Method for preparing a serum composition for preventing or treating mucous membrane-associated infectious diseases in young mammals, serum composition prepared by said method and use thereof

Abstract

The present invention relates to a method for producing serum containing protective antibodies with mucosal immunity capable of protecting against infection with a mucosal-associated infectious pathogen, which comprises the step of transmucosally administering a mucosal-associated infectious pathogen to adult pigs and cattle to induce the production of protective antibodies with mucosal immunity capable of protecting adult pigs and cattle against infection with the mucosal-associated infectious pathogen; and a method for preventing or treating young piglets and calves from infection with an infectious pathogen by administering the serum produced by the method to newborn or young piglets and calves. The method of the present invention successfully prevented and treated piglets and young calves from infection with a mucosal-associated infectious pathogen.

Description

[Technical field]

[0001] The present invention relates to a method for preventing and treating mucosal-associated infectious diseases in young mammals, and more particularly to a method for preventing or treating infection of young mammals with mucosal-associated infectious pathogens by transmucosally administering a mucosal-associated infectious pathogen to an adult mammal to produce serum containing protective antibodies having mucosal immunity capable of protecting the adult mammal from the mucosal-associated infectious pathogen, and administering the serum produced by the above method to the young mammal. [Background technology]

[0002] Mucous membrane-associated infectious diseases such as digestive, respiratory and reproductive infectious diseases in pigs, horses and ruminants cause mortality and growth retardation in young mammals, resulting in significant economic losses in the livestock industry.

[0003] One way that young mammals can be protected from infectious diseases is by acquiring protective antibodies from the mother that can protect against infectious pathogens. Such passive immunity transmitted from the mother is an important means of protecting young mammals from infection by infectious pathogens until their immune systems are fully developed.

[0004] Therefore, maternal vaccination, which induces humoral immunity against infectious pathogens from pregnant mothers and transmits protective antibodies to young animals via the placenta during gestation or via colostrum after parturition, is used as an effective method to protect young animals from infection with infectious pathogens.

[0005] For example, it has been reported that vaccination of the mother via the muscle or subcutaneous route with nonmucosa-related infectious pathogens such as Japanese B encephalitis virus (JEV) and classical swine fever virus (CSFV) induces humoral immunity that can protect the mother against infection with infectious pathogens, and that young mammals that ingest the mother's colostrum containing the protective antibodies can effectively protect against infection with nasal mucosa-related infectious pathogens (van Oirschot JT. 2003. Vet Microbiol 96:367-384; Fan YC et al., 2013. Vet Microbiol 163:248-256).

[0006] However, vaccines have not been developed against all infectious pathogens, and maternal vaccination via the muscle or subcutaneous route, which is performed to prevent infection with mucosa-related infectious pathogens that cause diseases in the mucosa of the digestive, respiratory or reproductive tracts, in particular, has only limited effectiveness in preventing mucosa-related infectious diseases in young animals. For example, in the case of mucosal-associated infectious pathogens such as Enterotoxigenic Escherichia coli (ETEC), Transmissible gastroenteritis virus (TGEV), Porcine epidemic diarrhea virus (PEDV), Group A porcine rotavirus (GAPRV), Bovine coronavirus (BCoV), and Bovine group A rotavirus (BGARV), serum from mothers vaccinated intramuscularly shows high viral neutralization (VN) or bacterial agglutination (AG) titers against these mucosal-associated infectious pathogens, but does not effectively protect piglets and calves that ingest colostrum from these mucosal-associated infectious pathogens.

[0007] As other methods to overcome this, egg yolk-derived IgY (immunoglobulin Y) and cow colostrum-derived antibodies have been tried in piglets, and hyperimmune serum against mucosal-associated infectious pathogens has been tried in calves, but these have not produced satisfactory results.

[0008] In the absence of effective methods for preventing and treating infections by mucosal-associated infectious pathogens, the continuous occurrence of mucosal-associated infectious diseases with high infection and mortality rates in piglets and calves, and considering the approximately 1 billion and 1.5 billion pig and cattle populations (Food and Agriculture Organization (FAO) of the United Nations), infections by mucosal-associated infectious pathogens in young mammals have become a major cause of huge economic losses in the world's livestock industry. For these reasons, there is an urgent need to develop more effective methods for preventing and treating mucosal-associated (respiratory, digestive, and reproductive) infectious diseases in young mammals, including piglets and calves.

[0009] Meanwhile, Russian Registered Patent No. 2438709C1 discloses "Serum against cattle diseases caused by viruses of infectious rhinotracheitis, paraflu, rota, corona and mucosa diarrhea-disease, polyspecific, hyperimmune, method of prevention and treatment of cattle diseases caused by viruses of infectious rhinotracheitis, parainfluenza, rota, corona and mucosa diarrhea-disease," and Russian Registered Patent No. 2396979C2 discloses "Hyperimmune polyvalent serum against mass viral disease of cattle," but there is no description of a serum containing protective antibodies with mucosal immune activity that can protect against infection by mucosa-associated infectious pathogens, as in the present invention, or a method for producing a serum composition containing protective antibodies with mucosal immune activity. Furthermore, when serum produced by the above method, i.e. serum produced by administering an infectious pathogen or a part of a pathogen to an adult cow via a non-mucosal route, was administered to a calf, experimental results confirmed that administration of serum produced in this manner was ineffective in protecting the calf from infection with a mucosa-associated infectious pathogen (Selim et al., 1995. Vaccine 13:1454-1459). [Prior art documents] [Patent documents]

[0010] [Patent Document 1] Russian Patent No. 2438709C1 [Patent Document 2] Russian Patent No. 2396979C2 [Non-patent literature]

[0011] [Non-Patent Document 1] van Oirschot JT. 2003. Vet Microbiol 96:367-384; Fan YC et al., 2013. Vet Microbiol 163:248-25 [Non-Patent Document 2] Selim et al., 1995. Vaccine 13:1454-1459 Summary of the Invention [Problem to be solved by the invention]

[0012] The present invention has been developed in response to the above-mentioned demand, and has an object to provide a serum containing protective antibodies having mucosal immunity capable of protecting against infection by mucosal-associated infectious pathogens in young mammals, a method for inducing and producing said serum from adult mammals, and a method for preventing or treating infection by mucosal-associated infectious pathogens in young mammals by administering the produced serum to newborn or young mammals. [Means for solving the problem]

[0013] As a means for achieving the above-mentioned objective, the present invention provides a method for producing serum containing protective antibodies having mucosal immunity capable of protecting adult mammals from infection with mucosal-associated infectious pathogens by transmucosally administering a mucosal-associated infectious pathogen from a young mammal to an adult mammal (PAMI serum donor).

[0014] The present invention provides a serum harboring protective antibodies with mucosal immunity against infectious pathogens (PAMI serum), more specifically, a serum that can protect against infection by mucosal-associated infectious pathogens and contains secretory immunoglobulin A (sIgA) having mucosal immunity secreted into the mucosa as a major protective antibody.

[0015] The present invention provides a method for preventing or treating infection of a newborn or young mammal with a mucosa-associated infectious pathogen by orally or intravenously administering an effective amount of serum induced and produced by the above method to the newborn or young mammal for an effective period of time. Effect of the Invention

[0016] Considering the growth inhibition and death of piglets and calves caused by infection with mucosa-associated infectious pathogens, and the number of pigs and cattle raised in Japan and overseas, the method for preventing and treating piglets and young calves from infection with mucosa-associated infectious pathogens according to the present invention is expected to have a very significant economic effect on the pig and cattle livestock industries not only in Japan but also around the world.

[0017] In the present invention, serum containing protective antibodies with mucosal immunity capable of protecting against infection by a number of different mucosal-associated infectious pathogens (PAMI serum) can be continuously induced, maintained and produced from a single individual (PAMI serum donor) by administering a number of different mucosal-associated infectious pathogens to the single individual.

[0018] In the examples of the present invention, the method of the present invention, which can prevent and treat infections of different mucosa-associated infectious pathogens in piglets and calves, can be applied not only to the mucosa-associated infectious pathogens of piglets and calves used in the examples (TGEV, PEDV, GAPRV, BCoV, BGARV, E. coli), but also to all mucosa-associated gradually contagious viral and bacterial pathogens of piglets and calves that cause diseases in the digestive, respiratory, and reproductive tracts, and can be used to prevent and treat infections of these mucosa-associated infectious pathogens.

[0019] In the present invention, the duration of passive immunity that protects piglets and young calves administered with the serum from infection by infectious pathogens can be adjusted by increasing or decreasing the volume of serum (PAMI serum) administered to piglets and calves or the titer of the serum against infectious pathogens.

