Introduction

Ticks have been recognized as important ectoparasites of livestock. They have been incriminated as voracious bloodsuckers, causing heavy blood losses resulting in low-quality hides (Jongejan and Uilenberg 2004), secondary bacterial infections (Ambrose et al. 1999), lowered productivity in terms of weight gain (Pegram and Oosterwijk 1990) and milk yield (Sajid et al. 2007), and increased mortality (Niyonzema and Kiltz 1986). In some countries, half of the earning from milk is spent on acaricidal treatment (Swai 2002). Hence, ticks and tick-borne diseases pose a serious impact on the individual and national economics of developing countries. Therefore, it has been suggested that developing countries like Pakistan should make tick control a priority (Bansal 2005). Although grooming (Mooring et al. 1996) and domestic poultry (Hassan et al. 1992) have been found to be effective for tick control, chemical control remains the cornerstone in developing countries (De Castro et al. 1997). The major constraint of chemical treatment is the selection for chemical-resistant strains of ticks (Ghosh et al. 2006). The predominant contributing factors in development of resistance may include misuse of drugs (Bianchi et al. 2003) and use of the wrong concentration of acaricide (Dolan 1999) leading to failure of the tick control program (Pegram et al. 2000). For an effective chemical control strategy, periodic monitoring of the effectiveness of drugs and identification of resistant strains is essential. In Pakistan, the over-the-counter availability of various brands of Ivermectin has led to the misuse of the drug by dairy farmers for the control of ecto- and endo-parasites (Sajid, personal observation), and this may be a predisposing factor in the development of ivermectin-resistant strains of ticks (Ghosh et al. 2006). The present study was designed in order to obtain data on the farmer's approach for the control of ticks and to determine the in vivo comparative efficacy of an avermectin (injectable ivermectin, IVM) and a pyrethroid (pour-on cypermethrin, CYM), against Hyalomma a. anatolicum, the most common cattle tick in lower Punjab, Pakistan (Sajid et al. 2008). The results of this study also provide a rough estimate of the efficacy of both the drugs in vivo.

Materials and methods

Survey

A preliminary survey of the small holder dairy farms of the selected areas was conducted using a pre-designed questionnaire (Thrusfield 1995), in order to record detailed relevant information about the current use of acaricides and/or other alternative therapies for the control of tick infestation in animals. The points emphasized were: (1) housing, management, and on-farm hygiene practices; (2) priority of treatment strategies for tick infestation; and (3) information about concentration, dosage, and administration of drugs for various species of animals.

Chemotherapeutic trials

The in vitro and in vivo trials were planned according to the recommendations of the World Association for the Advancement of Veterinary Parasitology (Holdsworth et al. 2006).

In vitro acaricidal efficacy

In vitro acaricidal efficacy was determined using live engorged female ticks collected from the study area. Ticks were exposed to IVM (Ivomec, Merial, France) or 5% CYM pour-on (Cipermetriven, Ivan) at various concentrations to estimate the acaricidal efficacy of these drugs. Ticks sham-treated with propylene glycol (Propandiol-(1, 2), Merck) acted as controls. The tick survival assay described by Mendes et al. (2001) was used. Briefly, 480 live engorged female Hyalomma a. anatolicum ticks were collected from the bovines of the selected area and separated into two equal groups of 240 ticks (1 and 2) for the efficacy trial of IVM and CYM, respectively. Each group of ticks was immersed in the different acaricides diluted in oil-based diluent for 5 min. Group 1 was further divided into four sub-groups (A through D) having 60 ticks in each (20 × 3 replicates). Sub-groups B, C, and D were immersed in 200, 400, and 600 µg of IVM concentrations, respectively, while sub-group A was treated with three concentrations of propylene glycol (sham treatment). Group 2 sub-groups B, C, and D were immersed in 1.0, 1.25, and 1.5 mg of CYM, while sub-group A was treated with corresponding doses of propylene glycol (sham treatment). After the immersion period, the engorged females were removed by passage through a plastic filter and dried on paper towels. These ticks were placed in Petri dishes and incubated at 27–28°C, 85–95% relative humidity, for 24 h. After this period, the number of live ticks in each group was counted in order to estimate the acaricidal efficacy of IVM and CYM. The live ticks (if any) were again provided the same conditions for 2 weeks in order to observe their reproductive potential.

In vivo acaricidal efficacy

Three hundred sixty bovines in the study area were selected for an in vivo acaricidal efficacy of the two compounds (IVM and CYM pour-on). The animals were selected based on the following criteria: (1) the animal had a tick burden of >100 ticks per animal, (2) the age of animal was more than 1 year, and (3) there was no history of application of acaricide to the animal.

