231Vet. Res. 34 (2003) 231–241© INRA, EDP Sciences, 2003DOI: 10.1051/vetres:2002070

Original article

Cellular and humoral local immune responsesin sheep experimentally infected with Oestrus ovis

(Diptera: Oestridae)

Guillaume TABOURET, Caroline LACROUX, Olivier ANDREOLETTI,Jean Paul BERGEAUD, Yacob HAILU-TOLOSA, Hervé HOSTE,Françoise PREVOT, Christelle GRISEZ, Philippe DORCHIES,

Philippe JACQUIET*

UMR 959 INRA/DGER de Physiopathologie Infectieuse et Parasitaire des Ruminants,École Nationale Vétérinaire, 23 chemin des Capelles, 31076 Toulouse, France

(Received 28 May 2002; accepted 12 November 2002)

Abstract – Cellular and humoral local responses were investigated following repetitive artificialOestrus ovis infections in lambs. The presence of larvae induced a huge local recruitment of eitherleucocytes (T and B lymphocytes, macrophages) or granulocytes (eosinophils, mast cells andglobule leucocytes). This cellular response was more pronounced in the ethmoid and sinus(development sites of second and third instar larvae) than in the septum or turbinates where firstinstar larvae migrate. Infected lambs produced Oestrus ovis specific IgG and IgA antibodies in theirmucus. This local humoral response was mainly directed against larval salivary gland antigens andnot against larval digestive tract antigens. Compared to the control animals, the sinusal mucosa ofinfected animals was extremely thickened and the epithelium exhibited hyperplasia, metaplasia andeosinophilic exocytosis. The possible roles of these local immune responses in the regulation ofO. ovis larvae populations in sheep are discussed.

Oestrus ovis / local immune response / lymphocyte / eosinophil / IgA and IgG immunoglobulins

1. INTRODUCTION

Grazing sheep are exposed to a widevariety of parasites such as gastrointestinalnematodes, liver flukes, cestodes andmyiasis agents. Oestrosis, caused byOestrus ovis (Linné 1758) larvae, is a com-mon myiasis of sheep affecting nasal andsinusal cavities. Female flies deposit firstinstar larvae on the sheep nostrils. These

larvae actively penetrate into the nasal cav-ities. L1 larvae migrate from the nasal sep-tum and turbinates to the ethmoid wherethe first moult to L2 larvae occurs. L2 lar-vae migrate towards the sinus where theymoult to L3 larvae. During the early phaseof L3 growth, an intense weight gainoccurs [3]. L3 larvae are then expelled bythe host sneezings onto the ground wherethey pupate [23]. Parasitic larval stages

* Correspondence and reprintsTel.: (33) 5 61 19 39 67; fax: (33) 5 61 19 39 44; e-mail: p.jacquiet@envt.fr

232 G. Tabouret et al.

might be considered as mesoparasitessince they evolve in open cavities and donot need any intra-tissular passage. Infec-tion in sheep, and to a lesser extent goats,induces a bilateral nasal discharge consec-utive to mucus hypersecretion. Necropsicobservations in naturally infected sheepshowed a strong inflammation of themucosae [4] which for a long time wasthought to be a consequence of mechanicaltrauma induced by larval spines and hooks.However, since histochemical studies ofseptal, turbinate and ethmoïd mucosaehave demonstrated that eosinophils andmast cells are more numerous in the chor-ion of naturally or experimentally infectedsheep and goats [14, 15], the inflammatoryprocess could result from the activation ofthese cellular subsets. The experimentalinfection of lambs with first instar larvaeinduces a systemic IgG response detectedby ELISA fifteen days after the L1 larvaewere deposited [5]. Nevertheless, manyelements of the local immune responseremain uncharacterised, especially therecruitment of mononuclear cells and thelocal production of immunoglobulins ininfected mucosae. The role of such a localresponse in the regulation of O. ovis popu-lations remains unknown, although epide-miological studies showed lower parasiticburdens in ewes than lambs (Dorchies,unpublished data) suggesting the progres-sive development of specific immunitywith flystrike exposure. Moreover, estab-lishment rates of O. ovis larvae afterexperimental infections are higher inimmunosupressed than in control or immu-nostimulated animals [11] suggesting thatlarval populations could be affected by thelocal immune response.

