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C-reactive protein quantification in porcine saliva: A minimally invasive test for pig health monitoring
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The Veterinary Journal The Veterinary Journal 181 (2009) 261–265 www.elsevier.com/locate/tvjl
C-reactive protein quantification in porcine saliva: A minimally invasive test for pig health monitoring A.M. Gutie´rrez a, S. Martı´nez-Subiela a, P.D. Eckersall b, J.J. Cero´n a,* b
a Department of Animal Medicine and Surgery, University of Murcia, 30100 Espinardo, Murcia, Spain Division of Animal Production and Public Health, Institute of Comparative Medicine, Faculty of Veterinary Medicine, University of Glasgow, Glasgow G61 1QH, UK
Accepted 28 March 2008
Abstract Study objectives were to investigate whether C-reactive protein (CRP) in pig saliva could be quantified using an adapted, timeresolved immunofluorometry assay (TR-IFMA), and to determine whether the assay could distinguish healthy from diseased animals. The test method had intra- and inter-assay coefficients of variation of 5.75% and 9.73%, respectively, the limit of detection was 0.47 ng/ mL and the coefficient of determination was 0.98. Analysis of CRP concentrations in paired serum and saliva samples from 50 pigs gave a positive correlation (r = 0.702, P < 0.01) and the salivary CRP concentration was able to distinguish healthy from diseased animals in 62 samples from pigs with naturally occurring or experimentally-induced inflammation. The results suggest that this minimally invasive, straightforward and sensitive assay may be useful in pig health and welfare monitoring. Ó 2008 Elsevier Ltd. All rights reserved. Keywords: C-reactive protein; Saliva; Time-resolved immunofluorometry assay (TR-IFMA); Pig
Introduction C-reactive protein (CRP) is an acute phase serum protein and a mediator of innate and adaptive immunity (Chomdej et al., 2004). The plasma concentration of CRP has been used clinically in pigs to monitor inflammation and infection (Kuji et al., 2007) with increases of up to 38-fold reported during acute inflammation (Parra et al., 2006). Non-invasive sampling methods, such as the collection of saliva, that cause minimal discomfort to the animal are in increasing demand given enhanced awareness of animal welfare, and such methods are particularly useful when studying animal stress (Guzik et al., 2006). In addition to being more straightforward and economical to obtain than blood, saliva has the added advantage of being easier to handle for diagnostic purposes because it does not clot (Wong, 2006). Saliva can be collected in geographically remote sites by per*
Corresponding author. Tel.: +34 968364722; fax: +34 968364147. E-mail address: [email protected] (J.J. Cero´n).
1090-0233/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2008.03.021
sonnel with limited training and, with appropriate collection devices, is stable at room temperatures for several weeks (Hofman, 2001). Multiple analytes such as haptoglobin (Hiss et al., 2003), cortisol (Ruis et al., 1997), oestrone sulfate (Ohtaki et al., 1997), progesterone (Moriyoshi et al., 1996), IgG (Devillers et al., 2004) and IgA (Van der Stede et al., 2001) have been measured in porcine saliva. The drawback that analytes in saliva are generally present in much smaller amounts than in serum (Wong, 2006) can be circumvented by using highly sensitive detection techniques such as a time-resolved immunofluorometry assay (TR-IFMA). This technology uses highly specific lanthanide chelate labels that facilitate the use of non-competitive immunoassays with superior detection limits and dynamic ranges (Lo¨vgren et al., 1996). The objectives of this study were to investigate if CRP in pig saliva could be quantified using an adapted TRIFMA, and whether the assay could be used to distinguish healthy from diseased animals under experimental and ‘field’ conditions.
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Materials and methods Sampling procedures Saliva was collected using Salivette tubes (Sarstedt) containing a sponge instead of a cotton swab (because these were less absorbent and released more saliva following centrifugation). The sampled pigs were allowed to chew the sponge, which was clipped to a flexible thin metal rod, until thoroughly moist. The sponges were then placed in test tubes and centrifuged at 3000 g for 10 min. The saliva samples (approximately 0.5– 1.0 mL per sponge) were removed and stored at 20 °C until analysis. To obtain serum, blood samples were allowed to clot for 1 h at room temperature and the serum was separated by centrifugation (2000 g for 15 min). Serum samples were frozen at 20 °C until time of analysis. All procedures involving animals were approved by the Murcia University Ethics Committee.
Immunoassay procedure C-reactive protein concentrations were measured in saliva and serum using a TR-IFMA, as previously described (Martı´nez-Subiela et al., 2007). In brief, serum and saliva samples were diluted 1:2000 and 1:4, respectively. Biotinylated antibodies were pipetted into DELFIA streptavidin microtitration strips (Perkin–Elmer Life and Analytical Sciences) and were incubated at room temperature for 45 min. After washing the plate four times with DELFIA wash buffer (Perkin–Elmer Life Sciences), samples or standards were added and a further 45 min incubation was carried out at room temperature. Following the addition of 200 lL of europium-labelled antibody solution to each well, a further 45 min incubation and a final washing step, 200 lL of enhancement solution (Perkin–Elmer Life Sciences) were added followed by 20 min incubation at room temperature. The enhanced fluorescence, that was proportional to the quantity of CRP in the sample, was then measured in a Victor 1420 multilabel counter (Perkin–Elmer Lifesciences). This immunoassay has a patent deposit (Spanish Patent Application No. P200401268).
