Veterinary Immunology and Immunopathology 163 (2015) 221–226
Contents lists available at ScienceDirect
Veterinary Immunology and Immunopathology journal homepage: www.elsevier.com/locate/vetimm
Short communication
Acute phase proteins in naturally occurring respiratory disease of feedlot cattle Ignacio Idoate ∗ , Brian Vander Ley ∗ , Loren Schultz, Meera Heller ∗ Department of Veterinary Medicine and Surgery, University of Missouri, 900 East Campus Dr., Columbia, MO 65211, USA
a r t i c l e
i n f o
Article history: Received 14 July 2014 Received in revised form 5 December 2014 Accepted 16 December 2014 Keywords: Bovine respiratory disease Acute phase proteins Haptoglobin Lipopolysaccharide binding protein Transferrin
a b s t r a c t The aim of this study was to evaluate three acute phase proteins (APP) [haptoglobin (HPT), lipopolysaccharide binding protein (LBP) and transferrin (Tf)] in feedlot cattle with naturally occurring respiratory disease diagnosed by a calf health scoring chart (CHSC). Seventy-seven beef calves were observed for signs of Bovine Respiratory Disease (BRD) during the first 28 days after arrival at the feedlot. Fourteen cases and pen matched controls were selected based on the CHSC. BRD cases were defined as a score of ≥5, while controls were defined as a score ≤4. The mean CHSC score in cases was 6.9 which was significantly greater than the controls 2.8 (P < 0.01). Mean plasma LBP and HPT concentrations were significantly greater in cases than controls (P < 0.01). Our study results show that measurement of HPT and LBP could be useful in detecting respiratory disease in feedlot conditions. Transferrin concentrations between the two groups were not statistically different. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Bovine respiratory disease complex (BRDC) is the most common disease among feedlot cattle in the United States, accounting for approximately 75% of feedlot morbidity and 50–70% of all feedlot deaths (Edwards, 2010). BRD causes between $800 million and $900 million annually in economic losses (Edwards, 2010; Wittum et al., 1996; USDA USDoA, 2007). Economic losses stem from prevention costs, treatment, death, associated costs and from diminished average daily gain, and decreased feed efficiency. The development of BRD is multifactorial and is influenced by a combination of host, environment and pathogen factors. Susceptibility to viral and bacterial pathogens is influenced by anatomy, physiology and management of beef cattle
∗ Corresponding authors at: University of Missouri, Department of Veterinary Medicine and Surgery, 900 East Campus Dr., Columbia, MO 65211, USA. Tel.: +1 573 882 6857; fax: +1 573 884 0173. E-mail addresses: [email protected] (I. Idoate), [email protected] (B. Vander Ley), [email protected] (M. Heller). http://dx.doi.org/10.1016/j.vetimm.2014.12.006 0165-2427/© 2014 Elsevier B.V. All rights reserved.
(Taylor et al., 2010). Respiratory disease diagnosis can be confirmed using an assortment of methods. Necropsy and detection of BRD pathogens remain the gold standard tests to diagnose BRD, however use of clinical scoring systems is widespread and useful for lay people to systematically evaluate and classify sick cattle. There have been six clinical scoring systems for BRD described in the literature which relies on evaluation of a variety of clinical signs and assignment of scores based on the evaluator’s impression (Thomas et al., 1977; McGuirk, 2008; Panciera and Confer, 2010; Love et al., 2014). Bacterial infections usually lead to a strong systemic acute phase response (APR) (Alsemgeest et al., 1994), due to the marked activation of monocytes and macrophages and release of inflammatory mediators, such as histamine, leukotriene’s, prostaglandins, and proinflammatory cytokines. TNF-␣, IL-1 and IL-6 play a pivotal role in activating hepatocyte receptors (HepG2 or Hep3B cells) to initiate the synthesis of various APPs (Heinrich et al., 1990, 1998; Gruys et al., 2005). Down-regulation of the hepatic APR is achieved by rapid hepatic removal of circulating cytokines (Heinrich et al., 1990, 1998) and release
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I. Idoate et al. / Veterinary Immunology and Immunopathology 163 (2015) 221–226
of IL-10 by the Kupffer cells which results in suppression of local IL-6 production (Knolle et al., 1995; Baumann et al., 1994). The initial APR is down regulated once the infective organisms are isolated, cellular debris is removed, and macrophages initiate tissue repair (Serhan and Savill, 2005). HPT is a positive APP, and is the principal scavenger of free hemoglobin in blood (Murata et al., 2004). HPT elicits bacteriostatic effects by binding free Hemoglobin, thus making iron unavailable to proliferating bacteria. Iron utilization is a conserved process that has been identified in multiple bacterial pathogens, including Vibrio sp., Pseudomonas aeruginosa, Escherichia coli, Shigella flexneri, and Bacillus subtilis. (Skaar, 2010). Hpt also plays a key role in the recruitment