Search

Viral nervous necrosis (VNN), or viral encephalopathy and retinopathy caused by nervous necrosis virus (NNV), is an acute viral disease of fish, resulting in high mortality globally in cultured marine fish. The pathogen, Betanodavirus belonging to the family Nodaviridae, has been widely documented in both farmed and wild fish, besides in invertebrate species across the habitat barriers. However, cichlid fish, in particular Etroplus species, have a low susceptibility or resistance to this disease. The present study was undertaken to check the susceptibility of a brackishwater cichlid, orange chromide (Etroplus maculatus) through intramuscular injection and immersion challenge by NNV (RGNNV genotype). Intramuscular injection (10 μl) with NNV (105 viral particles ml−1) resulted in cumulative mortality of 50% at 15 days of post-infection (dpi), while immersion challenge in NNV spiked sea water (105 TCID50 ml−1) resulting in a mortality of 25% at 19 dpi. Unlike many marine fish which showed 100% mortality within 3–5 days, E. maculatus challenged through both the routes exhibited severe clinical symptoms for 2–3 weeks followed by a recovery stage and moderate level of mortality. Reverse transcription polymerase chain reaction (RT-PCR) tests on live fish sampled during the course of the experiment and the dead fish exhibiting symptoms in both challenge groups tested NNV-positive. Histological examination of fish in challenged groups showed VNN-specific lesions characterized by vacuolations in the brain and eye with more severe lesions in the injected group than the immersion group. In survived fish, no clinical sign was observed, but NNV could be detected by RT-PCR with moderate to severe vacuolations in the brain and eyes until 30 dpi. Despite the virus entering the body and triggering typical symptoms of VNN, the fish appeared to exhibit moderate resistance with a survival of 50–75%. The results indicate that E. maculatus could serve as a brackishwater fish model to study host-pathogen interaction and development of resistance for potential application in NNV-vaccine studies.

Aim of the study: Vibrio harveyi bacteria are affecting shrimps during grow-out practices. The application of chemicals to control V. harveyi has resulted in antibiotic‐resistance among bacteria. An extract of the leaves of Rhizophora mucronata was tested to control the growth and virulences of V. harveyi. Area of study: This study was conducted in the Crustacean Culture Division of ICAR-CIBA, Chennai city, Tamil Nadu State, India. Material and methods: R. mucronata plants were collected from the Pitchavaram area, and the leaves contents extracted. The resultant extract was prepared and tested against the growth of V. harveyi and its virulence factors. The various functional compounds of R. mucronata were screened and volatile compounds were analyzed. Main results: When R. mucronata extract was treated against V. harveyi (350 µg/mL) an inhibitory zone of 14 ± 0.1 mm was observed. At 300 µg/mL, the extract was found to be active in decreasing the luciferase to a maximum of 76 counts per second in 30 days and a similar level of bioluminescence was reduced in 15 days. During, shrimp larviculture a reduction in the cumulative percent of mortality 15.70% (p

Enterocytozoon hepatopenaei (EHP), is an emerging microsporidian pathogen responsible for hepatopancreatic microsporidiasis (HPM) in shrimps and is associated with severe growth retardation. The disease causes economic losses in shrimp aquaculture. In this study, EHP spore germination was induced and demonstrated with a scanning electron microscope (SEM). The ions (cations and anions) generated by high-energy electrons during frozen water radiolysis in the SEM specimen chamber induce EHP spore germination. This study is the first to demonstrate the induction of a microsporidian spore germination by ions generated under SEM. This study will enhance our understanding of EHP biology, life cycle and lead to the development of prophylactics and therapeutics for EHP control. Also, this method will help standardize the study of germination in other microsporidians.

