Growth-rate retardation in porcine production is a problem that causes serious economic losses to stockbreeders. The weight gain in affected animals does not follow the expected time course. Therefore, they stay smaller and with lower weight than their fellow beings. The parameter average daily gain (ADG) is used to numerically assess animal growth and it gives the weight gained per day, expressed in grams/day. The ADG may be low, moderate or ideal, and there are different referential models that correspond to each one of these situations. In optimal conditions, the expected ADG is from 614 to 682 g/day during the fattening phase [1
In piglets, growth-rate retardation is due to a delay in their growth during the prenatal phase, due to a restriction in the intrauterine growth [2
]. In fattening animals, there are several causes that could act individually or in combination, such as non-infectious factors, that include genetics or immune status, and infectious factors, including pathogenic parasites, bacteria and viruses [3
]. There are methods to confirm the etiologic diagnosis for genetic and infectious causes, but in some animals with growth-rate retardation it is not an easy task to recognize the reasons for the process, due to their multifactorial character.
The number of pigs per pen, the type of feeder and the mix of animals of different origin or sex have been considered as the main parameters affecting the daily feed intake and the feed conversion ratio of pigs at growing phase [4
]. Low protein supply in growing pigs has been reported to have a retardation effect due to lower muscle growth [5
]. Moreover, it has been indicated that improving farm facilities and modifying management practice could reduce mortality and increase growth performance of grower-finishing pigs [6
]. A detrimental effect of low temperature has been evidenced on performance in pigs with restricted diets [7
The combination between infection with Porcine Circovirus type 2 (PCV2) and environmental stress has been reported to lead to decreased ADG [8
]. However, other studies have not obtained a connection between low growth and PCV2 virus in grower-finishing pigs but an influence of diarrhea [9
The present study is focused on the identification of proteins altered in pigs under growth-rate retardation using a gel-based proteomics approach in saliva samples, as they are supposed to show up affected pathways in this disease/syndrome.
2. Material and Methods
2.1. Characterization of Animals
20 commercial males ((Landrace x Large White) x Duroc) growing pigs were selected from a previous study [10
]. Two groups of animals were randomly selected based on clinical signs and availability of saliva samples: a group of 10 healthy animals without any clinical sign of disease (control pigs), and a group of 10 animals with exclusively growth-rate retardation (GRR) symptoms (GRR pigs).
Saliva samples were collected individually, as reported in the mentioned study, during clinical veterinary inspection of the animals by allowing them to chew a sponge. Afterwards the sponges were centrifuged in a specifically designed tube (Salivette tubes, Salimetrics, USA). Salivary supernatants were stored at −80 °C until analysis.
To exclude any subclinical infection against Porcine Circovirus type 2 (PCV2), which is a common cofactor in the development of several syndromes in the porcine production system, PCR analysis was performed on the saliva samples.
To obtain an overview of the possible mechanism that could be involved in the growth-rate retardation process, the acute phase reaction, the inflammatory and antioxidant status and the level of stress were determined in all pigs.
2.2. PCV2 Detection
The primers used for PCV2 detection in porcine saliva samples were reported previously [11
], specifically Forward primer: 5′-CGGATATTGTATTCCTGGTCGTA-3′ and Reverse primer: 5′-CCTGTCCTAGATTCCACTATTGATT-3′. The extraction of DNA from saliva samples was performed automatically using a commercial extraction kit in an automated DNA extraction instrument (Maxwell®
16 Blood DNA Purification Kit&Maxwell®
RSC Instrument for 16 samples, Promega Corporation, Madison, WI, USA, respectively). The extracted DNA samples were subjected to RT-PCR using an intercalating dye-based qPCR master mix (TB Green Premix Ex Taq (Tli RNase H Plus), Takara Bio Inc., Saint-Germain-en-Laye, France) and a real-time PCR system (Lightcycler 2.0. Roche Diagnostics, Indianapolis, IN, USA).
2.3. Salivary Marker Measurements
C-reactive protein (CRP) and haptoglobin (Hp) concentrations were determined in the saliva samples, for identification of acute and chronic acute phase reaction, respectively, using time-resolved immunofluorimetric assays described before [12
For the inflammatory and antioxidant status measurement, adenosine deaminase (ADA) activity and total antioxidant status (TAC) were determined, respectively, using enzymatic assays as reported in previous studies [10
The stress status in the selected animals was evaluated by measuring the alpha-amylase concentrations in the saliva samples with a commercial enzymatic assay for the human homologue, which had been previously optimized for pigs [15
The possible differences in the levels of the markers studied between control pigs and GRR pigs were statistically evaluated using a Mann-Whitney t-test, since data did not meet the normal distribution criteria. Any possible correlation between the studied analytes were detected by Spearman correlation test. The level of significance was set at p < 0.05 with the statistical program GraphPad Prism 6 (Graph Pad Software Inc., La Jolla, CA, USA).
