In vitro method for detecting intestinal barrier failure in animals
阅读说明：本技术 用于检测动物中的肠屏障衰竭的体外方法 (In vitro method for detecting intestinal barrier failure in animals ) 是由 M·佛卢格尔 S·佩尔泽 F·瑟尔曼 F·梵伊莫塞尔 R·杜卡特耶 E·高森斯 B·德沃 于 2019-02-28 设计创作，主要内容包括：本发明涉及用于检测禽类群体中的肠屏障衰竭的体外方法,该方法包括以下步骤：a)收集来源于禽类群体的合并的粪便样品,以及b)确定所述样品中包含的至少一种蛋白质标志物的量；其中所述至少一种蛋白质标志物包含急性期蛋白或其功能片段或由其组成,并且其中与参考样品相比,所述样品中包含的所述至少一种蛋白质标志物的量的增加表明肠屏障衰竭。(The present invention relates to an in vitro method for detecting intestinal barrier failure in a population of avians, the method comprising the steps of: a) collecting a pooled stool sample derived from a population of birds, and b) determining the amount of at least one protein marker contained in the sample; wherein the at least one protein marker comprises or consists of an acute phase protein or a functional fragment thereof, and wherein an increase in the amount of the at least one protein marker comprised in the sample compared to a reference sample is indicative of intestinal barrier failure.)
1. An in vitro method for detecting intestinal barrier failure in a population of avians, the method comprising the steps of:
a) collecting a pooled fecal sample derived from a population of birds, and
b) determining the amount of at least one protein marker comprised in the sample;
wherein
The at least one protein marker comprises or consists of an acute phase protein or a functional fragment thereof,
and wherein
An increase in the amount of the at least one protein marker comprised in the sample compared to a reference sample is indicative of intestinal barrier failure.
2. The method of claim 1, wherein the acute phase protein is selected from the group consisting of transferrin/Ovotransferrin (OTF), haptoglobin (HAPT, avian: PIT54), C-reactive protein (CRP), ceruloplasmin, fibrinogen, mannan-binding lectin, Serum Amyloid A (SAA), long-chain pentraxin PTX3, alpha 1 Acid Glycoprotein (AGP).
3. The method of any one of the preceding claims, wherein the acute phase protein is ovotransferrin.
4. The method according to any one of the preceding claims, wherein the reference sample is a species-specific control representing an intact intestinal barrier.
5. The method of any one of the preceding claims, wherein the acute phase protein is detected and quantified by enzyme-linked immunosorbent assay (ELISA).
6. The method of any one of the preceding claims, wherein the acute phase protein is detected and quantified by a lateral flow assay.
7. The method of any one of the preceding claims, wherein the combined fecal sample material is stabilized immediately after collection of the sample.
8. The method of any one of the preceding claims, wherein the sample is treated with a protease inhibitor.
9. Use of an acute phase protein or a functional fragment thereof as a stool marker for detecting intestinal barrier failure in a population of avians.
10. An in vitro method for detecting the extent of intestinal barrier failure in a population of avians, the method comprising the steps of:
a) collecting pooled fecal samples derived from the flock;
b) determining the amount of at least one protein marker comprised in the sample material;
wherein
The at least one protein marker comprises or consists of an acute phase protein or a functional fragment thereof,
and wherein
The amount of the at least one protein marker comprised in the sample indicates the extent of intestinal barrier failure.
11. An in vitro method for monitoring the intestinal barrier status in a flock of birds, said method comprising the steps of:
a) collecting pooled fecal samples derived from the flock at successive time points;
b) determining the amount of the at least one protein marker comprised in the sample obtained in step a); and
c) determining a deviation in the amount of the at least one protein marker comprised in the sample obtained in step a);
wherein
The at least one protein marker comprises or consists of an acute phase protein or a functional fragment thereof.
Technical Field
The present invention relates to an in vitro method for detecting intestinal barrier failure in an animal. More specifically, the present invention relates to Acute Phase Protein (APP) -based methods for assessing intestinal health status in individual animals and animal populations, respectively.
Background
The health of the intestines is crucial for the welfare and performance of livestock animals. Intestinal diseases that affect the structural integrity of the gastrointestinal tract (GIT) result in high economic losses due to reduced weight gain, poor feed conversion efficiency, increased mortality and higher costs of medication (M' Sadeq, s.a., Wu, s., Swick, R.A. & Choct, M. (2015.) labour the control of the genetic engineering in brouter chips with in-fed interactive reactivity-out working with in-animal Nutrition,1, 1-11; Timbermont, l., haeseborrhok, F., Ducatelle, R. & Van im reactor, F. (2011.) genetic engineering in broilers: an upper draft on the patient bed.
