Vaccine for protection against leptospira jaundice hemorrhagic serotypes

Abstract

The present invention relates to providing protective immunity to canine animals against leptospira jaundice hemorrhagic serotypes with vaccines comprising non-jaundice hemorrhagic leptospira serotypes.

Description

Technical Field

[0001] The present invention generally relates to immunogenic Leptospira compositions capable of eliciting a cross-protective immune response in animals, particularly canines. The present invention also relates to methods of providing animals, particularly canines, with a cross-protective immune response against Leptospira Icterohaemorrhagiae. Background Art

[0002] Leptospirosis is a global zoonotic disease caused by a Gram-negative spirochete of the genus Leptospira. Leptospirosis is common in humans, dogs, horses, cattle, and wildlife. Dogs are highly susceptible to infection and develop symptoms upon infection, including high fever, jaundice, hemorrhagic diathesis, abortion, and death within a few days. In addition, dogs may develop chronic symptoms including liver, kidney, and gastrointestinal symptoms. Domestic dogs live in close association with humans and livestock and can serve as a sentinel species for human environmental risks.

[0003] Seroprevalence studies have shown that the main and most prevalent serogroups in dogs are serogroups Canicola, Icterohaemorrhagiae, Australis and Grippotyphosa, and in addition to these serogroups there is the related Pomona serogroup in the United States and Hebdomadis serogroup in Japan. A range of vaccines against canine leptospirosis has been licensed in Europe, the earliest being a bivalent vaccine containing serotype Canicola and either Icterohaemorrhagiae or Copenhagen. Recently, quadrivalent and trivalent vaccines have been introduced into the European market, which also contain serotypes of either Grippotyphosa and Icterohaemorrhagiae or Grippotyphosa alone.

[0004] Literature reviews, such as the "Leptospirosis Fact Sheet" (World Health Organization Southeast Asia Regional Office, 2009) partially indicate that animals and humans can be immunized, but protection is mainly serotype specific. The lack of cross-protection is not surprising, especially in view of the significant genetic / genomic (e.g. gene organization in the lipopolysaccharide biosynthesis (rfb) locus) differences between serotypes (Pena-Moctezuma, A. et al, 2001 FEM S Immunology and Medical Microbiology 31 (2001) 73-81).

[0005] Thus, there is little evidence of "cross protection" between serotypes in canines. Cross protection or heterologous protection is defined herein as protection against a Leptospira serotype by administering an effective amount of a different serotype (e.g., protection against an icterohaemorrhagiae serotype by administering a homologous effective amount of a non-icterohaemorrhagiae serotype such as a Portland-vere serotype, a Dadas serotype, a Copenhagen serotype, and / or a Bratislava serotype).

[0006] EP2874653 discloses a method of providing protection against Leptospira interrogans Copenhagen serotypes using a multivalent vaccine comprising non-Copenhagen serotypes, canine serotypes, Grippotyphosa serotypes and Pomona serotypes.

[0007] This is the first disclosure of a Leptospira vaccine in dogs that provides protection against serotypes not present in the vaccine. The patent was published in January 2014 and no cross protection to additional Leptospira serotypes has been reported in dogs since then.

[0008] The article by Bouvet et al. (Veterinary Immunology and Immunopathology 219 (2020) 109985) is a scientific publication of the same findings and experiments described in EP2874653. It is confirmed in the article that the bacterin Leptospira vaccine only provides protection against the same serotype. In addition, it is disclosed that there is only very limited evidence for protection against other serotypes within the same serogroup, and only in rodent models. Evidence of the effectiveness of other Leptospira serotypes that provide cross-protection in dogs has not yet been shown in the prior art.

