Liver Flukes in Deer Parasites Pathogens-13-00922-V2

pathogens
Article
A Comprehensive Multi-Omics Study of Serum Alterations in
Red Deer Infected by the Liver Fluke Fascioloides magna
Josipa Kuleš 1, * , Miljenko Bujanić 2 , Ivana Rubić 3 , Karol Šimonji 3 and Dean Konjević 4
1 Department of Chemistry and Biochemistry, Faculty of Veterinary Medicine, University of Zagreb,

10000 Zagreb, Croatia
2 Educational Center for Game Management I/3 “Črnovšćak”, Faculty of Veterinary Medicine, University of
Zagreb, 10000 Zagreb, Croatia; mbujanic@vef.unizg.hr
3 Internal Diseases Clinic, Faculty of Veterinary Medicine, University of Zagreb, 10000 Zagreb, Croatia;
irubic@vef.unizg.hr (I.R.); ksimonji@vef.unizg.hr (K.Š.)
4 Department of Veterinary Economics and Epidemiology, Faculty of Veterinary Medicine, University of
Zagreb, 10000 Zagreb, Croatia; konjevic@vef.unizg.hr
* Correspondence: jkules@vef.unizg.hr
Abstract: Liver fluke infections are acknowledged as diseases with global prevalence and significant
implications for both veterinary and public health. The large American liver fluke, Fascioloides magna,
is a significant non-native parasite introduced to Europe, threatening the survival of local wildlife
populations. The aim of this study was to analyze differences in the serum proteome and metabolome
between F. magna-infected and control red deer. Serum samples from red deer were collected
immediately following regular hunting operations, including 10 samples with confirmed F. magna
infection and 10 samples from healthy red deer. A proteomics analysis of the serum samples was
performed using a tandem mass tag (TMT)-based quantitative approach, and a metabolomics analysis
of the serum was performed using an untargeted mass spectrometry-based metabolomics approach.
A knowledge-driven approach was applied to integrate omics data. Our findings demonstrated that
infection with liver fluke was associated with changes in amino acid metabolism, energy metabolism,
lipid metabolism, inflammatory host response, and related biochemical pathways. This study
Citation: Kuleš, J.; Bujanić, M.; Rubić, offers a comprehensive overview of the serum proteome and metabolome in response to F. magna
I.; Šimonji, K.; Konjević, D. A infection in red deer, unveiling new potential targets for future research. The identification of
Comprehensive Multi-Omics Study of
proteins, metabolites, and related biological pathways enhances our understanding of host–parasite
Serum Alterations in Red Deer
interactions and may improve current tools for more effective liver fluke control.
Infected by the Liver Fluke Fascioloides
magna. Pathogens 2024, 13, 922.
Keywords: proteomics; metabolomics; wildlife; host–pathogen interaction; liver fluke
https://doi.org/10.3390/
pathogens13110922
Academic Editors: Georgiana Deak,

Lavinia Ciucǎ and Harold Salant 1. Introduction
Received: 6 September 2024 For many years, wild animals have been reported as a potential source of infectious
Revised: 10 October 2024 and parasitic diseases for domestic animals and humans [1,2]. It is evident that the em-
Accepted: 17 October 2024 phasis has been on preserving the health of humans and domestic animals, while the
Published: 22 October 2024 impact of pathogens on wildlife has generally been a secondary concern, likely due to
past perspectives shaped by economic considerations and public health policies. The shift
to the integrated One Health approach recognizes that the health of humans, animals
(including wildlife), and ecosystems is interconnected and interdependent. Additionally,
Copyright: © 2024 by the authors.
the increasing role of emerging diseases in the wildlife population is well recognized [3].
Licensee MDPI, Basel, Switzerland.
This article is an open access article
Numerous factors, including invasive species, climate change, pollution, and resource
distributed under the terms and
overexploitation, negatively affect wildlife population health [4].
conditions of the Creative Commons Parasites can affect the behavior, health, and population dynamics of wildlife, and
Attribution (CC BY) license (https:// their presence in wild populations is often used as an indicator of ecosystem health. In-
creativecommons.org/licenses/by/ troducing non-native parasites to naïve hosts can result in losses in livestock production
4.0/). and negatively impact the survival of local wildlife populations [1]. The recent rising
Pathogens 2024, 13, 922. https://doi.org/10.3390/pathogens13110922 https://www.mdpi.com/journal/pathogens

Pathogens 2024, 13, 922 2 of 22
prevalence of fascioloidosis in Central Europe is an example of a non-native parasite being

introduced to naïve hosts. The giant liver fluke, Fascioloides magna (Digenea: Fasciolidae),
is originally a parasite of North American deer species, introduced to Europe through in-
fected white-tailed (Odocoileus virginianus) and wapiti deer (Cervus elaphus canadensis) [5,6].
Fascioloidosis, a parasitic disease caused by the non-native parasite F. magna, has significant
implications for wild and domestic animals, namely the management and conservation of
game and domestic animal husbandry.
The life cycle of F. magna begins with mature flukes releasing eggs, which are then
excreted in the feces of the mammalian host [6]. In water, miracidia hatch from the eggs
and actively seek out an aquatic snail, the intermediate host. Within the snail, the parasite
undergoes several developmental stages: sporocysts, rediae, and cercariae. The cercariae
then leave the snail and encyst as metacercariae on vegetation becoming an infective
stage. Once ingested by a mammalian host, the metacercariae excyst, and the juvenile
flukes penetrate the intestinal walls, migrate through the peritoneal cavity to reach the
liver, penetrate the Glisson’s capsule, and migrate through the liver parenchyma. Finally,
juvenile flukes encapsulate within pseudocysts and develop into mature flukes.
The pathological changes, clinical signs, and outcomes of F. magna infection are
strongly related to the three types of final hosts—definitive, aberrant and dead-end—and
their different tolerance to infection [5,6]. In definitive hosts like red deer, the parasite
reaches its sexual maturity placed within a pseudocyst in the liver parenchyma, and ani-
mals rarely show any clinical signs [7,8]. However, due to egg shedding through feces and
their large migration potential, red deer as definitive hosts are extremely important for the
maintenance and spread of F. magna in European nature.
The latest advances in omics approaches, particularly in relation to genomic and
transcriptomic sequencing, have provided important tools for the characterization of
pathogen proteomes. Proteomics, as the study of the proteome, involves connecting genes
with their functionally diverse protein products. Recently, there have been significant
efforts to identify and characterize the host–pathogen molecular interface, with particular
emphasis on secretomes and surface proteomes from blood and liver flukes, as well as
nematodes [9–15].
For the fluke F. magna, the transcriptome and the excretory and secretory proteome of
sexually mature flukes, obtained from naturally infected red deer livers, were characterized,
setting up a foundation for future research [16]. A comparative proteomics analysis of the
liver and serum of wild boar, a dead-end host for F. magna infection, provided insights
into proteome profile changes at both local and systemic levels [17]. This study revealed
associations with host immune response, oxidative stress, and metabolism changes. A
recent proteomics study also revealed alterations in the metabolism of proteins and fatty
acids, as well as modifications related to oxidative stress, fibrosis, and metabolism signaling
pathways in the liver of red deer infected by F. magna [18]. More studies can be found
related to infections with liver fluke Fasciola hepatica and Fasciola gigantica [19,20].
The metabolic effects of parasites on the host were studied before the ‘omics’ era.
Parasites and hosts compete for energy resources and, therefore, this profoundly affects
metabolic homeostasis. With the emergence of the new omics discipline of metabolomics,
parasitic helminth infections were among the first experimental models to which it was
applied [21,22]. Metabolomics involves the high-throughput characterization of metabolites,
small molecular compounds (<1.5 kDa) that are the end products of the cellular metabolism
and serve as direct indicators of biochemical activity [23].
Integrated omics approaches are required to obtain much deeper insights than any of
these techniques alone, providing a more comprehensive understanding of the molecular
mechanisms involved in disease development [24]. Combining individual omics data,
such as proteomics and metabolomics, emphasizes the relationships between the involved
biomolecules and their functions, and furthermore helps bridge the gap from genotype to
phenotype [25].
Pathogens 2024, 13, 922 3 of 22
The aim of this study was to analyze differences in serum proteome between F. magna-
infected and apparently healthy red deer (Cervus elaphus) that served as the control group,
using a label-based high-resolution liquid chromatography–tandem mass spectrometry
(LC-MS/MS) quantitative proteomics approach. Furthermore, an untargeted MS-based
metabolomics approach was employed to assess differences in the serum metabolome. The
bioinformatic functional analysis of proteins and metabolites with varying abundances
and intensities facilitated the understanding of the mechanisms underlying host–parasite
interactions. A knowledge-driven approach was applied to map data from each omics layer
to curated knowledge bases containing annotations in terms of pathways and interactions
for different types of biomolecules.
2. Materials and Methods

