Contribution of Hfq to gene regulation and virulence in Histophilus somni.

Histophilus somni is one of the predominant bacterial pathogens responsible for bovine respiratory and systemic diseases in cattle. Despite the identification of numerous H. somni virulence factors, little is known about the regulation of such factors. The post-transcriptional regulatory protein Hfq may play a crucial role in regulation of components that affect bacterial virulence. The contribution of Hfq to H. somni phenotype and virulence was investigated following creation of an hfq deletion mutant of H. somni strain 2336 (designated H. somni 2336Δhfq). A comparative analysis of the mutant to the wild-type strain was carried out by examining protein and carbohydrate phenotype, RNA sequence, intracellular survival in bovine monocytes, serum susceptibility, and virulence studies in mouse and calf models. H. somni 2336Δhfq exhibited a truncated lipooligosaccharide (LOS) structure, with loss of sialylation. The mutant demonstrated increased susceptibility to intracellular and serum-mediated killing compared to the wild-type strain. Transcriptomic analysis displayed significant differential expression of 832 upregulated genes and 809 downregulated genes in H. somni 2336Δhfq compared to H. somni strain 2336, including significant downregulation of lsgB and licA, which contribute to LOS oligosaccharide synthesis and sialylation. A substantial number of differentially expressed genes were associated with polysaccharide synthesis and other proteins that could influence virulence. The H. somni 2336Δhfq mutant strain was attenuated in a mouse septicemia model and somewhat attenuated in a calf intrabronchial challenge model. H. somni was recovered less frequently from nasopharyngeal swabs, endotracheal aspirates, and lung tissues of calves challenged with H. somni 2336Δhfq compared to the wild-type strain, and the percentage of abnormal lung tissue in calves challenged with H. somni 2336Δhfq was lower than in calves challenged with the wild-type strain. In conclusion, our results support that Hfq accounts for the regulation of H. somni virulence factors.

The role of uspE in virulence and biofilm formation by Histophilus somni.

UspE is a global regulator in Escherichia coli. To study the function of Histophilus somni uspE, strain 2336::TnuspE was identified from a bank of mutants generated with EZ::Tn5™ Tnp Transposome™ that were biofilm deficient. The 2336::TnuspE mutant was highly attenuated in mice, the electrophoretic profile of its lipooligosaccharide (LOS) indicated the LOS was truncated, and the mutant was significantly more serum-sensitive compared to the wildtype strain. In addition to forming a deficient biofilm, exopolysaccharide (EPS) production was also compromised, but the electrophoretic profile of outer membrane proteins was not altered. RNA sequence analysis revealed that the transcription levels of some stress response chaperones, transport proteins, and a large number of ribosomal protein genes in 2336::TnuspE were significantly differentially regulated compared to strain 2336. Therefore, uspE may differentially function in direct and indirect expression of H. somni genes, but its attenuation may be linked to poor biofilm formation and rapid clearance of the bacteria resulting from a compromised LOS structure. Our results support that uspE is a global stress regulatory gene in H. somni.

Histophilus somni: Antigenic and Genomic Changes Relevant to Bovine Respiratory Disease.

Histophilus somni is associated with several disease syndromes in cattle and plays an important role in the bovine respiratory disease complex. H somni isolates exhibit significant differences in terms of susceptibility to inactivation by normal serum corresponding to the general ability to cause clinical disease. Isolates possess a variety of virulence factors, and variation in virulence factor expression is well recognized and associated with antigenic differences. Sequencing of genes associated with known virulence factors has identified genetic variability between isolates. The antigenic and genomic differences represent significant challenges to the host immune system and are problematic for vaccine design.

Cattle Immunized with a Recombinant Subunit Vaccine Formulation Exhibits a Trend towards Protection against Histophilus somni Bacterial Challenge.

