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Effect of tea tree oil (Melaleuca alternifolia) on the longevity and immune response of rats infected by Trypanosoma evansi
Research in Veterinary Science 96 (2014) 501–506
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Research in Veterinary Science j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / r v s c
Effect of tea tree oil (Melaleuca alternifolia) on the longevity and immune response of rats infected by Trypanosoma evansi Matheus D. Baldissera a,b, Aleksandro S. Da Silva c,*, Camila B. Oliveira a, Rodrigo A. Vaucher b, Roberto C.V. Santos b, Thiago Duarte d, Marta M.M.F. Duarte e, Raqueli T. França f, Sonia T.A. Lopes f, Renata P. Raffin g, Aline A. Boligon h, Margareth L. Athayde h, Lenita M. Stefani c,h, Silvia G. Monteiro a,** a
Department of Microbiology and Parasitology, Universidade Federal de Santa Maria (UFSM), Santa Maria, RS, Brazil Laboratory of Microbiology, Centro Universitário Franciscano, Santa Maria, RS, Brazil c Department of Animal Science, Universidade do Estado de Santa Catarina (UDESC), Chapecó, SC, Brazil d Graduate Program in Pharmacology, UFSM, Santa Maria, Brazil e Lutheran University of Brazil (ULBRA), Santa Maria, RS, Brazil f Department of Small Animal, UFSM, Santa Maria, RS, Brazil g Laboratory of Nanotechnology, Centro Universitário Franciscano, Santa Maria, RS, Brazil h Animal Science Graduate Program, Universidade do Estado de Santa Catarina (UDESC), Lages, SC, Brazil b
A R T I C L E
I N F O
Article history: Received 1 November 2013 Accepted 25 March 2014 Keywords: TTO Immunology system Trypanocidal action Trypanosoma evansi
A B S T R A C T
This study aimed to evaluate the effect of tea tree oil (TTO – Melaleuca alternifolia) on hepatic and renal functions, and the immune response of rats infected by Trypanosoma evansi. A pilot study has shown that rats treated with TTO orally (1 ml kg−1) had increased survival rate without curative effect. In order to verify if increased longevity was related to a better immune response against T. evansi when using tea tree oil, a second experiment was conducted. Thus, twenty-four rats were divided into four groups. The groups A and B were composed of uninfected animals, and the groups C and D had rats experimentally infected by T. evansi. Animals from the groups B and D were treated orally with TTO (1 ml kg−1) for three days. Blood samples were collected to verify humoral response analysis for immunoglobulins (IgA, IgM, IgE, and IgG) and cytokines (TNF-α, INF-γ, IL-1, IL-6, IL-4, and IL-10) at days 0, 3, 5 and 15 post-infection (PI). TTO treatment caused changes in the immunoglobulins and cytokines profile, as well as the course of T. evansi infection in rats. It was found that the TTO was not toxic, i.e., hepatic and renal functions were not affected. Therefore, it is possible to conclude that TTO influences the levels of inflammatory mediators and has trypanocidal effect, increasing life expectancy of rats infected by T. evansi. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Trypanosoma evansi is a flagellate parasite and the etiological agent of the disease known as ‘Surra’ or ‘Mal das cadeiras’ in horses. This protozoan has a wide geographical distribution, and it has been found parasitizing various species of domestic and wild animals (Silva et al., 2002), and rarely humans (Joshi et al., 2005). The parasite is transmitted primarily by blood-sucking insects (Tabanus sp., Chrysops sp., Hematopota sp., and Stomoxys calcitrans), but also by vampire bats (Hoare, 1972). Therapy for tripanosomosis is based on chemotherapy with suramine, diminazene aceturate, quinapyramine,
* Corresponding author. Tel./fax: +55 55 3220-8958. E-mail address: [email protected] (A.S. Da Silva). ** Corresponding author. Tel./fax: +55 55 3220-8958 E-mail address: [email protected] (S.G. Monteiro). http://dx.doi.org/10.1016/j.rvsc.2014.03.013 0034-5288/© 2014 Elsevier Ltd. All rights reserved.
melarsoprol, homidium chloride and isometamidium chloride. However, some cases of parasite resistance to these drugs have been reported (Brum et al., 1998; Maudlin et al., 2004). Trypanosomosis due to T. evansi causes many clinical signs and pathological findings, such as an intense inflammatory response, but the pathogenesis of this disease is not fully elucidated yet. Infection by T. evansi causes an increase in the levels of immunoglobulins (Gressler et al., 2010) and also pro-inflammatory cytokines in mice experimentally infected (Paim et al., 2011). In trypanosomosis, the lymphocytes produce interferon-gamma (IFN-γ) in response to parasite antigens. IFN-γ activates macrophages, increasing their ability to kill phagocyted microorganisms (Gao and Pereira, 2002). Tea tree oil (TTO) is derived from an Australian plant, known scientifically by Melaleuca alternifolia. TTO has been used widely as antimicrobial and anti-inflammatory agent. The broad spectrum of these activities is mainly attributed to terpinen-4-ol and 1.8cineole, the major components of its essential oil. These sub-
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stances allow TTO to be used as therapeutic agent (Furneri et al., 2006). TTO treatment causes in vitro a 50% reduction of Leishmania major and Trypanosoma brucei growth (Mikus et al., 2000). Due to the inefficiency of some trypanocidal drugs, there is a need to find alternative treatments, thus this study was conducted to evaluate, for the first time, the effect of TTO on survival rate, immune response, and the hepatic and renal functions on T. evansi infected rats.
Silva et al., 2006). The TTO dose used in this study was based on literature findings (Russell, 1999), chosen to not be toxic to the rodents.
2. Materials and methods
2.4.1. Animal model Twenty-four 90-day-old male rats weighing 280 grams (± 11) were kept in cages with six rats each. The animals were kept in similar conditions to the pilot study, as described in section 2.3. All animals were submitted to cage adaptation for 8 days.
2.1. T. evansi isolate The isolate of T. evansi used in this experiment was from naturally infected dogs (Colpo et al., 2005). Two rats (R1 and R2) were inoculated intraperitoneally with trypomastigote-contaminated blood that was kept in liquid nitrogen. This procedure was performed to obtain a large amount of viable parasites to infect all experimental groups. 2.2. Essential oil preparation M. alternifolia oil was purchased from Importadora Química Delaware Ltda, Brazil. To adjust the dose to each animal, it was necessary to dilute the TTO in DMSO (1/10 v). Oil composition and yield were obtained by gas chromatography (GC) using an Agilent Technologies 6890N GC-FID system, equipped with DB-5 capillary column (30 m × 0.25 mm × 2.5 μm film thickness) connected to a flame ionization detector (FID). The injector and detector temperatures were set at 250 °C. The carrier gas was helium, at a flow rate of 1.3 ml/min. The thermal programmer was 100–280 °C at a rate of 10 °C/min. Component relative concentrations were calculated based on GC peak areas. The injection volume of the TTO was 1 μL (Boligon et al., 2013; Homer et al., 2000) and oil analysis was performed in duplicate. GC-Mass Spectroscopy (GC-MS) analyses were performed on an Agilent Technologies AutoSystem XL GC-MS system operating in the EI mode at 70 eV, equipped with a split/splitless injector (250 °C). The transfer line temperature was 280 °C. Helium was used as carrier gas (1.5 ml/ min) and the capillary columns used were an HP 5MS (30 m × 0.25 mm × 2.5 μ m film thickness) and an HP Innowax (30 m × 0.32 mm, i.e., film thickness 0.50 mm). The temperature programed was the same as that used for the GC analyses. The injected volume was 1 μL of the essential oil. Identification of the constituents of TTO was performed on the basis of retention index (RI), determined with reference of the homologous series of n-alkanes (C7-C30) under identical experimental conditions, comparing with the mass spectra library search (NIST and Wiley), and with the mass spectra from the literature date (Adams, 1995). The relative amounts of individual components were calculated based on the CG peak area (FID response). 2.3. Experiment I (a pilot study) Eight adult male rats were used in a pilot study designed to investigate the effect of TTO on T. evansi experimentally infected rats (used 0.2 ml of blood containing 1.3 × 106 trypomastigotes – Rat1). They were housed on a light/dark cycle of 12 h in an experimental room under controlled temperature and humidity (25 °C; 70%, respectively). They were fed with commercial feed and received water ad libitum. Five rats received orally 1.0 ml kg−1 of TTO for three days on 24hour intervals (the group T). The first dose was performed 2 hours after inoculation. Three rats were untreated, and used as positive control (the group P). The parasitemia evolution (quantitative) was monitored daily by microscopy examination of blood smears (Da
2.4. Experiment II (a clinical study) The experiment II was designed after a pilot study in order to determine if the humoral response could increase longevity of rats infected by T. evansi after TTO treament.
