EXPERIMENTAL
PARASITOLOGY
Trichinella
37,
108-116
spiralis:
(1975)
Growth of the Intracellular
(Muscle)
Larva
DICKSON DESPOMMIER Division
of Tropical
Columbia
Medicine, School of Public Health, College of Physicians and Surgeons, University, 630 West 168th Street, New York, New York 10032 LORNA
Division
of Tropical
Columbia
Medicine, University,
ARON
School of Public Health, College of Physicians and Surgeons, 630 West 168th Street, New York, New York 10032
LIVIA TURCEON Division of Biostatistics, School of Public Health, College of Physicians and Surgeons, Columbia Vniuersity, 630 West 168th Street, New York, New York 10032 (Submitted
for publication
March 27, 1974)
DESPOMMIER, DICKSON, AHON, LORNA, AND TURGEOX, LIVIA. 1975. Trichinella spiralis: Growth of the intracellular (muscle) larva. Experimental Parasitology 37, 108-116. Newborn larvae of Trichinella spiralis were infective when injected directly into the thigh muscle of mice and rats. Infections initiated in this manner resulted in synchronously growing populations of muscle larvae, thereby permitting a detailed study of larval growth to be carried out. In mice, the mean larval growth, as measured by increase in larval volume, occurred in three phases; an initial growth phase ( Day O-l ) , a lag phase ( Days l-3 ) , and an exponential growth phase (Days 3-19). Larvae grew an average of 39% per day during the exponential phase. No further increase in larval volume was noted after Day 19. There was no statistically significant difference found in the rate of larval growth among individual mice for any given day. The larval growth rate was the same in rats as in mice. INDEX DESCRIPTIONS: Trichinella spiralis; Mice, Rats; Growth; Life cycles; Synchrony.
larvae in order to define accurately the temporal nature of cytological, physiological, and biochemical changes in the infected host cell and the parasite which occur during the course of the parenteral phase of the life cycle. Attempts at limiting the age differences of developing larvae by the use of methyridine (Stewart and Read 1972a, 1973a, 1973b), have resulted in the elucidation of some important biochemical aspects of the intracellular phase. In the
INTRODUCTION
Infections of Trichinella spiralis initiated through the ingestion of muscle larvae ultimately result in populations of developing muscle larvae growing nonsynchronously. This lack of synchrony arises from the fact that larvae are born live, one at a time over a period of 10-14 days, and therefore establish themselves in host cells at various points in time. It would be advantageous to obtain synchronously developing muscle 108 Copyright 0 1975 by Academic Yress, Inc. All rights of reproduction in any form reserved.
GROWTH
OF
above mentioned studies, however, the difference in age between the youngest and oldest larvae was six days; therefore, it is likely that some changes that may have been seen, if the infections were synchronous, were missed. The growth curve of Trichinella spiralis during its parenteral phase in the host has not yet been satisfactorily determined, again, because of the nonsynchronous nature of growth of the larvae which occurs after a naturally induced infection. The purposes of the present study were to establish synchronous infections of T. spiralis larvae in muscle tissue and to describe the larval growth rate from the point of initiation to maturation.
Trichinella
109
FIG. 1. Schematic spiralis larva.
diagram
of
a Trichinella
gauge needle and the dose was slowly injected directly into the thigh muscles of either mice or rats. Several sites were used for the injection dose to insure uniform muscle infections. Only one injection site was used for the subcutaneous route. Measurement of Larvae
Larvae were measured every day for 20 days after intramuscular injection. Infected mice were killed each day by cervical dislocation. The injected thigh MATERIALS AND METHODS muscles were then immediately removed Infection with Newborn Larvae and placed into warm 0.85% NaCl. Pieces Newborn larvae were collected in vitro of muscle (3-5 mm3) were cut from the inby methods described by Dennis et al. fected thigh tissue and gently pressed be( 1970). During injection of newborn larvae, tween two slides. This procedure squeezed an 18-gauge needle was replaced with a 2% the developing larvae out of the infected tissue, and in no case did any larvae remain in the infected tissue being examined. TABLE I Warm saline was added between the glass Adjustment Factor for Worm Volume Determinations slides. Living larvae were then photographed, using microscopy, as they were V,IVP V,IVP Day Day encountered, so as to randomize the popu11 0.84 1.00 0 lation being sampled as much as possible. 12 0.84 1 0.99 Worms were photographed at a magnifica2 0.96 13 0.84 tion of x100. All negatives were printed 0.84 0.9s 14 s at the same magnification. The width and 0.84 4 0.90 15 length of each larva was then determined 16 0.84 5 0.87 0.85 0.83 17 6 from each photograph with the aid of a 18 0.85 0.80 7 map measuring device 1 and a set of cali0.85 0.78 19 8 phers. 0.85 0.80 20 9 A first approximation of the volume (Vi ) 10 0.83 for each worm was made using the length 0 V2/Vl represents a series of correction factors and width determination. The assumption derived from two different methods for calculating was made that the shape of the worm was worm values. VI represents worm volume as calthat of a cylinder (L x (+W) * x r). A better culated strictly from length and width measureapproximation of worm volume (V,) was ments and does not take into consideration anterior obtained by taking into account the degree tapering of the worm. VI represents worm volume as calculated by combining measurements of the of tapering. It was found that the percentcylindrical mid and posterior portion of the worm age of worm length involved in tapering with the conical anterior portion of the worm (Fig. 1). Vz, except for Day 0, was always less than VI.
1 Keuffel
and Esser Co., Switzerland.
110
DESPOMMIER,
AHON
TABLE II Infectivity
of Newborn Trichinella spiralis Larvae aj’ter Inoculation by various Routesa
Route of injection
Intravenous Intramuscular
Intraperitoneal Subcut,aneous Intraduodenal
Percentage recovery of muscle larvaeb
Remarks
SO-800/;, Dennis et al. 1970 25-35yo 60% of the recovered 1arva.e were from the thigh muscles-the rest were evenly distributed throughout the musculature of the carcass. 2-5 y. Dennis et al. 1970 0% 0% I)ennis et al. 1970
a The subcutaneous and intramuscular route infection rates were determined in the present study. Intravenous, intraperitoneal, and intraduodenal route infection rates were derived from the literature. b Thirty days after injection each mouse carcass was digested in pepsin-HCl and the total number of muscle larvae determined.
