Recommendations for environmental risk assessment of gene drive applications for malaria vector control.

Building on an exercise that identified potential harms from simulated investigational releases of a population suppression gene drive for malaria vector control, a series of online workshops identified nine recommendations to advance future environmental risk assessment of gene drive applications.

A Cross-Sectional Survey of Biosafety Professionals Regarding Genetically Modified Insects.

BACKGROUND: Genetic technologies such as gene editing and gene drive create challenges for existing frameworks used to assess risk and make regulatory determinations by governments and institutions. Insect genetic technologies including transgenics, gene editing, and gene drive may be particularly challenging because of the large and increasing number of insect species being genetically modified and the degree of familiarity with these organisms and technologies by biosafety officials charged with making containment decisions. METHODS: An anonymous online survey of biosafety professionals was distributed to the membership of ABSA International, a global society of biosafety professionals, to investigate their perspectives on their preparedness to meet these new challenges. RESULTS: Existing guidance used to make containment decisions for nongenetically modified insects was widely seen as adequate, and most respondents thought the available guidance for making containment decisions for genetically modified insects with and without gene drives was inadequate. Most respondents reported having less confidence in their decisions concerning containment of genetically modified insects compared to decisions involving genetically modified microbes, (noninsect) animals, and plants. CONCLUSIONS: These results reveal a need for additional support for biosafety professionals to improve the quality of and confidence in containment decisions regarding genetically modified insects with and without gene drive. These needs might be addressed by increasing training, updating existing guidance, creating new guidance, and creating a third-party accreditation entity to support institutions. Sixty percent of the respondents said they either would or might use a voluntary third-party accreditation service to support insect containment decisions.

Immunostimulatory responses to crude extracts of Warburgia ugandensis (Sprague) subsp ugandensis (Canellaceae) by BALB/c mice infected with Leishmania major.

INTRODUCTION: To determine the immunostimulatory potential of crude extracts of Warburgia ugandensis subsp. ugandensis with a soluble leishmanial antigen in vaccinating BALB/c mice. METHODS: Seventy two female BALB/c mice were randomly assigned into six groups. The mice were vaccinated with soluble leishmania antigens (SLA) alone, hexane, ethyl acetate, and dichloromethane extract co-administered with SLA. Unvaccinated mice formed the control group. The induction of cell-mediated immunity following vaccination was determined by measuring in vitro lymphocyte proliferation and the production of interleukin (IL)-4 and gamma interferon (IFN-γ) determined by flow cytometry. Protection against L. major was determined by quantifying parasite burdens in L. major infected footpads using a limiting dilution assay and by measuring lesion sizes of the infected footpad compared to the contralateral uninfected footpad. RESULTS: On vaccination with extracts of W. ugandensis subsp. ugandensis alone or as adjuvants when used in combination with Leishmania antigens, the hexane extract and the dichloromethane extract plus SLA stimulated moderate production of IFN-γ and low levels of IL-4.These mice were partially protected from cutaneous leishmaniasis as shown by the slow development of lesions and comparatively less parasite burdens. CONCLUSION: These data suggest that extracts of W. ugandensis subsp. ugandensis are suitable adjuvants for Leishmania vaccines. However, since W. ugandensis subsp. ugandensis has been shown to be effective against Leishmania parasites in vitro and in vivo, further studies ought to be conducted to determine its immunochemotherapeutic potential when co-administered with a soluble leishmanial antigen in vaccinating BALB/c mice.

The potential of the extracts of Tagetes minuta Linnaeus (Asteraceae), Acalypha fruticosa Forssk (Euphorbiaceae) and Tarchonanthus camphoratus L. (Compositae) against Phlebotomus duboscqi Neveu Lemaire (Diptera: Psychodidae), the vector for Leishmania major Yakimoff and Schokhor.

