Beyond bacteria: Large agents with analogies to Mimiviruses detected in canine cancers. Reexamining Gram staining in cancer diagnostics

Elena Angela LUSI^1*Federico Caicci²Viola Zappone^3*Marco Quartuccio³Ilaria Dragà³Antonio Ieni⁴Cornelia Mannarino³Giuseppe Mazzullo³Claudia Rifici³

• ¹St Vincent Health Care Group-UCD, Dublin, Ireland
• ²Department of Biology, Electon Microscopy Unit, University of Padua, Padua, Italy
• ³Department of Veterinary Sciences, University of Messina, Messina, Italy
• ⁴Department of Human Pathology in Adult and Developmental Age "Gaetano Barresi", Section of Pathology, University of Messina, Messina, Italy

While Gram staining is traditionally used for classifying bacteria based on their cell wall properties, Mimiviruses and large mammalian agents can also retain the Gram stain, despite not being typical bacteria. In fact, Mimivirus-like agents that exhibit Gram-positive staining were first found in human tissues, particularly in malignant samples, suggesting that these agents may be involved in a unique carcinogenic process. In order to translate the findings published in human medicine to animal models, we evaluated for the first time the presence of analogous Gram-positive agents in canine malignancies and differentiate them from traditional bacteria. Using Gram staining, we analyzed 35 canine tumors across various malignancy types, including 7 sarcomas, 15 carcinomas, and 13 round cell tumors such as mast cell tumors, transmissible venereal tumors and melanomas. Normal tissues and bacteria were used as controls. We were able to identify Gram-positive granulations, exhibiting intracytoplasmic, intra-nuclear and perinuclear patterns, measuring 1-2 µm that were distinct from traditional bacteria. This study, the first of its kind in the veterinary literature, supports comparable published findings in human research and advances our knowledge of the pathophysiology of cancer across species.