The aims of this study were (i) to inves-tigate the recruitment of lymphocytes andmacrophages to the mucosa of infectedanimals by immunohistochemistry, (ii) toconfirm the recruitment of granulocytes,especially, eosinophils, mast cells andglobule leucocytes and to extend the fieldof investigation to the sinus mucosa which

has never been studied. Furthermore, sincemature larvae are found in the sinus cavi-ties and since we suspected that theywere mostly responsible for lesions, ananatomo-pathological study of sinusmucosae was done, (iii) to assess the pres-ence of local IgG and IgA responsesagainst various larval antigens by ELISA.

2. MATERIALS AND METHODS

2.1. Infective O. ovis larvae recovery and preparation

Oestrus ovis first instar larvae (L1) werecollected from naturally infected sheep.L1 were washed in sterile PBS (pH 7.2) sixtimes and resuspended in a physiologicalsaline solution (pH 7). The viability of thelarvae was determined under a dissectingmicroscope.

2.2. Preparation of O. ovis larvae excretory secretory products (ES)

Second (L2) and third (L3) instar larvaewere collected from naturally infectedsheep heads. These larvae were washed sixtimes in PBS with Penicillin-Streptomycin5% (500 UI/mL and 500 µg/mL) and incu-bated in sterile PBS (500 µL/larvae)containing Penicillin-Streptomycin 1% for24 h at room temperature in the dark. Then,the supernatants were collected, centri-fuged (10 000 × g) and filtered through 0.8/0.2 µm sieves. Protein concentration wasdeterminated by the bicinchoninic acidassay method (Pierce, Rockford, Illinois,USA) and the ES solution was stored at–70 °C until use.

2.3. Preparation of digestive tractand salivary gland contents

The larvae of late second and thirdstages were dissected in ice cold PBS.The whole digestive tracts (DT) and the

Cellular recruitment during Oestrosis 233

salivary gland rings (SG) were collectedand centrifuged at 10 000 × g to expel theircontents (DTc and SGc). The proteinconcentration of the supernatants wasdetermined as above and these were storedfrozen (–70 °C) until use.

2.4. Animals and experimental design

Eleven 4 month old O. ovis free“Tarasconnais” lambs were randomlyallocated into two groups: 6 lambs in theinfected group and 5 in the control group.The animals were housed indoors onconcrete floors with straw bedding; thefeed was given ad libitum. Three separateinfections were made one week apart. Twobatches of 30 L1 (right and left nostrils)were administred during the first infection,2×15 L1 during the second one and2×10 L1 during the third and last one, sothat each animal received a total of 110 L1.Larvae were deposited in the nostril with aplastic pipette with a flexible tip.Necropsies were done nine weeks after thefirst infection. After splitting the heads,larvae were collected from the septal,turbinate and ethmoid surfaces as well asthe sinus cavities, were counted and theirstages (L1, L2 and L3) were identified.Immediately after death, two tissuesamples (1 cm²) were taken from each ofthe septal, turbinate, ethmoid and sinussurfaces and fixed either in 10% bufferedformaldehyde or Carnoy for histologicalstudies (see below).

2.5. Mucus sampling

Mucus samples were collected byabsorption onto filter paper strips of 4 cm2

each. PBS impregnated strips were depos-ited on the septal mucosa for five minuteson each side. The strips were then gentlyagitated in PBS for two hours at room tem-perature. The supernatants were centri-fuged and stored frozen at –70 °C untilELISA analysis.