[0009] Therefore, there is still a need to protect animals, especially dogs, from Leptospira serotypes. Since leptospirosis is also a potential zoonotic problem for humans, it is beneficial to have protection against additional Leptospira serotypes, and preferably using vaccines that have already been used. Therefore, it is beneficial to protect animals, especially dogs, from heterologous Leptospira serotypes. Until the present disclosure, methods of using non-icterohaemorrhagic serotypes to provide protection against Leptospira icterohaemorrhagic serotypes were unknown. Summary of the invention

[0010] The object of the present invention is to provide a method for protective immunity against a first Leptospira serotype, comprising the step of administering another Leptospira serotype of a different serotype compared to the first Leptospira serotype. In case the another Leptospira serotype is a combination of Leptospira serotypes (e.g. a combination / multivalent vaccine), the another Leptospira serotype must not comprise a Leptospira serotype of the same serotype as the first Leptospira serotype for which protective immunity is sought.

[0011] In one embodiment of the invention and / or embodiments thereof, the method provides protective immunity against a Leptospira icterohaemorrhagiae serotype and comprises the step of administering to an animal in need thereof an immunologically effective amount of a non-Leptospira icterohaemorrhagiae serotype.

[0012] In another embodiment, the method provides protective immunity against Leptospira icterohaemorrhagiae serotype by administering a combination / multivalent Leptospira vaccine. In a specific embodiment, the multivalent Leptospira vaccine comprises Leptospira serotypes Portland-vere, Dadas, Copenhagen, and Bratislava. Nobivac L4 (MSD Animal Health) is such a multivalent vaccine.

[0013] For a skilled person with current prior art knowledge in the field of leptospirosis, it is unexpected and surprising that a vaccine without Leptospira serotype icterohaemorrhagiae elicits protective immunity against Leptospira serotype icterohaemorrhagiae in canines. It is particularly surprising that a vaccine comprising Leptospira serotypes Portland-vere, Dadas, Copenhagen and Bratislava provides immunity against Leptospira serotype icterohaemorrhagiae.

[0014] The examples show that vaccines comprising Leptospira serotypes Portland-vere, Dadas, Copenhagen and Bratislava (eg Nobivac L4) provide protective immunity against Leptospira serotype icterohaemorrhagiae. DETAILED DESCRIPTION

[0015] The present invention encompasses methods of preventing or reducing infection caused by a specific serotype of Leptospira by administering a vaccine containing one or more different serotypes of Leptospira. The present invention relates to a vaccine composition comprising a Leptospira serotype icterohaemorrhagiae for providing protective immunity to a canine against a Leptospira serotype icterohaemorrhagiae. The vaccine does not contain a Leptospira serotype icterohaemorrhagiae.

[0016] In one embodiment of the invention and / or its embodiments, the invention provides a method of eliciting a protective immune response against a Leptospira icterohaemorrhagiae serotype in an animal, comprising the step of administering to the animal an effective amount of a non-Leptospira icterohaemorrhagiae serotype. "Non-icterohaemorrhagiae serotype" refers to a Leptospira serotype that is different from the icterohaemorrhagiae serotype. It may be from the same or different serogroup as the icterohaemorrhagiae serotype (icterohaemorrhagiae serogroup).

[0017] In one embodiment of the invention and / or embodiments thereof, the non-icterohaemorrhagiae Leptospira serotype belongs to the serogroup icterohaemorrhagiae. In a specific embodiment, the non-icterohaemorrhagiae Leptospira serotype is a Copenhagen serotype.

[0018] In one embodiment of the invention and / or embodiments thereof, the non-icterohemorrhagic Leptospira serotype is delivered as part of a multivalent / combination vaccine. In a specific embodiment, the non-icterohemorrhagic Leptospira serotype is a serotype selected from the group consisting of Leptospira Portland-vere serotype, Dadas serotype, Copenhagen serotype and Bratislava serotype, preferably Copenhagen serotype and one or more serotypes selected from the group consisting of Leptospira Portland-vere serotype, Dadas serotype and Bratislava serotype.

[0019] In one embodiment of the invention and / or embodiments thereof, the vaccine comprises a Leptospira Portland-vere serotype. In another embodiment, the vaccine comprises a Leptospira Dadas serotype. In another embodiment, the vaccine comprises a Leptospira Copenhagen serotype. In another embodiment, the vaccine comprises a Leptospira Bratislava serotype. In another embodiment, the vaccine comprises a Leptospira Portland-vere serotype, a Dadas serotype, a Copenhagen serotype, and a Bratislava serotype.

