EXPERIMENTAL

PARASITOLOGY

4,

181-189

( 1978)

Trichinella spiralis: Effects on the Host-Parasite in Mice of BCG (Attenuated Mycobacterium

Relationship bovis)

DAVID I. GROVE AND RICHARD H. CIVIL Division of Geographic Medicine, Department of Medicine and Department Pathology, Case Western Reserve University and University Hospitals of Cleveland, Cleveland, Ohio 44106, U.S.A. (Accepted

for publication

8 December

of

1977)

GROVE, D. I., AND CIVIL, IL H. 1978. Trichinella spiralis: Effects on the host-parasite relationship in mice of BCG (attenuated Mycobacterium bovis). Experimental Parasitology 44, 181-189. The effects of BCG (Bacillus Calmette-Guerin, i.e., attenuated Mycobacterium bovis) on the host-parasite relationship in murine trichinosis were examined. A total of 2 X 10’ colony forming units of BCG given iv 1 week prior to Trichirda spiralis infection delayed the expulsion of adult worms from the gut. The suppression of adult worm elimination was proportional to the dose of BCG given. This finding was associated with a reduction in the degree of partial’ villous atrophy induced in the small bowel by T. spiralis. Adult female worms were fecund when they were examined 1, 2, and 3 weeks after infection of mice with T. spiralis. Despite the prolongation of fecund adult worms in the gut, there were no significant differences in muscle larval counts 4 and 6 weeks after infection. When newborn larvae were cultivated in vitro and injected iv, there was a significant 25% reduction in larval numbers recovered from the muscles of BCG-treated mice 4 weeks later. The administration of BCG had no effect on the inflammatory reaction around larvae in the muscles 4 and 6 weeks after infection. It is conchrded that BCG alters the host-parasite relationship producing retention of adult worms in the gut, reduction in the severity of partial villous atrophy, and increased nonspecific resistance to the systemic larval phase of this parasite. Trichinella spiralis; Nematode; Adult worms; Newborn larvae; INDEX DESCRIPTORS: Trichinosis; Mouse; Partial vilbus atrophy; Nonspecific resistance; BCG = Bacillus Calmette-Guerin = attenuated Mycobacterium bovis.

INTRODUCTION

Administration of the attenuated strain of Mycobacterium bouis, Bacillus CalmetteG&in (BCG), stimulates nonspecific resistance in a wide variety of neoplastic (lead articles, British Medical Journal 1976, Lancet 1976) and infectious diseases. Evidence of protection may be seen in a number of protozoal infections such as those of Babe&a species, Pla.smodium species (Clark et al. 1976), Leishmunia species (Smrkovski and Larson 1977, Weintraub

and Weinbaum 1977), and Trypanosoma cruzi (Kuhn et al. 1975). Injection of living BCG also induces a high degree of resistance to multicellular helminths. BCG suppressed both the growth and metastasis of Echinococcus multiloculuris (Rau and Tanner 1975) and inhibited the development of secondary cysts of E. granulosus (Thompson 1976). Furthermore, BCG reduced both the number of schistosomula recovered from the lungs and the number of adult worms of Schistosoma munsoni in 181 0014-4894/78/0442-0181$02.00/0 All

Copyright 0 1978 by Academic Press, Inc. rights of reproduction in any form reserved.

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GROVE AND CIVIL

the portal veins (Fauve and Dodin 1976; R. H. Civil, K. S. Warren, and A. A. F. Mahmoud, in press). When the effect of another immunostimulant, Corynebacterium parvum, was investigated in rats infected with Trichinella spiralis, it was observed that the expulsion of adult worms from the gut was delayed (Ruitenberg and Steerenberg 1973). These observations suggested that BCG may alter the host-parasite relationship in trichinosis. Infection with T. spiralis offers the opportunity to observe the effects of BCG on several stages of the parasite life cycle such as the fecundity of female worms, expulsion of adult worms from the gut, migration of newborn larvae through the bloodstream, and the development of infective larvae in the muscles. Furthermore, other indices of the host response to the parasite may be determined by quantifying partial villous atrophy in the small intestine and the degree of inflammation around organisms in the muscles. We have therefore examined these parameters in mice injected with BCG then infected with T. spiralis. MATERIALS

