Isolation and characterization of Rhizopus stolonifer associated with otitis in cattle

Introduction

Otitis is an inflammatory condition affecting the external, middle, or inner ear and is associated with reduced animal welfare, decreased productivity, and economic losses in livestock production systems. Bacterial pathogens such as Staphylococcus spp., Streptococcus spp., Pseudomonas aeruginosa, and Proteus spp. are frequently implicated in bovine otitis; however, fungal pathogens are increasingly recognized as an important etiological agents, particularly in chronic and recurrent otitis cases (Duarte et al., 2004; Quinn et al., 2025 Elaiyaraja et. al., 2026) [1,7,3] . Fungal otitis is often underdiagnosed because routine diagnostic investigations primarily focus on bacterial pathogens. Opportunistic fungi may colonize the external auditory canal upon bacterial infection or following excessive moisture accumulation, tissue injury, or immunity compromised animals (Dworecka-Kaszak et. al., 2020) [2] . The warm and humid microenvironment of the ear canal provides favorable conditions for fungal proliferation and persistence. Rhizopus stolonifer, a member of the order Mucorales, is a ubiquitous environmental fungus commonly found in soil, decaying vegetation, animal bedding, and feed materials (Ribes et al., 2000) [8] . Although generally regarded as a saprophyte, Rhizopus species have been increasingly reported as opportunistic pathogens capable of causing localized and systemic infections in both humans and animals (Spellberg et al., 2005) [10] . The pathogenic potential of these fungi is primarily attributed to their rapid growth, thermotolerance, and ability to invade host tissues through broad aseptate hyphae. Members of Mucorales also possess a unique capacity for angioinvasion, resulting in vascular thrombosis, tissue ischemia, and necrosis, which are considered hallmarks of mucormycosis (Ibrahim et al., 2012) [4] . In animals, mucormycetes have been associated with respiratory infections, gastrointestinal disease, cutaneous lesions, mastitis, abortions, and systemic mycoses (Richardson, 2009) [9] . However, reports describing Rhizopus-associated otitis in cattle remain limited. The emergence of fungal pathogens in bovine otitis highlights the necessity of comprehensive microbiological investigations that include both bacteriological and mycological examinations. Accurate identification of fungal pathogens is essential for appropriate therapeutic interventions and for understanding their epidemiological significance in livestock populations. Therefore, the present study was undertaken to isolate and characterize Rhizopus stolonifer associated with otitis in cattle.

Materials and Methods

Study Animals and Sample Collection

Cattle presented with clinical signs suggestive of otitis, including discharge from the ear, irritation, head shaking, foul odor, and inflammation of the external ear canal, were selected for the study. Following clinical examination, ear swab samples from the affected ears were collected aseptically using sterile cotton swabs. The swabs were immediately transported to the department of veterinary microbiology laboratory for microbiological examination.

Fungal Isolation

Samples were inoculated onto Sabouraud dextrose agar (SDA) as well on brain heart infusion agar. The inoculated plates were incubated aerobically at 28±2 °C and monitored daily for fungal growth for up to seven days and at 37 °C for bacteriological growth. Emerging fungal colonies were subcultured onto fresh SDA plates to obtain pure cultures.

Cultural characteristics

The fungal isolates were characterized based on growth rate, pigmentation, surface and reverse colony appearance. The observations were recorded periodically during incubation.

Microscopic Characterization

A small portion of fungal growth was mounted in lactophenol cotton blue (LPCB) stain and examined under a light microscope for its microscopic features.

Results

Clinical Findings

Affected cattle exhibited varying degrees of otitic inflammation characterized by head shaking, ear scratching, purulent or serous ear discharge, foul-smelling exudate and pain upon manipulation of the ear.

Isolation of Fungal Organism

Pure fungal growth was obtained from affected ear samples following incubation on SDA.

Cultural characteristics

The fungal isolate produced rapidly spreading cottony colonies that initially appeared white and fluffy. As incubation progressed, colonies developed grayish-black pigmentation due to maturation of sporangia (Figure 1).

