Source: UNIV OF CALIFORNIA (VET-MED) submitted to
EXCRETED-SECRETED ANTIGENS OF NEOSPORA CANINUM: DIAGNOSTIC AND VACCINE POTENTIAL
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0200085
Grant No.
(N/A)
Project No.
CALV-AH-207
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2003
Project End Date
Sep 30, 2006
Grant Year
(N/A)
Project Director
Andrianarivo, A. G.
Recipient Organization
UNIV OF CALIFORNIA (VET-MED)
(N/A)
DAVIS,CA 95616
Performing Department
PATHOLOGY, MICROBIOLOGY & IMMUNOLOGY
Non Technical Summary
Neosporosis is a major cause of abortion and congenital infection in dairy and beef herds. Vertical transmission from an infected dam to her fetus is the major route of infection, and can occur in the dam in multiple pregnancies. Reactivation of a latent infection occurs during pregnancy, resulting in repeat fetal infections. The primary purpose of this study is to identify markers for acute and chronic infection
Animal Health Component
20%
Research Effort Categories
Basic
60%
Applied
20%
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3113310109040%
3113310110310%
3113410109040%
3113410110310%
Goals / Objectives
1)Evaluate the excreted-secreted antigens (ESA)-specific antibody responses in experimentally- and naturally-infected cattle using immunoblot analysis; Identify ESA(s) markers that are most frequently recognized during acute infection in experimentally infected cattle and following reactivation of a latent infection during pregnancy in naturally-infected cattle; 2)Prepare affinity-purified antibodies (Abs) from nitrocellulose blots immobilized with the ESA(s) markers identified in Objective 1; 3)Investigate the role of the ESA(s) markers identified in Objective 1 in host-cell invasion and intra-cellular development of the tachyzoites, using the affinity-purified Abs prepared in Objective 2; Identify the ESA(s) that are involved in host-cell invasion and/or intra-cellular development of tachyzoites; 4)Determine the nature of the ESA(s) involved in host-cell invasion that are identified in Objective 3, by screening an Neospora caninum lambda gt 11 cDNA library with the affinity purified Abs prepared in Objective 2.
Project Methods
We will assess the antibody (Ab) responses to excreted-secreted antigens (ESA) in experimentally and naturally infected cattle using immunoblot analysis. We will identify ESA(s) that most consistently react with Abs during acute infection in experimentally infected cattle, and following reactivation of a latent infection in naturally infected cattle. We will prepare affinity-purified Abs to these ESA(s) and use them to investigate the role of the ESA(s) in host-cell invasion. We will use the affinity purified Abs to screen cDNA library of Neospora caninum, and will determine the nature of the clones isolated. We will express the clones and develop recombinant antigen ELISA that will be able to differentiate acute from chronic infection.

Progress 10/01/03 to 09/30/06

Outputs
Neosporosis is a major cause of infectious bovine abortion. Most cattle acquire the infection as a fetus from their latently infected dam. Reactivation of the latent infection occurs during pregnancy, resulting in repeat fetal infections. The fetal outcome of the infection, i.e. abortion or congenital infection, depends on the timing of parasite reactivation during gestation. In this study we assessed the frequency of the excreted-secreted antigens (ESA) of Neospora caninum during active N. caninum infection in experimentally infected cattle and following reactivation of a latent infection during pregnancy in naturally-infected cattle. Neospora caninum ESA were separated by gel electrophoresis, transferred on nitrocellulose, and probed with serum samples collected during pregnancy from five experimentally-infected cattle and two uninfected controls, as well as serum samples from 21 naturally-infected cattle and five uninfected controls. Our data suggest that a 50-53 kDa doublet is a good marker of active infection in experimentally-infected cattle. In naturally-infected cattle which transmitted the infection to their progeny, a 31, a 38 and a 50-53 kDa antigens were detected at most time-points examined, with a significantly increased intensity at mid-gestation and/or during the third trimester of gestation. Serum samples from the only naturally-infected cow which did not vertically transmit, did not react with any N. caninum ESA. Moreover, serum samples from uninfected cattle did not recognize any N. caninum ESA. Therefore, these ESA appear to be good markers for reactivation of latent N. caninum infection during pregnancy in naturally-infected cattle.

