Research in VeterinaryScience 1995, 59, 110-113
Investigations of a killed dermatophyte cell-wall vaccine against infection with Microsporum canis in cats D. J. DeBOER, K. A. MORIELLO, Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, Wisconsin 53 706, USA
SUMMARY A laboratory-preparedkilled Microsporum canis cell-wall vaccine was evaluatedunder conditions simulating an accidental infection ofa cattery,by inoculating eight- to nine-week-oldcats with the vaccine or with a placebo control. The vaccinatedcats developed high titres of anti-dermatophyteIgG as measured by an ELISA,and a small cell-mediatedresponse against M canis as measured by a lymphocyteblastogenesis assay, using a whole ftmgus extract. After being inoculated the cats were challengedby the introduction of an infected cat into the same room. All the vaccinatedand control cats became culture-positivefor M canis within four weeks of the introductionof the infected cat. Four of the six control cats and all the vaccinatedcats developedlesions consistent with dermatophytosiswithin 16 weeks after exposureto the infected cat. CUTANEOUS infections with the dermatophytic fungus Microsporum canis are an endemic problem in multiple-cat households and breeding catteries worldwide (Quaife and Womar 1982, Zaror et al 1986). In such catteries, the shedding of infectious spores on hairs and scales during the relatively long course of the infection, combined with the durability of the fungal spores in the environment, make dermatophytosis exceptionally difficult to eradicate, and create a health hazard for human beings in contact with the animals. Most recommendations for the eradication of dermatophysis from catteries stress the aggressive treatment of all the cats with topical and systemic antifungal agents, together with vigorous environmental decontamination measures. Recently, a Microsporum canis killed vaccine has gained regulatory approval in the USA and is being marketed as an aid in the treatment of this disease. Vaccination against dermatophytic fungi is best documented in horses, fur-bearing animals and cattle. For these animals, live-spore fungal vaccines against various Trichophyton species were developed in Europe over 30 years ago (Segal 1989). Massive vaccination programmes in eastern Europe and the Scandinavian countries have dramatically reduced or eliminated the incidence of dermatophytosis in herds of cattle since the 1960s (Gudding and Naess 1986, Mackenzie et al 1986). On the basis of this success in other species, the development of Vaccines against M canis infection in cats has been advocated. Informal reports of the use of killed M canis mycelial preparations in cats have suggested therapeutic or prophylactic effects in some cases, and a worsening of the clinical signs of infection in others (Mosher et al 1977, Anonymous 1988, Smith et al 1992). A killed vaccine preparation Consisting principally of cell walls o f M canis was used to inoculate cats under laboratory conditions, followed by a challenge with large numbers of topically-applied spores (DeBoer and Moriello 1994). This vaccine induced antidermatophyte antibody titres similar to those produced during recovery from a natural infection, but only a small cell-mediated response against dermatophyte antigens. All the vaccinated cats developed dermatophytosis in response to the topical challenge, but the resulting lesions were sometimes smaller than the lesions produced in placebo-treated control cats.
Immunity to dermatophyte infection has generally been shown to be relative, ie, any state of immunity can be overcome by the application of a large enough challenge dose of the organism (Jones et al 1974). In the authors' previous study, it is likely that the large number of spores applied (105 per cat), and their direct application under occlusion to skin recently traumatised by close clipping, constituted a much more severe challenge than would be encountered by a naive cat exposed to an infected cat in a cattery. The purpose of the present study was to examine the prophylactic efficacy of the killed dermatophyte cell-wall vaccine under conditions that closely simulated natural transmission in a cattery.
