Previously unrecognised division within Moritella viscosa isolated from fish farmed in the North Atlantic.

Previously undocumented phenotypical and genetic variation was identified amongst isolates of Moritella viscosa collected from various geographical locations and from different fish species. The studied isolates could be split into 2 major phenotypically and genetically different clusters, one of which was consistent with the species type strain (NCIMB 13548). Isolates consistent with the type strain originated exclusively from Atlantic salmon farmed in Norway, Scotland and the Faroe Isles, although a single isolate from farmed Norwegian cod clustered closely with this group. The 'variant' cluster comprised isolates originating from Norwegian farmed rainbow trout, Icelandic farmed rainbow trout and salmon, Canadian farmed (Atlantic) salmon, Icelandic lumpsucker and only exceptionally from Norwegian salmon. With the exception of the single aforementioned cod isolate, all isolates from Norwegian farmed cod belonged to the variant cluster. Phenotypically, the clusters could be absolutely separated only by elevated haemolytic activity in the variant strain, although approximately half of these isolates also produced acid from mannose, in contrast to the typical (type) strain. While 16S rRNA gene sequencing was unable to separate the 2 clusters, Western blot analyses, plasmid profile analysis, pulsed field gel electrophoresis and gyrB gene sequence analysis produced clusters consistent with the phenotypic data. Macroscopically and histologically the disease in rainbow trout caused by the variant strain was consistent with that previously described in Atlantic salmon. The results of the present study may indicate a degree of host specificity of the typical strain for Atlantic salmon.

Real-time PCR detection of Moritella viscosa, the likely causal agent of winter-ulcer in Atlantic salmon Salmo salar and rainbow trout Oncorhynchus mykiss.

We report the development of a real-time PCR protocol for specific detection of Moritella viscosa. This bacterium is considered to be the main aetiological agent in development of winter-ulcer, a disease severely affecting salmonid aquaculture in Norway. From a newly elaborated draft version of the genome of M. viscosa, the tonB gene sequence was selected as a suitable basis for the development of the real-time PCR assay. The real-time PCR demonstrated the presence of M. viscosa DNA sequences in 88.1% of samples collected from 35 outbreaks of winter-ulcer in Norwegian fish farms. In contrast, standard culturing on blood agar identified M. viscosa in only 39.7% of fish. While the culturing method revealed a similar prevalence (26 to 27%) of M. viscosa in kidney and ulcer samples, substantially more ulcer (81.5%) than kidney (49.7%) samples were shown positive by real-time PCR.

Infectious salmon anaemia virus (ISAV) in experimentally challenged Atlantic cod (Gadus morhua).

Juvenile Atlantic cod, Gadus morhua, (6 g) were challenged with infectious salmon anaemia virus (ISAV) either by intraperitoneal (i.p.) injection or by cohabitation with ISA-diseased Atlantic salmon (Salmo salar). Samplings of cod were performed over a period of 45 days and various tissue samples were collected. The presence of ISAV RNA (segment 8) in samples was assessed by both conventional RT-PCR and a competitive quantitative real-time RT-PCR. In the i.p.-challenged group, ISAV RNA was detected in fish from all samplings, i.e. at days 7, 15, 21, 30 and 45 post-challenge. At day 7 post-challenge, all individual fish were positive, and so were the vast majority of individual tissue samples. At later samplings, the fraction of positive brain samples remained high (approximately 75%). In contrast, the positive fraction of other tissues/organs declined during the experiment. Analysis of positive brain samples by a quantitative real-time RT-PCR analysis showed that the level of ISAV RNA increased significantly (approximately 20 times) between days 7 and 30 post-challenge and remained high at day 45, indicating that a replication of ISAV had taken place. ISAV RNA was not detected in any control or cohabitation-challenged fish. No abnormal behaviour, clinical disease or, most notably, mortality was observed in any of the challenge or control groups.

Experimental infection of Atlantic halibut Hippoglossus hippoglossus with nodavirus: tissue distribution and immune response.

