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Echinococcus multilocularis: Responses to infection in cotton rats (Sigmodon hispidus)
Incernorionol Journalfor Parasirology Prinred in Great Brilain.
Vol. 15. No. 3 pp. 233-238.
I985 C
1985 Auwalllian
0020-7519185 53.00+0.00 Pergamon Press Ltd. Socrel.~ for Porasilology
ECHINUCOCCUS MULTILOCULARIS: RESPONSES TO INFECTION IN COTTON RATS (SIGMODON HISPIDWS) W. K. KROEZE and C. E. TANNER Institute of Parasitology, Macdonald College, McGill University, 21111 Lakeshore Road, Ste. Anne-de-Believue, P.Q., Canada HQX 1CO (Received 27 April 1984; in revisedform 28 August 1984) W. K. and TANNERC. E. 1985. Echinococcus multiiocularis: responses to infection in cotton rats (Sigmodon hispidus). International Journal for Parasitology 15: 233-238. Various aspects of the responses of cotton rats to intraperitoneal infections with Echinococcus multilocularis were examined to determine if they could be related to the progress of the infection. At 14 weeks post-infection, infected animals had enlarged spleens; there was a slight decrease in packed cell volume, but no reticulocytosis. The number of all four types of leukocytes in the peripheral blood (lymphocytes, monocytes, neutrophils and eosinophils) increased during the course of the infection. In the peritoneal fluid, the numbers of neutrophils increased, mono~ytes and basophils decreased, and lymphocytes and eosinophils remained unchanged. Antibodies to E. mu~tilocufuris were detected in the serum of infected cotton rats as early as 2 weeks post-infection. The mean levels of transaminases (SGOT and SGPT) in the serum of infected animals were higher than in controls, and 5’-nucleotidase levels were elevated in heavily infected animals. There were no differences in responses between male and female animals. Comparison with resuits previously obtained suggest that both the outcome of the infection, and responses to it, may be under host control. Abstract--KRoEzfi
INDEX KEY WORDS: Echinococcus multilocuiaris; cestode; multilocular hydatid; Sigmodon hispidus; cotton rat; anemia; reticulocytosis; leukocytes; antibody; splenomegaly; serum liver enzymes.
INTRODUCTION COTTON rats
(Sigmodon
have long been to infection with the
hispidus)
known to be susceptible metacestodes of Echinococcus multilocularis (see Sadun, Norman, Allain & King, 1957); these animals have provided stock infections of this parasite in our laboratory since 1960 (Lubinsky, 1960 a,b). Although macrophages have been implicated as effector cells in resistance stimulated by a previous infection with E. multifocularis (see Rau & Tanner, 1973, 1976; Baron & Tanner, 1977) or by treatment with BCG (Rau & Tanner, 1975; Reuben, Tanner & Rau, 1978; Reuben, Tanner & Portelance, 1979), little is known regarding the responses of cotton rats to primary infections with this parasite. The present study was done in order to follow r~ruitment of leukocytes from the bone marrow into the peripheral blood and the peritoneal cavity following infection, from where they might mediate resistance to challenge infections, or become involved in local inflammatory responses. Serum levels of specific anti-E. multilocularis antibodies were also measured during the course of the infection. In order to quantify some aspects of the pathology caused by this infection, spleen weight, packed cell volume (PCV), reticulocyte level and serum levels of three enzymes presumed to be indicators of pathology
[serum
glutamic
oxalacetic
transaminase
serum glutamic pyruvic transaminase 5’-nucleotidase] were also measured. MATERIALS
(SCOT), (SGPT) and
AND METHODS
Animals. Eight-to-ten-week-old cotton rats (Sigmodon hjspidus~ bred by brother-sister mating in our animal facility were used in all experiments. Infections. In five separate experiments, a total of 29 male and 19 female cotton rats were inoculated intraperitoneally with three small (less than 1 mm in diameter) acephalic cysts of E. multifocularis taken from stock infections in cotton rats. These experiments were done during the permissive (=susceptible) time of year, when the parasite grows most rapidly in these hosts (Rau & Tanner, 1972). In an additional experiment (Expt. 6), done during the non-permissive time of year, 11 male and nine female cotton rats were inoculated intra~ritoneally with 10 acephalic cysts, and five male and five female animals were kept as uninoculated controls. Hematologic studies. All animals were bled weekly for 14 weeks after inoculation from the retro-orbital plexus under light ether anesthesia using microcapillary tubes. For each animal, total leukocyte counts were done using a hemacytometer and differential counts of 500 leukocytes were done on Giemsa-stained blood smears. In Expt. 6, the blood was mixed at a 1 : i ratio with 1% brilliant cresyl blue in O-85% NaCl for 2 min before making smears to allow 233
234
W. K.
KROEZE
and C. E.
enumeration of reticulocytes (Humason, 1972). The number of reticulocytes per 500 randomly selected erythrocytes was counted and the data was expressed as % reticulocytes (= number of reticulocytes counted per 500 erythrocytes+S). Also in Expt. 6, microcapillary tubes containing blood were centrifuged in a microhematocrit rotor at about 600 g for 10 min. and packed cell volumes (PCV) were read in a microcapillary reader (Damon/IEC Division, Needham Hts., Massachusetts). Centrifuged microcapillary tubes containing blood were retained frozen at -20°C for use in the serological studies. In Expt. 6, animals at 14 weeks post-inoculation were bled from the retro-orbital plexus using a Pasteur pipette and then killed by cervical dislocation. To collect peritoneal cells, the skin of the abdomen was exposed and 10 ml of tissue culture medium RPM1 1640 (Flow Laboratories, McLean, Virginia), buffered with 10 mM HEPES (pH 7.2) and containing 10,000 units 1-t heoarin. 100.000 units 1-r penicillin and 100 mg 1-r streptomycin, was injected into the peritoneal cavity. After the abdomen was gently massaged, medium containing cells was drawn into a 10 ml syringe. Total peritoneal cell counts were done using a hemacytometer and data were expressed as cells ml-I of wash. Differential counts of 500 peritoneal cells were done for each animal on Giemsa-stained cytocentrifuge smears (Cytospin II, Shandon Southern Instrument Co., Sewickley, Pennsylvania). Cysts and spleens from these animals were weighed and the sera were retained for the serologic and enzyme studies. Serologic studies. Serum samples collected from animals in Expt. 6 were tested for anti-E. multi3oc~iar~ protein A-binding antibodies using a modified ELISA technique. For use as antigen, 100 ~1 of E. m~ltila~iaris cyst fluid from a cotton rat [initial protein concentration 2.8 mg ml-1 by the method of Bradford 1976)] diluted 1 : 800 in carbonate/bicarbonate buffer (pH 9.6) (Voller, Bidwell & Bartlett, 1979) was dried onto polystyrene microtiter plates (Flow Laboratories) overnight. Plates were then washed twice with 0.01 M phosphate-buffered saline (pH 7.0)+0.05% Tween-20 (PBS-Tween). To each well was added 100 ~1of serum diluted 1 : 80 in O-01 M PBS (pH 7.2). Plates were incubated in a moist chamber for 1 h at 37”C, followed by three washes in PBS-Tween. The conjugate used was 100 p1 well-l of peroxidase-labelled protein A (Flow Laboratories) diluted 1 : 1000 in 0.1 MPBS (pH 8-l); this was incubated at 37°C for 1 h, followed by three washes in PBS-Tween. o-phenylene diamine was used as substrate according to the method of Voller et al. (1979). Results were read after 1 h at room temperature using a Titertek Multiskan MC microplate reader (Flow Laboratories) at 492 nm. Each serum sample was tested in duplicate. Enzyme studies. Serum transaminases (SGOT and SGPT) were measured in fresh serum samples taken from animals at 14 weeks post-inoculation in Expt. 6 using diagnostic kits (Sigma Chemical Co., St. Louis) and results were read in a Unicam SPl800 spectrophotometer at 505 nm. Because the absorbance values at 505 nm (ABS505) obtained for some sera in the transaminase assays exceeded the limits of the standards provided by Sigma, results were not converted to units ml-t. 5’-nucleotidase levels were also measured in the same sera using a Sigma diagnostic kit, and results were read in a Beckman DU-7 spectophotom~er at 340 run. The results of this assay were expressed as units l-l of serum. Statistics. Data were analysed using analysis of variance (ANOVA) and linear regression; p-values less than 0.05
TANNER
I.J.P. VOL.
