RU2771793C1 - Method for early detection of necrotic enteritis outbreak in bird population - Google Patents

Abstract

FIELD: veterinary medicine.

SUBSTANCE: present invention relates to an in vitro method for early detection of an outbreak of necrotic enteritis in a bird population, wherein the method includes a) collection of fecal sample material obtained from a bird population at successive points in time and b) determination of the quantitative ratio of the marker genes netB and cpa contained in the sample material obtained at stage a); where the reversal of the quantitative ratio of netB and cpa over time is an early sign of an outbreak of necrotic enteritis.

EFFECT: expansion of the range of methods for diagnosing necrotic enteritis in birds.

11 cl, 5 dwg, 3 ex

Description

The field of technology to which the invention belongs

The present invention relates to an in vitro method for the early detection of an outbreak of necrotizing enteritis in a bird population. More specifically, the present invention provides a method for tracking the proportion of two toxin-coding genes (netB/cpa and cpa/netB, respectively) over time in a faecal sample material, thus providing an early indication of an outbreak of necrotizing enteritis.

Background of the invention

Clostridium perfringens is a common pathogen that uses complex toxins to cause histotoxic and intestinal infections in animals as well as humans. C. perfringens is a Gram-positive, rod-shaped, spore-forming, oxygen-tolerant anaerobe. Not all strains of C. perfringens are virulent. Virulent strains of C. perfringens have traditionally been classified into five toxin-associated types (A, B, C, D, and E) based on the production of four putative major toxins (alpha, beta, epsilon, and iota). Depending on the toxins produced (primary and secondary toxins such as NetB, Cpb2, and others), syndromes/diseases specific to subspecies of C. perfringens can be caused in different host organisms [Rood, J. I. (1998) "Virulence genes of Clostridium perfringens" ; Annual Review of Microbiology 52: 333-360]. Toxins are encoded by polynucleotide sequences located on the chromosome and/or on plasmids with toxin genes [Popoff, M. R. and P. Bouvet (2013). Genetic characteristics of toxigenic Clostridia and toxin gene evolution" Toxicon 75: 63-89].

As an animal pathogen, C. perfringens is responsible for several serious diseases, including avian necrotizing enteritis, which costs approximately US$6 billion a year in global agriculture [Wade, B., Keyburn, A.L. (2015), "The true cost of necrotic enteritis" World Poultry 31, 16–17].

Necrotizing enteritis (NE) is an intestinal disease of poultry that was first described in 1961. NE in chickens manifests as acute or chronic enterotoxemia. The acute form of the disease leads to a significant level of mortality due to the development of necrotic foci in the intestinal wall, while the chronic form of the disease leads to a significant loss of productivity and health. Early studies on NE suggested that the main disease-associated virulence factor was an alpha toxin (known as Cpa or Plc) with phospholipase C and sphingomyelinase activity [Keyburn, A. L. et al. (2006) Alpha-toxin of Clostridium perfringens is not an essential virulence factor in necrotic enteritis in chickens, Infection and Immunity 74(11): 6496-6500]. All strains of C. perfringens carry the gene encoding alpha toxin [Rood, J. I. (1998) "Virulence genes of Clostridium perfringens", Annual Review of Microbiology 52: 333-360; Titball, R.W., et al. (1999) "The Clostridium perfringens α-toxin." Anaerobe 5(2): 51-64]. However, recent studies have shown that alpha toxin does not appear to be an important virulence factor, as alpha toxin mutant strains were able to induce NE, casting doubt on the role of alpha toxin in the disease in general. In more recent studies, it has been suggested that the new pore-forming toxin NetB plays a major key role in the development of this disease [Keyburn, A. L. et al. (2008) "NetB, a new toxin that is associated with avian necrotic enteritis caused by Clostridium perfringens" PLoS Pathogens 4(2)].

NE is known to infect broilers, laying hens, turkeys and quails. The clinical form is most commonly seen in broilers between two and five weeks of age. This also typically occurs close to the time of diet transition from starter to growth food and close to the transition from maternal to adaptive immune system respectively, so opportunistic C. perfringens can take advantage of this transition period in the intestinal environment and proliferate. [Timbermont, L. et al. (2011) "Necrotic enteritis in broilers: An updated review on the pathogenesis" Avian Pathology 40(4): 341-347].

Because not all strains of Clostridium perfringens are capable of causing NE, differential assays are required to further distinguish strains that cause NE. Molecular methods such as PCR, AFLP (Amplified Fragment Length Polymorphism) and/or PFGE (Pulsed Field Gel Electrophoresis) are available to identify strains of Clostridium perfringens. Such methods, however, still have limited quantitative power in recognizing Clostridium perfringens strains that cause NE.

Moreover, a method for early detection of an NE outbreak in a bird population is of particular interest because such early detection would allow for early intervention and provide farmers with a few days advantage over traditional methods for detecting NE in a bird population. Thus, there was an urgent need to provide a fast and reliable non-invasive ante mortem method for the early detection of an outbreak of necrotizing enteritis in a bird population.

Brief description of the invention

Accordingly, one object of the present invention is to provide an in vitro method for the early detection of an outbreak of necrotizing enteritis in a bird population, the method comprising:

a) collecting faecal sample material from the bird population at successive time points, and

b) determining the quantitative ratio of the netB and cpa marker genes contained in the sample material obtained in step a);

where a reversal of the quantitative ratio of netB and cpa over time (“transition”) is an early sign of an outbreak of necrotizing enteritis.

A further object of the present invention is to provide an in vitro method for monitoring necrotizing enteritis status in a bird population, the method comprising monitoring the proportion of netB and cpa marker genes contained in fecal samples collected at successive time points, where

a) reversal of netB to cpa ratio over time (“transition”) indicates the need for nutritional or therapeutic intervention, and/or

b) A reversal of the proportion of netB and cpa over time (“reversal”) following the administration of a feed or therapeutic agent is indicative of the effectiveness of the feed or therapeutic intervention.

Additionally, the present invention provides a multiplex qPCR kit suitable for use in the method described above, which kit contains a netBα specific primer pair and a cpa specific primer pair.

Key aspects of the present invention are described in detail below.

Detailed description of the invention

The present inventors have found that a reversal of the netB and cpa marker gene ratios (ie, netB/cpa and cpa/netB ratios, respectively) is systematically observed prior to the morphological diagnosis of necrotizing enteritis in a bird population. Thus, this reversal of the quantitative ratio of the netB and cpa marker genes can be used as a diagnostic marker for predicting the onset and/or outbreak of necrotizing enteritis in the poultry population.

Accordingly, the present invention is directed to an in vitro method for early detection of an outbreak of necrotizing enteritis in a bird population, the method comprising:

a) collecting faecal sample material from the bird population at successive time points, and

b) determining the quantitative ratio of the netB and cpa marker genes contained in the sample material obtained in step a);

where a reversal of the quantitative ratio of netB and cpa over time (“transition”) is an early sign of an outbreak of necrotizing enteritis.

