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Density of itch mite, Sarcoptes scabiei (Acari: Sarcoptidae) and temporal development of cutaneous hypersensitivity in swine mange
Veterinary Parasitology, 36 (1990) 285-293 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
285
Density of Itch Mite, Sarcoptes scabiei (Acari: Sarcoptidae) and Temporal Development of Cutaneous Hypersensitivity in Swine Mange DAVID P. DAVIS 1 and ROGER D. MOON
Department of Entomology, University of Minnesota, St. Paul, M N 55108 (U.S.A.) (Accepted for publication 21 December 1989)
ABSTRACT Davis, D.P. and Moon, R.D., 1990. Density of itch mite, Sarcoptes scabiei (Acari: Sarcoptidae) and temporal development of cutaneous hypersensitivity in swine mange. Vet. Parasitol., 36: 285-293. Experimental infestations of Sarcoptes scabiei (De Geer) were established to study development of cutaneous hypersensitivity in pigs. Forty-eight pigs in six isolation rooms were used in two trials lasting 51 and 65 days, respectively. Treatments of 0 (control), 100 (low dose), and 1000 mites (high dose) per pig were randomly assigned to rooms. Intradermal skin tests with a sterile mite extract were done weekly to assess hypersensitivity. Control pigs never responded to the extract, whereas most infested pigs progressed through phases of ( 1 ) no response, (2) delayed hypersensitivity alone, (3) immediate and delayed hypersensitivity together, and (4) immediate hypersensitivity alone. High-dose pigs developed delayed and immediate hypersensitivity sooner than low-dose pigs (P < 0.005 ). When related to cumulative mite-days, a measure of exposure to mite antigens, low-dose and high-dose pigs developed delayed responses after experiencing the same level of exposure. Thereafter, immediate responses developed sooner in low-dose pigs, suggesting that immediate hypersensitivity develops at a rate that is independent of rate of antigen exposure.
INTRODUCTION
Itch mite, Sarcoptes scabiei (De Geer), resides in the skin of 40 mammalian species (Fain, 1978) causing mange in swine and other animals and scabies in humans. The mite secretes compounds that dissolve the epidermis (Arlian et al., 1984) and feeds on the cytoplasm of live skin cells (Van Neste, 1986). In the process, the mite, its secretory products, exuvia, feces, and eggs present antigens (Arlian et al., 1985, 1988) to which hosts produce hypersensitive reactions (Mellanby, 1944; Sheahan, 1975; Falk and Bolle, 1980). 1present address: Department of Entomology, 301 Funchess Hall, Auburn University, Auburn, AL 36849, U.S.A.
0304-4017/90/$03.50
© 1990 Elsevier Science Publishers B.V.
286
D.P. DAVIS AND R.D. MOON
Cutaneous hypersensitivity to ectoparasitic arthropods typically develops through five phases: (1) induction, where no response occurs; (2) delayed hypersensitivity; (3) both delayed and immediate hypersensitivity; (4) immediate hypersensitivity only; and finally (5) desensitization, where neither hypersensitive reaction remains detectable (Mellanby, 1946; Benjamini et al., 1960; Feingold et al., 1968). In human scabies, immediate hypersensitivity was detected in 18 of 24 non-treated cases and 9 of 18 treated cases (Prakken and Van Volten, 1949). Heilesen (1946) detected immediate hypersensitivity in 14 of 44 infested individuals; their symptoms persisted for an average of 7.6 weeks (SD = 4.6 weeks ). More recently, Falk and Bolle (1980) detected immediate hypersensitivity in 7 of 12 scabies patients who had symptoms for 2-8 months. Elevated levels of serum IgE, the principal immunoglobulin in immediate hypersensitivity in humans, have been detected in humans with scabies (Hancock and Ward, 1974; Falk, 1980; Rantanen et al., 1981). In experimental swine mange, Sheahan (1975) observed delayed and immediate hypersensitivity by Weeks 3 and 7, respectively, both of which continued to a lesser extent through Week 20. Further, Sheahan (1975) did not observe vasculo-necrosis, characteristic of Arthus reactions. Serum antibodies to S. scabiei appeared by Week 4 in another study (Wooten and Gaafar, 1984). Morsy and Gaafar (1989) observed immunoglobulin-secreting cells from ear pinnae skin biopsies within 1 week of artificially placing mites there. Numbers of IgA, IgM and IgG secreting cells peaked at 15, 28 and 35 days in pigs infested at 6 days of age. Pigs infested at 30 days responded sooner, demonstrating an age-dependent component of immunological responses to immunogens. Hypersensitivity may cause pathological changes that harm more than benefit the host, especially in cases of allergies and allergic dermatitis to inert substances (Altman, 1984; Dahl, 1987). Nevertheless, hypersensitivity is thought to protect the host from ectoparasitism. Hypersensitivity has been associated with decreased survival of ectoparasitic mites, including Ornithonyssus sylviarum on hens (DeVaney and Ziprin, 1980), Psoroptes ovis on cattle (Stromberg and Fisher, 1986; Guillot and Stromberg, 1987) and S. scabiei on humans (Mellanby, 1944 ) and swine (Sheahan, 1974 ). In swine mange, degree of hypersensitivity and subsequent pruritus have been associated with decreases in average daily gain and feed efficiency (Cargill and Dobson, 1979). Little is known about the quantitative relationship between mite density and development of hypersensitivity. Feingold et al. (1968) hypothesized that development of hypersensitivity depends on the amount of antigen that hosts encounter, which should be proportional to mite density. The present study was designed to test this hypothesis.