2.6. Immunohistochemistry (IHC)and histochemistry (HC)

Immunohistochemistry: tissue sections(2 microns thick) were laid onto adhesivetreated slides (ChemMate Capillary GapMicroscope Slides, S 2024, DAKO, Trap-pes, France) and dried overnight at 56 °Cbefore being deparaffinised and rehy-drated. A haematoxylin-eosin stained sec-tion was used as a quality control. For IHC,tissue sections were autoclaved for 20 minat 121 °C in 10 mM citrate buffer (pH 6.10)and cooled for 20 min. Endogenous perox-idase was inhibited using a 1:100 dilutionof hydrogen peroxide 30% (w/w) in methylalcohol for 30 min at room temperature.Washing steps were performed with tap-water. Tissue sections were then incubatedwith 20% normal goat serum in tris salinebuffer for 20 min to block non-specificbinding sites. Anti-CD antibodies wereeither mAbs specific for human CD68(clone Ki-M6 monocyte/macrophage,Serotec, 1:150 dilution), human CD20-like(clone BLA36, B lymphocytes, Novocas-tra, Le Perray en Yvelines, France, 1:50dilution), or a rabbit anti-human CD3 anti-body (A 0452, T lymphocytes, DAKO,1:100 dilution). An anti Ki67 monoclonalantibody (R521-7, DAKO) was diluted to1:50. Incubation with a primary antibody(60 min) was followed by 30 min incuba-tion with HRP labelled secondary antibody(1:100 diluted), specific for either rabbit ormouse immunoglobulin heavy chains.Revelation was performed using diaminobenzamidine (DAB). Each step was fol-lowed by washes with 1% skimmed milk-0.05% Tween 20 in TBS. Sections werecounterstained with Mayer haematoxylin.All steps were carried out at room temper-ature. Infected and control group sampleswere assessed by counting T lymphocytes(peri-vascular, chorionic and intra-epithe-lial) chorionic B lymphocytes and macro-phages. Each sample of mucosa wasanalysed by counting 5 different opticalfields at high magnification (×400).

234 G. Tabouret et al.

Histochemistry: in the nasal and sinusmucosae, eosinophils and globule leuco-cytes were counted after carbol chromo-trope staining. An alcian blue safraninstain was used to count the mast cells.Immunoglobulin-containing cells wereeasily identified by the presence of thearchoplasm (Golgi apparatus). Cellularcounts were done as described above.After hemalun eosin staining, sections ofturbinate, septal, ethmoïd and sinus muco-sae were examined and the lesions weresemi-quantitatively described. The criteriamentioned in Table I were graded from 1 to5 as follows: 1 = minimum, 2 = light,3 = moderate, 4 = marked, 5 = severe.

2.7. Enzyme-linked immunosorbent assay (ELISA)

An indirect ELISA was used. Briefly,each well of a flat-bottomed microtitreplate (Nunclon Distr. VWR International,Strasbourg, France) was coated with100 µL of ES, DTc or SGc at 5 µg/mL insodium carbonate buffer pH 9.6 for 2 h at37 °C. The plates were washed three timesin PBS pH 7.2 containing 0.05% Tween20 (PBS-T). To minimise non-specificbinding of the antibody, the plates werethen incubated for 2 h at room temperaturewith 200 µL of PBS-T with 4% skimmedmilk powder (PBS-TSM). Subsequentlythe plates were washed three times with

PBS-T before adding 100 µL of mucuspreparation (diluted 1:25 in PBS) to eachwell for 1 h at 37 °C. After three washeswith PBS-T, 100 µL of horseradish perox-idase labelled anti-ovine IgG (1:1000 inPBS-TSM, Sigma, Lisles d’AbeauChesnes, France) or 100 µL of mouse anti-bovine/ovine IgA (1:250 in PBS-TSM,Argene, Varilhes, France) were added toeach well and incubated for 30 min at37 °C before washing a further three timesin PBS-T. Then for IgA determination,100 µL of a HRP labelled polyclonal anti-mouse IgG was added to each well (1:750in PBS-TSM, Argene) and incubated for1 h at 37 °C. Finally 100 µL of the HRPsubstrate (o-phenylenediamine 0.4 mg/mL) was added to each well and incubatedfor 30 min at 37 °C for the IgA and IgGmethod. Optical densities were read witha Dynatech plate reader (Labsystem,Wynooski, Vermont, USA) at 450 nm.

2.8. Statistical analysis

The cellular counts were processed asfollows: the results of examinations(5 optical fields / anatomical site / animal)were pooled and the means and standarddeviations within the infected and thecontrol groups were calculated. These datawere compared by the Student test using atest correction since the variances betweenthe two groups were different.

Table I. Modifications and alterations of the sinus mucosa in infected and control animals.

