Bovine stem cell factor: production of a biologically active protein and mRNA analysis in cattle infected with Trypanosoma congolense.

The cDNA coding for the soluble form of bovine stem cell factor (boSCFAla165) was cloned and recombinant protein was produced in bacteria as a histidine tagged-protein. The protein was purified from the inclusion bodies in one step by metal chelation chromatography under denaturing conditions. Recombinant bovine SCF was shown to act synergistically with interleukin 3 (IL-3) and erythropoietin (EPO) in stimulating the growth of bone marrow progenitor cells such as colony forming units-granulocyte macrophage (CFU-GM) and burst forming units-erythroid (BFU-E). Analysis of SCF mRNA expression by reverse transcription-polymerase chain reaction (RT-PCR) revealed that the transcripts were detectable in bone marrow, lymph nodes and spleen of cattle, and that the level of transcription was upregulated in lymph nodes of cattle infected with Trypanosoma congolense. Two isoforms of SCF mRNA were amplified by RT-PCR. The availability of recombinant bovine SCF provides a valuable tool for studying the role of SCF in the development, growth and differentiation of bovine hematopoietic cells.

Biochemical analysis of preliminary clusters in the non-lineage panel.

After the preliminary clustering of the whole workshop panel using flowcytometry data, we selected those clusters that were composed of mAbs that bound a broad range of cells. To obtain evidence that mAbs in each of these preliminary clusters detected the same antigen, they were tested for their capacity to compete with each other for binding to a target cell and the molecular weights of their antigens were estimated after immunoprecipitation. Most preliminary clusters in the non-lineage panel contained control mAbs that had been characterised in one of the previous workshops, and this study therefore increased the number of mAbs available to each of these non-lineage antigens. One new interesting cluster, BoWC14, was described which defined an antigen on myeloid cells and a subpopulation of CD4, CD8 and WC1 T lymphocytes (BT3/8.12 and IL-A155). An additional cluster, which did not fit into any other panel, contained mAbs specific for an antigen restricted to the erythroid lineage and received the nomenclature BoWC15 (ANA8, IL-A135, IL-A137, IL-A138 and IL-A160).

Trypanosoma congolense: high erythropoietic potential in infected yearling cattle during the acute phase of the anemia.

N'Dama (Bos taurus) cattle are known to tolerate trypanosome infections, developing less severe anemia and lower parasitemia, compared to Boran (Bos indicus) cattle. Young calves were also reported to be more resistant to trypanosomiasis than adult cattle. To explore the basis for this resistance, the erythropoietic response was evaluated in four native yearling N'Dama calves and four age-matched Boran calves which developed anemia over a 140- day primary infection with Trypanosoma congolense clone IL 13E3. Similar levels of parasites were detected in the two breeds until 42 days postinfection (dpi). During the same period, a rapid and greater colony-forming units-erythroid response in the bone marrow of yearling Boran calves, compared with N'Dama calves, may explain the unusual absence of breed differences in mean packed cell volumes (PCV). However, this early erythropoietic response was transient and did not result in any rise in PCV from 70 dpi onward. In contrast, in the N'Dama calves, following the erythroid response, the mean PCV was gradually compensated from 56 dpi onward and reached 30% by 126 dpi. This period of PCV recovery coincided with low and intermittent parasitemia and a return of the erythroid progenitor levels to near preinfection values. Elevated levels of erythroid progenitors in the N'Dama calves, occurring 1 week after trypanocidal treatment, returned the PCV to preinfection values. These results suggest that the age of the Bos indicus cattle has an important impact on the early bone marrow response in primary T. congolense infection and confirmed previous suggestions of a high erythropoietic potential in trypanosome-infected young calves.

Trypanosoma congolense: comparative effects of a primary infection on bone marrow progenitor cells from N'Dama and Boran cattle.

