Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch

Year: 2004

Clinical development and serological antibody responses in and rabbits experimentally infested with ovis and Psoroptes cuniculi

Siegfried, Elena

Abstract: Für die experimentelle Infizierung wurden Psoroptes ovis vom Schaf und Psoroptes cuniculi vom Kaninchen verwendet. Im Experiment I wurden Gruppen, bestehend aus 4 Schafen oder 4 Kan- inchen, mit 50-100 Milben jedes Isolates auf die Haut des Rückens (skin infestation = SI) oder in den äusseren Gehörgang (aural infestation = AI) appliziert. Beim Kaninchen verursachte die AI und SI mit P. cuniculi, sowie die AI mit P.ovis typische Ohrensymptome und Antikörperreaktionen auf das P. cuniculi-Anitgen im ELISA. SI der Kaninchen mit P.ovis induzierte weder klinische Symptome noch An- tikörperreaktionen, noch konnten Milben reisoliert werden. Beim Schaf führte SI mit P.ovis zu Räude, während AI zu keinen klinischen Symptomen führte und keine Milben reisoliert werden konnten. In diesen beiden Tiergruppen zeigt der ELISA erhöhte und vergleichbare Werte. Nach SI und AI mit P.cuniculi bei den Schafen konnten weder klinische Symptome erzeugt werden, noch konnten Milben reisoliert wer- den. Trotzdem konnten geringe spezifische Antikörperreaktionen nachgewiesen werden. Im Experiment II wurden klinischer Verlauf und Antikörperreaktionen auf eine P. ovis Infektion an immunologisch naiven Tieren, sowie an Schafen die bereits einmal mit P. ovis und P. cuniculi befallen waren, beobachtet. Bei letzteren zeigte sich schon zwei Tage nach Reinfektion erste klinische Symptome und somit 3 Tage früher als bei den erstbefallenen Tieren. Demnach konnten keine offensichtlichen Unterschiede der klinischen Entwicklung in diesen drei Tiergruppen beobachtet werden. Die Resultate dieser Studie dokumentieren antigenetische Kreuzreaktionen zweier morphologisch und genetisch unterscheidbarer Psoroptes Spezies, aber sie dokumentieren auch Unterschiede in ihrem biologischen Verhalten und der Virulenz, welche beide epidemiologisch und taxonomisch wichtig sind. Psoroptes ovis of sheep origin, and Psoroptes cuniculi of rabbit origin were used in experimental infestations. In experiment I, groups of four rabbits and four sheep were infested with 50–100 of each isolate on the skin of the back (skin infestation, SI) or in the external auditory canal (aural infestation, AI). In rabbits, SI and AI with P. cuniculi and AI with P. ovis induced in all typical ear lesions and pronounced antibody reactions to P. cuniculi antigens in ELISA. After SI of rabbits with P. ovis no clinical signs were detected, no mites could be reisolated and no specific antibodies were detected. In sheep, P. ovis SI induced mange whereas AI did notinduce typical clinical signs and mites could not be reisolated. In both these groups, ELISA revealed pronounced and comparable specific antibody reactions. After SI and AI with P. cuniculi no clinical symptoms were observed and no mites could be reisolated. Nevertheless, low levels of specific antibody were detected. In experiment II, clinical progression and antibody reactions to P. ovis SI in naive sheep were compared with sheep previously exposed to P. ovis or P. cuniculi. In both preexposed groups of animals, clinical signs appeared within 2 days after challenge infestation and three days earlier than in primarily infested sheep. Subsequently, no obvious difference in the clinical progression was observed be- tween the three groups of animals. The results of this study document antigenetic crossreactivity of the two morphologically and genetically distinguishable Psoroptes species but differences in their biological behaviour and virulence which both are of epidemiological and taxonomic relevance. Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-163207 Dissertation Published Version

Originally published at: Siegfried, Elena. Clinical development and serological antibody responses in sheep and rabbits exper- imentally infested with Psoroptes ovis and Psoroptes cuniculi. 2004, University of Zurich, Vetsuisse Faculty.

2

Parasitologisches Institut der Vetsuisse-Fakultät Universität Zürich

Direktor: Peter Deplazes

Clinical development and serological antibody responses in sheep and rabbits experimentally infested with Psoroptes ovis and Psoroptes cuniculi

INAUGURAL-DISSERTATION

zur Erlangung der Doktorwürde der Vetsuisse-Fakultät Universität Zürich

vorgelegt von

Elena Siegfried

Tierärztin von Zofingen, AG

genehmigt auf Antrag von

Prof. Dr. Peter Deplazes, Referent Prof. Dr. Alfred Metzler, Korreferent

Zürich 2004

Veterinary Parasitology 124 (2004) 109–124 www.elsevier.com/locate/vetpar

Clinical development and serological antibody responses in sheep and rabbits experimentally infested with Psoroptes ovis and Psoroptes cuniculi

E. Siegfried, H. Ochs, P. Deplazes*

Institute of Parasitology, VetSuisse Faculty, University of Zurich, Winterthurerstrasse 266a, CH-805 7 Zurich, Switzerland

Received 13 January 2004; received in revised form 24 May 2004; accepted 4 June 2004

