İshalli Buzağılarda Serum Haptoglobin ve Sitokin Konsantrasyonunun Belirlenmesi

İshal buzağılar için önemli bir problem olması nedeniyle, bu çalışmada yangı medyatörü olarak bilinen Haptoglobin (Hp), Interleukin-1β (IL-1β), Interleukin-6 (IL-6) ve Tumor Necrosis Factor–α (TNF-α) seviyeleinin ishalli ve sağlıklı buzağılarda belirlenmesi amaçlanmıştır. Sunulan çalışmada 0-3 aylık yaşta 40 adet buzağı kullanıldı. Buzağıların klinik, sistemik ve hematolojik muayeneleri yapıldıktan sonra iki gruba ayrıldı. Klinik olarak ishal teşhisi (n=20) konulan buzağılar çalışma grubuna, sağlıklı buzağılar ise kontrol (n=20) grubuna dahil edildi. Yapılan çalışma sonucunda ishalli buzağılarda, Haptoglobin (Hp), Interleukin-1β (IL-1β), Interleukin-6 (IL-6) and Tumor Necrosis Factor–α (TNF-α) konsantrasyonları kontrol grubuna göre istatistiksel açıdan önemli düzeyde yüksek (P<0.001) olduğu tespit edildi. Ayrıca rektal vücut ısısı ve total lökosit sayısı (WBC), hematocrit değer (Hct), Gamma glutamyltransferase (GGT) seviyeleri ishalli buzağılarda yüksekti. Eritrosit sayısı (RBC), hemoglobin miktarı (HB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP) ve albumin (ALB) konsantrasyonların da gruplar arasında istatistiksel bir fark tespit edilmedi.

Since diarrhea is a significant problem in calves, the present study was undertaken to determine haptoglobin (Hp), Interleukin-1β (IL-1β), Interleukin-6 (IL-6) and Tumor Necrosis Factor–α (TNF-α) levels, which are known as inflammation mediators, in calves with diarrhea and healthy calves in order to identify the inflammatory condition caused by diarrhea. In the present study, 40 calves between 0-3 months of age were used. After clinical, systemic and haematological examinations, the calves were divided into two groups. Clinically diagnosed calves with diarrhea (n=20) composed the study group whereas healthy calves (n=20) were used as control group. As a result, the levels of Haptoglobin (Hp), Interleukin-1β (IL-1β), Interleukin-6 (IL-6) and Tumor Necrosis Factor–α (TNF-α) in the serum were statistically higher (P<0.001) in calves with diarrhea compared to the control group. Moreover, rectal body temperatures and the values of White blood cell (WBC), Hematocrit (Hct), Gamma glutamyltransferase (GGT) were also determined high in calves with diarrhea. Interms of Red blood cell (RBC), Hemoglobin (HB), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Total Protein (TP) and Albumin (ALB) concentrations, no statistical difference were found between the two groups.

Introduction

Diarrheal diseases lead to significant economic losses in that they cause high mortality and growth retardation in calves[1]. Diarrhea in calves adversely affects breeders economically by resulting in poor performance, increased medication costs and waste of efforts[2,3]. There is a wide variation in the incidence of calfhood diseases with substantial impacts on many commercial dairy operations. In addition to the cost of treating sick calves, the economic consequences may include increased mortality, reduced growth, and increased age and difficulty at first calving[4,5].

The synthesis of Acute Phase Proteins (APP) from the liver and their release into the bloodstream are initiated by the stimulation of proinflammatory cytokines[6]. Pro-inflammatory cytokines such as Tumor necrosis factor- α (TNF-α), interleukin-1 (IL-1) and interleukin-6 (IL-6) have been noted to be the main ingredients that are necessary to initiate systemic inflammatory response[7-9]. Acute Phase Response (APR) is a response which occurs after infection, inflammation, immunological disorders, trauma or neoplasia in the organism, and this response has been reported to characterize with metabolic and systemic changes[10,11]. Conducted researches have stated that haptoglobin (Hp) and Serum Amyloid A (SAA) are the most important acute phase proteins in ruminants as acid glycoprotein (AGP) is moderately important[9,12]. Whereas the amount of APP in plasma concentrations is associated with the severity and the activity of inflammation, determining the levels of APPs in circulation provides information about the ongoing inflammatory reaction.

