Journal of Medical and Veterinary Mycology (1992), 30, Supplement 1,307-316
Progress in veterinary mycology J. M. B. SMITH 1, R. AHO 2, R. MATTSSON 3 AND A. C. PIER 4
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1Department of Microbiology, University of Otago Medical School, Dunedin, New Zealand; 2Research Centerfor Pathogenic Fungi and Microbial Toxicoses, Chiba University Japan, National Veterinary Institute, Regional Laboratory, Oulu, Finland; 3National Veterinary Institute, Uppsala, Sweden; and 4Department of Veterinary Sciences, University of Wyoming, Laramie, USA The advent of human immunodeficiency virus (HIV) infection and AIDS, has given the study of fungal diseases in human medicine a tremendous boost. Unfortunately this is not so in veterinary medicine where mycotic diseases are still looked upon as unimportant and/or of little clinical relevance. Statements such as ' . . . that's far too important to be caused by a f u n g u s . . . ' still echo in the veterinary arena. This is rather unfortunate as fungal diseases are common and economically important in animals; indeed without some of the earlier veterinary studies, much of our knowledge on the causal agents and pathogenesis of mycoses in humans would still be fragmentary. For many years, laboratory animals have been utilized in the investigation of various fungal diseases. Earlier experimental animal models tended to be unsatisfactory in that they invariably employed massive inoculation of fungal elements, usually spores, by a completely foreign route to that involved in the disease situation; for example, 10s spores of Aspergillus fumigatus intravenously into a mouse devoid of functional white blood cells. This situation appears to be changing as more acceptable and logical animal models are developed; presently we have excellent experimental animal systems for diseases such as ringworm [13], candidosis, aspergillosis and cryptococcosis [39]. The rabbit subcutaneous chamber model would seem particularly amenable to a variety of studies [21]. How far results obtained in these experimental systems can be applied to natural human disease situations, however, is still a matter of some debate. Over the last 30 years or so, investigations into naturally occurring fungal diseases in animals have led to the recognition and clarification of the importance of these diseases and the causal fungi in man. Some examples are listed in Table 1. The purpose of this article is to highlight some of these animal studies, and to suggest ways in which they may benefit the study of human disease. TABLE 1. Examples of animal-linked studies relevant to the study of human fungal disease Aetiology of animal ringworm and its public health significance Potential of ringworm vaccines Sexual state (teleomorph) of dermatophytes Importance of fungal toxins in medicine Range and pathogenesis of opportunistic mycoses Improved diagnostic procedures, for example, induction of sporulation in apparently inert fungi.
Correspondence address: Dr John M. B. Smith, Department of Microbiology, University of Otago, P O Box 56, Dunedin, New Zealand. 307
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Dermatophyte vaccines Dermatophytes are the oldest known agents of infectious disease in animals and man and are responsible for a significant number of zoonotic infections. Perhaps the first report of ringworm as a zoonosis is that of a Swiss veterinary surgeon who in 1820 noted the infection of a young girl from cattle [38]. The importance of animals as a reservoir of the agents of human ringworm, however, was not fully appreciated until the pioneering work of Lucille Georg in the late 1950s; surprisingly little attempt has since been made to eliminate or at least reduce this animal reservoir which may in certain areas of the world contribute to 80-90% of all human ringworm [38]. The pattern of naturally occurring dermatophytosis in animals shows that infection is more frequent in the young; it is seldom recurrent and it is seldom caused by more than a single agent in a given animal. These facts suggest that an effective, lasting and broadbased immunity generally results from primary dermatophyte infection in animals and that vaccination is a potential control measure. Over the last decade, the clinical usefulness of a live Trichophyton verrucosum vaccine (LTF 130) in cattle has been aptly demonstrated in a number of areas of Europe and the USSR [17, 34, 43]. Experimental animal studies have demonstrated that cutaneous macrophages and T-lymphocyte populations participate in eliciting cell-mediated immune reactions associated with cutaneous hypersensitivity to dermatophyte antigens and resistance to primary infection and reinfection [11, 25, 33]. In addition, it has been possible, using experimental T. verrucosum infection in calves, to demonstrate an influx of macrophages, T4 and T8 lymphocytes and N cells into lesions 5 days post-inoculation which becomes well developed after 3 weeks (A. C. Pier, J. A. Ellis & K. W. Mills, Abstract 114, Conference for Research Workers in Animal Disease, Chicago, Illinois, 1990). Accumulation of immune globulins in the infected area was demonstrated and antibodies to culture filtrate antigens were found in the peripheral blood. These findings imply that a combination of cell-mediated and humoral events are involved in the immune response. Adoptive transfer of immunity to dermatophyte infection in mice has been demonstrated following injection of lymphocytes from infected donors [11]. Using guinea-pigs exposed to Trichophyton equinum or Trichophyton mentagrophytes by established methods [13], it has been possible to show that reduced infectivity results from both adoptive transfer of peripheral blood leukocytes and passive transfer of serum from donors hyperimmunized with killed fungal elements (A. C. Pier, unpublished results). In additional studies it was demonstrated (Pier et al., unpublished results) that live fungal elements of T. equinum did not persist in animal tissues following intramuscular injection; equal or better protection was achieved with killed preparations. The advantages of safety (no danger of inducing infection) and the ability to utilise a wide variety of adjuvant materials to enhance the immunological response, induced this group to develop killed preparations of mycelial and conidial elements mixed with adjuvants, as dermatophyte vaccines.
