Method for the early diagnosis of disease in graminivores

Abstract

The invention relates to a method for the early detection of the presence of disease in graminivores, comprising the following steps: a) determining the calcium isotope ratio δ44 / 42Ca in a blood serum sample from the animal, and / or b) determining the copper isotope ratio δ65 / 63Cu in a blood serum sample from the animal, and / or c) determining the calcium concentration in a blood serum sample from the animal, d) comparing the relevant determined sample value with a fixed threshold value specific to the determined sample type, wherein the presence of disease in the graminivore is ruled as unlikely if a1) the sample value of the calcium isotope ratio δ44 / 42Caserum is above the fixed threshold value, and / or b1) the sample value of the copper isotope ratio δ65 / 63Cu is below the threshold value, and / or c1) the sample value of the calcium concentration is above the fixed threshold value.

Description

[0001] Title: Method for the early diagnosis of disease in graminivores

[0002] The invention relates to a method for the early diagnosis of a disease in graminivores.

[0003] Many diagnostic procedures based on the analysis of blood or blood serum parameters are known, even for animals. However, the starting point is almost exclusively identified symptoms with a suspicion of a specific disease. Therefore, the known diagnostic procedures are always directed at a specific disease whose biological marker is known and determined as part of the procedure. Likewise, only a few diagnostic procedures are described specifically for graminivores, especially for horses. A good overview is provided, for example, in the specialist article "Biomarkers in Equine Medicine," by Svenja Möller, Laboklin aktuell, July 2020 issue. Another field for the application of blood tests in horses is doping analyses of sport horses. Test kits have already been developed for this purpose and are also used as part of a purchase medical examination. They test for substances that could potentially falsify the results of this test.The focus here is primarily on analgesic substances, which can mask, for example, mild lameness. However, sedatives and psychotropic drugs have also been shown to mask behavioral abnormalities.

[0004] Balter et al. (Metallomics, 2013, 5, 1470-1482) described various Cu, Fe, and Zn isotope determinations in mice and sheep, which provided evidence of isotope-preferential uptake in various organs. A correlation to disease was not investigated.

[0005] The LABOKLIN (Laboratory for Clinical Diagnostics GmbH & Co. KG) manual: Horse Service List 2023 / 2024, Bad Kissingen, 2023, 140 pages, describes the determination of calcium concentration in the blood serum of horses using photometry and provides a normal range. However, the detection of values ​​outside the normal range, which according to the manual can be below or above the normal range, does not provide a sufficient basis for a qualitative statement about the horse's health status. Accordingly, the manual does not provide any information on this.

[0006] DE 10 2018 214 660 A1 of the applicant describes a diagnostic method for humans with regard to diseases associated with reduced bone density and / or calcium loss, wherein the threshold value is selected independently of one or more factors attributable to the individual patient. The sample values ​​are determined in previously collected blood, urine, and / or stool samples. In addition to the calcium concentration, the isotope ratio of Ca isotopes and the ratio of calcium to strontium are specified as sample values. The threshold values ​​disclosed here refer exclusively to humans and cannot be transferred to animals, in particular due to the completely different metabolic processes and the different effects of diseases on the aforementioned parameters in humans and animals.

[0007] However, with regard to a rapid and initially qualitative assessment, for example, in the context of a sale of an animal, and especially in valuable animals such as horses, whether it is very likely that a disease is present or not, without targeting a specific disease, no method for early qualitative diagnosis or screening is currently available. However, in the diagnosis of cancerous tumors, other simple and rapid methods specifically for graminivores are still little known.

[0008] The object of the present invention is therefore to provide a method for early diagnosis in graminivores that provides reliable indications of the presence of a disease. The method should be minimally invasive, infinitely repeatable, and applicable in a decentralized manner. Furthermore, the method should be feasible without requiring clinical infrastructure. The use of the method for the detection of tumors and for monitoring therapy during tumor treatment is also desirable.

