Environmental population immune response

Abstract

A method for measuring and generating alerts for the response to vaccination of epidemiological units of production animals wherein said method includes the determination and evaluation of the environmental population immune response from aerial samples.

Description

Attorney Docket No.: 06500059.00016 PATENT ENVIRONMENTAL POPULATION IMMUNE RESPONSE FIELD OF INVENTION

[0001] The present invention is related to the field of animal health and uses a variety of molecular and epizootiological techniques to determine and evaluate the environmental population immune response to vaccines in environments of epidemiological units of production animals. BACKGROUND

[0002] In animal husbandry environments, proper monitoring and management of animal health and well-being is a critical aspect to ensure the production of safe, high-quality food, as well as disease prevention and control, especially in confined environments where pathogen transmission can be rapid and the risk of infection is high (US 2022026318 A1). In the field of epizootiology or veterinary epidemiology, the monitoring and analysis of animal populations is essential to understand disease dynamics, allowing the implementation of appropriate management strategies and the evaluation of the effectiveness of interventions such as vaccinations and treatments (Pardo MV, 2006).

[0003] In this context, the effectiveness of vaccination strategies in animal health is a fundamental pillar for evaluating interventions and treatments, which makes it essential to implement continuous surveillance of animal welfare, specifically focused on the evaluation of the immune response after vaccination. This approach allows us to determine how animals react to the introduced immunizing agents and their effectiveness in conferring protection against environmental pathogens. Therefore, the ability to monitor and assess the effect of vaccines is essential. This not only facilitates early detection of vaccine effectiveness, but also enables rapid and appropriate interventions to strengthen herd immunity and mitigate the spread of diseases within the animal population.

[0004] Traditionally, biomarker analysis, and serological methods have been used to evaluate the immune response in animals. However, these can be expensive, laborious, and limited in the diversity of pathogens evaluated, in addition to not offering a complete picture of the immune response of the animal population.

[0005] In prior art, analysis of biomarkers such as inflammatory proteins or cytokines has been used to evaluate the immune response in animals (Marcelo Ocamo, 2010) and other methods such as ELISA, SN or IPMA, which have high precision and are characterized byAttorney Docket No.: 06500059.00016 PATENT the detection of antibody activity in plasma or tissue samples from animals (Immunology Research Unit, 2018). ELISA, specifically, has been used to detect a wide variety of target analytes in different sample types. However, they can be expensive, laborious, and limited in terms of the diversity of pathogens evaluated (Hosseini et. al, 2018). Furthermore, these methods may not provide an accurate representation of the immune response of the entire animal population evaluated, due to biases in the selection of sampled animals and variations in the quality and quantity of samples obtained, especially at the beginning of an infection in an animal population where prevalence is low.

[0006] On the other hand, the understanding of immune responses against specific pathogens such as mycoplasmas is limited when only serology is used, and more advanced and less accessible methodologies are required for their comprehensive understanding, such as monitoring cellular responses. In this context, monitoring vaccine shedding emerges as a valuable strategy to infer protection levels in advance.

[0007] In parallel, the fight against highly prevalent pathogens in agricultural contexts does not focus solely on improving the immune response through vaccines, but also seeks to create interference between the attenuated vaccine strain and the endemic virus. This interference, between the vaccine strains and the field strains, is carried out through competition for cellular receptor sites, causing the vaccine strain, in high concentrations, to displace the pathogenic strains. Environmental monitoring of vaccine virus levels provides an indication of the degree of protection conferred by vaccine strain interference, which represents a significant advance in the management and understanding of animal herd immunity.

[0008] Recent research suggests non-invasive methods for diagnosing diseases through biomarkers in exhaled microdroplets of lung fluid, opening the possibility of applying similar techniques to monitor the immune response in animals (Morozov et al., 2018). Furthermore, it has been shown that air-dried saliva and fecal samples can be used for semiquantitative measurements of mucosal antibodies, which could simplify the collection and analysis of immunological data in agricultural settings (Vetvik et al., 1998). In parallel, the use of environmental eDNA / eRNA for pathogen detection and surveillance suggests a promising approach for non-invasive animal health monitoring (Bass et al., 2023).

[0009] In the field of patents, techniques have been explored to determine the immune response of birds after vaccination with attenuated vaccines, although these methods stillAttorney Docket No.: 06500059.00016 PATENT require invasive procedures (CZ309268 B6). Other work focuses on sequence-based measures to assess immune response, supporting the use of molecular biology for such purposes (US 2014356339 AA). Methods to predict immune responses at the population level have also been described, indicating a growing interest in collective immune assessment (US 2008220450 AA).

