Evaluating the efficacy of Mazao Tickoff (Metarhizium anisopliae ICIPE 7) in controlling natural tick infestations on cattle in coastal Kenya: Study protocol for a randomized controlled trial

Ticks and tick-borne diseases cause substantial economic losses to the livestock industry in sub-Saharan Africa. Mazao Tickoff is a novel bioacaricide developed for tick control and is based on the entomopathogenic fungus Metarhizium anisopliae sensu lato (s.l.) isolate ICIPE 7. To date, no randomized controlled study has been undertaken to demonstrate the efficacy of this bioacaricide in reducing natural tick infestation on cattle. To this end, this field trial is designed to evaluate the anti-tick efficacy of Mazao Tickoff on cattle in coastal Kenya compared to a standard chemical tick control protocol. In this prospective, multi-center randomized controlled trial, eligible herds will be randomized by the herd size to the intervention arm in a 1:1:1 ratio to either Triatix® (active ingredient: amitraz); Mazao Tickoff (active ingredient: M. anisopliae ICIPE 7); or placebo (excipients of the Mazao Tickoff), with a total enrollment target of 1,077 cattle. Treatments will be dispensed on Day 0 (defined individually as the day each animal receives the first treatment) and thereafter every two weeks until Day 182. Ticks will be counted on every animal in each herd (herds to be included have at least one animal bearing at least one tick on Day 0), and thereafter on bi-weekly intervals until Day 182. The primary efficacy assessments of Mazao Tickoff will be based on the mean percentage reduction in tick counts at each post-treatment follow-up visit compared to the placebo group and the Triatix® arm. Further, the effect of Mazao Tickoff on the prevalence of common cattle pathogens, Anaplasma marginale and Theileria parva, will be determined by assessing incidence and seroprevalence at four different time points. This protocol describes the first rigorous evaluation of the efficacy of Mazao Tickoff and its potential as a viable alternative non-chemical acaricide tool for tick control in Kenya and elsewhere. The study will be conducted in Kayafungo ward, Kaloleni sub-county in Kilifi County in coastal Kenya (Fig 1). The region is hot throughout the year with an average temperature range of 23°C—34°C. The altitude range is between 0 and 464 m above sea level. The region experiences two rainy seasons, i.e., from April to June and October to November, but some rain falls nearly every month, especially near the coastline. The total precipitation varies from 900 to 1500 mm per annum along the coastal belt to 500–600 mm in the backcountry [31–33]. The study area is suitable for the current trial as tick infestation is common in the selected sites. Further, previous reports have demonstrated that tick-borne infections are prevalent in the coastal region [5, 7, 34, 35]. This map is republished with data under CC BY licenses from the following sources: https://africaopendata.org/dataset/kenya-counties-shapefile from openAfrica, 2015 [36]; and https://gadm.org/download_country_v3.html from GADM, 2018 [37]. Cattle herds in the study area are typically small-holder herds (average herd size of seven cattle per herd) of the indigenous zebu (Bos indicus) breed. Most of these cattle are maintained under traditional extensive management systems. During the day, adult and weaner cattle of several herds graze together in a common pasture on fallow or communal grazing land. These animals also obtain drinking water in the same water pans that are scattered in the villages. Therefore, adults and weaner zebu of a village are likely exposed to a similar tick habitat with similar tick infestation levels. Young calves are usually tethered around the homestead or grazed separately from the dams until they are weaned. Before this trial, a cross-sectional survey was conducted during the dry season in 14 villages to assess the species diversity of ixodid ticks infesting cattle and their burden and prevalence of associated pathogens. This unpublished survey recorded a 25% (range 22–29.1%) tick infestation prevalence. All the examined herds (n = 95) had at least one animal infested by a tick. The median infestation intensity was four ticks per animal (Range 1–19). The corresponding figure at the herd level was 12.7 ticks (range: 8.7–19.4). As expected, the prevalence and intensity varied across the villages. For this trial, we identified four village clusters 3.5–5 km2 size with distinct animal grazing patterns and intensities. This study is a prospective, multi-center randomized controlled field trial designed to evaluate the efficacy of Mazao Tickoff. The study will be conducted during the dry and rainy season, from December 2021 to June 2022. The rainy period, and soon thereafter, is characterized by a high abundance of Rh. appendiculatus, Rh. decoloratus and Am. variegatum [3, 38–41] and favorable for high fungal growth [18]. The schedule of trial activities is presented in Fig 2. Local zebu cattle managed under an