Combined Phylogeographic Analyses and Epidemiologic Contact Tracing to Characterize Atypically Pathogenic Avian Influenza (H3N1) Epidemic, Belgium, 2019.

The high economic impact and zoonotic potential of avian influenza call for detailed investigations of dispersal dynamics of epidemics. We integrated phylogeographic and epidemiologic analyses to investigate the dynamics of a low pathogenicity avian influenza (H3N1) epidemic that occurred in Belgium during 2019. Virus genomes from 104 clinical samples originating from 85% of affected farms were sequenced. A spatially explicit phylogeographic analysis confirmed a dominating northeast to southwest dispersal direction and a long-distance dispersal event linked to direct live animal transportation between farms. Spatiotemporal clustering, transport, and social contacts strongly correlated with the phylogeographic pattern of the epidemic. We detected only a limited association between wind direction and direction of viral lineage dispersal. Our results highlight the multifactorial nature of avian influenza epidemics and illustrate the use of genomic analyses of virus dispersal to complement epidemiologic and environmental data, improve knowledge of avian influenza epidemiologic dynamics, and enhance control strategies.

Atypical Pathogenicity of Avian Influenza (H3N1) Virus Involved in Outbreak, Belgium, 2019.

In 2019, an outbreak of avian influenza (H3N1) virus infection occurred among commercial poultry in Belgium. Full-genome phylogenetic analysis indicated a wild bird origin rather than recent circulation among poultry. Although classified as a nonnotifiable avian influenza virus, it was associated with reproductive tropism and substantial mortality in the field.

In silico epitope analysis of unique and membrane associated proteins from Mycobacterium avium subsp. paratuberculosis for immunogenicity and vaccine evaluation.

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of paratuberculosis disease affecting ruminants worldwide. The aim of this study was to identify potential candidate antigens and epitopes by bio and immuno-informatic tools which could be later evaluated as vaccines and/or diagnosis. 110 protein sequences were selected from MAP K-10 genome database: 48 classified as putative enzymes involved in surface polysaccharide and lipopolysaccharide synthesis, as membrane associated and secreted proteins, 32 as conserved membrane proteins, and 30 as absent from other mycobacterial genomes. These 110 proteins were preliminary screened for Major Histocompatibility Complex (MHC) class II affinity and promiscuity using ProPred program. In addition, subcellular localization and host protein homology was analyzed. From these analyses, 23 MAP proteins were selected for a more accurate inmunoinformatic analysis (i.e. T cell and B cell epitopes analysis) and for homology with mycobacterial proteins. Finally, eleven MAP proteins were identified as potential candidates for further immunogenic evaluation: six proteins (MAP0228c, MAP1239c, MAP2232, MAP3080, MAP3131 and MAP3890) were identified as presenting potential T cell epitopes, while 5 selected proteins (MAP0232c, MAP1240c, MAP1738, MAP2239 and MAP3641c) harbored a large numbers of epitopes predicted to induce both cell- and antibody-mediated immune responses. Moreover, immunogenicity of selected epitopes from MAP1239c were evaluated in IFN-γ release assay. In summary, eleven M. avium subsp. paratuberculosis proteins were identified by in silico analysis and need to be further evaluated for their immunodiagnostic and vaccine potential in field and mice model.

Alfalfa Plants (Medicago sativa L.) Expressing the 85B (MAP1609c) Antigen of Mycobacterium avium subsp. paratuberculosis Elicit Long-Lasting Immunity in Mice.

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Paratuberculosis, a contagious, untreatable, and chronic granulomatous enteritis that results in diarrhea, emaciation, and death in farmed ruminants (i.e., cattle, sheep, and goats). In this study, the Ag85B antigen from MAP was expressed in transgenic alfalfa as an attractive vaccine candidate. Agrobacterium-mediated transformation allowed the rescue of 56 putative transformed plants and transgenesis was confirmed in 19 lines by detection of the Ag85B gene (MAP1609c) by PCR. Line number 20 showed the highest Ag85B expression [840 ng Ag85B per gram of dry weight leaf tissue, 0.062% Total Soluble Protein (TSP)]. Antigenicity of the plant-made Ag85B was evidenced by its reactivity with a panel of sera from naturally MAP-infected animals, whereas immunogenicity was assessed in mice immunized by either oral or subcutaneous routes. The plant-made Ag85B antigen elicited humoral responses by the oral route when co-administered with cholera toxin as adjuvant; significant levels of anti-85B antibodies were induced in serum (IgG) and feces (IgA). Long-lasting immunity was evidenced at day 180 days post-first oral immunization. The obtained alfalfa lines expressing Ag85B constitute the first model of a plant-based vaccine targeting MAP. The initial immunogenicity assessment conducted in this study opens the path for a detailed characterization of the properties of this vaccine candidate.

