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2 mice
ARTICLE IN PRESS Journal of Experimental Animal Science Journal of Experimental Animal Science 43 (2004) 29–38 www.elsevier-deutschland.de/jeas
Penetration and maturation of Schistosoma mansoni in suckling and adult Swiss Webster and DBA/2 mice A.A. Fidalgo-Neto, R.R. De-Carvalho, A.C.A.X. De-Oliveira, D.A. Manh*aes-Rocha, F.J.R. Paumgartten* Department of Biological Sciences, National School of Public Health, Oswaldo Cruz Foundation, Rio de Janeiro RJ 21040-361, Brazil
Abstract Murine schistosomosis is a widely used experimental model of the human disease. Different methods have been employed to infect mice with Schistosoma mansoni cercariae, such as subcutaneous or intraperitoneal injections, the tail immersion technique, and the use of a metal ring placed on the abdominal skin of anaesthetized animals. An alternative method of infection that requires no anaesthesia and no restraint is to place suckling mice (10 days old) on a Petri dish with a small volume of water containing cercariae. In this study, we compared the penetration and maturation of S. mansoni in mice infected by this alternative method with those noted in mice infected at an older age (45 days) by the tail immersion technique. Besides evaluating the effects of age and method of infection, we also compared the susceptibility of two strains of mice (Swiss Webster (SW) and DBA/2). Mice were exposed to 100 cercariae and worms (by portal–hepatic perfusion) as well as eggs were recovered in the liver and intestines on postinfection (PI) days 35, 55 and 90. Skin penetration was very efficient (about 100%) irrespective of the mouse strain, sex, age and method of infection. Worm and egg recoveries were higher in SW mice at any PI interval, but strain differences tended to be less pronounced on PI day 90. In both strains, recoveries of worms and eggs were clearly higher in mice infected at a younger age (10 days old). This study thus suggests that infection of free-moving suckling mice is a suitable alternative to other methods of infection with S. mansoni. r 2004 Elsevier GmbH. All rights reserved. Keywords: Schistosoma mansoni; Mus musculus; Animal models; Methods of infection; Trematodes; Schistosomosis; Swiss Webster; DBA/2
*Corresponding author. Laborat!orio de Toxicologia Ambiental, ENSP, FIOCRUZ, Av. Brasil 4036, Rio de Janeiro RJ 21040-361, Brazil. Tel.: +55-21-3882-9009; fax: +55-21-2564-8985. E-mail address: paum@ensp.fiocruz.br (F.J.R. Paumgartten). 0939-8600/$ - see front matter r 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.jeas.2004.02.001
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Introduction Schistosomosis remains a major health problem affecting more than 200 million people in Central and South America, Africa and Asia, and experimental infections of laboratory animals with Schistosoma trematodes have been widely used to investigate different pathophysiological features of this disease (Morgan et al., 2001; Cheever et al., 2002; McKerrow and Salter, 2002). Susceptibility to primary infection with Schistosoma mansoni has been shown to markedly vary among different species and strains of laboratory animals (Warren and Peters, 1967). Hamsters and mice are among the most susceptible species while rats are known to be rather resistant hosts (Warren and Peters, 1967). Since the disease progress in mice is to some extent similar to that seen in humans, murine schistosomosis has been the most studied experimental model (Cheever et al., 2002). Different methods have been used to infect mice and other laboratory rodents with S. mansoni. For instance, a metal ring has been employed to expose a limited area of the rodent abdominal skin to a certain volume of water containing cercariae (Ghandour and Webbe, 1973). Simpler methods, that do not require anaesthesia, have also been widely used to infect adult animals with S. mansoni such as subcutaneous or intraperitoneal injections and immersion of mouse’s tail in an assay tube containing cercariae. Injections are rather artificial methods of infection since the initial step in natural infections of mammalian hosts involves penetration of the intact skin by cercariae (McKerrow and Salter, 2002). The tail immersion technique, on the other hand, is a stressing procedure because it requires immobilization of the animal for a certain period of time. An alternative and more suitable method of infection that requires no anaesthesia and no restraint is to place very young mice (e.g. 10 days old) on a Petri dish with a small volume of water containing S. mansoni cercariae. Under those conditions pups can move freely in a warmed environment while a large skin area (paws, limbs and ventral abdominal skin) remains in contact with the water and allows cercariae penetration. The present study was undertaken to compare two different methods used to infect mice with S. mansoni: the ventral body skin exposure of young pups and the exposure of adult mice by the tail immersion technique. In addition to evaluating the effects of age and method of infection, we also compared the evolution of the experimentally induced S. mansoni infection in Swiss Webster (SW) mice with that observed in the inbred strain DBA/2.
Materials and methods Parasite: A ‘Belo-Horizonte’ (BH) strain of S. mansoni, maintained in Biomphalaria glabrata snails and SW mice (Paraense and Corr#ea, 1989), was used in this study. Infected snails were placed into a beaker with dechlorinated water and induced to shed cercariae by illuminating them with a 60 W tungsten lamp. S. mansoni cercariae were used for infection within 2 h of emergence.
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Mice: Male and female adult SW and DBA/2 mice from the FIOCRUZ Central Animal House breeding stock were used. All animals were housed in standard plastic cages with stainless-steel cover lids and wood shavings as bedding. Room temperature (2171 C), humidity (70%75) and dark/light cycle (lights on from 8:00 a.m. to 8:00 p.m.) were controlled. A pelleted diet for laboratory rodents (Nuvital, Nuvilab, Curitiba, PR, Brazil) and tap water were available ad libitum. All procedures were performed in accordance with Brazilian animal welfare protection laws and the research protocol was approved by the Ethics Committee on the Use of Animals of Oswaldo Cruz Foundation (CEUA-FIOCRUZ).
