Vaccine for protecting leptospira cold typhoid serotype

Abstract

The present invention relates to the use of a vaccine comprising a non-cold typhoid leptospira serotype to provide protective immunity to a dog against a cold typhoid leptospira serotype.

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 Grippotyphosa Leptospira. Background Art

[0002] Leptospirosis is a global zoonotic disease caused by a Gram-negative spirochete belonging to the genus Leptospira. Leptospirosis is common in humans, dogs, horses, cattle and wildlife. Dogs are highly susceptible to infection and become ill with symptoms such as high fever, jaundice, hemorrhagic diathesis, abortion and may die within a few days. In addition, dogs may develop chronic symptoms such as liver, kidney and gastrointestinal symptoms. Domestic dogs live in close contact 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 widespread serotypes in dogs are Canicola, Icterohaemorrhagiae, Australis and Grippotyphosa. In addition to these serotypes, Pomona is also present in the United States, and Hebdomadis in Japan. A series of vaccines against canine leptospirosis have been licensed in Europe, the earliest being a bivalent vaccine containing Canicola and either Icterohaemorrhagiae or Copenhageni serotypes. More recently, quadrivalent and trivalent vaccines have been introduced into the European market, which also contain serotypes of serogroups Grippotyphosa and Icterohaemorrhagiae or only Grippotyphosa serotypes.

[0004] Literature review, for example "Leptospirosis Fact Sheet" (WHO, Regional Office for South-East Asia, 2009) partially illustrates that animals and humans can be immunized, but the protection is mainly serotype specific. The lack of cross protection is not surprising, especially considering that there are significant genetic / genomic differences between serotypes, for example, between the gene organization in the lipopolysaccharide biosynthesis (rfb) locus (Pena-Moctezuma, A. et al., 2001 FEMS Immunology and Medical Microbiology 31 (2001) 73-81).

[0005] Thus, there is little evidence of "cross protection" between serotypes against dogs. 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 a Typhoid serotype by administering a homologous effective amount of a non-Typhoid serotype, such as Portland-vere, Dadas, Copenhagen, or Bratislava serotype).

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

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

[0008] An article by Bouvet et al. (Veterinary Immunology and Immunopathology 219 (2020) 109985) is a scientific publication of the same findings and experiments as described in EP2874653. It is confirmed that the bacterinary Leptospira vaccine only provides protection against the same serotype. It is also disclosed that the available evidence for protection against other serotypes within the same serogroup is very limited and only exists in rodent models. There is no evidence in the prior art that other Leptospira serotypes provide cross-protection in dogs.

[0009] Therefore, there is still a need to protect animals (especially dogs) against Leptospira serotypes. Since leptospirosis is also a potential zoonotic problem for humans, it would be beneficial to protect against additional Leptospira serotypes, and it is preferred to use vaccines that are already in use. It is therefore beneficial to protect animals (especially dogs) against heterologous Leptospira serotypes. Prior to the present disclosure, methods for using non-typhoid serotypes to provide protection against leptospira typhoid serotypes were unknown. Summary of the invention

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

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

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

[0013] It is unexpected and surprising to a skilled person with the current state of the art in the field of leptospirosis that a vaccine that does not contain a Leptospira Typhoid serotype elicits protective immunity against a Leptospira Typhoid serotype in dogs. It is particularly surprising that a vaccine containing a Leptospira Portland-vere, Dadas, Copenhagen and Bratislava serotype provides immunity against a Leptospira Typhoid serotype.

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

[0015] The present invention encompasses methods of preventing or reducing infection due to 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 for providing protective immunity to a dog against a Leptospira Typhoid serotype. The vaccine does not comprise a Leptospira Typhoid serotype.

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

[0017] In one embodiment of the present invention and / or its embodiments, the non-common typhoidal Leptospira serotype belongs to the common typhoidal serogroup. In a specific embodiment, the non-common typhoidal Leptospira serotype is Dadas type or Bananal / Liangguang type, preferably Dadas type.

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

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

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

[0021] Suitably, the vaccine provides dogs with protective immunity against Leptospira Typhi, Australasia, and Icterohaemorrhagiae serotypes.

[0022] The term "vaccine" as used herein refers to a pharmaceutical composition comprising at least one immunologically active component that induces an immune 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 contain other components typical of a pharmaceutical composition. In this specification, immunogenic compositions and vaccines are used interchangeably.