Molecular and virological characterization of the first poultry outbreaks of Genotype VII.2 velogenic avian orthoavulavirus type 1 (NDV) in North-West Europe, BeNeLux, 2018.

After two decades free of Newcastle disease, Belgium encountered a velogenic avian orthoavulavirus type 1 epizootic in 2018. In Belgium, 20 cases were diagnosed, of which 15 occurred in hobby flocks, 2 in professional poultry flocks and 3 in poultry retailers. The disease also disseminated from Belgium towards the Grand Duchy of Luxembourg by trade. Independently, the virus was detected once in the Netherlands, almost simultaneously to the first Belgian detection. As such Newcastle disease emerged in the entire BeNeLux region. Both the polybasic sequence of the fusion gene cleavage site and the intracerebral pathotyping assay demonstrated the high pathogenicity of the strain. This paper represents the first notification of this specific VII.2 subgenotype in the North-West of Europe. Time-calibrated full genome phylogenetic analysis indicated the silent or unreported circulation of the virus prior to the emergence of three genetic clusters in the BeNeLux region without clear geographical or other epidemiological correlation. The Dutch strain appeared as an outgroup to the Belgian and Luxembourgian strains in the time-correlated genetic analysis and no epidemiological link could be identified between the Belgian and Dutch outbreaks. In contrast, both genetic and epidemiological outbreak investigation data linked the G.D. Luxembourg case to the Belgian outbreak. The genetic links between Belgian viruses from retailers and hobby flocks only partially correlated with epidemiological data. Two independent introductions into the professional poultry sector were identified, although their origin could not be determined. Animal experiments using 6-week- old specific pathogen-free chickens indicated a systemic infection and efficient transmission of the virus. The implementation of re-vaccination in the professional sector, affected hobby and retailers, as well as the restriction on assembly and increased biosecurity measures, possibly limited the epizootic and resulted in the disappearance of the virus. These findings emphasize the constant need for awareness and monitoring of notifiable viruses in the field.

Genetic resistance of mice to Mycobacterium paratuberculosis is influenced by Slc11a1 at the early but not at the late stage of infection.

We have recently described the development of a luminescent Mycobacterium paratuberculosis strain of bovine origin expressing the luxAB genes of Vibrio harveyi. With this luminescent isolate, fastidious and costly enumeration of CFU by plating them on agar can be replaced by easy and rapid luminometry. Here, we have reevaluated the effect of Slc11a1 (formerly Nramp1) polymorphism on susceptibility to M. paratuberculosis, using this luminometric method. A series of inbred mouse strains were infected intravenously with luminescent M. paratuberculosis S-23 and monitored for bacterial replication in spleen, liver, and lungs for 12 weeks. The results indicate that, as for Mycobacterium avium subsp. avium, innate resistance to infection is genetically controlled by Slc11a1. In BALB/c, congenic BALB.B10-H2(b) (BALB/c background; H-2(b)), C57BL/6, and beige C57BL/6(bg/)(bg) mice (all Slc11a1(s)), bacterial numbers in spleen and liver remained unchanged during the first 4 weeks of infection, whereas in DBA/2 and congenic BALB/c.DBA/2 (C.D2) mice (both Slc11a1(r)) and in (C57BL/6 x DBA/2)F(1) mice (Slc11a1(s/r)), the bacterial numbers had decreased more than 10-fold at 4 weeks postinfection in both male and female mice. At later time points, additional differences in bacterial replication were observed between the susceptible mouse strains, particularly in the liver. Whereas bacterial numbers in the liver gradually decreased more than 100-fold in C57BL/6 mice between week 4 and week 12, bacterial numbers were stable in livers from BALB/c and beige C57BL/6(bg/)(bg) mice during this period. Mycobacterium-specific gamma interferon responses developed earlier and to a higher magnitude in C57BL/6 mice than in BALB/c mice and were lowest in resistant C.D2 mice.

