Marc Govaerts

Members of the 30- to 32-Kilodalton Mycolyl Transferase Family (Ag85) from Culture Filtrate of Mycobacterium avium subsp. paratuberculosis Are Immunodominant Th1-Type Antigens Recognized Early upon Infection in Mice and Cattle

ABSTRACT The characterization of protective antigens is essential for the development of an effective, subunit-based vaccine against paratuberculosis. Surface-exposed and secreted antigens, present abundantly in mycobacterial culture filtrate (CF), are among the well-known protective antigens of Mycobacterium tuberculosis and Mycobacterium bovis. Culture filtrate, prepared from Mycobacterium avium subsp. paratuberculosis ATCC 19698 grown as a surface pellicle on synthetic Sauton medium, was strongly and early recognized in experimentally infected B6 bg/bg beige mice and cattle, as indicated by elevated spleen cell gamma interferon (IFN-γ) secretion and lymphoproliferative responses of peripheral blood mononuclear cells, respectively. Strong proliferative and ex vivo IFN-γ responses against antigen 85 (Ag85) complex (a major protein component from M. bovis BCG culture filtrate) could be detected in cattle as early as 10 weeks after oral M. avium subsp. paratuberculosis infection. Synthetic peptides from the Ag85A and Ag85B components of this complex were strongly recognized, whereas T-cell responses were weaker against peptides from the Ag85C protein. A promiscuous T-cell epitope spanning amino acids 145 to 162 of Ag85B (identical sequence in M. bovis and M. avium subsp. paratuberculosis) was identified in experimentally infected cattle. Finally, young calves, born from cows with confirmed paratuberculosis, demonstrated proliferative responses to purified, recombinant Ag85A and Ag85B from M. avium subsp. paratuberculosis. These results indicate that the M. avium subsp. paratuberculosis Ag85 homologues are immunodominant T-cell antigens that are recognized early in experimental and natural infection of cattle.

Development and validation of a triplex real-time PCR for rapid detection and specific identification of M. avium sub sp. paratuberculosis in faecal samples.

A triplex real-time (TRT-PCR) assay was developed to ensure a rapid and reliable detection of Mycobacterium avium subsp. paratuberculosis (Map) in faecal samples and to allow routine detection of Map in farmed livestock and wildlife species. The TRT-PCR assay was designed using IS900, ISMAP02 and f57 molecular targets. Specificity of TRT-PCR was first confirmed on a panel of control mycobacterial Map and non-Map strains and on faecal samples from Map-negative cows (n=35) and from Map-positive cows (n=20). The TRT-PCR assay was compared to direct examination after Ziehl-Neelsen (ZN) staining and to culture on 197 faecal samples collected serially from five calves experimentally exposed to Map over a 3-year period during the sub-clinical phase of the disease. The data showed a good agreement between culture and TRT-PCR (kappa score=0.63), with the TRT-PCR limit of detection of 2.5 x 10(2)microorganisms/g of faeces spiked with Map. ZN agreement with TRT-PCR was not good (kappa=0.02). Sequence analysis of IS900 amplicons from three single IS900 positive samples confirmed the true Map positivity of the samples. Highly specific IS900 amplification suggests therefore that each single IS900 positive sample from experimentally exposed animals was a true Map-positive specimen. In this controlled experimental setting, the TRT-PCT was rapid, specific and displayed a very high sensitivity for Map detection in faecal samples compared to conventional methods.

Immunogenicity and protective efficacy of DNA vaccines encoding MAP0586c and MAP4308c of Mycobacterium avium subsp. paratuberculosis secretome.

Mycobacterium avium subsp. paratuberculosis (MAP), the etiological agent of chronic enteritis of the small intestine in domestic and wild ruminants, causes substantial losses to livestock industry. Control of this disease is seriously hampered by the lack of adequate diagnostic tools, vaccines and therapies. In this study, we have evaluated the vaccine potential of two MAP proteins, i.e. MAP0586c and MAP4308c, previously identified by postgenomic and immunoproteomic analysis of MAP secretome as novel serodiagnostic antigens. Immunizations of BALB/c and C57BL/6 mice with plasmid DNA encoding MAP0586c and MAP4308c induced strong Th1 type immune responses to both antigens, whereas antibody responses were only induced upon immunization with DNA encoding MAP4308c. Homologous boosting of DNA vaccinated mice with recombinant protein resulted in strong antibody responses against both proteins. Using synthetic overlapping peptides, immunodominant H-2(d) and H-2(b) restricted Th1 T cell epitopes were identified. Finally, MAP infected mice generated strong MAP0586c-specific T cell responses and MAP0586c DNA vaccination could protect BALB/c but not C57BL/6 mice against MAP challenge mice to the same extent as the Mycobacterium bovis BCG vaccine, indicating that this putative transglycosylase is an interesting vaccine candidate that warrants further investigation.

