Delivery of HUFA, probionts and biomedicine through bioencapsulated Artemia as a means to enhance the growth and survival and reduce the pathogenesity in shrimp Penaeus monodon postlarvae

One of the major problems in the shrimp culture industry is the difficulty in producing high-quality shrimp larvae. In larviculture, quality feeds containing a high content of highly unsaturated fatty acids (HUFA) and ingredients that stimulate stress and disease resistance are essential to produce healthy shrimp larvae. In the present study, Penaeus monodon postlarvae (PL15) were fed for 25 days on an unenriched Artemia diet (control; A) or on a diet of Artemia enriched with either HUFA-rich liver oil of the trash fish Odonus niger (B), probionts [Lactobacillus acidophilus (C1) or yeast-Saccharomyces cerevisiae (C2)] or biomedicinal herbal products (D) that have anti-stress, growth-promoting and anti-microbial characteristics. P. monodon postlarvae fed unenriched Artemia exhibited the lowest weight gain (227.9 ± 8.30 mg) and specific growth rate (9.95 ± 0.05%), while those fed the HUFA-enriched Artemia (B) exhibited the highest weight gain and specific growth rate (362.34 ± 12.56 mg and 11.77 ± 0.08%, respectively). At the end of the 25-day rearing experiment, the shrimp postlarvae (PL40) were subjected to a salinity stress study. At both low and high (0 and 50‰) salinities, the group fed the control diet (A) experienced the highest cumulative mortality indices (CMI) 935.7 ± 2.1 and 1270.7 ± 3.1, respectively. Those fed diet D showed the lowest stress-induced mortality, and CMI were reduced by 31.1 and 32.3% under conditions of low and high salinity stress, respectively. A 10-day disease challenge test was conducted with the P. monodon postlarvae (PL40–PL50) by inoculating the shrimp with the pathogen Vibrio harveyi at the rate of 10⁵–10⁷ CFU/ml in all rearing tanks. P. monodon postlarvae fed probiont-encapsulated Artemia diets (C1 and C2) exhibited the highest survival (94.3 and 82.3%, respectively) and lowest pathogen load (V. harveyi) in hepatopancreas (5.2 × 10² ± 9.0 × 10 and 4.6 × 10² ± 9.0 × 10 CFU g⁻¹, respectively) and muscle (2.0 × 10² ± 6 × 10 and 1.7 × 10² ± 8.6 × 10 CFU g⁻¹, respectively) tissues. The shrimp that were fed the unenriched Artemia (Control; A) showed the lowest survival (26.33%) and highest bacterial load in the hepatopancreas (1.0 × 10⁵ ± 5 × 10³ CFU g⁻¹) and muscle (3.6 × 10⁴ ± 6 × 10² CFU g⁻¹). The shrimp fed the herbal product (D)-enriched Artemia also exhibited enhanced survival and reduced V. harveyi load in the tissues tested compared to the control diet (A) group. The results are discussed in terms of developing a quality larval feed to produce healthy shrimp larvae.     

Comparative evaluation of live marker vaccine candidates "CP7_E2alf" and "flc11" along with C-strain "Riems" after oral vaccination

Due to the tremendous socio-economic impact of classical swine fever (CSF) outbreaks, emergency vaccination scenarios are continuously under discussion. Unfortunately, all currently available vaccines show restrictions either in terms of marker capacities or immunogenicity. Recent research efforts were therefore directed at the design of new modified live marker vaccines. Among the most promising candidates the chimeric pestiviruses "CP7_E2alf" and "flc11" were identified. Within an international research project, these candidates were comparatively tested in challenge experiments after a single oral vaccination. Challenge infection was carried out with highly virulent CSF virus strain "Koslov", 14 or 21 days post vaccination (dpv), respectively. Safety, efficacy, and marker potential were addressed. All assessments were done in comparison with the conventional "gold standard" C-strain "Riems" vaccine. In addition to the challenge trials, multiple vaccinations with both candidates were performed to further assess their marker vaccine potential. All vaccines were safe and yielded full protection upon challenge 21 days post vaccination. Neither serological nor virological investigations showed major differences among the three vaccines. Whereas CP7_E2alf also provided clinical protection upon challenge at 14 days post vaccination, only 50% of animals vaccinated with flc11, and 83% vaccinated with C-strain "Riems" survived challenge at this time point. No marked differences were seen in protected animals. Despite the fact that all multiple-vaccinated animals stayed sero-negative in the accompanying marker test, the discriminatory assay remains a weak point due to delayed or inexistent detection of some of the vaccinated and subsequently infected animals. Nevertheless, the potential as live marker vaccines could be confirmed for both vaccine candidates. Future efforts will therefore be directed at the licensing of "Cp7_E2alf" as the first live marker vaccine for CSF.     

