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nitrosative stress biomarkers and DNA damage in buffaloes naturally infected with Theileria annulata
Microbial Pathogenesis 138 (2020) 103821
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Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Evaluation of oxidative/nitrosative stress biomarkers and DNA damage in buffaloes naturally infected with Theileria annulata
T
Naeim Molayi-Jabdaragia, Bijan Esmaeilnejada,∗, Vahid Mohammadib a b
Department of Pathobiology, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran Department of Internal Medicine and Clinical Pathology, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran
A R T I C LE I N FO
A B S T R A C T
Keywords: T. annulata Buffalo Oxidative stress DNA damage Biomarker
Previously, we evaluated serum sialic acid (SA) levels in buffaloes naturally infected with T. annulata. In the current paper, we conducted a further study on oxidative/nitrosative stress biomarkers in erythrocyte lysate samples of the same buffaloes. DNA damage also was assessed. Additionally, we tested whether, there is any correlation between SA and the aforementioned indicators or not. To achieve these aims, several biomarkers including the activities of key antioxidant enzymes, superoxide dismutase, glutathione peroxidase and catalase, as well as malondialdehyde (MDA), protein carbonyl (PCO), nitric oxide contents (NO), total antioxidant capacity (TAC) and DNA damage levels were measured. The obtained results showed that the activities of the antioxidant enzymes and TAC levels decreased significantly as the percentage of parasitemia increased accordingly. Also, a significant increase in the levels of PCO, MDA, NO and DNA damage were recorded, depending on the degree of parasitemia. There was a significant correlation between oxidative/nitrosative stress indicators and SA. Conclusively, T. annulata infection in buffaloes is associated with a parasitic burden-dependent oxidative/nitrosative damages to erythrocytes and SA plays a crucial role in pathogenesis of the disease, as it is tightly correlated with oxidative/nitrosative indicators.
1. Introduction In Iran, arid and semi-arid geographical conditions are hospitable for faster propagation of the Ixodidae ticks, (Hyalomma anatolicum) and rapid spreading of tick-borne diseases such as bovine tropical theileriosis (BTT) caused by Theileria annulata [1–3]. It is estimated that millions of dollars are wasted in Iranian agricultural industry because of BTT [4]. The parasite undergoes a highly complex life cycle inside the mammalian hosts, initiating by the transformation of macroschizontinfected cells in the lymph nodes draining the site of inoculation of sporozoites by ticks. The sporozoites transform into schizonts in white blood cell of the mononuclear lineage leading to infection of cattle. Then, merozoites enter erythrocytes after being released upon lysis of the infected cells. Finally, intra-erythrocytic parasite becomes infective for the vector [5,6]. The most palpable signs and symptoms emerge because of the anemia and include: weakness, weight loss, anorexia, high body temperature, petechia on the conjunctival mucosa, swollen lymph nodes, and cough. At the final stages, animals become progressively feeble as they cannot tolerate their weight and body temperature drops below
∗
38.5 °C. Occasionally, ichterus, dehydration, and blood in feces are clinical symptoms [7,8] Following recovery, bovids can act as asymptomatic carriers for life, but they are reservoirs for subsequent tick infection [9,10]. Parasitic diseases are able to instigate oxidative stress because hosts produce reactive oxygen species (ROS), primarily to destroy invading pathogens [11–13]. However, these reactive molecules cannot discriminate between host cells and infectious agents, resulting in severe damage to the host cells and organs [14,15]. To alleviate the damages, the host activates protective mechanisms including anti-oxidative defenses. But, if the ROS is generated enormously, the defense systems can be overwhelmed leading to induction of oxidative stress [16,17]. The role of oxidative stress in the pathogenesis of bovine diseases has previously been described [18–22]. Free radicals and ROS target biomolecules such as lipids, proteins and DNA, causing irreversible changes [23]. Selective permeability of cell membrane is lost when it is progressively attacked by ROS which eventuates in lipid peroxidation. Oxidative damage to proteins can alter configuration, resulting in loss of biochemical functions, while oxidative DNA damage can cause a range of alterations including: mutations, replication errors, genomic instability and cell death [24].
Corresponding author. E-mail address: [email protected] (B. Esmaeilnejad).
https://doi.org/10.1016/j.micpath.2019.103821 Received 13 April 2019; Received in revised form 20 October 2019; Accepted 23 October 2019 Available online 23 October 2019 0882-4010/ © 2019 Elsevier Ltd. All rights reserved.
Microbial Pathogenesis 138 (2020) 103821
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[3-ethylbenzthiazoline sulfonate]; Randox Laboratories Ltd., UK as the substrate. The total nitrate/nitrite content of the blood samples was measured according to the routine method of Griess reaction. Briefly, 100 μl of the lysate was mixed with 100 μl of Griess reagent in a 96-well plate and the reaction was read at 570 nm after 10 min [35]. Protein carbonyls were measured based on the method of Levin and collaborators [36], using 2,4-dinitrophenylhydrazine, hydrochloric acid, ethanol–ethyl acetate (1:1, v/v) and guanidine hydrochloride solution (6 M). The carbonyl content was calculated based on the molar extinction coefficient of DNPH (ε = 2.2 × 104 cm/M).
There is evidence that indicates in small ruminants and cattle infected with piroplasmosis, oxidative stress is instigated and on the other side the concentrations of antioxidants are reduced [25–29]. However, the balance between oxidants/antioxidants has not yet been fully elucidated in buffaloes naturally infected with T. annulata and only a few studies are available [30]. Previously, we evaluated serum sialic acid (SA) levels in Azeri water buffaloes naturally infected with T. annulata and noticed that as the parasitemia increased accordingly, SA levels increased significantly [31]. Therefore, this study was performed because of two important reasons: first, to evaluate oxidative/nitrosative stress biomarkers and DNA damage in depth and detail to understand the balance between oxidants/antioxidants. Secondly, to investigate whether there is any relationship between SA and oxidative stress or not. To achieve the aims, our previous blood samples were used and several biomarkers including: protein carbonyl (PCO), total antioxidant capacity (TAC), activities of three key antioxidant enzymes, glutathione peroxidase (GSH-Px), catalase (CAT) and superoxide dismutase (SOD) as well as malondialdehyde (MDA) and nitric oxide (NO) contents in T. annulatainfected buffaloes were measured. Furthermore, DNA damage was assessed in lymphocytes with alkaline comet assay, because schizogony stage of parasite take place in lymphocytes.
