BIOMARKERS FOR USE IN DIAGNOSING THE SEVERITY STATUS OF CANINE PARVOVIRUS INFECTION.

Abstract

The present invention relates to a diagnostic method based on specific biomarkers for diagnosing the severity status of canine parvovirus (CPV-2) infection, which allows a specialist to provide the animal with the appropriate medication in a timely manner and prevent its death.

Description

The present invention relates to methods for diagnosing the severity of an infection, more specifically to a method for using biomarkers for diagnosing the severity of canine parvovirus infection. BACKGROUND OF THE INVENTION Canine parvovirus is a viral infection that affects canids worldwide, with morbidity rates as high as 100% and mortality rates as high as 91% in canines that do not receive timely treatment. Canine parvovirus (CPV-2) is the virus that causes parvovirus. The main route of transmission is fecal-oral, as the virus is excreted in feces. Viral replication occurs in rapidly dividing cells. After viral replication, the virus localizes in the epithelium of the gastrointestinal tract, bone marrow, and lymphoid tissue. CPV-2 causes destruction of the intestinal villi, increasing the risk of bacterial translocation, which can lead to sepsis and death. Following an incubation period of 3-7 days after infection, the characteristic clinical signs of parvovirus infection are gastroenteritis accompanied by signs such as anorexia, depression, vomiting, hemorrhagic diarrhea (and in some cases mucoid diarrhea), dehydration, and fever (Decaro 20 & Suoóavoglia, 2012). Due to fluid and protein loss from diarrhea and vomiting, hypothermic shock may develop. Affected canids may present with abdominal pain, caused by the gastroenteritis itself or, in some cases, by an intestinal intussusception. Clinical diagnosis is not always definitive, as various pathogens (bacteria, parasites, or other viruses) can cause the clinical signs described in a CPV-2 infection. Therefore, laboratory tests are essential for diagnosing parvovirus. In this regard, document KR211781O31 discloses a recombinant vector comprising a gene encoding a recombinant VP2 protein of canine parvovirus 2 with the ability to rapidly diagnose canine parvovirus infection from a large number of samples. On the other hand, document CN1040CK857S describes a PCR-HR.M primer and a method for rapidly distinguishing different canine parvovirus genotypes. The method is simple to operate and only requires the addition of fluorescently charged dyes before the PCR reaction; the detection rate is fast and high-throughput; the entire process takes only 3 hours, does not require virus cell culture, and significantly reduces the time required for typing.Although multiple molecular diagnostic methods have been identified with the ability to rapidly detect canine parvovirus, no method has been described with the ability to detect the stage of parvovirus infection, which is key to providing timely treatment and preventing death. Therefore, there is a need to find new molecular detection methods capable of detecting the severity of canine parvovirus infection for timely treatment and to prevent animal death. Taking into account the defects of the prior art, it is an object of the present invention to provide specific biomarkers for the diagnosis of the severity status of canine parvovirus infection. Likewise, the object of the present invention is the use of specific biomarkers for the diagnosis of the severity of canine parvovirus infection. An additional object of the present invention relates to a method for diagnosing the severity status of canine parvovirus infection, comprising the steps of: taking a sample and quantifying the concentration of specific markers. These and other objects are achieved through specific diagnostic biomarkers useful for detecting the severity of canine parvovirus infection. BRIEF NOTES ON THE INVENTION To that end, a first aspect of the present invention relates to a method for diagnosing the severity status of canine parvovirus infection, comprising the steps of: taking a blood serum sample from a canine, and quantifying at least one selected biomarker of IR-gamma, interukin 6 (IL-6), cytokine interukin 8 (IL-8), chemokine KC-like, MCP-1 factor and interukin 10 (IL-10). A second aspect of the present invention relates to a set of specific biomarkers for the diagnosis of the severity status of canine parvovirus infection, wherein the biomarkers are selected from the group of IFN-gamma, interleukin 6 (I1-6), interleukin 8 (I-8), chemokine KC-11, MCP-1 factor and interleukin 10 (IL-11). A third aspect of the present invention relates to the use of a set of specific biomarkers selected from the group of IFN-gamma, Interleukin γ (IL-6), Interleukin γ (IL-8), pure KQ-like, MCP-1 factor and Interleukin 16 (IL-1Q) for the diagnosis of the severity status of canine parvovirus