Praca oryginalna Original paper
Pneumonia is an important disease of the respira-tory system commonly seen in livestock. The disease is caused by various viruses and bacteria, either alone or in combination, as well as by various precipitating factors (16, 30). It is known that many inflammatory mediators and acute phase proteins are produced by the activation of various defense mechanisms in response to pathogens that play a role in the development of the disease entering the alveoli (27). The purpose of this inflammatory response is to control the progress of the infection and to kill microorganisms. The anti-inflammatory cytokines released in this process, such as tumor necrosis factor (TNF-α) and interleukin (IL-1, IL-6 and IL-8), are pro-inflammatory (5). The release of cytokines varies depending on the type of pathogens
(bacteria, viruses) (14). It has been reported that the re-leased cytokines are responsible for the release of acute phase proteins in the liver and that there is a positive correlation between cytokine levels and acute phase protein levels (16). Increase in cytokines, especially IL-1, IL-6, and TNF-α1, in acute and chronic inflam-matory events causes a release of acute phase proteins in the liver (3, 5, 7, 15, 29). It has been reported that haptoglobin (HPT) and serum amyloid A (SAA) are important acute phase proteins in cattle, while fibrino-gen is not specific in cattle (16). It has been reported, however, that there are significant changes in blood levels of fibrinogen, C-reactive protein (CRP), hapto-globin, ceruloplasmin, ferritin, and serum amyloid A in instances of infection, inflammation, and trauma in cattle (9, 11, 16, 29). Changes in the levels of acute phase proteins have been shown to provide important
1) This study was financed by Van Yuzuncu Yil University, Scientific Research Projects Coordination Unit, ID number 1762 and project number 2015-VF-B134.
Evaluation of acute phase protein levels
and some cytokine levels in pneumonic calves
1)
OMER AKGUL, SÜLEYMAN KOZAT*, CUMALI OZKAN*, ABDULLAH KAYA, YAKUP AKGUL
Department of Microbiology, Faculty of Pharmacy, Van Yuzuncu Yil University, Zeve Campus Van-Turkey *Department of Internal Diseases, Faculty of Veterinary Medicine, Van Yuzuncu Yil University, Zeve Campus Van-Turkey
Received 03.04.2018 Accepted 27.09.2018
Akgul O., Kozat S., Ozkan C., Kaya A., Akgul Y.
Evaluation of acute phase protein levels and some cytokine levels in pneumonic calves Summary
This study was carried out to determine the levels of various cytokines (TNF-α, IL-6, IL-8) and acute phase proteins (haptoglobin, C-reactive protein, serum amyloid A, alpha 1-acid glycoprotein, lactoferrin, and fibrinogen) in pneumonic calves, to reveal changes in these parameters depending on etiologic factors (bacterial, viral, bacterial + viral) in calf pneumonia, and to determine whether these parameters could be used for diagnosis and prognosis of the disease. The study was carried out on a total of 50 calves consisting of 10 healthy and 40 pneumonic animals aged 1 to 6 months. The pneumonic calves were classified into three groups: bacterial, viral and mixed (bacterial + viral). It was found that the levels of IL-6, IL-8, CRP, and TNF-α were statistically higher in the pneumonic calves than in the control group animals. It was found that the increase in levels of Serum IL-6, IL-8, and CRP were highest in the mixed infection group (bacterial + viral) and lowest in the virally infected group. Similarly, serum levels of Hp, CRP, SAA, α-1-AGP, and LF were found to be more statistically significant (P < 0.01) in the infected calves than in the control group animals, but changes in fibrinogen levels were not statistically significant. Although the TP and ALB levels in the pneumonic calves were numerically higher than the same parameters in the control group, this was not statistically significant (P > 0.05). As a result, this study showed that serum levels of cytokines (IL-6, IL-8, TNF-α) and serum concentrations of acute-phase proteins (Hp, CRP, SAA, α-1-AGP, and LF) are significantly higher in the pneumonic calves than in the control group calves. Furthermore, the assessment of cytokine and acute phase protein levels can play an important role in the early diagnosis of calf pneumonia. This early diagnosis can help prevent deaths caused by pneumonia, which is the leading cause of major losses, and can also be helpful in the early treatment of pneumonia in the veterinary clinical field.
clues about the acute phase response and the progres-sion of many diseases (27). It has been noted that changes in acute phase protein levels can be treated as indicators of various inflammatory, infectious and traumatic conditions in human medicine today (5).
