BRPI1103869A2 - means for laboratory diagnosis of leptospirosis and animal leptospirosis vaccine - Google Patents

Abstract

MATERIAL FOR LABORATORY DIAGNOSIS OF LEPTOSPIROSIS AND ANIMAL LEPTOSPIROSIS VACCINE. Especially from a medium and vaccine that use a pool of inactive leptospira strains (preventing the operator from being endangered) for diagnosing Leptospirosis disease in animals in general (cattle, pigs, horses and canines) as well as for diagnosis in man. The diagnostic medium works with inactive leptospires so that tests can be performed in the field, including culture media for maintaining the battery of leptospires strains; To obtain the antigenic suspensions of leptospires, that is, to perform the work, the leptospires serovars are used, causing Leptospirosis and of greater relevance in humans and bovine, swine, equine and canine animals; The vaccine has a higher immunogenicity power, by obtaining a pure mass of leptospiras integras, without the presence of other proteins from the culture medium itself; the vaccine contains 10 9 9 leptospires per ml of adjuvant (Aluminum Hydroxide); Under these conditions, the vaccine to be obtained may provide vaccine protection for animals between 9 and 12 months against such prevalence among animals.

Description

"MATERIAL FOR LABORATORY DIAGNOSIS OF LEPTOSPIROSIS AND ANIMAL LEPTOSPIROSIS VACCINE" BRIEF PRESENTATION

It addresses the present patent application for an unpublished "LEPTOSPIROSIS AND ANIMAL LEPTOSPIROSIS VACCINE MEDIA FOR LABORATORY DIAGNOSIS", especially a medium and vaccine that use a pool of inactive Ieptospira strains (preventing the operator from being placed in Leptospirosis disease, in animals in general (cattle, pigs, horses and canines) and in humans, as well as vaccine for animal use

and eventually in man.

Technically, the means used for laboratory diagnosis, leads to the vaccine against animal Leptospirosis, which has a higher immunogenicity (antibody formation), protecting more efficiently the animals in general (cattle, pigs, horses and canines).

APPLICATION FIELD

The invention is particularly suitable, in a first aspect, for the diagnosis of Leptospirosis in animals in general (bovine, porcine, equine and canine) as well as in humans (LEPTOSPIROSIS LABORATORY DIAGNOSIS).

In a second aspect, the invention is particularly suitable for

protection with greater efficiency of animals in general (cattle, pigs, horses and canines), using the developed method for laboratory diagnosis, which is also applicable to humans. BACKGROUND OF THE INVENTION Currently there are more than 220 Ieptospira serovars identified by techniques recommended by the World Health Organization (WHO). Among these serovarities, the vast majority are of pathogenic origin (cause disease).

There are several laboratory techniques for the exact determination of

which Ieptospira is present in the herd. Isolation would be ideal, but it is difficult and very costly. In the case of isolation, it may be used as animal urine and blood samples taken at the acute stage of the disease. And it turns out to be very complex, as mentioned above. io In this tuning fork, you have to imagine this kind of process in a

whole herd? That is, for obvious reasons, it is not practical, besides the cost that is very high. Therefore, it is not used in the diagnostic routine of any country in the world.

Thus, it is up to the researchers to confirm the findings of serological tests to guide vaccine producers to include those prevalent serovarities in each country.

What currently exists for the diagnosis of Leptospirosis is the methodology recommended by the WORLD HEALTH ORGANIZATION, comprising microagglutination serology (MAT) which uses a battery of at least 18 reference Ieptospira serovars.

To perform the test it is necessary to use culture of the various leptospiral serovars, dark field microscope, specialized technician, requiring at least 2 (two) hours to obtain the first results. The currently marketed Animal Ieptospirosis Vaccine is obtained from the cultivation of the most relevant Ieptospira strains among animals (cattle, pigs, horses and canines), containing 108 Ieptospiros per ml. This culture of Ieptospires is used to obtain the vaccine and adjuvant is added. The vaccine dose when inoculated in animals produces antibody formation against the leptospires themselves, as well as nonspecific antibodies against proteins in the culture medium used for the cultivation of the leptospires. BACKGROUND ART In the present state of the art is known document Pl 0407032-1,