2.4. Two-Dimensional Gel Electrophoresis (2DE)
To perform the protein profile comparison between control pigs and pigs with GRR, 2DE was used as previously described [16
]. Briefly, 30 μg of saliva proteins in rehydration buffer were rehydrated into immobilized pH gradient (IPG) strips with 11 cm long nonlinear gradients pH 3–11 (GE Healthcare Life Sciences, Munich, Germany) and subjected to isoelectric focusing in a Multiphor II electrophoresis chamber (GE Healthcare Life Sciences, Munich, Germany). For the second dimension, the IPG strips were equilibrated with a 2% DTT solution, followed with a 2.5% of iodoacetamide solution, and subjected to SDS-PAGE on homemade 10–15% polyacrylamide gradient gels of 140 × 140 × 1.5 mm in a vertical chamber (SE600 Chroma, Hoefer, INC., Holliston, MA, USA). Protein patterns were silver-stained according to general protocols [17
] and scanned in an image scanner (ImageScanner II, GE Healthcare Life Sciences. Uppsala, Sweden). Images were evaluated for spot detection and matching using specific software (ImageMaster 2D Platinum 7.0, GE Healthcare Life Sciences, Uppsala, Sweden). The relative percentage of spot volumes from each group of animals was statistically compared using a t
-test with the software mentioned above.
Selected spots with differential abundance between groups were subjected to mass spectrometric (MS) identification.
2.5. Sodiumdodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE)
One-dimensional SDS-PAGE was done in parallel to the 2DE approach in order to support those data and to see whether additional differences between the sample groups could be picked up. After protein determination [18
] 2.5 μg of total protein of each saliva sample were electrophoretically separated by SDS-PAGE as described for the second dimension of 2DE. Following silver staining and scanning, gel images were evaluated with a specific software (ImageQuant TL v2005, Amersham Biosciences Europe GmbH, Freiburg, Germany), to obtain the relative percentage in volume of the different bands in the gel. To evaluate any possible band volume differences between groups of animals, a t
-test was used with the statistical software detailed above. Corresponding bands differing by intensity were subjected to MS identification.
2.6. MS Identification of SDS-PAGE Bands and 2DE Spots
After washing and destaining, bands or spots were reduced with dithiothreitol and alkylated with iodoacetamide [19
]. In-gel digestion was performed with trypsin (Trypsin Gold, Mass Spectrometry Grade, Promega, Madison, WI) with a final trypsin concentration of 20 ng/µL in 50 mM aqueous ammonium bicarbonate and 5 mM CaCl2
]. Afterwards, peptides were extracted with four changes of 30 µL of 5% trifluoroacetic acid (TFA) in 50% aqueous acetonitrile (ACN) supported by ultrasonication for 10 min per change and dried down in a vacuum concentrator (Eppendorf, Hamburg, Germany).
2.7. Protein Identification by MALDI-TOF/TOF Mass Spectrometry
Dried peptides were concentrated and de-salted using Zip-Tips C18 (microbed) (Millipore, Billerica, MA) according to the manufacturer’s instructions.
After elution from the Zip-Tip 0.5 µL of the de-salted peptides were spotted with α-cyano-4-hydroxycinnamic acid onto a ground steel MALDI target plate (Bruker Daltonics, Bremen, Germany). MALDI-TOF/TOF mass spectrometry (Ultraflex II, Bruker Daltonics, Bremen, Germany) was used for spectra acquisition in MS and MS/MS modes. Spectra processing and peak annotation were carried out using FlexAnalysis 3.0 and Biotools 3.2 (both Bruker Daltonics, Bremen, Germany).
Processed spectra were searched via an in-house Mascot server version 2.4.1 (Matrix Science, Boston, MA) and the software ProteinScape 2.1 (Bruker Daltonics, Bremen, Germany) in the UniProt database of sus scrofa using the following search parameters: global modification carbamidomethylation on cysteine; variable modifications oxidation on methionine; deamidation on asparagine and glutamine as well as formation of pyroglutamic acid; enzyme specificity trypsin; charge state z = 1; MS tolerance 100 ppm; MS/MS tolerance 1 Da; two missed cleavages allowed; significance threshold p < 0.05.
