The Protective Effect of a Milk Diet on Toxoplasma gondii Infection in Mice C. H. Lai, 1. R. Tizard and D. G. Ingram*
ABSTRACT The effect of an all milk diet on resistance to Toxoplasma gondii infection in mice was studied. Mice fed an all milk diet were more resistant than conventionally fed mice to T. gondii. It was shown that this effect was due to a dietary deficiency para-aminobenzoic acid.
milk is deficient in PABA, containing less than 0.1 mg/liter (12). It appears that animals and man (7, 8, 9) which do not obtain PABA from their diet are resistant to P. berghei infection. Since Toxoplwsma gondii requires PABA for growth (11), and is susceptible to sulphonamides (4), it was the purpose of this investigation to determine whether mice fed on a diet consisting sole-ly of cow's milk were protected against T. gondii infection.
RASUMA Cette experience visait 'a tudier l'effet d'une diete lactee sur la resistance de la souris a l'infection par Toxoplasma gondii. Les souris qui ne requrent que du lait s'avererent plus resistantes que celles 'a qui on servait une ration conventionnelle. On detmontra que cet effet reoultait d'une d&ficience de la diete lactee en acide para-aminobenzoique.
INTRODUCTION In 1952 Maegraith, Deegan and Jones (13) reported that mice fed on an all milk diet were protected against Plasmodium berghei. Upon investigation it was demonstrated that the failure of the malaria parasites to develop under these circumstances was due to a dietary deficiency of para-aminobenzoic acid (PABA). Fresh *Department of Veterinary Microbiology and Immunology, Ontario Veterinary College, University of Guelph, Gueiph, Ontario. Submitted September 20, 1974.
Vol. 39-April, 1975
MATERIALS AND METHODS ANIMALS
Adult outbred, white swiss mice (30 to 40 g) were obtained from the Connaught Research Laboratories, Toronto. They were maintained in the isolation unit of the Ontario Veterinary College at an average temperature of 22°C and 40% relative humidity. Commercial laboratory feed (Purina Chow Company, Woodstock, Ontario) was fed ad libitum to mice which were not fed milk. MILK
Pasteurized and homogenized milk (Guelph Royal Dairy) was supplied to the test groups in their drinking bottle and was replaced frequently to minimize souring. It was determined that each mouse drank approximately 6 to 8 ml of milk in 24 hours. Mice subsisting on an all milk diet lost about 20% of their body weight within the first week but gained weight subsequently. They occasionally developed mild diarrhea.
191
SEROLOGICAL TEST
RESULTS
Milk and serum samples were examined for antibodies to T. goniii by the SabinFeldman dye test (14).
The milk did not contain antibodies to T. gondii when examined by the SabinFeldman dye test. No significant difference in survival time was observed between the groups of mice which were challenged with 1 x 103 Toxoplasma gondii or 5 x 103 organisms (Table I). However, Organisms of the RH strain of T. gondii milk fed mice which received 1 x 102 organwere harvested from the peritoneal fluid isms subcutaneously survived for a signifiof mice which had been infected three days cantly longer period than mice fed laborapreviously. Large numbers of relatively tory chow (P < 0.001). Addition of 1 pure organisms were found free in the peri- g/liter PABA to the milk completely toneal cavity at that time. The organisms abrogated the protective effect (Table I). were counted by using a hemocytometer. Two mice from the group which had reThe organisms were then used to infected ceived milk, survived for two months after mice by either intraperitoneal or subcutane- infection. The sera of these surviving mice did not ous inoculation. contain antibodies to T. gondii demonstrable by the Sabin-Feldman dye test and no Toxoplasma cysts were observed in tissue EXPERIMENTAL PROCEDURE sections of their brains. Homogenates of brain from these surviving mice were inGroups of mice were placed on a milk oculated into normal mice. The sera of the diet four days prior to infection and at the recipient mice were examined three weeks same time, control groups were maintained later and did not contain antibodies to T. on laboratory chow. Groups of mice were gondii. No Toxoplasma cysts were seen in challenged intraperitoneally with 1 x 102, tissue sections of the brains of these