ORIGINAL PAPER

Immunocytochemistry of Psoroptes cuniculi stained by serafrom naive and infested rabbits: preliminary results

Giacomo Rossi & Elena Donadio & Stefania Perrucci

Received: 26 September 2006 /Accepted: 21 November 2006 / Published online: 12 January 2007# Springer-Verlag 2007

Abstract Immunocytochemistry was used to identify pos-sible target antigens in the digestive system of Psoroptescuniculi. Sera from three recently acutely infested rabbits,from rabbits with a mild long lasting infestation, and from arabbit with repeated mite infestations and no longer able tomaintain a population of P. cuniculi were used to determineany antibody specificity to the mite digestive system. Thereactivity of these sera was compared with sera from threeun-infested animals. The different pool of sera targeteddifferent mite antigens; in particular, sera from the resistantrabbit and the chronically infested rabbits reacted with gutcells, faecal material and cuticle, while sera from therecently infested rabbits reacted with gut contents, faecalmaterial and cuticle of the parasites but not with gut cells.Finally, sera from un-infested rabbits did not demonstrateany specificity to P. cuniculi antigens reacting only withmite gut contents in a weak manner. These preliminary datasuggest the presence of antibodies induced in the host blood

by infection, which act against the parasite by binding toantigen at the surface of its gut.

Introduction

Psoroptic mange is a significant disease of wild andfarm animals; infested animals can lose condition anddie (Kirkwood 1986). The control of psoroptic and ofother animal manges relies largely on the use of acaricides.At the moment, several problems are associated with theuse of these drugs including toxicity for humans andanimals, potential and actual environmental damage (Halleyet al. 1993; O’ Brien 1999; Van den Broek and Huntley2003) and development of mite drug-resistance (Coles1995; Synge et al. 1995; Clark et al. 1996; Currie et al.2004). For these reasons, novel and safer control methodsare required. Among them, the possibility of an immuno-logically based control method might be explored (Smithet al. 2001; Coop et al. 2002). Effectively, nowadays, thesearch for a vaccination strategy for the control ofpsoroptic mange represents one of the main subject of thestudies on this mite genus (Nuttall et al. 2006). In previousstudies, sera from infested and resistant steers demonstrat-ed antibody specificity towards Psoroptes ovis digestivesystem (Beetham 1997), while ovine and rabbit immuno-globulins (IgG) were found immuno-localised to thesurface or cytoplasm of the gut cells of P. ovis and P.cuniculi respectively, (Pettit et al. 2000). These findingssuggest that P. ovis and P. cuniculi may be susceptible tovaccination by the gut antigen approach, a method usedsuccessfully for blood-feeding ectoparasites like Boophilusmicroplus (Willadsen et al. 1988, 1989; Wikel et al. 1996;Boué et al. 1999; de la Fuente et al. 1999; van den Broekand Huntley 2003). With the aim of establishing if sera

Parasitol Res (2007) 100:1281–1285DOI 10.1007/s00436-006-0400-z

G. RossiDipartimento di Scienze Veterinarie—Faculty of VeterinaryMedicine, University of Camerino,via Circonvallazione 93-95,62024 Matelica, MC, Italy

E. DonadioDipartimento di Anatomia, Biochimica e FisiologiaVeterinaria—Faculty of Veterinary Medicine, University of Pisa,Viale delle Piagge 2,56124 Pisa, Italy

S. Perrucci (*)Dipartimento di Patologia Animale Profilassi ed Igiene degliAlimenti—Faculty of Veterinary Medicine, University of Pisa,Viale delle Piagge 2,56124 Pisa, Italye-mail: perrucci@vet.unipi.it

from naive, acutely and chronically infested, and resistantrabbits recognise different antigens of P. cuniculi, in thepresent study, sera taken from variously infested rabbitswere compared by immnocytochemistry, performed onparaffin embedded mites to determine antibody specificitytowards P. cuniculi. As controls, pooled sera from un-infested animals were used.