Impacts
Results from this study lead to the identification of specific markers of acute/active and chronic/latent N. caninum infection in naturally-infected cattle. These markers could be used to develop assays to detect active infection and thereby to identify chronically infected cattle which are at high-risk of reactivation. This could enable veterinarians and farmers to more accurately predict the risk of abortion in endemic and epidemic situations.

Publications

  • No publications reported this period


Progress 01/01/05 to 12/31/05

Outputs
The protozoal parasite Neospora caninum is an important worldwide cause of bovine abortion. Most cattle acquire the infection as a fetus from their latently infected dam. Reactivation of the latent infection occurs during pregnancy, resulting in repeat fetal infections, some culminating in abortion, most leading to a new generation of persistently infected cattle. The timing of parasite reactivation during gestation is crucial, as it determines the fetal outcome of the infection, i.e. abortion or congenital infection. In this study, we assessed the potential of using the excreted-secreted antigens (ESA) of N. caninum as markers of active infection and of reactivation of a latent infection, based on similar studies in the closely related Toxoplasma gondii. N. caninum ESA were separated by gel electrophoresis, transferred on nitrocellulose, and probed with serum samples collected during pregnancy from five experimentally-infected cattle and two uninfected controls, as well as serum samples from 21 naturally-infected cattle and five uninfected controls. Our data suggest that a 50-53 kDa doublet is a good marker of active infection in experimentally-infected cattle. In naturally-infected cattle which transmitted the infection to their progeny, a 31, a 38 and a 50-53 kDa antigens were detected at most time-points examined, with a significantly increased intensity at mid-gestation and/or during the third trimester of gestation. Moreover, these ESA were not recognized by the only cow which did not vertically transmit, nor by the uninfected cattle. Therefore, these ESA appear to be good markers for reactivation of latent N. caninum infection during pregnancy in naturally-infected cattle. Studies are underway to characterize these antigens.

Impacts
Results from this study will lead to the identification of specific markers of acute/active and chronic/latent N. caninum infection in naturally-infected cattle. These markers will be used to develop methods to detect active infection and thereby to identify chronically infected cattle which are at high-risk of reactivation. This will enable veterinarians and farmers to more accurately predict the risk of abortion in endemic and epidemic situations. Moreover, these markers can be used as potential vaccine targets to prevent vertical transmission.

Publications

  • No publications reported this period


Progress 01/01/04 to 12/31/04

Outputs
Neosporosis is a worldwide major cause of bovine abortion. Most cattle acquire the infection as a fetus from their latently infected dam. Reactivation of the latent infection occurs during pregnancy, resulting in repeat fetal infections, some culminating in abortion, most leading to a new generation of persistently infected cattle. The timing of parasite reactivation during gestation is crucial, as it determines the fetal outcome of the infection, i.e. abortion or congenital infection. Excreted-secreted antigens (ESA) have been used as markers of active infection and of reactivation of a latent infection with the closely related parasite Toxoplasma gondii. Using immunoblot analysis of T. gondii-ESA, markers of acute and chronic T. gondii infection were identified in sera from T. gondii-infected humans. In the present study, we use the same approach to assess the frequency of ESA of Neospora caninum (NcESA) during active N. caninum infection in experimentally infected cattle and following reactivation of a latent infection during pregnancy in naturally-infected cattle. After optimizing the western blot analysis conditions, we began to examine the IgG immunoreactivity of serum samples from infected and uninfected control cattle. Our data suggest that a 38 kDa antigen and a 50-53 kDa doublet are good markers of active infection in experimentally-infected cattle. The 38 kDa antigen and a 15-17 kDa antigen, although present at all time points examined, had their intensity significantly increased starting at mid-gestation in naturally-infected cattle, and are therefore potential markers of reactivation of a latent N. caninum infection. So are a group of ESA bands between 45-65 kDa which were first detected at mid-gestation in naturally-infected cattle, with a maximum intensity between week 22-28 of gestation, and decreasing thereafter. Serum samples from uninfected cattle that were sofar analyzed were not reactive against any NcESA.

Impacts
The goal of the present study is to identify specific markers that can distinguish acute from chronic N. caninum infection. These markers can be used to develop methods to detect active infection and reactivation of latent N. caninum infection in chronically infected cattle. These markers can also be potential targets for the development of vaccines to prevent abortion and/or fetal infection.

Publications

  • No publications reported this period