ES2357487B1 - MYCOBACTERIUM AVIUM SUPESPECIES strain for TUBERCULOSIS TYPE II AND APPLICATIONS - Google Patents

Abstract

The present invention relates to a new strain of Mycobacterium avium subspecies paratuberculosis that has suffered a deletion of 16.3 Kb whereby it lacks mce (mammalian cell entry) genes. It also refers to a method developed based on the polymerase chain reaction to detect other strains of Mycobacterium avium paratuberculosis subspecies that lack such genes. In addition, the present invention reflects the validation of methodologies for the use of this strain of Mycobacterium avium subspecies paratuberculosis CECT 7530 in in vitro invasion assays in different cell lines of diverse origins. Additionally, the present invention refers to the development of methods for measuring the viability of Mycobacterium avium paratuberculosis subspecies inside eukaryotic cells during in vitro invasion processes by estimating the number of copies of the precursor gene of the 16S rRNA the pre -ARNr 16S.

Description

Mycobacterium avium subspecies paratuberculosis type II strain and applications.

Object of the invention

The present invention refers to the first and only strain of Mycobacterium avium subspecies mutant paratuberculosis for mammalian cell entry genes (mce). It is a microorganism that has a deletion of 16.3 Kpb in its genome that comprises mce genes, members of the PE / PPE family (so named due to the presence of proline-glutamic acid and proline-proline-glutamic acid motifs, respectively) and a transcriptional regulator. The strain object of the present invention was obtained from a herd of goats that had a paucibacillary form of Johne's disease or paratuberculosis (with a low bacterial load inside the macrophages). Therefore, the present application deals with the first and only strain of M. a. Natural mutant paratuberculosis of mce genes that can be used as a reference strain in pathogenicity and virulence assays. In turn, the strain object of the present invention can be applied in the design of new microarrays for comparative genomic hybridization (HGC) studies and as a work subject in the development of new live attenuated vaccines.

State of the art

Currently, the study of the virulence factors of bacteria belonging to the Mycobacterium genus is being carried out, in order to understand the mechanisms of pathogen-host interaction during the course of infection (Casali, N. et al., 2007, BMC Genomics, 8, 60; Haile, Y. et al., 2002, FEMS Immunol. Med. Microbiol. 33 (2), 125-132; Gioffre, A. et al., 2005, Microbes. Infect. 7 (3) , 325-334; Flesselles, B. et al., 1999, FEMS Microbiol. Lett. 177 (2), 237-242; El-Shazly, S. et al, 2007, Int. J. Tubero. Lung Dis. 11 (6), 676-682; Parker, SL et al., 1995, Clin. Diagn. Lab Immunol. 2 (6), 770-775). Of all the factors involved in this process, of special interest are those groups of genes called mammalian cell entry genes (mce), whose gene expression products (Mce proteins) are essential for cell entry and survival inside macrophages .

The importance of these mce genes was demonstrated by the invasion of host cells of the HeLa cell line with a non-pathogenic strain of Escherichia coli in whose genome the mce1A gene of Mycobacterium tuberculosis had been inserted (Arruda, S. et al., 1993, Science, 261 (5127), 1454-1457). Currently, all efforts are being directed to the creation of new mce mutants of M. tuberculosis and Mycobacterium bovis. These mutants have demonstrated, through different in vitro invasion experiments, an attenuation in their virulence and invasiveness (Flesselles, B. et al, 1999, FEMS Microbiol. Lett. 177 (2), 237-242). In addition, differences in the pathological features between mutant mce strains and wild strains of M. tuberculosis have also been observed, the first of which caused the formation of an aberrant granulomatous formation in mice, in addition to a greater proliferation of the lymphocytic population compared to the wild strain (Shimono, N. et al., 2003, Proc. Natl. Acad. Sci. US A, 100 (26), 15918-15923).

Within the bacteria of the Mycobacterium avium complex (MAC), which includes Mycobacterium avium subspecies avium (M. a. Avium), a direct correlation was observed between susceptibility to certain antibiotics, colony morphology and expression from different genes mce. In the same study they demonstrated that the most virulent phenotypes of M. a. avium presented an overexpression of the mce genes (Cangelosi, G. A. et al., 2006, Antimicrob. Agents Chemother. 50 (2), 461-468).

