SARS-CoV-2 B.1.1.7 Variant Infection in Malayan Tigers, Virginia, USA.

We report infection of 3 Malayan tigers with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) B.1.1.7 (Alpha) variant at a zoologic park in Virginia, USA. All tigers exhibited respiratory signs consistent with SARS-CoV-2 infection. These findings show that tigers are susceptible to infection with the SARS-CoV-2 B.1.1.7 variant.

Clinical evaluation of a multiplex real-time RT-PCR assay for detection of SARS-CoV-2 in individual and pooled upper respiratory tract samples.

The aim of this study was to identify and validate a sensitive, high-throughput, and cost-effective SARS-CoV-2 real-time RT-PCR assay to be used as a surveillance and diagnostic tool for SARS-CoV-2 in a university surveillance program. We conducted a side-by-side clinical evaluation of a newly developed SARS-CoV-2 multiplex assay (EZ-SARS-CoV-2 Real-Time RT-PCR) with the commercial TaqPath COVID-19 Combo Kit, which has an Emergency Use Authorization from the FDA. The EZ-SARS-CoV-2 RT-PCR incorporates two assays targeting the SARS-CoV-2 N gene, an internal control targeting the human RNase P gene, and a PCR inhibition control in a single reaction. Nasopharyngeal (NP) and anterior nares (AN) swabs were tested as individuals and pools with both assays and in the ABI 7500 Fast and the QuantStudio 5 detection platforms. The analytical sensitivity of the EZ-SARS-CoV-2 RT-PCR assay was 250 copies/ml or approximately 1.75 genome copy equivalents per reaction. The clinical performance of the EZ-SARS-CoV-2 assay was evaluated using NP and AN samples tested in other laboratories. The diagnostic sensitivity of the assay ranged between 94 and 96% across the detection platforms, and the diagnostic specificity was 94.06%. The positive predictive value was 94%, and the negative predictive value ranged from 94 to 96%. Pooling five NP or AN specimens yielded 93% diagnostic sensitivity. The overall agreement between these SARS-CoV-2 RT-PCR assays was high, supported by a Cohen's kappa value of 0.93. The EZ-SARS-CoV-2 RT-PCR assay performance attributes of high sensitivity and specificity with AN sample matrix and pooled upper respiratory samples support its use in a high-throughput surveillance testing program.

Infection and tissue distribution of highly pathogenic avian influenza A type H5N1 (clade 2.3.4.4b) in red fox kits (Vulpes vulpes).

Avian influenza H5N1 is a highly pathogenic virus that primarily affects birds. However, it can also infect other animal species, including mammals. We report the infection of nine juvenile red foxes (Vulpes vulpes) with Highly Pathogenic Avian Influenza A type H5N1 (Clade 2.3.4.4b) in the spring of 2022 in the central, western, and northern regions of New York, USA. The foxes displayed neurologic signs, and examination of brain and lung tissue revealed lesions, with brain lesions ranging from moderate to severe meningoencephalitis. Analysis of tissue tropism using RT-PCR methods showed a comparatively lower Ct value in the brain, which was confirmed by in situ hybridization targeting Influenza A RNA. The viral RNA labelling was highly clustered and overlapped the brain lesions, observed in neurons, and grey matter. Whole viral genome sequences obtained from the affected foxes were subjected to phylogenetic and mutation analysis to determine influenza A clade, host specificity, and potential occurrence of viral reassortment. Infections in red foxes likely occurred due to preying on infected wild birds and are unlikely due to transmission between foxes or other mammals.

Patterns and Ecological Mechanisms of Tick-Borne Disease Exposure Risk in Acadia National Park, Mount Desert Island, Maine, United States.

National parks are unique and significant vector-borne pathogen transmission settings, engaging over 300 million people in outdoor recreation per year. In this study, we integrated vector surveys and ecological habitat feature data in spatial models to characterize tick-borne disease exposure risk in Acadia National Park (ANP), Maine. To determine the broad-scale patterns of blacklegged tick Ixodes scapularis Say (Acari: Ixodidae) densities in ANP, we conducted host-seeking tick collections at 114 sites across the park over two years. Using these tick survey data and geospatial landscape feature data (i.e., land cover, elevation, forest patch size, and aspect) we developed a random forest model of nymphal tick density. We found that host-seeking tick density varies significantly across the park and is particularly high in areas characterized by deciduous forest cover and relatively low elevation. To explore potential fine-scale ecological drivers of tick density spatial patterns, we quantified microclimate conditions, host activity, and vegetation characteristics at a subset of 19 sites. We identified significant differences in microclimate conditions but not host activity or vegetation metrics across broad-scale landscape feature classes. Mean temperature and mean humidity were correlated to nymphal densities and therefore may provide a mechanistic link between landscape features and blacklegged tick densities. Finally, we detected multiple tick-borne pathogens in both ticks and small mammals sampled in ANP, including Borrelia burgdorferi, Babesia microti, and Anaplasma phagocytophilum. Our findings demonstrate the value of using ecological metrics to estimate vector-borne disease exposure risk and provide insight into habitat characteristics that may drive tick-borne disease exposure risk.

