Perspectives On Animal Research

Volume 1, Supplement

An Evaluation of Ten Randomly-Chosen Animal Models of Human Disease

Giardiasis (Mice)

(1)

Description of the Model:

In 1974, the protozoan Giardia lamblia was the leading cause of identifiable, epidemic, waterborne disease in America.(2) Roberts-Thomson et al. described experimental Giardia muris infection of CF-1 mice by peroral inoculation of G. muris cysts obtained from a naturally infected golden hamster. (1,2,2a)

Although the infection described in mice appears clinically more uniform than that described in man, sufficient similarities exist to suggest that study of this model will be relevant to an understanding of the interaction of host and parasite in this poorly understood disease ... this mouse model provides an opportunity to study the host's immune response to the organism, the role of the host's immune response in the natural history of giardiasis, and the infectivity and stability of Giardia trophozoites and cysts.(1)

Criterion I: Concordance between the Animal Model and the Human Disease

Clinical Presentation:

The natural history of G. muris in mice and G. lamblia in people is similar. People and mice require approximately the same number of cysts to cause infection. Infection in both is mild, self-limited, and approximately the same duration.

Pathogenesis:

There are important morphological and infectious differences between G. lamblia (G. duodenalis), the Giardia species that is pathogenic in man, and G. muris. Consequently, Meyer and Schaefer questioned whether the results of experiments using G. muris are relevant to people. They stated:

Although a great deal of information has been gained from the G. muris model research, it must be used with great caution. Over the past decade a number of striking differences have been documented between G. lamblia and G. muris. First, according to Filice, the two organisms belong to distinct morphological groups. G. lamblia belongs to the duodenalis group which is characterized by a size range of 9-21 microns long by 5-15 microns wide and having claw hammer-shaped median bodies. On the other hand, G. muris, which belongs to the muris group, is smaller, with a size range of 9.2-16.9 microns long by 6.6-10.8 microns wide, and is characterized by rounded median bodies. In vitro culture of the trophozoite has shown another difference; G. lamblia can be serially subcultivated. Attempts to culture G. muris have not been successful to date. This immediately suggests a physiological difference between the two species. Transmission electron microscopy done on Giardia trophozoites of the muris type by Nemanic et al. has demonstrated the presence of endosymbionts in some of these organisms. This could be one reason for the inability to culture G. muris, since endosymbionts have never been reported in C. lamblia trophozoites.(3)

The presence of endosymbionts (bacteria living inside the giardia trophozoites) within G. muris, but not G. lamblia, has important implications. Nemanic et al. suggested:

Endosymbionts within Giardia may be found to alter trophozoite pathogenicity, metabolism, range of infectivity, antigenic surface characteristics, and host specificity as they do in other protozoa.(4)

Meyer and Schaefer discussed other differences between the two species of Giardia:

...cyst maturation has been documented for G. lamblia but not for G. muris excystation. Excystation of G. lamblia seems to occur with mineral acids, but these are not required for G. muris. G. muris consistently excysts in the 90th percentile; however, G. lamblia cysts, depending on the cyst preparation and source, may excyst in a range anywhere from 0 to 90%. Empty cyst walls of G. muris are easily visualized by phase contrast microscopy while G. lamblia cyst walls are not. Giardia cysts from fecal material show differences as well.(3)

Further differences between G. muris and G. lamblia have been identified. Leahy et al. showed that G. muris is more resistant to treatment with chlorine than G. lamblia.(5) Edson et al. identified a 88,000 Mr antigen on G. lamblia from patients recovering from giardiasis. This protein was not found in G. muris, and it seems to be unique to G. lamblia.(6) Faubert et al. found that G. muris and G. lamblia show different patterns of cyst excretion. For example, in infected gerbils, they found:

The intermittent nature of cyst release observed in experiments with human and beaver isolates (G. lamblia) was similar to the pattern of cyst release reported in human infections by Rendtorff. However, the pattern of cyst release in animals infected with G. muris was continuous, which may indicate that the mode of cyst release is characteristic of the parasite and independent of the host.(7)

It is difficult to determine which of the above differences between G. muris and G. lamblia are clinically significant.

