Environmental Herd Immune Response

Abstract

A method for measuring and generating alerts on the response to vaccination of an epidemiological unit of production animals, comprising determining and assessing the environmental herd immune response from air samples.

Description

[Technical Field]

[0001] The present invention relates to the field of animal health and the determination and evaluation of environmental herd immune responses to vaccines in epidemiological units of production animals using a variety of molecular and zoonological techniques. [Background technology]

[0002] In livestock environments, proper monitoring and management of animal health and welfare are critical aspects for ensuring the production of safe, high-quality food and disease prevention and control, especially in confined environments where pathogen transmission can be rapid and the risk of infection high (US2022026318A1). In the field of zoonotic or veterinary epidemiology, monitoring and analysis of animal populations is essential for understanding disease dynamics, which allows for the implementation of appropriate control strategies and the evaluation of the effectiveness of interventions such as vaccination and treatment (Pardo MV, 2006).

[0003] In this context, the effectiveness of vaccination strategies in animal health is a fundamental pillar of intervention and treatment evaluation, making continuous monitoring of animal welfare essential, particularly focusing on the evaluation of post-vaccination immune responses. This approach allows for the determination of how animals respond to the introduced immunizing agent and its effectiveness in conferring protection against environmental pathogens. Therefore, the ability to monitor and evaluate vaccine effectiveness is essential. This not only facilitates early detection of vaccine efficacy, but also allows for prompt and appropriate intervention to strengthen herd immunity and mitigate the spread of disease within animal populations.

[0004] Traditionally, biomarker analysis and serological methods have been used to assess immune responses in animals, but these are expensive, labor-intensive, and limited in the diversity of pathogens they can assess, in addition to not providing a complete picture of the immune response of an animal population.

[0005] Previous studies have used biomarker analysis, such as inflammatory proteins or cytokines (Marcelo Ocamo, 2010), as well as other methods, such as ELISA, SN, or IPMA, which are highly accurate and characterized by the detection of antibody activity in plasma or tissue samples from animals (Immunology Research Unit, 2018). ELISAs, in particular, are used to detect a wide variety of target analytes in various sample types. However, they are expensive, labor-intensive, and limited in the diversity of pathogens they can evaluate (Hosseini et al., 2018). Furthermore, due to biases in the selection of animals sampled and qualitative and quantitative variations in the samples obtained, these methods may not provide an accurate representation of the immune response of the entire animal population, especially in the early stages of infection in animal populations with low disease prevalence.

[0006] On the other hand, our understanding of immune responses to specific pathogens, such as mycoplasmas, is limited using serology alone, and a comprehensive understanding requires more advanced and less accessible methods, such as monitoring cellular responses. In this context, monitoring vaccine shedding has emerged as a valuable strategy to estimate protection levels in advance.

[0007] In parallel, the fight against highly prevalent pathogens in agricultural settings has focused not only on improving immune responses through vaccines, but also on creating interference between attenuated vaccine strains and endemic viruses. This interference between vaccine and field strains occurs through competition for cellular receptor sites, thereby displacing high concentrations of the vaccine strain with the pathogenic strain. Environmental monitoring of vaccine virus levels provides an indication of the degree of protection conferred by vaccine strain interference, representing a significant advance in the management and understanding of animal herd immunity.

[0008] Recent studies have suggested noninvasive methods for disease diagnosis via biomarkers in exhaled microdroplets of lung fluid, opening up the possibility of applying similar techniques to monitor immune responses in animals (Morozov et al., 2018). Furthermore, it has been shown that air-dried saliva and fecal samples can be used for semi-quantitative measurement of mucosal antibodies, potentially simplifying the collection and analysis of immunological data in agricultural environments (Vetvik et al., 1998). In parallel, the use of environmental eDNA / eRNA for pathogen detection and surveillance has been suggested as a promising approach for non-invasive animal health monitoring (Bass et al., 2023).

