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A comparative study of the susceptibility of inbred strains of mice to infection with Mesocestoides corti
I~zternationnl Jourrznl for Parasitology. Printed in Great Britain
0020-7519/82/010029-05$03.00/O Pergamon Press Ltd. G 1982 Australian Society for Parasitology
Vo!. 12,No. 1,pp.29-33,1982.
A COMPARATIVE STUDY OF THE SUSCEPTIBILITY OF INBRED STRAINS OF MICE TO INFECTION WITH MESOCESTOIDES T. R. WHITE, R. C. A.
TWOMFSON
CO RTI and W. J. PENHALE
Division of Veterinary Biology, School of Veterinary Studies, Murdoch University, Murdoch, Western Australia 6150, Australia (Received 16 March I98 1) Abstract-WHr= T. R., THOMPSON R. C. A. and PENHALE W. J. 1982. A comparative study of the susceptibility of inbred strains of mice to infection with Mesocestoides corti. Zt~ternatiunnl Journal fir Parasitology 12: 29-33. The susceptibility of 6 strains of inbred mice to infection with Mesocrstoides corti was studied following both intraperitoneal and oral inoculation of tetrathyridia. The greatest degree of resistance was seen in C57BL/6 mice and this resistance was independent of route of inoculation. The proliferation of the parasite in C57BL/6 mice was compared with a more susceptible strain (CBA/H) on 7, 14, 21, 35 and 60 days post-infection. Although both strains harboured significantly different parasite burdens during the initial period following infection, these differences were no longer apparent by day 60. INDEX KEY WORDS:
Mrsoces~oi&.s corfi; larval cestode; tetrathyridium;
INTRODUCTION
mice; resistance.
in the susceptibility of inbred strains of mice to larval cestode infections are few and detailed investigations are restricted to Echinococcus muitilocularis (Yamashita, Ohbayashi, Kitamura, Suzuki & Okugi, 1958) and Taenia taeniuefbrmis (Olivier, 1962; Dow & Jarrett, 1960; Mitchell, Goding & Rickard, 1977). Although there is some evidence that resistance to E. multilocularis in the
ing the immunology of parasitic infections in the mouse, reported that no striking mouse strain variation in resistance to M. corti has been seen in mice, no detailed results have been published. Furthermore, there remains the possibility that host defences could be quickly overwhelmed by this rapidly proliferating parasite and strain differences may only be apparent in the initial weeks following infection. In addition there are no published
mouse has an immunological basis (Baron & Tanner, 1976; 1977; Kassis & Tanner, 1977; AK-Khan,
observations on the effects of varying the route of infection with M. corti on the overall susceptibility
STUDIES
on differences
1978) 7: t~eniaeformis remains the cestode where detailed observations
only
larva1
on
intraon the
of the host. The present
specific variation has yielded inforlnation inlmuno~ogical mechanism responsible for resistance
(Mitchell, Rajasekariah & Rickard, 1980). Mesocestoides corfi, a natural parasite of the mouse (Hoeppli, 1925) has gained increasing acceptance as a model for proliferating larval cestode infections in immunological and chemotherapeutic studies (Mitchell, Marchalonis, Smith, Nicholas & Warner, 1977; Eckert & Pohlenz, 1976; Bennett, Behm & Bryant, 1978). While some information about
immunological
study was therefore
designed
to assess the susceptibility of 6 strains of inbred mice to infection by different routes with M. corG.
MATERIALS
AND METHODS
A&n&. The breeding nuclei of 5 inbred strains of mice were obtained from the John Curtin School of Medicine (Canberra, A.C.T.). In addition a breeding nucleus of BALB/c mice was obtained from the Microbiology Section, School of Veterinary Studies, Murdoch University. Breeding colonies of the 6 strains of mice (BALB/c, A.J., A.TH, C57BL/6, CBA/H, and DBA/2) were established in a minimum disease area. Litters were weaned at 3 weeks and introduced into conventionally maintained experimental areas at 4 weeks of age. Mice were infected at 4-6 weeks of age and autopsied at 35 days post-infection unless otherwise specified. Pnvasife. Tetrathyridia of M. co& were maintained in
responses made by the mouse to this
parasite is available (Pollacco, Nicholas, Mitchell & Chaicharn Stewart, 1978; Mitchell, Marchalonis, Smith, Nicholas & Warner, 1977), to date there is no detailed study on intraspeci~c variation in the mouse to this cestode. Although Mitchell (1979), in review29
30
T. R.
WHITE,
R. C. A. TI~OMPSON and W. J. PENHALE
the laboratory in Quackenbush mice by serially passaging the larvae by intra~ritoneal injection. Tetrathyridia for ex~rimenta1 infections were removed from the hody cavity of donor mice and washed twice in Hanks balanced salt solution (HBSS). The optimal conditions for establishment of infection by the oral route were determined and the most important variable, temperature of the inocula vehicle (White & Thompson, in preparation), was maintained at 37°C prior to infection. Mice were infected orally with a stomach tube, and all animals in this study received 50 tetrathyridia by either oral or intraperitonea! inoculation. .&l~opsy. Tetrathyridia were recovered from infected mice by washing out the peritoneal cavity into a Petri dish of HBSS. Where necessary the tetrathyridia were suspended in 100 ml of saline and stirred m~hanically on a magnetic stirrer and the number of parasites estimated from the mean of 3 10 ml samples. Tetrathyridia were recovered from the livers following digestion of minced livers in freshly prepared 1 7; trypsin solution (pH 7.2) for 6 hours at 37°C and counted under a dissecting microscope. [The activity of the trypsin is given by the manufacturers (Hopkin & Williams) as one part trypsin will hydrolyse 75 parts casein.] Experimental design. The design of the experiments to determine the numbers of tetrathyridia in mice of different sex and strain following inoculation by the oral and intraperitoneal routes is given in Tables 1 and 2. Geometric means were used throughout together with the S.E.M. The students f-test was used in the statistical analysis of results, and a probability of more than 576 was not considered statistically significant.
