Pathological and microbiological investigations in mortality outbreaks involving doctor fish (Garra rufa, Heckel 1843) used for cosmetic treatment

Introduction: Doctor fish are widely used in fish spa; their presence as living animals prohibits the use of conventional methods for water and equipment sterilization and disinfection. This has led to concerns about the potential transmission of infection to human beings and on fish welfare. The susceptibility to microbiological agents in this species is not well known, only few reports focused on episodes of Garra rufa mortality are available. The aim is to describe disease outbreaks occurred in a fish spa Materials and Methods: In the period 2016-2017 two outbreaks resulted in a chronic mortality. Collected fish were euthanized and sampled immediately for pathological, bacteriological and molecular (Infectious Pancreatic Necrosis Virus-IPNV) investigations. A classification based on timing of granulomatous lesions, in association with an acid-fast staining and immunohistochemistry against Mycobacterium bovis were performed. Results: Some animals showed rapidly growing erosive and ulcerative skin lesions. The main histopathological finding was otherwise visceral, intermediate or late stage, granulomatosis. Immunoreactivity to Mycobacterium bovis was registered. Phenotypic and genetic characterizations of isolated bacteria showed the presence of Aeromonas veronii and Vibrio cholerae in one batch (2016), Aeromonas veronii and Shewanella putrefaciens in another batch (2017). The IPNV- RNA was not found. Conclusions: The attention to granulomatogenous bacteria in aquatic species has increased because of their zoonotic potential. In the cases described, microbiological investigation associated to the pathological features led to the final diagnosis of mycobacteriosis. The presence of other bacteria has led some concerns about the use of these animals for cosmetic treatment.