In studies of viral and bacterial respiratory infections in cattle, it was found that changes in levels of acute phase proteins were indicative of the level of inflam-mation (14, 16). It is believed that acute phase proteins can be used in the differentiation of bacterial and viral infections, in the differential diagnosis of clinical, sub-clinical, acute and chronic diseases, and in determining the prognosis of disease in infected animals throughout the course of treatment (16).
This study was carried out to determine the levels of various cytokines (TNF-α, IL-6, IL-8) and acute phase proteins (haptoglobin, C-reactive Protein, serum amyloid A, alpha 1-Acid glycoprotein, lactoferrin, and fibrinogen) in pneumonic calves, to reveal changes in these parameters depending on etiologic factors (bacte-rial, viral, and bacterial + viral) in calf pneumonia, and to determine whether these parameters could be used for diagnosis and prognosis of the disease.
Material and methods
The animal material for this study comprised a total of 50 calves: 40 pneumonic Simmental calves aged 1-6 months that had been brought to the clinic of the Veterinary Faculty of Internal Medicine at Van Yuzuncu Yil University with respiratory complaints and 10 healthy calves of the same age, making up the control group. Blood samples were taken on the first day of illness. General clinical examina-tions of the sick and control group calves were performed, and the body temperature, respiratory rate, and heart rate of all calves were determined. In addition, information was obtained through anamnesis about any previous diseases and antibiotic treatment applied. As a result of the anamne-sis, previously infected and antibiotic-treated calves were excluded from the study.
Blood samples were taken from the jugular vein of all calves included in the study using anticoagulant (EDTA, K3EDTA) and anticoagulant-free tubes so that their hema-tological and biochemical parameters could be analyzed prior to the study.
Hematological analysis. The blood samples collected
into anticoagulant tubes were taken to our Internal Medicine Department Laboratory on the same day, and hematological parameters (RBC, WBC, Lym, Mon, Neu, Eo, MCV, Hct, MCHC, Hb, PLT) were determined using a blood count device (Veterinary MS4-S-Melet Schloesing Laboratories in France).
Biochemical analysis. In order to analyze their
biochemi-cal parameters, the blood samples in the anticoagulant-free tubes were centrifuged at 1400 × g for 10 min (Rotofix32®
-Hettich), and their serums were removed. The serums were then kept at –20°C until measurements could be made. Bio-chemical parameters, that is, the levels of serum acute phase proteins (haptoglobulin, CRP, SAA, α1-AGP, lactoferrin (LF), fibrinogen) and cytokine (IL-6, IL-8, TNF-α), were
determined using an ELISA device (ELISA reader®-DAS)
according to the method described in commercial test kits. TP and albumin levels were measured with an autoanalyzer (BS-120 Vet-Mindray) in our Department.
The following commercial test kits were used: Bovine Haptoglobin ELISA Kit (Catalog No.: 201-04-0121) for haptoglobulin levels, Bovine Lactoferrin ELISA Kit (Cata-log No.: 201-04-0119) for lactoferrin levels, Bovine SAA ELISA Kit (Catalog No.: 201-04-0126) for serum amyloid A (SAA) levels, Bovine alpha1-AGP ELISA Kit (Catalog No.: 201-04-0120) for serum alpha1-acid glycoprotein (α1-AGP) levels, Bovine Fibrinogen ELISA Kit (Catalog No.: 201-04-0086) for serum levels of fibrinogen (FG), Bovine ELISA Kit (Catalog No.: CK-E91671) for C-reac-tive protein (CRP) levels, Bovine Interleukin 6 ELISA Kit (Catalog No.: CK-E90280) for interleukin (IL-6) levels, Bovine Interleukin 8 ELISA Kit (Catalog No.: CK-E90249) for interleukin (IL-8) levels, and Bovine TNF-α ELISA Kit (Catalog No.: CK-E90277) for TNF-α levels. As per the instructions in the test kits, they were measured and analyzed with the ELISA device.