ITCH MITE DENSITY IN SWINE MANGE
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METHODS AND MATERIALS
Experimental design Two trials were conducted, one in 1986 and the other in 1987 with infestations lasting 9 and 10 weeks, respectively. In each trial, 24 crossbred (Landrace×Yorkshire×Duroc) piglets were randomly placed in three isolation rooms, four barrows and four gilts per room. Treatments of 0 (control), 100 (low dose), and 1000 mites (high dose) per pig were randomly assigned to rooms. The trial in 1987 was double-blind, whereby no one knew where each treatment was assigned. Randomly selected pigs were periodically slaughtered to assess mite burdens (Davis and Moon, 1990), resulting in fewer animals for observation as the experiment progressed.
Establishment of infestations Mites were taken from crusted ear lesions of market hogs. Since mites kept at cooler temperatures in mineral oil survive well and are easy to handle (Davis and Moon, 1987), crusts were placed in petri dishes with mineral oil and transported in a cooler to the laboratory for refrigeration. Dishes were later warmed to room temperature for 1-3 h to allow mites to vacate crusts. Mites were counted under a stereomicroscope and pipetted into vials in lots of 100 and 1000 mites (25% females and 75% other life stages). Vials were then transported to isolation rooms and emptied on pigs. Control pigs received mineral oil placebos without mites in 1986 and with dead mites in 1987. Used vials and pipettes were examined to determine the exact number of mites each pig received.
Preparation of mite extract A sterile mite extract was prepared and used for intradermal injections. Mites from crusted lesions were placed in 10 ml phosphate buffered saline (PBS, pH 7.4), ground in a tissue homogenizer, and centrifuged at 500 g for 5 min. The pellet was sonicated to lyse cell membranes, centrifuged again, filtered through a 0.2-pm sterile filter into sterile vials, and stored at - 7 0 ° C. Protein content of the extract was determined (Biorad Laboratories, Richmond, CA). Vials were thawed and diluted to provide aliquots of 5 #g protein in 0.1 ml PBS. Fifty female mites were also processed as above to determine the crude protein content of the average female, and the number of females (female equivalent) required to produce 5/tg of protein injection.
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Injections and responses One-milliliter tuberculin syringes with 25 gauge needles were used. Two intradermal injections of extract in PBS and PBS alone (negative reference) were made into each pig each week, except for Week 6 in 1986 and Week 8 in 1987. Each pig was placed on its back in a cradle and the two injections were placed caudal to selected nipples. Injection sites were observed after 15 min for evidence of immediate hypersensitivity, defined as erythema (0.5-1.5 cm diameter) in excess of the paired PBS control. Injection sites were inspected again at 24 h for evidence of delayed hypersensitivity, defined as being edemous and indurated.
Data interpretation and analysis The number of pigs assayed per treatment group in Weeks 0-9 were: 2, 12, 16, 14, 12, 12, 4, 8, 0 and 4. The decline in numbers reflected removal of pigs for slaughter (see below). Pigs at each time of assay were categorized as showing (1) no response to date, (2) only delayed hypersensitivity, (3) immediate hypersensitivity and delayed hypersensitivity, or (4) immediate hypersensitivity only. Percent of responses in each category was averaged by week over the two trials. Categorical analysis (Bishop et al., 1975) was used to test for significant differences in frequency of responses among high- and low-dose pigs using each pig as an experimental unit. Control pigs were omitted from the analysis because they never responded. Because some pigs were slaughtered during the trials, observations were censored (Cox and Oaks, 1984), i.e. some pigs did not survive long enough to develop hypersensitivity. Accordingly, the program Lifetest (SAS, 1985 ) which accommodates right censored frequencies was used to calculate a Logrank )/2 test for significance between treatments. To relate development of hypersensitivity to mite antigen exposure, cumulative mite densities were derived from a series of estimates of mite densities on randomly selected pigs in each treatment group (Davis and Moon, 1990). Slaughtered pigs were skinned and then their hides were sampled to extract mites. Numbers of extracted females and other mite stages were first corrected for extraction efficiency, and then counts of other stages were converted to female equivalents, on the basis of relative size and presumed antigen presentation rate. Mean densities for pigs in each treatment group on each slaughter date were then calculated on the loglo scale, transformed back to the original scale, and finally summed across days by trapezoidal integration to estimate cumulative exposure for each group of pigs up to each time of assay.
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RESULTS
Procedures for establishing infestations were successful. Low-dose pigs received a mean of 25 +_1.5 (SD) female mites and 76.5 _+4.6 mites of other life stages. High-dose pigs received an average of 249.8 _+3.2 females and 753 + 12.7 mites of other life stages. During the course of the experiment, all infested pigs developed typical signs of hypersensitive mange, i.e. itching and reddened lesions. Control pigs never showed any signs of mange nor did they react to the mite extract. The extract from 50 female mites revealed that an average female mite contained 0.32 #g of extractable protein. Thus, the 5 #g injection contained the protein equivalent of 15.5 female mites. Low-dose pigs exhibited an induction phase (1) that lasted 3 weeks, ending when half of the pigs showed delayed hypersensitivity (Fig. 1A). All low-dose pigs showed delayed responses (Phase 2) by Week 6. Immediate responses (Phase 3 ) were first evident in Week 4 in individuals also showing concurrent delayed responses. In Weeks 5 and 7, some low-dose pigs had immediate reactions (Phase 4) after showing delayed reactions in earlier assays. Pigs that advanced into Phase 4 (having immediate reactions only) were slaughtered before their immediate responses could have diminished. Among high-dose pigs, hypersensitivity developed through all four phases at a faster rate than in low-dose pigs (Fig. 1B vs. 1A). The induction phase ended in less than 1 week, as evidenced by some pigs showing delayed responses at Week 1. All high dose pigs showed delayed responses by their fourth week. Phase 3 started in Week 3 when some pigs showed concurrent delayed and immediate reactions. Finally, some pigs were in Phase 4 in Weeks 5, 7, and 9. J
I00
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"~.~ 20 o
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i i i i i i!i!i i i
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0
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Fig. 1. Percent of pigs in low-dose (A) and high-dose (B) treatment groups showing hypersensitive phases of (1) induction, (2) delayed hypersensitivity (DH only), (3) delayed and immediate hypersensitivity (DH and IH ), and (4) immediate hypersensitivity alone (IH only).
