A Mixed Frigoribacterium faeni and Lactococcus garvieae Infection in Cultured Rainbow Trout (O.

: ocanak@istanbul.edu.tr ABSTRACT The aim of this study was to diagnose the bacterial pathogens of moribund rainbow trout (Oncorhynchus mykiss) reared in a dam-lake cage farm located in the Black Sea Region of Turkey and to determine their antibiotic susceptibility and histopathological effects by using routine bacteriological, histopathological and molecular methods. Besides possibility of the use of two probiotics against these pathogens for the prevention of further infections was investigated. In this study, a mixed bacterial infection case caused by Frigoribacterium faeni and Lactococcus garvieae was diagnosed in rainbow trout samples of 100-250 g with general clinical and histopathological symptoms of bacterial hemorrhagic septicemia. Pathogens were found to be resistant against most of the antibiotics tested and the possibility of the use of Bacillus subtilis as a probiotic to prevent diseases caused these

and in dam-lakes in Turkey since 1960's (Akbulut et al., 2009). Total rainbow trout production amount of Turkey in inland water facilities was cultured in dam lakes in Turkey.

Frigoribacterium faeni (fam: Microbacteriaceae) is a
Gram-positive, bacterium mainly associated with plants and dust (Kampfer et al., 2000;Evtushenko and Takeuchi, 2006), which was also reported in the intestinal flora of healthy fish (Carbajal-Gonzalez et al., 2011;Urtubia et al., 2017). Previously, there is no report on the infection cases or pathogenicity of F. faeni in fish.
As a result of misuse of antibiotics, pathogens have developed resistance recently. Studies on environment-friendly prevention and treatment programs which eliminate the use of chemicals, are increasing in numbers (Austin and Austin, 2016). It is possible to prevent bacterial diseases in aquaculture by using probiotics that previously showed antagonism against pathogens including the members of Vibrio, Aeromonas and Streptococcus in-vitro (Gomez-Gil et al., 2000;Kumar et al., 2006;Ng et al., 2014;Mingmongkolchai and Panbangred, 2018).
The aim of this study was the diagnosis of the bacterial pathogens of moribund rainbow trout reared in a damlake cage farm located in the Black Sea Region of Turkey and determination of their antibiotic susceptibility and histopathological effects by using routine bacteriological and histopathological methods and molecular tools. Besides, possibility of the use of two probiotics against these pathogens for the prevention of further infections was investigated.

Fish sampling:
Fish samples were collected during a field sampling of a one-year monitoring study. A rainbow trout cageculture rainbow trout facility located in a dam lake in Black Sea Region of Turkey was visited in April of 2017. Total of 7 fish samples (100-250 g) of slowly swimming on the water surface with some clinical disease symptoms were anaesthetized with 2phenoxyethanol (1 ml/l in culture water) and examined clinically.
This study was conducted with the permission of Istanbul University Animal Experiments Local Ethical Committee (approved on 23.02.2017).

Antibiotic susceptibility testing:
Antibiotic susceptibility testing was performed using modified Kirby-Bauer disc diffusion method (Bhunia et al., 1988). Fresh cultures of bacterial isolates grown in Nutrient Broth were spread onto Mueller-Hinton agar; commercial antibiotic discs were placed and three replicates of petri dishes were incubated at 22 ℃ for 48 h and inhibition zone diameters were measured. Tetracycline, kanamycin, florphenicol, furazolidone, sulphametaxozole trimethoprim, ciprofloxacin and enrofloxacin discs were used. Results were compared with the previous reports and NCCLS standards.
Antagonism testing: Lyophilized Bacillus subtilis (ATCC 6633TM) and Lactobacillus rhamnosus (ATCC 7469TM) were used as probiotic candidates and fresh cultures of them were prepared by streaking onto TSA (Tryptic soy agar) and incubated at 22 ℃ for 48 h. Modified Kirby-Bauer disc diffusion method was used for the determination of antagonism against pathogenic bacteria (Bhunia et al., 1988). Briefly, 200 µl of fresh cultures of pathogenic bacteria growth in Nutrient Broth were streaked onto TSA medium to cover all the surface. Later, blank antibiotic susceptibility paper-discs were dipped into fresh cultures of probiotic-candidates growth in Nutrient Broth and placed onto TSA medium. Three replicates of TSA medium containing petri dishes were incubated at 22 ℃ for 48 h and inhibition zone diameters were measured.

