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Lessons learned from research and surveillance directed at highly pathogenic influenza A viruses in wild birds inhabiting North America

Following detections of highly pathogenic (HP) influenza A viruses (IAVs) in wild birds inhabiting East Asia after the turn of the millennium, the intensity of sampling of wild birds for IAVs increased throughout much of North America. The objectives for many research and surveillance efforts were directed towards detecting Eurasian origin HP IAVs and understanding the potential of such viruses to be maintained and dispersed by wild birds. In this review, we highlight five important lessons learned from research and surveillance directed at HP IAVs in wild birds inhabiting North America: (1) Wild birds may disperse IAVs between North America and adjacent regions via migration, (2) HP IAVs can be introduced to wild birds in North America, (3) HP IAVs may cross the wild bird-poultry interface in North America, (4) The probability of encountering and detecting a specific virus may be low, and (5) Population immunity of wild birds may influence HP IAV outbreaks in North America. We review empirical support derived from research and surveillance efforts for each lesson learned and, furthermore, identify implications for future surveillance efforts, biosecurity, and population health. We conclude our review by identifying five additional areas in which we think future mechanistic research relative to IAVs in wild birds in North America are likely to lead to other important lessons learned in the years ahead.

Virology

H5N1 highly pathogenic avian influenza clade 2.3.4.4b in wild and domestic birds: Introductions into the United States and reassortments, December 2021–April 2022

Highly pathogenic avian influenza viruses (HPAIVs) of the A/goose/Guangdong/1/1996 lineage H5 clade 2.3.4.4b continue to have a devastating effect on domestic and wild birds. Full genome sequence analyses using 1369 H5N1 HPAIVs detected in the United States (U.S.) in wild birds, commercial poultry, and backyard flocks from December 2021 to April 2022, showed three phylogenetically distinct H5N1 virus introductions in the U.S. by wild birds. Unreassorted Eurasian genotypes A1 and A2 entered the Northeast Atlantic states, whereas a genetically distinct A3 genotype was detected in Alaska. The A1 genotype spread westward via wild bird migration and reassorted with North American wild bird avian influenza viruses. Reassortments of up to five internal genes generated a total of 21 distinct clusters; of these, six genotypes represented 92% of the HPAIVs examined. By phylodynamic analyses, most detections in domestic birds were shown to be point-source transmissions from wild birds, with limited farm-to-farm spread.

Virology

Molecular detection and characterization of highly pathogenic H5N1 clade 2.3.4.4b avian influenza viruses among hunter-harvested wild birds provides evidence for three independent introductions into Alaska

We detected and characterized highly pathogenic avian influenza viruses among hunter-harvested wild waterfowl inhabiting western Alaska during September–October 2022 using a molecular sequencing pipeline applied to RNA extracts derived directly from original swab samples. Genomic characterization of 10 H5 clade 2.3.4.4b avian influenza viruses detected with high confidence provided evidence for three independent viral introductions into Alaska. Our results highlight the utility and some potential limits of applying molecular processing approaches directly to RNA extracts from original swab samples for viral research and monitoring.

Alasak

Environmental surveillance and detection of infectious highly pathogenic avian influenza virus in Iowa wetlands

Avian influenza viruses (AIVs) infect both wild birds and domestic poultry, resulting in economically costly outbreaks that have the potential to impact public health. Currently, a knowledge gap exists regarding the detection of infectious AIVs in the aquatic environment. In response to the 2021–2022 Eurasian strain highly pathogenic avian influenza (HPAI) A/goose/Guangdong/1/1996 clade 2.3.4.4 lineage H5 outbreak, an AIV environmental outbreak response study was conducted using a One Health approach. An optimized method was used to temporally sample (April and May 2022) and analyze (culture and molecular methods) surface water from five water bodies (four wetlands and one lake used as a comparison location) in areas near confirmed HPAI detections in wild bird or poultry operations. Avian influenza viruses were isolated from water samples collected in April from all four wetlands (not from the comparison lake sample); HPAI H5N1 was isolated from one wetland. No virus was isolated from the May samples. Several factors, including increased water temperatures, precipitation, biotic and abiotic factors, and absence of AIV-contaminated fecal material due to fewer waterfowl present, may have contributed to the lack of virus isolation from May samples. Results demonstrate surface water as a plausible medium for transmission of AIVs, including the HPAI virus.