varied according to the age of the worm. (It should be noted that for the first three days of larval growth, the posterior region is more tapered than the anterior end. From that time on, the worm is tapered more in the anterior region. ) The percentage difference in volume between VI and V, is shown for each day in Table I. The percentage volume difference (V,/ V,) was tested experimentally using models of cylinders and worm shapes obtained with modelling clay. The volume (Ve,) of a given clay cylinder was determined by submerging the cylinder in a loo-ml burette filled to the 80-ml mark with distilled water and then recording the fluid volume increase. The appropriate degree of tapering for a given age of worm (2, 4, 6, 8, and 12day-old larvae) was then measured from the respective photographs and translated on to the same cylinder of clay by removing material from the cylinder according to these measurements. The new volume (Ve,) was then determined by displace-
AND
TURGEON
ment of water in the same burette. Less than a 5% difference was obtained between the calculated ( V2/V1 ) and the empirical ,clay model ( Vez/Vel ) for worms from any given day, thereby supporting the assumption that the solid geometric figures which best describe the shape of the developing larvae is the cylinder plus the cone. This point is further illustrated in Fig. 1. The cylindrical portion of the worm in Fig. 1 contains a small area at the anal region which does not conform to the straight end of the cylinder (shaded area). This results in an overestimation of the worm volume for this region. The oral region, however, extends beyond the limits of the cone region drawn inside the worm (shaded area). This results in an underestimation of worm volume for this region. These two errors in volume estimations counteract each other and thereby allow an estimate closely approximating the true worm volume. Histology Infected thigh muscle was fixed in 15% picric acid and 3% paraformaldehyde at pH 7.2 for 2 hr at room temperature, washed overnight in tap water, then dehydrated in a graded series of alcohols, and embedded into paraffin. Three to five micron thick serial sections were stained with hematoxylin and phloxin. 38 36
:z 30 28 26 24 22 2c
5 i ts L 162 14s 12 10 ; 4 I * FIG. 2. Length and width measurements of T. larvae obtained from im infected mice. O-O = length; A----A = width. The standard deviation is given for each determination.
spiralis
GROWTH
OF
TABLE
Trichinella
111
III
Means and Standard L)evialions 01 Trichinella xpiralis Larval Volume Determinations Mice for Days 6, 7, 8, 9, and 10 after Intramuscular
amuny
Injection
Days after intramuscular injection Mouse No.
6
1 2 3 4
11800”f 25476 12;723 f 2941 9989 f 1419 10,792 f 2407
One way analysis of varianced
NB
7 14,288 13,414 14,395 16,060
f 3113 f 3340 f 3527 f 3271
8 23,817 19,063 20,666 26,046
NS
f f f +
9 4841 10,977 7690 7680
NS
40,638 40,907 34,547 35,466
f 8921 z!z 6884 rk 5232 f 7220
10 53,783 57,384 54,646 59,655
NS
NS
~ ~- ~ .-__---~ a All volumes are expressed as pm3. b Each larval volume determination was made by measuring 10 worms/mouse/day. standard deviation is given. c NS = Not significant. d The means were compared by one \Fay analysis of variance.
f 10,730 f 9460 z!z 11,745 f 12,594
The mean and
Statistical Methodology
Animals
The distribution of the volumetric determinations for various selected days was examined. Each frequency distribution was skewed to the right. Furthermore, the standard deviations for these daily volume determinations were not constant, but varied proportionately with the daily mean volume. In the above case it is suggested (Snedecar and Cochran 1967) that the natural logarithms of the observations should be normally distributed with constant standard deviation. Therefore, all statistical analyses were performed on the natural logarithms of the volumetric determinations. Linear regression lines were calculated by the method of least squares: the slope for the natural logarithms of the volume of the worms grown in mice from Day 3 through 20 was estimated. The growth rates between Days 3 and 7 were computed for larvae grown in mice and rats and the two slopes were then compared by Student’s t test for independent samples. One-way analysis of variance was used to evaIuate the variation in the natural logarithms of the volumes for a given day of larval growth among several mouse hosts.
All mice used in these studies were male, CFW strain (random bred) two-monthold animals.2 Newborn larvae of T. spiralis were obtained from infections initiated in 200 gram male Wistar rats.3 Two hundred gram male WF strain (inbred) rats were used to determine the growth rate of larvae in the rat.4 RESULTS
Assessment of Larval Infectivity Various Routes
by
Newborn larvae injected intravenously (iv) into rats or mice do not develop synchronously with respect to growth (unpublished observation), although 60430% of all larvae injected iv eventually mature, and become muscle larvae (Dennis et al. 1970). The absence of synchronous larval growth in this instance probably results from the fact that some newborn larvae penetrate organs other than muscle prior to their penetration into striated muscle. Apparently, in intravenously administered infec2 Carworth Farms, Rockland, New York. 3 Bill Fabre, New City, New York. 4 Microbiological Associates, Baltimore, Maryland.
112
DESPOMMIER,
ARON
AND
TABLE Length, Width, and Volume
il~~nsurrrnrnfs
of Trichinella
N
Day
L
SD
SE
Range
w
48 41 36 50 42 5 50 58 50 48 82 47 49 45 44 6 9
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
83 105 110 100 116 126 121 133 164 202 250 314 385 499 461 507 623 682 820 860 890
9 9 10 6
2 2 3 1 3 6 3 2 4 4 5 9 9
99- 69 121- 82 121- 81 114- 72 138- 86 139-106 148- 98 157-108 228-108 258-136 327-181 412-213 510-210 620-330 620304 567432 715618 988-469 950-475 975-745 995-800
7.1 7.3 7.9 8.8 9.4 11.5 11.9 12.9 15.3 17.1 18.9 20.9 22.7 23.4 24.2 28.2 27.8 31.3 36.0 37.6 38.6
11
47 46 26
a N = number standard error.