In the context of the present invention, the term "marker gene" includes functional fragments of the corresponding marker gene; those. functional snippets of netB and cpa.

The term necrotizing enteritis/NE refers to both clinical and subclinical/latent conditions. Accordingly, the term "outbreak" should be understood as the sudden or spontaneous occurrence of a disease (necrotizing enteritis) in a normal bird population, as well as its induction by the introduction of Clostridium perfringens alone or in combination with the creation of predisposing conditions [Shojadoost, B., Vince, A.R., Prescott , J.F., 2012. The successful experimental induction of necrotic enteritis in chickens by Clostridium perfringens: a critical review. Vet. Res. 43, 74; Fernandes da Costa, S.P., Mot, D., Bokori-Brown, M., Savva, C.G., Basak, A.K., Van Immerseel, F., Titball, R.W., 2013. Protection against avian necrotic enteritis after immunization with NetB genetic or formaldehyde toxoids. Vaccine 31, 4003–4008; Williams, R.B., Marshall, R.N., La Ragione, R.M., Catchpole, J., 2003. A new method for the experimental production of necrotic enteritis and its use for studies on the relationships between necrotic enteritis, coccidiosis and anticoccidial vaccination of chickens. parasitol. Res. 90, 19–26; Wu, S.B., Rodgers, N., Choct, M., 2010. Optimized necrotic enteritis model producing clinical and subclinical infection of Clostridium perfringens in broiler chickens. Avian Dis. 54, 1058-1065; Wu S-B, Stanley D, Rodgers N, Swick RA, Moore RJ. Two necrotic enteritis predisposing factors, dietary fishmeal and eimeria infection, induce large changes in the caecal microbiota of broiler chickens. Vet Microbiol 2014;169:188e97].

The authors of the present invention found that the time interval between the moment of reversal of the quantitative ratio of the netB and cpa marker genes and the establishment of a morphological diagnosis of necrotizing enteritis using traditional methods is in the range from one day to five days.

An example of such a reversal or transition: the netB/cpa ratio may be < 1 (corresponding to a cpa/netB ratio > 1) one day, and the next day the netB/cpa ratio may be > 1 (corresponding to a cpa/netB ratio < 1) .

In accordance with these observations, the above method may further include

c) determining the point in time at which the reversal of the quantitative ratio of the marker genes netB and cpa ("transition points") occurs.

The specified transition point, for example, can be determined by graphical analysis. Therefore, it is necessary to construct two graphs. The first graph shows the amount of netB versus time of sample collection; while the second graph shows the amount of cpa depending on the point in time of sample collection. Based on the intersection point of these two graphs, you can register the transition point.

The polynucleotide sequences of netB and cpa are known in the art. However, for purposes of clarity and completeness, the netB consensus sequence is shown under SEQ ID NO: 1 and the cpa consensus sequence is shown under SEQ ID NO: 2. The cpa gene is located on the chromosome in all strains of C. perfringens (pathogenic and non-pathogenic); while the netB gene is located in a plasmid carrying genes for toxins, pathogenic (NE-inducing) strains of C. perfringens.

The faecal sample material from step a) may be a mixed faecal sample consisting of randomly selected individual samples.

In the context of the present invention, the term "faeces" should be understood as the product from avian subjects resulting from defecation through the cloaca. Thus, faecal sample material is obtained in a non-invasive manner and collected in accordance with the sampling procedures described below.

Fecal samples to be collected from a particular bird population are preferably collected from a number of locations within the animal housing facility in order to obtain a pooled sample that is representative of the animal population as a whole.

The sample size (i.e. the number of faecal samples to be collected, with each sample being collected from a specific location within the animal housing) should be determined based on the actual housing density of the animals, i.e. taking into account the actual number of animals belonging to the tested bird population.

The sample size can be calculated using the following formula

,

,

where

n ₀ is the recommended sample size,

Z is 1.96 at a 95% confidence level,

p is the estimated proportion of the population characterized by the presence of the attribute in question, q equals 1-p, and

e is a confidence interval expressed as a decimal.

As a general rule, at least 80 to 100 individual faecal samples are sufficient for most farm bird populations. For example, for a broiler population of 20,000 animals, 96 individual samples are required at a 95% confidence level.

Several sampling methods may be used to obtain pooled faecal sample material as required for step a). In one embodiment, a pooled fecal sample is obtained by systematic grid sampling (systematic random sampling). To implement this method, the animal housing or area containing the bird population is divided into uniformly sized cells or sub-areas forming a grid based on the required number of individual fecal samples (i.e., sample size). Then, a randomly selected sample collection area is determined within the first grid cell, and the first sample is collected in the specified area. Finally, additional samples are obtained sequentially from adjacent cells, for example, in a serpentine, corner or zigzag manner, using the same relative location within each cell. A randomly chosen starting point can be generated using dice or a random number generator.

The above procedure may optionally be repeated to obtain resamples. Those. a new random position is set for one collection point, which must be repeated in all cells. Using repeated sample analysis, you can determine the variability in the estimate of the mean obtained from the original samples.

Accordingly, the aforementioned methods may further comprise the following sub-steps:

(a1) dividing the animal housing or area containing the animal population into an equal number of uniformly sized cells forming a grid;

(a2) determining at least one randomly selected sampling site within the first cell and collecting one first sample from said randomly selected sampling site; and

(a3) collecting individual faecal samples sequentially from the remaining cells using sampling sites with the same relative location within each cell; and optionally (a4) repeating steps (a2) and (a3) to obtain at least one resampling.

The sample size corresponds to the number of cells forming a grid in the case of collecting one sample per cell. In general, if x samples are to be collected for each cell, the sample size is the number of cells divided by x.

The method of systematic grid sampling can be easily implemented in the field. In this way, over- or under-representation of sub-regions can be avoided. The patterns of systematic grid sampling in accordance with the present invention are exemplified in FIG. 1 and FIG. 2.

Another sampling method is stratified random sampling (i.e. random sampling in a grid). In this case, samples are obtained sequentially from adjacent cells of the grid, however, the location of the sampling site within each cell is random.

Alternatively, samples can be collected through simple random sampling, in which case samples are collected from random locations (not gridded) throughout the space in which the animals are kept. For this method, it is necessary to use a formal approach to determine random sampling locations, for example, based on a random number generator.

Samples can be collected manually with a spatula or similar instrument and immediately transferred to a sample collection vessel or tube.

In an alternative embodiment, a pooled faecal sample can be obtained using the "overshoe" method, performed by walking around the premises using a route that will provide representative samples for all parts of the premises or the corresponding sector. Such a route, for example, may be characterized by a uniform shape of serpentine or winding lines, angled lines or zigzag lines. Shoe covers for collecting samples from the floor, having sufficient absorbent properties to absorb moisture, are particularly suitable. However, tubular gauze stockings are also acceptable.

