GIARDIASIS IN THE MOUSE: AN' ANIMAL MODEL

Transcription

1 GASTROEl'TEROLOGY 71: by The Williams & Wilkins Co. Vol. 71, No.1 Printed n U.S.A. GARDASS N THE MOUSE: AN' ANMAL MODEL AN C. ROBERTS-THOMSON, M.D., DAVD P. STEVENS, M.D., ADEL A. F. MAHMOUD, M.D., Ph.D., AND KENNETH S. WARREN, M.D. Divisions of Gastroenterology and Geographic Medicine, Department of Medicine, Case Western Reserve University and University Hospitals, Cleveland, Ohio An animal model for giardiasis was developed using Giardia murls Swiss albino mice. ntraesophageal inoculation of G. muris cysts caused a reproducible pattern of infection, with trophozoite and cyst counts reaching a maximum on days 5 to 14 after cyst inoculation and thereafter showing a progressive decline. Spontaneous resolution of infection occurred in most mice after 21 to 28 days. When compared to uninfected controls, Giardia-infected mice had significant impairment of weight gain Bnd a significant reduction in the villus to crypt ratio of jejunal mucosa. Although maximal trophozoite and cyst counts were independent of the size of the cyst inoculum, those mice receiving inoculations of larger numbers of cysts showed earlier attainment of maximal counts, greater impairment of weight gain, and earlier and more severe small bowel changes than mice receiving inoculations of smaller numbers of cysts. This model offers unique opportunities for study of this poorly understood gastrointestinal parasite. n spite of a prevalence estimated to be 7% in the United States and higher in other areas of the world,1 giardiasis remains a poorly understood gastrointestinal infection.2 Clinical studies have identified infected persons who are asymptomatic while others demonstrate nonspecific gastrointestinal symptoms, diarrhea, and malabsorption. 3 ' 7 Similarly, studies of small bowel morphology have revealed a spectrum of changes from normal mucosa to severe mucosal lesions characterized by absence of villi, elongation of crypts, and chronic inflammatory cells in the lamina propria. 8, 8 Although persons with hypogammaglobulinemia appear more susceptible to giardiasis and to the development of severe mucosal lesions, the role of humoral immunity in the pathogenesis of disease is poorly understood. Moreover, the variable pattern of illness in persons with apparently normal immune function is unexplained. n this report is described an animal model of giardiasis. ntraesophageal inoculation of Giardia muris cysts into Swiss albino mice resulted in a gastrointestinal infection associated with impaired weight gain and morphological changes in jejunal mucosa. Materials and Methods Mice. Outbred female Swiss albino mice (CF-l), 16 to 20 gin weight, were obtained from Carworth Farms, nc., New City, New York. All animals were determined to be free of G. muris infection before use in experiments by demonstration of absence of cysts in stools. Cyst source. G. muris cysts were obtained initially from a naturally infected golden hamster in this laboratory's animal colony. The cysts were established as G. muris on the basis of cyst morphology (small median body) and the species from Received September 29, Accepted January 5, Address requests for reprints to: Dr. David P. Stevens, Department of MediCine, University H08pitals, Cleveland, Ohio which it was obtained. 1 Subsequently this strain of Giardia has been readily passaged in mice. Cyst inoculum. Methods for the isolation and quantitation of G. muris cysts were developed in this laboratory. Fresh stools from infected mice were broken up in tap water, and 3 ml of fecal suspension were layered on 2.5 ml of 1 M sucrose (specific gravity 1.11) in a 75- by 12-mm plastic tube and centrifuged at 400 x g for 15 min at 20 C. Cysts concentrated at the water-sucrose interface were carefully removed with a pasteur pipette, washed by res us pension in 4 ml of normal saline, and sedimented by centrifugation at 600 x g for 10 min. The supernatant was removed and the cysts were resuspended in normal saline and counted in a hemacytometer chamber. nocula were administered in 0.2 ml of normal saline by peroral inoculation into the esophagus with an l8-gauge blunt needle. Cysts were inoculated within 24 hr of isolation. Quantitation of cysts. Cysts in stools collected over a period of 2 hr were enumerated as follows. Nonfasted mice were isolated in individual cages for 2 hr. All stools obtained during this period were broken up in 3 ml of tap water and isolated on a sucrose gradient as previously described. Cysts were resuspended in 500 of normal saline and counted in a hemacytometer chamber, and the total