Evaluation of Oxidative Status and Inflammatory Changes in Naturally Occurring Canine Visceral Leishmaniasis

Tünay Kontaş Aşkar1,*, Şinasi Aşkar1, Olga Büyükleblebici2 and Murat Güzel3

1Department of Nutrition and Dietetic, Faculty of Health Sciences, University of Karatekin, 18200, Çankırı, Turkey

2Department of Biochemistry, Faculty of Veterinary, University of Aksaray, Aksaray, Turkey

3Department of Internal Medicine, Faculty of Veterinary, University of Ondokuz Mayıs, Samsun, Turkey

ABSTRACT

The aim of the study is to determine the oxidative stress and inflammatory changes in naturally occurring canine visceral leishmaniasis (CVL). A total of 30 dogs, comprising 10 clinically healthy and 20 dogs with leishmaniasis were enrolled in this study. The diagnosis of canine visceral leishmaniasis was performed by the immune fluorescence antibody test with antibody titter ≥1:128 and lymph node smear examination. In blood samples of dogs with visceral leishmaniasis, levels of malondialdehyde, total antioxidant capacity, glutathione, nitric oxide, myeloperoxidase, adenosine deaminase and cytokines (TNF-alpha, IL-1β) were determined and compared with the healthy controls. Significantly high levels of plasma malondialdehyde, nitric oxide, myeloperoxidase, adenosine deaminase and cytokines and significantly low levels in total antioxidant capacity and glutathione levels were found in dogs with leishmaniasis when compared with the healthy controls. According to the result of this study, oxidative stress and inflammatory changes occur in CVL. And this is the first report for myeloperoxidase (MPO) activity in CVL. Therefore investigation of this enzyme activity in dogs with leishmaniasis may be used for the diagnosis and inflammatory changes in dogs with VL.

Article Information

Received 09 April 2018

Revised 29 May 2018

Accepted 05 June 2018

Available online 21 December 2018

Authors’ Contribution

TKA and MG designed the study, collected and analysed the data and wrote the article. SA and OB helped in collection and analysis of data and article writing.

Key words

Dog, Inflammatory changes, Leishmaniasis, Oxidative stress.

DOI: http://dx.doi.org/10.17582/journal.pjz/2019.51.1.301.306

* Corresponding author: tunaykontas@yahoo.com

0030-9923/2019/0001-0301 $ 9.00/0

Copyright 2019 Zoological Society of Pakistan

Introduction

Visceral Leishmaniasis (VL) is a zoonosis in the Mediterranean basin which is very difficult to treat both humans and dogs. VL present in 88 countries on four continents, caused 2.4 million disability-adjusted life years (DALYs) and 59,000 death in 2001 (Alberola et al., 2004). The dog is the principal host for VL and in Turkey the infection caused by Leishmania infantum (Ozensoy et al., 1998).

Macrophages, neutrophils and other phagocytic cells are important compounds of host defense against parasites. These phagocytic inflammatory cells can produce highly toxic molecules such as reactive oxygen and nitrogen species (ROS and RNS) including superoxide radicals (O2), hydrogen peroxide (H2O), hydroxyl radicals (OH), nitric oxide (NO) and proinflammatory cytokines (Bogdan et al., 2000; Abdel-Maksoud et al., 2017). Nitric oxide is a significant cytotoxic and cytostatic tool for diverse cellular parasites with oxidative and prooxidative properties in inflammatory diseases. It has been shown that the interaction between NO and myeloperoxidase (MPO) could have a profound effect on the toxic capacity of neutrophils (Brunet, 2001; Knaapen et al., 2005).

Myeloperoxidase is an oxidative enzyme with antimicrobial activity that uses H2O2 to produce hypochloric acid (a potent cytotoxic compound) and other toxic substances in neutrophil phagolysosomes. Therefore, MPO is abundant in cytoplasmic granules of neutrophils, and 40-70% of H2O2 derived from neutrophils is consumed by MPO (Garça et al., 2013). Adenosine deaminase (ADA) is a purine catabolic enzyme with the highest concentration of lymphoid tissue in mammalian tissue. Detection of ADA activity in biological fluids is very useful for diagnosing diseases in which many pathological conditions and immune response are mediated by the cell (Kontaş and Salmanoğlu, 2006). Although the inflammatory and oxidative changes induced by Leishmania infantum has been the subject to many investigations (Pinelli et al., 1994; Erel et al., 1999; Heidarpour et al., 2012; Serarslan et al., 2005) the mechanisms that underlie host resistance and pathogenesis in CVL are not entirely understood. Therefore, we aimed to determine the inflammatory and oxidative changes in in naturally occurring CVL model.