Evaluation of mycobacteria-specific gamma interferon and antibody responses before and after a single intradermal skin test in cattle naturally exposed to M. avium subsp. paratuberculosis and experimentally infected with M. bovis.

This study reports on the diagnostic potential of IFN-γ release assays and serology for Mycobacterium bovis in six naturally M. avium subsp. paratuberculosis (Map) exposed bulls of which four were intratracheally infected with a Belgian field strain of M. bovis. Heparinized blood, serum and fecal samples were collected at regular time intervals for mycobacteria-specific IFN-γ release assays, antibody analysis and for Map culture respectively. Single intradermal skin test (SIT) with bovine tuberculin (PPD-B) was performed on day 115 and animals were sacrificed on day 133 after M. bovis infection. Organs were collected and stored for histopathological examination, modified Ziehl-Neelsen staining and bacteriological analysis of M. bovis and Map by culture and RT-PCR. Prior to infection five animals showed positive IFN-γ responses to avian PPD (PPD-A) and four were positive in Map PCR (IS900) on faeces. Three M. bovis infected animals reacted as early as day 14 with sustained higher PPD-B than PPD-A specific IFN-γ responses, whereas the fourth animal (with the strongest PPD-A response prior to infection) showed sustained higher PPD-B specific IFN-γ levels only a day 56 after infection. Two of the infected animals had a sustained positive IFN-γ response to the ESAT-6/CFP-10/TB7.7 (QuantiFERON®-TB Gold) peptide cocktail as early as day 14, among which the animal with the initial high PPD-A response. Later during infection, positive responses were found to ESAT-6 peptides in three infected bulls and to CFP-10 peptides in all four infected bulls. One of the control animals reacted intermittently to the ESAT-6/CFP10/TB7.7 cocktail. Prior to SIT, weak but positive MPB83/MBP70 specific antibody responses were detected in two of the infected bulls. All four M. bovis infected bulls reacted with a positive skin test and showed, as reported by others, increased mycobacteria specific IFN-γ production and increased positive responses in MPB83/MBP70 specific serology after SIT. At autopsy, M. bovis lesions were detected in all four experimentally infected bulls. Our results indicate that in Map exposed cattle, M. bovis diagnosis using IFN-γ assays needs a combination of PPD-B/A and ESAT-6/CFP10 for early and optimal sensitivity and that sensitivity of MPB83/MBP70 serodiagnosis is dramatically increased by prior skin testing. Map exposure did not interfere with the development of SIT in M. bovis infected animals, but resulted in a false positive M. bovis specific IFN-γ and antibody response after SIT in one of the two control animals (which remained negative in skin-test).

IFN-γ and IL-5 whole blood response directed against mycolactone polyketide synthase domains in patients with Mycobacterium ulcerans infection.

BACKGROUND: Buruli ulcer is a disease of the skin and soft tissues caused by infection with a slow growing pathogen, Mycobacterium ulcerans. A vaccine for this disease is not available but M. ulcerans possesses a giant plasmid pMUM001 that harbours the polyketide synthase (PKS) genes encoding a multi-enzyme complex needed for the production of its unique lipid toxin called mycolactone, which is central to the pathogenesis of Buruli ulcer. We have studied the immunogenicity of enzymatic domains in humans with M. ulcerans disease, their contacts, as well as non-endemic areas controls. METHODS: Between March 2013 and August 2015, heparinized whole blood was obtained from patients confirmed with Buruli ulcer. The blood samples were diluted 1 in 10 in Roswell Park Memorial Institute (RPMI) medium and incubated for 5 days with recombinant mycolactone PKS domains and mycolyltransferase antigen 85A (Ag85A). Blood samples were obtained before and at completion of antibiotic treatment for 8 weeks and again 8 weeks after completion of treatment. Supernatants were assayed for interferon-γ (IFN-γ) and interleukin-5 (IL-5) by enzyme-linked immunosorbent assay. Responses were compared with those of contacts and non-endemic controls. RESULTS: More than 80% of patients and contacts from endemic areas produced IFN-γ in response to all the antigens except acyl carrier protein type 3 (ACP3) to which only 47% of active Buruli ulcer cases and 71% of contacts responded. The highest proportion of responders in cases and contacts was to load module ketosynthase domain (Ksalt) (100%) and enoylreductase (100%). Lower IL-5 responses were induced in a smaller proportion of patients ranging from 54% after ketoreductase type B stimulation to only 21% with ketosynthase type C (KS C). Among endemic area contacts, the, highest proportion was 73% responding to KS C and the lowest was 40% responding to acyltransferase with acetate specificity type 2. Contacts of Buruli ulcer patients produced significantly higher IFN-γ and IL-5 responses compared with those of patients to PKS domain antigens and to mycolyltransferase Ag85A of M. ulcerans. There was low or no response to all the antigens in non-endemic areas controls. IFN-γ and IL-5 responses of patients improved after treatment when compared to baseline results. DISCUSSION: The major response to PKS antigen stimulation was IFN-γ and the strongest responses were observed in healthy contacts of patients living in areas endemic for Buruli ulcer. Patients elicited lower responses than healthy contacts, possibly due to the immunosuppressive effect of mycolactone, but the responses were enhanced after antibiotic treatment. A vaccine made up of the most immunogenic PKS domains combined with the mycolyltransferase Ag85A warrants further investigation.

