- Email: [email protected]
Trichinella spiralis: Recovery of newborn larvae from cardiac tissues
h~~~na~iOnd Journalfor
RESEARCH
Parasitology,
Vol. 24, No. 2, pp. 271-272, 1994
Copyright 0 1994 Australian Society for Parasitology Elwier Science Ltd Printed in Great Britain. All rights reserved 002&7519/94 %7.00+ 0.00
Pergamon
NOTE
TRICHINELLA
SPIRALIS: FROM MASAHIRO
RECOVERY OF NEWBORN CARDIAC TISSUES KUMAGAI
and
HARUO
LARVAE
KAMIYA*
Department of Parasitology, Hirosaki University School of Medicine, Hirosaki 036, Aomori, Japan (Received 31 August
1993; accepted 5 September 1993)
Abstract-KUMAGAl M. and KAMIYA H. 1994. Trichinelluspiralis: Recovery of newborn larvae from cardiac tissues. International Journalfor Parasitology 24: 271-212. Newborn larvae (NL) of Trichinella spiralis in cardiac tissues were recovered by incubating cardiac tissue blocks of mice and gerbils infected with T. spirnlis in RPMI- 1640 medium supplemented with antibiotics and 5% FCS for 24 h in a humidified atmosphere of 5% CO, in air at 37’C. This technique is effective for the recovery of migrating NL in visceral tissues which are en route to skeletal muscles, and may help to clarify the involvement of NL in the pathogenesis of cardiac or cerebral lesions in trichinellosis.
INDEX KEY WORDS: Trichinellu spiralis; newborn larvae; recovery technique; pathogenesis; cardiac tissue.
on the migrating pattern of newborn larvae (NL) of Trichinella spiralis in the tissues is important for studying the possible involvement of NL in the pathogenesis of trichinellosis, such as myocarditis or encephalitis (Gray, Morse & Phillips, 1962; Gould, 1970). The recovery of NL in the visceral tissues, however, has not been well demonstrated, since NL are so small (100 m long, 6 fl in diameter) that it is not easy to detect them in paraffin-embedded tissue sections or in compressed tissue preparations. Moreover, NL themselves are digested by artificial gastric juice and the Baermann technique has been reported to be unsuccessful for recovering NL from macerated tissues (Mauss & Otto, 1942). Therefore, we developed a simple method to recover NL in tissues such as hearts or brains, which the NL pass by en route to skeletal muscles of experimental animals. The Japanese isolate of T. spiralis originally isolated from Asiatic black bear (Selenarcros thibetanus) was maintained in our laboratory in ICR mice (Yamaguchi, Takada, Yagisawa, Inaba, Osanai, Hanada, Maruyama, Sasaki, Goto, Oobuchi, Endo & Terui, 1975) and was used throughout the experiment. Male BALB/c and female Mongolian gerbils (Meriones unguiculatus), bred in the Laboratory Animal Research Center, Hirosaki University, Japan, were used. Infective muscle larvae were obtained by INFORMATION
*To whom all correspondence
should be addressed.
digesting the muscle of ICR mice, infected with T. spiralis 11 weeks previously, in artificial gastric juice (0.8% pepsin and 0.8% HCI in 0.85% saline) for 3 hat 37 “C. Mice and gerbils were orally infected with 1000 and 500 larvae, respectively. The infected animals were killed at intervals under ethyl ether inhalation. The heart was removed and its ventricles were cut opened with scissors to wash out all the blood with the RPMI1640 medium (Nissui Pharmaceutical, Tokyo), pH 7.2, supplemented with 10 units ml-1 of heparin and 100 units ml-l of penicillin. The heart was further washed by passing through 3 successive 10 ml of media in plastic dishes (6 cm in diameter; Sumitomo Bakelite, Tokyo). These washing media were carefully examined under a dissection microscope for NL, but none was found. The heart was then cut as evenly as possible with fine-pointed scissors into 50 pieces for mouse or 150 pieces for gerbil in a tissue-culture dish with a 2 mm grid (6 cm in diameter; Nunc, Roskilde). Ten ml of the RPMI-1640 medium, containing 5% fetal calf serum (Sebak, Aidenbach), was added to the heart tissue, and the culture dish was incubated for 24 h in a humidified atmosphere of 5% CO, in air at 37°C. All procedures were carried out under aseptic conditions. Cardiac tissue blocks were then removed with forceps, so that NL which had migrated out into the media could easily be counted under the dissection microscope. As shown in Fig. 1, NL were first detected on day 6 post-infection (p.i.) in both mice and gerbils. Fewer 271
212
M. KUMAGAIand H. KAMIYA
0
4
8
12
l
Gerbil
0
Mouse
16
2.0
24
Days post-infection
FIG. 1. Recovery of T. spiralis newborn larvae by in vitro incubation of cardiac tissue of infected animals. Female Mongolian gerbils and male BALB/c mice received an oral dose of 500 and 1000 infective muscle larvae, respectively. Results are expressed as mean ~s.E.M. Five animals were examined on each day, except on days 14 and 21 p.i. for gerbils (n = 3). NL were recovered for the gerbils as compared with those from mice, although the difference in the infective dose of larvae must be considered. In mice, the NL number markedly decreased on day 10 p.i. and no NL was recovered from the heart on day 16 p.i. and thereafter. Contrary to this, NL were recovered from the heart of gerbils even on day 21 p.i. when the experiment was terminated. It is possible that the present method may not have exclusively detected only the NL which invaded the heart muscles, but might also have detected the NL which were retained in the capillaries of the cardiac tissue blocks although blood had been extensively washed out with the medium. The presence of the adult worms was examined by incubating the small intestine of infected animals in physiological saline for 4 hat 37 “C. A few adult worms were detected in the mouse intestine on day 14 p.i., but not on day 16 p.i., which corresponded to the fact that
no NL were recovered from heart. The adult worms were found in the small intestine of gerbils however even on day 21 pi., and this may be the reason why NL were still found in the heart of the gerbils. The presence of NL in the heart tissues may be transient. However, some NL on their way to skeletal muscles might have entered the cardiac tissues through the capillaries which might be damaged at the time of NL migration from arteries to venules. This notion was also supported by the finding that the migrating NL cause extensive haemorrhagic lesions in the brains of the same rodent hosts used in the experiments (Inaba, personal communication). To advance our knowledge on the pathogenesis of trichinellosis, it is important to know the migration kinetics of NL in the various organs. Therefore, the present technique can be applied to study the migration pattern of NL in those visceral organs in relation to the pathogenesis of trichinellosis.
