Browsing by Author "Bowen, Richard, advisor"
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Item Open Access Developing a murine model for Q fever(Colorado State University. Libraries, 2011) Benson, Jeret, author; Bowen, Richard, advisor; Duncan, Colleen, committee member; Jones, Robert, committee member; Van Metre, David, committee memberCoxiella burnetii is a gram-negative, intracellular bacterium that causes disease in humans and animals. The bacterium is commonly found in nature and humans and animals develop infections by inhaling infectious aerosols. Animal infections are generally asymptomatic, but the organism can induce abortion in pregnant sheep, goats, and cattle. Human infections, called Q fever, can induce mild to moderate disease, and lifelong infections may develop. Research to characterize this bacterium has been difficult due to its intracellular nature, and studying experimental infections in animal models has provided important information about the bacterial lifecycle and pathogenesis of the disease. The studies described here focused on evaluating a number of facets of C. burnetii infection in C57BL/6 inbred mice. Infections were determined through immunofluorescence detection, quantitative PCR assays, and histopathologic analysis. Mice developed similar histopathologic lesions as humans, specifically hepatitis, interstitial pneumonia, and myocarditis when infected by intranasal inoculation with the Nine Mile phase I strain of the bacterium. Detection of bacterial DNA in tissues and frequency of histopathologic lesions were highest two weeks after infection, with a significant decrease observed 42 and 59 days after infection. Mouse age and chemically induced immunosuppression with dexamethasone or cyclophosphamide were evaluated in C. burnetii infected mice to determine if these factors exacerbated disease. These studies revealed that neither age, (nine-weeks versus nine-months), or chemically induced immunosuppression (dexamethasone versus cyclophosphamide) significantly enhanced disease manifestations in infected mice. Additionally, antimicrobial treatment with doxycycline was evaluated in treating C. burnetii infections in mice. Such treatment reduced splenic pathology but did not significantly reduce the frequency of other histopathologic lesions or the amount of bacterial DNA detected in tissues. Overall, C57BL/6 mice infected by intranasal inoculation develop histopathologic lesions similar in many respects to what is observed in infected humans. However, disease manifestations were not exacerbated by host age or the immunosuppressive treatments investigated. Additionally, the dose of doxycycline received by mice was only marginally effective in treating bacterial infection in mice.Item Open Access Environmental maintenance and transmission of Francisella tularensis in cottontail rabbits, prairie voles, and amoebae(Colorado State University. Libraries, 2016) Brown, Vienna Rae, author; Bowen, Richard, advisor; Bachand, Annette, committee member; Pabilonia, Kristy, committee member; Petersen, Jeannine, committee memberTularemia is a zoonotic disease that is endemic in much of the Northern Hemisphere, capable of causing severe disease in a wide range of hosts. This disease is caused by the gram-negative bacterium Francisella tularensis and most human cases are caused by either subsp. tularensis (type A) or holarctica (type B). Genetic clustering has led to further differentiation within type A and type B strains; type A strains are currently classified as A1a, A1b, and A2. Due to the high virulence and low infectious dose of this pathogen, naïve immune status of the public, and previous weaponization, F. tularensis has been classified as a Tier 1 Select Agent by the Centers for Disease Control and Prevention. Although the Francisella bacterium was discovered over a century ago, understanding of ecological factors that contribute to environmental maintenance and transmission remains enigmatic. Extensive research has been performed in a variety of laboratory animal models to evaluate factors related to disease progression and vaccine and therapeutic options; however, very little is known about reservoir and/or amplification hosts in a natural setting. Reported here are a series of experimental studies performed in cottontail rabbits and voles as well as in vitro infections of amoebae with multiple strains of F. tularensis. The objectives of the in vivo studies were to characterize clinical disease, tissue dissemination and organ burden, and morbidity and mortality in a species believed to play an important role in naturally acquired infections. Rabbits were inoculated using a strain and dose of organism as well as a route of infection in accordance with what would be expected in nature. The initial experimental infections of cottontail rabbits involved intradermal inoculation with one of several strains of F. tularensis which resulted in varied patterns of clinical disease, gross pathology, and histopathology. Each of the type A strains was highly virulent, with rabbits requiring euthanasia or succumbing to infection 3-13 days post-infection. Gross lesions observed in infected rabbits included numerous microabscesses in the livers and spleens, suggesting high bacterial organ burdens. In contrast, most rabbits infected