Retrospective Single Nucleotide Polymorphism Analysis of Host Resistance and Susceptibility to Ovine Johne's Disease Using Restored FFPE DNA.

Johne's disease (JD), also known as paratuberculosis, is a chronic, untreatable gastroenteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP) infection. Evidence for host genetic resistance to disease progression exists, although it is limited due to the extended incubation period (years) and diagnostic challenges. To overcome this, previously restored formalin-fixed paraffin embedded tissue (FFPE) DNA from archived FFPE tissue cassettes was utilized for a novel retrospective case-control genome-wide association study (GWAS) on ovine JD. Samples from known MAP-infected flocks with ante- and postmortem diagnostic data were used. Cases (N = 9) had evidence of tissue infection, compared to controls (N = 25) without evidence of tissue infection despite positive antemortem diagnostics. A genome-wide efficient mixed model analysis (GEMMA) to conduct a GWAS using restored FFPE DNA SNP results from the Illumina Ovine SNP50 Bead Chip, identified 10 SNPs reaching genome-wide significance of p < 1 × 10-6 on chromosomes 1, 3, 4, 24, and 26. Pathway analysis using PANTHER and the Kyoto Encyclopedia of Genes and Genomes (KEGG) was completed on 45 genes found within 1 Mb of significant SNPs. Our work provides a framework for the novel use of archived FFPE tissues for animal genetic studies in complex diseases and further evidence for a genetic association in JD.

Survival of Tomato Outbreak Associated Salmonella Serotypes in Soil and Water and the Role of Biofilms in Abiotic Surface Attachment.

Salmonella serotypes linked to tomato-associated outbreaks were evaluated for survival in soil and water over a 40-day period. Salmonella enterica serotypes Anatum, Baildon, Braenderup, Montevideo, Newport, and Javiana were inoculated separately into sterile soil and water, followed by plating onto TSAYE and XLT4 at 10-day intervals. Biofilm production by Salmonella serotypes was measured on both quartz particles (soil surrogate) and glass coverslips, and was evaluated using a crystal violet dye assay. Salmonella populations in soil and water over 40 days indicated no significant differences between Salmonella serotypes tested (p > 0.05). Over a 40-day period, there was a 1.84 ± 0.22 log CFU/g and 1.56 ± 0.54 CFU/mL decrease in populations of Salmonella in soil and water, respectively. Enumeration indicated that Salmonella population fluctuated in water but decreased linearly in soil. All serotypes tested produced the "red dry and rough" morphotype on Congo Red agar. Biofilm produced by all the Salmonella serotypes tested was significantly different on quartz particles than on glass coverslips (p < 0.0001), indicating that material and surface characteristics could affect biofilm development. The ability of Salmonella serotypes to persist in soil or water and attach to abiotic surfaces through biofilm formation affirms that contact surfaces, soil, water, and sediment should be considered as possible sources of cross-contamination in the farm environment.

Investigation of Medium Chain Fatty Acid Feed Supplementation for Reducing Salmonella Typhimurium Colonization in Turkey Poults.

Studies indicate that persistent Salmonella colonization occurs in poultry that are infected early in life, leading to both food safety and public health concerns. Development of improved preharvest Salmonella management strategies is needed to reduce poultry product contamination. The objective of this study was to evaluate the efficacy of a product containing medium chain fatty acids (MCFA) for reducing early Salmonella colonization in turkey poults. Day-of-hatch turkeys were provided a standard starter diet supplemented with MCFA at 0 (negative and positive controls), 1.5, 3, 4.5, or 6 lbs/ton of feed. Positive control and MCFA treated birds were also crop-gavaged with 108 colony forming units (CFU) of bioluminescent Salmonella Typhimurium. Gastrointestinal tissue samples were collected at 3 days postinoculation for bioluminescence imaging (Meckel's diverticulum to the cloaca) and selective enumeration (cecal contents). Quantification of bioluminescence indicated that the 4.5 and 6 lbs/ton MCFA groups had significantly less colonization than the positive control group (p = 0.0412 and p < 0.0001, respectively). Similarly, significantly lower numbers (1-log10 CFU/g reduction) of Salmonella were observed in the ceca of the 6 lbs/ton MCFA group compared to the positive control group (p = 0.0153). These findings indicate that incorporation of MCFA in turkey diets can significantly reduce early Salmonella colonization. In addition, this study highlights the utility of bioluminescence imaging as a screening methodology for assessing the efficacy of treatments that may reduce Salmonella in poultry.