reci1 x 103 or 5 x 103 T. gondii of the RH strain pient mice. and were observed for 60 days. In one The experiment to determine the effect group of mice the all milk diet was supple- of an all milk diet on infection with the mented with 1 gm of PABA per liter of sulphadiazine resistant strain of T. go'ndii milk to determine whether this supple- showed that the all milk diet did not enmentation would reverse the effect of the hance resistance to this mutant strain all milk diet. The survival time of mice (Table I). No difference in survival time fed on normal chow was compared with the was observed between milk fed and chow survival of mice fed on milk alone. fed groups of mice infected with the sulIn the course of a series of experiments phadiazine resistant strain of T. gondii. in our laboratory, a sulphadiazine resistant strain of T. gondii was detected (10). The DISCUSSION effect of an all milk diet on the susceptibility of mice to this mutant strain of T. gondii was also determined. The results indicate that mice fed on a The mice which survived infection were diet consisting solely of milk had a reduced killed at 60 days after infection and their susceptibility to T. gondii infection. It is serum was examined for antibodies by the suggested that this protective effect was Sabin-Feldman dye test (14). Sections of due to a dietary deficiency of PABA. A the brains from the surviving mice were similar phenomenon has also been observed examined microscopically for cysts and in rats and monkeys infected with P. homogenates of brain tissue were inocu- berghei and Plasmodium knowlesi respeclated into susceptible mice in an attempt to tively (5). T. gondii requires PABA as a demonstrate the presence of T. gondii in nutrient to synthesize folic acid, folinic the brain. Three weeks after inoculation of acid and subsequently to form DNA (3). brain the recipient mice were killed, serum When infected animals are treated with samples were tested for the presence of sulfonamide this biosynthetic sequence is antibody and the brains of the recipient blocked in the parasites. However, a chronic mice were examined microscopically for T. infection may be established in sulfonamide treated infected animals (1), indicating gondii cysts.
192
Can. J. comp. Med.
TABLE I. Effect of an All Milk Diet on Resistance to Toxoplasma gondii Infection No. of No. of Challenge Route of Strain Organisms Challenge Feed Mice 5 x 103 10 RH ip chow 10 5 x 103 milk RH ip 1 x 103 10 chow RH ip 10 RH 1 x 103 milk ip 1 x 102 8 sc RH chow 1 x 102 8 sc RH milk + PABA 1 x 102 8 RH sc milk 7 1 x 102 sc SRb chow 1 x 102 7 milk sc SR
Day after Challenge 4
10a 10
10 10 8 8 8 7 7
5 6 7 8 10 8 1 0 10 10 0 0 10 10 10 3 10 10 10 10 8 8 8 8 8 8 8 8
8 7 7
8 7 7
8 7 7
8 7 7
9 10 11 12 13 60 0 2 8 8
0 0 3 5
1 1
1 0
0 0
0 0
8 7 7
5 3 7
2 2 7
2 1 2
2 0 0
2
,Number of surviving mice bSulfadiazine resistant strain of Toxoplasma gondii ip = Intraperitoneal sc = Subcutaneous
that T. gondii may not be as sensitive as Plasmodia spp. to sulfonamides and that T. gondii may possess other biosynthetic pathways (15). Examination of serum and tissues from milk fed mice which survived the challenge infection did not show any evidence that the challenge had established an infection in these mice. Antibodies against T. gondii were not demonstrable in the serum, the brain tissues were not infectious for susceptible mice and no Toxopkasma cysts were seen in brain tissue. These data indicate that T. gondii failed to survive in the milk fed animals. These findings may explain the lack of susceptibility of some young nursing animals to infection with T. gondii. The resistance of the milk fed mice to T. gondii could be influenced by antibodies to T. gondii in the milk. It is known that antibody present in the milk can be passively transferred through the small intestine, however, this situation occurs only during the first two to three weeks of life in mice (6). In addition it is known that antibody alone cannot protect an animal from Toxoplasma infection (2). In this study, only adult mice were used and the cow's milk employed was negative for antiToxoplasma antibodies by the Sabin-Feldman dye test showing that the presence of antibodies in the milk was not the explanation for the resistance to T. gondii in these
experiments.
