METHOD FOR DIAGNOSING CASEOUS LYMPHADENITIS IN GOATS USING THE ASSOCIATION OF CORYNEBACTERIUM PSEUDOTUBERCULOSIS ANTIGENS RCP01850 AND RCP40

Description

/ 10 Method for diagnosing caseous lymphadenitis in goats using the combination of Corynebacterium pseudotuberculosis rCP01850 and rCP40 antigens. Field of invention: Fundamentals of invention

[001] The present invention relates to a diagnostic method for the detection of caseous lymphadenitis (CL) in goats using the indirect ELISA methodology and having as antigens the recombinant acid phosphatase rCP01850 (SEQ ID No. 1) and the endoglucosidase rCP40 (SEQ ID No. 2), both from Corynebacterium pseudotuberculosis. The diagnosis presents an accuracy of 92%, sensitivity of 85% and specificity of 90%, these parameters being adequate for its use in the diagnosis of CL in the caprine species. The serological diagnosis was developed for application in the field of veterinary medicine, and to be produced by pharmaceutical and immunobiological companies. In the international patent classification, it falls within the fields of invention G01N 33 / 53, G01N 33 / 535, G01N 33 / 569. Description of the State of the Art

[002] CL is a disease that affects goats and sheep globally, caused by the gram-positive, pleomorphic bacterium Corynebacterium pseudotuberculosis, which does not form spores. The hallmark of this disease is the formation of purulent lesions in the superficial and internal lymph nodes, as well as internal organs of infected animals (BARNABÉ, et al. 2020).

[003] The spread of CL occurs mainly through contact with the secretion of superficial abscesses after natural rupture or during drainage of the caseous content, which releases a large quantity of bacteria that contaminate the environment (ANDRADE et al. 2012). Petition 870250021465, dated 03 / 19 / 2025, page 16 / 29 / 10

[004] LC, by affecting organs and tissues, causes significant losses for small and medium-sized producers, especially in the Northeast region of Brazil, where approximately 90% of the country's goat and sheep herds are concentrated. The disease can spread rapidly among herd animals, being transmitted mainly through the respiratory tract and by contact with healthy animals with secretions from infected animals. Thus, the presence of an animal carrying the silent form of the disease favors its spread (CORRÊA et al. 2017).

[005] Animals affected by CL may also exhibit symptoms such as progressive weight loss, respiratory disorders, and chronic ruminal tympanism, which impacts meat and milk production. CL can also lead to economic losses due to the condemnation of animal carcasses that are slaughtered, which, due to infection, become unfit for consumption.

[006] Once the disease is established within the herd, its control becomes difficult, as there is no effective treatment against this pathogen. Thus, the only way to eradicate it is to cull the contaminated animals, and for this, accurate diagnosis of those infected by C. pseudotuberculosis is necessary (ALGAABARY; OSMAN; OREIBY, 2009). In order to control transmission, and even eliminate the agent, it is necessary to accurately identify contaminated animals, thus avoiding contact with healthy animals (WINDSOR et al. 2011). For efficient identification, methods with good levels of sensitivity and specificity should be used, which is reflected in good overall accuracy.

[007] Over the years, numerous serological tests have been developed in an attempt to overcome the problem of animals with asymptomatic cases of CL, including the serum agglutination test (AWAD et al. 1960), gel immunodiffusion (ANDERSON; NAIRN 1984), hemolysis inhibition (BURREL et al. 1981), and different ELISA assays, such as indirect ELISA using various bacterial cell preparations, secreted proteins, or the PLD toxin of C. pseudotuberculosis, sandwich ELISA with PLD, and ELISA to detect interferon gamma (IFN-γ) as a marker of cell-mediated immunity against C. pseudotuberculosis. However, most of these tests do not have sufficiently satisfactory sensitivity and specificity. Petition 870250021465, dated 03 / 19 / 2025, page 17 / 29 / 10 The gold standard test for detecting LC is the isolation of the etiological agent of the disease; however, this is only possible when the external clinical form occurs. Although immunoassays such as enzyme-linked immunosorbent assay (ELISA) are able to detect animals carrying the internal form of the disease, many of the assays developed to date fail to provide high sensitivity and specificity values ​​(BARRAL, et al. 2019).

