EMULSIFIED VACCINE TO OBTAIN FORMULATIONS OF CONCENTRATED IGY IMMUNOGLOBULINS; PROCESSES AND USES FOR THE SAME

Abstract

The present invention relates to a therapy for treating or preventing several diseases in animals, based on the administration of a highly concentrated avian derived immunoglobulins formulation, obtained from the egg yolk from hens previously hiper-immunized with a vaccine formulation comprising infectious agents or toxins antigens, a light mineral oil and a particulate adjuvant.

Claims

1. An emulsified vaccine formulation comprising: (i) antigens, (ii) a light mineral oil and (iii) a particulate adjuvant selected from the group consisting of biodegradable polymer particles, micro-particles or nano-particles.

2. The emulsified vaccine formulation of claim 1, wherein the antigens are derived from one or more different strains of one or more viruses.

3. The emulsified vaccine formulation of claim 2, wherein the virus is the PRRS virus.

4. The emulsified vaccine formulation of claim 3, wherein the PRRS virus isolated strains have an ORF5 gen nucleotide sequence selected from the group consisting of SEQ. ID. NO: 1, SEQ. ID. NO: 2, SEQ. ID. NO: 3, SEQ. ID. NO: 4, SEQ. ID. NO: 5, SEQ. ID. NO: 6 and SEQ. ID. NO: 7.

5. The emulsified vaccine formulation of claim 2, wherein the virus is the Porcine Epidemic Diarrhea (PED) virus.

6. The emulsified vaccine formulation of claim 2, wherein the virus is the White Spot Syndrome Baculovirus Complex virus.

7. The emulsified vaccine formulation of claim 1, wherein the antigens are derived from one or more different strains or one or more bacteria.

8. The emulsified vaccine formulation of claim 7, wherein the antigens are derived from one or more different strains or one or more bacteria selected from the group consisting of: Staphylococcus aureus, Streptococcus agalactiae, Escherichia coli, Corynebacterium pyogenes y Mycoplasma bovis.

9. The emulsified vaccine formulation of claim 7, wherein the antigens are derived from one or more different strains of Actinobacillus pleuropneumoniae.

10. The emulsified vaccine formulation of claim 1, wherein the antigens are derived from one or more different strains of one or more protozoan.

11. The emulsified vaccine formulation of claim 10, wherein the antigens are derived from one or more different strains of one or more protozoan selected from the group consisting of: Eimeria tenella, Eimeria acervulina and Eimeria maxima.

12. The emulsified vaccine formulation of claim 1, wherein the antigens are derived from fungi-produced toxins.

13. The emulsified vaccine formulation of claim 1, wherein the antigens are derived from a mixture of antigens of one or more different strains of one or more viruses, one or more bacteria or one or more protozoan.

14. The emulsified vaccine formulation of claim 1, wherein the antigens are derived from Escherichia coli, Salmonella spp., Clostridium perfringens, Rotavirus spp and Coronavirus spp.

15. The emulsified vaccine formulation of claim 1, wherein the antigens are derived from Escherichia coli, Rotavirus spp. and Coronavirus spp.

16. The emulsified vaccine formulation of anyone of claims 1 to 4, wherein the antigens are derived from viral strains selected by having a serum titer lower than 1:160, assessed by crossed microneutralization using an immunoglobulins pool obtained with the current vaccine.

17. The emulsified vaccine formulation of anyone of claims 1 to 4, wherein it contains an antigens mixture with titers higher than DIE.sub.50% 10.sup.4/ml.

18. The emulsified vaccine formulation of anyone of claims 1 to 17, wherein the light mineral oil is Marcol.

19. The emulsified vaccine formulation of anyone of claims 1 to 18, wherein the particulate adjuvant is Chitosan.

20. The emulsified vaccine formulation of anyone of claims 1 to 19, further comprising an emulsifying agent.

21. The emulsified vaccine formulation of claim 20, wherein the emulsifying agent is selected from the group consisting of: polyoxyethilene monooleate Sorbitan (Tween 80), Sorbitan monooleate Sorbitan (Span 80), or a mixture thereof.

22. The use of the emulsified vaccine formulation of anyone of claims 1 to 4 in preparing a concentrated IgY immunoglobulins formulation useful for the treatment of Porcine Reproductive and Respiratory Syndrome (PRRS).

