WO2012149622A1

WO2012149622A1 - Skin test for diagnosing subclinical caseous lymphadenitis in goats and sheep

Info

Publication number: WO2012149622A1
Application number: PCT/BR2012/000134
Authority: WO
Inventors: Vasco Ariston De Carvalho Azevedo; Alessandro DE SÁ GUIMARÃES; Camila AZEVEDO ANTUNES DE OLIVEIRA; Núbia SEYFFERT; Fernanda Alves Dorella; Sintia SILA DE ALMEIDA; Anderson MYIOSHI; Aurora Maria GUIMARÃES GOUVEIA
Original assignee: Universidade Federal De Minas Gerais - Ufmg
Priority date: 2011-05-02
Filing date: 2012-05-02
Publication date: 2012-11-08
Also published as: BRPI1102202A2; BRPI1102202B1

Abstract

The present invention describes a skin test characterised in that it comprises protein antigens secreted by Corynebacterium pseudotuberculosis for diagnosing subclinical caseous lymphadenitis in goats and sheep. This test allows goats and sheep to be tested positive on the farm for caseous lymphadenitis (CL), assisting in combating the infection, since the test can be performed by field veterinarians.

Description

"Skin test for diagnosis of subclinical caseous lymphadenitis in goats and sheep"

The present invention describes a skin test comprising secreted Corynebacterium pseudotuberculosis protein antigens for the diagnosis of subclinical caseous lymphadenitis in goats and sheep. This test allows the evaluation of goats and sheep at farm level for positivity for caseous lymphadenitis (LC), favoring infection control, as it can be performed by veterinarians working in the field.

LC is a chronic disease caused by bacteria of the species.

Corynebacterium pseudotuberculosis, which mainly affects small ruminants and is responsible for significant economic losses related to reduced productivity and reproductive efficiency of infected animals (Arsenault, JO; Girard, C.; Dubreuil, P.; Daignault, DO; Galarneau, J.-R. Boisclair, J., Simard, C. Belanger, D. Prevalence of and carcass condemnation from maidis-visna, paratuberculosis and caseous lymphadenitis in culled sheep from Quebec, Canada, Prev. Vet. Med., V. 59, p. 67-81, 2003; Dorella, FA; Pacheco, LGC; Oliveira, SC; Miyoshi, A.; Azevedo, V. Corynebacterium pseudotuberculosis: microbiology, biochemical properties, pathogenesis and molecular studies of virulence, Vet. Res., V. 37 , pp. 201-218, 2006). This disease is prevalent in countries such as Australia, New Zealand, South Africa, the United States, Canada and Brazil, where there is intense activity of sheep and goats (Guimarães, AS, Carmo, FB Pauletti, RB Seyffert, N., Ribeiro, D ., Lage, AP Heinemann, MB Miyoshi, A., Azevedo, V. Gouveia, AMG Caseous Lymphadenitis: Epidemiology, Diagnosis, and Control, The IIOAB Journal, v. 2, pp. 33-43, 2011). There are few studies of caseous lymphadenitis seroprevalence in Brazil, but high prevalence has been detected in some states. In Minas Gerais were detected 75.8% prevalence in sheep and 95.9% of positive herds (Guimarães, AS Seyffert, N., Bastos, BL Portela, RWD Meyer, R., Carmo, FB, Cruz, JCM. McCulloch , JA, Lage, AP Heinemann, MB, Miyoshi, A., Azevedo, V. Gouveia, AMG Caseous lymphadenitis in sheep flocks of the state of Minas Gerais, Brazil: Prevalence and management surveys, Small Ruminant Research v. 87, p. 86-91, 2009). In goats animal prevalence of 78.9% and 98% of positive herds was detected (Seyffert, N., Guimarães, AS Pacheco, LG.C. Portela, RW Bastos, BL, Dorella, FA, Heinemann, MB, Lage, AP , Gouveia, AMG, Meyer, R., Miyoshi, A., Azevedo, V. High seroprevalence of caseous lymphadenitis in Brazilian goat herds revealed by Corynebacterium pseudotuberculosis secreted protein-based ELISA, Research in Veterinary Science, v. 88, p. -55, 2010).

Some diagnostic tests for LC have been tested, but have shown unsatisfactory results. In addition to needing to collect samples on the property, demanding time to perform, the cost is high and the tests require skilled labor (Dercksen, DP, Brinkhof, JMA, Dekker-Nooren, T., Van Maanen, K., Bode, CF, Baird, G., Kamp, EM Comparison of four serological tests for the diagnosis of caseous lymphadenitis in sheep and goats, Vet. Microbiol 75, 167-175, 2000; Williamson, LH Caseous lymphadenitis in small ruminants, Vet. Clin, North Am. Food Anim. Pract., v. 17, pp. 359-371, 2001; Menzies, PI; Hwang, T.-l .; Prescott, JF Comparison of an interferon-gamma to enzyme-linked phospholipase D immunosorbent assay for diagnosis of Corynebacterium pseudotuberculosis infection in experimentally infected goats, Vet. Microbiol., v. 100, p.129-137, 2004; Dorella, FA; Pacheco, LG C; Oliveira, S. C Myoshi, A. Azevedo, V. Corynebacterium pseudotuberculosis: microbiology, biochemical properties, pathogenesis and molecular studies of viru; lence. Veterinary Research, v. 37, no. 2, p. 201-218, 2006.).

A methodology is found in the literature, based on a reaction of

Multiplex PCR (mPCR) for faster and more efficient detection of C. pseudotuberculosis in clinical samples of animals with LC (Pacheco, LG.C, Pena, RR, Castro, TLP, Dorella, FA, Bahia, RC, Carminati, R ., Fleet, MNL, Oliveira, SC, Meyer, R., Alves, FSF, Miyoshi, A., AZEVEDO, V. Multiplex PCR Assay for Identification of Corynebacterium pseudotuberculosis from Pure Cultures and for Rapid Detection of this Pathogen in Clinical Samples. J. Med. Microbiol., V. 56, pp. 480-486, 2007). By using mPCR associated with a bacterial DNA extraction method directly from purulent material, C. pseudotuberculosis can be detected more rapidly and as specifically as would be done using bacteriological culture and biochemical identification of isolates, which is currently the gold standard for the diagnosis of LC. However, despite the high sensitivity and specificity, this laboratory technique demands high cost material and qualified personnel for its execution.