[0023] Typically, an "immune 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, specifically directed against one or more antigens contained in the composition or vaccine of the target. Suitably, the target will exhibit a therapeutic or prophylactic immune 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 disappearance of clinical signs normally exhibited 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 are, for example: 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 referred to herein, "reduction in the incidence and / or severity of clinical signs" or "reduction in the incidence and / or severity of clinical symptoms" refers to a reduction in the number of infected animals in a group, a reduction or elimination of the number of animals showing clinical signs of infection, or a reduction in 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 signs are reduced by 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 "drug 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. Such 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 explained, 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. Unless the context clearly indicates otherwise, the singular terms "a", "an" and "the" include plural referents.

[0029] Suitably, the vaccine is administered in a volume of about 0.05 to about 5.0 ml, such as 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. The vaccine is suitable for administration subcutaneously, intramuscularly, orally, intranasally, intraocularly and / or rectally. The vaccine is suitable for administration subcutaneously, intramuscularly, orally and / or intranasally. The vaccine is suitable for administration subcutaneously or intramuscularly.

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

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

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

[0034] Preferably, the at least two doses are renewed by revaccination with at least two doses per year. Suitably, the revaccination is performed every 10 to 15 months after the last dose of the at least two doses. Suitably, the revaccination is performed every 11 to 14 months, suitably the revaccination is performed every 12 to 13 months.

[0035] Suitably, the vaccine comprises additional antigens that provide immunity against additional non-Leptospira canine pathogens. Suitably, the additional antigens are selected from the group consisting of: canine parvovirus (CPV), canine parainfluenza virus (CPi2), canine distemper virus (CDV), adenovirus, herpes virus, rabies, canine coronavirus, Bordetella and combinations thereof.

[0036] The invention will now be further described by way of the following non-limiting examples.

[0037] Example

[0038] Overview

[0039] A total of 29 healthy puppies with undetectable serum antibodies to the canine, icterohaemorrhagic, typhoid and Australian serotypes were used. Three groups of puppies (7 in each group) were vaccinated. Groups 1 and 2 each received a different batch of L4-LV vaccine (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 was the volume of the dose. Group 4 was an unvaccinated control group.

[0040] L4-LV and L4 contain antigens from the following four serotypes:

[0041] Inactivated Leptospira strains:

[0042] Portland-vere serotype (strain Ca-12-000) 3550-7100U*

[0043] Serotype Copenhagen (strain Ic-02-001) 290-1000U*

[0044] Bratislava serotype (strain As-05-073) 500-1700U*

[0045] Dadas serotype (strain Gr-01-005) 650-1300U*

[0046] *Antigen mass ELISA units.

[0047] The first vaccination was performed at 6 weeks of age and the second vaccination was performed at 10 weeks of age. The Leptospira challenge was performed 3 weeks after the second vaccination. The dogs were challenged with Leptospira bacteria cultured from positive organs or homogenates of positive organs of experimentally infected hamsters.

[0048] To evaluate post-challenge efficacy, the following tests or measurements were performed:

[0049] Microscopic agglutination test (MAT) to determine serogroup-specific antibody titers against Australasian, Canine, Cold Typhoid, and Icterohaemorrhagia serotypes;

[0050] weight;

[0051] body temperature;

[0052] culture of the challenge organism from blood, urine, kidney, and liver;

[0053] Urinalysis

[0054] Clinical signs; and

[0055] Macroscopic and histopathological examinations after necropsy.

[0056] Serology

[0057] Blood samples for serology and platelet counts were collected from all pups five days before challenge (= day 16 after the second vaccination) and on days 3, 7, 14, 21 and 28 post-challenge (pc).

[0058] Sera were tested using the microscopic agglutination test (MAT) to determine the titers of serogroup-specific agglutinating serum antibodies against the Australian, Canine, Cold Typhoid, and Icterohaemorrhagical serogroups.

[0059] Briefly, serial two-fold dilutions of canine sera were incubated with live antigens of each of these serotypes. The titer was then determined as the log reciprocal of the highest dilution showing ≥50% agglutination of Leptospira in the serum-antigen mixture. 2 Value. 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 if the titer of the positive control serum was ≥5 (with homologous antigen). All test samples that showed agglutination at dilutions ≥2 were considered positive.

[0060] body temperature

[0061] Body temperature was measured three days before challenge and every day after challenge until the end of the study.

[0062] weight

[0063] Body weight of each dog was measured on the day of arrival (Day -12), before challenge (twice on Day -5 and Day 0), and every day thereafter until the end of the study.

[0064] Reisolation of the attacking organism from blood, urine, kidney and liver

[0065] *Blood: Blood samples for culture results were collected from all dogs 5 days prior to challenge and on days 1, 2, 3, 4, 7, 10, 14 and 21 pc. A 0.5 ml aliquot directly from a syringe was inoculated into 10 ml EMJH medium (containing 200 μg / ml 5-fluorouracil (5-FU) and 1% (v:v) rabbit serum that was negative for antibodies against the four relevant serogroups of Leptospira).