23. The use of the emulsified vaccine formulation of anyone of claims 1, 2 and 5 in preparing a concentrated IgY immunoglobulins formulation useful for the treatment of Porcine Epidemic Diarrhea (PED).

24. The use of the emulsified vaccine formulation of anyone of claims 1, 2 and 6 in preparing a concentrated IgY immunoglobulins formulation useful for the treatment of the white spot syndrome in shrimps.

25. The use of the emulsified vaccine formulation of anyone of claims 1 and 7 to 8 in preparing a concentrated IgY immunoglobulins formulation useful for the treatment of bovine mastitis.

26. The use of the emulsified vaccine formulation of anyone of claims 1, 7 and 9 in preparing a concentrated IgY immunoglobulins formulation useful for the treatment of Actinobacillus pleuropneumoniae derived infections in porcins.

27. The use of the emulsified vaccine formulation of anyone of claims 1, 10 and 11 in preparing a concentrated IgY immunoglobulins formulation useful for the treatment of coccidiosis in poultries.

28. The use of the emulsified vaccine formulation of anyone of claims 1 and 12 in preparing a concentrated IgY immunoglobulins formulation useful for the treatment of infections caused by thricothecenes.

29. The use of claim 28, wherein the thricothecene is 4-deoxinivalenol (DON).

30. The use of anyone of claims 21 to 29, wherein the emulsified vaccine formulation comprises Marcol as the light mineral oil.

31. The use of anyone of claims 21 to 29, wherein the emulsified vaccine formulation comprises Chitosan as particulate adjuvant.

32. The use of anyone of claims 1 to 31, wherein the emulsified vaccine formulation is subcutaneously administrable to the laying poultries.

33. A process for immunoglobulins extraction, delipidation and concentration from egg yolks from hens hiperimmunized with the emulsified vaccine formulation of claims 1 to 21, wherein it comprises the following steps: a) The yolk is diluted 1:4 or 1:8 (without the egg white) with 0.01% sodium azide and stored under refrigeration during at least 24 hours. b) The supernatant is separated and hydroxypropyl methylcellulose phtalate (HPMCP) is added at 1-15% in a 0.25 ml per each 100 ml of yolk ratio. It is allowed to rest during at least 24 hours. c) The lipid layer formed in the upper part is separated from the solution and the aqueous part is filtrated. d) PEG is added in a 5-30% P/V ratio, mixed and allowed to rest during at least 4 to 12 hours at 4° C. e) It is centrifuged during 20 to 30 minutes and the supernatant is removed. f) The resulting tablet is solubilized with PBS 1× or TRIS buffer, at a volume equivalent to 10% of the original volume.

34. The process for immunoglobulins extraction, delipidation and concentration of claim 33, wherein the egg yolk 1:4 or 1:8 (without the egg white) diluted with 0.01% sodium azide in step (a), is stored under refrigeration for 12 to 24 hours.

35. The process for immunoglobulins extraction, delipidation and concentration of claim 33, wherein in step (d), PEG8000 is added in a 5 to 30% P/V ratio, mixed and allowed to rest for at least 4 to 12 hours at 4° C.

36. An avian derived concentrated IgY immunoglobulins formulation obtained by the process of anyone of claims 33 to 35.

37. The avian derived concentrated IgY immunoglobulins formulation of claim 36, wherein it comprises 0.8% to 5% of IgY immunoglobulins neutralizing the virus causing PRRS.

38. The avian derived concentrated IgY immunoglobulins formulation of claim 37, wherein it comprises 1% of IgY immunoglobulins neutralizing the virus causing PRRS.

39. The avian derived concentrated IgY immunoglobulins formulation of anyone of claims 36 to 39, further comprising 70% to 85% of water.

40. The avian derived concentrated IgY immunoglobulins formulation of anyone of claims 36 to 39, further comprising 0.001% to 0.03% of preservatives.

41. The use of the avian derived concentrated IgY immunoglobulins formulation of anyone of claims 36 to 40 in preparing a medicament for the treatment of Porcine Reproductive and Respiratory Syndrome (PRRS).

42. The use of the avian derived concentrated IgY immunoglobulins formulation of claim 36 in preparing a medicament for the treatment of Porcine Epidemic Diarrhea (PED).

