Cellular responses and parasites kinetics associated with the development of local skin reactions were examined in
sheep following infection with metacyclic forms of Trypanosoma (Nannomonas) congolense. Leucocyte phenotypes in the
skin and regional draining lymph nodes were examined by indirect immunoperoxidase staining using monoclonal antibodies
specific for ovine leucocyte subsets. Immunofluorescence and flow cytometry was used to determine changes in the numbers
and proportions of different cell phenotypes in peripheral blood and in lymph from cannulated afferent and efferent lymphatic
ducts draining both the skin reaction and regional nodes respectively. Cellular reactions occurring in the skin prior to
development of local skin reactions were investigated by light and transmission electron microscopy.
Trypanosomes were observed in the skin during the first four days of infection. Following development of the local
skin reaction, and histological demonstration of trypanosomes in the skin and draining lymph nodes five to seven days after
infection, large numbers of parasites appeared in the afferent lymph, reaching peak numbers seven to 10 days after infection.
During this period, trypanosomes were also present in the efferent lymph. Trypanosomes were not detectable in skin or
draining lymph nodes after 13 days but persisted at a low level in both afferent and efferent lymph. Parasites did not appear
in peripheral blood until 15 days after infection.
Cellular responses varied according to the stage of development of local skin reactions and the presence of
trypanosomes in the various compartments. Prior to development of local skin reactions, the only cellular event observed
histologically by transmission electron microscopy was evidence of mast cell degranulation. Local skin reactions developed
at inoculation sites of sheep from day five after infection and were initially characterized by an infiltrate of mononuclear cells
and neutrophils. Mononuclear cell infiltrate comprised equal proportions of T cells (CD5+, CD4+ and CD8+) and B cells
(CD45R+), MHC Class II+ cells and macrophages. B cells and MHC Class II+ cells were found mainly in aggregates,
suggesting local proliferation and antibody production. There was a greater proportion of CD4+ cells than CD8+ cells, but
SBU-T19+ (tS T cells) were rarely present. The marked cellular response in afferent lymph during this time was
characterized by both an absolute and proportional increase in T cells, particularly those expressing CD4. Expansion of the B
cell population was observed in draining lymph nodes while a predominantly lymphoblastic, B cell (CD45R+, SIg+) and
MHC Class II+ cell response was observed in efferent lymph.
As the local skin reaction started to regress 10 days after infection, the cell infiltrate was predominantly
mononuclear, composed of equal numbers of CD4+ and CD8+ cells and a few B cells, MHC Class II+ cells and SBU-T19*
cells. The afferent lymph still contained high numbers but lower proportions of T cells (CD4+ and CD8+ cells) and also
increased numbers and proportions of lymphoblasts, B cells (CD45R+, SIg+ cells) and MHC Class II+ cells indicating that
these cells were leaving the skin. The B cell response persisted in draining regional nodes and efferent lymph. Changes in
peripheral blood leucocyte subpopulations did not occur until 15 to 38 days after infection when a gradual increase in B cells
and MHC Class II+ cells and decline in T cells was observed.
C Class II+ cells and decline in T cells was observed.
Observations of afferent lymph using light and transmission electron miscroscopy showed that trypanosomes were
attached to, and phagocytosed by macrophages/dendritic cells. Further evidence of the host-effector response was the
presence of numerous lysed trypanosomes in the skin, afferent and efferent lymph from day eight, suggesting that lytic
antibodies were being produced.
dies were being produced.
Sheep immunized by infection and trypanocidal drug therapy failed to develop local skin reactions following
challenge with an homologous serodeme: trypanosomes were absent from the afferent lymph and there was no evidence of a
cellular response. Similarly, following challenge of infected sheep with an heterologous serodeme, skin reactions failed to
develop, no trypanosomes were seen in draining lymphatics and there was no evidence of a cellular response in efferent
lymph. Hence, both homologous immunity and interference in establishment of secondary infection appears to operate at the
level of the skin.
Proliferation of trypanosomes in the skin is crucial for establishment of infection. The marked cellular responses
elicited in the skin and draining lymph nodes are however ineffective in preventing the onset of infection but are important in
subsequent development of homologous immunity following infection and therapy.