2.1. Animals
Serum samples were collected immediately following regular hunting operations.
Blood samples were obtained from large veins or the heart using a syringe. Samples
were centrifuged using a portable centrifuge (Hettich, EBA 20, Merck KGaA, Darmstadt,
Germany), the serum was transferred into tubes, properly labeled and frozen at −80 ◦ C
until further analysis. The health status of the red deer was assessed based on external
appearance, body condition, fur appearance, and behavior before shooting, and afterward,
based on the presence of any visible wounds, discharge from natural openings, and the
macroscopic appearance of the organs during evisceration. Age was determined according
to body development and tooth characteristics. All animals were females and young adults
older than 2 years.
A parasitological examination of the liver was carried out for fascioloidosis diag-
nosis [26]. Liver slices (approx. 2 cm thick) were thoroughly examined for traces of
iron-porphyrin, fluke migratory paths, pseudocysts, and juvenile or adult flukes. In
positive animals, numerous infection-specific features were present, while the livers of
uninfected control animals appeared normal, free of F. magna flukes, and without any
related gross lesions.
All animals had lungworms and strongyles. Considering that this is a common finding
in red deer and does not cause any symptoms, the only difference between the control
and case groups was the presence of the liver fluke F. magna. Thus, we hypothesize that
the observed alterations in the serum proteome and metabolome are attributable to the
F. magna infection.
In total, the study involved 20 animals, including 10 animals that served as the control
group and 10 animals with confirmed F. magna infection. The study was approved by the
Committee on the Ethics of the University of Zagreb, Faculty of Veterinary Medicine (Class:
640-01/18-17/60, Reg. number: 251-61-44-18-02).
2.2. Proteomics Analysis

2.2.1. Sample Preparation and LC-MS/MS Analysis
A proteomics analysis of the serum samples was performed using a tandem mass tag
(TMT)-based quantitative approach, as described previously [27]. Total protein concentra-
tion was measured with a BCA assay (Thermo Scientific, Rockford, IL, USA). Thirty-five
µg of total proteins were diluted to a volume of 50 µL using 0.1 M triethyl ammonium
bicarbonate (TEAB, Thermo Scientific, Rockford, IL, USA), reduced (200 mM DTT (Sigma
Aldrich, St. Louis, MO, USA), 60 min, 55 ◦ C), alkylated (375 mM IAA (Sigma Aldrich,
St. Lois, MO, USA), 30 min, room temperature in the dark) and acetone-precipitated
(overnight, −20 ◦ C). After centrifugation (9000× g, 4 ◦ C), the protein pellet was dissolved
in 0.1 M TEAB and digested using 1 µL of trypsin (1 mg/mL, (Promega, Madison, WI,
USA); trypsin-to-protein ratio 1:35, at 37 ◦ C overnight). Samples and internal standard
were labeled with TMT 6plex reagents (Thermo Scientific, Rockford, IL, USA) according to
the manufacturer’s procedure.
Pathogens 2024, 13, 922 4 of 22
LC-MS/MS analysis of TMT-labeled peptides was carried out using an Ultimate

3000 RSLCnano system (Dionex, Germering, Germany) coupled to a Q Exactive Plus mass
spectrometer (Thermo Fisher Scientific, Bremen, Germany). After dissolving in the loading
solvent (2% acetonitrile (ACN), 0.1% formic acid), TMT-labeled peptides were desalted on
the trap column (C18 PepMap100, 5 µm, 100 A, 300 µm × 5 mm; Thermo Fisher Scientific,
Waltham, MA, USA) for 12 min at a flow rate of 15 µL/min. Mobile phase A consisted
of 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in 80% ACN.
Separation was performed on the analytical column (PepMap™ RSLC C18, 50 cm × 75 µm;
Thermo Fisher Scientific, Waltham, MA, USA) using a linear gradient of 5–55% mobile
phase B over 120 min, followed by an increase to 95% for 1 min, held at 95% for 2 min, and
re-equilibrated at 5% B for 20 min at a flow rate of 300 nL/min. Ionization was achieved
using a nanospray Flex ion source (Thermo Fisher Scientific, Bremen, Germany) with a
SilicaTip emitter with an inner diameter of 10 µm (New Objective, Woburn, MA, USA).
The MS operated in positive ion mode using the DDA Top8 method. Full-scan MS spectra
were acquired in the range from m/z 350.0 to m/z 1800.0 with a resolution of 70,000, 120 ms
injection time, AGC target 1 × 106 , ± 2.0 Da isolation window, and a dynamic exclusion
of 30 s. High-energy collisional dissociation (HCD) fragmentation was performed at step
collision energy (29% and 35% NCE) at a resolution of 17,500 and an AGC target of 2 × 105 .
Precursor ions with an unassigned charge state, charge states of +1, or more than +7 were
excluded from fragmentation.
2.2.2. Data Processing

The acquired MS/MS spectra were analyzed for protein identification and quantifi-
cation using the SEQUEST algorithm implemented in Proteome Discoverer (version 2.3.,
Thermo Fisher Scientific). A database search against Cervus elaphus FASTA files (down-
loaded from Uniprot database on 5 July 2022, containing 19,262 sequences) was performed
using the following parameters: two trypsin missed cleavage sites, precursor mass toler-
ances of 10 ppm, a fragment mass tolerance of 0.02 Da, carbamidomethyl (C) as a fixed
peptide modification, and oxidation (M) and TMT sixplex (K, peptide N-terminus) as
dynamic modifications. The false discovery rate (FDR) for peptide identification was set at
1% and calculated using the Percolator algorithm in the Proteome Discoverer workflow.
To report confidently identified proteins, at least two unique peptides and a 1% FDR were
required. Protein abundances were normalized to the total protein amount and scaled
to the control average to enable comparison within one sixplex and between different
sixplexes, respectively.
2.2.3. Statistical and Bioinformatic Analysis

Statistical analysis was performed using R software v.4.1.2. [28], following the pre-
viously published in-house protocol [17]. Differences between groups were tested using
the non-parametric Mann–Whitney U test, with Benjamini–Hochberg false discovery rate
(FDR) correction, after removing proteins with abundances missing in more than half
of the samples. Principal component analysis (PCA) and volcano plots were generated
using the R package ggplot2 v3.1.1 [29]. For bioinformatic analysis, protein accession
numbers were converted to Gene IDs using the UniProt database conversion tool, and
replaced with the Bos taurus orthologue if an identity higher than 70% was found using
the UniProt BLAST tool [30]. Gene ontology (GO) classification was performed using the
Protein Analysis Through Evolutionary Relationship (PANTHER) classification tool [31].
Pathway enrichment analysis was conducted with the Reactome tool, using the human
genome as the background and an FDR-adjusted p-value < 0.05 to identify significantly
enriched pathways [32].
2.2.4. Validation of Proteomics Data

Frozen serum samples were thawed and analyzed using a sandwich enzyme-linked
immunosorbent assay (ELISA) to determine alpha-1-acid glycoprotein levels. A cattle
Pathogens 2024, 13, 922 5 of 22
a1AGP (Alpha-1-Acid Glycoprotein) ELISA kit (ELK Biotechnology, Denver, CO, USA) was
used according to the manufacturer’s instructions.
2.3. Untargeted Metabolomics Analysis

