Immunoproteomic Analysis of Trichinella britovi Proteins Recognized by IgG Antibodies from Meat Juice of Carnivores Naturally Infected with T. britovi

Infection with Trichinella nematodes elicits non-specific and specific immune responses; these depend on the dose of infection, the nematode, and the host species. Few studies have examined the presence of specific antibodies against Trichinella spp. in the meat juice of wild animals. The aims of the study were to determine the prevalence of antibodies against Trichinella spp. in meat juice and to identify the specific proteins reacting with the meat juice from free-living carnivores naturally infected with the parasite. Meat juice samples were taken from foxes, badgers, raccoon dogs, and martens and tested with indirect ELISA. Antibodies against Trichinella spp. were detected in 10% of foxes and 46% of raccoon dogs. The ELISA results were confirmed by immunoblot, which revealed different protein patterns in meat juice from red foxes, raccoon dogs, and badgers. The most frequently observed bands were sent for further analysis by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the detection of Trichinella britovi immunogenic proteins. The results confirm the presence of proteins such as serine protease and heat shock proteins associated with Trichinella infection. These findings provide that meat juice is a useful matrix for proteomic analysis.

All developmental stages of Trichinella elicit an immune response, including IgG antibodies, which can be used for the serological detection of Trichinella spp. infection [14]. Infection with nematodes of the genus Trichinella causes both non-specific and specific immune responses, which can vary depending on the dose of infection, and the species of Trichinella and host. The presence of anti-Trichinella antibodies and specific proteins or antigens may be observed in serum or plasma and meat juice. Both serum and plasma are easy to collect and are widely used as sources of proteins in a highly soluble form [15]; indeed, there are thousands of proteins that are released by cells and tissues [16]. Serum is a complex biological matrix, and the general health status of the specimen and the presence of parasitic, bacterial, or viral infections are reflected in changes in its proteomic pattern [16]. However, in many cases, it is not possible to collect serum samples from wild animals. In such cases, meat juice, consisting of a mixture of intra-and extracellular fluid, blood, and lymph, might provide an alternative matrix that can be easily obtained for post-mortem analysis from carcasses of free-living animals [17]. It has been demonstrated that meat juice samples may be successfully used as a matrix in immunological surveillance  Figure 1). sults concerning the intensity of infection in raccoon dogs were partially publis Cybulska et al. (2019) [12]. Moreover, the result was borderline by ELISA, but nega immunoblot in the case of one fox ( Figure 1, lane 1), which was also T. britovi-pos digestion. The intensity of infection was around one LPG. Immunoblot confirm the presence of anti-Trichinella antibodies in a meat juice sample from one badg was not positive in ELISA (Figure 1, lane 16). Interestingly, this badger was infect T. britovi, with a low intensity of infection (0.13 LPG).
Lanes 17-20 ( Figure 1) represent meat juice samples used as a negative con immunoblot. These samples were taken from badger, marten, fox, and raccoon d spectively), which were negative in digestion and in ELISA.

Immunoblot Analysis
Two meat juice samples from red foxes (F1, F2), and three from raccoon do R2, R3) were classified for further analysis to detect the specific proteins in the teristic bands ( Figure 2). The immunoreactive bands matched the corresponding bands on silver-stained gels and were selected for further LC-MS/MS analysis.  Figure 1). The results concerning the intensity of infection in raccoon dogs were partially published by Cybulska et al. (2019) [12]. Moreover, the result was borderline by ELISA, but negative by immunoblot in the case of one fox ( Figure 1, lane 1), which was also T. britovi-positive in digestion. The intensity of infection was around one LPG. Immunoblot confirmed also the presence of anti-Trichinella antibodies in a meat juice sample from one badger that was not positive in ELISA ( Figure 1, lane 16). Interestingly, this badger was infected with T. britovi, with a low intensity of infection (0.13 LPG).
Lanes 17-20 ( Figure 1) represent meat juice samples used as a negative control for immunoblot. These samples were taken from badger, marten, fox, and raccoon dog (respectively), which were negative in digestion and in ELISA.

Immunoblot Analysis
Two meat juice samples from red foxes (F1, F2), and three from raccoon dogs (R1, R2, R3) were classified for further analysis to detect the specific proteins in the characteristic bands ( Figure 2). The immunoreactive bands matched the corresponding protein bands on silver-stained gels and were selected for further LC-MS/MS analysis.

