Experimental Parasitology 135 (2013) 690–694
Contents lists available at ScienceDirect
Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr
Artemisone inhibits in vitro and in vivo propagation of Babesia bovis and B. bigemina parasites Monica L. Mazuz a,⇑, Jacob Golenser b,c, Leah Fish a, Richard K. Haynes d, Ricardo Wollkomirsky a, Benjamin Leibovich a, Varda Shkap a a
Division of Parasitology, Kimron Veterinary Institute, 50250 Bet Dagan, Israel Department of Microbiology and Molecular Genetics, The Kuvin Centre for the Study of Infectious and Tropical Diseases, The Hebrew University of Jerusalem, Jerusalem, Israel Department of Pathology and Bosch Institute, The University of Sydney, Sydney, Australia d Centre of Excellence for Pharmaceutical Sciences, North West University, Potchefstroom, South Africa b c
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Bovine babesiosis is an economically
important disease of cattle. Drugs with high specificity towards
the parasite and low toxicity are required. Artemisone a novel artemisinin derivative is effective against apicomplexa parasites. In vitro, artemisone reduced parasitemia in a dose-dependent manner. In calves artemisone was well tolerated and prevent development of acute babesiosis.
a r t i c l e
i n f o
Article history: Received 29 April 2013 Received in revised form 26 August 2013 Accepted 21 October 2013 Available online 30 October 2013 Keywords: Bovine babesiosis Babesia bovis Babesia bigemina Treatment Artemisinin Artemisone
a b s t r a c t Artemisone was evaluated, in in vitro and in vivo, for control of bovine babesiosis caused by Babesia bigemina and Babesia bovis parasites. In vitro, artemisone reduced parasitemia in a dose-dependent manner: the inhibitory effects increased gradually, reaching a maximum inhibition of 99.6% and 86.4% for B. bigemina and B. bovis, respectively 72 h after initiation of treatment with initial parasitemia of 0.5%. In calves infected with either B. bigemina or B. bovis artemisone treatment was well tolerated and prevented development of acute babesiosis in all animals except for one B. bovis-infected calf. The treatment did not eliminate all blood parasites, and recovered animals carried a persistent low-level infection. Treatment with artemisone may be useful as an alternative drug for preventing the pathology that results from babesiosis, without interfering with acquired immune protection following recovery from an acute babesiosis infection or vaccination. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction Bovine babesiosis, caused by hemoprotozoan parasites Babesia bovis and Babesia bigemina, is an economically important disease ⇑ Corresponding author. Address: Division of Parasitology, Kimron Veterinary Institute, P.O.B. 12, 50250 Bet Dagan, Israel. Fax: +972 3 9681678. E-mail addresses: [email protected], [email protected] (M.L. Mazuz). 0014-4894/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.exppara.2013.10.006
of cattle in tropical and subtropical regions. Some clinical symptoms associated with acute babesiosis are similar between the two species and include fever, anemia, and icterus. However, the disease differs significantly between B. bovis- and B. bigemina-infected cattle. In acute B. bigemina infection, parasitemia rises quickly, and the disease is characterized by high fever, hemoglobinuria and acute anemia. In B. bovis infection, although the peripheral blood parasitemia is relatively low, sequestration of
M.L. Mazuz et al. / Experimental Parasitology 135 (2013) 690–694
parasites into capillaries of internal organs leads to severe disease, resulting in cerebral babesiosis, respiratory distress syndrome, edema and death (Brown and Palmer, 1999; Bock et al., 2004). Efficient control of bovine babesiosis is based on integrated management, including tick control, preventive immunization – currently based on live attenuated parasites – and chemotherapy (Suarez and Noh, 2011). Successful treatment of bovine babesiosis depends on early diagnosis and prompt administration of effective drugs (Vial and Gorenflot, 2006), of which imidocarb dipropionate and diminazene diaceturate are considered the drugs of choice. Although