This study was undertaken to investigate the cause of mortality in the captive stock of silver moony Monodactylus argenteus. The fishes showed severe infection of dinoflagellate protozoan Amyloodinium sp. (Blastodinida, Oodiniaceae) on gills and skin with complete mortality of the stock within a week. Histopathological changes were evident in the gill tissues with severe lamellar epithelial cell hyperplasia and lamellar fusions with the presence of trophonts of Amyloodinium sp. Scanning electron microscopy (SEM) also revealed trophonts of Amyloodinium sp. of varying sizes in groups consisting of 3 to 5 trophonts tightly attached to gill lamellae. Source water contaminated with the tiny infectious form of the parasite (dinospores) favoured by higher salinity and low water temperature in the rearing tank could be the triggering factor for the spurt of infections. Proper quarantine and biosecurity protocols to prevent the potential sources of water-borne infection sources are likely to be far more effective than treatment. Keywords: Amyloodiniosis, Amyloodinium sp., Histopathology, Monodactylus argenteus, Scanning electron microscopy, Silver moony, Trophonts

Shrimp grow-out and hatchery systems are being affected by bacterial disease particularly Vibrios. The use of chemotherapeutic agents in aquaculture practices has to lead to the development of resistance among aquatic bacteria. Thus, health management becomes of major importance in aquaculture. Under this situation, progressing bio-inhibitors from marine resources are most appropriate to be considered against pathogenic bacteria. Molecular docking is an appropriate tool in structural biology and computer-assisted drug design to predict and neutralize a target protein of known diseases. In this study, marine macro-alga Ulva fasciata was aimed at developing inhibitors against luminescence disease-causing pathogenic bacteria Vibrio harveyi. U. fasciata was collected from Thoothukudi, Tamil Nadu, India. Extract of U. fasciata was tested against growth and virulence factors of V. harveyi during Penaeus monodon larviculture. Further U. fasciata extract was subjected to GC–MS analysis to identify the biomolecules. The homology modeling of virulent protein, hemolysin of V. harveyi was designed in this study. Hence, it was aimed for molecular docking against the biomolecules identified from U. fasciata extract. During shrimp larviculture, the extract of U. fasciata (200 μg mL−1) exhibited reduction on Cumulative Percentage of Mortality (32.40%) in postlarvae against challenge of V. harveyi infection. Biomolecule Methyl dehydroabietate had showed highest binding affinity among the compounds was evaluated in molecular docking study. Statistical analysis had revealed significant differences (p

Betanodaviruses are the causative agents of the disease known as viral nervous necrosis (VNN) or viral encephalopathy and retinopathy (VER) in a variety of marine and freshwater fish species. The aim of this study was to demonstrate experimental infection of an isolate of betanodavirus (RGNNV genotype) in freshwater fish, Gambusia affinis, for elucidation of transmission mechanism and potential use as a laboratory model. Morbidity and mortality rate was significantly higher by injection route of infection as compared to immersion by bath and resembled the natural infection of juvenile marine fish. The fish in disease affected group showed severe neurological disorders accompanied by extensive vacuolar degeneration and mild to moderate neuronal necrosis of the brain in comparison to control. Amplification of ~ 427 bp product in the variable region of the coat protein gene of betanodavirus was achieved by RT-PCR with 100% sequence homology to RGNNV genotype.

Economic loss due to Enterocytozoon hepatopenaei (EHP), white spot syndrome virus (WSSV) infections and other diseases to Penaeus vannamei farming in India was estimated through a questionnaire-based survey of shrimp farms in major shrimp farming states of India during 2018–2019. The survey covered an area of 7259 ha of shrimp farming in 23 coastal districts based on multistage stratified sampling (n = 909). Probability of disease occurrence (PDO) was calculated to estimate the economic loss due to diseases. Double log regression was employed to examine the relationship between economic loss with production, stocking density, culture period, average body weight, survival, FCR and incidence of disease. Loss of production (t ha−1crop−1) was highest in infections due to WSSV (2.58 ± 0.32), followed by EHP (1.80 ± 0.24), mixed infections of EHP and WSSV (1.89 ± 0.53), vibriosis (0.97 ± 0.42), running mortality syndrome (1.1 ± 0.39) and other diseases (1.72 ± 0.36). Although WSSV caused the highest loss of production (t ha−1 crop−1), EHP with a 17% probability of occurrence, accounted for a production loss of 0.77 M tons, with a corresponding revenue loss of Rs. 3977 crores (US$ 567.62 M). Although the probability of occurrence of WSSV was estimated (25%) to be higher than that of EHP, the estimated production loss due to WSSV was lower, 0.33 M tons, with a corresponding revenue loss of Rs. 1670 crores (US$ 238.33 M). National loss of revenue due to EHP was higher primarily because, in Andhra Pradesh, primary shrimp farming state had the PDO of 22% for EHP against 8% for WSSV. The total employment loss due to diseases was estimated to be 1.65 M man-days worth US$ 7.07 M. The study revealed that the overall probability of infectious disease occurrence in the country was at 49% leading to an annual loss of 0.21 M ton shrimp worth US$ 1.02 B. Economic loss due to shrimp diseases in Indian shrimp farming warrants prioritized implementation of better management practices (BMP) and biosecurity protocols along with policy interventions to reduce the direct and indirect losses.