extensive grazing system will be enrolled in the study based on evidence of infestation with live attached ticks, generally in good health, and the owners’ willingness to participate in the study. Zebu cattle are the predominant livestock species in the area and were therefore chosen for this study. Qualifying herds will be stratified by size and the study village clusters, and randomly allocated to receive either Triatix® (Cooper K-Brands Ltd); Mazao Tickoff; or placebo in a 1:1:1 ratio. If after randomization we note a large variability in tick counts among the intervention arms, then we will include tick infestation prevalence and intensity as part of the randomization plan. To reduce the likelihood of spillover effect with biopesticides, all cattle from the same herd will be treated using the same product and will remain with their owners under their natural grazing conditions throughout the trial. For logistical convenience, we will construct three crushes in each village cluster. Cattle will receive treatment on Day 0, and thereafter every two weeks until Day 182. Day 0 will be defined individually as the day an animal receives the first treatment. Whole-body tick counts will also be done on Day 0, and thereafter at two-week intervals until Day 182. To ensure chemical acaricide is not used after the experimental treatments, farmers will be incentivized for adhering to our trial protocol. This will be in the form of providing cattle with strategic deworming (twice during the project period) and free veterinary consultation and treatment in case any cattle show symptoms of trypanosomosis or tick-borne diseases. Study completion will be the day the animal will complete the study, normally on Day 182 unless it is prematurely withdrawn from the study. Individual animals will be the experimental units for statistical analysis of tick infestations. This trial proposes to assess the efficacy of Mazao Tickoff in reducing on-host tick counts compared to a commonly used chemical acaricide. Our previous small-scale field trial showed that, two weeks after the last application of Mazao Tickoff, the average number of ticks on the treated cattle (47.2 ± 8.8) was significantly lower than the untreated cattle (338.7 ± 12.8), representing 86.1% protection (https://patents.google.com/patent/WO2017216752A1/en). Similarly, a weekly application of a Triatix® acaricide for four weeks resulted in a 94.9% reduction of tick infestation counts on cattle [25]. Based on a significance level of 5%, and assuming a 95% efficacy in the reference Triatix® acaricide arm [25], a minimum sample size of 138 zebu cattle per intervention arm will provide 80% power to detect a difference in efficacy if the Mazao Tickoff bioacaricide is no more than 10% inferior to the reference arm. A non-inferiority margin of 10% below a Triatix® acaricide efficacy of 95% is considered acceptable given the limited availability of possible alternatives for tick control and the added advantages of M. anisopliae in biological control of ticks, i.e., selective and virulent against all tick stages [16–22, 42], pathogenic to both acaricide-resistant ticks [25], and is safe to humans, animals and the environment [43]. The sample size was calculated using a previously described formula [44]: where: n = sample size required in each intervention arm, p1 = protection efficacy of Triatix® = 0.95, p2 = protection efficacy of Mazao Tickoff = 0.85, p1—p2 = minimum worthwhile difference = 0.10, Zα/2: for 5% level of significance = 1.96, Zβ: for 80% power = 0.84. Herd-level treatments and repeated measurements are likely to enhance intra-herd clustering of measurements estimated at individual animal levels. To account for the variation that may occur among herds, i.e., clustering effect, inflating the sample size by two- to four folds can account for the potentially large variation among clusters [45]. We, therefore, inflated the sample size by two folds and obtained a total of 276 zebu cattle per intervention arm. A dropout rate of 30% was included in the calculation to account for potential dropouts during the trial, bringing the total number of cattle per study to 359. The total sample size for all the three treatments together being 1,077 zebu cattle. Enrolment in the study will be restricted to cattle herds fulfilling the following inclusion criteria: (i) all cattle in the herd are of local zebu breed managed under an extensive grazing system; (ii) at least one cattle in the herd is infested with live attached hard tick(s); (iii) all cattle in the herd are apparently healthy or have minor ailments judged not to interfere with the study; (iv) there is an informed consent given by the owner or by an authorized representative. A herd will be ineligible if the individual animals are severely ill, require intensive veterinary care before enrolment, or have pre-existing medical conditions judged to interfere with the study. The eligible herds will be enrolled in the study, and the following baseline characteristics recorded: age, sex, and live