Diagnosis of heart disease and degenerative mitral valve disease in canines

Abstract

The present invention relates to a method for diagnosing cardiac disease in canines, including early degenerative mitral valve disease, by using a microbiome comprising specific genera and species. In one embodiment, the method includes measuring the normalized relative abundance of fecal bacteria, including Faecalibacterium, Turicibacter, Streptococcus, Escherichia coli, Blautia, Fusobacterium, and C. hyranonis, calculating a dysbiosis index based on the fecal bacteria, and determining that the canine has cardiac disease if the dysbiosis index is greater than -1.0.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001]

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 127,247, filed December 18, 2020, the entire disclosure of which is incorporated herein by reference. [Background technology]

[0002] Canine degenerative mitral valve disease (DMVD) is characterized by slowly progressive valvular degeneration that leads to mitral regurgitation and, in some dogs, congestive heart failure (CHF). Dogs with early-stage DMVD typically have a long presymptomatic period, but once they progress to the stage of CHF, the disease progresses more rapidly, with a mean survival time of less than 12 months. Therefore, early intervention at the presymptomatic stage is crucial to extending the lifespan of affected dogs. Recently, a staging system for classifying canine DMVD has been adopted by a consensus committee established by the American College of Veterinary Internal Medicine (ACVIM). Dogs at risk for developing DMVD but who are otherwise healthy are considered to be in Stage A; dogs with a heart murmur due to mitral regurgitation but no clinical signs of CHF are classified as Stage B; and dogs with overt clinical signs of CHF are classified as Stage C. Dogs in stage B are further divided into stages B1 or B2 depending on the presence or absence of ventricular remodeling.

[0003]

[0003] Currently, the only drug proven to be effective in the early, presymptomatic stage of DMVD is pimobenden, which, like any other drug, is associated with side effects. Currently, the gold standard for diagnosing DMVD is echocardiography, which is not only expensive but also requires highly specialized veterinarians specializing in cardiac care. Therefore, effective diagnostic methods and treatments that overcome the shortcomings of current methods and treatments are still being sought. Summary of the Invention

[0004]

[0004] The present disclosure relates generally to diagnosing heart disease and degenerative mitral valve disease (DMVD) in canines, and in one aspect, to diagnosing early-stage DMVD. In one embodiment, a method for diagnosing heart disease in a canine includes measuring the normalized relative abundance of fecal bacteria, including Faecalibacterium, Turicibacter, Streptococcus, E. coli, Blautia, Fusobacterium, and C. hiranonis, calculating a dysbiosis index based on the fecal bacteria, and determining that the canine has heart disease if the dysbiosis index is greater than -1.0.

[0005] In another embodiment, the method for diagnosing early stage DMVD in canines includes the step of detecting and / or treating a canine with the use of a bacterial strain selected from the group consisting of Catenibacterium mitsuokai, Butyricicoccus pullicaecorum, Bacteroides coprocola, Bacteroides plebeius, Allobaculum stercolicanis, and / or the like. measuring the normalized relative abundance of a biomarker selected from the group consisting of Catenibacterium mitsuokai (Catenibacterium mitsuokai) and Bacteroides coprocola (Bacteroides plebeius) between 0.3 and 3, Butyricicoccus plicaecorum (Bacteroides plicaecorum) between 0.14 and 0.35, Bacteroides coprocola (Bacteroides plebeius) between 0.6 and 1.3, Bacteroides plebeius (Bacteroides plebeius) between 0.1 and 0.8, or Allobaculum stercoricanis (Bacteroides stercoricanis) between 0.1 and 1.5; and determining that the canid has early DMVD if the normalized relative abundance of Catenibacterium mitsuokai is between 0.3 and 3, the normalized relative abundance of Butyricicoccus plicaecorum is between 0.14 and 0.35, the normalized relative abundance of Bacteroides coprocola (Bacteroides plebeius) between 0.6 and 1.3, the normalized relative abundance of Bacteroides plebeius is between 0.1 and 0.8, or the normalized relative abundance of Allobaculum stercoricanis is between 0.1 and 1.5.