Antibacterial effect of medium-chain fatty acid: caprylic acid on gnotobiotic <Emphasis Type="Italic">Artemia franciscana</Emphasis> nauplii against shrimp pathogens <Emphasis Type="Italic">Vibrio harveyi</Emphasis> and <Emphasis Type="Italic">V. parahaemolyticus</Emphasis>

The present study was carried out to determine the antibacterial effect of caprylic acid in the culture system of Artemia franciscana nauplii inoculated with shrimp pathogens Vibrio harveyi and V. parahaemolyticus. To begin with, the antibacterial effect of different concentrations (1, 10 and 100 mM) of caprylic acid against V. harveyi and V. parahaemolyticus was assessed through bacterial growth study. This indicated that at 100 mM concentration, the growth of the pathogens was completely inhibited within 5 h, whereas, in 1.0 and 10 mM concentrations, effective inhibition was observed with the extension of time. Subsequently, the influence of pH variation on the growth inhibitory effect of 10 mM caprylic acid against V. harveyi and V. parahaemolyticus at different pH (5–7) was also made through bacterial growth study. At pH 5, the pathogen growth was very less, compared with pH 6 and 7. The Artemia nauplii (instar II) reared in 10 mM caprylic acid supplemented medium were challenged with V. harveyi and V. parahaemolyticus, and the mortality was recorded at an interval of 6 h up to 60 h. In this study, the mortality of Artemia nauplii reduced to 20.61 and 16.30% in V. parahaemolyticus and V. harveyi challenged groups, respectively. The present results provide evidence for the potential antibacterial activity of caprylic acid in aquaculture against luminescent vibrios.     

Immunostimulating influence of herbal biomedicines on nonspecific immunity in Grouper Epinephelus tauvina juvenile against Vibrio harveyi infection

Herbals such as Cynodon dactylon, Piper longum, Phyllanthus niruri, Tridax procumbens, and Zingiber officinalis were extracted with acetone, benzene, butanol, and petroleum ether and screened against the pathogen Vibrio harveyi isolated from the infected Grouper Epinephalus tauvina. Among the different solvent extractions screening to V. harveyi, petroleum ether extracts were suppressed significantly (P < 0.05). Equal proportions of the all-plant extracts were mixed with the artificial feeds at concentrations of 100, 200, 400, and 800 mg kg⁻¹ of diet and fed to grouper juveniles of 20 ± 2 g average weight for a period of 60 days. Every 20 days, fish juveniles were challenged with V. harveyi and the immune response was studied. The herbal diets significantly (P < 0.05) increased the survival, growth, and immune responses compared to the control group. The herbal diets were significantly improved (P < 0.01) in immune parameters such as phagocytic activity and albumin–globulin (A–G) ratio. Among the different concentrations of the herbals in the diet, the 400 mg kg⁻¹ diet was the most effective in the experiment.     

Characterization of C-strain "Riems" TAV-epitope escape variants obtained through selective antibody pressure in cell culture

ABSTRACT: Classical swine fever virus (CSFV) C-strain "Riems" escape variants generated under selective antibody pressure with monoclonal antibodies and a peptide-specific antiserum in cell culture were investigated. Candidates with up to three amino acid exchanges in the immunodominant and highly conserved linear TAV-epitope of the E2-glycoprotein, and additional mutations in the envelope proteins ERNS and E1, were characterized both in vitro and in vivo.It was further demonstrated, that intramuscular immunization of weaner pigs with variants selected after a series of passages elicited full protection against lethal CSFV challenge infection. These novel CSFV C-strain variants with exchanges in the TAV-epitope present potential marker vaccine candidates. The DIVA (differentiating infected from vaccinated animals) principle was tested for those variants using commercially available E2 antibody detection ELISA. Moreover, direct virus differentiation is possible using a real-time RT-PCR system specific for the new C-strain virus escape variants or using differential immunofluorescence staining.     

