1. Introduction
Human babesiosis is an emerging tick-born disease caused by members of the genus
Babesia, which are similar to
Plasmodium parasites that cause malaria as both of them are intraerythrocytic protozoan parasites.
Babesia divergens,
Babesia microti, and
Babesia duncani cause the most human babesiosis cases worldwide [
1]. Babesiosis is mostly transmitted by tick bite and also by blood transfusion and organ transplantation [
2,
3]. The clinical symptoms of babesiosis patients can range from flu-like mild to malaria-like severe symptoms, and patients who undergo immune disease (e.g., acquired immune deficiency syndrome (AIDS)), immunosuppressive therapies, and splenectomy can suffer more severe manifestations, and even death [
1]. To date, cases of human babesiosis in the USA have been increasing in the past two decades, and babesiosis was added to the list of nationally notifiable diseases by the Centers for Disease Control and Prevention (CDC) in 2011 [
4].
In the United States,
B. microti and
B. duncani are commonly recognized and identified in humans. Even though
B. duncani was firstly described in Washington State as strain WA1 in 1991 [
5], a total of 1119 cases infected by
B. duncani were identified in the U.S. and Canada during 2011–2017 [
6], indicating that
B. duncani had been detected and diagnosed widely in North America. Although intraerythrocytic stages of
B. duncani are morphologically indistinguishable from
B. microti, based on the phylogenetic analysis (
18S RNA gene,
ITS2 gene,
Cyt b gene, and
Cox I gene)
B. duncani n. sp. are in a distinct clade separated from
B. microti [
7,
8]. According to the treatment experience of physicians,
B. duncani was harder to treat than
B. microti, and patients typically required longer anti-babesiosis treatment [
6]. Moreover, in animal models,
B. duncani can give rise to more severe clinical and hematological presentation and then eventually death in hamsters and mice [
9].
To date, drugs and drug combinations are still the predominant weapons to treat human babesiosis due to the absence of vaccines. The current treatments for babesiosis are drug combinations of atovaquone plus azithromycin or clindamycin plus quinine [
10]. In some cases, babesia infections can be refractory, and recrudescence of infection may occur after therapy, even in persistence for more than two years [
11,
12]. Furthermore, clinical studies revealed that both treatment regimens are associated with significant adverse effects, and in the group treated with clindamycin + quinine, a much higher percentage suffered adverse effects than those who were treated with atovaquone + azithromycin (72% vs. 15%) [
9]. These deficiencies emphasize the need for more effective therapies that target persistence while having fewer side effects.
B. duncani culture can be established continuously and long-term ex vivo in hamster or human erythrocytes [
1,
13,
14], whereas only short-term culture can be achieved with
B. microti. Hence,
B. duncani is an appropriate species of choice for convenient drug screens for developing more effective human babesiosis treatment. However, there is no literature that reported results of drug screens on
B. duncani in vitro or in vivo so far. Furthermore, Abraham et al. (2018) revealed that, in human erythrocytes,
B. duncani showed high tolerance to routine therapies [
14]. Thus, it is necessary to develop new therapeutic options for human babesiosis.
Essential oils containing volatile chemical compounds extracted from plants are widely used in aromatherapy, food reservation, and also potentially in medical therapy, especially with recent concerns about antibiotic resistance [
1,
15,
16]. Certain essential oils have been discovered that have antibacterial activity against multidrug-resistant Gram-negative clinical isolates [
17]. In our previous studies, we have identified many essential oils with good activity against
Borrelia burgdorferi and
Bartonella henselae, which usually can cause co-infections with
Babesia sp. through tick transmission [
18,
19]. In this study, we screened a panel of 97 essential oils for activity against
B. duncani and identified some promising active hits. We showed that some active hits in combination with other drugs to be more potent than the current treatment for human babesiosis.
3. Discussion
Babesiosis has been classified as a nationally notifiable disease since 2011 and is recognized as an emerging health risk in several parts of the world [
21]. The actual number of cases caused by
B. microti and
B. duncani are supposed to be much greater because many are undetected and unreported, and some
B. duncani cases will be in the asymptomatic phase [
6,
22]. However, the current treatment options using atovaquone, azithromycin, clindamycin, quinine, and their combinations for human babesiosis are suboptimal as they were suggested based on their antimalarial activity, and these regimens are associated with significant side effects and treatment failures. On the other hand, no specific treatment has been proposed for babesiosis caused by
B. duncani, even though
B. duncani has been proved to have many unique characteristics in phylogeny, animal model, and clinical manifestations [
6,
7,
9]. Thus, it is necessary to find more effective and specific treatments for
B. duncani-related babesiosis. Our study demonstrated that from a screening of 97 essential oils, 10 essential oils (garlic, black pepper, tarragon, palo santo, coconut, pine, meditation, cajeput, moringa, and stress relief) had good activity in vitro against
B. duncani growth in vitro. In particular, garlic oil and black pepper oil, showed remarkably high activity at much lower concentrations than those reported for most essential oils in terms of their antibacterial activity [
23,
24].