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D.P. DAVISAND R.D. MOON
Immediate
Delayed
High Low
I
I I
I
I
I
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I
I
I
I
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Fig. 2. Estimated median {vertical bar) and interquantile (horizontal bar) times when high- and low-dose pigs displayed delayed and immediate hypersensitivity. Times to median delayed res p o n s e s were significantly different between treatments ( P < 0.0001 ); times to median immediate responses were different at P < 0.004, L o g r a n k X2 tests.
800
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~D
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/
~
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~:~ 400
~9
~9 +Z
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Fig. 3. Mean density of mites per pig at slaughter (A) from high and low dose treatment groups
(from Davis and Moon, 1990). Graph B is the cumulative mite burden (mite-days) for high and low dose pigs. Densities are in units of female equivalents.
Median times to develop delayed hypersensitivity in high- and low-dose pigs were 2 and 4 weeks (Fig. 2 ), and the difference was highly significant (X2 = 15.89, P < 0.0004, d f - - 1 ) . Median times to onset of immediate hypersensitivity for high and low dose pigs were 4.6 and 6.5 weeks, also significantly different (Z e -- 8.27, P < 0.004, df-- 1 ). Subtraction of the corresponding times to median onset of delayed hypersensitivity from times to median onset of immediate hypersensitivity indicated approximately 2.5 weeks was required by both treatment groups to develop the immediate response after first showing the delayed response. To separate the effects of time and mite density on the development of hypersensitivity, a comparison was made between high- and low-dose groups on the cumulative mite density scale (Figs. 3 and 4). The average low-dose and
ITCH MITE DENSITYIN SWINEMANGE
I00
o
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Immediat
Delayed High Low
so 60 40
bJ~ 20 o
0 L
i
i
, 2
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Fig. 4. Percent of high- and low-dose pigs responding with delayed and immediate hypersensitive reactions vs. loglo of cumulative mite-days (in female equivalents).
high-dose pigs both showed delayed hypersensitivity after experiencing approximately 300 mite-days. However, expression of immediate hypersensitivity in the average low-dose pig required an additional 700 mite-days, whereas the average high dose pig required about 5300 additional mite-days. DISCUSSION
These results confirm that S. scabiei infested pigs develop hypersensitive reactions through the first four phases as outlined by Mellanby (1946), Benjamini et al. (1960) and Feingold et al. (1968). More importantly, temporal development of the four phases depended on mite dose. These findings concur with those of Sheahan (1975), except that we observed the first three phases sooner, and we detected Phase 4 (immediate hypersensitivity only) which Sheahan did not detect. The sequence of phases presented here parallels development of flea bite hypersensitivity in guinea pigs (Benjamini et al., 1960). While those authors remarked that rate of hypersensitive development was independent of number of flea bites, their data showed that flea density and temporal development of hypersensitivity were related. Guinea pigs exposed to 300 vs. 2 fleas day -1 responded with delayed hypersensitivity by 4.8 and 6.3 days, respectively. Immediate hypersensitivity occurred by 9.8 and 11.0 days, respectively. In the present study, average mite densities in high- and low-dose groups during the course of the experiment differed by 7 fold (Fig. 3), 269 mites per high-dose pig and 39 mites per low-dose pig (Davis and Moon, 1990). The speed with which delayed hypersensitivity developed was determined by and was proportional to overall mite density, and presumably, to rate of antigen exposure. However, time from onset of the delayed response to onset of the immediate response appeared to be independent of mite density. Rather, time to immediate response was about 2 weeks from onset of delayed hypersensitivity.