[0009] In the field of patents, technologies for determining immune responses after vaccination with attenuated avian vaccines have been explored, but these methods still require invasive procedures (CZ309268B6). Other studies have focused on sequence-based indicators for the assessment of immune responses, supporting the use of molecular biology for such purposes (US2014356339AA). Methods for predicting immune responses at the population level have also been described, indicating a growing interest in population immunity assessment (US2008220450AA).

[0010] This observation highlights the need and potential for improved methods that allow accurate, non-invasive assessment of animal immune responses, which is essential for effective health management in production animal populations.

[0011] The present invention proposes a method to inform the level of protection of animals through the determination and evaluation of immune responses in populations of epidemiological units of production animals, through the environmental detection and quantification of vaccines, by the animal shedding effect from air samples. Summary of the Invention

[0012] The present invention relates to a method for determining and assessing the environmental herd immune response to a vaccine in the environment of an epidemiological unit of production animals, which allows for understanding and reporting the level of protection of the animals. The method involves several steps: DNA and RNA extraction, analysis of the environmental herd immune response associated with animal shedding after a vaccination event, and notification to users. [Brief explanation of the drawings]

[0013] [Figure 1] This is a chart of the SIR model adapted for vaccination (VIP).

[0014] [Figure 2] 1 is a chart of environmental monitoring of vaccine strains.

[0015] [Figure 3] 1 is a chart of immune responses in birds with environmental measurements of vaccine viruses.

[0016] [Figure 4] 1 is a tracking chart of immune system activity.

[0017] [Figure 5] 1 is an efficiency evaluation chart using the VIP model (SIR).

[0018] [Figure 6] 1 is a chart tracking peak and shedding times.

[0019] [Figure 7] 1 is an environmental interference and protection rating chart. DETAILED DESCRIPTION OF THE INVENTION

[0020] definition

[0021] "Environmental herd immunity" as used herein refers to the measurement and assessment of the direct immune system response in the environment, which is not differentiated by individual, but rather is a comprehensive measure of responding on a population basis.

[0022] "Environmental Herd Immune Response" (EPIR) herein refers to the assessment of the amount of vaccine DNA or RNA sequences in air samples over time, resulting from the shedding effect of animals once the vaccine has been administered.

[0023] "Shedding" here refers to the elimination of an attenuated pathogen or vaccine after successful replication in the host. This elimination into the environment is achieved through secretion or excretion of substances at the mucosal level (respiratory, intestinal, urogenital, etc.).

[0024] As used herein, the term "epidemiological unit" refers to one or more animal production facilities containing animals of the same type with the same hygiene, feeding, safety measures, etc.

[0025] The term "user" as used herein corresponds to a person or facility that uses the technology described herein.

[0026] The term "zoo epidemiology" refers to the branch of epidemiology that focuses on the study of diseases in animals, especially animal populations. It is responsible for the study of the distribution, risk factors, spread patterns and impact of diseases in animals. Zoo epidemiology also analyzes the interactions between pathogens, host animals and the environment, with the goal of better understanding animal diseases and developing appropriate prevention and response strategies.

[0027] The term "sample" as used herein refers to one or more filters or membranes of controlled pore size resulting from a filtration process and containing dust particles, microorganisms, traces of genetic material, etc., representative of the environment. To be unique, a sample must have information about the environment corresponding to the sampling time and duration, the name of the sampling environment, the location, the relevant production phase, the lot (or similar), the user, and other identifying factors of the sampling process.

[0028] The term "production phase" as used herein refers to one of the stages in the animal husbandry production process, which can be distinguished by the age of the animals and / or the processes carried out with them.

[0029] The term "predictive" as used herein refers to the ability to provide a forecast of health / productivity status based on data analysis techniques.

[0030] The term "sectored" as used herein refers to an item or sector in the sense of a type of animal production such as fattening pigs, fattening chickens or egg-laying or incubators.

