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Experimental Parasitology

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Short- and long-term effects of orally administered azithromycin onTrypanosoma brucei brucei-infected miceNthatisi I. Molefea,f, Peter S. Musinguzib, Daisuke Kondohe, Kenichi Watanabec,d,Oriel M.M. Thekisoef, Xuenan Xuana, Noboru Inoueg, Keisuke Suganumaa,c,∗aNational Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Nishi 2-11 Inada, Obihiro, Hokkaido, 080-8555, Japanb Faculty of Biomedical Sciences, Kampala International University, Western Campus, P.O. Box 71, Bushenyi, Ugandac Research Center for Global Agromedicine, Obihiro University of Agriculture and Veterinary Medicine, Nishi 2-11 Inada, Obihiro, Hokkaido, 080-8555, Japand Veterinary Pathology, Obihiro University of Agriculture and Veterinary Medicine, Nishi 2-11 Inada, Obihiro, Hokkaido, 080-8555, Japane Section of Anatomy and Pathology, Division of Veterinary Sciences, Department of Veterinary Medicine, Obihiro University of Agriculture and Veterinary Medicine, Nishi2-11 Inada, Obihiro, Hokkaido, 080-8555, JapanfUnit for Environmental Sciences and Management, North-West University, Private Bag X6001, Potchefstroom, 2520, South AfricagObihiro University of Agriculture and Veterinary Medicine, Nishi 2-11 Inada, Obihiro, Hokkaido, 080-8555, Japan

A R T I C L E I N F O

Keywords:AcidocalcisomesAzithromycinGlycosomesTrypanocidal effectTrypanosoma brucei brucei

A B S T R A C T

Human African trypanosomosis (HAT) and animal African trypanosomosis (AAT) are diseases of economic importancein humans and animals that affect more than 36 African countries. The currently available trypanocidal drugs areassociated with side effects, and the parasites are continually developing resistance. Thus, effective and safe drugs areneeded for the treatment of HAT and AAT. This study aimed to evaluate the effects of azithromycin (AZM) onTrypanosoma brucei brucei-infected mice. Mice were randomly divided into 7 groups consisting of a vehicle controlgroup, 5 test groups and a diminazene aceturate (DA)-treated group. Mice were treated orally for 7 and 28 days, asshort-term and long-term treatments, respectively. Short-term AZM treatment cured 23% (16 of 70) of the overalltreated mice whereas long-term treatment resulted in the survival of 70% of the mice in the groups that received AZMat doses of 300 and 400mg/kg. Trypanosomes treated in vitro with 25 μg/mL of AZM were subjected to transmissionelectron microscopy, which revealed the presence of increased numbers of glycosomes and acidocalcisomes incomparison to the vehicle group. The current study showed the trypanocidal effect of AZM on T. b. brucei in vivo. Thedemonstrated efficacy increased with an increase in treatment period and an increased concentration of AZM.

1. Introduction

The subgenus Trypanozoon is composed of Trypanosoma brucei rho-desiense and T. b. gambiense, which cause human African trypanoso-mosis (HAT), also known as sleeping sickness, and T. b. brucei, T. evansiand T. equiperdum, which cause animal African trypanosomosis (AAT),individually known as nagana, surra and dourine, respectively(Desquesnes et al., 2013; Morrison et al., 2016). T. brucei is divided intothree different sub-species, T. b. brucei, T. b. rhodesiense and T. b. gam-biense, based on their geographic origin, infectivity to humans and thedisease severity (Balmer et al., 2011). Previous studies have demon-strated the close relatedness of the three T. brucei sub-species, amongwhich only T. b. gambiense group 1 proved to be distinct from T. b.

