J. Vet. Med. B 46, 613–621 (1999)© 1999 Blackwell Wissenschafts-Verlag, BerlinISSN 0931–1793

Departamento de Patologıa Animal: Medicina Animal (Anatomıa Patologica), Facultad de Veterinaria, Universidadde Leon, Leon, Spain

Generalized Aspergillosis in Dairy Sheep

V. PEREZ1,4, J. M. CORPA1, J. F. GARCIA MARIN1, J. J. ADURIZ2 and H. E. JENSEN3

Addresses of authors: 1Departamento de Patologıa Animal: Medicina Animal (Anatomıa Patologica),Facultad de Veterinaria, Universidad de Leon, Campus de Vegazana, s/n, 24071 Leon, Spain; 2NEIKER

(Instituto Vasco de Investigacion y Desarrollo Agrario), Berreaga 1, 48160 Derio, Vizcaya, Spain;3Department of Pharmacology and Pathobiology, The Royal Veterinary and Agricultural University,

Copenhagen, Denmark; 4Corresponding author

With 5 figures

(Received for publication September 3, 1998)

Summary

Two cases of systemic aspergillosis are described in dairy sheep from a flock in which fungal mastitisappeared subsequent to the antibiotic treatment of animals before parturition. Lesions characterized bynecrosis and a pyogranulomatous exudate were observed in the mammary glands, supramammary andmediastinal lymph nodes, kidneys, lung, liver, heart, forestomachs and brain. The intense vasculitis withthrombosis observed in various organs, but especially in the mammary glands, suggested a haematogenousdissemination of the infection from this organ. The aetiological diagnosis was accomplished by theimmunohistochemical staining of the fungal structures seen in the histological sections by the specificAspergillus monoclonal antibody Mab-WF-AF-1 together with the isolation of Aspergillus fumigatus in pureculture from affected tissues.

Introduction

Filamentous fungi of the genus Aspergillus are ubiquitous in the environment but establishedinfections causing disease are uncommon in mammals (Dungworth, 1993) in which the differentpathological forms have not been sufficiently studied (Austwick, 1965; Nesbit, 1986). However,in cattle, Aspergillus fumigatus has been commonly incriminated as a cause of alimentary orsystemic aspergillosis (Cordes and Shortridge, 1968; Chihaya et al., 1991, 1992; Jensen et al.,1991; Jensen, 1994), diseases which have been estimated of great importance in cattle production.

In general, ovine aspergillosis has been regarded as a rare condition (Chauhan and Dwivedi,1974) and the majority of papers have reported cases of exclusive pulmonary affection (Austwicket al., 1960; Young, 1970; Chauhan and Dwivedi, 1974; Kamil and Parihar, 1991). However,studies concerning ovine generalized infections are scarce. As far as we are aware, only onenatural case in an adult sheep with severe lesions of the forestomachs (Chihaya et al., 1980) hasbeen reported, together with two cases in young lambs in which the lung was the main organinvolved (Gracey and Baxter, 1961; Krishna et al., 1981). Recently, some outbreaks of mastitisdue to A. fumigatus have been reported in dairy sheep with a few animals also having mycoticlesions in the lungs, kidney and liver (Perez et al., 1998). Other filamentous fungi, such as thoseof the class Zygomycetes, have also been involved in the development of mastitis and systemicmycosis in ruminants (Cordes and Shortridge, 1968; Jensen et al., 1989, 1996a; Carrasco et al.,1993; Jensen, 1994).

The clinical signs of the majority of systemic mycoses are unspecific and, in some cases,its development may be masked by symptoms of an underlying disease (Jensen et al., 1989).