11
13 1‘2 13 25 25 34 58 64 77 81 65 58 164 176 69 54
of larvae
11
12 26 19 48 28 12 11
measured/day;
IV spiralis I,nrtms.from
SD SE 0.8 0.6 0.7 0.9 0.9 1.3 1.9 1.1 l.Q 1.4 1.8 2.2 3.0 5.5 2.8 3.2 1.3 5.5 3.6 2.4 1.8
L = length;
tions, larval penetration of muscle can Occur at anytime up to three days after the since some newborn time of injection, larvae were observed in muscle tissue three days after iv infection. Preliminary experiments suggested that the intramuscular route of infection with newborn larvae resulted in a synchronously developing population of larvae. However, the infection rate for the intramuscular route was not resolved in that study. Therefore, the present experiment was designed to determine the percentage of newborn larvae capable of developing to mature muscle larvae after intramuscular injection. The subcutaneous route of injection was also tried. Two groups of five mice each, were individually infected either intramuscularly (im) or subcutaneously (subcut.) with 60,000 newborn larvae. Thirty days after infection, all mice were killed by cervical dislocation. In the im injection group re-
TURGEON
0.2 0.2 0.2 0.2 0.2 0.6 0.4 0.1 0.3 0.2 0.3 0.3 0.2 0.8 0.4 1.3 0.4 1.7 0.5 0.4 0.4
Intrarmcncular
Range
d
9.3- 5.6 8.4- 6.4 8.4- 6.4 12.6- 6.0 ll.O- 6.6 12.9- 9.6 15.6-10.0 15.3-10.6 23.5-11.5 20.5-15.2 22.0-14.0 27.6-15.2 26.6-18.0 26.6-19.0 32.2-20.0 34.2-25.8 32.0-27.6 44.4-24.7 48.7-25.5 42.5-32.5 41.5-34.5
W = width;
3243 4597 5298 4883 6946 9124 11,480 14,319 26,566 37,336 59,040 90,947 131,971 187,615 198,985 290,619 368,200 467,481 751,286 855,377 897,283
SD = standard
Infections
in ilfice’7
SD
SE
829 897 1193 758 1328 2044 2589 2840 8390 8136 14,144 25,232 31,417 36,498 93,461 114,450 55,377 301,995 243,517 367,699 181,530
179 254 440 219 346 796 749 728 2953 2745 3630 7283 7139 8965 19,286 46,724 18,459 91,053 77,330 54,214 35,601
deviation;
SE =
ceiving 60,000 larvae, the injected legs were removed, skinned, and minced. The rest of the carcass was also eviscerated, skinned, and minced. Both the minced legs and carcasses were separately digested in I$% HCI-1% pepsin at 37” C for 4 hr. The entire carcass (skinned and eviscerated) of each subcutaneously injected mouse was similarly digested for 4 hr. The number of larvae from each group was then determined. The results of these trials, together with earlier determinations in animals infected by intravenous, intraperitoneal, and intraduodenal routes are presented in Table II. Recovery rates of 2535% were obtained from im injections, whereas no larvae were recovered from subcutaneously injected mice. Sixty percent of larvae recovered from the im group came from the injected leg; the rest were found throughout the carcass. It was estimated from 5 pm thick serial sections that approximately 80% of the thigh muscle was infected when five im
GROWTH
OF TABLE
Comparison
of Trichinella
spiralis
Trichinella
113
V Larval Growth in Mice and Rats
Days after intramuscular
injection
Host
3
4
5
6
7
CFW mouse (randomly bred)
4883 f 75@ (50) h
6946 f 1328 (42)
9124 f 2044 (5)
11,480 f 2589 W)
14,319 f 2840 (58)
WF rat (inbred)
4634 It 776 (23)
5967 f 1215 (25)
955 7134 f (25)
11,129 f 1680 (27)
14,709 f 2484 (25)
Students’ 1 test
NRC
NS
NS
NS
NS
(1Mean and standard deviation. b Number of larvae/determination. c NS = Not significant. d All volumes in pm3.
injections, consisting of lO-12,000 larvae per site were each given 10-15 mm apart down the length of the leg. Variation in Larval Growth among Mice Prior to determining the growth curve for the larva of T. spiralis in the mouse, it was necessary to obtain data concerning the variation in worm growth among mice infected on the same day with the same pool of newborn larvae. These data allowed a selection of the appropriate number of mice per day from which to measure larval growth. Mice were infected im with 30,000 muscle larvae. On Days 6, 7, 8, 9, and 10 after injection, larval volume was determined for ten larvae from each of four mice. The results are presented in Table III. A one-way analysis of variance showed no significant variation in larval size among the infected mice for any given day. Growth of Larvae in Mice from Day 0 to 20 of Newborn Larvae
after im Injection
The purpose of this experiment was to determine the rate of larval growth in the thigh muscle of the mouse. Larval growth parameters measured included length, width, and volume. CFW, two-month-old, male mice were each injected with 60,000 newborn larvae.
Larvae were obtained from two mice per day until Day 20, when larval growth was completed. This experiment was repeated for Days 0, 1, 4, 10, 11, I.5 14, 18, 19 and 20. The results of these two experiments were pooled. The length, width, and volume measurements are shown in Table IV. For the first three days, little increase was seen in either length or width (Fig. 2). However, beginning on Day 3, and continuing until Day 20, there was an almost continuous increase in larval length and width. Worm volume data (Fig. 3) suggested that larval growth proceeded in three distinct phases. During phase 1 (Day O-l), worm volume increased about 20%. Throughout phase II (Days l-3), no significant change in volume occurred, and corresponded to a lag phase. Finally, d’uring phase III (Days 3-19) an exponential rate of volume increase was observed. No further increase in worm volume was noted after Day 19. An overall growth rate for Days 3 through 20 was determined by computing the slope of the least squares line fitted to the average natural logarithmic volume. This slope was .33 natural logarithmic volume per day, indicating that larval volume increased about 39% every day during this period.
114
DESPOMMIER,
ARON
FIG. 3. Mean larval volume (in pm3) determinations from im infected mice. Larval growth occurs in three phases; an initial growth phase (Day O-l), a lag phase (Days l-3), and an exponential growth phase (Days 3-19). The standard deviation is given for each day.
Variation of Larval Growth between Mice and Rats The purpose of this experiment was to determine whether the larval growth rate for a given period of time was the same in rats as in mice. Ten WF strain, 200-gram, male rats and ten CFW, two-month-old, male mice were each infected with 30,000 newborn larvae obtained from a single isolate. Worm volume determinations were made 3, 4, 5, 6, and 7 days after injection. The daily average volume did not differ significantly in the two hosts (Table V). Furthermore, slopes of the regression lines fitted to the average daily logarithmic volumes were also similar. The growth rate for larvae in rats during the five-day period was 34% while that for larvae in mice was 32%. However, this observed 2% difference in growth rates was not significant. The individual worm volumes within each of the five days were more variable for larvae recovered from mice than for those from rats. DISCUSSION
The intramuscular (im) route of injection was effective in establishing infections with Trichinella spiralis larvae.