T. equinum vaccination of horses. Since their earlier studies, Pier et al., have conducted a series of controlled vaccine experiments in vaccinated horses (killed vaccine plus adjuvant), a non-vaccinated control group of horses, and an actively infected group that acted as infection donors. The horses were penned together and contact
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exposure used to challenge the vaccine induced immunity. Microscopic and cultural examinations augmented periodic clinical examinations of the principal and donor animals. Vaccination was found to protect around 87% of the horses from detectable infection and those that did become infected had very arrested lesions. The exposure system was considered to be a stringent challenge to the vaccine induced immunity. In a field exposure study, where pen contact for 4 months was maintained between vaccinated and non-vaccinated groups in a chronically T. equinum infected facility, over 90% of the vaccinated animals remained free of detectable infection while 70% of the non-vaccinated animals became infected. Examination of lesions in infected vaccinated animals showed an arrested infection in comparison to the disease seen in non-vaccinated controls. Continued use of this T. equinum vaccine on all incoming animals (around 3500 head) in an equine assembly and holding facility, coupled with an improved sanitation programme, resulted in a reduction in the naturally occurring infection rate from over 40% to essentially zero over a 3-year period. Broad spectrum dermatophyte vaccine (Dermato-Vacc IV) use in laboratory animals and cats. Studies by Pier et al. (unpublished results), have shown extensive antigenic and immune cross-reactions between T. equinum, T. mentagrophytes, T. verrucosum, Microsporum canis, Microsporum equinum and Microsporum gypseum. Cross-reactions between antigens of different dermatophyte species have previously been observed [25, 32]. However, Pier et al. were able to demonstrate cross-protection to experimental infection as well. It was on this basis that they developed a killed multivalent dermatophyte vaccine to protect companion animals and livestock from dermatophytosis. The vaccine (Dermato-Vacc IV) contains killed material from M. canis, M. gypseum, T. equinum and T. mentagrophytes, plus adjuvant. It elicits both delayed cutaneous hypersensitivity and ELISA responses to antigens of the above dermatophyte species and also to M. equinum and T. verrucosum. This vaccine was administered to guinea pigs, and the animals immunity challenged by housing them with guinea pigs actively infected with M. canis. Dermato-Vacc IV induced a solid immunity in all vaccinated animals; none of the vaccinated animals became infected. In contrast, 70% of the non-vaccinated controls developed lesions. A field trial of Dermato-Vacc IV was instituted in a cooperating commercial cattery that had been plagued for several years with enzootic dermatophytosis of M. canis origin. Use of the vaccine in breeding adults and kittens for 1 year drastically reduced the incidence of new infection. In conclusion, immunity to dermatophytosis in animals appears to involve participation by both cell-mediated and humoral constituents of the animals immune system [16, 28]. Killed preparations of dermatophyte mycelial elements and conidia induce strong cell-mediated and humoral responses in immunized animals. Killed dermatophyte vaccines offer advantages of safety and the ability to utilise a variety of adjuvants to enhance the immune response. Antigenic cross-reactions between dermatophyte species and genera afford cross-protection to infection by other dermatophyte agents. Use of killed vaccines to T. equinum and a broad spectrum vaccine (Dermato-
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Vacc IV) have provided high levels of protection (around 90%) in controlled experiments and in field test situations involving companion animals and livestock. The use of effective dermatophyte vaccines to control dermatophytosis in companion animals and livestock is considered a realistic approach to reducing zoonotic dermatophytosis of man.
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Mating behaviour of T. mentagrophytes of human and animal origin Since the pioneering work of Drs Dawson, Gentles and Stockdale in the late 1950s, perfect states for many dermatophyte species have been discovered; it is of interest that much of this work entailed studies with zoophilic and geophilic dermatophytes, anthropophilic species being notoriously difficult to induce into a sexual phase. The taxonomy of T. mentagrophytes (Robin) Blanchard has a complicated history and although two perfect states have to date been recognized for this conidial state, namely, Arthroderma benhamiae Ajello & Chang and Arthroderma vanbreuseghemii Takashio, many isolates do not mate with known tester strains of these two fungi. Hironaga & Watanabe [19] found that Japanese isolates belonged in three categories: (i) compatible with A. vanbreuseghemii; (ii) incompatible with known testers - 'type Trichophyton interdigitale'; and (iii) incompatible - 'granulosum-asteroides form'. Takashio [40] divided A. benhamiae into an American-European race and an African race, and observed that intra-racial matings were very fertile whereas inter-racial matings were only moderately or poorly so, or even sterile. He also recognized two varieties, var. caviae and var. erinacei, which were sexually degenerate in comparison to the normal strains of A. benhamiae. Variety erinacei belonged to the African race and vat. caviae to the American-European one. The geographical differences in the occurrence of the mating types are interesting. While Ajello & Cheng [1] and Padhye & Carmichael [30] discovered that North American isolates of T. mentagrophytes represented the A. benhamiae type, Hironaga & Watanabe [19] in Japan obtained successful matings only with A. vanbreuseghemii. Takashio [41, 42] found both perfect states in his strains which originated from Europe, New Zealand and Africa, and Hejtmfinek & Hejtmfinkov~ [18] obtained the same result even though A. benhamiae was dominant in their Czechoslovakian material. The methods used in mating experiments have a great influence on the results obtained. The original mating of A. benl~amiae was performed on moistened soil baited with pieces of horse hair [1]. Later Takashio [40] introduced diluted Sabouraud agar with salts, which was favourable for the mating of T. mentagrophytes. Padhye and colleagues [31] compared several keratinous and non-keratinous media, such as a soil and hair medium, soil extract hair agar, oatmeal salts agar, diluted Sabouraud glucose agar with salts, and diluted Pablum cereal agar with salts. The A. benhamiae tester pair which they used mated successfully on all of the media; they also recommended that a conidial suspension originating from young colonies be used as the inoculum, as tester strains often lost their sexual activity after several passages on maintenance media. Honma & Nishimoto [20] have recently published the results of a successful mating of A. vanbreuseghemii using Eagle's Minimum Essential Medium as a basal medium; they considered this superior to diluted Sabouraud with salts. With the above observations in mind, a series of experiments were undertaken to determine the mating type of Finnish and Swedish strains of T. mentagrophytes
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of animal origin, and of Finnish strains of human extraction. Diluted Sabouraud agar with salts was used with spores being obtained from young cultures. Of the 72 strains studied, 29 mated with the ' - ' strain of A. benhamiae. These were therefore compatible with A. benhamiae ('+' type) suggesting that this is the prominent species in northern Europe. Most (23) of the compatible strains were recovered from guinea-pigs, with the others being isolated from rabbits (3), a cat, and two cases of tinea capitis in children. Whether these two latter strains originated from animals is unknown. These results tend to endorse the European dominance of A. benhamiae in the T. mentagrophytes complex.
