Elsevier

Veterinary Microbiology

Volume 232, May 2019, Pages 42-49
Veterinary Microbiology

Labeled quantitative mass spectrometry to study the host response during aspergillosis in the common bottlenose dolphin (Tursiops truncatus)

https://doi.org/10.1016/j.vetmic.2019.03.030Get rights and content

Highlights

  • A large number of host proteins changes were observed during aspergillosis in dolphins.

  • 106 proteins were dramatically increased or decreased during aspergillosis in dolphins.

  • prepronociceptin; protamine P1, MHC and NADPH-ubiquinone oxido-reductases were mostly involved.

  • iTRAQ® is a valuable mass spectrometry technique to address proteins identification and their relative changes between distinct samples.

Abstract

Aspergillosis is a fungal infection caused by Aspergillus molds that can affect both humans and animals. Despite advances in diagnostics and therapy, medical management of this disease remains difficult. Expansion of the basic knowledge regarding its pathophysiology in animals is critical to aid in the identification of new biomarkers of infection for diagnosis and therapeutic targets. For such a purpose, proteomics can be used by addressing protein changes during various disease processes. In the present study, a mass spectrometry analysis based on isobaric tagging for relative and absolute quantitation (iTRAQ®) was applied for direct identification and relative quantitation of proteins in blood collected from 32 Aspergillus-diseased common bottlenose dolphins (Tursiops truncatus, 32 samples) in comparison with blood from 55 other dolphins (55 samples from 41 clinically-normal controls and from 14 cetaceans with miscellaneous non-Aspergillus inflammation diseases) and ten convalescent dolphins (28 samples). Sixty-six and 40 proteins were found to be ≥2.0-fold over- and underrepresented versus miscellaneous non-Aspergillus inflammatory dolphins, respectively, and most were confirmed vs. clinically-normal controls and convalescents. Many proteins which play a role in the adaptive immune response were identified, including MHC proteins and others involved in catalytic activity like the NADPH-ubiquinone oxido-reductases. Overall, iTRAQ® appears to be a convenient proteomic tool greatly suited for exploratory ex vivo studies focusing on pathophysiology. This technique should be considered as a preliminary step before validation of new diagnostic markers.

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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  • 1

    Current address: Mystic Aquarium, Mystic, CT, 06355, USA.

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