AND
TURGEON
Although approximately 30% of the injected newborn larvae matured into muscle larvae under these conditions, this infection rate was well below that obtainable by intravenous infection (60-80s ). It is probable that the low infectivity rate obtained with the im route resulted from introducing too many larvae (30-60,000) into a given muscle mass. Support of this view comes from histological evidence (unpublished observations) which showed that a large number of muscle fibers were killed under these conditions. The above data indicate that there is an upper limit with respect to the number of larvae which can establish a viable infection in a given amount of muscle tissue, and further imply that the ability of newborn larvae to repeatedly penetrate into and out of muscle fibers while remaining infective may be limited, since if that were not the case, a higher infection rate should have been noted. Larval growth was characterized for im infections in the mouse by a constant rate of increase (39Cjo/day) in mean larval volume which occurred during Days 3-19 of the infection. The coefficient of variation (SD/mean) noted in the volume of the newborn larvae (26% ) persisted at the mean ratio of 27% for each day’s volume determinations (see Table IV ) . These data give strong evidence for synchrony of larval growth during im infection. Some parameters of growth for T. spiralis larvae have already been investigated by several groups (Richels 1955; Wu 1956; Thomas 1966; Ali Khan 1966; Villella 1970). However, in all of the above studies, only fixed worms were measured. Furthermore, since infections were initiated via the oral route with muscle larvae, only nonsynchronous infections were available for study. Villella (1970) measured 15 larvae at selected days after oral infection, but did not present a statistical analysis of the data. Finally, only length and width mea-
GROWTH
OF
surements were recorded for each time point. In the present work, only live worms were measured. The exact age of the larvae during infection was known, as they were all injected simultaneously. For most days, at least 40 larvae were measured for each determination. Finally, larval volume, as well as length and width, was determined each day after infection. Therefore, we feel that a detailed comparison of our data with the above mentioned studies is not possible. The use of larval volume as an index of worm growth proved more advantageous than either length or width, since living worms were used exclusively. Worm volume remained constant, even if the larvae being photographed at the moment of exposure moved in such a way as to become shorter and wider. By contrast, in those studies involving fixed worms, the true size of the larva was more than likely altered, since most fixation procedures usually result in some shrinkage or expansion. The lack of significant difference in average larval growth noted in the present study between individual mice, or between mice (randomly bred) and rats (inbred), is viewed as a reflection of the constancy of its intracellular environment. However, since the coefficient of variation in worm volume from day to day among mice was greater than among rats, it is reasonable to assume that there exists a more constant intracellular environment among inbred animals than among randomly bred ones. In the present work, larval growth was characterized by three phases; an initial growth phase, a lag phase, and an exponential growth phase. Ultrastructural studies and Rivera-Pomar 1968; ( Ribas-Mujal Backwinkel and Themann 1972; Purkerson and Despommier 1974) have served to outline the adaptive host cell changes leading to the formation of the Nurse cell (i.e., the infected muscle cell) during these crucial phases of larval growth. It is obvious, even from these preliminary fine structure studies, that the growth
Trichinella
115
pattern of the developing muscle larva coincides in some direct way with host muscle cell changes (Ribas-Mujal and RiveraPomar 1968 ) . Similarly, various biochemical alterations in host components have been studied in infected muscle tissue during the early stages of larval development, i.e., Days O-30 after infection (Stewart and Read 1972a, 1972b, 1973a, 1973b). These investigators noted radical increases in uptake rates for various precursors of some classes of macromolecules (i.e., DNA, RNA, protein). Similarly, decreases in certain host cell constituents such as myoglobulin and contractile proteins have also been found during the early stages of infection with 7’. spiralis ( Stewart, personal communication). There are, as yet no data which suggest that the developing larva during its intracellular life, feeds directly on host tissues (Purkerson and Despommier 1974; Despommier, unpublished data). There is, however, some indirect evidence suggesting that the Nurse cell imports at least some of the metabolites needed for growth and development of the larva, and also aids in the export of waste products and various antigens excreted and secreted by the worm (Stoner and Hanks 1955; Hanks and Stoner 1958; Stewart and Read 1972a, 1973a, 1973b). These biochemical and ultrastructural studies lead to the speculation by the present authors and others (Stewart and Read 1972a, 1973a, 1973b) that a permanent new metabolic balance between host and parasite has been established as a consequence of intracellular infection with T. spiralis. While the ultrastructural and biochemical data have served to outline areas for study, more temporally controlled structure and function studies are needed before the complex series of events leading to the formation of host cell-parasite unit (i.e., Nurse cell and muscle larva) can be described. Such studies are now possible using synchronously growing infections induced by intramuscular injection of newborn larvae,
116
DESPOMMIER,
ARON AND TURGEON
ACKNOWLEDGMENTS The work was supported, in part, by Crnnt No. 5-ROl-AI-10627-03 from the U.S. National Institutes of Health and, from the same source, the senior author is recipient of Career Development Award No. I-KO4-AI-70255.
ALI KAHN, Z. 1966. The post-embryonic development of Trichinellu spiralis with special reference to ecdysis. Journal of Parasitology 52, 248-259. BACKWINKEL, K. D., AND THEA~ANN, H. 1972. Electronmikroskopische Untersuchen iibrr die Pathomorphologie der Trichinellose. Beitraege xur Pathologie 146, 259-271. DENNIS, D., DESPO~MAIIER, D. D., AND DAVID, N. 1970. Infectivity of the newborn larva of Trichinella spiralis in the rat. Journal of Parasitology 56, 974-977. HANKS, L. V., AND STONER, R. D. 1958. Incorporation of DL-Tyrosine-2-C-14 and nL-Tryptophan-2-C-14 by encysted Trichinella spiralis Parasitology 7, 82-89. larvae. Experimental PURKERSON, M., AND DESPOX~UER, D. D. 1974. Fine structure of the muscle phase of Trichinellu spiralis in the mouse. In “Trichinellosis” (C. Kim, Ed.), Intext Publishers, New York, N.Y., in press. RIBAS-MUJAL, D., AND RIVERA-POXIAR, J. M. 1968. Biological significance of the early structural alterations in skeletal muscle fibers infected by Trichinella spiralis. Virchows Archiv: Abteilung A: Pathologische Ancitomie und Physiologie 345, 154-168.
RICHELS, I. 1955. Histologische Studien zu den Problemen der Zellkenstanz: Untersuchungen zur mikroskopischen Anatomic im Lebenszyklus von Trichinella spiralis. Zentralblatt Fur Bakteriologie, Parasitenkunde, Infectionskrankheinter und Hygiene 163, 46-84. SNEDECOR, G. W., AND COCHRAN, W. G. 1967. “Sta&tical Methods,” Iowa State Lrnivcrsity Press, Ames, Iowa. STEWART, G. L., AND READ, C. P. 1972a. Ribonucleic acid metabolism in mouse trichinosis. Journal of Parasitology 58, 252-256. STEWART, G. L., AND READ, C. P. 1972b. Some aspects of cyst synthesis in mouse trichinosis. Journal of Parasitology 58, 1061-1064. STEWART, G. L., AND READ, C. P. 1973a. Deoxyribonucleic acid metabolism in mouse trichinosis. Journal of Parasitology 59, 264-267. STEWART, G. L. AND READ, C. P. 1973b. Changes in RNA in mouse trichinosis. Journal of Parnsitology 59, 997-1005. STONER, R. D., AND HANKS, L. V. 1955. Incorporation of C”-labeled amino acids by Trichinellu spirulis. Experimental Parasitology 4, 435444. THOMAS, V. H. 1965. Beitrage zur Biologic uncl mikroskopischen Anatomie von Trichinella spiralis. Owen 1835. Zeitschrift fiir Tropenmedizin und Parasitologie 16, 148-180. VILLELLA, J. B. 1970. Life cycle and morphology. In. “Trichinosis in Man and Animals” (S. E. Gould, Ed.), pp. 19-60. Charles C Thomas, Springfield, Illinois. WU, L. W. 1955. The development of the stichosome and associated structures in Trichinella spiralis. Canadian Journal of Zoology 33, 44O466.