Methods of infection Tail immersion: Adult mice (4–6 weeks old) were infected by immersing their tails in assay tubes containing 10 ml of dechlorinated water with 100 cercariae for 20 min. After the exposure period, tails were gently rinsed with water and mice were placed back in their cages. The assay tube water content was then poured into a 5 cm plastic Petri dish. Pup ventral body skin exposure: After mating, impregnated female mice were housed in individual cages. From pregnancy day 18 on, female cages were inspected twice a day for births and the day on which the offspring was born was designated as postnatal day 1. On postnatal day 10, pups were taken from their mothers and placed individually into plastic Petri dishes (5 cm in diameter) containing 3 ml of dechlorinated water with 100 cercariae for 20 min. Pups were kept warm during infection procedure by means of 60 W tungsten lamps located close to Petri dishes. Under those circumstances, pups’ tail and paws and all the ventral part of their bodies were efficiently exposed to cercarial penetration. After having been infected, all pups returned to their mothers’ cages. After infection by either method, a few drops of Lugol’s iodine were added to each Petri dish and the number of cercariae that failed to penetrate mouse skin (i.e. intact cercariae plus separated cercarial heads) was counted under a stereomicroscope. Infected mice were killed 35, 55 or 90 days later. Control (sham-treated) mice were treated exactly as infected mice except that they were exposed to a water volume that did not contain any S. mansoni cercariae. Worm recovery and perfusion technique: Worms were recovered from mesenteric veins after a portal–hepatic perfusion carried out as described by Smithers and Terry (1965) with a few adaptations. Briefly, mice were killed by cervical dislocation and their abdominal and thoracic cavities were opened. The hepatic–portal vein was then sectioned and a perfusion needle (27 gauge) was positioned into the heart left ventricle. The perfusion solution (0.85% sodium chloride; 1.5% sodium citrate) was driven by a peristaltic pump (approx. 40 ml/min for 40–60 s until liver, intestines and kidneys became pale) and, after passing through a mesh that retained the worms, all perfusate was collected into a glass beaker. After switching off the pump, mesenteric veins and the liver were carefully checked for any remaining worm. Worms were then examined and counted under a stereomicroscope.
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Eggs recovery: Following portal–hepatic perfusions, livers and intestines (caecum plus large and small intestines) were removed and digested in separate conical flasks with a 5% potassium hydroxide solution at 37 C for 16 h (Cheever, 1968). Fifty ml aliquots of the digests were then placed under cover lids on microscope slides and eggs were counted with a 100 magnification. Three aliquots from each digest were examined. Statistical evaluation: Data were analysed by the Kruskal–Wallis test followed by the Mann–Whitney U test. Proportions were evaluated by the chi-square test or, alternatively, by the Fisher’s exact test. Statistical calculations were performed using a MINITAB software (MTB, University of Pennsylvania, 1984) and, in any case, a difference was considered as statistically significant at Po0:05:
Results and discussion As shown in Table 1, percutaneous penetration of S. mansoni cercariae was very efficient (almost 100%) irrespective of the mouse gender, strain (SW versus DBA/2)
Table 1. Skin penetration and subsequent maturation of S. mansoni in male and female Swiss Webster (SW) and DBA/2 mice exposed to 100 cercariae at two different ages (postnatal day 10 or 45) Strain
Sex
N
Age at infection (days)
Penetration (%)
Worms recovered at different time intervals after infectiona 35 days
55 days
90 days
SW
M F
5 5
10 10
99.570.7 99.570.6
35.2710.1 40.478.0
51.070.5 36.473.8
14.673.7e 21.872.6e
DBA/2
M F
5 5
10 10
99.570.5 99.570.8
18.675.8 19.272.9
27.273.6b 23.673.6
13.375.0 12.471.3be
SW
M F
5 5
45 45
99.670.7 99.470.9
15.073.0 11.473.0a
22.072.8a 11.672.0ac
11.871.8e 8.873.3
DBA/2
M F
5 5
45 45
98.073.2 97.872.5
8.472.2 6.471.3a
8.070.8ab 10.671.0a
10.670.8 4.671.2ace
M: male; F: female. N: number of infected mice. Values are shown as means7SE. Infection was either by pup ventral body skin exposure (postnatal day 10) or by tail immersion (postnatal day 45). a Number of worms recovered by portal–mesenteric venous system perfusion. Significant differences (Po0:05) are indicated by superscripts as follows; within the same column: aafrom mice of the same strain and sex infected at an earlier age (10 days old), bafrom SW mice of the same sex and age at infection, cafrom males of the same strain and age at infection; and within the same row: dafrom 35 PI days, eafrom 55 PI days.
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and age at infection (10 versus 45 days) or exposed skin area (ventral body skin versus tail). The subsequent worm maturation, however, seemed to markedly vary depending on the mouse strain and host age at infection. In any case (gender, strain and age at infection), on postinfection (PI) day 35, a substantial number of worms and worm pairs, but only a relatively small number of eggs were recovered (Tables 1 and 2). Twenty days later (on PI day 55), while the number of worms and worm pairs found in the portal–mesenteric system remained unchanged or was only slightly higher, the number of eggs trapped in the liver and intestines dramatically increased (Tables 1 and 2). On PI day 90, in comparison with PI day 55, there was, in almost all instances, a clear reduction of worms and worm pairs harboured in the liver. The number of eggs trapped in the liver and intestines on PI day 90, on the other hand, exhibited only slight changes the direction of which seemed to depend on the mouse strain and age at infection (Tables 1 and 2). The disease progress had no apparent effect on the body weight of SW and DBA/2 mice infected on postnatal day 45 (Fig. 1). Body weight gain of SW and DBA/2 mice infected on postnatal day 10 did not differ from that of uninfected controls either (Fig. 1). Some deaths, however, were observed among mice with infections lasting longer than 55 days. As shown in Table 3, among SW mice no deaths were noted up to 35 days after infection, only one death occurred between PI days 36 and 55, and a few more deaths were observed thereafter. No DBA/2 mice infected on PN day 10 died up to PI day 35, but one mouse died before PI day 55 and more than half of male and female survivors died before PI day 90 (Table 3). DBA/2 infected on PN day 45 seemed to be somewhat more resistant to S. mansoni-induced mortality and, in this group, no death occurred among infected males between PI days 56 and 90 (Table 3). The same number of animals of the same strain, sex and age were evaluated for the burden of worms and eggs at the end