Donatella Volpatti

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.

Dorsal arthrodesis of thoracic spine and effects on thorax growth in prepubertal New Zealand white rabbits.

Study Design. Dorsal arthrodesis of thoracic spine in a prepubertal New Zealand White rabbit model. Objective. Evaluating the consequences of dorsal arthrodesis on the growth of the spine, sternum, and thorax in prepubertal rabbits, through the study of CT scans. Summary of Background Data. Vertebral arthrodesis in the treatment of progressive idiopathic scoliosis in prepubertal patients is not ideal, but is still a choice in treating major deformities of the spine. Postoperative assessment of spinal deformity is essential, feasible, and recordable through CT scans. Methods. Twelve female rabbits, 9 weeks old, were subjected to surgery for dorsal arthrodesis of the upper thoracic spine. Surgery involved the implant of 2 “C”-shaped titanium bars, which were placed beside the spinous processes of the thoracic vertebrae. Three CT scans were performed, 10 (T1), 55 (T2), and 139 (T3) days after surgery. Measures were obtained by Myrian Pro software for 3 different groups: G1 with complete fusion, G2 with incomplete fusion, and G3 sham-operated. Results. The average of the dorsoventral/laterolateral thoracic diameter ratio at fused levels is lower than 1 in G1 as well as in G2; on the contrary, in G3 is higher than 1. The average growth of the sternum length between T1 and T2 and between T2 and T3 is minor in G1 than in G2 and G3. The dorsal and ventral lengths of thoracic vertebral bodies in the spinal segment D1-D6 is smaller in G1 and G2 than in G3, whereas no differences were observed between the 3 groups in the D7-D12 segment without arthrodesis. Conclusion. Dorsal arthrodesis in prepubertal rabbits changes thoracic growth patterns. In operated rabbits, the dorsoventral thoracic diameter grows more slowly than the laterolateral thoracic diameter. The sternum as well as the lengths of thoracic vertebral bodies in the spinal segment D1-D6 grow less. The crankshaft phenomenon is evident at the fused vertebral levels where there is a reduction of thoracic kyphosis.

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.

Development of an ELISA to detect Pasteurella piscicida in culture and in ‘spiked’ fish tissue

Abstract An enzyme linked immunosorbent assay (ELISA) was developed to detect the presence of Pasteurella piscicida. The test, after the preparation of the solid phase, takes less than 4 h to complete and the same technique can be applied for both bacteria in culture and in tissue. The limit of sensitivity for bacteria in culture was 103 cells ml−1 or 102 cells well−1, if a plate reader is available or 105 cells ml−1 (104 cells well−1) visually. Similar sensitivity was achieved (103 cells well−1 with a plate reader, 104 cells well−1 visually) when the ELISA was performed on tissue. The technique is specific and it offers a rapid, early and sensitive means of diagnosing Pasteurellosis during outbreaks and screening large populations of fish for the presence of P. piscicida.

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.

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.

In vivo morphological and antigenic characteristics of Photobacterium damselae subsp. piscicida

The present study was conducted to examine the morphology and antigenicity of Photobacterium damselae subsp. piscicida by culturing the bacterium in vivo in the peritoneal cavity of sea bass (Dicentrarchus labrax) within dialysis bags with either a low molecular weight (LMW) cut-off of 25 kDa or a high molecular weight (HMW) cut-off of 300 kDa. Differences were observed in the growth rate between the bacteria cultured in vivo or in vitro. Bacteria cultured in vivo were smaller and produced a capsular layer, which was more prominent in bacteria cultured in the HMW bag. Antigenicity was examined by Western blot analysis using sera from sea bass injected with live Ph. d. subsp. piscicida. The sera recognised bands at 45 and 20 kDa in bacteria cultured in vivo in the LMW bag. Bacteria cultured in vivo in the HMW bag did not express the 45 kDa band when whole cell extracts were examined, although the antigen was present in their extracellular products. In addition, these bacteria had a band at 18 kDa rather than 20 kDa. Differences in glycoprotein were also evident between bacteria cultured in vitro and in vivo. Bacteria cultured in vitro in LMW and HMW bags displayed a single 26 kDa band. Bacteria cultured in the LMW bag in vivo displayed bands at 26 and 27 kDa, while bacteria cultured in vivo in the HMW bag possessed only the 27 kDa band. These bands may represent sialic acid. The significance of the changes observed in the bacteriums structure and antigenicity when cultured in vivo is discussed.

Dorsal arthrodesis in prepubertal New Zealand white rabbits followed to skeletal maturity: effect on thoracic dimensions, spine growth and neural elements.

