Landless Monogastric Animal Production System

Abstract

The present invention relates to a preferably computer-implemented method of raising a group of monogastric animals Issues such as exposure of the animals to an initiator of a disease are recognized at an early stage by ex vivo analysis of blood samples. Said analysis is done where the animals are raised using point-of-care devices. Accuracy of separation between affected and non-affected animals is increased by measuring a set of pre-determined blood parameters. The landless monogastric animal production system of the invention facilitates implementation of the method of the invention.

Claims

1. A method of raising a group of monogastric animals of a single species and a single breed comprising the steps: receiving, from a point-of-care device, values of blood parameters from at least one member of the group of monogastric animals; analyzing values of at least two pre-determined blood parameters to determine if the values of the at least two pre-determined blood parameters deviate from pre-determined ranges in a pre-determined manner; and based on the analyzing, adapting feed that is fed to the group of monogastric animals if the values of the at least two pre-determined blood parameters deviate from the pre-determined ranges in the pre-determined manner.

2. The method of claim 1, wherein the group of monogastric animals consist of animals of a single sex and a single breed.

3. The method of claim 1, wherein the pre-determined ranges of the blood parameters originate from the group of monogastric animals having a pre-determined age range.

4. The method of claim 1, wherein the values of the blood parameters depend on a state of causal variable, and wherein the causal variable is selected from group consisting of heat stress, humidity, nutritional inadequacy, transport stress and exposure to an initiator of a disease.

5. The method of claim 4, wherein the exposure to the initiator of the disease causes a clinical or a sub-clinical disease, and wherein the disease is preferably coccidiosis.

6. A computer-implemented method comprising: providing values of blood parameters that have been measured ex vivo in blood of at least one member of a group of birds; providing a model which defines values of blood parameters that are out of range birds suffering from coccidiosis; using the model to determine if the group of birds is in need of an additive that treats or prevents coccidiosis; and after a determination that the group of birds is in need of the addictive, sending, to a user device, a recommendation for adding the additive to feed that treats or prevents coccidiosis.

7. The method of claim 6, wherein the additive is a feed additive that comprises one or more microbial muramidases obtained or obtainable from phylum Ascomycota, or subphylum Pezizomycotina.

8. The method of claim 6, wherein the group of birds is a group of chicken of a single breed and a single age.

9. The method of claim 6, wherein the values of the blood parameters have been measured ex vivo in the blood of chicken between 9 and 21 days old.

10. The method of claim 6, wherein the values of the blood parameters comprise a total amount of carotenoids in blood serum that has been separated from whole blood.

11. A landless monogastric animal production system comprising: at least 1,000 monogastric animals of a single species; an apparatus to draw venous blood from at least one of the monogastric animals; at least one point-of-care device suitable for measuring values of blood parameters from the venous blood; at least one empty blood collection tube into which the venous blood is deposited; the tube containing at least one additive comprising heparin or ethylenediaminetetraacetic acid; and at least one mobile device coupled to the at least one point-of-care device.

12. The landless monogastric animal production system of claim 11, wherein the system comprises a farm that introduces at least one ton of feed/year from outside the farm, preferably by buying feed from an external supplier.

13. The landless monogastric animal production system of claim 11, wherein the system further comprises an apparatus to separate blood serum from whole blood.

14. The landless monogastric animal production system of claim 11, the system further comprising: at least one point-of-care device that is suitable for ex vivo measuring of electrolytes in a blood sample; and at least one point-of-care device that is suitable for measuring a total amount of carotenoids in blood serum that has been separated from whole blood.

15. The landless monogastric animal production system of claim 11, further comprising: at least one supplier of feed additives, premixes and/or feed, wherein the at least one mobile device of the landless monogastric animal production system is in communication with the at least one supplier of feed additives, premixes and/or feed.

16. The method of claim 1, further comprising: analyzing values of at least three pre-determined blood parameters selected from a group consisting of sodium, potassium, globulin, carotenoid and anion gap.

17. The method of claim 6, wherein the blood parameters comprise at least three pre-determined blood parameters selected from a group consisting of sodium, potassium, globulin, carotenoid and anion gap.

18. The method of claim 6, wherein the group of birds is a group of chicken of a single breed, a single age, and a single sex.

19. The method of claim 9, wherein the blood parameters have been measured in the blood of chicken between 10 and 18 days old.

20. The method of claim 9, wherein the blood parameters have been measured in the blood of chicken of 13 days old.

Description

FIGURES

[0156] FIG. 1 illustrates how the pre-determined parameters for a given challenge can be found in a pre-study. Such pre-study was done in Example 2.

[0157] FIG. 2 illustrates the method of the invention. It is a method of raising a group of monogastric animals of same species and same breed.

[0158] FIG. 3 illustrates a preferred embodiment of the set-up of the invention. The point-of-care device is located where the animals are raised and is connected to a mobile device (e.g. to the chicken farmer's cell phone). If action is needed, the mobile device intervenes by ordering a feed additive that is suitable for mitigating the detected issue. The supplier executes the order by delivering the feed additive to where the animals are raised.