filed on 01/15/2004 presenting a canine vaccine against Bordetella

/

bronchiseptic. This invention relates to vaccines and methods for the protection of dogs against Bordebella bronchiseptica disease. This invention also relates to combination vaccines and methods for the protection of dogs against canine pathogen disease or disorder, for example infectious tracheobronchitis caused by Bordetella bronchiseptica, distemper caused by distemper virus (cd), infectious canine hepatitis ( ich) caused by canine adenovirus type 1 (cav-1), respiratory disease caused by canine adenovirus type 2 (cav-2), canine parainfluenza caused by canine parainfluenza virus (cpi), enteritis caused by canine coronavirus (ccv), and canine parvovirus (cpv), and Ieptospirosis caused by Leptospira bratislava, Leptospira canicola, Leptospira grippotyphosa, Leptospira cterohaemorrhagiae or Lepbospira pomona. The vaccines of the state-of-the-art invention include a Bordetella bronchiseptica p68 antigen, however it is noted that the document does not provide for a leptospiral culture to be used to obtain the vaccine and adjuvant is added. It is also known from the state of the art document Pl 0516253-0, filed on 23/09/2005, which consists of a multivalent canine vaccine against Leptospira bratislava and other pathogens. The invention relates to vaccines and methods for protecting dogs against disease caused by Leptospira bratislava. This prior art invention also relates to the combination of vaccines and methods to protect dogs against disease or disorder caused by pathogens, for example tracheobronchitis infections caused by Bordetella bronchiseptica, Canine Distemper caused by Canine Distemper (cc) virus, canine hepatitis (ihc) caused by canine adenovirus type 1 (act-1), respiratory disease caused by canine adenovirus type 2 (act-2), canine parainfluenza caused by canine parainfluenza virus (pie), canine coronavirus (eve) enteritis ) and Canine Parvovirus (pvc), and Leptospirosis caused by Leptospira bratislava, Leptospira canicola, Leptospira grippotyphosa, Leptospira icterohaemorrahagiae or Leptospira pomona. Therefore, the present document does not provide for formulation of the mentioned fungus, as in the present patent application.

It is also known from the prior art document P 0602669-9, filed July 7, 2006, which consists of a protein cloning, expression and purification process of Ieptospira sp. by recombinant DNA, process for obtaining polynucleotide sequence with potential vaccine and diagnosis, cloned into or not mammalian expression vectors, for DNA vaccine, composition with potential vaccine base of said protein, composition for diagnosis or for in vitro detection occurrence of Leptospira sp. infection, and kit for in vitro diagnosis of Leptospira sp infection, more specifically this previous invention relates to a given DNA sequence present in the genome of the bacterium Leptospira sp, said gene Iic12892 which encodes a amino acid sequence or a protein that can be used therapeutically as a vaccine or vaccine component, and also diagnostically, for example, as a composition for a kit for early diagnosis of leptospirosis, a protein called Isp29, which constitutes a surface protein of Ieptospira. to be used to detect antibodies in people as well as animals with m leptospirosis disease is suspected, and its utility is directed to both human and animal health.

P 9905780-8, filed on 12/1/1999, consisting of Leptospira vaccine antigens for prevention of Leptospirosis, is also known from the state of the art. Four antigenic preparations are provided, each containing a protein other than Leptospiras. , which may be used immunologically in vaccines against Leptospirosis caused by this microorganism, also provided by the invention are polynucleotides encoding these four proteins and protein-binding antibodies for use in the diagnosis of Leptospirosis.

From foreign documents, the present invention US 2004043391 (A1) deals with four provided antigenic preparations, each of which contains a different Leptospira protein, which can be used immunologically in Leptospirosis vaccines caused by this organism. Also provided in the invention are polynucleotides encoding these four proteins and protein-binding antibodies for use in the diagnosis of Leptospirosis, which does not occur in the formulation object of the present invention.

Document UA 20070000569 (U) provides for a strain of bacterium Leptospira interrogans yez bratislava serological group Australis, Sorovar bratislava, deposited in the official collection of the State and the Institute for Biotechnology Research and Control of Strains of Microorganisms under η 0 334, is used. for the manufacture of inactivated Leptospirosis vaccine in animals and for diagnosis, which does not occur in the formulation object of the present patent application.

