Biomedical subjects

A E Ellis

Publications and source records attributed to A E Ellis.

At least 19 recordsLinked to original sources

PubMed2006

Expression kinetics of interferon and interferon-induced genes in Atlantic salmon (Salmo salar) following infection with infectious pancreatic necrosis virus and infectious salmon anaemia virus.

Infectious pancreatic necrosis virus (IPNV) and infectious salmon anaemia virus (ISAV) are economically important pathogens of the salmonid aquaculture industry. Atlantic salmon were challenged by intraperitoneal injection (i.p.) with either virus followed by time-course sampling. Cohabiting fish in the IPNV challenge were also sampled. Kidney tissue was analysed using a TaqMan real-time PCR assay to measure the expression of a range of host immune genes in relation to the endogenous control, elongation factor 1 alpha (ELF). Host genes measured included Mx, type I and type II interferon (IFN), gammaIFN induced protein (gammaIP), interleukin-1 beta (IL-1beta) and tumour necrosis factor alpha (TNF-alpha). Viral levels were also measured. In i.p. injected fish, both viruses greatly induced expression of Mx, gammaIP, type I and type II IFN by day 6 post-infection, however only ISAV caused substantial mortality. Some differences between the expression kinetics produced by both viruses were noted. Infection with ISAV increased IL-1beta expression following day 6, but no effect was seen in fish infected with IPNV. Neither virus induced TNF-alpha expression. This study confirms the presence of both type I and type II IFN responses and their induced genes in Atlantic salmon upon infection with an orthomyxovirus and a birnavirus.

Animals↗

PubMed2006

Expression of Mx mRNA following infection with IPNV is greater in IPN-susceptible Atlantic salmon post-smolts than in IPN-resistant Atlantic salmon parr.

The Mx response was compared in parr and post-smolt Atlantic salmon following intra-peritoneal injection of the same dose of Infectious Pancreatic Necrosis Virus (IPNV) per g of fish. Mx gene expression, measured by quantitative RT-PCR in liver, showed a maximum level 3days after injection in parr with undetectable levels on day 7. In post-smolts, similar levels as in parr were attained on day 3, but levels then continued to rise on day 5 and 7 to about 10 times higher than the peak level in parr. Poly I:C injected parr showed Mx levels similar to IPNV injected post-smolts. Mortality from IPN in post-smolts occurred on days 6 and 7. Levels of IPN VP2 transcripts in parr were very low and did not increase with time, suggesting viral replication was low. Individual variation in levels of Mx and IPN VP2 gene transcripts was very high in post-smolts and although data is limited there was an inverse relationship between the levels of Mx and VP2, suggesting that individuals with high Mx levels on day 5 may be able to prevent viral replication. This contrasts with the response in parr, where IPN-resistance was not associated with a high Mx response.

Animals↗

PubMed2006

Expression of the glycoprotein of viral haemorrhagic septicaemia virus (VHSV) on the surface of the fish cell line RTG-P1 induces type 1 interferon expression in neighbouring cells.

In the present study using a luciferase/Mx promoter reporter system, it was shown that the rainbow trout gonad cell line (RTG-P1), a fibroblastic cell line, produces IFN when transfected with a plasmid encoding the glycoprotein of VHSV but not with plasmid vector alone. Only a small percentage of the cells expressed the G protein on the surface membrane as indicated by immunostaining of transfected cells. When transfection was performed in the presence of monoclonal antibodies (Mab) to the glycoprotein, the production of interferon mRNA transcripts was reduced by over 50%. This indicates that the surface expression of G protein was the major mechanism of interferon induction and that most of the interferon was being expressed by cells neighbouring the transfected cells.

Animals↗

PubMed2006

Improved purification of Piscirickettsia salmonis using Percoll gradients.

