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Structural and ultrastructural changes in the lungs of cats Felis catus (Linnaeus, 1758) experimentally infected with D. immitis (Leidy, 1856)
Veterinary Parasitology 176 (2011) 304–312
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Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Structural and ultrastructural changes in the lungs of cats Felis catus (Linnaeus, 1758) experimentally infected with D. immitis (Leidy, 1856) Frederico C.L. Maia a , John W. McCall b , Valdemiro A. Silva Jr a , Cristina A. Peixoto c , Prasit Supakorndej d , Nonglak Supakorndej d , Leucio C. Alves a,∗ a b c d
Departamento de Medicina Veterinária, Recife, PE, Brazil College of Veterinary Medicine, University of Georgia, Athens, GA, USA Centro de Pesquisa Aggeu Magalhaes, Recife, PE, Brazil TRS Labs Inc., Athens, GA, USA
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Keywords: Feline heartworm Light microscopy Transmission electron microscopy Pathology
a b s t r a c t Clinical signs are seldom observed in feline heartworm disease, and the pathophysiological changes in the lungs of infected animals remain undefined. The goal of this study was to evaluate the structural and ultrastructural changes in the lungs of cats experimentally infected with Dirofilaria immitis. Six healthy cats were each infected with two adult heartworms by intravenous transplantation (Receptor Group, RG). The control group consisted of two uninfected animals kept under the same conditions as the RG. At 42 days after transplantation, all cats were euthanized and necropsied for worm recovery and collection of lung samples for examination by light microscopy (LM) and transmission electron microscopy. By LM, lung sections from the six infected cats exhibited bronchial and bronchiolar lesions. Alterations in all tissues of the pulmonary arteries were observed in the infected animals. In conclusion, cats infected experimentally with D. immitis developed lesions in their lungs as a consequence of arterial disease and intense interstitial pneumonia. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Feline dirofilariasis, caused by the nematode parasite Dirofilaria immitis, has been diagnosed in many countries throughout the world and has been found in other animals, including humans. The infection in cats is occasionally discovered by radiographic examination, often with no clinical signs suggestive of a respiratory disease (McCall et al., 2008; Dillon, 2002). Recently, a pulmonary syndrome defined as heartworm-associated respiratory disease (HARD) has been associated with the presence of the parasite in cats (McCall et al., 2008). Despite the
∗ Corresponding author at: Leucio Camara Alves, Universidade Federal Rural de Pernambuco, Av. Dom Manoel de Medeiros s/n, Dois Irmãos, Recife/Pernambuco, CEP 52171-900, Brazil. Tel.: +55 81 96446060; fax: +55 81 33206404. E-mail address: [email protected] (L.C. Alves). 0304-4017/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2011.01.014
existence of extensive literature about clinical aspects of heartworm infection, treatment, and pathology of the disease in dogs, some aspects of feline heartworm remain obscure. The aim of this paper was to analyze and describe the structural and ultrastructural changes of lungs of cats experimentally infected with D. immitis. 2. Materials and methods Six healthy domestic cats (Felis catus), free of parasites, 8–10 months of age, that had been maintained at the College of Veterinary Medicine, University of Georgia were assigned to a group identified as the Receptor Group (RG). Two cats of similar age as those in the RG group were assigned to a control group (CG). All cats were housed in individual cages with controlled environmental temperature and 12-h light/dark cycles. The animals received cat food formulated to the nutritional needs of
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Fig. 1. Photomicrographs of cat lungs experimentally infected with Dirofilaria immitis. Intrapulmonary bronchus. Arrow designates partial obstruction of the lumen and star shows thickness of the pulmonary arterial wall next to bronchus (A); 15× magnification. Detail of bronquioli lumen within cellular debris is shown by the thin arrow, macrophages (arrow head) and deposit of fibrin above of respiratory epithelium (star) (B); 150× magnification. Intrapulmonary bronchiole. Partial obstruction of lumen (star) and hyperplasia of bronchiole gland (arrow) (C); 40× magnification. Transitional bronchus showing total obstruction of lumen (star) and bronchiole gland hyperplasia (large arrow) and Reisseissen muscle (D); 40× magnification.
cats of this age, and water was provided ad libitum. Cats in the RG were infected with D. immitis as described by Rawlings and McCall (1985). Before the transplantation, each animal was examined for heartworm disease using the modified method of Knott (1939), and for circulating D. immitis antigen by serology. Clinical examination was performed periodically for all animals during the study period. Euthanasia and necropsy were performed 42 days after transplantation of worms for worm recovery and collection of lung samples. Tissue samples were fixed in formalin and embedded in paraffin for examination by light microscopy (LM) and transmission electron microscopy (TEM). For the structural study, the sections of specimens were routinely stained with hematoxylin-eosin, Gomori methenamine silver stain (Behmer et al., 1976), and Gomori trichrome stain (McElroy, 1992). For the ultrastructural study by TEM, tissues were prepared in a 2.5% paraformaldehyde and 2.5% glutaraldehyde solution, embedded in EPONTM resin, and routinely contrasted by 5% uranila acetate solution and 1% lead citrate.
3. Results The histopathological analysis revealed the partial obstruction of some primary bronchi, with the nearly total obstruction of the lumen in some. There was a large amount of mucus containing sloughed epithelial cells, cell debris, a small number of eosinophils and neutrophils, and a moderate amount of macrophages (Fig. 1A and B). The goblet cells were hyperactive and demonstrated signs of hyperplasia and hypertrophy of the Reisseissen muscles (Fig. 1C and D). In the pulmonary arteries, regardless of the caliber of the vessel, there were alterations in the tunica intima, tunica media, and adventitia in all the experimentally infected animals. In the tunica intima of pulmonary arteries of a larger caliber, the initial lesions were characterized by hyperplastic thickening of the subendothelial connective tissue associated to discreet inflammatory infiltration consisting mainly of eosinophils and macrophages (Fig. 2A–D). The evolution of this process was characterized by a considerable increase in subendothelial conjunctive tissue and
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Fig. 2. Photomicrographs of pulmonary arteries of cat lungs experimentally infected with Dirofilaria immitis. Pulmonary artery (designated Ap ), showing thickness of tunica intima (arrows) (A); 15× magnification. Detail of pulmonary artery wall showing subendothelial connective tissue thickness (arrows) (B); 40× magnification. Pulmonary artery with thickness of tunica intima (Ti). Note fingerlike aspect of tunica intima (arrows) (C); 40× magnification. Detail of pulmonary artery wall with subendothelial inflammatory infiltrated in the tunica intima (Ti), hyperplasia and hypertrophy of tunica medium (star). Note adventitia (arrows) with inflammatory infiltratration (D); 40× magnification.