AND METHODS

Mice. Outbred female Swiss albino mice (CF-l), 18 to 20 g in weight, were obtained from Carworth Farms, Inc. (New City, New York, U.S.A.). BCG. The BCG strain of Mycobacterium bovis was obtained from the Research Foundation (Chicago, Ill., U.S.A.). Each ampoule of lyophilized BCG was reconstituted with distilled water and diluted to the appropriate concentration with 0.15 M (physiological) saline; then 0.4 ml was injected iv. Nematode infection. A colony of infected mice was established from a strain of Trichinella spiralis originally supplied by Dr. W. C. Campbell (Merck Institute for Medical Research, New Jersey, U.S.A.). Larvae were isolated by maceration of carcasses in a Waring Blendor, incubation in

1% acid-pepsin for 2 to 3 hr at 37 C, sedimentation in saline, and filtration through gauze (Gould 1970). Mice were infected with 300 larvae in 0.2 ml of saline by esophageal intubation with a 16-gauge needle. Intestinal worm burdens. Adult worms, i.e., nematodes, were recovered from the small intestines by a modification of the method of Campbell ( 1967). The small bowel was removed, slit longitudinally, cut into 2- to 3-cm pieces, and digested in 0.01 M NaOH in 100 ml of 0.15 M saline overnight at 4 C. The intestinal fragments were shaken vigorously and poured into a petri dish, the pieces were removed, and the fluid was transferred to 50-ml centrifuge tubes. The worms were spun down at 400g for 15 min and the total number in the sediment was counted. Muscle worm burdens. The number of larval nematodes in the musculature of each carcass was determined. Every animal was decapitated, skinned, eviscerated, macerated, and digested for 4 hr at 37 C in 1% acid-pepsin in a total volume of 100 ml. Each digestate was thoroughly shaken, 0.2-ml samples were taken, and the number of larvae was counted in duplicate. Newborn larvae. Newborn larvae of T. spiralis were obtained using a modification of the method of Dennis et al. (1970). Thirty mice were each infected with 800 larvae by esophageal intubation. Seven days later, the small intestines were removed, slit longitudinally, and layered on coarse wire mesh racks in beakers containing 0.15 M saline. After incubation for 2 to 3 hr at 37 C, the sediment was collected, and the adult worms were washed several times in 0.15 M saline by sedimentation. They were finally suspended in the following medium: RPMI-1640 with glutamine (Grand Island Biological Company, Grand Island, New York, U.S.A.) (70% by volume ), decomplemented fetal calf serum (KC Biological Inc., Lenexa, Kansas, U.S.A.) (29% by volume), and penicillin-

T.

[email protected]:

BCG

EFFECTS

streptomycin 5000 lU/ml and 5000 pg/ml (Flow Laboratories, Rockville, Maryland, U.S.A.) (1% by volume). Aliquots of 15 ml of medium containing approximately 100 worms were transferred to 30-ml tissue culture flasks (No. 3012, Falcon Plastics, Los Angeles, California, U.S.A.) and incubated for 36 hr at 35 C. Newborn larvae were separated from adult worms by passage through a sterile 325-gauge wire mesh (Tyler Co., Cleveland, Ohio, U.S.A.), reconstituted to the required concentration in 0.5 ml of medium, then injected into mice via the tail vein, Four weeks later, the numbers of muscle larvae were determined as described earlier, except that in view of the low numbers, multiple 0.2-ml samples were counted for each carcass. Fecundity. Mice were infected with 300 larvae of T. spirak The small intestines were removed, and adult worms were recovered 1, 2, and 3 weeks after infection as described above. Approximately 50 worms from each mouse were incubated in I5 ml of medium in tissue culture flasks at 35 C for 24 hr. Adult worms and newborn larvae were then centrifuged at 400g for 10 min, and the numbers of female adult worms and newborn larvae were counted. Small bowel pathology. Segments of small intestine 1.5 cm in length were excised 10 cm below the gastroduodenal junction, opened longitudinally, carefully orientated on filter paper, and fixed in 10% formalin. After routine processing, biopsies were stained with hematoxylin and eosin. The villus: crypt ratio of jejunal mucosa was determined by measurement of villus and crypt heights in 10 representative, well-oriented villi in at least two separate portions of each biopsy (Roberts-Thomson et al. 1976). Muscle pathology. The right hamstring muscles were removed, fixed in 10% formalin, sectioned, and stained with hematoxylin and eosin. The areas of inflammation around 10 isolated larvae cut longitudi-