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Immunohistochemical identification of fungal elements in tissue sections
Aspergilli and fungi belonging to the Zygomycotina, such as Absidia corymbifera, are significant pathogens of domestic and wild animals [39]. Recovery of these fungi from clinical specimens is usually difficult or impossible; tissues are often grossly contaminated or too autolytic for isolation procedures to be successful, or are received already fixed in formalin. This means that in many instances, the diagnosis and presumptive identification of the causal fungus can only be made following examination of stained histological sections. Indirect immunofluoresence (IIF) has been used successfully for the identification of fungal elements in tissue sections [26, 27]. It was therefore decided to compare this technique with a peroxidase-anti-peroxidase (PAP) staining method developed at the National Veterinary Institute, Uppsala, Sweden, by modifying a technique used for the detection of bacterial and parasitological antigens in formalin fixed tissue of domestic and wild animals [10]. Antisera were prepared in rabbits by inoculation of antigenic material obtained from a strain of A. fumigatus and a strain of A.corymbifera obtained from animal lesions. All antisera were tested for antibody levels and specificity on formalin-fixed paraffin-embedded tissue sections from organs of animals with mycoses verified as either aspergillosis or zygomycosis. All histological sections were routinely deparaffined and rehydrated. Two antisera with good specificity and without apparent crossreactivity were chosen for further study; one of these was raised against A. fumigatus somatic antigens and the other against A. corymbifera metabolic antigens. Both preparations were obtained from 4-week cultures. With the IIF technique, optimal fluorescence was obtained with 1:100 antiserum dilutions and a 1:10 dilution of a commercially available FITC-labeUed sheep antirabbit globulin (National Bacteriological Laboratory, Sweden). PAP staining was performed as described elsewhere [10] and the peroxidase visualised with 3, 3 diaminobenzidine tetrahydrochloride. All sections were counterstained with Mayer's haematoxylin and rinsed under running tap water for blueing, before finally being mounted. Both the IIF and PAP techniques revealed excellent staining of fungal elements in tissue sections; the specificity of the antisera for aspergilli other than A. fumigatus and for zygomycetes other than A. corymbifera, however, was undetermined. It seems that the latter antiserum is likely to react with most other zygomycetes [39]. It was apparent, however, that the PAP technique was superior to IIF in some important details. It was much more sensitive which allowed a higher antibody dilution to be used. This is important in order to minimize both non-specific back-
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ground staining and cross-reactivity. PAP staining also gave a permanent slide preparation which could also be examined histologically with an ordinary light microscope. Since 1988, PAP staining has been used routinely at the National Veterinary Institute, Uppsala, Sweden, in parallel with more conventional fungal staining techniques such as periodic acid - Schiff, for the examination of tissues potentially infected with fungi. In addition, reexamination of paraffin-embedded tissues from confirmed cases of fungal disease since 1980 has been undertaken. In all cases, the PAP technique has given excellent results in confirming cases of aspergillosis and zygomycosis [23, 24].
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Other aspects of veterinary mycology potentially relevant to human disease Large outbreaks of opportunistic fungal infections are seldom recorded in humans; in most instances cases are isolated and reflect an immunocompromised individual exposed to the causal fungus. In lower animals, however, the opposite has often been the case with most infections only being recognised following the death or obvious involvement of large numbers of animals [39]. Obviously isolated cases often go unrecognized. Candidosis has been recognised in lower animals since at least 1858, and it seems that Candida other than Candida albicans, for example Candida tropicalis, have considerable pathogenic potential for animals; indeed in some animal species, C. albicans would seem to be one of the least significant pathogens [36, 39]. Oral and oesophageal lesions involving C. albicans have been demonstrated in a number of young animals, such as piglets and calves; terminally ill patients with AIDS are now presenting with similar lesions. Another important pathogen of piglets is Candida slooffiae (Candida pintolopesii var. slooffiae), which has been found to be frequently associated with stomach lesions in pigs fed a diet low in solid roughage [35, 36]. The obvious predisposing effects of captivity (? immunologically based stress), and the susceptibility of young immature animals, emerges from many of the reported animal studies on candidosis. The potential for chronicity and latency in central nervous system disease involving Cryptococcus neoformans was demonstrated naturally in guinea-pigs in 1977 [7]; this study also revealed the ability of this yeast to form hyphae in naturally occurring lesions. C. neoformans also appears to be a significant pathogen of cats and dogs and a number of wild animals such as the koala bear, where the commonest form of disease involves lesions of the nasal cavity and cutaneous tissues of the head and neck [39]. Animals often present with a copious nasal discharge; the public health implications of this is unknown. Similar facial lesions are seldom seen, or at least reported, in humans. Does this represent some difference in the pathogenesis of animal and human disease, or is it simply a reflection of the predilection of C. neoformans for growth at temperatures in the 30°C range? Unfortunately none of the strains of C. neoformans recovered from koala bears appears to have been serotyped [9, 12]. One can only assume that some, if not most, of these were C. neoformans var. gattii, as it has recently been shown by Ellis & Pfeiffer [15] that the natural habitat of this variety is probably the flowering red river gum tree (Eucalyptus camaldulensis), a habitat favoured by koalas. A tremendous volume of literature exists concerning the veterinary aspects of aspergillosis. This was one of the first mycoses of animals to be described and helped
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focus attention on the microbial cause of disease at a time when bacteria were still microscopical curiosities. In poultry, captive/stressed water birds and captive birds of prey, outbreaks of aspergillosis can be explosive with high mortality (up to 90% of young birds) and a clear indication of environmental contagion, such as decaying litter and straw, as the reservoir of the AspergiUus [39]. Acute disease involving the respiratory tract (air sacs and lungs) is invariably seen; A. fumigatus is the usual causal fungus. In older, possibly less susceptible birds, the disease may take a more chronic and sporadic course. A. fumigatus and the zygomycete Mortierella wolfii are proven and important agents of abortion in cattle and to a lesser extent other domesticated animals [3, 5, 22, 39]. Surprisingly, these two fungi are unrecorded as pathogens in similar human situations; reasons for this are unknown atthough the most obvious explanation is a lack of high level exposure to the causative fungi, although lack of some essential placental 'nutrient' may also be involved. In cattle, A. fumigatus is considered to enter the body via inhalation [22], and M. wolfii by ingestion of contaminated food such as silage, in animals with