Roles of bacterial membrane vesicles.

Outer membrane vesicles (OMVs) are released from the outer membrane of Gram-negative bacteria. Moreover, Gram-positive bacteria also produce membrane-derived vesicles. As OMVs transport several bacterial components, especially from the cell envelope, their interaction with the host cell, with other bacteria or as immunogens, have been studied intensely. Several functions have been ascribed to OMVs, especially those related to the transport of virulence factors, antigenic protein composition, and development as acellular vaccines. In this work, we review some of the recent findings about OMVs produced by specific pathogenic bacterial species.

Recent advances in Brucella abortus vaccines.

Brucella abortus vaccines play a central role in bovine brucellosis control/eradication programs and have been successfully used worldwide for decades. Strain 19 and RB51 are the approved B. abortus vaccines strains most commonly used to protect cattle against infection and abortion. However, due to some drawbacks shown by these vaccines much effort has been undertaken for the development of new vaccines, safer and more effective, that could also be used in other susceptible species of animals. In this paper, we present a review of the main aspects of the vaccines that have been used in the brucellosis control over the years and the current research advances in the development of new B. abortus vaccines.

Interplay of degradation, dissolution and stabilization of clarithromycin and its amorphous solid dispersions.

Clarithromycin (CLA) is an aminomacrolide antibiotic whose physical properties are fascinating and challenging. It has very poor solubility at neutral intestinal pH, but much better solubility under acidic conditions due to amine protonation. The improved solubility in an acid environment is confounded by the poor chemical stability of clarithromycin that is quite labile toward acid-catalyzed degradation. This creates a complex system under gastrointestinal (GI) conditions: dissolution in the stomach, degradation, potential for precipitation in the small intestine, and interplay with the formulation components. We report herein a study of amorphous solid dispersion (ASD) of CLA with carboxyl-containing cellulose derivatives, which have recently been shown to be excellent ASD matrices for maximizing oral bioavailability. This approach was intended to improve CLA solubility in neutral media while minimizing release in an acid environment, and thereby increase its uptake from the small intestine. Amorphous polymer/CLA nanoparticles were also prepared by high-shear mixing in a multi-inlet vortex mixer (MIVM). Different extents of release were observed at low pH from the various formulations. Thus the solubility increase from nanosizing was deleterious to the concentration of intact CLA obtained upon reaching small intestine conditions; the high extent of release at gastric pH led to complete degradation of CLA. Using pH-switch experiments, it was possible to separate the effects of loss of CLA from solution by crystallization vs. that from chemical degradation. It was found that the hydrophobic cellulose derivative cellulose acetate adipate propionate (CAAdP) was effective at protecting CLA from dissolution in the stomach, and preventing CLA decomposition at low pH; 54% of CLA in CAADP ASD was released intact, vs. 0% and 6% from HPMCAS and CMCAB ASDs, respectively. We conclude that protection against degradation is central to enhancing overall release of intact CLA from ASD formulations; the formulations studied herein have great promise for simultaneous CLA solubility enhancement and protection from loss to chemical degradation, thereby reducing dose requirements and potentially decreasing colonic exposure to CLA (reduced colonic exposure is expected to minimize killing of beneficial colonic bacteria by CLA).

Successful restoration of archived ovine formalin fixed paraffin-embedded tissue DNA and single nucleotide polymorphism analysis.

Archived formalin fixed paraffin-embedded (FFPE) tissues are powerful tools in medicine, capable of harboring diagnostic and genetic answers to challenging clinical questions. Successful utilization of DNA derived from FFPE samples is dependent upon repairing DNA damage generated from the fixation process. Methods to repair FFPE DNA have been successful in human medicine for a variety of research and clinical applications, yet remain underutilized in veterinary medicine. Despite the available technology, our study is the first to evaluate the repair of FFPE derived DNA from veterinary species for single-nucleotide polymorphism (SNP) analysis using the Illumina OvineSNP50 BeadChip and Illumina FFPE QC and DNA Restore kit. To accomplish this, 48 ovine FFPE samples were run using the Illumina OvineSNP50 BeadChip with and without restoration. Compared to pre-restore data, we found increased sample call rates, SNP call frequency, and assay metrics for all samples post-restoration. Further, we utilized four sheep with available parallel fresh DNA and FFPE DNA to compare assay metrics and genotype calls between the two starting sample types. Although fresh samples generated increased call rates, we found 99% concordance in allele calls between restored FFPE and fresh DNA for all four samples. Our results indicate successful restoration and genotyping of ovine FFPE samples using this technology, with potential for utilization in other veterinary species.

