Abstract: Nemipterids are one of the important commercial and demersal resources of Visakhapatnam coast. Most of the Nemipterus japonicus caught in this coastal area were found to have lesions over the body surface along with a typical mucosal polyp in the buccal cavity, showing clinical signs of exophthalmia, skin discoloration with deep ulcers, frayed fins, damaged gills, distended abdomen with calcified gonads and hemorrhages. Hence, the present study is aimed to know the aetiological agent causing these lesions and polyps. Therefore, the smears were collected in the form of swabs from these lesions. The smear was examined by Grams method and inoculated into selective media. The organisms isolated and identified by biochemical and microbiological studies. Out of 50 Nemipterus japonicus, subjected to the above microbiological studies, Klebsiella pneumoniae were isolated from 40 fishes, Vibrio species from 5 fishes and Enterococci species from the remaining. It is concluded that the disease in these fishes is mainly due to infection by Klebsiella pneumoniae.
INTRODUCTION
Environmental awareness around the world in the past years increased interest in the study of fish diseases, mostly caused by bacteria. The bacterial diseases are caused mainly due to contaminated water and sea foods and their associated disease outbreaks affected by toxins, biotoxins and histamines reaching the higher trophic levels (Musa et al., 2008; Chen et al., 2010).
The threadfin breams of the genus Nemipterus have an important commercial value as good protein source. Large number of N. japonicus in the coastal area of Visakhapatnam is found to be suffering from buccal polyps and other associated manifestations. This fish is consumed by large number of economy class people and there is a potential risk, if such infected fish are consumed raw or improperly cooked. There is a threat of spreading infection to other species as well as decline of species due to disease (Diana, 2007).
In this area, there are few microbiological reports on Nemipterus japonicus. Petersen et al. (1993) reported an unidentified microspora infection in Nemipterus species from central Philippine water. Singh (1996) isolated S. typhosa in N. japonicus from Sapthagiri in Maharashtra. Rajapandiyan et al. (2009) reported on the prevalence and distribution of Vibrio vulnificus in N. japonicus from Chennai, Indian Ocean. Diana and Ramulu (2009, 2010) reported biochemical changes in Nemipterus japonicus infected with Klebsiella species as compared to the normal fish off Visakhapatnam Coast. Many scientists have reported on the Klebsiella infection in different fishes (Cann and Taylor, 1982; Daskalov et al., 1998; Rajkumar et al., 2007; Choudhury et al., 2008; Ogbonna et al., 2008; Ravichandran and Ajithkumar, 2008; Abdelhamid et al., 2009; Bragadeeswaran and Thangaraj, 2011; Mahin et al., 2011; Akinyemi and Buoro, 2011; Ayeloja et al., 2011). The present intention was to isolate the pathogenic microflora from mucosal buccal polyp of N. japonicus.
MATERIALS AND METHODS
The specimens were collected regularly from the harbour water of Visakhapatnam. The fish were examined for external disease characters for identification like skin ulcers and differentiation is done according as superficial or deep ulcerations. Skin scrapings and gill racker observations were done to identify the external etiology. The samples were brought to the laboratory and subjected to microbiological analysis under sterile conditions. Smears from the buccal polyp were collected by using swabs. These swabs were inoculated directly on Blood Agar, MacConkey agar and EMB agar and incubated at 30-32°C for 2 days. The subcultures were made from single colonies onto peptone water and incubated for 4 h. A wide range of biochemical tests were performed with this young culture to identify the incriminating pathogen.
Smears made from isolated colonies were stained by Grams method, motility was tested by hanging drop method followed by biochemical tests. Catalase production, oxidase, nitrate reduction, urea hydrolysis, Voges-Proskauer test, citrate utilization and gelatinase test were performed. Carbohydrate fermentation tests were done with glucose, lactose, sucrose, D-adonitol, arabinose, inositol, rhamnose, raffinose, salicin, D-sorbitol, trehalose and xylose. Aminoacid utilization tests were also done with arginine, lysine, ornithine and phenlyalanine deaminase as per standard procedures described by Collee et al. (2006).
RESULTS
In the present study, a typical mucosal polyp was detected in the buccal cavity of N. japonicus (Fig. 1). External aetiology showed exophthalmia, skin discoloration with deep ulcers, frayed fins, damaged gills, distended abdomen with calcified gonads, petechial haemorrhages on skin, mouth, operculum, base of pectoral, pelvic and anal fins over their body surface (Fig. 2, 3).
A total number of 50 N. japonicus were examined and subjected to microbiological studies. The direct smears from mucosal buccal polyp revealed thick Gram negative, non-motile bacilli with clear capsular hallows in large numbers. The culture plates were examined after 24 h incubation. On blood agar plates, large mucoid colonies measuring about 3 to 5 mm in size were observed.
| Fig. 1: | Polypoidal mass in the buccal cavity |
| Fig. 2: | Diseased skin with pale colouration, Exop: Exophthalmia (pop-eye), SU: Skin ulcer |
| Fig. 3: | The operculum and jaws are hyperemic |
| Fig. 4: | Encircled area shows Gram negative bacilli capsular with hallows, cultured on MacConkey’s agar |
Mucoid nature of the colony was observed by naked eye as well as by demonstration of mucous strings when lifted with a loop. The colonies were non-haemolytic.