Suscept Wity
RESULTS to irzfection
Tables 1 and 2 show that with the exception of the C57BL/6 strain, intra-strain variation exceeded
any inter-strain variation observed. C57BL/6 mice were the most resistant strain examined in this study and this resistance was independent of either sex or route of inoculation. Further, the 4 groups of C57BL/6 mice examined had the least variation as expressed by the S.E.M. in this study. A sex difference was observed following intraperitoneal mocufation in BALB/c and A.J. mice, however, these differences were not apparent follo~ving oral administration. Infection by this latter route resulted in lower parasite burdens in BALB/c, CBA/H and DBA/2, strains and female A.J. mice. In A.TH, C57BL/6 strains and male A.J. mice parasite burdens were unaffected by varying the route of inoculation. Kinetics qf infkction in CBA/H arm C57BL/6 rtrice C57BL/6 mice were selected for further study and CBA/H mice were chosen as a susceptible strain for comparative purposes. Groups of 10 male mice of each strain were infected intraperitoneally with 50 tetrathyridia and autopsied on days 7, 14,21,35 and 60 post infection. The respective kinetics of proliferation in the peritoneal cavity and liver, and the
I.J.P.
VOL.
12. 1982
increase in total parasite burden (Fig. I) was compared on the above days following infection. When the total numbers of tetrathyridia were estimated it was observed that in both strains an exponential increase occurred in numbers of tetrathyridia following inoculation, multiplication occurring at a lower rate in C57BL/6 mice. The rate of increase of tetrathyridial proliferation declined markedly in CBA/H mice between days 21 and 35. A similar decrease in the rate of proliferation was not evident in C57BL/6 mice during the same period (Fig. 1). Comparison of the respective rates of proliferation in the peritoneal cavity and liver revealed a lag phase in both strains during the first 7 days after inoculation when following division in the peritoneal cavity a significant but variable number of parasites entered the liver. In CBA/H mice this lag phase was followed by a rapid exponential increase in proliferation within the liver resulting in this organ containing a greater number of tetrathyridia on day 21 than the peritoneal cavity. A decline in parasite numbers was observed between days 21 and 35 in CBA/H mice followed by a further slight increase in numbers over the next 2.5 days. The increase in parasite numbers in the peritoneal cavity paralleled that in the liver between days 7 and 21 and this was followed by a decline in proliferative rate of less magnitude than the liver. There were several significant differences in C57BL/6 mice. The proliferative rate in the peritoneal cavity following inoculation was retarded in COWparison with CBA/l-I mice as revealed by the initial fall in parasite numbers following invasion of the liver. Although similar numbers of parasites had entered the liver by day 7, a marked increase in the numbers of tetrathyridia was not observed until day 21 (Fig. 1). This was followed by a stabilisation in numbers between days 21 and 35 followed by a slight increase during the next 35 days. In the peritoneal cavity a brisk increase in parasite numbers was followed by a slight decline in the rate of proliferation between days 14 and 21. This reduced resulting in signi~~ntly lower rate, although numbers of parasites on days 21 and 35 than CBA/H mice, was maintained for the subsequent period of observation without further reduction, resulting in comparable numbers of parasites being present in the peritoneal cavity at day 60 in both strains. DISCUSSION The inbred strains of mice examined in this study showed a uniform susceptibility to infection with M. corti with the single exception of C57BL/6 mice. There is no evidence in the results to suggest that susceptibility to M. corii is related to haplotype and thus of the influence of immune response genes associated with the major histocontpa~ibili~y complex. However, it remains to be determined whether
I.J.P. VOL.