In addition, antibody levels in the serum samples were determined in order to establish the presence of factors in the control group calves as well as those calves that had been brought in with respiratory complaints and diagnosed as having pneumonia on the basis of clinical findings. This was done by making a serological analysis of the serum samples with the ELISA reader®-DAS in our laboratory
following procedures described in the commercial test kits (Mycoplasma Bovis ELISA commercial test kit, Bio-X Diagnostics, Catalog No: BIO K 260/2; Adenovirus ELISA commercial test kit, Bio-X Diagnostics, Catalog No. BIO K 063/2) to isolate factors indicating the presence of myco-plasma or adenovirus. According to the adenovirus test kit method, any antibody titer of +2 and above is considered positive. Furthermore, the coefficient of each sample was calculated by the Mycoplasma bovis test kit method. Coef-ficient values higher than 37% were considered positive. Those that were lower than 37% were considered negative.
Microbiological analysis. In order to isolate certain
factors and identify them micriobiologically, blood was taken from both the control and pneumonic calves after shaving and cleaning the area of the jugular vein with 70% alcohol and then sterilizing it with an antiseptic solution (povidone-iodine). Wearing sterile gloves, 5 mg each of blood was drawn into aerobic and anaerobic culture vials, which were then sent to the Medical Faculty’s Department of Microbiology and Medicine. The aerobic vials were left to incubate for at least 24 hours in a blood culture device, while the anaerobic vials were left for seven days. Aerobic and anaerobic culture procedures were carried out on vials identified as positive.
Samples from the aerobic bottles were planted on Blood Agar, Eosin Methylene Blue Agar, and MacConkey Agar. They were left to incubate at 37°C for 24 hours. Colony morphology, gram staining, catalase, oxidase, and PYR (L pyronidonyl-beta- naphthylamide) tests were performed. The BD Phoenix Automated Identification Device was used to identify the isolated bacteria. Samples taken from the
anaerobic bottles were planted on the anaerobic Schaedler Broth medium. The mediums were left to incubate at 37°C for seven days. Samples from the tubes were added to anaerobic agar. They were left to incubate at 37°C for 24 hours. Gram staining was performed on the colonies. The isolates were identified on the BBLCRYSTAL ANR panel.
Statistical analysis. Descriptive statistics were made
for the hematological and biochemical parameters obtained from calves in the control and study groups. The mean and standard deviation values of these parameters were deter-mined. The one-way variance analysis (One-way) test was used to compare the parameters of the study group calves because some of the parameters in those calves (bacterial, viral, bacterial + viral) deviated from a normal distribution. An independent t-test was used to compare the parameters of the control group and the study group calves. Statisti-cal significance in the analysis was accepted as 5%. The SPSS 21 program was used to make the required statistical analysis in this study.
Results and discussion
Clinical findings. Clinical examinations of the sick
animals identified an increase in body temperature, heart rate, and respiratory rate. It was also seen that the animals had no appetite, were depressed and dis-interested in their surroundings. In addition, their hair was tangled and matted. Examination of the animals’
respiratory systems detected labored respiratory sounds and shallow breathing. In addition, costo-abdominal respiration was detected in most of the animals.
Microbiological findings. Blood samples were
drawn into aerobic and anaerobic culture bottles from the 40 calves diagnosed with pneumonia. The results of the microbiological plantation of these blood samples revealed bacterial agents in 20 calves, mixed factors (bacterial and viral) in 10 calves and only viral agents (adenovirus-3) in 10 calves. Microbiological analyses identified the following bacterial agents: Staphylococcus xylosus, Staphylococcus hominis, Staphylococcus klosii, Corynebacterium propiquum, Micrococcus varians, Bacillus brevis, Achronobacter spp., and Escherichia coli.
Hematologic findings. Statistical comparisons of
selected hematological parameters of the control group and the pneumonic calves (RBC, WBC, Lym, Mon, Neu, Eo, MCV, Hct, MCHC, Hb, PLT) are given in Table 1. Statistical comparisons revealed that WBC, LYM, Neu, Hct, Hb, MCV, and MCHC values in the pneumonic calves were higher than in the control group (P < 0.05), but PLT values were lower (P < 0.05) (Tab. 1).
Biochemical findings. It was determined that IL-6,
IL-8, and TNF-α levels were statistically higher in the pneumonic calves than in the control group animals. This study showed that among the pneumonic calves increases in IL-6 and IL-8 levels were highest in the mixed infection group (bacterial + viral) and that the lowest increase in these parameters was seen in the virally infected group (Tab. 2).