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Effects of mange on average daily gain and feed efficiency are quite variable, having been reported as positive or nil (Sheahan, 1974; Alva-Valdes et al., 1986; Wooten-Saadi et al., 1987), and negative by 3-6% (Hewett, 1985; AlvaValdes, 1986; Kofer et al., 1986) and 12% (Cargill and Dobson, 1979). Some of these studies involved natural infestations with unknown mite densities (Hewett, 1985; Alva-Valdes, 1986; Kofer et al., 1986), whereas the others involved experimental infestations. If mite density at time of infestation determines time to onset of hypersensitivity as shown here, and if severity of immunologically mediated mange symptoms is determined collectively by phase of hypersensitivity, extent of hypersensitivity, and mite density, t h e n some of the variation in reported effects of mange may be attributable to variation in the immune status and mite densities of the study animals. F u r t h e r work with herds larger t h a n the ones we used will be necessary to gauge the effects of mange severity on animal performance. ACKNOWLEDGEMENTS Drs. M. Dahl, T. Molitor and D. Ragsdale engaged in helpful discussions while planning this research. We t h a n k V. Cervenka, K. Kjer, D. Moe Nelson, R. Warzynski for help handling pigs; and Drs. D. Ragsdale and B. Stromberg commented on earlier drafts of the manuscript. Contribution No. 17,497 of the Minnesota Agricultural Experiment Station based on research supported by the Station and additional grants from the University of Minnesota Swine Center. REFERENCES Altman, L.C., 1984. ClinicalAllergyand Immunology.G.K. Hall MedicalPublishers, Boston, MA. Alva-Valdes, R., Wallace,D.H., Foster, A.G., Erickson, G.F. and Wooden,J.W., 1986. The effects of sarcoptic mange on the productivity of confinedpigs. Vet. Med., 81: 258-262. Arlian, L.G.,Runyan, R.A. and Estes, S.A., 1984. Cross infestivityof Sarcoptes scabiei. Am. Acad. Dermatol., 10: 979-986. Arlian, L.G., Runyan, R.A., Sorlie, L.B., Vyszenski-Moher,D.L. and Estes, S.A., 1985. Characterization of Sarcoptes scabiei vat. canis (Acari: Sarcoptidae) antigens and inducedantibodies in rabbits. J. Med. Entomol., 22: 321-323. Arlian, L.G., Vyszensky-Moher,D.L. and Gilmore, A.M., 1988. Cross-antigenicitybetween Sar coptes scabiei and the house dust mite, Dermatophagoides farinae (Acari: Sarcoptidae and Pyroglyphidae).J. Med. Entomol., 25: 240-247. Benjamini, E., Feingold,B.F. and Kartman, L., 1960. Allergyto flea bites. III. The experimental inductionof fleabite sensitivityin guineapigs by exposureto fleabites and by antigen prepared from whole flea extracts of Ctenocephalides felis felis. Exp. Parasitol., 10: 214-222. Bishop, Y.M.M.,Fienberg, S.E. and Holland, P.W., 1975. Discrete Multivariate Analysis;Theory and Practice. MIT Press, Cambridge,MA, 557 pp. Cargill, C.F. and Dobson, K.J., 1979. Experimental Sarcoptes scabiei infestation in pigs: (2) effects on production. Vet. Rec., 104: 33-36.
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Cox, D.R. and Oaks, D., 1984. Analysis of Survival Data. Chapman and Hall, London, 201 pp. Dahl, M.V., 1987. Clinical Immunodermatology, 2nd Ed. Year Book Medical Publishers Inc., Chicago, IL, 422 pp. Davis, D.P. and Moon, R.D., 1988. Survival of Sarcoptes scabiei (De Geer) in three media and three temperatures, J. Parasitol., 73: 661-662. Davis, D.P. and Moon, R.D., 1990. Density, location and sampling of itch mites, Sarcoptes scabiei (Acari: Sarcoptidae), on experimentally infested pigs. J. Med. Entomol., 27: 391-398. DeVaney, J.A. and Ziprin, R.L., 1980. Acquired immune response of white leghorn hens to populations of northern fowl mite, Ornithonyssus sylviarum (Canestrini and Fanzago). Poultry Sci., 59: 1742-1744. Fain, A., 1978. Epidemiological problems of scabies. Int. J. Dermatol., 17: 20-31. Falk, E.S., 1980. Serum immunoglobulin values in patients with scabies. Br. J. Dermatol., 102: 57-61. Falk, E.S. and Bolle, R., 1980. In vitro demonstration of specific immunological hypersensitivity to scabies mite. Br. J. Dermatol., 103: 367-373. Feingold, B.F., Benjamini, E. and Michaeli, D., 1968. The allergic responses to insect bites. Annu. Rev. Entomol., 13: 137-157. Guillot, F.S. and Stromberg, P.C., 1987. Reproductive success of Psoroptes ovis (Acari: Psoroptidae) on hereford calves with previous infestation of psoroptes mites. J. Med. Entomol., 24: 416-419. Hancock, B.W. and Ward, A.M., 1974. Serum immunoglobulin in scabies. J. Invest. Dermatol., 63: 482-484. Heilesen, B., 1946. Studies on Acarus scabiei and scabies. Acta Derm-Venereol. Suppl., 26: 1-370. Hewett, G.R., 1985. Phosmet for the systemic control of pig mange in growing pigs. Vet. Parasitol., 18: 265-268. Kofer, J., Glawischnig, E., Tockner, F. and Leitner, B., 1986. Therapy with Ivomec against ecto and endo parasites of swine. Wein. Tierarztl. Mschr. 73 Jahrgang Heft, 6: 196. Mellanby, K., 1944. The development of symptoms, parasitic infection and immunity in human scabies. Parasitology, 35: 197-206. Mellanby, K., 1946. Man's reactions to mosquito bites. Nature (London), 158: 554, 751. Morsy, G.H. and Gaafar, S.M., 1989. Response of immunoglobulin-secreting cells in skin of pigs during Sarcoptes scabiei infestation. Vet. Parasitol., 33: 165-175. Prakken, J.R. and Van Volten, T.J., 1949. Allergy in scabies. Dermatologica, 99: 124-131. Rantanen, T., Bjorksten, T.F., Reunala, T. and Salo, O.P., 1981. Serum IgE antibodies to scabies mites. Acta Dermatovener (Stockholm), 61: 358-360. SAS, 1985. User's guide. Version 5.0. SAS Institute Inc. Cary, NC. Sheahan, B.J., 1974. Experimental Sarcoptes scabiei infection in pigs: clinical signs and significance of infection. Vet. Rec., 94: 202-209. Sheahan, B.J., 1975. Pathology of Sarcoptes scabiei infection in pigs. II. Histological, histochemical and ultrastructural changes at skin test sites. J. Comp. Pathol., 85: 97-110. Stromberg, P.C. and 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: 1551-1559. Van Neste, D.J., 1986. Immunology of scabies. Parasitol. Today, 2: 194-196. Wooten, E.L. and Gaafar, S.M., 1984. Detection of serum antibodies to sarcoptic mange mite antigens by the passive hemagglutination assay in pigs infested with Sarcoptes scabiei var. suis. Vet. Parasitol., 15: 309-316. Wooten-Saadi, E., Broce, A.B., Stevenson, J.S. and Nelssen, J.L., 1987. Growth performance and behavioral patterns of pigs infested with sarcoptic mites (Acari: Sarcoptidae ). J. Econ. Entomol., 80: 625-628.