infection. The novel aspects that constitute the characteristics of this invention will be set forth in detail in the appended claims. However, its features and advantages will be better understood from the examples, when read in conjunction with the appended figures, where: Figure 1 shows a gei that represents the amplification of the gene associated with the W2 protein from fecal samples of different CPV-2 positive canids, where MPM is a molecular weight marker. Figure 2 shows a heat map where the concentration of the analyzed cytokines is shown, where according to the color code, purple is the lowest concentration and red is the highest concentration. Figure 3 shows a statistical analysis of the concentration of the most significant cytodnes in canids positive for CPV-2 infection and a CPV-negative control group, where a) is IFN-γ, b) is IL-6, c) is IL-8, d) is KÉ-like, e) is MCP-1, and f) is IL-10. Figure 4 shows a statistical analysis of the correlation between the concentration of KG-like and other atomics by means of a linear regression, where a) KCrílke and IL'6, b) KCHiké and ILS, c) KC-fe and IL-1Q, and d) KC-fe and MCP-L DETAILED DESCRIPTION OF THE INVENTION The present invention exhibits certain advantages over the state of the art, among which it can be mentioned that the diagnostic method based on specific biomarkers for the diagnosis of the severity status of canine parvovirus infection allows a specialist to provide the animal with the appropriate medication in a timely manner and prevent its death. Therefore, the present invention relates in the first place to a method for diagnosing the severity status of canine parvovirus infection in an infected canid, comprising the steps of: taking a serum sample from a canid, and quantifying at least one selected biomarker of IFN-gamma, interleukin 6 (IL-6), cytokine interleukin 8 (IL-8), chemokine K12, MCP-1 factor and interleukin 10 (IL-10). More preferably, the method for diagnosing the severity status of canine parvovirus infection comprises an additional step of: comparing the levels of one or more biomarkers to determine if the infected canine has presumed immunosuppression, severe inflammation, and / or a predisposition to death. In a preferred embodiment, the step of quantifying at least one biomarker comprises performing an immunohistochemistry, immunocytochemistry, immunofluorescence, immunoprescription, Western blot, ELISA, or some other assay that permits the detection of dioxins. More preferably, the step of quantifying at least one biomarker comprises combining an antibody with the biomarker, wherein the antibody binds specifically to the biomarker, or a fragment thereof containing a specific determinant of the biomarker. The antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a recombinant antibody fragment, a Fab fragment, a Fab fragment; an FCate fragment, an Fe fragment, and an scFa fragment In a preferred embodiment of the invention, a value less than or equal to 10 pg / ml for the quantification of autumn INFgamma in a canid infected with canine parvovirus is indicative of immunosuppression. In a preferred modality, a value greater than or equal to 1,700 pg / ml of fe cytodne IL- in a canid infected with canine parvovirus is indicative of severe inflammation. In a preferred modality, a value greater than or equal to 2,500 pg / ml of fe cytokine IL8 in a canine infected with canine parvovirus is indicative of severe inflammation. In a preferred modality, a KC15-like atocin value greater than or equal to 500 pg / mi in a canid infected with canine parvovirus is indicative of severe inflammation. In a preferred modality, a value greater than or equal to 1,500 pg / mt of the MCPi atocin in a canid infected with canine parvovirus is indicative of severe inflammation. In a preferred modality, a value greater than or equal to 100 pg / ml of the atocin XL-10 in a canid infected with canine parvovirus is indicative of severe inflammation. In a preferred modality, the levels of the cytokines IL-6 and KC-like together are indicative of severe inflammation and high predisposition to death of the canine infected by canine parvovirus. In a preferred modality, elevated levels of the cytokines 1L-8 and KC-lte together are indicative of severe inflammation and high predisposition to death in canine parvovirus 25 infected dogs. In a preferred modality, the levels of IL-10 and KC-like enzymes together are indicative of severe inflammation and high predisposition to death of the canine infected by canine parvovirus. In a preferred modality, the levels of the atokines MCP-1 and KC like 30 together are indicative of severe inflammation and high predisposition to death of the canine infected by canine parvovirus. A second aspect of the present invention relates to a set of specific biomarkers for the diagnosis of the severity status of canine parvovirus