MATERIALS AND METHODS Cats Fourteen barrier-reared domestic shorthaired cats, eight to nine weeks of age, were obtained from a colony with no history of dermatophytosis (Harlan Sprague Dawley). Six of the cats were inoculated with vaccine, six were inoculated with placebo, and two were retained for use as challenge animals. The cats were fed a dry kibbled feline growth ration and provided with water ad libitum. The housing conditions were chosen to resemble those in a typical breeding cattery. Each group of six cats was housed on the floor in individual biohazard containment rooms with rough-surfaced concrete floors, stainless steel open-fronted cages, and acrylic fleece sleeping mats. The cages and floors were cleaned weekly with hot water and 0.05 per cent sodium hypochlorite solution, and the sleeping mats were laundered weekly. The protocols and procedures used were reviewed and approved by the Animal Care and Use Committee before the experiment began. Killed dermatophyte cell-wall vaccine A crude killed M canis cell-wall antigen preparation was used for the inoculation of the cats. The method of preparation of this material has been described by DeBoer and
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Dermatophyte vaccine in Microsporum canis infection
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Moriello (1994). Briefly, bulk-cultured, killed M canis mycelium was subjected to mechanical homogenisation, followed by repeated washing by centrifugation in ice-cold 1 per cent sodium chloride solution and distilled water. The resulting suspension was filtered to remove large particles and lyophilised; it contained primarily cell-wall material, as assessed by electron microscopy. The lyophilised material was resuspended in buffered saline solution immediately before use. The batch of lyophilised antigen used as vaccine in the present study was identical to that used in the previous study.
Humoral immunity was measured by an enzyme-linked immunosorbent assay (ELISA) which measured IgG antibodies against dermatophyte glycoprotein antigens (DeBoer et al 1991, DeBoer and Moriello 1993). The sera were assayed at twofold serial dilutions from 1/16 to 1/2048, and the results were expressed as a titre, calculated as the -log 2 of the serum dilution that would theoretically yield an optical density of 0.200 in the test well. Samples with optical densities less than background (<0.100) at the 1/16 dilution were considered negative and were assigned a titre of O.
Inoculation and challenge exposure
Animal observations
Each of the six inoculated cats received 5 mg (0"5 ml) of the reconstituted vaccine preparation, given intradermally at five sites on both sides of the lateral thorax, when they were nine weeks old. The inoculation was repeated every two weeks, for a total of five doses. The six control cats each received an equivalent volume of buffered saline diluent, according to the same schedule. Two weeks after the last inoculation (week 10), all the cats in the vaccinated and control groups were challenged by exposure to an asymptomatically-infected cat. During the inoculation period, the two additional littermate eightweek-old kittens had been infected experimentally with a high fluorescent field strain o f M canis, using the procedure described by DeBoer and Moriello (1995). Because, under field conditions, it would be most likely that a cat breeder would unknowingly introduce a cat with a mild, inapparent infection rather than a florid, obvious infection, this experimental infection was timed so that when they were introduced the two cats had nearly resolved, grossly inapparent lesions that were visible only upon close examination with a Wood's light. At week 10, one of the infected cats was put into each of the rooms containing the control or vaccinated cats. For the first three days, the newly introduced cat was kept in a cage within the room to prevent fighting, and it was then released to associate freely with the uninfected cats. Neither the control nor the vaccinated cats were fitted with Elizabethan collars to restrict grooming. All the cats were monitored by visual observation, dermatophyte culture and immunological assays as described below, for 10 weeks after the introduction of the infected cats.
All the cats were examined for clinical evidence of dermatophytosis daily by a technician, and once or twice weekly by the investigators, throughout the study. Fungal cultures were prepared once weekly on dermatophyte test medium, using a sterile toothbrush for sampling the entire haircoat, as described by Moriello and DeBoer (1991). The culture plates were incubated for three weeks at 25°C, and any suspicious colony was examined microscopically to confirm the presence o f M canis. The fungal culture results were recorded semi-quantitatively on a 4-point scale, depending on the number of colonies present on a 90 mmdiameter plate, as follows: no colonies, score 0; one to five colonies, score 1; six to 10 colonies, score 2; more than 10 colonies, score 3. A careful dermatological examination was also made once a week, including an examination with an ultraviolet (Wood's) lamp, to reveal any lesions consistent with dermatophytosis. The ultraviolet examination was reported as either positive (glowing hair shafts found) or negative (no glowing hair shafts found). The gross visual examination was also reported as either positive (skin lesions found) or negative (no lesions found), and the nature and location of any lesions were recorded. Statistical analysis
The antibody titres, LBT stimulation indices and fungal culture scores were compared between the control and vaccinated cats by means of Student's t test. The results were considered significantly different at P<0.01.