Viral encephalopathy and retinopathy otherwise known as viral nervous necrosis (VNN) is a neuropathological condition affecting more than 50 fish species worldwide, mostly marine. Different PCR protocols with specific primers were reported from many countries for confirmation of VNN in fishes. In the present study, two pairs of primers were designed and evaluated for the diagnosis of clinical and subclinical cases of infections from field. These primers designated as BARL-F1/BARL-R1 amplified a 902 bp product in the variable region (T4) of the coat protein gene by first step PCR. Nested PCR primers BARL-F2/BARL-R2 amplified a fragment of 313 bp. The results were comparable with other commonly used primer sets such as F2/R3 and RG668f/RG919r primers. These new primers could detect betanodavirus in standard reference samples containing low, moderate and high viral load. Known positive and negative control samples of fish also revealed a predictive value of 100 % by RT-PCR diagnosis.

A 75-day 2 × 3 factorial experiment was conducted to evaluate the effect of two levels of dietary protein (32 and 40%) and two different carbon sources (rice flour–R and molasses-M), and without carbohydrate (control–C) in black tiger shrimp Penaeus monodon juveniles (3.37 ± 0.04 g) at 100 nos. m−3 in 100 L fibre reinforced plastic tanks. Biofloc volume and total suspended solid were higher in molasses added groups (32 + M and 40 + M) followed by rice flour (32 + R and 40 + R) and controls (32C and 40C). Molasses and rice flour addition significantly reduced (P

White spot syndrome virus (WSSV) replicates rapidly, can be extremely pathogenic and is a common cause of mass mortality in cultured shrimp. Variable number tandem repeat (VNTR) sequences present in the open reading frame (ORF)94, ORF125 and ORF75 regions of the WSSV genome have been used widely as genetic markers in epidemiological studies. However, reports that VNTRs might evolve rapidly following even a single transmission through penaeid shrimp or other crustacean hosts have created confusion as to how VNTR data is interpreted. To examine VNTR stability again, 2 WSSV strains (PmTN4RU and LvAP11RU) with differing ORF94 tandem repeat numbers and slight differences in apparent virulence were passaged sequentially 6 times through black tiger shrimp Penaeus monodon, Indian white shrimp Feneropenaeus indicus or Pacific white leg shrimp Litopenaeus vannamei. PCR analyses to genotype the ORF94, ORF125 and ORF75 VNTRs did not identify any differences from either of the 2 parental WSSV strains after multiple passages through any of the shrimp species. These data were confirmed by sequence analysis and indicate that the stability of the genome regions containing these VNTRs is quite high at least for the WSSV strains, hosts and number of passages examined and that the VNTR sequences thus represent useful genetic markers for studying WSSV epidemiology.

Jagadeesan, V.; Ezhil Praveena, P.; Otta, S.K., and Jithendran, K.P., 2019. Classical runt deformity syndrome cases in farmed Penaeus vannamei along the east coast of India. In: Jithendran...

Betanodavirus has been detected in Asian seabass in India. Molecular characterization of the isolates on the basis of the full-length viral RNA2 sequence was performed. Subsequent phylogenetic analysis with sequences from members of the four species in the genus Betanodavirus revealed that the present isolates are closely related to members of the species Redspotted grouper nervous necrosis virus. The analysis also revealed that the RNA2 sequence was not responsible for acute symptoms in seabass. This is the first attempt to characterize Indian isolates of fish nodaviruses, and the result will be useful for devising specific control and health-management strategies for this virus.