2.3.1. Sample Preparation and LC-MS/MS Analysis
Serum samples for untargeted metabolomics analysis were prepared using protein
precipitation, centrifugation, and supernatant filtration. For metabolite extraction, an
extraction solvent consisting of a chloroform/methanol/water (1:3:1, v/v/v) mixture was
used (chloroform, methanol (Honeywell, Charlotte, NC, USA), water (Merck, Darmstadt,
Germany)). Briefly, 1000 µL of ice-cold extraction mixture was added to 25 µL of each
serum sample and vortexed on a cooled mixer at 4 ◦ C for 5 min. A pooled sample was
prepared by mixing 10 µL of each individual sample, and 25 µL of the pooled sample was
also subjected to extraction. All assayed samples (individual samples, pooled samples,
matrix blank) were subsequently vortexed and centrifuged (13,000× g for 5 min at 4 ◦ C).
The analysis of metabolite extracts was performed on a Dionex UltiMate 3000 UHPLC
system (Thermo Fisher Scientific, Germering, Germany) coupled with a Thermo Orbitrap
Q Exactive (Thermo Fisher Scientific, Bremen, Germany). The metabolites were separated
using hydrophilic interaction liquid chromatography (HILIC) with a ZIC-pHILIC column
(150 mm × 4.6 mm, 5 µm column, Merck Sequant, Darmstadt, Germany). Metabolites
were eluted using a linear gradient from 80% of mobile phase B to 5% (mobile phase A:
20 mM ammonium carbonate in water; mobile phase B: 100% ACN), with a flow rate of
0.3 mL/min. The Orbitrap Q Exactive mass spectrometer was operated in both positive and
negative modes at a mass resolution of 70,000, covering the full scan range of m/z 70-1050.
The source voltage was set to 3.8 kV for positive mode and −3.8 kV for negative mode,
with sheath gas at 40 (arbitrary units), auxiliary gas at 5 (arbitrary units), and a capillary
temperature of 320 ◦ C. A standard mix (kindly provided by Glasgow Polyomics, United
Kingdom) consisted of 148 compounds (reference compounds for metabolite identification)
and was analyzed alongside the samples.
2.3.2. Data Processing

Metabolomics data pre-processing (e.g., alignment, batch correction, and identification)
was performed using the Polyomics integrated Metabolomics Pipeline (PiMP) available
at http://polyomics.mvls.gla.ac.uk (assessed on 7 February 2024), implemented as an
R pipeline based around XCMS for the feature detection and mzMatch.R for common
metabolomics data pre-processing tasks [33]. Metabolites were identified based on the
retention times/masses or masses of detected peaks matched to the standards, while
features were annotated using the metabolite databases HMDB (The Human Metabolome
Database) and/or KEGG (Kyoto Encyclopedia of Genes and Genomes) integrated within
the PiMP software.
2.3.3. Statistical and Bioinformatic Analysis

All statistical and pathway enrichment analyses were performed using online available
software MetaboAnalyst v.6.0 [34], on the combined positive and negative ion datasets
exported as a peak intensity table from PiMP.
Missing values (1.3%) were replaced by 1/5 of the minimum positive value for each
variable. The intensities of the extracted metabolites were normalized by median, log
transformed and then mean centered and divided by the square root of the standard
deviation of each variable (Pareto scaling). The differences in the metabolic profiles of two
groups, control and F. magna-infected group, were assessed by t-test with FDR correction
(p < 0.05). The RaMP-DB pathway-associated metabolite sets were selected as the pathway
library for Metabolite Set Enrichment Analysis (MSEA).
2.4. Integration of Proteomics and Metabolomics Data
Significant proteins and metabolites identified between the control and F. magna-
infected group were merged to form a network for visualization and functional analysis
Pathogens 2024, 13, 922 6 of 22
using OmicsNet [35]. A knowledge-driven multi-omics network was created and the
InfoMap algorithm was selected to search for densely connected subnetworks (modules)
by compressing
2.4. Integration the description
of Proteomics of information
and Metabolomics Dataflows based on random walks [36].
Significant proteins and metabolites identified between the control and F. magna-
3. Results
infected group were merged to form a network for visualization and functional analysis
3.1.using
Proteomics Analysis
OmicsNet [35]. A knowledge-driven multi-omics network was created and the
InfoMap algorithm was selected to search for densely connected subnetworks (modules)
In serum samples of red deer, 76 proteins were identified and quantified using a
by compressing the description of information flows based on random walks [36].
label-based quantitative proteomic approach (Supplementary Table S1). Significant
differences
3. Results in abundances between the F. magna-infected and control groups were
3.1. Proteomics
assessed by theAnalysis
Mann–Whitney test with FDR correction (FDR < 0.05), revealing
differences for samples
In serum 18 proteins
of red(Table
deer, 761). Of those,
proteins 5 exhibited
were identified higher abundance,
and quantified using a label-and 13
based quantitative proteomic approach (Supplementary Table
showed lower abundance in the F. magna-infected group compared to the S1). Significant differences
control group,
in abundances between the F. magna-infected
as depicted in the volcano plot (Figure 1). and control groups were assessed by the
Mann–Whitney test with FDR correction (FDR < 0.05), revealing differences for 18 proteins
The PCA plot showed clear discrimination between the groups (Figure 2). The
(Table 1). Of those, 5 exhibited higher abundance, and 13 showed lower abundance in the
clusters were separated
F. magna-infected groupbased
comparedon principal component
to the control group, as1depicted
(PC1), which captured
in the volcano 27.5% of
plot
the(Figure
variance1). in the dataset, while PC2 captured 10.4% of the variance.
Figure
Figure 1. Volcano
1. Volcano plotshowing
plot showing proteins
proteins with
withdifferential abundance
differential abundancein theinF.the
magna-infected group group
F. magna-infected
(N = 10) compared to the control group (N = 10). Red points indicate significantly
(N = 10) compared to the control group (N = 10). Red points indicate significantly increased abun-
increased
dances in the infected group and blue points indicate significantly decreased
abundances in the infected group and blue points indicate significantly decreased proteinprotein abundances.
Features that
abundances. do not meet
Features that the significance
do not meet thethreshold are shown
significance in gray.are shown in gray.
threshold
Table 1. Proteins with significantly differential abundances between control and F. magna-infected red
deer (Cervus elaphus) identified and quantified using a tandem mass tag (TMT) proteomics approach
in the serum.
Accession Gene Name Description p Value FDR log2FC