of each PI interval. Perfusion of mice found dead in their cages was not feasible so that it was not possible to determine their burden of worms. Therefore, the possibility that mice that died between PI days 56 and 90 could be harbouring the heaviest worm burdens cannot be ruled out. If deaths before PI day 90 were due to a heavier burden of worms, the longevity of S. mansoni until PI day 90 would be somewhat underestimated in our study. Nonetheless, since deaths were almost absent before PI day 35, and mortality was relatively low up to PI day 55, it seems fair to conclude that S. mansoni-induced host deaths had no influence on the average number of worms and eggs recovered on PI days 35 and 55. No consistent sex-related differences in the number of recovered worms and eggs were noted either in SW or in DBA/2 mice (Tables 1 and 2). Nonetheless, SW mice (an outbred strain) showed, at any of the PI intervals, numbers of recovered worms and eggs substantially greater than those presented by the inbred strain DBA/2 (Tables 1 and 2). Since the proportion of cercariae that successfully penetrated the skin did not differ between the two strains, the greater resistance of DBA/2 strain to infection may have resulted from a higher mortality of penetrating cercariae within the skin. Differences in the proportion of cercarial deaths within the host skin have been shown to explain most of the observed interspecies differences in susceptibility to infection with S. mansoni (Cheever, 1969). Lower susceptibility of rats to infection
34
Strain Sex N Age at Recovery of worm pairs and eggs in the liver infection and intestines at different time intervals after infection (days) 35 days 55 days
SW
M 5 10 F 5 10
Eggs Liva
Eggs Inta
15.475.2e 1.5870.33e 2.8570.48e 17.473.0 1.2370.22e 1.4170.2e
WP
Eggs Liva
Eggs Inta
22.872.1 16.672.0
23.673.4d 16.271.8d
69.075.7d 46.375.6cd
DBA/2 M 5 10 F 5 10
7.873.1 0.4370.08be 0.7570.28be 12.271.6b 6.870.9b 0.6370.23be 0.7170.08be 8.071.4b
SW
6.871.1 0.1070.01ae 0.1170.02ae 10.271.4a 10.671.3ad 21.472.4ad 5.271.6a 0.0170.06ae 0.0870.05ae 5.470.9ac 4.870.5acd 13.970.5ad
M 5 45 F 5 45
DBA/2 M 5 45 F 5 45
WP
Eggs Liva
6.271.4e 22.972.7d 9.471.6e 27.473.2de
7.970.9bd 27.973.3bd 6.072.3 7.371.9bd 11.371.8bcd 5.670.5
Eggs Inta 42.978.0de 36.574.2d
17.173.02de 23.270.9d 15.073.3d 22.573.2bde
5.471.0e 22.873.3de 3.871.4 18.773.0de
32.676.3de 27.474.3de
3.871.2 0.0870.04ae 0.0070.00abe 3.670.4ab 3.370.5abd 8.971.4abd 4.7870.4 13.470.9bde 15.271.6ad 2.870.6ae 0.0070.00ae 0.0070.00ae 5.070.5d 3.270.5ad 8.871.7bd 1.470.2ace 3.870.4abcd 6.471.0abcd
Values are shown as means7SE. M: male, F: female. Worm pair (WP): whenever a female was found within the gynecophorous canal of a male worm. Infection was performed either by pup ventral body skin exposure (postnatal day 10) or by tail immersion (postnatal day 45). Postinfection day 0: day on which mice were infected. For each parameter (WP, eggs in liver and eggs in intestines) significant differences (Po0:05) are indicated by superscripts as follows; within the same column: aafrom mice of the same strain and sex infected at an earlier age (10 days old), bafrom SW mice of the same sex and age at infection, cafrom males of the same strain and age at infection; within the same row: dafrom mice of the same sex, strain and age at infection at PI 35, eafrom mice of the same sex, strain and age at infection at PI 55. a Data are shown as thousands (103) of S. mansoni eggs.
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WP
90 days
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Table 2. Recovery of worm pairs (WP) and trapped eggs in the liver (Eggs Liv) and intestines (Eggs Int) of SW and DBA/2 mice exposed to 100 cercariae at two different ages (postnatal day 10 or 45)
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Fig. 1. Body weight gain of SW and DBA/2 mice infected with 100 S. mansoni cercariae either on postnatal day 10 (exposure of free-moving pups) or on postnatal day 45 (tail immersion method). PI=Postinfection day; SW C and DBA/2 C=Uninfected control groups; SW I and DBA/2 I=Infected groups.
as compared to hamsters and mice, however, have been demonstrated to preponderantly result from a more rapid elimination of schistosomules from other tissues—owing to an acquired immunity—at a subsequent phase of the disease evolution (Warren and Peters, 1967; McKerrow and Salter, 2002). In the infected rat, for instance, the number of recovered worms rapidly decreases from the 4th to the 8th PI week (Warren and Peters, 1967). Such a mechanism does not seem to explain the differences in susceptibilities to infection noted between the two strains of mice. The number of worms recovered from DBA/2 mice increased from PI day 35 to 55, and the rate of reduction in the number of recovered worms between PI days 55 and 90 apparently did not differ between DBA/2 and SW strains (Tables 1 and 2). In both mouse strains, recovery of worms and eggs on PI days 35 and 55 was markedly higher in animals that had been infected on postnatal day 10 (Tables 1 and 2). Later on, on PI day 90, differences in the burden of worms and eggs between mice infected on day 10 and those infected on day 45 were less evident (Tables 1 and 2). Since cercarial penetration did not differ among groups, the foregoing findings seemed to indicate that a greater number of S. mansoni cercariae developed into adult worms when mice are infected at a younger (10 days old) age. The effect of age on the susceptibility of mice to infection with S. mansoni was previously studied by a
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Table 3. Mortality of SW and DBA/2 mice infected with 100 S. mansoni cercariae at two different ages (postnatal day 10 or 45) and respective uninfected controls. Mortality (D/T) is shown as the number of mice that died (D) per total number of mice that were evaluated (T) during the postinfection interval Age at infection
10 days old
Postinfection interval (days)
0–35
36–55
56–90
0–35
36–55
56–90
SW
M F M+F M F M+F
0/9 0/9 0/18 0/9 0/9 0/18
0/9 0/9 0/18 0/9 0/9 0/18
0/9 0/9 0/18 3/9 3/9 6/18de
0/9 0/9 0/18 0/9 0/9 0/18
0/9 0/9 0/18 2/9 0/9 2/18
0/9 0/9 0/18 1/9 1/9 2/18
M F M+F M F M+F
0/9 0/9 0/18 0/9 0/9 0/18
0/9 0/9 0/18 0/9 1/9 1/18
0/9 0/9 0/18 6/8de 4/9 10/17de
0/9 0/9 0/18 0/9 1/8 1/17
0/9 0/9 0/18 2/9 0/9 2/18
0/9 0/9 0/18 0/9a 4/9 4/18a
Control
Infected
DBA/2
45 days old
Control
Infected
M: male; F: female. Infection was either by pup ventral body skin exposure (postnatal day 10) or by tail immersion (postnatal day 45). Proportions were analysed by the chi-square test or, alternatively, by the Fisher’s exact test, and differences (Po0:05) are indicated by superscripts as follows; aafrom mice of the same strain and sex infected at an earlier age (10 days old), bafrom SW mice of the same sex and age at infection, cafrom males of the same strain and age at infection, dafrom 35 PI days, eafrom 55 PI days.