[0010] This outlook highlights the need and potential for improved methods that enable accurate, non-invasive assessment of the animal immune response, crucial for effective health management in production animal populations.

[0011] The present invention proposes a method of notification of the level of protection of animals through the determination and evaluation of the immune response in populations of epidemiological units of production animals, through the detection and quantification in the environment of vaccines due to the shedding effect of animals from aerial samples. SUMMARY

[0012] The present invention refers to a method for determining the environmental population immune response to vaccines in environments of epidemiological units of production animals and its evaluation, which allows understanding and reporting the level of protection of the animals. The method includes several stages, including the extraction of DNA and RNA, the analysis of the environmental population immune response in relation to the shedding of animals after a vaccination event, and notification to users. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Fig.1 is a chart of the SIR model adapted to vaccination (VIP)

[0014] Fig.2 is a chart of the monitoring of vaccine strains in the environment.

[0015] Fig.3 is a chart of the immune response in birds by measurement of environmental vaccine virus.

[0016] Fig.4 is a tracking chart of the activity of the immune system.

[0017] Fig.5 is an efficiency evaluation chart with VIP Models (SIR).

[0018] Fig.6 is a chart tracking peaks and shedding times.

[0019] Fig.7 is an interference and protection evaluation chart in the environment.Attorney Docket No.: 06500059.00016 PATENT DETAILED DESCRIPTION OF THE INVENTION

[0020] Definitions

[0021] “Environmental population immunity” in this document refers to the measurement and evaluation of the response of the immune system directly in the environment, not discriminating by individual and making a general measurement that responds to the population unit.

[0022] The “Environmental Population Immune Response” (EPIR) herein refers to the evaluation over time of the quantification of vaccine DNA or RNA sequences in air samples caused by the shedding effect of animals once a vaccine was administered.

[0023] "Shedding" herein refers to the excretion of an attenuated pathogen or vaccine after it has successfully replicated in the host. This excretion to the environment is carried out through secretions or expulsion of material at the mucosal level (respiratory, intestinal, genitourinary, etc.).

[0024] The term “Epidemiological Unit” in this document refers to one or more animal production facilities containing the same lots of animals with the same sanitary management, feeding, safety measures, etc.

[0025] The term “User” in this document corresponds to a person or institution that makes use of the technology presented in this document.

[0026] The term “Epizootiology” herein refers to a branch of epidemiology that focuses on the study of animal diseases, specifically in animal populations. It is responsible for investigating the distribution, risk factors, spread patterns and impact of diseases in animals. Epizootiology also analyzes the interactions between pathogens, host animals and the environment, with the aim of better understanding animal diseases and developing appropriate prevention and control strategies.

[0027] The term “Sample” in this document refers to one or more filters or membranes of controlled pore size containing dust particles, microorganisms, traces of genetic material, etc., which are obtained after a filtration process and are representative of an environment. A sample must have information about the environment to which it corresponds, such as sampling time and duration, the nomination of the sampled environments, a location, an associated production phase, a lot (or similar), a user, and other identifiers of the environment sampling process that makes it unique.Attorney Docket No.: 06500059.00016 PATENT

[0028] The term “Production Phase” in this document refers to one of the stages of the production process of animal husbandry that can be differentiated by the age of the animals, and / or the processes that are carried out with them.

[0029] The term “Prediction” in this document refers to the ability to provide a forecast of the health / productive status, based on data analytical techniques.

[0030] The term “Sectorized” in this document refers to an item or sector in the sense of type of animal production, such as fattening of pigs, chickens, or egg production, or incubators, etc.

[0031] The term “Confined Animals” in this document refers to those groups of animals (generally for commercial use) that are in a barn or similar closed or semi-closed enclosure, during the production phases.

[0032] Introduction

[0033] The problem addressed by the present invention is the lack of non-invasive methods that provide population information on the specific response caused by vaccines. Currently, the immune response of animals is monitored by carrying out a representative sampling of animals by taking blood from them. Animal bleeding is an invasive method, which in some cases involves the sacrifice of young animals. In response, this invention proposes a non-invasive method to determine and evaluate the environmental population immune response, based on the quantification of DNA or RNA sequences of vaccines in air samples, which reflects the shedding effect of vaccinated animals.

