Reverse vaccinology-based identification of a novel surface lipoprotein that is an effective vaccine antigen against bovine infections caused by Pasteurella multocida.

Pasteurella multocida can infect a multitude of wild and domesticated animals, with infections in cattle resulting in hemorrhagic septicemia (HS) or contributing to bovine respiratory disease (BRD) complex. Current cattle vaccines against P. multocida consist of inactivated bacteria, which only offer limited and serogroup specific protection. Here, we describe a newly identified surface lipoprotein, PmSLP, that is present in nearly all annotated P. multocida strains isolated from cattle. Bovine associated variants span three of the four identified phylogenetic clusters, with PmSLP-1 and PmSLP-2 being restricted to BRD associated isolates and PmSLP-3 being restricted to isolates associated with HS. Recombinantly expressed, soluble PmSLP-1 (BRD-PmSLP) and PmSLP-3 (HS-PmSLP) vaccines were both able to provide full protection in a mouse sepsis model against the matched P. multocida strain, however no cross-protection and minimal serum IgG cross-reactivity was identified. Full protection against both challenge strains was achieved with a bivalent vaccine containing both BRD-PmSLP and HS-PmSLP, with serum IgG from immunized mice being highly reactive to both variants. Year-long stability studies with lyophilized antigen stored under various temperatures show no appreciable difference in biophysical properties or loss of efficacy in the mouse challenge model. PmSLP-1 and PmSLP-3 vaccines were each evaluated for immunogenicity in two independent cattle trials involving animals of different age ranges and breeds. In all four trials, vaccination with PmSLP resulted in an increase in antigen specific serum IgG over baseline. In a blinded cattle challenge study with a recently isolated HS strain, the matched HS-PmSLP vaccine showed strong efficacy (75-87.5% survival compared to 0% in the control group). Together, these data suggest that cattle vaccines composed of PmSLP antigens can be a practical and effective solution for preventing HS and BRD related P. multocida infections.

A Bovine Enteric Mycobacterium Infection Model to Analyze Parenteral Vaccine-Induced Mucosal Immunity and Accelerate Vaccine Discovery.

Mycobacterial diseases of cattle are responsible for considerable production losses worldwide. In addition to their importance in animals, these infections offer a nuanced approach to understanding persistent mycobacterial infection in native host species. Mycobacteriumavium ssp. paratuberculosis (MAP) is an enteric pathogen that establishes a persistent, asymptomatic infection in the small intestine. Difficulty in reproducing infection in surrogate animal models and limited understanding of mucosal immune responses that control enteric infection in the natural host have been major barriers to MAP vaccine development. We previously developed a reproducible challenge model to establish a consistent MAP infection using surgically isolated intestinal segments prepared in neonatal calves. In the current study, we evaluated whether intestinal segments could be used to screen parenteral vaccines that alter mucosal immune responses to MAP infection. Using Silirum® - a commercial MAP bacterin - we demonstrate that intestinal segments provide a platform for assessing vaccine efficacy within a relatively rapid period of 28 days post-infection. Significant differences between vaccinates and non-vaccinates could be detected using quantitative metrics including bacterial burden in intestinal tissue, MAP shedding into the intestinal lumen, and vaccine-induced mucosal immune responses. Comparing vaccine-induced responses in mucosal leukocytes isolated from the site of enteric infection versus blood leukocytes revealed substantial inconsistences between these immune compartments. Moreover, parenteral vaccination with Silirum did not induce equal levels of protection throughout the small intestine. Significant control of MAP infection was observed in the continuous but not the discrete Peyer's patches. Analysis of these regional mucosal immune responses revealed novel correlates of immune protection associated with reduced infection that included an increased frequency of CD335+ innate lymphoid cells, and increased expression of IL21 and IL27. Thus, intestinal segments provide a novel model to accelerate vaccine screening and discovery by testing vaccines directly in the natural host and provides a unique opportunity to interrogate mucosal immune responses to mycobacterial infections.

Draft Genome Sequences of 12 Histophilus somni Strains Isolated from Feedlot Cattle in Alberta, Canada.

Histophilus somni is a Gram-negative opportunistic pathogen associated with respiratory disease in cattle. Here, we report the draft genome sequences of 12 Histophilus somni strains isolated from feedlot cattle in Alberta, Canada, which were diagnosed with respiratory disease.

Integrative Conjugative Element ICEHs1 Encodes for Antimicrobial Resistance and Metal Tolerance in Histophilus somni.

