White Tail Research Articles

Genetic analysis of RNA1 and RNA2 of Macrobrachium rosenbergii nodavirus (MrNV) isolated from India

Macrobrachium rosenbergii nodavirus (MrNV) is responsible for the newly emerging catastrophic disease known as white tail disease (WTD) in M. rosenbergii. The complete sequence of RNA2 (1175 bp) and 3126 bp region of RNA1 of an Indian strain of MrNV was generated. Sequence analysis of RNA2 revealed the presence of a single ORF encoding a capsid protein of 371 amino acids with a predicted molecular mass and pI of 41.5 kDa and 8.97 respectively. RNA1 contained two ORFs, one encoding a partial RNA dependent RNA polymerase (RdRp) of length 1034 amino acids and another a B2-like protein with a length 133 amino acids. A phylogenetic analysis based on the amino acid sequence of the capsid protein, to related nodavirus sequences suggests the establishment of new genotypes within the Nodaviridae family and we suggest the name should be genus Gammanodavirus. A new reverse transcriptase-polymerase chain reaction (RT-PCR) assay has been developed and optimized for the detection of shrimp nodavirus with a sensitivity to detect up to 24 copy numbers of plasmid construct.

Non‐permissive C6/36 cell culture for the Australian isolate of Macrobrachium rosenbergii nodavirus

Macrobrachium rosenbergii nodavirus (MrNV) that causes white tail disease (WTD) is an emerging disease that contributes to serious production losses in Macrobrachium hatcheries worldwide. Mosquito cell lines (C6/36) have been reported to support the growth of MrNV and used to observe the cytopathic effects (CPE) in infected cells. This study determined the susceptibility of C6/36 mosquito cells to the Australian isolate of MrNV in order to use fewer animals in further investigations. Different staining methods were used to observe MrNV viral activity in C6/36 cells. Typical cytopathic effects such as vacuolation and viral inclusion bodies were observed in infected C6/36 cells with H&E and Giemsa staining. With acridine orange, it was easier to detect presumptive MrNV messenger ribonucleic acid in the infected cells. Using neutral red staining to measure mitochondrial activity showed light absorption of infected cells maximized at day 4 (O.D.=0.6) but was significantly lower (chi-square=41.265, df=1, P<0.05) than control groups (O.D.= 2) which maximized at day 12. Using trypan blue staining to count the number of cells with disrupted cell membranes, the maximum number of presumptively dead cells at day 8 (4×10(5) cells) in infected treatments was higher than the control treatment at day 10 (1.8×10(5) cells). However, TaqMan real-time PCR did not confirm the replication of MrNV in the cells over 14days. The mean viral copies and mean cycle times of positive samples were stable at 2.07×10(4) and 24.12, respectively. Limited evidence of viral replication was observed during four serial passages. This study determined the mortality of the C6/36 cell line to the Australian isolate of MrNV but suggests limited patent replication was occurring. Trying different cell lines or adapting the virus to the C6/36 cells may be necessary to successfully replicate Australian MrNV in cell lines.

Divergent tail and throat ornamentation in the barn swallow across the Japanese islands

AbstractThe geographic variations in male ornamentation provide insights into how different populations reach a different mean trait value under opposing forces of natural and sexual selection. Although the latitudinal cline of the elongated tail streamer, a sexually selected trait in the European subspecies of the barn swallowHirundo rustica rustica, is a classic example, it has recently been shown that other subspecies of swallows have different targets of sexual selection. Here, we studied the latitudinal cline of ornamentation in the Asian subspecies,H.r.gutturalis, in which not the tail length but the white tail spot and red throat patch are important sexually selected traits. After controlling for covariates, the size of the white tail spot increased with latitude, while the size of the red throat patch decreased with latitude. On the other hand, we could not find any clear pattern regarding the elongated tail streamer, measured as fork depth. The divergent ornamentation across populations could be explained by latitudinal clines of sexually selected advantages of each ornament.

