Veterinary Parasitology 233 (2017) 97–106

Contents lists available at ScienceDirect

Veterinary Parasitology

jou rnal homepage: www.elsevier.com/locate/vetpar

Research paper

Artesunate-tafenoquine combination therapy promotes clearance and

abrogates transmission of the avian parasite gallinaceum

a a b b

Suchada Tasai , Tawee Saiwichai , Morakot Kaewthamasorn , Sonthaya Tiawsirisup ,

a c d,∗

Prayute Buddhirakkul , Sirintip Chaichalotornkul , Sittiporn Pattaradilokrat

a

Department of Parasitology and Entomology, Faculty of Public Health, Mahidol University, Bangkok 10400, Thailand

b

Department of Pathology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand

c

Department of Dermatology, School of Anti-Aging and Regenerative Medicine, Mae Fah Luang University Hospital, Bangkok 10100, Thailand

d

Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand

a

r t a b

i c l e i n f o s t r a c t

Article history: Clinical manifestations of malaria infection in vertebrate hosts arise from the multiplication of the asexual

Received 12 August 2016

stage parasites in the blood, while the gametocytes are responsible for the transmission of the disease.

Received in revised form 2 December 2016

Antimalarial drugs that target the blood stage parasites and transmissible gametocytes are rare, but are

Accepted 12 December 2016

essentially needed for the effective control of malaria and for limiting the spread of resistance.

and its derivatives are the current first-line antimalarials that are effective against the blood stage par-

Keywords:

asites and gametocytes, but resistance to artemisinin has now emerged and spread in various malaria

Aedes aegypti

endemic areas. Therefore, a novel antimalarial drug, or a new drug combination, is critically needed to

8-Aminoquinoline

Artesunate overcome this problem. The objectives of this study were to evaluate the efficacy of a relatively new

Chemotherapy antimalarial compound, tafenoquine (TQ), and a combination of TQ and a low dose of artesunate (ATN)

Tafenoquine on the in vivo blood stage multiplication, gametocyte development and transmission of the

Plasmodium gallinaceum parasite Plasmodium gallinaceum to the vector Aedes aegypti. The results showed that a 5-d treatment

Oocysts with TQ alone was unable to clear the blood stage parasites, but was capable of reducing the mortality

rate, while TQ monotherapy at a high dose of 30 mg/kg was highly effective against the gametocytes and

completely blocked the transmission of P. gallinaceum. In addition, the combination therapy of TQ + ATN

completely cleared P. gallinaceum blood stages and sped up the gametocyte clearance from chickens,

suggesting the synergistic effect of the two drugs. In conclusion, TQ is demonstrated to be effective for

limiting avian malaria transmission and may be used in combination with a low dose of ATN for safe and

effective treatment.

© 2016 Elsevier B.V. All rights reserved.

1. Introduction and sub-tropical regions, mainly in Asia, Africa and South America,

with estimates of 200–300 million clinical cases annually. Life-

Malaria is a -borne disease caused by parasite proto- threatening complications, including coma and death, can develop

zoa in the genus Plasmodium, and is commonly found in a wide during a malaria infection, which frequently occur in children under

range of terrestrial vertebrates, including reptiles, birds and mam- 5 years old and pregnant women (Wassmer et al., 2015). The

mals (Clark et al., 2014; Pattaradilokrat et al., 2015; Schall, 1996; emergence of drug resistant malaria parasites also impedes global

Templeton et al., 2016). Human malaria diseases are considered malaria control and eradication efforts (Antony and Parija, 2016). It

major health problems due to their wide distribution in tropical has been recommended that all existing antimalarial drugs should

be evaluated for their transmission-blocking activities because this

has been advocated as a way for limiting the expansion of drug

resistant parasites (Teklehaimanot et al., 1985; White, 2008). Thus,

Abbreviations: ATM, artemisinin; ATN, artesunate; BW, body weight; CQ, chloro-

the development and screening of new antimalarial drugs with

quine; dpi, days post infection; MTD, maximum tolerant dose; PBS, phosphate

gametocytocidal properties has a high research priority.

buffered saline; PQ, ; TQ, tafenoquine.

∗ Like human malaria diseases, avian malaria in domestic chick-

Corresponding author.

ens (Gallus gallus domesticus) is highly pathogenic (Williams, 2005).

E-mail address: [email protected] (S. Pattaradilokrat).

http://dx.doi.org/10.1016/j.vetpar.2016.12.008

0304-4017/© 2016 Elsevier B.V. All rights reserved.

98 S. Tasai et al. / Veterinary Parasitology 233 (2017) 97–106

The main causative agent, Plasmodium gallinaceum, is prevalent bination therapy of TQ and other antimalarial drugs, such as CQ,

in a number of countries in Southeast and South Asia, where it mefloquine and ATM-lumefantrin, has been extensively tested for

often causes a high mortality rate, and contributes strongly to the prophylactic activities against pre-erythrocytic stages of human

economic loss in the poultry industry. The parasite itself can be with promising results (Dow et al., 2011; Llanos-Cuentas

transmitted by a number of mosquito species, including Aedes sp., et al., 2014; Ramharter et al., 2002). Whereas only a few studies

Anopheles sp. and Culex sp., allowing malaria to spread rapidly to have reported gametocytocidal activities of TQ, there has still been

new areas (Alavi et al., 2003; Pruck-Ngern et al., 2015). The life no report on the gametocytocidal activities of TQ in combination

cycle of P. gallinaceum in domestic chicken is complex and requires with other drugs (Coleman, 1990; Coleman et al., 1992; Ponsa et al.,

two rounds of pre-erythrocytic stage replication in the liver and 2003). It is also not known whether a combined treatment with

endothelial cells. After that, the resulting parasites are released into ATN + TQ will have a synergistic effect. Thus, this study aimed to

the blood vessels and develop inside the erythrocytes. During this evaluate the inhibitory effects of TQ monotherapy and ATN + TQ

erythrocytic stage development, avian malaria shares character- combination therapy on the blood stage development, gametocytes

istics with other malaria species (Macchi Bde et al., 2010; Nagao and transmission of P. gallinaceum to A. aegypti mosquitoes. The

et al., 2008; Paulman and McAllister, 2005), providing an alterna- outcome of the present study would likely be beneficial for design-

tive model for in vivo antimalarial drug tests that could be used to ing an effective treatment for avian malaria and may be applicable

complement human malaria research. for human malaria studies.

