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This study is an example of how applied genomic analysis can aid diagnostic test improvements. Detection of Mycobacterium avium subsp. paratuberculosis infection of livestock prior to the appearance of clinical disease signs is very difficult but essential for identifying animals shedding the bacterium to prevent transmission of Johne’s disease.

The RB51 vaccine strain of Brucella abortus, which confers safe and effective protection of cattle from B. abortus infection, was originally generated via serial passage of B. abortus 2308 to generate spontaneous, attenuating mutations. While some of these mutations have been previously characterized, such as an insertional mutation in the wboA gene that contributes to the rough phenotype of the strain, a comprehensive annotation of genetic differences between RB51 and B. abortus 2308 genomes has not yet been published. Here, the whole genome sequence of the RB51 vaccine strain is compared against two available 2308 parent sequences, with all observed single nucleotide polymorphisms, insertions, and deletions presented. Mutations of interest for future characterization in vaccine development, such as mutations in eipA and narJ genes in RB51, were identified. Additionally, protein homology modeling was utilized to provide in silico support for the hypothesis that the RB51 capD mutation is the second contributing mutation to the rough phenotype of RB51, likely explaining the inability of wboA-complemented strains of RB51 to revert to a smooth phenotype.

Leptospirosis is a global zoonotic disease affecting humans and livestock species. Bacterin vaccines lack cross protection between serogroups, and include multiple serovars propagated at 29 °C. Recent work demonstrated substantial variation in the transcriptome of identical species and serovars of Leptospira. Here, substantial differences in protein abundance profiles were identified in Leptospira borgpetersenii serovar Hardjo; strain HB203, which was isolated in the 1980s, compared to newer strains TC129 and TC273 isolated in 2016, and whether they were propagated at the routine temperature of 29 °C, compared to 37 °C which more closely emulates host infection. While 388 and 385 significantly differentially expressed (DE) proteins (FDR of 0.01) were identified in HB203 versus TC129, and HB203 versus TC273 when propagated at 29 °C respectively, only 66 and 4 DE proteins were identified in HB203 versus TC129, and HB203 versus TC273 when propagated at 37 °C respectively. Within each strain comparing temperatures, HB203 had 524 significantly DE proteins, TC129 had 347 DE proteins, and TC273 had 569 DE proteins. Data are available via ProteomeXchange with identifier PXD032831. Results highlight significant differential protein expression among identical serovars of L. borgpetersenii suggesting that bacterin vaccine design can benefit from consideration of strains employed and effects of temperature on growth. SIGNIFICANCE: Leptospirosis is a zoonotic disease caused by spirochete bacteria of the genus Leptospira. While leptospirosis affects over one million people per year, symptoms range vastly in severity from completely asymptomatic, to flu-like, to multi-organ failure and death in severe cases. Incidental hosts become infected after encountering pathogens directly from contact with another host, including domestic or wildlife animals, or indirectly from contaminated environments. Though animal vaccines exist, they lack cross protection across serogroups, and instead rely on inclusion of multiple carefully selected serovars from laboratory strains prepared at ~29 °C. Recent interest in gene expression at the Leptospira strain level, along with a newly achieved culture temperature of 37 °C (which more closely resembles host body temperature), led us to investigate the proteomic profiles of an older, established challenge strain HB203 in comparison to TC129 and TC273, two strains isolated in 2016 from abattoir cattle in the central United States. Herein, we identify substantial proteomic differences not only between strains of the same species and serovar, but notably between growth temperatures, collectively suggesting that bacterin vaccine composition may benefit from investigating strain selection and the temperature employed for growth of the bacteria used in bacterin preparation.

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Genome sequences of two type B and two type S strains of Mycobacterium avium subsp. paratuberculosis are presented. These strains were isolated in the United States from sheep, bison, and cattle suffering from Johne’s disease. These genomes will increase our understanding of the minor differences that exist among this genetically stable subspecies.

Plasmid-mediated polymyxin resistance encoded by mcr-1 has increased public health concerns due to the potential for rapid horizontal transfer. Here, we report the complete genome sequence of colistin-resistant Escherichia coli Antibiotic Resistance Isolate Bank number 0346, harboring a plasmid-borne mcr-1 gene.