body weight. Each animal will receive an ear tag containing a number of three-digit identification (ID) (village acronym/ herd number /individual number). All the animals will remain with their owners throughout the study and will be fed their usual diet with access to water according to their normal routine. The qualifying herds will be stratified by herd size and study village cluster and randomly allocated to either Triatix® (Cooper K-Brands Ltd), Mazao Tickoff (Real IPM Kenya Ltd), or placebo in a 1:1:1 ratio. If, after randomization, we note a large variability in tick infestation prevalence and intensity among the groups, tick intensity and prevalence will then be considered as part of the randomization plan. All cattle from the same herd will be randomized to the same treatment. The stock solution of Mazao Tickoff (4.05 × 1010 conidia/mL), Triatix® (Amitraz, 12.5% EC) and the placebo (excipients of Mazao Tickoff) will first be diluted at a dosage of 2 ml/L as recommended by the manufacturer before being applied on the cattle (4 liters per animal). The formulation of Mazao Tickoff will be prepared five to ten days right before application. Three treatments are deployed: Triatix®, Mazao Tickoff and Placebo. Triatix® contains 125 g of Amitraz. Mazao Tickoff is a formulation of M. anisopliae ICIPE 7 (4.05 × 1010 conidia/mL) prepared in canola oil (95%) and mixed with 0.05% Triton X-100 (1.5%) and Kerosene (3.5%). The placebo contains only the excipients of Mazao Tickoff. Treatments will be administered topically every two weeks from Day 0 to Day 182 (Fig 3), using a hand rocker sprayer with a cone-type nozzle and a pressure of 6 kg/cm. Day 0 will be defined individually as the day an animal receives the first treatment. The animals will be restrained in a crush and then sprayed from the bottom up and in the opposite direction to how the hair lies, giving greater attention to the areas most affected by ticks, such as the inner thighs, dewlap, tail, belly, inside ears, legs, and perineum. Animals will be treated in the morning (6–8 a.m.) to avoid the adverse effects of sunlight (UV radiation) on the fungus [46]. For logistical convenience, we will construct three crushes in each village cluster and all treatments will be administered (per herd) in each crush. Administration of treatments (red arrows), time-points of tick counting (green stars) and time-points for serum and whole blood sampling (blue triangles). The cattle will be restrained and whole-body tick counts will be done on each cattle at pre-treatment on Day 0, and thereafter on bi-weekly intervals until Day 182 (Fig 3). The on-host ticks will be counted just before each spraying. Further, we will determine the feeding status of the ticks in situ on all the experimental animals. The animals will be restrained in a crush and carefully examined for the presence of live attached ticks in five anatomical zones (Fig 4) as described by Rocha et al. [47]. The counting will be done sequentially from zone 1 to 5 while the ticks are still attached to the animal body. Z1: head, ears, neck and dewlap to the point of the sternum; Z2: back and loin; Z3: forelegs, shoulders and ribs; Z4: rear legs, udder/scrotum, fore and rear flank; Z5: rump and tail. Image adapted from [47]. To check on fungal activity and possible spillover effects, representative ten ticks of each species, life stage and feeding status will be collected (with forceps so as not to damage the mouthparts) from randomly selected treated animals from each intervention arm every two weeks. They will be placed in a sterile Petri dish (at most 10 ticks per dish) and maintained at a temperature of 26 ± 1°C and 85 ± 5% relative humidity (RH) for 2 weeks. Mortality will be recorded daily and dead ticks will be immediately removed and transferred to another sterile Petri dish lined with moistened filter paper to allow fungal growth on the cadaver [25]. For purposes of this experiment, we will use an already formulated product i.e., Mazao Tickoff and not the fungus as a stand-alone ingredient. However, the viability of conidia will be determined before the formulation of Mazao Tickoff by spray-platting 0.1 ml of the conidial suspension (titrated to 3 × 106 conidia/mL) on Sabouraud Dextrose Agar (SDA) plates. Plates will be incubated at 26 ± 2°C for 18 hours. Sterile microscope cover slip will then be placed on each plate and the percentage of germination will be determined by counting 100 spores for each plates, using a compound microscope at 40× magnification. Germination rates > 80% after 24 hours on SDA will be considered adequate for use in the field trials. To guarantee that the product will reach its highest efficacy in the field, Mazao Tickoff formulation will be freshly prepared and used within five to ten days. The product will also be stored and transported at room temperature. A cross-sectional survey will be done on all herds participating in the trial. Approximately 10 ml of blood will be collected from the cattle by jugular venipuncture using 20cc syringes