[0034] The present invention redefines the environmental population immune response as the process of quantifying specific genetic sequences of vaccine strains in air samples collected from an animal husbandry environment over a given period. This method is distinguished by its ability to detect the effect of post-vaccination shedding, which is the excretion of vaccine components by immunized animals. Unlike the DIVA (Differentiating Infected from Vaccinated Animals) approach, which is based on vaccines specifically designed to differentiate infected from vaccinated animals using serological markers, our innovation is not restricted to DIVA vaccines and is applicable to a broader spectrum of immunizers.

[0035] The present invention is different as it uses a population-based approach at the barn level, which allows detailed monitoring of the live virus of the vaccine strain, offering a collective perspective of the immune response that has not been explored until now.Attorney Docket No.: 06500059.00016 PATENT

[0036] The substantial contribution of this invention is the characterization of the dynamics of the vaccination response by interpreting the shape of the curve generated from quantitative data of the vaccine virus in the environment. This curve provides unprecedented information on vaccination effectiveness at the population level, allowing real-time adjustments of immunization strategies to optimize protection against communicable diseases.

[0037] The term environmental population immunity refers in the present invention to the measurement and evaluation of the response of the immune system in the environment directly, not discriminating by individual and making a general measurement that responds to the population unit. The information derived from the environment is therefore oriented towards the management needs of the facility by the user and therefore for decision making.

[0038] The environmental population immune response is the evaluation over time of the quantification of DNA or RNA sequences from vaccines in air samples caused by the shedding effect of animals once a vaccine was administered. It is essential to ensure that vaccines are being administered correctly at the appropriate times, and this technique allows its verification. In addition, specific monitoring can be carried out to evaluate the effectiveness of the vaccines.

[0039] The term "shedding" refers to the excretion of a pathogen or attenuated vaccine after it has successfully replicated in the host. This excretion to the outside is carried out through secretions or expulsion of material at the mucosal level (respiratory, intestinal, genitourinary, etc.).

[0040] This secretion of pathogens or attenuated vaccines to the outside can trigger infections through direct contact with other susceptible hosts, or indirectly through release into the environment (air, water, surfaces), which acts as a means of spread between individuals.

[0041] A typical example is shedding into the air environment by coughing / sneezing or other airway movements, which results in the dispersion of thousands of particles into the air and the potential for contagion to other susceptible organisms that share that environment.

[0042] Method description:

[0043] The method contains three stages: Determination of the environmental population immune response; Evaluation of the environmental population immune response and Generation of vaccination response alerts.Attorney Docket No.: 06500059.00016 PATENT

[0044] Determination of the population immune response

[0045] Given a vaccine A from an epidemiological unit, to determine its specific concentration (X) (XA) in the environment the procedure is:

[0046] Carrying out standardized sampling (a certain time and protocol) with air sampling equipment according to the type defined in patent application UY38805. Obtaining a filter (sample) that contains dust particles with microorganisms and genetic material of said associated microorganisms.

[0047] Storage and transportation of the sample at room temperature to the laboratory without the need to add stabilizing solution, which has stability for up to 72 hours.

[0048] The sample is subjected to a mixture of physical and chemical extractive processes that maximize the amount of genetic material extractable from the filter, which may include bead shaking, enzymatic digestion, centrifugation, and / or other validated / accessory extractive methods to ensure performance. A process consisting of these phases increases the sensitivity of subsequent analyzes from a small sample and ensures representativeness by minimizing bias and maximizing the chance of detecting pathogen diversity in the sample. Typically, a sample of less than 100 µl containing DNA or RNA is obtained to distribute among a variety of assays. DNA / RNA quantification is performed using fluorometry and / or absorbance methods. The information obtained from the sample is nanograms / m3.

[0049] In one representation, samples are used to extract DNA or RNA to apply qPCR with fluorescent probes in single or multiplex format in which a Ct value is obtained that correlates with the number of copies of A in the farm / facility. The information obtained from the sample is XA expressed as copies / m3.

[0050] Realization of chart XA (t), which establishes the concentration (XA) as a function of time.

[0051] Evaluation of the environmental population immune response

[0052] From the SIR (Susceptible, Infected, Recovered) model adapted to vaccination, a derived model called VIP was built whose 3 main populations are (Vaccinable, Infectious- Vaccinated, Protected) according to Figure 1.

[0053] Different indicators are calculated such as Total Population (PT), Secondary Vaccination Rate (SVR), Vaccine Conversion Rate (VCR), Incubation Rate (IR), VaccineAttorney Docket No.: 06500059.00016 PATENT Shedding (VS), Vaccine Shedding Rate (VSR) and Measurable Vaccine Shedding Rate (VSRm), among others.