Histophilosis, a mucosal and septicemic infection of cattle is caused by the Gram negative pathogen Histophilus somni (H. somni). As existing vaccines against H. somni infection have shown to be of limited efficacy, we used a reverse vaccinology approach to identify new vaccine candidates. Three groups (B, C, D) of cattle were immunized with subunit vaccines and a control group (group A) was vaccinated with adjuvant alone. All four groups were challenged with H. somni. The results demonstrate that there was no significant difference in clinical signs, joint lesions, weight change or rectal temperature between any of the vaccinated groups (B,C,D) vs the control group A. However, the trend to protection was greatest for group C vaccinates. The group C vaccine was a pool of six recombinant proteins. Serum antibody responses determined using ELISA showed significantly higher titers for group C, with P values ranging from < 0.0148 to < 0.0002, than group A. Even though serum antibody titers in group B (5 out of 6 antigens) and group D were significantly higher compared to group A, they exerted less of a trend towards protection. In conclusion, the vaccine used in group C exhibits a trend towards protective immunity in cattle and would be a good candidate for further analysis to determine which proteins were responsible for the trend towards protection.

Histophilus somni Stimulates Expression of Antiviral Proteins and Inhibits BRSV Replication in Bovine Respiratory Epithelial Cells.

Our previous studies showed that bovine respiratory syncytial virus (BRSV) followed by Histophilus somni causes more severe bovine respiratory disease and a more permeable alveolar barrier in vitro than either agent alone. However, microarray analysis revealed the treatment of bovine alveolar type 2 (BAT2) epithelial cells with H. somni concentrated culture supernatant (CCS) stimulated up-regulation of four antiviral protein genes as compared with BRSV infection or dual treatment. This suggested that inhibition of viral infection, rather than synergy, may occur if the bacterial infection occurred before the viral infection. Viperin (or radical S-adenosyl methionine domain containing 2--RSAD2) and ISG15 (IFN-stimulated gene 15--ubiquitin-like modifier) were most up-regulated. CCS dose and time course for up-regulation of viperin protein levels were determined in treated bovine turbinate (BT) upper respiratory cells and BAT2 lower respiratory cells by Western blotting. Treatment of BAT2 cells with H. somni culture supernatant before BRSV infection dramatically reduced viral replication as determined by qRT PCR, supporting the hypothesis that the bacterial infection may inhibit viral infection. Studies of the role of the two known H. somni cytotoxins showed that viperin protein expression was induced by endotoxin (lipooligosaccharide) but not by IbpA, which mediates alveolar permeability and H. somni invasion. A naturally occurring IbpA negative asymptomatic carrier strain of H. somni (129Pt) does not cause BAT2 cell retraction or permeability of alveolar cell monolayers, so lacks virulence in vitro. To investigate initial steps of pathogenesis, we showed that strain 129Pt attached to BT cells and induced a strong viperin response in vitro. Thus colonization of the bovine upper respiratory tract with an asymptomatic carrier strain lacking virulence may decrease viral infection and the subsequent enhancement of bacterial respiratory infection in vivo.

Reverse vaccinology as an approach for developing Histophilus somni vaccine candidates.

Histophilosis of cattle is caused by the Gram negative bacterial pathogen Histophilus somni (H. somni) which is also associated with the bovine respiratory disease (BRD) complex. Existing vaccines for H. somni include either killed cells or bacteria-free outer membrane proteins from the organism which have proven to be moderately successful. In this study, reverse vaccinology was used to predict potential H. somni vaccine candidates from genome sequences. In turn, these may protect animals against new strains circulating in the field. Whole genome sequencing of six recent clinical H. somni isolates was performed using an Illumina MiSeq and compared to six genomes from the 1980's. De novo assembly of crude whole genomes was completed using Geneious 6.1.7. Protein coding regions was predicted using Glimmer3. Scores from multiple web-based programs were utilized to evaluate the antigenicity of these predicted proteins which were finally ranked based on their surface exposure scores. A single new strain was selected for future vaccine development based on conservation of the protein candidates among all 12 isolates. A positive signal with convalescent serum for these antigens in western blots indicates in vivo recognition. In order to test the protective capacity of these antigens bovine animal trials are ongoing.

Association of Histophilus somni with spontaneous abortions in dairy cattle herds from Brazil.