2.4.2. Experimental design and parasitemia estimation The rats were divided into four groups (A to D). The uninfected animals from the groups A and B, composed of six healthy rats each, were intraperitoneally inoculated with 0.2 ml of blood from a healthy rat. Animals in the groups C and D were inoculated intraperitoneally with 0.2 ml of fresh blood obtained from a rat (R2), containing 2.3 × 106 trypanosomes (on day 1). The groups A and C represented the negative and positive control of the experiment and they were not treated with tea tree oil, respectively. Rats from the groups B and D received orally 1.0 ml kg−1 of TTO during three days, with dose intervals of 24 hours. The TTO had the first dose administered 2 hours after inoculation of the parasite. The number of trypomastigotes (parasitemia) and the treatment effect were monitored daily by microscopy examination of blood smears as described previously (Da Silva et al., 2006). 2.4.3. Sample collection On days 0, 3 and 6 post-infection (PI), survivor rats were anesthetized with isoflurane inside an anesthetic chamber to perform collection of blood samples (1.0 ml by intracardiac puncture). Approximately 10 min after this procedure all animals showed signs of recovery. The blood was used to obtain serum to measure the immune response. On the 15th day PI, all survivor rats were anesthetized following the same procedures prior cited, and humanely euthanized by decapitation. Blood samples (5 ml) were used to obtain serum to the measure liver and kidney functions, and immune response. 2.4.4. Influence of TTO treatment on the immune system The concentration of serum immunoglobulin G, M, A and E was determined using immunonephelometry on the Behring Nephelometer BN II (Dade Behring – USA) with reagents from Dade Behring. Samples were analyzed according to Dati et al. (1996). Briefly, all samples were diluted with specific diluents and measured after 10 min. Polystyrene particles were coated with a specific monoclonal antibody for each serum protein, forming an agglutinate that disperses the light irradiated in the presence of the protein. The intensity of scattered light depends on the amount of protein concentration in the sample, and the results are compared with known standard curves (Dati et al., 1996). Cytokines quantification (TNF-α, INF-γ, IL-1, IL-4, IL-6 and IL10) were assessed by ELISA assay using commercial Quantikine Immunoassay kits (R&D Systems, Minneapolis, MN), according to the manufacturer’s instructions. The concentration of the cytokines was determined by the intensity of the color measured spectrophotometrically using a microplate reader. 2.4.5. Influence of TTO treatment on liver and kidney functions The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea and creatinine were evaluated in a semiautomatic analyzer (TP Analyzer Plus®, Thermoplate, China) using
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Table 1 Qualitative and quantitative analyses of tea tree oil (TTO) used this study. Peak
Compounds
RIa
RIb
Amount (%)
ISO 4730 range (%)
Mol. Formula
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
α-pinene
937 976 1016 1025 1032 1037 1062 1178 1089 1190 1440 1521 1539 1583 1591
939 976 1018 1026 1031 1038 1062 1177 1088 1189 1439 1525 1538 1583 1590
3.51 0.46 9.85 2.27 1.39 6.03 20.15 41.98 4.17 2.43 1.04 0.80 0.63 0.97 0.18 95.86
1–6 Tr-3.5 5–13 0.5–8.0 0.5–1.5 Tr-15 10–28 30–48 1.5–5 1.5–8 Tr-3 Tr-3 Tr-3 Tr-1 Tr-1
C10H16 C10H16 C10H16 C10H14 C10H16 C10H18O C10H16 C10H18O C10H16 C10H18O C15H24 C15H24 C15H24 C15H26O C15H26O
sabinene
α-terpinene p-cymene limonene 1.8-cineole γ-terpinene terpinen-4-ol terpinolene α-terpineol aromadendrene ledene δ-cadinene globulol viridiflorol Total identified (%)
Relative proportions of the essential oil components were expressed as percentages. Tr = Trace amounts. a Retention indices experimental (based on homologous series of n-alkane C7-C30). b Retention indices from literature (Adams, 1995). International Organization for Standardization (ISO), standard number 4730.
commercial kits (Labtest® Diagnóstica S.A., Lagoa Santa, MG, Brazil). All tests were carried out in duplicate. 2.5. Statistic analysis Immunoglobulin, cytokine, and serum biochemistry data were submitted to analysis of variance followed by Duncan’s test (P < 0.05). 3. Results 3.1. Components present in the essential oil The results of the components present in the TTO are presented in Table 1. The top six compounds identified in TTO were dihydro carveol, myrtenol, cis-calamenene, t-pinocarveol, β-myrcene and α-terpineol. 3.2. Pilot study Untreated rats (the group P) showed increased parasitemia and died 5–7 days after inoculation. The animals treated with TTO (the group T) had prolonged survival rate; however, all died 13–21 days PI (Fig. 1). The experiment II was designed to determine whether the immune response could be related to the increased longevity in animals treated with TTO.
3.3. Experiment II 3.3.1. Disease course Data from the disease course are shown in Fig. 2. A significant increase in the pre-patent period in animal from the group D compared to the group C (similar pilot study) was observed. Trypomastigotes in the blood of infected animals treated with TTO were detected only on day 13 PI (group D) when compared with the group C, which showed trypanosomes on already on day 3 PI. At day 15 PI, there were no live animals in the positive control group (infected and untreated), unlike treated groups (Fig. 2). Based on the results from the pilot study and the increased parasitemia levels observed on rats from the group D (Fig. 2), the study was terminated on day 15 PI and blood samples were collected for biochemical and cytokine analysis. 3.3.2. Effect on the immune response Effect on the immune response is shown in Table 2. To investigate TTO immunomodulation effects, antibody and cytokine levels were quantified in serum 0, 3, 5 and 15 days PI using immunonephelometry and ELISA, respectively. On day 0, the levels of cytokines and immunoglobulins did not differ statistically. The TTO in healthy animals (the group B) did not alter IgG, IgE and IgA levels, but IgM was reduced when animals were TTO treated. Infected animals (the group C) had an increase in the levels of im-
Trypomastigotes/field
Experiment I - Pilot study
P1 P2 P3
140 120 100 80 60 40 20 0
T1 T2 T3 T4 T5
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 19 21
Days post-infection Fig. 1. Parasitemia of Trypanosoma evansi-infected rats in the groups P (not-treated) and T (treated with tea tree oil). All animals died after high parasitemia (more 100 trypanosomes/field).
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Fig. 2. Parasitemia of Trypanosoma evansi-infected rats at day 15 post-inoculation (PI) in the groups A to D. The treatment started at day 1 PI (three doses at 24-h intervals).
munoglobulins compared to four healthy rats. Infected rats treated with TTO (Group D) had increased levels of IgG, unlike the levels of IgM, IgA and IgE which were reduced compared to animals from the group C.
