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Observations on the experimental pathogenicity and toxigenicity of Mortierella wolfii strains of bovine origin
Br. vet . 7. (1991) . 147, 5 04
OBSERVATIONS ON THE EXPERIMENTAL PATHOGENICITY AND TOXIGENICITY OF MORTIERELLA WOLFII STRAINS OF BOVINE ORIGIN
M . J . CORBEL* and SUSAN M . EADES Ministry of Agriculture, Fisheries & Food, Central Veterinary Laboratory, Weybridge KT15 3NB
SUMMARY Four strains of Mortierella wolfii isolated from cattle in Britain were compared in pathogenicity and toxigenicity with a strain isolated from a cow with the mycotic abortion-pneumonia syndrome in New Zealand . All strains produced acute lethal infection in rabbits after intravenous inoculation of mycelial suspensions and all produced subacute mycotic encephalitis in mice after intracerebral injection . They also produced an acid-stable, heat- and trypsin-labile toxin in vitro . The action of the toxin was exerted mainly on the kidneys in rabbits and mice and produced effects distinct from those resulting from infection with M. wolfii.
INTRODUCTION Most cases of bovine mycotic abortion in Britain have been attributed to infection with Aspergillus fumigatus, related Aspergillus species or Absidia corymbifera (Hugh Jones & Austwick, 1967) . Hitherto, Mortierella wolfii or other Mortierella species have only rarely been identified as the cause of the syndrome (Harcourt & Thompson, 1969 ; Johnson et al., 1990) although implicated in a high proportion of cases in New Zealand (di Menna et al., 1972 ; Carter et al., 1973 ; Austwick, 1976) . The infrequency with which M. wolfii has been implicated as a cause of infection in this country probably reflects the difficulty of isolating it from contaminated material rather than its true significance as a pathogenic agent . Thus in recent years M. wolfii has been identified in a number of cases of infection in cattle, including several of mycotic abortion . Other studies have established the presence of M. wolfii in the British environment (Evans, 1971a, b) . Nevertheless, although it is clear that M. wolfii is present in this country and accounts for a proportion of bovine fungal infections, it is also apparent that the distinctive mycotic abortion-pneumonia syndrome associated with the fungus in New Zealand has been rarely recorded here . Indeed, a survey of the literature disclosed only a single reported case of the syndrome (Harcourt & Thompson, 1969) . *Present address : National Institute for Biological Standards & Control, Blanche Lane, South Mimms, Potters Bar, ENG 3QG.
MORTIERELLA WOLFII PATHOGENICITY
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As such a characteristic syndrome is unlikely to be widely overlooked it appears that the manifestations of M. wolfii infection in cattle in this country may differ somewhat from those seen in New Zealand . No reasons for such a difference have been disclosed but an obvious possibility is variation in the pathogenicity of the M. wolfii strains prevalent in the two countries . It may be significant in this respect that differences in the experimental pathogenicity of strains of the fungus obtained from different environments have been reported (Cordes et al., 1972a, b) . The determinants of the pathogenicity of M. wolfii for cattle have not been identified but the fungus has been shown to elaborate a _nephrotoxin in vitro and it has been suggested that this may play a role in natural infections (Davey et al., 1973, 1975) . Although direct evidence has not been presented to this effect, it is possible that toxin production could be a major factor in determining the virulence of individual M. wolfii strains . Variations in toxigenicity could also account for differences in the clinical syndromes by different strains of the fungus . This possibility has been examined for several strains of M. wolfii isolated from cattle in the UK .
MATERIALS AND METHODS Fungal strains M. wolfii strain V .73/414 in the laboratory collection was obtained from the Common-
wealth Mycological Institute, Kew as M. wolfii strain IMI 149020. It was originally isolated in New Zealand from the lung tissue of a cow with the mycotic abortionpneumonia syndrome . M. wolfii strains V .74/21 (ATCC .36820), V .75/125 and V .75/173 were isolated from the stomach contents of aborted fetuses in the UK . M. wolfii strain V .76/183 was isolated from the liver of a 2-week-old calf . Details of these cases have been described previously (MacDonald & Corbel, 1981) . Identification of Mortierella isolates
The cultural and morphological criteria used for the identification of isolates were those described in the literature (Zycha et al., 1969) . All strains identified as M. wolfii conformed to the description given by Mehrotra & Baijal (1963) . The isolates were submitted to the Commonwealth Mycological Institute, Kew, for confirmation of their identity . Preparation of inocula As all of the Mortierella strains used in this study sporulated poorly under laboratory
conditions, only mycelial suspensions were used for animal inoculation . These were prepared as described previously (Corbel & Eades, 1977) . Preparation of cellfree extracts The M. wolfii strains were grown in Difco glucose-neopeptone broth incubated at 37°C for 14 days, as described previously (Davey et al., 1973) . The culture supernatant from
each strain was decanted and concentrated by counterdialysis against polyethylene glycol 20 .%1 . The mycelial mats were collected separately and disrupted by four cycles of freeze pressing at -25°C in an Edebo X-press (LKB-Biotec, Croydon) . The mycelial extracts were freed of particulate material by centrifugation at 10 000 gfor 20 min and the
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supernatants collected and concentrated by counterdialysis against polyethylene glycol 20 M . These and the concentrated culture supernatants from each strain were pooled and sterilized by membrane filtration and stored at 4 °C until required . The protein concentration of each preparation was determined by the Folin-biuret method using a bovine serum albumin standard (Lowry et al., 1951). All cell-free extracts and the insoluble debris separated by centrifugation were examined for mycotoxins by thin layer chromatography of ethyl acetate extracts as described elsewhere (Roberts & Patterson, 1975) .