Viable preparations of intact Piscirickettsia salmonis, purified from host cell material, are necessary for studying characteristics associated with this bacterium. However, purification of the organism is difficult due to its obligate intracellular nature. A simple and effective method for isolating whole P. salmonis, which is quick and easy to perform, but still maintains the viability and antigenicity of the bacterium is described. P. salmonis was purified by differential pelleting and density gradient centrifugation using 30%, 40%, or 50% (v/v) Percoll gradients. Following fractionation, a band with a density of 1.056-1.080 was found to be composed entirely of rickettsiae, confirmed by fluorescent antibody technique (IFAT). Purity of the P. salmonis from different stages of the purification process was assessed by light and transmission electron microscopy, and the viability of yields determined from a plaque assay and a tissue culture infective dose (TCID(50) ml(-1)). P. salmonis recovered from the 30% Percoll gradient appeared to retain their intracellular structure better than P. salmonis obtained from the 40% and 50% Percoll gradients, and appeared to have a greater viability. Differences were seen between P. salmonis-infected CHSE-214 cells and purified P. salmonis when compared by SDS-PAGE and Western blotting, and less host cell contamination was present in preparations obtained from the 30% Percoll gradient. Finally ten different P. salmonis isolates obtained from three different geographical locations and four different fish species, were purified using the 30% Percoll gradient. When the morphology of these was compared by transmission electron microscopy (TEM), they appeared similar in size and appearance, although isolate R980769 was highly pleomorphic and isolate R-29 was larger than the other isolates examined.

Animals↗

PubMed2006

In infectious pancreatic necrosis virus carrier Atlantic salmon, Salmo salar L., post-smolts, almost all kidney macrophages ex vivo contain a low level of non-replicating virus.

The level of infection by infectious pancreatic necrosis virus (IPNV) of kidney macrophages from 12 asymptomatic carrier Atlantic salmon post-smolts was studied. Kidney leucocytes were fractionated on 34/51% Percoll gradients, allowed to adhere to plastic wells overnight, washed to remove non-adherent cells and cultured for up to 7 days with or without renewal of medium on day 3. On day 1, supernatants were harvested, macrophages were counted, lysed and IPNV in the supernatants and lysates was titred in chinook salmon embryo (CHSE-214) cells. The multiplicity of infection ranged between 1:2.2 and 1:7.4 (virus:macrophages). On day 3, the titres of IPNV in macrophage lysates decreased and in wells where the medium was renewed on day 3, IPNV was no longer detectable on day 7. In the supernatants, one fish was positive for IPNV on day 1, four fish on day 3 but none were detectably positive on day 7. In parallel wells in which the medium was not renewed, on day 7 IPNV was detected in macrophage lysates of three fish and the supernatants were also IPNV positive in two of these fish. This suggests that virus might be shed from infected macrophages and then reinfect other macrophages. When macrophages were serially diluted in wells and cultured for 24 h, IPNV could be cultured from macrophage lysates of wells containing between two and 70 macrophages. These results indicate that a very high proportion of the adherent kidney macrophages must be infected with very few non-replicating virions.

Animals↗

PubMed2006

Infectious pancreatic necrosis virus establishes an asymptomatic carrier state in kidney leucocytes of juvenile Atlantic cod, Gadus morhua L.

Juvenile Atlantic cod (10 g) were infected with infectious pancreatic necrosis virus (IPNV) by intraperitoneal injection and cohabitation. Fish showed no signs of disease but IPNV could be re-isolated from kidney tissue for up to 12 weeks. On weeks 2, 5, 8, 10, 11 and 12 following infection, kidney leucocytes were fractionated on Percoll gradients, and cells separated into plastic adherent and non-adherent cell populations after overnight incubation. IPNV was detectable in lysates of both cell populations and in supernatants by culture in CHSE-214 cells. Wells containing 10(5)-10(6) macrophages had an IPNV TCID(50) of about 10(3)/well and in serially diluted macrophages the minimum number of cells required to detect virus ranged from 10(1) to 10(4). These data indicate that about one in 10(4) macrophages were infected and the mean number of virus/infected cell was about 10. Replication of IPNV in the macrophages was low as the titre of the virus in macrophage lysates did not increase between days 1 and 3 of culturing the macrophages, but virus was released into the supernatant over this time.

