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Histological and immunohistochemical characterization of Hypoderma lineatum (Diptera: oestridae) warbles
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Histological and immunohistochemical characterization of Hypoderma lineatum (Diptera: oestridae) warbles E. Cabanelas a,∗ , R. Panadero a , M. Fuertes b , M. Fernández b , J. Benavides b , C. López a , ˜ a , V. Pérez b A. Pérez-Creo a , P. Díaz a , P. Morrondo a , P. Díez-Banos a Departamento de Patología Animal: Sanidad Animal (Grupo INVESAGA), Facultad de Veterinaria, Universidad de Santiago de Compostela, Lugo 27002, Spain b Departamento de Sanidad Animal, Instituto de Ganadería de Monta˜ na (CSIC-ULE). Facultad de Veterinaria. Universidad de León. Campus de Vegazana, s/n 24071 León, Spain
a r t i c l e
i n f o
Article history: Received 10 March 2015 Received in revised form 12 June 2015 Accepted 15 June 2015 Keywords: Cattle-arthropoda Hypoderma Warbles Subcutaneous larvae Cellular response
a b s t r a c t Hypoderma larvae are tissue invading parasites which spend several months migrating within the host tissues before completing their development in the sub-dermal tissues of the back. Subcutaneous stages of the parasite produce an inflammatory reaction in the skin called “warbles”, as well as holes through which larvae breathe. In order to elucidate the microscopical structure of the warbles, three hides from warbled cows were collected in a slaughterhouse in Lugo (NW, Spain) between March and May 2012. A total of 60 skin samples, including warbles at different phases of development, were chosen for histopathological and immunohistochemical examination. Microscopic lesions were classified into three groups, according to the predominance and distribution of different cell populations. In warbles containing living or recently dead larvae with apparently well preserved cuticle (type 1), plasma cells were observed in high number. However, macrophages and lymphocytes were the predominant cells in granulomas (type 2) formed in relation to remnants of the dead parasite, containing or not remains of the altered cuticle. Scars (type 3) were characterized by granulation tissue. Immunohistochemistry showed that B lymphocytes and IgG+ cells were predominant in the lesions, as long as the cuticle of the larvae is intact. On the other side, CD3+ lymphocytes increased once cuticle is destroyed and a granuloma is formed. Macrophages, revealed by CD68+ , MAC387+ and lysozyme immunolabelling, were detected in all types of lesions, but they were more abundant in type 2 and scarce in scars. These cells appeared isolated around the intact larvae or forming aggregates around its remains in the granuloma. Moreover, a strong immunolabelling against MAC387 antibody was registered in the squamous epithelium covering the breathing pore. This finding may be associated with the expression of calprotectin, a molecule involved on the healing process of the skin after larvae outcome. Our results suggest the predominance of a humoral response inside the warble as long as larvae are intact. Once they are destroyed, cellular response occurred, isolating and destroying the remains of the larvae until healing process completes and scars with low numbers of inflammatory cells appear. © 2015 Published by Elsevier B.V.
1. Introduction Hypoderma larvae are obligate parasites that migrate and feed for several months in host’s tissues. Newly hatched larvae penetrate unbroken bovine skin thanks to enzymatic secretions produced by their salivary glands (Boulard, 1970). Once the penetration is com-
∗ Corresponding author. Fax: +34 982822001. E-mail address: [email protected] (E. Cabanelas).
plete, the larva begins migration in subcutaneous tissue (Nelson and Weintraub, 1972), which leads to the oesophageal submucosa for Hypoderma lineatum and epidural fat for Hypoderma bovis. After that, larvae continue their migration towards subcutaneous tissue in the back, where they turn to second and third instar larvae successively. These larvae are aerobic and breathe through a hole bored in the skin (Hadwen and Bruce, 1916). After first instar molt, the expulsion of its content triggers an inflammatory response to encapsulate second and third instar larvae with connective tissue (Berkemkamp and Drummond, 1990),
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Please cite this article in press as: Cabanelas, E., et al., Histological and immunohistochemical characterization of Hypoderma lineatum (Diptera: oestridae) warbles. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.06.017
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2 Table 1 Primary antibodies used in immunohistochemistry. Specificity
Type or clone
Origin
Pretreatment
Dilution
Source
Myeloid/Histiocyte antigen (calprotectin) (Macrophages) CD68 (Macrophages) Lysozime CD79 (B cells) CD3 IgG Lambda Light Chains
MAC 387 PG-M1 Policlonal JCB117 Policlonal Policlonal
Mouse Mouse Rabbit Mouse Rabbit Rabbit
Trypsine Trypsine Trypsine Microwave Steamer Triton X-100
1:75 1:100 1:1000 1:25 1:200 1:6000
Dako Dako Dako Dako Sigma Dako
forming the characteristic swellings or subcutaneous furuncles called “warbles” (Beesley, 1974). According to Boulard (1975), fibroblasts are very active and produce lots of collagen fibers to isolate larvae, which stay in stable environment. Mature third instars exit the host and pupate within a short period of time. Larvae migrating within the host and in sub-dermal warbles provoke production losses and increased susceptibility to diseases (Drummond, 1987). Larvae are continuously exposed to host immune effector mechanisms trying to isolate and kill them. Gingrich (1980) reported that immunity mechanisms are especially active in early phases of larvae development, in which larvae mortality is higher. Nevertheless, Pruett and Kunz (1996) pointed out that larvae destruction is very intense in the last stages of development. While lesions caused by first instar larvae during penetration and migration in the host are well described (López et al., 2005; Dacal et al., 2009, 2011), not detailed descriptions on the different pathological responses associated with the presence of the larvae in the subcutaneous tissue have been carried out. Moreover, little information is also available on the cellular reactions responsible for larval isolation or destruction at sub-dermal sites. The aim of this study was to define and classify the microscopic lesions and to characterize, by using histopathology and immunohistochemistry, the cells that participate in the cutaneous reaction against subcutaneous larvae, including the response of macrophages, T cells, B cells and immunoglobulin G-producing plasma cells, during the stay of second or third instar H. lineatum in the back of the cattle.
Table 2 Evaluation of immunolabelled cell population numbers in the three histological lesions described.
1 2 3
CD3+
CD79+
CD68+
MAC387+
Lysozyme
IgG+
+ ++/+++ +
+++ + +
+ +++ +
++ +++ +
++ ++ +
++ 0 0
0: nil; +:mild; ++:moderate; +++:intense.