Field performance of six Mycobacterium avium subsp. paratuberculosis antigens in a 20h interferon gamma release assay in Belgium.

Paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis (Map), is a chronic granulomatous enteritis which primarily affects domestic and wild ruminants, resulting in serious economic losses for dairy and beef industry around the world. There is no satisfactory cure or vaccine, and actual diagnostic tests need improvement, particularly for the initial stages of the disease. Map specific cell-mediated immune responses may allow early detection of the infection at subclinical stages. In this study, over a period of 39 months, we collected 548 blood samples in two culture-confirmed Map-infected herds, 95 blood samples in five dairy herds that scored negative during 3 consecutive years of Map serology testing and 79 samples in three culture-confirmed M. bovis infected herds. Based on criteria of bacteriology, serology and ratio of IFN-γ induced with bovine and avian purified protein derivative of tuberculin (PPD-B/PPD-A), we classified the samples in four groups: 415 samples as Map-exposed/infected (MAP), 58 samples as aspecific reactors (AR), 179 samples as non-responders (NI) and 70 samples as M. bovis infected (TB). Age of the animals influenced the IFN-γ response in the MAP group, with PPD specific IFN-γ levels (but not PPD-B/PPD-A IFN-γ ratio) being significantly higher in animals <18 months of age. Map specific antibodies were detected by IDEXX ELISA in 13/415 (3%) sera of the MAP group, whereas fecal culture was positive for only 7/405 (1.7%) samples. Animals in the MAP group could therefore be considered being at the very early stage of Map infection. Six purified, recombinant Map antigens (Ag5, Ag6, MAP1637c, MAP0388, MAP3547c and MAP0586c), previously identified using combined advanced proteomic or reverse genomic approaches, were tested for their diagnostic potential in a 20h IFN-γ release assay. In the age group >18 months old, Ag5 and MAP0388 were recognized by only 10.1% and 7.7% of the animals in the MAP group, whereas a total of 38.6.%, 29.4%, 25.6% and 39.0% of the animals in the MAP group reacted to Ag6, MAP1637c, MAP3547c and MAP0586c respectively. None of the animals in the TB group reacted to Ag6, MAP1637c or MAP586c. Except for MAP0388, the % of reactors in the MAP group was significantly higher in animals <18 months old: 28.0%, 24.0%, 45.5%, 47.1%, 49.8% and 47.4% respectively. Further studies of these candidates and their combination are needed to confirm their diagnostic potential for the detection of early Map infection.

Virulence and immunogenicity of genetically defined human and porcine isolates of M. avium subsp. hominissuis in an experimental mouse infection.

