Metagenomics for Pathogen Detection During a Mass Mortality Event in Songbirds.

Mass mortality events in wildlife can be indications of an emerging infectious disease. During the spring and summer of 2021, hundreds of dead passerines were reported across the eastern US. Birds exhibited a range of clinical signs including swollen conjunctiva, ocular discharge, ataxia, and nystagmus. As part of the diagnostic investigation, high-throughput metagenomic next-generation sequencing was performed across three molecular laboratories on samples from affected birds. Many potentially pathogenic microbes were detected, with bacteria forming the largest proportion; however, no singular agent was consistently identified, with many of the detected microbes also found in unaffected (control) birds and thus considered to be subclinical infections. Congruent results across laboratories have helped drive further investigation into alternative causes, including environmental contaminants and nutritional deficiencies. This work highlights the utility of metagenomic approaches in investigations of emerging diseases and provides a framework for future wildlife mortality events.

Validation of a Field-Portable, Handheld Real-Time PCR System for Detecting Pseudogymnoascus destructans, the Causative Agent of White-Nose Syndrome in Bats.

White-nose syndrome (WNS), caused by the fungus Pseudogymnoascus destructans, has decimated bat populations across North America. Despite ongoing management programs, WNS continues to expand into new populations, including in US states previously thought to be free from the pathogen and disease. This expansion highlights a growing need for surveillance tools that can be used to enhance existing monitoring programs and support the early detection of P. destructans in new areas. We evaluated the feasibility of using a handheld, field-portable, real-time (quantitative) PCR (qPCR) thermocycler known as the Biomeme two3 and the associated field-based nucleic acid extraction kit and assay reagents for the detection of P. destructans in little brown bats (Myotis lucifugus). Results from the field-based protocol using the Biomeme platform were compared with those from a commonly used laboratory-based qPCR protocol. When using dilutions of known conidia concentrations, the lowest detectable concentration with the laboratory-based approach was 108.8 conidia/mL, compared with 1,087.5 conidia/mL (10 times higher, i.e., one fewer 10× dilution) using the field-based approach. Further comparisons using field samples suggest a high level of concordance between the two protocols, with positive and negative agreements of 98.2% and 100% respectively. The cycle threshold values were marginally higher for most samples using the field-based protocol. These results are an important step in establishing and validating a rapid, field-assessable detection platform for P. destructans, which is urgently needed to improve the surveillance and monitoring capacity for WNS and support on-the-ground management and response efforts.

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.

Multiple Introductions of SARS-CoV-2 Alpha and Delta Variants into White-Tailed Deer in Pennsylvania.

The SARS-CoV-2 pandemic began by viral spillover from animals to humans; today multiple animal species are known to be susceptible to infection. White-tailed deer, Odocoileus virginianus, are infected in North America at substantial levels, and genomic data suggests that a variant in deer may have spilled back to humans. Here, we characterize SARS-CoV-2 in deer from Pennsylvania (PA) sampled during fall and winter 2021. Of 123 nasal swab samples analyzed by RT-qPCR, 20 (16.3%) were positive for SARS-CoV-2. Seven whole genome sequences were obtained, together with six more partial spike gene sequences. These annotated as alpha and delta variants, the first reported observations of these lineages in deer, documenting multiple new jumps from humans to deer. The alpha lineage persisted in deer after its displacement by delta in humans, and deer-derived alpha variants diverged significantly from those in humans, consistent with a distinctive evolutionary trajectory in deer. IMPORTANCE Coronaviruses have been documented to replicate in numerous species of vertebrates, and multiple spillovers of coronaviruses from animals into humans have founded human epidemics. The COVID-19 epidemic likely derived from a spillover of SARS-CoV-2 from bats into humans, possibly via an intermediate host. There are now several examples of SARS-CoV-2 jumping from humans into other mammals, including mink and deer, creating the potential for new animal reservoirs from which spillback into humans could occur. For this reason, data on formation of new animal reservoirs is of great importance for understanding possible sources of future infection. Here, we identify extensive infection in white-tailed deer in Pennsylvania, including what appear to be multiple independent transmissions. Data further suggests possible transmission among deer. These data thus help identify a potential new animal reservoir and provide background information relevant to its management.

Transmission dynamics of an antimicrobial resistant Campylobacter jejuni lineage in New Zealand's commercial poultry network.

Understanding the relative contribution of different between-farm transmission pathways is essential in guiding recommendations for mitigating disease spread. This study investigated the association between contact pathways linking poultry farms in New Zealand and the genetic relatedness of antimicrobial resistant Campylobacter jejuni Sequence Type 6964 (ST-6964), with the aim of identifying the most likely contact pathways that contributed to its rapid spread across the industry. Whole-genome sequencing was performed on 167C. jejuni ST-6964 isolates sampled from across 30 New Zealand commercial poultry enterprises. The genetic relatedness between isolates was determined using whole genome multilocus sequence typing (wgMLST). Permutational multivariate analysis of variance and distance-based linear models were used to explore the strength of the relationship between pairwise genetic associations among the C. jejuni isolates and each of several pairwise distance matrices, indicating either the geographical distance between farms or the network distance of transportation vehicles. Overall, a significant association was found between the pairwise genetic relatedness of the C. jejuni isolates and the parent company, the road distance and the network distance of transporting feed vehicles. This result suggests that the transportation of feed within the commercial poultry industry as well as other local contacts between flocks, such as the movements of personnel, may have played a significant role in the spread of C. jejuni. However, further information on the historical contact patterns between farms is needed to fully characterise the risk of these pathways and to understand how they could be targeted to reduce the spread of C. jejuni.

