Isolation, Molecular Characterization and Virulence Study (Pathogenesis) of Photobacterium damselae subsp. damselae Isolated from Sea-cage and Wild Fishes

Sampling method and study area
 
Fish samples were collected from 5 cage culture units of 2 farms located at Mandapam, Tamilnadu, India. The species cultured under cages were seabass (n=37) (Lates calcarifer) and grouper (n=43) (Epinephelus spp.,) with a stocking density of 1000 fishes/m³. The water temperature and salinity of the cage culture farms were found to be 29±1°C and 32±1.5ppt, respectively during the time of sampling. Moribund/dead fishes and fishes showed lesions were collected using sterile polythene bags and the live fish samples were brought in oxygen filled polythene bags. All the samples were brought to the laboratory within 24 hr under iced condition. In addition, 3 species of wild caught marine fishes (Scarus globiceps; n=45, Lutjanus sebae; n=37, Brama brama; n=40) from 4 important landing centres and marine ornamental fish (n=10; Chaetodon octofasciatus) from one ornamental fish farm, southeast coast of India such as Tharuvaikulam, Theraspuram, Mandapam and Pamban were collected and transported to the bacteriology laboratory under iced condition.
 
Isolation, characterization and culture condition of bacterial strain
 
The detection of P. damselae and distinguishing the isolates at the subspecies level were achieved using the standard procedures of morphological and biochemical plate and tube tests as described in Finfish and shellfish diseases Practical Manual (Rijijohn and Rosalind George, 2004). All the dissection utensils and inoculation loop was sterilized using 70% alcohol and flame before isolating the bacteria. The surface of the animal was disinfected by wiping with sterile cotton swab soaked in 70% alcohol. Samples from the gill tissue was collected by inserting the sterile loop into it and immediately streaked on to the pre-dried TCBS agar plates by streaking method. Tissues like kidney, spleen and liver were dissected out aseptically and were scratched with the sterile loops and immediately streaked onto the pre-dried TCBS agar plates. The agar plates were kept under incubation at 27°C for 24 h at incubator (Kemi, India). P. damselae subsp. damselae grows on TCBS produces green colonies. Therefore, green colonies were selected randomly and streaked on to trypticase soya agar (TSA) slants with 2% NaCl for 24 hrs. All colonies having similar visual appearances were sub-cultured from a single colony for purity. Pure presumptive colonies isolated on plating media were used for biochemical analysis.
 
Biochemical characterization of P. damselae subsp. damselae isolates
 
For biochemical confirmation, colonies showed green colonies in TCBS + 2% NaCl were restreaked in the same medium. Then the reisolated single green colony was inoculated to 5ml of TSB broth. After 24 hrs of incubation, “the TSB broth contains bacteria was used for biochemical analysis”. The tests include: motility (hanging drop method), urease production (Urea hydrolysis test), oxidation/fermentation (O/F test), amino acid decarboxylase (Arginine, lysine and ornithine) and salt tolerance (Rijijohn and Rosalind George, 2004).
 
Molecular characterization of P. damselae subsp. damselae
 
Bacterial DNA extraction
 
The identities of the isolated bacteria were confirmed using PCR assay. Genomic DNA was extracted according to the protocols of Osorio et al., (2000). Briefly, individual green colour colonies grown in 5 ml of TSB broth were incubated at 27°C for 24hrs. After incubation, one ml of sample was transferred to a microcentrifuge tube with a capacity of 1.5ml. The cell suspension was centrifuged for 10 min at 10,000 x g in a refrigerated centrifuge (Sigma, Germany). The supernatant was discarded carefully and the pellet was air dried. Then the pellet was resuspended in 300 µl of DNA –Xpress^TM reagent (GeNei^TM HIMEDIA) in a dry bath at 60°C for 45 min. After that, the tube was centrifuged for 10 min at 10,000 x g. Then supernatant was carefully transferred to a new microcentrifuge tube and washed with 400 µl of 100% ethanol for 5 min at 10,000 x g. The supernatant was discarded carefully and then the pellet was washed twice with 400 µl of 95% ethanol as said above. After centrifugation, the supernatant was discarded and the tube was inverted for complete drying of pellet. After that, 100µl nuclease free water was added to dissolve the pellet. DNA purity and concentration were assessed using a Nano Drop^TM by measuring absorptions at 260 and 280 nm. The DNA concentration of each sample was adjusted to 40ng/µl.
 
