Proteomic analysis of porcine saliva

Saliva contains a number of proteins that may be useful as biomarkers of health and disease and can be easily obtained from large numbers of animals in a non-invasive, stress-free way. The objective of this study was to explore the protein composition of porcine saliva from 10 specific pathogen free pigs using first one-dimensional SDS–PAGE and then two-dimensional electrophoresis and mass spectrometry. A reference proteome pattern for porcine saliva was established with the identification of 13 different, mainly saliva-specific, proteins. These reference data will facilitate the investigation of salivary proteins potentially altered in disease and could serve as novel diagnostic biomarkers.     

Effect of condensed tannin ingestion in sheep and goat parotid saliva proteome

Saliva appears as a defence mechanism, against potential negative effects of tannins, in some species of animals which have to deal with these plant secondary metabolites in their regular diets. This study was carried out to investigate changes in parotid saliva protein profiles of sheep (Ovis aries) and goats (Capra hircus), induced by condensed tannin ingestion. Five Merino sheep and five Serpentina goats were maintained on a quebracho tannin enriched diet for 10 days. Saliva was collected through catheters inserted on parotid ducts and salivary proteins were separated by two-dimensional gel electrophoresis. Matrix-assisted Laser desorption ionization - time of flight (MALDI-TOF) and liquid chromatography tandem mass spectrometry (LC-MS/MS) were used to identify the proteins whose expression levels changed after tannin consumption. Although no new proteins appeared, quebracho tannin consumption increased saliva total protein concentration and produced changes in the proteome of both species. While some proteins were similarly altered in both species parotid salivary protein profile, sheep and goats also presented species-specific differences in response to tannin consumption.     

Porcine acute phase protein concentrations in different diseases in field conditions

Five acute phase proteins (APPs) [C-reactive protein (CRP), serum amyloid A (SAA), haptoglobin (Hp), pig-MAP and albumin] were measured in pigs with naturally occurring infections by porcine reproductive and respiratory syndrome virus (PRRSV), Aujeszky's disease virus (ADV), porcine circovirus type 2 (PCV2) and Mycoplasma hyopneumoniae, and in animals with tail and ear bites, arthritis and other acute inflammatory processes. Healthy specific pathogen-free (SPF) pigs were used as controls. In PRRSV-infected pigs, all APPs with the exception of pig-MAP exhibited significant changes compared with controls. In animals affected with ADV only Hp presented changes of statistical significance, whereas pigs with PCV2 showed marked modifications in all APPs tested. Animals affected with Mycoplasmosis showed concentrations of all positive APPs significantly above levels obtained in SPF pigs, though albumin concentrations did not differ from controls. Finally, all APPs studied showed substantial changes in pigs with acute inflammation. The results indicated that an acute phase response was developed in the different diseases studied, this response being higher in animals with clinical signs and concurrent bacterial processes. Haptoglobin would be the APP that better reflects pathological states; however, to get more complete and valuable information it might be advisable to perform APPs profiles including another APP, such as CRP or SAA.     

Porcine salivary analysis by 2-dimensional gel electrophoresis in 3 models of acute stress: A pilot study

The purpose of this research was to study changes in the salivary proteome of healthy pigs in stressful situations to identify any potential new salivary biomarker of stress. Three groups of animals were subjected to 3 stress models: snaring restraint followed by simulated sampling of vena cava blood; brief transport by road; and restriction of movement in a digestibility cage. Saliva was obtained from each animal before and 15 and 30 min after the induction of stress. The samples from the animals that showed the greatest increase in salivary cortisol concentration were pooled and run on 2-dimensional gels. Coomassie Brilliant Blue R-250 was used for spot detection and mass spectrometry for spot identification. Statistical analyses showed that 2 proteins had significant differences in expression before and after the induction of stress. These proteins were identified as odorant-binding protein and fragments of albumin. Further studies will be necessary to confirm the value of using these proteins as salivary biomarkers of stress in pigs.     

Critical differences of acute phase proteins in canine serum samples

The critical difference values for acute phase proteins in canine serum samples were established on a week-to-week basis. Blood samples from 11 apparently clinically healthy dogs were collected once weekly for five consecutive weeks. For each protein the total variance of analytical results was divided into intraindividual variance (S(Intra)(2)), interindividual variance (S(Inter)(2)), and analytical variance (S(Analytical)(2)). The critical difference (d(K)) was then calculated as d(k)=22(S(Intra)(2)+S(Analytical)(2)). The critical difference values were 1.95 g/L for haptoglobin, 4.85 mg/L for C-reactive protein, and 0.016 DeltaAbs/min for ceruloplasmin. When used in conjunction with the corresponding reference interval, critical difference values can be an aid in correctly interpreting acute phase protein results, by determining whether observed differences between two consecutive measurements in individual animals are due to natural variation or due to disease therapy or experimental procedures.     