[0066] To assess the total number of days each dog had a positive blood sample, in cases where euthanasia and necropsy occurred earlier than the planned necropsy day (pc day 28), the positive result of the missing blood sample was used. This was to avoid erroneous comparisons with the vaccinated group in which no dogs were euthanized and, therefore, no samples were missing.

[0067] * Urine: Urine samples (at least 2.5 ml) were collected from all dogs via bladder puncture 3 days before challenge and on days 3, 7, 14, 21 and 28 after challenge. 1 ml of urine was directly inoculated into 10 ml of EMJH medium as described above. The remaining 1.5 ml of urine from each dog was used for rapid urine analysis, see below (Rapid Urine Analysis).

[0068] To assess the total number of days each dog had positive urine + kidney samples, in the event of euthanasia and necropsy earlier than the scheduled necropsy day (day 28 post-challenge), the positive result of the missing urine sample was used. This was to avoid erroneous comparisons with the vaccinated group in which no dogs were euthanized and, therefore, no samples were missing.

[0069] * Kidney and liver: After euthanasia, a piece of kidney cortex and liver (1-2 grams) was removed from one of the kidneys and liver for culture. The (aseptically removed) pieces were placed in 10 ml of culture medium as described above.

[0070] Kidney and liver tissue fragments were homogenized in 10 ml of EMJH medium. A 100-fold dilution of each homogenate of kidney or liver in EMJH (containing 5-FU and "negative" rabbit serum) was used for the culture.

[0071] Homogenized cultures of blood, urine, kidney and liver in EMJH were incubated at 29°C and observed weekly for the presence of motile bacteria with typical Leptospiral shapes using dark field microscopy for at least 8 weeks, after which negative cultures were discarded. Leptospira in some positive cultures (at least one per treatment group) were tested to identify the serogroup by agglutination with MAT.

[0072] Clinical signs

[0073] 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.

[0074] When the results of one or more laboratory tests confirm the presence of leptospirosis, the dog will be assigned one or more clinical signs and a corresponding clinical score value. In this way, the chance of interference by concurrent (minor) clinical signs due to causes unrelated to the leptospiral challenge, such as concurrent diarrhea caused by, for example, enteric pathogenic bacteria or parasites, is significantly reduced.

[0075] The following clinical score values ​​were used for the following laboratory-confirmed clinical signs to assess the total clinical score value for each dog:

[0076] Table 1 Clinical scoring matrix

[0077]

[0078]

[0079] The total clinical score for a dog was defined as the sum of all individual scores for each day up to the study end date and for all days for dogs that were not euthanized prior to the end date. For dogs euthanized prior to the scheduled necropsy date (thus reaching the humane endpoint), a total clinical score value of 150 was used.

[0080] Autopsy, gross examination, and histopathology

[0081] In case of severe clinical signs after challenge, dogs were euthanized after adequate sedation and then immediately subjected to necropsy 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 from any organ / tissue with suspected lesions were examined histologically.

[0082] Tissue samples from liver, kidney, spleen and any organ / tissue with suspected lesions were processed according to standard procedures and sections were stained with hematoxylin and eosin (HE) for histopathological examination. In addition, sections of kidney and liver were stained with Warthin-Starry to detect leptospira in kidney and liver tissue. Based on microscopic examination of all slides of all sampled organs and tissues, the results were described and interpreted in the context of the specific pathology of canine leptospirosis.

[0083] Example 1 Protection against cold and typhoid serotypes

[0084] Table 2: Treatment groups

[0085]

[0086]

[0087] 1 The attack material contained 1.4×10 9 Bacterial cells / ml;

[0088] IP, intraperitoneal; IN, intranasal; Conj., instilled into the ventral conjunctival sac of each eye. Each dog received the challenge material in three administrations as indicated. The challenge was performed using an in vitro culture of the Typhimurium serotype strain Duyster.

[0089] Table 3 Results of blood and urine / kidney cultures from dogs challenged with strains of the common cold typhoid serotype

[0090]

[0091]

[0092] 1 - = negative, + = positive

[0093] 2 S, statistically significant difference between the test group and the control group (two-sided Wilcoxon test, P < 0.05)

[0094] 3 When both urine and kidney cultures were positive on day 28, only one positive result was used in the calculation of total positive days.