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INFECTIONS

I

3 2 Weeks of infection

4

FIG. 1. The numbers of adult worms recovered from the gut at various intervals after infection of mice with 300 larvae of Trichinellu spiralis. 0 - - - - - 0, control mice; O-O, mice given 2 X 10’ CFU of BCG iv 1 week before infection. Nine mice were in each group.

nally were measured with a rMC particle measurement computer system (Millipore Corp., Bedford, Massachusetts, U.S.A.) (Grove and Warren 1976). RESULTS

Intestinal

Worm Burdens

Forty-five mice were given 2 x lo* colony forming units (CFU) of BCG iv. One week later, these mice together with the same number of control mice were infected with 300 larvae of Trichidkz spiralis. Intestinal worm burdens were determined 4, 7, 12, 17, and 28 days later (Fig. 1). There were no significant differences in worm burdens between the two groups at 4 and 7 days. By I2 days after infection, worm expulsion had begun in the control mice as shown by the worm burden falling to 65% of the original value. In mice treated with BCG, however, no change had occurred; the difference between the two groups was statistically significant (P < 0.05, t test). Seventeen days after infection, worm expulsion had commenced in mice given BCG, the worm burden being 75% of the original value, but recovery of worms from control mice had fallen further to 30% of the initial level (P < 0.01, t test). Four

1x4

GROVE 300

1

T

Number

FIG. 2. The from the gut 300 larvae of given varying fection. Eight

of

B.C.G.organisms

I.V

numbers of adult worms recovered 14 days after infection of mice with Trichinella spiralis. Mice had been doses of BCG iv 1 week before inmice were in each group.

weeks after infection, very few worms (4% ) were present in control mice, while in mice treated with BCG, 25% of the original number of worms remained, the difference between the two groups still being significant (I’ < 0.005, t test). The effect of varying amounts of BCG was determined by giving groups of eight mice doses of BCG ranging from 4 x lo5 to 5 x lo7 CFU in serial fivefold dilutions. One week later, these mice plus a group of control mice were infected with 300 larvae of T. spiralis, then intestinal worm burdens were measured after a further 14 days. The number of worms recovered from the small intestines was proportional to the dose of BCG given (Fig. 2). Significantly increased numbers of worms were found in mice given 5 x 10’ CFU (P < 0.005, t test) and 1 x lo7 CFU (P < 0.025, t test) when compared with control mice. Mice receiving lower doses of BCG did not harbor significantly increased numbers of worms.

AND

CIVIL,

together with the same number of control mice were infected with 300 larvae of T. spiralis. The numbers of muscle larvae were counted 4 and 6 weeks after infection. Less larvae were recovered from BCG-treated mice (56,000 -t 6900 [ ItSE] ) than from control mice (72,000 -+ 4600 [*SE] ) 4 weeks after infection, but this difference was not statistically significant. Although adult worms persisted in the gut of mice given BCG, no significant differences in muscle larvae were found between the two groups 6 weeks after infection, the numbers recovered being 58,000 t 9700 ( &SE ) in control mice and 59,000 2 5800 (*SE) in BCG-treated animals. Fecundity Eighteen mice were given 2 x lo7 CFU of BCG iv. One week later, these mice together with the same number of control mice were infected with 300 larvae of T. spiralis. One week after infection, adult worms were recovered and their fecundity was measured. There were no significant differences between the two groups: Worms recovered from six control mice produced 34.4 ? 4.9 newborn larvae per female worm per 24 hr, while those obtained from six mice treated with BCG gave 35.8 + 3.3 newborn larvae per female worm per 24 hr. When worms were recovered from BCG-treated mice 2 and 3 weeks after infection with T. spiralis, they were still fecund; 50.4 newborn larvae per female worm and 29.6 newborn larvae per TABLE

I

Recovery of Trichindla spiralis Muscle Larva 4 Weeks after Intravenous Injection of 1250 Newborn Larvae in Normal and BCG-Treated Mice

Control

mice

No.