significant amount of inflammatory cells projecting the tunica intima toward the lumen of the vessels. This thickening determined digitate arrangements of the tunica intima in these arteries, thereby reducing the lumen of the vessels (Fig. 3A) and subsequently causing the complete obstruction of the vessels (Fig. 3B). Hypertrophy and hyperplasia were observed in the interior of some arteries, varying from moderate to intensive and followed by partial disorganization and cytoplasma vacuolization in smooth muscle cells (Fig. 3C). Some areas of the parenchyma exhibited alveolar dilation and rupture characteristic of emphysema (Fig. 4A). In the pulmonary interstice, there was considerable, diffuse infiltration of macrophages and eosinophils as well as macrophages, plasmocytes and eosinophils, and hyperplasia of type II pneumocytes, which caused the thickening of the alveolar walls and compression of the alveoli (Fig. 4B). The alveolar walls exhibited a large area of thickening and the proliferation of smooth muscle cells in the pulmonary parenchyma (Fig. 4C). Gomori’s trichrome stain revealed the presence of diffuse smooth muscle fibers and colla-
gen fibers, characterizing extensive fibrosis, hyperplasia, and hypertrophy of smooth muscle fibers in the pulmonary parenchyma (Fig. 4D and E). In some animals, the intensive chronic inflammatory process determined considerable loss of useful area of the pulmonary parenchyma, with an attempt at vascular neoformation. These alterations reflect a dramatic attempt at reconstituting the capacity for nutrition and oxygenation in the infected animals. Despite the intensive lesions in the arteries and pulmonary parenchyma throughout the experiment, there were no clinical signs of dirofilariasis in any of the animals in the RG. The ultrastructural analysis allowed a better visualization of the alterations and cell interactions as well as changes in the conjunctive stroma, which occurred in the pulmonary parenchyma of the infected cats as either a direct or indirect consequence of the presence of parasites in the arteries of the lungs. The main cell alteration was hyperplasia of type II pneumocytes, which produce surfactant substances. In the pulmonary parenchyma, cell “niche” were observed, which were characterized by the
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Fig. 3. Photomicrographs of lesions of pulmonary arteries cat lungs experimentally infected with Dirofilaria immitis. Detail of pulmonary artery wall, showing thickness and vacuolization of tunica medium (arrows). Tunica adventitia with inflammation infiltrated around of vasa vasorum (arrow head) (A) and total obstruction of pulmonary arteries lumen (stars) (B); 15× magnification. Pulmonary artery of small caliper (star), showing narrowing of lumen (thin arrow) and hyperplasia of tunica medium (arrows) (C); 150× magnification.
presence of type II pneumocytes in various stages of the maturation process of cyto-lysosomes, lymphocytes and bundles of collagen fiber in the extracellular matrix. Moreover, these cells exhibited an increase in cytoplasm volume as a consequence of the large amount of surfactant granules (cytolysosomes), also characterizing cell hypertrophy (Fig. 5A and B). Cell death of type II pneumocytes by apoptosis and necrosis was also observed (Fig. 5C and D). Next to an apoptotic type II pneumocyte, eosinophils with typical arrangements were observed, along with a normal type II pneumocyte in a state of degranulation. In the interstice of the pulmonary parenchyma, a large amount of eosinophils were found interacting with lymphocytes, fibroblasts, mastocytes, smooth muscle cells, and macrophages (Fig. 6A–D). Smooth muscle cells, fibroblasts, macrophages and their physical proximity to eosinophils were observed in the pulmonary parenchyma of infected animals (Fig. 7A–C). 4. Discussion Despite the histological findings reported in the literature on cats with dirofilariasis (heartworm), there is
little information on lesions in the bronchi and bronchioles in cats infected with D. immitis. Abbott (1966) reports thickening of the bronchiole wall and bronchiolitis, with hyperplasia of the epithelium and severe epithelial sloughing associated to the presence of histiocytes, eosinophils, and blood cells in the lumen of bronchioles in cats with heartworm. The pathological findings in the bronchi and bronchioles did not have a specific characteristic but stemmed from chronic interstitial pneumonia caused by other agents. These findings are compatible with those described by Labarthe (1997), who reports that the inflammatory process in the vessels of animals infected with D. immitis causes the degeneration of the endothelium, a reduction in the vascular lumen and even obstruction of the vessels. The author states that such lesions do not stem from the physical presence of the parasite alone. According to Calvert and Rawlings (2002), the hypertrophy of the tunica intima, with the formation of villosities, is due to the invasion of the intima by smooth muscle cells and collagen from muscle tunica media, stimulated by growth factors released by platelets and leukocytes. Complete obstruction of arteries by the formation of
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Fig. 4. Photomicrographs of pulmonary parenchyma of lung cats experimentally infected with Dirofilaria immitis. Pulmonary parenchyma within alveoli with rupture (arrow) and alveolar dilatation (star) (A); 40× magnification. Pulmonary parenchyma showing diffuse infiltration of macrophages (thin arrow) and eosinophils (large arrow) (B); 400× magnification. Diffuse presence of smooth muscle cell (arrow) (C). 400× magnification. Pulmonary parenchyma stained with Gomori’s trichromic, showing collagen fibers (arrow) stained in green (D); 150× magnification. Pulmonary parenchyma stained with Gomori’s trichromic. There are many smooth muscle cells (star) stained in dark blue (E); 40× magnification.
thromboses and granulomas in the tunica intima stemming from the death of parasites are frequent findings in canine and feline dirofilariasis (Dillon, 2002). The vacuolization of the muscle cells of the tunica media in the animals studied may be related to hydropic degenera-
tion as a consequence of alterations in the cell membrane due to a lack of nutrients and oxygenation stemming from lesions in the endothelium and the hypertrophy of this layer. Hamilton (1966) describes the vacuolization of muscle cells of hypertrophied, hyperplastic pulmonary
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Fig. 5. Photomicrographs of pulmonary parenchyma of lung cats experimentally infected with Dirofilaria immitis. Type II pneumocyte hypertrophy. Note several cytolisosomes inside the cytoplasm (arrow head) and degranulation of surfactant on cellular surface (arrow) (A); 7000× magnification. Pneumocytes II (P) with cytoplasm within several cytolisosomos (arrow) in association with lymphocytes (L). Interstitial macrophages (star), surrounded by collagens fibers (arrow head) (B); 3000× magnification. Eosinophils adjacent to type II pneumocytes in apoptosis (star). Arrow points to apoptotic vesicle (C); 3000× magnification. Pneumocytes II in necrosis. Nuclear chromatin condensation (star), stripping of nuclear membrane (thin arrow) and mitochondria without tubular crest (arrow head) (D). 7000× magnification.
arteries in cats with different lung diseases. According to the author, this vacuolization did not respond to the different staining methods used for the identification of neutral fats, cholesterol, mucopolysaccharides and glycogen. The vascular lesions reported in the present study resemble those described by Hamilton (1966), who found similar lesions in cats infected with A. abstrusus or with other lung diseases. The findings are also compatible with those described by a number of authors (Dillon, 1984; McCracken and Patton, 1993; Mupanomunda et al., 1997), according to whom, arterial lesions stem from the local anaphylactic reaction and inflammatory infiltration by mononuclear cells in the sub-endothelial tissue of the arteries. Dillon (1984) reports the similarity in lesions found in cats and dogs with dirofilariasis; within a week following the transplant of adult helminthes in cats, parasitic endarteritis, villous proliferation of the intima and severe vascular inflammatory response were evident. Labarthe (1997) found partial obstruction of the pulmonary arteries and arterioles stemming from hyperplasia and hypertro-
phy of the tunica muscularis and villous projections of the endothelium, suggesting that these lesions stem from the migration of the parasites to the pulmonary arteries. In cats experimentally infected with D. immitis, Holmes et al. (1992) describe moderate to severe eosinophilic endarteritis in pulmonary arteries of all sizes, followed by severe muscle hypertrophy of arteries of medium size and small calibers in all the lobes of the lungs, along with complete obstruction of some arterioles and the presence of thromboses. The morphological characteristics, intensity and type of inflammatory reaction in the arterial wall and pulmonary parenchyma suggest the lesions described in the present study likely occurred as a consequence of the mechanical action of the parasites on the endothelium of the arteries as well as the release of antigen factors by the helminths. Filoni et al. (2009) report fibromuscular proliferation, diffuse inter-alveolar fibrosis, perivascular eosinophilic inflammation, hemosiderosis, and the presence of microfilaria in small arteries. These factors may be responsible for triggering the type II hypersensitivity reac-
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Fig. 6. Active eosinophil (E) in interaction with another cell of pulmonary parenchyma (arrow) (A); 12000× magnification. Interstitial space of pulmonary parenchyma. Note interaction between plasma cell (P), mast cell (M), eosinophil (E), next to fibroblast (F) surrounded by collagen fibers (star) (B); 4400× magnification. Active eosinophil (E) in strict relationship with pneumocytes I (P) (C); 12000× magnification. Interstitial space of pulmonary parenchyma. Note smooth muscle cell (star) in relationship com eosinophils (arrow) (D); 3000× magnification.