43. The use of the avian derived concentrated IgY immunoglobulins formulation of claim 36 in preparing a medicament for the treatment of white spot syndrome in shrimps.

44. The use of the avian derived concentrated IgY immunoglobulins formulation of claim 36 in preparing a medicament for the treatment of bovine mastitis.

45. The use of the avian derived concentrated IgY immunoglobulins formulation of claim 36 in preparing a medicament for the treatment of Actinobacillus pleuropneumoniae derived infections in porcins.

46. The use of the avian derived concentrated IgY immunoglobulins formulation of claim 36 in preparing a medicament for the treatment of coccidiosis in poultries.

47. The use of the avian derived concentrated IgY immunoglobulins formulation of claim 36 in preparing a medicament for the treatment of intoxications caused by thricothecenes.

48. The use of claim 47, wherein the thricothecene is 4-deoxynivalenol (DON).

49. The use of anyone of claims 41, 42, 44, 45, 47 and 48, wherein the medicament is parenterally administrable.

50. The use of claim 49, wherein the medicament is intramuscularly administrable.

51. The use of anyone of claims 43 and 46, wherein the medicament is orally administrable and with food.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0044] FIG. 1 shows the MNT titration results of different samples of egg collected from poultries immunized with different vaccines. Group A was vaccinated with 2 vaccines made with mineral oil, each containing 3 PRRS virus strains and applied alternatively in a monthly base. Group B was vaccinated with 1 vaccine made with mineral oil and chitosan, which contains 6 different strains of the PRRS virus and applied monthly. Group C was vaccinated with a vaccine made with marcol and chitosan, containing 6 different strains of the PRRS virus and applied monthly.

[0045] These results indicate that the IgY antibodies titers produced by the vaccine containing six PRRS virus strains, and using a formulation with marcol and chitosan, is the one that induces the highest amount of antibodies against the Porcine Reproductive and Respiratory Syndrome.

[0046] FIG. 2 shows the IgY antibodies mean titer in the yolk using three kinds of vaccine formulations. Group A was vaccinated with 2 vaccines made with mineral oil, each of them containing 3 PRRS virus strains and applied alternatively and in a monthly base. Group B was vaccinated with 1 vaccine made with mineral oil and chitosan, which contains 6 different strains of the PRRS virus and was applied monthly. Group C was vaccinated with a vaccine made with marcol and chitosan, which contains 6 different strains of the PRRS virus and was applied monthly.

[0047] FIG. 3 shows the increase in the immunoglobulins titer after applying the concentration process. Yolk immunoglobulins were extracted, one part was quantified by the MNT method and the rest underwent the concentration process. Group A was vaccinated with 2 vaccines made with mineral oil, each containing 3 PRRS virus strains and applied alternatively in a monthly base. Group B was vaccinated with 1 vaccine made with mineral oil and chitosan, containing 6 different strains of the PRRS virus and was applied monthly. Group C was vaccinated with a vaccine made with marcol and chitosan, containing 5 different strains of the PRRS virus and applied monthly.

[0048] FIG. 4 shows the gradual decrease of the antibodies levels in the females of the reproductive herd after receiving the concentrated formulation.

[0049] FIG. 5 shows the monitoring of mortality from week 3 through 26. The groups that were treated with the avian derived IgY immunoglobulins concentrated formulation of the invention are highlighted in a more intense blue box (groups 7 to 16).

[0050] FIG. 6 shows a chart with the mortality percentages of each assessed group, wherein those that were treated with the avian derived concentrated formulation of the invention are highlighted.

DETAILED DESCRIPTION

Laying Poultries Immunization for the Obtention of Immunoglobulins (IgY) Neutralizing Against Infectious Agents or Toxins

[0051] An emulsified vaccine comprised by antigens for producing neutralizing antibodies and not only against a single strain of a determined infectious agent or toxin, but against several strains circulating in the field (epidemiological surveillance) or toxins is produced.

[0052] The above is relevant for the ARN viruses case, as from our research we noted that not all the strains of a circulating virus may be neutralized with antibodies produced from the antigens of an specific strain. The same happens with the vaccines against virus, as when the vaccination occurs with a heterologous strain, the protection is partial.