Animals↗

PubMed2005

Complement consumption by Photobacterium damselae subsp. piscicida in seabream, red porgy and seabass normal and immune serum. Effect of the capsule on the bactericidal effect.

A virulent strain of Photobacterium damselae subsp. piscicida (Pdp) was grown without (C form) or with (C+ form) glucose supplementation, the latter to enhance capsule formation. Both forms were resistant to killing by normal serum of seabream, red porgy and seabass. However, the C form was killed by immune serum of all three fish species while the C+ form was killed only by seabream and red porgy sera and to a lesser extent than the C form. Both C and C+ forms consumed complement in normal serum and this consumption was enhanced by precoating the bacteria in specific fish antibody. Complement consumption was greatest in seabass serum, especially with antibody-coated C+ form yet in this case the bacteria were not killed. The killing of the C form in immune serum of all three fish species was completely inhibited by EGTA/Mg(2+), indicating that the mechanism of complement activation leading to killing of the bacteria was by the classical pathway. The results suggest that immune serum killing by the classical complement pathway may provide some degree of protection against pasteurellosis, but enhanced expression of the capsule by Pdp in vivo may restrict complement-mediated killing, especially in immunised seabass.

Animals↗

PubMed2005

Confirmation of Piscirickettsia salmonis as a pathogen in European sea bass Dicentrarchus labrax and phylogenetic comparison with salmonid strains.

European sea bass Dicentrarchus labrax from the Mediterranean were diagnosed with a severe encephalitis. Rickettsia-like organisms (RLOs) were associated with brain lesions in routine paraffin sections. These were found to share common antigens with the Piscirickettsia salmonis type-strain, LF-89, by indirect fluorescent antibody test (IFAT) and by immunohistochemistry (IHC). In addition, we compared the DNA sequences of the 16S rDNA and 16S-23S internal transcribed spacer region (ITS) with those published for P. salmonis strains and found that the sea bass piscirickettsia-like organism (SBPLO) was another strain of P. salmonis, closely related to the salmonid pathogens. Furthermore, we showed that the SBPLO possessed at least 2 ITS regions, 1 of which contained tRNA genes.

Animals↗

PubMed2005

Influence of vaccination on the nitric oxide response of gilthead seabream following infection with Photobacterium damselae subsp. piscicida.

The nitric oxide (NO) response of vaccinated and non-vaccinated juvenile gilthead seabream was studied in vivo and the NO response of isolated kidney macrophages of fish was studied in vitro. Fish were vaccinated with formalin-killed Photobacterium damselae subsp. piscicida (Pdp) with or without Freund's incomplete adjuvant (FIA) and control fish received phosphate buffered saline (PBS). Thirty days later, fish were injected with a sublethal dose of Pdp and 3 fish/group were bled at time periods thereafter and serum nitrite and citrulline levels were determined as a measure of the NO response. All infected groups showed an increase in NO metabolites from 6h to 27 days, with peak levels at 24 h. However, the response in bacterin-vaccinated fish was significantly higher than in the non-vaccinated group and the bacterin plus FIA resulted in a further significant enhancement. Similarly enhanced NO responses were produced in vitro by isolated macrophages obtained from vaccinated compared with non-vaccinated fish 30 days after vaccination following infection, with the response in macrophages from fish vaccinated with the bacterin plus FIA being significantly higher than those from fish vaccinated with the bacterin alone. Thus, vaccination resulted in an enhanced NO response to infection with Pdp in vivo and in vitro. Furthermore, the level of protection of fish to experimental challenge with virulent Pdp correlated with the level of the NO responses in the different groups.