The lack of a satisfactory diagnosis for CL, especially in the subclinical phase of the disease, compromises the effective management of animals and contributes to the spread of the disease. The optimal diagnosis to prevent disease proliferation in a herd should be sensitive and specific enough to detect early infection and easily applied to field animals. For this purpose, a skin test was developed for use in goats using C. pseudotuberculosis Phospholipase D (PLD), but reactions were observed only after a period of 10 days (Alves, FSF; Olander, H. Vaccine Use Toxoid in the Control of Caseous Lymphadenitis in Goats (Journal of Veterinary Sciencen, v.5, n.1, pp. 69-74, 1999). A skin test using fragmented bacterial cells had previously been performed, and the results were similar (Brown, CC; Olander, HJ; Biberstein, EL; Morse, SM. Use of a toxoid vaccine to protect goats against intradermal challenge exposure to Corynebacterium pseudotuberculosis. Am. J. Vet. Res., V. 47, pp. 1116-1119, 1986).

Skin testing is a diagnostic method used to screen for infections caused by facultative intracellular microorganisms with cell-mediated immunity. This test demonstrated better efficiency in the diagnosis of bovine tuberculosis when performed with Mycobacterium bovis recombinant proteins ESAT6 and CFP10 than the traditional skin test using fragmented bacterial cells (Aagaard, C; Govaerts, M.; Meikle, V .; Vallecillo , AJ; Gutierrez-Pabello, JA; Suarez-Gu ^" Emes, F.; Mcnair, J .; Cataldi, A.; Espitia.C; Andersen, P.; Pollock, JM Optimizing Antigen Cocktails for Detection of Mycobacterium bovis in Herds with Different Prevalences of Beef Tuberculosis: ESAT6-CFP10 Mixture Shows Optimal Sensitivity and Specificity. J. Clin. Microbiol., V. 44, p. 4326-4335, 2006).

C. pseudotuberculosis virulence factors, genes and proteins such as: PLD, toxic cell wall lipids, fag A, B, C and D genes involved with iron absorption, Hsp60 heat shock protein and CP40 protease, have already been identified and tested in different experiments. (Lipsky, BA; Goldberger, AC; Tompkins, LS; Plorde, JJ Infections Caused By Nondiphtheria Corynebacteria, Rev. Infect Dis., V. 4, p. 1220-1235, 1982; Hodgson, ALM; Krywult, J. ; Eat, LA .; Rothel, JS; Radford, AJ. Rational attenuation of Corynebacterium pseudotuberculosis: potential cheesy gland vaccine and live delivery vehicle, Infect. Immun., V. 60, p.2900-2905, 1992; Carne, HR; Onon; , EO Action of Corynebacterium ovis exotoxin on endothelial cells of blood vessels, Nature, v. 271, pp. 246-248, 1978; Hard, GC Examination by electron microscopy of the interaction between peritoneal phagocytes and Corynebacterium ovis, J. Med. Microbiol , v. 5, pp. 483-491, 1972; Billington, SJ; Esmay, PA; Songer, JG; Jost, BH Identification and role in virulence of putative iron acquisition genes from Corynebacterium pseudotuberculosis, FEMS Microbiol. Lett., v 208, pp. 41-45, 2002; Wilson, MJ; Brandon, MR; Walker, J. Molecular and biochemical characterization of a pr otective 40-kilodalton antigen from Corynebacterium pseudotuberculosis, Infect. Immun., V. 63, p. 206-21, 1995). In addition, some researchers have analyzed and characterized soluble and insoluble C. pseudotuberculosis proteins that have immunodominant potential (Merchant, IA; Packer, RA. The Genus Corynebacterium, in: Merchant IA, Packer RA (Eds.), Veterinary bacteriology and virology, The Iowa State University Press, Iowa, pp 425-440 (1967).

In addition, other secreted-secreted immunogenic components have been described using the immunoblot technique, detected using infected goat serum, and may be useful as vaccine and diagnostic components (Paule, BJA; Meyer, R .; Moura-Costa, LF. ; Bahia, CR; Carminati, R; Regis, LF; Vale, VLC; Freire, SM; Nascimento, I .; Schaer, R .; Azevedo, V. Three-phase partitioning as an efficient method for extraction / concentration of immunoreactive excreted-secreted proteins of Corynebacterium pseudotuberculosis, Protein Expr. Purif., V. 34, p. 31 1-166. 2004; Paule, BJA Studies of Corynebacterium pseudotuberculosis antigens and their interactions with the goat host. 2003. 138p. Thesis, Doctorate in Immunology, Institute of Health Sciences of UFBA). Seyffert obtained six C. pseudotuberculosis secreted proteins that were evaluated for antigenicity, but were not field-tested in goats and sheep (Seyffert, N. 2009. Isolation, identification and molecular analysis of the cpsec39 gene encoding a Corynebacterium pseudotuberculosis exclusive protein : perspectives in the development of prophylactic methods for the control of caseous lymphadenitis (97pp. Master's thesis of the Institute of Biological Sciences of the Federal University of Minas Gerais).

Langenegger et al. (Langenegger, CH; Langenegger, J .; Costa, SG - Allergen for the diagnosis of caseous lymphadenitis in goats, Brazilian Veterinary Research, v.7, n.2, p.27-32, 1987) worked with allergen for the diagnosis of caseous lymphadenitis in goats by inoculating 0.1 ml lymphadenin at concentrations of 0.25 and 0.12 mg / ml intradermally in the goat shoulder blade region. The animals with the disease developed local allergic reactions, with greater intensity after 48 hours and with increased skin fold thickness ranging from 1.6 to 12.2 mm. Importantly, the protocol for obtaining the proteins used in this work (lymphadenin) was different from that proposed in this patent application, secretin. The culture medium used was tryptic soy broth, the final product consisted of water soluble bacterial proteins, autoclaved and precipitated by trichloroacetic acid, while secretin consists only of proteins secreted by Corynebacterium pseudotuberculosis. In addition, the lymphadenin bacterial mass was diluted in sodium phosphate and then diluted in a conservative solution consisting of glycerin, phenol and phosphate buffer while secretin is diluted in fully innocuous goat TrisHCI solution according to field tests. Patent document RU2053295, "Strain of bacterium corynebacterium pseudotuberculosis used as a reference for the preparation of caseous lymphadenitis", describes the use of a Corynebacterium pseudotuberculosis strain for identification of goat lymphadenitis in goats by serological, allergic and bacteriological diagnosis. Results for allergic diagnosis are obtained 24 h after intracutaneous inoculation of the allergen in the subcaudal region of goats. However, the allergen used herein differs from the secretin used in the present invention.

