Three consecutive cases of rhinocerebral mucormycosis in calves caused by Lichtheimia ramosa infection on a beef cattle farm

Abstract

Three Japanese Black calves died within a two-month period on a beef cattle farm. Necropsy consistently revealed liquefactive necrosis of the ventral brain (extending from the olfactory bulb to the midbrain) and thymic atrophy. Histopathologically, necrosuppurative encephalitis characterized by abundant fungal hyphae was observed in all three cases. Fungal lesions also involved the olfactory mucosa, olfactory nerves, and olfactory bulb. Fungi isolated from the cerebrum of all three calves were identified as Lichtheimia ramosa. These cases were diagnosed as rhinocerebral mucormycosis, originating in the olfactory mucosa and extending to the cerebrum. Although mucormycosis is typically sporadic, this report documents the first occurrence of consecutive cases of bovine rhinocerebral mucormycosis caused by L. ramosa.

Zygomycosis is a term used for infectious diseases caused by fungi within the phylum Zygomycota. Based on recent phylogenetic analyses, Zygomycota was reclassified into Mucoromycota (including Mucoromycotina, Mortierellomycotina, and Glomeromycotina) and Zoopagomycota (including Kickxellomycotina, Zoopagomycotina, and Entomophthoromycotina) [1, 28]. Infectious diseases caused by Mucorales—which encompasses the majority of pathogenic genera, such as Rhizopus, Rhizomucor, Mucor, and Lichtheimia—are termed mucormycosis [1, 28]. In human medicine, mucormycosis is classified into five clinical forms: rhinocerebral, pulmonary, gastrointestinal, cutaneous, and disseminated [21]. In cattle, mucormycosis has been reported to manifest as encephalitis [5, 9, 12, 13, 24], pneumonia [2], gastroenteritis [4, 10, 23], lymphadenitis [18, 19, 25], abortion [20], and systemic infection [6, 14, 16, 26]; however, these occurrences have typically been sporadic. In the present report, we describe three consecutive cases of rhinocerebral mucormycosis caused by Lichtheimia ramosa infection in Japanese Black calves on a beef cattle farm.

Three Japanese Black calves died on April 6 (Case 1; 14 days old), May 17 (Case 2; 8 days old), and May 19 (Case 3; 11 days old). Cases 1 and 3 exhibited nystagmus and opisthotonus antemortem. Case 3 was emaciated. None of the calves had a history of prior treatment, and all were raised in individual calf hutches. The calves had been transferred from another farm at 1–2 days of age. No abnormalities were observed in any of the three calves at birth, and all had been fed colostrum. Necropsies were performed on the day of death. Representative organs, including the liver, spleen, kidney, heart, lung, gastrointestinal tract, thymus, brain, and a portion of the cranium (encompassing the ethmoid bone), were fixed in 20% neutral-buffered formalin for pathological examination. Following fixation, cranial specimens were treated with a rapid decalcification solution (K-CX, Falma, Tokyo, Japan) at 4°C for 48 hr and processed to examine the olfactory mucosa, olfactory nerve, and olfactory bulb. Fixed tissues were embedded in paraffin, sectioned at a thickness of 3 µm, and stained with hematoxylin and eosin. Periodic acid-Schiff (PAS), Grocott’s methenamine silver (GMS), and immunohistochemical staining were performed to detect fungal hyphae. For immunohistochemistry, primary antibodies included anti-Rhizomucor mouse monoclonal antibody (clone: WSSA-RA-1, Dako, Carpinteria, CA, USA), anti-Aspergillus mouse monoclonal antibody (clone: WF-AF-1, Dako), and anti-Candida albicans rabbit polyclonal antibody (Biogenesis, Dorset, UK). The Histofine Simple Stain MAX-PO (Multi) (Nichirei Bioscience, Tokyo, Japan) was employed as the detection system, following the manufacturer’s instructions. Sections were visualized using a 3, 3’-diaminobenzidine (DAB) substrate solution (Histofine DAB substrate kit, Nichirei Bioscience). The anti-Rhizomucor mouse monoclonal antibody cross-reacts with Lichtheimia and Rhizopus in addition to Rhizomucor (manufacturer’s data sheet).

