Enzymatic formulation capable of degrading scrapie prion under mild digestion conditions.

The prion agent is notoriously resistant to common proteases and conventional sterilisation procedures. The current methods known to destroy prion infectivity such as incineration, alkaline and thermal hydrolysis are harsh, destructive, environmentally polluting and potentially hazardous, thus limit their applications for decontamination of delicate medical and laboratory devices, remediation of prion contaminated environment and for processing animal by-products including specified risk materials and carcases. Therefore, an environmentally friendly, non-destructive enzymatic degradation approach is highly desirable. A feather-degrading Bacillus licheniformis N22 keratinase has been isolated which degraded scrapie prion to undetectable level of PrP(Sc) signals as determined by Western Blot analysis. Prion infectivity was verified by ex vivo cell-based assay. An enzymatic formulation combining N22 keratinase and biosurfactant derived from Pseudomonas aeruginosa degraded PrP(Sc) at 65 °C in 10 min to undetectable level -. A time-course degradation analysis carried out at 50 °C over 2 h revealed the progressive attenuation of PrP(Sc) intensity. Test of residual infectivity by standard cell culture assay confirmed that the enzymatic formulation reduced PrP(Sc) infectivity to undetectable levels as compared to cells challenged with untreated standard scrapie sheep prion (SSBP/1) (p-value = 0.008 at 95% confidence interval). This novel enzymatic formulation has significant potential application for prion decontamination in various environmentally friendly systems under mild treatment conditions.

Evidence for varied aetiologies regulating the transmission of prion disease: implications for understanding the heritable basis of prion incubation times.

BACKGROUND: Transmissible Spongiform Encephalopathies (TSEs) are a group of progressive fatal neurodegenerative disorders, triggered by abnormal folding of the endogenous prion protein molecule. The encoding gene is a major biological factor influencing the length of the asymptomatic period after infection. It remains unclear the extent to which the variation between quantitative trait loci (QTLs) reported in mouse models is due to methodological differences between approaches or genuine differences between traits. With this in mind, our approach to identifying genetic factors has sought to extend the linkage mapping approach traditionally applied, to a series of additional traits, while minimising methodological variability between them. Our approach allows estimations of heritability to be derived, as well as predictions to be made about possible existence of genetic overlap between the various traits. METHODOLOGY/PRINCIPAL FINDINGS: Our data indicate a surprising degree of heritability (up to 60%). Correlations between traits are also identified. A series of QTLs on chromosomes 1, 2, 3, 4, 6, 11 and 18 accompany our heritability estimates. However, only a locus on chromosome 11 has a general effect across all 4 models explored. CONCLUSIONS/SIGNIFICANCE: We have achieved some success in detecting novel and pre-existing QTLs associated with incubation time. However, aside from the general effects described, the model-specific nature of the broader host genetic architecture has also been brought into clearer focus. This suggests that genetic overlap can only partially account for the general heritability of incubation time when factors, such as the nature of the TSE agent and the route of administration are considered. This point is highly relevant to vCJD (a potential threat to public health) where the route of primary importance is oral, while the QTLs being sought derive exclusively from studies of the ic route. Our results highlight the limitations of a single-model approach to QTL-mapping of TSEs.

Evidence of multiple pyrethroid resistance mechanisms in the malaria vector Anopheles gambiae sensu stricto from Nigeria.

Pyrethroid insecticide resistance in Anopheles gambiae sensu stricto is a major concern to malaria vector control programmes. Resistance is mainly due to target-site insensitivity arising from a single point mutation, often referred to as knockdown resistance (kdr). Metabolic-based resistance mechanisms have also been implicated in pyrethroid resistance in East Africa and are currently being investigated in West Africa. Here we report the co-occurrence of both resistance mechanisms in a population of An. gambiae s.s. from Nigeria. Bioassay, synergist and biochemical analysis carried out on resistant and susceptible strains of An. gambiae s.s. from the same geographical area revealed >50% of the West African kdr mutation in the resistant mosquitoes but <3% in the susceptible mosquitoes. Resistant mosquitoes synergized using pyperonyl butoxide before permethrin exposure showed a significant increase in mortality compared with the non-synergized. Biochemical assays showed an increased level of monooxygenase but not glutathione-S-transferase or esterase activities in the resistant mosquitoes. Microarray analysis using the An. gambiae detox-chip for expression of detoxifying genes showed five over-expressed genes in the resistant strain when compared with the susceptible one. Two of these, CPLC8 and CPLC#, are cuticular genes not implicated in pyrethroid metabolism in An. gambiae s.s, and could constitute a novel set of candidate genes that warrant further investigation.

