A strain of glaesserella parasuis, or a composition comprising thereof for use in the prevention and / or treatment of glÄsser's disease

Abstract

The present invention refers to the veterinary field. Particularly, it refers to a strain of Glaesserella parasuis, or a composition comprising thereof. Preferably, the strain or composition is for use in a method for the prevention and / or treatment of Glässer's disease or a condition caused by infection with a virulent Glaesserella parasuis strain. It also refers to a device suitable for administering a medicine to porcine animals characterized in that it comprises a non-virulent live strain of Glaesserella parasuis.

Description

[0001] A strain of Glaesserella parasuis, or a composition comprising thereof for use in the prevention and / or treatment of Glasser’s disease

[0002] TECHNICAL FIELD

[0003] The present invention refers to the veterinary field. Particularly, it refers to a strain of Glaesserella parasuis, or a composition comprising thereof. Preferably, the strain or composition is for use in a method for the prevention and / or treatment of Glasser’s disease or a condition caused by infection with a virulent Glaesserella parasuis strain. It also refers to a device suitable for administering a medicine to porcine animals characterized in that it comprises a non-virulent live strain of Glaesserella parasuis.

[0004] BACKGROUND ART

[0005] Glasser’s disease, is a bacterial infection that primarily affects young pigs, causing respiratory illness. The disease is caused by Glaesserella parasuis (also referred to as Haemophilus parasuis , a bacterium that is commonly found in the upper respiratory tract of healthy pigs. Glaesserella parasuis can cause a range of symptoms in infected pigs, including fever, difficulty breathing, nasal discharge, coughing, and sometimes lameness due to joint inflammation. In severe cases, the infection can lead to pneumonia, meningitis, and even death, particularly in piglets and young pigs with immature immune systems.

[0006] Economically, Glasser's disease poses significant challenges to the swine industry worldwide. The disease can lead to reduced growth rates, increased mortality rates, treatment costs for antibiotics, and expenses associated with veterinary care and management. Moreover, outbreaks of Glasser's disease can result in production losses, including decreased reproductive performance and lower market value of affected pigs. The economic impact extends beyond individual farms to affect regional and national swine production, influencing market stability and profitability.

[0007] Given the economic and welfare implications of Glasser’s disease, there is a pressing need to develop effective preventive strategies. By investing in preventive strategies, the swine industry can mitigate the economic losses associated with Glasser’s disease while promoting the health and welfare of pigs. Thus, there is a need to develop suitable and efficient strategies for preventing Glasser’s disease. The present invention solves this problem by providing a strain of Glaesserella parasuis that serves as a suitable and effective vaccine for the prevention of Glasser’s disease.

[0008] SUMMARY OF THE INVENTION

[0009] The present invention refers to a strain of Glaesserella parasuis, or a composition comprising thereof. Preferably, the strain or composition is for use in a method for the prevention and / or treatment of Glasser’s disease or a condition caused by infection with a virulent Glaesserella parasuis strain. It also refers to a device suitable for administering a medicine to porcine animals characterized in that it comprises a non-virulent live strain of Glaesserella parasuis.

[0010] The inventors evaluated the effects of inoculating piglets with a live non-virulent strain of Glaesserella parasuis, hereinafter referred to as “strain 94” of Glaesserella parasuis, prior to challenge with a virulent strain of Glaesserella parasuis. This strain was deposited before the Spanish Type Culture Collection (CECT) with accession number CECT 30960 on 24 November 2023.

[0011] Piglets vaccinated with the strain 94 displayed an increased weight gain at one week (p=0.01, Figure 5) and two weeks (p = 0.06, Figure 6) after exposure to the virulent Nagasaki strain compared to their control counterparts.

[0012] Moreover, piglets vaccinated with the strain 94 displayed an increased abundance of IgA antibodies that their control counterparts at two weeks after challenge with the virulent strain (p = 0.026, Figure 12), suggesting that the virulent Nagasaki strain is recognized better by the immune system that has been exposed to vaccination with strain 94, probably due to the presence of shared antigens between non-virulent and virulent strains.

[0013] Upon in vitro stimulation with the virulent Nagasaki Glaesserella parasuis strain, B cells extracted from pigs vaccinated with the strain 94 displayed an increased proliferation compared to their control counterparts (Figure 14), suggesting that vaccination with the 94 strain increases B cell reactivity against the virulent Nagasaki Glaesserella parasuis strain.

[0014] Pigs vaccinated with the strain 94 displayed a reduced nasal colonization by the virulent Nagasaki strain at two weeks after challenge compared to their control counterparts (p = 0.043, Figure 17), suggesting that the beneficial effect of the 94 strain may be due to competition / exclusion of the virulent Nagasaki strain.

[0015] Finally, histological analysis at two weeks after challenge with the virulent Nagasaki strain revealed that pigs vaccinated with the 94 strain displayed a tendency towards a lower inflammation in the upper respiratory track compared to their control counterparts, although the differences did not reach statistical significance (p=0.1, Figure 20).

[0016] Together, these results indicate that vaccination with strain 94 of Glaesserella parasuis of the invention confers protection against the virulent Nagasaki Glaesserella parasuis strain by increasing body weight upon challenge with the virulent strain, improving the immune response by increasing mucosal antibodies (IgAs) upon challenge with the virulent strain, increasing white blood cell reactivity against the virulent Glaesserella parasuis strain, reducing nasal mucosal colonization by the virulent Glaesserella parasuis strain and / or reducing inflammation in the upper respiratory track.

[0017] In the next phase of their research, the inventors evaluated the effect of intranasal administration of strain 94 of Glaesserella parasuis in a scenario designed to mimic a severe infection challenge with a virulent strain. To do so, the researchers first inoculated piglets intranasally with strain 94, then later challenged the animals with a very high dose of the pathogenic Nagasaki strain. To do so, the researchers first inoculated piglets intranasally with strain 94, then later challenged the animals with a very high dose of the pathogenic Nagasaki strain — a dose far greater than what piglets would ever naturally encounter under typical farm conditions.

[0018] The results showed a clear improvement in survival in the vaccinated groups compared to the control group (Figure 21). While all unvaccinated piglets succumbed to the severe challenge by day 2 post-challenge with the Nagasaki strain, vaccinated animals survived longer, with some lasting up to day 4. This extended survival time, even under an unnaturally high challenge dose, suggests that under normal farm conditions — where the infectious dose is much lower — vaccination could provide an even greater benefit, significantly increasing the chances of piglet survival,

[0019] In addition to improved survival, the vaccinated piglets showed a clear reduction in the severity of clinical signs associated with Glasser’s disease at day 2 post-challenge (Figure 22). Symptoms such as lack of appetite, lameness, and incoordination were significantly less severe in vaccinated animals (Table 5). Moreover, necropsy findings confirmed a notable decrease in the spread of the pathogenic G. parasuis strain in vaccinated animals, with a much lower bacterial presence detected in various organs (Table 6, Figure 23-25). This suggests that vaccination effectively limited bacterial colonization and systemic dissemination of the pathogen.

[0020] The intranasal administration of strain 94 also elicited a systemic humoral response. Specifically, vaccinated piglets developed higher levels of specific IgG antibodies against both the vaccine strain 94 and the virulent Nagasaki strain by day 26 after vaccination (Figure 26). This indicates the induction of both homologous protection (against the same strain) and heterologous protection (against a different, pathogenic strain). Such cross-protection is especially valuable in farm settings, where multiple G. parasuis strains may circulate simultaneously.

[0021] Furthermore, it was shown that the vaccine strain was able to effectively colonize the nasal mucosa of bacteria-free piglets and remain viable throughout the study period, supporting its capacity to induce a local immune response and providing further evidence of its suitability as an intranasal vaccine (Figure 27).

[0022] Because the challenge dose used in this study was intentionally much higher than the infective dose typically found on farms, any protective effect observed in this highly demanding experimental setup would likely be even more pronounced under real-world circumstances. In a farm environment, where piglets are not exposed to such high levels of the pathogen, the survival benefit could be even more significant, helping producers reduce losses, keep more animals healthy, and improve the overall profitability and sustainability of swine herds. Taken together, these results strongly support the potential of strain 94 as an effective intranasal vaccine candidate for controlling Glasser’s disease in commercial swine production.

[0023] Based on the above results, the invention provides:

[0024] 1. A strain of Glaesserella parasuis deposited under the accession number CECT 30960, or a composition comprising thereof.

[0025] 2. A live vaccine composition or a probiotic composition comprising the strain of item 1.

[0026] 3. The composition, according to any of the items 1 or 2, characterized in that it comprises a mucoadhesive solution. 4. The composition, according to item 3, wherein the mucoadhesive solution is selected from the list consisting of: chitosan, carbopol (carbomer), sodium alginate, hyaluronic acid, pectin, polyvinyl alcohol (PVA), hydroxypropyl methylcellulose (HPMC), methylcellulose, polycarbophil and xanthan gum.

[0027] 5. The strain or composition, according to any of the items 1 to 4, for use as a medicament.

[0028] 6. The strain or composition for use, according to item 5, in a method for the prevention and / or treatment of Glasser’s disease or a condition caused by infection with a virulent Glaesserella parasuis strain.