15.
1985
were considered significant. For clarity, only data collected on alternate weeks are shown in the figures. RESULTS In Expts. 1-5, 25 of 29 (86.2%) male cotton rats and 17 of 19 (89.5%) females became infected after inoculation with three acephalic cysts of E. ~~lr~loc~l~r~. In Expt. 6, nine of 11 males (8!*8%)
and eight of nine females {88*9~o) became infected after inoculation with ten cysts. In Expts. l-5, 18 of the 48 animals (37.5%) died before 14 weeks postinoculation and in Expt. 6, five of the 20 inoculated animals (25%) died (Fig. 1). All uninoculated control animals survived for the duration of the experiments.
90 -I 2 5 a 80 is
WEEKS
POST-INFECTION
FIG. 1. Survival of cotton rats following intraperitoneal inoculation of acephalic cysts of Echinococcus multilocularis. Shown are data from 48 cotton rats given three cysts during the permissive time of year (squares), 20 cotton rats given 10 cysts during the non-~rmissive time of year (closed circles) and 10 uninoculated control cotton rats (open circles).
Cysts were not weighed in Expts. l-5, but in Expt. 6, animals found to be infected at 14 weeks postinoculation had 20.52 f 2.36 g (X + S.E.) of cysts (Table I), mostly in the peritoneal cavity and liver, with a few cysts being found in the abdominal wall and in the kidneys. The heaviest infections comprised up to 21% of the host’s total body weight (xi S.E. = 9.0 rt 1.6%). Infected animals had enlarged spleens at 14 weeks post-inoculation (Table l), and there was a significant linear relationship between the size of the infection and the weight of the spleen (Fig. 2). The mean packed cell volume (PCV) of inoculated animals decreased gradually during the course of the infection, but was signi~cantly different from that in uninoculated control animals only at 14 weeks postinoculation (Table 1; Fig. 3). The decrease in PCV .was not accompanied by an increase in the level of circulating reticulocytes (Table 1).
I.J.P.VOL.15. 1985
Echinococcus multilocuiaris in cotton rats
235
TABLE I-VALUES OFVAMOUS PARAMETERS MEASURED IN COTTON RATS14 WEEKSAFTER INOCULATION WITH10 ACEPHALIC CYSTS OF Echinococcus multilocularis (EXPT. 6)
Uninoculated controls tyst weight (g) Spleen weight (g) Spleen index 5 Packed cell volume (070) % reticulocytes Circulating leukocytes % monocytes Vo lymphocytes % neutrophils % eosinophils Total/mm3 Monocytes/mm3 Lymphocytes/mm3 Neutrophils3 Eosinophils/mm3 Peritoneal leukocytes Total/ml wash x 10-6 % monocytes % lymphocytes % neutrophils % eosinophils % basophils ELBA (OD492) SCOT (ABS& SGPT (ASS& St-nucleotidase
(U/L)
10 0 0.295 f 1.469 f 42,98 2 2.66 2
Inoculated uninfected
Inoculated infected
P-t
0.026 0.127 0.60 0.13
3 0 0.283 f 1.358 * 43.07 * 2.87 2
0.020 0.045 1.13 0.43
20.5:: 1.067 6.558 39.68 3.87
+ + + ?
2.36+ 0.098 0.636 0.91 0.50
ns
2.56 f 88.46 + 5.14 * 3.84 + 12067k 309 + 10672 + 629 + 457 +
0.22 1.31 0.90 0.46 535 30 492 115 55
260 f 86.73 + 7.47 + 3.20 2 11092k 264? 9881 f 652 lr 294 +
0.41 4.71 3,34 1.00 2115 19 2224 169 29
15.13 f 54.23 f 20.57 + 10.13 * 33898 + 5351 * 18224 ? 6888 f 3454 +
2.94 3.79 3.22 1.54 1792 1199 1516 1125 528
** *** ** ** *** ** *** *** ***
2.265 92.50 2.94 0.14 3.82 060 0.007 0.413 0.362 29.81
0.214 1.25 0.94 0.05 0.45 0.12 0.006 0.011 0.006 1.00
0447 2.69 1.06 0.11 2.38 0.11 0.005 0.010 0.009 2.46
1.593 * 0.191 50.47 + 5.24 4.18 + 0.87 42.37 YIZ 5.70 290 + 0.69 0.08 + 0.05 0.304 + 0.036 0.493 + 0.014 0.474 r 0.025 31.94 * 3.94
ns *** ns *** ns ** *** *** ** ns
+ + + + * * + f + +
1.500 89.80 340 0.13 640 0.27 -0034 0.398 0.375 25.04
+ + + + + * + + + 2
*** *** *
TANOVA: n.s.-not significant; *--p < 0.05; **-p < 0.01; ***-P < OGOl *mean + one s.E., SSpleen index calculated as 1000 X spleen wt/(animal wt-cyst wt). Differential
counts
on peripheral
blood
.
