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Pen studies on the control of cattle tick (Rhipicephalus (Boophilus) microplus) with Metarhizium anisopliae (Sorokin)
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Veterinary Parasitology 156 (2008) 248–260 www.elsevier.com/locate/vetpar
Pen studies on the control of cattle tick (Rhipicephalus (Boophilus) microplus) with Metarhizium anisopliae (Sorokin) D.M. Leemon a, L.B. Turner a, N.N. Jonsson b,* a
Department of Primary Industries and Fisheries, Queensland. Animal Research Institute, Locked Mail Bag 4, Moorooka, Qld 4105, Australia b University of Queensland, School of Veterinary Science, St Lucia, Qld 4072, Australia Received 14 April 2008; received in revised form 5 June 2008; accepted 11 June 2008
Abstract Three field trials were conducted over 12 months to assess the pathogenicity of Metarhizium anisopliae to parasitic stages of Rhipicephalus (Boophilus) microplus on dairy heifers under different environmental conditions. Two isolates were selected based on their high optimal growth temperature (30 8C), good spore production characteristics and ability to quickly kill adult engorged ticks in the laboratory. Spores were formulated in an oil emulsion and applied using a motor driven spray unit. Surface temperatures of selected animals were monitored, as were the ambient temperature and relative humidity. Unengorged ticks sampled from each animal immediately after treatment were incubated in the laboratory to assess the efficacy of the formulation and application. Egg production by engorged ticks collected in the first 3 days after treatment was monitored. Side counts of standard adult female ticks were conducted daily, before and after treatment to assess the performance of the fungus against all tick stages on the animals. In each trial the formulation rapidly caused 100% mortality in unengorged ticks that were removed from cattle and cultured in the laboratory. A significant reduction in egg production was recorded for engorged ticks collected in the 3 days post-treatment. However, there was little effect of the formulation on the survival of ticks on cattle, indicating that there is an interaction between the environment of the ticks on the cattle and the biopesticide, which reduces its efficacy against ticks. # 2008 Elsevier B.V. All rights reserved. Keywords: Biological control; Metarhizium anisopliae; Rhipicephalus (Boophilus) microplus; Biopesticide; Tick control; Cattle
1. Introduction Although many studies have demonstrated high levels of efficacy of fungal biopesticides against ticks in vitro (Polar et al., 2005; Onofore et al., 2001; Sewify and Habib, 2001; Gindin et al., 2001; Frazzon et al., 2000; Monteiro et al., 1998; Zhioua et al., 1997; Mwangi et al., 1995; Castineiras et al., 1987), relatively few studies have shown acceptable efficacy when
* Corresponding author. Tel.: +61 7 3365 1279. E-mail addresses: [email protected] (D.M. Leemon), [email protected] (N.N. Jonsson). 0304-4017/$ – see front matter # 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2008.06.007
applied to cattle to control ticks (Polar et al., 2005; Kaaya, 2000; Benjamin et al., 2002; Bittencourt et al., 1999; Correia et al., 1998). Previous work using isolates derived from south eastern Queensland has shown that some isolates were able to achieve 100% mortality in vitro within 2 days (Leemon and Jonsson, 2008), more effective than achieved in the in vitro studies listed above. No previous study has simultaneously assessed the in vitro and in vivo performance of a fungal biopesticide, or the efficacy of a product against more than one stage of the life cycle. This paper describes three small pen trials designed to assess the potential of a fungal biopesticide based on Metarhizium anisopliae for the control of Rhipicephalus microplus, taking some
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account of the season and the effects of the on-animal microclimate on pathogenicity in the tick. 2. Materials and methods The studies made use of 3 small field enclosures and 15 young dairy heifers at the Queensland Department of Primary Industries and Fisheries (DPI&F) Dairy Research station at Mutdapilly, 60 km south west from Brisbane, South East Queensland. The first trial was carried out from the end of February until the end of March 2003. The second trial was carried out in the same year from the beginning of May until mid-June. A third trial was carried out in the next year (2004) during early-mid February. It was expected that carrying out the three trials at different times of the year might provide some indication of the effect of ambient temperature on the results, although it would not allow firm conclusions to be made regarding the effect of temperature on pathogenicity. The third trial in early February was scheduled to check the performance of the biopesticide under high environmental temperatures. 2.1. Animals Fifteen Holstein–Friesian and Friesian cross heifers were used in each of the trials. The ages of the animals ranged from 6 to 7 months in the first trial and their weights ranged from 118 to 176 kg (average 146 kg) at the start of the trial. In the second trial, the animals ranged from 3 to 5 months and their weights ranged
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from 56 to 109 kg (average 77 kg) at the start of the trial. In the third trial the ages on the animals ranged from 5 to 6 months and their weights ranged from 101 to 164 kg (average 119 kg). All heifers were vaccinated against clostridial diseases and leptospirosis. Animals were carefully observed daily during the trial for any adverse effects from the tick infestation and fungal treatments. Each group of five animals was held in one of three 12 m by 12 m tick-free field pens. Each pen had a 5-m by 0.7 m feeding trough, a 2-m by 0.5 m water trough and a shaded area of 2 m by 3 m. The heifers were fed ad libitum on a diet of high quality lucerne hay and a grain mix as well as oaten hay. 5000 tick larvae (0.25 g of eggs allowed to hatch) were applied along the back of each heifer using a small paint brush three times a week for 3 weeks. This approach ensured that all tick stages were present at the time of treatment. Standard tick side counts were conducted by one person only, at 9:00 a.m. on the 5 days prior to each treatment, according to the method of Wharton and Utech (1970). The heifers were then ranked according to their resistance to infestation for stratified random allocation to one of three groups: Trial 1: low = 42–65; medium = 78–125; high = 127–206; Trial 2: low = 43–100; medium = 108–145; high = 169–283; Trial 3: low = 10–30; medium = 42–89; high = 100–200. 2.2. Treatments In each trial, each of the five treatments was applied to one heifer in each pen. It was anticipated that
Table 1 Treatments applied to heifers in three groups in Trial 1 Treatment number
Description
Abbreviation
Trial 1 1 2 3 4 5
Control—no treatment at all 1 l of base/animal 1 l of base/(animal d) for 3 days 1 l of base + spores/animal 1 l of base + spores/(animal d) for 3 days
(C) (Oil 1) (Oil 3) (Spores 1) (Spores 3)
Trial 2 1 2 3 4 5
Control—no treatment at all 3 l of base/animal 1 l of base/(animal d) for 3 d 3 l of base + spores/animal 1 l of base + spores/(animal d) for 3 d
(C) (3 l (1 l (3 l (1 l
Trial 3 1 2 3 4 5
Control—no treatment at all 3 l of base/animal, low P (69 kPa) 3 l of base/(animal d) for 3 d, high P (276 kPa) 3 l of base + spores/animal, low P (69 kPa) 3 l of base + spores/animal, high P (276 kPa)
(C) (HP Oil) (LP Oil) (HP Spores) (LP Spores)
Oil) Oil 3) Spores) Spores 3)
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minimal effective cross over of treatments would occur, so the design enabled comparison of five treatments, each with three replicates. Treatments for Trial 1, Trial 2 and Trial 3 are shown in Table 1. The group sizes were smaller than we would have wished for a study of this nature and the number of different treatments too high, but this was unfortunately forced by the imperative to provide some proof of concept for future funding. 2.3. Formulation and application of treatments Spores for Trial 1 were produced on rice media as described by Goettel (1984). The rice was dried at 20 8C for 4 days then harvested by shaking through 1 mm; 300 and 150 mm Endicott sieves. Spores for Trials 2 and 3 were produced by Bio-Care technology. All spores were stored at 4 8C before use. The fungal spores were formulated in 10% Codacide, an emulsified canola oil made by Microcide in the United Kingdom and supplied by Kendon, Melbourne, Australia. In Trial 1 the fungal isolate ARIM16 was used at approximately 2 108 spores/ml. In Trial 2 spores from the isolates ARIM10 and ARIM16 produced by Bio-Care Technology Australia were mixed 3:2 (respectively) giving approximately 2.5 108 spores/ml. In Trial 3 spores of the isolate ARIM16 produced at the DPI&F Animal research institute were used at a concentration of 2.5 108 spores/ml. Spores were mixed with the Codacide oil in the laboratory and later diluted with tap water at the trial site to give the test formulation. The positive controls consisted of 10% Codacide oil diluted with tap water in the field. For the application of the formulation the animals were held in a crush. In the first two trials the
formulation was applied with a small volume (1 l) spray gun using compressed air from a 1.5-hp air compressor. The spray pressure varied from 276 kPa to below 69 kPa. The pressure was mostly at the lower end as the pressure dropped rapidly once the spray nozzle was opened. In Trial 3 the treatments were applied with a small volume (1 l) spray gun using compressed air from a 2.5-hp air compressor. The air compressor was larger than that used in the two previous trials, so the pressure did not drop as rapidly once the spray nozzle was opened. This allowed for more constant spray pressures and it was applied at two pressures, 69 and 276 kPa. All animals in all trials were sprayed in the late afternoon, finishing on dusk. 2.4. Formulation check Adult engorged female ticks supplied from the DPI&F ARI tick cultures were immersed in samples of the formulation that were collected immediately before and during application to animals (24 ticks per sample in Trial 1; 20 ticks per sample in Trial 2) for 1.5 min, blotted on absorbent paper, then incubated in 24 well microtitre trays at 28 8C. Tick mortality was assessed each day for 3 days. Ticks were considered to be dead when their Malphigian tubules ceased moving. 2.5. Application check Twenty-five incompletely engorged adult ticks between 4.5 and 8 mm were removed from each animal immediately after treatment. Twenty of these ticks were incubated in microtitre trays at 28 8C for daily assessment of mortality at optimal temperature. The other five ticks from each animal were used for spore recovery. To recover spores, ticks were agitated in 1 ml
Table 2 Mortality of adult ticks immersed in samples of the spray treatments taken before and during spraying in Trial 1 and before spraying in Trial 2 Sample
% Mortality d1 (S.E.)
% Mortality d2 (S.E.)
% Mortality d3 (S.E.)