BACKGROUND & OBJECTIVES: Harmful effects of synthetic chemical insecticides including vector resistance, environmental pollution and health hazards have necessitated the current significance in the search for plant-based insecticide products that are environmentally safe and effective to leishmaniases control. The insecticidal activity of Tagetes minuta Linnaeus (Asteraceae), Acalypha fruticosa Forssk (Euphorbiaceae) and Tarchonanthus camphoratus L. (Compositae) extracts were investigated against Phlebotomus duboscqi Neveu Lemaire (Diptera: Psychodidae). METHODS: The extracts were prepared from dried aerial parts soaked in methanol and ethyl acetate twice until the filtrates became clear, filtered and dried out by rotary evaporation at 30-35 degrees C. The solid extracts obtained were later prepared into 2.5, 5 and 10 mg/ml. Two millilitres of the solutions were blotted on filter papers, which were dried overnight and placed into jars where adult sandflies were aspirated. Males and females were assayed separately. RESULTS & CONCLUSION: The extracts had significant mortality (p<0.05) in both males and females bioassays but were not significantly different between sexes. The extracts of Acalypha fruticosa and Tagetes minuta had significantly higher mortality rates than those of Tarchonanthus camphoratus and the different concentrations used showed significantly different mortality rates and 10 mg/ml was the most effective concentration. Cent percent mortality was obtained at 96 h of exposure to 5 and 10 mg/ml concentrations except for Tarchonanthus camphoratus which had a mortality of only 46.7% in 10 mg/ml bioassay. These extracts were found to be insecticidal to adult sandflies.

In vitro effects of Warburgia ugandensis, Psiadia punctulata and Chasmanthera dependens on Leishmania major promastigotes.

Plant extracts from Warburgia ugandensis Sprague (Family: Canellaceae), Psiadia punctulata Vatke (Family: Compositae) and Chasmanthera dependens Hoschst (Family: Menispermaceae) were tested for activity on Leishmania major promastigotes (Strain IDU/KE/83 = NLB-144) and infected macrophages in vitro. Plants were collected from Baringo district, dried, extracted, weighed and tested for antileishmanial activity. Serial dilutions of the crude extracts were assayed for their activity against Leishmania major in cell free cultures and in infected macrophages in vitro. Inhibitory concentrations and levels of cytotoxicity were determined. Warburgia ugandensis, Psiadia punctulata and Chasmanthera dependens had an IC(50) of 1.114 mg/ml, 2.216 mg/ml and 4.648 mg/ml, respectively. The cytotoxicity of the drugs on BALB/c peritoneal macrophage cells was insignificant as compared to the highly toxic drug of choice Pentostam(®). The supernatants from control and Leishmania infected macrophages were analyzed for their nitrite contents by Griess reaction and nitrite absorbance measured at 540 nm. Warburgia ugandensis (stem bark water extract), Chasmanthera dependens (stem bark water extract) and Psiadia punctulata (stem bark methanol extract) produced 112.3%, 94% and 88.5% more nitric oxide than the untreated infected macrophages respectively. Plant crude extracts had significant (p<0.05) anti-leishmanial and immunomodulative effects but insignificant cytotoxic effects at 1mg/ml concentration. All experiments were performed in triplicate. Statistical analysis of the differences between mean values obtained from the experimental group compared to the controls was done by students't test. ANOVA was used to determine the differences between the various treatment groups. The analysis program Probit was used to determine IC(50)s.

Anti-parasitic activity and cytotoxicity of selected medicinal plants from Kenya.

Indigenous rural communities in the tropics manage parasitic diseases, like malaria and leishmaniasis, using herbal drugs. The efficacy, dosage, safety and active principles of most of the herbal preparations are not known. Extracts from 6 selected plant species, used as medicinal plants by indigenous local communities in Kenya, were screened for in vitro anti-plasmodial and anti-leishmanial activity, against 2 laboratory-adapted Plasmodium falciparum isolates (D6, CQ-sensitive and W2, CQ-resistant) and Leishmania major (IDU/KE/83=NLB-144 strain), respectively. The methanol extract of Suregada zanzibariensis leaves exhibited good anti-plasmodial activity (IC(50) 4.66+/-0.22 and 1.82+/-0.07 microg/ml for D6 and W2, respectively). Similarly, the methanol extracts of Albizia coriaria (IC(50) 37.83+/-2.11 microg/ml for D6) and Aspergillus racemosus (32.63+/-2.68 and 33.95+/-2.05 microg/ml for D6 and W2, respectively) had moderate anti-plasmodial activity. Acacia tortilis (IC(50) 85.73+/-3.36 microg/ml for W2) and Albizia coriaria (IC(50) 71.17+/-3.58 microg/ml for W2) methanol extracts and Aloe nyeriensis var kedongensis (IC(50) 87.70+/-2.98 and 67.84+/-2.12 microg/ml for D6 and W2, respectively) water extract exhibited mild anti-plasmodial activity. The rest of the extracts did not exhibit any anti-plasmodial activity. Although the leishmanicidal activity of extracts were lower than for pentosam (80%), reasonable activity was observed for Aloe nyeriensis methanol (68.4+/-6.3%), Albizia coriara water (66.7+/-5.0%), Maytenus putterlickoides methanol (60.0+/-6.23%), Asparagus racemosus methanol and water (58.3+/-8.22 and 56.8+/-6.58%, respectively), Aloe nyeriensis water (53.3+/-5.1%) and Acacia tortilis water (52.9+/-6.55%) extracts at 1000 microg/ml. Leishmania major infected macrophages treated with methanol extracts of Suregada zanzibariensis and Aloe nyeriensis var kedongensis and pentostam had infection rates of 28+/-2.11, 30+/-1.22 and 40+/-3.69%, respectively at 1000 microg/ml, indicating better anti-leishmanial activity for the extracts. The methanol extract of Albizia coriara (44.0+/-3.69%) and aqueous extracts of Asparagus racemosus (42+/-3.84%) and Acacia tortilis (44+/-5.59%) had similar activity to pentosam. Multiplication indices for Leishmania major amastigotes treated with methanol extracts of Albizia coriaria, Suregada zanzibariensis and Aloe nyeriensis var kedongensis, aqueous extract of Acacia tortilis and pentosam were 28.5+/-1.43, 29.4+/-2.15, 31.1+/-2.22, 35.9+/-3.49 and 44.0+/-3.27%, respectively, at 1000 microg/ml, confirming better anti-leishmanial activity for the extracts. Aqueous extracts of Aloe nyeriensis (46.7+/-3.28%) and Albizia coriaria (47.5+/-3.21%) had similar activity level to pentosam. The plant extracts have better inhibitory activity while pentosam has better leishmanicidal activity. All extracts exhibited very low cytotoxicity (CC(50) > 500 microg/ml) against human embryonic lung fibroblast (HELF) cells. The investigations demonstrated the efficacy and safety of some extracts of plants that are used by rural indigenous communities for the treatment of parasitic diseases.