Genomic tools have improved the ability to manage bison populations and enhanced efforts to conserve this iconic species. These tools have been particularly useful for detecting introgression of cattle genome within bison herds but are limited by the need to use the cattle genome as a surrogate for mapping reads. This complicates efforts to distinguish the species of origin of chromosomal segments in individual bison at the genomic level. An assembly (Bison_UMD1.0) based on 75X genome coverage by Illumina and 454 reads was generated using the MaSuRCA assembler, generating a 2.81 Gigbases de novo reference genome from American bison. Comparison of bison and domestic cattle references identified 28 443 364 single nucleotide variants and 2 627 645 insertions/deletions distinguishing the species. Sequence alignment of an additional 12 modern bison samples and two historic bison samples to domestic cattle and bison references provides a dataset of genomic variants defining the different species and within-species variation. This first annotated draft assembly represents a resource for the management and conservation of bison, as well as a means to study the effects on the genome of interspecies hybridization. The comparisons of historical bison sequences with the new bison reference identified genomic differences between modern and pre-population bottleneck bison. The results support the application of genomics to enhance future research on disease, the establishment of satellite conservation herds and insight into bison and cattle speciation. The first genome assembly for bison and dataset provides a foundation that can be built upon as genetic technologies improve over the years.

The complete genome sequence of a type III strain of Mycobacterium avium subsp. paratuberculosis was determined. The genome size for this pathogen of sheep is 4,895,755 bp with no plasmid DNA. The chromosome contains 19 copies of the hallmark IS900 element, which is routinely used to identify this subspecies.

Over a decade ago Mycobacterium avium subspecies paratuberculosis (Map) specific genes were initially identified in a whole genome context by comparing draft genome sequences of Map strain K-10 with Mycobacterium avium subspecies hominissuis (Mah) strain 104. This resulted in identification of 32 Map specific genes, not including repetitive elements, based on the two-genome comparison. The goal of this study was to define a more complete catalog of M. avium subspecies-specific genes. This is important for obtaining additional diagnostic targets for Johne's disease detection and for understanding the unique biology, evolution and niche adaptation of these organisms. There are now over 28 complete genome sequences representing three M. avium subspecies, including avium (Maa), Mah, and Map. We have conducted a comprehensive comparison of these genomes using two independent pan genomic comparison tools, PanOCT and Roary. This has led to the identification of more than 250 subspecies defining genes common to both analyses. The majority of these genes are arranged in clusters called genomic islands. We further reduced the number of diagnostic targets by excluding sequences having high BLAST similarity to other mycobacterial species recently added to the National Center for Biotechnology Information database. Genes identified as diagnostic following these bioinformatic approaches were further tested by DNA amplification PCR on an additional 20 M. avium subspecies strains. This combined approach confirmed 86 genes as Map-specific, seven as Maa-specific and three as Mah-specific. A single-tube PCR reaction was conducted as a proof of concept method to quickly distinguish M. avium subspecies strains. With these novel data, researchers can classify isolates in their freezers, quickly characterize clinical samples, and functionally analyze these unique genes.

Among more than twenty species belonging to the class Mollecutes, Mycoplasma bovis is the most common cause of bovine mycoplasmosis in North America and Europe. Bovine mycoplasmosis causes significant economic loss in the cattle industry. The number of M. bovis positive herds recently has increased in North America and Europe. Since antibiotic treatment is ineffective and no efficient vaccine is available, M. bovis induced mycoplasmosis is primarily controlled by herd management measures such as the restriction of moving infected animals out of the herds and culling of infected or shedders of M. bovis. To better understand the population structure and genomic factors that may contribute to its transmission, we sequenced 147 M. bovis strains isolated from four different countries viz. USA (n = 121), Canada (n = 22), Israel (n = 3) and Lithuania (n = 1). All except two of the isolates (KRB1 and KRB8) were isolated from two host types i.e., bovine (n = 75) and bison (n = 70). We performed a large-scale comparative analysis of M. bovis genomes by integrating 103 publicly available genomes and our dataset (250 total genomes). Whole genome single nucleotide polymorphism (SNP) based phylogeny using M. agalactiae as an outgroup revealed that M. bovis population structure is composed of five different clades. USA isolates showed a high degree of genomic divergence in comparison to the Australian isolates. Based on host of origin, all the isolates in clade IV was of bovine origin, whereas majority of the isolates in clades III and V was of bison origin. Our comparative genome analysis also revealed that M. bovis has an open pangenome with a large breadth of unexplored diversity of genes. The function based analysis of autogenous vaccine candidates (n = 10) included in this study revealed that their functional diversity does not span the genomic diversity observed in all five clades identified in this study. Our study also found that M. bovis genome harbors a large number of IS elements and their number increases significantly (p = 7.8x10-6) as the genome size increases. Collectively, the genome data and the whole genome-based population analysis in this study may help to develop better understanding of M. bovis induced mycoplasmosis in cattle.