Mycobacterium avium subsp. hominissuis (Mah) represents a health concern for humans and to a lesser extent for pigs, but its zoonotic potential remains elusive. Using multispacer sequence typing (MST) we previously identified 49 different genotypes of Mah among Belgian clinical and porcine isolates, with 5 MSTs shared by both hosts. Using experimental intranasal infection of BALB/c mice, we compared the virulence and immunogenicity of porcine and clinical human isolates with shared genotype or with a genotype only found in humans or pigs. Bacterial replication was monitored for 20 weeks in lungs, spleen and liver and mycobacteria specific spleen cell IFN-γ, IL-10 and IL-17 production as well as serum antibody responses were analyzed. Isolates varied in virulence, with human and porcine isolates sharing MST22 genotype showing a thousand fold higher bacterial replication in lungs and more dissemination to spleen and liver than the human and porcine MST91 isolates. Virulent MST22 type was also associated with progressive suppression of IFN-γ and IL-17 responses, and increased IL-10 production. Whole genome sequencing of the two virulent isolates with MST22 genotype and two avirulent isolates of genotype MST91 and comparison with two well-studied M. avium subsp. hominissuis reference strains i.e. Mah 104 and Mah TH135, identified in the two MST22 isolates nine specific virulence factors of the mammalian cell entry family, that were identical with Mah 104 strain. Despite the obvious limitations of the mouse model, a striking link of virulence and identity at the genome level of porcine and human isolates with the same multisequence type, for which no correlation of place of residence (humans) or farm of origin (pigs) was observed, seems to point to the existence in the environment of certain genotypes of Mah which may be more infectious both for humans and pigs than other genotypes.

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.

Analysis of the vaccine potential of plasmid DNA encoding nine mycolactone polyketide synthase domains in Mycobacterium ulcerans infected mice.

There is no effective vaccine against Buruli ulcer. In experimental footpad infection of C57BL/6 mice with M. ulcerans, a prime-boost vaccination protocol using plasmid DNA encoding mycolyltransferase Ag85A of M. ulcerans and a homologous protein boost has shown significant, albeit transient protection, comparable to the one induced by M. bovis BCG. The mycolactone toxin is an obvious candidate for a vaccine, but by virtue of its chemical structure, this toxin is not immunogenic in itself. However, antibodies against some of the polyketide synthase domains involved in mycolactone synthesis, were found in Buruli ulcer patients and healthy controls from the same endemic region, suggesting that these domains are indeed immunogenic. Here we have analyzed the vaccine potential of nine polyketide synthase domains using a DNA prime/protein boost strategy. C57BL/6 mice were vaccinated against the following domains: acyl carrier protein 1, 2, and 3, acyltransferase (acetate) 1 and 2, acyltransferase (propionate), enoylreductase, ketoreductase A, and ketosynthase load module. As positive controls, mice were vaccinated with DNA encoding Ag85A or with M. bovis BCG. Strongest antigen specific antibodies could be detected in response to acyltransferase (propionate) and enoylreductase. Antigen-specific Th1 type cytokine responses (IL-2 or IFN-γ) were induced by vaccination against all antigens, and were strongest against acyltransferase (propionate). Finally, vaccination against acyltransferase (propionate) and enoylreductase conferred some protection against challenge with virulent M. ulcerans 1615. However, protection was weaker than the one conferred by vaccination with Ag85A or M. bovis BCG. Combinations of these polyketide synthase domains with the vaccine targeting Ag85A, of which the latter is involved in the integrity of the cell wall of the pathogen, and/or with live attenuated M. bovis BCG or mycolactone negative M. ulcerans may eventually lead to the development of an efficacious BU vaccine.

Biological evaluation of bisbenzaldehydes against four Mycobacterium species.

A series of bisbenzaldehydes and structurally related analogs, conveniently synthesized via microwave-assisted reactions, were evaluated in vitro against drug susceptible and multi-drug resistant Mycobacterium tuberculosis, against virulent Mycobacterium bovis, against Mycobacterium ulcerans and against two Mycobacterium avium subspecies. Among the 33 substances that were tested, compound 12, i.e. 4,4'-[1,12-dodecanediyl(oxy)]bisbenzaldehyde, emerged as the most promising hit. Its activity was further confirmed in an intracellular growth inhibition assay of M. tb in murine J774 A.1 macrophages. None of the compounds showed significant cytotoxicity on human C3A hepatocytes in a neutral red dye uptake assay and no genotoxicity or mutagenicity was observed as demonstrated by a VITOTOX™ test and confirmed with a comet assay.

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.

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 discovery: a postgenomic approach to paratuberculosis diagnosis.

