The incidence of diseases has been increasing along with the global production of catfish in aquaculture. Vogesella perlucida has not been recognized as a fish pathogen. This study characterized the V. perlucida associated with diseased catfish, Clarias gariepinus (Burchell), with phenotypic and molecular methods. The ascites of diseased larvae had Aeromonas spp. and Pseudomonas spp. The translucent strain CGB1, phenotypically identified as Pseudomonas sp., was confirmed as V. perlucida with 99% nucleotide similarity. This strain was negative for gelatinase, amylase, lipase, DNAse, and hemolytic activities. No signs of external infection, behavioral abnormalities, or mortality were observed in catfish larvae when immersed challenged at 6.30 × 107 V. perlucida cells mL−1. The results of preliminary in-vitro virulence and immersion assays suggested that V. perlucida had no pathogenic potential to cause disease in catfish. Although the results provided the basic phenotypic and genotypic data on fish-borne V. perlucida (NCBI accession number KJ522788), establishing a host-pathogen relationship at the molecular level was needed to ascertain what kind of relationship this bacterium had with the diseased catfish. Nevertheless, reports on the increasing incidences of Vogesella spp. and V. perlucida with diseased fish in recent years is cause for concern.

Background: Viral diseases create one of the greatest challenges for the rapidly expanding ornamental fish culture sector. In recent years, the host-specific herpesviruses are receiving much attention due to the intensification in aquaculture globally. Cyprinid herpesvirus-2 (CyHV-2, goldfish hematopoietic necrosis virus), and Cyprinid herpesvirus-3 (CyHV-3, koi herpesvirus) are the lethal pathogens of koi carp Cyprinus carpio koi and goldfish Carassius auratus, respectively. In India, West Bengal is the leading state in ornamental fish production and export. Methods: The surveillance of CyHV-2 and CyHV-3 in koi carp and goldfish cultured in West Bengal was carried out between November 2014 and January 2017 as per Office International des Epizooties guidelines. Results: Both fish species were negative for CyHV-3. The CyHV-2 infection was detected in both gill and kidney tissues of apparently healthy and diseased C. auratus from December 2014 to March 2015. Severe necrosis was observed in the gills of infected C. auratus. Coinfection with members of the bacterial genera Aeromonas and Flavobacterium was also observed in the kidney of infected goldfish. The histopathological observations in the kidney of CyHV-2 infected C. auratus demonstrated the pathogenic potential of CyHV-2 and tissue tropism. Conclusions and Clinical relevance: Environmental stressors like low water temperature and the use of wastewater for culture played a vital role in the onset of CyHV-2 infection in the stressed goldfish. The spread of CyHV-2 can likely pose a certain degree of threat to aquaculture especially the unrestricted movement of goldfish. The results of the present study emphasize the necessity of an organized risk assessment to plan for the management strategies on the control and spread of CyHV-2 in Indian aquaculture.

The global shrimp aquaculture is impacted by episodes of viral diseases resulting in huge income losses. This communication presents the results of the polymerase chain reaction (PCR) based surveillance of white spot virus (WSV) and monodon baculovirus (MBV) in shrimp culture systems of West Bengal, India. The WSV was detected in 14.87% of the total samples (N = 121) by first PCR and 16.53% of samples by nested PCR. The WSV infection was noticed in 12 of 65 Penaeus monodon, 6 of 39 Litopenaeus vannamei and 2 of 11 Macrobrachium rosenbergii samples. The MBV was detected in 8 of 65 P. monodon samples by non-nested PCR and all were also positive for WSV, thus indicating concurrent infection of shrimp. The results emphasized the need to observe strict quarantine measures during the seed selection to prevent the introduction of viral pathogens in grow-out systems.

India is the second largest fish producing nation after China, contributing about 5.68% of the global fish production. The state West Bengal is occupying the second position in freshwater fish production after Andhra Pradesh. Although a number of constraints have been put forth, diseases and poor farm management are some of the most noticeable reasons for the reduced fish production in West Bengal. This study reports the prevalence of diseases caused by Flavobacterium spp. and other opportunistic bacteria in carps of sewage-fed farms in West Bengal. The bacteriological examination of the diseased carps revealed infections with different bacterial species and most of them were opportunistic pathogens. Flavobacteriosis, aeromoniasis, pseudomoniasis and other mixed bacterial infections, as confirmed by conventional biochemical tests and by VITEK 2 Compact system, were noted frequently. Antibiotic sensitivity of the opportunistic bacterial pathogens from the diseased carps indicated that some of these bacterial strains are resistant to potential human medicines like erythromycin, cotrimoxazole, oxytetracycline, nitrofurantoin, etc., which is a serious cause for concern. These results further present the fact that sewage-fed fish farms and the food fish from such systems may pose a serious public health risk from the antibiotic-resistant bacteria, if not handled properly.