(Fig. 4)
showed a progressive decrease in lymphocyte levels and an increase in the levels of neutrophils, monocytes and eosinophils. When these data were expressed as cells mm-3 blood (Fig. S), infected animals had higher levels of all four types of
I
c 45 5 3 P I! g 40 B 3
B 0 2
WEEKS
* 4 6 8 10 1214 POST-INFECTION
FIG. 3. Packed cell volume during infection with Echinococcus multilocularis in cotton rats (Expt. 6). Data is expressed as mean * S.E. Shown are values from inoculated animals (closed circles) and uninoculated controls (open circles). The single asterisk indicates a significant difference between the groups 0, < 0.05).
CYST
WEIGHT
(g)
FIG. 2. Relationship between cyst wt at 14 weeks postinfection and spleen index (Expt. 6). The open circle
represents the mean value in 10 uninoculated control cotton rats.
leukocytes than uninoculated control animals. The same trends were evident in both the 3-cyst and lo-cyst infections. In infected animals, linear regression analysis did not show a relationship between the size of the infection and the degree of change in the leukocyte counts. Differential counts of the peritoneal cells (Table 1)
W. K.
0-
0
2
4
6
8
KROEZE
and C. E.
TANNER
I.J.P.
VOL.15. 1985
10 12 14
WEEKS
POST-INFECTION WEEKS
FIN. 4. Differential counts of peripheral blood leukocytes during infection with Echinococcus multilocularis in cotton rats. Data is expressed as mean f S.E. Shown are values from Expts. l-5 (squares), Expt. 6 (closed circles) and uninoculated controls (open circles). Asterisks indicate values significantly different from controls (*--p < 0.05; **-p < 0.01; ***-Jr < 00X).
showed that infected animals had higher levels of neutrophils and lower levels of monocytes and basophils than uninoculated control animals. These same conclusions could be drawn when these data were expressed as cells ml-l of peritoneal wash. In infected animals, linear regression analysis did not show a relationship between the intensity of the infection and the peritoneal cell counts. Specific anti-E. multilocularis protein A-binding antibodies were detected in the serum of some animals as early as 2 weeks post-inoculation by ELBA. Mean antibody levels in inoculated animals were significantly higher than in controls by 4 weeks post-inoculation (Fig. 6). Specific antibody was not detected in inoculated animals that did not acquire the infection (Table 1). There was no linear relationship between the size of the infection and the level of circulating specific antibody 0, > 0.05 by linear regression). At 14 weeks post-inoculation, infected animals had significantly elevated SGOT and SGPT levels, but serum 5’-nucleotidase levels were not significantly different from controls (Table 1). However, the weight of cysts in the liver was linearly related to serum 5’-nucleotidase levels (Fig. 7), although not to either of the transaminases. No differences between male and female cotton rats were observed in any of the responses measured in the preceding experiments 0, > 0.05 by ANOVA).
POST-INFECTION
FIG. 5. Numbers of peripheral blood leukocytes during infection with Echinococcus multilocularis in cotton rats. Data is expressed as mean + S.E. Shown are values from Expts. l-5 (squares), Expt. 6 (closed circles) and uninoculated controls (open circles). Asterisks indicate values significantly different from controls (+--p < 0.05; **-p < 0.01; **t--p < O@Ol).
DISCUSSION
In the experiments
reported above, 59 of 68 (86.8%) cotton rats became infected after intraperitoneal inoculation of three or 10 acephalic cysts of Echinococcus multiloculark. This propor1
0.3-
5 0.28 ;: i WO.l-
-I’-I
0
2 WEEKS
4
6
8
10
12
14
POST-INFECTION
FIG. 6. Levels of specific anti-E. multilocularis protein A-binding antibodies during infection with E. multilocularis in cotton rats (Expt. 6). Data is expressed as mean * S.E. Shown are values from inoculated animals (closed circles) and uninoculated controls (open circles). Asterisks indicate values significantly higher than controls (*-p < 0.05; **-p < 0.01; ***-p < 0.001).
I.J.P. VOL.
15. 1985
Echinococcus multilocularisin cotton rats
WEIGHT OF LIVER CYSTS
237
(g)
FIO. 7. Relationship between weight of cysts m the liver of cotton rats at 14 weeks post-infection and serum S-nucleotidase activity (Expt. 6). The open circle represents the mean value of 10 uninoculated control cotton rats. tion is somewhat lower than the 100% infection rate reported by Sadun et al. (1957) for oral inoculations with eggs. A higher proportion of inoculated animals died before 14 weeks post-inoculation in Expts. 1-5, which were done during the permissive time of year, than in Expt. 6, done in the less permissive time of year (see Rau & Tanner, 1972), although the proportion of animals acquiring the infection was similar at both times of year. The total cyst weight in infected cotton rats at 14 weeks post-inoculation was similar to that in gerbils (Kroeze & Tanner, in press). However, in cotton rats the heaviest infections comprised up to 21% of the host’s total body wt (x + S.E. =9-O f 1.6r70), whereas in gerbils up to 57% of the body wt (x + S.E. =21.6 -t 4.0%) consisted of cyst material. The splenomegaly seen in cotton rats was similar in degree to that in gerbils (Kroeze & Tanner, in press); this phenomenon has also been reported in susceptible C57L/J mice (Ah-Khan, 1974, 1978) and in the relatively more resistant C57BL/6J mice (Devouge & Ali-Khan, 1983). However, in comparison to gerbils, infected cotton rats showed only slight decreases in packed cell volume, and little (if any) change in reticulocyte levels. In infected cotton rats, the proportion of circulating leukocytes that were lymphocytes decreased, whereas monocytes, neutrophils and eosinophils increased. However, when the actual number of circulating leukocytes was examined, all four types increased following infection. These results are in contrast with those in gerbils, in which there was a decrease in the number of circulating lymphocytes and eosinophils, but an increase in the other cell types, in the peripheral blood. Why cotton rats developed eosinophilia, and gerbils did not, remains unknown. Results obtained in mice (Sewell & Vadas, 1983) have suggested that whether or not an eosino-
