Discussion and conclusions
Cichlid fishes (Cichlidae) are one of the most worldwide species-rich and widespread families of vertebrates representing a substantial part of the ornamental fish trade and industry (Msukwa et al., 2021). Despite that, a paucity of information on the status of fish stock health is available.
Ornamental fish can display aspecific signs of disease such as emaciation, exophthalmia, keratitis and skin lesions, as nodules and black spots associated with differente causes.
Regarding viral diseases, in ornamental fish including cichlids, several infections due to iridovirus-like microorganisms are reported (Bucke, 2001). Particularly, lymphocystis outbreaks are characterized by a low mortality rate; however, the obvious cutaneous lesions make the subjects not suitable for sale (Noga, 2010). The lymphocystis virus generally infects dermal fibroblasts causing their hypertrophy; internal organs or gills are rarely affected (Russell, 1974). LCDV commonly presents with white to pigmented masses grossly visible mainly on the pectoral and dorsal fins (Volpatti and Ciulli, 2022), similar to those observed in some of the fish investigated in this study; however in our cases LCDV was not detected. The cutaneous nodules histologically were consistent with chronic dermatitis that matched with the negative molecular results. Chronic dermatitis was related with repeated post-traumatic events occurred. In fact, these fish species are extremely territorial and frequent fighting amongst each other.
Several bacteria that may cause damage to the fish are found naturally in their microbiota or environment and are usually in balance with their hosts. In cases of worsening environmental conditions, skin injuries and/or host immune system impairment, these bacteria may become pathogenic. Mycobacterial infections are the most common chronic disease affecting ornamental species (Noga, 2010), since so far is reported in more than 150 species (Decostere et al., 2004). This disease may cause also human infections, presenting as cutaneous ulcers that struggle to heal (Noga, 2010). As a matter of fact, fish mycobacteriosis is sustained most of the time by three species: M. marinum , M. fortuitum , M. chelonae (Decostere et al., 2004). Other less frequently isolated species are M. abscessus , M. gordonae ,M. conceptionense , M. parascrofulaceum and M. senegalense (Shukla et al., 2014). In this study, the results aligned with those reported, with both M. chelonae and M. parascrofulaceum detected in our fish subjectes. A previous study investigated the presence of mycobacteria in freshwater ornamental fish including six species of Cichlids with signs of chronic disease such as persistent cutaneous lesions, abdominal swelling and overall poor general health. This study pointed out the presence of a granulomatous inflammation associated with acid-fast bacteria in 41% of them. However, characterization of bacteria associated with granulomas was not conducted (Gómez, 2008).
Nevertheless, granulomas can be caused bu a variety of bacteria besides mycobacteria (Colquhoun and Duodu, 2011; Maekawa et al., 2018; He et al., 2020).
In our study, Mycobacterium infections were found in only two out of six subjects with granulomas. In seven cases, of whom four showed granulomas, C. acnes was identified from FFPE and/or frozen samples through molecular analysis and immunohistochemistry. This finding was interpreted as an intralesional presence, as it was found within visceral organs from fish placed in sterile conditions for tissue sampling. Recently, this bacterium has been found within fish and aquatic environment (Meron et al., 2020; Lorgen-Ritchie et al., 2021), however its pathological role in fish has not been so far investigated.
C. acnes (previously called Propionibacterium ) is an anaerobic, commensal, lipophilic Gram-positive bacterium. C. acnes , classically studied as human bacterial agent of acne vulgaris, is an opportunistic pathogen with a likely underestimated role in the development of disease. In addition to acne, it is associated with other human diseases including prosthetic joint infections, prostate cancer, intervertebral disks surgery and sarcoidosis (Fischer et al., 2020; Mayslich et al., 2021). It is now the second most frequent pathogen, after coagulase-negative staphylococci, isolated from infected internal cerebral ventricular bypasses, and rates of infection with this bacterium have increased from 1.5% to 38%. C. acnes has also been fould in blood cultures where it may represent up to 80% of the isolated anaerobes (Mayslich et al., 2021). However, its role in human diseases is still debated, its wide colonization of human organs suggests that the bacterium does not harm the human host, at least not under normal circumstances (Brüggemann et al., 2021).