The Genetic Relatedness and Antimicrobial Resistance Patterns of Mastitis-Causing Staphylococcus aureus Strains Isolated from New Zealand Dairy Cattle.

Staphylococcus aureus is one of the leading causes of bovine mastitis worldwide and is a common indication for use of antimicrobials on dairy farms. This study aims to investigate the association between on-farm antimicrobial usage and the antimicrobial resistance (AMR) profiles of mastitis-causing S. aureus. Whole-genome sequencing was performed on 57 S. aureus isolates derived from cows with either clinical or subclinical mastitis from 17 dairy herds in New Zealand. The genetic relatedness between isolates was examined using the core single nucleotide polymorphism alignment whilst AMR and virulence genes were identified in-silico. The association between gene presence-absence and sequence type (ST), antimicrobial susceptibility and dry cow therapy treatment was investigated using Scoary. Altogether, eight STs were identified with 61.4% (35/57) belonging to ST-1. Furthermore, 14 AMR-associated genes and 76 virulence-associated genes were identified, with little genetic diversity between isolates belonging to the same ST. Several genes including merR1 which is thought to play a role in ciprofloxacin-resistance were found to be significantly overrepresented in isolates sampled from herds using ampicillin/cloxacillin dry cow therapy. Overall, the presence of resistance genes remains low and current antimicrobial usage patterns do not appear to be driving AMR in S. aureus associated with bovine mastitis.

Using multiple data sources to explore disease transmission risk between commercial poultry, backyard poultry, and wild birds in New Zealand.

The movements of backyard poultry and wild bird populations are known to pose a disease risk to the commercial poultry industry. However, it is often difficult to estimate this risk due to the lack of accurate data on the numbers, locations, and movement patterns of these populations. The main aim of this study was to evaluate the use of three different data sources when investigating disease transmission risk between poultry populations in New Zealand including (1) cross-sectional survey data looking at the movement of goods and services within the commercial poultry industry, (2) backyard poultry sales data from the online auction site TradeMe®, and (3) citizen science data from the wild bird monitoring project eBird. The cross-sectional survey data and backyard poultry sales data were transformed into network graphs showing the connectivity of commercial and backyard poultry producers across different geographical regions. The backyard poultry network was also used to parameterise a Susceptible-Infectious (SI) simulation model to explore the behaviour of potential disease outbreaks. The citizen science data was used to create an additional map showing the spatial distribution of wild bird observations across New Zealand. To explore the potential for diseases to spread between each population, maps were combined into bivariate choropleth maps showing the overlap between movements within the commercial poultry industry, backyard poultry trades and, wild bird observations. Network analysis revealed that the commercial poultry network was highly connected with geographical clustering around the urban centres of Auckland, New Plymouth and Christchurch. The backyard poultry network was also a highly active trade network and displayed similar geographic clustering to the commercial network. In the disease simulation models, the high connectivity resulted in all suburbs becoming infected in 96.4 % of the SI simulations. Analysis of the eBird data included reports of over 80 species; the majority of which were identified as coastal seabirds or wading birds that showed little overlap with either backyard or commercial poultry. Overall, our study findings highlight how the spatial patterns of trading activity within the commercial poultry industry, alongside the movement of backyard poultry and wild birds, have the potential to contribute significantly to the spread of diseases between these populations. However, it is clear that in order to fully understand this risk landscape, further data integration is needed; including the use of additional datasets that have further information on critical variables such as environmental factors.

Genomic Analysis of Fluoroquinolone- and Tetracycline-Resistant Campylobacter jejuni Sequence Type 6964 in Humans and Poultry, New Zealand, 2014-2016.

In 2014, antimicrobial drug-resistant Campylobacter jejuni sequence type 6964 emerged contemporaneously in poultry from 3 supply companies in the North Island of New Zealand and as a major cause of campylobacteriosis in humans in New Zealand. This lineage, not previously identified in New Zealand, was resistant to tetracycline and fluoroquinolones. Genomic analysis revealed divergence into 2 major clades; both clades were associated with human infection, 1 with poultry companies A and B and the other with company C. Accessory genome evolution was associated with a plasmid, phage insertions, and natural transformation. We hypothesize that the tetO gene and a phage were inserted into the chromosome after conjugation, leaving a remnant plasmid that was lost from isolates from company C. The emergence and rapid spread of a resistant clone of C. jejuni in New Zealand, coupled with evolutionary change in the accessory genome, demonstrate the need for ongoing Campylobacter surveillance among poultry and humans.