[0159] FIG. 4 shows blood parameters that can be measured with i-Stag Alinity v handheld blood analyzer fitted with a Chem8+ cartridge. More detailed information is published in Cartridge and test information, Rev. Date: 15 Aug. 2016, Art: 714258-010, available at Abbott and in the brochure Introducing our easiest device yeti-STAT Alinity, available at Abbott and also downloadable at www.pointofcare.abbott. Additional information can also be found in The User Manual i-STAT Alinity v Analyzer, intended for veterinary use only, 650-7100 Rev. A, Rev Date: 19 Jan. 2018, available at Abaxis, Inc., 3240 Whipple Road, Union City, CA 94587, USA (www.abaxis.com).

[0160] FIG. 5 shows blood parameters that can be measured with Vetscan? VS2 Chemistry Analyzer (Abaxis, Inc) using the Avian/Reptilina Profile Plus cartridge. For details, see Operator's Manual VertScanVS2, dated February 2009, available at Abaxis, Inc., Union City, CA 94587 and in the publication VetScan? Avian Reptilian Profile Plus, dated March 2007, PN: 500-7131, Rev: D.

[0161] FIG. 6 shows the effect of age and a mixed-species coccidial challenge (introduced on d7) on plasma potassium (K) concentration in male Cobb 500 broiler chickens. A significant age*challenge interaction was observed which was generated by the transient increase around d13.

EXAMPLES

Example 1 (Effect of Age, Sex, and Breed on Blood Parameters)

[0162] The study of Example 1 shows which blood parameters of broilers are influenced by age, sex or breed.

[0163] A model which defines blood parameters that are out of range in case a chicken suffers from coccidiosis becomes more accurate if the results of the study of Example 1 are incorporated into the model.

[0164] Facilities and Rearing

[0165] Ross 708 and Cobb 500 chicks were sourced form a local commercial hatchery, vent-sex sorted, individually neck tagged, and allocated into 40 pens according to sex and breed to create a 2?2 factorial arrangement with 10 pens per treatment group. Pens were of uniform size (1.2 m?1.2 m; 1.82 m 2) with 16 chicks per pen in a closed, tunnel ventilated house. Each pen was supplied with one bell water drinker, one tube feeders, and bedded with fresh pine shavings (15 cm deep). All broiler chicks were assigned to the same corn-soy based diet in starter, grower, and finisher phases (cf. below Table 1).

TABLE-US-00002 TABLE 1 Composition of broiler starter, grower, and finisher diets Starter.sup.5 Grower.sup.6 Finisher.sup.7 Ingredients Corn 57.60 63.85 66.48 Soybean meal (48% CP) 32.02 23.89 21.55 Poultry by-product meal 5.00 5.86 5.22 Poultry fat 2.00 2.51 3.99 Dicalcium phosphate (18.5% P) 1.24 1.16 0.67 Limestone 0.61 0.82 0.66 Salt 0.50 0.50 0.50 Choline chloride (60%) 0.20 0.20 0.20 Vitamin premix.sup.1 0.05 0.05 0.05 Mineral premix.sup.2 0.20 0.20 0.20 Selenium premix.sup.3 0.05 0.05 0.05 DL-Methionine 0.23 0.17 0.11 L-Lysine 0.14 0.20 0.13 L-Threonine 0.11 0.09 0.14 Coccidiostat.sup.4 0.05 0.05 0.05 Total 100.00 100.00 100.00 Calculated nutrient content Crude protein 23.00 20.00 18.50 Calcium 0.90 0.90 0.70 Available phosphorus 0.45 0.45 0.35 Potassium 0.88 0.93 0.72 Total lysine 1.31 1.14 1.00 Total methionine 0.59 0.49 0.42 Total threonine 0.88 0.76 0.76 Total methionine + cysteine 0.95 0.81 0.72 Sodium 0.22 0.22 0.22 Metabolizable energy (kcal/g) 2,935 3,050 3,150 .sup.1Vitamin premix supplied the following per kg of diet: 13,200 IU vitamin A, 4,000 IU vitamin D.sub.3, 33 IU vitamin E, 0.02 mg vitamin B.sub.12, 0.13 mg biotin, 2 mg menadione (K.sub.3), 2 mg thiamine, 6.6 mg riboflavin, 11 mg d-pantothenic acid, 4 mg vitamin B.sub.6, 55 mg niacin, and 1.1 mg folic acid. .sup.2Mineral premix supplied the following per kg of diet: manganese, 120 mg; zinc, 120 mg; iron, 80 mg; copper, 10 mg; iodine, 2.5 mg; and cobalt, 1 mg. .sup.3Selenium premix provided 0.2 mg Se (as Na.sub.2SeO.sub.3) per kg of diet. .sup.4Coccidiostat supplied monensin sodium at 90 mg/kg of food. .sup.5Starter diet was fed to approximately 14 d of age, 910 g per bird. .sup.6Grower diet was fed from approximately 15 to 35 d of age, 2,750 g per bird. .sup.7Finisher diet was fed from approximately 36 to end of experiment.

[0166] Performance and Blood Physiology

[0167] Broiler BW and feed consumption were recorded at 1, 7, 14, 21, 28, 35, and 42 d.sup.1 of age and FCR calculated. At 14, 21, 28, 35, and 42 d of age two broilers per pen were selected for venous blood analysis. Blood was then analyzed as described in Example 2 (see below). .sup.1 d=day(s)

[0168] Statistical Analysis

[0169] Data were analyzed using JMP Pro 14 (available at SAS Institute, Cary, NC 27513-2414, USA GmbH).