tion (cytotoxic reaction), which is mediated by IgG, IgE, and eosinophils (Siqueira and Dantas, 2000). The type of cellular inflammatory reaction found in the present study allows the supposition that this reaction was a consequence of the presence of antigens released by the helminthes. Regarding the presence of alveolar emphysema, this may be interpreted as a consequence of the loss of alveolar expansion capacity in areas adjacent to the pneumonia and the intense pulmonary hypertension observed in the animals. Similar lesions to those found in the present study were reported by Dillon (1984, 2002), who referred to this lesion only as hyperplasia of type II pneumocytes in cats with dirofilariasis. Atwell et al. (1988) also report smooth muscle atrophy in alveolar ducts. The presence of smooth muscle fibers in the alveolar walls, especially in the intensive, disseminated form similar to those that occurred in the cats analyzed in the present study, should be considered hyperplasia followed by hypertrophy due to the intense pulmonary hypertension in the lungs. The main cell alteration in the ultrastructural analysis was hyperplasia of type II pneumocytes, which produce surfactant substances. Hyperplasia and hypertrophy of
pneumocytes are related to injury to the pulmonary parenchyma (Fenrenbach et al., 1999; Morikawa et al., 2000). According to Tomashefski (2000), hyperplastic lesions of type II pneumocytes frequently occur in acute respiratory distress syndrome, particularly 4–9 months following infection. Cell death of type II pneumocytes by apoptosis or necrosis may be attributed to complex mechanisms that involve the inflammatory response to D. immitis antigens, which are always accompanied by a variable quantity of type II pneumocytes and eosinophils. According to Gleich and Adolphson (1986) and Spry (1988), mature eosinophils can be stimulated by a large number of signals derived from various sources, particularly interleukin-5 (IL-5) (Rothenberg et al., 1989) and IL-4 (Coffman et al., 1988), to become active and migrate to specific sites, such as areas of acute or chronic inflammation. IL-5 induces chemotaxis and degranulation in eonsinophils in vivo and in vitro (Wong et al., 1990; Kita et al., 1992; Lampinen et al., 2004), and the proteins of eosinophils of granules are toxic to microfilariae in vivo, causing the death of the larvae (Hamann et al., 1991). The specific dependence of IL-5 on granulomatous eosinophilia has been corrobo-
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Fig. 7. Photomicrograph of pulmonary parenchyma showing cell association between eosinophil (E), macrophage (M) and pneumocyte II (P) in initial stage of necrosis (A); 4400× magnification. Eosinophil (E) next to capillary (C) edema peri-vascular (arrow head) and several collagen fibers (arrow) (B); 12000× magnification. Fibroblast (arrow) surrounded by collagen fibers bundles (F), next to eosinophils (E) (C); 4400× magnification.
rated by the complete ablation of eosinophilic response in mice infected with Schistosoma mansoni following treatment with specific antibodies for IL-5 (Sher et al., 1990; Rennick et al., 1990). This cytokine promotes chemotaxis selective for eosinophils and induces degranulation in vitro and in vivo (Wong et al., 1989; Kita et al., 1992; Terada et al., 1992). The increase in IL-5 expression significantly increases the number of eosinophils and antibody levels in vivo. On the other hand, rats with the absence of a functional gene for IL-5 or the alpha chain of the receptor for IL-5 (IL-5R) exhibit a series of alterations in the development and functioning of B cells in the eosinophil lineages (Kouro and Takatsu, 2009). Raible et al. (1992) and Shin et al. (2009) stress the importance of mastocytes and tissue inflammation mediated by eosinophils, respectively, in infection with helminthes. The interaction between these cells and stimulating factors may be responsible for the interstitial pneumonia found in the pulmonary parenchyma of the cats infected with D. immitis. According to Hamann et al. (1991), all proteins from the granules of eosinophils are toxic to microfilariae, causing their death in vivo. The findings of the authors confirm the existence of cell interaction between eosinophils, lymphocytes, and fibroblasts in the
production of interstitial pneumonia in response to the presence or not of D. immitis parasites in the pulmonary parenchyma. Among the inflammatory cells found in the pulmonary interstice, the presence of mastocytes can be explained by the fact that the factors produced by these cells, such as leukotriene B4, histamine, eosinophil chemotactic factor A and prostaglandin D2, are eosinophilic and are capable of activating eosinophils and increasing their helminthotoxic activity (Raible et al., 1992). The interaction of macrophages and eosinophils in the pulmonary parenchyma and alveolar lumen may be explained by the relation of dependence on factors derived from monocyte lymphocytes, such as tumor necrosis factor alpha, eosinophil-activating factor, and eosinophil cytotoxity-enhancing factor, which, together, influence the function of mature eosinophils (Thorne and Mazza, 1991). Horowitz and Thannickal (2006) describe phenotype alterations in type II alveolar epithelial cells, such as proliferation, bronchiolization, apoptosis, and regenerative hyperplasia related to idiopathic pulmonary fibrosis. It is therefore possible that type II pneumocytes and other inflammatory stimuli are implied in the development of fibrosis in cats experimentally infected with D. immitis.