The feeding of an all milk diet reduced susceptibility to T. gondii infection in mice. The practical significance of this observation to human and animal toxoplasmosis remains to be investigated.
Vol. 39 -April, 1975
REFERENCES 1. FRENKEL, J. K. Host, strain and treatment variatLion as factors in the pathogenesis of toxoplasmosis. Am. J. trop. Med. Hyg. 2: 390-416. 1953. 2. FRENKEL, J. K. Adoptive immunity to intracellular intection. J. Immun. 98: 1309-1319. 1967. 3. FRENKEL, J. K. Toxoplasmosis. In Pathology of Protozoal and Helminthic Diseases with Clinical Correlation. R. A. Marcial Rojas, Ed. pp. 254-290. Baltimore: Williams and Wilkins. 1971. 4. FRENKEL, J .K. and G. H. HITCHINGS. Relative reversal by vitamins (p-aminobenzoic acid, folic and folinic acids) of the effect of sulfadiazine and pyrimethamine on Toxoplasma in mouse and man. Antibiotic Chemother. 7: 630-638. 1957. 5. HAWKING, F. Milk, p-aminobenzoate and malaria of rats and monkeys. Br. med. J. 4: 425-429. 1954. 6. HUMPHREY, J. H. and R. G. WHITE. Immunology for Students of Medicine. Third Edition. p. 314. Oxford: Blackwell. 1970. 7. KRETSCHMAR, W. The significance of p-aminobenzoic acid for the course of infection and immunity in animal malaria (Plasmodium berghei) and that of man (Plasmnodium falciparum), L. Investigations with NMRI mice. Z. Tropenmed. Parasit. 17: 301-320. 1966. 8. KRETSCHMAR, W. The significance of p-aminobenzoic acid for the course of infection and immunity in animal malaria (Plasmodium berghei) and that of man (Plasmodium falciparum). II. Investigations on infants in Nigeria receiving a normal diet. Z. Tropenmed. Parasit. 17: 369-374. 1966. 9. KRETSCHMAR, W. The significance of p-aminobenzoic acid for the course of infection and immunity in animal malaria. (Plasmodium berghei) and that of man (Plasmodium falciparum). III. Investigations on those 'ed on milk. Z. Tropenmed. Parasit. 17: 375-390. 1966. 10. LAI, C. H., L. R. TIZARD and D. G. INGRAM. Development of a sulphonamide resistant strain of Toxoplasma gondii. Trans. R. Soe. trop. Med. Hyg. 68: 267-258. 1974. 11. LYCKE, E. and E. LUND. Studies on the reproduction of Texoplasma gendii in a cell culture system inhibition or stimulation of growth by change in the p-aminobenzoic acid and the folic acid metabolism. Acta. path. microbiol. seand. 67: 276-290. 1966. 12. OSER, B. L. Hawk's Physiological Chemistry, 14th Edition. Chapter 21, p. 684. New York: McGrawHill Book Company. 1965. 13. MAEGRAITH, B. G., T. DEEGAN and E. S. JONES. Suppression of malaria (P. brghei) by milk. Br. med. J. 2: 1382-1384. 1952. 14. SABIN, A. B. and H. A. FELDMAN. Dye as micro. chemical indicators of a new Immunity phenomenon affecting a protozoon parasite (Toxoplasma). Science, N.Y. 108: 660-663. 1948. 15. SUMMER, W. A. Antagonism of sulfonamide inhibition by para-aminobensoic acid and folic acid in Toxoplasma infected mioe. Proc. Soc. exp. Biol. Med. 66: 509-511. 1947.