Validation of three commercially available immunoassays for quantification of IgA, IgG, and IgM in porcine saliva samples

The objectives of this study were to perform the optimization and validation of three commercially available immunoassays for the measurement of IgA, IgG, and IgM (Igs) in porcine saliva samples and to determinate if their concentrations may be used to distinguish healthy from diseased animals. Intra and inter assay coefficients of variation were lower than 15% in all cases. All methods showed good linearity and recovery; and detection limits were low enough to detect Igs levels in healthy and diseased animals. The clinical validation showed an increase statistically significant (P<0.05) in the group of diseased animals versus healthy pigs. Therefore, these assays may be used in porcine saliva samples, in addition, the measurement of Igs in saliva could be a practical tool, simple and minimally invasive, to evaluate the humoral immune status of pigs.     

Identification of abundant proteins and potential allergens in Culicoides nubeculosus salivary glands

IgE-mediated type 1 hypersensitivity reactions to the bites of insects are a common cause of skin disease in horses. Insect bite hypersensitivity (IBH) is most frequently associated with bites of Culicoides spp. and occurs in all parts of the world where horses and Culicoides coexist. The main allergens that cause IBH are probably some of the abundant proteins in the saliva of Culicoides associated with blood feeding. Western blots of Culicoides proteins separated by 1D gel-electrophoresis detected strong IgE responses in all horses with IBH to antigens in protein extracts from wild caught Culicoides, but only weak responses to salivary antigens from captive bred C. nubeculosus which may reflect important differences among allergens from different species of Culicoides or differences between thorax and salivary gland antigens. 2D electrophoresis and mass spectrometry were used to identify several of the abundant proteins in the saliva of C. nubeculosus. These included maltase, members of the D7 family, and several small, basic proteins associated with blood feeding. The most frequently detected IgE-binding proteins were in a group of proteins with pI>8.5 and mass 40-50kDa. Mass spectrometry identified two of these allergenic proteins as similar to hyaluronidase and a heavily glycosylated protein of unknown function that have previously been identified in salivary glands of C. sonorensis.     

Production and characterization of monoclonal antibodies to (poly100)S1 protein of avian infectious bronchitis virus

Fragments within S1 genes ((poly100)S1) of infectious bronchitis virus (IBV) strains ZJ971, M41 and SC021202 (SC) were subcloned into a prokaryotic expression vector and expressed in Escherichia coli. Monoclonal antibodies (mAbs) against the recombinant (poly100)S1 proteins were produced, characterized and used to analyse epitopes on the S1 subunit of IBV. Nine mAbs raising from the three (poly100)S1 proteins recognized five different epitopes of the S1 subunit, designated as S1-A, B, C, D and E. Epitopes S1-C and S1-D are common for the three IBV strains, while S1-A and S1-B exist on ZJ971 and M41 strains, and S1-E was a strain-specific epitope for SC strain. Immunocytochemistry indicated that all the mAbs to the (poly100)S1 proteins can react with the homologous S1 glycoprotein expressed in Vero cells. Moreover neutralization test demonstrated that only mAbs 6E2, 4F9 and 6G4 had neutralization activity for the homologous IBV. These mAbs to (poly100)S1 protein were potential candidates for detecting and distinguishing IBV strains, and also used to examine antigenic variation of the S1 protein.     

Development of a cytometric bead array screening tool for the simultaneous detection of pro-inflammatory cytokines in porcine plasma

Lipopolysaccharide (LPS) has been widely used as a model of immune challenge in pigs as it induces the immediate synthesis of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6, which trigger the production of the acute phase proteins (APPs) C-reactive protein (CRP), haptoglobin (Hp) and pig-Major Acute Phase Protein (pig-MAP). To measure secreted proteins in porcine plasma, specific and sensitive Enzyme-Linked Immuno Sorbent Assays (ELISAs) are well-suited to perform single parameter analysis, yet this approach is time-consuming and expensive for multi-parameter analyses. During the last decade, multiplex bead-based flow cytometry has been increasingly applied as it offers the opportunity to estimate protein ratios in a small sample volume. Cytometric bead array (CBA) is a flow cytometric application using a diversity of beads with unique fluorescence intensities, covalently coupled to a capture antibody for each protein of interest. Detection antibodies, either directly or indirectly conjugated to a fluorochrome, are added to accomplish the desired sandwich format. The aim of the present study was to develop a CBA 3-plex assay for the major pro-inflammatory cytokines TNF-α, IL-1β and IL-6, and an additional CBA 2-plex assay for the major APPs, CRP and pig-MAP, in porcine plasma. Results were compared to commercial ELISA kits. For the CBA 3-plex assay, the limits of detection (LODs) varied between 0.005 and 0.363 ng/mL, the intra- and inter-assay coefficients of variation were <10% and <16%, respectively. For TNF-α, IL-1β, IL-6 and pig-MAP, CBA time-concentration profiles similar to those obtained with commercial ELISAs were observed. In conclusion, the novel validated CBA 3-plex assay provides a fast and economical screening tool for determination of pro-inflammatory cytokine profiles in limited porcine plasma volumes. This tool will be applied to study the immunomodulatory properties of drugs in a porcine LPS inflammation model. This study also demonstrated the applicability of CBA for measurement of APPs in pigs, although a different combination than pig-MAP with CRP is recommended.     