Chloroquine (CQ) and are the two most frequently

used drugs for the treatment of avian malaria (Sohsuebngarm et al.,

2. Materials and methods

2014). These drugs have been shown to target the erythrocyte stage

of P. gallinaceum, thereby reducing the morbidity and mortality

2.1. Malaria parasite and laboratory hosts

of the infected chickens. However, the drugs are poorly effective

against the gametocytes, and so they do not affect the rate of

A line of the blood stage P. gallinaceum strain Pg01/2013MUPH

malaria transmission. Identification of antimalarial drugs target-

was used. The parasite isolate originated from an infected chicken

ing both the blood stages and gametocytes would be preferable for

at a local farm in Chachoengsao, Thailand, in 2013 (Pruck-Ngern

long-term malaria control. To address this need, an in vivo transmis-

et al., 2015), and has been maintained by serial blood passage

sion blocking assay has been established, in which P. gallinaceum

and occasionally through mosquito feeding at the Department

is readily employed for screening gametocytocidal compounds

of Parasitology and Entomology, Faculty of Public Health, Mahi-

(Kumnuan et al., 2013). Briefly, the blood stage malarias are grown

dol University, Thailand. The malaria species was confirmed by

in domestic chickens (G. gallus domesticus). Once the parasites have

microscopic examination of Giemsa’s stain thin blood smears and

grown to approximate levels of 10% parasitemia and 1% gameto-

DNA sequencing analysis of the cytochrome oxidase subunit I and

cytemia, the infected chickens are treated with the compound(s)

cytochrome b genes (Pattaradilokrat et al., 2015). Experimental

of interest prior to mosquito feeding. The transmission dynamics

hosts were female domestic chickens. Chickens were allowed to

of the malaria parasites before and after treatment can be carefully

feed on commercial food and water ad labitum and were reared in

monitored. Recent studies demonstrated that a 7-d administration

cages covered with nets to prevent entry of insects. The mosquito

of 10 mg/kg artesunate (ATN) cleared the blood stage parasites and

vectors were female adults A. aegypti, 5–6 d post emergence from

gametocytes and also blocked transmission of the parasites to Aedes

pupae. The adults fed on 10% (w/v) sucrose in water and were

aegypti and Culex quinquefasciatus (Kumnuan et al., 2013; Pruck-

starved for 6 h before use in mosquito feeding experiments. All

Ngern et al., 2015). Hence, this assay has provided a platform for

laboratory animals were maintained in the animal facility in the

the rapid discovery of potential antimalarial drugs for avian and

Department of Pathology, Faculty of Veterinary Science, Chula-

human malaria diseases.

longkorn University, Thailand. The room was lit on a 12 h light:

The key antimalarial drug ATN, a derivative of artemisinin ◦

12 h dark cycle, with a temperature of 25–30 C and relative humid-

(ATM), is employed globally for malaria treatment (Noubiap, 2014),

ity of 40–60%. Animal use protocol was reviewed and approved

but parasites resistant to ATM and its derivatives have now

by the Chulalongkorn University Animal Care and Use Committee

emerged and spread in various malaria endemic areas (Ashley

(Approval No. 1431067).

et al., 2014; Dondorp et al., 2009). Critically, a novel antimalar-

ial drug, or a new drug combination, is needed to overcome this

problem. Tafenoquine (TQ), also known as WR238605 or etaquine, 2.2. In vivo drug test in chickens

is regarded as the most advanced candidate compound. It has

been tested in Phase III clinical trials for treatment of the hyp- Infected blood, with a parasitemia level of 5–10%, was with-

nozoite stages of , which are responsible for the drawn from an infected chicken donor and diluted in an appropriate

relapse of malaria (Campo et al., 2015). Although TQ belongs to the volume of phosphate buffered saline (PBS) to prepare an inocu-

6

group of 8-aminoquinolines, which also includes primaquine (PQ) lum containing 10 blood stages of P. gallinaceum per 100 ␮l. The

(Crockett and Kain, 2007), TQ has a better therapeutic index and inoculum was injected intravenously into a jugular vein of recipient

greater activities against liver stages of Plasmodium than PQ and 3-d old chickens. The blood stage infection was monitored daily by

so has the potential to replace PQ as a prophylactic agent (Li et al., microscopic examination of thin blood films stained with Giemsa’s

2014). In a pre-clinical study, TQ was well tolerated, with only mild, stain according to standard protocols. The numbers of parasitized

transient gastrointestinal side effects in healthy human volunteers erythrocytes, gametocytes and total number of erythrocytes were

(Brueckner et al., 1998a). Within animal models, including humans, counted from at least five microscopic fields (at 1000× magnifi-

TQ has a long half-life compared with that of PQ (Brueckner et al., cation) and the data were expressed as the % parasitemia and%

1998b; Charles et al., 2007; Li et al., 2014). While the greater in vitro gametocytemia, respectively.

potency and longer half-life of TQ may be attributed to its higher To evaluate the efficacy of the TQ treatment, the infected chick-

in vivo efficacy, TQ monotherapy may provide selective pressure ens were divided into four groups with four chickens per group.

for the development of drug resistance in areas with a high level One group (Control) was injected intramuscularly with 50 ␮l of PBS,

of malaria transmission. Accordingly, an appropriate combination while the other groups were injected intramuscularly with 50 ␮l of

of TQ with a more potent and fast acting blood schizontocidal drug TQ (Sigma-Aldrich) in PBS at a dose of 3, 10 and 30 mg/kg body

would yield a more rapidly acting and effective regimen. A com- weight (BW), respectively. The treatments were given once a day

S. Tasai et al. / Veterinary Parasitology 233 (2017) 97–106 99

for 5 d (between 4–8 d post infection, dpi). The levels of the blood until they were fully engorged (around 30 min). The unfed or par-

stage malaria parasites and gametocytes were monitored micro- tially engorged mosquitoes were then removed and the remaining

scopically. It should also be noted that an injection with a single (∼40) mosquitoes were maintained in the insectary for an addi-

dose of 60 or 100 mg/kg BW TQ was toxic and caused animal death, tional 9–10 d before dissection and examination for the presence

and so the efficacy of high TQ doses could not be evaluated in this of P. gallinaceum oocysts in their midguts. The mosquito midguts

study. The maximum tolerance dose (MTD) of TQ was 30 mg/kg BW. were stained with 0.01% (w/v) mercurochrome before examina-

