure, the variable sequences primarily mapped to putative surface-exposed loops, and a variable and surface-exposed epitope of MOMP-specific antibody was identified in the seventh-largest loop. These findings are useful for understanding the structural and immunological characteristics of H. somnus.

Differentiation between bovine and ovine strains of Histophilus somni based on the sequences of 16S rDNA and rpoB gene.

Nucleotide sequences of 16S rDNA and rpoB gene of 25 bovine and 6 ovine Histophilus somni strains were determined to detect subtle differences between the host animal species. The 1465 nucleotide residues of the 16S rDNA exhibited levels of sequence similarities of 99.4% or more. The high sequence similarity of the 16S rDNA of recently described species H. somni was confirmed in the 31 strains from cattle and sheep. These results suggested that the intra-specific diversity of 16S rDNA was limited in bovine and ovine strains of H. somni. The specific association of strains was also observed in the 311 bp region of rpoB gene which sequence similarities were 98.6% or more. However, the phylogenetic tree analysis of the rpoB gene showed that the ovine strains appeared to form a subgroup recovered in 70% of the bootstrap trees. In the 311 bp region of the ovine strains, a HincII restriction endonuclease site was detected. The PCR-amplified rpoB DNA of 46 bovine and 20 ovine H. somni strains were examined for the digestion with HincII. As the results, 17 strains of ovine strains were cleaved by the enzyme but none of the bovine strains appeared to possess the restriction site. The restriction enzyme analysis of rpoB gene may be useful to differentiate ovine strains from bovine strains of H. somni.

Improved rapid and efficient method for Staphylococcus aureus DNA extraction from milk for identification of mastitis pathogens.

A rapid and efficient DNA extraction method was developed for detecting mastitis pathogens in milk. The first critical step involved cell wall disruption by bead-beating, as physical disruption using beads was more effective for DNA extraction from Gram-positive bacteria, such as Staphylococcus aureus, than enzymatic disruption using proteinase K. The second critical step involves the use of acetic acid and ammonium sulfate in the purification process, as these reagents effectively and efficiently remove the lipids and proteins in milk. Using these methods, DNA suitable for loop-mediated isothermal amplification was obtained within 30 min. Also, the rapid and sensitive detection of S. aureus in milk was possible at levels as low as 200 cfu/ml.

Effect of tissue deterioration on postmortem BSE diagnosis by immunobiochemical detection of an abnormal isoform of prion protein.

Surveillance for bovine spongiform encephalopathy (BSE) in fallen stock in Japan is conducted with a commercial enzyme-linked immunosorbent assay (ELISA) for mass screening, with Western blotting (WB) and immunohistochemistry performed for confirmation of the ELISA. All tests are based on immunological detection of an abnormal isoform of the prion protein (PrP(Sc)) in brain tissues, which have sometimes deteriorated by the time samples from fallen stock reach a diagnostic laboratory. To evaluate BSE surveillance procedures for fallen stock, we examined PrP(Sc) detection from artificially deteriorated BSE-affected bovine brain tissues with a commercial ELISA kit and compared the results with those of WB. The optical density (OD) values of the ELISA decreased with advancing deterioration of the tissues, whereas no reduction in the signal for PrP(Sc) was observed in WB, even when performed after 4 days of incubation at 37 degrees C. The progressive decrease in the OD values in the ELISA appear to be caused by a partial loss of the N-terminal moiety of PrP(Sc) due to digestion by endogeneous and/or contaminated microbial enzymes, and by the presence of ELISA inhibitors that are generated in deteriorated tissues. These results suggest that WB is the most reliable test for fallen stock, especially for cattle brains within decaying carcasses.

Isolation and characterization of flagellar filament from zoospores of Dermatophilus congolensis.

