Determination of Blood Serum Protein Fractions of Calves with Clinically Diagnosed Pneumonia

Bovine Respiratory Diseases (BRD) is a common and important disease for cattle breeding. Pneumonia occurs in calves aged 1 to 5 months. Respiratory disease in calves breeding is major cause for economic losses in the beef industry. Treatment of pneumonia is likely to increase survival, economic and animal welfare of heifers. There are many different agents in the etiology of respiratory diseases such as H. Somni, M. haemolytica, P. Multocidaetc.
       
The diagnosis of calf pneumonia is based on clinical symptoms such as specific symptoms, depression and body temperature (Alacam and Sahal, 1997; Angen et al., 2009; Batra et al., 2003; Closs Jr. and Dechow, 2017; Snowder et al., 2006; Tóthová et al., 2013b; Turgut, 2000).
       
Serum proteins contain six protein fractions (albumin, α1 and α2, β1 and β2 and g-globulins) and have been identified for electrophoretic separation in cows and calves. There are some studies on the electrophoretic determination of serum protein fractions and the possibilities of using the obtained results as a useful and beneficial parameter in routine Veterinary Biochemistry laboratory medicine.With this method, it will be possible to evaluate dysproteinemia, protein pattern abnormalities and the presence of pathological features by evaluating the albumin and globulin concentrations (Apaydýn and Dede, 2010; Dede et al., 2014; Gar-Elnabi et al., 2020; Karagul et al., 2000; Tóthová et al., 2010 and Totova et al., 2013b).
       
The aim of this study, to determine the serum protein fractions and A/G ratio in calves with clinically diagnosed pneumonia and evaluate the possibility uses these parameters as clinical diagnostic parameters.

Animals
 
The 34 naturally infected calves with clinical pneumonia problem, between the ages of 6 months and 1 years, of different races and sex, were used as animal material. Ten calves without clinical pneumonia symptoms were determined as the healthy control group.
 
Clinical examination
 
In this study, the sick animals were identified as follows signs by lung auscultation of calves with clinical signs such as cough, fever and respiratory distress. Animals with these signs were detected as a patient group. Animals with normal body temperature, respiratory rate and normal lung auscultation without cough and breathing difficulty were separated as healthy group.
 
Samples preparation
 
After clinical examination, blood samples were collected taken from the Vena Cava of the calves according to procedure. Blood samples were added into serum tubes with gel. The tubes were centrifuged for 10 minutes at 3000 rpm at +4^oC and serum samples were obtained.
 
Biochemical analysis
 
The obtained serum samples were used for total protein and serum protein electrophoresis. The total protein levels was analyzed using the Biuret method. The serum protein fractions were obtained by Helena Lab-Titan III® Serum Protein Electrophoresis system (Helena, Bioscience Europe, UK). The determined bands obtained after electrophoresis were analyzed with Platinum 3.0 program.
 
Statistical analysis
 
The obtained data were analyzed with Independent samples test. Statistical differences were considered when p value was less than 0.05 using SPSS 22.0 statistical software.

The results for the serum samples are summarized in Table 1 and 2.
 

 

       
The percentages of other globulin fractions were higher in the patient group (p≤0.01).
       
According to concentration results, it was found that while albumin did not show a significant difference, α1-globulin (p≤0.05) and A/G ratio (p≤0.01) decreased in the patient group. In addition, other globulin fractions and total protein levels were significantly higher in patient groups (p≤0.01).
       
Determination of the serum protein electrophoretic profile may be an important diagnostic approach in clinical biochemistry and, in the recent years there are some studies about serum protein fractions in veterinary medicine. Serum protein electrophoresis can provide detailed information about pathological conditions related to protein profile. However, this method is rarely used in the diagnosis of bovine diseases (Apaydýn and Dede, 2010; Dede et al., 2014; Tóthová et al., 2010; Tóthová et al., 2013b).
       
Serum proteins frations provide useful information on pathological conditions and to predict abnormalities of clinical significance in ruminants and avians. There are some studies about serum protein fractions which described by electrophoresis in respiratory diseases (Alberghina et al., 2011; Piccione et al., 2014; Tóthová et al., 2013a; Werner et al., 1999).
       