2.3. Assessment of DNA damage with the alkaline comet assay Alkaline comet assay was performed just same as that of done by Ref. [13] which the method originally belongs to Ref. [37]. Briefly, lymphocytes were isolated by Ficoll-Hypaque lymphocyte separation medium and centrifugation (1900g 15 min) followed by a twice washing stage. After the each wash, centrifugation was done for 10 min at 1800g. Electrophoresis was done at 4 °C with electric current of 25 V and 300 mA for 30 min. Before analysis, slides were stained with ethidium bromide and analyzed under a fluorescence microscope (AxioImager Z1, Carl Zeiss; excitation filter, 515–560 nm; emission filter, 590 nm). Comets were scored visually and classified into five categories corresponding to the amounts of DNA in the tails (Class 0 = no tail (undamaged), Class 1 = tail shorter than the diameter of the head (nucleus), Class 2 = tail length 1 to 2x the diameter of the head, Class 3 = tail longer than 2x the diameter of the head, Class 4 = complete DNA destruction) [38]. Further details are available in Fig. 1. Comet scores were calculated by multiplying the number of damaged cells per specimen with the value of the respective comet class (0–4) and expressed as arbitrary unit.
2. Materials and methods 2.1. Samples The previous blood samples were used to evaluate oxidative/nitrosative stress biomarkers and DNA damage [31]. Briefly, the specimens were collected from 22 infected Azeri buffalos (2–3 year) in West Azerbaijan Province, Iran. Twenty healthy animals were selected as control. The infected animals were divided into four subgroups according to their parasitemia (low < 1%, moderate 1–3%, high 3–5%, very high > 5%). The parasitic identification and confirmation were performed based on the previously described method [31]. Very briefly, thin blood smears were made from peripheral blood (ear vein). After fixation with absolute methanol (5 min), the smears were stained with 10% Giemsa solution (30 min), and examined under oil immersion ( × 1000) to observe intraerythrocytic forms of T. annulata. More than 100 microscopic fields were examined to quantify the parasite and express the percentage of infected erythrocytes. The examined smear was recorded as negative if no parasites detected in 200 fields. The positive and negative results were further confirmed by species-specific nested-PCR assay.
2.4. Statistical analysis The packaged SPSS program for windows Version 22(SPSS, Chicago, IL, USA) was used for statistical analysis. Data was expressed as mean and standard deviation (means ± SD). The data normality was assessed using Shapiro-Wilk test and graphical assessment (histogram and Q-Q plot). Differences between groups were determined by one way analysis of variance (ANOVA1) followed by post hoc Bonferroni test. Pearson's correlation (r) and simple linear regression analysis (R2) were performed on the paired data obtained by individual infected cases. P < 0.05 was considered as statistically significant. 3. Results
2.2. Assessment of oxidative/nitrosative stress parameters As shown in Table 1, the activities of the three key antioxidant enzymes were significantly reduced in the diseased animals compared to the healthy control group. Interestingly, a significant negative relation was detected between the degree of parasitemia and the activities of SOD, GSH-Px and CAT (R2 = 0.974, 0.990 and 0.957, respectively), indicating that in “very high” parasitemia, the reduction rate is more remarkable compared to the other groups (Fig. 2). In particular, the activity of SOD decreased more than 2-fold in severely affected buffaloes (n = 4). PCO content as a marker of protein oxidation and MDA as a marker of lipid peroxidation were measured in lysate of erythrocytes. As can be seen from Table 1, the levels of the both biomarkers were significantly increased in the affected buffaloes as compared to the control group. Specifically, PCO contents were elevated more than 6-fold in severely affected buffaloes (n = 4). Furthermore, Fig. 2 shows a significant (p < 0.01) positive relation between parasitemia and PCO and MDA contents (R2 = 0.989 and 0.986, respectively), suggesting as parasitemia increases, the levels of lipid peroxidation and protein oxidation also increase in a parallel way. The NO levels were estimated in the both healthy and diseased animals and the obtained results showed
All of the oxidative/nitrosative stress biomarkers and indicators were measured in blood lysate according to the previous procedures [25,32]. We measured the end-points in RBC lysate based on the rational of previously published papers and the presence of merozoites in RBC [12,33]. Briefly, the activities of the three key antioxidant enzymes were evaluated spectrophotometrically, using high quality standard assay kits and automated biochemistry analyzer (BT1500, Italia). SOD activity was determined based on xanthine-xanthine oxidase assay using a commercially available standard kit (RanSod, RanDox Co., UK). The activity of GSH-Px was evaluated with GSH-Px detection kit (Ransel, RanDox Co., UK) according to the manufacturer's instructions. Catalase activity was determined using a standard kit (Catalase Assay Kit, Oxford Biomedical Research, Inc., USA). The activities of the enzymes were expressed as U/g Hb. Lipid peroxidation in the RBC hemolysate was determined as thiobarbituric acid reactive substance (TBARS) according to Ref. [34] with trifling alterations. The assay was done using trichloroacetic acid, thiobarbituric acid and l-1 hydrochloric acid. The total antioxidant capacity was determined in the blood lysate using ABTS 2, 2′azino-di2
Microbial Pathogenesis 138 (2020) 103821
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Fig. 1. Alkaline comet assay. Images were classified according to fluorescence intensity in the comet tail and were given a value of 0–4 (from undamaged class 0 to maximally damaged class 4).
data was used [31]. As indicated in Table 2, there is a significant negative correlation between TSA contents and the activities of antioxidant enzymes (p < 0.01). PCO, MDA, NO and DNA damage also have significant positive correlation with TSA contents but the correlation was reverse for TAC (Table 2).