Animal inoculation The pathogenicity of M. wolfii isolates for mice was determined essentially as described previously (Corbel & Eades, 1977) . Thus groups of six albino mice between 18 and 22 days old were injected by the intravenous, intramuscular, intraperitoneal or subcutaneous routes with 0 .1 ml volumes of mycelial suspensions containing between 1 and 10' viable units of M. wolfii. Similar groups of animals were injected by the intracerebral route with 0 .025 ml volumes of mycelial suspension . Subsequent treatment and observation of the animals was as described previously (Corbel & Eades, 1977) . The pathogenicity of M. wolfii strains for rabbits was examined by injecting groups of four Californian rabbits with 1 .0 ml volumes of graded dilutions of mycelial suspensions by the intravenous route . Equal numbers of male and female rabbits about 3 months old were used for each strain (see Table II) . The rabbits were inspected daily for abnormal signs . As soon as any evidence of disease became apparent they were examined at hourly intervals until death . Once the pattern of the disease had been established, rabbits in the terminal stages of infection were killed by intravenous injection of sodium pentobarbitone . Pathological and mycological examination This was done as described previously (Corbel & Eades, 1977) . Histological sections were stained with haematoxylin and eosin (H & E) or by the PAS-celestine blue method (Young, 1968) . Toxigenicity studies The toxicity of cell-free extracts of M. wolfii prepared from culture supernatants or from mycelium was assayed in mice and rabbits . Groups of six albino mice approximately 6 weeks old and matched for weight were given intravenous injections of 0 .1 ml volumes of M. wolfii extracts (see Table III) . These were used undiluted and at 1/10, 1/50, 1/100 and 1/1000 dilutions in phosphate buffered saline (PBS : 0 .15 mol/l NaCl in 0 .01 mol/l phosphate buffer, pH 7 .4) . The animals were observed at least once daily for up to 7 days . Those surviving at the end of this time were killed under ether anaesthesia . All mice were examined post mortem for both gross and microscopic lesions . Histological examinations were performed on H & E stained sections of formalin-fixed tissue . Samples of urine were collected by aspiration from the bladder with a disposable syringe and needle . Tests for protein concentration, glucose, haemoglobin and pH reaction were done using Hemacombistix (Ames Co . Ltd, Slough) . Similar tests were performed on groups of six albino mice given intracerebral injections of 0 .025 ml volumes of undiluted M. wolfii extracts . Toxin characterization was performed by a similar procedure by giving the mice intravenous injections of M. wolfii cell-
MORTIERELLA WOLFLI PATHOGENICITY
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free extracts which had been treated with trypsin (500 BAEE units/ml), heat (60 °C/ 1 h) or acid (0 .1 mol/1 HCl) (Davey et al., 1973) . Similar procedures were used for the toxicity tests in rabbits . For these, pairs of 3-month-old Californian rabbits were given intravenous injections of 1 .0 ml volumes of toxin preparation . The animals were observed for up to 7 days, their rectal temperatures were recorded at 8-hourly intervals for the first 3 days and at 12-hourly intervals for the remaining time . Post-mortem examinations were made as described for the mice . Toxin preparations were also examined for a dermonecrotic effect in guinea-pigs . Pairs of female albino guinea-pigs were depilated along both flanks and 0 .1 ml volumes of dilutions of M. wolfii extracts in PBS injected intradermally . Control injections of similar volumes of PBS were injected at intervals of 5 cm from the toxin preparations . On the following day I ml volumes of 1% w/v Evans' blue in saline were injected by the intracardiac route . The animals were killed 10 min later under anaesthesia and the skin removed and examined for infiltration of dye and other signs of local reaction . Histological examinations were made on H & E stained sections of formalin-fixed skin from the test and control sites .
RESULTS Comparative pathogenicity of M . wolfii strains None of the M. wolfii strains produced lethal infection in mice when inoculated by the intramuscular, intraperitoneal, intravenous or subcutaneous routes . Furthermore, the fungus was not recovered from the mice killed at the end of the observation period, nor did those animals show any gross lesions at necropsy . In contrast, intracerebral inoculation of all of the M. wolfii strains produced lethal infection in the majority of mice (Table I) . The number of deaths did not differ significantly between the groups but individual animals showed considerable variation in the interval between inoculation and death . This varied from a minimum of 4 days up to 48 days . It was not possible to determine any pattern characteristic of any one strain as variations occurred within the groups inoculated with different strains . Those mice developing acute infection showed clinical signs similar to those observed in acute zygomycosis produced by infection with other zygomycete species (Corbel & Eades, 1975) . Thus the animals became ataxic, refused food and water and developed continuous circling movements which preceded a terminal comatose state . This pattern was only seen in those animals which developed signs of disease less than 1 week after inoculation . In those developing the more chronic disease the pattern was similar to that previously described for M. wolfii strain V .74/21 (Corbel & Eades, 1977) . These mice developed granulomatous infiltrations surrounding the third cerebral ventricle and the aqueduct . This resulted in a chronic hydrocephalus with severe distension of both cerebral hemispheres . Some of the mice which developed subacute or chronic cerebral infection also had pulmonary lesions . These consisted of areas of greyish-yellow consolidation usually involving complete lobes of the lung and sometimes the whole lung . M. wolfii was readily recovered from both the cerebral and pulmonary lesions but not from other tissues . The characteristic narrow non-septate Mortierella hyphae were easily detected in histological sections of brain and lung tissue stained by the PAS-celestine blue method . In H & E
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MORTIERELLA WOLF11 PATHOGENICITY
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stained sections, the brain tissue showed pronounced granulomatous changes with whole areas of cerebral parenchyma replaced by infiltrations of mononuclear phagocytes and lymphocytes . The affected lung tissue showed extensive necrosis with an inflammatory response involving both mononuclear and polymorphonuclear leucocytes . Examination of the urine of mice dying after intracerebral inoculation with M. wolfii strains indicated no abnormality in pH, protein, glucose or haemoglobin content . The rabbits inoculated with M. wolfii suspension by the intravenous route developed acute lethal infection within 3-7 days (Table II) . The disease was rapid in onset, with'the animals developing signs of general loss of condition, anorexia, shivering, reduced mobility, respiratory distress and in some cases mucoid diarrhoea . Some of the animals developed corneal erosions which were usually unilateral and accompanied by release of a watery or purulent discharge from the affected eye . There was frequently a watery or blood-stained purulent discharge from the nose . Death usually occurred within a few hours of these signs becoming apparent . In the terminal phase, the body temperature was usually 1-2°C below normal . The urine was frequently blood-stained and semiquantitative tests indicated a reaction between pH 6 and pH 8, usually near pH 6, an absence of glucose, the presence of moderate to large quantities of haemoglobin and a protein concentration greater than 1000 mg/100 ml . At post-mortem examination, similar pathological changes were seen in all rabbits irrespective of the strain of M. wolfii used for inoculation . Generally these consisted of a large volume of serosanguineous fluid in the abdominal cavity, with a glassy fibrinous deposit over the dorsal abdominal muscles . Petechial haemorrhages were observed in the brain, heart, lungs, kidneys, spleen, large and small intestines, uterus and thymus . The most distinctive lesions were present in the liver which usually had a marbled appearance with multiple pale necrotic foci scattered throughout the parenchyma . These lesions were approximately spherical and consisted of a grey or greyish-yellow central region (57 mm in diameter, surrounded by a narrow haemorrhagic zone (Fig . 1) . In some cases complete lobes of liver or even the entire organ, were reduced to a friable grey necrotic mass . The spleen frequently contained several yellowish/white nodules about 3 mm in diameter, or in some cases a single large cauliform mass which completely distorted its structure . Multiple haemorrhages were often present in the wall of the duodenum, ileum and caecum . They usually consisted of numerous scattered petechiae but in some rabbits more extensive haemorrhages discharging into the intestinal lumen were present . Extensive haemorrhages were also present in the pancreas in the more severely affected animals . The kidneys often showed only slight subcapsular mottling but in some rabbits yellowish-grey necrotic patches were present on the poles of the kidneys where they had contacted similar lesions in the liver or spleen . Occasionally these necrotic changes extended locally to the dorsal abdominal muscles . On sectioning the kidneys, the medulla was usually very congested with numerous focal haemorrhages . Scattered small subcapsular infarcts were frequently present . In a few animals one of the kidneys was swollen to approximately twice the normal size . In such cases very large yellowish infarcts involving most of the renal parenchyma were seen on sectioning the organ . The lungs usually showed extensive areas of grey necrotic consolidation . In one animal extensive adhesions had formed between areas of affected lung and the pericardium . Petechiae were widespread throughout the skeletal muscles which also sometimes appeared oedematous . Histological examination of H & E and PAS-celestine blue-stained sections showed that the lesions in the liver, lungs and other organs usually consisted of extensive areas of
Cultural
and microscopic
examinations hnwo/i
v731414 v74121 V75/125 VW173 V761113
*Includes
KY I@ I@ lo" I@
212 212 212 212 212
3 6.5 5.5 3 3
012 012 012 012 012
results of histology and examination
112 o/2 112 012 012
012 012 012 012 012
012 012 012 012 012
of wet mounts.