[008] The recombinant protein rCP01850 is an acid phosphatase, containing 410 amino acids and an approximate molecular weight of 33.5 kDa, encoded by the gene cp1002_RS01850 (formerly cp1002_0369) (SANTOS, et al. 2012.). Patent BR 102016016891-0 A2 (RECOMBINANT ACID PHOSPHATASE RCP01850 FROM CORYNEBACTERIUM PSEUDOTUBERCULOSIS AND ITS USE IN SUBUNIT VACCINES FOR CASEOUS LYMPHADENITIS) uses this acid phosphatase as a vaccine antigen against CL associated with Brazilian red propolis, or saponin.

[009] The rCP40 protein is an endoglycosidase, which exhibits characteristics of a glycosidic hydrolase, specifically hydrolyzing certain glycans into immunoglobulins (SHADNEZHAD; NAEGELI; COLLIN, 2016). Patent BR 102018011672 0 (DIAGNOSTIC METHOD FOR CASEOUS LIFADENITIS IN SMALL RUMINANTS USING RECOMBINANT PROTEINS CP40 AND PLD AS ANTIGENS) uses the association of the rCP40 and rPLD proteins as antigens in indirect ELISA assays, but the use of rCP40 associated with rCP01850 in immunodiagnosis has not been described.

[0010] Rezende (2016) standardized an indirect ELISA using rCP01850 for sheep, where they obtained a specificity of 92.5% and a sensitivity of 90%, thus characterizing the test as suitable for identifying animals positive for LC (REZENDE, et al. 2016). However, the use of an ELISA using the combination of rCP01850 and rCP40 proteins in an indirect ELISA to be applied in the diagnosis of goats has not yet been described.

[0011] It is important to highlight that the same methodology cannot be used for different species, such as goats and sheep, taking into account the divergence Petition 870250021465, dated 03 / 19 / 2025, page 18 / 29 / 10 among the immunological responses elicited in each species. Furthermore, different strains of C. pseudotuberculosis infect goats and sheep (HOELZLE et al. 2013).

[0012] Silva et al. (2019) also employed the association of rPLD with rCP01850 for the diagnosis of CL in sheep, using 100 μL (100 ng) per well of recombinant protein in carbonate-bicarbonate buffer (pH: 9.6) for 16 h at 4°C, followed by washing and a blocking step using 100 μL of PBS-T supplemented with 5% skimmed milk powder for 1 h at 37°C. Following another wash, the positive and negative sera, and the controls, all in duplicate, diluted in a 1:100 ratio in PBS-T, were incubated for 1 h at 37°C. After another wash, 100 μL per well (1:4000) of anti-sheep IgG conjugated with peroxidase was incubated. After 1 hour of incubation at 37°C, a second wash was performed, followed by application of the substrate / chromogen (orthophenylenediamine, in 0.4 mg / mL citrate-phosphate buffer containing 0.04% hydrogen peroxide at 30%), and incubation at room temperature in the dark for 15 minutes.The reaction was stopped with 50 μL / well of sulfuric acid solution (4N), and the OD was measured in a plate spectrophotometer at a wavelength of 492 nm, obtaining 92.5% sensitivity and 72% specificity in the test, with an accuracy of 0.882 (SILVA, et al. 2019). Patent BR 10 2017 009371 9 (ASSOCIATION OF RECOMBINANT PROTEINS OF CORYNEBACTERIUM PSEUDOTUBERCULOSIS IN THE COMPOSITION OF IMMUNOASSAYS AND VACCINES FOR CASEOUS LYMPHADENITIS) requests protection for the combination of recombinant proteins rPLD and rCP01850 from Corynebacterium pseudotuberculosis for its use in immunodiagnostics and in the development of new vaccines against CL.