cyst output per 2 hr of stool collection was calculated. Quantitation of trophozoites. After the collection of stool for cysts, mice were killed after ether anesthesia by cervical dislocation, and the entire small bowel was carefully removed. A l -cm segment of small bowel, excised at a distance of 10 cm from the gastroduodenal junction, was taken for histological study. The remaining two segments were each flushed with 5 ml of normal saline. The perfusates from both segments were pooled and motile trophozoites were counted in a hemacytometer chamber. f trophozoites were not detected, the perfusates were reexamined after 5-fold concentration by centrifugation at 50 x g for 5 min and resuspension in 2 ml of normal saline. Small bowel morphology. The segment excised for histological study was opened by longitudinal incision, carefully oriented on filter paper, and fixed in 10% formalin. After routine processing and staining with hematoxylin and eosin,

2 58 ROBERTS-THOMSON ET AL. Vol. 71, No.1 the biopsy was examined with an Olympus Vanox microscope attached to._a Vidicon Scanner television camera and monitor. 11 The magnification achieved by this method (x 4(0) permitted accurate measurements of villus and crypt heights and, consequently, determination of villus to crypt ratios. The height of the villus and adjacent crypt was measured on 10 representative, well oriented villi in at last two separate portions of each biopsy. Statistical methods. Cyst recovery after addition of 10,000, 50,000, and 100,000 cysts to a fecal suspension without cysts was expressed as a percentage, and differences between groups were analysed by Student's t-test after arcsin transformation. Cyst counts in stools and trophozoite counts in small bowel were expressed as the geometric mean, and differences between groups of mice receiving inoculations of different cyst doses were analyzed by Student's t-test. A mean value for the villus to crypt ratio of small bowel was determined for each mouse, and differences between groups were determined by analysis of variance. Results Reproducibility of cyst recovery by sucrose gradient method. The reproducibility of cyst quantitation was determined by adding a known number of cysts to a fecal suspension without cysts. Mean cyst recovery was 0, 5, 64, 65, and 60% for sucrose solutions of specific gravity 1.05, 1.075, 1.10, 1.125, and 1.15, respectively. A sucrose solution of specific gravity 1.11 was chosen for maximum cyst recovery with minimal contamination by fecal debris. Using sucrose of this specific gravity, mean cyst recovery was independent of the number of cysts added, being 63, 66, and 63% after addition of 10,000, 50,000, and 100,000 cysts, respectively (fig. 1). Natural history of infection. Giardia infection was studied in mice receiving inoculations of 0, 100, 1,000 and 10,000 cysts. Two-hour cyst output in stools and total trophozoite counts in small bowel were determined in 8 mice in each of the four groups on days 3, 5, 7, 14, 21, and 28 after cyst inoculation. Twenty-two of 24 mice receiving inoculations of 100 cysts became infected as determined by the presence of cysts or trophozoites on days 5, 7, and 14. All mice receiving inoculations of 1,000 or 10,000 cysts became infected. No Giardia infection occurred in control animals. The pattern of cyst excretion in stools collected over 2 hr is shown in figure 2. On day 3 small numbers of cysts were detected in mice receiving inoculations of 1,000 and 10,000 cysts. Thereafter, cyst excretion increased in a113 infected groups, reaching a maximum on days 7, 7, and 14 for mice receiving inoculations of 10,000, 1,000, and 100 cysts, respectively. Maximal cyst excretion did not differ significantly in the three groups. On day 5 mice receiving inoculations of 10,000 cysts had significantly higher cyst counts than mice receiving inoculations of 100 cysts (P < 0.(05). On days 21 and 28, cyst excretion was low in all three infected groups and differences were not statistically significant. Cysts were detected in only 6 of 24 mice examined on day 28. The pattern of trophozoite counts in the small bowel of infected mice is shown in figure 3 and is similar to that of cysts in stools. Maximal trophozoite counts occurred on days 5, 7, and 14 for mice receiving inoculations of 10,000, 1,000, and 100 cysts. Maximal trophozoite counts did not differ significantly in the three groups. On day 5, 100..,. T ~ T.L t en > U 50 10,000 50, ,000 CYST CONTENT FG. 1. Cyst recovery (%) after addition of 10,000,50,000, and 100,000 G. muris cysts to a cyst-free suspension of mouse feces (mean ± SE).

