Int.J.Curr.Microbiol.App.Sci (2018) 7(7): 928-936 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 7 Number 07 (2018) Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2018.707.112 A Report of Centrocestus formosanus (Nishigori, 1924) (Digenea: Heterophyidae) in Intermediate Host (Fish) Amit Bharat Gamit*, Pramod Kumar Nanda, Ria Bhar, Samiran Bandyopadhyay and Subhasish Bandyopadhyay Eastern Regional Station, ICAR-Indian Veterinary Research Institute, 37-Belgachia Road, Kolkata-700 037, West Bengal, India *Corresponding author ABSTRACT Fish acts as an intermediate host to many helminth parasites, including trematodes of K e yw or ds public health significance. In this study, we screened freshwater fish samples (n=195) collected from different markets located in and around Kolkata, to isolate and identify Fish, Metacercariae, larval stage of minute intestinal fluke, Centrocestus formosanus employing a fast and Centrocestus accurate molecular approach. Processing of fish samples through pepsin digestion method formosanus , revealed an overall prevalence of 14.87 % infection in fish with encysted metacercariae of Polymerase chain trematode, Centrocestus. The prevalence rate was highest in Mystus tengara (40.00%) reaction followed by Puntius spp. (26.66%) and Awaous grammepomus (5.26 %), whereas screening fish species such as Labeo bata, Cirrhinus mrigala and Oreochromis niloticus Article Info did not harbor any such larval stage of this trematode. Polymerase chain reaction using Accepted: oligonucleotide primer sets from extracted genomic DNA of these metacercariae revealed 08 June 2018 amplicon of approx. 492 bp closely resembling to Centrocestus. Phylogenesis showed Available Online: maximum relationship with the Centrocestus spp. Therefore, implementation of control 10 July 2018 strategies, apart from awareness of public and adoption of good aquaculture practices by farmers, are required in order to minimize the risk of infection in fish. Introduction Thailand, Hawaii, Vietnam, Croatia, India, USA, Mexico, and Colombia (Srisawangwong There are numerous minute intestinal flukes et al., 1997; Mitchell et al., 2005; Salgado- belonging to the family Heterophyidae. Maldonado et al., 2005; Velasquez et al., Centrocestus formosanus (Nishigori, 1924) is 2006; Gjurcevic et al., 2007). one among them inhabiting the small intestine of fish eating birds and mammals, including It is digenean that requires two intermediate chickens, ducklings, dogs, cats, foxes, mice, hosts and one definitive host to complete the rats and rabbits. This trematode is considered life cycle. Red rimmed thiarid snail, as an Asian species but it has been introduced Melanoides tuberculatus act as first around the world and distributed in many intermediate host and many fish species, countries including Taiwan, China, Japan, harbouring metacercariae, have been reported 928 Int.J.Curr.Microbiol.App.Sci (2018) 7(7): 928-936 as a second intermediate host. Metacercariae infective metacercariae of trematode is a localize into the gills and sometimes under the concern for human health (WHO, 1995; scales of numerous fish species and finally Howgate, 1998). Fish borne zoonotic infects the fish eating animals and birds. This trematodes are mostly prevalent in East Asian trematode not only causes significant countries due to traditional aquaculture and morbidity but is often associated with food feeding habits, but many reports are available safety and quality problems in fish (Yu and from different parts of the world. Although Mott, 1994; Toledo et al., 2006). Infected fish this trematode is a species with pisciculture as can develop serious pathologies of gills well as medical importance, hardly any report including oedema, haemorrhage and loss of is available on identification of this trematode respiratory epithelium and cause serious through molecular means. Considering the weakness and sometimes mortality in case of public health importance of fish borne heavy infection. Furthermore, these zoonoses and to assure and accurate diagnosis trematodes can potentially infect humans as overcoming the deficiencies of microscopic reported from many countries of Asia examination, the present study was conducted including Taiwan, Japan and Lao PDR to identify C. formosanus in fish, the second (Nishigori, 1924; Waikagul et al., 1997; Chai intermediate host, employing both standard et al., 2013) through consumption of raw or and molecular (PCR) methods which could be improperly cooked fish containing useful to demonstrate its epidemiological metacercariae. situation and develop strategies so as to formulate and implement control programs. The detection of Centrocestus infection relies on the microscopic observation of