In this study, we assessed the effect of several fipronil dosing concentrations on the survivorship of adult mosquitoes (Anopheles arabiensis) and sand flies.
Treating Cattle for Mosquito And Sand Fly Control Richard Poche1, David Poche1, Larisa Polyakova1, Dylan Burres1, Trey Barresi1, Rajesh Garlapati1, Mutum Ingobi Singh2, Sachidananda Samantaray2, Saul Lozano-Fuentes1 1: Genesis Laboratories, Wellington, Colorado, USA; 2: Bihar Veterinary College, Patna, Bihar, India !
Introduction Hematophagous arthropods on many taxa are know to feed on livestock including cattle. Many of these insect transmit vector-borne diseases. Malaria and visceral leishmaniasis (VL) represent two such diseases that result in nearly one million fatalities each year. Since 2009 our research in India and Kenya has focused on the use of systemic insecticides to control sand flies and mosquitoes taking blood from cattle. Several candidate active ingredients have been examined and show promising effects on both adult and larval stages (data not presented).
In this study, we assessed the effect of several fipronil dosing concentrations on the survivorship of adult mosquitoes (Anopheles arabiensis) and sand flies (Phlebotomus argentipes), and measured fipronil residues in milk fat and plasma.
Objectives
Survival Statistical analysis
•Test different fipronil doses in semi-field conditions. •Evaluate the survivorship of adult sand flies and mosquitoes. •To measure the residue level for milk fat and blood.
The statistical analysis of the survival data obtained from the control and treatment groups was conducted using R (R core Team, 2014., Therneau, 2015). A survival curve is the plot of the survival function representing the survivorship of the target population. The statistical difference between the control and the treatment was assessed using the Mantel-Haenszel test as implemented in the survival package. The resulting survival functions were used to estimate the median survival time and 95% confidence intervals for the estimate and the size of the effect of the active ingredient.
Milk is an important part of the village economy, knowing the maximum residue levels (MRL) of active ingredients (AI) in milk are of paramount value to ensure human safety. Finding a balance between blood AI concentration and vector control efficacy is of extreme importance. We present data demonstrating the potential use of fipronil for reducing the numbers of cases of malaria and VL.
Photo: AP/Heng Sinith
Photo: CDC/James Gathany; Anopheles arabiensis
Photo: WHO/PKDL patient
Photo: Genesis/D. Poché
Residue Analysis
Mosquito Bioassays
Sand Fly Bioassays
Results and Discussion
To determine bioaccumulation, three consecutive dosings (dosing 1,2 and 3) were conducted at 8 week intervals. Plasma were taken once on 1, 4, 7 DPT while whole milk samples once daily for 14 days, then once per week. Residue analysis was conducted for milk and plasma samples to determine concentrations of fipronil and the metabolite fipronil sulfonate.
The full study consisted of 4 experiments testing ivermectin, eprinomectin and fipronil (Poché et al., in press). Here we present the results of the fipronil dosing. Each (Bos indicus) cow was randomly assigned to a control or treatment group. Each treatment group consisted of three lactating Zebu cattle , the control group was allocated two. Treatment groups were dosed with a gelatin capsule containing either no active ingredient or 0.25, 0.5 or 1.0 mg/Kg (body weight) of fipronil.
The study used 20 healthy lactating cows (Bos indicus x Bos taurus) randomly assigned to one of 5 treatment groups, each treatment group was composed of 4 cows.
•No fipronil bioaccumulation was observed in cattle as shown by the fipronil blood concentration between dosings. •Dosing cattle at 0.25mg/kg and 0.5 mg/kg creates a low risk for people based on residue levels. A recomendation for milk withdrawal of 14 days could be issued. •Fipronil in An. arabiensis, showed a significant difference between the control and treatment group for at least 7 DPT for the two lower concentrations and at least 21 DPT for 1.0 mg/kg concentration (Table 1). At 7 DPT, the median survival time for the 0.25 mg/kg concentration was 144h [144, 168] while 0.5 mg/kg showed a median survival of 48h [48, 48] (Table 1, Figure 3). •Fipronil remained consistenly effective (significantly different between the treatment and the control) for at least 7 DPT against Ph. argentipes for the two lower concentrations and at least 21 DPT for the highest concentration (Table 2). At 7 DPT the median survival time was 72h [72, 96] and 96h [72, 96] for the concentrations of 0.25mg/kg and 0.5mg/kg respectively (Table 2, Figure 4). •The concentration of 1.0 mg/kg remained efective against both species for at least 21 DPT, this dose could be sugested for cattle not use for milk production. •A comparison between between the sand fly mortality and fipronil blood concentration suggest a minimum concentration of 30 ppb is necessary for sand fly mortality.
Milk and plasma analysis were conducted by the Analytical Services of the Center for Environmental Medicine, Department of Environmental and Radiological Health Sciences at Colorado State University, CO USA.
