Correlation of lesion severity with bacterial changes in Treponeme-Associated Hoof Disease from free-roaming wild elk (Cervus canadensis).

BACKGROUND: Treponeme-Associated Hoof Disease (TAHD) is a polybacterial, multifactorial disease affecting free-ranging wild elk (Cervus canadensis) in the Pacific Northwest. Previous studies have indicated a bacterial etiology similar to digital dermatitis in livestock, including isolation of Treponema species from lesions. The lesions appear to progress rapidly from ulcerative areas in the interdigital space or along the coronary band to severe, ulcerative, necrotic, proliferative lesions under-running the hoof wall, perforating the sole, and contributing to hoof elongation, deformity, and overgrowth. Eventually the lesions undermine the laminal structure leading to sloughing of the hoof horn capsule. The objective of this study was to characterize the bacterial communities associated with hoof lesions, which were categorized into 5 stages or disease grade severities, with 0 being unaffected tissue and 4 being sloughed hoof capsule. We also wanted to determine if the etiology of TAHD through morphological changes was dominated by Treponema, as observed in hoof diseases in livestock. RESULTS: The bacterial 16S rRNA gene was sequenced from 66 hoof skin biopsy samples representing 5 lesion grades from samples collected by Washington Department of Fish and Wildlife as part of a voluntary hunter program. Analysis of the relative abundance of bacterial sequences showed that lesions were dominated by members of the bacterial phyla Proteobacteria, Firmicutes, Spirochaetes, Bacteroidetes and Actinobacteria. In lesion samples, members of the genus Treponema, Porphyromonas, and Mycoplasma increased with lesion severity. Association analysis indicated frequent identification of Treponema with Porphyromonas, Bacteroides and other anaerobic Gram-positive cocci. CONCLUSIONS: The bacterial 16S rRNA gene sequencing confirmed the presence of Treponema species at all stages of TAHD lesions, treponeme specie-specific PCR and histopathology, indicating that the morphological changes are a continual progression of disease severity with similar bacterial communities. Association and abundance of these other pathogenic genera within lesions may mean synergistic role with Treponema in hoof disease pathogenesis. Characterizing bacteria involved in lesion development, and their persistence during disease progression, provides evidence for science-based management decisions in TAHD infected elk populations.

Concurrent colonization of rodent kidneys with multiple species and serogroups of pathogenic Leptospira.

Rodents are important reservoir hosts of pathogenic leptospires in the US Virgin Islands. Our previous work determined that trapped rodents were colonized with Leptospira borgpetersenii serogroup Ballum (n = 48) and/or Leptospira kirschneri serogroup Icterohaemorrhagiae (n = 3). In addition, nine rodents appeared to be colonized with a mixed population comprising more than one species/serogroup. The aim of this study was to validate this finding by characterizing clonal isolates derived from cultures of mixed species. Cultures of presumptive mixed species (designated LR1, LR5, LR37, LR57, LR60, LR61, LR68, LR70, and LR72) were propagated in different media including Hornsby-Alt-Nally (HAN) media, incubated at both 29℃ and 37℃, and T80/40/LH incubated at 29℃. Polyclonal reference antisera specific for serogroup Ballum and Icterohaemorrhagiae were used to enrich for different serogroups followed by subculture on agar plates. Individual colonies were then selected for genotyping and serotyping. Of the nine cultures of mixed species/serogroups, a single clonal isolate was separated in five of them: L. borgpetersenii serogroup Ballum in LR1, LR5, and LR37, and L. kirschneri serogroup Icterohaemorrhagiae in LR60 and LR72. In four of the cultures with mixed species (LR57, LR61, LR68, and LR70), clonal isolates of both L. borgpetersenii serogroup Ballum and L. kirschneri serogroup Icterohaemorrhagiae were recovered. Our results definitively establish that rodents can be colonized with more than one species/serogroup of Leptospira concurrently. The identification and characterization of multiple species/serogroups of Leptospira from individual reservoir hosts of infection are essential to understand the epidemiology and transmission of disease to both human and domestic animal populations.IMPORTANCEPathogenic Leptospira, the causative agent of human and animal leptospirosis, comprise a diverse genus of species/serogroups which are inherently difficult to isolate from mammalian hosts due to fastidious growth requirements. Molecular evidence has indicated that reservoir hosts of Leptospira may shed multiple species concurrently. However, evidence of this phenomena by culture has been lacking. Culture is definitive and is essential for comprehensive characterization of recovered isolates by high-resolution genome sequencing and serotyping. In this work, a protocol using recently developed novel media formulations, in conjunction with reference antisera, was developed and validated to demonstrate the recovery of multiple species/serogroups of pathogenic Leptospira from the same host. The identification and characterization of multiple species/serogroups of Leptospira from individual reservoir hosts of infection are essential to understand the epidemiology and transmission of disease to both human and domestic animal populations.

