Use of canine red blood cell with high concentrations of potassium, reduced glutathione, and free amino acid as host cells for in vitro cultivation of Babesia gibsoni

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ABSTRACT. The present study was conducted to clarify the mechanism underlying the oxidative process in erythrocytes infected with Babesia gibsoni. The parasite B. gibsoni was cultured together with erythrocytes from normal dogs for 7 days. When parasitemia reached 12.0-13.4% at Day 7, the production of superoxide in erythrocytes was significantly higher in the parasitized culture than in the control culture (p<0.005). The concentration of thiobarbituric acid reactive substances (TBARS) in erythrocytes in parasitized culture was also significantly increased compared with the control culture (p<0.005), indicating that lipid peroxidation was greater in infected erythrocytes than in non-infected cells. In addition, the rates of superoxide generation in the blood of B. gibsoni-infected dogs were also significantly higher than in non-infected dogs (p<0.001). These results indicate that superoxide anions are increased in erythrocytes parasitized with B. gibsoni, and suggest that oxidative damage, due to lipid peroxidation, might be caused in host erythrocytes by the parasite. KEY WORDS: Babesia gibsoni, oxidative damage, superoxide.J. Vet. Med. Sci. 63(10): 1077-1081, 2001 Babesia spp. are well-known blood parasites that cause severe hemolytic anemia in domestic animals [4]. In general, Babesia parasites, as well as Theileria and malaria parasites, invade erythrocytes of infected animals, which results in the destruction of the parasitized erythrocytes [3]. However, severe anemia often occurs in animals infected with this parasite in spite of a low percentage of parasitized erythrocytes in their peripheral blood [4,9]. This phenomenon suggests the possibility of non-parasitized erythrocytes also being damaged due to the parasite infection, resulting in enhanced erythrocyte destruction. We previously reported that concentrations of both methemoglobin and malondialdehyde (MDA), an end product of lipid peroxidation, increase in erythrocytes cultured with B. gibsoni, and that B. gibsoni-infected dogs with high parasitemia also show significant increases of both methemoglobin and MDA in erythrocytes compared with those in non-infected dogs [10]. These results indicate that oxidative damage to erythrocytes is induced by B. gibsoni infection and suggest that active oxygen might be generated by the multiplication of the parasites. Furthermore, erythrocytes cultured with B. gibsoni are more susceptible to phagocytosis by bone marrow macrophages from normal dogs than erythrocytes cultured without B. gibsoni. In addition, 89 percent of erythrocytes phagocytized by macrophages were non-parasitized erythrocytes [10]. However, it has not been clarified whether the Babesia parasite is able to generate reactive oxygen species. The present study was carried out to elucidate the mechanisms underlying the oxidative process in erythrocytes infected with B. gibsoni. MATERIALS AND METHODSThree dogs were inoculated with B. gibsoni-infected blood from other carrier dogs, and used as B. gibsoniinfected dogs in the present study. Five normal do...

Section: Methodsmentioning

confidence: 99%

Increased Generation of Superoxide in Erythrocytes infected with Babesia gibsoni.

Otsuka

Yamato

Maede

2001

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“…Preparation of canine erythrocyte suspension: Canine erythrocytes were prepared by the method of Yamasaki et al [34] with some modifications. Venous blood was collected from the jugular vein of healthy adult dogs.…”

Section: Preparation Of Bruceine Amentioning

confidence: 99%

Evaluation of Efficacy of Bruceine A, a Natural Quassinoid Compound Extracted from a Medicinal Plant, Bruced javanica, for Canine Babesiosis

Mizukami

Kawamura

subeki

et al. 2009

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ABSTRACT. Bruceine A, a natural quassinoid compound extracted from the dried fruits of Brucea javanica (L.) Merr., was evaluated for its antibabesial activity in vitro and in vivo. Bruceine A inhibited the in vitro growth of Babesia gibsoni in canine erythrocytes at lower concentration compared with the standard antibabesial drug diminazene aceturate and killed the parasites within 24 hr at a concentration of 25 nM. Oral administration of bruceine A at a dosage of 6.4 mg/kg/day for 5 days resulted in no clinical findings in a dog with normal ranges of hematological and biochemical values in the blood. Three dogs were infected with B. gibsoni and two of them were treated with bruceine A at a dosage of 6.4 mg/kg/day for 6 days from day 5 post-infection. An untreated dog developed typical acute babesiosis symptoms including severe anemia, high fever, and complete loss of appetite and movement. However, the two bruceine A-treated dogs maintained their healthy conditions throughout the experimental period of 4 weeks although complete elimination of parasites from the peripheral blood was not achieved and decreases in the packed cell volume and the erythrocyte and platelet counts were observed. Since natural quassinoid compounds have been used as traditional medicines for the treatment of various ailments including cancer and malaria, the present results suggest that bruceine A or other related compounds are potential candidates for the treatment of canine babesiosis.

“…Culture of B. gibsoni in canine erythrocytes: B. gibsoni was cultivated according to the method reported by Yamasaki et al [24] with some modifications to examine the effect of B. gibsoni parasites on erythrocyte 5'-nucleotidase. Venous blood was collected from clinically healthy dogs with EDTA as an anticoagulant.…”

Section: Reagentsmentioning

confidence: 99%

Serum from Dogs Infected with Babesia gibsoni Inhibits Maturation of Reticulocytes and Erythrocyte 5'-Nucleotidase Activity in Vitro

Hossain

Yamato

Yamasaki

et al. 2003

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ABSTRACT. Erythrocyte 5'-nucleotidase is thought to be involved in the maturation of erythrocytes. In the present study, in vitro incubation of canine erythrocytes demonstrated that significant inhibition of 5'-nucleotidase activity occurred in the presence of serum from dogs infected with Babesia gibsoni, when the enzyme was assayed with cytidine 5'-monophosphate (5'-CMP) and inosine 5'-monophosphate (5'-IMP) as substrates. The multiplication of B. gibsoni in in vitro culture also resulted in a significant decrease in the enzyme activity of erythrocytes in the culture. Furthermore, the infected serum and 5'-CMP retarded the maturation of canine reticulocytes in vitro. These results suggested that nucleotides such as 5'-CMP and 5'-IMP might accumulate in young erythrocytes and/or serum in dogs infected with B. gibsoni as a result of decreased activity of erythrocyte 5'-nucleotidase, resulting in the delayed maturation of reticulocytes. KEY WORDS: Babesia gibsoni, canine erythrocyte, 5'-nucleotidase, pyrimidine 5'-monophosphate, reticulocyte.