Isolation of potential zoonotic Mycobacterium spp. from diseased freshwater angelfish (Pterophyllum scalare) from an aquarium

Nontuberculous mycobacteria infection is one of the most common chronic bacterial diseases in ornamental aquarium fish and appears to be directly related to stressful husbandry practices. Furthermore, it also represents zoonotic potential. Here we present the isolation and characterization of non-tuberculous mycobacteria from diseased freshwater angelfish ( Pterophyllum scalare ) in São Paulo, Brazil. Nine discarded breeding females with signs of disease were evaluated. The fish exhibited lethargy, loss of appetite, cachexia, skin ulcers, and exophthalmia. At necropsy, four fishes presented macroscopic granulomas in the spleen. Mycobacterium chelonae, M. fortuitum , M. gordonae , M. intracellulare and M. peregrinum were isolated and identified by hsp65 PCR restriction analysis. Histopathological analysis revealed microscopic lesions compatible with mycobacteriosis, and Mycobacterium bacillus were observed by Ziehl-Neelsen stain. Notably, all Mycobacterium species identified in this study have already been reported in human patients; therefore, diseased animals may be a source of infection for people who handle fish and aquariums.


Isolation of potential zoonotic Mycobacterium spp. from diseased freshwater angelfish (Pterophyllum scalare) from an aquarium
for Mycobacterium bacillus visualization.The slides were examined, and photomicrographs were taken using a DM 5000 B microscope equipped with a differential interference contrast (DIC) system and analyzed in a computerized system (Qwin Lite 3.1, Leica Microsystems).

Isolation and identification of Mycobacteria
From the nine studied animals, seven fish were too small to separate each structure; therefore, samples of macerated internal organs were prepared.One fish had a splenic granuloma, and another had a splenic granuloma and a skin lesion.Spleen granulomas, livers, and kidneys/ gonads were sampled from eight of the fish, and from the nineth the liver, kidneys, splenic granuloma, and skin lesion tissues were analyzed.
For mycobacteria isolation, all samples were decontaminated with 0.75% cetylpyridinium chloride (Ikuta et al., 2016).Thereafter samples were inoculated in duplicate into Stonebrink and Löwenstein-Jensen media and incubated at 25 °C and 37 °C for up to 60 days (CPZ, 1995).Obtained isolates were classified phenotypically according to the colony pigmentation and time required for colonies to be clearly visible to the naked eye (rapid growing < 7 days; slow growing > 7 days) (Kim et al., 2013).
The acid-fast bacilli isolated were selected by phenotypical characteristics in each culture flask and were subjected to DNA extraction as previously described by Boom et al. (1990), with initial lysozyme (100 mg/mL) and proteinase K (20 mg/mL) (US Biological, Swampscott, MA, USA) digestion at 37 ºC for 60 minutes.For molecular identification, PCR restriction analysis (PRA) of the hsp65 gene was performed according to Telenti et al. (1993) using BstEII (Promega) and HaeIII (Thermo Scientific) enzymes.Briefly, hsp65 gene amplification was performed using 20 pmoles of each primer Tb11 and Tb12 (Telenti et al., 1993), 1.5 mM MgCl 2 , 200 mM of dNTP, 1 U of Taq DNA polymerase (Fermentas Inc., Glen Burnie, MD, USA), 1 X PCR buffer and ultra-pure water.The amplification consisted of 95°C for 15 min followed by 35 cycles of 1 min at 94°C, 58ºC for 1 min, 72ºC for 1 min, and 10 min at 72ºC for final extension.From the amplified product, 10 µL were used for each enzyme digestion in which BstEII was incubated at 60ºC for 1 h and HaeIII at 37ºC for 1.5 h, according to the manufacturer's instructions.Electrophoresis in agarose gel (3.5%), stained with SYBR Safe (Invitrogen®), was performed at 90V for 4 h using the 50 bp DNA ladder (New EnglandBioLabs Inc) as molecular weight marker.The obtained restriction patterns were further analyzed within the PRAsite database (http://app.chuv.ch/prasite/index.html)for species identification.
Histopathological analysis showed as main lesions the inflammatory process by lymphocytes and macrophages in the brain, gills, gut, liver, muscle, and spleen; degeneration in the brain, gut, liver, muscle, and spleen; eosinophils in the gills and gut; hyperplasia in the gills, gut, and spleen; emaciation, retarded sexual maturation, or decreased reproductive performance.Other lesions can include skeletal deformities and chronic nonhealing shallow to deep ulcers or fin erosion.White nodules may be present on the viscera, especially on the hypertrophic kidney or spleen (Noga, 2010).The diagnosis of mycobacterial disease in aquarium fish is based on bacterial culture, histopathological, and molecular methods.
Angelfish Pterophyllum scalare (Schultze, 1823) is a common fish bred in captivity and traded in aquarium stores in Brazil.Due to the zoonotic potential of fish mycobacteriosis and the increasing reports of opportunistic NTM infections around the world, the aim of this study was to isolate and identify the species of non-tuberculous mycobacteria from diseased freshwater angelfish (P.scalare) from a breeder in São Paulo, Brazil.