That Hp, which is at low levels in healthy calves, is produced at high concentrations during APR, it is regarded as a specific APP for animals[13]. Serum or plasma Hp concentration has been reported to increase following the natural or experimental infection, inflammation or trauma in calves[14-16]. Previous studies have indicated that Hp concentration in calves is quite an important parameter for the diagnosis of bacterial[17] and viral[18] diseases, and it significantly increases during these diseases.

This study has aimed to determine serum Haptoglobin (Hp), Interleukin-1β (IL-1β), Interleukin-6 (IL-6) and TNF-α levels in clinically healthy and diarrheic calves.

Material and Method

Animal Material and Clinical Examination: The material in this study consisted of 40 calves at the age of 0-3 months. Twenty calves that are clinically diagnosed as diarrheic, formed the study group and the remaining 20 healthy ones were used as the control group.

Measurement of Haptoglobin and Cytokines: Haptoglobinn (Life Diagnostics Inc. Bovine Haptoglobin Test Kit), IL-1β (Cusabio Biotech CO., Ltd. Bovine Interleukin 1β), IL-6 (Cusabio Biotech CO., Ltd. BovineInterleukin 6 Test Kit) and Tumor Necrosis Factor-α (Cusabio Biotech CO., Ltd. Bovine TNF-α ELISA kit) measurements were made by using commercial kits and in ELISA device (AwarenessTechnology, Inc. U.S.A. Chem Well).

Hematological and Biochemical Examinations: Blood samples were taken from jugular vein. White blood cell (WBC), red blood cell (RBC), hemoglobin (HB), and hematocrit (Hct) were determined using cell counter (Compteur Analyseurd Hematologie MS9-3).

After the anticoagulant-free blood samples obtained for biochemical parameters, these samples centrifuged at 5000 rpm at room temperature, the serum were separated and stored at -20°C until the time of measurement. Alanine aminotransferase (BIOLABO SA ALT Test Kit), aspartate aminotransferase (BIOLABO SA AST Test Kit), gamma glutamyltransferase (BIOLABO SA GGT Test Kit), Total Protein (BIOLABO SA TP Test Kit), and Albumin (BIOLABO SA ALB test kit) measurements of the serum were made with commercial kits using ELISA reader (Awareness Technology, Inc. U.S.A. Chem Well).

Statistical Analyzes: In the statistical analysis of this study, PASW Statistics 18 software package program was used. Kolmogorov-Smirnov test and normal distribution test were applied on the data, and it was observed that a uniform distribution was not the case. Considering the number of the samples, Mann-Whitney U test was conducted for the intergroup comparisons of each parameter. The level of significance was determined as P<0.05.

Results

In our study, serum Hp, IL–1β, IL–6 and TNF-α concentrations were assessed. Serum Hp concentration was seen to be statistically lower in the control group than in the study group (P<0.001). Whereas serum Hp concentration was detected as 6.81±0.58 in the control group. It was seen to be higher in the study group than in the control group by reaching up to 195.88±32.24 levels (Table 1). In the study, serum IL–1β concentration was detected to be statistically lower in the control group than in the study group (P<0.001). Serum IL-1 concentration was at the level of 19.68±3.98 in the control group as it was 58.35±5.17 in the study group. Serum IL-6 concentration was statistically lower in the control group than in the study group (P<0.001). Serum IL-6 concentration was at the level of 8.02±1.49 in the control group as it was 28.75±4.21 in the study group (Table 1). Serum TNF-α concentration was found to be lower in the control group than in the study group. Serum TNF-α concentration was at the level of 0.14±0.02 in the control group while it was 0.36±0.05 in the study group (Table 1).