Abstract

Psoroptes ovis of sheep origin, and Psoroptes cuniculi of rabbit origin were used in experimental infestations. In experiment I, groups of four rabbits and four sheep were infested with 50–100 mites of each isolate on the skin of the back (skin infestation, SI) or in the external auditory canal (aural infestation, AI). In rabbits, SI and AI with P. cuniculi and AI with P. ovis induced in all animals typical ear lesions and pronounced antibody reactions to P. cuniculi antigens in ELISA. After SI of rabbits with P. ovis no clinical signs were detected, no mites could be reisolated and no specific antibodies were detected. In sheep, P. ovis SI induced mange whereas AI did not induce typical clinical signs and mites could not be reisolated. In both these animal groups, ELISA revealed pronounced and comparable specific antibody reactions. After SI and AI with P. cuniculi no clinical symptoms were observed and no mites could be reisolated. Nevertheless, low levels of specific antibody were detected. In experiment II, clinical progression and antibody reactions to P. ovis SI in naive sheep were compared with sheep previously exposed to P. ovis or P. cuniculi. In both pre- exposed groups of animals, clinical signs appeared within 2 days after challenge infestation and three days earlier than in primarily infested sheep. Subsequently, no obvious difference in the clinical progression was observed between the three groups of animals. The results of this study document antigenetic crossreactivity of the two morphologically and genetically distinguishable Psoroptes

* Corresponding author. Tel.: +41 1 635 8501; fax: +41 1 635 8907. E-mail address: [email protected] (P. Deplazes).

0304-4017/$ – see front matter # 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2004.06.014 110 E. Siegfried et al. / Veterinary Parasitology 124 (2004) 109–124 species but differences in their biological behaviour and virulence which both are of epidemiological and taxonomic relevance. # 2004 Elsevier B.V. All rights reserved.

Keywords: Mange; Sheep; Arthopoda; Rabbit; Isolates; Virulence; ELISA; Psoroptes spp.

1. Introduction

Psoroptic mites are cosmopolitan, non-burrowing, obligate ectoparasites, which may cause extensive pyodermatitis in the ear or on the body surface of sheep, goats, , horses and rabbits as well as wild ungulates. The taxonomic classification (host specificity and morphological variations) of the genus Psoroptes has been controversial for many years. Sweatman (1958) introduced a widely used taxonomic key based upon morphometric data of the outer opisthosomal setae (OOSL) of male mites, host species identity, and location of mites on the host. Consequently, species infesting the ears of rabbits was designated Psoroptes cuniculi, and species found on the body surface of sheep Psoroptes ovis. Subsequent investigators employing morphometric analysis of psoroptic mites, however, experienced difficulties in applying Sweatman’s taxonomic key (Boyce et al., 1990; Wright et al., 1984; Bates and Sayers, 2003). Transmission of psoroptic mites between different host species remains contentious (Shilston, 1915; Kirkwood, 1985). Experimental transmission of Psoroptes mites was achieved from the skin of cattle and wild sheep to the ears of rabbits (Meleney, 1967; DeLoach and Wright, 1981). However, when susceptible sheep were housed with infested goats or calves, there was no evidence of natural transmission between these hosts (O’Brien et al., 1994). Furthermore, interbreeding between P. ovis and P. cuniculi resulting in fertile off- spring as far as the F3 generation has been demonstrated by Wright et al. (1983). Accordingly, it was suggested that the ear of rabbit, P. cuniculi andthesheepscab mite, P. ovis are phenotypic and genetic variants of the same species (Bates, 1999; Zahler et al., 2000). Psoroptic sheep scab is of economic importance and a notifiable infestation in many countries including Switzerland, where the incidence of this disease has increased over the last years despite compulsory control measures (Falconi et al., 2002). Occasionally, aural P. cuniculi infestations of rabbits (Ochs et al., 1999) but also of goats and sheep have been observed in Switzerland (Ochs, unpublished data). A genetic study of Psoroptes isolates originating from sheep (n = 7) or from rabbits (n = 8) from the same region revealed a uniform single nucleotide difference in the ITS-2 (internal transcribed spacer 2) of rDNA between these isolates which correlated with morphometric differences of the OOSL (Ochs et al., 1999). These data with isolates collected in the same region strongly indicate that P. cuniculi and P. ovis are epidemiologically separated, but no information is available about immunological reactions of sheep to P. cuniculi infestations. Recent experimental studies have illustrated some protective effects in sheep after challenge infestation with P. ovis (Bates, 2000; Smith et al., 2002). van den Broek et al. (2000) and Smith et al. (2001) observed elevation in P. ovis antigen-specific IgE titres and E. Siegfried et al. / Veterinary Parasitology 124 (2004) 109–124 111 reduction of lesional areas in challenged as compared to primarily infested animals. Furthermore, Stromberg and Fisher (1986) reported on an early appearance of extensive dermatitis but with slow progression in cattle which had previously been exposed to P. ovis, comparable to an immediate hypersensitivity reaction. In the present study we performed experimental cross-infestation of sheep and rabbits with P. cuniculi and P. ovis to elucidate host specificity, behaviour of the mites on the different hosts, clinical manifestations and immune responses (IgG) after priming and challenge infestations.