The intact intestinal barrier provides a number of physiological and functional features including digestion and absorption of nutrients, host metabolism and energy production, stable microbiome, mucus layer development, barrier function and mucosal immune response (Kogut, m.h. and r.j. arsenault (2016. Editorial: Gut health: The new paramount in food and animal production. frontiers in vector Science 3 (AUG)). As the largest organ in the body, the intestine acts as a selective barrier to absorb nutrients and fluids into the body, while excluding unwanted molecules and pathogens. Therefore, proper intestinal barrier function is crucial for maintaining optimal health and balance throughout the body and represents a critical line of defense against foreign antigens from the environment.
Coccidiosis and Necrotic Enteritis (NE) are probably the most common intestinal diseases of Poultry (Dalloul, R.A. & Lillehoj, H.S. (2006.) Poultry coccoidosis: recovery enhancements in control measures and preservation details. Expert Rev Vaccines,5,143. 163.; Williams, R.B. (2005.) Intercurrent coccoidosis and genetic entitation of chicken: velocity, integrated disease management by main machinery of control interest. Avian Pathol,34, 159. 180). In poultry, coccidiosis may be caused by a number of species belonging to the genus Eimeria (Eimeria), of which Eimeria acervulina, Eimeria maxima (e.maxima) and Eimeria tenella (e.tenella) are the most common species among intensively bred broiler chickens. Depending on the species, the lesions can range from limited malabsorptive enteritis (eimeria acervulina) to more severe intestinal wall inflammation (eimeria maxima) and even extensive cecal bleeding and death (eimeria tenella) (Chapman, H.D. (2014.) milestone in avinocardiosis research: a review. Poult Sci,93, 501. 511). Furthermore, the presence of species of Eimeria can also exacerbate the consequences of co-infection with bacterial pathogens such as Clostridium perfringens (2016) (Moore, R.J. (2016.) Crytic organisms predisposing factors in british Pathols, 45, 275-. In fact, mucosal damage caused by these coccidiosis pathogens is an important cause of necrotic enteritis. NE is the most common clostridial enteropathy in poultry, which commonly occurs in broiler chickens. The disease is caused by clostridium perfringens and can occur in acute clinical or mild subclinical forms. Acute NE often results in a substantial increase in mortality in flocks. The most common subclinical form is characterized by multifocal necrosis and inflammation of the small intestine, with a marked decrease in growth performance. The performance decline is not only related to impaired growth rate and feed conversion rate during production, but also to increased rejection rate (consistency rate) in broilers due to hepatitis during processing (Paiva, D. & McElroy, A. (2014.) Cryteric entitis: Applications for the pore industry. journal of applied pore Research,23,557- & 566). Both coccidiosis and necrotic enteritis can occur in the flock without showing clinical symptoms. Therefore, several birds must be sacrificed to perform a macroscopic examination of the intestine to diagnose the disease.
Similar considerations apply to other intestinal diseases or conditions leading to mucosal damage in livestock animals, such as severe overgrowth of bacteria in the small intestine, various forms of excessive enteritis, exposure to mycotoxins, and every condition leading to failure of the intestinal barrier. In addition, intestinal barrier failure may enable the normal population of the GIT, such as Enterococcus cecal (Enterococcus caecorum), to invade the systemic circulation. This may lead to further diseases such as arthritis and osteomyelitis and ultimately to a reduced performance of the animal or herd, respectively.
Therefore, it would be highly desirable to be able to accurately detect markers or marker sets of intestinal barrier failure or intestinal inflammation and the accompanying perturbation of intestinal integrity at an early stage.
Recently, studies on the intestinal permeability of chickens have attracted increasing interest, leading to different strategies for measuring enteritis and concomitant intestinal barrier failure. However, none of these proposed strategies represent good markers for the broiler industry as they are not applicable under field conditions (e.g. oral administration of markers that can be measured in blood at some later point in time) (Gilani, s., Howarth, g.s., kit, s.m., Tran, c.d., form, r.e.a. & Hughes, R.J. (2017) New biomarkers for embedded expressed intracellular tissue induced by b.e. dextran sodium sulfate, d.a. Kuttappan, v.a., telez, g.oil, hernanz-veco, herx-veco, sars, 2015, e237-e 245; Vicuna, e.a., Kuttappan, v.a., telez, g.g., herz-veco, herx, sarx-vector, sars. iv. 1. g.b.g.g.g.g.g.g.g. et, sars. iv. et. sars. iv. et. sars. 1. t. gastrointestinal infection. Telez, G., Richards, J.D. & Escorbar, J. (2015.) Identification of Potential Biomarkers for Gut Barrier Failure in BroilerChikens. front Vet Sci,2, 14; o' Reilly, E.L. & Eckersall, P.D. (2014.) Acute phaseproteins a review of the ir function, a reviouur and measurement in chips, world Poultry Science Journal,70, 27-43; xie, h., Newberry, l., Clark, f.d., Huff, w.e., Huff, g.r., balg, j.m., et al (2002) Changes in a serovaractors level in chips with an experimental indication and diseases, avian Dis,46, 122-.