with type B strains developed a mild fever and became lethargic, but the disease was infrequently lethal. Those rabbits infected with type B strains that survived longer than 14 days post-infection developed a robust humoral immune response, and F. tularensis was not isolated from liver, spleen, or lungs of those animals. These findings depict a clear difference in virulence and immune kinetics between type A and B strains of F. tularensis in cottontail rabbits. Based on findings from the original study with cottontail rabbits, I evaluated the protection afforded against infection with a type A strain of F. tularensis by prior inoculation with a type B strain. Previous infection with a type B strain of the organism was found to lengthen survival time and, in some cases, prevented death following inoculation with a type A2 strain of F. tularensis. In contrast, inoculation of a type A1b strain was uniformly lethal in cottontail rabbits irrespective of a prior type B inoculation. These findings provide important insight about the role cottontail rabbits may play in environmental maintenance and transmission of this organism. Prairie voles are believed to acquire a natural infection with F. tularensis from contact with infected waterways or cannibalism of another vole that died from a tularemia infection. To evaluate such infection experimentally, I inoculated prairie voles orally with 107 organisms of type B F. tularensis and serially euthanized them to characterize organ burdens and pathology. The inoculated voles failed to show any clinical signs of disease and upon necropsy did not present with any gross lesions. Furthermore, organisms were not recovered from the liver and spleen, and antibodies were not detected, despite evaluation >14 days post-infection. Eight voles were then challenged intranasally with 350-650 organisms of one of two strains of F. tularensis. Infection with one strain (OR96-0246) resulted in all the animals succumbing to death or euthanasia between 6 and 7 days post-infection, whereas voles infected with the other strain (KY99-3387) survived to the end of the study period (10 days post-infection), with the exception of one vole which succumbed to infection. These findings were surprising and require further investigation to understand how voles become infected in nature and what role they may play in F. tularensis persistence and transmission. Free-living amoebae are capable of harboring pathogens and have been implicated in various disease outbreaks. I evaluated 3 strains of Acanthamoebae and 1 strain of Hartmannella as hosts for three bacterial pathogens. All strains of amoebae were propagated in culture with virulent strains of F. tularensis, Burkholderia pseudomallei and methicillin-resistant Staphylococcus aureus, with the aim of elucidating both general principles and pathogen-specific mechanisms associated with bacteria-amoebae interactions. F. tularensis and B. pseudomallei were recoverable from the lysate for all four strains of amoebae at both 4 and 24 hours post-inoculation, whereas MRSA was recoverable from the lysate of all four strains at the 4 hour time point and from only two of the strains at the 24 hour time point. Confocal microscopy allowed for the visualization of labeled bacteria of each strain and differentiation of amoebae morphology was possible. These findings provide intriguing evidence that amoebae are capable of phagocytosing pathogenic bacteria and that protozoa may play a role in environmental maintenance and persistence.Item Embargo Evaluation of feral swine as potential reservoirs, sentinels, and vectors of emerging and re-emerging zoonotic pathogens in the United States(Colorado State University. Libraries, 2023) Maison, Rachel Marie, author; Bosco-Lauth, Angela, advisor; Bowen, Richard, advisor; Borlee, Bradley, committee member; Brown, Vienna, committee member; Han, Sushan, committee memberFeral swine are an extremely adaptable and prolific invasive species present in many regions of the United States. A generalist diet, high reproductive capacity, and opportunistic nature make them a significant threat to native flora and fauna, and their destructive foraging behaviors have been attributed to substantial crop loss and property damage throughout their range. Feral swine have also been demonstrated as vectors and reservoirs for many diseases, some of which are transmissible to humans. Despite the efforts of government agencies to impede range expansion of feral swine and monitor populations for disease, human-mediated movements as well as continued natural dispersal in some regions have made management difficult. Subsequently, this invasive species continues to threaten human and animal health throughout its current range. Reported herein are a series of laboratory and field-based investigations that seek to address the potential role(s) of feral swine in the epidemiology of a few zoonotic pathogens that are emerging or re-emerging in the United States. As pigs interact closely with soils through their natural rooting and wallowing behaviors, we were primarily interested in soil-dwelling zoonoses, and identified three of particular interest. The two bacterial species Bacillus anthracis and Burkholderia pseudomallei, as well as the fungus Coccidioides, are the causative agents of