Table II on rabbits
inoculated
with
M. wolfii
mycelium
c%mninntibn*:
012 212 012 212 212
212 212 II2 212 212
Cullumltxaminalibn
012 212 212 212 212
212 212 112 I12 212
012 012 012 012 012
012 012 012 012 012
012 012 012 012 012
012 012 012 012 012
012 012 012 012 012
012 212 012 212 212
212 212 212 212 212
012 212 I12 112 112
112 212 112 112 212
012 o/z 012 012 012
MORTIERELLA
WOLFZI PATHOGENICITY
511
Fig. 1. Gross appearance of the liver of a rabbit which died 4 days after intravenous injection of a mycelial suspension of A4. wolfii. Numerous pale necrotic lesions are distributed throughout each lobe.
Fig. 2. Liver of a rabbit dying 6 days after intravenous injection of A4. woEfii mycelium. Severe focal parenchymal necrosis with predominantly polymorphonuclear cellular infiltration (H & EX 100).
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parenchymal necrosis surrounding developing A4. wolfii mycelium, often accompanied by local haemorrhage and with a variable degree of phagocytic and lymphocytic cellular reaction (Fig. 2). Examination of M. woltii strains for toxigenicity Undiluted culture supernatants of all the A4. wolfii strains killed mice within 2 days of injection by the intravenous, intracerebral or subcutaneous routes. Injection of dilutions of culture supernatant produced a graded response. When a correction was made for the different protein concentrations of each preparation, the LD,, values obtained were of comparable magnitude for most of the M. wolfii strains (Table III).
Comparative M. woliii
toxicity
Table III of mycelial extracts
of M. wolfii
Route of infection *
Toxin dilution
Toxin dose (mg potein)
i.c. i.v. i.v. i.v. i.v. iv.
l/i l/l l/10 l/50 l/100 l/1000
3.90 15.6 1.56 0.312 0.156 0.0156
i.c. i.v. i.v. i.v. i.v. i.v.
l/l
l/l l/10 l/50 l/100 l/1000
i.c. i.v. i.v. i.v. i.v. i.v.
v75/173
V76/183
strain
v73/414
V74/21
V75/125
*i.c., intracerebral;
Number Number
strains of deaths/ injected
in mice LD, (pg protein)
6/6 6/6 6/6 4/6 3/6 O/6
156
2.49 9.987 0.9987 0.1996 0.0998 0.00998
6/6 6/6 6/6 2/6 l/6 O/6
178
l/l l/l l/10 l/50 l/100 l/1000
1.64 6.575 0.6575 0.1350 0.06575 0.00657
6/6 6/6 4/6 O/6 O/6 O/6
376
i.c. i.v. i.v. i.v. i.v. i.v.
l/l l/l l/10 l/50 l/100 l/1000
1.43 5.712 0.5712 0.1142 0.0571 0.00571
6/6 6/6 2/6 O/6 O/6 O/6
i.c. i.v. i.v. i.v. i.v. i.v.
l/l l/l l/10 l/50 l/100 l/1000
1.51 6.025 0.6025 0.1205 0.0625 0.006025
6/6 6/6 5/6 O/6 O/6 O/6
i.v., intravenous.
243
513
WOLFZI PATHOGENICITY
MORTIERELLA
Irrespective of the route of injection or the fungal strain, each culture supernatant produced similar toxic effects. About 24 h after injection the affected mice developed severe incoordination characterized by a splay-legged gait with twitching movement in the upper half of the body. Righting reflexes were retained during this phase which was rapidly succeeded by a flaccid paralysis affecting all voluntary muscles. Death followed very quickly. A few animals died without having displayed any overt signs of disturbance. In all of the affected animals there was a reaction at the injection site consistent with a non-specific irritant effect. Post-mortem examination disclosed gross lesions only in the kidneys. The latter were enlarged and greyish white in colour. Histological examination disclosed cloudy swelling and necrosis of the renal tubular epithelium. Some of the surviving animals also had similar kidney lesions but these were generally less extensive. Urine samples collected from the affected mice shortly before death gave positive reactions for glucose and haemoglobin, had a pH value of 6 and contained > 1000 mg of protein/100 ml volume. No differences were observed either in the nature or the extent of lesions produced by culture supernatants from the various M. wolfii strains. The toxicity of M. wolfii culture supernatants for mice was drastically reduced by treatment with heat and to a lesser extent by trypsin, but not by acidification with dilute HCl (Table IV). No other known mycotoxins were detected in either the supernatant or mycelial residues. The stability
Toxin
source
M. wolfii strum
v73/414 V74/21 V75/125 v75/173 V76/183
of toxin
Number of LDso in untreated toxin dose
100 25 25 25 25
Table IV preparations from M. wolfii trypsin and acid Number
NO
strains
to heat,
of deaths/number in group for mice receiving toxin Preparations exposed to Heat
Tvpsin
HCI
treatment
6/6 6/6 6/6 6/6 6/6
O/6 O/6 O/6 O/6 O/6
3/6 l/6 O/6 2/6 O/6
6/6 6/6 6/6 6/6 O/6
Intravenous injection of M. wolfii culture extracts into rabbits produced effects broadly similar to those observed in mice. The concentrated culture supernatants killed the rabbits after l-6 (mean 3.5) days. The most concentrated preparation produced acute mucoid diarrhoea and respiratory distress within 15 min of injection. In most cases the onset of signs of intoxication was delayed for several days. The animals then became increasingly lethargic although hyper-reactive to stimuli, ataxic and anorexic. The body temperature was reduced, down to 35.6”C, and signs of increasing respiratory distress developed immediately before death. At post-mortem examination, the abdominal cavity was filled with clear non-purulent, serohbrinous exudate which formed a glassy deposit over the intestines and dorsal
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muscles. The liver was pale, the kidneys greyish-white and swollen and minute petechiae were present in the larger groups of skeletal muscles. Histological examination showed local haemorrhagic and oedematous changes in skeletal muscle and almost complete necrosis of the renal epithelium (Fig. 3). Similar effects were produced in rabbits given intravenous injections of A4. wolfii culture supernatants treated with dilute acid but not by those exposed to heat or trypsin. Intradermal injection of small volumes of M. wolfii culture supernatant into guineapigs given intracardiac doses of Evans’ blue disclosed increased local vascular permeability.