U. S. Copyright Clearance Center Code Statement: 0931-1793/99/4609-0613$14.00/0

614 PEREZ et al.

Therefore, these diseases can commonly be diagnosed only at postmortem and histologicalexamination (Cordes and Shortridge, 1968; Jensen, 1994). Attempts to isolate fungi in culturemay be impossible as, sometimes, only formalin-fixed tissue is available. The identification ofthe aetiological agent in tissue sections can be difficult and although the morphology ofAspergillus hyphae may offer a tentative diagnosis, the differentiation to other agents of systemicmycoses, e.g. zygomycosis, may be impossible (Cordes and Shortridge, 1968; Jensen, 1994;Jensen et al., 1996a, b).

In this paper, we report two cases of severe disseminated aspergillosis in sheep from aflock where an outbreak of mycotic mastitis had occurred.

Materials and Methods

Animals

All the ewes from a flock of 300 adult Assaf dairy sheep were treated with cloxacillin by theintramammary route before parturition in an attempt to prevent mastitis. Between 2 and 3 days afterparturition, ¼ 10% of the sheep developed a bilateral induration of the mammary glands which wastreated with different kinds of antibiotics. Approximately 60% of affected animals died and mastitis dueto A. fumigatus was diagnosed by pathological and mycological procedures (Perez et al., 1998). Two affectedsheep (nos 1 and 2) which presented fever, depression and loss of weight for 1 week were studied. Theywere killed by an intravenous injection of barbiturate due to their poor condition and a necropsy wasperformed.

Pathology

Macroscopic lesions were recorded and samples from affected tissues and from the intestine, spleen,uterus, brain and different lymph nodes were taken for histopathology. The tissues were fixed in 10%neutral-buffered formalin, dehydrated and embedded in paraffin wax. Several sections 4 mm thick were cutand stained with haematoxylin–eosin (HE), Van Gieson’s trichromic method, periodic acid-Schiff (PAS)and Grocott’s methenamine silver (GMS) method for fungi.

Immunohistochemistry

Selected tissues were mounted on glass slides coated with poly-L-lysine and processed for immu-nohistochemical staining. Endogenous peroxidase activity was removed by incubation in 0.3% hydrogenperoxide in methanol for 30min at room temperature. After rinsing in Tris-buffered saline (TBS), pH 7.5,twice for 10min each time, the sections were treated with a 0.1% trypsin–calcium chloride solution,pH 7.8, for 40min at 37°C. The sections were then rinsed in TBS (two 10min rinses) and incubated for30min with normal goat serum at room temperature. The slides were incubated overnight at roomtemperature with a 1:5 dilution of the primary murine monoclonal antibody (Mab), i.e. Mab-WF-AF-1 orMab-WSSA-RA-1, supplied by Dr H. E. Jensen (Copenhagen, Denmark), that specifically reacts with thefungal elements that cause aspergillosis and zygomycosis, respectively (Jensen et al., 1996c, d). After rinsingin TBS (two 10min rinses), the slides were incubated with biotinylated horse antimouse immunoglobulinG (Vector Laboratories, Burlingame, CA, USA) following the instructions of the manufacturer. Afterrinsing in TBS (two 10min rinses), the slides were incubated with the avidin–biotin–peroxidase (ABC)complex (Vector Laboratories) in TBS for 1 h at room temperature. Following rinsing in TBS (two 10minrinses), the sections were then incubated with the chromogen 3,3?-diaminobenzidine tetrahydrochloride(Sigma Chemicals, Madrid, Spain) diluted 0.05% in TBS. Before this incubation, each 100ml of thisdilution was mixed with 0.3ml of 3% hydrogen peroxide. Incubation was terminated by rinsing intap water, and the sections were counterstained with Mayer’s haematoxylin, dehydrated, and mounted.Simultaneously, and as positive controls, bovine and murine tissues infected with aspergillosis and zyg-omycosis were immunostained (Jensen et al., 1996c, d). The specific primary Mabs were substituted in thesame tests with murine preimmune serum or TBS, as negative controls. Furthermore, experiments withthe omission of the second biotinylated antibody, the ABC complex, and the chromogen were alsoperformed.

615Generalized Aspergillosis in Dairy Sheep

Mycological examination

For mycological examination, samples from all the macroscopic lesions were incubated on Sabouraud-chloramphenicol dextrose agar, pH 5.7 for 4 days at 24°C and 37°C.