The present study reports the first isolation of Chryseobacterium sp. and co-infection with Aeromonas hydrophila in pacu, Piaractus brachypomus fries cultured in West Bengal, India. The infected pacu fries showed white patches on gill, tail rot, skin discoloration, scale loss, skin peeling, emaciation, and kidney inflammation. The yellow pigmented colony from the kidney of pacu on selective cytophaga agar was identified as Chryseobacterium sp. based on colony pigmentation, cell morphology, phenotypic characterization, and 16S rDNA gene sequencing. Phylogenetically, the members of the genus Chryseobacterium clustered together as a separate clade and the strain PLI2 (NCBI accession number KP898212) branched with a non-type strain C. indologenes VIT-CMJ1 (accession number KJ437473) with high node value (100). Yet, the strain PLI2 was distinctly different from closely related type strain C. indologenes LMG 8337 (accession number LN681561) with 97% DNA homology and low (80) node value or other type strains of NCBI GenBank database. The challenge experiments indicated that Chryseobacterium sp. PLI2 can cause substantial mortalities (25%) in abraded and bath (2.2 x 10(7)/mL) treated pacu fries. The kidney of challenged pacu became pale and inflamed. Most likely, this report is a new record on the isolation of Chryseobacterium sp. from the kidney of P. brachypomus.Statement of relevance:Co-infection of Chryseobacterium sp. and A. hydrophila in Piaractus brachypomus caused nephropathy and large scale mortalities.The record of a newbacterial disease Chryseobacterium sp. infection in Indian aquaculture is a serious cause for concern. (C) 2017 Elsevier B.V. All rights reserved.

Aquaculture of tilapia is a new research venture in India. With intensification in farming practices, tilapia are increasingly susceptible to bacterial infections. This article describes the isolation and identification of pathogenic bacteria from cultured monosex Nile tilapia, Oreochromis niloticus (L.), that experienced moderate to severe mortalities in West Bengal, India between September and August 2014 and histopathological alterations in various organs. Gram-positive diplococci, identified as Streptococcus agalactiae with Streptococcus identification kits and 16S rDNA sequencing analysis, were isolated from the brain, operculum, and kidney. Other bacteria from the kidney were identified as Aeromonas sobria, A. caviae, Klebsiella pneumoniae ssp. pneumoniae, Escherichia coli, and Enterobacter cloacae. Staphylococcus epidermis was isolated from opercular hemorrhages. Histological sections of the infected tilapia brain revealed meningoencephalitis and granulomatous lesions. Sections from other organs indicated congestion, hemorrhagic and hyperplastic cells, necrosis, vacuolation, hemosiderin deposition, hypertrophic nuclei, melanomacrophage aggregation, and ruptured veins. This report is the first description of S. agalactiae as a primary pathogen causing meningoencephalitis in cultured tilapia in India.

This outbreak report details of a mortality event where Cyprinid herpes virus-2 (CyHV-2) was detected in association with multidrug-resistant Aeromonas hydrophila infection in goldfish, Carassius auratus, from commercial farms. The goldfish exhibited large scale haemorrhages on the body, fins and gills, lepidorthosis, necrosed gills, protruded anus and shrunken eyes. White nodular necrotic foci in spleen and kidneys were noticed, along with necrosis and fusion of gill lamellae. Transmission electron microscopy of affected tissues revealed the presence of mature virus particles. Involvement of CyHV-2 was confirmed by PCR, sequencing and observed cytopathic effect in koi carp fin cell line along with experimental infection study. A bacterium isolated from the internal organs of affected fish was found to be pathogenic Aeromonas hydrophila having resistance to more than 10 classes of antibiotics. We postulate that CyHV-2 was the primary etiological agent responsible for this outbreak with secondary infection by A. hydrophila. The experimental infection trials in Labeo rohita and koi carp by intraperitoneal challenge with CyHV-2 tissue homogenates failed to reproduce the disease in those co-cultured fish species. This is the first report of a viral disease outbreak in organised earthen ornamental fish farms in India and bears further investigation.