Iowa

North American wintering mallards infected with highly pathogenic avian influenza show few signs of altered local or migratory movements

Avian influenza viruses pose a threat to wildlife and livestock health. The emergence of highly pathogenic avian influenza (HPAI) in wild birds and poultry in North America in late 2021 was the first such outbreak since 2015 and the largest outbreak in North America to date. Despite its prominence and economic impacts, we know relatively little about how HPAI spreads in wild bird populations. In January 2022, we captured 43 mallards ( Anas platyrhynchos ) in Tennessee, USA, 11 of which were actively infected with HPAI. These were the first confirmed detections of HPAI H5N1 clade 2.3.4.4b in the Mississippi Flyway. We compared movement patterns of infected and uninfected birds and found no clear differences; infected birds moved just as much during winter, migrated slightly earlier, and migrated similar distances as uninfected birds. Infected mallards also contacted and shared space with uninfected birds while on their wintering grounds, suggesting ongoing transmission of the virus. We found no differences in body condition or survival rates between infected and uninfected birds. Together, these results show that HPAI H5N1 clade 2.3.4.4b infection was unrelated to body condition or movement behavior in mallards infected at this location during winter; if these results are confirmed in other seasons and as HPAI H5N1 continues to evolve, they suggest that these birds could contribute to the maintenance and dispersal of HPAI in North America. Further research on more species across larger geographic areas and multiple seasons would help clarify potential impacts of HPAI on waterfowl and how this emerging disease spreads at continental scales, across species, and potentially between wildlife and domestic animals.

Tennessee

Weak support for disappearance and restricted emergence/persistence of highly pathogenic influenza A in North American waterfowl

Krauss et al. (1) use lack of detection of highly pathogenic (HP) H5 clade 2.3.4.4 (henceforth "H5") influenza A viruses (IAVs) from >22,000 wild bird samples collected in North America in 2014–2015 to argue that HP H5 IAVs disappeared from waterfowl and that unresolved mechanisms restrict emergence and perpetuation of HP IAVs in natural reservoir species. Here we offer an alternative interpretation.

Proceedings of the National Academy of Sciences

The pathogenesis of a 2022 North American highly pathogenic clade 2.3.4.4b H5N1 avian influenza virus in mallards (Anas platyrhynchos)

Highly pathogenic (HP) avian influenza viruses (AIVs) of the clade 2.3.4.4 goose/Guangdong/1996 H5 lineage continue to be a problem in poultry and wild birds in much of the world. The recent incursion of a H5N1 clade 2.3.4.4b HP AIV from this lineage into North America has resulted in widespread outbreaks in poultry and consistent detections of the virus across diverse families of birds and occasionally mammals. To characterize the pathobiology of this virus in mallards ( Anas platyrhynchos ), which are a primary reservoir of AIV, a challenge study was conducted with 2 week-old birds. The 50% bird infectious dose was determined to be <2 log 10 50% egg infectious doses (EID 50 ) and all exposed ducks, including ducks co-housed with inoculated ducks, were infected. Infection appeared to be subclinical for 58.8% (20/34) of the ducks, 1 duck was lethargic, about 20% developed neurological signs and were euthanized, and 18% developed corneal opacity. The mallards shed virus by both the oral and cloacal routes within 24-48hr post-infection. Oral shedding substantially decreased by 6-7 days post-infection, but 65% of the ducks continued to shed virus cloacally through 14 days post-exposure (DPE) for the direct inoculate and 13DPE for contact exposed ducks. Based on the high transmissibility, high virus shed titers, and mild-to-moderate disease, mallards could serve as efficient reservoirs to amplify and disseminate recent North American clade 2.3.4.4b viruses.