[0020] In the present invention, the method of the present invention, which is applied to two different mammals (piglets and calves) to prevent and treat infections caused by mucosa-associated infectious pathogens, can be applied not only to pigs and cows but also to all mammals having similar immune systems, such as horses, swine, ruminants, and humans, to prevent and treat infections caused by mucosa-associated infectious pathogens in young mammals.

[0021] Piglets administered with the serum of the present invention can be isolated from their mothers at an early stage and grow accordingly, thereby solving problems such as piglet crushing by mothers and insufficient milk production in prolific sows, and increasing the number of weaned piglets (Piglets per Sow per Year, PSY).

[0022] The method of the present invention can be applied to all mammals having similar immune systems to protect young mammals against infection by mucosa-associated infectious pathogens, and can not only replace existing vaccines, but can also be used as a method for preventing and treating infection by mucosa-associated infectious pathogens that cannot be solved by existing vaccines. [Brief description of the drawings]

[0023] [Figure 1]Figure 1 shows the viral neutralization (VN) and bacterial agglutination (AG) titers of adult pig sera. Adult pigs were orally infected weekly for 26 weeks with infectious pathogens [TGEV(175L), TGEV-wt1, PEDV-sm98, KPEDV-9, PEDV-wt1, GAPRV-vac, GAPRV-wt1, E. coli (K88, K99, F18)], and pre-infection (week 0) and post-infection (week 26) sera (PAMI sera) were collected from the adult pigs. Pre-vaccination (control) and post-vaccination (vaccine control) sera were also collected from one adult pig vaccinated intramuscularly with infectious pathogens (PEDV-sm98 or KPEDV-9). The VN titers against TGEV (175L), TGEV-wt1, PEDV-sm98, KPEDV-9, PEDV-wt1, GAPRV-vac, and GAPRV-wt1, and the AG titers against E. coli (K88, K99, and F18) of adult pig sera before infection with infectious pathogens (control serum), adult pig sera 26 weeks after infection with infectious pathogens (PAMI serum), adult pig sera before vaccination (control serum), and adult pig sera after vaccination (vaccine control serum) were investigated according to the Materials and Methods. The numbers on each graph refer to the corresponding lane (column). FIG. 1A shows the VN titers against TGEV(175L), TGEV-wt1, PEDV-sm98, KPEDV-9, PEDV-wt1, GAPRV-vac, GAPRV-wt1, and the AG titers against E. coli (K88, K99, F18) of sera (control sera and PAMI sera, respectively) collected from adult pigs (castrated male pigs) before and after oral infection with TGEV(175L), TGEV-wt1, PEDV-sm98, KPEDV-9, PEDV-wt1, GAPRV-vac, GAPRV-wt1, or E. coli (K88, K99, F18).FIG. 1B shows the VN titers against TGEV(175L), TGEV-wt1, PEDV-sm98, KPEDV-9, PEDV-wt1, GAPRV-vac, GAPRV-wt1, and the AG titers against E. coli (K88, K99, F18) of sera (control sera and PAMI sera, respectively) collected from adult pigs (sows) before and after oral infection with TGEV(175L), TGEV-wt1, PEDV-sm98, KPEDV-9, PEDV-wt1, GAPRV-vac, GAPRV-wt1, and E. coli (K88, K99, F18). FIG. 1C shows the VN titers against PEDV-sm98, KPEDV-9, and PEDV-wt1 of adult pig sera after vaccination (vaccine control serum) and adult pig sera before vaccination (control serum), respectively. [Diagram 2] This figure shows the viral neutralization (VN) and bacterial agglutination (AG) titers of adult cattle sera. Adult cattle were orally infected with infectious pathogens [BCoV-wt1, BCoV-vac, BGARV-wt1, BGARV-vac, and E. coli (K99)] every week for 12 weeks, and serum (control serum) before infection with infectious pathogens (week 0) and serum (PAMI serum) after infection with infectious pathogens (week 12) were collected from adult cattle. The VN titers against BCoV-wt1, BCoV-vac, BGARV-wt1, and BGARV-vac and the AG titers against E. coli (K99) of adult cattle serum before infection with infectious pathogens (control serum) and adult cattle serum after infection with infectious pathogens (PAMI serum) 12 weeks after infection with infectious pathogens were investigated by Materials and Methods. The numbers on each graph indicate the corresponding lane (column). [Diagram 3]This is a diagram showing clinical symptoms observed in piglets administered with control serum and PAMI serum after challenge infection with infectious pathogens. In each piglet group, the control serum was orally administered to the piglets in the control serum-administered piglet group, and the PAMI serum was orally administered to the piglets in the PAMI serum-administered piglet group. Then, the piglets in group A were infected with TGEV-wt1, the piglets in group B with PEDV-wt1, the piglets in group C with GAPRV-wt1, and the piglets in group D with E. coli (K88) according to the materials and methods described by, and the clinical symptoms of the piglets were observed. The numbers on each graph refer to the corresponding lane (column). 3A shows the mortality rates of piglets in group A challenged with TGEV-wt1, piglets in group B challenged with PEDV-wt1, piglets in group C challenged with GAPRV-wt1, and piglets in group D challenged with E. coli (K88) in each piglet group administered with control serum and PAMI serum, and 3B shows the incidence of diarrhea in piglets in group A challenged with TGEV-wt1, piglets in group B challenged with PEDV-wt1, piglets in group C challenged with GAPRV-wt1, and piglets in group D challenged with E. coli (K88) in each piglet group administered with control serum and PAMI serum. [Figure 4]This is a diagram showing VN and AG titers against infectious pathogens in sow sera collected before and after farrowing. Sera were collected from four primiparous sows (sow 1, sow 2, sow 3, sow 4) before and after farrowing, and the VN titers against TGEV (175L), TGEV-wt1, PEDV-sm98, KPEDV-9, PEDV-wt1, GAPRV-vac, GAPRV-wt1, and the AG titers against E. coli (K88, K99, F18) of the collected sera were measured by the materials and methods described. The numbers on each graph refer to the corresponding lane (column). 4A shows VN titers against TGEV(175L), TGEV-wt1, PEDV-sm98, KPEDV-9, PEDV-wt1, GAPRV-vac, GAPRV-wt1, and AG titers against E. coli (K88, K99, F18) of serum from sow 1 collected before and after farrowing, and 4B shows VN titers against TGEV(175L), TGEV-wt1, PEDV-sm98, KPEDV-9, PEDV-wt1, GAPRV-vac, GAPRV-wt1, and AG titers against E. coli (K88, K99, F18) of serum from sow 2 collected before and after farrowing. 4C shows VN titers against TGEV(175L), TGEV-wt1, PEDV-sm98, KPEDV-9, PEDV-wt1, GAPRV-vac, GAPRV-wt1, and AG titers against E. coli (K88, K99, F18) of serum from sow 3 collected before and after farrowing, and 4D shows VN titers against TGEV(175L), TGEV-wt1, PEDV-sm98, KPEDV-9, PEDV-wt1, GAPRV-vac, GAPRV-wt1, and AG titers against E. coli (K88, K99, F18) of serum from sow 4 collected before and after farrowing. [Diagram 5]This is a diagram showing clinical symptoms observed in calves administered with control serum and PAMI serum after challenge infection with infectious pathogens. In each calf group, the control serum was orally administered to the calves in the control serum-administered calf group, and the PAMI serum was orally administered to the calves in the PAMI serum-administered calf group. Calves in group A were then infected with BCoV-wt1, calves in group B with BGARV-wt1, and calves in group C with E. coli (K99) according to the materials and methods described by, and the clinical symptoms of the calves were observed. The numbers on each graph refer to the corresponding lane (column). [Figure 6] This figure shows the time when PAMI serum was orally administered to piglets after farrowing and the clinical symptoms of piglets challenged with an infectious pathogen (PEDV-wt1). For piglets in the same litter, PAMI serum was orally administered to the "0-hour group piglets (n=3)" within 1 hour after farrowing, to the "12-hour group piglets (n=3)" within 12 hours after farrowing, and to the "18-hour group piglets (n=3)" within 18 hours after farrowing. Three days after farrowing, piglets in each group that had been administered PAMI serum were challenged with PEDV-wt1 (1×106 TCID50 / piglet) using the materials and methods described by, and the mortality and diarrhea incidence were observed until 28 days of age. The numbers on each graph refer to the corresponding lane (column). [Figure 7] This figure shows the recovery rate of diarrhea in calves infected with BCoV-wt1, BGARV-wt1, and E. coli (K99) after administration of control serum or PAMI serum. Calves (n=6) challenged with BCoV-wt1 were administered control serum (n=3) and PAMI serum (n=3), calves (n=6) challenged with BGARV-wt1 were administered control serum (n=3) and PAMI serum (n=3), and calves (n=6) challenged with E. coli (K99) were administered control serum (n=3) and PAMI serum (n=3) according to the materials and methods described, and the clinical symptoms of the calves were observed. The numbers on each graph refer to the corresponding lane (column). [Figure 8]This is a diagram showing clinical symptoms of a group of piglets administered with PAMI serum (n=280) and a group of piglets administered with vaccine control serum (n=9) in a farm where PEDV was occurring. PAMI serum was orally administered to the group of piglets administered with PAMI serum (n=280) and the group of piglets administered with vaccine control serum (n=9) in a farm where PEDV was occurring, within 1 hour after parturition, using the materials and methods described above, and the clinical symptoms of the piglets were observed until 28 days of age. The numbers on each graph indicate the corresponding lane (column). NA (not applicable): Not applicable. 8A is a diagram showing the mortality rate of piglets administered with PAMI serum and piglets administered with vaccine control serum in a farm where PEDV was occurring. 8B is a diagram showing the incidence of diarrhea in piglets administered with PAMI serum and piglets administered with vaccine control serum in a farm where PEDV was occurring. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] In piglets and calves, major mucosal-associated infectious pathogens such as TGEV, PEDV, GAPRV, BCoV, BGARV, and Enterotoxogenic E. coli induce severe diarrhea with various infection rates and mortality rates in piglets and calves. The resulting growth inhibition and death of piglets and calves are the cause of huge economic losses in the pig and cattle livestock industries, but an effective method for preventing infection by such mucosal-associated infectious pathogens has not yet been developed.