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Density of Itch Mite, Sarcoptes scabiei (Acari: Sarcoptidae) and Temporal Development of Cutaneous Hypersensitivity in Swine Mange DAVID P. DAVIS 1 and ROGER D. MOON
Department of Entomology, University of Minnesota, St. Paul, M N 55108 (U.S.A.) (Accepted for publication 21 December 1989)
ABSTRACT Davis, D.P. and Moon, R.D., 1990. Density of itch mite, Sarcoptes scabiei (Acari: Sarcoptidae) and temporal development of cutaneous hypersensitivity in swine mange. Vet. Parasitol., 36: 285-293. Experimental infestations of Sarcoptes scabiei (De Geer) were established to study development of cutaneous hypersensitivity in pigs. Forty-eight pigs in six isolation rooms were used in two trials lasting 51 and 65 days, respectively. Treatments of 0 (control), 100 (low dose), and 1000 mites (high dose) per pig were randomly assigned to rooms. Intradermal skin tests with a sterile mite extract were done weekly to assess hypersensitivity. Control pigs never responded to the extract, whereas most infested pigs progressed through phases of ( 1 ) no response, (2) delayed hypersensitivity alone, (3) immediate and delayed hypersensitivity together, and (4) immediate hypersensitivity alone. High-dose pigs developed delayed and immediate hypersensitivity sooner than low-dose pigs (P < 0.005 ). When related to cumulative mite-days, a measure of exposure to mite antigens, low-dose and high-dose pigs developed delayed responses after experiencing the same level of exposure. Thereafter, immediate responses developed sooner in low-dose pigs, suggesting that immediate hypersensitivity develops at a rate that is independent of rate of antigen exposure.
INTRODUCTION
Itch mite, Sarcoptes scabiei (De Geer), resides in the skin of 40 mammalian species (Fain, 1978) causing mange in swine and other animals and scabies in humans. The mite secretes compounds that dissolve the epidermis (Arlian et al., 1984) and feeds on the cytoplasm of live skin cells (Van Neste, 1986). In the process, the mite, its secretory products, exuvia, feces, and eggs present antigens (Arlian et al., 1985, 1988) to which hosts produce hypersensitive reactions (Mellanby, 1944; Sheahan, 1975; Falk and Bolle, 1980). 1present address: Department of Entomology, 301 Funchess Hall, Auburn University, Auburn, AL 36849, U.S.A.
0304-4017/90/$03.50
© 1990 Elsevier Science Publishers B.V.
286
D.P. DAVIS AND R.D. MOON
Cutaneous hypersensitivity to ectoparasitic arthropods typically develops through five phases: (1) induction, where no response occurs; (2) delayed hypersensitivity; (3) both delayed and immediate hypersensitivity; (4) immediate hypersensitivity only; and finally (5) desensitization, where neither hypersensitive reaction remains detectable (Mellanby, 1946; Benjamini et al., 1960; Feingold et al., 1968). In human scabies, immediate hypersensitivity was detected in 18 of 24 non-treated cases and 9 of 18 treated cases (Prakken and Van Volten, 1949). Heilesen (1946) detected immediate hypersensitivity in 14 of 44 infested individuals; their symptoms persisted for an average of 7.6 weeks (SD = 4.6 weeks ). More recently, Falk and Bolle (1980) detected immediate hypersensitivity in 7 of 12 scabies patients who had symptoms for 2-8 months. Elevated levels of serum IgE, the principal immunoglobulin in immediate hypersensitivity in humans, have been detected in humans with scabies (Hancock and Ward, 1974; Falk, 1980; Rantanen et al., 1981). In experimental swine mange, Sheahan (1975) observed delayed and immediate hypersensitivity by Weeks 3 and 7, respectively, both of which continued to a lesser extent through Week 20. Further, Sheahan (1975) did not observe vasculo-necrosis, characteristic of Arthus reactions. Serum antibodies to S. scabiei appeared by Week 4 in another study (Wooten and Gaafar, 1984). Morsy and Gaafar (1989) observed immunoglobulin-secreting cells from ear pinnae skin biopsies within 1 week of artificially placing mites there. Numbers of IgA, IgM and IgG secreting cells peaked at 15, 28 and 35 days in pigs infested at 6 days of age. Pigs infested at 30 days responded sooner, demonstrating an age-dependent component of immunological responses to immunogens. Hypersensitivity may cause pathological changes that harm more than benefit the host, especially in cases of allergies and allergic dermatitis to inert substances (Altman, 1984; Dahl, 1987). Nevertheless, hypersensitivity is thought to protect the host from ectoparasitism. Hypersensitivity has been associated with decreased survival of ectoparasitic mites, including Ornithonyssus sylviarum on hens (DeVaney and Ziprin, 1980), Psoroptes ovis on cattle (Stromberg and Fisher, 1986; Guillot and Stromberg, 1987) and S. scabiei on humans (Mellanby, 1944 ) and swine (Sheahan, 1974 ). In swine mange, degree of hypersensitivity and subsequent pruritus have been associated with decreases in average daily gain and feed efficiency (Cargill and Dobson, 1979). Little is known about the quantitative relationship between mite density and development of hypersensitivity. Feingold et al. (1968) hypothesized that development of hypersensitivity depends on the amount of antigen that hosts encounter, which should be proportional to mite density. The present study was designed to test this hypothesis.
