Marco Galeotti

Prevention of stress in rainbow trout (Oncorhynchus mykiss) fed diets containing different doses of glucan

An experiment was performed to determine the effects of stress on non-specific defence mechanisms in rainbow trout fed diets containing different doses of glucan. Fish were fed with 0, 0.1, 0.5 and 1.0% glucan concentration in food. After 4 weeks of feeding, fish were stressed by 2 h transportation. The effect of stress was investigated by measuring changes in blood levels of cortisol, glucose, total protein and composition of their leukocyte population, as well as by changes in non-specific defence mechanisms of the fish. After 4 weeks feeding with glucan, elevated phagocytosis and oxidative radical production were observed in treated fish, but the levels did not correlate with the different doses of glucan. Stress induced by 2 h transportation caused high cortisol levels in plasma and hyperglycaemia in all groups, but the lowest level of glucose was measured in the group fed the low (0.1%) dose of glucan. Respiratory burst activity, phagocytosis, serum protein and lyzosyme levels were found to be significantly reduced by stress. The most dramatic reduction was observed in the control group, but the changes were not affected by glucan doses. One week post stress, hyperglycaemia was still observed in control and fish fed medium and high doses of glucan. Further reductions of total protein and intracellular oxidative radical production, were measured in all groups, but in fish fed with low dose of glucan the changes were less dramatic. The phagocytosis ratio increased in all groups, but did not attain the levels measured before stress in control and in the group fed the high concentration of glucan. A spontaneous infection with Flexibacter columnaris caused mortality in all groups except the group fed the low level of glucan. The results of the present study show that feeding of glucan in low doses several weeks before transportation can help to prevent negative effects of stress.

Identification of a Novel RNA Virus Lethal to Tilapia

ABSTRACT Tilapines are important for the sustainability of ecological systems and serve as the second most important group of farmed fish worldwide. Significant mortality of wild and cultured tilapia has been observed recently in Israel. The etiological agent of this disease, a novel RNA virus, is described here, and procedures allowing its isolation and detection are revealed. The virus, denominated tilapia lake virus (TiLV), was propagated in primary tilapia brain cells or in an E-11 cell line, and it induced a cytopathic effect at 5 to 10 days postinfection. Electron microscopy revealed enveloped icosahedral particles of 55 to 75 nm. Low-passage TiLV, injected intraperitoneally in tilapia, induced a disease resembling the natural disease, which typically presents with lethargy, ocular alterations, and skin erosions, with >80% mortality. Histological changes included congestion of the internal organs (kidneys and brain) with foci of gliosis and perivascular cuffing of lymphocytes in the brain cortex; ocular inflammation included endophthalmitis and cataractous changes of the lens. The cohabitation of healthy and diseased fish demonstrated that the disease is contagious and that mortalities (80 to 100%) occur within a few days. Fish surviving the initial mortality were immune to further TiLV infections, suggesting the mounting of a protective immune response. Screening cDNA libraries identified a TiLV-specific sequence, allowing the design of a PCR-based diagnostic test. This test enables the specific identification of TiLV in tilapines and should help control the spread of this virus worldwide.

A preliminary histological and ultrastructural study of opercular anomalies in gilthead sea bream larvae (Sparus aurata) [Pagrus aurata].

The high prevalence of opercular deformities present in fish from Mediterranean marine hatcheries is an important problem, which is causing considerable economic losses. The aetiology of this syndrome is not yet well understood. In this study a histological and ultrastructural description of gilthead sea bream (Sparus aurata) larvae affected with anomalies of the opercular complex was carried out.Samples of larvae were obtained from a marine hatchery located in north-eastern Italy. For each sample, 100 larvae (age range day 0 to 90) were fixed for histological and ultrastructural observations. The first opercular alterations were detected 17 days after hatching, when the opercular support in the branchio-cranial area is still cartilaginous and the only structure of the opercle is a connective lamina composed of a dense connective tissue. Deformation could affect one or both opercles. No degenerative alterations or inflammatory processes were found in the opercular tissues.The electron microscopy (TEM) observations on 30 day old larvae allowed the identification of mineral deposition abnormalities within the dermal ossification in curled opercles. The mineralization process appears to be very heterogeneous and irregular, in some cases being less abundant than normally detected. These preliminary observations suggest that an early alteration in the connective tissue formation and an abnormal mineralization of the fibrous bone can predispose opercular folding.