Analytical validation Assessment of intra-assay precision The intra-assay precision, expressed as the coefficient of variation (CV), was calculated by measuring two pools of saliva samples (samples from six animals for each group) with high (h-CRP) and low (l-CRP) concentrations of CRP, respectively, six times in a single analytical run. The same two sample pools were used to determine the inter-assay precision by analysing them on five different days within a 15 day period. The samples were frozen in aliquots, and vials were only thawed as required for each analytical run in order to prevent potential variation as a result of repeated freeze–thaw cycles. Samples in the l-CRP pool (median of 69 lg/mL in serum) were obtained from pigs without clinical signs of disease and that were serologically negative for porcine reproductive and respiratory syndrome virus (PRRSV), Aujeszky’s disease virus and porcine circovirus type-2. Samples in the h-CRP pool (median of 106 lg/mL in serum) were taken from pigs exhibiting clinical signs of different inflammatory-based diseases (as described in clinical validation study below). To investigate the stability of CRP protein in saliva, the aliquoted pools of saliva used for the precision assay were measured 0 h, 3, 7 and 15 days and 1, 1.5 and 2 months following sample collection. All saliva samples were stored at 20 °C until assayed. Assessment of assay accuracy Linearity under dilution was used to evaluate assay accuracy. Two saliva samples with h-CRP levels (1136 ng/mL and 490 ng/mL for samples 1 and 2, respectively) were diluted by 0%, 6.25%, 12.5%, 25% and 50%
using assay buffer, and the CRP concentration was measured in duplicate for each dilution. Curves relating the measured to the expected CRP concentration were constructed. Fifty saliva and serum samples from the 20 healthy and 42 diseased pigs from the clinical validation study were used to assess the correlation between serum and salivary CRP concentrations. Assessment of limit of detection The limit of detection (LOD) was assessed by analysing a ‘zero calibrator’ (assay buffer) 10 times and was calculated as the mean concentration obtained plus two standard deviations (SD).
Clinical validation study To assess salivary CRP concentrations during the acute phase reaction, inflammation was experimentally induced in two pigs by giving each two, 4 mL subcutaneous injections of turpentine oil in each front flank, as previously described (Martı´nez-Subiela et al., 2007). The CRP concentrations in saliva and serum were compared at 0, 24, 48 and 72 h after injection. The ability of the assay to detect naturally occurring inflammation in pigs was assessed by collecting saliva from male, Duroc-cross animals from a local finishing unit of 1800 animals. These pigs were sampled at an average of 190 days of age and at approximately 100 kg bodyweight. The sampled pigs were divided into two groups. Group 1 consisted of 20 pigs without clinical signs of disease and serologically negative for PRRSV, Aujeszky’s disease virus, and porcine circovirus type-2. Group 2 consisted of 40 pigs that presented with disease conditions such as gastric ulceration (n = 13), depressed growth rate with diarrhoea (n = 15), bronchopneumonia with nasal discharge (n = 6) and multiple abscessation (n = 6).
Statistical analysis Intra- and inter-assay CVs and detection limits were calculated using routine descriptive statistical procedures with Microsoft Excel 2000. Ordinary regression analysis was used to investigate ‘linearity under dilution’ and CRP measurements in saliva and serum were compared using a statistical software programme (14th edition SPSS Inc.). C-reactive protein measurements obtained from the clinical validation study were evaluated for normality of distribution using the Kolmogorov– Smirnov statistic. As the results did not meet normal distribution criteria, a Mann–Whitney non-parametric test was used to compare salivary CRP concentrations from clinically normal pigs with those from diseased animals. Statistical significance for differences between diseased and healthy pigs was set at P < 0.05 and was calculated using the SPSS programme detailed above.
Results Analytical validation Assessment of intra-assay precision The intra-assay CV was 6.84% for the pool with h-CRP and 4.57% for the pool with l-CRP. The CVs obtained for inter-assay precision were higher than those for intra-assay precision, ranging from 7.63–11.83% (Table 1). When the stability study was carried out, no variations in salivary CRP levels were detected during the first 7 days. A decrease of 38.6% in salivary CRP values was found in the h-CRP pool (Fig. 1) at day 15 and the CRP concentration remained at this level until the end of the study 2 months later. No variations were found in the l-CRP pool.
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Table 1 Assessment of the precision of the time-resolved immunofluorometry assay (TR-IFMA) for the measurement of C-reactive protein (CRP) in porcine saliva Saliva pool
l-CRP h-CRP Mean
Intra-assay
Inter-assay
Mean (ng/mL)
SD
CV (%)
Mean (ng/mL)
SD
CV (%)
29 475 238
1.34 32.50 16.92
4.57 6.84 5.75
32 488 260
3.78 37.24 20.51
11.83 7.63 9.73
SD: standard deviation; CV: coefficient of variation.
Fig. 1. Stability of porcine CRP in saliva pools of high (h-CRP) and low (l-CRP) concentration, stored at 20 °C during a 2 month period as measured by time-resolved immunofluorometry assay.