Among the members of MAC is Mycobacterium avium subspecies paratuberculosis (M. a. Paratuberculosis), aetiological agent of Johne's disease or paratuberculosis. This disease is characterized by being widely distributed both geographically and as a host, although it is mainly affecting supply animals. In addition, several studies have described that M. a. Paratuberculosis is a possible contributing agent in Crohn's disease in man (Harris, N. B. et al., 2001, Veterinary Medicine. Clin. Microbiol. Rev., 14 (3), 489-512). For all this, the correct molecular characterization of M. a. Paratuberculosis is of crucial importance in order to implement the appropriate measures for its eradication.

In this microorganism, these mce genes are distributed throughout their genome, forming operons

or groups of six mce genes and two yrbE genes, whose functions are the production of substrate-binding proteins and permeases for transport across the cell membrane respectively (Casali, N. et al., 2007, BMC Genomics, 8, 60).

On the other hand, M. a. paratuberculosis has been divided into three broadly differentiated groups called I (previously cited as sheep or sheep group), II (previously referred to as cattle or bovine group) and III (intermediate), which have been subdivided through the use of various molecular techniques ( de Juan, L. et al., 2006, Vet. Microbiol. 115 (1-3), 102-110; Dohmann, K. et al., 2003, J. Clin. Microbiol. 41 (11), 5215-5223; Stevenson, K. et al, 2002, J. Clin. Microbiol., 43 (8), 3704-3712; Castellanos, E. et al, 2007, J. Clin. Microbiol. 45 (10), 3439-3442; Castellanos , E. et al., 2008, Proceedings of the 9th International Paratuberculosis Colloquium, 6-8; Grif fi ths, T. et al., 2008, J. Clin. Microbiol. 46 (4), 1207-1212) as well as nutritional requirements at the time of cultivation (de Juan, L. et al, 2006, Appl. Environ. Microbiol. 72 (9), 5927-5932).

Currently there are only two strains of M. a. Type II paratuberculosis available to laboratories conducting different invasion and pathogenicity studies in vivo and in vitro, among which is the only sequenced reference strain M. a. paratuberculosis K-10, and the vaccine strain M. a. paratuberculosis 316F. However, strains of M. a. paratuberculosis that are natural mutants mce (genes with an implication in the mechanisms of entry of the microorganism in the host cell), are not available to any laboratory since no one has obtained this type of mce mutants to date.

Description of the invention

The present invention relates firstly to obtaining, preserving and using a strain of M. a. Natural mutant mce paratuberculosis as a reference material to validate, standardize and carry out virulence and pathogenesis tests. The new strain has been deposited in the Spanish Type Culture Collection (CECT), University of Valencia, Burjassot Campus, Research Building, 46100 Burjassot (Valencia) where it has been assigned the provisional deposit number 7530.

With the term "natural mutant mce strain" in the present invention reference is made to all those strains of M.

to. paratuberculosis in whose genome a deletion of 16.3 Kb has been produced without any genetic engineering intervention. This deletion is made up of 19 consecutive open reading frames (ORFs) (MAP0108-MAP0126), including the mce7 operon (MAP0108-MAP0113), genes of the PE / PPE families (in the presence of proline-acid motifs glutamic and proline-proline-glutamic acid respectively) (MAP0122-MAP0124) and a transcriptional regulator (MAP0116) among others.

The strain of M. a. Paratuberculosis CECT 7530, object of the present invention, was obtained from a herd of goats of the Guadarrama breed that presented a paucibacillary form of Johne's disease or paratuberculosis (characterized by presenting a low bacterial load inside the macrophages). In this herd, only the strain of M. a. Paratuberculosis mce natural mutant object of the patent (monoclonal infection), so that the infected herd in turn presented a natural protection against the disease caused by other strains of M.

to. paratuberculosis. However, this strain deleted in virulence genes maintained its immunogenic capacity in the aforementioned herd. M. a. Paratuberculosis is characterized by being an intracellular pathogen and therefore, the development of live attenuated vaccines (not currently registered in the market) would be important to help control and eradicate this disease.

Therefore, the present application deals with the first and only strain of M. a. Natural mutant paratuberculosis of mce genes that can be used as a reference strain in pathogenicity and virulence assays.