Improved DNA Amplification of the Hallmark IS900 Element in Mycobacterium avium subsp. paratuberculosis: a Reexamination Based on Whole-Genome Sequence Analysis.

Amplification of the IS900 multicopy element is a hallmark nucleic acid-based diagnostic test for Mycobacterium avium subsp. paratuberculosis, which causes Johne's disease in ruminants. This assay is frequently used to determine the presence of the bacterium in feces of infected cattle and sheep. Two IS900 primer sets developed in the 1990s were widely used for decades, and their use has continued in current studies. However, these primers were developed prior to the availability of complete genome sequences. Recent sequence analysis of the binding locations for one primer pair (P90/P91) identified errors and binding inefficiencies that can be easily corrected to further increase detection sensitivity. The P90 primer is missing two nucleotides that should be present near the 3' end, and it does not bind all copies of IS900 due to 5' deletions at some IS900 loci. These IS900 primer pairs, along with newly developed primers, were tested by real-time PCR on purified genomic DNA to determine which primer set performed the best and how primer design errors affect amplification efficiencies. The newly designed PCR primer set (JB5) showed increased sensitivity by two to three quantification cycles using purified genomic DNA and was similar in efficiency to 150C/921. These tests were extended using DNA from feces and tissues of infected cows, which showed similar results. Finally, a 167-bp partial duplication of IS900 was found in type I strains. Although P90 and P91 primers successfully amplify M. avium subsp. paratuberculosis DNA, their use should be discontinued in favor of more efficient primer pairs in future studies. IMPORTANCE 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. Total M. avium subsp. paratuberculosis quantity in the feces as determined by real-time PCR (qPCR) using the IS900 target indicates bacterial shedding status and potential for transmission of the pathogen. However, legacy primers designed prior to the availability of complete genome sequences that are used in these tests to detect M. avium subsp. paratuberculosis were based on data from only a single copy of IS900 and not considering all copies collectively as a group. This approach resulted in primer design errors which can be easily corrected to improve test sensitivities. We tested original primers that contain these errors and their corrected versions by qPCR and showed improved sensitivity on purified genomic DNA as well as fecal and tissue samples. These findings may help detect the organism from environmental samples on farms where sensitivity is currently lacking.

Leptospira borgpetersenii serovar Hardjo and Leptospira santarosai serogroup Pyrogenes isolated from bovine dairy herds in Puerto Rico.

Leptospirosis is one of the most common zoonotic diseases in the world and endemic in the Caribbean Islands. Bovine leptospirosis is an important reproductive disease. Globally, cattle are recognized as a reservoir host for L. borgpetersenii serovar Hardjo, which is transmitted via urine, semen, and uterine discharges, and can result in abortion and poor reproductive performance. The dairy industry in Puerto Rico comprises up to 25% of agriculture-related income and is historically the most financially important agricultural commodity on the island. In this study, we report the isolation of two different pathogenic Leptospira species, from two different serogroups, from urine samples collected from dairy cows in Puerto Rico: L. borgpetersenii serogroup Sejroe serovar Hardjo and L. santarosai serogroup Pyrogenes. Recovered isolates were classified using whole-genome sequencing, serotyping with reference antisera and monoclonal antibodies, and immunoblotting. These results demonstrate that dairy herds in Puerto Rico can be concurrently infected with more than one species and serovar of Leptospira, and that bacterin vaccines and serologic diagnostics should account for this when applying intervention and diagnostic strategies.

Comparative virulence and antimicrobial resistance distribution of Streptococcus suis isolates obtained from the United States.