We isolated the infectious viral agent from infected Asian seabass, Lates calcarifer (Bloch, 1790) using SSN-1 cells. The isolated virus was cultivated in indigenous marine fish cell lines developed from ornamental sea anemone fish Amphiprion sebae and from Asian seabass. Reverse transcriptase PCR using betanodavirus-specific primers gave positive results for the RNA from virus-infected cells (hereinafter referred to as L. calcarifer nervous necrosis virus Indian strain – LCNNV-In01). Serum neutralization test indicated partial homogeneity between Atlantic halibut nervous necrosis virus (AHNNV 692/9/98) and LCNNV-In01. Transmission electron micrographs of the virus-infected cell culture showed large numbers of 25–30-nm particles in cellular vacuoles with features similar to betanodaviruses. Pathogenicity study in seabass fingerlings using cell culture grown virus revealed nervous necrosis in retinal cells following a 21-day challenge trial. The nodavirus was reisolated from the infected fish using clownfish and seabass cell lines. Sequence analysis of the coat protein gene showed that the virus belonged to RGNNV group (GenBank Acc. No. FR669249). The isolation of the nodavirus from seabass in India and its potential to grow in non-homologous cell lines indicate the serious threat of the Indian strain of betanodavirus (LCNNV-In01) to cultured and ornamental marine fish that have their hatchery production successfully standardized in India.

This study was carried out to evaluate the efficiency of the Flinders Technology Associates (FTA(®)) card (Whatman(®)) as a sampling device and storage platform for RNA from betanodavirus-infected biological samples (viz., larvae, broodstock, cell culture supernatants and rearing seawater spiked with infected materials). The study showed that FTA cards can be used to detect betanodaviruses by reverse transcription-polymerase chain reaction (RT-PCR). The diagnostic efficiency of RT-PCR from all sample types on FTA cards decreased after 21 days of storage at 4 °C, although the virus could be detected up to 28 days by nested RT-PCR. The FTA card protocol thus provides a supplementary method for quick and easy collection of samples, preservation of RNA on a dry storage basis, and detection of betanodavirus-infected fish.

We studied the natural progression of viral nerve necrosis (VNN) in larvae of Asian seabass Lates calcarifer Bloch from 0 to 40 days post-hatch (dph). The hatchlings were reared in the vicinity of a confirmed nodavirus-affected older batch. Using light and electron microscopy (EM), we made a sequential analysis of histopathological manifestations in nerve tissue and other organs. There were no changes from the day of hatching until 4 dph. Larvae at 4 dph had viral particles in the intramuscular spaces underlying the skin, but the nerve cells of the brain were normal. The first signs of necrosis of the brain cells were observed at 6 dph. EM observations revealed characteristic membrane-bound viral particles measuring 30 nm in the cytoplasm of nerve cells of the brain, spinal cord and retina. Histological samples of fry examined when group mortalities reached 20 to 35% revealed highly vacuolated brains, empty nerve cell cytoplasm and viral particles in the intercellular spaces. Viral particles occurred extensively in the intramuscular spaces and the epidermal layers. These observations were corroborated by positive immunostaining of the virus-rich intramuscular spaces. EM studies also revealed progressive necrotic changes in the cells harboring the virus. Results emphasize the need to maintain hygiene in the hatchery environment and to develop strategies for prevention of disease spread among cohabiting seabass and other susceptible fish larvae. Intramuscular localization of the nodavirus in both preclinical healthy-looking and post-clinical moribund larvae suggests that virus neutralization strategies during larval development could be effective in controlling VNN-associated mortalities.

A probable vertical mode of piscine nodavirus transmission is reported in the present investigation based on a case of nodavirus associated larval mortalities in hatchery produced Asian sea bass. Polyclonal rabbit anti-SJNNV antibodies (SGWak97) detected the viral antigens in the tissue sections from the eggs and the larvae at different time intervals from − 1 to 42 days post hatch (dph). Immunopositive ovarian connective tissue associated with the oocytes along with the progressive localization of the viral antigens in the brain, spinal cord, liver, stomach and dermal musculature during the larval development indicates a probable vertical transmission of nodavirus in the Asian sea bass. The surviving larger larvae, from the batch suffering mass mortalities, produced very intense immunofluorescent positivity in the liver, stomach and dermal musculature. Results of this investigation demonstrating a possibility of vertical transmission of the nodavirus emphasize the need for screening of eggs and larvae for evolving suitable preventive and prophylactic health management strategies.