Among more than twenty species belonging to the class Mollecutes,Mycoplasma bovisis the most common cause of bovine mycoplasmosis in North America and Europe. Bovine mycoplasmosis causes significant economic loss in the cattle industry. The number ofM. bovispositive herds has recently increased in North America and Europe. Since antibiotic treatment is ineffective and no efficient vaccine is available,M. bovis-induced mycoplasmosis is primarily controlled by herd management measures such as the restriction of moving infected animals out of the herds and culling of infected animals or shedders. To better understand the population structure and genomic factors that may contribute to its transmission, we sequenced 147M. bovisstrains isolated from four different countries and hosts, primarily cattle. We performed a large-scale comparative analysis ofM. bovisgenomes by integrating 104 publicly available genomes and our dataset (251 total genomes). A whole genome-single nucleotide polymorphism (SNP)-based phylogeny revealed thatM. bovispopulation structure is composed of five clades with one of the isolates clustering with the outgroupM. agalactiae. These isolates were found to cluster with those from Canada, Israel, Lithuania, and Switzerland, suggesting trans-continental transmission of the strains. We also validated a previous report suggesting minimum divergence in isolates of Australian origin, which grouped within a single clade along with strains from China and Israel. However, no observable pattern of host association inM. bovisgenomes was found in this study. Our comparative genome analysis also revealed thatM. bovishas an open pangenome with a large breadth of unexplored diversity of genes. Analysis ofvspgene-host association revealed a singlevspsignificantly associated with bovine isolates that may be targeted for diagnostics or vaccine development. Our study also found thatM. bovisgenome harbors a large number of IS elements, including a novel 1624 bp IS element, and ISMbov9. Collectively, the genome data and the whole genome-based population analysis in this study may help to develop control measures to reduce the incidence ofM. bovis-induced mycoplasmosis in cattle and/or to identify candidate genes for vaccine development.

International audience; Mycobacterium avium comprises four subspecies that contain both human and veterinary pathogens. At the inception of this study, twenty-eight M. avium genomes had been annotated as RefSeq genomes, facilitating direct comparisons. These genomes represent strains from around the world and provided a unique opportunity to examine genome dynamics in this species. Each genome was confirmed to be classified correctly based on SNP genotyping, nucleotide identity and presence/absence of repetitive elements or other typing methods. The Mycobacterium avium subspecies paratuberculosis (Map) genome size and organization was remarkably consistent, averaging 4.8 Mb with a variance of only 29.6 kb among the 13 strains. Comparing recombination events along with the larger genome size and variance observed among Mycobacterium avium subspecies avium (Maa) and Mycobacterium avium subspecies hominissuis (Mah) strains (collectively termed non-Map) suggests horizontal gene transfer occurs in non-Map, but not in Map strains. Overall, M. avium subspecies could be divided into two major subdivisions , with the Map type II (bovine strains) clustering tightly on one end of a phylogenetic spectrum and Mah strains clustering more loosely together on the other end. The most evolutionarily distinct Map strain was an ovine strain, designated Telford, which had >1,000 SNPs and showed large rearrangements compared to the bovine type II strains. The Telford strain clustered with Maa strains as an intermediate between Map type II and Mah. SNP analysis and genome organization analyses repeatedly demonstrated the conserved nature of Map versus the mosaic nature of non-Map M. avium strains. Finally, core and pangenomes were developed for Map and non-Map strains. A total of 80% Map genes belonged to the Map core genome, while only 40% of non-Map genes belonged to the non-Map core genome. These genomes provide a more complete and detailed comparison of these subspecies strains as well as a blueprint for how genetic diversity originated.

Here, we report the complete genome sequences of 12 Mycoplasma bovis isolates cultured from Canadian bison and 4 cultured from Canadian cattle. The sequences are of value for understanding the phylogenetic relationship between cattle and bison isolates and will aid in elucidating the genetic basis for virulence and host specificity.

Bovine viral diarrhea virus (BVDV) is one of the most economically important viruses of cattle, but this pathogen is also able to infect pigs, camelids, and a wide range of domestic and wild ruminants. BVDV isolates circulating in animal populations are genetically and antigenically highly diverse. Acute BVDV infections in cattle cause the introduction of many substitutions in the viral genome. Serial infection of pregnant sheep with a BVDV-1b isolate of bovine origin was also associated with great numbers of substitutions. To our knowledge, genomic changes arising during BVDV infections in swine have not been investigated. The purpose of this study was to investigate the changes occurring in the open reading frame (ORF) of BVDV during serial infection of pregnant swine with a BVDV isolate of bovine origin. The BVDV-1b isolate AU526 was serially passaged in six pregnant gilts, two of which gave birth to live piglets congenitally infected with BVDV. The complete ORF sequences of 14 BVDV isolates obtained from pregnant gilts and their piglets were determined. Their analysis revealed that serial transmission of AU526 in pregnant swine resulted in many genomic changes. All isolates of porcine origin shared 32 nucleotide and 12 amino acid differences with the virus inoculum AU526. These changes were detected after a single passage in pregnant swine and were conserved during the subsequent five passages. Amino acid changes occurred primarily in genomic regions encoding the BVDV structural proteins E2 and E rns . These results suggest that BVDV infections in pregnant swine may contribute significantly to the genetic variability of BVDV and lead to the appearance of adaptive changes.