[0170] Statistical analysis of the study of Example 1 shows that age has a highly significant impact on selected electrolytes (blood calcium, phosphorus, potassium, sodium, chloride), carotene, aspartate aminotransferase, creatine kinase, bile acids, uric acid, total protein, albumin, globulin, total carbon dioxide, hematocrit, and malondialdehyde.

[0171] Statistical analysis of the study of Example 1 also shows that sex has a significant impact: male broilers had reduced blood sodium and chloride (electrolytes), carotene, uric acid, albumin, and increased total protein, glucose, and total carbon dioxide.

[0172] Statistical analysis of the study of Example 1 also shows that breed has a significant impact: Ross broilers had greater blood potassium and sodium (electrolytes), as well as reduced uric acid, total protein, globulin, and malondialdehyde, compared with Cobb birds.

[0173] The model of the present invention can be used to determine if a group of chicken is in need of an additive that treats or prevents coccidiosis. Such model becomes more accurate if the results of the study of Example 1 are taken into account. This can be done by raising a group of chicken of same sex, same breed and same age and by ex vivo analysis of a blood sample that is drawn when the chicken have reached a pre-determined age.

[0174] Details of the study of Example 1 are shown in below Tables 2, 3, and 4:

TABLE-US-00003 TABLE 2 Effects of breed (Cobb or Ross), sex (Male or Female) and age on blood mineral .sup.vCa .sup.vPHOS .sup.vK .sup.vNa .sup.iNa .sup.iK .sup.iCl .sup.iCa .sup.iAnGap Breed Sex Age (mg/dL) (mmol/L) Cobb 11.52 7.27 7.36 148.1 143.6 4.93 108.3 1.36 16.2 Ross 11.48 7.32 8.12 149.2 144.1 4.96 108.4 1.35 16.4 P< NS NS <0.001 NS 0.011 NS NS NS NS Male 11.48 7.35 7.78 148.3 143.6 5.01 107.6 1.35 16.4 Female 11.53 7.23 7.70 149.0 144.1 4.88 109.1 1.36 16.3 P< NS NS NS NS 0.015 NS 0.009 NS NS 14 d 11.43bc 6.11.sup.c 6.69 146.1 140.9.sup.c 5.55.sup.a 110.6.sup.a 1.26.sup.c 12.9.sup.b 21 d 11.97.sup.a 7.45.sup.ab 7.14 149.6 144.0.sup.b 5.11.sup.b 108.3.sup.ab 1.39.sup.a 17.2.sup.a 28 d 11.26.sup.bc 7.91.sup.a 8.17 148.1 143.5.sup.b 4.63.sup.cd 107.0.sup.b 1.33.sup.b 16.9.sup.a 35 d 11.72.sup.ab 7.26.sup.b 9.07 149.5 145.5.sup.a 4.52.sup.d 106.7.sup.b 1.39.sup.a 16.8.sup.a 42 d 11.13.sup.c 7.74.sup.ab 7.64 149.8 145.3.sup.a 4.94.sup.bc 108.9.sup.ab 1.39.sup.a 17.8.sup.a P< <0.001 <0.001 <0.001 0.022 <0.001 <0.001 <0.001 <0.001 <0.001 Linear P< 0.032 <0.001 <0.001 0.012 <0.001 <0.001 0.021 <0.001 <0.001 Quadratic P< 0.027 <0.001 <0.001 NS <0.001 <0.001 <0.001 0.010 <0.001 Model Pooled SEM 0.25 0.32 0.48 1.9 0.5 0.19 1.3 0.02 0.5 Interaction Terms Breed*Age NS NS 0.005 NS NS NS NS NS NS Sex*Age NS NS NS NS NS 0.017 NS NS NS Breed*Sex NS 0.08 NS NS NS NS NS NS NS Breed*Age*Sex NS 0.023 NS NS NS NS NS NS NS composition of broiler chickens fed a nutritionally adequate corn/soy-based diet. .sup.abMeans within a column of 3 or more independent variables lacking a common superscript differ significantly (P < 0.05) Main effect means of breed and sex calculated using n = 200, main effect means of age calculated using n = 80. SEM = Standard error of mean

TABLE-US-00004 TABLE 3 Effects of breed (Cobb or Ross), sex (Male or Female) and age on broiler blood carotene, aspartate aminotransferase (AST), creatine kinase (CK), bile acids (BA), uric acid (UA), total protein (TP), albumin (ALB), globulin (GLOB). Carotene AST CK BA UA TP ALB GLOB Breed Sex Age (mg/kg) (U/L) (?mol/L) (mg/dL) (g/L) Cobb 2.30 393.0 4359 11.37 6.3 3.06 2.33 0.73 Ross 2.30 392.3 4017 10.10 5.7 2.96 2.31 0.64 P< NS NS NS NS 0.003 <0.001 NS <0.001 Male 2.17 386.7 4064 10.28 5.6 3.04 2.28 0.68 Female 2.42 398.0 4313 11.19 6.4 2.97 2.35 0.69 P< <0.001 NS NS NS <0.001 0.011 0.002 NS 14 d 1.49.sup.d 193.7.sup.d 2401 12.6.sup.a 6.2.sup.b 2.67.sup.c 2.20.sup.b 0.45.sup.c 21 d 2.10.sup.c 225.7.sup.d 4908 13.4.sup.a 6.2.sup.b 2.95.sup.b 2.36.sup.a 0.59.sup.b 28 d 2.26.sup.bc 335.6.sup.c 5255 11.4.sup.ab 6.3.sup.b 2.97.sup.b 2.31.sup.a 0.64.sup.b 35 d 3.09.sup.a 494.5.sup.b 9.1.sup.ab 6.9.sup.b 3.25.sup.a 2.39.sup.a 0.87.sup.a 42 d 2.55.sup.b 713.6.sup.a 7.2.sup.b 4.4.sup.a 3.21.sup.a 2.34.sup.a 0.87.sup.a P< <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 Linear P < <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 Quadratic P< <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 NS Model Pooled SEM 0.140 42.79 727 727 727 2.6 0.05 0.06 Interaction Terms Breed*Age NS NS NS NS NS 0.012 NS 0.100 Sex*Age NS NS NS 0.068 0.002 NS 0.016 NS Breed*Sex 0.058 NS NS NS NS <0.001 0.050 0.048 Breed*Age*Sex NS 0.023 0.020 NS NS NS NS NS .sup.abMeans within a column of 3 or more independent variables lacking a common superscript differ significantly (P < 0.05) Main effect means of breed and sex calculated using n = 200, main effect means of age calculated using n = 80. SEM = Standard error of mean