Fig. 2. Investigation of linearity under dilution of two saliva samples containing high levels of CRP (1136 ng/mL and 490 ng/mL, respectively). r: correlation coefficient.
Assessment of assay accuracy The dilution of two saliva samples with h-CRP concentrations resulted in linear regression equations (Fig. 2), where ‘x’ represents the expected CRP level at the particular dilution and ‘y’ represents the measured CRP level. The correlation coefficients were 0.991 and 0.990, respectively. When the CRP concentrations in the saliva and serum of the 20 healthy and 30 diseased pigs were compared, a significant positive correlation was identified with a coefficient of correlation of r = 0.726 (P < 0.001) (Fig. 3). Serum CRP levels were almost 323 times higher than those in saliva, as indicated by the regression equation y = 0.0031x 0.06.
Fig. 3. Correlation of CRP concentrations (lg/mL) in serum and saliva as measured by time-resolved immunofluorometry assay (n = 50). r: correlation coefficient.
Assessment of limit of detection The LOD of the assay was 0.47 ng/mL which corresponded to the mean value of the CRP concentration plus two SD of 10 replicates of the assay buffer. Clinical validation study The pigs with experimentally-induced inflammation had significantly increased serum and salivary CRP (Fig. 4). The median serum CRP concentration for both animals increased from 53 lg/mL before injection to 275 lg/mL at 24 h, a 5.21-fold increase. The original value had increased 9.8-fold at 48 h and peaked at 570 lg/mL, a 10.80-fold increase at 72 h. The median salivary CRP level
Fig. 4. Salivary and serum CRP concentrations in two pigs (1 and 2) with experimentally-induced inflammation.
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Fig. 5. Salivary CRP levels in healthy pigs (n = 20) and in pigs with inflammatory disease (n = 40). The plot shows median (line within box), 25th and 75th percentiles (box), 5th and 95th percentiles (whiskers) and outliers (°).
in pigs increased from 22 ng/mL to 86 ng/mL, 24 h after injection, a 3.93-fold increase. At 48 h this value had increased 6.39-fold to 140 ng/mL and peaked at 393 ng/ mL, a 17.95-fold increase, at 72 h. Significantly higher (P < 0.001) salivary CRP concentrations were observed in diseased relative to clinically normal animals (Fig. 5). Median CRP concentrations of 31 ng/mL (25th and 75th percentiles of 24.24 and 38.14, respectively) and of 123 ng/mL (25th and 75th percentiles of 74.37 and 241.94, respectively) were found in healthy and diseased animals, respectively. Discussion The scientific assessment of the welfare status of animals has gained increasing prominence in recent years (OIE, 2007). In this context the measurement of indicators of an animal’s welfare status such as acute phase protein (APP) levels in samples such as saliva that can be obtained with minimal stress on the sampled animal are particularly attractive (Eckersall, 2004). Furthermore, given the minimally invasive nature of saliva sampling, such an approach has the further advantages that samples can be collected by individuals following modest levels of training, and repeated sampling over short time-intervals can be carried out which facilitates ongoing animal monitoring (Kaufman and Lamster, 2002). A number of disease conditions in humans result in increases in analytes such as CRP in saliva that can be used in clinical monitoring (Christodoulides et al., 2005; Wong, 2006). The measurement of APPs such as canine CRP (Parra et al., 2005) and porcine haptoglobin (Hiss et al., 2003) in saliva have been used in a similar context in veterinary medicine. The main limitation to the widespread use of this approach is the insufficient sensitivity of detection of most
current commercial assay systems for salivary APP detection. For example, CRP could not be detected in pig saliva using a commercial enzyme-linked immunosorbent assay (ELISA) (Llamas Moya et al., 2003). However the development of new, more sensitive detection systems such as TRIFMA can overcome these limitations. The results of the present study indicate that salivary CRP concentrations are some 322-fold lower than in serum with values in the order of ng/mL. In this study, the problems associated with the detection of low levels of CRP in porcine saliva were resolved by using europium chelate labels and timeresolved fluorometry. The three standard criteria for immunoassay validation, namely precision, accuracy and limit of detection, were determined for the TR-IFMA in this study, while assessment of the specificity of the assay was as described previously (Martı´nez-Subiela et al., 2007). Analytical validation indicated that porcine salivary CRP concentrations could be reliably measured using the TR-IFMA and both intraand inter-assay CVs were <12%. As far as the authors are aware, there is no previous validated method for the detection of CRP in porcine saliva, however the precision assessment results were equivalent to those found for CRP quantification in dog saliva by TR-IFMA (Parra et al., 2005). Furthermore, the CVs for the porcine salivary CRP measurements were lower than those obtained in the validation of commercially available assays for serum CRP (Tecles et al., 2007). The LOD of the method was very low, demonstrating the high sensitivity