few authors. Lewert and Mandllowitz (1963) found that recovery of adult worms from CF1 mice infected at a younger age (19–28 days old) was higher than that obtained from animals infected at an older age (about 1 year). Clegg and Smithers (1968), on the other hand, found no difference in the susceptibility to infections in comparisons of young mice (28–35 days) with old mice (10–12 months) of the Parkes and CBA strain. The apparent discrepancy between these two studies seems to be explained by Purnell’s findings (Purnell, 1966) showing that, during the first weeks of life, the susceptibility of mice to infection with S. mansoni is initially very high but thereafter it decreases with age and reaches a steady level about 1 month after birth. The influence of host age on the death of S. mansoni cercariae in the skin was investigated by Ghandour and Webbe (1973). These authors infected mice up to 7 days old, by taping them over a circular well attached to a Petri dish so that the abdominal surface was in contact with a suspension of cercariae in the well, and adult mice (anaesthetized) by employing an abdominal metal ring. They were thus able to demonstrate that the number of cercariae which died in the skin of very young mice (2 days old) was less than one-third of those that died in the adult animal, and that losses in the skin steadily increases with host age up to about 28–35
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days (Ghandour and Webbe, 1973). Results from the present study with 10 days old mice therefore seem to support the view that suckling pups are more susceptible to infection with S. mansoni than older animals. In conclusion, data provided by the present study indicated that penetration of mouse skin by S. mansoni cercariae did not differ either between the two strains (SW and DBA/2) or between the two methods of infection, i.e. infection of free-moving 10 days old pups and infection of restrained adults by the tail immersion technique. Nonetheless, SW proved to be more susceptible than DBA/2 mice and, in both strains, the proportion of penetrating cercariae that developed into adult worms as well as the number of eggs trapped in the liver and intestines were clearly higher when 10 days old pups were infected. The foregoing results suggested that 10 days old pups are more susceptible than adult mice to infection with S. mansoni, and that infection at this very young age—which requires no anaesthesia and no restraint—is an interesting alternative to the widely used tail immersion method.
Acknowledgements The authors wish to thank Prof. Wladimir Lobato-Paraense and all the staff of the Department of Malacology of the Oswaldo Cruz Institute for having kindly provided the S. mansoni cercariae (BH strain) used in this study. The research project was supported by grants from FAPERJ (Bolsa Cientistas do Nosso Estado), CNPq and PAPES III-FIOCRUZ. FJRP was the recipient of a research fellowship from CNPq (Brazilian National Research Council).
References Cheever, A.W., 1968. Conditions affecting the accuracy of potassium hydroxide digestion techniques for counting Schistosoma mansoni eggs in tissue. Bull. World Health Organ. 39, 328. Cheever, A.W., 1969. Quantitative comparison of the intensity of Schistosoma mansoni infections in man and experimental animals. Trans. R. Soc. Trop. Med. Hyg. 63, 781–795. Cheever, A.W., Lenzi, J.A., Lenzi, H.L., Andrade, Z.A., 2002. Experimental models of Schistosoma mansoni infection. Mem. Inst. Oswaldo Cruz 97, 917–940. Clegg, J.A., Smithers, S.R., 1968. Death of schistosome cercariae during penetration of the skin. II. Penetration of mammalian skin by Schistosome mansoni. Parasitology 58, 111–128. Ghandour, A.M., Webbe, G., 1973. A study of the death of Schistosoma mansoni cercariae during penetration of mammalian host skin: the influence of the ages of the cercariae and of the host. Int. J. Parasitol. 3, 789–794. Lewert, R.M., Mandllowitz, S., 1963. Innate immunity to Schistosoma mansoni relative to the state of connective tissue. Ann. NY Acad. Sci. 113, 54–62. McKerrow, J.H., Salter, J., 2002. Invasion of skin by Schistosoma cercariae. Trends Parasitol. 18, 193–195. Morgan, J.A., Dejong, R.J., Snyder, S.D., Mkoji, G.M., Loker, E.S., 2001. Schistosoma mansoni and Biomphalaria: past and future trends. Parasitology 123, S211–218. Paraense, W.L., Corr#ea, L., 1989. A potential vector of Schistosoma mansoni in Uruguay. Mem. Inst. Oswaldo Cruz 84, 281–288.
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Purnell, R.E., 1966. Host parasite relationships in schistosomiasis. II. The effects of age and sex on the infection of mice and hamsters with cercariae of Schistosoma mansoni and of hamsters with cercariae of Schistosoma haematobium. Ann. Trop. Med. Parasitol. 60, 94–99. Smithers, S.R., Terry, R.J., 1965. The infection of laboratory hosts with cercariae of Schistosoma mansoni and the recovery of adult worms. Parasitology 55, 695–700. Warren, K.S., Peters, P.A., 1967. Comparison of penetration and maturation of Schistosoma mansoni in the hamster, mouse, guinea pig, rabbit, and the rat. Am. J. Trop. Med. Hyg. 16 (6), 718–722.