Fig. 3. Kidney of rabbit injected 4 days previously with A4. zuolfii mycelial extract. Degeneration tubular epithelium. (H & EXlOO).
of
DISCUSSION The results of the inoculation experiments with mycelial suspensions failed to disclose any qualitative difference in the pathogenicity of the five M. wolfii strains examined. Thus each strain demonstrated a similar degree of pathogenicity in either rabbits or mice, although the syndromes produced in the two host species were quite distinct. Rabbits were clearly much more susceptible to acute systemic M. wolfii infection than mice although both species showed similar sensitivity to the mycelial toxin. The reason for this difference was not identified but presumably it reflected the greater capacity of the murine immune system to control the fungal infection. This conclusion was consistent with the localization of progressive infection to the central nervous system of the mouse and by its slow development even at this site. In those few animals in which extraneural infection did develop it was confined to the lungs.
MORTZERELLA
WOLF11
PATHOGENICI’I‘E
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The growth of the fungus in the central nervous system was probably a consequence of the limited immune responses at this site but its growth in pulmonary lesions suggested a specific tropism for lung tissue. The basis for this was not determined but it could have been related to a preference for tissues with a high oxygen tension. As a capacity to produce pulmonary infection was shown by all the M. wolfii strains, this suggested that the development of lung lesions in individual animals was determined by host factors and not simply the properties of the infecting strain. This probably also applies to the post-abortion-pneumonia syndrome in cattle, although the factors predisposing animals to this condition have not been identified (Cordes et al, 1972a, b). The toxigenicity of the infecting fungal strain was evidently not a factor in determining the distribution of lesions. Thus, all of the M. wolfii strains, irrespective of source. elaborated toxic material. The nature of this was clearly similar in each case and on the basis of heat and chemical stability was probably identical with that described by Davey and colleagues (Davey et al, 1973, 1975). The role of this toxin in the pathogenesis of the disease resulting from M. wolfii infection was not clear. Certainly in mice, the lesions produced by infection were totally dissimilar from those resulting from administration of toxin. Indeed, no evidence was obtained that M. wolfii elaborated the toxin in uiuo in the mouse. Thus, no renal lesions developed in animals with chronic infection of the central nervous system although they were readily produced by intracerebral injection of toxin formed in vitro. A closer similarity between the effects of toxic extracts and those of M. woljii infection was evident in the rabbit, however. Thus, signs of increased vascular permeability were prominent in animals given toxic extracts and in those developing acute infection. Nevertheless, the two syndromes were not identical, as shown by the predominance of focal hepatic rather than renal lesions in the infected rabbits. This suggested that the determinants of pathogenicity of M. woljii infection were more complex than simply elaboration of acid-stable toxin. These results did not disclose any difference in pathogenic potential between UK isolates and the single New Zealand strain of M. wolfii examined in the experimental models used. This does not mean, of course, that such a difference would not be evident in studies conducted in cattle. Such studies are indicated to elucidate the mechanisms of pathogenicity of M. wolfii in this species.
REFERENCES AL 51 ~\K.K, P. K. C. (1976). New Zealand Journal of Agricultural Research 19, 25. C.AKI F.K, hl. E., COKIIES, D. O., III kf~h.\i\, kl. E. & Ht \ I CK, R. (1973). Research in Veterinary Sczence 14, 201. COKHCL, &I. J. & E.um, S. M. (1975). ournal of Medical Mzcrobiology 8, 551. Cwmt., %I. J. & Emta, S. M. (1977). Mycopathologia 60, 129. COKII~S, D. O., C~KIEK, M. E. & III MESU, Iz;I. E. (1972a). Veterinary PatholoQ 9, 190. COKIX+ D. O., III &hxr\, M. E. & &Kim, M. E. (197213). Veterinary Pathology 9, 131. DA\E\I, G., SMIIII, J. M. B. & ~L.\IAKO~~, J. (1973). Infection and Immunity& 882. DA\ ~1, G., SIWSOS, L. 0. & KALMKOFF, J. (1975). Journal of Pathology 117, 33. III Mt;xs.+, M. E., CAKIER, M. E. & COKDt5, D. 0. (1972). Research in Veterinary Science 13, 439. ELANS, H. C. (1971a). Transactions of the British Mycological Society 57, 241. EL os, H. C. (1971b). Transactions of the Brutish Mycological Society 57, 255. H;\KCOI~KI., R. A. & T~W~WSON, F. G. A. (1969). Veterinary Record 85, 199. H( (;I]-JONES, bl. E. & ALSIWICK, P. K. C. (1967). Veterinary Recordal, 273.
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JOHNSON, C . T., LLPSON, G . R. & LAWRENCE, K. E . (1990) . Veterinary Record 127, 363 . (1951) . Journal of Biological LOWRY, O . H ., ROSEBROUGII, N . J ., FARR, A . L . & RANDELL, R .
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MACDONALD, S . M . & CORBEL, M . (1981) . Veterinary Record 109, 419 . MEHROTRA, B . S . & BAIJAL, U . (1963) . Mycopathologia et Mycologia Applicata 20, 49 . ROBERTS, B . A. & PAT1'ERSON, D . S . P . (1975) . Journal of the Association of Official Analytical Chemists
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J.
YOUNG, B . (1968) . Journal of Medical Laboratory Technology 25, 343 . ZYCHA, H ., SIEPn1ANN, R . & LINNEb1ANN, G . (1969) . Mucorales : Eine Beschreibung aller Gattungen und Arten dieser Pilzgruppe . Lehre : Cramer Verlag .