KR 20100011770 (A) deals with an antigen protein

recombinant to prevent canine eptospirosis provided for use as a subunit vaccine antigen and massively produce OmpLI LipL41 and protein. The recombinant antigen protein contains Leptospira outer membrane protein and MBP (Maltose-binding protein). The outer membrane protein is OmpLI or LipL41. The recombinant expression vector contains sequence encoding outer membrane and leptospira maltose-binding protein (MBP). One host cell that is transformed with the recombinant expression vector is E.coli deposited in a deposition number KCTC 11363BP or 11019BP KCTC. A canine Leptospirosis diagnostic kit contains a recombinant Leptospira antigen. The vaccine composition for prevention Canine Leptospirosis contains the recombinant antigen protein as an active ingredient, however the prior invention does not provide formulation as in the formulation of the present patent application.

Document RU 2395299 relates to biotechnology. The vaccine contains an active substance and target additives. The active substance is presented with inactivated suspensions of cattle strains of infectious rhinotracheitis virus, SVC (State Collection Virus) η 0 2333, family Herpesviridae, subfamily Alphaherpesviridae, inactivated suspension of cattle diarrhea. The SVC virus η 0 2,336, Flaviviridae family, genus Pestivirus, inactivated suspension of cattle rotavirus strain, SVC η 0 2,337, Reoviridae family, genus rotavirus, inactivated cattle coronavirus strain, SVC η 0 2,338, Coronaviridae1 genus coronavirus with activity of each virus strain not less than 106.5 "ó" 50 per 1 cm3 of vaccine. As said active substance, the vaccine also contains mixed inactivated suspension of bacteria Leptospira interrogans serogroup Pomona lineage cells of VGNKI-6, inactivated suspension of Leptospira bacterial cells interrogates strain tarassovi serogroup VGNKI-4, inactivated suspension of Leptospira bacteria interrogates Grippotyphosa serogroup cells of strain VGNKI-1, the inactivated suspension of Leptospira bacteria interrogans Sejroe serogroup cells of the Hardjo strain and inaktivirovannoj suspensions of Leptospira bacterium cells taken Sejroe strain VGNKI-5 strains taken in proportion equal to the final concentration of each Leptospira strain no less than 270 million microbial cells per Icm3 of the vaccine. The vaccine is intended for the prevention of infectious diseases of cattle accompanied by reproductive disorders of various severities. The vaccine is better than common vaccines in the spectrum of specific protection and efficacy for the tolerant animal group, duration of intense immunity, safety for incalvers and newborn calves, stability of biological properties including antigen and whole immunogen activity. 18 months, which does not occur in the formulation object of the present patent application.

OF THE INVENTION

Considering the importance of Ieptospirosis in our country, the

This application aims to produce, industrialize and commercialize LEPTOSPIROSIS LABORATORY DIAGNOSTIC MEDIA (qualitative test), using a "poof of different strains of leptospires, by the technique of glass plate agglutination, with results in 10 (ten) minutes. As it is a qualitative test, it detects the presence of IgM and IgG It is important to point out that it can be used to perform field tests, not requiring specific equipment, nor a specialized technician to perform the test and several strains of Leptospiras. , used in the micro agglutination test.

The medium is presented with a flask containing antigenic suspension, with different leptospiral serovars of predetermined concentrations of the leptospires most relevant to each animal species.

Therefore, it can be stated that the proposal of the present invention is the standardization of a rapid test, which will bring benefits as a result response (10 minutes) with a much lower cost when compared to the traditional method (does not put the operator at risk, as it works with inactive leptospires), the tests can be performed in the field, without the need of sophisticated equipment, specialized technicians and culture media for the maintenance of the battery of leptospires strains.

The currently marketed Animal Ieptospirosis Vaccine is obtained from the cultivation of the most prevalent leptospires strains among the cited animals, containing 108 leptospires per ml. The leptospiral culture thus obtained, containing the culture medium used, will be used to obtain the vaccine, added with the adjuvant. The vaccine dose when inoculated in animals will produce antibody formation against the leptospires themselves, as well as nonspecific antibodies against proteins in the culture medium used for the cultivation of the leptospires.

In view of this, the inventor proposes a new methodology for obtaining the animal leptospirosis vaccine, which consists of a vaccine with greater immunogenicity, from obtaining a pure mass of intact leptospires, from the centrifugation of leptospires culture, neglecting the supernatant (culture medium used), therefore not containing the presence of other proteins from the culture medium itself, when compared to the vaccines currently produced and marketed.

