Immune protection in carp, Cyprinus carpio L., after immunization with Aeromonas hydrophila crude lipopolysaccharide

Abstract. Vaccination with crude lipopolysaccharide (LPS) induced better protection against infection with Aeromonas hydrophila in carp than vaccination with formalin killed vaccine. Dipping fish in vaccine for 2 h at 25°C was more effective than intraperitoneal injection of the vaccine in procedural simplicity, lower stress loading and the degree of protection acquired. In carp immunized with crude LPS by the dip method, antibodies were not detected by bacteriai agglutination, passive haemagglutination and the agar diffusion tests. The results indicate that the protection against A. hydrophila infection in carp is not dependent on humoral immunity.     

Protective immunity of sevenband grouper, Epinephelus septemfasciatus Thunberg, against experimental viral nervous necrosis

This paper describes the protective immune responses of sevenband grouper, Epinephelus septemfasciatus Thunberg, immunized with live piscine nodavirus, the causative agent of viral nervous necrosis (VNN), or the Escherichia coli – expressed recombinant coat protein. Nodavirus-neutralizing antibodies were detected at titres ranging from 1:158 to 1:1257 in serum of sevenband grouper which survived intramuscular injection with the virus, by a cell culture assay system. The virus-neutralizing ability of immune serum was also confirmed by injecting virus previously treated with serum into fish. This indicates establishment of acquired immunity in survivors and thus explains why survivors from natural infection are resistant to recurrence of the disease. Young sevenband grouper were immunized twice by intramuscular injections with the recombinant coat protein. Immunized fish produced neutralizing antibodies at high titres for at least 110 days and showed significantly lower mortalities in virus challenge tests. These results suggest the potential for vaccination against VNN in sevenband grouper, which is susceptible to piscine nodavirus at all life-stages.     

Compensatory growth in Atlantic halibut: Effect of starvation and subsequent feeding on growth, maturation, feed utilization and flesh quality

A group of juvenile Atlantic halibut (mean, SD, 199.5 g, 44.7) was split into two experimental groups: one group was fed in excess twice a day and later five days a week (i.e. Control group), and the other group was starved for 5 weeks and then subsequently re-fed for 10 weeks (i.e. 5/10 starvation/re-fed group). This treatment was repeated for three years until the fish were harvested (mean final weight, 4.4 kg). A size +specific compensatory response was seen; partial compensation was observed in halibut weighing less than 500 g whereas full recovery growth was seen in halibut over 2 kg. Food consumption and feed conversion efficiency was measured during two full 5/10 starvation/re-fed cycles. The Control group consumed 86% more food in the measurement period, whereas the FCE 40% higher in the 5/10 starvation/re-fed group (FCE = 0.95) compared to the Control group (FCE = 0.68). In males lower gonadosomatic index (2.2 vs. 3.0) and plasma 11-ketotestosterone (0.22 vs. 1.26 ng ml^− 1) levels in the 5/10 starvation/re-fed group were observed during late winter and spring of 2006 (age 3 years) indicating higher age at 1st maturity. Starvation had a significant effect for improving the quality as starved fish had higher muscle pH (6.4 vs. 6.2), less gaping (0.1 vs. 0.3) and harder texture (61.4 vs. 57.6 N). Overall, the rearing on a repeated 5/10 starvation/re-fed regime for 3 years led to full growth compensation, higher feed conversion efficiency, lower male maturation and improved flesh quality.     

Validation of diagnostic assays to screen broodstock for Flavobacterium psychrophilum infections

It is hypothesized that the frequency of bacterial coldwater disease outbreaks can be reduced through the detection of the aetiologic agent, Flavobacterium psychrophilum, in broodstock followed by culling of eggs from heavily infected broodstock. Before a culling programme can be instituted, however, it is necessary to determine the sensitivity and specificity of existing assays for the detection of F. psychrophilum. In this study, tissue and ovarian fluid samples were collected from 224 fish at five hatcheries and screened using an enzyme‐linked immunosorbent assay (ELISA), a membrane‐filtration fluorescent antibody test (MF‐FAT), bacteriological culture and nested PCR. Latent class analysis was used to estimate sensitivity and specificity of kidney culture, kidney ELISA, nested PCR and MF‐FAT. Analytical sensitivity of the ELISA varied but was greatest when bacteria were cultured under iron‐limiting conditions. Diagnostic sensitivity estimates ranged from 0.02 (kidney culture) to 0.97 (kidney ELISA). Specificity estimates ranged from 0.02 (MF‐FAT) to 0.98 (kidney ELISA). In a separate challenge experiment, the ELISA confirmed the presence of F. psychrophilum in sub‐clinically infected fish. Results from this study demonstrate that the ELISA is an appropriate tool to screen broodstock and provides an indication of infection severity, which is crucial for implementation of a screening/culling programme.     

