Ingrid Olsen

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.

Bovine NK cells can produce gamma interferon in response to the secreted mycobacterial proteins ESAT-6 and MPP14 but not in response to MPB70

ABSTRACT Bovine NK cells have recently been characterized and the present study describes the interaction between NK cells, antigen-presenting cells, and secreted mycobacterial proteins. Gamma interferon (IFN-γ) production by NK cells was seen in approximately 30% of noninfected calves in response to the Mycobacterium tuberculosis complex-specific protein ESAT-6, MPP14 from Mycobacterium avium subsp. paratuberculosis, and purified protein derivative (PPD) from M. tuberculosis. In contrast, no response was induced by MPB70, which is another M. tuberculosis complex-specific secreted antigen. The production of IFN-γ by NK cells in whole blood in response to ESAT-6 and MPP14 was demonstrated using intracellular staining together with surface labeling for the NK cell-specific receptor, NKp46, or CD3. Furthermore, the depletion of NK cells from peripheral blood mononuclear cells completely abolished the IFN-γ production. The response was mediated through stimulation of adherent cells and was largely independent of contact between adherent cells and the NK cells. Neutralization of interleukin-12 only partly inhibited IFN-γ production, showing that other cytokines were also involved. The demonstration of NK cell-mediated IFN-γ production in young cattle provides an explanation for the nonspecific IFN-γ response frequently encountered in young cattle when using the IFN-γ test in diagnosis of mycobacterial infections.

The Protozoan Neospora caninum Directly Triggers Bovine NK Cells To Produce Gamma Interferon and To Kill Infected Fibroblasts

ABSTRACT Natural killer (NK) cells are considered to be key players in the early innate responses to protozoan infections, primarily indirectly by producing gamma interferon (IFN-γ) in response to cytokines, like interleukin 12 (IL-12). We demonstrate that live, as well as heat-inactivated, tachyzoites of Neospora caninum, a Toxoplasma-like protozoan, directly trigger production of IFN-γ from purified, IL-2-activated bovine NK cells. This response occurred independently of IL-12 but was increased by the addition of the cytokine. A similar IFN-γ response was measured in cocultures of NK cells and N. caninum-infected autologous fibroblasts. However, no NK cell-derived IFN-γ response was detected when cells were cultured with soluble antigens from the organism, indicating that intact tachyzoites or nonsoluble components are necessary for NK cell triggering. Furthermore, N. caninum-infected autologous fibroblasts had increased susceptibility to NK cell cytotoxicity compared to uninfected fibroblasts. This cytotoxicity was largely mediated by a perforin-mediated mechanism. The activating receptor NKp46 was involved in cytotoxicity against fibroblasts but could not explain the increased cytotoxicity against infected targets. Interestingly, N. caninum tachyzoites were able to infect cultured NK cells, in which tachyzoites proliferated inside parasitophorous vacuoles. Together, these findings underscore the role of NK cells as primary responders during a protozoan infection, describe intracellular protozoan infection of NK cells in vitro for the first time, and represent the first functional study of purified bovine NK cells in response to infection.

New probes used for IS1245 and IS1311 restriction fragment length polymorphism of Mycobacterium avium subsp. avium and Mycobacterium avium subsp. hominissuis isolates of human and animal origin in Norway

BackgroundMycobacterium avium is an environmental mycobacterium that can be divided into the subspecies avium, hominissuis, paratuberculosis and silvaticum. Some M. avium subspecies are opportunistic pathogens for animals and humans. They are ubiquitous in nature and can be isolated from natural sources of water, soil, plants and bedding material. Isolates of M. avium originating from humans (n = 37), pigs (n = 51) and wild birds (n = 10) in Norway were examined by IS1245 and IS1311 RFLP using new and specific probes and for the presence of IS901 and ISMpa1 by PCR. Analysis and generation of a dendrogram were performed with the software BioNumerics.ResultsIS1311 RFLP provided clear results that were easy to interpret, while IS1245 RFLP generated more complex patterns with a higher discriminatory power. The combination of the two methods gave additional discrimination between isolates. All avian isolates except one were M. avium subsp. avium with two copies of IS1311 and one copy of IS1245, while the isolates of human and porcine origin belonged to M. avium subsp.hominissuis. The isolates from human patients were distributed randomly among the clusters of porcine isolates. There were few identical isolates. However, one isolate from a human patient was identical to a porcine isolate. Regional differences were detected among the porcine isolates, while there was no clustering of human isolates according to type of clinical symptoms or geographical location of the patients home addresses.ConclusionThe results demonstrate that a wide range of M. avium subsp.hominissuis are present in pigs and humans in Norway, and that some of these isolates are very similar. It remains to be determined whether humans are infected from pigs or if they are infected from common environmental sources.

Elevated Antibody Responses in Patients with Crohn's Disease against a 14-kDa Secreted Protein Purified from Mycobacterium avium subsp. paratuberculosis

Patients with Crohns 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 (rs = − 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)‐γ 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.