ITCH MITE DENSITY IN SWINE MANGE
287
METHODS AND MATERIALS
Experimental design Two trials were conducted, one in 1986 and the other in 1987 with infestations lasting 9 and 10 weeks, respectively. In each trial, 24 crossbred (Landrace×Yorkshire×Duroc) piglets were randomly placed in three isolation rooms, four barrows and four gilts per room. Treatments of 0 (control), 100 (low dose), and 1000 mites (high dose) per pig were randomly assigned to rooms. The trial in 1987 was double-blind, whereby no one knew where each treatment was assigned. Randomly selected pigs were periodically slaughtered to assess mite burdens (Davis and Moon, 1990), resulting in fewer animals for observation as the experiment progressed.
Establishment of infestations Mites were taken from crusted ear lesions of market hogs. Since mites kept at cooler temperatures in mineral oil survive well and are easy to handle (Davis and Moon, 1987), crusts were placed in petri dishes with mineral oil and transported in a cooler to the laboratory for refrigeration. Dishes were later warmed to room temperature for 1-3 h to allow mites to vacate crusts. Mites were counted under a stereomicroscope and pipetted into vials in lots of 100 and 1000 mites (25% females and 75% other life stages). Vials were then transported to isolation rooms and emptied on pigs. Control pigs received mineral oil placebos without mites in 1986 and with dead mites in 1987. Used vials and pipettes were examined to determine the exact number of mites each pig received.
Preparation of mite extract A sterile mite extract was prepared and used for intradermal injections. Mites from crusted lesions were placed in 10 ml phosphate buffered saline (PBS, pH 7.4), ground in a tissue homogenizer, and centrifuged at 500 g for 5 min. The pellet was sonicated to lyse cell membranes, centrifuged again, filtered through a 0.2-pm sterile filter into sterile vials, and stored at - 7 0 ° C. Protein content of the extract was determined (Biorad Laboratories, Richmond, CA). Vials were thawed and diluted to provide aliquots of 5 #g protein in 0.1 ml PBS. Fifty female mites were also processed as above to determine the crude protein content of the average female, and the number of females (female equivalent) required to produce 5/tg of protein injection.
288
D.P. DAVIS AND R.D. MOON
Injections and responses One-milliliter tuberculin syringes with 25 gauge needles were used. Two intradermal injections of extract in PBS and PBS alone (negative reference) were made into each pig each week, except for Week 6 in 1986 and Week 8 in 1987. Each pig was placed on its back in a cradle and the two injections were placed caudal to selected nipples. Injection sites were observed after 15 min for evidence of immediate hypersensitivity, defined as erythema (0.5-1.5 cm diameter) in excess of the paired PBS control. Injection sites were inspected again at 24 h for evidence of delayed hypersensitivity, defined as being edemous and indurated.
Data interpretation and analysis The number of pigs assayed per treatment group in Weeks 0-9 were: 2, 12, 16, 14, 12, 12, 4, 8, 0 and 4. The decline in numbers reflected removal of pigs for slaughter (see below). Pigs at each time of assay were categorized as showing (1) no response to date, (2) only delayed hypersensitivity, (3) immediate hypersensitivity and delayed hypersensitivity, or (4) immediate hypersensitivity only. Percent of responses in each category was averaged by week over the two trials. Categorical analysis (Bishop et al., 1975) was used to test for significant differences in frequency of responses among high- and low-dose pigs using each pig as an experimental unit. Control pigs were omitted from the analysis because they never responded. Because some pigs were slaughtered during the trials, observations were censored (Cox and Oaks, 1984), i.e. some pigs did not survive long enough to develop hypersensitivity. Accordingly, the program Lifetest (SAS, 1985 ) which accommodates right censored frequencies was used to calculate a Logrank )/2 test for significance between treatments. To relate development of hypersensitivity to mite antigen exposure, cumulative mite densities were derived from a series of estimates of mite densities on randomly selected pigs in each treatment group (Davis and Moon, 1990). Slaughtered pigs were skinned and then their hides were sampled to extract mites. Numbers of extracted females and other mite stages were first corrected for extraction efficiency, and then counts of other stages were converted to female equivalents, on the basis of relative size and presumed antigen presentation rate. Mean densities for pigs in each treatment group on each slaughter date were then calculated on the loglo scale, transformed back to the original scale, and finally summed across days by trapezoidal integration to estimate cumulative exposure for each group of pigs up to each time of assay.
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RESULTS
Procedures for establishing infestations were successful. Low-dose pigs received a mean of 25 +_1.5 (SD) female mites and 76.5 _+4.6 mites of other life stages. High-dose pigs received an average of 249.8 _+3.2 females and 753 + 12.7 mites of other life stages. During the course of the experiment, all infested pigs developed typical signs of hypersensitive mange, i.e. itching and reddened lesions. Control pigs never showed any signs of mange nor did they react to the mite extract. The extract from 50 female mites revealed that an average female mite contained 0.32 #g of extractable protein. Thus, the 5 #g injection contained the protein equivalent of 15.5 female mites. Low-dose pigs exhibited an induction phase (1) that lasted 3 weeks, ending when half of the pigs showed delayed hypersensitivity (Fig. 1A). All low-dose pigs showed delayed responses (Phase 2) by Week 6. Immediate responses (Phase 3 ) were first evident in Week 4 in individuals also showing concurrent delayed responses. In Weeks 5 and 7, some low-dose pigs had immediate reactions (Phase 4) after showing delayed reactions in earlier assays. Pigs that advanced into Phase 4 (having immediate reactions only) were slaughtered before their immediate responses could have diminished. Among high-dose pigs, hypersensitivity developed through all four phases at a faster rate than in low-dose pigs (Fig. 1B vs. 1A). The induction phase ended in less than 1 week, as evidenced by some pigs showing delayed responses at Week 1. All high dose pigs showed delayed responses by their fourth week. Phase 3 started in Week 3 when some pigs showed concurrent delayed and immediate reactions. Finally, some pigs were in Phase 4 in Weeks 5, 7, and 9. J
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Fig. 1. Percent of pigs in low-dose (A) and high-dose (B) treatment groups showing hypersensitive phases of (1) induction, (2) delayed hypersensitivity (DH only), (3) delayed and immediate hypersensitivity (DH and IH ), and (4) immediate hypersensitivity alone (IH only).