193
ABSTRACT The effect of an all milk diet on resistance to Toxoplasma gondii infection in mice was studied. Mice fed an all milk diet were more resistant than conventionally fed mice to T. gondii. It was shown that this effect was due to a dietary deficiency para-aminobenzoic acid.
milk is deficient in PABA, containing less than 0.1 mg/liter (12). It appears that animals and man (7, 8, 9) which do not obtain PABA from their diet are resistant to P. berghei infection. Since Toxoplwsma gondii requires PABA for growth (11), and is susceptible to sulphonamides (4), it was the purpose of this investigation to determine whether mice fed on a diet consisting sole-ly of cow's milk were protected against T. gondii infection.
RASUMA Cette experience visait 'a tudier l'effet d'une diete lactee sur la resistance de la souris a l'infection par Toxoplasma gondii. Les souris qui ne requrent que du lait s'avererent plus resistantes que celles 'a qui on servait une ration conventionnelle. On detmontra que cet effet reoultait d'une d&ficience de la diete lactee en acide para-aminobenzoique.
INTRODUCTION In 1952 Maegraith, Deegan and Jones (13) reported that mice fed on an all milk diet were protected against Plasmodium berghei. Upon investigation it was demonstrated that the failure of the malaria parasites to develop under these circumstances was due to a dietary deficiency of para-aminobenzoic acid (PABA). Fresh *Department of Veterinary Microbiology and Immunology, Ontario Veterinary College, University of Guelph, Gueiph, Ontario. Submitted September 20, 1974.
Vol. 39-April, 1975
MATERIALS AND METHODS ANIMALS
Adult outbred, white swiss mice (30 to 40 g) were obtained from the Connaught Research Laboratories, Toronto. They were maintained in the isolation unit of the Ontario Veterinary College at an average temperature of 22°C and 40% relative humidity. Commercial laboratory feed (Purina Chow Company, Woodstock, Ontario) was fed ad libitum to mice which were not fed milk. MILK
Pasteurized and homogenized milk (Guelph Royal Dairy) was supplied to the test groups in their drinking bottle and was replaced frequently to minimize souring. It was determined that each mouse drank approximately 6 to 8 ml of milk in 24 hours. Mice subsisting on an all milk diet lost about 20% of their body weight within the first week but gained weight subsequently. They occasionally developed mild diarrhea.
191
SEROLOGICAL TEST
RESULTS
Milk and serum samples were examined for antibodies to T. goniii by the SabinFeldman dye test (14).
The milk did not contain antibodies to T. gondii when examined by the SabinFeldman dye test. No significant difference in survival time was observed between the groups of mice which were challenged with 1 x 103 Toxoplasma gondii or 5 x 103 organisms (Table I). However, Organisms of the RH strain of T. gondii milk fed mice which received 1 x 102 organwere harvested from the peritoneal fluid isms subcutaneously survived for a signifiof mice which had been infected three days cantly longer period than mice fed laborapreviously. Large numbers of relatively tory chow (P < 0.001). Addition of 1 pure organisms were found free in the peri- g/liter PABA to the milk completely toneal cavity at that time. The organisms abrogated the protective effect (Table I). were counted by using a hemocytometer. Two mice from the group which had reThe organisms were then used to infected ceived milk, survived for two months after mice by either intraperitoneal or subcutane- infection. The sera of these surviving mice did not ous inoculation. contain antibodies to T. gondii demonstrable by the Sabin-Feldman dye test and no Toxoplasma cysts were observed in tissue EXPERIMENTAL PROCEDURE sections of their brains. Homogenates of brain from these surviving mice were inGroups of mice were placed on a milk oculated into normal mice. The sera of the diet four days prior to infection and at the recipient mice were examined three weeks same time, control groups were maintained later and did not contain antibodies to T. on laboratory chow. Groups of mice were gondii. No Toxoplasma cysts were seen in challenged intraperitoneally with 1 x 102, tissue sections of the brains of these reci1 x 103 or 5 x 103 T. gondii of the RH strain pient