Use of ethanol extract of Mycobacterium bovis for detection of specific antibodies in sera of farmed red deer (Cervus elaphus) with bovine tuberculosis.

BACKGROUND: Bovine tuberculosis (bTB) in wildlife species poses a threat to domestic livestock in many situations. Control programs for bTB in livestock depend on testing and slaughtering the positive animals; however, the currently available diagnostic tests often have poor specificity. In our previous study, we developed a specific and sensitive enzyme linked immunosorbent assay (ELISA) for another mycobacterial disease - Johne's disease, using surface antigens of Mycobacterium avium ssp. paratuberculosis (MAP) extracted by briefly agitating the bacilli in 80% ethanol solution. The ELISA test was named ethanol vortex ELISA (EVELISA). The objective of this study is to examine whether EVELISA technique could be used to specifically detect anti-Mycobacterium bovis (M. bovis) antibodies in the serum of M. bovis-infected farmed red deer (Cervus elaphus). We tested a total of 45 red deer serum samples, divided in 3 groups - uninfected animals (n = 15), experimentally infected with M. bovis (n = 15) and experimentally infected with MAP (n = 15). RESULTS: The presence of anti-M. bovis antibodies was tested using an ethanol extract of M. bovis. Without absorption of anti-MAP cross reactive antibodies, it was found that 13 out of the 15 MAP-infected animals showed high antibody binding. Using heat killed MAP as an absorbent of cross reactive antibodies, anti-M. bovis antibodies were detected in 86.7% of M. bovis-infected animals with minor false positive results caused by MAP infection. CONCLUSIONS: The results from this study suggest that EVELISA may form a basis for a sensitive and specific test for the diagnosis of bTB in farmed red deer.

Global gene expression profiling of monocyte-derived macrophages from red deer (Cervus elaphus) genotypically resistant or susceptible to Mycobacterium avium subspecies paratuberculosis infection.

Mycobacterium avium subspecies paratuberculosis (MAP) can cause a chronic inflammatory bowel disease, Johne's disease (JD), in ruminant animals. This study has explored the molecular basis of resistance and susceptibility to this disease in red deer breeds previously confirmed to express polarised phenotypes by experimental infection trials and following natural infection. Monocyte-derived macrophage cultures were obtained from uninfected red deer selected for either a resistant or susceptible phenotype. Cells were infected with MAP in vitro and gene expression analysed by RNA-Seq. Transcriptome analysis revealed a more disrupted gene expression profile in macrophages from susceptible animals compared with cells from resistant animals in terms of the number of genes up- or downregulated. Highly upregulated genes were related to chemotaxis (CXCL10, CSF3, and CCL8) and type 1 interferon signalling (RSAD2, IFIT1, IFIT2, ISG12, ISG15, USP18, and HERC6). Upregulation of these genes was observed to be greater in macrophages from susceptible animals compared to cells from resistant animals in response to in vitro MAP infection. These data support the use of transcriptomic approaches to enable the identification of markers associated particularly with susceptibility to MAP infection.

Innate immune markers that distinguish red deer (Cervus elaphus) selected for resistant or susceptible genotypes for Johne's disease.

While many factors contribute to resistance and susceptibility to infectious disease, a major component is the genotype of the host and the way in which it is expressed. Johne's disease is a chronic inflammatory bowel disease affecting ruminants and is caused by infection with Mycobacterium avium subspecies paratuberculosis (MAP). We have previously identified red deer breeds (Cervus elaphus) that are resistant; have a low rate of MAP infection and do not progress to develop Johne's disease. In contrast, susceptible breeds have a high rate of MAP infection as seen by seroconversion and progress to develop clinical Johne's disease. The aim of this study was to determine if immunological differences exist between animals of resistant or susceptible breeds. Macrophage cultures were derived from the monocytes of deer genotypically defined as resistant or susceptible to the development of Johne's disease. Following in vitro infection of the cells with MAP, the expression of candidate genes was assessed by quantitative PCR as well as infection rate and cell death rate. The results indicate that macrophages from susceptible animals show a significantly higher upregulation of inflammatory genes (iNOS, IL-1α, TNF-α and IL-23p19) than the macrophages from resistant animals. Cells from resistant animals had a higher rate of apoptosis at 24 hours post infection (hpi) compared to macrophages from susceptible animals. The excessive expression of inflammatory mRNA transcripts in susceptible animals could cause inefficient clearing of the mycobacterial organism and the establishment of disease. Controlled upregulation of inflammatory pathways coupled with programmed cell death in the macrophages of resistant animals may predispose the host to a protective immune response against this mycobacterial pathogen.