Antigen Mining with Iterative Genome Screens Identifies Novel Diagnostics for the Mycobacterium tuberculosis Complex

ABSTRACT The definition of antigens for the diagnosis of human and bovine tuberculosis is a research priority. If diagnosis is to be used alongside Mycobacterium bovis BCG-based vaccination regimens, it will be necessary to have reagents that allow the discrimination of infected and vaccinated animals. A list of 42 potential M. bovis-specific antigens was prepared by comparative analysis of the genomes of M. bovis, M. avium subsp. avium, M. avium subsp. paratuberculosis, and Streptomyces coelicolor. Potential antigens were tested by applying them in a high-throughput peptide-based screening system to M. bovis-infected and BCG-vaccinated cattle and to cattle without prior exposure to M. bovis. A response hierarchy of antigens was established by comparing responses in infected animals. Three antigens (Mb2555, Mb2890, and Mb3895) were selected for further study, as they were strongly recognized in experimentally infected animals but with low or no frequency in BCG-vaccinated and naïve cows. Interestingly, all three antigens were recognized in animals vaccinated against Johnes disease, suggesting the presences of epitopes cross-reacting with M. avium subsp. paratuberculosis antigens. Eight peptides from the three antigens studied in detail were identified as immunodominant and were characterized in terms of major histocompatibility complex class II restriction element usage and shown to be restricted through both DR and DQ molecules. Reasons for antigenic cross-reactivity with M. avium subsp. paratuberculosis and refinement of the in silico strategy to predict such cross-reactivity from the primary protein sequence will be discussed. Evaluation of the peptides identified from the three dominant antigens by use of larger field studies is now a priority.

Use of Mycobacterium avium subsp. paratuberculosis specific coding sequences for serodiagnosis of bovine paratuberculosis

In this study, the finished complete genome of Mycobacterium avium subsp. paratuberculosis (Map) was screened for specific coding sequences that could be very valuable in the design of a sensitive and specific Map detection serological assay. Eighty-seven Map-specific sequences were retained. Among these, three candidate antigens have been analysed for their serodiagnostic potential. These antigens were selected on the basis of their putative immunogenicity as predicted by in silico analysis. The antigens were cloned in Escherichia coli, expressed, and purified before testing in an antibody detection ELISA test, using a well characterized panel of 18 and 48 sera from Map infected and uninfected cattle, respectively. Two of these antigens, antigen 6 and MAP1637c, yielded in our conditions a sensitivity of 72% and 82%, respectively, for a specificity of 98%. It is particularly noticeable that, when probed with the same serum panel, the most widely used European paratuberculosis commercial seroassay (Pourquier test) yielded a sensitivity of 72% for a specificity of only 92%.

Homologues of natural killer cell receptors NKG2-D and NKR-P1 expressed in cattle

Partial transcripts of the homologues in cattle, of the genes encoding the NKR-P1 and NKG2-D natural killer cell lectin-like receptor families, were cloned by reverse transcriptase-PCR from bovine spleen. Three different cDNAs were partially sequenced for the NKG2-D homologue, and two for the NKR-P1 homologue. Identity to human nucleotide sequences was of 90 and 75%, respectively, and all structural residues of C-type lectin carbohydrate recognition domains were conserved. The identification of two of its members allows to hypothesise the existence of a bovine NK gene complex, prospectively located on chromosome 5.

Immunogenicity of eight Mycobacterium avium subsp. paratuberculosis specific antigens in DNA vaccinated and Map infected mice.

Mycobacterium avium subsp. paratuberculosis (Map), the etiological agent of chronic enteritis of the small intestine in domestic and wild ruminants, causes substantial losses to livestock industry. Control of this disease is seriously hampered by the lack of adequate diagnostic tools and vaccines. Here we report on the immunogenicity of eight Map specific antigens, i.e. MAP1693c, Ag3, MAP2677c (identified by post-genomic and immunoproteomic analysis of Map secretome) and Ag5, Ag6, MAP1637c, MAP0388 and MAP3743 (identified by bioinformatic in silico screening of the Map genome). Strong, antigen-specific IFN-γ responses were induced in mice vaccinated with plasmid DNA encoding MAP1693c, MAP1637c, MAP0388 and MAP3743. In contrast, T cell responses in Map infected mice were directed preferentially against Ag5 and to a lesser extent against MAP3743. None of the tested DNA vaccines conferred protection against subsequent challenge with Map.