Tissue Alteration Infected sheep(N = 6)

Control sheep(N = 5)

Epithelium HyperplasiaMetaplasiaGoblet cells

Cilia reduction

Eosinophilic exocytosis

2.671.33

04

2.33

0.250

3.6700

Lamina propria Multifocal irregularities 1.33 0

Chorion Oedema 3 0.5

1 = minimum, 2 = light, 3 = moderate, 4 = marked, 5 = severe.The results are expressed as the means of the infected and control sheep.

Cellular recruitment during Oestrosis 235

3. RESULTS

3.1. Larval burden

No larvae were recovered from thecontrol group. The percentages of larvalestablishment varied from 6.5% to 17%(mean: 11.2% and standard deviation:3.8%) in the infected group. Larval bur-dens were not significantly differentbetween the infected animals (m = 12.3 ±2.1) (data not shown). Moreover therewere no significant differences in the pro-portions of different instar larvae betweenthe infected animals with the presence ofL1 and developing stages (L2 and L3) inall infected animals.

3.2. Mucosa alterations in infected animals

General post mortem examination didnot reveal any pathology other than oestro-sis. All six infected lambs presented amoderate mucus discharge. The macro-scopic observation of the nasal and sinusmucosae revealed strong inflammatorychanges and the sinus epithelium wasextremely thickened compared to the con-trol animals. In the epithelium of infectedanimals, Ki67 stained cells were 2.5 (p <0.05), 2.0 (p < 0.01), 8.1 (p < 0.01) and

40.0 (p < 0.001) times more numerous thanin the control lambs on the turbinate, sep-tal, ethmoïd and sinus surfaces respec-tively (Fig. 1). The epithelium of the sinusmucosa showed hyperplasia and metapla-sia as well as reductions in goblets cellsand cilia and an eosinophilic exocytosis.Multifocal irregularities and oedema wereseen in the lamina propria and the chorion,respectively (Tab. I).

3.3. Local recruitment of leucocytes

Intra-epithelial and perivascular stain-ing of CD3+ cells did not show any differ-ences between infected and controlanimals (Data not shown). In contrast, sig-nificant differences (p < 0.001) wereobserved between the chorion of twogroups especially in the ethmoïd and sinuscompartments (Fig. 2A). CD20+ cell num-bers were increased in the infected group(p < 0.001) (Fig. 2B). CD3+ and CD20+

cell numbers increased gradually from theturbinates to the sinus. Plasma cells werelocalised near secretory glands in the sub-epithelial region. Their number was mod-erate in the septal and turbinate mucosa butthe ethmoïd and sinus mucosae showed ahuge recruitment of plasma cells (Fig. 2D).CD68+ cells were significantly morenumerous in all locations of the infectedanimals, except in the turbinate region

Figure 1. Ki-67 epitope expression inturbinates, septum, ethmoïd and sinusmucosae of the control (white bars)and infected lambs (black bars).

236 G. Tabouret et al.

Fig

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Cellular recruitment during Oestrosis 237

where no difference between the infectedand control lambs was observed (Fig. 2C).The presence of O. ovis larvae also induceda high recruitment of granulocytes in alllocations: eosinophils, mast cells and glob-ule leucocytes were more numerous ininfected than in control animals. Moreo-ver, as for leucocytes, their number wasdramatically increased in the ethmoïd andsinus compartments (Figs. 3A, 3B and 3C).

3.4. Local humoral response

Infected lambs showed Oestrus ovisspecific IgG (Fig. 4A) and IgA (Fig. 4B)antibodies in their mucus when ELISAplates were coated with excretory secre-tory products or salivary gland contents.No differences in optical densities wereobserved between the infected and controlanimals when the ELISA plates werecoated with digestive tube contents. Coat-ing with SGc resulted in higher OD valuesthan with ESP as the antigen (Figs. 4Aand 4B).

4. DISCUSSION

The larval establishment rate (11.2%)resulting from three infections with a totalof 110 L1 per lamb was comparable to thatobtained previously [22]. However a betterestablishment (39%) was observed whenrepeated infections were made with L1directly harvested ex utero of gravidO. ovis females [5]. Possibly, the L1 col-lected from fly abdomens are more viablethan those taken from naturally infectedsheep. Another explanation could be thatthe L1 collected from sheep might havealready been exposed to the complement orimmunoglobulin and therefore might bemore quickly targeted when re-implantedin a naive sheep. Larval development(assessed by the presence of L2 and L3)occurred in all infected animals.