In order to evaluate the efficacy of the ATN + TQ combination tion under a light microscope at 100× magnification. The number of

treatment, the infected chickens were divided into four groups, mosquitoes with an infected midgut and the number of oocysts per

each of four chickens. The control group received 50 ␮l of PBS intra- midgut were recorded. After awakening from the anesthesia, the

muscularly, while the other groups received 50 ␮l of 1 mg/kg BW chickens were maintained for subsequent treatments. Mosquito

ATN, 30 mg/kg BW TQ or a combination of the two drugs at those feedings were performed again on 6, 8 and 16 dpi. The numbers

doses in PBS, respectively, in PBS. All animals were treated for 5 d of infected mosquitoes with oocysts were counted and expressed

between 4–8 dpi and the % parasitemia and % gametocytemia of as the % infectivity.

the infected chickens were recorded. Likewise, to evaluate the efficacy of the ATN + TQ combina-

tion treatment on P. gallinaceum transmission, 16 chickens were

6

infected with 10 blood stage P. gallinaceum and divided into four

2.3. In vivo transmission blocking assay of P. gallinaceum

groups (n = 4). At 4 dpi, the infected chickens were given PBS only

or with 1 mg/kg BW ATN, 30 mg/kg BW TQ, and a combination

The mosquito feeding was performed as previously reported

of 1 mg/kg BW ATN + 30 mg/kg BW TQ, respectively, as described

(Kumnuan et al., 2013; Pruck-Ngern et al., 2015). In brief, 12 chick-

6 above (Section 2.2). Then, 90 min after the respective treatment,

ens were infected with 10 blood stage P. gallinaceum and divided

three chickens were chosen in each group for blood feeding on 4

into three groups (n = 4). To evaluate the inhibitory properties of

dpi. After that, mosquito feedings were performed again on 6, 9, 12

TQ on P. gallinaceum transmission, the infected chickens with the

and 18 dpi (we were unable to perform the mosquito feeding on

desired parasitemia levels at 4 dpi (% parasitemia of 11.8 ± 0.7%

8 and 16 dpi due to technical reasons). The numbers of infected

and% gametocytemia of 1.62 ± 0.68%) were weighed and given PBS

mosquitoes with oocysts were counted and expressed as the %

with 0, 10 or 30 mg/kg BW TQ as described in Section 2.2. Blood

infectivity.

infections were monitored daily (data not shown). The blood feed-

ing was performed 90 min after the treatments. The chickens were

anesthetized by an intramuscular injection with 50 ␮l of Tiletamine 3. Results

HCl/Zolazepam HCl (Zoletil, Virbac, Thailand) at a dose of 10 mg/kg

into the thigh muscle. Once anesthetized, three chickens in each 3.1. Effects of TQ on blood stage infection and gametocyte

group were randomly chosen for mosquito feeding to compromise production of P. gallinaceum

variation in the levels of blood stage parasites and gametocyte

production. They were brought into contact with three cages con- Sixteen chickens were infected with the blood stage of P. gal-

taining 50 female adult A. aegypti mosquitoes each, called R1, R2 linaceum as described in the Materials and Methods. The malaria

and R3. The mosquitoes were then allowed to feed on the chickens parasites were microscopically detected in the blood at 3 dpi, and

Fig. 1. Tafenoquine (TQ) suppresses the blood stage multiplication of Plasmodium gallinaceum in chickens. Data given is the mean % parasitemia of four chickens per group

with error bars indicating the standard errors of the mean (SEM). PBS or TQ treatment was carried out on days 4 to 8 post infection. Letter “d” represents the deaths of two

animals in the control group.

100 S. Tasai et al. / Veterinary Parasitology 233 (2017) 97–106

Fig. 2. Tafenoquine (TQ) inhibits gametocyte production of Plasmodium gallinaceum in chickens. Data given is the mean % gametocytemia of four chickens per group, with

error bars indicating the standard errors of the mean (SEM). PBS or TQ treatment was carried out on days 4 to 8 post infection. Letter “d” represents the deaths of two animals

in the control group.

± ±

at 4 dpi the levels of parasitemia reached 10.6 1.5% (mean SEM) dpi (Fig. 2C and D). Thus, TQ was an effective gametocytocidal drug

parasitemia. The animals were then divided into four groups of four with the low dose of 10 mg/kg BW as the minimum effective dose

chickens per group. For the control group, which received injections against P. gallinaceum gametocytes.

of PBS for 5 d between 4 and 8 dpi, the level of blood stage para-

sites rose rapidly and reached a peak parasitemia of 73.3 ± 3.8% at

3.2. Effects of TQ on the transmission of P. gallinaceum to the

6 dpi before then declining to 5.7 ± 5.0% at 18 dpi (Fig. 1A). In two

vector Aedes aegypti

of the chickens in the control group, the parasitemia level did not

decline but remained steady at 15.9% and 13.8%, respectively, and

As shown in Fig. 3, the P. gallinaceum malaria parasites were

they then died at 17 dpi. This illustrated that P. gallinaceum infec-

highly infectious to A. aegypti mosquitoes. When fed on infected

tion could be lethal to the avian hosts. The other two chickens in

chickens in the control group at 4 dpi, the overall% infectivity of

the control group survived to the study end point (17 dpi).