3 July 1976 ANMAL MODEL FOR GARDASS , ~ V t.-.j..", : jl : /,,, " 5 7 ~~. 11 TME (DAYS) CYSTS FG. 2. Cyst excretion in stool per 2 hr after inoculation of 100, 1,000, and 10,000 G. muris cysts (mean ± SE). mice receiving inoculations of 10,000 cysts had significantly higher trophozoite counts than mice receiving inoculations of 1,000 (P < 0.005) or 100 (P < 0.005) cysts, whereas on day 21, mice receiving inoculations of 10,000 cysts had significantly lower trophozoite counts than mice receiving inoculations of 1,000 (P < 0.001) and 100 (P < 0.001) cysts. On day 28, trophozoites were detected in only 5 of 24 mice examined, and differences between the three groups were not statistically significant. n a second experiment to establish the duration of infection, 8 mice received inoculations of 1,000 cysts, and cyst output per 2 hr was determined at intervals of 4 to 7 days for a period of 3 months after cyst inoculation. A typical pattern of maximal cyst output was observed on days 7 through 14. Small numqers of cysts were detected by day 28 but were undetected on day 32 and thereafter throughout the remainder of the 3-month period. Animal weight. Mice receiving inoculations of 0, 100, 1,000, and 10,000 cysts were weighed at the time of inoculation and after a period of 28 days. Eight mice were included in each group. The mean weight gain (± SE) in control mice was 8.25 g (±0.4). Mean weight gain in mice receiving inoculations of 100, 1,000, and 10,000 cysts was 6.88 (±0.3), 6.13 (±0.7) and 5.00 (±0.5) g, respectively. When these gains were compared to those in un infected control animals, the differences were statistically significant for all three infected groups: P < 0.02 for mice receiving inoculations of 100 cysts; P < 0.02 for mice receiving inoculations of 1,000 cysts; and P < for mice receiving inoculations of 10,000 cysts. Furthermore, weight gain in mice receiving inoculations of 10,000 was significantly less than weight gain in mice receiving inoculations of 100 cysts (P < 0.01). Small bowel morphology. No macroscopic changes Were observed in small bowel removed from animals harboring Giardia. n preliminary histological studies to determine the site of small bowel changes, mice receiving inoculations of 0, 1,000, and 10,000 cysts were killed on days 7, 14, 21, and 28. Small bowel biopsies were taken 5 to 10 cm from the gastroduodenal junction (upper jejunum), 15 to 20 cm from the gastroduodenal junction (mid-small bowel), and 30 to 35 cm from the gastroduodenal junction (lower ileum). Two mice were killed in each group on each day of study. n control mice the mean villus to crypt ratio (± SE) in upper jejunum, mid-small bowel, and lower ileum was 3.5 (±O.1), 2.5 (±0.2), and 1.9 (±0.2), respectively. For infected mice the villus to crypt ratio of upper jejunum decreased significantly to a mean of2.9 on day 7 and 2.7 on day 14. No significant changes occurred in the villus to crypt ratio of mid-small bowel and lower ileum. On the basis of these observations subsequent studies of small bowel morphology were confined to jejunum, 10 cm from t~e gastroduodenal junction. Jejunal morphology at this level in a control and Giardia-infected mouse is shown in figure 4. Villus to crypt ratios in jejunum were determined in mice receiving inoculations of 100, 1,000, and 10,000 cysts on days 3, 5, 7, 14,21, and 28 after cyst inoculation. Eight mice were killed from each of the three infected groups on each day of study. Villus to crypt ratios for infected mice were compared to villus to crypt ratios for a total of 16 control mice killed on days 14 and 28. The mean villus to crypt ratio (±SE) for control mice was 3.1 (±O.1). The villus to crypt ratio decreased during the course of infection, as shown in figure 5. Significant differences between control and infected mice occurred on day 3 for mice receiving inoculations of 10,000 cysts and on day 5 for mice receiving inoculations of 1,000 and 10,000 cysts. On day 7 the villus to crypt ratio in all three Z ::> o v. o J S :z: A ;--... '. J - /.. /} -' J ~ r ' ,000 CYSTS TME (DAYS) FG. 3. Trophozoite counts in small bowel after inoculation of 100, 1,000, and 10,000 G. muris cysts (mean ± SE).