wish to thank Drs H. K. Ooi and T. Inaba for their valuable suggestions. This study was supported by a Fellowship of the Japan Society for Promotion of Science for Japanese Junior Scientists, and by the Karoji Memorial Fund for Medical Research in Hirosaki University. Acknowledgemenrs-We
REFERENCES GOULD S. E. 1970. Trichinosis in Man and Animals. Thomas,
Springfield, IL. GRAY D. F., MORSEB. S. &PHILLIPSW. F. 1962. Trichinosis with neurologic and cardiac involvement. Annals of Internal Medicine 57: 230-244.
MAUSS E. A. & Oreo G. F. 1942. The occurrence. of Trichinella spiralis larvae in tissues other than skeletal muscles. Journal of Laboratory Clinical Medicine 27: 1384 1387. YAMAGUCHIT., TAKADA T., YAGISAWAM., INABA T., OSANAI H., HANADAK., MARUYAMA Y., SASAKIY., GOTO S., OOBUCHIH. & TERUI Y. 1975. The first outbreak of trichinosis in Japan. Japanese Medical Journal 2668: 162 1 (in Japanese).
RESEARCH
Parasitology,
Vol. 24, No. 2, pp. 271-272, 1994
Copyright 0 1994 Australian Society for Parasitology Elwier Science Ltd Printed in Great Britain. All rights reserved 002&7519/94 %7.00+ 0.00
Pergamon
NOTE
TRICHINELLA
SPIRALIS: FROM MASAHIRO
RECOVERY OF NEWBORN CARDIAC TISSUES KUMAGAI
and
HARUO
LARVAE
KAMIYA*
Department of Parasitology, Hirosaki University School of Medicine, Hirosaki 036, Aomori, Japan (Received 31 August
1993; accepted 5 September 1993)
Abstract-KUMAGAl M. and KAMIYA H. 1994. Trichinelluspiralis: Recovery of newborn larvae from cardiac tissues. International Journalfor Parasitology 24: 271-212. Newborn larvae (NL) of Trichinella spiralis in cardiac tissues were recovered by incubating cardiac tissue blocks of mice and gerbils infected with T. spirnlis in RPMI- 1640 medium supplemented with antibiotics and 5% FCS for 24 h in a humidified atmosphere of 5% CO, in air at 37’C. This technique is effective for the recovery of migrating NL in visceral tissues which are en route to skeletal muscles, and may help to clarify the involvement of NL in the pathogenesis of cardiac or cerebral lesions in trichinellosis.
INDEX KEY WORDS: Trichinellu spiralis; newborn larvae; recovery technique; pathogenesis; cardiac tissue.
on the migrating pattern of newborn larvae (NL) of Trichinella spiralis in the tissues is important for studying the possible involvement of NL in the pathogenesis of trichinellosis, such as myocarditis or encephalitis (Gray, Morse & Phillips, 1962; Gould, 1970). The recovery of NL in the visceral tissues, however, has not been well demonstrated, since NL are so small (100 m long, 6 fl in diameter) that it is not easy to detect them in paraffin-embedded tissue sections or in compressed tissue preparations. Moreover, NL themselves are digested by artificial gastric juice and the Baermann technique has been reported to be unsuccessful for recovering NL from macerated tissues (Mauss & Otto, 1942). Therefore, we developed a simple method to recover NL in tissues such as hearts or brains, which the NL pass by en route to skeletal muscles of experimental animals. The Japanese isolate of T. spiralis originally isolated from Asiatic black bear (Selenarcros thibetanus) was maintained in our laboratory in ICR mice (Yamaguchi, Takada, Yagisawa, Inaba, Osanai, Hanada, Maruyama, Sasaki, Goto, Oobuchi, Endo & Terui, 1975) and was used throughout the experiment. Male BALB/c and female Mongolian gerbils (Meriones unguiculatus), bred in the Laboratory Animal Research Center, Hirosaki University, Japan, were used. Infective muscle larvae were obtained by INFORMATION
*To whom all correspondence
should be addressed.