Mycoplasma bovis causes pneumonia, pharyngitis, otitis, arthritis, mastitis, and reproductive disorders in cattle and bison. Two multilocus sequence typing (MLST) schemes have been developed for M. bovis, with one serving as the PubMLST reference method, but no comparison of the schemes has been undertaken. Although the PubMLST scheme has proven to be highly discriminatory and informative, the recent discovery of isolates missing one of the typing loci, adh-1, raises concern about its suitability for continued use. The goal of our study was to compare the performance of the two MLST schemes and identify a new reference scheme capable of fully typing all isolates. We evaluated 448 isolates from diverse geographic and anatomic sites that collectively represent cattle, bison, deer, and a goat. The discrimination indexes (DIs) for the PubMLST and the alternative scheme are 0.909 (91 sequence types [STs]) and 0.842 (77 STs), respectively. Although the PubMLST scheme outperformed the alternative scheme, the adh-1 locus must be retired from the PubMLST scheme if it is to be retained as a reference method. The DI obtained using the six remaining PubMLST loci (0.897, 79 STs) fails to reach the benchmark recommended for a reference method (0.900), mandating the addition of a seventh locus. Comparative analysis of genome sequences from the isolates used here identified the dnaA locus from the alternative scheme as the optimal replacement for adh-1. This revised scheme, which will be implemented as the new PubMLST reference method, has a DI of 0.914 and distinguishes 88 STs from the 448 isolates evaluated.

Bordetella bronchiseptica isolate KM22 has been used in experimental infections of swine as a model of clinical B. bronchiseptica infection and to study host-to-host transmission. The draft genome sequence of KM22 was reported in 2014. Here, we report the complete genome sequence of KM22.

Mycobacterium bovis, the causative agent of bovine tuberculosis, is a pathogen that impacts both animal and human health. Consequently, there is a need to improve understanding of disease dynamics, identification of infected animals, and characterization of the basis of immune protection. This study assessed the transcriptional changes occurring in cattle during the early weeks following a M. bovis infection. RNA-seq analysis of whole blood-cell transcriptomes revealed two distinct transcriptional clusters of infected cattle at both 4- and 10-weeks post-infection that correlated with disease severity. Cattle exhibiting more severe disease were transcriptionally divergent from uninfected animals. At 4-weeks post-infection, 25 genes had commonly increased expression in infected cattle compared to uninfected cattle regardless of disease severity. Ten weeks post-infection, differential gene expression was only observed when severely-affected cattle were compared to uninfected cattle. This indicates a transcriptional divergence based on clinical status following infection. In cattle with more severe disease, biological processes and cell type enrichment analyses revealed overrepresentation of innate immune-related processes and cell types in infected animals. Collectively, our findings demonstrate two distinct transcriptional profiles occur in cattle following M. bovis infection, which correlate to clinical status.

Background Brucella melitensis bacteria cause persistent, intracellular infections in small ruminants as well as in humans, leading to significant morbidity and economic loss worldwide. The majority of experiments on the transcriptional responses of Brucella to conditions inside the host have been performed following invasion of cultured mammalian cells, and do not address gene expression patterns during long-term infection. Results Here, we examine the application of the previously developed coincidence cloning methodology to recover and characterize B. melitensis RNA from the supramammary lymph node of experimentally-infected goats. Using coincidence cloning, we successfully recovered Brucella RNA from supramammary lymph nodes of B. melitensis-infected goats at both short-term (4 weeks) and long-term (38 weeks) infection time points. Amplified nucleic acid levels were sufficient for analysis of Brucella gene expression patterns by RNA-sequencing, providing evidence of metabolic activity in both the short-term and the long-term samples. We developed a workflow for the use of sequence polymorphism analysis to confirm recovery of the inoculated strain in the recovered reads, and utilized clustering analysis to demonstrate a distinct transcriptional profile present in samples recovered in long-term infection. In this first look at B. melitensis gene expression patterns in vivo, the subset of Brucella genes that was highly upregulated in long-term as compared to short-term infection included genes linked to roles in murine infection, such as genes involved in proline utilization and signal transduction. Finally, we demonstrated the challenges of qPCR validation of samples with very low ratios of pathogen:host RNA, as is the case during in vivo brucellosis, and alternatively characterized intermediate products of the coincidence cloning reaction. Conclusions Overall, this study provides the first example of recovery plus characterization of B. melitensis RNA from in vivo lymph node infection, and demonstrates that the coincidence cloning technique is a useful tool for characterizing in vivo transcriptional changes in Brucella species. Genes upregulated in long-term infection in this data set, including many genes not previously demonstrated to be virulence factors in mice or macrophage experiments, are candidates of future interest for potential roles in Brucella persistence in natural host systems. Electronic supplementary material The online version of this article (10.1186/s12867-018-0111-x) contains supplementary material, which is available to authorized users.