Analogously, C. acnes was recently detected through NGS analysis in internal organs of marine fish without external or internal pathological changes and could suggest its harmless nature; however, three well-known bacterial pathogens, Photobacterium damselae ,Vibrio harveyi and Streptococcus iniae, were also found in these organs (Meron et al., 2020). Despite previous studies stating that healthy fish internal organs should be sterile, lately, studies have reported that bacteria are being foundin healthy kidneys and livers (Sevellec et al., 2014; Meron et al., 2020). However, assuming the organs were macroscopically healthy, a histopathological investigation was not performed in these studies, so the presence of potential tissue reactions as granuloma in association with the bacterial presence was not possible to exclude.
To demonstrate how C. acnes causes infection or colonization suggestions have been made to use a combination of techniques including immunohistochemistry (Capoor et al., 2019). In our study, complementary techniques like PCR, histochemical stainings and IHC were performed to detect bacterial components. According to the experience of these authors (Volpe et al., 2019), the sampling of tissues using combined methods, such as IHC and molecular techniques, is highly recommended to maximize the results. Gram-positive bacterial aggregates, that were concurrently immunoreactive to the anti-PAB antibody and identified by molecular analysis, were detected within granulomas. As a whole, these findings make C. acnes a possible advocate that is able to elicit a granulomatous reaction. The immunohistochemical results obtained in our cases were similar to those described in human patients affected by sarcoidosis and represented as “Hamazaki-Wesenberg bodies”. Electron microscopy findings suggest that these bodies are intact forms of intracellular bacteria lacking a cell wall structure and occasionally exhibit protrusions (Negi et al., 2012).
Sarcoidosis is an enigmatic multi-systemic human disease of unknown origin: insights into the etiology and pathogenesis have been elusive (Casanova et al., 2020). It is postulated to be a multifactorial disease caused by chronic antigenic stimulation (Mousapasandi et al., 2021). Genetic background may have a predisposing role, and pine pollen, microbial infection, specifically C. acnes andMycobacteria spp., as well as air pollutants are increasingly regarded as strong environmental trigger candidates (Wilson et al., 2019). Sarcoidosis is characterized by the development and accumulation of epithelioid, non-caseating granulomas typically found in the lungs; however, sarcoid granulomas can be present almost anywhere in the body (Wilson et al., 2019).
Considering animal models of sarcoidosis, zebrafish (Danio rerio ) has been employed to study its pathogenesis. Nevertheless, there is no universally accepted animal model for human sarcoidosis, largely because animals other than horses, do not develop spontaneous sarcoidosis. and the link between human gene polymorphisms and disease prevalence has not been established with the aim to be recapitulated in the animal genetic manipulation (Locke et al., 2020).
Regarding the use of the term sarcoidosis in fish, it was used in a recent paper by He et al. (2020) to describe multiple granulomas, dispersed through several organs in a species of commercial interest, the largemouth bass (Micropterus salmoides) , and the association with the bacterium Nocardia seriolae , a well-known agent able to induce granulomatous reaction in fish.
By using the term sarcoidosis, He et al., (2020) placed emphasis on the relationship among the granuloma and bacteria, other than mycobacteria, trying to identify and recognize this condition.
In this respect, the association of granulomas with C. acnesfound in some of our cases can be described as a form of sarcoidosis.
Concluding, the detection of bacteria, which are able to elicit granulomatous reaction, represents a significant but still debatable finding in this study. We advise adding C. acnes , as well as the yet widely established Mycobacterium spp., to the list of bacteria that are associated with granulomatous reaction. The well-known zoonotic role of mycobacteria found in aquarium fish still raises concern into proper management by fish handlers, aquarists, and dedicated personnel.
Due to zoonotic disease potential and impossibility of elimination of mycobacteria from the aquatic environment, the depopulation and aquaria disinfection are recommended as they are the only current measure for containment disease outbreaks (Decostere et al., 2004). The role of the newly detected C. acnes needs to be investigated more in depth, with combined laboratory tecnhiques and on several fish samples.
Several deepenings are necessary to understand pathogenetic mechanisms and the yet unknown, highly probable multifactorial etiologies, that underline the granulomatous condition affecting ornamental fish.