Both garlic oil and black pepper oil are traditional diet food supplements across the world. Garlic has been used in herbal medicine for thousands of years, and fresh garlic has been proved to have wide spectrum of antimicrobial activities against many bacteria, fungi, and viruses [
25,
26,
27,
28]. In this study, garlic oil was the top hit among 97 essential oils against the growth of
B. duncani at a concentration that was extremely lower than that against bacterial growth in previous reports [
29,
30], but comparable to that of allicin against other parasites, such as
Plasmodium and
Entamoeba [
31,
32]. Salama et al. indicated that allicin had inhibitory effect in vitro on
Babesia bovis,
Babesia bigemina, and
Babesia caballi, but the IC
50 values of allicin were quite high (470–818 μM) [
33]. This indicated that allicin might not be the most active anti-
Babesia constituent in garlic. Allicin is the compound that contributes to antibacterial activity in crushed garlic clove, a part of unstable allicin yielded by self-condensation of sulfenic acid molecules then transformed into diallyl sulfide (DAS), DADS, and diallyl trisulfide (DATS) [
34]. DADS and DATS are the major constituents of garlic oil, and DADS is reported to comprise about 60% of garlic oil [
26]. In our study, DADS showed similar considerable inhibitory effect as garlic oil, at a slightly higher IC
50 value than that of garlic oil (0.00084% vs. 0.00030%) and may attribute to the activity of other sulfides (DAS and DATS) contained in garlic oil as well. Previous studies have reported that black pepper essential oil and its major compounds terpenoids had potential antibacterial activity [
35,
36]. To our best knowledge, the antiparasite activity of black pepper identified in this study was not previously reported. A study indicated that black pepper extract showed good inhibitory activity against
Blastocystis hominis in vitro at the concentration of 100 μg/mL [
37]. In this study, we found black pepper essential oil had an in vitro IC
50 value of 0.00075% concentration against
B. duncani, and the value of its major terpenoid BCP was 0.0014% (13 μg/mL). It demonstrated that BCP as an active continent of black pepper essential oil has incredible inhibitory efficacy against
B. duncani and may also explain the inhibitory efficacy of other essential oils including cloves, oregano, rosemary, and ylang ylang, which contain BCP, in our screening.
Previous clinical study indicated
Babesia infections can be refractory to drug treatment, and recrudescence or relapse of infection may occur after current treatment and can persist even for more than two years [
11,
12]. In an immunodeficient mouse model, Lawres et al. have proved that current treatment with azithromycin up to 50 mg/kg, clindamycin up to 50 mg/kg, or quinine up to 100 mg/kg had no significant effect on
B. microti, only atovaquone showed potent activity against
B. microti during treatment period, but recrudescence appeared in a few days after treatment [
38]. Atovaquone also showed potent activity against
B. bovis (18 nM; IC
50) and
B. divergens (32 nM; IC
50) [
39]. However, in a human erythrocyte culture system, Abraham et al. have reported that
B. duncani revealed unusually high tolerance to the current recommended therapies [
14]. These results are consistent with our findings with
B. duncani, which demonstrated that atovaquone had incredible inhibitory effect at relatively low concentration (0.25 μM), but relapse was observed in subculture test after up to 20 μM atovaquone exposure for 72 h. We also confirmed the better killing effect of
B. duncani by atovaquone plus azithromycin combination than their monotreatment, but relapse could still transiently arise when they were combined in high dose for unclear reasons. In contrast, in this study
B. duncani did not regrow after garlic oil and DADS treatment at concentrations that did not lyse host cells, except for the concentrations of the 1× IC
50 values. In the combination tests, the killing activity of atovaquone plus azithromycin can be improved when one of them was replaced with garlic oil or DADS. Interestingly, as a pure compound, DADS showed better eradication effect than garlic oil, whether used singly or combined with atovaquone or with azithromycin. Our results imply that in light of the good anti-
B. duncani activity of DADS, garlic oil is a promising alternative to treat human babesiosis when used alone or in combination with azithromycin, considering resistance to atovaquone may emerge as it has been emerging rapidly in malaria treatment.