A statistical difference in rectal body temperature was detected between the study group and the control group (P<0.001). In the hematological examination, WBC (x10⁹/L), RBC (x10¹²/L), HB (g/dL) and Hct(%) concentrations were evaluated. In this study, WBC and Hct concentration was detected to be statistically (P<0.01) lower in the control group than in the study group. No significant differences were determined in diarrheic calves compared to the control group with concern to RBC and HB concentrations (Table 2). In the presented study, serum ALT, AST, GGT, TP, ALB levels were also evaluated. Serum GGT concentration was determined to be statistically lower (P<0.001) in the control group than in the study group. No significant differences were determined in diarrheic calves compared to the control group with concern to ALT, AST, TP and ALB concentrations (Table 3).

Discussion

In a study conducted on diarrheic calves, it was ascertained that rectal body temperature increased depending on the infection and inflammation[19]. In our study as well, rectal body temperature was determined to be higher in the diarrheic calves compared to the control group. This incident might be related to the fact that the diarrhea result from infections.

It has been reported that serum or plasma Hp concentration has increased in calves after naturally or experimentally generating infection or inflammation[14-16,20]. A statistical increase in Hp concentration was noted in the study group compared to the control group in our study as well. In our study, while serum Hp concentration was measured as 6.81±0.58 μg/mL in the control group, it was 195.88±32.24 μg/mL in the study group. We believe that the high concentration in the study group arises due to infectious factors. Some researchers reported that serum Hp concentration has increased during infectious and inflammatory diseases[21,22].

It has been reported that TNFα, IL-1β and IL-6 play a key role in APR[9,23,24]. It has been reported that the synthesis of APPs from the liver cells is initiated by the pro-inflammatory cytokines (TNFα, IL-1β and IL-6) released from monocytes and macrophages during inflammation[7]. In the presented study, serum IL-1β, IL-6 and TNFα concentrations in calves were detected to be statistically higher in the study group (IL-1β: 58.35±5.17, IL-6: 28.75±4.21 TNFα: 0.36±0.05) in comparison to the control group (IL-1β: 19.68±3.98, IL-6: 8.02±1.49, TNFα: 0.14±0.02). Serum IL-1β concentration in calves which Bovine viral diarrhea (BVD) disease generated experimentally was seen to be higher in the study group than in the control group on the 9th day of the disease. In the same study, serum TNFα concentration was determined to decrease in the study group in comparison to the control group on the 9th day of the disease[25]. In an another study, calves were experimentally administered with intravenous endotoxin, and after the administration; serum IL-1β, IL-6 and TNFα concentrations were seen to be higher in comparison to the control group[20]. In a previous study, it has been reported that IL-1β concentration in calves diagnosed with pneumonia could increase 6-fold more than the healthy ones[26]. Risalde et al.[19] have determined that IL-1β, TNFα concentrations in diarrheic calves were higher than the control group. In our study, serum IL-1β, IL-6 and TNFα concentrations were determined to be higher in the study group than in the control group. We suggest that this incident resulted from infectious agents which caused diarrhea.

Some researchers have reported an increase in WBC values in the calves with diarrhea[27,28]. In another study, it has been noted that WBC figures markedly increased in the calves with diarrhea; and leukocytosis stemming from the relative increase of neutrophil granulocytes has occured as a result of the reaction of the body against gastrointestinal infection[29]. In our study we have also believed that the increase in WBC values in the calves with diarrhea arises from gastrointestinal infection. It has been reported that in calf diarrhea serum Hct and plasma protein levels increase owing to the decrease in extracellular fluid volume[30]. In our study, Hct concentration was determined to be higher in the study group diagnosed with diarrhea than in the control group, as well. In the study, no statistical difference (P>0.05) was detected between the study and the control groups in terms of RBC and Hb concentrations.