Highly motile zoospores from Dermatophilus congolensis bovine isolates from clinical dermatophilosis in Japan were obtained by culturing at 27°C in an ambient atmosphere on heart infusion agar supplemented with 5% defibrinated sheep blood for 72h or in heart infusion broth for 48h with gentle shaking. After vigorous mechanical agitation of the zoospore suspension, the flagellar filaments detached from motile zoospores and were isolated in the clear gelatinous part of the final pellet by differential centrifugation. Typical morphology of a flagellar filament, with a width of approximately 15nm, was observed in the isolated flagellar filament by electron microscopy. A single major protein (flagellin) band with an apparent molecular mass of 35kDa was detected in the flagellar filament of D. congolensis strain AM-1 and that of 33kDa was detected in strain IT-2 by SDS-PAGE. In immunoblot analysis of whole-cell proteins from seven isolates of D. congolensis, antiserum to strain AM-1 zoospores reacted with the 35-kDa antigen band of strain AM-1, but not with any antigen band of other strains in a similar molecular mass range. In contrast, antiserum to strain IT-2 zoospores reacted with antigen bands at 33kDa from six strains, except strain AM-1. Similar strain-specific reactions of these anti-zoospore sera with isolated flagellar filaments from strains AM-1 and IT-2 were confirmed by immunoblot, indicating the presence of antigenic variations of flagellins of D. congolensis zoospores.

Mutations in the major outer membrane protein gene from Histophilus somni by an allelic exchange method.

Functional roles of the major outer membrane protein (MOMP) gene from the bovine pathogen Histophilus somni have remained to be elucidated due to lack of mutagenesis methods easily applicable to this gene. In this study, the direct use of PCR-amplified mutated DNA flanking by an antibiotic selection marker for transformation of H. somni was applied to accomplish the site-directed mutagenesis via homologous recombination in H. somni non-pathogenic strain 129Pt and pathogenic strain 2336. A protocol for unmarking the antibiotic resistance gene from the created MOMP mutant by using a temperature-sensitive plasmid vector was also established. In both strains, no significant phenotypic difference was observed between the wild-type strain and its isogenic mutant expressing the exchanged MOMP in growth in broth medium and antibiotic susceptibility. However, the mutant 129Pt expressing MOMP from strain 2336 was significantly more susceptible to bacterial killing by fresh normal bovine serum than its wild-type parent strain. Serum susceptibility of the mutant 2336 expressing MOMP from strain 129Pt was significantly lower than its wild-type parent strain although the susceptibility difference was considerably less than that found between the 129Pt wild-type and MOMP mutant strains. From further assays using MOMP mutants that express chimeric MOMP consisting of the amino-terminal part that contains loops L1-L3 from one strain and the carboxyl-terminal part that contains loops L4-L8 from another strain, the C-terminal part of MOMP was found to affect serum susceptibility of H. somni.

Molecular-based identification of yeasts isolated from bovine clinical mastitis in Japan.

This study analyzed molecular-based identification of yeasts that associated with bovine clinical mastitis in Japan. Over 3,200 quarter milk samples from Holstein dairy cows collected in 2011 on Hokkaido and Honshu islands were examined. Yeast isolates were characterized by polymerase chain reaction amplification and sequencing of the D1/D2 region of the 26S rDNA. Molecular characterization confirmed that Candida spp. and Pichia spp. were most frequently isolated species. Our molecular analysis of mastitic milk samples demonstrated the prevalence of Pichia kudriavzevii(22/58) and Candida tropicalis(14/58). In addition, we demonstrated that molecular analysis of the D1/D2 region of the 26S rDNA is a rapid and reliable method for identifying clinically significant yeasts in dairy hygiene, including potentially new or emerging pathogenic species.

Genetics and infectivity of H5N1 highly pathogenic avian influenza viruses isolated from chickens and wild birds in Japan during 2010-11.

Outbreaks of H5N1 subtype highly pathogenic avian influenza virus (HPAIV) were recorded in chickens, domesticated birds and wild birds throughout Japan from November 2010 to March 2011. Genetic analysis of the Japanese isolates indicated that all gene segments, except the PA gene, were closely related to Japanese wild bird isolates in 2008 and belonged to clade 2.3.2.1 classified by the WHO/OIE/FAO H5N1 Evolution Working Group. Direct ancestors of the PA gene segment of all Japanese viruses analyzed in this study can be found in wild bird strains of several subtypes other than H5N1 isolated between 2007 and 2009. The PA gene of these wild bird isolates share a common ancestor with H5N1 HPAIVs belonging to clades 2.5, 7 and 9, indicating that wild birds were involved in the emergence of the current reassortant 2.3.2.1 viruses. To determine how viruses were maintained in the wild bird population, two isolates derived from chickens (A/chicken/Shimane/1/2010, Ck10 and A/chicken/Miyazaki/S4/2011, CkS411) and one from a wild bird (A/mandarin duck/Miyazaki/22M-765/2011, MandarinD11) were compared in their ability to infect and be transmitted to chickens. There was a significant difference in the survival of chickens that were infected with 10(6)EID(50) of CkS411 compared to those with MandarinD11 and the transmission efficiency of CkS411 was greater than the other viruses. The increased titer of CkS411 excreted from infected chickens contributed to the improved transmission rates. It was considered that reduced virus excretion and transmission of MandarinD11 could have been due to adaptation of the virus in wild birds.