The Probiotic Bacillus subtilis MB40 Improves Immunity in a Porcine Model of Listeriosis.

Probiotics for humans and direct-fed microbials for livestock are increasingly popular dietary ingredients for supporting immunity. The aim of this study was to determine the effects of dietary supplementation of Bacillus subtilis MB40 (MB40) on immunity in piglets challenged with the foodborne pathogen Listeria monocytogenes (LM). Three-week-old piglets (n = 32) were randomly assigned to four groups: (1) basal diet, (2) basal diet with LM challenge, (3) MB40-supplemented diet, and (4) MB40-supplemented diet with LM challenge. Experimental diets were provided throughout a 14-day (d) period. On d8, piglets in groups 2 and 4 were intraperitoneally inoculated with LM at 108 CFU/mL per piglet. Blood samples were collected at d1, d8, and d15 for biochemical and immune response profiling. Animals were euthanized and necropsied at d15 for liver and spleen bacterial counts and intestinal morphological analysis. At d15, LM challenge was associated with increased spleen weight (p = 0.017), greater circulating populations of neutrophils (p = 0.001) and monocytes (p = 0.008), and reduced ileal villus height to crypt depth ratio (p = 0.009), compared to non-challenged controls. MB40 supplementation reduced LM bacterial counts in the liver and spleen by 67% (p < 0.001) and 49% (p < 0.001), respectively, following the LM challenge, compared to the basal diet. MB40 supplementation was also associated with decreased circulating concentrations of monocytes (p = 0.007). Altogether, these data suggest that MB40 supplementation is a safe and well-tolerated approach to enhance immunity during systemic Listeria infection.

Characterization of outer membrane vesicles from Brucella melitensis and protection induced in mice.

The outer membrane vesicles (OMVs) from smooth B. melitensis 16 M and a derived rough mutant, VTRM1 strain, were purified and characterized with respect to protein content and induction of immune responses in mice. Proteomic analysis showed 29 proteins present in OMVs from B. melitensis 16 M; some of them are well-known Brucella immunogens such as SOD, GroES, Omp31, Omp25, Omp19, bp26, and Omp16. OMVs from a rough VTRM1 induced significantly higher expression of IL-12, TNFα, and IFNγ genes in bone marrow dendritic cells than OMVs from smooth strain 16 M. Relative to saline control group, mice immunized intramuscularly with rough and smooth OMVs were protected from challenge with virulent strain B. melitensis 16 M just as well as the group immunized with live strain B. melitensis Rev1 (P < 0.005). Additionally, the levels of serum IgG2a increased in mice vaccinated with OMVs from rough strain VTRM1 consistent with the induction of cell-mediated immunity.

Infection cycle of Salmonella enterica serovar Enteritidis in latent carrier mice.

This work reports the distribution of an oral dose of Salmonella enterica serovar Enteritidis (SE) in C57Bl/6-Bcgr mice, to study its pathogenesis in a latent carrier animal. Mice orally inoculated with a high dose of SE developed a latent infection characterized by the absence of clinical symptoms in which the cecum is functioning as a "strategic site" of SE proliferation, releasing bacteria into feces intermittently over the 4-week study. A sequence of disruptions occurred in the small intestine at 1 day postinculation (PI). The microvilli exhibited different degrees of degeneration, which were reversible as the cells became vacuolated. From 2 days PI, SE was detected in the mononuclear phagocytic system, and an exponential growth of the remaining bacteria in tissues was observed until 4 days PI. The production of interferon gamma from 3 days PI is restricting the SE growth, and a plateau phase was observed from 4 to 15 days PI. A recurrence of the bacterial growth in tissue occurred from 15 to 28 days PI, especially in the cecum. Increasing our knowledge about the host-pathogen interaction of adapted pathogens with the ability to develop latency is essential for the development of an efficient strategy for Salmonella control.

