Characterization of the adaptive cellular and humoral immune responses to persistent colonization of Brucella abortus strain RB51 in a Jersey cow.

Brucella abortus strain RB51 is the commercial cattle vaccine used in the United States (US) and many parts of the world against bovine brucellosis. RB51 was licensed for use in 1996, and it has been shown to be safe and efficacious in cattle, eliciting humoral and cellular responses in calves and adult animals. In 2017, an epidemiological trace-back investigation performed by the Centers for Disease Control and Prevention (CDC) identified human cases of brucellosis caused by infection with RB51. These infections resulted from the consumption of unpasteurized dairy products, which were traced back to otherwise healthy animals that were shedding RB51 in their milk. At the current time, six adult Jersey cows have been identified in the U.S. that are shedding RB51 in milk. One of the RB51 shedding cattle was obtained and housed at the National Animal Disease Center (NADC) for further study. Improved understanding of host cellular and humoral immune responses to RB51 in persistently colonized cattle may be achieved by the characterization of responses in shedding animals. We hypothesized, based on the lack of RB51 clearance, that the RB51 shedder animal has a diminished adaptive cellular immune response to RB51. Our data demonstrate that in the presence of persistent RB51 infection, there is a lack of peripheral anti-RB51 CD4+ T cell responses and a concurrently high anti-RB51 IgG humoral response. By understanding the mechanisms that result in RB51 persistence, the development of improved interventions or vaccinations for brucellosis may be facilitated, which would provide public health benefits, including reducing the risks associated with the consumption of non-pasteurized milk products.

CRISPR-prime editing, a versatile genetic tool to create specific mutations with a single nucleotide resolution in Leptospira.

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.IMPORTANCELeptospirosis 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.

An injectable subunit vaccine containing Elongation Factor Tu and Heat Shock Protein 70 partially protects American bison from Mycoplasma bovis infection.

Mycoplasma bovis (M. bovis) is the etiologic agent of high mortality epizootics of chronic respiratory disease in American bison (Bison bison). Despite the severity of the disease, no efficacious commercial vaccines have been licensed for the prevention of M. bovis infection in bison. Elongation factor thermal unstable (EFTu) and Heat Shock Protein 70 (Hsp70, DnaK) are highly conserved, constitutively expressed proteins that have previously been shown to provide protection against M. bovis infection in cattle. To assess the suitability of EFTu and Hsp70 as vaccine antigens in bison, the immune response to and protection conferred by an injectable, adjuvanted subunit vaccine comprised of recombinantly expressed EFTu and Hsp70 was evaluated. Vaccinates developed robust antibody and cellular immune responses against both EFTu and Hsp70 antigens. To assess vaccine efficacy, unvaccinated control and vaccinated bison were experimentally challenged with bovine herpes virus-1 (BHV-1) 4 days prior to intranasal infection with M. bovis. Vaccinated bison displayed reductions in joint infection, lung bacterial loads, and lung lesions compared to unvaccinated controls. Together, these results showed that this subunit vaccine reduced clinical disease and bacterial dissemination from the lungs in M. bovis challenged bison and support the further development of protein subunit vaccines against M. bovis for use in bison.

Persistence of viral RNA in North American elk experimentally infected with an ancestral strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

White-tailed deer (Odocoileus virginianus) have emerged as a reservoir host for SARS-CoV-2 given their susceptibility to infection and demonstrated high rates of seroprevalence and infection across the United States. As SARS-CoV-2 circulates within free-ranging white-tailed deer populations, there is the risk of transmission to other wildlife species and even back to the human population. The goal of this study was to determine the susceptibility, shedding, and immune response of North American elk (Cervus elaphus canadensis) to experimental infection with SARS-CoV-2, to determine if another wide-ranging cervid species could potentially serve as a reservoir host for the virus. Here we demonstrate that while North American elk do not develop clinical signs of disease, they do develop a neutralizing antibody response to infection, suggesting the virus is capable of replicating in this mammalian host. Additionally, we demonstrate SARS-CoV-2 RNA presence in the medial retropharyngeal lymph nodes of infected elk three weeks after experimental infection. Consistent with previous observations in humans, these data may highlight a mechanism of viral persistence for SARS-CoV-2 in elk.

