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Haptoglobin and Amyloid A Levels in Milk of Clinical and Subclinical Mastitic Cows in Turkey

PJZ_55_1_01-09

Haptoglobin and Amyloid A Levels in Milk of Clinical and Subclinical Mastitic Cows in Turkey

Nurdan Urvaylioglu1*, Ogunc Meral1, Serkan Sayiner2, Ulvi Reha Fidanci1 and Arif Altintas1

1Department of Biochemistry, Faculty of Veterinary Medicine, Ankara University, 06610, Ankara, Turkey

2Department of Biochemistry, Faculty of Veterinary Medicine, Near East University, 99138 Nicosia, Northern Cyprus, Mersin-10, Turkey

ABSTRACT

Mastitis is one of the most common diseases in dairy cows and causes significant economic losses globally. We aimed to investigate whether haptoglobin (Hp) and amyloid A levels in milk may be an alternative method for diagnosing subclinical mastitis (SM) in dairy cows. Ten subclinical-, 16 clinical mastitis and 39 healthy Holstein cows were allocated to the study following California Mastitis Test (CMT). Thus, a total of 65 Holstein cows participated in the study. In the study, Staphylococcus aureus (n = 10) was isolated as the most dominant bacterial species seen in clinical and subclinical mastitis samples. Somatic cell counts (SCC) in cows with clinical mastitis were significantly higher than the milk of cows with subclinical mastitis and healthy cows. Milk serum haptoglobin (Hp) and amyloid A levels were not determined statistically different between groups (p> 0.05). No correlation was found between CMT scores, SCC values, Hp and milk amyloid A levels in milk serum. There were no significant differences in Hp, MAA levels and SCC in the milk of clinically healthy cows and cows with clinical and subclinical mastitis (p> 0.05). As a result, Hp and amyloid A levels in milk showed no proper parameters for diagnosing subclinical mastitis in dairy cattle and monitoring treatment efficacy. Acute-phase proteins have not yet been adequately studied and clinically used in dairy animals, routinely diagnostic and prognostic, but they are used in human medicine for these purposes.


Article Information

Received 16 June 2021

Revised 01 August 2021

Accepted 12 August 2021

Available online 08 February 2022

(early access)

Published 14 October 2022

Authors’ Contribution

NU and OM designed the study and wrote the article. NU, OM and SS performed the experiments and analyzed the data. SS, URF and AA critically reviewed and revised the manuscript.

Key words

Acute phase proteins, Mastitis, Milk, Amyloid A, Haptoglobin

DOI: https://dx.doi.org/10.17582/journal.pjz/20210616100638

* Corresponding author: feyza.nurdan@hotmail.com

0030-9923/2023/0001-01 $ 9.00/0

Copyright 2023 by the authors. Licensee Zoological Society of Pakistan.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).



INTRODUCTION

Mastitis is a multifactorial disease characterized by decreased milk yield and a change in its composition in dairy animals. It is an inflammatory response of the mammary gland to physiological and metabolic alterations, injuries, allergies and, more often, damage caused by microorganisms. The loss of yield and economic losses caused by the disease still is a devastating problem in the worlds’ dairy cow breeding (Pir Yağcı, 2008; Deb et al., 2013; Ismail et al., 2018).

Accurate and precise diagnosis of mastitis determines the fate of the udder health, specifically at early stages. Although several methods are routinely used in the indirect diagnosis of mastitis, the most practical application is the California Mastitis Test (CMT) (Schalm and Noorlander, 1957; Ashraf and Imran, 2018). Despite the use of many chemical and microbiological tests in diagnosing subclinical mastitis, tests based on determining the somatic cell count (SCC) per ml of milk have gained more importance in recent years (Rişvanlı and Kalkan, 2002; Duarte et al., 2015). However, studies for new diagnostic approaches continue.

Determination of the levels of positive acute-phase proteins (APP), serum amyloid-A (SAA) and haptoglobin (Hp) in differentiating acute and chronic inflammations in cattle are more valuable than using haematological tests, and the SAA levels are higher in acute inflammation than in chronic inflammation (Alsemgeest et al., 1994; Thomas et al., 2015). The APP levels in body fluids are related to the extent of tissue damage and the severity of the problem in affected animals and can provide clinical diagnostic and prognostic information (Coşkun and Şen, 2011; Dhama et al., 2019).