Innovative vaccination protocol against vibriosis in Dicentrarchus labrax (L.) juveniles: Improvement of immune parameters and protection to challenge

The effect of vaccination on immune parameters of European sea bass, Dicentrarchus labrax, is not fully established, as well as surveyed throughout rearing till the commercial size. Furthermore, available information on the possible role of booster treatments is scarce. Sea bass juveniles were vaccinated against Listonella anguillarum using a commercial bivalent formulation administered by immersion (priming: 95 dph; booster: 165 dph) or by immersion (priming: 95 dph; booster: 165 dph) and subsequent i.p. injection (booster: 233 dph). Serum specific IgM and numbers of IgM(+) cells in head kidney and spleen evidenced B-cell responses mainly after the immersion booster, accompanied by increased TcR-β transcripts and leucocyte respiratory burst. Immune enhancement was confirmed by the protection towards i.p. challenges with a virulent strain. RPS accounted for >70% in fish immersion-boosted and near 100% in fish further boosted i.p. Differently from usual farm practices, this innovative vaccination protocol proved to be highly effective. Booster treatments are therefore strongly recommended.

Differential characterization of emerging skin diseases of rainbow trout - a standardized approach to capturing disease characteristics and development of case definitions

Farmed and wild salmonids are affected by a variety of skin conditions, some of which have significant economic and welfare implications. In many cases, the causes are not well understood, and one example is cold water strawberry disease of rainbow trout, also called red mark syndrome, which has been recorded in the UK since 2003. To date, there are no internationally agreed methods for describing these conditions, which has caused confusion for farmers and health professionals, who are often unclear as to whether they are dealing with a new or a previously described condition. This has resulted, inevitably, in delays to both accurate diagnosis and effective treatment regimes. Here, we provide a standardized methodology for the description of skin conditions of rainbow trout of uncertain aetiology. We demonstrate how the approach can be used to develop case definitions, using coldwater strawberry disease as an example.

Development and application of a real-time PCR assay for the detection and quantitation of lymphocystis disease virus

Lymphocystis disease virus (LCDV) is responsible for a chronic self-limiting disease that affects more than 125 teleosts. Viral isolation of LCDV is difficult, time-consuming and often ineffective; the development of a rapid and specific tool to detect and quantify LCDV is desirable for both diagnosis and pathogenic studies. In this study, a quantitative real-time PCR (qPCR) assay was developed using a Sybr-Green-based assay targeting a highly conserved region of the MCP gene. Primers were designed on a multiple alignment that included all known LCDV genotypes. The viral DNA segment was cloned within a plasmid to generate a standard curve. The limit of detection was as low as 2.6DNA copies/μl of plasmid and the qPCR was able to detect viral DNA from cell culture lysates and tissues at levels ten-times lower than conventional PCR. Both gilthead seabream and olive flounder LCDV has been amplified, and an in silico assay showed that LCDV of all genotypes can be amplified. LCDV was detected in target and non-target tissues of both diseased and asymptomatic fish. The LCDV qPCR assay developed in this study is highly sensitive, specific, reproducible and versatile for the detection and quantitation of Lymphocystivirus, and may also be used for asymptomatic carrier detection or pathogenesis studies of different LCDV strains.

Preliminary study on expression of antimicrobial peptides in European sea bass (Dicentrarchus labrax) following in vivo infection with Vibrio anguillarum. A time course experiment.

Antimicrobial polypeptides (AMPPs) are humoral components of the vertebrates and invertebrates innate immune system. Their potent broad spectrum antimicrobial activities have drawn the attention of the scientific community to their potential use not only as an alternative to antibiotics but also as functional targets for immunostimulants in order to enhance the host immunity. Fish synthesize a great number of these peptides but in European sea bass, an important fish species in the Mediterranean aquaculture, only a few AMPPs have been studied and these surveys have highlighted their functional role as predictive markers of stressful conditions. Many aspects concerning AMPP mode of action in the host during bacterial infections are still unknown. In this work a 72 h time course experiment, performed on juvenile sea bass intraperitoneally (i.p.) injected with a sub-lethal dose of Vibrio anguillarum, was aimed to investigate the mRNA expression of four specific AMPP genes and interleukin-1β (IL-1β) in skin, gills, spleen, and head kidney. AMPP genes were: dicentracin (DIC), histone-like protein 1 (HLP-1), histone-like protein 2 (HLP-2) and hemoglobin-like protein (Hb-LP). The delta-delta C(T) method in real-time RT-PCR allowed to gain more knowledge about temporal dynamics, preferential sites of expression as well as immunological and physiological role of these molecular markers. DIC was significantly up-regulated mainly in head kidney at 1.5-3 h post-infection (p.i.). HLP-1 showed an extended-time overexpression in gills and a significant up-regulation in spleen. HLP-2 was interestingly overexpressed in gills at 24 h p.i., while Hb-LP showed a significant up-regulation in skin for all the 72 h trial as well as lower but always significant values either in gills or in spleen. Different was the response of IL-1β that showed a dramatic up-regulation in spleen and head kidney at 8 h p.i. whilst in gills it displayed a severe inhibition. During this survey the i.p. stimulus surely conditioned the AMPP expression in skin and gills, especially as regards the DIC that as piscidin-related gene has an important defensive role in the mucosal tissues. However, two unconventional AMPP genes such as HLP-2 and Hb-LP, strictly related to the physiological mechanisms of fish, were less affected in terms of expression by the route of infection, being more evident in peripheral loci. These findings might suggest them as potential markers to be analyzed within plans of health survey in fish farms.

