The bovine nasal fungal community and associations with bovine respiratory disease.

Introduction: Effective identification and treatment of bovine respiratory disease (BRD) is an ongoing health and economic issue for the dairy and beef cattle industries. Bacteria pathogens Pasteurellamultocida, Mycoplasmabovis, Mannheimia haemolytica, and Histophilus somni and the virus Bovine herpesvirus-1 (BHV-1), Bovine parainfluenza-3 virus (BPIV-3), Bovine respiratory syncytial virus (BRSV), Bovine adenovirus 3 (BAdV3), bovine coronavirus (BoCV) and Bovine viral diarrhea virus (BVDV) have commonly been identified in BRD cattle; however, no studies have investigated the fungal community and how it may also relate to BRD. Methods: The objective of this study was to understand if the nasal mycobiome differs between a BRD-affected (n = 56) and visually healthy (n = 73) Holstein steers. Fungal nasal community was determined by using Internal Transcribed Spacer (ITS) sequencing. Results: The phyla, Ascomycota and Basidiomycota, and the genera, Trichosporon and Issatchenkia, were the most abundant among all animals, regardless of health status. We identified differences between healthy and BRD animals in abundance of Trichosporon and Issatchenkia orientalis at a sub-species level that could be a potential indicator of BRD. No differences were observed in the nasal fungal alpha and beta diversity between BRD and healthy animals. However, the fungal community structure was affected based on season, specifically when comparing samples collected in the summer to the winter season. We then performed a random forest model, based on the fungal community and abundance of the BRD-pathobionts (qPCR data generated from a previous study using the same animals), to classify healthy and BRD animals and determine the agreement with visual diagnosis. Classification of BRD or healthy animals using ITS sequencing was low and agreed with the visual diagnosis with an accuracy of 51.9%. A portion of the ITS-predicted BRD animals were not predicted based on the abundance of BRD pathobionts. Lastly, fungal and bacterial co-occurrence were more common in BRD animals than healthy animals. Discussion: The results from this novel study provide a baseline understanding of the fungal diversity and composition in the nasal cavity of BRD and healthy animals, upon which future interaction studies, including other nasal microbiome members to further understand and accurately diagnose BRD, can be designed.

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.

A paper-based colorimetric molecular test for SARS-CoV-2 in saliva.

Herein, we describe the development of a paper-based device to detect nucleic acids of pathogens of interest in complex samples using loop-mediated isothermal amplification (LAMP) by producing a colorimetric response visible to the human eye. To demonstrate the utility of this device in emerging public health emergencies, we developed and optimized our device to detect SARS-CoV-2 in human saliva without preprocessing. The resulting device was capable of detecting the virus within 60 min and had an analytical sensitivity of 97% and a specificity of 100% with a limit of detection of 200 genomic copies/µL of patient saliva using image analysis. The device consists of a configurable number of reaction zones constructed of Grade 222 chromatography paper separated by 20 mil polystyrene spacers attached to a Melinex® backing via an ARclean® double-sided adhesive. The resulting device is easily configurable to detect multiple targets and has the potential to detect a variety of pathogens simply by changing the LAMP primer sets.