eggs in the Materials and Methods faeces of infected definitive host (animal or human). However, accurate diagnosis with Isolation of metacercariae microscopy is not always possible due to similar morphology with the eggs of other For this study, commercially important heterophyids and opisthorchiids. Identification freshwater fish (n=195) of six different species of metacercariae at species level is also (Mystus tengara, Labeo bata, Cirrhinus difficult due to similar morphology with other mrigala, Puntius spp., Awaous grammepomus Centrocestus metacercariae. To overcome and Oreochromis niloticus) were collected such problems, molecular approaches through from nine different fish markets located in and amplification and sequencing of some around Kolkata. As per the history, all the conserved DNA regions, especially utilizing samples were from fish farms with traditional genetic markers in nuclear ribosomal DNA culture methods. Following collection, fish (rDNA) and mitochondrial DNA are often species were placed in ice box and transported employed to diagnose helminth parasites to the laboratory for further processing. After (Blair et al., 1996). The spacer regions of thorough washing under distilled water, all rDNA (ITS1 and ITS2) are particularly useful fish were measured and weighed. The in the diagnosis of metazoan parasites at the metacercariae were isolated employing the species level (Morgan and Blair, 1995; Leon- standard pepsin digestion method. Briefly, the Regagnon et al., 1999; Nolan and Cribb, finely ground fish were digested by artificial 2005). digestion solution containing 0.5% pepsin (1:10,000, Hi-Media, India) and 1% HCl (Hi- As freshwater fish act as second intermediate Media, India) and incubated for 2-3 hrs at 370 host, cultured or farmed fish harbouring C with occasional shaking. Digestion was 929 Int.J.Curr.Microbiol.App.Sci (2018) 7(7): 928-936 followed by straining and sedimentation The PCR reaction was carried out in 25µl repeated for 7-8 times using solution of 0.85% volume with 8-10 pmol of each primers from saline, until the supernatant became clear. The Xcelris genomics (Ahmedabad, India), 1unit metacercariae were isolated from the bottom of Taq DNA polymerase (Thermo Scientific, sediment and the rate of prevalence and USA), 10x Taq buffer with KCl (Thermo intensity of infection were calculated out. The Scientific, USA), dNTPs mix (10 mM, isolated metacercariae were examined under Thermo Scientific, USA). PCR was performed stereomicroscope and microscope for by initial denaturation at 94˚C temperature for identifying the morphological features 2 min, followed by 35 cycles each at 95˚C for including shape and size of cyst, circumoral 30 sec, 50˚C for 30 sec and 72˚C for 2 min spines and shape of excretory bladder using and final extension at 72˚C for 7 min. The the morphological criteria (Chai et al., 2012) amplified PCR products were separated by and stored in 70% ethanol at -20°C until DNA electrophoresis through 1% (w/v) agarose gel extraction. in TAE buffer and visualized as described earlier. For DNA sequencing, amplified PCR Total genomic DNA extraction products were purified using gel purification kit (Sigma-Aldrich, USA) according to the Genomic DNA extraction was done separately manufacturer’s instructions and sequenced in from all the isolated metacercarial samples both forward and reverse directions using PCR using GF-1 Tissue Extraction Kit (Vivantis, primers on an automated sequencer by DNA Malaysia) according to the manufacturer’s sequencing services of Scigenom labs, Kerala, instructions. DNA was eluted in 70-100 µl of India. elution buffer and stored at -20°C until further use. The integrity of DNA was checked by 1% Sequence analysis and phylogenic tree agarose gel electrophoresis in 0.5X TAE reconstruction buffer. A 2µl of genomic DNA was loaded in the gel after proper mixing with 6X loading The DNA sequences were further analyzed dye (Thermo Scientific, USA) and gel was run using basic local alignment search tool at 70V for 1hr and visualized under ultraviolet (BLAST; http://blast.ncbi.nlm.nih.gov/Blast. light in a Wealtech apparatus (Wealtech, cgi) to check the homology with published USA). sequence databases. Phylogenic tree reconstruction was carried out by MEGA Primer design, PCR amplification and software version 7.0 using published ITS2 sequencing sequences of Centrocestus and other related heterophyids available in the Genbank In this study, we selected published primer set databases by maximum likelihood (ML) as (Van et al., 2009) for amplification of well as neighborhood joining (NJ) method complete ITS2 segment of ribosomal DNA of (Table 1). Centrocestus spp. using genomic DNA extracted from the metacercariae. All the