Corrigendum: Fecal and vaginal microbiota of vaccinated and non-vaccinated pregnant elk challenged with Brucella abortus.

[This corrects the article DOI: 10.3389/fvets.2024.1334858.].

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.

Neurodevelopmental Outcomes of Extremely Preterm Infants Fed Donor Milk or Preterm Infant Formula: A Randomized Clinical Trial.

Importance: Maternal milk feeding of extremely preterm infants during the birth hospitalization has been associated with better neurodevelopmental outcomes compared with preterm formula. For infants receiving no or minimal maternal milk, it is unknown whether donor human milk conveys similar neurodevelopmental advantages vs preterm formula. Objective: To determine if nutrient-fortified, pasteurized donor human milk improves neurodevelopmental outcomes at 22 to 26 months' corrected age compared with preterm infant formula among extremely preterm infants who received minimal maternal milk. Design, Setting, and Participants: Double-blind, randomized clinical trial conducted at 15 US academic medical centers within the Eunice Kennedy Shriver National Institute of Child Health and Human Development Neonatal Research Network. Infants younger than 29 weeks 0 days' gestation or with a birth weight of less than 1000 g were enrolled between September 2012 and March 2019. Intervention: Preterm formula or donor human milk feeding from randomization to 120 days of age, death, or hospital discharge. Main Outcomes and Measures: The primary outcome was the Bayley Scales of Infant and Toddler Development (BSID) cognitive score measured at 22 to 26 months' corrected age; a score of 54 (score range, 54-155; a score of ≥85 indicates no neurodevelopmental delay) was assigned to infants who died between randomization and 22 to 26 months' corrected age. The 24 secondary outcomes included BSID language and motor scores, in-hospital growth, necrotizing enterocolitis, and death. Results: Of 1965 eligible infants, 483 were randomized (239 in the donor milk group and 244 in the preterm formula group); the median gestational age was 26 weeks (IQR, 25-27 weeks), the median birth weight was 840 g (IQR, 676-986 g), and 52% were female. The birthing parent's race was self-reported as Black for 52% (247/478), White for 43% (206/478), and other for 5% (25/478). There were 54 infants who died prior to follow-up; 88% (376/429) of survivors were assessed at 22 to 26 months' corrected age. The adjusted mean BSID cognitive score was 80.7 (SD, 17.4) for the donor milk group vs 81.1 (SD, 16.7) for the preterm formula group (adjusted mean difference, -0.77 [95% CI, -3.93 to 2.39], which was not significant); the adjusted mean BSID language and motor scores also did not differ. Mortality (death prior to follow-up) was 13% (29/231) in the donor milk group vs 11% (25/233) in the preterm formula group (adjusted risk difference, -1% [95% CI, -4% to 2%]). Necrotizing enterocolitis occurred in 4.2% of infants (10/239) in the donor milk group vs 9.0% of infants (22/244) in the preterm formula group (adjusted risk difference, -5% [95% CI, -9% to -2%]). Weight gain was slower in the donor milk group (22.3 g/kg/d [95% CI, 21.3 to 23.3 g/kg/d]) compared with the preterm formula group (24.6 g/kg/d [95% CI, 23.6 to 25.6 g/kg/d]). Conclusions and Relevance: Among extremely preterm neonates fed minimal maternal milk, neurodevelopmental outcomes at 22 to 26 months' corrected age did not differ between infants fed donor milk or preterm formula. Trial Registration: ClinicalTrials.gov Identifier: NCT01534481.

Fecal and vaginal microbiota of vaccinated and non-vaccinated pregnant elk challenged with Brucella abortus.