Multiplex PCR assay
 
A multiplex PCR assay using P. damselae specific 16S rRNA gene directed primers and primers designed from the partial urease C (ureC) gene sequence are employed to discriminate between the two subspecies of P. damselae (Osorio et al., 2000) are shown in Table 1. The PCR condition includes 25 μL reaction included 2 μL forward and reverse primers, 6.5 μL ultrapure water, 12.5 μL master mix and 2 μL template DNA. The thermal cycle (Biorad, USA) program comprised 30 cycles of 4 min at 95°C, 1min at 95°C, 1 min at 60°C and 40 s at 72°C and a final 5 min extension at 72°C. 10 µl of amplified products were separated by electrophoresis on a 1% (w/v) agarose at 100V gel run for 50-90min in Trisborate –EDTA (TBE) buffer (0.89 M tris, 0.89 M boric acid, 0.02 M EDTA, pH 8.0) and a 100bp DNA ladder was used as a size reference. This gel was then stained with 0.5µg/ml ethidium bromide solution for 20 min and visualized by UV – induced fluorescence (Biorad, USA).
 

 
Dendrogram analysis
 
The fingerprints obtained for selected 30 isolates based on band patterns of PCR product after using IS and ERIC PCR were statistically analyzed by unweighted pair group method using mathematic averages (UPGMA) and coefficient cluster analysis (Nei and Li, 1979) by UVI bandmap software in the UVI Doc gel documentation system.
 
Pathogenesis of P. damselae subsp. damselae isolates in damselfish
 
For challenge study, healthy and uniform sized damsel fish (Pomacentrus similis) fingerlings (80nos) (average size as 6 ± 2 cm and 5 ± 2 g) were obtained from local aquarium at Thoothukudi, India. The fishes were acclimatized before experimentation. The study was conducted in the wet laboratory of Department of Fish Pathology, Tamilnadu Dr. J. Jayalalitha Fisheries University, India. The study was conducted period of 2018-2019. Experiment was conducted in a glass aquarium tanks of 40L capacity with the aerated sea water temperature of 29 ± 1°C, salinity 30 ± 2ppt and pH 8.4 ± 0.2 and the fishes were fed with pellet feed twice daily. Water was replaced daily at a rate of 20% in each tank. All the fishes were acclimated for 3 days prior to the initiation of the experiment. The fish health and water quality were monitored by observing swimming behavior of fishes and appearance of water, respectively. The third day of acclimatization, 8 fingerlings (one from each tank) was randomly collected and their internal organs were checked using microbiological tests. The fourth day, tanks were grouped to 8 Groups [6 test groups and 2 control groups; All treatments in duplicate] were formed and 10 number of fishes were introduced in each groups. One strain of P. damselae subsp. damselae isolates from natural outbreak was cultured on TCBS agar plate with 2% NaCl and incubated at 27°C for 24hrs. Then after 24 hrs, the culture was centrifuged at 10,000 x g for 10min, supernatant discarded carefully. The pellet was taken and suspended with sterile saline solution. The fishes were intraperitoneally injected with 50 µl of bacterial suspension per fish for test groups and 50 µl of saline for control fishes. For damsel fish challenge study, inoculum strength of bacterial suspension was 3.0x10⁴, 3.0x10³ and 3.0x10² CFU fish^-1 (tenfold dilution from LD₅₀ value). The mortality of the fishes was recorded daily up to 7 days post challenge and fish behaviour also recorded. The dead fishes were collected from the tank and tissues such as kidney, spleen and liver were dissected out and bacteria were reisolated for confirmation purpose. The reisolation of bacteria has been done to confirm the mortality due to bacteria. The LD₅₀ value was calculated by the Reed and Muench, (1938).
 