White tail disease is a recently reported disease of giant freshwater prawn, Macrobrachium rosenbergii associated with M. rosenbergii nodavirus (MrNV). The present study is the first report on RT-PCR and sequence analysis of MrNV of an Indian isolate confirming the nodavirus infection in M. rosenbergii from India. Total RNA was extracted and the cDNA was synthesized from the infected post larvae collected from a hatchery located in Chennai, India. An 850-bp amplified product could be obtained by RT-PCR. The nucleotide sequence analysis of this 850-bp segment showed 98% nucleotide and 99% amino acid sequence identity with the reported sequence of an MrNV isolate from the West Indies.

Histopathology of Y-organ (moulting gland) in Indian white shrimp Fenneropenaeus indicus was studied after challenging the shrimp with virulent white spot syndrome virus (WSSV). Histopathological investigation on the Y-organ collected from moribund shrimps revealed the presence of intranuclear basophilic inclusions, characteristic of WSSV. More than 70% of the Y-organ cells were infected, suggesting the degenerated state of the organ. The cellular integrity of the Y-organ was completely destroyed by the WSSV. Further, Y-organ tissue samples collected from all the 16 experimental shrimp were positive by one-step polymerase chain reaction (PCR), confirming severe WSSV infection. The infected Y-organ in F. indicus with majority of the organ cells observed in the lysed condition suggests a physiological dysfunction of the organ. In uninfected and healthy controls, the lobulated Y-organ showed closely packed normal cells with prominent nuclei and sparse cytoplasm. Physiological implication of a degenerated Y-organ in the moulting and reproduction of the penaeid shrimp is discussed.

Aquaculture is growing by leaps and bounds and is one of the world’s fastest-growing industries in food production. Unlike other terrestrial farm animals and plants, aquatic animals require more attention in order to monitor their health. They live in a complex and dynamic environment and are not readily visible except under tank-holding conditions. Similarly, feed consumption and mortalities are also equally well hidden under water (Bondad-Reantaso et al. 2001). So the problems faced by the aquatic animals are also species and system specific. The complexity of the aquatic ecosystem makes it difficult to understand the difference between health, suboptimal performance, and disease. The range of diseases found in aquaculture is one among the major problems faced by aquaculturists all over the world. Diseases in aquaculture are caused by the outcome of a series of linked events involving the interactions between the host, the environment, and the presence of a pathogen (Snieszko 1974). Environment includes not only the water and its components (such as oxygen, pH, temperature, toxins, and wastes) but also the kind of management practices (e.g., handling, drug treatments, transport procedures, etc.). There are three factors such as stocking density, innate susceptibility, and immunity which are particularly important in affecting host’s susceptibility to diseases. The intensive shrimp aquaculture has parallely brought disease problems leading to great economic loss. Diseases may be caused by a single or combinations of multifarious factors. Generally, diseases are broadly classified in to infectious and noninfectious. The former is caused either by virus, bacteria, fungi, parasites, or rickettsia, while the latter is due to environmental stresses, genetic factors, and nutritional deficiencies. The most important steps to reduce or prevent losses due to diseases in aquaculture are monitoring as regularly as possible and appropriate action at the first sign(s) of suspicious behavior, lesions, or mortalities. These fundamental approaches should be followed in many aquatic animal production sectors as in animal husbandry and agricultural production. Some farmers hesitate to reveal the disease problems due to their ignorance that it may result in failure in the competitive market price. It should be made understood that hiding or denying health problems can be as destructive to aquatic animals as it is elsewhere.

A systematic survey was conducted during two spells of 5 years each (1986–1990 and 1993–1997) to study the prevalence of parasitoses with particular reference to gastrointestinal (GI) nematodes and flukes in dairy animals (crossbred and native cattle, and buffaloes) belonging to 12 villages in the Kangra valley (Himachal Pradesh). Flukes (Fasciola, amphistomes and Dicrocoelium) and strongyles were the most important parasitic infections. Fasciola was endemic throughout the year, with a higher percentage infection in buffaloes than in cattle. Other fluke and nematode infections showed a seasonal pattern in prevalence, with a small peak in March–April followed by a high peak in July–September. The faecal egg counts (eggs per gram, epg) of flukes (Fasciola/amphistomes) ranged from 50 to 300 in cattle and 50 to 400 in buffaloes, with high loads during the rainy and post-rainy seasons. The GI nematode egg counts (excluding Toxocara) revealed a similar trend, with the overall monthly mean epg ranging from 85 to 1720 in cattle and 90 to 1625 in buffaloes, with a high peak during the months of July to September. On coproculture of positive samples, the nematode infections in order of prevalence were: Strongyloides, Trichostrongylus, Haemonchus, Oesophagostomum, Bunostomum and Mecistocirrus. The prevalence of most of the parasites was lower during the second 5-year period.