M. corti infection in mice
12. 1982
TABLE ~-NUMBER
OF TETRATHYRIDIA
IN
THEPERITONEAL
CAVITY AND
POST-INFEC'UON FOLLOWI~O
Strain
BALB/C
H-2 haplotype H-2d H-2“ H-2” H-2b H-2” H-2k H-2” H-29 H_2”jd H-2”f H-2d H-2d
C57B1/6 CBA/H A.J. A.TH DRA/2
TARLE
Z-NUMBEROF
Sex
No. per group
F M
13 11 12 9 13 14 10
L F M F M F RI F M
BALB/c C57B1/6 CBA/H A.J. A.TH DBAj2 -
STRAINS OF MICE AT 35 DAYS
Mean No. tetrathyridia 35 days p.i. (mean i S.E.M.) Liver Total Body cavity 745 + 68 15685 153 170+4 168+9 802 + 70 950562 1015+104 594 + 94 9971133 718&97 575 & 93 7542 137
1: 10 10 9
6 INBRED
ORAL INOCI~LATION
343+140 624+82 63+7 127+_29 319+_48 412k3.5 305+58 184&28 160& 22 217235 279 J- 34 4344 53
952 + 72 21951172 2342 26 295+32 1124+81 1335+_79 1323’1146 700_+ 13s 1159+ 133 938&122 858 + 93 1189tlS6
TETRATHYRIOIA IN THEPERITONEAL CAVITY AND LIVER OF 6 INBRED STRAINS OF MICEAT35DAYS POST-INFECTION FOLLOWING ORAL INOCULATION Mean
Strain
LIVER OF
31
H-2 haplotype H-26 H-2d H-2b H-2* H-2k H-2k H-2” H-2” H-2k/d H-2k/d H-2* H-2d
Sex
No. per group
F M F
9 11 10 12 14 14 10 9 11 14 8 9
: r M F M F M
other strains of mice sharing the H-2h heplotype with C57BL/6 mice show the same degree of redstante to infection with M. corti. Varying the route of inoculation did affect the resultant parasite burdens in certain strains of mice but not in others. Further work is required to determine what factors may be responsible for these differences. Enhanced resistance in certain strains of mice folIowing oral inoculation as a result of antigenic stimulation by the parasite passing through the gut wall does not appear to be the reason. This follows from studies in which in certain strains of mice autopsied at 48 h after infection, only 6@-70% of the tetrathyridia in the inocula were successful in penetrating the gut wall and reaching the peritoneal cavity (White & Thompson, in preparation). Consequently the lower parasite burdens found in the present study appear to reflect differences in the number of parasites initially establishing following oral or intra~ritoneal inoculation. During a 6 month period following establishment
No. tetrathyridia35 days p.i. (meankS.E.M.)
Body cavity 367+ 52 452 + 99 253 + 24 1902 13 397 + 72 452+%3 405+f28 594 t 94 7162 102 760+ 58 284 + 68 281+67
Liver
Total
244 I 54 219,42 83+8 144f15 210+44 161~45 68+12 184+28 162~36 445 + 92 233 + 56 289+66
613+60 633kllO 339+28 338+27 610_+9S 615& t15 545* 127 778 + 104 880+ 127 1211t111 508rf-118 570+113
of a breeding colony of C57BL/6 mice in this laboratory a statistically significant decrease in resistance (P~0.01)occurred to infection with M. cork The magnitude of this decrease in resistance can be seen by comparing the value in Table 1 for male (C57BL/6 mice (total parasite burden 295 -t 321, with Fig. 1 on day 35 (tota parasite burden 540 + 49). No changes in resistance were observed in other strains of mice used in this study. The change in resistance coincided with expansion of the C57BL/6 breeding colony which suggests that environmental factors were responsible for the observed change in resistance, although genetic drift cannot be ruled out. Figure 1 indicates that even with this change in resistance the kinetics of the infection in C57BL/6 mice, when compared with a susceptible strain (CBA/H) differ significantly during the initial postinfection period. The most striking differences between the two strains is the overall lower rate of proliferation of tetrathyridia in the peritoneal cavity and the absence of the rapid proliferation in the
32
T. R.
WHITE,
R. C. A. THOMPSON
Tetrathyridio p
in
both
Liver
W. J. PENHALE
and
Peritoneal
J.J.P.
VOL.
Cavity N.S.
co.001
N5.
and
T
,
I-I
1
Tetrathyridia p co.05
co.05
L
*
Peritoneal
in
0
N.S.
co.01
10
FIG.
1.
infection
N.S.
in
Liver
<0.03
30
20
DAYS
Cavity
co.001
co.001:
Trztrathyridia a
8
OF
N.S.
40
50
co
INFECTION
Numbers of tetrathyridia in C57B1/6 (0) and CBA/H (01 mice after intraperitoneal with 50 tetratbyr~d~a of b4esoees~uidos co,+. Values were expressed as meatI+ S.E.M.
12. 1982
M. corti infection in mice
I.J P. VOL. 12. 1982
liver of C57BL/6
mice during the first 20 days of infection. Resistance to ~esocesfai~es corfi in the mouse in contrast to the rat, is incomplete and parasite burdens show a progressive increase (Specht & Voge, 1965). This partial resistance in the mouse appears to be mediated by a soluble factor or factors inhibiting the proliferation of tetrathyridia in that inhibition of proliferation can be passively transferred with 1972; immune serum (Kowalski & Thorson, Pollacco et ai., 197s). It is of note in this respect that while serving as a good host for nematode infections (Wakelin, 1980) C57BL/6 mice are a poor host for E. mt&iloclrfaris and T. taenia&rmis (Ydmashita et crl., 1958: Mitchell, Coding & Rickard, 1977). Mitchell er al. (1980) have correlated, resistance to the Iatter cestode with the ability of C57BL/6 mice to react swiftly to the parasite by producing host protective antibodies. Indeed, an IgC, hypergammaglobulinaemia is associated with M. carti infections (MitchelI, Marchalonis, Smith, Nicholas & Warner, 1977) a portion of which is specific for surface antigens on the cestode but although antibodies have been postulated to restrain parasite proliferation in the rat, their role in the mouse is controversial (Mitchell, Marchalonis, Smith, Nicholas & Warner, 1977; SogandaresBernal & Voge, 1978). In addition, it is not clear how antibody binding to the surface of the cestode can result in the inhibition of division, although some workers have observed a decrease in metabolic rate following the immersion of tetrathyridia in immune serum (Sogandares-Bernal & Voge, 1978). Comparative studies on C57BL/6 and CEA/H mice are at present taking place in this laboratory with the aim of analysing the mechanisms which attempt to control M. corti in the naive mouse and also in the naive immuno-modulator primed animal. Acknowledgements-We thank Dr. J. B. Smith and Dr. I. A. Clark of the John Curtin School of Medical Research for the breeding nuclei of the colonies of inbred mice used in this study. The expert skills of Dr. K. E. White in the preparation of the figure are deeply apprecia-
ted.
