Bikash Ranjan Mohanty

Expression of immune-related genes in rohu Labeo rohita (Hamilton) by experimental freshwater lice Argulus siamensis (Wilson) infection.

The crustacean ectoparasite, Argulus poses one of the major threats to carp culture due to absence of any suitable control measure. The study was undertaken to determine the expression of immune-related genes in three major immunocompetent organs viz., kidney, skin and liver of rohu (Labeo rohita) during experimental freshwater lice Argulus siamensis infection. Results showed that the expression of TLR 22-like, lysozyme G and β2-microglobulin genes in kidney was significantly (P ≤ 0.05) down-regulated in lice-infected fish. On the other hand, no significant difference (P>0.05) in CXCa, lysozyme C, TNFα and complement component 3 (C3) expression was found between uninfected control and different degrees of lice infected fish. In the skin, the expression of TLR 22-like and TNFα genes were significantly up-regulated whereas that of C3 was significantly (P ≤ 0.05) down-regulated in lice-infected fish with respect to control fish. The expression of CXCa, lysozyme C and transferrin was not detected in the skin samples of fish. In the liver, the expression of CXCa, lysozyme G, β2-microglobulin and transferrin was significantly (P≤0.05) up-regulated in lice-infected fish with respect to control fish whereas expression of C3 was significantly (P ≤ 0.05) down-regulated in lice-infected fish. The expression of TLR 22-like, lysozyme C, TNFα genes was not detected in the liver samples of fish. This study indicates that majority of the genes showed down-regulation in kidney tissue whereas up-regulation in liver and skin tissues except C3 in Argulus-infected fish. We show that infection with this parasite irrespective of intensity can also result in immune gene expression changes in tissues situated away from the site of parasite attachment and feeding. The information obtained here could be useful towards understanding the susceptibility of rohu to argulosis and mechanisms involved in protection of rohu to ectoparasitic infections, which is causing immense economic losses to freshwater aquaculture sector.

Ontogeny and tissue-specific expression of innate immune related genes in rohu, Labeo rohita (Hamilton).

The innate immune response in fish represents an early and rapid defense against pathogens. The present study aims at looking into ontogeny of innate immune system in the teleost, Labeo rohita using RT-PCR based approach. Total RNA extracted from unfertilized and fertilized eggs, and hatchlings (hatched at 28 ± 2 °C) at 0, 1, 3, 6, 12, 24 h, and 3, 7, 16, 21, 31 days post-fertilization were subjected to RT-PCR using self-designed or earlier published primers to amplify some innate immune relevant genes (lysozyme C, lysozyme G, beta-2 microglobulin, toll-like receptor 22-like and transferrin). The constitutive expression of β-actin was detected in unfertilized eggs and further developmental stages. Transferrin and TLR22-like mRNA transcripts were detected by RT-PCR from 6 h post-fertilization to 31 day post-fertilization, whereas β-2 microglobulin transcripts were detected only from 7 day post-fertilization onwards. Lysozyme C mRNA transcripts were detected from 24 h post-fertilization to 31 day post-fertilization. Lysozyme G mRNA transcripts were detected early from unfertilized egg stage onwards. Similarly, tissues viz. intestine, heart, ovary, gill, spleen, muscle, liver, brain, skin, anterior kidney, posterior kidney, and blood collected from juveniles of rohu were subjected to detection of all above mentioned gene transcripts by RT-PCR. β2-microglobulin mRNA transcript was expressed in all tissues. Lysozyme C mRNA expression is confined to blood and posterior kidney only whereas lysozyme G mRNA is expressed in all tissues. TLR22-like mRNA is expressed in all tissues except ovary and liver whereas transferrin mRNA transcript is detected only in liver. Finally, all these information taken are likely to shed light on the ontogeny of innate immunity in L. rohita, which offers new insights to developmental biology when compared to higher vertebrates and also helpful in the development of preventive measures against problems concerning infectious diseases.

Pathophysiology of experimental Aeromonas hydrophila infection in Puntius sarana: early changes in blood and aspects of the innate immune-related gene expression in survivors.

