Development of a novel multiplex-PCR technology for simultaneous detection of five major aquaculture pathogens

379-388Early and precise pathogen identification and corresponding disease management are primary concerns in aquaculture. Here, we attempted at diagnostic methods that can simultaneously identify multiple pathogens, where many samples, several pathogens, and concurrent infections are to be handled. Hence, a multiplex PCR assay targeting five major aquaculture pathogens, viz. Vibrio parahaemolyticus, Vibrio anguillarum, Vibrio alginolyticus, Vibrio vulnificus and Vibrio harveyi was developed for the first time. The primers targeting toxR of V. parahaemolyticus, amiB of V. anguillarum, col of V. alginolyticus, vvhA of V. vulnificus, and topA of V. harveyi were applied. Furthermore, the reaction included an internal amplification control against prokaryotic 16S rRNA to perceive false-negative results. The assay showed 100% specificity against 56 unique bacteria. The sensitivity was 0.25 ng for V. harveyi, 0.5 ng for V. vulnificus, 1 ng for V. parahaemolyticus and V. anguillarum, and 2 ng for V. alginolyticus DNA per μL assay. Sensitivity regarding CFU was 1.2, 5.2, 10, 5.6×101 and 3.8×102 per μL, for V. harveyi, V. vulnificus, V. anguillarum, V. parahaemolyticus and V. alginolyticus, respectively. The results suggest that the optimized method can be applied for sensitive and specific identification of five aquaculture pathogens through a single PCR

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Not AvailableThe present study reports a case of hepatic microsporidiosis caused by Microgemma sp in brackishwater sh, Boleophthalmus dussumieri (Valenciennes, 1837) (n = 60), from north-west coast of India. An eightmonth study from September 2017 to April 2018 revealed a prevalence of 11.6% for this parasite. The microsporidian showed tissue-specic infection and did not reveal any gross pathology in infected sh. Large whitish cysts containing microspores of size 0.3–0.5 mm were observed in the liver of sh. The range of pyriform microsporidian spore size varied from 2.9–3.77 X 1.85–2.67 µm. Histological observations of infected liver revealed large xenoma of the microsporidian lled with spores and encircled by a cyst wall-like layer. Scanning electron microscopy of the spores showed a distinct groove on the anterior end of the spore for polar tube extrusion. Polymerase chain reaction (PCR) amplication of the DNA extracted from the microsporidian spores using primers targeting small ribosomal subunit DNA (SSU rDNA) yielded ~ 1340 bp amplicon and the genetic distance analysis showed a 0.2% variation with the reported M. tilanpasiri. Accordingly, in the phylogenetic tree, the present species of Microgemma clustered with M. tilanpasiri. Even though, the morphomeristic characters of the present Microgemma sp. was marginally different from the reported M. tilanpsasiri; the SSU rDNA showed considerably higher similarity with M. tilanpasiri. Thus, we report the species of Microgemma as Microgemma aff. tilanpasiri from a new host. This is the rst report of a microsporidian from B. dussumieri and the rst record of the genus Microgemma from India.Not Availabl

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Not AvailableA novel myxozoan parasite is identified and described from mudskipper, Boleophthalmus dussumieri, collected from a brackishwater ecosystem in Maharashtra, India. Ellipsomyxa boleophthalmi sp. nov. was found in the gallbladder of 58 of 60 fish examined (96.7%). The parasite formed disporous plasmodia that varied in size and shape, and the thin-walled, ellipsoidal and elongated myxospores measured 9.0–10.7 × 6.0–7.8 μm. The two, spherical polar capsules measured 2.7 μm in diameter and enclosed 3–4 coils of polar tubules. Histological observations of infected gallbladder revealed the attachment of disporous plasmodial stages of the parasite to the gallbladder wall with fine pseudopodia. Under the scanning electron microscope (SEM), the myxospores showed a distinct central sutural line and two distinct depressions on the opposite sides at the openings of polar capsules. SEM also revealed the engulfment of microvilli of gallbladder wall by pseudopodia of the plasmodial stages. Analysis of the partial fragment of the SSU rDNA region (1386 bp) showed less than 98% sequence similarity with the other reported Ellipsomyxa spp. In the phylogenetic tree, the present species formed as a distinct subclade within the major clade of Ellipsomyxa spp. The unique morphological and morphometric features of the myxospore, together with the molecular analysis, allowed us to conclude that the present myxozoan is a new species and is named Ellipsomyxa boleophthalmi sp. nov., after the generic name of the host. This is the first report on the occurrence of the genus Ellipsomyxa in B. dussumieri. Introduction Myxozoans are the most abundant, obligate, microscopiNot Availabl

Furcocercous cercariae infecting freshwater snails in Malabar: two new species from Lymnea luteola Lamarck and Gyraulus convexiusculus (Hutton)

The present paper describes two new species of furcocercous cercariae, Cercaria sp. XVIII Malabar n. sp. and Cercaria sp. XIX Malabar n. sp. infecting the freshwater snails, Lymnea luteola and Gyraulus convexiusculus respectively in the Malabar region of Kerala. Cercaria sp. XVIII Malabar n. sp., is a distome, pharyngeate, nonocellate, longifurcate furcocercous cercaria with two pairs of penetration glands and 16 pairs of flame cells and recovered from L. luteola. Cercaria sp. XIX Malabar n. sp., is also a distome, pharyngeate, longifurcate cercaria with a pair of unpigmented eyespots and 20 pairs of flame cells and recovered from G. convexiusculus. Sporocysts of both the cercariae developed in the digestive glands. The present paper describes the cercariae and compares them with related species to establish their systematic position