The present study attempted sequencing the 18S rRNA gene of Myxobolus catmrigalae infecting the gill lamellae of carp, Cirrhinus mrigala and compared its genetic homology and phylogenetic characteristics with 18S rRNA genes of other Myxobolus spp. The infected fish had up to 3 small, creamy white plasmodia per gill filament with 30–50 spores each. The spore size was 17.90 ± 0.70 × 7.40 ± 0.40 μm. The sporoplasm contained two large nuclei of size 0.57 ± 0.09 μm and no iodinophilous vacuole. The DNA sequence of M. catmrigalae was clustered phylogenetically with other Myxobolus spp. infecting the gills of cyprinids available in GenBank, which showed 77–87 % homogeneity. On the phylogenetic tree, M. catmrigalae (KC933944) was clustered with M. pavlovskii (HM991164) infecting the gill lamellae of silver carp, Hypophthalmichthys molitrix . The species most closely related to M. catmrigalae in GenBank was M. pavlovskii (AF507973) infecting the gill lamellae of big head carp, Aristichthys nobilis with 87 % homogeneity. This is the first report on molecular characterization of gill lamellae infecting M. catmrigalae.

"Gill rot" caused by the Gram negative, long, rod shaped bacterium Flavobacterium sp. in a sewage fed farm growing Indian major carps in West Bengal, India, is described. The phenotypic and molecular characteristics, virulence and pathology of Flavobacterium sp. strain KG3 are also documented. The infected catla Catla catla had cutaneous haemorrhages, white patches on body and gill, gill rot and excess mucus secretion. On selective cytophaga agar, the isolate produced non-rhizoid yellow pigmented colonies. Phylogenetically, the strain KG3 was distinctly different from the closely related type strains of Flavobacterium species, though it branched with type strain F. hydatis DSM 2063 (NCBI accession number AM230487) with low node value. Histologically, the naturally infected catla gills showed cartilaginous tissue inflammation, mucus secretion, necrosis of gill filament with associated reduction in the number of lamellae per filament, obliteration of interlamellar water channels, and fusion of lamellae. Catla catla fingerlings experienced about 57% moralities within 5 days of abrasion and immersion in a bath containing 4.7 x 10(6) Flavobacterium sp. cells/mt. The abraded and unchallenged fingerlings recorded 20% mortality in 3 days. These results demonstrated the virulence of Flavobacterium sp. in catla especially during the winter season, which warrants its management measures.Statement of relevance: Flavobacterium spp. are emerging as major pathogens of Indian freshwater fish. They can cause chronic mortalities in fish at low water temperature. Correct diagnosis and understanding the pathology would help manage flavobacterial infection in fish. (C) 2016 Elsevier B.V. All rights reserved.

This study describes rapid detection of Flavobacterium columnare induced columnaris disease in cultured freshwater fish, viz., Labeo rohita, Ctenopharyngodon idella, Puntius sp. and Anabas testudineus by species-specific polymerase chain reaction. Gill discolouration, yellow necrotic areas, white patches on gill, saddle back and erosion of scales were the prominent clinical signs in all diseased fish, except Puntius sp., which had typical signs of ulcer at the base of dorsal fin. Of the nine disease cases, eight were found columnaris positive through culture independent species-specific PCR. The two sets of F. columnare specific primers such as ColF, ColR and Col72F, Col1260R yielded amplicons of around 675 bp and 1000 bp, respectively in all positive samples. Phylogenetically, the nucleotide sequences of the positive samples namely, C1 and RG1 formed monophyletic group with F. columnare, thus confirmed the infection as columnaris.

Indian catfish production is increasing over the years so as the frequency of diseases. In this report, the isolation of Stenotrophomonas maltophilia from the kidney of Clarias gariepinus juvenile with distended abdomen along with its phenotypic, molecular and phylogenetic characteristics was described. The diseased catfish juveniles in an earthen pond (1500 m(3)) with high stocking density (approximate to 60 nos/m(3)) had body discoloration, fin/tail rot, focal cutaneous hemorrhage, distended abdomen, inflammation of internal organs and visceral hemorrhage. The abdominal cavity was filled with transparent jelly-like mass. The bacterial strain isolated from the kidney of catfish formed amonophyletic group with other strains of S. maltophilia based on genotypic characters and phylogenetic analyses. The strain S. maltophilia SK1 (NCBI accession number KP898215) was alpha-hemolytic, protease and lipase positive and weakly pathogenic to C. gariepinus juveniles. It caused only 35 +/- 5% mortality at a level of 1.40 x 10(7) cells/fish via intraperitonial injection. While the intramuscular injection caused only 10% mortality. The dead fish up on challenge had distended abdomen with jelly-like mass. Understanding the pathology and pathogenesis of this emerging pathogen in cultured catfish would help manage the disease. (C) 2015 Elsevier B.V. All rights reserved.