It has been suggested that the SAA levels are an indicator in the diagnosis of chronic subclinical mastitis (Grönlund et al., 2005). Serum Hp and SAA levels below the detection limit were evaluated as healthy nipple markers (Grönlund et al., 2003). Haptoglobin is also locally synthesized in the mammary gland (Hiss et al., 2004; Thielen et al., 2007). However, Hp in milk has been determined to have originated from the circulation (Pedersen et al., 2003; Jain et al., 2011). Since the APP levels in milk increase significantly with increasing SCC, it has been stated that milk APPs can indicate the severity of infection (Dalanezi et al., 2020). Due to the direct synthesis of milk amyloid-A (MAA), a specific isoform of SAA, from breast epithelial cells, it is stated that MAA is a more sensitive indicator in mastitis cases and can be used in the diagnosis of clinical and subclinical mastitis (Hussein et al., 2018). Studies conducted on milk Hp (mHp) and the MAA levels with acute clinical mastitis have revealed that APPs in milk can be determined more accurately than in serum and thus can be used as a valuable tool in the diagnosis of mastitis and can be an indicator of milk quality (Nazifi et al., 2008; Gerardi et al., 2009; Kováč et al., 2011; Alkan et al., 2014).

Since mastitis is a global health issue for dairy cows, this study aimed (i) to measure and assess mHp, MAA, SCC together with microbiological results in detecting various degrees of subclinical and clinical mastitis, and (ii) to compare the biochemical and microbiologic result with the SCC.

MATERIALS AND METHODS

Animals

In this study, 65 Holstein cows in the postpartum period (lactation period) between 2-8 years of age, were selected from family-type farms in Çankırı Center and Eldivan District and were used as healthy animals and animals with mastitis. Cows with the same diets were included in the study. Milk samples of 25 healthy cows (those with no signs of mastitis and no microbiological growth as a result of microbiological examinations), 14 healthy controls (those with no signs of mastitis, but bacterial growth detected in microbiological cultivation), 16 cows with clinical mastitis and 10 cows with subclinical mastitis, were allocated into four groups.

California mastitis test (CMT)

According to the previously described method, the CMT was conducted (Schalm and Noorlander, 1957). Milk samples obtained from each 4-udder group were collected into a test container with four separate compartments. Equal amounts of milk were mixed with the test solution containing “bromocresol purple” an anionic detergent. Changes such as color changes or gel formation were evaluated by rotating the test plate slowly. The CMT scores were graded as follows: 0 for no reaction, 1 for weak positive, 2 for a significant positive and 3 for a strong positive reaction.

Bacteriological examinations

Milk samples were incubated at +37°C for 1-3 days in an aerobic environment after inoculation on MacConkey’s agar and blood agar medium. For isolation, bacteria were also cultivated from each milk sample in broth medium with serum and incubated at +37°C for 48 h under microaerophilic conditions. After the media on which the growth was observed were examined microscopically, pure cultures were formed by passaging from single falling colonies in mixed growing cultures. Based on the obtained analysis results, routine microbiological examinations were used to see other biochemical properties for identification.

Measurement of somatic cell count (SCC)

The SCC was calculated with the Bentley Bactocount IBC-M (Bentley Instruments, MERKİM®, USA) device.

Determination of haptoglobin and amyloid-A levels

The milk samples were brought to the laboratory and centrifuged at 26.000 x g for 30 minutes. The fat layer accumulated over the tubes was removed, milk serum was collected, placed into centrifuge tubes and kept at -80°C until the analysis. Quantification of MAA and mHp was performed using commercially available ELISA kits (respectively, Serum Amyloid A PhaseTM Analysis and Bovine Haptoglobin PhaseTM Enyme Immunassay, Tridelta Ltd. Company, Ireland).

Statistical analysis

The data obtained from our study were statistically analyzed using SPSS 11 program. The control of the difference between the groups, analysis of variance and multiple comparison tests were performed. The results were given as X ± Sx: mean ± standard error. P<0.05 was considered statistically different.

RESULTS

The mean number of SCC, mHp and MAA values and standard error rates in healthy Holstein cows with clinical and subclinical mastitis are presented in Table I. As a result of the variance analysis, the SCC difference between the groups was detected significant (p<0.001), while the difference between the groups was not significant for mHp and MAA (p>0.05). The analysis of variance for SCC showed similarity in the healthy and the healthy control groups, while SCC was significantly lower than in the subclinical and clinical mastitis groups. The mean SCC was highest in clinical mastitis, followed by the mean SCC in subclinical mastitis.