[0020] Suitably, the Leptospira Portland-vere strain is Ca-12-000. Suitably, the Leptospira Dadas strain is Gr-01-005. Suitably, the Leptospira Copenhagen strain is Ic-02-001. Suitably, the Leptospira Bratislava strain is As-05-073.

[0021] Suitably, the vaccine provides canines with protective immunity against Leptospira serovars icterohaemorrhagiae, Australasia and Gastroenteritis Typhimurium.

[0022] As used herein, the term "vaccine" refers to a pharmaceutical composition comprising at least one immunologically active component that mediates an immunogenic response in an animal and a pharmaceutically acceptable carrier. In this specification, a vaccine may also be referred to as an immunogenic composition. A vaccine may additionally include further components conventional to pharmaceutical compositions. In this specification, immunogenic compositions and vaccines are used interchangeably.

[0023] Typically, an "immunogenic response" includes, but is not limited to, one or more of the following effects: the production or activation of antibodies, B cells, helper T cells, suppressor T cells, and / or cytotoxic T cells and / or gamma-delta T cells specific for one or more antigens included in the composition or vaccine of interest. Suitably, the target will exhibit a therapeutic or prophylactic immunogenic response, thereby enhancing resistance to new infections and / or reducing the clinical severity of the disease. Such protection will be demonstrated by a reduction or absence of clinical symptoms normally displayed by an infected host, a faster recovery time and / or a reduced duration or bacterial titer in the tissues or body fluids or excreta of the infected host.

[0024] “Clinical signs” or “clinical symptoms or clinical reactions” of leptospirosis include: loss of appetite, slow or stiff gait, weakness, vomiting, diarrhea, decreased skin turgor (indicating dehydration), pale or yellow mucous membranes (conjunctiva or oral mucosa) and hunched back.

[0025] As used herein, "reduction in the incidence and / or severity of clinical signs" or "reduction in the incidence and / or severity of clinical signs" refers to reducing the number of infected animals in a group, reducing or eliminating the number of animals showing clinical signs of infection, or reducing the severity of any clinical signs present in an animal, compared to infection with a wild-type pathogen. For example, such clinical signs include body temperature, general health score, loss of appetite, slow or stiff gait, weakness, vomiting, diarrhea, decreased skin turgor (indicating dehydration), pale or yellow mucous membranes (conjunctiva or oral mucosa), and hunched back. Suitably, these reductions are at least 10% in animals receiving the vaccine composition of the invention compared to animals infected when not receiving the vaccination.

[0026] As used herein, "pharmaceutically acceptable carrier" or "pharmaceutical carrier" includes any and all excipients, solvents, growth media, dispersion media, coatings, adjuvants, stabilizers, diluents, preservatives, inactivators, antimicrobial agents, antibacterial agents and antifungal agents, isotonic agents, adsorption delay agents, etc. These ingredients include those that are safe and suitable for veterinary applications. Suitably, stabilizers for use in the present invention include stabilizers for lyophilization or freeze drying.

[0027] "Diluents" may include water, saline, glucose, ethanol, glycerol, etc. Isotonic agents may include sodium chloride, glucose, mannitol, sorbitol, lactose, etc. Stabilizers may include albumin and alkali metal salts of ethylenediaminetetraacetic acid, etc.

[0028] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs.The singular terms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0029] Suitably, the vaccine is administered in a volume of about 0.05 to about 5.0 ml, for example 0.1 to 2.5 ml. Suitably, the vaccine is administered in a volume of 0.2 to 2.0 ml, or 0.25 to 1.5 ml, or 0.3 to 1.2 ml, or 0.4 to 1.0 ml, or 0.5 to 0.9 ml, or 0.6 to 0.8 ml.

[0030] The vaccine can be administered subcutaneously, intramuscularly, intraperitoneally, orally, intranasally, intraocularly and / or rectally. Suitably, the vaccine is administered subcutaneously, intramuscularly, orally, intranasally, intraocularly and / or rectally. Suitably, the vaccine is administered subcutaneously, intramuscularly, orally and / or intranasally. Suitably, the vaccine is administered subcutaneously or intramuscularly.