In the prior art there is no standardized Corynebacterium pseudotuberculosis secreted total protein product for use in skin testing. The present invention allows diagnosis of caseous lymphadenitis within 24-72 h, similar to the diagnosis of bovine tuberculosis by skin testing. In addition, the present invention will make it possible to standardize test values for positive or negative animal classification for caseous lymphadenitis, fundamental for sheep and goat species.

BRIEF DESCRIPTION OF THE FIGURES

FIGURE 1 - Measurement of skin thickness with cutimeter of male Saanen animals, approximately 6 months old, prior to secretin inoculation, both from group B (artificially infected)

FIGURE 2 - Measurement of skin thickness with cutimeter of male Saanen animals, approximately 6 months old, 24 h after secretin inoculation, both from group B (artificially infected)

DETAILED DESCRIPTION OF TECHNOLOGY

The development of the present technology involved the following steps: (1) obtaining the stress-free C. pseudotuberculosis secretin through the three-phase fractionation technique, (2) evaluating the antigenicity of proteins obtained using goat sera. and sheep at different stages of C. pseudotuberculosis infection, (3) skin test development in C. infected goats and sheep pseudotuberculosis, (4) determination of the classification parameters of the animals tested in the skin test in negative, inconclusive and positive.

The present invention may be better understood, but not limited to, by the following examples:

EXAMPLE 1 - Obtaining Secretin

The bacterial strain MIC 6 of C. pseudotuberculosis biovar ovis was grown at 37 ° C in "Brain-Heart Infusion" (BHI) medium according to Pacheco and collaborators (Pacheco, LG; Pena, RR; Castro, TL; Dorella, FA; Bahia , RC; Carminati, R.; Fleet, MN ;; Oliveira, SC; Meyer, R.; Alves, FS; Miyoshi, A.; Azevedo, V .. Multiplex PCR assay for Identification of Corynebacterium pseudotuberculosis from pure cultures and for rapid detection of this pathogen in clinical samples J Med Microbiol v. 56, pp. 480-486, 2007. and Pacheco, LG; Slade SE; Seyffert N.; Santos, AR; Castro, TL; Silva, WM; Santos, AV; Santos, SG; Farias, LM; Carvalho, MA; Pimenta, AM; Meyer, R.; Silva, A.; Scrivens, JH; Oliveira, SC; Miyoshi, A.; Dowson, CG; Azevedo, V. A combined approach for comparative exoproteome analysis of Corynebacterium pseudotuberculosis (BMC Microbiol., V.1 1-12; p. 1-14,201).

Secretin was obtained through the three-phase fractionation technique (TPP), according to Paule and collaborators (Paule, BJ; Meyer, R.; Moura-Costa, LF; Bahia, RC; Carminati, R.; Régis, LF; Vale , VL; Freire, SM; Nascimento, I .; Schaer, R.; Azevedo, V. Three-phase partitioning as an efficient method for extraction / concentration of excreted-secreted immunoreactive proteins of Corynebacterium pseudotuberculosis Protein Expr Purif., V. 34, pp 311-166, 2004). Overnight precultures (~ 2-18hrs) of C. pseudotuberculosis MIC 6 strain were re-inoculated (1: 200) in BHI (Calf Brain Infusion, Bovine Heart Infusion, Peptone, Dextrose, Sodium Chloride and Fostate) media. preheated to 37 ° C, and incubated for an additional 24 hours. h, 500 ml until they reach the beginning of the exponential growth phase (D0 ₅₄ nm = -0.3-0.4, LabSystems iEMS Absorbance Plate Reader).

The cultures were centrifuged for 20 minutes at 4000 rpm to lower the cells and the supernatants were transferred to new tubes. Samples were filtered through 0.22 μιτι filters and then 30% (w / v) ammonium sulfate and n-butanol were added in the same volume as the supernatant, the pH of the mixture adjusted to 4.0. The mixed solution was incubated at room temperature for one hour and centrifuged at 1,350 X g for 10 minutes for three phase separation. The interfacial precipitation was collected and dissolved in 1 ml of 20 mM Tris (pH 7.4) plus protease inhibitor cocktail (Sigma) in the ratio of 1 μ to 100 μΙ of the final product obtained (Paule et al, 2004). The protein extract was then diafiltered with Amicon columns, 5 kDa exclusion factor (Millipore).

EXAMPLE 2 - Secretin Antigenicity Tests

Serological samples of goats and sheep comprised the following groups: uninfected animals (negative control); vaccinated (artificially infected) animals and animals at different stages of natural C. pseudotuberculosis infection.

Protein immunogenicity was assessed by indirect enzyme-linked immunosorbent assay (ELISA) in which they were used as an antigen, according to Carminati and colleagues (Carminati, R .; Bahia, R .; Moura-Costa, LF; Paule). , BJA; Vale, V. L; Régis, L; Freire, SM; Nascimento, I .; Schaer, R .; Meyer, R. Determination of the sensitivity and specificity of an indirect ELISA test for the diagnosis of caseous lymphadenitis in goats, R. Ci. Med. Biol., v. 2, pp. 88-93, 2003).

Polystyrene 96 well microplates (Nunc Plate-ImmunoTM PolySorpTM Surface NUNCTM Brand Products) were coated with proteins at 4 C overnight, diluted in carbonate bicarbonate buffer pH 9.8. Plates were washed 3 times with phosphate buffered saline pH 8.0 plus 0.05% Tween 20 (PBS-T) and blocked with 0.25% casein for 1 h at 37 ° C. After 3 more washes with PBS-T , the sera of the animals mentioned in item above were diluted in PBS-T and added to the plates. Samples were incubated for 1 h at 37 ° C and, after rewashing the plates, peroxidase-conjugated goat or sheep anti-immunoglobulin serum (Sigma) diluted in PBS-T was added and incubated for an additional 1 h at 37 ° C. C. After 5 washes with PBS-T, ELISA-OPD Peroxidase (Sigma) substrate was added and the plate incubated for 15 minutes at room temperature in a dark place. Optical density reading was performed at 450 nm on a Multiskan MCC / 340 ELISA reader (Titertek).

Serum samples from each animal were evaluated in triplicate in the ELISA test according to Carminatti et al, 2003 (Renato Carminati, Robson Bahia, Lilia Ferreira de Moura Costa, Bruno Jean Adrien Paule, Vera Lucia Valley, Lia Régis, Songeli Menezes Freire, Ivana Nascimento, Robert Schaer, Roberto Meyer, Determination of the sensitivity and specificity of an indirect ELISA test for the diagnosis of caseous lymphadenitis in goats (R. CL Méd. Biol., 2, 1, 88-93, 2003).

