The Diversity of Causative Agent Associated With Bacterial Diseases on Catfish ( Clarias gariepinus ) with Molecular Based from Demak , Indonesia

Bacterial diseases is frequently occur in catfish culture. The aim of this research was to find out the diversity of causative agent associated with bacterial diseases in catfish based on 16S rDNA gene sequences. The combination between exploratory in the field and experiment, method were applied. Seventeen isolates (D01–D17) were gained from kidney and external wound of moribound catfish with NA and GSP medium that were collected from fish pond of Demak Regency, Indonesia. Based on the postulat results showed that four isolates (D07, D10, D11 and D14) that were caused 10–55% of fishes get sick and 0–30% fishes mortal. On the other hand, there were 13 isolates do not cause both sick and mortality of fish. On the basis of sequence 16S rDNA analysis, the result showed that D07, D10, D11 and D14 were closely related to Aeromonas caviae (96%), Aeromonas veronii (97%.), Plesiomonas shigelloides (97%) and Pseudomonas putida (96%) respectively. The sensitivity test result indicated that these causative agents have not sensitively to some fish drugs test.


Introduction
Catfish (Clarias gariepinus) is an important aquacultural species which has been cultured in Demak Regency, Central Java, Indonesia. This area was known as a central catfish producer in central java, Indonesia. However, the decreasing of the production from 21.967,06 tonnes in 2006 to 14.432 tonnes in 2013 (Marine and Fisheries Ministry of Central Java Province, 2014) due to the diseases infection. Therefore increasing the production and development on the sustainable cat fish aquaculture in order to full fill the catfish demand in domestic markets, the fish farmers were applied the intensive culture. However inappropriate managementmay cause some negative impacts, such as out break of bacterial disease (Nguyen et al., 2014).
Bacterial disease is still a major problem diseases in catfish culture. Bacterial diseases in the catfish was characterized by pale or blacken of the skin, haemorrhagic surround the mouth, fins, and tails, exopthalmia, fin root, body wounds and pale, darken liver and kidneys, red and root antenna (Anyanwu et al., 2015;Thanh et al., 2009).
Preced researches were done by some researchers in attempt to discover the bacterial pathogen on catfish using Polymerase Chain Reaction (PCR) with 16S rDNA gene sequences. However, identification of bacterial pathogen using molecular approach was derived from intensive pond of Demak Regency, Central Java, Indonesia was still need to create the certain design of prevention system and strategy againts this disease.

Sampling of catfish and bacterial isolation
The catfish presumed infected by bacteria collected from production ponds in  (Brock and Madigan, 1996).

Postulat koch and characterization
Seventeen bacteria isolates tested with Postulate Koch to 360 healthy catfish as experimental fish. Acclimatization done dividing the fish into 36 aquarium so there was 10 fishes in every aquarium. Liquid bacteria culture done using zobell liquid medium, injected to fish as many as 0.1 ml with of 10 8 CFU/ml. The fish injected on the intramuscular and observed for 96 hours to know clinical signs may appear.
From the seventeen bacteria isolates, four isolates were characterised with molecularly approach based on methods Radjasa et al (2001). Bacteria extracted from agar plate then suspended in steril water (Sigma, Germany). The Polymerase Chain Reaction was run using Eppendorf Mastercycler (Eppendorf Inc. Germany)with five freezing cycles (-80ºC) and thaw (95ºC). The primer were used to amplify are GM3F (5'AGAGTTTGATCMTGGC-3') and GM4R (5'-TACCTTGTTACGACTT-3') nearly complete 16S rDNA gene. Big Dye Terminator V3.1 dyes and automatic DNA sequencer ABI3130 Genetic Analyzer XL Applied Biosystemsat Macrogen Korea used for sequencing the bacteria DNA. DNA sequences of the bacteria forward was campared to the BLAST (Basic Local Alignment Search Tool) on National Center for Biotechnology Information, National Institute for Health database USA to gain the homology (Atschul et al. 1997;Radjasa et al, 2001). Wheras the phylogentic was constructed with Mega 6 programme (Sarjito et al, 2017).