Acute phase proteins assessment for an early selection of treatments in growing calves suffering from bronchopneumonia under field conditions

Blood samples were taken from calves with respiratory disease the first day of examination for determination of the serum concentration of haptoglobin, fibrinogen, alpha-2- and gamma-globulins, and albumin. A clinical examination was performed daily for the duration of the disease. The animals were retrospectively classified in two categories: those animals requiring no treatment or antibiotics alone (group A), and antibiotics associated to anti-inflammatory drugs (group B). The serum proteins were tested in order to check whether they were able to distinguish, on the first day of clinical examination, between calves requiring anti-inflammatory treatment (group B) or not (group A). About 80% of calves were properly classified in both groups by the combined use of the two serum proteins haptoglobin and fibrinogen: these two proteins, and especially haptoglobin, were useful for the identification of calves requiring an anti-inflammatory treatment.     

Analytical validation of commercially available methods for acute phase proteins quantification in pigs

The aim of this study was to validate commercially available methods for porcine haptoglobin (Hp), C-reactive protein (CRP), serum amyloid A (SAA) and major acute phase protein (Pig-MAP) determinations. Intra and inter assay coefficients of variation (CVs) were lower than 20% in all cases with exception of inter assay CVs for CRP and Pig-MAP assays with samples of low acute phase proteins concentration, and for SAA assay at any acute phase proteins concentration. All methods showed good linearity and detection limits were low enough to detect APPs levels in healthy animals. Hp and SAA were very affected by haemolysis. Lipaemia influenced mainly on SAA determination. Over 15-fold increase was observed in CRP and SAA concentrations after artificially induced inflammation by a single subcutaneous dose of turpentine, whereas Hp and Pig-MAP increased less than 5-fold.     

Acute phase protein concentrations in colostrum-deprived pigs immunized with subunit and commercial vaccines against Glässer's disease

Haemophilus parasuis is the etiological agent of Gl?sser's disease, which is characterized by fibrinous polyserositis, polyarthritis and meningitis in pigs. This study was focused on the characterization of the acute-phase response after immunization and infection of colostrum-deprived pigs with H. parasuis serovar 5, by measuring serum concentrations of three positive acute-phase proteins (APPs) (pig major acute-phase protein pig, MAP; haptoglobin, HPG; C-reactive protein, CRP) and one negative APP (apolipoprotein A-I, ApoA-I). Six experimental groups were established: a non-immunized but infected control group (CTL); two groups immunized with either a recombinant transferrin-binding protein (Tbp) A or TbpB fragment from H. parasuis Nagasaki strain (rTbpA and rTbpB, respectively); two groups immunized with native outer membrane proteins with affinity to porcine transferrin (NPAPT), one of them inoculated intramuscularly (NPAPTim) and the other intratracheally (NPAPTit), and the last group receiving a commercially available bacterin (PG). The greatest concentrations of the three positive APPs and the lowest concentration of the negative APP were detected in CTL group, as well as in those animals belonging to rTbpA or rTbpB groups that died in response to challenge. Significant differences (P<0.005) were found in these groups when comparing challenge with the following days after it. However, no significant differences were seen for the remaining vaccinated groups (NPAPTim, NPAPTit and PG), which were effectively protected against Gl?sser's disease. Therefore, APPs could be used as useful biomarkers for both evaluating disease progression and determining vaccination effectiveness.     