of neutrophils in the early phase of inflammation (Riollet et al., 2000). LBP is a soluble polypeptide that binds to bacterial lipopolysaccharide (LPS) and presents the LPS to pattern recognition receptors CD14 and TLR4, found on monocytes, macrophages and granulocytes. The presentation of LPS via LBP enhances the pro-inflammatory activity of these innate immune cells by 100 to 1000 fold (Fierer et al., 2002). LBP can also bind Lipoteichoic acid (LTA), a pathogen recognition molecule exposed on the cell wall of Gram-positive bacteria. The interaction between LBP and LTA triggers a pro-inflammatory cascade via TLR-2 activation (Mogensen, 2009). Transferrin (Tf) is an iron-binding blood plasma glycoprotein found in mucosa. Tf has a single polypeptide chain of about 700 amino acids and contains two specific highaffinity Fe3+ binding sites, for the transport of iron in the circulation (Ceron et al., 2005; Oliveira et al., 2014). Tf concentrations fall during an acute phase response, making Tf a negative APP (Nguyen, 1999). Acute phase response to infection or inflammation can lead to marked anemia (Feldman et al., 1981a,b). Tf elicits indirect bactericidal effects by binding to free iron, thus making iron unavailable to proliferating bacteria, in a response that may be mediated by lipocalin (Flo et al., 2004). Previous studies using challenge models have shown that all three of these APPs may be useful biomarkers for bacterial pneumonia in calves, however no studies have been done to assess APP levels in naturally occurring pneumonia in a feedlot setting (Gånheim et al., 2003; Conner et al., 1989; Schroedl et al., 2001; Dowling et al., 2002; Heegaard et al., 2000; McNair et al., 1998). The purpose of this study was to evaluate three acute phase proteins (Haptoglobin, Lipopolysaccharide binding protein and Transferrin) in naturally occurring respiratory disease of feedlot cattle diagnosed by a calf health scoring chart (CHSC). The hypothesis was that there would be a significant difference between the acute phase protein levels evaluated, between the clinical cases and their matched healthy controls, as diagnosed by a CHSC. 2. Materials and methods 2.1. Animals 77 mixed breed beef steer calves of similar age and weight were purchased at three area livestock auction
Table 1 Adapted from McGuirk SM. 2008. Calf health scoring chart 1 0
2
3
Rectal temperature 100–100.9 ◦ F 101–101.9 ◦ F
102–102.9 ◦ F
≥103 ◦ F
Induced repeated coughs or occasional spontaneous cough
Repeated spontaneous cough
Bilateral, cloudy or excessive mucus discharge
Copious bilateral mucopurulent discharge
Small amounts of discharge
Moderate amount of bilateral discharge
Heavy ocular discharge
Ear flick or head shake
Slight unilateral droop
Head tilt or bilateral droop
Cough None
Induce single cough
Nasal discharge Normal serous Small amount discharge of unilateral cloudy discharge Eye scores Normal
Ear scores Normal
barns for enrollment in a nutritional study. Animals were transported 30–230 mile to and housed at the University of Missouri Beef Research and Teaching Farm (BRTF). At arrival the animals were weighed, ear tagged [both Allflex visual identification (VID) & Radio-frequency identification (RFID)], vaccinated with Bovi-Shield Gold® 5 and One Shot Ultra® 8 (Zoetis, Florham Park, NJ, USA) and treated with Cydectin® Pour-On (Boehringer Ingelheim Vetmedica, Inc., St. Joseph, MO, USA). All procedures were approved by the University of Missouri Institutional 116 Animal Care and Use Committee. The calves were part of a post-weaning feedlot performance and feed efficiency tests using the GrowSafe automated feeding system (GrowSafe Systems Ltd., Airdrie, Alberta, Canada). All calves were housed six per pen. Pens were of open construction, measuring 4.9 × 8.84 m. Frost-free waterers were shared between two pens, and feed bunks and slabs were under sloped roof shades (4.9 × 6.7 m). 2.2. Clinical examination Bovine respiratory disease was diagnosed by an experienced animal technician based on a CHSC health scoring chart developed at the University of Wisconsin. The CHSC assigns each calf the sum of the nasal discharge, rectal temperature, cough scores and the greater one of the two scores from the ocular discharge and head/ear carriage (Table 1, McGuirk, 2008). Calves whose total score was ≥5 were categorized “BRD positive”, while controls were defined as a score ≤4. The cattle were monitored closely on a daily basis and the scoring based on the CHSC was done in the mornings to mimic common practices in commercial in feedlots. Fourteen steers were selected as cases based on their score and were not diagnosed with any other diseases during the study. Control animals (n = 14) were selected from contemporary pen mates and sampled at the same time point.
I. Idoate et al. / Veterinary Immunology and Immunopathology 163 (2015) 221–226
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Fig. 1. The figures represent higher BRD scores and LBP and HPT concentrations for cases versus controls in the study.