[0031] Suitably, the vaccine is administered in a single dose. Suitably, the vaccine is administered in at least 2 doses. The at least 2 doses are administered at intervals of 2 to 100 days, preferably 5 to 60 days, more preferably 7 to 50 days, more preferably 10 to 40 days, more preferably 14 to 30 days, and more preferably 15 to 25 days. Preferably, the at least 2 doses are administered at intervals of 5 to 40 days, preferably 6 to 35 days, preferably 7 to 32 days, preferably 8 to 30 days, preferably 9 to 28 days, preferably 10 to 25 days, preferably 11 to 22 days, preferably 12 to 20 days, preferably 13 to 18 days, preferably 14 to 16 days. Also suitably, the at least 2 doses are administered at intervals of 1 to 12 weeks, more suitably 2 to 10 weeks, more suitably 2 to 8 weeks, more suitably 3 to 6 weeks, and more suitably 3 to 5 weeks.

[0032] Preferably, the single dose is renewed by annual revaccination. Suitably, after the single dose, revaccination is every 10 to 15 months. Suitably, revaccination is every 11 to 14 months, suitably, revaccination is every 12 to 13 months.

[0033] Preferably, the at least two doses are renewed with annual revaccination of a single dose. Suitably, after the last dose of the at least two doses, the revaccination is every 10 to 15 months. Suitably, the revaccination is every 11 to 14 months, suitably, the revaccination is every 12 to 13 months.

[0034] Suitably, the vaccine comprises additional antigens that provide immunity against additional non-Leptospira canine pathogens. Suitably, the additional antigens are selected from canine parvovirus (CPV), canine parainfluenza virus (CPi2), canine distemper virus (CDV), adenovirus, herpes virus, rabies, canine coronavirus, Bordetella and combinations thereof.

[0035] The present invention will now be further described by the following non-limiting examples.

[0036] Example

[0037] General

[0038] A total of twenty-nine healthy puppies with undetectable levels of serum antibodies against serogroup canis, serogroup icterohaemorrhagic, serogroup influenzae typhoid, and serogroup australia were used. Three groups of seven puppies were vaccinated each. Group 1 and Group 2 each received a different batch of L4-LV (a dose of 0.5 ml). Group 3 received a batch of L4 (a dose of 1 ml). The amount of antigen in L4 and L4-LV was the same, the only difference being the volume of the dose. Group 4 was an unvaccinated control group.

[0039] L4-LV and L4 contain antigens from the following four serotypes:

[0040] Inactivated Leptospira strains:

[0041] Portland-vere serotype (Ca-12-000 strain) 3550-7100U*

[0042] Copenhagen serotype (Ic-02-001 strain) 290-1000U*

[0043] Bratislava serotype (As-05-073 strain) 500-1700U*

[0044] Dadas serotype (Gr-01-005 strain) 650-1300U*

[0045] *Antigen mass ELISA units.

[0046] The first vaccination was performed at 6 weeks of age and the second vaccination was performed at 10 weeks of age. Leptospira challenge was performed 3 weeks after the second vaccination. Dogs were challenged with Leptospira bacteria cultured from positive organs or homogenates of positive organs of experimentally infected hamsters.

[0047] To assess post-challenge effectiveness, the following tests or measurements are performed:

[0048] Microscopic agglutination test (MAT) to determine serogroup-specific antibody titers against serogroup Australasian, serogroup Canine, serogroup Influenza Typhoid, and serogroup Icterohaemorrhagiae;

[0049] weight;

[0050] body temperature;

[0051] Culture of the offending organism from blood, urine, kidney, and liver;

[0052] Urinalysis

[0053] Clinical signs; and

[0054] Macroscopic and histopathological examinations after autopsy.

[0055] Serology

[0056] Blood samples were collected from all pups five days before challenge (= 16 days after the second vaccination) and on days 3, 7, 14, 21 and 28 post-challenge (pc) for serology and platelet counts.

