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Histopathology of the Lungs of Rabbits Experimentally Infected with Dirofilaria immitis
J. Comp. Path. 1994Vol. 110,403M.06
SHORT PAPERS
Histopathology of the LUngs of Rabbits Experimentally Infected with Dirofilaria immitis T. Sato, S. N o g a m i * , K. Nakagaki~, I. Inoue*, W. Shlrai and K. Araki++ Departments of VeterinaryPathology and *Medical Zoology, School of VeterinaryMedicine, Nihon University, 1866 Kameino, Fujisawa, Kanagawa252, *iDepartmentof Veterinary Medicine, Nippon Jui Chikusan University,Musashino 180 and +National Institute of Public Health, Minato-ku 108, Japan Summary The lungs of rabbits experimentally infected with Dirofilaria inzmitis were examined histopathologically, to compare the changes with those seen in human pulmonary dirofilariasis. Eight rabbits were infected subcutaneously with two to eight immature worms to induce pulmonary dirofilariasis. Obstructive changes, similar to those reported in human pulmonary dirofilariasis, were observed in the blood vessels surrounding the worms. A form of arteritis, similar to occlusive arteritis, and periarteritis were also observed in the lungs of the rabbits. These results suggest that experimentaIly induced dirofilariasis in the rabbit is a useful animal model for human pulmonary dirofilariasis.
Introduction A reliable diagnostic technique is required to distinguish cases of human pulmonary dirofilariasis from lung cancer and tuberculosis in areas where Dirofilaria immitis is endemic, such as Japan, Australia and the United States (Beaver and Orihel, 1965; Navarrette-Reyna and Noon, 1968; Moorhouse et aI., 1971; Ciferri, 1982; Yoshimura, 1989). In man, the lesion associated with pulmonary dirofilariasis is large, and consists of eosinophils, neutrophils, histiocytes, giant cells and fibroblasts surrounding an immature worm, and is described in chest radiography as being a "coin lesion" (Harrison and Thompson, 1965). For a definitive diagnosis, however, a surgical biopsy is required. Immunological diagnostic methods for this disease have been reported, but they are unreliable, due to cross-reactivity with other organisms. To establish a reliable diagnostic technique, it is necessary to produce a suitable animal model for human pulmonary dirofilariasis. The dog, a definitive host,of. D. immitis, is not a suitable animal, because the parasite does not produce a coin lesion in the lung. In addition, neither mice nor rabbits are susceptible to the infective larvae. However, Nakagaki and co-workers (unpublished) found that transplanted immature worms migrate into the pulmonary arteries and right ventricle in the rabbit. In the present study immature worms were transplanted into rabbits, and the lungs were examined histopathologically to evaluate the 0021-9975/94/040403+ 04 $08.00/0
O 1994AcademicPressLimited
404
T. Sato
et
al.
use of pulmonary dirofilariasis in the rabbit as an experimental model for the human disease. Materials and M e t h o d s
Animals Eight female Japanese white rabbits (Nippon Bio-Supply Center, Tokyo), weighing between 2"8 and 3"0 kg and aged 14 weeks, were infected with two to eight immature D. immitis worms, obtained t?om dogs 140 days after experimental infection. The rabbits were infected by transplanting the worms surgically into the subcutaneous tissues of the posterior region. The animals were killed and necropsied 196 days later. Pathological Examination The rabbits were killed with an overdose of pentobarbital. The lungs were removed and fixed in phosphate-buffered saline containing fbrmaldehyde 3'5%. Thin sections, prepared for microscopical examination from paraffin wax-embedded specimens, were stained with hae~natoxylin and eosin and elastic van Gieson. Results
Macroscopical lesions in the lungs consisted of nodules. These were associated with varying degrees of congestion, haemorrhage, oedema and emphysema in the parenchyma. Out of the eight animals, five harboured fragmented worms in the nodules. Microscopical examination showed degenerating worms in the blood vessels, sometimes blocking the pulmonary arteries (Fig. 1). Coagulation necrosis had formed around the worms and macrophages, lymphocytes, plasma cells and eosinophils had infiltrated into the centre of the lesion (Fig. 2). These findings were characteristic of parasitic granulomas. The elastic fibres in the infarcted vascular wall had ruptured, and this change was accompanied by cellular infiltration (Fig. 3). Varying degrees of hyperaemia and haemorrhage were observed in the pulmonary parenchyma, and emphysema was apparent in the lung margin. Some rabbits showed severe arteritis and periarteritis, with marked thickening and oedema, dissociation of myofibres, calcium deposition, hyperaemia, and infiltration of plasma cells, lymphocytes and eosinophils (Fig.