Suitable masses of individual samples when collected are, for example, 0.1 to 20 g, in particular 0.2 to 10 g, preferably 0.5 to 5 g. similar device.

After sampling is completed, the sample material is to be homogenized. A person skilled in the art will be aware of suitable commonly used homogenization methods. The pooled sample thus obtained may be diluted and/or stabilized. Sample stabilization in this context means protecting the nucleic acid material contained in the sample from attack by nucleases in solution, for example by using a buffer solution containing nuclease inhibitors.

Sample material is required to be collected at successive time points. Fecal sample material can be collected and analyzed weekly, daily or hourly. For example, test faecal samples can be collected and analyzed daily from birth to slaughter.

The bird population is preferably a bird population. The bird population according to the present invention is preferably poultry. Preferred poultry according to the present invention are chickens, turkeys, ducks and geese. Poultry may have characteristics that are optimal for producing young stock. This type of poultry is also called parent and grandparent animals. Preferred parents and grandparents respectively are (great)parent broilers, (great)parent ducks, (great)parent turkeys and (great)parent geese.

Poultry according to the present invention may also be selected from ornamental birds and game birds. Preferred ornamental birds or game birds are peacocks, pheasants, partridges, guinea fowls, quails, capercaillie, geese, pigeons and swans. In addition, the preferred poultry in accordance with the present invention are ostriches and parrots. The most preferred poultry in accordance with the present invention are broilers.

For broiler flocks, faecal samples may be collected and analyzed daily during the initial growth phase (starter phase, day 5 to day 10) and/or during the boost phase (day 11 to day 18), and optionally at a later stage. stages.

In one embodiment, faecal sample material, specifically fecal sample material from a broiler population, is collected and analyzed daily starting on day 10.

The netB and cpa marker genes can be isolated from faecal samples prior to quantification. Isolation of polynucleotides, for example, can be carried out by extraction with the cetyltrimethylammonium bromide (CTAB) method or by various commercial nucleic acid extraction kits, in which cell lysis is achieved by chemical lysis and/or mechanical disruption of the cells and the nucleic acid is captured silica matrices or magnetic beads coated with silica. Commercial extraction kits designed specifically for faecal material or coarse material are particularly suitable.

Marker genes can be detected and/or quantified by conventional methods such as sequencing, hybridization or various PCR techniques known in the art.

In an alternative embodiment, marker genes contained in a sample from an animal can be quantified directly, for example, using PCR, qPCR, sequencing, or hybridization techniques.

In one specific embodiment, the quantitative ratio of the netB and cpa marker genes or homologues or functional fragments of these marker genes contained in the sample material obtained in step a) is determined by qPCR.

In the above methods of the present invention, one or more oligonucleotides selected from the group consisting of

a) oligonucleotides having at least 80%, preferably at least 85%, 90% or 95%, most preferably 100% sequence identity with the polynucleotide shown under SEQ ID NO:3;

b) oligonucleotides having at least 80%, preferably at least 85%, 90% or 95%, most preferably 100% sequence identity with the polynucleotide shown under SEQ ID NO: 4;

c) oligonucleotides having at least 80%, preferably at least 85%, 90% or 95%, most preferably 100% sequence identity with the polynucleotide shown under SEQ ID NO: 5;

d) oligonucleotides having at least 80%, preferably at least 85%, 90% or 95%, most preferably 100% sequence identity with the polynucleotide shown under SEQ ID NO: 6;

e) oligonucleotides having at least 80%, preferably at least 85%, 90% or 95%, most preferably 100% sequence identity with the polynucleotide shown under SEQ ID NO: 7;

f) oligonucleotides having at least 80%, preferably at least 85%, 90% or 95%, most preferably 100% sequence identity with the polynucleotide shown under SEQ ID NO:8;

g) oligonucleotides complementary to oligonucleotides according to (a) - (f);

h) oligonucleotides containing any of the oligonucleotides according to (a) - (g) and extended by no more than 5 base pairs compared to the oligonucleotides according to (a) - (g);

can be used as a PCR primer and/or as a PCR probe.

While the polynucleotide presented under SEQ ID NO: 3, is a primer for PCR (forward) to detect netB. The polynucleotide shown under SEQ ID NO: 4 is a primer for PCR (reverse) to detect netB. The polynucleotide shown under SEQ ID NO: 5 is a PCR probe for detecting netB.

In addition, the polynucleotide shown under SEQ ID NO: 6 is a PCR (forward) primer for cpa detection. The polynucleotide shown under SEQ ID NO: 7 is a PCR (reverse) primer for cpa detection. The polynucleotide shown under SEQ ID NO: 8 is a PCR probe for cpa detection.

In accordance with the foregoing, the present invention is further directed to the use of oligonucleotides selected from the group consisting of

a) oligonucleotides having at least 80%, preferably at least 85%, 90% or 95%, most preferably 100% sequence identity with the polynucleotide shown under SEQ ID NO:3;

b) oligonucleotides having at least 80%, preferably at least 85%, 90% or 95%, most preferably 100% sequence identity with the polynucleotide shown under SEQ ID NO: 4;

c) oligonucleotides having at least 80%, preferably at least 85%, 90% or 95%, most preferably 100% sequence identity with the polynucleotide shown under SEQ ID NO: 5;

d) oligonucleotides having at least 80%, preferably at least 85%, 90% or 95%, most preferably 100% sequence identity with the polynucleotide shown under SEQ ID NO: 6;

e) oligonucleotides having at least 80%, preferably at least 85%, 90% or 95%, most preferably 100% sequence identity with the polynucleotide shown under SEQ ID NO: 7;

f) oligonucleotides having at least 80%, preferably at least 85%, 90% or 95%, most preferably 100% sequence identity with the polynucleotide shown under SEQ ID NO:8;

g) oligonucleotides complementary to oligonucleotides according to (a)-(f);

h) oligonucleotides containing any of the oligonucleotides according to (a) - (g) and extended by no more than 5 base pairs compared to the oligonucleotides according to (a) - (g);

for early detection of an outbreak of necrotizing enteritis in a bird population.

The present invention provides the non-invasive methods for early detection of an NE outbreak as described above, which can be performed ante mortem. This allows the farmer to take action against an outbreak of necrotic enteritis at an early stage.

Accordingly, the present invention also relates to the use of any of the aforementioned methods for determining the need for nutritional or therapeutic interventions.

Such interventions or measures include feeding or administering substances with therapeutic properties, such as zootechnical feed additives, or therapeutic agents. The term "administration" or related terms includes oral administration. Oral administration can be by drinking water, oral tube, spray aerosol, or animal feed. The term "zootechnical feed additive" refers to any additive used to positively affect the performance of healthy animals or used to positively impact the environment. Examples of zootechnical feed additives are digestibility enhancers, i. substances that, when fed to animals, increase the digestibility of the diet by affecting the target feed materials; stabilizers of intestinal microflora; microorganisms or other substances with a chemically determined composition, which, when fed to animals, show a positive effect on the intestinal microflora; or substances that have a positive effect on the environment. Preferably, the therapeutic agents are selected from the group consisting of probiotics, prebiotics, botanicals, organic/fatty acids, bacteriophages, and bacteriolytic enzymes, or any combination thereof.