Thus, there remains a need to provide a fast, reliable, ideal non-invasive pre-mortem method that can determine whether an individual animal or animal population, particularly a poultry population, is suffering from intestinal barrier failure under field conditions at low cost and with minimal effort.
Summary of The Invention
It is therefore an object of the present invention to provide an in vitro method for detecting intestinal barrier failure in a population of avians, the method comprising the steps of:
a) collecting a pooled fecal sample derived from a population of birds, and
b) determining the amount of at least one protein marker comprised in the sample;
wherein
At least one protein marker comprises or consists of an acute phase protein or a functional fragment thereof,
and wherein
An increase in the amount of the at least one protein marker comprised in the sample compared to a reference sample is indicative of intestinal barrier failure.
Another aspect of the present application is the use of an acute phase protein or a functional fragment thereof as a stool marker for detecting intestinal barrier failure in a population of avians.
It is another object of the present invention to provide an in vitro method for detecting the extent of intestinal barrier failure in a flock of birds, the method comprising the steps of:
a) collecting pooled fecal samples derived from the flock;
b) determining the amount of at least one protein marker comprised in the sample material;
wherein
At least one protein marker comprises or consists of an acute phase protein or a functional fragment thereof,
and wherein
The amount of the at least one protein marker in the sample indicates the extent of intestinal barrier failure.
Finally, the present invention provides an in vitro method for monitoring the intestinal barrier status in a flock of birds, the method comprising the steps of:
a) collecting pooled fecal samples derived from the flock at successive time points;
b) determining the amount of at least one protein marker comprised in the sample obtained in step a); and
c) determining a deviation in the amount of at least one protein marker comprised in the sample obtained in step a);
wherein
The at least one protein marker comprises or consists of an acute phase protein or a functional fragment thereof.
The key aspects of the invention are described in detail below.
Detailed Description
The inventors have surprisingly found that the amount of Acute Phase Protein (APP) -based markers comprised in sample material of animal origin is correlated with the manifestation of intestinal barrier failure. More specifically, the amount of these APP-based markers contained in pooled stool samples derived from a population of avians was found to correlate with the manifestation of intestinal barrier failure in said population of avians.
In the context of the present invention, the term "APP-based marker" refers to a protein comprising or consisting of APP; or to a polypeptide/protein comprising or consisting of a functional fragment of APP.
More specifically, the inventors have found that an increase in the amount of APP-based marker comprised in an intestinal sample material of animal origin compared to a reference sample is indicative of intestinal barrier failure.
Accordingly, the present invention relates to an in vitro method for detecting intestinal barrier failure in an animal, comprising the steps of:
a) collecting intestinal sample material from an individual animal or a population of animals; and
b) determining the amount of at least one protein marker comprised in the sample material;
wherein
At least one protein marker comprises or consists of an acute phase protein or a functional fragment thereof,
and wherein
An increase in the amount of the at least one protein marker comprised in the sample compared to a reference sample is indicative of intestinal barrier failure.
Furthermore, the present invention provides an in vitro method for detecting intestinal barrier failure in a population of avians, the method comprising the steps of:
a) collecting pooled fecal samples derived from a population of birds, and
b) determining the amount of at least one protein marker comprised in the sample;
wherein
At least one protein marker comprises or consists of an acute phase protein or a functional fragment thereof,
and wherein
An increase in the amount of the at least one protein marker comprised in the sample compared to a reference sample is indicative of intestinal barrier failure.
As used in the context of the present invention, the term "intestinal barrier failure" refers to a condition in which the intestinal barrier function is significantly impaired (e.g. due to oxidative stress, non-digestible proteins, coccidiosis, etc.); and include conditions of intestinal barrier dysfunction, intestinal leakage/permeability, and conditions resulting from histopathological injury. Intestinal barrier failure is often associated with inflammatory processes. The terms "intestinal barrier failure" and "intestinal barrier failure" may be used interchangeably.
As mentioned above, the at least one protein marker comprises or consists of an acute phase protein or a functional fragment thereof. Acute Phase Proteins (APP) are known in the art.
In one embodiment of the invention, the aforementioned method is applied using only one specific protein marker. Alternatively and also according to the invention, two or more protein markers may be analyzed simultaneously.