2.8. Protein Identification by Q Exactive HF Orbitrap Mass Spectrometry
Dried peptides were resuspended in 0.1% TFA for the LC-MS/MS analysis and separated on a nano-HPLC Ultimate 3000 RSLC system (Dionex). Sample pre-concentration and desalting was accomplished with a 5 mm Acclaim PepMap μ-Precolumn (Dionex). For sample loading and desalting 2% ACN in ultra-pure H2O with 0.05% TFA was used as a mobile phase with a flow rate of 5 µL/min. Separation of peptides was performed on a 25 cm Acclaim PepMap C18 column with a flow rate of 300 nL/min. The gradient started with 4% B (80% ACN with 0.08% formic acid) and increased to 9% B in 7 min, to 31% in 30 min and to 44% in additional 5 min. It was followed by a washing step with 95% B, Mobile Phase A consisted of ultra-pure H2O with 0.1% formic acid.
For MS analysis, the LC was directly coupled to a high-resolution Q Exactive HF Orbitrap mass spectrometer. MS full scans were performed in the ultrahigh-field Orbitrap mass analyzer in ranges m/z 350−2000 with a resolution of 60,000, the maximum injection time (MIT) was 50 ms and the automatic gain control (AGC) was set to 3e6. The top 10 intense ions were subjected to Orbitrap for further fragmentation via high energy collision dissociation (HCD) activation over a mass range between m/z 200 and 2000 at a resolution of 15,000 with the intensity threshold at 4e3. Ions with charge state +1, +7, +8 and >+8 were excluded. Normalized collision energy (NCE) was set at 28. For each scan. the AGC was set at 5e4 and the MIT was 50 ms. Dynamic exclusion of precursor ion masses over a time window of 30 s was used to suppress repeated peak fragmentation.
Database search was performed using an in-house Mascot server (version 2.4.1., Matrix Science, Boston, MA) with following settings:
The protein database consisted of amino acid sequences downloaded from UniProt for taxonomy “Sus scrofa
” (TaxID 9823) as well as a common contaminant database (http://www.thegpm.org/crap/
, identified contaminant proteins were removed from results file). Search settings were as described above adapted to the QExactive MS system: global modification carbamidomethylation on cysteine; variable modifications oxidation on methionine; deamidation on asparagine and glutamine as well as formation of pyroglutamic acid; enzyme specificity trypsin; charge state z = 2+, 3+, 4+; MS tolerance 10 ppm; MS/MS tolerance 0.05 Da; two missed cleavages allowed; significance threshold p
Finally, taking into account the identified proteins, function information was annotated from UniProt database [21
2.9. Protein Quantification by Western Blot
One of the proteins found regulated in SDS-PAGE, S100A12, was subjected to validation analysis. Since the alignment between the human and porcine protein sequences gave an identity of 70.6% according to UniProt database (identification numbers P80511 and P80310, respectively) a commercial human antibody was selected to perform the validation study. Saliva samples of the 10 control pigs and the 10 GRR animals and an additional group of 7 pigs with GRR from the same farm were used for validation. A total protein content of 2.5 μg was applied for SDS-PAGE. Control sample number 6 was randomly selected and used as internal control sample in all gels.
Immunodetection was performed after SDS-PAGE on small-size home-made 12% polyacrylamide mini gels using a vertical electrophoresis chamber (Mini-PROTEAN Tetra Vertical Electrophoresis Cell, Bio-Rad, Hercules, CA, USA) and protein transfer to PVDF membranes using the Trans-Blot Turbo Transfer System (Bio-Rad, Hercules, CA, USA). The primary antibody applied was goat anti-human S100A12 (Thermo Scientific, Rockford, IL, USA) followed by horseradish peroxidase conjugated rabbit anti-goat IgG (Sigma Aldrich, St. Louis, MO, USA). Positive signal was detected by enhanced chemiluminescence (ECL 2 Western Blotting Substrate, Thermo Scientific, Rockford, IL, USA) in an imager LAS 600 (GE Healthcare, Uppsala, Sweden). The comparison between the signals of the different samples was performed using an image analysis software (ImageQuant TL v2005, Amersham Biosciences Europe GmbH, Freiburg, Germany). For comparison of results from different gels, the signal intensity of the internal control sample was used for data normalization.
The focus for our study was on obtaining additional information about growth-rate retarded pigs without infectious disease, as too little biological data is available for this pathophysiological condition.
No differences in the level of acute phase proteins nor in the concentrations of ADA or TAC activity were observed between the two groups of animals. Similar results were also previously described in another study that involved a higher number of animals [10
]. A possible subclinical infection against PCV2 could be excluded. Moreover, a trend to increased salivary alpha-amylase concentrations in animals with GRR was observed in comparison to the control pigs but without statistical significance. A higher level of stress could be postulated in animals suffering from GRR and could be considered as one of the factors affecting the animals, in accordance to [6
To search for biological pathways that could be involved in GRR development, proteins changed in abundance were studied in saliva samples by a 2DE gel-based proteomic approach. Among the regulated proteins, Salivary lipocalin (SAL), double-headed protease inhibitor submandibular gland-like protein and Odorant binding protein (OBP) were detected as down-regulated proteins, whereas lambda light chain of immunoglobulins as well as a fragment of IgA were up-regulated in GRR.