infertility in an infected canid, wherein the biomarkers are selected from the group of IFN-gamma, interferon 6 (IL-6), interferon 8 (IL-8), KC-like chemokine, MCP-1 factor and interferon 10 (IL-10). A third aspect of the present invention relates to the use of a set of specific markers selected from the group of IFN-gamma, interleukin 6 (IL-6), interleukin 8 (IL-8), chemokine KC-hkeffactor MCP-1 and interleukin 10 (IL-1) for the diagnosis of the severity status of canine parvovirus infection in an infected canid, In a preferred modality, the use of the specific set of biomarkers determines whether the canine infected with canine parvovirus exhibits immunosuppression, severe inflammation, and / or a high predisposition to death The advantages of the present invention will be better understood from the following examples, which are presented solely for illustrative purposes to allow a better understanding of the preferred embodiments of the present invention, without implying that there are no other unillustrated embodiments that can be implemented based on the detailed description above. EXAMPLE 1 In the present example, the process of selecting and collecting blood and fecal samples from canines infected with canine parvovirus (CPV-2) and healthy canines is described. The canines with canine parvovirus selected for the study were those that presented with clinical signs consistent with CPV-2 infection and were diagnosed by a positive SAP2 test at the time of the study. The patient's medical history was recorded: breed, sex, age, weight, eating habits, cohabitation with other pets, walks, contact with animals outside the home, previous illnesses, and vaccination and deworming protocol. A general physical examination of the patient was performed, recording the following physiological constants: heart rate, respiratory rate, temperature, capillary refill time, and pulse, as well as attitude, body condition, hydration, mucous membrane color, and the condition of palpable lymph nodes. Each patient was assigned a code and followed up, recording whether the patient recovered from the infection or died.Table 1 shows the occlusion and exclusion criteria for the selection of canids infected with canine parvovirus. Likewise, the medical history of the healthy canines as a control group was obtained, and a general physical examination was performed on 30 of them to ascertain their health status. Table 2 shows the Once the canines were selected, a rectal swab was used for the fecal samples, which were placed in a sterile tube labeled with a code, which was recorded in the patient's medical history and stored at -20^0 for later analysis. The blood sample was obtained by venipuncture of the cerebral vein, specifically the jugular vein. Two to three milliliters of blood were collected in EDTA tubes without anticoagulant and centrifuged at 1500 rpm for 10 minutes to obtain the blood serum. Subsequently, at least four aliquots of the serum obtained were prepared in a laminar flow hood, labeled, and stored frozen at -20°C for later analysis. The entire procedure was performed within the first three hours after sample collection. EXAMPLE 2 An assay was performed to extract DNA from the collected fecal samples and to confirm the diagnosis of canine parvovirus; a PCR test was performed. For DNA extraction, the Vivantis GF-1 kit was used, following the manufacturer's instructions. First, the samples were thawed, 1 ml of water for injection was added, they were sterilized in a vortex mixer for 1 minute, and centrifuged at 12,000 rpm for 5 minutes. Subsequently, 200 µl of the clarified sample were taken and placed in a microcentrifuge tube, to which 10 µl of proteinase K and 200 µl of BB were added. The sample was incubated at 65°C for 10 minutes, then 200 µl of absolute ethanol were added, and the sample was pulverized on a silica column, centrifuged at 6,000 rpm for 1 minute, and the contents of the tube were decanted.500 µL of solution 81 Wash1 were added, the column was centrifuged again at 6,000 rpm for 1 min, and the contents of the tube were decanted. The column was washed again with 500 µL of solution 82 Wash2, centrifuged at 12,000 rpm for 3 min, and the microcentrifuge tube was discarded. The column was placed in a new tube, 100 µL of hot solution 68 (65 eQ) was added to the column, and it was centrifuged at 6,000 rpm for 1 min. The DNA obtained was labeled and stored at -20°C. In the fine-point polymerase chain reaction (PCR) assay, a partial amplification of the vp2 gene was first performed using the oligonucleotides F-5'-GGAAACCAACCATACCAACTCC'3' and R-5'-GGATTCCMGTAT^. To the reaction, 1 µl (10 µmol / ml) of each of the oligonucleotides, 2.5 µl of 10x PCR buffer (Vivántis, LJSA), 0.3 µl (5 U / ml) of Taq polymerase enzyme, 0.5 µl of dNTP mixture (10 mM), 2 µl of magnesium chloride (25 mM), 14.7 µl of sterile LW, and 3 µl of DNA previously extracted from the sample were added, to reach a final volume of 25 µl. A monovalent, attenuated virus CPV vaccine (Holiand Laboratories, Mexico) was used as a positive control and distilled H?.Q was used as a negative control. The amplification program used consisted of a first denaturation step at 94°C for 5 minutes, followed by 30 cycles with denaturation at 50°C for 30 seconds, an annealing step at 50°C for 1 minute and an extension step at 72°C for 10 minutes, and a final step at 4°C. The amplified samples were visualized by electrophoresis. 