On MacConkey’s medium, similar type of large mucoid colonies were observed which were pink in colour denoting that they were lactose fermentors. The Gram stained smears from the culture plates are morphologically similar to those seen in the direct smears (Fig. 4).
| Table 1: | Pathogenic bacterial species isolated from fish |
| Table 2: | Biochemical characters of Klebsiella pneumoniae strains isolated, n = 40 |
Out of the fifty N. japonicus examined, Klebsiella pneumoniae strains were isolated from forty specimens, Vibrio species from five specimens and Enterococci species from the remaining five specimens (Table 1).
All the strains of Klebsiella pneumoniae isolated (40) from the lesions, exhibited typical biochemical characters. They were catalase positive, Voges-Proskauer positive and utilised citrate, melanate and lysine, reduced nitrate to nitrites and hydrolysed urea. All the sugars tested were fermented with production of acid and gas. They were non motile, oxidase and indole negative, arginine, ornithine utilisation and phenylalanine deaminase tests were negative (Table 2).
DISCUSSION
Harbour is therefore, a typical example of ecosystem that experiences chronic pollution due to untreated pollutants discharged it. These become stagnant and aggravate heavy loads of bacteria, resulting in chronic infectious diseases and mass mortality of fish. As Nemipterids are bottom dwellers, these fish are mostly affected by the polluted environment in which they live (Diana, 2007). These demersal species are particularly susceptible to physical abnormalities and diseases, which appear to be associated with contaminated sediments (Stehr et al., 1997).
In the present study, heavy bacterial loads have been isolated from the mucosal buccal polyp of N. japonicus. Since mucous surfaces are important defense barriers against bacterial infection, mucous layers in fish are related to a number of activities including the prevention of colonization by pathogens. These observations are supported by few scientists. Interaction of bacterial pathogens with subjacent tissues, the gut, the gills and the intestines have been described as possible sites of entry for pathogens into the fish (Chabrillon et al., 2003). Success of any pathogen is dependent on its ability to elude host immune responses. Bacteria often overcome physical barriers by secreting enzymes that digest the barrier (Cianciotto, 2005).
In the present study, the fish collected from harbour waters showed high prevalence of ulcers and fin erosion. Microbial studies revealed that Klebsiella pneumoniae isolated from forty N. japonicus is the most common pathogen causing these lesions. In the other ten fishes tested, Vibrio species were isolated in five fishes and Enterococci species in the other five fishes. These observations are in correlation with the earlier studies made by several investigators (Cann and Taylor, 1982). Daskalov et al. (1998) reported Klebsiella pneumoniae causing fin and tail disease in Rainbow trout (Oncorhynchus mykiss Walbaum), Rajkumar et al. (2007) studied on the secondary infection caused by Vibrio, Salmonella and Pseudomonas loads from Stolephorus commersonnii. Choudhury et al. (2008) isolated Vibrio species along with other histamine bacteria from Indian mackerel fish Rastrelliger kanagurta. Ogbonna et al. (2008) isolated Klebsiella species from gills and intestine of Tilapia zilli from creeks around Port Harcourt, Nigeria. Ravichandran and Ajithkumar (2008) observed lesioned spots with heavy load of Vibrio parahaemolyticus and V. anguillarum in Ilisha melastoma. Abdelhamid et al. (2009) worked on pathogenic strains of Gram negative bacteria like Vibrio species and Klebsiella species isolated from African catfish. Bragadeeswaran and Thangaraj (2011) isolated Vibrio parahaemolyticus, Klebsiella pneumonia, Klebsiella oxytoca from skin mucus of eel fish, Anguilla Anguilla. Mahin et al. (2011) identified Klebsiella ozaenae, Klebsiella edwardsii from fresh water Mola fish Amblypharyngodon mola. Akinyemi and Buoro (2011) reported Klebsiella sp. and Vibrio parahaemolyticus from gills, skin and buccal cavity of Lutjanus agennes, Pseudotolithus elongatus and Sphyraena barracuda from Lagos Lagoon, Nigeria. Ayeloja et al. (2011) isolated Klebsiella pneumoniae from smoked African catfish, Clarias gariepinus.
In the present study, Klebsiella pneumoniae which belong to the family Enterobacteriaceae is the predominant pathogen isolated in most of the fishes examined. These results correlate with many investigators, Taylor et al. (1979) isolated histamine-producing Klebsiella pneumoniae strain T2 from spoiled Tuna Sashimi. Enteric bacteria have been reported to be the dominant Histamine-Producing Bacteria (HPB) in fish (Taylor and Eitenmiller, 1986). In a previous study, Diana and Ramulu (2009, 2010) observed abnormal variations in biochemical composition in different tissues of N. japonicus in relation to Klebsiella infection. Hence, the manifestations in the infected fishes with Klebsiella pneumoniae may be due to direct affect of endotoxins with associated abnormal immunological responses.
The significance and possible role of these pathogens, in relation to physiological process and immunological response of the fish, sets a platform for further studies in relation to pathology, pollution and stress conditions in fish habitats (natural and polluted environments), prevalent in the world today. With relation to the other organisms isolated in this study, no conclusion can be drawn since their numbers are too small. Further, wider studies are required to attribute any significance of these organisms in their pathogenic role.
ACKNOWLEDGMENT
The corresponding author is grateful to Department of Science and Technology, Andhra University, Visakhapatnam, Andhra Pradesh, New Delhi, India for financial support of the project.