System, device, kit, and method for identifying salivary biomarkers of gastric ulcer detection in equine subject

Abstract

Biomarkers associated with the disease state equine gastric ulcer syndrome (EGUS), in an equine animal are provided. Methods for using the biomarkers to diagnose gastric ulcer syndrome in a non-invasive test along with an article for detecting gastric ulcer in an equine animal are disclosed.

Description

Attorney Docket No.: 76980-00004SYSTEM, DEVICE, KIT, AND METHOD FOR IDENTIFYING SALIVARY BIOMARKERS OF GASTRIC ULCER DETECTION IN EQUINE SUBJECTCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 718,379, filed on November 8, 2024, which is incorporated herein by reference in its entirety.FIELD

[0002] The disclosure relates to proteomics and animal analytical medicine, particularly identifying an animal as being in a gastric ulcer state.BACKGROUND

[0003] Equine gastric ulcer syndrome (EGUS) is a worldwide disease in equids. It is considered the most common stomach disease in horse clinical practice and associated with colic, decreased appetite, failure to thrive, and poor performance. This syndrome is a disease having clinical signs that are rather unspecific; the most common signs that can be observed are associated with colic, decreased appetite, loss of weight, yawning, bruxism, anorexia, salivation, abdominal discomfort, failure to thrive, and poor performance (Lester et al, 2008; Padalino et al, 2019; Shawaf et al., 2020). Equine gastric ulcer syndrome has been recommended as an encompassing term for all erosive and ulcerative conditions of the equine stomach including diseases of the squamous (ESGD) and glandular (EGGD) mucosa (Sykes et al, 2015).

[0004] For proper diagnosis, gastroscopy is the only reliable ante mortem method used nowadays in horses and is considered the gold standard against which all other possible diagnostic tests should be compared (Niedzwiedz et al, 2013; Hewetson et al, 2017). However, this diagnostic technique has several limitations in daily practice, such as the high cost, the need for experience to develop the technique correctly, or that not all veterinarians have access to gastroscopy. The gastroscopy is expensive. It is not readily available to most veterinarians and it requires a minimum level of expertise to perform and interpret. Moreover, there is little evidence to support the notion that lesion grade (as assessed visually) correlates with clinical signs (Sykes et al, 2015).SUMMARY

[0005] The present disclosure provides or identifies a set of biomarkers associated with a disease state, such as equine gastric ulcer syndrome (EGUS), in a subject, such as an equineAttorney Docket No.: 76980-00004 animal, and methods for discovering or using the biomarkers to characterize the pathological condition and to facilitate treatment of the subject. The present disclosure also provides a related article, such as a diagnosis strip or paper, for detecting gastric ulcer in an equine animal, and a kit comprising such an article. The present disclosure also provides a device, a computer implemented system, and software, which are configured to implement the method as described herein. In example embodiments, the device includes a user-friendly saliva swab that is simple and painless to utilize in a stable or clinical setting. Together, the device, the computer implemented system, and the software provide a comprehensive view of the health status of the animal, allowing for convenient evaluation without using invasive techniques. As such, continual monitoring of the animal, as it relates to gastric health, is possible and rapid resolution of an issue is enabled.

[0006] In one aspect, the present disclosure provides a method of treatment. Such a method comprises steps as described herein. In some embodiments, a biofluid sample is obtained from a subject suspected of having a gastric ulcer state. The subject is an equine animal. The biofluid sample is analyzed to obtain a testing data set comprising protein information, which includes concentrations of a set of biomarkers comprising IL-1F5, carbonic anhydrase VI (CA VI), prolactin inducible protein (PIP), albumin, and serotransferrin. The testing data set is compared to a comparative data set to determine whether the subject is in a gastric ulcer state based on a set of criteria. The comparative data set comprises pre-determined ranges of IL-1F5, CA VI, PIP, albumin, and serotransferrin when the animals of the same type are in a healthy state. In certain embodiments, a treatment can be prescribed and / or administered to the subject when the subject is identified to be in a gastric ulcer state depending, at least in part, on the identified state.

[0007] The subject may include, without limitation, a horse, a donkey, or a zebra. The biofluid sample may be a saliva or a serum sample. In some example embodiments, the biofluid sample is a saliva sample. The saliva sample is collected using a sponge coupled to a rod. The sponge is positioned within the mouth of the subject and directed purposefully on the bars of the mouth and along the gums and front lip pouch of the subject where saliva typically collects for a pre-determined time, and then removed from within the mouth of the subject and placed into a vial.Attorney Docket No.: 76980-00004

[0008] In the set of criteria, one or more of the biomarkers including IL-1F5, CA VI, PIP, albumin, and serotransferrin have concentrations outside the predetermined ranges in the comparative data set. In some example embodiments, in the set of criteria, one or more of biomarkers including IL-1F5, CA VI, PIP, albumin, and serotransferrin have concentrations higher, for example, 1- to 100-fold higher, than corresponding upper limits in the predetermined ranges in the comparative data set. Both the testing data set and the comparative data set include information of additional biomarkers, which are selected from the proteins or the biomarkers, as described herein.

[0009] In another aspect, the present disclosure provides an article for detecting equine gastric ulcer. The article includes a sample receiving zone and a membrane comprising a testing zone. The sample receiving zone is configured to receive a biofluid sample from a subject suspected of having a gastric ulcer state. In example embodiments, the subject is an equine animal. The testing zone includes at least one antibody to detect a presence and a relative concentration of at least one respective biomarker selected from the group consisting of IL-1F5, CA VI, PIP, albumin, and serotransferrin compared to a corresponding reference indicating a healthy state. The membrane is configured to provide an observable positive result to indicate the presence of the relative concentration of the at least one respective biomarker so as to indicate that the subject is in the gastric ulcer state. In some example embodiments, the article is a diagnosis strip or paper. In some example embodiments, the observable positive result is provided in a line or a dot, which has a larger size and / or a higher darkness than that of the corresponding reference.

[0010] In another aspect, the present disclosure provides a kit. In example embodiments, the kit includes a sponge attached with a pole and the article, as described herein.

[0011] In another aspect, the present disclosure also provides a device that includes the article, as described herein.

[0012] In another aspect, the present disclosure provides a method. An example method includes providing or obtaining a biofluid sample from a subject suspected of having a gastric ulcer state, wherein the subject is an equine animal, analyzing the biofluid sample to obtain a testing data set including protein information, wherein the protein information includes concentrations of a set of biomarkers including IL-1F5, CA VI, PIP, albumin, and serotransferrin, comparing the testing data set with a comparative data set to determine whetherAttorney Docket No.: 76980-00004 the subject is in a gastric ulcer state based on a set of criteria, wherein the comparative data set includes pre-determined ranges of IL-1F5, CA VI, PIP, albumin, and serotransferrin in a healthy state, and providing an instruction to administer a treatment to the subject when the subject is identified to be in a gastric ulcer state.

[0013] In another aspect, the present disclosure provides a device configured to implement the method as described herein.

[0014] In another aspect, the present disclosure provides a computer implemented system including one or more processors, and at least one tangible, non-transitory machine readable medium encoded with one or more programs, to be executed by the one or more processors, to perform steps of the method as described herein.

[0015] In another aspect, the present disclosure provides one tangible, non-transitory machine readable medium encoded with one or more programs, to be executed by one or more processors, to perform steps of the method as described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The present disclosure is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not necessarily to scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Like reference numerals denote like features throughout specification and drawings.

[0017] FIGS, la-lf show concentrations of salivary analytes studied in healthy and ulcer gastric horses. A box plot shows median (central horizontal line within the plot), 25th and 75th percentiles (non-central horizontal lines within the plot), maxima and minima (edges of the plot) and statistical significance (*=p<0.05; ***=p<0.001);

[0018] FIGS. 2a-2d show concentrations of serum blood analytes studied in healthy and ulcer gastric horses. A box plot shows median (central horizontal line within the plot), 25th and 75th percentiles (non-central horizontal lines within the plot), maxima and minima (edges of the plot) and statistical significance (*=p<0.05; **=p<0.01);

[0019] FIGS. 3 a and 3b show representative images of the salivary protein pattern (2- DE) on samples from healthy horses (a) and horses with gastric ulcer (b). Coloured circle spots show statistically significant increases in abundance between the two health status conditionsAttorney Docket No.: 76980-00004(red = ulcer; green = healthy). More information about spot regulation and protein identification is described in Tables 5 and 6;

[0020] FIGS. 4a and 4b show representative images of the serum protein pattern (2-DE) on samples from healthy horses (a) and horses with gastric ulcer (b). Colored circle spots show statistically significant increases in abundance between the two health status conditions (red = ulcer; green = healthy). More information about spot regulation and protein identification is described in Tables 7 and 8;

[0021] FIGS. 5a and 5b show western blot images of proteins in saliva. Representation of the protein bands of each protein validated by western blotting, with indication of its molecular weight. The images indicate: a) increased proteins in the group of horses with gastric ulcer; and b) validated proteins increased in the group of healthy horses. Gels 1 and 2 are distributed in a “Healthy Horses” group between wells 2 and 4, and a “Horses with Gastric Ulcer” group between wells 5 and 9;

[0022] FIGS. 6a and 6b show western blot images of proteins in serum. Representation of the protein bands of each protein validated by western blotting, with indication of its molecular weight. The images indicate: a) increased proteins in the group of horses with gastric ulcer; and b) proteins increased in the group of healthy horses. Gels 1 and 2 are distributed in a “Healthy Horses” group between wells 2 and 4, and a “Horses with gastric ulcer” group, between wells 5 and 9;