Investigations on the digestibility and metabolizability of the major nutrients and energy of maize leaves and barnyard grass in grass carp (Ctenopharyngodon idella)

In the uplands of northern Vietnam, culture of grass carp contributes significantly to income and household food security of Black Thai farmers. Maize is one of the most important upland crops and barnyard grass is the most important weed in the paddy rice fields. Thus, these are frequently used by small‐scale fish farmers as fish feed. An 8‐week feeding trial was conducted simultaneously in a recirculation and in a respirometric system to determine the digestibility and metabolizability of the nutrients of maize leaves and barnyard grass, to assess their crude protein, lipid and energy conversion and to estimate the energy allocation in grass carp. The following diets were used: diet ‘A’ (reference diet) containing 39% crude protein with 19.8 MJ kg^?1 gross energy; diets ‘B’, ‘C’ and ‘D’, which contained the same amount of the reference diet as the control diet, supplemented with a known amount of dried barnyard grass, dried maize leaves or fresh maize leaves, respectively. Five fish were assigned to each treatment in each experimental unit. Reference and test diets were fed to fish and faecal samples were collected and the oxygen consumption was measured in order to set up an energy budget of the fish over the whole experimental period. The weekly development of the body weight was recorded. The body weight gain of the fish fed diet D was significantly higher than that of the group fed diet A, which also in turn was significantly higher than that of fish fed diet B or C. The apparent digestibility coefficients (ADC) of nutrients and gross energy for the different experimental diets in fish kept in the aquaria were, for diets A, B, C and D respectively, 94.1%, 60.9%, 70.5% and 84.7% for protein, 91.3%, 60.7%, 76.8% and 71.8% for lipid; 95.9%, 44.5%, 60.6% and 69.1% for gross energy. The partial ADC of plant leaf ingredients were determined and barnyard grass and dried maize leaves were found to be not only poorly digestible but having negative impact on the digestibility of the reference diet, while fresh maize leaves were well digested. The results of the present study indicated that fresh maize leaves have a good potential to be used as supplement in diets for grass carp.     

Relative potencies of natural estrogens on vitellogenin and choriogenin levels in the Indian freshwater spotted snakehead, <Emphasis Type="Italic">Channa punctata</Emphasis>: in vivo and in vitro studies

The relative efficacies of three natural estrogens viz., estrone (E₁), estradiol-17β (E₂) and estriol (E₃) to induce synthesis of vitellogenin (Vg) and choriogenin (Chg) were assessed in primary hepatocyte cultures of the Indian freshwater spotted snakehead, Channa punctata. Hepatocytes were isolated from the spotted snakehead liver by a non-enzymatic protocol. Optimum culture conditions were standardized for ensuring their viability and functioning. Isolated hepatocytes were cultured for 48 h for monolayer formation and then exposed to various concentrations (0.001–10 μM) of the three estrogens. Competitive homologous ELISAs, developed and validated for spotted snakehead Vg and Chg were employed to determine the amounts of these two proteins secreted into the culture medium after 48 h of incubation. The results reveal that although all the three estrogens were effective in inducing the production of Vg and Chg in a dose-dependent manner, there were differences in their relative potencies. Of three estrogens, E₁ was the least potent and could induce synthesis of Vg and Chg only at a minimum concentration of 0.5 μM; whereas significant levels of both the proteins were quantified in culture medium by exposing the hepatocytes to E₂ or E₃ even at a concentration of 0.001 μM. All three estrogens were effective in inducing synthesis of Vg and Chg in vivo also. These results suggest the possibility of employing the above in vitro experimental design to monitor the presence of estrogens/estrogen-like chemicals in natural waters, which could interfere with the estrogen receptor system of fish. This study further points to the possibility of using Chg, in addition to Vg, as a parameter for screening various chemicals for their estrogenic activity.     

Differences in uptake and killing of pathogenic and non‐pathogenic bacteria by haemocyte subpopulations of penaeid shrimp,Litopenaeus vannamei, (Boone)