Pacific white shrimp, Litopenaeus vannamei has been introduced recently for culture practice in India. Though SPF stocks are imported for larval production and thereafter culture practice, these are prone to infection with the existing viruses in the environment. Here we report mortality of L.vannamei in several farms in India with minimum biosecurity. The shrimp were harvested early within 50–72 days of culture due to the onset of disease and consequent mortality. As per the analysis carried out, the shrimp were infected with two virus, white spot syndrome virus (WSSV) and infectious hypodermal and hematopoietic necrosis virus (IHHNV). About 80 % of the samples collected had either or both of the viruses. A majority of these samples (60 %) had dual infection with WSSV and IHHNV. Infection of shrimp with WSSV and IHHNV could be detected both by PCR and histopathology. Some of the samples had either exclusively WSSV infection or IHHNV infection and were also harvested before the completion of the required culture period. All the samples analyzed were negative for taura syndrome virus, yellow head virus and infectious myonecrosis virus. While it is difficult to point out the exact etiological agent as the cause of mortality, strict biosecurity measures are advisable for the continuity of L. vannamei culture in India.

The classification of betanodavirus into four species was reviewed including newer and well-characterised isolates. Six major clusters were identified, four of which were similar to the classic species. Two single isolate clusters were worth consideration as new species.

[EN] Coccidiosis causad by Eimeria species, is a majar parasitic disease of rabbits and is responsible for a high incidence of morbidity and mortalitv. The incidence of th1s disease in EuroP.ean countries in 21-60% and in India 13-64%. In commercially rearad rabbits; coccidiosis occurs .in a subclinical form witti growth retardation and alterad feed conversion. The disease occurs in two forms, hepatic and intestinal, the latter being more common than the formar. Presently, the control of rabbit coccidiosis relies almost entirely on chemical coccidiostats. However, extensiva use of such arugs in commercial rabbitries has already started creating problems of drug resistance. In view of the limitations of chemotherapy, it is imperativa to develop immunoprophylactic measures against rabbit coccidiosis. These incluae development of live-attenuated and recombinant vaccines against the parasite., [FR] La coccidíose provoquée par des esp_eces du genre Emeiria est la principafe maladie parasitaire affectant le lapin. Elle est respqnssble de la morb1dité et de la mortalité a fréquence élevée. Dans les pays européens, son incidence est de 21 a 60% dans les troupeaux, ene est de 13 a 64% en lnde. Dans les élevages commerciaux de lapins la coccidiose se manifeste sous fonne subc/inique p_rovoquant des retareis de croissance et une réduction de l'effícacité alimentaire. Cette maladie se manifeste sous deux formes, hépatique et intestina/e, la seconde étant plus fréquente que la premíere. Actuellement le contr6/e de la coccidiose chez le lapín est presque exclusivement basé sur l'emploí de coccidiostats chim1ques. Cependant l'usage intensif des ces médicaments commence a créer des problemes de résistance aux anticoccidiens. Afín de limtter la chimíothérapie 11 devient ímpératif de déve/opper des mesures ímmunoprophylactíques pour lutter contre la coccidiose. Celles-ci íncluent le développement de líg_nées atténuées et de vaccíns recombínants utiltsables contre Je parasite.