Materials and methods

Mites

Mixed populations of live P. cuniculi were collected fromthe ears of three infested rabbits; mites were separated fromcrusts overnight (Perrucci et al. 2005). Approximately 50mites per rabbit were placed in 1.0 ml of Bouin’s fixative(Sigma, Poole, UK) at room temperature. The mites werewashed with distilled water until the fluid was clear, thenthey were dehydrated using an ethanol series and, after axylene wash, embedded in paraffin. At the same time, thecrusts and superficial skin layers, collected from a justdeceased infested rabbit, were fixed in 10% bufferedformalin, dehydrated and embedded in paraffin wax. Thisrabbit harboured a large number of living mites. All the 200embedded mites, contained in or isolated from thepathologic material, were sectioned forming 3-μm serialribbons, and the sections were allowed to adhere onto pre-coated microscope slides (Bio-Optica, Milan, Italy) forimmunocytochemistry protocol.

Sera

Whole blood samples (5 ml) were collected from: 1) threerecently infested rabbits with a severe acute form, 2) threerabbits with a chronic mild infestation (about 12 months),and 3) a rabbit with repeated mite infestations on which a

population of P. cuniculi no longer survived (resistantrabbit). Pooled sera from naive un-infested rabbits wereused as control sera. After centrifugation (3,000×g, 10′),samples were filtered and kept at −20°C until used.

Immunocytochemistry

After re-hydration, the sections were allowed to stand for 1 hin 10% H2O2 solution to block endogenous peroxidases.Then, non-specific antibody reactions were blocked withnormal swine serum diluted 1:10 in Tris-buffered saline(TBS) containing 1% bovine serum albumin (BSA). Thesera from infested and resistant rabbits were diluted 1:800 inTBS + BSA and employed as primary antibody. Pooled serafrom un-infested rabbits were also employed at the samedilution on control slides. After overnight incubation at 4°Cin a moist chamber, slides were rinsed four times withbuffer. Biotinylated secondary antibody (swine anti-rabbitIgG, Sigma) was added and allowed to incubate on the slidefor 45 min. The slides were rinsed and allowed to incubatewith the avidin–biotin complex (ABC Vector, Burlingame,CA) for 45′ and then, after various rinses, incubated forshort time with the substrate 3,3′-diaminobenzidine tetrahy-drochloride (DAB Vector, Burlingame, CA). The sectionswere counter-stained with Meyer haematoxylin, dehydratedand mounted in Permount (Bio-Optica).

Results

Immunocytochemistry performed on purified, Bouin’s fixedand embedded mites gave poor results because of themorphological alterations that the procedure produced onmites organs. The best results were obtained with the slideson which the mites were embedded when within theorganic material (crusts and superficial layer of the skin).For a correct evaluation of stain, a total of 100 mites, 20

Fig. 1 Schematic draw ofa coronal, and b sagittal sectionsof a P. cuniculi mite; these twoviews indicate the morphologyand the different portions of thealimentary apparatus of theparasite

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mites per pool of sera, belonging to un-infected, recentlyinfested, chronically infested rabbits and to the resistantrabbit, respectively, were microscopically analysed. InFig. 1, a schematic draw of the alimentary apparatus ofthe parasite is shown. In these sections, the serum from therecently infested rabbits with the acute form of psoroptic

mange stained only the gut contents, in a weak manner, andfaecal pellets observed among the mites in crusts or inepidermis (Fig. 2b). On the same slides, sometimes, a weakcuticular positivity was observed especially in the mouth-parts (Fig. 2b). The pooled sera from the chronicallyinfested rabbit targeted also some portions of the digestive

Fig. 2 a Immunocytochemical stain of mite incubated with sera fromuninfested rabbits: a weak unspecific chromogen reaction associatewith the cuticle is evident associated with only faint and a specificstain of gut cells (20×). b Positive immunocytochemical reactioncharacterized by P. cuniculi faecal pellets and mouthpart targeted bythe light infested rabbit serum; note the absence of internal organs staininduced by the serum (20×). c Immunocytochemical reaction of P.cuniculi ventricular cell line targeted by the chronic infested rabbitserum (20×). d particular view of ventricular cells specifically stained

by the serum from the chronic infested rabbit; note the only superficialreaction characterized by a brown margin above gut’s epithelial cells(100×). e Immunocytochemical reaction of P. cuniculi ventricular cellline targeted by the resistant rabbit serum (20×). f Particular view ofventricular cells stained by the serum from the resistant infested rabbit;a very strong and diffuse reaction is observed in the cytoplasm of allthe mite’s enterocytes (100×). Immunohistochemical stain (avidin–biotin complex), Mayer’s haematoxylin counterstain