philic response occurs is probably affected by the genetic make-up of the host. Other metacestode infections are known to produce eosinophilia in rodents (Freeman, 1964; Ansari & Williams, 1976; Johnson, Nicholas, Metcalf, McKenzie & Mitchell, 1979). In the peripheral blood of infected cotton rats, the numbers of both monocytes and neutrophils increases more than IO-fold, i.e., to a much higher degree than in gerbils (Kroeze & Tanner, in press). However, in the peritoneal cavity of infected cotton rats, there was a 200-fold increase in the number of neutrophils, and a significant decrease in the number of monocytes. Taken together, these data suggest that infected cotton rats undergo a generalized inflammation, but that the cysts in the peritoneal cavity act as a ‘sink’ for the peritoneal monocytes. Specific anti-E. multilocularis protein A-binding antibodies were found in the serum of infected cotton rats. These presumably were mostly IgG, the principal class of antibody binding to protein A. The kinetics of the antibody response were similar to that seen in gerbils (Kroeze & Tanner, in press). Whether antibodies are involved in control of primary infection remains unknown; however parasite-specific IgG was not found at any time in the serum of the three animals in Expt. 6 that did not become infected following inoculation of the parasite, suggesting that IgG may not have a role in controlling the infection, or that other factors may have prevented establishment of the cysts. Serum transaminases @GOT and SGPT) were significantly elevated in infected cotton rats, but the mean 5’-nucleotidase level in these animals were not significantly different from controls. However, the 5’-nucleotidase activity showed a significant relationship to the weight of cysts in the liver of infected cotton rats, whereas the tranaminases did not. Since
W. K. KROEZEand C. E. TANNER
238
all three of these enzymes are indicators of hepatobiliary disease (Widmann, 1979; Combes & Schenker, 1982), the results of the present study are somewhat equivocal in that neither enzyme showed both a significant increase in the mean level in infected animals and a relationship to the amount of parasite in the liver. The studies presented here show a number of responses to infection with E. multilocularls in cotton rats. The role of these changes in control of the infection remains uncertain. The differences in responses to the infection between cotton rats (present study) and gerbils (Kroeze & Tanner, in press), which are both susceptible hosts, suggest that the genetic constitution of the host may be important in determining both the outcome of an infection, as well as the responses to it. Studies to examine these questions using mice are currently in progress. Acknowledgements-This research was supported by the Medical Research Council of Canada. Research at the Institute of Parasitology is supported by the Natural Sciences and Engineering Research Council of Canada and the Fonds FCAC pour 1’Aide et le Soutien a la Recherche. The technical assistance of Suzanne Blanchard, Aileen McMillan and Miriam Staudt is gratefully acknowledged. Our appr~iation is also extended to Terry O’Reiily (Department of Microbiology, Macdonald College), who performed the 5’-nucleotidase assays, and to Linda Houlihan, who typed the manuscript. RJiFERENCES
ALI-KHANZ. 1974. Host-parasite reIationship in echinococcosis. II. Cyst weight, hematologic alterations, and gross changes in the spleen and lymph nodes of C57L mice against graded doses of Echinococcus multilocularis cysts. Journal of Parasitology 60: 236-242. AU-KHAN Z. 1978. Cellular changes in the lymphoreticular tissues of C57L/J mice infected with Echinococcus multilocuiaris cysts. fntmunology 34: 831-839. ANS~ A. and WILLIAMSJ. F. 1976. The ~sinop~ic response of the rat to infection with Taenia taeniaeformls. Journal of Purasitology 62: 728-736. BARON R. W. and TANNER C. E. 1977. Echinococcus multilocularis in the mouse: the in vitro protoscolicidal activity of peritoneal macrophages. International Journal for Parasitology 7: 489-495. BIUDFORDM. M. 1976. A rapid and sensitive method for the qu~titation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72: 248-254. CO~~BES B. and SCHENKERS. 1982. Laboratory tests. In: Diseases of the Liver (Edited by SCHIPPL. and Scrnsr E. R.), 5th ed., pp. 259-302. Lippincott, Philadelphia. D~vouoa M. and ALI-KHAN Z. 1983. Intraperitoneal murine alveolar hydatidosis: relationship between the
I.J.P.VOL.
15. 1985
size of the larval cyst mass, immigrant inflammatory cells, splenomegaly and thymus involution. Tropenmedizin und Parasitologie 34: H-20. FREEMANR. S. 1964. Studies on responses of intermediate hosts to infection with Taenia crassiceps (Zeder, 1800) (Cestoda). Canadian Journal of Zoologv __ 42: 367~383. HVMA~~NG. L. 1972. Animal Tissue Techniques, 3rd ed. Freeman, San Francisco. JOHNSONG. R., NICHOLAS W. L., METCALFD., MCKENZIE I. F. C. and MITCHELLG. F. 1979. Peritoneal cell population of mice infected with Mesocestoides corti as a source of eosinophils. international Archives of Allergy and Applied immunology 59: 3 1S-322. KROEZEW. K. & TANNERC. E. In press. Echinococcus multiiocularis: responses to infection in Mongolian gerbils. Experimental Parasitology. LTJBINSKY G. 196Oa. The maintenance of Echinococcus multilocularis sibiricensis without the definitive host. Canadian Journal of Zoology 3%: 149-151. LUBINSKYG. 1960b. -A vegetatively propagated strain of larval Echinococcus multil~ular~. Canadian Journal of Zoology 3%: 1117-l 12s. RAU M. E. and TANNERC. E. 1972. Echinococcus multiIocularis in the cotton rat. Asexual proliferation following the intraperitoneal inoculation of graded doses of protoscolices. Canadian Journal of Zoology 50: 941-946. RAu M. E. and TANNERC. E. 1973. Echinococcus multiIocularis in the cotton rat. The effect of pr~~sting subcutaneous cysts on the development of a subsequent intraperitoneal inoculum of protoscolices. Canadian Journal of Zoology 51: 55-59. RAU M. E. and TANNER C. E. 1975. BCG suppresses growth and metastasis of hydatid infections. Nature 256: 318-319. RAU M. E. and TANNER C. E. 1976. Echinococcus multicularis in the cotton rat: the in vitro protoscolicidal activity of peritoneal cells. International Journal for Parasitology 6: 195-198. REUBEN J. M., TANNERC. E. and PORTELANC~ V. 1979. Protection of cotton rats against experimental Echinococcus multilocularis infection with BCG cell walls. Infection and Immunity 23: 582-586. REUBEN J. M., TANNER C. E. and Uu M. E. 1978. Immunoprophyl~s with BCG of experimental Echinococcus multilocularis infections. Infection and Immunity 21: 135-139. SA~UN E. H., NORMANL., ALLAIN D. S. and Kmo N. M. 1957. Observations on the susceptibilities of cotton rats to Echinococcus multilocularis (Leuckart, 1863). Journal of Infectious Diseases 100: 273-277. SEWSLLW. A. and VADM M. A. 1983. Evidence for the control of eosinophilia by the major histocompatibility complex in mice. Immunogenetics 17: 167..177. VOLLERA., BIDWELLD. E. and BARTLETTA. 1979. The enzyme-linked immunosorbent assay (ELISA). Dynatech Laboratories, Alexandria, Virginia. WEDMANN F. K. 1979. &linitatbtterpretation of Laboratoty Tests, 8th ed. Davis, Philadelphia.
