Introduction

Stenotrophomonas maltophilia, a motile, aerobic, non-fermenting, asporogenus, Gram-negative bacillus, previously known as Xanthomonas maltophilia, has got its current name S. maltophilia based on DNA-rRNA hybridization studies and sequencing and mapping of PCR-amplified 16S rRNA genes (Palleroni and Bradbury, 1993). Though is not a highly virulent pathogen, it has emerged as an important nosocomial pathogen associated with crude mortality rates in patients with bacteremia (Brooke, 2012) and infections such as pneumonia, bloodstream infections, as well as urinary tract infections, intra-abdominal infections, meningitis and ocular infections (Falagas et al., 2009). It has been recovered from soils and plant roots, animals, invertebrates, water treatment and distribution systems, wastewater plants, sinkholes, lakes, rivers, biofilms on fracture surfaces in aquifers, washed salads, hemodialysis water and dialysate samples, faucets, tap water, bottled water, contaminated chlorhexidine-cetrimide topical antiseptic, hand-washing soap, contact lens solutions, ice machines, and sink drains (see the review by Brooke, 2012). Ugur et al. (2002) classified S. maltophilia as one of the dominant bacterial species in the skin of Anguilla anguilla, constituting 12.05% of the total microflora. S. maltophilia was isolated from channel catfish Ictalurus punctatus with infectious intussusception syndrome (IIS) in Sichuan Province, China (Geng et al., 2007; 2010a, b) as a virulent pathogen.

In India, catfish production has been increasing over the years due to high economic return with low input. The state West Bengal holds the second position in catfish production, which contributed about 16-20% of the total catfish production of India since 2007 (Dahdf, 2012). With the increase in cultured catfish production, there has been increased incidence of infectious diseases (Kumar et al., 2013). Earlier we reported the isolation, and phenotypic and molecular characterization of this organism as opportunistic pathogen in cultured African catfish Clarias gariepinus juveniles (Abraham et al., 2016). In this communication, we report the association of S. maltophilia in the mortalities of C. gariepinus (Burchell, 1822) fries with distended abdomen and its phenotypic characterization.

1 Materials and Methods

1.1 Sampling and bacteriology

During the routine fish disease surveillance in February 2016, the African catfish Clarias gariepinus (Burchell, 1822) fries (≈1.50-2.50 cm) with typical symptoms of dropsy (Figure 1) in a nursery located in Ramchandrapur (Lat 22°54’01” N; Long 88°24’48” E), North 24 Parganas district, West Bengal, India were examined as per Heil (2009). At site, the behavioural abnormalities, gross and clinical signs of diseased C. gariepinus fries were recorded. Morbid C. gariepinus fries with typical disease symptoms (n=60) and also apparently healthy fries (n=20) were brought to the laboratory in oxygen filled polythene bags separately for bacteriological analysis. Prior to bacteriology, the morbid and healthy catfish fries (5 each) were first rinsed in sterile saline, wiped the adhering saline with sterile paper towels. Aseptically, the abdomen was punctured with a sterile needle and inocula from the ascites were streaked onto brain heart infusion agar (BHIA), glutamate starch phenol red agar supplemented with penicillin G sodium salt, 100 IU/ml (GSPA) (HiMedia, 2009) and Edwardsiella ictaluri agar (EIA) (Shotts and Waltman II, 1990) plates, and incubated at 30±2°C for 24-48 h. Apparently healthy fries had no abdominal fluid accumulation. Based on dominance and definite colony morphology, representative pink coloured colonies from GSPA were picked, purified on BHIA and maintained on BHIA slants at room temperature and also as glycerol stock at -20°C.

Figure 1  African catfish Clarias gariepinus fries with typical symptoms of dropsy. The abdominal cavity was filled with transparent jelly-like viscous ascites

1.2 Phenotypic characterization of bacterial strains

A series of biochemical reactions were performed to identify the randomly picked bacteria up to genus level (Collins et al., 2004). The initial characterization of ten isolates indicated that the bacterial strains were presumptive pseudomonds. Further, characterization of two strains EGC and RGC, showing pink colouration on GSPA, was done by Vitek 2 compact system (BioMerieux, France). Proteinase, lipase and haemolytic activity of the strains were tested on gelatin agar, tributryin agar and sheep blood agar (HiMedia, Mumbai), respectively (Collins et al., 2004).