gambiense group 2, T. b. rhodesiense and T. b. brucei. Furthermore, al-though T. b. gambiense group 2 and T. b. brucei are closely related, T. b.rhodesiense appears to be just a variant of T. b. brucei, with a differentphenotype regarding human infectivity (Gibson and Bailey, 2003;Balmer et al., 2011).The currently available drugs used for the treatment of sleeping

sickness, especially those used for late-stage infection, are associatedwith severe side effects. Furthermore, these drugs are expensive, andthe parasites have widely developed resistance to the drugs. Most im-portantly, these drugs can only be administered parenterally, whichrequires hospitalization and technical assistance (Wilkinson and Kelly,2009). Thus, a search for safe drugs that are easy to administer andhave fewer side effects is currently underway.

https://doi.org/10.1016/j.exppara.2019.02.018Received 25 June 2018; Received in revised form 4 February 2019; Accepted 23 February 2019

∗ Corresponding author. Research Center for Global Agromedicine, National Research Center for Protozoan Diseases, Obihiro University of Agriculture andVeterinary Medicine, Nishi 2-11 Inada, Obihiro, Hokkaido, 080-8555, Japan. Tel.: +81 155 49 5697; fax: +81 155 49 5643.

E-mail addresses: nthatisimolefe@gmail.com (N.I. Molefe), pmusinguzi@yahoo.com (P.S. Musinguzi), kondoh-d@obihiro.ac.jp (D. Kondoh),knabe@obihiro.ac.jp (K. Watanabe), Oriel.Thekisoe@nwu.ac.za (O.M.M. Thekisoe), gen@obihiro.ac.jp (X. Xuan), ircpmi@obihiro.ac.jp (N. Inoue),k.suganuma@obihiro.ac.jp (K. Suganuma).

Experimental Parasitology 199 (2019) 40–46

Available online 03 March 20190014-4894/ © 2019 Elsevier Inc. All rights reserved.

T

Azithromycin (AZM), C38H72N2O12·2H2O, is a semi-synthetic 15-membered azalide antibacterial agent isolated from Streptomyces spp.,which is used for the treatment of various bacterial infections (Jelić andAntolović, 2016). We recently demonstrated the efficacy of orally ad-ministered AZM on T. congolense-infected mice (Molefe et al., 2017). T.congolense has a wide range of hosts, including camels, horses, donkeys,goats, pigs, cattle and dogs. Although the infection is highly pathogenicin dogs, it mainly affects cattle (Steverding, 2010; Lisulo et al., 2014;Simukoko et al., 2007). The effect of AZM is not influenced by thepresence of food; however, in ruminants, food is primarily absorbedinto the rumen in large volumes and is digested very slowly, thus re-sulting in the slow absorption of compounds and/or drugs in compar-ison to the fast absorption in monogastric animals such as dogs and pigsor humans (Sawaguchi et al., 2016). Due to the recently reported effi-cacy of AZM on T. congolense-infected mice (Molefe et al., 2017), thepresent study sought to repurpose the usage of AZM as a treatment oftrypanosomosis in humans and/or monogastric animals by assessing theefficacy of AZM using a T. b. brucei model, which is phenotypically andgenetically related to the HAT-causative trypanosomes and to furtherdetermine the possible morphological changes in treated trypanosomesin comparison to non-treated controls.

2. Materials and methods

2.1. Animal experiments

Healthy female C57BL/6 mice (body weight, 16–25 g) were used inthis study (CLEA Japan Inc., Tokyo, Japan). The mice were kept in theanimal facility of the National Research Center for Protozoan Diseases,Obihiro University of Agriculture and Veterinary Medicine, Japan. Theanimals were acclimatized in plastic cages in an air-conditioned en-vironment and were maintained at 25 ± 2 °C with 60 ± 10% relativehumidity under a 12-h light and dark cycle for one week before com-mencing the experiments. All of the animals had ad libitum access tonormal feed and water. This study was conducted according to theRegulations and Operation of Animal Experiments approved by theanimal ethics committee of Obihiro University of Agriculture andVeterinary Medicine (Approval Nos. 28–129 and 28–169).

