The polarity of tyrosine 67 in yeast iso-1-cytochrome c monitored by second derivative spectroscopy.

The average tyrosine polarity of 10 yeast iso-1-cytochrome c proteins and two horse heart cytochrome c proteins was assayed by second derivative spectroscopy. Yeast iso-1-cytochrome c contains five tyrosines, one of which (tyrosine 67) is in the heme pocket. The wild-type protein and the Y67F, N52V Y67F, and N521 Y67F proteins were used to differentiate events that were occurring in or near the heme pocket from those occurring closer to the protein's surface. The wild-type protein shows a substantial change in the second derivative spectrum as the protein goes from oxidized to reduced; mutants lacking tyrosine 67 do not show this change. This indicates that it is primarily the spectrum of tyrosine 67 that changes as the protein cycles between the oxidized and reduced state. One thing that contributes to the overall polarity of the heme pocket is a water molecule hydrogen bonded to several of the nearby residues. The wild-type protein has one water molecule in the heme pocket but this can be increased or decreased by introducing mutations into the protein. N52A has two water molecules and N52I has no water molecule in the pocket. The three proteins allowed us to assess the contribution of water to the inferred heme crevice polarity. The number of water molecules in the crevice correlates with the perceived polarity of the pocket when one takes account of the fact that the second water molecule in the crevice of the N52A mutant takes the position and hydrogen bonding pattern of the amide it replaces.

The effect of Trypanosoma brucei brucei infection on rabbit plasma iron and zinc concentrations.

Changes in plasma iron and zinc concentration were studied in rabbits following a needle challenge with Trypanosoma brucei brucei clone ILTat 2.1. The infection resulted in a decrease of the concentration of both trace elements. Plasma iron concentrations decreased gradually and were decreased maximally to 52.3% of pre-infection levels on day 18 post-inoculation. Plasma zinc concentrations, on the other hand, decreased more rapidly and were decreased maximally to 27.4% of pre-infection levels on day 3 post-inoculation. The onset of these decreases coincided with the appearance of parasites in the peripheral blood. Furthermore, the magnitude of their decrease correlated closely with the level and duration of the parasitaemia. Other abnormal findings, namely, anaemia and periods of leucocytosis and leukopenia, were also observed. This study therefore demonstrates that depression in plasma iron and zinc concentrations is part of the acute phase response in rabbits infected with this clone of T. b. brucei.

A colorimetric assay for trypanosome viability and metabolic function.

We have adapted a tetrazolium salt (MTT) colorimetric cytotoxicity assay to the assessment of viability and metabolic function in cultured African trypanosomes. Trypomastigotes of Trypanosoma congolense and T. brucei rhodesianse were harvested from the blood of parasitemic rats and cultured under axenic conditions that support trypanosome viability and growth. Analysis of serial dilutions of these bloodstream forms indicated that the assay could detect 10(4) parasites. To assess the effect of lymphoid cytokines on trypomastigote viability, 10(5) freshly harvested parasites were cultured with a wide range of dilutions of human recombinant IL-1, IL-3, IL-6, interferon-gamma (IFN gamma) or tumor necrosis factor-alpha (TNF alpha), or bovine recombinant IFN gamma or TNF alpha for 24, 48 or 96 h. These cytokines had no apparent growth enhancing or inhibitory effect on the trypomastigotes compared with growth in supplemented medium alone. This assay has several advantages over traditional counting methods, including increased sensitivity and rapid, repeatable quantitation. This adaptation of the MTT colorimetric assay should be useful in screening drugs and host-derived factors for growth-modulating effects on trypanosomes and other extracellular protozoan parasites.

Purification of sn-glycerol-3-phosphate dehydrogenase from Trypanosoma brucei brucei.

A protein has been purified from the membranes of bloodstream forms of Trypanosoma brucei brucei. The purified material contained a single polypeptide chain of molecular mass 67 kilodaltons as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; under "native" conditions it migrated through a Sephacryl S-300 column with a similar molecular mass. The purified protein catalysed electron transfer from sn-glycerol 3-phosphate to oxygen with the subsequent formation of water. Electron transfer by the purified enzyme to O2 was dependent on the presence of low concentrations of the mediator phenazine methosulfate. This protein is clearly the major membrane-bound sn-glycerol-3-phosphate dehydrogenase, but it also has some characteristics suggestive of the trypanosome alternative oxidase activities.

Experimental infection with a haemorrhage-causing Trypanosoma vivax in N'Dama and Boran cattle.

N'Dama cattle control experimental infections with clones of Trypanosoma congolense of varying degrees of virulence, but nothing is known about their capacity to control infections caused by highly virulent, East African stocks of T. vivax. Thus four N'Damas and four trypanosusceptible Borans were infected with a tsetse-transmitted stock of T. vivax IL2337. In Ayrshire cattle this stock is known to cause severe haemorrhagic disease. No differences were observed in the parasitaemia between the two groups. Both groups became anaemic. The mean packed cell volume fell to 16.8 +/- 5.0% in the N'Dama cattle and to 24.2 +/- 2.2% in the Borans on day 26 post infection. These differences were not significant. Antibody responses to invariant trypanosome antigens were analysed. No differences were observed between the groups in the pattern of recognition or the isotype elicited. Antibody bound to the surface of erythrocytes was occasionally detected. No anti-platelet activity was observed. The results show that N'Dama cattle, which are known to be resistant to disease caused by T. congolense and by T. vivax stocks from West Africa, were highly susceptible to an infection of T. vivax which causes acute haemorrhagic disease.

Susceptibility of N'Dama and Boran cattle to tsetse-transmitted primary and rechallenge infections with a homologous serodeme of Trypanosoma congolense.