LC-MS/MS Analysis
Three gel pieces were obtained for both examined meat juice samples from red foxes (bands: A, B, C), four gel pieces for the two meat juice samples from raccoon dogs (bands: A, B, C, D), and seven gel pieces for the single meat juice sample from another raccoon dog (bands: A, B, C, D, E, F, G) ( Figure 2). LC-MS/MS analysis revealed the presence of 279 proteins in the bands recognized by meat juice from fox F1; 209 proteins in the bands recognized by meat juice from fox F2; 357 proteins in the bands recognized by meat juice from raccoon dog R1; 339 proteins in the bands recognized by meat juice from raccoon dog R2; and 393 proteins in the bands recognized by meat juice from raccoon dog R3. More detailed information is presented in Supplementary Material Table S1 (foxes) and Table S2 (raccoon dogs). A comparative analysis of the proteins identified from the five examined meat juice samples indicates that all samples share 117 proteins, including heat shock proteins or paramyosin, which are known immunomodulators (Table 1). In addi-

LC-MS/MS Analysis
Three gel pieces were obtained for both examined meat juice samples from red foxes (bands: A, B, C), four gel pieces for the two meat juice samples from raccoon dogs (bands: A, B, C, D), and seven gel pieces for the single meat juice sample from another raccoon dog (bands: A, B, C, D, E, F, G) ( Figure 2). LC-MS/MS analysis revealed the presence of 279 proteins in the bands recognized by meat juice from fox F1; 209 proteins in the bands recognized by meat juice from fox F2; 357 proteins in the bands recognized by meat juice from raccoon dog R1; 339 proteins in the bands recognized by meat juice from raccoon dog R2; and 393 proteins in the bands recognized by meat juice from raccoon dog R3. More detailed information is presented in Supplementary Material Table S1 (foxes) and shock proteins or paramyosin, which are known immunomodulators (Table 1). In addition, samples from foxes share 136 proteins, and those from raccoon dogs share 238 proteins. Several proteins were identified from multiple bands; these may correspond to protein isoforms or post-translational modifications (Tables S1 and S2).

Gene Ontology (GO) Analysis
Gene Ontology (GO) analysis was used to identify proteins reacting with antibodies present in examined meat juice samples, and these proteins were categorized according to their molecular function, cellular component, and biological process. The percentage share of the number of identified proteins by categories is shown in Figure 3. The data indicates that 222 proteins are associated with biological processes for F1, 159 for F2, 284 for R1, 282 for R2, and 312 for R3. Regarding biological processes, the highest number of proteins are involved in translation and protein folding processes. Briefly, 220 proteins were associated with cellular components for F1, 145 for F2, 295 for R1, 186 for R2, and 317 for R3. The identified proteins are mainly associated with organelles (nucleus, ribosome) and cell parts (cytoplasm, components of membrane). Molecular functions were also found: 469 proteins for F1, 357 for F2, 608 for R1, 571 for R2, and 651 for R3. The predominant types are related to binding (e.g., ion binding, ATP binding, RNA binding) and catalytic activity (e.g., ATP hydrolysis activity, GTPase activity).
were associated with cellular components for F1, 145 for F2, 295 for R1, 186 for R2, an 317 for R3. The identified proteins are mainly associated with organelles (nucleus, ribo some) and cell parts (cytoplasm, components of membrane). Molecular functions wer also found: 469 proteins for F1, 357 for F2, 608 for R1, 571 for R2, and 651 for R3. Th predominant types are related to binding (e.g., ion binding, ATP binding, RNA binding and catalytic activity (e.g., ATP hydrolysis activity, GTPase activity).

Discussion
Trichinellosis is a disease with a global range. Unsurprisingly, there is a great inte est in identifying peptide or protein biomarkers that could be used as markers to identif early Trichinella infection in humans and domestic animals (diagnostic properties) or a vaccines against Trichinella infection, e.g., in pigs (immunoprotective properties). Al hough recent studies have focused on the immunoreactive proteins in host serum an plasma during Trichinella infection, they have mainly been based on 2DE-electrophores examination coupled with LC-MS/MS, or 2DE electrophoresis followed by MALDI-TO mass spectrometry of human, mouse, or pig samples [30,[36][37][38][39][47][48][49][50][51][52][53]. In contrast, th present paper employs standard 1DE electrophoresis coupled with LC-MS/MS analys to obtain preliminary findings; it is the first proteomic analysis of proteins recognized b meat juice samples collected from wildlife. These findings provide new insight into th immune response to Trichinella infection in wild animals. Free-living animals constitute natural reservoir for various parasites, including Trichinella nematodes, and unlike la boratory-based studies, the infection dose and the number of potential re-infections ar