It is also proposed to obtain a vaccine containing 109 Ieptospires per ml of adjuvant (Aluminum Hydroxide). Under these conditions, the vaccine to be obtained may give vaccination protection in animals between 9 to 12 months against such infection (another advantage over vaccines marketed that gives protection for 06 months). It is proposed to prepare the vaccine using strains of Ieptospiras more prevalent among animals, from scientific references. Separately grown Ieptospira strains, after centrifugation, should be washed in NaCl solution (sodium chloride), then pooled (all Ietospira cultures together), and resuspended in 0.9% NaCl solution. , containing from 0.1 to 1.0% formalin, adjusting the concentration to 109 Ieptospiras per ml, using aluminum hydroxide solution as an adjuvant. DETAILED DESCRIPTION OF THE INVENTION

The present invention with respect to the LEPTOSPIROSIS LABORATORY DIAGNOSTIC MEASURE, aims at a standardization of a rapid test that will have the benefits of a response of results in 10 (ten) minutes with a much lower cost when compared to the traditional method mentioned above, It does not put the operator at risk, because working with inactive Ieptospiras, the tests can be performed in the field without the need for sophisticated equipment, specialized technicians and culture media to maintain the battery of leptospira strains.

To obtain the antigenic suspensions of leptospires (MEDIUM and VACCINE), that is, to perform the work, Ieptospiras1 serovars that cause Leptospirosis are used and most relevant in humans and cattle, pigs, horses and canines: L. icterohaemorrhagiae; L. copenhageni; L. canicola; L. pomona; L. grippotyphosa; L. hardjo and L. wolffi. These leptospiral serovars were obtained from the Leptospirosis Reference Center at FIOCRUZ / RJ. These strains of Leptospiras are kept in semi-solid medium with peaks every three months.

Leptospira samples are grown in liquid medium. This liquid medium is prepared in a first phase, ten (10) times concentrated, fully sterile and can be conditioned at room temperature. The culture medium thus prepared contains for each 1000 ml 1 Og of Disodium Phosphate; 0.3g Monopotassic Phosphate; 1, Og of Sodium Chloride; 0.25g of Ammonium Chloride; from 0.1 mg to 0.8 mg of thiamine; 0.1g to 0.7g Sodium Pyruvate; 0.1 ml Glycerin; Calcium Chloride and Magnesium Chloride 0.5mg to 15mg; 4mg ZnSO4 ■ 7H2oi Bovine Hermitages between 0.1 to 1.4mg; 2mg Cyanocobalamin and Tween-80 between 0.7 to 1.5ml. The culture medium should be sterilized by filtration system, having the final pH 6.0 to 8.7 before use. Cultivation of leptospira strains will be performed in screw-capped glass tubes containing 10 ml of culture medium, adding 2 to 10% of Ieptospira culture and maintained at 15-30 ° C for up to 10 (10 ) days for growth.

After days of incubation, the tubes showing growth

bacterial, are examined for purity, density and the presence or absence of self-agglutination. Tubes with satisfactory bacterial growth will be used for a second pass, inoculated into 100 ml flasks of the same culture medium and incubated with shaking at 15 - 3 ° C for further growth for one week. After this period, the assessment is again made for t 11/15

to purity, density and self-agglutination. The flasks with satisfactory aspects to the visual examination (homogeneous growth with turbidity in the superior third of the tube and without the presence of lumps) will be inoculated in a 2 (two) liter erlenmeyer, containing between 200 ml to 100 ml of the same culture medium and incubated with stirring, the temperature between 15 - 30 ° C. This procedure will be used for the 7 (seven) different Ieptospiras serovars mentioned above. After this period, proceed to culture evaluation for purity, growth density and self-agglutination. With satisfactory conditions, inactivate the additional formaldehyde culture and verify the absence of live Ieptospira by 450-fold dark field microscopy and centrifugation at 5000 rpm for 30 to 60 minutes.

After centrifugation and discarding the supernatant, the Ieptospiras are washed with brine (0.15 M NaCl) and finally resuspended in small volume of 0.01 M buffered saline (PBS). The bacteria thus prepared will be left overnight. at 4 ° C for stabilization. For each leptospira strain, antigenic suspensions should be obtained, used in the preparation of the medium used for the serological diagnosis of animal and human Ieptospirosis, as well as in obtaining the animal Ieptospirosis vaccine.