2. Material and methods
For IHC studies skin sections, 4 m thick, were placed onto polyL-Lysine coated slides. Endogenous peroxidase activity was blocked in deparaffinised sections by incubation with 3% hydrogen peroxide for 30 min in darkness at room temperature (RT). Rehydrated slides were rinsed twice in PBS pH 7.4. To optimize the immunoreaction, the antigen retrieval was performed using enzymatic or heat-based methods, depending on the primary antibody (Table 1). Sections were incubated with the primary antibodies diluted in PBS overnight at 4 ◦ C in a humidified chamber. After extensive washing with PBS, sections were incubated for 40 min at RT with EnVision® +/HRP solution (Dako North America Inc., Carpinteria, USA) for the appropriate monoclonal or polyclonal antibodies. After washing in PBS, antibody localization was determined using 3,3-diaminobenzidine (DAB, Sigma–Aldrich Corp.) as chromogenic substrate for peroxidase. Sections were counterstained with haematoxylin for 30 s. Evaluation of immunostaining was performed under a light microscope with final magnification of 500×. Labelled cells taking part into the lesion were examined and a qualitative classification of the number of positively immunostained cells, from nil (0) to intense (+++), was established (Table 2).
2.1. Animal samples
3. Results
The hides from three intensely warbled cows were collected in a local slaughterhouse in Lugo (NW, Spain). The outer side of the hides was examined to confirm the absence of other relevant lesions (mange, ticks, lice, etc.) and the inner side was inspected recording warbles at different stages of development. The first hide (H1) was collected in March 2012 and presented 16 viable warbles and 13 non-viable warbles; the second hide (H2) was collected in April 2012 and exhibited 4 viable warbles and 38 non-viable warbles; the last hide (H3) was obtained in May and presented 19 non-viable warbles. A total of 45 skin samples (15 from H1, 31 from H2 and 9 from H3), including warbles containing living (n = 10), dead larvae (n = 20) and scars (n = 15) were taken for histopathological and immunohistochemical (IHC) examination. Samples were fixed in 10% formaldehyde and embedded in paraffin wax using standard protocols.
3.1. Gross lesions
2.2. Histopathological and immunohistochemical examination For histopathological examination samples were processed by routine methods and sections, 4 m thick, were stained with haematoxylin and eosin (HE).
Macroscopically, 4 mm diameter breathing holes (n = 20) and 2 mm diameter scars (n = 70) (Fig. 1a) were visible in the outer side of the skin. In the inner side, warbles at different stages of development were observed in the hides examined: viable warbles (n = 20) were composed of a layer of fibrous connective tissue that creates a cavity containing the larvae and necrotic material (Fig. 1b); in non-viable warbles (n = 40), larvae seemed to be dark, crushed and dehydrated (Fig. 1c). The remaining 30 samples showed healing tissue without visible larvae remains. 3.2. Histopathology In all the examined samples, the superficial dermis showed a mild to moderate perivascular inflammatory infiltrate, composed mainly of lymphocytes, plasma cells and occasional polymorphonuclear eosinophils. In the deep dermis lesions were variable and were classified into three different groups, according to the presence and stage of preservation of the larvae, the characteristics of the inflammatory infiltrate and the predominant cells present in the lesions:
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Fig. 1. (a) Outer side of the skin of one affected cattle showing a breathing hole (continuous arrow) and a scar (discontinuous arrow). (b) Viable larva, surrounded by necrotic debris, inside a fibrous cavity. (c) Non-viable larvae in the inner side of the skin.
Type 1 lesion (18 samples): In the centre of the lesion, viable or recently dead larvae could be seen. In all of them, a well-preserved chitinized cuticle was always clearly distinguishable. When the larvae were viable, skeletal muscle, tracheal rings and intestinal cells were detected while an amorphous, eosinophilic material, surrounded by the cuticle, was seen where they were dead (Fig. 2a). Larvae were surrounded by the deposition of variable amounts of fibrin and necrotic material (Fig. 2a). Externally, a cellular infiltrate composed mainly of plasma cells (Fig. 2b), together with a moderate amount of macrophages and lower numbers of eosinophils and lymphocytes was seen infiltrating the surrounding connective tissue. A squamous layered epithelium was found covering the internal side of the breathing hole. Type 2 lesion (12 samples): In all the cases, larvae were always dead and cuticle was not visible in any case. The morphology of dead larvae varied from cases in which they hold its shape (Fig. 3a) to the presence of remnants where the only identifiable structures were fragments of the cuticle and spicules visible among necrotic material. The inflammatory infiltrate differed significantly from the former type. It was composed of numerous macrophages with abundant eosinophilic cytoplasm consistent with epithelioid cells together with some multinucleated giant cells, both foreignbody and Langhan’s types, forming clearly defined granulomas (Figs. 3 and 4). Externally, the infiltrate was surrounded by a layer composed of variable numbers of lymphocytes, sporadically forming aggregates (Fig. 4). Occasionally, small foci of mineralization were seen in groups of necrotic epithelioid cells. At the periphery, the entire lesion was demarcated by a capsule of connective tissue (Figs. 3 and 4). In some samples, no remains of the larvae could be detected. Type 3 lesion (15 samples): Perivascular mononuclear infiltrates and granulation tissue conformed by fibroblasts and neovascularization were the only lesions detected. Focal aggregates of lymphocytes with occasional macrophages between them were also observed (Fig. 5). Considering the correlation between gross lesions and histopathology, 100% of viable warbles were categorized as Type 1 lesion. All the 20 examined warbles containing non-viable
Fig. 2. Type 1 lesion. (a) Non viable larvae (note the eosinophilic, amorphous material inside the relatively well-preserved cuticle) in relation a cellular infiltrate of the dermis with fibrin and necrotic debris. HE. Bar 100 m. (b) Detail of the inflammatory infiltrate, mainly formed by plasma cells, found in the dermis. HE. Bar 50 m. Inset: Immunohistochemical labelling of plasma cells in this infiltrate. IgG IHC. Bar 25 m.
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Fig. 4. Type 2 lesion. Granuloma characterized by a central area of multinucleated giant cells, surrounded by lymphocytes forming aggregates and demarcated by a fibrous capsule. HE. Bar 100 m.
Fig. 3. Type 2 lesion. (a) Granulomatous lesions where larvae remnants, with no visible cuticle, are present in the centre and surrounded by an inflammatory infiltrate and externally demarcated by fibrous tissue. HE. Bar 200 m. (b) Detail of the granulomatous infiltrate where epithelioid and multinucleated giant cells are prominent. HE. Bar 100 m.
larvae were categorized as Type 2. Finally, 100% of healing lesions were classified as Type 3. 3.3. Immunohistochemistry 3.3.1. T lymphocytes While these cells were scarce in the infiltrate of type 1 lesions, the aggregates observed in type 2 lesions were composed mainly of CD3+ lymphocytes (Fig. 4). In type 3, CD3+ cells were very scarce. 3.3.2. B lymphocytes CD79+ lymphocytes were present in large numbers (about 60% of those forming the infiltrate) in type 1 lesions. They were scarce in the remaining types of lesion (Fig. 6). 3.3.3. Macrophages CD68+ cells were found in all the types of lesion. In type 1, isolated immunolabelled cells were detected among the inflammatory infiltrate, especially in the vicinity of the necrotic areas around the larvae. In type 2 lesions all the epithelioid and giant cells forming the granuloma showed positive immunolabelling with variable intensity. Finally, few and scattered CD68+ cells observed in type 3 lesions as well as in the superficial and deep dermis in areas unrelated to the lesion. MAC387+ cells were observed in the inflammatory infiltrate that surrounded the necrotic areas around the larvae in type 1 lesions (Fig. 6). The epithelium covering the warble opening was positive
Fig. 5. Type 3 lesion. Granulation tissue with abundance of collagen and scattered small aggregates of inflammatory cells, mainly lymphocytes and macrophages. HE. Bar 100 m.