Suitable protein markers for use in the methods of the invention are, for example, transferrin/Ovotransferrin (OTF), haptoglobin (HAPT, avian: PIT54), C-reactive protein (CRP), ceruloplasmin, fibrinogen, mannan-binding lectin, Serum Amyloid A (SAA), long-chain pentraxin PTX3, alpha 1 Acid Glycoprotein (AGP).
For example, Ovotransferrin (OTF) and/or the long-chain pentraxin PTX3 may be used as markers of intestinal barrier failure in birds.
Alternatively, functional fragments of the above proteins may be used as protein markers. The term "fragment" refers to a polypeptide or protein derivative of a particular protein having a reduced amino acid chain length. The fragments according to the invention have epitopes that can be specifically recognized by antibodies.
In one embodiment, the at least one protein marker is ovotransferrin or a functional fragment thereof.
The reference sample is a species-specific control representing an intact intestinal barrier. Suitable reference samples are samples obtained from individual animals or from a population of animals of the same species or subspecies, wherein the animal or the population of animals has a proven intact intestinal barrier.
As one example, a reference sample may be obtained in an animal trial from an animal that is not a treated control, the reference sample being examined by pathology, histopathology, and/or other measures to confirm that there is no evidence of intestinal barrier failure.
Protein-based markers may be detected and quantified using well-known conventional techniques, such as immunoassays like ELISA (enzyme linked immunosorbent assay), lateral flow assays, Mass Spectrometry (MS) analysis or any method that enables the detection of a protein or functional fragment thereof.
In a specific embodiment, the at least one protein marker is detected and quantified by ELISA.
The use of monoclonal antibodies enables specific detection in a complex sample matrix for analysis.
The methods of the invention can be used to determine whether an individual animal is suffering from intestinal barrier failure. In this case, the intestinal sample material is derived from an individual animal.
The individual animal may for example be a pet or livestock animal, a farm animal in the form of a poultry animal, a wild animal or a zoo animal. In addition, individual animals that are transported for slaughter or removal can be examined using the methods described above.
In one embodiment, the individual animal is an avian subject.
The avian subject to be tested is preferably poultry. Preferred poultry according to the invention are chickens, turkeys, ducks and geese. Poultry may be optimized for production of young animals. This type of poultry is also referred to as parental and progenitor parental animals. Thus, preferred parental and ancestral parent animals are (ancestral) parent broiler chickens, (ancestral) parent ducks, (ancestral) parent turkeys and (ancestral) parent geese.
The poultry according to the invention may also be selected from ornamental and wild birds. Preferred ornamental poultry or pheasants are peacock, pheasant, pine, guinea fowl, quail, thunderbird, goose, pigeon and swan. Further preferred poultry according to the invention are ostriches and parrots. The most preferred poultry according to the invention are broiler chickens.
The intestinal sample material obtained from the individual animal may be selected from the group consisting of an intestinal content sample, a bodily waste sample, and solutions or suspensions thereof; and from materials contaminated with bodily wastes. The term "intestinal content" is to be understood as the content of the small intestine, the content of the large intestine and/or the content of the cecum. Methods of collecting such intestinal content samples are known in the art.
As used in the context of the present invention, bodily waste is feces or cecal waste. Materials contaminated with bodily discharges are, for example, dust samples, wiping samples, urine and feces samples, liquid manure samples, fur samples, feather samples and skin samples.
In general, the term "toilet pad" is understood to mean a mixture of animal excrement and bedding material.
As used in the context of this embodiment, the term "urine collection pad sample" refers to the excretory drippings from an individual animal. Furthermore, in the context of this embodiment, the term "liquid manure sample" refers to a fecal sample comprising feces and urine from an individual animal.
Samples from individual animals may be taken directly from the animal, for example using a swab. Alternatively and especially in the case of single-caged animals, sample material may be collected from the floor of the pen, cage or slat. The sample material must be dispensable to the animal under study.
In one embodiment, the intestinal sample material used to determine whether the individual animal suffers from intestinal barrier failure is stool.
For certain applications, it is also useful to analyze samples of intestinal contents (e.g., samples from the small intestine, samples from the large intestine, and/or samples from the cecum).
Suitable sample volumes are, for example, from 0.05ml to 20ml or from 0.1 to 20ml, in particular from 0.2 to 10ml, preferably from 0.5 to 5 ml. Suitable sample masses are, for example, from 0.05g to 20g or from 0.1 to 20g, in particular from 0.2 to 10g, preferably from 0.5 to 5 g.
In alternative embodiments, the method is used to determine whether an animal population is suffering from intestinal barrier failure. In this case, the sample material is derived from the group of animals to be tested.
As used herein, the term "animal population" refers to a group of individual animals belonging to the same species. The animal population may be, for example, a group of pets or livestock, as found in animal breeding, a group of farm animals, as found in livestock production or livestock breeding, or a group of wild or zoo animals.