exactly similar pattern to those of observed in PCO and MDA. The TAC amounts were assessed in the lysate samples and the results were recorded in Table 1. As can be seen, the TAC levels significantly decreased in the diseased animals compared to those of controls. Besides, a significant negative relation (R2 = 0.952) was noticed between parasitemia and TAC (Fig. 2). Table 1 also demonstrates a significant elevation of DNA damage in the affected buffaloes. Particularly, the DNA damage was recorded approximately 8-fold higher in severely infected group (n = 4) compared to the healthy animals. There was also a positive relation between DNA damage and the degree of parasitemia (R2 = 0.946). To test whether there is any correlation between oxidative/nitrosative stress biomarkers and serum sialic acid contents, our previous
4. Discussion Previously, we studied SA levels in Azeri water buffaloes naturally infected with T. annulata. We found that following the infection, red blood cells (RBCs), packed cell volume (PCV) and hemoglobin (Hb) significantly decreased in the diseased animals. We also noticed that as the parasitemia increased accordingly, a significant increase in SA
Table 1 Effect of various degrees of parasitemia on oxidative/nitrosative stress parameters and DNA damage. Control (n = 20) SOD (U g−1 Hb) GSH-Px (U g−1 Hb) CAT (U g−1 Hb) PCO (nmol mg−1Hb) MDA (nmol mg−1Hb) TAC (nmol mg−1Hb) NO (nmol mg−1Hb) DNA damage (arbitrary unit)
10.04 ± 0.83a 74.35 ± 1.60a 56.86 ± 2.93a 1.01 ± 0.09a 25.38 ± 1.48a 4.02 ± 0.31a 18.57 ± 0.95a 8.02 ± 0.16a
Low (n = 5)
Moderate (n = 7)
8.68 ± 0.33b 68.35 ± 1.16b 53.62 ± 1.57b 2.47 ± 0.24b 43.35 ± 3.97b 3.26 ± 0.18b 27.41 ± 1.02b 13.55 ± 0.41b
7.02 ± 0.24c 59.55 ± 1.03c 48.44 ± 1.67c 4.08 ± 0.31c 61.49 ± 2.43c 2.47 ± 0.16c 34.83 ± 1.15c 24.45 ± 1.52c
Values within a row carrying different superscript letter (a–e) denote significant differences (P < 0.05). 3
High (n = 6) 6.02 ± 0.10d 49.76 ± 1.65d 41.45 ± 2.57d 5.20 ± 0.27d 92.25 ± 2.61d 1.87 ± 0.15d 47.50 ± 1.90d 40.02 ± 1.23d
Very high (n = 4) 4.85 ± 0.23e 41.35 ± 1.75e 37.77 ± 1.02e 6.77 ± 0.93e 108.35 ± 4.15e 0.94 ± 0.05e 55.13 ± 1.29e 61.79 ± 2.38e
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Fig. 2. The estimated relation between various degrees of parasitemia and oxidative/nitrosative stress biomarkers as well as DNA damage. (Low < 1%, moderate 1–3%, high 3–5%, very high > 5%).
in these cells. The probability of any relation between oxidative/nitrosative stress biomarkers and DNA damage with SA content was also investigated. Our experiments clearly show that the activities of the three key antioxidant enzymes are significantly decreased as the parasitemia increases. This finding can be related to destruction of antioxidant enzymes or depletion of their cofactors, including minerals or vitamins. In fact, when ROS and other free radicals are produced excessively, they attack and damage protein molecules including antioxidant enzymes and thereby reduce their activities [39]. Consistent
content was evident. Therefore, we assumed that SA might play a role in the pathogenesis of T. annulata infection as it stimulates the host immune response and influences the parasite-host cell adhesion [31]. In the current research, the pathogenesis of theileriosis was studied in further detail by assessing oxidative/nitrosative stress biomarkers in RBC lysate. DNA damage also was probed. Considering that merozoites migrate to RBC, we used erythrocyte lysate samples to evaluate oxidative/nitrosative stress status. In addition, DNA damage was studied in lymphocytes, because the schizogony stage of the life cycle takes place 4
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Table 2 Correlation between activities of antioxidant enzymes, oxidative stress markers, DNA damage, nitric oxide and sialic acid in buffalos naturally infected with theileriosis (Pearson correlation). parameter
SOD
GSH-Px
CAT
PCO
MDA
TAC
NO
DNA
TSA
SOD GSH-Px CAT PCO MDA TAC NO DNA TSA
1
0.983a 1
0.969a 0.992a 1
−0.982a −0.988a −0.967a 1
−0.974a −0.996a −0.990a 0.985a 1
0.983a 0.980a 0.972a −0.973a −0.969a 1
−0.977a −0.995a −0.986a 0.988a 0.999a −0.967a 1
−0.948a −0.983a −0.978a 0.963a 0.978a −0.952a 0.977a 1
−0.937a −0.955a −0.941a 0.941a 0.957a −0.930a 0.960a 0.941a 1
SOD: superoxide dismutase, GSH-Px: glutathione peroxidase, CAT: catalase, PCO: protein carbonyl, MDA: malondialdehyde, TAC: total antioxidant capacity, NO: nitric oxide, DNA: DNA damage, TSA: total sialic acid. a Correlation is significant at the 0.01 level (2-tailed).