mosquitoes was 46.7% (range 37.5–55.0%), with a mean oocyst

Treatments as above but with 3, 10 or 30 mg/kg BW TQ sup-

count of 2.85, whereas in the groups treated with 10 and 30 mg/kg

pressed the blood stage infections, resulting in a lower peak

BW TQ, the overall% infectivity of mosquitoes was 45.8% (range

parasitemia of 62.6 ± 5.4%, 53.0 ± 4.5% and 55.1 ± 4.1%, respec-

32.5–60.0%) and 40.8% (range 30–62.5%), respectively, with mean

tively, at 5–6 dpi (Fig. 1B–D). Thereafter, the malaria parasites in

oocyst counts of 3.49 and 3.34, respectively. Thus, the first dose of

the treated groups declined, but were maintained at parasitemia

10 or 30 mg/kg BW TQ did not markedly affect the transmission of

levels of 0.01% or above until 17 dpi. Although the blood stage

P. gallinaceum.

malaria parasites in the treated groups were not completely elim-

Subsequently, the mosquito feeding experiments were repeated

inated, all TQ-treated animals survived the infections. While the

with the infected chickens administrated with three or five doses of

5-d treatment with 30 mg/kg BW TQ was ineffective in clearing the

PBS or TQ, that is also at 6 and 8 dpi. The % infectivity of mosquitoes

blood stage P. gallinaceum parasites, TQ treatments could reduce

remained high in the control group, but mosquitoes that fed on

the parasite load and mortality rate of the infected chickens.

chickens treated with 10 or 30 mg/kg BW TQ showed a very low%

We next evaluated the effects of TQ on the production of P. gal-

infectivity (<5%) and low mean oocyst numbers (<1 oocyst per

linaceum gametocytes. In the control group, the gametocytes were

midgut). Interestingly, only the mosquitoes fed on the chickens

detectable from 3 dpi, reached 1.68 ± 0.72% at 4 dpi and increased

treated with 30 mg/kg BW TQ had no detectable oocysts (at 8 dpi).

to a peak gametocytemia of 7.83 ± 1.11% at 8 dpi before declining

Regardless, the 5-d treatment with 30 but not 10 mg/kg BW TQ

to undetected levels (<0.01% gametocytemia) at 17 dpi (Fig. 2A). In

could completely block the transmission of P. gallinaceum to A.

contrast, the numbers of gametocytes were significantly reduced aegypti.

in the three TQ treatment groups. The peak% gametocytemia of the

In addition, in order to determine the recrudescence of the

chickens treated with 3 mg/kg BW was reduced to 3.78 ± 0.36% at 6

gametocytes in the treated chickens, the mosquito feeding experi-

dpi, declined and then disappeared by 14 dpi (Fig. 2B). In the chick-

ment was performed again with the infected chickens at 16 dpi,

ens treated with the high doses of 10 and 30 mg/kg BW TQ, the

8 d after the complete TQ treatment. Once again, the infected

levels of gametocytes were rapidly reduced to undetectable levels

mosquitoes showed no detectable oocysts when fed on the infected

after administration of four and three consecutive doses (by 8 and

chickens treated with 30 mg/kg BW TQ, but had detectable oocysts

7 dpi), respectively, and remained at undetectable levels until 17

when fed on chickens treated with 10 or 0 mg/kg BW TQ. Thus, the

S. Tasai et al. / Veterinary Parasitology 233 (2017) 97–106 101

Fig. 3. Infectivity of Plasmodium gallinaceum to Aedes aegypti mosquitoes after tafenoquine (TQ) treatment. In each mosquito feeding, three chickens designated as replicate

(R)1, R2 and R3 were used. Red bar indicates the mean number of oocysts per mosquito midgut (n = 40) with error bars indicating SEM. The number above the error bar

indicates the percent infectivity of the mosquitoes.

5-d treatment with 30 mg/kg BW TQ was the only effective regi- by 18 dpi, although one of the chickens in the untreated group

men for the complete inhibition of gametocyte development and died at 18 dpi (Fig. 4A). In contrast, the 5-d treatment with 1 mg/kg

transmission of P. gallinaceum. BW ATN or 30 mg/kg BW TQ was able to suppress the blood stage

growth rate of P. gallinaceum, with a lower peak parasitemia level

± ±

3.3. Effects of the ATN + TQ combination treatment on the P. of 55.2 8.1% and 64.8 5.5%, respectively (Fig. 4B and C). How-

gallinaceum blood stage infection and gametocytes production ever, recrudescence of the parasite occurred in both groups and

the parasites were maintained at low levels (<1%) until the exper-

Given that the TQ monotherapy was poorly effective against imental endpoint (18 dpi). This data confirmed that monotherapy

the blood stages of P. gallinaceum, the effect of a combination of with either a low dose of ATN or a high dose of TQ was ineffective

ATN + TQ treatment on the blood stage multiplication and game- in clearance of the blood stage parasites. The 5-d combined treat-

tocyte production was investigated. A total of 16 chickens were ment with 1 mg/kg BW ATN plus 30 mg/kg BW TQ not only strongly

±

injected with an inoculum of P. gallinaceum blood stages and reduced the peak parasitemia level to 17.0 2.9% but also promoted

were then monitored for signs of infection. At 4 dpi, the mean% the rapid clearance of the blood stage parasites (Fig. 4D), where the

parasitemia and% gametocytemia in the infected chickens were levels of the blood stage parasites in the ATN + TQ treated group

10.8 ± 1.8% and 0.11 ± 0.08%, respectively. The infected chickens were reduced to undetectable levels at 9 dpi and remained unde-

were then divided into four groups of four chickens per group for tectable until the experimental end point. This finding revealed for

treatment with PBS, 1 mg/kg BW ATN, 30 mg/kg BW TQ and a com- the first time the synergistic effect of ATN and TQ in the accelerated

bination of 1 and 30 mg/kg BW ATN and TQ, respectively. The level clearance of P. gallinaceum from the chicken’s blood.

of the blood stage parasites in the control group reached a peak In addition, the analysis of the gametocytes also showed that

parasitemia level of 78.9 ± 1.6% at 6 dpi and then declined to 5% monotherapy treatments with 1 mg/kg BW ATN or 30 mg/kg BW

102 S. Tasai et al. / Veterinary Parasitology 233 (2017) 97–106

Fig. 4. A combination of tafenoquine (TQ) and artesunate (ATN) promotes clearance of the blood stage infection of Plasmodium gallinaceum. Data given is the mean %

parasitemia of four chickens per group with error bars indicating the standard errors of the mean (SEM). PBS or drug treatment was performed between days 4 to 8 post

infection. Letter “d” represents the death of one animal in the control group.