 

Materials and methods

Animals and clinical examination

In this study, outdoor dogs (n=30) were enrolled in the study. The animals were between 2-6 years old age and 15-35 kg body weight in different breed and both of sex. For all dogs, the clinical history and physical examinations were performed. These dogs with two or more obvious clinical signs of leishmaniosis such as weight loss (16/20), lymphadenopathy (8/20), fever, epistaxis (4/20), fatigue, conjunctivitis (14/20), dermatitis, hair loss, mouth and skin ulcers, onychogryphosis. After clinical examination, the animal with two or more obvious symptoms of the disease was considered as clinical positive and investigated further parasitological analysis to VL. In addition to, suspected or clinical positive animals were evaluated both serological and lymph node smear examination.

Parasitological analysis

Lymph node aspiration

Popliteal lymph node aspirates were taken from the dogs with extended lymph nodes from the clinical positive and negative animals according to the clinical history and physical examinations. After aspiration and fixing in methanol, prepared smears were stained with Giemsa 10% for 10 min at room temperature and examined with light microscope for the presence of amastigotes. Parasitological examination was regarded as positive based on smear positivity.

Immune fluorescence test (IFAT)

The diagnosis of CVL was performed by the immune fluorescence test with antibody titter ≥1:128 and lymph node smear examination. Briefly, the in-house antigen consisted of promastigotes of local L. infantum zymodeme MON-1 was used in IFAT. Following incubation at 37°C for 30 min slides were washed and stained with FITC-labeled anti-dog IgG conjugate (Sigma, A9042) for dog sera. Titers ≥1:128 were considered as seropositive (Abranches et al., 1991).

Biochemical analysis

Blood samples were collected from dogs and added to lithium heparin and silicone gel-coated test tubes. Serum/plasma samples were separated by centrifugation at 2500 rpm for 10 min at room temperature and kept at -80°C until processing. Blood samples were analyzed for the changes in malondialdehyde (MDA), total antioxidant capacity (TAC), glutathione (GSH), NO and cytokines (TNF-α an IL-1β) levels, in addition to MPO and ADA enzyme activities.

MDA levels in the plasma samples were defined by a formerly defined method (Yoshioka et al., 1979), and reduced GSH levels were determined by the method of Beutler et al. (1963). Plasma TAC levels were measured by using Rel Assay Diagnostics kit (Mega Tıp, Turkey). This method was based upon the bleaching of the distinct color of the 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radical cation via the action of antioxidants. The production of nitric oxide (NO) was determined indirectly by measuring the nitrite levels based on Griess reaction (Cortas and Wakid, 1990). MPO activity was measured using the method of Kruidenier et al. (2003). Serum samples were incubated for 30 min with 0.5 % hexadecyltrimethylammonium bromide (HTAB) solution (pH 5.5) and 0.026 % ortho-dianisidinedihydrochloride plus 0.018 % H2O2. The reaction was specifically confirmed by sodium azide (0.1 mM). MPO activity was expressed as U/L. Serum total ADA activity was determined with Giusti (1974) method. It is a colorimetric manual method based on the principle of measuring absorbance of the coloredindophenole complex at 628 nm. Canine interleukin-1β (Eastbiopharm, Cat No. CK-E90800), and canine tumor necrosis factor-α (Eastbiopharm, Cat No. CK-E90806) levels were measured using the ELISA method. Measurable sensitivity of IL-1β was 0.1 pg/mL, and the test interval of IL-1β level was 0.2 pg/mL and 50 pg/mL, measurable sensitivity of TNF-α is 0.01 ng/L and test interval of TNF-α level was 0,02 ng/L and 8 ng/L.

 

Statistical analysis

Data for biochemical analyses are presented as mean±SD. Statistical analysis was performed using Student’s t-test; P values <0.05 were considered statistically significant. The correlation of the MDA concentration with TAC and GSH concentrations, besides the correlation of TNF-α concentration with NO, MPO, ADA, and IL-1β concentrations were analyzed using Pearson’s rank correlation coefficient.

 

Results

In the study, the clinical status of the dogs was carefully assessed, and leishmaniasis dogs showed two or more open clinical findings of leishmaniasis.

The results of oxidative stress and antioxidant status parameters of leishmaniasis and healthy control group dogs are presented in Figure 1. We observed a significant increase in MDA (P <0.001) levels in the CVL group (Fig. 1); and as seen in Figure 1B, there is a significant decrease in TAC (P<0.05) and GSH (P<0.01) levels in CVL group when compared with the control group.

