Steven C. Olsen

Gene duplication is a major source of evolutionary novelty, yet recurrent duplication of the same genomic locus across independent lineages remains understudied. Using a chromosome-scale assembly and de novo annotation of the North American bison (Bison bison), we investigated the mechanisms underlying immune-system diversification in ungulates. Comparative genomics of bison, cattle, elk, and 13 mammalian species identified a large segmental duplication encompassing the regulatory genes REST and NOA1. Although similar duplications were present in 14/16 species examined, phylogenetic analyses revealed that duplicated genes cluster within species rather than across taxa, indicating repeated independent origins rather inheritance from a common ancestor. To characterize the evolutionary consequences of this duplication, we integrated transcriptomics from six tissues with weighted gene co-expression and comparative protein clustering. We identified extensive structural divergence among duplicated copies of REST and NOA1, including domain losses, truncations, and expression partitioning across tissues, consistent with functional divergence following duplication. Comparative analyses revealed substantial remodeling of immune-related genes in bison, including BOLA class I molecules, T-cell receptor genes, leukocyte immunoglobulin-like receptors, and regulators of immune signaling. These modifications included domain rearrangements, novel sequence insertions, and lineage-specific duplications associated with immune-related expression networks. Together, these findings identify the REST-NOA1 locus as a recurrent hotspot of segmental duplication in ungulates and demonstrate how repeated duplication, structural divergence, and regulatory partitioning contribute to genome evolution. These results suggest that immune system diversification in bison and related species has been shaped not only by sequence evolution but also by recurrent remodeling of regulatory pathways and antigen-recognition pathways.

Background: Brucella melitensis is considered one of the most widespread zoonotic pathogens worldwide. Vaccination remains the most cost-effective strategy for controlling B. melitensis infection in small ruminants. Methods: In this study, we evaluated the immunologic responses and protection against experimental challenge in 18 goats vaccinated with either lipopolysaccharide (LPS) from B. melitensis strain 16M (LPS alone), LPS of B. melitensis strain 16M and MONTANIDE™ ISA 61 VG adjuvant (Seppic; 50 Bd national, 92250 La Garenne-Colombes, France) (LPS + ISA 61 VG), or saline as a control. Results: Goats (n = 6) vaccinated with LPS + ISA 61 VG had greater (p < 0.05) antibody responses than those that were nonvaccinated. Our data demonstrate that goats vaccinated with LPS + ISA 61 VG exhibited greater lymphocyte proliferative responses (p < 0.05) to the LPS antigen than those vaccinated with LPS alone at week 12 after vaccination. However, proliferative responses of peripheral blood mononuclear cell (PBMC) from goats vaccinated with LPS + ISA61VG did not differ (p > 0.05) from responses of PBMC from control goats. CD4+, CD8+, and γδ T cells from all vaccinated goats had negligible proliferation and failed to induce antigen-specific IFN-γ production. Control and vaccinated goats did not differ (p > 0.05) in their protection against abortion, uterine, fetal, mammary, or maternal infection. Conclusions: Our data suggests that LPS + ISA 61 VG induces a robust humoral response but negligible cellular responses. Our data also suggest that LPS + ISA 61 VG or LPS alone would not be efficacious for use as a vaccine in goats, but the LPS + ISA 61 VG inoculum may be beneficial as a booster. Additional trials would be necessary to evaluate the vaccine’s efficacy as a booster inoculation for small ruminants.

Background/Objectives: White-tailed deer (Odocoileus virginianus) (WTD) play a central role at the human–livestock–wildlife interface, given their contribution to the spread of diseases that can affect livestock. These include a variety of bacterial, viral, and prion diseases with significant economic impact. Given the implications for WTD as potential reservoirs for a variety of diseases, methods for prevention and disease control in WTD are an important consideration. Methods: Using commercial livestock vaccines against bovine viral diarrhea virus (BVDV) in killed and modified live formulations, we test the ability of WTD to develop humoral and cellular immune responses following vaccination. Results: We demonstrate that, similar to cattle, WTD develop humoral immune responses to both killed and modified live formulations. Conclusions: As the farmed deer industry and the use of livestock vaccines in non-approved species grow, this type of information will help inform and develop improved husbandry and veterinary care practices. Additionally, while we were unable to detect cell-mediated immune responses to the vaccine, we established PrimeFlow as a method to detect IFN-γ responses in specific T cell populations, adding another level of resolution to our ability to understand WTD immune responses.