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5. Conclusions The results of the present study demonstrate that the alterations induced by experimental dirofilariasis experimental in felines 42 days following infection were related to arterial lesions, interstitial pneumonia, hypertrophy, and hyperplasia of type II pneumocytes, the proliferation of smooth muscle cells, fibrosis, and emphysema. The ultrastructural analysis revealed that interstitial pneumonia was characterized by the presence of eosinophils, macrophages, plasma cells, mastocytes, and fibroblasts. The interaction between these cells involves complex defense mechanisms in an active response on the part of the host to antigens derived from the helminthes in blood vessels and the pulmonary parenchyma. Funding/Conflict of interest The cats used for this study were provided by Dr. J. McCall and had been used a project funded by TRS Labs. No other funding was provided. The authors report no financial or personal interests or relationships or other potential conflicts in this research. References Abbott, P.K., 1966. Feline dirofilariasis in Papua. Aust. Vet. J. 42, 247–249. Atwell, R.B., Sutton, R.H., Moodie, E.W., 1988. Pulmonary changes associated with dead filariae (Dirofilaria immitis) and concurrent antigenic exposure in dogs. J. Med. Chem. 31, 774–779. Behmer, O.A., Tolosa, E.M.C., Neto, A.G.F., 1976. Manual de técnicas para histologia normal e patológica. Edart, São Paulo, 257 pp. Calvert, C.A., Rawlings, C.A., 2002. Dirofilariose canina. In: Tilley, L.P., Goodwin, J. (Eds.), Manual de cardiologia para cães e gatos. , 3rd ed. Roca, São Paulo, 489 pp. Coffman, R.L., Seymour, B.W.P., Lebman, D.A., Hiraki, D.D., Christiansen, J.A., Shrader, B., Cherwinski, H.M., Savelkoul, H.F.B., Finkelman, F.D., Bond, M.A., Mosmann, T.R., 1988. The role of helper T-cell products in mouse B-cell differentiation and isotype regulation. Immunol. Rev. 102, 5–28. Dillon, R., 1984. Feline dirofilariasis. Vet. Clin. N. Am. Small Anim. Pract. 14, 1185–1199. Dillon, R., 2002. Dirofilariose felina. In: Tilley, L.P., Goodwin, J.K. (Eds.), Manual de cardiologia para cães e gatos. , 3rd ed. Roca, São Paulo, 489 pp. Fenrenbach, H., Kasper, M., Tschernig, T., Pan, T., Schuh, D., Shannon, J.M., Muller, M., Mason, R.J., 1999. Keratinocytes growth factor-induced hyperplasia of rat alveolar type II cell in vivo is resolved by differentiation into type I cells and by apoptosis. Eur. Resp. J. 14, 534–544. Filoni, C., Pena, H.F.J., Gennari, S.M., Cristo, D.S., Torres, L.N., Catão-Dias, J.L., 2009. Heartworm (Dirofilaria immitis) disease in a Brazilian oncilla (Leopardus tigrinus). Pesq. Vet. Bras. 29, 474–478. Gleich, G.J., Adolphson, C.R., 1986. The eosinophilic leukocyte: structure and function. Adv. Immunol. 39, 177–253. Hamann, K.J., Barker, R.L., Ten, R.M., Gleich, G.J., 1991. The molecular biology of eosinophil granule proteins. Int. Arch. Allergy Appl. Immunol. 94, 202–209. Hamilton, J.M., 1966. Pulmonary arterial disease of the cat. J. Comp. Pathol. 76, 133–245. Holmes, R.A., Clarck, J.N., Casey, H.W., Henk, W., Plue, R.E., 1992. Histopathologic and radiographic study of the development of heartworm pulmonary vascular disease in experimentally infected cats. In:
Soll, M.D. (Ed.), Proceedings of the Heartworm Symposium’92. American Heartworm Society, Batavia, IL, pp. 81–89. Horowitz, J.C., Thannickal, V.J., 2006. Idiopathic pulmonary fibrosis: new concepts in pathogenesis and implications for drug therapy. Treat. Respir. Med. 5, 325–342. Kita, H., Weiler, D.A., Abu-Ghazaleh, R., Sanderson, C.J., Gleich, G.J., 1992. Release of granule proteins from eosinophils cultured with IL-5. J. Immunol. 149, 629–635. Knott, J., 1939. A method for making microfilarial surveys on dog blood. Trans. R. Soc. Trop. Med. Hyg. 33, 191–196. Kouro, T., Takatsu, K., 2009. IL-5- and eosinophil-mediated inflammation: from discovery to therapy. Int. Immunol. 21, 1303–1309. Labarthe, N.V., 1997. Dirofilariose canina: diagnóstico, prevenc¸ão e tratamento adulticida. Clin. Vet. 10, 10–16. Lampinen, M., Carlson, M., Hakansson, L.D., Venge, P., 2004. Cytokineregulated accumulation of eosinophils in inflammatory disease. Allergy 59, 793–805. McCall, J.W., Genchi, C., Kramer, L.H., Guerrero, J., Venco, L., 2008. Heartworm disease in animals and humans. Adv. Parasitol. 66, 193–285. McCracken, M.D., Patton, S., 1993. Pulmonary arterial changes in feline dirofilariasis. Vet. Pathol. 30, 64–69. McElroy, D.A., 1992. Connective tissue. In: Prophet, E.B., Mills, B., Arrington, J.B., Sobin, L.H. (Eds.), AFIB Laboratory Methods in Histotechnology, vol. 18. American Registry of Pathology, Washington, DC, pp. 149–174. Morikawa, Y., Katsumoto, Y., Okada, T., Sasaki, F., 2000. Pulmonary hypoplasia induced by liquid paraffin injection into fetal thoracic cavity with special reference to renal development in rats. J. Vet. Med. Sci. 62, 135–140. Mupanomunda, M., Williams, J.F., Mackenzie, C.D., Kaiser, L., 1997. Dirofilaria immitis: heartworm infection alters pulmonary artery endothelial cell behavior. J. Appl. Physiol. 82, 389–398. Raible, D.G., Schulman, E.S., DiMuzio, J., Cardilo, R., Post, T.J., 1992. Mast cell mediators, prostaglandin D2 and histamine activate humaneosinophils. J. Immunol. 148, 3536–3542. Rawlings, C.A., McCall, J.W., 1985. Surgical transplantation of adult Dirofilaria immitis to study heartworm infection and disease in dogs. Am. J. Vet. Res. 46, 221–224. Rennick, D.M., Thompson-Snipes, L., Coffman, R.L., Seymour, B.W., Jackson, J.D., Hudak, S., 1990. In vivo administration of antibody to interleukin5 inhibits increased generation of eosinophils and their progenitors in bone marrow of parasitized mice. Blood 76, 2311–2316. Rothenberg, M.E., Petersen, J., Stevens, R.L., Silberstein, D.S., McKenzie, D.T., Austen, K.F., Owen, W.F., 1989. IL-5-dependent conversion of normo-dense human eosinophils to the hipodense phenotype uses 3T3 fibroblasts for enhanced viability, accelerated hypodensity and sustained antibody-dependent cytotoxicity. J. Immunol. 143, 2311–2316. Sher, A., Coffman, R.L., Hieny, S., Scott, P., Cheever, A.W., 1990. Interleukin5 is required for the blood and tissue eosinophilia but not granuloma formation induced by infection with Schistosoma mansoni. Proc. Natl. Acad. Sci. 87, 61–65. Shin, M.H., Lee, Y.A., Min, D.Y., 2009. Eosinophil-mediated tissue inflammatory responses in helminth infection. J. Parasitol. 47, 125–131. Siqueira, J.F., Dantas, C.J.C. (Eds.), 2000. Mecanismos celulares e moleculares da inflamac¸ão. Medsi, Rio de Janeiro, 238 pp. Spry, C.J.F., 1988. Eosinophils: A Comprehensive Review and Guide to the Scientific and Medical Literature. Oxford University Press, Oxford, pp. 10–28. Terada, N., Konno, A., Shirotori, K., Togawa, K., 1992. In human nasal mucosa, interleukin-5 accumulates and degranulates eosinophils, as well as increases responsiveness to histamine. Rhinol. Suppl. 14, 57–61. Thorne, K.J.I., Mazza, G., 1991. Eosinophilia, activated eosinophils and human schistosomiasis. J. Cell. Sci. 98, 265–270. Tomashefski, J.F., 2000. Pulmonary pathology of acute respiratory distress syndrome. Clin. Chest Med. 21, 435–466. Wong, D.T., Weller, P.F., Galli, S.J., Elovic, A., Rand, T.H., Gallagher, G.T., Chiang, T., Chou, M.Y., Matossian, K., McBride, J., Todd, R., 1990. Human eosinophils express transforming growth factor alpha. J. Exp. Med. 172, 673–681.