In addition, the authors of the present invention found that the reverse reversion ("reverse transition") of the quantitative ratio of netB and cpa over time indicates the regression or disappearance of necrotizing enteritis in the bird population. Said regression or disappearance may occur naturally (in the form of spontaneous recovery) or may be due to therapeutic or nutritional intervention.

Accordingly, the present invention provides an in vitro method for monitoring necrotizing enteritis status in a population of birds, the method comprising monitoring the proportion of netB and cpa marker genes contained in fecal samples collected at successive time points, where

a) reversal of netB to cpa ratio over time (“transition”) indicates the need for nutritional or therapeutic intervention, and

b) A reversal of the proportion of netB and cpa over time (“reversal”) following the administration of a feed or therapeutic agent is indicative of the effectiveness of the feed or therapeutic intervention.

The point in time at which the reverse reversion occurs (the "reverse transition point") can be determined graphically, in a similar manner as described above for the transition point.

The term "status control for necrotizing enteritis" should be understood as an assessment of the presence or absence of any sign of an outbreak of necrotizing enteritis or regression/disappearance of necrotizing enteritis, respectively.

Suitable sample materials and sample collection methods for this method are as described above.

The nutritional or therapeutic intervention may include the administration of substances selected from the group consisting of probiotic agents, prebiotic agents, herbal formulations, organic/fatty acids, bacteriophages, and bacteriolytic enzymes, or any combination thereof. Particularly preferred are probiotics.

Accordingly, the present invention further relates to probiotic agents for use in the treatment of necrotizing enteritis, wherein an outbreak of necrotizing enteritis is detected by any of the aforementioned methods.

As an example of the above-described methods for monitoring necrotizing enteritis status in a bird population, necrotizing enteritis is diagnosed based on the reversal of the quantitative ratio of netB and cpa over time; for example, the netB/cpa ratio is observed to go from < 1 to > 1. Immediately after the diagnosis is made, the farmer intervenes, for example by administering probiotics. The proportion of netB and cpa contained in faecal samples collected at successive time points continues to be further monitored. If the intervention is effective, the ratio of netB and cpa changes again, for example, the transition of the netB/cpa ratio from a value > 1 to a value < 1 is observed.

In one embodiment of the methods described above for monitoring necrotizing enteritis status in a bird population, one or more oligonucleotides selected from the group consisting of

a) oligonucleotides having at least 80%, preferably at least 85%, 90% or 95%, most preferably 100% sequence identity with the polynucleotide shown under SEQ ID NO: 3;

b) oligonucleotides having at least 80%, preferably at least 85%, 90% or 95%, most preferably 100% sequence identity with the polynucleotide shown under SEQ ID NO: 4;

c) oligonucleotides having at least 80%, preferably at least 85%, 90% or 95%, most preferably 100% sequence identity with the polynucleotide shown under SEQ ID NO: 5;

d) oligonucleotides having at least 80%, preferably at least 85%, 90% or 95%, most preferably 100% sequence identity with the polynucleotide shown under SEQ ID NO: 6;

e) oligonucleotides having at least 80%, preferably at least 85%, 90% or 95%, most preferably 100% sequence identity with the polynucleotide shown under SEQ ID NO: 7;

f) oligonucleotides having at least 80%, preferably at least 85%, 90% or 95%, most preferably 100% sequence identity with the polynucleotide shown under SEQ ID NO: 8;

g) oligonucleotides complementary to oligonucleotides according to (a) - (f);

h) oligonucleotides containing any of the oligonucleotides according to (a) - (g) and extended by no more than 5 base pairs compared to the oligonucleotides according to (a) - (g);

used as a PCR primer and/or as a PCR probe.

In accordance with the foregoing, the present invention further relates to the use of oligonucleotides selected from the group consisting of

a) oligonucleotides having at least 80%, preferably at least 85%, 90% or 95%, most preferably 100% sequence identity with the polynucleotide shown under SEQ ID NO:3;

b) oligonucleotides having at least 80%, preferably at least 85%, 90% or 95%, most preferably 100% sequence identity with the polynucleotide shown under SEQ ID NO: 4;

c) oligonucleotides having at least 80%, preferably at least 85%, 90% or 95%, most preferably 100% sequence identity with the polynucleotide shown under SEQ ID NO: 5;

d) oligonucleotides having at least 80%, preferably at least 85%, 90% or 95%, most preferably 100% sequence identity with the polynucleotide shown under SEQ ID NO: 6;

e) oligonucleotides having at least 80%, preferably at least 85%, 90% or 95%, most preferably 100% sequence identity with the polynucleotide shown under SEQ ID NO: 7;

f) oligonucleotides having at least 80%, preferably at least 85%, 90% or 95%, most preferably 100% sequence identity with the polynucleotide shown under SEQ ID NO:8;

g) oligonucleotides complementary to oligonucleotides according to (a) - (f);

h) oligonucleotides containing any of the oligonucleotides according to (a) - (g) and extended by no more than 5 base pairs compared to the oligonucleotides according to (a) - (g);

to determine the effectiveness of nutritional or therapeutic interventions.

The present invention further provides a multiplex qPCR diagnostic kit for determining the ratio of netB and cpa and for tracking the ratio of netB and cpa over time, respectively.

Said set contains a primer pair for detecting netB and a primer pair for detecting cpa, where the primer pair for netB provides

oligonucleotides having at least 80%, preferably at least 85%, 90% or 95%, most preferably 100% sequence identity with the polynucleotide represented by SEQ ID NO:3, and

oligonucleotides having at least 80%, preferably at least 85%, 90% or 95%, most preferably 100% sequence identity with the polynucleotide shown under SEQ ID NO: 4;

and a couple of primers for cpa provides

oligonucleotides having at least 80%, preferably at least 85%, 90% or 95%, most preferably 100% sequence identity with the polynucleotide represented by SEQ ID NO: 6, and

oligonucleotides having at least 80%, preferably at least 85%, 90% or 95%, most preferably 100% sequence identity with the polynucleotide shown under SEQ ID NO: 7.

Optionally, the multiplex qPCR kit of the present invention may further comprise one or more netB detection probes and/or one or more cpa detection probes.

In one embodiment, the multiplex qPCR kit contains, in addition to the aforementioned netB and cpa primer pair, a netB detection probe and a cpa detection probe,

where the probe to detect netB provides

oligonucleotides having at least 80%, preferably at least 85%, 90% or 95%, most preferably 100% sequence identity with the polynucleotide shown under SEQ ID NO: 5;

and cpa detection probe provides

oligonucleotides having at least 80%, preferably at least 85%, 90% or 95%, most preferably 100% sequence identity with the polynucleotide shown under SEQ ID NO: 8.

