Mountain Scholar :: Browsing by Author "Huyvaert, Kathryn P., committee member"

Browsing by Author "Huyvaert, Kathryn P., committee member"

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Open Access
An eco-epidemiological approach to management of tuberculosis in free-ranging and captive wildlife
(Colorado State University. Libraries, 2018) Rosen, Laura Elizabeth, author; Olea-Popelka, Francisco J., advisor; Miller, Michele A., committee member; Huyvaert, Kathryn P., committee member; Hobbs, N. Thompson, committee member
Tuberculosis (TB) is a disease of global importance affecting millions of humans, livestock, and wildlife. Control and eventual eradication of TB depends on dedicated management actions for all species. Accurately diagnosing TB can be challenging in wildlife species, for which validated tests may be unavailable or of limited sensitivity or specificity. Managing TB in wildlife poses additional difficulties, requiring considerable time and resources to implement at an appropriately broad scale. Each unique ecosystem where TB occurs requires management interventions designed to meet the area's conservation, ecological, social, and financial needs. In this dissertation, I explored the diagnosis and management of tuberculosis in wildlife in three different settings: free-ranging European badgers (Meles meles) in Ireland, working African elephants (Loxodonta africana) in Zimbabwe, and captive African and Asian elephants (Elephas maximus) in North America. Badgers are a reservoir of bovine TB in Ireland, while captive elephants around the world are at risk of TB from their human handlers. Badgers have historically been managed by culling, but there is a current transitioning to vaccination as the primary management tool. In contrast, captive elephants in high-resource settings are typically treated for TB upon diagnosis, although this option may be limited in low-income countries. The first objective of this research was to assess the impact of environmental factors in management of TB over three different studies. I explored how biotic and abiotic factors influence trapping success of badgers being managed for bovine TB in Ireland. In a second study of badgers, I estimated density of a population undergoing vaccination in relation to environmental variables and prior management history. Underlying badger density is an important driver in the TB disease dynamics between cattle and badgers, and can be used in predictions about and assessment of outcomes under vaccination. Finally, I examined potential risk factors for TB seropositive status in working African elephants in Zimbabwe, and identified unique potential exposures from the environment. The second objective of this dissertation was to study the performance of diagnostic tests in a novel setting and interpret the results in the context of exposures within the ecosystem. This study employed two serological tests, STAT-PAK and DPP, for the first time in working African elephants in a range country. I interpreted the results suggestive of exposure to mycobacteria in some elephants based on possible interactions with the complex community of humans, livestock, wildlife, and mycobacteria. The third objective of this dissertation was to develop recommendations for TB management programs based on surveys, capture data, and consideration of individual, population, and community factors. The results from our badger trapping study in Ireland formed the basis of suggested conditions under which vaccine delivery can be increased, because captures are most likely. We used mark-recapture data to estimate badger density in a vaccination area, which adds an important dimension to the Irish TB management program that includes badgers and cattle. Population density is an important factor in pathogen transmission and estimating density using these methods may be a priority for other wildlife populations being managed for TB. Our study of TB treatment in elephants provided a compilation of empirical data for elephant managers and veterinarians to inform clinical decision making. It also underscores the need for improved diagnostics to more confidently identify when animals are no longer infectious. For working African elephants, we documented other wildlife species with host potential on and around facilities, and considered these as possible sources for mycobacterial transmission. Our management guidelines for TB prevention specifically include measures to reduce direct and indirect contact with potential host species. Management of TB across humans and animal species remains a challenging prospect. A One Health approach that incorporates data and techniques across disciplines to build a complete picture of disease control is ideal for TB in wildlife. I drew from ecology and epidemiology to implement a holistic approach to diagnosing and managing TB in species of conservation concern, provide insight into the challenges of diagnosing and managing TB in free-ranging and captive wildlife, describe the benefits of a transdisciplinary approach, and expose areas in need of further research.
Open Access
Bacterial coldwater disease investigations
(Colorado State University. Libraries, 2021) Avila, Brian Walter, author; Winkelman, Dana L., advisor; Huyvaert, Kathryn P., committee member; Fetherman, Eric R., committee member; Hobbs, N. Thompson, committee member
To view the abstract, please see the full text of the document.