Trial 1 Control Oil pre-spray Spore pre-spray Spore spray
0 (0) 0 (0) 1.7 (1.5) 5.0 (2.5)
0 (0) 1.7 (1.5) 82.7 (14) 100 (0)
0 (0) 3.3 (3) 100 (0) 100 (0)
Trial 2 Control 3 l spores 1 l spores 1 (d1) 1 l spores 2 (d2) 1 l spores 3 (d3)
0 0 1 6 3
0 100 100 82 85
0 100 100 100 100
(0) (0) (1) (1) (1)
(0) (0) (0) (4) (2)
(0) (0) (0) (0) (0)
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of 1.0% Tween 80 for 10 min. A haemocytometer was used to count spores.
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3. Results 3.1. Formulation checking
2.6. Mortality and egg production in vitro Fully engorged female ticks were collected from animals during the early hours of the morning following treatment, before the ticks dropped from the animals. The aim was to collect 24 ticks from each animal, but for some animals this number could not be collected, possibly due to engorged ticks dropping earlier than anticipated. Ticks were incubated in preweighed microtitre trays in a dark cupboard, subject to ambient temperature fluctuations for 10 days. Tick mortality was assessed at 4 and 10 days in Trial 1, at 7 and 15 days in Trial 2, and at 5 and 12 days in Trial 3. Egg weights were also determined after the last inspection for each incubation period. Egg production, expressed as the weight of eggs per weight of ticks for each treatment, was determined for each day of collection. An analysis of variance (GenStat, 2007) was conducted using treatment and collection day and their interaction as terms and egg production as the outcome variable. 2.7. Temperature The ambient temperatures and the surface temperatures of selected animals were recorded. A data logger recording the relative humidity and temperature every 20 min was placed in the shade near the pens holding the animals. Remote sensor data loggers were attached to the base of the tail of three animals in Trial 1, four animals in Trial 2 and Trial 3. The sensors were secured in the coat at the base of the tail of each animal. The data loggers (Tinytag, Hastings data loggers) recorded the surface temperature of these animals every 20 min. 2.8. Assessment of treatment efficacy Side counts of adult female ticks (4.5–8 mm) were carried out daily on each animal at 9:00 a.m. The counts began 16 days after the first batch of ticks was applied (5 days before treatment) and continued for 3 weeks. A one-way analysis of variance (GenStat, 2007) with randomised blocking was performed on the data using the day 0 counts as a covariate. In Trial 3 the counts were conducted from day 3 (4 days before treatment) until 8 days after treatment. This trial was discontinued after this time as there were few ticks left on the control or treatment animals due to extreme conditions of very high temperatures and relative humidity.
The mortalities among engorged ticks immersed in the different spray samples are shown in Table 2. In Trial 1 there were no mortalities in the control ticks and the mortality of ticks immersed in the formulation base (Oil pre-spray) was only 3.3% after 3 days. However, ticks immersed in the spore formulation sampled before (spore pre-spray) and during spraying (spore spray) were rapidly affected by the formulation with most dead by 2 days, and all dead by 3 days. There was no significant difference ( p 0.05) between the spore formulation samples taken before and during spraying, indicating that the pressure of the spray apparatus did not affect the virulence of the spores in the formulation. In Trial 2 after 3 days of incubation all ticks immersed in the formulation samples were dead. In Trial 3 formulation samples were taken and stored to be used only if the results of the application check were poor. 3.2. Application check In Trial 1, only the treatment with three applications of 1 l of spore formulation (3 l/d for 3 days) caused 100% mortality in ticks removed from animals (Fig. 1), and the mortality in ticks removed from animals receiving only one application (1 l) of spore formulation reached 58% after 3 days. There were no mortalities in the control ticks and mortalities in ticks removed from animals receiving the oil base ranged from 5 to 15% after 3 days. These results indicate that the spray equipment was effective in delivering lethal doses of spores to ticks but 3 l of spore formulation was needed. In Trial 2 spore application caused 100% mortality after 2 days in all ticks removed from spore treated animals (Fig. 1). The mortality in ticks removed from control animals was 1% after 3 days. The mortality in ticks removed from animals receiving 3 l of formulation base in one application was 3% after 3 days but 37% in ticks removed from the animals that received the 3 l of formulation base over 3 days. In Trial 3 both the formulation and application were effective, all ticks removed from treated animals were dead after 2 days while ticks removed from control animals showed very low mortalities (0–6%) after 3 days. The mean numbers of spores collected from ticks removed from cattle immediately after spraying for all treatments in Trial 1 are listed in Table 3. Due to the inconsistent results in Trial 1 this procedure was not carried out during Trials 2 and 3.
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Fig. 1. Average mortality (%) of unengorged ticks (4.5–8 mm) removed from cattle immediately after treatment and incubated for 3 days at 28 8C. Trial 1 (top), Trial 2 (middle) and Trial 3 (bottom).
3.3. Engorged tick mortality and egg production in vitro The mortalities (%) in engorged ticks collected from animals in the 3 days after treatment are shown in Table 4 and the egg production expressed as the average weight of eggs per average weight of ticks is shown in Fig. 2.
The results of the ANOVAs conducted on the egg production data (Tables 5 and 6) show that the treatment term was a dominant and highly significant effect ( p < 0.001). In all three trials egg production of ticks removed from spore treated animals was significantly different ( p < 0.05) from the egg production of ticks removed from the control animals or animals receiving
D.M. Leemon et al. / Veterinary Parasitology 156 (2008) 248–260 Table 3 Number of spores recovered from unengorged ticks removed from heifers after spraying in Trial 1 only Treatment
Mean spores/tick (S.E.)
Spores 1 Spores 1 (d1) Spores 2 (d2) Spores 3 (d3)
597,917 568,750 420,833 352,083
(57,310) (70,434) (49,047) (34,422)
only the oil base without the spores. In Trials 1 and 3 the interaction of treatment and day of collection from the animal was significant ( p < 0.05), and for all three trials these means are shown in Fig. 2. This shows that the main reason for the interactions in Trials 1 and 3 was within the ‘spore’ treatments—these tended to be much more effective on day 1, decreasing in efficacy by days 2 and 3. In Trial 2 there was no significant difference in egg production attributable to day. However, in Trial 1, egg production by ticks removed in the first 2 days posttreatment was significantly different ( p < 0.05) from egg production by ticks removed on day 3. In Trial 3 ticks removed in the first day post-treatment produced significantly fewer eggs ( p < 0.05) than the ticks removed on days 2 and 3 post-treatment. The data summarised in Table 4 show the spore treatments in the three trials were effective in causing high mortality in engorged ticks removed from the animals after treatment. In addition the same effect of time can be seen with tick mortality as that seen with the reduction in egg production in Trials 1 and 3. The longer the ticks stayed on the animal before incubation, the lower the tick mortality and higher the egg production in these two trials. In Trial 1, mortalities in ticks removed from animals treated with the spore formulation varied widely after 4 days of incubation, from 91 to 94% in ticks removed the day after treatment down to 4–22% in ticks removed 3 days after treatment. Most of these dead ticks were covered in fungal mycelium. Ticks removed from control animals and animals treated with the formulation base all showed very low rates of mortality (0–2%) after 4 days of incubation except for the ticks collected from one control animal on day 2 (45%). Some of these dead ticks exhibited fungal growth indicating that some cross contamination might have occurred at the time of collection of ticks or among animals after application. In Trial 2 there was less variation in mortalities in ticks removed from treated animals. Because the ambient conditions were cooler than those in Trial 1 ticks were incubated for longer (up to 15 days) to allow for completion of egg production. Many of the ticks removed from animals treated with the spore formula-
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Table 4 Mortality (%) of female engorged ticks collected from cattle over the 3 days post-treatment and then incubated at ambient temperatures in the dark Trial 1
Day collected
Mean % mortality after 4 d incubation (S.E.)
Mean % mortality after 10 d incubation (S.E.)
Control
1 2 3
2 (1) 45 (25) 0 (0)
25 (18) 46 (25) 1 (1)
Oil 1
1 2 3
1 (0) 0 (0) 1 (1)
42 (9) 5 (2) 1 (1)
Oil 3
1 2 3
1 (1) 1 (1) 1 (1)
64 (10) 6 (3) 6 (1)
Spores 1
1 2 3
92 (4) 3 (2) 4 (3)
100(0) 50 (12) 36 (10)
Spores 3
1 2 3
91 (5) 14 (3) 22 (2)
100 (0) 66 (13) 83 (7)
Trial 2
Day Mean % mortality after Mean % mortality after collected 7 d incubation (S.E.) 15 d incubation (S.E.)
Control
1 2 3
0 (0) 0 (0) 0 (0)
16 (7) 6 (1) 3 (1)
1 l Oil 3
1 2 3
0 (0) 4 (4) 18 (31)
37 (21) 47 (10) 68 (18)
3 l Oil
1 2 3
4 (3) 0 (0) 0 (0)
14 (6) 22 (10) 3 (2)
1 l Spores 3 1 2 3
84 (6) 97 (3) 99 (1)
100 (0) 100 (0) 100 (0)
98 (1) 62 (22) 48 (31)
100 (0) 99 (1) 88 (9)
3 l Spores
1 2 3
Trial 3
Day collected
Mean % mortality after 5 d incubation (S.E.)
Control
1 2 3
1 (1) 0 (0) 0 (0)
56 (25) 0 (0) 11 (9)
Low press oil
1 2 3
0 (1) 0 (0) 1 (0)
5 (19) 20 (13) 1 (0)
High press oil
1 2 3
0 (1) 0 (0) 0 (0)
27 (17) 5 (2) 2 (0)
Low press spores
1 2 3
100 (0) 29 (0) 3 (1)
100 (0) 100 (0) 51(1)
High press spores
1 2 3
83 (0) 44 (0) 8 (1)
100 (0) 100 (0) 77 (1)
Mean % mortality after 12 d incubation (S.E.)