Hp, C-RP, SAA, α-1-AGP, and LF levels were found to be more statistically significant in the pneu-monic calves than in the control group animals, but changes in fibri- nogen levels were not statistically significant. A comparison of the different groups with pneumonia showed that only changes in LF lev-els were statistically significant and that the highest LF and CRP levels existed in the mixed infection group (bacterial + viral) (Tab. 3).
Statistical comparisons revealed some differences in TP and ALB levels between the control group and the pneumonic group, but these differences were not statistically significant (Tab. 4).
With their high morbidity and mortality rates, respiratory system diseases in calves cause significant
Tab. 1. Some hematological parameters in healthy and pneumonic calves (x ± SD)
Parameter (n = 10)Control Pneumonic Bacterial (n = 20) (n = 10)Viral Mixed (Bacterial + Viral) (n = 10) RBC (m/mm3) 9.69 ± 2.52a 10.24 ± 2.36a 9.95 ± 1.38a 10.80 ± 2.20a WBC (m/mm3) 13.79 ± 3.41a 27.67 ± 19.56b 25.06 ± 19.02b 28.22 ± 18.10b Lym (m/mm3) 17.06 ± 9.73a 10.59 ± 5.76b 8.08 ± 2.67c 13.18 ± 8.78b Mon (m/mm3) 0.78 ± 0.20a 0.68 ± 0.28a 0.62 ± 0.24a 0.70 ± 0.18a Neu (m/mm3) 3.64 ± 1.14a 10.27 ± 4.51b 10.10 ± 4.80b 10.58 ± 6.54b Eo (m/mm3) 1.26 ± 0.40a 1.10 ± 0.60a 0.90 ± 0.40a 1.52 ± 0.40a MCV (fl) 38.40 ± 3.30a 41.67 ± 5.46b 38.18 ± 7.12a 42.80 ± 3.80b Hct (%) 37.88 ± 3.86a 41.49 ± 11.10b 41.70 ± 10.80b 42.30 ± 10.30b MCHC (g/dl) 31.53 ± 1.38a 33.39 ± 5.14a 32.48 ± 5.70a 34.54 ± 3.66b Hb (g/dl) 11.96 ± 1.27a 13.63 ± 3.12b 13.10 ± 3.10b 14.40 ± 3.13b PLT (m/mm3) 603.00 ± 211.90a 364.51 ± 195.88b 296.70 ± 116.47b 406.32 ± 272.10c
Explanations: a, b, c – means with different superscript letters in the same line differ significantly at p < 0.05
Tab. 2. Some cytokine levels in control group calves and pneumonic calves (x ± SD)
Parameter (n = 10)Control Pneumonic Bacterial (n = 20) (n = 10)Viral Mixed (Bacterial + Viral) (n = 10) IL-6 (pg/ml) 538.77 ± 259.98a 943.52 ± 52.85b 768.86 ± 63.04b 1434.76 ± 564.65c IL-8 (ng/L) 66.40 ± 46.37a 210.60 ± 7.20b 174.60 ± 20.32b 258.92 ± 164.40b TNF-α (ng/L) 302.42 ± 86.16a 430.00 ± 18.45b 500.02 ± 9.40b 640.40 ± 285.26c Explanations: as in Tab. 1
(11, 33). It can be interpreted that the significant increase in WBC and neutrophil counts in pneumonic calves is a result of inflammation caused by the disease.
Studies (6, 23, 34) have reported that excessive increases in pro-inflammatory cytokines, such as serum TNF-α or IL-6, in condi-tions such as pneumonia and septic shock are indicative of the severity of the disease and that the increase in serum cytokine levels is as-sociated with the prognosis of the disease. It has also been reported that the measurement of cytokines (TNF-α, IL-6, IL-8) and some acute phase proteins is important for the bacterial or viral differentiation of infection and for guiding the an-timicrobial therapy to be applied. Furthermore, increases in cytokine and acute phase protein levels are reported to be higher in bacterial pneumonia than in viral pneumonia (6, 16, 34). It has also been reported that IL-1, IL-6, and TNF-α con-centrations are higher in sick animals than in control animals in many infectious diseases (15, 16).