The strain of M. a. Natural mutant paraceuberculosis mce was characterized by the microarray technique. Within the field of molecular characterization in M. a. paratuberculosis, the use of microarrays is currently being a widely disseminated technique given the large amount of information it provides, for which it is only necessary to use a small amount of high quality DNA (Semret, M. et al, 2005, J. Clin. Microbiol. 43 (8), 37043712; Paustian, ML et al., 2008, BMC Genomics 9, 135; Wu, CW et al., 2006, J. Bacteriol. 188 (2), 711- 723). This microarray technique is based on the creation of a microarray upholstered with the DNA of the microorganism genes to study on a slide. The plates are then hybridized with complementary DNA of the test sample, which is compared with complementary DNA of the reference strain (in this case M. a. Paratuberculosis K-10, a type II strain, with GeneBank accession number AE016958; reference sequence number NC_002944) marked each one (sample and control) with a chromogen each (Cy3 and Cy5) and, subsequently, by using a specific Software (Imagene 7. 5, BlueFuse v. 3. 5 and Gene Spring v.7.2) a genomic profile of the analyzed sample is obtained. In this way we can carry out comparative studies between the different strains of M. a. paratuberculosis, that is, the completion of a complete study of HGC.

In order to confirm the findings obtained with the microarray, a PCR ampli fi cation reaction was designed. The aforementioned PCR was directed to the fluorescent genes (ie, to the MAP0107 and MAP1027 genes) of the deleted region (MAP0108-MAP0126). In the case of the non-presence of the deletion in the genome of the strains of M. a. paratuberculosis, the fragment between both genes was too large (16.3 Kb) to be ampli fi ed, so the result of the PCR was negative. Only when the deletion had taken place, the fragment was amplified by PCR, resulting in a product of 881 bp.

In order to standardize this methodology, an exhaustive analysis was carried out on a total of 99 M. isolates.

to. paratuberculosis obtained in the different geographical areas of Spain, Scotland and Denmark, representing the three types of M. a. paratuberculosis (I, II and III), from different animal hosts (goats, cattle, sheep) and obtained over a period of seven years (2000-2007), and among which two of the strains analyzed in turn by microarray were included . PCR analysis revealed a total of 53 strains of M. a. paratuberculosis whose genome presented this deletion. Of the previous ones, 49 strains belonged to the same herd and the rest to herds that were geographically close and among which there had been an exchange of animals in the past. The size of the PCR product for these 53 strains was as expected, ie 881 bp, which was evidenced in a 2% electrophoresis gel. To carry out the validation of this PCR technique, the sequencing of the PCR products (881 bp) obtained in three of the strains of M. a. paratuberculosis in which the deletion was present (CECT 7530, MI05 / 00483-2 and MI05 / 00484-2) and for each of the strands of DNA separately [from both the strand (+) and the strand (-) ]. Subsequent sequencing confirmed that the ampli fi ed product corresponded with part of the sequence of the fluorescent genes (ie, MAP0107 and MAP0127) to the deleted region (MAP0108-MAP0126).

Brief description of the fi gures

Figure 1. Starry morphology of the colonies of M. a. paractuberculosis CECT 7530.

Figure 2. Image obtained with Gene Spring Software v.7. 2, and after the normalization of the data obtained in the first instance with the microarray. In the image, each of the common genes between the strain M. a. paratuberculosis CECT 7530 and M. a. K-10 paratuberculosis. The points reflected in dark gray represent all those genes present in M. a. K-10 paratuberculosis but absent in strain CECT 7530.

Figure 3. The dendrogram shows the comparison between strains of M. a. paratuberculosis types I, II and III for that specific region of the genome (delimited by the genes MAP0108 and MAP0126). Represented in dark gray are all those genes that have been deleted in strain CECT 7530 but, on the contrary, are present in the other strains of M. a. Paratuberculosis subjected to this analysis of genomic hybridization compared by microarray.