To isolate bacteria, tissue samples from the cerebrum, midbrain, cerebellum, liver, spleen, kidney, heart, and lung of all three calves—as well as spinal cord (Cases 1 and 2), cerebrospinal fluid, and nasal/tympanic swabs (Case 1)—were inoculated onto 5% sheep blood agar and deoxycholate-hydrogen sulfide-lactose (DHL) agar plates. Cultures were incubated at 37°C for 24 hr under 5% CO₂ (blood agar) or aerobic conditions (DHL agar). Fungal colonies isolated on 5% sheep blood agar from all cerebrum samples, the midbrain samples of Case 1 and 3 and the lung sample of Case 2 were subcultured on potato dextrose agar at 37°C for 3–5 days under aerobic conditions. Bedding material from the calf hutch of Case 3 was also submitted for mycological examination, despite the absence of visible mold. Colony morphology and lactophenol cotton blue-stained preparations were examined using stereomicroscopy and light microscopy, respectively. Genomic DNA was extracted from fungal isolates of Cases 2 and 3 via bead beating. Subsequently, the internal transcribed spacer (ITS) regions were amplified by polymerase chain reaction (PCR) using the primer pair ITS1 and ITS4 [29]. PCR amplicons were directly sequenced, and the resulting sequences were analyzed using the Basic Local Alignment Search Tool (Nucleotide BLAST, https://blast.ncbi.nlm.nih.gov/) against the GenBank database. Phylogenetic analysis of the ITS region sequences was performed as previously described [9].

Reverse transcription-PCR and PCR assays were performed to detect bovine viral diarrhea virus (BVDV) and bovine adenovirus (BAdV), respectively [27]. Tissue samples (cerebrum, liver, spleen, kidney, heart, and lung) from all three calves, as well as rectal contents from Case 1, were tested to detect BVDV. The same tissue set and rectal contents from Case 2 and 3 were examined for BAdV. For virus isolation, the same tissue set and rectal contents from all three calves were passaged three times on MDBK cells.

Gross examination revealed liquefactive necrosis of the ventral brain (extending from the olfactory bulb to the midbrain) and thymic atrophy in all three cases (Fig. 1). On the cut surface, dark red lesions were observed in the midbrain and pons of Case 1, and in the midbrain of Case 3. Additionally, Case 1 presented with dark red, edematous lungs. Case 2 exhibited several ulcers (0.5–1 cm in diameter) on the ruminal mucosa. In Case 3, adhesions were noted between the cecum and a segment of the colon, accompanied by dark green discoloration. Hemorrhagic lesions were also noted in the connective tissue surrounding the umbilical artery and urinary bladder (Cases 2 and 3), as well as on the abomasal serosa (Case 3).

Fig. 1.

Macroscopic findings on the ventral aspect of the brains. Liquefactive necrosis is present in the ventral brain, extending from the olfactory bulb to the midbrain. (a) Case 1. (b) Case 2. (c) Case 3.

Histopathologically, all cases were characterized by liquefactive necrosis and necrosuppurative encephalitis, accompanied by multifocal necrotizing vasculitis and thrombosis containing numerous fungal hyphae (Fig. 2). Neuronal ischemic changes were evident within the lesions. Fungal hyphae were broad (4–12 µm in width), pauciseptate, and irregularly branched, with thin cell walls. Pyogranulomatous inflammation with intralesional fungal hyphae was observed in the olfactory mucosa and nerves, and necrosuppurative lesions involving the olfactory bulb were present in all three cases (Figs. 3

Fig. 4.

Pyogranulomatous inflammation with intralesional fungal hyphae is observed in the olfactory mucosa of Case 3. Arrows indicate fungal hyphae. Decalcified tissue, HE staining.