Eight trypanotolerant N'Dama cattle controlled an infection of Trypanosoma congolense ILNat 3.1 transmitted by Glossina morsitans centralis, more efficiently than a group of similarly infected trypanosusceptible Boran cattle. All eight N'Damas maintained their PCV above 15% throughout the primary infection whereas the PCV of six of the eight Borans dropped below 15%; these latter animals were treated with diminazene aceturate to prevent possible death. Lymphocyte, neutrophil and platelet counts also decreased in the Boran during the primary infection. In contrast, a lymphocytosis was observed in the N'Dama; and although the neutrophil and platelet counts decreased, the drop was less severe than in the Boran. Two years after the primary infection and immediately prior to a homologous rechallenge infection, all eight N'Damas had neutralizing anti-metacyclic trypanosome variant-specific antibodies present in their sera compared to five of the eight Borans. Following the homologous rechallenge infection the eight N'Damas became parasitaemic but there were no alterations in their erythrocyte or leukocyte counts. The Borans became highly parasitaemic and developed severe, chronic anaemia and leukopaenia. Thus, the trypanotolerant N'Damas controlled a primary infection of T. congolense more efficiently than trypanosusceptible Boran cattle and eliminated a homologous rechallenge infection without the pathology associated with the disease.

Susceptibility of N'Dama and Boran cattle to sequential challenges with tsetse-transmitted clones of Trypanosoma congolense.

The susceptibility of N'Dama cattle (Bos taurus) to four consecutive infections with different tsetse-transmitted clones of Trypanosoma congolense was compared with that of Borans (Bos indicus). All animals were aged 13 months at the start of the study and had been born and raised free from trypanosomiasis under the same management and nutritional conditions, thereby limiting environmental factors that could have influenced susceptibility. While cattle of both breeds were equally susceptible to the establishment of trypanosome infections, the N'Damas exhibited superior resistance. Despite infection with virulent parasites, the N'Damas gained weight at the same rate as uninfected control animals, they did not develop anaemia to the extent that trypanocidal drug treatment was required, and all made a spontaneous recovery to normal haematological values within two to four months. In contrast, all the Borans needed treatment during the course of the four infections because of severe anaemia and showed markedly reduced liveweight gains. These clinical differences in the N'Damas were associated with two repeatable characteristics, namely, the ability to control parasitaemia and to 'resist' anaemia, processes that did not appear to be linked. Also in contrast to the Borans, the N'Damas were able to mount accelerated haemopoietic responses, resulting in the reduced severity of anaemia following a primary infection. These findings pose the question as to whether the ability to control parasitaemia and to 'resist' anaemia could be used as criteria for identifying resistant or trypanotolerant cattle.

Analysis of peripheral leucocyte populations in N'Dama and Boran cattle following a rechallenge infection with Trypanosoma congolense.

Monoclonal antibodies, flow cytometry and routine haematological techniques were used to analyse circulating leucocyte populations in trypanotolerant (N'Dama) and trypanosusceptible (Boran) cattle following a homologous rechallenge with Trypanosoma congolense clone IL13-E3. The N'Damas developed a low, transient parasitaemia and did not develop anaemia. The Borans became parasitaemic and developed chronic anaemia but three of the five animals eventually self-cured, whilst, a group of primary-challenged Borans experienced a severe infection characterized by high levels of parasitaemia and acute anaemia. During infection the numbers of circulating B-cells increased in all three groups from day 21 onwards. The proportion of B-cells expressing the CD5 antigen increased from pre-infection levels of 5-10% of B-cells to 49-90% by day 19 post infection in all three groups. The neutrophil count declined in both Boran groups but not in the N'Damas. The CD4+ T-cell and gamma delta T-cell populations decreased in both Boran groups but did not alter significantly in the N'Damas. Although it was not possible to infer from the data, that the CD4+, gamma delta T-cell, neutrophil and erythrocyte populations were directly responsible for the differential control of the disease by the two breeds, it was possible to correlate alterations in these cell populations with the severity of the disease.

The effect of temperature and storage on the infectivity and motility of African animal trypanosomes in the blood of different hosts.

Blood from mice, rats, goats or cattle infected with Trypanosoma congolense, T. vivax or T. brucei was stored at 0-4 degrees C, 20-25 degrees C, 30-35 degrees C or 36-40 degrees C. Each sample was examined after set intervals to determine the maximum period the trypanosomes could remain motile and infective. T. brucei in blood remained motile for 96 h at 0-4 degrees C, being the longest period that was observed, but remained infective for only 8 h. T. vivax survived poorly in rodent blood, but did well in ruminant blood, especially at 20-25 degrees C, whereas T. congolense and T. brucei survived well in rodent blood. The infectivity and motility of the three species of trypanosomes were adversely reduced at temperatures above 36 degrees C.

A comparative evaluation of the parasitological techniques currently available for the diagnosis of African trypanosomiasis in cattle.

The parasitological techniques currently in use for the diagnosis of African trypanosomiasis were compared in a series of experiments for their capacity to detect Trypanosoma congolense, T. vivax and T. brucei in the blood of cattle. The darkground/phase contrast buffy coat method proved to be more sensitive than the haematocrit centrifugation technique, thick, thin and wet blood films in detecting T. congolense and T. vivax. On the other hand with T. brucei, mouse inoculation was the most sensitive method, followed by the haematocrit centrifugation technique. In a further series of experiments involving cattle infected with either T. congolense or T. vivax, the darkground/phase contrast buffy coat method was consistently more sensitive in detecting parasites than haematocrit centrifugation, capillary concentration using glycerol and miniature anion-exchange/centrifugation techniques. As well as showing superior sensitivity, the darkground/phase contrast buffy coat method allowed species identification, estimation of parasitaemia and simultaneous assessment of anaemia (packed red cell volume).