2. Material and methods

2.1. Animals

Twenty New Zealand White SPF-laboratory female rabbits, and 20 Swiss White Alpine sheep (10 castrated males and 10 females), all animals between one and two years of age, were used in the study. All experimental animals were born and raised in isolated groups indoors. No animals had previous exposure to either P. ovis nor P. cuniculi. Sheep were fed on a diet consisting of hay, and rabbits were fed with pellets. The experiments were approved by the animal experiment commission in accordance with the Swiss legislation on animal welfare. One week before experimental infestation, all animals were tested serologically for Psoroptes antibodies.

2.2. Psoroptes-isolates

The P. ovis isolate originating from skin samples of sheep (Central Veterinary Laboratory, New Haw, Addlestone, Surerey, UK) and raised on cattle, was generously provided by Novartis Animal Health (St. Aubin, Switzterland). The P. cuniculi isolate, originating from rabbit ears in Switzerland, was propagated on rabbits (Ochs et al., 1999).

2.3. Infestation and monitoring

Primary infestations were carried out with two applications of mites, four weeks apart in the first experiment and with one application in the second experiment. Skin infestations (SI) of sheep or rabbits were performed by placing 25–50 active mites directly on the skin between the shoulder blades and additional 25–50 mites on the croup. Aural infestations (AT) were done by placing 25–50 mites deeply in each external auditory canal, which then was 100 closed with cotton for two days. Animal behaviour and skin irritations were recorded daily. Progression of ear scabs in rabbits was scored semi-quantitatively. Briefly, small skin lesions, only identifiable with an otoscope, were recorded with one cross; skin lesions involving 1/4 of the auricle with two crosses; skin lesions involving 1/3 to 1/2 of the auricle with three crosses; a completely occluded auricle with crusts and puss with four crosses. In sheep, the area (cm2) of skin lesions and discoloured areas on the body were recorded. 112 E. Siegfried et al. / Veterinary Parasitology 124 (2004) 109–124

2.4. Experimental design

In experiment I, sheep and rabbits were divided into five groups of four animals. Animals of four groups were experimentally infested (twice within one month) and animals of one group remained as uninfested controls. Rabbits and sheep were infested aurally (AI) or on the skin (SI) with P. ovis or P. cuniculi and the animals were monitored for clinical manifestations and specific antibody reactions. Experiment II started 24 weeks after the infestations of experiment I and was carried out to assess clinical progression and antibody reactions of sheep previously exposed to P. ovis or P. cuniculi after challenge infestation with P. ovis. Sheep were selected from experiment I and were regrouped into three groups: Groups one and two included six sheep each exposed either to P. ovis (two without and one with moderate clinical signs from AI and three animals from SI with mange) or to P.cuniculi (all without clinical signs, three from SI and three from AI), respectively. Group three was composed of four Psoroptes-free, naive sheep, which served as controls. All three groups were infested simultaneously with P. ovis (SI). Animals were monitored clinically and serologically for 6 weeks.

2.5. ELISA

For serological testing blood samples were collected weekly from the jugular vein of sheep and the ear arteries of rabbits. ELISA with sheep sera was performed as described (Ochs et al., 2001). Rabbit sera were tested for specific IgG against P. cuniculi crude extract prepared as described (Boyce et al., 1991). P. cuniculi extract at 5 mg/ml in carbonate buffer (NaHCO3 70 mM, Na2CO3 30 mM, NaN3 0.2 mg/ml; pH 9.6) was directly coated to 96-well ELISA plates (Nunc Maxisorb); 100 ml/well were incubated overnight at 4 8C. As a secondary antibody, goat anti-rabbit IgG (H + L) conjugated to alkaline phosphatase (SigmaNr. A- 7778, diluted 1:1000 with PBS–hemoglobin–Tween) (NaCl 138 mM, Na2HPO4 8 mM, KC1 2.5 mM, KH2PO4 1.5 mM, MgCl2 1 mM, NaN3 0.2 mg/ml, bovine hemoglobin 0.5 mg/ml, Tween 20 (Fluka) 0.3% (v/v); pH 7.2) was used. All other steps were carried out as previously described (Ochs et al., 2001). The cut-off levels were determined as the mean optical densities (OD) ELISA values plus three standard deviations (S.D.) of the ELISA values of 20 rabbit or 20 sheep sera, respectively, taken one week before experimental infestation. The serological results are expressed as antibody units (AU), which were calculated as percentages of the OD of a positive reference serum minus the cut-off value (Ochs et al., 2001).

2.6. Examination of skin scrapings and ear swabs

Skin scrapings and ear swabs were collected 12 weeks post-infestation (p.i.). In sheep, supplementary to the ear swabs, each ear was flushed with approximately 20 ml prewarmed (37 8C) water to examine the external auditory canal for live mites. Skin debris were digested in 10% (w/v) potassium hydroxide (KOH) for at least four hours at room temperature and examined in petri dishes using a stereomicroscope with transillumination (50Â). Swabs and flushing samples were examined for live mites using a stereomicroscope E. Siegfried et al. / Veterinary Parasitology 124 (2004) 109–124 113 with overhead lighting (25Â). Detected mites were transferred to microscopic slides for morphological differentiation (l00Â) of larvae, nymphs and adults.