concurrent trauma to the mucosa of the alimentary tract [3]. In other animals, such as cattle and sheep, which are exposed to large numbers of airborne spores, asteroid lung lesions may be a feature [2, 39, 44]; in such lesions swollen distorted hyphae and/or condida are present which apparently represent arrested conidial development in otherwise normal hosts. This sort of lesion has not been recorded in humans. Localized involvement of the nasal cavity is an often recorded presentation of aspergillosis in animals such as dogs and horses [39]. This .sort of infection is now being recognized more frequently in man and seems to follow the traumatic implantation of spores, usually of A. fumigatus, into the mucosal surface of the nasal turbinates. An extreme form is the invasive paranasal granuloma due to Aspergillus flavus seen in hot, dry, geographical areas such as the Sudan [39]. An unusual form of disseminated aspergillosis has been recorded in dogs [14, 39]. This involves Aspergillus terreus and appears to be non-pulmonary in origin; at least a lack of respiratory involvement is a feature of the disease which also shows a predilection for a single breed of dog, the German shepherd. Does this indicate that genetic factors are important in pathogenesis of the infection? Another feature is that culture of urine sediment from affected dogs is invariably positive for the fungus. Should we be culturing urine in the attempted isolation of aspergilli from cases of invasive/disseminated disease in humans? Most mycological texts suggest that spores and the lungs are primarily involved in the pathogenesis of zygomycosis. Studies in animals would suggest otherwise with the finger being pointed more towards hyphal fragments and the alimentary tract mucosa [37]. Certainly mucocutaneous trauma appears to be a significant predisposing factor and has been noted regularly with Rhizopus microsporus (var. rhizopodiformis) and Saksenaea vasiformis infections in man, and with lesions caused by fungi of the order Entomophthorales in a variety of animals, including humans [39]. Animal studies have revealed the importance of the oomycete Pythium insidiosum as a pathogen of horses [39]; probably the first recognition of this disease was by Theobold Smith in the early 1890s. It now seems that this fungus is also capable of causing natural disease in many other animals such as dogs, cattle, cats, sheep and even man, and that lesions can occur in deep-seated organs as well as the
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more common cutaneous regions [8, 39]. Investigations [4] with this aquatic fungus have resulted in the development of media and techniques adequate for the induction of both asexual and sexual spores in a number of oomycetes and zygomycetes previously found to be inert from the point of view of sporulation [29]. To conclude, mycotic diseases are common in lower animals, and it is obvious that the study of veterinary mycology over the last 100 years or so has contributed vastly to our knowledge and understanding of many fungal diseases in man. As far as dermatology is concerned, much of our present day knowledge on the ecology, epidemiology, pathogenesis and taxonomy of dermatophytes has veterinary roots; hopefully the next decade will see the development of vaccines suitable for the control of animal ringworm become a distinct reality. Identification and characterization of the antigen(s) associated with immunity to dermatophyte infection, would now seem a priority area in medical mycology; ringworm is clearly an important zoonosis. In lower animals, mycoses often occur in explosive outbreaks with pathogenesis involving exposure of immature or stressed animals to massive amounts of environmental contagion. This situation is seldom seen in man, but it does appear that some lessons learnt in the veterinary area are applicable to humans; for instance, the observation that the occurrence of aspergillosis can be eliminated by careful environmental control of spore levels [6, 39]. In addition, our knowledge of the potential pathogenicity for man of many fungi such as P. insidiosum, has been heightened by the study of sporadic natural disease situations in animals. Some diagnostic and/or laboratory techniques now being used for the investigation of fungal infections in humans arose from earlier animal studies. More significant examples include the perfect states of dermatophytes, the use of serology in the investigation of opportunistic mycoses such as oomycosis and zygomycosis [8], the identification of fungal elements in tissue sections using immunohistochemical techniques, and the formulation of procedures for the induction of sporulation in what have been basically considered non-sporulating fungi, for example P. insidiosum. CONTRIBUTORS The contributors to this symposium were: A. C. Pier, Dermatophyte vaccines to protect livestock and companion animals; R. Aho Mating behaviour of T. mentagrophytes of human and animal origin; R. Mattsson, Immunohistochemical identification of fungal elements in tissue sections; J. M. B. Smith, Aspects of veterinary mycology relevant to human disease. The co-convenors were R. Aho and J. M. B. Smith. REFERENCES 1. AJELLO,L. & CHEN6.S. L. 1967. The perfect state of Trichophyton mentag~'ophytes. Sabouraudia, 5, 230-234. 2. AUSTWICK,P. K. C. 1962. The presence of Aspergillusfumigatus in the lungs of dairy cows. Laboratory Investigation, 11, 1065-1072. 3. AUSTWtCK,P. K. C- 1976. Environmentalaspects of MortiereUawolfii infectionin cattle. New Zealand Journal of Agricultural Research, 19, 25-33. 4. AUSTWICK,P. K. C. & COPLAND,J. W. 1974. Swamp cancer. Nature, 250, 84. 5. Ausvw~cK,P. K. C. & VENN,J. A. J. 1961. Mycoticabortion in England and Wales 1954-1960. In:
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31. PADHYE,A. A., SEKHON,A. S. & CARMICHAEL,J. W. 1973. Ascocarp production by Nannizzia and Arthroderma on keratinous and non-keratinous media. Sabouraudia, 11, 109-114. 32. PHILPOT, C. M. 1978. Serological differences among the dermatophytes. Sabouraudia, 16, 247-256. 33. POULAIN, D., TRONCHIN, G., VERNES, A., DELABRE, M. & BIGUET, J. 1980. Experimental study of resistance to infection by Trichophyton mentagrophytes: demonstration of memory skin cells. Journal of Investigative Dermatology, 74, 205-209. 34. SARK1SOV,A. K., PETROVITCH, S. V., NIKIFOROV, L. I., YABLOCHNIK, L. M. & KOROLEV, V. P. 1971. Immunization of cattle against ringworm. Veterinaria, No. 2, 54-56. 35. SMITH,J. M. B. 1967. Candidiasis in animals in New Zealand. Sabouraudia, 5, 220-225. 36. SMITH, J. M. B. 1968. Mycoses of the alimentary tract of animals. New Zealand Veterinary Journal, 16, 89-100. 37. SMITH, J. M. B. 1968. Experimental mycotic ulceration. Mycopathologia, 34, 353-358. 38. SMITH, J. M. B. 1975. Superficial and cutaneous mycoses. In: W. T. HUBBERT, W. F. McCuLLOCH & P. R. SCHNURRENBERGER(Eds) Diseases Transmitted From Animals to Man, pp. 469--487. Charles C. Thomas, Springfield, I1. 39. SMITH, J. M. B. 1989. Opportunistic Mycoses of Man and Other Animals. C.A.B. International, Wallingford. 40. TAKASHIO, M. 1972. Sexual reproduction of some Arthroderma and Nannizzia on diluted Sabouraud agar with or without salts. Mykosen, 15, 11-17. 41. TAr.ASHIO, M. 1977. The Trichophyton mentagrophytes complex. In: K. IWATA(Ed.) Recent Advances in Medical and Veterinary Mycology, pp. 271-276, University of Tokyo Press, Tokyo. 42. TAr.ASHIO,M. 1979. Taxonomy of dermatophytes based on their sexual states. Mycologia, 71,968-975. 43. WHO/ISHAM LIAISON COMMITTEE. 1987. Proceedings 1SHAM Consultation of Prevention and Control of Dermatophytosis with special reference to immunity and immunization. National Veterinary Institute, Oslo, Norway. 44. YouYG, N. E. 1970. Pulmonary aspergillosis in the lamb. Veterinary Record, 86, 790.