TQ strongly suppressed the peak gametocytemia to 1.60 ± 0.53% with 1 mg/kg BW ATN and 30 mg/kg BW TQ greatly reduced the

and 1.70 ± 0.35%, respectively, compared to that of 7.56 ± 1.02% in peak gametocyte level to 1.20 ± 0.10% at 4 dpi and to undetectable

the control group (Fig. 5A–C). The levels of gametocytes in both levels after two doses of the combination therapy (Fig. 5D). Thus,

treated groups were further reduced to <0.01% and maintained at this combined treatment effectively inhibited the in vivo develop-

this level until the endpoint. Interestingly, the combined treatment

Fig. 5. A combination therapy of tafenoquine (TQ) and artesunate (ATN) abolishes gametocyte production in Plasmodium gallinaceum. Data given is the mean % gametocytemia

of four chickens per group with error bars indicating the standard errors of the mean (SEM). PBS or drug treatment was performed between days 4 to 8 post infection. Letter

“d” represents the deaths of two animals in the control group.

S. Tasai et al. / Veterinary Parasitology 233 (2017) 97–106 103

ment of the gametocytes, resulting in their rapid clearance from the infectivity of P. gallinaceum in the untreated group was dramat-

the blood circulation. ically decreased but maintained at low levels at the late infection

stage (18 dpi). The 5-d treatment with 1 mg/kg BW ATN strongly

reduced the number of gametocytes and also suppressed the %

3.4. Effects of the ATN + TQ combination treatment on the

infectivity to zero, but around 2.5% of the mosquitoes in one of the

transmission of P. gallinaceum to A. aegypti

three batches that fed on the treated chickens at 18 dpi (10 d after

the complete ATN treatment) had oocysts in their midgut (Fig. 6B).

Fig. 6 shows that at least 62.5% (range 62.5%–100%) of the

In contrast, the 5-d treatment with TQ alone or TQ in combina-

mosquitoes were infected with P. gallinaceum when allowed to feed

tion with ATN blocked the oocyst development completely, since

on the untreated chickens between 4, 6 and 9 dpi (Fig. 6A). Later,

Fig. 6. A combination therapy of tafenoquine (TQ) and artesunate (ATN) blocks transmission of Plasmodium gallinaceum to vectors. In each mosquito feeding, three chickens

designated as replicate (R)1, R2 and R3 were used. Red bar indicates the mean number of oocysts per mosquito midgut (n = 40) with error bars indicating SEM. The number

above the error bar indicates the % infectivity of the mosquitoes.

104 S. Tasai et al. / Veterinary Parasitology 233 (2017) 97–106

no oocysts were detected in any mosquitoes until the experimen- could be used as a combination therapy for malaria treatment and

tal endpoint (Fig. 6C and D). It should be noted that the complete prevention of malaria transmission. In addition to ATN, in vitro stud-

removal of the oocysts occurred after the chickens received five ies have also demonstrated a synergism of blood schizontocidal

doses of TQ alone but after as few as three doses of the ATN + TQ activity between CQ and ATM (Ramharter et al., 2002; Vollnberg

combination. Therefore, these results support the view that a com- et al., 2003). It would be of interest to investigate the action of

bination of ATN and TQ had a synergistic effect on inhibiting the TQ + CQ and TQ + ATM combinations on gametocytes and sporo-

development of blood stage malaria parasites and gametocytes and gonic development of the malaria parasites, and this will require

blocking the transmission of P. gallinaceum parasites to its insect further investigation.

hosts. To date, PQ and ATN are the few antimalarial drugs with the

capacity to interrupt transmission of P. falciparum (Abay, 2013).

Although widespread resistance to PQ has not been reported,

4. Discussion laboratory culture resistance to PQ has been induced in the

human malaria parasite P. vivax (Fernando et al., 2011), the avian

The first goal of the present study was to determine the effi- malaria P. gallinaceum (Bishop, 1967) and rodent malaria parasites

cacy of TQ against blood stages and gametocytes of P. gallinaceum (Merkli and Peters, 1976). Furthermore, resistance to ATN has been

and its transmission to the mosquito vector A. aegypti. Treatment reported widely in field populations in Southeast Asia (Ashley et al.,

with the MTD of TQ (30 mg/kg BW), which was safe and tolera- 2014). It would be of interest in testing the efficacy of the ATN + TQ

ble to chickens, could potentially markedly reduce the mortality combination on the drug resistant parasites. Should the combina-

rate of P. gallinaceum infected chickens. However, TQ monother- tion treatment prove to be effective, it would imply that TQ could

apy at the MTD dose was inefficient in clearing the blood stage be used in combination with ATN to replace a standard treatment

infections. It has previously been shown that 10 mg/kg BW ATN (PQ or ATN monotherapy) for the treatment of multidrug resistance

exerted a marked blood schizontocidal activity in P. gallinaceum malaria.

(Kumnuan et al., 2013), and so the blood schizontocidal activity So far little is known about the blood schizontocidal and game-

of TQ was lower than that of ATN. This is in agreement with an tocytocidal modes of action of TQ. Like PQ, TQ has a quinoline

in vitro study in which the blood schizontocidal activities of TQ core structure, but it also has a 5-substituted phenylether group

and ATM were compared in 43 clinical isolates of P. falciparum. The that helps protect the core from oxidation, thereby improving the

mean effective concentration at 50% effect (EC50) of TQ and ATM metabolic stability of TQ. Due to the structural similarity between

were 209 and 12.1 nmol/L, respectively, (Ramharter et al., 2002). TQ and PQ, there is the possibility that both compounds may share

Therefore, these results indicate that TQ monotherapy was less the same mechanism of action, at least in part (Campo et al.,

effective in eliminating blood stage parasites than ATN monother- 2015). The antiplasmodial activity of PQ has been attributed to

apy. its competition with ubiquinone, an essential component in the

However, the TQ monotherapy at 30 mg/kg BW was highly mitochondria transport chain (Lanners, 1991; Tekwani and Walker,

effective in blocking the development of the gametocytes, which 2006). Likewise, TQ was shown to impair the metabolic functions

in turn led to the complete blockage of the malarial transmis- of the mitochondria of Leishmania promastigotes, causing a rapid

sion to the mosquitoes. This result clearly demonstrated that TQ drop in intracellular ATP levels and inducing apoptosis (Carvalho

had strong gametocytocidal and sporontogonic activities. Simi- et al., 2010). It was reported that the liver cytochrome P450 (CYP)

lar results were observed in an in vivo study using the rodent 2D6 enzyme metabolize TQ to generate active metabolites (Vuong

malaria parasite P. berghei strain ANKA, in which treatment with et al., 2015), since TQ showed an excellent efficacy in preventing P.