In one embodiment, the animal population is an animal population as found in livestock production processes. For example, the animal population or herd may be a poultry population; sheep, goats or cattle herds, horses or pigs.
In a specific embodiment, the animal population is an avian population.
The animal population is preferably an avian population. The flock of birds according to the invention is preferably poultry. Preferred poultry according to the invention are chickens, turkeys, ducks and geese. Poultry may be optimized for production of young animals. This type of poultry is also referred to as parental and progenitor parental animals. Thus, preferred parental and ancestral parent animals are (ancestral) parent broiler chickens, (ancestral) parent ducks, (ancestral) parent turkeys and (ancestral) parent geese.
The poultry according to the invention may also be selected from ornamental and wild birds. Preferred ornamental poultry or pheasants are peacock, pheasant, pine, guinea fowl, quail, thunderbird, goose, pigeon and swan. Further preferred poultry according to the invention are ostriches and parrots. The most preferred poultry according to the invention are broiler chickens.
The method of the invention is particularly suitable for determining the health status of a population of animals by a large number of tests. As used herein, the term "bulk testing" refers to a testing method in which the sample material is a pooled sample of a population of animals. In the context of this embodiment, "pooled sample" is understood to be a composite sample from randomly selected individual samples, one sample taken with one or several wetted fabric swabs, or a pooled sample consisting of individual samples of fresh samples taken randomly from many sites in a house or space where a population of animals or fauna are kept. It may be necessary to homogenize the sample material prior to sample analysis. Suitable homogenization techniques are known in the art.
The pooled samples reflect the amount of APP-based protein marker present in the animal population.
The sample material obtained from the individual animal may be selected from a sample of intestinal content, a sample of bodily waste, and solutions or suspensions thereof; and from materials contaminated with bodily waste. Materials contaminated with bodily discharges are, for example, dust samples, wiping samples, urine and feces samples, liquid manure samples, fur samples, feather samples and skin samples.
As used in the context of this embodiment, the term "urine bedding sample" refers to mixed drainage drippings in a pen, cage or slat. Furthermore, in the context of this embodiment, the term "liquid manure sample" refers to a fecal sample containing a mixture of feces and urine.
For example, these fecal pad samples can be collected from a population of animals using an overshoe method at various locations of the pen or using a fecal pad swab.
Boot swabs that are sufficiently absorbent to absorb moisture are particularly suitable for use in collecting pooled animal samples. Cop socks are also acceptable.
If the animal population is housed in a cage or a slat, a fecal sample can be collected by a conveyor belt.
In one embodiment, the sample material used to determine whether the animal population is suffering from intestinal barrier failure is stool. Preferably, the sample material is a pooled fecal sample derived from a flock of birds.
For certain applications, it is also useful to analyze pooled intestinal content samples, such as pooled samples from the small intestine, pooled samples from the large intestine, and/or pooled samples from the cecum.
Suitable sample volumes are, for example, from 0.1 to 20ml, in particular from 0.2 to 10ml, preferably from 0.5 to 5 ml. Suitable sample masses are, for example, from 0.1 to 20g, in particular from 0.2 to 10g, preferably from 0.5 to 5 g.
Depending on the sample material and storage conditions, it may be helpful to stabilize the collected sample, for example by treating the sample with a protease inhibitor to avoid enzymatic degradation of the APP contained in the sample. Preferably, the stabilizer is added to the sample immediately after sample collection.
In accordance with the above, a particular embodiment of the present application relates to an in vitro method for detecting intestinal barrier failure in a flock of birds, the method comprising the steps of:
a) collecting and combining fecal sample material derived from said flock of birds;
b) optionally stabilizing the combined sample material; and
c) determining the amount of ovotransferrin contained in the pooled sample material;
wherein
An increase in the amount of ovotransferrin contained in the sample as compared to the reference sample is indicative of intestinal barrier failure.
In addition to the above, the inventors have unexpectedly found that the amount of a particular APP-based marker comprised in an intestinal sample material derived from an individual animal or animal population correlates with the degree of intestinal barrier failure. Accordingly, the present invention provides an in vitro method for detecting the extent of intestinal barrier failure in an animal, the method comprising the steps of:
a) collecting intestinal sample material of a specific animal or animal population; and
b) determining the amount of at least one protein marker comprised in the sample material;
wherein
At least one protein marker comprises or consists of an acute phase protein or a functional fragment thereof,
and wherein
The amount of the at least one protein marker comprised in the sample indicates the extent of intestinal barrier failure.
