Avian influenza A virus susceptibility, infection, transmission, and antibody kinetics in European starlings.

Avian influenza A viruses (IAVs) pose risks to public, agricultural, and wildlife health. Bridge hosts are spillover hosts that share habitat with both maintenance hosts (e.g., mallards) and target hosts (e.g., poultry). We conducted a comprehensive assessment of European starlings (Sturnus vulgaris), a common visitor to both urban and agricultural environments, to assess whether this species might act as a potential maintenance or bridge host for IAVs. First, we experimentally inoculated starlings with a wild bird IAV to investigate susceptibility and replication kinetics. Next, we evaluated whether IAV might spill over to starlings from sharing resources with a widespread IAV reservoir host. We accomplished this using a specially designed transmission cage to simulate natural environmental transmission by exposing starlings to water shared with IAV-infected mallards (Anas platyrhynchos). We then conducted a contact study to assess intraspecies transmission between starlings. In the initial experimental infection study, all inoculated starlings shed viral RNA and seroconverted. All starlings in the transmission study became infected and shed RNA at similar levels. All but one of these birds seroconverted, but detectable antibodies were relatively transient, falling to negative levels in a majority of birds by 59 days post contact. None of the contact starlings in the intraspecies transmission experiment became infected. In summary, we demonstrated that starlings may have the potential to act as IAV bridge hosts if they share water with IAV-infected waterfowl. However, starlings are unlikely to act as maintenance hosts due to limited, if any, intraspecies transmission. In addition, starlings have a relatively brief antibody response which should be considered when interpreting serology from field samples. Further study is needed to evaluate the potential for transmission from starlings to poultry, a possibility enhanced by starling's behavioral trait of forming very large flocks which can descend on poultry facilities when natural resources are scarce.

Persistence of maternal antibodies to influenza A virus among captive mallards (Anas platyrhynchos).

Wild waterfowl are maintenance hosts of most influenza A virus (IAV) subtypes and are often the subjects of IAV surveillance and transmission models. While maternal antibodies have been detected in yolks and in nestlings for a variety of wild bird species and pathogens, the persistence of maternal antibodies to IAVs in mallard ducklings (Anas platyrhynchos) has not been previously investigated. Nonetheless, this information is important for a full understanding of IAV transmission dynamics because ducklings protected by maternal antibodies may not be susceptible to infection. In this study, we examined the transfer of IAV-specific maternal antibodies to ducklings. Blood samples were collected approximately every five days from ducklings hatched from hens previously infected with an H6 strain of IAV. Serum samples were tested for antibodies to IAV by an enzyme-linked immunosorbent assay. The median persistence of maternal antibodies in ducklings was 12.5 days (range: 4-33 days) post-hatch. The majority of ducklings (71%) had detectable maternal antibodies from 4 to 17 days post-hatch, while a small subset of individuals (29%) had detectable maternal antibodies for up to 21-33 days post-hatch. Antibody concentrations in hens near the time of egg laying were correlated with maternal antibody concentrations in the initial blood sample collected from ducklings (0-4 days post-hatch). Knowledge of the duration of maternal antibodies in ducklings will aid in the interpretation of IAV serological surveillance results and in the modeling of IAV transmission dynamics in waterfowl.

Influenza A virus surveillance, infection and antibody persistence in snow geese (Anser caerulescens).

Some snow geese (Anser caerulescens) migrate between Eurasia and North America and exhibit high seroprevalence for influenza A viruses (IAVs). Hence, these birds might be expected to play a role in intercontinental dispersal of IAVs. Our objective in this manuscript was to characterize basic incidence and infection characteristics for snow geese to assess whether these birds are likely to significantly contribute to circulation of IAVs. Thus, we 1) estimated snow goose infection prevalence by summarizing > 5,000 snow goose surveillance records, 2) experimentally infected snow geese with a low pathogenic IAV (H4N6) to assess susceptibility and infection dynamics and 3) characterized long-term antibody kinetics. Infection prevalence based on surveillance data for snow geese was 7.88%, higher than the infection rates found in other common North American goose species. In the experimental infection study, only 4 of 7 snow geese shed viral RNA. Shedding in infected birds peaked at moderate levels (mean peak 102.62 EID50 equivalents/mL) and was exclusively associated with the oral cavity. Serological testing across a year post-exposure showed all inoculated birds seroconverted regardless of detectable shedding. Antibody levels peaked at 10 days post-exposure and then waned to undetectable levels by 6 months. In sum, while broad-scale surveillance results showed comparatively high infection prevalence, the experimental infection study showed only moderate susceptibility and shedding. Consequently, additional work is needed to assess whether snow geese might exhibit higher levels of susceptibility and shedding rates when exposed to other IAV strains.

Validation of a screening method for the detection of colistin-resistant E. coli containing mcr-1 in feral swine feces.