 

Table I. Somatic cell count (SCC), haptoglobin and amyloid A mean values and statistical significance of groups in clinical mastitis, subclinical mastitis and healthy cows.

Healthy

Healthy control

Subclinical mastitis 

Clinical mastitis

SCC

n

23

14

10

9

Mean ± SD

61,60 ± 59,39a

36,14 ± 35,21a

517,30 ± 284,87b

2519 ± 934,11c

SEM

12,38

9,41

90,08

311,37

Haptoglobin

(mg/ml)

n

25

14

10

16

Mean ± SD

0,3409 ± 0,0314

0,3429 ± 0,0386

0,3370 ± 0,0213

0,3396 ± ,0215

SEM

0,0062

0,0103

0,0067

0,0053

Amyloid A

(ng/ml)

n

15

8

6

12

Mean ± SD

136,40 ± 126,89

207,50 ± 159,64

126,66 ± 137,88

228,75 ± 115,57

SEM

32,76

56,44

56,29

33,36

 

a, b, c, The difference between the averages of the groups with these different media in the same row is significant. As a result of the correlation analysis, there was no statistically significant correlation between SCC, haptogloblin and amyloid A variables. (p>0,05).

 

Table II. Somatic cell count, CMT and microbiological analysis results in clinical mastitis, subclinical mastitis and healthy cow milk.

No

Somatic cell count (SCC) x 1000 cells/ml)

Group

California mastitis test (CMT)

Microbiological analysis

1

48

Healthy

negative (-)

Corynebacterium spp.

2

29

Healthy control

negative (-)

No microbiological growth

3

1302

Clinical mastitis

positive (+++) 3

Staphylococcus aureus

4

480

Subclinical mastitis

suspicious

Staphylococcus aureus /Micrococcus spp.

5

24

Healthy

negative (-)

Bacillus spp. / Pseudomonas spp.

6

21

Healthy control

negative (-)

No microbiological growth

7

2948

Clinical mastitis

positive (+++) 3

Bacillus spp. / Streptococcus spp.

8

10

Healthy

negative (-)

Bacillus spp. / Staphylococcus spp.

9

12

Healthy

negative (-)

Staphylococcus spp.

10

22

Healthy

negative (-)

Bacillus spp. / Staphylococcus intermedius

11

12

Healthy

negative (-)

Escherichia coli /Staphylococcus aureus

12

31

Healthy control

negative (-)

No microbiological growth

13

51

Healthy

negative (-)

Staphylococcus aureus / Micrococcus spp.

14

68

Healthy

negative (-)

Alcaligenes spp. /Streptococcus dysgalactiae

15

8

Healthy control

negative (-)

No microbiological growth

16

581

Subclinical mastitis

positive (+) 1

Staphylococcus aureus

17

18

Healthy

negative (-)

Staphylococcus spp.

18

9

Healthy control

negative ( - )

No microbiological growth

19

333

Subclinical mastitis

suspicious

Staphylococcus aureus / Escherichia coli

20

8

Healthy control

negative (-)

No microbiological growth

21

10

Healthy control

negative (-)

No microbiological growth

22

193

Healthy

negative (-)

Staphylococcus aureus / Micrococcus spp.

23

674

Subclinical mastitis

positive (+) 1

Pasteurella haemolytica

24

183

Healthy

negative (-)

No microbiological growth

No

Somatic cell count (SCC) x 1000 cells/ml)

Group

California mastitis test (CMT)

Microbiological analysis

25

55

Healthy

negative (-)

Escherichia coli

26

36

Healthy control

negative (-)

No microbiological growth

27

39

Healthy control

negative (-)

No microbiological growth

28

201

Subclinical mastitis

suspicious

Staphylococcus aureus / Staphylococcus spp.

29

68

Healthy control

negative (-)

No microbiological growth

30

6

Healthy

negative (-)

Staphylococcus spp.

31

271

Subclinical mastitis

suspicious

Staphylococcus spp.

32

443

Subclinical mastitis

suspicious

Staphylococcus spp.

33

76

Healthy

negative (-)

Staphylococcus intermedius

34

194

Healthy

negative (-)

Staphylococcus intermedius / Staphylococcus spp.