Cell-mediated immunity is an important component of the immune response for intracellular pathogens. Live vaccines containing different pathogens are used concurrently in the field but are generally licensed individually. Concurrent administration of these vaccines has led to concerns about immune interference. The goal of this study was to characterize BVDV-specific responses to vaccination and determine the effect of concurrent Brucella abortus strain RB51 (RB51) vaccine administration. Peripheral blood mononuclear cells (PBMCs) from cattle vaccinated with a modified-live viral (MLV) vaccine containing BVDV, both RB51 and an MLV, or unvaccinated controls were utilized to evaluate antibody titers and the frequency of interferon gamma (IFN-γ) production within CD4⁺, CD8⁺ T cells, and NK cell populations via flow-cytometry. Our data demonstrated the lack of vaccine interference following concurrent administration of two common bovine MLVs and may even suggest some level of enhanced IFN-γ production with concurrent administration.

Background/Objectives: White-tailed deer (Odocoileus virginianus) (WTD) play a central role at the human-livestock-wildlife interface, given their contribution in the spread of diseases, that can affect livestock. These include a variety of both bacterial, viral and prion diseases with significant economic impact. Given the implications for WTD as potential reservoirs for a variety of diseases, methods for prevention and disease control in WTD are an important consideration. Methods: Using commercial livestock vaccines against bovine viral diarrhea virus (BVDV), in killed and modified live formulations, we test the ability of WTD to develop humoral and cellular immune responses following vaccination. Results: We demonstrate that similar to cattle, WTD develop humoral immune responses to both killed and modified live formulation. While we were unable to detect cell-mediated immune responses, the assay used in this manuscript demonstrate that WTD can produce IFN-gamma in response to stimulation. Conclusions: While we were unable to detect cell-mediated immune responses, the assay used in this manuscript demonstrate that WTD can produce IFN-gamma in response to stimulation.

Introduction: Brucellosis is endemic in bison and elk in Yellowstone National Park and surrounding areas.Methods: A comparative study was conducted using data from naive (n = 82 and 67, respectively) and Brucella abortus strain RB51 (RB51) vaccinated (n-99 and 29, respectively) bison and elk experimentally challenged with virulent B. abortus strain during pregnancy.Results: The incidence of abortion, fetal infection, uterine or mammary infection, or infection in maternal tissues after experimental challenge was greater (p < 0.05) in naïve and vaccinated bison when compared to similar groups in elk. Vaccinated bison had lower (p < 0.002) abortion rates and recovery of Brucella from fetal or uterine/mammary tissues when compared to naïve bison. Vaccinated elk had reduced (p < 0.01) rates of maternal infection, but rates of abortion and fetal or uterine/mammary infection did not differ (p > 0.05) from naïve elk. Naïve and vaccinated bison had greater (p < 0.05) Brucella colonization in placentomes, and parotid and supramammary lymphatic tissues when compared to elk. In elk or bison that aborted, mean colonization in placentome tissues were typically more than 5 logs higher than in animals that did not abort.Discussion: The results of our study suggest differences in disease pathogenesis between these two wildlife reservoirs of B. abortus.

Leptospirosis, caused by pathogenic bacteria from the genus Leptospira , is a global zoonosis responsible for more than one million human cases and 60,000 deaths annually. The disease also affects many domestic animal species. Historically, genetic manipulation of Leptospira has been difficult to perform, resulting in limited knowledge on pathogenic mechanisms of disease and the identification of virulence factors. The application of CRISPR/Cas9 and its variations have helped fill these gaps but the generation of knockout mutants remains challenging because double-strand breaks (DSBs) inflicted by Cas9 nuclease are lethal to Leptospira cells. The novel CRISPR prime editing (PE) strategy is the first precise genome-editing technology that allows deletions, insertions, and base substitutions without introducing DSBs. This revolutionary technique utilizes a nickase Cas9 that cleaves a single strand of DNA, coupled with an engineered reverse transcriptase and a modified single-guide RNA (termed prime editing guide RNA) containing an extended 3′ end with the desired edits. We demonstrate the application of CRISPR-PE in both saprophytic and pathogenic Leptospira from multiple species and serovars by introducing deletions or insertions into target DNA with a remarkable precision of just one nucleotide. Additionally, we demonstrate the ability to genetically manipulate Leptospira borgpetersenii , a prevalent pathogenic species of humans, domestic cattle, and wildlife animals. Rapid plasmid loss by mutated strains in liquid culture allows for the generation of knockout strains without selective markers, which can be readily used to elucidate virulence factors and develop optimized bacterin and/or live vaccines against leptospirosis. IMPORTANCE: Leptospirosis is a geographically widespread bacterial zoonosis. Genetic manipulation of pathogenic Leptospira spp. has been laborious and difficult to perform, limiting our ability to understand how leptospires cause disease. The application of the CRISPR/Cas9 system to Leptospira enhanced our ability to generate knockdown and knockout mutants; however, the latter remains challenging. Here, we demonstrate the application of the CRISPR prime editing technique in Leptospira , allowing the generation of knockout mutants in several pathogenic species, with mutations comprising just a single nucleotide resolution. Notably, we generated a mutant in the Leptospira borgpetersenii background, a prevalent pathogenic species of humans and cattle. Our application of this method opens new avenues for studying pathogenic mechanisms of Leptospira and the identification of virulence factors across multiple species. These methods can also be used to facilitate the generation of marker-less knockout strains for updated and improved bacterin and/or live vaccines.

