Levels of detection of Cryptosporidium oocysts in mussels (Mytilus galloprovincialis) by IFA and PCR methods

Cryptosporidium spp. are monoxenous protozoan parasites that cause gastrointestinal diseases in humans and animals. Shellfish harvesting areas can become contaminated by the infectious stage of the parasite and humans are therefore at risk of infection either by consumption of shellfish, or by taking part in recreational activities in these areas. In the present study we determined the levels of detection, by IFA and PCR techniques, of Cryptosporidium oocysts in mussels experimentally contaminated with a theoretical number of oocysts. There was a significant correlation between the results obtained by both techniques (P<0.05). IFA and PCR were also applied to a total of 222 samples of mussels (Mytilus galloprovincialis) destined for human consumption. In the naturally contaminated samples, we detected a 31.1% of contamination and only Cryptosporidium parvum (previously denominated C. parvum genotype II) was identified.     

A survey for BVDV antibodies in cattle farms in Slovakia and genetic typing of BVDV isolates from imported animals

A serological survey for bovine viral diarrhoea virus (BVDV) antibodies on a collection of 1295 serum samples obtained from 6-12 months old cattle originating from 45 farms in Slovakia was carried out. On 13 farms more than 90% of the examined animals were seropositive, on 14 farms 71-90% seroprevalence was observed, on 13 farms only 50-70% animals were found to be positive for BVDV antibodies, while the remaining 5 farms showed fewer than 50% seropositive animals. The average incidence of BVDV antibodies (around 70%) was similar as determined 30 years ago. Of 84 serum samples from seronegative animals originating from 14 farms in which 70-98% seropositivity was observed, six were positive in Ag-BVDV ELISA indicating persistently infected (PI) cattle. On a farm to which animals were imported from abroad, a BVD outbreak was observed. Of 110 animals tested, four were positive in Ag-ELISA indicating the presence of PI cattle on this farm. Genetic typing of two isolates from imported animals performed by RT-PCR (324/326 primers from 5'-UTR), sequencing of PCR products and computer-assisted phylogenetic analysis revealed that they belong to BVDV-1 h group.     

Effect of increasing docosahexaenoic acid content in weaning diets on survival,growth and skeletal anomalies of longfin yellowtail (Seriola rivoliana,Valenciennes 1833)

Five isoproteic (54.8%) and isolipidic (24.1%) microdiets, which varied in their docosahexaenoic acid (DHA) content (0.25%, 0.75%, 1.64%, 1.99% and 3.17%; dw), were manufactured to determine its effects on longfin yellowtail Seriola rivoliana larvae in terms of fish biological performance, whole body fatty acid profile and incidence of skeletal anomalies from 30 dah (11.31 ± 1.79 Total Length, TL) to 50 dah (19.80 ± 0.58 mm TL). The inclusion of dietary DHA up to 3.17% (dw) improved larval resistance to air exposure, although DHA did not significantly affect fish final growth or final survival. Indeed, high levels of dietary DHA (1.99% and 3.17%, dw) tended to increase the incidence of skeletal anomalies in S. rivoliana larvae, albeit no significant differences were observed. Furthermore, the occurrence of severe anomalies such as kyphosis and lordosis, was mainly associated to the larvae fed the highest levels of dietary DHA. In terms of survival, increasing dietary DHA levels did not significantly affect longfin yellowtail survival rate, despite a tendency for enhanced survival. The results of the present study proved that the inclusion of dietary DHA in inert diets up to a 3.17% (dw) and a DHA/EPA ratio above 3.1 increased the final survival and stress resistance in S. rivoliana larvae.     

Live prey first feeding regimes for short‐snouted seahorse Hippocampus hippocampus (Linnaeus, 1758) juveniles

As with many species of seahorses, Hippocampus hippocampus wild populations are being subjected to uncontrolled exploitation in their natural environment. Thus, aquaculture could contribute to satisfy the commercial demand for animals while promoting the recovery of wild stocks. The present study was conducted to compare the effect of the substituting Artemia nauplii with rotifers for first feeding seahorse juveniles. Survival, growth and biochemical composition of prey organisms and fish were studied during the feeding trial. In addition, to help the biometric study, an anaesthetic test was also carried out using clove oil. The results showed excellent survival (average 60%) in juveniles exclusively fed with Artemia, with better values than those reported previously obtained by other authors for this species. By comparison, high mortality and poor growth were observed during first feeding with seahorses fed on rotifers. This could have been related to the lower energy intake and poorer nutritional value of the rotifers. Furthermore, clove oil concentrations of 25 ppm were found to work well as an anaesthetic for seahorse juveniles. Overall, first feeding Artemia alone was found to be an efficient and simplified method for feeding young H. hippocampus fry, building the principles for their culture for ornamental or re‐stocking purposes.     

High‐quality spontaneous spawning in greater amberjack (Seriola dumerili,Risso 1810) and its comparison with GnRHa implants or injections

Production of sufficient high‐quality eggs of greater amberjack (Seriola dumerili) still constitutes the main bottleneck for commercial production of this species. The main objective of this study was to compare the quality of spontaneous spawn of greater amberjack with those obtained by either GnRHa injection or GnRHa implant protocols. Captive amberjack broodstock were distributed in three circular tanks of 40 m³. Broodstock from Tank 1 were not hormonally induced and spawned spontaneously, whereas those of Tank 2 were intramuscularly injected with GnRHa (20 µg/kg body weight) and those of Tank 3 were given EV‐500 µg GnRHa implants. The number of eggs per spawn obtained in the broodstock without hormonal treatment was larger than in those obtained with injections or implants. Egg quality was best in broodstock with spontaneous spawn, followed by GnRHa‐injected fish and then GnRHa implants. Besides, size of larvae from control and injected broodstock was similar between them and significantly higher (p < 0.01) than those from GnRHa implant spawn. Overall, this study showed that it is possible to obtain very high‐quality spontaneous spawn in greater amberjack, providing the adequate conditions. Furthermore, GnRHa weekly injections lead to similar egg viability and hatching rates than spontaneous spawn and higher fertilization rates than GnRHa hormonal implants, which is better than in previous studies.     