5 g of the PCR product were taken and 3 µl of loading buffer were added; they were loaded onto a 2% agarase gel in TAE 1x, to which SY8R Green (1 µl SY8R Green / 10 µl TAE 1x) was added, and the samples were run with an electrical current of 90 V for 30 minutes. The heads were observed on a transilluminator (MaestroGen, Singapore) and the presence of a band of approximately 1,042 bp, coinciding with the positive center (lane 8) as shown in figure 1, was considered to be a positive diagnosis of parvovirus. 5: A total of 41 samples tested positive for canine parvovirus. The diagnosis was initially made with an immunoglobulin test and confirmed by PCR. Three of the samples tested negative by SNAP, even though the patients presented signs consistent with parvovirus; subsequently, PCR was retested and the samples tested positive. It has been previously described that a disadvantage of the SNAP test is its low sensitivity, since it detects the virus in stool for only a few days (Decaro et al, 2010).Genotyping was performed using enzymatic digestion. The cutoff pattern was compatible with variant 2c for all samples, which coincides with previous studies by Pedroza-Roldán et al. in 2015 and Hernández-Almaraz in 2017, where it is reported that this variant is the only one circulating in the Guadalajara Metropolitan Area. The mortality rate observed in patients with parvovirus who do not receive any treatment is 91%, whereas in referral hospitals where treatments are aggressive, mortality is less than 1%. In this study, 39% of the patients died. The factors that are related to mortality rates are the presence of phleukopenia, lymphopenia or initial neutropenia, presence of signs of RSV or sepsis, as well as inadequate treatment (Goddard et al., 2008; Kaíli et al., 2010). Sykes, 2014).In the present study, the 20 blood count data were not obtained from the patients; however, the results of the atokine concentration suggest a state of sepsis in the deceased patients, as described below. EXAMPLE 3 An assay was performed: to determine the quantification of cytokines, the assay was performed with the Millipiex Canine Cytokine Magnetic Bead Panel4 kit following the manufacturer's instructions. First, the reagents for the immunoassay were prepared. The immunoassay comprised: (a) preparing the antibody-immobilized beads: each bead vial was settled for 30 seconds, 60 ml from each bead vial was added to the 30 ml mixing vial, brought to a final volume of 3 ml with assay buffer and the mixture was brought to the vertex for 30 seconds; (b) preparing the quality controls: Control 1 and Control 2, which were reconstituted with 250 ml of deionized water, the mixture was shaken, brought to the vertex, and the vial was allowed to settle for 10 minutes; (e) Prepare the wash buffer: the Wash buffer solution was allowed to reach room temperature and mixed, 30 ml of the solution were diluted in 270 ml of deionized water; (d) Preparation of the serum matrix: 1 ml of deionized water was added to the serum matrix mixing bottle, mixed well and left to stand for 10 minutes.No dilution of the matrix serum was performed, as the serum samples were not diluted. (e) To prepare the standard canine cytokine panel, the standard canine cytokine panel was reconstituted with 250 ml of deionized water, the flask was vortexed for 10 seconds, allowed to settle for 10 minutes, and vortexed again for 30 seconds. Six tubes were labeled (standard). 6, 5, 4, 3, 2, and 1), 150 ml of assay buffer were added to each and the corresponding dilutions were made. The assay buffer was taken as the 0 standard (bac^raun^ Subsequently, all reagents were allowed to reach room temperature (2-25°C) before starting the assay. As reagents and samples were added to the plate, the data were recorded on a chart. Samples were not added in duplicate. 200 µL of assay buffer were added to each well of the plate, which was then sealed and shaken for 10 minutes at room temperature and subsequently decanted. 25 µL of each standard and control were added to the corresponding wells, and 25 µL of assay buffer were added to the sample wells. 