[0054] Total population (TP): Sum of all the populations of an Epidemiological Unit

[0055] TP = VP + IVP + PP

[0056] Where:

[0057] TP is the total population

[0058] VP is the vaccinable population

[0059] IVP is the infectious-vaccinated population

[0060] PP is the protected population

[0061] Secondary vaccination rate (SVR): Number of new vaccinations that occur due to direct or indirect transmission from infectious-vaccinated population to vaccinable animals in a unit of time.

[0062] SVR = dIVP

[0063] dt

[0064] Where:

[0065] SVR is secondary vaccination rate.

[0066] dIVP = represents the rate of change of infectious vaccinated population as a function of time

[0067] dt = time differential

[0068] Vaccine conversion rate (VCR): number of new individuals who are fully vaccinated in a unit of time.

[0069] VCR = dPP

[0070] dt

[0071] Where:

[0072] VCR is vaccine conversion rate.

[0073] dPP = represents the differential of change of protected population as a function of timeAttorney Docket No.: 06500059.00016 PATENT

[0074] dt = time differential

[0075] Incubation rate (IR): time it takes for a vaccinable individual to become infectious- vaccinated after first contact.

[0076] IR = 1

[0077] λ

[0078] Where:

[0079] IR is incubation rate

[0080] λ is the vaccine transmission rate

[0081] Vaccine Shedding (VS): excretion of vaccine into the environment. It is the release into the environment of vaccine-infective particles from an infectious- vaccinated population (or individual), capable of infecting the vaccinable population.

[0082] VS. ∝ XA

[0083] Where:

[0084] VS is vaccine shedding

[0085] XA is the measurable vaccine shedding, which is proportional to VS, with XA being the analyzable fraction of this at an environmental level.

[0086] Vaccine Shedding Rate (VSR): represents the rate of change of vaccine shedding over time

[0087] VSR = dVS

[0088] dt

[0089] Where:

[0090] VSR is vaccine shedding rate

[0091] dVS is the differential of change of vaccine shedding

[0092] dt is the temporal variation

[0093] Measurable Vaccine Shedding Rate (VSRm): represents the rate of change in vaccine shedding measurable over time

[0094] VSRm = dXAAttorney Docket No.: 06500059.00016 PATENT

[0095] dt

[0096] Where:

[0097] VSRm is measurable vaccine shedding rate

[0098] dXAis the differential of change of measurable vaccine shedding

[0099] dt the temporal variation

[0100] Generation of vaccination response alerts

[0101] A vaccinable animal population can reach different levels of vaccination efficiency after the administration of a vaccine. Although several vaccination conditions are standardized, the result of a vaccination process can be variable, depending on different operational, management or biological conditions. This results in a population that can have a protective response against pathogens earlier in time, more powerful, with greater coverage (herd effect) or longer depending on the vaccination process in the population.

[0102] We propose that the environmental monitoring of vaccine shedding reflects the vaccination dynamics, and that this can be parameterized to measure the level of vaccination that confined animal populations undergo.

[0103] In the present invention, it is proposed to generate different alerts from indicators such as Total Population (TP), Secondary Vaccination Rate (SVR), Vaccine Conversion Rate (VCR), Incubation Rate (IR), Vaccine Shedding (VS), Vaccine Shedding Rate (VSR) and Measurable Vaccine Shedding Rate (VSRm), among other indicators, including their combinations which can give rise to different alerts such as: a) Vaccination efficiency, b) Vaccine memory efficiency, c) Protection by competition with vaccine strain and d) Other alerts built with the information obtained from the stages I - Determination of the environmental population immune response and II - Evaluation of the environmental population immune response.

[0104] Some of these possible alerts included in this invention are described below:Attorney Docket No.: 06500059.00016 PATENT

[0105] Vaccination efficiency To determine the percentage of animals that have developed protection at a specific time post-vaccination, reflecting the g isAttorney Docket No.: 06500059.00016 PATENT

[0106] Vaccine memory efficiency To evaluate and understand the ability of an animal population to remember an immune response to a ) at aAttorney Docket No.: 06500059.00016 PATENT

[0107] Protection by competition with vaccine strain (PBCWV) To determine the period during which an animal population Objective is protected from infections by field strains through s s ) n l [01

[0109] Vaccines are not only sources of protective benefits but occasionally, alone or in combination, they can cause problems such as revertant vaccine viruses (vaccines carrying de novo pathogenic mutations) or circulating vaccine-pathogenic recombinant strains (strains that circulate with a mixed identity between vaccine and pathogenic but that cause diseases, aAttorney Docket No.: 06500059.00016 PATENT product of recombination in some virus families). Environmental population monitoring of vaccine strain dynamics, combined with sequence identity analysis, may serve to alert of these risks “undercover” under vaccine identity.