TABLE-US-00005 TABLE 4 Effects of breed (Cobb or Ross), sex (Male or Female) and age on blood mineral composition of broiler chickens fed a nutritionally adequate corn/soy-based diet. .sup.vGlu .sup.iGlu TCO2 HCT MDA Breed Sex Age (mg/dL) (mmol/L) (%) (?M) H L H:L Cobb 235 240 24.9 20.1 0.84 2175 8743 0.25 Ross 239 241 24.7 19.8 0.78 2421 9583 0.25 P< NS NS NS NS 0.047 0.10 0.02 NS Male 239 243 25.1 19.5 0.81 2180 8711 0.25 Female 234 237 24.4 20.3 0.81 2416 9615 0.25 P< 0.033 0.008 0.006 0.052 0.883 0.11 0.010 NS 14 d .sup.235.sup.ab 243 23.5 18.9 1.14 1978 6924 0.28.sup.ab 21 d .sup.240.sup.ab 239 24.5 19.0 1.12 2912 9165 0.32.sup.a 28 d .sup.242.sup.a 244 25.1 20.5 0.63 2777 10720 0.26.sup.bc 35 d .sup.238.sup.ab 240 26.3 21.6 0.48 1182 6388 0.15.sup.d 42 d .sup.231.sup.b 235 24.6 19.7 0.69 2640 12620 0.21.sup.c P< 0.039 0.082 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 Linear P< NS 0.042 <0.001 0.005 <0.001 NS <0.001 <0.001 Quadratic P< 0.003 NS <0.001 0.0121 <0.001 NS NS NS Model Pooled SEM 6 5 0.6 0.9 0.07 332 774 0.03 Interaction Terms Breed*Age NS 0.074 NS 0.089 <0.001 NS NS NS Sex*Age NS NS 0.023 NS 0.77 NS NS NS Breed*Sex NS NS NS <0.001 NS NS NS NS Breed*Age*Sex NS NS NS NS NS NS NS NS .sup.abMeans within a column of 3 or more independent variables lacking a common superscript differ significantly (P < 0.05) Main effect means of breed and sex calculated using n = 200, main effect means of age calculated using n = 80. SEM = Standard error of mean

Example 2 (Early Diagnosis of Coccidiosis by Use of Blood Biomarker Combination)

[0175] The study of example 2 shows that, in a healthy environment, coccidiosis vaccinated birds perform poorer than unvaccinated birds. The possibility of early diagnosis of coccidiosis renders unnecessary vaccination superfluous and thereby boosts performance to the desired level. The study of example 2 also shows that the combination of Na, K, GLOB and/or carotenoid biomarker allows for a precise and early identification of the occurrence of coccidiosis in a broiler population. Early diagnosis allows for early intervention such that performance losses associated with coccidiosis can be successfully mitigated.

[0176] Facilities and Rearing

[0177] A total of 960 Cobb 500 male broiler chicks were obtained from the resident broiler breeder flock (North Carolina State University, Raleigh, NC) and randomly allocated to 48 pens (20 chicks per pen). The experiment comprised two factors, being without or with coccidial vaccination (d1.sup.2; vaccination available at Merck B-52 Coccivac, Merck & Co, Kenilworth, NJ) and without or with a coccidial challenge (d7.sup.3; 10? dose of the same vaccine used on d1 delivered via oral gavage), generating a total of four experimental treatments. The Merck B-52 coccidial vaccine contains live oocysts from Eimeria acervulina, Eimeria maxima, Eimeria maxima MFP, Eimeria mivati and Eimeria tenella. A common starter (d1-14) and grower (d15-35) diet was fed to all pens (cf. below Table 5) on an ad libitum basis. Water was also available ad libitum. Temperature was set at 95? F. for the first 12 hours and was subsequently reduced by 1? F. per day until 70? F. was reached, at which point this temperature was maintained for the remainder of the experiment. Photoperiod was 23 hours light for the first 6 days and was gradually reduced to 16 hours by the end of the experiment. .sup.2 vaccination at day 1, abbreviated as d1; this applies mutatis mutandis the other uses of the abbreviation d for day in this document.sup.3 challenge at day 7, abbreviated as d7; this applies mutatis mutandis the other uses of the abbreviation d for day in this document