[0006] In yet another embodiment, a method for diagnosing early stage DMVD in a canine comprises the steps of: measuring the normalized relative abundance of bacteria in the genus, wherein the genus is selected from the group consisting of Catenibacterium, Prevotella, Butyricicoccus, Faecalibacterium, Clostridium, Allobaculum, or a combination thereof; and determining whether the normalized relative abundance of bacteria in the genus Catenibacterium is between 0.3 and 3. determining that the canine has early DMVD if the normalized relative abundance of bacteria in the genus Prevotella is between 0.5 and 4, the normalized relative abundance of bacteria in the genus Butyricicoccus is between 0.14 and 0.4, the normalized relative abundance of bacteria in the genus Faecalibacterium is between 0.012 and 0.04, the normalized relative abundance of bacteria in the genus Clostridium is between 2 and 4, or the normalized relative abundance of bacteria in the genus Allobaculum is between 0.2 and 1.5.

[0007]

[0007] Additional features and advantages are described herein, and will be apparent from the detailed description that follows. DETAILED DESCRIPTION OF THE INVENTION

[0008] definition

[0008] As used in this disclosure and the appended claims, the singular forms "a," "an," and "the" include plural references unless otherwise indicated. Thus, for example, reference to "a fecal bacteria or bacterium" or "the fecal bacteria or bacterium" includes two or more such bacteria or bacterium. The term "and / or" used in the context of "X and / or Y" should be interpreted as "X" or "Y" or "X and Y." As used herein, the terms "example" and "for example," particularly when followed by a list of terms, are used merely exemplary and illustratively and are not exclusive or inclusive.

[0009] As used herein, "about" is understood to refer to a number within a numerical range, e.g., within -10% to +10%, -5% to +5%, or in one embodiment, within -1% to +1% of the referenced number, and in a specific embodiment, within -0.1% to +0.1% of the referenced number. Furthermore, all numerical ranges herein should be understood to include all integers, whole numbers, or fractions within that range. Furthermore, these numerical ranges should be interpreted as supporting claims directed to any number or subset of numbers within that range. For example, a disclosure of 1 to 10 should be interpreted as supporting ranges of 1 to 8, 3 to 7, 1 to 9, 3.6 to 4.6, 3.5 to 9.9, etc.

[0010] As used herein, "between" is inclusive of the endpoints. For example, a dysbiosis index between -1 and 0.5 (-1 to 0.5) includes cases where dysbiosis is -1 or 0.5.

[0011] All percentages expressed herein are by weight of the composition on a dry matter basis unless otherwise stated. Those skilled in the art will understand that the term "dry matter basis" means that the concentration or percentage of an ingredient in a composition is measured or specified after all free moisture in the composition has been removed. When reference is made to pH, the value corresponds to the pH measured at 25°C with standard equipment. "Amount" may be the total amount of the referenced ingredient per serving of composition or per discrete unit of composition, and / or may be a weight percent of the referenced ingredient by dry weight. Furthermore, "amount" includes zero; for example, reciting the amount of a compound does not necessarily mean that the compound is present unless accompanied by a range excluding zero.

[0012]

[0012] As used herein, "normalized relative abundance" refers to the amount of each microorganism calculated by taking its respective count in each sample, dividing by the total sequence count, and then taking the square root.

[0013] As used herein, "early degenerative mitral valve disease" refers to Stage B of degenerative mitral valve disease.

[0014] As used herein, "Stage A" refers to dogs that are at risk for developing degenerative mitral valve disease but have otherwise healthy hearts.

[0015] As used herein, "Stage B" refers to dogs that have a heart murmur due to mitral regurgitation but no clinical signs of congestive heart failure. "Stage B" includes Stage B1 (without ventricular remodeling) and Stage B2 (with ventricular remodeling).

[0016] As used herein, "Stage C" refers to dogs with congestive heart failure.

