Identification of pathogens in fish polyculture systems in southern Minas Gerais , Brazil

Article history Fish diseases represent a significant limiting factor in aquaculture systems. Among the many pathogens, bacteria are probably the most significant group of etiological agents. Thus, the objective of this work was to isolate and to report the incidence of streptococcosis and other possible pathogens in fish polyculture systems in the region of Lavras, state of Minas Gerais, Brazil. Fish samples were randomly collected from seven fish polyculture systems in the region, including the following municipalities: Lavras, Itutinga, Itumirim, Nepomuceno, Carrancas, São Sebastião da Vitória, and Ingaí. The specimens analyzed were: Tilapia (Oreochromis niloticus), Dourado (Salminus brasiliensis), Cará (Geophagus brasiliensis), Curimba (Prochilodus lineatus), Surubim (Pseudoplatystoma corruscans), and Piracanjuba (Brycon orbignyanus). Samples of kidney, brain, liver, spleen, and intestinal tract, and skin scraping was collected and sown in a culture medium. After the incubation period, the microorganisms were identified according to the culture, morphology, dyeing, and biochemical characteristics of the bacteria. Colonies of Streptococci, Aeromonas, and Edwardsiella were identified in Tilapia, Cara, Curimba, and Surubim fish species. The results showed the identification of the bacteria in fish polyculture systems in the region of Lavras, southern Minas Gerais. Therefore, greater sanitary control and the development of other studies to treat diseases related to these pathogens are necessary. Received 30 April 2018 Received in revised form 14 February 2019 Accepted 03 March 2019


INTRODUCTION
Aquaculture is the fastest growing sector of animal protein production in the world, with a total production increase from nearly 1 million tons in 1950 to approximately 52.5 million tons in 2008 (FAO, 2014).In this context, Brazil occupies the 12 th position worldwide, with a production of approximately 707 thousand tons in 2012 (FAO, 2014); the southeast and center-west regions account for 30% of the national production (BRASIL, 2012;KUBITZA et al., 2012).
With the increase in productivity, there is also the increase in management difficulties related to water quality increased, stocking densities, and poor fish handling, which result in disease outbreaks that have caused severe economic losses for the producers (LI et al., 2018;BENHAMED et al., 2014;LEIRA et al., 2016) The transmission of Streptococcosis occurs horizontally by the direct contact between the infected and healthy fish, as well as indirect contact by the bacteria present in the aquatic environment.This transmission causes the disease to gradually spread in the same property or through sources such as any inanimate object or substance capable of absorbing, retaining, and carrying infectious pathogens from one location to another, such as buckets and dip nets (BOTREL et al., 2010;EVANS;KLESIUS;GILBERT, 2000;SALVADOR, 2008).The virulence factors of the bacteria are related to the invasion, replication, and evasion of the host immune system, causing lesions during the disease pathogenesis (VILCHES et al., 2004).These rod cells, which are adapted to growth at temperatures ranging from 5 °C to 37 °C, are considered Gram negative, oxidative positive, facultative anaerobic, and are present on the skin and gills of fish (KOZINSKA, 2007).
The objective of this work was to isolate and to report the incidence of streptococcosis and other possible pathogens in fish polyculture systems in the region of Lavras, state of Minas Gerais, Brazil.

MATERIAL AND METHODS
Fish samples from seven fish polyculture systems in the region, including the municipalities of Lavras, Itutinga, Itumirim, Nepomuceno, Carrancas, São Sebastião da Vitória, and Ingaí, in southern Minas Gerais, Brazil, were collected.The study was accepted by the Bioethics Committee n°017/13 on April 18, 2013.
Fish samples were desensitized according to Zahl; Samuelsen; Kiessling (2012), followed by external physical examination and necropsy for macroscopic changes recording.Samples of kidney, brain, liver, spleen, and digestive tract were collected in each fish.Skin scraping was also carried out.
The organ samples were initially seeded on BHI (Brain Heart Infusion -Difco) agar plates, supplemented with 5% defibrinated sheep blood, and incubated at 27 °C under aerophilic atmosphere for seven days.After the incubation period, the microorganisms were identified according to the bacteria culture, morphology, dyeing, and biochemical characteristics (KRIEG; HOLT, 1984).
The morphological analyses conducted in the plates containing BHI agar supplemented with blood suggested that Streptococcus agalactiae presented gray, translucent, circular, slightly convex, and punctate colonies instead of non-hemolytic colonies.
Subsequently, the cultures were submitted to the latexagglutination test, in which the presence of the carbohydrate antigen indicates that the colonies belong to the B Lancefield group.The bacterial colonies were characterized by catalase, oxidase, KOH, and growth proof in methylene blue, 6.5% NaCl, with confirmation through PCR.
The bacterial genomic DNA were extracted from 20-50 mg of the organs using the Qiagen Kit for tissue extraction according to the manufacturer's protocol.The samples were diluted in 50 μL of autoclaved ultrapure water.A sequence of the S. agalactiae 16S rRNA target gene was amplified using a real-time PCR assay for quantification of equivalent cells/bacterial DNA.All realtime PCR reactions were performed in triplicates.