Fig. 5.

Pyogranulomatous inflammation with intralesional fungal hyphae is observed in the olfactory nerves of Case 3. Arrows indicate fungal hyphae. Decalcified tissue, HE staining.

–6). In Case 2, neutrophilic infiltration associated with fungal hyphae and thrombi was observed in the ruminal lamina propria. In Case 3, thrombi and perivascular neutrophilic infiltration accompanied by fungal hyphae were present in the cecal lamina propria. Hyphae were visualized using PAS and GMS stains (Fig. 7). Immunohistochemically, hyphae reacted positively with anti-Rhizomucor antibodies but were negative for anti-Aspergillus and anti-Candida antibodies (Fig. 7). The distribution of brain lesions is depicted in Fig. 8. The thymus exhibited severe atrophy in all cases. Additionally, focal suppurative bronchopneumonia with epithelial desquamation and alveolar fibrin exudation was observed in Case 1. In Case 2, alveolar and bronchiolar spaces contained serous fluid, meconium, and desquamated epithelium, consistent with meconium aspiration; however, fungal hyphae were absent in these pulmonary lesions. In lung sections from Case 2, fungal elements were not detected by PAS, GMS, or immunohistochemical staining. Abomasal submucosal hemorrhage was noted in Case 3.

Fig. 2.

Necrosuppurative encephalitis, accompanied by necrotizing vasculitis and thrombosis, is evident in the cerebrum of Case 1. Hematoxylin and eosin (HE) staining.

Fig. 3.

Sagittal section of the cribriform plate in Case 3. Yellow boxes indicate the regions corresponding to the higher-magnification views shown in Figs. 4–6. Decalcified tissue, HE staining. Inset: The red box indicates the sampling site of the ethmoid bone for histopathological examination.

Fig. 6.

Necrosuppurative encephalitis, concurrent with necrotizing vasculitis, thrombosis, and intralesional fungal hyphae, is present in the olfactory bulb of Case 3. Arrows indicate fungal hyphae. Decalcified tissue, HE staining.

Fig. 7.

The hyphae are visualized using Periodic acid-Schiff (PAS) and Grocott’s methenamine silver (GMS) stains. Immunohistochemically, the hyphae react positively with anti-Rhizomucor antibodies but negatively with anti-Aspergillus and anti-Candida antibodies. Bar=20 μm.

Fig. 8.

Distribution of brain lesions across the three cases. Circled numbers 1–7 represent cross-sections of the brain at the indicated locations.

Zygomycetes were isolated from the cerebrum and midbrain of Case 1; the cerebrum (Isolate 1) and lung (Isolate 2) of Case 2; the cerebrum (Isolate 3) and midbrain (Isolate 4) of Case 3; and bedding material from the hutch of Case 3. In lactophenol cotton blue preparations, a cup-shaped apophysis was observed subtending the sporangium (Fig. 9a). Stereomicroscopy revealed round sporangia and branched sporangiophores (Fig. 9b). Molecular analysis of Isolates 1–4 indicated that the ITS region nucleotide sequences shared 100% (Isolates 1, 3, and 4) or 99.9% (Isolate 2) identity with Lichtheimia ramosa strain D35097Fukushima2241 (Accession No. LC643024). Identical results (100% identity) were obtained for the isolate from the bedding material. Phylogenetic analysis of the ITS sequences further confirmed that the isolates clustered with L. ramosa. Based on these findings, the isolated fungi were identified as L. ramosa. Escherichia coli was isolated from the lung of Case 1, but no pathogenic bacteria were isolated from Cases 2 and 3. All PCR results for BVDV and BAdV were negative, and no viruses were isolated.

Fig. 9.

Morphology of the isolated fungi. (a) In lactophenol cotton blue preparations, a cup-shaped apophysis is observed subtending the sporangium. Bar=25 μm. (b) Stereomicroscopy reveals round sporangia and branched sporangiophores (yellow circle). Bar=0.128 mm.