After the stabilization period, in the preparation of the medium for laboratory diagnosis, determine the concentration of the suspension of Ieptospiras in photocholorimeter with wavelength between 230nm to 850nm, between 10 and 38% Transmittance, using as a diluent, a solution containing NaCI, Glycerol and Formalin. The suspensions thus obtained will be stored in rubber stoppered glass vials for one week in a 4 ° C refrigerator for stabilization, for subsequent evaluation with the positive and negative Leptospirosis control sera.

In performing the quality control test (sensitivity and specificity) of antigenic suspensions of leptospires, human and animal sera considered suspicious for Leptospirosis are performed, all previously evaluated by microscopic serum agglutination (WHO's Gold1 test) and submitted to the test. macroscopic agglutination. For this purpose a glass plate with divisions will be used.

The present invention, with regard to the ANIMAL LEPTOSPIROSIS VACCINE, aims at a vaccine with greater immunogenicity, by obtaining a pure mass of whole leptospires, without the presence of other proteins from the culture medium, when compared. with the vaccines currently produced and marketed.

It is also proposed to obtain a vaccine containing 109 leptospires per ml of adjuvant (Aluminum Hydroxide). Under these conditions, the vaccine to be obtained may provide vaccine protection for animals between 9 and 12 months against such prevalence among animals, based on scientific references.

Separately grown leptospiral strains after centrifugation should be washed in NaCl solution (Sodium Chloride), then pooled (all leptospiral cultures together) and resuspended in 0.9% NaCl solution. containing 0.1 to 1.0% formalin, adjusting the concentration to 109 leptospires per ml using Aluminum Hydroxide solution as an adjuvant.

The effectiveness of leptospires inactivation by formalin is checked by inoculating the vaccine in liquid culture medium, maintained at 15 - 30 ° C. Vaccine sterility testing is performed by inoculating 0.5ml to 2.5ml of the vaccine in 5 0.01 to 20.0 ml of Thioglycolate culture medium.

The test to determine the potency of the vaccine (immunogenicity) will be performed in hamster (animal 21 days old), after determining the LD50, according to Reed & Muench method (standard way to reach the LD50 result).

After carrying out the quality control of the batches of Ieptospiras vaccines produced ("pool"), they will be packed and released for commercialization.

As previously described and repeated below obtaining follows the following steps:

- Leptospira serovars, which cause Leptospirosis and are most relevant to humans and bovine, porcine, equine and canine animals, are used for this work: L. icterohaemorrhagiae; L. copenhageni; L. canicola; L. pomona; L. grippotyphosa; L. hardjo and L. wolffi. These leptospiral serovars were,

obtained from the Leptospirosis Reference Center, FIOCRUZ / RJ. These strains of Leptospiras are kept in semi-solid medium with peaks every three months.

- Samples of leptospires are grown in liquid medium. This liquid medium is prepared in a first phase, ten (10) times concentrated, fully sterile and can be conditioned at room temperature. The culture medium like that

elaborated, contains for each 1000 ml, 10g Disodium Phosphate; 0.3g Monopotassic Phosphate; 1.0g Sodium Chloride; 0.25g of Ammonium Chloride; from 0.1 mg to 0.8 mg of thiamine; 0.1g to 0.7g Sodium Pyruvate; 0.1ml Glycerin; Calcium Chloride and Magnesium Chloride 0.5mg to 15mg; 4mg ZnSO4.7H20; Bovine Hermitages between 0.1 to 1.4mg; 2mg Cyanocobalamin and Tween-80 between 0.7 to 1.5ml. The culture medium should be sterilized by filtration system, having the final pH 6.0 to 8.7 before use. Cultivation of leptospira strains is performed in screw-capped glass tubes containing 10ml of culture medium, adding 2 to 10% of Ieptospira culture and maintained at 15-30 ° C for up to 10 (ten) days for growth.