This study investigated the participation of infectious agents in spontaneous abortions and reproductive problems at eight dairy cattle herds from three geographical regions of Brazil. Fourteen aborted fetuses and the organ sections of one cow with history of repeated abortions were received for pathological evaluations and molecular diagnostics. PCR/RT-PCR assays targeted specific genes of abortifacient agents of cattle: bovine viral diarrhea virus (BVDV), bovine herpesvirus 1 (BoHV-1), Listeria monocytogenes, Neospora caninum, Leptospira spp., Brucella abortus, and Histophilus somni. Six fetuses were adequate for pathological investigations; one of these did not demonstrate remarkable pathological alterations. Significant histopathological findings included vasculitis, hemorrhage, and fibrinous thrombosis of the cerebrum (n = 4); necrotizing myocarditis (n = 3); and hemorrhagic enteritis (n = 3). The placenta and uterus of the cow as well as the kidney, pancreas, and liver of her aborted fetus contained H. somni DNA and demonstrated histopathological evidence of histophilosis. All fetuses contained H. somni DNA in multiple organs. Coinfections of H. somni with B. abortus (n = 2), N. caninum (n = 2), BVDV (n = 1), and BoHV-1 (n = 1) were identified; two fetuses demonstrated three pathogens. These findings suggest that H. somni was associated with the spontaneous abortions and reproductive problems of these herds. However, the exact cause of fetal death might not be attributed only to H. somni in all aborted fetuses, since some of these were infected with other abortifacient agents.

Natural competence in Histophilus somni strain 2336.

Histophilus somni is an etiologic agent of shipping fever pneumonia, myocarditis, and other systemic diseases of bovines. Virulence factors that have been identified in H. somni include biofilm formation, lipooligosaccharide phase variation, immunoglobulin binding proteins, survival in phagocytic cells, and many others. However, to identify the genes responsible for virulence, an efficient mutagenesis system is needed. Mutagenesis of H. somni using allelic exchange is difficult, likely due to its tight restriction modification system. Mutagenesis by natural transformation in Haemophilus influenzae is well established and shows a strong bias for fragments containing specific uptake signal sequences (USS) within the genome. We hypothesized that natural transformation may also be possible in H. somni strain 2336 because its genome is over-represented with H. influenzae USS (5'-AAGTGCGGT-3') and contains most of the genes necessary for competence. H. somni strain 2336 was successfully transformed and mutated with genomic linear DNA from an H. somni mutant (738Δlob2a), which contains a kanamycin-resistance (Kan(R)) gene and the USS within lob2A. Although most of the competence genes found in H. influenzae were present in H. somni, comD and the 5' portion of comE were absent, which may account for the low transformation efficiency. The transformation efficiency of strain 2336 was greatest during mid-log growth phase and when cyclic adenosine monophosphate was added to the transformation medium. However, mutants were not isolated when strain 2336 was transformed with genomic DNA containing the same Kan(R) gene from H. somni luxS or uspE mutants, which lack the USS in these specific genes. Shuttle vector pNS3K was also naturally transformed into strain 2336, though at a lower efficiency. However, natural transformation with either H. somni linear DNA (2336Δlob2A) or pNS3K was unsuccessful with H. somni commensal strain 129Pt and several other disease isolates.

Histophilus somni-induced infections in cattle from southern Brazil.

The sudden death of three calves, one diarrheic calf, and one aborted fetus from four farms in southern Brazil was investigated. Two Histophilus somni-associated syndromes were identified: systemic histophilosis (n = 4) and abortion (n = 1). The principal pathological findings included vasculitis, meningoencephalitis with thrombosis, necrotizing myocarditis, renal infarctions, hepatic abscesses, and bronchopneumonia. PCR assays were used to amplify specific amplicons of the ovine herpesvirus 2, bovine herpesvirus 1 and -5, Listeria monocytogenes, H. somni, and pestivirus; bovine group A rotavirus (BoRV-A) and bovine coronavirus (BCoV) were investigated in calves with diarrhea. H. somni DNA was amplified in tissues from all calves and the brain of the aborted fetus with pathological alterations consistent with histophilosis. All other PCR assays were negative; BoRV-A and BCoV were not identified. These findings confirm the participation of H. somni in the pathological alterations observed in this study and represent the first description of histophilosis in cattle from Brazil.

Diversity of bacterial species in the nasal cavity of sheep in the highlands of Ethiopia and first report of Histophilus somni in the country.