Bovine CD2 - /NKp46 + cells are fully functional natural killer cells with a high activation status

BackgroundNatural killer (NK) cells in the cow have been elusive due to the lack of specific NK cell markers, and various criteria including a CD3-/CD2+ phenotype have been used to identify such cells. The recent characterization of the NK-specific NKp46 receptor has allowed a more precise definition of bovine NK cells. NK cells are known as a heterogeneous cell group, and we here report the first functional study of bovine NK cell subsets, based on the expression of CD2.ResultsBovine CD2- NK cells, a minor subset in blood, proliferated more rapidly in the presence of IL-2, dominating the cultures after a few days. Grown separately with IL-2, CD2- and CD2+ NK cell subsets did not change CD2 expression for at least two weeks. In blood, CD2- NK cells showed a higher expression of CD44 and CD25, consistent with a high activation status. A higher proportion of CD2- NK cells had intracellular interferon-gamma in the cytoplasm in response to IL-2 and IL-12 stimulation, and the CD2- subset secreted more interferon-gamma when cultured separately. Cytotoxic capacity was similar in both subsets, and both carried transcripts for the NK cell receptors KIR, CD16, CD94 and KLRJ. Ligation by one out of two tested anti-CD2 monoclonal antibodies could trigger interferon-gamma production from NK cells, but neither of them could alter cytotoxicity.ConclusionThese results provide evidence that bovine CD2- as well as CD2+ cells of the NKp46+ phenotype are fully functional NK cells, the CD2- subset showing signs of being more activated in the circulation.

Distribution of IS1311 and IS1245 in Mycobacterium avium Subspecies Revisited

ABSTRACT We demonstrated that IS1245 is not present in Mycobacterium avium subsp. paratuberculosis by restriction fragment length polymorphism and that the designated three-banded bird pattern of IS1245 in M. avium subsp. avium consists of one copy of IS1245 and two copies of IS1311. Cross hybridization between the two elements can be avoided by using more specific probes.

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

ABSTRACT Antigens characteristic for Mycobacterium aviumsubspecies 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 aMycobacterium gordonae strain, against which a weak cross-reactive band was produced. Goats experimentally infected withM. avium subsp. paratuberculosis had strong gamma interferon (IFN-γ) responses toward both AhpC and AhpD, and they also had antibodies against AhpC. The ability of AhpC and AhpD to induce IFN-γ 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. aviumsubsp. 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.

Biofilm formation by Mycobacterium avium isolates originating from humans, swine and birds

BackgroundMycobacterium avium includes the subspecies avium, silvaticum, paratuberculosis and hominissuis, and M. avium subspecies has been isolated from various environments all over the world including from biofilms in water distribution systems. The aim of this study was to examine isolates of M. avium subsp. avium and M. avium subsp. hominissuis of different origin for biofilm formation and to look for correlations between biofilm formation and RFLP-types, and to standardise the method to test for biofilm formation. In order to determine the best screening method, a panel of 14 isolates of M. avium subsp. avium and M. avium subsp. hominissuis, were tested for their ability to form biofilm in microtiter plates under different conditions. Subsequently, 83 additional isolates from humans, swine and birds were tested for biofilm formation. The isolates were tested for the presence of selected genes involved in the synthesis of glycopeptidolipids (GPLs) in the cell wall of M. avium, which is believed to be important for biofilm formation. Colony morphology and hsp65 sequvar were also determined.ResultsNine isolates from swine produced biofilm. There was a significant higher frequency of porcine isolates forming biofilm compared to human isolates. All isolates were previously characterised by IS1311- and IS1245-RFLP typing. The ability to form biofilm did not correlate with the RFLP-type, hsp65 sequevar, colony morphology or the presence of gene sequences related to GPL synthesis.ConclusionThe observed differences in biofilm forming abilities between porcine and human isolates raises questions regarding the importance of biofilm formation for infectious potential. The optimised method worked well for screening of multiple isolates.

Innate IFN-gamma production in cattle in response to MPP14, a secreted protein from Mycobacterium avium subsp. Paratuberculosis.

Calves experimentally infected with Mycobacterium avium subsp. paratuberculosis and uninfected calves were tested for interferon(IFN)‐γ production after stimulation with purified protein derivative from M. avium subsp. paratuberculosis (PPDp) or a secreted 14 kDa protein (MPP14) specific for the M. avium‐intracellulare‐scrofulaceum (MAIS) complex. Several calves in both groups responded strongly up to about 5 months to both antigens. Two uninfected calves responded repeatedly, but not always, to MPP14 and PPDp throughout the study. The responses in the uninfected animals seemed to be independent of cell contact between the antigen presenting cells (APC) and the responding population. The supernatant from adherent cells stimulated with MPP14 induced similar levels of IFN‐γ production in CD14+/B‐cell depleted peripheral blood mononuclear cells (PBMC) as when the antigen was used directly on PBMC. In contrast, APC/T‐cell contact was necessary to induce the IFN‐γ production in infected animals, suggesting that both innate and adaptive IFN‐γ production in response to MPP14 could occur. CD8+ cells contributed to some of the IFN‐γ production in response to MPP14, but the rest could not be explained, while CD4+ cells were responsible for the adaptive response to PPDp. This study showed that secreted proteins could induce innate IFN‐γ production that interferes with diagnostic testing using the IFN‐γ‐test.