Background: Brucella melitensis is recognized as one of the predominant zoonotic pathogens globally. Live-attenuated vaccine Rev 1 is currently the most effective vaccine for controlling B. melitensis in small ruminants. While Brucella inactivated, nanoparticle, and subunit vaccines are less effective and require multiple doses, live-attenuated vaccines are less expensive and more efficacious. Several drawbacks are associated with the administration of current attenuated B. melitensis vaccines, including interference with serological diagnostic tests, inducing abortion in pregnant animals, shedding in milk, and zoonotic infections in humans. In this systematic review, we summarize the current literature (1970–2022) on B. melitensis vaccines and review their advantages and disadvantages in order to support the rationale for a need for new or improved small ruminant brucellosis vaccines. Methods: A systematic search was carried out in Web of Science, CAB Abstracts, and PubMed. The original articles describing the B. melitensis vaccines were included. Review articles, articles not published in English, articles that did not offer full text, editorials, correspondences, case reports, case series, diagnostic tests, duplicate publications, and other Brucella vaccines (e.g., B. abortus and B. suis) were excluded. Results: Out of 3700 studies, we identified 18 articles that evaluated B. meltensis vaccines, including recombinant B. melitensis strains (16MΔhfq, 16MΔTcfSR, M5-90ΔmanB, LVM31, M5-90ΔvjbR, 16MΔmucR, ΔznuA, M5-90Δpgm, M5-90ΔwboA), live B. melitensis strain (Rev 1), nanoparticle vaccines (B. melitensis 16M, B. melitensis OMP 31, FliC protein—Mannosylated Chitosan Nanoparticles (FliC and FliC-MCN), B. melitensis and B. abortus combined, and B. melitensis 16M nanoparticles combined with oligopolysaccharide), subunit vaccines (outer membrane vesicles or outer membrane proteins), and a DNA vaccine based on B. melitensis outer membrane proteins (Omp25 and Omp31). The results from these studies revealed that these vaccines can induce humoral and cellular responses and reduce macrophage survival. However, most of these vaccines were evaluated only in murine models, which may not accurately reflect how they work in natural hosts. Conclusions: The high prevalence of B. melitensis in humans and animals remains an issue in many parts of the world. Human brucellosis can be prevented by controlling brucellosis in livestock using vaccination and test-and-removal strategies. Prospective vaccines have limitations, including interference with serodiagnostics after vaccination, virulence in humans and animals, the requirement of booster vaccinations, and insufficient efficacy in preventing infection or abortion. Moreover, most of these vaccines have been assessed in mice models, which have failed to predict immunogenicity or efficacy in natural hosts. Because of these limitations and the re-emergence of B. melitensis worldwide with a high incidence of human infection, our review suggests a need for additional research into the molecular pathology and immunological properties of B. melitensis infection and the identification of protective epitopes or genes that would allow for the development of improved vaccines for small ruminants.

The coronavirus disease pandemic has highlighted significant gaps in community mental health services, placing vulnerable individuals at greater risk for mental health and substance use difficulties via disrupting their wellness journey. Guided by a wellness framework, a needs assessment was conducted among adult consumers of behavioral health services to understand their needs during the pandemic and to help develop and strengthen service delivery strategies. A team of three university researchers and four Consumer Researchers, who receive services at a publicly funded community mental health center, engaged in a community‐based participatory project in which 13 focus groups were conducted with 51 consumers. Several themes emerged from a thematic analysis of transcripts regarding consumer well‐being and healthcare needs, coping strategies employed, and the accessibility, benefits, and perception of clinical and support services during the pandemic. Results highlighted strengths in service delivery and areas in need of enhancement. Findings may inform similar community services that seek to enhance delivery of care among vulnerable populations.