Vol. 15. No. 3 pp. 233-238.
I985 C
1985 Auwalllian
0020-7519185 53.00+0.00 Pergamon Press Ltd. Socrel.~ for Porasilology
ECHINUCOCCUS MULTILOCULARIS: RESPONSES TO INFECTION IN COTTON RATS (SIGMODON HISPIDWS) W. K. KROEZE and C. E. TANNER Institute of Parasitology, Macdonald College, McGill University, 21111 Lakeshore Road, Ste. Anne-de-Believue, P.Q., Canada HQX 1CO (Received 27 April 1984; in revisedform 28 August 1984) W. K. and TANNERC. E. 1985. Echinococcus multiiocularis: responses to infection in cotton rats (Sigmodon hispidus). International Journal for Parasitology 15: 233-238. Various aspects of the responses of cotton rats to intraperitoneal infections with Echinococcus multilocularis were examined to determine if they could be related to the progress of the infection. At 14 weeks post-infection, infected animals had enlarged spleens; there was a slight decrease in packed cell volume, but no reticulocytosis. The number of all four types of leukocytes in the peripheral blood (lymphocytes, monocytes, neutrophils and eosinophils) increased during the course of the infection. In the peritoneal fluid, the numbers of neutrophils increased, mono~ytes and basophils decreased, and lymphocytes and eosinophils remained unchanged. Antibodies to E. mu~tilocufuris were detected in the serum of infected cotton rats as early as 2 weeks post-infection. The mean levels of transaminases (SGOT and SGPT) in the serum of infected animals were higher than in controls, and 5’-nucleotidase levels were elevated in heavily infected animals. There were no differences in responses between male and female animals. Comparison with resuits previously obtained suggest that both the outcome of the infection, and responses to it, may be under host control. Abstract--KRoEzfi
INDEX KEY WORDS: Echinococcus multilocuiaris; cestode; multilocular hydatid; Sigmodon hispidus; cotton rat; anemia; reticulocytosis; leukocytes; antibody; splenomegaly; serum liver enzymes.
INTRODUCTION COTTON rats
(Sigmodon
have long been to infection with the
hispidus)
known to be susceptible metacestodes of Echinococcus multilocularis (see Sadun, Norman, Allain & King, 1957); these animals have provided stock infections of this parasite in our laboratory since 1960 (Lubinsky, 1960 a,b). Although macrophages have been implicated as effector cells in resistance stimulated by a previous infection with E. multifocularis (see Rau & Tanner, 1973, 1976; Baron & Tanner, 1977) or by treatment with BCG (Rau & Tanner, 1975; Reuben, Tanner & Rau, 1978; Reuben, Tanner & Portelance, 1979), little is known regarding the responses of cotton rats to primary infections with this parasite. The present study was done in order to follow r~ruitment of leukocytes from the bone marrow into the peripheral blood and the peritoneal cavity following infection, from where they might mediate resistance to challenge infections, or become involved in local inflammatory responses. Serum levels of specific anti-E. multilocularis antibodies were also measured during the course of the infection. In order to quantify some aspects of the pathology caused by this infection, spleen weight, packed cell volume (PCV), reticulocyte level and serum levels of three enzymes presumed to be indicators of pathology
[serum
glutamic
oxalacetic
transaminase
serum glutamic pyruvic transaminase 5’-nucleotidase] were also measured. MATERIALS
(SCOT), (SGPT) and
AND METHODS
Animals. Eight-to-ten-week-old cotton rats (Sigmodon hjspidus~ bred by brother-sister mating in our animal facility were used in all experiments. Infections. In five separate experiments, a total of 29 male and 19 female cotton rats were inoculated intraperitoneally with three small (less than 1 mm in diameter) acephalic cysts of E. multifocularis taken from stock infections in cotton rats. These experiments were done during the permissive (=susceptible) time of year, when the parasite grows most rapidly in these hosts (Rau & Tanner, 1972). In an additional experiment (Expt. 6), done during the non-permissive time of year, 11 male and nine female cotton rats were inoculated intra~ritoneally with 10 acephalic cysts, and five male and five female animals were kept as uninoculated controls. Hematologic studies. All animals were bled weekly for 14 weeks after inoculation from the retro-orbital plexus under light ether anesthesia using microcapillary tubes. For each animal, total leukocyte counts were done using a hemacytometer and differential counts of 500 leukocytes were done on Giemsa-stained blood smears. In Expt. 6, the blood was mixed at a 1 : i ratio with 1% brilliant cresyl blue in O-85% NaCl for 2 min before making smears to allow 233
234
W. K.
KROEZE
and C. E.