25 µL of matrix solution were added to the wells of the standards and controls. After all reagents were added, 25 µL of each sample were added to the corresponding wells and recorded on the chart. The bead mixing bottle was vortexed, and 25 µL were added to each well. The plate was sealed and incubated at 4°C for 18 hours. After incubation, the contents of the wells were removed and the plate was washed twice. The plate was placed on a handheld magnet and allowed to settle for 1-20 minutes. The contents of the wells were then decanted and the plate washed with 200 µL of wash buffer. 25 µL of detection antibodies (Detectlon antibodies) were added to each well, the plate was sealed, and it was incubated for 1 hour with shaking at room temperature. Then, 25 µL of streptavidin-3-c were added to each well, the plate was sealed, and it was incubated for 30 minutes at room temperature with shaking. The contents of the piara were decanted and two layadas were performed, with the same procedure 25 previously described. 150 pl of Drive Huid were added to all the wells, the pruneds were resuspended in an agitator for 5 minutes and the plate was then placed. Cytokine quantification was performed using a Luminexi® MAGPIX® instrument with xP0NENT® software. The data, obtained from the Mean Intensity of Fluorescence (MR), were saved for later analysis. EXAMPLE 4 This example presents the results of the quantification of toxins. Figure 2 graphically shows the concentrations of the 13 toxins analyzed in each of the canids studied. Of the aforementioned markers analyzed, six of them, IFNγ, IL-6, IL-8, KC4α, MCP-1, and IL-4β, had statistically significant values ​​between the groups. The data obtained and analyzed show that canines with moderate clinical signs and 100% survival presented processes of suppression (in graph a) of Figure 3, where there is a significant decrease in the presence of IFNγ (group 1 vs. group 2 and control). However, group 2 and the control group did not show a significant difference, which indicates that at this stage of the infection, suppression is the most relevant factor. The concentration of IL-6 was higher in group 2 compared to group 1 (p<0.01) and compared to... control group (p<0.05) as shown in graph b) of figure 3. As well as in IL-8, whose concentration in group 2 was higher compared to group 1 (p<0.00i) and compared to the control (p<0.0001) as shown in graph c) of figure 3. The same occurs in the case of KC as shown in graph d) of figure 3 and MCP-1 in graph e) of figure 3, the concentration is higher in group 1 compared to the other two (p<0.9001). Finally, in the case of IL-10 the concentration in group 2 was higher than in group 1 (p <O.0E) como se muestra en ei gráfico Ó de la figura 3. In the remaining cytokines analyzed in this study, GM-CSF, IL-3,11-7,11-15, IP- The difference in concentration between groups for IL-10, IL-18, and TNF-κB was not statistically significant; therefore, these data are not presented. Based on the findings described above, a linear regression analysis was performed to determine the correlation of KC-like activity with IL-6, IL-8, IL-10, and MCP-1, which showed significant increases in severely affected canines (groups 20 and 2) compared to healthy canines and those with moderate signs (group 1). The correlation was statistically significant with IL-1 (p < 0.24%), IL-6 (p < 0.136), and IL-10 (p < 0.01), while the correlation was not significant with MCP (p < 0.14), as shown in Figure 4. In canines with severe disease due to parvovirus infection, levels of KG-12, as well as IL-8, IL-6, and IL-10, are elevated. Knowing that there is a correlation between these cytokines makes it possible to have a prognostic biomarker of disease severity in canines with parvovirus, determining that if the concentration of KG-12 is elevated, the concentrations of IL-6, IL-8, and IL-10 will also be elevated, and the patient will have a higher risk of complications. In veterinary clinical practice, determining serum cytokine levels would be useful to predict the onset of complications in canines infected with parvovirus. ranino (CPV) and identify the 30 key points in therapeutics to limit the effects of the disease. In accordance with the foregoing description, it will be observed that the diagnostic method based on specific markers for diagnosing the severity of parvovirus infection has been devised for application in the veterinary industry, and it will be evident to any expert in the field that the embodiments of the invention as described above and illustrated in the accompanying drawings are merely illustrative and not limiting to the present invention, since numerous changes in their details are possible without departing from the scope of the invention; for example, it is possible to use different methodologies, protocols, assays, and particular reagents described herein, as these may vary, for their proper application in molecular diagnosis. Therefore, the present invention shall not be considered restricted except as required by prior art and by the scope of the appended claims.