REFERENCES ALI-KNAN Z.
197X. Echinacorcus
multifocufaris:
cell-
mediated immune response in early and chronic alveolar murine hydatidosis. Exgerirrtental Parasitology 46: 157.--165. BARON R. W. & TANNER C. E. 1976. The effect of immunosuppression on secondary Eehinococcus multifocularis infections in mice. International Journal for Parasitology 6 : 37-42. BARON R. W. & TANNER C. E. 1977. Ectiinococcus maltifo~ufaris in the mouse: the in uitro protoscolicidal activity of peritoneal macrophages. Z~?teraatio~af Journaf for Parasitafog~~ 7: 489-495. BENNETT E. M., BEHM C. & BRYANT C. 1978. Effects of mebendazole and Ievamisole on tetrathyridia of
33
~esocestoides carti in the mouse. Zaternatiailal Journal for Parusitofogy 8: 463-466. Dow C. & JARRETTW. F. H. 1960. Age, strain and sex differences in susceptibility to C.v~licerczrsfasci~faris in the mouse. Experimental Parasitology 10: 72-X. ECKERTJ. & POHLENZJ. 1976. Zur Wirkung von Mebendazol auf Metazestoden von Mesocestoides corti und Echinococcus muftifocufaris. Tropenmedizin und Parr;sitologie 27 : 247-262. HOEPPLIR. J. C. 1925. Mesocestoides corti, a new species of cestode from the mouse. Jourrzaf of Parasitology 12: 91-96. KASSIS A. I. &TANNERC. E. 1977. Host serum proteins in Echinococcus muliilocularis: complement activation via the classical pathway. Immunology 33: l-9. KOWALSKI J. C. & THORSON R. E. 1972. Protective immunity against tetra~hyridia of ~esu~e~r~j~~s corti by passive transfer of serum in mice. Jui~r~ai of Parasitfff~g~l 58: 244-246. MITCHELLG. F. 1979. Responses to infection with metazoan and protozoan parasites in mice. Aduanws in Immunology 28: 45 l-5 I 1. MITCHELLG. F., GOUING J. W. & RICKARD M. D. 1977. Studies on immune responses to parasite antigens in mice. Increased susceptibility of certain mouse strains and hypothymic mice to Taenia taeniaefbrmis and analysis of passive transfer of resistance with serum. Australian Journal of Experimental Biology and Medical Science 55: 165-l 86. MITCHELL G. F., MARCHALONISJ. J., SMITH P. M., NICHOLAS W. L. & WARNER N. 1977. Studies on immune responses to larval cestodes in mice. Immunoglobulins associated with the larvae of ~~esoeestoides corti. Australian Journaf qf E~.~erime?~tal Biology a~7d Medicaf Science 55 : I 87-2 I 1. MITCHELLG. F., RAJ~SEKARIAH G. R. & RICKARD M. D. 1980. A mechanism to account for mouse strain variation in resistance to the larval cestode Tuenia taeniaeformis. Immunology 39: 48 I-459. OLIVIER L. 1962. Natural resistance to Tarniu tueuiueformis. I. Strain differences in susceptibility of rodents. Journal of Parasitalagy 48: 373-378. POLLACCO S., NICHOLAS, W. L., MITCHELL G. F. & CHAICHARN STEWART A. 1978. T-cell dependent coliagenous encapsulating response in the mouse liver to Mesocertoides corti. I~fernatianaf Journai for Parasit0fag.v 8 : 457-462. S~AN~ARES-BERNAL F. & Voo~ M. 1978. Immunoglobulins on the surfaces of tetrathyridia of &&sQcesfoides co&, Hoeppli I925 (Cestodal from faboratory infections of ICR mice. Journal of Parasitofagy 64: 62CL624. SPECHTD. & VOCE M. 1965. Asexual multiplication of Mesocestoides tetrathyridia in laboratory animals. Journal of Parasitology 51: 268-272. WAKELIN D. 1980. Genetic control of immunity to parasites. Infection with Trichinelfa spiralis in inbred and cogenic mice showing rapid and slow response to infection. Parasite Zmmunology 2: 85-98. YAMASHITAJ., OHBAYASHIM., KITAMURAY., SUZUKI K. & GKUGI M. 1958. Studies on Echinococcusis. VIII. Ex~~ri~~e~tal E&nococcasis ~laitifa~l~furis in various rodents; especiahy on the difference of susceptibility among uniform strains of mouse. Japanese Jo~~r~a~of Veterinary Research 6: 3 1 S-322.