Using in vitro clonogenic assays, the changes in haemopoietic progenitor cell levels were compared in the bone marrow of three adult trypanotolerant N'Dama cattle and three age-matched trypanosusceptible Boran cattle over 17 weeks (119 days) of a primary Trypanosoma congolense (clone IL 1180) infection. As the infection progressed, a clear tendency of the parasitaemia to decrease was seen in the N'Damas, while it remained high throughout the infection in the Borans. The decline in the colony-forming units-granulocyte macrophage (CFU-GM) between 7 and 42 days postinfection (dpi) corresponded with the decreased numbers of neutrophils and monocytes in the blood observed in both breeds. Thereafter, a further significant drop in the CFU-GM levels was observed in the Borans which may partially explain the continued decrease in the numbers of neutrophils and monocytes in blood. In contrast, a significant peak of CFU-GM above preinfection levels was observed in the N'Damas on 49 dpi, which could partially explain the subsequent recovery of the numbers of neutrophils and monocytes in blood. When compared to the N'Damas, the Borans had a more dramatic drop in the packed cell volume (PCV) from 25 dpi onwards, resulting in significantly lower PCV. From 46-49 dpi onwards, the mean PCV stabilised at significantly lower levels in the Borans than in the N'Damas. The mean corpuscular volume (MCV) levels increased in both breeds, but at a much faster rate in the Borans. The clonogenic assays demonstrated an erythropoietic response, characterised by peaks above pre-infection levels of both the early and late erythroid progenitor cells (respectively, burst-forming units-erythroid, BFU-E, and colony-forming units-erythroid, CFU-E), occurring between 35 and 70 dpi in both breeds of cattle. However, despite a more severe anaemia in the Borans, the magnitude of their erythroid response was similar to that of the N'Damas, suggesting that the response of the Borans was insufficient to compensate for the greater degree of anaemia. Moreover, the mean PCV did not improve in the Borans, indicating the ineffectiveness of their erythropoietic response. An increased rate of erythrocyte destruction and/or a defective differentiation and maturation of erythroid precursors have also been shown to be partially responsible for this persistent anaemia. From 98 dpi onwards, despite the persistent low PCV, the MCV decreased to preinfection levels and low CFU-E numbers were observed in the Borans. Over the same period, in the N'Damas the mean PCV progressively increased to reach 25%, which fell within the low normal range for cattle.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression of antigens on haemopoietic progenitor cells in bovine bone marrow.

We analysed the surface phenotype of bovine bone marrow erythroid and myeloid progenitor cells with monoclonal antibodies (mAbs) from the Second Workshop. For the antibodies tested, no difference could be observed in burst-forming unit (erythroid) and colony-forming unit (erythroid) both are positive for BoCD44, BoWC9, MHC Class I, transferrin receptor and the p150/158 antigen detected by BT3/8.12, but neither express BoCD11a, BoCD45, BoWC5 or the antigen recognized by mAb Bo116. The myeloid progenitor cells, colony-forming unit (granulocyte/macrophage), can be discriminated from the erythroid progenitors by the absence of a transferrin receptor and the expression of BoCD11a and BoWC5 antigens. By selecting the right panel of mAbs, it should now be possible to enrich bone marrow cells for erythroid and/or myeloid progenitor cells.

Characterization of bovine haemopoietic progenitor cells using monoclonal antibodies and fluorocytometry.

Monoclonal antibodies against bovine leucocyte cell surface differentiation antigens were used in combination with a fluorescence activated cell sorter to enrich bovine haemopoietic progenitor cells present in bone marrow cell populations prior to in vitro culture. After two sequential centrifugations of the bone marrow cell suspension through Ficoll-Paque, the interface fraction was stained with a cocktail of monoclonal antibodies directed against mature monocytes/macrophages, granulocytes and lymphocytes. Using appropriate electronic window settings on a FACStar Plus, cells with a high 90 degrees light scattering property (granular cells), a low forward light scattering property (erythrocytes and reticulocytes) and cells positive for monoclonal antibodies specific for lineage-restricted leucocyte markers were removed and the negative cell fraction collected. These negatively-selected cells were stained with monoclonal antibodies specific for a pan-leucocyte or a MHC class II marker and the positive cell population was collected in a second sort and subsequently submitted to culture. All erythroid and granulocyte/macrophage colony forming cells expressed MHC class II antigens, as well as the pan-leucocyte antigen. These same progenitors did not bind any of a variety of monoclonal antibodies directed against lineage-specific antigens on lymphocytes, granulocytes or monocytes/macrophages, although they did bind monoclonal antibodies recognizing MHC class I antigens. Between 85% and 91% of the isolated cells seeded were capable of forming erythroid or granulocyte/macrophage colonies within 5 to 10 days, thus increasing the plating efficiency of these cell types in bone marrow populations by at least 60 fold.