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Fig. 2. Egg production by engorged ticks removed from Trial 1 (top); Trial 2 (middle) and Trial 3 (bottom) heifers in the 3 days post-treatment expressed as an average weight of eggs per average weight of ticks.
tion were dead after 7 days and nearly all were dead after 15 days of incubation. Fungal mycelium was present on most of these dead ticks. There were no mortalities in the ticks removed from control animals after 7 days, but after 15 days mortalities ranged from 3 to16%. There was a wide range in mortalities among ticks removed from animals receiving the formulation base after 7 days (0–18%) and 15 days (3–68%). In Trial 3, conducted with higher ambient temperatures, mortalities in ticks removed from treated animals showed a wide variation after 5 days of incubation, ranging from 83 to 100% in ticks collected the day after
treatment down to 3–8% in ticks removed from animals 3 days post-treatment. Ticks removed from control animals or those receiving the formulation base had very low mortalities after 5 days of incubation (0–1%). 3.4. Temperature The ambient and animal surface temperatures recorded by the data loggers in the three trials are shown in Table 7. In Trial 1 only one of the three loggers attached to the animals recorded any data. The sensor cables on the other two loggers became detached from
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Table 5 ANOVA of egg production by ticks removed from animals in the 3 days post-treatment in Trials 1, 2 and 3 showing main effects and interaction terms Source of variation
d.f
s.s
m.s.
v.r
F pr.
Trial 1 Rep stratum Treatment Day Treatment day Residual
2 4 2 8 28
0.002784 0.766469 0.039716 0.166238 0.076278
0.001392 0.191617 0.019858 0.020780 0.002724
0.51 70.34 7.29 7.63
<0.001 0.003 <0.001
Trial 2 Rep stratum Treatment Day Treatment day Residual
2 4 2 8 28
0.042934 0.915062 0.035278 0.045510 0.268715
0.021467 0.228766 0.017639 0.005689 0.009597
2.24 23.84 1.84 0.59
<0.001 0.178 0.775
Trial 3 Rep stratum Treatment Day Treatment day Residual
2 4 2 8 27
0.10349 1.31052 0.17506 0.39804 0.36362
0.05175 0.32763 0.04975 0.04975 0.01347
3.84 24.33 6.50 3.69
<0.001 0.005 0.005
Table 6 ANOVA of adjusted means of egg production showing the main effects Treatment
Control
1 l oil
3 1 l oil
1 l spores
3 1 l spores
(l.s.d.)
0.409 a 1 0.2607 a
0.3980 a 2 0.2702 a
0.3710 a 3 0.3279 b
0.1753 b
0.0862 c
(0.0504)
Control
3 l oil
3 1 l oil
3 l spores
3 1 l spores
(l.s.d.)
0.355 a 1 0.2607 a
0.349 a 2 0.2702 a
0.269 a 3 0.3279 a
0.082 b
0.007 b
(0.0946)
Control
LP oil
HP oil
LP spores
HP spores
(l.s.d.)
0.614 a 1 0.343 a
0.539 a 2 0.483 b
0.550 a 3 0.465 b
0.223 b
0.224 b
(0.1122)
Trial 1 Day
Treatment
(0.03904)
Trial 2 Day
Treatment
(0.07330
Trial 3 Day
(0.0869)
Within rows, means followed by the same letter are not significantly different ( p 0.05).
the loggers within a day of attachment to the animals. The lowest recorded ambient temperature was 17 8C and the highest was 33 8C. The lowest recorded temperature on the animal was 25 8C and the highest was 40 8C. During the first 7 days after spraying surface temperatures on the animal were between 25 and 34 8C for at least half of each day although there were a number of hours each day above 34 8C. In Trial 2 ambient temperatures ranged from 5 to 25 8C while animal surface temperatures ranged from 16 to 38 8C. The temperatures on the four animals were
mostly consistent except for the heifer with logger 3; the night-time temperatures on this animal were always higher than those on the other animals. The ambient temperature was below 20 8C for most of each day for the first 7 days and only went above 25 8C for 2 h during day 7. The average surface temperatures on the four heifers were correspondingly low with more than half of each day below 25 8C, with little time above 34 8C. In Trial 3 because the data logger recording the ambient temperature and humidity failed, the ambient temperature data recorded at the nearest weather station
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Table 7 Ambient temperatures (range and hours below 20 8C and 25 8C), and corresponding average surface temperatures (range, hours below 25 8C, between 25 and 34 8C, and hours above 34 8C) recorded on animals during the first 7 days after treatment in the three trials Days after treatment
Ambient temperature range (8C)
Hours ambient 20 8C
Hours ambient 25 8C
Temperature range on animal (8C)
Hours on animal 25 8C
Trial 1 (n = 1) 1 23–28 2 18–33 3 18–31 4 18–31 5 17–31 6 18–32 7 17–30
0 9 6.5 11 17.5 9 11
12 15 15 16 13 16 16
32–38 32–40 24–38 25–40 31–38 30–38 30–38
0 0 2 2 0 0 0
Trial 2 (n = 4) 1 5–23 2 10–21 3 13–21 4 14–22 5 11–22 6 10–23 7 12–25
18 18 17 17 18 16 14
24 24 24 24 24 24 22
16–38 16–31 18–34 19–34 18–34 20–35 14–39
15.5 17 13.5 14.5 12.5 13 12
Hours on animal 25 8C 34 8C
Hours on animal 34 8C
13 14 12 13 14 17 17
11 10 10 9 10 7 7
6.9 6.9 9.8 8.5 11.3 8.9 9.1
1.5 0 0.5 1 0 2 2.5
Days after treatment
Ambient temperature range (8C)
Av. Ambient temp (8C)
Temperature range on animal (8C)
Hours on animal 25 8C
Hours on animal 25 8C 34 8C
Hours on animal 34 8C
Trial 3 (n = 4) 1 2 3 4 5 6 7
21–37 19–31 21–35 23–38 24–37 24–42 23–42
28 25 27 29 29 33 30
23–38 28–41 32–44 33–42 33–45 33–45 24–41
3 0 0.25 0 0 0 1.5
15.9 12 4.5 4.9 6.5 5.5 12.8
5.1 12 19.4 19.1 17.5 18.5 10.5
was used. This data only included a range and average daily temperatures. Ambient temperatures were high during this trial, with daytime temperatures exceeding 35 8C on most days. By the end of the first 7 days daily temperatures were exceeding 40 8C. The ambient temperature ranged from 19 to 42 8C. Surface temperatures on the animals ranged from 23 to 45 8C. After the first day the surface temperatures on the animals were above 34 8C for more than half of each day. 3.5. Assessment of treatment efficacy The average counts of adult ticks between 4.5 and 8 mm on the right side of each animal recorded each morning are shown in Fig. 3. The ANOVAs conducted on the tick counts are presented in Table 8. In Trial 1 counts began 5 days before treatment and continued for 15 days after treatment. There was no significant effect of any treatment on the tick count on any day. In Trial 2 counts recorded from 5 days before treatment to 23 days after treatment show that there was
a reduction in ticks on animals 9–10 days after treatment with the spore formulation but this effect did not differ significantly from the effect of the 3 l oil application. In Trial 3 there were no significant differences in tick counts in any of the treatment groups. The side counts on all animals in this trial were far lower than those in the two previous trials despite all three trials having the same number of tick larvae applied over the same time period. The trial was discontinued after 8 days because it was clear that the extreme weather conditions were affecting tick numbers on all animals. 4. Discussion The fungal biopesticide was highly efficacious when applied directly to ticks that were subsequently incubated in the laboratory, or when ticks that were exposed to the biopesticide while feeding on cattle were removed from the cattle and incubated in the laboratory. However, its ability to kill ticks on the cattle was
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Fig. 3. Average daily side counts of ticks before and after treatment in Trial 1 (top), Trial 2 (middle) and Trial 3 (bottom).
consistently poor, most markedly so under the extremely hot conditions of Trial 3. This suggests that some factor in the environment on the surface of the animals negatively affected the pathogenicity of the fungus to ticks. Given the known optimal growth temperatures of M. anisopliae (Leemon and Jonsson, 2008), we suggest that the factor most likely limiting the performance of this biopesticide is high temperature on the skin surface.
Extensive sampling and testing was undertaken to provide data to differentiate the performance of the fungal biopesticide on ticks under conditions on the animal or under optimal conditions in the laboratory. Testing in Trials 1 and 2 showed that the formulations were highly virulent to engorged female ticks under laboratory conditions. The application of the formulation to animals in all three trials resulted in the rapid mortality of most unengorged ticks removed post-
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Table 8 ANOVA of adjusted means of tick side counts on selected days Trial 1
Day 0
Day 4
Day 11
Day 15
Control Oil 1 Oil 3 Spores 1 Spores 3
129 107 185 181 117
141 190 113 129 71
171 287 135 168 109
191 217 68 138 52
F-probability Standard error of mean
0.066 19.91
0.254 38.9
0.427 73.2
0.186 56.6
Trial 2
Day 0
Day 4
Day 11
Day 18
Control 1 l Oil 3 3 l Oil 1 l Spores 3 3 l Spores
123 212 155 181 174
260 243 195 142 199
402 230 227 144 147
519 193 293 62 83
F-probability Standard error of mean
0.509 34.5
a a ab b ab
0.034 21.83
a b b b b
0.027 44.2
a bc b c c
< 0.001 39.6
Trial 3
Day 0
Day 4
Day 8
Control High press oil Low press oil High press spores Low press spores
39.3 42.0 68.0 52 65.3
36.4 49.2 38.2 34.3 30.7
35.6 60.1 58.6 38.2 38.4
F-probability Standard error of mean
0.518 13.97
0.435 7.64
0.660 16.05
Means followed by the same letter for each day are not significantly different ( p 0.05).