Another problem with the murine-G. muris model is that the results are dependent upon the choice of experimental animal. Vasudev et al. expressed caution in extrapolating results from one type of mouse to another. They stated:

Animal experiments have shown that Giardia infection in nude (athymic) mice is more severe and prolonged. In this study however, the intensity of infection in the nude mice was compared with that in the normal animals, which is strictly not comparable since species susceptibility is an important variable factor.(8)

Rosenberg and Bowman noted that experiments have shown significant mucosal change in mice but none in rats.(9) Kanwar et al. showed that 1000 G. lamblia cysts are sufficient to infect 100 percent of adult mice, but 10,000 are actually needed to achieve this infection rate in rats.(10) Smith et al. noted that, "rat and mouse peritoneal exudate macrophages are as spontaneously cytotoxic for G. lamblia as human MNL (mononuclear lymphocytes), but guinea pig peritoneal macrophages are not."(11) All of these experiments suggest that each host reacts differently to Giardia infection. This view is supported by Stevens, who stated:

Studies in the murine model of giardiasis have indicated that intensity and duration of infection can be measured in this setting by counting trophozoites in the small intestine and cysts in the faeces; however extrapolation from these results to protozoal infections in man is not yet justified.(12)

Finally, Kanwar et al. showed that Swiss albino mice acquire a resistance to secondary infection after three days when infected with G. muris, but they need nine days to acquire resistance when infected with G. lamblia.(13) Thus, G. muris has different surface antigens, different patterns of infection, and different induced immune response from G. lamblia.

In summary, the murine G. muris model seems to be of questionable relevance to the human giardiasis. As Meyer et al. concluded:

Because ... the G. muris organisms 1) fail to infect humans and 2) differ demonstrably from the G. duodenalis (lamblia) organisms that do, a key question arises as to the validity of the murine model as a basis for explaining human giardiasis.(14)

Fortunately, human clinical investigation is an acceptable alternative to studies with G. muris. Besides research with the large number of people who are unintentionally infected with Giardia, some investigators have claimed that human experimental infection is both possible and safe. The first such studies were done in 1954 by Rendtorff.(15) In 1987, Nash et al. performed similar experimental studies on 15 human volunteers with full informed consent and concluded:

The most direct way to answer many basic biological questions concerning Giardia infection in humans is by experimental infections of humans Human experimental infections with Giardia are safe and are a useful, direct approach to answer some of these questions.(16)

Criterion II: Citations

After Robert-Thomson et al. defined the animal model, (1,2) there has been extensive research with G. muris infection in mice. The authors found 154 of 165 papers in the Science Citation Index from 1976 to 1988 that cited at least one of these two original reports. (See Appendix A.) Of these, 16 were experiments involving human subjects or blood components.(17-32.32a)

Eleven of these papers referred to the animal model as evidence of acquired resistance to secondary infection.(17-27) For example, Jokipii et al. cited the 1976 paper by Roberts-Thomson et al. as evidence for acquired resistance in mice, but they also cited a 1975 human clinical study as evidence for acquired resistance in people.(21) In fact, Roberts-Thomson et at. cited two epidemiological studies from 1969 and 1970, which showed, "Prior exposure to G. lamblia might decrease susceptibility to reinfection."(2) Furthermore, in 1972, Ament and Rubin found that hypogammaglobulinemic patients appeared to be more susceptible to giardiasis.(33) Gillon, citing this work, concluded, "...the importance of the host-immune response in giardiasis only became clear when the high incidence of the disease in hypogammaglobulinaemic patients was recognized."(34) Finally, Farthing and Goka noted that the Rendtorff study on prisoners in 1954 "...demonstrated that protective immunity is acquired following exposure to Giardia."(35) Thus, the first evidence for acquired resistance in humans came from human studies.

Kasim et al. cited Roberts-Thomson et al. in an abstract on giardiasis in Saudi Arabia, but they did not elaborate on the relevance of the animal model.(28) Erlandsen et al. cited the methodology for obtaining G. muris described by Roberts-Thomson et al.(32a) Roberts-Thomson et al. reported an association of HLA Al and Bl2 genotypes with giardiasis in humans, but mice showed no correlation between their susceptibility to giardiasis and their H-2 genotypes.(29) Woo and Paterson evaluated the incidence of giardiasis in a day care center. They found:

Experimentally established Giardia-free colonies of mice, hamsters, rats, cats, and dogs could not be infected with Giardia cysts from clinical and non-clinical patients. Also, kittens, hamsters, and mice could not be infected by trophozoites from an axenic culture. However, five-day-old suckling rats can be infected with G. lamblia.(30)

Nayak et al. found that IgA against G. lamblia in milk seemed to protect suckling infants from infection.(31) This was found previously in the mouse model by Andrews and Hewett, and it is a finding of possible clinical significance.(36) However, some recent experiments indicated that the mechanism of Giardia prevention by milk may differ between mice and people. In studies using human milk in an in vitro assay, Gillin et at. showed that human milk was toxic to G. lamblia, and this process did not depend on secretory IgA. They demonstrated that bile salt-stimulated lipase, an enzyme not found in non-primates, appeared to be at least partly responsible for the Giardia-cidal action of human milk.(37) Furthermore, Gillon later questioned the importance of secretory antibodies in the milk of mice. He noted that, "...immunity may have been transferred by primed gut-derived lymphocytes as well as by specific autoantibodies."(34) Thus, while milk confers some protection against Giardia in both infant mice and humans, the mechanisms may differ.