of the TR-IFMA. This LOD was significantly lower than that reported for other commercially available kits (Kjelgaard-Hansen et al., 2007; Tecles et al., 2007). The median concentrations of CRP in the saliva of both healthy and diseased pigs were lower than the LOD of commercial kits but greater than the LOD of the method validated in this study, so that this technique has applications in quantifying salivary CRP concentrations in this species. In human medicine, conventional ELISAs are unable to detect salivary CRP levels in the majority of the samples so that other techniques such as microchip assay systems have been used as an alternative (Christodoulides et al., 2005). Analysis of assay accuracy using linearity under dilution indicated a linear relationship over a wide range of salivary CRP concentrations and the assay response. Despite the differences in the CRP concentrations in saliva and serum, the correlations found, suggest measurement of salivary CRP may be a suitable alternative to the measurement of serum CRP. The study found that saliva samples could be stored at 20 °C for 7 days after collection without compromising result validity. To the authors’ knowledge there are no previous studies of the stability of CRP in porcine saliva and this stability gives an assay based on saliva more flexibility than one performed on whole blood where porcine CRP is stable for 3 days if refrigerated (Martı´nez-Subiela et al., 2007). The fall in salivary CRP concentration after 15 days at 20 °C suggests analysis should be undertaken within
A.M. Gutie´rrez et al. / The Veterinary Journal 181 (2009) 261–265
this timescale, however, it may be possible to prolong the stability of CRP in saliva by adding protease inhibitors or serum albumin. The results of the clinical validation study showed significant differences in the median salivary CRP concentrations between healthy pigs and pigs with a variety of inflammatory conditions. Further studies of larger number of animals will be necessary to establish the normal range of CRP concentrations of healthy animals at different ages and of varying production status, and to evaluate the sensitivity of salivary CRP in detecting animals with inflammatory disease. Although the injection of turpentine oil increased both serum and salivary CRP concentrations, these differences could not be given statistical significance as for ethical reasons only two pigs were used. The greatest increases occurred in serum between 24 and 48 h after injection and between 48 and 72 h in saliva. This increase in salivary CRP was almost 18-fold relative to the almost 11-fold increase in serum. These differences could be due to several factors that influence the composition of saliva such as flow rate, degree of hydration, body composition, plasma composition or even possible local synthesis of salivary CRP. Further studies will be required to elucidate this issue. Conclusions The results of this study indicate that if highly sensitive detection systems such as the TR-IFMA are used, measurement of salivary CRP in pigs can provide a straightforward, flexible and minimally invasive method of detecting inflammatory-based disease in this species that can be used as a tool in animal health and welfare monitoring. Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper. Acknowledgement This work was supported by a grant from the Spanish Ministry of Education and Science (AGL 2006-05701). References Christodoulides, N., Mohanty, S., Miller, C.S., Langub, M.C., Floriano, P.N., Dharshan, P., Ali, M.F., Bernard, B., Romanovicz, D., Anslyn, E., Fox, P.C., McDevitt, J.T., 2005. Application of microchip assay system for the measurement of C-reactive protein in human saliva. Lab on a Chip 5, 261–269. Chomdej, S., Ponsuksili, S., Schellander, K., Wimmers, K., 2004. Detection of SNPs and linkage and radiation hybrid mapping of the porcine C-reactive protein (CRP) gene. Animal Genetics 35, 469–470. Devillers, N., Farmer, C., Mounier, A.M., Le Dividich, J., Prunier, A., 2004. Hormones, IgG and lactose changes around parturition in
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plasma, and colostrum or saliva of multiparous sows. Reproduction, Nutrition, Development 44, 381–396. Eckersall, P.D., 2004. The time is right for acute phase protein assays. The Veterinary Journal 168, 3–5. Guzik, A.C., Matthews, J.O., Kerr, B.J., Bidner, T.D., Southern, L.L., 2006. Dietary tryptophan effects on plasma and salivary cortisol and meat quality in pigs. Journal of Animal Science 84, 2251–2259. Hiss, S., Knura-Deszcza, S., Regula, G., Hennies, M., Gymnich, S., Peterse, B., Sauerwein, H., 2003. Development of an enzyme immuno assay for the determination of porcine haptoglobin in various body fluids: testing the significance of meat juice measurements for quality monitoring programs. Veterinary Immunology and Immunopathology 96, 73–82. Hofman, L.F., 2001. Human saliva as a diagnostic specimen. The Journal of Nutrition 131, 1621S–1625S. Kaufman, E., Lamster, I.B., 2002. The diagnostic applications of saliva – a review. Critical Reviews in Oral Biology and Medicine 13, 197–212. Kjelgaard-Hansen, M., Martı´nez-Subiela, S., Petersen, H.H., Jensen, A.L., Cero´n, J.J., 2007. Evaluation and comparison of two immunoturbidimetric assays for