A study was conducted to isolate bacterial species/pathogens from the nasal cavity of apparently healthy and pneumonic sheep. Nasal swabs were collected aseptically, transported in tryptose soya broth and incubated for 24 h. Then, each swab was streaked onto chocolate and blood agar for culture. Bacterial species were identified following standard bacteriological procedures. Accordingly, a total of 1,556 bacteria were isolated from 960 nasal swabs collected from three different highland areas of Ethiopia, namely Debre Berhan, Asella, and Gimba. In Debre Berhan, 140 Mannheimia haemolytica, 81 Histophilus somni, 57 Staphylococcus species, and 52 Bibersteinia trehalosi were isolated. While from Gimba M. haemolytica, Staphylococcus, Streptococcus, and H. somni were isolated at rates of 25.2, 15.9, 11.4, and 5.9 %, respectively, of the total 647 bacterial species. In Asella from 352 bacterial species isolated, 93 (26.4 %) were M. haemolytica, 48 (13.6 %) were Staphylococcus species, 26 (7.4 %) were B. trehalosi, and 17 (4.8 %) H. somni were recognized. Further identification and characterization using BIOLOG identification system Enterococcus avium and Sphingomonas sanguinis were identified at 100 % probability, while, H. somni and Actinobacillus lignerisii were suggested by the system. The study showed that a variety of bacterial species colonize the nasal cavity of the Ethiopian highland sheep with variable proportion between healthy and pneumonic ones. To our knowledge, this is the first report on isolation of H. somni, an important pathogen in cattle, from the respiratory tract of a ruminant species in the country.

Decoration of Histophilus somni lipooligosaccharide with N-acetyl-5-neuraminic acid enhances bacterial binding of complement factor H and resistance to killing by serum and polymorphonuclear leukocytes.

The incorporation of N-acetyl-5-neuraminic acid (Neu5Ac), or sialic acid, onto surface components of some bacterial species may enhance their virulence. We have previously shown that Neu5Ac can be incorporated onto the lipooligosaccharide (LOS) of the bovine pathogen Histophilus somni, resulting in diminished antibody binding and enhanced serum resistance (Inzana et al., 2002. Infect. Immun. 70, 4870). In the present study, we assessed the effect of sialylation of H. somni LOS on the interaction with bovine innate host defenses. Incubation of non-sialylated H. somni with pre-colostral calf serum (PCS) resulted in dose-dependent, complement-mediated killing of the bacteria by the alternative pathway. However, sialylated H. somni was significantly more resistant to killing at any of the concentrations of PCS used. Sialylated H. somni LOS activated and consumed less complement than non-sialylated LOS, as determined by reduction in hemolysis of opsonized red blood cells, and by Western blotting of C(3) activation products. Sialylated H. somni bound more factor H and iC(3)b and less C(3) than non-sialylated bacteria, as determined by enzyme-linked immunosorbent assay, supporting the deficiencies observed in complement activation and consumption by sialylated LOS. Sialylation of H. somni LOS inhibited both polymorphonuclear leukocyte phagocytosis of (3)H-thymidine-labeled bacteria and intracellular killing of the bacteria, compared to non-sialylated bacteria. Furthermore, sialylated H. somni bound less non-specific antibodies in normal bovine sera than non-sialylated bacteria. Therefore, sialylation of H. somni LOS had profound effects on resistance of the bacteria to innate bovine host defenses, which should be taken into consideration during in vitro studies of H. somni.

The role of lipooligosaccharide phosphorylcholine in colonization and pathogenesis of Histophilus somni in cattle.

Histophilus somni is a Gram-negative bacterium and member of the Pasteurellaceae that is responsible for respiratory disease and other systemic infections in cattle. One of the bacterium's virulence factors is antigenic phase variation of its lipooligosaccharide (LOS). LOS antigenic variation may occur through variation in composition or structure of glycoses or their substitutions, such as phosphorylcholine (ChoP). However, the role of ChoP in the pathogenesis of H. somni disease has not been established. In Haemophilus influenzae ChoP on the LOS binds to platelet activating factor on epithelial cells, promoting bacterial colonization of the host upper respiratory tract. However, ChoP is not expressed in the blood as it also binds C-reactive protein, resulting in complement activation and killing of the bacteria. In order to simulate the susceptibility of calves with suppressed immunity due to stress or previous infection, calves were challenged with bovine herpes virus-1 or dexamethazone 3 days prior to challenge with H. somni. Following challenge, expression of ChoP on the LOS of 2 different H. somni strains was associated with colonization of the upper respiratory tract. In contrast, lack of ChoP expression was associated with bacteria recovered from systemic sites. Histopathology of cardiac tissue from myocarditis revealed lesions containing bacterial clusters that appeared similar to a biofilm. Furthermore, some respiratory cultures contained substantial numbers of Pasteurella multocida, which were not present on preculture screens. Subsequent biofilm experiments have shown that H. somni and P. multocida grow equally well together in a biofilm, suggesting a commensal relationship may exist between the two species. Our results also showed that ChoP contributed to, but was not required for, adhesion to respiratory epithelial cells. In conclusion, expression of ChoP on H. somni LOS contributed to colonization of the bacteria to the host upper respiratory tract, but phase variable loss of ChoP expression may help the bacteria survive systemically.