25 mg/kg BW TQ showed strong inhibitory effects against the game- berghei liver stage development in wild type mice, but not in CYP2D

tocytes and oocyst formation of P. berghei (Coleman et al., 1992). knockout mice. While TQ is reported to undergo C-oxidation on the

Furthermore, evaluation of the in vivo efficacy of TQ against the 8-amino alkyl chain, the overall metabolic products of TQ remain

human malaria parasite, P. vivax, infected mosquitoes was per- unexplored (Crockett and Kain, 2007). Metabolites of TQ are also

formed in mice treated with a single dose of 25 mg/kg BW TQ. thought to kill blood schizonts through their rapid accumulation

The TQ treatment not only affected the oocyst production of P. in erythrocytes (Edstein et al., 2001; McCarthy and Price, 2014).

vivax but also reduced the number of sporozoites in the salivary High levels of TQ act to inhibit hematin polymerization, thereby

glands of the mosquitoes (Ponsa et al., 2003). Altogether, these interrupting the conversion of toxic heme to non-toxic hemozoin.

findings consistently showed that TQ is an antimalarial drug with However, because the detoxification of heme to hemozoin does

a potent gametocytocidal activity and could be used to interrupt not occur in the gametocytes, it is highly likely that TQ may target

sporogonic development of the malaria parasites to mosquito vec- the gametocytes via an alternative pathway. Alternatively, the pro-

tors. oxidant properties of TQ metabolites may contribute to its action.

In order to apply TQ for the treatment of chickens in the field, In the pneumonia-causing fungus Pneumocystis carinii, TQ caused

it is necessary to combine TQ with other antimalarial drugs to ultra structural damages to the cell membrane, suggesting that the

promote absolute clearance of the blood stage malaria parasites. oxidative impact may be a major mechanism of action (Goheen

Because ATN has a more potent and fast acting blood schizon- et al., 1993). Having shown here that P. gallinaceum gametocytes

tocidal activity against P. gallinaceum, the combined treatment can be targeted by TQ, this may provide an alternative model to

of ATN + TQ would have the potential to yield a more effective elucidate the mechanism of action of TQ.

regimen. Thus, the next goal of this study was to determine the In conclusion, this study showed that TQ monotherapy was

effects of an ATN + TQ combination therapy on the blood stage effective against the gametocytes and sporogonic stages of P. gal-

parasites, gametocytes and transmission of P. gallinaceum. A com- linacaeum, although it is not recommended for the treatment of

bination of a low dose (1 mg/kg BW) of ATN with a MTD dose of blood stage infections of the avian malaria. This study demon-

TQ (30 mg/kg BW) was more effective in eliminating the blood strated for the first time that the TQ plus ATN combination

stage P. gallinaceum parasites than treatments with ATN or TQ treatment was synergistic and could promote the clearance of the

alone. Furthermore, it was noted that fewer doses of the ATN + TQ blood stage P. gallinaceum, elimination of gametocytes and block-

combined treatment were required for complete removal of game- ing the transmission of P. gallinaceum to the A. aegypti vector. This

tocytes and blockage of the malaria transmission to A. aegypti. provides a new regimen for treatment of avian malaria that could

These results demonstrated that TQ and ATN were synergistic and be applicable for malaria control in the field.

S. Tasai et al. / Veterinary Parasitology 233 (2017) 97–106 105

Authors’ contributions Crockett, M., Kain, K.C., 2007. Tafenoquine: a promising new antimalarial agent.

Expert Opin. Investig. Drugs 16, 705–715.

Dondorp, A.M., Nosten, F., Yi, P., Das, D., Phyo, A.P., Tarning, J., Lwin, K.M., Ariey, F.,

SuT, TS and MK performed the in vivo transmission blocking

Hanpithakpong, W., Lee, S.J., Ringwald, P., Silamut, K., Imwong, M.,

assay. TS was responsible for parasite maintenance. SoT, PB and SC Chotivanich, K., Lim, P., Herdman, T., An, S.S., Yeung, S., Singhasivanon, P., Day,

N.P.J., Lindegardh, N., Socheat, D., White, N.J., 2009. Artemisinin resistance in

provided guidance for data interpretation. SP provided conceptual

Plasmodium falciparum malaria. New Engl. J. Med. 361, 455–467.

framework for the project, guidance for the interpretation of data,

Dow, G.S., Gettayacamin, M., Hansukjariya, P., Imerbsin, R., Komcharoen, S.,

participated in the manuscript preparation, revision and coordina- Sattabongkot, J., Kyle, D., Milhous, W., Cozens, S., Kenworthy, D., Miller, A.,

tion. Veazey, J., Ohrt, C., 2011. Radical curative efficacy of tafenoquine combination

regimens in Plasmodium cynomolgi-infected Rhesus monkeys (Macaca

mulatta). Malar. J. 10, 212.

Competing interests Edstein, M.D., Kocisko, D.A., Brewer, T.G., Walsh, D.S., Eamsila, C., Charles, B.G.,

2001. Population pharmacokinetics of the new antimalarial agent tafenoquine

in Thai soldiers. Br. J. Clin. Pharmacol. 52, 663–670.

The authors declare that there are no competing interests.

Fernando, D., Rodrigo, C., Rajapakse, S., 2011. Primaquine in vivax malaria: an

update and review on management issues. Malar. J. 10, 351.

Acknowledgements Goheen, M.P., Bartlett, M.S., Shaw, M.M., Queener, S.F., Smith, J.W., 1993. Effects of

8-aminoquinolines on the ultrastructural morphology of Pneumocystis carinii.

Int. J. Exp. Pathol. 74, 379–387.

We are grateful to Dr. Robert Butcher for editing. This research Kumnuan, R., Pattaradilokrat, S., Chumpolbanchorn, K., Pimnon, S., Narkpinit, S.,

Harnyuttanakorn, P., Saiwichai, T., 2013. In vivo transmission blocking

was supported by a grant from the Thailand Research Fund (Grant

activities of artesunate on the avian malaria parasite Plasmodium gallinaceum.

number MRG-5680134) and a Research Grant for Development and

Vet. Parasitol. 197, 447–454.