[0017]

[0017] As used herein, "degenerative mitral valve disease," "DMVD," "chronic valvular disease," "CVD," "myxomatous mitral valve disease," and "MMVD" can be used interchangeably and refer to progressive valvular degeneration that causes mitral regurgitation and / or congestive heart failure (CHF), including Stage A, Stage B, and Stage C.

[0018]

[0018] As used herein, "dysbiosis index" or "DI" is quantified as a single number measuring the closeness (l2-normalized) of a test sample to the mean (prototype) of each class, as disclosed in "A Dysbiosis Index to Assess Microbial Changes in Fecal Samples of Dogs with Chronic Inflammatory Enteropathy" by AlShawaqfeh et al, FEMS Microbiology Ecology, vol. 93, no. 11, pp 1-8 (2017) (doi:0.1093 / femsec / fix136). As discussed in the above paper, DI is defined as the difference between (the Euclidean distance between the test sample centroid and the healthy class centroid) and (the Euclidean distance between the test class centroid and the diseased class centroid). DI is mathematically calculated as follows: The DI of a text sample z is defined as:

[0019]

number

number

number

[0020]

[0019] The methods disclosed herein may be absent any step not specifically disclosed herein. Thus, disclosure of an embodiment using the term "comprising" includes disclosure of embodiments "essentially comprising" the specified step, as well as embodiments "comprising" the specified step. Unless otherwise specified and directly stated, any embodiment disclosed herein can be combined with any other embodiment disclosed herein.

[0021]

[0020] Unless otherwise defined, all technical and scientific terms and any abbreviations used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any compositions, methods, products, or other means or materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred compositions, methods, products, or other means or materials are described herein.

[0022] All patents, patent applications, publications, and other references cited or referred to in this specification are hereby incorporated by reference to the extent permitted by law. The discussion of these references is intended only to summarize the assertions made in the references. No admission is made that such patents, patent applications, publications, or references, or any portion thereof, are relevant prior art to the present invention, and the right to challenge the accuracy and pertinence of such patents, patent applications, publications, and other references is expressly reserved.

[0023] Embodiment The present inventors have discovered that cardiac disease can be diagnosed based on a dysbiosis index and a specific microbiome, including specific genera and species. Furthermore, the present inventors have discovered that this microbiome can be used to diagnose early degenerative mitral valve disease. Such methods allow for inexpensive and efficient diagnosis of a variety of conditions that can be difficult and expensive to diagnose.

[0024]

[0023] In one embodiment, a method for diagnosing heart disease in a canine may include measuring the normalized relative abundance of fecal bacteria including Faecalibacterium, Turicibacter, Streptococcus, Escherichia coli, Blautia, Fusobacterium, and C. hyranonis, calculating a dysbiosis index based on the fecal bacteria, and determining that the canine has heart disease if the dysbiosis index is greater than -1.0.

[0025]

[0024] Generally, a canine may be diagnosed with DMVD if the dysbiosis index is greater than -1.0. However, in one embodiment, the determining step may also include determining that the canine has early DMVD if the dysbiosis index is between -1 and 0.5. Additionally, in another embodiment, the method may further determine that the canine has early DMVD B1 if the dysbiosis index is between -1 and -0.25. In yet another embodiment, the method may further determine that the canine has early DMVD B2 if the dysbiosis index is between -0.25 and 0.5. In yet another embodiment, the method may further determine that the canine has congestive heart failure if the dysbiosis index is greater than 0.5.

[0026] In another embodiment, a method for diagnosing early stage DMVD in canines includes the steps of measuring the normalized relative abundance of a biomarker selected from the group consisting of Catenibacterium mitsuokai, Butyricicoccus plicaecorum, Bacteroides coprocola, Bacteroides plebeius, Allobaculum stercolicanis, or a combination thereof; and detecting a biomarker in which the normalized relative abundance of Catenibacterium mitsuokai is between 0.3 and 3. and determining that the canid has early DMVD if the normalized relative abundance of Butyricicoccus plicaecorum is 0.14 to 0.35, the normalized relative abundance of Bacteroides coprocola is 0.6 to 1.3, the normalized relative abundance of Bacteroides plebeius is 0.1 to 0.8, or the normalized relative abundance of Allobaculum stercolicanis is 0.1 to 1.5.