to this antibody (Fig. 7); on the contrary, the remaining epithelium in the skin sample was negative. In type 2 lesions, most of macrophages were positive, with differences in staining intensity according to the location inside the granuloma: while epithelioid cells at the centre of the granuloma were positive, the number of immunolabelled cells was lower among the peripheric ones (Fig. 6). In type 3 lesion, MAC387+ labelled cells were barely detected. They were not seen either in the normal skin. Macrophages immunolabelled for lysozyme, with no evident variation in the intensity of the labelling, were found in all the types of lesions. They were scarce in type 3. 3.3.4. IgG+ cells IgG+ cells were numerous in type 1 lesions (Fig. 3). They were not detected in the remaining types. 4. Discussion This study has shown the existence of different microscopic lesions that are related to the stage and viability of the larvae, during back phase of hypodermosis in cattle. They are characterized by the existence of a chronic, proliferative dermatitis in the internal dermis with variations in the cell populations present in the infiltrate according to the different types of lesion. During the
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Fig. 6. Comparison between 1 and 2 type lesions of the immunohistochemical labelling (gold-brown colour) for CD3 (T lymphocytes), CD79 (B lymphocytes) and calprotectin (freshly recruited macrophages) in the inflammatory infiltrate. IHC. Bar 200 m. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
Fig. 7. Immunohistochemical labelling (gold-brown colour) of reactive epithelia cells at the larvae opening in type 1-2 lesions. Note the scattered positive cells (freshly recruited macrophages) among the inflammatory infiltrate in the dermis (left side). Calprotectin IHC. Bar 100 m. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
penetration phase, and associated with the first instar, an inflammation characterized by abundant neutrophils and eosinophils in the skin is recognized (Lello and Rosis, 2003; López et al., 2005; Oryan and Bahrami, 2012). However, in our study, inflammation has been predominantly mononuclear where neutrophils have not been recorded. Moreover, the presence of eosinophils around the larvae was reduced. This finding contrasts strongly with the high number of eosinophils observed by Nelson and Weintraub (1972) and López et al. (2005) at the entry point of the larvae and by Boulard (1975) and Arru et al. (1985) in the resting phase of the oesophageal submucosa. Probably this finding is a result of the differences in larvae activity in the skin. During the penetration stage, the first instar larvae eroded the subcutaneous tissue by the collagenolytic enzymes secreted by their salivary and intestinal glands (Madel and Nahif, 1971; Tassi et al., 1989) causing necrosis and attracting neutrophils. In the back phase, after migration, third
instar larvae are located in subcutaneous nodules where they last for four to six weeks (Beesley, 1974). Although necrosis was present in relation to the larvae, this was always mild and seen together with fibrin (type 1 lesions) with no neutrophilic infiltrate. It is likely that, at the time of sampling, large areas of necrosis caused by second and third instar larvae, eliciting neutrophilic infiltration have already disappeared. The possibility that the different larvae could secrete different substances with different chemoattractability for neutrophils or eosinophils could be also considered. Type 1 lesions are consistent with the classical warble nodules characterized by the presence of intact third instar larvae, an inflammatory infiltrate and a central pore layered by a squamous epithelium from surrounding epidermis proliferation in the internal side of the opening, similar to that described by Marcato (1990) and consistent with the respiratory pore of the warble (Hadwen and Bruce, 1916). This pore has not been detected in the other types of lesion described. That could be due to the fact that after the out coming of the third instar larvae, opening closes with scar tissue, although in some occasions the remains of the parasite may delay the process. A similar pattern of cicatrization was reported by Rietschel (1979) in the evolution of Oestromya leporine warbles; fibroblasts surround the hole left by the larvae, containing exudate and remains of the parasite. The epidermis proliferates between both tissues and the content is discharged to the outside thanks to muscular pressure and new tissue growing. A striking feature of this study was the existence of a strong positive immunolabelling for MAC387 antibody of the squamous epithelial cells in the pore, in contrast with the epithelium seen in normal epidermis. It has been reported that “reactive” epithelial cells can express high levels of calprotectin, the protein that MAC387 antibody recognizes (Brandtzaeg et al., 1992). Calprotectin has been shown to play important roles in infection, inflammation and wound healing (Dhas et al., 2012) and their expression in these particular cells would suggest that they could play a significant role in the local host response against Hypoderma larvae. Particularly, production of calprotectin has been associated with a decrease in neutrophil migration and release of cytokines (Averill et al., 2012). This fact could be the cause that could explain the
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absence of neutrophils in this type of lesion although necrosis was present, in contrast to the damage caused by first instar larvae penetration, above discussed. Future studies addressing calprotectin expression during first instar larvae migration could help to understand the role of this protein in the pathological response against this parasite. Regarding to larvae structure, the absence of mouthparts had been previously described by Boulard (1969). In this phase, larvae are sedentary and feed on the surrounding materials. Reaction around viable larvae was characterised by the predominance of plasma cells. This finding is consistent with the increase of systemic serum antibodies simultaneously to the appearance of the warbles in the back reported by Panadero et al. (2013). The progressive reduction of plasma cells in the other types of lesion together with the increase in macrophages and T lymphocytes, may be related to the exposure to different antigens released after the death of the larvae, and rupture of cuticle, or its out coming from the dermis. The high levels of B lymphocytes (CD79+ cells) were in relationship with the number of IgG+ plasma cells in the infiltrate, with the highest values in type 1 lesion. The reduction of both type of cells, plasma cells and B-lymphocytes, in types 2 and 3 lesions, may explain the decrease of serological