2.3. Samples Blood samples were collected from BRD clinical cases and their matched healthy controls selected from the same pens, using the coccygeal veins at the time of the classification based on the CHSC. The blood was allowed to clot at 4 ◦ C. After clotting, the samples were centrifuged at 2800 × g for 20 min at 4 ◦ C. Serum was harvested and frozen at −80 ◦ C for later analyses. 2.4. Acute phase proteins analysis Concentration of LBP was assayed using a commercially available Human LBP ELISA kit (Cell Sciences Inc., Canton, MA) that recognizes both human and bovine LBP, as described previously (Bannerman et al., 2003). Plasma samples were diluted 1:100 and assayed according to the manufacturer’s instructions. The detection range for the LBP ELISA kit is 0.05–2.5 g/ml. HPT concentrations were determined with a commercially available Bovine Haptoglobin ELISA kit (Immunology Consultants Laboratory, Inc., Portland, OR) according to the manufacturer instructions. Samples were diluted at 1:50, 1:200. 1:1000 and 1:2000 ratios and assayed according to manufacturer’s instructions. The detection range for
the HPT ELISA kit is 15.6–1000 ng/ml. Concentrations were converted from ng/ml to mg/ml to make the numbers more manageable. Transferrin concentrations were determined with a commercially available Bovine Transferrin ELISA kit (Immunology Consultants Laboratory, Inc., Portland, OR) according to the manufacturer’s instructions. Samples were diluted at 1:40,000 and assayed according to manufacturer’s instructions. The detection range for the Tf ELISA kit is 18.75–6000 ng/ml. Concentrations were converted from ng/ml to mg/ml to make the numbers more manageable. 2.5. Statistical analysis Differences in BRD scores, LBP, HPT and Tf between cases and contemporary control animals were analyzed and carried out using Stata® 13 software analysis systems (StataCorp LP, College Station, Texas, USA). The Shapiro test was used to appraise normal distribution. Since all values were not normally distributed or in the case of LBP and BRD scores were normally distributed but had unequal variances, a non-parametric test, Kruskal–Wallis equalityof-populations rank test was performed. A P-value 0.001 was used as the level of significance for all tests.
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Fig. 2. The two graphs show ROC curves for LBP and HPT shows ROC curves in comparison to a calf heath scoring chart. The tests were able to accurately separate BRD cases from controls. An area of 1 represents a perfect test; an area of 0.5 represents a worthless test.
3. Results and discussion BRD scores, serum LBP and HPT concentrations were considerably higher for BRD clinical cases than for their matched healthy controls (see Fig. 1) as diagnosed by a CHSC scoring chart. The differences were statistically significant between cases and controls (P = 0.001). The negative APP Tf evaluated in our study, was not significantly different between cases (8.93 mg/ml) and controls (9.64 mg/ml) (P = 0.5656). Our study result shows that measurement of HPT and LBP is associated with clinically diagnosed respiratory disease under field conditions, and
the levels correlate with those previously described in challenged experimental studies (Gånheim et al., 2003; Conner et al., 1989; Schroedl et al., 2001; Dowling et al., 2002; Heegaard et al., 2000; McNair et al., 1998). Schroedl et al. (2001) showed an early significant increase in LBP concentration in a group of 20 calves experimentally infected with Gram-negative Mannheimia haemolytica 6 h after bacterial inoculation. In this same study, Hpt levels did not significantly rise before 12 h. Gånheim et al. (2003) studied HPT levels in calves experimentally challenged with bovine viral diarrhea virus (BVDV) and/or M. haemolytica, demonstrating HPT levels similar to the ones found in our study.
I. Idoate et al. / Veterinary Immunology and Immunopathology 163 (2015) 221–226 Table 2 The table represents the ultimate cut point concentrations 512 for HPT and LBP. APP
Cut point
Sensitivity (%)
Specificity (%)
Correctly classified
Hpt LBP
≥0.81 (mg/ml) ≥0.33 (g/ml)
92.86 92.86
85.71 92.86
89.29 92.86
Conner et al., 1989 and Dowling et al., 2002, challenged calves experimentally with M. haemolytica and Pasteurella multocida, respectively. In both studies, the concentration levels were significantly higher in the challenged animals versus the study controls. Receiver operating characteristic (ROC) curves were calculated in our study to determine an ideal cutoff value for LBP and HPT concentrations. The ROC curve is created by plotting the fraction of true positives out of the total actual positives (TPR = true positive rate) vs. the fraction of false positives out of the total actual negatives (FPR = false positive rate), at various threshold settings. Sensitivities, specificities and percent correct classifications are listed as a function of cutoff value in Table 2. Another important measure of the accuracy of the clinical test is the area under the ROC curve. A perfect classification is an area that equals to 1.0. This test is 100% accurate because both the sensitivity and specificity are 1.0 so there are no false positives and no false negatives. The area under ROC curve for HPT in our study was 0.9235 and for LBP was 0.9668 (Fig. 2). This data suggests a very accurate classification for both LBP and HPT ideal concentration cutoffs for our study. On the other hand a test that cannot discriminate between normal and abnormal corresponds to an ROC curve 304 that is 0.5. The calculated area under ROC curve for Tf was 0.4674, suggesting that Tf concentrations were not accurate in differentiating BRD cases from healthy controls. In our study, the WI calf health scoring chart used to diagnose BRD ended up being user friendly and efficient at differentiating BRD cases from controls, even in a feedlot setting, even though it was originally designed for neonatal dairy calves. The system proved efficient discriminating cases and controls in our study, as it did in the study by Love et al. (2014) where they looked at developing a novel clinical scoring system for on-farm diagnosis of bovine respiratory disease in pre-weaned dairy calves. Other clinical scoring systems are also available for diagnosing BRD. DART (Depression, Appetite, Respiration and Temperature) is a clinical scoring system developed to identify beef cattle for BRD treatment in feedlots, but it is difficult to standardize because the clinical sign weights and decision points are not defined (Panciera and Confer, 2010). Love et al. (2014) developed three additional scorning systems where the individual assesses the presence or absence of ocular discharge, nasal discharge, ear droop or head tilt, respiratory quality and spontaneous coughing. This scoring system determines an animal to be BRD positive, when it has an abnormal ear or head carriage, or calves with nasal discharge and one other clinical sign, or calves that have any three clinical signs. These clinical scoring tools are of practical interest in the cattle industry because they are based on clinical signs that
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can be easily identified by producers. Buczinski et al. (2014) showed that adding the clinical score assessment to thoracic auscultation and thoracic ultrasonography, increased sensitivity of BRD diagnosis in pre-weaned Dairy Calves, although it decreases specificity when compared with lung consolidation findings. The recommendation of the WI CHSC is to treat calves because of high BRD presumption if the score is ≥5, and to monitor calves with scores of 4. Calves with scores of ≤3 are considered healthy. Although APPs appear to be very sensitive, we must also recognize that they are also non-specific, and potential confounding factors, such stress, age of the animal, and presence of concurrent diseases should be considered while deciphering results. When bovine respiratory disease (BRD) outbreaks occur, veterinarians might steer away from expensive or complex ante mortem diagnostics requiring long turnaround times. Instead, the initial actions are often directed toward electing the appropriate therapy. There is a need for the development and optimization of rapid diagnostic field tests to guide decisions regarding vaccination, metaphylaxis, or treatment programs. Monitoring of LBP or HPT could be a useful tool to help direct these on farm decisions.