0020-7519/82/010029-05$03.00/O Pergamon Press Ltd. G 1982 Australian Society for Parasitology
Vo!. 12,No. 1,pp.29-33,1982.
A COMPARATIVE STUDY OF THE SUSCEPTIBILITY OF INBRED STRAINS OF MICE TO INFECTION WITH MESOCESTOIDES T. R. WHITE, R. C. A.
TWOMFSON
CO RTI and W. J. PENHALE
Division of Veterinary Biology, School of Veterinary Studies, Murdoch University, Murdoch, Western Australia 6150, Australia (Received 16 March I98 1) Abstract-WHr= T. R., THOMPSON R. C. A. and PENHALE W. J. 1982. A comparative study of the susceptibility of inbred strains of mice to infection with Mesocestoides corti. Zt~ternatiunnl Journal fir Parasitology 12: 29-33. The susceptibility of 6 strains of inbred mice to infection with Mesocrstoides corti was studied following both intraperitoneal and oral inoculation of tetrathyridia. The greatest degree of resistance was seen in C57BL/6 mice and this resistance was independent of route of inoculation. The proliferation of the parasite in C57BL/6 mice was compared with a more susceptible strain (CBA/H) on 7, 14, 21, 35 and 60 days post-infection. Although both strains harboured significantly different parasite burdens during the initial period following infection, these differences were no longer apparent by day 60. INDEX KEY WORDS:
Mrsoces~oi&.s corfi; larval cestode; tetrathyridium;
INTRODUCTION
mice; resistance.
in the susceptibility of inbred strains of mice to larval cestode infections are few and detailed investigations are restricted to Echinococcus muitilocularis (Yamashita, Ohbayashi, Kitamura, Suzuki & Okugi, 1958) and Taenia taeniuefbrmis (Olivier, 1962; Dow & Jarrett, 1960; Mitchell, Goding & Rickard, 1977). Although there is some evidence that resistance to E. multilocularis in the
ing the immunology of parasitic infections in the mouse, reported that no striking mouse strain variation in resistance to M. corti has been seen in mice, no detailed results have been published. Furthermore, there remains the possibility that host defences could be quickly overwhelmed by this rapidly proliferating parasite and strain differences may only be apparent in the initial weeks following infection. In addition there are no published
mouse has an immunological basis (Baron & Tanner, 1976; 1977; Kassis & Tanner, 1977; AK-Khan,
observations on the effects of varying the route of infection with M. corti on the overall susceptibility
STUDIES
on differences
1978) 7: t~eniaeformis remains the cestode where detailed observations
only
larva1
on
intraon the
of the host. The present
specific variation has yielded inforlnation inlmuno~ogical mechanism responsible for resistance
(Mitchell, Rajasekariah & Rickard, 1980). Mesocestoides corfi, a natural parasite of the mouse (Hoeppli, 1925) has gained increasing acceptance as a model for proliferating larval cestode infections in immunological and chemotherapeutic studies (Mitchell, Marchalonis, Smith, Nicholas & Warner, 1977; Eckert & Pohlenz, 1976; Bennett, Behm & Bryant, 1978). While some information about
immunological
study was therefore
designed
to assess the susceptibility of 6 strains of inbred mice to infection by different routes with M. corG.
MATERIALS
AND METHODS
A&n&. The breeding nuclei of 5 inbred strains of mice were obtained from the John Curtin School of Medicine (Canberra, A.C.T.). In addition a breeding nucleus of BALB/c mice was obtained from the Microbiology Section, School of Veterinary Studies, Murdoch University. Breeding colonies of the 6 strains of mice (BALB/c, A.J., A.TH, C57BL/6, CBA/H, and DBA/2) were established in a minimum disease area. Litters were weaned at 3 weeks and introduced into conventionally maintained experimental areas at 4 weeks of age. Mice were infected at 4-6 weeks of age and autopsied at 35 days post-infection unless otherwise specified. Pnvasife. Tetrathyridia of M. co& were maintained in
responses made by the mouse to this
parasite is available (Pollacco, Nicholas, Mitchell & Chaicharn Stewart, 1978; Mitchell, Marchalonis, Smith, Nicholas & Warner, 1977), to date there is no detailed study on intraspeci~c variation in the mouse to this cestode. Although Mitchell (1979), in review29
30
T. R.
WHITE,
R. C. A. TI~OMPSON and W. J. PENHALE
the laboratory in Quackenbush mice by serially passaging the larvae by intra~ritoneal injection. Tetrathyridia for ex~rimenta1 infections were removed from the hody cavity of donor mice and washed twice in Hanks balanced salt solution (HBSS). The optimal conditions for establishment of infection by the oral route were determined and the most important variable, temperature of the inocula vehicle (White & Thompson, in preparation), was maintained at 37°C prior to infection. Mice were infected orally with a stomach tube, and all animals in this study received 50 tetrathyridia by either oral or intraperitonea! inoculation. .&l~opsy. Tetrathyridia were recovered from infected mice by washing out the peritoneal cavity into a Petri dish of HBSS. Where necessary the tetrathyridia were suspended in 100 ml of saline and stirred m~hanically on a magnetic stirrer and the number of parasites estimated from the mean of 3 10 ml samples. Tetrathyridia were recovered from the livers following digestion of minced livers in freshly prepared 1 7; trypsin solution (pH 7.2) for 6 hours at 37°C and counted under a dissecting microscope. [The activity of the trypsin is given by the manufacturers (Hopkin & Williams) as one part trypsin will hydrolyse 75 parts casein.] Experimental design. The design of the experiments to determine the numbers of tetrathyridia in mice of different sex and strain following inoculation by the oral and intraperitoneal routes is given in Tables 1 and 2. Geometric means were used throughout together with the S.E.M. The students f-test was used in the statistical analysis of results, and a probability of more than 576 was not considered statistically significant.