35

16

Healthy control

negative (-)

No microbiological growth

36

3254

Clinical mastitis

positive (+++) 3

Streptococcus spp.

37

314

Subclinical mastitis

suspicious

Micrococcus spp.

38

142

Healthy control

negative (-)

No microbiological growth

39

3460

Clinical mastitis

positive (+++) 3

Staphylococcus aureus

40

107

Healthy

negative (-)

Corynebacterium spp.

41

40

Healthy control

negative (-)

No microbiological growth

42

129

Healthy

negative (-)

Streptecoccus dysagalactia

43

45

Healthy

negative (-)

Corynebacterium spp.

44

58

Healthy

negative (-)

Staphylococcus spp.

45

32

Healthy

negative (-)

Corynebacterium spp. / Bacillus spp.

46

2270

Clinical mastitis

positive (+++) 3

Staphylococcus spp.

47

1167

Subclinical mastitis

positive (+) 1

Alcaligenes spp. / Streptococcus dysgalactiae

48

28

Healthy

negative (-)

Bacillus spp. / Enterococcus spp.

49

1455

Clinical mastitis

positive (+) 1

Corynebacterium spp.

50

28

Healthy

negative (-)

Bacillus spp.

51

18

Healthy

negative (-)

Staphylococcus intermedius

52

49

Healthy control

negative (-)

No microbiological growth

53

1336

Clinical mastitis

positive (+++) 3

Staphylococcus intermedius

54

709

Subclinical mastitis

positive (+) 1

Streptococcus spp.

55

3364

Clinical mastitis

positive (+++) 3

Staphylococcus aureus

56

3282

Clinical mastitis

positive (+++) 3

Staphylococcus intermedius / Staphylococcus spp.

57

Could not be measured

Clinical mastitis

positive (+++) 3

Streptococcus dysagalactia / Corynebacterium spp.

58

Could not be measured

Healthy

suspicious

Staphylococcus spp.

59

Could not be measured

Healthy

suspicious

Staphylococcus aureus/Micrococcus spp.

60

Could not be measured

Clinical Mastitis

positive (++) 2

Microbiological analysis could not be done

61

Could not be measured

Clinical Mastitis

positive (++) 2

Microbiological analysis could not be done

62

Could not be measured

Clinical Mastitis

positive (++) 2

Microbiological analysis could not be done

63

Could not be measured

Clinical Mastitis

positive (+) 1

Microbiological analysis could not be done

64

Could not be measured

Clinical Mastitis

positive (++) 2

Microbiological analysis could not be done

65

Could not be measured

Clinical Mastitis

positive (++) 2

Microbiological analysis could not be done

 

In this study, 65 cows in their lactation period were examined with CMT. As a result of the microbiological evaluation of milk samples obtained from CMT positive cows, inoculation was not possible in 6 of 65 samples, and in the remaining 59 samples, isolation and identification were carried out. No growth was observed in 20 of 59 samples, and no aerobic microorganism was isolated. While aerobic microorganisms were isolated from 39 specimens with bacterial growth, Staphylococcus aureus was identified in 15 of them. Besides, mixed infections were determined in 24 of 39 milk samples (Table II).

DISCUSSION

Bovine milk is an essential nutrient for human health. Besides, it is a critical production element in terms of the country’s economy. Therefore, the health of the mammary gland and protection against possible diseases, diagnosis and treatment regimes are significant efforts of veterinary medicine (Wiley, 2008; Mahmood et al., 2017).

During inflammation of the mammary gland, the SCC in milk increases, which is a good indicator of the degree of inflammation in mastitis (Souza et al., 2016; Mahmood et al., 2017; Darbaz et al., 2019). The presented study revealed a significant difference in milk SCC between the groups (p<0.001), while the highest mean values were found in the clinical mastitis group, followed by the subclinical mastitis group. The mean values of the healthy and the control groups were low and close to each other. The significant increases in SCC values in milk with clinical and subclinical mastitis were remarkable and consistent with the literature.

Detection of Staphylococcus aureus in 15 of 39 specimens with positive CMT and bacterial growth and mixed infection was determined in 24. This result may be due to that all milk samples were obtained from the family-type farm, and the farmers in the region did not know or did not apply milking hygiene and milking rules well due to lack of knowledge of methods of protection from mastitis such as disinfecting the udders before milking, and lack of the dry period treatment habit.