The kit may further contain buffer solutions such as PCR buffer; magnesium salts; deoxynucleotide triphosphates (dNTPs). The kit may also contain items such as sample collection tubes, nucleic acid extraction reagents, and/or instructions for their use.

Embodiments of the methods of the present invention are, for example, (i) assisting in the diagnosis and/or prognosis of avian necrotizing enteritis, (ii) monitoring the progression or recurrence of avian necrotizing enteritis, (iii) facilitating the evaluation of treatment efficacy in a population of animals that are being treated or for which treatment is intended; or (iv) monitoring the efficacy of (therapeutic) vaccination against C. perfringens avian necrotizing enteritis.

Embodiments of the methods of the present invention, in particular, help to avoid declining animal performance indicators such as, for example, body weight gain and feed conversion.

The present invention is further illustrated by non-limiting examples and illustrative embodiments.

Examples

Approximately 20,000 broilers were randomly assigned to broiler houses as part of the company's normal broiler placement procedures in accordance with an American Humane Association certified program that limits stocking density to 6.2 lb/square foot at slaughter, including meeting basic housing and control requirements. . All flocks were maintained according to standard company protocols that followed the breeders' recommendations for light, temperature and ventilation. Feeds consisted of the main diet (corn and soy) adjusted to the needs of the birds for starter, grower and finisher feeds. The general conditions of the livestock were monitored daily: the availability of food and water, temperature control, and any non-standard conditions. Dead birds were removed and dissected to determine the cause of death, and emaciated birds were killed to save them from further suffering.

Sample Collection

Fecal samples and livestock performance data for several standard live broiler production processes were collected daily from day 10/11 to day 24/25 over a period of 2 years. During this time period, three flocks (Examples 1, 2 and 3) were diagnosed as positive for necrotizing enteritis (outbreak flocks) based on the spike in mortality during the disease window for NE and the observation of typical NE intestinal lesions in dead birds. opened by a veterinarian. All faecal samples collected from these NE outbreak flocks were processed individually according to Evonik's proprietary sample processing and qPCR protocol.

At each time point or collection event, 24 separate samples were collected from each quadrant of the room using plastic tongs, passing through each quadrant in a zigzag path. To avoid cross-contamination of specimens, new sterile tongs were used for each room and the prescribed biosecurity measures were followed. In addition, debris such as wood shavings, droppings, etc. were removed from the samples before all samples from 4 quadrants were mixed to produce one pooled sample (consisting of 96 individual fecal samples) in a sterile sample collection bag. The samples were placed on ice and transferred to the laboratory for storage at -80°C.

DNA extraction

Each bag of 96 pooled samples was allowed to thaw slowly at room temperature; then the faeces were transferred to a sterile container and thoroughly mixed with a sterile spatula to squeeze the tongue. Five (5) grams of the homogenized sample was transferred to a proprietary sample collection tube containing 20 ml of stabilizing buffer and glass beads. Faecal samples in collection tubes are stable for a period of no more than 7 days at temperatures between +15°C and +30°C.

The tube containing the faecal sample was incubated at 70° C. for 20 minutes in a water bath. The tube was then transferred to a Poly Mix mill (granule beater) for homogenization at 20 Hz for 15 minutes. At the end of homogenization, the sample was centrifuged at 2000g for 5 minutes and 500 µl of the supernatant was used for DNA extraction. DNA extraction was performed using a King Fisher Flex system (Thermo Fisher, USA) according to the protocol of the proprietary fecal extraction kit available from Evonik.

The King Fisher device was prepared by loading a predefined program ("Cper_Extraction_01") defining the various steps of the extraction procedure; sampling tips, DNA elution plate, wash plates and sample plate were prepared as described below.

The 96-tip comb was inserted into an empty deep well plate and placed in the device. Thereafter, 100 µl of elution buffer was added to the elution plate and this plate was also placed in the device. In addition, 500 μl of wash buffers 3, 2 and 1 were placed in each well of 3 different wash plates, respectively, and these plates were placed in the device in the same order. Finally, 300 µl lysis buffer, 25 µl magnetic beads, 20 µl enhancer, 10 µl internal control, and 500 µl faecal sample supernatant were added to each well of the sample plate. After placing the sample plate in the device, the extraction was started by pressing the start button.

Quantification of DNA

To quantify markers in DNA, 20 µl of a master mix consisting of 5 µl of component A of the master mix, 15 µl of component B of the master mix and 1 µl of IC (internal control) was prepared according to the instructions for the patented PCR assay kit in real time from Evonik Nutrition & Care GmbH for every reaction. Received enough master mix to ensure the analysis of all samples, controls without template (NTC) and 4 standards (from S1 to S4) in duplicate. 20 μl of the master mix was added to individual wells of a 96-well plate. Then, 10 μl of the extracted DNA sample was transferred to each well. 10 µl of the respective standard and 1 µl of IC were transferred to each standard well, respectively. To obtain NTC, 10 μl of sterile nuclease-free water and 1 μl of IC were transferred to each well for NTC. The contents of the plate were mixed thoroughly with a multichannel pipette and the plate was sealed with Clear Weld Seal Mark II foil. The tablet was centrifuged for 30 seconds at 1000 g (~3000 rpm). Finally, the plate was analyzed in a real-time PCR device CFX96 (Bio Rad, Germany) with the following PCR conditions: 45 cycles of denaturation at 95°C for 15 seconds, annealing at 58°C for 45 seconds and extension at 72°C C for 15 seconds. Data were obtained during the amplification phase of the qPCR cycle. At the end of the run, data acquired with BioRad's CFX96 was pre-processed with Bio-Rad's CFX Manager 3.1 and exported to Excel 2013 for further analysis. Quantitative determination of markers in the samples was carried out using a standard curve constructed using standard solutions (from S1 to S4) containing equal concentrations of both targets. The concentrations of netB and cpa in S1, S2, S3 and S4 were 10 ⁴ copies/µl, 10 ³ copies/µl, 10 ² copies/µl and 10 ¹ copies/µl, respectively. Logarithmic values for standards were plotted on the x-axis, while Ct (threshold cycle values) were plotted on the y-axis. The resulting linear regression line [y = mx + b or Ct = m (log number) + b] was used to determine the target concentrations in the test sample.