Penetration and maturation of Schistosoma mansoni in suckling and adult Swiss Webster and DBA/2 mice A.A. Fidalgo-Neto, R.R. De-Carvalho, A.C.A.X. De-Oliveira, D.A. Manh*aes-Rocha, F.J.R. Paumgartten* Department of Biological Sciences, National School of Public Health, Oswaldo Cruz Foundation, Rio de Janeiro RJ 21040-361, Brazil
Abstract Murine schistosomosis is a widely used experimental model of the human disease. Different methods have been employed to infect mice with Schistosoma mansoni cercariae, such as subcutaneous or intraperitoneal injections, the tail immersion technique, and the use of a metal ring placed on the abdominal skin of anaesthetized animals. An alternative method of infection that requires no anaesthesia and no restraint is to place suckling mice (10 days old) on a Petri dish with a small volume of water containing cercariae. In this study, we compared the penetration and maturation of S. mansoni in mice infected by this alternative method with those noted in mice infected at an older age (45 days) by the tail immersion technique. Besides evaluating the effects of age and method of infection, we also compared the susceptibility of two strains of mice (Swiss Webster (SW) and DBA/2). Mice were exposed to 100 cercariae and worms (by portal–hepatic perfusion) as well as eggs were recovered in the liver and intestines on postinfection (PI) days 35, 55 and 90. Skin penetration was very efficient (about 100%) irrespective of the mouse strain, sex, age and method of infection. Worm and egg recoveries were higher in SW mice at any PI interval, but strain differences tended to be less pronounced on PI day 90. In both strains, recoveries of worms and eggs were clearly higher in mice infected at a younger age (10 days old). This study thus suggests that infection of free-moving suckling mice is a suitable alternative to other methods of infection with S. mansoni. r 2004 Elsevier GmbH. All rights reserved. Keywords: Schistosoma mansoni; Mus musculus; Animal models; Methods of infection; Trematodes; Schistosomosis; Swiss Webster; DBA/2
*Corresponding author. Laborat!orio de Toxicologia Ambiental, ENSP, FIOCRUZ, Av. Brasil 4036, Rio de Janeiro RJ 21040-361, Brazil. Tel.: +55-21-3882-9009; fax: +55-21-2564-8985. E-mail address: paum@ensp.fiocruz.br (F.J.R. Paumgartten). 0939-8600/$ - see front matter r 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.jeas.2004.02.001
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Introduction Schistosomosis remains a major health problem affecting more than 200 million people in Central and South America, Africa and Asia, and experimental infections of laboratory animals with Schistosoma trematodes have been widely used to investigate different pathophysiological features of this disease (Morgan et al., 2001; Cheever et al., 2002; McKerrow and Salter, 2002). Susceptibility to primary infection with Schistosoma mansoni has been shown to markedly vary among different species and strains of laboratory animals (Warren and Peters, 1967). Hamsters and mice are among the most susceptible species while rats are known to be rather resistant hosts (Warren and Peters, 1967). Since the disease progress in mice is to some extent similar to that seen in humans, murine schistosomosis has been the most studied experimental model (Cheever et al., 2002). Different methods have been used to infect mice and other laboratory rodents with S. mansoni. For instance, a metal ring has been employed to expose a limited area of the rodent abdominal skin to a certain volume of water containing cercariae (Ghandour and Webbe, 1973). Simpler methods, that do not require anaesthesia, have also been widely used to infect adult animals with S. mansoni such as subcutaneous or intraperitoneal injections and immersion of mouse’s tail in an assay tube containing cercariae. Injections are rather artificial methods of infection since the initial step in natural infections of mammalian hosts involves penetration of the intact skin by cercariae (McKerrow and Salter, 2002). The tail immersion technique, on the other hand, is a stressing procedure because it requires immobilization of the animal for a certain period of time. An alternative and more suitable method of infection that requires no anaesthesia and no restraint is to place very young mice (e.g. 10 days old) on a Petri dish with a small volume of water containing S. mansoni cercariae. Under those conditions pups can move freely in a warmed environment while a large skin area (paws, limbs and ventral abdominal skin) remains in contact with the water and allows cercariae penetration. The present study was undertaken to compare two different methods used to infect mice with S. mansoni: the ventral body skin exposure of young pups and the exposure of adult mice by the tail immersion technique. In addition to evaluating the effects of age and method of infection, we also compared the evolution of the experimentally induced S. mansoni infection in Swiss Webster (SW) mice with that observed in the inbred strain DBA/2.
Materials and methods Parasite: A ‘Belo-Horizonte’ (BH) strain of S. mansoni, maintained in Biomphalaria glabrata snails and SW mice (Paraense and Corr#ea, 1989), was used in this study. Infected snails were placed into a beaker with dechlorinated water and induced to shed cercariae by illuminating them with a 60 W tungsten lamp. S. mansoni cercariae were used for infection within 2 h of emergence.
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Mice: Male and female adult SW and DBA/2 mice from the FIOCRUZ Central Animal House breeding stock were used. All animals were housed in standard plastic cages with stainless-steel cover lids and wood shavings as bedding. Room temperature (2171 C), humidity (70%75) and dark/light cycle (lights on from 8:00 a.m. to 8:00 p.m.) were controlled. A pelleted diet for laboratory rodents (Nuvital, Nuvilab, Curitiba, PR, Brazil) and tap water were available ad libitum. All procedures were performed in accordance with Brazilian animal welfare protection laws and the research protocol was approved by the Ethics Committee on the Use of Animals of Oswaldo Cruz Foundation (CEUA-FIOCRUZ).