Pathogenic leptospires colonize the renal tubules of reservoir hosts of infection, including cattle, and are excreted via urine. In order to identify circulating serovars of pathogenic leptospires in beef cattle, and their associated rates of urinary excretion, a cross sectional study was performed. Fifty urine samples were collected one day each month over 12 consecutive months (N = 600), directly from the bladder of beef cattle at a single slaughter facility and assessed for the presence of leptospires by culture and the fluorescent antibody test (FAT). Where possible, a matched serum sample was also collected for the microscopic agglutination test (MAT). Forty-three urine samples were either culture positive or FAT positive, indicating that 7.2% of sampled beef cattle were actively excreting leptospires in urine. Twenty-three urine samples were culture positive. Sequence analysis of 16S ribosomal DNA and secY indicated that all isolates were Leptospira borgpetersenii. Typing by serology indicated that all isolates were serogroup Sejroe. An overall seroprevalence of 20% (MAT ≥ 1:25) was determined; positive bovine sera was most reactive to serogroup Sejroe (serovar Hardjo) (8.1%), and serogroup Australis (serovar Bratislava) (6.7%). There was poor correlation between seroprevalence and excretion of leptospires since 18/43 (41.9%) cattle, which were positive by culture or FAT, were seronegative. The virulence of two selected isolates of L. borgpetersenii was confirmed by experimental infection in small animal models of infection. Results confirm that L. borgpetesenii continues to circulate in beef cattle and that multiple diagnostic assays are required to detect active shedding. These findings also highlight beef cattle as a reservoir host for the potential zoonotic transmission of leptospires.

'HoBi'-like virus is an atypical group within the Pestivirus genus that is implicated in economic losses for cattle producers due to both acute and persistent infections. Pestivirus strains exist as quasispecies (swarms of individual viruses) in infected animals and the viral populations making up the quasispecies differ widely in size and diversity in each animal. In the present study the viral quasispecies circulating in persistently infected (PI) calves, generated and maintained under experimental conditions using two different 'HoBi'-like strains, was observed over time. An increase in genetic variability and the development of certain mutations was observed over time. Mutations observed included the loss of a putative N-linked glycosylation site in the E2 region and the change of specific residues in E1/E2. It is hypothesized that these changes may be the results on continued adaption of the pestivirus to individual hosts. This is the first study characterizing variation in the viral swarms of animals persistently infected with HoBi-like viruses over time. Studies of the shifts in PI viral swarms will contribute to our understanding of the host and viral mechanisms that function in the maintenance of pestivirus persistent infections.

Glaesserella parasuis is the cause of Glӓsser's disease in pigs and is a significant contributor to post-weaning mortality in the swine industry. Prevention of G. parasuis disease relies primarily on bacterin vaccines, which have shown good homologous protection and variable heterologous protection. Bacterin production involves large scale growth of the bacteria and proteins produced during the proliferation phase of production become important antigens that stimulate the immune response. In order to evaluate genes activated during G. parasuis growth on different media substrates, the transcriptome of broth and agar grown G. parasuis strain 29755 were sequenced and compared. The transcription of most purported virulence genes were comparable between broth and agar grown G. parasuis; however, four virulence-associated genes, including ompA and vapD, had elevated expression under agar growth, while six virulence-associate genes had elevated expression during broth growth, including several protease genes. Additionally, there were metabolic shifts toward increased protein and lipid production and increased cellular division in broth grown G. parasuis. The results contribute to the understanding of how growth substrate alters gene transcription and protein expression, which may impact vaccine efficacy if immunogens important to the protective immune response are not produced under specific in vitro conditions. While the results of this work are unable to fully elucidate which growth medium presents a transcriptome more representative of in vivo samples or best suited for bacterin production, it forms a foundation that can be used for future comparisons and provides a better understanding of the metabolic differences in broth and agar grown bacteria.