PCR Multiplex fluorescente para detecção de bactérias em sêmen bovino / Fluorescent Multiplex PCR for detection of bacteria in semen bovine

Este estudo teve como objetivo avaliar o limiar de detecção da técnica de PCR multiplex fluorescente aliada a eletroforese capilar na detecção de agentes infecciosos em amostras de sêmen experimentalmente contaminadas com concentrações decrescentes das bactérias Brucella abortus, Leptospira interrogans sorovar pomona, Campylobacter fetus e Haemophilus somnus. Amostras de sêmen bovino foram experimentalmente contaminadas com concentrações decrescentes de bactérias obtidas através de diluições seriadas na base 10 de modo a obter-se amostras contendo desde 1 vez até 10-7 bactérias/mL a partir da concentração inicial de Leptospira pomona, Brucella abortus, Campylobacter fetus e Haemophilus somnus. As diluições foram efetuadas individualmente para cada bactéria, bem como nas diferentes concentrações necessárias para a padronização do teste de multiplex PCR. As extrações de DNA de todas as soluções contendo espermatozóides e bactérias analisadas no presente estudo foram realizadas segundo protocolo descrito por Heinemann et al. (2000). Os produtos de PCR multiplex foram avaliados por eletroforese em gel de poliacrilamida 8% e separação eletroforética por sistema capilar em equipamento automático de análise de fragmentos de DNA MegaBace. Observou-se a amplificação de fragmentos de 193pb, 330pb, 400pb e 415pb a partir do DNA de B. abortus, L. pomona, H. somnus, C. fetus, respectivamente. Na análise por eletroforese capilar de produtos da PCR multiplex do DNA para detecção simultânea dos quatro patógenos observou-se a sinal de positividade até a diluição de 10-3 bactérias/mL vezes da concentração inicial da solução estoque de cada bactéria. A técnica de PCR multiplex aliada à eletroforese capilar foi usada pela primeira vez para o diagnóstico direto de quatro bactérias patogênicas no sêmen, demonstrando ser um método rápido na detecção de bactérias causadoras de doenças reprodutivas.

This study aimed to evaluate the threshold of detection of fluorescent multiplex PCR coupled with capillary electrophoresis for detection of infectious agents in semen samples from experimentally infected with decreasing concentrations of the bacteria Brucella abortus, Leptospira interrogans serovar pomona, Campylobacter fetus and Haemophilus somnus. Samples of bovine semen were experimentally infected with decreasing concentrations of bacteria obtained from serial dilutions in the base 10 so as to obtain samples containing a long time until 10-7 bacteria/mL from the initial concentration of Lepstospira pomona, Brucella abortus, Haemophilus somnus and Campylobacter fetus. The dilutions were made individually for each bacterium, as well as in different concentrations needed to standardize the multiplex PCR test. DNA extractions of all solutions containing sperm and bacteria analyzed in this study were performed according to protocol described by Heinemann et al. (2000). The multiplex PCR products were analyzed by electrophoresis on 8% polyacrylamide gel and capillary electrophoretic separation system for automated equipment in the analysis of DNA fragments MegaBACE. We observed amplification of fragments of 193pb, 330pb, 415pb and 400bp from the DNA of B. abortus, L. pomona, H. somnus, C. fetus respectively. The analysis by capillary electrophoresis of multiplex PCR products of DNA for simultaneous detection of the four pathogens was observed by detecting the dilution of 10-3 bacteria / mL times the initial concentration of the stock solution of each bacterium. The multiplex PCR coupled with capillary electrophoresis was first used for the direct diagnosis of four pathogenic bacteria in semen, proving to be a rapid method to detect bacteria that cause reproductive disorders.