[0023] FIG. 7 shows western blot images in saliva. Representation of the protein bands of each serum protein validated by western blotting in saliva, with indication of its molecular weight. Gels 1 and 2 are distributed in a “Healthy Horses” group between wells 2 and 4, and a “Horses with gastric ulcer” group, between wells 5 and 9;

[0024] FIGS. 8al-8a9 and 8bl-8b3 show quantification of levels of band area from the WB analysis according to approach 1 of potential biomarkers in saliva, specifically IL1-F5, IL1- F6, PIP, FABP5, CA-VI, albumin, Apo-A4, a-1- antitrypsin, serotransferrin (a) and serum Apo- A4, a-1- antitrypsin, serotransferrin (b);

[0025] FIGS. 9a-9i show quantification of levels of band area from the WB analysis according to approach 2 of potential biomarkers in saliva, specifically, IL1-F5, IL1-F6, PIP, FABP5, CA-VI, albumin, Apo-A4, a-1- antitrypsin, serotransferrin;Attorney Docket No.: 76980-00004

[0026] FIGS. lOa-l Oe show concentrations of IL-1F5, PIP, CA VI, serotransferrin and albumin in healthy clean horses (n = 8) and in horses with experimentally induced gastric ulcer (n = 7);

[0027] FIGS. 1 la-1 le show concentrations of IL-1F5, PIP, CA VI, serotransferrin and albumin in clinically healthy horses (n = 20) and in horses with gastric ulcer in field conditions (n = 37);

[0028] FIG. 12 is a flow chart illustrating an exemplary method of analyzing a biofluid sample to provide instructions on administering a treatment to a subject identified to be in a gastric ulcer state in accordance with example embodiments;

[0029] FIG. 13 is a block diagram of an exemplary system in accordance with example embodiments;

[0030] FIG. 14 is a block diagram of an exemplary system of sample collection in accordance with example embodiments;

[0031] FIG. 15 shows schematically a YES / NO model to differentiate YES from NO animals with a sensitivity and a specificity of at least 85% in accordance with example embodiments;

[0032] FIGS. 16a-16e show concentrations of salivary analytes studied (IL-1F5, PIP, CA VI, serotransferrin and albumin) in clean horses (n = 20) and horses with gastric ulceration (n = 37) in accordance with example embodiments;

[0033] FIG. 17 shows a radar cart of all variables measured in saliva of clean horses and horses with gastric ulceration in accordance with example embodiments;

[0034] FIGS. 18a-18e show concentrations of salivary analytes studied (IL-1F5, PIP, CA VI, serotransferrin and albumin) in subclinical horses (n = 11) and horses with gastric ulceration (n = 37) in accordance with example embodiments;

[0035] FIG. 19 shows a radar cart of all variables measured in saliva of subclinical horses (n = 11) and horses with gastric ulceration (n = 37) in accordance with example embodiments;

[0036] FIGS. 20a-20e show concentrations of salivary analytes studied (IL-1F5, PIP, CA VI, serotransferrin and albumin) in clean horses (n = 20) and subclinical horses (n = 11);

[0037] FIG. 21 shows a radar cart of all variables measured in saliva of clean horses, and subclinical horses in accordance with example embodiments;Attorney Docket No.: 76980-00004

[0038] FIG. 22 shows a radar cart of all variables measured in saliva of clean horses, subclinical horses, and horses with gastric ulceration in accordance with example; and

[0039] FIGS. 23 and 24 show an example saliva collection apparatus or device in accordance with example embodiments.DETAILED DESCRIPTION

[0040] For purposes of the description hereinafter, it is to be understood that the embodiments described below may assume alternative variations and embodiments. It is also to be understood that the specific articles, compositions, and / or processes described herein are exemplary and should not be considered as limiting.

[0041] In the present disclosure the singular forms “a,” “an,” and “the” include the plural reference, and reference to a particular numerical value includes at least that particular value, unless the context clearly indicates otherwise. Thus, for example, a reference to “a nano structure” is a reference to one or more of such structures and equivalents thereof known to those skilled in the art, and so forth. When values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. As used herein, “about X” (where X is a numerical value) preferably refers to ±1% of the recited value, inclusive. For example, the phrase “about 8” preferably refers to a value of 7.2 to 8.8, inclusive; as another example, the phrase “about 8%” preferably (but not always) refers to a value of 7.2% to 8.8%, inclusive. Where present, all ranges are inclusive and combinable. For example, when a range of “1 to 5” is recited, the recited range should be construed as including ranges “1 to 4”, “1 to 3”, “1-2”, “1-2 & 4-5”, “1-3 & 5”, “2-5”, and the like. In addition, when a list of alternatives is positively provided, such listing can be interpreted to mean that any of the alternatives may be excluded, e.g., by a negative limitation in the claims. For example, when a range of “1 to 5” is recited, the recited range may be construed as including situations whereby any of 1, 2, 3, 4, or 5 are negatively excluded; thus, a recitation of “1 to 5” may be construed as “1 and 3-5, but not 2”, or simply “wherein 2 is not included.” It is intended that any component, element, attribute, or step that is positively recited herein may be explicitly excluded in the claims, whether such components, elements, attributes, or steps are listed as alternatives or whether they are recited in isolation.

[0042] The present disclosure provides a set of biomarkers associated with a disease state, such as equine gastric ulcer syndrome (EGUS), in a subject, such as an equine animal, andAttorney Docket No.: 76980-00004 methods for discovering or using the biomarkers. The present disclosure also provides a related method of treatment, an article, such a diagnosis strip or paper, for detecting a gastric ulcer in an equine animal, and a kit including the article. The present disclosure also provides a device, a computer implemented system, and a software, each configured to implement the example methods described herein.

[0043] In accordance with some example embodiments, a method of treatment is provided. The method includes one or more of the steps described herein. In some example embodiments, a biofluid sample is obtained from a subject suspected of having a gastric ulcer state. The subject is an equine animal, for example. The biofluid sample is analyzed to obtain a testing data set comprising protein information, which includes concentrations of a set of biomarkers comprising one or more biomarkers selected from IL-1F5, carbonic anhydrase VI (CA VI), albumin, and serotransferrin. Additional biomarkers as described herein may be included.

[0044] The testing data set is compared to a comparative data set to determine whether the subject is in a gastric ulcer state based on a set of criteria. The comparative data set comprises pre-determined ranges of IL-1F5, carbonic anhydrase VI (CA VI), prolactin inducible protein (PIP), albumin, and serotransferrin when the animals of the same type are in a healthy state. A treatment is prescribed and / or administered to the subject when the subject is identified to be in a gastric ulcer state. The treatment of gastric ulcers in horses may be dependent on the severity and / or the type of ulcerations. Treatment may include the use of pharmaceuticals, dietary changes, environment changes and / or supplementation with non-prescription oral products, with prescription acid suppressive therapies the most prevalent. These include proton pump inhibitors, such as omeprazole.

[0045] In example embodiments, the subject is a horse, a donkey, an ass, or a zebra, or any member of the genus Equus. The biofluid sample may be a saliva or a serum sample. In some embodiments, the biofluid sample is a saliva sample. The saliva sample is collected using a sponge attached with a pole. The sponge is applied into the mouth of the subject and chewed by the subject for a predetermined time, and then taken out and placed into a vial.

[0046] In the set of criteria, one or more of the biomarkers including IL-1F5, CA VI, PIP, albumin, and serotransferrin have concentrations outside the predetermined ranges in the comparative data set. In some embodiments, in the set of criteria, one or more of biomarkersAttorney Docket No.: 76980-00004 including IL-1F5, CA VI, PIP, albumin, and serotransferrin have concentrations higher, for example, 1-fold to 100-fold higher, than corresponding upper limits in the predetermined ranges in the comparative data set.

[0047] In some example embodiments, both the testing data set and the comparative data set include information of additional biomarkers, which are selected from the proteins or the biomarkers as described herein.

[0048] In another aspect, the present disclosure provides an article for detecting equine gastric ulcer. The article includes a sample receiving zone and a membrane including a testing zone. The sample receiving zone is configured to receive a biofluid sample from a subject suspected of having a gastric ulcer state. In example embodiments, the subject is an equine animal. The testing zone includes at least one antibody to detect presence and relative concentrations of at least one respective biomarker selected from the group consisting of IL-1F5, CA VI, PIP, albumin, and serotransferrin compared to a corresponding reference indicating a healthy state. The membrane is configured to provide an observable positive result to indicate a presence of a relative concentration of the at least one respective biomarker to indicate that the subject is in the gastric ulcer state. In some example embodiments, the article is a diagnosis strip or paper. In some example embodiments, the observable positive result is provided in a line or a dot, which has a larger size and / or a higher darkness than that of the corresponding reference.

[0049] In another aspect, the present disclosure provides a kit including a sponge attached with a pole and the article, as described herein. In another aspect, the present disclosure also provides a device including the article, as described herein.

[0050] In another aspect, the present disclosure provides a method that includes providing a biofluid sample from a subject suspected of having a gastric ulcer state, wherein the subject is an equine animal, analyzing the biofluid sample to obtain a testing data set comprising protein information, wherein the protein information includes concentrations of a set of biomarkers comprising IL-1F5, CA VI, PIP, albumin, and serotransferrin; comparing the testing data set with a comparative data set to determine whether the subject is in a gastric ulcer state based on a set of criteria, wherein the comparative data set comprises pre-determined ranges of IL-1F5, CA VI, PIP, albumin, and serotransferrin in a healthy state, and providing instruction to administer a treatment to the subject when the subject is identified to be in a gastric ulcer state.Attorney Docket No.: 76980-00004In another aspect, the present disclosure provides a device configured to implement the method, as described herein.Examples

[0051] 1. Material and Methods

[0052] 1.1. Animals and Experimental Procedures

[0053] All procedures involving animals were approved by the Institutional Animal Care and Use Committee of Clemson University (AUP #2020-013).