Open Access
Factors affecting flea densities in prairie dog colonies: implications for the maintenance and spread of plague
(Colorado State University. Libraries, 2014) Eads, David A., author; Antolin, Michael F., advisor; Biggins, Dean E., advisor; Gage, Kenneth L., committee member; Huyvaert, Kathryn P., committee member
Plague is a re-emerging, rodent-associated disease caused by the primarily flea-borne bacterium Yersinia pestis. The bacterium likely originated 1,500-20,000 years ago in Asia but has been transported by humans to multiple additional continents and islands where it degrades populations of a wide array of rodents. In the western United States, there is an urgent need to acquire a deeper understanding of plague because over half the rodent species of conservation concern occur within its introduced range. This dissertation describes research on fleas in colonies of black-tailed prairie dogs (Cynomys ludovicianus), colonial rodents that amplify Y. pestis in the Great Plains. Adult fleas were combed from live-trapped prairie dogs during June-August 2010-2012 in the short-grass prairie of Vermejo Park Ranch, New Mexico, USA. We evaluated correlations between flea densities and the attributes of soils, prairie dog colonies, and weather. Adult fleas were most abundant in portions of prairie dog colonies with coarse surface-soils and moderately textured subsurface-soils. Coarse surface-soils may allow precipitation to infiltrate to the depth of prairie dog nests, where the moisture could create humid microclimates that are preferred by fleas. Inside burrows, moderately textured soils may hold considerable amounts of water, some of which could evaporate into prairie dog nests, thereby creating humid microclimates. Although fleas tend to fare best under humid conditions, they were scarce in areas with very wet subsurface-soils, presumably because sodden soils can facilitate the accumulation of fungi and mites, some of which are lethal to fleas. We also studied the abundance of fleas in old colonies (initially 8-11 years-old) and young colonies (3-6 years). Fleas were 110% more abundant in old colonies and their abundance was positively correlated with the number of years since a colony was established. Fleas may accumulate to high densities in old colonies because prairie dogs have created deep burrows there, and deep burrows provide ectothermic fleas with humid microclimates and stable temperatures. Moreover, older burrows presumably contain a wealth of organic matter upon which flea larvae feed. Fleas desiccate under dry conditions and, consequently, their densities are thought to decline during droughts. At Vermejo, February-June precipitation was relatively plentiful in 2010 and 2012 but scarce in 2011, the driest spring-summer on record for New Mexico. Unexpectedly, fleas were 250% more abundant in 2011 than in other years. During the dry 2011 field season, prairie dogs were in poor condition and devoted little time to grooming. In contrast, during 2010 and 2012, prairie dogs were in 27% better condition and, when controlling for month and observer variation, devoted 450% more time to grooming. Prairie dogs provided with supplemental food and water during March-May 2012 were in 18% better condition and carried 40% fewer fleas during June-August. Increased flea densities during droughts may provide context for the maintenance and spread of plague. Three additional studies are presented herein. First, we developed a new method for combing fleas from hosts. The method and resulting data can be used with occupancy models to estimate prevalence rates for ectoparasites while accounting for imperfect detection. Second, we used the combing new method to estimate prevalence rates for the generalist flea Pulex simulans during June-August 2012. Prevalence estimates were >30% higher than indices from studies with substantial sample sizes for prairie dogs. If P. simulans can attain high prevalence on prairie dogs, the species may commonly serve as a bridge vector between Cynomys and other mammalian hosts of Y. pestis, and even function as a reservoir of plague. Third, a case study is presented to describe how Y. pestis can transform grassland ecosystems by devastating populations of prairie dogs and, thereby, causing (1) declines in native species abundance and diversity, including threatened and endangered forms, (2) alterations in food web connections, (3) alterations in the import/export of nutrients, (4) loss of ecosystem resilience to encroaching invasive plants, and (5) modifications of prairie dog burrows.
Open Access
Identifying blood meals in cat fleas (Ctenocephalides felis) from a plague-endemic region of Uganda using a SYBR Green real-time polymerase chain reaction-based assay
(Colorado State University. Libraries, 2012) Graham, Christine B., author; Black, William C., advisor; Eisen, Rebecca J., committee member; Karkhoff-Schweizer, RoxAnn R., committee member; Huyvaert, Kathryn P., committee member