EXAMPLE 3 - Sequencing of Secretin Proteins

Protein sequencing was performed according to Pacheco and colleagues (Pacheco, LGC, Susan and Slade, Seyffert, N., Santos, AR Castro, TLP, Silva, WM, Santos, AV, Santos, SG, Farias, LM., Carvalho, MAR, Pepper, AMC, Meyer, R., Silva, A., Scrivens, JH Oliveira, SC, Miyoshi, A., Dowson, CG, Azevedo, V. A combined approach for comparative exoproteome analysis of Corynebacterium pseudotuberculosis. BMC Microbiology 201 1,11: 12). Table 1 lists the sequenced Corynebacterium pseudotuberculosis proteins from secretin.

Table 1 - Proteins sequenced from the secretin of

Corynebacterium pseudotuberculosis

SEQ

Protein Identity Accession Number ID N ^e

Conserved hypothetical protein

1 . [Corynebacterium pseudotuberculosis gi | 302329728 | gb | ADL19922.11

1002]

Putative membrane protein

2. [Corynebacterium pseudotuberculosis gi | 302331646 | gb | ADL21840.11 Putative secreted protein

[Corynebacterium pseudotuberculosis> gi | 302330576 | gb | ADL20770.11 1002]

Cell-surface hemin receptor

[Corynebacterium pseudotuberculosis> gi | 302331647 | gb | ADL2 ^' 1841.11 1002]

Putative cytochrome c oxidase subunit

2 [Corynebacterium> gi | 302331108 | gb | ADL21302.11 pseudotuberculosis 1002]

Putative cell-surface hemin receptor

[Corynebacterium pseudotuberculosis> gi | 302331 44 | gb | ADL21338.11 1002]

Putative membrane protein

[Corynebacterium pseudotuberculosis> gi | 302330157 | gb | ADL20351.1 | 1002]

Conserved hypothetical protein

[Corynebacterium pseudotuberculosis> gi | 302330855 | gb | ADL21049.11 1002]

NLP / P60 protein [Corynebacterium

> gi | 302331099 | gb | ADL21293.1 pseudotuberculosis 1002] 1

Trehalose corynomycolyl transferase

C [Corynebacterium> gi | 302331618 | gb | ADL21812.11 pseudotuberculosis 1002] Putative secreted protein

[Corynebacterium pseudotubercuiosis> gi | 302206205 | gb | ADL10547.11 C231]

Putative HtaA Family protein

[Corynebacterium pseudotubercuiosis> gi | 302331143 | gb | ADL21337.11 002]

Putative NPIJP60 family secreted

protein [Corynebacterium> gi | 302331100 | gb | ADL21294.11 pseudotubercuiosis 1002]

Substrate-binding protein

[Corynebacterium pseudotubercuiosis> gi | 302331720 | gb | ADL21914.11 1002]

Hypothetical protein CpC231_0129

[Corynebacterium pseudotubercuiosis> gi | 302205284 | gb | ADL09626.11 C231]

Putative transglycosylase

[Corynebacterium pseudotubercuiosis> gi | 302330293 | gb | ADL20487.11 1002]

Penicillin-binding protein

[Corynebacterium pseudotubercuiosis> gi | 302331696 | gb | ADL21890.11 1002]

Putative secreted protein

[Corynebacterium pseudotubercuiosis> gi | 302330094 | gb | ADL20288.11 1002] Putative surface-anchored protein

[Corynebacterium pseudotuberculosis> gi | 302331717 | gb | ADL21911.11 002]

Conserved hypothetical protein

[Corynebacterium pseudotuberculosis> gi | 302330028 | gb | ADL20222.11 1002]

Putative secreted protein

[Corynebacterium pseudotuberculosis> gi | 302331731 | gb | ADL21925.11 1002]

Poly (3-hydroxybutyrate)

depolymerase [Corynebacterium> gi | 302330594 | gb | ADL20788.11 pseudotuberculosis 1002]

Putative membrane protein

[Corynebacterium pseudotuberculosis> gi | 302331081 | gb | ADL21275.11 1002]

Cell-surface hemin receptor

[Corynebacterium pseudotuberculosis> gi | 302330 53 | gb | ADL20347.11 1002]

Putative efflux system protein

[Corynebacterium pseudotuberculosis> gi | 302331553 | gb | ADL21747.11 1002]

putative phosphatase

[Corynebacterium pseudotuberculosis> gi | 302205522 | gb | ADL09864.11 C231] Putative surface-anchored membrane

protein [Corynebacterium> gi | 302329880 | gb | ADL20074. 1 pseudotuberculosis 1002]

resuscitation-promoting factor RpfB

[Corynebacterium pseudotuberculosis> gi | 302330380 | gb | ADL20574.11 1002]

Putative secreted hydrolase

[Corynebacterium pseudotuberculosis> gi | 302330235 | gb | ADL20429.11 1002]

Trehalose corynomycolyl transferase

B [Corynebacterium> gi | 302331620 | gb | ADL218 4.11 pseudotuberculosis 1002]

Putative secreted hydrolase

[Corynebacterium pseudotuberculosis> gi | 302329940 | gb | ADL20134.1 | 1002]

surface layer protein A

[Corynebacterium pseudotuberculosis> gi | 302329946 | gb | ADL20140.11 1002]

Putative secreted protein

[Corynebacterium pseudotuberculosis> gi | 302331520 | gb | ADL21714.11 1002]

Corynomycolyl transferase

[Corynebacterium pseudotuberculosis> gi | 302331416 | gb | ADL21610.11 1002] Precursor Sialidase [Corynebacterium

> gi | 302330093 | gb | ADL20287.1 pseudotuberculosis 1002] 1

Putative trypsin-like serine protease

[Corynebacterium pseudotuberculosis> gi | 302330459 | gb | ADL20653.11 1002]

Putative peptide transport system

[Corynebacterium pseudotuberculosis> gi | 302330456 | gb | ADL20650.11 002]

Putative twin-arginine translocation

pathway signal protein

> gi | 302330314 | gb | ADL20508.1 [Corynebacterium pseudotuberculosis 1 1002]

Putative peptidoglycan recognition

protein [Corynebacterium> gi | 302331634 | gb | ADL21828.11 pseudotuberculosis 1002]

Peptidase, S8A (subtilisin) family

protein [Corynebacterium> gi | 302331305 | gb | ADL21499.11 pseudotuberculosis 1002]