TTO administration in uninfected rats (the group B) was able to significantly reduce the levels of TNF, INF, IL-1, IL-4 and IL-6, and increase the levels of IL-10 when compared to animals from the group A. Infected rats (the group C) showed increased pro-
Table 2 Mean and standard deviation of the immunoglobulins [IgG, IgM, IgA and IgE] and cytokines [TNF-α, INF-γ, IL-1, IL-4, IL-6 and IL-10] results after treatment with tea tree oil (TTO – Melaleuca alternifolia) in rats experimentally infected by T. evansi. Parameters
IgG (mg/dL)
IgM (mg/dL)
IgE (Ul/ml)
IgA (mg/dL)
TNF-α (pg/ml)
INF-γ (µg/ml)
IL-1 (pg/ml)
IL-4 (pg/ml)
IL-6 (pg/ml)
IL-10 (pg/ml)
Days
0 3 5 15 0 3 5 15 0 3 5 15 0 3 5 15 0 3 5 15 0 3 5 15 0 3 5 15 0 3 5 15 0 3 5 15 0 3 5 15
Means ± standard deviations of groups* Group A
Group B
Group C#
Group D
132.5 ± 133.1 ± 14.7ab 131.6 ± 21.8a 130.2 ± 4.1a 51.3 ± 9.4a 44.5 ± 8.7b 43.5 ± 7.4b 43.4 ± 5.7a 74.1 ± 9.4a 70.5 ± 7.7a 68.8 ± 6.2a 68.2 ± 6.6a 71.0 ± 15.3a 67.8 ± 8.4b 65.1 ± 4.4b 64.0 ± 7.5a 102.8 ± 10.4a 104.0 ± 12.3c 102.6 ± 9.7c 99.8 ± 15.4b 118.0 ± 21.1a 114.6 ± 11.7b 114.1 ± 7.4c 112.8 ± 15.2a 40.5 ± 7.6a 39.1 ± 4.7c 39.5 ± 6.0c 39.2 ± 2.4b 69.6 ± 11.2a 65.6 ± 2.2c 59.0 ± 4.4c 56.8 ± 7.2b 82.3 ± 18.0a 78.6 ± 15.0b 79.5 ± 8.8c 79.0 ± 10.7b 64.8 ± 11.8a 71.5 ± 10.2b 74.6 ± 8.0b 77.4 ± 9.7b
131.6 ± 125.6 ± 16.9a 131.2 ± 20.4a 137.1 ± 7.7a 47.3 ± 12.4a 23.2 ± 6.3c 20.8 ± 3.9c 20.0 ± 2.8b 80.8 ± 11.1a 75.9 ± 5.4a 72.6 ± 8.2a 66.8 ± 8.3a 71.4 ± 10.9a 71.6 ± 8.7b 77.2 ± 4.4b 72.8 ± 13.7a 108.2 ± 12.1a 77.2 ± 8.8d 73.4 ± 8.4d 63.8 ± 7.1c 96.8 ± 17.2a 91.2 ± 7.4c 86.8 ± 10.1d 80.6 ± 12.1a 42.6 ± 9.4a 20.0 ± 7.1d 16.2 ± 3.2d 14.8 ± 4.1c 67.8 ± 9.2a 42.8 ± 6.8d 35.8 ± 4.4d 33.2 ± 3.0c 89.9 ± 10.4a 51.8 ± 5.9c 45.6 ± 9.1d 40.0 ± 2.2c 74.0 ± 14.0a 102.8 ± 13.9a 120.4 ± 21.4a 137.4 ± 25.2a
134.8 ± 10.1a 139.0 ± 11.1b 158.2 ± 23.6b – 56.3 ± 7.6a 61.8 ± 13.2a 78.0 ± 6.6a – 83.7 ± 12.3a 95.2 ± 9.1b 149.0 ± 14.6b – 74.6 ± 12.8a 84.4 ± 4.2a 98.0 ± 4.6a – 115.2 ± 20.4a 252.8 ± 28.4a 265.0 ± 21.2a – 107.3 ± 23.6a 294.4 ± 31.7a 314.0 ± 40.8a – 48.3 ± 8.9a 119.2 ± 23.2a 158.1 ± 32.6a – 79.8 ± 14.3a 127.6 ± 12.4a 177.3 ± 4.6a – 99.3 ± 21.6a 210.2 ± 32.1a 228.2 ± 18.2a – 58.0 ± 13.6a 53.8 ± 9.2c 44.0 ± 3.6c –
140.2 ± 15.8a 157.6 ± 23.4c 171.2 ± 17.2c 184.3 ± 12.9b 57.8 ± 14.9a 52.6 ± 10.3ab 51.2 ± 12.2b 43.7 ± 3.9a 77.8 ± 17.2a 80.8 ± 5.9a 74.6 ± 9.1a 64.3 ± 6.8a 72.0 ± 17.3a 74.4 ± 5.9b 66.2 ± 7.2b 64.8 ± 11.7a 109.4 ± 18.7a 215.2 ± 23.2b 180.6 ± 19.1b 170.3 ± 17.9a 105.8 ± 14.7a 273.8 ± 24.9a 217.3 ± 20.2b 217.0 ± 28.9a 47.2 ± 10.2a 93.0 ± 2.2b 95.2 ± 13.0b 93.1 ± 7.4a 72.6 ± 10.8a 106.6 ± 8.4b 108.2 ± 7.2b 102.3 ± 19.2a 96.1 ± 20.0a 191.0 ± 27.2a 178.0 ± 24.4b 173.2 ± 32.2a 72.0 ± 17.0a 80.6 ± 12.2b 78.2 ± 9.2b 70.3 ± 8.9b
9.2a
16.4a
* Means followed by the same letter in the same row do not differ significantly by the Duncan test. On day 15, no measurements were performed on animals from the group C, due to trypanosomiasis animal death.
#
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Fig. 3. Alanine aminotranferase, ALT (A), aspartate aminotranferase, AST (B), urea (C) and creatinine levels (D) in uninfected mice (group A); uninfected and treated with tea tree oil (group B); and infected with T. evansi and treated with tea tree oil at 1.0 mg kg−1 (group D) in day 15 post-infection. The animals of group C (infected and untreated) died 6 days post-infection. The same letters in columns indicate that there is no significant difference between groups.