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Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Structural and ultrastructural changes in the lungs of cats Felis catus (Linnaeus, 1758) experimentally infected with D. immitis (Leidy, 1856) Frederico C.L. Maia a , John W. McCall b , Valdemiro A. Silva Jr a , Cristina A. Peixoto c , Prasit Supakorndej d , Nonglak Supakorndej d , Leucio C. Alves a,∗ a b c d
Departamento de Medicina Veterinária, Recife, PE, Brazil College of Veterinary Medicine, University of Georgia, Athens, GA, USA Centro de Pesquisa Aggeu Magalhaes, Recife, PE, Brazil TRS Labs Inc., Athens, GA, USA
a r t i c l e
i n f o
Keywords: Feline heartworm Light microscopy Transmission electron microscopy Pathology
a b s t r a c t Clinical signs are seldom observed in feline heartworm disease, and the pathophysiological changes in the lungs of infected animals remain undefined. The goal of this study was to evaluate the structural and ultrastructural changes in the lungs of cats experimentally infected with Dirofilaria immitis. Six healthy cats were each infected with two adult heartworms by intravenous transplantation (Receptor Group, RG). The control group consisted of two uninfected animals kept under the same conditions as the RG. At 42 days after transplantation, all cats were euthanized and necropsied for worm recovery and collection of lung samples for examination by light microscopy (LM) and transmission electron microscopy. By LM, lung sections from the six infected cats exhibited bronchial and bronchiolar lesions. Alterations in all tissues of the pulmonary arteries were observed in the infected animals. In conclusion, cats infected experimentally with D. immitis developed lesions in their lungs as a consequence of arterial disease and intense interstitial pneumonia. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Feline dirofilariasis, caused by the nematode parasite Dirofilaria immitis, has been diagnosed in many countries throughout the world and has been found in other animals, including humans. The infection in cats is occasionally discovered by radiographic examination, often with no clinical signs suggestive of a respiratory disease (McCall et al., 2008; Dillon, 2002). Recently, a pulmonary syndrome defined as heartworm-associated respiratory disease (HARD) has been associated with the presence of the parasite in cats (McCall et al., 2008). Despite the
∗ Corresponding author at: Leucio Camara Alves, Universidade Federal Rural de Pernambuco, Av. Dom Manoel de Medeiros s/n, Dois Irmãos, Recife/Pernambuco, CEP 52171-900, Brazil. Tel.: +55 81 96446060; fax: +55 81 33206404. E-mail address: [email protected] (L.C. Alves). 0304-4017/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2011.01.014
existence of extensive literature about clinical aspects of heartworm infection, treatment, and pathology of the disease in dogs, some aspects of feline heartworm remain obscure. The aim of this paper was to analyze and describe the structural and ultrastructural changes of lungs of cats experimentally infected with D. immitis. 2. Materials and methods Six healthy domestic cats (Felis catus), free of parasites, 8–10 months of age, that had been maintained at the College of Veterinary Medicine, University of Georgia were assigned to a group identified as the Receptor Group (RG). Two cats of similar age as those in the RG group were assigned to a control group (CG). All cats were housed in individual cages with controlled environmental temperature and 12-h light/dark cycles. The animals received cat food formulated to the nutritional needs of
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Fig. 1. Photomicrographs of cat lungs experimentally infected with Dirofilaria immitis. Intrapulmonary bronchus. Arrow designates partial obstruction of the lumen and star shows thickness of the pulmonary arterial wall next to bronchus (A); 15× magnification. Detail of bronquioli lumen within cellular debris is shown by the thin arrow, macrophages (arrow head) and deposit of fibrin above of respiratory epithelium (star) (B); 150× magnification. Intrapulmonary bronchiole. Partial obstruction of lumen (star) and hyperplasia of bronchiole gland (arrow) (C); 40× magnification. Transitional bronchus showing total obstruction of lumen (star) and bronchiole gland hyperplasia (large arrow) and Reisseissen muscle (D); 40× magnification.
cats of this age, and water was provided ad libitum. Cats in the RG were infected with D. immitis as described by Rawlings and McCall (1985). Before the transplantation, each animal was examined for heartworm disease using the modified method of Knott (1939), and for circulating D. immitis antigen by serology. Clinical examination was performed periodically for all animals during the study period. Euthanasia and necropsy were performed 42 days after transplantation of worms for worm recovery and collection of lung samples. Tissue samples were fixed in formalin and embedded in paraffin for examination by light microscopy (LM) and transmission electron microscopy (TEM). For the structural study, the sections of specimens were routinely stained with hematoxylin-eosin, Gomori methenamine silver stain (Behmer et al., 1976), and Gomori trichrome stain (McElroy, 1992). For the ultrastructural study by TEM, tissues were prepared in a 2.5% paraformaldehyde and 2.5% glutaraldehyde solution, embedded in EPONTM resin, and routinely contrasted by 5% uranila acetate solution and 1% lead citrate.
3. Results The histopathological analysis revealed the partial obstruction of some primary bronchi, with the nearly total obstruction of the lumen in some. There was a large amount of mucus containing sloughed epithelial cells, cell debris, a small number of eosinophils and neutrophils, and a moderate amount of macrophages (Fig. 1A and B). The goblet cells were hyperactive and demonstrated signs of hyperplasia and hypertrophy of the Reisseissen muscles (Fig. 1C and D). In the pulmonary arteries, regardless of the caliber of the vessel, there were alterations in the tunica intima, tunica media, and adventitia in all the experimentally infected animals. In the tunica intima of pulmonary arteries of a larger caliber, the initial lesions were characterized by hyperplastic thickening of the subendothelial connective tissue associated to discreet inflammatory infiltration consisting mainly of eosinophils and macrophages (Fig. 2A–D). The evolution of this process was characterized by a considerable increase in subendothelial conjunctive tissue and
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Fig. 2. Photomicrographs of pulmonary arteries of cat lungs experimentally infected with Dirofilaria immitis. Pulmonary artery (designated Ap ), showing thickness of tunica intima (arrows) (A); 15× magnification. Detail of pulmonary artery wall showing subendothelial connective tissue thickness (arrows) (B); 40× magnification. Pulmonary artery with thickness of tunica intima (Ti). Note fingerlike aspect of tunica intima (arrows) (C); 40× magnification. Detail of pulmonary artery wall with subendothelial inflammatory infiltrated in the tunica intima (Ti), hyperplasia and hypertrophy of tunica medium (star). Note adventitia (arrows) with inflammatory infiltratration (D); 40× magnification.