Microscopic Characteristics

The LPCB staining results revealed that the broad ribbonlike aseptate hyphae, long sporangiophores, spherical sporangia, abundant sporangiospores, well-developed rhizoids and these microscopic features were consistent with Rhizopus stolonifer (Figure 2).

Associated Bacterial Isolates

Gram staining of bacterial isolates recovered from otitic samples revealed predominantly Gram-positive cocci arranged in pairs and short chains.

Discussion

The present study reports the isolation and characterization of Rhizopus stolonifer from bovine otitis cases. The recovery of this fungus from clinical ear samples suggests that opportunistic fungal pathogens may contribute to the pathogenesis of otitis in cattle. Although bacterial agents remain the predominant causes of bovine otitis, fungal involvement should be considered, particularly in chronic, recurrent, or treatment-refractory cases. The cultural characteristic observed in the present study was characterized by rapidly growing cottony white colonies that subsequently developed gray to black pigmentation upon maturation. These findings are consistent with previous descriptions of Rhizopus species reported by Larone (1994) [6] , Kidd et al., (2022) [5] who described Rhizopus colonies as fast-growing, fluffy, and initially white before becoming grayish-black due to the development of mature sporangia (Quinn et al., 2025) [7] . Microscopic examination revealed broad aseptate hyphae, sporangiophores, sporangia, and rhizoids, which are considered characteristic features of Rhizopus stolonifer (Walther et al., 2019, Winn 2006) [11,12] . The presence of well-developed rhizoids directly beneath sporangiophores is an important distinguishing feature that differentiates Rhizopus species from other members of the Mucorales. The isolation of Rhizopus stolonifer from otitic lesions suggests a potential role in chronic inflammatory processes of the ear. The moist microenvironment within the external auditory canal may facilitate fungal colonization and proliferation, particularly in the presence of tissue damage or concurrent bacterial infection. Rhizopus spores are commonly present in soil, feed, and organic debris and can readily contaminate damaged epithelial surfaces (Ribes et al., 2000) [8] . Conditions such as prolonged antimicrobial treatment, excessive moisture, trauma, or concurrent bacterial infections may facilitate fungal colonization and subsequent infection (Spellberg et al., 2005) [10] . The pathogenesis of Rhizopus infections is largely attributed to the ability of the fungus to germinate rapidly and produce invasive hyphae capable of penetrating host tissues leading to local inflammation and tissue destruction. Mucorales fungi exhibit marked angioinvasive properties that promote vascular thrombosis, tissue infarction, and necrosis (Ibrahim et al., 2012) [4] . Although invasive mucormycosis is uncommon in cattle, localized infections can occur when host defenses are compromised.

The concurrent isolation of Gram-positive cocci from affected ears suggests the possibility of polymicrobial infections. Mixed bacterial-fungal infections have been reported in otitis and may contribute to disease chronicity and reduced therapeutic efficacy (Dworecka-Kaszak et. al., 2020) [2] . The inflammatory environment created by bacterial infection may facilitate fungal establishment, while fungal colonization may further aggravate tissue damage and inflammation.

The findings of the present study emphasize the importance of incorporating mycological investigations into routine diagnostic protocols for bovine otitis. Failure to recognize fungal involvement may result in prolonged disease duration and inappropriate antimicrobial usage.

Conclusion

The present study confirms the isolation and characterization of Rhizopus stolonifer from bovine otitis cases based on cultural characteristics and microscopic examination. The findings highlight the potential role of opportunistic fungal pathogens in bovine ear infections and emphasize the importance of including mycological investigations in chronic or recurrent otitis cases. Early diagnosis and appropriate therapeutic interventions may improve clinical outcomes and reduce economic losses associated with otitic diseases in cattle.

Declaration Conflict of Interest

On behalf of all author declare that no conflict of interest with the publication

Ethics Statement

The animals used in this study were in accordance with the ethics of the TANUVAS institutional animal ethics committee (IAEC).

Acknowledgement

I gratefully acknowledge and thankful to the TANUVAS University for funding and support during entire study period.

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