THE FIRST OUTBREAK OF LACTOCOCCOSIS CAUSED BY LACTOCOCCUS GARVIEAE IN SERBIA

Th e outbreak of lactococcosis aff ecting rainbow trout, weighing 70 120 g, occurred at a trout aquaculture facility in Central-West Serbia during July, 2018. Th is outbreak lasted for three weeks, and cumulative mortality attributed to the disease was around 40%. Th e diseased fi sh showed erratic swimming, dark discoloration and exophthalmia, with the cumulative mortality of around 40%. A pure Gram-positive cocci isolates were obtained from the eye and kidney samples. Based on colony morphology, phenotypic and biochemical characteristics, the isolated bacterium was presumably identifi ed as Lactococcus garvieae. Using the BBL CRYSTAL GP ID system, the isolate was identifi ed as L. garvieae and the identity of the isolate was confi rmed with MALDI-TOF Mass Spectrometry. Blast analysis of 16S rRNA sequence of our isolate had a 99.4 to 99.6% similarity to the L. garvieae strains previously isolated from diseased fi sh. Th e antibacterial activity of 15 antimicrobials against L. garvieae was evaluated using disc diff usion. In this paper, we report the fi rst case of lactococcosis in rainbow trout in Serbia, isolation and characterization of causative agent, Lactococcus garvieae from diseased rainbow trout.


INTRODUCTION
Lactococcosis is a signifi cant fi sh disease caused by the Lactococcus garvieae bacteria. It is a systemic hyper-acute infection with the occurrence of widespread haemorrhaging (Austin & Austin 2016), described for the fi rst time at the end of the 1950s in Japan, where the fi rst cases were diagnosed in rainbow trout (Vendrell et al. 2006). Now, the disease is present in many parts of the world, aff ecting sea fi sh and freshwater fi sh in aquaculture (Meyburgh et al. 2017). Rainbow trout is an important species for aquaculture in Serbia, with more than 100 farms in business. Trout farms are mainly concentrated in South-East part of the country. In Europe, the fi rst outbreak of this disease in rainbow trout was reported in Spain in 1989 (Palacios et al., 1993). Aft er that, L. garvieae was isolated in Italy (Reimundo et al. 2011), the UK (Algöet et al. 2009), France (Eyngor et al. 2004), Portugal (Pereira et al. 2004), Greece (Savvidis et al. 2007), Spain (Aguado-Urda et al. 2011), Turkey (Diler et al. 2002) and Bulgaria (Eyngor et al. 2004). Lactococcosis is the single most important risk factor in the European trout industry, with losses approximating 50% of the total annual production (Eyngor et al., 2004). Th e impact of lactococcosis is particularly signifi cant as losses oft en occur when fi sh reach market size (Ceschia et al. 1998). Th e disease causes signifi cant losses at temperatures above 15°C. Th e oral route is the main route of L. garvieae transmission (Nakai et al. 1999), but the results of a study conducted by Avci et al. (2010) suggest that the gills and eyes are major spots of attachment and the proliferation of L. garvieae during infection period. Infected fi sh exhibit a variety of clinical signs, such as anorexia, exophthalmia, melanosis, conjunctivitis, erect swimming, severe internal haemorrhage and congestion of blood vessel, peritonitis, abscess of spleen and liver, meningoencephalitis, and bacterial septicemia (Eldaret al.1999, Evans et al.2009, Pereira et al. 2004. Th e causative agent, L. garvieae is one of the most important bacterial fish pathogens indiff erent freshwater and marine fish species in many countries (Vendrell et al. 2006;Evans et al. 2009, Meyburgh et al. 2017, with the highest economic impact in rainbow trout aquaculture. L. garvieae is a nonmotile, non-sporulating, facultative anaerobic, catalase and cytochrome oxidase negative, Gram-positive coccus. It is a lactic acid bacterium, fi rst isolated from a case of bovine mastitis it the UK (Collins et al. 1983), and later from other animal hosts, such as cows, buff alos, pigs, dolphins, water buff alos, cats and dogs (Aguado-Urda et al. 2013, Tejedor et al. 2011). Th e bacterium was isolated from rivers and sewage waters, vegetables, meat and dairy products (Aguado-Urda et al. 2013). L. garvieae strains of dairy origin have been found to be free from virulence determinants, such as haemolysins and gelatinase (Fortina et al., 2007), suggesting that L. garvieae dairy strains are unrelated to the pathogenic ones (Foschino et al., 2008). Also, L. garvieae was involved in an increasing number of human clinical cases including infective endocarditis, septicemia, urinary and skin infections (Aguado-Urda et al. 2011), giving rise to the status of an emerging zoonotic pathogen. Although L. garvieae is a well-known fi sh pathogen, human infections are usually related to a contact with raw fi sh. It has gained recognition as an emerging zoonotic opportunistic pathogen, with the ingestion of contaminated foodstuff s being a likely route of infection. Handling and of raw fi sh is reported as a source or risk factor in the majority of clinical cases (Gibello et al. 2016). High levels of antibiotic resistance and resistance genes in L. garvieae strains should be considered as a potential danger for trout culture as well as for public health (Raissy and Moumeni, 2016).
In this paper, we report the fi rst case of lactococcosis in rainbow trout in Serbia, isolation and characterization of causative agent, L. garvieae from diseased rainbow trout (Oncorhynchus mykiss, Walbaum).