The importance of visual markings in night birds. The case of the Eagle Owl, Bubo bubo

This work brings together the results of a series of researches on the importance of the visual communication for nocturnal birds, carried out using the Eagle Owl as a model species. We analysed the main characteristics of the white feathers of the adults and the offspring and we tested the function of the achromatic plumage in territorial behaviour and parental care. Furthermore, by means of radio-tracking, we monitored the hunting activity of the Eagle Owl and, by analysing the white tail of its main prey - the Rabbit - we highlighted the possible role of the visual communication for nocturnal birds also in the contest of the predator-prey interactions.

Production and Application of Polyclonal Antibodies Against Recombinant Capsid Protein of Extra Small Virus of Macrobrachium rosenbergii

Macrobrachium rosenbergii nodavirus along with a satellite virus, extra small virus (XSV) causes white tail disease (WTD) in the giant freshwater prawn M. rosenbergii. Infected M. rosenbergii postlarvae were collected from a hatchery in Kakinada, Andhra Pradesh. The gene coding the capsid protein of XSV was cloned in a bacterial expression vector pRSET A and the recombinant protein was expressed in Escherichia coli BL21(DE3)pLysS cells. The recombinant protein was purified by Nickel affinity chromatography. Polyclonal antibodies were produced in mice against the recombinant protein and the antibodies reacted specifically with the recombinant protein and XSV in WTD-infected tissues. This is the first report of detection of XSV using antibodies against recombinant capsid protein.

The complete sequence of the Australia recognizate of Macrobrachium rosenbergii nodavirus which causes white tail disease

White tail disease (WTD) caused by Macrobrachium rosenbergii nodavirus (MrNV) has been found in the giant freshwater prawn (Macrobrachium rosenbergii) and has recently been the cause of high mortalities in many countries such as India, China, Taiwan and Thailand. In mid 2004, the index case of WTD in Australia presented in adult broodstock M. rosenbergii from Flinders River in western Queensland. In order to understand the phylogenetic relationship of the Australian recognizate of MrNV to other MrNV recognizates, the complete sequences of the Australian MrNV (RNA1 and RNA2) were determined. Nucleotide and phylogenetic analysis revealed that the Australian strain of MrNV (RNA1) was between 94% and 97% identical to Malaysian, the French West Indies, Chinese and the Thai recognizates. Also, the nucleotide sequence of the Australian MrNV (RNA2) was 92% identical to the French West Indies, Chinese and the Thai recognizates. The nucleotide comparison of protein B2 showed a different relationship between MrNV and fish nodaviruses. However, the phylogenetic tree of protein B2 determined that some fish nodaviruses are closely related to insect nodavirus. These results can be used to develop effective diagnostic tests and design specific RNA interference (RNAi) against protein B2 to control other nodaviruses in the future.

White Tail Disease of Freshwater Prawn, Macrobrachium rosenbergii

Macrobrachium rosenbergii is the most important cultured freshwater prawn in the world and it is now farmed on a large scale in many countries. Generally, freshwater prawn is considered to be tolerant to diseases but a disease of viral origin is responsible for severe mortalities in larval, post-larval and juvenile stages of prawn. This viral infection namely white tail disease (WTD) was reported in the island of Guadeloupe in 1995 and later in Martinique (FrenchWest Indies) in Taiwan, the People's Republic of China, India, Thailand, Australia and Malaysia. Two viruses, Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus-like particle (XSV) have been identified as causative agents of WTD. MrNV is a small icosahedral non-enveloped particle, 26-27nm in diameter, identified in the cytoplasm of connective cells. XSV is also an icosahedral virus and 15nm in diameter. Clinical signs observed in the infected animals include lethargy, opaqueness of the abdominal muscle, degeneration of the telson and uropods, and up to 100% within 4days. The available diagnostic methods to detect WTD include RT-PCR, dot-blot hybridization, in situ hybridization and ELISA. In experimental infection, these viruses caused 100% mortality in post-larvae but failed to cause mortality in adult prawns. The reported hosts for these viruses include marine shrimp, Artemia and aquatic insects. Experiments were carried out to determine the possibility of vertical transmission of MrNV and XSV in M. rosenbergii. The results indicate that WTD may be transferred from infected brooders to their offspring during spawning. Replication of MrNV and XSV was investigated in apparently healthy C6/36 Aedes albopictus and SSN-1 cell lines. The results revealed that C6/36 and SSN-1cells were susceptible to these viruses. No work has been carried out on control and prevention of WTD and dsRNA against protein B2 produced RNAi that was able to functionally prevent and reduce mortality in WTD-infected redclaw crayfish.