[0013] The rCP01850 protein has already been patented (BR 10 2023 006284 9 METHOD AND KIT FOR DIAGNOSIS OF CASEOUS LYMPHADENITIS IN GOATS, USING THE RCP01850 ANTIGEN), however its use in association with the rCP40 protein for the diagnosis of CL in goats has not yet been described. Similarly, the rCP40 protein has also been patented (BR 102015014074-6 A2 SERINE PROTEASE RECOMBINANT 40KDa FROM CORYNEBACTERIUM PSEUDOTUBERCULOSIS). However, the cited patent describes the use of the CP40 protein as a vaccine antigen, therefore, its use in association with the rCP01850 protein in immunodiagnostics has not yet been described. Petition 870250021465, dated 03 / 19 / 2025, page 19 / 29 / 10

[0014] Thus, unlike what has already been reported in the state of the art, we developed an indirect ELISA methodology using the combination of recombinant antigens rC01850 and rCP40 for the diagnosis of CL in goats. The inventive activity is evident, since it is not possible to infer that a diagnosis applied to one species will be equally applicable to another, requiring rigorous experimental work to standardize (different reagent concentrations), validate (test different serum samples and perform repetitions to assess reproducibility) and apply the developed methodology specifically for goats. The industrial application is clear, considering the possibility of continuous and standardized production of reagents by the industry, in addition to the interest of goat breeders in an accurate diagnosis that helps them in the sanitary management of their herds. Brief description of the drawings

[0015] Figure 1 - Checkerboard titration method in a 96-well plate (1st step), in columns 1 to 12, different concentrations of the proteins rCP01850 and rCP40 (1:1 ratio) were tested, grouping three columns and using dilutions of 0.25, 0.5, 1 and 2 μg / mL. And in rows A to H, grouping two rows, goat serum at concentrations of 1:400, 1:200, 1:100 and 1:50.

[0016] Figure 2 - Checkerboard titration method in a 96-well plate (2nd step). Once the concentration of the rCP01850 and rCP40 proteins (1:1 ratio) was established, fixed at 2 μg / mL, different dilutions of goat serum were tested in columns 1 to 12, separated into groups of 3, with concentrations of 1:400, 1:200, 1:100, and 1:50. Following the standard blank (Br, without serum, only diluent), positive (+) and negative (-) serum for each concentration. And in rows A to H, separated into groups of two rows, the different concentrations of the conjugated antibody were tested: 1:40,000, 1:20,000, 1:10,000, and 1:5,000.

[0017] Figure 3 - ROC curve of the indirect ELISA result using the rCP01850 protein. The Y-axis represents the sensitivity scale, and the X-axis represents the 1-minus scale for the specificity of the test.

[0018] Figure 4 - Distribution of absorbance results of the tested sera in relation to the test cutoff point. Negative sera are shown in red, and positive sera are shown in blue. Petition 870250021465, dated 03 / 19 / 2025, p. 20 / 29 / 10 positive sera. On the left, the DO scale, measured at a wavelength of 492 nanometers. Description of the invention

[0019] The present invention describes a diagnostic enzyme-linked immunosorbent assay based on the recombinant acid phosphatase rCP01850 (SEQ ID No. 1) and the endoglucosidase CP40 (SEQ ID No. 2) from C. pseudotuberculosis, with the aim of detecting anti-C. pseudotuberculosis antibodies in goats in order to diagnose CL.