mechanism involved in the pathophysiology of piroplasmosis. In other word, piroplasms generate various kinds of free radicals and ROS which attack biomolecules culminating in histopathological changes and damages in tissues and organs. Inconsistent with our findings, El-Deeb and Younis (2009) have reported decreased levels of NO in blood samples of Egyptian buffaloes (Bubalus bubalis) infected with T. annulata [30]. The observed discrepancy can partly be related to the different levels of parasitemia, since the authors did not categorize the infected animals based on the degree of parasitemia like done here. In fact the severity of infection is not clear in their study. Additionally, there are racial differences between the buffalos used in their study and ours. Based on the results depicted in Tables 1 and 2, a significant correlation between oxidative stress indicators and total SA (TSA) was detected. The role of SA and its relation with oxidative stress have not yet been fully understood in veterinary medicine and only a limited number of reports are available. Particularly, no study has been done on theileriosis in buffalo. With this aspect, Guzel et al. (2007) measured increased levels of TSA in serum samples of cattle with theileriosis and detected a significant negative correlation between TSA and TAC [45]. Moreover, Uzlu and collaborators (2016) reported increased levels of SA, MDA and NO in saliva and serum samples of bulls with Foot and Mouth Disease (FMD) [46]. Both of these studies support our results of increased TSA, MDA, NO. Mehdi et al. (2012) reported increased levels of plasma SA as a function of age in humans. Interestingly, they also noticed that SA content decreases significantly in RBC membrane in older people. They have also demonstrated that lipid peroxidation measured in the form of hydroperoxides increases significantly in plasma and RBC membranes with age [47]. Mucin is a high molecular weight glycoprotein which contains SA in its structure and functions as a strong hydroxyl radical scavenger. This ability is lost when mucin is desialylated, thus it is capable of protecting cells from oxidants [48]. The decreased levels of SA in the membrane of RBCs as reported by Mehdi et al. (2012), make the erythrocyte vulnerable to oxidative damages. They also have concluded that significant elevation of SA level in plasma is not only due to loss from the RBC membrane through oxidative damage but also due to damages in other organ cells [47]. Consistent with this conclusion, Hasanpour et al. (2008) have reported abnormal electrocardiographic changes in buffaloes naturally infected with T. annulata [10]. Therefore, the increased levels of serum SA in our previous study [31] can be related to oxidative damages to RBC membrane and cardiovascular system. In conclusion, our findings showed that T. annulata infection in buffaloes is associated with a parasitic burden-dependent oxidative/ nitrosative damages to erythrocytes as indicated by lipid peroxidation, carbonylated protein, increased NO production and DNA damage as well as reduction of the activities of antioxidant enzymes and TAC. Seems like antioxidant mechanisms of erythrocytes that protect them against oxidative damages may be overwhelmed by Theileria infection.
with this hypothesis, elevated levels of PCO were recorded in the diseased buffaloes. Cathcart (1985) has suggested that under oxidative stress condition, glutathione (GSH) is consumed by the glutathionerelated enzymes (such as GSH-Px) to detoxify peroxides produced due to increased lipid peroxidation eventuating in the decreased activity of GSH-Px as we observed here [40]. Our data of increased concentrations of MDA are supporting of this hypothesis. Furthermore, copper is an essential trace element for proper function of SOD. In a comprehensive study, Zaeemi et al. (2016) evaluated significantly lower levels of copper in blood samples of Turkoman horses and donkeys infected with Theileria equi compared to those of healthy controls [41]. Besides, zinc and selenium are not only part of antioxidant enzymes, but also they possess direct antioxidant properties. In our previous study, the levels of the aforementioned trace elements were significantly lower in sheep infected with Babesia ovis [12]. Similar results in cattle infected with Anaplasma marginale were also recorded [42]. The aforementioned results suggest that piroplasms interfere with the activities of antioxidant enzymes probably by decreasing the required cofactors and trace elements. Previously, Esmaeilnejad et al. (2012) measured several biomarkers of oxidative stress including the activities of SOD, GSH-Px and CAT in RBC lysate of sheep naturally infected with Babesia ovis [33] and observed similar pattern in the activities of the enzymes as recorded here. These findings suggest that piroplasms destroy and lysate RBCs by induction of severe oxidative stress and overwhelming of the antioxidant enzymes which eventuate in anemia and other clinical outcomes. Our data of decreased SOD and GSH-Px activities are also consistent with those reported in cattle naturally infected with T. annulata [25]. Total antioxidant capacity is a tool to investigate the delicate balance in vivo between oxidants and antioxidants. In fact the capacity of enzymatic and none-enzymatic antioxidants are thereby measured, giving an insight about oxidative stress status in body [39]. According to our results, the TAC was significantly reduced in the affected animals. This finding is consistent with our previous study [33] and clearly indicates inundation of antioxidative mechanisms in RBCs. Our experiments on the blood samples show a significant increase in NO concentrations and DNA damage in the infected buffaloes. Furthermore, a positive relation among parasitemia, nitric oxide and DNA damage was detected. These observations are in agreement with those of reported from horses naturally infected with T. equi [13]. NO is a key participant in many physiological pathways; however, its reactivity gives it the potential to cause considerable damage to cells and tissues in its vicinity, including DNA. Once produced, subsequent conversion of NO to nitrous anhydride and/or peroxynitrite can lead to the nitrosative deamination of DNA bases such as guanine and cytosine [43]. With this aspect, Esmaeilnejad and colleagues (2018) reported, elevated levels of MDA, PCO, NO and DNA damage in hepatic tissue of rats experimentally infected with Babesia bigemina [44]. These data clearly demonstrates that oxidative/nitrosative stress is the major 5
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Furthermore, serum SA plays a crucial role in pathogenesis of the disease and is tightly correlated with oxidative/nitrosative indicators.
[21]
Declaration of competing interest
[22]
None.
[23] [24]
Acknowledgement
[25]
The authors would like to thank the Office of the Vice Chancellor for Research of Urmia University for financial support of this study. This paper is part of DVM thesis of Naeim Molayi-Jabdaragi, numbered 1591, under supervision of Drs. Esmaeilnejad and Mohammadi.
[26]
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Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.micpath.2019.103821.