treatment and incubated in the laboratory (Fig. 1). However, there was some variation in Trial 1 that may have been due to an insufficient volume of formulation being applied to some animals. In these trials the application check showed that the spray equipment was delivering enough spores to kill ticks under optimal conditions so any failure of the fungal formulation to kill ticks in vivo was not due to inadequate formulation and application of the fungal biopesticide. Engorged ticks collected in the first 3 days posttreatment in each trial and incubated under ambient temperature conditions had a higher mortality than ticks collected from control animals and showed a significant reduction in egg production. However, in Trials 1 and 3 there was a significant interaction between day collected post-treatment and the treatment on egg production. This may indicate an adverse effect from the animal microclimate on the spores on ticks before they dropped from the animal. One putative factor that could account for the variation in in vivo tick control is temperature. Extensive in vitro assays showed that no M. anisopliae isolates grew at 40 8C and few showed much growth at
35 8C (Leemon and Jonsson, 2008). The two isolates used in the formulations in the present trials, ARIM10 and ARIM16, were selected for their vigour and ability to grow well at 30 8C with some growth capability at 34 8C. In the hottest conditions of Trial 3 ambient temperatures exceeded 41 8C and recordings showed that surface temperatures remained above 34 8C for more than half of the time over the first 8 days. In Trial 2, where some degree of tick control was evident, ambient temperatures were much lower and surface temperatures on animals remained below 34 8C for nearly all of the time. Previous animal trials (Polar et al., 2005; Correia et al., 1998; Bittencourt et al., 1999) investigating in vivo fungal control of R. microplus have also shown limited success, although unfavourable temperature were not shown to be a contributing factor. Polar et al. (2005) recognised the importance of temperature for fungal performance and selected an isolate that performed best between 31 and 35 8C. In addition, the ambient temperatures during the trials of both Correia et al. (1998) and Polar et al. (2005) were quite low (18–24.5 8C and 23–34 8C, respectively). However,
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the fungal isolates and formulations used in the in vivo trials by both Polar et al. (2005) and Correia et al. (1998) seemed to differ in virulence to those used in these trials. The highest spore concentration used by Correia et al. (1998), (7.5 108 spores/ml), was much higher than the concentration used in our trials and with ambient temperatures similar to those in our Trial 2 there was no evidence at all of tick control on animals. Polar et al. (2005) used a similar spore concentration to us and reported a mean survival time of 6.7 days for ticks incubated at 28 8C. In contrast, ticks removed from our treated animals in Trials 2 and 3 and incubated at 28 8C were all dead after 2 days. This suggests that the isolates we used had a higher virulence to R. microplus. In all three of our trials larvae were applied three times a week for 3 weeks before treatment to ensure that all stages would be present at treatment; tick counts were also made before treatment so that animals could be assigned to treatment groups in a randomly stratified design. Additionally, in two of the three studies posttreatment side counts were conducted daily over the period of time that it was thought that female ticks that were present as larvae at the time of treatment would have completed their on-animal stages. However, the studies conducted by Polar et al. (2005) and Bittencourt et al. (1999) relied upon natural infestations of ticks and did not report on side counts prior to treatment to allow for variation in tick susceptibility among the animals, nor did they conduct daily tick counts after treatment, so it is possible that their procedures for assessing tick control might have missed some evidence of control. Correia et al. (1998) did infest animals with ticks for 3 weeks prior to treatment, but did not appear to have recognised any natural variation in susceptibility to ticks. Despite the poor overall efficacy of the product in our trials, care must be taken with the interpretation of the lack of significant results, which might be due in part to inadequate statistical power. For evaluations of efficacy for the registration of new acaricides, it is recommended that each treatment group should generally include at least three animals (Holdsworth et al., 2006). Although the treatment groups in the present study did each include three animals, and although groups were assigned after ranking on tick counts, there was a wider than expected variation in tick counts, resulting in a lack of statistical power. The present trials highlight the importance of adequate replication when conducting animal trials, especially when using a product of unknown or unpredictable efficacy. Three animals might be mostly adequate when the benchmark is 95% control, as per conventional acaricides (Holds-
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worth et al., 2006) but is likely to be inadequate with biological control strategies. There is a natural variation in tick susceptibility of cattle both within breed and between breed (Utech et al., 1978; Seifert, 1971). The results from this study suggest that future trials of this fungal biopesticide are warranted to evaluate its efficacy under conditions of controlled temperature, humidity and air flow to establish a proof of concept for in vivo tick control by a fungal biopesticide. 5. Conclusion The primary aim in each of these animal trials was to investigate whether spores of M. anisopliae formulated as a biopesticide and applied to dairy heifers could kill all parasitic stages of R. microplus. The trials were carried out at different times of the year under different weather conditions. It was anticipated that positive results in the trials would provide a proof of concept for fungal biopesticide control of R. microplus on cattle. The fungal biopesticide caused high mortalities in unengorged ticks incubated in vitro and had a significant effect in reducing egg production by engorged ticks collected in the first 3 days post-treatment. These trials did not demonstrate a statistically significant effect on tick mortality in vivo. However, the trials did provide information for the design of further trials to assess the role of ambient conditions in affecting the ability of fungal spores to control ticks in vivo. Acknowledgements We would like to thank Peter Green, Senior Parasitologist, Animal Research Institute for supply of ticks and thoughtful advice, the staff of Mutdapilly Research Station for animals and facilities, Dave Mayer and Pat Pepper, Principal Biometricians, Animal Research Institute for statistical advice, John Allan of DPI&F Actest, for advice on acaricide trial protocols and BioCare Technology (now Becker Underwood Australia) for the supply of spores. References Benjamin, M.A., Zhioua, E., Ostfeld, R.S., 2002. Laboratory and field evaluation of the entomopathogenic fungus Metarhizium anisopliae (Deuteromyces) for controlling questing adult Ixodes scapularis (Acari: Ixodidae). J. Med. Entomol. 39, 723–728. Bittencourt, V.R.E.P., Souza, E.J., Peralva, S.L.F.S., Reis, R.C.S., 1999. Efficacy of the fungus Metarhizium anisopliae (Metschnikoff, 1887) Sorokin, 1883 in field test with bovines naturally infested with the tick Boophilus microplus (Canestrini 1887) Acari: Ixodidae). Rev. Bras. Med. Vet. 21, 78–81.
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Castineiras, A., Jimeno, G., Lopez, M., Sosa, L., 1987. Effect of Beauveria bassiana Metarhizium anisopliae (Fungi Imperfecti) and Pheidole megacephala (Hymenoptera: Formicidae) against eggs of Boophilus microplus. Revista Salud Anim. 9, 288– 384. Correia, A.C.B., Fiorin, A.C., Monteiro, a.C., Verissimo, C.J., 1998. Effect of Metarhizium anisopliae on the tick Boophilus microplus (Acari: Ixodidae) in stable cattle. J. Invertebr. Pathol. 71, 189–191. Frazzon, G.A.P., Vaz Jr., S.I., Masuda, A., Schrank, A., Vainstein, H.M., 2000. In vitro assessment of Metarhizium anisopliae isolates to control the cattle tick Boophilus microplus. Vet. Parasitol. 94, 117–125. GenStat, 2007. GenStat for Windows, Release 9.1, 9th ed. VSN International, Oxford. Goettel, M.S., 1984. A simple method for mass culturing entomopathogenic Hyphomycete fungi. J. Microbiol. Methods 3, 15–20. Gindin, G.M., Samish, M., Alekseev, E., Glazer, I., 2001. The susceptibility of Boophilus annulatus (Ixodidae) ticks to entomopathogenic fungi. Biocontrol. Sci. Technol. 11, 111–118. Holdsworth, P.A., Kemp, D.H., Green, P., Peter, R.J., de Bruin, C., Jonsson, N.N., Letonja, T., Rehbein, S., Vercruysse, J., 2006. World association for the advancement of veterinary parasitology (WAAVP) guidelines for evaluating the efficacy of acaricides against ticks (Ixodidae) on ruminants. Vet. Parasitol. 136, 29–43. Kaaya, G.P., 2000. Laboratory and field evaluation of entomogenous fungi for tick control. Ann. N. Y. Acad. Sci. 916, 559–564. Leemon, D.M., Jonsson, N.N., 2008. Laboratory studies on Australian isolates of Metarhizium anisopliae as a biopesticide for the cattle tick Boophilus microplus. J. Invert. Pathol. 97, 40–49. Mwangi, E.N., Kaaya, G.P., Essuman, S., 1995. Experimental infections of the tick Rhipicephalus appendiculatus with entomopathogenic fungi, Beauveria bassiana and Metarhizium anisopliae, and
natural infections of some ticks with bacteria and fungi. J. Afr. Zool. 109, 151–160. Monteiro, S.G., Bittencourt, V.R., Daemon, Faccini, J.L., 1998. Pathogenicity under laboratory conditions of the fungi Beauveria bassiana and Metarhizium anisopliae on larvae of the tick Rhipicephalus sanguineus (Acari: Ixodidae). Braz. J. Vet. Parasitol. 72, 113–116. Onofore, S.B., Miniuk, C.B., De Barros, N.M., Azevedo, J.L., 2001. Pathogenicity of four strains of entomopathogenic fungi against the bovine tick Boophilus microplus. Am. J. Vet. Res. 62 (9), 1478–1480. Polar, R., Aquino de Muro, M., Kairo, T.K., Moore, D., Pegram, R., John, S., Roach-Benn, C., 2005. Thermal characteristics of Metarhizium anisopliae isolates important for the development of biological pesticides for the control of cattle ticks. Vet. Parasitol. 134, 159–167. Seifert, G.W., 1971. Variations between and within breeds of cattle in resistance to field infestations of the cattle tick (Boophilus microplus). Aust. J. Agric. Res. 22, 159–168. Sewify, G.H., Habib, S.M., 2001. Biological control of the tick fowl Argas persicargus persicus by the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae. J. Pest Sci. 74, 121– 123. Utech, K.B., Wharton, R.H., Kerr, J.D., 1978. Resistance to Boophilus microplus in different breeds of cattle. Aust. J. Agric. Res. 29, 411–422. Wharton, R.H., Utech, K.B.W., 1970. The relation between engorgement and dropping of Boophilus microplus (canestrini) (Ixodidae) to assessment of tick number on cattle. J. Aust. Entomol. Soc. 9, 171–182. Zhioua, E., Browning, M., Johnson, P.W., Ginsberg, H.S., LeBrun, R.A., 1997. Pathogenicity of the entomopathogenic fungus Metarhizium anisopliae (Deuteromycetes) to Ixodes scapularis (Acar: Ixodidae). J. Parasitol. 83, 815–818.