A method was developed and validated for the detection of colistin-resistant Escherichia coli containing mcr-1 in the feces of feral swine. Following optimization of an enrichment method using EC broth supplemented with colistin (1 µg/mL) and vancomycin (8 µg/mL), aliquots derived from 100 feral swine fecal samples were spiked with of one of five different mcr-1 positive E. coli strains (between 100 and 104 CFU/g), for a total of 1110 samples tested. Enrichments were then screened using a simple boil-prep and a previously developed real-time PCR assay for mcr-1 detection. The sensitivity of the method was determined in swine feces, with mcr-1 E. coli inocula of 0.1-9.99 CFU/g (n = 340), 10-49.99 CFU/g (n = 170), 50-99 CFU/g (n = 255), 100-149 CFU/g (n = 60), and 200-2200 CFU/g (n = 175), which were detected with 32%, 72%, 88%, 95%, and 98% accuracy, respectively. Uninoculated controls (n = 100) were negative for mcr-1 following enrichment.

Gulls as Sources of Environmental Contamination by Colistin-resistant Bacteria.

In 2015, the mcr-1 gene was discovered in Escherichia coli in domestic swine in China that conferred resistance to colistin, an antibiotic of last resort used in treating multi-drug resistant bacterial infections in humans. Since then, mcr-1 was found in other human and animal populations, including wild gulls. Because gulls could disseminate the mcr-1 gene, we conducted an experiment to assess whether gulls are readily colonized with mcr-1 positive E. coli, their shedding patterns, transmission among conspecifics, and environmental deposition. Shedding of mcr-1 E. coli by small gull flocks followed a lognormal curve and gulls shed one strain >101 log10 CFU/g in their feces for 16.4 days, which persisted in the environment for 29.3 days. Because gulls are mobile and can shed antimicrobial-resistant bacteria for extended periods, gulls may facilitate transmission of mcr-1 positive E. coli to humans and livestock through fecal contamination of water, public areas and agricultural operations.

Influenza infection in wild raccoons.

Raccoons (Procyon lotor) are common, widely distributed animals that frequently come into contact with wild waterfowl, agricultural operations, and humans. Serosurveys showed that raccoons are exposed to avian influenza virus. We found antibodies to a variety of influenza virus subtypes (H10N7, H4N6, H4N2, H3, and H1) with wide geographic variation in seroprevalence. Experimental infection studies showed that raccoons become infected with avian and human influenza A viruses, shed and transmit virus to virus-free animals, and seroconvert. Analyses of cellular receptors showed that raccoons have avian and human type receptors with a similar distribution as found in human respiratory tracts. The potential exists for co-infection of multiple subtypes of influenza virus with genetic reassortment and creation of novel strains of influenza virus. Experimental and field data indicate that raccoons may play an important role in influenza disease ecology and pose risks to agriculture and human health.

Serologic evidence of West Nile virus exposure in North American mesopredators.

Sera from 936 mammalian mesopredators (Virginia opossums, gray foxes, striped skunks, hooded skunks, raccoons, a bobcat, and a red fox) were collected during 2003 and 2004 in California, Arizona, Texas, Louisiana, Ohio, and Wyoming and screened for flavivirus-specific antibodies by an epitope-blocking enzyme-linked immunosorbent assay (blocking ELISA). Serum samples positive for antibodies against flaviviruses were screened for West Nile virus (WNV)-specific antibodies by blocking ELISA and selectively confirmed with plaque-reduction neutralization tests. High prevalence rates were observed in raccoons (45.6%) and striped skunks (62.9%). The high WNV antibody prevalence noted in mesopredators, their peridomestic tendencies, and their overall pervasiveness make these species potentially useful sentinels for monitoring flaviviruses in defined areas.

Impact of body condition on influenza A virus infection dynamics in mallards following a secondary exposure.

Migratory waterfowl are often viewed as vehicles for the global spread of influenza A viruses (IAVs), with mallards (Anas platyrhynchos) implicated as particularly important reservoir hosts. The physical demands and energetic costs of migration have been shown to influence birds' body condition; poorer body condition may suppress immune function and affect the course of IAV infection. Our study evaluated the impact of body condition on immune function and viral shedding dynamics in mallards naturally exposed to an H9 IAV, and then secondarily exposed to an H4N6 IAV. Mallards were divided into three treatment groups of 10 birds per group, with each bird's body condition manipulated as a function of body weight by restricting food availability to achieve either a -10%, -20%, or control body weight class. We found that mallards exhibit moderate heterosubtypic immunity against an H4N6 IAV infection after an infection from an H9 IAV, and that body condition did not have an impact on shedding dynamics in response to a secondary exposure. Furthermore, body condition did not affect aspects of the innate and adaptive immune system, including the acute phase protein haptoglobin, heterophil/lymphocyte ratios, and antibody production. Contrary to recently proposed hypotheses and some experimental evidence, our data do not support relationships between body condition, infection and immunocompetence following a second exposure to IAV in mallards. Consequently, while annual migration may be a driver in the maintenance and spread of IAVs, the energetic demands of migration may not affect susceptibility in mallards.