References Alsemgeest, S.P.M., Kalsbeek, H.C., Wensing, T., Koeman, J.P., van Ederen, A.M., Gruys, E., 1994. Concentrations of serum amyloid-A (SAA) and haptoglobin (Hpt) as parameters of inflammatory diseases in cattle. Vet. Q. 16, 21–23. Bannerman, D.D., Paape, M.J., Hare, W.R., Eun, J.S., 2003. Increased levels of LPS-binding protein in bovine blood and milk following bacterial lipopolysaccharide challenge. J. Dairy Sci. 86 (10), 3128–3137. Baumann, H., Gauldie, J., 1994. The acute phase protein response. Immunology Today. 15 (2), 74–80. Buczinski, S., Forte, G., Francoz, D., Belanger, A.M., 2014. Comparison of 368 thoracic auscultation, clinical score, and ultrasonography as indicators of bovine respiratory disease in pre-weaned dairy calves. J. Vet. Intern. Med. 28, 234–242. Ceron, J.J., Eckersall, P.D., Martynez-Subiela, S., 2005. Acute phase proteins in dogs and cats: current knowledge and future perspectives. Vet. Clin. Pathol. 34, 85–99. Conner, J.G., Eckersall, P.D., Wiseman, A., Bain, R.K., Douglas, T.A., 1989. Acute phase response in calves following infection with Pasteurella haemolytica, Ostertagia ostertagi and endotoxin administration. Res. Vet. Sci. 47 (2), 203–207. Dowling, A., Hodgson, J.C., Schock, A., Donachie, W., Eckersall, P.D., McKendrick, I.J., 2002. Experimental induction of pneumonic pasteurellosis in calves by intratracheal infection with Pasteurella multocida biotype A: 3. Res. Vet. Sci. 73, 37–44. Edwards, T.A., 2010. Control methods for bovine respiratory disease for 388 feedlot cattle. Vet. Clin. N. Am., Food Anim. Pract. 26, 273–284. Feldman, B.F., Kaneko, J.J., Farver, T.B., 1981a. Anemia of inflammatory disease in the dog-clinical characterization. Am. J. Vet. Res. 42, 1109–1113. Feldman, B.F., Keen, C.L., Kaneko, J.J., 1981b. Anemia of inflammatory disease in the dog measurement of hepatic superoxide-dismutase, hepatic nonheme iron, copper, zinc and ceruloplasmin and serum iron, copper and zinc. Am. J. Vet. Res. 42, 1114–1117. Fierer, J., Swancutt, Heumann, D., Golenback, D., 2002. The role of lipopolysaccharide binding in mice protein in resistance to salmonella infections. J. Immunol. 168, 6396–6403. Flo, T.H., Smith, K.D., Sato, S., 2004. Lipocalin 2 mediates an innate immune response to bacterial infection by sequestrating iron. Nature 432, 917–921. Gånheim, C., Hultén, C., Carlsson, U., Kindahl, H., Niskanen, R., Waller, K.P., 2003. The acute phase response in calves experimentally infected with bovine viral diarrhea virus and/or Manheimia haemolytica. J. Vet. Med. Ser. B 50, 183–190.