Use of Microbacterium sp. and Exiguobacterium mexicanum to improve the survival and development of Artemia under xenic conditions

The effect of Microbacterium sp. strain 8L and Exiguobacterium mexicanum strain 8N was evaluated in the diet of Artemia under xenic conditions. Viable cultures of bacteria were provided to xenic cultures of Artemia in combination with Sacharomyces cerevisiae, cornflour or Spirulina, and the effect on the survival and growth was recorded. The use of these bacterial strains improves significantly the survival of Artemia independently of the used food (P < 0.05), and variable results were observed in the growth.     

Immune response to Encephalitozoon cuniculi infection in laboratory mice.

The study was performed to determine the immune response to Encephalitozoon cuniculi infection in immunocompetent mice during 120 days of experiment. Mice infected with E. cuniculi had an increased number of CD4+ T cells up to Day 20 post infection (p.i.), but counts of CD8+ T lymphocytes were significantly lower up to Day 90 p.i. in peripheral blood. Blood monocytes were significantly increased on the Day 60 and Day 120 of infection. A lack of significant decrease of CD4+ T cells may be considered as an important event in the immune response to E. cuniculi infection in immunocompetent mice.     

Evaluation of Aqueous Biphasic Electrophoresis System Based on Halide-Free Ionic Liquids for Direct Recovery of Keratinase

Aqueous biphasic electrophoresis system (ABES) incorporates electric fields into the biphasic system to separate the target biomolecules from crude feedstock. Ionic liquid (IL) is regarded as an excellent candidate as the phase-forming components for ABES because of the great electrical conductivity, which can promote the electromigration of biomolecules in ABES, and thereby enhances the separation efficiency of the target biomolecules from crude feedstock. The application of electric fields to the conventional biphasic system expedites the phase settling time of the biphasic system, which eases the subsequent scaling-up steps and reduces the overall processing time of the recovery process. Alkyl sulphate-based IL is a green and economical halide-free surfactant when compared to the other halide-containing IL. The feasibility of halide-free IL-based ABES to recover Kytococcus sedentarius TWHK01 keratinase was studied. Optimum partition coefficient (K_e = 7.53 ± 0.35) and yield (Y_T = 80.36% ± 0.71) were recorded with IL-ABES comprised of 15.0% (w/w) [EMIM][ESO₄], 20.0% (w/w) sodium carbonate and 15% (w/w) crude feedstock. Selectivity (S) of 5.75 ± 0.27 was obtained with the IL-ABES operated at operation time of 5 min with 10 V voltage supplied. Halide-free IL is proven to be a potential phase-forming component of IL-ABES for large-scale recovery of keratinase.     

Effect of Different Live Prey on Spawning Quality of Short‐Snouted Seahorse,Hippocampus hippocampus (Linnaeus, 1758)

The importance of a suitable diet for reproduction has been recognized as one of the major factors in ornamental aquaculture. In seahorses, mysids have been described as preys in the wild. Also, Artemia has been usually employed for rearing fish, including syngnathids. Therefore, the aim of this work was to evaluate the effect of these live preys on the spawning quality of Hippocampus hippocampus. After 108 d, no differences were found in adults concerning all biological parameters evaluated, but broodstock fed on mysids showed better results than Artemia regarding number of spawning events (12 vs. 3), brood sizes (233.50 ± 59.04 vs. 68.00 ± 57.97 juveniles), and newborn seahorses standard length (10.61 ± 0.64 vs. 8.75 ± 1.32 mm). The better nutritional quality of mysids, overall in Docosahexanoic acid, could be one of the main responsible factors. However, mysids stock is conditioned by natural catches and rearing techniques are little known. Another alternative would be to combine them with Artemia in mixed diet. Further research must be done concerning mysids breeding techniques to delineate their employment as a sustainable prey for seahorse aquaculture. This trial showed for the first time the effect of mysids to enhance reproduction efficiency in H. hippocampus.     

Isolation and control of the functional quality of mRNA of bovine leukocyte interferon]

For the construction of the cDNA library total cellular RNA was separated from bovine leucocytes induced with NDV (inductor of interferons) for five hours. Guanidinisothiocyanate and ultracentrifugation in the gradient of CsCl were used for the separation of the total RNA (Chirgwin et al., 1979). Bands 18S and 28S were detected in the samples of RNA by electrophoresis in an MOPS buffer (Fig. 1). mRNA was separated from the mixture of RNA via affinity chromatography on oligo(dT)-cellulose (Aviv and Leder, 1972). The function quality of mRNA BoIFN-alpha was verified by microinjection into the oocytes of Xenopus laevis using a microinjector of our own construction (Fig. 2). The microinjector was calibrated by injection of a radioactive 51Cr solution (Fig. 3). It was found out on the basis of CPE inhibition measurements that the injected oocytes synthesized 320-640 U/ml BoIFN-alpha (16-32 U/50 microliters; Tab. I).