EXPERIMENTAL DERMATOPHYTIC INFECTION OF GUINEA-PIGS AND CALVES

Rybnikaf, A., Chumela, J., Vrzal, V.: Experimental Dermatophytic Injection of Guinea-pigs and Calves. Acta vet. Brno, 54, 1984: 73-78. Typical mycotic lesions occurred in all infected guinea-pigs after application of ca 103 viable units of Microsporum canis onto 1 em2 of shaved and scarified skin. However, similar lesions on shaved but non-scarified skin were only observed with inocula of the same culture containing 105 units/cm2 skin, i.e. 100 times higher doses. Minimum infective doses of Trichophyton equinum and T. verrucosum necessary to produce experimental clinical skin trichophytosis in guinea-pigs amounted ca 100 units/em2 of shaved scarified skin (1 animal showed signs of the disease after application of 10 units/cm2 of T. verrucosum). Susceptibility of calves to the infective agent T. verrucosum was lower than that of guinea-pigs. Trichophytosis occurred only after inoculation of 104 units/em2 T. verrucosum in shaved and scarified skin, i.e. 100 times higher dose than that used with guinea-pigs. M. canis, T. equinum, T. verrucosum, inoculation doses. Experimental infection of animals has been an important component of efficacy testing of biologicals in veterinary medicine. The results of these tests may be influenced by a number of factors, e.g. the mode of experimental infection and the amount of inoculum. These factors also play an important role in challenge experiments with dermatophytic cultures. Experimental inoculation of pathogenic skin fungi is usually carried out by rubbing suspensions or pastes of the fungal culture into the shaved and often scarified skin of animals (Cox and Moore 1968; English et a!. 1979; Veronese et a!. 1981). Some workers inoculated the infective agent into non-scarified skin (Lepper 1972; Poulain et a!. 1980 and others). Various doses of infective material have been inoculated in such studies. However, no optimum inoculation doses for the individual dermatophyte cultures have been recommended. Therefore the present work was aimed at completion of these data. Materials and Methods In the experiment, lyophilized samples of 3 dermatophytes were employed: Microsporum canis Bodin, 1902, Trichophyton equinum (Matruchot et Dassonville) Gedoelst, 1902, Trichophyton verrucosum Bodin, 1902. The lyophilized samples were diluted in saline up to the selected concentrations based on previously determined density of viable microorganisms. Their density was determined both microscopically and using the plate dilution method. The culture was inoculated by rubbing 0.2 em3 of its suspension into the shaved and scarified skin of the right side of the bodies of 3-4 guinea-pigs using a rubber stopper. The size of the inoculation area was about 10 cm2 in all animals. The same animals were then inoculated with M. canis into the shaved non-scarified skin on the left side of the body. The number of inoculated organisms was verified by seeding of the respective diluted culture onto Sabouraud glucose-peptone agar. The experimental animals were examined from day 7 until day 12 after inoculation. Mycotic changes at the inoculation site were evaluated macroscopically. In an exploratory study also skin scrapings were seeded on Sabouraud's agar and the isolated cultures were identified. Similar experimental design was also employed with calves which were inoculated with various

Experimental infection of animals has been an important component of efficacy testing of biologicals in veterinary medicine.The results of these tests may be influenced by a number of factors, e.g. the mode of experimental infection and the amount of inoculum.These factors also play an important role in challenge experiments with dermatophytic cultures.Experimental inoculation of pathogenic skin fungi is usually carried out by rubbing suspensions or pastes of the fungal culture into the shaved and often scarified skin of animals (Cox and Moore 1968;English et a!. 1979;Veronese et a!. 1981).Some workers inoculated the infective agent into non-scarified skin (Lepper 1972;Poulain et a!. 1980 and others).Various doses of infective material have been inoculated in such studies.However, no optimum inoculation doses for the individual dermatophyte cultures have been recommended.Therefore the present work was aimed at completion of these data.

Materials and Methods
In the experiment, lyophilized samples of 3 dermatophytes were employed: Microsporum canis Bodin, 1902, Trichophyton equinum (Matruchot et Dassonville) Gedoelst, 1902, Trichophyton  verrucosum Bodin, 1902.The lyophilized samples were diluted in saline up to the selected concentrations based on previously determined density of viable microorganisms.Their density was determined both microscopically and using the plate dilution method.The culture was inoculated by rubbing 0.2 em3 of its suspension into the shaved and scarified skin of the right side of the bodies of 3-4 guinea-pigs using a rubber stopper.The size of the inoculation area was about 10 cm 2 in all animals.The same animals were then inoculated with M. canis into the shaved non--scarified skin on the left side of the body.The number of inoculated organisms was verified by seeding of the respective diluted culture onto Sabouraud glucose-peptone agar.The experimental animals were examined from day 7 until day 12 after inoculation.Mycotic changes at the inoculation site were evaluated macroscopically.In an exploratory study also skin scrapings were seeded on Sabouraud's agar and the isolated cultures were identified.
Similar experimental design was also employed with calves which were inoculated with various doses of T. verrucosum.Two cm 3 of suspension of T. verrucosum microconidia was _ rubbed into shaved scarified skin of the left side the body, on an area of approximately 100 cm2.Mycotic lesions were evaluated both macroscopically and by cultivation.