Avian Pathology

Quantitative polymerase chain reaction (PCR) for detection of aquatic animal pathogens in a diagnostic laboratory setting

Real-time, or quantitative, polymerase chain reaction (qPCR) is quickly supplanting other molecular methods for detecting the nucleic acids of human and other animal pathogens owing to the speed and robustness of the technology. As the aquatic animal health community moves toward implementing national diagnostic testing schemes, it will need to evaluate how qPCR technology should be employed. This review outlines the basic principles of qPCR technology, considerations for assay development, standards and controls, assay performance, diagnostic validation, implementation in the diagnostic laboratory, and quality assurance and control measures. These factors are fundamental for ensuring the validity of qPCR assay results obtained in the diagnostic laboratory setting.

Journal of Aquatic Animal Health

Early detection of wildlife disease pathogens using CRISPR-Cas system methods

Wildlife diseases are a considerable threat to human health, conservation, and the economy. Surveillance is a critical component to mitigate the impact of animal diseases in these sectors. To monitor human diseases, CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein) biosensors have proven instrumental as diagnostic tools capable of detecting unique DNA and RNA sequences related to their associated pathogens. However, despite the significant advances in the general development of CRISPR-Cas biosensors, their use to support wildlife disease management is lagging. In some cases, wildlife diseases of concern could be rapidly surveyed using these tools with minimal technical, operational, or cost requirements to end users. This review explores the potential to further leverage this technology to advance wildlife disease monitoring and highlights how concerted standardization of protocols can help to ensure data reliability.

The CRISPR Journal

Management of arthropod pathogen vectors in North America: Minimizing adverse effects on pollinators

Tick and mosquito management is important to public health protection. At the same time, growing concerns about declines of pollinator species raise the question of whether vector control practices might affect pollinator populations. We report the results of a task force of the North American Pollinator Protection Campaign (NAPPC) that examined potential effects of vector management practices on pollinators, and how these programs could be adjusted to minimize negative effects on pollinating species. The main types of vector control practices that might affect pollinators are landscape manipulation, biocontrol, and pesticide applications. Some current practices already minimize effects of vector control on pollinators (e.g., short-lived pesticides and application-targeting technologies). Nontarget effects can be further diminished by taking pollinator protection into account in the planning stages of vector management programs. Effects of vector control on pollinator species often depend on specific local conditions (e.g., proximity of locations with abundant vectors to concentrations of floral resources), so planning is most effective when it includes collaborations of local vector management professionals with local experts on pollinators. Interventions can then be designed to avoid pollinators (e.g., targeting applications to avoid blooming times and pollinator nesting habitats), while still optimizing public health protection. Research on efficient targeting of interventions, and on effects on pollinators of emerging technologies, will help mitigate potential deleterious effects on pollinators in future management programs. In particular, models that can predict effects of integrated pest management on vector-borne pathogen transmission, along with effects on pollinator populations, would be useful for collaborative decision-making.

Journal of Medical Entomology

Pathogenicity of Steinernema carpocapsae and S. glaseri (Nematoda: Steinernematidae) to Ixodes scapularis (Acari: Ixodidae)

The entomopathogenic nematodes Steinernema carpocapsae (Weiser) and S. glaseri (Steiner) are pathogenic to engorged adult, blacklegged ticks, Ixodes scapularis (Say), but not to unfed females, engorged nymphs, or engorged larvae. Nematodes apparently enter the tick through the genital pore, thus precluding infection of immature ticks. The timing of tick mortality, and overall mortality after 17 d, did not differ between infections by S. carpocapsae and S. glaseri . These nematodes typically do not complete their life cycles or produce infective juveniles in I. scapularis . However, both species successfully produced infective juveniles when the tick body was slit before nematode infection. Mortality of engorged I. scapularis females infected by S. carpocapsae was greater than uninfected controls, but did not vary significantly with nematode concentration (50–3,000 infective juveniles per 5-cm-diameter petri dish). The LC 50 was 347.8 infective juveniles per petri dish (5 ticks per dish). Hatched egg masses of infected ticks weighed less than those of uninfected controls. Mortality of infected ticks was greatest between 20 and 30°C, and was lower at 15°C.