Introduction: Brucella abortus is the causative agent of brucellosis in cattle and in humans, resulting in economic losses in the agricultural sector and representing a major threat to public health. Elk populations in the American Northwest are reservoirs for this bacterium and transmit the agent to domestic cattle herds. One potential strategy to mitigate the transmission of brucellosis by elk is vaccination of elk populations against B. abortus; however, elk appear to be immunologically distinct from cattle in their responses to current vaccination strategies. The differences in host response to B. abortus between cattle and elk could be attributed to differences between the cattle and elk innate and adaptive immune responses. Because species-specific interactions between the host microbiome and the immune system are also known to affect immunity, we sought to investigate interactions between the elk microbiome and B. abortus infection and vaccination. Methods: We analyzed the fecal and vaginal microbial communities of B. abortus-vaccinated and unvaccinated elk which were challenged with B. abortus during the periparturient period. Results: We observed that the elk fecal and vaginal microbiota are similar to those of other ruminants, and these microbial communities were affected both by time of sampling and by vaccination status. Notably, we observed that taxa representing ruminant reproductive tract pathogens tended to increase in abundance in the elk vaginal microbiome following parturition. Furthermore, many of these taxa differed significantly in abundance depending on vaccination status, indicating that vaccination against B. abortus affects the elk vaginal microbiota with potential implications for animal reproductive health. Discussion: This study is the first to analyze the vaginal microbiota of any species of the genus Cervus and is also the first to assess the effects of B. abortus vaccination and challenge on the vaginal microbiome.

Effect of selection genotype on immune response to Brucella abortus RB51 in Holstein cattle.

Genetic selection for milk production traits in US Holsteins has affected numerous genes associated with reproduction and immunity. This study compares the transcriptomic response of peripheral blood mononuclear cells to an in vitro Brucella abortus strain RB51 (RB51) bacterial challenge between contemporary Holsteins and Holsteins that have not been selected for milk production traits since the mid-1960s. Total RNA was extracted from peripheral blood mononuclear cells from four contemporary and four unselected lactating, primiparous cows following 24-h incubation with or without stimulation with RB51 bacteria. RNA was sequenced and reads analyzed using tools from galaxy.scinet.usda.gov. A total of 412 differentially expressed genes (false discovery rate p < 0.05, log fold change > |1|) were identified. The upregulated genes (genes with higher expression in contemporary than unselected cattle) were enriched for 19 terms/pathways, including alanine, aspartate, and glutamate metabolism, indicating a cellular stress response. Downregulated genes (genes with higher expression in unselected than contemporary cows) were enriched for 37 terms/pathways, representing diverse immune responses, including natural killer cell-mediated immunity, interferon-γ production, negative regulation of interleukin-10 production, and cytokine receptor activity indicating a broad immune response with an emphasis on immune defense. These results provide evidence that differences exist between the two genotypes in response to in vitro bacterial challenge. This suggests that contemporary cows, genetically selected for milk production, may have reduced immune function, including limitations in response to intracellular bacteria.

Wildlife Immune Responses to Mycobacterium bovis and to Bacille of Calmette-Guerin.

Bovine tuberculosis (bTB) is a zoonotic bacterial disease presenting public health, veterinary, and economic threats around the globe. Although cattle producers rely on regular testing and management practices to minimize domestic herd exposure, wildlife species around the world continue to be the main reservoirs for disease. Wildlife reservoirs for bTB include the Eurasian badger (Meles meles) in Great Britain and Ireland, the brushtail possum (Trichosurus vulpecula) in New Zealand, wild boar (Sus scrofa) in Spain, as well as white-tailed deer (Odocoileus virginianus) in the United States and red deer (Cervus elaphus) in Spain. Although all reservoir species share the ability to infect cattle, they differ in transmission capability, disease pathogenesis, diagnostic detection, and vaccination strategies. In this review, bTB interactions with these wildlife reservoirs are discussed, illustrating the need to address bTB disease in wildlife hosts to achieve eradication in domestic livestock.

Effects of concurrent administration of modified live viral vaccines with RB51 on immune responses to RB51.