Peptidyl-prolyl cis-trans isomerase A

[Corynebacterium pseudotuberculosis> gi | 302329734 | gb | ADL19928.11 1002]

Hemin precursor receptor

[Corynebacterium pseudotuberculosis> gi | 302330154 | gb | ADL20348.1 | 1002]

metalloendopeptidase-like membrane

protein [Corynebacterium> gi | 302330342 | gb | ADL20536.11 pseudotuberculosis 1002] Putative serine protease

[Corynebacterium pseudotuberculosis> gi | 302330361 | gb | ADL20555.11 1002]

putative secreted protein

[Corynebacterium pseudotuberculosis> gi | 302330272 | gb | ADL20466. 1 1002]

Lipoprotein LpqE [Corynebacterium

> gi | 30233 434 | gb | ADL2 628. pseudotuberculosis 1002] 1

Putative secreted protein

[Corynebacterium pseudotuberculosis> gi | 302329903 | gb | ADL20097. 1 1002]

Putative lysozyme precursor

[Corynebacterium pseudotuberculosis> gi | 302329779 | gb | ADL19973.11 1002]

putative phosphatase

[Corynebacterium pseudotuberculosis> gi | 302205522 | gb | ADL09864. 1 C231]

putative secreted protein

[Corynebacterium pseudotuberculosis> gi | 302330462 | gb | ADL20656.11 1002]

Putative secretory lipase

[Corynebacterium pseudotuberculosis> gi | 302331473 | gb | ADL21667.11 1002]

putative secreted protein

[Corynebacterium pseudotuberculosis> gi | 302330261 | gb | ADL20455.11 1002]

Putative amino deoxychorismate lyase

[Corynebacterium pseudotuberculosis> gi | 302330834 | gb | ADL21028.11 1002] Putative secreted protein

[Corynebacterium pseudotuberculosis> gi | 302331045 | gb | ADL21239.11 1002]

ABC superfamily ATP binding cassette

transporter, binding protein

> gi | 302330024 | gb | ADL20218.1 [Corynebacterium pseudotuberculosis 1 1002]

Co-chaperonin GroES

[Corynebacterium pseudotuberculosis> gi | 302330124 | gb | ADL20318.11 1002]

phosphoglyceromutase

[Corynebacterium pseudotuberculosis> gi | 302329967 | gb | ADL20161.11 1002]

Elongation factor Tu [Corynebacterium

> gi | 302330046 | gb | ADL20240. pseudotuberculosis 1002] 1 elongation factor Ts [Corynebacterium

> gi | 302330993 | gb | ADL21187.1 pseudotuberculosis 1002] 1 transcription elongation factor GreA

[Corynebacterium pseudotuberculosis> gi | 302330418 | gb | ADL20612.11 1002]

Conserved hypothetical protein

[Corynebacterium pseudotuberculosis> gi | 302330028 | gb | ADL20222.11 1002]

Thiol peroxidase [Corynebacterium

> gi | 302330853 | gb | ADL21047.1 | pseudotuberculosis 1002]

Phosphoglycerate kinase

[Corynebacterium pseudotuberculosis> gi | 302330796 | gb | ADL20990. 1 1002]

Triosephosphate Isomerase

[Corynebacterium pseudotuberculosis> gi | 302330795 | gb | ADL20989.11 1002]

Conserved hypothetical protein

[Corynebacterium pseudotuberculosis> gi | 302331148 | gb | ADL21342.11 1002]

Putative Phosphocarrier protein HPr

[Corynebacterium pseudotuberculosis> gi | 302330920 | gb | ADL21114.11 1002] Putative inhibitor of ODH activity

[Corynebacterium pseudotuberculosis> gi | 302330687 | gb | ADL20881.11 1002]

Putative methylmalonyl-CoA

epimerase [Corynebacterium> gi | 302330545 | gb | ADL20739.11 pseudotuberculosis 1002]

Putative lipoprotein [Corynebacterium

> gi | 302329778 | gb | ADL19972. pseudotuberculosis 002] 1

Hypothetical protein CpC231_1797

[Corynebacterium pseudotuberculosis> gi | 3022069 | gb | ADL 1253. 1 C231]

Phospholipase D Precursor (PLD)

[Corynebacterium pseudotuberculosis> gi | 302205182 | gb | ADL09524.1 | C231]

Serine proteinase precursor

[Corynebacterium pseudotuberculosis> gi | 302206997 | gb | ADL 1339.11 C231]

Hypothetical protein CpC231_0514

[Corynebacterium pseudotuberculosis> gi | 302205659 | gb | ADL10001.11 C231]

Putative secreted protein

[Corynebacterium pseudotuberculosis> gi | 302205529 | gb | ADL09871.11 C231]

Putative secreted protein

[Corynebacterium pseudotuberculosis> gi | 302206042 | gb | ADL 0384.11 C231]

Hypothetical protein CpC231_1756

[Corynebacterium pseudotuberculosis> gi | 302206871 | gb | ADL11213.11 C231]

Putative secreted protein

[Corynebacterium pseudotuberculosis> gi | 302331343 | gb | ADL21537.11 1002]

Putative secreted protein

[Corynebacterium pseudotuberculosis> gi | 302206147 | gb | ADL10489.11 C231]

Secreted penicillin-binding protein

[Corynebacterium pseudotuberculosis> gi | 302205190 | gb | ADL09532.1 | C231] Putative DsbG protein ¹

[Corynebacterium pseudotuberculosis> gi | 302331361 | gb | ADL21555.11 1002]

Putative surface-anchored protein

(fimbrial subunit) [Corynebacterium> gi | 302207002 | gb | ADL11344.11 pseudotuberculosis C231]

Putative penicillin-binding secreted

protein [Corynebacterium> gi | 302205355 | gb | ADL09697.11 pseudotuberculosis C231]

Putative membrane anchored protein

[Corynebacterium pseudotuberculosis> gi | 302206996 | gb | ADL11338.11 C231]

Conserved hypothetical protein

[Corynebacterium pseudotuberculosis> gi | 302206984 | gb | ADL11326.11 C231]

Transcriptional regulator

[Corynebacterium pseudotuberculosis> gi | 302205648 | gb | ADL09990.11 C231]

Putative serine / threonine protein

kinase [Corynebacterium> gi | 302206538 | gb | ADL10880.11 pseudotuberculosis C231]

Extracellular putative solute-binding

protein [Corynebacterium> gi | 302205510 | gb | ADL09852.11 pseudotuberculosis C231]

maltotriose-binding protein

[Corynebacterium pseudotuberculosis> gi | 302205530 | gb | ADL09872.11 C231]

