Influence of pathogens and pollution on Mugilidae health: first evidence of mycobacteriosis and intersex condition in extensively reared mullets from Sardinian lagoons

The multifactorial nature of fish health and diseases are generally linked to an imbalance of pathogen, resistance of the fish and environmental stress. Pollution is considered an anthropogenic factor that can affect fish life leading to immunosuppression that increases fish susceptibility to pathogens affecting their survival and growth rates. Coastal waters and lagoons are typical environments devoted to extensive aquaculture. In Italy, which annual production of extensive reared fish is nearly 5.250 tons, mullets represent the most important cultured species with an average production of about 3.000 tons per year. The coastal environments in which they grow are constantly exposed to high levels of urbanization and, consequently, to the action of increasing amounts of contaminants discharged in waters. This fact can play a central role in some emerging issues like mycobacteriosis and gonadal abnormalities in cultured mullets, representing a real concern for fish health and reproduction. The term “mycobacteriosis” or “fish tuberculosis” decribes a chronic systemic and progressive disease caused by mycobacteria belonging to the genus Mycobacterium. In particular, Mycobacterium marinum is a slow-growing non-tuberculous mycobacterium and it is considered the most common etiologic agent of mycobacteriosis in wild and cultured fish. This disease is considered a real risk for fishermen and aquarists that manipulate infected fish. The diagnosis of mycobacteriosis is principally made by histology when positive Ziehl-Neelsen stain granulomas are detected. The aim of the first part of this study was to investigate the occurrence of mycobacteriosis in extensively cultured Mugilidae (Chelon labrosus, Liza aurata, Liza ramada and Mugil cephalus) of four lagoons from Sardinia by the use of histology, microbiology, PCR and DNA sequencing. Twenty-five out of 495 mullets (148 specimens from Cabras, 120 from Calich, 89 from Marceddì, and 138 from San Teodoro), collected during summer and autumn of the years 2013, 2014 and 2015, were suspected of being infected with mycobacteriosis revealing granulomas containing acid-fast bacilli at histopathological examination. In particular, 10 out of 25 mullets were certainly affected by mycobacteriosis and Mycobacterium marinum was identified in 6 out of 10 as the primary cause, and the concordance obtained by histology, cultural evaluation and sequence analysis of the hsp65 gene was 100%. In the remaining 4 specimens, Mycobacterium spp. were detected and the concordance obtained by histology and molecular method showed 100% of positivity. In the remaining 15 specimens, granulomas with acid-fast bacilli were detected although culture confirmed the positivity for Mycobacterium spp. only in 6 cases, with an accordance of 43% with histology. In all of these cases, PCR-hsp65 and sequencing failed to identify atypical mycobacteria. Mullets affected by mycobacteriosis were mainly sampled in the Calich (10%) and San Teodoro (8%) lagoons. Only 2 subjects were positive for mycobacteriosis and no cases were observed in Cabras and Marceddì lagoons, respectively. This study confirms that histopathological examination is a very important diagnostic screening tool for the detection of mycobacteriosis in fish and PCR-hsp65 is a valid and easy method to identify atypical mycobacteria, expecially for M. marinum. Our finding are worthy of attention because mycobacteriosis in mullets has been evidenced for the first time in Sardinia, suggesting that this disease may be underestimated also in other cultured fish species. These results confirm our expectation that mullets living in San Teodoro and Calich lagoons, both characterized by critical environmental conditions, could play a central role in understanding the occurrence of fish mycobacteriosis which, if not properly monitored, could represent a serious concern for public health. Fish are among the most studied organisms for the effects of chemical contaminants on the development and reproductive processes. In coastal and estuarine ecosystems, euryhaline fish living in polluted waters like Mugilidae can frequently show sexual anomalies like intersex. This term describes alterations in gonadal development with the simultaneous presence of male and female reproductive stages in the same gonad of a gonochoristic species. In the second part of this study, adult specimens of three species of euryhaline mullets (Chelon labrosus, Liza aurata, and Mugil cephalus), from two Sardinian lagoons (Marceddì and San Teodoro) devoted to extensive aquacultural practices, were analyzed in order to identify putative alterations in gonads and in gamete development. Overall, 13 of the 158 mullets examined (8.2%) were affected by gonadal disorders: four subjects (one C. labrosus, two L. aurata and one M. cephalus) exhibiting an intersex condition were found in the Marceddì lagoon and the other nine (five C. labrosus, two L. aurata and two M. cephalus) in the San Teodoro lagoon. Twelve of these gonads were classified as testis-ova (TOs) and one, belonging to a C. labrosus specimen, was a mixed gonadal tissue (MGT). Intersex condition was evaluated using an intersex index and all the recorded values showed a mild ovotestis severity index (OSI). However, our findings suggest that fish gonadal disorders may be underestimated in extensive reared fish species, particularly in coastal brackish environments polluted by intensive agriculture and animal husbandry activities. In conclusion, further research on emerging fish disorders and diseases in Sardinian coastal lagoons could confirm the hypothesis that species like mullets have to be considered as biological sentinels to detect the degree of pollution in extensive aquaculture systems, with the purpose of reducing risks to animal and human health.