[0029] 7. The strain or composition for use, according to item 6, wherein the prevention and / or treatment of Glasser’s disease, or a condition caused by the infection with a virulent Glaesserella parasuis strain, comprises: increasing body weight, improving the immune response by increasing mucosal antibodies (IgAs), increasing white blood cell reactivity against the virulent Glaesserella parasuis strain, reducing nasal mucosal colonization by the virulent Glaesserella parasuis strain and / or reducing inflammation in the upper respiratory track.

[0030] 8. The strain or composition for use, according to any of the items 5 to 7, wherein the method comprises administering the strain or composition intranasally, preferably using an intranasal applicator.

[0031] 9. The strain or composition for use, according to any of the items 5 to 8, wherein the method comprises administering the strain or composition at a dose of at least 2xl05total CFU / dose.

[0032] 10. The strain or composition for use, according to any of the items 5 to 9, wherein the method comprises administering the strain or composition at a concentration of between 105and 109total CFU / ml.

[0033] 11. The strain or composition for use, according to any of the items 5 to 10, wherein the method comprises administering the strain or composition in a total volume of between 0.2 and 2 ml / animal.

[0034] 12. The strain or composition for use, according to any of the items 5 to 11, wherein the method comprises administering the strain or composition in a total volume of between 0.1 and 1 ml per nostril. 13. The strain or composition for use, according to any of the items 5 to 12, in a method for the prevention and / or treatment of pigs, preferably piglets, more preferably piglets during the first two weeks of life.

[0035] 14. A device pre-loaded with a composition comprising a non-virulent live strain of Glaesserella parasuis. wherein the device comprises an intranasal applicator which in turn comprises: a) connector means to connect the intranasal applicator with the device and b) a cannula suitable for insertion into the nasal orifice which has a tapered tapering towards the blunt or rounded end and at least one orifice to facilitate the exit of the medicine.

[0036] DESCRIPTION OF THE FIGURES

[0037] Figure 1: Rectal temperature after vaccination in placebo and 94 vaccinated groups.

[0038] Figure 2: Rectal temperature after challenge in placebo and 94 vaccinated groups.

[0039] Figure 3: Maximal rectal temperature measured in the piglets after intranasal challenge with the virulent G. parasuis Nagasaki at 28 days of life (SD27). Piglets were previously treated by intranasal inoculation at 1 day of life of vaccine strains 94.

[0040] Figure 4: Necropsy findings in piglet 44, which needed to be euthanised at 36 days of life (study day -SD-35), 8 days after the challenge with the Nagasaki strain. A) Purulent abscess in mandibular area; B) closeup picture of the opened abscess; C) peritonitis

[0041] Figure 5: Weight gain after challenge with virulent G. parasuis Nagasaki. Challenge was performed intranasally at 28 days of life (study day SD27). Weight was measured at SD26 and SD34 and the difference is plotted as the average of the group and error bars are the standard deviation.

[0042] Figure 6: Weight gain after challenge with virulent G. parasuis Nagasaki. Challenge was performed intranasally at 28 days of life (study day SD27). Weight was measured at SD26 and SD40 and the difference is plotted as the average of the group and the standard.

[0043] Figure 7: Recovery of the G. parasuis (Nagasaki strain) from bronchoalveolar lavage (BALF) semiquantified as a bacterial score from 0 (no isolation) to 3 (lawn of bacteria) on agar plates. Figure 8: Serum antibody levels in the piglets from the different groups at the time of challenge (SD27). Antibody levels were measured with the commercial kit Ingezim-Haemophilus and it is expressed as an index following the manufacturer’s instructions.

[0044] Figure 9: Serum antibody levels in the piglets from the different groups at the time of challenge (SD27) and after 3 days (SD30) and 2 weeks (SD41). Antibody levels were measured with the commercial kit Ingezim-Haemophilus and it is expressed as an index following the manufacturer’ s

[0045] Figure 10: Serum antibodies against the F4 fragments of the VtaA proteins from virulent G. parasuis measured by ELISA.

[0046] Figure 11: ELISA indexes using the Ingezim kit for BALF samples taken at necropsy, which was performed 2 weeks after the challenge with the Nagasaki strain.

[0047] Figure 12: Evaluation by ELISA of the antibodies in BALF against F4. IgG (Left) and IgA (Right).

[0048] Figure 13: Proliferation of CD4+ cells measured by Ki67 marker by cytometry in Peripherial blood mononuclear cells (PBMC) at SD27 (before challenge) upon stimulation with strain 94 and Roswell Park Memorial Institute culture Medium (RPMI).

[0049] Figure 14: Higher proliferation of B cells from the group 94 than the PBS control upon stimulation with the 94 strain (left panel) or the Nagasaki strain (right panel).

[0050] Figure 15: Level of activation markers detected in the surface of porcine alveolar macrophages (PAMs) from the group vaccinated with strain 94 and the PBS control group.

[0051] Figure 16 Phagocytosis assay with alveolar macrophages (PAMs) collected from control piglets or piglets inoculated with strain 94. PAMs were incubated with fluorescent G. parasuis Nagasaki (resistant to phagocytosis) or SW114 (sensible to phagocytosis) and the percentage of PAMs with associated bacteria was measured by cytometry. The population of dead and alive PAMs was analysed, since PAMs lose viability after phagocytosis.

[0052] Figure 17: Detection of virulent strains of G. parasuis by PCR in nasal samples taken at necropsies, indicating colonization by the challenge strain Nagasaki.

[0053] Figure 18: Inflammation / lesion score in nasal mucosa. Samples were obtained at necropsy, two weeks after the challenge with the virulent G. parasuis strain Nagasaki. Figure 19: Inflammation score in tracheal mucosa. Samples were obtained at necropsy, two weeks after the challenge with the virulent G. parasuis strain Nagasaki.

[0054] Figure 20: Inflammation score in nasal mucosa and trachea. Samples were obtained at necropsy, two weeks after the challenge with the virulent G. parasuis strain Nagasaki.

[0055] Figure 21: Survival rate of piglets challenged with Glaesserella parasuis Nagasaki strain (serovar 5).

[0056] Figure 22: Score of severity of Glasser disease clinical signs two days postchallenge.

[0057] Figure 23: Microbiological evaluation of pathogenic Glaesserella parasuis Nagasaki challenge strain dissemination (culture).

[0058] Figure 24: Molecular evaluation of organs dissemination of the pathogenic Glaesserella parasuis Nagasaki challenge strain by qPCR.

[0059] Figure 25: Global body dissemination of the pathogenic Glaesserella parasuis^ sa i strain (challenge) by qPCR.

[0060] Figure 26: A) Specific IgG for 94 Glaesserella parasuis strain in a homemade ELISA at Study Days 0, 6 and 26. B) Specific IgG for Nagasaki strain (serotype 5) in a homemade ELISA at Study Days 0, 6 and 26.

[0061] Figure 27: Nasal colonization with the strain 94 of Glaesserella parasuis in newborn piglets by qPCR.

[0062] DESCRIPTION OF EMBODIMENTS

[0063] General considerations

[0064] Each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed embodiments. Thus, all combinations of the various elements described herein are within the scope of the invention.

[0065] Unless otherwise stated, the terms used in the present application have the meanings indicated herein. Terms that are not defined herein should be given the meanings that would be given to them by a person skilled in the art in the context of this disclosure. For the purposes of the present invention, any ranges given include both the lower and the upper end-points of the range.

[0066] In the context of the present invention the following terms are defined:

[0067] • The term "comprising" means including, but it is not limited to, whatever follows the word "comprising". Thus, use of the term "comprising" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present.

[0068] • The term "consisting of’ means including, and it is limited to, whatever follows the phrase “consisting of’. Thus, the phrase "consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present.

[0069] • The term “strain” refers to a group of organisms that belong to the same species but share certain genetic characteristics not found in other members of the species. Microorganisms, such as viruses, bacteria, and fungi, have many strains within a single species. Different strains of an organism may have different biological characteristics, such as the ability to cause more severe disease.

[0070] • The term “composition” refers to a formulation comprising live, or attenuated bacteria intended for pharmaceutical purposes. This composition may include one or more strains of bacterial along with excipients, stabilizers, and other components necessary to maintain viability, stability, and / or efficacy of the bacteria. Such compositions are used for various applications, including bacterial vaccines, probiotics, or bacterialbased therapies. In addition, the term “composition” also refers to a formulation comprising dead bacteria or bacterial fragments. These components may be intentionally included for various purposes, such as enhancing the immune response, delivering specific antigens, or serving as adjuvants to stimulate the immune system. The composition may also contain other additives and excipients to stabilize the formulation and optimize its therapeutic or diagnostic properties.

[0071] • The term “mucoadhesive solution” refers to a formulation designed to adhere to mucosal surfaces within the body, such as the lining of the nasal passages, oral cavity, gastrointestinal tract, or vaginal tract. These solutions contain mucoadhesive polymers or other compounds that have the ability to bind mucins, glycoproteins present in the mucus, allowing the solution to stick to the mucosal surface upon application. Mucoadhesive solutions are used for various purposes, including drug delivery, sustained release of medications, or creating protective barriers against pathogens or irritants. They are often utilized in pharmaceuticals, medical devices, or oral care products.

[0072] • The term “adjuvant” refers to a substance that enhances the body’s immune response to an antigen, typically a vaccine, making it more effective. It helps stimulate the immune system to produce a stronger a longer-lasting response to the vaccine.