Viral RNA Load and Infectivity of SARS-CoV-2 in Paired Respiratory and Oral Specimens from Symptomatic, Asymptomatic, or Postsymptomatic Individuals.

In the present study, we assessed the diagnostic sensitivity and determined the viral RNA load and infectivity of SARS-CoV-2 in paired respiratory (nasopharyngeal and anterior nares) and oral samples (saliva and sublingual swab). Samples were collected from 77 individuals of which 75 were diagnosed with COVID-19 and classified as symptomatic (n = 29), asymptomatic (n = 31), or postsymptomatic (n = 15). Specimens were collected at one time point from each individual, between day 1 and 23 after the initial COVID-19 diagnosis, and included self-collected saliva (S), or sublingual (SL) swab, and bilateral anterior nares (AN) swab, followed by health care provider collected nasopharyngeal (NP) swab. Sixty-three specimen sets were tested using five assay/platforms. The diagnostic sensitivity of each assay/platform and specimen type was determined. Of the 63 specimen sets, SARS-CoV-2 was detected in 62 NP specimens, 52 AN specimens, 59 saliva specimens, and 31 SL specimens by at least one platform. Infectious SARS-CoV-2 was isolated from 21 NP, 13 AN, 12 saliva, and one SL specimen out of 50 specimen sets. SARS-CoV-2 isolation was most successful up to 5 days after initial COVID-19 diagnosis using NP specimens from symptomatic patients (16 of 24 positives, 66.67%), followed by specimens from asymptomatic patients (5 of 17 positives, 29.41%), while it was not very successful with specimens from postsymptomatic patients. Benefits of self-collected saliva and AN specimens balance the loss of sensitivity relative to NP specimens. Therefore, saliva and AN specimens are acceptable alternatives for symptomatic SARS-CoV-2 diagnostic testing or surveillance with increased sampling frequency of asymptomatic individuals. IMPORTANCE The dynamics of infection with SARS-CoV-2 have a significant impact on virus infectivity and in the diagnostic sensitivity of molecular and classic virus detection tests. In the present study we determined the diagnostic sensitivity of paired respiratory (nasopharyngeal and anterior nares swabs) and oral secretions (saliva and sublingual swab) and assessed infectious virus shedding patterns by symptomatic, asymptomatic, or postsymptomatic individuals. Understanding the diagnostic performance of these specimens and the patterns of infectious virus shedding in these bodily secretions provides critical information to control COVID-19, and may help to refine guidelines on isolation and quarantine of positive individuals and their close contacts identified through epidemiological investigations.

Infection and Transmission of SARS-CoV-2 B.1.617.2 Lineage (Delta Variant) among Fully Vaccinated Individuals.

The emergence of the SARS-CoV-2 B.1.617.2 lineage (Delta variant) in 2021 was associated with increased case numbers and test positivity rates, including a large number of infections in fully vaccinated individuals. Here, we describe the findings of an investigation conducted in Tompkins County, New York, to evaluate factors underlying a significant uptick in the number of coronavirus disease 2019 (COVID-19) cases observed in the months of July and August 2021. We performed genomic surveillance and genotyping as well as virological assessments to determine infectivity of the virus in a select number of clinical diagnostic samples. Genomic sequence analyses revealed complete replacement of the B.1.1.7 lineage (Alpha variant) with the B.1.617.2 lineage (Delta variant) between July 1 and August 4 2021. We observed a strong association between viral RNA loads detected by real-time reverse transcriptase PCR and infectious virus detected in respiratory secretions by virus titration. A marked increase in positive cases among fully vaccinated individuals was observed. The sequence divergence between two index Delta variant cases in April and May, and the cases after July 1st, revealed independent Delta variant introductions in Tompkins County. Contact tracing information enabled the detection of clusters of connected cases within closely related phylogenetic clusters. We also found evidence of transmission between vaccinated individuals and between vaccinated and unvaccinated individuals. This was confirmed by detection and isolation of infectious virus from a group of individuals within epidemiologically connected transmission clusters, confirming shedding of high viral loads and transmission of the virus by fully vaccinated individuals. IMPORTANCE The SARS-CoV-2 lineage B.1.617.2 (Delta variant) emerged in Asia and rapidly spread to other countries, becoming the dominant circulating lineage. Worldwide infections with B.1.617.2 peaked at a time in which vaccination rates were increasing. In this study, we present data characterizing the emergence of SARS-CoV-2 lineage B.1.617.2 (Delta variant) in Tompkins County, New York, which has one of the highest vaccination rates in the state. We present evidence demonstrating infection, replication, and transmission of SARS-CoV-2 lineage B.1.617.2 (Delta variant) between fully vaccinated individuals. Importantly, infectious virus loads were determined in a subset of samples and demonstrated shedding of high viral titers in respiratory secretions of vaccinated individuals.

