Labeled quantitative mass spectrometry to study the host response during aspergillosis in the common bottlenose dolphin (Tursiops truncatus)
Introduction
Aspergillosis is an airborne fungal infection caused by saprophytic ubiquitous molds that belong to the Aspergillus genus (Latgé, 1999). It is responsible for several distinct respiratory diseases in both humans and animals. In marine mammals, Aspergillus-related disease is assumed to be rare, but it has been reported with increasing incidence: 66.7% of the 18 reported cases have been published after the year 2000 (Dagleish et al., 2008; Abdo et al., 2012). Aspergillosis can involve the lungs and brain in cetaceans (Latgé, 1999; Seyedmousavi et al., 2015). The major species responsible for infection belong to the Fumigati section (Lamoth, 2016), Nigri section and Terrei section in which A. fumigatus stricto sensu (ss), A. niger ss and A. terreus ss are the most frequently isolated (Balajee, 2009), respectively.
In marine mammals, and especially in dolphins, diagnostic tools are less developed than those for humans (Dagleish et al., 2008; Cassle et al., 2016). For instance, quantitative polymerase chain reaction (qPCR) has not been widely implemented in veterinary laboratories, and advanced medical imaging (computed tomography (CT), magnetic resonance imaging (MRI)) may not be accessible. Moreover, a positive culture from respiratory specimens does not definitively diagnose a true infection as it may reflect a simple colonization of the upper airways or represent an environmental contaminant (Desoubeaux et al., 2014a). Also, as reported in humans, the sensitivity of blood cultures is very low for Aspergillus spp (Desoubeaux et al., 2014b). Furthermore, detection of galactomannan antigen (GM) was demonstrated to be unreliable (Desoubeaux et al., 2017a). Conversely, serologic testing by western blot has been demonstrated to accurately detect anti-Aspergillus antibody in dolphin blood (Desoubeaux et al., 2017a). Thus, additional and novel biomarkers for aspergillosis should be investigated to improve the basic understanding of the pathophysiology of this disease. Evaluation of biomarkers in the host response may be able to provide such information.
Within the last decade, proteomics has been largely used for addressing significant protein changes within diseased organisms or pathologic fluids (Fekkar et al., 2012; Desoubeaux et al., 2014b). Innovative mass spectrometry (MS) tools were initially developed for identification and qualitative characterization; however, some are now able to directly quantitate the relative amount of proteins identified. For instance, iTRAQ® (isobaric tags for relative and absolute quantitation) is an isobaric labeling method to identify and to determine the amount of proteins from different multiplexed sources within a single-run experiment (Ross et al., 2004; Desoubeaux et al., 2017b). This technique uses stable isotope-tagged molecules that can be covalently bonded to the N-terminus and side chain amines of trypsin-digested peptides (Bourassa et al., 2015; Desoubeaux et al., 2018). In the present study, through an original MS approach based on iTRAQ®, we attempted to bring new insight to the host protein response against Aspergillus in diseased common bottlenose dolphins (Tursiops truncatus).
Section snippets
Study population: inclusion and samples
Over 15 years, 115 blood samples were opportunistically collected during routine or clinical health assessments from 87 common bottlenose dolphins (Tursiops truncatus) under human care (from 2002 to 2016 (except for four samples obtained in 1991, 1993, 1993, and 1995), but 62.8% obtained over the last three years). The cetaceans were hosted in several different facilities throughout the United States of America (U.S.A.). All blood samples were centrifuged and plasma specimens were stored at
Study population and samples
Characteristics of included dolphins are detailed in Table 1. Measurement of GM antigen in blood was not diagnostic, displaying the following mean values: 0.2 ± 0.1 versus 0.2 ± 0.2 ng/mL for Aspergillus-diseased cases and controls. There was a significant difference between anti-Aspergillus antibody titers in diseased and control dolphins (p < 0.0001).
Proteomic analysis
Overall, MS analysis achieved identification of 1385 proteins in dolphin blood. Sixty-six proteins were statistically overexpressed in
Discussion
In marine mammals, development of aspergillosis varies fundamentally in comparison to the infection in humans. For instance, in dolphins, it appears to be associated with a chronic invasive process; generally suggestive of another disease and/or (sub-)acute physiologic stress (Desoubeaux et al., 2017a) rather than associated with severe immunosuppression and profound neutropenia as observed in humans (Seyedmousavi et al., 2015). Underlying pulmonary disease may affect host defense mechanisms,
Conflicts of interest
The authors have no conflicts of interest to declare.
Funding
This work was supported by internal laboratory funding and partly by NIH center grant P30-EY14801. There is no disclosure of conflict of interest.
Acknowledgments
The authors thank all the veterinarians and practitioners that enrolled their animal patients. They are grateful to all the multi-institutional animal care and biological staff dedicated to the care and monitoring of the bottlenose dolphins in this study.
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Current address: Mystic Aquarium, Mystic, CT, 06355, USA.