[0053] In the case of microorganisms, the strains were selected by crossed MNT (microneutralization) tests. This test consists in testing the neutralization level of the immunoglobulins, obtained using a vaccines with defined strains, against the new isolation of the infectious agent. In our work in the lab, we have determined that if the new isolation has titers lower than 1:160 in the MNT, then said strain is considered as a candidate to be included in a new vaccine. Thus, to date our vaccine has contained some strains conferring a wide neutralization spectrum against the strains of the infectious agents circulating in Mexico. Nevertheless, this does not limits the invention and it must not be understood that the invention refers to a vaccine including only the selected strains, as the strains of the infectious agent may vary as the circulation in field of the new strains is being monitored, which may be included lately in the vaccine for maintaining the protection in the animals. The neutralization spectrum of the present invention could also be widened and suited against other strains of infectious agents circulating in other countries or in other regions.

[0054] The strains of the infectious agent may be field isolations obtained from sick animal's serum or tissue. The base for the selection, as above cited, lays on the cross microneutralization test between the different isolated strains with the Ig's contained in the product and produced from the selected strains.

[0055] In the case of the PRRS virus, the cellular line MARC-145 may be replicated and not more than five passes of the working seed are performed, so as to avoid attenuation. The selected antigens are inactivated with 0.01% formalin or by any other method described in the prior art. In our lab, we have determined that the antigens mixture must have each one a minimum DIE.sub.50% 10.sup.4/ml titer.

[0056] Considering the above, the emulsified vaccine formulation for immunizing poultries further comprises antigens from one or more carefully selected strains, a light mineral oil and particulate adjuvant consisting of a biodegradable polymer particles, micro-particles or nano-particles so as to enhance the vaccination, particularly, a lineal polysaccharide comprised of randomly distributed chains of β-(1-4)D-glucosamine (deacetylated units) and N-acetyl-D-glucosamine (acetylated units), such as Chitosan, is preferred.

[0057] The formulation of the water in oil type vaccination may comprise around 50% to 70% and preferably around 60% to 67% of Marcol, around 20% to 40% and preferably around 28% to 35% of water with PRRS virus antigens; around 5% to 15% of each strain of the PRRS virus selected based on the above explained criteria, around 2% to 5% of polyoxyethilene monooleate Sorbitan (Tween 80), around 2% to 8% of Sorbitan monooleate Sorbitan (Span 80) and an adjuvant such as a lineal polysaccharide comprised of randomly distributed chains of β-(1-4)D-glucosamine (deacetylated units) and N-acetyl-D-glucosamine (acetylated units), particularly 4% to 6% of chitosan. For preparing the vaccination, each component is added one by one under a stirring system during 1 to 5 minutes, the chitosan being added at the end.

[0058] The vaccine is applied to the pathogen-free laying poultries, 0.5 ml, once a month, subcutaneously starting on week 8 and until week 60 of age, so as to finally obtain a formulation of immunoglobulins (IgY) neutralizing of the infectious agents or toxins from the egg yolk.

[0059] Particularly, the antibodies level for each of the strains of the infectious agent is assessed by the microneutralization (MNT) of the egg yolks samples collected from the vaccinated flock. The minimum serum titer must comply with the 1:160 criteria.

[0060] After immunization, the eggs are collected from the immunized poultries and undergo an extraction, delipidation and immunoglobulins concentration process from the egg yolk.

Extraction, Delipidation and Immunoglobulins IgY Concentration Process

[0061] The following steps are performed for the extraction and concentration of immunoglobulins IgY neutralizing of infectious agents or toxins from the egg yolk.

[0062] The egg yolk is diluted 1:4 or 1:8 (without the egg white) with 0.01% sodium azide and is stored under refrigeration during at least all day, preferably from 12 to 24 hours. Then, the supernatant is separated and 1 to 15% hydroxypropylmethilcellulose phtalate (HPMCP) is added in a ratio of 0.25 ml per each 100 ml of water. It is allowed to rest for at least 24 hours. The lipids layer formed in the upper part of the solution is separated and the aqueous part is filtered, then PEG is added, preferably 8000, in a 5-30% P/V ratio, mixed and allowed to rest during at least 4 hours, but preferably it is allowed to rest over night at 4° C. Then, it is centrifuged during 20 to 30 minutes and the supernatant removed. The resulting tablet is solubilized with PBS 1× or TRIS buffer, at a volume equivalent to 10% of the original volume.