enumeration of reticulocytes (Humason, 1972). The number of reticulocytes per 500 randomly selected erythrocytes was counted and the data was expressed as % reticulocytes (= number of reticulocytes counted per 500 erythrocytes+S). Also in Expt. 6, microcapillary tubes containing blood were centrifuged in a microhematocrit rotor at about 600 g for 10 min. and packed cell volumes (PCV) were read in a microcapillary reader (Damon/IEC Division, Needham Hts., Massachusetts). Centrifuged microcapillary tubes containing blood were retained frozen at -20°C for use in the serological studies. In Expt. 6, animals at 14 weeks post-inoculation were bled from the retro-orbital plexus using a Pasteur pipette and then killed by cervical dislocation. To collect peritoneal cells, the skin of the abdomen was exposed and 10 ml of tissue culture medium RPM1 1640 (Flow Laboratories, McLean, Virginia), buffered with 10 mM HEPES (pH 7.2) and containing 10,000 units 1-t heoarin. 100.000 units 1-r penicillin and 100 mg 1-r streptomycin, was injected into the peritoneal cavity. After the abdomen was gently massaged, medium containing cells was drawn into a 10 ml syringe. Total peritoneal cell counts were done using a hemacytometer and data were expressed as cells ml-I of wash. Differential counts of 500 peritoneal cells were done for each animal on Giemsa-stained cytocentrifuge smears (Cytospin II, Shandon Southern Instrument Co., Sewickley, Pennsylvania). Cysts and spleens from these animals were weighed and the sera were retained for the serologic and enzyme studies. Serologic studies. Serum samples collected from animals in Expt. 6 were tested for anti-E. multi3oc~iar~ protein A-binding antibodies using a modified ELISA technique. For use as antigen, 100 ~1 of E. m~ltila~iaris cyst fluid from a cotton rat [initial protein concentration 2.8 mg ml-1 by the method of Bradford 1976)] diluted 1 : 800 in carbonate/bicarbonate buffer (pH 9.6) (Voller, Bidwell & Bartlett, 1979) was dried onto polystyrene microtiter plates (Flow Laboratories) overnight. Plates were then washed twice with 0.01 M phosphate-buffered saline (pH 7.0)+0.05% Tween-20 (PBS-Tween). To each well was added 100 ~1of serum diluted 1 : 80 in O-01 M PBS (pH 7.2). Plates were incubated in a moist chamber for 1 h at 37”C, followed by three washes in PBS-Tween. The conjugate used was 100 p1 well-l of peroxidase-labelled protein A (Flow Laboratories) diluted 1 : 1000 in 0.1 MPBS (pH 8-l); this was incubated at 37°C for 1 h, followed by three washes in PBS-Tween. o-phenylene diamine was used as substrate according to the method of Voller et al. (1979). Results were read after 1 h at room temperature using a Titertek Multiskan MC microplate reader (Flow Laboratories) at 492 nm. Each serum sample was tested in duplicate. Enzyme studies. Serum transaminases (SGOT and SGPT) were measured in fresh serum samples taken from animals at 14 weeks post-inoculation in Expt. 6 using diagnostic kits (Sigma Chemical Co., St. Louis) and results were read in a Unicam SPl800 spectrophotometer at 505 nm. Because the absorbance values at 505 nm (ABS505) obtained for some sera in the transaminase assays exceeded the limits of the standards provided by Sigma, results were not converted to units ml-t. 5’-nucleotidase levels were also measured in the same sera using a Sigma diagnostic kit, and results were read in a Beckman DU-7 spectophotom~er at 340 run. The results of this assay were expressed as units l-l of serum. Statistics. Data were analysed using analysis of variance (ANOVA) and linear regression; p-values less than 0.05
TANNER
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were considered significant. For clarity, only data collected on alternate weeks are shown in the figures. RESULTS In Expts. 1-5, 25 of 29 (86.2%) male cotton rats and 17 of 19 (89.5%) females became infected after inoculation with three acephalic cysts of E. ~~lr~loc~l~r~. In Expt. 6, nine of 11 males (8!*8%)
and eight of nine females {88*9~o) became infected after inoculation with ten cysts. In Expts. l-5, 18 of the 48 animals (37.5%) died before 14 weeks postinoculation and in Expt. 6, five of the 20 inoculated animals (25%) died (Fig. 1). All uninoculated control animals survived for the duration of the experiments.
90 -I 2 5 a 80 is
WEEKS
POST-INFECTION
FIG. 1. Survival of cotton rats following intraperitoneal inoculation of acephalic cysts of Echinococcus multilocularis. Shown are data from 48 cotton rats given three cysts during the permissive time of year (squares), 20 cotton rats given 10 cysts during the non-~rmissive time of year (closed circles) and 10 uninoculated control cotton rats (open circles).
Cysts were not weighed in Expts. l-5, but in Expt. 6, animals found to be infected at 14 weeks postinoculation had 20.52 f 2.36 g (X + S.E.) of cysts (Table I), mostly in the peritoneal cavity and liver, with a few cysts being found in the abdominal wall and in the kidneys. The heaviest infections comprised up to 21% of the host’s total body weight (xi S.E. = 9.0 rt 1.6%). Infected animals had enlarged spleens at 14 weeks post-inoculation (Table l), and there was a significant linear relationship between the size of the infection and the weight of the spleen (Fig. 2). The mean packed cell volume (PCV) of inoculated animals decreased gradually during the course of the infection, but was signi~cantly different from that in uninoculated control animals only at 14 weeks postinoculation (Table 1; Fig. 3). The decrease in PCV .was not accompanied by an increase in the level of circulating reticulocytes (Table 1).
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TABLE I-VALUES OFVAMOUS PARAMETERS MEASURED IN COTTON RATS14 WEEKSAFTER INOCULATION WITH10 ACEPHALIC CYSTS OF Echinococcus multilocularis (EXPT. 6)
Uninoculated controls tyst weight (g) Spleen weight (g) Spleen index 5 Packed cell volume (070) % reticulocytes Circulating leukocytes % monocytes Vo lymphocytes % neutrophils % eosinophils Total/mm3 Monocytes/mm3 Lymphocytes/mm3 Neutrophils3 Eosinophils/mm3 Peritoneal leukocytes Total/ml wash x 10-6 % monocytes % lymphocytes % neutrophils % eosinophils % basophils ELBA (OD492) SCOT (ABS& SGPT (ASS& St-nucleotidase
(U/L)
10 0 0.295 f 1.469 f 42,98 2 2.66 2
Inoculated uninfected
Inoculated infected
P-t
0.026 0.127 0.60 0.13
3 0 0.283 f 1.358 * 43.07 * 2.87 2
0.020 0.045 1.13 0.43
20.5:: 1.067 6.558 39.68 3.87
+ + + ?
2.36+ 0.098 0.636 0.91 0.50
ns
2.56 f 88.46 + 5.14 * 3.84 + 12067k 309 + 10672 + 629 + 457 +
0.22 1.31 0.90 0.46 535 30 492 115 55
260 f 86.73 + 7.47 + 3.20 2 11092k 264? 9881 f 652 lr 294 +
0.41 4.71 3,34 1.00 2115 19 2224 169 29
15.13 f 54.23 f 20.57 + 10.13 * 33898 + 5351 * 18224 ? 6888 f 3454 +
2.94 3.79 3.22 1.54 1792 1199 1516 1125 528
** *** ** ** *** ** *** *** ***
2.265 92.50 2.94 0.14 3.82 060 0.007 0.413 0.362 29.81
0.214 1.25 0.94 0.05 0.45 0.12 0.006 0.011 0.006 1.00
0447 2.69 1.06 0.11 2.38 0.11 0.005 0.010 0.009 2.46
1.593 * 0.191 50.47 + 5.24 4.18 + 0.87 42.37 YIZ 5.70 290 + 0.69 0.08 + 0.05 0.304 + 0.036 0.493 + 0.014 0.474 r 0.025 31.94 * 3.94
ns *** ns *** ns ** *** *** ** ns
+ + + + * * + f + +
1.500 89.80 340 0.13 640 0.27 -0034 0.398 0.375 25.04
+ + + + + * + + + 2
*** *** *
TANOVA: n.s.-not significant; *--p < 0.05; **-p < 0.01; ***-P < OGOl *mean + one s.E., SSpleen index calculated as 1000 X spleen wt/(animal wt-cyst wt). Differential
counts
on peripheral
blood
.