Acute phase protein synthesis is regulated by pro-inflammatory cytokines, such as TNF-α, IL-1, and IL-6, released from monocytes and macrophages after tissue damage (29). It is reported that during illness and inflammation cytokines start the production of AFPs in the liver by activating and altering hepatocyte recep-tors (3). CRP is an important component of the acute protein response (32). CRP occurs in many infection processes, with CRP levels reflecting the severity of the infection. CRP can be used as a biomarker for the diagnosis of pneumonia (28). As a matter of fact, in this study, it was determined that CRP levels were higher in all three groups of pneumonic calves than in the control group. CRP levels were highest in the mixed infection (bacterial + viral) group in particular. In this study, it was found that IL-6, IL-8, and TNF-α levels were statistically significantly higher in the pneumonic calves than in the control group animals. The study showed that the increase in IL-6, IL-8, and CRP levels in the pneumonic calves was highest in the mixed infection group (bacterial + viral), and lowest in the virally infected group. It was found that these results are in parallel with those of other studies (6, 12, 16, 24, 28, 32, 34).
Acute phase proteins are produced by both hepa-tocytes and peripheral tissues (4). The task of these proteins is to protect the organism from further injury, to eliminate infectious agents, to remove molecules and residues that are harmful for the organism, and to
Tab. 3. Levels of selected acute phase proteins in control group calves and pneu-monic calves (x ± SD) Parameter (n = 10)Control Pneumonic Bacterial (n = 20) (n = 10)Viral Mixed (Bacterial + Viral) (n = 10) Hp (ng/L) 27.99 ± 10.56a 78.10 ± 9.77b 56.93 ± 6.17c 157.31 ± 69.42b CRP (ng/L) 13.64 ± 3.68a 20.93 ± 0.32b 17.64 ± 0.52b 28.68 ± 12.40b SAA (ng/L) 1.24 ± 0.98a 4.86 ± 1.14b 4.44 ± 1.11b 5.23 ± 1.23b α-1-AGP (ng/L) 0.47 ± 0.13a 2.49 ± 0.12b 2.03 ± 0.12b 3.69 ± 2.49b LF (ng/L) 338.68 ± 94.07a 369.41 ± 107.68b 358.96 ± 227.85b 518.32 ± 376.96c Fibrinogen (ng/L) 9.48 ± 2.89a 10.09 ± 5.30a 9.80 ± 3.80a 11.17 ± 5.20a Explanations: as in Tab. 1
Tab. 4. Serum albumin and total protein concentrations in control group calves and pneumonic calves (x ± SD)
Parameter (n = 10)Control Pneumonic Bacterial (n = 20) (n = 10)Viral Mixed (Bacterial + Viral) (n = 10) TP (g/dl) 5.74 ± 0.74a 6.10 ± 1.11a 5.95 ± 0.33a 6.11 ± 0.20a ALB(g/dl) 2.37 ± 0.42a 2.71 ± 0.45a 2.70 ± 0.10a 2.96 ± 0.17a
economic loss, and they still exist in the modern world (2, 10, 12). As well as bacterial and viral factors, en-vironmental and stress factors play an important role in the formation of the disease (31). Because of the physiological and anatomical characteristics of the respiratory system in cattle, the risk of respiratory disease is higher than in other farm animals (13). This study was carried out to determine the levels of various cytokines (TNF-α, IL-6, IL-8) and acute phase proteins (haptoglobin, CRP serum amyloid A, alpha 1-acid glycoprotein, lactoferrin, and fibrinogen) in pneumonic calves, to reveal changes in these parameters depending on etiologic factors (bacterial, viral, bacterial + viral) in calf pneumonia, and to determine whether these parameters could be used for diagnosis and prognosis of the disease.
It has been reported that there is a positive correla-tion between the severity of the clinical findings of the disease and cytokine and acute phase protein activities in pneumonic calves. It has also been reported that cy-tokine and acute phase protein activities are detected at the highest levels in mixed infection (bacterial + viral) groups and that assessing changes in acute phase pro-teins and hematological parameter values is important in evaluating the animals’ state of health (12, 24).