significant amount of inflammatory cells projecting the tunica intima toward the lumen of the vessels. This thickening determined digitate arrangements of the tunica intima in these arteries, thereby reducing the lumen of the vessels (Fig. 3A) and subsequently causing the complete obstruction of the vessels (Fig. 3B). Hypertrophy and hyperplasia were observed in the interior of some arteries, varying from moderate to intensive and followed by partial disorganization and cytoplasma vacuolization in smooth muscle cells (Fig. 3C). Some areas of the parenchyma exhibited alveolar dilation and rupture characteristic of emphysema (Fig. 4A). In the pulmonary interstice, there was considerable, diffuse infiltration of macrophages and eosinophils as well as macrophages, plasmocytes and eosinophils, and hyperplasia of type II pneumocytes, which caused the thickening of the alveolar walls and compression of the alveoli (Fig. 4B). The alveolar walls exhibited a large area of thickening and the proliferation of smooth muscle cells in the pulmonary parenchyma (Fig. 4C). Gomori’s trichrome stain revealed the presence of diffuse smooth muscle fibers and colla-
gen fibers, characterizing extensive fibrosis, hyperplasia, and hypertrophy of smooth muscle fibers in the pulmonary parenchyma (Fig. 4D and E). In some animals, the intensive chronic inflammatory process determined considerable loss of useful area of the pulmonary parenchyma, with an attempt at vascular neoformation. These alterations reflect a dramatic attempt at reconstituting the capacity for nutrition and oxygenation in the infected animals. Despite the intensive lesions in the arteries and pulmonary parenchyma throughout the experiment, there were no clinical signs of dirofilariasis in any of the animals in the RG. The ultrastructural analysis allowed a better visualization of the alterations and cell interactions as well as changes in the conjunctive stroma, which occurred in the pulmonary parenchyma of the infected cats as either a direct or indirect consequence of the presence of parasites in the arteries of the lungs. The main cell alteration was hyperplasia of type II pneumocytes, which produce surfactant substances. In the pulmonary parenchyma, cell “niche” were observed, which were characterized by the
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Fig. 3. Photomicrographs of lesions of pulmonary arteries cat lungs experimentally infected with Dirofilaria immitis. Detail of pulmonary artery wall, showing thickness and vacuolization of tunica medium (arrows). Tunica adventitia with inflammation infiltrated around of vasa vasorum (arrow head) (A) and total obstruction of pulmonary arteries lumen (stars) (B); 15× magnification. Pulmonary artery of small caliper (star), showing narrowing of lumen (thin arrow) and hyperplasia of tunica medium (arrows) (C); 150× magnification.
presence of type II pneumocytes in various stages of the maturation process of cyto-lysosomes, lymphocytes and bundles of collagen fiber in the extracellular matrix. Moreover, these cells exhibited an increase in cytoplasm volume as a consequence of the large amount of surfactant granules (cytolysosomes), also characterizing cell hypertrophy (Fig. 5A and B). Cell death of type II pneumocytes by apoptosis and necrosis was also observed (Fig. 5C and D). Next to an apoptotic type II pneumocyte, eosinophils with typical arrangements were observed, along with a normal type II pneumocyte in a state of degranulation. In the interstice of the pulmonary parenchyma, a large amount of eosinophils were found interacting with lymphocytes, fibroblasts, mastocytes, smooth muscle cells, and macrophages (Fig. 6A–D). Smooth muscle cells, fibroblasts, macrophages and their physical proximity to eosinophils were observed in the pulmonary parenchyma of infected animals (Fig. 7A–C). 4. Discussion Despite the histological findings reported in the literature on cats with dirofilariasis (heartworm), there is
little information on lesions in the bronchi and bronchioles in cats infected with D. immitis. Abbott (1966) reports thickening of the bronchiole wall and bronchiolitis, with hyperplasia of the epithelium and severe epithelial sloughing associated to the presence of histiocytes, eosinophils, and blood cells in the lumen of bronchioles in cats with heartworm. The pathological findings in the bronchi and bronchioles did not have a specific characteristic but stemmed from chronic interstitial pneumonia caused by other agents. These findings are compatible with those described by Labarthe (1997), who reports that the inflammatory process in the vessels of animals infected with D. immitis causes the degeneration of the endothelium, a reduction in the vascular lumen and even obstruction of the vessels. The author states that such lesions do not stem from the physical presence of the parasite alone. According to Calvert and Rawlings (2002), the hypertrophy of the tunica intima, with the formation of villosities, is due to the invasion of the intima by smooth muscle cells and collagen from muscle tunica media, stimulated by growth factors released by platelets and leukocytes. Complete obstruction of arteries by the formation of
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Fig. 4. Photomicrographs of pulmonary parenchyma of lung cats experimentally infected with Dirofilaria immitis. Pulmonary parenchyma within alveoli with rupture (arrow) and alveolar dilatation (star) (A); 40× magnification. Pulmonary parenchyma showing diffuse infiltration of macrophages (thin arrow) and eosinophils (large arrow) (B); 400× magnification. Diffuse presence of smooth muscle cell (arrow) (C). 400× magnification. Pulmonary parenchyma stained with Gomori’s trichromic, showing collagen fibers (arrow) stained in green (D); 150× magnification. Pulmonary parenchyma stained with Gomori’s trichromic. There are many smooth muscle cells (star) stained in dark blue (E); 40× magnification.
thromboses and granulomas in the tunica intima stemming from the death of parasites are frequent findings in canine and feline dirofilariasis (Dillon, 2002). The vacuolization of the muscle cells of the tunica media in the animals studied may be related to hydropic degenera-
tion as a consequence of alterations in the cell membrane due to a lack of nutrients and oxygenation stemming from lesions in the endothelium and the hypertrophy of this layer. Hamilton (1966) describes the vacuolization of muscle cells of hypertrophied, hyperplastic pulmonary
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Fig. 5. Photomicrographs of pulmonary parenchyma of lung cats experimentally infected with Dirofilaria immitis. Type II pneumocyte hypertrophy. Note several cytolisosomes inside the cytoplasm (arrow head) and degranulation of surfactant on cellular surface (arrow) (A); 7000× magnification. Pneumocytes II (P) with cytoplasm within several cytolisosomos (arrow) in association with lymphocytes (L). Interstitial macrophages (star), surrounded by collagens fibers (arrow head) (B); 3000× magnification. Eosinophils adjacent to type II pneumocytes in apoptosis (star). Arrow points to apoptotic vesicle (C); 3000× magnification. Pneumocytes II in necrosis. Nuclear chromatin condensation (star), stripping of nuclear membrane (thin arrow) and mitochondria without tubular crest (arrow head) (D). 7000× magnification.
arteries in cats with different lung diseases. According to the author, this vacuolization did not respond to the different staining methods used for the identification of neutral fats, cholesterol, mucopolysaccharides and glycogen. The vascular lesions reported in the present study resemble those described by Hamilton (1966), who found similar lesions in cats infected with A. abstrusus or with other lung diseases. The findings are also compatible with those described by a number of authors (Dillon, 1984; McCracken and Patton, 1993; Mupanomunda et al., 1997), according to whom, arterial lesions stem from the local anaphylactic reaction and inflammatory infiltration by mononuclear cells in the sub-endothelial tissue of the arteries. Dillon (1984) reports the similarity in lesions found in cats and dogs with dirofilariasis; within a week following the transplant of adult helminthes in cats, parasitic endarteritis, villous proliferation of the intima and severe vascular inflammatory response were evident. Labarthe (1997) found partial obstruction of the pulmonary arteries and arterioles stemming from hyperplasia and hypertro-
phy of the tunica muscularis and villous projections of the endothelium, suggesting that these lesions stem from the migration of the parasites to the pulmonary arteries. In cats experimentally infected with D. immitis, Holmes et al. (1992) describe moderate to severe eosinophilic endarteritis in pulmonary arteries of all sizes, followed by severe muscle hypertrophy of arteries of medium size and small calibers in all the lobes of the lungs, along with complete obstruction of some arterioles and the presence of thromboses. The morphological characteristics, intensity and type of inflammatory reaction in the arterial wall and pulmonary parenchyma suggest the lesions described in the present study likely occurred as a consequence of the mechanical action of the parasites on the endothelium of the arteries as well as the release of antigen factors by the helminths. Filoni et al. (2009) report fibromuscular proliferation, diffuse inter-alveolar fibrosis, perivascular eosinophilic inflammation, hemosiderosis, and the presence of microfilaria in small arteries. These factors may be responsible for triggering the type II hypersensitivity reac-
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Fig. 6. Active eosinophil (E) in interaction with another cell of pulmonary parenchyma (arrow) (A); 12000× magnification. Interstitial space of pulmonary parenchyma. Note interaction between plasma cell (P), mast cell (M), eosinophil (E), next to fibroblast (F) surrounded by collagen fibers (star) (B); 4400× magnification. Active eosinophil (E) in strict relationship with pneumocytes I (P) (C); 12000× magnification. Interstitial space of pulmonary parenchyma. Note smooth muscle cell (star) in relationship com eosinophils (arrow) (D); 3000× magnification.