[0073] • The term “potentiating the host’s mucosal immunity” refers to enhancing or strengthening the body’s immune response at mucosal surfaces, such as those found in the respiratory tract, gastrointestinal tract, or urogenital tract. This enhancement can involve various mechanisms, including increasing the production of antibodies (such as IgA), promoting the activity of immune cells (such as T cells and dendritic cells), and stimulating the secretion of protective molecules (such as mucins and defensins) that help defend against pathogens and maintain mucosal health. Potentiating mucosal immunity is important for protecting against infections and maintaining overall health at mucosal surfaces, which are common sites of entry for pathogens.

[0074] • The term “non-virulent” refers to something that is not harmful or not capable of causing disease. In the context of microbiology and medicine, it describes organisms (such as bacteria) that do not produce or cause illness or harm to their host or environment.

[0075] • The term “sensitive”, in the context of a strain that is sensitive to an antibiotic, refers to a bacterial strain that is susceptible to the inhibitory or bactericidal effects of a specific antibiotic. In other words, the bacteria can be effectively killed or inhibited by the antibiotic when exposed to it. This sensitivity indicates that the antibiotic is capable of exerting its intended therapeutic effect against the bacterial strain, leading to suppression of bacterial growth or eradication of the infection. It’s important to note that antibiotic sensitivity can vary among different strains of bacteria.

[0076] • The term “provides mucosal immunity” refers to enhancing the body’s immune defences at mucosal surfaces, such as those found in the respiratory, gastrointestinal, or urogenital tracts. Mucosal immunity plays a crucial role in protecting the body from pathogens that enter through these surfaces, which are common points of entry for infectious agents. Mucosal immunity involves several components, including: o Mucous membranes: these serve as physical barriers that trap pathogens and prevent them from entering deeper tissues. o Mucins: mucins are glycoproteins found in mucus secretions that help to trap and neutralize pathogens. o Antimicrobial peptides: these are small proteins and can directly kill or inhibit the growth of pathogens. o Secretory IgA antibodies: these antibodies are produced locally in mucosal tissues and help to neutralize pathogens and prevent them from adhering to mucosal surfaces.

[0077] • The term “virulence genes” refers to specific genes within the genome of a pathogenic microorganism that contributes to its ability to cause disease or harm to the host organisms. These genes encode various factors, such as toxins, adhesins, invasins, or secretion systems, that enable the pathogen to colonize host tissues, evade the immune system, and cause damage to host cells or tissues. The presence or expression of virulence genes can influence the severity of the infection and the outcome for the host. Studying virulence genes is important for understanding the mechanisms of pathogenesis, developing diagnostic tests, vaccines, and therapeutic interventions, and tracking the spread of infectious diseases. Of note, virulence factors can be easily retrieved from databases (e.g. DBvfdb). The presence of these genes can be evaluated using genome sequencing techniques.

[0078] • The term “antibiotic resistance genes” refers to specific genes within the genome of bacteria that provide resistance to antibiotics, allowing the bacteria to survive and proliferate in the presence of these drugs. These genes can encode various mechanisms of resistance, such as enzymes that inactivate antibiotics, efflux pumps that remove antibiotics from the bacterial cell, or modifications to antibiotic targets that prevent the drugs from binding effectively. Of note, antibiotic resistance genes can be easily retrieved from databases (e.g. ResFinder, AMRFinderPlus, CARD Resistance Gene Identifier (RGI) or ARGminer). The presence of these genes can be evaluated using genome sequencing techniques.

[0079] • The term “live vaccine composition” refers to a formulation that contains live microorganisms, such as bacteria, that are used to stimulate an immune response in the host. Live vaccine compositions typically include: live microorganisms (generally attenuated pathogens or avirulent strains, used to elicit an immune response), stabilizers (ingredients that help maintain the viability and effectiveness of the live microorganisms during storage and handling), diluent (a liquid solution, often saline, used to reconstitute the vaccine if it is provided in a lyophilized form), and optionally adjuvants (substances that may be added to enhance the body's immune response to the vaccine).

[0080] • The term “probiotic composition” refers to refers to a formulation that contains live organisms which, when administered in adequate amounts, confer a health benefit on the host. These compositions are designed to support and maintain a healthy balance of microbiota in a given part of the body (such as in the gut, nasal cavity or in the respiratory mucosa), or to provide other health benefits. Probiotic compositions are used to support digestive health, enhance the immune system, prevent or treat infections, improving gut health, and promoting overall well-being. These compositions can be found in various forms, including dietary supplements, fermented foods, and functional foods fortified with probiotic strains.

[0081] • The term “respiratory” in the context of a “live respiratory vaccine composition” or a “respiratory probiotic composition” refers to refers to the system or area of the body related to breathing and the respiratory tract. o A “live respiratory vaccine composition” refers to a vaccine formulation containing live organisms that are specifically designed to stimulate an immune response against pathogens affecting the respiratory tract. Administration is typically via inhalation or nasal delivery, targeting the respiratory tract where the pathogens primarily infect and cause disease. This route allows the vaccine to induce both systemic and mucosal immune responses. o A “respiratory probiotic composition” refers to a formulation containing live beneficial microorganisms (probiotics) that are targeted to influence the microbial balance and immune responses in the respiratory tract. Probiotics in respiratory compositions may be used to support respiratory health, modulate immune responses, or compete with pathogenic bacteria in the upper or lower respiratory tract. Administration of respiratory probiotics can vary; it may include inhalation or nasal delivery to target the nasal passages, sinuses, or lower respiratory tract.

[0082] • The term “nasal” in the context of a “live nasal vaccine composition” or a “nasal probiotic composition” refers to the route of administration or delivery method targeted at the nasal cavity. o A “live nasal vaccine composition” refers to a vaccine formulation containing live organisms that are administered directly into the nasal cavity. The live microorganisms are intended to stimulate an immune response against specific pathogens, thereby providing protection against infectious diseases. Administration is typically through nasal sprays or drops. The nasal route offers advantages such as easy administration, potential for mucosal immune response induction, and reduced need for needles or injections. o A “nasal probiotic composition:” refers to a formulation containing live beneficial microorganisms (probiotics) that are specifically designed to colonize and exert their beneficial effects in the nasal cavity. Probiotics in nasal compositions may be used to modify the nasal microbiota, enhance local immune responses, or compete with pathogenic bacteria to prevent infections or alleviate symptoms of conditions like allergic rhinitis. Administration of nasal probiotics is typically through nasal sprays or nasal drops, allowing direct application to the nasal mucosa where they can exert their beneficial effects.

[0083] • The term “Glasser’s disease” refers to a bacterial infection primarily affecting pigs. It is caused by Glaesserella parasuis (also referred to as Hemophilus parasuis), a bacterium commonly found in the upper respiratory tract of pigs. Glasser’s disease typically manifests as polyserositis, inflammation of multiple serous membranes such as the pleura, pericardium, and peritoneum, leading to symptoms such as fever, lameness, difficulty breathing, and sometimes sudden death in severe cases. It can occur in pigs of all ages, but it is most commonly seen in young pigs, particularly those under six months old. Proper management and vaccination strategies are important for prevention and control of Glasser’s disease in pig populations.

[0084] • The term “condition” refers to an abnormal state of health affecting an organism, typically characterized by specific signs, symptoms, or features. These conditions can range from mild to severe and may be caused by various factors such as infections, genetic disorders, environmental factors, or lifestyle choices. Medical professionals diagnose and treat conditions based on their underlying causes and the manifestations they produce in the affected individual.

[0085] • The term “virulent” refers to the ability of a microorganism, such as virus, bacterium, or other pathogen, to cause disease or harm to its host. A virulent organism typically exhibits a high degree of pathogenicity, meaning it has the capability to invade tissues, replicate within the host, and cause significant damage or illness. In the context of an infectious disease, a virulent strain of a pathogen is one that causes severe or aggressive symptoms and is often associated with higher rates of morbidity and mortality.

[0086] • The term “pig” refers to a domesticated mammal belonging to the species Sus scrofa domesticus. The term "pig" does not specify the age of the animal, encompassing all stages from young piglets to fully grown adults.

[0087] • The term “piglets” refers to a young pig that is under eight weeks old. During this period, piglets are usually still nursing or have recently been weaned from their mother's milk.

[0088] • The term “device” refers to a piece of equipment or an apparatus designed to perform a specific task or function. This can encompass a wide range of objects, from simple tools to complex machinery.

[0089] • The term “pre-loaded” to a state where the device is already filled with the composition comprising a strain of Glaesser ella parasuis at the time of manufacture or prior to its distribution. This ensures that the device is ready for immediate use without the need for the end-user to load the composition into the device themselves.

[0090] • The term “intranasal applicator” refers to a device specifically designed for the administration of medications, solutions, or other substances into the nasal cavity. It typically consists of a nozzle or tip that can be attached or is attached to ’’the device”, allowing for precise and controlled delivery of the substance into the nostrils. Intranasal applicators are commonly used for the delivery of nasal sprays, nasal drops, or nasal gels for various purposes, including medication administration, nasal irrigation, or nasal moisturization. They are designed to ensure accurate dosing and optimal distribution of the substance within the nasal passages for effective absorption or action. In the context of this invention the term “intranasal applicator” refers to the part of a device that facilitates the delivery of a drug into the nasal cavity, and not to the part that releases the drug upon activation of a mechanism (which we have referred to as the “mechanism suitable for administering the medicine”).