(Fig. 4)
showed a progressive decrease in lymphocyte levels and an increase in the levels of neutrophils, monocytes and eosinophils. When these data were expressed as cells mm-3 blood (Fig. S), infected animals had higher levels of all four types of
I
c 45 5 3 P I! g 40 B 3
B 0 2
WEEKS
* 4 6 8 10 1214 POST-INFECTION
FIG. 3. Packed cell volume during infection with Echinococcus multilocularis in cotton rats (Expt. 6). Data is expressed as mean * S.E. Shown are values from inoculated animals (closed circles) and uninoculated controls (open circles). The single asterisk indicates a significant difference between the groups 0, < 0.05).
CYST
WEIGHT
(g)
FIG. 2. Relationship between cyst wt at 14 weeks postinfection and spleen index (Expt. 6). The open circle
represents the mean value in 10 uninoculated control cotton rats.
leukocytes than uninoculated control animals. The same trends were evident in both the 3-cyst and lo-cyst infections. In infected animals, linear regression analysis did not show a relationship between the size of the infection and the degree of change in the leukocyte counts. Differential counts of the peritoneal cells (Table 1)
W. K.
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0
2
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6
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KROEZE
and C. E.
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I.J.P.
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10 12 14
WEEKS
POST-INFECTION WEEKS
FIN. 4. Differential counts of peripheral blood leukocytes during infection with Echinococcus multilocularis in cotton rats. Data is expressed as mean f S.E. Shown are values from Expts. l-5 (squares), Expt. 6 (closed circles) and uninoculated controls (open circles). Asterisks indicate values significantly different from controls (*--p < 0.05; **-p < 0.01; ***-Jr < 00X).
showed that infected animals had higher levels of neutrophils and lower levels of monocytes and basophils than uninoculated control animals. These same conclusions could be drawn when these data were expressed as cells ml-l of peritoneal wash. In infected animals, linear regression analysis did not show a relationship between the intensity of the infection and the peritoneal cell counts. Specific anti-E. multilocularis protein A-binding antibodies were detected in the serum of some animals as early as 2 weeks post-inoculation by ELBA. Mean antibody levels in inoculated animals were significantly higher than in controls by 4 weeks post-inoculation (Fig. 6). Specific antibody was not detected in inoculated animals that did not acquire the infection (Table 1). There was no linear relationship between the size of the infection and the level of circulating specific antibody 0, > 0.05 by linear regression). At 14 weeks post-inoculation, infected animals had significantly elevated SGOT and SGPT levels, but serum 5’-nucleotidase levels were not significantly different from controls (Table 1). However, the weight of cysts in the liver was linearly related to serum 5’-nucleotidase levels (Fig. 7), although not to either of the transaminases. No differences between male and female cotton rats were observed in any of the responses measured in the preceding experiments 0, > 0.05 by ANOVA).
POST-INFECTION
FIG. 5. Numbers of peripheral blood leukocytes during infection with Echinococcus multilocularis in cotton rats. Data is expressed as mean + S.E. Shown are values from Expts. l-5 (squares), Expt. 6 (closed circles) and uninoculated controls (open circles). Asterisks indicate values significantly different from controls (+--p < 0.05; **-p < 0.01; **t--p < O@Ol).
DISCUSSION
In the experiments
reported above, 59 of 68 (86.8%) cotton rats became infected after intraperitoneal inoculation of three or 10 acephalic cysts of Echinococcus multiloculark. This propor1
0.3-
5 0.28 ;: i WO.l-
-I’-I
0
2 WEEKS
4
6
8
10
12
14
POST-INFECTION
FIG. 6. Levels of specific anti-E. multilocularis protein A-binding antibodies during infection with E. multilocularis in cotton rats (Expt. 6). Data is expressed as mean * S.E. Shown are values from inoculated animals (closed circles) and uninoculated controls (open circles). Asterisks indicate values significantly higher than controls (*-p < 0.05; **-p < 0.01; ***-p < 0.001).
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Echinococcus multilocularisin cotton rats
WEIGHT OF LIVER CYSTS
237
(g)
FIO. 7. Relationship between weight of cysts m the liver of cotton rats at 14 weeks post-infection and serum S-nucleotidase activity (Expt. 6). The open circle represents the mean value of 10 uninoculated control cotton rats. tion is somewhat lower than the 100% infection rate reported by Sadun et al. (1957) for oral inoculations with eggs. A higher proportion of inoculated animals died before 14 weeks post-inoculation in Expts. 1-5, which were done during the permissive time of year, than in Expt. 6, done in the less permissive time of year (see Rau & Tanner, 1972), although the proportion of animals acquiring the infection was similar at both times of year. The total cyst weight in infected cotton rats at 14 weeks post-inoculation was similar to that in gerbils (Kroeze & Tanner, in press). However, in cotton rats the heaviest infections comprised up to 21% of the host’s total body wt (x + S.E. =9-O f 1.6r70), whereas in gerbils up to 57% of the body wt (x + S.E. =21.6 -t 4.0%) consisted of cyst material. The splenomegaly seen in cotton rats was similar in degree to that in gerbils (Kroeze & Tanner, in press); this phenomenon has also been reported in susceptible C57L/J mice (Ah-Khan, 1974, 1978) and in the relatively more resistant C57BL/6J mice (Devouge & Ali-Khan, 1983). However, in comparison to gerbils, infected cotton rats showed only slight decreases in packed cell volume, and little (if any) change in reticulocyte levels. In infected cotton rats, the proportion of circulating leukocytes that were lymphocytes decreased, whereas monocytes, neutrophils and eosinophils increased. However, when the actual number of circulating leukocytes was examined, all four types increased following infection. These results are in contrast with those in gerbils, in which there was a decrease in the number of circulating lymphocytes and eosinophils, but an increase in the other cell types, in the peripheral blood. Why cotton rats developed eosinophilia, and gerbils did not, remains unknown. Results obtained in mice (Sewell & Vadas, 1983) have suggested that whether or not an eosino-