Background Escherichia coli O157:H7 (O157) has been linked to numerous foodborne disease outbreaks. The ability to rapidly sequence and analyze genomes is important for understanding epidemiology, virulence, survival, and evolution of outbreak strains. In the current study, we performed comparative genomics to determine structural and functional features of the genome of a foodborne O157 isolate NADC 6564 and infer its evolutionary relationship to other O157 strains. Results The chromosome of NADC 6564 contained 5466 kb compared to reference strains Sakai (5498 kb) and EDL933 (5547 kb) and shared 41 of its 43 Linear Conserved Blocks (LCB) with the reference strains. However, 18 of 41 LCB had inverse orientation in NADC 6564 compared to the reference strains. NADC 6564 shared 18 of 19 bacteriophages with reference strains except that the chromosomal positioning of some of the phages differed among these strains. The additional phage (P19) of NADC 6564 was located on a 39-kb insertion element (IE) encoding several hypothetical proteins, an integrase, transposases, transcriptional regulators, an adhesin, and a phosphoethanolamine transferase (PEA). The complete homologs of the 39-kb IE were found in E. coli PCN061 of porcine origin. The IE-encoded PEA showed low homology (32–33%) to four other PEA in NADC 6564 and PEA linked to mobilizable colistin resistance in E. coli but was highly homologous (95%) to a PEA of uropathogenic, avian pathogenic, and enteroaggregative E. coli. NADC 6564 showed slightly higher minimum inhibitory concentration of colistin compared to the reference strains. The 39-kb IE also contained dndBCDE and dptFGH operons encoding DNA S-modification and a restriction pathway, linked to oxidative stress tolerance and self-defense against foreign DNA, respectively. Evolutionary tree analysis grouped NADC 6564 with lineage I O157 strains. Conclusions These results indicated that differential phage counts and different chromosomal positioning of many bacteriophages and genomic islands might have resulted in recombination events causing altered chromosomal organization in NADC 6564. Evolutionary analysis grouped NADC 6564 with lineage I strains and suggested its earlier divergence from these strains. The ability to perform S-DNA modification might affect tolerance of NADC 6564 to various stressors. Electronic supplementary material The online version of this article (10.1186/s12864-019-5568-6) contains supplementary material, which is available to authorized users.

Background Leptospirosis is a zoonotic, bacterial disease, posing significant health risks to humans, livestock, and companion animals around the world. Symptoms range from asymptomatic to multi-organ failure in severe cases. Complex species-specific interactions exist between animal hosts and the infecting species, serovar, and strain of pathogen. Leptospira borgpetersenii serovar Hardjo strains HB203 and JB197 have a high level of genetic homology but cause different clinical presentation in the hamster model of infection; HB203 colonizes the kidney and presents with chronic shedding while JB197 causes severe organ failure and mortality. This study examines the transcriptome of L. borgpetersenii and characterizes differential gene expression profiles of strains HB203 and JB197 cultured at temperatures during routine laboratory conditions (29°C) and encountered during host infection (37°C). Methodology/Principal findings L. borgpetersenii serovar Hardjo strains JB197 and HB203 were isolated from the kidneys of experimentally infected hamsters and maintained at 29°C and 37°C. RNAseq revealed distinct gene expression profiles; 440 genes were differentially expressed (DE) between JB197 and HB203 at 29°C, and 179 genes were DE between strains at 37°C. Comparison of JB197 cultured at 29°C and 37°C identified 135 DE genes while 41 genes were DE in HB203 with those same culture conditions. The consistent differential expression of ligB, which encodes the outer membrane virulence factor LigB, was validated by immunoblotting and 2D-DIGE. Differential expression of lipopolysaccharide was also observed between JB197 and HB203. Conclusions/Significance Investigation of the L. borgpetersenii JB197 and HB203 transcriptome provides unique insight into the mechanistic differences between acute and chronic disease. Characterizing the nuances of strain to strain differences and investigating the environmental sensitivity of Leptospira to temperature is critical to the development and progress of leptospirosis prevention and treatment technologies, and is an important consideration when serovars are selected and propagated for use as bacterin vaccines as well as for the identification of novel therapeutic targets., Author summary Leptospirosis is a global zoonotic, neglected tropical disease. Interestingly, a high level of species specificity (both bacteria and host) plays a major role in the severity of disease presentation which can vary from asymptomatic to multi-organ failure. Pathogenic Leptospira colonize the kidneys of infected individuals and are shed in urine into the environment where they can survive until they are contracted by another host. This study looks at two strains of L. borgpetersenii, HB203 and JB197 which are genetically very similar, and identical by serotyping as serovar Hardjo, yet HB203 causes a chronic infection in the hamster while JB197 causes organ failure and mortality. To better characterize bacterial factors causing different disease outcomes, we examined the gene expression profile of these strains in the context of temperatures that would reflect natural Leptospira life cycles (environmentally similar 29°C and 37°C which is more indicative of host environment). We found vast differences in gene expression both between the strains and within strains between temperatures. Characterization of the transcriptome of L. borgpetersenii serovar Hardjo strains JB197 and HB203 provides insights into factors that can determine acute versus chronic disease in the hamster model of infection. Additionally, these studies highlight strain to strain variability within the same species, and serovar, at different growth temperatures, which needs to be considered when serovars are selected and propagated for use as bacterin vaccines used to immunize domestic animal species.

