Development and Characterization of a Strain of Babesia gibsoni Resistant to Diminazene Aceturate In Vitro

Abstract

ABSTRACT. We attempted to develop a strain of Babesia gibsoni resistant to diminazene aceturate (DA), an anti-babesial drug, in vitro. Since the DA-sensitive B. gibsoni strain could survive and proliferate in culture medium containing 1 ng/ml DA, the concentration of DA was gradually increased from 1 to 200 ng/ml. The results showed that the parasites could survive and proliferate in the medium containing 200 ng/ml DA, which was much higher than the 50% inhibitory concentration (IC 50 ) of DA for B. gibsoni. Subsequently, these parasites were removed from erythrocytes and exposed directly to 200 ng/ml DA. They were able to survive and invade fresh erythrocytes, though the DA-sensitive B. gibsoni strain did not survive. Based on these results, the parasites cultured within 200 ng/ml DA were determined to be a DA-resistant B. gibsoni strain. In addition, the IC 50 levels of clindamycin, doxycycline and pentamidine for the DA-resistant B. gibsoni strain were determined. The IC 50 levels of clindamycin, doxycycline and pentamidine for the DA-resistant strain were higher than those for the DA-sensitive strain. The IC 50 of pentamidine for the resistant strain was much greater than that for the DA-sensitive strain. These results indicated that the DA-resistant B. gibsoni strain could have resistance not only to DA, but also to other anti-babesial drugs. In conclusion, we successfully developed a DA-resistant B. gibsoni strain in vitro. Canine babesiosis, a tick-borne hematozoan disease, is caused by Babesia gibsoni and B. canis. This disease is characterized by fever, lethargy, anemia and, in severe cases, death [9,11]. Diminazene aceturate (DA), an antibabesial drug, is an aromatic diamidine derivative. Currently, the mechanism of action of DA on B. gibsoni and B. canis is unknown. DA can temporarily improve the clinical signs of canine babesiosis [4,13]. However, this drug is unable to eliminate the parasites from infected dogs, and relapses often occur [11]. We believe that this is due to the development of drug resistance of B. gibsoni against DA. Collett [8] considered that B. canis surviving DA treatment could develop drug resistance against DA clinically. However, there is no report proving DA resistance of B. gibsoni and B. canis. In trypanosomiasis and leishmaniasis, it has been reported that DA can inhibit the DNA replication and mitochondrial respiratory activity of these pathogens [3,16]. The loss of P2 nucleoside transporter function in Trypanosoma brucei brucei has been implicated in resistance to DA [6]. Likewise, it is possible that B. gibsoni could develop drug resistance against DA.In reports about other anti-babesial drugs, including atovaquone, clindamycin, metronidazole, doxycycline and pentamidine, almost no single drug treatment or combined treatment could eliminate the parasites from the peripheral blood at the dosages used, and the possibility of relapse and development of resistant variants remained [11,13,24,27,32]. From those previous reports, it seems to be difficult to eliminate B. gibson...

Involvement of Mitochondrial Genes of <i>Babesia gibsoni</i> in Resistance to Diminazene Aceturate

“…To treat canine babesiosis with Da effectively, these problems have to be clarified. The DA-resistant B. gibsoni isolate developed by Hwang et al was more resistant to pentamidine, clindamycin and doxycycline than the wild-type [8]. Furthermore, the transcription levels of the heat shock protein 70 (hsp70) gene in this isolate decreased during the development of the Da-resistant isolate, suggesting that the hsp70 gene is involved but does not play a major role in the development of the Da-resistant isolate.…”

“…It is difficult to eliminate this parasite from infected dogs, although a number of drugs, including diminazene aceturate (Da), clindamycin, metronidazole, pentamidine, and atovaquone combined with azithromycin, are used for treatment of the disease [9,16,22,29]. in the treatment of canine babesiosis, possible relapses and the development of drug-resistant isolates are matters of concern.Da, an aromatic diamidine derivative, has been used as a first-line agent for the treatment of B. gibsoni infection in dogs [22]; however, Da cannot eliminate B. gibsoni from infected dogs, and relapses often occur [5,8,17,29]. in addition, a Da-resistant B. gibsoni isolate was previously developed in vitro in our laboratory [8].…”

“…Da, an aromatic diamidine derivative, has been used as a first-line agent for the treatment of B. gibsoni infection in dogs [22]; however, Da cannot eliminate B. gibsoni from infected dogs, and relapses often occur [5,8,17,29]. in addition, a Da-resistant B. gibsoni isolate was previously developed in vitro in our laboratory [8].…”

“…in addition, a Da-resistant B. gibsoni isolate was previously developed in vitro in our laboratory [8]. However, the mechanism of action of Da on B. gibsoni has not been elucidated, and the mechanism of the development of Da resistance in B. gibsoni is still not known.…”

Aspectos epidemiológicos, clínicos e anatomopatológicos da intoxicação por aceturato de diminazeno em cães

“…Nos Estados Unidos, aceturato de diminazeno tem uso proibido pelo FDA (Food and Drug Administration) (Taboada & Lobetti 2006), mas, em outros países, principalmente naqueles da África, o fármaco é ainda hoje bastante utilizado no tratamento da babesiose (Collett 2000) e da tripanossomíase (Obi et al 2013) caninas, duas dentre as causas mais comuns de morte de cães na África do Sul (Collett 2000) e Nigéria (Anene et al 2006), respectivamente. Nesses dois países, aceturato de diminazeno ainda permanece sendo o agente antiprotozoário mais comumente utilizado (Naidoo et al 2009), apesar de haver relatos de resistência por determinadas cepas de Babesia gibsoni (Hwang et al 2010) e Trypanosoma brucei brucei (Anene et al 2006). Embora a intoxicação por aceturato de diminazeno tenha sido descrita em cães desde a década de 1970, existem apenas cinco citações sobre essa toxicose na literatura internacional, a saber: 1) um resumo em anais de congresso (Boyt et al 1968), 2) um relato curto de alguns casos (Losos & Crockett 1969), 3) um trabalho científi-co experimental (Naudé et al 1970), 4) um relato de um caso (van Heerden 1981) e 5) uma descrição acerca de um caso submetido ao seminário semanal (Wednesday Slide Conference) da AFIP (Armed Forces Institute of Pathology) em 2000 (AFIP 2000).…”