Phagocytosis is an important function of both invertebrate and vertebrate blood cells. In this study, the phagocytic activity of haemocyte subpopulations of penaeid shrimp, Litopenaeus vannamei, (Boone), against pathogenic and non‐pathogenic particles was investigated in vitro. The haemocytes of penaeid shrimp were firstly separated by centrifugation on a continuous density gradient of iodixanol into four fractions with five subpopulations (sub), of which sub 1 (hyalinocytes) and sub 4 (semi‐granulocytes) have the main function in phagocytosis of both pathogenic and non‐pathogenic bacteria as well as fluorescent polystyrene beads. It was found that these haemocyte subpopulations engulfed virulent Vibrio campbellii and Vibrio harveyi at a higher rate than non‐virulent Escherichia coli and polystyrene beads. When these bacteria were mixed with shrimp haemocyte subpopulations and incubated for 180 min, the percentage of viable intracellular V. campbellii (25.5 ± 6.0%) recovered was significantly higher than the percentage recovered from V. harveyi (13.5 ± 1.1%). No viable intracellular E. coli was observed in this study. In contrast to V. harveyi and E. coli, V. campbellii containing endosomes did not acidify in time. Incubation of haemocyte subpopulations with the most virulent V. campbellii strain resulted in a significant drop in haemocyte viability (41.4 ± 6.3% in sub 1 and 30.2 ± 15.1% in sub 4) after 180 min post‐inoculation in comparison with the less virulent V. harveyi (84.1 ± 5.6% in sub 1 and 83.4 ± 4.1% in sub 4) and non‐virulent E. coli (92.7 ± 2.8% in sub 1 and 92.3 ± 5.6% in sub 4) and polystyrene beads (91.9 ± 1.6% in sub 1 and 84.4 ± 3.4% in sub 4). These findings may be a valuable tool for monitoring shrimp health and immunological studies.     

Description and phylogeny of Ceratomyxa anko sp. n. and Zschokkella lophii sp. n. from the Japanese anglerfish, Lophius litulon (Jordan)

Two new species of myxozoans from the Japanese anglerfish, Lophius litulon, are described using myxospore morphology and small subunit rDNA sequences. Ceratomyxa anko sp. n. is a parasite of the gall bladder and had a prevalence of 57%. Mature spores of C. anko sp. n. are arcuate to crescent shaped with valves tapering to rounded tips. A prominent sutural line runs centrally between the round adjacent polar capsules containing the polar filament coiled two to three times. Spore measurements: length 10.8 (9.7-11.9) microm, width 41.9 (36.9-47.2) microm, polar capsule diameter 4.6 (4.1-5.3) microm. Ceratomyxa anko sp. n. can be distinguished from other Ceratomyxa spp. due to its spore dimensions and shape. Zschokkella lophii sp. n. is a parasite of the urinary bladder and had a prevalence of 70%. Mature spores are ellipsoidal to semicircular with bluntly pointed ends. The sutural line is curved or sinuous and the valves have no discernable surface ornamentation. Two almost spherical polar capsules are located separately in the ends of the spore, opening in almost opposite directions and contain the polar filament with five coils. Spore measurements: length 20.1 (16.8-24.0) microm, width 14.9 (12.7-16.8) microm, polar capsule diameter 5.1 (3.6-5.8) microm. Zschokkella lophii sp. n. can be distinguished from other Zschokkella spp. due to the terminal opening of the polar capsules within the spores and the site of infection within the host fish. In the phylogenetic analyses, C. anko sp. n. grouped with other members of the same genus forming a monophyly. Zschokkella lophii sp. n. forms a discrete clade with another Zschokkella sp. that infects the urinary bladder of marine fish. This grouping forms a sister clade to one containing members of the genus Parvicapsula, all of which are parasites of the urinary system in marine fish.     

Biochemical composition and quality of captive-spawned cobia Rachycentron canadum eggs

Interest in the commercial production of cobia Rachycentron canadum continues to rise as additional insight is gained into the hardy and fast growing nature of this species. However, research regarding the biochemical composition of captive-spawned eggs and egg and larval quality remains scarce. Such data is essential as a common bottleneck to production is a steady supply of fingerlings for grow-out. This study quantified the biochemical composition and quality of cobia eggs produced over 2 spawning seasons by broodstock on a traditional ‘trash fish’ diet which is commonly fed to tank spawning cobia. Throughout the study, batch fecundity, proportion of floating eggs and percent hatch averaged > 1 million eggs,  0.8 and 70%, respectively. Batch fecundity was significantly higher during the second spawning season as a result of the increased size of the females which weighed 18/22 kg and 22/26 kg at the beginning of each season. A positive correlation was found between the proportion of floating eggs and hatch rate for both spawning seasons. No correlations were found between egg composition (total lipid (30.0 ± 1.1% dry wt), protein (25.4 ± 2.2% dry wt), carbohydrate (2.4 ± 0.3% dry wt), vitamin E (10.2 ± 0.6 μg/g wet wt) or dry weight (119.1 ± 5.5 μg/egg)) and egg quality (proportion of floating eggs, hatch rate, larval growth and larval survival). Further, no differences in egg composition were noted between seasons or over the course of each season. The fatty acid composition of cobia eggs varied between seasons possibly due to changes in the quality of frozen feed (fish, shrimp, squid) given to the broodstock. The only correlation between the fatty acid profile and egg quality was a decrease in the proportion of floating eggs as the total amount of n-3 highly unsaturated fatty acids increased. No relationship between egg quality and amino acid content was noted with the most prominent amino acids being glutamate, leucine, alanine, proline, lysine and aspartate nor were any differences detected between spawning seasons.