PCR Amplification and Sequence of the p33 Piroplasm Surface Antigen Gene of a Theileria Species Isolated from Cattle in West Java, Indonesiaa

ABSTRACT: Oriental theileriosis, a parasitic disease of cattle caused by protozoa of the Theileria orientalis/sergenti/buffeli group, has been reported in Indonesia but its causal agent had not yet been characterized. This study was carried out to isolate and characterize the parasite through comparison of its p33 piroplasm surface antigen gene sequence, with known p32 sequences of T. sergenti and T. buffeli isolates. A Theileria spp. isolate was collected from an Ongole cow in Jonggol, West‐Java, and transferred into a splenectomized calf for antigen production. Piroplasms were extracted from erythrocytes by ammonium chloride‐lysis, separated from unlysed leukocytes and parasitic DNA was phenol‐extracted. Polymerase chain reaction (PCR) was carried out on genomic DNA with a pair of 20 bp primers showing consensus for the p32‐35 nucleotide sequence of 7 known T. orientalis/sergenti/buffeli isolates. An 875 bp fragment was amplified, and further sequenced on both strands by the dye‐labeled terminators method. It showed an 88% homology with the p33 nucleotide sequence of the Japanese T. sergenti Ikeda stock and a lesser homology with 6 other sequences of Australian T. buffeli or Japanese T. sergenti stocks. It was shown to share the presence of the Pst 1 and the absence of the HindIII restriction sites of the T. sergenti Ikeda stock and of one Australian T. buffeli stock, respectively. In conclusion, the affiliation to and the relative position of this Indonesian isolate within the T. orientalis/sergenti/buffeli group has been elucidated.

Serologic screening for 13 infectious agents in roe deer ( Capreolus capreolus ) in Flanders

Introduction In order to investigate the role of roe deer in the maintenance and transmission of infectious animal and human diseases in Flanders, we conducted a serologic screening in 12 hunting areas. Materials and methods Roe deer sera collected between 2008 and 2013 (n=190) were examined for antibodies against 13 infectious agents, using indirect enzyme-linked immunosorbent assay, virus neutralisation, immunofluorescence, or microagglutination test, depending on the agent. Results and discussion High numbers of seropositives were found for Anaplasma phagocytophilum (45.8%), Toxoplasma gondii (43.2%) and Schmallenberg virus (27.9%), the latter with a distinct temporal distribution pattern following the outbreak in domestic ruminants. Lower antibody prevalence was found for Chlamydia abortus (6.7%), tick-borne encephalitis virus (5.1%), Neospora caninum (4.8%), and Mycobacterium avium subsp paratuberculosis (4.1%). The lowest prevalences were found for Leptospira (1.7%), bovine viral diarrhoea virus 1 (1.3%), and Coxiella burnetii (1.2%). No antibodies were found against Brucella sp., bovine herpesvirus 1, and bluetongue virus. A significant difference in seroprevalence between ages (higher in adults >1 year) was found for N. caninum. Four doubtful reacting sera accounted for a significant difference in seroprevalence between sexes for C. abortus (higher in females). Conclusions Despite the more intensive landscape use in Flanders, the results are consistent with other European studies. Apart from maintaining C. abortus and MAP, roe deer do not seem to play an important role in the epidemiology of the examined zoonotic and domestic animal pathogens. Nevertheless, their meaning as sentinels should not be neglected in the absence of other wild cervid species.

Characterisation of the 33 kDa piroplasm surface antigen of Theileria orientalis/sergenti/buffeli isolates from West Java, Indonesia.

The immunodominant 33/35kDa antigen of a Theileria isolate from West Java, Indonesia, was characterised and immuno-affinity purified by use of a monoclonal antibody, KUL-a4, and was shown to be representative of the T. orientalis/sergenti/buffeli group. The aminoterminal sequence of the purified 35kDa peptide (20 residues) was determined by automated Edman degradation and found to correspond to the predicted amino acid sequence of a prospective p33 gene previously sequenced from the same isolate. The cleavage site of a putative signal peptide was identified and conforms the (-3, -1) rule for signal peptidases. The existence of dimeric and trimeric forms of the p33/35 antigen is hypothesised from Western blot profiles. KUL-a4 appeared specific for the T. orientalis/sergenti/buffeli group. It did not recognise in indirect fluorescence antibody test (IFAT), intraerythrocytic bodies of Anaplasma marginale or piroplasms and schizonts of T. mutans, T. parva and T. annulata, whereas cattle antisera raised to these species showed cross-reactivity in IFAT. It however, appeared weakly cross-reactive in Western blot and ELISA, with the 34kDa piroplasm antigen of one T. annulata (Gharb) isolate. The present study indicates that the isolated antigen belongs to the p33/34 antigen family described within the T. sergenti/orientalis/buffeli group, and documents the group-specificity of one of its epitopes.