these drugs are effective in treatment of acute babesiosis, they are not available worldwide. Furthermore, the problems of residues left in the food chain by these drugs are documented, and parasite resistance to both drugs is increasing (Goo et al., 2010; Suarez and Noh, 2011; Mosqueda et al., 2012). Therefore, alternative anti-Babesia drugs with high specificity towards the parasite and low toxicity to the host are required, in order to control bovine babesiosis (Vial and Gorenflot, 2006; Mosqueda et al., 2012). Mosqueda et al. (2012) have listed current and novel drugs for the treatment of bovine babesiosis; among the novel drugs, artemisinin derivatives, which are the most potent anti-malaria drugs available, are listed as potential drugs for treatment of babesiosis. The artemisinin derivative artesunate inhibits in vitro growth of B. bovis and B. gibsoni; it was effective in treatment of mice infected by B. microti (Goo et al., 2010). It also inhibits in vivo development of Theileria equi, a closely related protozoan, but is not active against B. caballi (Nagai et al., 2003). Artemisone is a novel ‘‘second-generation’’ semisynthetic artemisinin derivative that is effective against other apicomplexan parasites: Plasmodium falciparum (Haynes et al., 2006; WaknineGrinberg et al., 2010), Toxoplasma gondii (Dunay et al., 2009) and Neospora caninum (Mazuz et al., 2011). Artemisone has a longer half-life, lower curative dose and superior bioavailability than commercial artemisinins (Nagelschmitz et al., 2008). It is apparent that it has potential for treatment of babesiosis, and we have now evaluated artemisone for this purpose by screening it both in vitro and in vivo against B. bigemina and B. bovis. 2. Materials and methods 2.1. Babesia parasites The in vitro assays were performed with culture-adapted parasites (Fish et al., 2008). For in vivo assays, the virulent field isolates B. bovis (isolate 767) and B. bigemina (isolate 871) were used. Virulent isolates were obtained from naturally infected cows with acute babesiosis. Because of the relatively low number of parasites in blood of naturally infected cattle, blood from such animals was sub-inoculated into splenectomized calves, to obtain sufficient number of parasites. When parasitemia reached 4.5% in B. bovis infection and 7% for B. bigemina blood was drawn and cryopreserved stabilates in 3.6 ml vials were frozen with 15% DMSO until used for infection of cattle in vivo experiments. 2.2. Cattle The experiments with cattle were performed in compliance with the requirements of the Animal Welfare Committee of the Kimron Veterinary Institute. A total of 13 Holstein-Friesian calves aged 3–6 months, seronegative for Babesia parasites and kept on zero grazing were used for the in vivo experiments.
691
2.3. Effect of artemisone on B. bovis and B. bigemina multiplication in vitro For in vitro experiments Babesia parasites were grown in a microaerophilous stationary phase (MASP) culture according to Levy and Ristic (1980) with some modifications. Erythrocytes were suspended at 10% packed cell volume (PCV) in complete medium (CM) consisting of 60% Medium 199 with Earle salts and 40% normal bovine adult serum and supplemented (per milliliter) with 25 mM TES buffer, 100 lg streptomycin, 100 U penicillin and 0.25 mg amphothericin B. One milliliter of suspension containing the erythrocytes was placed in each well of 24-well tissue-culture plates. The cultures were kept at 37 °C under a gas mixture consisting of 2% O2, 5% CO2 and 93% nitrogen. CM was replaced daily and parasitemia was monitored. Ongoing Babesia cultures at 3–6% of parasitized erythrocytes (PPE) were diluted with normal red blood cells (nRBCs) to obtain parasitemia of 0.5%. The diluted cultures were aliquoted into 24-well plates and four duplicates were prepared for each concentration of the drug, or controls containing DMSO alone. Artemisone, synthesized from dihydroartemisinin and purified by flash column chromatography, followed by recrystallization (Haynes et al., 2006), was added to the cultures dissolved in DMSO to obtain final concentrations of 300, 100, 33, and 11 lg/ml. The DMSO concentration in the cultures did not exceed 0.25%. The medium containing the drug or DMSO alone was changed daily and thin blood smears stained with Giemsa were prepared to evaluate the parasitemia. Drug efficacy was evaluated daily, by counting the PPE in at least 1000 RBCs. The mean parasitemia from each quadruplet was determined, and the growth inhibition calculated as:
½ðmean PPE of control wells mean PPE of treated wellsÞ=ðmean PPE of control wellsÞ 100 Statistical analyses were performed by applying the Chi-square (x2) test, and results were considered significant at p < 0.05. 2.4. In vivo effect of artemisone in Babesia infected cattle To assess the compatibility and safety of artemisone in cattle, two healthy uninfected calves were treated with artemisone by twice daily intraperitoneal administration of artemisone at 5 mg/ kg diluted in 5 ml of DMSO, for four consecutive days. Physical veterinary examinations and laboratory tests, including hematological and biochemical analyses were conducted prior to and during drug administration. The amount of artemisone was based on previous experiments in which mammals infected with other Apicomplexan parasites were treated (detailed in Waknine-Grinberg et al., 2010 and Mazuz et al., 2011). After the in vivo safety evaluation, 11 additional calves were randomly infected by intra-venous inoculation of parasites. Four calves were infected with B. bigemina and the remaining seven with B. bovis. Two calves infected with B. bigemina, and five calves infected with B. bovis were treated with artemisone. The remaining four infected calves, two carrying B. bovis and two carrying B. bigemina, were injected with DMSO only and served as controls. Following infection, all calves were monitored daily for up to 3 weeks for rectal temperature, packed-cells volume (PCV), and peripheral blood parasitemia.
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M.L. Mazuz et al. / Experimental Parasitology 135 (2013) 690–694
Treatment with artemisone was initiated when parasites were first observed in the blood smears. The drug was administered to infected animals during three consecutive days. Control animals were left untreated until they had developed acute babesiosis. Then, in order to prevent suffering and foreseeable death, the acutely infected calves were treated with diminazene aceturate (Berenil) at 3.5 mg/kg for two consecutive days.
3. Results 3.1. Effect of artemisone on B. bigemina and B. bovis multiplication in vitro Artemisone reduced parasitemia in a dose-dependent manner. The effect against B. bigemina propagation in vitro was observed as early as 24 h after treatment with artemisone at 11–300 lg/ml (Table 1), when the percentage inhibition of parasite propagation ranged from 61.4% to 80% with IC50 of 8.9 lg/ml (22.5 lM). The inhibitory effect increased gradually in the following days in culture, and reached a maximum of 99.6% after 72 h of treatment. In untreated cultures, the parasitemia reached a maximum after 48 h and maintained about the same level through 72 h, ranging from 3.5% to 6.5%. The inhibitory effect of artemisone on B. bovis was observed within 24 h; it also was dose-dependent, although the only statistically significant effect was obtained at the highest dose tested – 300 lg/ml (Table 2) and the IC50 dose was 180.4 lg/ml (449 lM). The parasitemia was significantly inhibited after 72 h of treatment at all doses, compared with the untreated control cultures (IC50 of about 20 lg/ml–50 lM). However, a low level of parasitemia was still recorded at the end of the experiments even at high artemisone dosage. 3.2. In vivo effect of artemisone in cattle Inoculation of artemisone into uninfected calves did not produce any side effect except for a transient pronounced abdominal pain that lasted a few seconds, after which the animals appeared normal. No hematological or biochemical changes were observed in blood tests performed during the treatment (data not shown). 3.3. Effect of artemisone on Babesia-infected cattle The B. bigemina-infected calves treated with artemisone did not develop acute signs of babesiosis: there was no fever, the minimum PCV level decreased to 27%, the parasitemia reached a maximum of 1.3%, both calves recovered, and minor parasitemia