Studying the host-associated butyrate-producing bacterial community is important, because butyrate is essential for colonic homeostasis and gut health. Previous research has identified the butyryl coenzyme A (CoA):acetate-CoA transferase (EC 2.3.8.3) as a gene of primary importance for butyrate production in intestinal ecosystems; however, this gene family ( but ) remains poorly defined. We developed tools for the analysis of butyrate-producing bacteria based on 12 putative but genes identified in the genomes of nine butyrate-producing bacteria obtained from the swine intestinal tract. Functional analyses revealed that eight of these genes had strong But enzyme activity. When but paralogues were found within a genome, only one gene per genome encoded strong activity, with the exception of one strain in which no gene encoded strong But activity. Degenerate primers were designed to amplify the functional but genes and were tested by amplifying environmental but sequences from DNA and RNA extracted from swine colonic contents. The results show diverse but sequences from swine-associated butyrate-producing bacteria, most of which clustered near functionally confirmed sequences. Here, we describe tools and a framework that allow the bacterial butyrate-producing community to be profiled in the context of animal health and disease. IMPORTANCE Butyrate is a compound produced by the microbiota in the intestinal tracts of animals. This compound is of critical importance for intestinal health, and yet studying its production by diverse intestinal bacteria is technically challenging. Here, we present an additional way to study the butyrate-producing community of bacteria using one degenerate primer set that selectively targets genes experimentally demonstrated to encode butyrate production. This work will enable researchers to more easily study this very important bacterial function that has implications for host health and resistance to disease.

Mycobacterium avium subsp. paratuberculosis (MAP), the aetiological agent of Johne's disease, is one of the most important bacterial pathogens in ruminants. A thorough understanding of MAP pathogenesis is needed to develop new vaccines and diagnostic tests. The generation of comprehensive random transposon mutant libraries is a fundamental genetic technology to determine the role of genes in physiology and pathogenesis. In this study, whole MAP genome analysis compared the insertion sites for the mycobacterial transposon Tn5367 derived from the Mycobacterium smegmatis insertion sequence IS1096 and the mariner transposon MycoMarT7 carrying the Himar1 transposase. We determined that only MycoMarT7 provides a random representation of insertions in 99 % of all MAP genes. Analysis of the MAP K-10 genome indicated that 710 of all ORFs do not possess IS1096 recognition sites, while only 37 do not have the recognition site for MycoMarT7. Thus, a significant number of MAP genes remain underrepresented in insertion libraries from IS1096-derived transposons. Analysis of MycoMarT7 and Tn5367 mutants showed that Tn5367 has a predilection to insert within intergenic regions, suggesting that MycoMarT7 is the more adequate for generating a comprehensive library. However, we uncovered the novel finding that both transposons have loci-dependent biases, with Tn5367 being the most skewed. These loci-dependent transposition biases led to an underestimation of the number of independent mutants required to generate a comprehensive mutant library, leading to an overestimation of essential genes. Herein, we also demonstrated a useful platform for gene discovery and analysis by isolating three novel mutants for each transposon.

Haemophilus parasuis is a respiratory pathogen of swine and the etiological agent of Glasser's disease. H. parasuis isolates can exhibit different virulence capabilities ranging from lethal systemic disease to subclinical carriage. To identify genomic differences between phenotypically distinct strains, we obtained the closed whole-genome sequence annotation and genome-wide methylation patterns for the highly virulent Nagasaki strain and for the non-virulent D74 strain. Evaluation of the virulence-associated genes contained within the genomes of D74 and Nagasaki led to the discovery of a large number of toxin-antitoxin (TA) systems within both genomes. Five predicted hemolysins were identified as unique to Nagasaki and seven putative contact-dependent growth inhibition toxin proteins were identified only in strain D74. Assessment of all potential vtaA genes revealed thirteen present in the Nagasaki genome and three in the D74 genome. Subsequent evaluation of the predicted protein structure revealed that none of the D74 VtaA proteins contain a collagen triple helix repeat domain. Additionally, the predicted protein sequence for two D74 VtaA proteins is substantially longer than any predicted Nagasaki VtaA proteins. Fifteen methylation sequence motifs were identified in D74 and fourteen methylation sequence motifs were identified in Nagasaki using SMRT sequencing analysis. Only one of the methylation sequence motifs was observed in both strains indicative of the diversity between D74 and Nagasaki. Subsequent analysis also revealed diversity in the restriction-modification systems harbored by D74 and Nagasaki. The collective information reported in this study will aid in the development of vaccines and intervention strategies to decrease the prevalence and disease burden caused by H. parasuis.