[0025] As an example of solving such problems, the present invention induces and produces serum with mucosal immunity (PAMI serum) containing protective antibodies capable of protecting against the mucosal-associated infectious pathogens by transmucosally (orally) administering different mucosal-associated infectious pathogens (TGEV, PEDV, GAPRV, ETEC, etc.) to adult pigs. When the produced PAMI serum was orally administered to newborn piglets early after birth, the piglets administered with PAMI serum were protected from challenge infection by the different mucosal-associated infectious pathogens. In addition, when the PAMI serum was administered to newborn piglets at a farm where PEDV was occurring, the piglets administered with PAMI serum were protected from PEDV infection, successfully ending the spread of PEDV at the farm where PEDV was occurring.

[0026] In order to examine whether the method of the present invention capable of preventing infection by mucosa-associated infectious pathogens can be applied to other mammals having similar immune systems, another example was carried out on calves. Different mucosa-associated infectious pathogens BCoV, BGARV, and E. coli (K99) were administered mucosally (orally) to adult cattle to induce and produce serum (i.e., PAMI serum) containing protective antibodies capable of preventing infection by mucosa-associated infectious pathogens, and the produced PAMI serum was orally administered to newborn calves, and then challenged with mucosa-associated infectious pathogens [BCoV-wt1, BGARV-wt1, and E. coli (K99)]. Calves administered with PAMI serum were protected from infection by the different mucosa-associated infectious pathogens. In addition, when PAMI serum was administered intravenously to calves that had diarrhea after challenge with different mucosal-associated infectious pathogens [BCoV-wt1, BGARV-wt1, and E. coli (K99)], the diarrhea in the calves that were administered PAMI serum was cured.

[0027] Thus, the present invention was completed by confirming through experiments that the method of the present invention can be applied to two different mammals and to various different mucosa-associated infectious pathogens to prevent and treat mucosa-associated infectious pathogen infections in young mammals. These results suggest that the method of the present invention can be applied to all mammals having similar immune systems, including ruminants, horses, humans, etc., in addition to the pigs and cows used as examples, in preventing and treating mucosa-associated infectious pathogen infections, and can be applied to all mucosa-associated infectious pathogens of young mammals, not only the piglets and calves used as examples.

[0028] In the present invention, the term "serum" refers to the residue obtained after removing fibrinolysis from plasma, and the serum according to the present invention (PAMI serum) contains protective antibodies having mucosal immunity capable of protecting against infection by mucosal-associated infectious pathogens induced by an immune response to an infectious pathogen administered to an adult mammal.

[0029] In the method for producing serum according to the present invention, the mucosa-associated infectious pathogen is a wild type or attenuated virus or bacterium that is infectious but induces asymptomatic or mild clinical symptoms in adult pigs (or cows) and induces severe clinical symptoms in piglets (or young calves), and is a mucosa-associated viral and bacterial pathogen that induces diseases in the digestive, respiratory, reproductive, etc. tracts of piglets (or young calves).

[0030] In the method for producing serum according to the present invention, the administration amount of the mucosa-associated infectious pathogen is an amount that is infectious and immunogenic in adult pigs (or cows) but does not cause mortality, and may be an amount that is capable of sufficiently inducing and producing protective antibodies against the infectious pathogen in the serum of the adult pigs (or cows). Those skilled in the art may select and use an appropriate amount depending on the type and condition of the mucosa-associated infectious pathogen, and the number of administrations may be adjusted so as to induce and produce an effective level of protective antibodies in the serum.

[0031] In addition, in the method for producing serum according to the present invention, the mucosal route administration may be preferably oral or nasal administration, and protective antibodies having mucosal immunity are produced when infectious pathogens are administered via the mucosal route. Conventional vaccine compositions induce antibody production via a non-mucosal route such as intramuscular injection of an immunogen, and serum containing protective antibodies induced and produced by non-mucosal administration lacks mucosal immunity, and is therefore not very effective as a passive immunogen for preventing and treating infection by mucosal-associated infectious pathogens.

[0032] In the method for producing serum according to the present invention, the adult pig (or cow) refers to a mammal whose immune system is sufficiently developed to induce and produce serum containing protective antibodies having mucosal immunity that can protect against infection by an infectious pathogen when administered with the infectious pathogen or its protein, and may be a mammal including, preferably, pigs, horses, ruminants, humans, etc.

[0033] In the method for producing serum according to the present invention, PAMI serum capable of protecting against different mucosal-associated infectious pathogens (TGEV, PEDV, GAPRV, BCoV, BGARV, and E. coli) was induced and produced from a single individual (pig or cow). This means that PAMI serum capable of preventing and treating infections from multiple mucosal-associated infectious pathogens can be induced, maintained, and produced from a single individual (PAMI serum donor) by administering multiple different mucosal-associated infectious pathogens to the single individual (PAMI serum donor).

[0034] In addition, in the method for producing serum according to one embodiment of the present invention, the protective antibody includes an immunoglobulin specific to an infectious pathogen or its protein, and is preferably a protective antibody having a mucosally secreted antibody (secretory IgA, sIgA) as a major immunoglobulin, which has mucosal immunity against mucosal-associated infectious pathogens and can protect against mucosal-associated infectious pathogens.

[0035] The present invention also provides a serum containing protective antibodies having mucosal immunity capable of preventing infection by mucosa-associated infectious pathogens, produced by the above-mentioned production method.

[0036] In this specification, the serum containing protective antibodies with mucosal immunity that can protect against infection by infectious pathogens is called the PAMI serum. The PAMI serum is characterized by a mixture of protective antibodies that recognize infectious pathogens or various epitopes of their proteins and have different antigen recognition sites.

[0037] Furthermore, the PAMI serum of the present invention can be stored for a long period of time when powdered by lyophilization, and can be easily prepared by the simple step of adding a water-soluble solvent, such as water, before administration to young mammals.

[0038] The serum containing the protective antibody having mucosal immunity capable of protecting against the infection of the mucosa-associated infectious pathogen according to the present invention can be used for passive immunity, and the term "passive immunity" refers to immunity acquired by injecting an antibody or sensitized lymphocyte produced by an immune response to an antigenic stimulus from another individual into an individual who has not been stimulated with the antigen. Passive immunity has the advantage that it can obtain a protective effect in a short time for an individual who has no immunity against a pathogen, compared to active immunity, which induces antibody production by injecting a vaccine.

[0039] The present invention also provides a method for preventing or treating a piglet (or young calf) from infection with a mucosa-associated infectious pathogen, comprising administering to the newborn or piglet (or young calf) an effective amount of serum containing a protective antibody having mucosal immunity capable of preventing infection with the mucosa-associated infectious pathogen.