The correlations of MDA level with TAC and GSH levels were analyzed in this study. By using Pearson’s rank correlation coefficient, a potent correlation was seen with TAC level (r=-0.618, P<0.05), but there was a low correlation with GSH level (r=-0.69, P<0.001). The negative correlations of MDA level with TAC, and GSH is shown in Figure 2A and B.

Whereas, the levels of NO, MPO, ADA, and cytokines in the dogs with VL and the healthy controls are shown in Table I. NO and TNF-α, IL-1β levels were found significantly higher (P<0.01, P<0.001, and P<0.001, respectively) in CVL in comparison to healthy animals. Serum enzyme activities of ADA and MPO in the dogs with VL were found significantly higher (P<0.001) than that of the healthy animals.

 

Table I.- Serum cytokine, NO, MPO and ADA levels in dogs with VL and healthy controls.

	Control group (n=10)	CVL group(n=20)	P
NO (µmol/L)	20.87±1.01a	29.37±2.62b	P<0.01
MPO (ng/mL)	12.02±3.47a	24.16±8.59b	P<0.001
ADA (IU/L)	5.24±0.24a	8.24±0.26b	P<0.05
IL-1ß pg/mL	15.24±1.87a	27.8±5.12b	P<0.001
TNF-α ng/L	0.39±0.83a	3.12±0.71b	P<0.001

*Data are expressed as mean±standart error of the mean values; a,bdifferences are statistically significant in groups marked with different letters in the same row (P<0.05). NO, nitric oxide; MPO, miyeloperoxidase; ADA, adenosine deaminase; IL-1β, ınterleukine-1β; TNF-α, tumour necrosis factor- α.

 

However, we analyzed the correlations between the TNF-α and NO, MPO, and ADA concentrations. Pearson’s rank correlation coefficient for TNF-α and NO (r=0.812, P<0.001), MPO (r=0.674, P<0.01), ADA (r=0.432, P<0.05), and IL-1β (r=0.801, P<0.001) concentrations, indicating a strong positive correlation was observed. The positive correlation was seen between the TNF-α and NO, MPO, ADA, and IL-1β concentrations as shown in Figure 3.

 

Discussion

Haematological and serum biochemical measurements in dogs with VL have limited applications for diagnosis, but can be important in understanding the VL pathogenesis (Reis et al., 2010). This study was designed to evaluate the oxidative and inflammatory changes in CVL.

Oxidative stress is defined as the inability of the organ or the cell to defend against ROS and to cause oxidative injury (Ozcan et al., 2004). Organisms have developed various antioxidant defenses, including non-enzymatic and specific antioxidant enzymes, to provide protection against oxidative damage (Zelko et al., 2002). One of the most commonly used ROS biomarkers that is indicative of general lipid peroxidation levels is MDA, a product of lipid peroxidation (Nielsen et al., 1997). There are many reports about the increased levels of MDA in cutaneous leishmaniasis (Serarslan et al., 2005). In this study, increased levels of MDA and decreased levels of TAC and GSH in CVL (Fig. 1) indicated the presence of oxidative stress and lipid peroxides (Bildik et al., 2004; Heidarpour et al., 2012); and decreases in TAC and GSH levels during CVL may be related with the prevention of the synthesis of antioxidant enzymes and glutathione by oxidant-induced DNA damage in the chronic stage of the disease (Garça et al., 2013).

NO is an important cytotoxic and cytostatic mediator for various intracellular parasites, and the role of NO in Leishmania infection has been evaluated. NO has been shown to be the main effector molecule responsible for mediating the intracellular killing of Leishmania parasites (Sarkar et al., 2011). Studies have shown that there is an interaction between NO and MPO (Brunet, 2001; Knaapen et al., 2005). MPO is abundantly present within the cytoplasmatic granules of neutrophils. To the best of our knowledge, no previous report about the serum MPO activity in CVL has been published. In this study, we observed that serum MPO activity was influenced by oxidative stress and NO. Therefore, increased MPO activity may play an important role with NO in the pathogenesis of CVL, and may be related to parasite stimulation of macrophages (Heidarpour et al., 2012).