Histopathology of experimentally infected fish
 
To study the histopathological changes provoked by P. damselae subsp. damselae in fish tissues, parts of gills and internal organs (liver, kidney and spleen) of dead fishes were dissected out and fixed overnight in 10% buffered formalin. Then the samples were dehydrated in 70 to 100% alcohol, placed in two changes of xylene (10 min each) and then embedded in paraffin (56°C melting point). The parts were sliced transversely into 4-mm-thick sections with the help of microtome (Thermo) and stained with hematoxylin and eosin (Roberts, 2001). Then the stained slides were analyzed and photographed by Nikon microscope (Nikon, Japan) installed with digital camera.
     
 The fish study including animal experimentation, tissue collection and sacrifice was carried out as per the ethical guidelines and adheres to the legal requirements of the study country.

P. damselae subsp. damselae is an autochthonous member of aquatic ecosystems. It is considered as primary pathogen of several species of wild fish (damselfish, catfish, shark, stingray, etc.) as well as commercially important fish species in mariculture, causing wound infections and hemorrhagic septicemia (Fouz et al., 1992). The present study hypothesized that there exist potential for the bacteria to transmit from wild caught fishes to cage cultured fishes. The pathogenic potential of transmitted bacteria on cage culture fishes also been revealed.
 
Bacterial identification
 
The bacterial colonies isolated from spleen, kidneys and liver of wild caught and cage culture fishes was inoculated on TCBS agar at 24°C for 1-2 days. In total, 289 green colonies were isolated on TCBS agar. Out of which sixty one isolates were found gram negative, rod-cocci, motile organisms, oxidation/Fermentation test positive and arginine and lysine decarboxylase production, growing in 3% and 6% Nacl and urease production (Table 2). The previous researcher also confirmed that the presence of P. damselae subsp. damselae when three or more positive results were obtained in the following tests: LDC, motility, TCBS growth and urease production (Holt et al., 1994; Zorrilla et al., 1999; Rajan et al., 2003; Thyssen et al.,1998).
 

 
Molecular identification
 
The multiplex PCR assay was carried out with 16S rRNA gene and ureC gene binding primers. A total of 289 isolates were selected from infected fish, representing different organs and different locations. The expected amplicon sizes were 267bp and 448bp (corresponding to an internal fragment of the 16S rRNA gene and one of the ureC genes respectively). A total of 61 isolates were positive for both 16S rRNA gene and ureC gene. No isolates were positive for the 16S rRNA gene (267bp) alone indicating the absence of P. damselae subsp. piscicida (Fig 1). Osorio et al., (2000) reported the multiplex-PCR using two primer pairs directed to internal regions of the 16S rRNA and ureC genes, was employed to differentiate between the subspecies of P. damselae. Labella et al., (2006) observed two amplification bands of 267bp and 448bp in the multiplex- PCR assay demonstrated that the isolates from redbanded seabream are members of the P. damselae subsp. damselae. The 30 PCR product selected based on species wise thickness of PCR generated by the 16S rRNA (1500bp) gene primers was sequenced by outsourcing to MWG, Eurofins Genomics India Pvt Lt Bangalore, India.
 

 
Prevalence of P. damselae subsp. damselae isolates from wild and cage culture fishes
 