RNA-seq based transcriptional map of bovine respiratory disease pathogen "Histophilus somni 2336".

Genome structural annotation, i.e., identification and demarcation of the boundaries for all the functional elements in a genome (e.g., genes, non-coding RNAs, proteins and regulatory elements), is a prerequisite for systems level analysis. Current genome annotation programs do not identify all of the functional elements of the genome, especially small non-coding RNAs (sRNAs). Whole genome transcriptome analysis is a complementary method to identify "novel" genes, small RNAs, regulatory regions, and operon structures, thus improving the structural annotation in bacteria. In particular, the identification of non-coding RNAs has revealed their widespread occurrence and functional importance in gene regulation, stress and virulence. However, very little is known about non-coding transcripts in Histophilus somni, one of the causative agents of Bovine Respiratory Disease (BRD) as well as bovine infertility, abortion, septicemia, arthritis, myocarditis, and thrombotic meningoencephalitis. In this study, we report a single nucleotide resolution transcriptome map of H. somni strain 2336 using RNA-Seq method.The RNA-Seq based transcriptome map identified 94 sRNAs in the H. somni genome of which 82 sRNAs were never predicted or reported in earlier studies. We also identified 38 novel potential protein coding open reading frames that were absent in the current genome annotation. The transcriptome map allowed the identification of 278 operon (total 730 genes) structures in the genome. When compared with the genome sequence of a non-virulent strain 129Pt, a disproportionate number of sRNAs (∼30%) were located in genomic region unique to strain 2336 (∼18% of the total genome). This observation suggests that a number of the newly identified sRNAs in strain 2336 may be involved in strain-specific adaptations.

Identification, structure, and characterization of an exopolysaccharide produced by Histophilus somni during biofilm formation.

BACKGROUND: Histophilus somni, a gram-negative coccobacillus, is an obligate inhabitant of bovine and ovine mucosal surfaces, and an opportunistic pathogen responsible for respiratory disease and other systemic infections in cattle and sheep. Capsules are important virulence factors for many pathogenic bacteria, but a capsule has not been identified on H. somni. However, H. somni does form a biofilm in vitro and in vivo, and the biofilm matrix of most bacteria consists of a polysaccharide. RESULTS: Following incubation of H. somni under growth-restricting stress conditions, such as during anaerobiosis, stationary phase, or in hypertonic salt, a polysaccharide could be isolated from washed cells or culture supernatant. The polysaccharide was present in large amounts in broth culture sediment after H. somni was grown under low oxygen tension for 4-5 days (conditions favorable to biofilm formation), but not from planktonic cells during log phase growth. Immuno-transmission electron microscopy showed that the polysaccharide was not closely associated with the cell surface, and was of heterogeneous high molecular size by gel electrophoresis, indicating it was an exopolysaccharide (EPS). The EPS was a branched mannose polymer containing some galactose, as determined by structural analysis. The mannose-specific Moringa M lectin and antibodies to the EPS bound to the biofilm matrix, demonstrating that the EPS was a component of the biofilm. The addition of N-acetylneuraminic acid to the growth medium resulted in sialylation of the EPS, and increased biofilm formation. Real-time quantitative reverse transcription-polymerase chain reaction analyses indicated that genes previously identified in a putative polysaccharide locus were upregulated when the bacteria were grown under conditions favorable to a biofilm, compared to planktonic cells. CONCLUSIONS: H. somni is capable of producing a branching, mannose-galactose EPS polymer under growth conditions favorable to the biofilm phase of growth, and the EPS is a component of the biofilm matrix. The EPS can be sialylated in strains with sialyltransferase activity, resulting in enhanced density of the biofilm, and suggesting that EPS and biofilm formation may be important to persistence in the bovine host. The EPS may be critical to virulence if the biofilm state is required for H. somni to persist in systemic sites.

Genetics and molecular specificity of sialylation of Histophilus somni lipooligosaccharide (LOS) and the effect of LOS sialylation on Toll-like receptor-4 signaling.