[0057] Sera were tested using the microscopic agglutination test (MAT) to determine the titers of serogroup-specific agglutinating serum antibodies against serogroups Australasian, Canine, Influenza Typhoid, and Icterohaemorrhagiae.

[0058] Briefly, serial two-fold dilutions of canine sera were incubated with live antigens from these serotype groups. The titer was then determined as the log2 value of the reciprocal of the highest dilution in the serum-antigen mixture that agglutinated ≥50% of the Leptospira. Positive and negative rabbit antisera were used as control sera. The test was considered valid if the titer of the negative control serum was ≤1 and the titer of the positive control serum was ≥5 (with the homologous antigen). All test samples that produced agglutination at dilutions ≥2 were considered positive.

[0059] body temperature

[0060] Body temperature was measured three days before challenge and daily thereafter until the end of the study.

[0061] weight

[0062] Body weight was measured for each dog on the day of arrival (Day -12), prior to challenge (twice on Day -5 and Day 0), and then daily until the end of the study.

[0063] Reisolation of the offending organism from blood, urine, kidneys and liver

[0064] *Blood: Blood samples for culture results were collected from all dogs 5 days before challenge and on days 1, 2, 3, 4, 7, 10, 14 and 21 pc. A 0.5 ml aliquot directly from the syringe was inoculated into 10 ml of EMJH medium (containing 200 μg / ml 5-fluorouracil (5-FU) and 1% (v:v) rabbit serum negative for antibodies to the four relevant serogroups of Leptospira).

[0065] To assess the total number of days with positive blood samples per dog, in the case of euthanasia and necropsy earlier than the planned necropsy date (pc day 28), the positive results of the missing blood samples were used. This was done to avoid false comparisons with the vaccinated groups where no dogs were euthanized (and therefore no samples were missing).

[0066] * Urine: Urine samples (at least 2.5 ml) were collected by bladder puncture from all dogs 3 days before challenge and on days 3, 7, 14, 21 and 28 after challenge. One ml of urine was inoculated directly into 10 ml of EMJH medium as described above. The remaining 1.5 ml of urine per dog was used for rapid urinalysis, see below (Rapid Urinalysis below).

[0067] To assess the total number of days with positive urine + kidney samples for each dog, positive results from missing urine samples were used in cases prior to the scheduled necropsy date (day 28 post-challenge). This was done to avoid false comparisons with the vaccinated group where no dogs were euthanized (and therefore no samples were missing).

[0068] *Kidney and liver: After euthanasia, 1-2 gram fragments were removed from the cortex of one kidney and the liver for culture. The aseptically removed fragments were placed in 10 ml of EMJH medium as described above.

[0069] Kidney and liver tissue fragments were homogenized in 10 ml of EMJH medium. Each kidney or liver homogenate was diluted 100-fold in EMJH (containing 5-FU and "negative" rabbit serum) for culture.

[0070] Blood, urine, kidney, and liver homogenates were incubated in EMJH at 29°C and observed weekly for the presence of motile bacteria that were typical of Leptospira using dark-field microscopy for at least 8 weeks before discarding negative cultures. Some positive cultures (at least one per treatment group) were tested for Leptospira and serogroups were identified by agglutination with MAT.

[0071] Clinical signs

[0072] Dogs were examined twice daily for clinical signs on days 1-30 after challenge, with particular attention paid to the following nonspecific signs associated with canine leptospirosis: loss of appetite, slow or stiff gait, weakness, vomiting, diarrhea, decreased skin turgor (indicating dehydration), pale or yellow mucous membranes (conjunctiva or oral mucosa), and hunched back.

[0073] When one or more laboratory tests confirm the presence of leptospirosis, the dog is assigned one or more clinical signs and a corresponding clinical score value. In this way, the likelihood of interference caused by concurrent (mild) clinical signs due to reasons unrelated to the leptospiral challenge (e.g. concurrent diarrhea due to intestinal opportunistic bacteria or parasites, etc.) is significantly reduced.