Ecological routes of avian influenza virus transmission to a common mesopredator: an experimental evaluation of alternatives.

BACKGROUND: Wild raccoons have been shown to be naturally exposed to avian influenza viruses (AIV). However, the mechanisms associated with these natural exposures are not well-understood. METHODOLOGY/PRINCIPAL FINDINGS: We experimentally tested three alternative routes (water, eggs, and scavenged waterfowl carcasses) of AIV transmission that may explain how raccoons in the wild are exposed to AIV. Raccoons were exposed to 1) water and 2) eggs spiked with an AIV (H4N6), as well as 3) mallard carcasses experimentally inoculated with the same virus. Three of four raccoons exposed to the high dose water treatment yielded apparent nasal shedding of >10(2.0) PCR EID50 equivalent/mL. Little to no shedding was observed from the fecal route. The only animals yielding evidence of serologic activity during the study period were three animals associated with the high dose water treatment. CONCLUSIONS/SIGNIFICANCE: Overall, our results indicate that virus-laden water could provide a natural exposure route of AIV for raccoons and possibly other mammals associated with aquatic environments. However, this association appears to be related to AIV concentration in the water, which would constitute an infective dose. In addition, strong evidence of infection was only detected in three of four animals exposed to a high dose (e.g., 10(5.0) EID50/mL) of AIV in water. As such, water-borne transmission to raccoons may require repeated exposures to water with high concentrations of virus.

Shedding of a low pathogenic avian influenza virus in a common synanthropic mammal--the cottontail rabbit.

BACKGROUND: Cottontails (Sylvilagus spp.) are common mammals throughout much of the U.S. and are often found in peridomestic settings, potentially interacting with livestock and poultry operations. If these animals are susceptible to avian influenza virus (AIV) infections and shed the virus in sufficient quantities they may pose a risk for movement of avian influenza viruses between wildlife and domestic animals in certain situations. METHODOLOGY/PRINCIPAL FINDINGS: To assess the viral shedding potential of AIV in cottontails, we nasally inoculated fourteen cottontails with a low pathogenic AIV (H4N6). All inoculated cottontails shed relatively large quantities of viral RNA both nasally (≤ 10(6.94) PCR EID50 equivalents/mL) and orally (≤ 10(5.09) PCR EID50 equivalents/mL). However, oral shedding tended to decline more quickly than did nasal shedding. No animals showed any obvious signs of disease throughout the study. Evidence of a serological response was found in all infected rabbits at 22 days post infection in convalescent sera. CONCLUSIONS/SIGNIFICANCE: To our knowledge, cottontails have not been previously assessed for AIV shedding. However, it was obvious that they shed AIV RNA extensively via the nasal and oral routes. This is significant, as cottontails are widely distributed throughout the U.S. and elsewhere. These mammals are often found in highly peridomestic situations, such as farms, parks, and suburban neighborhoods, often becoming habituated to human activities. Thus, if infected these mammals could easily transport AIVs short distances.

Extended viral shedding of a low pathogenic avian influenza virus by striped skunks (Mephitis mephitis).

BACKGROUND: Striped skunks (Mephitis mephitis) are susceptible to infection with some influenza A viruses. However, the viral shedding capability of this peri-domestic mammal and its potential role in influenza A virus ecology are largely undetermined. METHODOLOGY/PRINCIPAL FINDINGS: Striped skunks were experimentally infected with a low pathogenic (LP) H4N6 avian influenza virus (AIV) and monitored for 20 days post infection (DPI). All of the skunks exposed to H4N6 AIV shed large quantities of viral RNA, as detected by real-time RT-PCR and confirmed for live virus with virus isolation, from nasal washes and oral swabs (maximum ≤ 10(6.02) PCR EID50 equivalent/mL and ≤ 10(5.19) PCR EID50 equivalent/mL, respectively). Some evidence of potential fecal shedding was also noted. Following necropsy on 20 DPI, viral RNA was detected in the nasal turbinates of one individual. All treatment animals yielded evidence of a serological response by 20 DPI. CONCLUSIONS/SIGNIFICANCE: These results indicate that striped skunks have the potential to shed large quantities of viral RNA through the oral and nasal routes following exposure to a LP AIV. Considering the peri-domestic nature of these animals, along with the duration of shedding observed in this species, their presence on poultry and waterfowl operations could influence influenza A virus epidemiology. For example, this species could introduce a virus to a naive poultry flock or act as a trafficking mechanism of AIV to and from an infected poultry flock to naive flocks or wild bird populations.