[0054] Eight mature horses, with ages ranging from 10 to 20 years, housing in Clemson, United States, in 10 x 12 ft stalls during the day and turning out into an 8-acre pasture at night, were used in a gastric ulcer study. To sum up, the animals were fed 1.5% body weight (BW) in hay and 0.25% BW in concentrates daily and were weighed weekly and the fed amounts were adjusted accordingly, and horses had ad libitum water access. Gastric ulcers were induced by the scientific team alternating five 12-hour periods of feed privations and normal feeding routines as well as two 24-hour feed deprivations for a total of 108 hours of feed deprivation. To determine the extent of gastric ulcer presence in the stomach, horses were submitted to endoscopy after ulcer induction. Prior to these examinations, horses were fasted and water was withheld for a minimum of 3 hours before the endoscopy to ensure visibility during the procedure. All horses under ulcer condition show gastric lesion scores above 3 in a 0-4 scale (Sykes and Jokisalo, 2014).

[0055] 1.2. Sample Collection

[0056] Samples of saliva and blood were collected before the induction of gastric ulcer and prior to endoscopic procedure after induction. Though horses were in a fasted state, the mouth was rinsed of any remaining feedstuffs as a precautionary measure to avoid contamination. Saliva collection was performed by insertion of a 6.0 x 10'4m2compressed sponge (Arcliber® cellulose sponge) through the bars of each horse’s mouth to be chewed until saturated with saliva. The procedure lasted approximately 90 seconds. Then, sponges were placed into salivette tubes (Salivette Tubes, Fisher Scientific®) and were stored in a cooler until transported to the laboratory and centrifuged at 3200 rpm for 15 minutes. Blood samples were collected by jugular venepuncture into Vacutainer® rapid serum test (RST) tubes via a double- ended bleeding needle (BD Vacutainer® Rapid Serum Tubes, Mississauga, ON). Blood samplesAttorney Docket No.: 76980-00004 were stored in a cooler until arrival to the laboratory to be centrifuged at 1300 rpm for 15 minutes. All saliva and serum samples were aliquoted and stored at -80°C.

[0057] 1.3. Health Status Assessment

[0058] Both groups of animals were analytically characterized to obtain a general overview of the health status of the animals in each condition. The analysis included the quantification of immune parameters (adenosine deaminase (ADA), IgG and total protein content (TP)), psychological markers (cortisol, a- amylase and IgA) and total antioxidant capacity (TAC).

[0059] The levels of ADA were measured in both saliva and serum samples using a microplate adaptation of a human commercial colorimetric assay (Biosystems, SA) after optimization for its use in horse specimens. The method of the assay is based on the measurement of the decrease in absorbance (OD) per minute of a coupled reaction initially catalysed by ADA. The reaction is measured at 340 nm. The levels of ADA activity were calculated according to the manufacturer’s instructions (AOD / min x 3333 = U / L). The optimal sample dilutions were 1 :8 for saliva and 1 :4 for serum samples.

[0060] For the measurement of IgG in serum samples, a dilution of 1 :200000 was required according to the manufacturer’s instructions of the commercial ELISA assay used (IgG Horse ELISA kit, Abnova).

[0061] All the psychological markers were quantified in saliva samples using commercial assays. The cortisol was measured using an optimized competitive ELISA (Extended range high sensitivity salivary cortisol Enzyme immunoassay kit, Salimetrics, USA). No dilution of horse saliva was applied under the optimal conditions of the assay. Cortisol levels were calculated according to the manufacturer’s instructions (3 pg / dL = 82.77 nmol / L). Salivary alpha-amylase was quantified using a kinetic enzyme assay (Salivary alpha-amylase kinetic enzyme assay kit, Salimetrics, USA), without any saliva dilution. The levels of alpha-amylase activity were calculated according to the manufacturer’s instructions (AOD / min = U / L) under the optimal conditions for horse saliva measurements (15 minutes of incubation time within the two absorbance readings). Finally, the amount of salivary IgA was measured using an optimized ELISA (IgA Horse ELISA kit, Abnova) with a saliva dilution of 1 : 5000.

[0062] The total TAC (total antioxidant capacity) was measured in the saliva and serum samples by the ferric reducing antioxidant power assay (Benzi et al, 1996), which was optimizedAttorney Docket No.: 76980-00004 for the two-horse body-fluids. The method consisted in the reduction of the ferric complex tripiridil triazine (Fe3+TPTZ) to form Fe2+in an acidic medium. Prior to its implementation, the optimal sample dilution was calculated using a standard curve of Trolox (a water-soluble analog of vitamin E employed as a control antioxidant agent for assay calibration), ranging from 1.5 to 100 pM Trolox equivalents. No sample dilution was required for any body fluid.

[0063] Total protein content (TP) in saliva and serum was determined according to the standard protocol (Bradford, 1976). Saliva and serum samples were diluted (1 :20 and 1 :1000, respectively) for their proper quantification using a curve with a 5-100 ng / ml concentration range of bovine serum albumin (Sigma Aldrich, Darmstandt, Germany).

[0064] 1.4. Protein Identification by Gel-Based Method followed MS

[0065] Two-dimensional Gel Electrophoresis (2DE)

[0066] After total protein quantifications (Bradford, 1976) of saliva and serum samples, three pools of each body fluid were performed per condition (Ulcer and Health).

[0067] Each pool consisted of 30 micrograms (pg) of total protein for the saliva samples and 300 pg of total protein for serum samples. Each pool was composed of a mix of 3 samples randomly selected from animals within each condition. A total of 20 pg of total protein from each serum and saliva pool was subjected to 2DE. In brief, proteins were initially separated by isoelectric point using immobilized pH gradient (IPG) strips with 11cm long nonlinear gradients pH 3-11 (GE Healthcare Life Sciences, Munich, Germany) in an isoelectric focusing unit (Ettan™ IPGphor™ 3, GE Healthcare Life Sciences, Munich, Germany). Afterward, the IPG strips were equilibrated with 2% DTT solution, followed by 2.5% of iodoacetamide solution, and subjected to SDS-PAGE on homemade 10-15% poly-acrylamide gradient gels of 140 x 140 x 1.5mm in a vertical chamber (SE600 Chroma, Hoefer, INC., Holliston, USA). Gels were silver- stained for protein visualization and scanned in an image scanner (AI600 RGB GEL Imaging System, GE Healthcare Life Sciences. Uppsala, Sweden). Images were evaluated for spot detection and matching using specific software (ImageMaster 2D Platinum 7.0, GE Healthcare Life Sciences, Uppsala, Sweden). The relative percentage of spot volumes from each group of animals within each body fluid was statistically compared using a t-test.

[0068] MS Analyses

[0069] For MS analyses, spots of interest were cut from the submitted gel, and after washing and destaining proteins fixed in the gel were reduced with dithiothreitol and alkylatedAttorney Docket No.: 76980-00004 with iodoacetamide (Jimenez et al, 2001). In-gel digestion was performed with trypsin (Trypsin Gold, Mass Spectrometry Grade, Promega, Madison, WI) with a final trypsin concentration of 20 ng / pl in 50 mM aqueous ammonium bicarbonate and 5 mM CaCh. Digest proceeded for 8 hours at 37°C (Shevchenko et al, 1996). Afterward, peptides were extracted with three changes of 50 pL of 5% trifluoro acidic acid in 50% aqueous acetonitrile supported by ultrasonication for 10 min per change. Extracted peptides were dried down in a vacuum concentrator (Eppendorf, Hamburg, Germany). Dried peptides were re-suspended in 0.1% TFA for LC-MS / MS analysis. Samples were dissolved in 8 pl and 6 pl were injected depending on the protein concentration visible on the gels. Peptides were separated on a nano-HPLC Ultimate 3000 RSLC system (Dionex). Sample pre-concentration and desalting was accomplished with a 5 mm Acclaim PepMap p-Precolumn (300 pm inner diameter, 5 pm particle size, and 100 A pore size) (Dionex). For sample loading and desalting 2% ACN in ultra-pure H2O with 0.05% TFA was used as a mobile phase with a flow rate of 5 pl / min. Separation of peptides was performed on a 25 cm Acclaim PepMap C18 column (75 pm inner diameter, 2 pm particle size, and 100 A pore size) with a flow rate of 300 nl / min. The gradient started with 4% B (80% ACN with 0.08% formic acid) for 7 min, increased to 31% in 30 min and to 44% in additional 5 min. It was followed by a washing step with 95% B. Mobile Phase A consisted of ultra-pure H2O with 0.1% formic acid. For mass spectrometric analysis the LC was directly coupled to a high-resolution Q Exactive HF Orbitrap mass spectrometer. MS full scans were performed in the ultrahigh-field Orbitrap mass analyzer in ranges m / z 350-2000 with a resolution of 60 000, the maximum injection time (MIT) was 50 ms and the automatic gain control (AGC) was set to 3eA6. The top 10 intense ions were subjected to Orbitrap for further fragmentation via high energy collision dissociation (HCD) activation over a mass range between m / z 200 and 2000 at a resolution of 15 000 with the intensity threshold at 4eA3. Ions with charge state +1, +7, +8 and >+8 were excluded. Normalized collision energy (NCE) was set at 28. For each scan, the AGC was set at 5eA4 and the MIT was 50 ms. Dynamic exclusion of precursor ion masses over a time window of 30s was used to suppress repeated peak fragmentation.