Monoclonal antibodies against extra small virus show that it co-localizes with Macrobrachium rosenbergii nodavirus

The capsid protein (CP) gene of extra small virus (XSV) expressed in Escherichia coli as a 42 kDa glutathione S-transferase (GST)-fusion protein (GST-XCP) or a 20 kDa His6-fusion protein (His6-XCP) were purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), combined, and used to immunize Swiss mice to produce monoclonal antibodies (MAbs). Using dot blot, Western blot, and immunohistochemistry (IHC) methods, 4 MAbs specific to the XSV CP detected XSV in the freshwater prawn Macrobrachium rosenbergii without cross-reaction to host proteins or to proteins of Macrobrachium rosenbergii nodavirus (MrNV) or 5 of the most pathogenic viruses of penaeid shrimp. In dot blots, the combined MAbs could detect down to ~10 to 20 fmol µl-1 of purified GST-XCP protein, which was somewhat more sensitive compared to any single MAb. Used in conjunction with an MrNV-specific MAb, white tail disease (WTD) was diagnosed more effectively. However, the sensitivity at which the combined 4 MAbs detected XSV CP was 1000-fold lower than XSV RNA detected by RT-PCR. IHC analysis of M. rosenbergii tissue sections using the MAbs showed XSV infection to co-localize at variable loads with MrNV infection in heart and muscle cells as well as cells of connective tissues in the hepatopancreas. Since XSV histopathology remained prominent in tissues of some prawns in which MAb reactivity for MrNV was low compared to MAb reactivity for XSV, XSV might play some role in WTD severity.

A review on the diseases of freshwater prawns with special focus on white tail disease of Macrobrachium rosenbergii

Freshwater prawns, particularly, the Giant Freshwater prawn Macrobrachium rosenbergii, are one of the most economically important farmed species in the world. Faced with increasing disease problems in penaeid shrimp culture, farmers turned to freshwater prawn farming. Freshwater prawns were considered relatively less susceptible to diseases. However, with intensification of culture and increased world trade of the farmed species, emerging diseases are beginning to constitute an increasingly serious health problem in freshwater prawn culture. This article is a review of the important diseases reported in freshwater prawns, with particular emphasis on the white tail disease of M. rosenbergii as it is the most important disease recorded to date and also the most well studied among the diseases of freshwater prawns. Steps to be taken for proper health management in the farming of this species is also touched upon.

A Nonluminescent and Highly Virulent Vibrio harveyi Strain Is Associated with “Bacterial White Tail Disease” of Litopenaeus vannamei Shrimp

Recurrent outbreaks of a disease in pond-cultured juvenile and subadult Litopenaeus vannamei shrimp in several districts in China remain an important problem in recent years. The disease was characterized by “white tail” and generally accompanied by mass mortalities. Based on data from the microscopical analyses, PCR detection and 16S rRNA sequencing, a new Vibrio harveyi strain (designated as strain HLB0905) was identified as the etiologic pathogen. The bacterial isolation and challenge tests demonstrated that the HLB0905 strain was nonluminescent but highly virulent. It could cause mass mortality in affected shrimp during a short time period with a low dose of infection. Meanwhile, the histopathological and electron microscopical analysis both showed that the HLB0905 strain could cause severe fiber cell damages and striated muscle necrosis by accumulating in the tail muscle of L. vannamei shrimp, which led the affected shrimp to exhibit white or opaque lesions in the tail. The typical sign was closely similar to that caused by infectious myonecrosis (IMN), white tail disease (WTD) or penaeid white tail disease (PWTD). To differentiate from such diseases as with a sign of “white tail” but of non-bacterial origin, the present disease was named as “bacterial white tail disease (BWTD)”. Present study revealed that, just like IMN and WTD, BWTD could also cause mass mortalities in pond-cultured shrimp. These results suggested that some bacterial strains are changing themselves from secondary to primary pathogens by enhancing their virulence in current shrimp aquaculture system.