In vivo efficacy of oral and intraperitoneal administration of extracts of Warburgia ugandensis (Canellaceae) in experimental treatment of Old World cutaneous leishmaniasis caused by Leishmania major.

The antileishmanial activity of extracts of Warburgia ugandensis Spraque (Canellaceae), a known traditional therapy in Kenya was evaluated in vivo. Treatment of infected BALB/c mice with W. ugandensis extracts orally resulted in a reduction of the size of lesions compared to the untreated control. The lesion sizes differed significantly for the four extracts (p=0.039) compared to the untreated control. For mice treated by intraperitoneal injection, the lesion sizes increased initially for the hexane, dichloromethane and ethyl acetate extracts and healed by day 42. The lesion sizes for mice treated with methanol increased steadily from 2.47mm to 3.57mm. The parasitic burden was significantly higher (p<0.001) in mice treated with methanol extracts and PBS compared to those treated with hexane, dichloromethane and ethyl acetate. This study demonstrated the antileishmanial potential of extracts of W. ugandensis.

Cross-protection against Leishmania donovani but not L. Braziliensis caused by vaccination with L. Major soluble promastigote exogenous antigens in BALB/c mice.

Vaccinating with soluble Leishmania major promastigote exogenous antigens (LmSEAgs) protects mice against challenge with L. major. To explore the potential of LmSEAgs to cross-protect against infection with other species of Leishmania, BALB/c mice were immunized with LmSEAgs prior to challenge with either L. donovani or L. braziliensis promastigotes. Such mice were protected against L. donovani but not L. braziliensis infection. Leishmania braziliensis-infected mice developed lesions that were not significantly different from those of controls and that contained 13-fold more parasites. In contrast, immunized mice infected with L. donovani were protected as illustrated by low splenic parasite loads (as much as 4,913-fold fewer parasites). This protection corresponded to significant increases in gamma interferon and low production of interleukin-4 (IL-4) IL-4 or IL-10, which suggested an enhanced type 1 response.

Protection of susceptible BALB/c mice from challenge with Leishmania major by nucleoside hydrolase, a soluble exo-antigen of Leishmania.

Leishmania major culture-derived, soluble, exogenous antigens have been shown to be a source of vaccine targets for the parasite. We have previously reported that L. major culture-derived, soluble, exogenous antigens can immunize BALB/c mice against challenge with L. major. However, the molecule(s) involved in this protection was not known. We describe the potential of one component of soluble exogenous antigens (recombinant nucleoside hydrolase) to vaccinate mice against challenge with L. major. We found that recombinant nucleoside hydrolase vaccinated BALB/c mice against a subsequent challenge with L. major. Protection was manifested by a significant decrease in lesion size (as much as a 30-fold reduction) and parasite burden (as much as a 71-fold reduction). Protection was achieved whether recombinant nucleoside hydrolase was administered to mice in the presence or absence of adjuvant (interleukin-12). Finally, protection was accompanied by an increase in interferon-gamma production but a decrease in interleukin-10 production by vaccinated animals in response to challenge with L. major.