The selected animals were divided into four equal groups (A through D) and treated with IVM or CYM, or with a sham treatment of propylene glycol (Propandiol-(1, 2), Merck) vehicle as a control. A layout of the treatment protocol is shown in Table 1.

Table 1 Treatment protocol for in vivo acaricidal resistance in ticks of bovine and/or caprines against IVM and CYM
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After a single treatment with either of the above-mentioned acaricides, the animals were examined quantitatively every 24 h through “finger counting” (Rugg and Hair 2007) of the ticks on one side of the body and multiplying the number by 2 to get the whole body count (Knopf et al. 2002). The number of ticks shed after the first 24 h and the duration for which the treatment remained effective was calculated from the data. The graphs show the post-treatment tick burden on days 0, 5, 10, 15, and 20.

Data analyses

The results of in vitro and in vivo acaricidal efficacy trials were analyzed using a general linear model with repeated measure analysis of variance and least square means of interaction (StatSoft 1999).

Results

Ninety percent (27/30) of the selected small holder dairy farm owners in the current trial had a history of inappropriate and blind treatment of animal tick infestation through self-administration of various brands of IVM (easily available at local markets) rather than consulting a veterinarian. Only 14% of these farmers were using correct doses and formulations of the drugs. All the selected farms farmed both small and large ruminants simultaneously. Only 10% (03/30) of farm owners were consulting veterinarians and had acceptable management and hygiene practices that included: (1) well-constructed sheds with cemented floors and an open housing system, (2) proper cleanliness and management of wastes, and (3) regular showering/bathing and deworming practices. IVM was the only drug used on all the farms, with various local and international brands being used. Good management and hygienic practices were found only in 10% of the farms surveyed.

The tick survival assay indicated the in vitro efficacy of 5% CYM pour-on and IVM against H. a. anatolicum. The arc transformed mean number of ticks surviving 24 h after treatment in IVM-treated groups was higher (P = 0.00) than that of the CYM-treated groups. However, a dose-dependent decrease in the mean number of ticks was observed in both of the treated groups (Figs. 1 and 2). All the live ticks found 24 h after treatment died during the 2-week incubation period except those in the sham-treated groups. The ticks of the control group not only survived, but they also laid eggs during incubation. The mean number of H. a. anatolicum surviving 24 h is given in Table 2. The results showed that H. a. anatolicum is susceptible to both of the test drugs. However, roughly, the death of the ticks treated with IVM was delayed as compared to those treated with CYM.

Fig. 1
figure 1

Linear regression between the log doses of IVM and arc transformed means of surviving ticks 24 h after in vitro application of the drug. The trend shows the dose-dependent decrease in mean tick survival as compared to vehicle control group

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Fig. 2
figure 2

Linear regression between the log doses of CYM and arc-transformed means of surviving ticks 24 h after in vitro application of drug. The trend shows the dose-dependent decrease in mean tick survival as compared to vehicle control

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Table 2 In vitro tick survival assay of Hyalomma spp. after treatment with various doses of IVM and CYM
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The in vivo post-treatment quantitative assessment of tick burden revealed that the sham-treated animals maintained a tick infestation throughout the study period. Both the IVM- and CYM-treated groups resulted in significantly lower (P < 0.05) tick counts relative to controls on all post-treatment counting days. The finger counts were significantly higher (F 12, 32 = 48.6; P = 0.00) in group A (IVM-treated group) than in Group C (5% CYM pour-on) as shown in Fig. 3. From day 0 (pre-treatment) to day 5 (post-treatment), the reduction in the mean number of ticks was not significant (P > 0.05) in the IVM-treated group. The maximum reduction in mean number of ticks in the IVM-treated group was found from day 5 to day 10, followed by day 10 to day 15. IVM was not found to be effective in controlling the tick burden after 15 days post-treatment. In the CYM-treated group, reduction in the mean number of ticks was significant (P < 0.05) even in the day 0 (pre-treatment) to day 5 (post-treatment) period. CYM 5% pour-on was found to be effective even after 15 days post-treatment; however, the rate of reduction in mean tick numbers was a little lower than in earlier periods. The lowest tick burden in the IVM-treated group was significantly higher (P < 0.05) than that of the CYM-treated group, the latter being close to zero. Hence, the in vivo efficacy trials of injectable IVM and CYM pour-on revealed better results for the latter.