An important finding of this study is that both garlic oil and DADS plus azithromycin at doses as low as 1× their IC
50 value resulted in total eradication with no relapse, compared with monotreatment of atovaquone or azithromycin and their combination. Treatment failure of antimicrobial drugs is a serious global health threat, and the non-replicating but viable bacterial cells with drug tolerance were firstly termed “persisters” by Joseph Bigger in 1944 [
40]. Besides bacteria, eukaryotic cells such as fungi and parasites can also enter into this dormant phase of drug-tolerant persister-like phenotype [
41,
42]. Malaria may recrudesce years after completion of therapy because of the persister-like cells hypnozoites of
Plasmodium ovale or
Plasmodium vivax hiding in the liver, or small non-replicating ring-stage parasites of
Plasmodium falciparum persisting in erythrocytes induced by artemisinin derivatives during treatment [
43,
44]. For
Babesia, no such extraerythrocytic stage has been described, however, we also found many “parasite dots” of
B. duncani with reduced cytoplasm and condensed nucleus under microscope after atovaquone exposure, and merozoite and tetrad form was hard to find (
Figure 2B). These dormant persister-like parasites may explain the recrudescence of
B. duncani even after high-dose atovaquone treatment. In contrast, after the exposure in garlic oil or DADS, the morphology of
B. duncani did not show significant difference from that of the control group, as both merozoites and trophozoites were clearly observed (
Figure 2D,F). DADS and other garlic-derived sulfur compounds have been demonstrated to have anticancer effects in vitro and in vivo with some potential mechanisms including inducing cell cycle arrest, growth inhibition, differentiation, and apoptosis [
45]. Because the
Babesia parasite is a eukaryotic cell that also has these cellular processes as a cancer cell, they may share equivalent response mechanisms to DADS and other garlic-derived sulfur compounds. Strong evidence is needed in the future to explain why
B. duncani was extremely susceptible to garlic oil and DADS. However, it is worth noting that both garlic oil and DADS even at the low concentration studied in our current culture system can significantly reduce the lifespan of hamster erythrocytes in vitro, whether the adverse effect occurs in vivo needs to be addressed in future. In addition, longer subculture study is hampered by the limited lifespan of erythrocytes in this culture system if no fresh erythrocytes are added, indicating that a more effective and convenient approach for determining the viability of
Babesia needs to be developed.
It has been reported that garlic oil presented direct toxic effects in high dose in mouse and rat models [
46]. The median lethal dose (LD
50) of garlic extract is higher than 30 mg/kg in rodents [
47]. DADS is toxic at the dose of 400 mg/kg but well tolerated at the dose of 200 mg/kg in rats [
48]. In humans, the reported maximum of tolerated dose of garlic extract was 25 mL [
49]. In vitro experiment indicated that active oxygen generated by these sulfides derived from garlic and onions could cause oxidative damage to erythrocytes [
50]. Thus, it is necessary to determine whether the anti-babesiosis therapeutic effect presents without side effect in vivo. However, Hiroyuki et al. demonstrated that DADS administrated by intraperitoneal injection at the dose of 1 or 2 mg three times a week was effective in inhibiting the growth of breast cancer cells without serious side effects [
37]. BCP is generally recognized as safe (GRAS) by the Flavor and Extract Manufacturers Association, and a 90 day oral gavage study at the dose up to 700 mg/kg/day in rats confirmed the safety of BCP used in medical foods [
51]. Interestingly, both garlic oil and BCP could form inclusion complexes with β-cyclodextrin to improve their physicochemical stability and bioavailability [
52,
53]. Future studies are needed to test the efficacy of DADS and BCP in eradicating
B. duncani infection in animal models.
In summary, we identified 10 essential oils that showed good inhibitory activity against B. duncani in vitro at 0.001% (v/v). Among them, garlic oil and black pepper oil performed best, as well as DADS and BCP, which are their potential active constituents, respectively. We further demonstrated that the combination of garlic oil or DADS and azithromycin could eradicate B. duncani at low concentrations in vitro. Future studies are needed to test the activity of DADS and other garlic-derived sulfur compounds alone and in combination with current drugs to eradicate babesiosis in animal models and their activity against related Babesia parasites, and potential mechanisms of action of these compounds against Babesia parasites.