Ovine immune parameters following immunisation against Mycobacterium avium ssp. paratuberculosis using a lipid-based live-cell vaccine.

Current commercial sheep vaccines against Mycobacterium avium subspecies paratuberculosis (MAP) are based on the use of live or killed cells from avirulent MAP strains. These stimulate a mixed immune response, featuring both antibody-based and cell-mediated immunity, and can only confer partial protection against Johne's disease but do not prevent infection. This study aimed to identify immune responses in sheep following immunisation with a novel lipid-based live-cell vaccine, drawing comparisons against responses observed to a commercial killed-cell vaccine (Gudair). The live vaccine was administered either subcutaneously or intra-peritoneally, as either a single-dose vaccine or in an homologous prime-boost protocol. A single-dose of the live vaccine was found to stimulate a cellular immune response similar to that of single-dose Gudair, but with markedly lower levels of antibody; however, homologous boosting with the live vaccine, by either s.c. or i.p. routes, generated higher levels of MAP-specific antibody. All immunisation regimes tended to decrease the proportion of CD4(+) T cells but increase the proportions of gammadeltaTCR(+) T cells and CD25(+) cells in antigen-stimulated ex vivo blood samples. The CD8(+):gammadeltaTCR(+) T cell ratio, thought to represent a reduced regulatory capacity among T cells responding to MAP, was increased among animals receiving either Gudair or a single i.p. dose of the live vaccine; however, only the Gudair vaccine simultaneously increased the level of lymph node IFNgamma mRNA expression, and this treatment also caused a significant elevation in the IFNgamma:IL-10 (effector:regulatory) cytokine expression ratio. Thus, among these immunisation regimes, the responses generated by a single s.c. dose of the novel live-cell vaccine appeared to selectively target the CMI-based immune profile thought necessary for control of MAP infection; in contrast, homologous prime-boosting with the live vaccine stimulated a mixed immune response similar to that produced by immunisation with Gudair.

Development of the thymus and Peyer's patches in the first year of life in red deer (Cervus elaphus).

In young ruminants, as in most animals, the thymus and Peyer's patches (PP) play a key role in immune function. In sheep and cattle it has been shown that they follow a recognisable pattern of development throughout early life but a study of these tissues in red deer (Cervus elaphus) had not been previously conducted. Red deer are undergoing increasing domestication thus it was important to determine whether there were any significant differences between species. Samples from the thymus, jejunal PP and ileal PP were obtained from deer at 1, 1.5, 2, 3, 6, 9 and 12 months of age. Tissues were measured, processed and comparisons between the equivalent tissues in cattle and sheep were made. The thymus and Peyer's patches of red deer were observed to follow similar patterns of development and involution as in other ruminant species. The thymus was also observed to show a seasonal decrease in weight and cellular composition possibly due to nutritional and environmental stresses. Moreover, evidence for the prevailing idea that the ileal PP acts as primary lymphoid tissue in ruminants was obtained.

Peripheral blood mononuclear cell population changes associated with age and Mycobacterium avium subspecies paratuberculosis infection in red deer (Cervus elaphus).

In this study we aimed to document the maturation of the immune system in red deer and investigate the hypothesis that an immature immune system may predispose young red deer (Cervus elaphus) to an increased susceptibility to Johne's disease. Peripheral blood mononuclear cell populations were analysed using flow cytometry techniques to monitor changes associated with age and severity of infection with Mycobacterium avium subspecies paratuberculosis in red deer. The percentage of cells expressing cell population markers CD4, CD8, WC1, gammadelta TCR, CD14 and B-B4 as well as the cell activation markers CD25, CD44, ACT1 and ACT31 were analysed in relation to age and M. avium subsp. paratuberculosis infection status and disease severity. Significant changes in both cell surface markers and cell activation markers were observed as animals matured irrespective of their response to infection. The levels of CD4 and CD8 increased with age and the levels of B-B4, WC1, CD14 and gammadelta TCR decreased similar to previous studies in cattle. No differences were observed in cell surface markers or cell activation markers in relation to M. avium subsp. paratuberculosis infection status and disease severity. This research demonstrates that peripheral blood cell populations change with age in red deer and suggests that studies of cell surface markers and activation markers in peripheral blood samples do not provide information on the age-related susceptibility to Johne's disease.

Identification of immune parameters to differentiate disease states among sheep infected with Mycobacterium avium subsp. paratuberculosis.