The objectives of this study were to determine antimicrobial resistance and metal tolerance, and identify associated genes and mobile genetic elements in clinical strains of Histophilus somni isolated from feedlot cattle in Alberta during years 2012-2016 (contemporary isolates, n = 63) and years 1980-1990 (historical isolates, n = 31). Comparison of antimicrobial resistance (AMR) showed a significant increase in resistance among contemporary isolates compared to historical isolates (P < 0.001). Tolerance to copper (Cu) and zinc (Zn) concentrations above 1 mM was observed in 68 and 52% of the contemporary isolates, respectively. The tet(H) gene associated with oxytetracycline resistance and multicopper oxidase (mco) and cation efflux (czcD) genes associated with Cu and Zn tolerance were identified. An integrative conjugative element; ICEHs1, was identified in whole genome sequences of strains resistant to oxytetracycline, which had Cu and Zn minimum inhibitory concentrations (MIC) >1 mM. The length of ICEHs1 was 64,932 bp and it contained 83 genes, including tetracycline resistance gene tetH, a multidrug efflux pump gene ebrB, and metal tolerance genes mco, czcD, and acr3. Comparative genomics of ICEs revealed that ICEHs1 shares high homology with previously described ICEs of Histophilus somni, Pasteurella multocida, and Mannheimia haemolytica. The ICEHs1 is an active element capable of intra- and inter-genus transfer as demonstrated by successful transfer to H. somni and P. multocida recipients. All isolates carrying ICEHs1 were resistant to tetracycline, a commonly used antibiotic in feedlots, and had Cu and Zn MIC higher than 1 mM. Since Cu and Zn are routinely used in feedlots, there is the possibility of co-selection of AMR in H. somni due to selection pressure created by Cu and Zn. Based on results of in-vitro conjugation experiments, ICEHs1 mediated transmission of antimicrobial and metal resistance genes is possible between BRD pathogens in the respiratory tract, potentially undermining treatment options available for histophilosis and BRD.

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.

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.

Single nucleotide polymorphisms in the bovine Histophilus somni genome; a comparison of new and old isolates.

Histophilus somni, a causative agent of the bovine respiratory disease complex, can also cause a variety of systemic disorders, including bronchopneumonia, myocarditis, pericarditis, arthritis, pleuritis, and infectious thrombotic meningoencephalitis. The purpose of this study was to determine if currently circulating strains differ from those of the 1980s by identifying genomic changes. Single nucleotide polymorphisms (SNPs) and insertion and deletion (INDEL) sites were examined by whole-genome sequencing in 12 samples, 6 old and 6 new. The 31 028 SNP/INDELs recorded were compared against the reference genome sequence of the pathogenic H. somni strain 2336. The distribution of about 75% of these SNPs within a specified gene differed between old and new isolates and did not follow any particular pattern. The other 25% clustered into 2 groups containing the same SNPs in various genes: group I included 5 old isolates and 1 new isolate; group II included 5 new isolates and 1 old isolate. For putative virulence genes there were more SNPs in group I compared with strain 2336, itself an older isolate, than in group II. Although only 25% of all the SNPs formed 2 clusters, the results suggest some genetic difference in various genes between old and new strains.

Histophilus somni est l'un des agents majeurs du complexe respiratoire bovin (CRB), qui peut aussi causer diverses pathologies dont de la bronchopneumonie, myocardite, péricardite, arthrite, pleurésie et de la méningo-encéphalite thrombotique. L'objectif général de l'étude était de comparer les souches actuellement en circulation avec les souches isolées dans les années 80. Plus spécifiquement les changements génétiques survenus entre des isolats récents et des isolats collectés il y a une trentaine d'années ont été analysés. Les polymorphismes d'un seul nucléotide (single nucleotide polymorphism, SNP) ont été examinés en utilisant une approche de séquençage global de tout le génome pour 12 échantillons, six anciens et six nouveaux. Un total de 31 028 SNPs a été identifié et une analyse comparative de ces SNPs avec la séquence génomique de référence de la souche pathogène 2336 de H. somni a été effectuée. La distribution génique d'environ 75 % de ces SNPs entre les souches anciennes et récentes est différente et ne suit pas de tendance particulière. Toutefois, 25 % des SNPs se répartissent rapidement en deux groupes distincts. Le groupe I inclut cinq isolats anciens et un récent alors que le groupe II comprend cinq isolats récents et un isolat ancien qui se regroupent ensemble pour de mêmes SNPs dans plusieurs gènes. La présence des SNPs dans des gènes potentiellement liés à la virulence est plus manifeste dans le groupe I, comparé à l'ancien isolat 2336, que dans le groupe II. Bien que seulement 25 % des SNPs totaux se répartissent en deux groupes, les résultats suggèrent des variations génétiques significatives entre souches anciennes et récentes dans les séquences de nombreux gènes.(Traduit par Docteur François Meurens).

Identification of genomic differences between Escherichia coli strains pathogenic for poultry and E. coli K-12 MG1655 using suppression subtractive hybridization analysis.

Diseases of poultry caused by Escherichia coli result in significant economic loss every year. Specific virulence factors associated with E. coli strains pathogenic for poultry have been identified, but it is likely that others remain to be identified. To identify unique DNA fragments associated with avian strains we used suppression subtractive hybridization. The genome of E. coli K-12 strain MG1655 was subtracted from the genomes of two avian E. coli strains resulting in the identification of 62 fragments specific to the two avian strains. Sequence homology analysis was done and four types of fragments were identified: plasmid sequences, phage sequences, sequences with known function and sequences without any currently known function. Two E. coli collections, a reference collection of diverse strains (ECOR) and a collection of 41 avian isolates, were screened for the presence of 25 of the 62 fragments. We identified nine fragments present in significantly more of the avian strains than of the ECOR strains. Five fragments were in significantly more of the ECOR strains than the avian strains. These results suggested that the nine fragments could play a role in the pathogenesis of E. coli as it relates to diseases of poultry.