A zoonotic disease that has killed millions over the course of at least three pandemics, plague remains a threat in regions where the etiologic agent, Yersinia pestis, persists in natural cycles involving small mammals and their fleas. Numerous flea species have been implicated as Y. pestis vectors, and some provide a "bridge" from zoonotic hosts to humans, particularly during the epizootics that decimate susceptible small mammal populations. In order to serve as a bridging vector, a flea species must be able to transmit Y. pestis, it must feed on infectious zoonotic hosts, and it must feed on humans. Identifying bridging vector species in plague-endemic regions can aid in the development of vector-control activities aimed at reducing the incidence of human plague. The West Nile region is an established plague focus in northwest Uganda. Since 1999, more than 2400 suspect human plague cases have been reported from Vurra and Okoro counties. The most likely source of infection for humans in this region is the black rat, Rattus rattus, which commonly infests human habitations and is highly susceptible to Y. pestis infection. Other potential zoonotic hosts include other rodent and shrew species that predominate in the peridomestic environment and occasionally enter huts. Two rat flea species, Xenopsylla cheopis and X. brasiliensis, both among the most efficient flea vectors of Y. pestis, are very likely to serve as bridging vectors to humans in Vurra and Okoro counties. Recent investigations, however, have found that the cat flea, Ctenocephalides felis, comprises more than 88% of host-seeking (off-host) fleas captured in huts in this region. Though an inefficient vector, this species is capable of transmitting Y. pestis. Given its dominance in human habitations and its catholic feeding habits in other regions, we hypothesized that C. felis might serve as a secondary bridging vector in Vurra and Okoro counties. In order to address this hypothesis, we sought to determine what proportion of blood meals in off-host cat fleas collected in huts in this region come from humans, and what proportion come from potentially-infectious small mammal species. Blood meal assays have long been used to examine the feeding behavior of a wide variety of disease vectors, but existing blood meal assays were deemed inadequate for our purposes because they were either not sensitive enough to detect the very small amounts of host DNA in field-collected fleas, or they were unable to capture the wide range of potential cat flea hosts in the West Nile region. Therefore, we developed a blood meal assay that takes advantage of the exquisite sensitivity of SYBR Green I-based real-time polymerase chain reaction (PCR) and combines it with the specificity and flexibility afforded by sequencing. We found that this highly-sensitive assay was subject to human DNA contamination, so we analyzed vertebrate DNA detection in artificially-fed and unfed fleas to establish a threshold cycle (Ct) cutoff that would optimize specificity without completely sacrificing sensitivity. Specifically, we identified a Ct cutoff that maximized positive predictive value. Using the established cutoff, our assay was 94 percent specific, detecting contaminating human DNA in 3 of 50 unfed fleas, and it detected and correctly identified the source of human and rat blood meals in 100 percent of artificially fed fleas held alive for up to 4 hours post feeding. Assay sensitivity declined as the time between feeding and collection increased, but we were able to detect and identify human and rat DNA in a proportion of artificially-fed fleas held alive for up to 72 hours post feeding. Using this assay, we detected and identified vertebrate DNA in 148 off-host C. felis collected in human habitations in Vurra and Okoro counties, none of it from wild rodents or shrews. Our findings indicate that cat fleas infesting huts in the West Nile region probably feed on humans, but the majority of off-host C. felis blood meals came from domesticated species that are unlikely to play a significant role in perpetuating transmission of Y. pestis. We concluded that C. felis is unlikely to serve as a bridging vector for Y. pestis in the West Nile region.
Open Access
Ivermectin mass drug administration to humans for malaria parasite transmission control
(Colorado State University. Libraries, 2011) Kobylinski, Kevin, author; Foy, Brian D., advisor; Eisen, Lars, committee member; Huyvaert, Kathryn P., committee member; Rosenberg, Ronald, committee member
Every year, an estimated 500 million people are afflicted with malaria worldwide, killing more than one million people, most of whom are children in sub-Saharan Africa. The current malaria eradication program requires novel vector control methods to reduce the transmission of Plasmodium, the causative agent of malaria. These new methods must: target exophagic and exophilic Plasmodium vectors, integrate with current vector control efforts, be evaluated in the field in combination with other interventions, evade potential behavioral mechanisms that mosquitoes may evolve to avoid the intervention, new agents should have different modes of action, reduce the risk of physiological resistance development, and affect vector population age structure. This dissertation addresses how ivermectin mass drug administration, meets and exceeds all of these issues. Laboratory-based experiments demonstrated that ivermectin at human relevant pharmacokinetics affects Anopheles gambiae s.s. survivorship and that cumulative ivermectin blood meals compound mortality, blood feeding frequency, knockdown, and recovery. Field-based experiments demonstrate that ivermectin mass drug administration to humans reduces the survivorship of wild-caught Anopheles gambiae s.s. and probably Anopheles arabiensis up to six days post-administration. Most importantly, ivermectin mass drug administration to humans was shown to reduce the proportion of field-caught Plasmodium falciparum-infectious Anopheles gambiae s.s. for at least 12 days post-treatment. Ivermectin mass drug administration could be a powerful addition to malaria eradication campaign efforts.