[0020] Initially, in a 96-well polystyrene plate, the sensitization step is performed using a mix of rCP01850 and rCP40 proteins in a 1:1 ratio, at a concentration of 2.0 μg diluted in carbonate-bicarbonate buffer (pH 9.6). 100 μl of the diluted mix is ​​used in each well, and the plate is incubated at 4°C for 16 hours. Then, a PBS-T solution (1x PBS, pH 7.2 and 0.05% Tween 20) is used to wash the plate, repeated 3 times. Subsequently, a blocking step is performed, where 200 μl of a PBS-T solution with 5% skimmed milk powder is used in each well and incubated for 2 hours at 37°C, followed by 3 washes with PBS-T. Immediately afterwards, 50 μl of goat serum diluted in PBS-T at a ratio of 1:50 is added, and the mixture is incubated at 37°C for 1 hour, followed by 3 washes of the plate with PBS-T.In the subsequent step, 50 μl of peroxidase-conjugated goat anti-IgG antibody at a 1:5000 dilution is added, followed by incubation of the plate for 1 hour at 37°C, with the plate being washed 3 times. Finally, the development step is performed where 100 μl of the development solution (10 ml of citrate-phosphate buffer, pH 4.0; 0.03g of orthophenylenediamine; and 50 μl of H2O2) is added and incubated in a dark environment for 15 minutes. The reaction should be stopped using a stopping solution (25 μL / well of 5% sulfuric acid). Finally, the plate is read in a spectrophotometer, verifying the absorbance at a wavelength of 492 nm.

[0021] EXAMPLE 1 - Checkerboard Titration Method for standardization of indirect ELISA using recombinant proteins rCP01850 and rCP40 Petition 870250021465, dated 03 / 19 / 2025, page 21 / 29 / 10

[0022] To standardize the concentrations to be used in the serological diagnostic steps, the checkerboard titration method was used, which consists of testing different dilutions of recombinant proteins with different serum dilutions, keeping the concentration of the conjugated antibody constant, in order to define the best concentration of antigens to be used. For this standardization step, a pool of 3 proven positive sera (from animals with clinical CL) and another pool of 3 proven negative sera (from animals from non-endemic areas for CL, Southern Region of Brazil) were used as positive and negative samples at different dilutions. A mix of rCP01850 and rCP40 proteins (1:1 ratio) was used, tested at concentrations of 0.25, 0.5, 1, and 2 μg / mL, diluted in carbonate-bicarbonate buffer, and serum was used at concentrations of 1:400 and 1:200.1:100 and 1:50 in PBS-T + 1% skimmed milk powder. The different protein concentrations were distributed in the columns of the 96-well plate, while the serum concentrations were distributed in the rows, according to Figure 1. The concentration of the conjugated antibody remained fixed at 1:10,000 in this step.

[0023] Once the optimal protein concentration was defined, it was fixed at 2μg / mL and the combination of different concentrations of goat serum was tested, using the same concentrations mentioned above, along with different concentrations of the conjugated antibody, which was tested at concentrations of 1:40,000, 1:20,000, 1:10,000 and 1:5,000, as shown in Figure 2. The columns showed the different serum concentrations, while the rows showed the different concentrations of the conjugated antibody.

[0024] Thus, based on the experiments previously conducted, the present invention will be described below, considering that our indirect ELISA diagnostic method uses the steps and reagent concentrations mentioned below. 96-well, flat-bottomed plates were used, sensitized with rCP01850 and rCP40 diluted in carbonate-bicarbonate buffer (pH 9.6) in a 1:1 ratio and a concentration of 2 μg / mL, with incubation for 16 hours at 4 °C. This was followed by three washes with PBS-T (PBS 1x pH 7.2, plus Tween 20 at 0.05%), and blocking of the plate using 200 μL of PBS-T + 5% skim milk, followed by incubation at Petition 870250021465, dated 03 / 19 / 2025, page 22 / 29 / 10 37°C for another 2 hours. The plate was washed three times with PBS-T, and then 50 μL of goat serum at a 1:50 dilution (in PBS-T) was added and incubated again at 37°C for 1 hour. Then the plate was washed three times with PBS-T, and 50 μL of peroxidase-labeled anti-goat IgG antibody (Sigma-Aldrich) at a 1:5,000 dilution was applied and incubated at 37°C for 45 minutes. The plate was washed five more times with PBS-T, and the developing solution (10 mL of citrate-phosphate buffer pH 4.0, 0.01 g of OPD and 50 μL of H2O2) was added. The plate was left in the dark for 15 min, and then the reaction was stopped with 25 μL / well of 5% sulfuric acid, and the optical density of the wells was measured using a plate reader at a length of 492 nanometers. EXAMPLE 2 - Definition of Cutoff Point, Sensitivity, Specificity, and Accuracy of the indirect ELISA test