anthrax, melioidosis, and coccidioidomycosis respectively, and each is understudied as they relate to feral swine. While each of these organisms is unique in their biology and the relative threat(s) they've posed to humans and animals throughout their respective histories, they are similar in that much of their ecology remains undescribed. The often-complex ecological relationships exhibited by pathogenic organisms are vital to their continued survival and evolution and can play an important role in their dissemination to human populations. The presence of high-density populations of feral swine in many regions of the United States, as well as their ability to occupy a range of habitats, may be playing a significant role in the persistence and dissemination of these organisms. Furthermore, the interactions of feral swine with contaminated substrates within their environment, as well as high levels of interspecies interactions throughout their current range could make them a likely source of additional emerging or novel pathogens. After a summary of the literature concerning feral swine in the United States, followed by that for the causative agents of anthrax, coccidioidomycosis, melioidosis, and a few select emerging disease threats, each respective chapter within this work details investigations largely by pathogen. Within Chapter 2, we present the results of a field serosurvey in feral swine residing across regions of known anthrax endemicity in Texas, and retrospectively document bacterial exposure via enzyme-linked immunosorbent assay (ELISA). This study was performed principally to evaluate the biosentinel utility of feral swine for anthrax contaminated environments, and the ELISA utilized was developed and optimized for detecting antibodies of swine origin to B. anthracis protective antigen. We additionally report on a laboratory-based experimental infection study where which a group of juvenile feral swine were either intranasally or subcutaneously exposed to varying amounts of B. anthracis strain Sterne 34F2 spores, and seroconversion as well as bacterial shedding through the nasal passages documented over time. Through both the field-based serosurvey, as well as the experimental inoculation study, we report that feral swine serology may be used as a management strategy to indirectly identify regions contaminated with anthrax bacteria. Moreover, we report that some feral swine intranasally exposed to high amounts of B. anthracis Sterne contained detectable and viable spores within their nasal passages up to 56 days past their initial exposure event as demonstrated by bacterial culture. The presence of viable B. anthracis within the nasal mucosa well after an exposure event suggests that feral swine may be capable of spreading infectious anthrax bacteria as mechanical vectors. Chapter 3 of this work details an experimental infection study similar to that reported in the previous chapter, however, instead intranasally exposes a group of juvenile feral swine to Coccidioides posadasii strain Silveira spores. This study was performed to evaluate the pathogenesis and immune response of feral swine to one of the causative agents of coccidioidomycosis. Fungal culture of tissues collected from inoculated individuals at necropsy was performed to assess whether animals might act as reservoirs for the fungus in addition to describing the characteristics of infection. Feral swine utilized for this study did not display overt clinical signs of disease, and despite being inoculated with a large dose of fungal spores, also did not seroconvert based on the results of porcine agar gel immunodiffusion assays. Fungal culture of lung and mediastinal lymph node tissues of a subset of pigs revealed the presence of viable C. posadasii, however, examination of additional sections of the same tissues did not reveal the presence of the granulomatous lesions often associated with coccidioidomycosis. Most individuals had significant comorbidities as illustrated via histology, most notably Metastrongylus nematodes in the lungs. Despite an absence of lesions and organisms histologically, isolation of the fungus from tissues by culture indicated active infection and imply that feral swine are mildly susceptible to acute Coccidioides infection. These results suggest that in some instances, feral swine may aid in fungal dispersal on the landscape due to the presence of spherules in tissues post-mortem. Chapter 4, concerning melioidosis, reports on laboratory-based experiments evaluating an indirect ELISA for measuring antibodies to B. pseudomallei in domestic and wild swine. Described previously to test human sera, this ELISA utilized whole-cell lysate derived from B. pseudomallei strain Bp82 and was optimized and preliminarily validated by testing sera from domestic goats and domestic swine experimentally inoculated with virulent and avirulent strains of B. pseudomallei, respectively. Further evaluation of assay specificity was performed by testing sera against similar antigen preparations of the closely related bacterial species Pseudomonas aeruginosa strain PAO1. Examination of sera from laboratory animals against Bp82 and PAO1 whole-cell lysate antigens revealed that most animals seroconverted and displayed B. pseudomallei antibodies, and that little cross-reactivity