inflammatory cytokines (TNF, INF, IL-1, IL-4 and IL-6) and reduction of anti-inflammatory (IL-10) compared to healthy animals (group A and B). The pro-inflammatory cytokines in infected/treated animals (the group D) increased when compared to healthy animals (the groups A and B), but was lower than the rats from the group C. The IL-10 levels did not differ between groups A and D (Table 2). 3.3.3. Serum biochemistry Serum biochemistry data are shown in Fig. 3. ALT activity increased significantly in all groups infected by T. evansi and treated orally with TTO at 1.0 ml kg−1 when compared to the other groups. AST, urea, and creatinine results did not differ between all groups. 4. Discussion TTO treatment was able to control parasitemia and thus to prolong the longevity of rats in this study. Compounds present in the TTO essential oil, such as terpin-4-ol, were responsible for the in vitro death of T. brucei trypomastigotes (Mikus et al., 2000), which may also have occurred in this study. Associated with the effects of the TTO oil, the inflammatory response (immunoglobulin and cytokine increased) mounted by rats to fight T. evansi infection may also have prolonged their lives. IgG and IgM antibodies have been described in different studies at high levels, and IgM has been directly responsible for fighting trypomastigotes infection (Costa et al., 2013; Gressler et al., 2010). During experimental infection by T. evansi, rats showed an increase on IFN, TNF, IL-1, and IL-6 levels in serum (Paim et al., 2011), similarly to what occurred in this study. Therefore, our hypothesis is that the trypanocidal effect of TTO combined with the inflammatory response was responsible for the increase in longevity by controlling the parasitemia (the group D),
despite the fact that TTO oil has anti-inflammatory effect. However, it is noteworthy that the immune response alone (antibodies and cytokines) is not sufficient to control the disease, since the animals died within a few days PI (the group C). TTO anti-inflammatory effect on cellular responses is well documented (Hart et al., 2000). However, the effect of this oil on the humoral response is scarce in the literature. In our study, we found that the treatment of healthy rats did not affect the levels of IgG, IgA, and IgE, unlike what happened with IgM levels that were reduced in the serum (the group B). An explanation for IgM reduction is unknown at the moment, but our hypothesis is that some of the components present in TTO oil may be responsible for this fact, but further studies are needed. As already mentioned, in this study we observed a progressive increase in the serum levels of IFN-γ, TNF-α, IL-1, IL-4, and IL-6 on animals infected by T. evansi, and according to the literature, these findings are correlated to the inflammatory response against infection (Gao and Pereira, 2002; Sileghem et al., 1989; Silva et al., 1995). The inflammatory response is considered the first host response against protozoa infection (Titus et al., 1991), a fact observed also in this study. Healthy animals (the group B) and infected (the group D) treated with TTO showed reduced levels of serum cytokines, which could be considered a positive finding for rats. That is because excessive amounts of these pro-inflammatory cytokines can be harmful to the host, leading to tissue injury, therefore exacerbating clinical signs of the disease (Abbas et al., 2012). IL-10 serum levels were low in infected compared to uninfected animals. The animals of groups receiving TTO showed increased serum levels of IL-10, a cytokine with important anti-inflammatory response. This agrees with the literature that reports IL-10 increase after TTO treatment of mononuclear cells from the periph-
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eral blood (Caldefie-Chézet et al., 2006). There are numerous studies in vivo and in vitro supporting the evidence of TTO anti-inflammatory effect. The components of TTO may inhibit the lipopolysaccharideinduced production of inflammatory mediators such as the tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), and prostaglandin E2 by human peripheral blood monocytes (Hart et al., 2000). TTO also decreases the production of reactive oxygen species by stimulated neutrophils and monocytes (Caldefie-Chézet et al., 2004). TTO also modulates the vasodilation and plasma extravasation associated with histamine-induced inflammation in humans (Khalil et al., 2004). The dose of TTO used in this study was able to modulate the inflammatory response against T. evansi infection (the group D), which is maintained at moderate levels when compared to the positive control, reducing the typical cellular and tissue lesions often occurred during inflammation. The TTO therapeutic protocol caused no increase in serum levels of AST, ALT, creatinine, and urea in healthy animals. These results show that treatment with TTO does not cause liver and kidney damage in healthy animals treated with a dose of 1.0 ml kg−1. However, the rats from the group D (infected/treated) showed increased ALT levels, which is a typical finding in animals infected by T. evansi (Monzón and Villavicencio, 1990; Barr, 1991; Arora and Pathak, 1995; Aquino et al., 2002). There are a significant progress in the antimicrobial and anti-inflammatory studies of TTO, but only a few studies on the safety and toxicity of this oil. TTO can be toxic if ingested, as evidenced by studies with animals and from cases of human poisoning. The 50% lethal dose for TTO in a rat model is 1.9 to 2.6 ml kg−1 and rats dosed with ≤1.5 g kg−1 TTO appeared lethargic and ataxic (Russell, 1999). Based on these results, we conclude that treatment with TTO is able to prolong the life of rats infected by T. evansi, maintaining low parasitemia. The TTO treatment caused an anti-inflammatory effect on rats, which may have been beneficial for the animals. The therapeutic protocol with TTO has neither curative effect nor renal and hepatic toxicity on rats. These promising results show that the TTO has compounds with trypanocidal activity and it could be used in future trypanosomosis chemotherapy. Ethical approval The procedure was approved by the Animal Welfare Committee of Universidade do Estado de Santa Catarina, number 1.51.13. References Abbas, A.K., Lichtman, A.H., Pillai, S., 2012. Cellular and Molecular Immunology, seventh ed. WB Saunders Elsevier, Philadelphia, p. 527. Adams, R.P., 1995. Identification of Essential Oil Components by Gas Chromatography/ Mass Spectroscopy. Allured Publishing Corporation, Illinois, USA, 456 pp. Aquino, L.P.C.T., Machado, R.Z., Alessi, A.C., Santana, A.E., Castro, M.B., Marques, L.C., et al., 2002. Aspectos hematológicos, bioquímicos e anatomopatológicos da infecção de Trypanosoma evansi em cães. Arquivo Brasileiro de Medicina Veterinária e Zootecnia 54, 8–18. Arora, J.K., Pathak, M.L., 1995. Clinico-hematological and biochemical changes associated with Trypanosoma evansi infection in dogs. Indian Journal of Animal Health 34, 33–38. Barr, S.C., 1991. American trypanosomiasis in dogs. Compendium on Continuing Education for the Practicing Veterinarian 13, 745–752. Boligon, A.A., Kubiça, T.K., Mario, D.B., Brum, T.F., Piana, M., Weiblen, R., et al., 2013. Antimicrobial and antiviral activity-guided fractionation from Scutia buxifolia Reissek extracts. Acta Physiologiae Plantarum 35, 2229–2239.
Brum, R., Hecker, H., Lun, Z., 1998. Trypanosoma evansi and T. equiperdum: distribuition, biology, treatment and phylogenetic relationship (a review). Veterinary Parasitology 72, 95–107. Caldefie-Chézet, F., Guerry, M., Chalchat, J.C., Fusilier, C., Vasson, M.P., Guillot, J., 2004. Anti-inflammatory effects of Melaleuca alternifolia essential oil on human polymorphonuclear neutrophils and monocytes. Free Radical Research 38, 805–811. Caldefie-Chézet, F., Fusillier, C., Jarde, T., Laroye, H., Damez, M., Vasson, M.P., et al., 2006. Potential anti-inflammatory effects of Melaleuca alternifolia essential oil on human peripheral blood leukocytes. Phytotherapy Research 20, 364– 370. Colpo, C.B., Monteiro, S.G., Stainki, D.R., 2005. Natural infection by Trypanosoma evansi in dogs. Ciência Rural 35, 717–719. Costa, M.M., Dos Anjos Lopes, S.T., França, R.T., da Silva, A.S., Paim, F.C., Palma, H.E., et al., 2013. Role of acute phase proteins in the immune response of rabbits infected with Trypanosoma evansi. Research in Veterinary Science 95, 182– 188. Da Silva, A.S., Doyle, R.L., Monteiro, S.G., 2006. Método de contenção e confecção de esfregaço sanguíneo para pesquisa de hemoparasitas em ratos e camundongos. Revista de Zootecnia, Veterinária e Agronomia 13, 83–87. Dati, F., Schumann, G., Thomas, L., 1996. Consensus of a group of professional societies and diagnostic companies on guidelines for interim reference ranges from 14 patients in serum based on the