Proinflammatory caspase-2-mediated macrophage cell death induced by a rough attenuated Brucella suis strain.

Brucella spp. are intracellular bacteria that cause an infectious disease called brucellosis in humans and many domestic and wildlife animals. B. suis primarily infects pigs and is pathogenic to humans. The macrophage-Brucella interaction is critical for the establishment of a chronic Brucella infection. Our studies showed that smooth virulent B. suis strain 1330 (S1330) prevented programmed cell death of infected macrophages and rough attenuated B. suis strain VTRS1 (a vaccine candidate) induced strong macrophage cell death. To further investigate the mechanism of VTRS1-induced macrophage cell death, microarrays were used to analyze temporal transcriptional responses of murine macrophage-like J774.A1 cells infected with S1330 or VTRS1. In total 17,685 probe sets were significantly regulated based on the effects of strain, time and their interactions. A miniTUBA dynamic Bayesian network analysis predicted that VTRS1-induced macrophage cell death was mediated by a proinflammatory gene (the tumor necrosis factor alpha [TNF-α] gene), an NF-κB pathway gene (the IκB-α gene), the caspase-2 gene, and several other genes. VTRS1 induced significantly higher levels of transcription of 40 proinflammatory genes than S1330. A Mann-Whitney U test confirmed the proinflammatory response in VTRS1-infected macrophages. Increased production of TNF-α and interleukin 1ß (IL-1ß) were also detected in the supernatants in VTRS1-infected macrophage cell culture. Hyperphosphorylation of IκB-α was observed in macrophages infected with VTRS1 but not S1330. The important roles of TNF-α and IκB-α in VTRS1-induced macrophage cell death were further confirmed by individual inhibition studies. VTRS1-induced macrophage cell death was significantly inhibited by a caspase-2 inhibitor but not a caspase-1 inhibitor. The role of caspase-2 in regulating the programmed cell death of VTRS1-infected macrophages was confirmed in another study using caspase-2-knockout mice. In summary, VTRS1 induces a proinflammatory, caspase-2- and NF-κB-mediated macrophage cell death. This unique cell death differs from apoptosis, which is not proinflammatory. It is also different from classical pyroptosis, which is caspase-1 mediated.

Contaminated heparin associated with adverse clinical events and activation of the contact system.

BACKGROUND: There is an urgent need to determine whether oversulfated chondroitin sulfate (OSCS), a compound contaminating heparin supplies worldwide, is the cause of the severe anaphylactoid reactions that have occurred after intravenous heparin administration in the United States and Germany. METHODS: Heparin procured from the Food and Drug Administration, consisting of suspect lots of heparin associated with the clinical events as well as control lots of heparin, were screened in a blinded fashion both for the presence of OSCS and for any biologic activity that could potentially link the contaminant to the observed clinical adverse events. In vitro assays for the activation of the contact system and the complement cascade were performed. In addition, the ability of OSCS to recapitulate key clinical manifestations in vivo was tested in swine. RESULTS: The OSCS found in contaminated lots of unfractionated heparin, as well as a synthetically generated OSCS reference standard, directly activated the kinin-kallikrein pathway in human plasma, which can lead to the generation of bradykinin, a potent vasoactive mediator. In addition, OSCS induced generation of C3a and C5a, potent anaphylatoxins derived from complement proteins. Activation of these two pathways was unexpectedly linked and dependent on fluid-phase activation of factor XII. Screening of plasma samples from various species indicated that swine and humans are sensitive to the effects of OSCS in a similar manner. OSCS-containing heparin and synthetically derived OSCS induced hypotension associated with kallikrein activation when administered by intravenous infusion in swine. CONCLUSIONS: Our results provide a scientific rationale for a potential biologic link between the presence of OSCS in suspect lots of heparin and the observed clinical adverse events. An assay to assess the amidolytic activity of kallikrein can supplement analytic tests to protect the heparin supply chain by screening for OSCS and other highly sulfated polysaccharide contaminants of heparin that can activate the contact system.

The Paratuberculosis Paradigm Examined: A Review of Host Genetic Resistance and Innate Immune Fitness in Mycobacterium avium subsp. Paratuberculosis Infection.