Cytokines play an important role in the development and the regulation of immune response. IL-1β and TNF-α, important inflammatory cytokines, are usually increased in inflammatory diseases of dogs. According to the previous studies, cellular immune response in CVL is associated with activation of Th1 cells producing IFN-γ, IL-1β and TNF-α. IFN-γ, IL-1β and TNF-α are key factors for the initiation, maintenance and persistence of inflammation in leishmaniasis. And these cytokines activate macrophages to generate toxic molecules and ROS that destroy Leishmania parasites inside the macrophages (Erel et al., 1999; Reis et al., 2010; De Melo and Fortelaza, 2013). ADA enzyme levels are high in many diseases where cellular immunity is stimulated. ADA has been considered as a marker of cell-mediated immunity (Kontaş and Salmanoğlu, 2006) and high levels of ADA activities were well documented in human leishmaniasis (Raziuddin et al., 1994). Therefore, we found significantly increase in the levels of serum ADA, IL-1β and TNF-α as MPO and NO levels in dogs with VL in our study (Table I). And this may be related with the phagocytic activity of macrophages and the increased cellular immunity (Erel et al., 1999).

 

Conclusıon

In the present study, we demonstrated the status of oxidative stress and inflammatory changes in CVL. Decreases in the antioxidant defence and the over production of MDA showed the presence of oxidative stress in CVL. And we consider that free radical causing oxidative stress play an active role in the pathogenesis and immun disorders of CVL. This study is the first report for MPO activity in CVL. Therefore investigation of this enzyme activity in dogs with leishmaniasis may be used for the diagnosis of CVL and inflammatory changes in dogs with VL. Also the increase of activities of MPO and ADA, besides proinflammatory cytokines and NO, can be attributed to respovation of the immune response and parasitemia control. But there is need for further studies on these enzyme activities after different treatment models in CVL.

 

Statament of conflict of interest

Authors have declared no conflict of interest.

 

References

Abdel-Maksoud, M.A., Abdel-Ghaffar, F.A., El-Amir, A., Gamal, B. and Al-Quraishy, S., 2017. Increased oxidative stress and apoptosis in splenic tissue of lupus-prone (NZB/NZW) F1 Mice infected with live but not gamma irradiated Plasmodium chabaudi. Pakistan J. Zool., 49: 351-357.

Abranches, P., Silva-Pereria, M.C., Conceicao-Silva, F.M., Santos-Gomes, G.M. and Janz, J.G., 1991. Canine leishmaniasis: Pathological and ecological factors influencing transmission of infection. J. Parasitol., 77: 557-561. https://doi.org/10.2307/3283159

Alberola, J., Rodriguez, A., Francino, O., Roura, X., Rivas, L. and Andreu, D., 2004. Safety and efficacy of antimicrobial peptides against naturally acquired leishmaniasis. Antimicrob. Agents Chemother., 48: 641-643. https://doi.org/10.1128/AAC.48.2.641-643.2004

Beutler, E., Olga, D. and Barbara, M.R., 1963. Improved method for the determination of blood glutatione. J. Lab. clin. Med., 61: 882-888.

Bildik, A., Kargin, F., Seyrek, K., Pasa, S. and Ozensoy, S., 2004. Oxidative stress and non-enzymatic antioxidative status in dogs with visceral Leishmaniasis. Res. Vet. Sci., 77: 63-66. https://doi.org/10.1016/j.rvsc.2004.01.005

Bogdan, C., Rollinghoff, M. and Diefenbach, A., 2000. Reactive oxygen and reactive nitrogen intermediates in innate and specific immunity. Curr. Opin. Immunol., 12: 64-76. https://doi.org/10.1016/S0952-7915(99)00052-7

Brunet, L.R., 2001. Nitric oxide in parasitic infections. Int. Immunopharmacol., 1: 1457-1467. https://doi.org/10.1016/S1567-5769(01)00090-X

Cortas, N.K. and Wakid, N.W., 1990. Determination of inorganic nitrate in serum and urine by a kinetic cadmium-reduction method. Clin. Chem., 36: 440-443.

de Melo, E.C. and Fortaleza, C.M.C.B., 2013. Challenges in the therapy of visceral leishmaniasis in Brazil: A public health perspective. J. Trop. Med., 1: 1-5. https://doi.org/10.1155/2013/319234

Erel, O., Kocyigit, A., Bulut, V. and Gurel, M.S., 1999. Reactive nitrogen and oxygen intermediates in patients with cutaneous leishmaniasis. Mem. Inst. Oswaldo Cruz, 94: 179-183. https://doi.org/10.1590/S0074-02761999000200009

Garça, M.F., Aslan, M., Tuna, B., Kozan, A. and Cankaya, H., 2013. Serum myeloperoxidase activity, total antioxidant capacity and nitric oxide levels in patients with chronic otitis media. J. Membr. Biol., 246: 519-524. https://doi.org/10.1007/s00232-013-9561-8