A0A212DAA2 FGA Fibrinogen alpha chain 1× 10−5 0.0002 1.380
A0A212D7J2 FGB Fibrinogen beta chain 1 × 10−5 0.0002 1.296
A0A212D8V0 FGG Fibrinogen gamma chain 2 × 10−5 0.0003 0.940
A0A212D4K0 Histone H4 1 × 10−2 0.0440 0.860
2 FGA Fibrinogen alpha chain 1 × 10−5 0.0002 1.380
FGB Fibrinogen beta chain 1 × 10−5 0.0002 1.296
FGG Pathogens 2024, 13,
Fibrinogen
922
gamma chain 2 × 10 −5 0.0003 0.940
7 of 22
Histone H4 1 × 10−2 0.0440 0.860
6 ORM1 Alpha-1-acid glycoprotein 1 × 10−5 0.0002 0.727
Table 1. Cont.
ALB Albumin 8 × 10−3 0.0373 −0.173
C8A Accession Gene Name Description
Complement C8 alpha chain p Value
6 × 10 −3 FDR0.0301 log2FC
−0.188
A0A212CNC6 ORM1 Alpha-1-acid glycoprotein 1 × 10−5 −2 0.0002 0.727
ITIH1 A0A212D5P0Inter-alpha-trypsin
ALB
inhibitor heavy chain H1
Albumin
1 × 10 0.0373
8 × 10−3
0.0519 −0.173 −0.190
A0A212CG89Uncharacterized
C8A protein (BLAST:
Complement Pregnancy zone
C8 alpha chain 6 × 10 − 3 0.0301 −0.188
LOC506828
A0A212C922 ITIH1 Inter-alpha-trypsin inhibitor heavy chain H1 1×310×−10 0.0183 −0.190
2 −3 0.0519 −0.221
protein) Uncharacterized protein (BLAST: Pregnancy
A0A212CDE1 LOC506828 3 × 10−3 −3 0.0183 −0.221
CFH Complement factor H
zone protein) 4 ×−10 3
0.0225 −0.228
A0A212CQC9 CFH Complement factor H 4 × 10 0.0225 −0.228
ITIH2 A0A212CC12Inter-alpha-trypsin
ITIH2 inhibitor heavy
Inter-alpha-trypsin inhibitor chain 2 2
heavy chain 1×110×−103 −3 0.0105 −0.241
0.0105 −0.241
− 2
HRG A0A212CIM7Histidine-richHRG Histidine-rich glycoprotein
glycoprotein 1× 0.0519 −0.309
110× 10−2 0.0519 −0.309
A0A212CMY9 IGHM Ig-like domain-containing protein 1 × 10−2 0.0430 −0.321
9 IGHMA0A212CIC4Ig-like domain-containing
FETUB Fetuin-B protein 3×110×−103 −2 0.0430 −0.321
0.0183 −0.321
A0A212DHP9 APOA1 Apolipoprotein A-I 2 × 10−3 −3 0.0183 −0.374
FETUBA0A212CI17 Fetuin-B CD5L CD5 molecule like
3 × 10 0.0374
8 × 10−3
0.0183 −0.432 −0.321
APOA1 A0A212CQ10Apolipoprotein
CFH A-I
Complement factor H 3×210× 10 0.0183
− 3 −3 0.0183 −0.434 −0.374
A0A212D5R7 JCHAIN Joining chain of multimeric IgA and IgM 7 × 10−4 −3 0.0092 −0.447
CD5L CD5 molecule like 8 × 10 0.0374 −0.432
CFH Complement factor H plot showed clear discrimination between the
The PCA 3 ×groups
10 (Figure
−3 0.0183 −0.434
2). The clusters
JCHAIN Joining chainwere separated basedIgA
of multimeric on principal
and IgM component 1 (PC1), which
7 ×captured
10−4 27.5% of the variance
0.0092 −0.447
in the dataset, while PC2 captured 10.4% of the variance.
Figure 2. Principal
Figurecomponent analysisanalysis
2. Principal component (PCA)(PCA)
score plot
score plotshowing the
showing the distribution
distribution of samples
of samples from fro
the control (red dots) and F. magna-infected (green dots) group.
the control (red dots) and F. magna-infected (green dots) group.
According to PANTHER GO Slim analysis, serum proteins with significan

differences in abundances between the control and F. magna-infected group were involve
in biological regulation (N = 11), response to stimulus (N = 8), and metabolic processes (
= 7), among others (Figure 3). The molecular function of proteins with significantl
Pathogens 2024, 13, x FOR PEER REVIEW 8 of 23
Pathogens 2024, 13, 922 7), among others (Figure 3). The molecular function of proteins with significantly differ- 8 of 22
ential abundances included binding (N = 8), molecular function regulator activity (N = 5),
and structural molecule activity (N = 5).
Reactome
Accordingpathway
to PANTHERanalysis
GO revealed 24 significant
Slim analysis, pathways
serum proteins with (Supplementary Table
significant differences
S2). Most of thebetween
in abundances the serum
significant controlproteins were involvedgroup
and F. magna-infected in platelet
were degranulation
involved in biolog-(N =
7),
icalresponse to elevated
regulation (N = 11),platelet
responsecytosolic
to stimulus (N == 8),
Ca2+ (N 7), and
platelet activation,
metabolic signaling
processes (N =and7),
among others
aggregation (N(Figure 3). The molecularprotein
= 7), post-translational functionphosphorylation
of proteins with (Nsignificantly differential
= 5), regulation of in-
abundances
sulin-like included
growth binding
factor (N = 8), molecular
(IGF) transport and uptake function regulatorgrowth
by insulin-like activityfactor
(N = binding
5), and
structural molecule activity (N = 5).
proteins (IGFBPs) (N = 5), and others (Figure 4).
Figure
Figure 3.
3. Gene
Gene ontology analysisfor
ontology analysis forproteins
proteinswith
withdifferent
different abundance
abundance in in serum
serum between
between the the con-
control
trol and F. magna-infected red deer using the PANTHER GO-Slim analysis: (A) molecular function;
and F. magna-infected red deer using the PANTHER GO-Slim analysis: (A) molecular function;
(B) biological process; (C) cellular component; (D) protein class.
(B) biological process; (C) cellular component; (D) protein class.
Validating assays such

Reactome pathway as ELISA
analysis revealedor24
Western blotpathways
significant for non-model species, like
(Supplementary Tabledeer,
S2).
poses significant challenges due to the specificity of antibodies. In the
Most of the significant serum proteins were involved in platelet degranulation (N absence of cervid-
= 7),
specific
response kits, we employed
to elevated a cattle-specific
platelet cytosolic Ca2+ ELISA,
(N = 7),given that
platelet cattle aresignaling
activation, the closest
andrelatives
aggre-
to deer.(N
gation Although we successfully
= 7), post-translational generated
protein a calibration(N
phosphorylation curve
= 5),using the kit’s
regulation standards,
of insulin-like
the deer serum
growth samples
factor (IGF) did notand
transport produce
uptakea detectable signal.
by insulin-like This highlights
growth the limitations
factor binding proteins
of using cross-species reagents and
(IGFBPs) (N = 5), and others (Figure 4). underscores the need for species-specific assay devel-
opment in future
Validating studies.
assays such as ELISA or Western blot for non-model species, like deer, poses
significant challenges due to the specificity of antibodies. In the absence of cervid-specific
kits, we employed a cattle-specific ELISA, given that cattle are the closest relatives to deer.
Although we successfully generated a calibration curve using the kit’s standards, the deer
serum samples did not produce a detectable signal. This highlights the limitations of using
cross-species reagents and underscores the need for species-specific assay development in
future studies.
Pathogens 2024,
Pathogens 13, 922
2024, 13, x FOR PEER REVIEW 9 of 23
9 of 22
Figure 4. Reactome pathways enriched from serum proteins with differential abundance between
Figure 4. Reactome pathways enriched from serum proteins with differential abundance between the
the control and F. magna-infected red deer (FDR < 0.05).
control and F. magna-infected red deer (FDR < 0.05).
3.2.Metabolomics
3.2. MetabolomicsAnalysis
Analysis
The metabolomics analysis resulted in the detection of 2744 features in serum
The metabolomics analysis resulted in the detection of 2744 features in serum samples
samples of red deer (Supplementary Table S3). Among them, a total of 67 metabolites were
of red deer (Supplementary Table S3). Among them, a total of 67 metabolites were identified
identified based on mass/retention time matching with known standards (Supplementary
based on mass/retention time matching with known standards (Supplementary Table S4).
Table S4).
A t-test with FDR correction (p < 0.05) showed that 1789 features were significantly
A t-test with FDR correction (p < 0.05) showed that 1789 features were significantly
altered among the groups, while 44 metabolites were identified by reference to standards
altered among the groups, while 44 metabolites were identified by reference to standards
(Table 2). Most of these metabolites belonged to carboxylic acids, hydroxy acids and
(Table 2). Most of these metabolites belonged to carboxylic acids, hydroxy acids and
derivatives,
derivatives,organooxygen compounds,and
organooxygen compounds, andfatty
fattyacyls
acyls(Figure
(Figure
5).5). When
When applying
applying a fold
a fold
change
change threshold of 2.0, 329 features had higher intensity, and 224 had lower intensity in in
threshold of 2.0, 329 features had higher intensity, and 224 had lower intensity
theF. F.
the magna-infected
magna-infectedgroup
groupcompared
comparedtotothe
thecontrol
controlgroup,
group,asasdepicted
depictedininthe
thevolcano
volcanoplot
(Figure 6).
plot (Figure 6).
Very clear discrimination of the groups is shown using PCA plot (Figure 7). The
clusters were separated based on the principal component 1 (PC1), which captured 43.6%
of the variance in the dataset, while PC2 captured 12.3% of the variance.
Pathogens 2024, 13, x FOR PEER REVIEW
Pathogens 2024, 13, 922 1010of of
2223
Figure 5. Distribution of main chemical classes of identified differential metabolites between the
Figure 5. Distribution of main5.chemical
Figure classes
Distribution of of identified
main chemicaldifferential
classes ofmetabolites between the
identified differential metabolites between the
controlcontrol and F. magna-infected
and F. magna-infected
controlred
anddeer. red deer. red deer.
F. magna-infected
FigureFigure
6. Volcano plot Figure
showing
6. Volcano plot6. features
Volcano with
showing differential
features
plot showing with intensities
with between
differential
features the intensities
intensities
differential control (Nbetween
between = 10)
the control (N (N
the control = 10)
= 10)
and F. magna-infected and
groupF. (N = 10). Red points indicate
magna-infected significantly
pointsincreased levels in theincreased levels in the
and F. magna-infected group (N = group (N = points
10). Red 10). Redindicate indicate significantly
significantly increased levels in the
infected group and blueinfected
points indicate
group andsignificantly decreased
blue points indicate levels. Features
significantly that do levels.
decreased not meet the
Features that do not meet the
infected
significance groupare
threshold and blueinpoints
shown
significance gray. indicate
threshold
significantly decreased levels. Features that do not meet the
are shown in gray.
significance threshold are shown in gray.
Table 2. A list of the identified and significantly changed metabolites in the serum of red deer infected
with F. magna versus control red deer, obtained using the untargeted LC-MS metabolomics approach.
Peak ID Name Mass RT p Value FDR log2(FC)