J.
(Accepted for publication 17 January 1991)
OBSERVATIONS ON THE EXPERIMENTAL PATHOGENICITY AND TOXIGENICITY OF MORTIERELLA WOLFII STRAINS OF BOVINE ORIGIN
M . J . CORBEL* and SUSAN M . EADES Ministry of Agriculture, Fisheries & Food, Central Veterinary Laboratory, Weybridge KT15 3NB
SUMMARY Four strains of Mortierella wolfii isolated from cattle in Britain were compared in pathogenicity and toxigenicity with a strain isolated from a cow with the mycotic abortion-pneumonia syndrome in New Zealand . All strains produced acute lethal infection in rabbits after intravenous inoculation of mycelial suspensions and all produced subacute mycotic encephalitis in mice after intracerebral injection . They also produced an acid-stable, heat- and trypsin-labile toxin in vitro . The action of the toxin was exerted mainly on the kidneys in rabbits and mice and produced effects distinct from those resulting from infection with M. wolfii.
INTRODUCTION Most cases of bovine mycotic abortion in Britain have been attributed to infection with Aspergillus fumigatus, related Aspergillus species or Absidia corymbifera (Hugh Jones & Austwick, 1967) . Hitherto, Mortierella wolfii or other Mortierella species have only rarely been identified as the cause of the syndrome (Harcourt & Thompson, 1969 ; Johnson et al., 1990) although implicated in a high proportion of cases in New Zealand (di Menna et al., 1972 ; Carter et al., 1973 ; Austwick, 1976) . The infrequency with which M. wolfii has been implicated as a cause of infection in this country probably reflects the difficulty of isolating it from contaminated material rather than its true significance as a pathogenic agent . Thus in recent years M. wolfii has been identified in a number of cases of infection in cattle, including several of mycotic abortion . Other studies have established the presence of M. wolfii in the British environment (Evans, 1971a, b) . Nevertheless, although it is clear that M. wolfii is present in this country and accounts for a proportion of bovine fungal infections, it is also apparent that the distinctive mycotic abortion-pneumonia syndrome associated with the fungus in New Zealand has been rarely recorded here . Indeed, a survey of the literature disclosed only a single reported case of the syndrome (Harcourt & Thompson, 1969) . *Present address : National Institute for Biological Standards & Control, Blanche Lane, South Mimms, Potters Bar, ENG 3QG.
MORTIERELLA WOLFII PATHOGENICITY
505
As such a characteristic syndrome is unlikely to be widely overlooked it appears that the manifestations of M. wolfii infection in cattle in this country may differ somewhat from those seen in New Zealand . No reasons for such a difference have been disclosed but an obvious possibility is variation in the pathogenicity of the M. wolfii strains prevalent in the two countries . It may be significant in this respect that differences in the experimental pathogenicity of strains of the fungus obtained from different environments have been reported (Cordes et al., 1972a, b) . The determinants of the pathogenicity of M. wolfii for cattle have not been identified but the fungus has been shown to elaborate a _nephrotoxin in vitro and it has been suggested that this may play a role in natural infections (Davey et al., 1973, 1975) . Although direct evidence has not been presented to this effect, it is possible that toxin production could be a major factor in determining the virulence of individual M. wolfii strains . Variations in toxigenicity could also account for differences in the clinical syndromes by different strains of the fungus . This possibility has been examined for several strains of M. wolfii isolated from cattle in the UK .
MATERIALS AND METHODS Fungal strains M. wolfii strain V .73/414 in the laboratory collection was obtained from the Common-
wealth Mycological Institute, Kew as M. wolfii strain IMI 149020. It was originally isolated in New Zealand from the lung tissue of a cow with the mycotic abortionpneumonia syndrome . M. wolfii strains V .74/21 (ATCC .36820), V .75/125 and V .75/173 were isolated from the stomach contents of aborted fetuses in the UK . M. wolfii strain V .76/183 was isolated from the liver of a 2-week-old calf . Details of these cases have been described previously (MacDonald & Corbel, 1981) . Identification of Mortierella isolates
The cultural and morphological criteria used for the identification of isolates were those described in the literature (Zycha et al., 1969) . All strains identified as M. wolfii conformed to the description given by Mehrotra & Baijal (1963) . The isolates were submitted to the Commonwealth Mycological Institute, Kew, for confirmation of their identity . Preparation of inocula As all of the Mortierella strains used in this study sporulated poorly under laboratory
conditions, only mycelial suspensions were used for animal inoculation . These were prepared as described previously (Corbel & Eades, 1977) . Preparation of cellfree extracts The M. wolfii strains were grown in Difco glucose-neopeptone broth incubated at 37°C for 14 days, as described previously (Davey et al., 1973) . The culture supernatant from
each strain was decanted and concentrated by counterdialysis against polyethylene glycol 20 .%1 . The mycelial mats were collected separately and disrupted by four cycles of freeze pressing at -25°C in an Edebo X-press (LKB-Biotec, Croydon) . The mycelial extracts were freed of particulate material by centrifugation at 10 000 gfor 20 min and the
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supernatants collected and concentrated by counterdialysis against polyethylene glycol 20 M . These and the concentrated culture supernatants from each strain were pooled and sterilized by membrane filtration and stored at 4 °C until required . The protein concentration of each preparation was determined by the Folin-biuret method using a bovine serum albumin standard (Lowry et al., 1951). All cell-free extracts and the insoluble debris separated by centrifugation were examined for mycotoxins by thin layer chromatography of ethyl acetate extracts as described elsewhere (Roberts & Patterson, 1975) .