2.2. The in vivo trypanocidal efficacy of AZM on T. b. brucei

2.2.1. Short-term treatmentThe virulent T. b. brucei GUTat 3.1 strain was passaged twice in mice

before the experiment. Each experimental mouse was intraperitoneallyinoculated with 100 μL of T. b. brucei (1×102 parasites/mouse) withphosphate buffered saline – 1% glucose. The mice (n=70) were ran-domly divided into 7 groups as follows: Group I (vehicle control group),the mice were infected but not treated; Group II (positive control group),the mice were infected and treated intraperitoneally with diminazeneaceturate (DA) at 3.5mg/kg (Sigma Aldrich, Japan); Groups III, IV, V, VIand VII (test groups), the infected mice were orally-treated using afeeding needle with 50, 100, 200, 300 or 400mg/kg AZM, respectively.Treatment was performed once a day from 24 h post-infection and wasmaintained for 7 consecutive days. The treatments were freshly preparedeach day. Mice that survived for 30 days were further monitored for anadditional 60 days, and the experiments were terminated at 90 days post-infection (dpi). Each day, parasitemia was evaluated and the effects oftreatment were monitored using wet blood smears. Each slide was pre-pared with fresh blood collected from the tail vein (magnification:400× ). The experiments were conducted in duplicate.

2.2.2. Long-term treatmentThe long-term trypanocidal efficacy of AZM was investigated in T. b.

brucei (GUTat 3.1)-infected mice (n=70). The mice were divided into7 treatment groups as described in section 2.2.1. Treatment for T. b.brucei was performed once a day from 24 h post-infection and was

maintained for 28 consecutive days. Parasitemia and the body weight ofthe mice were recorded twice a week, whereas the blood parameterswere evaluated once a week. The objective of this treatment was todetermine the duration for which Trypanosoma-infected mice dependedon AZM. These experiments were conducted in duplicate.

2.2.3. Cerebrospinal fluid (CSF) analysisThe CSF samples were collected at 90 dpi from mice that survived

for the entire study period, as described by Zarghami et al. (2013) withminor modifications. The objective of conducting the CSF examinationwas to confirm the presence or absence of T. b. brucei in the centralnervous system (CNS). Briefly, the mice were anaesthetized via theintraperitoneal administration of ketamine (50mg/kg) and xylazine(10mg/kg). Using a stereotaxic frame, a midline scalp incision wasmade to expose the atlanto-occipital membrane. A capillary tube wascarefully placed to pierce the membrane and was inserted into themouse brain under direct vision. CSF started flowing into the capillaryand was transferred into Eppendorf tubes. The collected CSF was sub-jected to a microscopic examination.

2.3. Morphological analysis of AZM-treated trypanosomes by transmissionelectron microscopy (TEM)

The T. b. brucei parasites were incubated in vitro in HMI-9 mediumsupplemented with 25 μg/mL of AZM for 7 and 24 h. Subsequently, thesamples were centrifuged twice at 15 000 g for 10min with 0.1M phos-phate buffer (PB) to wash away the medium. The samples were then fixedin 2.5% glutaraldehyde, kept for 1 h at 4 °C and then centrifuged at15 000 g for 10min. The samples were resuspended in 0.1MPB post-fixedin 1% OsO4 for 30min. After washing three times with PB, the sampleswere dehydrated and then embedded in LR white resin. Ultrathin sections(approximately 90-nm thick) were cut using a diamond knife and ex-amined using an HT7700 transmission electron microscope (Hitachi,Tokyo, Japan) without uranyl acetate or lead citrate staining.

2.4. Statistical analysis

The results are expressed as the mean ± standard deviation (S.D.)for the number of repeated trials in each experiment. The statisticalanalyses were conducted in the acute phase of infection. The t-test wasused for intergroup comparisons between the treated and non-treatedgroups. Survival curves were constructed using the Kaplan-Meiermethod, and the curves were compared using a log-rank test. TheMann-Whitney U test was conducted to quantify the differences be-tween the control and the treated groups from TEM imaging. A p valueof< 0.05 was considered to indicate statistical significance. All datawere compiled using the GraphPad Prism software program (version5.0, GraphPad Software Inc., CA, USA).