Paratuberculosis, or Johne's Disease (JD) is a debilitating chronic enteritis mainly affecting ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). This organism causes worldwide economic losses to the livestock industry, and is of public health importance due to the potential zoonotic risk between MAP and Crohn's disease (CD) in humans. Without economical treatments, or a vaccine capable of preventing infection without causing cross-reactions with bovine tuberculosis, test-and-cull methods for disease control are imperative. Unfortunately, difficulties in diagnostics and long subclinical stage hinder adequate control and is further complicated by variation in MAP exposure outcome. Interestingly, the majority of infections result in asymptomatic presentation and never progress to clinical disease. One contributing factor is host genetics, where polymorphisms in innate immune genes have been found to influence resistance and susceptibility to disease. Candidate genes identified across studies overlap with those found in CD and tuberculosis including; Solute carrier family 11 member 1 gene (SLC11A1), Nucleotide-binding-oligomerization domain containing gene 2 (NOD2), Major histocompatibility complex type II (MHC-II), and Toll-like receptor (TLR) genes. This review will highlight evidence supporting the vital role of these genes in MAP infection outcome, associated challenges, and implications for the future of JD research.

Brucella abortus RB51 ΔleuB expressing Salmonella FliC conjugated gonadotropins reduces mouse fetal numbers: A possible feral swine brucellosis immunocontraceptive vaccine.

Population and health management of wildlife is a key to environmental health, domestic herd health, and ultimately public health. Many different methods including: surgical sterilization, poison baits, and sponsored hunting programs have been used in the attempt to control populations of various nuisance animal species. Particular interest has been given to immunocontraception through wildlife vaccination protocols. This study specifically looked at the potential immunocontraceptive and protective properties of a Brucella abortus RB51 ΔleuB vaccine expressing Salmonella typhimurium FliC conjugated to porcine follicle stimulating hormone beta subunit (FSHß) or gonadotropin releasing hormone (GnRH) DNA sequences. B. abortus RB51 ΔleuB pNS4-TrcD-FliC- FSH ß (RB51LFSHß) and B. abortus RB51 ΔleuB pNS4-TrcD-FliC-GnRH (RB51LGnRH) were tested in a pilot breeding study with BALB/c mice, and a significant reduction in fertility characteristics was observed in both male and female mice. Ultimately, this study provides support to test these vaccine candidates in feral swine, a destructive invasive species in the United States of America.

Tightly regulated expression vectors for Ochrobactrum anthropi.

Genetic studies of Ochrobactrum anthropi, a bacterial species important in bioremediation and biopesticide degradation, are hindered by the lack of suitably regulated gene expression system. A tightly regulated gene-expression system was developed for O. anthropi using the lacI(q) gene and a re-engineered coliphage T5 promoter containing completely symmetrical DNA segment that binds more efficiently to the lactose repressor. The beta-galactosidase activity was increased 57-fold when the expression of the re-engineered T5 promoter was induced. The degree of induction was controllable by varying the concentration of inducer isopropyl-beta-D: -thiogalactopyranoside.

Silica-antibiotic hybrid nanoparticles for targeting intracellular pathogens.

We investigated the capability of biodegradable silica xerogel as a novel carrier of antibiotic and the efficacy of treatment compared to that with the same dose of free drug against murine salmonellosis. The drug molecules (31%) entrapped in the sol-gel matrix remained in biologically active form, and the bactericidal effect was retained upon drug release. The in vitro drug release profiles of the gentamicin from the xerogel and that from the xerogel-polyethylene glycol (PEG) were distinctly different at pH 7.4. A delayed release of gentamicin was observed from the silica xerogel network (57% in 33 h), and with the addition of 2% PEG, the release rate reached 90% in 33 h. Administration of two doses of the silica xerogel significantly reduced the Salmonella enterica serovar Typhimurium load in the spleens and livers of infected AJ 646 mice. The silica xerogel and xerogel-PEG achieved a 0.45-log and a 0.41-log reduction in the spleens, respectively, while for the free drug there was no reduction. On the other hand, silica xerogel and xerogel-PEG achieved statistically significant 1.13-log and 1.15-log reductions in the livers, respectively, while for the free drug the reduction was a nonsignificant value of 0.07 log. This new approach, which utilizes a room-temperature synthetic route for incorporating therapeutic drugs into the silica matrix, should improve the capability for targeting intracellular pathogens.