[0009] The object is achieved by a method according to claim 1. The invention relates to a method for the early detection of the presence of a disease in graminivores, comprising the following steps: a) Determining the calcium isotope ratio 8 44 / 42 Ca in a blood serum sample of the animal, and / or b) determining the copper isotope ratio 8 65 / 63Cu in a blood serum sample of the animal, and / or c) determining the calcium concentration in a blood serum sample of the animal, d) comparing the respective determined sample value with a fixed threshold value specific for the particular sample type, whereby the presence of a disease of the graminivore is excluded as unlikely if al) the sample value of the calcium isotope ratio 8 44 / 42 Ca Serum is above the fixed threshold, and / or bl) the sample value of the copper isotope ratio 8 65 / 63 Cu Serum is below the threshold value, and / or cl) the sample value of the calcium concentration is above the fixed threshold value.

[0010] Graminivory is a technical term that refers to the diet of herbivorous animals that feed primarily on grasses. These herbivores have specialized in digesting grasses and have developed anatomical and physiological adaptations to efficiently extract nutrients from the grasses.

[0011] An example of a graminivorous animal is cattle. Cattle have a specialized digestive system that helps them break down the cellulose in grasses. They have a multi-chambered stomach known as the rumen. Fermentation occurs in the rumen, where microorganisms break down the cellulose into simpler compounds that can be digested by the cow. Another important example of a graminivore is the horse, which, unlike the cow, is a enteric digester. Graminivorous herbivores have longer digestive tracts to give food materials more time to be digested. This allows them to absorb the nutrients from the grasses more efficiently. Some graminivorous animals also have specialized teeth or tooth structures that help them grind and chew grasses.

[0012] Overall, graminivory is a specialized form of herbivorous nutrition in which animals focus on digesting grasses. These adaptations allow them to efficiently utilize the nutrients from grasses and meet their nutritional needs. Some examples of animals that specialize in graminivory are cows, horses, and sheep.

[0013] Some herbivores specialize in both graminivory and other types of diet. They have a wider range of foods they can consume and are able to adapt their diets depending on availability. This makes them generalists and allows them to survive in diverse environments.

[0014] Some herbivores have also evolved a specific preference for certain types of grasses. For example, some species of horses may have a preference for lush pasture grasses, while other species of herbivores prefer dry steppe grasses. These preferences may be due to the varying nutrient levels and availability of grasses in different habitats. Specializing in a grass diet can be advantageous for herbivores because grasses are often rich in energy and nutrients. However, it can also present challenges because grasses are sometimes difficult to digest and require specialized digestive mechanisms. To support their specialized diet of grasses, some herbivores have evolved a complex digestive system to help them efficiently extract nutrients from grasses.For example, ruminants such as cattle and sheep have a rumen that allows them to ferment grass and better utilize the nutrients it contains.

[0015] The term "fixed threshold" in the context of the present invention is understood in particular to mean that neither an animal-related baseline with regard to the sample values ​​to be determined needs to be determined beforehand, nor are further factors such as a nutritional plan to be followed before the actual diagnosis or similar factors or measurements of previous values ​​of the individual animal carried out or included in the diagnostic or screening procedure. In other words, the fixed threshold is a respective fixed numerical value depending on the sample type of the calcium isotope ratio, the copper isotope ratio, or the calcium concentration. In particular, the respective threshold is not simply an average from a plurality of samples; rather, within the scope of the invention, it was surprisingly possible to determine a fixed threshold for each type of determination through extensive studies and validations.

[0016] According to the invention, the presence of a disease of the graminivore is excluded as unlikely if al) the sample value of the calcium isotope ratio 8 44 / 42 Ca Serum is above the fixed threshold, and / or bl) the sample value of the copper isotope ratio 8 65 / 63 Cu Se rum is below the threshold value, and / or cl) the sample value of the calcium concentration is above the fixed threshold value.

[0017] If the above procedure gives a value which a2) is considered as sample value of the calcium isotope ratio 8 44 / 42 Ca Serum is below the fixed threshold, and / or b2) as sample value of the copper isotope ratio 8 65 / 63 Cu Serumis above the threshold value, and / or c2) the sample value of the calcium concentration is below the fixed threshold value, testing for a specific disease and / or therapy for the graminivore is carried out.

[0018] Within the scope of the present invention, a method is thus provided for the first time for early diagnosis or screening in graminivores, providing reliable indications of the presence of an as yet unspecified disease. The method is minimally invasive, requiring only a single blood draw, is infinitely repeatable, and can be applied decentrally. Furthermore, the method can be carried out without clinical infrastructure. The method can also be used to detect tumors and monitor therapy during the treatment of tumors. In particular, the Ca isotope ratio allows for early detection of tumors of the musculoskeletal system and metastases affecting the bones of various types of cancer.