Animal inoculation The pathogenicity of M. wolfii isolates for mice was determined essentially as described previously (Corbel & Eades, 1977) . Thus groups of six albino mice between 18 and 22 days old were injected by the intravenous, intramuscular, intraperitoneal or subcutaneous routes with 0 .1 ml volumes of mycelial suspensions containing between 1 and 10' viable units of M. wolfii. Similar groups of animals were injected by the intracerebral route with 0 .025 ml volumes of mycelial suspension . Subsequent treatment and observation of the animals was as described previously (Corbel & Eades, 1977) . The pathogenicity of M. wolfii strains for rabbits was examined by injecting groups of four Californian rabbits with 1 .0 ml volumes of graded dilutions of mycelial suspensions by the intravenous route . Equal numbers of male and female rabbits about 3 months old were used for each strain (see Table II) . The rabbits were inspected daily for abnormal signs . As soon as any evidence of disease became apparent they were examined at hourly intervals until death . Once the pattern of the disease had been established, rabbits in the terminal stages of infection were killed by intravenous injection of sodium pentobarbitone . Pathological and mycological examination This was done as described previously (Corbel & Eades, 1977) . Histological sections were stained with haematoxylin and eosin (H & E) or by the PAS-celestine blue method (Young, 1968) . Toxigenicity studies The toxicity of cell-free extracts of M. wolfii prepared from culture supernatants or from mycelium was assayed in mice and rabbits . Groups of six albino mice approximately 6 weeks old and matched for weight were given intravenous injections of 0 .1 ml volumes of M. wolfii extracts (see Table III) . These were used undiluted and at 1/10, 1/50, 1/100 and 1/1000 dilutions in phosphate buffered saline (PBS : 0 .15 mol/l NaCl in 0 .01 mol/l phosphate buffer, pH 7 .4) . The animals were observed at least once daily for up to 7 days . Those surviving at the end of this time were killed under ether anaesthesia . All mice were examined post mortem for both gross and microscopic lesions . Histological examinations were performed on H & E stained sections of formalin-fixed tissue . Samples of urine were collected by aspiration from the bladder with a disposable syringe and needle . Tests for protein concentration, glucose, haemoglobin and pH reaction were done using Hemacombistix (Ames Co . Ltd, Slough) . Similar tests were performed on groups of six albino mice given intracerebral injections of 0 .025 ml volumes of undiluted M. wolfii extracts . Toxin characterization was performed by a similar procedure by giving the mice intravenous injections of M. wolfii cell-
MORTIERELLA WOLFLI PATHOGENICITY
507
free extracts which had been treated with trypsin (500 BAEE units/ml), heat (60 °C/ 1 h) or acid (0 .1 mol/1 HCl) (Davey et al., 1973) . Similar procedures were used for the toxicity tests in rabbits . For these, pairs of 3-month-old Californian rabbits were given intravenous injections of 1 .0 ml volumes of toxin preparation . The animals were observed for up to 7 days, their rectal temperatures were recorded at 8-hourly intervals for the first 3 days and at 12-hourly intervals for the remaining time . Post-mortem examinations were made as described for the mice . Toxin preparations were also examined for a dermonecrotic effect in guinea-pigs . Pairs of female albino guinea-pigs were depilated along both flanks and 0 .1 ml volumes of dilutions of M. wolfii extracts in PBS injected intradermally . Control injections of similar volumes of PBS were injected at intervals of 5 cm from the toxin preparations . On the following day I ml volumes of 1% w/v Evans' blue in saline were injected by the intracardiac route . The animals were killed 10 min later under anaesthesia and the skin removed and examined for infiltration of dye and other signs of local reaction . Histological examinations were made on H & E stained sections of formalin-fixed skin from the test and control sites .
RESULTS Comparative pathogenicity of M . wolfii strains None of the M. wolfii strains produced lethal infection in mice when inoculated by the intramuscular, intraperitoneal, intravenous or subcutaneous routes . Furthermore, the fungus was not recovered from the mice killed at the end of the observation period, nor did those animals show any gross lesions at necropsy . In contrast, intracerebral inoculation of all of the M. wolfii strains produced lethal infection in the majority of mice (Table I) . The number of deaths did not differ significantly between the groups but individual animals showed considerable variation in the interval between inoculation and death . This varied from a minimum of 4 days up to 48 days . It was not possible to determine any pattern characteristic of any one strain as variations occurred within the groups inoculated with different strains . Those mice developing acute infection showed clinical signs similar to those observed in acute zygomycosis produced by infection with other zygomycete species (Corbel & Eades, 1975) . Thus the animals became ataxic, refused food and water and developed continuous circling movements which preceded a terminal comatose state . This pattern was only seen in those animals which developed signs of disease less than 1 week after inoculation . In those developing the more chronic disease the pattern was similar to that previously described for M. wolfii strain V .74/21 (Corbel & Eades, 1977) . These mice developed granulomatous infiltrations surrounding the third cerebral ventricle and the aqueduct . This resulted in a chronic hydrocephalus with severe distension of both cerebral hemispheres . Some of the mice which developed subacute or chronic cerebral infection also had pulmonary lesions . These consisted of areas of greyish-yellow consolidation usually involving complete lobes of the lung and sometimes the whole lung . M. wolfii was readily recovered from both the cerebral and pulmonary lesions but not from other tissues . The characteristic narrow non-septate Mortierella hyphae were easily detected in histological sections of brain and lung tissue stained by the PAS-celestine blue method . In H & E
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MORTIERELLA WOLF11 PATHOGENICITY
5 09
stained sections, the brain tissue showed pronounced granulomatous changes with whole areas of cerebral parenchyma replaced by infiltrations of mononuclear phagocytes and lymphocytes . The affected lung tissue showed extensive necrosis with an inflammatory response involving both mononuclear and polymorphonuclear leucocytes . Examination of the urine of mice dying after intracerebral inoculation with M. wolfii strains indicated no abnormality in pH, protein, glucose or haemoglobin content . The rabbits inoculated with M. wolfii suspension by the intravenous route developed acute lethal infection within 3-7 days (Table II) . The disease was rapid in onset, with'the animals developing signs of general loss of condition, anorexia, shivering, reduced mobility, respiratory distress and in some cases mucoid diarrhoea . Some of the animals developed corneal erosions which were usually unilateral and accompanied by release of a watery or purulent discharge from the affected eye . There was frequently a watery or blood-stained purulent discharge from the nose . Death usually occurred within a few hours of these signs becoming apparent . In the terminal phase, the body temperature was usually 1-2°C below normal . The urine was frequently blood-stained and semiquantitative tests indicated a reaction between pH 6 and pH 8, usually near pH 6, an absence of glucose, the presence of moderate to large quantities of haemoglobin and a protein concentration greater than 1000 mg/100 ml . At post-mortem examination, similar pathological changes were seen in all rabbits irrespective of the strain of M. wolfii used for inoculation . Generally these consisted of a large volume of serosanguineous fluid in the abdominal cavity, with a glassy fibrinous deposit over the dorsal abdominal muscles . Petechial haemorrhages were observed in the brain, heart, lungs, kidneys, spleen, large and small intestines, uterus and thymus . The most distinctive lesions were present in the liver which usually had a marbled appearance with multiple pale necrotic foci scattered throughout the parenchyma . These lesions were approximately spherical and consisted of a grey or greyish-yellow central region (57 mm in diameter, surrounded by a narrow haemorrhagic zone (Fig . 1) . In some cases complete lobes of liver or even the entire organ, were reduced to a friable grey necrotic mass . The spleen frequently contained several yellowish/white nodules about 3 mm in diameter, or in some cases a single large cauliform mass which completely distorted its structure . Multiple haemorrhages were often present in the wall of the duodenum, ileum and caecum . They usually consisted of numerous scattered petechiae but in some rabbits more extensive haemorrhages discharging into the intestinal lumen were present . Extensive haemorrhages were also present in the pancreas in the more severely affected animals . The kidneys often showed only slight subcapsular mottling but in some rabbits yellowish-grey necrotic patches were present on the poles of the kidneys where they had contacted similar lesions in the liver or spleen . Occasionally these necrotic changes extended locally to the dorsal abdominal muscles . On sectioning the kidneys, the medulla was usually very congested with numerous focal haemorrhages . Scattered small subcapsular infarcts were frequently present . In a few animals one of the kidneys was swollen to approximately twice the normal size . In such cases very large yellowish infarcts involving most of the renal parenchyma were seen on sectioning the organ . The lungs usually showed extensive areas of grey necrotic consolidation . In one animal extensive adhesions had formed between areas of affected lung and the pericardium . Petechiae were widespread throughout the skeletal muscles which also sometimes appeared oedematous . Histological examination of H & E and PAS-celestine blue-stained sections showed that the lesions in the liver, lungs and other organs usually consisted of extensive areas of
Cultural
and microscopic
examinations hnwo/i
v731414 v74121 V75/125 VW173 V761113
*Includes
KY I@ I@ lo" I@
212 212 212 212 212
3 6.5 5.5 3 3
012 012 012 012 012
results of histology and examination
112 o/2 112 012 012
012 012 012 012 012
012 012 012 012 012
of wet mounts.