[0091] • The term “connector means” refers to a component or mechanism that facilitates the joining or linking of two or more parts, systems, or entities to enable communication, transfer of power, data, or physical connection. This term is often used in technical contexts such as engineering, electronics, and telecommunications. • The term “mechanism suitable for administering the medicine” refers to a device or system designed to deliver medication accurately and safely to a patient, ensuring effective treatment while considering factors such as dosage, route of administration, and patient comfort. In the context of the present invention, this term refers to the component or mechanism of a device that, upon activation, dispenses or releases the medication, and not to adaptors that may be used to modify the manner in which the drug is released so that it fits a specific delivery method, such as an intranasal applicator that may be used to create a specific stream of the drug that is compatible with intranasal administration. Some examples of “mechanisms suitable for administering the medicine” include a syringe or injector (e.g., a Hauptner gun), a nebulizer and an infusion pump.

[0092] • The term “cannula” refers to a thin tube that can be inserted into the body, often for the delivery or removal of fluid or for the collection of data. Cannulas are commonly used in medical settings and come in various forms depending on their specific application.

[0093] • The term “tapered tapering” refers to a gradual decrease in thickness, diameter, or width of an object. This term is often used in various contexts, including medical instruments, engineering, and design, to describe the shape or design of an item that narrows progressively from one end to the other.

[0094] Detailed description of embodiments

[0095] A first aspect of the present invention refers to a strain of Glaesserella parasuis deposited before the CECT under the accession number CECT 30960 on 24 November 2023, or a comprising thereof.

[0096] In a preferred embodiment the composition is characterized in that it comprises a mucoadhesive solution. Preferably, the mucoadhesive solution is selected from the list consisting of: chitosan, carbopol (carbomer), sodium alginate, hyaluronic acid, pectin, polyvinyl alcohol (PVA), hydroxypropyl methylcellulose (HPMC), methylcellulose, polycarbophil and xanthan gum.

[0097] The composition may further comprise an adjuvant that potentiates the host’s mucosal immunity. Some options of adjuvants that potentiate the host’s mucosal immunity are: chitosan, CpG oligodeoxynucleotides (ODNs), monophosphoryl lipid A (MLA), MF59, aluminum salts, polyinosinic:polycytidylic acid (poly EC), immunostimulating complexes (ISCOMs), liposomes, heat-labile enterotoxin (LT) derivates, cholera toxin (CT) derivatives, guanylyl cyclase C agonist peptide, beta-glucans, resquimod (R848) and virosomes. A second aspect of the invention refers to a non-virulent live strain of Glaesserella parasuis, or a composition comprising thereof.

[0098] In a preferred embodiment the strain is a single strain.

[0099] In a preferred embodiment the composition is characterized in that it comprises a mucoadhesive solution. The mucoadhesive solution is the same as for the first aspect of the invention. Thus, all the embodiments of the first aspect of the invention applies herein. As for the first aspect of the invention, the composition may further comprise an adjuvant that potentiates the host’s mucosal immunity.

[0100] In a preferred embodiment, the strain is sensitive to at least one antibiotic selected from the list consisting of: ciprofloxacin, ceftiofur, enrofloxacin, tetracycline, ampicillin, kanamycin, gentamicin, sulfamethoxazole trimethoprim, amoxicillin, erythromycin, streptomycin, colistin and fosfomycin. Preferably, the strain is sensitive to all the antibiotics of the list consisting of: ciprofloxacin, ceftiofur, enrofloxacin, tetracycline, ampicillin, kanamycin, gentamicin, sulfamethoxazole trimethoprim, amoxicillin, erythromycin, streptomycin, colistin and fosfomycin.

[0101] In a preferred embodiment, the strain or composition provides mucosal immunity. Preferably, the mucosal immunity is nasal mucosal immunity.

[0102] In a preferred embodiment the strain lacks virulence genes.

[0103] In a preferred embodiment the strain lacks antibiotic resistance genes.

[0104] In a preferred embodiment the strain is the strain of Glaesserella parasuis deposited under the accession number CECT 30960 (depositor: AQ08-Gp94; date of original deposit: 24 November 2023).

[0105] A third aspect of the invention refers to a live vaccine composition or a probiotic composition comprising the strain of any of the first or second aspect of the invention. Thus, all the embodiments of the first and second aspects of the invention apply herein.

[0106] Preferably, the live vaccine composition is a live respiratory vaccine composition, more preferably a nasal vaccine composition.

[0107] Preferably, the probiotic composition is a respiratory probiotic composition, more preferably a nasal probiotic composition. A fourth aspect of the invention refers to the strain of any of the first or second aspects of the invention or the composition of any of the first to third aspects of the invention for use as a medicament.

[0108] In a preferred embodiment, the strain or composition is for use in a method for the prevention and / or treatment of Glasser’s disease or a condition caused by infection with a virulent Glaesserella parasuis strain.

[0109] In a preferred embodiment, the prevention and / or treatment of Glasser’s disease, or a condition caused by the infection with a virulent Glaesserella parasuis strain, comprises: increasing body weight, improving the immune response by increasing mucosal antibodies (IgAs), increasing white blood cell reactivity against the virulent Glaesserella parasuis strain, reducing nasal mucosal colonization by the virulent Glaesserella parasuis strain and / or reducing inflammation in the upper respiratory track.

[0110] In another preferred embodiment, the method comprises administering the strain or composition intranasally, preferably using a device suitable for administering a medicine to porcine animals comprising an intranasal applicator, more preferably using the device of the fifth aspect of the invention. Thus, all the embodiments of the fifth aspect of the invention apply herein.

[0111] In another preferred embodiment, the method comprises administering the strain or composition at a dose of at least 2xl05total CFU / dose, preferably at least 5xl05total CFU / dose, more preferably at least 8xl05total CFU / dose, even more preferably at least l. lxlO6total CFU / dose, even more preferably at least 1.4xl06, even more preferably at least 1.7xl06total CFU / dose, even more preferably at least 2xl06total CFU / dose, even more preferably 2xl06total CFU / dose.

[0112] In another preferred embodiment, the method comprises administering the strain or composition at a concentration of between 105and 109total CFU / ml, preferably between 106and 108CFU / ml, more preferably 107total CFU / ml.

[0113] In another preferred embodiment, the method comprises administering the strain or composition in a total volume of between 0.2 and 2 ml / animal, preferably between 0.2 and 1.6 ml / animal, more preferably between 0.2 and 1.2 ml / animal, even more preferably between 0.2 and 0.8 ml / animal, even more preferably between 0.2 and 0.4 ml / animal, even more preferably 0.2 ml / animal. In another preferred embodiment, the method comprises administering the strain or composition in a total volume of between 0.1 and 1 ml per nostril, preferably between 0.1 and 0.8 ml per nostril, more preferably between 0.1 and 0.6 ml per nostril, even more preferably between 0.1 and 0.4 ml per nostril, even more preferably between 0.1 and 0.2 ml per nostril, even more preferably 0.1 ml per nostril.

[0114] In another preferred embodiment, method for the prevention and / or treatment of pigs, preferably piglets, more preferably piglets during the first two weeks of life.

[0115] A fifth aspect refers to a device pre-loaded with a composition comprising a strain of Glaesserella parasuis. wherein the device comprises an intranasal applicator which in turn comprises: a) connector means to connect the intranasal applicator with the device and b) a cannula suitable for insertion into the nasal orifice which has a tapered tapering towards the blunt or rounded end and at least one orifice to facilitate the exit of the medicine.

[0116] In a preferred embodiment, the connector means consists of a thread on one of the ends of the intranasal applicator that screws into the device.

[0117] In a preferred embodiment, the device is a Hauptner gun.

[0118] In a preferred embodiment, the cannula is between 5mm and 40 mm long.

[0119] In a preferred embodiment, the cannula has a diameter of between 2.2 mm and 4.2 mm, preferably between 2.7 mm and 3.7 mm, more preferably 3.2 mm.

[0120] In a preferred embodiment, the orifice to facilitate the exit of the medicine has a diameter of between 0.7 mm and 1.7 mm, preferably between 0.95 mm and 1.45 mm, more preferably 1.2 mm.

[0121] In a preferred embodiment, the intranasal applicator comprises an orifice at the end of the cannula.

[0122] In a preferred embodiment, the intranasal applicator comprises at least two orifices on the side of the cannula.

[0123] In a preferred embodiment, the intranasal applicator comprises at least four orifices on the side of the cannula.

[0124] In a preferred embodiment, the intranasal applicator comprises an orifice at the end of the cannula and at least two orifices on the side of the cannula. In a preferred embodiment, the intranasal applicator comprises an orifice at the end of the cannula and at least four orifices on the side of the cannula.

[0125] In a preferred embodiment, the lateral orifices of comprises cannula are located opposite each other on both sides.

[0126] In a preferred embodiment, the lateral orifices of comprises cannula are located 3mm from the end of the cannula.

[0127] In a preferred embodiment, the strain of Glaesserella parasuis is the strain of the first aspect of the invention.

[0128] In another preferred embodiment, the strain of Glaesserella parasuis is a non-virulent live strain of strain of Glaesserella parasuis, preferably the strain of the second aspect of the invention.

[0129] A sixth aspect of the invention refers to a method for treating and / or preventing Glasser’s disease or a condition caused by infection with a virulent Glaesserella parasuis comprising the administration of the strain of any of the first or second aspects of the invention, or the composition of any of the first to third aspects of the invention. Thus, all the embodiments of the first to third aspects of the invention apply herein.