standardization against the IFCC/BCR/CAP Reference Material (CRM 470). European Journal of Clinical Chemistry and Clinical Biochemistry 34, 517–520. Furneri, P.M., Paolino, D., Saija, A., Marino, A., Bisignano, G., 2006. In vitro antimycoplasmal activity of Melaleuca alternifolia essential oil. Journal of Antimicrobial Chemotherapy 58, 706–707. Gao, W., Pereira, M.A., 2002. Interleukin – 6 is required for parasite specific for parasite response and host resistance to Trypanosoma cruzi. International of Journal Parasitology 32, 167–170. Gressler, L.T., Da Silva, A.S., Otto, M.A., Azevedo, M.I., Zanette, R.A., Monteiro, S.G., et al., 2010. Níveis de imunoglobulinas em ratos infectados por Trypanosoma evansi. Ciência Animal Brasileira 11, 201–204. Hart, P.H., Brand, C., Carson, C.F., Riley, T.V., Prager, R.H., Finlay-Jones, J.J., 2000. Terpin-4-ol, the main component of the essential oil of Melaleuca alternifolia (tea tree oil), suppresses inflammatory mediator production by activated human monocytes. Inflammation Research 49, 619–626. Hoare, C.A., 1972. The Trypanosomes of Mammals a Zoological Monograph. Blackwell, Oxford, p. 546. Homer, L.E., Leach, D.N., Lea, D., Lee, L.S., Henry, R.J., Baverstock, P.R., 2000. Natural variation in the essential oil content of Melaleuca alternifolia Cheel (Myrtaceae). Biochemical Systematics and Ecology 28, 367–382. Joshi, P.P., Shegokar, V.R., Powar, R.M., Herder, S., Katti, R., Salkar, H.R., et al., 2005. Human trypanosomiasis caused by Trypanosoma evansi in India: the first case report. American Journal of Tropical Medicine and Hygiene 73, 491–495. Khalil, Z., Pearce, A.L., Satkunanathan, N., Storer, E., Finlay-Jones, J.J., Hart, P., 2004. Regulation of wheal and flare by tea tree oil: complementary human and rodent studies. Journal of Investigate Dermatology Symposium 123, 683–690. Maudlin, I., Holmes, P., Miles, M.A., 2004. The Trypanosomiases. CABI Publishing, Wallingford, p. 1150. Mikus, J., Harkenthal, M., Steverding, D., Reichling, J., 2000. In vitro effect of essential oils and isolated mono – and sesquiterpenes on Leishmania major and Trypanosna brucei. Planta Medica 66, 366–368. Monzon, C.M., Villavicencio V.I., 1990. Serum proteins in guinea-pigs and horse infected with Trypanosoma evansi (Steel, 1885). Veterinary Parasitology 36, 295–301. Paim, F.C., Duarte, M.M., Costa, M.M., Da Silva, A.S., Wolkmer, P., Silva, C.B., et al., 2011. Cytokines in rats experimentally infected with Trypanosoma evansi. Experimental Parasitology 128, 365–370. Russell, M., 1999. Toxicology of tea tree oil. In: Southwell, I., Lowe, R. (Eds.), Tea Tree: The Genus Melaleuca. Harwood Academic Publishers, Amsterdam, The Netherlands, 9, pp. 191–201. Sileghem, M.R., Darji, A., Hamers, R., De Baetselier, P., 1989. Modulation of IL−1 production and IL-1 release during experimental trypanosomose infections. Immunology 68, 137–139. Silva, J.S., Vespa, G.N.R., Cardoso, M.A.G., Aliberti, J.C.S., Cunha, F.Q., 1995. Tumor necrosis mediate resistance to Trypanosoma cruzi infection in mice by inducing nitric oxide production in infected gamma interferon – activated macrophages. Infection and Immunology 63, 4862–4867. Silva, R.A.M.S., Seidl, A., Ramirez, L., Dávila, A.M.R., 2002. Trypanosoma evansi e Trypanosoma vivax: Biologia, Diagnóstico e Controle. Embrapa Pantanal, Corumbá, p. 162. Titus, R.G., Sherry, B., Cerami, A., 1991. The involvement of TNF – α and IL-6 in the response to protozoan parasites. Trends in Immunology 12, 13–16.
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Research in Veterinary Science j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / r v s c
Effect of tea tree oil (Melaleuca alternifolia) on the longevity and immune response of rats infected by Trypanosoma evansi Matheus D. Baldissera a,b, Aleksandro S. Da Silva c,*, Camila B. Oliveira a, Rodrigo A. Vaucher b, Roberto C.V. Santos b, Thiago Duarte d, Marta M.M.F. Duarte e, Raqueli T. França f, Sonia T.A. Lopes f, Renata P. Raffin g, Aline A. Boligon h, Margareth L. Athayde h, Lenita M. Stefani c,h, Silvia G. Monteiro a,** a
Department of Microbiology and Parasitology, Universidade Federal de Santa Maria (UFSM), Santa Maria, RS, Brazil Laboratory of Microbiology, Centro Universitário Franciscano, Santa Maria, RS, Brazil c Department of Animal Science, Universidade do Estado de Santa Catarina (UDESC), Chapecó, SC, Brazil d Graduate Program in Pharmacology, UFSM, Santa Maria, Brazil e Lutheran University of Brazil (ULBRA), Santa Maria, RS, Brazil f Department of Small Animal, UFSM, Santa Maria, RS, Brazil g Laboratory of Nanotechnology, Centro Universitário Franciscano, Santa Maria, RS, Brazil h Animal Science Graduate Program, Universidade do Estado de Santa Catarina (UDESC), Lages, SC, Brazil b
A R T I C L E
I N F O
Article history: Received 1 November 2013 Accepted 25 March 2014 Keywords: TTO Immunology system Trypanocidal action Trypanosoma evansi
A B S T R A C T
This study aimed to evaluate the effect of tea tree oil (TTO – Melaleuca alternifolia) on hepatic and renal functions, and the immune response of rats infected by Trypanosoma evansi. A pilot study has shown that rats treated with TTO orally (1 ml kg−1) had increased survival rate without curative effect. In order to verify if increased longevity was related to a better immune response against T. evansi when using tea tree oil, a second experiment was conducted. Thus, twenty-four rats were divided into four groups. The groups A and B were composed of uninfected animals, and the groups C and D had rats experimentally infected by T. evansi. Animals from the groups B and D were treated orally with TTO (1 ml kg−1) for three days. Blood samples were collected to verify humoral response analysis for immunoglobulins (IgA, IgM, IgE, and IgG) and cytokines (TNF-α, INF-γ, IL-1, IL-6, IL-4, and IL-10) at days 0, 3, 5 and 15 post-infection (PI). TTO treatment caused changes in the immunoglobulins and cytokines profile, as well as the course of T. evansi infection in rats. It was found that the TTO was not toxic, i.e., hepatic and renal functions were not affected. Therefore, it is possible to conclude that TTO influences the levels of inflammatory mediators and has trypanocidal effect, increasing life expectancy of rats infected by T. evansi. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Trypanosoma evansi is a flagellate parasite and the etiological agent of the disease known as ‘Surra’ or ‘Mal das cadeiras’ in horses. This protozoan has a wide geographical distribution, and it has been found parasitizing various species of domestic and wild animals (Silva et al., 2002), and rarely humans (Joshi et al., 2005). The parasite is transmitted primarily by blood-sucking insects (Tabanus sp., Chrysops sp., Hematopota sp., and Stomoxys calcitrans), but also by vampire bats (Hoare, 1972). Therapy for tripanosomosis is based on chemotherapy with suramine, diminazene aceturate, quinapyramine,
* Corresponding author. Tel./fax: +55 55 3220-8958. E-mail address: [email protected] (A.S. Da Silva). ** Corresponding author. Tel./fax: +55 55 3220-8958 E-mail address: [email protected] (S.G. Monteiro). http://dx.doi.org/10.1016/j.rvsc.2014.03.013 0034-5288/© 2014 Elsevier Ltd. All rights reserved.
melarsoprol, homidium chloride and isometamidium chloride. However, some cases of parasite resistance to these drugs have been reported (Brum et al., 1998; Maudlin et al., 2004). Trypanosomosis due to T. evansi causes many clinical signs and pathological findings, such as an intense inflammatory response, but the pathogenesis of this disease is not fully elucidated yet. Infection by T. evansi causes an increase in the levels of immunoglobulins (Gressler et al., 2010) and also pro-inflammatory cytokines in mice experimentally infected (Paim et al., 2011). In trypanosomosis, the lymphocytes produce interferon-gamma (IFN-γ) in response to parasite antigens. IFN-γ activates macrophages, increasing their ability to kill phagocyted microorganisms (Gao and Pereira, 2002). Tea tree oil (TTO) is derived from an Australian plant, known scientifically by Melaleuca alternifolia. TTO has been used widely as antimicrobial and anti-inflammatory agent. The broad spectrum of these activities is mainly attributed to terpinen-4-ol and 1.8cineole, the major components of its essential oil. These sub-
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stances allow TTO to be used as therapeutic agent (Furneri et al., 2006). TTO treatment causes in vitro a 50% reduction of Leishmania major and Trypanosoma brucei growth (Mikus et al., 2000). Due to the inefficiency of some trypanocidal drugs, there is a need to find alternative treatments, thus this study was conducted to evaluate, for the first time, the effect of TTO on survival rate, immune response, and the hepatic and renal functions on T. evansi infected rats.