Brandborg et al. and Balsazs and Szatlocz cited the murine model as proof of the pathogenicity of Giardia.(17,32) Previously, other investigators had demonstrated Giardia pathogenicity in humans. Rendtorff induced G. lamblia infection in volunteer prisoners in 1954,(16) and Burke cited three papers from the nineteenth century, stating that G. lamblia caused diarrhea in people.(38)

Finally, in a study involving human intestinal biopsies, Phillips found differences between human and murine infection:

There is no evidence of surface lesions as reported with infected mice. In the jejunum of the rat, G. muris is reported to cover the villi promoting the suggestion that Giardia forms a competitive barrier against absorption of foodstuffs. This theory would not be applicable to childhood infections in our experience. Indeed, in 9 recent cases (in children) G. lamblia appeared to have little overt effect on mucosal structure.(27)

Criterion III Historical Impact

A 1987 review by Farthing and Goka entitled "Immunology of giardiasis" provided a broad overview of Giardia immunology and diagnosis. They noted that the first evidence for acquired resistance to Giardia was obtained by clinical and epidemiological studies. Animal studies, they continued, were consistent with these clinical observations.

Involvement of both the humoral and cellular immune systems was also first found in human patients. Farthing and Goka wrote:

The earliest report of serum antibodies in giardiasis was by Halita and Isaicu in 1946 when they demonstrated the presence of complement-fixing antibody in patients with giardiasis ... In 1974 Radulescu et al, using indirect immunofluorescence with Giardia trophozoites obtained from the small intestine, demonstrated the presence of specific anti-Giardia antibody in the serum ... Serum antibody responses have also been detected in the mouse model of giardiasis. (35)

Demonstration of intestinal secretory antibody to Giardia in human patients preceded similar observations in infected mice. Farthing and Goka observed:

Although several reports have suggested that patients with secretory IgA deficiency are more susceptible to Giardiasis (Zinneman and Kaplan, 1972; Popovic et al, 1974) there have been few studies directed towards determining the importance of the secretory immune response in human giardiasis. Griaud et al (1981) demonstrated by indirect immunofluorescence that secretory IgA antibody was present on the surface of Giardia lamblia trophozoites in human jejunal biopsies The presence and importance of secretory immunity in giardiasis has been clearly demonstrated in the mouse model. Both anti-Giardia sIgA and IgG antibodies can be detected in mouse intestinal secretions (Anders et a1, 1982; Snider et al, 1985; Snider and Underdown, 1986; Heyworth, 1986).(35)

Regarding cellular immune response to giardiasis, Farthing and Goka cited human studies from 1976, 1977, and 1981:

...several studies have shown that there is a marked increase in the number of intraepithelial lymphocytes in the jejunal mucosa of patients with giardiasis to a level similar to that observed in untreated coeliac disease ... The numbers of intraepithelial lymphocytes decrease following treatment and clearance of the parasite.(35)

These clinical observations preceded the animal studies, first reported in 1978, that demonstrated a cellular immune response in the animal model. Using nude T-cell-deficient mice, investigators demonstrated the importance of T-cells in the elimination of Giardia in mice. Based on these findings, Farthing and Goka claimed, "Most of our understanding of the cellular immune response in giardiasis has been derived from work in the mouse model of experimental infection with Giardia muris."(35) However, it seems that the participation of the cellular immune system had already been suggested by clinical biopsy studies cited above, and it was then verified in the animal model. Thus, it is questionable whether or not this insight was derived from the animal model.

Studies of the G. lamblia parasite have been facilitated by the development of in vitro cultures. While G. muris cannot be cultured, G. lamblia is amenable to this technique. Farthing and Goka commented:

The ability to isolate and culture Giardia lamblia in the laboratory has allowed the characterization of parasite antigens both with respect to their diversity between strains and their importance in the human immune response.(35)

In terms of diagnosis of Giardia infection, Farthing and Goka contended, "Conventional approaches to diagnosis of giardiasis by microscopy are labour-intensive, relatively insensitive and depend heavily of the skill of the observer."(35) They claimed that a specific anti-Giardia IgM response appeared to be a sensitive method for the detection of acute infection. Because this test identifies human antibodies, it was developed by clinical investigation.(39) Another effective diagnostic approach is counterimmunoelectrophoresis, which was also developed with clinical specimens.(40)