3. Results

3.1. In vivo trypanocidal efficacy of short-term treatment

There was no significant difference in parasitemia between theshort-term-treated groups and the non-treated groups. The prepatentperiod, marked by the presence of the parasites in the peripheral cir-culation, of the non-treated group was 2 dpi, that of the 50 and 100mg/kg groups was 3 dpi, and that of the 200, 300 and 400mg/kg groupswere each 4 dpi (Fig. 1). The parasitemia reached the first peak wavebetween 6 and 8 dpi in all groups. Approximately 23% (16 of 70) of allmice that made it through the peak wave survived until the experimentswere terminated at 90 dpi (Fig. 1). The parasitemia of the AZM-treatedgroups did not differ to a statistically significant extent from that in thecontrol group. Nonetheless, the survival rates of the AZM-treated micein the 50, 100, 200 and 300mg/kg groups were 10%, 10%, 20% and20%, respectively. None (0%) of the mice in the 400mg/kg group

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survived; in contrast, all mice (100%) in the DA group survived. Thesurvival duration of the mice in the 400mg/kg group was up to 12 dpi,whereas the mice in the control group survived for up to 11 dpi (Fig. 2).

3.2. Long-term treatment

3.2.1. Changes in body weight during Trypanosoma infection and AZMtreatmentThe body weight of the T. b. brucei-infected mice that received 50mg/

kg of AZM did not differ significantly from that of the control group. Incontrast, the mice in the 100, 300 and 400mg/kg groups (p < 0.001) andthe 200mg/kg group (p < 0.05) showed a significant increase in bodyweight in comparison to the non-treated control group (Fig. 3).

3.2.2. ParasitemiaThe long-term treatment of T. b. brucei with AZM resulted in sig-

nificant differences in the parasitemia levels of the 100, 200 and300mg/kg groups (p < 0.05) and of the 400mg/kg and DA-treatedgroups (p < 0.01) in comparison to the control group (Fig. 4). The firstparasitemia peak was reached on the first week of the experiment. Inthe vehicle control and in the 50, 100 and 200mg/kg groups, the peakwas maintained with a steady increase of parasitemia for a period of 2weeks before the parasites could be cleared from the peripheral circu-lation. There was a reduction in the parasitemia of the 400 mg/kg-treated mice, even though the parasites had not been cleared, whereastreatment at a dose of 300mg/kg resulted in the rapid clearance of theparasites on the second week (Fig. 4). Approximately 50% of the mice

that made it through the peak wave survived until the experimentswere terminated at 90 dpi; no cases of relapse were observed. The micein the 200 (p < 0.001) and 300 and 400 (p < 0.01) mg/kg-treatedgroups showed significantly better survival as compared to the vehiclecontrol. The survival rates in the control and 50, 100, 200, 300 and400mg/kg groups were 10%, 20%, 30%, 40%, 70% and 70%, respec-tively [Fig. 5]. All of the mice in the DA group survived (Fig. 5).

3.2.3. Cerebrospinal fluid analysisNo parasites were recovered from the CSF of the mice that survived.

Blood samples were also examined for the presence of parasites; noparasites were observed in the peripheral circulation of the mice.Additionally, no traces of parasites were found in the blood or the CSFof control group mice that survived.

3.3. Ultrastructural analysis by TEM

Glycosome-like structures were observed in both the control(Fig. 6A) and AZM-treated (Fig. 6B and C) specimens; however, thesestructures were numerous in the AZM-treated specimens. In addition,the trypanosomes treated for 7 h (p=0.041) and for 24 h (p=0.025)possessed acidocalcisome-like structures, which were absent in thecontrol specimens (Table 1). Furthermore, trypanosomes incubatedwith AZM for 24 h showed the presence of a vacuole-like structurecontaining digested cellular materials (Fig. 6C).