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D.P. DAVISAND R.D. MOON
Immediate
Delayed
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Fig. 2. Estimated median {vertical bar) and interquantile (horizontal bar) times when high- and low-dose pigs displayed delayed and immediate hypersensitivity. Times to median delayed res p o n s e s were significantly different between treatments ( P < 0.0001 ); times to median immediate responses were different at P < 0.004, L o g r a n k X2 tests.
800
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Fig. 3. Mean density of mites per pig at slaughter (A) from high and low dose treatment groups
(from Davis and Moon, 1990). Graph B is the cumulative mite burden (mite-days) for high and low dose pigs. Densities are in units of female equivalents.
Median times to develop delayed hypersensitivity in high- and low-dose pigs were 2 and 4 weeks (Fig. 2 ), and the difference was highly significant (X2 = 15.89, P < 0.0004, d f - - 1 ) . Median times to onset of immediate hypersensitivity for high and low dose pigs were 4.6 and 6.5 weeks, also significantly different (Z e -- 8.27, P < 0.004, df-- 1 ). Subtraction of the corresponding times to median onset of delayed hypersensitivity from times to median onset of immediate hypersensitivity indicated approximately 2.5 weeks was required by both treatment groups to develop the immediate response after first showing the delayed response. To separate the effects of time and mite density on the development of hypersensitivity, a comparison was made between high- and low-dose groups on the cumulative mite density scale (Figs. 3 and 4). The average low-dose and
ITCH MITE DENSITYIN SWINEMANGE
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o
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so 60 40
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i
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Fig. 4. Percent of high- and low-dose pigs responding with delayed and immediate hypersensitive reactions vs. loglo of cumulative mite-days (in female equivalents).
high-dose pigs both showed delayed hypersensitivity after experiencing approximately 300 mite-days. However, expression of immediate hypersensitivity in the average low-dose pig required an additional 700 mite-days, whereas the average high dose pig required about 5300 additional mite-days. DISCUSSION
These results confirm that S. scabiei infested pigs develop hypersensitive reactions through the first four phases as outlined by Mellanby (1946), Benjamini et al. (1960) and Feingold et al. (1968). More importantly, temporal development of the four phases depended on mite dose. These findings concur with those of Sheahan (1975), except that we observed the first three phases sooner, and we detected Phase 4 (immediate hypersensitivity only) which Sheahan did not detect. The sequence of phases presented here parallels development of flea bite hypersensitivity in guinea pigs (Benjamini et al., 1960). While those authors remarked that rate of hypersensitive development was independent of number of flea bites, their data showed that flea density and temporal development of hypersensitivity were related. Guinea pigs exposed to 300 vs. 2 fleas day -1 responded with delayed hypersensitivity by 4.8 and 6.3 days, respectively. Immediate hypersensitivity occurred by 9.8 and 11.0 days, respectively. In the present study, average mite densities in high- and low-dose groups during the course of the experiment differed by 7 fold (Fig. 3), 269 mites per high-dose pig and 39 mites per low-dose pig (Davis and Moon, 1990). The speed with which delayed hypersensitivity developed was determined by and was proportional to overall mite density, and presumably, to rate of antigen exposure. However, time from onset of the delayed response to onset of the immediate response appeared to be independent of mite density. Rather, time to immediate response was about 2 weeks from onset of delayed hypersensitivity.
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Effects of mange on average daily gain and feed efficiency are quite variable, having been reported as positive or nil (Sheahan, 1974; Alva-Valdes et al., 1986; Wooten-Saadi et al., 1987), and negative by 3-6% (Hewett, 1985; AlvaValdes, 1986; Kofer et al., 1986) and 12% (Cargill and Dobson, 1979). Some of these studies involved natural infestations with unknown mite densities (Hewett, 1985; Alva-Valdes, 1986; Kofer et al., 1986), whereas the others involved experimental infestations. If mite density at time of infestation determines time to onset of hypersensitivity as shown here, and if severity of immunologically mediated mange symptoms is determined collectively by phase of hypersensitivity, extent of hypersensitivity, and mite density, t h e n some of the variation in reported effects of mange may be attributable to variation in the immune status and mite densities of the study animals. F u r t h e r work with herds larger t h a n the ones we used will be necessary to gauge the effects of mange severity on animal performance. ACKNOWLEDGEMENTS Drs. M. Dahl, T. Molitor and D. Ragsdale engaged in helpful discussions while planning this research. We t h a n k V. Cervenka, K. Kjer, D. Moe Nelson, R. Warzynski for help handling pigs; and Drs. D. Ragsdale and B. Stromberg commented on earlier drafts of the manuscript. Contribution No. 17,497 of the Minnesota Agricultural Experiment Station based on research supported by the Station and additional grants from the University of Minnesota Swine Center. REFERENCES Altman, L.C., 1984. ClinicalAllergyand Immunology.G.K. Hall MedicalPublishers, Boston, MA. Alva-Valdes, R., Wallace,D.H., Foster, A.G., Erickson, G.F. and Wooden,J.W., 1986. The effects of sarcoptic mange on the productivity of confinedpigs. Vet. Med., 81: 258-262. Arlian, L.G.,Runyan, R.A. and Estes, S.A., 1984. Cross infestivityof Sarcoptes scabiei. Am. Acad. Dermatol., 10: 979-986. Arlian, L.G., Runyan, R.A., Sorlie, L.B., Vyszenski-Moher,D.L. and Estes, S.A., 1985. Characterization of Sarcoptes scabiei vat. canis (Acari: Sarcoptidae) antigens and inducedantibodies in rabbits. J. Med. Entomol., 22: 321-323. Arlian, L.G., Vyszensky-Moher,D.L. and Gilmore, A.M., 1988. Cross-antigenicitybetween Sar coptes scabiei and the house dust mite, Dermatophagoides farinae (Acari: Sarcoptidae and Pyroglyphidae).J. Med. Entomol., 25: 240-247. Benjamini, E., Feingold,B.F. and Kartman, L., 1960. Allergyto flea bites. III. The experimental inductionof fleabite sensitivityin guineapigs by exposureto fleabites and by antigen prepared from whole flea extracts of Ctenocephalides felis felis. Exp. Parasitol., 10: 214-222. Bishop, Y.M.M.,Fienberg, S.E. and Holland, P.W., 1975. Discrete Multivariate Analysis;Theory and Practice. MIT Press, Cambridge,MA, 557 pp. Cargill, C.F. and Dobson, K.J., 1979. Experimental Sarcoptes scabiei infestation in pigs: (2) effects on production. Vet. Rec., 104: 33-36.