Furthermore, the present invention provides an in vitro method for detecting the extent of intestinal barrier failure in a flock of birds, the method comprising the steps of:
a) collecting pooled fecal samples derived from the flock;
b) determining the amount of at least one protein marker in the sample material;
wherein
At least one protein marker comprises or consists of an acute phase protein or a functional fragment thereof,
and wherein
The amount of the at least one protein marker comprised in the sample indicates the extent of intestinal barrier failure.
Suitable sample materials, protein markers, and test parameters and conditions are as defined above.
The above method can be performed using only one specific protein marker. Alternatively and also according to the invention, two or more protein markers may be analyzed simultaneously.
In a particularly preferred embodiment, the intestinal sample material is a pooled fecal sample derived from a flock of birds and the at least one protein marker is ovotransferrin or a functional fragment thereof.
The present invention provides the above-described methods for detecting and determining the extent of intestinal barrier failure, respectively. This enables farmers to make eligibility decisions as to whether or not to take measures to improve gut health.
Measures against the occurrence of and/or against the progression of intestinal barrier failure include feeding or administering substances that promote health, such as animal feed additives or therapeutic agents. The term "administering" or related terms includes oral administration. Oral administration can be via drinking water, oral gavage, aerosol spray, or animal feed. The term "livestock feed additive" refers to any additive used to favorably influence the performance of animals in good health conditions or to favorably influence the environment. Examples of livestock feed additives are digestibility enhancers, i.e. substances that increase the digestibility of the diet by acting on the target feed material when fed to animals; an intestinal flora stabilizer; microorganisms or other chemically defined substances which, when fed to an animal, have a positive effect on the intestinal flora; or substances that favorably affect the environment. Preferably, the health promoting substance is selected from the group consisting of probiotics, prebiotics, phytotherapeutic agents, organic/fatty acids, zeolites, bacteriophages and lysozyme or any combination thereof.
The inventors have found that the test procedure of the present invention can also be used to monitor the intestinal health status of animals.
As used in the context of this embodiment, the term "intestinal health state" refers to the state of the intestinal barrier.
By the above method, the occurrence or progression of intestinal barrier failure can be detected. On the other hand, the effectiveness of the measures taken against the occurrence and/or progression of intestinal barrier failure can be controlled.
Accordingly, the present invention also relates to an in vitro method for monitoring the intestinal health status in an animal, comprising the steps of:
a) collecting intestinal sample material of a particular animal or animal population at successive time points;
b) determining the amount of at least one protein marker comprised in the sample obtained in step a); and
c) determining a deviation in the amount of at least one protein marker comprised in the sample obtained in step a);
wherein
The at least one protein marker comprises or consists of an acute phase protein or a functional fragment thereof.
Furthermore, the present invention provides an in vitro method for monitoring the status of the intestinal barrier in a flock of birds, the method comprising the steps of:
a) collecting pooled fecal samples derived from the flock at successive time points;
b) determining the amount of at least one protein marker comprised in the sample obtained in step a); and
c) determining a deviation in the amount of the at least one protein marker comprised in the sample obtained in step a);
wherein
The at least one protein marker comprises or consists of an acute phase protein or a functional fragment thereof.
Wherein an increase in the amount of the at least one protein marker over time is indicative of the occurrence or progression of intestinal barrier failure. Conversely, a decrease in the amount of the at least one protein marker over time indicates an improvement in the intestinal health status, which may be caused by the natural healing process or by specific measures taken against the occurrence or development of intestinal barrier failure.
An "increase" or "decrease" in the amount of a protein marker generally refers to a statistically relevant amount.
The above method can be performed using only one specific protein marker. Alternatively and also according to the invention, two or more protein markers may be analyzed simultaneously.
Suitable sample materials, protein markers, and test parameters and conditions are as defined above. In a specific embodiment, the intestinal sample material is a pooled sample derived from a flock of birds and the at least one protein marker is ovotransferrin or a functional fragment thereof.
As an example, after the initial determination of the amount of the protein marker in the intestinal sample, the amount of the at least one specific biomarker may be monitored in the test sample collected and analyzed on a weekly, daily or hourly basis. In one embodiment, fecal samples are collected and analyzed on consecutive days. Fecal test samples are collected and analyzed daily from birth to slaughter.
In a specific embodiment directed to poultry, it is preferred that the first test sample is collected and analyzed during the initial growth phase (initial phase, days 5 to 10), the second test sample is collected and analyzed during the enhanced growth phase (days 11 to 18), and optionally, the third test sample is collected and analyzed at a later stage.
In an alternative embodiment, a first test sample is collected and analyzed in an initial growth phase, and additional test samples are collected and analyzed daily, for example in an enhanced growth phase, optionally until slaughter.