In a 1984 review, Gillon addressed treatment of giardiasis, as well as many of the aspects of Giardia immunology discussed by Farthing and Goka. Gillon noted:

Quinacrine is the most commonly prescribed drug in the USA ... Metronidazole has been regarded as the drug of choice in the UK ... More recently it has been shown that a newer drug, tinidazole, appears to be more effective than metronidazole and to have fewer side-effects ... Perhaps this should now be regarded as the treatment of choice.(34)

All three drugs were used for the treatment of human giardiasis before the development of the murine C. muris model in 1976.(41,42)

Conclusions:

The murine-G. muris model of human giardiasis, although extensively studied, does not appear to have made any significant contributions to the understanding, diagnosis, or treatment of the human disease. Differences between the species of parasites and differences in the host response make extrapolation from the animal model to the human disease difficult. A large body of knowledge about human giardiasis has been gained through investigations of humans with naturallyoccurring disease. It appears that further studies in human patients offer the greatest hope for understanding the immunology, diagnosis, and treatment of this infection. Furthermore, in vitro culture of G. lamblia provides the opportunity for immunological research in a controlled environment and facilitates screening for new anti-Giardia medications.

References

1. Roberts-Thomson IC, Stevens DP, Mabmoud AAF, Warren KS: Giardiasis in the mouse: an animal model Gastroenterol 1976,71:57-61.

2. Roberts-Thomson IC, Stevens DP, Mahmoud AAF, Warren KS: Acquired resistance to infection in an animal model of giardiasis. J Immunol 1976;117:2036-2037.

2a. Stevens DP, Roberts-Thomson IC: Giardiasis, Model No. 149, in Jones TC, Hackel DB, Migaki G (eds): Handbook Animal Models of Human Disease, Fasc 7. Washington DC Registry of Comparative Pathology, Armed Forces Institute of Pathology, 1978.

3. Meyer EA, Schaeffer FW III: Models of excystation, in, Erlandsen SL, Meyer EA (eds): Giardia and Giardiasis. New York, Plenum, 1984.

4. Nemanic PC, Owen RL, Stevens DP, Mueller JC: Ultrastructural observations on giardiasis in a mouse model II. Endosymbiosis and organelle distribution in Giardia muris and Giardia lamblia. J Infect Dis 1979;140:222-228.

5. Leahy JG, Robin AJ, Sproul OJ: Inactivation of Giardia muris cysts by free chlorine. Appl Environ 1987;53:1448-1453.

6. Edson CM, Farthing MJ, Thorleyl DA, Keusch GT: An 88,000Mr Giardia lamblia surface protein which is immunogenic in humans. Infect Immunol 1986;54:621-625.

7. Faubert GM, Belosevic M, Walker TS, Maclean JD, Meerovit E: Comparative studies on the pattern of infection with Giardia sp in Mongolian gerbils. J Parasitol 1983;69:802-805.

8. Vasudev V, Ganguly NK, Anand BS. Krishna NR, Dilawari JB, Mahajan RC: A study of Giardia infection in irradiated and thymectomized mice. J Trop Med 1982;85:119-122.

9. Rosenberg IH, Bowman BB; Impact of intestinal parasites on digestive function in humans. Fed Proc 1984;43:246-250.

10. Kanwar SS, Samra H, Ganguly NK, Mahajan RC: Comparative evaluation of Giardia lamblia infection in mouse and rat. Int J Med Res 198684:577-581.

11. Smith PD, Elson CO. Keister DB, Nash TE: Human host response to Giardia lamblia 1. Spontaneous Wiling by mononuclear leukocytes in vitro. J Immunol 1985;134:4153-4162. (sic)

12. Stevens DP: Quantitative techniques. Clin Gastroenterol 1978;7:231-238.

13. Kanwar SS, Ganguly NK, Walia BNS, Mahajan RC: Acquired resistance to Giardiasis lamblia infection in mice. Trop Geograph Med 1985;37:32-36.

14. Meyer EA, Erlandsen SL, Radulescu S: Animal models for giardiasis, in, Erlandsen SL, Meyer EA (eds): Giardia and Giardiasis. New York, Plenum, 1984.

15. Rendtorff RC: The experimental transmission of human intestinal protozoan parasites II. Giardia lamblia cysts given in capsules. Am J Hygiene 1954;59:209-220.

16. Nash TE, Herrington DA, Losonsky GA, Levine MM: Experimental human studies with Giardia lamblia. J Infect Dis 1987;156:974-984.