Results

Gross pathology

Gross lesions were observed in the mammary glands, supramammary and mediastinallymph nodes, kidneys, lung, heart, liver, rumen and omasum of both sheep. In addition, sheepno. 1 had lesions in the reticulum and the abomasum.

The mammary glands were notably enlarged, hard to the touch and, on the cut surface,extensive necrotic and haemorrhagic areas were seen. The lymph nodes were also swollen andshowed small pale spots on the cut surface. Lesions of the kidneys were characterized bymultiple, pale and necrotic foci surrounded by a haemorrhagic area, i.e. renal infarcts, distributedacross the cortex (Fig. 1). They ranged from 0.5 to 2 cm in diameter and some extended intothe medulla. In sheep no. 1 these foci were numerous and widespread in both kidneys whichappeared pale and had a purulent exudate in the pelvis (Fig. 1). The lung presented multiplesmall, hard and grey foci, between 0.5 and 3 cm in diameter, scattered both superficially anddeeply in the parenchyma (Fig. 2). Along the periphery of the nodules, a narrow reddish zonewas present. In large nodules (× 1 cm), a creamy necrotic material was observed in the centre.Small white necrotic areas were found throughout the liver. In the heart, the myocardium fromthe right ventricle of sheep no. 1 and from the left ventricle of sheep no. 2 showed large, paleand well-defined areas which could be seen deeply on the cut surface. Similar small lesions werealso present elsewhere in the myocardium. Sheep no. 1 presented a valvular endocarditis in theleft ventricle. In all the forestomachs, large and deep necrotic and ulcerative areas surroundedby a haemorrhagic zone, in number ranging from 2 to 6, were seen with a thickening of theaffected zone. In the rumen of sheep no. 1, the lesion had perforated the wall and a focal area offibrinous peritonitis was seen on the serosa; the abomasum presented ulcerative and necrotizing

Fig. 1. Cross-section of a kidney from sheep no. 1 showing two renal infarcts and a purulent exudate intothe pelvis. Scale shown is in mm.

616 PEREZ et al.

Fig. 2. Nodules of variable size scattered throughout the lung of sheep no. 2. Note the presence of a clearreddish zone surrounding those of larger size (arrow) (bar = 1 cm).

lesions throughout all the mucosa which had resulted in the loss of parts of some abomasalfolds.

Histopathology

Microscopical examination revealed the presence of lesions in the brain of sheep no. 1and confirmed the gross changes already described. The mammary glands had lost their normalhistological architecture and multiple necrotic foci of variable size were scattered throughoutthe parenchyma. They consisted of a central area of necrosis surrounded by neutrophils,lymphocytes and some macrophages. Numerous PAS- and GMS-positive fungal structureswere seen in these foci. Septate and branched hyphae were arranged in a radial pattern.Throughout affected areas, vasculitis with fungal thrombi was a frequent observation. Thesupramammary and mediastinal lymph nodes showed a marked hyperplasia of lymphoid nodulesand an infiltration by neutrophils in addition to necrotic areas in the medullary sinuses wherehyphae could be noticed.

The kidneys presented large areas of coagulative necrosis together with necrotic lesionssimilar to those observed in the mammary gland. In sheep no. 1, large groups of neutrophilsshowing a high number of hyphae among them, were seen in the medulla. Thrombosis andfungal invasion of small blood vessels were also common features in this area.

Pulmonary lesions were consistent with a multifocal pneumonia and typical necrotizingmycotic granulomata, forming well-defined nodules, were observed. They were composed of acentral area of necrosis with PAS- and GMS-positive hyphae surrounded by neutrophils andan external layer of mononuclear cells, predominantly macrophages. However, in some lesionsgiant cells containing fungal structures (Fig. 3) were occasionally seen. Such nodules provokeda band of atelectasis in the surrounding alveoli (Fig. 3).