RESULTS
In this study, infections caused by F. faeni and L. garvieae in rainbow trout cultured in a dam lake was diagnosed by using bacteriologic and molecular methods, pathological effects of the disease in the infected fish tissues were demonstrated, antibiotics which can be used for the treatment were determined and a possibility of the use of a probiotic bacterial species was proposed.
Fish samples examined in this study were chosen from the individuals that are swimming slowly on the water surface which are lethargic with loss of appetite. Moribund fish samples showed mass skin pigmentation, darkening of the skin, loss of scales, melting of the dorsal fin and erosion in the upper jaw ( Figure 1a). Mass hemorrhages in the eyes and severe exophthalmos in some samples were observed ( Figure  1a). Internally, hemorrhagic lesions on the anemic liver, splenomegaly and enlargement of the bile duct was observed ( Figure 1b). Also, accumulation of a bloody fluid in the peritoneal cavity was noted in some fish samples ( Figure 1b).
Anemia, slight atrophy, cell necrosis and hyperemia were observed in the liver (Figure 2a). Hemosiderin accumulation, slight liquefactive necrosis of the interrenal haemopoietic tissue and tubular deformation were observed in the kidney (Figure 2b). Slight necrosis and depletion of the pulps were noted in the spleen where the hemosiderin accumulation was rarely seen (Figure 2c). Epithelial and connective tissues were weakened in the primer and secondary lamellae of the gills (Figure 2d). Also, there were mass hyperemia in the supportive tissue of the exophthalmic eyes and deformation of the microvilli were observed in the intestines.
Two types of colonies were recovered from the visceral organs of fish samples; creamy-white colonies with a diameter of 1-2 mm (Figure 3a) and yellowish colonies with a diameter of 3-4 mm (Figure 3b). Creamy colonies that consist of Gram-positive fermentative non-motile cocci-shapes cells in short chains were oxidase, catalase, lactose and VP negative; MR and arginine positive and α-hameolytic on blood agar and hence they were identified as Lactococcus sp.
Yellowish colonies that consist of Gram-positive motile cocci-shapes cells in small clusters were oxidase, MR, VP and indole negative; catalase positive and possessed variable results in citrate and nitrate tests and hence they were identified as Frigoribacterium sp.
Results of the conventional bacteriologic tests were shown in Table 1. An 880 bp region was obtained with the PCR amplification conducted with the universal primers 27F and 907R. The obtained 16S RNA sequence analysis was processed in the BioEdit software and after the GeneBank nucleotide blasting,    F. faeni was found to be sensitive or semi-sensitive against all antibiotics used (Table 2). In contrast, as a well-known fish pathogen, L. garvieae isolates were found to be resistant against kanamycin, florphenicol, and sulphametaxozole trimethoprim. They were also semi-resistant against furazolidone, ciprofloxacin and enrofloxacin, but only sensitive to tetracycline (Table  2). Bacillus subtilis showed weak antagonistic effect against the secondary pathogen F. faeni with a mean inhibition zone diameter of 1.5 cm (Figure 4a). Also, this species showed strong positive antagonistic effect against the main pathogen L. garvieae isolates recovered from internal organs the diseased fish samples with inhibition zone diameters between 3.0 and 4.3 cm (Figure 4b). In contrast, L. rhamnosus showed no antagonistic effect against both pathogens in vitro.

DISCUSSION
Fish samples showed similar clinical external and internal symptoms such as darkening of the skin, hemorrhages, and splenomegaly as reported in previous lactococcosis cases (Kang et al., 2004;Altun et al., 2005;Vendrell et al., 2006;Özer et al., 2008;Avcı et al., 2010;Öztürk et al., 2013;Avcı et al., 2014;Didinen et al., 2014;Ürkü and Timur, 2014;Balta and Balta, 2019) with slight variations. As an expectation, similar symptoms in the eyes such as exophthalmos, hemorrhages and opacification of the cornea was observed in the fish samples but not the loss of eyes as reported by Timur et al., (2011) and Öztürk et al. (2013). Also lesions on the skin reported by Öztürk et al. (2013) were not observed in our fish samples.
Similar to the previous lactococcosis cases, fish samples showed various histopathological symptoms such as tubular degeneration, periglomerular edema and melanomacrophage centers in the kidney Timur et al., 2011;Avcı et al., 2014;Didinen et al., 2014;Ürkü and Timur 2014). But, liquefactive necrosis in the liver and kidney that were demonstrated previously (Timur et al., 2011;Ürkü and Timur, 2014;Korun et al., 2017) were in a more advanced stage than our samples.
Many species of the genus Frigoribacterium (Microbacteriaceae family) were previously thought to be pshcyrophilic bacteria that can be isolated from air and soil (Kampfer et al., 2000;Evtushenko and Takeuchi, 2006 Lactococcosis is a well-known disease of rainbow trout worldwide (Austin and Austin, 2016) and previously reported in Turkish trout culture sector in warm seasons (Diler et al., 2002;Altun et al., 2005, Kav andErganis, 2007;Akşit and Kum, 2008;Avcı et al., 2010;Timur et al., 2011;Didinen et al., 2014;Durmaz and Kılıçoğlu, 2015;Korun et al., 2017;Balta and Balta, 2019). In most of the reports on Lactococcosis cases of KSU J. Agric Nat 23 (6) Öztürk et al. (2013) and Balta and Balta (2019) described rainbow trout lactococcosis cases in dam lakes located in the Blacksea Region in April and May similar to our study with mostly similar clinical signs.
Due to the problems in antibiotic treatment in term of selection as described above, recent research on fish diseases has aimed to improve diagnostics by use of sensitive and specific molecular methods and disease control especially by vaccination, probiotics and plant products (Austin and Austin, 2016). Various Lactic acid bacteria such as Lactobacillus species and members of the genus Bacillus, especially B. subtilis were determined to have antagonistic effect against many fish pathogens including Aeromonas hydrophila (Kumar et al., 2006), Yersinia ruckeri (Raida et al., 2003) and Streptococcus agalactiae (Ng et al., 2014). Lactocbacillus rhamnosus was used as a probiotic bacterium especially against Gram-negative pathogens of marine fishes (Gomez-Gil et al., 2000;Ashraf, 2000;Katırcıoğlu, 2001) but it was insufficient to inhibit Gram-positive pathogens (Ringo and Gatesoupe, 1998;Burr and Gathlin, 2005). In this study, B. subtilis was determined as a promising probiotic-candidate with in-vitro studies for the prevention of lactococcosis in rainbow trout. Long-term and repetitive use of this probiotic-candidate bacterium in the consecutive production seasons, would possibly increase the antagonistic effect against this pathogen and protection.
In conclusion, the results of this study showed that F. faeni and L. garvieae are important fish pathogens affecting rainbow trout culture with important clinical and histopathological symptoms. Since these bacteria causes mortalities and can raise resistance against some of the most popular antibiotics used in aquaculture, protection via vaccines and/or probiotics is of crucial importance. In-vitro results of this study showed that, B. subtilis is a promising probioticcandidate for the protection of rainbow trout in aquaculture from bacterial infections.