SAL is synthetized mainly in the boar submaxillary gland and binds reversibly odorants and the endogenous ligands androstenol and androstenone [22
]. The protein has two isoforms that are glycosylated at a single position [23
]. SAL is a member of the lipocalin family in which OBP is also included. OBPs are assumed to be directly involved in chemical communication and in the pre-mating recognition process [24
]. Since those proteins were observed down-regulated in GRR pigs it could be supposed that sexual communication may be sub-optimal in those animals. Our SDS-PAGE results support the 2DE findings of altered SAL concentrations; this protein was found as a main component of two bands with down-regulated intensities in GRR. Previous own experiments to detect pig salivary lipocalin more specifically with an antibody against the human homologue failed (the antibody was produced against a synthetic peptide with a sequence close to the C-terminal end of the protein; Sigma Aldrich). The reaction of the antibody was not stable over time, though the reason could not be traced back to either the immunoreagent or the sample (in)stability [25
Carbonic anhydrase VI was another differentially regulated protein of our study, found in different locations of the gel and in altered abundance. It has been reported that CA VI participates in the regulation of salivary pH and protects oral tissue against excess acidity [26
]. Two different forms of 41 kDa and 37 kDa have been reported in rat whole saliva corresponding to carbonic anhydrase VI and a partially deglycosylated form of CA VI [27
]. We have also obtained two forms of CA VI in porcine saliva with similar molecular weights in the present study. Moreover, a higher level of CA VI in its bigger form appeared in animals with GRR while a lower level of the small molecular weight CA VI form was also observed. Further studies should be necessary to understand the different forms of CA VI and its relation to GRR. It could be postulated that the decrease in the smaller, assumed partially deglycosylated form in pigs with GRR might be related to a higher stress status since it has been reported that stress conditions produce an intracellular form of CA VI in rats [28
]. In saliva samples from animals under stress conditions this protein form could be (almost completely) lacking.
Both, 2DE and SDS-PAGE, detected upregulation of immunoglobulin light chains or parts of heavy chains. This could reveal a humoral immune activation in GRR pigs as reported in other porcine diseases [29
]. However, markers of innate immune response such as acute phase proteins, adenosine deaminase and S100A12 levels appeared decreased in those pigs with GRR, so further studies should be performed to explain the immune reaction status in pigs with GRR.
Evaluation of band 49 of the SDS-PAGE pattern suggested regulation of two S100 proteins, S100A8 and S100A12. Only for S100A12 a cross-reactive antibody was available, confirming its downregulation in animals with GRR. This was found both when investigating the 10 saliva samples originally included in the 1DE and 2DE sets and when adding 7 samples from other GRR animals of the previous trial [10
]. S100A12 has proinflammatory activity and is involved in recruitment of leukocytes, promotion of cytokine and chemokine production, and regulation of leukocyte adhesion and migration (UniProt database). According to these results it could indicate that animals with GRR may be more susceptible to infections with an increase in stress response. Validation of S100A8 data was also tried by western blot in our study. However, the available human S100A8 antibody (Thermo Scientific, Rockford, IL, USA) used did not show any cross-reactivity against the porcine protein. This may be due to differences of the human and porcine sequences (identity of 68.8% between the identification numbers P05109 and C3S7K5, respectively, UniProt database).
Developing targeted proteomic assays (selected reaction monitoring, SRM) for the proteins found regulated in this study could be a way to avoid searching for well-reacting commercial pig-specific or cross-reactive antibodies. SRMs are based on MS detection of unique peptide sequences and their fragmentation pattern (transitions). Candidate peptides may be selected either from the peptides identified in the respective study, or in comparison with dedicated database collections [30
], and, once successfully set up, could also in our case help to verify protein candidates or screen larger numbers of samples.
In conclusion, we have detected two possible proteins that could be used as markers of growth-rate retardation in pigs, S100A12 and CA VI, and should be explored in-depth for routine quantification. The behavior of S100A12 in the conditions studied confirmed that the innate immune response is decreased in pigs with GRR. However, on the other hand, concentration of some immunoglobulin chains increased—suggesting some imbalance of the immune system. The role of CA VI in the disease should be clarified in future studies. It may additionally be advisable to set up a specific detection system for the different forms of this protein as well as characterizing them in more details. Moreover, a stress influence was also suggested in animals suffering from GRR that should be taken into account in further studies.