In the heart, a diffuse necrotizing myocarditis composed of an infiltrate of neutrophils,macrophages and some giant cells was present (Fig. 4). In the lesions, fungal structures werecommonly observed. The majority of muscle fibres had undergone necrosis and showed an

617Generalized Aspergillosis in Dairy Sheep

Fig. 3. Nodule composed of a central area of necrosis surrounded by neutrophils, macrophages and giantcells. Note the presence of Grocott’s methenamine silver (GMS)-positive hyphae. Lung. Sheep no. 1.

GMS, haematoxylin–eosin (HE) counterstain, × 240.

Fig. 4. Diffuse necrotizing myocarditis with the presence of degenerated muscle fibres and fungal structuresbetween them. Heart. Sheep no. 2. Grocott’s methenamine silver (GMS), haematoxylin–eosin (HE)

counterstain, × 240.

618 PEREZ et al.

eosinophilic and hyaline appearance (Fig. 4); mineralization was observed in some of them.Thrombosing vasculitis was also a common finding. In the areas corresponding to the grosslyvisible plaques, the majority of the fibres had disappeared and were substituted by an intensepyogranulomatous infiltrate and granulation tissue. As in the myocardium, PAS- and GMS-positive fungal structures were also demonstrated in the endocardial valves which presentedabundant fibrin and cellular debris together with granulation tissue.

The hepatic lesions consisted of small areas of necrosis of hepatocytes, associated withneutrophils and a few lymphocytes, and occasional fungal organisms.In the forestomachs necrotizing lesions similar to those already described were present in allthe layers, showing a higher intensity in the muscular wall. This layer was disrupted in therumen of sheep no. 1, leading to a perforation and the presence of deposits of fibrin on theserosa in which neutrophils were also seen. However, the abomasal lesions were characterizedby laminar coagulative necrosis of the lamina propria which showed a band of mineralization.In the abomasum, fungi were not detected.

Finally, in the diencephalon, at the level of the thalamus, of sheep no. 1, a necrotic focussurrounded by neutrophils and, externally, macrophages and giant cells, was present (Fig. 5). Itwas located in the vicinity of the choroid plexus of the lateral ventricles. PAS- and GMS-positive hyphae, as seen in other organs, were also demonstrated in the brain lesions.

Fungal identification

All PAS- and GMS-positive fungal elements revealed a strong and uniform reactivity withthe Mab (Mab-WF-AF-1) reacting with Aspergillus spp., whereas no reactivity was obtainedwhen the zygomycosis-specific antibody (Mab-WSSA-RA-1) was used or when the controlswere run. Identical green to blue-green fungal colonies were obtained from all the macroscopiclesions of both sheep. The fungus was identified as A. fumigatus, on the basis of the morphologyof fungal structures, according to Swatek et al. (1985).

Fig. 5. Detail of a necrotic focus surrounded by neutrophils and macrophages in the periphery, located inthe vicinity of the choroid plexus (right). Fungal structures (arrows) are seen. Brain. Sheep no. 1. Grocott’s

methenamine silver (GMS), haematoxylin–eosin (HE) counterstain, × 240.

619Generalized Aspergillosis in Dairy Sheep

Discussion

The present study describes two cases of generalized aspergillosis in adult sheep with avariety of organs affected. There are few reports concerning disseminated aspergillosis in sheep.Two cases have been reported in young lambs with a predominant respiratory lesion (Graceyand Baxter, 1961; Krishna et al., 1981); regarding adult sheep, systemic aspergillosis has beenseen associated with mastitis (Perez et al., 1998) and, in one case, with a primary alimentary andrespiratory involvement and subsequent spreading to the heart and liver (Chihaya et al., 1980).In our cases, lesions were also found in the kidney and, in one animal, in the brain, which, asfar as we are aware, is the first time this location has been reported in sheep. However, lesionsdue to aspergillosis in nervous tissues have been previously observed in man (Boon et al., 1990)and in cattle (Chihaya et al., 1988).