Mean

SE

8

966

85

12

740

49

Muscle Worm Burdens Eighteen mice were given 2 x 10’ CFU of BCG iv. One week later, these mice

Probabilitya

< 0.05 BCG-treated u t test’,

mice

T.

S@?di~:

BCG

EFFECTS

female worm were produced at each time point, respectively. When worms were recovered from control mice 2 weeks after infection with T. spiralis, they also were fecund, producing 23.5 newborn larvae per female. Fecundity could not be measured 3 weeks after infection of control mice because insufficient numbers of adult worms were recovered. Resistance to Newborn

Larvae

Twelve mice were given 2 x lo7 CFU of BCG iv. Two weeks later, these mice together with the same number of control mice were injected with 8500 newborn larvae iv. All the mice given BCG plus newborn larvae died within 24 hr while none of the control mice given newborn larvae died. Histological examination of the lungs removed from one of the mice given BCG and newborn larvae showed gross hemorrhagic pneumonitis. In a second experiment, 12 mice were given 1 x lo7 CFU of BCG iv. One week later, these mice together with eight control mice were injected with 1250 newborn larvae by the same route. Four weeks later, muscle larval counts were performed. There was a significant 2,!$% reduction in the number of muscle larvae in mice treated with BCG compared with control mice (Table I). Small Bowel Pathology When compared with normal uninfected mice, there was a moderate reduction in the villus: crypt ratio of intestinal biopsies from both mice treated with BCG and from control mice when these groups were examined 4 days after infection with 300 larvae of T. spiralis (P < 0.005, t test) (Fig. 3). The mean value *SE for this ratio in 16 normal, uninfected mice was 3.1 * 0.1. During Days 7 through 17, the degree of partial villous atrophy did not change in mice given BCG, but it became progressively more severe in the control

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INFECTIONS

-*

.....JJ; .......p.,__.,.,,,,,,, ~../’ (-o”,ro,

-

m,ce

BCG mice

OWeeks of Infectron

FIG. 3. The villus:crypt ratio of the small bowel at various intervals after infection of mice with 300 larvae of TM&aeZlu spiralis. 0 - - - - - 0, control mice; O-0, mice given 2 X 10’ CFU of BCG iv 1 week before infection. Nine mice were in each group.

mice given T. spiralis only. When the values of Days 12 and 17 were pooled, the difference between the two groups was statistically significant (P < 0.005, t test). By 28 days after infection, however, the villus: crypt ratio in the mice given T. spidis only was returning toward normal. The mucosa had become less atrophic than in the BCG-treated mice in which the partial villous atrophy remained unchanged (P < 0.02, t test). Muscle Inflammation The degree of inflammation around muscle larvae was measured 4 and 6 weeks after infection. No significant differences were found between the two groups. The geometric mean areas and ranges 4 weeks after infection were 2800 (570 to 19,900) and 3480 pm* (520 to 22,900) for control and BCG-treated mice, respectively. The corresponding values 6 weeks after infection were 4000 (710 to 22,400) and 4770 pm2 (1170 to 19,500). DISXJSSION

The administration of BCG produced a number of alterations in the host-parasite relationship in murine trichinosis. The most

186

GROVE

striking changes were persistence of adult worms in the gut and reduced numbers of muscle larvae after the iv injection of newborn larvae. The presence of Trichindu spiralis adult worms in the gut is associated with intestinal mucosal inflammation (Larsh and Race 1975). This inflammatory reaction may be initiated by a variety of mechanical, chemical, enzymatic, and antigenic stimuli (Castro 1976). There is infiltration of the intestinal mucosa with white cells and development of a moderately severe partial villous atrophy. This hostile environment presumably leads to expulsion of the adult worms after which there is resolution of the inflammation and restoration of the architecture and physiology to normal (Larsh and Race 1975; Castro 1976). We have observed two effects of the administration of BCG on the intestinal phase of Trichinella infection. First, the spontaneous elimination of adult worms, which normally occurs about 2 weeks after the first exposure to T. spiralis, was delayed approximately 1 week. A similar phenomenon has been observed in rats treated with the adjuvant, Corynebacterium parvum, then infected with T. spiralis (Ruitenberg and Steerenberg 1973). Furthermore, we were able to show that the degree of suppression of worm elimination was directly proportional to the dose of BCG administered. Second, the administration of BCG reduced the severity of partial villous atrophy induced by T. spiralis infection but delayed the restoration of the mucosal architecture to normal. This latter observation is not surprising as a number of chronic infections have been associated with prolonged partial villous atrophy ( Shiner 1976). It is unclear whether the retention of adult worms results from this amelioration of partial villous atrophy or whether both of these findings are merely epiphenomena. Ruitenberg and Steerenberg (1973), who