[0110] Examples

[0111] Work example 1: Monitoring of Vaccine Strains in the Environment

[0112] In one embodiment, animals in confinement are monitored regarding the evolution of load levels of attenuated vaccine strains in the environment, as an indicator of immunization. Live attenuated viruses and bacteria are capable of infecting and disseminating in the environment. In this example, a good vaccination gives a more intense and shorter peak of detection of vaccine strain levels, and a mediocre vaccination tends to flatten the curve, since the animals become infected asynchronously due to the variation in immunization.

[0113] Working example 2: Immune Response in Birds using Environmental Vaccine Virus Curves

[0114] In another example, series of vaccinations with attenuated virus against the same respiratory pathogen in birds give rise to peaks of vaccine virus in the environment, which correspond to the times (week) of vaccination. Given these successive vaccinations, the virus peaks in the environment are increasingly lower, flattened or delayed if the animal's immune response is adequate, given that the virus could replicate less in a host with a better capacity to eliminate it, and likewise in a better immunized population. In this way, by observing the shape of the environmental attenuated virus curves we can evaluate the immune response to vaccines.

[0115] Prophetic Example 1: Tracking Immune System Activity

[0116] In another use of the technology, animal immunity involves activation and inhibition of genes related to immune system activity, and traces of that activity could be found in environmental samples. In the same way that we find pathogens and vaccines that derive from animals in the environment, we can also find other traces of animals that may have indicators of the condition of the animals.

[0117] In this example, the possibility of finding groups of gene activation / inhibition by qPCR / transcriptomics is explored and the patterns associated with the vaccination or infection phases would be analyzed.Attorney Docket No.: 06500059.00016 PATENT

[0118] In another embodiment of this example, the environment would be searched for traces at the DNA level due to methylation, or changes in the sequence associated with the activity of cells that participate in the immune response, or inflammation, stress, or other physiological indicators. In this way, specific changes could be associated from DNA analysis, which would correspond to physiological conditions of the immune system in relation to infections or vaccinations.

[0119] Prophetic Example 2: Graphical Efficiency Evaluation with VIP Models (SIR)

[0120] For an instance of initial vaccination with a live attenuated vaccine, the peak vaccine shedding for a standard population, with a standardized vaccine dose, should give a shedding curve, with a higher and earlier peak the more synchronous and efficient the vaccination.

[0121] The graphic descriptors being peak height, time to peak (from vaccine administration), shape and route of the shedding curve and area under the curve are the indicators that define vaccination efficiency. Then we can define vaccination efficiency at a percentage level (percentage of vaccinated animals protected at a given time) according to these parameters, using the shedding and functions of the VIP model (Vaccinable, Infectious- Vaccinated, Protected).

[0122] An example of this is seen in the chart of Figure 5 where different barns of animals vaccinated at greater or lesser efficiency evolve a different shedding curve, which results in protection states over time (time to reach 50% protected) different in each case.

[0123] Prophetic Example 3: Tracking Peaks and Shedding Times

[0124] To monitor multiple attenuated vaccinations of the same type, in the event of successive vaccinations, the peaks of the repetitions of administration would appear more delayed and lower as the immunization becomes stronger, being able to assign reference height differences between the "initial peak” and “repetitions.”

[0125] Being t(Vx) the vaccination time and t(Px) the shedding peak time corresponding to that vaccination x, then in successive successful vaccinations V1, V2 and V3, the following will be true: SV(P1) >> SV(P2) >> SV(P3) and t(V1-P1) < t(V2-P2) < t(V3-P3).

[0126] In a vaccination schedule, applied in a standard way to animals, the differences in heights and times between peaks can be standardized, and constitute an expected response. AAttorney Docket No.: 06500059.00016 PATENT report of significant deviation from this pattern may mean insufficient administrations, suboptimal spacing, etc., and would allow corrective actions to be taken.

[0127] Prophetic Example 4: Evaluation of Interference and Protection in the Environment

[0128] Live attenuated vaccines generate interference with field strains via immunology and by blocking entry to infection sites by site competition (for example cellular receptors). Certain vaccination schedules meet this objective, with the intention of providing extra protection in vulnerable periods. Given this, the user may want to monitor the levels of vaccine virus present in the environment as an indicator of “interference / blocking potential.”