TABLE-US-00006 TABLE 5 Composition (%) and calculated nutrient provision (%, unless otherwise stated) of the starter and grower diets. Starter.sup.4 Grower.sup.5 Ingredients Corn 57.65 63.90 Soybean meal (48% CP) 32.02 23.89 Poultry by-product meal 5.00 5.86 Poultry fat 2.00 2.51 Dicalcium phosphate (18.5% P) 1.24 1.16 Limestone 0.61 0.82 Salt 0.50 0.50 Choline chloride (60%) 0.20 0.20 Vitamin premix.sup.1 0.05 0.05 Mineral premix.sup.2 0.20 0.20 Selenium premix.sup.3 0.05 0.05 DL-Methionine 0.23 0.17 L-Lysine 0.14 0.20 L-Threonine 0.11 0.09 Total 100.00 100.00 Calculated nutrient content Crude protein 23.00 20.00 Calcium 0.90 0.90 Available phosphorus 0.45 0.45 Potassium 0.88 0.93 Total lysine 1.31 1.14 Total methionine 0.59 0.49 Total threonine 0.88 0.76 Total methionine + cysteine 0.95 0.81 Sodium 0.22 0.22 Metabolizable energy (kcal/g) 2,935 3,050 .sup.1Vitamin premix supplied the following per kg of diet: 13,200 IU vitamin A, 4,000 IU vitamin D.sub.3, 33 IU vitamin E, 0.02 mg vitamin B.sub.12, 0.13 mg biotin, 2 mg menadione (K.sub.3), 2 mg thiamine, 6.6 mg riboflavin, 11 mg d-pantothenic acid, 4 mg vitamin B.sub.6, 55 mg niacin, and 1.1 mg folic acid. .sup.2Mineral premix supplied the following per kg of diet: manganese, 120 mg; zinc, 120 mg; iron, 80 mg; copper, 10 mg; iodine, 2.5 mg; and cobalt, 1 mg. .sup.3Selenium premix provided 0.2 mg Se (as Na.sub.2SeO.sub.3) per kg of diet. .sup.4Starter diet was fed to 14 d of age. .sup.5Grower diet was fed from 15 to 35 d of age.

[0178] Negative Effect of Vaccination on Performance of Healthy Birds

[0179] Body weight (BW) and feed conversion ratio (FCR) per pen was recorded on d 7, 14, 21, 28 and 35. Mortality was monitored daily and the weights of any dead birds were used to correct the measured FCR values. The effect of coccidial challenge and vaccination on the feed conversion ratio is shown in Table 6. Feed conversion ratio (FCR) is a rate measuring the efficiency with which an animal convert animal feed into the desired output (e.g. the flesh). FCR is the mass of the input divided by the output. Thus, a high FCR is an indication for poor performance, i.e. a high FCR is undesirable. As shown in Table 6, administering a coccidial vaccination, without subsequent challenge, increased feed conversion ratio (FCR).

TABLE-US-00007 TABLE 6 Effect of coccidial challenge and vaccination on the feed conversion ratio (FCR; g:g) and mortality (%) of broiler chickens from d 1-35. FCR FCR FCR FCR FCR Mortality Vaccine Challenge d 1-7 d 1-14 d 1-21 d 1-28 d 1-35 d 1-35, % Treatment Effects ? + 0.048 1.51 1.57 1.58 1.63 19.1 ? ? 0.048 1.23 1.31 1.42 1.51 2.4 + + 0.048 1.38 1.41 1.48 1.55 2.4 + ? 0.048 1.24 1.40 1.49 1.56 8.9 P< NS 0.001 0.001 0.001 0.001 0.05 SEM 0.001 0.030 0.022 0.017 0.011 0.06 Interaction Terms Vaccine*Challenge NS 0.05 0.001 0.001 0.001 0.05 Abbreviation d is used for day. The lower the P value the better; any P value below 0.05 is considered statistically significant. NS = not statistically significant. The notation with * means interaction, i.e. describes a situation in which the effect of one causal variable on an outcome depends on the state of a second causal variable. g:g refers to grams of feed intake per gram of weight gain

[0180] Birds with vaccine (+) but without challenge (?) had an FCR d1-35 (cumulative FCS, total feed intake and total weight gain over 1-35d) of 1.56 whereas birds without vaccine (?) and without challenge (?) a had a lower (i.e. better) FCR d1-35 of 1.51. Thus, in the absence of a challenge, a vaccination has a negative effect on performance. This also applies to mortality. The poorer performance of the vaccinated birds in the current study is in-line with previous observations and is clearly undesirable. According to a preferred embodiment of the present invention, unnecessary vaccination of healthy birds is preferably replaced by early intervention triggered by early diagnosis of coccidiosis.

[0181] Analysis of Blood Biochemistry

[0182] On day 6, 8, 13, 20, 27 and 34 one bird per pen was randomly selected, removed from the pen, euthanized and subjected to blood sampling via the vena cava.

[0183] Heparinized blood (approximately 0.2 ml) was analyzed in the i-Stat? Alinity v handheld blood analyzer fitted with a Chem8+ cartridge (Abbott Point of Care Inc., Princeton, NJ), which measured hematocrit (HCT), ionized calcium (Ca), glucose (GLU), chloride (Cl), sodium (Na), potassium (K), total carbon dioxide (TCO.sub.2) and anion gap (AnGap).