4). Discussion
The heartworm, D. immitis, infects Canidae and is found in the right ventricle and pulmonary arteries. This parasite may accidentally infect man by the bite of a mosquito harbouring inl~ctive larvae. Parasitizing worms migrate into tile pulmonary arteries and block these vessels (Yoshimura, 1983). The lesion formed may be observed as a nodular mass by chest radiography, and pulmonary dirofilariasis can be confirmed histopathologically by biopsy (Ciferri, 1982; Yoshimura, 1983). Diagnosis by serological tests has been attempted but is unreliable. We have established a rabbit model for pulmonary dirofilariasis in man. White-gray nodules were formed in the lungs of rabbits infected with transplanted D. immitis. A few lesions were accompanied by haemorrhage, but
Rabbit Pulmonary Dirofira~iasis
Fig. Fig. Fig, Fig.
405
1. Blocking of pulmonary arteries by worms. HE. x 60. 2. Infiltration of macrophages, lymphocytes, plasma cells and eoslnophils. HE. x 120. 3. Rupture of elastic fibres in the wall o[' the arteries. Elastic van Gieson. x 120. 4. A section showing arterltls and periarteritis, Tylosis with hyaline degeneration of the endarterlum~ luminal arctation, and divaric-adon of leiomyofibrils of arterial walls. HE. x 60.
406
T. Sato et al.
this macroscopical feature was less severe than that described for h u m a n cases (Hayashi et al., 1985). In rabbits infected with the worms, the main microscopical manifestation was pulmonary infarction, as in h u m a n cases. In contrast to changes described in man (Navarrette-Reyna and Noon, 1968; Y a m a n e et al., 1977; Caravelli et al., 1981), rabbits showed greater infiltration by eosinophils but no Langhans-type giant cells. The presence of torn arterial dastic fibres in the infarct was as observed by Harrison and Thompson (1965). Navarrette-Reyna and Noon (1968) reported that infarction from pulmonary dirofilariasis could be distinguished from the more usual type of infarction. One rabbit showed severe obstructive arteritis and periarteritis in multiple areas of coagulation necrosis. This manifestation was consistent with that seen in human cases described by Hayashi et al. (1985); in brief, two out of five human cases showed severe obstructive arteritis and endarteritis pulmonale. These authors speculated that the lesions might be induced by excretory or secretory products from the worms, or by i m m u n e complexes. The rabbit model described should prove helpful in resolving the pathogenesis of obstructive arteritis.
Acknowledgment This study was supported in part by a Grant-in-Aid for scientific research from the Ministry of Education, Science and Culture, J a p a n (No. 01570227).
References Beaver, P. C. and Orihel, T. C. (1965). Human infection with filariae of animals in the United States. American Journal of Tropical Medicine and Hygiene, 14, 1010-1029. Caravelli, J. F., Zaman, M. B. and Bains, M. S. (1981). Human pulmonary dirofilariasis mimicking metastatic disease. Clinical Bulletin, 11, 88-90. Ciferri, F. (1982). Human pulmonary dirofilariasis in the United States: a critical review. American Journal of Tropical Medicine and Hygiene, 31, 302-308. Harrison, E. G. and Thompson, J. H. (1965). Dirofilariasis of human lung. American Journal of Clinical Pathology, 43, 224-234. Hayashi, K., Mizobuchi, K., Taguchi, K., Motoi, M., Tsutsumi, A., Mori, M., Tongu, K., Ishii, A., Tanaka, N., Nakai, H., Mondori, E., Morisaki, F., Ohta, T. and Nakanishi, Y. (1985). Five cases of human pulmonary dirofilariasis. Pathology and Clinical Medicine, 3, 925-931. Moorhouse, D. E., Abrahams, E. W. and Stephens, B.J. (1971). Human pulmonary dirofilariasis in Queensland. Medical Journal of Australia, 2, 1230-1233. Navarrette-Reyna, A. and Noon, G. (1968). Pulmonary dirofilariasis manifested as a coin lesion: report of a case and review of the literature. Archives of Pathology, 85, 266-271. Yamane, Y., Yazaki, S. and Fukumoto, S. (1977). A human case of pulmonary dirofilariasis. Yonago Acta Medica, 21, 1ll-118. Yoshimura, H. (1983). Larva migrans and clinico-parasitology of toxocariasis, dirofilariasis, angiostrongylosis, sparganosis, cysticercosis, and echinococcosis. Pathology and Clinical Medicine, 1, 1389-1406. Yoshimura, H. (1989). Control of filariasis and increase in Dirofilaria immitis infection in Japan. Saishin Igaku, 44, 815-826.