Detection and phylogenetic profiling of nodavirus associated with white tail disease in Malaysian Macrobrachium rosenbergii de Man

White tail disease (WTD) is a serious viral disease in the hatcheries and nursery ponds of Macrobrachium rosenbergii in many parts of the world. A new disease similar to WTD was observed in larvae and post larvae of M. rosenbergii cultured in Malaysia. In the present study, RT-PCR assay was used to detect the causative agents of WTD, M. rosenbergii nodavirus (MrNV) and extra small virus (XSV) using specific primers for MrNV RNA2 and XSV. The results showed the presence of MrNV in the samples with or without signs of WTD. However, XSV was only detected in some of the MrNV-positive samples. Phylogenetic analysis showed that the RNA2 of our Malaysian isolates were significantly different from the other isolates. Histopathological studies revealed myofiber degeneration of the tail muscles and liquefactive myopathy in the infected prawns. This was the first report on the occurrence of MrNV in the Malaysian freshwater prawn.

B2 or not B2: RNA interference reduces Macrobrachium rosenbergii nodavirus replication in redclaw crayfish ( Cherax quadricarinatus)

Macrobrachium rosenbergii nodavirus (MrNV) which causes white tail disease (WTD) is one of the most serious viral infections in M. rosenbergii. In less than one week mortality has been reported in hatcheries. RNA interference (RNAi) is an innate immune response which can be triggered by the recognition of intracellular dsRNA and can inhibit viral RNA in a sequence specific manner in cells. RNAi has been used against viral infections such as yellow head virus and white spot syndrome virus in prawns. However, no study of RNAi against MrNV has been reported. In this study, RNAi against viral protein B2 and MrNV were inoculated into redclaw crayfish (Cherax quadricarinatus) which was developed as an experimental animal model. Mortalities were 10% with stealth RNAi plus MrNV and 60% with control stealth RNAi and MrNV exposure. Moreover, length and weight analyses of control stealth RNAi with MrNV were significantly smaller (8.7 cm) and lighter (17.69 g) (F = 8.106, df = 5, 54, p < 0.05 and F = 2.943, df = 5, 54, p < 0.05), respectively compared to other treatments (average 10.3 cm in length and 22.9 g in weight). In this study, clinical signs of MrNV and histopathological lesions such as myolysis with haemocytic infiltration in the muscle were found in infected redclaw crayfish. Using qPCR, two out of ten redclaw crayfish in the stealth RNAi with MrNV treatment and eight out of ten in control stealth RNAi with MrNV treatment were detected to have MrNV. Protein B2 is anti-RNAi mechanism of nodavirus. Down regulating the viral defence by targeting protein B2 gene with RNAi could be an effective tool to decrease mortality and limit MrNV infection. This approach could be very effective against the economical very important viral encephalopathy and retinopathy in fish caused by nodavirus.

T-DNA integration site analysis in transformed barley ( Hordeum vulgare L.) by Tail-PCR

A multiplex reverse transcriptase polymerase chain reaction (MRT-PCR) assay was developed and applied to the detection of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) associated with field and hatchery outbreaks of white tail disease (WTD) in M. rosenbergii. Primers targeted regions of the capsid protein genes of the two viruses and produced amplicons of 1136 and 447 bp, for MrNV and XSV, respectively, with sensitivity to the level of 1 fg of total RNA for both the viruses. Assays could be completed within 7 to 9 h after samples were received. This is of great importance for epidemiological analysis and screening of large numbers of samples. MRT-PCR assay on samples collected from different hatcheries and farms of Nellore, India revealed the presence of both the viruses in all. Experimental infections produced typical clinical signs of white muscle in juveniles and mortality up to 20%. MrNV could be detected up to 1 month in surviving prawns, both by histology and RT-PCR. Sequence analysis of capsid protein gene amplicons of XSV revealed minor variations. By contrast, sequence analysis of MrNV amplicons showed no variation in amino acid sequence of the capsid protein gene.