Immunological assays
Assays to evaluate the degree of cell-mediated and humoral immunity against M canis were conducted at the beginning of the study, at the end of the inoculation period (week 10) and at the end of the study (week 20). Dermatophyte-specific cell-mediated immunity was assessed by means of a lymphocyte blastogenesis test (LBT) with soluble, whole-fungus M canis extract (DeBoer and Moriello 1993). Peripheral blood mononuclear cells were isolated by centfifugation over Ficoll-Hypaque and then cultured in the presence of an aqueous extract o f M canis whole fungus for 138 hours. Blastogenesis was assessed by measuring the incorporation of [3H]-thymidine during the final 18 hours of incubation. Each cat's general state of cellmediated immtmity was assessed by using the same test and the mitogen concanavalin A. The results of the LBTS were expressed as a stimulation index, calculated as the ratio of the counts per minute (CPM) in the test (antigen-containing) wells to the CPM in the control (unstimulated) wells.
RESULTS Immunological assays
The anti-dermatophyte IgG titres in the control and vaccinated cats are shown in Fig 1. Inoculation with killed dermatophyte cell-wall antigen resulted in the development of antibody titres in all the cats in the group. These titres were similar to those induced with a similar vaccination protocol in a previous study, and were of similar magnitude to the titres measured in cats which had recovered from an experimental M canis infection (DeBoer and Moriello 1994). Three of six control cats had developed low antibody titres by week 20, presumably as a result of the challenge exposure to the fungus. At the beginning of the study, the results of the LBT with concanavalin A were within the expected normal range, but throughout the remainder of the study they were variable, with no consistent pattern apparent between the groups after vaccination or challenge exposure (data not shown).
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FIG 1: Humeral (anti-dermatophyte IgG) and cellular immune response induced in cats by inoculation with killed dermatophyte antigen. Left, anti-dermatophyte antibody titres in vaccinated and control groups at weeks O, 10 and 20 of the study. Right, lymphocyte stimulation indices obtained with whole fungal antigens in the same cats. • vaccinated group, [ ] control group, * significantly different from control values (Student's t test, P<0.01). Error bars denote standard deviation
The results of the LBT with fungal antigen (Fig 1) showed that a cell-mediated immune response to the organism had developed in all the vaccinated cats by the end of the inoculation period (week 10), but no such response was observed in the control cats. The stimulation indices recorded with fungal antigen in the vaccinated cats were similar to those obtained previously, and approximately 60 per cent lower than the stimulation indices observed after recovery from infection (DeBoer and Moriello 1994). Animal observations
The majority of the cats in both the vaccinated and control groups developed lesions consistent with dermatophytosis after their exposure to an infected cat at week 10 (Fig 2). The lesions first appeared six weeks after exposure and consisted of scaling, crusting and focal alopecia; fluol
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rescing hair shafts could be observed upon examination with the Wood's lamp. The great majority of the lesions were on the head, ears, periorbital area and muzzle, with an occasional lesion on the dorsal thorax and no lesions on the limbs or other portions of the trunk. In several of the cats the earliest lesions appeared as scaling, comedones and Wood's lamp-positive hairs on the chin, clinically reminiscent of 'feline acne'. At the conclusion of the study, all six cats in the vaccinated group and four of the six cats in the control group had visible lesions. The weekly fungal cultures were first positive at week 12 in the control group and at week 14 in the vaccinated group (Fig 3). All six cats in each group became culture-positive at the same examination. Once positive, all the cats remained positive throughout the study. Because all the cats in the control group became culture-positive at week 12, and the vaccinated cats did not become positive until week 14, the control cats had a significantly higher fungal culture score than the vaccinated cats at weeks 12 and 13.