Results
The local mycotic skin lesions in guinea-pigs after inoculation of M. canis are indicated in Table 1.Typical lesions developed after inoculation of a minimum of 1000 elements/l cm 2 of scarified skin; Inoculation of smaller doses caused typical mycotic changes only in one animal infected with 100 units M. canis/l cm 2 of scarified skin.No changes in the rest of the animals were found.Inoculation of into non-scarified skin led to solitary mycotic lesions only at a dose as high as 10 5 units/cm 2 whereas the same dose inoculated into scarified skin resulted in a confluent mycotic crust on the whole scarified skin surface.The dose of 10 5 units/cm 2 caused confluent mycotic lesions also with T. equinum (Table 2) and'r.verrucosum (Table 3).Minimum concentration leading to trichophytic lesions was ca 100 units/cm 2 with both cultures (in one guinea-pig a mycotic lesion developed after only 10 units/cm 2 of T. verrucflsum).
meter.Inoculation of ca 10 5 units/cm 2 resulted in formation of confluent trichophytic lesions on the inoculated skin surface similar to those found in guinea-pigs.The crusts were prominent up to I em above the skin surface, th.ey broke.i~.~.kil!.fol<i §Il1!d after they had fallen off, haemorrhagic bottom was exposed.
The inoculated cultures were re-isolated from the cutaneous lesions in all guinea-pigs and calves with these lesions.

Discussion
Attempted experimental inoculation infections of guinea-pigs with 6 strains of T. verrucosum isolated from cattle, horses and sheep failed (Cox and Mo~r.e 1968).
However, inoculations of rabbits and calves with the same doses of these strains resulted in formation of typical trichophytic lesions.The minimum infective dose for rabbits was ca 10 3 units/cm 2 • Our results are at variance with data of the above-mentioned authors.Whereas successful experimental infection with T. verrucosum in guinea-pigs was achieved with as little as 100 viable units/cm 2 skin, in calves no lesions occurred unless a 100 times higher infective dosis of the same strain (i.e. 10 4 units/cm 2 skin) was used.Also virulence of various strains of the same dermatophyte species may differ considerably for one species of experimental animals, and appropriate inoculation doses should be selected.Determination of minimum infective doses for the individual strains of dermatophytes at standard modes of application is essential for successful experimental infection.For cattle, a minimum infective dose of T. verrucosum 10 3 viable units has been recommended (Lepper 1972).In our experiment, the disease in calves occurred only after application of 10 4 viable units/cm 2 • Hence the virulence of our T. verrucosum strain was lower than that reported by the above-mentioned author.
The minimum infective doses of T. equinum and T. verrucosum for guinea-pigs were practically identical (about 100 units/cm 2 ).However, with M. canis the minimum infective dose for guinea-pigs was 1000 units/cm 2 (in I of 3 animals it was only 100 units/cm 2 ).Similar infective doses of M. canis have been reported by English et a1. (1979).However, these data are only valid for inoculation into shaved and scarified skin.Our results show that the same M. canis culture inoculated into shaved but not scarified skin caused the disease only at doses 100 tiines higher.These results indicate that impaired skin integrity as achieved by scarification is a factor enhancing successful experimental dermatophytic infection.
It is noteworthy that a certain error is involved in the determination of minimum infective doses as after inoculation of the culture the animals often rub and scratch the' inoculated skin sO that a portion of the inoculum may be removed.In. spite of this disadvantage we consider the method employed in our trials closer to natural conditions than e.g.protection of the inoculum by tapes (J ones et al. 1974) or by a polyethylene chamber (Weigl 1976) even if these methods enable application of precise doses of inoculums with no losses.
In practice, our results may be used for estimation of optimum challenge doses in testing the efficacy of antimycotic vaccines.In our earlier experiments a breakdown of immunity occurred after application of extremely high challenge inoculum.Thus biased efficiency tests of otherwise high-quality preparations were obtained.Therefore, for challenge tests we recommend doses 10 to 100 IDso, i.e. 10 3 to 10 4 fungus elements/ cm 2 in guinea.,pigsand lOs to 10 6 fungus elements/cm 2 skin in calves when using M. canis~ T. verrucosum and T. equinum strains.

Table 1
Experimrntal epicutaneous inoculations of guinea-pigs with various doses of Microsporum canis