and sterile 18G needles. Four milliliters of whole blood will be transferred to vacutainer tubes coated with ethylenediaminetetraacetic acid (EDTA) and another 4 ml transferred into plain tubes coated with serum clot activator. The samples will be transported in cool boxes to the laboratory for further processing and analysis. The serum will be processed and the antibodies against T. parva and A. marginale will be detected by enzyme-linked immunosorbent assay (ELISA) using standard methodology [48, 49]. For accurate interpretation of serological data, calves below 21 weeks old will not be sampled. This is because maternal T. parva antibodies can be detected in calves until 21 weeks of age [47] and may compromise the accuracy of the analysis. Genomic DNA will be extracted from the EDTA whole blood samples using the DNeasy Blood & Tissue kit following the manufacturer’s protocol (Qiagen, Hilden, Germany) and then screened for the presence of T. parva and A. marginale by HRM-PCR [50] using primers described by Georges et al. [51] and De La Fuente et al. [52], respectively. Owing to the nature of the interventions, it will be impossible to blind the livestock owners to their intervention arm randomization. However, the identity of the intervention arm will be concealed from the owners until the completion of enrolment and randomization to eliminate participation bias. To maintain appropriate blinding, non-blinded trained personnel will be responsible for administering treatments to cattle and not participating in outcome assessment. Outcome assessors will neither have access to the database nor a preview of the previous tick count as the outcome will be recorded and transmitted electronically. The blinding of the statistician will be done using intervention arm identification codes. The primary outcome measure is the average number of ticks per individual cattle at each post-treatment follow-up visit in each intervention arm. The personnel responsible for counting ticks and administering the study products will receive training on the study protocol. Additional training sessions on tick feeding state (fully engorged, partially engorged and unfed) and live state (live or dead) will be provided to personnel performing tick counts. Moreover, they will be trained on differentiating the cattle breeds (indigenous zebu, exotic, and cross-breeds), age groups (calves, yearlings, and adults), sex (male and female) and how to measure the live-weight of the cattle. All consent forms will be initially prepared in English and translated into Kiswahili. To promote participant retention and minimize any loss to follow-up, contact details of the farmers will be maintained in a database for ease of follow-up. General climate data (rainfall, temperature and relative humidity) for the study area covering the experimental period will be obtained from Kenya’s Department of Meteorology. The study data will be collected and managed using Research Electronic Data Capture (REDCap) tools hosted at the International Centre of Insect Physiology and Ecology (ICIPE) [53, 54]. To ensure the confidentiality of the collected data, the database will be password protected and only accessed by the investigators. All consent forms containing names and contacts of livestock owners will be kept in a locked cabinet and the key kept by the principal investigator. Authorized representatives from the funding agency or regulatory bodies may inspect all documents and records of the trial. All data will be summarized and analyzed using R statistical software version 3.6.1 (http://cran.r-project.org/). Baseline demographic variables (age, sex, and body-weight) will be tabulated. All efficacy analyses will be conducted on both per-protocol (PP) population, consisting of all cattle without major protocol violations, and the intent-to-treat (ITT) population consisting of all cattle randomized to an intervention arm. Efficacy will be calculated using geometric and arithmetic means at each post-treatment day as the percentage reduction in live tick-counts in treated animals compared to the untreated controls using the following formula [55]: Where and Generalized linear mixed models (GLMM) for negative binomial data using the logarithmic link function (log-linear modeling or regression, or Poisson regression) will be used to assess the effect of the treatments at the different follow-up measurements. The model will include the treatment variable (A, B, C), time-point and the interaction between the treatment variable and time-point [56] as fixed effects [option to supplement with age and sex]. We will account for village and herds effects by including herd nested within village as a random effect. Standard errors for the means will be calculated, and 95% confidence intervals will be constructed by time-point. Testing will be two-sided at the significance level of α = 0.05. The non-inferiority of the Mazao Tickoff will be evaluated to the commercial Triatix® acaricide at each post-treatment time-point using a margin (δ) of 