Streptococcus suis is a zoonotic bacterial swine pathogen causing substantial economic and health burdens to the pork industry worldwide. Most S. suis genome sequences available in public databases are from isolates obtained outside the United States. We sequenced the genomes of 106 S. suis isolates from the U.S. and analyzed them to identify their potential to function as zoonotic agents and/or reservoirs for antimicrobial resistance (AMR) dissemination. The objective of this study was to evaluate the genetic diversity of S. suis isolates obtained within the U.S., for the purpose of screening for genomic elements encoding AMR and any factors that could increase or contribute to the capacity of S. suis to transmit, colonize, and/or cause disease in humans. Forty-six sequence types (STs) were identified with ST28 observed as the most prevalent, followed by ST87. Of the 23 different serotypes identified, serotype 2 was the most prevalent, followed by serotype 8 and 3. Of the virulence genes analyzed, the highest nucleotide diversity was observed in sadP, mrp, and ofs. Tetracycline resistance was the most prevalent phenotypic antimicrobial resistance observed followed by macrolide-lincosamide-streptogramin B (MLSB) resistance. Numerous AMR elements were identified, many located within MGE sequences, with the highest frequency observed for ble, tetO and ermB. No genes encoding factors known to contribute to the transmission, colonization, and/or causation of disease in humans were identified in any of the S. suis genomes in this study. This includes the 89 K pathogenicity island carried by the virulent S. suis isolates responsible for human infections. Collectively, the data reported here provide a comprehensive evaluation of the genetic diversity among U.S. S. suis isolates. This study also serves as a baseline for determining any potential risks associated with occupational exposure to these bacteria, while also providing data needed to address public health concerns.

Proteomic dataset comparing strains of Leptospira borgpetersenii serovar Hardjo cultured at different temperatures.

Leptospirosis is a global zoonotic bacterial disease which is a threat for humans and most mammals. Bacterin vaccines for leptospirosis are available however they are severely limited in cross protection between serogroups. Leptospira typically colonize the kidneys of reservoir hosts where they are subsequently shed in the urine and persist in the environment and can thus be indirectly or directly transmitted to incidental hosts. Leptospira borgpetersenii serovar Hardjo is the primary cause of leptospirosis in cattle which can result in abortion, unhealthy calves, and rebreed problems. This dataset comprises proteomic profiles of four strains of L. borgpetersenii serovar Hardjo propagated at the routinely utilized culture temperature of 29 °C, and a newly achieved culture temperature of 37 °C, which more closely emulates the temperature of an infected host. The strains analyzed include JB197 (established strain that causes Hardjo atypical acute disease in the hamster model of leptospirosis), HB203 (established strain, causes typical chronic disease in hamsters), as well as TC129 and TC273 (recently isolated strains from the central United States). Differential expression profiles were detected not only between strains but also within strains between culture temperatures. Mass spectrometry data are available via ProteomeXchange with identifier PXD032831.

Some like it hot, some like it cold; proteome comparison of Leptospira borgpetersenii serovar Hardjo strains propagated at different temperatures.

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.

Bovine Leptospirosis Due to Persistent Renal Carriage of Leptospira borgpetersenii Serovar Tarassovi.

Leptospirosis is a global zoonotic disease that causes significant morbidity and mortality in human and animal populations. Leptospira interrogans is a leading cause of human disease, and L. borgpetersenii is a leading cause of animal disease. Cattle are reservoir hosts of L. borgpetersenii serovar Hardjo, which is transmitted via urine, semen, and uterine discharges resulting in abortion and poor reproductive performance. Bovine bacterin vaccines can only protect against those serovars included in vaccine formulations and typically include serovar Hardjo among others. Genotyping and serotyping represent two different and unique methods for classifying leptospires that do not always correlate well; comprehensive characterization using either method requires recovery of isolates from infected animals. In this study, we report for the first time, isolation of L. borgpetersenii serovar Tarassovi from the urine of a dairy cow in the U.S. The classification of the isolate, designated strain MN900, was confirmed by whole-genome sequencing, serotyping with reference antisera and monoclonal antibodies, Matrix Assisted Laser Desorption/Ionization (MALDI), and immunoblotting with reference antisera. Strain MN900 was excreted in urine samples for 18 weeks even as the cow was seronegative for serovar Tarassovi. Strain MN900 has an unusual morphology since it is not as motile as other leptospires and lacks hooked ends. Serovar Tarassovi is not included in U.S. bacterin vaccines. These results demonstrate the importance of culture and concomitant genotyping and serotyping to accurately classify leptospires, and as required to design efficacious vaccine and diagnostic strategies to not only limit animal disease but reduce zoonotic risk.

Complete Genome Sequence of Four Strains of Leptospira borgpetersenii serovar Hardjo isolated from Cattle in the Central United States.

Pathogenic species of Leptospira cause leptospirosis, a global zoonotic disease affecting humans and all major livestock species. Cattle act as a reservoir host for L. borgpetersenii serovar Hardjo which colonize the kidneys and reproductive tract from which they are excreted and transmitted to other cattle via urine, semen or uterine discharges. Bovine leptospirosis results in reproductive failure, abortion, stillbirth and loss of milk production, and is an occupational risk for those working with infected animals. A recent study determined that 7.2% of cattle from an abattoir in the central United States were actively shedding pathogenic Leptospira. Here, we report and compare the complete genome sequence of four recent isolates of L. borgpetersenii serovar Hardjo designated strain TC112, TC147, TC129, and TC273.