[0184] Remaining heparinized blood (0.1 ml) was analyzed in the Vetscan? VS2 Chemistry Analyzer (Abaxis, inc) using the Avian/Reptilina Profile Plus cartridge (Abbott Point of Care Inc., Princeton, NJ). This resulted in aspartate aminotransferase (AST), creatine kinase (CK), uric acid (UA), GLU, Ca, phosphorus (P), total protein (TP), albumin (ALB), albumin/globulin (GLOB), potassium (K) and sodium (Na).

[0185] By selection of the above-mentioned cartridges, some of the blood parameter can be measured by both devices (i-Stat? and Vetscae?; superscripts for refer to either VetScan v or iStat i devices that were used). Other blood parameters can be measured with one of two devices only, given the indicated selection of cartridges. Below Table 7 gives an overview of parameters that can be measured with both devices:

TABLE-US-00008 TABLE 7 i-Stat? Alinity.sup.4 Vetscan? VS2.sup.5 v handheld blood Chemistry Analyzer (Abaxis, Blood analyzer fitted with a Inc) using the Avian/ Parameter Chem8+ cartridge Reptilina Profile Plus cartridge calcium + + (electrolyte) (.sup.iCa) (.sup.vCa) glucose + + (.sup.iGlu) (.sup.vGlu) chloride + + (electrolyte) (.sup.iCl) (.sup.vCl) sodium + + (electrolyte) (.sup.iNa) (vNa) potassium + + (electrolyte) (.sup.iK) (.sup.vK) .sup.4For details, see cartridge and test information, Rev. Date: 15, Aug. 2016, Art: 714258-01O, available at Abbott .sup.5For details, see Operators Manual VertScanVS2, dated February 2009, available at Abaxis, Inc., Union City, CA 94587; Customer and Technical Service: 1-800-822-2947

[0186] Even though not explicitly mentioned, it is be understood that in the context of the present invention, Ca, Cl, Na and K refers to electrolytes, i.e. to ions.

[0187] Where the same blood parameter was measured with more than one device (e.g. Na or K), there was generally close agreement with respect to the observed treatment effect trajectory although the absolute values differed slightly. The units recorded by the two devices may differ though.

[0188] Precisely 1.0 ml of EDTA blood was mixed with 0.20 ml cellular fixant (Transfix?, MBL International), stored and shipped on wet ice to Cayman Analytical Laboratories (Ann Arbor, MI) for heterophil (HET) to lymphocyte (LYM) ratio analysis using flow cytometry as described by Lentfer et al. and Bilkova et al. (Lentfer, T. L., H. Pendl, E. K. F. Fr?hlich, E. Von Borell, H. Pendl, and E. K. F. Fr?hlich. 2015. H/L ratio as a measurement of stress in laying hensmethodology and reliability. Br. Poult. Sci. 56:157-163; B?lkov?, B., Z. Bainova, J. Janda, and M. Vinkler. 2017. Different breeds, different blood: Cytometric analysis of whole blood cellular composition in chicken breeds. Vet. Immunol. Immunopathol. 188:71-77).

[0189] Whole blood (3 ml) was spun and serum removed and stored on wet ice. 0.40 ml serum was used for total carotenoids (CAR) using the iCheck? carotene photometer device and test kit (BioAnalyt GmbH, Potsdam, Germany) as described by Kawashima et al. (Kawashima, C., S. Nagashima, K. Sawada, F. J. Schweigert, A. Miyamoto, and K. Kida. 2010. Effect of ?-Carotene Supply During Close-up Dry Period on the Onset of First Postpartum Luteal Activity in Dairy Cows. 287:282-287). Remaining serum was frozen on dry ice and shipped to Cayman Analytical Laboratories (Ann Arbor, MI) for thiobarbituric acid (TBARS) analysis (Wills, E. D. 1966. Mechanisms of lipid peroxide formation in animal tissues. Biochem. J. 99: 667-676), which measures malondialdehyde (MDA) in the blood.

[0190] An overview of the analysis of blood biochemistry done in example 2 is shown in below Table 8:

TABLE-US-00009 TABLE 8 Sample Type of category sample tested blood parameters Device/Laboratory 1 heparinized Ca, P, Cl, Na, K, TP, Vetscan? VS2 Chemistry blood GLOB, TCO2 and others Analyzer and/or i-Stat? Alinity v handheld blood analyzer 2 EDTA HET, LYM and Cayman Analytical treated HET:LYM Laboratories blood (Ann Arbor, MI) 3 serum total carotenoids (mg/kg); iCheck? carotene photometer collection TBARS (malondialdehyde; Cayman Analytical Laboratories tube MDA) (Ann Arbor, MI)

[0191] Age dependent blood parameters useful for early diagnosis of coccidiosis Statistical analysis of the study of Example 2 revealed that day 13 (13 days post-hatch) is the age at which the biomarker responses to coccidiosis challenge were most acute. At ages below day 7 and at ages above day 21, the biomarker responses to coccidiosis challenge was not useful for early diagnosis of coccidiosis. Ages from day 10 and day 16 are expected to be very useful for early diagnosis of coccidiosis. Statistically, the highest diagnostic precision is achieved at day 13.

[0192] Blood parameters useful for early diagnosis of coccidiosis Statistical analysis of study of Example 2 shows that on day 13, there are specific biomarkers that are significantly influenced by coccidial challenge.