[0129] The chart in Figure 7 shows how vaccine shedding, in an example of two vaccinations V1 and V2, has areas of high protection due to interference that are shaded. On the other hand, this overlaps with the protection given by vaccines, in which each vaccination generates greater protection. Between the two curves, areas of greater and lesser combined protection between immunization and interference can be visualized, which allow planning the use of vaccines in this type of schedule.

[0130] References

[0131] Hosseini, S., Vázquez-Villegas, P., Rito-Palomares, M., Martinez-Chapa, S.O. (2018). Advantages, Disadvantages and Modifications of Conventional ELISA. In: Enzyme- linked Immunosorbent Assay (ELISA). SpringerBriefs in Applied Sciences and Technology(). Springer, Singapore. https: / / doi.org / 10.1007 / 978-981-10-6766-2_5.

[0132] Pardo Cobas, M.V. (November 2006). Compendio de Epidemiología de la Universidad Nacional Agraria. Recovered from https: / / repositorio.una.edu.ni / 2439 / 1 / nl73p226.pdf.

[0133] “Non-invasive approach to diagnosis of pulmonary tuberculosis using microdroplets collected from exhaled air” Victor N. Morozov et al 2018 J. Breath Res.12 036010. Recovered from DOI: 10.1088 / 1752-7163 / aab3f2.

[0134] “Mucosal antibodies can be measured in air-dried samples of saliva and feces” Vetvik H, Grewal HM, Haugen IL, Ahrén C, Haneberg B J Immunol Methods.1998 Jun 1;215(1-2):163-72. Recovered from DOI: 10.1016 / s0022-1759(98)00089-1.

[0135] Environmental DNA / RNA for pathogen and parasite detection, surveillance, and ecology. David Bass, Kevin W. Christison, Grant D. Stentiford, Lauren S.J. Cook, HannaAttorney Docket No.: 06500059.00016 PATENT Hartikainen, Trends in Parasitology, 39, 4, 2023, 285-304. Recovered from https: / / doi.org / 10.1016 / j.pt.2022.12.010.

Claims

Attorney Docket No.: 06500059.00016 PATENT Claims:

1. A method for measuring and generating alerts for the response to vaccination of epidemiological units of production animals, comprising the determination and evaluation of the environmental population immune response from aerial samples.

2. The method of claim 1, wherein the determination of the environmental population immune response comprises the detection and quantification of DNA or RNA fragments of vaccines caused by the shedding effect of animals once a vaccine was administered.

3. The method of claim 2, wherein the generating of alerts on the response to vaccination of epidemiological units of production animals comprises alerts on vaccination efficiency, vaccine memory efficiency, protection due to competition with vaccine strain, pathogenic revertant vaccine risk and circulating recombinant strains.

4. The method of claim 2, wherein the environmental population immune response comprises the analysis of the behavior of the values and graphs associated with the Measurable Vaccine Shedding " VSm", Measurable Vaccine Shedding Change Rate " VSRm " and their combinations, wherein, the “VS” value expresses the amount of vaccine strain measured in copies / m3and is obtained from the application of qPCR on DNA or RNA extracted from aerial samples of animal shedding once a vaccine was administered and the value “VSRm” represents the rate of change of measurable vaccine shedding “dVS” in time “dt” and is expressed as VSRm = dVS / dt.

5. The method of claim 3, wherein the generating of alerts on the response to vaccination of epidemiological units of production animals comprises alerts on vaccination efficiency, vaccine memory efficiency, protection due to competition with vaccine strain, pathogenic revertant vaccine risk and circulating recombinant strains.

6. The method of claim 1, wherein the environmental population immune response comprises the analysis of the behavior of the values and graphs associated with the Measurable Vaccine Shedding " VSm", Measurable Vaccine Shedding Change Rate " VSRm " and their combinations, wherein, the “VS” value expresses the amount of vaccine strain measured in copies / m3and is obtained from the application of qPCR on DNA or RNA extracted from aerial samples of animal shedding once a vaccine was administered and the value “VSRm” represents the rate of change of measurable vaccine shedding “dVS” in time “dt” and is expressed as VSRm = dVS / dt.Attorney Docket No.: 06500059.00016 PATENT 7. The method of claim 1, wherein the generating of alerts on the response to vaccination of epidemiological units of production animals comprises alerts on vaccination efficiency, vaccine memory efficiency, protection due to competition with vaccine strain, pathogenic revertant vaccine risk and circulating recombinant strains.

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