antibody levels observed by Baron and Colwell (1991) as larvae leaves the host and tissue heals. These findings would suggest that the local inflammatory infiltrate could play an important role in the antibody production detected in sera. The regional lymph nodes were not examined, but they will probably also contribute. As plasma and B cells decreased in lesions type 2 and 3, there was an increase in the number of macrophages, to form clearly defined granulomas. Their role is probably related to the phagocytosis (Fukiwara and Kobayashi, 2005) of necrotic larval tissue remnants leading to the formation of granulomas when they are persistent and poorly degradable substances (Agostini and Semenzato, 2003). Actually, this type of lesion was always in relation to the presence of dead larvae or remnants or absence of the parasite. The fact that the most dramatic increase in macrophages was found between type 1, where cuticle was intact or with very little discontinuities, and type 2, where cuticle was absent, suggest that Hypoderma cuticle would play a role in the inflammatory response, probably containing antigens that would trigger the local infiltration of plasma cells rather than macrophages. Once the cuticle has disappeared (type 2), the inflammatory infiltrate would torn into a predominantly macrophage type, surrounding remnants of dead larvae or remains of cuticle sloughed during ecdysis that are difficult to eliminate (Ginn et al., 2007). According to previous studies (Pruett and Kunz, 1996), host acquired resistance provoked considerable larval mortality occurring predominantly after their arrival to the back. The predominance of a Th2 response, characterized by high levels of IgG1 and IL-4, at the latest stages of the infestation (Vázquez et al., 2012) could be responsible for larval destruction. Type 3 lesions are formed mainly by granulation tissue that proliferates to eventually form the scar after the larval emergence, as it has been classically recognized (Marcato, 1990). Concerning the macrophages present in the lesions, all of them were immunolabelled with antibodies against lysozyme and C68 marker. The latter antigen is a glycoprotein associated with lysosomes and phagocytic function (Ackermann et al., 1994; Ramprasad et al., 1996). The positive immunoreaction detected with the two antibodies suggests that macrophages present in the lesions will show an activated status related to increased amounts of lysosomes (Valheim et al., 2004). Macrophages positively immunolabelled for MAC387 antibody have been considered as freshly recruited cells since they lose its expression with further differentiation (Kipar et al., 2003; Soulas et al., 2011). In this study, positive immunolabelling for MAC 387 was detected in the small amounts of macrophages seen in type 1 lesion, and in the epitheloid cells
present in type 2 forms. Interestingly, in the well-formed granulomas present the latter, freshly recruited macrophages MAC387 positive were present in the center of the granulomas, where the remnants of the parasite were located, while those at the periphery were mostly negative. This result would suggest that macrophages located in the center of the granulomas are continuously replaced by new macrophages while those that have already phagocytosed move to the periphery. T lymphocytes were present in remarkable amounts in types 2 and 3 lesions, always in relation with higher amounts of macrophages. This finding would suggest that once the larvae are dead and after antigen processing, the macrophages could present antigens (Fujiwara and Kobayashi, 2005) and mount a cellmediated immune response specific to Hypoderma antigens. The presence of T lymphocytes in the inflammatory infiltrate associated with first instar penetration has been already demonstrated (López et al., 2005). In lesions caused by third instar larvae of Przhevalskiana silenus in goats, it has been reported as a common finding the formation of pyogranulomatous abscesses in the vicinity of some live L3 of even long time after the L3 emergence (Oryan and Bahrami, 2012) that have not been found in our study. They are probably due to secondary bacterial contamination of the infested area through the orifice of the perforated skin (Oryan and Bahrami, 2012) that would have not occurred in our samples. This study has characterized the pathological response and the cells present in the inflammatory infiltrate related to second or third instar larvae of H. lineatum in the subcutaneous tissue and has shown a variability of forms associated with the parasite stage and viability, suggesting different roles of the inflammatory cells in the pathogenesis of the lesions. Acknowledgements This study was supported by the Research Project AGL-200908939 and by a grant for Consolidating and Structuring Competitive Research Groups (Xunta de Galicia CN2012/326). The authors also thank the Xunta de Galicia government for pre-doctoral grant to E. Cabanelas. References Ackermann, M.R., DeBey, B.M., Stabel, T.J., Gold, J.H., Register, K.B., Meehan, J.T., 1994. Distribution of anti-CD68 (EBM11) immunoreactivity in formalin-fixed paraffin-embedded bovine tissues. Vet. Pathol. 31, 340–348. Agostini, C., Semenzato, G., 2003. Biology and immunology of the granuloma. In: James, D.G., Zumlaed, A. (Eds.), The Granulomatous Disorders. Cambridge University Press, 1999, Cambridge UK, pp. 3–16. Arru, E., Leoni, A., Garippa, G., Rassu, A., 1985. Ipodermosi bovina: localizacione stagionale delle larve e lesion anatomo-patologiche. Atti de la Societá Italiana di Buiatria 17, 697–707. Averill, M.M., Kerkhoff, C., Bornfeldt, K.E., 2012. S100A8 and S100A9 in cardiovascular biology and disease. Arterioscler. Thromb. Vasc. Biol. 32, 223–229. Baron, R.W., Colwell, D.D., 1991. Mammalian immune responses to miasis. Parasitol. Today 7, 353–355. Beesley, W.N., 1974. Datos económicos y progresos en la erradicación de la ˜ Noticias Médico-Veterinarias 4, hipodermosis bovina en Gran Bretana. 342–355. Berkemkamp, S.D., Drummond, R.O., 1990. Hypodermosis-part I + part II. Compendium on continuing education for the practicing veterinarian, 12: 740–746 + 881–888. Boulard, C., 1969. Anatomie et histologie du tube digestif de la larve d’Hypoderma bovis (Diptères, Oestriformes). Ann. Soc. Entomol. Fr. N.S. 5, 371–387. Boulard, C., 1970. Etude préliminaire d’une collagénase brute extraite de la larve de premier stade d’Hypoderma lineatum (De Villers). C.R. Acad. Sci. Paris 270, 1349–1351. Boulard, C., 1975. Evolution des anticorps circulants chez les bovins traités contre l´ıhypodermose. Ann. Rech. Vét. 6, 143–154. Brandtzaeg, P., Dale, I., Gabrielsen, T.O., 1992. The leucocyte protein L1 (calprotectin): usefulness as an immunohistochemical marker antigen and putative biological function. Histopathol. 21, 191–196.