Promotion of Science and Technology Talents Project (DPST) grad- Lanners, H.N., 1991. Effect of the 8-aminoquinoline primaquine on culture-derived

gametocytes of the malaria parasite Plasmodium falciparum. Parasitol. Res. 77,

uate with First Placement, Royal Thai Government.

478–481.

Li, Q., O’Neil, M., Xie, L., Caridha, D., Zeng, Q., Zhang, J., Pybus, B., Hickman, M.,

References Melendez, V., 2014. Assessment of the prophylactic activity and

pharmacokinetic profile of oral tafenoquine compared to primaquine for

inhibition of liver stage malaria infections. Malar. J. 13, 141.

Abay, S.M., 2013. Blocking malaria transmission to Anopheles mosquitoes using

Llanos-Cuentas, A., Lacerda, M.V., Rueangweerayut, R., Krudsood, S., Gupta, S.K.,

artemisinin derivatives and primaquine: a systematic review and

Kochar, S.K., Arthur, P., Chuenchom, N., Mohrle, J.J., Duparc, S., Ugwuegbulam,

meta-analysis. Parasit Vectors 6, 278.

C., Kleim, J.P., Carter, N., Green, J.A., Kellam, L., 2014. Tafenoquine plus

Alavi, Y., Arai, M., Mendoza, J., Tufet-Bayona, M., Sinha, R., Fowler, K., Billker, O.,

for the treatment and relapse prevention of Plasmodium vivax

Franke-Fayard, B., Janse, C.J., Waters, A., Sinden, R.E., 2003. The dynamics of

malaria (DETECTIVE): a multicentre, double-blind, randomised, phase 2b

interactions between Plasmodium and the mosquito: a study of the infectivity

dose-selection study. Lancet 383, 1049–1058.

of Plasmodium berghei and Plasmodium gallinaceum, and their transmission by

Macchi Bde, M., Quaresma, J.A., Herculano, A.M., Crespo-Lopez, M.E., DaMatta, R.A.,

Anopheles stephensi, Anopheles gambiae and Aedes aegypti. Int. J. Parasitol. 33,

933–943. do Nascimento, J.L., 2010. Pathogenic action of Plasmodium gallinaceum in

chickens: brain histology and nitric oxide production by blood

Antony, H.A., Parija, S.C., 2016. Antimalarial drug resistance: an overview. Trop.

monocyte-derived macrophages. Vet. Parasitol. 172, 16–22.

Parasitol. 6, 30–41.

McCarthy, J.S., Price, R.N., 2014. Antimalarial drugs. In: Bennett, J.E., Dolin, R.,

Ashley, E.A., Dhorda, M., Fairhurst, R.M., Amaratunga, C., Lim, P., Suon, S., Sreng, S.,

Blaser, M.J. (Eds.), Mandell, Douglas and Bennett’s Principles and Practice of

Anderson, J.M., Mao, S., Sam, B., Sopha, C., Chuor, C.M., Nguon, C., Sovannaroth,

Infectious Diseases. , 8th Edition. Elsevier Saunders, Philadelphia, pp. 495–509.

S., Pukrittayakamee, S., Jittamala, P., Chotivanich, K., Chutasmit, K.,

Merkli, B., Peters, W., 1976. A comparison of two different methods for the

Suchatsoonthorn, C., Runcharoen, R., Hien, T.T., Thuy-Nhien, N.T., Thanh, N.V.,

selection of primaquine resistance in Plasmodium berghei. Ann. Ann. Trop. Med.

Phu, N.H., Htut, Y., Han, K.T., Aye, K.H., Mokuolu, O.A., Olaosebikan, R.R.,

Parasitol. 70, 473–474.

Folaranmi, O.O., Mayxay, M., Khanthavong, M., Hongvanthong, B., Newton,

Nagao, E., Arie, T., Dorward, D.W., Fairhurst, R.M., Dvorak, J.A., 2008. The avian

P.N., Onyamboko, M.A., Fanello, C.I., Tshefu, A.K., Mishra, N., Valecha, N., Phyo,

malaria parasite Plasmodium gallinaceum causes marked structural changes on

A.P., Nosten, F., Yi, P., Tripura, R., Borrmann, S., Bashraheil, M., Peshu, J., Faiz,

the surface of its host erythrocyte. J. Struct. Biol. 162, 460–467.

M.A., Ghose, A., Hossain, M.A., Samad, R., Rahman, M.R., Hasan, M.M., Islam, A.,

Noubiap, J.J., 2014. Shifting from to artesunate as first-line treatment of

Miotto, O., Amato, R., MacInnis, B., Stalker, J., Kwiatkowski, D.P., Bozdech, Z.,

severe malaria in children and adults: saving more lives. J. Infect. Public Health

Jeeyapant, A., Cheah, P.Y., Sakulthaew, T., Chalk, J., Intharabut, B., Silamut, K.,

7, 407–412.

Lee, S.J., Vihokhern, B., Kunasol, C., Imwong, M., Tarning, J., Taylor, W.J., Yeung,

Pattaradilokrat, S., Tiyamanee, W., Simpalipan, P., Kaewthamasorn, M., Saiwichai,

S., Woodrow, C.J., Flegg, J.A., Das, D., Smith, J., Venkatesan, M., Plowe, C.V.,

T., Li, J., Harnyuttanakorn, P., 2015. Molecular detection of the avian malaria

Stepniewska, K., Guerin, P.J., Dondorp, A.M., Day, N.P., White, N.J., 2014. Spread

parasite Plasmodium gallinaceum in Thailand. Vet. Parasitol. 210, 1–9.

of artemisinin resistance in Plasmodium falciparum malaria. New Engl. J. Med.

Paulman, A., McAllister, M.M., 2005. Plasmodium gallinaceum: clinical

371, 411–423.

progression, recovery, and resistance to disease in chickens infected via

Bishop, A., 1967. Resistance to primaquine in Plasmodium gallinaceum, and the

mosquito bite. Am. J. Trop. Med. Hyg. 73, 1104–1107.

problem of resistance to quinoline compounds in malaria parasites.

Ponsa, N., Sattabongkot, J., Kittayapong, P., Eikarat, N., Coleman, R.E., 2003.