Johne's disease, a chronic enteritis of ruminants, is caused by infection with Mycobacterium avium subsp. paratuberculosis. Three distinct forms have been observed in sheep: paucibacillary disease (PB), multibacillary disease (MB), and asymptomatic infection (AS). In this study, immune parameters for animals naturally infected with M. avium subsp. paratuberculosis and identified postmortem as having PB, MB, or AS were compared to provide a further understanding of the immunological reactivity contributing to or resulting from these different disease states in sheep. PB was associated with strong ex vivo M. avium subsp. paratuberculosis antigen-stimulated gamma interferon responses, pronounced increases in CD25(+) T-cell frequencies in circulation, antibody production, and a B-cell population that expanded significantly upon ex vivo antigenic stimulation. The MB group featured the highest antibody levels and a lack of cellular immune responsiveness to the M. avium subsp. paratuberculosis antigen. The AS group expressed an immunological phenotype intermediate between that for noninfected control animals and that for the PB group. The relationship between immune responses and disease severity within the PB group was investigated more closely; significant positive correlations were observed between disease severity and both the CD8(+) population in the circulating blood and the expression of interleukin-4 mRNA in antigen-stimulated blood samples ex vivo. Together, these data point toward distinct immune profiles in sheep that correspond to different Johne's disease states, which can be determined from circulating blood and/or from localized intestinal tract tissue samples.

Efficacy of novel lipid-formulated whole bacterial cell vaccines against Mycobacterium avium subsp. paratuberculosis in sheep.

Mycobacterium avium subsp. paratuberculosis [MAP], the causative agent of enteric Johne's disease, incurs significant economic losses to the livestock industry. Prophylactic vaccination can be employed as a control means, however mineral oil-based vaccines currently in practice have limited efficacy, produce strong antibody responses that confound serological diagnostic testing, and cause severe injection site reactions. In the present study, the safety and efficacy of a commercial mineral oil-adjuvanted vaccine (Gudair) was compared with novel parenteral-route vaccines in sheep; these comprised live or heat-killed (HK) whole cell preparations of MAP strain 316F, formulated into a food-grade lipid vaccine delivery matrix. Subcutaneous administration of lipid-formulated live or HK 316F-induced significantly fewer adverse injection site reactions than Gudair; adverse injection site reactions were eliminated altogether by intraperitoneal (i.p.) injection of lipid-formulated live 316F. Injections of lipid-formulated 316F-induced significant peripheral blood cell-mediated immune (CMI) responses in the absence of antibody, while Gudair-induced strong antibody and CMI reactivity. Vaccinated and non-vaccinated control sheep were challenged via oral inoculation of a virulent MAP isolate, and disease progress was monitored for 16 months, followed by necropsy. All vaccine regimes reduced the overall pathological grading of biopsied intestinal tract (IT) tissues; among these, only Gudair promoted a significant reduction in the incidence of histopathological IT lesions, while only i.p. injection of lipid-formulated live 316F significantly reduced the incidence of gross IT lesions. All lipid-formulated vaccines (but not Gudair) significantly reduced the incidence of bacteriological culture-confirmed MAP infection. This study identifies a new vaccination strategy against Johne's disease in sheep using conventional MAP vaccine strains formulated in a metabolisable lipid delivery matrix.

Autoreactive antibodies are present in sheep with Johne's disease and cross-react with Mycobacterium avium subsp. paratuberculosis antigens.

Mycobacterial infection has been linked to the generation of autoantibodies, including anti-neutrophil cytoplasmic autoantibodies (ANCA), in clinical studies and small animal models. In an attempt to identify antibodies that react with both self and mycobacterial antigens in naturally infected ruminants, we generated a phage display library comprising single chain antibody fragments (scFv) from sheep with Johne's disease (JD). The library was screened simultaneously against ovine small intestinal tissue and Mycobacterium avium subsp. paratuberculosis (MAP). A clone (termed AutoH1) reacted strongly with host tissue and MAP, recognizing a proteinase-susceptible 32 kDa determinant in ovine gut tissues and lymphatics, and in blood granulocytes but not mononuclear cells. In granulocytes, binding was to cytoplasmic granules and cell membranes; in MAP, AutoH1 bound bacterial cell wall determinants. We further identified a synthetic peptide sequence recognized by AutoH1, using this to generate a carrier:peptide fusion protein (paH1). Sera of normal and JD sheep were screened for AutoH1-like autoantibody activity; 7/11 JD animals showed autoreactivity that could be blocked by paH1, while 0/21 normal animals showed no such serological reactivity. It is possible that the severe pathology observed in ruminant JD may have an autoimmune component, possibly due to ANCA-type binding; this remains to be further investigated.

Production and characterization of monoclonal antibodies against a major membrane protein of Mycobacterium avium subsp. paratuberculosis.