Silva et al., 2006). The TTO dose used in this study was based on literature findings (Russell, 1999), chosen to not be toxic to the rodents.
2. Materials and methods
2.4.1. Animal model Twenty-four 90-day-old male rats weighing 280 grams (± 11) were kept in cages with six rats each. The animals were kept in similar conditions to the pilot study, as described in section 2.3. All animals were submitted to cage adaptation for 8 days.
2.1. T. evansi isolate The isolate of T. evansi used in this experiment was from naturally infected dogs (Colpo et al., 2005). Two rats (R1 and R2) were inoculated intraperitoneally with trypomastigote-contaminated blood that was kept in liquid nitrogen. This procedure was performed to obtain a large amount of viable parasites to infect all experimental groups. 2.2. Essential oil preparation M. alternifolia oil was purchased from Importadora Química Delaware Ltda, Brazil. To adjust the dose to each animal, it was necessary to dilute the TTO in DMSO (1/10 v). Oil composition and yield were obtained by gas chromatography (GC) using an Agilent Technologies 6890N GC-FID system, equipped with DB-5 capillary column (30 m × 0.25 mm × 2.5 μm film thickness) connected to a flame ionization detector (FID). The injector and detector temperatures were set at 250 °C. The carrier gas was helium, at a flow rate of 1.3 ml/min. The thermal programmer was 100–280 °C at a rate of 10 °C/min. Component relative concentrations were calculated based on GC peak areas. The injection volume of the TTO was 1 μL (Boligon et al., 2013; Homer et al., 2000) and oil analysis was performed in duplicate. GC-Mass Spectroscopy (GC-MS) analyses were performed on an Agilent Technologies AutoSystem XL GC-MS system operating in the EI mode at 70 eV, equipped with a split/splitless injector (250 °C). The transfer line temperature was 280 °C. Helium was used as carrier gas (1.5 ml/ min) and the capillary columns used were an HP 5MS (30 m × 0.25 mm × 2.5 μ m film thickness) and an HP Innowax (30 m × 0.32 mm, i.e., film thickness 0.50 mm). The temperature programed was the same as that used for the GC analyses. The injected volume was 1 μL of the essential oil. Identification of the constituents of TTO was performed on the basis of retention index (RI), determined with reference of the homologous series of n-alkanes (C7-C30) under identical experimental conditions, comparing with the mass spectra library search (NIST and Wiley), and with the mass spectra from the literature date (Adams, 1995). The relative amounts of individual components were calculated based on the CG peak area (FID response). 2.3. Experiment I (a pilot study) Eight adult male rats were used in a pilot study designed to investigate the effect of TTO on T. evansi experimentally infected rats (used 0.2 ml of blood containing 1.3 × 106 trypomastigotes – Rat1). They were housed on a light/dark cycle of 12 h in an experimental room under controlled temperature and humidity (25 °C; 70%, respectively). They were fed with commercial feed and received water ad libitum. Five rats received orally 1.0 ml kg−1 of TTO for three days on 24hour intervals (the group T). The first dose was performed 2 hours after inoculation. Three rats were untreated, and used as positive control (the group P). The parasitemia evolution (quantitative) was monitored daily by microscopy examination of blood smears (Da
2.4. Experiment II (a clinical study) The experiment II was designed after a pilot study in order to determine if the humoral response could increase longevity of rats infected by T. evansi after TTO treament.
2.4.2. Experimental design and parasitemia estimation The rats were divided into four groups (A to D). The uninfected animals from the groups A and B, composed of six healthy rats each, were intraperitoneally inoculated with 0.2 ml of blood from a healthy rat. Animals in the groups C and D were inoculated intraperitoneally with 0.2 ml of fresh blood obtained from a rat (R2), containing 2.3 × 106 trypanosomes (on day 1). The groups A and C represented the negative and positive control of the experiment and they were not treated with tea tree oil, respectively. Rats from the groups B and D received orally 1.0 ml kg−1 of TTO during three days, with dose intervals of 24 hours. The TTO had the first dose administered 2 hours after inoculation of the parasite. The number of trypomastigotes (parasitemia) and the treatment effect were monitored daily by microscopy examination of blood smears as described previously (Da Silva et al., 2006). 2.4.3. Sample collection On days 0, 3 and 6 post-infection (PI), survivor rats were anesthetized with isoflurane inside an anesthetic chamber to perform collection of blood samples (1.0 ml by intracardiac puncture). Approximately 10 min after this procedure all animals showed signs of recovery. The blood was used to obtain serum to measure the immune response. On the 15th day PI, all survivor rats were anesthetized following the same procedures prior cited, and humanely euthanized by decapitation. Blood samples (5 ml) were used to obtain serum to the measure liver and kidney functions, and immune response. 2.4.4. Influence of TTO treatment on the immune system The concentration of serum immunoglobulin G, M, A and E was determined using immunonephelometry on the Behring Nephelometer BN II (Dade Behring – USA) with reagents from Dade Behring. Samples were analyzed according to Dati et al. (1996). Briefly, all samples were diluted with specific diluents and measured after 10 min. Polystyrene particles were coated with a specific monoclonal antibody for each serum protein, forming an agglutinate that disperses the light irradiated in the presence of the protein. The intensity of scattered light depends on the amount of protein concentration in the sample, and the results are compared with known standard curves (Dati et al., 1996). Cytokines quantification (TNF-α, INF-γ, IL-1, IL-4, IL-6 and IL10) were assessed by ELISA assay using commercial Quantikine Immunoassay kits (R&D Systems, Minneapolis, MN), according to the manufacturer’s instructions. The concentration of the cytokines was determined by the intensity of the color measured spectrophotometrically using a microplate reader. 2.4.5. Influence of TTO treatment on liver and kidney functions The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea and creatinine were evaluated in a semiautomatic analyzer (TP Analyzer Plus®, Thermoplate, China) using
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Table 1 Qualitative and quantitative analyses of tea tree oil (TTO) used this study. Peak
Compounds
RIa
RIb
Amount (%)
ISO 4730 range (%)
Mol. Formula
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
α-pinene
937 976 1016 1025 1032 1037 1062 1178 1089 1190 1440 1521 1539 1583 1591
939 976 1018 1026 1031 1038 1062 1177 1088 1189 1439 1525 1538 1583 1590
3.51 0.46 9.85 2.27 1.39 6.03 20.15 41.98 4.17 2.43 1.04 0.80 0.63 0.97 0.18 95.86
1–6 Tr-3.5 5–13 0.5–8.0 0.5–1.5 Tr-15 10–28 30–48 1.5–5 1.5–8 Tr-3 Tr-3 Tr-3 Tr-1 Tr-1
C10H16 C10H16 C10H16 C10H14 C10H16 C10H18O C10H16 C10H18O C10H16 C10H18O C15H24 C15H24 C15H24 C15H26O C15H26O
sabinene
α-terpinene p-cymene limonene 1.8-cineole γ-terpinene terpinen-4-ol terpinolene α-terpineol aromadendrene ledene δ-cadinene globulol viridiflorol Total identified (%)
Relative proportions of the essential oil components were expressed as percentages. Tr = Trace amounts. a Retention indices experimental (based on homologous series of n-alkane C7-C30). b Retention indices from literature (Adams, 1995). International Organization for Standardization (ISO), standard number 4730.
commercial kits (Labtest® Diagnóstica S.A., Lagoa Santa, MG, Brazil). All tests were carried out in duplicate. 2.5. Statistic analysis Immunoglobulin, cytokine, and serum biochemistry data were submitted to analysis of variance followed by Duncan’s test (P < 0.05). 3. Results 3.1. Components present in the essential oil The results of the components present in the TTO are presented in Table 1. The top six compounds identified in TTO were dihydro carveol, myrtenol, cis-calamenene, t-pinocarveol, β-myrcene and α-terpineol. 3.2. Pilot study Untreated rats (the group P) showed increased parasitemia and died 5–7 days after inoculation. The animals treated with TTO (the group T) had prolonged survival rate; however, all died 13–21 days PI (Fig. 1). The experiment II was designed to determine whether the immune response could be related to the increased longevity in animals treated with TTO.