[0027] Generally, canines can be diagnosed as having early-stage DMVD using various biomarkers, i.e., bacteria, discussed herein, with specific normalized relative abundances. However, in one embodiment, diagnosis can be based on at least two biomarkers. In another embodiment, diagnosis can be based on at least three biomarkers. In yet another embodiment, diagnosis can be based on at least four biomarkers. In yet another embodiment, diagnosis can be based on all five biomarkers.

[0028]

[0027] In yet another embodiment, a method for diagnosing early-stage DMVD in a canine comprises measuring the normalized relative abundance of bacteria in a genus selected from the group consisting of Catenibacterium, Prevotella, Butyricicoccus, Faecalibacterium, Clostridium, Allobaculum, or a combination thereof; and determining that the canine has early-stage DMVD if the normalized relative abundance of bacteria in the genus Catenibacterium is 0.3 to 3, the normalized relative abundance of bacteria in the genus Prevotella is 0.5 to 4, the normalized relative abundance of bacteria in the genus Butyricicoccus is 0.14 to 0.4, the normalized relative abundance of bacteria in the genus Faecalibacterium is 0.012 to 0.04, the normalized relative abundance of bacteria in the genus Clostridium is 2 to 4, or the normalized relative abundance of bacteria in the genus Allobaculum is 0.2 to 1.5.

[0029] Generally, canines can be diagnosed as having early-stage DMVD using bacteria in the various genera discussed herein with specific normalized relative abundances. However, in one embodiment, the diagnosis can be based on at least two genera. In another embodiment, the diagnosis can be based on at least three genera. In yet another embodiment, the diagnosis can be based on at least four genera. In another embodiment, the diagnosis can be based on at least five genera. In yet another embodiment, the diagnosis can be based on all six biomarkers. [Example]

[0030] The following non-limiting examples are illustrative of embodiments of the present disclosure.

[0031] Example 1 - Canine DMVD Study Clinically healthy dogs aged 7 years or older without heart murmurs or concurrent systemic disease were prospectively enrolled as controls (Group A, N = 29). Groups B1 (N = 34), B2 (N = 25), and C / D (N = 25) were cohorts of dogs aged 7 years or older with an echocardiographic diagnosis of left apical systolic murmur, thickened and prolapsed mitral valve leaflets, and mitral regurgitation, as well as a medical history and physical examination consistent with stage B1, stage B2, stage C, or stage D DMVD, respectively. Any dogs with severe concurrent systemic disease, including diabetes, cancer, or renal failure, or any congenital heart disease, were excluded. Dogs with signs of gastrointestinal disease, such as vomiting or diarrhea, and dogs that had received antibiotics within 30 days were also excluded. Fecal samples were obtained from these dogs.

[0032] Preclinical dietary intervention study in dogs with DMVD Dogs with preclinical DMVD and weighing less than 15 kilograms were considered for inclusion in this study. Dogs were randomly assigned to two dietary groups: a control diet (CON, N=9) and a diet supplemented with a cardioprotective blend (CPB, N=10). The two diets have been described in detail previously. Dogs were fed their assigned diet as their sole source of nutrition for 6 months. Clinical measurements and fecal samples were collected at baseline, 3 months, and 6 months.