Analysis of Variance↗

PubMed2005

Kinetics of Mx expression in rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar L.) parr in response to VHS-DNA vaccination.

The duration of the Mx mRNA response to an intramuscular injection of the viral haemorrhagic septicaemia virus (VHSV) glycoprotein (G) gene DNA vaccine as well as to the control plasmid was determined in rainbow trout at 14 degrees C over a period of 11 weeks. The Mx response was detectable on day 7, peaked on day 14 and returned to pretreatment levels on day 21 and thereafter. No increase in Mx expression was detectable to the control plasmid. In further experiments, the kinetics of the Mx response were compared in rainbow trout and Atlantic salmon parr kept at 10 degrees C and injected with the DNA vaccine or the synthetic double-stranded RNA, poly I:C. In both species there was a rapid response to poly I:C detectable from day 1, reaching maximum from days 3 to 9 and decreasing to background level by day 12. The peak level and return to background was reached slightly later in salmon. In both species the response to the VHS/DNA vaccine was slower to begin, not being detectable on days 1 and 3, but elevated levels were found on day 6. However, in the salmon parr, the peak level was on day 6 and the signal disappeared by day 12, while in the rainbow trout, the response peaked at day 12 and lasted until day 21. The kinetics of the Mx response to the VHS/DNA vaccine in rainbow trout correlate with the early non-specific protection against VHS in this species following vaccination. It is speculated that the more transient Mx response in Atlantic salmon parr to the DNA vaccine may be related to the innate resistance of salmon to VHS.

Analysis of Variance↗

PubMed2005

Iron acquisition mechanisms of Flavobacterium psychrophilum.

Forty strains of Flavobacterium psychrophilum were tested for the production of siderophores using the universal Chrome Azurol S (CAS) assay. The majority of the strains (85%) were CAS positive (CAS+) and some (15%) were CAS negative (CAS-). The cryptic plasmid pCP1 was carried by all positive strains and was lacking from negative strains. While a weak catechol reaction was detectable in CAS+ culture supernatants, the CAS reaction was, to some extent, heat sensitive, questioning whether the positive reaction was caused only by siderophores. The ability to grow in vitro under iron-restricted conditions did not correlate with the CAS reactivity, as growth of both CAS+ and CAS- strains was similarly impaired under iron restriction induced by 2,2 dipyridyl. Suppressed growth under these conditions was restored by addition of FeCl3, haemoglobin and transferrin for both CAS+ and CAS- strains.

2,2'-Dipyridyl↗

PubMed2005

Update on bacterial vaccines: Photobacterium damselae subsp. piscicida.

Photobacterium damselae subsp. piscicida is the causative agent of pasteurellosis in wild and farmed marine fish worldwide. Although serologically homogeneous, recent molecular advances have led to the discovery of distinct genetic clades, depending on geographical origin. Further details of the strategies for host colonisation have arisen including information on the role of capsule, susceptibility to oxidative stress, confirmation of intracellular survival in host epithelial cells, and induced apoptosis of host macrophages. This improved understanding has given rise to new ideas and advances in vaccine technologies, which are reviewed in this paper.

Animals↗

PubMed2004

Activation of the nitric oxide response in gilthead seabream after experimental infection with Photobacterium damselae subsp. piscicida.

