Progress 10/01/98 to 09/30/04
Outputs
In vitro studies of Leptospira interrogans have revealed that synthesis of several proteins involved in equine infection is regulated in response to temperature change. Two of these proteins, Qlp42 and Hsp15 are a lipoprotein and an alpha crystallin stress response protein respectively. Another LipL36, is an outer membrane lipoprotein. Two strongly immunoreactive proteins, Lk75.3 and Lig A were expressed only in vivo with demonstrated utility in differentiating vaccine from infection serum antibody responses. Lk75.3, a sphingomyelinase, damages cultured endothelial cells, an indication it may have a virulence function. *The signals that trigger responses in the pathogenic leptospira as they accommodate to conditions within the host and to the external environment following urinary shedding are largely unknown. **Immunoreactive proteins expressed only in vivo are by definition absent from cultured organisms and so commercial vaccines lack these potentially useful
immunogens. Since such proteins are likely to have virulence functions, specific antibodies would be predicted to be protective.
Impacts
Qlp42, Lk75.3 and Lig A are strongly immunoreactive and therefore have potential as components of new generation vaccines and immunodiagnostics. Both Lk75.3 and Lig A are of value in differentiating vaccine from infection antibody responses. These discoveries should eventually, directly or indirectly, reduce losses caused by Leptospira spp in livestock.
Publications
- Artiushin, S., Timoney, J.F., Nally, J. and Verma, A. 2004. Host-inducible immunogenic sphingomyelinase-like protein Lk73.5 of Leptospira interrogans. Infection & Immunity 72:742-749.
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Progress 01/01/03 to 12/31/03
Outputs
This phase of the project was undertaken to identify immunoreactive leptospiral proteins expressed within the infected eye. A genomic DNA library of L. interrogans serovar pomona type kennewicki was screened with a pool of eye fluids from uveitic horses. Sequences of inserts of reactive phage were identified using L. interrogans genomic sequence at http://www.chgc.org.cn/gn/. Sequences homologous to ligA/ligB, ligC, grpE/dnaK/dnaJ and qlp42, were detected as well as genes for hypothetical proteins and a gene for an iron regulation protein. At least 2 proteins appeared to be expressed only within the ocular environment. Genes for most of the identified proteins were present in all serovars of L. interrogans but absent from non-pathogenic Leptospira.
Impacts
Knowledge of the leptospira proteins involved in the immunopathogenesis of uveitis will be valuable in vaccine development and in design of therapeutic interventions.
Publications
- Verma, A., Artiushin, S. and Timoney, J.F. 2003. Intraocular expression of proteins of Leptospira interrogans in uveitic horses. Proceedings of the 84th Annual Meeting of the Conference of Research Workers in Animal Diseases. Chicago, IL. Abstract #13P.
- Verma, A., Artiushin S., Shuck, K.M. and Timoney, J.F. 2003. Cytotoxic effects of the sphingomyelinase-like Lk73.5 on equine pulmonary endothelial cells. Proceedings of the 103rd General Meeting of the American Society for Microbiology. Washington, D.C. Abstract #D-167.
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Progress 01/01/02 to 12/31/02
Outputs
The host inducible protein Lk73.5 of Leptospira interrogans was identified for the first time. This protein shares 63% homology with a sphingomyelinase of serovar hardjo and lesser homology with other bacterial phospholipases. All serovars of L. interrogans have a single copy of the gene which varies in number of N-terminal repeats in different serovars. Lk73.5 reacts strongly with uveitic eye fluids and with convalescent but not post-vaccinal sera. Equine pulmonary endothelial cells exposed to Lk73.5 released lactate dehydrogenase in a dose dependent manner indicating that Lk73.5 was cytotoxic for endothelial cells. Rabbit erythrocytes were hemolysed by the protein providing further evidence of a sphingomyelinase action.
Impacts
The sphingomyelinase-like Lk73.5 is possibly an important virulence factor which may have value in immunodiagnosis and as a vaccine component.
Publications
- Raghavan, P.U.M., Y. Chang, S.S.D. Jusuf, S. Artiushin, J.F. Timoney, S.P. McDonough, S.C. Barr, T.J. Divers, K.W. Simpson, P.L. McDonough and H.O. Mohammed. 2002. Cloning and molecular characterization of an immunogenic LIG A protein of Leptospira interrogans. Infect. Immun. 70:5924-5930.