Contents lists available at ScienceDirect
Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr
Artemisone inhibits in vitro and in vivo propagation of Babesia bovis and B. bigemina parasites Monica L. Mazuz a,⇑, Jacob Golenser b,c, Leah Fish a, Richard K. Haynes d, Ricardo Wollkomirsky a, Benjamin Leibovich a, Varda Shkap a a
Division of Parasitology, Kimron Veterinary Institute, 50250 Bet Dagan, Israel Department of Microbiology and Molecular Genetics, The Kuvin Centre for the Study of Infectious and Tropical Diseases, The Hebrew University of Jerusalem, Jerusalem, Israel Department of Pathology and Bosch Institute, The University of Sydney, Sydney, Australia d Centre of Excellence for Pharmaceutical Sciences, North West University, Potchefstroom, South Africa b c
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Bovine babesiosis is an economically
important disease of cattle. Drugs with high specificity towards
the parasite and low toxicity are required. Artemisone a novel artemisinin derivative is effective against apicomplexa parasites. In vitro, artemisone reduced parasitemia in a dose-dependent manner. In calves artemisone was well tolerated and prevent development of acute babesiosis.
a r t i c l e
i n f o
Article history: Received 29 April 2013 Received in revised form 26 August 2013 Accepted 21 October 2013 Available online 30 October 2013 Keywords: Bovine babesiosis Babesia bovis Babesia bigemina Treatment Artemisinin Artemisone
a b s t r a c t Artemisone was evaluated, in in vitro and in vivo, for control of bovine babesiosis caused by Babesia bigemina and Babesia bovis parasites. In vitro, artemisone reduced parasitemia in a dose-dependent manner: the inhibitory effects increased gradually, reaching a maximum inhibition of 99.6% and 86.4% for B. bigemina and B. bovis, respectively 72 h after initiation of treatment with initial parasitemia of 0.5%. In calves infected with either B. bigemina or B. bovis artemisone treatment was well tolerated and prevented development of acute babesiosis in all animals except for one B. bovis-infected calf. The treatment did not eliminate all blood parasites, and recovered animals carried a persistent low-level infection. Treatment with artemisone may be useful as an alternative drug for preventing the pathology that results from babesiosis, without interfering with acquired immune protection following recovery from an acute babesiosis infection or vaccination. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction Bovine babesiosis, caused by hemoprotozoan parasites Babesia bovis and Babesia bigemina, is an economically important disease ⇑ Corresponding author. Address: Division of Parasitology, Kimron Veterinary Institute, P.O.B. 12, 50250 Bet Dagan, Israel. Fax: +972 3 9681678. E-mail addresses: [email protected], [email protected] (M.L. Mazuz). 0014-4894/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.exppara.2013.10.006
of cattle in tropical and subtropical regions. Some clinical symptoms associated with acute babesiosis are similar between the two species and include fever, anemia, and icterus. However, the disease differs significantly between B. bovis- and B. bigemina-infected cattle. In acute B. bigemina infection, parasitemia rises quickly, and the disease is characterized by high fever, hemoglobinuria and acute anemia. In B. bovis infection, although the peripheral blood parasitemia is relatively low, sequestration of
M.L. Mazuz et al. / Experimental Parasitology 135 (2013) 690–694
parasites into capillaries of internal organs leads to severe disease, resulting in cerebral babesiosis, respiratory distress syndrome, edema and death (Brown and Palmer, 1999; Bock et al., 2004). Efficient control of bovine babesiosis is based on integrated management, including tick control, preventive immunization – currently based on live attenuated parasites – and chemotherapy (Suarez and Noh, 2011). Successful treatment of bovine babesiosis depends on early diagnosis and prompt administration of effective drugs (Vial and Gorenflot, 2006), of which imidocarb dipropionate and diminazene diaceturate are considered the drugs of choice. Although these drugs are effective