[0074] Use the following clinical score values ​​for the following laboratory-confirmed clinical signs to assess an overall clinical score for each dog.

[0075] Table 1 Clinical scoring matrix

[0076]

[0077]

[0078] The total clinical score for a dog was defined as the sum of the individual scores for each day and all days up to the study end date for dogs that were not euthanized prior to the end date. For dogs euthanized prior to the scheduled necropsy date (because a humane endpoint was reached), a total clinical score of 150 was used.

[0079] Autopsy, gross examination, and histopathology

[0080] In case of severe clinical signs after challenge, dogs were euthanized after adequate sedation and immediately followed by post-mortem examination. The same procedure was performed on all surviving dogs on the scheduled necropsy date. Macroscopic examination was performed, especially for the lungs, liver, kidneys and spleen. Tissue samples (taken from lesions) from the liver, kidneys, spleen and any organ / tissue with suspected lesions were examined histologically.

[0081] Tissue samples from the liver, kidney, spleen, and any organ / tissue with suspected lesions were processed and sections were stained with hematoxylin and eosin (HE) according to standard procedures for histopathological examination. In addition, Warthin-Starry staining was performed on kidney and liver sections to detect leptospira in kidney and liver tissues. The results were described and interpreted in the specific pathological context of canine leptospirosis based on microscopic examination of all slides of all sampled organs and tissues.

[0082] Example 1 Protection against icterohaemorrhagiae serotype

[0083] Table 2 Treatment groups

[0084]

[0085] 1 The attack material contains 6.3x 10 8 Bacterial cells / ml;

[0086] IP, intraperitoneal; IN, intranasal; Conj., instilled into the anterior conjunctival sac of both eyes. Each dog received the challenge material in the three administrations described. The challenge was performed using an in vitro culture of the serotype Verdun strain of icterohaemorrhagiae.

[0087] Table 3 Total score of clinical symptoms of laboratory-confirmed leptospirosis in each dog after challenge with serotype icterohaemorrhagic strain

[0088]

[0089]

[0090] Table 4 Blood and urine / kidney culture results from dogs challenged with serotype icterohaemorrhagic strains

[0091]

[0092]

[0093]

[0094]

[0095] 1 - = negative, + = positive

[0096] 2 S, statistically significant difference between the test group and the control group (two-sided Wilcoxon test, P < 0.05)

[0097] 3 When both urine and kidney cultures were positive on day 28, only one positive result was used to calculate the total number of positive days

[0098] 4 Euthanasia, although these samples were not available after euthanasia, was still counted as a result "+" to avoid false comparisons between groups with and without euthanasia due to incomplete numbers of blood or urine samples from euthanized dogs.

[0099] Table 5 Overview of histopathological lesions associated with serovar Leptospira icterohaemorrhagiae infection

[0100]

[0101]

[0102] E. Euthanasia

[0103] - = no lesions ++ = moderate multifocal lesions

[0104] + / - = mild / focal disease +++ = severe / diffuse disease

[0105] + = mild focal to multifocal disease NS = not sampled

[0106] **There was no significant difference in the frequency of moderate / severe nephropathy (score ++ or above) between the control and vaccinated groups (Fisher's exact test).

[0107] Example 2 Protection against influenza typhoid serotypes

[0108] Table 6 Treatment groups

[0109]

[0110] 1 Attack material includes 1.4x10 9 Bacterial cells / ml;

[0111] IP, intraperitoneal; IN, intranasal; Conj., instilled into the anterior conjunctival sac of both eyes. Each dog received the challenge material in the three administrations described. The challenge was performed using an in vitro culture of the serotype Duyster strain of the influenza Typhoid type.