Open Access
Ivermectin-treated bird feed to control West Nile virus transmission
(Colorado State University. Libraries, 2018) Nguyen, Chilinh, author; Foy, Brian, advisor; Ebel, Greg, committee member; Bowen, Richard, committee member; Huyvaert, Kathryn P., committee member
West Nile virus is the leading cause of arboviral fever and encephalitis in the United States. The highest WNV disease incidence occurs along the Great Plains region of the United States, as the ecology and land use provide a supportive habitat for the main WNV enzootic and bridge vector of the region, Culex tarsalis. However, due to the lack of dense human population, this area often does not benefit current WNV control measures as applied by conventional mosquito control districts. Based on the ecology of WNV transmission in the Great Plains region, a strategy that targets Cx. tarsalis through its ornithophilic blood feeding behavior could disrupt WNV transmission. Given that the majority of Cx. tarsalis blood meals on the northern Colorado plains may come from doves and passerine species during the WNV transmission season, effective targeting of these or other local preferred hosts with endectocide-treated bird feed could result control of WNV transmission. This study develops and characterizes the effects of IVM-treated bird feed in birds and biting Cx. tarsalis mosquitoes in both a laboratory and field setting. In Chapter 2, the effects of IVM on Cx. tarsalis survival were examined using both in vitro membrane blood meals and direct blood feeding on IVM-treated birds. Chickens and wild Eurasian Collared Doves fed solely on IVM-treated bird feed concentrations up to 200 mg IVM/kg feed exhibited no signs of toxicity, and most Cx. tarsalis that blood fed on these birds died compared to controls. Mosquito survivorship following blood feeding correlated with IVM serum concentrations at the time of blood feeding, which dropped rapidly after the withdrawal of treated feed. These results suggested IVM-treated bird feed should be further explored as a hyper-localized control strategy for WNV transmission. Chapter 3 presents the development of a method to detect and quantify IVM in individual blood meals of Anopheles gambiae and Cx. tarsalis, which will be important in measuring the coverage of this intervention in the field, and accurately assessing IVM's mosquitocidal effects in field situations. This ability to detect IVM in mosquito blood meals was similar between blood fed Cx. tarsalis and An. gambiae, and between sampling times of 0 or 12 hours post blood feed. The quantity of IVM ingested in individual mosquitoes was also compared to the venous serum concentrations of live animals. Chapter 4 presents promising results from two separate pilot field trials of IVM-treated bird feed that were conducted during the summers of 2016 and 2017. Results from 2016 showed that wild birds frequently visit the IVM-treated feeders. In addition, there was an observable trend where "far" traps that are expected to be beyond the zone of control had more WNV-positive pools compared to "near" traps at both ELC and ARDEC South sites. Results from the 2017 study continued to be promising, where birds were again visiting IVM-treated feeders and IVM could be detected in the sera of birds sampled by IVM feeders. There was also a trend of higher VI for the control sites compared to IVM sites for the 2017 season. The efficacy of IVM-treated bird feed was evaluated in two pilot trials where natural WNV transmission cycles occurred in wild birds and Cx. tarsalis, but should be followed up with field seasons with many control and IVM sites to allow for a robust analysis of IVM effects. This study introduces the novel concept of using systemic endectocides for controlling WNV transmission, and this concept could be explored for other arboviruses.
Open Access
Molecular ecology and hierarchical models elucidate chronic wasting disease dynamics
(Colorado State University. Libraries, 2018) Galloway, Nathan L., author; Antolin, Michael F., advisor; Hobbs, N. Thompson, committee member; Miller, Michael W., committee member; Huyvaert, Kathryn P., committee member
Prions present a unique evolutionary scenario because a single gene codes for both a disease agent and a functionally constrained native protein. The prion precursor gene, Prnp, codes for the prion precursor protein, PrP, which is constitutively expressed as a native isoform within all mammals. Upon misfolding to the disease isoform, known as prions, the same protein causes fatal neurodegenerative diseases known as transmissible spongiform encephalopathies. We review the literature and available data for the genetics of Prnp in order to examine its molecular evolutionaryhistory, and the likely force of natural selection acting on it, by analyzing genetic diversity both within and between species within Class Mammalia. We accessed Prnp nucleotide sequences of a large number of mammalian species from GenBank. We undertook three distinct analyses of these molecular data to characterize the force of selection acting on Prnp through comparisons of gene sequences and allele frequencies within and between species. Our analyses include: 1.) comparisons of genetic and amino acid polymorphisms across protein domains within Prnp, 2.) a within and between species comparison of nucleotide diversity within Prnp to characterize natural selection acting on the gene, and 3.) observed frequencies of genetic and amino acid polymorphisms from natural populations of animals. We show that amino acid substitutions reported to correlate with prion disease risk within species do not aggregate within particular protein structural domains, but rather are disparately located throughout. Branch model estimates using Phylogenetic Analysis by Maximum Likelihood across mammals show that Prnp undergoes strong purifying selection at the broad scale, that purifying selection is stronger between species than it is within species but no evidence that species orally susceptible to prion disease experience unique positive selection. We do show, however, that amino acid substitutions occur at higher frequencies than synonymous substitutions within Prnp, in direct conflict with the expectations for purifying selection. This evidence suggests that Prnp is experiencing balancing selection in opposition to the purifying selection observed at the large scale; this unique selective pressure may be due to the presence of prion disease. Ecological processes such as reproduction, habitat use, and disease epizootiology contribute to the growth or decline of wildlife populations, but many of these processes go directly unobserved. We set out to describe gene flow and disease transmission to better understand the ecological role of chronic wasting disease (CWD) in a northern Colorado population of mule deer (Odocoileus hemionus). CWD, a fatal prion disease, has been affecting this population for many decades. It has not caused extirpation of the deer, but may play an important limiting role in population growth and resilience. We employed genetic methods to analyze