- After days of incubation, tubes showing bacterial growth will be examined for purity, density and the presence or absence of self-agglutination. Tubes with satisfactory bacterial growth will be used for a second pass, inoculated into 100 ml flasks of the same culture medium and incubated with shaking at 15 - 3 ° C for further growth for one week. After this period, the purity, density and self-agglutination are again evaluated. Vials with satisfactory visual examination aspects (homogeneous growth with turbidity in the upper third of the tube and no lumps) will be inoculated in a 2 (two) liter conical flask containing between 200ml to 1000ml of the same culture medium and incubated with stirring, the temperature between 15 - 30 ° C. This procedure is used for the 7 (seven) different leptospira serovars mentioned above. After this period, proceed to culture evaluation for purity, growth density and self-agglutination. With satisfactory conditions, inactivating the additional formaldehyde culture to verify the absence of live leptospires by 450-fold dark field microscopy, centrifugation is then performed at 5000 rpm for 30 to 60 minutes.

- After centrifugation and discarding the supernatant, the leptospires will be washed with brine (0.15 M NaCl) and finally resuspended in small volume of 0.01 M buffered saline (PBS). overnight at 4 ° C for stabilization. For each Ieptospira strain should be performed individually, the entire process described above. The dose of inoculated vaccine comprises: 1. Large animals: 02 to 03 ml of vaccine. 2.Small animals: 01 ml of vaccine.

Claims (20)