Veterinary Parasitology 156 (2008) 248–260 www.elsevier.com/locate/vetpar
Pen studies on the control of cattle tick (Rhipicephalus (Boophilus) microplus) with Metarhizium anisopliae (Sorokin) D.M. Leemon a, L.B. Turner a, N.N. Jonsson b,* a
Department of Primary Industries and Fisheries, Queensland. Animal Research Institute, Locked Mail Bag 4, Moorooka, Qld 4105, Australia b University of Queensland, School of Veterinary Science, St Lucia, Qld 4072, Australia Received 14 April 2008; received in revised form 5 June 2008; accepted 11 June 2008
Abstract Three field trials were conducted over 12 months to assess the pathogenicity of Metarhizium anisopliae to parasitic stages of Rhipicephalus (Boophilus) microplus on dairy heifers under different environmental conditions. Two isolates were selected based on their high optimal growth temperature (30 8C), good spore production characteristics and ability to quickly kill adult engorged ticks in the laboratory. Spores were formulated in an oil emulsion and applied using a motor driven spray unit. Surface temperatures of selected animals were monitored, as were the ambient temperature and relative humidity. Unengorged ticks sampled from each animal immediately after treatment were incubated in the laboratory to assess the efficacy of the formulation and application. Egg production by engorged ticks collected in the first 3 days after treatment was monitored. Side counts of standard adult female ticks were conducted daily, before and after treatment to assess the performance of the fungus against all tick stages on the animals. In each trial the formulation rapidly caused 100% mortality in unengorged ticks that were removed from cattle and cultured in the laboratory. A significant reduction in egg production was recorded for engorged ticks collected in the 3 days post-treatment. However, there was little effect of the formulation on the survival of ticks on cattle, indicating that there is an interaction between the environment of the ticks on the cattle and the biopesticide, which reduces its efficacy against ticks. # 2008 Elsevier B.V. All rights reserved. Keywords: Biological control; Metarhizium anisopliae; Rhipicephalus (Boophilus) microplus; Biopesticide; Tick control; Cattle
1. Introduction Although many studies have demonstrated high levels of efficacy of fungal biopesticides against ticks in vitro (Polar et al., 2005; Onofore et al., 2001; Sewify and Habib, 2001; Gindin et al., 2001; Frazzon et al., 2000; Monteiro et al., 1998; Zhioua et al., 1997; Mwangi et al., 1995; Castineiras et al., 1987), relatively few studies have shown acceptable efficacy when
* Corresponding author. Tel.: +61 7 3365 1279. E-mail addresses: [email protected] (D.M. Leemon), [email protected] (N.N. Jonsson). 0304-4017/$ – see front matter # 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2008.06.007
applied to cattle to control ticks (Polar et al., 2005; Kaaya, 2000; Benjamin et al., 2002; Bittencourt et al., 1999; Correia et al., 1998). Previous work using isolates derived from south eastern Queensland has shown that some isolates were able to achieve 100% mortality in vitro within 2 days (Leemon and Jonsson, 2008), more effective than achieved in the in vitro studies listed above. No previous study has simultaneously assessed the in vitro and in vivo performance of a fungal biopesticide, or the efficacy of a product against more than one stage of the life cycle. This paper describes three small pen trials designed to assess the potential of a fungal biopesticide based on Metarhizium anisopliae for the control of Rhipicephalus microplus, taking some
D.M. Leemon et al. / Veterinary Parasitology 156 (2008) 248–260
account of the season and the effects of the on-animal microclimate on pathogenicity in the tick. 2. Materials and methods The studies made use of 3 small field enclosures and 15 young dairy heifers at the Queensland Department of Primary Industries and Fisheries (DPI&F) Dairy Research station at Mutdapilly, 60 km south west from Brisbane, South East Queensland. The first trial was carried out from the end of February until the end of March 2003. The second trial was carried out in the same year from the beginning of May until mid-June. A third trial was carried out in the next year (2004) during early-mid February. It was expected that carrying out the three trials at different times of the year might provide some indication of the effect of ambient temperature on the results, although it would not allow firm conclusions to be made regarding the effect of temperature on pathogenicity. The third trial in early February was scheduled to check the performance of the biopesticide under high environmental temperatures. 2.1. Animals Fifteen Holstein–Friesian and Friesian cross heifers were used in each of the trials. The ages of the animals ranged from 6 to 7 months in the first trial and their weights ranged from 118 to 176 kg (average 146 kg) at the start of the trial. In the second trial, the animals ranged from 3 to 5 months and their weights ranged
249
from 56 to 109 kg (average 77 kg) at the start of the trial. In the third trial the ages on the animals ranged from 5 to 6 months and their weights ranged from 101 to 164 kg (average 119 kg). All heifers were vaccinated against clostridial diseases and leptospirosis. Animals were carefully observed daily during the trial for any adverse effects from the tick infestation and fungal treatments. Each group of five animals was held in one of three 12 m by 12 m tick-free field pens. Each pen had a 5-m by 0.7 m feeding trough, a 2-m by 0.5 m water trough and a shaded area of 2 m by 3 m. The heifers were fed ad libitum on a diet of high quality lucerne hay and a grain mix as well as oaten hay. 5000 tick larvae (0.25 g of eggs allowed to hatch) were applied along the back of each heifer using a small paint brush three times a week for 3 weeks. This approach ensured that all tick stages were present at the time of treatment. Standard tick side counts were conducted by one person only, at 9:00 a.m. on the 5 days prior to each treatment, according to the method of Wharton and Utech (1970). The heifers were then ranked according to their resistance to infestation for stratified random allocation to one of three groups: Trial 1: low = 42–65; medium = 78–125; high = 127–206; Trial 2: low = 43–100; medium = 108–145; high = 169–283; Trial 3: low = 10–30; medium = 42–89; high = 100–200. 2.2. Treatments In each trial, each of the five treatments was applied to one heifer in each pen. It was anticipated that
Table 1 Treatments applied to heifers in three groups in Trial 1 Treatment number
Description
Abbreviation
Trial 1 1 2 3 4 5
Control—no treatment at all 1 l of base/animal 1 l of base/(animal d) for 3 days 1 l of base + spores/animal 1 l of base + spores/(animal d) for 3 days
(C) (Oil 1) (Oil 3) (Spores 1) (Spores 3)
Trial 2 1 2 3 4 5
Control—no treatment at all 3 l of base/animal 1 l of base/(animal d) for 3 d 3 l of base + spores/animal 1 l of base + spores/(animal d) for 3 d
(C) (3 l (1 l (3 l (1 l
Trial 3 1 2 3 4 5
Control—no treatment at all 3 l of base/animal, low P (69 kPa) 3 l of base/(animal d) for 3 d, high P (276 kPa) 3 l of base + spores/animal, low P (69 kPa) 3 l of base + spores/animal, high P (276 kPa)
(C) (HP Oil) (LP Oil) (HP Spores) (LP Spores)
Oil) Oil 3) Spores) Spores 3)
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minimal effective cross over of treatments would occur, so the design enabled comparison of five treatments, each with three replicates. Treatments for Trial 1, Trial 2 and Trial 3 are shown in Table 1. The group sizes were smaller than we would have wished for a study of this nature and the number of different treatments too high, but this was unfortunately forced by the imperative to provide some proof of concept for future funding. 2.3. Formulation and application of treatments Spores for Trial 1 were produced on rice media as described by Goettel (1984). The rice was dried at 20 8C for 4 days then harvested by shaking through 1 mm; 300 and 150 mm Endicott sieves. Spores for Trials 2 and 3 were produced by Bio-Care technology. All spores were stored at 4 8C before use. The fungal spores were formulated in 10% Codacide, an emulsified canola oil made by Microcide in the United Kingdom and supplied by Kendon, Melbourne, Australia. In Trial 1 the fungal isolate ARIM16 was used at approximately 2 108 spores/ml. In Trial 2 spores from the isolates ARIM10 and ARIM16 produced by Bio-Care Technology Australia were mixed 3:2 (respectively) giving approximately 2.5 108 spores/ml. In Trial 3 spores of the isolate ARIM16 produced at the DPI&F Animal research institute were used at a concentration of 2.5 108 spores/ml. Spores were mixed with the Codacide oil in the laboratory and later diluted with tap water at the trial site to give the test formulation. The positive controls consisted of 10% Codacide oil diluted with tap water in the field. For the application of the formulation the animals were held in a crush. In the first two trials the
formulation was applied with a small volume (1 l) spray gun using compressed air from a 1.5-hp air compressor. The spray pressure varied from 276 kPa to below 69 kPa. The pressure was mostly at the lower end as the pressure dropped rapidly once the spray nozzle was opened. In Trial 3 the treatments were applied with a small volume (1 l) spray gun using compressed air from a 2.5-hp air compressor. The air compressor was larger than that used in the two previous trials, so the pressure did not drop as rapidly once the spray nozzle was opened. This allowed for more constant spray pressures and it was applied at two pressures, 69 and 276 kPa. All animals in all trials were sprayed in the late afternoon, finishing on dusk. 2.4. Formulation check Adult engorged female ticks supplied from the DPI&F ARI tick cultures were immersed in samples of the formulation that were collected immediately before and during application to animals (24 ticks per sample in Trial 1; 20 ticks per sample in Trial 2) for 1.5 min, blotted on absorbent paper, then incubated in 24 well microtitre trays at 28 8C. Tick mortality was assessed each day for 3 days. Ticks were considered to be dead when their Malphigian tubules ceased moving. 2.5. Application check Twenty-five incompletely engorged adult ticks between 4.5 and 8 mm were removed from each animal immediately after treatment. Twenty of these ticks were incubated in microtitre trays at 28 8C for daily assessment of mortality at optimal temperature. The other five ticks from each animal were used for spore recovery. To recover spores, ticks were agitated in 1 ml
Table 2 Mortality of adult ticks immersed in samples of the spray treatments taken before and during spraying in Trial 1 and before spraying in Trial 2 Sample
% Mortality d1 (S.E.)
% Mortality d2 (S.E.)
% Mortality d3 (S.E.)