neutral genetic markers, which provide information about gene flow. Further, we examined allelic variation in the functional prion precursor gene, Prnp, which codes for the disease-causative prion of CWD and has alternative alleles with one (225F) that confers some resistance to prion disease within individual deer. The study in northern Colorado included sampling across four winter ranges. Genetic analysis identified four genetic lineages of deer, but lineages were distributed throughout the study area and did not correspond with winter ranges used annually with high fidelity by groups of females. Further, we show that males drive gene flow across genetic lineages. In contrast, CWD prevalence was spatially segregated: CWD-positive female deer were located in two of the winter ranges and absent from the others. This suggests that neither breeding sites nor natal dispersal are the primary means of disease spread in females across the winter ranges. Furthermore, we found, as previously reported, that an individual deer's Prnp genotype predicts the likelihood of a positive disease test. Even so, the frequency of the Prnp 225F allele was similar across winter ranges, and was notably higher than that reported in neighboring populations a decade earlier. Thus, it appears that gene flow spreads the favored allele across the study area despite different selective regimes in winter ranges. Our work shows the benefit of using population genetics to gain insight into ecological processes that go directly unobserved, such as the epizootiology of chronic wasting disease. Chronic wasting disease is a fatal neurodegenerative prion disease that infects members of the deer family in North America and Scandinavia. We conducted a five-year mark recapture study of a northern Colorado population of mule deer (Odocoileus hemionus) with endemic disease, including 217 females. All study animals were also genotyped at the prion precursor gene, Prnp, which has alternative alleles in many species to express amino acid differences that alter prion disease dynamics. Mark-recapture analysis revealed decreased disease incidence for individuals expressing genotypes with at least one copy of the minor allele, including heterozygotes, Prnp 225SF (expressing both a serine and phenylalanine at amino acid position 225) , and rare homozygotes, 225FF.We found no evidence for an evolutionary trade-off of decreased survival of CWD-negative deer for this group but emphasize the difficulty in estimating dynamic rates for the rare homozygotes alone. We employed estimates of annual disease risk and survival from this study as well as recruitment estimates from the literature, to forecast the expected future minor allele frequency in the population under the observed disease risk. This forecast revealed a clear expected evolutionary increase in the Prnp minor allele (225F) frequency given our model and field data.
Open Access
Mucosal immunization of mice with a recombinant Salmonella choleraesuis that expresses a multimeric gonadotropin releasing hormone fusion protein
(Colorado State University. Libraries, 2011) Kemp, Jeffrey M., author; Graham, James, advisor; Huyvaert, Kathryn P., committee member; Bowen, Richard, committee member; Miller, Lowell, committee member; Rhyan, Jack, committee member
To view the abstract, please see the full text of the document.
Open Access
Of toads and tolerance: intraspecific variation in host persistence when challenged by disease
(Colorado State University. Libraries, 2023) Hardy, Bennett, author; Bailey, Larissa, advisor; Funk, W. Christopher, advisor; Huyvaert, Kathryn P., committee member; Muths, Erin, committee member; Hoke, Kimberly, committee member
Infectious diseases are increasingly known to drive population declines and extinctions and ultimately contribute to the loss of global biodiversity. This phenomenon is none more apparent than in the extinctions and extirpations of over 500 amphibian species worldwide due to a disease caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd). This ongoing loss of amphibian diversity is concerning given the important roles amphibians serve for ecosystem function, ecosystem services, and pharmacology, among others. To mitigate these declines, scientists must understand the pathogen, environmental, and host factors that interact to affect disease outcomes. Despite over 25 years of research on amphibian declines and Bd, there is still much we can learn that can crucially inform our conservation actions. For instance, while Bd is undoubtedly a lethal pathogen of severe threat to many amphibians around the world, researchers have observed substantial variation in host responses to Bd infection or presence in the wild and in the laboratory. While many host populations are extirpated by Bd, some persist at lower abundances or rebound completely. Understanding variation in the mechanisms of host population persistence may provide vital hints for how to better conserve vulnerable amphibian populations. This leads me to the central question of my dissertation: "Why do some populations persist with Bd while others do not?". I address this question in three chapters by studying variation in boreal toad (Anaxyrus boreas boreas) responses to Bd at several locations across the toad's range. The boreal toad occurs from coastal southern Alaska down into Colorado. Boreal toads are susceptible to Bd and have experienced dramatic declines and extirpations at the southern edge of its range in New Mexico, Colorado, and southwestern Wyoming, which together are considered the southern Rocky Mountain population. Populations in the northern portion of its range, however, appear to be persisting despite the confirmed presence, and high prevalence, of Bd. Two major hypotheses for the difference in apparent persistence of boreal toad populations with Bd across regions include a) differences in demographic compensation and b) differences in host susceptibility. Therefore, I used observational and experimental studies to investigate these hypotheses to better understand the contexts in which boreal toads persist with Bd. In my first chapter, I leveraged a long-term mark-recapture dataset from multiple populations of boreal toads across a high-elevation gradient in Colorado to test for the existence of compensatory recruitment. Compensatory recruitment is a hypothesized mechanism of host persistence when challenged by disease where hosts increase their baseline recruitment, which compensates for decreased survival attributed to disease, and ultimately stabilizes or slows population declines. Limitations from prior investigations of this phenomenon in other amphibian-Bd systems include the lack of pre-Bd monitoring data, the lack of population replication, and studies that primarily examine low elevation populations. I found a life history trade-off between survival and recruitment across elevations, where high-elevation toads have high survival but low recruitment and vice versa at lower elevations. Once Bd arrived, however, recruitment was reduced across all populations and survival was reduced to