Alkan et al. (2014) collected 109 milk samples from 112 udders of 28 Holstein cows and detected a difference between the mean SCC values of the groups with and without bacterial growth was not statistically significant (p>0.05). The SCC results of the present study were also compatible with the literature. Besides, Alkan et al. (2014) suggested that CMT alone was insufficient in determining subclinical intramammary infections in dry cows and that the CMT results should be evaluated together with bacteriological examination results. In our study, evaluation was made based on CMT and the bacteriological examination results. It was determined that although there was bacterial growth in samples obtained from some udders, the CMT test was negative, and although the CMT test was positive in some samples, no bacteria were isolated. It is thought that the main reasons for this situation may be factors such as weak sensitivity and specificity of the CMT test, which may not be determinative for intra-mammary infections in all cases, as well as the difference in SCC values in mastitis cases due to microorganisms, the number of bacterial colonies and the inoculation technique.

Rişvanlı and Kalkan (2002) has been determined that the mean SCC in milk samples obtained from CMT (+) lobes with microbiological growth was 313.001; the mean SCC in milk samples obtained from CMT (++) lobes with microbiological growth was 559.007, and the mean SCC in milk samples obtained from CMT (+++) lobes with microbiological growth was 1.563.618. Results of our study were that the mean value of milk SCC of the CMT (+) (subclinical mastitis) group with microbiological growth was 517.000 ± 90.000 / ml; the mean value of milk SCC in the CMT (++) and (+++) (clinical mastitis) group with microbiological growth was 2.519.000 ± 311.000 / ml (Table I). Thus, our results were compatible with the literature, and the SCCs in milk with clinical and subclinical mastitis were significantly higher than those in healthy milk (p <0.001).

SCC was found to be 379,000 in Staphylococcal-infected lobes and 63,000 in non-infected lobes (Hillerton and Walton, 1991). In milk samples obtained from lobes infected with S. aureus, SCC was determined as 364,866 in CMT (+) cows, 504,306 in CMT (++) cows and 1,675,008 in CMT (+++) cows (Rişvanlı and Kalkan, 2002). In the presented study, High SCC values of S. aureus or Staphylococcus spp. infected lobes were determined to be remarkable and compatible with the literature.

The acute phase response is an essential process against inflammation and infection. Hepatic adaptation responds by synthesizing acute-phase proteins, such as Hp and amyloid A, which restores homeostasis (Eckersall et al., 2006; Suojala et al., 2008). Many studies have shown that S. aureus is in the first place among microorganisms isolated from clinical and subclinical mastitis cases (Alaçam et al., 1989; Shitandi and Kihumbu, 2004; Schröder et al., 2005; Pumipuntu et al., 2019).

Increasing levels of MAA in the mammary gland with mastitis were compared with healthy breasts, and it was determined that some udders be affected by infection processes and result in a negative CMT (Nazifi et al., 2008). It has been established that milk and serum Hp and SAA increases rapidly in the acute phase response in cows with mastitis caused by Staphylococcus spp. (Grönlund et al., 2003). Besides, mHp and MAA levels were reported to increase significantly with the increase of SCC, which may indicate clinical mastitis severity (Nielsen et al., 2004). In the presented study, there was no significant difference between the groups for mHp and MAA (p>0.05), and no statistically significant relationship was found between the variables SCC, mHp and MAA (p>0.05) (Table I). Contrary to our findings, Eckersall (2004) reported a significant difference in udder Hp and SAA concentrations in cows with chronic subclinical mastitis. We believe that the difference between the results of the two studies may be related to the difference in the severity of mastitis and whether it is acute or chronic. It has been reported that the difference between the APP levels determined in serum and milk may be caused by the duration and severity of breast infection (Nielsen et al., 2004) and that the increases in Hp, MAA and SCC may address the severity of clinical mastitis.

MAA and mHp levels below the detection limit were evaluated as healthy nipple markers, and significant increases were observed in Hp and SAA levels in the milk of cows with chronic subclinical mastitis (Grönlund et al., 2005). The disease stages are suggested to be evaluated by monitoring more than one APP, and in this way, chronic and acute conditions can be evaluated and characterized with an APP profile (Eckersall, 2004; Thomas et al., 2015). Our results were consistent with to study of Gultiken et al. (2012) that reported no relationship between the CMT scores, SCC values, plasma Hp and milk Hp levels presence in subclinical mastitis.