The prevalence of P. damselae subsp. damselae isolates are presented in Table 3. A total of 61 P. damselae subsp. damselae were identified from 289 isolates of bacteria collected from different fish samples. Out of all P. damselae subsp. damselae 86.91% isolates were found in wild caught fishes and 13.09% in cage cultured fishes collected from South east coast of India. Highest number of P. damselae subsp. damselae were obtained from wild caught parrot fish samples (55.73%) followed by wild Lutjanus sp. (22.95%). Of the 61 isolates obtained, 9.83% were from seabass cultured in cages. The lowest number of P. damselae subsp. damselae (0.35%) was isolated from cage grouper and eightband butterflyfish. The more amounts of P. damselae subsp. damselae isolates present in parrot fish when compared to cage culture fishes was noticed. Similarly, studies noticed high bacterial loads in parrot fish tissues when compared to other marine fish species (Tarnecki et al., 2016). This suggests that their feeding behavior found correlated with the abundance of P. damselae subsp. damselae in parrot fish. It has been noticed that gut microbiota of coral reef associated fishes contains more amount of Photobacterium. The parrot fish feeding on coral reef, seagrass and algae (Smriga et al., 2010). Eissa et al., (2018) also reported total prevalence of P. damselae subsp. damselae among naturally infected marine fishes was 45.04%. Essam et al., (2016) reported that total prevalence of P. damselae subsp. damselae in naturally infected fishes was 20.76%. The low bacterial presence in cage culture fishes might be due to the reason that hatchery produced seeds may devoid of P. damselae subsp. damselae. Once the fishes were introduced into the cage environment, it becomes more vulnerable to P. damselae subsp. damselae infection (Essam et al., 2016). The reason for Cross-contagion between cage and wild fish species with shared pathogens may occur either through movement of individual fish or through species-specific migrations. Unlike parasitic pathogens, bacteria seem to exhibit higher potential to spread between wild and cage fish. Infected wild fish might also transfer pathogens to the cage fish. This co-infection process leads to a large variety of shared pathogens among wild and cage fish, while the various pathways of pathogen transmission increase the potential for infection and render epidemiological risk management difficult (Diamant et al., 2007) Therefore, the present study suggests that proper site selection and screening of P. damselae subsp. damselae in the cage culture environment before incorporating cages in the natural environment is very much necessary to avoid the P. damselae subsp. damselae infection and associated fish mortality.
 

 
Dendrogram analysis of P. damselae subsp. damselae isolates from wild caught and cage culture fishes
 
A cluster analysis of the pairwise distance matrix among patterns was performed using the unweighted pair group method with average linkage (UPGMA) (Sneath and Sokal, 1973). The dendrogram obtained after numerical analysis with the Dice coefficient and UPGMA method (Fig 2, 3, 4 and 5) shown that all patterns shared more than 50% similarity. The isolates (MP66, MP147 and MP157) showed 87% similarity, will belong to same genogroup. The isolate no MP66 isolated from eightband butterflyfish collected at marine ornamental fish farm, Mandapam. MP147 and MP 157 isolate in wild caught parrot fish collected from south east coast of India. The dendrogram obtained after numerical analysis with the Dice coefficient and UPGMA method shows that all patterns shared more than 45% similarity (Botella et al., 2002). The isolates (MP189, MP190, MP191, MP193 and MP194) are one genogroups showed 85% similarity. All isolates were collected from wild caught parrot fish. The isolate (MP162, MP189 and MP190) belongs to one genogroup and showed 70% similarity. MP162 isolate collected from cage culture seabass in Mandapam. MP189 and MP190 isolate collected from wild caught fishes. So, these isolates showed abundance in both wild caught and cage culture fishes. Regev et al., (2020) revealed that all detected Vibrio strains were divided into four different genogroups of Vibrio sp., with an overlap in one group between the wild and the cultured species. This may suggest a spontaneous transmission between the wild and the cage fish. The first group showed high similarity to V. parahaemolyticus and V. alginolyticus. The second group showed high similarity to V. harveyi. The third group contained only one strain that belonged to the Lessepsian fish S. lessepsianus, which showed similarity to uncultured Vibrionaceae bacterium isolated from pinfish (Lagodon rhomboids), with a 95% homology. In addition, the fourth group contained three identical strains all belonging to the cultured S. aurata from 2017 without any similar references. The thirty out of sixty isolates (20% or 50%) showed their presence in both wild caught and cage cultured fishes. The presence of similar genogroup in both wild and cage cultured fishes confirmed the horizontal transfer of these bacteria from wild caught fishes to cage cultured fishes. Similarly reports from various countries also showed horizontal transfer (Chiu et al., 2013). Therefore, it is suggested that while selecting sites for cage culture, the place which showed abundance in reef associated fishes should be neglected to avoid P. damselae subsp. damselae infection.
 