[0070] 1.5. Validation of Potential Biomarkers of Gastric Ulcer

[0071] Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

[0072] Individual saliva and serum samples were prepared for electrophoresis by protein denaturation with DTT, 10% SDS, Tris and 0.02% bromophenol blue.Attorney Docket No.: 76980-00004

[0073] Saliva and serum samples were analysed separately. 2.5 pg of total protein of each sample were electrophoretically separated by SDS-PAGE on small-size home-made mini gels using a vertical electrophoresis chamber (Mini-PROTEAN Tetra Vertical Electrophoresis Cell, Bio-Rad, Hercules, CA, USA). A 12% polyacrylamide resolving gel and a 5% stacking gel was used to separate the proteins. In each electrophoresis, 16 samples were run in parallel in two mini gels. Each gel consists of 4 samples from “healthy horses” and 4 samples from “ulcer horses”. The electrophoresis ran at 180V for 45 min.

[0074] Once the electrophoresis was finished, the proteins were transferred to PDVF membranes using the Trans-Blot TurboTransfer System (Bio-Rad, Hercules, CA, USA) for 20 minutes at 20mV. After protein transference, the membranes were blocked with blocking solution (5% non-fat dried milk in PBS solution) overnight at 4°C.

[0075] Protein Quantification by Western Blot

[0076] From the previous step, from 19 salivary proteins and 5 serum protein candidates of gastric ulcer biomarkers, only 13 were analysed based on the availability of commercial antibodies for proper analysis as detailed below. Specifically, in saliva, the analysis was performed in seven of the twelve up-regulated in the group of horses with gastric ulcer and in two of the seven up-regulated proteins in the group of healthy horses. In serum, the analysis includes three out of four proteins identified as up-regulated in horses with gastric ulcer and the protein identified as up-regulated in the group of healthy animals.

[0077] To develop the validation study, the first step was to obtain commercially available antibodies for proper semi-quantification by western blotting. Because only one antibody was found specifically designed against the equine protein, specifically the one against serotransferrin protein, the alignment between the horse and human protein sequences was performed for the rest of the protein candidates. The alignments between the human and the equine proteins according to Uniprot should be seen in Table 1.Table 1. Percentage of Alignments between Equine and Human Protein Sequences according to UniprotAttorney Docket No.: 76980-00004

[0078] Once primary antibodies against the different protein candidates and the correspondent secondary antibodies were selected (Table 2), western blotting procedures were used for validation. In brief, blocked membranes were washed three times, 5 minutes incubation each, with Tris saline buffer with 0.1% of tween 20 ph = 7.5 (TBST). After washing, membranes were incubated with the corresponding primary antibody in Tris buffer saline with 1% milk (TBS 1%) for one and a half hours. An initial concentration of 5 pg / mb of primary antibody was tested and, when necessary, according to the results obtained, an individual optimization was performed. Prior to the incubation of the membranes with the corresponding secondary antibody, a new wash step was performed with TBST three times as before. Finally, they were washed twice with TBST, and once with TBS prior to the addition of substrate (Pierce™ ECL Plus Western Blotting Substrate, ThermoFisher Scientific, Carlsbad, USA) for the development of band’s chemiluminescence. Band signal was detected in an imager LAS 600 (GE Healthcare, Uppsala, Sweden) and quantified using an image analysis software (ImageQuant TL v2005, Amersham Biosciences Europe GmbH, Freiburg, Germany) to obtain the relative percentage in area of the different bands in the gel.Table 2. List of Primary and Secondary Antibodies used for Validation of Biomarkers of Gastric Ulcer by Western Blotting in Saliva and Serum SamplesAttorney Docket No.: 76980-00004

[0079] 1.6. Statistical Analysis

[0080] All statistical procedures were performed with statistical software (Graph Pad Prism 10.2). Data were checked for a normal distribution with the Saphiro-Wilk test.

[0081] For parameters with normal distribution, a t-test was used, while for those with a non-normal distribution a non-parametric Man-Whitney statistical test was used. The parametersAttorney Docket No.: 76980-00004 normally distributed were ADA, in serum and saliva, TP, cortisol and TgA in saliva, and TAC in serum. Non-normally distributed parameters were a- amylase and TAC in saliva samples and TP and IgG in serum.

[0082] Correlations between the different biomarkers in serum and saliva samples were evaluated using Pearson or non-parametric Spearman correlation coefficients according to the data distribution since no normality distribution of all data was observed.

[0083] The criterion for statistical significance was established at P < 0.05.

[0084] Two different approaches were developed for evaluating salivary area band differences between healthy horses and gastric ulcer horses. The first approach (approach 1) used salivary band area per mg of protein, considering 2.5 pg of protein. The second approach (approach 2) used salivary band area per ml of saliva because the protein concentrations between the two conditions are very different. In serum, however, this second approach is not necessary because the total protein level is similar in both conditions, so only approach 1 was used, serum bad area per mg of protein.

[0085] The biomarker validation study in horses with experimentally induced gastric ulceration calculated the size effect of the proposed biomarkers, with statistical differences significant, and calculated the minimum sample size to be used in the future for the measurement of biomarker concentrations in individual samples. For this purpose, the software G*Power 3.1.9.7 was used.

[0086] 2. Results

[0087] 2.1. Health Status Assessment

[0088] Descriptive statistical measures for all markers studied in each condition in saliva and serum samples are shown in Table 3.Attorney Docket No.: 76980-00004Table 3. Descriptive Statistics of Marker Values in Serum and Saliva Samples of 8 horses before (Healthy) and after Gastric Ulcer Induction (Ulcer)

[0089] There were statistically significant differences in saliva concentrations of adenosine deaminase (ADA) (FIG. la), total protein content (TP) (FIG. lb), also total antioxidant capacity (TAC) (FIG. If) between the healthy and ulcer group. The mean levels of salivary ADA in animals with gastric ulcer were about three times higher than in healthy animals and in the case of TP almost twice as high as in healthy animals. Statistically significant differences in the mean salivary TAC levels were found between animals with gastric ulcer and healthy animals, with concentrations three times higher in the ulcer condition. The psychological status markers analysed in saliva did not show statistically significant differences in any of the three parameters (FIGS. lc-le).Attorney Docket No.: 76980-00004

[0090] Serum ADA analysis showed statistically significant differences between the two groups of animals (302.7U / L in healthy vs. 236.0 U / L in ulcer animals) as shown in FIG. 2a. This was not the case for serum TP and IgG, where no statistically significant differences were observed (see FIGS. 2b-2c). As seen in saliva, serum TAC concentrations in ulcerated animals were statistically higher than in the healthy group (20.44 pM / L vs 14.56 pM / L trolox equivalents, respectively; FIG. 2d).

[0091] The overall correlation coefficients between the different salivary and serum biomarkers could be observed in Table 4. The highest correlation coefficients were observed between salivary cortisol and serum TAC levels (r = -0.64), salivary TAC and serum IgG concentrations (r = 0.60) and salivary TP and serum IgG concentrations (r = 0.60). However, only the pair salivary cortisol-serum TAC has statistical significance.Table 4. Correlation Coefficients between Salivary (ADA, TP, cortisol, amylase, IgA and TAC) and Serum (ADA, TP, IgG and TAC) Biomarkers Studied for Health Status Assessment in 8 Horses before and after Gastric Ulcer Induction* Represent statistically significant correlation coefficients (p < 0.05).

[0092] 2.2. Protein Identification

[0093] Salivary Proteomic Analysis

[0094] The salivary protein patterns in healthy horses and in horses with gastric ulcer displayed several visual differences (FIGS. 3a-3b). Among the 142 matched spot sets evaluated, 28 showed statistically significant differences with 16 spots significantly up-regulated in horses suffering from gastric ulcer in comparison to healthy condition and 12 spots up-regulated inAttorney Docket No.: 76980-00004 healthy horses (Table 5). The increases in spot abundance range from 1 .19 to 7.35 (zero values of the medians have been excluded).Table 5. Saliva Spots Differentially Regulated in Healthy Horses Compared to Horses Suffering from Gastric Ulcer (by p value)Attorney Docket No.: 76980-00004

[0095] The MS analysis revealed a total of 16 unique protein identities (Table 6). 12 proteins were up-regulated in horses with gastric ulcer, while 7 proteins were identified as up- regulated in healthy animals. According to uniprot database, the proteins up-regulated in ulcer condition are involved in important biological functions such as immune response and inflammatory response (IL-36A, IL-36RA, S100-A9, Cytokeratin-1, immunoglobulin a, PIP, Parotid secretory protein), redox processes (thioredoxin), regulation of steroid hormones (secretoglobin), protein transport and defense response (ZG 16B), lipid metabolism process (FABP5).Table 6. MS Identification of Spots using a Q Exactive HF Orbitrap Mass Spectrometer and Data Base NCBI_horse_tx9796_210421.fastaAttorney Docket No.: 76980-00004*Spot number correspondence at FIG. 3 and Table 5.