Claims

1. A method for the diagnosis of the severity status of canine parvovirus infection in an infected canid, characterized in that it comprises the steps of: taking a blood serum sample from a canid, and quantifying at least one selected biomarker from IL-gamma, interleukin 6 (IL-6), interleukin 8 (IL-8), KC-Hke chemokine, MCP factor 1 and interleukin 10 (IL-10), 2. B method according to claim 1, further characterized in that it comprises an additional step of comparing the levels of one or more biomarkers to determine whether the canine infected with canine parvovirus exhibits immunosuppression, severe inflammation, and / or a high predisposition to death, 3, Θ method according to claim 1, further characterized in that the step of quantifying at least one biopark comprises performing an immunohistochemical, immunocytochemical, immunofluorescence, immunoprecipitation, Western blot, ELISA or: some other assay that allows the detection of cytokines, 4. The method according to claim 1, further characterized in that the step of quantifying at least one biomarker comprises: contacting an antibody with the biomarker, wherein the antibody binds specifically to the biomarker, or a fragment thereof that binds an antigenic determinant of the biomarker.

5. The method according to claim 4, further characterized in that the antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a recombinant antibody fragment, a Fab fragment, a Fab' fragment, an F(ab') 2 fragment, an Fv fragment, and an scFv fragment.

5. The method according to claim 1, further characterized in that a value less than or equal to 10 pg / mL of the quantification of the cytokine IFN-gamma in a canid infected with canine parvovirus is indicative of immunosuppression, 7. The method according to claim 1, further characterized in that a value greater than or equal to 1,700 pg / mL of the cytocin IL-6 in a canine infected with canine parvovirus is indicative of severe inflammation, 30 8. The method in conformity with claim 1, further characterized in that a value greater than or equal to 2,500 pg / mL of the IL-8 toxin in a canine infected with canine parvovirus is indicative of severe damage.

9. The method according to claim 1, further characterized in that a value greater than or equal to 500 pg / ml of the cytokine KC-fe in a canid infected with canine parvovirus 35 is indicative of severe inflammation.

10. A method according to claim 1, further characterized in that a value greater than or equal to 1,500 pg / mi of the cytochrome MCP 1 in a canid infected with canine parvovirus is indicative of severe inflammation.

11. The method according to claim 1, further characterized in that a value greater than or equal to 109 pg / mi of te atocin in an infected canine with canine parvovirus is indicative of severe inflammation.

12. The method according to claim 1, further characterized in that the levels of the cytokines IL-6 and KC-10 are indicative of severe inflammation and a high predisposition to death in the canid infected with canine parvovirus. 10 13. The method according to claim 1, further characterized in that the levels of the toxins IL-8 and KC-1 are indicative of severe inflammation and a high predisposition to death of the canine infected with canine parvovirus, 14. The method according to claim 1, further characterized in that the levels of the toxins IL-10 and KC-Hfe together are indicative of severe inflammation and high 15 predisposition to death of the canid infected by canine fever.

15. The method of conformity with claim 1, further characterized in that the levels of the cytokines MCfel and RC-fe together are indicative of severe inflammation and high predisposition to death of the canine infected by canine parvovirus.

16. A set of specific biomarkers for the diagnosis of the severity status of canine parvavirus infection in an infected canid, characterized in that the biomarkers are selected from the FN-gamma group, interteukin 6 (IL-11.-6), interteukin 8 (IL-8), KCdite chemokine, MCP-1 factor and interteukin 10 (IL-10).

17. Use of a set of specific biomarkers, characterized in that they are selected from the IWgamma group, intereucin 6 (11-6) / iritereucin 8 (11-8), RC-ite chemokine, MCP-1 factor 25 and intereucin 10 (IL-10) for the diagnosis of the severity status of canine parvovirus infection in an infected canid. 1S. The use in accordance with claim 17, further characterized in that it determines whether the canine infected with canine parvovirus presents immunosuppression, severe inflammation, and / or a high predisposition to death, the present invention relates to a diagnostic method based on specific biomarkers for the diagnosis of the severity status of canine parvovirus (CPV-2) infection, which allows a specialist to provide the animal with the appropriate medication in a timely manner and prevent its death.