Genomewide transcriptional response of Escherichia coli O157:H7 to norepinephrine.

BACKGROUND: Chemical signaling between a mammalian host and intestinal microbes is health and maintenance of 'healthy' intestinal microbiota. Escherichia coli O157:H7 can hijack host- and microbiota-produced chemical signals for survival in a harsh and nutritionally competitive gastrointestinal environment and for intestinal colonization. Norepinephrine (NE) produced by sympathetic neurons of the enteric nervous system has been shown in vitro to induce expression of genes controlling E. coli O157:H7 swimming motility, acid resistance, and adherence to epithelial cells. A previous study used a microarray approach to identify differentially expressed genes in E. coli O157:H7 strain EDL933 in response to NE. To elucidate a comprehensive transcriptional response to NE, we performed RNA-Seq on rRNA-depleted RNA of E. coli O157:H7 strain NADC 6564, an isolate of a foodborne E. coli O157:H7 strain 86-24. The reads generated by RNA-Seq were mapped to NADC 6564 genome using HiSat2. The mapped reads were quantified by htseq-count against the genome of strain NADC 6564. The differentially expressed genes were identified by analyzing quantified reads by DESeq2. RESULTS: Of the 585 differentially expressed genes (≥ 2.0-fold; p < 0.05), many encoded pathways promoting ability of E. coli O157:H7 strain NADC 6564 to colonize intestines of carrier animals and to produce disease in an incidental human host through increased adherence to epithelial cells and production of Shiga toxins. In addition, NE exposure also induced the expression of genes encoding pathways conferring prolonged survival at extreme acidity, controlling influx/efflux of specific nutrients/metabolites, and modulating tolerance to various stressors. A correlation was also observed between the EvgS/EvgA signal transduction system and the ability of bacterial cells to survive exposure to high acidity for several hours. Many genes involved in nitrogen, sulfur, and amino acid uptake were upregulated while genes linked to iron (Fe3+) acquisition and transport were downregulated. CONCLUSION: The availability of physiological levels of NE in gastrointestinal tract could serve as an important cue for E. coli O157:H7 to engineer its virulence, stress, and metabolic pathways for colonization in reservoir animals, such as cattle, causing illness in humans, and surviving outside of a host.

Isolation of Leptospira kirschneri serovar Grippotyphosa from a red panda (Ailurus fulgens) after antimicrobial therapy: Case report.

A 1-year-old female red panda started showing symptoms of illness, including lethargy, anorexia, abdominal discomfort, and vomiting, shortly after transfer to a new zoo. Serum was tested for leptospirosis using the microscopic agglutination test, and a titer of 1:25,600 to serogroup Grippotyphosa was detected. Antimicrobial treatment with doxycycline was initiated. After completion of treatment and resolution of clinical symptoms, a urine sample was collected to ensure clearance of leptospires and cessation of urinary shedding prior to co-housing with other red pandas. A repeat serum sample taken 13 days later had a lower titer of 1:6,400 to serogroup Grippotyphosa. A sample of the animal's urine was cultured in HAN media and was culture positive for Leptospira. The recovered isolate was completely characterized by whole genome sequencing and serotyping with reference antisera, and the isolate was classified as Leptospira kirschneri serogroup Grippotyphosa serovar Grippotyphosa strain RedPanda1.

Proteomic dataset comparing strains of Leptospira borgpetersenii serovar Hardjo cultured at different temperatures

Leptospirosis is a global zoonotic bacterial disease which is a threat for humans and most mammals. Bacterin vaccines for leptospirosis are available however they are severely limited in cross protection between serogroups. Leptospira typically colonize the kidneys of reservoir hosts where they are subsequently shed in the urine and persist in the environment and can thus be indirectly or directly transmitted to incidental hosts. Leptospira borgpetersenii serovar Hardjo is the primary cause of leptospirosis in cattle which can result in abortion, unhealthy calves, and rebreed problems. This dataset comprises proteomic profiles of four strains of L. borgpetersenii serovar Hardjo propagated at the routinely utilized culture temperature of 29 °C, and a newly achieved culture temperature of 37 °C, which more closely emulates the temperature of an infected host. The strains analyzed include JB197 (established strain that causes Hardjo atypical acute disease in the hamster model of leptospirosis), HB203 (established strain, causes typical chronic disease in hamsters), as well as TC129 and TC273 (recently isolated strains from the central United States). Differential expression profiles were detected not only between strains but also within strains between culture temperatures. Mass spectrometry data are available via ProteomeXchange with identifier PXD032831.