Fig. 3
figure 3

In vivo comparative (F 12, 32 = 48.6; P = 0.00) acaricidal efficacy of IVM and CYM against H. anatolicum in bovines; A IVM-treated group, B sham treatment for IVM, C CYM-treated group, D sham treatment for CYM

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Discussion

A number of tick control strategies have so far been used by the livestock farmers and animal practitioners. These include grooming (Mooring et al. 1996), genetic manipulation through increasing Bos indicus content in the progeny (Sutherst and Utech 1981; Frisch et al. 2000), biological control through domestic poultry (Hassan et al. 1992), entomopathogenic fungi (Bittencourt et al. 1994; Samish and Rehacek 1999; Gindin et al. 2001), immunological control through production of vaccines against some of the tick species (Willadsen 1987; Rodriguez et al. 1995; Brossard 1998), and ethnoveterinary practices (Sutherst et al. 1982; Carol et al. 1989; Regassa 2000); however, chemotherapeutic control remains the foundation of tick control programs for eradication of livestock infestations in the developing world (De Castro et al. 1997; Bianchi et al. 2003). However, a progressive decrease in efficiency of acaricidal drugs through the development of resistance (Beugnet et al. 1994) would undermine this method. Epidemiological investigations have suggested that a reduction in acaricide-treatment frequency that permits high tick-attachment rates allows the development of endemic stability (Norval et al. 1992). To this end, a regular screening of compounds is required for the determination of their efficacy. So far, various groups of insecticides and acaricides have been found to have significant efficacy for tick control, including pyrethroides (Miller 1987; Zerba 1988), avermectins (Miller 1987), organophosphates (Fiedler 1958; Miller 1988), organochlorines, carbamates, and insect growth regulators (Miller 1987).

The present research indicated that the vehicles of IVM and CYM do not have any acaricidal activity (Rugg and Hair 2007). Previous reports of comparative chemotherapeutic trials of IVM showed better efficacy (Khan et al. 1997; 1998; George et al. 1998). Our results are surprisingly different from the above-mentioned previous comparative studies indicating better in vivo efficacy of CYM (a pyrethroid) than IVM (an avermectin). The probable reason may be less exposure of domestic animals to pyrethroids for the control of ecto- and endo-parasites in this region of Pakistan. In a recent study, the impact of CYM on the vittelongenesis-inducing factor was determined by Friesen and Kaufman (2003), who showed that, instead of stimulating vitellogenesis, it has an inhibiting effect on egg development. The results of the tick survival assay do not allow the comparison of the efficacy of the two drugs because the doses used in the experiment may not be having physiological equivalence with each other. However, these results allow us to estimate the dose needed to kill all the ticks for each of the drugs and provide some tools to help manage the tick problem in the testing area.

According to reported speculation (Ghosh et al. 2006), the principal use of a limited number of chemicals (e.g., IVM) for tick control leads to the selection of chemical-resistant strains of ticks, along with environmental contamination. We suspect that over-the-counter availability and misuse of IVM in the selected farms may be the cause of the apparent decrease in its acaricidal efficacy and development of resistance as compared to 5% CYM pour-on, as previously reported in other countries by Bianchi et al. (2003), Beugnet et al. (1994), Dolan (1999), and Ogden et al. (2005). However, standardization of the permethrin hydrolytic assay (Jamroz et al. 2000) and larval packet test (LPT) (FAO 1984) still needs to be done in Pakistan for confirmatory screening of acaricide-resistant strains of ticks. Various studies have been conducted on the mechanism of acaricidal resistance. Organophosphate resistance mechanisms include the following: (1) Acetylcholine esterase affinity is changed in resistant tick populations (Pruett 2002), and (2) a link to cytochrome P450 monooxygenase activity (Foil et al. 2004). Pyrethroid resistance was found to be due to two mechanisms: (1) a mutation of Na+ channels (He et al. 1999), confirmed by Guerrero et al. (2001) through PCR, and (2) involvement of some metabolic activity leading to a much higher CzEst9 esterase activity in resistant populations (Jamroz et al. 2000). Avermectin resistance has also been suggested to be due to this mechanism by Jamroz et al. (2000), but this needs to be scientifically proved.

The application of integrated pest management (IPM), e.g., the use of seasonal treatment at the peak of tick activity, accompanied by good management and sanitary conditions, may be a prophylactic approach for tick control in small holder dairy farming systems of Pakistan. Hence, the future plans of research should not only be directed towards the development of modern drugs and searching of new drug targets with different modes of application, but also towards finding some feasible alternative strategies using ethnoveterinary medicine, immunotherapy, and genetic manipulation in order to get some useful tools in future tick control programs.