RESULTS AND DISCUSSION
Streptococcal colonies were found on the BHI agar.The colonies presented gray, translucent, circular, slightly convex, punctate, and nonhemolytic appearance.All strains presented negative results for the catalase tests, growth in 6.5% NaCl, 40% bile, esculin, and methylene blue.However, colonies with characteristics that are distinct from the streptococci characterized by PCR, such as Aeromonas and Edwardsiella, occurred (Table 1).Eight cases of S. agalactiae streptococcus were confirmed in two farms.Only three systems had the presence of pathogenic bacteria in fish: three in Tilapia and one in Curimba, which were contaminated with Streptococcus in the fish system A. Two Tilapia and one Cara were contaminated with Streptococcus in fish system C, while in the fish system F, three Surubim fish were contaminated by Edwardsiella.Bacteria were also found in Tilapia, Cara, and Curimba.A possible cause may be the high amount of fish placed in the tanks, excess organic matter, and the different species in the same tank.Streptococci are bacteria widely found in the aquatic environment, and their emergence is connected to stressful conditions (FIGUEIREDO, 2012).
Bacteria seem to be more prevalent in hot waters with large amounts of organic material associated with stressing factors.These factors include high stock densities, high temperatures, abrupt temperature changes, trauma from handling, fish transfer and low oxygen levels, nutritional deficiency, external skin lesions, fungal or parasitic infections that contribute to physiological changes, and the increase of susceptibility to contamination (AOKI, 1999;HAWKE et al., 2013;PARK;AOKI;JUNG, 2012).Thus, when conditions are favorable, the occurrence of streptococcosis in fish may occur (BOTREL et al., 2010;PEREIRA;MIAN;OLIVEIRA, 2010).
Aeromonas and Edwardsiella are naturally present in the environment and typically inhabit the intestine of fish, which explain their occurrence in the studied fish.The diseases caused by these bacteria are common in fish farming throughout the world (HOLLIMAN et al., 1993).
The impact of these bacteria must be considered since several researchers affirm that the exposure to the aquatic ecosystem and its habitats can be precursors of diseases caused by Aeromonas and Edwardsiella (KIM et al., 2018;WIEDENMAYER;EVANS;KLESIUS;GILBERT, 2006).These are facultative pathogens that target hosts weakened or affected by other etiological agents, identified as secondary invaders, and establish themselves in the same way as other bacterial, viral, or parasitic infections, as well as those caused by nutritional or stress-related factors (BOTREL et al., 2010;PAVANELLI;MACHADO, TAKEMOTO, 1997).
The adequate characterization and identification of the diseases and their causal agents are essential to better understand their behavior, as well as to define the epidemiology and implement control methods.With such knowledge, it is possible to develop preventive measures, avoiding low fish production and high economic losses, expanding and consolidating the aquaculture activity, in addition to allowing the identification and selection of fish more resistant to pathogens (HAWKE et al., 2013).
Immunoprophylaxis, used as a preventive measure by vaccination, is the most appropriate form to control some bacterial diseases such as streptococcosis.It consists of integrated sanitary management, which combines the correct maintenance of the nurseries and biosafety in production.In order to achieve this goal, it is necessary to adjust the structure and production system, to train employees, and to organize the entire team.The management of larvae and water sources should also be monitored (PÁDUA; MENEZES FILHO; CRUZ, 2012).

CONCLUSION
This study showed the presence of Streptococcus, Aeromonas, and Edwardsiella in fish systems in the region of Lavras, state of Minas Gerais, Brazil, indicating the need for greater sanitary control and the development of epidemiological research at the national level.

Table 1 -
Identification and microbiological analyses of the bacteria found in the fish species reared in Southern Minas Gerais, Brazil (fish systems from A to G).