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- J Vet Med Sci
- v.83(2); 2021 Feb
- PMC7972874
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J Vet Med Sci. 2021 Feb; 83(2): 329–332.
Published online 2020 Dec 29. doi:10.1292/jvms.20-0550
PMCID: PMC7972874
PMID: 33390361
Marina OTSUKA,1 Mieko SUGIYAMA,2 Takaaki ITO,3 Kenji TSUKANO,4 Shin OIKAWA,1 and Kazuyuki SUZUKI1,*
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Abstract
This study established the precision and accuracy of a modified latex agglutinationturbidimetric immunoassay (LATIA) reagent, and evaluated the ability of the measurement ofserum amyloid A (SAA) compared to haptoglobin and α1-acid glycoprotein, which are acutephase proteins (APPs), for diagnosis of clinical mastitis. Concentrations of APPs in cowswith mastitis were significantly higher than those in healthy cow. Only the plasma SAAconcentration in cows with clinical mastitis (44.90 mg/l; n=15) was significantly higherthan that in those with subclinical mastitis (10.70 mg/l; n=16), enabling their diagnosisin contrast to other APPs. Thus, the SAA assay using a LATIA reagent is useful inassessing mastitis severity due to its higher sensitivity and specificity than other APPassays.
Keywords: acute phase protein, bovine, inflammation, mastitis, serum amyloid A
Bovine mastitis is a major cause of economic loss in the dairy industry [12, 13]. Adiagnostic tool for mastitis that is quick and easy to apply is necessary because the severityof inflammation affects the prognosis of cows. A diagnosis of prognosis is important becauseacute mastitis can be fatal due to severe inflammation. Additionally, subclinical mastitiscauses milk losses due to persistent inflammation, but it is difficult to assess the extent ofinflammation and subsequent productivity by clinical signs. If an efficient acute phaseproteins (APPs) assay is available in bovine practice, the severity of inflammation ofmastitis can be estimated.
APPs, such as C-reactive protein (CRP), haptoglobin (HPT), α1-acid glycoprotein (AGP) andserum amyloid A (SAA), are well used as markers to estimate the severity of inflammation inhuman and companion animal practice [6, 7, 15]. CRP isgenerally not considered an APP in bovine practice because it does not change due toinflammation in cows [24]. On the other hand, theusefulness of HPT and AGP in the evaluation of inflammation severity was previously reportedin bovine mastitis [10].
SAA is clinically applied as an inflammatory marker in horses [23] and cows [4, 14, 19, 21]. SAA responded most rapidly to infection; therefore,the plasma SAA concentration has potential as a marker to distinguish the severity of mastitis[2, 3, 8, 9, 22]. It is known that there are four SAA protein isoforms(SAA1, SAA2, SAA3 and SAA4) in human, and classic proteins for acute phase response is SAA1.Bovine SAA proteins are SAA 1 to 4, like humans, and bovine SAA 1 and 2 are proteins for acutephase response [4].
In bovine SAA assays, the ELISA system is mainly used, but time and cost may be disadvantagesof this method for general purposes use as a bovine biomarker. A latex agglutinationturbidimetric immunoassay (LATIA) has advantages in that measurement is quick using anautomated clinical chemistry analyzer, regardless of the number of samples. When LATIA wasapplied to cows, a human-specific reagent was used for evaluation of bovine SAA because acow-specific reagent for the SAA assay using LATIA was not commercially available. However,accurate evaluation of inflammation was difficult because the human-specific reagent was lesssensitive for bovine mastitis. Accordingly, a modified reagent for bovine SAA assay usingLATIA was prepared for accurate and quick measurement of bovine samples.
To apply this new reagent in bovine practice, a study comparing SAA measured by the modifiedreagent with that measured by other APP assays previously validated in cattle is needed.Therefore, the aim of this study was to clarify the usefulness of the modified reagent forbovine SAA, and to evaluate the diagnostic ability of the SAA assay of mastitis in comparisonwith HPT and AGP.
This animal study was performed in accordance with the Guide for the Care and Use ofLaboratory Animals of the School of Veterinary Medicine at Rakuno Gakuen University(Approval#: VH18C10). Seventy-four Holstein-Friesian lactating cows consisting of 43 healthyand 31 mastitic cows kept at 4 commercial dairy farms in Japan were enrolled in this study.All cows were milked twice daily and fed total mixed rations and provided free access towater.
Blood samples (10 ml) were withdrawn from the jugular vein, and stored in serum separator andheparine-2K-coated vacuum tubes. Serum and plasma were obtained after centrifugation at 1,500× g at room temperature for 15 min, and stored at −80°C until assay to avoidrepeated thawing and freezing. Refrozen samples were not used in the SAA assay because the SAAconcentration is affected by freeze-thaw cycles.