10% at the one-sided α of 0.025 significance level. If the lower 97.5% confidence limit of the Mazao Tickoff is within the margin of non-inferiority, it will indicate that it is not less effective (non-inferior) to Triatix® at that time-point. If the lower confidence limit is above the margin of non-inferiority, it will indicate that Mazao Tickoff is superior to Triatix® and it is inferior if the upper bound is below the margin of non-inferiority. The study will be inconclusive if the upper and lower bounds of the confidence intervals are outside the margin of non-inferiority. We will use the stochastic susceptible-infectious-recovered (SIR)-based model to understand the transmission dynamics of T. parva and A. marginale in cattle, and to model the epidemiological impact of Mazao Tickoff. In this model, the cattle population will be classified as either susceptible (Sc), infected and infectious (Ic), or recovered and carrier (Cc). The tick population will be divided into three subclasses according to their lifecycle stage: larvae, nymph and adult classes, with each stage containing a susceptible (ST) and infectious (IT) class. During the study, the numbers of infectious and susceptible cattle in each herd will be observed and recorded at the start of each observation interval (four-time intervals i.e., Days 0, 60, 120 and 182 of the trial). Animals will be registered as a new case (incidence) on the date they are reported with TBD (either anaplasmosis or East Coast fever and confirmed using HRM-PCR and sequencing). Further, a mathematical model will be developed to simulate the epidemiological and entomological impact of Mazao Tickoff, using the obtained trial data as critical model input. The trial protocol has been approved by Kenya’s Veterinary Medicines Directorate (Approval reference: MOALF/SDL/VMD/TRIALS/VOL1/14), Directorate of Veterinary Services (no objection ref: MOALF/SDL/DVS/DS/RES/74), Pwani University Ethics Review (approval number ERC/EXT/002/2020), National Commission for Science, Technology and Innovation (NACOSTI; License No: NACOSTI/P/21/6726), and ICIPE’s Institutional Animal Care and Use Committee (IACUC, Reference No. Oundo-icipeACUC-Mar2020). Any deviation from the protocol that will impact the conduct of the study will also be immediately reported to VMD and DVS, ICIPE’s IACUC, and NACOSTI as appropriate. Before the implementation of interventions, consultative meetings will be held to explain the objectives, study design, details of products under investigation, implementation procedures and expected outcomes of the trial. This will include the DVS and the various administrative levels from the County level up to the village level. Permission through official letters will be obtained from the various administrative levels. Participating cattle owners will then be required to sign an informed consent form (S1 File) allowing enrolment of their cattle into the study. If the signatory is illiterate, a thumbprint will be obtained and confirmed by an independent witness. All consent forms will be countersigned by the staff member obtaining consent and a copy will be left at the household. No data safety and monitoring board will be installed for this trial. Triatix® forms part of the routine tick control program in Kenya and will be undertaken in collaboration with Kenya’s Directorate of Veterinary Services in the locality of the trial. There are no apparent risks to safety for humans, animals, and the environment in the use of Mazao Tickoff, as per the toxicity and eco-toxicity results of the M. anisopliae isolate ICIPE 7 (https://patents.google.com/patent/WO2017216752A1/en). Kaaya et al. [18] also reported no adverse reactions in cattle treated with fungal formulations of M. anisopliae at any time during the experiment. It was also observed that all cattle gained significant body weight during the experimental period, suggesting that fungal entomopathogens are safe for cattle. Further, the fungus M. anisopliae in aqueous or oil formulations is known to pose negligible risk to humans, animals and the environment [43, 57]. Therefore, we do not anticipate any adverse effects on the cattle or humans from the Mazao Tickoff bioacaricide. However, all personnel responsible for spraying the cattle with acaricides will be provided with the appropriate personal protective equipment (PPE). During the trial, the principal investigator or a delegated person will make regular field visits for quality control of the work done in the field. During these visits, the investigator will monitor all aspects of the trial, including adherence to the standard operating procedures (SOP), documentation, and record-keeping. Herds could be withdrawn prematurely from the study due to the owner’s decision to withdraw consent; or at the discretion of the investigator for reasons that include non-compliance with the study protocol (for instance, treatment with a study-proscribed acaricidal product), or development of a serious illness that is incompatible with continuation in the study.