Atlantic halibut Hippoglossus hippoglossus, age 8 mo and weighing 20 g, were challenged by either intraperitoneal injection (i.p.) or by bath exposure using nodavirus isolated from Atlantic halibut. Fish were sampled at intervals over a 41 d period, starting on Day 5 post-challenge. Although no clinical disease or mortality was recorded, the data show that nodavirus did successfully propagate in i.p.-challenged fish. Using conventional end-point reverse transcription (RT)-PCR, nodavirus was detected in the kidney of all examined i.p.-challenged fish, and further in the head, heart, liver and posterior intestine of most of these individuals. Quantitative real-time RT-PCR revealed that the amount of virus in head samples from the i.p.-challenged group increased during the experiment. The presence of nodavirus in nervous tissue of i.p.-challenged fish was detected by immunohistochemistry from Day 13 post-challenge. In the retina, virus positive cells were found adjacent to the circumferential germinal zone at the ciliary margin towards the iris. In the brain, a few positive cells were detected in the tectum opticum. An ELISA was developed to detect anti-nodavirus activity in plasma. The method included an optimized coating procedure, which allowed the use of non-purified nodavirus as the coating antigen in a simple indirect ELISA. An anti-nodavirus antibody response was detected from Day 19 post-challenge in i.p.-challenged fish, while a response was not detected in the bath-challenged or control fish. This experiment demonstrates a subclinical nodavirus infection in Atlantic halibut at a post-juvenile stage induced by i.p. injection of virus.

DNA injection in combination with electroporation: a novel method for vaccination of farmed ruminants.

Injection of plasmid DNA encoding antigens into rodents followed by electroporation improved the immune response when compared with injection without electroporation (Widera et al. J Immunol 2000;164:4635-40; Zucchelli et al. J Virol 2000;74:11598-607; Kadowaki et al. Vaccine 2000;18:2779-88). The present study describes the extension of this technology to farm animals, by injecting plasmid DNA encoding mycobacterial antigens (MPB70, Ag85B and Hsp65) into the muscles of goats and cattle using two different types of electrodes, both allowing DNA delivery at the site of electroporation. The animals were vaccinated under local anaesthesia without any observed immediate or long-term distress or discomfort, or any behavioural signs of muscle damage or pathological changes after the electroporation. DNA-injected and electroporated goats showed increased humoral response after the primary vaccination when compared with nonelectroporated animals. Improved T-cell responses following electroporation were observed in hsp65 DNA-vaccinated cattle. DNA injection with or without electroporation did not compromise the specificity of the tuberculin skin test. In conclusion, a protocol applying in vivo electroporation free of side effects to farmed ruminants was established. In addition, we show that DNA vaccination in combination with electroporation can improve the primary immune responses to the encoded antigens.

Antibody responses in sheep vaccinated against Staphylococcus aureus mastitis: a comparison of two experimental vaccines containing different adjuvants.

A double-blind, placebo-controlled study was conducted to compare two vaccines using different adjuvants with regard to their ability to stimulate antibody production against the alpha- and beta-toxins and the exopolysaccharide of Staphylococcus aureus. The vaccines contained identical antigens, consisting of inactivated whole bacteria of two strains of S. aureus in addition to alpha- and beta-toxoid. One vaccine contained mineral oil, while the other used a water-soluble acrylic acid polymer resin (Carbopol) as adjuvant. Saline served as the placebo. One hundred and forty ewes were vaccinated twice before lambing, by subcutaneous injection with vaccine or placebo in the region of the supramammary lymph node, and were observed and sampled over a period of 6 months. The vaccine containing mineral oil as adjuvant induced significantly greater immune responses to the alpha- and beta-toxins than did the vaccine containing Carbopol. The latter vaccine induced higher levels of antibodies to exopolysaccharide. The degree of local adverse reactions did not differ between the two groups. The results indicate differences between the oil-adjuvanted and Carbopol-adjuvanted vaccines with regard to their ability to stimulate antibody production against S. aureus protein antigens in sheep.

AhpC, AhpD, and a secreted 14-kilodalton antigen from Mycobacterium avium subsp. paratuberculosis distinguish between paratuberculosis and bovine tuberculosis in an enzyme-linked immunosorbent assay.

Sera from cattle naturally infected with Mycobacterium avium subsp. paratuberculosis (n = 56) and naturally (n = 4) and experimentally (n = 8) infected with Mycobacterium bovis were tested for the presence of antibodies against paratuberculosis antigens. An enzyme-linked immunosorbent assay (ELISA) was established based on absorption of M. avium subsp. paratuberculosis antigens on a hyperimmune antiserum against M. avium subsp. avium proteins in order to remove cross-reacting antigens. This absorbed-antigen ELISA recognized 66% of animals with paratuberculosis (37 of 56), while none of the animals with naturally occurring bovine tuberculosis (TB) had detectable antibodies. However, the animals with experimental bovine TB also responded in this ELISA. Similar results were found in a commercial ELISA, showing that neither of these tests was able to distinguish between paratuberculosis and bovine TB. The sera were further tested for antibody activities against purified AhpC and AhpD, which are proteins constitutively expressed by M. avium subsp. paratuberculosis, and against a secreted 14-kDa protein present in culture filtrates from the M. avium complex. Elevated antibody levels to AhpC, AhpD, and the 14-kDa antigen were found in 27% (13 of 48), 15% (7 of 48), and 27% (13 of 48), respectively, of the cattle with paratuberculosis. Together these ELISAs were positive with 35% (17 of 48) of the animals. None of the animals with bovine TB had detectable antibodies against any of the purified proteins despite their high levels of cross-reacting antibodies. These results show that purified specific antigens are needed to differentiate between paratuberculosis and bovine TB in ELISA.