Progress in veterinary mycology J. M. B. SMITH 1, R. AHO 2, R. MATTSSON 3 AND A. C. PIER 4
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1Department of Microbiology, University of Otago Medical School, Dunedin, New Zealand; 2Research Centerfor Pathogenic Fungi and Microbial Toxicoses, Chiba University Japan, National Veterinary Institute, Regional Laboratory, Oulu, Finland; 3National Veterinary Institute, Uppsala, Sweden; and 4Department of Veterinary Sciences, University of Wyoming, Laramie, USA The advent of human immunodeficiency virus (HIV) infection and AIDS, has given the study of fungal diseases in human medicine a tremendous boost. Unfortunately this is not so in veterinary medicine where mycotic diseases are still looked upon as unimportant and/or of little clinical relevance. Statements such as ' . . . that's far too important to be caused by a f u n g u s . . . ' still echo in the veterinary arena. This is rather unfortunate as fungal diseases are common and economically important in animals; indeed without some of the earlier veterinary studies, much of our knowledge on the causal agents and pathogenesis of mycoses in humans would still be fragmentary. For many years, laboratory animals have been utilized in the investigation of various fungal diseases. Earlier experimental animal models tended to be unsatisfactory in that they invariably employed massive inoculation of fungal elements, usually spores, by a completely foreign route to that involved in the disease situation; for example, 10s spores of Aspergillus fumigatus intravenously into a mouse devoid of functional white blood cells. This situation appears to be changing as more acceptable and logical animal models are developed; presently we have excellent experimental animal systems for diseases such as ringworm [13], candidosis, aspergillosis and cryptococcosis [39]. The rabbit subcutaneous chamber model would seem particularly amenable to a variety of studies [21]. How far results obtained in these experimental systems can be applied to natural human disease situations, however, is still a matter of some debate. Over the last 30 years or so, investigations into naturally occurring fungal diseases in animals have led to the recognition and clarification of the importance of these diseases and the causal fungi in man. Some examples are listed in Table 1. The purpose of this article is to highlight some of these animal studies, and to suggest ways in which they may benefit the study of human disease. TABLE 1. Examples of animal-linked studies relevant to the study of human fungal disease Aetiology of animal ringworm and its public health significance Potential of ringworm vaccines Sexual state (teleomorph) of dermatophytes Importance of fungal toxins in medicine Range and pathogenesis of opportunistic mycoses Improved diagnostic procedures, for example, induction of sporulation in apparently inert fungi.
Correspondence address: Dr John M. B. Smith, Department of Microbiology, University of Otago, P O Box 56, Dunedin, New Zealand. 307
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Dermatophyte vaccines Dermatophytes are the oldest known agents of infectious disease in animals and man and are responsible for a significant number of zoonotic infections. Perhaps the first report of ringworm as a zoonosis is that of a Swiss veterinary surgeon who in 1820 noted the infection of a young girl from cattle [38]. The importance of animals as a reservoir of the agents of human ringworm, however, was not fully appreciated until the pioneering work of Lucille Georg in the late 1950s; surprisingly little attempt has since been made to eliminate or at least reduce this animal reservoir which may in certain areas of the world contribute to 80-90% of all human ringworm [38]. The pattern of naturally occurring dermatophytosis in animals shows that infection is more frequent in the young; it is seldom recurrent and it is seldom caused by more than a single agent in a given animal. These facts suggest that an effective, lasting and broadbased immunity generally results from primary dermatophyte infection in animals and that vaccination is a potential control measure. Over the last decade, the clinical usefulness of a live Trichophyton verrucosum vaccine (LTF 130) in cattle has been aptly demonstrated in a number of areas of Europe and the USSR [17, 34, 43]. Experimental animal studies have demonstrated that cutaneous macrophages and T-lymphocyte populations participate in eliciting cell-mediated immune reactions associated with cutaneous hypersensitivity to dermatophyte antigens and resistance to primary infection and reinfection [11, 25, 33]. In addition, it has been possible, using experimental T. verrucosum infection in calves, to demonstrate an influx of macrophages, T4 and T8 lymphocytes and N cells into lesions 5 days post-inoculation which becomes well developed after 3 weeks (A. C. Pier, J. A. Ellis & K. W. Mills, Abstract 114, Conference for Research Workers in Animal Disease, Chicago, Illinois, 1990). Accumulation of immune globulins in the infected area was demonstrated and antibodies to culture filtrate antigens were found in the peripheral blood. These findings imply that a combination of cell-mediated and humoral events are involved in the immune response. Adoptive transfer of immunity to dermatophyte infection in mice has been demonstrated following injection of lymphocytes from infected donors [11]. Using guinea-pigs exposed to Trichophyton equinum or Trichophyton mentagrophytes by established methods [13], it has been possible to show that reduced infectivity results from both adoptive transfer of peripheral blood leukocytes and passive transfer of serum from donors hyperimmunized with killed fungal elements (A. C. Pier, unpublished results). In additional studies it was demonstrated (Pier et al., unpublished results) that live fungal elements of T. equinum did not persist in animal tissues following intramuscular injection; equal or better protection was achieved with killed preparations. The advantages of safety (no danger of inducing infection) and the ability to utilise a wide variety of adjuvant materials to enhance the immunological response, induced this group to develop killed preparations of mycelial and conidial elements mixed with adjuvants, as dermatophyte vaccines.