1.) "LEPTOSPYROSIS LABORATORY DIAGNOSTIC MEANS", which promotes the standardization of a rapid test that will have the benefits of a ten (10) minute response, CHARACTERIZED by working with inactive Ieptospiras, so that testing can be performed. in the field, including culture media for battery maintenance of leptospira strains; To obtain the antigenic suspensions of leptospires, that is, to perform the works, the leptospiros serovars are used, causing Leptospirosis and of greater relevance in humans and bovine, swine, equine and canine animals; Leptospiras strains are kept in semi-solid medium with peaks every three months. 2.) "LEPTOSPIROSIS LABORATORY DIAGNOSTIC MEDIA" according to Claim 1, characterized in that the leptospires are preferably selected from: L. icterohaemorrhagiae; L. copenhageni; L. canicola; L. pomona; L. grippotyphosa; L. hardjo and L. wolffi. 3.) "LEPTOSPIROSIS LABORATORY DIAGNOSTIC MEDIA" according to claims 1 and 2, characterized in that the leptospiral samples are grown in liquid medium. 4.) "LEPTOSPYROSIS LABORATORY DIAGNOSTIC MEDIA" according to claim 1 or 3, characterized in that the liquid medium is prepared in a first stage, ten (10) times concentrated, fully sterile, conditioned at room temperature. 5.) "LEPTOSPYROSIS LABORATORY DIAGNOSTIC MEDIA" according to claim 1 or 4, characterized in that the culture medium contains for each 1000 ml, 1, Og Disodium Phosphate; 0.3g Monopotassic Phosphate; 110g Sodium Chloride; 0.25g of Ammonium Chloride; from 0.1 mg to 0.8 mg of thiamine; 0.1 g to 0.7 g Sodium Pyruvate; 0.1 ml Glycerin; Calcium Chloride and Magnesium Chloride 0.5mg to 15mg; 4mg ZnSO4.7H20; Bovine Hermitages between 0.1 to 1.4mg; 2mg Cyanocobalamin and Tween-80 between 0.7 to 1.5ml. 6.) "LEPTOSPYROSIS LABORATORY DIAGNOSTIC MEDIA" according to claim 1 or 5, characterized in that the culture medium is sterilized by a filtration system having a final pH 6.0 to 8.7 prior to use. 7.) "LEPTOSPYROSIS LABORATORY DIAGNOSIS MEDIA" means "LEPTOSPIROSIS LABORATORY DIAGNOSIS MEDIA" according to claim 1 or 6, after days of incubation, tubes showing bacterial growth are examined for purity, density and presence or absence of self-agglutination. 8.) "LEPTOSPIROSIS LABORATORY DIAGNOSIS MEDIA", "LEPTOSPIROSIS LABORATORY DIAGNOSIS MEDIA" according to claim 1 or 7, CHARACTERIZED BY Tubes with satisfactory bacterial growth are used for a second pass, being inoculated into vials with 100 ml of the same culture medium and incubated with shaking at 15 - 3 ° C for growth for a further week; after this period, the purity, density and self-agglutination are again evaluated; the vials with satisfactory aspects to the visual examination (homogeneous growth with turbidity in the superior third of the tube and without the presence of lumps) will be inoculated in a 2 (two) liter erlenmeyer, containing between 200 ml to 100 ml of the same culture medium and incubated with stirring, the temperature between 15 - 30 ° C. 9.) "LEPTOSPIROSIS LABORATORY DIAGNOSTIC MEDIA" according to claim 1, 2 or 8, characterized in that the procedure is used for the seven (7) different Ieptospira related serovars. 10.) "LEPTOSPYROSIS LABORATORY DIAGNOSTIC MEDIA" according to claim 1, 2 or 8, characterized in that after this period the culture is evaluated for purity, growth density and self-agglutination; presenting satisfactory conditions, inactivating the additional formaldehyde culture and verification of the absence of live Ieptospiras by dark field microscopy with a 450-fold increase, and centrifugation at 5000 rpm for 30 to 60 minutes. 11.) "LEPTOSPYROSIS LABORATORY DIAGNOSTIC MEDIA" according to Claim 1 or 10, characterized by, after centrifugation and discarding the supernatant, the Ieptospiras are washed with saline (0.15M NaCl) and finally suspended in small volume of 0.01 M buffered saline (PBS); The bacteria thus prepared will be left overnight at 4 ° C for stabilization. 12.) "LEPTOSPYROSIS LABORATORY DIAGNOSTIC MEDIA" according to claim 1 or 11, characterized in that for each leptospira strain the antigenic suspensions used in the preparation of the medium used for the serological diagnosis of animal eptospirosis should be obtained. as well as in obtaining the animal Ieptospirosis vaccine. 13.) "LEPTOSPIROSIS LABORATORY DIAGNOSTIC MEDIA" according to claim 1 or 12, characterized by, after the stabilization period, in the preparation of the medium for laboratory diagnosis, the concentration of the Ieptospira suspension in photocolorimeter with wavelength between 230nm and 850nm, between 10 and 38% Transmittance, using as a diluent solution containing NaCl1 Glycerol and Formol; The suspensions obtained are stored in glass bottles with rubber stoppers for one week in a refrigerator at 4o C for stabilization, for later evaluation with the positive and negative Leptospirosis control sera. 14.) "ANIMAL LEPTOSPIROSIS VACCINE", CHARACTERIZED FOR A HIGHER IMMUNOGENITY, from obtaining a pure mass of Ieptospiras integras, without the presence of other proteins from the culture medium itself; The vaccine contains 109 Ieptospires per ml of adjuvant (Aluminum Hydroxide); Under these conditions, the vaccine to be obtained may give vaccine protection in animals between 9 and 12 months against such prevalence among animals and possibly in humans. 15.) "ANIMAL LEPTOSPIROSIS VACCINE" according to claim 14, characterized in that the separately cultivated strains of Ieptospira, after centrifugation, are washed in NaCl solution (Sodium Chloride) and then mixed ("pool" - all Ieptospira cultures together) and resuspended in 0.9% NaCl solution containing 0.1 to 1.0% formalin, adjusting the concentration to 109 Ieptospiras per ml using Aluminum Hydroxide solution as an adjuvant. 16.) "ANIMAL LEPTOSPIROSIS VACCINE" according to claims 14 and 15, characterized in that the efficacy of Ieptospira inactivation by formalin is checked by inoculating the vaccine in liquid culture medium maintained at 15 - 30 ° C. 17.) "ANIMAL LEPTOSPIROSIS VACCINE" according to claim 14 or 15, characterized in that the sterility test of the vaccine is performed by inoculating 0.5 ml to 2.5 ml of the vaccine into 5.0 ml to 20 ml. Thioglycolate culture medium. 18.) "ANIMAL LEPTOSPIROSIS VACCINE" according to claim 14 or 15, characterized in that the test to determine the potency of the vaccine (immunogenicity) is to be performed on a 21 day old animal after determining the LD50. 19.) "ANIMAL LEPTOSPIROSIS VACCINE" according to claims 14, 15, or 16 to 18, CHARACTERIZED BY after performing quality control of batches of the produced Ieptospira vaccines ("pool"), they will be packed and released for marketing. 20.) "ANIMAL LEPTOSPIROSIS VACCINE" according to claims 14, 15, or 16 to 19, characterized in that the inoculated vaccine dose comprises: 1. Large animals: 02 to 03 ml of the vaccine; 2.Small animals: 01 ml of vaccine.