Effect of Sargassum wightii fucoidan on growth and disease resistance to Vibrio parahaemolyticus in Penaeus monodon post‐larvae

The polysaccharide – fucoidan was extracted from brown seaweed Sargassum wightii and its antibacterial activity was screened by agar well diffusion method. The maximum zone of inhibition observed was 15.66 mm in 20 mg mL^?1 concentration against Vibrio parahaemolyticus. The Minimum Inhibitory Concentration (MIC) of the fucoidan was 12 mg mL^?1 against V. parahaemolyticus. The fucoidan was then enriched with Artemia nauplii at four different concentrations such as 100, 200, 300 and 400 mg L^?1 for 12 h. The enriched Artemia nauplii were fed to Penaeus monodon post‐larvae for 20 days and the growth performance was assessed. The weight gain and SGR of the control group were 0.2432 g and 15.78%, respectively. But, in experimental groups fed with fucoidan enriched Artemia nauplii, the weight gain and SGR were increased and were respectively ranged from 0.2602 to 0.3161 g and from 16.11 to 17.05%. The P. monodon post‐larvae were challenged with V. parahaemolyticus for a period of 30 days showed a reduction in mortality percentage of experimental groups over the control group and it was ranged between 36.97 and 89.86%. During the challenge test, the V. parahaemolyticus load was also enumerated from the infected shrimp at every 10 day intervals. In the control group, the Vibrio load showed a linear increase in hepatopancreas and muscle tissues from 10th to 30th days of challenge test, whereas in the experimental groups, the Vibrio load established a declining trend with the advancement of challenged test.     

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Antibacterial effect of short‐chain fatty acids on gnotobiotic Artemia franciscana nauplii against Vibrio parahaemolyticus

The present study was undertaken to determine the antibacterial effect of short‐chain fatty acids (SCFA) such as acetic, propionic and butyric acids on gnotobiotic Artemia franciscana nauplii against Vibrio parahaemolyticus. An initial experiment was undertaken to determine the antibacterial effect of different concentrations (1, 10 and 100 mM) of individual SCFA against V. parahaemolyticus through bacterial growth study at pH 6. In this study, the pathogen growth was completely inhibited at 100 mM concentration of all the tested SCFA. In another investigation, the different pH (5–7) on growth inhibitory effect of 20 mM SCFA was tested against V. parahaemolyticus through bacterial growth study. In this study, the pathogen growth was highly inhibited at pH 5 in all the tested SCFA. The Artemia nauplii were gnotobiotically reared in 20 mM SCFA supplemented medium and the mortality was recorded at an every 6 h interval up to 60 h after challenged with V. parahaemolyticus. The reduction in mortality (%) of A. franciscana nauplii over control was ranged from 25.36% to 38.56% in all the tested SCFA.     

Effect of hot water extracts of brown seaweeds Sargassum spp. on growth and resistance to white spot syndrome virus in shrimp Penaeus monodon postlarvae

An experiment was conducted to evaluate the effect of a hot water extract of brown seaweeds Sargassum duplicatum and Sargassum wightii on the growth and white spot syndrome virus (WSSV) resistance in shrimp Penaeus monodon postlarvae (PL). Artemia nauplii (instar II) were enriched with both seaweed extracts at various concentrations (250, 500 and 750 mg L^?1) and fed to the respective P. monodon (PL15–35) group for 20 days. A control group was also maintained without seaweed extract supplementation. The weight gain of the experimental groups was significantly higher (0.274–0.323 g) than the control group (0.261 g). Similarly, the specific growth rate was also significantly higher (16.27–17.06%) in the experimental groups than in the control group (16.03%). After 20 days of the feeding experiment, the shrimp PL were challenged with WSSV for 21 days. During the challenge test, the control shrimp displayed 100% mortality within 8 days. In contrast, the mortality percentage of the highest concentration (750 mg L^?1) of seaweed extract enriched Artemia nauplii fed shrimp was 54–79%. Comparatively, low mortality was observed in S. wightii extract‐enriched Artemia nauplii fed shrimp. The polymerase chain reaction analysis indicated the concentration‐dependent infection of WSSV in P. monodon PL.     

Antiviral Effect of Fucoidan Extracted from the Brown Seaweed,Sargassum wightii,on Shrimp Penaeus monodon Postlarvae against White Spot Syndrome Virus

This study was carried out to determine the effect of seaweed polysaccharide, fucoidan from brown seaweed, Sargassum wightii, on Penaeus monodon postlarvae (PL) against white spot syndrome virus (WSSV). The fucoidan was extracted from S. wightii, and the yield was observed as 2.832 ± 0.204%. Artemia franciscana nauplii were enriched with extracted fucoidan at four different concentrations (100, 200, 300, and 400 mg/L) and fed to shrimp P. monodon PL for 20 d. After feeding experiment, the P. monodon PL were challenged with WSSV, and the mortality percentage was recorded daily up to 21 d. During the challenge test, the control PL showed 100% cumulative mortality within 9 d, but the fucoidan‐enriched Artemia nauplii fed groups of PL exhibited 60–94% cumulative mortality within 21 d. The reduction in mortality percentage of experimental groups of PL over control PL was ranged between 33.71 and 61.65%. The polymerase chain reaction analysis confirmed the concentration‐dependent variation in WSSV infection.