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Gruys, E., Toussaint, M.J.M., Niewold, T.A., Koopmans, S.J., 2005. Acute phase reaction and acute phase proteins. J. Zhejiang Univ. Sci. B 6 (11), 1045–1056. Heegaard, P.M.H., Godson, D.L., Toussaint, M.J.M., Tjørnehøj, K., Larsena, L.E., Viuff, B., Rønsholt, L., 2000. The acute phase response of haptoglobin and serum amyloid A (SAA) in cattle undergoing experimental infection with bovine respiratory syncytial virus. Vet. Immunol. Immunopathol. 77 (1–2), 151–159. Heinrich, P.C., Castell, T.A., Andus, T., 1990. Interleukin-6 and the acute phase response. Biochem. J. 265, 621L 636. Heinrich, P.C., Behrmann, I., Müller-Newen, G., Schaper, F., Graeve, L., 1998. Interleukin-6-type cytokine signaling through the gp130/Jak/STAT pathway. Biochem. J. 334, 297L 314. Knolle, P., Lohr, H., Treichel, U., Dienes, H.P.T., Lohse, A., Schlaack, J., Gerken, G., 1995. Parenchymal and nonparenchymal liver cells and their interaction in the local immune response. Z. Gastroenterol. 33, 613L 620. Love, W.J., Lehenbauer, T.W., Kass, P.H., Van Eenennaam, A.L., Aly, S.S., 2014. Development of a novel clinical scoring system for on-farm diagnosis of bovine respiratory disease in pre-weaned dairy calves. Peer J. 2, e238, http://dx.doi.org/10.7717/peerj.238. McGuirk, S.M., 2008. Disease management of dairy calves and heifers. Vet. Clin. N. Am.: Food Anim. Pract. 24, 139–153. McNair, J., Elliott, C., Bryson, D.G., Mackie, D.P., 1998. Bovine serum Tf concentration during acute infection with Haemophilus somnus. Vet. J. 155, 251–255. Mogensen, T.H., 2009. Pathogen recognition and inflammatory signaling in innate immune defenses. Clin. Microbiol. Rev. 22 (2), 240–273. Murata, H., Shimada, N., Yoshioka, M., 2004. Current research on acute phase proteins in veterinary diagnosis: an overview. Vet. J. 168, 28–40.
Nguyen, H.T., 1999. Transport proteins. In: Loeb, W.F., Quimby, F.W. (Eds.), Clinical Chemistry of Laboratory Animals. , second ed. Taylor & Francis, Philadelphia, PA, pp. 309–335. Oliveira, F., Rocha, S., Fernandes, R., 2014. Iron metabolism: from health to disease. J. Clin. Lab. Anal. 00, 1–9. Panciera, R.J., Confer, A.W., 2010. Pathogenesis and pathology of bovine pneumonia. Vet. Clin. N. Am.: Food Anim. Pract. 26, 191–214. Riollet, C., Rainard, P., Poutrel, B., 2000. Kinetics of cells and cytokines during immune-mediated inflammation in the mammary gland of cows systemically immunized with Staphylococcus aureus alphatoxin. Inflamm. Res. Sep. 49 (9), 486–496. Schroedl, W., Fuerll, B., Reinhold, P., Krueger, M., Schuett, C., 2001. A novel acute phase marker in cattle: lipopolysaccharide binding protein (LBP). J. Endotoxin. Res. 7, 49–52. Serhan, C.N., Savill, J., 2005. Resolution of inflammation: the beginning programs the end. Nat. Immunol. 6, 1191–1197. Skaar, E.P., 2010. The battle for iron between bacterial pathogens and their vertebrate hosts. PLoS Pathog. 6 (8), e1000949, http://dx.doi.org/10.1371/journal.ppat.1000949. Taylor, J.D., Fulton, R.W., Lehenbauer, T.W., Step, D.L., Confer, A.W., 2010. The epidemiology of bovine respiratory disease: what is the evidence for predisposing factors? Can. Vet. J. 51 (10), 1095–1102. Thomas, L.H., Stott, E.J., Collins, A.P., Jebbett, N.J., Stark, A.J., 1977. Evaluation of respiratory disease in calves: comparison of disease response to different viruses. Res. Vet. Sci. 23, 157–164. USDA USDoA, 2007. Heifer calf health and management practices on U.S. dairy operations. In: APHIS (Ed.), Dairy 2007. USDA-APHIS-VS, CEAH. Wittum, T.E., Young, C.R., Stanker, L.H., Griffin, D.D., Perino, L.J., Littledike, E.T., 1996. Haptoglobin response to clinical respiratory tract disease in feedlot cattle. Am. J. Vet. Res. 57, 646–649.