tion (cytotoxic reaction), which is mediated by IgG, IgE, and eosinophils (Siqueira and Dantas, 2000). The type of cellular inflammatory reaction found in the present study allows the supposition that this reaction was a consequence of the presence of antigens released by the helminthes. Regarding the presence of alveolar emphysema, this may be interpreted as a consequence of the loss of alveolar expansion capacity in areas adjacent to the pneumonia and the intense pulmonary hypertension observed in the animals. Similar lesions to those found in the present study were reported by Dillon (1984, 2002), who referred to this lesion only as hyperplasia of type II pneumocytes in cats with dirofilariasis. Atwell et al. (1988) also report smooth muscle atrophy in alveolar ducts. The presence of smooth muscle fibers in the alveolar walls, especially in the intensive, disseminated form similar to those that occurred in the cats analyzed in the present study, should be considered hyperplasia followed by hypertrophy due to the intense pulmonary hypertension in the lungs. The main cell alteration in the ultrastructural analysis was hyperplasia of type II pneumocytes, which produce surfactant substances. Hyperplasia and hypertrophy of
pneumocytes are related to injury to the pulmonary parenchyma (Fenrenbach et al., 1999; Morikawa et al., 2000). According to Tomashefski (2000), hyperplastic lesions of type II pneumocytes frequently occur in acute respiratory distress syndrome, particularly 4–9 months following infection. Cell death of type II pneumocytes by apoptosis or necrosis may be attributed to complex mechanisms that involve the inflammatory response to D. immitis antigens, which are always accompanied by a variable quantity of type II pneumocytes and eosinophils. According to Gleich and Adolphson (1986) and Spry (1988), mature eosinophils can be stimulated by a large number of signals derived from various sources, particularly interleukin-5 (IL-5) (Rothenberg et al., 1989) and IL-4 (Coffman et al., 1988), to become active and migrate to specific sites, such as areas of acute or chronic inflammation. IL-5 induces chemotaxis and degranulation in eonsinophils in vivo and in vitro (Wong et al., 1990; Kita et al., 1992; Lampinen et al., 2004), and the proteins of eosinophils of granules are toxic to microfilariae in vivo, causing the death of the larvae (Hamann et al., 1991). The specific dependence of IL-5 on granulomatous eosinophilia has been corrobo-
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Fig. 7. Photomicrograph of pulmonary parenchyma showing cell association between eosinophil (E), macrophage (M) and pneumocyte II (P) in initial stage of necrosis (A); 4400× magnification. Eosinophil (E) next to capillary (C) edema peri-vascular (arrow head) and several collagen fibers (arrow) (B); 12000× magnification. Fibroblast (arrow) surrounded by collagen fibers bundles (F), next to eosinophils (E) (C); 4400× magnification.
rated by the complete ablation of eosinophilic response in mice infected with Schistosoma mansoni following treatment with specific antibodies for IL-5 (Sher et al., 1990; Rennick et al., 1990). This cytokine promotes chemotaxis selective for eosinophils and induces degranulation in vitro and in vivo (Wong et al., 1989; Kita et al., 1992; Terada et al., 1992). The increase in IL-5 expression significantly increases the number of eosinophils and antibody levels in vivo. On the other hand, rats with the absence of a functional gene for IL-5 or the alpha chain of the receptor for IL-5 (IL-5R) exhibit a series of alterations in the development and functioning of B cells in the eosinophil lineages (Kouro and Takatsu, 2009). Raible et al. (1992) and Shin et al. (2009) stress the importance of mastocytes and tissue inflammation mediated by eosinophils, respectively, in infection with helminthes. The interaction between these cells and stimulating factors may be responsible for the interstitial pneumonia found in the pulmonary parenchyma of the cats infected with D. immitis. According to Hamann et al. (1991), all proteins from the granules of eosinophils are toxic to microfilariae, causing their death in vivo. The findings of the authors confirm the existence of cell interaction between eosinophils, lymphocytes, and fibroblasts in the
production of interstitial pneumonia in response to the presence or not of D. immitis parasites in the pulmonary parenchyma. Among the inflammatory cells found in the pulmonary interstice, the presence of mastocytes can be explained by the fact that the factors produced by these cells, such as leukotriene B4, histamine, eosinophil chemotactic factor A and prostaglandin D2, are eosinophilic and are capable of activating eosinophils and increasing their helminthotoxic activity (Raible et al., 1992). The interaction of macrophages and eosinophils in the pulmonary parenchyma and alveolar lumen may be explained by the relation of dependence on factors derived from monocyte lymphocytes, such as tumor necrosis factor alpha, eosinophil-activating factor, and eosinophil cytotoxity-enhancing factor, which, together, influence the function of mature eosinophils (Thorne and Mazza, 1991). Horowitz and Thannickal (2006) describe phenotype alterations in type II alveolar epithelial cells, such as proliferation, bronchiolization, apoptosis, and regenerative hyperplasia related to idiopathic pulmonary fibrosis. It is therefore possible that type II pneumocytes and other inflammatory stimuli are implied in the development of fibrosis in cats experimentally infected with D. immitis.