Brucella abortus is a gram negative, zoonotic pathogen that can cause abortions and stillbirths in the cattle industry and has contributed to significant economic losses to cow-calf producers. Cell mediated immunity (CMI) is an important component of the immune response associated with protection against Brucella abortus and other intracellular pathogens. Brucellosis and viral modified live vaccines (vMLV) are licensed individually but may be used concurrently under field conditions. Peripheral blood mononuclear cells (PBMC) from non-vaccinated cattle and cattle vaccinated with either Brucella abortus strain RB51, a vMLV or both RB51 and a vMLV vaccine were isolated. The frequency of CD4+, CD8+ and γδ+ T cell populations within PBMC, and the frequency of interferon gamma (IFN-γ) production within these cell types was characterized via flow-cytometry. The goal of this study was to characterize immune responses to RB51 vaccination and determine the effect of concurrent vaccine administration. Although immune responses were greatest in PBMC from cattle vaccinated with only RB51, cattle vaccinated with both RB51 and vMLV demonstrated measurable T cell responses associated with protective immunity. Data suggests a lack of significant biological differences between the groups in protective immune responses. Collectively, our data demonstrated a lack of vaccine interference following concurrent administration of vMLV and RB51. Although concurrent administration of individually licensed vaccines may influence immune responses and contribute to vaccine interference, potential vaccine combinations should be evaluated for biological effects.

Upper Extremity Musculoskeletal Injuries Presenting to an Emergency Department Before and During the Initial COVID-19 Pandemic Surge.

Study Objective: The SARS-CoV-2 (COVID-19) pandemic significantly impacted emergency department volume and acuity. The Delta and Omicron variants contributed to additional surges. We describe the impact that the initial pandemic phase had on frequency and severity of typically non-life-threatening emergencies using upper extremity injuries as a model for other potentially emergent presentation as compared to pre-pandemic times. We do this using the epidemiology of pre-defined significant upper extremity injuries at our facility as a specific example of what occurred at an urban trauma center. Methods: We conducted a comparison of two 6-month periods: between March 2019 and August 2019 (prior to COVID-19) and between March 2020 and August 2020 after the onset of the initial COVID-19 wave. We performed a retrospective chart review of patients who presented with upper extremity injury chief complaints using analysis of the electronic medical record at a single urban tertiary care trauma center in the Midwestern United States. We investigated examination findings, imaging, frequency of surgical procedures and final diagnosis. Results: In the 2019 study period, there were 31,157 ED patients, including 429 with upper extremity injuries, of which 108 patients had significant injuries. In the 2020 study period, there were 24,295 patient presentations, of which 118 of 296 upper extremity presentations were significant. We a priori defined significant injury as follows: fractures, dislocations, neurovascular injuries, or need for operative intervention within 24 hours of ED presentation. Specifically, 25.2% of injuries were significant pre-COVID-19 and 39.9% (p < 0.001) during the initial COVID-19 surge. The absolute number and percentage of significant injuries increased from pre-COVID-19 compared to the initial COVID-19 surge despite an overall 22% decrease in total patient volume. Conclusion: The incidence of significant upper extremity musculoskeletal injuries increased during the pandemic even though the overall number of ED presentations for upper extremity musculoskeletal injuries decreased.

Pulmonary hamartoma in an elk calf.

Hamartomas are benign tumor-like lesions composed of disorganized growth of mature mesenchymal or epithelial tissues indigenous to the organ involved. Sporadically observed in ruminants, vascular, fibrous, nasal, and pulmonary hamartomas have been reported in calves; pulmonary and cutaneous forms have been reported in sheep. A full-term elk calf found dead had a large intrathoracic mass replacing the left caudal lung lobe and compressing other thoracic organs. Histologically, cross- and tangential sections of bronchi were separated by collagenous mesenchyme and irregularly shaped canaliculi and saccules resembling terminal bronchioles. Rarely present were regions in which saccules, lined by simple cuboidal epithelium, transitioned into attenuated epithelium lining fully developed alveoli. These findings are consistent with a pulmonary hamartoma. To our knowledge, pulmonary hamartoma has not been reported previously in a non-domestic ruminant.