Histophilus somni is an etiologic agent of bovine respiratory and systemic diseases. Most pathogenic strains of H. somni that have been tested (36 of 42) are able to utilize N-acetyl-5-neuraminic acid (Neu5Ac) to sialylate their lipooligosaccharide (LOS). Homologs of all the genes required for transport, metabolism, and regulation of Neu5Ac in Haemophilus influenzae were identified in the sequenced genomes of H. somni. Three open reading frames (ORFs) in H. somni strain 2336 were identified that contained homology to genes required for LOS sialylation in related bacteria. ORF-1 (hssT-I), ORF-2 (hssT-II), and ORF-3 (neuA(Hs)) were predicted to encode for putative proteins with 37% amino acid homology to an α-(2-3)-sialyltransferase in H. influenzae, 43% amino acid homology to an Haemophilus ducreyi sialyltransferase, and 72% amino acid homology to an H. influenzae CMP-Neu5Ac synthetase, respectively. The specific enzyme activity of each ORF was determined using synthetic acceptor substrates. The HssT-I sialyltransferase primarily sialylated N-acetyllactosamine (LacNAc, Gal-ß-[1-4]-GlcNAc-R), which is expressed on strain 2336, whereas HssT-II preferentially sialylated lacto-N-biose (LNB, Gal-ß-[1-3]-GlcNAc-R), which is expressed on a phase variant of strain 2336: strain 738. Phase variation of the terminal galactose linkage in strain 738 from ß-(1-3)-(LNB) to ß-(1-4)-(LacNAc) was confirmed using monoclonal antibody reactivity and nuclear magnetic resonance spectroscopy. Sialylated LOS induced significantly less chemokine response from macrophages derived from Toll-like receptor (TLR)-4 knockout mice than from de-sialylated LOS. Furthermore, sialylated LOS induced significantly less NF-κB activity from mouse-derived bone marrow macrophages than de-sialylated LOS. Therefore, sialylation inhibited LOS signaling through TLR-4. In conclusion, H. somni utilizes linkage-specific sialyltransferases to sialylate its LOS to avoid innate host defense mechanisms despite simultaneous epitope phase variation.

Isolation of Histophilus somni from the nasal exudates of a clinically healthy adult goat.

METHODS: The nasal exudate from 42 goats of the Mixteca Region in the state of Puebla, Mexico, was evaluated. A strain was isolated after 4 days of incubation. This strain was identified according to its phenotypic characteristics and by means of a species-specific polymerase chain reaction (PCR), as well as by sequencing of the amplified product. RESULTS: The species-specific PCR amplified a 407-bp fragment of 16S RNAr subunit, and the product sequencing revealed 100% homology with Histophilus somni 129PT. The nucleotide sequence was deposited in the GenBank under accession number HM032735. CONCLUSION: This is the first worldwide isolation of H. somni from nasal exudates of a clinically healthy goat.

Histophilus somni IbpA Fic cytotoxin is conserved in disease strains and most carrier strains from cattle, sheep and bison.

Histophilus somni causes bovine pneumonia, septicemia, myocarditis, thrombotic meningoencephalitis and arthritis, as well as a genital or upper respiratory carrier state in normal animals. However, differences in virulence factors among strains are not well studied. The surface and secreted immunoglobulin binding protein A (IbpA) Fic motif of H. somni causes bovine alveolar type 2 (BAT2) cells to retract, allowing virulent bacteria to cross the alveolar monolayer. Because H. somni IbpA is an important virulence factor, its presence was evaluated in different strains from cattle, sheep and bison to define whether there are syndrome specific markers and whether antigenic/molecular/functional conservation occurs. A few preputial carrier strains lacked IbpA by Western blotting but all other tested disease or carrier strains were IbpA positive. These positive strains had either both IbpA DR1/Fic and IbpA DR2/Fic or only IbpA DR2/Fic by PCR. IbpA Fic mediated cytotoxicity for BAT2 cells and sequence analysis of IbpA DR2/Fic from selected strains revealed conservation of sequence and function in disease and IbpA positive carrier strains. Passive protection of mice against H. somni septicemia with antibody to IbpA DR2/Fic, along with previous data, indicates that the IbpA DR1/Fic and/or DR2/Fic domains are candidate vaccine antigens for protection against many strains of H. somni. Since IbpA DR2/Fic is conserved in most carrier strains, they may be virulent if introduced to susceptible animals at susceptible sites. Conservation of the protective IbpA antigen in all disease isolates tested is encouraging for development of protective vaccines and diagnostic assays.