Methods of infection Tail immersion: Adult mice (4–6 weeks old) were infected by immersing their tails in assay tubes containing 10 ml of dechlorinated water with 100 cercariae for 20 min. After the exposure period, tails were gently rinsed with water and mice were placed back in their cages. The assay tube water content was then poured into a 5 cm plastic Petri dish. Pup ventral body skin exposure: After mating, impregnated female mice were housed in individual cages. From pregnancy day 18 on, female cages were inspected twice a day for births and the day on which the offspring was born was designated as postnatal day 1. On postnatal day 10, pups were taken from their mothers and placed individually into plastic Petri dishes (5 cm in diameter) containing 3 ml of dechlorinated water with 100 cercariae for 20 min. Pups were kept warm during infection procedure by means of 60 W tungsten lamps located close to Petri dishes. Under those circumstances, pups’ tail and paws and all the ventral part of their bodies were efficiently exposed to cercarial penetration. After having been infected, all pups returned to their mothers’ cages. After infection by either method, a few drops of Lugol’s iodine were added to each Petri dish and the number of cercariae that failed to penetrate mouse skin (i.e. intact cercariae plus separated cercarial heads) was counted under a stereomicroscope. Infected mice were killed 35, 55 or 90 days later. Control (sham-treated) mice were treated exactly as infected mice except that they were exposed to a water volume that did not contain any S. mansoni cercariae. Worm recovery and perfusion technique: Worms were recovered from mesenteric veins after a portal–hepatic perfusion carried out as described by Smithers and Terry (1965) with a few adaptations. Briefly, mice were killed by cervical dislocation and their abdominal and thoracic cavities were opened. The hepatic–portal vein was then sectioned and a perfusion needle (27 gauge) was positioned into the heart left ventricle. The perfusion solution (0.85% sodium chloride; 1.5% sodium citrate) was driven by a peristaltic pump (approx. 40 ml/min for 40–60 s until liver, intestines and kidneys became pale) and, after passing through a mesh that retained the worms, all perfusate was collected into a glass beaker. After switching off the pump, mesenteric veins and the liver were carefully checked for any remaining worm. Worms were then examined and counted under a stereomicroscope.
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Eggs recovery: Following portal–hepatic perfusions, livers and intestines (caecum plus large and small intestines) were removed and digested in separate conical flasks with a 5% potassium hydroxide solution at 37 C for 16 h (Cheever, 1968). Fifty ml aliquots of the digests were then placed under cover lids on microscope slides and eggs were counted with a 100 magnification. Three aliquots from each digest were examined. Statistical evaluation: Data were analysed by the Kruskal–Wallis test followed by the Mann–Whitney U test. Proportions were evaluated by the chi-square test or, alternatively, by the Fisher’s exact test. Statistical calculations were performed using a MINITAB software (MTB, University of Pennsylvania, 1984) and, in any case, a difference was considered as statistically significant at Po0:05:
Results and discussion As shown in Table 1, percutaneous penetration of S. mansoni cercariae was very efficient (almost 100%) irrespective of the mouse gender, strain (SW versus DBA/2)
Table 1. Skin penetration and subsequent maturation of S. mansoni in male and female Swiss Webster (SW) and DBA/2 mice exposed to 100 cercariae at two different ages (postnatal day 10 or 45) Strain
Sex
N
Age at infection (days)
Penetration (%)
Worms recovered at different time intervals after infectiona 35 days
55 days
90 days
SW
M F
5 5
10 10
99.570.7 99.570.6
35.2710.1 40.478.0
51.070.5 36.473.8
14.673.7e 21.872.6e
DBA/2
M F
5 5
10 10
99.570.5 99.570.8
18.675.8 19.272.9
27.273.6b 23.673.6
13.375.0 12.471.3be
SW
M F
5 5
45 45
99.670.7 99.470.9
15.073.0 11.473.0a
22.072.8a 11.672.0ac
11.871.8e 8.873.3
DBA/2
M F
5 5
45 45
98.073.2 97.872.5
8.472.2 6.471.3a
8.070.8ab 10.671.0a
10.670.8 4.671.2ace
M: male; F: female. N: number of infected mice. Values are shown as means7SE. Infection was either by pup ventral body skin exposure (postnatal day 10) or by tail immersion (postnatal day 45). a Number of worms recovered by portal–mesenteric venous system perfusion. Significant differences (Po0:05) are indicated by superscripts as follows; within the same column: aafrom mice of the same strain and sex infected at an earlier age (10 days old), bafrom SW mice of the same sex and age at infection, cafrom males of the same strain and age at infection; and within the same row: dafrom 35 PI days, eafrom 55 PI days.
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and age at infection (10 versus 45 days) or exposed skin area (ventral body skin versus tail). The subsequent worm maturation, however, seemed to markedly vary depending on the mouse strain and host age at infection. In any case (gender, strain and age at infection), on postinfection (PI) day 35, a substantial number of worms and worm pairs, but only a relatively small number of eggs were recovered (Tables 1 and 2). Twenty days later (on PI day 55), while the number of worms and worm pairs found in the portal–mesenteric system remained unchanged or was only slightly higher, the number of eggs trapped in the liver and intestines dramatically increased (Tables 1 and 2). On PI day 90, in comparison with PI day 55, there was, in almost all instances, a clear reduction of worms and worm pairs harboured in the liver. The number of eggs trapped in the liver and intestines on PI day 90, on the other hand, exhibited only slight changes the direction of which seemed to depend on the mouse strain and age at infection (Tables 1 and 2). The disease progress had no apparent effect on the body weight of SW and DBA/2 mice infected on postnatal day 45 (Fig. 1). Body weight gain of SW and DBA/2 mice infected on postnatal day 10 did not differ from that of uninfected controls either (Fig. 1). Some deaths, however, were observed among mice with infections lasting longer than 55 days. As shown in Table 3, among SW mice no deaths were noted up to 35 days after infection, only one death occurred between PI days 36 and 55, and a few more deaths were observed thereafter. No DBA/2 mice infected on PN day 10 died up to PI day 35, but one mouse died before PI day 55 and more than half of male and female survivors died before PI day 90 (Table 3). DBA/2 infected on PN day 45 seemed to be somewhat more resistant to S. mansoni-induced mortality and, in this group, no death occurred among infected males between PI days 56 and 90 (Table 3). The same number of animals of the same strain, sex and age were evaluated for the burden of worms and eggs at the end of each PI interval. Perfusion