2 Results and Discussion

In the surveyed nursery, the C. gariepinus fries were stocked in three cemented tanks of size length 6 m x breadth 5 m x water depth 0.3 m at high densities (≈200 000 nos/tank). Water circulation and aeration were adequate. They were fed with commercial pellet feed of 1.0 mm size on demand. Morbidity was about 60% of the total population. In about 10 days, 20-25% of the population died with dropsy in all three tanks. The gross and clinical signs observed in diseased fries include lethargy, body discolouration, mild fin/tail rot, distended abdomen, inflammation of internal organs, swelled intestine and visceral haemorrhages. The abdominal cavity of affected fries was filled with transparent jelly-like viscous ascites. Inacula from ascites samples yielded predominantly pink colour colonies on GSPA. The absence of green translucent colonies of 0.5-1.0 mm size on EIA or absence and/or scattered presence of few yellow colour colonies on GSPA after 48 h, thus ruled out the involvement of Aeromonas spp. and Edwardsiella tarda in the diseased condition. The bacterial strains (n=10) isolated from the ascites on GSPA were presumptively identified as pseudomonds. Two strains EGC and RGC were further characterized by BioMerieux Vitek system, which identified the strains as Stenotrophomonas maltophilia with 99% probability (Table 1). These strains were positive for protease and lipase activity and exhibited alpha haemolytic on sheep blood agar. Phenotypically, only minor variations were noticed among the present strains and the reference S. maltophilia strain SK1 (Abraham et al., 2016), especially in malonate utilization, Glu-Gly-Arg-arylamidase, and L-lactate alkalinisation (Table 1)

Table 1  Phenotypic characteristics of Stenotrophomonas maltophilia strains EGC and RGC isolated from Clarias gariepinus fries with dropsy Note: a: The results of the strain SK1 was from Abraham et al. (2016)

ports on S. maltophilia as fish pathogen are emerging since 2006 (Kapetanoviæ et al., 2006; Geng et al., 2007; 2010a, b; Musa et al., 2008; Abraham et al., 2016). The isolation of S. maltophilia strains with alpha-haemolytic, protease and lipase activities from C. gariepinus fries with 60% morbidity and 20-25% mortalities in the cemented tanks indicated the fact that S. maltophilia is emerging as a virulent pathogen capable of causing significant mortalities in C. gariepinus fries in the nursery phase. In our earlier study, 5% mortalities at the farm level, and 35±5% and 10% mortalities in challenge experiments with S. maltophilia at 1.40x10⁷ cells/fish via intraperitonial and intramuscular injections, respectively were observed with the production of viscous jelly mass in the abdomen (Abraham et al., 2016). While, the virulence tests by Geng et al. (2010a) via intraperitoneal injection of channel catfish identified S. maltophilia as a virulent pathogen. The pathology and pathogenesis of S. maltophilia in fish is still poorly understood. It has been proposed that extracellular enzymes play a role in the pathogenesis of S. maltophilia associated infection (Bottone et al., 1986). Few earlier results, however, confirmed that the extracellular products (ECP) of S. maltophilia exhibited strong virulence to channel catfish (Geng et al., 2010b) and mice (Du et al., 2011). According to Geng et al. (2010b), the ECP is an important pathogenic factor of S. maltophilia, which could induce apoptosis in the lymphocytes, multiple organ damnification and death. Although no detailed pathogenesis investigation was made in this study, the production of putative virulence factors such as protease, lipase and alpha haemolysin could be associated with catfish fry mortalities involving S. maltophilia. Further, our continued observations on the involvement of S. maltophilia with 5% mortalities in C. gariepinus juveniles in grow-out phase (Abraham et al., 2016) and 20-25% mortalities in fries in nursery phase indicated that S. maltophilia in emerging as a pathogen of C. gariepinus, which is a serious cause for concern. Further studies are necessary to understand the pathology as well as pathogenesis of this emerging pathogen in cultured catfish and its control measures.

Conflict of interest

The authors declare that there is no conflict of interest.

Acknowledgements

The research work was supported by the Indian Council of Agricultural Research, Government of India, New Delhi under the Niche Area of Excellence programme. The authors thank the Vice-Chancellor, West Bengal University of Animal and Fishery Sciences, Kolkata for providing necessary infrastructure facility to carry out the work.

Authors’ Contributions

TJA contributed during conception and design, analysis and interpretation of results and write-up of the manuscript. HA contributed during sample collection and analysis as well as acquisition of data. All the authors read and approved the final manuscript.

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