Table II on rabbits
inoculated
with
M. wolfii
mycelium
c%mninntibn*:
012 212 012 212 212
212 212 II2 212 212
Cullumltxaminalibn
012 212 212 212 212
212 212 112 I12 212
012 012 012 012 012
012 012 012 012 012
012 012 012 012 012
012 012 012 012 012
012 012 012 012 012
012 212 012 212 212
212 212 212 212 212
012 212 I12 112 112
112 212 112 112 212
012 o/z 012 012 012
MORTIERELLA
WOLFZI PATHOGENICITY
511
Fig. 1. Gross appearance of the liver of a rabbit which died 4 days after intravenous injection of a mycelial suspension of A4. wolfii. Numerous pale necrotic lesions are distributed throughout each lobe.
Fig. 2. Liver of a rabbit dying 6 days after intravenous injection of A4. woEfii mycelium. Severe focal parenchymal necrosis with predominantly polymorphonuclear cellular infiltration (H & EX 100).
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parenchymal necrosis surrounding developing A4. wolfii mycelium, often accompanied by local haemorrhage and with a variable degree of phagocytic and lymphocytic cellular reaction (Fig. 2). Examination of M. woltii strains for toxigenicity Undiluted culture supernatants of all the A4. wolfii strains killed mice within 2 days of injection by the intravenous, intracerebral or subcutaneous routes. Injection of dilutions of culture supernatant produced a graded response. When a correction was made for the different protein concentrations of each preparation, the LD,, values obtained were of comparable magnitude for most of the M. wolfii strains (Table III).
Comparative M. woliii
toxicity
Table III of mycelial extracts
of M. wolfii
Route of infection *
Toxin dilution
Toxin dose (mg potein)
i.c. i.v. i.v. i.v. i.v. iv.
l/i l/l l/10 l/50 l/100 l/1000
3.90 15.6 1.56 0.312 0.156 0.0156
i.c. i.v. i.v. i.v. i.v. i.v.
l/l
l/l l/10 l/50 l/100 l/1000
i.c. i.v. i.v. i.v. i.v. i.v.
v75/173
V76/183
strain
v73/414
V74/21
V75/125
*i.c., intracerebral;
Number Number
strains of deaths/ injected
in mice LD, (pg protein)
6/6 6/6 6/6 4/6 3/6 O/6
156
2.49 9.987 0.9987 0.1996 0.0998 0.00998
6/6 6/6 6/6 2/6 l/6 O/6
178
l/l l/l l/10 l/50 l/100 l/1000
1.64 6.575 0.6575 0.1350 0.06575 0.00657
6/6 6/6 4/6 O/6 O/6 O/6
376
i.c. i.v. i.v. i.v. i.v. i.v.
l/l l/l l/10 l/50 l/100 l/1000
1.43 5.712 0.5712 0.1142 0.0571 0.00571
6/6 6/6 2/6 O/6 O/6 O/6
i.c. i.v. i.v. i.v. i.v. i.v.
l/l l/l l/10 l/50 l/100 l/1000
1.51 6.025 0.6025 0.1205 0.0625 0.006025
6/6 6/6 5/6 O/6 O/6 O/6
i.v., intravenous.
243
513
WOLFZI PATHOGENICITY
MORTIERELLA
Irrespective of the route of injection or the fungal strain, each culture supernatant produced similar toxic effects. About 24 h after injection the affected mice developed severe incoordination characterized by a splay-legged gait with twitching movement in the upper half of the body. Righting reflexes were retained during this phase which was rapidly succeeded by a flaccid paralysis affecting all voluntary muscles. Death followed very quickly. A few animals died without having displayed any overt signs of disturbance. In all of the affected animals there was a reaction at the injection site consistent with a non-specific irritant effect. Post-mortem examination disclosed gross lesions only in the kidneys. The latter were enlarged and greyish white in colour. Histological examination disclosed cloudy swelling and necrosis of the renal tubular epithelium. Some of the surviving animals also had similar kidney lesions but these were generally less extensive. Urine samples collected from the affected mice shortly before death gave positive reactions for glucose and haemoglobin, had a pH value of 6 and contained > 1000 mg of protein/100 ml volume. No differences were observed either in the nature or the extent of lesions produced by culture supernatants from the various M. wolfii strains. The toxicity of M. wolfii culture supernatants for mice was drastically reduced by treatment with heat and to a lesser extent by trypsin, but not by acidification with dilute HCl (Table IV). No other known mycotoxins were detected in either the supernatant or mycelial residues. The stability
Toxin
source
M. wolfii strum
v73/414 V74/21 V75/125 v75/173 V76/183
of toxin
Number of LDso in untreated toxin dose
100 25 25 25 25
Table IV preparations from M. wolfii trypsin and acid Number
NO
strains
to heat,
of deaths/number in group for mice receiving toxin Preparations exposed to Heat
Tvpsin
HCI
treatment
6/6 6/6 6/6 6/6 6/6
O/6 O/6 O/6 O/6 O/6
3/6 l/6 O/6 2/6 O/6
6/6 6/6 6/6 6/6 O/6
Intravenous injection of M. wolfii culture extracts into rabbits produced effects broadly similar to those observed in mice. The concentrated culture supernatants killed the rabbits after l-6 (mean 3.5) days. The most concentrated preparation produced acute mucoid diarrhoea and respiratory distress within 15 min of injection. In most cases the onset of signs of intoxication was delayed for several days. The animals then became increasingly lethargic although hyper-reactive to stimuli, ataxic and anorexic. The body temperature was reduced, down to 35.6”C, and signs of increasing respiratory distress developed immediately before death. At post-mortem examination, the abdominal cavity was filled with clear non-purulent, serohbrinous exudate which formed a glassy deposit over the intestines and dorsal
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muscles. The liver was pale, the kidneys greyish-white and swollen and minute petechiae were present in the larger groups of skeletal muscles. Histological examination showed local haemorrhagic and oedematous changes in skeletal muscle and almost complete necrosis of the renal epithelium (Fig. 3). Similar effects were produced in rabbits given intravenous injections of A4. wolfii culture supernatants treated with dilute acid but not by those exposed to heat or trypsin. Intradermal injection of small volumes of M. wolfii culture supernatant into guineapigs given intracardiac doses of Evans’ blue disclosed increased local vascular permeability.