Therapies with the Concentrated IgY Immunoglobulins Formulation of the Invention

[0063] The formulation resulting from the extraction, delipidation and concentration process of the IgY immunoglobulins neutralizing of infectious agents or toxins, obtained from the egg yolk of hens previously immunized with an emulsified vaccine comprising antigens, a light mineral oil and a particulate adjuvant consisting of biodegradable polymer particles, such as a lineal polysaccharide comprised of randomly distributed chains of β-(1-4)D-glucosamine (deacetylated units) and N-acetyl-D-glucosamine (acetylated units), (chitosan), may comprise: around 0.8% o 5% of concentrated IgY immunoglobulins against pathogens or toxins of the invention, preferably around 1% of concentrated immunoglobulins, around 80% to 90% of water and around 0.001% to 0.03% of preservatives. The treatment dose of the concentrated formulation of the invention may be of 1 ml and of 3 ml at most, which achieves the neutralization of the field varieties of the infectious agents or toxins of the invention, unexpectedly providing a protection of up to 100% in animals, besides regularizing the S/P level in infected farms, as shown below.

[0064] Considering the above, in a first embodiment, the present invention refers to an emulsified vaccine formulation comprising antigens, a light mineral oil, preferably Marcol, and a particulate adjuvant consisting of biodegradable polymer particles, such as a lineal polysaccharide comprised of randomly distributed chains of β-(1-4)D-glucosamine (deacetylated units) and N-acetyl-D-glucosamine (acetylated units), preferably Chitosan. Further, the vaccine formulation of the present invention comprises one or more emulsifiers, such as polyoxyethilene monooleate Sorbitan (Tween 80), Sorbitan monooleate Sorbitan (Span 80), or a mixture thereof.

[0065] The antigens may come from one or more different strains from one or more infectious agents or toxins, such as for example viruses, bacteria or protozoa, more particularly from the PRRS virus, the PED virus, the White Spot Syndrome Baculovirus Complex virus, Rotavirus spp. or Coronavirus spp., and more particularly from the PRRS viruses having a nucleotide sequence of the ORF5 gen selected from the group consisting of SEQ. ID. NO:1, SEQ. ID. NO: 2, SEQ. ID. NO: 3, SEQ. ID. NO: 4, SEQ. ID. NO: 5, SEQ. ID. NO: 6 and SEQ. ID. NO: 7, and also from the Porcine Epidemic Diarrhea (PED) virus. Also, the antigens may come from one or more bacteria, such as Salmonella spp., from the selected ones from the group consisting of: Staphylococcus aureus, Streptococcus agalactiae, Escherichia coli, Corynebacterium pyogenes and Mycoplasma bovis, or also from Actinobacillus pleuropneumoniae. The antigens may further come from one or more protozoan selected from the group consisting of: Eimeria tenella, Eimeria acervuiina and Eimeria maxima, or from toxins produced by fungi, such as thricothecenes and more particularly from 4-deoxynivalenol (DON). Moreover, the antigens may come from one or more different strains of one or more viruses, one or more bacteria, or one or more protozoan, such as for example from antigens derived from Eschericihia coli, Salmonella spp., Clostridium perfringens, Rotavirus spp. and Coronavirus spp. Particularly, the antigens may also come from the group consisting of Escherichia coil, Rotavirus spp. and Coronavirus spp.

[0066] The emulsified vaccine formulation of the invention may contain viral strains selected by having a serum titer lower than 1:160, assessed by crossed microneutralization using an immunoglobulins pool obtained with the current vaccine.

[0067] Likewise, the emulsified vaccine formulation of the invention may contain an antigens mixture with titers higher than DIE.sub.50% 10.sup.4/ml.

[0068] According to the selected antigen or antigens, the present invention also refers to the use of the emulsified vaccine formulation in preparing a concentrated IgY immunoglobulins formulation composition useful for the treatment of the Porcine Reproductive and Respiratory Syndrome (PRRS), the treatment of the Porcine Epidemic Diarrhea (PED), the treatment of the white spot syndrome in shrimps, the treatment of bovine mastitis, the treatment of infections produced by Actinobacillus pleuropneumoniae in porcins, the treatment of coccidiosis in poultry, the treatment of intoxications caused by trycothecenes, particularly wherein the trycothecene is 4-deoxynivalenol (DON), respectively, wherein the emulsified vaccine formulation is subcutaneously administrable to the laying poultries.