2.7. Identification of mites

For morphological characterisation, the OOSL of the adult male mites was measured microscopically (200Â) using a digital picture analysis system (Quantimet 500, Leica, Glattbrug, Switzerland). Mites originating from the skin scrapings and ear swabs were subjected to PCR as previously described (Ochs et al., 1999).

3. Results

3.1. Experiment I

In rabbits, the appearance of localized, but characteristically extensive, exudative dermatitis in the ears with accumulation of serous material forming a dense crust, was first detected three to six weeks p.i. (Table 1). These clinical signs were induced by aural (AI) and skin infestation (SI) with P. cuniculi and also by AI with P. ovis. No clinical signs were observed in rabbits after SI with P. ovis and no mites could be reisolated. Six weeks p.i., a more pronounced scab formation in the ears was induced by P. ovis AI as compared to P. cuniculi infestations. Twelve weeks p.i., clinical manifestations of rabbits were most pronounced with P. cuniculi SI and all four rabbits in this group showed heavyear skin crusts and pus in the external auditory canal. At the same time, profound clinical manifestations of psoroptosis were also observed in rabbits with P. ovis AI and less pronounced ear mange was induced by P. cuniculi AI (Table 1). At twelve weeks p.i., ear swabs taken from symptomatic animals of any group (no mites nor scab could be identified on the body of any rabbit) contained all stages including eggs, larvae, nymphs and adults. Within these three groups, morphological differentiation of male psoroptic mites revealed no significant difference (P > 0.05) in the OOSL between reisolated mites and the isolates used for initial infestation (Fig. 1). Furthermore, PCR and sequence analyses of these isolates confirmed the morphological results (Table 1). However, morphometric and molecular differences between isolates of P. ovis and P. cuniculi were demonstrated. The serological specific antibody reactions of experimentally infested rabbits are shown in Fig. 2. Because of the homogeneity of the antibody reactions within the rabbit groups (n =4,S.D.< 10.3 AU), the mean AU values only are presented. Aural infestation with P. cuniculi or P. ovis in rabbits produced comparable specific antibody reactions during the whole experiment with no significant differences (P > 0.05). Skin infestation with P. ovis did not induce specific antibody reaction. However, the mean antibody units of rabbits with P. cuniculi SI were significantly lower (P < 0.05) only at weeks one and two p.i., compared to P. cuniculi AI. The uninfected control group (data not shown) remained clinically and serologically negative during the experiment. Sheep with P. ovis SI exhibited abnormal behaviour including restlessness, rubbing, and biting at the flanks starting 9–12 days p.i. These activities were associated with discoloured areas (white areas of the fleece without irritation of the skin) of about 100 cm2 at the site of 114 E. Siegfried et al. / Veterinary Parasitology 124 (2004) 109–124 b Species confirmation P. ovis P. cuniculi P. cuniculi ). Ochs et al., 1999 c c c Numbers of rabbits with alive mites reisolated mites P. cuniculi or ) a P. ovis 100 – No. of rabbits withinfested clinical rabbits signs/no. (lesion of classification 6 weeks p.i. 12 weeks p.i. 12 weeks p.i. Number of infested rabbits; time p.i. of first clinical appearance 1; 4 weeks 1; 5 weeks 1; 6 weeks SI 2; 4 weeks 4/4 (+, ++) 4/4 (+++, ++++) 4, all stages AI 4; 4 weeks 4/4 (+, ++) 4/4 (+, ++) 4, all stages AI 3; 3 weeks 4/4 (++, +++) 4/4 (++, +++) 4, all stages SI 0 0/4 0/4 0 – Species characterisation was performed by measuring the OOSL of the adult male mites and by PCR as previously described ( (+) Small lesions only identifiable with an otoscope, (++) lesions involving 1/4 of the auricle, (+++) lesions involvingEggs, 1/3 larvae, to 1/2 nymphs of and the auricle, adults. (++++) ear completely a b c Species and location of infestation Table 1 Clinical signs of rabbits with experimental skin (SI) or aural (AI) infestations with 50 occluded with skin crusts and pus. The mean score for skin lesions over the entire group is described. P. ovis, P. cuniculi, P. ovis, P. cuniculi, Uninfected 0 0/4 0/4 0 – E. Siegfried et al. / Veterinary Parasitology 124 (2004) 109–124 115

Fig. 1. Box plot of the outer opisthosomal setae lenghts (OOSL) from initial P.ovis (A) and P.cuniculi (D) isolates and from mites (B, C, E, F) reisolated 12 weeks p.i. (n gives number of male mites investigated). Isolates from skin of sheep after skin infestation (SI) with P. ovis (B), isolates from ears of rabbits after aural infestation (AI) with P. ovis(C), isolates from ears of rabbits after SI with P. cuniculi (E), isolates from ears of rabbits after AI with P. cuniculi (F). The mean values and the standard deviations (S.D.) of the groups were calculated. OOSL lenghts of A, B and C differed significantly from D, E, F (P < 0.05, x2-test). initial inoculation of mites (Table 2). Abnormal behaviour intensified and discoloured areas increased until twelve weeks p.i. Sheep with P. cuniculi AI and SI and three of four sheep with P. ovis AI did not show any clinical manifestations during the whole experiment. However, sheep no. 5, which initially was infested aurally with P. ovis, exhibited three small discoloured non-pruritic areas on the croup that lasted ten days. Twelve weeks p.i., ear swabs were taken from all sheep and ears were flushed (Table 2) but no mites could be retrieved. In symptomatic sheep, skin scrapings were taken at the edge of lesion sites. Morphological differentiation of male psoroptic mites from skin samples (Fig. 1) revealed no significant difference (P > 0.05) in the OOSL between the P. ovis isolate used for initial infestation and reisolated mites. Furthermore, PCR results confirmed morphological data (Table 2). Psoroptes specific antibody reactions (Fig. 3) showed more individual variation within the groups of sheep as compared to the rabbits. Sheep with P. ovis AI without clinical manifestation showed comparable immune response to P. ovis SI with clinical appearance. 116 E. Siegfried et al. / Veterinary Parasitology 124 (2004) 109–124

