• 1.

    Luo J, Yin H. Theileriosis of sheep and goats in China. Trop Anim Health Prod 1997;29 (suppl 4):810.

  • 2.

    Schnittger L, Yin H & Luo J, et al. Ribosomal small-subunit RNA gene-sequence analysis of Theileria lestoquardi and a Theileria species highly pathogenic for small ruminants in China. Parasitol Res 2000;86:352358.

    • Search Google Scholar
    • Export Citation
  • 3.

    Yin H, Luo J & Schnittger L, et al. Phylogenetic analysis of Theileria species transmitted by Haemaphysalis qinghaiensis. Parasitol Res 2004;92:3642.

    • Search Google Scholar
    • Export Citation
  • 4.

    Yin H, Guan G & Ma M, et al. Haemaphysalis qinghaiensis ticks transmit at least two different Theileria species: one is infective to yaks, one is infective to sheep. Vet Parasitol 2002;107:2935.

    • Search Google Scholar
    • Export Citation
  • 5.

    Yin H, Luo J & Guan G, et al. Experiments on transmission of an unidentified Theileria sp. to small ruminants with Haemaphysalis qinghaiensis and Hyalomma anatolicum anatolicum. Vet Parasitol 2002;108:2130.

    • Search Google Scholar
    • Export Citation
  • 6.

    Nagore D, Garcia-Sanmartin J & Garcia-Perez AL, et al. Identification, genetic diversity and prevalence of Theileria and Babesia species in a sheep population from Northern Spain. Int J Parasitol 2004;34:10591067.

    • Search Google Scholar
    • Export Citation
  • 7.

    Gao YL, Yin H & Luo JX, et al. Development of an enzyme-linked immunosorbent assay for the diagnosis of Theileria sp. infection in sheep. Parasitol Res 2002;88:S810.

    • Search Google Scholar
    • Export Citation
  • 8.

    Katende J, Morzaria S & Toye P, et al. An enzyme-linked immunosorbent assay for detection of Theileria parva antibodies in cattle using a recombinant polymorphic immunodominant molecule. Parasitol Res 1998;84:408416.

    • Search Google Scholar
    • Export Citation
  • 9.

    Yin H, Liu J & Luo J, et al. Observation on the schizont stage of an unidentified Theileria sp. in experimentally infected sheep. Parasitol Res 2003;91:3439.

    • Search Google Scholar
    • Export Citation
  • 10.

    Schnittger L, Yin H & Luo J, et al. Characterisation of a polymorphic gene of T. lestoquardi and of a recently identified Theileria species pathogenic for small ruminants in China. Parasitol Res 2002;88:553556.

    • Search Google Scholar
    • Export Citation
  • 11.

    Schnittger L, Yin H & Luo J, et al. Phylogenetic analysis by rRNA comparison of the highly pathogenic sheep-infecting parasites Theileria lestoquardi and a Theileria species identified in China. Ann N Y Acad Sci 2000;916:271275.

    • Search Google Scholar
    • Export Citation
  • 12.

    Holman PJ, Chieves L & Frerichs WM, et al. Babesia equi erythrocytic stages continuously cultured in an enriched medium. J Parasitol 1994;80:232236.

    • Search Google Scholar
    • Export Citation
  • 13.

    Zweygarth E, Just MC, de Waal DT. Continuous in vitro cultivation of erythrocytic stages of Babesia equi. Parasitol Res 1995;81:355358.

  • 14.

    Zweygarth E, Just MC & de Waal DT, et al. In vitro diagnosis of Babesia equi, in Proceedings. Equine Infect Dis 8th Int Conf 1999;232236.

  • 15.

    Levy MG, Ristic M. Babesia bovis: continuous cultivation in a microaerophilous stationary phase culture. Science 1980;207:12181220.

  • 16.

    Vega CA, Buening GM & Green TJ, et al. In vitro cultivation of Babesia bigemina. Am J Vet Res 1985;46:416420.

  • 17.

    Rodriguez SD, Buening GM & Green TJ, et al. Cloning of Babesia bovis by in vitro cultivation. Infect Immun 1983;42:1518.

  • 18.

    Zintl A, Skerrett HE & Gray JS, et al. Babesia divergens (Phylum Apicomplexa) in vitro growth in the presence of calf serum. Vet Parasitol 2004;122:127130.

    • Search Google Scholar
    • Export Citation
  • 19.

    Zweygarth E, Lopez-Rebollar LM. Continuous in vitro cultivation of Babesia gibsoni. Parasitol Res 2000;86:905907.

  • 20.

    Sunaga F, Namikawa K, Kanno Y. Continuous in vitro culture of erythrocytic stages of Babesia gibsoni and virulence of the cultivated parasite. J Vet Med Sci 2002;64:571575.

    • Search Google Scholar
    • Export Citation
  • 21.

    Zweygarth E, van Niekerk CJ, de Waal DT. Continuous in vitro cultivation of erythrocytic stages of Babesia caballi in serum-free medium. Parasitol Res 1999;85:413416.