Salt bridges in prion proteins are necessary for high-affinity binding to the monoclonal antibody T2.

We studied the role of the 2 salt bridges (Asp143-Arg147 and Asp146-Arg150) in helix 1 of mouse prion protein (PrP) on the formation of the complex between PrP and the monoclonal antibody T2. We introduced 6 charge-changing mutations to the amino acid residues associated with the salt bridges. Analysis of the circular dichroism spectra of the mutant PrPs showed that the salt bridge mutations did not change the secondary structures. We analyzed the kinetics of the association and dissociation of the PrPs with the T2 antibody. The results showed that the association kinetics were not significantly different among the variants except Arg150Lys, while the dissociation rate of the neutralized-charge variants was 2 orders of magnitude higher than that of the wild type. These results indicate that salt bridges make the interaction of PrP with T2 tighter by slowing down dissociation.

A novel anti-prion protein monoclonal antibody and its single-chain fragment variable derivative with ability to inhibit abnormal prion protein accumulation in cultured cells.

mAbs T1 and T2 were established by immunizing PrP gene ablated mice with recombinant MoPrP of residues 121-231. Both mAbs were cross-reactive with PrP from hamster, sheep, cattle and deer. A linear epitope of mAb T1 was identified at residues 137-143 of MoPrP and buried in PrP(C) expressed on the cell surface. mAb T1 showed no inhibitory effect on accumulation of PrP(Sc) in cultured scrapie-infected neuroblastoma (ScN2a) cells. In contrast, mAb T2 recognized a discontinuous epitope ranged on, or structured by, residues 132-217 and this epitope was exposed on the cell surface PrP(C). mAb T2 showed an excellent inhibitory effect on PrP(Sc) accumulation in vitro at a 50% inhibitory concentration of 0.02 microg/ml (0.14 nM). The scFv form of mAb T2 (scFv T2) was secreted in neuroblastoma (N2a58) cell cultures by transfection through eukaryotic secretion vector. Coculturing of ScN2a cells with scFv T2-producing N2a58 cells induced a clear inhibitory effect on PrP(Sc) accumulation, suggesting that scFv T2 could potentially be an immunotherapeutic tool for prion diseases by inhibition of PrP(Sc) accumulation.

Protection of mice against H. somni septicemia by vaccination with recombinant immunoglobulin binding protein subunits.

Histophilus somni causes bovine pneumonia as well as septicemia and its sequelae but mechanisms of virulence and protective immunity are poorly understood. Since surface immunoglobulin binding proteins are virulence factors, we addressed their role as protective antigens in a mouse model of H. somni septicemia. Immunoglobulin binding protein A (IbpA), has homology to Bordetella pertussis filamentous hemagglutinin and other large bacterial exoproteins. IbpA is a major surface antigen encoded by the ibpA gene with many domains that may be important in pathogenesis and immune protection. Three IbpA recombinant protein subunits, IbpA3, IbpA5 and IbpADR2 were chosen for study because of putative functional domains and motifs. These recombinant GST fusion subunit proteins were compared with GST (negative control), formalin-killed H. somni (commercial vaccine control), live H. somni (to induce convalescent immunity) and H. somni culture supernatant (containing IbpA shed from the bacterial surface). In vaccination/challenge studies, both live H. somni (convalescent immunity) and supernatant protected equally but formalin-killed H. somni and GST did not protect against septicemia. The DR2 and A3 subunits protected moderately well and induced antibody responses against supernatant antigen and the homologous subunit in ELISA but not against whole cell antigens. Supernatant immunization protected better than the IbpA subunit antigens and induced high antibody activity against both whole cells and supernatant antigens. The results indicate that culture supernatant antigens or perhaps recombinant IbpA subunits may be useful in H. somni vaccines. These studies also provide insight into the contribution of IbpA domains to pathogenesis of H. somni septicemia.