T. equinum vaccination of horses. Since their earlier studies, Pier et al., have conducted a series of controlled vaccine experiments in vaccinated horses (killed vaccine plus adjuvant), a non-vaccinated control group of horses, and an actively infected group that acted as infection donors. The horses were penned together and contact
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exposure used to challenge the vaccine induced immunity. Microscopic and cultural examinations augmented periodic clinical examinations of the principal and donor animals. Vaccination was found to protect around 87% of the horses from detectable infection and those that did become infected had very arrested lesions. The exposure system was considered to be a stringent challenge to the vaccine induced immunity. In a field exposure study, where pen contact for 4 months was maintained between vaccinated and non-vaccinated groups in a chronically T. equinum infected facility, over 90% of the vaccinated animals remained free of detectable infection while 70% of the non-vaccinated animals became infected. Examination of lesions in infected vaccinated animals showed an arrested infection in comparison to the disease seen in non-vaccinated controls. Continued use of this T. equinum vaccine on all incoming animals (around 3500 head) in an equine assembly and holding facility, coupled with an improved sanitation programme, resulted in a reduction in the naturally occurring infection rate from over 40% to essentially zero over a 3-year period. Broad spectrum dermatophyte vaccine (Dermato-Vacc IV) use in laboratory animals and cats. Studies by Pier et al. (unpublished results), have shown extensive antigenic and immune cross-reactions between T. equinum, T. mentagrophytes, T. verrucosum, Microsporum canis, Microsporum equinum and Microsporum gypseum. Cross-reactions between antigens of different dermatophyte species have previously been observed [25, 32]. However, Pier et al. were able to demonstrate cross-protection to experimental infection as well. It was on this basis that they developed a killed multivalent dermatophyte vaccine to protect companion animals and livestock from dermatophytosis. The vaccine (Dermato-Vacc IV) contains killed material from M. canis, M. gypseum, T. equinum and T. mentagrophytes, plus adjuvant. It elicits both delayed cutaneous hypersensitivity and ELISA responses to antigens of the above dermatophyte species and also to M. equinum and T. verrucosum. This vaccine was administered to guinea pigs, and the animals immunity challenged by housing them with guinea pigs actively infected with M. canis. Dermato-Vacc IV induced a solid immunity in all vaccinated animals; none of the vaccinated animals became infected. In contrast, 70% of the non-vaccinated controls developed lesions. A field trial of Dermato-Vacc IV was instituted in a cooperating commercial cattery that had been plagued for several years with enzootic dermatophytosis of M. canis origin. Use of the vaccine in breeding adults and kittens for 1 year drastically reduced the incidence of new infection. In conclusion, immunity to dermatophytosis in animals appears to involve participation by both cell-mediated and humoral constituents of the animals immune system [16, 28]. Killed preparations of dermatophyte mycelial elements and conidia induce strong cell-mediated and humoral responses in immunized animals. Killed dermatophyte vaccines offer advantages of safety and the ability to utilise a variety of adjuvants to enhance the immune response. Antigenic cross-reactions between dermatophyte species and genera afford cross-protection to infection by other dermatophyte agents. Use of killed vaccines to T. equinum and a broad spectrum vaccine (Dermato-
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Vacc IV) have provided high levels of protection (around 90%) in controlled experiments and in field test situations involving companion animals and livestock. The use of effective dermatophyte vaccines to control dermatophytosis in companion animals and livestock is considered a realistic approach to reducing zoonotic dermatophytosis of man.
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Mating behaviour of T. mentagrophytes of human and animal origin Since the pioneering work of Drs Dawson, Gentles and Stockdale in the late 1950s, perfect states for many dermatophyte species have been discovered; it is of interest that much of this work entailed studies with zoophilic and geophilic dermatophytes, anthropophilic species being notoriously difficult to induce into a sexual phase. The taxonomy of T. mentagrophytes (Robin) Blanchard has a complicated history and although two perfect states have to date been recognized for this conidial state, namely, Arthroderma benhamiae Ajello & Chang and Arthroderma vanbreuseghemii Takashio, many isolates do not mate with known tester strains of these two fungi. Hironaga & Watanabe [19] found that Japanese isolates belonged in three categories: (i) compatible with A. vanbreuseghemii; (ii) incompatible with known testers - 'type Trichophyton interdigitale'; and (iii) incompatible - 'granulosum-asteroides form'. Takashio [40] divided A. benhamiae into an American-European race and an African race, and observed that intra-racial matings were very fertile whereas inter-racial matings were only moderately or poorly so, or even sterile. He also recognized two varieties, var. caviae and var. erinacei, which were sexually degenerate in comparison to the normal strains of A. benhamiae. Variety erinacei belonged to the African race and vat. caviae to the American-European one. The geographical differences in the occurrence of the mating types are interesting. While Ajello & Cheng [1] and Padhye & Carmichael [30] discovered that North American isolates of T. mentagrophytes represented the A. benhamiae type, Hironaga & Watanabe [19] in Japan obtained successful matings only with A. vanbreuseghemii. Takashio [41, 42] found both perfect states in his strains which originated from Europe, New Zealand and Africa, and Hejtmfinek & Hejtmfinkov~ [18] obtained the same result even though A. benhamiae was dominant in their Czechoslovakian material. The methods used in mating experiments have a great influence on the results obtained. The original mating of A. benl~amiae was performed on moistened soil baited with pieces of horse hair [1]. Later Takashio [40] introduced diluted Sabouraud agar with salts, which was favourable for the mating of T. mentagrophytes. Padhye and colleagues [31] compared several keratinous and non-keratinous media, such as a soil and hair medium, soil extract hair agar, oatmeal salts agar, diluted Sabouraud glucose agar with salts, and diluted Pablum cereal agar with salts. The A. benhamiae tester pair which they used mated successfully on all of the media; they also recommended that a conidial suspension originating from young colonies be used as the inoculum, as tester strains often lost their sexual activity after several passages on maintenance media. Honma & Nishimoto [20] have recently published the results of a successful mating of A. vanbreuseghemii using Eagle's Minimum Essential Medium as a basal medium; they considered this superior to diluted Sabouraud with salts. With the above observations in mind, a series of experiments were undertaken to determine the mating type of Finnish and Swedish strains of T. mentagrophytes
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of animal origin, and of Finnish strains of human extraction. Diluted Sabouraud agar with salts was used with spores being obtained from young cultures. Of the 72 strains studied, 29 mated with the ' - ' strain of A. benhamiae. These were therefore compatible with A. benhamiae ('+' type) suggesting that this is the prominent species in northern Europe. Most (23) of the compatible strains were recovered from guinea-pigs, with the others being isolated from rabbits (3), a cat, and two cases of tinea capitis in children. Whether these two latter strains originated from animals is unknown. These results tend to endorse the European dominance of A. benhamiae in the T. mentagrophytes complex.