Received, March 8th, 1993 Accepted, December 21 st, 1993_J
SHORT PAPERS
Histopathology of the LUngs of Rabbits Experimentally Infected with Dirofilaria immitis T. Sato, S. N o g a m i * , K. Nakagaki~, I. Inoue*, W. Shlrai and K. Araki++ Departments of VeterinaryPathology and *Medical Zoology, School of VeterinaryMedicine, Nihon University, 1866 Kameino, Fujisawa, Kanagawa252, *iDepartmentof Veterinary Medicine, Nippon Jui Chikusan University,Musashino 180 and +National Institute of Public Health, Minato-ku 108, Japan Summary The lungs of rabbits experimentally infected with Dirofilaria inzmitis were examined histopathologically, to compare the changes with those seen in human pulmonary dirofilariasis. Eight rabbits were infected subcutaneously with two to eight immature worms to induce pulmonary dirofilariasis. Obstructive changes, similar to those reported in human pulmonary dirofilariasis, were observed in the blood vessels surrounding the worms. A form of arteritis, similar to occlusive arteritis, and periarteritis were also observed in the lungs of the rabbits. These results suggest that experimentaIly induced dirofilariasis in the rabbit is a useful animal model for human pulmonary dirofilariasis.
Introduction A reliable diagnostic technique is required to distinguish cases of human pulmonary dirofilariasis from lung cancer and tuberculosis in areas where Dirofilaria immitis is endemic, such as Japan, Australia and the United States (Beaver and Orihel, 1965; Navarrette-Reyna and Noon, 1968; Moorhouse et aI., 1971; Ciferri, 1982; Yoshimura, 1989). In man, the lesion associated with pulmonary dirofilariasis is large, and consists of eosinophils, neutrophils, histiocytes, giant cells and fibroblasts surrounding an immature worm, and is described in chest radiography as being a "coin lesion" (Harrison and Thompson, 1965). For a definitive diagnosis, however, a surgical biopsy is required. Immunological diagnostic methods for this disease have been reported, but they are unreliable, due to cross-reactivity with other organisms. To establish a reliable diagnostic technique, it is necessary to produce a suitable animal model for human pulmonary dirofilariasis. The dog, a definitive host,of. D. immitis, is not a suitable animal, because the parasite does not produce a coin lesion in the lung. In addition, neither mice nor rabbits are susceptible to the infective larvae. However, Nakagaki and co-workers (unpublished) found that transplanted immature worms migrate into the pulmonary arteries and right ventricle in the rabbit. In the present study immature worms were transplanted into rabbits, and the lungs were examined histopathologically to evaluate the 0021-9975/94/040403+ 04 $08.00/0
O 1994AcademicPressLimited
404
T. Sato
et
al.
use of pulmonary dirofilariasis in the rabbit as an experimental model for the human disease. Materials and M e t h o d s
Animals Eight female Japanese white rabbits (Nippon Bio-Supply Center, Tokyo), weighing between 2"8 and 3"0 kg and aged 14 weeks, were infected with two to eight immature D. immitis worms, obtained t?om dogs 140 days after experimental infection. The rabbits were infected by transplanting the worms surgically into the subcutaneous tissues of the posterior region. The animals were killed and necropsied 196 days later. Pathological Examination The rabbits were killed with an overdose of pentobarbital. The lungs were removed and fixed in phosphate-buffered saline containing fbrmaldehyde 3'5%. Thin sections, prepared for microscopical examination from paraffin wax-embedded specimens, were stained with hae~natoxylin and eosin and elastic van Gieson. Results
Macroscopical lesions in the lungs consisted of nodules. These were associated with varying degrees of congestion, haemorrhage, oedema and emphysema in the parenchyma. Out of the eight animals, five harboured fragmented worms in the nodules. Microscopical examination showed degenerating worms in the blood vessels, sometimes blocking the pulmonary arteries (Fig. 1). Coagulation necrosis had formed around the worms and macrophages, lymphocytes, plasma cells and eosinophils had infiltrated into the centre of the lesion (Fig. 2). These findings were characteristic of parasitic granulomas. The elastic fibres in the infarcted vascular wall had ruptured, and this change was accompanied by cellular infiltration (Fig. 3). Varying degrees of hyperaemia and haemorrhage were observed in the pulmonary parenchyma, and emphysema was apparent in the lung margin. Some rabbits showed severe arteritis and periarteritis, with marked thickening and oedema, dissociation of myofibres, calcium deposition, hyperaemia, and infiltration of plasma cells, lymphocytes and eosinophils (Fig.
