Self-Assessment of Gram Staining Skill Using Video Recording in a Classroom Environment for Veterinary Nursing Students.

Practical skills are essential in the veterinary nursing curriculum. Given the increasing implementation of video recording in higher education, this study explored the feasibility and benefits of video recording as a classroom tool in professional education. Concerns regarding the inability to monitor individual students' performance during their laboratory course promoted the implementation of video recording-a blended learning method-in a veterinary nursing course. The approach was personalized for this study, particularly for the Gram staining skill. Students submitted video recordings demonstrating the progression of their skills development, and the instructor reviewed the recordings for assessment. The Participant Perception Indicator, a self-assessment, was used to determine students' experience, knowledge, and confidence gained after performing the skill. Video recording helped students to identify areas for self-improvement. It is also a helpful tool for instructors to ensure that students are meeting the learning standards. The results suggest that the use of video recording in learning Gram staining skills was effective. The evidence-based approach maximized students' learning and engagement, and it improved individualized assessment by the instructor and enabled the instructor to provide feedback on students' performance. During this period of increasing reliance on online teaching and learning, video recording in a classroom environment could be more widely used by instructors.

On-farm colorimetric detection of Pasteurella multocida, Mannheimia haemolytica, and Histophilus somni in crude bovine nasal samples.

This work modifies a loop-mediated isothermal amplification (LAMP) assay to detect the bovine respiratory disease (BRD) bacterial pathogens Pasteurella multocida, Mannheimia haemolytica, and Histophilus somni in a colorimetric format on a farm. BRD causes a significant health and economic burden worldwide that partially stems from the challenges involved in determining the pathogens causing the disease. Methods such as polymerase chain reaction (PCR) have the potential to identify the causative pathogens but require lab equipment and extensive sample processing making the process lengthy and expensive. To combat this limitation, LAMP allows accurate pathogen detection in unprocessed samples by the naked eye allowing for potentially faster and more precise diagnostics on the farm. The assay developed here offers 66.7-100% analytical sensitivity, and 100% analytical specificity (using contrived samples) while providing 60-100% concordance with PCR results when tested on five steers in a feedlot. The use of a consumer-grade water bath enabled on-farm execution by collecting a nasal swab from cattle and provided a colorimetric result within 60 min. Such an assay holds the potential to provide rapid pen-side diagnostics to cattle producers and veterinarians.

Leukotoxin production by Fusobacterium necrophorum strains in relation to severity of liver abscesses in cattle.

Fusobacterium necrophorum, a Gram-negative anaerobe, is the primary etiologic agent of liver abscesses of beef cattle. The bacterium, a member of the microbial community of the rumen, travels to the liver via portal circulation to cause abscesses. The severity of liver abscesses vary from mild with one or two small abscesses to severe with medium to large multiple abscesses. Leukotoxin, a secreted protein, is the critical virulence factor involved in the infection. Our objective was to compare leukotoxin production between strains of F. necrophorum isolated from mild and severe liver abscesses collected from slaughtered cattle. The quantification of leukotoxin was based on assays to measure cytotoxicity and protein antigen concentration. One-hundred strains, 50 from mild and 50 from severe abscesses, were utilized in the study. Cell-free supernatants were prepared from cultures grown in anaerobic broth at 9 and 24 h incubations. The leukotoxic activity was quantified by measuring cytotoxicity based on the release of lactic dehydrogenase from bovine lymphocyte cells, BL3, treated with the culture supernatant. Leukotoxin protein concentration was quantified by a sandwich ELISA assay with a leukotoxin-specific monoclonal antibody as the capture antibody. The leukotoxin activity and concentration were highly variable among the strains within each severity of liver abscesses. Although the leukotoxic activity was unaffected by incubation time, leukotoxin protein concentration was consistently higher at 24 h compared to 9 h incubation. Strains from severe liver abscesses had significantly higher leukotoxic activity and higher protein concentration compared to strains from mild liver abscesses (P < 0.0001) at both 9 and 24 h culture supernatants. Across all strains, the correlation coefficients between leukotoxic activity and leukotoxin concentration at 9 and 24 h were 0.14 (P = 0.17) and 0.47 (P < 0.0001), respectively. In conclusion, strains isolated from severe liver abscesses had significantly higher leukotoxic activities and leukotoxin protein concentrations compared to strains isolated from mild liver abscesses.

Leukotoxic activity of Fusobacterium necrophorum of cattle origin.