Fig. 2. Serological follow-up by ELISA for specific IgG against P. cuniculi crude antigens in rabbits experi- mentally infested with 50–100 live Psoroptes ovis or P. cuniculi mites (aural infestations AI, skin infestations SI). Antibody units were calculated as percentages of the OD of positive reference serum minus the cut-off value (OD value + 3 S.D. of 20 uninfected rabbits). Mean values of antibody units (n = 4, S.D. < 10.3) are indicated. * and ** significant difference in values (P < 0.05, x2-test).

There was a strong and persistent immune response of two sheep (no. 6 and 10) with P. ovis SI that also presented severe clinical mange (Table 2). ELISA values of the other two sheep (no. 7 and 8) of the same group declined at 6 and 9 weeks p.i., and these two animals presented moderate clinical symptoms. Low specific antibody responses were observed in 3 of the 4 sheep with P. cuniculi AI (no. 11, 12 and 13) starting between one and two weeks after the second infestation. P. cuniculi SI only induced a low specific antibody response in one of four sheep (no. 20) three weeks p.i. (Fig. 3) but without clinical symptoms. No clinical signs were observed in any animal of the uninfected control group and all these animals presented no specific antibody reaction throughout the experiment.

3.2. Experiment II

After P. ovis challenge infestation of sheep, previously exposed to P. ovis or P. cuniculi, a continuous progression of clinical signs over 21 days was observed (Table 3), beginning with scratching and rubbing one and two days p.i. Discoloured areas on the croup, where mites had been placed, developed five to seven days later. Within three weeks p.i., these lesions developed into extensive exudative dermatitis with massive development of 0.3– 0.5 cm thick crusts or scabs and slight petechiation. Primarily infested sheep as controls presented clinical sings 5–7 days p.i. Clinical manifestations were similar to the previously exposed groups. However, there was an E. Siegfried et al. / Veterinary Parasitology 124 (2004) 109–124 117 a Species confirmation P. ovis ). b Ochs et al., 1999 Numbers of sheep with alive mites reisolated 2, all stages ) 2 mites c ) 2 –625 cm P. cuniculi during 10 days 0 – or ) with oedema c 2 ) 2 ) and on the lateral 2 P. ovis No. 7 and 8, (225–400 cm No. 10, skin lesions, (400 shoulder (625 cm No. 6, skin lesions(729–900 on cm the croup c ) 2 4/4, scratching and rubbing (225 cm c ) 2 No. of sheep with(lesion clinical size signs/no. on of the infested body) sheep l0 days p.i. 4 weeks p.i. 12 weeks p.i. 12 weeks p.i. rubbing (100 cm AISI 0/4 0/4 0/4 0/4 0/4 0/4 0 0 – – , AI 0/4 0/4 No. 5, (9–25 cm , SI 4/4, scratching and Eggs, larvae, nymphs and adults. Species characterisation was performed by measuring the OOSLDiscoloured of areas the of adult the male fleece mites = and white by areas PCR but as without previously irritation describe of ( skin. a b c Species and location of infestation Table 2 Clinical signs of sheep with experimental skin (SI) or aural (AI) infestations with 50–100 P. ovis P. ovis P. cuniculi, P. cuniculi, Uninfected 0/4 0/4 0/4 0 – 118 E. Siegfried et al. / Veterinary Parasitology 124 (2004) 109–124 P. 100 live skin infestation. Antibody units were P. cuniculi crude antigens after experimental infestation with 50– aural infestation, (d) P. cuniculi P. cuniculi skin infestation, (c) P. ovis = 20) by ELISA for specific IgG against n aural infestation, (b) P. ovis ELISA. mites, (a) Psoroptes- P. cuniculi and ovis Fig. 3. Serological follow-up of individual sheep ( calculated as percentages of the ODreaction of positive in reference the serum minus the cut-off value (mean OD value + 3 S.D. of 20 uninfected sheep). Antibody units indicate specific E. Siegfried et al. / Veterinary Parasitology 124 (2004) 109–124 119 extensive extensive extensive 2 2 2 6/6; 625–900 cm 6/6; 625–900 cm 4/4; 400–729 cm dermatitis, wool loss and petechiae. Restless behaviour dermatitis, wool loss and petechiae. Restless behaviour dermatitis and wool loss. Restless behaviour . 2 area with area with 100 cm 2 2 as compared to the clinical outcome of primarily infested P.ovis 6/6; 225–400 cm 6/6; 225–400 cm skin lesions and woolRestless loss. behaviour skin lesions and woolRestless loss. behaviour 4/4; pruritus, 25– Restless behaviour ) 2 , , a a 2 2 after challenge infestation with a 2 P.cuniculi, and restlessness restlessness 25 cm P.ovis 2 days p.i. 5–7 days p.i. 14 days p.i. 21–33 days p.i. pre-exposed 6/6; pruritus 6/6; pruritus, 25–100 cm pre-exposed 6/6; pruritus 6/6; pruritus, 25–100 cm Discoloured, white wool areas without obvious irritation of skin. a Table 3 Progression of clinical signs of sheep, pre-exposed to sheep Group description Numbers of sheep with clinical signs, lesion size (cm P. ovis P. cuniculi Primary infestation 0/4 3/4; pruritus, 1/4; pruritus, 120 E. Siegfried et al. / Veterinary Parasitology 124 (2004) 109–124 = 6), (b) n ( ELISA. P. ovis Psoroptes- = 4). Antibody units indicate specific reaction in the n crude antigens upon first or challenge exposure of (a) sheep pre-exposed to infestation ( P. ovis P. cuniculi = 6), (c) sheep with primary n ( P. cuniculi sheep pre-exposed to Fig. 4. Serological follow-up by ELISA for specific IgG against E. Siegfried et al. / Veterinary Parasitology 124 (2004) 109–124 121 absence of petechiae at the end of the experiment. Between 21 days p.i. and the end of the experiment, all infested sheep became increasingly weakened (Table 3). All six sheep exposed to P. cuniculi and five of the sheep exposed to P. ovis with low specific antibody reactions (0-33 AU) at the beginning of the experiment displayed strongly increased specific antibody reactions after challenge infestations. The sheep with initial high antibody units persistently remained at high ELISA levels (Fig. 4). At the end of the experiment individual specific antibody reactions of both exposed groups, showed a more homogenous range of antibody units (60–102 AU), compared with primarily infested animals with a range of AU between 14 and 90.