Contents lists available at ScienceDirect
Veterinary Immunology and Immunopathology journal homepage: www.elsevier.com/locate/vetimm
Short communication
Acute phase proteins in naturally occurring respiratory disease of feedlot cattle Ignacio Idoate ∗ , Brian Vander Ley ∗ , Loren Schultz, Meera Heller ∗ Department of Veterinary Medicine and Surgery, University of Missouri, 900 East Campus Dr., Columbia, MO 65211, USA
a r t i c l e
i n f o
Article history: Received 14 July 2014 Received in revised form 5 December 2014 Accepted 16 December 2014 Keywords: Bovine respiratory disease Acute phase proteins Haptoglobin Lipopolysaccharide binding protein Transferrin
a b s t r a c t The aim of this study was to evaluate three acute phase proteins (APP) [haptoglobin (HPT), lipopolysaccharide binding protein (LBP) and transferrin (Tf)] in feedlot cattle with naturally occurring respiratory disease diagnosed by a calf health scoring chart (CHSC). Seventy-seven beef calves were observed for signs of Bovine Respiratory Disease (BRD) during the first 28 days after arrival at the feedlot. Fourteen cases and pen matched controls were selected based on the CHSC. BRD cases were defined as a score of ≥5, while controls were defined as a score ≤4. The mean CHSC score in cases was 6.9 which was significantly greater than the controls 2.8 (P < 0.01). Mean plasma LBP and HPT concentrations were significantly greater in cases than controls (P < 0.01). Our study results show that measurement of HPT and LBP could be useful in detecting respiratory disease in feedlot conditions. Transferrin concentrations between the two groups were not statistically different. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Bovine respiratory disease complex (BRDC) is the most common disease among feedlot cattle in the United States, accounting for approximately 75% of feedlot morbidity and 50–70% of all feedlot deaths (Edwards, 2010). BRD causes between $800 million and $900 million annually in economic losses (Edwards, 2010; Wittum et al., 1996; USDA USDoA, 2007). Economic losses stem from prevention costs, treatment, death, associated costs and from diminished average daily gain, and decreased feed efficiency. The development of BRD is multifactorial and is influenced by a combination of host, environment and pathogen factors. Susceptibility to viral and bacterial pathogens is influenced by anatomy, physiology and management of beef cattle
∗ Corresponding authors at: University of Missouri, Department of Veterinary Medicine and Surgery, 900 East Campus Dr., Columbia, MO 65211, USA. Tel.: +1 573 882 6857; fax: +1 573 884 0173. E-mail addresses: [email protected] (I. Idoate), [email protected] (B. Vander Ley), [email protected] (M. Heller). http://dx.doi.org/10.1016/j.vetimm.2014.12.006 0165-2427/© 2014 Elsevier B.V. All rights reserved.
(Taylor et al., 2010). Respiratory disease diagnosis can be confirmed using an assortment of methods. Necropsy and detection of BRD pathogens remain the gold standard tests to diagnose BRD, however use of clinical scoring systems is widespread and useful for lay people to systematically evaluate and classify sick cattle. There have been six clinical scoring systems for BRD described in the literature which relies on evaluation of a variety of clinical signs and assignment of scores based on the evaluator’s impression (Thomas et al., 1977; McGuirk, 2008; Panciera and Confer, 2010; Love et al., 2014). Bacterial infections usually lead to a strong systemic acute phase response (APR) (Alsemgeest et al., 1994), due to the marked activation of monocytes and macrophages and release of inflammatory mediators, such as histamine, leukotriene’s, prostaglandins, and proinflammatory cytokines. TNF-␣, IL-1 and IL-6 play a pivotal role in activating hepatocyte receptors (HepG2 or Hep3B cells) to initiate the synthesis of various APPs (Heinrich et al., 1990, 1998; Gruys et al., 2005). Down-regulation of the hepatic APR is achieved by rapid hepatic removal of circulating cytokines (Heinrich et al., 1990, 1998) and release
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of IL-10 by the Kupffer cells which results in suppression of local IL-6 production (Knolle et al., 1995; Baumann et al., 1994). The initial APR is down regulated once the infective organisms are isolated, cellular debris is removed, and macrophages initiate tissue repair (Serhan and Savill, 2005). HPT is a positive APP, and is the principal scavenger of free hemoglobin in blood (Murata et al., 2004). HPT elicits bacteriostatic effects by binding free Hemoglobin, thus making iron unavailable to proliferating bacteria. Iron utilization is a conserved process that has been identified in multiple bacterial pathogens, including Vibrio sp., Pseudomonas aeruginosa, Escherichia coli, Shigella flexneri, and Bacillus subtilis. (Skaar, 2010). Hpt also plays a key role in the recruitment of neutrophils in the early phase of inflammation (Riollet et al., 2000). LBP is a soluble polypeptide that binds to bacterial lipopolysaccharide (LPS) and presents the LPS to pattern recognition receptors CD14 and TLR4, found on monocytes, macrophages and granulocytes. The presentation of LPS via LBP enhances the pro-inflammatory activity of these innate immune cells by 100 to 1000 fold (Fierer et al., 2002). LBP can also bind Lipoteichoic acid (LTA), a pathogen recognition molecule exposed on the cell wall of Gram-positive bacteria. The interaction between LBP and LTA triggers a pro-inflammatory cascade via TLR-2 activation (Mogensen, 2009). Transferrin (Tf) is an iron-binding blood plasma glycoprotein found in mucosa. Tf has a single polypeptide chain of about 700 amino acids and contains two specific highaffinity Fe3+ binding sites, for the transport of iron in the circulation (Ceron et al., 2005; Oliveira et al., 2014). Tf concentrations fall during an acute phase response, making Tf a negative APP (Nguyen, 1999). Acute phase response to infection or inflammation can lead to marked anemia (Feldman et al., 1981a,b). Tf elicits indirect