[0019] A study demonstrated that for each of the claimed biomarkers—i.e., for the Ca isotope ratio, the Cu isotope ratio, and the blood calcium concentration—a fixed threshold exists that is independent of individual factors such as natural feeding, and that is not derived from a mean value for a comparison group, which would change accordingly if the comparison group were structured differently. It can therefore be stated that the identification of such a fixed threshold overcomes a previously existing prejudice that the aforementioned threshold values ​​for the Ca and Cu isotope ratios and the blood calcium concentration depend on individual factors such as natural feeding or other comparisons.Excluded are targeted influences, for example on calcium or copper intake through corresponding nutritional supplements, which are intentionally carried out by the animal's owner and therefore cause the animal's corresponding values ​​to deviate from those values ​​that the animal would exhibit without this targeted intake of supplements. In such a case, such deviations are also due to the fact that the isotope ratios of calcium and copper in the nutritional supplements can differ from the isotope ratios of the natural feed. However, the method according to the invention can also be applied to animals taking artificial nutritional supplements. However, additional measurements would have to be taken to correct the fixed threshold value according to the deviation caused by the unnatural food addition.

[0020] Overall, each of the threshold values ​​mentioned exhibits such good specificity and sensitivity that it can be used as the sole diagnostic or screening value to reliably provide a rapid determination of the presence or absence of disease in a grazing animal. To achieve further improved diagnostic performance, it is equally possible to compare any combination of two or all three sample values ​​with the respective threshold values ​​and evaluate them according to the criteria specified in the claim.

[0021] The term "excluded as unlikely" is also to be understood in the aforementioned sense of the good specificity and sensitivity of the claimed diagnostic or screening values. It is well known that in medical statistics, a complete "exclusion" of the presence or absence of a disease is very difficult. Therefore, in medicine, one speaks of a "positive predictive value" (PPV) and a "negative predictive value" (NPV), which indicate how many of the tested subjects (here animals) are actually ill if the diagnostic value lies in the corresponding "ill" range, or how many animals are actually not ill if the diagnostic value lies in the corresponding "healthy" range. If these statistical values ​​PPV and NPV are high, the probability that the diagnostic value indicates the correct finding is correspondingly high. As will be shown below using the example, for all claimed diagnostic or screening values,Screening values ​​or biomarkers, i.e., for the Ca isotope ratio, the Cu isotope ratio, and the calcium concentration, yield very good PPV and NPV values. Therefore, the term "excluded as unlikely" should be understood in this sense.

[0022] For the purposes of the present invention, the term disease is understood in particular to mean a disease that lies within the scope of the organic and metabolism-related aspects of the animal and in particular does not relate to the mechanical musculoskeletal system. In other words, the term disease includes in particular diseases of the organs, the metabolism and above all tumor diseases, but not diseases due to disturbed mechanical processes caused, for example, by bone fractures, bruises, tendon injuries, etc. The significance of the claimed biomarkers therefore relates in particular to diseases that are related to the calcium and / or copper balance of the animal or interact with it. Important examples include all diseases caused by tumors metastasizing in the bones or non-specific organ failure, including liver or kidney failure.

[0023] In other words, the present method differs from the previously known

[0024] Analysis of the calcium concentration in the blood of horses is characterized by the fact that the sample value is not compared with a normal interval, such as the interval of 2.50 to 3.40 mmol / L as described in the Laboklin laboratory manual, but with a single fixed threshold. According to the invention, the fixed threshold is significant for ruling out disease if the sample value is above the threshold. This significance is also surprising compared to previous expert knowledge, as no corresponding statement has yet been made as to whether a sample value above or below the normal range can even indicate disease.

[0025] The determination of copper isotope ratios in sheep has so far been purely scientific research aimed at investigating isotope-preferential uptake in various organs and has not provided any screening or early diagnostic information regarding the presence or exclusion of disease. Against this background, the method of the present invention differs significantly from previous expertise.

[0026] In a preferred embodiment of the invention, the isotope ratios or quantity ratios of calcium and copper isotopes are determined using mass spectrometry.