[0130] EXAMPLES

[0131] The present invention is illustrated by means of the Examples set below without the intention of limiting its scope of protection.

[0132] Example 1. Experimental design

[0133] Description of the Experiment

[0134] Twenty-four male and female newborn piglets from antibiotic treated sows with irrelevant amounts of Glaesserella parasuis antibodies were enrolled in the study and randomly distributed in two groups: a group that was inoculated with strain 94 of Glaesserella parasuis and a placebo group.

[0135] Animals were purchased from a commercial farm (Rega ES171090020866, Montagut, in Catalonia, Spain) after farrowing and colostrum intake (birth on the 03 / 05 / 22, over the course of the morning). Piglets were transported to CReSA’s facilities on the 04 / 05 / 22 at 10:30 a.m. (study day 0, SD0).

[0136] Each animal was inoculated with a dose of 200 pl (100 pl in each nasal orifice) of a vaccine formulation containing life bacteria (Glaesserella parasuis) or placebo in the form of an intranasal spray (within the next 4 hours after arrival; from 11 :30 to 13:30 approximately). Temperature for safety evaluation was measured before inoculation and daily for 4 days at the same time frame.

[0137] Piglets were daily observed for general clinical signs. Nasal swabs were collected before challenge (SD26, 30 / 05 / 22), which was performed intranasally at SD27 (31 / 05 / 22) with a virulent Nagasaki G. parasuis collection strain. The end of the study was set at SD41 (14 / 06 / 22). Temperature was controlled daily from the day before challenge until the end of the study. Blood samples were collected before challenge, 3 days post-challenge and the last day of the study.

[0138] Piglets were weighted at challenge, one week after challenge (SD35; 07 / 06 / 22) and one day before the end of the experiment (SD40; 13 / 06 / 22).

[0139] At the end of the trial all piglets were necropsied. Potential macroscopical and microscopical lesions were evaluated. Furthermore, samples from systemic organs, bronchoalveolar lavage fluid (BALF) and nasal swabs were analyzed for the presence of G. parasuis. Humoral and cellular responses were also assessed.

[0140] A summary of events is shown in Table 1.

[0141] Table 1: Summary of events

[0142] Justification of the selected schedule

[0143] This schedule was designed according to previous studies performed by the research group.

[0144] Intranasal vaccination of piglets just after farrowing was intended to colonize the nasal cavity of the animals and to cause both a microbiota modulation with an early colonization of avirulent strains and immune stimulation to prepare the piglets against the potential contact with pathogenic G. parasuis strains.

[0145] Challenge was performed at SD27, which can be considered as equivalent to the weaning moment by intensive porcine farming and the most sensitive period in the porcine breeding. This point has been considered as the most suitable to test the protection of the intranasal vaccination against a potential respiratory infection caused by G. parasuis.

[0146] Sampling schedule and laboratory analysis were aimed to the safety and efficacy evaluation of the vaccine and immune response of the piglets at the most suitable points.

[0147] Randomization and blinding Animals were included in each group at the farm trying to distribute piglets from different litters in the different groups.

[0148] The study was blind for the personnel assessing the clinical signs and necropsies, and for the personnel in the laboratory which only had knowledge of the ID of the piglets.

[0149] Example 2. Materials and methods Products

[0150] Vaccine

[0151] • Active substance: Lyophilizate (life bacteria) o Concentration of strain 94 of Glaesserella parasuis'. 1.00E+08 UCF / g o Date of manufacture: 19 / 04 / 22 o Manufacturer: BDi Biotech o Storage conditions: -20°C

[0152] • Diluent o Mucoadhesive aqueous solution (ID: F2) o pH: 7-8 o Date of manufacture: 30 / 03 / 22 o Manufacturer: Bionanoplus o Storage conditions: 2-8°C

[0153] Placebo

[0154] • Mucoadhesive aqueous solution (ID: F2)

[0155] • pH: 7-8

[0156] • Date of manufacture: 30 / 03 / 22

[0157] • Manufacturer: Bionanoplus

[0158] • Storage conditions: 2-8°C

[0159] Product preparation and administration

[0160] Before application, 0.5 g of the strain / lyophilizate was diluted in a mucoadhesive aqueous solution to reach a concentration of 107CFU / ml. A total of 5 ml was prepared of strain 94.

[0161] Doses of 0.1 ml were applied in each nasal orifice (0.2 ml / piglet) by means of a Hauptner 5 ml pistol and an intranasal application device (Intranasal Mucosal Atomization Device, MAD Nasal™). Thus, a total of 2xl06CFU were administered to each piglet.

[0162] Challenge with virulent G. parasuis

[0163] • Description of inoculum: Glaesserella parasuis serotype 5, Nagasaki

[0164] • Preparation date: Day of challenge

[0165] • Target titre: 109CFU / piglet

[0166] • Route of infection: Intranasal Challenge strain was plated on chocolate agar plates the day before the challenge. On the day of the challenge, the growth on the chocolate plates was resuspended in PBS + 20% glycerol to give a bacterial concentration of 1.82 x 109CFU / mL (1 mL per piglet; 0.5 mL per nostril). Inoculum was transported to the animal facilities at room temperature in glass bottles labelled with the study number and the strain name. A small aliquot of the inoculum (approximately 1 mL) remained in the laboratory for titration.

[0167] The intranasal inoculation with 1 mL (0.5 mL per nostril) of the Nagasaki strain was performed with a sterile syringe attached to a nasal nozzle.

[0168] Animals

[0169] Description, justification and source of the animals used

[0170] Piglets were used in the present study since it is the target species of Glaesserella parasuis. The experimental unit was the individual.

[0171] The experiment was to be conducted with 24 male and female newborn piglets (Batalle breed, Duroc x Landrace genetics). Animals were purchased from the source farm within the 24 hours period after farrowing and after colostrum intake from their mothers. Sows were tested in advance for the presence of Glaesserella parasuis antibodies by the commercial Ingezim- Haemophilus ELISA (Ingenasa). Negative sows in the Ingezim-Haemophilus ELISA were used as source of the piglets to avoid the maternal passive immunization of the piglets via colostrum.

[0172] Furthermore, sows were treated with ceftiofur to reduce transfer of bacteria and colonization of the offspring.

[0173] Intranasal inoculation was performed in the next 4 hours after arrival to CReSA’s facilities. Each piglet received two spray inoculations of 100 microlitres of the corresponding product (one for each nasal orifice).

[0174] Table 2. Experimental groups. Animal identification

[0175] At inclusion, ear tags with the corresponding ID number (from 1 to 24) were used to unequivocally identify each single animal.

[0176] Management and Housing of the Animals

[0177] Animals were housed in the BSL3 Animal facilities of CReSA (ES082660037069).

[0178] Due to the vulnerable immunological status of the piglets, they were kept under severe conditions of hygiene and sanitary isolation. Animals were allocated in a room (box 8) in pens of 12 individuals corresponding to each treatment group, which allowed a suitable interaction between them at this stage.

[0179] Animals were controlled by four visits / day at the first week, 3 visits / day from SD8 to 11, 2 daily visits from SD12 to 16 and once a day from this moment until the end of the experiment.

[0180] The biosafety measures applied were:

[0181] Shower required at the entrance and exit from the boxes in the ABSL3.

[0182] - Restricted staff.

[0183] - Personal protective equipment (PPE) and exclusive material for each group of the study.

[0184] - Feed entrance was controlled and with a prior disinfection of the packaging.

[0185] All the material was disinfected at the entrance and exit of the boxes.

[0186] Animal feeding and watering

[0187] At arrival, piglets were feed individually with feeding bottles with an artificial replacement milk for swine. Bottle feeding was performed during the first 7 days; 4 times / day, every 4-5 hours until 23:00h, then the animals were left with milk plates and containers overnight until next feeding starting at approximately 7:00h. In addition, from arrival, a container with Patavie milk replacement and a separate floor feeder with Neopig power feed were available to the piglets in each pen, including during the night.

[0188] From day 8 to 14 piglets were fed with milk containing increasing quantities of Neopig using floor feeders until day 15.

[0189] From this point, feed (dry Neopig) was available ad libitum by means of regular feeding procedures (feeders). Water was available ad libitum from the beginning of the study.

[0190] Inclusion Criteria Only piglets from sows with low antibodies against Glaesserella parasuis were recruited (indexes in the Ingezim-Haemophilus ELISA of less than 0.4 (negative) or from 0.4 to 0.6 (doubtful)).

[0191] Besides, all piglets showed an optimal health status:

[0192] Good general aspect

[0193] - No locomotor, nervous, digestive or / and respiratory symptoms

[0194] Correct colostrum intake after farrowing

[0195] Confirmation of the optimal health status of each piglet was carried out at the selection of the animals (observation of the litters in the farm of origin).

[0196] Exclusion Criteria

[0197] Any animals showing an abnormal health status were discarded.

[0198] Procedures

[0199] Product administration

[0200] Animals were properly restrained and treated by an intranasal inoculation of one spray-shot of 100 microlitres to each nasal orifice after distribution in the corresponding group in CReSA’s facilities. Vaccine formulations and placebo were inoculated by means of a 5 ml Hauptner syringe and an intranasal applicator device (Intranasal Mucosal Atomization Device, MAD Nasal™).

[0201] Blood collection and sample manipulation

[0202] Blood was collected from the cava vein of each study animal by direct puncture with a needle and a vacutainer system. Samples were collected in plain tubes without anticoagulant from all animals just before challenge (SD27), 3 days post-challenge (SD30) and the last day of the study (SD41). In addition, samples taken with tubes with anticoagulant were taken for peripheral blood mononuclear cells (PBMCs) isolation.