Parasitology 57, 755–770.

Transmission-blocking activity of tafenoquine (WR-238605) and artelinic acid

Brueckner, R.P., Coster, T., Wesche, D.L., Shmuklarsky, M., Schuster, B.G., 1998a.

against naturally circulating strains of Plasmodium vivax in Thailand. Am. J.

Prophylaxis of Plasmodium falciparum infection in a human challenge model

Trop. Med. Hyg. 69, 542–547.

with WR 238605, a new 8-aminoquinoline antimalarial. Antimicrob. Agents

Pruck-Ngern, M., Pattaradilokrat, S., Chumpolbanchorn, K., Pimnon, S., Narkpinit,

Chemother. 42, 1293–1294.

S., Harnyuttanakorn, P., Buddhirakkul, P., Saiwichai, T., 2015. Effects of

Brueckner, R.P., Lasseter, K.C., Lin, E.T., Schuster, B.G., 1998b. First-time-in-humans

artesunate treatment on Plasmodium gallinaceum transmission in the vectors

safety and pharmacokinetics of WR 238605, a new antimalarial. Am. J. Trop.

Aedes aegypti and Culex quinquefasciatus. Vet. Parasitol. 207, 161–165.

Med. Hyg. 58, 645–649.

Ramharter, M., Noedl, H., Thimasarn, K., Wiedermann, G., Wernsdorfer, G.,

Campo, B., Vandal, O., Wesche, D., Wells, T.N., Burrows, J.N., 2015. Killing the

Wernsdorfer, W.H., 2002. In vitro activity of tafenoquine alone and in

hypnozoite: drug discovery approaches to prevent relapse in Plasmodium

combination with artemisinin against Plasmodium falciparum. Am. J. Trop. Med.

vivax. Pathog. Glob. Health 109, 107–122.

Hyg. 67, 39–43.

Carvalho, L., Luque-Ortega, J.R., Manzano, J.I., Castanys, S., Rivas, L., Gamarro, F.,

Schall, J.J., 1996. Malarial parasites of lizards: diversity and ecology. Adv. Parasitol.

2010. Tafenoquine, an antiplasmodial 8-aminoquinoline, targets leishmania

37, 255–333.

respiratory complex III and induces apoptosis. Antimicrob. Agents Chemother.

Sohsuebngarm, D., Sasipreeyajan, J., Nithiuthai, S., Chansiripornchai, N., 2014. The

54, 5344–5351.

efficacy of artesunate, chloroquine, doxycycline, primaquine and a

Charles, B.G., Miller, A.K., Nasveld, P.E., Reid, M.G., Harris, I.E., Edstein, M.D., 2007.

combination of artesunate and primaquine against avian malaria in broilers. J.

Population pharmacokinetics of tafenoquine during in

Vet. Med. Sci. 76, 813–817.

healthy subjects. Antimicrob. Agents Chemother. 51, 2709–2715.

Teklehaimanot, A., Nguyen-Dinh, P., Collins, W.E., Barber, A.M., Campbell, C.C.,

Clark, N.J., Clegg, S.M., Lima, M.R., 2014. A review of global diversity in avian

1985. Evaluation of sporontocidal compounds using Plasmodium falciparum

haemosporidians (Plasmodium and : ): new

gametocytes produced in vitro. Am. J. Trop. Med. Hyg. 34, 429–434.

insights from molecular data. Int. J. Parasitol. 44, 329–338.

Tekwani, B.L., Walker, L.A., 2006. 8-Aminoquinolines: future role as

Coleman, R.E., Clavin, A.M., Milhous, W.K., 1992. Gametocytocidal and

drugs. Curr. Opin. Infect. Dis. 19, 623–631.

sporontocidal activity of antimalarials against Plasmodium berghei ANKA in ICR

Templeton, T.J., Asada, M., Jiratanh, M., Ishikawa, S.A., Tiawsirisup, S., Sivakumar,

mice and Anopheles stephensi mosquitoes. Am. J. Trop. Med. Hyg. 46, 169–182.

T., Namangala, B., Takeda, M., Mohkaew, K., Ngamjituea, S., Inoue, N.,

Coleman, R.E., 1990. Sporontocidal activity of the antimalarial WR-238605 against

Sugimoto, C., Inagaki, Y., Suzuki, Y., Yokoyama, N., Kaewthamasorn, M.,

Plasmodium berghei ANKA in Anopheles stephensi. Am. J. Trop. Med. Hyg. 42,

196–205. Kaneko, O., 2016. Ungulate malaria parasites. Sci. Rep. 6, 23230.

106 S. Tasai et al. / Veterinary Parasitology 233 (2017) 97–106

Vollnberg, A., Prajakwong, S., Sirichaisinthop, J., Wiedermann, G., Wernsdorfer, G., Wassmer, S.C., Taylor, T.E., Rathod, P.K., Mishra, S.K., Mohanty, S., Arevalo-Herrera,

Wernsdorfer, W.H., 2003. In-vitro interaction of tafenoquine and chloroquine M., Duraisingh, M.T., Smith, J.D., 2015. Investigating the pathogenesis of severe

in Plasmodium falciparum from northwestern Thailand. Wien. Klin. malaria: a multidisciplinary and cross-geographical approach. Am. J. Trop.

Wochenschr. 115, 28–32. Med. Hyg. 93, 42–56.

Vuong, C., Xie, L.H., Potter, B.M., Zhang, J., Zhang, P., Duan, D., Nolan, C.K., Sciotti, White, N.J., 2008. The role of anti-malarial drugs in eliminating malaria. Malar. J. 7,

R.J., Zottig, V.E., Nanayakkara, N.P., Tekwani, B.L., Walker, L.A., Smith, P.L., Paris, S8.

R.M., Read, L.T., Li, Q., Pybus, B.S., Sousa, J.C., Reichard, G.A., Smith, B., Marcsisin, Williams, R.B., 2005. Avian malaria: clinical and chemical pathology of Plasmodium

S.R., 2015. Differential cytochrome P450 2D metabolism alters tafenoquine gallinaceum in the domesticated fowl Gallus gallus. Avian Pathol. 34, 29–47.

pharmacokinetics. Antimicrob. Agents Chemother. 59, 3864–3869.