[0027] In an alternative embodiment of the invention, the isotope ratios or quantity ratios of calcium isotopes are determined using laser-induced fluorescence.

[0028] The procedure for determining the isotope ratios or quantity ratios of calcium isotopes using laser-induced fluorescence can be found, for example, in US10302565B2.

[0029] In addition to mass spectrometric methods, spectroscopic methods can also be used to determine the isotopic ratios or quantity ratios of calcium isotopes. These methods utilize the mass dependence of the hyperfine structure of the spectral lines by means of light emission. m3 / m 2

[0030] The o Ca values ​​can also be applied to other Ca isotope ratios (for example, involving 40 Approx. 41 Approx. 46 Approx. 48 Approx. 43 Ca) transferred: with m3 > m2 > ml.

[0031] In this way, other calcium isotope ratios can also be determined in accordance with the invention, which can then be applied to the claimed 8 44 / 42 Ca Serum value of step ii).

[0032] In one embodiment of the method according to the invention, the fixed threshold value of the calcium isotope ratio 8 44 / 42 Ca Serum -0.065%o.

[0033] In a further embodiment of the method according to the invention, the fixed threshold value of the copper isotope ratio is 5 65 / 63 Cu Ser by -0.955%o.

[0034] In a further embodiment of the method according to the present invention, the fixed threshold value of the calcium concentration in the blood serum is 2.835 mmol / L.

[0035] In a preferred embodiment of the invention, the calcium isotope ratio ö 44 / 42 Ca Serum and the copper isotope ratio 5 65 / 63 Cu Ser and compared with the respective threshold values.

[0036] This configuration proves to be particularly advantageous, since, on the one hand, it significantly increases the probability of prediction and, on the other hand, because the two isotope ratios primarily capture the disease process in different affected areas of the animal better than the other. While the calcium isotope ratio is particularly good at diagnosing tumor diseases of various types of cancer, especially cancers metastasizing to bones, the copper isotope ratio appears to have a particularly good predictive value for non-specific organ insufficiency, especially for liver and / or kidney diseases and metabolic disorders. In a preferred embodiment of the invention, the calcium isotope ratio 6 44 / 42 Ca Serum and the calcium concentration was determined and compared with the respective threshold values.

[0037] In this design, the reliability of the prediction is also increased compared to recording and comparing only one value.

[0038] In a preferred embodiment of the invention, the copper isotope ratio ö 65 / 63 Cu Serum and the calcium concentration was determined and compared with the respective threshold values.

[0039] This design also proves to be particularly advantageous, both because it further increases the probability of prediction and because the two sample values ​​primarily capture the disease process in different affected areas of the animal better than the other. While the calcium concentration is particularly effective in diagnosing tumors in the bones of metastatic cancers, the copper isotope ratio appears to have a particularly good predictive value for liver and kidney diseases and metabolic disorders.

[0040] In a further embodiment, the method of the present invention is preferably used for the early detection of a tumor disease, a metabolic disease or a liver or kidney disease.

[0041] In the case of sick animals undergoing treatment, the procedure also allows therapy and medication monitoring, which allows the veterinarian to adapt the treatment to the individual animal.

[0042] With regard to calcium-related metabolic disorders, the use of biomarkers allows for early detection of bone loss, potentially eliminating the need for imaging and invasive procedures using electromagnetic (X-ray) radiation. Unlike invasive imaging procedures, minimally invasive biomarkers can be used according to veterinary guidelines.

[0043] The procedure is environmentally and climate-friendly, as it minimizes animal transport to specialized veterinary clinics. Due to its disruptive nature, the number of specialized diagnostic devices will be reduced.

[0044] The graminivore is preferably a horse, a cattle, a sheep, a goat or a donkey, and especially a horse.

[0045] In particular, by analyzing calcium and copper isotope ratios, the method according to the invention can:

[0046] Subtle metabolic changes can be detected that are not visible with conventional concentration measurements.

[0047] Of the 8 measured 44 / 42 Ca Ser um values, the fixed threshold for "sick" and "healthy" can be

[0048] -0.065 %o with a sensitivity of 96.0% and a specificity of 85.7% (see also the following examples).