4. Discussion

The in vitro trypanocidal effect of AZM on T. congolense, T. b. bruceiand T. evansi was published in our previous study (Molefe et al., 2017)at IC50 (concentration inhibiting 50% of the population) values of0.19 ± 0.17, 3.69 ± 2.26 and 1.81 ± 1.82 μg/mL. The IC50 value ofAZM for T. congolense meets the activity criteria for drug candidates forAfrican trypanosomosis recommended by Nwaka and Hudson (2006)whereas those of T. b. brucei and T. evansi were slightly higher.

T. congolense-infected mice that were treated with AZM showed abetter response to treatment, which manifested as significantly sup-pressed parasitemia in all treated groups and a significantly prolongedsurvival rate in comparison to the vehicle control group (Molefe et al.,2017). The survival rates in the present study were lower than thoseobserved in the T. congolense study, in which the survival rates of micethat received doses of 300mg/kg and 400mg/kg were 80% and 100%,respectively (Molefe et al., 2017).None of the mice undergoing short-term treatment with AZM 400mg/

kg in the present study survived the infection. AZM results in a reversiblebinding of the 50S subunits of bacteria, thereby resulting in a

Fig. 1. Evaluation of parasitemia in mice infectedwith T. b. brucei and orally-treated with differentconcentrations of AZM for 7 days. There were nosignificant differences between the treated groupsand the control group (p > 0.05). The data are ex-pressed as the mean ± S.D. The 1× 100 value in-dicates parasitemia below the limit of detection. DA,diminazene aceturate.

Fig. 2. Survival curves of mice infected with T. b. brucei and orally treated withdifferent concentrations of AZM for 7 days. The survival rate did not differ fromthat of the control group to a statistically significant extent (n = 10).**p < 0.001 in the DA group and * p < 0.05 at 200mg/kg (log-rank test). DA,diminazene aceturate.

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bacteriostatic effect (Haworth et al., 2014). The short-term treatment oftrypanosomes with AZM suggests that a trypanostatic effect was observedin the present study. Furthermore, AZM possesses immunomodulatoryeffects. Maintenance of the immunomodulatory compound is necessary todelay relapse and prolong progression of the pathogens; however, relapseand recurrence of the infection remain inevitable (Li et al., 2018). Ac-cording to Serisier and Martin (2011), AZM persists at a sub-inhibitoryconcentration, which could explain the better efficacy of the compound atlower concentration during the short-term treatment as compared to the400mg/kg concentration. The efficacy of the 400mg/kg dose was im-proved by increasing the treatment period to 28 days.Antiparasitic drugs are developed to target and/or inhibit a specific

intracellular metabolic pathway that is vital to the pathogen, i.e., thepathway responsible for the metabolism of nutrients, the survival of thepathogen in the hosts, or cell replication. The metabolic pathways,however, are mostly species-specific (Botero et al., 2017). Sahin et al.(2014) demonstrated the susceptibility of T. congolense to iso-metamidium (active ingredient in the commercial drugs Veridium® andSamorin®), whereas T. b. brucei was 15 times less susceptible to thecompound, which was attributed to differences in the transporter usedby the parasites to take up the drug (Sahin et al., 2014).

T. congolense is a strictly intravascular parasite that is constrained inthe blood vessels, which means that any effective compound that is welldistributed in the bloodstream is likely to affect and/or clear thisparasite. Furthermore, T. congolense is continuously exposed to host

circulatory factors (Kuriakose et al., 2012), whereas T. b. brucei, isbroadly tropic with the potential to invade extracellular sites such asadipose tissues and, most importantly, the CNS. Drug efficacy against T.b. brucei in the later stage is determined by the ability of the drugs topenetrate tissues (McCall et al., 2016). The parasites take refuge in sites,mainly in the CNS that is not accessible by drugs, which is the leadingcause of post-treatment relapse in T. b. brucei infection (Poltera et al.,1981; Moulton, 1986; Ezeh et al., 2016).

Fig. 3. Effect of 28 days of oral treatment with AZM on the body weight of T. b. brucei-infected mice (n=10). DA, diminazene aceturate.