1447 Methylmalonic acid 117.0196 700.59 4× 10−8 1.25 × 10−6 4.87
72 Isonicotinic acid 124.0394 436.77 2 × 10−3 0.004722 4.03
42 4-Methoxybenzyl propanoate 258.1101 646.48 6 × 10−7 8.05 × 10−6 3.71
1551 L-Aspartate 132.0305 690.35 2 × 10−5 9.79 × 10−5 3.67
1497 Inosine 267.074 554.37 2 × 10−3 0.004966 3.43
1441 Malate 133.0145 734.56 2 × 10−10 1.92 × 10−8 3.38
1855 Glycerol 3-phosphate 171.0068 646.54 6 × 10−7 8.49 × 10−6 3.35
298 Imidazole-4-acetate 127.0502 584.57 2 × 10−3 0.004722 3.25
6 Inosine 269.0881 555.54 1 × 10−3 0.004086 3.18
95 2-Hydroxyadenine 152.0568 634.21 8 × 10−8 2.15 × 10−6 3.08
Pathogens 2024, 13, 922 11 of 22
Table 2. Cont.
Peak ID Name Mass RT p Value FDR log2(FC)

364 Pantothenic acid 220.1179 463.76 2× 10−7 4.23 × 10−6 2.76
1844 Pantothenic acid 218.1038 463.33 2 × 10−7 4.55 × 10−6 2.74
20 Choline 104.107 1037.39 3 × 10−7 5.93 × 10−6 2.47
1433 Pseudouridine 243.0627 522 3 × 10−8 8.99 × 10−7 2.39
1948 Citraconic acid 129.0197 700.79 5 × 10−8 1.52 × 10−6 2.32
199 Taurine 126.022 729.49 4 × 10−7 7.08 × 10−6 2.26
2256 Cytidine 242.0789 597.61 6 × 10−3 0.012562 2.15
1671 Ureidopropionic acid 131.0465 709.18 1 × 10−3 0.003059 2.15
1486 Taurine 124.0076 730 2 × 10−7 4.20 × 10−6 2.06
3-Deoxy-D-glycero-D-galacto-2-
363 310.1132 647.3 5 × 10−5 0.000255 1.94
nonulosonic acid
1561 N-Acetylneuraminic acid 308.0994 628.17 2 × 10−5 0.000128 1.93
1683 N-Acetylglutamic acid 188.0569 654.1 1 × 10−4 0.000591 1.60
50 L-Glutamate 148.0604 674.5 2 × 10−6 1.74 × 10−5 1.54
83 3-Dehydroxycarnitine 146.1176 616.16 3 × 10−5 0.000162 1.49
1573 Galactonic acid 195.0514 655.33 1 × 10−5 9.32 × 10−5 1.38
1458 L-Glutamate 146.0462 674.96 2 × 10−6 1.75 × 10−5 1.38
1576 L-Alanine 88.0406 688.94 5 × 10−5 0.000248 1.04
682 Citramalic acid 190.0709 653.62 1 × 10−3 0.002955 0.90
2455 Malonate 103.0039 727.57 5 × 10−4 0.001558 0.82
5 Betaine 118.0862 557.5 4 × 10−5 0.000196 0.78
43 3-Methylhistidine 170.0924 610.68 3 × 10−5 0.000158 0.73
1687 Pterolactam 114.0563 618.23 8 × 10−5 0.000381 0.71
24 L-Proline 116.0706 618.02 1 × 10−5 9.31 × 10−5 0.69
1814 3-Methylhistidine 168.0782 611.63 1 × 10−6 1.12 × 10−5 0.67
282 L-Serine 106.0499 730.75 1 × 10−2 0.02085 0.54
1657 L-Phenylalanine 164.0721 515.76 2 × 10−4 0.000915 0.50
195 L-Methionine 150.0584 573.73 4 × 10−3 0.008503 0.49
57 L-Citrulline 176.1029 719.55 2 × 10−2 0.032129 0.48
1599 L-Leucine 130.0876 550.18 7 × 10−4 0.002242 0.46
1964 L-Valine 116.072 555.79 2 × 10−4 0.000882 0.39
1565 L-Leucine 130.0876 540.2 7 × 10−3 0.014649 0.36
55 L-Isoleucine 132.1019 540.6 5 × 10 − 3 0.010758 0.33
70 D-Alloisoleucine Very clear discrimination132.1019 560.86
of the groups is shown using PCA plot1× 10−27). The
(Figure 0.021533 0.32
2459 L-Kynurenine 207.0779 548.26 9 × 10−3 43.6%
clusters were separated based on the principal component 1 (PC1), which captured
0.018014 –0.48
of the variance in the dataset, while PC2 captured 12.3% of the variance.
RT—retention time. FC—fold change.
Figure
Figure 7. Principal
7. Principal componentcomponent analysis
analysis (PCA) score (PCA)
plot showing score plot
the distribution showing
of samples from the distribution of samples from
the control group (green dots), and the F. magna-infected group (red dots), with the green and red
the control
backgrounds group95%
representing (green dots),
confidence and the F. magna-infected group (red dots), with the green and red
intervals.
backgrounds representing 95% confidence intervals.
The distribution of the 100 most significant features separating the investigated groups
was visualized by hierarchical cluster analysis using Euclidean distance as the measure, the
Ward method as the clustering algorithm, and a t-test for feature ranking. The heat map
showed a clear distinction between the control and infected group (Figure 8).
Pathogens 2024, 13, 922 12 of 22
The distribution of the 100 most significant features separating the investigated groups
was visualized by hierarchical cluster analysis using Euclidean distance as the measure, the
Pathogens 2024, 13, x FOR PEERWard
REVIEWmethod as the clustering algorithm, and a t-test for feature ranking. The heat 13 ofmap
23
showed a clear distinction between the control and infected group (Figure 8).
Figure 8. Hierarchical
Figure 8. Hierarchicalcluster
clusteranalysis
analysis(HCA)
(HCA)based
based on on the
the top
top 100
100 features with significantly
features with significantly
differential
differential intensities
intensities between
between thethe control(green
control (greenpanel)
panel)andand F.F.magna-infected
magna-infected (red
(redpanel)
panel)group
group
usingusing Euclidean
Euclidean as aasdistance
a distance measure
measure and
and Ward
Ward asasa aclustering
clusteringalgorithm.
algorithm. Each
Each colored
coloredcell
cellon
onthe
the
map corresponds to the intensity value, with the red color indicating increased, and blue a decreased
map corresponds to the intensity value, with the red color indicating increased, and blue a decreased
level of a specific feature.
level of a specific feature.
To identify biological patterns associated with significant metabolites, pathway
To identify biological patterns associated with significant metabolites, pathway en-
enrichment analysis was based on 3694 metabolites and lipid pathways from RaMP-DB
richment analysis was based on 3694 metabolites and lipid pathways from RaMP-DB
(integrating KEGG via HMDB, Reactome and WikiPathways). The enrichment analysis
(integrating KEGG via HMDB, Reactome and WikiPathways). The enrichment analysis
revealed 184 significant pathways (FDR > 0.05) (Supplementary Table S5), while 25 of the
revealed 184 significant
most significant pathways
pathways (FDR
are shown in >Figure
0.05) 9.(Supplementary Table S5),
Among these, pathways withwhile 25 of
the most
the most significant pathways are shown in Figure 9. Among these, pathways with
identified metabolites included biochemical pathways—part I, the transport of small the
molecules, the metabolism of amino acids and derivatives, and SLC-mediated
Pathogens 2024, 13,Pathogens
922 2024, 13, x FOR PEER REVIEW 13 of 22 14 of 23
most identified metabolites included