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system; they included the ventriculum, the two ventraldiverticula and the colon (Fig. 2c and d). The oesophagusand postcolon were unstained (Fig. 2c and d). The serafrom the resistant rabbit targeted with high intensity thesame portions of the digestive system mentioned above forthe chronically infested rabbits and also the postcolon(Fig. 2e and f); at high power fields magnification, stainwas concentrated at the surface of the lumen (Fig. 2f) anddispersed throughout the adjacent gut cells. In somesections, examination of the gut cells at high magnificationshowed the chromogen to be particularly concentrated inthe inter-cellular spaces. Finally, mites incubated with serafrom un-infested rabbits demonstrate a weak unspecificchromogen reaction associate with the cuticle, particularlywhere striations were visible. Additionally, in this group,the gut contents of the mites were only faintly stained, andany specific gut cells reaction was observed (Fig. 2a).These latter results demonstrated the lack of any specificitytowards P. cuniculi antigens of pooled sera belonging to theun-infested rabbits.

Discussion

The present study demonstrated, for the first time, that thesera of chronically infested and resistant rabbits reactedstrongly with the digestive system of P. cuniculi. In herstudy, Beetham (1997), using immunocytochemistry, ob-served the presence of different antibodies to P. ovis gutantigens both in the sera from infested naive steers and inmultiply infested animals. Pettit et al. (2000) demonstratedthe presence of host IgG localised at the surface orcytoplasm of the gut lumen of P. ovis taken from infestedsheep and of P. cuniculi collected from infested rabbits,thus indicating that the mites ingest host immunoglobulin.Beetham (1997) observed that all infested animals, naiveand resistant, produced antibodies to antigens throughoutthe digestive tract. In the present study, it was observed thatsera from the naive and acutely infested rabbits recognisedonly the faecal material and the cuticle, especially of themouthparts, of the mite. On the contrary, it was observedthat the sera from chronically infested and resistant rabbitsreact with the ventriculum, the two diverticula, the colonand also with the postcolon in the case of the resistantrabbit. The observation of only a faint stain of the gutcontents, and of only a weak cuticle reaction in the absenceof gut cells stain of the mites incubated with pooled serafrom naive un-infected rabbits, indicated that P. cuniculiingested rabbit IgG, confirming results obtained in previousstudies (Pettit et al. 2000; Beetham 1997). The absence ofhost IgG detection in gut cells surface and/or cytoplasm orinter-cellular spaces observed in mites incubated with serafrom naive and acutely infested rabbits and its observation

in mites incubated with sera from chronically infested andresistant rabbits suggests the presence of IgG direct tocellular antigens of mite digestive tract only in sera fromchronically infested and resistant rabbits. These findingslead to hypothesise that high levels of circulating antibodiesdirected to mite gut surface antigens could be induced inhost by multiple or long lasting infections. Thus, thereexists the possibility to acquire a resistant status to theinfestation. In previous studies in which attempts weremade to induce immunity against Psoroptes mites, solubleantigens resulted more protective than membrane-boundantigens (Nisbet et al. 2006). Among them, Psoroptes mitesallergen “Pso o 1”, which has significant homology togroup I allergen cysteine proteinases of dust mites, isconsidered one of the possible vaccine candidates (Nisbet etal. 2006). However, gut concealed antigens of some tickspecies, such as Boophilus microplus (Willadsen et al.1988, 1989; Opbebeeck et al. 1988) and Rhipicephaluszambiensis (Fivaz et al. 1991), have the ability to elicitprotection in vaccinated animals; in addition, vaccinesderived from Bm86 (a midgut membrane-bound protein ofthe cattle tick B. microplus) are the only ectoparasitevaccines commercially available (Nuttall et al. 2006).Preliminary results obtained in the present study suggestthat gut antigens may have a possible role in host resistancealso towards P. cuniculi and encourage further studies on alarger number of animals to confirm these findings and toidentify the specific proteins, related to the gut cells, whichmay have an immunogenic role. Recently, for controllingticks, the concept of a vaccine that targets both secreted andconcealed antigens (dual-action vaccine) is considered thekey for new and more effective anti-tick vaccines (Nuttall etal. 2006). In this prospective, in our opinion both studies onsoluble and membrane-bound antigens might be coupledfor the search of an effective vaccination strategy.

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