Trial 1 Control Oil pre-spray Spore pre-spray Spore spray
0 (0) 0 (0) 1.7 (1.5) 5.0 (2.5)
0 (0) 1.7 (1.5) 82.7 (14) 100 (0)
0 (0) 3.3 (3) 100 (0) 100 (0)
Trial 2 Control 3 l spores 1 l spores 1 (d1) 1 l spores 2 (d2) 1 l spores 3 (d3)
0 0 1 6 3
0 100 100 82 85
0 100 100 100 100
(0) (0) (1) (1) (1)
(0) (0) (0) (4) (2)
(0) (0) (0) (0) (0)
D.M. Leemon et al. / Veterinary Parasitology 156 (2008) 248–260
of 1.0% Tween 80 for 10 min. A haemocytometer was used to count spores.
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3. Results 3.1. Formulation checking
2.6. Mortality and egg production in vitro Fully engorged female ticks were collected from animals during the early hours of the morning following treatment, before the ticks dropped from the animals. The aim was to collect 24 ticks from each animal, but for some animals this number could not be collected, possibly due to engorged ticks dropping earlier than anticipated. Ticks were incubated in preweighed microtitre trays in a dark cupboard, subject to ambient temperature fluctuations for 10 days. Tick mortality was assessed at 4 and 10 days in Trial 1, at 7 and 15 days in Trial 2, and at 5 and 12 days in Trial 3. Egg weights were also determined after the last inspection for each incubation period. Egg production, expressed as the weight of eggs per weight of ticks for each treatment, was determined for each day of collection. An analysis of variance (GenStat, 2007) was conducted using treatment and collection day and their interaction as terms and egg production as the outcome variable. 2.7. Temperature The ambient temperatures and the surface temperatures of selected animals were recorded. A data logger recording the relative humidity and temperature every 20 min was placed in the shade near the pens holding the animals. Remote sensor data loggers were attached to the base of the tail of three animals in Trial 1, four animals in Trial 2 and Trial 3. The sensors were secured in the coat at the base of the tail of each animal. The data loggers (Tinytag, Hastings data loggers) recorded the surface temperature of these animals every 20 min. 2.8. Assessment of treatment efficacy Side counts of adult female ticks (4.5–8 mm) were carried out daily on each animal at 9:00 a.m. The counts began 16 days after the first batch of ticks was applied (5 days before treatment) and continued for 3 weeks. A one-way analysis of variance (GenStat, 2007) with randomised blocking was performed on the data using the day 0 counts as a covariate. In Trial 3 the counts were conducted from day 3 (4 days before treatment) until 8 days after treatment. This trial was discontinued after this time as there were few ticks left on the control or treatment animals due to extreme conditions of very high temperatures and relative humidity.
The mortalities among engorged ticks immersed in the different spray samples are shown in Table 2. In Trial 1 there were no mortalities in the control ticks and the mortality of ticks immersed in the formulation base (Oil pre-spray) was only 3.3% after 3 days. However, ticks immersed in the spore formulation sampled before (spore pre-spray) and during spraying (spore spray) were rapidly affected by the formulation with most dead by 2 days, and all dead by 3 days. There was no significant difference ( p 0.05) between the spore formulation samples taken before and during spraying, indicating that the pressure of the spray apparatus did not affect the virulence of the spores in the formulation. In Trial 2 after 3 days of incubation all ticks immersed in the formulation samples were dead. In Trial 3 formulation samples were taken and stored to be used only if the results of the application check were poor. 3.2. Application check In Trial 1, only the treatment with three applications of 1 l of spore formulation (3 l/d for 3 days) caused 100% mortality in ticks removed from animals (Fig. 1), and the mortality in ticks removed from animals receiving only one application (1 l) of spore formulation reached 58% after 3 days. There were no mortalities in the control ticks and mortalities in ticks removed from animals receiving the oil base ranged from 5 to 15% after 3 days. These results indicate that the spray equipment was effective in delivering lethal doses of spores to ticks but 3 l of spore formulation was needed. In Trial 2 spore application caused 100% mortality after 2 days in all ticks removed from spore treated animals (Fig. 1). The mortality in ticks removed from control animals was 1% after 3 days. The mortality in ticks removed from animals receiving 3 l of formulation base in one application was 3% after 3 days but 37% in ticks removed from the animals that received the 3 l of formulation base over 3 days. In Trial 3 both the formulation and application were effective, all ticks removed from treated animals were dead after 2 days while ticks removed from control animals showed very low mortalities (0–6%) after 3 days. The mean numbers of spores collected from ticks removed from cattle immediately after spraying for all treatments in Trial 1 are listed in Table 3. Due to the inconsistent results in Trial 1 this procedure was not carried out during Trials 2 and 3.
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Fig. 1. Average mortality (%) of unengorged ticks (4.5–8 mm) removed from cattle immediately after treatment and incubated for 3 days at 28 8C. Trial 1 (top), Trial 2 (middle) and Trial 3 (bottom).
3.3. Engorged tick mortality and egg production in vitro The mortalities (%) in engorged ticks collected from animals in the 3 days after treatment are shown in Table 4 and the egg production expressed as the average weight of eggs per average weight of ticks is shown in Fig. 2.
The results of the ANOVAs conducted on the egg production data (Tables 5 and 6) show that the treatment term was a dominant and highly significant effect ( p < 0.001). In all three trials egg production of ticks removed from spore treated animals was significantly different ( p < 0.05) from the egg production of ticks removed from the control animals or animals receiving
D.M. Leemon et al. / Veterinary Parasitology 156 (2008) 248–260 Table 3 Number of spores recovered from unengorged ticks removed from heifers after spraying in Trial 1 only Treatment
Mean spores/tick (S.E.)
Spores 1 Spores 1 (d1) Spores 2 (d2) Spores 3 (d3)
597,917 568,750 420,833 352,083
(57,310) (70,434) (49,047) (34,422)
only the oil base without the spores. In Trials 1 and 3 the interaction of treatment and day of collection from the animal was significant ( p < 0.05), and for all three trials these means are shown in Fig. 2. This shows that the main reason for the interactions in Trials 1 and 3 was within the ‘spore’ treatments—these tended to be much more effective on day 1, decreasing in efficacy by days 2 and 3. In Trial 2 there was no significant difference in egg production attributable to day. However, in Trial 1, egg production by ticks removed in the first 2 days posttreatment was significantly different ( p < 0.05) from egg production by ticks removed on day 3. In Trial 3 ticks removed in the first day post-treatment produced significantly fewer eggs ( p < 0.05) than the ticks removed on days 2 and 3 post-treatment. The data summarised in Table 4 show the spore treatments in the three trials were effective in causing high mortality in engorged ticks removed from the animals after treatment. In addition the same effect of time can be seen with tick mortality as that seen with the reduction in egg production in Trials 1 and 3. The longer the ticks stayed on the animal before incubation, the lower the tick mortality and higher the egg production in these two trials. In Trial 1, mortalities in ticks removed from animals treated with the spore formulation varied widely after 4 days of incubation, from 91 to 94% in ticks removed the day after treatment down to 4–22% in ticks removed 3 days after treatment. Most of these dead ticks were covered in fungal mycelium. Ticks removed from control animals and animals treated with the formulation base all showed very low rates of mortality (0–2%) after 4 days of incubation except for the ticks collected from one control animal on day 2 (45%). Some of these dead ticks exhibited fungal growth indicating that some cross contamination might have occurred at the time of collection of ticks or among animals after application. In Trial 2 there was less variation in mortalities in ticks removed from treated animals. Because the ambient conditions were cooler than those in Trial 1 ticks were incubated for longer (up to 15 days) to allow for completion of egg production. Many of the ticks removed from animals treated with the spore formula-
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Table 4 Mortality (%) of female engorged ticks collected from cattle over the 3 days post-treatment and then incubated at ambient temperatures in the dark Trial 1
Day collected
Mean % mortality after 4 d incubation (S.E.)
Mean % mortality after 10 d incubation (S.E.)
Control
1 2 3
2 (1) 45 (25) 0 (0)
25 (18) 46 (25) 1 (1)
Oil 1
1 2 3
1 (0) 0 (0) 1 (1)
42 (9) 5 (2) 1 (1)
Oil 3
1 2 3
1 (1) 1 (1) 1 (1)
64 (10) 6 (3) 6 (1)
Spores 1
1 2 3
92 (4) 3 (2) 4 (3)
100(0) 50 (12) 36 (10)
Spores 3
1 2 3
91 (5) 14 (3) 22 (2)
100 (0) 66 (13) 83 (7)
Trial 2
Day Mean % mortality after Mean % mortality after collected 7 d incubation (S.E.) 15 d incubation (S.E.)
Control
1 2 3
0 (0) 0 (0) 0 (0)
16 (7) 6 (1) 3 (1)
1 l Oil 3
1 2 3
0 (0) 4 (4) 18 (31)
37 (21) 47 (10) 68 (18)
3 l Oil
1 2 3
4 (3) 0 (0) 0 (0)
14 (6) 22 (10) 3 (2)
1 l Spores 3 1 2 3
84 (6) 97 (3) 99 (1)
100 (0) 100 (0) 100 (0)
98 (1) 62 (22) 48 (31)
100 (0) 99 (1) 88 (9)
3 l Spores
1 2 3
Trial 3
Day collected
Mean % mortality after 5 d incubation (S.E.)
Control
1 2 3
1 (1) 0 (0) 0 (0)
56 (25) 0 (0) 11 (9)
Low press oil
1 2 3
0 (1) 0 (0) 1 (0)
5 (19) 20 (13) 1 (0)
High press oil
1 2 3
0 (1) 0 (0) 0 (0)
27 (17) 5 (2) 2 (0)
Low press spores
1 2 3
100 (0) 29 (0) 3 (1)
100 (0) 100 (0) 51(1)
High press spores
1 2 3
83 (0) 44 (0) 8 (1)
100 (0) 100 (0) 77 (1)
Mean % mortality after 12 d incubation (S.E.)