[0025] For validation of the diagnostic test, 80 goat blood serum samples were used, collected by jugular vein puncture, where 40 were characterized as positive for CL, taken from animals that presented hypertrophied lymph nodes, with microbiological characterization of C. pseudotuberculosis performed, and 40 characterized as negative, from animals up to 1 year of age, without visible signs of CL, and originating from properties with a history of adequate disease control. An indirect ELISA was also performed using antigens secreted from the 1002 strain of C. pseudotuberculosis to confirm the seronegative status of the disease.

[0026] After performing the test, absorbance was measured and the results were expressed as mean ± standard deviation. This was then analyzed using SPSS software (v. 23), where an ROC curve was constructed using IBM SPSS® (Statistical Package for Social Sciences) to demonstrate the overall performance of the test and establish a cutoff point, considering all sensitivity and specificity values ​​for each variable in the test. The cutoff point that generated maximum specificity and sensitivity values ​​was 0.254. Using this cutoff point, a sensitivity value of 90% and a specificity value of 85% were obtained. The area under the curve was 0.90, reflecting an overall accuracy of 90% for the indirect ELISA diagnosis with rCP01850 and rCP40 in goats. Petition 870250021465, dated 03 / 19 / 2025, page 23 / 29 / 10 Bibliographic References

[0027] ANDERSON, M.; NAIRN, M. E. Control of caseous lymphadenitis in goats by vaccination. Colloques de l'INRA, [S.l.], vol. 28, pp. 605-609, 1984.

[0028] ANDRADE, J. S. L. et al. Occurrence and risk factors associated with Corynebacterium pseudotuberculosis infection in goats and sheep from the semiarid region of Paraíba. Pesquisa Veterinária Brasileira, Soropédica - RJ, vol. 32, pp. 116-120, 2012.

[0029] AL-GAABARY, M. H.; OSMAN, S. A.; OREIBY, A. F. Caseous lymphadenitis in sheep and goats: Clinical, epidemiological and preventive studies. Small Ruminant Research, vol. 87, no. 1-3, pp. 116-121, Nov. 2009. Available at: < https: / / www.sciencedirect.com / science / article / pii / S092144880 <9002156>.

[0030] AWAD, F.J. Serological investigation of Corynebacterium pseudotuberculosis in sheep. I. Agglutination test. Am. J. Vet. Res. [S.I], vol. 81, pp. 251-253, 1960.

[0031] AZEVEDO, VAC et al. Method and immunodiagnostic kit for caseous lymphadenitis in small ruminants using recombinant proteins CP40 and PLD as antigens. Owner: Federal University of Minas Gerais (BR / MG); São Paulo State University “Júlio Mesquita Filho” (BR / SP); Federal University of Pelotas (BR / RS); Federal University of Bahia (BR / BA). BR 102018011672-0 A2. Filing date: June 8, 2018.

[0032] BARNABÉ, et al. Assessment of caseous lymphadenitis in goats in a slaughterhouse in the Brazilian semi-arid region and estimates of economic losses due to carcass condemnation, Semina: Ciências Agrárias, v. 41, no. 6, p. 2655-2668, Nov. / Dec. 2020.