List of primers and probes used during qPCR to quantify target expression levels:

Target Primers and probes (where applicable) Probe reporter

netB Direct: 5'-TATACTTCTAGTGATACCGC-3'

(SEQ ID NO: 3)

Reverse: 5'-ATCAGAATGAGGATCTTCAA-3'

(SEQ ID NO: 4)

Probe: 5'-TCACATAAAGGTTGGAAGGCAAC-3' FAM

(SEQ ID NO: 5)

cpa Straight: 5'-TACATATCAACTAGTGGTGA-3'

(SEQ ID NO: 6)

Reverse: 5'-ATTCTTGAGTTTTTCCATCC-3'

(SEQ ID NO: 7)

Probe: 5'-TGGAACAGATGACTACATGTATTTTGG-3' Cy5

(SEQ ID NO: 8)

Example 1

Day Marker cq Mean Cq Initial amount per 1 g of faeces Log10 Mean netB/cpa
(for log 10 values) thirteen netB 28.05 27.955 4.49E+04 4.65E+00 4.68E+00 0.99 27.86 5.14E+04 4.71E+00 spa 28.86 28.795 5.04E+04 4.70E+00 4.72E+00 28.73 5.50E+04 4.74E+00 fourteen netB 28.41 28.365 3.48E+04 4.54E+00 4.55E+00 0.83 28.32 3.70E+04 4.57E+00 spa 26.32 26.26 2.94E+05 5.47E+00 5.49E+00 26.2 3.19E+05 5.50E+00 fifteen netB 25.19 25.195 3.42E+05 5.53E+00 5.53E+00 1.04 25.2 3.40E+05 5.53E+00 spa 26.88 26.875 1.99E+05 5.30E+00 5.30E+00 26.87 2.00E+05 5.30E+00 sixteen netB 29.02 29.035 2.26E+04 4.35E+00 4.35E+00 1.04 29.05 2.20E+04 4.34E+00 spa 30.65 30.64 14570 4.16E+00 4.17E+00 30.63 14690 4.17E+00 17 netB 29.55 29.395 1.54E+04 4.19E+00 4.24E+00 1.08 29.24 1.93E+04 4.28E+00 spa 31.64 31.42 7.32E+03 3.86E+00 3.93E+00 31.2 9.92E+03 4.00E+00 20 netB 28.79 28.75 2.66E+04 4.42E+00 4.44E+00 1.06 28.71 2.82E+04 4.45E+00 spa 30.59 30.585 1.52E+04 4.18E+00 4.18E+00 30.58 1.53E+04 4.18E+00 21 netB 24.46 24.28 5.75E+05 5.76E+00 5.81E+00 1.05 24.1 7.41E+05 5.87E+00 spa 26.21 26.06 3.16E+05 5.50E+00 5.55E+00 25.91 3.89E+05 5.59E+00 22 netB 28.37 28.285 3.58E+04 4.55E+00 4.58E+00 1.05 28.2 4.03E+04 4.60E+00 spa 30.09 29.985 2.14E+04 4.33E+00 4.36E+00 29.88 2.49E+04 4.40E+00 23 netB 25.96 26.09 1.98E+05 5.30E+00 5.26E+00 1.08 26.22 1.65E+05 5.22E+00 spa 28.12 28.26 8.38E+04 4.92E+00 4.88E+00 28.4 6.93E+04 4.84E+00 24 netB 24.39 24.405 6.05E+05 5.78E+00 5.78E+00 1.05 24.42 5.93E+05 5.77E+00 spa 26.08 26.155 3.46E+05 5.54E+00 5.52E+00 26.23 3.12E+05 5.49E+00

In this example, the reversal of the quantitative ratio of netB and cpa (transition point) occurred from day 14 to day 15. The presence of an outbreak of necrotizing enteritis was established based on the diagnosis of the veterinarian (at autopsy) on day 16.

A graphical representation of the data from Example 1 can be found in FIG. 3.

Example 2

Day Marker cq Mean Cq Initial quantity per 1 g of faeces Log10 Mean netB/cpa
(for log 10 values) thirteen netB 33.55 33.73 8.98E+02 2.95E+00 2.90E+00 0.75 33.91 6.97E+02 2.84E+00 spa 31.94 31.62 5.92E+03 3.77232171 3.86977098 31.3 9.27E+03 3.96722026 fourteen netB 27.71 27.705 5.73E+04 4.76E+00 4.76E+00 0.92 27.7 5.77E+04 4.76E+00 spa 27.24 27.225 1.55E+05 5.19E+00 5.19E+00 27.21 1.58E+05 5.20E+00 fifteen netB 27.28 27.33 7.76E+04 4.89E+00 4.87E+00 1.02 27.38 7.22E+04 4.86E+00 spa 28.6 28.57 6.05E+04 4.78E+00 4.79E+00 28.54 6.26E+04 4.80E+00 sixteen netB 24.54 24.505 5.45E+05 5.74E+00 5.75E+00 1.05 24.47 5.72E+05 5.76E+00 spa 26.33 26.26 290400 5.46E+00 5.49E+00 26.19 321900 5.51E+00 17 netB 22.49 22.5 2.34E+06 6.37E+00 6.37E+00 1.06 22.51 2.30E+06 6.36E+00 spa 24.55 24.485 1.00E+06 6.00E+00 6.02E+00 24.42 1.10E+06 6.04E+00 20 netB 21.74 21.6 3.99E+06 6.60E+00 6.64E+00 1.07 21.46 4.87E+06 6.69E+00 spa 24.16 23.95 1.32E+06 6.12E+00 6.18E+00 23.74 1.75E+06 6.24E+00 21 netB 20.76 20.715 8.00E+06 6.90E+00 6.92E+00 1.05 20.67 8.50E+06 6.93E+00 spa 22.7 22.665 3.61E+06 6.56E+00 6.57E+00 22.63 3.81E+06 6.58E+00 22 netB 18.1 18.12 5.31E+07 7.73E+00 7.72E+00 1.06 18.14 5.15E+07 7.71E+00 spa 20.41 20.405 1.78E+07 7.25E+00 7.25E+00 20.4 1.79E+07 7.25E+00 23 netB 22.4 22.295 2.50E+06 6.40E+00 6.43E+00 1.03 22.19 2.89E+06 6.46E+00 spa 23.8 23.725 1.68E+06 6.23E+00 6.25E+00 23.65 1.87E+06 6.27E+00 24 netB 20.3 20.27 1.11E+07 7.05E+00 7.05E+00 1.03 20.24 1.16E+07 7.06E+00 spa 21.8 21.825 6.77E+06 6.83E+00 6.82E+00 21.85 6.54E+06 6.82E+00

In this example, the reversal of the quantitative ratio of netB and cpa (transition point) occurred from day 14 to day 15. The presence of an outbreak of necrotizing enteritis was established based on the diagnosis of the veterinarian (at autopsy) on day 16.

A graphical representation of the data from Example 1 can be found in FIG. 4.