Multi-laboratory evaluation of the Illumina iSeq platform for whole genome sequencing of Salmonella, Escherichia coli and Listeria.

There is a growing need for public health and veterinary laboratories to perform whole genome sequencing (WGS) for monitoring antimicrobial resistance (AMR) and protecting the safety of people and animals. With the availability of smaller and more affordable sequencing platforms coupled with well-defined bioinformatic protocols, the technological capability to incorporate this technique for real-time surveillance and genomic epidemiology has greatly expanded. There is a need, however, to ensure that data are of high quality. The goal of this study was to assess the utility of a small benchtop sequencing platform using a multi-laboratory verification approach. Thirteen laboratories were provided the same equipment, reagents, protocols and bacterial reference strains. The Illumina DNA Prep and Nextera XT library preparation kits were compared, and 2×150 bp iSeq i100 chemistry was used for sequencing. Analyses comparing the sequences produced from this study with closed genomes from the provided strains were performed using open-source programs. A detailed, step-by-step protocol is publicly available via protocols.io (https://www.protocols.io/view/iseq-bacterial-wgs-protocol-bij8kcrw). The throughput for this method is approximately 4-6 bacterial isolates per sequencing run (20-26 Mb total load). The Illumina DNA Prep library preparation kit produced high-quality assemblies and nearly complete AMR gene annotations. The Prep method produced more consistent coverage compared to XT, and when coverage benchmarks were met, nearly all AMR, virulence and subtyping gene targets were correctly identified. Because it reduces the technical and financial barriers to generating WGS data, the iSeq platform is a viable option for small laboratories interested in genomic surveillance of microbial pathogens.

Method comparison of targeted influenza A virus typing and whole-genome sequencing from respiratory specimens of companion animals.

Epidemics of H3N8 and H3N2 influenza A viruses (IAVs) in dogs, along with recognition of spillover infections from IAV strains typically found in humans or other animals, have emphasized the importance of efficient laboratory testing. Given the lack of active IAV surveillance or immunization requirements for dogs, cats, or horses imported into the United States, serotype prediction and whole-genome sequencing of positive specimens detected at veterinary diagnostic laboratories are also needed. The conserved sequences at the ends of the viral genome segments facilitate universal amplification of all segments of viral genomes directly from respiratory specimens. Although several methods for genomic analysis have been reported, no optimization focusing on companion animal strains has been described, to our knowledge. We compared 2 sets of published universal amplification primers using 26 IAV-positive specimens from dogs, horses, and a cat. Libraries prepared from the resulting amplicons were sequenced using Illumina chemistry, and reference-based assemblies were generated from the data produced by both methods. Although both methods produced high-quality data, coverage profiles and base calling differed between the 2 methods. The sequence data were also used to identify the subtype of the IAV strains sequenced and then compared to standard PCR assays for neuraminidase types N2 and N8.

DNA Extraction and Host Depletion Methods Significantly Impact and Potentially Bias Bacterial Detection in a Biological Fluid.