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Younis, Clinical and biochemical studies on Theileria annulata in Egyptian buffaloes (Bubalus bubalis) with particular orientation to oxidative stress and ketosis relationship, Vet. Parasitol. 164 (2009) 301–305. B. Esmaeilnejad, S.M. Froushani, Evaluation of serum sialic acid level in buffaloes naturally infected with Theileria annulata, Trop. Anim. Health Prod. 48 (2016) 1381–1386. B. Esmaeilnejad, A. Samiei, Y. Mirzaei, F. Farhang-Pajuh, Assessment of oxidative/ nitrosative stress biomarkers and DNA damage in Haemonchus contortus, following exposure to zinc oxide nanoparticles, Acta Parasitol. 63 (2018) 563–571. B. Esmaeilnejad, M. Tavassoli, S. Asri-Rezaei, B. Dalir-Naghadeh, Evaluation of antioxidant status and oxidative stress in sheep naturally infected with Babesia ovis, Vet. Parasitol. 185 (2012) 124–130. J.A. Buege, S.D. Aust, Microsomal lipid peroxidation, in: S. Fleischer, L. Packer (Eds.), Methods in Enzymology, Academic Press, 1978, pp. 302–310. A.H. Ding, C.F. 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Cathcart, Vitamin C: the nontoxic, nonrate-limited, antioxidant free radical scavenger, Med. Hypotheses 18 (1985) 61–77. M. Zaeemi, G.R. Razmi, G.R. Mohammadi, V. Abedi, S. Yaghfoori, Evaluation of serum biochemical profile in Turkoman horses and donkeys infected with Theileria equi, Rev. Med. Vet. (Toulouse) 167 (2016) 301–309. B. Esmaeilnejad, M. Tavassoli, A. Samiei, N. Hajipour, A. Imani-Baran, F. FarhangPajuh, Evaluation of oxidative stress and antioxidant status, serum trace mineral levels and cholinesterases activity in cattle infected with Anaplasma marginale, Microb. Pathog. 123 (2018) 402–409. S. Burney, J.L. Caulfield, J.C. Niles, J.S. Wishnok, S.R. Tannenbaum, The chemistry of DNA damage from nitric oxide and peroxynitrite, Mutat. Res. Fundam. Mol. Mech. Mutagen. 424 (1999) 37–49. B. Esmaeilnejad, M. Tavassoli, A. Samiei, A. Abbasi, A. Shafipour, N. 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Contents lists available at ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Evaluation of oxidative/nitrosative stress biomarkers and DNA damage in buffaloes naturally infected with Theileria annulata
T
Naeim Molayi-Jabdaragia, Bijan Esmaeilnejada,∗, Vahid Mohammadib a b
Department of Pathobiology, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran Department of Internal Medicine and Clinical Pathology, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran
A R T I C LE I N FO
A B S T R A C T
Keywords: T. annulata Buffalo Oxidative stress DNA damage Biomarker
Previously, we evaluated serum sialic acid (SA) levels in buffaloes naturally infected with T. annulata. In the current paper, we conducted a further study on oxidative/nitrosative stress biomarkers in erythrocyte lysate samples of the same buffaloes. DNA damage also was assessed. Additionally, we tested whether, there is any correlation between SA and the aforementioned indicators or not. To achieve these aims, several biomarkers including the activities of key antioxidant enzymes, superoxide dismutase, glutathione peroxidase and catalase, as well as malondialdehyde (MDA), protein carbonyl (PCO), nitric oxide contents (NO), total antioxidant capacity (TAC) and DNA damage levels were measured. The obtained results showed that the activities of the antioxidant enzymes and TAC levels decreased significantly as the percentage of parasitemia increased accordingly. Also, a significant increase in the levels of PCO, MDA, NO and DNA damage were recorded, depending on the degree of parasitemia. There was a significant correlation between oxidative/nitrosative stress indicators and SA. Conclusively, T. annulata infection in buffaloes is associated with a parasitic burden-dependent oxidative/nitrosative damages to erythrocytes and SA plays a crucial role in pathogenesis of the disease, as it is tightly correlated with oxidative/nitrosative indicators.
1. Introduction In Iran, arid and semi-arid geographical conditions are hospitable for faster propagation of the Ixodidae ticks, (Hyalomma anatolicum) and rapid spreading of tick-borne diseases such as bovine tropical theileriosis (BTT) caused by Theileria annulata [1–3]. It is estimated that millions of dollars are wasted in Iranian agricultural industry because of BTT [4]. The parasite undergoes a highly complex life cycle inside the mammalian hosts, initiating by the transformation of macroschizontinfected cells in the lymph nodes draining the site of inoculation of sporozoites by ticks. The sporozoites transform into schizonts in white blood cell of the mononuclear lineage leading to infection of cattle. Then, merozoites enter erythrocytes after being released upon lysis of the infected cells. Finally, intra-erythrocytic parasite becomes infective for the vector [5,6]. The most palpable signs and symptoms emerge because of the anemia and include: weakness, weight loss, anorexia, high body temperature, petechia on the conjunctival mucosa, swollen lymph nodes, and cough. At the final stages, animals become progressively feeble as they cannot tolerate their weight and body temperature drops below
∗
38.5 °C. Occasionally, ichterus, dehydration, and blood in feces are clinical symptoms [7,8] Following recovery, bovids can act as asymptomatic carriers for life, but they are reservoirs for subsequent tick infection [9,10]. Parasitic diseases are able to instigate oxidative stress because hosts produce reactive oxygen species (ROS), primarily to destroy invading pathogens [11–13]. However, these reactive molecules cannot discriminate between host cells and infectious agents, resulting in severe damage to the host cells and organs [14,15]. To alleviate the damages, the host activates protective mechanisms including anti-oxidative defenses. But, if the ROS is generated enormously, the defense systems can be overwhelmed leading to induction of oxidative stress [16,17]. The role of oxidative stress in the pathogenesis of bovine diseases has previously been described [18–22]. Free radicals and ROS target biomolecules such as lipids, proteins and DNA, causing irreversible changes [23]. Selective permeability of cell membrane is lost when it is progressively attacked by ROS which eventuates in lipid peroxidation. Oxidative damage to proteins can alter configuration, resulting in loss of biochemical functions, while oxidative DNA damage can cause a range of alterations including: mutations, replication errors, genomic instability and cell death [24].
Corresponding author. E-mail address: [email protected] (B. Esmaeilnejad).
https://doi.org/10.1016/j.micpath.2019.103821 Received 13 April 2019; Received in revised form 20 October 2019; Accepted 23 October 2019 Available online 23 October 2019 0882-4010/ © 2019 Elsevier Ltd. All rights reserved.
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[3-ethylbenzthiazoline sulfonate]; Randox Laboratories Ltd., UK as the substrate. The total nitrate/nitrite content of the blood samples was measured according to the routine method of Griess reaction. Briefly, 100 μl of the lysate was mixed with 100 μl of Griess reagent in a 96-well plate and the reaction was read at 570 nm after 10 min [35]. Protein carbonyls were measured based on the method of Levin and collaborators [36], using 2,4-dinitrophenylhydrazine, hydrochloric acid, ethanol–ethyl acetate (1:1, v/v) and guanidine hydrochloride solution (6 M). The carbonyl content was calculated based on the molar extinction coefficient of DNPH (ε = 2.2 × 104 cm/M).