the heterologous determination of porcine serum C-reactive protein. The Veterinary Journal 173, 571– 577. Kuji, T., Masaka, T., Li, Li, Cheung, A.K., 2007. Expression of C-reactive protein in myointimal hyperplasia in a porcine arteriovenous graft model. Nephrology, Dialysis, Transplantation 22, 2469–2475. Lo¨vgren, T., Merio¨, L., Mitrunen, K., Ma¨kinen, M.L., Ma¨kela¨, M., Blomberg, K., Palenius, T., Pettersson, K., 1996. One-step all-in-one dry reagent immunoassays with fluorescent europium chelate label and time-resolved fluorometry. Clinical Chemistry 42, 1196–1201. Llamas Moya, S., Boyle, L.A., Lynch, P.B., Arkins, S., 2003. Age-related changes in pro-inflammatory cytokines, acute phase proteins and cortisol concentrations in neonatal piglets. Neonatology 91, 44–48. Martı´nez-Subiela, S., Eckersall, P.D., Campbell, F.M., Parra, M.D., Fuentes, P., Cero´n, J.J., 2007. A time-resolved immunofluorometric assay for porcine C-reactive protein quantification in whole blood. Luminescence 22, 171–176. Moriyoshi, M., Tanaka, Y., Nakao, T., Kawata, K., 1996. Early pregnancy diagnosis in the sow by saliva progesterone measurement using a bovine milk progesterone qualitative test EIA kit. The Journal of Veterinary Medical Science 58, 737–741. Ohtaki, T., Moriyoshi, M., Nakada, K., Kawata, K., 1997. Radioimmunoassay of saliva estrone sulfate in pregnant sows. The Journal of Veterinary Medical Science 59, 759–763. OIE, 2007. Terrestrial Animal Health Code. (accessed 02.10.07.) Parra, M.D., Tecles, F., Martı´nez-Subiela, S., Cero´n, J.J., 2005. C-reactive protein measurement in canine saliva. Journal of Veterinary Diagnostic Investigation 17, 139–144. Parra, M.D., Fuentes, P., Tecles, F., Martı´nez-Subiela, S., Martı´nez, J.S., Mun˜oz, A., Cero´n, J.J., 2006. Porcine acute phase protein concentration in different diseases in field conditions. Journal of Veterinary Medicine, B 53, 488–493. Ruis, M.A.W., Te Brake, J.H.A., Engel, B., Dinand Ekkel, E., Buist, W.G., Blokhuis, H.J., Koolhaas, J.M., 1997. The circadian rhythm of salivary cortisol in growing pigs: effects of age, gender and stress. Physiology and Behavior 62, 623–630. Tecles, F., Fuentes, P., Martı´nez-Subiela, S., Parra, M.D., Mun˜oz, A., Cero´n, J.J., 2007. Analytical validation of commercially available methods for acute phase proteins quantification in pigs. Research in Veterinary Science 83, 133–139. Van der Stede, Y., Cox, E., Van der broeck, W., Goddeeris, B.M., 2001. Enhanced induction of the IgA response in pigs by calcitriol alters intramuscular immunization. Vaccine 19, 1870–1878. Wong, D.T., 2006. Salivary diagnostics powered by nanotechnologies, proteomics and genomics. Journal of the American Dental Association 137, 313–321.
The Veterinary Journal The Veterinary Journal 181 (2009) 261–265 www.elsevier.com/locate/tvjl
C-reactive protein quantification in porcine saliva: A minimally invasive test for pig health monitoring A.M. Gutie´rrez a, S. Martı´nez-Subiela a, P.D. Eckersall b, J.J. Cero´n a,* b
a Department of Animal Medicine and Surgery, University of Murcia, 30100 Espinardo, Murcia, Spain Division of Animal Production and Public Health, Institute of Comparative Medicine, Faculty of Veterinary Medicine, University of Glasgow, Glasgow G61 1QH, UK
Accepted 28 March 2008
Abstract Study objectives were to investigate whether C-reactive protein (CRP) in pig saliva could be quantified using an adapted, timeresolved immunofluorometry assay (TR-IFMA), and to determine whether the assay could distinguish healthy from diseased animals. The test method had intra- and inter-assay coefficients of variation of 5.75% and 9.73%, respectively, the limit of detection was 0.47 ng/ mL and the coefficient of determination was 0.98. Analysis of CRP concentrations in paired serum and saliva samples from 50 pigs gave a positive correlation (r = 0.702, P < 0.01) and the salivary CRP concentration was able to distinguish healthy from diseased animals in 62 samples from pigs with naturally occurring or experimentally-induced inflammation. The results suggest that this minimally invasive, straightforward and sensitive assay may be useful in pig health and welfare monitoring. Ó 2008 Elsevier Ltd. All rights reserved. Keywords: C-reactive protein; Saliva; Time-resolved immunofluorometry assay (TR-IFMA); Pig
Introduction C-reactive protein (CRP) is an acute phase serum protein and a mediator of innate and adaptive immunity (Chomdej et al., 2004). The plasma concentration of CRP has been used clinically in pigs to monitor inflammation and infection (Kuji et al., 2007) with increases of up to 38-fold reported during acute inflammation (Parra et al., 2006). Non-invasive sampling methods, such as the collection of saliva, that cause minimal discomfort to the animal are in increasing demand given enhanced awareness of animal welfare, and such methods are particularly useful when studying animal stress (Guzik et al., 2006). In addition to being more straightforward and economical to obtain than blood, saliva has the added advantage of being easier to handle for diagnostic purposes because it does not clot (Wong, 2006). Saliva can be collected in geographically remote sites by per*
Corresponding author. Tel.: +34 968364722; fax: +34 968364147. E-mail address: [email protected] (J.J. Cero´n).