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Marcato, P.S. 1990. Anatomía e histología patológica especial de los mamíferos domésticos. McGraw Hill, Madrid, 2a edición. Nelson, W.A., Weintraub, J., 1972. Hypoderma lineatum (De Vill.) (Diptera: Oestridae): Invasion of the bovine skin by newly hatched larvae. J. Parasitol. 58, 614–624. Oryan, A., Bahrami, S., 2012. Pathology of natural Przhevalskiana silenus infestation in goats. Trop. Biomed. 29, 524–531. Panadero, R., López, C., Vázquez, L., Díaz, P., Pérez, A., Cabanelas, E., Morrondo, P., ˜ P., 2013. Effect of reinfestations on systemic immune responses in Díez-Banos, cattle naturally infested by Hypoderma sp. (Diptera: Oestridae). Vet. Parasitol 193, 238–244. Pruett, J.H., Kunz, S.E., 1996. Thermal requirements for Hypoderma lineatum (Diptera:Oestridae) egg development. J. Med. Entomol. 33, 976–978. Rietschel, G., 1979. Histologischereaktionen des wirtes auf den Befall durch die Dasselfliege Oestromya leporine Pall. (Diptera, Hypodermatidae). Z. Parasintenkd 60, 277–289. Ramprasad, M.P., Terpstra, V., Kondratenko, N., Quehenberger, O., Steinberg, D., 1996. Cell surface expression of mouse macrosialin and human CD68 and their role as macrophage receptors for oxidized low density lipoprotein. Proc. Natl. Acad. Sci. U. S. A. 93, 14833–14838. Soulas, C., Conerly, C., Kim, W.K., Burdo, T.H., Alvarez, X., Lackner, A.A., Williams, K.C., 2011. Recently infiltrating MAC387+ monocytes/macrophages A third macrophage population involved in SIV and HIV encephalitic lesion formation. Am. J. Pathol. 178, 2121–2135. Tassi, P., Puccini, V., Giangaspero, A., 1989. Infection by the warble fly Przhevalskiana silenus Brauer, 1858, in the Italian goats. An update up to 1988. Parassitologia 31, 239–250. Valheim, M., Sigurdardóttir, O.G., Storset, A.K., Aune, L.G., 2004. Characterization of macrophages and occurrence of T cells in intestinal lesions of subclinical paratuberculosis in goats. J. Comp. Pathol. 131, 221–232. ˜ P., Panadero, R., Vázquez, L., Dacal, V., López, C., Díaz, P., Morrondo, P., Díez-Banos, 2012. Antigen-specific antibody isotypes, lymphocyte subsets and cytokine profiles in cattle naturally infested by Hypoderma sp. (Diptera: Oestridae). Vet. Parasitol. 184, 230–237.
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Histological and immunohistochemical characterization of Hypoderma lineatum (Diptera: oestridae) warbles E. Cabanelas a,∗ , R. Panadero a , M. Fuertes b , M. Fernández b , J. Benavides b , C. López a , ˜ a , V. Pérez b A. Pérez-Creo a , P. Díaz a , P. Morrondo a , P. Díez-Banos a Departamento de Patología Animal: Sanidad Animal (Grupo INVESAGA), Facultad de Veterinaria, Universidad de Santiago de Compostela, Lugo 27002, Spain b Departamento de Sanidad Animal, Instituto de Ganadería de Monta˜ na (CSIC-ULE). Facultad de Veterinaria. Universidad de León. Campus de Vegazana, s/n 24071 León, Spain
a r t i c l e
i n f o
Article history: Received 10 March 2015 Received in revised form 12 June 2015 Accepted 15 June 2015 Keywords: Cattle-arthropoda Hypoderma Warbles Subcutaneous larvae Cellular response
a b s t r a c t Hypoderma larvae are tissue invading parasites which spend several months migrating within the host tissues before completing their development in the sub-dermal tissues of the back. Subcutaneous stages of the parasite produce an inflammatory reaction in the skin called “warbles”, as well as holes through which larvae breathe. In order to elucidate the microscopical structure of the warbles, three hides from warbled cows were collected in a slaughterhouse in Lugo (NW, Spain) between March and May 2012. A total of 60 skin samples, including warbles at different phases of development, were chosen for histopathological and immunohistochemical examination. Microscopic lesions were classified into three groups, according to the predominance and distribution of different cell populations. In warbles containing living or recently dead larvae with apparently well preserved cuticle (type 1), plasma cells were observed in high number. However, macrophages and lymphocytes were the predominant cells in granulomas (type 2) formed in relation to remnants of the dead parasite, containing or not remains of the altered cuticle. Scars (type 3) were characterized by granulation tissue. Immunohistochemistry showed that B lymphocytes and IgG+ cells were predominant in the lesions, as long as the cuticle of the larvae is intact. On the other side, CD3+ lymphocytes increased once cuticle is destroyed and a granuloma is formed. Macrophages, revealed by CD68+ , MAC387+ and lysozyme immunolabelling, were detected in all types of lesions, but they were more abundant in type 2 and scarce in scars. These cells appeared isolated around the intact larvae or forming aggregates around its remains in the granuloma. Moreover, a strong immunolabelling against MAC387 antibody was registered in the squamous epithelium covering the breathing pore. This finding may be associated with the expression of calprotectin, a molecule involved on the healing process of the skin after larvae outcome. Our results suggest the predominance of a humoral response inside the warble as long as larvae are intact. Once they are destroyed, cellular response occurred, isolating and destroying the remains of the larvae until healing process completes and scars with low numbers of inflammatory cells appear. © 2015 Published by Elsevier B.V.
1. Introduction Hypoderma larvae are obligate parasites that migrate and feed for several months in host’s tissues. Newly hatched larvae penetrate unbroken bovine skin thanks to enzymatic secretions produced by their salivary glands (Boulard, 1970). Once the penetration is com-
∗ Corresponding author. Fax: +34 982822001. E-mail address: [email protected] (E. Cabanelas).
plete, the larva begins migration in subcutaneous tissue (Nelson and Weintraub, 1972), which leads to the oesophageal submucosa for Hypoderma lineatum and epidural fat for Hypoderma bovis. After that, larvae continue their migration towards subcutaneous tissue in the back, where they turn to second and third instar larvae successively. These larvae are aerobic and breathe through a hole bored in the skin (Hadwen and Bruce, 1916). After first instar molt, the expulsion of its content triggers an inflammatory response to encapsulate second and third instar larvae with connective tissue (Berkemkamp and Drummond, 1990),
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2 Table 1 Primary antibodies used in immunohistochemistry. Specificity
Type or clone
Origin
Pretreatment
Dilution
Source
Myeloid/Histiocyte antigen (calprotectin) (Macrophages) CD68 (Macrophages) Lysozime CD79 (B cells) CD3 IgG Lambda Light Chains
MAC 387 PG-M1 Policlonal JCB117 Policlonal Policlonal
Mouse Mouse Rabbit Mouse Rabbit Rabbit
Trypsine Trypsine Trypsine Microwave Steamer Triton X-100
1:75 1:100 1:1000 1:25 1:200 1:6000
Dako Dako Dako Dako Sigma Dako
forming the characteristic swellings or subcutaneous furuncles called “warbles” (Beesley, 1974). According to Boulard (1975), fibroblasts are very active and produce lots of collagen fibers to isolate larvae, which stay in stable environment. Mature third instars exit the host and pupate within a short period of time. Larvae migrating within the host and in sub-dermal warbles provoke production losses and increased susceptibility to diseases (Drummond, 1987). Larvae are continuously exposed to host immune effector mechanisms trying to isolate and kill them. Gingrich (1980) reported that immunity mechanisms are especially active in early phases of larvae development, in which larvae mortality is higher. Nevertheless, Pruett and Kunz (1996) pointed out that larvae destruction is very intense in the last stages of development. While lesions caused by first instar larvae during penetration and migration in the host are well described (López et al., 2005; Dacal et al., 2009, 2011), not detailed descriptions on the different pathological responses associated with the presence of the larvae in the subcutaneous tissue have been carried out. Moreover, little information is also available on the cellular reactions responsible for larval isolation or destruction at sub-dermal sites. The aim of this study was to define and classify the microscopic lesions and to characterize, by using histopathology and immunohistochemistry, the cells that participate in the cutaneous reaction against subcutaneous larvae, including the response of macrophages, T cells, B cells and immunoglobulin G-producing plasma cells, during the stay of second or third instar H. lineatum in the back of the cattle.