philic response occurs is probably affected by the genetic make-up of the host. Other metacestode infections are known to produce eosinophilia in rodents (Freeman, 1964; Ansari & Williams, 1976; Johnson, Nicholas, Metcalf, McKenzie & Mitchell, 1979). In the peripheral blood of infected cotton rats, the numbers of both monocytes and neutrophils increases more than IO-fold, i.e., to a much higher degree than in gerbils (Kroeze & Tanner, in press). However, in the peritoneal cavity of infected cotton rats, there was a 200-fold increase in the number of neutrophils, and a significant decrease in the number of monocytes. Taken together, these data suggest that infected cotton rats undergo a generalized inflammation, but that the cysts in the peritoneal cavity act as a ‘sink’ for the peritoneal monocytes. Specific anti-E. multilocularis protein A-binding antibodies were found in the serum of infected cotton rats. These presumably were mostly IgG, the principal class of antibody binding to protein A. The kinetics of the antibody response were similar to that seen in gerbils (Kroeze & Tanner, in press). Whether antibodies are involved in control of primary infection remains unknown; however parasite-specific IgG was not found at any time in the serum of the three animals in Expt. 6 that did not become infected following inoculation of the parasite, suggesting that IgG may not have a role in controlling the infection, or that other factors may have prevented establishment of the cysts. Serum transaminases @GOT and SGPT) were significantly elevated in infected cotton rats, but the mean 5’-nucleotidase level in these animals were not significantly different from controls. However, the 5’-nucleotidase activity showed a significant relationship to the weight of cysts in the liver of infected cotton rats, whereas the tranaminases did not. Since
W. K. KROEZEand C. E. TANNER
238
all three of these enzymes are indicators of hepatobiliary disease (Widmann, 1979; Combes & Schenker, 1982), the results of the present study are somewhat equivocal in that neither enzyme showed both a significant increase in the mean level in infected animals and a relationship to the amount of parasite in the liver. The studies presented here show a number of responses to infection with E. multilocularls in cotton rats. The role of these changes in control of the infection remains uncertain. The differences in responses to the infection between cotton rats (present study) and gerbils (Kroeze & Tanner, in press), which are both susceptible hosts, suggest that the genetic constitution of the host may be important in determining both the outcome of an infection, as well as the responses to it. Studies to examine these questions using mice are currently in progress. Acknowledgements-This research was supported by the Medical Research Council of Canada. Research at the Institute of Parasitology is supported by the Natural Sciences and Engineering Research Council of Canada and the Fonds FCAC pour 1’Aide et le Soutien a la Recherche. The technical assistance of Suzanne Blanchard, Aileen McMillan and Miriam Staudt is gratefully acknowledged. Our appr~iation is also extended to Terry O’Reiily (Department of Microbiology, Macdonald College), who performed the 5’-nucleotidase assays, and to Linda Houlihan, who typed the manuscript. RJiFERENCES
ALI-KHANZ. 1974. Host-parasite reIationship in echinococcosis. II. Cyst weight, hematologic alterations, and gross changes in the spleen and lymph nodes of C57L mice against graded doses of Echinococcus multilocularis cysts. Journal of Parasitology 60: 236-242. AU-KHAN Z. 1978. Cellular changes in the lymphoreticular tissues of C57L/J mice infected with Echinococcus multilocuiaris cysts. fntmunology 34: 831-839. ANS~ A. and WILLIAMSJ. F. 1976. The ~sinop~ic response of the rat to infection with Taenia taeniaeformls. Journal of Purasitology 62: 728-736. BARON R. W. and TANNER C. E. 1977. Echinococcus multilocularis in the mouse: the in vitro protoscolicidal activity of peritoneal macrophages. International Journal for Parasitology 7: 489-495. BIUDFORDM. M. 1976. A rapid and sensitive method for the qu~titation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72: 248-254. CO~~BES B. and SCHENKERS. 1982. Laboratory tests. In: Diseases of the Liver (Edited by SCHIPPL. and Scrnsr E. R.), 5th ed., pp. 259-302. Lippincott, Philadelphia. D~vouoa M. and ALI-KHAN Z. 1983. Intraperitoneal murine alveolar hydatidosis: relationship between the
I.J.P.VOL.
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size of the larval cyst mass, immigrant inflammatory cells, splenomegaly and thymus involution. Tropenmedizin und Parasitologie 34: H-20. FREEMANR. S. 1964. Studies on responses of intermediate hosts to infection with Taenia crassiceps (Zeder, 1800) (Cestoda). Canadian Journal of Zoologv __ 42: 367~383. HVMA~~NG. L. 1972. Animal Tissue Techniques, 3rd ed. Freeman, San Francisco. JOHNSONG. R., NICHOLAS W. L., METCALFD., MCKENZIE I. F. C. and MITCHELLG. F. 1979. Peritoneal cell population of mice infected with Mesocestoides corti as a source of eosinophils. international Archives of Allergy and Applied immunology 59: 3 1S-322. KROEZEW. K. & TANNERC. E. In press. Echinococcus multiiocularis: responses to infection in Mongolian gerbils. Experimental Parasitology. LTJBINSKY G. 196Oa. The maintenance of Echinococcus multilocularis sibiricensis without the definitive host. Canadian Journal of Zoology 3%: 149-151. LUBINSKYG. 1960b. -A vegetatively propagated strain of larval Echinococcus multil~ular~. Canadian Journal of Zoology 3%: 1117-l 12s. RAU M. E. and TANNERC. E. 1972. Echinococcus multiIocularis in the cotton rat. Asexual proliferation following the intraperitoneal inoculation of graded doses of protoscolices. Canadian Journal of Zoology 50: 941-946. RAu M. E. and TANNERC. E. 1973. Echinococcus multiIocularis in the cotton rat. The effect of pr~~sting subcutaneous cysts on the development of a subsequent intraperitoneal inoculum of protoscolices. Canadian Journal of Zoology 51: 55-59. RAU M. E. and TANNER C. E. 1975. BCG suppresses growth and metastasis of hydatid infections. Nature 256: 318-319. RAU M. E. and TANNER C. E. 1976. Echinococcus multicularis in the cotton rat: the in vitro protoscolicidal activity of peritoneal cells. International Journal for Parasitology 6: 195-198. REUBEN J. M., TANNERC. E. and PORTELANC~ V. 1979. Protection of cotton rats against experimental Echinococcus multilocularis infection with BCG cell walls. Infection and Immunity 23: 582-586. REUBEN J. M., TANNER C. E. and Uu M. E. 1978. Immunoprophyl~s with BCG of experimental Echinococcus multilocularis infections. Infection and Immunity 21: 135-139. SA~UN E. H., NORMANL., ALLAIN D. S. and Kmo N. M. 1957. Observations on the susceptibilities of cotton rats to Echinococcus multilocularis (Leuckart, 1863). Journal of Infectious Diseases 100: 273-277. SEWSLLW. A. and VADM M. A. 1983. Evidence for the control of eosinophilia by the major histocompatibility complex in mice. Immunogenetics 17: 167..177. VOLLERA., BIDWELLD. E. and BARTLETTA. 1979. The enzyme-linked immunosorbent assay (ELISA). Dynatech Laboratories, Alexandria, Virginia. WEDMANN F. K. 1979. &linitatbtterpretation of Laboratoty Tests, 8th ed. Davis, Philadelphia.