[0069] In another embodiment, the present invention refers to a process for the extraction and concentration of immunoglobulins from egg yolk of hens previously immunized with the vaccine formulation of the present invention, characterized by the following steps:

[0070] a) The yolk is diluted 1:4 or 1:8 (without the egg white) with 0.01% sodium azide and stored under refrigeration during at least 24 hours.

[0071] b) The supernatant is separated and hydroxypropyl methylcellulose phtalate (HPMCP) is added at 1-15% in a 0.25 ml per each 100 ml of yolk ratio. It is allowed to rest during at least 24 hours.

[0072] c) The lipid layer formed in the upper part is separated from the solution and the aqueous part is filtrated.

[0073] d) PEG is added, preferably PET 8000, in a 5-30% P/V ratio, mixed and allowed to rest during at least 4 to 12 hours at 4° C.

[0074] e) It is centrifuged during 20 to 30 minutes and the supernatant is removed.

[0075] f) The resulting tablet is solubilized with PBS 1× or TRIS buffer, at a volume equivalent to 10% of the original volume, maintain pH at 7.

[0076] In an additional embodiment, the present invention is aimed to an avian derived concentrated IgY immunoglobulins formulation obtained by the extraction, delipidation and immunoglobulins concentration from egg yolk derived from hens previously hiper-immunized with the emulsified vaccine of the invention. Said avian derived concentrated formulation of the invention may further comprise around 70% to 85% of water and/or around 0.001% to 0.03% of preservatives. Particularly, the concentrated IgY immunoglobulins formulation is characterized in because it comprises 0.8% to 5% of concentrated IgY immunoglobulins neutralizing the virus causing PRRS.

[0077] Further, depending on the selected antigen or antigens, the present invention also refers to the use of the avian derived concentrated IgY immunoglobulins formulation of the invention in preparing a medicament for treating the Porcine Reproductive and Respiratory Syndrome (PRRS), the Porcine Epidemic Diarrhea (PED), the white spot syndrome in shrimps, bovine mastitis, infections produced by Actinobacillus pleuropneumoniae in porcins, coccidiosis in poultry, intoxications caused by trycothecenes, particularly wherein the ones caused by trycothecenes such as 4-deoxynivalenol (DON), wherein the medicament may be parenterally administered, preferably intramuscularly administered, or orally administered and with food, as applicable.

[0078] The following examples show the results of the application of the concentrated formulation of the invention in porcins. Nevertheless, said examples are provided merely as illustration and are not to be considered as limitations.

Example 1

[0079] Two vaccines, each containing three strains of the PRRS virus, were prepared, using a formulation without marcol and chitosan. These vaccines were applied monthly and in an alternate way to the poultries; this means, one vaccine was administered one month and the other one the next month, and so on.

[0080] After the immunization, the eggs from the immunized poultries were collected and underwent an immunoglobulin extraction process from the egg yolk, as above described.

[0081] As a result of the above immunization, a formulation with IgY immunoglobulins neutralizing of the PRRS virus was obtained, but with a low concentration.

[0082] FIG. 1 shows the results of this experiment (group A), wherein the low performance of the IgY immunoglobulins neutralizing the PRRS virus of the previously obtained formulation may be noted, which is incapable of carrying an efficient treatment in porcins suffering the Porcine Reproductive and Respiratory Syndrome, as the use of volumes higher than 5 mL and which protect 10.sup.4 titers is necessary. Further, this experiment was complicated and troublesome due to the handling of two vaccines.

Example 2

[0083] One vaccine containing six different strains of the PRRS virus was prepared, using a formulation without marcol and chitosan, which was administered to the poultries in a monthly immunization schedule.

[0084] After the immunization, the eggs from the immunized poultries were collected and underwent an immunoglobulin extraction process from the egg yolk, as above described.

[0085] As a result of the above immunization, the performance as compared to the experiment of Example 1 was improved, but did not suffice (See FIG. 1, group B).