 

 

 

 
Pathogenicity study
Clinical signs and mortality
 
Pathogenicity of P. damselae subsp. damselae strain to damsel fish was investigated using standard protocols and the LD₅₀ was calculated. After injection, the first three days of post challenge no fishes showed symptoms, but fourth and fifth days of post challenge, reduced feed intake was noticed. Six days of post challenge, infected fish developed clinical signs such as lethargy, discolouration of skin, followed by abnormal swimming before mortality. By day 7, the 100% cumulative mortality was observed in damselfish injected with two higher concentrations of bacteria (Fig 7). As compared to infected fish, no clinical signs and mortality were noticed in control fish injected with saline solution. Mahmoud et al., (2018) has done the pathogenicity assay and revealed that P. damselae subsp. damselae, were pathogenic for seabass at LD50 of (1.5×10⁸ CFU/g body weight), causing 66.67% mortality. Infected seabass showed ulcers and hemorrhages all over the body with fin erosions and pale gills (Mahmoud et al., 2018). Love et al., (1981) discussed with ulcers typically occur in the region of the pectoral fin and caudal peduncle of naturally infected damselfish and may reach 5-20 mm in diameter. LD₅₀ value of this injected culture of P. damselae subsp. damselae strain was calculated to be 2.48x10³ CFU/mL. Bacteria were re-isolated from the kidney, spleen and liver of moribund fish (Fig 6) and the tissue extracts were directly analysed for the presence of injected pathogen by means of multiplex PCR and the results are given in Fig 8. It has confirmed that mortality and the clinical sign developed in the experimental fish was due to P. damselae subsp. damselae.
 

 

 

 
Histopathological findings
 
Histopathology was carried out from moribund fish tissues such as kidney, liver, spleen and gill (Fig 9). In kidney, tubular epithelial fusion and hyperplasia of haematopoietic tissue, hyperactivity of melanomacrophage centers and coagulative necrosis of epithelial lining of some renal tubules and glomerulonephritis was noticed (Fig 7). Similarly, P. damselae subsp. damselae infected fish kidneys showed congestion of blood vessels, necrosis of renal tubules and hyperplasia of melanomacrophage centres (Eissa et al., 2018, Mladineo et al., 2006). The presence of highly dense clusters of bacteria in the kidney in seabass suggested that kidney should be microbiologically examined in a first attempt (Avci et al., 2013). Gills of infected damselfish showed destructive changes in both primary and secondary lamellae, collapsed and curled secondary lamellae, lamellar fusion and hyperplasia. Essam et al., (2016) investigated the histological alterations in gills were characterized by changes in both primary and secondary lamellae, collapsed and curled secondary lamellae, edema in filaments, severe lamellar aneurism and hyper activation of goblet cells. These sever lesions in the gills suggests their primary role in the bacterial entrance into the fish body. Hence, the study suggest that P. damselae subsp. damselae isolates from wild caught fishes found virulent to the culture fishes and cause severe tissue damage and mortality to the individuals.
 

Photobacterium damselae subsp. damselae is the most devastating bacterial pathogen and a global health issue. Photobacteriosis found to be one of the major causes for the failure of cage culture industry. The present study revealed higher prevalence of P. damselae subsp. damselae in wild caught parrot fish when compared to the other wild and cage culture fishes. Among cage culture fishes, seabass found more infected with P. damselae subsp. damselae when compared to grouper. There exists possibility for transmission of P. damselae subsp. damselae from wild caught to cage culture fishes and could cause severe tissue damage, lesions and mortality of the individuals. Therefore proper site selection, screening of P. damselae subsp. damselae presence in cage sites proximity area and good management practices during cage culture is very much necessary for the prevention of P. damselae subsp. damselae infection in fishes and their associated economic loss.