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Immunohistochemical identification of fungal elements in tissue sections
Aspergilli and fungi belonging to the Zygomycotina, such as Absidia corymbifera, are significant pathogens of domestic and wild animals [39]. Recovery of these fungi from clinical specimens is usually difficult or impossible; tissues are often grossly contaminated or too autolytic for isolation procedures to be successful, or are received already fixed in formalin. This means that in many instances, the diagnosis and presumptive identification of the causal fungus can only be made following examination of stained histological sections. Indirect immunofluoresence (IIF) has been used successfully for the identification of fungal elements in tissue sections [26, 27]. It was therefore decided to compare this technique with a peroxidase-anti-peroxidase (PAP) staining method developed at the National Veterinary Institute, Uppsala, Sweden, by modifying a technique used for the detection of bacterial and parasitological antigens in formalin fixed tissue of domestic and wild animals [10]. Antisera were prepared in rabbits by inoculation of antigenic material obtained from a strain of A. fumigatus and a strain of A.corymbifera obtained from animal lesions. All antisera were tested for antibody levels and specificity on formalin-fixed paraffin-embedded tissue sections from organs of animals with mycoses verified as either aspergillosis or zygomycosis. All histological sections were routinely deparaffined and rehydrated. Two antisera with good specificity and without apparent crossreactivity were chosen for further study; one of these was raised against A. fumigatus somatic antigens and the other against A. corymbifera metabolic antigens. Both preparations were obtained from 4-week cultures. With the IIF technique, optimal fluorescence was obtained with 1:100 antiserum dilutions and a 1:10 dilution of a commercially available FITC-labeUed sheep antirabbit globulin (National Bacteriological Laboratory, Sweden). PAP staining was performed as described elsewhere [10] and the peroxidase visualised with 3, 3 diaminobenzidine tetrahydrochloride. All sections were counterstained with Mayer's haematoxylin and rinsed under running tap water for blueing, before finally being mounted. Both the IIF and PAP techniques revealed excellent staining of fungal elements in tissue sections; the specificity of the antisera for aspergilli other than A. fumigatus and for zygomycetes other than A. corymbifera, however, was undetermined. It seems that the latter antiserum is likely to react with most other zygomycetes [39]. It was apparent, however, that the PAP technique was superior to IIF in some important details. It was much more sensitive which allowed a higher antibody dilution to be used. This is important in order to minimize both non-specific back-
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ground staining and cross-reactivity. PAP staining also gave a permanent slide preparation which could also be examined histologically with an ordinary light microscope. Since 1988, PAP staining has been used routinely at the National Veterinary Institute, Uppsala, Sweden, in parallel with more conventional fungal staining techniques such as periodic acid - Schiff, for the examination of tissues potentially infected with fungi. In addition, reexamination of paraffin-embedded tissues from confirmed cases of fungal disease since 1980 has been undertaken. In all cases, the PAP technique has given excellent results in confirming cases of aspergillosis and zygomycosis [23, 24].
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Other aspects of veterinary mycology potentially relevant to human disease Large outbreaks of opportunistic fungal infections are seldom recorded in humans; in most instances cases are isolated and reflect an immunocompromised individual exposed to the causal fungus. In lower animals, however, the opposite has often been the case with most infections only being recognised following the death or obvious involvement of large numbers of animals [39]. Obviously isolated cases often go unrecognized. Candidosis has been recognised in lower animals since at least 1858, and it seems that Candida other than Candida albicans, for example Candida tropicalis, have considerable pathogenic potential for animals; indeed in some animal species, C. albicans would seem to be one of the least significant pathogens [36, 39]. Oral and oesophageal lesions involving C. albicans have been demonstrated in a number of young animals, such as piglets and calves; terminally ill patients with AIDS are now presenting with similar lesions. Another important pathogen of piglets is Candida slooffiae (Candida pintolopesii var. slooffiae), which has been found to be frequently associated with stomach lesions in pigs fed a diet low in solid roughage [35, 36]. The obvious predisposing effects of captivity (? immunologically based stress), and the susceptibility of young immature animals, emerges from many of the reported animal studies on candidosis. The potential for chronicity and latency in central nervous system disease involving Cryptococcus neoformans was demonstrated naturally in guinea-pigs in 1977 [7]; this study also revealed the ability of this yeast to form hyphae in naturally occurring lesions. C. neoformans also appears to be a significant pathogen of cats and dogs and a number of wild animals such as the koala bear, where the commonest form of disease involves lesions of the nasal cavity and cutaneous tissues of the head and neck [39]. Animals often present with a copious nasal discharge; the public health implications of this is unknown. Similar facial lesions are seldom seen, or at least reported, in humans. Does this represent some difference in the pathogenesis of animal and human disease, or is it simply a reflection of the predilection of C. neoformans for growth at temperatures in the 30°C range? Unfortunately none of the strains of C. neoformans recovered from koala bears appears to have been serotyped [9, 12]. One can only assume that some, if not most, of these were C. neoformans var. gattii, as it has recently been shown by Ellis & Pfeiffer [15] that the natural habitat of this variety is probably the flowering red river gum tree (Eucalyptus camaldulensis), a habitat favoured by koalas. A tremendous volume of literature exists concerning the veterinary aspects of aspergillosis. This was one of the first mycoses of animals to be described and helped