4. Discussion

Two genetically and morphologically distinct Psoroptes isolates, P. ovis of sheep origin and P. cuniculi of rabbit origin, were used for experimental infestations of sheep and rabbits. At the end of the experiment, all mites recovered corresponded morphologically and genetically to the isolates initially used to infest the respective animals. In addition, none of the control rabbits and sheep became infected. Therefore, cross contamination of the animals with mites during the experiment can be excluded. In the first experiment host specificity and local behaviour on body environments typically preferred by the isolates, the croup and shoulder part for P. ovis and the aural environment for P. cuniculi, were investigated. Sheep with P. ovis SI developed typical signs of mange on the back. Intensity of clinical signs of these animals were associated with high specific antibody levels in the ELISA. Correlation between presentation of clinical mange and ELISA values has been presented earlier (Ochs et al., 2001). Surprisingly, strong specific antibody responses in all four sheep with P.ovis AI lasted until the end of the experiment without clinical manifestation. The long lasting immune response strongly indicates that the mites survived for some time on these animals, although no mites could be isolated 12 weeks p.i. This observation also supports the hypothesis that sheep with positive antibody reactions but without clinical signs may be latent carrier of mites. Bates (1991) and Morgan (1991) described cases of otocariasis in the ears of sheep naturally infested with Psoroptes mites, which were not further characterised. The suggestion of Bates (1996) that psoroptic sheep ear mites might be classical scab mites adapted to an aural environment could not be confirmed in our study as no P. ovis mites were identified in the ears of sheep at the end of the experiment. The P. cuniculi isolate did not establish itself on sheep in our experiment, neither in the ears nor on the back. No clinical sings and only low specific IgG reactions were observed. These findings are in accordance with those reported by Shilston (1915) and Kirkwood (1985) that P. cuniculi originating from rabbits does not survive on sheep but caused only transient disease on sheep and then died off. Recently, however, in three unrelated flocks of sheep in Switzerland, mites which were genetically identified as P. cuniculi (Ochs et al., 1999) were isolated from the ears of naturally infested, Psoroptes-seropositive sheep (Ochs, unpublished data). In the present study, four of eight sheep infested with P. cuniculi exhibited at least once positive specific antibody reactions in the Psoroptes-ELISA during the experiment. 122 E. Siegfried et al. / Veterinary Parasitology 124 (2004) 109–124