Anti-inflammatory properties of cerium oxide nanoparticles.

The valence and oxygen defect properties of cerium oxide nanoparticles (nanoceria) suggest that they may act as auto-regenerative free radical scavengers. Overproduction of the free radical nitric oxide (NO) by the enzyme inducible nitric oxide synthase (iNOS) has been implicated as a critical mediator of inflammation. NO is correlated with disease activity and contributes to tissue destruction. The ability of nanoceria to scavenge free radicals, or reactive oxygen species (ROS), and inhibit inflammatory mediator production in J774A.1 murine macrophages is investigated. Cells internalize nanoceria, the treatment is nontoxic, and oxidative stress and pro-inflammatory iNOS protein expression are abated with stimulation. In vivo studies show nanoceria deposition in mouse tissues with no pathogenicity. Taken together, it is suggested that cerium oxide nanoparticles are well tolerated in mice and are incorporated into cellular tissues. Furthermore, nanoceria may have the potential to reduce ROS production in states of inflammation and therefore serve as a novel therapy for chronic inflammation.

Plasmid-based system for high-level gene expression and antisense gene knockdown in Bartonella henselae.

Six broad-host-range plasmid vectors were developed to study gene expression in Bartonella henselae. The vectors were used to express a beta-galactosidase reporter gene in B. henselae and to generate antisense RNA for gene knockdown. When applied to ompR, a putative transcription response regulator of B. henselae, this antisense RNA gene knockdown strategy reduced bacterial invasion of human endothelial cells by over 60%.

The putative penicillin-binding proteins 1 and 2 are important for viability, growth and cell morphology of Brucella melitensis.

The penicillin-binding proteins (PBPs) are enzymes that regulate the assembly of the peptidoglycan layer of the bacterial cell wall. The genome of Brucella melitensis strain 16M possesses seven pbp genes: three in pbp-1 family (designated as 1A, 1B, and 1C); one in pbp-2 family; and three in pbp-6 family (designated as 6A, 6B, and 6C). We investigated the importance of pbp-1 and pbp-2 genes to viability, cell morphology and infectivity of B. melitensis. A recombinant B. melitensis strain (designated 16MDeltapbp1C) was generated by disrupting the pbp-1C of strain 16M by allelic exchange. This strain produced nearly 20% smaller colonies on trypticase soy agar plates, and grew slower in trypticase soy broth compared to the strain 16M. Electron microscopy revealed that strain 16M exhibited native cocco-bacillus morphology, while 16MDeltapbp1C possessed a spherical morphology. Strain 16MDeltapbp1C did not differ from strain 16M in terms of recovery from infected mouse macrophage cell line J774.1, or recovery from spleens of infected BALB/c mice, suggesting that pbp-1C is dispensable for intracellular persistence of B. melitensis. Expression of mRNA of fixR, the gene downstream of pbp-1C was similar between the strains 16M and 16MDeltapbp1C suggesting that disruption of pbp-1C did not induce any polar effects. Multiple attempts to mutate pbp-1A, pbp-1B, or pbp-2 genes failed, most probably because these genes are indispensable for viability of B. melitensis. Our findings suggest that pbp-1C regulates in vitro growth and cell morphology, whereas pbp-1A, pbp-1B, and pbp-2 are essential for viability of B. melitensis.

Intracellular invasion and survival of Brucella neotomae, another possible zoonotic Brucella species.

In 1967, Brucella neotomae was first isolated from Neotoma lepida, the dessert wood rat, in Utah. With little infection data since its discovery, the zoonotic potential of this Brucella species is largely unknown. Recent reports of isolation from human cerebrospinal fluid, along with current literature suggest that B. neotomae has the ability to infect various hosts and cell types. In this report we extend the knowledge of B. neotomae ATCC 23459's intracellular invasion and survival abilities to a variety of cell lines through gentamicin protection assays. Some of the phagocytic and epithelial cell lines from various mammalian species represent characteristics of some cell types that could be encountered by Brucella in potential hosts. It was found that B. neotomae ATCC 23459 exhibits generally lower intracellular bacterial CFUs compared to the mouse-passaged strain of B. neotomae ATCC 23459, B. suis 1330, and B. abortus 2308. Ultimately, these observations provide a small piece of the puzzle in the investigation of the breadth of B. neotomae's pathogenic potential.