Occurrence of widespread epizootics among cultured stock of shrimp has put research programmes on preventive approaches such as application of probiotics on a high priority in aquaculture. In the present study two bacteria, Pseudomonas sp. PM 11 and Vibrio fluvialis PM 17 were selected as candidate probionts from a pool of bacteria isolated from gut of farm reared sub-adult shrimp and tested for their effect on the immunity indicators of tiger shrimp. Sub-adult shrimp, weighing 14 to 22 g were treated in separate experiments with Pseudomonas sp. PM 11 and V. fluvialis PM 17 at 10(3) bacterial cells ml(-1) in the experimental shrimp culture tanks. One set of experimental animals was treated every 3 days and another set of animals every 7 days with each of the candidate probionts. Estimation of immunological indicators such as haemocyte counts, phenol oxidase and antibacterial activity showed declining trends. The haemocyte counts dropped from 31 x 10(3) to 65 x 10(3) ml(-1) on the first day to 4-16 x 10(3) ml(-1) on the 45th day. Similarly, the phenol oxidase activity declined from 12-32 units on the first day to 11-14 units on 45th day of the experiment. Antibacterial activity of haemolymph reduced to 46-67 percent on the 45th day of the experiment. The results of the study suggest that, the criteria used for the selection of putative probiotic strains in the present study, such as predominant growth on primary isolation media, ability to produce extracellular enzymes and siderophores, did not bring about the desired effect in vivo and improve the immune system in shrimp. Hence, new protocols have to be evolved for selection of microbe(s) as putative probiotics and that, detailed understanding of proven probiotics, employed presently on empirical basis may provide a clue on the selection procedure.

A systematic survey was conducted during two spells of 5 years each (1986-1990 and 1993-1997) to study the prevalence of parasitoses with particular reference to gastrointestinal (GI) nematodes and flukes in dairy animals (crossbred and native cattle, and buffaloes) belonging to 12 villages in the Kangra valley (Himachal Pradesh). Flukes (Fasciola, amphistomes and Dicrocoelium) and strongyles were the most important parasitic infections. Fasciola was endemic throughout the year, with a higher percentage infection in buffaloes than in cattle. Other fluke and nematode infections showed a seasonal pattern in prevalence, with a small peak in March April followed by a high peak in July September. The faecal egg counts (eggs per gram, epg) of flukes (Fasciola/amphistomes) ranged from 50 to 300 in cattle and 50 to 400 in buffaloes, with high loads during the rainy and post-rainy seasons. The GI nematode egg counts (excluding Toxocara) revealed a similar trend, with the overall monthly mean epg ranging from 85 to 1720 in cattle and 90 to 1625 in buffaloes, with a high peak during the months of July to September. On coproculture of positive samples, the nematode infections in order of prevalence were: Strongyloides, Trichostrongylus, Haemonchus, Oesophagostomum, Bunostomum and Mecistocirrus. The prevalence of most of the parasites was lower during the second 5-year period.

Cysticercus fasciolaris, the larval form of Taenia taeniaformis is commonly encountered in rodents most often in mice and rats through contaminated feed and bedding materials. The infection is asymptomatic and is considered harmless, but its presence in the laboratory mice/rats could lead to misinterpretation of results for biological experiments. The course of infection and pathogenesis of induced C. fasciolaris was studied in Swiss albino mice. The number of established cysts were not significantly different during the course of infection. The mean diameter of the cysts and the metacestode were significantly different during the course of infection reaching a maximum size of 8.1 +/- 2.2 mm and 80.4 +/- 20.2 mm, respectively on 45 DPI. Histopathologically, on 15 DPI, the duodenum of the affected mice revealed cross sections of early larval stage of C. fasciolaris. On 30 and 45 DPI, the liver showed tract of migration of C. fasciolaris larvae with a thick zone of inflammatory reaction and encapsulation against mature larvae in liver. The routine spontaneous Cystucercus infection is clinically asymptomatic in these animals and is considered harmless. The present experimental infection also followed the same course resulting only in asymptomatic colonisation of the parasites.