[0031] As used herein, the term "confined animals" refers to a group of animals (typically for commercial use) that are housed in a pen or similar closed or semi-closed enclosure during the production phase.

[0032] Preface

[0033] The problem to be solved by the present invention is the lack of a non-invasive method that provides population information on the specific responses induced by a vaccine. Currently, animal immune responses are monitored by performing representative sampling by bleeding the animals. Animal bleeding is an invasive method that, in some cases, involves the slaughter of young animals. In response, this invention proposes a non-invasive method for determining and assessing environmental population immune responses based on the quantification of vaccine DNA or RNA sequences in air samples, which reflects the shedding effect of vaccinated animals.

[0034] This invention redefines environmental herd immune responses by quantifying vaccine strain-specific gene sequences in air samples collected from livestock environments over a period of time. This method is distinguished by its ability to detect the effects of post-vaccination shedding from the excretion of vaccine components by immunized animals. Unlike vaccine-based DIVA (Differentiating Infected from Vaccinated Animals) approaches, which are specifically designed to distinguish infected from vaccinated animals using serological markers, this method is innovative in that it is not limited to DIVA vaccines but is applicable to a wide range of immunization agents.

[0035] The present invention is different in that it uses a population-based approach at the shed level, which allows detailed monitoring of viable virus in the vaccine strain and provides a population view of the immune response that has not been explored to date.

[0036] This invention makes a significant contribution to characterizing the dynamics of vaccination responses through the interpretation of the shape of the curves generated from quantitative data on vaccine viruses in the environment. These curves provide unprecedented information on vaccination efficacy at the population level and allow real-time adjustment of immunization strategies for optimal protection against infectious diseases.

[0037] The term "environmental herd immunity" as used herein refers to the direct measurement and assessment of the immune system's response in the environment, which does not distinguish between individuals and provides a general measure of response on a population basis. Thus, the information provided by the environment is relevant to the management needs of the facility by the user and therefore beneficial for decision making.

[0038] Environmental herd immune response is a time-dependent assessment of the quantification of DNA or RNA sequences from the vaccine in air samples, which is triggered by the animal shedding effect once the vaccine has been administered. It is essential to ensure that the vaccine is administered at the correct time, and this technology allows for this verification. Furthermore, specific monitoring can be performed to assess the effectiveness of the vaccine.

[0039] The term "shedding" refers to the elimination of a pathogen or attenuated vaccine after sufficient replication within the host. This elimination to the outside is achieved through secretion or excretion of substances at the mucosal level (respiratory, intestinal, urogenital, etc.).

[0040] This external secretion of the pathogen or attenuated vaccine can induce infection either through direct contact with other susceptible hosts or indirectly through release into the environment (air, water, ground), acting as a means of spread between individuals.

[0041] A typical example is shedding into the air environment by coughing / sneezing or other airway movement, which results in the dispersion of countless particles into the air and the potential for transmission to other susceptible organisms sharing the environment.

[0042] Method description:

[0043] The method includes three steps: determining the environmental herd immune response; assessing the environmental herd immune response; and generating a vaccination response alert.

[0044] Determining the herd immunity response

[0045] Given a vaccine A from an epidemiological unit, its specific concentration (X) in the environment (X A ) is determined as follows:

[0046] Carrying out standardized sampling (defined time and protocol) of air sampling with an apparatus of the type defined in patent application UY38805. Obtaining a filter (sample) containing dust particles containing microorganisms and the genetic material of said microorganisms.

[0047] Samples are stable for up to 72 hours during laboratory storage and transport at room temperature without the addition of stabilizing solution.

[0048] The sample is subjected to a combination of physical and chemical extraction methods that maximize the amount of genetic material extractable from the filter, which may include bead shaking, enzymatic digestion, centrifugation, and / or other validated / complementary extraction methods to ensure performance. This process, consisting of these phases, increases the sensitivity of subsequent analyses from small samples, minimizes bias in the sample, and maximizes the chance of detecting pathogen diversity, thereby ensuring representativeness. Typically, less than 100 μl of sample containing DNA or RNA is obtained for distribution among various assays. DNA / RNA quantification is performed using fluorometry and / or absorbance. Information obtained from the sample is expressed in nanograms / ml. 3 is.