Example 3

Day Marker cq Mean Cq Initial quantity per 1 g of faeces Log10 Mean netB/cpa
(for log 10 values) eleven netB 30.08 30.15 1.06E+04 4.03E+00 4.00E+00 0.81 30.22 9.58E+03 3.98E+00 spa 28.07 28.01 8.72E+04 4.94E+00 4.96E+00 27.95 9.47E+04 4.98E+00 12 netB 31.33 31.295 4.37E+03 3.63998425 3.65061443 0.70 31.26 4.58E+03 3.66124461 spa 27.29 27.255 1.50E+05 5.17E+00 5.19E+00 27.22 1.57E+05 5.20E+00 thirteen netB 29.21 29.25 1.96E+04 4.29E+00 4.28E+00 0.81 29.29 1.85E+04 4.27E+00 spa 26.86 26.91 2.02E+05 5.31E+00 5.29E+00 26.96 1.88E+05 5.27E+00 fourteen netB 30.6 30.53 7.33E+03 3.87E+00 3.89E+00 0.97 30.46 8.09E+03 3.91E+00 spa 31.15 31.15 10230 4.01E+00 4.01E+00 31.15 10240 4.01E+00 17 netB 22.17 22.13 2.94E+06 6.47E+00 6.48E+00 1.05 22.09 3.11E+06 6.49E+00 spa 24.13 24 1.34E+06 6.13E+00 6.17E+00 23.87 1.60E+06 6.20E+00 eighteen netB 27.52 27.58 6.56E+04 4.82E+00 4.80E+00 1.06 27.64 5.99E+04 4.78E+00 spa 29.33 29.445 3.62E+04 4.56E+00 4.52E+00 29.56 3.09E+04 4.49E+00 nineteen netB 25.08 24.865 3.71E+05 5.57E+00 5.64E+00 1.09 24.65 5.02E+05 5.70E+00 spa 27.54 27.25 1.26E+05 5.10E+00 5.19E+00 26.96 1.88E+05 5.27E+00 21 netB 22.27 21.87 2.73E+06 6.44E+00 6.56E+00 1.07 21.47 4.84E+06 6.68E+00 spa 24.99 24.075 7.39E+05 5.87E+00 6.14E+00 23.16 2.63E+06 6.42E+00

In this example, the reversal of the quantitative ratio of netB and cpa (transition point) occurred from day 14 to day 17. The presence of an outbreak of necrotizing enteritis was established based on the diagnosis of the veterinarian (at autopsy) on day 17.

A graphical representation of the data from Example 1 can be found in FIG. 5.

Generalized results

The above experiments demonstrate that reversal of the relative abundance of the netB and cpa marker genes is systematically observed before establishing a morphological diagnosis of necrotizing enteritis in a bird population. Thus, this reversal of the quantitative ratio of the netB and cpa marker genes is suitable as a diagnostic marker for predicting an imminent outbreak of necrotizing enteritis in the poultry population.

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SEQUENCE LIST

<110> Evonik Degussa GMBH

<120> METHOD FOR THE EARLY DETECTION OF A NECROTIC ENTERITIS OUTBREAK IN

BIRD POPULATIONS

<130> 201800116

<160> 8

<170> PatentIn version 3.5

<210> 1

<211> 969

<212> DNA

<213> Clostridium perfringens

<220>

<221> other_characteristic

<222> (10)..(10)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (391)..(396)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (418)..(420)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (443)..(443)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (497)..(497)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (502)..(502)

<223> n is a, c, g or t

<400> 1

ttgaaaagan taaaaattat ttcaattaca ctagttctta caagtgtaat tagtacaagc 60

cttttttcaa ctcaaactca agtttttgca agtgaattaa atgacataaa caaaattgag 120

ttgaaaaatc taagtggaga aataataaaa gaaaatggaa aggaagctat taaatatact 180

240

gatcctcatt ctgataagaa aactgcttta ttaaatttag aaggatttat accttctgat 300

360

aatgtaaaaa gtgctgatgt aaataataat nnnnnnatag caaattctat tcctaaannn 420

actatagata aaaaagatgt atntaattca attggttatt ctataggcgg taatatatctct 480

gttgaaggaa aaactgntgg tnctggaata aatgcttcat ataatgtcca aaatactata 540

agctatgaac aacctgattt tagaacaatt caaagaaaag atgatgcaaa tttagcatca 600

tgggatataa aatttgttga gactaaggac ggttataata tagattctta tcatgctatt 660

tatggaaatc aattattcat gaaatcaaga ttgtataata atggtgataa aaatttcaca 720

gatgatagag atttatcaac attaatttct ggtggatttt cacccaatat ggctttagca 780

ttaacagcac ctaaaaatgc taaagaatct gtaataatag ttgaatatca aagatttgat 840

aatgactata ttttaaattg ggaaactact caatggcgag gaacaaacaa actttcgtca 900

acaagtgaat ataacgaatt tatgtttaaa ataaattggc aagatcataa aatagaatat 960

tatctgtaa 969

<210> 2

<211> 1197

<212> DNA

<213> Clostridium perfringens

<220>

<221> other_characteristic

<222> (21)..(21)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (23)..(24)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (28)..(28)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (36)..(37)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (40)..(40)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (44)..(44)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (64)..(65)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (78)..(78)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (81)..(81)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (96)..(96)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (102)..(102)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (128)..(128)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (138)..(139)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (147)..(147)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (160)..(160)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (165)..(165)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (167)..(167)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (170)..(170)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (213)..(213)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (225)..(225)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (264)..(264)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (267)..(267)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (274)..(274)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (288)..(288)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (307)..(307)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (318)..(318)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (360)..(360)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (393)..(393)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (420)..(420)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (423)..(423)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (429)..(429)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (453)..(453)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (510)..(510)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (540)..(540)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (552)..(552)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (558)..(558)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (567)..(567)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (584)..(584)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (600)..(600)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (604)..(605)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (613)..(613)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (621)..(621)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (633)..(633)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (680)..(680)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (684)..(684)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (738)..(738)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (741)..(742)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (757)..(757)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (759)..(759)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (783)..(783)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (807)..(807)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (825)..(826)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (840)..(840)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (843)..(843)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (885)..(885)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (963)..(963)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (978)..(978)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (985)..(985)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (993)..(993)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (999)..(999)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (1014)..(1014)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (1020)..(1020)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (1087)..(1087)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (1094)..(1094)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (1117)..(1117)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (1124)..(1124)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (1143)..(1143)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (1149)..(1149)

<223> n is a, c, g or t

<220>

<221> other_characteristic

<222> (1158)..(1158)