[0040] In the preventive or therapeutic method according to the present invention, the piglet (or young calf) (PAMI serum recipient) means a piglet (or young calf) whose immune system is not sufficiently developed to produce protective antibodies against infection with an infectious pathogen, or which needs to acquire protective antibodies capable of preventing infection with an infectious pathogen.

[0041] In the preventive or therapeutic method according to the present invention, the preferred administration of serum containing protective antibodies having mucosal immunity capable of preventing infection by the mucosa-associated infectious pathogen may be, but is not limited to, oral or intravenous administration.

[0042] In addition, the oral administration can be applied to newborn piglets (or calves) to prevent the piglets (or young calves) from infection with mucosa-associated infectious pathogens, and the oral administration is preferably performed within one hour after the birth of the newborn mammal. The time window of the oral administration is a time region in which the serum of the present invention can be administered to the newborn mammal to absorb protective antibodies capable of preventing infection with mucosa-associated infectious pathogens and exhibit the most significant passive immune effect.

[0043] In addition, the intravenous administration of the serum can be used for the purpose of preventing and treating infection of newborn and young piglets (or calves) from mucosa-associated infectious pathogens. The effective time of the intravenous administration means the time required for administration of serum containing protective antibodies capable of protecting against infection of mucosa-associated infectious pathogens from the outside in order to prevent or treat infection of mucosa-associated infectious pathogens in young piglets (or calves).

[0044] The serum according to the present invention may be administered in an immunologically effective amount. The term "immunologically effective amount" refers to an amount sufficient to prevent or treat infection by an infectious pathogen and not causing side effects or severe or excessive immune reactions, and the exact administration concentration varies depending on the titer of an antibody against a specific immunogen and pathogen, and can be easily determined by a person skilled in the art depending on factors well known in the medical field such as the age, weight, health, sex, administration route, administration method, etc. of the subject, and may be administered once to several times.

[0045] In addition, in the preventive or therapeutic method of the present invention, serum containing protective antibodies having mucosal immunity capable of preventing infection by the mucosal-associated infectious pathogens can be further mixed with one or more immune enhancers and administered to newborn or young mammals.

[0046] The immune enhancer that may be included in the present invention refers to a substance that enhances the immune response of an animal to which the serum of the present invention is administered, and many different immune enhancers are known to those skilled in the art, including, but not limited to, Proint complete and incomplete immune enhancers, Vitamin E, Quil A, mineral oil and non-mineral oil and Carbopol, water-in-oil emulsion immune enhancers, and the like.

[0047] In the present invention, PAMI serum against mucosal-associated infectious pathogens is induced and produced from two different mammalian species, pigs and cows, and the serum can be used to successfully prevent and treat mucosal-associated infectious pathogen infections. This shows that the method of the present invention can be applied to animals such as pigs, horses, ruminants, and humans, which have similar immune systems.

[0048] It was confirmed in the examples that the method of the present invention can be applied to different mammals and various different mucosa-associated infectious pathogens to protect against mucosa-associated infectious pathogens. For this reason, pathogens applicable to the method of the present invention include not only the pathogens used in the examples (TGEV, PEDV, GAPRV, BCoV, BGARV, enterotoxigenic E. coli), but also all mucosa-associated viruses and bacterial infectious pathogens that cause diseases in the digestive, respiratory and reproductive tracts of piglets and calves. In addition, pathogens applicable to the method of the present invention include all mucosa-associated viruses and bacterial infectious pathogens that cause diseases in the digestive, respiratory and reproductive tracts of all mammals, including pigs, horses, ruminants, and humans, that have similar immune systems.

[0049] In addition, the infectious pathogen may be a virus or bacteria that induces a gastrointestinal or respiratory disease, and examples of the infectious pathogen include rhinovirus, coronavirus, parainfluenza virus, respiratory syncytial virus, influenza virus, adenovirus, norovirus, enterovirus, rotavirus, calicivirus, Streptococcus pneumonia, Mycoplasma pneumonia, Haemophilus influenza, Klebsiella pneumonia, Chlamydia pneumonia, Vibrio cholera, enterotoxigenic Escherichia coli, and the like. coli), Salmonella spp., Clostridium spp., Shigella spp., Yersinia spp., Porcine rotavirus, transmissible Gastroenteritis virus, Porcine epidemic diarrhea virus, Porcine parvovirus, Porcine reproductive and respiratory syndrome virus, Porcine respiratory corona virus, Swine Influenza virus, Swine vesicular disease virus, Porcine circovirusvirus, Streptococcus suis, Pasteurella multocida, Actinobacillus pleuropneumoniae, Brachyspira hyodysenteriae, Bordetella bronchiseptica, Haemophilus parasuis, Mycoplasma hyopneumoniae, Erysipelothrix rhusiopathiae, malignant catarrhal fever virus, bovine rotavirus, bovine influenza virus, bovine corona virus, bovine herpes virus, bovine parainfluenza virus, Bovine respiratory syncytial virus, Bovine viral diarrhea virus, Vesicular stomatitis virus, Infectious Bovine Rhinotracheitis Virus, Bovine clostridium spp., Mannheimia haemolytica, African horse sickness virus, Equine Influenza virus, Equine coronavirus, Streptococcus equi, Rhodococcus equi, and the like, but are not limited to these.

[0050] The present invention also provides a pharmaceutical composition for preventing or treating diseases caused by infection with infectious pathogens, comprising as an active ingredient a serum containing a protective antibody having mucosal immunity capable of preventing infection with the infectious pathogens.

[0051] The pharmaceutical composition of the present invention may further contain a pharma- ceutically acceptable carrier, excipient, or diluent in addition to the serum. It may also be formulated into oral dosage forms such as suspensions, emulsions, and syrups, and sterile injectable solutions by conventional methods, but is not limited thereto. Examples of carriers, excipients, and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, water, and magnesium stearate. When formulated, it is prepared using a diluent or excipient that is commonly used, such as a filler, extender, binder, wetting agent, disintegrant, or surfactant. Liquid preparations for oral administration include suspensions, liquid preparations for internal use, emulsions, syrups, etc., and may contain various excipients such as wetting agents, sweeteners, flavoring agents, preservatives, etc. in addition to water, which is a commonly used simple diluent. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, and emulsions.

[0052] The appropriate dosage of the pharmaceutical composition according to the present invention can be formulated in various ways depending on factors such as the formulation method, the mode of administration, the patient's age, weight, sex, pathological condition, food, administration route, antibody half-life, antibody titer, and reaction sensitivity.

[0053] The concentration of the active ingredient contained in the pharmaceutical composition of the present invention can be determined taking into consideration the purpose of treatment, the condition of the subject to be treated, the required period, etc., and is not limited to a specific concentration range.

[0054] In the pharmaceutical composition of the present invention, the disease caused by infection with the infectious pathogen may be, but is not limited to, coronavirus infection, flu (acute respiratory disease caused by influenza virus), adenovirus infection, coronavirus infection, parainfluenza virus infection, respiratory syncytial virus infection, rhinovirus infection, metapneumovirus infection, pneumococcus infection, mycoplasma infection, chlamydia infection, rotavirus infection, norovirus infection, enteric adenovirus infection, astorvirus infection, sapovirus infection, Escherichia coli infection, or salmonella infection.

[0055] The present invention also provides a veterinary composition for preventing or treating infection of newborn or young mammals with mucosa-associated infectious pathogens, comprising as an active ingredient a serum containing protective antibodies with mucosal immunity capable of preventing infection with the infectious pathogens.

[0056] The serum containing protective antibodies having mucosal immunity capable of preventing infection by infectious pathogens according to the present invention has an infection suppressing and therapeutic effect against infectious pathogens, and therefore can enhance the immunity of livestock, thereby maintaining and improving the health of livestock.

[0057] The veterinary compositions of the present invention may further comprise suitable excipients and diluents commonly used in the manufacture of veterinary compositions.

[0058] The veterinary dosage forms of serum according to the present invention may be used alone or in suitable associations as well as in combination with other veterinary active compounds.

[0059] Excipients and diluents that may be included in the veterinary compositions containing the serum of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water and magnesium stearate.

[0060] The veterinary composition containing the serum according to the present invention may further contain a filler, an anticoagulant, a lubricant, a wetting agent, spices, an emulsifier, a preservative, etc. The veterinary composition according to the present invention may be formulated by a method well known in the art so as to provide rapid absorption of the active ingredient and safety after administration to an animal, and the formulation may be in the form of a suspension, emulsion, solution, syrup, sterile injection solution, etc.

[0061] In the veterinary composition according to the present invention, the infectious pathogen is a virus or a bacterium that induces diseases in the digestive, respiratory and reproductive tracts, as described above.