Suscept Wity
RESULTS to irzfection
Tables 1 and 2 show that with the exception of the C57BL/6 strain, intra-strain variation exceeded
any inter-strain variation observed. C57BL/6 mice were the most resistant strain examined in this study and this resistance was independent of either sex or route of inoculation. Further, the 4 groups of C57BL/6 mice examined had the least variation as expressed by the S.E.M. in this study. A sex difference was observed following intraperitoneal mocufation in BALB/c and A.J. mice, however, these differences were not apparent follo~ving oral administration. Infection by this latter route resulted in lower parasite burdens in BALB/c, CBA/H and DBA/2, strains and female A.J. mice. In A.TH, C57BL/6 strains and male A.J. mice parasite burdens were unaffected by varying the route of inoculation. Kinetics qf infkction in CBA/H arm C57BL/6 rtrice C57BL/6 mice were selected for further study and CBA/H mice were chosen as a susceptible strain for comparative purposes. Groups of 10 male mice of each strain were infected intraperitoneally with 50 tetrathyridia and autopsied on days 7, 14,21,35 and 60 post infection. The respective kinetics of proliferation in the peritoneal cavity and liver, and the
I.J.P.
VOL.
12. 1982
increase in total parasite burden (Fig. I) was compared on the above days following infection. When the total numbers of tetrathyridia were estimated it was observed that in both strains an exponential increase occurred in numbers of tetrathyridia following inoculation, multiplication occurring at a lower rate in C57BL/6 mice. The rate of increase of tetrathyridial proliferation declined markedly in CBA/H mice between days 21 and 35. A similar decrease in the rate of proliferation was not evident in C57BL/6 mice during the same period (Fig. 1). Comparison of the respective rates of proliferation in the peritoneal cavity and liver revealed a lag phase in both strains during the first 7 days after inoculation when following division in the peritoneal cavity a significant but variable number of parasites entered the liver. In CBA/H mice this lag phase was followed by a rapid exponential increase in proliferation within the liver resulting in this organ containing a greater number of tetrathyridia on day 21 than the peritoneal cavity. A decline in parasite numbers was observed between days 21 and 35 in CBA/H mice followed by a further slight increase in numbers over the next 2.5 days. The increase in parasite numbers in the peritoneal cavity paralleled that in the liver between days 7 and 21 and this was followed by a decline in proliferative rate of less magnitude than the liver. There were several significant differences in C57BL/6 mice. The proliferative rate in the peritoneal cavity following inoculation was retarded in COWparison with CBA/l-I mice as revealed by the initial fall in parasite numbers following invasion of the liver. Although similar numbers of parasites had entered the liver by day 7, a marked increase in the numbers of tetrathyridia was not observed until day 21 (Fig. 1). This was followed by a stabilisation in numbers between days 21 and 35 followed by a slight increase during the next 35 days. In the peritoneal cavity a brisk increase in parasite numbers was followed by a slight decline in the rate of proliferation between days 14 and 21. This reduced resulting in signi~~ntly lower rate, although numbers of parasites on days 21 and 35 than CBA/H mice, was maintained for the subsequent period of observation without further reduction, resulting in comparable numbers of parasites being present in the peritoneal cavity at day 60 in both strains. DISCUSSION The inbred strains of mice examined in this study showed a uniform susceptibility to infection with M. corti with the single exception of C57BL/6 mice. There is no evidence in the results to suggest that susceptibility to M. corii is related to haplotype and thus of the influence of immune response genes associated with the major histocontpa~ibili~y complex. However, it remains to be determined whether
I.J.P. VOL.