The Mycobacterium avium subsp. paratuberculosis 35-kDa major membrane protein (MMP) encoded by MAP2121c is an important membrane antigen recognized in cattle with Johne's disease. In this study, purified recombinant MMP was used to produce two stable monoclonal antibodies, termed 8G2 and 13E1, which were characterized by immunoblotting, epitope mapping, and immunofluorescence microscopy.

Isolation of high-affinity single-chain antibodies against Mycobacterium avium subsp. paratuberculosis surface proteins from sheep with Johne's disease.

Johne's disease, caused by infection with Mycobacterium avium subsp. paratuberculosis, causes significant economic losses to the livestock farming industry. Improved investigative and diagnostic tools-necessary to understand disease processes and to identify subclinical infection-are much sought after. Here, we describe the production of single-chain antibodies with defined specificity for M. avium subsp. paratuberculosis surface proteins. Single-chain antibodies (scFv) were generated from sheep with Johne's disease by cloning heavy-chain and lambda light-chain variable regions and expressing these in fusion with gene III of filamentous phages. Two scFv clones (designated SurfS1.2 and SurfS2.2) were shown to be immunoreactive against M. avium subsp. paratuberculosis surface targets by flow cytometry, and immunoblotting identified specificity for a 34-kDa proteinase-susceptible determinant. Both antibodies were cross-reactive against Mycobacterium avium subsp. avium but nonreactive against Mycobacterium bovis or Mycobacterium phlei cells and were shown to be capable of enriching M. avium subsp. paratuberculosis cells by a factor of approximately 10(6)-fold when employed in magnetic bead separation of mixed Mycobacterium sp. cultures. Further, magnetic bead separation using SurfS1.2 and SurfS2.2 was capable of isolating as few as 10(3) M. avium subsp. paratuberculosis cells from ovine fecal samples, indicating the diagnostic potential of these reagents. Finally, inclusion of SurfS1.2 or SurfS2.2 in in vitro broth culture with M. avium subsp. paratuberculosis indicated that surface binding activity did not impede bacterial growth, although colony clumping was prevented. These results are discussed in terms of the potential use of single-chain phage display monoclonal antibodies as novel diagnostic reagents.

Immunoglobulin G1 enzyme-linked immunosorbent assay for diagnosis of Johne's Disease in red deer (Cervus elaphus).

This study was designed to develop a customized enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of Johne's disease (JD) in farmed deer. Two antigens were selected on the basis of their superior diagnostic readouts: denatured purified protein derivative (PPDj) and undenatured protoplasmic antigen (PpAg). ELISA development was based on the antigen reactivity of the immunoglobulin G1 (IgG1) isotype, which is a highly specific marker for mycobacterial disease seroreactivity in deer. Sensitivity estimates and test parameters were established using 102 Mycobacterium paratuberculosis-infected animals from more than 10 deer herds, and specificity estimates were determined using 508 uninfected animals from 5 known disease-free herds. A receiver-operated characteristic analysis determined that at a cut point of 50 ELISA units, there was a specificity of 99.5% and sensitivities of 84.0% with PPDj antigen, 88.0% with PpAg, and 91.0% when the antigens were used serially in a composite test. Estimated sensitivity was further improved using recombinant protein antigens unique for M. paratuberculosis, which identified infected animals that were unreactive to PPDj or PpAg. While 80% of animals that were seropositive in the IgG1 ELISA had detectable histopathology, the assay could also detect animals with subclinical disease. The test was significantly less sensitive (75%) for animals that were culture positive for M. paratuberculosis but with no detectable pathology than for those with pathological evidence of JD (>90%). When the IgG1 ELISA was used annually over a 4-year period in a deer herd with high levels of clinical JD, it eliminated clinical disease, increased production levels, and reduced JD-related mortality.

A strategic approach to vaccine development: animal models, monitoring vaccine efficacy, formulation and delivery.

One of the most pressing issues in animal and human medicine currently is the need to develop new and more effective vaccines, against an ever increasing range of infectious diseases. Traditional vaccines have involved the use of killed microorganisms, live attenuated cultures or antigenic extracts. In spite of extensive research, there have been very few newly developed vaccines for humans or animals, over the past 20 years. Recent developments in our understanding of the pathways of immunity required to produce protection against different infections, allow immunological principles to be incorporated into the design new and better vaccines. This paper outlines the key variables that need to be taken into consideration when developing a new vaccine. Three critical areas will be considered in the review: (1) use of an appropriate experimental animal model of infection; (2) targeting appropriate immune markers for protection; (3) design of optimal vaccine formulation and delivery systems.