transmembrane transport.biochemical pathways—part
Of special interest I, theantibiotic
were various transportaction
of small
pathways,
molecules, the metabolism
which were alsoofenriched.
amino acids and derivatives, and SLC-mediated transmem-
brane transport. Of special interest were various antibiotic action pathways, which were
also enriched.
Figure 9. Pathway enrichment (top 25) of significant metabolites identified by the untargeted
Figure 9. Pathway enrichment (top 25) of significant metabolites identified by the untargeted
metabolomic approach between control and F. magna-infected group based on 3694 metabolites and
lipid pathwayslipid
from RaMP-DB.
pathways from RaMP-DB.
A knowledge-driven multi-omics network was created with OmicsNet using signifi-
A knowledge-driven multi-omics network was created with OmicsNet using
cantly altered proteins and metabolites between the control and F. magna-infected groups
significantly altered proteins and metabolites between the control and F. magna-infected
to provide a groups
comprehensive
to providevisual representation.
a comprehensive The
visual multi omics-network
representation. The multiwas further
omics-network was
searched by further
the InfoMap
searched by the InfoMap algorithm to identify densely connected in
algorithm to identify densely connected modules, resulting modules,
eight significant modules.
resulting These
in eight modules
significant were These
modules. subsequently
modules searched and annotated
were subsequently searched and
with the top integrative
annotated with the top integrative KEGG pathways (Figure 10). In thisnetwork
KEGG pathways (Figure 10). In this way, the multi-omics way, the multi-
demonstratesomics
the interplay
networkamong significant
demonstrates the proteins
interplayand metabolites,
among highlighting
significant altered
proteins and metabolites,
pathways between the control and F. magna-infected groups across different omics layers.
Pathogens 2024, 13, 922 14 of 22