Fig. 4. Evaluation of parasitemia in mice infectedwith T. b. brucei and orally treated with differentconcentrations of AZM for 28 days. *p < 0.05 at100, 200 and 300 mg/kg; **p < 0.01 at 400mg/kgat week one. The data are expressed as themean ± S.D. The 1× 100 value represents para-sitemia below the limit of detection. DA, diminazeneaceturate.

Fig. 5. Survival curves of mice infected with T. b. brucei and orally treated withdifferent concentrations of AZM for 28 days. The survival rate was significantlydifferent from that of the control group (n = 10), **p < 0.001;***p < 0.0001 (log-rank test). DA, diminazene aceturate.

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The peak parasitemia period differed between the short-term andlong-term treatment even though infection and initiation of treatmentcommenced at the same time. The complexity of the parasitemia peaksdepends mainly on the antibody-mediated immune response of the hostand the antigenic variation. Additionally, trypanosome proliferationrate, differentiation and cell death also contribute to the increase anddecrease of the parasites in the bloodstream. Trypanosome proliferationis controlled by the clearance of the parasites by the immune systemand by antigenic variation (Tyler et al., 2001). The authors thereforebelieve that the immunity of the mice played a major role in the dif-ferent parasitemia peaks as the experiments were conducted in seriesrather than simultaneously.None of the groups of AZM-treated T. b. brucei-infected mice showed

100% survival. Furthermore, only 10% of the mice in the control groupsurvived the initial parasitemia wave and further survived for 28 dayspost-infection prior to euthanasia (Fig. 5). Several studies have de-monstrated the resistance of C57BL/6 mice to various pathogens, in-cluding Leishmania spp., Plasmodium spp., Babesia spp. and bacterialinfections (Yap and Stevenson, 1994; Aguilar-Delfin et al., 2001;Breitbach et al., 2006; Colpitts and Scott, 2011). According to Trindade

et al. (2016), C57BL/6 mice are a good laboratory model to study theweight loss and emaciation associated with T. brucei infections. How-ever, the concept differed in the current study, in which body weightswere not significantly different between the treated, non-treated andnon-infected mice.No trypanosomes were observed in the CSF of either the non-treated

or the treated groups; however, it is suspected that the absence of theparasites in the control group was due to the short interval before eu-thanasia (30 dpi), whereas the other mice were allowed a sufficientamount of time (90 dpi) for the infection to develop to the chronic stageassuming that the infection is progressive and that 3 weeks to 2 monthsare required for the parasites to invade the CSF (Mogk et al., 2014).AZM efficacy in bacteria occurs through the inhibition of protein

synthesis by binding to the 50S subunit complex; however, eukaryoticcells possess no 50S but 60S and 40S subunits. The main action of AZMon protozoa from the genera Plasmodium, Toxoplasma and Babesia hasnot yet been shown and therefore remains unclear (Weiss et al., 1993;Derouin, 1995; Castro-Filice et al., 2014). For this reason, trypanosomeswere incubated with AZM for different periods of time to determine themode of action of the compound on T. b. brucei.

T. b. brucei samples incubated with AZM for 7 and 24 h showed thepresence of glycosomes-like and acidocalcisomes-like structures, which,according to literature, are vital for the survival of the parasites indifferent environmental conditions. Glycosomes and acidocalcisomesare peculiar to trypanosomatids. Glycosomes contain the majority ofthe enzymes of the glycolytic pathway and are therefore used by thebloodstream form of T. b. brucei for glycolysis as this form dependsentirely on glucose metabolism for its supply of ATP (Haanstra et al.,2016). Acidocalcisomes, lysosome-related organelle, are a major sto-rage compartment for phosphate compounds and cations such as cal-cium. Their acidification is driven by either vacuolar H+-ATPase or avacuolar H+-pyrophosphatase in T. brucei (Huang and Docampo,2015).