Fusobacterium necrophorum is a Gram negative, rod-shaped and aero tolerant anaerobe. In animals, it is an opportunistic pathogen frequently associated with necrotic infections, generally called necrobacillosis, such as calf diphtheria, foot rot and liver abscesses in cattle. Two subspecies exist: subsp. necrophorum and subsp. funduliforme. Among several virulence factors, leukotoxin (Lkt) is considered to be a major factor and a protective antigen. The objective of the study was to utilize BL3 cells and measure the release of lactic dehydrogenase to quantify Lkt activity of F. necrophorum. The assay was used to examine the effects of storage and handling conditions, growth media, polymyxin B addition on the cytotoxicity and evaluate Lkt activities of F. necrophorum strains isolated from bovine liver abscesses and foot rot. The Lkt activity peaked at 9 h of incubation. There was a significant decrease in the cytotoxicity measured in the samples after each freeze and thaw cycle. No difference was observed in the cytotoxicity for the samples handled aerobically versus anaerobically. Lkt activities of strains grown in anaerobic Brain-Heart Infusion broth were higher compared to Vegitone broth. A small reduction in the cytotoxicity activity was observed after the addition of polymyxin. The Lkt activity was consistently higher in strains of subsp. necrophorum than subsp. funduliforme of liver abscess origin. Among the strains isolated from cattle foot rot, Lkt activities of subsp. necrophorum strains appear to be much more variable. Use of BL3 cells in combination of lactic acid dehydrogenase assay appears to be a simple and valid assay to measure Lkt activity of F. necrophorum.

Outer-Membrane Vesicles of Fusobacterium necrophorum: A Proteomic, Lipidomic, and Functional Characterization.

Outer-membrane vesicles (OMVs) are extruded nanostructures shed by Gram-negative bacteria, containing periplasmic contents, and often including virulence factors with immunogenic properties. To assess their potential for use in vaccine development, we purified OMVs from the Fusobacterium necrophorum subspecies necrophorum, an opportunistic necrotic infection-causing pathogen, and characterized these structures using proteomics, lipid-profiling analyses, and cytotoxicity assays. A proteomic analysis of density-gradient-purified F. necrophorum OMVs identified 342 proteins, a large proportion of which were outer-membrane proteins (OMPs), followed by cytoplasmic proteins, based on a subcellular-localization-prediction analysis. The OMPs and toxins were among the proteins with the highest intensity identified, including the 43-kDa-OMP-, OmpA-, and OmpH-family proteins, the cell-surface protein, the FadA adhesin protein, the leukotoxin-LktA-family filamentous adhesin, the N-terminal domain of hemagglutinin, and the OMP transport protein and assembly factor. A Western blot analysis confirmed the presence of several OMPs and toxins in the F. necrophorum OMVs. The lipid-profiling analysis revealed phospholipids, sphingolipids, and acetylcarnitine as the main lipid contents of OMVs. The lactate-dehydrogenase-cytotoxicity assays showed that the OMVs had a high degree of cytotoxicity against a bovine B-lymphocyte cell line (BL-3 cells). Thus, our data suggest the need for further studies to evaluate the ability of OMVs to induce immune responses and assess their vaccine potential in vivo.

Characterization of Three New Outer Membrane Adhesion Proteins in Fusobacterium necrophorum.

Fusobacterium necrophorum, an anaerobic Gram-negative pathogen, causes necrotic cattle infections, impacting livestock health and the US feedlot industry. Antibiotic administration is the mainstay for treating F. necrophorum infections, although resistance hampers their effectiveness. Vaccination, especially targeting outer membrane proteins (OMPs) due to their antigenic properties and host specificity, offers an alternative to antibiotics. This study identified high-binding-affinity adhesion proteins from F. necrophorum using binding and pull-down assays with bovine adrenal gland endothelial cells (EJG). Four OMP candidates (17.5 kDa/OmpH, 22.7 kDa/OmpA, 66.3 kDa/cell surface protein (CSP), and a previously characterized 43 kDa OMP) were expressed as recombinant proteins and purified. Rabbit polyclonal antibodies to recombinant OMPs were generated, and their ability to inhibit bacterial binding in vitro was assessed. The results show that treatment with individual polyclonal antibodies against 43 kDa significantly inhibited bacterial adhesion, while other antibodies were less potent. However, combinations of two or more antibodies showed a more prominent inhibitory effect on host-cell adhesion. Thus, our findings suggest that the identified OMPs are involved in fusobacterial attachment to host cells and may have the potential to be leveraged in combination for vaccine development. Future in vivo studies are needed to validate their roles and test the feasibility of an OMP-based subunit vaccine against fusobacterial infections.

Comparative Genomic Analysis of Fusobacterium necrophorum Provides Insights into Conserved Virulence Genes.