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The repeated inoculation of young cats with unadjuvanted cell-wall material from M canis induced a measurable humoral and cell-mediated immune response to the organism. The anti-dermatophyte antibody titres induced were similar to those recorded in cats which had recovered from an infection, but the cell-mediated response induced was considerably less than that typically seen after recovery (DeBoer and Moriello 1994). Despite the induction of this immune response, the cats were not protected from infection by challenge exposure to a cat with active dermatophytosis. These results provide fi~rther evidence that the induction of high antibody titres against M canis is not sufficient to provide protection against infection. The authors' earlier study demonstrated that the immune response produced by a similar vaccination protocol did not protect cats from contracting an infection when they were challenged by the topical application of 105 M canis spores
Dermatophyte vaccine in Microsporum canis infection
under occlusion. All the vaccinated cats in this previous study developed lesions, but the lesions were sometimes smaller than in unvaccinated animals, suggesting that partial immunity had been conferred. This method of topical challenge is considerably more severe than what one might expect to occur in a breeding cattery, when there would probably be fewer spores, which would contact the haircoat rather than the skin and not be under occlusion; furthermore, they would be subject to being removed by the cat's own grooming behaviour. The present study demonstrates that the vaccination protocol was not able to protect against even the more limited challenge provided by casual contact with an asymptomatically-infected cat. Fungal cultures from the naive cats became positive within two to four weeks after exposure to the infected cat, and visible lesions of dermatophytosis developed two to 16 weeks later. The lag period between the positive cultures and the lesions was most likely a period during which there was gradually increasing contamination of the environment and the hairs of the naive cats with infective spores. From a clinical perspective, this lag period also emphasises the difficulty in differentiating between a truly infected cat, and a cat that is merely carrying spores on its haircoat. Airborne or direct-contact transmission of spores to naive cats is tempered by the cats' grooming behaviour which can result in the failure of the infection to become established (DeBoer and Moriello 1995). The possible influence of grooming on the transmission of the infection is further evidence by the finding that nearly all the lesions that developed in both the vaccinated and control cats were on the head and face, which are relatively difficult areas for a cat to groom efficiently. In one study of experimental dermatophytosis in guinea pigs, the air and animals in separate cages in the same room became contaminated within 10 days of the introduction of an infected animal. Such enviroumental contamination was particularly noticeable with M canis, less so with Trichophyton mentagrophytes, and negligible with other Microsporum, Trichophyton or Epidermophyton species (Chittasobhon and Smith 1979). Thus, M canis becomes disseminated particularly rapidly upon the introduction of an infected animal, and decontamination is a major challenge when eradicating the fungus from an infected cattery. Positive fungal cultures were observed in the vaccinated cats two weeks later than in the control group (Fig 3); this result was unexpected, and remains unexplained. It is possible that the difference could be attributed to an effect of the vaccine, but the authors consider that it was more likely to have been due to some difference between the cats that were used as sources of infection in the two rooms. Though the two littermate cats were infected experimentally at the same time, by the same protocol and with the same inoculure, and they appeared to be equally infected when they were introduced, it is possible that the cat in the control room was shedding more spores than the cat in the room containing the vaccinated cats. Alternatively, it is possible that the cat introduced into the group of vaccinated cats was less socially interactive with the naive cats, thus delaying the transmission of the infection. Most previous studies of prophylactic vaccination against dermatophytosis have been carried out in cattle, horses, fur-bearing animals and laboratory rodents. The studies with rodents have invariably used direct topical application of spores as an experimental challenge. Such models have the advantage of constant experimental conditions, making them suitable for mechanistic studies and
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vaccine development. For example, such a model was useful for demonstrating that live-spore and fungal cell-wallbased vaccines induced a more pronounced immune response than fungal cytoplasmic extracts (Hussin and Smith 1983). However, from a practical standpoint, a vaccine must ultimately be tested under conditions that will simulate its use. Thus, after their initial development, commercial live-spore dermatophyte vaccines for horses and cattle were tested by using systems incorporating natural transmission from infected to uninfected animals (Segal 1989, Smith et al 1992). The results of such studies have not previously been reported for vaccines against feline dermatophytosis. Vaccination still holds promise as a method of controlling feline dermatophytosis. By the proper formulation of the fungal antigen and the use of adjuvants, it is possible that a stronger cell-mediated immune response to M canis may be induced, and/or even higher antibody titres, and it is possible that these responses may confer prophylactic immunity. However, as dermatophyte vaccine products for cats are introduced on the commercial market, it is important that their prophylactic and therapeutic efficacy should be well documented, and related to their ability to induce humoral and cell-mediated immune responses in the host. ACKNOWLEDGEMENTS The authors thank Laura Ayers, Michelle Fintelmann, Xinmin Yue and Robin Donner for technical assistance. This study was supported by a grant from the Robert H. Winn Foundation.