Table 2 Evaluation of immunolabelled cell population numbers in the three histological lesions described.
1 2 3
CD3+
CD79+
CD68+
MAC387+
Lysozyme
IgG+
+ ++/+++ +
+++ + +
+ +++ +
++ +++ +
++ ++ +
++ 0 0
0: nil; +:mild; ++:moderate; +++:intense.
2. Material and methods
For IHC studies skin sections, 4 m thick, were placed onto polyL-Lysine coated slides. Endogenous peroxidase activity was blocked in deparaffinised sections by incubation with 3% hydrogen peroxide for 30 min in darkness at room temperature (RT). Rehydrated slides were rinsed twice in PBS pH 7.4. To optimize the immunoreaction, the antigen retrieval was performed using enzymatic or heat-based methods, depending on the primary antibody (Table 1). Sections were incubated with the primary antibodies diluted in PBS overnight at 4 ◦ C in a humidified chamber. After extensive washing with PBS, sections were incubated for 40 min at RT with EnVision® +/HRP solution (Dako North America Inc., Carpinteria, USA) for the appropriate monoclonal or polyclonal antibodies. After washing in PBS, antibody localization was determined using 3,3-diaminobenzidine (DAB, Sigma–Aldrich Corp.) as chromogenic substrate for peroxidase. Sections were counterstained with haematoxylin for 30 s. Evaluation of immunostaining was performed under a light microscope with final magnification of 500×. Labelled cells taking part into the lesion were examined and a qualitative classification of the number of positively immunostained cells, from nil (0) to intense (+++), was established (Table 2).
2.1. Animal samples
3. Results
The hides from three intensely warbled cows were collected in a local slaughterhouse in Lugo (NW, Spain). The outer side of the hides was examined to confirm the absence of other relevant lesions (mange, ticks, lice, etc.) and the inner side was inspected recording warbles at different stages of development. The first hide (H1) was collected in March 2012 and presented 16 viable warbles and 13 non-viable warbles; the second hide (H2) was collected in April 2012 and exhibited 4 viable warbles and 38 non-viable warbles; the last hide (H3) was obtained in May and presented 19 non-viable warbles. A total of 45 skin samples (15 from H1, 31 from H2 and 9 from H3), including warbles containing living (n = 10), dead larvae (n = 20) and scars (n = 15) were taken for histopathological and immunohistochemical (IHC) examination. Samples were fixed in 10% formaldehyde and embedded in paraffin wax using standard protocols.
3.1. Gross lesions
2.2. Histopathological and immunohistochemical examination For histopathological examination samples were processed by routine methods and sections, 4 m thick, were stained with haematoxylin and eosin (HE).
Macroscopically, 4 mm diameter breathing holes (n = 20) and 2 mm diameter scars (n = 70) (Fig. 1a) were visible in the outer side of the skin. In the inner side, warbles at different stages of development were observed in the hides examined: viable warbles (n = 20) were composed of a layer of fibrous connective tissue that creates a cavity containing the larvae and necrotic material (Fig. 1b); in non-viable warbles (n = 40), larvae seemed to be dark, crushed and dehydrated (Fig. 1c). The remaining 30 samples showed healing tissue without visible larvae remains. 3.2. Histopathology In all the examined samples, the superficial dermis showed a mild to moderate perivascular inflammatory infiltrate, composed mainly of lymphocytes, plasma cells and occasional polymorphonuclear eosinophils. In the deep dermis lesions were variable and were classified into three different groups, according to the presence and stage of preservation of the larvae, the characteristics of the inflammatory infiltrate and the predominant cells present in the lesions:
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Fig. 1. (a) Outer side of the skin of one affected cattle showing a breathing hole (continuous arrow) and a scar (discontinuous arrow). (b) Viable larva, surrounded by necrotic debris, inside a fibrous cavity. (c) Non-viable larvae in the inner side of the skin.
Type 1 lesion (18 samples): In the centre of the lesion, viable or recently dead larvae could be seen. In all of them, a well-preserved chitinized cuticle was always clearly distinguishable. When the larvae were viable, skeletal muscle, tracheal rings and intestinal cells were detected while an amorphous, eosinophilic material, surrounded by the cuticle, was seen where they were dead (Fig. 2a). Larvae were surrounded by the deposition of variable amounts of fibrin and necrotic material (Fig. 2a). Externally, a cellular infiltrate composed mainly of plasma cells (Fig. 2b), together with a moderate amount of macrophages and lower numbers of eosinophils and lymphocytes was seen infiltrating the surrounding connective tissue. A squamous layered epithelium was found covering the internal side of the breathing hole. Type 2 lesion (12 samples): In all the cases, larvae were always dead and cuticle was not visible in any case. The morphology of dead larvae varied from cases in which they hold its shape (Fig. 3a) to the presence of remnants where the only identifiable structures were fragments of the cuticle and spicules visible among necrotic material. The inflammatory infiltrate differed significantly from the former type. It was composed of numerous macrophages with abundant eosinophilic cytoplasm consistent with epithelioid cells together with some multinucleated giant cells, both foreignbody and Langhan’s types, forming clearly defined granulomas (Figs. 3 and 4). Externally, the infiltrate was surrounded by a layer composed of variable numbers of lymphocytes, sporadically forming aggregates (Fig. 4). Occasionally, small foci of mineralization were seen in groups of necrotic epithelioid cells. At the periphery, the entire lesion was demarcated by a capsule of connective tissue (Figs. 3 and 4). In some samples, no remains of the larvae could be detected. Type 3 lesion (15 samples): Perivascular mononuclear infiltrates and granulation tissue conformed by fibroblasts and neovascularization were the only lesions detected. Focal aggregates of lymphocytes with occasional macrophages between them were also observed (Fig. 5). Considering the correlation between gross lesions and histopathology, 100% of viable warbles were categorized as Type 1 lesion. All the 20 examined warbles containing non-viable
Fig. 2. Type 1 lesion. (a) Non viable larvae (note the eosinophilic, amorphous material inside the relatively well-preserved cuticle) in relation a cellular infiltrate of the dermis with fibrin and necrotic debris. HE. Bar 100 m. (b) Detail of the inflammatory infiltrate, mainly formed by plasma cells, found in the dermis. HE. Bar 50 m. Inset: Immunohistochemical labelling of plasma cells in this infiltrate. IgG IHC. Bar 25 m.
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Fig. 4. Type 2 lesion. Granuloma characterized by a central area of multinucleated giant cells, surrounded by lymphocytes forming aggregates and demarcated by a fibrous capsule. HE. Bar 100 m.