Another aspect of the invention is the use of an acute phase protein as an intestinal marker for detecting intestinal barrier failure in an animal subject or a population of animals. A particular embodiment of the invention is the use of ovotransferrin as a fecal marker for detecting intestinal barrier failure in an avian subject or an avian population.
Applications of the method according to the invention are, for example: (i) aiding in the diagnosis and/or prognosis of intestinal barrier failure due to intestinal disease; (ii) (ii) monitoring the progression or recurrence of intestinal barrier failure, or (iii) aiding in the assessment of the efficacy of treatment in a population of animals receiving or considered to receive treatment.
The use of the invention is particularly useful in avoiding loss of animal performance like weight gain and feed conversion ratio.
In the following, the invention is illustrated by means of non-limiting examples and exemplary embodiments.
Examples
Coccidiosis and necrotic enteritis in broiler chickens were used as models of intestinal barrier failure. Ovotransferrin serves as a protein marker.
Necrotic bowelInflammation test-sample Collection
A group of 271 day old Ross308 broiler chickens were fed a diet rich in protein and non-starch polysaccharides, which was susceptible to induction of necrotic enteritis. A detailed diet composition is described by Gholoaminoandehkorri et al (Gholoaminoandehkorri, A.R., Timbermont, L., Lanckiet, A., Van Den Broeck, W., Pedersen, K., Dewulf, J., et al (2007) a Quantification of g's details in a subclinical genetic scientific. Avian Pathol,36, 375-382). Other inducing factors consisted of administering a Gumboro vaccine to induce mild immunosuppression and ten times the dose of coccidiosis vaccine (Parabox-8 or Hiprabox, depending on the trial) to induce induced intestinal injury. For induction of necrotic lesions, approximately 4X 10 was used for three consecutive days8The animals were challenged with the netB positive clostridium perfringens strain CP56 of CFU and then euthanized. Lesion scoring in the small intestine (duodenum, jejunum and ileum) at necropsy was performed as described in Keyburn et al (Keyburn, a.l., Sheedy, s.a., Ford, m.e., Williamson, m.m., Awad, m.m., hood, j.i., et al (2006), Alpha-toxin of Clostridium perfringens not instant animal viral factor in genetic materials in chips, infection im, 74,6496 @ 6500) as follows: score 0 ═ no lesions, score 1 ═ congested intestinal mucosa, score 2 ═ focal necrosis or ulcers (1-5 lesions), score 3 ═ focal necrosis or ulcers (6-15 lesions), score 4 ═ focal necrosis or ulcers (greater than or equal to 16 lesions), score 5 ═ 2-3 cm long necrotic patches, score 6 ═ diffuse necrosis. Birds with lesion scores of 2 or higher were classified as necrotic enteritis positive. Fresh cloacal samples were collected from all birds and frozen at-70 ℃. In addition, mixed urine pads were collected from each pen and frozen at-70 ℃.
After lesion scoring, samples were grouped according to disease severity of the animals, resulting in the following disease severity groups: all elicitors were received but not challenged with clostridium perfringens: birds with negative controls; challenge with clostridium perfringens without necrosis: score 0 or challenge with clostridium perfringens and with varying degrees of severity: birds scoring 2 (mild), 3-4 (moderate), or 5-6 (severe).
Coccidiosis test-sample Collection
A15 day old Ross308 broiler was challenged orally with Eimeria acervulina and Eimeria tenella. 7 days after challenge, one mixed fecal pad sample from each pen and cloaca sample from all birds were collected at the time the chickens were euthanized and lesion scoring was performed using the method of Johnson and Reid (Johnson, J. & Reid, W.M. (1970). anticancer drugs: dispensing techniques in batteries and floor-pen experiments with chips. exp Parasitiol, 28, 30-36). On the same day, mixed urinating pad and cloacal content samples were collected from their age-of-day matched controls. All birds challenged showed macroscopically visible eimeria-infected lesions with an average coccidiosis score of 5.11 ± 0.51, whereas in the non-challenged control group only one of ten birds was coccidiosis positive (coccidiosis score ═ 1). All samples were stored at-70 ℃.
Detection of ovotransferrin by enzyme-linked immunosorbent assay (ELISA)
Eight samples from negative control birds (not challenged with clostridium perfringens) and eight samples from each necrosis scoring group of challenged birds were selected. Also included are the stool pad samples collected on the day of necropsy (one stool pad sample per pen, with a total of 3 samples from pens with non-challenged birds and 3 samples from pens with challenged birds). In addition, ovotransferrin concentrations were determined in both cloacal and fecal bedding samples of the coccidiosis test. Thus, 1 litter sample was used per pen, with a total of 5 samples from pens with non-challenged birds and 6 samples from pens with eimeria-challenged birds. In addition, 20 cloacal samples from eimeria-challenged birds (n-10) or their non-challenged controls (n-10) were selected.