There is evidence that indicates in small ruminants and cattle infected with piroplasmosis, oxidative stress is instigated and on the other side the concentrations of antioxidants are reduced [25–29]. However, the balance between oxidants/antioxidants has not yet been fully elucidated in buffaloes naturally infected with T. annulata and only a few studies are available [30]. Previously, we evaluated serum sialic acid (SA) levels in Azeri water buffaloes naturally infected with T. annulata and noticed that as the parasitemia increased accordingly, SA levels increased significantly [31]. Therefore, this study was performed because of two important reasons: first, to evaluate oxidative/nitrosative stress biomarkers and DNA damage in depth and detail to understand the balance between oxidants/antioxidants. Secondly, to investigate whether there is any relationship between SA and oxidative stress or not. To achieve the aims, our previous blood samples were used and several biomarkers including: protein carbonyl (PCO), total antioxidant capacity (TAC), activities of three key antioxidant enzymes, glutathione peroxidase (GSH-Px), catalase (CAT) and superoxide dismutase (SOD) as well as malondialdehyde (MDA) and nitric oxide (NO) contents in T. annulatainfected buffaloes were measured. Furthermore, DNA damage was assessed in lymphocytes with alkaline comet assay, because schizogony stage of parasite take place in lymphocytes.
2.3. Assessment of DNA damage with the alkaline comet assay Alkaline comet assay was performed just same as that of done by Ref. [13] which the method originally belongs to Ref. [37]. Briefly, lymphocytes were isolated by Ficoll-Hypaque lymphocyte separation medium and centrifugation (1900g 15 min) followed by a twice washing stage. After the each wash, centrifugation was done for 10 min at 1800g. Electrophoresis was done at 4 °C with electric current of 25 V and 300 mA for 30 min. Before analysis, slides were stained with ethidium bromide and analyzed under a fluorescence microscope (AxioImager Z1, Carl Zeiss; excitation filter, 515–560 nm; emission filter, 590 nm). Comets were scored visually and classified into five categories corresponding to the amounts of DNA in the tails (Class 0 = no tail (undamaged), Class 1 = tail shorter than the diameter of the head (nucleus), Class 2 = tail length 1 to 2x the diameter of the head, Class 3 = tail longer than 2x the diameter of the head, Class 4 = complete DNA destruction) [38]. Further details are available in Fig. 1. Comet scores were calculated by multiplying the number of damaged cells per specimen with the value of the respective comet class (0–4) and expressed as arbitrary unit.
2. Materials and methods 2.1. Samples The previous blood samples were used to evaluate oxidative/nitrosative stress biomarkers and DNA damage [31]. Briefly, the specimens were collected from 22 infected Azeri buffalos (2–3 year) in West Azerbaijan Province, Iran. Twenty healthy animals were selected as control. The infected animals were divided into four subgroups according to their parasitemia (low < 1%, moderate 1–3%, high 3–5%, very high > 5%). The parasitic identification and confirmation were performed based on the previously described method [31]. Very briefly, thin blood smears were made from peripheral blood (ear vein). After fixation with absolute methanol (5 min), the smears were stained with 10% Giemsa solution (30 min), and examined under oil immersion ( × 1000) to observe intraerythrocytic forms of T. annulata. More than 100 microscopic fields were examined to quantify the parasite and express the percentage of infected erythrocytes. The examined smear was recorded as negative if no parasites detected in 200 fields. The positive and negative results were further confirmed by species-specific nested-PCR assay.
2.4. Statistical analysis The packaged SPSS program for windows Version 22(SPSS, Chicago, IL, USA) was used for statistical analysis. Data was expressed as mean and standard deviation (means ± SD). The data normality was assessed using Shapiro-Wilk test and graphical assessment (histogram and Q-Q plot). Differences between groups were determined by one way analysis of variance (ANOVA1) followed by post hoc Bonferroni test. Pearson's correlation (r) and simple linear regression analysis (R2) were performed on the paired data obtained by individual infected cases. P < 0.05 was considered as statistically significant. 3. Results
2.2. Assessment of oxidative/nitrosative stress parameters As shown in Table 1, the activities of the three key antioxidant enzymes were significantly reduced in the diseased animals compared to the healthy control group. Interestingly, a significant negative relation was detected between the degree of parasitemia and the activities of SOD, GSH-Px and CAT (R2 = 0.974, 0.990 and 0.957, respectively), indicating that in “very high” parasitemia, the reduction rate is more remarkable compared to the other groups (Fig. 2). In particular, the activity of SOD decreased more than 2-fold in severely affected buffaloes (n = 4). PCO content as a marker of protein oxidation and MDA as a marker of lipid peroxidation were measured in lysate of erythrocytes. As can be seen from Table 1, the levels of the both biomarkers were significantly increased in the affected buffaloes as compared to the control group. Specifically, PCO contents were elevated more than 6-fold in severely affected buffaloes (n = 4). Furthermore, Fig. 2 shows a significant (p < 0.01) positive relation between parasitemia and PCO and MDA contents (R2 = 0.989 and 0.986, respectively), suggesting as parasitemia increases, the levels of lipid peroxidation and protein oxidation also increase in a parallel way. The NO levels were estimated in the both healthy and diseased animals and the obtained results showed
All of the oxidative/nitrosative stress biomarkers and indicators were measured in blood lysate according to the previous procedures [25,32]. We measured the end-points in RBC lysate based on the rational of previously published papers and the presence of merozoites in RBC [12,33]. Briefly, the activities of the three key antioxidant enzymes were evaluated spectrophotometrically, using high quality standard assay kits and automated biochemistry analyzer (BT1500, Italia). SOD activity was determined based on xanthine-xanthine oxidase assay using a commercially available standard kit (RanSod, RanDox Co., UK). The activity of GSH-Px was evaluated with GSH-Px detection kit (Ransel, RanDox Co., UK) according to the manufacturer's instructions. Catalase activity was determined using a standard kit (Catalase Assay Kit, Oxford Biomedical Research, Inc., USA). The activities of the enzymes were expressed as U/g Hb. Lipid peroxidation in the RBC hemolysate was determined as thiobarbituric acid reactive substance (TBARS) according to Ref. [34] with trifling alterations. The assay was done using trichloroacetic acid, thiobarbituric acid and l-1 hydrochloric acid. The total antioxidant capacity was determined in the blood lysate using ABTS 2, 2′azino-di2
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Fig. 1. Alkaline comet assay. Images were classified according to fluorescence intensity in the comet tail and were given a value of 0–4 (from undamaged class 0 to maximally damaged class 4).