Reports identifying specific fungal species in cases of bovine mucormycosis are limited. Lichtheimia (formerly Absidia) infection in cattle has been associated with necrotic granulomatous encephalitis [9, 24], granulomatous lesions in the liver and gastrointestinal tract [14], granulomatous lymphangitis [25], abortion [20], and systemic infection [16], although occurrences are sporadic. The rhinocerebral form is the most frequent type of human mucormycosis; however, only one such case has been previously reported in cattle [24]. In the present report, we describe three cases of rhinocerebral mucormycosis in calves caused by L. ramosa. The distribution of lesions was characteristic: specifically, liquefactive necrosis in the ventral brain (extending from the olfactory bulb to the midbrain), and pyogranulomatous inflammation containing fungal hyphae in the olfactory mucosa and nerves were observed in all three cases. These findings are consistent with the previous report [24]. The fungal infection is hypothesized to have originated in the olfactory mucosa and extended to the frontal cerebrum via the olfactory nerves. Angioinvasion and infarction are hallmark features of invasive mucormycosis [3, 7]. These characteristic lesions were consistently observed in the present cases. Although fungal lesions with thrombi were also identified in the rumen (Case 2) and cecum (Case 3), these likely resulted from hematogenous dissemination from the primary cerebral foci rather than representing the primary portal of entry. Alternatively, given the size of gastrointestinal lesions, they may have arisen independently via infection through the gastrointestinal mucosa.

Lichtheimia ramosa is a ubiquitous saprophyte found in soil, pasture and rumen flora [15, 17, 22] and is recognized as a causative agent of human mucormycosis [8]. Fungal isolates from Cases 2 and 3 exhibited apophyses and branched sporangiophores, characteristic features of the genus Lichtheimia. Species identification as L. ramosa was confirmed via phylogenetic analysis of the ITS region. Although molecular identification was not performed for the isolate from Case 1, the causative agent was presumed to be identical to that in Cases 2 and 3, based on hyphal morphology and immunohistochemical results. Since L. ramosa was isolated from bedding material in the hutch of Case 3, it is suspected that the contaminated bedding might be the source of infection.

Immunosuppression, diabetes, and neoplasia are established risk factors for mucormycosis; however, infections have also been reported in immunocompetent individuals [3, 8, 21]. In the present study, all three calves exhibited severe thymic atrophy, suggesting potential immunocompromise, although the etiology of the atrophy remains undetermined. Severe thymic atrophy may have been related to an underlying condition, such as weak calf syndrome. The administration of glucocorticoids and prolonged broad-spectrum antibiotic therapy has been implicated in L. ramosa invasion and proliferation in a reported case of disseminated calf infection [14]; however, the calves in the present study had no history of prior treatment. Four cases of rhinocerebral zygomycosis (conidiobolomycosis) in sheep caused by Conidiobolus incongruus—a member of the Zoopagomycota (formerly Zygomycota)—were reported in Australia [11]. These sheep grazed in a low-lying paddock adjoining a tidal river, and the resulting humid environmental conditions were considered predisposing factors [11].

We hypothesize that the onset of disease in the present cases involved multiple factors, including host immune status, spore concentration in bedding material, and ventilation within the calf hutches. Notably, all reported bovine cases of necrotic encephalitis caused by L. ramosa, including the present cases, have occurred in Japanese Black calves [9, 24]. Furthermore, the previously reported cases occurred at 5 and 12 days of age, and the present cases were similarly observed in neonatal calves. Further investigation is warranted to determine whether breed-specific predisposing factors, such as genetic background and specific husbandry conditions, are involved.

In conclusion, this is the first report of consecutive cases of rhinocerebral mucormycosis in calves caused by L. ramosa. Given the rarity of rhinocerebral mucormycosis in cattle, the accumulation of further case data is essential to elucidate the pathogenic characteristics of L. ramosa and identify predisposing factors.

Conflicts of Interest

The authors have no conflicts of interest to declare.