3.3. Experiment II 3.3.1. Disease course Data from the disease course are shown in Fig. 2. A significant increase in the pre-patent period in animal from the group D compared to the group C (similar pilot study) was observed. Trypomastigotes in the blood of infected animals treated with TTO were detected only on day 13 PI (group D) when compared with the group C, which showed trypanosomes on already on day 3 PI. At day 15 PI, there were no live animals in the positive control group (infected and untreated), unlike treated groups (Fig. 2). Based on the results from the pilot study and the increased parasitemia levels observed on rats from the group D (Fig. 2), the study was terminated on day 15 PI and blood samples were collected for biochemical and cytokine analysis. 3.3.2. Effect on the immune response Effect on the immune response is shown in Table 2. To investigate TTO immunomodulation effects, antibody and cytokine levels were quantified in serum 0, 3, 5 and 15 days PI using immunonephelometry and ELISA, respectively. On day 0, the levels of cytokines and immunoglobulins did not differ statistically. The TTO in healthy animals (the group B) did not alter IgG, IgE and IgA levels, but IgM was reduced when animals were TTO treated. Infected animals (the group C) had an increase in the levels of im-
Trypomastigotes/field
Experiment I - Pilot study
P1 P2 P3
140 120 100 80 60 40 20 0
T1 T2 T3 T4 T5
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 19 21
Days post-infection Fig. 1. Parasitemia of Trypanosoma evansi-infected rats in the groups P (not-treated) and T (treated with tea tree oil). All animals died after high parasitemia (more 100 trypanosomes/field).
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Fig. 2. Parasitemia of Trypanosoma evansi-infected rats at day 15 post-inoculation (PI) in the groups A to D. The treatment started at day 1 PI (three doses at 24-h intervals).
munoglobulins compared to four healthy rats. Infected rats treated with TTO (Group D) had increased levels of IgG, unlike the levels of IgM, IgA and IgE which were reduced compared to animals from the group C.
TTO administration in uninfected rats (the group B) was able to significantly reduce the levels of TNF, INF, IL-1, IL-4 and IL-6, and increase the levels of IL-10 when compared to animals from the group A. Infected rats (the group C) showed increased pro-
Table 2 Mean and standard deviation of the immunoglobulins [IgG, IgM, IgA and IgE] and cytokines [TNF-α, INF-γ, IL-1, IL-4, IL-6 and IL-10] results after treatment with tea tree oil (TTO – Melaleuca alternifolia) in rats experimentally infected by T. evansi. Parameters
IgG (mg/dL)
IgM (mg/dL)
IgE (Ul/ml)
IgA (mg/dL)
TNF-α (pg/ml)
INF-γ (µg/ml)
IL-1 (pg/ml)
IL-4 (pg/ml)
IL-6 (pg/ml)
IL-10 (pg/ml)
Days
0 3 5 15 0 3 5 15 0 3 5 15 0 3 5 15 0 3 5 15 0 3 5 15 0 3 5 15 0 3 5 15 0 3 5 15 0 3 5 15
Means ± standard deviations of groups* Group A
Group B
Group C#
Group D
132.5 ± 133.1 ± 14.7ab 131.6 ± 21.8a 130.2 ± 4.1a 51.3 ± 9.4a 44.5 ± 8.7b 43.5 ± 7.4b 43.4 ± 5.7a 74.1 ± 9.4a 70.5 ± 7.7a 68.8 ± 6.2a 68.2 ± 6.6a 71.0 ± 15.3a 67.8 ± 8.4b 65.1 ± 4.4b 64.0 ± 7.5a 102.8 ± 10.4a 104.0 ± 12.3c 102.6 ± 9.7c 99.8 ± 15.4b 118.0 ± 21.1a 114.6 ± 11.7b 114.1 ± 7.4c 112.8 ± 15.2a 40.5 ± 7.6a 39.1 ± 4.7c 39.5 ± 6.0c 39.2 ± 2.4b 69.6 ± 11.2a 65.6 ± 2.2c 59.0 ± 4.4c 56.8 ± 7.2b 82.3 ± 18.0a 78.6 ± 15.0b 79.5 ± 8.8c 79.0 ± 10.7b 64.8 ± 11.8a 71.5 ± 10.2b 74.6 ± 8.0b 77.4 ± 9.7b
131.6 ± 125.6 ± 16.9a 131.2 ± 20.4a 137.1 ± 7.7a 47.3 ± 12.4a 23.2 ± 6.3c 20.8 ± 3.9c 20.0 ± 2.8b 80.8 ± 11.1a 75.9 ± 5.4a 72.6 ± 8.2a 66.8 ± 8.3a 71.4 ± 10.9a 71.6 ± 8.7b 77.2 ± 4.4b 72.8 ± 13.7a 108.2 ± 12.1a 77.2 ± 8.8d 73.4 ± 8.4d 63.8 ± 7.1c 96.8 ± 17.2a 91.2 ± 7.4c 86.8 ± 10.1d 80.6 ± 12.1a 42.6 ± 9.4a 20.0 ± 7.1d 16.2 ± 3.2d 14.8 ± 4.1c 67.8 ± 9.2a 42.8 ± 6.8d 35.8 ± 4.4d 33.2 ± 3.0c 89.9 ± 10.4a 51.8 ± 5.9c 45.6 ± 9.1d 40.0 ± 2.2c 74.0 ± 14.0a 102.8 ± 13.9a 120.4 ± 21.4a 137.4 ± 25.2a
134.8 ± 10.1a 139.0 ± 11.1b 158.2 ± 23.6b – 56.3 ± 7.6a 61.8 ± 13.2a 78.0 ± 6.6a – 83.7 ± 12.3a 95.2 ± 9.1b 149.0 ± 14.6b – 74.6 ± 12.8a 84.4 ± 4.2a 98.0 ± 4.6a – 115.2 ± 20.4a 252.8 ± 28.4a 265.0 ± 21.2a – 107.3 ± 23.6a 294.4 ± 31.7a 314.0 ± 40.8a – 48.3 ± 8.9a 119.2 ± 23.2a 158.1 ± 32.6a – 79.8 ± 14.3a 127.6 ± 12.4a 177.3 ± 4.6a – 99.3 ± 21.6a 210.2 ± 32.1a 228.2 ± 18.2a – 58.0 ± 13.6a 53.8 ± 9.2c 44.0 ± 3.6c –
140.2 ± 15.8a 157.6 ± 23.4c 171.2 ± 17.2c 184.3 ± 12.9b 57.8 ± 14.9a 52.6 ± 10.3ab 51.2 ± 12.2b 43.7 ± 3.9a 77.8 ± 17.2a 80.8 ± 5.9a 74.6 ± 9.1a 64.3 ± 6.8a 72.0 ± 17.3a 74.4 ± 5.9b 66.2 ± 7.2b 64.8 ± 11.7a 109.4 ± 18.7a 215.2 ± 23.2b 180.6 ± 19.1b 170.3 ± 17.9a 105.8 ± 14.7a 273.8 ± 24.9a 217.3 ± 20.2b 217.0 ± 28.9a 47.2 ± 10.2a 93.0 ± 2.2b 95.2 ± 13.0b 93.1 ± 7.4a 72.6 ± 10.8a 106.6 ± 8.4b 108.2 ± 7.2b 102.3 ± 19.2a 96.1 ± 20.0a 191.0 ± 27.2a 178.0 ± 24.4b 173.2 ± 32.2a 72.0 ± 17.0a 80.6 ± 12.2b 78.2 ± 9.2b 70.3 ± 8.9b
9.2a
16.4a
* Means followed by the same letter in the same row do not differ significantly by the Duncan test. On day 15, no measurements were performed on animals from the group C, due to trypanosomiasis animal death.