in treatment of acute babesiosis, they are not available worldwide. Furthermore, the problems of residues left in the food chain by these drugs are documented, and parasite resistance to both drugs is increasing (Goo et al., 2010; Suarez and Noh, 2011; Mosqueda et al., 2012). Therefore, alternative anti-Babesia drugs with high specificity towards the parasite and low toxicity to the host are required, in order to control bovine babesiosis (Vial and Gorenflot, 2006; Mosqueda et al., 2012). Mosqueda et al. (2012) have listed current and novel drugs for the treatment of bovine babesiosis; among the novel drugs, artemisinin derivatives, which are the most potent anti-malaria drugs available, are listed as potential drugs for treatment of babesiosis. The artemisinin derivative artesunate inhibits in vitro growth of B. bovis and B. gibsoni; it was effective in treatment of mice infected by B. microti (Goo et al., 2010). It also inhibits in vivo development of Theileria equi, a closely related protozoan, but is not active against B. caballi (Nagai et al., 2003). Artemisone is a novel ‘‘second-generation’’ semisynthetic artemisinin derivative that is effective against other apicomplexan parasites: Plasmodium falciparum (Haynes et al., 2006; WaknineGrinberg et al., 2010), Toxoplasma gondii (Dunay et al., 2009) and Neospora caninum (Mazuz et al., 2011). Artemisone has a longer half-life, lower curative dose and superior bioavailability than commercial artemisinins (Nagelschmitz et al., 2008). It is apparent that it has potential for treatment of babesiosis, and we have now evaluated artemisone for this purpose by screening it both in vitro and in vivo against B. bigemina and B. bovis. 2. Materials and methods 2.1. Babesia parasites The in vitro assays were performed with culture-adapted parasites (Fish et al., 2008). For in vivo assays, the virulent field isolates B. bovis (isolate 767) and B. bigemina (isolate 871) were used. Virulent isolates were obtained from naturally infected cows with acute babesiosis. Because of the relatively low number of parasites in blood of naturally infected cattle, blood from such animals was sub-inoculated into splenectomized calves, to obtain sufficient number of parasites. When parasitemia reached 4.5% in B. bovis infection and 7% for B. bigemina blood was drawn and cryopreserved stabilates in 3.6 ml vials were frozen with 15% DMSO until used for infection of cattle in vivo experiments. 2.2. Cattle The experiments with cattle were performed in compliance with the requirements of the Animal Welfare Committee of the Kimron Veterinary Institute. A total of 13 Holstein-Friesian calves aged 3–6 months, seronegative for Babesia parasites and kept on zero grazing were used for the in vivo experiments.
691
2.3. Effect of artemisone on B. bovis and B. bigemina multiplication in vitro For in vitro experiments Babesia parasites were grown in a microaerophilous stationary phase (MASP) culture according to Levy and Ristic (1980) with some modifications. Erythrocytes were suspended at 10% packed cell volume (PCV) in complete medium (CM) consisting of 60% Medium 199 with Earle salts and 40% normal bovine adult serum and supplemented (per milliliter) with 25 mM TES buffer, 100 lg streptomycin, 100 U penicillin and 0.25 mg amphothericin B. One milliliter of suspension containing the erythrocytes was placed in each well of 24-well tissue-culture plates. The cultures were kept at 37 °C under a gas mixture consisting of 2% O2, 5% CO2 and 93% nitrogen. CM was replaced daily and parasitemia was monitored. Ongoing Babesia cultures at 3–6% of parasitized erythrocytes (PPE) were diluted with normal red blood cells (nRBCs) to obtain parasitemia of 0.5%. The diluted cultures were aliquoted into 24-well plates and four duplicates were prepared for each concentration of the drug, or controls containing DMSO alone. Artemisone, synthesized from dihydroartemisinin and purified by flash column chromatography, followed by recrystallization (Haynes et al., 2006), was added to the cultures dissolved in DMSO to obtain final concentrations of 300, 100, 33, and 11 lg/ml. The DMSO concentration in the cultures did not exceed 0.25%. The medium containing the drug or DMSO alone was changed daily and thin blood smears stained with Giemsa were prepared to evaluate the parasitemia. Drug efficacy was evaluated daily, by counting the PPE in at least 1000 RBCs. The mean parasitemia from each quadruplet was determined, and the growth inhibition calculated as:
½ðmean PPE of control wells mean PPE of treated wellsÞ=ðmean PPE of control wellsÞ 100 Statistical analyses were performed by applying the Chi-square (x2) test, and results were considered significant at p < 0.05. 2.4. In vivo effect of artemisone in Babesia infected cattle To assess the compatibility and safety of artemisone in cattle, two healthy uninfected calves were treated with artemisone by twice daily intraperitoneal administration of artemisone at 5 mg/ kg diluted in 5 ml of DMSO, for four consecutive days. Physical veterinary examinations and laboratory tests, including hematological and biochemical analyses were conducted prior to and during drug administration. The amount of artemisone was based on previous experiments in which mammals infected with other Apicomplexan parasites were treated (detailed in Waknine-Grinberg et al., 2010 and Mazuz et al., 2011). After the in vivo safety evaluation, 11 additional calves were randomly infected by intra-venous inoculation of parasites. Four calves were infected with B. bigemina and the remaining seven with B. bovis. Two calves infected with B. bigemina, and five calves infected with B. bovis were treated with artemisone. The remaining four infected calves, two carrying B. bovis and two carrying B. bigemina, were injected with DMSO only and served as controls. Following infection, all calves were monitored daily for up to 3 weeks for rectal temperature, packed-cells volume (PCV), and peripheral blood parasitemia.
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M.L. Mazuz et al. / Experimental Parasitology 135 (2013) 690–694
Treatment with artemisone was initiated when parasites were first observed in the blood smears. The drug was administered to infected animals during three consecutive days. Control animals were left untreated until they had developed acute babesiosis. Then, in order to prevent suffering and foreseeable death, the acutely infected calves were treated with diminazene aceturate (Berenil) at 3.5 mg/kg for two consecutive days.
3. Results 3.1. Effect of artemisone on B. bigemina and B. bovis multiplication in vitro Artemisone reduced parasitemia in a dose-dependent manner. The effect against B. bigemina propagation in vitro was observed as early as 24 h after treatment with artemisone at 11–300 lg/ml (Table 1), when the percentage inhibition of parasite propagation ranged from 61.4% to 80% with IC50 of 8.9 lg/ml (22.5 lM). The inhibitory effect increased gradually in the following days in culture, and reached a maximum of 99.6% after 72 h of treatment. In untreated cultures, the parasitemia reached a maximum after 48 h and maintained about the same level through 72 h, ranging from 3.5% to 6.5%. The inhibitory effect of artemisone on B. bovis was observed within 24 h; it also was dose-dependent, although the only statistically significant effect was obtained at the highest dose tested – 300 lg/ml (Table 2) and the IC50 dose was 180.4 lg/ml (449 lM). The parasitemia was significantly inhibited after 72 h of treatment at all doses, compared with the untreated control cultures (IC50 of about 20 lg/ml–50 lM). However, a low level of parasitemia was still recorded at the end of the experiments even at high artemisone dosage. 3.2. In vivo effect of artemisone in cattle Inoculation of artemisone into uninfected calves did not produce any side effect except for a transient pronounced abdominal pain that lasted a few seconds, after which the animals appeared normal. No hematological or biochemical changes were observed in blood tests performed during the treatment (data not shown). 3.3. Effect of artemisone on Babesia-infected cattle The B. bigemina-infected calves treated with artemisone did not develop acute signs of babesiosis: there was no fever, the minimum PCV level decreased to 27%, the parasitemia reached a maximum of 1.3%, both calves recovered, and minor parasitemia