The SAA concentration was measured using an automated LATIA assay kit for animal SAA. Thisassay kit is a modified human SAA assay kit (LZ test ‘Eiken’ SAA, Eiken Chemical Co., Tokyo,Japan) and has improved specificity for bovine SAA by changing the antibody. The hom*ologies ofamino acid sequences between human SAA 1 and, bovine SAA 1 or 2 is low as the hom*ology are53.1 or 76.2%. This is due to the deletion of some amino acid sequences in human SAA 1relative to bovine SAA 1 and 2. However, the hom*ology in other regions is high. In this study,the antibody prepared to targeting the 80th to 90th from the N-terminus of amino acid sequencein the human SAA1 protein and its corresponding amino acid sequences in bovine SAA1 and 2proteins. Therefore, its accuracy and validation were assessed and performed before theclinical trial. The assay was performed using an automated clinical chemical analyzer (Hitachi7170S, Hitachi Ltd., Tokyo, Japan) [5].
The detection limit (DL) of the LATIA, which was estimated as the mean ± 2.6 standarddeviation (SD) of SAA determination of blank samples, was 2.90 mg/l(P<0.01). Linearity under dilution was investigated by linear regressionmodel analysis. The Pearson correlation coefficient was used to assess the strength anddirection of association.
Precision was assessed by inter-assay and dilution linearity test. For the inter-assay test,validation was assessed by the coefficient of validation (CV) from the mean and SD of 10replicate determinations of three pooled bovine sera. The mean SAA concentrations of threebovine sera used in the inter-assay were 11.61, 55.83 and 168.57 mg/l, respectively. The CVranged from 1.08 to 1.31%.
The dilution linearity test was performed as serial dilutions of two pooled bovine sera,which were diluted to obtain sample volume percentages of 20, 40, 60, 80 and 100%. Expectedconcentrations of SAA were calculated by dividing the maximum measured value with the modifiedreagent by the dilution ratio. The mean SAA concentrations of them were 11 and 158 mg/l.Observed concentrations by the modified SAA assay correlated well with the expectedconcentration. The regression equations and r2 of the twoconcentration ranges were as follows; y=0.96 × −0.45 and 0.988, and y=0.98 × −8.68 and 0.979,respectively.
To evaluate the effects of blood sample types, the correlation of bovine SAA concentrationmeasured by the modified SAA assay between serum and plasma samples was evaluated by linearapproximation. Bovine SAA concentrations of serum and plasma samples were well positivelycorrelated. The regression equation and r2 were y=0.97 × −0.23 and0.992, respectively, which suggests that both serum and plasma can be used for the measurementand compared. Since plasma samples are generally used in biochemical exams, plasma was usedfor assays in the study.
Based on the results of the physical examination and modified California Mastitis Test (CMT;P.L tester, Nippon Zenyaku Kogyo Co., Ltd., f*ckushima, Japan), cows with and withoutabnormality were designated as the mastitis group (n=31) and control group (n=43),respectively. The mastitis group was further divided into subclinical (n=16) and clinicalmastitis (n=15) groups according to the absence and presence of clinical symptoms such asfever, anorexia, udder swelling, redness and/or hardness [20]. No symptoms suggesting diseases other than mastitis were observed. All cowswere examined more than 10 days after parturition and APP levels were not considered to beaffected by parturition based on reports of APP levels being higher the week after calving[11].
The milk bacterial test was also carried out for all mastitic milk harvested from cows in themastitis group in accordance with previously published guidelines [17]. The breakdown of bacteria in each group was as follows: thesubclinical group: Staphylococcus aureus (7/16),Streptococcus spp. (3/16), coagulase-negative staphylococci (1/16),Streptococcus uberis (1/16), Corynebacterium bovis (1/16),others (2/16) and not detected (1/16); the clinical mastitis group: Klebsiellapneumoniae (7/15), Escherichia coli (3/15), Trueperellapyogenes (2/15), Streptococcus spp. (1/15), Enterobactercloacae (1/15) and unknown species (1/15).
As APPs are produced mainly in the liver, blood biochemical tests were carried out toevaluate liver function. The results of blood biochemical tests are summarized in Table 1, with no significant difference between the groups. The plasma HPT (BovineHaptoglobin ELISA, Immunology Consultants Laboratory, Inc., Portland, OR, USA) and AGP (Bovineα1-glycoprotein ELISA, Immunology Consultants Laboratory, Inc.) concentrations were measuredusing commercial ELISA kits according to the instructions. The plasma SAA concentration wasmeasured as above.
