J Vet Sci.  2007 Jun;8(2):169-174. 10.4142/jvs.2007.8.2.169.

Suppressive effect of culture supernatant of erythrocytes and serum from dogs infected with Babesia gibsoni on the morphological maturation of canine reticulocytes in vitro

• Hossain MA
• Yamato O
• Kim G
• Yamasaki M
• Maede Y

• KMID: 1089669
• DOI: http://doi.org/10.4142/jvs.2007.8.2.169

Abstract

The present study evaluated the effects of infected culture supernatant of erythrocytes, fractionation of culture supernatant and serum from dogs infected with Babesia gibsoni (B. gibsoni) on the maturation of canine reticulocytes in vitro. The SDS-PAGE demonstrated that significantly broader bands were generated by both the infected culture supernatant of erythrocytes and the serum from dogs chronically infected with B. gibsoni. The culture supernatant of erythrocytes infected with B. gibsoni strongly suppressed the maturation of reticulocytes. Prior studies showed that chronically infected serum had inhibitory effects on both the maturation of reticulocytes and the canine pyrimidine 5'-nucleotidase subclass I and purine-specific 5'-nucleotidase activity. In addition, serum free infected culture supernatant of erythrocytes had an inhibitory effect on the morphological maturation of reticulocytes. These results suggest that infected serum and culture supernatant of erythrocytes might accumulate excess proteins and/or metabolites as a result of the inhibited maturation of reticulocytes and decreased activity of erythrocyte 5'-nucleotidase. Furthermore, the fractions observed at >150 kDa- and 150-70 kDa- in the infected culture supernatant and serum retarded the maturation of canine reticulocytes in vitro. The results obtained from the in vitro examinations, in the present study, suggested that B. gibsoni itself and/or its metabolites might release certain proteins in the infected culture supernatant and serum from infected dogs and as a result delay morphological maturation of canine reticulocytes.

Keyword

Babesia gibsoni; fraction; infected serum; reticulocyte; supernatant

MeSH Terms

Animals
Babesia/*immunology
Babesiosis/blood/immunology/parasitology/*veterinary
Cell Differentiation/immunology
Dog Diseases/*blood/immunology/*parasitology
Dogs
Electrophoresis, Polyacrylamide Gel
Erythrocytes/*immunology
Reticulocytes/*immunology

Figure

• 

  Fig. 1 SDS-PAGE analysis of serum from dogs infected with B. gibsoni and culture supernatant of erythrocytes infected with B. gibsoni compared with serum from normal dogs. Lane 1; molecular mass markers, Lane 2; serum from normal dogs, Lane 3; serum from dogs chronically infected with B. gibsoni, Lane 4; culture supernatant of erythrocytes collected after 6 days of culture infected with B. gibsoni. Serum and sample buffer were dissolved as a ratio of 1 : 10; infected supernatant and sample buffer were dissolved as ration of 1 : 1. Then 10 micrograms of sample buffer were loaded onto each lane. Arrows indicate the bands with apparent molecular mass and letters indicate the standard molecular bands.

• 

  Fig. 2 The in vitro effect of culture supernatant of erythrocytes infected with B. gibsoni on the maturation of canine reticulocytes. Reticulocyte-rich erythrocytes were incubated at 37℃ for 24 h in a humidified atmosphere containing 5% CO2 and 95% air in the incubation media, with 20% incubated supernatant collected after 6 days of incubation of the erythrocytes without infection (control) and 20% of the culture supernatant of erythrocytes collected after 6 days cultured of erythrocytes infected with B. gibsoni (infected). Vertical bars indicate the mean ± SD (n = 4), *p < 0.005, compared with the value obtained for the incubation medium with the incubated erythrocyte supernatant (control) using Student's t-test.

• 

  Fig. 3 The in vitro effect of the serum free culture supernatant of erythrocytes infected with B. gibsoni on the maturation of the canine reticulocytes. Reticulocyte-rich erythrocytes were incubated at 37℃ in a humidified atmosphere containing 5% CO2 and 95% air in the incubation media for 24 h. These conditions were used with: 20% serum from normal dogs (control); 20% incubated supernatant collected after 6 days incubation of erythrocytes from serum free media of normal dogs (normal); and 20% serum free infected culture supernatant of erythrocytes collected after 6 days cultured with B. gibsoni in the erythrocytes (infected). Vertical bars indicate the mean ± SD (n = 3), *p < 0.005 compared with values obtained from the incubation medium with 20% serum from normal dogs (control) and analyzed by the Student's t-test.

• 

  Fig. 4 The in vitro effect of serum from dogs infected with B. gibsoni and their fractions on the maturation of canine reticulocytes. Reticulocyte-rich erythrocytes were incubated at 37℃ for 24 h in a humidified atmosphere containing 5% CO2 and 95% air in the incubation media with: 20% serum from normal dogs (1); 20% serum from dogs chronically infected with B. gibsoni (2); 20% serum from the >150 kDa fraction infected with B. gibsoni (3); 20% serum from the 150-70 kDa fraction infected with B. gibsoni (4); and 20% serum <70 kDa fraction infected with B. gibsoni (5). Vertical bars indicate the mean ± SD (n = 3), *p < 0.005 and **p < 0.001 compared with the value obtained from the incubation medium with 20% serum from normal dogs (1) analyzed by the Student's t-test.