[0112] Table 7 Blood and urine / kidney culture results from dogs challenged with serotype influenza typhoid strains

[0113]

[0114]

[0115]

[0116]

[0117] 1 - = negative, + = positive

[0118] 2 S, statistically significant difference between the test group and the control group (two-sided Wilcoxon test, P < 0.05)

[0119] 3 When both urine and kidney cultures were positive on day 28, only one positive result was used to calculate the total number of positive days

[0120] Table 8 Number of dogs with renal lesions in each group

[0121]

[0122]

[0123] E. Concurrent euthanasia

[0124] - = no lesions ++ = moderate multifocal lesions

[0125] + / - = mild / focal disease +++ = severe / diffuse disease

[0126] + = mild focal to multifocal disease NS = not sampled

[0127] **The frequency of moderate / severe nephropathy (score ++ or above) in the control group was significantly higher than that in the vaccinated group. P values ​​for each group < 0.05 (Fisher's exact test).

[0128] Example 3 Protection against Australian serotypes

[0129] Table 9 Treatment groups

[0130]

[0131] 1 Attack material contains 1.2x10 9 Bacterial cells / ml;

[0132] IP, intraperitoneal; IN, intranasal; Conj., instilled into the anterior conjunctival sac of both eyes. Each dog received the challenge material in the three administrations described. The challenge was performed using an in vitro culture of the Australian serotype Dohhilo strain.

[0133] Table 10 Total score of clinical symptoms of laboratory-confirmed leptospirosis in each dog after challenge with Australian serotype strain

[0134]

[0135]

[0136] ^Using two-sided Wilcoxon test

[0137] Table 11 Blood and urine / kidney culture results from dogs challenged with Australian serotype strains

[0138]

[0139]

[0140]

[0141]

[0142] 1 - = negative, + = positive

[0143] 2 S, statistically significant difference between the test group and the control group (two-sided Wilcoxon test, P < 0.05)

[0144] 3 When both urine and kidney cultures were positive on day 28, only one positive result was used to calculate the total number of positive days

[0145] 4 Euthanasia, although these samples were not available after euthanasia, was still counted as a result "+" to avoid false comparisons between groups with and without euthanasia due to incomplete numbers of blood or urine samples from euthanized dogs.

[0146] Table 12 Overview of histopathological lesions associated with infection with Australian serotype Leptospira

[0147]

[0148]

[0149] E. Euthanasia

[0150] - = no lesions ++ = moderate multifocal lesions

[0151] + / - = mild / focal disease +++ = severe / diffuse disease

[0152] + = mild focal to multifocal disease NS = not sampled

[0153] **The frequency of moderate / severe nephropathy (score ++ or above) in the control group was significantly higher than that in the vaccinated group. P values ​​for each group < 0.05 (Fisher's exact test).

Claims

1. A vaccine composition comprising a non-icterohaemorrhagiae serotype of Leptospira for use in providing protective immunity to canines against an icterohaemorrhagiae serotype of Leptospira.

2. The vaccine for use according to claim 1, wherein the vaccine is a multivalent vaccine.

3. The vaccine for use according to claim 1 or 2, wherein the vaccine does not contain the Leptospira icterohaemorrhagiae serotype.

4. The vaccine for use according to any one of claims 1 to 3, wherein the vaccine comprises Leptospira serotype Portland-vere, Dadas, Copenhagen and / or Bratislava.

5. The vaccine for use according to any one of claims 1 to 4, which provides protective immunity to canines against Leptospira serovars icterohaemorrhagiae, Australasia and Gastroenteritis Typhimurium.

6. The vaccine for use according to any one of claims 1 to 5, wherein 0.1 to 2.5 ml of the vaccine is administered.

7. The vaccine for use according to any one of claims 1 to 6, wherein the vaccine is administered subcutaneously.

8. The vaccine for use according to any one of claims 1 to 7, wherein the vaccine is administered in at least 2 doses.

9. The vaccine for use according to any one of claims 1 to 8, wherein the vaccine is administered in at least 2 doses, and the doses are administered at least 5 days apart.

10. The vaccine for use according to any one of claims 1 to 9, wherein the vaccine comprises additional antigens which provide immunity against additional non-Leptospira canine pathogens.

11. The vaccine for use according to claim 10, wherein the additional antigen is selected from canine parvovirus (CPV), canine parainfluenza virus (CPi2), canine distemper virus (CDV), adenovirus, herpes virus, rabies, canine coronavirus and combinations thereof.

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