Development of a Targeted NGS Assay for the Detection of Respiratory Pathogens including SARS-CoV-2 in Felines.

Acute respiratory diseases in felines can be attributed to a diverse range of pathogens. The recent emergence of novel viruses, particularly SARS-CoV-2 and its variants, has also been associated with respiratory ailments in cats and other pets, underscoring the need for a highly sensitive diagnostic assay capable of concurrently detecting multiple respiratory pathogens. In this study, we developed a targeted next generation sequencing panel using Ion Torrent Ampliseq technology to detect multiple respiratory pathogens, including recent SARS-CoV-2 variants and Feline herpesvirus-1, Feline calicivirus, Bordetella bronchiseptica, Mycoplasmopsis (previously Mycoplasma) felis, and Chlamydia felis. A PCR amplification-based library preparation, employing primers designed for pathogen target regions, was synthesized and divided into two pools, followed by sequencing and assembly to a repertoire of target pathogen genomes. Analytical sensitivity was assessed based on Ct values from real-time PCR for the corresponding pathogens, indicating an equivalent detection limit. Most of the pathogens under study were positively identified to a limit of approximately Ct 36, whereas for Feline herpesvirus-1 and SARS-CoV-2, positive reads were observed in samples with a Ct of 37. Based on a limited number of samples, the diagnostic sensitivity values for the SARS-CoV-2, Feline herpesvirus-1, and M. felis samples were 100% with no false negative results. The diagnostic specificity of SARS-CoV-2, Feline herpesvirus-1, Feline calicivirus, and C. felis were 100%. Importantly, none of the target primers exhibited non-specific amplification, ensuring the absence of false positive results for other pathogens within the study. Additionally, the assay's specificity was validated by cross-referencing the raw sequencing data with established databases like BLAST, affirming the high specificity of the targeted Next-Generation Sequencing (tNGS) assay. Variations in the sequencing reads of different pathogens were observed when subjected to diverse extraction methods. Rigorous assessment of the assay's reliability involved reproducibility across testing personnel and repeated runs. The developed assay's clinical applicability was tested using samples submitted to the diagnostic laboratory from cat shelters and suspected cases. The developed targeted next-generation sequencing methodology empowers the detection of multiple respiratory pathogens manifesting similar clinical symptoms while offering confirmation of results through genome sequencing.

Moraxella oculi sp. nov., isolated from a cow with infectious bovine keratoconjunctivitis.

A novel species of the genus Moraxella was isolated from an ocular swab from a cow with infectious bovine keratoconjunctivitis. 16S rRNA gene sequencing suggested this species was Moraxella bovis (99.59 % nucleotide identity). Average nucleotide identity was calculated using a draft whole genome sequence of this strain compared with type strains of closely related Moraxella species and results established that it represents a new species. The genome size was 2 006 474 nucleotides and the G+C content was 42.51 mol%. The species could not be identified by matrix assisted laser desorption/ionization-time of flight mass spectrometry using a commercial database, confirming the novelty of the strain. We propose the name Moraxella oculi sp. nov. for this new species. The type strain is Tifton1T and has been deposited into the American Type Culture Collection (TSD-373T) and the National Collection of Type Cultures (NCTC), UK Health Security Agency (NCTC 14942T).

Investigating the rise of Omicron variant through genomic surveillance of SARS-CoV-2 infections in a highly vaccinated university population.

Novel variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to emerge as the coronavirus disease 2019 (COVID-19) pandemic extends into its fourth year. Understanding SARS-CoV-2 circulation in university populations is vital for effective interventions in higher education settings and will inform public health policy during pandemics. In this study, we generated 793 whole-genome sequences collected over an entire academic year in a university population in Indiana, USA. We clearly captured the rapidity with which Delta variant was wholly replaced by Omicron variant across the West Lafayette campus over the length of two academic semesters in a community with high vaccination rates. This mirrored the emergence of Omicron throughout the state of Indiana and the USA. Further, phylogenetic analyses demonstrated that there was a more diverse set of potential geographic origins for Omicron viruses introduction into campus when compared to Delta. Lastly, statistics indicated that there was a more significant role for international and out-of-state migration in the establishment of Omicron variants at Purdue. This surveillance workflow, coupled with viral genomic sequencing and phylogeographic analyses, provided critical insights into SARS-CoV-2 transmission dynamics and variant arrival.

Targeted next-generation sequencing assay to detect 3 Moraxella spp. directly from bovine ocular swabs.

Infectious bovine keratoconjunctivitis (IBK) is associated with 2 species of Moraxella: M. bovis and M. bovoculi. A third novel Moraxella spp., designated tentatively as M. oculi, has been identified from the eyes of cattle with and without pinkeye. These 3 Moraxella spp. can be found in various combinations within the same clinical sample, making speciation of this genus directly from a sample impossible with Sanger sequencing. Assessing Moraxella diversity found in IBK- and non-IBK-affected cattle eyes, independent of culture, may provide additional information about IBK by avoiding the selectivity bias of culturing. We developed a targeted NGS panel to detect and speciate these 3 Moraxella spp. directly from bovine ocular swabs. Our targeted panel amplifies bacterial essential genes and the 16S-23S ribosomal RNA intergenic spacer region (ITS) of the 3 Moraxella spp. and speciates based on these sequences. Our panel was able to differentiate the 3 species directly from DNA extracted from 13 swabs (6 from healthy animals, 7 from animals with IBK), and every swab except one (clinically healthy eye) had the 3 Moraxella spp. Targeted NGS with sequencing of Moraxella spp. housekeeping genes appears to be a suitable method for speciation of Moraxella directly from ocular swabs.

Investigation of the pathogens contributing to naturally occurring outbreaks of infectious bovine keratoconjunctivitis (pinkeye) using Next Generation Sequencing.