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Fig. 2. Egg production by engorged ticks removed from Trial 1 (top); Trial 2 (middle) and Trial 3 (bottom) heifers in the 3 days post-treatment expressed as an average weight of eggs per average weight of ticks.
tion were dead after 7 days and nearly all were dead after 15 days of incubation. Fungal mycelium was present on most of these dead ticks. There were no mortalities in the ticks removed from control animals after 7 days, but after 15 days mortalities ranged from 3 to16%. There was a wide range in mortalities among ticks removed from animals receiving the formulation base after 7 days (0–18%) and 15 days (3–68%). In Trial 3, conducted with higher ambient temperatures, mortalities in ticks removed from treated animals showed a wide variation after 5 days of incubation, ranging from 83 to 100% in ticks collected the day after
treatment down to 3–8% in ticks removed from animals 3 days post-treatment. Ticks removed from control animals or those receiving the formulation base had very low mortalities after 5 days of incubation (0–1%). 3.4. Temperature The ambient and animal surface temperatures recorded by the data loggers in the three trials are shown in Table 7. In Trial 1 only one of the three loggers attached to the animals recorded any data. The sensor cables on the other two loggers became detached from
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255
Table 5 ANOVA of egg production by ticks removed from animals in the 3 days post-treatment in Trials 1, 2 and 3 showing main effects and interaction terms Source of variation
d.f
s.s
m.s.
v.r
F pr.
Trial 1 Rep stratum Treatment Day Treatment day Residual
2 4 2 8 28
0.002784 0.766469 0.039716 0.166238 0.076278
0.001392 0.191617 0.019858 0.020780 0.002724
0.51 70.34 7.29 7.63
<0.001 0.003 <0.001
Trial 2 Rep stratum Treatment Day Treatment day Residual
2 4 2 8 28
0.042934 0.915062 0.035278 0.045510 0.268715
0.021467 0.228766 0.017639 0.005689 0.009597
2.24 23.84 1.84 0.59
<0.001 0.178 0.775
Trial 3 Rep stratum Treatment Day Treatment day Residual
2 4 2 8 27
0.10349 1.31052 0.17506 0.39804 0.36362
0.05175 0.32763 0.04975 0.04975 0.01347
3.84 24.33 6.50 3.69
<0.001 0.005 0.005
Table 6 ANOVA of adjusted means of egg production showing the main effects Treatment
Control
1 l oil
3 1 l oil
1 l spores
3 1 l spores
(l.s.d.)
0.409 a 1 0.2607 a
0.3980 a 2 0.2702 a
0.3710 a 3 0.3279 b
0.1753 b
0.0862 c
(0.0504)
Control
3 l oil
3 1 l oil
3 l spores
3 1 l spores
(l.s.d.)
0.355 a 1 0.2607 a
0.349 a 2 0.2702 a
0.269 a 3 0.3279 a
0.082 b
0.007 b
(0.0946)
Control
LP oil
HP oil
LP spores
HP spores
(l.s.d.)
0.614 a 1 0.343 a
0.539 a 2 0.483 b
0.550 a 3 0.465 b
0.223 b
0.224 b
(0.1122)
Trial 1 Day
Treatment
(0.03904)
Trial 2 Day
Treatment
(0.07330
Trial 3 Day
(0.0869)
Within rows, means followed by the same letter are not significantly different ( p 0.05).
the loggers within a day of attachment to the animals. The lowest recorded ambient temperature was 17 8C and the highest was 33 8C. The lowest recorded temperature on the animal was 25 8C and the highest was 40 8C. During the first 7 days after spraying surface temperatures on the animal were between 25 and 34 8C for at least half of each day although there were a number of hours each day above 34 8C. In Trial 2 ambient temperatures ranged from 5 to 25 8C while animal surface temperatures ranged from 16 to 38 8C. The temperatures on the four animals were
mostly consistent except for the heifer with logger 3; the night-time temperatures on this animal were always higher than those on the other animals. The ambient temperature was below 20 8C for most of each day for the first 7 days and only went above 25 8C for 2 h during day 7. The average surface temperatures on the four heifers were correspondingly low with more than half of each day below 25 8C, with little time above 34 8C. In Trial 3 because the data logger recording the ambient temperature and humidity failed, the ambient temperature data recorded at the nearest weather station
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Table 7 Ambient temperatures (range and hours below 20 8C and 25 8C), and corresponding average surface temperatures (range, hours below 25 8C, between 25 and 34 8C, and hours above 34 8C) recorded on animals during the first 7 days after treatment in the three trials Days after treatment
Ambient temperature range (8C)
Hours ambient 20 8C
Hours ambient 25 8C
Temperature range on animal (8C)
Hours on animal 25 8C
Trial 1 (n = 1) 1 23–28 2 18–33 3 18–31 4 18–31 5 17–31 6 18–32 7 17–30
0 9 6.5 11 17.5 9 11
12 15 15 16 13 16 16
32–38 32–40 24–38 25–40 31–38 30–38 30–38
0 0 2 2 0 0 0
Trial 2 (n = 4) 1 5–23 2 10–21 3 13–21 4 14–22 5 11–22 6 10–23 7 12–25
18 18 17 17 18 16 14
24 24 24 24 24 24 22
16–38 16–31 18–34 19–34 18–34 20–35 14–39
15.5 17 13.5 14.5 12.5 13 12
Hours on animal 25 8C 34 8C
Hours on animal 34 8C
13 14 12 13 14 17 17
11 10 10 9 10 7 7
6.9 6.9 9.8 8.5 11.3 8.9 9.1
1.5 0 0.5 1 0 2 2.5
Days after treatment
Ambient temperature range (8C)
Av. Ambient temp (8C)
Temperature range on animal (8C)
Hours on animal 25 8C
Hours on animal 25 8C 34 8C
Hours on animal 34 8C
Trial 3 (n = 4) 1 2 3 4 5 6 7
21–37 19–31 21–35 23–38 24–37 24–42 23–42
28 25 27 29 29 33 30
23–38 28–41 32–44 33–42 33–45 33–45 24–41
3 0 0.25 0 0 0 1.5
15.9 12 4.5 4.9 6.5 5.5 12.8
5.1 12 19.4 19.1 17.5 18.5 10.5
was used. This data only included a range and average daily temperatures. Ambient temperatures were high during this trial, with daytime temperatures exceeding 35 8C on most days. By the end of the first 7 days daily temperatures were exceeding 40 8C. The ambient temperature ranged from 19 to 42 8C. Surface temperatures on the animals ranged from 23 to 45 8C. After the first day the surface temperatures on the animals were above 34 8C for more than half of each day. 3.5. Assessment of treatment efficacy The average counts of adult ticks between 4.5 and 8 mm on the right side of each animal recorded each morning are shown in Fig. 3. The ANOVAs conducted on the tick counts are presented in Table 8. In Trial 1 counts began 5 days before treatment and continued for 15 days after treatment. There was no significant effect of any treatment on the tick count on any day. In Trial 2 counts recorded from 5 days before treatment to 23 days after treatment show that there was
a reduction in ticks on animals 9–10 days after treatment with the spore formulation but this effect did not differ significantly from the effect of the 3 l oil application. In Trial 3 there were no significant differences in tick counts in any of the treatment groups. The side counts on all animals in this trial were far lower than those in the two previous trials despite all three trials having the same number of tick larvae applied over the same time period. The trial was discontinued after 8 days because it was clear that the extreme weather conditions were affecting tick numbers on all animals. 4. Discussion The fungal biopesticide was highly efficacious when applied directly to ticks that were subsequently incubated in the laboratory, or when ticks that were exposed to the biopesticide while feeding on cattle were removed from the cattle and incubated in the laboratory. However, its ability to kill ticks on the cattle was
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257
Fig. 3. Average daily side counts of ticks before and after treatment in Trial 1 (top), Trial 2 (middle) and Trial 3 (bottom).
consistently poor, most markedly so under the extremely hot conditions of Trial 3. This suggests that some factor in the environment on the surface of the animals negatively affected the pathogenicity of the fungus to ticks. Given the known optimal growth temperatures of M. anisopliae (Leemon and Jonsson, 2008), we suggest that the factor most likely limiting the performance of this biopesticide is high temperature on the skin surface.
Extensive sampling and testing was undertaken to provide data to differentiate the performance of the fungal biopesticide on ticks under conditions on the animal or under optimal conditions in the laboratory. Testing in Trials 1 and 2 showed that the formulations were highly virulent to engorged female ticks under laboratory conditions. The application of the formulation to animals in all three trials resulted in the rapid mortality of most unengorged ticks removed post-
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Table 8 ANOVA of adjusted means of tick side counts on selected days Trial 1
Day 0
Day 4
Day 11
Day 15
Control Oil 1 Oil 3 Spores 1 Spores 3
129 107 185 181 117
141 190 113 129 71
171 287 135 168 109
191 217 68 138 52
F-probability Standard error of mean
0.066 19.91
0.254 38.9
0.427 73.2
0.186 56.6
Trial 2
Day 0
Day 4
Day 11
Day 18
Control 1 l Oil 3 3 l Oil 1 l Spores 3 3 l Spores
123 212 155 181 174
260 243 195 142 199
402 230 227 144 147
519 193 293 62 83
F-probability Standard error of mean
0.509 34.5
a a ab b ab
0.034 21.83
a b b b b
0.027 44.2
a bc b c c
< 0.001 39.6
Trial 3
Day 0
Day 4
Day 8
Control High press oil Low press oil High press spores Low press spores
39.3 42.0 68.0 52 65.3
36.4 49.2 38.2 34.3 30.7
35.6 60.1 58.6 38.2 38.4
F-probability Standard error of mean
0.518 13.97
0.435 7.64
0.660 16.05
Means followed by the same letter for each day are not significantly different ( p 0.05).