Putative zinc metallopeptidase

[Corynebacterium pseudotuberculosis> gi | 302206284 | gb | ADL10626.11 C231]

Putative iron transport system binding

(secreted) protein [Corynebacterium> gi | 302206118 | gb | ADL10460.1 | pseudotuberculosis C231]

Glycerophosphoryl diester

phosphodiesterase [Corynebacterium> gi | 302207068 | gb | ADL11410.11 pseudotuberculosis C231] Hypothetical protein CpC231_1187

[Corynebacterium pseudotuberculosis> gi | 302206321 | gb | ADL10663.11 C231]

Iron siderophore binding protein-FagD

[Corynebacterium pseudotuberculosis> gi | 302205186 | gb | ADL09528.1 | C231]

Hypothetical protein CpC231_0022

[Corynebacterium pseudotuberculosis> gi | 302205179 | gb | ADL09521.1 | C231]

FHA-domain-containing protein

[Corynebacterium pseudotuberculosis> gi | 302205193 | gb | ADL09535.1 | C231]

Conserved hypothetical protein

[Corynebacterium pseudotuberculosis> gi | 302205253 | gb | ADL09595.11 C231]

Conserved hypothetical protein

[Corynebacterium pseudotuberculosis> gi | 302205266 | gb | ADL09608.11 C231]

Hypothetical protein CpC231_0115

[Corynebacterium pseudotuberculosis> gi | 302205271 | gb | ADL09613.11 C231]

Putative Sdr-family related adhesin

[Corynebacterium pseudotuberculosis> gi | 302205321 | gb | ADL09663.11 C231]

Iron (Fe3 +) ABC superfamily ATP

binding cassette transporter, binding

> gi | 302205338 | gb | ADL09680. protein [Corynebacterium 1 pseudotuberculosis C231] Two-component sensor protein

[Corynebacterium pseudotuberculosis> gi | 302205341 | gb | ADL09683.11 C231]

Putative secreted protein

[Corynebacterium pseudotuberculosis> gi | 302205349 | gb | ADL09691.11 C231]

Putative secreted phosphohydrolase

protein [Corynebacterium> gi | 302205353 | gb | ADL09695.11 pseudotuberculosis C231]

Thiol disulfide isomerase, secreted

protein [Corynebacterium> gi | 302205363 | gb | ADL09705.11 pseudotuberculosis C231]

Putative secreted protein

[Corynebacterium pseudotuberculosis> gi | 302205376 | gb | ADL09718.11 C231]

Putative secreted protein

[Corynebacterium pseudotuberculosis> gi | 302205404 | gb | ADL09746.11 C231]

Putative secreted protein with DSBA- like thioredoxin domain

> gi | 302205424 | gb | ADL09766.1 [Corynebacterium pseudotuberculosis 1 C231]

Uroporphyrinogen decarboxylase

[Corynebacterium pseudotuberculosis> gi | 302205434 | gb | ADL09776.11 C231]

Putative secreted conserved protein

[Corynebacterium pseudotuberculosis> gi | 302205447 | gb | ADL09789.11 C231] Putative secreted amino acid

hydrolase [Corynebacterium> gi | 302205521 | gb | ADL09863.11 pseudotuberculosis C231]

Putative secreted protein

[Corynebacterium pseudotuberculosis> gi | 302205566 | gb | ADL09908.11 C231]

NPL / P60-family secreted protein

[Corynebacterium pseudotuberculosis> gi | 302205615 | gb | ADL09957.11 C231]

Putative secreted protein

[Corynebacterium pseudotuberculosis> gi | 302205684 | gb | ADL 0026.11 C231]

PDZ domain family, secreted protein

[Corynebacterium pseudotuberculosis> gi | 302205697 | gb | ADL10039.11 C231]

Putative secreted protein

[Corynebacterium pseudotuberculosis> gi | 302205714 | gb | ADL10056.11 C231]

putative secreted protein

[Corynebacterium pseudotuberculosis> gi | 302205720 | gb | ADL10062.11 C231]

Hypothetical protein CpC231_ 0593

[Corynebacterium pseudotuberculosis> gi | 302205736 | gb | ADL 0078.11 C231]

Putative secreted protein

[Corynebacterium pseudotuberculosis> gi | 302205740 | gb | ADL10082.11 Putative secreted protein

[Corynebacterium pseudotubercuiosis> gi | 302205806 | gb | ADL10148.11 C231]

resuscitation-promoting factor

[Corynebacterium pseudotubercuiosis> gi | 302205821 | gb | ADL10163.11 C231]

Putative secreted protein

[Corynebacterium pseudotubercuiosis> gi | 302205826 | gb | ADL10168.1 | C231]

Putative 2-nitropropane dioxygenase

[Corynebacterium pseudotubercuiosis> gi | 302205838 | gb | ADL10180.11 C231]

Putative LpqU family protein

[Corynebacterium pseudotubercuiosis> gi | 302205852 | gb | ADL10194.1 | C231]

Putative secreted protein

[Corynebacterium pseudotubercuiosis> gi | 302205859 | gb | ADL10201.11 C231]

putative secreted protein

[Corynebacterium pseudotubercuiosis> gi | 302205965 | gb | ADL10307.11 C231]

Putative secreted protein

[Corynebacterium pseudotubercuiosis> gi | 302206032 | gb | ADL10374.11 C231]

Putative secreted protein

[Corynebacterium pseudotubercuiosis> gi | 302206052 | gb | ADL10394.11 Putative secreted protein

[Corynebacterium pseudotuberculosis> gi | 302206135 | gb | ADL10477.11 C231]

Putative SPFH domain, band 7

integral membrane protein

> gi | 302206200 | gb | ADL10542.1 [Corynebacterium pseudotuberculosis 1 C231]

NADPH-dependent FMN reductase

[Corynebacterium pseudotuberculosis> gi | 302206289 | gb | ADL1063 .11 C231]

NADPH-dependent FMN reductase

[Corynebacterium pseudotuberculosis> gi | 302206366 | gb | ADL10708.1 | C231]

Putative penicillin-binding protein 2

(PBP-2) [Corynebacterium> gi | 302206428 | gb | ADL10770.11 pseudotuberculosis C231]

M23 domain-containing peptidase

protein [Corynebacterium> gi | 302206439 | gb | ADL10781.11 pseudotuberculosis C231]

Putative secreted protein

[Corynebacterium pseudotuberculosis> gi | 302206473 | gb | ADL10815.11 C231]