[0096] Serum Proteomic Analysis

[0097] Serum protein patterns showed significant differences (FIGS. 4a-4b). Of the total 125 matched sets analysed, 6 showed statistically significant differences (Table 7) with 5 spots up-regulated in horses with gastric ulcer and 1 spot up-regulated in healthy condition. TheAttorney Docket No.: 76980-00004 variations in spot quantity range from 1 .45 to 1 .96-fold change. MS analysis identified a total of 5 unique protein identities involved in the spot change with 4 unique up-regulated proteins in horses with gastric and only a single up-regulated protein in healthy animals (Table 8). The identified upregulated proteins in ulcer condition are mainly involved in 3 biological functions: lipid regulation (AP0A4), immune response (Alpha- 1 -antitrypsin, clusterin and immunoglobulin), regulation of endopeptidase activity (Alpha- 1 -antitrypsin) according to uniprot database.Table 7. Serum Spots Differentially Up-Regulated in Horses Suffering from Gastric Ulcer Compared to Healthy Condition (by p value)*Spot correspondence at FIGS. 4a-4b and Table 8.Table 8. MS Identification of Spots using a Q Exactive HF Orbitrap Mass Spectrometer and Data Base NCBI_horse_tx9796_210421.fastaAttorney Docket No.: 76980-00004*Spot number correspondence at FIGS. 4a-4b.

[0098] 2.3. Validation of Potential Biomarkers of Gastric Ulcer

[0099] Western Blot

[0100] The band signal detection for saliva and serum samples was obtained for 9 out of 13 candidate biomarkers evaluated (FIGS. 5a-5b, 6a-6b, and 7).

[0101] In saliva samples (FIGS. 5a-5b), a band signal was detected for four out of seven proteins that showed up-regulation in the group of horses with gastric ulcer: IL1-F5, IL1-F6, PIP, and FABP5 (FIG. 5a). Specifically, neither thioredoxin, nor zymogen-16B, nor S100-A9 showed a signal. Moreover, the two protein candidates that appeared up-regulated in the group of healthy horses showed specific band signal: CA-VI and Albumin (FIG. 5b).

[0102] In serum samples (FIGS. 6a-6b) of the three proteins validated in the serum of the group of horses with gastric ulcer only clusterin showed no signal, whereas Apo-A4 and alpha- 1- antitrypsin showed a signal band (FIG. 6a). The serotransferrin, up-regulated in the group of healthy animals, showed a signal (FIG. 6b).

[0103] Moreover, the serum candidates of biomarkers were also validated in the saliva samples. Apo-A4, Alpha- 1 -antitrypsin and serotransferrin showed signal as shown in FIG. 7. Apo-A4 was assessed with a colorimetric technique, as the intensity of the chemiluminescence signal band was very high, and the accuracy of the analysis needed to be checked.

[0104] Validation AnalysisAttorney Docket No.: 76980-00004

[0105] Once the statistical analysis was finished, an outlier was found, and it was decided not to use it in the analysis, so the statistic shows the results based on n = 7 for each condition (healthy and ulcer).

[0106] In approach 1, the analysis take into account the variation in the band protein area normalized per mg of total protein content in the sample (Table 9 and FIG. 8), serotransferrin showed statistically significant differences in saliva and serum from healthy horses, while statistical differences in IL1-F5, FABP5, and Apo-A4 were observed in saliva from animals with gastric ulcer. Only a-1- antitrypsin showed statistically higher in serum from ulcer condition.

[0107] On the other hand, in approach 2, the analysis takes into account the variation in the band protein area normalized per mb of saliva because in this body fluid the total protein content differs between conditions (healthy and ulcered horses) (Table 10 and FIG. 9), all potential biomarkers studied showed statistically significant differences in saliva from horses with gastric ulcer, especially relevant were increases in albumin, PIP, CA-VI, and Apo-A4.

[0108] The effect size was calculated in all biomarkers with statistical relevance, in both approaches (Tables 9 and 10), whose saliva values were within the range of 1.38-3.25 in approach 1, and 2.07-5.1 in approach 2. There is an exception in the latter approach, IL-1F6 had an effect size of 0.2.Table 9. Descriptive Statistics for Each Band Protein Area in Saliva and Serum Samples of 7Horses at Health and Ulcer Condition according to Approach 1Attorney Docket No.: 76980-00004Table 10. Descriptive Statistics for Each Band Protein Area in Saliva and of 7 Horses at Health and Ulcer Condition according to Approach 2Attorney Docket No.: 76980-00004

[0109] 2.4. Quantification of biomarkers in saliva samples using immunoassays.

[0110] The selection of biomarkers for proper quantification was performed based on two added inclusion criteria: biomarkers with “easy” commercially available analytical solutions + the “novelty” concept (the selected biomarkers have not been related to gastric ulceration before). From the eight biomarkers validated in the western blotting experiment that fulfilled the inclusion criteria, five were finally selected, as shown in Table 11. Those biomarkers were initially quantified in the saliva of the horses used for the proteomics analysis, defined as the “experimental trial” in advance, as could be considered as a double validation because statistically significant differences were observed for all the salivary biomarkers quantified (FIG. 10)(for more information see Section 4 EXEMPLARY BIOMARKERS & APPLICATIONS below).Table 11. List of Proteins Subjected to Validation as Gastric Ulcer Biomarkers.Difference in WBProtein Analytical Solution Found Selected in ExperimentalTrialIL1-F5 Human ELISA YESIL1-F6 No available (human ELISA failed) NoFABP5 No available (human ELISA failed) NoPIP Human ELISA YESELISA in-house developed usingAlbumincommercial horse-specific antibodiesCA VI Human ELISA***Attorney Docket No.: 76980-00004„ j, . ELISA in-house developed using >Serotransfemn . . .0,. YES ** commercial horse-specitic antibodiesAlpha- 1 -antitrypsin No available (human ELISA failed) No **. .. . . . No available (human antibodies ***Apolipoprotein A4 faj]ec[ No ***

[0111] Development of a YES / NO Answer Analytical Tool

[0112] Concepts to Consider for the Development of an Analytical Tool

[0113] The definition of the groups of animals: YES and NO includes the following inclusion criteria. YES: Horses with clinical signs of gastric ulcer & positive gastroscopy scope; and NO: Horses with no clinical signs of gastric ulcer & negative gastroscopy scope.

[0114] Minimal sample size required: In this example, a minimal number of YES and NO animals is calculated according to statistical tools using a pilot study. For this calculation, the data from our experimental trial showed that the number of animals in each group should be 26. From the 26 YES and the 26 NO horses, all the analytes from each sample should be analyzed to obtain proper statistical power and construct a model for discrimination.

[0115] Characteristics of the Analytical Tool: the analytical tool solves to define an answer of the YES / NO model that should differentiate YES animals from NO animals with a sensitivity and a specificity of at least 85%. The answer will be obtained by quantifying a panel of biomarkers and including the results in an statistical model that differentiate animals with gastric ulceration from clean animals.

[0116] 2.5. A first attempt to develop a Salivary Analytical Tool for Differentiation ofYES and NO Horses using the original definitions of YES and NO.

[0117] The samples used for statistical analysis include (a) YES: 37 horses with clinical signs of disease and positive gastroscopy scope; all analytes were measured in a total of 24 horses, and (b) NO: 20 clean horses without clinical signs of disease and negative gastroscopy scope; all analytes were measured in a total of 15 horses.

[0118] We analyzed individually all the YES and NO saliva samples and calculated the statistically significant differences, as shown in FIG. 16. All analytes showed modifications.

[0119] Afterwards, the highest pairs sensitivity and specificity were calculated from those analytes that showed differences between groups in order to obtain an individual approach of each analyte to differentiate between YES and NO horses, as shown in Table 12.Table 12. ROC Individual Analysis for Each Biomarker of Gastric Ulcer Detection in HorsesAttorney Docket No.: 76980-00004

[0120] We need to perform a statistical model in which the behavior of all the biomarkers will be analyzed to obtain an algorithm that differentiate between YES and NO and with this model we could calculate the sensitivity and specificity of the model. As a first approach we see that the behavior of all the biomarkers show a tendency of lower values of all analytes in NO (Clean horses) vs. YES (Ulcer horses) (FIG. 17).

[0121] 2.6. The development of a salivary analytical tool for de differentiation of YES and NO using an alternative definition.

[0122] The alternative definition includes YES: 37 horses with clinical signs of disease and positive gastroscopy scope. All analytes were measured in a total of 24 horses. NO SUBCLINICAL: 11 animals without clinical signs of disease and positive gastroscopy scope. We have analyzed individually all the YES and NO saliva samples from this new definition and calculate the statistically significant differences (FIG. 18). Only three analytes showed modifications, specifically IL1-F5, CA VI and albumin.

[0123] The highest pairs sensitivity and specificity were calculated from those analytes that showed differences between groups in order to obtain an individual approach of each analyte to differentiate between YES and NO horses (Table 13).Attorney Docket No.: 76980-00004Table 13. ROC Individual Analysis for Each Biomarker of Gastric Ulcer Detection in Horses

[0124] Some biomarkers do not change in two biomarkers, specifically, PIP and serotransferrin, while the other biomarkers show a tendency of lower values in NO (Sub clinic horses) vs. YES (Ulcer horses) (IL1-F5, CA VI and albumin) (FIG. 19).

[0125] 2.7. The development of a salivary analytical tool for de differentiation of NO and NO using an alternative definition.

[0126] The alternative definitions include NOc (Clean): Horses without clinical signs of gastric ulcer and negative gastroscopy scope; and NOs (Subclinic): Horses with no clinical signs of gastric ulcer and positive gastroscopy scope. We analyzed individually all NOc and NOs saliva samples using this alternative definition and calculated the statistically significant differences as shown in FIG. 20. Two analytes showed modifications, specifically PIP and serotransferrin.