1090-0233/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2008.03.021
sonnel with limited training and, with appropriate collection devices, is stable at room temperatures for several weeks (Hofman, 2001). Multiple analytes such as haptoglobin (Hiss et al., 2003), cortisol (Ruis et al., 1997), oestrone sulfate (Ohtaki et al., 1997), progesterone (Moriyoshi et al., 1996), IgG (Devillers et al., 2004) and IgA (Van der Stede et al., 2001) have been measured in porcine saliva. The drawback that analytes in saliva are generally present in much smaller amounts than in serum (Wong, 2006) can be circumvented by using highly sensitive detection techniques such as a time-resolved immunofluorometry assay (TR-IFMA). This technology uses highly specific lanthanide chelate labels that facilitate the use of non-competitive immunoassays with superior detection limits and dynamic ranges (Lo¨vgren et al., 1996). The objectives of this study were to investigate if CRP in pig saliva could be quantified using an adapted TRIFMA, and whether the assay could be used to distinguish healthy from diseased animals under experimental and ‘field’ conditions.
262
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Materials and methods Sampling procedures Saliva was collected using Salivette tubes (Sarstedt) containing a sponge instead of a cotton swab (because these were less absorbent and released more saliva following centrifugation). The sampled pigs were allowed to chew the sponge, which was clipped to a flexible thin metal rod, until thoroughly moist. The sponges were then placed in test tubes and centrifuged at 3000 g for 10 min. The saliva samples (approximately 0.5– 1.0 mL per sponge) were removed and stored at 20 °C until analysis. To obtain serum, blood samples were allowed to clot for 1 h at room temperature and the serum was separated by centrifugation (2000 g for 15 min). Serum samples were frozen at 20 °C until time of analysis. All procedures involving animals were approved by the Murcia University Ethics Committee.
Immunoassay procedure C-reactive protein concentrations were measured in saliva and serum using a TR-IFMA, as previously described (Martı´nez-Subiela et al., 2007). In brief, serum and saliva samples were diluted 1:2000 and 1:4, respectively. Biotinylated antibodies were pipetted into DELFIA streptavidin microtitration strips (Perkin–Elmer Life and Analytical Sciences) and were incubated at room temperature for 45 min. After washing the plate four times with DELFIA wash buffer (Perkin–Elmer Life Sciences), samples or standards were added and a further 45 min incubation was carried out at room temperature. Following the addition of 200 lL of europium-labelled antibody solution to each well, a further 45 min incubation and a final washing step, 200 lL of enhancement solution (Perkin–Elmer Life Sciences) were added followed by 20 min incubation at room temperature. The enhanced fluorescence, that was proportional to the quantity of CRP in the sample, was then measured in a Victor 1420 multilabel counter (Perkin–Elmer Lifesciences). This immunoassay has a patent deposit (Spanish Patent Application No. P200401268).
Analytical validation Assessment of intra-assay precision The intra-assay precision, expressed as the coefficient of variation (CV), was calculated by measuring two pools of saliva samples (samples from six animals for each group) with high (h-CRP) and low (l-CRP) concentrations of CRP, respectively, six times in a single analytical run. The same two sample pools were used to determine the inter-assay precision by analysing them on five different days within a 15 day period. The samples were frozen in aliquots, and vials were only thawed as required for each analytical run in order to prevent potential variation as a result of repeated freeze–thaw cycles. Samples in the l-CRP pool (median of 69 lg/mL in serum) were obtained from pigs without clinical signs of disease and that were serologically negative for porcine reproductive and respiratory syndrome virus (PRRSV), Aujeszky’s disease virus and porcine circovirus type-2. Samples in the h-CRP pool (median of 106 lg/mL in serum) were taken from pigs exhibiting clinical signs of different inflammatory-based diseases (as described in clinical validation study below). To investigate the stability of CRP protein in saliva, the aliquoted pools of saliva used for the precision assay were measured 0 h, 3, 7 and 15 days and 1, 1.5 and 2 months following sample collection. All saliva samples were stored at 20 °C until assayed. Assessment of assay accuracy Linearity under dilution was used to evaluate assay accuracy. Two saliva samples with h-CRP levels (1136 ng/mL and 490 ng/mL for samples 1 and 2, respectively) were diluted by 0%, 6.25%, 12.5%, 25% and 50%
using assay buffer, and the CRP concentration was measured in duplicate for each dilution. Curves relating the measured to the expected CRP concentration were constructed. Fifty saliva and serum samples from the 20 healthy and 42 diseased pigs from the clinical validation study were used to assess the correlation between serum and salivary CRP concentrations. Assessment of limit of detection The limit of detection (LOD) was assessed by analysing a ‘zero calibrator’ (assay buffer) 10 times and was calculated as the mean concentration obtained plus two standard deviations (SD).
Clinical validation study To assess salivary CRP concentrations during the acute phase reaction, inflammation was experimentally induced in two pigs by giving each two, 4 mL subcutaneous injections of turpentine oil in each front flank, as previously described (Martı´nez-Subiela et al., 2007). The CRP concentrations in saliva and serum were compared at 0, 24, 48 and 72 h after injection. The ability of the assay to detect naturally occurring inflammation in pigs was assessed by collecting saliva from male, Duroc-cross animals from a local finishing unit of 1800 animals. These pigs were sampled at an average of 190 days of age and at approximately 100 kg bodyweight. The sampled pigs were divided into two groups. Group 1 consisted of 20 pigs without clinical signs of disease and serologically negative for PRRSV, Aujeszky’s disease virus, and porcine circovirus type-2. Group 2 consisted of 40 pigs that presented with disease conditions such as gastric ulceration (n = 13), depressed growth rate with diarrhoea (n = 15), bronchopneumonia with nasal discharge (n = 6) and multiple abscessation (n = 6).
