, Volume 162, Issue 6, pp 421–426 | Cite as

Effect of Prototheca zopfii on neutrophil function from bovine milk

  • Luciane T. Cunha
  • Silvana P. Pugine
  • Claudia R. Valle
  • Andrea R. Ribeiro
  • Ernane J. X. Costa
  • Mariza P. De MeloEmail author


This study was carried to investigate neutrophil function in the presence of Prototheca zopfii. For this purpose, bovine milk neutrophils were incubated in the absence (control) of and presence of P. zopfii, and then they were examined hydrogen peroxide (H2O2) production, antioxidant enzyme activities, and phagocytic capacity. Milk was collected from negative “California Mastitis Test” (CMT) quarter from three lactating Holstein cows after induction of leukocytosis with an intramammary infusion of oyster glycogen. H2O2 production was measured using the phenol red method. Catalase activity was measured following H2O2 reduction at 240 nm and the activity of glutathione reductase was determined by measuring the rate of NADPH oxidation at 340 nm. P. zopfii death was assessed by fluorescent microscopy using acridine orange assay and by colony forming units (CFUs). Comparisons between the groups were initially performed by analysis of variance (ANOVA). Significant differences were then compared using Tukey’s test with a significance coefficient of 0.05. Hydrogen peroxide production, catalase and glutathione reductase activities by neutrophils incubated in presence of P. zopfii were stimulated five times, 21% and 27% respectively, compared to the unstimulated-neutrophils. Neutrophils did not affect P. zopfii death as shown by microscopy and CFUs. These observations led to the conclusion that the P. zopfii promote a high increase of H2O2 production by neutrophils from bovine milk during algae exposition accompanied by increase of antioxidant enzyme activities; however, this process did not affect P. zopfii death.


