Browsing by Author "Leon, Ariel E."

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    Development and validation of a house finch interleukin-1β (HfIL-1β) ELISA system
    Kim, Sungwon; Park, Myeongseon; Leon, Ariel E.; Adelman, James S.; Hawley, Dana M.; Dalloul, Rami A. (2017-08-30)
    Background A unique clade of the bacterium Mycoplasma gallisepticum (MG), which causes chronic respiratory disease in poultry, has resulted in annual epidemics of conjunctivitis in North American house finches since the 1990s. Currently, few immunological tools have been validated for this songbird species. Interleukin-1β (IL-1β) is a prototypic multifunctional cytokine and can affect almost every cell type during Mycoplasma infection. The overall goal of this study was to develop and validate a direct ELISA assay for house finch IL-1β (HfIL-1β) using a cross-reactive chicken antibody. Methods A direct ELISA approach was used to develop this system using two different coating methods, carbonate and dehydration. In both methods, antigens (recombinant HfIL-1b or house finch plasma) were serially diluted in carbonate-bicarbonate coating buffer and either incubated at 4 °C overnight or at 60 °C on a heating block for 2 hr. To generate the standard curve, rHfIL-1b protein was serially diluted at 0, 3, 6, 9, 12, 15, 18, 21, and 24 ng/mL. Following blocking and washing, anti-chicken IL-1b polyclonal antibody was added, plates were later incubated with detecting antibodies, and reactions developed with tetramethylbenzidine solution. Results A commercially available anti-chicken IL-1β (ChIL-1β) polyclonal antibody (pAb) cross-reacted with house finch plasma IL-1β as well as bacterially expressed recombinant house finch IL-1β (rHfIL-1β) in immunoblotting assays. In a direct ELISA system, rHfIL-1β could not be detected by an anti-ChIL-1β pAb when the antigen was coated with carbonate-bicarbonate buffer at 4°C overnight. However, rHfIL-1β was detected by the anti-ChIL-1β pAb when the antigen was coated using a dehydration method by heat (60°C). Using the developed direct ELISA for HfIL-1β with commercial anti-ChIL-1β pAb, we were able to measure plasma IL-1β levels from house finches. Conclusions Based on high amino acid sequence homology, we hypothesized and demonstrated cross-reactivity of anti-ChIL-1β pAb and HfIL-1β. Then, we developed and validated a direct ELISA system for HfIL-1β using a commercial anti-ChIL-1β pAb by measuring plasma HfIL-1β in house finches.
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    Differing house Finch cytokine expression responses to Original and evolved isolates of Mycoplasma gallisepticum
    Vinkler, Michal; Leon, Ariel E.; Kirkpatrick, Laila T.; Dalloul, Rami A.; Hawley, Dana M. (Frontiers, 2018-01-22)
    The recent emergence of the poultry bacterial pathogen Mycoplasma gallisepticum (MG) in free-living house finches (Haemorhous mexicanus), which causes mycoplasmal conjunctivitis in this passerine bird species, resulted in a rapid coevolutionary arms-race between MG and its novel avian host. Despite extensive research on the ecological and evolutionary dynamics of this host–pathogen system over the past two decades, the immunological responses of house finches to MG infection remain poorly understood. We developed seven new probe-based one-step quantitative reverse transcription polymerase chain reaction assays to investigate mRNA expression of house finch cytokine genes (IL1B, IL6, IL10, IL18, TGFB2, TNFSF15, and CXCLi2, syn. IL8L). These assays were then used to describe cytokine transcription profiles in a panel of 15 house finch tissues collected at three distinct time points during MG infection. Based on initial screening that indicated strong pro-inflammatory cytokine expression during MG infection at the periorbital sites in particular, we selected two key house finch tissues for further characterization: the nictitating membrane, i.e., the internal eyelid in direct contact with MG, and the Harderian gland, the secondary lymphoid tissue responsible for regulation of periorbital immunity. We characterized cytokine responses in these two tissues for 60 house finches experimentally inoculated either with media alone (sham) or one of two MG isolates: the earliest known pathogen isolate from house finches (VA1994) or an evolutionarily more derived isolate collected in 2006 (NC2006), which is known to be more virulent. We show that the more derived and virulent isolate NC2006, relative to VA1994, triggers stronger local inflammatory cytokine signaling, with peak cytokine expression generally occurring 3–6 days following MG inoculation. We also found that the extent of pro-inflammatory interleukin 1 beta signaling was correlated with conjunctival MG loads and the extent of clinical signs of conjunctivitis, the main pathological effect of MG in house finches. These results suggest that the pathogenicity caused by MG infection in house finches is largely mediated by host pro-inflammatory immune responses, with important implications for the dynamics of host–pathogen coevolution.
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    Host exposure history modulates the within-host advantage of virulence in a songbird-bacterium system
    Leon, Ariel E.; Fleming-Davies, Arietta E.; Hawley, Dana M. (Springer Nature, 2019)
    The host immune response can exert strong selective pressure on pathogen virulence, particularly when host protection against reinfection is incomplete. Since emerging in house finch populations, the bacterial pathogen Mycoplasma gallisepticum (MG) has been increasing in virulence. Repeated exposure to low-doses of MG, a proxy for what birds likely experience while foraging, provides significant but incomplete protection against reinfection. Here we sought to determine if the within-host, pathogen load advantage of high virulence is mediated by the degree of prior pathogen exposure, and thus the extent of immune memory. We created variation in host immunity by experimentally inoculating wildcaught, MG-naïve house finches with varying doses and number of exposures of a single pathogen strain of intermediate virulence. Following recovery from priming exposures, individuals were challenged with one of three MG strains of distinct virulence. We found that the quantitative pathogen load advantage of high virulence was strongly mediated by the degree of prior exposure. The greatest within-host load advantage of virulence was seen in hosts given low-dose priming exposures, akin to what many house finches likely experience while foraging. Our results show that incomplete host immunity produced by low-level prior exposure can create a within-host environment that favors more virulent pathogens.