4). Discussion
The heartworm, D. immitis, infects Canidae and is found in the right ventricle and pulmonary arteries. This parasite may accidentally infect man by the bite of a mosquito harbouring inl~ctive larvae. Parasitizing worms migrate into tile pulmonary arteries and block these vessels (Yoshimura, 1983). The lesion formed may be observed as a nodular mass by chest radiography, and pulmonary dirofilariasis can be confirmed histopathologically by biopsy (Ciferri, 1982; Yoshimura, 1983). Diagnosis by serological tests has been attempted but is unreliable. We have established a rabbit model for pulmonary dirofilariasis in man. White-gray nodules were formed in the lungs of rabbits infected with transplanted D. immitis. A few lesions were accompanied by haemorrhage, but
Rabbit Pulmonary Dirofira~iasis
Fig. Fig. Fig, Fig.
405
1. Blocking of pulmonary arteries by worms. HE. x 60. 2. Infiltration of macrophages, lymphocytes, plasma cells and eoslnophils. HE. x 120. 3. Rupture of elastic fibres in the wall o[' the arteries. Elastic van Gieson. x 120. 4. A section showing arterltls and periarteritis, Tylosis with hyaline degeneration of the endarterlum~ luminal arctation, and divaric-adon of leiomyofibrils of arterial walls. HE. x 60.
406
T. Sato et al.
this macroscopical feature was less severe than that described for h u m a n cases (Hayashi et al., 1985). In rabbits infected with the worms, the main microscopical manifestation was pulmonary infarction, as in h u m a n cases. In contrast to changes described in man (Navarrette-Reyna and Noon, 1968; Y a m a n e et al., 1977; Caravelli et al., 1981), rabbits showed greater infiltration by eosinophils but no Langhans-type giant cells. The presence of torn arterial dastic fibres in the infarct was as observed by Harrison and Thompson (1965). Navarrette-Reyna and Noon (1968) reported that infarction from pulmonary dirofilariasis could be distinguished from the more usual type of infarction. One rabbit showed severe obstructive arteritis and periarteritis in multiple areas of coagulation necrosis. This manifestation was consistent with that seen in human cases described by Hayashi et al. (1985); in brief, two out of five human cases showed severe obstructive arteritis and endarteritis pulmonale. These authors speculated that the lesions might be induced by excretory or secretory products from the worms, or by i m m u n e complexes. The rabbit model described should prove helpful in resolving the pathogenesis of obstructive arteritis.
Acknowledgment This study was supported in part by a Grant-in-Aid for scientific research from the Ministry of Education, Science and Culture, J a p a n (No. 01570227).
References Beaver, P. C. and Orihel, T. C. (1965). Human infection with filariae of animals in the United States. American Journal of Tropical Medicine and Hygiene, 14, 1010-1029. Caravelli, J. F., Zaman, M. B. and Bains, M. S. (1981). Human pulmonary dirofilariasis mimicking metastatic disease. Clinical Bulletin, 11, 88-90. Ciferri, F. (1982). Human pulmonary dirofilariasis in the United States: a critical review. American Journal of Tropical Medicine and Hygiene, 31, 302-308. Harrison, E. G. and Thompson, J. H. (1965). Dirofilariasis of human lung. American Journal of Clinical Pathology, 43, 224-234. Hayashi, K., Mizobuchi, K., Taguchi, K., Motoi, M., Tsutsumi, A., Mori, M., Tongu, K., Ishii, A., Tanaka, N., Nakai, H., Mondori, E., Morisaki, F., Ohta, T. and Nakanishi, Y. (1985). Five cases of human pulmonary dirofilariasis. Pathology and Clinical Medicine, 3, 925-931. Moorhouse, D. E., Abrahams, E. W. and Stephens, B.J. (1971). Human pulmonary dirofilariasis in Queensland. Medical Journal of Australia, 2, 1230-1233. Navarrette-Reyna, A. and Noon, G. (1968). Pulmonary dirofilariasis manifested as a coin lesion: report of a case and review of the literature. Archives of Pathology, 85, 266-271. Yamane, Y., Yazaki, S. and Fukumoto, S. (1977). A human case of pulmonary dirofilariasis. Yonago Acta Medica, 21, 1ll-118. Yoshimura, H. (1983). Larva migrans and clinico-parasitology of toxocariasis, dirofilariasis, angiostrongylosis, sparganosis, cysticercosis, and echinococcosis. Pathology and Clinical Medicine, 1, 1389-1406. Yoshimura, H. (1989). Control of filariasis and increase in Dirofilaria immitis infection in Japan. Saishin Igaku, 44, 815-826.
Received, March 8th, 1993 Accepted, December 21 st, 1993_J