Putative secreted protein

[Corynebacterium pseudotuberculosis> gi | 302206506 | gb | ADL10848.11 C231]

cell division protein FtsQ

[Corynebacterium pseudotuberculosis> gi | 302206517 | gb | ADL10859.11 C231] Putative secreted protein

[Corynebacterium pseudotuberculosis> gi | 302206603 | gb | ADL10945.11 C231]

Putative guanyl-specific ribonuclease

Sa3 [Corynebacterium> gi | 302206633 | gb | ADL10975.11 pseudotuberculosis C231]

Putative secreted protein

[Corynebacterium pseudotuberculosis> gi | 302206772 | gb | ADL 1114.11 C231]

Hypothetical protein CpC231_1762

[Corynebacterium pseudotuberculosis> gi | 302206877 | gb | ADL1 1219.11 C231]

D-Ala-D-Ala carboxypeptidase 3 (S13)

[Corynebacterium pseudotuberculosis> gi | 302206901 | gb | ADL11243.11 C231]

Multisubunit Na + / H + antiporter,

subunit E [Corynebacterium> gi | 302206921 | gb | ADL1 1263.11 pseudotuberculosis C231]

Putative secreted protein

[Corynebacterium pseudotuberculosis> gi | 302206926 | gb | ADL1 1268.11 C231]

Thiol disulfide oxidoreductase resA

[Corynebacterium pseudotuberculosis> gi | 302206950 | gb | ADL1 1292.11 C231]

Cytochrome c-552 [Corynebacterium

> gi | 302206954 | gb | ADL11296.1 pseudotuberculosis C231] 1

Putative secreted protein

[Corynebacterium pseudotuberculosis> gi | 302206971 | gb | ADL1 3 3.11 C231] DedA family protein [Corynebacterium

> gi | 302207052 | gb | ADL11394.1 pseudotuberculosis C231] 1

Putative secreted protein

[Corynebacterium pseudotuberculosis> gi | 302207079 | gb | ADL1142 .11 C231]

ABC-type dipeptide / oligopeptide / nickel

transport system, secreted component

> gi | 302207165 | gb | ADL11507.1 [Corynebacterium pseudotuberculosis 1 C231]

Phytoene Dehydrogenase

(Desaturase) [Corynebacterium> gi | 302207181 | gb | ADL11523.11 pseudotuberculosis C231]

Putative secreted protein

[Corynebacterium pseudotuberculosis> gi | 302205906 | gb | ADL10248.11 C231]

Putative secreted protein

[Corynebacterium pseudotuberculosis> gi | 302205923 | gb | ADL10265.11 C231]

Putative secreted protein

[Corynebacterium pseudotuberculosis> gi | 302205955 | gb | ADL10297.1 | C231]

Putative secreted protein

[Corynebacterium pseudotuberculosis> gi | 302206027 | gb | ADL10369.11 C231]

Putative amino deoxychorismate lyase

[Corynebacterium pseudotuberculosis> gi | 302206277 | gb | ADL10619.11 C231] Putative secreted protein

[Corynebacterium pseudotuberculosis> gi | 302206398 | gb | ADL 0740.11 C231]

NLP / P60 family protein

[Corynebacterium pseudotuberculosis> gi | 302206545 | gb | ADL 0887.11 C231]

Metal-dependent hydrolase

[Corynebacterium pseudotuberculosis> gi | 302206778 | gb | ADL11120.11 C231]

Flagellar hook protein flgE

[Corynebacterium pseudotuberculosis> gi | 302206869 | gb | ADL 1211.11 C231]

Putative secretory lipase

[Corynebacterium pseudotuberculosis> gi | 302206906 | gb | ADL1 248.11 C231]

Putative secreted protein

[Corynebacterium pseudotuberculosis> gi | 302206917 | gb | ADL11259.11 C231]

Hypothetical protein CpC231_1862

[Corynebacterium pseudotuberculosis> gi | 302206975 | gb | ADL11317.11 C231]

Hypothetical protein CpC231_1880

[Corynebacterium pseudotuberculosis> gi | 302206992 | gb | ADL 1334.11 C231]

Hypothetical protein CpC231_1899

[Corynebacterium pseudotuberculosis> gi | 302207009 | gb | ADL 1351.11 C231] iron siderophore binding protein-FagD

[Corynebacterium pseudotuberculosis> gi | 302329745 | gb | ADL19939.11 1002]

Putative secreted penicillin-binding

protein [Corynebacterium> gi | 302329749 | gb | ADL19943.11 pseudotuberculosis 1002]

Zn2 + ABC superfamily ATP binding

cassette transporter, binding protein

> gi | 302329825 | gb | ADL20019.1 [Corynebacterium pseudotuberculosis 1 1002]

Putative secreted protein with LPxTG

motif [Corynebacterium> gi | 302329874 | gb | ADL20068. 1 pseudotuberculosis 1002]

Putative penicillin-binding secreted

protein [Corynebacterium> gi | 302329909 | gb | ADL20103.11 pseudotuberculosis 1002]

Hypothetical protein Cp1002_0376

[Corynebacterium pseudotuberculosis> gi | 302330083 | gb | ADL20277.11 1002]

putative peptidoglycan synthesis

related protein [Corynebacterium> gi | 302330165 | gb | ADL20359.11 pseudotuberculosis 1002]

Putative secreted protein

[Corynebacterium pseudotuberculosis> gi | 302330479 | gb | ADL20673.11 1002]

Putative secreted protein - Ycel

[Corynebacterium pseudotuberculosis> gi | 302330704 | gb | ADL20898.11 1002] Putative DsbG protein

[Corynebacterium pseudotuberculosis> gi | 302331361 | gb | ADL2 555.11 1002]

Putative membrane anchored protein

[Corynebacterium pseudotuberculosis> gi | 302331558 | gb | ADL21752.11 1002]

Glycosyl hydrolase family protein

[Corynebacterium pseudotuberculosis> gi | 30233 559 | gb | ADL21753.11 1002]

Putative conserved integral membrane

protein [Corynebacterium> gi | 302331759 | gb | ADL21953.11 pseudotuberculosis 1002]

Elongation factor P [Corynebacterium

> gi | 302330827 | gb | ADL21021.1 pseudotuberculosis 1002] 1

Hypothetical protein Cp1002_1841

[Corynebacterium pseudotuberculosis> gi | 30233 509 | gb | ADL21703.11 1002]

Hypothetical protein Cp1002_1840

[Corynebacterium pseudotuberculosis> gi | 302331508 | gb | ADL21702.11 1002]

