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.

Susceptibility of rock doves to low-pathogenic avian influenza A viruses.

Following a 2008 outbreak of North American low-pathogenic H5N8 influenza A virus at an upland gamebird farm, we sero-sampled rock doves (pigeons, Columba livia) at the outbreak site and conducted experimental inoculations of wild-caught pigeons using the H5N8 virus and another low-pathogenic virus (H4N6). While 13% of pigeons at the outbreak site were seropositive, none were positive for exposure to H5, and one was positive for N8. Challenged pigeons exhibited low susceptibility and limited viral RNA excretion for both viruses tested, but at least one individual had RNA loads indicative of the potential for viral transmission to other birds.

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.

Low-pathogenic avian influenza viruses in wild house mice.

BACKGROUND: Avian influenza viruses are known to productively infect a number of mammal species, several of which are commonly found on or near poultry and gamebird farms. While control of rodent species is often used to limit avian influenza virus transmission within and among outbreak sites, few studies have investigated the potential role of these species in outbreak dynamics. METHODOLOGY/PRINCIPAL FINDINGS: We trapped and sampled synanthropic mammals on a gamebird farm in Idaho, USA that had recently experienced a low pathogenic avian influenza outbreak. Six of six house mice (Mus musculus) caught on the outbreak farm were presumptively positive for antibodies to type A influenza. Consequently, we experimentally infected groups of naïve wild-caught house mice with five different low pathogenic avian influenza viruses that included three viruses derived from wild birds and two viruses derived from chickens. Virus replication was efficient in house mice inoculated with viruses derived from wild birds and more moderate for chicken-derived viruses. Mean titers (EID(50) equivalents/mL) across all lung samples from seven days of sampling (three mice/day) ranged from 10(3.89) (H3N6) to 10(5.06) (H4N6) for the wild bird viruses and 10(2.08) (H6N2) to 10(2.85) (H4N8) for the chicken-derived viruses. Interestingly, multiple regression models indicated differential replication between sexes, with significantly (p<0.05) higher concentrations of avian influenza RNA found in females compared with males. CONCLUSIONS/SIGNIFICANCE: Avian influenza viruses replicated efficiently in wild-caught house mice without adaptation, indicating mice may be a risk pathway for movement of avian influenza viruses on poultry and gamebird farms. Differential virus replication between males and females warrants further investigation to determine the generality of this result in avian influenza disease dynamics.

Shedding light on avian influenza H4N6 infection in mallards: modes of transmission and implications for surveillance.

BACKGROUND: Wild mallards (Anas platyrhychos) are considered one of the primary reservoir species for avian influenza viruses (AIV). Because AIV circulating in wild birds pose an indirect threat to agriculture and human health, understanding the ecology of AIV and developing risk assessments and surveillance systems for prevention of disease is critical. METHODOLOGY/PRINCIPAL FINDINGS: In this study, mallards were experimentally infected with an H4N6 subtype of AIV by oral inoculation or contact with an H4N6 contaminated water source. Cloacal swabs, oropharyngeal swabs, fecal samples, and water samples were collected daily and tested by real-time RT-PCR (RRT-PCR) for estimation of viral shedding. Fecal samples had significantly higher virus concentrations than oropharyngeal or cloacal swabs and 6 month old ducks shed significantly more viral RNA than 3 month old ducks regardless of sample type. Use of a water source contaminated by AIV infected mallards, was sufficient to transmit virus to naïve mallards, which shed AIV at higher or similar levels as orally-inoculated ducks. CONCLUSIONS: Bodies of water could serve as a transmission pathway for AIV in waterfowl. For AIV surveillance purposes, water samples and fecal samples appear to be excellent alternatives or additions to cloacal and oropharyngeal swabbing. Furthermore, duck age (even within hatch-year birds) may be important when interpreting viral shedding results from experimental infections or surveillance. Differential shedding among hatch-year mallards could affect prevalence estimates, modeling of AIV spread, and subsequent risk assessments.