Embodiment of the invention

Obtaining and characterizing the strain of M. a. paractuberculosis CECT 7530

The strain of M. a. paratuberculosis CECT 7530 was initially obtained from an homogenate of the ileoceal valve, mesenteric lymph nodes and intestine of a goat of the native Guadarrama breed, located in the Community of Madrid. The sample was cultured in specific media for the isolation of this microorganism and by previously described methods (de Juan, L. et al., 2006, Vet. Microbiol. 115 (1-3), 102-110) and incubated at 37 ° C in an oven until visible growth is obtained (Figure 1).

The aforementioned strain was initially typed as type II strain by the PCR described by Desmond Collins (Collins, DM et al., 2002, J. Clin. Microbiol. 40 (12), 4760-4762) and other rapid molecular techniques mentioned above ( Castellanos, E. et al., 2008, Proceedings of the 9th International Paratuberculosis Colloquium, 6-8). For the study of the complete genome of this strain of M. a. for paratuberculosis CECT 7530 the MA-PAC microarray was used, developed by the Microarray Group (BμG @ S) of the University of Saint George’s in London, United Kingdom.

In the HGC study, strains of M. a. paratuberculosis representatives of type I and III and the strain subject to the patent application (CECT 7530).

As for the microarray (MAPAC) that was used in the study, it is a 60-mer oligonucleotide microarray in which genomes of both M. a. K-10 paratuberculosis as of Mycobacterium avium subspecies hominissuis 104. The present microarray comprises 4132 oligonucleotides targeting genes shared between the genomes of both microorganisms, 218 genes specific to M. a. paratuberculosis K-10, 952 specifics for M. a. hominissuis 104, 18 unique genes for M. a. paratuberculosis types I and III, 19 sequences MIRU (mycobacterial interspersed repetitive units) in M. a. paratuberculosis, 58 intergenic regions in

M. a. K-10 paratuberculosis and 7 genes transported in plasmid pVT2 from M. a. hominissuis 104. All oligonucleotides that formed the microarray were synthesized (Operon, Biotechnologies, Germany), supplied in 384 well plates and resuspended at a concentration of 50 mM in 50% DMSO. The oligonucleotides were then placed in a microarray by using the MicroGrid II robot (BioRobotics) and subsequently post-processed according to the manufacturer's recommendations for rehydration and fixation processes. The design of this microarray is available in BμG @ Sbase (Accession number A-BUGS-35; http://bugs.sgul.ac.uk/A-BUGS-35) and also in ArrayExpress (Accession number: A -BUGS35; http://www.ebi.ac.uk/microarray-as/ae/).

The DNA hybridization protocol of the strains of M. a. CECT 7530 paratuberculosis and reference M.

to. K-10 paratuberculosis was the same as that described by Dorrell et. to the. (Dorrell, N. et al. 2001, Genome Res. 11, 1706-1715). Then, and as previously described (Castellanos, E. et al, 2009, Appl. Environ. Microbiol. 75 (3), 676-686) the microarrays were scanned (Affymetrix 428) and the signal strength data was extracted with the BlueFuse program for Microarrays v 3.5 (BlueGenome, Cambridge UK). Finally, the data analysis and the normalization of the data was performed with the GeneSpring 7.3.1 (Agilent Technologies) and Eisen Cluster (Eisen, JA, et al., 1998, Genome Res. 8 (3), 163-167 ).

The results of the microarray for strain CECT 7530 reflected an important deletion with respect to the reference strain of M. a. K-10 paratuberculosis. This deletion implies a set of 19 consecutive ORFs between the MAP0108 and MAP0126 genes, both inclusive, among which the mce7 virulence operon is framed, as well as genes of the PE / PPE family, and transcriptional regulators among others (Table 1 ), (Figures 2 and 3).

TABLE 1

List of the ORFs deleted in M. a. paractuberculosis CECT 7530 compared to the genome of

M. a. K-10 paratuberculosis after analysis of the data obtained through the study of genomic hybridization compared by microarray

The confirmation of the deletion was performed by PCR directed to the fluorescent genes of the region deleted in the strain of M. a. paratuberculosis CECT 7530, which are the MAP0107 and MAP0127 genes. The oligonucleotides developed for this purpose (SEQ ID no. 1 and 2) were designed using the Primer 3 software (http://biotools.umassmed.edu/bioapps/primer3_www.cgi). In addition, as a double quality control, another PCR directed to the F57 gene was performed (Poupart, P. et al, 1993, J. Clin. Microbiol. 31 (6), 1601-1605) (SEQ ID no. 3 and 4) to confirm the presence of DNA from M. a. paratuberculosis. With this other PCR it was thus possible to confirm that the absence of PCR ampli fi cation was due to the large size of the fragment to be amplified (16.3 Kb) in the case of the non-existence of the deletion in this genome fragment and not to the insufficient quality and quantity of DNA from M. a. paratuberculosis (Table 2).