Bovine viral diarrhea virus (BVDV), a ubiquitous pathogen of cattle, causes subclinical to severe acute disease. Two species of BVDV are recognized, BVDV1 and BVDV2 with BVDV1 divided into at least 21 subgenotypes and BVDV2 into 3–4 subgenotypes, most commonly using sequences from the 5’ untranslated region (5’ UTR). We report genomic sequencing of 8 BVDV2 isolates that did not segregate into the 2a subgenotype; but represented two additional BVDV2 subgenotypes. One BVDV2 subgenotype was previously recognized only in Asia. The other seven viruses fell into a second subgenotype that was first reported in Brazil and the U.S. in 2002. Neutralization assays using antiserum raised against vaccine strain BVDV2a 296c revealed varying degrees of neutralization of genetically diverse BVDV2 isolates. Neutralization titers decreased from 1.8 to more than a four log(2) decrease. This study illustrated the considerable genetic and antigenic diversity in BVDV2 circulating in the U.S.

Exposure to bovine viral diarrhea viruses (BVDV) results in acute and persistent infections. Persistent infections result from in utero exposure during the first trimester of gestation. Clinical presentation, in persistently infected cattle (PI), is highly variable. The reasons for this variation is largely unknown. The BVDV circulating in PI exist as quasispecies (swarms of individual viruses). An outbreak resulting in 34 PI cattle presented an opportunity to compare a large number of PI׳s. Methods were developed to compare the circulating viral populations within PI animals. It was found that PI animals generated in the same outbreak carry circulating viral populations that differ widely in size and diversity. Further, it was demonstrated that variation in PI viral populations could be used as a quantifiable phenotype. This observation makes it possible to test the correlation of this phenotype to other phenotypes such as growth rate, congenital defects, viral shed and cytokine expression.

Highlights • The KM22 genome sequence is similar to other B. bronchiseptica strains isolated from animals. • bopN, bvgA, fimB, and fimC were the most highly conserved BvgAS-regulated genes present in all strains analyzed. • BvgAS-regulated genes with the highest sequence divergence werefimN, fim2, fhaL, and fhaS. • A total of eight major fimbrial subunit genes were identified in KM22. • qPCR data demonstrated that seven of the eight fimbrial subunit genes are expressed and regulated by BvgAS., The well-characterized Bordetella bronchiseptica strain KM22, originally isolated from a pig with atrophic rhinitis, has been used to develop a reproducible swine respiratory disease model. The goal of this study was to identify genetic features unique to KM22 by comparing the genome sequence of KM22 to the laboratory reference strain RB50. To gain a broader perspective of the genetic relationship of KM22 among other B. bronchiseptica strains, selected genes of KM22 were then compared to five other B. bronchiseptica strains isolated from different hosts. Overall, the KM22 genome sequence is more similar to the genome sequences of the strains isolated from animals than the strains isolated from humans. The majority of virulence gene expression in Bordetella is positively regulated by the two-component sensory transduction system BvgAS. bopN, bvgA, fimB, and fimC were the most highly conserved BvgAS-regulated genes present in all seven strains analyzed. In contrast, the BvgAS-regulated genes present in all seven strains with the highest sequence divergence werefimN, fim2, fhaL, andfhaS. A total of eight major fimbrial subunit genes were identified in KM22. Quantitative real-time PCR data demonstrated that seven of the eight fimbrial subunit genes identified in KM22 are expressed and regulated by BvgAS. The annotation of the KM22 genome sequence, coupled with the comparative genomic analyses reported in this study, can be used to facilitate the development of vaccines with improved efficacy towards B. bronchiseptica in swine to decrease the prevalence and disease burden caused by this pathogen.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) is a bacterium carried by or obtained from swine and other livestock. The initial and predominant swine-associated LA-MRSA sequence type (ST) identified is ST398. Here, we present 14 draft genome sequences from LA-MRSA ST398 isolates found in the United States.