Infectious bovine keratoconjunctivitis (IBK), commonly known as pinkeye, has a marked negative impact on the economy of the cattle industry. Moraxella species, including Mor. bovis and Mor. bovoculi, which have been associated with this disease, colonize clinically healthy eyes as well, suggesting that there are intrinsic changes that may occur to the ocular microbiota or the involvement of additional unrecognized organisms that contribute to IBK. To evaluate this, 104 ocular swabs collected from eyes with IBK or clinically healthy eyes from 16 different cattle herds were subjected to 16 S rRNA gene PCR and next generation sequencing (NGS) analysis. Organisms detected were similar across the herds and there was no difference in the total number of bacterial groups detected among IBK cases and controls. However, the percentages of the different organisms detected varied between the two groups, including Moraxella spp., with more Moraxella spp. in eyes with IBK than controls. Further, using culture and whole genome NGS, a new species of Moraxella (suggested name Mor. oculobovii) was detected from the eyes of cattle from two farms. This strain is non-hemolytic on blood agar, is missing the RTX operon, and is likely a non-pathogenic strain of the bovine ocular microbiome. Alteration of the ocular microbiota composition may have a predisposing role, enhancing bacterial infection and the occurrence of clinical IBK. Future studies are required to evaluate if these changes are permanent or if there is a shift in the microbiome following recovery from the infection and how antibiotics might affect the microbiome.

Targeted Next-Generation Sequencing for Comprehensive Testing for Selected Vector-Borne Pathogens in Canines.

The standard for detecting vector-borne pathogens is real-time PCR (rtPCR). However, this requires many individual tests to obtain an accurate diagnosis. The purpose of this study was to develop and validate a targeted next-generation sequencing (NGS) assay for vector-borne pathogens. Pathogen target regions were amplified via PCR using two primer pools that were developed in conjunction with ThermoFisher Scientific, and barcoded DNA libraries were prepared and sequenced with the Ion Torrent S5 system. Data were assembled using SPAdes and mapped to a reference file containing sequences from the pathogens. The raw reads were analyzed to confirm the results. Test feasibility and analytical specificity were evaluated with type strains or validated positive clinical samples from dogs. The analytical sensitivity of the method was compared to Ct values obtained by rtPCR testing. Diagnostic sensitivity and specificity were assessed with a set of known positive and negative clinical samples based on rtPCR testing. Positive and negative percent agreements and Cohen's kappa were calculated. The primer sets were specific for the intended targets, based on sequence analysis of the amplified products, and the method detected 17 different pathogens. Analytical sensitivity was equivalent to an rtPCR Ct value of approximately 35-36. The positive percent agreement was 92%, and the negative percent agreement was 88%. Cohen's kappa was 0.804, which indicates almost perfect agreement between the rtPCR assays and the targeted NGS assay. Using a targeted method reduces the costs associated with NGS sequencing and allows for a 2-3 day turn-around time, making this a viable method for detection of vector-borne pathogens in canine whole blood samples.

Genomic Surveillance of SARS-CoV-2 in a University Community: Insights Into Tracking Variants, Transmission, and Spread of Gamma (P.1) Variant.

Background: Using a combination of data from routine surveillance, genomic sequencing, and phylogeographic analysis, we tracked the spread and introduction events of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants focusing on a large university community. Methods: Here, we sequenced and analyzed 677 high-quality SARS-CoV-2 genomes from positive RNA samples collected from Purdue University students, faculty, and staff who tested positive for the virus between January 2021 and May 2021, comprising an average of 32% of weekly cases across the time frame. Results: Our analysis of circulating SARS-CoV-2 variants over time revealed periods when variants of concern (VOC) Alpha (B.1.1.7) and Iota (B.1.526) reached rapid dominance and documented that VOC Gamma (P.1) was increasing in frequency as campus surveillance was ending. Phylodynamic analysis of Gamma genomes from campus alongside a subsampling of >20 000 previously published P.1 genomes revealed 10 independent introductions of this variant into the Purdue community, predominantly from elsewhere in the United States, with introductions from within the state of Indiana and from Illinois, and possibly Washington and New York, suggesting a degree of domestic spread. Conclusions: We conclude that a robust and sustained active and passive surveillance program coupled with genomic sequencing during a pandemic offers important insights into the dynamics of pathogen arrival and spread in a campus community and can help guide mitigation measures.

Analysis of structure-function relationship in porcine rotavirus A enterotoxin gene.

Rotavirus (RV)-infected piglets are presumed to be latent sources of heterologous RV infection in humans and other animals. In RVs, non-structural protein 4 (NSP4) is the major virulence factor with pleiotropic properties. In this study, we analyzed the nsp4 gene from porcine RVs isolated from diarrheic and non-diarrheic cases at different levels of protein folding to explore correlations to diarrhea-inducing capabilities and evolution of nsp4 in the porcine population. Full-length nsp4 genes were amplified, cloned, sequenced, and then analyzed for antigenic epitopes, RotaC classification, homology, genetic relationship, modeling of NSP4 protein, and prediction of post-translational modification. RV presence was observed in both diarrheic and non-diarrheic piglets. All nsp4 genes possessed the E1 genotype. Comparison of primary, secondary, and tertiary structure and the prediction of post-translational modifications of NSP4 from diarrheic and non-diarrheic piglets revealed no apparent differences. Sequence analysis indicated that nsp4 genes have a multi-phyletic evolutionary origin and exhibit species independent genetic diversity. The results emphasize the evolution of the E9 nsp4 genotype from the E1 genotype and suggest that the diarrhea-inducing capability of porcine RVs may not be exclusively linked to its enterotoxin gene.