Isolation and characterization of a strain of Lichtheimia corymbifera (ex Absidia corymbifera) from a case of bovine abortion

Abstract

Background: Lichtheimia corymbifera (previously Absidia corymbifera) is a filamentous zygomycetes belonging to the order Mucorales and to the family Lichtheimiaceae. Members of genus Lichtheimia spp. are cosmopolitan and ubiquitous in nature. Lichtheimia corymbifera is a recognized agent of diseases in man and animals. In cattle it causes abortion and mastitis. Three cases of bovine abortion occurred in a herd located in the Po Valley. Serological examinations were performed on fetal and mother's blood. One of the aborted fetus was referred to our laboratory. The paper describes the isolation and characterization of Lichtheimia corymbifera from a bovine aborted fetus.

Methods: Serological examinations were performed on fetal and mother's blood. Lesions on fetal tissues and placenta leaded the diagnostic suspect towards a mycotic aetiology. Tissues were then put in culture, and at the same time an histological examination was performed, together with bacteriological and virological tests. The isolate from placenta and fetal tissues was identified and characterized by PCR and RFLP, using the ITS region as a target sequence and AclI restriction site within the amplicon to distinguish Lichtheimia corymbifera among the other fungi.

Results: Serological, bacteriological and virological tests gave aspecific results. Histological examination evidenced numerous PAS positive hyphae within the necrotic cotiledons and numerous fungal nonseptate hyphae to the GMS stain. Colonies with typical morphological features of fungi grew up on Sabouraud agar from fetal skin and placenta. On the developed colonies the microscopic examination has shown a large number of nonseptate hyphae and sporangia consistent with Mucorales. PCR and RFLP allowed the identification of the isolate as Lichtheimia corymbifera.

Conclusion: The present report describes the isolation and the molecular characterisation of a fungal isolate from bovine aborted fetus and placenta. The diagnostic protocol allowed to identify and characterise the strain. This is the first isolation in Italy of Lichtheimia corymbifera in a bovine aborted fetus.

Figure 1

Gross lesions on the fetus and placenta. A) The placenta is thickened, cotyledons appear necrotic and firm. Hyperemia of intercotyledonar areas is also visible. B) Aborted calf. White and irregular lesions localized in the head (periorbital region) are visible in the picture.

Figure 2

Histological findings on tissue sections. Histological section of the placenta. A) Numerous PAS positive hyphae are present within the necrotic cotyledons (PAS reaction, magnification ×200). B) Numerous fungal hyphae (black), irregular in diameter and without regular septation, are present. (GMS stain, magnification ×200).

Figure 3

Cultural and microscopic aspect of the fungus. A) Fungal culture. The whitish, woolly or cottony aspect of the colonies is evident. The colony raises 1.5 cm high. B) Microscopic aspect of sporangia, pyriform with a conical-shaped columella (indicated by arrow) and pronounced apophysis (Cotton Blue stain, magnification ×400). C) Nonseptate hyphae arising in groups of three at the internode (arrow, magnification ×400). D) Ellipsoidal sporangiospores (magnification ×1000).

Figure 4

Molecular characterization of Lichtheimia corymbifera. A) Electrophoresis patterns of amplicons obtained with specific sets of primers for Lichtheimia corymbifera, Rhizopus spp., Rhizomucor spp., Mucor spp. and a set of primers for all other filamentous fungi used as a positive control (840 bp). B) Diagram showing the amplicon (not on scale) and the AclI restriction enzyme predicted fragments of 518 and 306 bp. C) Diagram showing the sub-cloned 824 bp amplicon (not on scale) into the pTZ57R/T vector and AclI restriction sites within the vector (2194 bp and 2567 bp) and within the amplicon. D) Electrophoresis patterns of AclI uncut 824 bp amplicon, AclI cut 824 bp amplicon and AclI cut sub-cloned 824 bp amplicon.