Journal of Medical Entomology

Pathogenicity of Bacillus thuringiensis variety kurstaki to Ixodes scapularis (Acari: Ixodidae)

Pathogenicity of the entomopathogenic bacterium Bacillus thuringiensis var. kurstaki de Barjac & Lemille was tested against the black-legged tick, Ixodes scapularis Say. Engorged larvae dipped in a solution of 108 spores per ml showed 96% mortality, 3 wk post-infection. The LC50 value for engorged larvae (concentration required to kill 50% of ticks) was 107 spores/ml. Bacillus thuringiensis shows considerable potential as a microbial control agent for the management of Ixodes scapularis .

Journal of Medical Entomology

Crossroads of highly pathogenic H5N1: overlap between wild and domestic birds in the Black Sea-Mediterranean impacts global transmission

Understanding transmission dynamics that link wild and domestic animals is a key element of predicting the emergence of infectious disease, an event that has highest likelihood of occurring wherever human livelihoods depend on agriculture and animal trade. Contact between poultry and wild birds is a key driver of the emergence of highly pathogenic avian influenza (HPAI), a process that allows for host-switching and accelerated reassortment, diversification and spread of virus between otherwise unconnected regions. This study addresses questions relevant to the spillover of HPAI at a transmission hotspot: what is the nature of the wild bird-poultry interface in Egypt and adjacent Black Sea-Mediterranean countries and how has this contributed to outbreaks occurring worldwide? Using a spatio-temporal model of infection risk informed by satellite tracking of waterfowl and viral phylogenetics, this study identified ecological conditions that contribute to spillover in this understudied region. Results indicated that multiple ducks (Northern Shoveler and Northern Pintail) hosted segments that shared ancestry with HPAI H5 from both clade 2.2.1 and clade 2.3.4 supporting the role of Anseriformes in linking viral populations in East Asia and Africa over large-distances. Quantifying the interface between wild ducks and H5N1-infected poultry revealed an increasing interface in late winter peaking in early spring when ducks expanded their range before migration, with key differences in the timing of poultry contact risk between local and long-distance migrants.

Virus Evolution

Ecosystem-engineered infections: Beaver-modified wetlands are associated with conflicting drivers of amphibian pathogen prevalence

Beavers are ecosystem engineers and keystone species that protect freshwater resources and increase biodiversity. Beaver reintroductions are promoted for amphibian conservation, yet their impact on Batrachochytrium dendrobatidis (Bd), a pathogen linked with amphibian population declines worldwide, remains unclear. We investigated the abiotic and biotic drivers of Bd prevalence in Columbia spotted frogs ( Rana luteiventris ) and western toads ( Anaxyrus boreas ) in 20 beaver-modified and 23 non-beaver wetlands in Glacier National Park, USA. We found that beavers increased wetland hydroperiod, which was associated with higher Bd prevalence. However, beavers also reduced wetland canopy cover, which was associated with lower Bd prevalence. Our models also predicted higher Bd prevalence associated with higher adult density of both species of amphibians, although species’ densities were similar in beaver-modified and non-beaver wetlands. These results suggest that beavers have a cumulatively negligible net effect on Bd prevalence owing to their effects on both hydroperiod and canopy cover, which is encouraging for amphibian conservation. Our findings also suggest that decreasing canopy cover may be a potential management option to reduce Bd prevalence. In addition, these findings indicate that beaver-mimicking restoration projects may harm amphibian populations if they increase wetland hydroperiods without reducing canopy cover.