[0127] Afterwards, the highest pairs sensitivity and specificity were calculated from those analytes that showed differences between groups in order to obtain an individual approach of each analyte to differentiate between NOc and NOs horses, as shown in Table 14.Table 14. ROC Individual Analysis for Each Biomarker of Gastric Ulcer Detection in HorsesAttorney Docket No.: 76980-00004

[0128] We note that some biomarkers do not change, specifically IL1-F5, CA VI and albumin, while other biomarkers show a tendency of lower values in NOc (Clean horses) vs. NOs (Subclinic horses)(PIP and serotransferrin), as shown in FIG. 21.

[0129] A summary including the analytical modifications observed in the three groups of horses is shown in Table 15 below and the visual tendency of the changes could be seen in FIG. 22.Table 15. Analytical Modifications Observed in the Horse Groups

[0130] 3. Discussion

[0131] Researchers have been trying for years to find methods of rapid and reliable diagnosis of such common and relevant diseases as gastric ulcer, whose final diagnosis requires endoscopy. Furthermore, obtaining biomarkers in non-invasive fluids is a goal that has been pursued over time, especially important is saliva, because it is economical, easy to collect and addresses animal welfare. A correct evaluation of proteins that are differentially expressed during gastric ulcer in biological fluids would offer valuable biological information as well as facilitate the diagnosis of disease using protein biomarkers.

[0132] This study applied a gel-based proteomic approach for the identification of new biomarkers of gastric ulcer in horses in serum and in saliva samples followed by the validation of potential biomarkers. To that end, a group of 8 healthy horses were used for gastric ulcer induction, and the protein profile before and after the induction were compared in both body fluids. Afterwards the quantification of a total of 5 identified and validated biomarkers were quantified in field conditions to obtain a first diagnostic power.Attorney Docket No.: 76980-00004

[0133] Prior to proteomics analysis, the overall immune, psychological, and oxidative status of the horses before (healthy condition) and after ulcer induction (ulcer condition) was established using a panel of salivary and serum parameters. An inflammatory condition was detected in horses with gastric ulcer since the levels of ADA activity in the saliva of horses with gastric ulcer were increased in comparison to the healthy condition. This behavior in the levels of salivary ADA activity agrees with previous reports in horses with acute abdominal disease (Contreras-Aguilar et al., 2020), and also in pigs (Gutierrez et al., 2017), and dairy cows (Castillo et al., 2023) with inflammatory diseases.

[0134] In addition to the inflammatory condition, a possible dysregulation in the antioxidant-oxidant balance was detected in horses with gastric ulcer in the inventors’ study by an increase in the antioxidant capacity level. The observed increase in antioxidants could be a first response to reduce a high level of oxidants to defend the organism (Kirschvink et al, 2007). Similar TAC increases have been observed in diseased pigs (Sanchez et al, 2021) which was also correlated to the levels of an acute phase protein haptoglobin. The implication of oxidative stress in gastric ulceration in horses have been also documented before (Contreras Aguilar et al., 2022). On the other hand, an influence of phycological stress on the induced gastric ulcer was discarded according to the salivary stress panel biomarkers analysed.

[0135] The comparison between 2-D protein patterns from healthy and gastric ulcer status showed several protein spots that were differentially expressed between groups and could be considered as potential biomarkers of gastric ulcer. A total of 15 salivary protein identities were differentially regulated in saliva of horses with gastric ulcer in comparison to healthy condition (IL36 receptor antagonist protein, IL36 alpha, Thioredoxin, Zymogen 16 homolog B, Prolactin-inducible protein, Parotid precursor protein, Immunoglobulin alpha constant heavy chain, Secretoglobin, Fatty acid-binding protein, Keratin type II and cytoskeletal 1, Carbonic anhydrase 6, Albumin, S100-A9 isoform X2, Polymeric immunoglobulin receptor precursor, Keratin 5 ). Furthermore, 5 serum proteins showed statistically significant differences between groups (Serotransferrin, Apolipoprotein, Immunoglobulin gamma 5, Clusterin, Alpha-1- anti trypsin).

[0136] Most of the proteins identified as candidates for gastric ulcer markers were involved in inflammatory processes such as interleukins but also negative acute phase proteins such as serotransferrin or albumin. However, other proteins have a role in the lipid metabolismAttorney Docket No.: 76980-00004(FABP5 and Apo-A4) or redox reactions (Thioredoxin). 13 of the 20 proteins identified in saliva and serum as potential biomarkers of gastric ulcer in horses were validated by western blotting. One of the limitations of this procedure is that specific antibodies are needed for each protein. Four of the candidate proteins analysed were identified in serum samples, Apolipoprotein A4, Alpha 1 antitrypsin, Clusterin, and Serotransferrin, and were evaluated by western blotting in both body fluids, serum, and saliva while the other nine biomarkers were identified in saliva samples and validated exclusively in saliva (IL36 receptor antagonist protein, IL36 alpha, Prolactin-inducible protein, Fatty acid-binding protein 5, Carbonic anhydrase 6, Albumin, Thioredoxin, S100A9 and Zymogen granule protein 16 homolog B)Due to the protein salivary concentration changes between health and disease conditions, it is considered that the second approach may be more accurate for interpreting the obtained results. Finally, 9 proteins were validated as biomarkers of gastric ulcer in horses: alpha 1 antitrypsin, apolipoprotein A4, serotransferrin, IL36 receptor antagonist protein, IL36 alpha, Carbonic anhydrase 6, Albumin, Prolactin-inducible protein and Fatty acid-binding protein 5.

[0137] Alpha 1 antitrypsin (AAT) is a glycoprotein also related to the inflammatory process. It is very resistant to proteolysis in the gastrointestinal tract, is not metabolised by the microbes in the intestine, it is excreted intact in faeces where it can be measured in humans, similar observations have been made in cats and dogs but there aren't reports on the fecal measurement of AAT in farm animals (Celi et al, 2019). Furthermore, AAT is a well-established biomarker of intestinal permeability in humans (Kosek et al, 2013), characterised by protein loss. In horses, al -antitrypsin was validated as a biomarker of gastric ulcer in foal serum (Taharaguchi et al., 2007) and proposed a protective role against proteolytic activities in the damaged tissues. In that study, the high level of ATT in diseased horses could be related to the inflammatory condition and even to protein loss due to gastric tissue damage. Our increase in the level of serum ATT in animals with gastric ulceration was in concordance with the latest report.

[0138] Apolipoprotein A4 (Apo-A4) is an anti-inflammatory molecule with antioxidant activity and a key role in lipid metabolism (Steelman et al, 2012). The tissue sources and physiological functions of Apo-A4 have not yet been explored in horses, nor its role in saliva. However, it has been reported a protective gastric action by the inhibition of gastric acid secretion in rats (Okumura et al., 1995). No statistically significant differences were observed in the serum analyzed, but a trend to reduced values in horses with gastric ulcer was observed thatAttorney Docket No.: 76980-00004 could be related to a more susceptibility to gastric ulcer. However, in saliva, increased levels of Apo-A4 were observed in our horses with gastric ulcer condition (5.7 times higher in ulcer than in healthy condition), so the quantification of this protein in saliva should be further studied to understand its connection to the gastric disease.

[0139] Serotransferrin is a negative acute phase protein (Heinrich et al, 1990). According to the literature, it is an iron transportation protein that may be associated with ulcerative conditions when haemorrhage occurs, resulting in a reduction of its serum levels (Poltep et al, 2018). This theory could be also application to our results in serum samples because we found lower values of serotransferrin in horses with gastric ulcer than in the healthy condition, which it could be coherent with blood loss. However, similar to the results of Apo-A4, the salivary results were the opposite because an up-regulation was observed in animals with gastric ulcer that should be explained in further studies. In human medicine, its presence in saliva had been considered a reliable marker of blood contamination, as it was understood not to originate from the salivary glands. However, in subsequent studies, its presence has been associated with the salivary gland, and increased levels of transferrin have been associated with head and neck tumours (by association with the metabolism of growing cells), changes in mastication (by increased gingival blood vessel transudation) and oral microorganisms (whose mechanism is still unknown) (Kang et al., 2019). The role that this protein has in the saliva of horses with gastric ulcer is still to be defined, however, the use of serotransferrin as a biomarker of this disease in horses should be in-depth explored in both serum and saliva body fluids.

[0140] IL-36A or IL-1-F6 is a proinflammatory cytokine implicated in inflammatory and immune responses (De Siqueiral & Fernandes, 2016). While normal IL-1 signalling helps maintain tissue homeostasis by promoting wound healing and tissue repair, aberrantly elevated signalling has been associated with diverse inflammatory diseases (Queen et al., 2019). IL-1F5 or IL-36RN has been shown negatively to regulate TLR / IL-1 signalling pathway through the antagonism of IL-1R related proteins 2, hence inhibiting the activation of NF-KB (Chustz et al., 2011). As shown in Table 10 and FIG. 9, both IL1-F5 and IL1-F6 showed statistically higher values in the diseased group, 60 times higher than in healthy animals in the case of IL1-F5 and almost 5 times higher in IL1-F6, which calls for further study of these biomarkers as a diagnostic tool for gastric ulcer.Attorney Docket No.: 76980-00004

[0141] CA-VT may participate in the regulation of salivary pH and buffer capacity and protects the mouth and upper alimentary canal against excess acidity (Nishita et al, 2014). The observed presence of CA-VI in the airway surface liquid indicated that it protects mucous not only in the gastrointestinal tract, but also in the respiratory tract as a pH neutralizer. Moreover, the presence of CA VI in stomach, small and large intestine indicate that that CA VI is secreted in upper and lower alimentary tract of horses (Ochiai et al., 2009). Therefore, depending on how CA-VI acts at the level of buffering the pH and protecting from damage by excess acid, we could find its levels decreased or increased in horses with gastric ulcer. In the protein identification step, at first, CA-VI was up-regulated in health condition, which could be consistent with an extra protection and buffering against the disease. However, at the validation moment, the increase was four times higher in the ulcer gastric group than in healthy horses, so it could be an attempt to buffer excess stomach acidity derived from gastric ulcer. Further studies on this protein and its practical application in the diagnosis of EGUS are needed.