REFERENCES ANONYMOUS (1988) Comments on the use of autogenous fungal vaccines in feline dermatophytosis. Cat Tracks Winter Issue, 42-45 CHITTASOBHON, N. & SMITH, L M. B, (1979) The production of experimental dermatophyte lesions in guinea pigs. Journal of Investigative Dermatology 73, I98-201 DeBOER, D. J., MORIELLO, K. A. & COOLEY, A. J. (1991) Immunological reactivity to intradermal dermatophyte antigens in cats with dermatophytosis. Veterinary Dermatology 2, 59-67 DeBOER, D. J. & MORIELLO, K. A. (1993) Humoral and cellular irmnune responses ofMierosporum canis in naturally occurring feline dermatophytosis. Journal of Medical and Veterinary Mycology 31, 121-132 DeBOER, D. J. & MORIELLO, K. A. (1994) The immune response to Mierosporum canis induced by a fungal cell wall vaccine. Veterinary Dermatology 5, 47-55 DeBOER, D. J. & MORIELLO, K. A. (1995) Development of an experimental model of Microsporum canis infection in cats. Veterinary Microbiology 44, 105-113 GUDDING, R. & NAESS, B. (1986) Vaccination of catfle against ringworm caused by Trichophyton verrucosum. American Journal of Veterinary Research 11, 2415-2417 HUSS1N, Z. & SMITH, J. M. B. (1983) Vaccination procedures and the infectivity of dermatophyte lesions. Mycopathologia 81, 71-76 JONES, H. E., REINHARDT, J. H. & RINALDI, M. G. (1974) Acquired immunity to dermatophytes. Archives of Dermatology 109, 840-848 MACKENZIE, D. W. R., LOEFFLER, W., MANTOVANI, A. & FUJIKURA, T. (1986) Guidelines for the diagnosis, prevention and control of dermatophytoses in man and animals. Geneva, World Health Organization. pp 55-68 MORIELLO, K. A. & DeBOER, D. J. (1991) Fungal flora of the haircoat of cats with and without dermatophytosis. Journal of Medical and Veterinary Mycology 29, 285-292 MOSHER, C. L., LANGENDOEN, K. & STODDARD, P. (1977) Treatment of ringworm (Microsporum canis) with inactivated fungal vaccine. Veterinary Medicine/Small Animal Clinician 72, 1343-1345 QUAIFE, R. A. & WOMAR, S. M. (1982) Microsporum canis isolations from show cats. Veterinary Record 110, 333-334 SEGAL, E. (1989) Vaccines for the management of dermatophyte and superficial yeast infections. Current Topics in Medical Mycology 3, 36-49 SMITH, J. M., AHO, R., MATTSSON, R. & PIER, A. C. (1992) Progress in veterinary mycology. Journal of Medical and Veterinary Mycology 31} (Supplement 1), 307-316 ZAROR, L., FISCHMANN, O., BORGES, M., VILANOVA, A. & LEVITES, J. (1986) The role of cats and dogs in the epidemiological cycle of Microsporum canis. Mykosen 29, 185-188
Received September 21, 1994 Accepted March 6, 1995