treatment and incubated in the laboratory (Fig. 1). However, there was some variation in Trial 1 that may have been due to an insufficient volume of formulation being applied to some animals. In these trials the application check showed that the spray equipment was delivering enough spores to kill ticks under optimal conditions so any failure of the fungal formulation to kill ticks in vivo was not due to inadequate formulation and application of the fungal biopesticide. Engorged ticks collected in the first 3 days posttreatment in each trial and incubated under ambient temperature conditions had a higher mortality than ticks collected from control animals and showed a significant reduction in egg production. However, in Trials 1 and 3 there was a significant interaction between day collected post-treatment and the treatment on egg production. This may indicate an adverse effect from the animal microclimate on the spores on ticks before they dropped from the animal. One putative factor that could account for the variation in in vivo tick control is temperature. Extensive in vitro assays showed that no M. anisopliae isolates grew at 40 8C and few showed much growth at
35 8C (Leemon and Jonsson, 2008). The two isolates used in the formulations in the present trials, ARIM10 and ARIM16, were selected for their vigour and ability to grow well at 30 8C with some growth capability at 34 8C. In the hottest conditions of Trial 3 ambient temperatures exceeded 41 8C and recordings showed that surface temperatures remained above 34 8C for more than half of the time over the first 8 days. In Trial 2, where some degree of tick control was evident, ambient temperatures were much lower and surface temperatures on animals remained below 34 8C for nearly all of the time. Previous animal trials (Polar et al., 2005; Correia et al., 1998; Bittencourt et al., 1999) investigating in vivo fungal control of R. microplus have also shown limited success, although unfavourable temperature were not shown to be a contributing factor. Polar et al. (2005) recognised the importance of temperature for fungal performance and selected an isolate that performed best between 31 and 35 8C. In addition, the ambient temperatures during the trials of both Correia et al. (1998) and Polar et al. (2005) were quite low (18–24.5 8C and 23–34 8C, respectively). However,
D.M. Leemon et al. / Veterinary Parasitology 156 (2008) 248–260
the fungal isolates and formulations used in the in vivo trials by both Polar et al. (2005) and Correia et al. (1998) seemed to differ in virulence to those used in these trials. The highest spore concentration used by Correia et al. (1998), (7.5 108 spores/ml), was much higher than the concentration used in our trials and with ambient temperatures similar to those in our Trial 2 there was no evidence at all of tick control on animals. Polar et al. (2005) used a similar spore concentration to us and reported a mean survival time of 6.7 days for ticks incubated at 28 8C. In contrast, ticks removed from our treated animals in Trials 2 and 3 and incubated at 28 8C were all dead after 2 days. This suggests that the isolates we used had a higher virulence to R. microplus. In all three of our trials larvae were applied three times a week for 3 weeks before treatment to ensure that all stages would be present at treatment; tick counts were also made before treatment so that animals could be assigned to treatment groups in a randomly stratified design. Additionally, in two of the three studies posttreatment side counts were conducted daily over the period of time that it was thought that female ticks that were present as larvae at the time of treatment would have completed their on-animal stages. However, the studies conducted by Polar et al. (2005) and Bittencourt et al. (1999) relied upon natural infestations of ticks and did not report on side counts prior to treatment to allow for variation in tick susceptibility among the animals, nor did they conduct daily tick counts after treatment, so it is possible that their procedures for assessing tick control might have missed some evidence of control. Correia et al. (1998) did infest animals with ticks for 3 weeks prior to treatment, but did not appear to have recognised any natural variation in susceptibility to ticks. Despite the poor overall efficacy of the product in our trials, care must be taken with the interpretation of the lack of significant results, which might be due in part to inadequate statistical power. For evaluations of efficacy for the registration of new acaricides, it is recommended that each treatment group should generally include at least three animals (Holdsworth et al., 2006). Although the treatment groups in the present study did each include three animals, and although groups were assigned after ranking on tick counts, there was a wider than expected variation in tick counts, resulting in a lack of statistical power. The present trials highlight the importance of adequate replication when conducting animal trials, especially when using a product of unknown or unpredictable efficacy. Three animals might be mostly adequate when the benchmark is 95% control, as per conventional acaricides (Holds-
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worth et al., 2006) but is likely to be inadequate with biological control strategies. There is a natural variation in tick susceptibility of cattle both within breed and between breed (Utech et al., 1978; Seifert, 1971). The results from this study suggest that future trials of this fungal biopesticide are warranted to evaluate its efficacy under conditions of controlled temperature, humidity and air flow to establish a proof of concept for in vivo tick control by a fungal biopesticide. 5. Conclusion The primary aim in each of these animal trials was to investigate whether spores of M. anisopliae formulated as a biopesticide and applied to dairy heifers could kill all parasitic stages of R. microplus. The trials were carried out at different times of the year under different weather conditions. It was anticipated that positive results in the trials would provide a proof of concept for fungal biopesticide control of R. microplus on cattle. The fungal biopesticide caused high mortalities in unengorged ticks incubated in vitro and had a significant effect in reducing egg production by engorged ticks collected in the first 3 days post-treatment. These trials did not demonstrate a statistically significant effect on tick mortality in vivo. However, the trials did provide information for the design of further trials to assess the role of ambient conditions in affecting the ability of fungal spores to control ticks in vivo. Acknowledgements We would like to thank Peter Green, Senior Parasitologist, Animal Research Institute for supply of ticks and thoughtful advice, the staff of Mutdapilly Research Station for animals and facilities, Dave Mayer and Pat Pepper, Principal Biometricians, Animal Research Institute for statistical advice, John Allan of DPI&F Actest, for advice on acaricide trial protocols and BioCare Technology (now Becker Underwood Australia) for the supply of spores. References Benjamin, M.A., Zhioua, E., Ostfeld, R.S., 2002. Laboratory and field evaluation of the entomopathogenic fungus Metarhizium anisopliae (Deuteromyces) for controlling questing adult Ixodes scapularis (Acari: Ixodidae). J. Med. Entomol. 39, 723–728. Bittencourt, V.R.E.P., Souza, E.J., Peralva, S.L.F.S., Reis, R.C.S., 1999. Efficacy of the fungus Metarhizium anisopliae (Metschnikoff, 1887) Sorokin, 1883 in field test with bovines naturally infested with the tick Boophilus microplus (Canestrini 1887) Acari: Ixodidae). Rev. Bras. Med. Vet. 21, 78–81.
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Castineiras, A., Jimeno, G., Lopez, M., Sosa, L., 1987. Effect of Beauveria bassiana Metarhizium anisopliae (Fungi Imperfecti) and Pheidole megacephala (Hymenoptera: Formicidae) against eggs of Boophilus microplus. Revista Salud Anim. 9, 288– 384. Correia, A.C.B., Fiorin, A.C., Monteiro, a.C., Verissimo, C.J., 1998. Effect of Metarhizium anisopliae on the tick Boophilus microplus (Acari: Ixodidae) in stable cattle. J. Invertebr. Pathol. 71, 189–191. Frazzon, G.A.P., Vaz Jr., S.I., Masuda, A., Schrank, A., Vainstein, H.M., 2000. In vitro assessment of Metarhizium anisopliae isolates to control the cattle tick Boophilus microplus. Vet. Parasitol. 94, 117–125. GenStat, 2007. GenStat for Windows, Release 9.1, 9th ed. VSN International, Oxford. Goettel, M.S., 1984. A simple method for mass culturing entomopathogenic Hyphomycete fungi. J. Microbiol. Methods 3, 15–20. Gindin, G.M., Samish, M., Alekseev, E., Glazer, I., 2001. The susceptibility of Boophilus annulatus (Ixodidae) ticks to entomopathogenic fungi. Biocontrol. Sci. Technol. 11, 111–118. Holdsworth, P.A., Kemp, D.H., Green, P., Peter, R.J., de Bruin, C., Jonsson, N.N., Letonja, T., Rehbein, S., Vercruysse, J., 2006. World association for the advancement of veterinary parasitology (WAAVP) guidelines for evaluating the efficacy of acaricides against ticks (Ixodidae) on ruminants. Vet. Parasitol. 136, 29–43. Kaaya, G.P., 2000. Laboratory and field evaluation of entomogenous fungi for tick control. Ann. N. Y. Acad. Sci. 916, 559–564. Leemon, D.M., Jonsson, N.N., 2008. Laboratory studies on Australian isolates of Metarhizium anisopliae as a biopesticide for the cattle tick Boophilus microplus. J. Invert. Pathol. 97, 40–49. Mwangi, E.N., Kaaya, G.P., Essuman, S., 1995. Experimental infections of the tick Rhipicephalus appendiculatus with entomopathogenic fungi, Beauveria bassiana and Metarhizium anisopliae, and
natural infections of some ticks with bacteria and fungi. J. Afr. Zool. 109, 151–160. Monteiro, S.G., Bittencourt, V.R., Daemon, Faccini, J.L., 1998. Pathogenicity under laboratory conditions of the fungi Beauveria bassiana and Metarhizium anisopliae on larvae of the tick Rhipicephalus sanguineus (Acari: Ixodidae). Braz. J. Vet. Parasitol. 72, 113–116. Onofore, S.B., Miniuk, C.B., De Barros, N.M., Azevedo, J.L., 2001. Pathogenicity of four strains of entomopathogenic fungi against the bovine tick Boophilus microplus. Am. J. Vet. Res. 62 (9), 1478–1480. Polar, R., Aquino de Muro, M., Kairo, T.K., Moore, D., Pegram, R., John, S., Roach-Benn, C., 2005. Thermal characteristics of Metarhizium anisopliae isolates important for the development of biological pesticides for the control of cattle ticks. Vet. Parasitol. 134, 159–167. Seifert, G.W., 1971. Variations between and within breeds of cattle in resistance to field infestations of the cattle tick (Boophilus microplus). Aust. J. Agric. Res. 22, 159–168. Sewify, G.H., Habib, S.M., 2001. Biological control of the tick fowl Argas persicargus persicus by the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae. J. Pest Sci. 74, 121– 123. Utech, K.B., Wharton, R.H., Kerr, J.D., 1978. Resistance to Boophilus microplus in different breeds of cattle. Aust. J. Agric. Res. 29, 411–422. Wharton, R.H., Utech, K.B.W., 1970. The relation between engorgement and dropping of Boophilus microplus (canestrini) (Ixodidae) to assessment of tick number on cattle. J. Aust. Entomol. Soc. 9, 171–182. Zhioua, E., Browning, M., Johnson, P.W., Ginsberg, H.S., LeBrun, R.A., 1997. Pathogenicity of the entomopathogenic fungus Metarhizium anisopliae (Deuteromycetes) to Ixodes scapularis (Acar: Ixodidae). J. Parasitol. 83, 815–818.