Freshwater clams as bioconcentrators of avian influenza virus in water.

We report experimental evidence for bioconcentration of a low-pathogenicity avian influenza virus (H6N8) in the tissue of freshwater clams. Our results support the concept that freshwater clams may provide an effective tool for use in the early detection of influenza A viruses in aquatic environments.

Experimental infection of raccoons (Procyon lotor) with West Nile virus.

To characterize the responses of raccoons to West Nile virus (WNV) infection, we subcutaneously exposed them to WNV. Moderately high viremia titers (≤ 10(4.6) plaque forming units [PFU]/mL of serum) were noted in select individuals; however, peak viremia titers were variable and viremia was detectable in some individuals as late as 10 days post-inoculation (DPI). In addition, fecal shedding was prolonged in some animals (e.g., between 6 and 13 DPI in one individual), with up to 10(5.0) PFU/fecal swab detected. West Nile virus was not detected in tissues collected on 10 or 16 DPI, and no histologic lesions attributable to WNV infection were observed. Overall, viremia profiles suggest that raccoons are unlikely to be important WNV amplifying hosts. However, this species may occasionally shed significant quantities of virus in feces. Considering their behavioral ecology, including repeated use of same-site latrines, high levels of fecal shedding could potentially lead to interspecies fecal-oral WNV transmission.

Antibody responses of raccoons naturally exposed to influenza A virus.

An investigation was performed to describe the responses of naturally acquired antibodies to influenza A virus in raccoons (Procyon lotor) over time. Seven wild raccoons, some of which had been exposed to multiple subtypes of influenza A virus, were held in captivity for 279 days, and serum samples were collected on 10 occasions during this interval. Serum samples from 9 of 10 bleeding occasions were tested using an epitope-blocking enzyme-linked immunosorbent assay for the presence of antibodies to influenza A virus. Although titer declines were noted in most animals over time, all animals maintained detectable antibodies for the duration of the study. These data indicate that naturally acquired antibodies to influenza A virus can remain detectable in raccoons for many months, with the actual duration presumably being much longer because all animals had been exposed to influenza A virus before this study commenced. This information is important to surveillance programs because the duration of naturally acquired antibodies to influenza A virus in wildlife populations is largely unknown.

Evaluation of an epitope-blocking enzyme-linked immunosorbent assay for the detection of antibodies to influenza A virus in domestic and wild avian and mammalian species.

An epitope-blocking enzyme-linked immunosorbent assay (bELISA) was developed for the detection of antibodies to influenza A virus in taxonomically diverse domestic and wild vertebrate species. In contrast to the bELISAs published previously that require reagent production, manipulation by the end-user, or have not been evaluated for use with both mammalian and avian species, this assay is performed using commercially available recombinant nucleoprotein antigen and corresponding nucleoprotein-specific monoclonal antibody and has been shown to work with multiple avian and mammalian species. The efficacy of the bELISA as a serum screening assay was compared to the agar gel immunodiffusion (AGID) assay using 251 serum samples obtained from experimentally infected mallards (Anas platyrhynchos) and raccoons (Procyon lotor). The concordance between the AGID assay and bELISA was 94.1% (95% CI=89.9, 98.3) for raccoons, and 71.2% (95% CI=63.5, 78.9) for mallards and 82.8% (95% CI=78.2, 87.3) overall. The bELISA was more sensitive than the AGID assay as demonstrated by the detection of antibodies to influenza A virus at earlier time points in experimental infection studies and at higher serial dilutions. The efficacy of the bELISA to monitor natural influenza A virus exposure was also compared to the AGID assay using an additional 745 serum samples from six avian species and six mammalian species. This bELISA provides a rapid, reliable, and inexpensive technique for large-scale surveillance of influenza A virus exposure in taxonomically diverse vertebrate species.

Experimental infection of cliff swallows (Petrochelidon pyrrhonota) with varying doses of West Nile virus.

Cliff swallows (Petrochelidon pyrrhonota) were inoculated with differing doses of West Nile virus (WNV) to evaluate their potential role as reservoir hosts in nature. Swallows often nest in large colonies in habitats and months associated with high mosquito abundance and early WNV transmission in North America. Additionally, cliff swallow diet consists of insects, including mosquitoes, leading to an additional potential route of WNV infection. The average peak viremia titer among infected cliff swallows was 10(6.3) plaque-forming units (PFU)/mL serum and the reservoir competence index was 0.34. There was no correlation between dose and probability of becoming infected or viremia peak and duration. Oral shedding was detected from 2 to 14 days post-inoculation with an average peak titer of 10(4.4) PFU/swab. These results suggest that cliff swallows are competent reservoir hosts of WNV and therefore, they may play a role in early seasonal amplification and maintenance of WNV.