Discussion
Trichinellosis is a disease with a global range. Unsurprisingly, there is a great interest in identifying peptide or protein biomarkers that could be used as markers to identify early Trichinella infection in humans and domestic animals (diagnostic properties) or as vaccines against Trichinella infection, e.g., in pigs (immunoprotective properties). Although recent studies have focused on the immunoreactive proteins in host serum and plasma during Trichinella infection, they have mainly been based on 2DE-electrophoresis examination coupled with LC-MS/MS, or 2DE electrophoresis followed by MALDI-TOF mass spectrometry of human, mouse, or pig samples [30,[36][37][38][39][47][48][49][50][51][52][53]. In contrast, the present paper employs standard 1DE electrophoresis coupled with LC-MS/MS analysis to obtain preliminary findings; it is the first proteomic analysis of proteins recognized by meat juice samples collected from wildlife. These findings provide new insight into the immune response to Trichinella infection in wild animals. Free-living animals constitute a natural reservoir for various parasites, including Trichinella nematodes, and unlike laboratory-based studies, the infection dose and the number of potential re-infections are unknown. Therefore, a thorough understanding of the protein profiles present in free-living animals is needed to provide an accurate picture of the course of a parasite infection.
The entire life cycle of the Trichinella nematodes takes place in a single host, following the ingestion of meat containing infectious Trichinella larvae. Trichinella displays three main antigenic stages: muscle larvae (ML), adult worms (Ad), and newborn larvae (NBL) [14]. The muscle larvae are released in the stomach, from where they migrate to the epithelial cells of the small intestine, where they molt and transform into adult worms. After coupling, the females start to deliver NBLs, which move mainly through the blood circulation to reach the striated muscle, where they finally develop into MLs [1]. All developmental stages of Trichinella stimulate an immune response and produce antigens which can be used for the serological detection of Trichinella spp. infection. Additionally, some research suggests that the Trichinella antigens produced by the three developmental stages are stage-specific [14] and they stimulate or suppress the host immune response to varying degrees.
Most studies have used serum/plasma samples to determine the protein profile characteristic of Trichinella infection [14,30,[36][37][38][39][47][48][49][51][52][53]. However, while serum or plasma are not always available for analysis, meat juice samples provide an adequate, alternative matrix that can be easily obtained for post-mortem analysis, especially from Pathogens 2022, 11, 1155 9 of 16 free-living animals. The use of meat juice also offers the added benefit of studying protein profiles at the exact location where Trichinella larvae localize, thus providing potential new insight into parasite infection, especially when investigating stage-specific antigens for muscle larvae.
In the present studies, different proteins recognized by meat juice samples were observed not only between two of the examined animal species but also between those of the same species (Figure 2A, Table 1). The obtained results may suggest that the examined animals were infected with different doses of the parasite with a different duration of infection. However, in the present paper, examined meat juice samples were taken from animals infected with T. britovi in the late stage of infection, with larvae present in the muscle. It needs to be underlined that the intensity of infection in examined animals differs between specimens. Additionally, in immunoblot, the characteristic bands typically migrated between 50 and 250 kDa, with some differences between animals (Figure 1). In another study, performed with sera of raccoon dogs infected with T. spiralis or T. nativa the bands were observed at around 100 kDa, with a group of bands between 76 and 40 kDa [40]. Contrary to this, research conducted with the use of the serum of foxes infected with Trichinella nativa, revealed that most bands migrate between 40 and 60 kDa [41].
Although most trichinellosis in humans is caused by T. spiralis, some cases of T. britovi infection have recently been observed [54][55][56][57]. Furthermore, several papers have described the analysis of crude and excretory-secretory proteins characteristic of T. britovi [14,30,36,38,39,48]. These studies, conducted using human or T. britovi-infected pig serum, identified some proteins characteristic of T. britovi infection, including those that play a role in the migration of NBL in host tissue or those with immunomodulatory potential.