[0062] In the present invention, the method of inducing, maintaining and producing serum, i.e., PAMI serum, capable of preventing and treating infections by different mucosal-associated infectious pathogens by administering different mucosal-associated infectious pathogens to different mammals, and administering the produced PAMI serum to young mammals to prevent and treat infections by mucosal-associated infectious pathogens, is proposed to be applicable to all mammals having similar immune systems and the mucosal-associated infectious pathogens of these mammals to protect against mucosal-associated infectious pathogens, and to be a new method for preventing or treating infections by mucosal-associated infectious pathogens that cannot be solved by existing methods, or to replace existing methods such as vaccines.

[0063] The present invention will be described in detail below with reference to examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

[0064] Example 1. Materials and Methods 1-1. Cells, viruses, bacteria Vero cells, swine testis cells (ST cells), and fetal rhesus monkey kidney (MA104) cells were cultured in EMEM (Eagle's minimum essential medium) or DMEM (Dulbecco's Modified Eagle's Medium) containing 5-10% fetal bovine serum (FBS), penicillin (100 units / ml), and streptomycin (100 μg / ml) in a CO2 incubator at 37°C.The following viruses were identified: wild-type porcine epidemic diarrhea virus (PEDV-wt1), wild-type transmissible gastroenteritis virus (TGEV-wt1), wild-type group A porcine rotavirus (GAPRV-wt1), vaccine strains [KPEDV-9 (Korea BNP, Korea), PEDV-sm98 (Green Cross Veterinary Products Co., LTD, Korea), TGEV(175L) (Green Cross Veterinary Products Co., LTD, Korea) and GAPRV-vac (Green Cross Veterinary Products Co., LTD, Korea)], enterotoxigenic Escherichia coli (ETEC) strains K88, K99 and F18 (Veterinary Science Research Institute, Korea), wild-type bovine coronavirus (bovine rotavirus) and ... The viruses used were bovine coronavirus (BCoV-wt1), vaccine strain bovine coronavirus (BCoV-vac), wild type bovine group A rotavirus (BGARV-wt1), vaccine strain bovine rotavirus (Korea, BNP), BGARV-vac), etc. PEDV and BCoV were grown in Vero cells using DMEM containing trypsin (10 μg / ml), TGEV in ST cells using DMEM, and GAPRV and BGARV in MA104 cells using EMEM (or DMEM) containing trypsin (0.5-1.5 μg / ml). ETEC was cultured in LB broth (Affymetrix, Inc., Ohio, USA).

[0065] 1-2. Isolation of field virus strains Field strains TGEV (Zhenhui S et al., 2015. Israel Journal of Veterinary Medicine 70:22-30), PEDV and BCoV (Chen Q et al., 2014. J Clin Microbiol 52:234-243), GAPRV and BGARV (Arnold M et al., 2009. Curr Protoc Microbiol Chapter 15:Unit 15C 13) were isolated from feces of infected piglets and calves according to previously described methods and named TGEV-wt1, PEDV-wt1, BCoV-wt1, GAPRV-wt1 and BGARV-wt1, respectively.

[0066] 1-3.Virus titer measurement Viral titration was performed as previously described. For titration of PEDV and BCoV (Thomas JT et al., 2015. PLoS One 10:e0139266), 80-90% monolayer Vero cells cultured in 96-well culture dishes were washed three times with PBS. Then, PEDV activated by trypsin (10 μg / ml) was serially diluted in DMEM at 37°C for 1 hour, and 0.1 ml of each diluted PEDV (or BCoV) was inoculated into Vero cells washed with PBS. After reacting the Vero cells inoculated with the virus for 1 hour at 37°C, the virus inoculum was replaced with 0.2 ml DMEM containing trypsin (10 μg / ml), and the Vero cells were cultured in a CO2 incubator at 37°C for 3-5 days. For TGEV titration (He L et al., 2012. Virol J 9:176), 80-90% monolayer ST cells cultured in 96-well culture dishes were washed three times with PBS. Then, TGEV was serially diluted stepwise in DMEM, and 0.1 ml of each diluted TGEV was inoculated into ST cells washed with PBS. ST cells inoculated with the virus were reacted at 37°C for 1 hour, after which the virus inoculation solution was replaced with 0.2 ml DMEM (5% FBS), and ST cells were cultured in a CO2 incubator at 37°C for 3-5 days. For GAPRV and BGARV titration (Otto PH et al., 2015. J Virol Methods 223:88-95), 80-90% monolayer MA104 cells cultured in 96-well culture dishes were washed three times with PBS. Then, GAPRV (or BGARV) activated by trypsin (10 μg / ml) for 1 hour at 37°C was serially diluted in EMEM (or DMEM), and 0.1 ml of each diluted GAPRV (or BGARV) was inoculated into MA104 cells washed with PBS. After reacting the virus-inoculated MA104 cells at 37°C for 1 hour, the virus inoculation solution was replaced with 0.2 ml EMEM (or DMEM) containing trypsin (0.5-1.5 μg / ml), and the MA104 cells were cultured in a CO2 incubator at 37°C for 5-7 days.When the cytopathic effect (CPE) due to the virus inoculation became evident, the virus-inoculated cells were fixed with 10% formalin for 30 min and then stained with 0.5% (w / v) crystal violet. The virus titer was expressed as the fifty percent tissue culture infective dose (TCID). 50 ) was calculated by inversely expressing the highest virus dilution that showed CPE in cells infected with each diluted virus.

[0067] 1-4. Inoculation of experimental animals and infectious pathogens Five pregnant primiparous pigs (sow 1, sow 2, sow 3, sow 4, sow 5) purchased from the farm were kept in separate areas. To induce and produce PAMI serum, which contains protective antibodies with mucosal immunity that can protect against infection by infectious pathogens in pigs, four adult pigs, two female pigs and two castrated male pigs, were kept in the same area. Then, 40 ml of each virus (1 × 10 6 TCID 50 400 ml of each E. coli (K88, K99, and F18) cultured for 18 h were mixed with pig feed and administered weekly to four adult pigs for 26 weeks. To produce serum from pigs vaccinated with the vaccine strains (vaccine control serum), one adult pig was inoculated twice with the vaccine strains (KPEDV-9 and PEDV-sm98) via intramuscular route according to the manufacturer's instructions, with an interval of 2 weeks. After the final inoculation, two booster inoculations were administered with an interval of 2 weeks.

[0068] To induce and produce PAMI serum, which contains protective antibodies with mucosal immunity that can protect against infection with mucosal-associated infectious pathogens in cattle, one 15-month-old adult cow was used. 40 ml of each virus (1 × 10 6 TCID50 / ml) [BCoV-vac, BCoV-wt1, BGARV-vac and BGARV-wt1] and 100 ml of E. coli (K99) cultured for 18 h were orally administered to adult cows weekly for 12 weeks.

[0069] 1-5. Serum and titer measurements Sera from four primiparous sows (sow 1, sow 2, sow 3, sow 4) before and after farrowing, sera from four adult pigs before and 26 weeks after infection with infectious pathogens (PEDV, TGEV, GAPRV, and E. coli) (control sera), and sera from pigs before and after vaccination with vaccine strains (KPEDV-9 and PEDV-sm98) (vaccine control sera) were collected via the milk vein or carotid artery, respectively. Sera from adult cows before and 12 weeks after infection with BCoV-vac, BCoV-wt1, BGARV-vac, BGARV-wt1, and E. coli (K99) were collected via the jugular vein. All collected porcine sera were tested for VN titers against PEDV, TGEV, and GAPRV, and for AG titers against E. coli, and all collected bovine sera were tested for VN titers against BCoV-vac, BCoV-wt1, BGARV-vac, and BGARV-wt1, and for AG titers against E. coli.