Due to the ability to synthesis of Hp from udder tissue (Eckersall et al., 2006), Hp has been evaluated as a mastitis marker in many studies (Grönlund et al., 2005; Akerstedt et al., 2007, 2009). Our study result on milk Hp was not found to be consistent with these literature data. We think that the fact that the milk Hp values in mastitis cases were not as high as expected could be associated with the mild severity of the mastitis cases because the Hp level in cattle has been reported to be higher in moderate and severe cases compared to mild cases of clinical mastitis (Nielsen et al., 2004; Wenz et al., 2010). According to the presented study results, the milk MAA levels were not a significant indicator in the diagnosis of mastitis. However, Grönlund et al. (2005) found significant differences in the mHp and MAA levels in cows with chronic subclinical mastitis. It is thought that the chronic and acute nature of the mastitis cases examined in our study may have affected the results.

Increased mHp and MAA levels were found in cows with mastitis, and the levels were significantly higher in cows with moderate mastitis than those with mild mastitis. Besides, it was stated that MAA had a higher potential in detecting mastitis than mHp, because of high sensitivity, specificity and efficiency in differentiating healthy cows and cows with mastitis (Eckersall et al., 2001; Taghdiri et al., 2018). In our study, no significant increase was found in the mHp and MAA levels of cows with clinical and subclinical mastitis, and the mild course of mastitis can explain this.

In chronic subclinical mastitis cases, only the milk MAA levels were significantly higher than the pre-infection status and significantly higher than in healthy controls (Grönlund et al., 2003; Tomazi et al., 2015). Subsequent studies have shown that increased APP levels in milk significantly altered the Hp and SAA levels in cows with chronic subclinical mastitis (Grönlund et al., 2005). Our Hp and MAA levels findings showed a difference between the healthy milk group and milk with various clinical and subclinical mastitis groups. However, there was no statistical significance (p>0.05), incompatible with the literature data. This may be due to the stage and severity of the disease because it has been reported that the disease’s being acute or chronic may affect the milk Hp and MAA values (Nielsen et al., 2004; Kumar et al., 2017)

Our study could not establish a significant relationship between the SCC, mHp and the MAA values. In contrast to our findings, Akerstedt et al. (2007) reported a significant relationship between Hp and SCC in dairy cattle with subclinical mastitis 4-udder groups and the composite milk levels. Similar results have also been reported previously (Kovac et al., 2007; Safi et al., 2009). Gultiken et al. (2012) reported that although the SCC results and the CMT scores decreased in the third week of mastitis, they could not find a significant relationship between these parameters and the plasma Hp and mHp. Based on these findings, it can be said that studies in cattle with clinical and subclinical mastitis should be carried out, taking into account some details such as the disease stage and the severity in order to establish a clear relationship between the parameters studied.

It has been reported that the MAA levels in milk can only function as a sign of mastitis in the initial stage of asymptomatic mastitis in dairy cows. The MAA levels were evaluated as an indicator of mastitis (Vasiľ et al., 2012). The Hp and MAA levels and the SCC values in the milk of cows with clinical mastitis were significantly higher than those of healthy cows and cows with subclinical mastitis (Gerardi et al., 2009; Dalanezi et al., 2020). Similarly, in our study, the SCC and MAA values were detected higher in clinical mastitis than in healthy and subclinical mastitis group, but while the increase in SCC values was found to be statistically significant (p<0.001), the difference in the MAA values was not statistically significant (p>0.05).

In conclusion, mHp and MAA levels may not be valuable parameters for diagnosing subclinical mastitis in dairy cattle, as well as monitoring the efficacy of the treatment. In future studies, it is thought that APP-based diagnostic tests that are useful for cattle and that will yield results in a short time following the collection of blood or milk samples should be developed and optimized to be used in the field. Besides, details such as the stage and course of the disease, whether the previous treatment has been applied or not should be determined in more detail by monitoring more than one APP and considering these parameters.

ACKNOWLEDGEMENTS

Ankara University Scientific Research Grant supported this study (Grant no: 16H0239004). The presented study was summarized from the first author’s master of science thesis.

Statement of conflict of interest

The authors have declared no conflict of interest.

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