Serum proteins have many important functions. Albumin acts as the active protein and carrier protein of osmotic events. Globulins include antibodies, especially inflammatory molecules, hemostatic and fibrinolytic proteins and also includes carrier proteins for lipids, vitamins and hormones (Alberghina et al., 2011; Dede et al., 2014; Karagul et al., 2000; Tóthová et al., 2013a).
       
In the present study, total serum protein in calves with chronic bronchopneumonia was significantly higher (p≤0.01) than in healthy animals. This result is consistent with the literature data (Alberghina et al., 2011; Piccione et al., 2014; Tóthová et al., 2013a).
       
There is a concomitant decrease in albumin as a result of its decreased synthesis (Tóthová et al., 2013b). In the present study, the albumin level of the patient group was found to be decreased in terms of both gram % and concentration. However, the decrease was significant only for g % (p≤0.01).
       
Tóthová et al., (2013b) reported that the sick calves have significantly higher values of α1-, β1- β2- and g-globulins than controls. The α-globulin fraction includes many of the acute phase proteins (ceruloplasmin, haptoglobin, α1-acid glycoprotein, some lipoproteins). In various causes cases pneumonia, the affected animals had significantly higher values of haptoglobin, serum amyloid A, fibrinogen, ceruloplasmin, α1-antitrypsin and acidglyco protein, (Angen, 2009; Fagliari et al., 2003; Katoh et al., 1999; Kumar et al., 2015; Yamamoto et al., 1998) and these parameters might be best choice to diagnose for pneumonia in field conditions (Snowder et al., 2006).
       
In the earlier studies reported that immunoglobulin (Ig) concentrations in serum were correlated with pneumonia in calves (Corbeil et al., 1984). Serum g-globulins and haptoglobin levels were closely related becoming illness with respiratory tract infections (Furman-Fratczak et al., 2011). As in acute phase, chronic infections may be associated with changes in the α-globulin fraction significantly higher compared to healthy animals and significantly higher concentrations also for β1 and β2 globulins (Tóthová et al., 2013b).
       
In present study, although the level of α1-globulin was slightly lower in the patient group, both globulins, including g-globulin, were found to be higher in sick calves, like previous reports that mentioned at below (Tóthová et al., 2013b).
       
A/G ratio is useful parameter to allow electrophoretic profile classification, identification and evaluation of the dysproteinemias. In the evaluation of some other diseases and chronic respiratory diseases, the A/G ratio appears to be an important parameter as evidence of dysproteinemia. While the concentrations of α1-globulin (p≤0.05), TP, β2 and g-globulins were significantly higher (p≤0.01) in calves with respiratory diseases, the albumin values and A/G ratios were significantly lower (p≤0.01) in the affected calves (Alberghina et al., 2011; Tóthová et al., 2013b). These results explain why the A/G ratio was low in the patient group, just as in this presented study.
       
In our previous studies, we have identified that the serum protein electrophoretic fractions were affected in parasitic diseases, such as sheep babesiosis (Apaydýn and Dede, 2010) and cattle with theileriosis (Dede et al., 2014).

As a result, from our present study we can conclude that all protein fractions except albumin were found to be consistent in terms of both gr % and concentration. While the α1- globulin and A/G ratio decreased in the patient group, other globulin fractions increased in the patient group. Total protein level was also increased in the patient group. Albumin was found to be low in the patient group. Based on these results, it can be suggested that serum protein fractions should be evaluated as a useful biochemical blood parameter for diagnosis and follow up of lung diseases especially in calves.

The authors declare that they have no financial or personal relationship (s) which may have inappropriately influenced them in writing this article.

Semiha Dede and Pýnar Tanritanir Ekici were responsible for the conceptualisation of the research. Pýnar Tanritanir Ekici was responsible for data collection and Semiha Dede, Veysel Yüksek, Sedat Çetin and Ayşe Usta were responsible for blood analysis, evaluation of data, statistical analysis. Writing of the original draft, review and editing were carried out by Semiha Dede, Pýnar Tanritanir Ekici, Veysel Yüksek, Sedat Çetin, Ayşe Usta.