PARASITOLOGY
Trichinella
37,
108-116
spiralis:
(1975)
Growth of the Intracellular
(Muscle)
Larva
DICKSON DESPOMMIER Division
of Tropical
Columbia
Medicine, School of Public Health, College of Physicians and Surgeons, University, 630 West 168th Street, New York, New York 10032 LORNA
Division
of Tropical
Columbia
Medicine, University,
ARON
School of Public Health, College of Physicians and Surgeons, 630 West 168th Street, New York, New York 10032
LIVIA TURCEON Division of Biostatistics, School of Public Health, College of Physicians and Surgeons, Columbia Vniuersity, 630 West 168th Street, New York, New York 10032 (Submitted
for publication
March 27, 1974)
DESPOMMIER, DICKSON, AHON, LORNA, AND TURGEOX, LIVIA. 1975. Trichinella spiralis: Growth of the intracellular (muscle) larva. Experimental Parasitology 37, 108-116. Newborn larvae of Trichinella spiralis were infective when injected directly into the thigh muscle of mice and rats. Infections initiated in this manner resulted in synchronously growing populations of muscle larvae, thereby permitting a detailed study of larval growth to be carried out. In mice, the mean larval growth, as measured by increase in larval volume, occurred in three phases; an initial growth phase ( Day O-l ) , a lag phase ( Days l-3 ) , and an exponential growth phase (Days 3-19). Larvae grew an average of 39% per day during the exponential phase. No further increase in larval volume was noted after Day 19. There was no statistically significant difference found in the rate of larval growth among individual mice for any given day. The larval growth rate was the same in rats as in mice. INDEX DESCRIPTIONS: Trichinella spiralis; Mice, Rats; Growth; Life cycles; Synchrony.
larvae in order to define accurately the temporal nature of cytological, physiological, and biochemical changes in the infected host cell and the parasite which occur during the course of the parenteral phase of the life cycle. Attempts at limiting the age differences of developing larvae by the use of methyridine (Stewart and Read 1972a, 1973a, 1973b), have resulted in the elucidation of some important biochemical aspects of the intracellular phase. In the
INTRODUCTION
Infections of Trichinella spiralis initiated through the ingestion of muscle larvae ultimately result in populations of developing muscle larvae growing nonsynchronously. This lack of synchrony arises from the fact that larvae are born live, one at a time over a period of 10-14 days, and therefore establish themselves in host cells at various points in time. It would be advantageous to obtain synchronously developing muscle 108 Copyright 0 1975 by Academic Yress, Inc. All rights of reproduction in any form reserved.
GROWTH
OF
above mentioned studies, however, the difference in age between the youngest and oldest larvae was six days; therefore, it is likely that some changes that may have been seen, if the infections were synchronous, were missed. The growth curve of Trichinella spiralis during its parenteral phase in the host has not yet been satisfactorily determined, again, because of the nonsynchronous nature of growth of the larvae which occurs after a naturally induced infection. The purposes of the present study were to establish synchronous infections of T. spiralis larvae in muscle tissue and to describe the larval growth rate from the point of initiation to maturation.
Trichinella
109
FIG. 1. Schematic spiralis larva.
diagram
of
a Trichinella
gauge needle and the dose was slowly injected directly into the thigh muscles of either mice or rats. Several sites were used for the injection dose to insure uniform muscle infections. Only one injection site was used for the subcutaneous route. Measurement of Larvae
Larvae were measured every day for 20 days after intramuscular injection. Infected mice were killed each day by cervical dislocation. The injected thigh MATERIALS AND METHODS muscles were then immediately removed Infection with Newborn Larvae and placed into warm 0.85% NaCl. Pieces Newborn larvae were collected in vitro of muscle (3-5 mm3) were cut from the inby methods described by Dennis et al. fected thigh tissue and gently pressed be( 1970). During injection of newborn larvae, tween two slides. This procedure squeezed an 18-gauge needle was replaced with a 2% the developing larvae out of the infected tissue, and in no case did any larvae remain in the infected tissue being examined. TABLE I Warm saline was added between the glass Adjustment Factor for Worm Volume Determinations slides. Living larvae were then photographed, using microscopy, as they were V,IVP V,IVP Day Day encountered, so as to randomize the popu11 0.84 1.00 0 lation being sampled as much as possible. 12 0.84 1 0.99 Worms were photographed at a magnifica2 0.96 13 0.84 tion of x100. All negatives were printed 0.84 0.9s 14 s at the same magnification. The width and 0.84 4 0.90 15 length of each larva was then determined 16 0.84 5 0.87 0.85 0.83 17 6 from each photograph with the aid of a 18 0.85 0.80 7 map measuring device 1 and a set of cali0.85 0.78 19 8 phers. 0.85 0.80 20 9 A first approximation of the volume (Vi ) 10 0.83 for each worm was made using the length 0 V2/Vl represents a series of correction factors and width determination. The assumption derived from two different methods for calculating was made that the shape of the worm was worm values. VI represents worm volume as calthat of a cylinder (L x (+W) * x r). A better culated strictly from length and width measureapproximation of worm volume (V,) was ments and does not take into consideration anterior obtained by taking into account the degree tapering of the worm. VI represents worm volume as calculated by combining measurements of the of tapering. It was found that the percentcylindrical mid and posterior portion of the worm age of worm length involved in tapering with the conical anterior portion of the worm (Fig. 1). Vz, except for Day 0, was always less than VI.
1 Keuffel
and Esser Co., Switzerland.
110
DESPOMMIER,
AHON
TABLE II Infectivity
of Newborn Trichinella spiralis Larvae aj’ter Inoculation by various Routesa
Route of injection
Intravenous Intramuscular
Intraperitoneal Subcut,aneous Intraduodenal
Percentage recovery of muscle larvaeb
Remarks
SO-800/;, Dennis et al. 1970 25-35yo 60% of the recovered 1arva.e were from the thigh muscles-the rest were evenly distributed throughout the musculature of the carcass. 2-5 y. Dennis et al. 1970 0% 0% I)ennis et al. 1970
a The subcutaneous and intramuscular route infection rates were determined in the present study. Intravenous, intraperitoneal, and intraduodenal route infection rates were derived from the literature. b Thirty days after injection each mouse carcass was digested in pepsin-HCl and the total number of muscle larvae determined.