MATERIAL AND METHODS
A total of 20 rainbow trout (weighing 70-120g) that showed the clinical signs of the disease were collected from a trout aquaculture facility in Central-West Serbia in July, 2018. Th e samples for bacterial isolation were obtained from the kidney, liver, spleen and eye of the diseased fi sh. Th ey were streaked on blood agar plates containing 10% defi brinated sheep blood (BA), Mueller-Hinton (MH) agar and trypticase-soy agar (TSA) plates, and incubated at 20°C for 48h. Single colonies were restreaked on the same media to obtain pure isolates. Pure colonies were subjected to Gram staining, followed by catalase and oxidase tests. Th e routine tests for the determination of biochemical characteristics were carried out as previously described (Austin & Austin 2016). Additionally, the BBL CRYSTAL™ Gram-Positive (GP) Identifi cation (ID) system was used for the biochemical identifi cation of isolated bacterium.
Th e isolate was tested for antimicrobial susceptibility by the disc diff usion method on MH agar. Aft er 24h of the incubation, incubation zone diameters were measured and evaluated. Th e isolates were classifi ed as sensitive (S), intermediary sensitive (I), or resistant (R), on the basis of the size of the zone of bacterial growth inhibition, according to the National Committee for Clinical Laboratory Standards (CLSI, 2019).

MALDI-TOF
Protein mass spectroscopy analysis was carried out by matrix-assisted laser desorption/ionization time-of-fl ight mass spectrometry (MALDI-TOF MS) as previously described (Heras Cañas et al. 2015) using a VITEK MS Mass spectrometry microbial identifi cation system (VITEK MS, bioMerieux, France).

16S rRNA sequence analysis
Total bacterial DNA of the isolate was extracted using the Cador Pathogen Mini Kit (Qiagen, Germany), following the manufacturer's instructions. Th e extracted DNA from the isolate was used as a template to amplify a 1419bp segment of the 16S rRNA gene by the polymerase chain reaction (PCR) technique using the universal prokaryotic primers 27F (5'-AGAGTTTGATC-CTGGCTCAG-3') and 1492R (5' GGTTACCTTGTTACGACTT-3'). Th e composition of PCR mixture was as recommended by manufacturer (Hot-StarTaq Master Mix Kit, Qiagen, Germany). Th e PCR cycling regime was the following: one cycle of 15 min denaturation and Taq activation at 95°C, 35 cycles of 40 s at 95°C, 40 s at 55°C, and 90 s at 72°C, and fi nal extension cycle of 10 min at 72°C. A small quantity of PCR products was verifi ed by standard agarose gel electrophoresis. Th e amplicons were purifi ed using QIAquick PCR Purifi cation kit (Qiagen, Germany) and sequenced using the primers 27F and 1492R. Th e sequencing of PCR product was performed by Macrogen. Th e isolate sequence was compared to sequences from the NCBI database, using the BLASTn algorithm. Th e limit fi xed for identifi cation of a bacterial species was 98% nucleotide identity for the 16S rRNA gene. Th e phylogenetic relationships of the isolates were determined by comparative 16S rRNA gene sequence analysis. Genetic distances matrix was obtained using Kimura's two-parameter model, and an evolutionary tree was created using the Neighbour-Joining method with Mega X (Kumar et al.2018).