digesting the muscle of ICR mice, infected with T. spiralis 11 weeks previously, in artificial gastric juice (0.8% pepsin and 0.8% HCI in 0.85% saline) for 3 hat 37 “C. Mice and gerbils were orally infected with 1000 and 500 larvae, respectively. The infected animals were killed at intervals under ethyl ether inhalation. The heart was removed and its ventricles were cut opened with scissors to wash out all the blood with the RPMI1640 medium (Nissui Pharmaceutical, Tokyo), pH 7.2, supplemented with 10 units ml-1 of heparin and 100 units ml-l of penicillin. The heart was further washed by passing through 3 successive 10 ml of media in plastic dishes (6 cm in diameter; Sumitomo Bakelite, Tokyo). These washing media were carefully examined under a dissection microscope for NL, but none was found. The heart was then cut as evenly as possible with fine-pointed scissors into 50 pieces for mouse or 150 pieces for gerbil in a tissue-culture dish with a 2 mm grid (6 cm in diameter; Nunc, Roskilde). Ten ml of the RPMI-1640 medium, containing 5% fetal calf serum (Sebak, Aidenbach), was added to the heart tissue, and the culture dish was incubated for 24 h in a humidified atmosphere of 5% CO, in air at 37°C. All procedures were carried out under aseptic conditions. Cardiac tissue blocks were then removed with forceps, so that NL which had migrated out into the media could easily be counted under the dissection microscope. As shown in Fig. 1, NL were first detected on day 6 post-infection (p.i.) in both mice and gerbils. Fewer 271
212
M. KUMAGAIand H. KAMIYA
0
4
8
12
l
Gerbil
0
Mouse
16
2.0
24
Days post-infection
FIG. 1. Recovery of T. spiralis newborn larvae by in vitro incubation of cardiac tissue of infected animals. Female Mongolian gerbils and male BALB/c mice received an oral dose of 500 and 1000 infective muscle larvae, respectively. Results are expressed as mean ~s.E.M. Five animals were examined on each day, except on days 14 and 21 p.i. for gerbils (n = 3). NL were recovered for the gerbils as compared with those from mice, although the difference in the infective dose of larvae must be considered. In mice, the NL number markedly decreased on day 10 p.i. and no NL was recovered from the heart on day 16 p.i. and thereafter. Contrary to this, NL were recovered from the heart of gerbils even on day 21 p.i. when the experiment was terminated. It is possible that the present method may not have exclusively detected only the NL which invaded the heart muscles, but might also have detected the NL which were retained in the capillaries of the cardiac tissue blocks although blood had been extensively washed out with the medium. The presence of the adult worms was examined by incubating the small intestine of infected animals in physiological saline for 4 hat 37 “C. A few adult worms were detected in the mouse intestine on day 14 p.i., but not on day 16 p.i., which corresponded to the fact that
no NL were recovered from heart. The adult worms were found in the small intestine of gerbils however even on day 21 pi., and this may be the reason why NL were still found in the heart of the gerbils. The presence of NL in the heart tissues may be transient. However, some NL on their way to skeletal muscles might have entered the cardiac tissues through the capillaries which might be damaged at the time of NL migration from arteries to venules. This notion was also supported by the finding that the migrating NL cause extensive haemorrhagic lesions in the brains of the same rodent hosts used in the experiments (Inaba, personal communication). To advance our knowledge on the pathogenesis of trichinellosis, it is important to know the migration kinetics of NL in the various organs. Therefore, the present technique can be applied to study the migration pattern of NL in those visceral organs in relation to the pathogenesis of trichinellosis.
wish to thank Drs H. K. Ooi and T. Inaba for their valuable suggestions. This study was supported by a Fellowship of the Japan Society for Promotion of Science for Japanese Junior Scientists, and by the Karoji Memorial Fund for Medical Research in Hirosaki University. Acknowledgemenrs-We
REFERENCES GOULD S. E. 1970. Trichinosis in Man and Animals. Thomas,
Springfield, IL. GRAY D. F., MORSEB. S. &PHILLIPSW. F. 1962. Trichinosis with neurologic and cardiac involvement. Annals of Internal Medicine 57: 230-244.
MAUSS E. A. & Oreo G. F. 1942. The occurrence. of Trichinella spiralis larvae in tissues other than skeletal muscles. Journal of Laboratory Clinical Medicine 27: 1384 1387. YAMAGUCHIT., TAKADA T., YAGISAWAM., INABA T., OSANAI H., HANADAK., MARUYAMA Y., SASAKIY., GOTO S., OOBUCHIH. & TERUI Y. 1975. The first outbreak of trichinosis in Japan. Japanese Medical Journal 2668: 162 1 (in Japanese).