[0033] Fecal DNA extraction and metagenomic sequencing Fecal genomic DNA (input: 450–600 ng) was fragmented using a Covaris LE220 instrument targeting a 375-bp insert. Automated Illumina libraries were generated using the KAPA Hyper PCR-free Library Preparation Kit (KAPA Biosystems / Roche) on the SciClone NGS platform (Perkin Elmer). The fragmented genomic DNA was size-selected using AMPure XP beads on the SciClone instrument to narrow the DNA fragment distribution and ensure that the average insert of the library was 350–375 bp. The manufacturer's protocol provided by Perkin Elmer was followed with the following exception: after ligation, the library was purified twice at a 0.7× AMPure bead / sample ratio to remove any remaining adapters. An aliquot of the final library was diluted 1:5 and quantified using a Caliper GX instrument (Perkin Elmer). The concentration of each library was accurately determined by qPCR using the KAPA Library Quantification Kit according to the manufacturer's protocol (KAPA Biosystems / Roche) to generate the appropriate number of clusters for the Illumina NovaSeq6000 instrument. Libraries were pooled and processed using the XP workflow and a 150 x 10 x 10 x 150 sequencing recipe according to the manufacturer's protocol, running on a 0.1 NovaSeq6000 S4 flow cell. Approximately 5 Gb of paired-end sequence was generated for each sample.

[0034] Dysbiosis Index Fecal DNA samples from four groups of dogs with DMVD: Group A (N = 31), Group B1 (N = 35), Group B2 (N = 25), and Group C / D (N = 30) were assessed for the abundance of eight bacterial groups, including total bacteria, Faecalibacterium, Turicibacter, Escherichia coli, Streptococcus, Blautia, Fusobacterium, and Clostridium hiranonis. The qPCR primer set, protocol, and method for the dysbiosis index (DI) were previously described in AlShawaqfeh et al. (2017) FEMS Microbiology Ecology, vol. 93, no. 11, pp. 1–8. A negative DI indicates normobiosis, whereas a positive DI indicates dysbiosis. The reference interval for the beneficial bacterium C. hyranonis was 5.1-7.1.

[0035] Bioinformatics analysis

[0034] Sequence quality was assessed using fastQC. Paired-end sequences were stitched together using PEAR with default settings. Trimmomatic was used to filter out low-quality sequences, while Bowtie2 was run to exclude contaminating sequences that mapped to the canid (CanFam3.1) or PhiX reference genomes from the selection. Low-quality sequences were scanned with a 4-base-wide sliding window and excluded if the average quality score per base fell below 20. Sequences shorter than 50 bases were also excluded. Phylogenetic analysis was performed using MetaPhlAn 2.0, and UniRef90 gene family abundances and MetaCyc pathways were calculated using HUMAN 2.0.

[0036] statistical analysis For cross-sectional studies, multiple group comparisons were performed using the Kruskal-Wallis test. Dunn's multiple comparisons were performed for significant groups. For dietary intervention studies, changes from baseline at 3 and 6 months were calculated for each group. Differences between group means (CPB vs. CON) were then calculated. Positive numbers indicate an increase in CPB from CON, while negative numbers indicate a decrease.

[0037] result Table 1 presents bacterial species with differential abundance among groups A, B1, B2, and C / D, which refer to the four stages of DMVD in canines. Table 2 presents the changes between CPB and CON bacterial abundance at 3 and 6 months, which were normalized to baseline values ​​using calculated mean values. Twenty-three bacterial species varied among the four groups of dogs with DMVD (Table 1). Seven bacteria whose abundance decreased with DMVD severity across groups: B. plebeiu, A. stercolicanis, E. biforme, B. coprocola, B. pluricorum, C. mitsuoka, and P. copri, were increased in abundance in CPB-fed dogs compared with CON-fed dogs, whereas the abundance of Bacteroides vulgatus was decreased in CPB-fed dogs compared with CON-fed dogs (Table 2).

[0038] [Table 1]

[0039] [Table 2]

[0040] Table 3 presents bacterial genera with differential abundance among groups A, B1, B2, and C / D, which represent the four stages of DMVD in canines. Table 3 presents the changes between CPB and CON bacterial abundance at 3 and 6 months, which were normalized to baseline values ​​using calculated mean values. Three bacterial genera, Prevotella, Catenibacterium, and Allobaculum, decreased in abundance with DMVD severity (Table 3), but their abundance increased in dogs fed the CPB diet compared with the CON diet (Table 4).