RESULTS
Th e disease outbreak aff ecting rainbow trout weighing 70 -120 g occurred at a trout aquaculture facility in Zaovine Lake (43°52'46.3"N 19°24'08.4"E) during July, 2018. Th is outbreak lasted for three weeks, and cumulative mortality attributed to this pathogen was around 40%. Th e water temperature during the outbreak was consistently higher than 14°C (with highest temperature of 20°C). Th e infected fi sh exhibited lethargy, anorexia, dark skin coloration, marked unilateral and bilateral exophthalmos with the presence of generalized hemorrhaging or blood spots in the eye, eyeball disruption and loss of eye or eyes ( Figure 1).    Based on colony morphology, phenotypic and biochemical characteristics, the isolated bacterium was presumably identifi ed as L. garvieae (Table 1). Using the BBL CRYSTAL GP ID system, the isolate was identifi ed as L. garvieae ID: 3440571723 (Table 2). Table 2. Th e BBL Crystal profi le for L. garvieae isolated from diseased rainbow trout L-phenylalanine-AMC + p-nitrophenyl-β-D-cellobioside + L-tryptophan-AMC + p-nitrophenyl-α-D-maltoside + Trehalose + Esculin + Sucrose + L-valine-AMC +-Arabinose pyroglutamic acid-AMC +- p-nitrophenyl-β-D-glucoside + 4MU-N-acetyl-β-D-glucosaminide +- Th e L. garvieae isolate was tested in the present study with 15 diff erent antimicrobials in terms of antimicrobial sensitivity and results are shown in Table 3. To further confi rm our result, the L. garvieae strain SRB NIVS-1 was identifi ed with MALDI-TOF, which also confi rmed the identity of our isolate. To Arhiv veterinarske medicine, Vol. 13, No. 1, 53 -68, 2020 Radosavljevic V. … et al.: Th e fi rst outbreak of lactococcosis … determine the genotype identity, we have extracted and sequenced the 16S rRNA (1419 bp, NCBI Genbank accession number MT000099) of strain SRB NIVS-1 and blasted using BLAST search program in the GenBank of NCBI, which revealed 99.6% sequence identity with L. garvieae. Th e sequence was compared with the sequences of reference species in Genebank data library by the BLAST program. Blast analysis of 16S rRNA sequence of our isolate presented a 99.4 to 99.6% similarity in sequences to the L. garvieae strains previously isolated from diseased fi sh. Th e isolate showed high percentage sequence similarity to other strains of L. garvieae: 99.6 % sequence similarity to MG016446 from China, 99.5 % sequence similarity to KT428591 from Turkey and 99.4 % sequence similarity to KU360715 from Iran. Th e relationship between the strain SRB NIVS-1 and the closest taxonomic species based on 16S rDNA sequences is described in the phylogenetic tree ( Figure 5). Th e evolutionary history was inferred using the Neighbor-Joining method. Th e optimal tree with the sum of branch length = 0.15577609 is shown. Th e percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) is shown next to the branches. Th e tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.