zero. Estimates of population abundance and population growth rates were also variable prior to Bd arrival, but dramatically declined after. I did not find evidence for compensatory recruitment in these high elevation boreal toad populations. My findings highlight that demographic responses to disease may be environmentally context-dependent, and that high elevation amphibian populations are particularly vulnerable to the effects of Bd. In Chapter 2, I used a laboratory exposure experiment to identify an appropriate isolate of Bd to use in a future experiment designed to investigate differential susceptibility of boreal toad populations to Bd (Chapter 3). While researchers have known about the potential for laboratory-maintained Bd cultures to lose pathogenicity over time (i.e., pathogen attenuation), most exposure studies use isolates that are available to them, regardless of how long they have been maintained in the laboratory, or are unaware of their chosen isolate's culture history. I exposed wild-caught, captive-reared boreal toads to three different isolates of Bd that varied in the amount of time maintained in the lab (old vs. new) and the geographic origin of the isolate compared to the host (local vs. novel) to determine the best isolate for use in Chapter 3. I found that boreal toads exposed to the older isolates had higher weekly survival probabilities than those exposed to the new isolate, indicating pathogen attenuation for older isolates. This effect was also mediated by individual body mass, where larger toads had higher survival. My findings indicate that newer, local isolates are likely better choices when exposing amphibian hosts to Bd and that isolate age and host weight can dramatically affect our inferences from exposure studies. In my third chapter, I tested the hypothesis that boreal toads exhibit intraspecific variation in susceptibility to Bd. I expected that the host defense strategies of tolerance and resistance are stronger in boreal toads from Wyoming, and weaker in boreal toads from Colorado, and are primarily responsible for our observations of boreal toad population declines in Colorado, and relative population persistence in Wyoming. Previous studies investigating variation in amphibian host tolerance and resistance to Bd are predominantly focused on species-level comparisons, with fewer focusing on intraspecific variation. Most studies also lack replication among strata of interest (e.g., geography, disease prevalence, host genotypes), and none use a robust methodological framework that can reveal host and pathogen dynamics throughout experimental exposures. Therefore, I conducted a laboratory experimental exposure of boreal toads to Bd, informed by the results of Chapter 2. I included toads from two populations in Colorado and two populations in Wyoming, representing replicates from our strata of interest (i.e., differences in decline severity). Using a multistate modeling approach, I modeled the effects of static covariates (e.g., host population origin, treatment dose of Bd, etc.) and dynamic, individual, time-varying covariates (e.g., weekly individual Bd load, weekly change in individual body mass, etc.) on boreal toad weekly survival and state transition probabilities. State transitions included the weekly probability an individual would clear their infection, or whether a cleared individual would re-gain infection, providing insight into typically hidden infection dynamics. I found that boreal toads from Colorado populations had lower weekly survival probabilities than those from Wyoming when comparing identical Bd loads. This is evidence of increased tolerance to Bd in Wyoming toad populations. As in Chapter 2, individual mass was also important at predicting the effects of Bd on weekly survival probabilities of boreal toads. Boreal toads from Colorado had similar peak Bd loads and cleared Bd at the same probabilities as Wyoming. Colorado boreal toads, however, were on average quicker to reach their peak Bd infection loads and had increased probabilities of re-gaining their infections. These results provide some support for increased resistance among boreal toads in Wyoming compared to those in Colorado. My findings highlight that differential susceptibility to Bd among boreal toads from different regions may play a crucial role in generating the disparity in decline severity across the region. In conclusion, my dissertation provides evidence that intraspecific variation in persistence, when challenged by disease, is an important driver of host-pathogen dynamics. My research has filled vital research gaps for an imperiled amphibian species with the goal of helping wildlife managers make tough conservation decisions about host translocations, reintroductions, and captive breeding. I hope my work aids in the persistence of an iconic Rocky Mountain amphibian for years to come.
Open Access
Out with the old and in with the new? Investigating competition between Barred Owls (Strix varia) and Northern Spotted Owls (Strix occidentalis caurina) in northwestern California with a playback experiment
(Colorado State University. Libraries, 2010) Van Lanen, Nicholas J., author; Franklin, Alan B., advisor; Noon, Barry R., committee member; Reiser, Raoul Frederick, II, committee member; Huyvaert, Kathryn P., committee member
The Northern Spotted Owl (Strix occidentalis caurina) is a controversial species in the Pacific Northwest that is listed as threatened under the Endangered Species Act. The Barred Owl (Strix varia), a species historically restricted to eastern North America, has recently expanded its range to completely overlap that of the Northern Spotted Owl. Recent evidence suggests that Barred Owls may displace Northern Spotted Owls from their territories. The focus of my study was to determine whether Barred Owls have the potential to competitively exclude Northern Spotted Owls from their territories. I used a playback experiment to observe and quantify aggressive vocal and physical behavior of Barred and Northern Spotted Owls during territorial defense. Trials consisted of displaying a Northern Spotted or Barred Owl taxidermy mount, and broadcasting recorded vocalizations of the corresponding species, in both Barred and Northern Spotted Owl territories. The frequency and intensity of residents' responses to playbacks were digitally recorded as was the acceleration experienced by the mount's head during physical attacks by the residents. When agonistic interspecific interactions occurred in this study I found that Barred Owls responded with higher levels of vocal and physical aggression than Northern Spotted Owls. However, the frequency of interspecific interactions was lower compared to intraspecific interactions among Northern Spotted Owls alone. This study suggests that Barred Owls are likely to assume the dominant role during interspecific interactions with Northern Spotted Owls and indicates that competitive exclusion is a plausible mechanism by which Barred Owls could contribute to the observed population declines of Northern Spotted Owls in areas of co-occurrence.