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Progress 01/01/01 to 12/31/01
Outputs
In vitro studies of Leptospira interrogans serovar pomona type kennewicki have shown that synthesis of several proteins is regulated in response to temperature change. In order to specifically identify antigenic proteins upregulated at 37 degrees Celsius, antisera were prepared separately in ponies with organisms cultured at 30 degrees Celsius and at 37 degrees Celsius. A genomic DNA library was screened with the latter antiserum. Clones reactive with this serum but unreactive with serum raised against organisms cultured at 30 degrees Celsius were selected. Sequence analysis of 2 of these clones identified open reading frames for proteins designated Qlp42, a putative lipoprotein and Hsp15, a stress response protein and a member of the Hsp20/2-crystallin family. Homologues of qlp42 and hsp15 were detected in pathogenic serovars but not in the non-pathogenic Leptospira biflexa. Convalescent sera contained antibody to Qlp42, indicating expression of the protein during
infection.
Impacts
The Qlp42 protein is potentially useful in new generation leptospira vaccines and in assays of antibody responses. A leptospira vaccine targeted to horses would reduce abortion losses in Kentucky.
Publications
- Nally, J.E., J.F. Timoney and B. Stevenson. 2001. Temperature-regulated protein synthesis by Leptospira interrogans. Infect. Immun. 69:400-404.
- Molecular characterization of thermoinduced immunogenic proteins Qlp42 and Hsp15 of Leptospira interrogans. 2001. Infect. Immun. 69:7616-7624.
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Progress 01/01/00 to 12/31/00
Outputs
The effects of temperature on protein synthesis in Leptospira interrogans serovar pomona have been investigated. Bacteria were grown for several days after culture temperatures were shifted from 30 to 37 degrees Celsius. Triton X-114 cellular fractionation identified several proteins of the cytoplasm, periplasm, and outer membrane for which synthesis was dependent on the culture temperature. Synthesis of a cytoplasmic protein of 20 kDa was switched off at 37 degrees Celsius, whereas synthesis of a 66-kDa periplasmic protein was increased at the higher temperature. Increased synthesis of a 25-kDa outer membrane protein was observed when the organisms were shifted from 30 to 37 degrees Celsius. A 36-kDa protein synthesized at 30 but not at 37 degrees Celsius was identified as LipL36, an outer membrane lipoprotein. In contrast, expression of another lipoptotein, LipL41 was the same at either temperature. Immunoblotting with convalescent equine sera revealed that some
proteins exhibiting thermoregulation of synthesis elicited antibody responses during infection. Another gene (Lk74.3), encoding a novel sphingomyelinase , has also been sequenced and expressed in E. coli. Finally, the antibody responses of late-term equine fetuses have been characterized and shown to be qualitatively different to those of their dams.
Impacts
These studies will be helpful in design of better immuno-diagnostics and vaccines against leptospirosis in horses and other Kentucky livestock.
Publications
- Sheoran, A.S., J.E. Nally, J.M. Donohue, B.J. Smith, and J.F. Timoney. 2000. Antibody isotypes in sera of equine fetuses aborted due to Leptospira interrogans serovar pomona-type kennewicki infection. Vet. Immunol. Immunopath. 77: 301-309.
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Progress 01/01/99 to 12/31/99
Outputs
Sera of 23 mares collected immediately after abortion caused by Leptospira pomona serovar kennewicki reacted strongly with antigen bands of 23, 34-39, 41, 51 and 75 kDa in a sonicate of serovar kennewicki. Rabbit antisera have been prepared against each band. All had strong agglutinating and opsonic activity. A library of serovar kennewicki DNA fragments (3-5 Kb) was prepared in Lambda ZAPII from a Tsp 5091 partial digest of chromosomal DNA and amplified. Twenty two plagues reactive with convalescent serum AWO were selected and subcloned. Two genes (Lk90 and Lk74) encoding 89,466 and 74,000 MW proteins were sequenced and expressed in E. coli. Lk90 appears to be a cytoplasmic protein of unknown function. Lk74 is a heat shock DNA K protein. Convalescent serum antibodies to Lk74 and Lk90 were not correlated with agglutinating antibodies. Uveitic eye fluids have been shown to have Lk90 and 74 specific antibody levels substantially higher than in companion sera suggesting
local stimulation and expansion of the appropriate B cell populations. Low and high molecular weight thermoregulated proteins have also been identified and are currently being sequenced and characterized. These proteins may be significant during in vivo infection at body temperature.
Impacts
These studies will be helpful in design of better immuno-diagnostics and vaccines for leptospirosis in horses and other Kentucky livestock
Publications
- No publications reported this period
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