M. corti infection in mice
12. 1982
TABLE ~-NUMBER
OF TETRATHYRIDIA
IN
THEPERITONEAL
CAVITY AND
POST-INFEC'UON FOLLOWI~O
Strain
BALB/C
H-2 haplotype H-2d H-2“ H-2” H-2b H-2” H-2k H-2” H-29 H_2”jd H-2”f H-2d H-2d
C57B1/6 CBA/H A.J. A.TH DRA/2
TARLE
Z-NUMBEROF
Sex
No. per group
F M
13 11 12 9 13 14 10
L F M F M F RI F M
BALB/c C57B1/6 CBA/H A.J. A.TH DBAj2 -
STRAINS OF MICE AT 35 DAYS
Mean No. tetrathyridia 35 days p.i. (mean i S.E.M.) Liver Total Body cavity 745 + 68 15685 153 170+4 168+9 802 + 70 950562 1015+104 594 + 94 9971133 718&97 575 & 93 7542 137
1: 10 10 9
6 INBRED
ORAL INOCI~LATION
343+140 624+82 63+7 127+_29 319+_48 412k3.5 305+58 184&28 160& 22 217235 279 J- 34 4344 53
952 + 72 21951172 2342 26 295+32 1124+81 1335+_79 1323’1146 700_+ 13s 1159+ 133 938&122 858 + 93 1189tlS6
TETRATHYRIOIA IN THEPERITONEAL CAVITY AND LIVER OF 6 INBRED STRAINS OF MICEAT35DAYS POST-INFECTION FOLLOWING ORAL INOCULATION Mean
Strain
LIVER OF
31
H-2 haplotype H-26 H-2d H-2b H-2* H-2k H-2k H-2” H-2” H-2k/d H-2k/d H-2* H-2d
Sex
No. per group
F M F
9 11 10 12 14 14 10 9 11 14 8 9
: r M F M F M
other strains of mice sharing the H-2h heplotype with C57BL/6 mice show the same degree of redstante to infection with M. corti. Varying the route of inoculation did affect the resultant parasite burdens in certain strains of mice but not in others. Further work is required to determine what factors may be responsible for these differences. Enhanced resistance in certain strains of mice folIowing oral inoculation as a result of antigenic stimulation by the parasite passing through the gut wall does not appear to be the reason. This follows from studies in which in certain strains of mice autopsied at 48 h after infection, only 6@-70% of the tetrathyridia in the inocula were successful in penetrating the gut wall and reaching the peritoneal cavity (White & Thompson, in preparation). Consequently the lower parasite burdens found in the present study appear to reflect differences in the number of parasites initially establishing following oral or intra~ritoneal inoculation. During a 6 month period following establishment
No. tetrathyridia35 days p.i. (meankS.E.M.)
Body cavity 367+ 52 452 + 99 253 + 24 1902 13 397 + 72 452+%3 405+f28 594 t 94 7162 102 760+ 58 284 + 68 281+67
Liver
Total
244 I 54 219,42 83+8 144f15 210+44 161~45 68+12 184+28 162~36 445 + 92 233 + 56 289+66
613+60 633kllO 339+28 338+27 610_+9S 615& t15 545* 127 778 + 104 880+ 127 1211t111 508rf-118 570+113
of a breeding colony of C57BL/6 mice in this laboratory a statistically significant decrease in resistance (P~0.01)occurred to infection with M. cork The magnitude of this decrease in resistance can be seen by comparing the value in Table 1 for male (C57BL/6 mice (total parasite burden 295 -t 321, with Fig. 1 on day 35 (tota parasite burden 540 + 49). No changes in resistance were observed in other strains of mice used in this study. The change in resistance coincided with expansion of the C57BL/6 breeding colony which suggests that environmental factors were responsible for the observed change in resistance, although genetic drift cannot be ruled out. Figure 1 indicates that even with this change in resistance the kinetics of the infection in C57BL/6 mice, when compared with a susceptible strain (CBA/H) differ significantly during the initial postinfection period. The most striking differences between the two strains is the overall lower rate of proliferation of tetrathyridia in the peritoneal cavity and the absence of the rapid proliferation in the
32
T. R.
WHITE,
R. C. A. THOMPSON
Tetrathyridio p
in
both
Liver
W. J. PENHALE
and
Peritoneal
J.J.P.
VOL.
Cavity N.S.
co.001
N5.
and
T
,
I-I
1
Tetrathyridia p co.05
co.05
L
*
Peritoneal
in
0
N.S.
co.01
10
FIG.
1.
infection
N.S.
in
Liver
<0.03
30
20
DAYS
Cavity
co.001
co.001:
Trztrathyridia a
8
OF
N.S.
40
50
co
INFECTION
Numbers of tetrathyridia in C57B1/6 (0) and CBA/H (01 mice after intraperitoneal with 50 tetratbyr~d~a of b4esoees~uidos co,+. Values were expressed as meatI+ S.E.M.
12. 1982
M. corti infection in mice
I.J P. VOL. 12. 1982
liver of C57BL/6
mice during the first 20 days of infection. Resistance to ~esocesfai~es corfi in the mouse in contrast to the rat, is incomplete and parasite burdens show a progressive increase (Specht & Voge, 1965). This partial resistance in the mouse appears to be mediated by a soluble factor or factors inhibiting the proliferation of tetrathyridia in that inhibition of proliferation can be passively transferred with 1972; immune serum (Kowalski & Thorson, Pollacco et ai., 197s). It is of note in this respect that while serving as a good host for nematode infections (Wakelin, 1980) C57BL/6 mice are a poor host for E. mt&iloclrfaris and T. taenia&rmis (Ydmashita et crl., 1958: Mitchell, Coding & Rickard, 1977). Mitchell er al. (1980) have correlated, resistance to the Iatter cestode with the ability of C57BL/6 mice to react swiftly to the parasite by producing host protective antibodies. Indeed, an IgC, hypergammaglobulinaemia is associated with M. carti infections (MitchelI, Marchalonis, Smith, Nicholas & Warner, 1977) a portion of which is specific for surface antigens on the cestode but although antibodies have been postulated to restrain parasite proliferation in the rat, their role in the mouse is controversial (Mitchell, Marchalonis, Smith, Nicholas & Warner, 1977; SogandaresBernal & Voge, 1978). In addition, it is not clear how antibody binding to the surface of the cestode can result in the inhibition of division, although some workers have observed a decrease in metabolic rate following the immersion of tetrathyridia in immune serum (Sogandares-Bernal & Voge, 1978). Comparative studies on C57BL/6 and CEA/H mice are at present taking place in this laboratory with the aim of analysing the mechanisms which attempt to control M. corti in the naive mouse and also in the naive immuno-modulator primed animal. Acknowledgements-We thank Dr. J. B. Smith and Dr. I. A. Clark of the John Curtin School of Medical Research for the breeding nuclei of the colonies of inbred mice used in this study. The expert skills of Dr. K. E. White in the preparation of the figure are deeply apprecia-
ted.