highlighting altered pathways between the control and F. magna-infected groups across
different omics layers.
Figure10.
Figure 10. The
The multi-omics
multi-omics network
networkcreated
createdwith
withOmicsNet.
OmicsNet.Yellow nodes
Yellow represent
nodes metabolites,
represent metabolites,
while salmon colored nodes represent proteins. Significant modules of connected metabolites and
while salmon colored nodes represent proteins. Significant modules of connected metabolites and
proteins are highlighted in different colors and annotated with their most significant top integrative
proteins are highlighted in different colors and annotated with their most significant top integrative
KEGG pathways.
KEGG pathways.
4. Discussion
4. Discussion
Red deer are economically and culturally significant wildlife and game species in
Red deer are economically and culturally significant wildlife and game species in
Europe and worldwide. They are scientifically important for phylogenetic studies,
Europe and worldwide. They are scientifically important for phylogenetic studies, partic-
particularly in relation to population decline and genetic diversity influenced by new
ularly in relation
settlers. In to population
medicine, deer antlers decline andfor
are notable genetic diversity
their potential ininfluenced by new and
organ regeneration settlers.
In medicine, deer antlers are notable for their potential in organ regeneration
cancer studies. In addition, deer are studied for infectious and parasitic diseases that can and cancer
studies. In addition, deer are studied for infectious and parasitic diseases
affect humans and domestic animals. Liver fluke infections are considered a globally that can affect
humans and domestic animals. Liver fluke infections are considered a globally
neglected disease with a significant impact on public health [37]. Fascioloidosis has neglected
disease withimplications
significant a significant
forimpact on public
both wild health [37].
and domestic Fascioloidosis
animals, as F. magna has significant
parasitizes impli-
in the
cations
liver of for both hosts,
infected wild and domestic
causing animals,
macroscopic F. magna parasitizes
andasmicroscopic lesions thatincan
thelead
liver
to of infected
severe
hosts, causingand
liver damage macroscopic
dysfunction. and microscopic lesions that can lead to severe liver damage
and dysfunction.
4.1. Proteomics
4.1. Proteomics
The TMT-based proteomics approach was applied to determine differences in protein
The TMT-based
abundances betweenproteomics approach was
the F. magna-infected and applied to determine
control groups, differences
resulting in protein
in 18 proteins
abundances between
with significantly the F. abundances.
different magna-infected and control
According groups,
to gene resulting
ontology, theseinproteins
18 proteins
werewith
significantly different abundances. According to gene ontology, these proteins were linked
to response to stimulus (e.g., immune response and response to stress), immune system
processes, biological regulation, and cellular processes (e.g., cell activation, cell adhesion,
and cell metabolic processes).
Pathogens 2024, 13, 922 15 of 22
The central role of the host response to the parasite is evidenced by the significantly
abundant proteins associated with the immune system, such as acute phase proteins and
components of complement cascade.
Among the differentially abundant serum proteins, acute phase proteins (APPs)
such as fibrinogen, alpha-1-acid glycoprotein, albumin, inter-alpha-trypsin inhibitor, and
histidine-rich glycoprotein stand out. Despite the uniform nature of the acute phase
response—a non-specific and complex reaction of an organism triggered by various stimuli
including infection—there are numerous differences in acute phase characteristics between
different animal species [38]. Studies on APP in cervids are relatively scarce. Fibrinogen
has been shown to act as an APP; increased fibrinogen concentrations have been observed
in sick reindeer [39], after an experimental infection of red deer with herpesvirus causing
malignant catarrhal fever [40], in red deer following tuberculine testing [41], and after
inoculation with Yersinia pseudotuberculosis [42]. In our study, fibrinogen chains (alpha, beta,
and gamma) were among the top three identified proteins, with the highest increase in
the F. magna-infected group, which could indicate an acute phase response due to chronic
inflammation. An increased abundance of alpha-1-acid glycoprotein (AGP), previously
unreported in red deer, was also found in the F. magna-infected group. Besides its im-
munomodulating effects, AGP binds and carries basic and neutral lipophilic drugs from
both endogenous and exogenous sources [43].
Lower abundances of inter-alpha-trypsin inhibitor heavy chains H1 and H2 (ITIH1,
ITIH2) were found in the F. magna-infected group compared to controls. Inter-alpha-trypsin
inhibitors are a family of plasma serine protease inhibitors that play a role in the acute phase
response and contribute to extracellular matrix stability by covalent linkage to hyaluronan.
A previous study exploring the liver proteome in F. magna-infected red deer reported
an alteration in different fibrosis-related proteins, suggesting the remodeling of the actin
cytoskeleton due to the parasite [18]. Liver fibrosis occurs as a result of parasite migration
through the liver and the continuous remodeling process, which is marked by the excessive
deposition of extracellular matrix in the hepatic parenchyma. Lower abundances of ITIH1
and ITIH2 may contribute to reduced protease inhibitor activity, potentially preventing
excessive protease-mediated liver injury. The rapid consumption of protease inhibitors
limits the potentially deleterious effects of protease activation on endothelial and epithelial
tissues, as reported in sepsis [44]. Additionally, lower abundances of fetuin-B, another
plasma proteinase inhibitor, were also found in the F. magna-infected group.
The identification of complement-related proteins, such as complement component C8
and complement factor H, in the serum of red deer infected with F. magna supports the role
of the complement cascade in the innate immune response to the pathogen. The comple-
ment system may activate platelets or induce biochemical and morphological changes in the
endothelium, thereby enhancing coagulation and contributing to homeostasis in response
to stimuli [45]. Our work further emphasizes this by highlighting platelet-associated
pathways, namely platelet degranulation, response to elevated platelet cytosolic Ca2+ ,
and platelet activation-, signaling-, and aggregation-enriched pathways. The coagulation
system plays a crucial role in host–pathogen interactions and the host’s immune responses.
Another highlighted pathway in this study was the regulation of insulin-like growth
factor (IGF) transport and uptake by IGF. Its expression in the vasculature is affected by
interactions with cytokines, lipoproteins, growth factors, reactive oxygen species, and
hemodynamic forces [46]. The roles of this pathway and the proteins involved, such as
fibrinogen, albumin, apolipoprotein A1, and inter-alpha-trypsin inhibitor, suggest the
importance of vascular interactions in the pathogenesis of fascioloidosis.
We believe that, on their own, none of the obtained proteins are likely specific enough
as markers for F. magna infection in deer. However, this study suggests that fibrinogen
and alpha-1-acid glycoprotein may be valuable as acute-phase proteins in deer and could
be considered as indicators of inflammatory responses, while the roles of other proteins
should be further investigated.
Pathogens 2024, 13, 922 16 of 22
4.2. Metabolomics
An untargeted MS-based metabolomics analysis of F. magna infection in red deer
resulted in the generation of a serum metabolic signature reflective of the pathogen.
Previous nuclear magnetic resonance (NMR)-based metabolomics studies of helminth
infections revealed the following three major metabolic traits: alteration in amino acid
metabolism, an infection-driven shift in lipid metabolism, and changes in metabolites
associated with the gut microbiota [22]. Recently, four parasite–rodent models were used
to investigate the metabolic profile of the host using NMR spectroscopy [47]. Additionally,
the integrated response of tissue and biofluids in a rat host to the liver fluke F. hepatica
was investigated in a comprehensive study utilizing NMR and multiplex cytokine mark-
ers [48]. Although altered metabolic signatures due to pathogen infections have been
observed in rodent models, little is known about the metabolic fingerprints of infections
in other mammals. More recently, metabolomic analysis and systematic biochemical tests
on sheep infected with F. hepatica showed alterations in energy metabolism and nutrient
deprivation [49].
An alteration in amino acid metabolism was one of the prominent findings in the F. magna-
infected group, consistent with previous studies on helminth-infected animals [22,48,50]. Ten
amino acids had higher intensities in the F. magna group, including essential amino acids
such as valine, phenylalanine, methionine, leucine, and isoleucine. These amino acids
are considered general markers for protein degradation, as variations in their levels are
due to the catabolism of existing proteins resulting from tissue destruction, apoptosis,
or autophagy [51]. In our case, we can hypothesize that the degradation of proteins is
due to the presence of flukes, which cause damage to liver tissue and modulate energy
requirements.
Two N-linked glycoproteins (N-acetylneuraminic acid and N-acetylglutamic acid)
were found to be elevated in the F. magna-infected group, consistent with increased levels
of N- and O-acetylated glycoproteins observed in the plasma of Trypanosoma brucei brucei-
and F. hepatica-infected rodents [47]. These metabolites serve as ligands for cell adhesion
molecules and play an important role in cell stability and direct interactions with pathogens
and toxins [52,53]. Therefore, the increased intensities likely reflect an inflammatory
response to F. magna infection in red deer.
Another significant metabolic pattern observed in the group infected with F. magna
was related to energy metabolism. The host and parasite compete for available metabolic
building blocks and energy resources, so the utilization of alternative metabolic pathways
maintains the balance of host metabolism. Hence, the host’s adaptations to F. magna infec-
tion in energy metabolism are reflected in the serum metabolome. Higher intensities of
methylmalonic acid, malate, pantothenic acid, malonate, citraconic acid, and citramalic acid
were found in the serum of infected red deer. Malate is a metabolic intermediate in the citric
acid cycle and is also important for the malate–aspartate shuttle system, which enables
the transport of electrons across the membrane between the cytosol and the mitochondrial
matrix [54]. Malonate acts as a competitive inhibitor of succinate dehydrogenase, the only
enzyme involved in both the citric acid cycle and the electron transport chain. Methyl-
malonic acid, a derivative of malonic acid, serves as part of the anaplerotic reaction for the
citric acid cycle. Pantothenic acid is a B5 vitamin required for the synthesis of coenzyme A,
which is essential for cellular energy production and for the synthesis and breakdown of
proteins, carbohydrates and lipids [54]. These changes in energy metabolism are also sup-
ported by an earlier proteomics study that found altered abundances of various enzymes
involved in carbon metabolism, the citric acid cycle, and oxidative phosphorylation in the
liver of red deer infected with F. magna [18]. The main biochemical effects of S. japonicum
infection in hamsters included reduced levels of urinary citric acid cycle intermediates,
including citrate and succinate, alongside increased levels of pyruvate, indicating stimu-
lated glycolysis [55]. Similarly, in buffaloes infected with F. gigantica, the downregulation
of metabolism-related processes in the liver was prominent across all time points [20].
Pathogens 2024, 13, 922 17 of 22
Changes in metabolites related to energy metabolism, particularly in the citric acid

cycle, support the hypothesis of a disruption of energy metabolism in fascioloidosis as a
result of infection and the switch to alternative energy pathways. The parasite consumes
large amounts of host glucose and has a greatly reduced lipid metabolism due to its
inability to process beta-oxidation [56,57]. The modulation of energy metabolism in the
host is further supported by increased protein degradation, as evidenced by an altered
amino acid pattern and the increased intensity of methylhistidine, a marker for protein
breakdown in skeletal muscle [54]. However, further and more detailed studies are needed
to decipher the exact alternative pathways and the adaptive response of the host.
Changes in lipid metabolism are also evident in this study, with higher intensities
of choline and its derivative betaine observed in the F. magna-infected group. These
metabolites can be further converted into phosphocholine, a fundamental component of
membrane synthesis, or utilized for producing polyunsaturated fatty acids like arachidonic
acid, leading to the synthesis of pro-inflammatory eicosanoid mediators [58]. Choline
is associated with the inflammatory host response to the parasite demonstrating anti-
inflammatory activity by restraining excessive inflammation through the activation of the
cholinergic anti-inflammatory pathway [59,60].
Higher glycerol-3-phosphate intensity in the F. magna-infected group further indicates
increased lipid degradation and enhanced gluconeogenesis.
Several pathways related to transcription/translation, tRNA aminoacylation, and
metabolites related to nucleotide derivatives were highlighted in this study. Inosine is a
product of the intracellular conversion of adenosine by nucleotide-degrading enzymes
secreted by the fluke [61,62]. The shift to inosine in the F. magna-infected group suggests
an attenuated inflammatory response that suppresses pro-inflammatory cytokines such as
IFN-γ, TNF-α, and IL-12, ultimately prolonging fluke survival [63].