[0033] BARRAL, TD et al. A panel of recombinant proteins for the serodiagnosis of caseous lymphadenitis in goats and sheep. Microb Biotechnol. 2019 Nov;12(6):13131323. doi: 10.1111 / 1751-7915.13454. Epub 2019 Jul 9. PMID: 31287241; PMCID: PMC6801148. Petition 870250021465, dated 03 / 19 / 2025, page 24 / 29 / 10

[0034] BORSUK, S. et al. Recombinate acid phosphatase rCP01850 from Corynebacterium pseudotuberculosis and its use in recombinant subunit vaccines for caseous lymphadenitis. Owner: Federal University of Pelotas (BR / RS). BR 102016016891-0 A2. Filing date: 07 / 21 / 2016.

[0035] BORSUK, S. et al. Recombinant serine protease 40kDa from Corynebacterium pseudotuberculosis. Owner: Tiradentes University - Institute of Technology and Research (BR / SE). ) BR 102015014074-6 A8. Filing date: 06 / 15 / 2015.

[0036] BURREL, DH Caseous lymphadenitis in goats. Aust. Vet. J., [Sl], n. 57, p. 105-110, 1981.

[0037] CORRÊA, JI et al. Expression of six possible virulence factors of C. pseudotuberculosis in murine macrophage. PUBVET, v. 12, no. 12, 2017. Available at: https: / / www.cabdirect.org / cabdirect / abstract / 20193055066.

[0038] HOELZLE, L. E. et al. Differences in the antigen structures of Corynebacterium pseudotuberculosis and the induced humoral immune response in sheep and goats. Veterinary Microbiology, v. 164, n. 3-4, p. 359-365, jun. 2013.

[0039] REZENDE, A. DE F. S. et al. In sílico identification of Corynebacterium pseudotuberculosis antigenic targets and application in immunodiagnosis. Journal of Medical Microbiology, v. 65, n. 6, p. 521-529, 1 jun. 2016.

[0040] SANTOS, A. R. et al. The Corynebacterium pseudotuberculosis in silico predicted pan exoproteome. BMC Genomics, v. 13, n. S5, out. 2012.

[0041] SILVA, M. T. DE O. et al. The combination of Corynebacterium pseudotuberculosis recombinant proteins rPLD, rCP01850 and rCP09720 for improved detection of caseous lymphadenitis in sheep by ELISA. Journal of Medical Microbiology, v. 68, n. 12, p. 1759-1765, 1 dez. 2019.

[0042] WINDSOR, PA. Control of Caseous Lymphadenitis. Veterinary Clinics of North America: Food Animal Practice, v. 27, n. 1, p. 193-202, Mar. 2011. Available at: <https: / / pubmed.ncbi.nlm.nih.gov / 21215903 / >. Petition 870250021465, dated 03 / 19 / 2025, pages 25 / 29

Claims

CLAIMS 1. METHOD FOR DIAGNOSING CASEOUS LYMPHADENITIS IN GOATS, characterized by comprising an immunoassay based on the indirect ELISA methodology using recombinant proteins CP01850, as per SEQ ID NO 1, and CP40, as per SEQ ID NO 2, used in an equimolar ratio of 1:1 and at a total concentration of 2.0 μg / mL in the sensitization step of the solid phase, presenting a cutoff point corresponding to an absorbance value of 0.254 for the detection of specific antibodies against caseous lymphadenitis in goats.

2. METHOD FOR DIAGNOSING CASEOUS LYMPHADENITIS IN GOATS, according to claim 1, characterized by using a biological sample comprising serum or plasma from goats.

3. METHOD FOR DIAGNOSING CASEOUS LYMPHADENITIS IN GOATS, according to claims 1 and 2, characterized by comprising the use of a secondary anti-caprine IgG antibody conjugated to an enzymatic marker.

4. METHOD FOR DIAGNOSING CASEOUS LYMPHADENITIS IN GOATS, according to claims 1, 2 and 3, characterized by comprising a development step using a chromogenic system for detecting the immune complex formed in the immunoassay.

5. METHOD FOR DIAGNOSING CASEOUS LYMPHADENITIS IN GOATS, according to claims 1 to 4, characterized by being performed on a solid support for antigenic immobilization and immunological detection. Petition 870260077288, dated 04 / 08 / 2026, page 5 / 7

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