[0193] Alternative biomarkers were not significantly influenced (e.g. there was no effect of coccidial challenge on plasma Ca.sup.6, P.sup.7, and Cl.sup.8). Below Table 9 shows the responses to coccidial challenge, on d13, for a sub-set of specific blood .sup.6 calcium (Ca); analysis of heparinized blood.sup.7 phosphorus (P); analysis of heparinized blood.sup.8 chloride (Cl); analysis of heparinized blood parameters. These are the blood parameters that can be used for early diagnosis of coccidiosis. Any P value below 0.05 is considered statistically significant.

TABLE-US-00010 TABLE 9 AnGap.sup.9 Blood (calculated Car parameter Na K GLOB Anion Gap) (Carotenoids) Age of bird Day 13 Day 13 Day 13 Day 13 Day 13 when blood sample was taken Device used i-Stat? Alinity i-Stat? Alinity Vetscan? VS2 i-Stat? Alinity iCheck? carotene for analysis with Chem8+ with Chem8+ Chemistry with Chem8+ photometer of blood cartridge cartridge Analyzer with cartridge device sample Avian/Reptilina Profile Plus cartridge non- 144.4 mmol/l 5.06 mmol/l 0.43 g/l 19.6 mmol/l 0.91 ppm challenge coccidiosis 140.7 mmol/l 6.10 mmol/l 0.73 g/l 17.2 mmol/l 0.40 ppm challenge P value P < 0.001 P < 0.001 P < 0.001 P < 0.01 P < 0.001 Model for Value for Na Value for K Value for GLOB Value for AnGap Value for clinical or parameter parameter parameter parameter Carotenoids pre-clinical measured at measured at measured at measured at parameter diagnosis of d 13 post-hatch d 13 post-hatch d 13 post-hatch d 13 post-hatch measured at coccidiosis lower than higher than higher than lower than d 13 post-hatch pre-determined pre-determined pre-determined pre-determined lower than range range range range pre-determined range .sup.9Anion Gap is calculated in CHEM8+ cartridges as follows: Anion Gap (CHEM8+) = (Na + K) ? (Cl + (TCO2 ? 1)) cf. publication PCO.sub.2 AND CALCULATED VALUES FOR HCO.sub.3, BASE EXCESS AND ANION GAP available at Abaxis, Art: 714182-00V, Rev. Date 29 Sep. 2017; https://www.abaxis.com/sites/default/files/resource-packages/PCO2%20CTI%20Sheet%20714182-00V.pdf

[0194] Thus, (i) measuring higher than normal (baseline) Na, K and/or GLOB on d13 and/or (ii) measuring a lower than normal (baseline) AnGap and/or Car on d13 is an indication of coccidiosis in the respective broiler population. After such indication, a suitable intervention is recommended such that performance losses associated with coccidiosis can be successfully mitigated. Surprisingly, the same conclusion could not have been reached with the same precision or could not have been reached at all if the blood sample had been taken when the bird was younger than 7 days or older than 21 day. At ages from day 10 and day 18, the same conclusion could have been reached, although not with the same precision as on day 13.

[0195] Improved Precision of Early Diagnosis of Coccidiosis

[0196] Statistical analysis of study of Example 2 shows that prediction of coccidiosis using blood analysis can be improved by the simultaneous use of more than one biomarker. For example, when using K as only pre-determined blood parameter, a Receiver Operating Characteristic (ROC) analysis of the interaction between bird age and K delivers an area under the curve (AUC) of 0.62 (i.e. 62% separation between challenged and non-challenged birds). A mixed model with multiple pre-determined blood parameters including carotenoids, GLOB, ANGAP and K with the age of the bird as an interactive term delivers a ROC AUC of 0.82 (i.e. 82% accurate separation between challenged and non-challenged birds).

[0197] In the most preferred embodiment of the present invention, a very high ROC AUC of 0.89 is achieved; in the most preferred embodiment, the pre-determined following blood parameters are used: AST.sup.10, PHOS, GLOB, carotenoids, K, ANGAP and TCO2, including the interaction with age. .sup.10 blood aspartate aminotransferase (AST)

[0198] Statistical analysis of study of Example 2 shows very clearly that when taking into consideration (i) more than 1 pre-determined blood parameter (preferably 3 to 6 pre-determined blood parameters) and (ii) the age of the bird, coccidiosis can be diagnosed at early stage with accurate diagnostic precision.

[0199] Thus, Example 2 shows that collection of specific data at a specific moment in time offers a biochemical fingerprint of coccidiosis allowing to achieve an objective early diagnosis.

[0200] Further Evidence for the Age Dependency (Veterinary Necropsy)

[0201] The bird randomly selected on day 6, 8, 13, 20, 27 and 34 was not only subjected to blood sampling. After euthanization, it was also subjected to veterinary necropsy. Veterinary post-mortem analysis was specifically oriented toward metrics that were known to be associated with coccidiosis, specifically gross acervulina (GAc), gross tenella (GTn), gross micro max (GMx) and micro max (mMx). These observations have a score from 0 to 4 with 0 being absent and 4 being severe as described by Conway et al. (Conway, D. P. and M. E. McKenzie. 2007. Poultry coccidiosis: diagnostic and testing procedures. 3rd Edition. Blackwell Publishing Professional, Ames, Iowa, USA. pp. 7-20).

[0202] In the study of Example 2, birds that were not vaccinated but challenged had higher GAc scores on day 13 than was the case for alternative treatment groups, resulting in a significant challenge*vaccination*age interaction. This confirms the importance of day 13. The same challenge*vaccination*age interaction could not be observed on or before day 8. Similarly, the same challenge*vaccination*age interaction could not be observed on or after day 20.