[0142] Albumin is a negative acute phase protein well studied in animals by other authors (Fischer et al, 1998; Messana et al, 2008; Cray et al, 2009; Ceciliani et al, 2014; Assuncao et al, 2019; Poltep et al, 2018), especially in horse serum or plasma. There are studies that suggest the use of serum albumin as a biomarker of animal welfare in pigs (Fuentes- Rubio et al, 2014). In human medicine, its presence in saliva, not being a normal component of the salivary gland, is thought to be the result of contamination by exudates resulting from gingival inflammation or other infections related to the destruction of the oral mucosal integrity (Kang et al, 2019). Although a study of the albumin pattern in horse tears has been done (Terhaar et al, 2009), in saliva it is still unknown. In the study described herein, although saliva protein identification was found to be increased in healthy animals, subsequent validation showed a statistically significant increase in saliva protein levels in horses with gastric ulcer, which could be a potential biomarker of the disease. It would be necessary in-depth study. Its high level could be a consequence of damage to gastric tissue, even oral mucosa.

[0143] PIP is a glycoprotein biosynthesized and secreted by apocrine cell types that produce milk, seminal fluid, tears, and saliva (Hassan et al, 2009). The exact biological functions are still uncertain, but it is highly expressed in breast cancer tumours (Hassan et al, 2009; Baniwal et al, 2013) and is a protein known to play a major role in immunoregulation, fertility, antimicrobial activity, apoptosis, and tumour progression (Hassan et al, 2009(Ngounou et al,Attorney Docket No.: 76980-000042015). The higher levels observed in this study in horses with gastric ulcer led us to continue with further studies to understand its role as a potential biomarker.

[0144] FABP5 is a protein involved in the regulation of sensory perception of pain (tool from https: / / www.uniprot.org / ), has a role in the coordination of lipid response in cells, and has been considered as a promising marker of tissue injury (Ishimura et al, 2013). The link between FABP and lipid metabolism is based on the close association with the metabolic and inflammatory processes by modulating lipid pathways in adipocytes and macrophages (Celi et al, 2019). Salivary FABP5 has been previously proposed as a biomarker of equine squamous gastric disease in horses in a clinical study (Munoz-Prieto et al., 2022), but it was not validated. The present study supports the use of FABP5 as biomarker of gastric ulcer and showed increases of in comparison to healthy animals.

[0145] Four proteins, clusterine, S100-A9, zymogen-16B, and thioredoxin, are also possible biomarkers of gastric ulcer.

[0146] In brief, the proteins validated as up-regulated in the gastric ulcer group were related to the immune response (CA-VI, albumin, serotransferrin, IL1-F5, IL1F6) and inflammatory processes (PIP, FABP5, Apo-A4, AAT) mainly, although some of them have more specific functions, as explained above.

[0147] Therefore, these biomarkers validated in serum and saliva could be used in the diagnosis of equine gastric ulcer. Their quantification and the development of their diagnostic power has been performed in 5 out of the 9 biomarkers validated and could be seen in Section 4.

[0148] Overall, the analysis of the serum and salivary identified and validated proteins from healthy horses and horses with gastric ulcer reflected a largely inflammatory condition in which antioxidant defences were involved.

[0149] 4. EXEMPLARY BIOMARKERS & APPLICATIONS

[0150] Five proteins are proposed as biomarkers of gastric ulcer in horses: IL-1F5, Carbonic anhydrase VI (CA VI), Prolactin inducible protein (PIP), albumin, and serotransferrin. None of these proposed biomarkers have been defined as biomarkers of gastric ulcer in saliva samples before.

[0151] As described above, the results support the use of at least these five biomarkers in saliva samples for gastric ulcer of horses. Further analysis and summary of the results are described below.Attorney Docket No.: 76980-00004

[0152] 4.1 . Diagnostic Power Analysis of Proposed Biomarkers under ControlledExperimental ConditionsThe inventors have identified, by two-dimensional gel electrophoresis, and validated afterwards, by western blotting in horses under experimental ulcer induction, a total of 9 proteins as biomarkers of gastric ulcer. Moreover, the inventors have developed and / or optimized immunoassays for the proper quantification of 5 out of 9 biomarkers in saliva samples. Specifically, the validated and quantified proteins were IL-1F5, CA VI, PIP, albumin and serotransferrin. The biomarker validation study performed by western blotting in the experimental trial, induced gastric ulceration horses showed a high size effect, higher than 2, from all the 5 proposed biomarkers. The minimal sample size to be used for the quantification of the concentrations of biomarkers in individual saliva samples was calculated using the GPower statistical tool (Table 16).Table 16. Size Effect of Validation Results performed by Western Blotting in Induced Gastric Ulceration Experiment in 8 HorsesBiomarkers Size Effect Total Minimal Sample SizeIL-1F5 2.91 10PIP 5.1 6CA VI 3.07 10Serotransferrin 2.11 14Albumin 3.37 8

[0153] The immunoassays for the quantification of the concentration of biomarkers in saliva samples were optimized from commercially available human assays or in-house developed, when horse-specific antibodies were commercially available. For that end, the commercially available human assays optimized were against IL-1F5 (Invitrogen), CA VI (Cusabio) and PIP (abeam). For the measurements of albumin and serotransferrin, in-house sandwich enzyme-linked immunosorbent assays (ELISA) were developed using commercially available antibodies (Bethyl).

[0154] Statistically significant differences are noted between the concentrations in healthy horses and horses with gastric ulcer for the 5 biomarkers in the experimental trial (FIG.Attorney Docket No.: 76980-0000410). The prediction power to detect gastric ulcer for the 5 biomarkers was higher than 0.8 for all the biomarkers (Table 17).Table 17. Diagnostic Power Analysis of Proposed Biomarkers of Gastric Ulcer under Experimental Conditions (n = 15; 8 healthy clean and 7 ulcer), SE = sensitivity, SP = specificity, AUC = area under the curveBiomarkers Cut-off value Se Sp AUC Power (required n)IL1-F5 (ng / mL) 3.281 87.5% 85.7% 0.928 2.42 (n= 6)PIP (ng / mL) 2.25 87.5% 100% 0.946 1.04 (n = 25)CA VI (pg / mL) 159.9 85.7% 100% 0.964 2.41 (n = 6)Serotransferrin (pg / mL) 3.802 75% 85.7% 0.857 1.06 (n = 25)Albumin (pg / mL) 51.40 87.5% 62.5% 0.797 1.21 (n = 19)

[0155] 4.2. Diagnostic Power Analysis of Proposed Biomarkers under ClinicalConditions.

[0156] A total of 37 horses with clinical signs compatible with gastric ulcer and a positive confirmation by endoscopy were sampled at USA in a veterinary hospital that received animals with signs of disease for diagnosis. Moreover, 20 clinically healthy horses from a local Horse’s competition Centre (Centro Ecuestre Oliva Nova, Valencia; https: / / metoliva.com) were sampled in Spain to have the possibility to compare the levels of biomarkers in both health conditions while waiting for a new USA sampling procedure. Statistically significant differences were observed for IL-1F5, CA VI, serotransferrin and albumin but not for PIP (FIG. 11). The diagnostic power analysis is reported in Table 18.Table 18. Diagnostic Power Analysis of Proposed Biomarkers of Gastric Ulcer under Clinical Conditions (n = 57) *required number of animals / group, with an error of 0.05 and a power of analysis of 90%Biomarkers Cut-Off Value Se Sp AUC n n Size Effect (%) (%) Healthy Ulcer (required n*)IL1-F5 (ng / mL) 2.429 69.2 60 0.74 15 26 0.88 (28)CA VI (pg / mL) 189 80 73.3 0.83 15 30 0.65 (52)Attorney Docket No.: 76980-00004Serotransferrin 5.815 83.3 80 0.90 20 36 1.98 (7)(pg / mL)Albumin (pg / mL) 189.8 78.8 75 0.87 20 33 1.33 (13)

[0157] Next step was to analyze the diagnostic power of each biomarker in a bigger number of animals including a group of healthy clean horses (clinically healthy animals plus negative to gastric ulcer by endoscopy). For that end a total of 20 healthy clean animals were obtained in two extra sampling procedures in USA. When compared to the diseased horses suffering from gastric ulcer, the expected statistically significant differences were observed for all the biomarkers (FIG. 16).

[0158] The diagnostic power analysis is reported in Table 19.Table 19. Diagnostic Power Analysis of Proposed Biomarkers of Gastric Ulcer under Clinical Conditions (n = 51) *required number of animals / group, with an error of 0.05 and a power of analysis of 90%Biomarkers Cut-off Se Sp AUC n n Size effect value (%) (%) Healthy Ulcer (required n*)IL1-F5 (ng / mL) 2.44 73.7 64.3 0.74 19 28 0.84 (21)PIP (ng / mL) 2.08 66.7 63.3 0.66 18 30 0.58 (63)CA VI 187 83.3 80 0.85 18 30 0.66 (49)Serotransferrin 6.46 75 77.8 0.85 20 36 1.48 (11)Albumin 145.2 65 84.8 0.76 20 33 0.72 (42)

[0159] 4.3. Methods and Products

[0160] Referring to FIG. 13, a method, such as a method of treatment, includes steps 102, 104, 106, and 108.