Inoculation of small gilthead seabream (Sparus aurata) (30-75 g body weight) with a sublethal dose of different Photobacterium damselae subsp. piscicida (Pdp) strains (DI-21 and 94/99) induced an increase in serum concentrations of stable nitric oxide (NO) metabolites lasting from 6 h to six days post-infection, with a peak at 24 h. In contrast, no such response was detected in larger fish (150-600 g). Since the virulence of Pdp correlates with the presence of a polysaccharide capsular layer which can be induced by growing the bacteria in medium supplemented with 1% glucose (C+ forms), the effect of the presence of an enhanced capsular layer on the NO response in small fish was also evaluated. Although, all bacteria induced a similar rapid (6 h) and sustained (up to six days) NO response, serum concentrations of nitrites and citrulline were significantly increased in fish infected with the Pdp strains grown in glucose-supplemented medium. When the NO response of fish infected with the C+ form of Pdp was blocked by prior injection of the inhibitor L-NAME, the LD(50) was reduced by over 10-fold and the mean time to death was also markedly reduced. Considering that (i) pasteurellosis only affects gilthead seabream with body weights below 100 g; (ii) capsulated Pdp are more resistant to the bactericidal action of NO and peroxynitrites than non-capsulated strains; and (iii) blocking the NO response of the fish results in greater susceptibility to Pdp, it seems reasonable to propose that the sustained NO response reported in this study represents a relevant protective mechanism of juvenile gilthead seabream against pasteurellosis.

Analysis of Variance↗

PubMed2004

An assessment of immunostimulants as Mx inducers in Atlantic salmon (Salmo salar L.) parr and the effect of temperature on the kinetics of Mx responses.

The aims of this study were (i) to identify alternative Mx stimulatory compounds in Atlantic salmon (Salmo salar L.) and to characterise the kinetics and intensity of the stimulated responses and (ii) to investigate the effect of temperature on such responses by semi-quantitative RT-PCR. Mx transcripts were measured in Atlantic salmon parr kept at 14 degrees C and injected with either LPS, the synthetic double-stranded polyribonucleotide poly I:C, Vibrio anguillarum serotypes I and II-ordalii bacterin, beta-glucan, whole yeast cells or yeast RNA. Sampling periods lasted until transcripts were undetectable or up to three weeks after immunisation. The effect of temperature on poly I:C-induced Mx response was studied by injecting parr kept at 6 degrees C. Newly hatched salmon fry were immersed once, twice or three times in the Vibrio bacterin diluted five or 10 times and sampled for three weeks. None of the yeast compounds induced Mx expression in Atlantic salmon parr. LPS induced a very low Mx response 2 and 3 days after injection. The Vibrio bacterin administered by injection in parr (but not by immersion in fry) resulted in strong Mx induction on days 2 and 3, disappearing by day 6. Poly I: C-induced Mx responses that were more intense and longer lasting than those induced by the bacterin, peaking on day 3 and lasting over 6 days, disappearing by day 9 at 14 degrees C. Lower temperature caused a longer lasting Mx response to poly I:C (at least 21 days), which peaked on days 7-14, with a similar intensity and no delayed onset as compared with the response at 14 degrees C. However, some toxicity of the poly I:C was indicated in treatments at 6 degrees C.

Adjuvants, Immunologic↗

PubMed2004

Poly I:C-induced Mx responses in Atlantic salmon parr, post-smolts and growers.

Intraperitoneal injection of 500 microg poly I:C/fish into Atlantic salmon parr in freshwater and post-smolts and growers in seawater (all at 11 degrees C) induced enhanced expression of Mx mRNA in liver tissue 24 h post-injection. The level of Mx transcripts peaked at day 3 (Mx:beta-actin ratio of about 0.8) and the response disappeared by day 7. In post-smolts, mortalities occurred up to day 14 post-injection, which was dose-dependent. Histological examination of tissues revealed severe pathological changes in the liver of poly I:C injected post-smolts resulting from apoptosis and necrosis of hepatocytes. All other organs appeared histologically normal. Levels of Mx mRNA expression on day 3 post-injection were similar for fish with normal and pathological livers. In untreated or control fish injected with PBS, low levels of Mx transcripts (Mx:beta-actin ratio about 0.1) were sometimes detectable in parr but not in growers. Constitutive Mx expression was variable in post-smolts. Some populations had no detectable transcripts while in others moderate ratios (about 0.3) were detectable over a 3-week period of sampling. Poly I:C administered to parr by bath or orally did not induce upregulation of Mx expression.