varied according to the age of the worm. (It should be noted that for the first three days of larval growth, the posterior region is more tapered than the anterior end. From that time on, the worm is tapered more in the anterior region. ) The percentage difference in volume between VI and V, is shown for each day in Table I. The percentage volume difference (V,/ V,) was tested experimentally using models of cylinders and worm shapes obtained with modelling clay. The volume (Ve,) of a given clay cylinder was determined by submerging the cylinder in a loo-ml burette filled to the 80-ml mark with distilled water and then recording the fluid volume increase. The appropriate degree of tapering for a given age of worm (2, 4, 6, 8, and 12day-old larvae) was then measured from the respective photographs and translated on to the same cylinder of clay by removing material from the cylinder according to these measurements. The new volume (Ve,) was then determined by displace-
AND
TURGEON
ment of water in the same burette. Less than a 5% difference was obtained between the calculated ( V2/V1 ) and the empirical ,clay model ( Vez/Vel ) for worms from any given day, thereby supporting the assumption that the solid geometric figures which best describe the shape of the developing larvae is the cylinder plus the cone. This point is further illustrated in Fig. 1. The cylindrical portion of the worm in Fig. 1 contains a small area at the anal region which does not conform to the straight end of the cylinder (shaded area). This results in an overestimation of the worm volume for this region. The oral region, however, extends beyond the limits of the cone region drawn inside the worm (shaded area). This results in an underestimation of worm volume for this region. These two errors in volume estimations counteract each other and thereby allow an estimate closely approximating the true worm volume. Histology Infected thigh muscle was fixed in 15% picric acid and 3% paraformaldehyde at pH 7.2 for 2 hr at room temperature, washed overnight in tap water, then dehydrated in a graded series of alcohols, and embedded into paraffin. Three to five micron thick serial sections were stained with hematoxylin and phloxin. 38 36
:z 30 28 26 24 22 2c
5 i ts L 162 14s 12 10 ; 4 I * FIG. 2. Length and width measurements of T. larvae obtained from im infected mice. O-O = length; A----A = width. The standard deviation is given for each determination.
spiralis
GROWTH
OF
TABLE
Trichinella
111
III
Means and Standard L)evialions 01 Trichinella xpiralis Larval Volume Determinations Mice for Days 6, 7, 8, 9, and 10 after Intramuscular
amuny
Injection
Days after intramuscular injection Mouse No.
6
1 2 3 4
11800”f 25476 12;723 f 2941 9989 f 1419 10,792 f 2407
One way analysis of varianced
NB
7 14,288 13,414 14,395 16,060
f 3113 f 3340 f 3527 f 3271
8 23,817 19,063 20,666 26,046
NS
f f f +
9 4841 10,977 7690 7680
NS
40,638 40,907 34,547 35,466
f 8921 z!z 6884 rk 5232 f 7220
10 53,783 57,384 54,646 59,655
NS
NS
~ ~- ~ .-__---~ a All volumes are expressed as pm3. b Each larval volume determination was made by measuring 10 worms/mouse/day. standard deviation is given. c NS = Not significant. d The means were compared by one \Fay analysis of variance.
f 10,730 f 9460 z!z 11,745 f 12,594
The mean and
Statistical Methodology
Animals
The distribution of the volumetric determinations for various selected days was examined. Each frequency distribution was skewed to the right. Furthermore, the standard deviations for these daily volume determinations were not constant, but varied proportionately with the daily mean volume. In the above case it is suggested (Snedecar and Cochran 1967) that the natural logarithms of the observations should be normally distributed with constant standard deviation. Therefore, all statistical analyses were performed on the natural logarithms of the volumetric determinations. Linear regression lines were calculated by the method of least squares: the slope for the natural logarithms of the volume of the worms grown in mice from Day 3 through 20 was estimated. The growth rates between Days 3 and 7 were computed for larvae grown in mice and rats and the two slopes were then compared by Student’s t test for independent samples. One-way analysis of variance was used to evaIuate the variation in the natural logarithms of the volumes for a given day of larval growth among several mouse hosts.
All mice used in these studies were male, CFW strain (random bred) two-monthold animals.2 Newborn larvae of T. spiralis were obtained from infections initiated in 200 gram male Wistar rats.3 Two hundred gram male WF strain (inbred) rats were used to determine the growth rate of larvae in the rat.4 RESULTS
Assessment of Larval Infectivity Various Routes
by
Newborn larvae injected intravenously (iv) into rats or mice do not develop synchronously with respect to growth (unpublished observation), although 60430% of all larvae injected iv eventually mature, and become muscle larvae (Dennis et al. 1970). The absence of synchronous larval growth in this instance probably results from the fact that some newborn larvae penetrate organs other than muscle prior to their penetration into striated muscle. Apparently, in intravenously administered infec2 Carworth Farms, Rockland, New York. 3 Bill Fabre, New City, New York. 4 Microbiological Associates, Baltimore, Maryland.
112
DESPOMMIER,
ARON
AND
TABLE Length, Width, and Volume
il~~nsurrrnrnfs
of Trichinella
N
Day
L
SD
SE
Range
w
48 41 36 50 42 5 50 58 50 48 82 47 49 45 44 6 9
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
83 105 110 100 116 126 121 133 164 202 250 314 385 499 461 507 623 682 820 860 890
9 9 10 6
2 2 3 1 3 6 3 2 4 4 5 9 9
99- 69 121- 82 121- 81 114- 72 138- 86 139-106 148- 98 157-108 228-108 258-136 327-181 412-213 510-210 620-330 620304 567432 715618 988-469 950-475 975-745 995-800
7.1 7.3 7.9 8.8 9.4 11.5 11.9 12.9 15.3 17.1 18.9 20.9 22.7 23.4 24.2 28.2 27.8 31.3 36.0 37.6 38.6
11
47 46 26
a N = number standard error.