[0049] Of the 8 measured 65 / 63 Cu Ser um values, the threshold for "sick" and "healthy" could be

[0050] -0.955 %o with a sensitivity of 76% and a specificity of 85.7%.

[0051] The threshold value for Ca concentration in blood serum is 2.835 mmol / L with a sensitivity of 76.0% and a specificity of 78.6%.

[0052] Dynamic processes such as bone remodeling or mineralization can be directly assessed using calcium isotope values, which is particularly important in chronic diseases or during therapy.

[0053] In addition, general organ failure can be assessed in a differentiated manner based on copper isotopy, as this manifests itself in changes in copper isotopy.

[0054] Due to the high sensitivity, particularly for calcium isotopes, equine diseases can be diagnosed early, before they become apparent clinically or due to concentration deviations from a normal range. Treatment can therefore begin very early. Furthermore, the invention, particularly in embodiments that combine the determination of calcium concentration with a calcium or copper isotope ratio measurement, enables differentiated diagnostics (differential diagnostics) that distinguish between different equine diseases that may have the same effect on calcium concentration. This is crucial because similar concentration values ​​in standard tests can be attributed to completely different disease mechanisms. Subsequently, a targeted therapy can be selected and implemented.

[0055] EXAMPLES Test animals and method: 39 horses were included in the study. Of the animals, the age of 23 and the health status of 39 are known. With regard to their health status, the horses were characterized either as "apparently healthy," which was the case for 14 animals, or as "confirmed sick," which was the case for 25 animals. With regard to "confirmed sick," various other diseases were specified - in addition to various types of cancer - and known prior to the blind determination of the samples. In the blood serum of 39 animals, Ca and Cu isotope values ​​of the blood serum were determined as 5 44 / 42 Ca Serum in Kiel or as ö 65 / 63 Cu in Lyon (France) was determined using mass spectrometry. Additionally, the Ca concentration in blood serum was determined (see Table 1).

[0056] Table 1 (measurements of the tested horses, age, sex as far as known, and status):

[0057] Descriptive statistics of calcium and copper isotope measurements The descriptive statistics were based on five characteristics: age, serum Ca concentration, 8 44 / 42 Ca Serum and 8 65 / 63 Cu Se rum values ​​were applied. All data are normally distributed according to the Kolgorov-Smirnova and Shapiro-Will tests. The main result is that the mean values ​​of the horses characterized as "confirmed sick" are similar for both the Ca concentration in the blood sera and in the 5 44 / 42 Ca Se around and 8 65 / 63 Cu S erum values ​​differ significantly from the animals characterized as "apparently healthy" (both p<0.001). There is no distinction between the two groups with regard to age (p<0.543). The results of the descriptive statistics are shown in Figures 1a to 1d.

[0058] Thresholds for the 8 44 / 42 Ca Se rum - and the 8 65 / 63 Cu Se rum values

[0059] Of the 8 measured 44 / 42 Ca Serum -values, the fixed threshold was determined to be -0.065%o with a sensitivity of 96.0% and a specificity of 85.7%. Of the 8 measured 65 / 63 Cu Serum The threshold value was determined to be -0.955% with a sensitivity of 76% and a specificity of 85.7%. The threshold value for serum Ca concentration is 2.835 mmol / L with a sensitivity of 76.0% and a specificity of 78.6%.

[0060] Figures 2 and 3: this figure shows the sorted distribution of 8 44 / 42 Ca Se rum values ​​and 8 65 / 63 Cu Serum -values ​​and the characteristic "apparently healthy" and "confirmed sick" specified by cancer and other diseases ("other disseases").

[0061] The relationship between the isotope ratio values, the different diseases and the respective threshold value is shown graphically in Figures 2 and 3. It can be seen in Figure 2 that, with the exception of one horse, all horses diagnosed as "apparently healthy" were above the 8 44 / 42 Ca Serum - threshold and all but two of the horses characterised as "confirmed sick" are below the threshold.