Table 1.
Concentrations of acute phase proteins and blood biochemistry in control andmastitis groups
| Parameter | Unit | Control | Mastitis | P-value | Cut-off value | Sensitivity | Specificity | AUC |
|---|---|---|---|---|---|---|---|---|
| (n=43) | (n=31) | (%) | (%) | |||||
| SAA | (mg/l) | 1.00 [0.10–8.00] | 20.70 [0.80–157.50] | <0.001 | 6.60 mg/l | 80.6 | 95.3 | 0.931 |
| HPT | (µg/ml) | 0.25 [0.10–0.80] | 32.15 [0.07–303.25] | <0.001 | 0.96 µg/ml | 80.6 | 100 | 0.871 |
| AGP | (mg/ml) | 0.32 [0.16–0.72] | 0.35 [0.18–0.88] | 0.019 | 0.34 mg/ml | 71.0 | 60.5 | 0.661 |
| ALT | (U/l) | 29.5 ± 10.2 | 25.6 ± 9.9 | 0.078 | - | - | - | - |
| AST | (U/l) | 85.0 [52.0–175.0] | 7.0 [48.0–204.0] | 0.097 | - | - | - | - |
| γGTP | (U/l) | 29.0 [16.0–60.0] | 26.0 [14.0–60.0] | 0.383 | - | - | - | - |
| BUN | (mg/dl) | 12.0 [6.3–17.2] | 13.1 [3.7–26.7] | 0.095 | - | - | - | - |
| CRE | (mg/dl) | 0.72 [0.55–0.98] | 0.73 [0.52–1.36] | 0.961 | - | - | - | - |
| T-Bil | (mg/dl) | 0.05 [0.00–0.15] | 0.05 [0.00–1.47] | 0.493 | - | - | - | - |
Open in a separate window
The normally distributed and non-normally distributed data are expressed the means ± SDand medians [minimum-maximum], respectively. SAA: serum amyloid A, HPT: haptoglobin,AGP: α1-acid glycoprotein, ALT: alanine transaminase, AST: aspartate aminotransferase,γGTP: γ-glutamyltransferase, BUN: blood urea nitrogen, CRE: creatinin, T-Bil: totalbilirubin, AUC: area under the curve.
All statistical analyses were performed by IBM SPSS Statistics, v.23 (IBM Co, Somers, NY,USA). The normally distributed and non-normally distributed data are expressed the means ± SDand medians [minimum-maximum], respectively. Differences between groups were analyzed with theMann-Whitney U test. A proposed cut-off value of each APP concentrations todiagnose mastitis was determined using a receiver operating characteristic (ROC) curve. Theoptimal cut-off point for each APP was calculated by the Youden index [1, 16]. The significance level wasP<0.05.
Concentrations of plasma HPT, AGP and SAA in control groups were 0.25 [0.10–0.80] µg/ml, 0.32[0.16–0.72] mg/ml and 1.00 [0.10–8.00] mg/l, respectively (Table 1). In the mastitis group, APP concentrations weresignificantly higher than those in control group in plasma HPT (P<0.001),AGP (P<0.05) and SAA (P<0.001). The proposeddiagnostic cut-off points for plasma HPT, AGP and SAA concentrations to identify dairy cattlewith mastitis based on analyses of ROC curves were set at >0.96 µg/ml, >0.34 mg/ml and>6.60 mg/l, respectively. All APPs were able to diagnose mastitis. The reason for thesignificant changing in APPs values despite the unchanged biochemical test results wereconsidered to be due to the lack of strong systemic inflammation of mastitic cattle used inthis study.
The plasma SAA concentration was significantly higher in the clinical group (44.90[7.40–157.50] mg/l) than in the subclinical group (10.70 [0.80–59.90] mg/l,P<0.001, Fig. 1A), although no significant differences between subclinical and clinical groups wereobserved in plasma HPT and AGP concentrations. The proposed diagnostic cut-off point forplasma SAA concentrations to identify dairy cattle with clinical mastitis based on analyses ofROC curves was set at >14.05 mg/l (Fig. 1B). Theresults of this study are consistent with a previous study using an ELISA system todemonstrate significant differences in the blood SAA level by severity of inflammation [18].
Fig. 1.
Comparison of serum amyloid A (SAA) concentrations of subclinical and clinical mastitisgroups. A: The plasma SAA concentration in the subclinical and clinical groups. Thehorizontal line in each box represents the median value. The boxes represent theinterquartile range (25 to 75 percentiles). *Values with asterisks are significantlydifferent between groups (P<0.05). B: The receiver operatingcharacteristic (ROC) curves of the plasma SAA concentration to identify dairy cattlewith clinical mastitis.
