No abstract

An immunofluorescence staining system was developed for the identification of mycelial fungi causing systemic mycoses in cattle. Antisera to Absidia corymbifera, Aspergillus fumigatus, Aspergillus flavus, and Aspergillus niger were raised in rabbits. Tissue sections of experimentally infected mice were used to determine the specificity of immune staining with antisera that had been absorbed with heterologous antigens. When 46 lesions from 22 cattle with systemic mycosis were studied, the infecting agent could be classified as a zygomycete, possibly A. corymbifera, in 18 lesions (15 cattle), A. fumigatus in i0 lesions (7 cattle) and both a zygomycete and A. fumigatus in a single lesion from one animal. Diagnosis was established by light microscopy in 13 (72%) of the 18 lesions classified as zygomycosis. Conversely, a dual infection with both a zygomycete and an Aspergillus species was suspected on the basis of morphology in nine of the 10 lesions reacting exclusively and uniformly with anti-A. fumigatus antiserum. A. fumigatus may be a more frequent aetiological agent of systemic mycosis in cattle than hitherto recognized on the basis of light microscopy and mycological culture.In Scandinavia most cases of systemic mycosis in domestic animals are caused by fungi of the subclass Zygomycetidae or the genus Aspergillus (11,14). Identification of the aetiological agent normally requires mycological culture, but this cannot be accomplished in cases where hyphae are found unexpectedly on microscopical examination at autopsy. Moreover, attempts to isolate zygomycetes and aspergilli from tissues often fail [3,4,9,15].Hyphae may be seen in sections stained with haematoxylinandeosin (HE), but visualization can be improved markedly with special stains such as periodic acidSchiff (PAS) or Grocott's methenamine-silver stain (GMS) [2]. Distinctive morphological details may provide a tentative mycological diagnosis but the appearance of hyphae in sections can be affected by steric orientation, age of hyphae, type of infected tissue and the host response [3,16]. Identification of Absidia corymbifera (A. lichtheimii) and

SUMMARYFrom each of two flocks (A and B) of poults comprising 14,100 females and 11,300 males, respectively, 15 poults were examined pathologically. Poults of flock A had signs of neurological disturbances whereas birds from flock B showed respiratory symptoms. Gross lesions were observed only in two poults from flock A in which minute circular areas of cerebral malaria were seen. Histopathologically, the brain lesions contained fungal elements, and so did some of the pulmonary granulomas detected in three and six poults out of four and six birds examined from flock A and B, respectively. Mycological cultivation was attempted from the brains and lungs of five poults from flock A. However, only from the brain of a single bird a fungus, identified as Aspergillus fumigatus, was grown. Immunohistochemistry was applied because the histomorphology of fungal elements within some lesions did not offer any characteristics allowing an assessment of the identity of the infective fungi. Moreover, as fungi could not be detected within all lesions, immunohistochemistry accomplished the screening of tissues. For immunostaining of tissues a panel of monoclonal and polyclonal antibodies identifying agents of aspergillosis, candidosis, fusariosis, scedosporiosis, and zygomycosis, was used. Due to a strong and uniform reactivity of all fungal elements with immunoreagents to Aspergillus spp. an unequivocal diagnosis of aspergillosis was established in all mycotic lesions. Apart from the establishment of an aetiological diagnosis, the application of immunohistochemistry also disclosed fungal fragments in granulomas which could not be identified with conventional histochemical stains.

Background-IgG and IgG subclass antibodies to Aspergillus fumigatus (A fumigatus) were measured in a large population of patients with cystic fibrosis to elucidate a putative antibody pattern specific for allergic bronchopulmonary aspergillosis (ABPA). Conclusions-In a large number of unselected patients with cystic fibrosis significantly increased levels of Af-specific antibodies belonging to total IgG and all four subclasses were found in all groups of patients compared with healthy controls. In patients persistently colonised with A fumigatus these levels were significantly higher than in non-colonised patients, and the significantly highest levels (with the exception of IgG 3 ) were found in patients with ABPA. Using a sensitive ELISA technique, measurements of IgG and IgG subclass antibodies to A fumigatus might be of importance in the management of ABPA, especially as a screening test to exclude the presence of ABPA; other tests are needed to confirm the diagnosis. Methods-An

Abstract. Murine monoclonal antibodies (MAbs) against water-soluble somatic antigens (WSSA) and the wall fraction (WF) from Aspergillus fumigatus were produced by fusion of splenocytes from immunized BALB/c mice with mouse myeloma X63-Ag 8.653 cells. The supernatants of in vitro cultured hybridomas were initially screened for reactivity with the WSSA and the WF from A. fumigatus and WSSA of other fungi in an enzymelinked immunosorbent assay (ELISA). Supernatants reacting only with A. fumigatus antigens were subsequently screened for homologous and heterologous reactivity with immunohistochemical techniques using formalinfixed, paraffin-embedded tissues from experimentally infected mice. Because of a high immunohistochemical reactivity with homologous fungi, 4 MAbs raised against A. fumigatus WSSA and WF were selected for a further evaluation of cross-reactivity (diagnostic specificity) in immunohistochemical and immunoblotting assays. In immunohistochemical assays, all MAbs raised against WSSA cross-reacted heavily with a number of other fungal species. All 4 MAbs (MAb-WF-AF-1-4) raised against the WF reacted strongly with hyphae of Aspergillus spp.; hyphae of Scedosporium apiospermum were also strongly labeled by MAb-WF-AF-3 and-4. The 2 specifically reacting MAbs (MAb-WF-AF-1 and-2) were of the IgM biotype and were precipitating, and in immunoblotting experiments both bound to a 106-kD antigen of the WF, whereas they did not bind to WSSA of A. fumigatus. One of the 2 aspergillosis-specific MAbs (MAb-WF-AF-1) was used to screen 145 mycotic lesions of cattle. The diagnoses on bovine lesions obtained by MAb-WF-AF-1 were compared with results based on reactivity with heterologously absorbed polyclonal antibodies and, for some lesions, to culture results. In the vast majority of lesions (n = 133), the MAb-WF-AF-1 and the polyclonal anti-Aspergillus antibodies reacted in a similar pattern, i.e., positively in 41 aspergillosis lesions and negatively in 92 zygomycotic lesions. Hyphae in 3 of 12 lesions that were not stained by the polyclonal antibodies reacted with the specific MAb-WF-AF-1; i.e., aspergillosis was diagnosed. The characteristics of the 2 MAbs (MAb-WF-AF-1 and-2) raised against the WF of A. fumigatus in ELISA and immunoblotting and immunohistochemical assays justify their application for the in situ diagnosis of systemic aspergillosis of cattle.Aspergillus fuimigatus is the main cause of systemic bovine aspergillosis, a disease occuring worldwide and of great importance in cattle production. [2][3][4]14,[18][19][20][21]27,31,37 Because systemic mycoses including aspergillosis are difficult to diagnose clinically, most cases are not diagnosed until histopathologic evaluation is completed as part of a postmortem examination. 2-4,14,18-2l,27,3l,37 Attempts to culture the etiologic agents of systemic mycoses including Aspergillus spp. may give false results because of several difficulties, e.g., growth failure, overgrowth by contaminants, recovery of a fungus different from that suggested by the morpholo...