Montana

Pathogenic prion protein is degraded by a manganese oxide mineral found in soils

Prions, the aetiological agents of transmissible spongiform encephalopathies, exhibit extreme resistance to degradation. Soil can retain prion infectivity in the environment for years. Reactive soil components may, however, contribute to the inactivation of prions in soil. Members of the birnessite family of manganese oxides (MnO 2 ) rank among the strongest natural oxidants in soils. Here, we report the abiotic degradation of pathogenic prion protein (PrP TSE ) by a synthetic analogue of naturally occurring birnessite minerals. Aqueous MnO 2 suspensions degraded the PrP TSE as evidenced by decreased immunoreactivity and diminished ability to seed protein misfolding cyclic amplification reactions. Birnessite-mediated PrP TSE degradation increased as a solution's pH decreased, consistent with the pH-dependence of the redox potential of MnO 2 . Exposure to 5.6 mg MnO2 ml -1 (PrP TSE :MnO 2 =1 : 110) decreased PrPTSE levels by ???4 orders of magnitude. Manganese oxides may contribute to prion degradation in soil environments rich in these minerals. ?? 2009 SGM.

Journal of General Virology

Host contact and shedding patterns clarify variation in pathogen exposure and transmission in threatened tortoise Gopherus agassizii : implications for disease modelling and management

Summary Most directly transmitted infections require some form of close contact between infectious and susceptible hosts to spread. Often disease models assume contacts are equal and use mean field estimates of transmission probability for all interactions with infectious hosts. Such methods may inaccurately describe transmission when interactions differ substantially in their ability to cause infection. Understanding this variation in transmission risk may be critical to properly model and manage some infectious diseases. In this study, we investigate how varying exposure and transmission may be key to understanding disease dynamics in the threatened desert tortoise Gopherus agassizii . We created heterogeneity in Mycoplasma agassizii exposure (the putative bacterial agent of a respiratory disease) by varying the duration of interactions between naturally infected and uninfected captive desert tortoises. Using qPCR, we identified new infections and compared models of transmission probability as a function of contact duration and pathogen load. We then examined the contact patterns of a wild tortoise population using proximity loggers to identify heterogeneity in contact duration. The top-ranked model predicting M. agassizii transmission included a dose term defined as the product of the number of days in proximity to an infected host and the infection level of that host. Models predicted low transmission probability for short interactions, unless the infectious host had a high load of M. agassizii : such hosts were predicted to transmit infection at higher rates with any amount of contact. We observed predominantly short-lived interactions in a free-ranging tortoise population and thus, expect transmission patterns in this population to vary considerably with the frequency and duration of high infection levels. Mean field models may misrepresent natural transmission patterns in this and other populations depending on the distribution of high-risk contact and shedding events. Rapid outbreaks in generally solitary species may result from changes to their naturally low-risk contact patterns or due to increases in the frequency of severe infections or super-shedding events &ndash; population characteristics that should be further investigated to develop effective management strategies.

Nevada

Highly pathogenic avian influenza virus H5N2 (Clade 2.3.4.4) challenge of mallards age appropriate to the 2015 midwestern poultry outbreak

Background The 2015 highly pathogenic avian influenza virus (HPAIV) H5N2 clade 2.3.4.4 outbreak in upper midwestern U.S. poultry operations was not detected in wild birds to any great degree during the outbreak, despite wild waterfowl being implicated in the introduction, reassortment, and movement of the virus into North America from Asia. This outbreak led to the demise of over 50 million domestic birds and occurred mainly during the northward spring migration of adult avian populations. Objectives There have been no experimental examinations of the pathogenesis, transmission, and population impacts of this virus in adult wild waterfowl with varying exposure histories—the most relevant age class. Methods We captured, housed, and challenged adult wild mallards ( Anas platyrhynchos ) with HPAIV H5N2 clade 2.3.4.4 and measured viral infection, viral excretion, and transmission to other mallards. Results All inoculated birds became infected and excreted moderate amounts of virus, primarily orally, for up to 14 days. Cohoused, uninoculated birds also all became infected. Serological status had no effect on susceptibility. There were no obvious clinical signs of disease, and all birds survived to the end of the study (14 days). Conclusions Based on these results, adult mallards are viable hosts of HPAIV H5N2 regardless of prior exposure history and are capable of transporting the virus over short and long distances. These findings have implications for surveillance efforts. The capture and sampling of wild waterfowl in the spring, when most surveillance programs are not operating, are important to consider in the design of future HPAIV surveillance programs.

Wisconsin