Serum haptoglobin: an objective indicator of experimentally-induced Salmonella infection in calves

Experimental models of Salmonella -induced gastroenteritis have previously relied on crude subjective clinical markers of infection to assess disease severity. The aim of this study was to investigate the possibility that changes in serum levels of the acute phase protein, haptoglobin, may be used as an objective, quantitative measurement of infection. Eight 3- to 4-week-old animals were challenged with a mixture of three Salmonella serotypes containing 6 x 10(10)bacteria and compared with five animals given a placebo preparation. Animals were monitored and characteristic clinical symptoms of infection; diarrhoeal scores, morbidity scores and rectal temperature, were recorded. Serum samples, from both animal groups, taken prior to challenge and again on days 1, 3, and 5 post-challenge, were analysed for haptoglobin levels using a direct serum binding assay. Prior to challenge, all 13 animals had normal levels of haptoglobin in their serum. By day 3 post-challenge six of eight animals challenged with Salmonella had abnormal serum haptoglobin levels (median level = 212 microg ml(-1)), while haptoglobin levels remained normal in placebo-challenged animals (median level = 0 microg ml(-1)). The change in haptoglobin levels during the 5-day observation period was statistically significant in the Salmonella -challenged animals (P = 0.0003, H = 16.477). Serum haptoglobin levels showed a statistical correlation with clinical measures of disease severity; diarrhoeal scores (P = 0.0015, H =8. 988), morbidity scores (P = 0.0004, H = 15.711) and rectal temperature (P = 0.0001, Z = 4.304). Thus, serum haptoglobin levels closely reflect the clinical symptoms of infection and are therefore a useful marker of infection severity in salmonellosis in calves.     

Comparison of methods used to diagnose generalized inflammatory disease in manatees (Trichechus manatus latirostris).

Manatees (Trichechus manatus latirostris) are afflicted with inflammatory and infectious disease secondary to human interaction, such as boat strike and entanglement, as well as "cold stress syndrome" and pneumonia. White-blood-cell count and fever, primary indicators of systemic inflammation in most species, are insensitive in diagnosing inflammatory disease in manatees. Acute phase-response proteins, such as haptoglobin and serum amyloid A, have proven to be sensitive measures of inflammation/infection in domestic large animal species. This study assessed diagnosis of generalized inflammatory disease by different methods including total white-blood-cell count, albumin: globulin ratio, gel electrophoresis analysis, C-reactive protein, alpha, acid glycoprotein, haptoglobin, fibrinogen, and serum amyloid A. Samples were collected from 71 apparently healthy and 27 diseased animals during diagnostic medical examination. Serum amyloid A, measured by ELISA, followed by albumin:globulin ratio, measured by plasma gel electrophoresis, were most sensitive in diagnosing inflammatory disease, with diagnostic sensitivity and specificity of approximately 90%. The reference interval for serum amyloid A is <10-50 microg/ml with an equivocal interval of 51-70 microg/ml. The reference interval for albumin:globulin ratio by plasma gel electrophoresis is 0.7-1.1. Albumin: globulin ratio, calculated using biochemical techniques, was not accurate due to overestimation of albumin by bromcresol green dye-binding methodology. Albumin:globulin ratio, measured by serum gel electrophoresis, has a low sensitivity of 15% due to the lack of fibrinogen in the sample. Haptoglobin, measured by hemoglobin titration, had a reference interval of 0.4-2.4 mg/ml, a diagnostic sensitivity of 60%, and a diagnostic specificity of 93%. The haptoglobin assay is significantly affected by hemolysis. Fibrinogen, measured by heat precipitation, has a reference interval of 100-400 mg/dl, a diagnostic sensitivity of 40%, and a diagnostic specificity of 95%.     

C-reactive protein measurement in canine saliva.

An established time-resolved immunofluorometric assay designed for measurement of C-reactive protein (CRP) in canine blood was evaluated and validated for use in canine saliva. C-reactive protein was measured in saliva specimens from 5 healthy dogs before and after the injection of casein and in 37 dogs with different disease conditions. The analytical and functional limits of detection were 0.000053 microg/ml and 0.0091 microg/ml, respectively, and intra- and interassay coefficients of variation ranged between 6.7-9.9% and 8.5-16.5%, respectively. A recovery experiment showed no significant disagreement between detected values and expected ones, and saliva CRP concentration was measured in a linear and proportional manner. A positive correlation was found between CRP levels obtained in saliva and serum samples in the experimental (R2 = 0.76) and clinical studies (R2 = 0.70). The assay was able to detect significant differences between salivary CRP levels in healthy dogs and dogs with inflammatory processes. These results suggest that saliva can be used for CRP measurement in dogs. The use of saliva presents the advantage of an easier and less stressful sampling method for the animals, which might be performed outside of hospital environments.     