Elevated antibody responses in patients with Crohn's disease against a 14-kDa secreted protein purified from Mycobacterium avium subsp. paratuberculosis.

Patients with Crohn's disease (CD) (n = 10) and ulcerative colitis (UC) (n = 10) were tested for immune responses against various antigens from Mycobacterium avium subsp. paratuberculosis; alkyl hydroperoxide reductase C (AhpC) and alkyl hydroperoxide reductase D (AhpD), which are constitutively expressed in this species as opposed to other mycobacteria, a 14-kDa secreted antigen and PPD-J. The CD patients had significantly elevated antibody levels against the 14 kDa protein (P < 0.05) that were negatively correlated with the duration of the disease (r(s) = - 0.85). They also seemed to have increased antibody levels against AhpC and AhpD, but the differences between the two groups were not significant. However, taken together, the antibody responses to three individual mycobacterial antigens in CD patients strengthen the possibility that the observed responses are caused by mycobacterial infection. No significant differences in the interferon (IFN)-gamma production, the interleukin (IL)-10 production and the ability to proliferate upon stimulation with these antigens were observed. These results show that measuring antibody responses against purified specific antigens is a suitable and simple approach when assessing the connection between CD and mycobacteria in patients with clinical CD. Another important aspect in such studies is to have well defined patient groups tested at the onset of clinical symptoms.

Distinct differences in repertoires of low-molecular-mass secreted antigens of Mycobacterium avium complex and Mycobacterium tuberculosis.

Antigens in a 4-week-old culture filtrate (CF) of Mycobacterium avium subsp. avium were separated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by Western blotting. The culture had minimal lysis of bacilli, giving a CF preparation consisting mainly of secreted proteins. Comparison with a similar CF of Mycobacterium tuberculosis with almost no contamination with intracellular proteins showed the presence of cross-reactive antigens homologous to the four components of the antigen 85 complex, as well as MPT32. These were major constituents of the M. avium subsp. avium CF. In addition, there were several low-molecular-mass bands (<15 kDa) in both species that did not cross-react with polyclonal and polyvalent rabbit antibodies in Western blotting. Furthermore, these bands were not detected in corresponding sonicate preparations, indicating high localization indexes, which is typical of soluble secreted proteins. A 14-kDa protein was selected for purification and more detailed characterization. The N-terminal amino acid sequence was determined, and a matching gene was found within the genomic sequence of M. avium subsp. avium which was highly homologous to Rv0455c of M. tuberculosis. The gene encoded a signal peptide typical of secreted mycobacterial proteins. A rabbit antiserum was raised against the purified protein, and the antigen was demonstrated by Western blotting in CFs of M. avium subsp. avium, Mycobacterium avium subsp. paratuberculosis, Mycobacterium intracellulare, and Mycobacterium scrofulaceum but was not detected in M. tuberculosis. This is a new example of a highly homologous gene being differentially expressed by different mycobacterial species.

Alkyl hydroperoxide reductases C and D are major antigens constitutively expressed by Mycobacterium avium subsp. paratuberculosis.