Paratuberculosis is a chronic enteritis caused in domestic and wild ruminant species by Mycobacterium avium subsp. paratuberculosis (MAP) that is responsible for major economic losses to the agricultural industry. To date, no satisfactory therapeutic, vaccine, or diagnostic tools are available, globally impairing all control programs. In this study, we have undertaken a large-scale postgenomic analysis of MAP proteins, to identify specific antigens that could potentially improve the diagnosis of paratuberculosis. Two complementary approaches were implemented, the first one consisting in the systematic proteomic identification of proteins present in MAP culture filtrates (CFs), followed by the selection of MAP-specific proteins by BLAST query on available mycobacterial genomes. The resulting database represents the first established secretome of MAP and a useful source of potentially specific antigens. The second approach consisted in the immunoproteomic analysis of both MAP extracts and CFs, using sera from MAP-infected and uninfected cattle. Combining results obtained with both approaches resulted in the identification of 25 candidate diagnostic antigens. Five of these were tested in an ELISA assay for their diagnostic potential, on a limited panel of field sera, and the combination of three of them competed in performance with available commercial assays, reaching a test sensitivity of 94.74% and specificity of 97.92%.

Immunogenicity of eight dormancy regulon-encoded proteins of Mycobacterium tuberculosis in DNA-vaccinated and tuberculosis-infected mice.

Hypoxia and low concentrations of nitric oxide have been reported to upregulate in vitro gene expression of 48 proteins of the dormancy (DosR) regulon of Mycobacterium tuberculosis. These proteins are thought to be essential for the survival of bacteria during persistence in vivo and are targeted by the immune system during latent infection in humans. Here we have analyzed the immunogenicity of eight DosR regulon-encoded antigens by plasmid DNA vaccination of BALB/c and C57BL/6 mice, i.e., Rv1733c, Rv1738, Rv2029c (pfkB), Rv2031c/hspX (acr), Rv2032 (acg), Rv2626c, Rv2627c, and Rv2628. Strong humoral and/or cellular Th1-type (interleukin-2 and gamma interferon) immune responses could be induced against all but one (Rv1738) of these antigens. The strongest Th1 responses were measured following vaccination with DNA encoding Rv2031c and Rv2626c. Using synthetic 20-mer overlapping peptides, 11 immunodominant, predicted major histocompatibility complex class II-restricted epitopes and one K(d)-restricted T-cell epitope could be identified. BALB/c and (B6D2)F(1) mice persistently infected with M. tuberculosis developed immune responses against Rv1733c, Rv2031c, and Rv2626c. These findings have implications for proof-of-concept studies in mice mimicking tuberculosis (TB) latency models and their extrapolation to humans for potential new vaccination strategies against TB.

Evaluation of the immunogenicity of pBudCE4.1 plasmids encoding mycolyl-transferase Ag85A and phosphate transport receptor PstS-3 from Mycobacterium tuberculosis.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb), remains a major health problem. The only currently available vaccine, BCG, confers only variable protection and an improved vaccine is urgently needed. Administration of DNA vaccines encoding the secreted mycolyl-transferase Ag85A and the surface-exposed phosphate transport receptor PstS-3 elicit an immune response capable of protecting mice challenged with Mtb. In order to combine the protection against Mtb infection induced by these two DNA vaccines, we have cloned Ag85A and PstS-3 in pBudCE4.1 vector, in which antigenic expression is controlled by two independent promoters. (BALB/cxC57BL/6)F1 mice were vaccinated with this combination vaccine and immune responses were compared to those induced by vaccination with plasmids encoding the single antigens on pBudCE4.1 or pV1J.ns-tPA backbone. Antibody and Th1 type cytokine responses against Ag85A were comparable, whereas responses against PstS-3 were clearly lower in mice vaccinated with the combination plasmid, suggesting antigenic competition with the mycolyl-transferase being the dominant antigen.

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.

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-gamma) secretion and lymphoproliferative responses of peripheral blood mononuclear cells, respectively. Strong proliferative and ex vivo IFN-gamma 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 of luminescent Mycobacterium avium subsp. paratuberculosis for rapid screening of vaccine candidates in mice.

Mycobacterium avium subsp. paratuberculosis is a slowly growing mycobacterial species, requiring 6 to 8 weeks of culture before colonies can be counted visually. Here, we describe the development of luminescent M. avium subsp. paratuberculosis expressing luxAB genes of Vibrio harveyi and its use for vaccine testing in an experimental mouse model, replacing fastidious CFU counting by rapid luminometry.