AND

CIVIL

did not study the pathological changes in the gut, interpreted their results to indicate that C. parvum exerted an immunosuppressive action on T. spiralis infection. While both C. parvum and BCG are usually immunostimulants, under certain conditions of dose, timing, and route of administration they may act as immunosuppressants (Mathe et al. 1973; Mitchell et al. 1973; Doft ‘et al. 1976). It is also possible that they could delay the elimination of adult worms by altering the traffic of lymphocytes through the gut (Zatz 1976). Alternatively, these agents may, in some other as yet undetermined way, create a more favorable millieu which is manifested by a less distrubed intestinal mucosa and prolonged retention of the adult worms. A large number of factors determine the number of infective larvae which can be isolated from the muscles. These include the fecundity of the female worms, the length of time which they persist in the intestines, the largely unknown elements which influence the migration of newborn larvae through the gut and bloodstream, their penetration into the muscles, and maturation into infective larvae. Prior exposure to T. spiralis, either by infection or by immunization with antigenic fractions, hastens the expulsion of adult worms from the gut (Grove et al. 1977) and reduces their fecundity (Despommier et al. 1977). In contrast, the administration of BCG nonspecifically delays the expulsion of adult worms and has no effect on their fecundity. Since the adult worms persisting in BCG-treated mice were fecund, the greater production of newborn larvae should have been reflected in increased numbers of muscle larvae. The numbers recovered from BCG-treated and from control mice 6 weeks after infection, however, were the same despite persistence of adult worms. This suggests that the systemic migration of these larvae or their maturation in the

T. S+?'&S:

BCG EFFECTS ON MOUSE INFECTIONS

muscles was impaired in mice treated with BCG. This was confirmed when the effects of BCG on the systemic phase of Trichinella infection were studied in isolation by the injection of newborn larvae directly into the veins. When newborn larvae were injected iv in the first experiment, a severe hemorrhagic pneumonitis was produced and all the mice died. This is an artificial situation, however, for large numbers of larvae were injected over an extremely short period. In natural infections, it is probable that much smaller numbers of larvae are passing through the lungs at any $ven time. Furthermore, the lungs were probably already involved with heavy granulomatous inflammation and were less resistant to this insult (Youmans and Youmans 1964). Since the granulomatous reaction is directly proportional to the dry weight of BCG administered (Youmans and Youmans 1964), mice were given a reduced dose of BCG and a smaller number of newborn larvae were injected after a shorter interval. In this case, no mice died and there were significantly less larvae in the muscles of BCG-treated animals 4 weeks later. There was no difference in the inflammatory response around larvae in the muscles 4 and 6 weeks after infection and it is difficult to envisage that BCG alters the maturation of larvae once they are within the muscle cells. It seems more likely that BCG exerts its effects on newborn larvae as they migrate through the circulatory system. It is possible that a major site of such impairment is in the lungs which are involved with granulomatous inflammation. BCG induces hyperplasia of the reticuloendothelial system (Blanden et al. 1969) and activation of macrophages (Ratzan et al. 1972) in mice. Furthermore, this agent increases the magnitude and duration of delayed hypersensitivity reactions (Mackaness et al. 1974), the production and release of non-antibody mediators into

187

the circulation (Younger and Salvin 1973)) and the generation of “natural killer cells” (Wolfe et al. 1976). In addition, BCG can increase antibody production under some circumstances (Miller et al. 1973). Nevertheless, the precise mechanism of action of BCG immunization in trichinosis is uncertain. It is unclear whether lung inflammation ,per se is related to the increased resistance to the systemic phase of T. spir&s. It has been shown that an intact cellular immune system (Walls et al. 1973; Gore et al. 1974) and eosinophils (Grove @ al. 1977) are important factors in resistance to the systemic phase of Trichin&la infection. While there is no evidence that BCG stimulates eosinophils, the activation of macrophages and stimulation of cytotoxic lymphocytes by BCG could account for the increased protection observed. The location and timing of this effect and the mechanism of such an activity, however, remain to be determined. Other studies have shown that BCG nonspecifically stimulates resistance to a wide variety of infectious agents. This report supports this concept by extending observations to another helminth species. It provides a model in which the host-parasite relationship can be studied. In particular, it illustrates the opportunity to dissect and define the separate effects of BCG on the intestinal and systemic phases of this helminth infection. ACKNOWLEDGMENTS This study was supported in part by a grant from the Rockefeller Foundation and by Training Grant 5T32GM07250 from the U.S. National Institutes of Health.