Molecular characterization of a myosin alkali light chain-like protein, a "concealed" antigen from the hard tick Haemaphysalis qinghaiensis

There should be some differences between antibodies generated by feeding ticks on animals and those derived by immunizing animals with tick extracts. Here, we found serum collected from sheep immunized with Haemaphysalis qinghaiensis salivary gland extracts could detect two more protein bands with molecular weights of 22 and 37 kDa (P22 and P37) on Western blots of extracts of tick salivary glands than serum from tick infected animals. Rabbit anti-H. qinghaiensis differential protein immune serum was then generated from P22 and P37 and was used to immunoscreen a cDNA library constructed from salivary glands, Malpighian tubules and ovaries of partially engorged H. qinghaiensis. A cDNA contains an open reading frame of 483 bp that codes for 160 amino acid residues with a coding capacity of 18 kDa was cloned and designated Hq02. Expression analysis by RT-PCR showed that this gene is expressed in salivary glands, midguts, other organs and different developmental stages of H. qinghaiensis. The predicted amino acid sequence of the Hq02 gene had high homology to some known myosin alkali light chain (MLC) proteins. A fusion protein consisting of 130 amino acids of Hq02 protein and 335 amino acids of T7 gene 10 protein was expressed in Escherichia coli and used to immunize sheep. Western blot showed that only rabbit anti-H. qinghaiensis differential protein immune serum could recognize the expressed Hq02 protein, while rabbit anti-H. qinghaiensis saliva immune could not. This proved Hq02 protein was a "concealed" antigen. Immunization with the recombinant Hq02 conferred a 21.8% reduction of engorgement weight for adult female ticks that fed on the immunized sheep. This is the first report of tick myosin alkali light chain and the function of this protein is discussed.     

Non-immunoglobulin defense factors in canine saliva and effects of a tooth gel containing antibacterial enzymes

Selected innate: non-immunoglobulin defense factors in canine saliva were characterized and quantitated. The samples from dogs showed increased pH, higher lysozyme and salivary peroxidase activities, but lower hypothiocyanite concentration and myeloperoxidase activity when compared with human saliva. Secondly, a 1-month clinical pilot study was performed using a commercial tooth gel to determine acute and long-term changes in salivary host defense proteins. Daily application of the tooth gel did not substantially affect the concentrations of these factors in dogs with normal salivation. Our results suggest that canine saliva may be similar to human saliva, comprised of both immune and non-immune antimicrobial factors. However, as in humans, oral administration of antimicrobial proteins as reported here does not seem to benefit dogs with normal saliva secretion. Products such as the tooth gel evaluated in this study may benefit dogs with xerostomia or other clinical conditions causing decreased saliva production.     

Cloning of pig parotid secretory protein gene upstream promoter and the establishment of a transgenic mouse model expressing bacterial phytase for agricultural phosphorus pollution control

This study examined the feasibility of using the promoter of the pig parotid secretory protein (PSP) gene for expression of the phytase transgene in mouse models. The pig parotid secretory protein gene is specifically expressed at high levels in the salivary glands. The 10-kb upstream promoter region of the gene necessary for tissue-specific expression has been identified. We have constructed phytase transgenes composed of the appA phytase gene from Escherichia coli driven by the upstream promoter region of the pig PSP gene with a 3' tail of either bovine growth hormone or the pig PSP gene polyadenylation signal. Transgenic mouse models with the construct showed that the upstream region of the pig PSP gene is sufficient for directing the expression of phytase transgenes in the saliva. Expression of salivary phytase reduced fecal phytate by 8.5 and 12.5% in 2 transgenic mouse lines, respectively. These results suggest that the expression of phytase in salivary glands of monogastric animals offers a promising biological approach to relieve the requirement for dietary phosphate supplements and to reduce phosphorus pollution from animal agriculture.     

Dual-label time-resolved fluoroimmunoassay for simultaneous quantification of haptoglobin and C-reactive protein in meat juice from pigs

A new method was developed to simultaneously measure 2 acute-phase proteins (APPs) by time-resolved immunofluorometry. The assay, based on double-label quantification of haptoglobin (Hp) and C-reactive protein (CRP) in meat juice samples from pigs, was constructed by use of a combination of europium and samarium chelate lanthanides as labels. Meat juice samples from 154 pigs were used for analytic and clinical validation of the assay through determination of precision, accuracy, limit of detection, and quantification. The analytic performance of the assay was satisfactory, with good intra-assay and interassay precision and accuracy. The levels of Hp and CRP were increased in the meat juice samples of diseased animals compared with healthy ones. According to the results, higher sensitivity could be achieved if the cut-off values of both proteins were taken into account for clinical relevance rather than used individually. Since the dual assay saved both time and sample, it could be used as a rapid and sensitive screening test in porcine production.