4 60 ROBERTS-THOMSON ET AL. Vol. 71, No.1 FG. 4. Jejunal morphology in representative sections from a control (left) and Giardia-infected (right) mouse. nfection with G. muris is 8880Ciated with reduction of the villus to crypt ratio and an apparent increase in chronic inflammatory cells in the lamina propria. Surface epithelial cells appear normal (H & E. x SO). 4 2 > 3 U "" V ::;, > 2, : : o i 3 S TME (DAYS) 10,000 CYSTS FG. 5. Villus to crypt ratio of jejunal mucosa (mean ± SE) after inoculation of 100, 1,000, and 10,000 G. muris cysts. Mean value for un infected controls was 3.1 ± O.l. groups was significantly reduced. On day 14 significant reduction in the villus to crypt ratio was confined to mice receiving inoculations of 1,000 cysts, and on day 21 to mice receiving inoculations of 100 and 1,000 cysts. By day 28 the villus to crypt ratio in all three groups had returned to normal. Discussion nfection with Giardia has been observed in man, lower mammals, and amphibia. 1 The species which infects man has been designated Giardia lamblia. n mice, rats, and hamsters, the species has been designated G. muris. G. muris cysts differ morphologically 21 from G. Lam blia cysts in having a small median body. Furthermore, host specificity exists, as attempts to infect rodents with G. Lamblia cysts have been unsuccessful in this laboratory, and largely unsuccessful in other centers However, until further crossinfection studies have been performed the possibility of an animal reservoir for human giardiasis cannot be excluded. The present data indicate that G. muris infection in mice is characterized by proliferation of trophozoites. in small bowel, almost simultaneous excretion of cysts in stools, impaired weight gain, and mor,>hological changes in jejunal mucosa. Spontaneous resolution of infection occurred in most mice after 21 to 28 days. nfection could be induced by inoculation of 100 cysts into most mice and by inoculation of 1,000 or 10,000 cysts into all mice. The number of cysts required to induce infection and the duration of infection were similar to the finding of Rendtorff,14 who used G. lamblia cysts to infect male volunteers. Methods for quantitating trophozoites and cysts permitted an evaluation of the relationship between the number of cysts in the inoculum and the subsequent pattern of total trophozoite counts in small bowel and cyst counts in stools. Larger cyst inocula resulted in early maximal trophozoite and cyst counts, whereas with smaller cyst inocula the time of maximal trophozoite and cyst counts was delayed. However, there were no significant differences among maximal trophozoite or cyst counts attained in the three infected groups. Thus it appears that trophozoite reproduction progresses in the small bowel until numbers reach a consistently reproducible level, at which time the host clears the infection. Mechanisms for this clearance of Giardia are unknown. The association of chronic giardia-

5 July 1976 ANMAL MODEL FOR GARDASS 61 sis with hypogammaglobulinemia in man" 10 and the report of clinically significant chronic giardiasis in congenitally athymic (nude) and neonatally thymectomized micel~ support a role for immunological processes. f this is so, then the relationship between the size of the cyst inoculum and the time of maximal trophozoite and cyst counts might be explained on the basis that large cyst inocula increase the antigenic stimulus earlier in the course of infection, leading to earlier elimination of the parasite. Associated with gastrointestinal infection were significant impairment in weight gain and mild but significant changes in jejunal mucosa. mpairment of weight gain might reflect anorexia associated with infection, competition for exogenous nutrient, or malabsorption of exogenous nutrient. Of interest was the finding that mice receiving inoculations of larger numbers of cysts had earlier and more severe small bowel changes and significantly more impairment of weight gain than did mice receiving inoculations of small numbers of cysts. As the maximal trophozoite and cyst counts achieved were independent of the size of the cyst inoculum, the trophozoite load in small bowel could not be the sole determinant of small bowel changes. This observation of delayed development of maximal infection with less severe small bowel changes in mice receiving inoculations of fewer cysts suggests a dose-response effect on small bowel morphology, but a mechanism for such an obseveration is undetermined by these studies. 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. For example, the present data suggest that the size of the cyst inoculum may influence the severity of symptoms and the severity of small bowel changes in the host. Furthermore, 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. REFERENCES 1. Levine NO: Protozoan Parasites of Domestic Animals and of Man. Second edition. Minneapolis, Minnesota, Burgess Publishing, 1973, p Mahmoud AAF, Warren KS: Algorithms in the diagnosis and management of exotic diseases.. Giardiasis. J nfect Dis 131 : , Petersen H: Giardiasis (Lambliasis). Scand J Gastroenterol 14 (Suppl.): 1-44, Hoskins LC, Winawer SJ, Broitman SA, et al: Clinical giardiasis and intestinal malabsorption. Gastroenterology 53: , Walzer PO, Wolfe MS, Schultz MG: Giardiasis in travelers. J nfect Dis 124: , Thompson RG, Karandikar OS, Leek J: Giardiasis. An unusual cause of epidemic diarrhea. Lancet 1: , Center for Disease Control. Morbidity and Mortality Weekly Rep. 23: , Yardley JH, Bayless TM: Giardiasis. Gastroenterology 52: , Ament ME, Rubin CE: Relation of giardiasis to abnormal intestinal structure and function in gastrointestinal immunodeficiency syndromes. Gastroenterology 62: , Hermans PE, Huizenga KA, Hoffman HN, et al: Dysgammaglobulinemia associated with nodular lymphoid hyperplasia of the small intestine. Am J Med 40:78-89, Mahmoud AAF, Mandel MA, Warren KS, et al: Niridazole. A potent long-acting suppressant of cellular hypersensitivity. J mmunol 114: , Simon CE: A critique of the supposed rodent origin of human giardiasis. Am J Hyg 2: , Armaghan V: Biological studies on the giardia of rats. Am J Hyg 26: , Rendtorff RC: The experimental transmission of human intestinal protozoan parasites.. Giardia lamblia cysts given in capsules. Am J Hyg 59: , Boorman GA, Lina PHC, Zurcher C, et al: Hexamita and Giardia as a cause of mortality in congenitally thymus-less (nude) mice. Clin Exp mmunol 13: , 1973