The integrated omics approach based on the parallel and integrated assessment of
metabolite and protein profiles enabled the extraction of biochemical pathways directly
and indirectly associated with F. magna infection in red deer.
The most highlighted functional annotations from the integrated proteomics and
metabolomics data were related to amino acid metabolism. Despite the fact that amino
acids always appear on the list of metabolites affected by infection, several similarities
were observed in helminth-infected animals. Higher levels of essential amino acids, such
as valine, are consistent with the findings from Schistosoma and Fasciola models [48,64],
suggesting that the parasite disrupts the insulin-stimulated uptake of essential amino acids
by tissues, in order to benefit from an increased pool of this important carbon and energy
source in the blood. Furthermore, taurine is another amino acid metabolite frequently
affected by helminth infections [48,64]. Taurine and hypotaurine metabolism are closely
linked to glutathione metabolism, which plays a role in the protection against oxidative
stress [65]. Previous studies have reported reduced activity of antioxidant enzymes due to
parasite infections. Infection with F. hepatica reduces antioxidant capacity in humans and
rats [66–68]. Alterations in the components and pathways associated with oxidative stress
were also found in red deer and wild boar infected with F. magna [17,18], indicating that
infection with F. magna is accompanied by changes in antioxidant capacity.
One of the top functional annotations of the integrated proteomics and metabolomics
data was related to ATP-binding cassette (ABC) transporters. ABC transporters form a ubiq-
uitous superfamily of integral membrane proteins responsible for the ATP-driven translo-
cation of various substrates across membranes, ranging from ions to macromolecules [69].
ABC transporters are associated with various host–pathogen interactions and have been
linked to reduced drug susceptibility in parasitic helminths [70].
Several flukicide drugs are available, but the treatment of choice for both juvenile
and adult stages in final hosts is the benzimidazole, triclabendazole (TCBZ). The control
of liver fluke infections has predominantly relied on treatment with anthelmintic drugs
Pathogens 2024, 13, 922 18 of 22
in endemic areas. As part of the animal disease control program, medicated baits con-
taining triclabendazole have been made available in the free-ranging deer population [71].
Unfortunately, the intensive use of strong flukicides has led to the development of re-
sistance. Reports of TCBZ-resistant F. hepatica populations in cattle, sheep, and humans,
could undermine fluke control and necessitate research into new treatment options [72–74].
Several in vitro and in vivo experiments have elucidated the role of the CYP450 system in
resistance and susceptibility to antiparasitic drugs [75–77]. As a cellular stress response
to parasite presence, the host’s response includes the upregulation of a suite of protective
and detoxifying genes, including ABC transporters and cytochrome P450 detoxification
proteins [78]. The enzymes involved in the metabolism of xenobiotics showed significant
changes in red deer and wild boar infected with F. magna, as well as in sheep infected
with the fluke F. hepatica [17,18,79]. Additionally, different drugs’ action pathways were
significantly affected in our metabolomics pathways’ enrichment analysis. Therefore, our
results support the hypothesis of the involvement of these pathways and proteins in the
development of drug resistance, suggesting a need for further research.
Purine metabolism and aminoacyl-tRNA biosynthesis were also emphasized in this
study. Purine metabolism synthesizes key structural elements for nucleic acid synthesis, as
well as energy metabolism molecules, such as coenzymes, allosteric modulators, energy
intermediates, and intracellular and extracellular messengers. Extracellular nucleosides
and nucleotides are involved in various cellular responses, including proliferation, migra-
tion, differentiation, and the secretion of growth factors and inflammatory mediators [80].
Aminoacyl-tRNA synthetases (ARSs) are a vital and ubiquitously present family of en-
zymes essential for protein synthesis. However, the growing body of evidence shows that
ARS are involved in a variety of physiological and pathological processes beyond trans-
lation [81]. ARSs participate in the maturation, transcription, activation, and recruitment
of immune cells, functioning as regulators and signaling molecules in various infectious
diseases. Considering the above, exploring the biological functions of these pathways in an
immune context represents a promising field for future studies.
4.4. Strengths and Limitations of the Study

Studying wild animals is often more complex due to challenges in study design, moni-
toring, data collection, and interventions in free-ranging populations and open habitats.
As we studied naturally infected free-ranging deer with varying stages of infection and
individual variability, caution is necessary for interpretation, thereby representing potential
limitations of the study.
The strengths of this research include the use of two complementary, up-to-date omics
approaches based on high-resolution mass spectrometry. A major challenge in untargeted
metabolomics is metabolite identification, influenced by technological limitations, including
the analytical coverage of the employed platform, biases favoring the detection of more
abundant molecules, and differing fragmentation patterns of the same metabolite. While
the small sample size may be considered a limitation, the strong effects of infection were
sufficient to create a clear picture of the dominant proteins and metabolites associated with
the disease.
5. Conclusions
Public health programs and emerging infectious disease surveillance must prioritize
diseases at the wildlife–livestock interface, especially zoonotic ones. Understanding wildlife
and their roles is crucial for grasping the epidemiology and ecology of these diseases.
Understanding host–parasite interactions in wild animals is essential for predicting the
impact of both the spread of existing parasites and the emergence of new ones on population
management and dynamics.
In conclusion, we observed a pattern of significantly altered proteins and metabolites
referring to the inflammatory host response and energy metabolism modulation in deer
fascioloidosis. Our findings demonstrated that infection with the giant liver fluke F. magna
Pathogens 2024, 13, 922 19 of 22
in red deer was associated with changes in amino acid metabolism, energy metabolism,
lipid metabolism, immune system, and related biochemical pathways. This study provides
a global overview of the serum proteome and metabolome in response to F. magna infection
in red deer, highlighting new targets for further investigation. The identification of proteins,
metabolites and associated biological pathways represents a valuable contribution to
the understanding of host–parasite interactions and may enhance current tools for more
effective liver fluke control.
Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/pathogens13110922/s1, Supplementary data: Supplemental
Table S1. List of all identified and quantified proteins in serum samples. Supplemental Table S2.
Reactome pathways enriched from serum proteins significantly differential in abundance between
control and F. magna-infected groups. Supplemental Table S3. Peak intensity table with mass,
retention time and annotations of features identified by untargeted metabolomics approach in the
serum of control and F. magna-infected red deer. Supplemental Table S4. List of all identified
features on the basis of the mass/retention time matched to known standards in the PiMP software.
Supplemental Table S5. Pathway enrichment of the significant metabolites identified by untargeted
lipid pathways from RaMP-DB.
Author Contributions: Conceptualization, D.K. and J.K.; methodology, I.R. and J.K.; software, M.B.
and K.Š.; validation, J.K., M.B. and I.R.; formal analysis, I.R. and J.K.; investigation, J.K. and K.Š.;
resources, M.B.; data curation, I.R.; writing—original draft preparation, J.K.; writing—review and
editing, J.K., M.B., I.R., K.Š. and D.K.; visualization, K.Š. and I.R.; supervision, J.K. and D.K.; project
administration, M.B.; funding acquisition, D.K. All authors have read and agreed to the published
version of the manuscript.
Funding: Study was fully supported by the Croatian Science Foundation grant HRZZ-IP-2018-01-8963
“Host-parasite interactions: a relation between three types of hosts and Fascioloides magna infection”.
Institutional Review Board Statement: The animal study protocol was approved by the Committee
on the Ethics of the University of Zagreb, Faculty of Veterinary Medicine (Class: 640-01/18-17/60,
Reg. number: 251-61-44-18-02).
Informed Consent Statement: Not applicable.
Data Availability Statement: The original contributions presented in the study are included in the
article/Supplementary Materials, further inquiries can be directed to the corresponding author.
Acknowledgments: Part of the findings of the study was presented at the 4th Nordic Metabolomics
Conference (Turku, Finland, 2024).
Conflicts of Interest: The authors declare no conflicts of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or
in the decision to publish the results.
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pathogens

1 Department of Chemistry and Biochemistry, Faculty of Veterinary Medicine, University of Zagreb,

Academic Editors: Georgiana Deak,

Pathogens 2024, 13, 922. https://doi.org/10.3390/pathogens13110922 https://www.mdpi.com/journal/pathogens

prevalence of fascioloidosis in Central Europe is an example of a non-native parasite being

2. Materials and Methods

2.2. Proteomics Analysis

LC-MS/MS analysis of TMT-labeled peptides was carried out using an Ultimate

2.2.2. Data Processing

2.2.3. Statistical and Bioinformatic Analysis

2.2.4. Validation of Proteomics Data

2.3. Untargeted Metabolomics Analysis

2.3.2. Data Processing

2.3.3. Statistical and Bioinformatic Analysis

Accession Gene Name Description p Value FDR log2FC

According to PANTHER GO Slim analysis, serum proteins with significan

Validating assays such

Peak ID Name Mass RT p Value FDR log2(FC)

Peak ID Name Mass RT p Value FDR log2(FC)

most identified metabolites included

Pathogens 2024, 13, 922 14 of 22

Changes in metabolites related to energy metabolism, particularly in the citric acid

4.3. Integration of Proteomics and Metabolomics Data

4.4. Strengths and Limitations of the Study

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