Specific anti-PrP mucosal and systemic response in white tail deer vaccinated with attenuated salmonella expressing deer PrP

Specific anti-PrP mucosal and systemic response in white tail deer vaccinated with attenuated salmonella expressing deer PrP

Experimental infection of redclaw crayfish ( Cherax quadricarinatus) with Macrobrachium rosenbergii nodavirus, the aetiological agent of white tail disease

Macrobrachium rosenbergii nodavirus (MrNV) or white tail disease has been reported as a new disease of crustacea in western Queensland, Australia. In Australia, Macrobrachium can be hard to source due to their need for a saltwater environment for breeding. No alternative animal experimental model for MrNV has been identified, so redclaw crayfish (Cherax quadricarinatus) were tested as a potential experimental animal in order to study MrNV infection. The highest mortality (35%) was in the groups injected with MrNV and the lowest mortality (0%) was in the control groups. Necrotic muscle and muscle degeneration with haemocytic infiltration were observed in infected crayfish. For the first time, a quantitative real-time polymerase chain reaction (qPCR) on clinical material was developed and it confirmed MrNV infection in infected animals. The mean viral titres (2.73 × 102 copies) and cycle times (Ct = 31.33) lead us to hypothesize that MrNV only poorly replicates in juvenile C. quadricarinatus. However, C. quadricarinatus may be a less than perfect but useable experimental animal model for MrNV infection in the future because of clinical signs, gross lesions, histopathological changes and qPCR titres present in experimentally infected C. quadricarinatus. This study determined that redclaw crayfish (C. quadricarinatus) had low susceptibility and were limited carriers of white tail disease.

The White-collared Kite (Leptodon forbesi Swann, 1922) and a Review of the Taxonomy of the Grey-headed Kite (Leptodon cayanensis Latham, 1790)

The White-collared Kite (Leptodon forbesi Swann, 1922), previously known by the holotype and three specimens from northeastern Brazil from the late 1980s, is considered by many as a juvenile variant of the Grey-headed Kite (L. cayanensis Latham, 1790). We present new morphological evidence from museum specimens of both species, including a previously misidentified specimen of L. forbesi, and field study to support the validity of the White-collared Kite as a species, now seen as endemic and severely threatened in northeastern Brazil. This species occurs only in remnants of the Atlantic Forest in the states of Alagoas and Pernambuco. It is distinguished from its congener by its white hind-collar, underwing coverts, and leading edge of the wings. The under surface of the secondaries show reduced black barring. The number of white and black tail bands is variable, and not a good diagnostic character. We also review all taxa described for L. cayanensis and show the described subspecies are not valid.

Virus-like particles of Macrobrachium rosenbergii nodavirus produced in bacteria

Macrobrachium rosenbergii nodavirus (MrNv) infects giant freshwater prawns and causes white tail disease (WTD). The coding region of the capsid protein of MrNv was amplified with RT-PCR and cloned into the pTrcHis2-TOPO vector. The recombinant plasmid was introduced into Escherichia coli and protein expression was induced with IPTG. SDS-PAGE showed that the recombinant protein containing the His-tag and myc epitope has a molecular mass of about 46 kDa and it was detected by the anti-His antibody in Western blotting. The protein was purified using immobilized metal affinity chromatography (IMAC) and transmission electron microscopic analysis revealed that the recombinant protein assembled into virus-like particles (VLPs) with a diameter of about 30±3 nm. The size of the particles was confirmed by dynamic light scattering. Nucleic acids were extracted from the VLPs and treatment with nucleases showed that they were mainly RNA molecules. This is the first report describing the production of MrNv capsid protein in bacteria and its assembly into VLPs.