[0095] Table 4 Number of dogs with renal lesions in each group

[0096]

[0097] E. Simultaneous euthanasia

[0098] - = no lesions ++ = moderate multifocal lesions

[0099] + / - = minor / focal lesions +++ = severe / diffuse lesions + = mild focal to multifocal disease NS = not sampled

[0100] **The frequency of moderate / severe nephropathy (score ++ or higher) in the control group was significantly higher than that in the vaccinated group. P-value < 0.05 for each group (Fisher's exact test)

[0101] Example 2 Protection against Australian serotypes

[0102] Table 5: Treatment groups

[0103]

[0104] 1 The attack material contained 1.2×10 9 Bacterial cells / ml;

[0105] IP, intraperitoneal; IN, intranasal; Conj., instilled into the ventral conjunctival sac of each eye. Each dog received the challenge material by three administrations as indicated. The challenge was performed using an in vitro culture of the Australian serotype strain Dohhilo.

[0106] Table 6 Total score of laboratory-confirmed clinical signs of leptospirosis per dog after challenge with strains of the Australian serotype

[0107]

[0108] ^Using two-sided Wilcoxon test

[0109] Table 7 Results of blood and urine / kidney cultures of dogs challenged with strains of the Australian serotype

[0110]

[0111]

[0112] 1 - = negative, + = positive

[0113] 2 S, statistically significant difference between the test group and the control group (two-sided Wilcoxon test, P < 0.05)

[0114] 3 When both urine and kidney cultures were positive on day 28, only one positive result was used in the calculation of total positive days

[0115] 4 euthanasia, although these samples were not available after euthanasia, were still counted as a result of “+” to avoid false comparisons between groups with and without euthanized dogs due to incomplete numbers of blood or urine samples from euthanized dogs.

[0116] Table 8 Overview of histopathological lesions associated with leptospirosis with Australian serotypes

[0117]

[0118] E. Euthanasia

[0119] - = no lesions ++ = moderate multifocal lesions

[0120] + / - = minor / focal lesions +++ = severe / diffuse lesions

[0121] + = mild focal to multifocal disease NS = not sampled

[0122] **The frequency of moderate / severe nephropathy (score ++ or higher) in the control group was significantly higher than that in the vaccinated group. P-value < 0.05 for each group (Fisher's exact test).

[0123] Example 3 Protection against icterohaemorrhagiae serotype

[0124] Table 9: Treatment Groups

[0125]

[0126] 1 The attack material contained 6.3×10 8 Bacterial cells / ml;

[0127] IP, intraperitoneal; IN, intranasal; Conj., instilled into the ventral conjunctival sac of each eye. Each dog received the challenge material in three administrations as indicated. The challenge was performed using an in vitro culture of the serotype Verdun strain of icterohaemorrhagiae.

[0128] Table 10 Total score for laboratory-confirmed clinical signs of leptospirosis per dog after challenge with strains of serovar Icterohaemorrhagiae

[0129]

[0130] Table 11 Results of blood and urine / kidney cultures of dogs challenged with serotype icterohaemorrhagic

[0131]

[0132]

[0133] 1 - = negative, + = positive

[0134] 2 S, statistically significant difference between the test group and the control group (two-sided Wilcoxon test, P < 0.05)

[0135] 3 When both urine and kidney cultures were positive on day 28, only one positive result was used in the calculation of total positive days

[0136] 4 euthanasia, although these samples were not available after euthanasia, were still counted as a result of “+” to avoid false comparisons between groups with and without euthanized dogs due to incomplete numbers of blood or urine samples from euthanized dogs.

[0137] Table 12 Overview of histopathological lesions associated with leptospirosis with serovar icterohaemorrhagiae

[0138]

[0139] E. Simultaneous euthanasia

[0140] - = no lesions ++ = moderate multifocal lesions

[0141] + / - = minor / focal lesions +++ = severe / diffuse lesions

[0142] + = mild focal to multifocal disease NS = not sampled

[0143] **There was no significant difference in the frequency of moderate / severe nephropathy (score ++ or higher) between the control and vaccinated groups (Fisher's exact test).

Claims

1. A vaccine composition comprising a non-common typhoid serotype of Leptospira for use in providing protective immunity to dogs against common typhoid serotypes of Leptospira.

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

3. A vaccine for use according to claim 1 or 2, wherein the vaccine does not contain Leptospira Typhi serotypes.

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

5. A vaccine for use according to any one of claims 1 to 4, which provides dogs with protective immunity against Leptospira Typhi, Australasia and Icterohaemorrhagiae serotypes.

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 intervals of at least 5 days.

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 the group consisting of canine parvovirus (CPV), canine parainfluenza virus (CPi2), canine distemper virus (CDV), adenovirus, herpes virus, rabies, canine coronavirus and combinations thereof.

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