• 

  Fig. 5 The in vitro effect of the fractionated culture supernatant of erythrocytes infected with B. gibsoni on the maturation of canine reticulocytes. Reticulocyte-rich erythrocytes were incubated at 37℃ for 24 h in a humidified atmosphere containing 5% CO2 and 95% air in incubation media with: 20% serum from normal dogs (1); 20% >150 kDa fraction of the culture supernatant of erythrocytes infected with B. gibsoni (2); 20% 150-70 kDa fraction of the culture supernatant of erythrocytes infected with B. gibsoni (3); and 20% <70 kDa fraction of the culture supernatant of erythrocytes infected with B. gibsoni (4). Vertical bars indicate the mean ± SD (n = 3), *p < 0.005 compared with values obtained in the incubation medium with 20% serum from normal dogs (1) analyzed by the Student's t-test.

Reference

1. Botros BAM, Moch RW, Barsoum IS. Some observations on experimentally induced infection of dogs with Babesia gibsoni. Am J Vet Res. 1975. 36:293–296.

2. Carson CA, Phillips RS. Ristic M, Kreier JP, editors. Immunologic response of the vertebrate host to Babesia. Babesiosis. 1981. New York: Academic Press;411–443.

3. Carcy B, Precigout E, Valentin A, Gorenflot A, Schrevel J. A 37-kilodalton glycoprotein of Babesia divergens is a major component of a protective fraction containing low-molecular-mass cultured-derived exoantigens. Infect Immun. 1995. 63:811–817.

4. Farwell GE, LeGrand EK, Cobb CC. Clinical observations on Babesia gibsoni and Babesia canis infections in dogs. J Am Vet Med Assoc. 1982. 180:507–511.

5. Groves MG, Dennis GL. Babesia gibsoni: field and laboratory studies of canine infections. Exp Parasitol. 1972. 31:153–159.

6. Hossain MA, Yamato O, Yamasaki M, Jeong JR, Chang HS, Maede Y. Serum from dogs infected with Babesia gibsoni inhibits maturation of reticulocytes and erythrocyte 5'-nucleotidase activity in vitro. J Vet Med Sci. 2003. 65:1281–1286.

7. Hossain MA, Yamato O, Yamasaki M, Maede Y. Inhibitory effect of pyrimidine and purine nucleotides on the multiplication of Babesia gibsoni: possible cause of low parasitemia and simultaneous reticulocytosis in canine babesiosis. J Vet Med Sci. 2004. 66:389–395.

8. Hossain MA, Yamato O, Yamasaki M, Otsuka Y, Maede Y. Relation between reticulocyte count and characteristics of erythrocyte 5'-nucleotidase in dogs, cats, cattle and humans. J Vet Med Sci. 2003. 65:193–197.

9. Jain NC. Hematological techniques. Schalm's Veterinary Hematology. 1986. 4th ed. Philadelphia: Lea & Febiger;20–86.

10. Kawamura M, Maede Y, Namioka S. Mitogenic responsibilities of lymphocytes in canine babesiosis and the effects of splenectomy on it. Jpn J Vet Res. 1987. 35:1–10.

11. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970. 227:680–685.

12. Maede Y, Inaba M. (Na,K)-ATPase and ouabain binding in reticulocytes from dogs with high K and low K erythrocytes and their changes during maturation. J Biol Chem. 1985. 260:3337–3343.

13. Mons B. Preferential invasion of malarial merozoites into young red blood cells. Blood Cells. 1990. 16:299–312.

14. Murase T, Iwai M, Maede Y. Direct evidence for preferential multiplication of Babesia gibsoni in young erythrocytes. Parasitol Res. 1993. 79:269–271.

15. Murase T, Maede Y. Increased erythrophagocytic activity of macrophages in dogs with Babesia gibsoni infection. Nippon Juigaku Zasshi. 1990. 52:321–327.

16. Murase T, Ueda T, Yamato O, Tajima M, Maede Y. Oxidative damage and enhanced erythrophagocytosis in canine erythrocytes infected with Babesia gibsoni. J Vet Med Sci. 1996. 58:259–261.

17. Mya MM, Roy A, Saxena RK, Roy KB. Isolation, part characterization, immunogenicity, and specificity study of Plasmodium falciparum culture supernatant. Jpn J Infect Dis. 2002. 55:150–156.

18. Otsuka Y, Yamasaki M, Yamato O, Maede Y. The effect of macrophages on the erythrocyte oxidative damage and the pathogenesis of anemia in Babesia gibsoni-infected dogs with low parasitemia. J Vet Med Sci. 2002. 64:221–226.

19. Otsuka Y, Yamasaki M, Yamato O, Maede Y. Increased generation of superoxide in erythrocytes infected with Babesia gibsoni. J Vet Med Sci. 2001. 63:1077–1081.

20. Ray K, Rao TS, Sivaraman CA, Basu RN. Studies on in vitro culture supernatant of P. falciparum. Isolation of antigens for serology. Ann Trop Med Parasitol. 1987. 81:101–103.

21. Valentine WN, Fink K, Paglia DE, Harris SR, Adams WS. Hereditary hemolytic anemia with human erythrocyte pyrimidine 5'-nucleotidase deficiency. J Clin Invest. 1974. 54:866–879.

22. Yamasaki M, Asano H, Otsuka Y, Yamato O, Tajima M, Maede Y. 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. Am J Vet Res. 2000. 61:1520–1524.

23. Yamasaki M, Otsuka Y, Yamato O, Tajima M, Maede Y. The cause of the predilection of Babesia gibsoni for reticulocytes. J Vet Med Sci. 2000. 62:737–741.