Some like it hot, some like it cold: proteome comparison of Leptospira borgpetersenii serovar Hardjo strains propagated at different temperatures

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 amongst identical serovars of L. borgpetersenii suggesting that bacterin vaccine design can benefit from consideration of strains employed and effects of temperature on growth.

Draft Genome Sequences of Two Bison-Type and Two Sheep-Type Strains of Mycobacterium avium subsp. paratuberculosis.

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 strains of this genetically stable subspecies.

Complete Genome Sequence of Escherichia coli Antibiotic Resistance Isolate Bank Number 0346.

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.

A pseudomolecule assembly of the Rocky Mountain elk genome.

Rocky Mountain elk (Cervus canadensis) populations have significant economic implications to the cattle industry, as they are a major reservoir for Brucella abortus in the Greater Yellowstone area. Vaccination attempts against intracellular bacterial diseases in elk populations have not been successful due to a negligible adaptive cellular immune response. A lack of genomic resources has impeded attempts to better understand why vaccination does not induce protective immunity. To overcome this limitation, PacBio, Illumina, and Hi-C sequencing with a total of 686-fold coverage was used to assemble the elk genome into 35 pseudomolecules. A robust gene annotation was generated resulting in 18,013 gene models and 33,422 mRNAs. The accuracy of the assembly was assessed using synteny to the red deer and cattle genomes identifying several chromosomal rearrangements, fusions and fissions. Because this genome assembly and annotation provide a foundation for genome-enabled exploration of Cervus species, we demonstrate its utility by exploring the conservation of immune system-related genes. We conclude by comparing cattle immune system-related genes to the elk genome, revealing eight putative gene losses in elk.

Distinct transcriptional profiles of Leptospira borgpetersenii serovar Hardjo strains JB197 and HB203 cultured at different temperatures.

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.

Complete Genome Sequence of a Type III Ovine Strain of Mycobacterium avium subsp. paratuberculosis.

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.

Lesion Material From Treponema-Associated Hoof Disease of Wild Elk Induces Disease Pathology in the Sheep Digital Dermatitis Model.

A hoof disease among wild elk (Cervus elaphus) in the western United States has been reported since 2008. Now present in Washington, Oregon, Idaho, and California, this hoof disease continues to spread among elk herds suggesting an infectious etiology. Causing severe lesions at the hoof-skin junction, lesions can penetrate the hoof-horn structure causing severe lameness, misshapen hooves, and in some cases, sloughed hooves leaving the elk prone to infection, malnutrition, and premature death. Isolated to the feet, this disease has been termed treponeme-associated hoof disease due to the numerous Treponema spp. found within lesions. In addition to the Treponema spp., treponeme-associated hoof disease shares many similarities with digital dermatitis of cattle and livestock including association with several groups of anaerobic bacteria such as Bacteroides, Clostridia, and Fusobacterium, neutrophilic inflammatory infiltrate, and restriction of the disease to the foot and hoof tissues. To determine if there was a transmissible infectious component to this disease syndrome, elk lesion homogenate was used in a sheep model of digital dermatitis. Ten animals were inoculated with lesion material and lesion development was followed over 7 weeks. Most inoculated feet developed moderate to severe lesions at 2- or 4-weeks post-inoculation timepoints, with 16 of 18 feet at 4 weeks also had spirochetes associated within the lesions. Histopathology demonstrated spirochetes at the invading edge of the lesions along with other hallmarks of elk hoof disease, neutrophilic inflammatory infiltrates, and keratinocyte erosion. Treponema-specific PCR demonstrated three phylotypes associated with elk hoof disease and digital dermatitis were present. Serum of infected sheep had increased anti-Treponema IgG when compared to negative control sheep and pre-exposure samples. Analysis of the bacterial microbiome by sequencing of the bacterial 16S rRNA gene showed a community structure in sheep lesions that was highly similar to the elk lesion homogenate used as inoculum. Bacteroidies, Fusobacterium, and Clostridia were among the bacterial taxa overrepresented in infected samples as compared to negative control samples. In conclusion, there is a highly transmissible, infectious bacterial component to elk treponeme-associated hoof disease which includes several species of Treponema as well as other bacteria previously associated with digital dermatitis.

Distinct transcriptional profiles of Leptospira borgpetersenii serovar Hardjo strains JB197 and HB203 cultured at different temperatures

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 DE 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.