[0049] In one representative example, samples are used to extract DNA or RNA and subjected to qPCR using fluorescent probes in a simple or multiplex format to obtain Ct values ​​that correlate with the copy number of A in the farm / facility. 3 X expressed as A is.

[0050] Concentration (X) as a function of time A ) to establish the chart X A The embodiment of (t).

[0051] Assessment of environmental herd immune responses

[0052] From the SIR (susceptible, infected, recovered) model adapted to vaccination, an inductive model called VIP was constructed, whose three main populations are (vaccine-able, susceptible-vaccinated, protected) according to Figure 1.

[0053] Various indicators such as total population (PT), secondary vaccination coverage (SVR), vaccine seroconversion rate (VCR), incubation rate (IR), vaccine shedding (VS), vaccine shedding rate (VSR) and measurable vaccine shedding rate (VSRm) among others are calculated.

[0054] Total Population (TP): Total for the entire population of an epidemiological unit TP=VP+IVP+PP [During the ceremony: TP is the total population; VP is the vaccineable population; IVP is the infectious vaccinated population; PP is a protected group.

[0055] Secondary vaccination rate (SVR): The number of new vaccinations resulting from direct or indirect transmission from an infectious vaccinated population to vaccinated animals per hour.

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[0056] Vaccine conversion rate (VCR): The number of new individuals that become fully vaccinated in a unit of time.

number

[0057] Incubation rate (IR): It is the time it takes for a vaccine-eligible individual to become infectiously vaccinated after first exposure.

number

[0058] Vaccine Shedding (VS): Vaccine shedding into the environment: Release of vaccine infectious particles from an infectious vaccinated population (or individual) into the environment that can infect the vaccinated population. VS ∝ X A [During the ceremony: VS is vaccine shedding X A is the measurable vaccine shedding proportional to VS, X A is the proportion of this that can be analyzed at the environmental level.

[0059] Vaccine Shedding Rate (VSR): Figure 1. Percent change in vaccine shedding over time.

number

[0060] Measurable Vaccine Shedding Rate (VSRm): Represents measurable vaccine shedding over time.

number

[0061] Creating vaccination response alerts

[0062] Vaccine-capable animal populations can achieve different levels of vaccination efficiency after vaccine administration. Although some vaccination conditions are standardized, the results of the vaccination process can vary depending on differences in operational, management, or biological conditions. This can result in earlier, stronger, and greater coverage (herd effect) or longer-lasting protective responses of the population against pathogens, depending on the vaccination process in the population.

[0063] We propose that environmental monitoring of vaccine shedding reflects vaccination dynamics and that this can be parameterized to measure vaccination levels received in confined animal populations.

[0064] In this invention, we propose to generate different alerts from indicators such as Total Population (TP), Secondary Vaccination Coverage (SVR), Vaccine Conversion Rate (VCR), Incubation Rate (IR), Vaccine Shedding (VS), Vaccine Shedding Rate (VSR) and Measurable Vaccine Shedding Rate (VSRm), among others, including combinations of these, which can give rise to different alerts such as a) vaccination efficiency, b) vaccine memory efficiency, c) protection due to competition with vaccine strains and d) other alerts constructed with information obtained from Phase I - Determination of Environmental Herd Immune Response and II - Evaluation of Environmental Herd Immune Response.

[0065] Some of these possible alerts encompassed by this invention are listed below:

[0066] Vaccination Efficiency [Table 1]

[0067] Vaccine Memory Efficacy [Table 2]

[0068] Protection by competition with vaccine strain (PBCWV) [Table 3]

[0069] Other alarms

[0070] Vaccines are not only a source of protective benefit, but can sometimes, alone or in combination, cause problems such as revertant vaccine viruses (vaccines carrying de novo pathogenic mutations) or circulating vaccine-pathogenic recombinant strains (strains that circulate with mixed vaccine and pathogenic identity but cause disease, which are the product of recombination in a virus family). Environmental population monitoring of vaccine strain dynamics, combined with sequence identity analysis, can help alert to these risks "masked" under vaccine identity.