[0062] It is clearly visible that the respective threshold value (gray horizontal line) separates these features. It should be noted that the Ca and Cu isotope ratio thresholds separate differently. For the 8 44 / 42 Ca Serum All values ​​higher than the threshold are considered healthy. For the 8 65 / 63 Cu Se For values, the opposite is true; all values ​​below the threshold are considered healthy. Positive (PPV) and Negative Predictive (NPV) values ​​with regard to all unspecified diseases

[0063] The positive predictive value (PPV) indicates how many individuals in a group actually have a particular tested trait. The negative predictive value (NPV) indicates how many individuals actually do not have a tested trait. With regard to the tested horse population, the following values ​​result for unspecified diseases:

[0064] Table 2: PPV and NPV for all unspecified diseases

[0065] The table shows that the Ca isotope has the highest PPV value of 93% with regard to the characteristic "apparently sick". This means that the animals tested as positive, i.e. sick, by the Ca isotopes are also sick with a probability of 93%. The same applies to the NPV. This means that all animals tested as non-positive, i.e. "apparently healthy" by the Ca isotope, are also healthy with a probability of 93%. In addition to the calcium isotopes, the copper isotope and the calcium concentration also show relatively high PPV and NPV values.

[0066] Positive and Negative Predictive Values ​​with Regard to Unspecified Cancer Only In the following, the PPV and NPV values ​​were specified with regard to the characteristic cancer, but applied without differentiating between cancer types.

[0067] Table 3: PPV and NPV for all unspecified diseases

[0068] Table 3 shows that the Ca isotope has the highest PPV value of 94% with regard to the characteristic "cancer." This means that the animals tested as positive for the Ca isotopes, i.e., suffering from cancer, are also sick with a 94% probability. The same applies to the NPV. This means that all animals tested as non-positive, i.e., healthy and not suffering from cancer, using the Ca isotope are also healthy with a 93% probability. In addition to the calcium isotopes, the copper isotopes also show relatively high PPV and NPV values. The PPV and NPV values ​​of the Ca concentration, on the other hand, are lower, particularly with regard to the PPV.

Claims

-22- Patent claims 1. A method for the early detection of the presence of a disease in graminivores comprising the following steps: a) Determining the calcium isotope ratio 5 44 / 42 Ca in a blood serum sample of the animal, and / or b) determining the copper isotope ratio ö 65 / 63 Cu in a blood serum sample of the animal, and / or c) determining the calcium concentration in a blood serum sample of the animal, d) comparing the respective determined sample value with a fixed threshold value specific for the particular sample type, whereby the presence of a disease of the graminivore is excluded as unlikely if al) the sample value of the calcium isotope ratio 6 44 / 42 Ca Serum is above the fixed threshold, and / or bl) the sample value of the copper isotope ratio ö 65 / 63 Cu Serumis below the threshold value, and / or cl) the sample value of the calcium concentration is above the fixed threshold value.

2. The method according to claim 1, wherein the fixed threshold value of the calcium isotope ratio is 5 44 / 42 Ca Serum -0.070 ± 0.06 %o.

3. The method according to claim 1, wherein the fixed threshold value of the copper isotope ratio is 5 65 / 63 Cu is -0.960 ± 0.06 %o.

4. The method of claim 1, wherein the fixed threshold calcium concentration is 2.835 mmol / L.

5. A method according to any one of the preceding claims, wherein the calcium isotope ratio is 544 / 42ca Serum and the copper isotope ratio 6 65 / 63 Cu and compared with the respective threshold values.

6. A method according to any one of claims 1 to 4, wherein the calcium isotope ratio ö 44 / 42 Ca Se . r umand the calcium concentration are determined and compared with the respective threshold values.

7. A method according to any one of claims 1 to 4, wherein the copper isotope ratio ö 65 / 63 Cu and calcium concentration are determined and compared with the respective threshold values.

8. Method according to one of the preceding claims, wherein the method is used for the early detection of a tumor disease, a metabolic disease or a liver or kidney disease.

9. Method according to one of the preceding claims, wherein the graminivore is a horse, a cattle, a sheep, a goat or a donkey, in particular a horse.

10. Method according to one of the preceding claims, wherein a test for a specific disease and / or a therapy of the graminivore is carried out if a2) the sample value of the calcium isotope ratio 6 44 / 42 Ca Serumis below the fixed threshold, and / or b2) the sample value of the copper isotope ratio ö 65 / 63 Cu Serum is above the threshold value, and / or c2) the sample value of the calcium concentration is below the fixed threshold value.

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