bactericidal effects by binding to free iron, thus making iron unavailable to proliferating bacteria, in a response that may be mediated by lipocalin (Flo et al., 2004). Previous studies using challenge models have shown that all three of these APPs may be useful biomarkers for bacterial pneumonia in calves, however no studies have been done to assess APP levels in naturally occurring pneumonia in a feedlot setting (Gånheim et al., 2003; Conner et al., 1989; Schroedl et al., 2001; Dowling et al., 2002; Heegaard et al., 2000; McNair et al., 1998). The purpose of this study was to evaluate three acute phase proteins (Haptoglobin, Lipopolysaccharide binding protein and Transferrin) in naturally occurring respiratory disease of feedlot cattle diagnosed by a calf health scoring chart (CHSC). The hypothesis was that there would be a significant difference between the acute phase protein levels evaluated, between the clinical cases and their matched healthy controls, as diagnosed by a CHSC. 2. Materials and methods 2.1. Animals 77 mixed breed beef steer calves of similar age and weight were purchased at three area livestock auction
Table 1 Adapted from McGuirk SM. 2008. Calf health scoring chart 1 0
2
3
Rectal temperature 100–100.9 ◦ F 101–101.9 ◦ F
102–102.9 ◦ F
≥103 ◦ F
Induced repeated coughs or occasional spontaneous cough
Repeated spontaneous cough
Bilateral, cloudy or excessive mucus discharge
Copious bilateral mucopurulent discharge
Small amounts of discharge
Moderate amount of bilateral discharge
Heavy ocular discharge
Ear flick or head shake
Slight unilateral droop
Head tilt or bilateral droop
Cough None
Induce single cough
Nasal discharge Normal serous Small amount discharge of unilateral cloudy discharge Eye scores Normal
Ear scores Normal
barns for enrollment in a nutritional study. Animals were transported 30–230 mile to and housed at the University of Missouri Beef Research and Teaching Farm (BRTF). At arrival the animals were weighed, ear tagged [both Allflex visual identification (VID) & Radio-frequency identification (RFID)], vaccinated with Bovi-Shield Gold® 5 and One Shot Ultra® 8 (Zoetis, Florham Park, NJ, USA) and treated with Cydectin® Pour-On (Boehringer Ingelheim Vetmedica, Inc., St. Joseph, MO, USA). All procedures were approved by the University of Missouri Institutional 116 Animal Care and Use Committee. The calves were part of a post-weaning feedlot performance and feed efficiency tests using the GrowSafe automated feeding system (GrowSafe Systems Ltd., Airdrie, Alberta, Canada). All calves were housed six per pen. Pens were of open construction, measuring 4.9 × 8.84 m. Frost-free waterers were shared between two pens, and feed bunks and slabs were under sloped roof shades (4.9 × 6.7 m). 2.2. Clinical examination Bovine respiratory disease was diagnosed by an experienced animal technician based on a CHSC health scoring chart developed at the University of Wisconsin. The CHSC assigns each calf the sum of the nasal discharge, rectal temperature, cough scores and the greater one of the two scores from the ocular discharge and head/ear carriage (Table 1, McGuirk, 2008). Calves whose total score was ≥5 were categorized “BRD positive”, while controls were defined as a score ≤4. The cattle were monitored closely on a daily basis and the scoring based on the CHSC was done in the mornings to mimic common practices in commercial in feedlots. Fourteen steers were selected as cases based on their score and were not diagnosed with any other diseases during the study. Control animals (n = 14) were selected from contemporary pen mates and sampled at the same time point.
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Fig. 1. The figures represent higher BRD scores and LBP and HPT concentrations for cases versus controls in the study.
2.3. Samples Blood samples were collected from BRD clinical cases and their matched healthy controls selected from the same pens, using the coccygeal veins at the time of the classification based on the CHSC. The blood was allowed to clot at 4 ◦ C. After clotting, the samples were centrifuged at 2800 × g for 20 min at 4 ◦ C. Serum was harvested and frozen at −80 ◦ C for later analyses. 2.4. Acute phase proteins analysis Concentration of LBP was assayed using a commercially available Human LBP ELISA kit (Cell Sciences Inc., Canton, MA) that recognizes both human and bovine LBP, as described previously (Bannerman et al., 2003). Plasma samples were diluted 1:100 and assayed according to the manufacturer’s instructions. The detection range for the LBP ELISA kit is 0.05–2.5 g/ml. HPT concentrations were determined with a commercially available Bovine Haptoglobin ELISA kit (Immunology Consultants Laboratory, Inc., Portland, OR) according to the manufacturer instructions. Samples were diluted at 1:50, 1:200. 1:1000 and 1:2000 ratios and assayed according to manufacturer’s instructions. The detection range for
the HPT ELISA kit is 15.6–1000 ng/ml. Concentrations were converted from ng/ml to mg/ml to make the numbers more manageable. Transferrin concentrations were determined with a commercially available Bovine Transferrin ELISA kit (Immunology Consultants Laboratory, Inc., Portland, OR) according to the manufacturer’s instructions. Samples were diluted at 1:40,000 and assayed according to manufacturer’s instructions. The detection range for the Tf ELISA kit is 18.75–6000 ng/ml. Concentrations were converted from ng/ml to mg/ml to make the numbers more manageable. 2.5. Statistical analysis Differences in BRD scores, LBP, HPT and Tf between cases and contemporary control animals were analyzed and carried out using Stata® 13 software analysis systems (StataCorp LP, College Station, Texas, USA). The Shapiro test was used to appraise normal distribution. Since all values were not normally distributed or in the case of LBP and BRD scores were normally distributed but had unequal variances, a non-parametric test, Kruskal–Wallis equalityof-populations rank test was performed. A P-value 0.001 was used as the level of significance for all tests.