Fusobacterium necrophorum is a Gram-negative, filamentous anaerobe prevalent in the mucosal flora of animals and humans. It causes necrotic infections in cattle, resulting in a substantial economic impact on the cattle industry. Although infection severity and management differ within F. necrophorum species, little is known about F. necrophorum speciation and the genetic virulence determinants between strains. To characterize the clinical isolates, we performed whole-genome sequencing of four bovine isolates (8L1, 212, B17, and SM1216) and one human isolate (MK12). To determine the phylogenetic relationship and evolution pattern and investigate the presence of antimicrobial resistance genes (ARGs) and potential virulence genes of F. necrophorum, we also performed comparative genomics with publicly available Fusobacterium genomes. Using up-to-date bacterial core gene (UBCG) set analysis, we uncovered distinct Fusobacterium species and F. necrophorum subspecies clades. Pangenome analyses revealed a high level of diversity among Fusobacterium strains down to species levels. The output also identified 14 and 26 genes specific to F. necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme, respectively, which could be essential for bacterial survival under different environmental conditions. ClonalFrameML-based recombination analysis suggested that extensive recombination among accessory genes led to species divergence. Furthermore, the only strain of F. necrophorum with ARGs was F. necrophorum subsp. funduliforme B35, with acquired macrolide and tetracycline resistance genes. Our custom search revealed common virulence genes, including toxins, adhesion proteins, outer membrane proteins, cell envelope, type IV secretion system, ABC (ATP-binding cassette) transporters, and transporter proteins. A focused study on these genes could help identify major virulence genes and inform effective vaccination strategies against fusobacterial infections. IMPORTANCE Fusobacterium necrophorum is an anaerobic bacterium that causes liver abscesses in cattle with an annual incidence rate of 10% to 20%, resulting in a substantial economic impact on the cattle industry. The lack of definite biochemical tests makes it difficult to distinguish F. necrophorum subspecies phenotypically, where genomic characterization plays a significant role. However, due to the lack of a good reference genome for comparison, F. necrophorum subspecies-level identification represents a significant challenge. To overcome this challenge, we used comparative genomics to validate clinical test strains for subspecies-level identification. The findings of our study help predict specific clades of previously uncharacterized strains of F. necrophorum. Our study identifies both general and subspecies-specific virulence genes through a custom search-based analysis. The virulence genes identified in this study can be the focus of future studies aimed at evaluating their potential as vaccine targets to prevent fusobacterial infections in cattle.

Identification of bovine respiratory disease through the nasal microbiome.

BACKGROUND: Bovine respiratory disease (BRD) is an ongoing health and economic challenge in the dairy and beef cattle industries. Multiple risk factors make an animal susceptible to BRD. The presence of Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and Mycoplasma bovis in lung tissues have been associated with BRD mortalities, but they are also commonly present in the upper respiratory tract of healthy animals. This study aims to compare the cattle nasal microbiome (diversity, composition and community interaction) and the abundance of BRD pathogens (by qPCR) in the nasal microbiome of Holstein steers that are apparently healthy (Healthy group, n = 75) or with BRD clinical signs (BRD group, n = 58). We then used random forest models based on nasal microbial community and qPCR results to classify healthy and BRD-affected animals and determined the agreement with the visual clinical signs. Additionally, co-occurring species pairs were identified in visually BRD or healthy animal groups. RESULTS: Cattle in the BRD group had lower alpha diversity than pen-mates in the healthy group. Amplicon sequence variants (ASVs) from Trueperella pyogenes, Bibersteinia and Mycoplasma spp. were increased in relative abundance in the BRD group, while ASVs from Mycoplasma bovirhinis and Clostridium sensu stricto were increased in the healthy group. Prevalence of H. somni (98%) and P. multocida (97%) was high regardless of BRD clinical signs whereas M. haemolytica (81 and 61%, respectively) and M. bovis (74 and 51%, respectively) were more prevalent in the BRD group than the healthy group. In the BRD group, the abundance of M. haemolytica and M. bovis was increased, while H. somni abundance was decreased. Visual observation of clinical signs agreed with classification by the nasal microbial community (misclassification rate of 32%) and qPCR results (misclassification rate 34%). Co-occurrence analysis demonstrated that the nasal microbiome of BRD-affected cattle presented fewer bacterial associations than healthy cattle. CONCLUSIONS: This study offers insight into the prevalence and abundance of BRD pathogens and the differences in the nasal microbiome between healthy and BRD animals. This suggests that nasal bacterial communities provide a potential platform for future studies and potential pen-side diagnostic testing.

Role of the stress-associated chemicals norepinephrine, epinephrine and substance P in dispersal of Mannheimia haemolytica from biofilms.

Bovine respiratory disease (BRD) is a major problem for the cattle industry that is triggered by various environmental stressors, pathogens and host responses. Mannheimia hemolytica, an important bacterial component of BRD, are present within the nasopharayngeal region of normal calves as commensal biofilm communities. However, following stress there are changes in the nasopharyngeal microenvironment that triggers the transition of the commensal M. haemolytica into a pulmonary pathogen. The factors responsible for this transition in- vivo are unknown. In this study we developed an in-vitro biofilm model and investigated the effect of three stress- related compounds: norepinephrine (NE), epinephrine (E), and substance P (SP) on M. haemolytica biofilms. Biofilm formation was demonstrated for 3 bovine nasal isolates of M. haemolytica by growing them in basal culture media, basal media with additional glucose, and basal media with a reduced pH. Increased glucose enhanced biofilm biomass for 2/3 isolates, but acidic media did not increase biofilm biomass when compared to biofilm biomass in basal media. When the biofilm was exposed to NE, E and SP, there was a dispersal of the biofilm which was most effective with E, followed by NE, and SP being the least effective. Using high - throughput scanning electron microscopy and confocal-imaging we confirmed our experimental data that treatment with NE, E and SP cause dispersion of M.haemolytica from biofilms.