REFERENCES BLANDEN, R.V., LEFFORD, M.J., AND MACKANESS, G. B. 1969. The host response to CalinetteGuerin Bacillus in mice. Joz~rnaZ of Experimental Medicine 129, 1079-1107. CAMPBELL, W. C. 1967. Distribution of Ttichinella spiralis in the small intestine of young mice. Journal of Parasitology 53, 395-397.

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CASTRO, G. A. 1976. Spatial and temporal integration of host responses to intestinal changes of Trichinella spiralis: Retro- and prospective views. In “Biochemistry of Parasites and HostParasite Relationships” (H. Van den Bossche, ed.), pp. 343358. North-Holland, Amsterdam. CIVIL, R. H., WARREN, K. S., AND MAHA~OUD, A. A. F. Characterization of BCG-induced resistance to Schistosoma mansoni infection. J. Infect. Dis., in press. CLARK, I. A., ALLISON, A. C., and Cox, F. E. 1976. Protection of mice against Babesia and Plasmo&urn with BCG. Nature (London) 259, 309311. DENNIS, D. T., DESPOMMIER, D. D., AND DAVIS, N. 1970. Infectivity of the newborn larva of Trichinella spiralis in the rat. Journal of Parasitology 56, 974-977. DESPOMMIER, D. D., CA~~PBELL, W. C., AND BLAIR, L. S. 1977. The in vivo and in vitro analysis of immunity to Trichinella spiralis in mice and rats. Parasitology 74, 109-119. DOFT, B. H., MERCHANT, B., JOHANNESSEN, L., CHAPAI(AS, S. T., AND SHER, N. A. 1976. Contrasting effects of BCG on spleen and lymph node antibody responses in nude and normal mice. Journal of Immunology 117, 1638-1643. FAUVE, R. M., AND DOUIN, A. 1976. Influence dune reaction inflammatoire provoquee par le BCG ou par un irritant non biodegradable sur la resistance des souris a la bilharziose. Comptes Rendus Hebdomadaires des Seances de I’Academie des Sciences 282, 131-134. GORE, R. W., BURGER, H. J., AND SADUN, E. H. 1974. Humoral and cellular factors in the resistance of rats to Trichinella spiralis. In “Trichinellosis” (C. W. Kim, ed.), pp. 367382. Intext Educational Publishers, New York. GROVE, D. I., HAMBURGER, J., AND WARREN, K. S. 1977. Kinetics of immunological responses, resistance to reinfection and pathological reactions to infection with Trichinella spiralis. .lournal of Infectious Diseases 136, 562-570. GROVE, D. I., MAHXXOUD, A. A. F., AND WARREN, K. S. 1977. Eosinophils and resistance to Trichinella spiralis. Jourrzal of Experimental Medicine 145, 755-759. GHOVE, D. I., AND WARREN, K. S. 1976. Effects on murine trichinosis of niridazole, a suppressant of cellular but not humoral immunological Medicine and responses. Annals of Tropical Parasitology 70, 449-453. GOULD, S. E. 1970. Clinical pathology: Diagnostic laboratory procedures. In “Trichinosis in Man and Animals” (S. E. Gould, ed.), pp. 190-221. Charles C Thomas, Springfield, Ill. KUHN, R. E., VAUGHN, R. T., AND HEHBST, G. A. 1975. The effect of BCG on the course of ex-

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Trichinella spiralis: effects on the host-parasite relationship in mice of BCG (attenuated Mycobacterium bovis).

EXPERIMENTAL PARASITOLOGY 4, 181-189 ( 1978) Trichinella spiralis: Effects on the Host-Parasite in Mice of BCG (Attenuated Mycobacterium Relatio...
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