Bacterial sensitivity test
Bacteria sensitivity test further was applied to the bacteria with in vitro method, the drugs are A ™, B ™, C ™ and D ™. The activity of each drugs showed with clear zone emerge on the bacterial colonies area, afterward the sensitivity test result compared to stantard by National Committee for Clinical Laboratory Standards (NCCLS, 2001)

Clinical signs of moribound catfish
Clinical signs of moribound catfish which infected by bacterial diseases from intensive pond of Demak were body wound and pale, haemorhagic surround the mouth, tail, fins and fins root; red and root antenna and dark color in liver and kidneys This morpholocical symptom (pale and body wound, haemorrhagic surrounds the mouths, fins, and tails; red and root antenna), the behaviour abnormalities such as decreased appetite, lethargy, body upsite down, swimming imbalance were also observed in tested catfish. The clinical signs above have also been reported by Areerat (1987); Anyanwu et al., (2015) and Sarjito et al. (2017). These clinical sigs found in the present study has also been observed in naturallydiseased catfish cultured (Areerat, 1987). These clinical symtoms were observed in the present study may due to attachment and colonization of consorsium opportunistic bacterials to fish skin of cat fish (Anyanwu et al., 2015).
Seventeen bacterial isolates were gained from kidney, fins root, and body wound of moribound catfish (Table 1). Postulate Koch test results showed that four isolates, namely that D07, D10, D11 and D14 were able caused disease symptom up to 55% of the experimentlly catfish, whilst the three isolates was caused mortality range of 0-30 % ( Table  2). The present study also showed that challenged catfish were injected by others isolates and PBS had 100% survival rate and normal behavior. Therefore, these isolates (D07, D10, D11, and D14) were positively confirmed as a causative agent assosiate with bacterial diseases in catfish from Demak. These result also revealed that these causative agent was higher pathogenicity compare to causative agent that was found in catfish from Kendal (Sarjito et al., 2017).  et al., 2013), and walking catfish culture Clarias bratachus (Areerat, 1987). A. caviae was also reported as causative agent of bacterial diseases in walking catfish in India (Thomas et al., 2013) and Rhamdia quelen (Baldissera et al., 2018). While, A. veronii was also found in Malaysian red hybrid tilapia (Amal et al., 2018), Gold Fish, Carasius auratur, in Srilangka (Jagoda et al., 2017), catfish (Nawaz et al., 2010). Futhermore, A. veronii biovar sobria was found as a causative agent of Epizootic Ulcerative Syndrome in fish in Bangladesh (Rahman et al., 2002).  (Krovacek et al., 2000), Grouper (Herrera et al., 2006). Plesiomonas sp. have been isolated from freshwater fish and bivalve (Niedziela et al., 2002). Pseudomonas spp. has been reported to be an important fish pathogen that has endangered aquaculture (Mao et al., 2012).
P. putida was found as causastive agent of bacterial diseases in rainbow trout (Altinok et al., 2006), Yellow Croaker (Pseudoschiaena crocea) (Mao et al., 2012); Pleurotus eryngii (Wang et al., 2016). This species was also reported as dominace bacteria in aquatic environment in Congo, India and switzerland (Devarajan et al., 2017). In these present study also confirmed that P. Putida was found as a causative agent associated with bacterial diseases on catfish that was intensively cultured in pond of Demak regency.
The phylogenetic of bacteria associated with bacterial diseases of catfish from Demak was seen in figure 1. The relationship between query strain in present study and other related members of the genus Aeromonas, Pseudomonas and Plesiomonas. The sensitivity test results (Table 5) showed that four causative agents assosiate with bacterial disease in catfish from Demak. Tabel 5. The result of senstivity test four causative agent of bacterial diseases on catfish in Demak regency Table 5 shows that A. caviae (D07), A. veronii (D10), Plesiomonas shigelloides (D11, P. putida (D14), were not sensitively to drug A ™, B ™, C ™ and D ™. This was indicated by the clear zone around the paper discs on all the bacteria with diameter of 0-1,2 mm. Based on the criteria of National Committee for Clinical Laboratory Standards (2001), the resistant bacteria was characterized by the diameter of clear zone ranged between 0-10 mm. The present researchrevealed that the four causative agent associated with bacterial dieseases in catfish from Demak were resistence to some fish drug tested. The previous study also reported that genus Aeromonas (Shinha et al., 2004;Ashiru et al., 2011), A. caviae (Motyl et al., 1985) and Pseudomonas spp (Devarajan et al., 2017) were reported a resistence to antibiotic. This resistance occurs may caused by irrational dosage administration during culture process (Sukenda et al., 2008) in order to prevent and combat the bacterial diseases. The similar result also reported by Sarjito et al . (2017) that the causative agent assosiate with bacterial diseases with catfish from kendal was not sensitively to some fish drug.