Untargeted sequencing of nucleic acids present in food can inform the detection of food safety and origin, as well as product tampering and mislabeling issues. The application of such technologies to food analysis may reveal valuable insights that are simply unobtainable by targeted testing, leading to the efforts of applying such technologies in the food industry. However, before these approaches can be applied, it is imperative to verify that the most appropriate methods are used at every step of the process: gathering of primary material, laboratory methods, data analysis, and interpretation. The focus of this study is on gathering the primary material, in this case, DNA. We used bovine milk as a model to (i) evaluate commercially available kits for their ability to extract nucleic acids from inoculated bovine milk, (ii) evaluate host DNA depletion methods for use with milk, and (iii) develop and evaluate a selective lysis-propidium monoazide (PMA)-based protocol for host DNA depletion in milk. Our results suggest that magnetically based nucleic acid extraction methods are best for nucleic acid isolation of bovine milk. Removal of host DNA remains a challenge for untargeted sequencing of milk, highlighting the finding that the individual matrix characteristics should always be considered in food testing. Some reported methods introduce bias against specific types of microbes, which may be particularly problematic in food safety, where the detection of Gram-negative pathogens and hygiene indicators is essential. Continuous efforts are needed to develop and validate new approaches for untargeted metagenomics in samples with large amounts of DNA from a single host. IMPORTANCE Tracking the bacterial communities present in our food has the potential to inform food safety and product origin. To do so, the entire genetic material present in a sample is extracted using chemical methods or commercially available kits and sequenced using next-generation platforms to provide a snapshot of the microbial composition. Because the genetic material of higher organisms present in food (e.g., cow in milk or beef, wheat in flour) is around 1,000 times larger than the bacterial content, challenges exist in gathering the information of interest. Additionally, specific bacterial characteristics can make them easier or harder to detect, adding another layer of complexity to this issue. In this study, we demonstrate the impact of using different methods for the ability to detect specific bacteria and highlight the need to ensure that the most appropriate methods are being used for each particular sample.

Active surveillance of pathogens from ticks collected in New York State suburban parks and schoolyards.

Schoolyards and suburban parks are two environments where active tick surveillance may inform local management approaches. Even in a state such as New York with a robust active tick surveillance programme operated by the state Department of Health, these settings are not routinely covered. The goal of this study was to highlight the importance of active surveillance for tick-borne pathogens by describing their prevalence in ticks collected from schoolyards and suburban parks and to guide the use of integrated pest management in these settings. Tick dragging was performed in three regions of New York State: Long Island, the Lower Hudson Valley and the Capital Region. A total of 19 schoolyards and 32 parks were sampled. The location, habitat and weather at the time of tick collection were recorded. Ticks were speciated and tested for the presence of 17 pathogens with a novel application of nanoscale real-time PCR. The causative agents of Lyme disease, anaplasmosis, babesiosis and Powassan virus disease were all detected from Ixodes scapularis in various sites throughout the capital region and south-eastern counties of New York state. The most common agent detected was Borrelia burgdorferi, and coinfection rates were as high as 36%. This surveillance study also captured the first of the invasive Asian longhorned tick species, Haemaphysalis longicornis, in New York state (collected 2 June 2017). Results from this study highlight the importance of collaborative efforts and data sharing for improvement of surveillance for tick-borne disease agents.

From People to Panthera: Natural SARS-CoV-2 Infection in Tigers and Lions at the Bronx Zoo.

Despite numerous barriers to transmission, zoonoses are the major cause of emerging infectious diseases in humans. Among these, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and ebolaviruses have killed thousands; the human immunodeficiency virus (HIV) has killed millions. Zoonoses and human-to-animal cross-species transmission are driven by human actions and have important management, conservation, and public health implications. The current SARS-CoV-2 pandemic, which presumably originated from an animal reservoir, has killed more than half a million people around the world and cases continue to rise. In March 2020, New York City was a global epicenter for SARS-CoV-2 infections. During this time, four tigers and three lions at the Bronx Zoo, NY, developed mild, abnormal respiratory signs. We detected SARS-CoV-2 RNA in respiratory secretions and/or feces from all seven animals, live virus in three, and colocalized viral RNA with cellular damage in one. We produced nine whole SARS-CoV-2 genomes from the animals and keepers and identified different SARS-CoV-2 genotypes in the tigers and lions. Epidemiologic and genomic data indicated human-to-tiger transmission. These were the first confirmed cases of natural SARS-CoV-2 animal infections in the United States and the first in nondomestic species in the world. We highlight disease transmission at a nontraditional interface and provide information that contributes to understanding SARS-CoV-2 transmission across species.IMPORTANCE The human-animal-environment interface of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important aspect of the coronavirus disease 2019 (COVID-19) pandemic that requires robust One Health-based investigations. Despite this, few reports describe natural infections in animals or directly link them to human infections using genomic data. In the present study, we describe the first cases of natural SARS-CoV-2 infection in tigers and lions in the United States and provide epidemiological and genetic evidence for human-to-animal transmission of the virus. Our data show that tigers and lions were infected with different genotypes of SARS-CoV-2, indicating two independent transmission events to the animals. Importantly, infected animals shed infectious virus in respiratory secretions and feces. A better understanding of the susceptibility of animal species to SARS-CoV-2 may help to elucidate transmission mechanisms and identify potential reservoirs and sources of infection that are important in both animal and human health.