[0070] VN titers of sera against TGEV, PEDV, GAPRV, BCoV, and BGARV were performed according to published methods (Arnold M et al, 2009. Curr Protoc Microbiol Chapter 15:Unit 15C 13; Woods RD et al., 1988. Am J Vet Res 49:300-304; Song DS et al., 2007. Res Vet Sci 82:134-140). Briefly, each serum was heat-treated at 56°C for 30 min and serially diluted two-fold in DMEM (or EMEM) in a round-bottomed micro-titer 96 well plate. 50 μl of virus (200 TCID 50 100 μl of PEDV / serum (or BCoV / serum) mixture was added to a monolayer of Vero cells cultured in a 96-well culture dish, 100 μl of TGEV / serum mixture was added to a monolayer of ST cells cultured in a 96-well culture dish, and 100 μl of GAPRV / serum (or BGARV / serum) mixture was added to a monolayer of MA104 cells cultured in a 96-well culture dish, and then reacted at 37°C for 2 hours. After virus adsorption, the TGEV / serum mixture was replaced with 0.2 ml DMEM containing 5% FBS, the PEDV / serum (or BCoV / serum) mixture was replaced with 0.2 ml DMEM containing 10 μg / ml trypsin, and the GAPRV / serum (or BGARV / serum) mixture was replaced with 0.2 ml EMEM (or DMEM) containing 0.5-1.5 μg / ml trypsin, respectively. The virus-infected cells in the 96-well culture dishes were then cultured for 3-7 days in a CO2 incubator at 37°C. When significant CPE phenomenon appeared in the virus-infected cells, the virus-inoculated cells in the 96-well culture dishes were fixed with 10% formalin for 30 minutes at room temperature and stained with 0.5% (w / v) crystal violet. The VN titer of the serum against the virus was expressed as the inverse of the highest dilution of the serum that did not show CPE in the monolayer cells inoculated with the virus / serum mixture. To investigate the AG titer of serum against E. coli, an AG assay was performed (To SC et al., 1984. Infect Immun 43:1-5). E. coli was cultured in LB broth at 37°C for 18 hours, and then treated with 1% formalin for 12 hours. The formalin-treated E. coli was washed three times with PBS, and the OD 600 The serum was diluted in PBS to a value of 0.7 and used as E. coli antigen for measuring the AG titer of the serum. 100 μl of serum serially diluted 2-fold was mixed with 100 μl of E. coli antigen on a 96U plate and incubated at 37°C for 3 hours. The AG titer of the serum against E. coli was expressed as the inverse of the highest dilution of the serum that showed significant agglutination in reaction with the E. coli antigen.

[0071] 1-6. Mucosal-associated infectious pathogen challenge infection and serum treatment To investigate whether piglets administered with PAMI serum could be protected from infection with infectious pathogens, challenge infection with infectious pathogens was performed on piglets born from four primiparous sows (sow 1, sow 2, sow 3, and sow 4). Group A piglets born from sow 1 (control serum-treated piglets (n = 7) and PAMI serum-treated piglets (n = 3)) were used for TGEV-wt1 infection, group B piglets born from sow 2 (control serum-treated piglets (n = 8) and PAMI serum-treated piglets (n = 3)) were used for PEDV-wt1 infection, group C piglets born from sow 3 (control serum-treated piglets (n = 7) and PAMI serum-treated piglets (n = 3)) were used for GAPRV-wt1 infection, and group D piglets born from sow 4 (control serum-treated piglets (n = 8) and PAMI serum-treated piglets (n = 3)) were used for E. coli (K88) infection. Within 1 hour after farrowing, 10 ml of PAMI serum and 10 ml of control serum were orally administered to each piglet. Two days after farrowing, the piglets of each group (piglets of group A, piglets of group B, piglets of group C, and piglets of group D) were separated from the sow and kept in an isolated place with milk replacer (Purina) until the end of the experiment. Three days after farrowing, the piglets of group A were fed TGEV-wt1 (1 × 106 TCID 50 / piglet), and piglets in group B were PEDV-wt1 (1 × 10 6 TCID 50 / piglet), and piglets in group C were GAPRV-wt1 (1 × 10 6 TCID 50 / piglet), and piglets in group D were infected with E. coli (K88) (1 × 10 8 After oral infection with each of the infectious pathogens, the piglets were observed for clinical symptoms for four weeks.

[0072] To investigate the relationship between the time when PAMI serum was administered to newborn piglets and the protective ability of the piglets against infection with infectious pathogens, an infection experiment with infectious pathogens was conducted on piglets from the same litter. Piglets from group E born to sow 5 were divided into three groups [0 hour group (n=3), 12 hour group (n=3), and 18 hour group (n=3)]. 10 ml of PAMI serum was orally administered to the piglets within 1 hour after parturition (0 hour group), within 12 hours after parturition (12 hour group), and within 18 hours after parturition (18 hour group). All piglets that received PAMI serum were infected with PEDV-wt1 (1×10 6 TCID 50 After oral infection with PEDV-wt1 (piglets), the piglets infected with PEDV-wt1 were observed for clinical symptoms for 4 weeks.

[0073] In addition, to confirm whether the calves administered with PAMI serum could be protected from infection with infectious pathogens, an infectious pathogen infection experiment was conducted on the calves. The calves were divided into calf group A for BCoV-wt1 infection experiment [calves administered with control serum (n = 3) and calves administered with PAMI serum (n = 3)], calf group B for BGARV-wt1 infection experiment [calves administered with control serum (n = 3) and PAMI serum (n = 3)], calf group C for E. coli (K88) experimental infection [calves administered with control serum (n = 3) and calf administered with PAMI serum (n = 3)], etc. 200 ml of PAMI serum was orally administered to the calves administered with PAMI serum, and 200 ml of control serum was orally administered to the calves administered with control serum, respectively, within 1 hour after parturition. Three days after parturition, calf groups A, B, and C were orally administered with BCoV-wt1 (1 × 10 6 TCID 50 / calf), BGARV-wt1(1×10 6 TCID 50 / calf) and E. coli (K88) (1 × 10 8 Calves were orally infected with each of the infectious pathogens (100 CFU / calf) and the clinical symptoms of the infected calves were observed for 5 days.

[0074] To examine whether administration of PAMI serum could treat calves with diarrhea caused by infection with BoCoV-wt1, BGARV-wt1, or E. coli (K99), PAMI serum was administered to calves with diarrhea caused by the infectious pathogens. Each group of calves was administered 1×10 6 TCID 50 Calf group D [calf group administered with control serum (n = 3) and calf group administered with PAMI serum (n = 3)] exhibiting diarrhea due to challenge infection with BGARV-wt1 (1 × 10 6 TCID 50 Group E of calves exhibiting diarrhea due to E. coli (K88) (1 × 10 8Calves showing diarrhea due to challenge infection were divided into group F [calf group administered with control serum (n=3) and calf group administered with PAMI serum (n=3)], etc. 200 ml of PAMI serum was intravenously administered to the calves showing diarrhea in the PAMI serum-administered calf group, and 200 ml of control serum was intravenously administered to the calves in the control serum-administered calf group, respectively, and the clinical symptoms of the calves were observed up to 5 days after administration.

[0075] 1-7. Use of PAMI serum at PEDV outbreak farms To investigate whether PAMI serum can prevent infection with mucosal-associated infectious pathogens in the field, PAMI serum was orally administered to newborn piglets at a PEDV outbreak farm (200 sows), and the clinical symptoms of the piglets administered with PAMI serum were observed for 4 weeks. 10 ml of PAMI serum collected from 4 adult pigs infected with infectious pathogens was orally administered to the PAMI serum-administered piglets (n=280), and 10 ml of vaccine control serum collected from adult pigs vaccinated against PEDV was orally administered to the vaccine control serum-administered piglets (n=9) within 1 hour after parturition. The clinical symptoms of the piglets administered with PAMI serum and vaccine control serum were then observed for 4 weeks.

[0076] Example 2. VN and AG titer analysis of sera from adult pigs and cattle orally infected with mucosal-associated infectious pathogens One important factor that determines the protective ability of antibodies against infectious pathogen infection is the neutralizing ability of antibodies against infectious pathogens. Therefore, to examine whether sera collected from adult pigs orally infected with infectious pathogens (TGEV, PEDV, GAPRV, E. coli) and sera from adult cattle orally infected with infectious pathogens (BCoV, BGARV, E. coli) have protective ability against infectious pathogen infection, we investigated the VN and AG titers of the sera against each infectious pathogen.

