Differential effects of alloherpesvirus CyHV-3 and rhabdovirus SVCV on apoptosis in fish cells.

Whilst Herpesviridae, which infect higher vertebrates, actively influence host immune responses to ensure viral replication, it is mostly unknown if Alloherpesviridae, which infect lower vertebrates, possess similar abilities. An important antiviral response is clearance of infected cells via apoptosis, which in mammals influences the outcome of infection. Here, we utilise common carp infected with CyHV-3 to determine the effect on the expression of genes encoding apoptosis-related proteins (p53, Caspase 9, Apaf-1, IAP, iNOS) in the pronephros, spleen and gills. The influence of CyHV-3 on CCB cells was also studied and compared to SVCV (a rhabdovirus) which induces apoptosis in carp cell lines. Although CyHV-3 induced iNOS expression in vivo, significant induction of the genetic apoptosis pathway was only seen in the pronephros. In vitro CyHV-3 did not induce apoptosis or apoptosis-related expression whilst SVCV did stimulate apoptosis. This suggests that CyHV-3 possesses mechanisms similar to herpesviruses of higher vertebrates to inhibit the antiviral apoptotic process.

C-reactive protein and complement as acute phase reactants in common carp Cyprinus carpio during CyHV-3 infection.

Cyprinid herpesvirus 3 (CyHV-3) is the aetiological agent of a highly virulent and lethal disease of common carp Cyprinus carpio and its ornamental koi varieties. However, specific knowledge about immune mechanisms behind the infection process is very limited. We aimed to evaluate the effect of the CyHV-3 infection on the profile of 2 major components of the common carp immune acute phase response: the C-reactive protein (CRP) and the complement system. Common carp were infected with CyHV-3 by bath immersion. Fish were sampled before the infection and at 6, 12, 24, 72, 120 and 336 h post-infection for serum and head kidney, liver, gill and spleen tissues. CRP levels and complement activity were determined from the serum, whereas CRP- and complement-related genes (crp1, crp2, c1rs, bf/c2, c3, masp2) expression profiles were analysed in the tissues by quantitative PCR. Both CRP levels and complement activity increased significantly up to 10- and 3-fold, respectively, in the serum of infected fish during the challenge. Analysis revealed distinct organ- and time-dependent expression profile patterns for all selected genes. These results suggest that CRP and complement behave as acute phase reactants to CyHV-3 infection in common carp with an organ- and time-dependent response.

Feeding common carp Cyprinus carpio with ß-glucan supplemented diet stimulates C-reactive protein and complement immune acute phase responses following PAMPs injection.

The effect of ß-glucan as a feed additive on the serum and gene profile of C-reactive protein (CRP) and complement acute phase responses was ascertained in common carp Cyprinus carpio. In addition effects of subsequent intraperitoneal injections of pathogen-associated molecular patterns (PAMPs), i.e. LPS or poly(I:C), to mimic bacterial or viral infection respectively, were studied. Carp were first orally fed with ß-glucan (MacroGard®) with a daily ß-glucan intake of 6 mg per kg body weight or with control food for 25 days and then injected with PBS containing either LPS (4 mg/kg) or poly(I:C) (5 mg/kg) or PBS alone. Fish were sampled during the 25 days of the feeding period and up to 7 days post-PAMPs injections for serum and liver, head kidney and mid-gut tissues. Oral administration of ß-glucan for 25 days significantly increased serum CRP levels and alternative complement activity (ACP). In addition, the subsequent LPS and poly(I:C) challenges significantly affected CRP and complement related gene expression profiles (crp1, crp2, c1r/s, bf/c2, c3 and masp2), with the greatest effects observed in the ß-glucan fed fish. However, in fish fed ß-glucan the PAMPs injections had less effects on CRP levels and complement activity in the serum than in control fed fish, suggesting that the 25 days of ß-glucan immunostimulation was sufficient enough to reduce the effects of LPS and poly(I:C) injections. Results suggest that MacroGard® stimulated CRP and complement responses to PAMPs immunological challenges in common carp thus highlighting the beneficial ß-glucan immunostimulant properties.

Identification of apoptosis-related genes and transcription variations in response to microcystin-LR in zebrafish liver.

There is growing evidence that the effects of microcystin-LR (MC-LR) are closely related to apoptosis. This study utilized microarray to identify the apoptosis-related genes induced by MC-LR in zebrafish liver. The messenger RNA abundance of some apoptosis-related genes was found to be increased, including five tumor necrosis factor (TNF)-related members (apoptosis regulatory protein siva, tumor necrosis factor-α (tnfa) TNF (ligand) superfamily member 10 (tnfsf10), TNF-inducible protein 6 (tnfaip6) and TNF receptor associated factor 2 binding protein (traf2bp)), three p53-related genes (tumor protein p53 inducible nuclear protein 1 (tp53inp1), p53-induced protein phosphatase 1 (ppm1d) and a novel apoptosis stimulating protein of p53 (aspp2)), bcl 2 family members (proapoptosis gene bax and antiapoptosis gene mcl 1), caspases (caspase y (caspy) and a PYD and CARD domain-containing protein (pycard)) and the transforming growth factor beta (TGF-ß) induced apoptosis protein 2 (taip2). Real-time polymerase chain reaction was used to study the kinetic transcriptional changes in seven apoptosis-related genes. Elevated transcription of p53, tp53inp1, mcl 1 and taip2 could only be detected at 6 h, increased transcription of the antagonist molecules, bcl 2 and bax could be detected at most time points and the significant change of caspy could be found at 48 h and 72 h after stimulation. Taken together, the results obtained in the present study clearly demonstrate that large amount of apoptosis-related genes are involved in the regulation of MC-LR-induced apoptosis.