The untreated cloacal material or the homogenized urine pad material was thawed at room temperature. 150mg of cloacal contents or urine bedding material was diluted in 1500. mu.l TBS (50mM Tris, 150mM NaCl pH 7.2) together with protease inhibitor cocktail (P2714, Sigma-Aldrich). The samples were mixed by vortexing (2 × 1 min). The protein (supernatant) was collected after centrifugation (13,000 Xg, 10', 4 ℃) and used in duplicate (1/50 dilution) for ELISA (ChickenOvotransferrin ELISA, KT-530, Kamiya Biomedical Company, Tukwila, USA). The ELISA was performed according to the manufacturer's instructions.
Statistical analysis
The normality of the data was examined by the D' Agostino-Pearson normality test.
The difference in ovotransferrin levels (determined by ELISA) between the necrotic enteritis severity groups was calculated using Kruskal-Wallis test followed by Dunn's post test.
The difference in ovotransferrin levels between the eimeria-challenged group and the non-challenged control group was calculated using the mann-whitney test.
Spearman scale correlation was used to assess the relationship between ovotransferrin concentration in cloacal samples and necrotic enteritis lesion score or coccidiosis score. Results are reported as mean and standard error of the mean (SEM).
Correlation of fecal ovotransferrin concentration with the severity of necrotic enteritis
Most samples from birds suffering from mild necrotic enteritis (score 2) or no intestinal lesions (challenged animals and negative control animals) showed low signal. In samples from birds with more severe necrotic enteritis (necrosis score ≧ 3), significantly more ovotransferrin was detected than in samples from challenged birds that did not show intestinal disease (score 0), see Table 1. Furthermore, there was a positive correlation between necrotic enteritis disease severity and ovotransferrin concentration in fecal samples from the NE in vivo test (p ═ 0.0004).
TABLE 1
Table 1 represents the transferrin concentration (mean ± standard error of the mean) in faeces from birds receiving all the causative factors but not challenged with clostridium perfringens (negative control; n ═ 8) or from birds challenged with clostridium perfringens, which resulted in necrotic enteritis of varying degrees: no necrotizing lesions (score 0; n ═ 8); mild intestinal necrosis (score 2; n-8); moderate necrotic enteritis (score 3-4; n ═ 8) or severe necrosis (score 5-6; n ═ 8). P < 0.05.
Correlation of fecal ovotransferrin concentration with coccidiosis severity
Birds challenged with Eimeria acervulina and Eimeria tenella were used as a second model of intestinal barrier failure. Ovotransferrin levels are increased in samples from coccidiosis positive birds compared to non-challenged controls (p ═ 0.0029). Furthermore, there was a positive correlation between coccidiosis score and ovotransferrin concentration in feces (p ═ 0.0082). This difference in ovotransferrin levels is also reflected in the bedding samples, where significantly higher ovotransferrin levels were detected in the bedding samples from eimeria-challenged poultry than in the bedding samples from the non-challenged control group (p ═ 0.0043), see table 2.
TABLE 2
Table 2 represents the ovotransferrin concentration (mean ± standard error of mean) in faeces (grey) or mixed pads (white) from experimental coccidiosis infected birds (coccidiosis; individual faeces sample: n ═ 10 or mixed pad sample: n ═ 6) or non-challenged control birds (negative control; individual faeces: n ═ 10 or mixed pad sample: n ═ 5). Significant differences between the coccidiosis positive group and the non-challenged control group were expressed as p < 0.01.
Results
As shown above, elevated fecal ovotransferrin levels are measured in avians with experimental coccidiosis or necrotic enteritis (both of which cause intestinal barrier failure) using different methods. ELISA analysis samples from different in vivo NE tests showed that ovotransferrin was more abundant in samples from birds suffering from necrotic enteritis compared to non-challenged birds. Furthermore, an increased ovotransferrin concentration was measured in samples of coccidiosis positive birds compared to their non-challenged controls.
Fecal ovotransferrin levels are significantly associated with the severity of intestinal barrier failure caused by coccidiosis or necrotic enteritis.
The extent of intestinal barrier failure can be classified according to the severity of symptoms at the affected site (e.g., necrosis due to clostridium perfringens-induced necrotic enteritis) and the extent of the affected surface area. The extent of intestinal barrier failure of NE is more severe as this is associated with necrosis and the extent (in terms of surface area) of coccidiosis is higher.
As shown in the above experiments, the measurement of specific APP (ovotransferrin) is a valuable tool for measuring inflammation and accompanying intestinal barrier failure, as it can provide information about specific biological disease processes and is a useful tool for assessing the efficacy of molecules that reduce gastrointestinal disorders.