Ovine aspergillosis is a predominantly respiratory disease as it has been shown in thedifferent natural cases reported with only pulmonary involvement (Austwick et al., 1960; Young,1970; Chauhan and Dwivedi, 1974) and in experimentally infected sheep that constantlypresented lesions in the lungs without a link to the route of infection (Chattopadhyay et al.,1996). Although the lungs were severely affected in both sheep, it seems most likely that theprimary portal of entry for fungal invasion was the mammary gland, as mammary aspergillosisoccurred in sheep with occasional involvement of the lung and kidneys in some animals (Perezet al., 1998). The fact that other necropsied animals in the flock showed mammary lesionswithout spreading of the infection supports this hypothesis. The administration of intra-mammary antibiotics before parturition could be an important factor in the development ofmycotic mastitis (Thompson et al., 1978; Jensen et al., 1996a; Perez et al., 1998), especially whenit is performed with the lack of proper sanitary procedures as seemed to occur in the presentflock, where the treatment was carried out by untrained farmers. Due to the ubiquity of fungifrom the genus Aspergillus in the environment, the contamination of syringes used for theintramammary treatments could have been the source of infection, as has been suggested inother studies (Jensen et al., 1996a; Perez et al., 1998). However, the mechanism responsible forthe susceptibility of the mammary gland to fungal infections remains obscure (Dungworth,1993) and needs further investigations.

Although the mammary route of infection is the most suitable hypothesis, the presenceof severe lesions in the lungs and the forestomachs does not permit the complete exclusion ofa primary respiratory or alimentary lesion with subsequent spreading, as this has been describedboth in sheep and cattle (Gracey and Baxter, 1961; Chihaya et al., 1980, 1992; Jensen et al.,1991; Jensen, 1994).

Concerning the dissemination of the Aspergillus infection, several routes have been pro-posed (Jensen, 1994). Although the lymphatic spreading probably occurs due to the involvementof the lymph nodes, the haematogenous spread would be the most probable route for developinglesions in the different organs, as has been previously observed (Krishna et al., 1981; Jensenet al., 1991; Chihaya et al., 1992; Perez et al., 1998). This is supported by the common findingof thrombosing vasculitis in several organs.

The necrotizing features and the inflammatory reaction observed in the mammary glandand other organs are compatible with those previously observed in cows and sheep sufferingfrom aspergillosis (Austwick et al., 1960; Gracey and Baxter, 1961; Young, 1970; Chauhan andDwivedi, 1974; Chihaya et al., 1980, 1991, 1992; Krishna et al., 1981; Jensen et al., 1989, 1991,1996a; Kamil and Parihar, 1991; Jensen, 1994; Perez et al., 1998). They had the characteristicsof acute- to subacute-type lesions. However, the coagulative necrosis and mineralization withno fungal elements observed in the abomasum of sheep no. 1 would be consistent with lesionsassociated to uremia as a result of renal failure (Confer and Panciera, 1995). Supporting this,sheep no. 1 showed a severe lesion affecting both kidneys, although the blood level of ureacould not be tested.

The morphology of fungal hyphae present in tissue sections suggested a presumptiveaetiological diagnosis of aspergillosis. However, this typical morphology is shared by otherfungal species, including those responsible for cases of zygomycosis (Dungworth, 1993; Jensenet al., 1996a, b, c, d) which can also occur in sheep (Carrasco et al., 1993). To confirm thediagnosis, immunohistochemistry was performed and the only positive staining was obtained

620 PEREZ et al.

with Mab-WF-AF-1 confirming that the lesions were due to aspergillosis alone. Finally, therecovery of A. fumigatus in pure culture from several lesions helped to confirm the aetiologicaldiagnosis.

In conclusion, our observations demonstrate that aspergillosis may be an important andsevere disseminated disease in sheep. A correct manipulation of syringes and proper proceduresshould be considered in the intramammary administration of antibiotics to dairy sheep, otherwisesevere cases of mastitis and systemic aspergillosis may occur.

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