DISCUSSION
Th e outbreak of the lactococcosis aff ecting rainbow trout occurred at a trout aquaculture facility in Zaovine Lake, an artifi cial lake in the Central-West Serbia, on the Tara Mountain, created on the Beli Rzav River as a reservoir for the Bajina Bašta II reversible hydro power plant. Th is outbreak with a total mortality of around 40% lasted for three weeks. Rainbow trout mortality due to L. garvieae infection can be up to 60%, depending on the water temperature, stress for fi sh and strain type of the bacterium (Shahi et al, 2018). Th e water temperature during the outbreak was consistently higher than 14°C (with highest temperature of 20°C). Such high temperature was a critical environmental condition for the appearance of the disease with a high mortality (Pereira et al., 2004, Castro et al. 2017. Clinical fi ndings during the course of the disease, including characteristic symptoms as hyperpigmentation and bilateral exophthalmia, were similar to those previously described in other studies (Evans et al. 2009, Algöet et al.2009, Austin and Austin, 2016. Pure colonies of the gram positive streptococcus, non-motile, negative for oxidase and catalase, with properties typical for the Lactococcus garvieae, as previously described by Collins et al. (1983), were recovered from eye and kidney samples from the diseased rainbow trout. L. garvieae strains are phenotypically homogeneous, regardless of their geographic location or the aquatic host from which they were isolated (Buller, 2014). Th e biochemical properties of the isolated bacteria from rainbow trout were analogous to those described by Austin & Austin (2016). Th e taxonomy based on a molecular level was used to establish the phylogenesis and taxonomic position of the bacterium. Preferred methods for the identifi cation of L. garvieae are MALDI-TOF MS and 16s rRNA gene PCR (Heras Cañas et al. (2015). It is important to emphasize that this pathogen causes serious economic losses due to increased mortality rates (up to 50%), decreasing the growth rates. Th e appearance of sick fi sh makes them unmarketable (Vendrell et al 2006). L. garvieae is a major fi sh pathogen of rainbow trout in Europe. To our knowledge, this is the fi rst report of L. garvieae associated with trout diseases in Serbia. However, it is likely that the lactococcosis in rainbow trout will become more frequent in the future.

CONCLUSION
Th is study confi rmed that L. garvieae was the etiological agent of a hemorrhagic septicemia in farmed rainbow trout and that the lactococcosis of rainbow trout caused by L. garvieae emerged in Serbia. Th e occurrence could be attributed to the signifi cant increase in water temperature during summer months, since water temperature is described as the most important environmental factor in the development of the L. garvieae infections in trout. In addition, variations in water temperature can aff ect fi sh immune response against bacterial infection. Lactococcosis is a limiting problem for rainbow trout culture in many South European countries. Aft er the fi rst occurrence in Spain and Italy, the pathogen and the associated disease has spread rapidly throughout the South Europe, and further to the South-Eastern part of the continent, with disease outbreaks in Greece, Bulgaria and Turkey. In the aff ected countries, lactococcosis is a major threat to trout culture, especially during awarm period. Th e rapid spread of the pathogen is a result of the multiple routes of dissemination and transmission of this pathogen. Th is includes direct spread though the movement of infected fi sh or asymptomatic carriers and transmission via contaminated water (Savvidis et al., 2007). Since L. garvieae have the ability to adapt and survive in many environmental conditions including a wide range of pH, temperatures, salinity concentrations and nutrient sources (Kusuda et al. 1991), the occurrence of the disease in Serbia is a warning for the neighboring countries with trout aquaculture. It is evident that this agent spreads to the new geographic areas, causing the disease with high mortality in susceptible population. Th e source of outbreak is not known, but since the disease is present in the region and susceptible species are in a river basin which is shared between countries, under adequate conditions, further spread of the causative agent and concomitant disease is inevitable. Due to the ability of L. garvieae to colonize multiple, diverse environments, and because it causes infection in a broad range of diff erent hosts, it is reasonable to expect further spread of the bacterium and the disease. Since vaccination is considered the best option to control lactococcosis in rainbow trout, we hope that an appropriate strategy to prevent this infection on Serbian trout farms will be available in the future.

ACKNOWLEDGEMENT
Th is study was fi nancially supported by a grant from the Ministry of Education, Science and Technology Development of the Republic of Serbia.

Author's contributions:
VR, OR, NZ and MS made contributions to concept and design of the study, they collected data and draft ed the manuscript. JMZ and BS carried out the molecular diagnostic tests and prepared the alignment of nucleotide sequences and conducted the molecular genetic analysis. LJV carried out the data analysis. KN revised the manuscript critically and together with VR prepared the fi nal draft of the manuscript. All the authors read and approved the fi nal manuscript.