Mutations of the active site residues F87 and Y96 greatly enhanced the activity of cytochrome P450(cam) (CYP101) from Pseudomonas putida for the oxidation of the polycyclic aromatic hydrocarbons phenanthrene, fluoranthene, pyrene and benzo[a]pyrene. Wild-type P450(cam) had low (<0.01 min(-1)) activity with these substrates. Phenanthrene was oxidized to 1-, 2-, 3- and 4-phenanthrol, while fluoranthene gave mainly 3-fluoranthol. Pyrene was oxidized to 1-pyrenol and then to 1,6- and 1,8-pyrenequinone, with small amounts of 2-pyrenol also formed with the Y96A mutant. Benzo[a]pyrene gave 3-hydroxybenzo[a]pyrene as the major product. The NADH oxidation rate of the mutants with phenanthrene was as high as 374 min(-1), which was 31% of the camphor oxidation rate by wild-type P450(cam), and with fluoranthene the fastest rate was 144 min(-1). The oxidation of phenanthrene and fluoranthene were highly uncoupled, with highest couplings of 1.3 and 3.1%, respectively. The highest coupling efficiency for pyrene oxidation was a reasonable 23%, but the NADH turnover rate was slow. The product distributions varied significantly between mutants, suggesting that substrate binding orientations can be manipulated by protein engineering, and that genetic variants of P450(cam) may be useful for studying the oxidation of polycyclic aromatic hydrocarbons by P450 enzymes.

In the absence of national control programmes against Rhodesian human African trypanosomiasis, farmer-led treatment of cattle with pyrethroid-based insecticides may be an effective strategy for foci at the edges of wildlife areas, but there is limited evidence to support this. We combined data on insecticide use by farmers, tsetse abundance and trypanosome prevalence, with mathematical models, to quantify the likely impact of insecticide-treated cattle. Sixteen percent of farmers reported treating cattle with a pyrethroid, and chemical analysis indicated 18% of individual cattle had been treated, in the previous week. Treatment of cattle was estimated to increase daily mortality of tsetse by 5-14%.

23In the absence of national control programmes against Rhodesian human African 24 trypanosomiasis, farmer-led treatment of cattle with pyrethroid-based insecticides may be an 25 effective strategy for foci at the edges of wildlife areas, but there is limited evidence to 26 support this. 27 We combined data on insecticide use by farmers, tsetse abundance and trypanosome 28 prevalence with mathematical models to quantify the likely impact of insecticide-treated 29 cattle. 30 Sixteen percent of farmers reported treating cattle with a pyrethroid, and chemical analysis 31 indicated 18% of individual cattle had been treated, in the previous week. Treatment of cattle 32 was estimated to increase daily mortality of tsetse by 5 -14%. Trypanosome prevalence in 33 tsetse, predominantly from wildlife areas, was 1.25% for T. brucei s.l. and 0.03% for T. b. 34 rhodesiense. For 750 cattle sampled from 48 herds, 2.3% were PCR positive for T. brucei s.l. 35 and none for T. b. rhodesiense. Using mathematical models, we estimated there was 8 -29% 36 increase in mortality of tsetse in farming areas and this increase can explain the relatively low 37 prevalence of T. brucei s.l. in cattle. 38Farmer-led treatment of cattle with pyrethroids is likely, in part, to be limiting the spill-over 39 of human-infective trypanosomes from wildlife areas. 40 41 Author summary 42 The acute form of sleeping sickness in Africa is caused by the parasite Trypanosoma brucei 43 rhodesiense. It is transmitted by tsetse flies and can be maintained in cycles involving both 44 livestock and wildlife as hosts. Humans are incidentally infected and are particularly at risk 3 45 of infection near protected areas where there are both wildlife and suitable habitat for tsetse. 46In these regions, the tsetse vector cannot be eradicated, nor can infection be prevented in 47 wildlife. Here we use field studies of tsetse and livestock in combination with mathematical 48 models of tsetse population change and trypanosome transmission to show that use of 49 pyrethroid-based insecticides on cattle by farmers at the edge of protected areas could be 50 contributing to lowering the risk of sleeping sickness in Serengeti District, Tanzania. To our 51 knowledge, our study is the first to report farmer-led tsetse control, co-incident with tsetse 52 decline and relatively low prevalence of T. brucei s.l. in cattle. 53 54 Introduction 55 In East and Southern Africa, tsetse flies (Glossina spp) transmit Trypanosoma brucei 56 rhodesiense, which causes Rhodesian human African trypanosomiasis (r-HAT). Tsetse also 57 transmit T. congolense, T. vivax and T. brucei, the causative agents of animal African 58 trypanosomiasis (AAT) in livestock. 59 Trypanosoma brucei s.l., T. congolense and T. vivax can circulate in transmission cycles 60 involving livestock or wild mammals [1]. The extensive conservation areas of East and 61 Southern Africa that support tsetse, as well as wildlife, can therefore be foci for r-HAT and 62 AAT. At the interface of wildlife-and livestock areas, ...

East Coast fever (ECF) in cattle is caused by the protozoan parasite Theileria parva , transmitted by Rhipicephalus appendiculatus ticks. In cattle ECF is often fatal, causing annual losses >$500 million across its range. The African buffalo ( Syncerus caffer ) is the natural host for T. parva but the transmission dynamics between wild hosts and livestock are poorly understood. This study aimed to determine the prevalence of T. parva in cattle, in a 30 km zone adjacent to the Serengeti National Park, Tanzania where livestock and buffalo co-exist, and to ascertain how livestock keepers controlled ECF and other vector-borne diseases of cattle. A randomised cross-sectional cattle survey and questionnaire of vector control practices were conducted. Blood samples were collected from 770 cattle from 48 herds and analysed by PCR to establish T. parva prevalence. Half body tick counts were recorded on every animal. Farmers were interviewed (n = 120; including the blood sampled herds) using a standardised questionnaire to obtain data on vector control practices. Local workshops were held to discuss findings and validate results. Overall prevalence of T. parva in cattle was 5.07% (CI: 3.70−7.00%), with significantly higher prevalence in older animals. Although all farmers reported seeing ticks on their cattle, tick counts were very low with 78% cattle having none. Questionnaire analysis indicated significant acaricide use with 79% and 41% of farmers reporting spraying or dipping with cypermethrin-based insecticides, respectively. Some farmers reported very frequent spraying, as often as every four days. However, doses per animal were often insufficient. These data indicate high levels of acaricide use, which may be responsible for the low observed tick burdens and low ECF prevalence. This vector control is farmer-led and aimed at both tick- and tsetse-borne diseases of livestock. The levels of acaricide use raise concerns regarding sustainability; resistance development is a risk, particularly in ticks. Integrating vaccination as part of this community-based disease control may alleviate acaricide dependence, but increased understanding of the Theileria strains circulating in wildlife-livestock interface areas is required to establish the potential benefits of vaccination.