11
13 1‘2 13 25 25 34 58 64 77 81 65 58 164 176 69 54
of larvae
11
12 26 19 48 28 12 11
measured/day;
IV spiralis I,nrtms.from
SD SE 0.8 0.6 0.7 0.9 0.9 1.3 1.9 1.1 l.Q 1.4 1.8 2.2 3.0 5.5 2.8 3.2 1.3 5.5 3.6 2.4 1.8
L = length;
tions, larval penetration of muscle can Occur at anytime up to three days after the since some newborn time of injection, larvae were observed in muscle tissue three days after iv infection. Preliminary experiments suggested that the intramuscular route of infection with newborn larvae resulted in a synchronously developing population of larvae. However, the infection rate for the intramuscular route was not resolved in that study. Therefore, the present experiment was designed to determine the percentage of newborn larvae capable of developing to mature muscle larvae after intramuscular injection. The subcutaneous route of injection was also tried. Two groups of five mice each, were individually infected either intramuscularly (im) or subcutaneously (subcut.) with 60,000 newborn larvae. Thirty days after infection, all mice were killed by cervical dislocation. In the im injection group re-
TURGEON
0.2 0.2 0.2 0.2 0.2 0.6 0.4 0.1 0.3 0.2 0.3 0.3 0.2 0.8 0.4 1.3 0.4 1.7 0.5 0.4 0.4
Intrarmcncular
Range
d
9.3- 5.6 8.4- 6.4 8.4- 6.4 12.6- 6.0 ll.O- 6.6 12.9- 9.6 15.6-10.0 15.3-10.6 23.5-11.5 20.5-15.2 22.0-14.0 27.6-15.2 26.6-18.0 26.6-19.0 32.2-20.0 34.2-25.8 32.0-27.6 44.4-24.7 48.7-25.5 42.5-32.5 41.5-34.5
W = width;
3243 4597 5298 4883 6946 9124 11,480 14,319 26,566 37,336 59,040 90,947 131,971 187,615 198,985 290,619 368,200 467,481 751,286 855,377 897,283
SD = standard
Infections
in ilfice’7
SD
SE
829 897 1193 758 1328 2044 2589 2840 8390 8136 14,144 25,232 31,417 36,498 93,461 114,450 55,377 301,995 243,517 367,699 181,530
179 254 440 219 346 796 749 728 2953 2745 3630 7283 7139 8965 19,286 46,724 18,459 91,053 77,330 54,214 35,601
deviation;
SE =
ceiving 60,000 larvae, the injected legs were removed, skinned, and minced. The rest of the carcass was also eviscerated, skinned, and minced. Both the minced legs and carcasses were separately digested in I$% HCI-1% pepsin at 37” C for 4 hr. The entire carcass (skinned and eviscerated) of each subcutaneously injected mouse was similarly digested for 4 hr. The number of larvae from each group was then determined. The results of these trials, together with earlier determinations in animals infected by intravenous, intraperitoneal, and intraduodenal routes are presented in Table II. Recovery rates of 2535% were obtained from im injections, whereas no larvae were recovered from subcutaneously injected mice. Sixty percent of larvae recovered from the im group came from the injected leg; the rest were found throughout the carcass. It was estimated from 5 pm thick serial sections that approximately 80% of the thigh muscle was infected when five im
GROWTH
OF TABLE
Comparison
of Trichinella
spiralis
Trichinella
113
V Larval Growth in Mice and Rats
Days after intramuscular
injection
Host
3
4
5
6
7
CFW mouse (randomly bred)
4883 f 75@ (50) h
6946 f 1328 (42)
9124 f 2044 (5)
11,480 f 2589 W)
14,319 f 2840 (58)
WF rat (inbred)
4634 It 776 (23)
5967 f 1215 (25)
955 7134 f (25)
11,129 f 1680 (27)
14,709 f 2484 (25)
Students’ 1 test
NRC
NS
NS
NS
NS
(1Mean and standard deviation. b Number of larvae/determination. c NS = Not significant. d All volumes in pm3.
injections, consisting of lO-12,000 larvae per site were each given 10-15 mm apart down the length of the leg. Variation in Larval Growth among Mice Prior to determining the growth curve for the larva of T. spiralis in the mouse, it was necessary to obtain data concerning the variation in worm growth among mice infected on the same day with the same pool of newborn larvae. These data allowed a selection of the appropriate number of mice per day from which to measure larval growth. Mice were infected im with 30,000 muscle larvae. On Days 6, 7, 8, 9, and 10 after injection, larval volume was determined for ten larvae from each of four mice. The results are presented in Table III. A one-way analysis of variance showed no significant variation in larval size among the infected mice for any given day. Growth of Larvae in Mice from Day 0 to 20 of Newborn Larvae
after im Injection
The purpose of this experiment was to determine the rate of larval growth in the thigh muscle of the mouse. Larval growth parameters measured included length, width, and volume. CFW, two-month-old, male mice were each injected with 60,000 newborn larvae.
Larvae were obtained from two mice per day until Day 20, when larval growth was completed. This experiment was repeated for Days 0, 1, 4, 10, 11, I.5 14, 18, 19 and 20. The results of these two experiments were pooled. The length, width, and volume measurements are shown in Table IV. For the first three days, little increase was seen in either length or width (Fig. 2). However, beginning on Day 3, and continuing until Day 20, there was an almost continuous increase in larval length and width. Worm volume data (Fig. 3) suggested that larval growth proceeded in three distinct phases. During phase 1 (Day O-l), worm volume increased about 20%. Throughout phase II (Days l-3), no significant change in volume occurred, and corresponded to a lag phase. Finally, d’uring phase III (Days 3-19) an exponential rate of volume increase was observed. No further increase in worm volume was noted after Day 19. An overall growth rate for Days 3 through 20 was determined by computing the slope of the least squares line fitted to the average natural logarithmic volume. This slope was .33 natural logarithmic volume per day, indicating that larval volume increased about 39% every day during this period.
114
DESPOMMIER,
ARON
FIG. 3. Mean larval volume (in pm3) determinations from im infected mice. Larval growth occurs in three phases; an initial growth phase (Day O-l), a lag phase (Days l-3), and an exponential growth phase (Days 3-19). The standard deviation is given for each day.
Variation of Larval Growth between Mice and Rats The purpose of this experiment was to determine whether the larval growth rate for a given period of time was the same in rats as in mice. Ten WF strain, 200-gram, male rats and ten CFW, two-month-old, male mice were each infected with 30,000 newborn larvae obtained from a single isolate. Worm volume determinations were made 3, 4, 5, 6, and 7 days after injection. The daily average volume did not differ significantly in the two hosts (Table V). Furthermore, slopes of the regression lines fitted to the average daily logarithmic volumes were also similar. The growth rate for larvae in rats during the five-day period was 34% while that for larvae in mice was 32%. However, this observed 2% difference in growth rates was not significant. The individual worm volumes within each of the five days were more variable for larvae recovered from mice than for those from rats. DISCUSSION
The intramuscular (im) route of injection was effective in establishing infections with Trichinella spiralis larvae.