Fig. 3. Type 2 lesion. (a) Granulomatous lesions where larvae remnants, with no visible cuticle, are present in the centre and surrounded by an inflammatory infiltrate and externally demarcated by fibrous tissue. HE. Bar 200 m. (b) Detail of the granulomatous infiltrate where epithelioid and multinucleated giant cells are prominent. HE. Bar 100 m.
larvae were categorized as Type 2. Finally, 100% of healing lesions were classified as Type 3. 3.3. Immunohistochemistry 3.3.1. T lymphocytes While these cells were scarce in the infiltrate of type 1 lesions, the aggregates observed in type 2 lesions were composed mainly of CD3+ lymphocytes (Fig. 4). In type 3, CD3+ cells were very scarce. 3.3.2. B lymphocytes CD79+ lymphocytes were present in large numbers (about 60% of those forming the infiltrate) in type 1 lesions. They were scarce in the remaining types of lesion (Fig. 6). 3.3.3. Macrophages CD68+ cells were found in all the types of lesion. In type 1, isolated immunolabelled cells were detected among the inflammatory infiltrate, especially in the vicinity of the necrotic areas around the larvae. In type 2 lesions all the epithelioid and giant cells forming the granuloma showed positive immunolabelling with variable intensity. Finally, few and scattered CD68+ cells observed in type 3 lesions as well as in the superficial and deep dermis in areas unrelated to the lesion. MAC387+ cells were observed in the inflammatory infiltrate that surrounded the necrotic areas around the larvae in type 1 lesions (Fig. 6). The epithelium covering the warble opening was positive
Fig. 5. Type 3 lesion. Granulation tissue with abundance of collagen and scattered small aggregates of inflammatory cells, mainly lymphocytes and macrophages. HE. Bar 100 m.
to this antibody (Fig. 7); on the contrary, the remaining epithelium in the skin sample was negative. In type 2 lesions, most of macrophages were positive, with differences in staining intensity according to the location inside the granuloma: while epithelioid cells at the centre of the granuloma were positive, the number of immunolabelled cells was lower among the peripheric ones (Fig. 6). In type 3 lesion, MAC387+ labelled cells were barely detected. They were not seen either in the normal skin. Macrophages immunolabelled for lysozyme, with no evident variation in the intensity of the labelling, were found in all the types of lesions. They were scarce in type 3. 3.3.4. IgG+ cells IgG+ cells were numerous in type 1 lesions (Fig. 3). They were not detected in the remaining types. 4. Discussion This study has shown the existence of different microscopic lesions that are related to the stage and viability of the larvae, during back phase of hypodermosis in cattle. They are characterized by the existence of a chronic, proliferative dermatitis in the internal dermis with variations in the cell populations present in the infiltrate according to the different types of lesion. During the
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Fig. 6. Comparison between 1 and 2 type lesions of the immunohistochemical labelling (gold-brown colour) for CD3 (T lymphocytes), CD79 (B lymphocytes) and calprotectin (freshly recruited macrophages) in the inflammatory infiltrate. IHC. Bar 200 m. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
Fig. 7. Immunohistochemical labelling (gold-brown colour) of reactive epithelia cells at the larvae opening in type 1-2 lesions. Note the scattered positive cells (freshly recruited macrophages) among the inflammatory infiltrate in the dermis (left side). Calprotectin IHC. Bar 100 m. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
penetration phase, and associated with the first instar, an inflammation characterized by abundant neutrophils and eosinophils in the skin is recognized (Lello and Rosis, 2003; López et al., 2005; Oryan and Bahrami, 2012). However, in our study, inflammation has been predominantly mononuclear where neutrophils have not been recorded. Moreover, the presence of eosinophils around the larvae was reduced. This finding contrasts strongly with the high number of eosinophils observed by Nelson and Weintraub (1972) and López et al. (2005) at the entry point of the larvae and by Boulard (1975) and Arru et al. (1985) in the resting phase of the oesophageal submucosa. Probably this finding is a result of the differences in larvae activity in the skin. During the penetration stage, the first instar larvae eroded the subcutaneous tissue by the collagenolytic enzymes secreted by their salivary and intestinal glands (Madel and Nahif, 1971; Tassi et al., 1989) causing necrosis and attracting neutrophils. In the back phase, after migration, third
instar larvae are located in subcutaneous nodules where they last for four to six weeks (Beesley, 1974). Although necrosis was present in relation to the larvae, this was always mild and seen together with fibrin (type 1 lesions) with no neutrophilic infiltrate. It is likely that, at the time of sampling, large areas of necrosis caused by second and third instar larvae, eliciting neutrophilic infiltration have already disappeared. The possibility that the different larvae could secrete different substances with different chemoattractability for neutrophils or eosinophils could be also considered. Type 1 lesions are consistent with the classical warble nodules characterized by the presence of intact third instar larvae, an inflammatory infiltrate and a central pore layered by a squamous epithelium from surrounding epidermis proliferation in the internal side of the opening, similar to that described by Marcato (1990) and consistent with the respiratory pore of the warble (Hadwen and Bruce, 1916). This pore has not been detected in the other types of lesion described. That could be due to the fact that after the out coming of the third instar larvae, opening closes with scar tissue, although in some occasions the remains of the parasite may delay the process. A similar pattern of cicatrization was reported by Rietschel (1979) in the evolution of Oestromya leporine warbles; fibroblasts surround the hole left by the larvae, containing exudate and remains of the parasite. The epidermis proliferates between both tissues and the content is discharged to the outside thanks to muscular pressure and new tissue growing. A striking feature of this study was the existence of a strong positive immunolabelling for MAC387 antibody of the squamous epithelial cells in the pore, in contrast with the epithelium seen in normal epidermis. It has been reported that “reactive” epithelial cells can express high levels of calprotectin, the protein that MAC387 antibody recognizes (Brandtzaeg et al., 1992). Calprotectin has been shown to play important roles in infection, inflammation and wound healing (Dhas et al., 2012) and their expression in these particular cells would suggest that they could play a significant role in the local host response against Hypoderma larvae. Particularly, production of calprotectin has been associated with a decrease in neutrophil migration and release of cytokines (Averill et al., 2012). This fact could be the cause that could explain the
