Gonad recrudescence and annual sex steroid patterns in female goldspot mullet, Liza parsia reared in brackishwater pond.

Goldspot mullet, Liza parsia is a commercially important fish of South East Asia, where its farming depends on wild seed resources due to unavailability of hatchery technology. It, therefore, is important to understand the annual reproductive cycle of female L. parsia in captivity. In this study, adult male and female L. parsia (body weight ranges: 45-90 g; total length ranges: 100-125 mm, age >1 year) were collected from the wild and reared in a brackishwater pond. Thereafter, fish were randomly sampled at monthly intervals to measure the hepatosomatic index (HSI), gonadosomatic index (GSI), levels of serum steroids (testosterone, T; 17ß-estradiol, E2 and 17α-hydroxyprogesterone, 17-OHP), and oocyte growth. Results exhibited that female L. parsia undergoes six different maturation stages, namely I (oocyte diameter, OD: <100 µm), II (OD: 100-350 µm), III (OD: 350-400 µm), IV (vitellogenic oocyte, OD: 400-450 µm), V (ripe oocyte, OD: 450-550 µm) and VI (atretic oocyte, OD: 60-150 µm), with synchronous oocyte development. The highest (P < 0.05) HSI (1.96 ± 0.24) and GSI (12.01 ± 0.73) were recorded in December and January, respectively. Concentration of E2 gradually increased from August and reached its peak (807.67 ± 25.98 pg mL-1, P < 0.05) in December. The level of 17-OHP (85.87 ± 0.91 pg mL-1) was at its peak during the normal spawning month (January) (P < 0.05). Overall, the results indicated that L. parsia attains maturity in brackishwater pond, which is consistent with previous observations, and altogether provide the basis to develop a breeding technology in captivity through hormonal and environmental manipulations.

Artificial germination of Enterocytozoon hepatopenaei (EHP) spores induced by ions under the scanning electron microscope.

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.

Design and evaluation of reverse transcription nested PCR primers for the detection of betanodavirus in finfish.

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.

Paper-based archiving of biological samples from fish for detecting betanodavirus.

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.

White spot syndrome virus (WSSV) genome stability maintained over six passages through three different penaeid shrimp species.

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.

Association of dual viral infection with mortality of Pacific white shrimp (Litopenaeus vannamei) in culture ponds in India.

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.

Genomic classification of betanodavirus by molecular phylogenetic analysis of the coat protein gene.

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.

Genetic characterization of betanodavirus isolates from Asian seabass Lates calcarifer (Bloch) in India.

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.

A short note on heavy infection of acanthocephalan worm (Neoechinorhynchus agilis) in grey mullet, Mugil cephalus.

Infection with acanthocephalan parasite, Neoechinorhynchus agilis (Acanthocephala: Neoechinorhynchidae) is reported from adult mullet (Mugil cephalus). Heavy infections with the parasites were observed in the posterior region of the intestine, almost blocking the lumen. At the site of parasite attachment, the surface of the intestine appeared thickened and the mucosal epithelium. A description of the parasites and its clinicopathology is discussed.

Viral nerve necrosis in hatchery-produced fry of Asian seabass Lates calcarifer: sequential microscopic analysis of histopathology.

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.

Evaluation of Pseudomonas sp. PM 11 and Vibrio fluvialis PM 17 on immune indices of tiger shrimp, Penaeus monodon.

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.

Epidemiology of parasitoses in dairy animals in the North West Humid Himalayan Region of India with particular reference to gastrointestinal nematodes.

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.

Experimental infection of mice with Taenia taeniaformis eggs from cats--course of infection and pathological studies.

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.

Prevalence of dicrocoeliosis in sheep and goats in Himachal Pradesh, India.

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.

Comparative evaluation of agar gel precipitation, counterimmunoelectrophoresis and passive haemagglutination tests for the diagnosis of Dicrocoelium dendriticum infection in sheep and goats.

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

Experimental studies on anaemia in sheep infected with Dictyocaulus filaria.

The pathogenesis of Dictyocaulus filaria induced anaemia was experimentally investigated. Nineteen Dorset-Muzaffarnagri male lambs were divided into two groups of 13 and six. The lambs in the former group received a primary infection dose of 2000 D. filaria infective larvae, whereas animals in the latter group were kept as uninfected controls. The haematological alterations and faecal larval output were monitored weekly until day 220 post-infection. The infection caused an increase in erythrocyte sedimentation rate (ESR) and total leukocyte count (TLC). However, a decrease in packed cell volume (PCV), haemoglobin, total erythrocyte count (TEC) and blood pH was observed in acute infection. The shape and size of the erythrocytes, serum bilirubin level, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) remained unaffected In carriers, except for TEC, the values of these parameters returned to near-normal levels. The analysis of the results suggests that the infected animals developed a normocytic normochromic anaemia which persisted during the later stage of infection.