existed between antigens. After preliminary assay validation, serum from wild pig populations originating from Arizona, California, and Puerto Rico were assessed using both antigens to document B. pseudomallei exposure in wild pigs residing in regions where bacterial absence or endemicity can be confidently inferred at this time. In stark contrast to the serology displayed by laboratory animals, analysis of field sera collected from these regions demonstrated high levels of cross-reactivity between Bp82 and PAO1 antigens, suggesting the assay is not suitable for use in wild pig populations. Lastly, Chapter 5 describes a general survey of feral swine removed from the Aransas National Wildlife Refuge in Texas for each of the organisms investigated in the proceeding chapters, as well as additional bacterial and viral pathogens; added pathogens that were included within this survey included Francisella tularensis, and SARS-CoV-2, the causative agents of tularemia and COVID-19, respectively. A suite of samples, including external parasites (e.g., ticks), nasal swabs, blood, and a series of tissues were collected from each feral swine in the field and analyzed by bacterial and fungal culture, serology, and viral assays to determine if any pigs were actively infected or previously exposed to a range of pathogens. General and selective culture of tissue samples did not reveal active infection with B. anthracis, Coccidioides spp., B. pseudomallei, or other pathogenic bacteria, however, serology illustrated that a subset of pigs were previously exposed to B. anthracis. Further examination of tissues histologically illustrated a high degree of parasitic infection in the majority of pigs, particularly with Metastrongylus nematodes. Finally, three ixodid tick species, all adults, were collected off of the majority of feral swine sampled, none of which appeared to be carrying infectious virus based on the results of general cytopathic effects assays. Results generated for this study further confirm that feral swine are hosts for a range of ixodid ticks present in Texas and include species that are known to pose a risk to the health of livestock, wildlife, and humans. Moreover, serological results indicating previous exposure to anthrax-causing bacteria agree with past ecological modeling studies that have suggested environmental suitability for B. anthracis in the region of Aransas National Wildlife Refuge. Taken together, these investigations demonstrate that feral swine in the United States very likely are contributing to the ecology of anthrax and coccidioidomycosis by being mechanical vectors and possible reservoirs of B. anthracis and Coccidioides, respectively. Serological data demonstrated from field and laboratory studies additionally support the use of feral swine as biosentinels within landscapes undergoing invasive species management and that may be contaminated with B. anthracis. While the serological data generated through the B. pseudomallei investigations reported here currently do not support use of a crude whole-cell lysate-based ELISA to evaluate samples collected from the field, we hypothesize that feral swine may be reservoirs and/or vectors of melioidosis-causing bacteria. Future investigations should continue to examine additional antigens for serologic testing, as laboratory-infected swine demonstrated a measurable antibody response after intranasal exposure with Bp82. Additional follow-up studies should also be conducted to further describe the relative role(s) that feral swine may play in the ecology and epidemiology of B. anthracis and Coccidioides, particularly if regions with high incidence of animal or human disease correspond with high densities and activity of feral swine. Future research should continue to screen feral swine as well as the ectoparasites that feed upon them for emerging or re-emerging pathogens, especially in areas with high biodiversity or species of conservation concern, as well as in areas with high levels of human activity.Item Open Access Japanese encephalitis virus: pathogenesis and immunity(Colorado State University. Libraries, 2010) Bosco-Lauth, Angela M., author; Bowen, Richard, advisor; Quackenbush, Sandra, advisorJapanese encephalitis virus (JEV) is a vector-borne disease of Asian origin that has the potential to spread into temperate regions across the globe. The recent incursion of the virus into Northern Australia illustrates its ability to replicate in different vectors and has led to an increase in the need for disease surveillance in the U.S. and other locations. The focus of this dissertation was to investigate the potential of some common North American mosquitoes to transmit JEV and to study the pathogenesis and immunity in animal models, namely horses and hamsters.Item Open Access The role of domestic ducks in the maintenance and spread of avian influenza viruses in Indonesia(Colorado State University. Libraries, 2012) Pabilonia, Kristy L., author; Bowen, Richard, advisor; Aboellail, Tawfik, committee member; Hyatt, Doreene, committee member; Knight, Anthony, committee memberWild waterfowl and aquatic birds serve as the natural reservoir host for influenza A viruses. As the reservoir, wild waterfowl play an important role in the persistence and transmission of influenza viruses among bird