In O. ovis infected lambs, the hyperpla-sia and metaplasia of the sinus epithelium

suggested the existence of a highly regen-erative process. This was corroborated bythe expression of the Ki67 cellular prolifer-ation marker, which was 40 times moreabundant in the sinus epithelium ofinfected compared to control lambs, and bythe ultrastructural changes observed,namely the epithelial cells were dissoci-ated, rounded and showed signs of degen-eration [8].

The expression of the transmembraneprotein CD20 is restricted to pre-B lym-phocytes in bone marrow and matureB cells; plasma cells do not express CD20[2, 12] which is why these cells were iden-tified on a morphological basis in histo-chemistry. CD68 is a glycoprotein mostlylocalised in lysosomes and specific formacrophages and cells of the mononuclearphagocytic system [7, 16]. Infection ofnasal and sinus cavities by O. ovis larvaeled to a strong mucosal cellular immuneresponse and significantly more leucocytesthan control lambs. The presence of largenumbers of macrophages suggested activeantigen presentation to recruited T lym-phocytes. CD3+, CD20+ and CD68+ cellnumbers increased gradually from the tur-binates to the sinus suggesting that T andB lymphocytes and phagocytic mononu-clear cells were essentially recruited towhere larval moults take place i.e. whereL2 and L3 larvae are found. Similar obser-vations could be made with eosinophils,mast cells and globule leucocytes. Moreo-ver, a significant eosinophilic exocytosiswas shown in the sinus cavities, possiblysuggesting that these cells were passingthrough the epithelium to reach the larvae.Similar local cellular responses have beenobserved in the skin of sheep infected withLucilia cuprina larvae [1] or in the gutmucosae of sheep infected with gastro-intestinal nematodes [13, 19]. The numer-ous plasma cells found in the O. ovisinfected animals were likely to be respon-sible for the local IgG and IgA responses,even though specific staining to character-ise the immunoglobulin isotypes was not

238 G. Tabouret et al.

Fig

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Cellular recruitment during Oestrosis 239

carried out. Larval salivary gland antigensare strongly recognised by local IgG andIgA antibodies and by circulating IgG anti-bodies [9, 21]. In contrast, mucus andserum immunoglobulins reacted weaklywith O. ovis gut soluble proteins (this studyand [21]). Recently we found that theO. ovis larval gut secretes serine proteases[20] but it is known that the proteases ofthe pathogens are frequently weaklyimmunogenic because of their structuralhomology with the host ones [10]. LocalIgG and IgA humoral responses have beenobserved in the abomasal mucosa of sheepparasitised by Haemonchus contortus orTeladorsagia circumcincta [6, 17]. What is

the role of such anti-parasitic cellular andhumoral local immune responses? Localantibodies, especially IgA, have beenclearly associated with worm stunting andreduced fecundity with Teladorsagia cir-cumincta infections in lambs [18]. InO. ovis infections, local IgG and IgA rec-ognise mainly salivary gland antigenswhose functions are not known.Conversely, serine-proteases from thedigestive tract which are essential for thenutrition and the growth of larvae [20] arepoorly recognised by these antibodies.This could explain why the immunisationof lambs with excretory-secretory productsof Oestrus ovis larvae leads only to slight

Figure 4. Mucus IgG (A) and IgA (B)antibodies (at the necropsy date).Control lambs: white bars; infectedlambs: black bars. ESP: excretory /secretory products, DTc: digestive tubecontent of L3 larvae, SGc: salivarygland content of L3 larvae.

240 G. Tabouret et al.

larval growth inhibition [5]. Eosinophilscan adhere to, and immobilise the invasiveL3 larval stage of H. contortus in in vitrocultures in the presence of specific anti-parasitic antibodies suggesting thathelminth parasites can be compromisedthrough the co-operative action of antibod-ies and eosinophils. Since exocytosis ofeosinophils and the presence of specificIgG and IgA antibodies in the sheep upperairways have been demonstrated in ourstudy, a similar mechanism could be pro-posed for the regulation of O. ovis larvae.

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