Animals
A total of nine discarded breeding females of freshwater angelfish (P.scalare) with signs of disease, obtained from a fish breeder, were used in the study.The fish were maintained in pairs (female and male) in individual aquariums of 0.1 m 3 (with individual filtration), fed twice a day ad libitum with mixed commercial diet (Tetra ® Min flakes and Color bits granules), and without rest between one spawning and another.Water quality parameters were daily measured and maintained at: temperature 28-30ºC, dissolved oxygen 5 mg L-1 , pH 6.8-7.2, total ammonia <0.1 mgL-1 and nitrite < 0.05 mgL-1 .The females presenting reduced reproduction rate or clinical signs of disease were separated from the pair and submitted to the Department of Preventive Veterinary Medicine and Animal Health, School of Veterinary Medicine and Animal Science, University of São Paulo, for diagnostic purposes in May 2019.
Fish were sedated with a solution of Eugenol at 75 mg/L (Roubach et al., 2005) -for 2 to 6 min and then euthanized by the spinal cord section technique (Noga, 2010).They were then sanitized with 70% alcohol to remove possible environmental contaminants and necropsied for sample collection.This study was approved by the institutional Ethics Committee (approval number CEUA Nº 6289090519).

Histopathology
The gill, gut, kidney, liver, skin, stomach, and spleen of three fish were fixed in buffered 10% formalin solution for histopathological analysis.Fragments of lesions were carefully removed, embedded in paraffin, cut into posterior cross sections of 5 µm, and stained with hematoxylineosin to observe microscopic lesions and Ziehl Neelsen  replaced them with apparently healthy females.The constant high pressure of stress during reproduction may have induced the females to be more predisposed to infection by environmental Mycobacterium spp., leading to the appearance of clinical signs and granulomas in the internal organs.
Due the different infection profiles identified, it was not possible to correlate the presence of one Mycobacterium species and lesions observed at studied animals.Nevertheless, the clinical description corroborates previous studies in fishes worldwide (Kušar et al., 2017;Novotny et al., 2010;Shukla et al., 2013;Zanoni et al., 2008).In a previous study in Slovenia, Pate et al. (2005) found M. chelonae, M. fortuitum, M. gordonae, and M. peregrinum in Goldfish (Carassius auratus), Guppy (Poecilia reticulata), freshwater angelfish (P.scalare), and three-spot gourami (Tricogaster trichopterus).In addition to these species, we also detected M. intracellulare in fish with spleen granulomatosis.Our histopathological findings corroborate those of Lescenko et al. (2003) who observed that more than 60% of granulomatous lesions in infected aquarium fish, from the Czech Republic, were caused by bacterial colonies in the gills, liver, muscle and spleen; and numerous granulomas in the liver and spleen (Figure 2).In the Ziehl-Neelsen stain, acid-fast bacilli were observed in the spleen.

Discussion
Mycobacteriosis caused by NTM appears to be causally related to stressful husbandry practices.The females of freshwater angelfish (P.scalare) in the present study were subjected to high chronic reproduction stress by the breeder, who did not allow time for rest between spawning.As time passed, the reproduction rate of the females decreased, and when the first clinical signs of the disease appeared, the owner discarded the females from the system and Mycobacterium.In addition, similar to these authors, we also noted a low quantity of this bacterium in tissues with granulomas stained with Ziehl-Neelsen.This could be due to granulomatous tissue changes through direct microscopy, which could have led to the destruction of the mycobacteria (Zanoni et al., 2008).Rallis and Koumantaki-Mathioudaki (2007) in histopathological examinations, showed that fish infected by Mycobacterium had nonspecific inflammatory infiltration of epithelioid cells, lymphocytes, and Langhan's giant cells without caseation, which was also observed in this study.
The NTM infection also represents a public health risk due to its zoonotic potential.Contact of injured areas with contaminated environments may be responsible for infection in humans and animals (Tortoli, 2009).Therefore, it is common for people to become infected while working with sick fish or cleaning aquariums (Yacisin et al., 2017).Despite the fact that NTM is relatively avirulent in healthy hosts, all species identified in the present study have already been reported in human patients (Silva Neto et al., 2019;Napaumpaiporn and Katchamart, 2019;Dodiuk-Gad et al., 2007;Gharbi et al., 2019;Ko et al., 2017).Considering that these mycobacteria are part of the environment as well as of the fish microbiota (Cowman et al., 2018;Kušar et al., 2017).

Conclusion
All studied angelfish with chronic reproduction stress were diagnosed with spleen granulomatosis caused by one or more Mycobacterium species.The detected M. chelonae, M. fortuitum, M. gordonae, M. intracellulare and M. peregrinum species present zoonotic potential, according to the literature, and therefore sick animals may be a potential source of infection for humans who manipulate and /or work with fish and aquariums.

Figure 1 .
Figure 1.Freshwater angelfish (Pterophyllum scalare) with skin ulcers (a, b); exophthalmia (c); macroscopic granulomas of the spleen (d); and granulomas found on an internal wet mount exam of a fish with mycobacteriosis (e).Note the dark brown center with surrounding lighter capsule visible on a wet mount spleen biopsy [4x magnification] (f).

Figure 2 .
Figure 2. Microscopic lesions in the spleen and liver were stained with hematoxylin-eosin and Ziehl-Neelsen.Presence of numerous fibrous granulomas (thin arrow) in the spleen (A, B, E, F) and liver (C, D) containing a necrotic center (N) in the spleen (A), melanomacrophages (arrow head) in the spleen (B), bacteria (large arrow) in the spleen (B), and melanomacrophages-center (MC) in the liver (C).In the Ziehl-Neelsen stain, acid-fast bacilli (large arrow) were observed in the spleen (E, F).

Table 1 .
Identified Mycobacterium species according to analyzed animals and respective samples.