Antigens characteristic for Mycobacterium avium subspecies paratuberculosis were identified by crossed immunoelectrophoresis (CIE) and by absorbing out cross-reactive antigens by using a polyclonal and polyvalent Mycobacterium avium subspecies avium antiserum. Two antigens were present in M. avium subsp. paratuberculosis and not detected in Mycobacterium avium subsp. avium. They were identified as antigens 17 and 20 in a CIE reference system for M. avium subsp. paratuberculosis antigens. Purified antigen 20 was identified as alkyl hydroperoxide reductase C (AhpC) while the N-terminal part of purified antigen 17 showed 80% homology with alkyl hydroperoxide reductase D (AhpD) of Mycobacterium tuberculosis. AhpC had a nonreduced mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to a molecular mass of 45 kDa and is probably a homodimer linked with disulfide bridges in its native form. AhpD had a mobility corresponding to 19 kDa. Monospecific rabbit antiserum against AhpC and AhpD reacted with 9 strains of M. avium subsp. paratuberculosis but not with 20 other mycobacterial strains except for a Mycobacterium gordonae strain, against which a weak cross-reactive band was produced. Goats experimentally infected with M. avium subsp. paratuberculosis had strong gamma interferon (IFN-gamma) responses toward both AhpC and AhpD, and they also had antibodies against AhpC. The ability of AhpC and AhpD to induce IFN-gamma production shows that these proteins potentially could be used in future vaccines or in diagnostic assays. These results further show that AhpC and AhpD are immunologically important proteins which are constitutively and highly expressed in M. avium subsp. paratuberculosis without the bacteria being submitted to oxidative stress and that the specificities of antigens can be a matter of different levels of protein expression in various species as well as distinct structural differences.

In vitro methods for vaccine evaluation.

This paper reviews aspects related to the establishment of in vitro assays for fish vaccine evaluation and presents mainly assays for potency testing where specific immune responses are monitored. Aspects related to the evaluation of currently available bacterial vaccines are focused on, and results are mainly presented where both immune responses and protection are assessed in the same trial. Although there is no agreement on which antigens are protective in such vaccines, results show that there are candidate antigens from the various pathogens that are strongly immunogenic in fish, and a correlation between the immune responses they elicit and protection is striking. For some antigens, a clear relationship between immunizing dose, antibody response and protection is evident, which is an important basis for a potency test. Results presented on the evaluation of live vaccines show that assays to measure specific T-cell responses are required. These assays, like lymphocyte proliferation and detection of macrophage activation factor, are considered to be less specific than antibody assays and are hampered by greater variations between individual responses. However, when reagents become available for the assessment of cytokine release in an ELISA based assay, this will allow easier assessment of cellular immune responses in fish and could form the basis for potency testing of vaccines where protection is correlated with cellular immunity.

Effects of diets with graded levels of naturally deoxynivalenol-contaminated oats on immune response in growing pigs.

A trial was conducted to evaluate the effect of including different levels of deoxynivalenol (DON)-contaminated oats in the complete diets of growing pigs on immune response and performance. The diets contained 0.6, 1.8 and 4.7 mg DON/kg, and both restricted and ad libitum feeding were used. Performance was recorded as weight gain, feed intake, efficiency of feed utilization and carcass quality. Immune response parameters recorded included primary and secondary antibody titres after injections of five different antigens: Human serum albumin (HSA), sheep red blood cells (SRBC), paratuberculosis vaccine (MPT), tetanus toxoid (TT) and diphteria toxoid (DT). A johnin test was also performed. Lymphocyte stimulation response was measured with three different mitogens (PWM, ConA and PHA). A significant, DON dose-dependent reduction in secondary antibody response to tetanus toxoid was observed. A slightly higher mitogen response after PHA stimulation in lymphocytes from the medium and high DON groups compared to the low DON group after 9 weeks was considered inconclusive. No other indication of dose-dependent immune response inhibition or stimulation was found. Significantly reduced feed intake with increased levels of DON was observed in groups fed restricted rations according to weight, but not in animals fed ad libitum.

Characterization of the immune response to an epitope on Mycobacterium leprae antigen 7 defined by a monoclonal antibody.

A mouse monoclonal antibody (038D-C6) was shown by crossed immunoelectrophoresis and radioimmunoassay to react with an epitope on the Mycobacterium leprae antigen 7. This epitope was highly crossreactive with BCG/M. tuberculosis and of a non-arabinogalactan-arabinomannan nature. A solid-phase radioimmunoassay (SPRIA) was applied, based on competitive inhibition by human sera of antigen binding by this anti-M. leprae monoclonal antibody. Inhibitory activity determined by this assay decreased markedly upon treatment in both lepromatous and tuberculoid leprosy patients. A correlation was found between the bacterial load of the patient and the inhibitory activity measured in the SPRIA assay. Serum-inhibitory activity could therefore perhaps be used as a follow-up test for patients on treatment or as a screening method to detect infective cases. A dot enzyme-linked immunosorbent assay based, like the SPRIA assay, on competitive inhibition by human sera, was explored as an inexpensive and technically simple alternative also applicable under field conditions.

Further characterization including preliminary chemical analysis of antigen MLW1 from Mycobacterium leprae.