A decline in production of Puntius sarana worldwide and subsequent intensification of its culture as a measure for conservation and increase food fish production have led to emergence of diseases. Investigations on innate immunological changes in fish that survives an acute bacterial infection are a few or lacking. An attempt was made to study the detailed changes in innate immune responses in P. sarana following an intraperitoneal challenge with Aeromonas hydrophila at 2.24 × 10(7)colony-forming unit (CFU)/fish. Sequential changes in transcription of immune related genes in liver and kidney, innate immune status, haematological profile and histopathology of major organs were studied after different time intervals post-challenge in the survivors. A significant increase in serum myeloperoxidase, ceruloplasmin activities and total leucocyte count, and decline in alternative complement activity were observed at early infection periods. A significant decline in plasma glucose level, total erythrocyte count, haemoglobin content and packed cell volume in blood was noticed after challenge. Superoxide radical production and serum antiprotease activity increased significantly at later period of challenge. In addition, few immune related genes pertinent to acute phase reactants and inflammatory response viz., complement component 3 (C3), lysozyme G, lysozyme C, β2-microglobulin (B2M), transferrin, interleukin 1β, interleukin 8, manganese superoxide dismutase (MnSOD), natural killer cell enhancing factor-B and chemokine CXCa were modulated after bacterial challenge. In liver, a significant up-regulation of C3 and transferrin, and down-regulation of lysozyme G, interleukin 8, MnSOD and B2M were noticed in survivors. On the contrary, lysozyme G, lysozyme C, interleukin 1β, interleukin 8, CXCa, and MnSOD were found to be significantly up-regulated and C3 was down-regulated in the kidney. All these data suggest that the innate immune factors were highly modulated during infection process to render protection of P. sarana to this bacterium. This study also generated the primary information on many innate immune molecules/genes for this important medium carp species for the first time.

Cavernous haemangioma in cultured olive barb, Puntius sarana (Hamilton)

Neoplasms of fish are classified similarly to those of higher animals (Hochwartner et al. 2010). The primary neoplasms of the skin of cultured fish are uncommon (Schoniger et al. 2008). Haemangiomas are benign tumours of vascular endothelial cells or their progenitors. In humans, they are most frequently located in the skin, but have also been also described in other tissues and organs (Weiss & Goldblum 2001). These tumours are common in dogs and rare in cats, horses and other domestic animals (Goldschmidt & Hendrick 2002). They may be classified histologically as capillary, cavernous, arteriovenous, lobular, spindle cell or epithelioid subtypes (Vos et al. 1986; Calonje & Fletcher 2007; Gross et al. 2007; Warren & Summers 2007). Tumours of vascular origin have also been reported from a number of fish species (Fournie, Overstreet & Bullock 1985). Rare occurrences of capillary, cavernous haemangiomas, haemangioendotheliomas and haemangio-endotheliosarcomas affecting integument and visceral organs have been recorded in fish from the USA by Harshbarger (1965). Microscopically, haemangiomas are characterized by proliferating, vasoformative mesenchymal tissue forming capillary and cavernous vessels and, less often, arterial and venous structures. Endothelial cells in these tumours may be surrounded by pericytes and fibroblasts (Calonje & Fletcher 2007; Gross et al. 2007). Cavernous haemangioma is a frequent benign vascular neoplasm that can occur in multiple tissues in humans and animals including fish (Harshbarger 1965–1981; Goldschmidt & Hendrick 2002; Zhang et al. 2009). Cavernous haemangioma consists of vascular malformation, that is, the vascular walls with no basement membranes and rare pericytes, singlelayer lining endothelial cells (ECs) with fenestrated luminal surfaces and large gaps at intercellular junctions between ECs (Tu et al. 2005). This communication reports cases of multiple cavernous haemangiomas from the olive barb, Puntius sarana (Hamilton). The first case observed was in a 2-year-old, pondcultured Puntius sarana, which developed mediumsized, exophytic dermal masses at the base of the pelvic fin and caudal fin. The masses (1–4 cm size) were dark red and fluctuant on palpation. Subsequently, similar lesions were observed in three specimens out of a total of 250 one-year-old fish, maintained in a wet laboratory for experimental purpose. The lesions were observed as small nodular lesions at the base of the fin and operculum. Following physical examination, the fish were killed by overdose of anaesthesia (MS222). A complete post-mortem examination was carried out in all cases. All tumours were well-demarcated, multilobular reddish brown masses mostly occurring at the base of the fins and lower jaw (Fig. 1). The tumours were soft, friable and contained a small amount of sero-sanguinous fluid. The excised tumour masses were removed, fixed in 10% neutral buffered formalin and processed for Journal of Fish Diseases 2012 doi:10.1111/j.1365-2761.2012.01370.x