[0203] All blood samples were identified with: study number, animal number, study day and date and they transfer immediately to the CReSA laboratory facilities under refrigeration.

[0204] In the analysis facilities, blood tubes for sera obtention were centrifuged at 4000 g during 15 minutes. Sera were collected with a micropipette and a 1 mL sterilized tip while the blood tubes were discarded. The quantity of sera collected was equally distributed into a sterilized microtubes. Each microtube was identified with the same details as its original blood sample (study number, animal number, study day and date).

[0205] All sera samples were stored in plastic boxes, labelled with the study name and frozen at -20 °C until performance of the analysis.

[0206] For peripheral blood mononuclear cells (PBMC) collection a Ficoll-based density gradient was used.

[0207] Weight record

[0208] All piglets were weighed individually in a verified scale the day before challenge (SD26; 30 / 05 / 22), one week after challenge (SD34; 07 / 06 / 22), and one day before the end of the experiment (SD40; 13 / 06 / 22).

[0209] Nasal swab sampling

[0210] Sterile swabs were used to collect nasal samples from each study animal at SD26 (one day before challenge) and at the end of the study (SD41). Briefly, animals were properly restrained and the tip of a swab was softly rubbed in the inside of each nasal orifice. Nasal swabs were placed in 500 pl of PBS and stored frozen until processed for DNA extraction for PCR.

[0211] Rectal temperature

[0212] For determination of the rectal temperature, a digital thermometer was used. The sensor was placed in the rectum until the number on the display was constant.

[0213] Rectal temperature was registered for each piglet before vaccine inoculation and on the following 4 days at approximately the same hour frame of the day for all animals. It was also recorded at challenge (starting one day before) and until euthanasia (SD41) as indication of invasive disease.

[0214] Evaluation of fever was established by means of a numerical score: 0 (less than 39.5°C), 1 (between 39.5 and 40°C), 2 (between 40.1 and 41°C) and 3 (higher than 41°C). Scores 2 and 3 were considered fever.

[0215] Observation of General Clinical Signs

[0216] General clinical signs were observed daily in all piglets.

[0217] For efficacy evaluation, observations were scored according to the following parameters: External aspect, skin and mucosae, behaviour (lethargy), respiratory signs (dyspnoea, nasal 1 discharge, coughing, sneezing), digestive signs (vomiting), ocular signs (conjunctivitis), nervous and locomotory signs (paralysis, lack of coordination).

[0218] Each clinical sign was evaluated following these criteria:

[0219] Score 0: Not altered

[0220] Score 1 : Mild alteration (specify)

[0221] Score 2: Moderate alteration (specify)

[0222] Score 3 : Severe alteration (specify)

[0223] Euthanasia

[0224] Animals were euthanized at the scheduled moment (end of study D41), or at the required moment according to welfare criteria using an intravenous overdose of sodium pentobarbital in the cranial cava. Piglets were necropsied and carcasses were destroyed according to CReSA’s protocols.

[0225] Necropsy and macroscopical evaluation.

[0226] Study animals were necropsied for observation of macroscopical lesions and sample collection.

[0227] Macroscopical observation of potential polyserositis lesions associated to the acute development of Glasser’s disease were performed in pericardium, pleura, peritoneum, and respiratory organs, recorded, scored from 0 to 3 depending on the severity, and described.

[0228] Samples of bronchoalveolar lavage (BALF) and swabs from different organs: pericardium, peritoneum, thorax, joints (2 anterior and 2 posterior) and cerebrospinal fluid (CFS) were taken for microbiological culture and immune response evaluation (BALF samples).

[0229] Histology

[0230] If macroscopical lesions were observed in lung, lung lesions were histological evaluated according to a 0-3 score.

[0231] Score 0: absence of lesions.

[0232] Score 1 : mild infiltration of lymphoplasmacytic cells of bronchiolar wall and surrounding alveolar septa (broncho-interstitial pneumonia).

[0233] Score 2: moderate infiltration of lymphoplasmacytic cells of bronchiolar wall with moderate presence of neutrophils in bronchiolar lumen and alveoli (moderate suppurative bronchopneumonia). Score 3 : severe infiltration of lymphoplasmacytic cells of bronchiolar and alveolar septa with extensive infiltration of neutrophils in bronchiolar lumen and alveoli (severe suppurative bronchopneumonia).

[0234] Besides, tissue samples from the upper respiratory tract (nasal and tracheal) were taken for histological observation and inflammation assessment. A 0-3 score was used to evaluate and quantify inflammation in these tissues at necropsy (SD41).

[0235] Laboratory analysis

[0236] ELISA

[0237] Collected sera were analysed by an indirect ELISA test to assess general level of antibodies against G. parasuis (IgG) in the study piglets by the commercial Ingezim-Haemophilus kit, following manufacturer’s instructions.

[0238] Besides, an ELISA using an antigen specific of virulent strains of G. parasuis, the fragment F4 from the VtaAs, was also performed.

[0239] ALF samples were also analysed for presence of total IgG antibodies and IgG and IgA antibodies against F4 from VtaAs antigens

[0240] PCR for G. parasuis virulence identification

[0241] Nasal colonization of virulent / non-virulent strains was evaluated by means of a specific PCR using the leader sequence of the vtaA genes, followed by an evaluation with the score:

[0242] Positive = 3

[0243] Low positive = 2

[0244] Rest / remaining = 1

[0245] This analysis indicated the presence of the strain of the vaccine and / or the virulent Nagasaki strain used in the challenge model and was used to assess differences in the colonization of this strain in control vs vaccinated groups. Microbiology

[0246] Swab samples from pericardium, thorax, peritoneum, joints (2 anterior and 2 posterior), cerebrospinal fluid and bronchoalveolar lavage fluid were taken at necropsy and plated on chocolate agar.

[0247] Swabs were spread using half the surface of the plate (area 1) to semiquantify G. parasuis growth by assigning a score from 0 to 3 to the samples: Score 0: no bacterial growth.

[0248] Score 1 : samples yielding 1 to 20 colonies.

[0249] Score 2: samples yielding more than 21 isolated colonies.

[0250] Score 3 : confluent growth.

[0251] The second half of the plate was used to assess purity of the culture by streaking from area 1 with a loop to obtain isolated colonies. Plates were incubated at 37°C for a maximum of 4 days to assess G. parasuis in the samples.

[0252] In the case of growth of G. parasuis-\ e. colonies, a colony, or equivalent of bacterial growth, per sample was selected for G. parasuis strain determination by ERIC-PCR. In addition, different strains in the ERIC-PCR were analysed by the specific G. parasuis PCR indicated above.

[0253] Analysis of vaccine-induced memory immune responses

[0254] To investigate if vaccination with the vaccine strains induced systemic memory T cell responses against the virulent strain, the activation and proliferation of CD4 T cells after in vitro stimulation were analysed by flow cytometry.

[0255] Briefly, peripheral blood mononuclear cells (PBMC) from blood collected before challenge of the animals (SD27) were isolated from blood by Ficoll-based density gradient and stimulated in vitro with a lysate of the virulent G. parasuis strain (heat-inactivated at 65 °C for 1 hour, followed by 3 freeze / thaw cycles).

[0256] Responsive CD4+ T cells were quantified by flow cytometry after:

[0257] (i) the intracellular staining of the activation marker CD 154 and the cytokine TNF alpha after 8-16 hours of stimulation

[0258] (ii) the intracellular staining of the proliferation marker Ki67 after 5 days of stimulation.

[0259] Analysis of blood immune cell percentages and activation status of alveolar macrophages after challenge

[0260] Kinetics of T and B cell percentages in blood after challenge were also analysed.

[0261] Thus, previously frozen porcine alveolar macrophages (PAM) form BALF obtained at the end of the study were thaw and stained with fluorescently-labelled antibodies (CD 163, CD 14, SWC3, SLA-II) to analyse their activation status by flow cytometry and their phagocytosis capacity was assessed with a non-virulent reference strain, SW114, and the virulent strain Nagasaki

[0262] Example 3. Assessment of safety

[0263] Rectal temperature

[0264] The difference between rectal temperature before and post-vaccination was calculated for each experimental group. Fever record was evaluated and the relation with the inoculation of the products was analysed.

[0265] General clinical signs observation

[0266] The general aspect and clinical signs were evaluated in the animals from all groups from product inoculation until SD14 to evaluate potential adverse events related to the intranasal inoculation of the tests’ products.

[0267] Example 4. Assessment of efficacy

[0268] Rectal temperature

[0269] Rectal temperature was taken daily after challenge to assess the impact of the infection on this clinical parameter. Temperature evolution was assessed and compared between experimental groups.

[0270] General clinical signs observation

[0271] For efficacy evaluation of the test products, general clinical signs of the study animals were scored from 0 to 3 after challenge with the virulent Glaesserella parasuis Nagasaki strain and compared between experimental groups.

[0272] Average daily gain (ADG)

[0273] Average daily weight after the challenge with the Nagasaki strain was analysed in order to detect differences among the groups.

[0274] Evaluation of macroscopical lesions

[0275] After necropsy (or sacrifice / dead of an experimental animal), macroscopical evaluation was performed. Macroscopical observation and description of potential polyserositis lesions associated to the acute development of Glasser’s disease was performed in pericardium, pleura, peritoneum, and respiratory organs by vaccinated and placebo animals. Besides, lung lesions were evaluated according to a 0-3 score and results were compared.