In the present study, plasma HPT, AGP and SAA concentrations in cows with mastitis weresignificantly higher than those in healthy cows, and these APP levels reflect inflammationcaused by mastitis. Furthermore, the plasma SAA concentration was able to distinguishsubclinical and clinical mastitis. Thus, the plasma SAA concentration using a modified reagentfor animals was superior in assessing the severity of inflammation in mastitis due to itshigher sensitivity and specificity than other APP assays. Therefore, the plasma SAAconcentration measured by LATIA is useful to asses bovine mastitis quickly and accurately thanother APP assays. Furthermore, the plasma SAA concentration measured by the present LATIA mayassist in assessing the outcomes of cows with mastitis.
POTENTIAL CONFLICTS OF INTEREST. The authors have nothing to disclose.
Acknowledgments
We thank Dr. Kikuchi, T., Dr. Asahi, Y. and Dr. Inamori, S. for support inthe measurement of SAA.
REFERENCES
1. Akobeng A. K.2007. Understanding diagnostic tests 3:Receiver operating characteristic curves. ActaPaediatr. 96: 644–647.doi: 10.1111/j.1651-2227.2006.00178.x [PubMed] [CrossRef] [Google Scholar]
2. Alsemgeest S. P., Kalsbeek H. C., Wensing T., Koeman J. P., van Ederen A. M., Gruys E.1994. Concentrations of serum amyloid-A (SAA)and haptoglobin (HP) as parameters of inflammatory diseases in cattle.Vet. Q. 16:21–23. doi: 10.1080/01652176.1994.9694410 [PubMed] [CrossRef] [Google Scholar]
3. Boosman R., Niewold T. A., Mutsaers C. W., Gruys E.1989. Serum amyloid A concentrations in cowsgiven endotoxin as an acute-phase stimulant. Am. J. Vet.Res. 50: 1690–1694. [PubMed] [Google Scholar]
4. Ceciliani F., Ceron J. J., Eckersall P. D., Sauerwein H.2012. Acute phase proteins inruminants. J.Proteomics 75:4207–4231. doi: 10.1016/j.jprot.2012.04.004 [PubMed] [CrossRef] [Google Scholar]
5. Christensen M., Jacobsen S., Ichiyanagi T., Kjelgaard-Hansen M.2012. Evaluation of an automated assay based onmonoclonal anti-human serum amyloid A (SAA) antibodies for measurement of canine,feline, and equine SAA. Vet.J. 194: 332–337. doi: 10.1016/j.tvjl.2012.05.007 [PubMed] [CrossRef] [Google Scholar]
6. Cray C., Zaias J., Altman N. H.2009. Acute phase response in animals: areview. Comp. Med. 59:517–526. [PMC free article] [PubMed] [Google Scholar]
7. Eckersall P. D., Bell R.2010. Acute phase proteins: Biomarkers ofinfection and inflammation in veterinary medicine. Vet.J. 185: 23–27. doi: 10.1016/j.tvjl.2010.04.009 [PubMed] [CrossRef] [Google Scholar]
8. Heegaard P. M., Godson D. L., Toussaint M. J., Tjørnehøj K., Larsen L. E., Viuff B., Rønsholt L.2000. The acute phase response of haptoglobinand serum amyloid A (SAA) in cattle undergoing experimental infection with bovinerespiratory syncytial virus. Vet. Immunol.Immunopathol. 77:151–159. doi: 10.1016/S0165-2427(00)00226-9 [PMC free article] [PubMed] [CrossRef] [Google Scholar]
9. Horadagoda A., Eckersall P. D., Hodgson J. C., Gibbs H. A., Moon G. M.1994. Immediate responses in serum TNF α andacute phase protein concentrations to infection with Pasteurella haemolytica A1 incalves. Res. Vet.Sci. 57: 129–132. doi: 10.1016/0034-5288(94)90094-9 [PubMed] [CrossRef] [Google Scholar]
10. Horadagoda N. U., Knox K. M. G., Gibbs H. A., Reid S. W. J., Horadagoda A., Edwards S. E. R., Eckersall P. D.1999. Acute phase proteins in cattle:discrimination between acute and chronic inflammation.Vet. Rec. 144:437–441. doi: 10.1136/vr.144.16.437 [PubMed] [CrossRef] [Google Scholar]
