Immunohistochemical Diagnosis of Systemic Bovine Zygomycosis by Murine Monoclonal Antibodies

Autor: Bent Aalbæk, Henrik Jeldtoft Jensen, H. V. Krogh, Peter Lind
Rok vydání: 1996
Předmět:
Zdroj: University of Copenhagen
ISSN: 1544-2217
0300-9858
DOI: 10.1177/030098589603300207
Popis: Murine monoclonal antibodies (Mabs) against water-soluble somatic antigens (WSSA) and the wall fraction (WF) from Rhizopus arrhizus (Rhizopus oryzae) were produced in vitro by fusion of splenocytes from immunized BALB/c mice with mouse myeloma X63-Ag 8.653 cells. Supernatants reacting only with homologous antigens in an enzyme-linked immunosorbent assay were subsequently screened for reactivity with homologous fungi in immunohistochemical techniques. All four Mabs raised against the WF of A. arrhizus failed to react on tissues. However, four of the Mabs raised against the WSSA of R. arrhizus (Mab-WSSA-RA- 1 through Mab- WSSA-RA-4) revealed a high homologous reactivity on tissues and the cross-reactivity ofthese were subsequently evaluated on tissues containing other members of the family Mucoraceae and other unrelated fungi. On tissues and on immunoblots all four Mabs reacted identically and specifically with members of the family Mucoraceae, i.e., Absidia corymbifera, R. arrhizus, and Rhizomucor pusillus. The Mabs were all isotyped as IgM antibodies, were nonprecipitating, and reacted with homologous antigens with molecular masses from 14 to 1 10 kDa. With WSSA from A. corymbifera and R. pusillus the four Mabs were bound to antigens from 14 to 52 kDa and from 20 to 28 kDa, respectively. The diagnosis of 145 bovine lesions obtained by one of the specific Mabs (Mab- WSSA-RA- 1) were compared to results obtained by heterologously absorbed polyclonal antibodies. In most lesions (n = 140 (- 97%)) the Mab and the polyclonal antibodies reacted in a similar pattern, i.e., positively for zygomycosis in 89 lesions, negatively in 4 1 aspergillosis lesions, and negatively in 10 undiagnosed lesions. Hyphae within two of four lesions in lymph nodes, which were not stained by the polyclonal antibodies, reacted with the specific Mab. However, in another three lesions of lymph nodes stained by the polyclonal antibodies no reactivity was seen with the Mab-WSSA-RA- 1. The immunoreactivity of the Mabs (Mab-WSSA-RA-1 through Mab-WSSA-RA-4) raised against WSSA of R. arrhizus justify their application for the in situ diagnosis of systemic bovine zygomycosis. Zygomycosis and aspergillosis are the main causes of systemic bovine mycoses due to filamentous fungi.2-6,13-20,25,27,30 Zygomycosis is predominantly caused by members of the family Mucoraceae of which especially Absidia corymbifera, Rhizopus spp., and Rhizomucorpusillus (syn. Mucorpusillus) are frequent- ly associated with systemic infections.2,3,6J3J6-25~30 As- pergillus fumigatus is the most common etiological agent of systemic bovine aspergill~sis.~~ ~~~~,'~ Systemic bovine mycoses are usually not diagnosed before histopathology is applied.24,'3-20,25,27~30 More- over, attempts to culture, or to judge the identity of a fungal agent within tissues on the basis of morpholog- ical criteria, are in many cases ~nrewarding.'-~,~,'~~~~z~~,~~ During recent years a number of immunohistochem- ical techniques have contributed significantly to the diagnosis of systemic aspergillosis and zygomycosis of man and animals. In the majority of these techniques polyclonal antibodies have been used as the primary
Databáze: OpenAIRE
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