Chaperone protein DnaK

[Corynebacterium pseudotuberculosis> gi | 302331563 | gb | ADL21757.11 1002]

chaperonin GroEL [Corynebacterium

> gi | 302331479 | gb | ADL21673.1 pseudotuberculosis 1002] 1

Enolase [Corynebacterium

> gi | 302330411 | gb | ADL20605.1 pseudotuberculosis 1002] 1 Glyceraldehyde-3-phosphate

dehydrogenase (GAPDH)

184.> gi | 302330797 | gb | ADL20991 .1 [Corynebacterium pseudotuberculosis 1 1002]

ABC-type transporter

185. [Corynebacterium pseudotuberculosis> gi | 302330197 | gb | ADL20391 .1 | 1002]

Carbonate dehydratase

186. [Corynebacterium pseudotuberculosis> gi | 302330283 | gb | ADL20477.11

1002]

Manganese Superoxide Dismutase

187. [Corynebacterium pseudotuberculosis> gi | 302331655 | gb | ADL2 849.11

1002]

EXAMPLE 4 - Infection Protocol

The animals used in the Skin Test experiments were all male, three Boer, four Alpine and sixteen Saanen. These animals were artificially infected with Corynebacterium pseudotuberculosis MIC 6 strain.

The animals were randomly divided into 2 groups (A and B). Group A with 11 animals: 2 were part of the control group and 9 animals were challenged with strain MIC 6 (10 ⁶ bacteria per dose). Group B with 12 animals: 2 in the control group and 10 animals were challenged with strain MIC 6 (10 ⁶ bacteria per dose).

Prior to infection, the animals had their serum tested by indirect ELISA. All were negative for LC.

Preliminary serologic results (ELISA) before infection are reported in Tables 2 and 3. Table 2 - Indirect Elisa test of serum from group A animals before skin test application.

Table 3 - Indirect Elisa test of group B animals serum before skin test application

5 In group A, 4 animals received the first dose of Corynebacterium pseudotuberculosis (10 ⁶ bacteria), MIC 6 strain, subcutaneously in the shoulder blade region. In group B, 5 animals received the same dose of Corynebacterium pseudotuberculosis strain MIC 6.

EXAMPLE 5 - Evaluation of skin test efficacy in goats and sheep The animals underwent clinical examination, evaluating the general condition, coloration of conjunctival mucosa and vital functions (body temperature, heart rate, respiratory rate and ruminal movements). The application of the simple cervical test (TCS) was performed after the detailed examination of each animal.

15 The skin test was applied intradermally, at a concentration of 0.1 mg / mL secretin, at a dose of 0.1 mL in the shoulder area, in an area previously shaved about 4x4 cm. Afterwards, skin thickness measurement was performed with Hauptner cutimeter. The allergen was inoculated into the center of the area shaved with Hauptner tuberculin syringe.

The first reading was taken before inoculation. The other skin measurements and evaluation of edema and local pain were performed every 24 h for the next ten days, always by the same professional to avoid individual measurement variations. Figures 1 and 2 exemplify the results obtained for two animals from group B, 24 h after inoculation.

Absence of reaction or increase in skinfold thickness (AEP) up to 1.0 mm was considered negative, reactions with ESA between 1.0 and 3.8 mm were considered inconclusive and reactions of 3.9 mm or greater were considered negative. positive (Paulo Eduardo Gomes da Silva, Sônia Regina Pinheiro, Marta Lizandra from Rego Leal, Heloisa Godói Bertagnom, Pedro Moacyr Pinto Coelho Motta, Idercio Luiz Sinhorini, Silvio Arruda Vasconcellos, Fernando José Benesi, Rural Science, v. 36, no. 3 , pp. 880-886, 2006).

Initially, a skin dose of I mg / mL was used, based on a protocol from the Ministry of Agriculture and Supply (MAPA) (http://www.agricultura.gov.br/), with application of 0, 1 mL per animal, subcutaneously, as used for cattle in the diagnosis of bovine tuberculin. The results were promising, with detectable responses at 24h, with peaks at 48-72h. Given these results, new tests were performed and the initial dose was gradually decreased in order to obtain the lowest effective dose. Results are shown in Table 4. The lowest effective dose was 0.1 mg / mL. Table 4 - Skinfold thickness (AEP), in mm, 24h after inoculation of 0.1 mL of the secretin solution subcutaneously in the shoulder blade region in four animals. Different concentrations were used to detect the lowest effective dose.

EXAMPLE 6 - Desensitization Assessment of Tested Animals

Among the animals tested in Table 4, two were retested 62 days after the first application of the Skin Test, in order to evaluate the desensitization period. The result was positive on the second test, that is, the animals were desensitized to the previously inoculated secretin.

New tests were performed even shorter, 42 days after the first test, with animals 7, 1, 9 and 4 and the results were also positive. During this period there was a skin reaction, that is, the animals were already desensitized.

Further testing will be needed to confirm the exact period in which animals will be able to receive a second dose of secretin, but it is evident that this period in goats is shorter than in cattle (60 days).

Claims

1) SKIN TEST, characterized in that it comprises secreted-secreted protein antigens of Corynebacterium pseudotuberculosis and pharmaceutically acceptable excipients. SKIN TEST according to Claim 1, characterized in that the protein antigens are obtained from C. pseudotuberculosis not subjected to any type of stress by the three-phase fractionation technique.

SKIN TEST according to Claims 1 and 2, characterized in that the protein antigens are preferably selected from the group comprising the proteins identified by SEQ ID NOS. 1 to 187.

Skin test according to Claims 1 and 2, characterized in that it comprises at least one of the proteins identified by SEQ ID NOS. 1 to 187, or their corresponding epitopes.

5) SKIN TEST, characterized by the following steps:

A. Obtaining protein antigens according to claims 1 to 4;

B. Application of protein antigens intradermally, at a concentration of 0.1 mg / mL to 1 mg / mL of secreted proteins, at a dose of 0.1 mL, in the shoulder region, in a previously shaved area, after measuring the thickness. of skin made with Hauptner cutimeter;

C. Skin measurement and assessment of edema and local pain between 24 and 72 h.

D. Classification of animals as follows:

I. Absence of reaction or increased skinfold thickness (AEP) up to 1.0 centimeter: negative;

II. Reactions with PEA between 1, 0 and 3.8 mm: inconclusive; III. Reactions with AEP of 3.9 or greater: positive.

SKIN TEST according to any of claims 1 to 5, characterized in that it is for the diagnosis of subclinical caseous lymphadenitis in goats and sheep.