[0077] In the sera of adult pigs, sera collected from adult pigs before infection with the infectious pathogens [TGEV(L75), TGEV-wt1, PEDV-sm98, KPEDV-9, PEDV-wt1, GAPRV-vac, GAPRV-wt1, and E. coli (K88, K99, and F18)] (control sera) showed negative VN titers against the infectious pathogens [TGEV(L75), TGEV-wt1, PEDV-sm98, KPEDV-9, PEDV-wt1, GAPRV-vac, and GAPRV-wt1] (Fig. 1A and 1B, lanes 1, 3, 5, 7, 9, 11, and 13), respectively, and negative AG titers against E. coli (K88, K99, and F18), respectively (Fig. 1A and 1B, lanes 15, 17, and 19). However, sera collected from adult pigs 26 weeks after infection with infectious pathogens [TGEV(L75), TGEV-wt1, PEDV-sm98, KPEDV-9, PEDV-wt1, GAPRV-vac, GAPRV-wt1, E. coli (K88, K99, F18)] (PAMI sera) were positive for TGEV(L75), TGEV-wt1, PEDV-sm98, KPEDV-9, PEDV-wt1, GAPRV-vac, GAPRV-wt1, E. coli (K88, K99, F18), and PEDV-sm98. The sera collected from adult pigs vaccinated with the PEDV-sm98 and KPEDV-9 vaccines (vaccine control sera) showed high VN titers of 1024 or more against EDV-wt1, GAPRV-vac, and GAPRV-wt1, respectively (Fig. 1A and 1B, lanes 2, 4, 6, 8, 10, 12, and 14), and AG titers of 64 or more against E. coli (K88), E. coli (K99), and E. coli (F18), respectively (Fig. 1A and 1B, lanes 16, 18, and 20). In addition, sera collected from adult pigs vaccinated with the PEDV-sm98 and KPEDV-9 vaccines (vaccine control sera) showed high VN titers of 1024 or more against PEDV-sm98, KPEDV-9, and PEDV-wt1, respectively (Fig. 1C, lanes 2, 4, and 6). However, prevaccinated adult pig sera (control sera) showed negative VN titers against PEDV-sm98, KPEDV-9, and PEDV-wt1, respectively (Fig. 1C , lanes 1, 3, and 5).

[0078] In adult cattle sera, control sera collected from adult cattle prior to infection with infectious pathogens [BCoV-wt1, BCoV-vac, BGARV-wt1, BGARV-vac, and E. coli (K99)] showed negative VN titers against pathogens BCoV-wt1, BCoV-vac, BGARV-wt1, and BGARV-vac, respectively (Fig. 2 , lanes 1, 3, 5, and 7), and negative AG titers against E. coli (K99) (Fig. 2 , lane 9). However, sera collected from adult cattle 12 weeks after infection with the infectious pathogens [BCoV-wt1, BCoV-vac, BGARV-wt1, BGARV-vac, and E. coli (K99)] showed VN titers of 256 or more against BCoV-wt1, BCoV-vac, BGARV-wt1, and BGARV-vac, respectively (Fig. 2, lanes 2, 4, 6, and 8), and AG titers of 64 or more against E. coli (K99), respectively (Fig. 2, lane 10).

[0079] Generally, serum titers against infectious pathogens are a method for measuring the protective ability against infection by infectious pathogens. Therefore, the above experimental results showing that PAMI sera collected from adult pigs and adult cows show high VN titers against mucosa-associated infectious viruses and high AG titers against E. coli suggest that when serum collected from adult pigs and adult cows, i.e., PAMI serum, is administered to piglets and calves, the young piglets and calves administered with PAMI serum may acquire protective antibodies of PAMI serum and be able to protect against infection by mucosa-associated infectious pathogens.

[0080] Example 3. Analysis of the protective ability of piglets and calves orally administered with PAMI serum against pathogen challenge infection To investigate whether PAMI sera induced from adult pigs showing high VN and AG titers against infectious pathogens (PEDV, TGEV, GAPRV, E. coli) could protect piglets from infection with PEDV-wt1, TGEV-wt1, GAPRV-wt, and E. coli (K88), and whether PAMI sera induced from adult cows showing neutralizing and agglutinating activity against infectious pathogens [BCoV-wt1, BGARV-wt1, E. coli (K88)] could protect calves from infection with BCoV-wt1, BGARV-wt, and E. coli (K99), challenge experiments with infectious pathogens were performed on piglets and calves, respectively.

[0081] In the piglets of groups A, B, C, and D, the PAMI serum and the control serum were orally administered to the PAMI serum-treated piglets and the control serum, respectively, within 1 hour after parturition. The piglets were then kept separately from the sows at 2 days of age and orally infected at 3 days of age with TGEV-wt1 in group A, PEDV-wt1 in group B, GAPRV-wt1 in group C, and E. coli (K88) in group D. The piglets were then observed for clinical signs until 28 days of age.

[0082] As a result, in piglets in group A challenged with TGEV-wt1, piglets administered with PAMI serum showed 0% mortality (Fig. 3A, lane 1) and 33% diarrhea incidence (Fig. 3B, lane 1), whereas piglets administered with control serum showed 100% mortality (Fig. 3A, lane 2) and 100% diarrhea incidence (Fig. 3B, lane 2). In piglets in group B challenged with PEDV-wt1, piglets administered with PAMI serum showed 0% mortality (Fig. 3A, lane 3) and 33% diarrhea incidence (Fig. 3B, lane 3), whereas piglets administered with control serum showed 100% mortality (Fig. 3A, lane 4) and 100% diarrhea incidence (Fig. 3B, lane 4). In piglets in group C challenged with GAPRV-wt1, piglets administered with PAMI serum showed 0% mortality (Fig. 2A, lane 5) and 0% diarrhea incidence (Fig. 3B, lane 5), whereas piglets administered with control serum showed 29% mortality (Fig. 3A, lane 6) and 100% diarrhea incidence (Fig. 3B, lane 6). In piglets in group D challenged with E. coli (K88), piglets administered with PAMI serum showed 0% mortality (Fig. 3A, lane 7) and 33% diarrhea incidence (Fig. 3B, lane 7), whereas piglets administered with control serum showed 25% mortality (Fig. 3A, lane 8) and 100% diarrhea incidence (Fig. 3B, lane 8).

[0083] The fact that piglets administered PAMI serum were protected against challenge with infectious pathogens [PEDV-wt1, TGEV-wt1, GAPRV-wt1, E. coli (K88)] raises the possibility that sows before farrowing may have been infected with infectious pathogens [PEDV-wt1, TGEV-wt1, GAPRV-wt1, E. coli (K88)], which may have led to the formation of protective antibodies in the sows. To exclude this possibility, we investigated the VN and AG titers against infectious pathogens in sow sera before and after farrowing. Sera from sows before and after farrowing showed negative VN titers against infectious pathogens [TGEV(L75), TGEV-wt1, PEDV-sm98, KPEDV-9, PEDV-wt1, GAPRV-vac, GAPRV-wt1] (Fig. 4A, B, C, and D, lanes 1–14) and negative AG titers against infectious pathogens E. coli(K88), E. coli(K99), and E. coli(F18) (Fig. 4A, B, C, and D, lanes 15–20), respectively.

[0084] We investigated whether the method of the present invention, which shows protective ability against mucosa-associated infectious pathogens in piglets, can be applied to other mammals, such as cattle. Immediately after parturition, the calves of groups A, B, and C were orally administered PAMI serum, which has neutralizing and agglutinating ability against infectious pathogens [BCoV-wt1, BGARV-wt1, and E. coli (K99)], to the calves administered with PAMI serum, and the calves of group C were orally administered control serum to the calves administered with control serum. At 3 days after birth, the calves of group A were orally infected with BCoV-wt1, the calves of group B with BGARV-wt1, and the calves of group C with E. coli (K99), and clinical symptoms were observed. In group A calves challenged with BCoV-wt1, calves administered with control serum showed 0%, 33%, and 100% diarrhea incidence up to 1, 3, and 5 days after challenge, respectively (Fig. 5, lanes 1, 7, and 13), and calves administered with PAMI serum showed 0% diarrhea incidence up to 1, 3, and 5 days after challenge (Fig. 5, lanes 2, 8, and 14). In group B calves challenged with BGARV-wt1, calves administered with control serum showed 0%, 33%, and 100% diarrhea incidence up to 1, 3, and 5 days after challenge, respectively (Fig. 5, lanes 3, 9, and 15), and calves administered with PAMI serum showed 0% diarrhea incidence up to 1, 3, and 5 days after challenge (Fig. 5, lanes 4, 10, and 16). In group C calves challenged with E. coli (K99), calves administered control serum showed diarrhea incidences of 0%, 67%, and 100%, respectively, up to 1, 3, and 5 days after pathogen challenge (Fig. 5, lanes 5, 11, and 17), while calves administered PAMI serum showed diarrhea incidences of 0% up to 1, 3, and 5 days after pathogen challenge (Fig. 5, lanes 6, 12, and 18).

[0085] These infectious pathogen challenge infection results clearly show that PAMI serum containing protective antibodies with mucosal immunity that can protect against infection by mucosal-associated infectious pathogens can be induced and produced from adult pigs and adult cows (PAMI serum donors), and when the PAMI serum induced and produced in this manner is orally administered to newborn piglets or calves at an early stage, the protective antibodies with mucosal immunity of the PAMI serum can be transmitted to the piglets or calves by passive immunity, thereby protecting the piglets and calves, respectively, from infection by mucosal-associated infectious pathogens.