Open Access
Pathogenesis and immunological response of Yersina pestis in carnivores
(Colorado State University. Libraries, 2019) Baeten, Laurie Ann, author; Bowen, Richard A., advisor; Pabilonia, Kristy L., advisor; Huyvaert, Kathryn P., committee member; VandeWoude, Susan, committee member; Gidlewski, Thomas, committee member
Yersinia pestis is the causative agent of plague. The pathogen is endemic in rodent populations in the western United States where humans and other mammals become infected with this highly virulent organism when exposed to infected rodents or their fleas. Coyotes (Canis latrans) have been used in animal-based surveillance efforts for the detection of plague foci for over 40 years. Coyotes are likely exposed via flea bite or oral routes and are presumed to be refractory to the development of clinical disease. Historical data have been useful in establishing models of the geographic distribution of Y. pestis in the landscape. Because the pathological and immunological response of coyotes to Y. pestis infection had not been thoroughly characterized, I conducted experimental inoculations of captive-reared, juvenile coyotes (n=12) with Y. pestis CO92 via oral and intradermal routes. No clinical signs of disease were observed, and minimal changes were noted in body temperature, and white blood cell counts during the 7 days following exposure. Gross pathology was unremarkable and minimal histopathological changes were noted at days 3 and 7 post-inoculation. The innate immune response was characterized by a brief peak in acute phase proteins between day 2 and 5 after oral exposure but little to no response was noted in the intradermal exposure group. The humoral response to Y. pestis fraction 1 capsular protein (anti-F1) was significantly different between inoculation groups in magnitude and duration of antibody production. The anti-F1 titers were measured by passive hemagglutination assay (PHA) and animals inoculated by the intradermal route peaked at day 10 post-inoculation (range = 1:32 to 1:128) with titers remaining stable through day 84. In contrast, orally inoculated animals developed higher titers (range =1:125 to 1:1024) that remained stable through day 42. Re-challenge at day 98 post-inoculation using the same dosage and routes did not result in changes in clinical behavior, body temperature, or white blood cell counts. Anti-F1 titers in the oral challenge group produced a striking increase (up to 1:4096) within three days, whereas there was minimal to no increase in antibody response noted in the intradermal challenge group. Using western blot, the antibody profile against known immunogenic Y. pestis antigens was evaluated. In pre-infection samples, antibodies against components of the Yersinia type III secretory system (LcrV, YopD, YopH, YpkA) were detected indicating a possible mechanism for acquired resistance to plague. Post-inoculation samples were found to contain antibodies against all of the antigens in the testing profile (F1, LcrV, Pla, Pst, YopD, YopH, YpkA). Serum samples generated from this experimental trial were used to develop an enzyme-linked immunosorbent assay (ELISA) using recombinant Y. pestis antigens where isotypic immunoglobulin production (IgG, IgM) was subsequently compared to PHA. The assay was subsequently optimized for use in testing of whole blood collected from free-ranging coyotes using Nobuto filter paper strips. ELISA was also evaluated for use in the testing of other wildlife species commonly encountered in plague endemic regions. Information gathered from this experimental work may provide additional insight into the use of coyotes for plague serosurveillance programs.