MLW1, an antigen preparation from Mycobacterium leprae previously shown to have a high content of M. leprae antigen No. 7 (ML7), was found to contain the typical cell wall constituents arabinose, galactose and mannose. The fatty acid composition of MLW1 was largely comparable to that of undisrupted cells. The capacity of MLW1 to stimulate lymphocytes was further studied. Good correlation was obtained between the in vitro lymphocyte responses to MLW1 and human-derived M. leprae, indicating similar specificity of the two antigen preparations in this test. The stimulatory activity of MLW1 was not significantly influenced by batch to batch variations, was well-preserved during storage and most of it was heat-stable. Attempts to remove the ML7 antigen indicate that this component plays a dominant role in inducing in vitro lymphocyte stimulation.

In vitro stimulation of lymphocytes with an antigen fraction prepared from Mycobacterium leprae and tuberculin PPD in contacts and non-contacts of leprosy patients.

An antigen fraction from Mycobacterium leprae, called MLW1, was used as stimulator in the lymphocyte stimulation test, for comparison with tuberculin PPD. The test was performed in three groups of contacts of leprosy patients with various degree of exposure: (1) close contacts, (2) healthy occupational contacts and (3) non-close contacts and, in addition, in a group of BCG vaccinated and non-exposed controls. The MLW1 preparation induced moderate to strong responses in all three groups of contacts. Although the close contact group showed the highest median responses to all three doses tested, there were no significant differences between the contact groups. At all three doses levels the non-exposed group showed markedly and significantly lower median responses than the contact groups. The responses to tuberculin PPD was markedly and significantly lower in the close contact group than in the other groups. Both when individual responses to the two antigens MLW1 and PPD are compared and when the delta ct/min' estimator is used, the results indicate that the intensity of the specific response increases with the closeness of contact with leprosy patients.

In vitro lymphocyte stimulation in patients with lepromatous and borderline tuberculoid leprosy. The effect of dapsone treatment on the response to Mycobacterium leprae antigens, tuberculin purified protein derivative and non-mycobacterial stimulants.

Lymphocytes from peripheral blood were isolated from leprosy patients and healthy contacts (HC) of leprosy patients and stimulated in vitro with: Mycobacterium leprae and a M. leprae cell wall antigen, MLW 1; tuberculin purified protein derivative (PPD); antigens prepared from Candida albicans, Entamoeba histolytica, Leishmania aethiopica, and parotitis virus; the non-specific mitogens phytohemagglutinin (PHA) and concanavalin A (Con-A). Lymphocytes from patients with untreated lepromatous leprosy failed to respond to the M. leprae antigens, and the median response to PPD was also significantly (p less than 0.005) lower than in the HC group. They responded almost as well as the other groups to non-mycobacterial antigens, PHA, and Con-A. In LL patients who had been treated with dapsone for several (median 10) years, the failure to respond to M. leprae antigens remained, but the depression of the PPD response and the slight non-specific depression of the lymphocyte stimulation test (LST) responsiveness had been reversed. Our results confirm that the major defect in the cell-mediated immune response of LL patients is M. leprae-specific and permanent. The possibility that the defect may be due to a continuous, antigen-induced suppression of the immune response is discussed. That the defect also affected the response to PPD is important since it points to a clear antigenic relationship between M. leprae and BCG/M. tuberculosis. Evidence is presented suggesting that an antigen induced suppressor mechanism may be operating in vitro with cells from patients with borderline tuberculoid leprosy.

In vitro stimulation of lymphocytes in leprosy patients, healthy contacts of leprosy patients, and subjects not exposed to leprosy. Comparison of an antigen fraction prepared from Mycobacterium leprae and tuberculin-purified protein derivative.

In vitro lymphocyte stimulation was performed on peripheral blood lymphocytes from 48 leprosy patients, 15 healthy contacts of leprosy patients, and 16 normal controls who lived in a leprosy-free area and who had not been exposed to leprosy. Tuberculin PPD and an antigen fraction. MLW 1, prepared from M. leprae, were used as stimulants. The MLW 1 preparation contained one antibody-precipitable component when tested in crossed immunoelectrophoresis against a polyvalent anti-M. leprae immunoglobulin preparation, namely the ML 7 antigen. MLW 1 induced strong lymphocyte responses in patients with tuberculoid leprosy and healthy contacts of leprosy patients, but only a weak or no responses in lepromatous leprosy patients and non-exposed controls. A marked depression of the response to tuberculin PPD was observed in lepromatous leprosy patients. The specificity of the MLW 1 antigen is discussed, and a new estimator of specific lymphocyte stimulation, the delta cpm', is introduced.