Dietary ß-glucan stimulate complement and C-reactive protein acute phase responses in common carp (Cyprinus carpio) during an Aeromonas salmonicida infection.

The effect of ß-glucans as feed additive on the profile of C-reactive protein (CRP) and complement acute phase responses was studied in common carp Cyprinus carpio after exposition to a bacterial infection with Aeromonas salmonicida. Carp were orally administered with ß-glucan (MacroGard®) for 14 days with a daily ß-glucan intake of 6 mg per kg body weight. Fish were then intraperitoneally injected with either PBS or 1 × 108 bacteria per fish and sampled at time 0, 6, 12, 24, 48, 72, 96 and 120 h post-injection (p.i.) for serum and head kidney, liver and mid-gut tissues. CRP levels and complement activity were determined in the serum samples whilst the gene expression profiles of CRP and complement related genes (crp1, crp2, c1r/s, bf/c2, c3 and masp2) were analysed in the tissues by quantitative PCR. Results obtained showed that oral administration of ß-glucan for 14 days significantly increased serum CRP levels up to 2 fold and serum alternative complement activity (ACP) up to 35 fold. The bacterial infection on its own (i.e. not combined with a ß-glucan feeding) did have significant effects on complement response whilst CRP was not detectably induced during the carp acute phase reaction. However, the combination of the infection and the ß-glucan feeding did show significant effects on both CRP and complement profiles with higher serum CRP levels and serum ACP activity in the ß-glucan fed fish than in the control fed fish. In addition, a distinct organ and time dependent expression profile pattern was detected for all the selected genes: a peak of gene expression first occurred in the head kidney tissue (6 h p.i. or 12 h p.i.), then an up-regulation in the liver several hours later (24 h p.i.) and finally up- or down-regulations in the mid-gut at 24 h p.i. and 72 h p.i. In conclusion, the results of this study suggest that MacroGard® stimulated CRP and complement responses to A. salmonicida infection in common carp.

X-ray microanalysis (EDXMA) of cadmium-exposed eggs of Bothriocephalus acheilognathi (Cestoda: Bothriocephalidea) and the influence of this heavy metal on coracidial hatching and activity.

Over recent years it has been established that pollutants can have a significant impact on host-parasite systems in the aquatic environment, so much so that it has been proposed that parasite fauna may be a useful parameter to monitor water quality. Surprisingly, with perhaps the exception of trematodes and bioaccumulation in adult acanthocephalans, detailed observations on the interaction between helminths, particularly cestodes, and pollutants such as heavy metals, are lacking. In this study, eggs of the carp tapeworm, Bothriocephalus acheilognathi were exposed to a range of cadmium concentrations (0.1, 10, 100 and 10,000 mcirog/L) and coracidial hatching and survival assessed. Results indicated that the egg is highly resistant to heavy metal pollution and hatching occurs even at 10,000 microg/L. In contrast, the activity of the liberated coracidium significantly decreased after 1h exposure to cadmium at 10 and 100 microg/L. Electron microscopic X-ray microanalysis of parasite eggs exposed to 1000 and 10,000 microg/L cadmium revealed that cadmium accumulates on the surface of the egg and does not penetrate detectably into the enclosed coracidium. This means that the parasite eggs may be able to withstand a heavy metal pollutant incident.

The role of apoptosis in non-mammalian host-parasite relationships.

It is clear that the roles of apoptosis in the interactions between the parasite and their non-mammalian hosts are multifaceted and highly dependent on individual associations between the two organisms involved. Whilst there are instances where both organisms appear to gain from the apoptotic mechanism induced, in the majority of cases apoptosis appears to favour only one of the parties. In the instances when the parasite benefits, the apoptosis has been related to infectivity and virulence, an interruption of the killing mechanism of the host, and liberation of the pathogen. However, there are occasions where the apoptotic process benefits the host, as controlled cell death has been associated with limiting the pathogen population, parasite migration within the host and, in some instances, actually killing the invading organism. Apoptosis thus appears to play several fundamental roles within the host-parasite relationship which is ultimately reflected in an effect on the host population either mediated through an alteration in host fecundity or reduction in host numbers. The next decade promises to be both exciting and productive with respect to our knowledge of the relationship between apoptosis in non-mammalian animals and infection. Over the last few years the information obtained from studies on the apoptotic process in mammals and invertebrates (i.e. C. elegans and Drosophila) have been effectively used to increase our understanding of the apoptotic process in other animals such as insects, fish and amphibians. Such knowledge has paved the way for extensive studies on the effect of infections to be carried out.