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5. Conclusions The results of the present study demonstrate that the alterations induced by experimental dirofilariasis experimental in felines 42 days following infection were related to arterial lesions, interstitial pneumonia, hypertrophy, and hyperplasia of type II pneumocytes, the proliferation of smooth muscle cells, fibrosis, and emphysema. The ultrastructural analysis revealed that interstitial pneumonia was characterized by the presence of eosinophils, macrophages, plasma cells, mastocytes, and fibroblasts. The interaction between these cells involves complex defense mechanisms in an active response on the part of the host to antigens derived from the helminthes in blood vessels and the pulmonary parenchyma. Funding/Conflict of interest The cats used for this study were provided by Dr. J. McCall and had been used a project funded by TRS Labs. No other funding was provided. The authors report no financial or personal interests or relationships or other potential conflicts in this research. References Abbott, P.K., 1966. Feline dirofilariasis in Papua. Aust. Vet. J. 42, 247–249. Atwell, R.B., Sutton, R.H., Moodie, E.W., 1988. Pulmonary changes associated with dead filariae (Dirofilaria immitis) and concurrent antigenic exposure in dogs. J. Med. Chem. 31, 774–779. Behmer, O.A., Tolosa, E.M.C., Neto, A.G.F., 1976. Manual de técnicas para histologia normal e patológica. Edart, São Paulo, 257 pp. Calvert, C.A., Rawlings, C.A., 2002. Dirofilariose canina. In: Tilley, L.P., Goodwin, J. (Eds.), Manual de cardiologia para cães e gatos. , 3rd ed. Roca, São Paulo, 489 pp. Coffman, R.L., Seymour, B.W.P., Lebman, D.A., Hiraki, D.D., Christiansen, J.A., Shrader, B., Cherwinski, H.M., Savelkoul, H.F.B., Finkelman, F.D., Bond, M.A., Mosmann, T.R., 1988. The role of helper T-cell products in mouse B-cell differentiation and isotype regulation. Immunol. Rev. 102, 5–28. Dillon, R., 1984. Feline dirofilariasis. Vet. Clin. N. Am. Small Anim. Pract. 14, 1185–1199. Dillon, R., 2002. Dirofilariose felina. In: Tilley, L.P., Goodwin, J.K. (Eds.), Manual de cardiologia para cães e gatos. , 3rd ed. Roca, São Paulo, 489 pp. Fenrenbach, H., Kasper, M., Tschernig, T., Pan, T., Schuh, D., Shannon, J.M., Muller, M., Mason, R.J., 1999. Keratinocytes growth factor-induced hyperplasia of rat alveolar type II cell in vivo is resolved by differentiation into type I cells and by apoptosis. Eur. Resp. J. 14, 534–544. Filoni, C., Pena, H.F.J., Gennari, S.M., Cristo, D.S., Torres, L.N., Catão-Dias, J.L., 2009. Heartworm (Dirofilaria immitis) disease in a Brazilian oncilla (Leopardus tigrinus). Pesq. Vet. Bras. 29, 474–478. Gleich, G.J., Adolphson, C.R., 1986. The eosinophilic leukocyte: structure and function. Adv. Immunol. 39, 177–253. Hamann, K.J., Barker, R.L., Ten, R.M., Gleich, G.J., 1991. The molecular biology of eosinophil granule proteins. Int. Arch. Allergy Appl. Immunol. 94, 202–209. Hamilton, J.M., 1966. Pulmonary arterial disease of the cat. J. Comp. Pathol. 76, 133–245. Holmes, R.A., Clarck, J.N., Casey, H.W., Henk, W., Plue, R.E., 1992. Histopathologic and radiographic study of the development of heartworm pulmonary vascular disease in experimentally infected cats. In:
Soll, M.D. (Ed.), Proceedings of the Heartworm Symposium’92. American Heartworm Society, Batavia, IL, pp. 81–89. Horowitz, J.C., Thannickal, V.J., 2006. Idiopathic pulmonary fibrosis: new concepts in pathogenesis and implications for drug therapy. Treat. Respir. Med. 5, 325–342. Kita, H., Weiler, D.A., Abu-Ghazaleh, R., Sanderson, C.J., Gleich, G.J., 1992. Release of granule proteins from eosinophils cultured with IL-5. J. Immunol. 149, 629–635. Knott, J., 1939. A method for making microfilarial surveys on dog blood. Trans. R. Soc. Trop. Med. Hyg. 33, 191–196. Kouro, T., Takatsu, K., 2009. IL-5- and eosinophil-mediated inflammation: from discovery to therapy. Int. Immunol. 21, 1303–1309. Labarthe, N.V., 1997. Dirofilariose canina: diagnóstico, prevenc¸ão e tratamento adulticida. Clin. Vet. 10, 10–16. Lampinen, M., Carlson, M., Hakansson, L.D., Venge, P., 2004. Cytokineregulated accumulation of eosinophils in inflammatory disease. Allergy 59, 793–805. McCall, J.W., Genchi, C., Kramer, L.H., Guerrero, J., Venco, L., 2008. Heartworm disease in animals and humans. Adv. Parasitol. 66, 193–285. McCracken, M.D., Patton, S., 1993. Pulmonary arterial changes in feline dirofilariasis. Vet. Pathol. 30, 64–69. McElroy, D.A., 1992. Connective tissue. In: Prophet, E.B., Mills, B., Arrington, J.B., Sobin, L.H. (Eds.), AFIB Laboratory Methods in Histotechnology, vol. 18. American Registry of Pathology, Washington, DC, pp. 149–174. Morikawa, Y., Katsumoto, Y., Okada, T., Sasaki, F., 2000. Pulmonary hypoplasia induced by liquid paraffin injection into fetal thoracic cavity with special reference to renal development in rats. J. Vet. Med. Sci. 62, 135–140. Mupanomunda, M., Williams, J.F., Mackenzie, C.D., Kaiser, L., 1997. Dirofilaria immitis: heartworm infection alters pulmonary artery endothelial cell behavior. J. Appl. Physiol. 82, 389–398. Raible, D.G., Schulman, E.S., DiMuzio, J., Cardilo, R., Post, T.J., 1992. Mast cell mediators, prostaglandin D2 and histamine activate humaneosinophils. J. Immunol. 148, 3536–3542. Rawlings, C.A., McCall, J.W., 1985. Surgical transplantation of adult Dirofilaria immitis to study heartworm infection and disease in dogs. Am. J. Vet. Res. 46, 221–224. Rennick, D.M., Thompson-Snipes, L., Coffman, R.L., Seymour, B.W., Jackson, J.D., Hudak, S., 1990. In vivo administration of antibody to interleukin5 inhibits increased generation of eosinophils and their progenitors in bone marrow of parasitized mice. Blood 76, 2311–2316. Rothenberg, M.E., Petersen, J., Stevens, R.L., Silberstein, D.S., McKenzie, D.T., Austen, K.F., Owen, W.F., 1989. IL-5-dependent conversion of normo-dense human eosinophils to the hipodense phenotype uses 3T3 fibroblasts for enhanced viability, accelerated hypodensity and sustained antibody-dependent cytotoxicity. J. Immunol. 143, 2311–2316. Sher, A., Coffman, R.L., Hieny, S., Scott, P., Cheever, A.W., 1990. Interleukin5 is required for the blood and tissue eosinophilia but not granuloma formation induced by infection with Schistosoma mansoni. Proc. Natl. Acad. Sci. 87, 61–65. Shin, M.H., Lee, Y.A., Min, D.Y., 2009. Eosinophil-mediated tissue inflammatory responses in helminth infection. J. Parasitol. 47, 125–131. Siqueira, J.F., Dantas, C.J.C. (Eds.), 2000. Mecanismos celulares e moleculares da inflamac¸ão. Medsi, Rio de Janeiro, 238 pp. Spry, C.J.F., 1988. Eosinophils: A Comprehensive Review and Guide to the Scientific and Medical Literature. Oxford University Press, Oxford, pp. 10–28. Terada, N., Konno, A., Shirotori, K., Togawa, K., 1992. In human nasal mucosa, interleukin-5 accumulates and degranulates eosinophils, as well as increases responsiveness to histamine. Rhinol. Suppl. 14, 57–61. Thorne, K.J.I., Mazza, G., 1991. Eosinophilia, activated eosinophils and human schistosomiasis. J. Cell. Sci. 98, 265–270. Tomashefski, J.F., 2000. Pulmonary pathology of acute respiratory distress syndrome. Clin. Chest Med. 21, 435–466. Wong, D.T., Weller, P.F., Galli, S.J., Elovic, A., Rand, T.H., Gallagher, G.T., Chiang, T., Chou, M.Y., Matossian, K., McBride, J., Todd, R., 1990. Human eosinophils express transforming growth factor alpha. J. Exp. Med. 172, 673–681.