[0086] As a result of this experiment, we learned that the IgY immunoglobulins neutralizing the PRRS virus performance is inappropriate for carrying out an effective treatment in porcins suffering the Reproductive and Respiratory Syndrome.

Example 3

[0087] Again, one vaccine was formulated, but now using Marcol as the light mineral oil and chitosan as the particulate adjuvant. This time, antigens of 6 strains of the PRRS virus were included in one single vaccine.

[0088] After the immunization, the eggs from the immunized poultries were collected and underwent an immunoglobulin extraction process from the egg yolk, as above described.

[0089] Our results indicate that the antibodies titers increase up to 2 fold regarding that obtained with the formulation of the experiment in Example 2 (see FIG. 2, group C).

[0090] FIGS. 1 and 2 show the results of this experiment, wherein it may be noted that the performance of the IgY immunoglobulins neutralizing of the PRRS virus relevantly increases when a formulation with marcol and chitosan is used, which is appropriate for carrying on an effective treatment in porcins suffering of the Reproductive and Respiratory Syndrome.

Example 4

[0091] In this example, the immunoglobulins finished product was analyzed. Laying poultries were immunized, the eggs of the immunized poultries were collected and the egg yolks underwent an extraction and immunoglobulins concentration process, as above described. The vaccines used to immunize the flocks were the same used in the Examples 1 to 3, but in this case, the size of the batch was increased to 10,000 eggs per flock.

[0092] Our results indicate that the above described concentration process increases the antibodies titers regarding that obtained without said process. The concentration increase goes from 1.6 to 2.1 fold (FIG. 3). As a result of this experiment, we found that the performance of IgY immunoglobulins neutralizing the PRRS virus is appropriate for carrying on an effective treatment in porcins suffering the Reproductive and Respiratory Syndrome, using a lower volume to reach the protection.

Example 5

Immunoglobulins (IgY Type) Application in the Females of the Reproductive Herd

[0093] A longitudinal monitoring of the presence of antibodies against the PRRS virus was performed by assessing the sera of 30 randomly selected sows from a full exploitation cycle farm with 500 sows, located in Western Mexico. This farm produces suckling pigs which are negative to PRRS virus, and which pollute through their productive life.

[0094] The reproductive herd was treated with a 1 ml application of the avian derived concentrated IgY immunoglobulins formulation of the invention, intramuscularly, each 4 months and one repetition within 15 days. Then, weekly applications were made at days 70 and 85 of the gestation.

[0095] The broodstock sows were bimonthly monitored so as to determine the viral movement. Blood samples were taken from the same 30 animals initially selected. For assessment, ELISA technique was used (IDEXX, PRRS X3), which is an indirect way to measure the effectiveness of the treatment with concentrated IgY. The kit measures the levels of IgG type antibodies produced by the pig in response to a viremia. Thus, if the pig is infected, it will produce IgG type antibodies as a response and S/P values higher than the cut-off point will be obtained, but if there is no viremia, the kit will yield S/P values lower than the cut-off point. The cut-off point of ELISA was 0.4 S/P.

[0096] FIG. 4 shows that the treatment applied to the herd gradually reduces the antibodies levels as the bimonthly monitoring is being performed and thus, it is shown that the PRRS virus has been neutralized by the applied immunoglobulins (IgY type), thereby limiting the infection. It is worth to note that, surprisingly, our product neutralized the virus in such a way that after 6 months there was no record of a PRRS virus reinfection. This is shown as a substantial difference between the productive parameters of negative and positive farms was recorded.

Example 6

Concentrated Immunoglobulins (IgY) Application in Suckling Pigs

[0097] The assessment results of the viremia decrease in the PRRS virus clinically affected suckling pigs are shown in this example. The suckling pigs did not show viremia during the maternity stage (first 3 weeks). Nevertheless, there was clinical evidence of viremia around 6-8 weeks of age. This was proved by the PCR technique in real time, wherein 5 pools of viremic animal sera were generated; each pool comprised by sera of 5 different porcins (a total of 25 porcins assessed). As it is shown in the table, the viral load reached titers between 6.16×10.sup.5 and 2.24×10.sup.7.