Sing and Sodhi[31] have reported that there was an increase in serum ALT and AST levels in the calves with diarrhea. In other studies, it has also been ascertained that an increase in serum ALT and AST concentrations was found in the calves clinically diagnosed with diarrhea comparing to the healthy ones[32,33]. In this study, even though an increase in serum ALT and AST concentrations was determined in the study group in comparison to the control group, this increase was noted as not statistically significant. In our study, serum GGT concentration was determined to be statistically higher in the study group (34.29±3.59 IU/L) than in the control group (17.62±1.51 IU/L). It has been reported that serum GGT concentration could be higher than 200 IU/L in both diarrheic calves and the healthy ones, and this could result from the amount of colostrum that the calves take[34]. In the presented study, no statistically significant increase in serum TP and ALB concentrations was detected in the study group compared to the control group.

In conclusion, regarding the results derived from our study, it has been determined that the increase in the serum Hp concentration in the calves with diarrhea has accompanied clinical symptoms of the calves naturally infected with diarrhea. Moreover, it has been detected that the serum Hp in the healthy animals of the control group was at low concentrations. In the light of these results, we suggest that routine measurement of serum Hp IL-1β, IL-6, and TNF-α levels in the serum and are the most valuable parameters to evaluate the course of the disease and prognosis in the field of veterinary medicine.