antioxidant mastitis oxygen peroxide phagocytosis 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Peng, SL 2006Neutrophil apoptosis in autoimmunityJ. Mol. Med.84122125PubMedCrossRefGoogle Scholar
  2. 2.
    Kobayashi, SD, Voyich, JM, Burlak, C, DeLeo, FR 2005Neutrophils in the innate immune responseArch. Immunol. Ther. Exp.53505517Google Scholar
  3. 3.
    Pore, RS, Barnett, EA, Barnes, WC,Jr, Walker, JD 1983Prototheca ecologyMycopathologia814962PubMedCrossRefGoogle Scholar
  4. 4.
    Davies, RR, Spencer, H, Wakelin, PO 1964A case of human protothecosisTrans. Roy. Soc. Trop. Med. Hyg.58448451PubMedCrossRefGoogle Scholar
  5. 5.
    Chan, JC, Jeffers, LJ, Gould, EW, Hutson, D, Martinez, OV, Reddy, KR, Hassan, F, Schiff, ER 1990Visceral protothecosis mimicking sclerosing cholangitis in an immunocompetent host: Successful antifungal therapyRev. Infect. Dis.12802807PubMedGoogle Scholar
  6. 6.
    Heney, C, Greeff, M, Davis, V 1991Hickman catheter-related protothecal algaemia in an immunocompromised childJ. infect. Dis.163930931PubMedGoogle Scholar
  7. 7.
    Laeng, RH, Egger, C, Schaffner, T, Borish, B, Pedrinis, E 1994Protothecosis in HIV-positive patientAm. J. Surg. Pathol.1812611264PubMedGoogle Scholar
  8. 8.
    Gómez-Hernando, C, Palacio, A, Nevado, M, Carabias, E, González, A, Cuétera, MS, Carpintero, Y 1996Colonización transitoria por un alga (Prototheca wickerhamii) en un enfermo de cancerRev Theroam Microbiol135658Google Scholar
  9. 9.
    Kirk, JH 1991Diagnosis and treatment of difficult mastitis casesAgri-practice121520Google Scholar
  10. 10.
    Costa, EO, Carciofi, AC, Melville, PA, Prada, MA, Ribeiro, AR, Watanabe, ET 1996Bovine mastitis due to algae of the genus Prototheca spMycopathologia1338588PubMedCrossRefGoogle Scholar
  11. 11.
    Taniyama, H, Okamoto, F, Kurosawa, T, Furuoka, H, Kaji, Y, Okada, H, Matsukawa, K 1994Disseminated protothecosis caused by Prototheca zopfii in a cowVet. Pathol.31123125PubMedCrossRefGoogle Scholar
  12. 12.
    Gaunt, SD, McGrath, RK, Cox, HU 1984Disseminated protothecosis in a dogJ. Am. Vet. Med. Assoc.185906907PubMedGoogle Scholar
  13. 13.
    Migaki, G, Font, RL, Sauer, RM, Kaplan, W, Miller, RL 1982Canine protothecosis: Review of the literature and report of an additional caseJ. Am. Vet. Med. Ass.181794797Google Scholar
  14. 14.
    Coloe, PJ, Allison, JF 1982Protothecosis in a catJ. Am. Vet. Med. Assoc.1807879PubMedGoogle Scholar
  15. 15.
    Kaplan, W, Chandler, FW, Holzinger, EA, Plue, RE, Dickinson, RO,3rd 1976Protothecosis in a cat: First recorded caseSabouraudia14281286PubMedGoogle Scholar
  16. 16.
    Smith, CG 1980Ultrastructural study of Prototheca salmonis and comparison with known isolates of Prototheca Mycopathologia7195101CrossRefGoogle Scholar
  17. 17.
    Costa, EO, Melville, PA, Ribeiro, AR, Watanabe, ET, Parolari, MC 1997Epidemiologic study of environmental sources in a Prototheca zopfii outbreak of bovine mastitisMycopathologia1373336PubMedCrossRefGoogle Scholar
  18. 18.
    Costa, EO, Carciofi, AC, Melville, PA, Prada, MS, Schaich, U 1996Prototheca sp. Outbreak of Bovine MastitisJ. Vet. Med. B.43321324CrossRefGoogle Scholar
  19. 19.
    Bueno, VFF, Mesquita, AJ, Neves, RB, Souza, MA, Ribeiro, AR, Nicolau, ES, Oliveira, AN 2006Epidemiological and clinical aspects of the first outbreak of bovine mastitis caused by Prototheca zopfii in Goias State, BrazilMycophatologia161141145CrossRefGoogle Scholar
  20. 20.
    Melville, PA, Watanabe, ET, Benites, NR, Rbeiro, AR, Silva, JAB, Garino, F, Costa, E 1999Evaluation of the susceptibility of Prototheca zopfii to milk pasteurizationMycopathologia1477982CrossRefGoogle Scholar
  21. 21.
    Melville, PA, Benites, NR, Sinhorini, IL, Costa, EO 2002Susceptibility and features of the ultrastructure of Prototheca zopfii following exposure to copper sulphate, silver nitrate and chlorexidineMycophatologia15617CrossRefGoogle Scholar
  22. 22.
    Green, MJ, Leach, KA, Breen, JE, Green, LE, Bradley, AJ 2005Where are we with mastitis control? Current thoughts and preliminary findings from a national research projectCatt. Pract.13127129Google Scholar
  23. 23.
    Seegers, H, Fourichon, C, Beaudeau, F 2003Production effects related to mastitis and mastitis economics in dairy cattle herdsVet. Res.34475491PubMedCrossRefGoogle Scholar
  24. 24.
    Akira, S, Takeda, K, Kaisho, T 2001Toll-like receptors: Critical proteins linking innate and acquired immunityNat. Immunol.2675680PubMedCrossRefGoogle Scholar
  25. 25.
    Dinarello, CA 1996Cytokines as mediators in the pathogenesis of septic shockCurr. Top. Microbiol. Immunol.216133165PubMedGoogle Scholar
  26. 26.
    Thijs, LG, Groeneveld, AB, Hack, CE 1996Multiple organ failure in septic shockCurr. Top. Microbiol. Immunol.216209237PubMedGoogle Scholar
