Putrescine: Essential factor for in vitro proliferation of Babesia bovis.
This study reports the effect of putrescine addition, either alone or in combination with insulin, transferrin and selenite (ITS), to serum-free Advanced DMEM/F12 (A-DMEM/F12) medium, on the in vitro culture of Babesia bovis and using a perfusion bioreactor to improve efficiency of the process. A B. bovis strain previously adapted to proliferate in serum-free medium (Bbovis-SF) was evaluated using eight increasing concentrations of putrescine. The percentage of parasitized erythrocytes (PPE) obtained from cultures supplemented with 0.101 mg/L was 6.23% compared with 2.3% for control cultures with M199 with Earle's salts (M199) and 40% serum. The combination of putrescine (0.101 mg/L) and a mixture of ITS (2000, 1100, and 1.34 mg/L, respectively) (Pu-ITS), in A-DMEM/F12 culture medium without serum yielded a maximum PPE of 17.26% compared to 2.58% in the control medium. This new formulation of culture medium, together with the use of a hollow-fiber perfusion bioreactor system (HFPBS), caused a substantial increase in the proliferation of B. bovis, yielding a maximum cumulative PPE of 118.8% after five days, compared to 58.6% in cultures treated with control medium M199 and 40% serum. We concluded that the addition of the ITS mixture and putrescine to the culture medium stimulated the proliferation of B. bovis in vitro. This new medium formulation, used in a HFPBS culture system, can be an effective, automated-prone system that can induce massive proliferation of B. bovis for use as a source of parasite antigens and immunogens.
CENID-Parasitología Veterinaria INIFAP, Carr. Fed. Cuernavaca-Cuautla No. 8534, Col. Progreso, C.P. 62550, Jiutepec, Morelos, Mexico; Campus de Ciencias Biológicas y Agropecuarias, FMVZ, Universidad Autónoma de Yucatán, km. 15.5 Carretera Mérida-Xmatkuil, Mérida, Yucatán, Mexico.
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Rojas-Martínez
Campus de Ciencias Biológicas y Agropecuarias, FMVZ, Universidad Autónoma de Yucatán, km. 15.5 Carretera Mérida-Xmatkuil, Mérida, Yucatán, Mexico.
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Rodríguez-Vivas
CENID-Parasitología Veterinaria INIFAP, Carr. Fed. Cuernavaca-Cuautla No. 8534, Col. Progreso, C.P. 62550, Jiutepec, Morelos, Mexico.
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Figueroa Millán
Centro de Investigaciones Regionales "Dr. Hideyo Noguchi", Universidad Autónoma de Yucatán, Av. Itzáes No. 490 x 59, Col. Centro, C.P.97000, Mérida, Yucatán, Mexico.
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Acosta Viana
Campus de Ciencias Biológicas y Agropecuarias, FMVZ, Universidad Autónoma de Yucatán, km. 15.5 Carretera Mérida-Xmatkuil, Mérida, Yucatán, Mexico.
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Gutiérrez Ruiz
CENID-Parasitología Veterinaria INIFAP, Carr. Fed. Cuernavaca-Cuautla No. 8534, Col. Progreso, C.P. 62550, Jiutepec, Morelos, Mexico. Electronic address: alvarez.jesus@inifap.gob.mx.
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Álvarez Martínez
D000818:Animals; D016793:Babesia bovis / Q000254:growth & development*; D019149:Bioreactors / Q000469:parasitology / Q000662:veterinary; D002417:Cattle; D015925:Cryopreservation / Q000662:veterinary; D016895:Culture Media, Serum-Free; D004912:Erythrocytes / Q000469:parasitology*; D007328:Insulin / Q000378:metabolism; D011700:Putrescine / Q000378:metabolism*; D020887:Selenious Acid / Q000378:metabolism; D014168:Transferrin / Q000378:metabolism
D016895:Culture Media, Serum-Free; D007328:Insulin; D014168:Transferrin; D020887:Selenious Acid; D011700:Putrescine
B. bovis; In vitro culture; Perfusion bioreactor; Putrescine; Serum-free medium