All An. arabiensis used in this study were reared at the KEMRI/CDC, Kisian station, Kenya. The day of application, 11-12 plastic capsules were filled with ~50 female mosquitoes. Efficacy of each treatment was assessed by comparing survivorship of fully blood fed An. arabiensis at 1, 3, 7, 14 and 21 DPT. Unfed females were removed from the study. Mortality was recorded daily for 9 days after the first 24 hours.
Total Fipronil Concentration in Bovine Milk Fat (Dosing 1-3) 5.5 0.25 mg/kg D1 5 0.5 mg/kg D1
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Table 1. Median survival time and 95% confidence intervals for An. arabiensis exposed to fipronil. Exp./Days 1d 3d 7d 10d 14d 21d Control 144 [120, 144] 168 [168, 216] 192 [168, 240] n/a n/a¹ [216, ∞] 240 [216, ∞] 0.25 mg/Kg 48 [48, 72]* 120 [120, 144]* 144 [144, 168]* n/a 216 [144, ∞] n/a¹ [216, ∞] 0.5 mg/Kg 48 [48, 48]* 120 [96, 144]* 48 [48, 48]* n/a 204 [144, ∞] 216[192, ∞] Control n/a¹ [-∞, ∞] n/a 216 [216, 240] 168 [144, 168] 192 [192, 240] n/a¹ [240, ∞] 1.0 mg/Kg 24 [24, 24]* n/a 48 [48, 48]* 72 [72, 96]* 132 [72, 192]* 144 [72, 240]* d =days; Exp. =experiment; Values in square brackets represent the 95% confidence intervals for the estimated median lethal time; n/a =time point not tested; n/a¹ =denotes estimate nor applicable because the survival function did not reach 0.5, therefore there is not a median value estimate; “∞” =infinity, the survival function did not reach the corresponding to 95% limit value; * Comparison statistically different from the control at an adjusted α of 0.05.
3 0.25 mg/kg D3 2.5 0.5 mg/kg D3
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Each treatment group was dosed with a single gelatin capsule containing either no active ingredient or 0.25, 0.5 or 1.0 mg/Kg (body weight) of fipronil. ~50 Sand flies were placed in two cups, and directly applied to the skin of a cow on days 1, 2, 3, 4, 7 and 14 DPT. Sand flies were separated in fully and partially blood fed. Mortality was assessed only on fully fed sand flies at 0, 3, 6, 24, 48, 72, 96 and 120 hours. Table 2. Median survival time and 95% confidence intervals for Ph. argentipes exposed to fipronil Exp./Days 1d 2d 4d 7d 14d 21d Control 96 [72, 120] 120 [96, ∞] n/a¹ 96 [96, 120] 96 [72, ∞] 120 [120, ∞] 0.25 mg/Kg 48 [48, 72]* 72 [48, 96]* 72 [72, 96]* 72 [72, 96]* 96 [72, 120] 120 [120, ∞] 0.5 mg/Kg 24 [24, 24]* 72 [48, 96]* 72 [72, 72]* 96 [72, 96]* 96 [72, 96] 96 [96, 96]* 1.0 mg/Kg 0 [0, ∞]* 24 [-∞, ∞]* 24 [24, 24]* 72 [72, 72]* 72 [72, 72]* 96 [72, 96]* d =days; Exp. =experiment; Values in square brackets represent the 95% confidence intervals for the estimated median lethal time; n/a =time point not tested; n/a¹ =denotes estimate nor applicable because the survival function did not reach 0.5, therefore there is not a median value estimate; “∞” =infinity, the survival function did not reach the corresponding to 95% limit value; * Comparison statistically different from the control at α =0.05;
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Figure 1: Residue analysis in repited cow dosing. Samples taken in days 1, 7 and 14 for dosing 3. Blue dashed line represents the MRL of 1.5 ppm (US EPA), that relates to a 0.05 ppm in whole milk.
Cited Literature
Total Fipronil Concentration in Bovine Plasma (Dosing 1 and 2)
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Figure 4: Survivorship plots for An. arabiensis fed on cows treated with fipronil at 0.25mg/Kg and 0.5 mg/Kg at 7 DPT and 14 DPT. The dashed lines denote the 95% confidence intervals.
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Figure 3: Survivorship plots for An. arabiensis fed on cows treated with fipronil at 0.25mg/Kg and 0.5 mg/Kg at 7 DPT and 14 DPT. The dashed lines denote the 95% confidence intervals.
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Poché et al. "Treatment of livestock with systemic insecticides for control of Anopheles arabiensis in western Kenya." Malaria Journal, in press. R Core Team. R: A Language and Environment for Statistical Computing. Vienna, Austria: R Foundation for Statistical Computing, 2014. http://www.R-project.org/. Therneau, Terry M. A Package for Survival Analysis in S, 2015. http://CRAN.R-project.org/package=survival.
These projects is founded by the Bill and Melinda Gates Foundations (OPP1032369 and OPP1053271)
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Figure 2: Residue analysis in blood samples.
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Genesis Laboratories http://www.genesislabs.com/