The present study characterized Argulus spp. infecting the cultured carps using 18S rRNA gene sequences, estimated the genetic similarity among Argulus spp. and established their phylogenetic relationship. Of the 320 fish samples screened, 34 fish (10.6%) had Argulus infection. The parasitic frequency index (PFI) was observed to be high (20%) in Hypophthalmichthys molitrix and Labeo bata. The frequency of infection was high in September (PFI: 17%) and October (PFI: 12.9%). The 18S rRNA sequences of five A. bengalensis (KF583878, KF192316, KM016968, KM016969, and KM016970) and one A. siamensis (KF583879) of this study showed genetic heterogeneity and exhibited 77-99% homology among the 18S rRNA gene sequences of Argulus spp. of NCBI GenBank database. Among the Indian Argulus spp. the sequence homology was 87-100%. Evolutionary pair-wise distances between Indian Argulus spp. and other Argulus spp. ranged from 0 to 20.20%. In the phylogenetic tree, all the crustaceans were clustered together as a separate clade with two distinct lineages. The lineage-1 comprised exclusive of Branchiura (Argulus spp.). All Argulus bengalensis clustered together and A. siamensis (KF583879) was closely related to Argulus sp. JN558648. The results of the present study provided baseline data for future work on population structure analysis of Indian Argulus species.

Isolation of Flavobacterium sp. from the gill of telescopic eye goldfish Carassius auratus with white patches on gill along with its phenotypic, molecular and phylogenetic characteristics are described in this report. The diseased goldfish from a cemented tank (6.75 m3) had gill rot, white patches on gill with excess mucus secretion. On selective cytophaga agar, inocula from the gills yielded yellow pigmented colonies. A bacterial strain (WPGT1) isolated from the gill of goldfish formed a monophyletic group with other strains of Flavobacterium sp. based on phylogenetic analyses. The strain Flavobacterium sp. WPGT1 (NCBI accession number KP997191) was Gram negative long rod and weakly pathogenic to C. auratus. It caused only 14.29% mortality at a level of 3.50x106 cells/ml through immersion challenge after skin wounding; while no mortality was recorded in intraperitonially injected goldfish even at 3.50x108 cfu/fish. Understanding the pathology and pathogenesis of this emerging pathogen in cultured goldfish would help manage flavobacteriosis.

Myxozoans are an economically important group of microscopic parasites best known for the diseases they cause in commercially important fish hosts. The classification of myxosporeans is generally based on the morphology of their myxospores. Without molecular data, it is very difficult to identify new or existing species. DNA sequence information is therefore, a prerequisite to taxonomic and phylogenic studies of myxosporeans. In the present study, a myxozoan parasite, Myxobolus carnaticus, infecting the gill lamellae of mrigal carp, Cirrhinus mrigala, was characterized by the 18S rRNA gene sequence. The DNA sequence of M. carnaticus clustered phylogenetically with other gill infecting Myxobolus spp. of freshwater clades, forming a dichotomy with closely related M. pavlovskii (HM991164) that infects the gill lamellae epithelium of silver carp, Hypophthalmichthys molitrix with 95% similarity. Evolutionary pair-wise distances among M. carnaticus and other species of myxosporeans indicated high genetic diversity among myxosporeans. The present study demonstrated that tissue tropism, host specificity and habitat play important roles in phylogenetic relationships among myxozoan species.

A new bacterial disease (flectobacillosis) caused by Gram negative, long rod shaped Flectobacillus roseus in captive held carp, rohu (Labeo rohita) in West Bengal, India is described. Besides the phenotypic and molecular characterization, histopathology of F. roseus of naturally infected and challenged rohu were studied. The affected rohu had loss of mucus and scale, discoloured body, peeled skin and rotten tail, but no signs of gill damage. On selective cytophaga agar, F. roseus produced pink to rose pigmented colonies. Phylogenetically, F. roseus RF1 formed a monophyletic branch with F. roseus EU921645 (96% sequence similarity) and Flectobacillus sp. KC588923 (94% sequence similarity). It caused significant mortalities in rohu when challenged. The naturally infected rohu showed epithelial disruption, inflammation and necrosis of skeletal muscle tissue accompanied by haemocyte infiltration. The kidney of challenged rohu showed proximal and distal tubule degeneration, necrosis, thickening of epithelial layer of proximal and distal tubules, necrotised haematopoietic tissue, nephritic tubule with widened lumen, vacuolation of tubular epithelium, glomerular congestion, desquamation, glomerulopathy with dilated Bowman's space, cellular and nuclear hyperprophy, pycnotic nuclei, melano-macrophage aggregates and granuloma-like inflammatory response. These results suggest that F. roseus can produce systemic pathology in kidney similar to many other known fish pathogens.