TABLE 2

Specific oligonucleotides designed to confirm the deletion (MAP0108-MAP0126) and those used for amplification of the F57 gene. Their position is re fl ected with respect to the strain

M. a. paratuberculosis K-10, sequenced reference strain

* In the event that the strain analyzed presents the deletion of the fragment between the MAP0108 and MAP0126 genes, the PCR size is 881 bp. However, in the case of the presence of this fragment (16.3 Kb), since it is too large for PCR amplification, a product is not obtained in this amplification reaction.

PCR reactions were carried out in a final volume of 50 μl, which contained 5 μl of the DNA sample, 1x Standard Reaction Buffer with 1.5 mM MgCl2, 200 μM dNTPs (Biotools, B&M Labs, SA, Madrid), oligonucleotides F (SEQ ID No. 1 and 3) and R (SEQ ID No. 2 and 4) (3 μM), 10% DMSO (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland) and 2.5 U of Biotools DNA Taq polymerase (Biotools, Spain). The PCR conditions were: a single denaturation step at 94 ° C for 3 minutes and then 35 cycles of denaturation at 94 ° C for 30 seconds, hybridization at 58 ° C for 45 seconds, and extension at 72 ° C for 1 minute 30 seconds, with a final cycle of extension at 72 ° C for 5 minutes.

In the case of the presence of the mce operon, the PCR product was too large to be ampli fi ed (16.3 Kb). Therefore, in the case of strain M. a. paratuberculosis CECT 7530 a visible 881 bp PCR product was obtained on a 2% agarose gel. After the completion of the PCR technique, a total of 99 strains of M. a. paratuberculosis, isolated from different hosts, regions of Spain, Denmark and Scotland, obtained over a period of seven years (2000-2007) and representatives of types I, II and III. The PCR results revealed the presence of this deletion (MAP0108-MAP0126) in a total of 53 M. isolates.

to. paratuberculosis, 49 of which were obtained from the same herd as M. a. paractuberculosis CECT 7530 and the rest of herds located in nearby geographical areas, which had previously shared animals.

Next, the PCR products (881 bp) of the CECT 7530, MI05 / 00483-2 and MI05 / 00484-2 strains obtained with the oligonucleotides (SEQ ID No. 1 and SEQ ID No. 2) directed to the MAP0107 and MAP0127 genes. The aforementioned products of PCR ampli fi cation were purified using the QIAquick PCR puri fi cation kit (Qiagen, GmbH), and sequenced in an ABI Prism 3730 (Applied Biosystems) DNA sequencer (Sequencing Service, CIB, Madrid, Spain). The sequences of both strands of DNA [strand (+) and strand (-)] were then aligned using Biological Sequence Aligment Editor Software and analyzed with Artemis Software (www.sanger.ac.uk/Software/Artemis) ( Rutherford, K. et al., 2000, Bioinformatics, 16 (10), 944-945) and with the tool available in GenBank's Basic Local Alignment Search Tool (http://blast.ncbi.nlm.nih.gov/Blast .cgi). The result of the sequence analysis confirmed that the ampli fi ed PCR product corresponded only to the sequences of the fluorescent genes (MAP0107 and MAP0127), and not the sequence of the genes included in the deletion (MAP108 to MAP0126).

The sequences of the three strains of M. a. paratuberculosis mce sequenced natural mutants (CECT 7530, MI05 / 00483-2 and MI05 / 00484-2) are characterized by SEQ ID NO: 5, 6 and 7, respectively. It should be noted that the sequences reflected in the present invention correspond to the interpretable part of the chromatograms of the sequences obtained after sequencing of the PCR products resulting from the PCR amplification of the 881 bp fragment, corresponding to the gene sequences Floating (MAP0107 and MAP0127) to the deleted region (MAP0108-MAP0126).