mice. and were observed for 60 days. In one The experiment to determine the effect group of mice the all milk diet was supple- of an all milk diet on infection with the mented with 1 gm of PABA per liter of sulphadiazine resistant strain of T. go'ndii milk to determine whether this supple- showed that the all milk diet did not enmentation would reverse the effect of the hance resistance to this mutant strain all milk diet. The survival time of mice (Table I). No difference in survival time fed on normal chow was compared with the was observed between milk fed and chow survival of mice fed on milk alone. fed groups of mice infected with the sulIn the course of a series of experiments phadiazine resistant strain of T. gondii. in our laboratory, a sulphadiazine resistant strain of T. gondii was detected (10). The DISCUSSION effect of an all milk diet on the susceptibility of mice to this mutant strain of T. gondii was also determined. The results indicate that mice fed on a The mice which survived infection were diet consisting solely of milk had a reduced killed at 60 days after infection and their susceptibility to T. gondii infection. It is serum was examined for antibodies by the suggested that this protective effect was Sabin-Feldman dye test (14). Sections of due to a dietary deficiency of PABA. A the brains from the surviving mice were similar phenomenon has also been observed examined microscopically for cysts and in rats and monkeys infected with P. homogenates of brain tissue were inocu- berghei and Plasmodium knowlesi respeclated into susceptible mice in an attempt to tively (5). T. gondii requires PABA as a demonstrate the presence of T. gondii in nutrient to synthesize folic acid, folinic the brain. Three weeks after inoculation of acid and subsequently to form DNA (3). brain the recipient mice were killed, serum When infected animals are treated with samples were tested for the presence of sulfonamide this biosynthetic sequence is antibody and the brains of the recipient blocked in the parasites. However, a chronic mice were examined microscopically for T. infection may be established in sulfonamide treated infected animals (1), indicating gondii cysts.
192
Can. J. comp. Med.
TABLE I. Effect of an All Milk Diet on Resistance to Toxoplasma gondii Infection No. of No. of Challenge Route of Strain Organisms Challenge Feed Mice 5 x 103 10 RH ip chow 10 5 x 103 milk RH ip 1 x 103 10 chow RH ip 10 RH 1 x 103 milk ip 1 x 102 8 sc RH chow 1 x 102 8 sc RH milk + PABA 1 x 102 8 RH sc milk 7 1 x 102 sc SRb chow 1 x 102 7 milk sc SR
Day after Challenge 4
10a 10
10 10 8 8 8 7 7
5 6 7 8 10 8 1 0 10 10 0 0 10 10 10 3 10 10 10 10 8 8 8 8 8 8 8 8
8 7 7
8 7 7
8 7 7
8 7 7
9 10 11 12 13 60 0 2 8 8
0 0 3 5
1 1
1 0
0 0
0 0
8 7 7
5 3 7
2 2 7
2 1 2
2 0 0
2
,Number of surviving mice bSulfadiazine resistant strain of Toxoplasma gondii ip = Intraperitoneal sc = Subcutaneous
that T. gondii may not be as sensitive as Plasmodia spp. to sulfonamides and that T. gondii may possess other biosynthetic pathways (15). Examination of serum and tissues from milk fed mice which survived the challenge infection did not show any evidence that the challenge had established an infection in these mice. Antibodies against T. gondii were not demonstrable in the serum, the brain tissues were not infectious for susceptible mice and no Toxopkasma cysts were seen in brain tissue. These data indicate that T. gondii failed to survive in the milk fed animals. These findings may explain the lack of susceptibility of some young nursing animals to infection with T. gondii. The resistance of the milk fed mice to T. gondii could be influenced by antibodies to T. gondii in the milk. It is known that antibody present in the milk can be passively transferred through the small intestine, however, this situation occurs only during the first two to three weeks of life in mice (6). In addition it is known that antibody alone cannot protect an animal from Toxoplasma infection (2). In this study, only adult mice were used and the cow's milk employed was negative for antiToxoplasma antibodies by the Sabin-Feldman dye test showing that the presence of antibodies in the milk was not the explanation for the resistance to T. gondii in these
experiments.