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Fig. 3. Alanine aminotranferase, ALT (A), aspartate aminotranferase, AST (B), urea (C) and creatinine levels (D) in uninfected mice (group A); uninfected and treated with tea tree oil (group B); and infected with T. evansi and treated with tea tree oil at 1.0 mg kg−1 (group D) in day 15 post-infection. The animals of group C (infected and untreated) died 6 days post-infection. The same letters in columns indicate that there is no significant difference between groups.
inflammatory cytokines (TNF, INF, IL-1, IL-4 and IL-6) and reduction of anti-inflammatory (IL-10) compared to healthy animals (group A and B). The pro-inflammatory cytokines in infected/treated animals (the group D) increased when compared to healthy animals (the groups A and B), but was lower than the rats from the group C. The IL-10 levels did not differ between groups A and D (Table 2). 3.3.3. Serum biochemistry Serum biochemistry data are shown in Fig. 3. ALT activity increased significantly in all groups infected by T. evansi and treated orally with TTO at 1.0 ml kg−1 when compared to the other groups. AST, urea, and creatinine results did not differ between all groups. 4. Discussion TTO treatment was able to control parasitemia and thus to prolong the longevity of rats in this study. Compounds present in the TTO essential oil, such as terpin-4-ol, were responsible for the in vitro death of T. brucei trypomastigotes (Mikus et al., 2000), which may also have occurred in this study. Associated with the effects of the TTO oil, the inflammatory response (immunoglobulin and cytokine increased) mounted by rats to fight T. evansi infection may also have prolonged their lives. IgG and IgM antibodies have been described in different studies at high levels, and IgM has been directly responsible for fighting trypomastigotes infection (Costa et al., 2013; Gressler et al., 2010). During experimental infection by T. evansi, rats showed an increase on IFN, TNF, IL-1, and IL-6 levels in serum (Paim et al., 2011), similarly to what occurred in this study. Therefore, our hypothesis is that the trypanocidal effect of TTO combined with the inflammatory response was responsible for the increase in longevity by controlling the parasitemia (the group D),
despite the fact that TTO oil has anti-inflammatory effect. However, it is noteworthy that the immune response alone (antibodies and cytokines) is not sufficient to control the disease, since the animals died within a few days PI (the group C). TTO anti-inflammatory effect on cellular responses is well documented (Hart et al., 2000). However, the effect of this oil on the humoral response is scarce in the literature. In our study, we found that the treatment of healthy rats did not affect the levels of IgG, IgA, and IgE, unlike what happened with IgM levels that were reduced in the serum (the group B). An explanation for IgM reduction is unknown at the moment, but our hypothesis is that some of the components present in TTO oil may be responsible for this fact, but further studies are needed. As already mentioned, in this study we observed a progressive increase in the serum levels of IFN-γ, TNF-α, IL-1, IL-4, and IL-6 on animals infected by T. evansi, and according to the literature, these findings are correlated to the inflammatory response against infection (Gao and Pereira, 2002; Sileghem et al., 1989; Silva et al., 1995). The inflammatory response is considered the first host response against protozoa infection (Titus et al., 1991), a fact observed also in this study. Healthy animals (the group B) and infected (the group D) treated with TTO showed reduced levels of serum cytokines, which could be considered a positive finding for rats. That is because excessive amounts of these pro-inflammatory cytokines can be harmful to the host, leading to tissue injury, therefore exacerbating clinical signs of the disease (Abbas et al., 2012). IL-10 serum levels were low in infected compared to uninfected animals. The animals of groups receiving TTO showed increased serum levels of IL-10, a cytokine with important anti-inflammatory response. This agrees with the literature that reports IL-10 increase after TTO treatment of mononuclear cells from the periph-
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eral blood (Caldefie-Chézet et al., 2006). There are numerous studies in vivo and in vitro supporting the evidence of TTO anti-inflammatory effect. The components of TTO may inhibit the lipopolysaccharideinduced production of inflammatory mediators such as the tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), and prostaglandin E2 by human peripheral blood monocytes (Hart et al., 2000). TTO also decreases the production of reactive oxygen species by stimulated neutrophils and monocytes (Caldefie-Chézet et al., 2004). TTO also modulates the vasodilation and plasma extravasation associated with histamine-induced inflammation in humans (Khalil et al., 2004). The dose of TTO used in this study was able to modulate the inflammatory response against T. evansi infection (the group D), which is maintained at moderate levels when compared to the positive control, reducing the typical cellular and tissue lesions often occurred during inflammation. The TTO therapeutic protocol caused no increase in serum levels of AST, ALT, creatinine, and urea in healthy animals. These results show that treatment with TTO does not cause liver and kidney damage in healthy animals treated with a dose of 1.0 ml kg−1. However, the rats from the group D (infected/treated) showed increased ALT levels, which is a typical finding in animals infected by T. evansi (Monzón and Villavicencio, 1990; Barr, 1991; Arora and Pathak, 1995; Aquino et al., 2002). There are a significant progress in the antimicrobial and anti-inflammatory studies of TTO, but only a few studies on the safety and toxicity of this oil. TTO can be toxic if ingested, as evidenced by studies with animals and from cases of human poisoning. The 50% lethal dose for TTO in a rat model is 1.9 to 2.6 ml kg−1 and rats dosed with ≤1.5 g kg−1 TTO appeared lethargic and ataxic (Russell, 1999). Based on these results, we conclude that treatment with TTO is able to prolong the life of rats infected by T. evansi, maintaining low parasitemia. The TTO treatment caused an anti-inflammatory effect on rats, which may have been beneficial for the animals. The therapeutic protocol with TTO has neither curative effect nor renal and hepatic toxicity on rats. These promising results show that the TTO has compounds with trypanocidal activity and it could be used in future trypanosomosis chemotherapy. Ethical approval The procedure was approved by the Animal Welfare Committee of Universidade do Estado de Santa Catarina, number 1.51.13. References Abbas, A.K., Lichtman, A.H., Pillai, S., 2012. Cellular and Molecular Immunology, seventh ed. WB Saunders Elsevier, Philadelphia, p. 527. Adams, R.P., 1995. Identification of Essential Oil Components by Gas Chromatography/ Mass Spectroscopy. Allured Publishing Corporation, Illinois, USA, 456 pp. Aquino, L.P.C.T., Machado, R.Z., Alessi, A.C., Santana, A.E., Castro, M.B., Marques, L.C., et al., 2002. Aspectos hematológicos, bioquímicos e anatomopatológicos da infecção de Trypanosoma evansi em cães. Arquivo Brasileiro de Medicina Veterinária e Zootecnia 54, 8–18. Arora, J.K., Pathak, M.L., 1995. Clinico-hematological and biochemical changes associated with Trypanosoma evansi infection in dogs. Indian Journal of Animal Health 34, 33–38. Barr, S.C., 1991. American trypanosomiasis in dogs. Compendium on Continuing Education for the Practicing Veterinarian 13, 745–752. Boligon, A.A., Kubiça, T.K., Mario, D.B., Brum, T.F., Piana, M., Weiblen, R., et al., 2013. Antimicrobial and antiviral activity-guided fractionation from Scutia buxifolia Reissek extracts. Acta Physiologiae Plantarum 35, 2229–2239.
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