populations and to other mammalian species. In many Asian countries, domestic ducks are raised for meat and egg production. Some of these domestic ducks are ranged on rice paddies or post-harvest rice fields. The ducks provide service to the rice fields by fertilizing the field with feces and aerating the field by swimming and walking through the ground cover. Additionally, the ducks serve as a form of insect control through their natural grazing behaviors. The role that domestic ducks play in the ecology of influenza viruses is poorly understood. Highly pathogenic avian influenza H5N1 virus (HPAI H5N1) originated in Guangdong Province, China in 1996, which was followed by global dissemination of the virus that began in 2003. This virus is unprecedented in geographical spread, economic consequences and public health significance. At the present time, HPAI H5N1 virus is endemic six countries, including Indonesia. Indonesia has experienced the highest incidence of human infections with HPAI H5N1 virus and one of the highest case fatality rates. Control of the virus in Indonesia has proven extremely challenging, due to its diverse and complex poultry and domestic duck production systems. HPAI H5N1 virus is highly virulent in chickens and turkeys and causes severe systemic disease. Outbreaks of HPAI H5N1 in poultry populations are accompanied by high mortality. In contrast, HPAI H5N1 virus is typically nonpathogenic or mildly pathogenic in ducks and mortality in duck flocks during outbreaks of the virus is absent or limited. This allows ducks to serve as silent carriers of the virus, as they may shed large quantities of virus without displaying clinical signs of illness allowing infected ducks to evade detection by flock owners or government livestock officials. Domestic duck production is common in Southeast Asia. Indonesia has a large domestic duck population, estimated at more than 34 million ducks. Because HPAI H5N1 induces only mild disease in domestic ducks, outbreaks of the virus are difficult to detect and are rarely reported by domestic duck flock owners. Thus, domestic duck flocks have been left out of many government HPAI H5N1 surveillance and control programs. While a number of studies have demonstrated that the presence of domestic ducks in a country or at a specific location may be a risk factor for the presence of HPAI H5N1 virus, few studies have been conducted evaluating the role that domestic ducks play in the ecology of HPAI H5N1 virus. The objectives of the studies described in this dissertation were to elucidate the role of domestic ducks in the maintenance and spread of avian influenza viruses, particularly HPAI H5N1 virus, by evaluating domestic duck flock characteristics and behaviors, estimating the prevalence and incidence of avian influenza viruses in these flocks and characterizing HPAI H5N1 viruses detected in the field. To meet the objectives, two studies were conducted in West Java, Indonesia. The first study was a cross-sectional study aimed at characterizing domestic duck flocks and estimating the point prevalence and seroprevalence of avian influenza viruses, particularly HPAI H5N1 virus. This study was followed by a 7 month longitudinal study, aimed at estimating the incidence of avian influenza viruses, particularly HPAI H5N1 virus, in domestic duck flocks and evaluating flock illness and mortality during avian influenza virus outbreaks. A subset of samples from each of the studies was transported to the United States for virus characterization. The findings of the studies conducted demonstrate that domestic duck flocks are raised in complex production systems, are highly mobile, have significant contact with wild and domestic birds and mammals, are frequently ill and are provided with little formal veterinary care. The prevalence and incidence of avian influenza virus, including HPAI H5N1 virus, are high in domestic duck flocks in Indonesia. Clinical signs of illness and increased mortality did not correlate with the presence of avian influenza virus in the flock. Interestingly, there was also no correlation between increased flock mortality and the presence of HPAI H5N1 virus in the flock, demonstrating that domestic duck flocks can be asymptomatically infected with HPAI H5N1 virus while shedding high quantities of virus. Characterization of some of the viruses isolated from domestic duck flocks demonstrated that the flocks can be infected with more than one avian influenza virus at one time, as demonstrated by one flock that was positive for HPAI H5N1, as well as H3 and H7 avian influenza viruses. These situations are concerning, as domestic duck flocks may serve as mixing vessels for avian influenza viruses and co-infections in these flocks may result in the emergence of novel influenza viruses that may have capabilities for human-to-human transmission. It is likely that domestic ducks play an important role in the maintenance and spread of avian influenza viruses, including HPAI H5N1 virus. A number of domestic duck flock practices, including extensive flock movement, frequent introduction and sale of ducks, free-ranging of ducks in areas where they have contact with wild birds and animals and continual contact of duck flocks with other duck and poultry flocks, increasingly adds to the difficulty of control of