of mice found dead in their cages was not feasible so that it was not possible to determine their burden of worms. Therefore, the possibility that mice that died between PI days 56 and 90 could be harbouring the heaviest worm burdens cannot be ruled out. If deaths before PI day 90 were due to a heavier burden of worms, the longevity of S. mansoni until PI day 90 would be somewhat underestimated in our study. Nonetheless, since deaths were almost absent before PI day 35, and mortality was relatively low up to PI day 55, it seems fair to conclude that S. mansoni-induced host deaths had no influence on the average number of worms and eggs recovered on PI days 35 and 55. No consistent sex-related differences in the number of recovered worms and eggs were noted either in SW or in DBA/2 mice (Tables 1 and 2). Nonetheless, SW mice (an outbred strain) showed, at any of the PI intervals, numbers of recovered worms and eggs substantially greater than those presented by the inbred strain DBA/2 (Tables 1 and 2). Since the proportion of cercariae that successfully penetrated the skin did not differ between the two strains, the greater resistance of DBA/2 strain to infection may have resulted from a higher mortality of penetrating cercariae within the skin. Differences in the proportion of cercarial deaths within the host skin have been shown to explain most of the observed interspecies differences in susceptibility to infection with S. mansoni (Cheever, 1969). Lower susceptibility of rats to infection
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Strain Sex N Age at Recovery of worm pairs and eggs in the liver infection and intestines at different time intervals after infection (days) 35 days 55 days
SW
M 5 10 F 5 10
Eggs Liva
Eggs Inta
15.475.2e 1.5870.33e 2.8570.48e 17.473.0 1.2370.22e 1.4170.2e
WP
Eggs Liva
Eggs Inta
22.872.1 16.672.0
23.673.4d 16.271.8d
69.075.7d 46.375.6cd
DBA/2 M 5 10 F 5 10
7.873.1 0.4370.08be 0.7570.28be 12.271.6b 6.870.9b 0.6370.23be 0.7170.08be 8.071.4b
SW
6.871.1 0.1070.01ae 0.1170.02ae 10.271.4a 10.671.3ad 21.472.4ad 5.271.6a 0.0170.06ae 0.0870.05ae 5.470.9ac 4.870.5acd 13.970.5ad
M 5 45 F 5 45
DBA/2 M 5 45 F 5 45
WP
Eggs Liva
6.271.4e 22.972.7d 9.471.6e 27.473.2de
7.970.9bd 27.973.3bd 6.072.3 7.371.9bd 11.371.8bcd 5.670.5
Eggs Inta 42.978.0de 36.574.2d
17.173.02de 23.270.9d 15.073.3d 22.573.2bde
5.471.0e 22.873.3de 3.871.4 18.773.0de
32.676.3de 27.474.3de
3.871.2 0.0870.04ae 0.0070.00abe 3.670.4ab 3.370.5abd 8.971.4abd 4.7870.4 13.470.9bde 15.271.6ad 2.870.6ae 0.0070.00ae 0.0070.00ae 5.070.5d 3.270.5ad 8.871.7bd 1.470.2ace 3.870.4abcd 6.471.0abcd
Values are shown as means7SE. M: male, F: female. Worm pair (WP): whenever a female was found within the gynecophorous canal of a male worm. Infection was performed either by pup ventral body skin exposure (postnatal day 10) or by tail immersion (postnatal day 45). Postinfection day 0: day on which mice were infected. For each parameter (WP, eggs in liver and eggs in intestines) significant differences (Po0:05) are indicated by superscripts as follows; within the same column: aafrom mice of the same strain and sex infected at an earlier age (10 days old), bafrom SW mice of the same sex and age at infection, cafrom males of the same strain and age at infection; within the same row: dafrom mice of the same sex, strain and age at infection at PI 35, eafrom mice of the same sex, strain and age at infection at PI 55. a Data are shown as thousands (103) of S. mansoni eggs.
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WP
90 days
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Table 2. Recovery of worm pairs (WP) and trapped eggs in the liver (Eggs Liv) and intestines (Eggs Int) of SW and DBA/2 mice exposed to 100 cercariae at two different ages (postnatal day 10 or 45)
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Fig. 1. Body weight gain of SW and DBA/2 mice infected with 100 S. mansoni cercariae either on postnatal day 10 (exposure of free-moving pups) or on postnatal day 45 (tail immersion method). PI=Postinfection day; SW C and DBA/2 C=Uninfected control groups; SW I and DBA/2 I=Infected groups.
as compared to hamsters and mice, however, have been demonstrated to preponderantly result from a more rapid elimination of schistosomules from other tissues—owing to an acquired immunity—at a subsequent phase of the disease evolution (Warren and Peters, 1967; McKerrow and Salter, 2002). In the infected rat, for instance, the number of recovered worms rapidly decreases from the 4th to the 8th PI week (Warren and Peters, 1967). Such a mechanism does not seem to explain the differences in susceptibilities to infection noted between the two strains of mice. The number of worms recovered from DBA/2 mice increased from PI day 35 to 55, and the rate of reduction in the number of recovered worms between PI days 55 and 90 apparently did not differ between DBA/2 and SW strains (Tables 1 and 2). In both mouse strains, recovery of worms and eggs on PI days 35 and 55 was markedly higher in animals that had been infected on postnatal day 10 (Tables 1 and 2). Later on, on PI day 90, differences in the burden of worms and eggs between mice infected on day 10 and those infected on day 45 were less evident (Tables 1 and 2). Since cercarial penetration did not differ among groups, the foregoing findings seemed to indicate that a greater number of S. mansoni cercariae developed into adult worms when mice are infected at a younger (10 days old) age. The effect of age on the susceptibility of mice to infection with S. mansoni was previously studied by a
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Table 3. Mortality of SW and DBA/2 mice infected with 100 S. mansoni cercariae at two different ages (postnatal day 10 or 45) and respective uninfected controls. Mortality (D/T) is shown as the number of mice that died (D) per total number of mice that were evaluated (T) during the postinfection interval Age at infection
10 days old
Postinfection interval (days)
0–35
36–55
56–90
0–35
36–55
56–90
SW
M F M+F M F M+F
0/9 0/9 0/18 0/9 0/9 0/18
0/9 0/9 0/18 0/9 0/9 0/18
0/9 0/9 0/18 3/9 3/9 6/18de
0/9 0/9 0/18 0/9 0/9 0/18
0/9 0/9 0/18 2/9 0/9 2/18
0/9 0/9 0/18 1/9 1/9 2/18
M F M+F M F M+F
0/9 0/9 0/18 0/9 0/9 0/18
0/9 0/9 0/18 0/9 1/9 1/18
0/9 0/9 0/18 6/8de 4/9 10/17de
0/9 0/9 0/18 0/9 1/8 1/17
0/9 0/9 0/18 2/9 0/9 2/18
0/9 0/9 0/18 0/9a 4/9 4/18a
Control
Infected
DBA/2
45 days old
Control
Infected
M: male; F: female. Infection was either by pup ventral body skin exposure (postnatal day 10) or by tail immersion (postnatal day 45). Proportions were analysed by the chi-square test or, alternatively, by the Fisher’s exact test, and differences (Po0:05) are indicated by superscripts as follows; aafrom mice of the same strain and sex infected at an earlier age (10 days old), bafrom SW mice of the same sex and age at infection, cafrom males of the same strain and age at infection, dafrom 35 PI days, eafrom 55 PI days.