Plasmodium berghei: efficacy and safety of combinations of chloroquine and promethazine in chloroquine resistant infections in gravid mice.

Efficacy and safety of combinations ofChloroquine (CQ) and doses of Promethazine (PR) against CQ resistant Plasmodium berghei infections in gravid mice was evaluated. Parasites were cleared faster in mice treated with CQ combined with doses of PR ranging from 20mg/kg to 50mg/kg (3.4 +/- 0.5 to 2.7 +/- 0.7) compared with CQ alone (4.7 +/- 0.8) (P<0.5). Parturition resulting in live pups in animals treated with CQ and 20mg/ kg and 30mg/kg of PR (81%) was significantly higher than in animals treated with CQ alone (44%) or saline (13%). Mean birth weight of pups delivered by infected gravid animals treated with CQ and 30mg/kg or 40mg/kg of PR (1.51 +/- 0.16 or 1.56 +/- 0.16) was significantly higher than animals treated with CQ alone (1.33 +/- 0.13) (P=0.00004, 0.0014 respectively). No gross malformations were observed in pups delivered by infected or non-infected animals treated with the combinations of chloroquine and Promethazine.

Cultural categorization of febrile illnesses in correlation with herbal remedies used for treatment in Southwestern Nigeria.

The ethnographic study was conducted in two communities in Oyo State in Southwestern Nigeria. The study sites consisted of a rural and an urban local government area located in the tropical rain forest zone of Nigeria. The study was designed to obtain information on febrile illnesses and herbal remedies for treatment with the aim of identifying potential antimalarial drugs. The study revealed that fever is a general term for describing illnesses associated with elevated body temperature. The indigenous Yoruba ethnic population has categorized fever based on symptoms and causes. The present communication is the result of focus group discussion and semi-structured questionnaire administered to traditional healers, herb sellers, elders and mothers. This was on types of fevers, symptoms and causes of febrile illnesses. The investigation also included use of traditional herbs in the prevention and treatment of the illnesses in the two communities.A total of 514 respondents were interviewed. This was made up of 266 (51.8%) from Atiba local government area (LGA), an urban centre while 248 (48.2%) respondents were interviewed from Itesiwaju LGA, a rural community. The LGAs are located in Oyo State of Nigeria. The respondents proffered 12 types of febrile illnesses in a multiple response answering system in Yoruba language. The most common ones (direct translation into English) were: yellow fever (39.1%), typhoid (34.8%), ordinary (28.8%), rainy season (20.8%) and headache (10.5%) fevers, respectively. Perceived causes of each of the febrile illnesses included stress, mosquito bites, unclean water, rains and over exposure to the sun. Methods of fever prevention were mainly with the use of herbal decoctions, powdered herbs, orthodox medications and maintenance of proper hygiene. Of a total of 112 different herbal remedies used in the treatment of the febrile illnesses compiled from the study, 25 recipes are presented. Recipes consisted of 2-7 ingredients. Oral decoctions (84%), oral powders (63%), use as soaps and creams (40%) in a multiple response system, were the most prevalent routes of administration of prepared herbs used in the treatment of the fevers. Boiling in water or alcohol was the most common method used in the preparation of the remedies. The four most frequently mentioned (multiple response system) plants in the Southwest ethnobotany for fevers were Azadirachta indica (87.5%), Mangifera indica (75.0%), Morinda lucida (68.8%) and Citrus medica (68.8%).

Profound sex-specific effects on incubation times for transmission of bovine spongiform encephalopathy to mice.

Four strains of mice were inoculated intracerebrally with a primary isolate of bovine spongiform encephalopathy (BSE) and the cloned mouse-adapted scrapie strain ME7. Clinical prion disease diagnosis was made at the appearance of three or more neurological symptoms and their persistence for 3 consecutive weeks and confirmed by neuropathological criteria. For BSE, incubation periods were profoundly different between the sexes in all four mouse strains, being longer in the females. In contrast, ME7 scrapie incubation times were similar between the sexes. Our results indicate that sex-specific processes are involved in the course of primary BSE transmission. Research into this phenomenon may provide clues to the prophylaxis of BSE and have possible implications for new variant Creutzfeldt-Jakob disease in humans.