HPAI H5N1 virus within this production system and makes eradication of the virus within a country extremely challenging.Item Open Access Transmission and shedding of Middle East respiratory syndrome coronavirus from dromedary camels, alpacas, and domestic livestock(Colorado State University. Libraries, 2016) Adney, Danielle, author; Bowen, Richard, advisor; Mathiason, Candace, committee member; Olea-Popelka, Francisco, committee member; Zabel, Mark, committee memberThe Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel betacoronavirus discovered in 2012 that causes severe respiratory disease that can result in death in infected humans. Human-to-human transmission can occur, although zoonotic transmission from dromedary camels plays an important role in transmission. A large percentage of dromedaries in the Middle East and Africa have antibodies specific for MERS-CoV, indicating widespread transmission among camels. In vitro studies indicate that other livestock may be susceptible to infection and transmit virus, however, field studies have not detected any seropositive goats, horses, sheep, or horses. Due to the requirement for specific high-containment facilities research on the role of dromedaries has primarily been limited to field surveys. Here we report experimental infection of dromedary camels with a human isolate of MERS-CoV. The objectives of this study were to characterize clinical disease, shedding, and tissue burdens in infected animals. Experimental infection with the virus resulted in a mild, transient upper respiratory tract infection accompanied by shedding of large amounts of infectious virus. While infectious virus was only detected for a short time viral RNA was detected for much longer, indicating that field studies only using PCR may not be sampling animals able to transmit virus. Due to their cost, size, and temperament, dromedaries are not conducive to high-containment studies, and we hypothesized that alpacas may be a suitable replacement for some studies. This dissertation reports shedding and transmission of MERS-CoV in experimentally infected alpacas (n = 3) or those infected by direct contact (n = 3). Infectious virus was detected in all experimentally infected animals and in 2 of 3 in-contact animals. All alpacas seroconverted and were rechallenged 70 days after the original infection in order to understand if previous infection results in protective immunity. Experimentally infected animals were protected against reinfection, and those infected by contact were partially protected. Necropsy specimens from immunologically naive animals (n = 3) obtained on day 5 postinfection showed virus in the upper respiratory tract. These data demonstrate efficient virus replication and animal-to-animal transmission and indicate that alpacas might be useful surrogates for camels in laboratory studies. Current understanding of MERS-CoV infection suggests that camels become infected as calves or during transport to slaughterhouses, and we hypothesized that vaccinating naïve animals could decrease viral shedding upon exposure. We vaccinated three dromedary camels and two alpaca with a MERS-CoV S protein subunit vaccine. Vaccinated animals developed neutralizing antibodies to MERS-CoV with titers ranging from <10 to 2560 at week 16. Vaccinated animals, as well as two control camels and two control alpaca were challenged with a human isolate of MERS-CoV and were monitored for clinical signs and viral shedding. Vaccinated animals displayed partial protection associated with low or moderate antibody titers whereas the vaccinated alpaca developed high levels of antibody and were protected against infection following viral challenge. In vitro assays indicate that sheep, goats, and horses are susceptible to infection with MERS-CoV. However, unlike dromedaries and alpacas, infected sheep, goats, and horses all failed to shed more than trivial quantities of virus and are therefore unlikely to serve as a source of transmission to humans or other animals. The research presented in this dissertation provides experimental evidence linking dromedaries to MERS-CoV infection. Here, we present the first experimental infection of dromedary camels with MERS-CoV. We describe clinical disease, viral shedding, organ burden, and seroconversion in these animals. It is hoped that these studies will help shape camel sampling as well as allow for a more complete understanding of current field sampling data. Because of the difficulties associated with dromedary camels, we developed a more tractable model of reservoir infection. We found that alpaca model can be a useful substitution for camels in some studies and as well as demonstrate its strengths and weaknesses in a vaccination study. Finally, the data presented here indicate that sheep, goats, and horses do not shed large amounts of infectious virus after challenge with MERS-CoV. This indicates that these animals do not play an important role in viral transmission and that surveillance and vaccine efforts should focus primarily on dromedary camels.Item Open Access Viral shedding and antibody response of mallard ducks to avian influenza viruses(Colorado State University. Libraries, 2012) Muth, Jack P., author; Bowen, Richard, advisor; Landolt, Gabriele, committee member; Mason, Gary, committee