data was used [31]. As indicated in Table 2, there is a significant negative correlation between TSA contents and the activities of antioxidant enzymes (p < 0.01). PCO, MDA, NO and DNA damage also have significant positive correlation with TSA contents but the correlation was reverse for TAC (Table 2).
exactly similar pattern to those of observed in PCO and MDA. The TAC amounts were assessed in the lysate samples and the results were recorded in Table 1. As can be seen, the TAC levels significantly decreased in the diseased animals compared to those of controls. Besides, a significant negative relation (R2 = 0.952) was noticed between parasitemia and TAC (Fig. 2). Table 1 also demonstrates a significant elevation of DNA damage in the affected buffaloes. Particularly, the DNA damage was recorded approximately 8-fold higher in severely infected group (n = 4) compared to the healthy animals. There was also a positive relation between DNA damage and the degree of parasitemia (R2 = 0.946). To test whether there is any correlation between oxidative/nitrosative stress biomarkers and serum sialic acid contents, our previous
4. Discussion Previously, we studied SA levels in Azeri water buffaloes naturally infected with T. annulata. We found that following the infection, red blood cells (RBCs), packed cell volume (PCV) and hemoglobin (Hb) significantly decreased in the diseased animals. We also noticed that as the parasitemia increased accordingly, a significant increase in SA
Table 1 Effect of various degrees of parasitemia on oxidative/nitrosative stress parameters and DNA damage. Control (n = 20) SOD (U g−1 Hb) GSH-Px (U g−1 Hb) CAT (U g−1 Hb) PCO (nmol mg−1Hb) MDA (nmol mg−1Hb) TAC (nmol mg−1Hb) NO (nmol mg−1Hb) DNA damage (arbitrary unit)
10.04 ± 0.83a 74.35 ± 1.60a 56.86 ± 2.93a 1.01 ± 0.09a 25.38 ± 1.48a 4.02 ± 0.31a 18.57 ± 0.95a 8.02 ± 0.16a
Low (n = 5)
Moderate (n = 7)
8.68 ± 0.33b 68.35 ± 1.16b 53.62 ± 1.57b 2.47 ± 0.24b 43.35 ± 3.97b 3.26 ± 0.18b 27.41 ± 1.02b 13.55 ± 0.41b
7.02 ± 0.24c 59.55 ± 1.03c 48.44 ± 1.67c 4.08 ± 0.31c 61.49 ± 2.43c 2.47 ± 0.16c 34.83 ± 1.15c 24.45 ± 1.52c
Values within a row carrying different superscript letter (a–e) denote significant differences (P < 0.05). 3
High (n = 6) 6.02 ± 0.10d 49.76 ± 1.65d 41.45 ± 2.57d 5.20 ± 0.27d 92.25 ± 2.61d 1.87 ± 0.15d 47.50 ± 1.90d 40.02 ± 1.23d
Very high (n = 4) 4.85 ± 0.23e 41.35 ± 1.75e 37.77 ± 1.02e 6.77 ± 0.93e 108.35 ± 4.15e 0.94 ± 0.05e 55.13 ± 1.29e 61.79 ± 2.38e
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Fig. 2. The estimated relation between various degrees of parasitemia and oxidative/nitrosative stress biomarkers as well as DNA damage. (Low < 1%, moderate 1–3%, high 3–5%, very high > 5%).
in these cells. The probability of any relation between oxidative/nitrosative stress biomarkers and DNA damage with SA content was also investigated. Our experiments clearly show that the activities of the three key antioxidant enzymes are significantly decreased as the parasitemia increases. This finding can be related to destruction of antioxidant enzymes or depletion of their cofactors, including minerals or vitamins. In fact, when ROS and other free radicals are produced excessively, they attack and damage protein molecules including antioxidant enzymes and thereby reduce their activities [39]. Consistent
content was evident. Therefore, we assumed that SA might play a role in the pathogenesis of T. annulata infection as it stimulates the host immune response and influences the parasite-host cell adhesion [31]. In the current research, the pathogenesis of theileriosis was studied in further detail by assessing oxidative/nitrosative stress biomarkers in RBC lysate. DNA damage also was probed. Considering that merozoites migrate to RBC, we used erythrocyte lysate samples to evaluate oxidative/nitrosative stress status. In addition, DNA damage was studied in lymphocytes, because the schizogony stage of the life cycle takes place 4
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Table 2 Correlation between activities of antioxidant enzymes, oxidative stress markers, DNA damage, nitric oxide and sialic acid in buffalos naturally infected with theileriosis (Pearson correlation). parameter
SOD
GSH-Px
CAT
PCO
MDA
TAC
NO
DNA
TSA
SOD GSH-Px CAT PCO MDA TAC NO DNA TSA
1
0.983a 1
0.969a 0.992a 1
−0.982a −0.988a −0.967a 1
−0.974a −0.996a −0.990a 0.985a 1
0.983a 0.980a 0.972a −0.973a −0.969a 1
−0.977a −0.995a −0.986a 0.988a 0.999a −0.967a 1
−0.948a −0.983a −0.978a 0.963a 0.978a −0.952a 0.977a 1
−0.937a −0.955a −0.941a 0.941a 0.957a −0.930a 0.960a 0.941a 1
SOD: superoxide dismutase, GSH-Px: glutathione peroxidase, CAT: catalase, PCO: protein carbonyl, MDA: malondialdehyde, TAC: total antioxidant capacity, NO: nitric oxide, DNA: DNA damage, TSA: total sialic acid. a Correlation is significant at the 0.01 level (2-tailed).