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absence of neutrophils in this type of lesion although necrosis was present, in contrast to the damage caused by first instar larvae penetration, above discussed. Future studies addressing calprotectin expression during first instar larvae migration could help to understand the role of this protein in the pathological response against this parasite. Regarding to larvae structure, the absence of mouthparts had been previously described by Boulard (1969). In this phase, larvae are sedentary and feed on the surrounding materials. Reaction around viable larvae was characterised by the predominance of plasma cells. This finding is consistent with the increase of systemic serum antibodies simultaneously to the appearance of the warbles in the back reported by Panadero et al. (2013). The progressive reduction of plasma cells in the other types of lesion together with the increase in macrophages and T lymphocytes, may be related to the exposure to different antigens released after the death of the larvae, and rupture of cuticle, or its out coming from the dermis. The high levels of B lymphocytes (CD79+ cells) were in relationship with the number of IgG+ plasma cells in the infiltrate, with the highest values in type 1 lesion. The reduction of both type of cells, plasma cells and B-lymphocytes, in types 2 and 3 lesions, may explain the decrease of serological antibody levels observed by Baron and Colwell (1991) as larvae leaves the host and tissue heals. These findings would suggest that the local inflammatory infiltrate could play an important role in the antibody production detected in sera. The regional lymph nodes were not examined, but they will probably also contribute. As plasma and B cells decreased in lesions type 2 and 3, there was an increase in the number of macrophages, to form clearly defined granulomas. Their role is probably related to the phagocytosis (Fukiwara and Kobayashi, 2005) of necrotic larval tissue remnants leading to the formation of granulomas when they are persistent and poorly degradable substances (Agostini and Semenzato, 2003). Actually, this type of lesion was always in relation to the presence of dead larvae or remnants or absence of the parasite. The fact that the most dramatic increase in macrophages was found between type 1, where cuticle was intact or with very little discontinuities, and type 2, where cuticle was absent, suggest that Hypoderma cuticle would play a role in the inflammatory response, probably containing antigens that would trigger the local infiltration of plasma cells rather than macrophages. Once the cuticle has disappeared (type 2), the inflammatory infiltrate would torn into a predominantly macrophage type, surrounding remnants of dead larvae or remains of cuticle sloughed during ecdysis that are difficult to eliminate (Ginn et al., 2007). According to previous studies (Pruett and Kunz, 1996), host acquired resistance provoked considerable larval mortality occurring predominantly after their arrival to the back. The predominance of a Th2 response, characterized by high levels of IgG1 and IL-4, at the latest stages of the infestation (Vázquez et al., 2012) could be responsible for larval destruction. Type 3 lesions are formed mainly by granulation tissue that proliferates to eventually form the scar after the larval emergence, as it has been classically recognized (Marcato, 1990). Concerning the macrophages present in the lesions, all of them were immunolabelled with antibodies against lysozyme and C68 marker. The latter antigen is a glycoprotein associated with lysosomes and phagocytic function (Ackermann et al., 1994; Ramprasad et al., 1996). The positive immunoreaction detected with the two antibodies suggests that macrophages present in the lesions will show an activated status related to increased amounts of lysosomes (Valheim et al., 2004). Macrophages positively immunolabelled for MAC387 antibody have been considered as freshly recruited cells since they lose its expression with further differentiation (Kipar et al., 2003; Soulas et al., 2011). In this study, positive immunolabelling for MAC 387 was detected in the small amounts of macrophages seen in type 1 lesion, and in the epitheloid cells
present in type 2 forms. Interestingly, in the well-formed granulomas present the latter, freshly recruited macrophages MAC387 positive were present in the center of the granulomas, where the remnants of the parasite were located, while those at the periphery were mostly negative. This result would suggest that macrophages located in the center of the granulomas are continuously replaced by new macrophages while those that have already phagocytosed move to the periphery. T lymphocytes were present in remarkable amounts in types 2 and 3 lesions, always in relation with higher amounts of macrophages. This finding would suggest that once the larvae are dead and after antigen processing, the macrophages could present antigens (Fujiwara and Kobayashi, 2005) and mount a cellmediated immune response specific to Hypoderma antigens. The presence of T lymphocytes in the inflammatory infiltrate associated with first instar penetration has been already demonstrated (López et al., 2005). In lesions caused by third instar larvae of Przhevalskiana silenus in goats, it has been reported as a common finding the formation of pyogranulomatous abscesses in the vicinity of some live L3 of even long time after the L3 emergence (Oryan and Bahrami, 2012) that have not been found in our study. They are probably due to secondary bacterial contamination of the infested area through the orifice of the perforated skin (Oryan and Bahrami, 2012) that would have not occurred in our samples. This study has characterized the pathological response and the cells present in the inflammatory infiltrate related to second or third instar larvae of H. lineatum in the subcutaneous tissue and has shown a variability of forms associated with the parasite stage and viability, suggesting different roles of the inflammatory cells in the pathogenesis of the lesions. Acknowledgements This study was supported by the Research Project AGL-200908939 and by a grant for Consolidating and Structuring Competitive Research Groups (Xunta de Galicia CN2012/326). The authors also thank the Xunta de Galicia government for pre-doctoral grant to E. Cabanelas. References Ackermann, M.R., DeBey, B.M., Stabel, T.J., Gold, J.H., Register, K.B., Meehan, J.T., 1994. Distribution of anti-CD68 (EBM11) immunoreactivity in formalin-fixed paraffin-embedded bovine tissues. Vet. Pathol. 31, 340–348. Agostini, C., Semenzato, G., 2003. Biology and immunology of the granuloma. In: James, D.G., Zumlaed, A. (Eds.), The Granulomatous Disorders. Cambridge University Press, 1999, Cambridge UK, pp. 3–16. Arru, E., Leoni, A., Garippa, G., Rassu, A., 1985. Ipodermosi bovina: localizacione stagionale delle larve e lesion anatomo-patologiche. Atti de la Societá Italiana di Buiatria 17, 697–707. Averill, M.M., Kerkhoff, C., Bornfeldt, K.E., 2012. S100A8 and S100A9 in cardiovascular biology and disease. Arterioscler. Thromb. Vasc. Biol. 32, 223–229. Baron, R.W., Colwell, D.D., 1991. Mammalian immune responses to miasis. Parasitol. Today 7, 353–355. Beesley, W.N., 1974. Datos económicos y progresos en la erradicación de la ˜ Noticias Médico-Veterinarias 4, hipodermosis bovina en Gran Bretana. 342–355. Berkemkamp, S.D., Drummond, R.O., 1990. Hypodermosis-part I + part II. Compendium on continuing education for the practicing veterinarian, 12: 740–746 + 881–888. Boulard, C., 1969. Anatomie et histologie du tube digestif de la larve d’Hypoderma bovis (Diptères, Oestriformes). Ann. Soc. Entomol. Fr. N.S. 5, 371–387. Boulard, C., 1970. Etude préliminaire d’une collagénase brute extraite de la larve de premier stade d’Hypoderma lineatum (De Villers). C.R. Acad. Sci. Paris 270, 1349–1351. Boulard, C., 1975. Evolution des anticorps circulants chez les bovins traités contre l´ıhypodermose. Ann. Rech. Vét. 6, 143–154. Brandtzaeg, P., Dale, I., Gabrielsen, T.O., 1992. The leucocyte protein L1 (calprotectin): usefulness as an immunohistochemical marker antigen and putative biological function. Histopathol. 21, 191–196.
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Please cite this article in press as: Cabanelas, E., et al., Histological and immunohistochemical characterization of Hypoderma lineatum (Diptera: oestridae) warbles. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.06.017