11. Humblet M. F., Guyot H., Boudry B., Mbayahi F., Hanzen C., Rollin F., Godeau J. M.2006. Relationship between haptoglobin, serumamyloid A, and clinical status in a survey of dairy herds during a 6-monthperiod. Vet. Clin.Pathol. 35: 188–193.doi: 10.1111/j.1939-165X.2006.tb00112.x [PubMed] [CrossRef] [Google Scholar]
12. Kandasamy S., Green B. B., Benjamin A. L., Kerr D. E.2011. Between-cow variation in dermalfibroblast response to lipopolysaccharide reflected in resolution of inflammation duringEscherichia coli mastitis. J. DairySci. 94: 5963–5975. doi: 10.3168/jds.2011-4288 [PubMed] [CrossRef] [Google Scholar]
13. Kerro Dego O., Oliver S. P., Almeida R. A.2012. Host-pathogen gene expression profilesduring infection of primary bovine mammary epithelial cells with Escherichia colistrains associated with acute or persistent bovine mastitis.Vet. Microbiol. 155:291–297. doi: 10.1016/j.vetmic.2011.08.016 [PubMed] [CrossRef] [Google Scholar]
14. Lehtolainen T., Røntved C., Pyörälä S.2004. Serum amyloid A and TNF α in serum andmilk during experimental endotoxin mastitis. Vet.Res. 35: 651–659. doi: 10.1051/vetres:2004043 [PubMed] [CrossRef] [Google Scholar]
15. Murata H., Shimada N., Yoshioka M.2004. Current research on acute phase proteinsin veterinary diagnosis: an overview. Vet.J. 168: 28–40. doi: 10.1016/S1090-0233(03)00119-9 [PubMed] [CrossRef] [Google Scholar]
16. Nakas C. T., Dalrymple-Alford J. C., Anderson T. J., Alonzo T. A.2013. Generalization of Youden index formultiple-class classification problems applied to the assessment of externally validatedcognition in Parkinson disease screening. Stat.Med. 32: 995–1003.doi: 10.1002/sim.5592 [PubMed] [CrossRef] [Google Scholar]
17. National MastitisCouncil.2017. Laboratory handbook on bovine mastitis, 3rd ed. Nationalmastitis Council, New Prague. [Google Scholar]
18. Nazifi S., Haghkhah M., Asadi Z., Ansari-Lari M., Tabandeh M. R., Esmailnezhad Z., Aghamiri M.2011. Evaluation of Sialic Acid and Acute PhaseProteins (Haptoglobin and Serum Amyloid A) in Clinical and Subclinical BovineMastitis. Pak. Vet.J. 31:353–358. [Google Scholar]
19. Nielsen B. H., Jacobsen S., Andersen P. H., Niewold T. A., Heegaard P. M. H.2004. Acute phase protein concentrations inserum and milk from healthy cows, cows with clinical mastitis and cows with extramammaryinflammatory conditions. Vet.Rec. 154: 361–365. doi: 10.1136/vr.154.12.361 [PubMed] [CrossRef] [Google Scholar]
20. Ruegg P. L., Erskine R. J.2020. Mammary gland health and disorders. pp. 1118–1150. In:Large Animal Internal Medicine, sixth ed. (Smith, B. P., Van Metre, D. C., Pusterla, N.eds.), Elsevier, St. Louis. [Google Scholar]
21. Thomas F. C., Geraghty T., Simões P. B. A., Mshelbwala F. M., Haining H., Eckersall P. D.2018. A pilot study of acute phase proteins asindicators of bovine mastitis caused by different pathogens.Res. Vet. Sci. 119:176–181. doi: 10.1016/j.rvsc.2018.06.015 [PubMed] [CrossRef] [Google Scholar]
22. Werling D., Sutter F., Arnold M., Kun G., Tooten P. C., Gruys E., Kreuzer M., Langhans W.1996. Characterisation of the acute phaseresponse of heifers to a prolonged low dose infusion oflipopolysaccharide. Res. Vet.Sci. 61: 252–257. doi: 10.1016/S0034-5288(96)90073-9 [PubMed] [CrossRef] [Google Scholar]
23. Witkowska-Piłaszewicz O. D., Żmigrodzka M., Winnicka A., Miśkiewicz A., Strzelec K., Cywińska A.2019. Serum amyloid A in equine health anddisease. Equine Vet.J. 51: 293–298. doi: 10.1111/evj.13062 [PMC free article] [PubMed] [CrossRef] [Google Scholar]
24. Yogeshpriya S., Selvaraj P.2019. C-Reactive Protein in VeterinaryPractice. Dairy and Vet. Sci.J. 13: 555858. [Google Scholar]
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