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.