The feeding of an all milk diet reduced susceptibility to T. gondii infection in mice. The practical significance of this observation to human and animal toxoplasmosis remains to be investigated.
Vol. 39 -April, 1975
REFERENCES 1. FRENKEL, J. K. Host, strain and treatment variatLion as factors in the pathogenesis of toxoplasmosis. Am. J. trop. Med. Hyg. 2: 390-416. 1953. 2. FRENKEL, J. K. Adoptive immunity to intracellular intection. J. Immun. 98: 1309-1319. 1967. 3. FRENKEL, J. K. Toxoplasmosis. In Pathology of Protozoal and Helminthic Diseases with Clinical Correlation. R. A. Marcial Rojas, Ed. pp. 254-290. Baltimore: Williams and Wilkins. 1971. 4. FRENKEL, J .K. and G. H. HITCHINGS. Relative reversal by vitamins (p-aminobenzoic acid, folic and folinic acids) of the effect of sulfadiazine and pyrimethamine on Toxoplasma in mouse and man. Antibiotic Chemother. 7: 630-638. 1957. 5. HAWKING, F. Milk, p-aminobenzoate and malaria of rats and monkeys. Br. med. J. 4: 425-429. 1954. 6. HUMPHREY, J. H. and R. G. WHITE. Immunology for Students of Medicine. Third Edition. p. 314. Oxford: Blackwell. 1970. 7. KRETSCHMAR, W. The significance of p-aminobenzoic acid for the course of infection and immunity in animal malaria (Plasmodium berghei) and that of man (Plasmnodium falciparum), L. Investigations with NMRI mice. Z. Tropenmed. Parasit. 17: 301-320. 1966. 8. KRETSCHMAR, W. The significance of p-aminobenzoic acid for the course of infection and immunity in animal malaria (Plasmodium berghei) and that of man (Plasmodium falciparum). II. Investigations on infants in Nigeria receiving a normal diet. Z. Tropenmed. Parasit. 17: 369-374. 1966. 9. KRETSCHMAR, W. The significance of p-aminobenzoic acid for the course of infection and immunity in animal malaria. (Plasmodium berghei) and that of man (Plasmodium falciparum). III. Investigations on those 'ed on milk. Z. Tropenmed. Parasit. 17: 375-390. 1966. 10. LAI, C. H., L. R. TIZARD and D. G. INGRAM. Development of a sulphonamide resistant strain of Toxoplasma gondii. Trans. R. Soe. trop. Med. Hyg. 68: 267-258. 1974. 11. LYCKE, E. and E. LUND. Studies on the reproduction of Texoplasma gendii in a cell culture system inhibition or stimulation of growth by change in the p-aminobenzoic acid and the folic acid metabolism. Acta. path. microbiol. seand. 67: 276-290. 1966. 12. OSER, B. L. Hawk's Physiological Chemistry, 14th Edition. Chapter 21, p. 684. New York: McGrawHill Book Company. 1965. 13. MAEGRAITH, B. G., T. DEEGAN and E. S. JONES. Suppression of malaria (P. brghei) by milk. Br. med. J. 2: 1382-1384. 1952. 14. SABIN, A. B. and H. A. FELDMAN. Dye as micro. chemical indicators of a new Immunity phenomenon affecting a protozoon parasite (Toxoplasma). Science, N.Y. 108: 660-663. 1948. 15. SUMMER, W. A. Antagonism of sulfonamide inhibition by para-aminobensoic acid and folic acid in Toxoplasma infected mioe. Proc. Soc. exp. Biol. Med. 66: 509-511. 1947.
193