Example 3 (Administration of a Microbial Muramidase to Feed as a Response to an Avian Disease)

[0203] The results obtained in the study of Example 3 shows that broiler chickens which were infected by Eimeria and Clostridium perfringes can effectively be treated by the inclusion of microbial muramidase in animal feed. Adding microbial muramidase to animal feed is therefore one manner to adapt nutrition after early diagnosis of an avian disease.

[0204] Materials and Methods

[0205] Center of Immune Response in Poultry (CERIA) at Federal University of Parana. The birds were housed in the experimental room with negative pressure. Each replicate was in cages with sterilized litter, nipple drinkers, and automatic temperature control. Birds were raised with water and feed ad libitum.

[0206] Experimental DesignTreatments and Animals

[0207] The amount of 256 male broilers (01 to 28 days of age) were distributed in a completely randomized design divided into 04 treatments with 7 repetitions each, starting 8 birds in each repetition and 1 group control without challenge.

[0208] The birds were allocated in different rooms: with or without challenge.

[0209] 8 rooms with 4 cages each: [0210] 1 room without challenge (experimental negative control)4 cages with 8 birds (1 cage per treatment)total: 32 birds; [0211] 7 rooms with a challenge (Eimeria and C. perfringens): In each room, 4 treatments were allocated and at the end, there is 7 replicates with 8 birds eachtotal: 224 birds.

TABLE-US-00011 TABLE 10 Treat- Eimeria Clostridium ment challenge challenge Product CN No No None T1 Yes Yes None T2 Yes Yes Muramidase (25 000 LSU/kg, 476 mg/kg) T3 Yes Yes Muramidase (35 000 LSU/kg, 667 mg/kg) T4 Yes Yes Enramycin 10 ppm (Enradin? 8%) Muramidase: activity 52 500 FSU(F)/g

[0212] Challenge

[0213] In the first day of the trial, animals from T1, T2, T3, and T4 have received the anticoccidial vaccine 15 times the manufactured recommendation dose. In the 10th, 11th and 12th day of the experiment, they were inoculated with Clostridium perfringens (108 CFU/mlisolated from the field Necrotic enteritis case) by gavage.

[0214] Performance

[0215] At 7, 14, 21 and 28 days of age, the feed and birds were weighted, and was evaluated feed intake (FI), body weight gain (BWG) and feed conversion ratio (FCR). The data were submitted to analysis of variance (ANOVA) and Tukey's test for the means with a significant difference (P<0.05). Data are listed as Tables 11 and 12.

TABLE-US-00012 TABLE 11 Mean ? standard deviation of BWGBody Weight Gain (g) at all periods. Treatment 1-7 D CV % 1-14 D CV % 1-21 D CV % 1-28 D CV % CN 154.04 ? 16.23 a 21.973 563.13 ? 26.32 a 9.348 1207.4 ? 107.08 a 17.737 1858.1 ? 52.83 a 5.687 T1 88.114 ? 6.60 b 19.846 412.95 ? 27.88 b 17.866 923.61 ? 41.83 b 11.985 1551.9 ? 49.55 b 8.448 T2 89.99 ? 5.47 b 16.086 394.62 ? 18.70 b 12.544 905.47 ? 25.39 b 7.420 1566.7 ? 55.38 b 9.352 T3 107.96 ? 12.11 b 29.684 389.94 ? 13.20 b 8.956 939.34 ? 58.64 b 16.519 1570.3 ? 37.43 b 6.307 T4 90.95 ? 5.11 b 14.890 413.37 ? 12.89 b 8.251 926.22 ? 23.58 b 6.738 1595.5 ? 42.62 b 7.067 P value 0.0005 0.0001 0.0049 0.0097 ab Mean with different letters in the same column are significantly different at P < 0.05 Tukey's test

TABLE-US-00013 TABLE 12 Mean ? standard deviation of FCR (Feed Conversion Ratio) at all periods. Treatment 1-7 D CV % 1-14 D CV % 1-21 D CV % 1-28 D CV % CN 1.101 ? 0.139 22.999 0.832 ? 0.017 4.097 1.052 ? 0.071 b 13.503 1.150 ? 0.019 b 3.362 T1 1.504 ? 0.099 17.470 1.074 ? 0.062 15.439 1.345 ? 0.054 a 10.628 1.332 ? 0.034 a 6.791 T2 1.496 ? 0.155 27.440 1.080 ? 0.044 10.992 1.322 ? 0.058 a 11.601 1.290 ? 0.050 ab 10.370 T3 1.427 ? 0.195 36.284 1.035 ? 0.063 16.302 1.314 ? 0.060 a 12.212 1.285 ? 0.020 ab 4.143 T4 1.568 ? 0.123 20.815 1.070 ? 0.049 12.256 1.255 ? 0.036 ab 7.735 1.228 ? 0.024 ab 5.240 P value 0.3816 0.0811 0.0250 0.0229 ab Mean with different letters in the same column are significantly different at P < 0.05 Tukey's test

[0216] From the above tables, Eimeria and Clostridium perfringens challenge significantly reduce the weight gain (16%) and Feed intake (3%) compared to the control (CN) without challenge. The groups T2 and T3 supplemented with muramidase had improved weight gain and FCR.