References

1. Ranja R, Naresh R, Patra RC, Swarup D. Erythrocyte lipid peroxides and blood zinc vs copper concentrations in acute undifferentiated diarrhoea in calves. Vet Res Commun 2006; 30: 154-249.
2. Mohri M, Sharifi K, Eidi S. Hematology and serum biochemistry of Holstein dairy calves: Age related changes and comparison with blood composition in adults. Res Vet Sci 2007; 83: 30-39.
3. Smith DR. Field disease diagnostic investigation of neonatal calf diarrhea. Vet Clin North Am Food Anim Pract 2012; 28: 465-481.
4. Sivula NJ, Ames TR, Marsh WE, Werdin RE. Descriptive epidemiology of morbidity and mortality in Minnesota dairy heifer calves. Prev Vet Med 1996; 27: 155-171.
5. Stanton AL, Kelton DF, LeBlanc SJ, Wormuth J, Leslie KE. The effect of respiratory disease and a preventative antibiotic treatment of growth, survival, age at first calving, and milk production of dairy heifers. J Dairy Sci 2013; 95: 4950-4960.
6. Ceron JJ, Eckersall PD, Martinez-Subiela S. Acute phase proteins in dogs and cats: Current knowledge and future perspectives. Vet Clin Pathol 2005; 34: 85-99.
7. Baumann H, Gauldie J. The acute phase response. Immunol Today 1994; 15: 74-80.
8. Murtaugh MP, Baarsch MJ, Zhou Y, Scamurra RW, Lin G. Inflammatory cytokines in animal health and disease. Vet Immunol Imminopathol 1996; 54: 45-55.
9. Ceciliani F, Ceron JJ, Eckersall PD, Sauerwein H. Acute phase proteins in ruminants. J Proteomics 2012; 75: 4207-4231.
10. Gruys E, Obwolo MJ, Toussaint M. Diagnostic significance of the major acute phase proteins in veterinary clinical chemistry. J Zhejiang Univ Sci B 1994; 64: 1009-1018.
11. Petersen HH, Neilsen JP, Heegard PMH. Application of acute phase protein measurements in veterinary clinical chemistry. Vet Res 2004; 35: 163-187.
12. Eckersall PD, Bell R. Acute phase proteins: Biomarkers of infection and inflammation in veterinary medicine. Vet J 2010; 185: 2-27.
13. Nazifi S, Rezakhani A, Koohimoghadam M, Ansari-Lari M, Esmailnezhad Z. Evaluation of serum haptoglobin in clinically healthy cattle and cattle with inflammatory diseases in shiraz, a tropical area in Southern Iran. Bulg J Vet Med 2008; 11: 95-101.
14. Alsemgeest SPM, Kalsbeek HC, Wensing TH, et al. Concentrations of serum amyloid A (SAA) and haptoglobin (Hp) as parameters of inflammatory disease in cattle. Vet Q 1994; 16: 21-23.
15. Heegard PMH, Godson DL, Toussaint MJM. The acute phase response of haptoglobin and serum amyloid A in cattle undergoing experimental infection with bovine respiratory syncytial virus. Vet Immunol Immunopathol 2000; 77: 151-159.
16. Fisher AD, Knight TW, Cosgrove GP, Death AF. Effects of surgical or banding castration on stress responses and behaviour of bulls. Aust Vet J 2000; 79: 279-284.
17. Skinner JG, Brown RAL, Roberts L. Bovine haptoglobin response in clinically defined field conditions. Vet Rec 1991; 128: 147-149.
18. Höfner MC, Fosbery MW, Eckersall PD, Donaldson AL. Haptoglobin response of cattle infected with foot-mouth disease virus. Res Vet Sci 1994; 57:125-128.
19. Risalde MA, Molinaa V, Sánchez-Cordóna PJ, et al. Response of proinflammatory and anti-inflammatory cytokines in calves with subclinical bovine viral diarrhea challenged with bovine herpesvirus-1. Vet Immunol Immunopathol 2011; 144: 135-143.
20. Carroll JA, Arthington JD, Chase CC. Early weaning alters the acute-phase reaction to an endotoxin challenge in beef calves. J Anim Sci 2009; 87: 4167-4172.
21. Makimura S, Usui M. Correlation between haptoglobin and sialic acid or mucoprotein in dieased bovine serum. J Vet Med Sci 1990; 44: 15-21.
22. Skinner JG, Roberts L. Haptoglobin as an indicator of infection in sheep. Vet Rec 1994; 134: 33-36.
23. Ingenbleek M, Young V. Transthyretin (prealbumin) in health and disease: nutritional implications. Annu Rev Nutr 1994; 14: 495-533.
24. Heinrich PC, Behrmann I, Muller-Neven G, Schaper F, Graeve L. Interleukin-6-type cytokine signalling through the gp130/Jak/STAT pathway. Biochem J 1998; 334: 297-314.
25. Molina V, Risalde MA, Sanchez-Cordon PJ, et al. Cell-Mediated immune response during experimental acute infection with bovine viral diarrhoea virus: evaluation of blood parameters. Transbound Emerg Dis 2014; 61: 44-59.
26. El-Bahr SM, El-Deeb WM. Acute phase proteins, lipid profile and proinflammatory cytokines in healthy and bronchopneumonic water buffalo calves. Am J Biochem Biotechnol 2013; 9: 34-40.
27. Şahal M, Kurtdede A, Börkü MK, et al. Treatment of newborn diarrheic calves with sodium bicarbonate and saline solution with respect to clinical signs and acid-base balance. Ankara Univ Vet Fak J 1994;41: 509-525.
28. Öcal N, Duru SY, Yağcı BB, Gazyağcı S. Field condition diagnosis and treatment of acid-base balance disorders in calves with diarrhea. Kafkas Univ Vet Fak J 2006; 12: 175-183.
29. Coles EH. Veterinary Clinical Pathology. 2nd Edition, Philadelphia, USA: W.B. Saunders Company,1986.
30. Vermunt JJ. Rearing and management of diarrhea in calves to weaning. Aust Vet J 1994; 71: 33-41.
31. Sing OY, Sodhi SP. Effect of shock of certain biochemical profiles of blood in calves. Ind J Anim Sci 1992; 62: 1031-1036.
32. Karademir B, Şendil Ç. Clinical, haemotological and biochemical findings of diarrhea caused by E.Coli and the efficancy of treatment applications calf. Kafkas Univ Vet Fak J 2001; 7: 175-183.
33. Pekcan M, Altintas A, Karagul H, et al. Serum biochemistry and native protein electrophoresis in diarrheic calves with arthritis. Acta Vet (Beograd) 2012; 62: 261-269.
34. Braun RK, Tennant BC. The relationship of serum gamma globulin levels of assembled neonatal calves to mortality caused by enteric diseases. Agri-Practice 1938; 4: 14-24.