The aim of the present study was to determine the pattern of proteins reacting with IgG antibodies in meat juice samples collected from free-living animals infected with T. britovi. It was found that antibodies present in meat juice samples react with proteins that are associated with T. britovi infection; in addition, the majority of them appear to be involved in metabolic processes and host-parasite interaction (e.g., the mechanisms of the parasite invasion of host tissue, larval migration, larval molting, immunomodulation), such as heat shock proteins, a serine protease, paramyosin, a poly-cysteine tailed protein, and deoxyribonuclease-2-alpha [14,30,36,38,39]. The identified proteins are compared with the literature data on proteins specific to T. britovi infection in Table 2.   LC-MS/MS analysis allowed to reveal the presence of a wide range of proteins known to take part in tissue invasion, larval migration, larval molting, immune modulation, and metabolic processes. For example, the subunits of 26S protease have been found to be involved in cell cycle and transcription regulation, apoptosis, and the oxidative stress response [38]. Deoxyribonuclease-2-alpha plays a role in the invasion of host tissue and in evading host defense [58,59]. In addition, intermediate filament proteins were observed in all examined samples. These proteins are known as structural elements of the cellular cytoskeleton and are responsible for worm growth [60]. As well as the filament proteins, myosin-4 and paramyosin also play a role in parasite growth. The first is responsible for cellular component organization and actin filament depolymerization, thus facilitating parasite growth and development [14], while the second influences muscle length and stability. It has been observed that paramyosin from helminths serves not only as a structural protein but also as an immunomodulatory agent [61].
In addition, peroxiredoxin-2 protects nematodes from hazardous reactive oxygen species (ROS) during invasion [62], and serine protease 30 plays a crucial role in the host tissues during cell invasions and larval molting [14]. Interestingly, all examined samples were found to contain hypothetical protein T03_17187; this protein has been found to share more than 90% identity with the multi-cystatin-like domain protein (CLP), which is a promising immunoreactive protein [48,63,64].
These proteins are known to be involved in the biological processes of the parasite, such as intracellular protein transport, stress response, glycolytic processes, tricarboxylic acid cycle, protein folding, and translation; they also form parts of important cellular components, such as the cytosol, mitochondrion, proteasome complex, small ribosomal subunit, ribosome, nucleus, cytoplasm, and integral membrane component. They are also involved in significant molecular functions, mainly as catalytic activity (ATP hydrolysis activity, GTPase activity, transferase activity, hydrolase activity, kinase activity) and binding activity to ions, ATP, and RNA. However, it has to be emphasized that differences in the protein profile were observed not only between two examined animal species but also between specimens of the same species (Table S3). This is not surprising, as it has been proposed that the host immune response may vary depending on the dose and phase of infection, parasite, and host species [65][66][67]. Interestingly, the percentage share of the number of proteins derived by three categories, viz. biological process, cellular component, and molecular function, remained comparable between the five examined meat juice samples (Figure 3).
The present study addresses the previous lack of data regarding the presence of T. britovi antibodies in meat juice from free-living animals. T. britovi is known to be widespread among wild animal species and can be transmitted to humans. Hence, there is a pressing need to identify T. britovi-specific antigens which may be used to diagnose infection in humans or support vaccine development [64]. Indeed, searching for markers or proteins with vaccine potential among free-living animals infected with the parasite is in line with the One Health Concept. Nevertheless, there is a need to extend the study using more advanced proteomics techniques such as 2DE electrophoresis coupled with LC-MS/MS analysis.

