Histological and immunohistochemical characterization of Hypoderma lineatum (Diptera: oestridae) warbles

- This study has characterized for the first time the pathological response and the cells present in the inflammatory infiltrate related to second or third instar larvae of Hypoderma lineatum.
- An important variability in host immune response was observed in the different stages of parasite development, suggesting different roles of the inflammatory cells in the pathogenesis of the lesions.
- A striking feature of this study was the existence of a strong positive immunolabelling for MAC387 antibody of the squamous epithelial cells in the larvae pore. This finding was associated to the expression of calprotectin, which may play a significant role in the local immune response against Hypoderma larvae.

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.