Abstract Book of EAVLD2012 - eavld congress 2012

S1 - P - 38
DETECTION OF BOVINE LEUKAEMIA VIRUS PROVIRAL DNA IN DENDRITIC CELLS BY IN SITU PCR
Maria Szczotka, Jacek Kuźmak, Ewelina Iwan
National Veterinary Research Institute, Biochemistry Department, Pulawy, Poland
Bovine leukemia, dendritic cells, PCR in situ
Introduction
Bovine leukaemia virus (BLV), like human and simian T-
lymphotropic viruses (HTLV-I/II, STLV-I/II, STLV-L), belongs to
the Deltaretrovirus genus of Retroviridae family and causes
enzootic bovine leukosis (EBL), a disease of the
lymphoreticular system.
Dendritic cells (DCs) are most potent antigen presenting cells
(APCs) with unique ability take up and process antigens in the
peripheral blood and tissues. They subsequently migrate to
draining lymph nodes, where they present antigen to resting
lymphocytes. They express higher levels of MHC class II and
accesory molecules on their surface than other professional
APCs. DCs are generally considered as relatives of monocytes
and macrophages and to be of myeloid origin. They appear to
derive from progenitor also capable of forming granulocytes
and macrophages, although more recently a committed DC
progenitor, possibly a downstream precursor, has been
identified. The myeloid mature DCs emphasize by the direct
development of a form of DCs from blood monocytes. All these
lines of evidence for a myeloid origin of DC derive from culture
studies using GM-CSF. The aim of presented study was to
develop in situ PCR for the detection of proviral DNA of BLV in
DCs of naturally infected cattle. The results of provirus
detection by in situ PCR were compared with those obtained
using conventional PCR i.e. solution phase PCR
Materials & methods
Animals. Investigations were performed on the group of 9
naturally infected with BLV leukaemic and 5 healthy cows.
Samples. Blood samples, bone marrow, spleen and lymph
nodes were examined in this experiment. Blood was collected,
centrifuged in Histopaque gradient, density 1.077. Cells from
interphase were collected and monocytes were separated with
the use of magnetic microbeads labelled with MoAb CD14.
Samples of lymphoid organs were cut, digested with
collagenase-DNase, centrifuged and incubated with
immunomagnetic microbeads coated with monoclonal
antibody. Cells were passed through the magnetic sorter, the
cells retendent on the magnet were eluted, washed and placed
in cell culture for 2 weeks in RPMI in the presence of IL-4 and
GM-CSF. Cells were grown in special labteks in incubator at
37 o C in 5% of CO 2 . After that cells were washed, fixed and
PCR in situ was performed in termocycler. Detection was
performed with the use of anti-DIG peroxidase conjugate and
AEC or alkaline phosphatase substrates.
Immunofluorescence test (IF) and flow cytometry were
performed with the use of MoAbs anti gp51 –BLV glycoprotein
for determination of gp51 expression. Flow cytometry was
used for determination of DCs immunophenotype in healthy
and naturally infected with BLV cattle. Expression of cytokines:
IL-6, IL-10, IL-12p40 and IL-12p70 was measured with
commercial ELISA sets.
Control. As positive control the foetal lamb kidney
permanently infected with BLV - (FLK-BLV) cell line was used
Results
Generated from the blood and inner organs dendritic cells had
typical shape, veils and processes. The expression of gp51
glycoprotein was determined by flow cytometry.
In dendritic cells infected with BLV we observed very high
percentage of determinants: CD11a, CD11b, CD11c and
MHC-II class and very high expression of IL-6, IL-10. IL-12p40
and IL-12p70 in culture fluids. Leukemic DCs exhibited DCs
morphology and had a phenotype of mature DCs. The
expression of gp51 glycoprotein of BLV on leukaemic DCs was
detected in flow cytometry and immunofluorescence. The cells
of samples with positive reaction in in situ PCR were dark
brown stained. The negative controls were unstained. In situ
PCR results agreed with those of provirus detection using
conventional PCR and localization of proviral material in cells
was visible.
The results of PCR in situ:
DCs-blood DCs - spleen DCs bone marrow
DCs – blood
T
The results of IF:
DCs - spleen
Discussion & conclusions
In the present study, DCs generated from peripheral blood and
different lymphoid tissues of cattle naturally infected with BLV,
were examined for the presence of proviral DNA. In the first
approach, called also direct in situ PCR, dNTP’s, labelled with
digoxygenin, biotin or other markers, are incorporated into
amplification products; the second approach involves two
stage process which combines in situ PCR plus in situ
hybridization with DNA probe. In our study we have used the
first strategy and digoxigenin labelled dNTP’s. The presence of
gp51 expression was determined as well in IF as in flow
cytometry. We obtained agreement in results of in situ PCR
with results of other test. The DCs infected with BLV had more
delicate morphology with many vacuoles in cytoplasm.
Infection with BLV caused an increase in cytokines expression
and changes in the percentage of surface molecules on
dendritic cells. With the use of in situ PCR the localization of
proviral load could be detected.
Application of in situ PCR would be recommended as a
method to confirmation of infection with BLV.
Acknowledgements
The research was supported by the Polish State Committee for
Scientific Research, grant No N N 308 622 138. The authors
thank the Polish State Committee for Scientific Research for
financial support.
References
1.Mirsky M.L., Olmstead C.A., Da Y., Lewin H.A. 1996: The prevalence
of proviral bovine leukemia virus in peripheral blood mononuclear cells
at two subclinical stages of infection. J Virol, 70, 2178-2183.
2. Naif H.M., Brandon R.B., Daniel R.C.W., Lavin M.F. 1998: Bovine
leukaemia proviral DNA detection in cattle using the polymerase chain
reaction. Vet Microbiol, 1, 25, 117-129.
3. Steinman R.M. 1991. The dendritic cell system and its role in
immunogenicity. Annu Rev Immunol. 9, 271-296.
4. Ballagi-Pordany A., Klintevall K., Merza M., Klingeborn B., Belak S.
1992 : Direct detection of bovine leukemia virus infection: Practical
applicability of a double polymerase chain reaction. J Vet Med B, 39,
69-77.