AND
TURGEON
Although approximately 30% of the injected newborn larvae matured into muscle larvae under these conditions, this infection rate was well below that obtainable by intravenous infection (60-80s ). It is probable that the low infectivity rate obtained with the im route resulted from introducing too many larvae (30-60,000) into a given muscle mass. Support of this view comes from histological evidence (unpublished observations) which showed that a large number of muscle fibers were killed under these conditions. The above data indicate that there is an upper limit with respect to the number of larvae which can establish a viable infection in a given amount of muscle tissue, and further imply that the ability of newborn larvae to repeatedly penetrate into and out of muscle fibers while remaining infective may be limited, since if that were not the case, a higher infection rate should have been noted. Larval growth was characterized for im infections in the mouse by a constant rate of increase (39Cjo/day) in mean larval volume which occurred during Days 3-19 of the infection. The coefficient of variation (SD/mean) noted in the volume of the newborn larvae (26% ) persisted at the mean ratio of 27% for each day’s volume determinations (see Table IV ) . These data give strong evidence for synchrony of larval growth during im infection. Some parameters of growth for T. spiralis larvae have already been investigated by several groups (Richels 1955; Wu 1956; Thomas 1966; Ali Khan 1966; Villella 1970). However, in all of the above studies, only fixed worms were measured. Furthermore, since infections were initiated via the oral route with muscle larvae, only nonsynchronous infections were available for study. Villella (1970) measured 15 larvae at selected days after oral infection, but did not present a statistical analysis of the data. Finally, only length and width mea-
GROWTH
OF
surements were recorded for each time point. In the present work, only live worms were measured. The exact age of the larvae during infection was known, as they were all injected simultaneously. For most days, at least 40 larvae were measured for each determination. Finally, larval volume, as well as length and width, was determined each day after infection. Therefore, we feel that a detailed comparison of our data with the above mentioned studies is not possible. The use of larval volume as an index of worm growth proved more advantageous than either length or width, since living worms were used exclusively. Worm volume remained constant, even if the larvae being photographed at the moment of exposure moved in such a way as to become shorter and wider. By contrast, in those studies involving fixed worms, the true size of the larva was more than likely altered, since most fixation procedures usually result in some shrinkage or expansion. The lack of significant difference in average larval growth noted in the present study between individual mice, or between mice (randomly bred) and rats (inbred), is viewed as a reflection of the constancy of its intracellular environment. However, since the coefficient of variation in worm volume from day to day among mice was greater than among rats, it is reasonable to assume that there exists a more constant intracellular environment among inbred animals than among randomly bred ones. In the present work, larval growth was characterized by three phases; an initial growth phase, a lag phase, and an exponential growth phase. Ultrastructural studies and Rivera-Pomar 1968; ( Ribas-Mujal Backwinkel and Themann 1972; Purkerson and Despommier 1974) have served to outline the adaptive host cell changes leading to the formation of the Nurse cell (i.e., the infected muscle cell) during these crucial phases of larval growth. It is obvious, even from these preliminary fine structure studies, that the growth
Trichinella
115
pattern of the developing muscle larva coincides in some direct way with host muscle cell changes (Ribas-Mujal and RiveraPomar 1968 ) . Similarly, various biochemical alterations in host components have been studied in infected muscle tissue during the early stages of larval development, i.e., Days O-30 after infection (Stewart and Read 1972a, 1972b, 1973a, 1973b). These investigators noted radical increases in uptake rates for various precursors of some classes of macromolecules (i.e., DNA, RNA, protein). Similarly, decreases in certain host cell constituents such as myoglobulin and contractile proteins have also been found during the early stages of infection with 7’. spiralis ( Stewart, personal communication). There are, as yet no data which suggest that the developing larva during its intracellular life, feeds directly on host tissues (Purkerson and Despommier 1974; Despommier, unpublished data). There is, however, some indirect evidence suggesting that the Nurse cell imports at least some of the metabolites needed for growth and development of the larva, and also aids in the export of waste products and various antigens excreted and secreted by the worm (Stoner and Hanks 1955; Hanks and Stoner 1958; Stewart and Read 1972a, 1973a, 1973b). These biochemical and ultrastructural studies lead to the speculation by the present authors and others (Stewart and Read 1972a, 1973a, 1973b) that a permanent new metabolic balance between host and parasite has been established as a consequence of intracellular infection with T. spiralis. While the ultrastructural and biochemical data have served to outline areas for study, more temporally controlled structure and function studies are needed before the complex series of events leading to the formation of host cell-parasite unit (i.e., Nurse cell and muscle larva) can be described. Such studies are now possible using synchronously growing infections induced by intramuscular injection of newborn larvae,
116
DESPOMMIER,
ARON AND TURGEON
ACKNOWLEDGMENTS The work was supported, in part, by Crnnt No. 5-ROl-AI-10627-03 from the U.S. National Institutes of Health and, from the same source, the senior author is recipient of Career Development Award No. I-KO4-AI-70255.
ALI KAHN, Z. 1966. The post-embryonic development of Trichinellu spiralis with special reference to ecdysis. Journal of Parasitology 52, 248-259. BACKWINKEL, K. D., AND THEA~ANN, H. 1972. Electronmikroskopische Untersuchen iibrr die Pathomorphologie der Trichinellose. Beitraege xur Pathologie 146, 259-271. DENNIS, D., DESPO~MAIIER, D. D., AND DAVID, N. 1970. Infectivity of the newborn larva of Trichinella spiralis in the rat. Journal of Parasitology 56, 974-977. HANKS, L. V., AND STONER, R. D. 1958. Incorporation of DL-Tyrosine-2-C-14 and nL-Tryptophan-2-C-14 by encysted Trichinella spiralis Parasitology 7, 82-89. larvae. Experimental PURKERSON, M., AND DESPOX~UER, D. D. 1974. Fine structure of the muscle phase of Trichinellu spiralis in the mouse. In “Trichinellosis” (C. Kim, Ed.), Intext Publishers, New York, N.Y., in press. RIBAS-MUJAL, D., AND RIVERA-POXIAR, J. M. 1968. Biological significance of the early structural alterations in skeletal muscle fibers infected by Trichinella spiralis. Virchows Archiv: Abteilung A: Pathologische Ancitomie und Physiologie 345, 154-168.
RICHELS, I. 1955. Histologische Studien zu den Problemen der Zellkenstanz: Untersuchungen zur mikroskopischen Anatomic im Lebenszyklus von Trichinella spiralis. Zentralblatt Fur Bakteriologie, Parasitenkunde, Infectionskrankheinter und Hygiene 163, 46-84. SNEDECOR, G. W., AND COCHRAN, W. G. 1967. “Sta&tical Methods,” Iowa State Lrnivcrsity Press, Ames, Iowa. STEWART, G. L., AND READ, C. P. 1972a. Ribonucleic acid metabolism in mouse trichinosis. Journal of Parasitology 58, 252-256. STEWART, G. L., AND READ, C. P. 1972b. Some aspects of cyst synthesis in mouse trichinosis. Journal of Parasitology 58, 1061-1064. STEWART, G. L., AND READ, C. P. 1973a. Deoxyribonucleic acid metabolism in mouse trichinosis. Journal of Parasitology 59, 264-267. STEWART, G. L. AND READ, C. P. 1973b. Changes in RNA in mouse trichinosis. Journal of Parnsitology 59, 997-1005. STONER, R. D., AND HANKS, L. V. 1955. Incorporation of C”-labeled amino acids by Trichinellu spirulis. Experimental Parasitology 4, 435444. THOMAS, V. H. 1965. Beitrage zur Biologic uncl mikroskopischen Anatomie von Trichinella spiralis. Owen 1835. Zeitschrift fiir Tropenmedizin und Parasitologie 16, 148-180. VILLELLA, J. B. 1970. Life cycle and morphology. In. “Trichinosis in Man and Animals” (S. E. Gould, Ed.), pp. 19-60. Charles C Thomas, Springfield, Illinois. WU, L. W. 1955. The development of the stichosome and associated structures in Trichinella spiralis. Canadian Journal of Zoology 33, 44O466.