[0276] Other parameters

[0277] Evaluation of nasal colonization

[0278] Nasal colonization of virulent strains was evaluated before challenge (SD26) and at the end of the study (SD41) by means of PCR analysis of nasal swab samples.

[0279] Evaluation of bacterial re-isolation

[0280] Microbiological analysis of pericardium, thorax, peritoneumjoints (2 anterior and 2 posterior), cerebrospinal fluid (CSF) and bronchoalveolar lavage fluid (BALF) were performed for bacterial colonization analysis.

[0281] Results of the semi quantitative analysis of the swab samples according to a 0-3 score were evaluated and compared between experimental groups in order to assess the potential prevention of the Glaesserella parasuis virulent strain invasion in the analysed organs within the vaccinated vs placebo groups.

[0282] Humoral response assessment

[0283] Evaluation of the humoral response against Glaesserella parasuis were evaluated by means of antibody titres evaluation and F4 response, specific from virulent strains, in ELISA tests.

[0284] Evaluation of immune responses

[0285] Analysis of vaccine-induced memory immune responses and activation status of alveolar macrophages after challenge were performed to define the immune response by vaccinated and placebo groups.

[0286] Example 3. Results

[0287] Safety results

[0288] Rectal temperature

[0289] Piglets were vaccinated at 1 day of life (SDO) by intranasal inoculation of G. parasuis strain 94. Rectal temperature was monitored for 4 days after inoculation. Vaccination did not increase the temperature of the piglets (Figure 1).

[0290] General clinical signs observation after inoculation General clinical signs were observed in all experimental groups from arrival to SD15 for safety evaluation. The main clinical sign observed in the piglets was diarrhea (Table 3), which is very common in young piglets, particularly when performing very early weaning. Some piglets also showed small injuries in the skin and some abscesses. In case of suffering, piglets were euthanised for welfare reasons. The group inoculated with strain 94 showed a reduced number of piglets affected by diarrhea compared to the PBS-incoculated group.

[0291] Table 3: Frequency of diarrhea after vaccination with the G. parasuis live strain 94. A group inoculated with PBS served as control, (b) 1 piglet very weak, it is euthanized for welfare reasons, (e) 1 piglet had soft feces.

[0292] Mortality

[0293] Mortality of the animals was remarkable at this stage: 1 piglet in the group inoculated with strain 94 (at SD2). The animal from group 94 (Piglet ID 45) was not necropsied since it was not due necessary by the responsible veterinarian.

[0294] Efficacy results

[0295] Rectal temperature

[0296] Rectal temperature was taken daily after challenge with the virulent Nagasaki strain to assess the impact of the infection on this clinical parameter. No differences due to the challenge were observed among groups (Figure 2).

[0297] In addition, when the maximal temperature in each piglet after challenge with Nagasaki was studied no differences were detected (Figure 3).

[0298] General clinical signs observation No clinical signs compatible with Glasser disease were observed after the challenge with the Nagasaki strain. However, animal 44 from group 94 needed to be euthanized for welfare reasons due to lameness and unnormal behaviour. Necropsy revealed a submandibular purulent abscess (see Figure 4), and moderate peritonitis and arthritis. No G. parasuis was isolated from the samples taken from this animal, while white haemolytic colonies were isolated from the abscess.

[0299] Weight gain

[0300] Weight was measured one day before the challenge with the Nagasaki strain (SD26) and then 1 and 2 weeks after the challenge (SD34 and SD40).

[0301] Initial weights at SD26 were similar among the groups (not significantly different), although the PBS group had the lowest (average of 7.46 kg). Piglets in groups 94 had an average weight of 8.09 Kg.

[0302] Weight gain after 1 week from the challenge (SD34-SD26) was significantly higher in piglets from group 94 in compared to the control PBS group (Student T test, p=0.01, Figure 5).

[0303] When considering weight gain during the 2 weeks after the challenge (SD40-SD26), this difference to control group was maintained in group 94 as a statistical tendency (Figure 6).

[0304] Considering this period, statistical significance was confirmed for the group inoculated with strain 94, which weight gain was significantly higher in comparison with placebo group (Student T test, p=0.06).

[0305] Evaluation of macroscopic lesions

[0306] No macroscopic lesions associated to the challenge were observed, suggesting that the challenge resulted in a subclinical infection. However, as in some animals that needed to be euthanized before the challenge with G. parasuis, some abscesses were observed (as indicated above for piglet 44).

[0307] Other parameters

[0308] Evaluation of bacterial re-isolation

[0309] A microbiological study was performed in swab samples taken from pericardium, thorax, peritoneum, joints (2 anterior and 2 posterior), cerebrospinal fluid and bronchoalveolar lavage fluid (BALF) at necropsy. G. parasuis was not isolated from samples taken from systemic organs. BALF samples were the only ones that yielded G. parasuis, and specifically the Nagasaki strain, but the score of Nagasaki isolated from BALF was not different across groups (p = 0.71, Figure 7).

[0310] Humoral response assessment

[0311] Sera samples

[0312] The level of G. parasuis antibodies at the moment of the challenge (SD27) with the Nagasaki strain was measured by the commercial kit Ing ezim-Haem ophilus. No major differences were found in the level of antibodies across groups (Figure 8).

[0313] Antibody titres of both experimental groups at challenge can be considered as negative (below 0.4), except for animal 38 belonging to group 94, which presented a titre of 0.419.

[0314] Just after the challenge (SD30), the antibody levels showed a negative slope in the PBS and 94 groups (Figure 9). This initial reduction after the challenge may be associated to opsonization of the challenge strain and, therefore, capture of circulating antibodies.

[0315] In addition, an ELISA using an antigen specific of virulent strains of G. parasuis, the fragment F4 from the VtaAs, was also performed. Since the vaccine strains were selected as non-virulent strains, it is not expected that they induce antibodies against F4, but we cannot rule out that they play a role in protection. At the moment of the challenge, SD27, a few piglets showed high antibody levels anti-F4, independently of the group (Figure 10), probably due to maternal transfer through the colostrum.

[0316] In summary, the level of serum total antibodies against Glaesserella parasuis, measured with the Ingezim ELISA, does not seem to be involved in the action of the nasal vaccination.

[0317] The possible interference of the F4 antibodies transferred by the colostrum does not seem to explain the results observed in weight gain, as indication of better clinical status.

[0318] Bronchoalveolar Lavage Fluid (BALF) samples

[0319] Furthermore, the level of G. parasuis antibodies were also measured by ELISA in the BALF samples at necropsy (SD41) to assess the mucosal response to the challenge. When we used the Ingezim ELISA to analyze these samples, no differences were observed (Figure 11). Additionally, antibodies against F4, both IgG and IgA were evaluated. While the level of IgG antibodies was similar across groups and very low, the IgA levels were significatively higher in the 94 group than the PBS control group (Student T test, =0.026; Figure 12)

[0320] Since the non-virulent strains of G. parasuis are not expected to elicit antibodies against F4 because they lack this antigen, this result may indicate that the virulent strain Nagasaki is recognized better by the immune system that has been exposed to the vaccine strain 94, probably due to the presence of shared antigens between non-virulent and virulent strains.

[0321] Evaluation of the vaccine-specific memory T cells

[0322] PBMC cells

[0323] The presence of vaccine-specific memory T cells was evaluated at the time of the challenge (SD27). Peripheral Blood Mononuclear Cells (PBMC) were stimulated in vitro with strain 94 and the Nagasaki strain.

[0324] Proliferation of CD4+ cells was measured with the Ki67 marker by cytometry. PBMCs from the piglets were taken at SD27, just before challenge, and were stimulated with strain 94 or RPMI as control.

[0325] The percentage of proliferating (Ki67+) CD4+ cells observed in group 94 when stimulated with the homologous strain (strain 94) was lower than in the PBS group (p=0.043, Figure 13). No differences were observed in the proliferation of CD4+ cells after stimulation with Nagasaki with the control.

[0326] On the other side, an increase in the percentages of proliferating (Ki67+ marker) B cells was observed in the group 94 when stimulated with the homologous strain (strain 94), which was higher than in the PBS group (Student T test, SigmaPlot; p=0.048) (Figure 14, left panel). The same trend was observed upon stimulation with the virulent Nagasaki strain, but it was not statistically significative (Figure 14, right panel).

[0327] PAMs

[0328] On the other side, previously frozen porcine alveolar macrophages (PAM) form BALF obtained at the end of the study from groups PBS and 94 were thaw and stained with fluorescently-labelled antibodies (SWC3, CD163, SLA-II) to analyse their activation status by flow cytometry (Figure 15). These analyses did not reveal a significantly different activation of the macrophages in the different groups, but the marker SLAII (involved in antigen presentation) was slightly higher in the PAM from group 94 (VassarStats: Statistical Computation Web Site: With all the data p=0.07 in Mann Whitney, unidirectional; P=0.045 non-directional when eliminating the outlier value).

[0329] The phagocytosis capacity of the PAMs was similar across groups and no differences were detected after incubation with Nagasaki (resistant to phagocytosis) or strain SW114 (susceptible to phagocytosis) (Figure 16). Levels of phagocytosis were quite variable within groups.