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Cox, D.R. and Oaks, D., 1984. Analysis of Survival Data. Chapman and Hall, London, 201 pp. Dahl, M.V., 1987. Clinical Immunodermatology, 2nd Ed. Year Book Medical Publishers Inc., Chicago, IL, 422 pp. Davis, D.P. and Moon, R.D., 1988. Survival of Sarcoptes scabiei (De Geer) in three media and three temperatures, J. Parasitol., 73: 661-662. Davis, D.P. and Moon, R.D., 1990. Density, location and sampling of itch mites, Sarcoptes scabiei (Acari: Sarcoptidae), on experimentally infested pigs. J. Med. Entomol., 27: 391-398. DeVaney, J.A. and Ziprin, R.L., 1980. Acquired immune response of white leghorn hens to populations of northern fowl mite, Ornithonyssus sylviarum (Canestrini and Fanzago). Poultry Sci., 59: 1742-1744. Fain, A., 1978. Epidemiological problems of scabies. Int. J. Dermatol., 17: 20-31. Falk, E.S., 1980. Serum immunoglobulin values in patients with scabies. Br. J. Dermatol., 102: 57-61. Falk, E.S. and Bolle, R., 1980. In vitro demonstration of specific immunological hypersensitivity to scabies mite. Br. J. Dermatol., 103: 367-373. Feingold, B.F., Benjamini, E. and Michaeli, D., 1968. The allergic responses to insect bites. Annu. Rev. Entomol., 13: 137-157. Guillot, F.S. and Stromberg, P.C., 1987. Reproductive success of Psoroptes ovis (Acari: Psoroptidae) on hereford calves with previous infestation of psoroptes mites. J. Med. Entomol., 24: 416-419. Hancock, B.W. and Ward, A.M., 1974. Serum immunoglobulin in scabies. J. Invest. Dermatol., 63: 482-484. Heilesen, B., 1946. Studies on Acarus scabiei and scabies. Acta Derm-Venereol. Suppl., 26: 1-370. Hewett, G.R., 1985. Phosmet for the systemic control of pig mange in growing pigs. Vet. Parasitol., 18: 265-268. Kofer, J., Glawischnig, E., Tockner, F. and Leitner, B., 1986. Therapy with Ivomec against ecto and endo parasites of swine. Wein. Tierarztl. Mschr. 73 Jahrgang Heft, 6: 196. Mellanby, K., 1944. The development of symptoms, parasitic infection and immunity in human scabies. Parasitology, 35: 197-206. Mellanby, K., 1946. Man's reactions to mosquito bites. Nature (London), 158: 554, 751. Morsy, G.H. and Gaafar, S.M., 1989. Response of immunoglobulin-secreting cells in skin of pigs during Sarcoptes scabiei infestation. Vet. Parasitol., 33: 165-175. Prakken, J.R. and Van Volten, T.J., 1949. Allergy in scabies. Dermatologica, 99: 124-131. Rantanen, T., Bjorksten, T.F., Reunala, T. and Salo, O.P., 1981. Serum IgE antibodies to scabies mites. Acta Dermatovener (Stockholm), 61: 358-360. SAS, 1985. User's guide. Version 5.0. SAS Institute Inc. Cary, NC. Sheahan, B.J., 1974. Experimental Sarcoptes scabiei infection in pigs: clinical signs and significance of infection. Vet. Rec., 94: 202-209. Sheahan, B.J., 1975. Pathology of Sarcoptes scabiei infection in pigs. II. Histological, histochemical and ultrastructural changes at skin test sites. J. Comp. Pathol., 85: 97-110. Stromberg, P.C. and 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: 1551-1559. Van Neste, D.J., 1986. Immunology of scabies. Parasitol. Today, 2: 194-196. Wooten, E.L. and Gaafar, S.M., 1984. Detection of serum antibodies to sarcoptic mange mite antigens by the passive hemagglutination assay in pigs infested with Sarcoptes scabiei var. suis. Vet. Parasitol., 15: 309-316. Wooten-Saadi, E., Broce, A.B., Stevenson, J.S. and Nelssen, J.L., 1987. Growth performance and behavioral patterns of pigs infested with sarcoptic mites (Acari: Sarcoptidae ). J. Econ. Entomol., 80: 625-628.