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197X. Echinacorcus
multifocufaris:
cell-
mediated immune response in early and chronic alveolar murine hydatidosis. Exgerirrtental Parasitology 46: 157.--165. BARON R. W. & TANNER C. E. 1976. The effect of immunosuppression on secondary Eehinococcus multifocularis infections in mice. International Journal for Parasitology 6 : 37-42. BARON R. W. & TANNER C. E. 1977. Ectiinococcus maltifo~ufaris in the mouse: the in uitro protoscolicidal activity of peritoneal macrophages. Z~?teraatio~af Journaf for Parasitafog~~ 7: 489-495. BENNETT E. M., BEHM C. & BRYANT C. 1978. Effects of mebendazole and Ievamisole on tetrathyridia of
33
~esocestoides carti in the mouse. Zaternatiailal Journal for Parusitofogy 8: 463-466. Dow C. & JARRETTW. F. H. 1960. Age, strain and sex differences in susceptibility to C.v~licerczrsfasci~faris in the mouse. Experimental Parasitology 10: 72-X. ECKERTJ. & POHLENZJ. 1976. Zur Wirkung von Mebendazol auf Metazestoden von Mesocestoides corti und Echinococcus muftifocufaris. Tropenmedizin und Parr;sitologie 27 : 247-262. HOEPPLIR. J. C. 1925. Mesocestoides corti, a new species of cestode from the mouse. Jourrzaf of Parasitology 12: 91-96. KASSIS A. I. &TANNERC. E. 1977. Host serum proteins in Echinococcus muliilocularis: complement activation via the classical pathway. Immunology 33: l-9. KOWALSKI J. C. & THORSON R. E. 1972. Protective immunity against tetra~hyridia of ~esu~e~r~j~~s corti by passive transfer of serum in mice. Jui~r~ai of Parasitfff~g~l 58: 244-246. MITCHELLG. F. 1979. Responses to infection with metazoan and protozoan parasites in mice. Aduanws in Immunology 28: 45 l-5 I 1. MITCHELLG. F., GOUING J. W. & RICKARD M. D. 1977. Studies on immune responses to parasite antigens in mice. Increased susceptibility of certain mouse strains and hypothymic mice to Taenia taeniaefbrmis and analysis of passive transfer of resistance with serum. Australian Journal of Experimental Biology and Medical Science 55: 165-l 86. MITCHELL G. F., MARCHALONISJ. J., SMITH P. M., NICHOLAS W. L. & WARNER N. 1977. Studies on immune responses to larval cestodes in mice. Immunoglobulins associated with the larvae of ~~esoeestoides corti. Australian Journaf qf E~.~erime?~tal Biology a~7d Medicaf Science 55 : I 87-2 I 1. MITCHELLG. F., RAJ~SEKARIAH G. R. & RICKARD M. D. 1980. A mechanism to account for mouse strain variation in resistance to the larval cestode Tuenia taeniaeformis. Immunology 39: 48 I-459. OLIVIER L. 1962. Natural resistance to Tarniu tueuiueformis. I. Strain differences in susceptibility of rodents. Journal of Parasitalagy 48: 373-378. POLLACCO S., NICHOLAS, W. L., MITCHELL G. F. & CHAICHARN STEWART A. 1978. T-cell dependent coliagenous encapsulating response in the mouse liver to Mesocertoides corti. I~fernatianaf Journai for Parasit0fag.v 8 : 457-462. S~AN~ARES-BERNAL F. & Voo~ M. 1978. Immunoglobulins on the surfaces of tetrathyridia of &&sQcesfoides co&, Hoeppli I925 (Cestodal from faboratory infections of ICR mice. Journal of Parasitofagy 64: 62CL624. SPECHTD. & VOCE M. 1965. Asexual multiplication of Mesocestoides tetrathyridia in laboratory animals. Journal of Parasitology 51: 268-272. WAKELIN D. 1980. Genetic control of immunity to parasites. Infection with Trichinelfa spiralis in inbred and cogenic mice showing rapid and slow response to infection. Parasite Zmmunology 2: 85-98. YAMASHITAJ., OHBAYASHIM., KITAMURAY., SUZUKI K. & GKUGI M. 1958. Studies on Echinococcusis. VIII. Ex~~ri~~e~tal E&nococcasis ~laitifa~l~furis in various rodents; especiahy on the difference of susceptibility among uniform strains of mouse. Japanese Jo~~r~a~of Veterinary Research 6: 3 1 S-322.