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The bovine immune response to leptospira borgpetersenii serovar hardjo

posted on 17.02.2017, 00:17 authored by Deveson Lucas, Deanna Suzanne
Leptospirosis, caused by the spirochaete Leptospira, is a widespread disease that affects virtually all known mammals. Pathogenic Leptospira is classified into a number of diverse species and further subdivided into over 250 serovars, based on surface antigens. As classification of Leptospira includes both antigenic and genetic systems, certain serovars, such as Hardjo, may include organisms from two different species (i.e. L. borgpetersenii serovar Hardjo subtype Hardjobovis and L. interrogans subtype Hardjoprajitno). Subtype Hardjobovis is the most common cause of bovine leptospirosis. Infection can result in mastitis, abortion and foetal death. Cattle are the maintenance host for Hardjobovis and are responsible for the spread and shed of the organism, which may result in zoonotic infections. Historically, protective immunity to Leptospira spp. was thought to be exclusively humoral, due to its serovar-specific nature and the ability to transfer passive immunity between some leptospiral animal models such as mice to guinea pigs. However, protective immunity against subtype Hardjobovis in cattle requires a cellular immune response. Vaccination of cattle with a protective subtype Hardjobovis bacterin stimulates a Th1 response characterised by IFN-γ production. Production of IFN-γ closely correlates with protective immunity in cattle. However, the antigens involved in stimulating this immune response have not been identified. The genome of L. borgpetersenii serovar Hardjo subtype Hardjobovis strain L550 has been fully sequenced. Bioinformatic analysis of the genome was used to identify 260 potential surface exposed, candidate vaccine antigens. These proteins included: predicted secreted proteins, predicted outer membrane proteins, leucine-rich repeat proteins, TPR repeat proteins, proteases, iron acquisition proteins, homologs of known bacterial virulence factors and homologs of known protective antigens in Pasteurella multocida. The list also includes 45 conserved hypothetical proteins and 103 unique hypothetical proteins. Of the 260 proteins identified, 238 proteins were successfully cloned, expressed in E. coli and purified. These proteins were then screened for their ability to stimulate a cell-mediated immune response in vaccinated cattle blood. In order to screen these proteins cattle were vaccinated with a monovalent L. borgpetersenii serovar Hardjo subtype Hardjobovis vaccine. The cattle were periodically bled and the whole blood was initially cultured with L. borgpetersenii serovar Hardjo subtype Hardjobovis (Hardjo WCL), to determine whether the vaccine stimulated the desired immune response. Once this was established the recombinant proteins and additional potential antigens, such as whole cell sonicate fractions of Hardjo WCL were also screened for the ability to stimulate IFN-γ in vaccinated cattle blood. From this study it was found that LipL32, the major outer-membrane protein of pathogenic Leptospira spp., is involved in stimulation of IFN-γ in the blood of vaccinated cattle. Sera from the vaccinated cattle also recognised recombinant LipL32, indicating that anti-LipL32 antibodies were produced. Therefore, LipL32 can stimulate both humoral and cell-mediated immune responses in vaccinated cattle. In an attempt to identify the regions of LipL32 involved in T-cell interactions, a number of truncations were prepared and screened using the whole blood assay. Data from this study indicates that amino acids 20-106 are involved in T-cell interactions, as this peptide stimulated equal or greater levels of IFN-γ in the blood of vaccinated animals when compared to LipL32 or Hardjo WCL alone. Further characterisation of the bovine immune response to Hardjo WCL, recombinant LipL32 and the soluble master pool was also undertaken. Transcript levels of a number of Th1 and Th2 cytokine genes were measured. FACS analysis was used to determine the T-cell populations present in blood cultured with Hardjo WCL and recombinant LipL32. It has also been demonstrated that the strain of Hardjobovis used in vaccine formulation and challenge studies can affect the severity of disease. Therefore, genomic and proteomic comparative analysis of three Hardjobovis isolates was undertaken to identify any potential differences that may contribute to stimulating a cell mediated immune response. These isolates included L550, a Queensland isolate with a fully annotated and closed genome. Strain L683, an isolate from Victoria, Australia, used in the formulation of SpirovacTM and other commercial vaccines produced by Pfizer Animal Health (Australia), and strain 93U, an isolate from the United States, used in vaccine studies but was unable to protect animals from infection. Finally, a small-scale vaccine trial was performed to assess the potential for recombinant LipL32 to stimulate protective immunity in hamsters, the accepted small animal model of leptospirosis. However, due to the nature of hamster immunity towards pathogenic Leptospira spp. the rLipL32 vaccine failed to protect the hamsters from leptospi


Principal supervisor

Ben Adler

Year of Award


Department, School or Centre


Campus location



Doctor of Philosophy

Degree Type



Faculty of Medicine Nursing and Health Sciences