member; Zabel, Mark, committee memberWild ducks are a key reservoir for avian influenza (AI) viruses. Their long distance migrations, coupled to frequent contact with domestic poultry enhances risk for spread of highly pathogenic avian influenza (HPAI) viruses. Despite years of study, our understanding of how AI viruses are maintained and transmitted in nature remains poorly understood. The work described here examines several aspects of avian influenza virus infections that play a role in perpetuation and spread of this disease, including persistence of virus in duck feces, effect of prior exposure to AI viruses on subsequent infections and the passage of maternal antibodies between hen and duckling. In recent years, the emergence of H5N1 HPAI virus stimulated establishment of massive international surveillance programs to detect that virus in wild waterfowl. One deficit in these efforts was a lack of data on the stability of AI virus and AI virus RNA in bird feces under different environmental conditions. Consequently, an experiment was designed to address this knowledge gap. Feces were collected from mallards infected with a low pathogenic avian influenza (LPAI) virus (H5N2) on days 3 and 4 post infection and kept in environmental chambers for 21 days under the following conditions: 32°C/20% relative humidity (RH), 32°C/50%RH, 32°C/90%RH, 4.5°C/50%RH, 4.5°C/90%RH, and 0°C/50%RH. Sensitivity of detection of infectious virus in fresh fecal material was equivalent to that from cloacal swab samples, while time and environmental conditions did not significantly affect detection of AI virus RNA by PCR. Infectious virus was isolated from feces for considerably shorter intervals than RNA could be detected and was isolated for longer periods of time when feces were maintained under cold conditions. High relative humidity also had a negative effect on virus isolation at 4.5°C. Use of quantitative reverse transcriptase PCR to detect AI virus in fecal samples is as a valuable tool in limiting the labor involved in surveying wild ducks for AI virus. Few prior studies have examined virus shedding over the course of short interval, sequential infections of ducks with LPAI viruses, as likely occurs in natural settings such as breeding grounds. We characterized such infections by sequential inoculation of ducks with homosubtypic versus heterosubtypic with H5N2 and H3N8 LPAI viruses. We found that prior infection with either virus reduced the duration of viral shedding during a subsequent infection initiated 14 or 28 days later. Further, shedding was significantly shorter when the secondary infection occurred 28 days following the initial infection compared to 14 days. No difference in rate of shedding for the secondary infection were noted based on the viral subtype causing the initial infection, suggesting induction of some degree of heterosubtypic immunity. As reported from previous studies, some ducks shed virus but did not develop detectable antibody titers. There was no evidence of subtype cross-reactivity by antibodies as demonstrated by hemagglutination inhibition testing. The antibody response to a heterosubtypic virus was not improved by a prior infection while a second infection with the same virus was capable of boosting the antibody response to that virus. This information should be useful in parameterizing models examining the ecology of avian influenza infection. Another factor of significance in understanding transmission of AI viruses among wild ducks is the influence of passive immunity. A third study was performed to evaluate the magnitude of passive transfer of anti-influenza virus antibodies in mallard ducks and to determine their rate of decay in ducklings. Since not all ducks develop antibodies following natural infection with AI virus and the antibody titers are typically low, a vaccine was used to induce consistent seroconversion. Four, 11 month-old mallard hens were inoculated with a recombinant H5 protein in adjuvant. Specifically, hens received a single injection of 20 μg of hemagglutinin protein derived from A/Vietnam/1203/2004 emulsified in Freund's incomplete adjuvant. Beginning two weeks post-vaccination, eggs were collected daily. Yolk was harvested from eggs laid at one-week intervals and the remainder of the eggs incubated for hatching. All hens developed detectable antibody titers with an average log 2 hemagglutination inhibition titer (HI) of 6.4. Maternal-origin antibodies were detected in the yolk of eggs laid by all hens. Antibody titers peaked in yolks three weeks post vaccination for two hens and were still rising four weeks post vaccination for the other two hens. The highest yolk HI antibody titer was 32. Serum samples from the ducklings hatched from vaccinated hens were collected between days 0 and 22 post-hatch. The calculated mean half-life of maternal antibody in ducklings was 2.3 days with a range of 1.6 to 4.0 days. The short duration of passive immunity in ducks is similar to what has been reported for other species of birds and suggests that maternal antibodies may not play a major role in modulating protection against AI virus infection in natural populations. The strong immune response elicited by the H5 protein suggested that further evaluation should be performed to determine the viability of this vaccine for ducks.