Background: Several studies have shown that severe spinal deformity and early arthrodesis can adversely affect the development of the spine and thorax by changing their shape and reducing their normal function. This article analyzes the consequences of posterior fusion on the growth of spine, thorax and neural elements in New Zealand white rabbits and compares with similar human data. Materials and Methods: The first section of the article analyzes the consequences of T1-T6 dorsal arthrodesis on the growth of the spine, sternum, thorax volume and neural elements in 12 prepubertal female New Zealand white rabbits, through a study of CT scans and histology specimens. The second part, evaluates thoracic dimensions in 21 children with spinal arthrodesis for treatment of deformity performed prior to nine years of age. Results: Dorsal arthrodesis in prepubertal rabbits changes thoracic growth patterns. In operated rabbits thoracic depth grows more slowly than thoracic width. The sternum as well as length of thoracic vertebral bodies in the spinal segment T1-T6 show reduced growth. Children undergoing spinal arthrodesis before nine years of age were noted to have shortened height, short trunk and disproportionate body habitus at skeletal maturity. Observed spine height and chest dimension values were reduced compared to the expected norms. The ratio between chest width and chest depth was below normal values. Conclusions: The first part of the study shows that thoracic dorsal arthrodesis in prepubertal New Zealand white rabbit influences thoracic, spine growth and affects the shape of pseudo unipolar neurons of the dorsal root ganglia. The second part demonstrates that children treated before nine years of age have significantly reduced spine height and thoracic dimensions. The thorax becomes elliptical as chest depth grows less than chest width. Both experimental and clinical findings contribute to explain reduced chest growth and subsequent thoracic growth disturbance in patients treated with early arthrodesis.

Variation in the molecular weight of Photobacterium damselae subsp. piscicida antigens when cultured under different conditions in vitro.

The antigenicity of Photobacterium damselae (Ph. d.) subsp. piscicida, cultured in four different growth media [tryptone soya broth (TSB), glucose-rich medium (GRM), iron-depleted TSB (TSB + IR-), and iron-depleted GRM (GRM + IR-)] was compared by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis using sera obtained from sea bass (Dicentrarchus labrax) raised against live or heat-killed Ph. d. subsp. piscicida. The antigenic expression of Ph. d. subsp. piscicida was found to differ depending on the culture medium used. A significantly higher antibody response was obtained with iron-depleted bacteria by ELISA compared with non-iron depleted bacteria obtained from the sera of sea bass raised against live Ph. d. subsp. piscicida. The sera from sea bass raised against live bacteria showed a band at 22 kDa in bacteria cultured in TSB + IR- or GRM+ IR- when bacteria that had been freshly isolated from fish were used for the screening, while bands at 24 and 47 kDa were observed with bacteria cultured in TSB or GRM. When bacteria were passaged several times on tryptic soya agar prior to culturing in the four different media, only bands at 24 and 47 kDa were recognized, regardless of the medium used to culture the bacteria. It would appear that the molecular weight of Ph. d. subsp. piscicida antigens change in the presence of iron restriction, and sera from sea bass infected with live bacteria are able to detect epitopes on the antigens after this shift in molecular weight.

Expression of infection-related immune response in European sea bass (Dicentrarchus labrax) during a natural outbreak from a unique dinoflagellate Amyloodinium ocellatum

In the Mediterranean area, amyloodiniosis represents a major hindrance for marine aquaculture, causing high mortalities in lagoon-type based rearing sites during warm seasons. Amyloodinium ocellatum (AO) is the most common and important dinoflagellate parasitizing fish, and is one of the few fish parasites that can infest several fish species living within its ecological range. In the present study, A. ocellatum was recorded and collected from infected European sea bass (Dicentrarchus labrax) during a summer 2017 outbreak in north east Italy. Histological observation of infected ESB gill samples emphasized the presence of round or pear-shaped trophonts anchored to the oro-pharingeal cavity. Molecular analysis for small subunit (SSU) rDNA of A. ocellatum from gill genomic DNA amplified consistently and yielded 248 bp specific amplicon of A. ocellatum, that was also confirmed using sequencing and NCBI Blast analysis. Histological sections of ESB gill samples were addressed to immunohistochemical procedure for the labelling of ESB igm, inos, tlr2, tlr4, pcna and cytokeratin. Infected gills resulted positive for igm, inos, pcna and cytokeratin but negative to tlr-2 and tlr-4. Furthermore, ESB immune related gene response (innate immunity, adaptive immunity, and stress) in the course of A. ocellatum infection using quantitative polymerase chain reaction (qpcr) for infected gills and head kidney was analysed. Among the twenty three immune related gene molecules tested, cc1, il-8, il-10, hep, cox-2, cla, cat, casp9, and igt were significantly expressed in diseased fish. Altogether, these data on parasite identification and expression of host immune-related genes will allow for a better understanding of immune response in European sea bass against A. ocellatum and could promote the development of effective control measures.