Statistical analysis Intra- and inter-assay CVs and detection limits were calculated using routine descriptive statistical procedures with Microsoft Excel 2000. Ordinary regression analysis was used to investigate ‘linearity under dilution’ and CRP measurements in saliva and serum were compared using a statistical software programme (14th edition SPSS Inc.). C-reactive protein measurements obtained from the clinical validation study were evaluated for normality of distribution using the Kolmogorov– Smirnov statistic. As the results did not meet normal distribution criteria, a Mann–Whitney non-parametric test was used to compare salivary CRP concentrations from clinically normal pigs with those from diseased animals. Statistical significance for differences between diseased and healthy pigs was set at P < 0.05 and was calculated using the SPSS programme detailed above.
Results Analytical validation Assessment of intra-assay precision The intra-assay CV was 6.84% for the pool with h-CRP and 4.57% for the pool with l-CRP. The CVs obtained for inter-assay precision were higher than those for intra-assay precision, ranging from 7.63–11.83% (Table 1). When the stability study was carried out, no variations in salivary CRP levels were detected during the first 7 days. A decrease of 38.6% in salivary CRP values was found in the h-CRP pool (Fig. 1) at day 15 and the CRP concentration remained at this level until the end of the study 2 months later. No variations were found in the l-CRP pool.
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Table 1 Assessment of the precision of the time-resolved immunofluorometry assay (TR-IFMA) for the measurement of C-reactive protein (CRP) in porcine saliva Saliva pool
l-CRP h-CRP Mean
Intra-assay
Inter-assay
Mean (ng/mL)
SD
CV (%)
Mean (ng/mL)
SD
CV (%)
29 475 238
1.34 32.50 16.92
4.57 6.84 5.75
32 488 260
3.78 37.24 20.51
11.83 7.63 9.73
SD: standard deviation; CV: coefficient of variation.
Fig. 1. Stability of porcine CRP in saliva pools of high (h-CRP) and low (l-CRP) concentration, stored at 20 °C during a 2 month period as measured by time-resolved immunofluorometry assay.
Fig. 2. Investigation of linearity under dilution of two saliva samples containing high levels of CRP (1136 ng/mL and 490 ng/mL, respectively). r: correlation coefficient.
Assessment of assay accuracy The dilution of two saliva samples with h-CRP concentrations resulted in linear regression equations (Fig. 2), where ‘x’ represents the expected CRP level at the particular dilution and ‘y’ represents the measured CRP level. The correlation coefficients were 0.991 and 0.990, respectively. When the CRP concentrations in the saliva and serum of the 20 healthy and 30 diseased pigs were compared, a significant positive correlation was identified with a coefficient of correlation of r = 0.726 (P < 0.001) (Fig. 3). Serum CRP levels were almost 323 times higher than those in saliva, as indicated by the regression equation y = 0.0031x 0.06.
Fig. 3. Correlation of CRP concentrations (lg/mL) in serum and saliva as measured by time-resolved immunofluorometry assay (n = 50). r: correlation coefficient.
Assessment of limit of detection The LOD of the assay was 0.47 ng/mL which corresponded to the mean value of the CRP concentration plus two SD of 10 replicates of the assay buffer. Clinical validation study The pigs with experimentally-induced inflammation had significantly increased serum and salivary CRP (Fig. 4). The median serum CRP concentration for both animals increased from 53 lg/mL before injection to 275 lg/mL at 24 h, a 5.21-fold increase. The original value had increased 9.8-fold at 48 h and peaked at 570 lg/mL, a 10.80-fold increase at 72 h. The median salivary CRP level
Fig. 4. Salivary and serum CRP concentrations in two pigs (1 and 2) with experimentally-induced inflammation.
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Fig. 5. Salivary CRP levels in healthy pigs (n = 20) and in pigs with inflammatory disease (n = 40). The plot shows median (line within box), 25th and 75th percentiles (box), 5th and 95th percentiles (whiskers) and outliers (°).