few authors. Lewert and Mandllowitz (1963) found that recovery of adult worms from CF1 mice infected at a younger age (19–28 days old) was higher than that obtained from animals infected at an older age (about 1 year). Clegg and Smithers (1968), on the other hand, found no difference in the susceptibility to infections in comparisons of young mice (28–35 days) with old mice (10–12 months) of the Parkes and CBA strain. The apparent discrepancy between these two studies seems to be explained by Purnell’s findings (Purnell, 1966) showing that, during the first weeks of life, the susceptibility of mice to infection with S. mansoni is initially very high but thereafter it decreases with age and reaches a steady level about 1 month after birth. The influence of host age on the death of S. mansoni cercariae in the skin was investigated by Ghandour and Webbe (1973). These authors infected mice up to 7 days old, by taping them over a circular well attached to a Petri dish so that the abdominal surface was in contact with a suspension of cercariae in the well, and adult mice (anaesthetized) by employing an abdominal metal ring. They were thus able to demonstrate that the number of cercariae which died in the skin of very young mice (2 days old) was less than one-third of those that died in the adult animal, and that losses in the skin steadily increases with host age up to about 28–35
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days (Ghandour and Webbe, 1973). Results from the present study with 10 days old mice therefore seem to support the view that suckling pups are more susceptible to infection with S. mansoni than older animals. In conclusion, data provided by the present study indicated that penetration of mouse skin by S. mansoni cercariae did not differ either between the two strains (SW and DBA/2) or between the two methods of infection, i.e. infection of free-moving 10 days old pups and infection of restrained adults by the tail immersion technique. Nonetheless, SW proved to be more susceptible than DBA/2 mice and, in both strains, the proportion of penetrating cercariae that developed into adult worms as well as the number of eggs trapped in the liver and intestines were clearly higher when 10 days old pups were infected. The foregoing results suggested that 10 days old pups are more susceptible than adult mice to infection with S. mansoni, and that infection at this very young age—which requires no anaesthesia and no restraint—is an interesting alternative to the widely used tail immersion method.
Acknowledgements The authors wish to thank Prof. Wladimir Lobato-Paraense and all the staff of the Department of Malacology of the Oswaldo Cruz Institute for having kindly provided the S. mansoni cercariae (BH strain) used in this study. The research project was supported by grants from FAPERJ (Bolsa Cientistas do Nosso Estado), CNPq and PAPES III-FIOCRUZ. FJRP was the recipient of a research fellowship from CNPq (Brazilian National Research Council).
References Cheever, A.W., 1968. Conditions affecting the accuracy of potassium hydroxide digestion techniques for counting Schistosoma mansoni eggs in tissue. Bull. World Health Organ. 39, 328. Cheever, A.W., 1969. Quantitative comparison of the intensity of Schistosoma mansoni infections in man and experimental animals. Trans. R. Soc. Trop. Med. Hyg. 63, 781–795. Cheever, A.W., Lenzi, J.A., Lenzi, H.L., Andrade, Z.A., 2002. Experimental models of Schistosoma mansoni infection. Mem. Inst. Oswaldo Cruz 97, 917–940. Clegg, J.A., Smithers, S.R., 1968. Death of schistosome cercariae during penetration of the skin. II. Penetration of mammalian skin by Schistosome mansoni. Parasitology 58, 111–128. Ghandour, A.M., Webbe, G., 1973. A study of the death of Schistosoma mansoni cercariae during penetration of mammalian host skin: the influence of the ages of the cercariae and of the host. Int. J. Parasitol. 3, 789–794. Lewert, R.M., Mandllowitz, S., 1963. Innate immunity to Schistosoma mansoni relative to the state of connective tissue. Ann. NY Acad. Sci. 113, 54–62. McKerrow, J.H., Salter, J., 2002. Invasion of skin by Schistosoma cercariae. Trends Parasitol. 18, 193–195. Morgan, J.A., Dejong, R.J., Snyder, S.D., Mkoji, G.M., Loker, E.S., 2001. Schistosoma mansoni and Biomphalaria: past and future trends. Parasitology 123, S211–218. Paraense, W.L., Corr#ea, L., 1989. A potential vector of Schistosoma mansoni in Uruguay. Mem. Inst. Oswaldo Cruz 84, 281–288.
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Purnell, R.E., 1966. Host parasite relationships in schistosomiasis. II. The effects of age and sex on the infection of mice and hamsters with cercariae of Schistosoma mansoni and of hamsters with cercariae of Schistosoma haematobium. Ann. Trop. Med. Parasitol. 60, 94–99. Smithers, S.R., Terry, R.J., 1965. The infection of laboratory hosts with cercariae of Schistosoma mansoni and the recovery of adult worms. Parasitology 55, 695–700. Warren, K.S., Peters, P.A., 1967. Comparison of penetration and maturation of Schistosoma mansoni in the hamster, mouse, guinea pig, rabbit, and the rat. Am. J. Trop. Med. Hyg. 16 (6), 718–722.