Response of rainbow trout gill (Na++K+)-ATPase and chloride cells to T3 and NaCl administration

With the aim of comparing the effects of oral T3 and NaCl administration on trout hypoosmoregulatory mechanisms, three groups of rainbow trout (Oncorhynchus mykiss Walbaum) held in freshwater (FW) were fed a basal diet (C), the same diet containing 8.83 ppm of 3,5,3′-triiodo-L-thyronine (T3) (T) or 10% (w/w) NaCl (N) respectively for 30 d. They were then transferred to brackish water (BW) for 22 d and fed on diet C. Gill (Na++K+)-ATPase activity and its dependence on ATP, Na+ and pH, number of gill chloride cells (CC), serum T3 level as well as fish growth, condition factor (K) and mortality were evaluated. During the FW phase, as compared to C trout, T trout showed a two fold higher serum T3 level, had unchanged gill (Na++K+)-ATPase activity and increased CC number, whereas N trout showed higher gill (Na++K+)-ATPase activity and CC number. At the end of the experiment the enzyme activity was in the order T>N>C groups and all groups showed similar CC number. Both treatments changed the enzyme activation kinetics by ATP and Na+. A transient increase in K value occurred in N group during the period of salt administration. In BW, T and N groups had higher and lower survival than C group respectively. Other parameters were unaffected by the treatments. This trial suggests that T3 administration promotes the development of hypoosmoregulatory mechanisms of trout but it leaves the (Na++K+)-ATPase activity unaltered till the transfer to a hyperosmotic environment.

Defensive response of European sea bass (Dicentrarchus labrax) against Listonella anguillarum or Photobacterium damselae subsp. piscicida experimental infection.

Sea bass were experimentally infected with Listonella anguillarum or Photobacterium damselae subsp. piscicida (Phdp). At 24 and 72h post-infection, the expression analysis of immune-relevant genes (IL-1β, IL-6, IL-8, Hepcidin), the transcriptional level and detection of HSP70, and the quantification of serum iron were investigated in association with the histological analysis and the bacterial recognition in tissues by immunohistochemistry. At 15 days post-infection, the specific antibody response was detected in surviving fish, as well as the transcriptional levels of TcR and BcR sequences. Both experimental infections were characterized by a similar acute response, whereas different histological and immunohistochemistry evidences were observed. In particular, the early reaction appeared suitable for the clearance of L. anguillarum, thus limiting the histological lesions, the bacterial dissemination and the further development of acquired immunity in surviving fish. On the contrary, the innate response appeared not enough to resolve the Phdp infection, which was characterized by tissue damage, bacterial widespread and substantial detection of specific humoral immunity in surviving fish, also associated to lymphocytes clonal expansion. Besides the opportunistic conditions involved in fish vibriosis and pasteurellosis, the comparison between these experimental infection models seems to suggest that the rate of development of the acquired immunity is strictly linked to the activation of the host innate response combined to the degree of bacterial virulence.

Quality and Safety of Beef Produced in Central African Sub-Region

Aim of this research is to provide a general situation of cattle slaughtered in Cameroon, as a representative example for the Central African Sub-region. The quality and safety of beef from the abattoir of Yaoundé, the largest in Cameroon, were considered. From January 2009 to March 2012, the pre-slaughter conditions and characteristics of 1953 cattle carcasses were recorded, as well as the pH of m. longissimus thoracis 24 h after slaughter. From these carcasses, 60 were selected to represent the bulls slaughtered. The quality parameters and composition of m. longissimus thoracis were carried out. The origin of most of the cattle was the Guinea High Savannah (74.6%), and transhumance was the common production system (75.5%). Gudali (45.6%), White Fulani (33.3%) and Red Mbororo (20.3%) breeds were predominant. Carcass weight was affected by rearing system and cattle category, and it markedly varied during year. Considering meat quality, the fat content was low (1.2%) and similar between breeds, moreover Gudali showed the toughest meat. Of the cows slaughtered, 27% were pregnant and the most common abnormal conditions encountered were ectoparasites, fatigue, lameness, fungal-like skin lesions, enlarged lymph nodes, respiratory distress, nodular lesions. More than 20% of the carcasses had some organs condemned, mainly for liver flukes (5.17%), and 1.0% of them were completely condemned due to tuberculosis, that also affected 3.28% of lungs. These data could aid authorities draw up programmes with the aim to strengthen cattle production, improve beef supply, control and prevent the observed diseases, and promote the regional trade.