    • Search Google Scholar
    • Export Citation
  • 22.

    Zweygarth E, Lopez-Rebollar LM, Meyer P. In vitro isolation of equine piroplasms derived from Cape Mountain zebra (Equus zebra zebra) in South Africa. Onderstepoort J Vet Res 2002;69:197200.

    • Search Google Scholar
    • Export Citation
  • 23.

    Zweygarth E, Lopez-Rebollar LM & Nurton J, et al. Culture, isolation and propagation of Babesia caballi from naturally infected horses. Parasitol Res 2002;88:460462.

    • Search Google Scholar
    • Export Citation
  • 24.

    Levy MG, Clabaugh G, Ristic M. Age resistance in bovine babesiosis: role of blood factors in resistance to Babesia bovis. Infect Immun 1982;37:11271131.

    • Search Google Scholar
    • Export Citation
  • 25.

    Zweygarth E, Just MC & van Niekerk C, et al. Continuous in vitro cultivation of erythrocytic stages of Babesia equi in serum-free medium. In: Maromorosch K, Mitsuhashi J, eds. Invertebrate cell culture: novel directions and biotechnology applications. Enfield, NH: Science Publishers Inc, 1997;271278.

    • Search Google Scholar
    • Export Citation

Advertisement

Establishment of optimal conditions for long-term culture of erythrocytic stages of Theileria uilenbergi

View More View Less
  • 1 Experimental and Technological Biology Institute-Chemical and Biological Technology Institute, Universidade Nova de Lisboa, 2780-901 Oeiras, Portugal.
  • | 2 Experimental and Technological Biology Institute-Chemical and Biological Technology Institute, Universidade Nova de Lisboa, 2780-901 Oeiras, Portugal.
  • | 3 Experimental and Technological Biology Institute-Chemical and Biological Technology Institute, Universidade Nova de Lisboa, 2780-901 Oeiras, Portugal and ECBio SA, Lab 4.11, ITQB Building, 2780-901 Oeiras, Portugal
  • | 4 Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Xujiaping 11, Lanzhou, Gansu 730046, People's Republic of China.
  • | 5 Molecular Biology Division, Onderstepoort Veterinary Institute, Onderstepoort 0110, South Africa.
  • | 6 Experimental and Technological Biology Institute-Chemical and Biological Technology Institute, Universidade Nova de Lisboa, 2780-901 Oeiras, Portugal

Abstract

Objective—To establish optimal conditions for long-term culture of the erythrocytic stage of Theileria uilenbergi.

Sample Population—Red blood cells from 3 splenectomized sheep experimentally infected with a blood stabilate of T uilenbergi.

Procedures—Cultures of T uilenbergi were initiated by use of blood from experimentally infected sheep collected when parasites were detected in Giemsa-stained thin blood smears. Different culture conditions were tested to optimize in vitro growth of the organisms. Subcultures were performed at a ratio of 1:2, 1:4, and 1:8 when the percentage of parasitized erythrocytes (PPE) was at least 1% or when the initial PPE was doubled.

Results—The optimal culture medium was HL-1 medium (a complete chemically defined medium) supplemented with 20% sheep serum and 0.75% chemically defined lipids. Optimal culture conditions included incubation in a humidified 2% O2, 5% CO2, and 93% N2 atmosphere at 37°C. Cultures of the merozoite stage of the parasite were continuously propagated in vitro for > 1 year. The PPE reached values of up to 3%.

Conclusions and Clinical Relevance—Optimization of culture conditions to reach a high PPE seems worthwhile. The continuous propagation of T uilenbergi in culture allows the production of parasite material without infecting animals and provides a continuous laboratory source of parasites for further studies.

Abstract

Objective—To establish optimal conditions for long-term culture of the erythrocytic stage of Theileria uilenbergi.

Sample Population—Red blood cells from 3 splenectomized sheep experimentally infected with a blood stabilate of T uilenbergi.

Procedures—Cultures of T uilenbergi were initiated by use of blood from experimentally infected sheep collected when parasites were detected in Giemsa-stained thin blood smears. Different culture conditions were tested to optimize in vitro growth of the organisms. Subcultures were performed at a ratio of 1:2, 1:4, and 1:8 when the percentage of parasitized erythrocytes (PPE) was at least 1% or when the initial PPE was doubled.

Results—The optimal culture medium was HL-1 medium (a complete chemically defined medium) supplemented with 20% sheep serum and 0.75% chemically defined lipids. Optimal culture conditions included incubation in a humidified 2% O2, 5% CO2, and 93% N2 atmosphere at 37°C. Cultures of the merozoite stage of the parasite were continuously propagated in vitro for > 1 year. The PPE reached values of up to 3%.

Conclusions and Clinical Relevance—Optimization of culture conditions to reach a high PPE seems worthwhile. The continuous propagation of T uilenbergi in culture allows the production of parasite material without infecting animals and provides a continuous laboratory source of parasites for further studies.

Contributor Notes

Supported in part by the European Union and the Portuguese Foundation for Science and Technology.

Address correspondence to Dr. Miranda.