Fig. 3. Kidney of rabbit injected 4 days previously with A4. zuolfii mycelial extract. Degeneration tubular epithelium. (H & EXlOO).
of
DISCUSSION The results of the inoculation experiments with mycelial suspensions failed to disclose any qualitative difference in the pathogenicity of the five M. wolfii strains examined. Thus each strain demonstrated a similar degree of pathogenicity in either rabbits or mice, although the syndromes produced in the two host species were quite distinct. Rabbits were clearly much more susceptible to acute systemic M. wolfii infection than mice although both species showed similar sensitivity to the mycelial toxin. The reason for this difference was not identified but presumably it reflected the greater capacity of the murine immune system to control the fungal infection. This conclusion was consistent with the localization of progressive infection to the central nervous system of the mouse and by its slow development even at this site. In those few animals in which extraneural infection did develop it was confined to the lungs.
MORTZERELLA
WOLF11
PATHOGENICI’I‘E
5 15
The growth of the fungus in the central nervous system was probably a consequence of the limited immune responses at this site but its growth in pulmonary lesions suggested a specific tropism for lung tissue. The basis for this was not determined but it could have been related to a preference for tissues with a high oxygen tension. As a capacity to produce pulmonary infection was shown by all the M. wolfii strains, this suggested that the development of lung lesions in individual animals was determined by host factors and not simply the properties of the infecting strain. This probably also applies to the post-abortion-pneumonia syndrome in cattle, although the factors predisposing animals to this condition have not been identified (Cordes et al, 1972a, b). The toxigenicity of the infecting fungal strain was evidently not a factor in determining the distribution of lesions. Thus, all of the M. wolfii strains, irrespective of source. elaborated toxic material. The nature of this was clearly similar in each case and on the basis of heat and chemical stability was probably identical with that described by Davey and colleagues (Davey et al, 1973, 1975). The role of this toxin in the pathogenesis of the disease resulting from M. wolfii infection was not clear. Certainly in mice, the lesions produced by infection were totally dissimilar from those resulting from administration of toxin. Indeed, no evidence was obtained that M. wolfii elaborated the toxin in uiuo in the mouse. Thus, no renal lesions developed in animals with chronic infection of the central nervous system although they were readily produced by intracerebral injection of toxin formed in vitro. A closer similarity between the effects of toxic extracts and those of M. woljii infection was evident in the rabbit, however. Thus, signs of increased vascular permeability were prominent in animals given toxic extracts and in those developing acute infection. Nevertheless, the two syndromes were not identical, as shown by the predominance of focal hepatic rather than renal lesions in the infected rabbits. This suggested that the determinants of pathogenicity of M. woljii infection were more complex than simply elaboration of acid-stable toxin. These results did not disclose any difference in pathogenic potential between UK isolates and the single New Zealand strain of M. wolfii examined in the experimental models used. This does not mean, of course, that such a difference would not be evident in studies conducted in cattle. Such studies are indicated to elucidate the mechanisms of pathogenicity of M. wolfii in this species.
REFERENCES AL 51 ~\K.K, P. K. C. (1976). New Zealand Journal of Agricultural Research 19, 25. C.AKI F.K, hl. E., COKIIES, D. O., III kf~h.\i\, kl. E. & Ht \ I CK, R. (1973). Research in Veterinary Sczence 14, 201. COKHCL, &I. J. & E.um, S. M. (1975). ournal of Medical Mzcrobiology 8, 551. Cwmt., %I. J. & Emta, S. M. (1977). Mycopathologia 60, 129. COKII~S, D. O., C~KIEK, M. E. & III MESU, Iz;I. E. (1972a). Veterinary PatholoQ 9, 190. COKIX+ D. O., III &hxr\, M. E. & &Kim, M. E. (197213). Veterinary Pathology 9, 131. DA\E\I, G., SMIIII, J. M. B. & ~L.\IAKO~~, J. (1973). Infection and Immunity& 882. DA\ ~1, G., SIWSOS, L. 0. & KALMKOFF, J. (1975). Journal of Pathology 117, 33. III Mt;xs.+, M. E., CAKIER, M. E. & COKDt5, D. 0. (1972). Research in Veterinary Science 13, 439. ELANS, H. C. (1971a). Transactions of the British Mycological Society 57, 241. EL os, H. C. (1971b). Transactions of the Brutish Mycological Society 57, 255. H;\KCOI~KI., R. A. & T~W~WSON, F. G. A. (1969). Veterinary Record 85, 199. H( (;I]-JONES, bl. E. & ALSIWICK, P. K. C. (1967). Veterinary Recordal, 273.
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JOHNSON, C . T., LLPSON, G . R. & LAWRENCE, K. E . (1990) . Veterinary Record 127, 363 . (1951) . Journal of Biological LOWRY, O . H ., ROSEBROUGII, N . J ., FARR, A . L . & RANDELL, R .
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MACDONALD, S . M . & CORBEL, M . (1981) . Veterinary Record 109, 419 . MEHROTRA, B . S . & BAIJAL, U . (1963) . Mycopathologia et Mycologia Applicata 20, 49 . ROBERTS, B . A. & PAT1'ERSON, D . S . P . (1975) . Journal of the Association of Official Analytical Chemists
58, 1178 .
J.
YOUNG, B . (1968) . Journal of Medical Laboratory Technology 25, 343 . ZYCHA, H ., SIEPn1ANN, R . & LINNEb1ANN, G . (1969) . Mucorales : Eine Beschreibung aller Gattungen und Arten dieser Pilzgruppe . Lehre : Cramer Verlag .
J.
(Accepted for publication 17 January 1991)