Collection of Material and Research Scheme
The study was conducted on 20 red foxes (Vulpes vulpes), 26 raccoon dogs (Nyctereutes procyonoides), 24 martens (Martes spp.), and 18 badgers (Meles meles). The animals were acquired between 2013 and 2016 through hunting activities within Project Life+ (NAT/PL/428). Meat juice samples were collected on the occasion of the digestion method for the presence of Trichinella spp. larvae according to EC Regulation No. 2075/2005 [68], and the Trichinella species was determined using multiplex PCR described by Zarlenga et al. 1999 [69], with some modification by Cybulska et al. 2019 [12]. Carcasses were transported to the Witold Stefański Institute of Parasitology, Polish Academy of Sciences (Warsaw, Poland). Muscle samples were taken during dissection and then stored at −20 • C for further analysis. The day before digestion, muscle samples (diaphragms and limb muscles) were thawed at room temperature (RT) and meat juice samples were collected individually into sterile tubes. Next, the obtained samples were stored at −20 • C for further tests: ELISA and immunoblotting.

ELISA
The presence of antibodies against Trichinella from the meat juice samples was detected using a commercial ELISA kit (ID Screen Trichinella Indirect Multi-species, IDvet, Grabels, France), according to the manufacturer's instructions. The meat juice sample dilution was 1:2, according to the protocol. The optical density (O.D.) was measured at a wavelength of 450 nm using an EL*800 ELISA automated plate reader (BioTek, Winooski, VT, USA). The cutoff was calculated based on the sample-to-positive (S/P) percentage according to the formula S/P = [optical density (OD) sample − OD negative control (NC)/OD positive control (PC) − OD NC] × 100. Samples with S/P ≥ 30% were considered as positive; with 25% ≤ S/P% ≤ 30% were considered as borderline; and with S/P% ≤ 25% were considered as negative.

Preparation of Crude Antigens from Muscle Larvae of T. britovi
The Balb/C mice were orally infected with 500 T. britovi (ISS002) muscle larvae (ML), then maintained for at least three months, and then euthanized. Next, to prepare muscle larvae crude antigens (ML C antigens), the MLs were isolated using digestion. The recovered MLs were subsequently purified several times with water through succeeding steps of sedimentation in cylinders. After the final sedimentation, the MLs were collected into 1.5 mL tubes. The larval pellet was extensively washed three times in phosphate-buffered saline (PBS). After that, the larval pellet was washed three times and mixed with lysis buffer as described previously by Bień et al. 2013 [50]. The mixture was homogenized in a Potter-Elvehjem Tissue Homogenizer and disintegrated by sonication (OMNI International Tissue Homogenizer). Next, the solution was centrifuged at 14,000 RPM at 4 • C for 10 min.

SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Immunoblot
The ML C antigens (approximately 10 µg per well) were run on a 12% SDS-PAGE gel with 4% stacking gel at 180 V constant voltage for 50 min. After electrophoresis, the proteins were transferred from the gel to a nitrocellulose sheet (Bio-Rad, Hercules, CA, USA) at 95V for 55 min using Mini-Protean Tetra Cell (Bio-Rad, Hercules, CA, USA). Nitrocellulose strips (NCS) were blocked with Pierce Protein-Free T20 Blocking Buffer (Thermo Fisher Scientific, Waltham, MA, USA) for one hour at RT, and then washed three times in PBS-Tween buffer (pH = 7.2). Following this, the NCSs were incubated with shaking for one hour at RT with meat juice samples diluted 1:20 in Pierce Protein-Free T20 Blocking Buffer (Thermo Fisher Scientific, Waltham, MA, USA). Afterward, the NCSs were washed and then incubated with shaking for one hour at RT with secondary antibody, i.e., horse radish-peroxidase-conjugated Anti-dog IgG (Sigma-Aldrich, Saint Louis, MO, USA), diluted by 1:25,000. Finally, after washing, the NCSs were developed using SIGMAFAST™ 3,3 -diaminobenzidine (Sigma-Aldrich, Saint Louis, MO, USA) and visualized with the Chemi Doc MP Imaging System (Bio-Rad, Hercules, CA, USA).

Proteomic Analyses-Mass Spectrometry (MS) and Protein Identification
Bands of interest were manually excised from the gel and analyzed by liquid chromatography coupled to a mass spectrometer (LC-MS/MS) in the Laboratory of Mass Spectrometry, Institute of Biochemistry and Biophysics, Polish Academy of Sciences (Warsaw, Poland). The obtained peptide masses and fragmentation spectra were matched to the NCBIProt database, with a Nematoda filter using the Mascot search engine. All proteins identified in the Mascot search were compared withthe UniProtKB database (https://www.uniprot.org/; accessed on 29 August 2022) and QuickGO (http://www.ebi.ac.uk/QuickGO/; accessed on 29 August 2022) and classified in gene ontology (GO) in accordance with their molecular functions, biological processes, and cellular component information.

Conclusions
The findings show that meat juice is a useful material for studying antibodies from animals naturally infected with T. britovi. The use of proteomic analysis reveals the presence of proteins known to take part in tissue invasion, larval migration, larval molting, immune modulation, and metabolic processes. Additionally, the mentioned proteins are immunogenic, and they are recognized by host antibodies. The results indicate also that the IgG-response may differ between host species in wildlife. A comprehensive understanding of immune response in free-living animals may provide an accurate picture of parasite infection.