RBC, WBC, Lym, Mon, Neu, Eo, MCV, Hct, MCHC, Hb, and PLT values of the control group calves and pneumonic calves were evaluated in this study, and it was shown that WBC, Lym, Neu, Hct, Hb, MCV, and MCHC values in the pneumonic calves were higher than in the control group (P < 0.05), but PLT values were lower (P < 0.05). Changes in the hematological parameters support data reported in related studies
activate the repair process necessary for the organism to return to its normal function and restore homeosta-sis (4, 7, 8, 18, 21). In many studies, the acute phase response has been reported as important in evaluating animal health (15). On the basis of these data, it has been reported that both cytokines and AFPs can be used as markers in the diagnosis and differential diagnosis of many diseases. Cytokines and AFPs, when used for these purposes, strengthen the diagnosis and provide more accurate information for determining prognosis (8, 18). Studies have emphasized that the acute phase response is important in both bacterial and viral respi-ratory system infections (14, 16).
It has been reported that Hp concentrations increase in many bovine diseases (1, 17) and are not detected in healthy cattle (1, 26). Ganheim et al. (14) reported Hp concentrations of 60-123 µg/ml in healthy calves. Many studies note that Hp concentrations can also be used for diagnosis and prognosis of pneumonia (4, 16, 33). Nikunen et al. (27) reported that serum fibrino-gen, Hp, and SAA levels increased more rapidly in Mannheima haemolytica infections as compared to BVDV infections and that Hp levels were significantly elevated in bacterial infections and can be used as a marker for distinguishing viral and bacterial diseases.
Indeed, it has been stated that haptoglobin, SAA, and fibrinogen levels are high and the acute phase re-sponse is significant in the respiratory system disease caused by bovine viral diarrhea virus (BVDV) and Mannheima haemolytica factors in calves (16). That same study also reported that increases in haptoglobin, SAA, and fibrinogen concentrations in calves infected by Mannheima haemolytica are higher than in those infected by bovine viral diarrhea virus (16).
In the present study, Hp, SAA, α-1-AGP, and LF levels were more statistically significant (P < 0.01) in the pneumonic calves than in the control group animals, but changes in fibrinogen levels were not statistically significant. A comparison of the groups with pneu-monia revealed that only changes in LF levels were statistically significant and the highest LF levels oc-curred in the mixed infection (bacterial + viral) group. In addition, it appears that the Hp level is highest in the mixed infection group (bacterial + viral) calves and that the acute phase response to pneumonia-causing agents may be the cause of this increase. The increases in AFP levels in this study were similar and parallel to data reported by many other researchers (1, 9, 22).
Fibrinogen is the first AFP used routinely in the diag-nosis of bovine diseases (27). Fibrinogen is synthesized by the liver during inflammation in response to the acute phase response and is reported to be in the range of 200-700 mg/dL in healthy cattle (19). Fibrinogen is typically increased in infectious, irritable, traumatic, and neoplastic diseases, and when measured together with the erythrocyte sedimentation rate, it is used as a nonspecific parameter in determining tissue damage
and inflammation (20). It was also observed in this study that, although there were some differences in se-rum fibrinogen levels between the pneumonic groups, these differences were not statistically significant (P > 0.05). However, the highest score was observed in the mixed infection (bacterial + viral) group. This data supports the findings of other researchers (8, 19, 20).
Studies have shown that TP and Alb levels increase during the acute phase reaction (9). It was determined in the present study that there were some differences in TP and ALB levels in the control group and in the pneumonic calves, but these differences were not statis-tically significant. These findings support the findings of some researchers (8), but do not support those of some other researchers (25).
In conclusion, this study showed that serum cytokine (IL-6, IL-8, TNF-α) levels and serum acute-phase pro-tein (Hp, CRP, SAA, α-1-AGP, and LF) concentrations were significantly higher in the pneumonic calves than in the control group calves. Furthermore, the assess-ment of cytokine and acute phase protein levels can play an important role in the early diagnosis of calf pneumonia. Early diagnosis can help prevent deaths caused by pneumonia, which is the leading cause of major losses, and can also be helpful in the early treat-ment of pneumonia in the veterinary clinical field.
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Corresponding author: Prof. Dr. Suleyman Kozat, Department of Internal Diseases, Faculty of Veterinary Medicine, University of Yuzuncu Yil, 65080 Van, Turkey; e-mail: skozat@hotmail.com