Screening the post-larvae of Macrobrachium rosenbergii for early detection of Macrobrachium rosenbergii nodavirus ( MrNV) and extra small virus (XSV) by RT–PCR and immunological techniques

Abstract White tail disease (WTD) was found to be a serious problem in hatcheries and nursery ponds of freshwater prawn ( Macrobrachium rosenbergii ) . The causative organisms have been identified as Macrobrachium rosenbergii nodavirus ( Mr NV) and extra small virus (XSV). RT–PCR and immunological techniques such as Western blot and ELISA were used for early detection of Mr NV and XSV in post-larval samples obtained from time-course experiments at different time intervals. Two viruses were purified from diseased post-larvae of M. rosenbergii by a combination of low and high speed centrifugation using sucrose and CsCl gradients to raise the antisera separately. One structural protein with molecular weight of 43 kDa (CP-43) was identified from the purified preparation of Mr NV, and two overlapping polypeptides of about 17 kDa (CP-17) and 16 kDa (CP-16) were found in XSV particles by SDS-PAGE. The antisera raised against CP-43 of Mr NV, CP-16 and CP17 of XSV in mice were used to detect Mr NV and XSV by Western blot and ELISA. Published primers specific to Mr NV and XSV were used for the early detection of these viruses by RT–PCR and nested RT–PCR. The post-larval samples collected at 3 h post infection (h p.i.) showed positive for both viruses by nested RT–PCR and negative by RT–PCR, Western blot and ELISA techniques. The samples collected at 24 h p.i. and thereafter were found to be positive for Mr NV and XSV by RT–PCR, ELISA and Western blot analyses.

A Survey of ocular abnormalities in Miniature Horses

To determine the incidence of ocular abnormalities in miniature horses. Descriptive study. Fifty-three miniature horses. Ophthalmic examinations were performed using diffuse and focal illumination, slit-lamp biomicroscopy and indirect ophthalmoscopy. Coat color, mane and tail color, age and gender were recorded with results of ophthalmic examination. Ocular abnormalities detected most frequently consisted of cysts arising from the posterior iris, ciliary body, and peripheral retina, curvilinear streaks of retinal pigmented epithelium extending from the peripheral temporal retina, and retinal dysplasia (folds). One miniature horse also had multiple ocular abnormalities (cornea globosa, goniosynechiae, decreased or absent and pupillary light reflexes, miotic and dyscoric pupils, iris hypoplasia, and epinuclear cataract). Ocular abnormalities were observed in horses that had a flaxen or white mane and tail color and a chocolate coat color. Abnormal ocular phenotype detected in eyes of Miniature Horses was similar to multiple congenital and hereditary ocular abnormalities reported in the Rocky Mountain Horse. Phenotypic ocular developmental abnormalities and coat, mane and tail color were most compatible with a heterozygous or homozygous effect of the Silver Dapple locus.

Effect of isobaric breathing gas shifts from air to heliox mixtures on resolution of air bubbles in lipid and aqueous tissues of recompressed rats

Deep tissue isobaric counterdiffusion that may cause unwanted bubble formation or transient bubble growth has been referred to in theoretical models and demonstrated by intravascular gas formation in animals, when changing inert breathing gas from nitrogen to helium after hyperbaric air breathing. We visually followed the in vivo resolution of extravascular air bubbles injected at 101 kPa into nitrogen supersaturated rat tissues: adipose, spinal white matter, skeletal muscle or tail tendon. Bubbles were observed during isobaric breathing-gas shifts from air to normoxic (80:20) heliox mixture while at 285 kPa or following immediate recompression to either 285 or 405 kPa, breathing 80:20 and 50:50 heliox mixtures. During the isobaric shifts, some bubbles in adipose tissue grew marginally for 10–30 min, subsequently they shrank and disappeared at a rate similar to or faster than during air breathing. No such bubble growth was observed in spinal white matter, skeletal muscle or tendon. In spinal white matter, an immediate breathing gas shift after the hyperbaric air exposure from air to both (80:20) and (50:50) heliox, coincident with recompression to either 285 or 405 kPa, caused consistent shrinkage of all air bubbles, until they disappeared from view. Deep tissue isobaric counterdiffusion may cause some air bubbles to grow transiently in adipose tissue. The effect is marginal and of no clinical consequence. Bubble disappearance rate is faster with heliox breathing mixtures as compared to air. We see no reason for reservations in the use of heliox breathing during treatment of air-diving-induced decompression sickness.