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focus attention on the microbial cause of disease at a time when bacteria were still microscopical curiosities. In poultry, captive/stressed water birds and captive birds of prey, outbreaks of aspergillosis can be explosive with high mortality (up to 90% of young birds) and a clear indication of environmental contagion, such as decaying litter and straw, as the reservoir of the AspergiUus [39]. Acute disease involving the respiratory tract (air sacs and lungs) is invariably seen; A. fumigatus is the usual causal fungus. In older, possibly less susceptible birds, the disease may take a more chronic and sporadic course. A. fumigatus and the zygomycete Mortierella wolfii are proven and important agents of abortion in cattle and to a lesser extent other domesticated animals [3, 5, 22, 39]. Surprisingly, these two fungi are unrecorded as pathogens in similar human situations; reasons for this are unknown atthough the most obvious explanation is a lack of high level exposure to the causative fungi, although lack of some essential placental 'nutrient' may also be involved. In cattle, A. fumigatus is considered to enter the body via inhalation [22], and M. wolfii by ingestion of contaminated food such as silage, in animals with concurrent trauma to the mucosa of the alimentary tract [3]. In other animals, such as cattle and sheep, which are exposed to large numbers of airborne spores, asteroid lung lesions may be a feature [2, 39, 44]; in such lesions swollen distorted hyphae and/or condida are present which apparently represent arrested conidial development in otherwise normal hosts. This sort of lesion has not been recorded in humans. Localized involvement of the nasal cavity is an often recorded presentation of aspergillosis in animals such as dogs and horses [39]. This .sort of infection is now being recognized more frequently in man and seems to follow the traumatic implantation of spores, usually of A. fumigatus, into the mucosal surface of the nasal turbinates. An extreme form is the invasive paranasal granuloma due to Aspergillus flavus seen in hot, dry, geographical areas such as the Sudan [39]. An unusual form of disseminated aspergillosis has been recorded in dogs [14, 39]. This involves Aspergillus terreus and appears to be non-pulmonary in origin; at least a lack of respiratory involvement is a feature of the disease which also shows a predilection for a single breed of dog, the German shepherd. Does this indicate that genetic factors are important in pathogenesis of the infection? Another feature is that culture of urine sediment from affected dogs is invariably positive for the fungus. Should we be culturing urine in the attempted isolation of aspergilli from cases of invasive/disseminated disease in humans? Most mycological texts suggest that spores and the lungs are primarily involved in the pathogenesis of zygomycosis. Studies in animals would suggest otherwise with the finger being pointed more towards hyphal fragments and the alimentary tract mucosa [37]. Certainly mucocutaneous trauma appears to be a significant predisposing factor and has been noted regularly with Rhizopus microsporus (var. rhizopodiformis) and Saksenaea vasiformis infections in man, and with lesions caused by fungi of the order Entomophthorales in a variety of animals, including humans [39]. Animal studies have revealed the importance of the oomycete Pythium insidiosum as a pathogen of horses [39]; probably the first recognition of this disease was by Theobold Smith in the early 1890s. It now seems that this fungus is also capable of causing natural disease in many other animals such as dogs, cattle, cats, sheep and even man, and that lesions can occur in deep-seated organs as well as the
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more common cutaneous regions [8, 39]. Investigations [4] with this aquatic fungus have resulted in the development of media and techniques adequate for the induction of both asexual and sexual spores in a number of oomycetes and zygomycetes previously found to be inert from the point of view of sporulation [29]. To conclude, mycotic diseases are common in lower animals, and it is obvious that the study of veterinary mycology over the last 100 years or so has contributed vastly to our knowledge and understanding of many fungal diseases in man. As far as dermatology is concerned, much of our present day knowledge on the ecology, epidemiology, pathogenesis and taxonomy of dermatophytes has veterinary roots; hopefully the next decade will see the development of vaccines suitable for the control of animal ringworm become a distinct reality. Identification and characterization of the antigen(s) associated with immunity to dermatophyte infection, would now seem a priority area in medical mycology; ringworm is clearly an important zoonosis. In lower animals, mycoses often occur in explosive outbreaks with pathogenesis involving exposure of immature or stressed animals to massive amounts of environmental contagion. This situation is seldom seen in man, but it does appear that some lessons learnt in the veterinary area are applicable to humans; for instance, the observation that the occurrence of aspergillosis can be eliminated by careful environmental control of spore levels [6, 39]. In addition, our knowledge of the potential pathogenicity for man of many fungi such as P. insidiosum, has been heightened by the study of sporadic natural disease situations in animals. Some diagnostic and/or laboratory techniques now being used for the investigation of fungal infections in humans arose from earlier animal studies. More significant examples include the perfect states of dermatophytes, the use of serology in the investigation of opportunistic mycoses such as oomycosis and zygomycosis [8], the identification of fungal elements in tissue sections using immunohistochemical techniques, and the formulation of procedures for the induction of sporulation in what have been basically considered non-sporulating fungi, for example P. insidiosum. CONTRIBUTORS The contributors to this symposium were: A. C. Pier, Dermatophyte vaccines to protect livestock and companion animals; R. Aho Mating behaviour of T. mentagrophytes of human and animal origin; R. Mattsson, Immunohistochemical identification of fungal elements in tissue sections; J. M. B. Smith, Aspects of veterinary mycology relevant to human disease. The co-convenors were R. Aho and J. M. B. Smith. REFERENCES 1. AJELLO,L. & CHEN6.S. L. 1967. The perfect state of Trichophyton mentag~'ophytes. Sabouraudia, 5, 230-234. 2. AUSTWICK,P. K. C. 1962. The presence of Aspergillusfumigatus in the lungs of dairy cows. Laboratory Investigation, 11, 1065-1072. 3. AUSTWtCK,P. K. C- 1976. Environmentalaspects of MortiereUawolfii infectionin cattle. New Zealand Journal of Agricultural Research, 19, 25-33. 4. AUSTWICK,P. K. C. & COPLAND,J. W. 1974. Swamp cancer. Nature, 250, 84. 5. Ausvw~cK,P. K. C. & VENN,J. A. J. 1961. Mycoticabortion in England and Wales 1954-1960. In:
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