<223> n is a, c, g or t

<400> 2

atgaaaagaa agatttgtaa ngnncttntt tgtgcnncgn tagnaactag cctatgggct 60

gggnnatcaa ctaaagtnta ngcttgggat ggaaanattg anggaacagg aactcatgct 120

tgtcnanaan tgaaccagaa 180

agtgtaagaa aaaacttaga gattttaaaa ganaacatgc atgancttca attaggttct 240

acttatccag attatgataa gaangcntat gatntatatc aagatcantt ctgggatcct 300

gatacanata ataatttntc aaaggataat agttggtatt tagcttattc tatacctgan 360

acaggggaat cacaaataag aaaattttca gcnttagcta gatatgaatg gcaaagaggn 420

aantataanc aagctacatt ctatcttgga gangctatgc actattttgg agatatagat 480

actccatatc atcctgctaa tgttactgcn gttgatagcg caggacatgt taagtttgan 540

acttttgcag angaaagnaa agaacantat aaaataaaca cagnaggttg caaaactaan 600

gagnnttttt atnctgatat nttaaaaaac aangatttta atgcatggtc aaaagaatat 660

gcaagaggtt ttgctaaaan aggnaaatca atatactata gtcatgctag catgagtcat 720

agttgggatg attggganta nncagcaaag gtaactntng ctaactctca aaaaggaaca 780

gcnggatata tttatagatt cttacangat gtatcagagg gtaannatcc atcagttggn 840

aanaatgtaa aagaactagt agcttacata tcaactagtg gtganaaaga tgctggaaca 900

gatgactaca tgtattttgg aatcaaaaca aaggatggaa aaactcaaga atgggaaatg 960

ganaacccag gaaatgantt tatgnctgga agnaaagana cttatacttt caanttaaan 1020

1080

gcattcncag atgnttataa gccagaaaac ataaagntaa tagnaaatgg aaaagttgta 1140

gtngacaang atataaanga gtggatttca ggaaattcaa cttataatat aaaataa 1197

<210> 3

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 3

tatacttcta gtgataccgc 20

<210> 4

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 4

atcagaatga ggatcttcaa 20

<210> 5

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 5

tcacataaag gttggaaggc aac 23

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 6

tacatatcaa ctagtggtga 20

<210> 7

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 7

attcttgagt ttttccatcc 20

<210> 8

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Probe

<400> 8

tggaacagat gactacatgt attttgg 27

<---

Claims (45)

1. An in vitro method for early detection of an outbreak of necrotizing enteritis in a bird population, where the bird population is a broiler population, the method comprising: a) collecting faecal sample material from the bird population at successive time points; and b) determining the quantitative ratio of the netB and cpa marker genes contained in the sample material obtained in step a); where reversal of netB to cpa ratio over time is an early sign of an outbreak of necrotizing enteritis. 2. The method of claim 1, wherein the fecal sample material from step a) is a mixed fecal sample consisting of randomly selected individual samples. 3. The method according to claim 2, where the number of samples to be collected is determined using the following formula

, where n ₀ is the recommended sample size, Z is 1.96 at a 95% confidence level, p is the estimated proportion of the population characterized by the presence of the attribute in question, q equals 1-p, and e is a confidence interval expressed as a decimal. 4. The method according to any one of paragraphs. 2 or 3 where a mixed faecal sample is obtained by (a1) dividing the animal housing or area containing the animal population into an equal number of uniformly sized cells forming a grid; (a2) determining at least one randomly selected sampling site within the first cell and collecting one first sample from said randomly selected sampling site; and (a3) collecting individual faecal samples sequentially from the remaining cells using sampling sites with the same relative location within each cell; and optional (a4) repeating steps (a2) and (a3) to obtain at least one resampling. 5. The method of claim 1 wherein faecal sample material from the broiler population is collected and analyzed daily from day 10. 6. The method according to any one of the preceding claims, wherein the quantitative ratio of the netB and cpa marker genes contained in the sample material obtained in step a) is determined by qPCR. 7. The method according to any of the preceding paragraphs, where one or more oligonucleotides selected from the group consisting of a) oligonucleotides having at least 80%, preferably at least 85%, 90% or 95%, most preferably 100% sequence identity with the polynucleotide shown under SEQ ID NO: 3; b) oligonucleotides having at least 80%, preferably at least 85%, 90% or 95%, most preferably 100% sequence identity with the polynucleotide shown under SEQ ID NO: 4; c) oligonucleotides having at least 80%, preferably at least 85%, 90% or 95%, most preferably 100% sequence identity with the polynucleotide shown under SEQ ID NO: 5; d) oligonucleotides having at least 80%, preferably at least 85%, 90% or 95%, most preferably 100% sequence identity with the polynucleotide shown under SEQ ID NO: 6; e) oligonucleotides having at least 80%, preferably at least 85%, 90% or 95%, most preferably 100% sequence identity with the polynucleotide shown under SEQ ID NO: 7; f) oligonucleotides having at least 80%, preferably at least 85%, 90% or 95%, most preferably 100% sequence identity with the polynucleotide shown under SEQ ID NO: 8; g) oligonucleotides complementary to oligonucleotides according to (a) - (f); h) oligonucleotides containing any of the oligonucleotides according to (a) - (g) and extended by no more than 5 base pairs compared to the oligonucleotides according to (a) - (g); used as a PCR primer and/or as a PCR probe. 8. Application of the method according to any one of paragraphs. 1-7 for early detection of an outbreak of necrotizing enteritis. 9. An in vitro method for monitoring the status of necrotizing enteritis in a population of birds, the method comprising monitoring the quantitative ratio of the netB and cpa marker genes contained in fecal samples collected at successive time points, where a) reversal of netB to cpa ratio over time indicates the need for nutritional or therapeutic intervention, and b) A reversal of the quantitative ratio of netB and cpa over time after the introduction of feed or therapeutic agents indicates the effectiveness of the feed or therapeutic intervention. 10. The method of claim 9, wherein the nutritional or therapeutic intervention comprises administering substances selected from the group consisting of probiotic agents, prebiotic agents, herbal formulations, organic/fatty acids, bacteriophages, and bacteriolytic enzymes, or any combination thereof. 11. The method according to any one of paragraphs. 9 and 10, where one or more oligonucleotides selected from the group consisting of a) oligonucleotides having at least 80%, preferably at least 85%, 90% or 95%, most preferably 100% sequence identity with the polynucleotide shown under SEQ ID NO: 3; b) oligonucleotides having at least 80%, preferably at least 85%, 90% or 95%, most preferably 100% sequence identity with the polynucleotide shown under SEQ ID NO: 4; c) oligonucleotides having at least 80%, preferably at least 85%, 90% or 95%, most preferably 100% sequence identity with the polynucleotide shown under SEQ ID NO: 5; d) oligonucleotides having at least 80%, preferably at least 85%, 90% or 95%, most preferably 100% sequence identity with the polynucleotide shown under SEQ ID NO: 6; e) oligonucleotides having at least 80%, preferably at least 85%, 90% or 95%, most preferably 100% sequence identity with the polynucleotide shown under SEQ ID NO: 7; f) oligonucleotides having at least 80%, preferably at least 85%, 90% or 95%, most preferably 100% sequence identity with the polynucleotide shown under SEQ ID NO: 8; g) oligonucleotides complementary to oligonucleotides according to (a) - (f); h) oligonucleotides containing any of the oligonucleotides according to (a) - (g) and extended by no more than 5 base pairs compared to the oligonucleotides according to (a) - (g); used as a PCR primer and/or as a PCR probe.

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