Giusti, G., 1974. Adenosine deaminase. In: Methods of enzyme analysis (ed. H.U. Bergmeyer). Academic Press, New York, pp. 1092-1099. https://doi.org/10.1016/B978-0-12-091302-2.50108-0

Heidarpour, M., Soltani, S., Mohri, M. and Khoshnegah, J., 2012. Canine visceral leishmaniasis: Relationships between oxidative stress, liver and kidney variables, trace elements, and clinical status. Parasitol. Res., 111: 1491-1496. https://doi.org/10.1007/s00436-012-2985-8

Knaapen, A.M., Schins, R.P., Borm, P.J. and van Schooten F.J., 2005. Nitrite enhances neutrophil-induced DNA strand breakage in pulmonary epithelial cells by inhibition of myeloperoxidase. Carcinogenesis, 26: 1642-1648. https://doi.org/10.1093/carcin/bgi116

Kontaş, T. and Salmanoğlu, B., 2006. Tumour necrosis factor-alpha, adenosine deaminase and nitric oxide levels in cattle babesiosis before and after treatment. Bull. Vet. Inst. Pulawy, 50: 485-487.

Kruidenier, L., Kuiper, I., van Duijn, W., Mieremet-Ooms, M.A., van Hogezand, R.A., Lamers, C.B. and Verspaget, H.W., 2003. Imbalanced secondary mucosal antioxidant response in inflammatory bowel disease. J. Pathol., 201: 17-27. https://doi.org/10.1002/path.1408

Nielsen, F., Mikkelsen, B.B. and Nielsen, J.B., 1997. Plasma malondialdehyde as biomarker for oxidative stress: reference interval and effects of life-style factors. Clin. Chem., 43: 1209-1214.

Ozcan, M.E., Gulec, M., Ozerol, E., Polat, R. And Akyol, O., 2004. Antioxidant enzyme activities and oxidative stress in affective disorders. Int. Clin. Psychopharmacol., 19: 89-95. https://doi.org/10.1097/00004850-200403000-00006

Ozensoy, S., Ozbel, Y. and Turgay, N., 1998. Serodiagnosis and epidemiology of visceral leishmaniasis in Turkey. Am. J. Trop. Med. Hyg., 59: 363-369. https://doi.org/10.4269/ajtmh.1998.59.363

Pinelli, E., Killick-Kendrick, R.,Wagenaar, J., Bernadina, W., del Real, G. and Ruitenberg, J., 1994. Cellular and humoral immune responses in dogs experimentally and naturally infected with leishmania infantum. Infect. Immun., 62: 229-235.

Raziuddin, S., Abdalla, R., El-Awad, E. and Al-Janadi, M., 1994. Immunoregulatory and proinflammatory cytokine production in visceral and cutaneous leishmaniasis. J. Infect. Dis., 170: 1037-1040. https://doi.org/10.1093/infdis/170.4.1037

Reis, A.B., Giunchetti, R.C., Carrillo, E., Martins-Filho, O.A. and Moreno, J., 2010. Immunity to leishmania and the rational search for vaccines against canine leishmaniasis. Trends Parasitol., 26: 341-349. https://doi.org/10.1016/j.pt.2010.04.005

Sarkar, A., Saha, P., Mandal, G., Mukhopadhyay, D., Roy, S., Singh, S.K., Das, S., Goswami, R.P., Saha, B., Kumar, D., Das, P. and Chatterjee, M., 2011. Monitoring of intracellular nitric oxide in leishmaniasis: İts applicability in patients with visceral leishmaniasis. Cytometry Part A, 79: 35-45. https://doi.org/10.1002/cyto.a.21001

Serarslan, G., Yılmaz, H.R. and Sogut, S., 2005. Serum antioxidant activities, malondialdehyde and nitric oxide levels in human cutaneous leishmaniasis. Clin. exp. Dermatol., 30: 267-271. https://doi.org/10.1111/j.1365-2230.2005.01758.x

Yoshioka, T., Kawada, K. and Shimada, T., 1979. Lipid peroxidation in maternal and cord blood and protective mechanism aganist actived-oxygen toxicity in the blood. Am. J. Obstet. Gynecol., 135: 372-376. https://doi.org/10.1016/0002-9378(79)90708-7

Zelko, I.N., Mariani, T.J. and Folz, R.J., 2002. Superoxide dismutase multigene family: A comparison of the CuZn-SOD (SOD1), Mn-SOD (SOD2), and EC-SOD (SOD3) gene structures, evolution, and expression. Free Rad. Biol. Med., 33: 337-349. https://doi.org/10.1016/S0891-5849(02)00905-X