Myxosporeans are best known for the diseases they cause in commercially important fish species. Identification of myxosporeans at the species-level is mainly based on conventional methods. The 18S rRNA gene sequence of morphologically identified Myxobolus orissae infecting the gill lamellae of mrigal carp Cirrhinus mrigala was characterized in the present study. The plasmodia of M. orissae were small, elongated and white to pale in colour. Phylogenetically, the 18S rDNA nucleotide sequence of M. orissae was clustered with other gill-infecting Myxobolus spp. of cyprinids. The species closely related to M. orissae was M. koi (FJ841887) infecting the gill lamellae of Cyprinus carpio with 96% similarity. The carp fin-infecting Thelohanellus caudatus (KC865607) from India exhibited only 78% DNA sequence similarity with M. orissae. Low level of M. orissae infection on gill caused thickening of epithelial cells surrounding the plasmodium. Under stressful conditions, it is likely that such infection can easily spread in confined fish and may cause serious disease outbreaks and economical losses.

Myxosporean taxonomy which is traditionally based on the morphology of the myxospore stage, is in a state of flux given new insights provided by the expanding dataset of DNA sequences. To date, more than 40 species of Thelohanellus from India have been described according to morphometric characteristics. Nevertheless, molecular data on these histozoic myxosporean parasites of freshwater fish are scarce. In the present study, molecular characterizations of Thelohanellus qadrii infecting the secondary gill epithelium of Indian major carp Catla catla (Hamilton, 1822) and its phylogenetic relationship is reported. The sub-adult cultured catla were observed to have low to moderate gill myxosporean infections. The morphometry of mature spores was in compliance with original descriptions of T. qadrii. Based on the analysis of 18S rRNA gene, phylogenetic clusters which were established according to a consensus sequence, illustrated the taxonomic placement of a series of myxobolids. The DNA sequence homogeneity of T. qadrii (KF170928) with other Thelohanllus spp. ranged from 78% to 95% and formed a dichotomy with cyprinid gill lamellae infecting T. toyamai (HQ338729). Distance matrix results indicated a high genetic diversity among myxosporeans. The present report is the first on the molecular and phylogenetic characterizations of T. qadrii.

As new pathogenic strains are emerging and threatening aquaculture development, myxosporeans (Myxozoa) are receiving much attention in recent years. Myxosporean taxonomy is traditionally based on morphology of the myxospore stage. Molecular data on Indian myxosporeans are rare. In this report, the 18S rRNA gene sequence of Myxobolus cuttacki infecting gill lamellae of minor carp Labeo bata (Ham.) and its phylogenetic relationship with other myxobolids are described for the first time. The plasmodia of M. cuttacki were 0.5-0.9 mm in size and whitish with a round to oval shape. The mean mature spore size was 16.10×7.05 μm. The 18S rRNA nucleotide sequence with 1703 bp of M. cuttacki (accession number KF465682) clustered phylogenetically with other Myxobolus spp. infecting cyprinid gills with 78-90% homogeneity. The gill lamellae infecting M. catmrigalae (KC933944) and M. orissae (KF448527) of Indian major carp Cirrhinus mrigala from India, exhibited 86% and 81% homogeneity with M. cuttacki, respectively. The infection rate was low to moderate on the gills which can have a negative impact on respiratory and physiological functions and subsequently on fish production.

To study the association of Aeromonas salmonicida subsp. achromogenes in the haemorrhagic blister of cultured exotic carp Cyprinus carpio in West Bengal, India, its cellular fatty acid composition and antibiotic sensitivity. The phenotypic characterization and antibiotic sensitivity of bacterial flora of diseased fish were performed. The whole cellular fatty acid composition of Aeromonas salmonicida (A. salmonicida) was determined by MIS Sherlock automatic identification system. Aeromonas hydrophila, A. salmonicida and Pseudomonas alcaligenes were isolated from the haemorrhagic blister. The fatty acid methyl esters pattern confirmed the A. salmonicida strain as A. salmonicida subsp. achromogenes. Unsaturated fatty acid 16:1 w7c/16:1 w6c (39.09%), saturated fatty acid 16:0 (26.84%) and the mono-unsaturated fatty acids 18:1 w7c (8.89%) and 16:1 iso I/14:0 3OH (8.49%) were the most common fatty acids, which accounted for 83.31% of the total fatty acids. A. salmonicida subsp. achromogenes was highly susceptible to broad spectrum antibiotics such as chloramphenicol, ciprofloxacin, co-trimoxazole, gentamycin, nitrofurantoin and oxytetracycline. The finding on the presence of A. salmonicida in carps necessitates molecular level study on establishing the prevalence of this bacterium in Indian aquaculture systems especially on its free-living viable but non-culturable state.