TABLE-US-00001 Initial Viral Load After Identification Viral Load Treatment Weaning 6 6.16 × 10.sup.5 NEGATIVE Weaning 6 4.98 × 10.sup.6 NEGATIVE Weaning 7-8 5.54 × 10.sup.4 NEGATIVE Weaning 7-8 2.24 × 10.sup.7 NEGATIVE Weaning 7-8 1.21 × 10.sup.7 NEGATIVE

[0098] Once the viremia was confirmed, they were applied with the avian derived concentrated formulation of the invention treatment. A 3 mL dose was intramuscularly applied. In order to assess the efficiency of the treatment, the clinical data were observed and the amount of circulating virus in the porcins was quantified.

[0099] The monitoring results after 10 days of the application of the avian derived concentrated IgY immunoglobulins formulation of the invention showed that 100% of the samples were negative to the presence of PRRS virus genetic material in serum.

[0100] With the obtained results, it is shown that an immunoglobulin schedule application in the broodstock prevents the viral circulation and may neutralize the viremia in weaning suckling pigs with loads of 10.sup.7 viral particles per mL.

Example 7

The Use of Concentrated IgY Immunoglobulines Reduces Suckling Pigs Mortality

[0101] The avian derived concentrated IgY immunoglobulins formulation of the invention was applied to weaned and feedlot-aimed porcins. During all the production time, the mortality under different treatment systems was recorded.

[0102] The studied farm is located at the Midwest region of Mexico and has 1100 sow in its reproductive herd and a weekly production of 500 suckling pigs. The farm has antecedents of PRRS endemic infection, even when several control strategies have been deployed. There is an auto-replace system, which considers the introduction of sows every four months. The broodstock includes the application of the modified live virus vaccine against PRRS every three months.

[0103] In this example, a weekly monitoring was performed and the mortality percentage was calculated both in the weaning (weeks 3 to 9) and in the fattening period (week 10 to 26).

[0104] Two treatments were compared. One based in live vaccine and the other with the avian derived concentrated IgY immunoglobulins formulation of the invention, further, a naive control block was also considered. Each treatment was comprised by different groups which were independently assessed; groups 01 to 06 correspond to conventionally treated suckling pigs (live vaccine) and groups 07 to 16 were those that received two doses of the avian derived concentrated IgY immunoglobulins formulation of the invention, while groups 17, 18 and 19 were not treated.

TABLE-US-00002 Treatment Groups that Treatment type descriptions received treatment Modified live Vaccine application 6, from 01 to 06 virus vaccine at 21 days of age and 3 months. PRRS-specific Two 3 ml each 10, from 07 to 16 avian derived applications, week highly concentrated 1 and 10 after immunoglobulines weaning. (IgY type) Naive control No treatment 3, from 17 to 19

[0105] FIG. 5 shows the mortality monitoring starting on week 3 and until week 26. The groups treated with the avian derived concentrated IgY immunogloblins formulation of the invention are highlighted in a more intense blue box (groups 07 to 16).

[0106] The percentage of mean mortality of the naive groups was the highest one (25.06), followed by the groups that received the conventional treatment (average of 21.2%) and the best treatment was the one of the avian derived concentrated IgY immunoglobulins formulation of our invention, where a mean mortality percentage of 10.27% was obtained. This means, using the IgY immunoglobulins of the invention, a survival of almost 90% of the animals treated is reached, which represents 10% of that reached by the vaccination method.

[0107] FIG. 6 shows a chart of the cumulative mortality percentages of each assessed group, wherein those which were treated with the avian derived concentrated formulation of the invention are highlighted.

[0108] The results from the use of concentrated IgY type immunoglobulins show a relevant decrease in the mortality percentages associated to the PRRS virus. Moreover, it was noted that the health conditions and productivity of these animals was also improved, unlike the groups that received the vaccine. It is important to note that this is the first time that the use of avian immunoglobulins for the treatment of suckling pigs is proved.

[0109] These results are very relevant as the schedule comprising the avian derived concentrated IgY immunoglobulins formulation of the invention not only decreases the mortality percentage, but it also provides the possibility of obtaining other improvements in the productive parameters.

[0110] Thus, it will be appreciated that although the specific embodiments of the invention have been described here in for illustrative purposes, modifications may be made without departing from the nature and scope of the invention. Accordingly, the invention is not limited except by the appended claims.