Claims (5)

one.

Mycobacterium avium subspecies paratuberculosis mutant type II strain for cellular and virulence entry genes: mce (MAP0108-MAP0113) and PE / PPE (MAP0122-MAP0124).

2.

Use of the Mycobacterium avium subspecies paratuberculosis CECT 7530 strain in cell line invasion assays as a reference for invasion and pathogenicity studies in vivo and in vitro.

3.

Use of the Mycobacterium avium subspecies paratuberculosis CECT 7530 strain in the design of new microarrays for application in comparative genomic hybridization (HGC) studies.

Four.

Use of the Mycobacterium avium subspecies paratuberculosis strain CECT 7530 as a work subject to obtain new live attenuated vaccine strains for Johne's disease or paratuberculosis.

SEQUENCE LIST <110> Complutense University of Madrid <120> Mycobacterium avium strain subspecies paratuberculosis type II and applications. <160> 7 <210> 1 <211> 20 <212> DNA <213> Mycobacterium avium subspecies paratuberculosis <400> 1 <210> 2 <211> 18 <212> DNA <213> Mycobacterium avium subspecies paratuberculosis <400> 2 <210> 3 <211> 20 <212> DNA <213> Mycobacterium avium subspecies paratuberculosis <400> 3 <210> 4 <211> 21 <212> DNA <213> Mycobacterium avium subspecies paratuberculosis <400> 4 <210> 5 <211> 690 <212> DNA <213> Mycobacterium avium subspecies paratuberculosis CECT 7530 <400> 5 <210> 6 <211> 688 <212> DNA <213> Mycobacterium avium subspecies paratuberculosis MI05 / 00483-2 <400> 6 <210> 7 <211> 648 <212> DNA <213> Mycobacterium avium subspecies paratuberculosis MI05 / 00484-2 <400> 7 <210> 8 <211> 18 <212> DNA <213> Mycobacterium avium subspecies paratuberculosis <400> 8 <210> 9 <211> 20 <212> DNA <213> Mycobacterium avium subspecies paratuberculosis <400> 9 <210> 10 <211> 19 <212> DNA <213> Mycobacterium avium subspecies paratuberculosis <400> 10 <210> 11 <211> 20 <212> DNA <213> Mycobacterium avium subspecies paratuberculosis <400> 11 SPANISH OFFICE OF THE PATENTS AND BRAND Application no .: 200901635 SPAIN Date of submission of the application: 23.07.2009 Priority Date: REPORT ON THE STATE OF THE TECHNIQUE 51 Int. Cl.: C12N1 / 20 (2006.01) C12R1 / 325 (2006.01) RELEVANT DOCUMENTS

Category

Documents cited Claims Affected

TO

SENARATNE, R.H. et al., 'Mycobacterium tuberculosis strains disrupted in mce3 and mce4 operons are attenuated in mice', JOURNAL OF MEDICAL MICROBIOLOGY, 2008, Vol. 57, No. 2, pages 164-170, ISSN: 0022-2615, the whole document. 1-4

TO

GIOFFRE, A. et al., 'Mutation in mce operons attenuates Mycobacterium tuberculosis virulence', MICROBES AND INFECTION, 2005, Vol. 7, No. 3, pages 325-334, ISSN: 1286-4579, the whole document. 1-4

TO

WO 99/10475 A2 (CONNAUGHT LAB.), The whole document. 1-4

TO

JOSHI, SM. et al., 'Characterization of mycobacterial virulence genes through genetic interaction mapping', PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE USA, 2006, Vol. 103, No. 31, pages 11760-11765, ISSN: 0027-8424, all document. 1-4

TO

CASALI, N. et al., 'A phylogenomic analysis of the Actinomycetales mce operons', BMC GENOMICS, 2007, Vol. 8, page 60, ISSN: 1471-2164, the whole document. 1-4

Category of the documents cited X: of particular relevance Y: of particular relevance combined with other / s of the same category A: reflects the state of the art O: refers to unwritten disclosure P: published between the priority date and the date of priority submission of the application E: previous document, but published after the date of submission of the application

This report has been prepared • for all claims • for claims no:

Date of realization of the report 06.04.2011

Examiner J. Vizán Arroyo Page 1/5

SPANISH OFFICE OF THE PATENTS AND BRAND Application no .: 200901635 SPAIN Date of submission of the application: 23.07.2009 Priority Date: REPORT ON THE STATE OF THE TECHNIQUE 51 Int. Cl.: C12N1 / 20 (2006.01) C12R1 / 325 (2006.01) RELEVANT DOCUMENTS

Category

Documents cited Claims Affected

TO

Li, L. et al., 'The complete genome sequence of Mycobacterium avium subspecies paratuberculosis', PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE USA, 2005, Vol. 102, No.35, pages 12344-12349, ISSN: 0027- 8424, the whole document. 1-4

Category of the documents cited X: of particular relevance Y: of particular relevance combined with other / s of the same category A: reflects the state of the art O: refers to unwritten disclosure P: published between the priority date and the date of priority submission of the application E: previous document, but published after the date of submission of the application

This report has been prepared • for all claims • for claims no:

Date of realization of the report 06.04.2011

Examiner J. Vizán Arroyo Page 2/5

REPORT OF THE STATE OF THE TECHNIQUE Application number: 200901635 Minimum documentation sought (classification system followed by classification symbols) C12N, C12R Electronic databases consulted during the search (name of the database and, if possible, terms of search used) INVENES, EPODOC, BIOSIS, MEDLINE State of the Art Report Page 3/5  WRITTEN OPINION Application number: 200901635 Date of Completion of Written Opinion: 06.04.2011 Statement

Novelty (Art. 6.1 LP 11/1986)

Claims Claims 1-4 IF NOT

Inventive activity (Art. 8.1 LP11 / 1986)

Claims Claims 1-4 IF NOT

The application is considered to comply with the industrial application requirement. This requirement was evaluated during the formal and technical examination phase of the application (Article 31.2 Law 11/1986).  Opinion Base.- This opinion has been made on the basis of the patent application as published. State of the Art Report Page 4/5  WRITTEN OPINION Application number: 200901635 1. Documents considered.- The documents belonging to the state of the art taken into consideration for the realization of this opinion are listed below.

Document

Publication or Identification Number publication date

D01

Senaratne, R.H. et al., J. Med. Microbiol., (2008), 57 (Pt 2): 164-70. 2008

D02

Gioffre, A. et al., Microbes Infect., (2005), 7 (3): 325-34. 2005

D03

WO 99/10475 A2 (CONNAUGHT LAB.) 04.03.1999

D04

Joshi, SM. et al., Proc. Natl Acad. Sci. U S A., (2006), 103 (31): 11760-5. 2006

D05

Casali, N. et al., BMC Genomics, (2007), 8: 60. 2007

D06

Li, L. et al., Proc. Natl Acad. Sci. U S A., (2005), 102 (35): 12344-9. 2005

In D1-D4 different attenuated strains of Mycobacterium tuberculosis are described. In D5 the mce operons are analyzed in Actinomycetales. In D6 the complete genome sequence of Mycobacterium avium subspecies paratuberculosis is disclosed. 2. Statement motivated according to articles 29.6 and 29.7 of the Regulations for the execution of Law 11/1986, of March 20, on Patents on novelty and inventive activity; quotes and explanations in support of this statement 1. NEW (Art. 4.1. And Art. 6.1. Of the Patent Law) and INVENTIVE ACTIVITY (Art. 4.1. And Art. 8.1. Of the Patent Law).  1.1. Independent claim 1 The object of claim 1 is a mutant strain of Mycobacterium avium subspecies paratuberculosis that is basically characterized in that it is affected in the mce genes (MAP0108-MAP0113) and PE / PPE (MAP0122-MAP0124). In the closest state of the art, constituted by documents D1-D6, no attenuated mutant of Mycobacterium avium subspecies paratuberculosis has been disclosed that shares the same technical characteristics of the mutant of the invention. In addition, obtaining said mutant is not deduced in an obvious way by combining the information contained in D1-D6. On the basis of the foregoing, the uses of claims 1-4 are also considered new and inventive. 1.2. This application satisfies the criteria established in Art. 4.1. of the Patent Law, as the object of claims 1-4 is new and has inventive activity in accordance with Arts. 6.1. and 8.1. of the Patent Law. State of the Art Report Page 5/5