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Fig. 2. The two graphs show ROC curves for LBP and HPT shows ROC curves in comparison to a calf heath scoring chart. The tests were able to accurately separate BRD cases from controls. An area of 1 represents a perfect test; an area of 0.5 represents a worthless test.
3. Results and discussion BRD scores, serum LBP and HPT concentrations were considerably higher for BRD clinical cases than for their matched healthy controls (see Fig. 1) as diagnosed by a CHSC scoring chart. The differences were statistically significant between cases and controls (P = 0.001). The negative APP Tf evaluated in our study, was not significantly different between cases (8.93 mg/ml) and controls (9.64 mg/ml) (P = 0.5656). Our study result shows that measurement of HPT and LBP is associated with clinically diagnosed respiratory disease under field conditions, and
the levels correlate with those previously described in challenged experimental studies (Gånheim et al., 2003; Conner et al., 1989; Schroedl et al., 2001; Dowling et al., 2002; Heegaard et al., 2000; McNair et al., 1998). Schroedl et al. (2001) showed an early significant increase in LBP concentration in a group of 20 calves experimentally infected with Gram-negative Mannheimia haemolytica 6 h after bacterial inoculation. In this same study, Hpt levels did not significantly rise before 12 h. Gånheim et al. (2003) studied HPT levels in calves experimentally challenged with bovine viral diarrhea virus (BVDV) and/or M. haemolytica, demonstrating HPT levels similar to the ones found in our study.
I. Idoate et al. / Veterinary Immunology and Immunopathology 163 (2015) 221–226 Table 2 The table represents the ultimate cut point concentrations 512 for HPT and LBP. APP
Cut point
Sensitivity (%)
Specificity (%)
Correctly classified
Hpt LBP
≥0.81 (mg/ml) ≥0.33 (g/ml)
92.86 92.86
85.71 92.86
89.29 92.86
Conner et al., 1989 and Dowling et al., 2002, challenged calves experimentally with M. haemolytica and Pasteurella multocida, respectively. In both studies, the concentration levels were significantly higher in the challenged animals versus the study controls. Receiver operating characteristic (ROC) curves were calculated in our study to determine an ideal cutoff value for LBP and HPT concentrations. The ROC curve is created by plotting the fraction of true positives out of the total actual positives (TPR = true positive rate) vs. the fraction of false positives out of the total actual negatives (FPR = false positive rate), at various threshold settings. Sensitivities, specificities and percent correct classifications are listed as a function of cutoff value in Table 2. Another important measure of the accuracy of the clinical test is the area under the ROC curve. A perfect classification is an area that equals to 1.0. This test is 100% accurate because both the sensitivity and specificity are 1.0 so there are no false positives and no false negatives. The area under ROC curve for HPT in our study was 0.9235 and for LBP was 0.9668 (Fig. 2). This data suggests a very accurate classification for both LBP and HPT ideal concentration cutoffs for our study. On the other hand a test that cannot discriminate between normal and abnormal corresponds to an ROC curve 304 that is 0.5. The calculated area under ROC curve for Tf was 0.4674, suggesting that Tf concentrations were not accurate in differentiating BRD cases from healthy controls. In our study, the WI calf health scoring chart used to diagnose BRD ended up being user friendly and efficient at differentiating BRD cases from controls, even in a feedlot setting, even though it was originally designed for neonatal dairy calves. The system proved efficient discriminating cases and controls in our study, as it did in the study by Love et al. (2014) where they looked at developing a novel clinical scoring system for on-farm diagnosis of bovine respiratory disease in pre-weaned dairy calves. Other clinical scoring systems are also available for diagnosing BRD. DART (Depression, Appetite, Respiration and Temperature) is a clinical scoring system developed to identify beef cattle for BRD treatment in feedlots, but it is difficult to standardize because the clinical sign weights and decision points are not defined (Panciera and Confer, 2010). Love et al. (2014) developed three additional scorning systems where the individual assesses the presence or absence of ocular discharge, nasal discharge, ear droop or head tilt, respiratory quality and spontaneous coughing. This scoring system determines an animal to be BRD positive, when it has an abnormal ear or head carriage, or calves with nasal discharge and one other clinical sign, or calves that have any three clinical signs. These clinical scoring tools are of practical interest in the cattle industry because they are based on clinical signs that
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can be easily identified by producers. Buczinski et al. (2014) showed that adding the clinical score assessment to thoracic auscultation and thoracic ultrasonography, increased sensitivity of BRD diagnosis in pre-weaned Dairy Calves, although it decreases specificity when compared with lung consolidation findings. The recommendation of the WI CHSC is to treat calves because of high BRD presumption if the score is ≥5, and to monitor calves with scores of 4. Calves with scores of ≤3 are considered healthy. Although APPs appear to be very sensitive, we must also recognize that they are also non-specific, and potential confounding factors, such stress, age of the animal, and presence of concurrent diseases should be considered while deciphering results. When bovine respiratory disease (BRD) outbreaks occur, veterinarians might steer away from expensive or complex ante mortem diagnostics requiring long turnaround times. Instead, the initial actions are often directed toward electing the appropriate therapy. There is a need for the development and optimization of rapid diagnostic field tests to guide decisions regarding vaccination, metaphylaxis, or treatment programs. Monitoring of LBP or HPT could be a useful tool to help direct these on farm decisions.
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