  27. 27.
    Corbellini, LG, Driemeier, D, Cruz, C, Dias, MM, Ferreiro, L 2001Bovine mastitis due to Prototheca zopfii: Clinical, epidemiological and pathological aspects in a Brazilian dairy herdTrop. Anim. Health Prod.33463470PubMedCrossRefGoogle Scholar
  28. 28.
    Pore, RS 1985Prototheca taxonomyMycopathologia90129139CrossRefGoogle Scholar
  29. 29.
    Camargo, ZP, Fischman, O 1979Use of morpho-physiological characteristics for differentiation of the species of Prototheca Sabouraudia17275278Google Scholar
  30. 30.
    Schalm, OW, Noorlander, DO 1957Experimental and observations leading to developing of California mastitis testJ. Am. Vet. Med. Assoc.130199204PubMedGoogle Scholar
  31. 31.
    Weber, L, Peterhans, E, Wyler, R 1983The chemiluminescent response of bovine polymorphonuclear leucocytes isolated from milk and bloodVet. Immunol. Immunopathol.4397412PubMedCrossRefGoogle Scholar
  32. 32.
    Absolom, DR 1986Basic methods for the study of phagocytosisMeth. Enzymol.13295180PubMedGoogle Scholar
  33. 33.
    Pick, E, Mizel, D 1981Rapid microassays for the measurement of superoxide and hydrogen peroxide production by macrophages in culture using na automatic enzyme immunoassay readerJ. Immunol. Meth.46211226CrossRefGoogle Scholar
  34. 34.
    Beers, RF, Sizer, JW 1952A spectrophotometric method of measuring the breakdown of hidrogen peroxide by catalaseJ. Biol.Chem.195133140PubMedGoogle Scholar
  35. 35.
    Carlberg, I, Mannervick, B 1985Glutathione reductaseMeth. Enzymol.113484490PubMedCrossRefGoogle Scholar
  36. 36.
    Russ JC. The Image Processing Handbook. CRC Press, 1998Google Scholar
  37. 37.
    Babior, BM 1992The respiratory burst oxidaseAdv. Enzymol. Rel.654995Google Scholar
  38. 38.
    Segal, A W, Abo, A 1993The biochemical basis of the NADH oxidase of phagocytosisTrends Biochem. Sci.184347PubMedCrossRefGoogle Scholar
  39. 39.
    Halliwell, B, Gutteridge, MC 1999Free radicals in biology and medicineOxford University Press. IncNew York389395Google Scholar
  40. 40.
    Foucaud, L, Bennasroune, A, Klestadt, D, Laval-Gilly, P, Falla, J 2006Oxidative stress induction by short time exposure to ozone on THP-1 cellsToxicol. In Vitro.20101108PubMedCrossRefGoogle Scholar
  41. 41.
    Valentine, JS, Doucette, PA, Potter, SZ 2005Copper-zinc superoxide dismutase and amyotrophic lateral sclerosisRev. Biochem.74563593CrossRefGoogle Scholar
  42. 42.
    Rodriguez, C, Mayo, JC, Sainz, RM, Antolin, I, Herrera, F, Martin, V, Reiter, RJ 2004Regulation of antioxidant enzymes: A significant role for melatoninJ. Pineal Res.3619PubMedCrossRefGoogle Scholar
  43. 43.
    Florea, NF, Nightingale, CH 2004Review of the pharmacodynamics of antibiotic use in animal food productionDiagn. Microbiol. Infect. Dis.49105108PubMedCrossRefGoogle Scholar
  44. 44.
    Permpanich, P, Kowolik, MJ, Galli, DM 2006Resistance of fluorescent-labelled Actinobacillus actinomycetemcomitans strains to phagocytosis and killing by human neutrophilsCell. Microbiol.87284PubMedCrossRefGoogle Scholar
  45. 45.
    Phair, JP, Williams, JE, Bassaris, HP, Zeiss, CR, Morlock, BA 1981Phagocytosis and algicidal activity of human polymorphonuclear neutrophils against Prototheca wickerhamii J. Infect. Dis.1447277PubMedGoogle Scholar
  46. 46.
    Boyd, AS, Langley, M, King, LE,Jr 1995Cutaneous manifestations of Prototheca infectionsJ. Am. Acad. Dermatol.32758764PubMedCrossRefGoogle Scholar
  47. 47.
    Walsh, SV, Johnson, RA, Tahan, SR 1998Protothecosis: An unusual cause of chronic subcutaneous and soft tissue infectionAmer. J. Derm.20379382CrossRefGoogle Scholar
  48. 48.
    Carey, WP, Kaykova, Y, Bandres, JC, Sidhu, GS, Brau, N 1997Cutaneous protothecosis in a patient with AIDS and a severe functional neutrophil defect: Successful therapy with amphotericin bClin. Infec. Dis.2512651266CrossRefGoogle Scholar
  49. 49.
    Wirth, FA, Passalacqua, JA, Kao, G 1999Disseminated cutaneous protothecosis in an immunocompromised host: A case report and literature review Cutis63185188Google Scholar
  50. 50.
    Jensen, HE, Aalbaek, B, Bloch, B, Huda, A 1998Bovine mammary protothecosis due to Prototheca zopfii Med. Mycol.368995PubMedCrossRefGoogle Scholar

Copyright information

© Springer 2006

Authors and Affiliations

  • Luciane T. Cunha
    • 1
  • Silvana P. Pugine
    • 1
  • Claudia R. Valle
    • 2
  • Andrea R. Ribeiro
    • 2
  • Ernane J. X. Costa
    • 1
  • Mariza P. De Melo
    • 1
    Email author
  1. 1.Departament of Basic Science, Faculty of Zootechny and Food Engineering-FZEAUniversidade de São PauloPirassunungaBrazil
  2. 2.Faculty of Medicine VeterinaryPUCPocos de CaldasBrazil

Personalised recommendations