Analysis of Variance↗

PubMed2004

Mx expression in Atlantic salmon (Salmo salar L.) parr in response to Listonella anguillarum bacterin, lipopolysaccharide and chromosomal DNA.

Mx genes are inducible by Type I interferons and are involved in antiviral defences. A commercially available vibrio bacterin, intended for immersion vaccination, was shown to be a potent inducer of Mx gene expression in Atlantic salmon parr following intraperitoneal injection. The response was dose and temperature dependent. At 10 degrees C and 10 times concentration the bacterin induced Mx response kinetics similar to that induced by poly I:C. At 10 degrees C, enhanced Mx responses were detected from days 1 to 9 with both 1 times (1x) and 10 times (10x) concentrated bacterin, with a tendency for a higher response to the concentrated bacterin on days 1 and 3. Basal levels of Mx mRNA were detected on day 12 after injection to both concentrations. The response induced by poly I:C was higher on day 1 and it was still present at day 12, with basal levels being reached on day 18. At 6 degrees C, there was a more definitive dose effect of the vibrio bacterin and the Mx response was delayed in comparison to that at 10 degrees C. Increased Mx expression did not appear until day 6 and with the 1x dose it had disappeared by day 9. However, the 10x dose continued to induce Mx at day 12, disappearing by day 18. The Mx response to the purified Listonella anguillarum lipopolysaccharide (LPS) and DNA in fish held at 10 degrees C showed some differences in the rate of onset. The response to DNA was faster, beginning on day 1 compared with day 3 for the LPS. The response to DNA peaked on day 3 while for LPS the peak was on day 9. However, the response to both components had disappeared by day 12. The response kinetics to the L. anguillarum DNA was essentially similar to the 10x dose of the vibrio bacterin and to poly I:C at 10 degrees C.

Animals↗

PubMed2004

IPNV carrier Atlantic salmon growers do not express Mx mRNA and poly I:C-induced Mx response does not cure the carrier state.

Injection of infectious pancreatic necrosis virus (IPNV) in post-smolt Atlantic salmon induced a rapid and persistent expression of Mx mRNA from day 1 to at least day 11 when Mx:beta actin ratios were still at peak values of about 1.0. In contrast, an Atlantic salmon grower population, shown to be carriers of IPNV by culture of the virus from plastic adherent kidney leucocytes, showed no evidence of the expression of Mx transcripts. Nevertheless, IPNV-carrier growers showed a typical Mx response following injection of poly I:C, beginning on day 1, peaking on day 3 (mean Mx:beta actin ratio 0.82) and disappearing by day 7. Notwithstanding such treatment, IPNV continued to persist in growers as the virus could still be isolated 14 days after poly I:C injection.

Actins↗

PubMed2004

A sensitive non-destructive method for detecting IPNV carrier Atlantic salmon, Salmo salar L., by culture of virus from plastic adherent blood leucocytes.

In populations of Atlantic salmon in sea water, infectious pancreatic necrosis virus (IPNV) could be detected by standard virological culture methods in sonicated kidney homogenates and in mucus samples (gill, skin and rectum) from 14 and nine of 25 fish, respectively, but all fish were positive by virus culture from lysates of kidney macrophages and adherent blood leucocytes. In fish which tested negative for IPNV by the standard method of detection, the virus could be detected using adherent blood leucocytes isolated on a Percoll gradient from as little as 10 microL of blood. The blood sample could be stored for at least 3 days in a heparinized tube on ice before preparing the plastic adherent leucocytes. Furthermore, the latter could be prepared without prior fractionation on Percoll simply by incubating whole blood (33 microL) in cell culture medium (66 microL) in 96-well plates overnight and washing away the non-adherent cells before lysing the adherent cells and inoculation of the lysate onto CHSE-214 cells. This highly sensitive method for detecting IPNV-carriers is therefore very suitable for non-destructive sampling of fish in the field.

Animals↗