[0330] Nasal colonization by the challenge strain

[0331] The challenge strain was detected in the nose of the piglets by PCR specific of virulent strains.

[0332] In nasal samples taken the same day of the challenge (SD27), but before inoculation, G. parasuis virulent strains were not detected in any of the samples by PCR.

[0333] At necropsy (SD41), the situation changed, and virulent positive samples were detected, indicating that the Nagasaki strain used for the challenge was able to colonize some of the piglets.

[0334] Taking into consideration the intensity of the amplification in the PCR, a score was applied to evaluated potential differences between experimental groups. A reduction in the nasal colonization in the group 94 was detected (Student T test, p=0.043; Figure 17).

[0335] This indicates that the beneficial effect of the 94 strain may be due to competition / exclusion of the virulent Nagasaki strain, interfering in this way in the initial steps of the infection in a direct or indirect (through mucosal immunity) way.

[0336] Inflammation in the upper respiratory tract

[0337] The inflammation in the nasal and tracheal mucosa was assessed at necropsy (SD41) by histological analysis using a 0-3 score, results of the evaluation are presented in Table 4, Figures 18-20.

[0338] Table 4: Nasal (up) and tracheal (down) inflammation scores.

[0339] Nasal inflammation was slightly lower in the 94 group than in the PBS, but the difference was not statistically significant (Figure 18). Similar results were obtained in the tracheal mucosa (Figure 19).

[0340] Taking together the results from both sites (nasal score + tracheal score) a tendency to show less inflammation in the upper respiratory tract was observed in the piglets inoculated with strain 94 (Student T test, p=0.1; Figure 20).

[0341] Validation phase

[0342] Survival

[0343] Vaccine formulation was based on a life strain of Glaesserella parasuis (strain AQ08-Gp94) deposited under the accession number CECT 30960. The active substance was based on broth culture of Glaesserella parasuis in a dilution solution containing at least 2 x 106CUF / ml.

[0344] Animals were treated by intranasal inoculation of one spray-shot of 1 ml to each nasal orifice once all piglets were identified and allocated in each experimental group.

[0345] For the challenge, 107CFUs of a Nagasaki (pathogenic Glaesserella parasuis) broth culture in a volume of 500 ul was administered drop by drop into both piglets’ turbinate (250 per nostril) of all the piglets at D27. The results showed a clear improvement in the survival rate in the vaccinated groups compared to the control group (Figure 21).

[0346] It is important to note that the Nagasaki challenge faced in this experiment is much higher than the field infective dose that usually infects piglets on farms. Even under these conditions of such aggressive challenge, the effect of vaccination was demonstrated, since an improvement in the survival rate of vaccinated animals was clearly observed (Figure 21). At lower infectious doses, a better vaccine efficacy is expected with this intranasal Glasser disease vaccine.

[0347] Glasser Clinical Sings

[0348] Severe clinical signs compatible with Glasser disease were observed after the challenge with Nagasaki strain.

[0349] The clinical signs were measured using the following score: 0 = No clinical signs, 1 = Mild clinical signs, 2 = Moderate clinical signs and 3 = Severe clinical signs.

[0350] The control group presented an increase in the severity in most of the Glasser symptoms evaluated compared with vaccinated group (Table 5), as well in the average severity score (Table 5, Figure 22).

[0351] Two days after challenge SD29 (22-03-2025) there were significant differences between vaccinated and no vaccinated group for the symptoms lack of appetite, lameness and incoordination (Table 5).

[0352] Table 5: Glasser clinical signs score and average in no vaccinated and vaccinated groups.

[0353] Pathogen Dissemination

[0354] At necropsy, swab samples were collected in different piglet's organs to evaluate pathogenic Glaesserella parasuis dissemination by microbiology. The results showed a lower presence of the pathogenic Glaesserella parasuis strain in vaccinated animals compared to the unvaccinated control (Figure 23-25, Table 6). No pathogenic Glaesserella parasuis could be isolated in liver, neither brain of vaccinated piglets. Vaccination with the avirulent Glaesserella parasuis strain decreased the spread of the pathogenic Nagasaki strain in 7 of 8 organs evaluated.

[0355] Table 6: Post-challenge comparison of percentages of positives piglets to Glaesserella parasuis in different organs between the animals vaccinated with Glaesserella avirulent and the control group (microbiologically).

[0356] Systemic Humoral Response The serum of the samples was collected and placed into a labelled sterile 1 ml tube and stored at 4°C or freeze (-20°C) analyzed by commercial and homemade ELISA. The level of specific IgG in sera was evaluated by homemade ELISA to the 94 strain (vaccine G. parasuis) and with a Nagasaki ELISA (challenge G. parasuis).

[0357] The level of specific IgG antibodies was higher in the vaccinated groups compared with the control, in the 94 strain and in the Nagasaki homemade ELISAs (Figure 26).

[0358] IgG levels against 94 G. parasuis and Nagasaki strains antigen were quantified on serum samples using two in-house ELISA. Briefly, ELISA plates were coated by the addition of 100 pl of complete inactivated G. parasuis antigens diluted in carbonate buffer (5 pg per well). This was followed by a 2 h incubation at 37 °C and then an overnight incubation at 4 °C. Plates were subsequently washed three times with 300 pl / well of PBS containing 0.05% Tween 20 (PBS- T) (Sigma-Aldrich, USA). Non-specific binding sites were blocked using 300 pl / well of 3% (w / v) skim milk powder (Sigma- Aldrich, USA) in PBS-T. Plates were then washed three times with 300 pl / well of PBST. After coating, 100 pl of diluted porcine serum (1 : 100) was added to each well and incubated for 60 min at 37 °C. Plates were then washed three times with 300 pl / well of PBS-T. Following this, 100 pl / well of peroxidase-conjugated goat anti-swine IgG (Sigma-Aldrich, USA) was then applied, followed by another 60 min incubation at 37 °C. Plates were subsequently washed three times with 300 pl / well of PBS-T. This was succeeded by the addition of 100 pl / well TMB substrate (Sigma-Aldrich, USA) containing 0.002% H2O2 for 10-15 min. The reaction was ended with 100 pl / well of 3 M HC1, and absorbance was measured at 450 nm.

[0359] Nasal colonization

[0360] Nasal colonization of the vaccine strain of Glaesserella parasuis (avirulent) was evaluated in bacteria-free piglets under biomedical research conditions. Microbiological culture and qPCR were used. In both cases, the vaccine strain was able to survive in the piglets' respiratory tract and remain viable throughout the study.

[0361] The microbiological analysis consisted of isolation of the bacteria by microbiological culture of nasal swabs and subsequent identification by MALDI-TOF analysis. The vaccine 94 strain of Glaesserella parasuis was shown to be isolated from the nasal mucosa two weeks after inoculation. Nasal colonization was also evaluated by qPCR. The results showed that the bacteria survived in the upper respiratory tract of vaccinated piglets throughout the study, remaining at a concentration of approximately 4 log units (Figure 27).

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[0364] PCT

[0365] (Original in Electronic Form) (This sheet is not part of and does not count as a sheet of the international application)

[0366] FOR RECEIVING OFFICE USE ONLY

[0367] FOR INTERNATIONAL BUREAU USE ONLY

Claims

1. 42CLAIMS1. A strain of Glaesserella parasuis deposited under the accession number CECT 30960, or a composition comprising thereof.

2. A live vaccine composition or a probiotic composition comprising the strain of claim 1.

3. The composition, according to any of the claims 1 or 2, characterized in that it comprises a mucoadhesive solution.

4. The composition, according to claim 3, wherein the mucoadhesive solution is selected from the list consisting of: chitosan, carbopol (carbomer), sodium alginate, hyaluronic acid, pectin, polyvinyl alcohol (PVA), hydroxypropyl methylcellulose (HPMC), methylcellulose, polycarbophil and xanthan gum.

5. The strain or composition, according to any of the claims 1 to 4, for use as a medicament.

6. The strain or composition for use, according to claim 5, in a method for the prevention and / or treatment of Glasser’s disease or a condition caused by infection with a virulent Glaesserella parasuis strain.

7. The strain or composition for use, according to claim 6, wherein the prevention and / or treatment of Glasser’s disease, or a condition caused by the infection with a virulent Glaesserella parasuis strain, comprises: increasing body weight, improving the immune response by increasing mucosal antibodies (IgAs), increasing white blood cell reactivity against the virulent Glaesserella parasuis strain, reducing nasal mucosal colonization by the virulent Glaesserella parasuis strain and / or reducing inflammation in the upper respiratory track.

8. The strain or composition for use, according to any of the claims 5 to 7, wherein the method comprises administering the strain or composition intranasally, preferably using an intranasal applicator.

9. The strain or composition for use, according to any of the claims 5 to 8, wherein the method comprises administering the strain or composition at a dose of at least 2xl05total CFU / dose.

10. The strain or composition for use, according to any of the claims 5 to 9, wherein the method comprises administering the strain or composition at a concentration of between 105and 109total CFU / ml.

11. The strain or composition for use, according to any of the claims 5 to 10, wherein the method comprises administering the strain or composition in a total volume of between 0.2 and 2 ml / animal.

12. The strain or composition for use, according to any of the claims 5 to 11, wherein the method comprises administering the strain or composition in a total volume of between 0.1 and 1 ml per nostril.

13. The strain or composition for use, according to any of the claims 5 to 12, in a method for the prevention and / or treatment of pigs, preferably piglets, more preferably piglets during the first two weeks of life.

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