17. Brandborg LL, Owen R, Fogel R, et al.: Giardiasis and traveler’s diarrhea. Gastroenterol 1980;78:1602-1604.

18. Istre GR, Dunlop TS, Gaspard GB, Hopkins RS: Waterborne giardiasis at a mountain resort: Evidence for acquired immunity. Am J Pub Health 1984;74:602-604.

19. Keystone JS, Krajden S, Warren MR: Person to person transmission of Giardia lamblia in day-care nurseries. Can Med Ass J 1978;119:241-248.

20. Abdelhafez MM, Elkady N, Bolbol AS, Baknina MH: Prevelance of intestinal parasite infections in Riyadh district, Saudi-Arabia, Ann Trop Med 1986;80:631-634.

21. Jokipii L, Jokipli AMM: Is predisposition to giardiasis associated with the ABO blood groups. Am J Trop Med 1980;29:5-7.

22. Lopez CE, Dykes AC, Juranek DD, et al.: Waterborne giardiasis - a community wide outbreak of disease and a high rate of asymptomatic infection. Am J Epidemiol 1980;112:495-507.

23. Agarwal A, Nash TE: Lack of cellular cytotoxicity by human mononuclear cells to Giardia. J Immunol 1986;136:3486-348&

24. Smith PD, Olson CO, Keister DB, Nash TE: Human host response to Giardia lamblia 1. spontaneous killing by mononuclear leukocytes in vitro. J Immunol 1982;128:1372-1376. (sic)

25. Walia BNS, Ganguly NK, Mahajan RC, et al.: Morbidity in preschool giardia cyst excretors Trop Geograph Med 1986;38:367-370.

26. Hill DR, Pearson RD: Ingestion of Giardia lamblia trophozoites by human mononuclear phagocytes. Infect Immunol 1987;55:3155-3161.

27. Phillips AD: Small intestine mucosa in childhood in health and disease. Sc J Gastro1981;16(s70):65-85.

28. Kasim AA, Elhelu MA: Giardiasis in Saudi-Arabia. ACT Trop 1983;40:155-158.

29. Roberts-Thomson IC, Mitchell CF, Anders RF, et at.: Genetic studies in human and murine giardiasis. Gut 198021:397-401.

30. Woo PTK, Paterson WB: Giardia lamblia in children in day-care centers in southern Ontario, and susceptibility of animals to Giardia lamblia. TRS Trop Med 1986;80:56-59.

31. Nayak N, Ganguly NK, Walia BNS, Wahi V, Kanwar SS, Mahajan RC: Specific secretory IgA in the milk of Giardia lamblia-infected and uninfected women. J Infect Dis 1987;155:724-727.

32. Balazs M, Szatlocz E: Electron microscopic examination of the mucosa of the small intestine in infection due to Giardia lamblia. Path Res Proc 1978;163:251-260.

32a. Erlandsen SL, Sherlock LA, Januschka M, Schupp DG, Shaefer FW, Jakubowski W, Bemrick WJ: Cross-species transmission of Giardia spp.: Inoculation of beavers and muskrats with cysts of human, beaver, mouse, and muskrat origin. Appl Environ Microbiol 1988; 54:2777-2785.

33. Ament ME, Rubin CE: Relation of giardiasis to abnormal intestinal structure and function in gastrointestinal immunodeficiency syndromes. Gastroenterol 1972;62:216-226.

34. Gillon J: Giardiasis: review of epidemiology, pathogenic mechanisms, and host responses. Quart J Med 1984;53:29-39.

35. Farthing MJG, Goka AJK Immunology of giardiasis. Bail Clin Gastroenterol 1987;1:589-603.

36. Andrews JS, Hewlett EL Protection against infection with Giardia must by milk containing antibody to Giardia. J Infect Dis 1981;143:242-246.

37. Gillin FD, Reiner DS, Wang CS: Human milk kills parasitic intestinal protozoa. Science 1983;221:1290-1291

38. Burke JA: Clinical and laboratory diagnosis of giardiasis. CRC Crit Rev Lab 1977;7:373-391.

39. Goka AJK, Rolston DDK, Mathan VI, Farthing MJG: Diagnosis of giardiasis by specific IgM antibody enzyme linked immunosorbent assay. Lancet 1986ii:184-186.

40. Craft JC Nelson JD: Diagnosis of giardiasis by counterimmunoelectrophoresis of feces. J Infect Dis 145:499-504.

41. Botero DR Chemotherapy of human intestinal parasitic diseases. Ann Rev Pharmacol Toxicol 1978;18:1-15.

42. Jokipii L, Jokipii AMM: Singie-dose metronidazole and tinidazole as therapy for giardiasis: Success rates, side effects, and drug absorption and elimination. J Infect Dis 1979;140:984-988.