[0086] Example 4. Time window analysis of PAMI serum on protection of piglets against infectious pathogens In animals such as horses, pigs, and ruminants, which acquire passive immunity through maternal colostrum after parturition, the time of colostrum intake is a factor that affects the level of passive immunity of the offspring. Therefore, to investigate the relationship between the time of oral administration of PAMI serum to newborn mammals and the protective effect of young mammals against infection with infectious pathogens, the following experiment was carried out on piglets.

[0087] In group E piglets, PAMI serum was orally administered to the piglets within 1 hour ("0"), 12 hours ("12"), and 18 hours ("18") after farrowing. Then, all piglets administered with PAMI serum at 3 days after farrowing were challenged with PEDV-wt1 and observed for clinical signs until 28 days of age. As a result, piglets administered with PAMI serum within 1 hour after farrowing did not show any clinical signs such as mortality or diarrhea (Figure 6, lanes 1 and 2). However, piglets administered with PAMI serum within 12 hours after farrowing showed 66% mortality and 100% diarrhea incidence, respectively (Figure 6, lanes 3 and 4), and piglets administered with PAMI serum within 18 hours after farrowing showed 100% mortality and 100% diarrhea incidence, respectively (Figure 6, lanes 5 and 6).

[0088] These results indicate that effective protection against infectious pathogen infections in piglets can be achieved by early oral administration of PAMI serum to newborn piglets, and suggest that the method of protecting young animals against infectious pathogen infections by early oral administration of PAMI serum to young animals is applicable not only to piglets but also to young animals such as horses and ruminants, in which maternal antibodies are transmitted via colostrum.

[0089] Example 5. Therapeutic effect of PAMI serum against diarrhea in calves caused by infection with BCoV-wt1, BGARV-wt1, and E. coli (K99) To investigate whether PAMI serum could be administered intravenously as well as orally to young piglets and calves against infection with mucosa-associated infectious pathogens, an experiment was conducted on calves. Calves with diarrhea caused by BCoV, BGARV, and E. coli (K99) were administered PAMI serum intravenously, and clinical signs in the calves that received the serum were observed.

[0090] In group D calves that developed diarrhea due to challenge infection with BCoV-wt1, the calves in the control serum-treated calf group that received the control serum showed a recovery rate of 0% on days 1, 3, and 5 after serum administration (Fig. 7, lanes 1, 7, and 13). However, the calves in the PAMI serum-treated calf group that received the PAMI serum showed a recovery rate of 0%, 67%, and 100%, respectively, on days 1, 3, and 5 after serum administration (Fig. 7, lanes 2, 8, and 14). In group E calves that developed diarrhea due to challenge infection with BGARV-wt1, the calves in the control serum-treated calf group that received the control serum showed a recovery rate of 0% on days 1, 3, and 5 after serum administration (Fig. 7, lanes 3, 9, and 15). However, the diarrhea symptoms of the calves in the PAMI serum-treated calf group, which were administered PAMI serum, showed recovery rates of 0%, 67%, and 100%, respectively, on days 1, 3, and 5 after serum administration (Fig. 7, lanes 4, 10, and 16). The diarrhea symptoms of the calves in the control serum-treated calf group, which were administered control serum in the calves of group E, which developed diarrhea by challenge infection with E. coli (K99), showed recovery rates of 0%, 33%, and 100%, respectively, on days 1, 3, and 5 after serum administration (Fig. 7, lanes 5, 11, and 17). However, the diarrhea symptoms of the calves in the PAMI serum-treated calf group, which were administered PAMI serum, showed recovery rates of 0%, 33%, and 100%, respectively, on days 1, 3, and 5 after serum administration (Fig. 7, lanes 6, 12, and 18).

[0091] Thus, the method of delivering protective antibodies from PAMI serum intravenously to young animals, unlike oral administration, which is affected by gut closure in newborns and the administration time, can be applied to young animals when necessary, indicating that it can be used not only to prevent infection with infectious pathogens but also for therapeutic purposes.

[0092] Example 6. Analysis of PEDV infection protection ability of PAMI sera at PEDV outbreak farms To investigate whether the PAMI serum of the present invention can effectively prevent infection by infectious pathogens in the field, the effect of the serum was evaluated after orally administering the PAMI serum to newborn piglets in a PEDV outbreak farm (200 sow units). PAMI serum was orally administered to piglets (n=280) in the PAMI serum administration group, and vaccine control serum was orally administered to piglets (n=9) in the vaccine control administration group. The clinical symptoms of piglets administered with PAMI serum and vaccine control serum were observed until 28 days after birth. All piglets administered with PAMI serum showed no mortality (Figure 8A, lanes 1, 3, 5, 7, and 9) or diarrhea symptoms (Figure 8B, lanes 1, 3, 5, 7, and 9) until 28 days after birth. However, piglets administered vaccine control serum collected from adult pigs vaccinated intramuscularly showed mortality rates of 44% at 7 days of age (Fig. 8A, lane 4), 89% at 14 days of age (Fig. 8A, lane 6), and 100% at 21 days of age (Fig. 8A, lane 8). Also, piglets administered vaccine control serum showed 33% diarrhea at 3 days of age (Fig. 8B, lane 2), 100% diarrhea at 7 days of age (Fig. 8B, lane 4), and 100% diarrhea at 14 days of age (Fig. 8B, lane 6).

[0093] These results indicate that the method of the present invention can effectively protect young mammals from infection with mucosal-associated infectious pathogens not only in infectious pathogen challenge infection experiments but also in infectious disease-affected farms, and that PAMI serum containing protective antibodies capable of protecting against infection with mucosal-associated infectious pathogens can be induced and produced regardless of the gender of the PAMI serum donor. In addition, it indicates that serum containing protective antibodies with mucosal immunity capable of protecting against infection with mucosal-associated infectious pathogens, i.e., PAMI serum, can be induced and produced from the PAMI serum donor when the mucosal-associated infectious pathogen is administered to the PAMI serum donor via a mucosal route (oral, intranasal), not via a non-mucosal route (subcutaneous, intradermal, intramuscular, etc.).

[0094] Furthermore, the fact that the examples of the method of the present invention were able to prevent and treat different species of young mammals from infection with a variety of different mucosa-associated infectious pathogens indicates that the method of the present invention for preventing and treating infection with infectious pathogens can be applied not only to pigs and cows used in the examples but also to all mammals having similar immune systems, and can be applied not only to the mucosa-associated infectious pathogens used in the examples but also to all mucosa-associated infectious pathogens that cause diseases in the digestive, respiratory, reproductive, etc. tracts of young mammals.

Claims

1. A method for producing serum containing protective antibodies having mucosal immunity capable of protecting against infection by a mucosal-associated infectious pathogen, the method comprising the step of orally or intranasally administering a mucosa-associated infectious pathogen to an adult pig or adult cow, thereby inducing production of protective antibodies having mucosal immunity capable of protecting against infection by the mucosa-associated infectious pathogen from the adult pig or adult cow, The mucosa-associated infectious pathogen is Porcine epidemic diarrhea virus (PEDV), Transmissible gastroenteritis virus (TGEV), Bovine coronavirus (BCoV), Bovine group A rotavirus (BGARV) or Enterotoxigenic Escherichia coli (ETEC); The method for producing serum, wherein the main protective antibody having mucosal immunity is secretory IgA, which is secreted into the mucosa and can protect against infection by mucosa-associated infectious pathogens.

2. A method for preventing or treating a newborn piglet, newborn calf, young piglet or young calf from infection with a mucosa-associated infectious pathogen, comprising the step of administering to the newborn piglet, newborn calf, young piglet or young calf an effective amount of serum containing protective antibodies having mucosal immunity capable of preventing infection with the mucosa-associated infectious pathogen, produced by the method of claim 1, The mucosa-associated infectious pathogen is Porcine epidemic diarrhea virus (PEDV), Transmissible gastroenteritis virus (TGEV), Bovine coronavirus (BCoV), Bovine group A rotavirus (BGARV) or Enterotoxigenic Escherichia coli (ETEC).

3. A method for producing a veterinary composition for preventing or treating newborn piglets, newborn calves, young piglets or young calves from infection by an infectious pathogen, comprising as an active ingredient a serum containing protective antibodies with mucosal immunity capable of protecting against infection by a mucosal-associated infectious pathogen, produced by the method described in claim 1.

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