[0161] At step 102, a biofluid sample is obtained from a subject suspected of having a gastric ulcer state. In example embodiments, the subject is an equine animal. Such a biofluid is provided in some example embodiments.

[0162] At step 104, the biofluid sample is analyzed to obtain a testing data set including protein information, which includes concentrations of a set of biomarkers comprising one or more selected from IL-1F5, PIP, CA VI, albumin, and serotransferrin.Attorney Docket No.: 76980-00004

[0163] At step 106, the testing data set is compared to a comparative data set to determine whether or not the subject is in a gastric ulcer state based on a set of criteria. The comparative data set comprises pre-determined ranges of IL-1F5, PIP, CA VI, albumin, and serotransferrin in a healthy state.

[0164] At step 108, an instruction to administer a treatment to the subject is provided when the subject is identified to be in a gastric ulcer state. In some example embodiments, step 108 includes a step of administering the treatment to the subject.

[0165] In another aspect, the present disclosure provides a computer implemented system comprising: one or more processors; and at least one tangible, non-transitory machine readable medium encoded with one or more programs, to be executed by the one or more processors, to perform steps of the method, as described herein.

[0166] In another aspect, the present disclosure provides one tangible, non-transitory machine readable medium encoded with one or more programs, to be executed by one or more processors, to perform steps of the method, as described herein.

[0167] The methods and system described herein may be at least partially embodied in the form of computer-implemented processes and apparatus for practicing those processes. The disclosed methods may also be at least partially embodied in the form of tangible, non-transient machine readable storage media encoded with computer program code. The media may include, for example, RAMs, ROMs, CD-ROMs, DVD-ROMs, BD-ROMs, hard disk drives, flash memories, or any other non-transient machine-readable storage medium, or any combination of these mediums, wherein, when the computer program code is loaded into and executed by a computer, the computer becomes an apparatus for practicing the method. The methods may also be at least partially embodied in the form of a computer into which computer program code is loaded and / or executed, such that, the computer becomes an apparatus for practicing the methods. When implemented on a general -purpose processor, the computer program code segments configure the processor to create specific logic circuits. The methods may alternatively be at least partially embodied in a digital signal processor formed of application specific integrated circuits for performing the methods.

[0168] Referring to FIG. 14, an exemplary system 200 is illustrated. System 200 includes a sample and information input tool 202, which may include or may be attached with an analyzer (not shown). The input tool 202 may include a set of instructions 204, a module 206 for inputtingAttorney Docket No.: 76980-00004 sample information or receiving a sample, and a data set input module 208 to provide testing results or data set. The modules 206 and 208 provide information to the diagnosis models 226.

[0169] System 200 includes one or more processors 221, and at least one tangible, non- transitory machine readable medium encoded with one or more programs 234, to be executed by the one or more processors, to perform the functions or the methods as described herein. The processor(s) 221 may include a diagnosis and prescription control 222, which includes instruction files 224, diagnosis models 226, comparative data set 228, and a prescription and information module 230. The processors 221 may be connected with one or more displays 236. The information including the detection of gastric ulcer in a subject and related treatment prescription are output and displayed in the display 236.

[0170] 5.0. Description of Sample Collection Apparatus for EGUS Prediction

[0171] Referring to FIGS. 23 and 24, in example embodiments, saliva is collected via a collection apparatus 100. Collection apparatus 100 includes a compressed cellulose-based sponge 102, for example, measuring approximately 6.0 x 10'4m2, attached to a plastic rod 104, and enclosed securely in a cylindrical tube 106 to prevent damage or contamination to sponge 102 prior to use. In example embodiments, sponge 102 and rod 104 can be utilized by unscrewing or otherwise removing a portion from cylindrical tube 106, rotating, and screwing or otherwise reattaching. Sponge 102, now attached by plastic rod 104 to cylindrical tube 106, becomes instantly ready to use. Though of course any configuration of a sponge, or other similar material is encompassed by the invention. Similarly, while a plastic rod is suggested for ease of use to apply the saliva sample to the sponge, any means for delivery of a sponge or similar material to the mouth of a subject to obtain a saliva sample.

[0172] To collect the fluid, sponge 102 is inserted onto the bars of the mouth for 30 to 90 seconds, until sponge 102 is fully expanded and saturated with fluid. Mouth must be free of debris including, but not limited to, feed, forage, or hay. After collection, the top portion of collection apparatus 100 is then removed from cylindrical tube 106, rotated 180 degrees (turned upside down), inserted into cylindrical tube 106 and closed securely by screwing or otherwise fastening this portion of collection apparatus 100 to cylindrical tube 106 of collection apparatus 100.

[0173] The testing kit may consist of an immunoassay testing strip housed within a rectangular or square plastic casing. The strip may be in the form of a membrane or paper basedAttorney Docket No.: 76980-00004 material. The purpose of the test is to determine the presence and / or concentration of the bioactive compounds described herein. Upon detection of the bioactive compounds, an indication is given upon the surface of the testing strip or kit. An included guide is provided to the user to interpret the result of the test. See FIG. 15.

[0174] Another form of the test is combining a small, portable Instrument with reader; microfluidic Test Strip; simple, standardized workflow; and seamless, secure digital connectivity to the Cloud and clinic IT systems results in a high sensitivity point of care diagnostic test.Taking the saliva and dropping a small amount directly onto the test strip which is then fed into the portable instrument which will present results in several minutes. Both the Instrument and Test Strip use a number of analytical electronic quality controls to ensure the integrity and validity of the test, automated functionality checks and Test Strip performance controls are made throughout the operating steps of the test. The immunoassay is programmed on the electronic test strip that is read by the portable instrument that the test strip card is inserted into.

[0175] The methods and the products provided in the present disclosure have significant advantages. For example, the methods and the products provided in the present disclosure can be used to readily detect gastric ulcer in a subject such as a horse through biomarkers in a biofluid sample such as saliva without using invasive endoscopy. It is a great advance in the diagnosis of the disease, facilitating prevention, the initiation of treatment and overall, the welfare of the animals.

[0176] Although the subject matter has been described in terms of exemplary embodiments, it is not limited thereto. Rather, the appended claims should be construed broadly, to include other variants and embodiments, which may be made by those skilled in the art.

Claims

Attorney Docket No.: 76980-00004WHAT TS CLAIMED IS:

1. A method, comprising: obtaining a biofluid sample from a subject suspected of having a gastric ulcer state, wherein the subject is an equine animal; analyzing the biofluid sample to obtain a testing data set comprising concentration ranges of protein biomarkers comprising IL-1F5, carbonic anhydrase VI (CA VI), Prolactin inducible protein (PIP), albumin, and serotransferrin; comparing the testing data set with a comparative data set to determine if the subject is in a gastric ulcer state, wherein the comparative data set comprises pre-determined ranges of IL-1F5, carbonic anhydrase VI (CA VI), Prolactin inducible protein (PIP), albumin, and serotransferrin of a healthy state; and when the testing data set is outside the range determined by the comparative data set for one or more of the protein biomarkers further providing instructions for administering a treatment to the subject for a gastric ulcer state.

2. The method of claim 1, wherein the subject is a horse, a donkey, an ass, a zebra, or a member of the genus Equus.

3. The method of claim 1, wherein the biofluid sample is a saliva sample.

4. The method of claim 3, wherein the saliva sample is collected using a sponge attached with a pole, wherein the sponge is applied into the mouth of the subject and chewed by the subject for a predetermined time, and then taken out and placed into a vial.

5. The method of claim 1, further comprising evaluation of concentration range of the protein clusterine, S100-A9, zymogen- 16B, or thioredoxin as indicative of the gastric ulcer state.

6. The method of claim 1, wherein the testing data set comprises a range of protein concentration that is 1-fold to 100-fold higher than the predetermined range in the comparative data set.

7. The method of claim 1, wherein each of the testing data set and the comparative data set includes information of additional biomarkers.Attorney Docket No.: 76980-000048. An article for detecting equine gastric ulcer, the article comprising: a sample receiving zone configured to receive a biofluid sample from a subject suspected of having a gastric ulcer state, wherein the subject is an equine animal; and a membrane comprising a testing zone, wherein the testing zone comprises at least one antibody to detect a presence and a relative concentration of at least one biomarker that is IL-1F5, carbonic anhydrase VI (CA VI), Prolactin inducible protein (PIP), albumin, or serotransferrin, wherein the membrane is configured to provide an observable positive result to indicate the presence and the relative concentration of the at least one respective biomarker, and the concentration of the at least one biomarker indicates that the subject is in the gastric ulcer state.

9. The article of claim 8, wherein the article is one of a diagnosis strip or a paper.

10. The article of claim 8, wherein the observable positive result is provided in a line or a dot having at least one of a larger size or a higher darkness than that of a corresponding reference.

11. A kit comprising a sponge attached with a pole and the article of claim 8.

12. A device comprising the article of claim 8.

14. A device configured to implement the method of claim 1.

15. A computer implemented system, comprising: one or more processors; and at least one tangible, non-transitory machine readable medium encoded with one or more programs, to be executed by the one or more processors, to perform steps of the method of claim 1.

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