[0071] Example

[0072] Work example 1: Environmental monitoring of vaccine strains

[0073] In one embodiment, animals in the enclosure are monitored for the development of attenuated vaccine strain load levels in the environment as an indicator of immunization. Live attenuated viruses and bacteria can infect and disseminate in the environment. In this example, successful vaccination results in a more intense and shorter peak in vaccine strain level detection, while poor vaccination results in a flatter curve as animals become infected asynchronously due to fluctuations in immunization.

[0074] Work example 2: Immune responses in birds using environmental vaccine virus curves

[0075] In another example, a series of vaccinations with attenuated viruses against the same respiratory pathogen in birds results in a peak of vaccine virus in the environment corresponding to the time (weeks) of vaccination. Given these successive vaccinations, the virus peak in the environment will gradually become lower, flatter, or delayed because it replicates less in hosts with a good ability to clear the virus if the animal's immune response is adequate, as well as in well-immunized populations. In this way, the immune response to the vaccine can be assessed by observing the shape of the environmental attenuated virus curve.

[0076] Prediction 1: Tracking immune system activity

[0077] In other uses of the technique, animal immunization involves the activation and inhibition of genes associated with immune system activity, and signatures of that activity are found in environmental samples. In the same way that we have found animal-derived pathogens and vaccines in the environment, we have also found other signatures of animals that may be indicators of the animal's condition.

[0078] In this example, the possibility of discovering the activation / inhibition of a group of genes by qPCR / transcriptomics is explored, and patterns associated with vaccination or infection phases are analyzed. In another embodiment of this example, DNA-level signatures of methylation or sequence changes associated with immune response or inflammation, stress, or other physiological indicators are searched for in the environment. In this way, specific changes are associated with DNA analysis that correspond to the physiological state of the immune system associated with infection or vaccination.

[0079] Prediction 2: Graphical efficiency evaluation using the VIP model (SIR)

[0080] In the case of initial vaccination with a live attenuated vaccine, the vaccine shedding peak of a standard population at a standardized vaccine dose results in a shedding curve where the higher and earlier the peak, the more synchronous and efficient the vaccination.

[0081] The graphical descriptors are the peak height, the time to peak (from vaccine administration), the shape and path of the shedding curve, and the area under the curve is an index that defines the vaccination efficiency. Then, using the shedding and VIP model (vaccine available, infectious vaccinated, protected) functions, the vaccination efficiency can be defined according to these parameters at a percentage level (percentage of vaccinated animals that are protected at a certain time).

[0082] An example of this can be seen in the chart in Figure 5, where different sheds of highly or less efficiently vaccinated animals produce different shedding curves, in each case resulting in different protection states over time (time to reach 50% protection).

[0083] Prediction 3: Tracking peak and shedding times

[0084] To monitor multiple attenuated vaccinations of the same type, a reference height difference between the "initial peak" and the "repeat" can be assigned, since in the case of sequential vaccinations the peak of the repeat administration is expected to be later and lower the stronger the immunization.

[0085] For the success of consecutive vaccinations V1, V2 and V3, where t(Vx) is the vaccination time and t(Px) is the shedding peak time corresponding to vaccination x, the following applies: SV(Pl) >> SV(P2) >> SV(P3) and t(V1-Pl). <t(V2-P2)<t(V3-P3)。

[0086] With a vaccination schedule applied to animals in a standardized manner, the differences in height and time between peaks can be normalized to establish an expected response. Reporting significant deviations from this pattern can indicate insufficient dosing, suboptimal intervals, etc., allowing corrective action to be taken.