Utilizing a CBPR approach to assess the impact of COVID-19 on individuals who receive publicly funded behavioral health services.

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.

Real-World Clinical Outcomes after Genomic Profiling of Circulating Tumor DNA in Patients with Previously Treated Advanced Non-Small Cell Lung Cancer.

Comprehensive genomic profiling for advanced non-small cell lung cancer (NSCLC) can identify patients for molecularly targeted therapies that improve clinical outcomes. We analyzed data from 3084 patients (median age 65 years, 72.9% with adenocarcinoma) with advanced NSCLC registered in a real-world healthcare claims database (GuardantINFORMTM, Guardant Health) who underwent next-generation sequencing (NGS)-based circulating tumor DNA (ctDNA) testing (Guardant360®, Guardant Health) after first-line therapy (28.0% with agents targeted against genomic alterations). ctDNA was detected in 2771 samples (89.9%), of which 41.9% harbored actionable alterations, most commonly EGFR (epidermal growth factor receptor) mutations (29.7%). Actionable alterations were detected in 26.7% of patients (534/2001) previously treated with non-targeted agents. Emerging potentially targetable mutations were found in 40.1% (309/770) of patients previously treated with targeted therapies. Among patients with qualifying alterations detected by ctDNA testing, the time to treatment discontinuation (median 8.8 vs. 4.2 months; hazard ratio 1.97, p < 0.001) and overall survival (median 36.1 vs. 16.6 months; hazard ratio 2.08, p < 0.001) were longer for those who received matched second-line therapy versus unmatched second-line therapy. In real-world practice, results of a blood-based NGS assay prior to second-line treatment inform therapeutic decisions that can improve clinical outcomes for patients with advanced NSCLC.

Comprehensive Genomic Profiling of Circulating Tumor DNA in Patients with Previously Treated Metastatic Colorectal Cancer: Analysis of a Real-World Healthcare Claims Database.

We used a real-world database (GuardantINFORMTM) to analyze the treatment choices for patients with mCRC who underwent next-generation sequencing of circulating tumor DNA (ctDNA) using a commercially available test (Guardant360®) after first- or second-line therapy. From 18,875 patients with claims for CRC, 1064 had confirmed metastatic disease and sufficient histories for analysis (median age 59 years, 44.8% female, 44.5% left-sided). ctDNA was detectable for 997/1064 (93.7%) patients. Clinically actionable molecular profiles were present for 507/1064 (47.7%) patients, including those who had not received targeted therapy in the previous line (410/926, 44.3%). Second- or third-line targeted therapies were administered to 338/1064 patients (31.8%) and were considered matched for 193/338 (57.1%) patients. Therapies administered after testing were informed by the ctDNA results in 56.7% of patients overall (603/1064). Time to treatment discontinuation was most favorable for patients with a clinically actionable ctDNA profile who received matched therapy. This analysis demonstrates the real-world clinical value of plasma-based comprehensive genomic profiling for selecting appropriate molecular-targeted therapies in mCRC patients with disease progression after first- or second-line therapy.

Behavioral economics in neonatology-balancing provider wellness and departmental finances.

High work relative value units (wRVU) per clinical full-time-equivalent (cFTE) productivity by Neonatologists have played a key role in enhancing departmental revenue in Pediatrics. However, such high productivity is not sustainable due to recent changes in trainee schedules and global daily codes and is likely to impact physician morale and wellness. Incentives based on wRVU benchmarks have the capacity to promote desirable behavior such as better documentation and in-person attendance in delivery room resuscitation and consults but comes at a cost of physician time providing care. An alternate method of funding academic Pediatric departments using time- or point-based staffing models, a reduction in productivity benchmarks for academic neonatologists through more accurate reporting of effort and physician leadership that promotes transparency and mutual respect are warranted to improve neonatologist well-being and morale.