[0041] [Table 3]

[0042] [Table 4]

[0043] The DI (log DNA / gram of feces) in healthy dogs in group A was -1.48, but increased to -0.6 and -0.07 in dogs with preclinical DMVD in groups B1 and B2, respectively, and to 1.47 in dogs in groups C / D with congestive heart failure. Therefore, the DI can be used as an early indicator of DMVD in dogs (Table 5). In addition, the abundance of Clostridium hyranonis, a beneficial bacterium that converts primary bile acids to secondary bile acids, was within the reference range in healthy dogs but outside the range in dogs with DMVD (Table 5).

[0044] [Table 5]

[0045] It should be understood that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the present subject matter and without diminishing its intended advantages. Accordingly, it is intended that such changes and modifications be covered by the appended claims.

Claims

1. 1. A method for diagnosing heart disease in a canine, comprising: measuring the normalized relative abundance of fecal bacteria, including Faecalibacterium, Turicibacter, Streptococcus, Escherichia coli, Blautia, Fusobacterium, and C. hyranonis; calculating a dysbiosis index based on the fecal bacteria; determining that the canine has heart disease if the dysbiosis index is greater than -1.0; A method comprising:

2. 10. The method of claim 1, further determining that the canine has early degenerative mitral valve disease if the dysbiosis index is between -1 and 0.

5.

3. 3. The method of claim 2, further determining that the early degenerative mitral valve disease is B1 if the dysbiosis index is between -1 and -0.

25.

4. The method of claim 2, further determining that the early degenerative mitral valve disease is B2 if the dysbiosis index is between -0.25 and 0.

5.

5. 10. The method of claim 1, further determining that the canine has congestive heart failure if the dysbiosis index is greater than 0.

5.

6. 1. A method for diagnosing early degenerative mitral valve disease in a canine, comprising: measuring the normalized relative abundance of a biomarker selected from the group consisting of Catenibacterium mitsuokai, Butyricicoccus plicaecorum, Bacteroides coprocola, Bacteroides plebeius, Allobaculum stercolicanis, or a combination thereof; determining that the canine has early degenerative mitral valve disease if the normalized relative abundance of the Catenibacterium mitsuokai is 0.3 to 3, the normalized relative abundance of the Butyricicoccus plicaecorum is 0.14 to 0.35, the normalized relative abundance of the Bacteroides coprocola is 0.6 to 1.3, the normalized relative abundance of the Bacteroides plebeius is 0.1 to 0.8, or the normalized relative abundance of the Allobaculum stercolicanis is 0.1 to 1.5; A method comprising:

7. 7. The method of claim 6, wherein said determining step is based on at least two biomarkers.

8. 7. The method of claim 6, wherein said determining step is based on at least three biomarkers.

9. 7. The method of claim 6, wherein said determining step is based on at least four biomarkers.

10. 1. A method for diagnosing early degenerative mitral valve disease in a canine, comprising: measuring the normalized relative abundance of bacteria selected from the group consisting of Catenibacterium, Prevotella, Butyricicoccus, Faecalibacterium, Clostridium, Allobaculum, or a combination thereof; determining that the canine has early degenerative mitral valve disease if the normalized relative abundance of the Catenibacterium bacteria is 0.3 to 3, the normalized relative abundance of the Prevotella bacteria is 0.5 to 4, the normalized relative abundance of the Butyricicoccus bacteria is 0.14 to 0.4, the normalized relative abundance of the Faecalibacterium bacteria is 0.012 to 0.04, the normalized relative abundance of the Clostridium bacteria is 2 to 4, or the normalized relative abundance of the Allobaculum bacteria is 0.2 to 1.5; A method comprising:

11. The method of claim 10 , wherein the determination is based on at least two genera.

12. The method of claim 10 , wherein the determination is based on at least three genera.

13. The method of claim 10 , wherein the determination is based on at least four genera.

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