Temporal and spatial spread of Hypoderma actaeon infection in roe deer from peninsular Spain determined by an indirect enzyme-linked immunosorbent assay.

The host switching of Hypoderma actaeon (Diptera: Oestridae), a specific parasite of red deer (Cervus elaphus), towards roe deer (Capreolus capreolus) has been recently reported in Spain. To provide information about the temporal and spatial spreading of H. actaeon infection in roe deer, 244 serum samples from animals hunted in Spain between 2013 and 2018 were analysed by an indirect enzyme-linked immunosorbent assay. The overall seropositivity was 13.9%. Seropositivity was higher in continental (27.7%) and mountainous (12%) areas from central Spain, followed by southern-Mediterranean (11.2%) and northern-oceanic regions (3.5%). Differences were significant between central-continental and northern-oceanic regions (P = 0.003). No differences were found according to the sex and age of roe deer (P > 0.05). In 2013, all seropositive animals were concentrated in two distant areas in central and southern Spain, suggesting that the host switch could have occurred independently in both regions. Changes in the pattern of distribution of red deer and roe deer could have favoured the spreading of this myiasis towards roe deer, indicating that roe deer may become infested by H. actaeon in areas where both cervids coexist at high densities.

Modulatory effects of interferon-γ and interleukin-4 on cellular immune responses against Hypoderma lineatum antigens.

This study investigates the in vitro modulatory effects of interferon-γ (IFN-γ) and interleukin-4 (IL-4) on both proliferative bovine T cell responses and IL-10 production induced by different antigens [crude larval extract and the purified fractions hypodermin A, B and C (HyA, HyB, HyC)] obtained from first instars of Hypoderma lineatum (Diptera: Oestridae), alone or in the presence of the mitogen concanavalin A. Incubation with the different parasitic antigens resulted in significant inhibition of T cell proliferation and IL-10 production, which, in general, did not revert after the addition of IFN-γ and IL-4. In the absence of antigens, IL-4 induced significant inhibition of mitogen-induced T cell responses. Exogenous IFN-γ exhibited an inhibitory effect on cell proliferation in the presence of the purified fractions HyB and HyC. These in vitro data suggest that far from neutralizing the effects of larval antigens, the addition of IFN-γ potentiates their anti-proliferative activity; by contrast, IL-4 had no consistent effects on proliferative responses to Hypoderma. IL-4 provoked an increment of IL-10 levels in supernatants of HyB-stimulated cells. In conclusion, exogenous IFN-γ and IL-4 were unable to counteract the suppressor effects of H. lineatum antigens.

Cytokine expression in bovine PBMC cultures stimulated with Hypoderma lineatum antigens.

Hypoderma antigens are involved in host inflammation and immune response, conditioning larvae survival. In this study, peripheral blood mononuclear cell (PBMC) cultures from Hypoderma sensitized and unsensitized cattle were performed to determine the effect of H. lineatum antigens and incubation time (18, 24, 48 h) on IFN-γ, TNF-α, IL-10 and IL-4 mRNA gene expression determined by RT-qPCR. TNF-α and IL-4 gene expression were higher in Hypoderma previously sensitized PBMCs, suggesting that a mixed Th1/Th2 response may play a significant role in host defence reactions against Hypoderma exhibited by previously infested cattle. Incubation time had a significant effect on IL-10 and TNF-α gene expression, which decreased over time. Regarding to H. lineatum antigens, the crude larval extract and the purified fraction hypodermin B (HB) produced a significant reduction of the mRNA expression levels of the proinflammatory cytokine, IFN-γ; moreover, the HB had a stimulating effect on the mRNA gene expression of the anti-inflammatory cytokine IL-10, demonstrating that the parasite would modulate the host defence mechanisms by avoiding harmful immune responses that would limit its survival into the host tissues.

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

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.

Effect of reinfestations on systemic immune responses in cattle naturally infested by Hypoderma sp. (Diptera: Oestridae).

Systemic humoral and cellular immune responses were studied during natural infestations by Hypoderma lineatum in cattle at their first (G-1) and second exposure (G-2). Four out of seven animals in G-1 were palpation positive, with a mean intensity of 11.2 (12.81SD) warbles; the same proportion (4/7) presented warbles in G-2 but the intensity was 3.7 (2.21SD). The evolution of total IgG levels was characterized by a noticeable increment coinciding with the presence of warbles on the back, especially in G-2. The IgG1 isotype displayed a parallel evolution in both groups, with peak values prior to the appearance of first warbles. The IgG2 subclass followed an irregular pattern in both groups and IgM maintained low and constant levels throughout the study, mainly in G-1. CD4/CD8 ratios showed a predominance of CD4(+) throughout the infestation, principally in G-2 during the warble season. The evolution of IFN-γ in G-2 was constant, whereas in G-1 there was a gradual descent until warble emergence. The dynamics of the IL-10 differed between G-1 and G-2, although both groups showed a significant drop after the exit of the larvae that could be implicated in the termination of the inflammatory response. IL-4 and TNF-α levels did not show differences between groups. Our results suggest that the resistance mechanisms would become more apparent at the latest stages of the infestation by Hypoderma, supporting the hypothesis that considerable larval destruction in sensitized animals might take place after their arrival to the back.