Open Access
Sinus tumors of Rocky Mountain bighorn sheep: investigation of an infectious etiology
(Colorado State University. Libraries, 2013) Fox, Karen A., author; Quackenbush, Sandra L., advisor; Miller, Michael W., committee member; Wootton, Sarah K., committee member; Huyvaert, Kathryn P., committee member
Rocky Mountain bighorn sheep are an icon in Colorado. As our state animal, bighorn sheep are a well-recognized symbol of the wildlife, wildlands, and wilderness-centric people that Colorado is famous for. Efforts to manage and conserve this species are a priority in Colorado and throughout western North America. As part of those efforts a great deal of research has been conducted to understand bighorn sheep respiratory disease, the leading infectious cause of death in these animals. In the process of investigating respiratory disease in bighorn sheep in Colorado, we discovered a surprisingly high occurrence of sinus tumors within the upper respiratory tracts of many animals. This disease had not been described previously and became the focus of work for this dissertation. Here, I have compiled our findings regarding the characterization of bighorn sheep sinus tumors and the results of our efforts to identify an infectious etiology for this disease. Through the examination of naturally-occurring cases, we identified characteristic histologic and gross features of bighorn sheep sinus tumors to define this disease. We also analyzed factors associated with sinus tumors at a population level. The results of this study suggest that bighorn sheep sinus tumors are an infectious disease, maintained within specific geographic areas corresponding to distinct populations of animals. Our results also suggest a role for bighorn sheep sinus tumors in predisposing animals to secondary infections by bacterial agents that can cause pneumonia. To specifically test the hypothesis that bighorn sheep sinus tumors are a transmissible disease, we experimentally inoculated bighorn sheep and domestic sheep lambs with a cell-free filtrate derived from a naturally-occurring bighorn sheep sinus tumor and its associated exudates. Within 18 months post-inoculation we demonstrated transmission of the disease to both bighorn sheep and domestic sheep species, supporting our hypothesis that bighorn sheep sinus tumors represent an infectious process. This experiment also provided an opportunity to examine tumors early in development, further characterize the cells comprising the tumors, and suggest mechanisms for pathogenesis. With evidence that bighorn sheep sinus tumors are caused by an infectious agent, we also attempted to identify a specific etiology for this disease. We primarily used PCR methods with degenerate PCR primers to evaluate samples from bighorn sheep sinus lining tissues for the presence of herpesviruses and retroviruses, which are well-known causes of infectious tumors. We successfully identified the presence of herpesviral and (likely endogenous) retroviral sequences in our samples, but we were unable to find an association between these viruses and the occurrence of sinus tumors. Based on similarities between bighorn sheep sinus tumors and oncogenic retroviral diseases of domestic sheep and goats, we specifically screened our samples for the presence of Jaagsiekte sheep retrovirus (JSRV), and enzootic nasal tumor viruses (ENTV-1 and ENTV-2). We successfully identified ENTV-2-specific sequences from some of our samples, but an association between this virus and bighorn sheep sinus tumors was not clear. We found an association between ENTV-2 and early tumor cases, but not well-defined tumors. While our PCR data alone did not definitively identify ENTV-2 as the cause of bighorn sheep sinus tumors, our histologic, histochemical, and immunohistochemical results have helped us to develop a hypothesis for the pathogenesis of bighorn sheep sinus tumors, and provided additional support for the hypothesis that this disease is caused by ENTV-2. Our working hypothesis for the pathogenesis of bighorn sheep sinus tumors is that epithelial cells of the sinus lining are infected by ENTV-2, but that uninfected periosteal pluripotent cells are stimulated to replicate, resulting in predominantly stromal tumors. This hypothesis is based on histologic observations, histochemical stains used to differentiate cell types, and IHC results specifically identifying the presence of ENTV antigen within surface epithelial cells of experimentally-induced tumors, but not within the predominating stromal cells of the tumors. These results help to explain why detection of the virus is uncommon in well-developed stromal tumors, but more easily detected in early tumor cases with less stromal proliferation. Additional research will help to further elucidate the pathogenesis of bighorn sheep sinus tumors, and the potential role that tumors may play in predisposing bighorn sheep to fatal respiratory disease. The definitive identification of an etiologic agent for bighorn sheep sinus tumors, and the development of an antemortem diagnostic assay will greatly enhance efforts to understand and manage this disease.
Open Access
We want our view and eat it too
(Colorado State University. Libraries, 2011) Brazee, Tammi Lynn, author; Yust, David E., advisor; Sullivan, Patrice M., committee member; Coronel, Patricia D., committee member; Huyvaert, Kathryn P., committee member
How do we amuse ourselves in America's most beautiful places? The relationship that many Americans have with the natural world is one of awkwardness and detachment that is manifested in the way we vacation in and tour around National Parks and other naturally beautiful places. Culturally instilled perceptions of place and a frantic pace to see it all keep many circulating around the edges of the natural world rather than experiencing it in more intimate ways. Many have a distanced appreciation for a beautiful natural landscape, especially those iconic views that are recognizable from ubiquitous travel brochures, postcards, posters, books, and calendars. They inspire awe and appreciation, but we soon shoot our photograph and quickly move to the next panorama so the view becomes a film, flashing by frame by frame through our vehicle's windows. Kitschy tourist stops, amusement parks, golf courses, shopping centers, restaurants, or funky little coffee shops and pubs bring urban pleasures and comforts to our experience of the natural world. Our behavior exposes several underlying tensions that exist in our individual and collective psyches: the tensions between conservation and consumption, observation and immersion, and the natural and artificial. My intention is to visually investigate these tensions by exploring the roads we build, the parks we set aside, the objects we place within the natural landscape, and the activities in which we participate, often pushing these into the realm of the ridiculous in order to raise questions about what we might do if we could. As a society we simultaneously want a world filled with beautiful landscapes and a comfortable lifestyle. However, our current way of life demands a high rate of natural resource consumption that destroys precious ecosystems, which by association destroys the beautiful view. We want the best of both worlds; we want our view and to eat it too. My work is aimed at visually exploring this paradox and the tension that exists when a society tries to reconcile competing desires.

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