[0087] Prophetic Example 4: Assessment of interference and protection in the environment

[0088] Live attenuated vaccines interfere with field strains immunologically by blocking entry to the site of infection through locational competition (e.g., cellular receptors). Some vaccination schedules aim to achieve this goal and provide extra protection during vulnerable periods. With this in mind, users may wish to monitor the level of vaccine virus present in the environment as an indicator of "interference / blocking ability."

[0089] The chart in Figure 7 shows how, in the example of two vaccinations V1 and V2, vaccine shedding has an area of ​​high protection with shaded interference. On the other hand, this overlaps with the protection provided by the vaccine, where each vaccination results in a large amount of protection. Between the two curves, the areas of high or low combined protection between immunization and interference are visualized, which allows planning of vaccine use in this type of schedule.

[0090] References

[0091] Hosseini, S., Vazquez-Villegas, P., Rito-Palomares, M., Martinez-Chapa, SO (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 / 978-981-10-6766-25 .

[0092] Pardo Cobas, MV (November 2006). Compendio de Epidemiologia de la Universidad Nacional Agraria. https: / / repositorio.una.edu.ni / 2439 / 1 / nl73p226.pdf Recovered from.

[0093] "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. DOI: 10.1088 / l 752-7163 / aab3f2 retrieved from.

[0094] "Mucosal antibodies can be measured in air-dried samples of saliva and feces" Vetvik H, Grewal HM, Haugen IL, Ahren C, Haneberg B J Immunol Methods. 1998 Jun 1;215(1-2): 163-72. DOI: 10.1016 / s0022-l759(98)00089-l retrieved from.

[0095] Environmental DNA / RNA for pathogen and parasite detection, surveillance, and ecology. David Bass, Kevin W. Christison, Grant D. Stentiford, Lauren S.J. Cook, Hanna Hartikainen, Trends in Parasitology, 39, 4, 2023, 285-304. https: / / doi.org / 10.1016 / j.pt.2022.12.010 Recovered from.

Claims

1. A method for measuring and generating alerts on the response to vaccination of an epidemiological unit of production animals, comprising determining and assessing the environmental herd immune response from air samples.

2. 2. The method of claim 1, wherein determining the environmental herd immune response comprises detecting and quantifying DNA or RNA fragments of the vaccine caused by the shedding effect of animals once the vaccine has been administered.

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

4. The environmental herd immune response includes analysis of values ​​and graph behavior associated with measurable vaccine shedding "VSm," measurable vaccine shedding rate of change "VSRm," and combinations thereof, where the "VS" value is the number of copies / m ３ and represents the amount of vaccine strain obtained by application of qPCR of DNA or RNA extracted from air samples of shedding animals once the vaccine has been administered, and the value "VSRm" represents the rate of change in measurable vaccine shedding "dVS" over time "dt", expressed as VSRm=dVS / dt.

5. 4. The method of claim 3, wherein generating alerts of the response to vaccination of an epidemiological unit of production animals includes alerts on vaccination efficiency, vaccine memory efficiency, protection by competition with vaccine strains, pathogenic revertant vaccine risk, and circulating recombinant strains.

6. The environmental herd immune response includes analysis of values ​​and graph behavior associated with measurable vaccine shedding "VSm," measurable vaccine shedding rate of change "VSRm," and combinations thereof, where the "VS" value is the number of copies / m ３ and represents the amount of vaccine strain obtained by application of qPCR of DNA or RNA extracted from air samples of shedding animals once the vaccine has been administered, and the value "VSRm" represents the rate of change in measurable vaccine shedding "dVS" over time "dt", expressed as VSRm=dVS / dt.

7. 2. The method of claim 1, wherein generating alerts of the response to vaccination of an epidemiological unit of production animals includes alerts on vaccination efficiency, vaccine memory efficiency, protection by competition with vaccine strains, pathogenic revertant vaccine risk, and circulating recombinant strains.

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