Leishmania major soluble exo-antigens (LmSEAgs) protect neonatal BALB/C mice from a subsequent challenge with L. major and stimulate cytokine production by Leishmania-naïve human peripheral blood mononuclear cells.

The vaccine potential and immunogenicity of soluble Leishmania major exo-antigens (LmSEAgs), a potentially novel source for vaccine candidates for leishmaniasis, were evaluated in neonatal BALB/c mice and with human peripheral blood mononuclear cells. Vaccinated neonatal BALB/c mice resisted infection with L. major, and lymphoid cells from the mice proliferated when restimulation with LmSEAgs and produced interferon-gamma and some interleukin-4. In addition, LmSEAgs stimulated human peripheral blood mononuclear cells to produce large amounts of interferon-gamma and some interleukin-5. This finding suggests that LmSEAgs may be a vaccine candidate for leishmaniasis in humans.

Situational analysis of leishmaniases research in Kenya.

Leishmania spp are protozoan parasites of the Trypanosomatidae family that cause disease in humans and animals. In general, infections with these parasites can be divided into three main forms namely, cutaneous, mucocutaneous, and visceral leishmaniases. The disease is prevalent in many tropical and subtropical regions of the world, where it is transmitted via the bite of an infected sand fly. Leishmaniasis has been known to be endemic in parts of Kenya from as far back as early in the 20th century. These endemic areas include Turkana, Baringo, Kitui, Machakos, Meru, West Pokot and Elgeyo Marakwet districts which have been reported to be endemic for kala-azar. Recent outbreaks of VL have been reported in the previously non-endemic districts of Wajir and Mandera in North Eastern Kenya between May 2000 and August 2001. The vector for VL in Kenya is Phlebotomus martini though other vectors including P. orientalis have been reported. Baringo district is the only foci reported where both VL and CL are known to occur in Kenya. The aetiological agents for CL which include L. major which has been reported in Baringo; L. tropica in Laikipia, Samburu, Isiolo, Nakuru and Nyandarua districts while L. aethiopica has been reported in the Mt Elgon area. In Kenya, P. duboscqi, P. guggisbergi have been shown to be the vectors of L. major and L. tropica, respectively, while P. pediffer, P. longipes and P. elgonensis have been implicated as vectors of L. aethiopica. Since 1980, the Kenya Medical Research Institute (KEMRI) has spearheaded research on leishmaniases research in Kenya focusing on various aspects including characterization of Leishmania species, biology, and ecology of sand fly vectors, development of biological strategies for sand fly control, identification of animal reservoirs, diagnosis, new treatment strategies, new chemotherapeutic agents, and vaccine-related studies. KEMRI, a founding partner of the Drugs for Neglected Disease Initiative (DNDi), whose overall aim is to address lack of new or improved drugs for neglected diseases (which include leishmaniases, malaria, trypanosomiasis and Chagas disease) has made major contributions in leishmaniases research and control in Kenya and the eastern Africa region.

Immunization with Leishmania major exogenous antigens protects susceptible BALB/c mice against challenge infection with L. major.

The potential of Leishmania major culture-derived soluble exogenous antigens (SEAgs) to induce a protective response in susceptible BALB/c mice challenged with L. major promastigotes was investigated. Groups of BALB/c mice were immunized with L. major SEAgs alone, L. major SEAgs coadministered with either alum (aluminum hydroxide gel) or recombinant murine interleukin-12 (rmIL-12), L. major SEAgs coadministered with both alum and rmIL-12, and L. major SEAgs coadministered with Montanide ISA 720. Importantly and surprisingly, the greatest and most consistent protection against challenge with L. major was seen in mice immunized with L. major SEAgs alone, in the absence of any adjuvant. Mice immunized with L. major SEAgs had significantly smaller lesions that at times contained more than 100-fold fewer parasites. When lymphoid cells from L. major SEAg-immunized mice were stimulated with leishmanial antigen in vitro, they proliferated and secreted a mixed profile of type 1 and type 2 cytokines. Finally, analyses with Western blot analyses and antibodies against three surface-expressed and secreted molecules of L. major (lipophosphoglycan, gp46/M2/PSA-2, and gp63) revealed that two of these molecules are present in L. major SEAgs, lipophosphoglycan and the molecules that associate with it and gp46/M2/PSA-2.