mechanism involved in the pathophysiology of piroplasmosis. In other word, piroplasms generate various kinds of free radicals and ROS which attack biomolecules culminating in histopathological changes and damages in tissues and organs. Inconsistent with our findings, El-Deeb and Younis (2009) have reported decreased levels of NO in blood samples of Egyptian buffaloes (Bubalus bubalis) infected with T. annulata [30]. The observed discrepancy can partly be related to the different levels of parasitemia, since the authors did not categorize the infected animals based on the degree of parasitemia like done here. In fact the severity of infection is not clear in their study. Additionally, there are racial differences between the buffalos used in their study and ours. Based on the results depicted in Tables 1 and 2, a significant correlation between oxidative stress indicators and total SA (TSA) was detected. The role of SA and its relation with oxidative stress have not yet been fully understood in veterinary medicine and only a limited number of reports are available. Particularly, no study has been done on theileriosis in buffalo. With this aspect, Guzel et al. (2007) measured increased levels of TSA in serum samples of cattle with theileriosis and detected a significant negative correlation between TSA and TAC [45]. Moreover, Uzlu and collaborators (2016) reported increased levels of SA, MDA and NO in saliva and serum samples of bulls with Foot and Mouth Disease (FMD) [46]. Both of these studies support our results of increased TSA, MDA, NO. Mehdi et al. (2012) reported increased levels of plasma SA as a function of age in humans. Interestingly, they also noticed that SA content decreases significantly in RBC membrane in older people. They have also demonstrated that lipid peroxidation measured in the form of hydroperoxides increases significantly in plasma and RBC membranes with age [47]. Mucin is a high molecular weight glycoprotein which contains SA in its structure and functions as a strong hydroxyl radical scavenger. This ability is lost when mucin is desialylated, thus it is capable of protecting cells from oxidants [48]. The decreased levels of SA in the membrane of RBCs as reported by Mehdi et al. (2012), make the erythrocyte vulnerable to oxidative damages. They also have concluded that significant elevation of SA level in plasma is not only due to loss from the RBC membrane through oxidative damage but also due to damages in other organ cells [47]. Consistent with this conclusion, Hasanpour et al. (2008) have reported abnormal electrocardiographic changes in buffaloes naturally infected with T. annulata [10]. Therefore, the increased levels of serum SA in our previous study [31] can be related to oxidative damages to RBC membrane and cardiovascular system. In conclusion, our findings showed that T. annulata infection in buffaloes is associated with a parasitic burden-dependent oxidative/ nitrosative damages to erythrocytes as indicated by lipid peroxidation, carbonylated protein, increased NO production and DNA damage as well as reduction of the activities of antioxidant enzymes and TAC. Seems like antioxidant mechanisms of erythrocytes that protect them against oxidative damages may be overwhelmed by Theileria infection.
with this hypothesis, elevated levels of PCO were recorded in the diseased buffaloes. Cathcart (1985) has suggested that under oxidative stress condition, glutathione (GSH) is consumed by the glutathionerelated enzymes (such as GSH-Px) to detoxify peroxides produced due to increased lipid peroxidation eventuating in the decreased activity of GSH-Px as we observed here [40]. Our data of increased concentrations of MDA are supporting of this hypothesis. Furthermore, copper is an essential trace element for proper function of SOD. In a comprehensive study, Zaeemi et al. (2016) evaluated significantly lower levels of copper in blood samples of Turkoman horses and donkeys infected with Theileria equi compared to those of healthy controls [41]. Besides, zinc and selenium are not only part of antioxidant enzymes, but also they possess direct antioxidant properties. In our previous study, the levels of the aforementioned trace elements were significantly lower in sheep infected with Babesia ovis [12]. Similar results in cattle infected with Anaplasma marginale were also recorded [42]. The aforementioned results suggest that piroplasms interfere with the activities of antioxidant enzymes probably by decreasing the required cofactors and trace elements. Previously, Esmaeilnejad et al. (2012) measured several biomarkers of oxidative stress including the activities of SOD, GSH-Px and CAT in RBC lysate of sheep naturally infected with Babesia ovis [33] and observed similar pattern in the activities of the enzymes as recorded here. These findings suggest that piroplasms destroy and lysate RBCs by induction of severe oxidative stress and overwhelming of the antioxidant enzymes which eventuate in anemia and other clinical outcomes. Our data of decreased SOD and GSH-Px activities are also consistent with those reported in cattle naturally infected with T. annulata [25]. Total antioxidant capacity is a tool to investigate the delicate balance in vivo between oxidants and antioxidants. In fact the capacity of enzymatic and none-enzymatic antioxidants are thereby measured, giving an insight about oxidative stress status in body [39]. According to our results, the TAC was significantly reduced in the affected animals. This finding is consistent with our previous study [33] and clearly indicates inundation of antioxidative mechanisms in RBCs. Our experiments on the blood samples show a significant increase in NO concentrations and DNA damage in the infected buffaloes. Furthermore, a positive relation among parasitemia, nitric oxide and DNA damage was detected. These observations are in agreement with those of reported from horses naturally infected with T. equi [13]. NO is a key participant in many physiological pathways; however, its reactivity gives it the potential to cause considerable damage to cells and tissues in its vicinity, including DNA. Once produced, subsequent conversion of NO to nitrous anhydride and/or peroxynitrite can lead to the nitrosative deamination of DNA bases such as guanine and cytosine [43]. With this aspect, Esmaeilnejad and colleagues (2018) reported, elevated levels of MDA, PCO, NO and DNA damage in hepatic tissue of rats experimentally infected with Babesia bigemina [44]. These data clearly demonstrates that oxidative/nitrosative stress is the major 5
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Furthermore, serum SA plays a crucial role in pathogenesis of the disease and is tightly correlated with oxidative/nitrosative indicators.
[21]
Declaration of competing interest
[22]
None.
[23] [24]
Acknowledgement
[25]
The authors would like to thank the Office of the Vice Chancellor for Research of Urmia University for financial support of this study. This paper is part of DVM thesis of Naeim Molayi-Jabdaragi, numbered 1591, under supervision of Drs. Esmaeilnejad and Mohammadi.
[26]
[27]
Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.micpath.2019.103821.
[28]
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