The converse part of the experiment, namely the infestation of rabbits with P. ovis, demonstrated that this isolate, when placed directly in the external auditory canal, can survive, reproduce and induce ear scab as well as a long lasting immune response. Appearance of clinical signs in rabbits was similar to P.cuniculi infestation. Accordingly, Kirkwood (1985) reported that psoroptic mites originating from sheep could be estab- lished in rabbit ears and transferred back and forth between the hosts for three generations. However, in our study, no mites could be detected in the ears of rabbits with P. ovis SI. This strongly indicates that this species is not able to migrate on rabbit skin. Therefore, we conclude that rabbits are of only marginal epidemiological importance as a reservoir of P. ovis. This observation further supports the conclusions of Kirkwood (1985) that there is no reservoir of sheep scab mites other than sheep and cattle. In contrast, migration of mites was observed after P. c un i cu li SI, as skin lesions and crusts developed only in the external auditory canal, where live mites could be isolated. Specific antibody reactions were delayed until three weeks p.i., as compared to those of rabbits infested directly into the ears. The evident differences of host specificity, virulence and behaviour on the hosts of the two isolates used in this experiment strongly suggest that these genetically and morpho- logically distinguishable species within the genus Psoroptes belong to epidemiologically separated populations. Therefore, from the medical and epidemiological point of view, the existing is of practical relevance even if these species are claimed to be conspecific(Zahler et al., 2000). Primarily infested sheep in experiments I and II showed comparable progression of sheep scab symptoms, with a subclinical phase followed by a rapid growth of the lesion as earlier described (Bates, 1997). Clinical development of pre-exposed sheep, to P. ovis or P. cuniculi differed to clinical development of naive sheep within the first week. In previously exposed sheep pruritus occurred within two days p.i., three to five days earlier than in naive sheep. Similar observations have been reported on cattle by Stromberg and Fisher (1986), who discribed early appearance of extensive dermatitis but with slow progression in cattle previously exposed to P. ovis. They concluded that, after reinfestation, a hypersensitivity to mite antigens occur, which influence the mite microenvironment and contribute to acquire resistance by controlling mite reproduction. Bates (2000) and van den Broek et al. (2000) described considerably smaller lesion in reinfested sheep, whereas our experiment revealed that lesions were quantitatively comparable in naive and pre-exposed sheep at the end of the study. However, at this point we observed only differences in the severity of skin lesions by the absence of petechia in the naive, primarily infested group. For welfare reasons, sheep had to be treated 33 days p.i. Therefore, development of skin lesions could not be further observed as described by Stromberg and Fisher (1986) for seven weeks and by van den Broek et al. (2000) for thirteen weeks. Antibody response in previously exposed sheep was more homogenous compared to the variable immune responses in naive sheep of both experiments. Surprisingly, sheep previously exposed to P. cuniculi with low or no antibody response, presented similar clinical development and serological progression as sheep previously exposed to P. ovis with clinical sings and specific antibody response. Crossreactivity of crude mite antigens of Psoroptes isolates from different hosts have been observed in Western blot studies (Boyce and Brown, 1991). These data strongly indicate that immunological reactions to subclinical E. Siegfried et al. / Veterinary Parasitology 124 (2004) 109–124 123

P. cuniculi infestations in sheep may influence early development of skin lesions and specific antibody reactions after challenge infestation with P. ovis.

Acknowledgements

The authors would like to thank Dr. P.R. Torgerson and Dr. A. Mathis (Institute of Parasitology, Universitiy of Zurich, Switzerland) and Dr. A. Metzler (Institute of Virology, Universitiy of Zurich, Switzerland) for the fruitful discussions and comments of the manuscript. We kindly like to thank the animal keepers Hanspeter Mu¨ller and Armin 320 Ru¨demann for their intensive care of the animals. This work represents the dissertation of Elena Siegfried, veterinarian.

References

Bates, P.G., 1991. Ear mites in sheep. Vet. Rec. 128, 555–555. Bates, P.G., 1996. Ovine psoroptic otoacariasis: an abattoir survey. Vet. Rec. 139, 235–236. Bates, P.G., 1997. The pathogenesis and ageing of sheep scab lesions. Part two. State Vet. J. 7, 13–16. Bates, P.G., 1999. Inter- and intra-specific variation within the genus Psoroptes (: Psoroptidae). Vet. Parasitol. 83, 201–217. Bates, P.G., 2000. Differences between primary and secondary infestations with the sheep scab mite, Psoroptes ovis. Vet. Rec. 146, 528–529. Bates, P., Sayers, R., 2003. Proceedings of the final conference of COST Action 833.Bari, Italy, 19th to 22nd September 2002, The male L4 outer opisthosomal seta (L4OOS) of psoroptic mites as a marker for speciation and virulence 84–91. Boyce, W.M., Brown, R.N., 1991. Antigenic characterization of Psoroptes spp. (Acari: Psoroptidae) mites from different hosts. J. Parasitol. 77, 675–679. Boyce, W.M., Elliott, L., Clark, R., Jessup, D., 1990. Morphometric analysis of Psoroptes spp. mites from bighorn sheep, mule deer, cattle and rabbits. J. Parasitol. 76, 823–828. Boyce, W.M., Mazet, J.A., Mellies, J., Gardner, I., Clark, R.K., Jessup, D.A., 1991. Kinetic ELISA for detection of antibodies to Psoroptes sp. (Acari: Psoroptidae) in bighorn sheep (Ovis canadensis). J. Parasitol. 77, 692–696. Deloach, J.R., Wright, F.C., 1981. Ingestionof rabbit erythrocytes containing 51Cr-labeled hemoglobin by Psoroptes spp. (Acari: Psoroptidae) that originated on cattle, mountain sheep, or rabbits. J. Med. Entomol. 18, 345–348. Falconi, F., Ochs, H., Deplazes, P., 2002. Serological cross-sectional survey of psoroptic sheep scab in Switzerland. Vet. Parasitol. 109, 119–127. Kirkwood, A.C., 1985. Some observations on the biology and control of the sheep scab mite Psoroptes ovis (Hering) in Britain. Vet. Parasitol. 18, 269–279. Meleney, W.P., 1967. Experimentally induced bovine psoroptic acariasis in rabbits. Am. J. Vet. Res. 28, 892–894. Morgan, K.L., 1991. Aural haematomata, cauliflower ears and Psoroptes ovis in sheep. Vet. Rec. 128, 459–460. O’Brien, D.J., Gray, J.S., O’Reilly, P.F., 1994. Examination of possible transmission of sheep scab mite Psoroptes ovis between host species. Vet. Res. Commun. 18, 113–117. Ochs, H., Mathis, A., Deplazes, P., 1999. Single nucleotide variation in rDNA ITS-2 differentiates Psoroptes isolates from sheep and rabbits from the same geographical area. Parasitology 119, 419–424. Ochs, H., Lonneux, J.F., Losson, B.J., Deplazes, P., 2001. Diagnosis of psoroptic sheep scab with an improved enzyme-linked immunosorbent assay. Vet. Parasitol. 96, 233–242. Shilston, A.W., 1915. Sheep scab. Observations on the life history of Psoroptes communis var. ovis and some points connected with the epizootiology on the disease in South Africa. 3rd and 4th Reports of the Director of Veterinary Research, Union of South Africa, Departament of Agriculture, Pretoria, pp. 69–98. 124 E. Siegfried et al. / Veterinary Parasitology 124 (2004) 109–124