Fig. 6. Effects of AZM on the cellular morphology of T. b. brucei cells grown in HMI-9 medium. Transmission electron microscopy images of (A) non-treatedtrypanosomes (control) and specimens treated with 25 μg/mL AZM for (B) 7 h and (C) 24 h. Black arrows, glycosome-like structures; red arrows, acidocalcisome-likestructures; red circles, vacuole-like structures containing digested materials. (For interpretation of the references to colour in this figure legend, the reader is referredto the Web version of this article.)

Table 1Glycosomes-like and acidocalcisome-like structure counts.

Structures Counts (Μm2) p-values

Glycosome-likemControl p 1.056 ± 0.203 –7 h 1.347 ± 0.120 0.13124 h 1.255 ± 0.144 0.257Acidocalcisome-likeControl 0.101 ± 0.053 –7 h 0.356 ± 0.115 0.041*24 h 0.349 ± 0.100 0.025*

*p values < 0.05 (significantly different). Data presented as Mean ± S.D.

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Previous studies have shown that glycosomes and acidocalcisomesare affected by environmental stressors such as starvation, hyper-os-motic stress and hypo-osmotic stress and play a major role in theparasite's response to environmental stress (Moreno and Docampo,2009; Brennand et al., 2012). External stress has an effect on the gly-cosome composition, enzymatic composition, and cellular metabolism,and therefore contributes greatly to the response of the parasites tovariable conditions. The results of external stress include rapid hydro-lysis leading to the synthesis of acidocalcisomes. Furthermore, acid-ocalcisomes are associated with autophagy of the trypanosomes, espe-cially in T. brucei (Li and He, 2014). According to Docampo et al.(2010), starvation and chemically induced autophagy are accompaniedby acidocalcisome acidification. They concluded that the blocking ofacidocalcisomes blocks autophagy because lysosomes are needed toinitiate the process of autophagy. In the present study, acidocalcisome-like structures were only observed in AZM-exposed trypanosomes in-dicating that the trypanocidal action of AZM is most likely due totriggering autophagy in the parasites.

5. Conclusion

The present study showed that AZM treatment—especially long-term treatment—has trypanocidal activity against T. b. brucei. Thepresence of the glycosome- and acidocalcisome-like structures observedin T. b. brucei by TEM proved that the parasites experience externalstress when exposed to AZM. Additional studies should be performed tofurther clarify the efficacy of AZM and its mode of action in naturallyinfected hosts.

Authors’ contributions

NIM designed and performed the study. PSM participated in thestudy design and drafting of the paper. DK conducted the TEM analysisof the samples. KW assisted with the CSF collection. OMMT participatedin the drafting and revision of the manuscript. XX was responsible forthe administration of the research study. NI and KS participated in thestudy design and coordination of the research study from its onset. Allauthors read and approved the manuscript.

Conflicts of interest

The authors have declared no conflict of interest.

Consent of publication

Not applicable.

Funding

The authors would like to express their gratitude to the Ministry ofEducation, Culture, Sports, Science and Technology for providing fi-nancial support for the present study. This study was also supported bygrants from the Japan Society for the Promotion of Science KAKENHIGrant Number 16K18793 (Grants-in-Aid for Young Scientists [B]), andthe “International Collaborative Research Program for Tackling theNeglected Tropical Disease (NTD) Challenges in African Countries”from the Japan Agency for Medical Research and Development(AMED), and AMED/JICA SATREPS (17jm0110006h0005).

Acknowledgements

We would like to thank Obihiro University of Agriculture andVeterinary Medicine for granting us the opportunity to conduct theexperiments in their facility. The experiments were conducted with theassistance of the laboratory members of the Research Unit for VectorBiology of the National Research Center of Protozoan Diseases.

Abbreviations

AAT animal African trypanosomosisAZM azithromycinCNS central nervous systemCSF cerebrospinal fluidDA diminazene aceturatedpi days post-infectionHAT human African trypanosomosisIC50 concentration inhibiting 50% of the populationPB phosphate bufferTEM transmission electron microscopy

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