in pigs increased from 22 ng/mL to 86 ng/mL, 24 h after injection, a 3.93-fold increase. At 48 h this value had increased 6.39-fold to 140 ng/mL and peaked at 393 ng/ mL, a 17.95-fold increase, at 72 h. Significantly higher (P < 0.001) salivary CRP concentrations were observed in diseased relative to clinically normal animals (Fig. 5). Median CRP concentrations of 31 ng/mL (25th and 75th percentiles of 24.24 and 38.14, respectively) and of 123 ng/mL (25th and 75th percentiles of 74.37 and 241.94, respectively) were found in healthy and diseased animals, respectively. Discussion The scientific assessment of the welfare status of animals has gained increasing prominence in recent years (OIE, 2007). In this context the measurement of indicators of an animal’s welfare status such as acute phase protein (APP) levels in samples such as saliva that can be obtained with minimal stress on the sampled animal are particularly attractive (Eckersall, 2004). Furthermore, given the minimally invasive nature of saliva sampling, such an approach has the further advantages that samples can be collected by individuals following modest levels of training, and repeated sampling over short time-intervals can be carried out which facilitates ongoing animal monitoring (Kaufman and Lamster, 2002). A number of disease conditions in humans result in increases in analytes such as CRP in saliva that can be used in clinical monitoring (Christodoulides et al., 2005; Wong, 2006). The measurement of APPs such as canine CRP (Parra et al., 2005) and porcine haptoglobin (Hiss et al., 2003) in saliva have been used in a similar context in veterinary medicine. The main limitation to the widespread use of this approach is the insufficient sensitivity of detection of most
current commercial assay systems for salivary APP detection. For example, CRP could not be detected in pig saliva using a commercial enzyme-linked immunosorbent assay (ELISA) (Llamas Moya et al., 2003). However the development of new, more sensitive detection systems such as TRIFMA can overcome these limitations. The results of the present study indicate that salivary CRP concentrations are some 322-fold lower than in serum with values in the order of ng/mL. In this study, the problems associated with the detection of low levels of CRP in porcine saliva were resolved by using europium chelate labels and timeresolved fluorometry. The three standard criteria for immunoassay validation, namely precision, accuracy and limit of detection, were determined for the TR-IFMA in this study, while assessment of the specificity of the assay was as described previously (Martı´nez-Subiela et al., 2007). Analytical validation indicated that porcine salivary CRP concentrations could be reliably measured using the TR-IFMA and both intraand inter-assay CVs were <12%. As far as the authors are aware, there is no previous validated method for the detection of CRP in porcine saliva, however the precision assessment results were equivalent to those found for CRP quantification in dog saliva by TR-IFMA (Parra et al., 2005). Furthermore, the CVs for the porcine salivary CRP measurements were lower than those obtained in the validation of commercially available assays for serum CRP (Tecles et al., 2007). The LOD of the method was very low, demonstrating the high sensitivity of the TR-IFMA. This LOD was significantly lower than that reported for other commercially available kits (Kjelgaard-Hansen et al., 2007; Tecles et al., 2007). The median concentrations of CRP in the saliva of both healthy and diseased pigs were lower than the LOD of commercial kits but greater than the LOD of the method validated in this study, so that this technique has applications in quantifying salivary CRP concentrations in this species. In human medicine, conventional ELISAs are unable to detect salivary CRP levels in the majority of the samples so that other techniques such as microchip assay systems have been used as an alternative (Christodoulides et al., 2005). Analysis of assay accuracy using linearity under dilution indicated a linear relationship over a wide range of salivary CRP concentrations and the assay response. Despite the differences in the CRP concentrations in saliva and serum, the correlations found, suggest measurement of salivary CRP may be a suitable alternative to the measurement of serum CRP. The study found that saliva samples could be stored at 20 °C for 7 days after collection without compromising result validity. To the authors’ knowledge there are no previous studies of the stability of CRP in porcine saliva and this stability gives an assay based on saliva more flexibility than one performed on whole blood where porcine CRP is stable for 3 days if refrigerated (Martı´nez-Subiela et al., 2007). The fall in salivary CRP concentration after 15 days at 20 °C suggests analysis should be undertaken within
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this timescale, however, it may be possible to prolong the stability of CRP in saliva by adding protease inhibitors or serum albumin. The results of the clinical validation study showed significant differences in the median salivary CRP concentrations between healthy pigs and pigs with a variety of inflammatory conditions. Further studies of larger number of animals will be necessary to establish the normal range of CRP concentrations of healthy animals at different ages and of varying production status, and to evaluate the sensitivity of salivary CRP in detecting animals with inflammatory disease. Although the injection of turpentine oil increased both serum and salivary CRP concentrations, these differences could not be given statistical significance as for ethical reasons only two pigs were used. The greatest increases occurred in serum between 24 and 48 h after injection and between 48 and 72 h in saliva. This increase in salivary CRP was almost 18-fold relative to the almost 11-fold increase in serum. These differences could be due to several factors that influence the composition of saliva such as flow rate, degree of hydration, body composition, plasma composition or even possible local synthesis of salivary CRP. Further studies will be required to elucidate this issue. Conclusions The results of this study indicate that if highly sensitive detection systems such as the TR-IFMA are used, measurement of salivary CRP in pigs can provide a straightforward, flexible and minimally invasive method of detecting inflammatory-based disease in this species that can be used as a tool in animal health and welfare monitoring. Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper. Acknowledgement This work was supported by a grant from the Spanish Ministry of Education and Science (AGL 2006-05701). References Christodoulides, N., Mohanty, S., Miller, C.S., Langub, M.C., Floriano, P.N., Dharshan, P., Ali, M.F., Bernard, B., Romanovicz, D., Anslyn, E., Fox, P.C., McDevitt, J.T., 2005. Application of microchip assay system for the measurement of C-reactive protein in human saliva. Lab on a Chip 5, 261–269. Chomdej, S., Ponsuksili, S., Schellander, K., Wimmers, K., 2004. Detection of SNPs and linkage and radiation hybrid mapping of the porcine C-reactive protein (CRP) gene. Animal Genetics 35, 469–470. Devillers, N., Farmer, C., Mounier, A.M., Le Dividich, J., Prunier, A., 2004. Hormones, IgG and lactose changes around parturition in
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