The classification of betanodavirus into four species was reviewed including newer and well-characterised isolates. Six major clusters were identified, four of which were similar to the classic species. Two single isolate clusters were worth consideration as new species. Betanodaviruses are small (37 nm average) icosahedral viruses with two positive-stranded non-polyadenylated RNAs as its genome. RNA1 (3.1 kb) codes for the viral RNA-dependent RNA polymerase (protein A), while RNA2 (1.4 kb) codes for the coat protein [16]. A subgenomic RNA3 synthesized from RNA1 during early viral replication codes for protein B2 [17], which functions against the host RNA interference system [3, 8]. These viruses cause viral nervous necrosis (VNN) or viral encephalopathy and retinopathy (VER) in fin fishes. They were first identified as picorna-like virus particles [5] and later described as members of the family Nodaviridae [12]. Taxonomically, these viruses belong to the genus Betanodavirus of family Nodaviridae, with four species recognised so far [16] based on similarities in the variable region of the viral coat protein (CP) gene (nt 604-1030) [14]. The currently recognized species are Redspotted grouper nervous necrosis virus (RGNNV), Striped jack nervous necrosis virus (SJNNV), Tiger puffer nervous necrosis virus (TPNNV) and Barfin flounder nervous necrosis virus (BFNNV), with SJNNV as the type species. The preferred temperature ranges of members of these species are 25-30 C, 20-25 C, 20 C and 15-20 C, respectively. Over 40 fish species have been reported to be susceptible to betanodaviruses, and the number is steadily increasing [10, 13]. Since the first description 15 years ago, the taxonomy of betanodavirus has remained more or less the same irrespective of the huge number of virus isolates being reported from different geographical environments and from different fish hosts. Some modifications were proposed to the system [4, 11, 15, 19], but these have been largely restricted to the geographical area of the researcher. The study presented here is an attempt to analyze the global phylogenetic relationships among the viruses by comparing all isolates for which full-length coat protein (CP) gene ORF sequence information is available in the NCBI GenBank database. A short variable region in the CP gene is shown to be sufficient for determining the phylogenetics of these viruses [14], but the full-length CP gene ORF is used in this study to obtain more reliable results. Sequences of the full-length coat protein gene ORF of betanodaviruses were retrieved from the NCBI GenBank database with due care to include members of the species described in Eighth Report of the International Committee on Taxonomy of Viruses [16]. A total of 78 sequences were retrieved from the database. The sequences were labelled with their geographical location of isolation for ease of compilation for analysing the distribution pattern of the isolates included in this study. Betanodaviruses show high temperature specificity [6] and low host specificity [19], and this is expected to be reflected in their geographical distribution. The nucleotide and deduced amino acid sequences were aligned using the MUSCLE program [2] in MEGA software [18]. Neighbour-joining trees were C. P. Binesh (&) Sacred Heart College, Thevara, Cochin 682013, Kerala, India e-mail: bineshkanayi@gmail.com C. Greeshma Department of Life Sciences, Calicut University, Thenjippalam, Malappauram, Kerala, India e-mail: greeshmakongatta@gmail.com 123 Arch Virol (2013) 158:1589–1594 DOI 10.1007/s00705-012-1549-7

A survey incorporating field and abattoir studies was carried out on the prevalence of Dicrocoelium dendriticum in sheep and goats in the Kangra valley of Himachal Pradesh (India). Coprological studies on these migratory animals revealed that 8.1% of sheep and 4.1% of goats were positive for dicrocoeliosis, with a mean number of eggs per gram of faeces (EPG) of 702.1 for sheep and 566.7 for goats. The necropsy liver examination of slaughtered animals revealed that 24.1% of sheep and 12.3% of goats were infected with D. dendriticum and they had mean fluke burdens of 247.2 (sheep) and 298.1 (goats). No significant differences in fluke burden, liver lesion score and EPG count were observed between sheep and goats. The seasonal distribution of dicrocoeliosis indicated a higher percentage of infection in autumn and winter as compared with spring and summer. The potential role of migratory sheep and goats in the epizootiology of dicrocoeliosis is discussed.

The sensitivity and specificity of the agar gel precipitation test (AGPT), counter immunoelectrophoresis (CIEP) and passive haemagglutination test (PHT) were evaluated for the diagnosis of Dicrocoelium dendriticum infection in naturally infected sheep and goats. Two hundred and forty five sheep and goat sera samples were tested using phosphate buffered saline, pH 7.2 extracted adult fluke antigen. CIEP detected 69.8% of the infected animals and was found to be the most sensitive, followed by PHT which detected 50.0% of the infected sheep and goats. AGPT was found to be the least sensitive, detecting only 23.8% of the infected animals. The specificity of AGPT, CIEP and PHT were 93.3%, 84.0% and 93.3%, respectively. CIEP was found to be the most sensitive, specific and rapid test for the seroepidemiological survey of dicrocoeliosis in sheep and goats.