Smith, W.D., van den Broek, A., Huntley, J., Pettit, D., Machell, J., Miller, H.R., Bates, P.G., Taylor, M., 2001. Approaches to vaccines for Psoroptes ovis (sheep scab). Res. Vet. Sci. 70, 87–91. Smith, W.D., Bates, P.G., Pettit, D.M., van Den Broek, A., Taylor, M.A., 2002. Attempts to immunize sheep against the scab mite, Psoroptes ovis. Parasite Immunol. 24, 303–310. Stromberg, P.C., Fisher, W.F., 1986. Dermatopathology and immunity in experimental Psoroptes ovis (Acari: Psoroptidae) infestation of naive and previously exposed Hereford cattle. Am. J. Vet. Res. 47, 1551–1560. Sweatman, G.K., 1958. On the life history and validity of the species in Psoroptes, a genus of mange mites. Can. J. Zool. 36, 905–929. van den Broek, A.H.M., Huntley, J.F., Machell, J., Taylor, M., Bates, P.G., Groves, B., Miller, H.R.P., 2000. Cutaneous and systemic responses during primary and challenge infestations of sheep with the sheep scab mite, Psoroptes ovis. Parasite Immun. 22, 407–414. Wright, F.C., Riner, J.C., Guillot, F.S., 1983. Cross mating studies with Psoroptes ovis (Hering) and Psoroptes cuniculi (Delafond) (Acarina: Psoroptidae). J. Parasitol. 6, 696–700. Wright, F.C., Riner, J.C., Fisher, W.F., 1984. Comparison of lengths of outer opisthosomal setae of male psoroptic mites collected from different hosts. J. Parasitol. 7, 141–143. Zahler, M., Hendrix, W.M., Essig, A., Rinder, H., Gothe, R., 2000. Species of the genus Psoroptes (Acari: Psoroptidae): a taxonomic consideration. Exp. Appl. Acarol. 24, 213–225. Lebenslauf

Name Elena Siegfried Geburtsdatum 24.05.1974 Geburtsort Zürich Nationalität Schweizerin Heimatort Zofingen, AG

1981 – 1987 Primarschule, Zürich 1987 – 1990 Sekundarschule Bostetten 1990 – 1995 Gymnasium Seminar Unterstrasse, Zürich 1995 Matura Typus L am Seminar Unterstrasse, Zürich 01.04.1995-01.04.1995 Sprachaufenthalt an der „Universita per Stranieri“ in Perugia, Italien August 1995 Fremdenmatura Typus D, Zürich

1995 – 2001 Studium der Veterinärmedizin an der Veterinärmedizinischen Fakultät Universität Zürich, Schweiz Juni 2001 Schlussprüfung an der Veterinärmedizinischen Fakultät Universität Zürich, Schweiz

2001 – 2003 Doktorandenstelle Institut für Parasitologie Universität Zürich, Schweiz (100%) 01.09.2003 – 31.01.2004 Assistentenstelle Pferdeklinik Neugraben AG in Niederlenz, Schweiz (100%) seit 01.05.2004 Assistentenstelle Kleintierpraxis Schwäntenmoos in Zumikon, Schweiz Weiteres: Februar 1998 Praktikum in der Pferdeklinik Rossdale & Partners in Newmarket, England Juli 1998 Obligatorisches Praktikum bei Drs. med. vet. Jenny und Bohli in Au Wädenswil, Schweiz Februar 2000 Praktikum am Tierspital Zürich auf den Abteilungen Rindermedizin, Rinderchirurgie und Ambulanz, Schweiz Juli/August 2002 Praktikum an der Klinik für Kleintiere und Pferde in Montheron VD, Schweiz Oktober 2000 Praktikum am Pathologischen Institut des Tierspital Zürich, Schweiz

Zürich, 23.9.2004