Poultry red mite eradication potential of ivermectin and allicin combination treatment

Abstract Background Existing treatments against poultry red mite (PRM; Dermanyssus gallinae) infestation have reduced efficacy or exhibit hazardous effects on chickens. Considering the economic importance of chickens, development of a safe and effective method for exterminating PRMs is necessary. Ivermectin and allicin are effective against some ectoparasites; however, their acaricidal efficacies against PRMs remain unknown. Objective To evaluate individual and combined efficacies of ivermectin and allicin in exterminating PRMs. Methods Different concentrations (0.10–1.0 mg/mL) of ivermectin (1 mL) were applied via dropping method in different insect culture dishes (ICDs), prior to transferring PRMs. For the spraying method, PRMs were transferred to ICDs, before spraying ivermectin (1 mg/mL) solution (1 mL). Further, the acaricidal effect of allicin on PRMs was evaluated by applying different concentrations (0.25–1.0 mg/mL) of allicin (1 mL). The combined acaricidal effects of ivermectin and allicin were analysed using four concentration combinations. PRM death rates were determined after 2 h, 24 h, 2 days, 5 days and 7 days of drug application. Results Ivermectin application (1 mg/mL) exterminated 64% and 100% of PRMs on 1 and 5 days, respectively, and prevented their revival. Further, 0.5 mg/mL ivermectin and 1 mg/mL allicin individually exterminated 98% and 44% of PRMs, respectively, within 7 days of treatment. In combination, 0.5 mg/mL ivermectin and 0.5 mg/mL allicin exterminated 100% of PRMs within 5 d of treatment. The most effective combination was 0.25 mg/mL ivermectin + 1.00 mg/mL allicin. Conclusions The efficacy of ivermectin–allicin combination in exterminating PRMs was demonstrated. This novel approach could be optimised for industrial applications.

sucked blood, mate, and lay their eggs (Abbas et al., 2014;Hearle, 1938). Nymphs and females suck blood, whereas males do so occasionally; larvae do not suck blood (Abbas et al., 2014). Severe PRM infestation may result in considerable blood loss (anaemia) and other diseases caused by transmitted viruses, bacteria and parasites (Abdel-Ghaffar et al., 2009;Hungerford & Hart, 1937;Zeman et al., 1982), possibly leading to host mortality. Some of the other adverse effects of PRM infections in chickens are reduced egg quality due to blood spots, stressful behaviour and body weight reduction (Chauve, 1998). Therefore, PRM extermination is crucial for maintaining the good health of poultry and preventing gross agroeconomic losses.
Acaricide application is the main approach often employed to exterminate PRMs, and some acaricides have been approved globally for this purpose. The most widely used acaricides are organophosphates, carbamates, amidines and pyrethroid-based acaricides. However, many acaricides are not recommended against PRMs and illegally used in poultry farms in various countries (Chauve, 1998;Beugnet et al., 1997;Marangi et al., 2009;Nordenfors et al., 2001;. For example, fipronil is an acaricide that is not classified as an 'allowed substance' for use as a veterinary medicinal product in food-producing animals and birds (European Food Safety Authority (EFSA) 2018).
Although some acaricides are effective against PRMs, they affect nontargets such as humans, poultry and eggs. Moreover, PRMs have developed resistance to different classes of acaricides in various regions worldwide. Therefore, several new alternative solutions, including biological compounds, essential oils, heat treatments, predator mites, inert dust, intermittent lighting programs and even vaccines, have been developed (Mozafar & Tierzucht, 2014). Furthermore, the anthelmintic drug ivermectin (0.5% lotion) can kill head lice (a human ectoparasite) (Deeks et al., 2013). Garlic has a lethal effect on northern fowl mites (Birrenkott et al., 2000), and its main component believed to be responsible for its biological activity is allicin (Cho et al., 2006).
Thus, we hypothesised that ivermectin and allicin may interfere with the viability of PRMs. Accordingly, we aimed to evaluate the individual and combined efficacies of ivermectin and allicin in exterminating PRMs.

MATERIALS AND METHODS
Cardboard traps (100 mm × 70 mm × 3 mm) were installed in a poultry farm in Munkyeong City, Korea, to collect PRMs in accordance with a previously reported method (Chirico & Tauson, 2002); PRMs were collected in Ziploc plastic bags. A 90 mm filter paper (Advantech, Tokyo, 100-0011, Japan) was placed in each insect culture dish (ICD), and 1 mL of different concentrations (5, 1, 0.5, 0.25, 0.1 and 0.05 mg/mL) of ivermectin solutions was applied to the filter papers in different ICDs by using a micropipette. On a working bench, the filter papers were left to soak the drug solution for 30 min and then partially dried ( Figure 1).
PRMs (20 individuals) were transferred to each ICD. The PRMs and the drug-containing ICDs were stored in iceboxes, and the death rates of PRMs were recorded after 2 h, 24 h, 2 days, 5 days and 7 days of treatment.
F I G U R E 1 Schematic representation of the treatment method of the ivermectin-allicin combination to exterminate poultry red mites in this study.
For the spraying method, a 90 mm filter paper (Advantec, Tokyo, Japan) was kept in each ICD, and 20 PRMs were transferred to each of these ICDs. Approximately 1 mL (5 puffs) of ivermectin solution (1000 ppm) was sprayed onto the PRMs in the ICDs. The percentage of dead PRMs was determined after 2 h, 24 h, 2 days, 5 days and 7 days of ivermectin application.
Furthermore, the acaricidal effect of allicin on PRMs was evaluated by applying 1 mL of allicin (1, 0.5 and 0.25 mg/mL) with a micropipette.
The percentage of dead PRMs after allicin treatment was determined using similar methods to ivermectin treatment. The combined acaricidal effects of ivermectin and allicin on PRMs were also analysed. Four combinations (0.25 mg/mL ivermectin + 0.25 mg/mL allicin; 0.25 mg/mL ivermectin + 0.5 mg/mL allicin; 0.25 mg/mL ivermectin + 1 mg/mL allicin; and 0.5 mg/mL ivermectin + 0.5 mg/mL allicin) were applied to 20 PRMs in ICDs by using a micropipette, and their effects were examined in accordance with the procedure described earlier in this section.

RESULTS AND DISCUSSION
Chemicals used to control PRMs may adversely affect workers through direct exposure and indirect consumption of pesticide residuecontaining eggs (Hamscher et al., 2003). We aimed to develop a more effective and convenient treatment for the successful extermination of PRMs without causing any harm to target/non-target animals and humans by using ivermectin, which is an efficient and safe treatment for controlling the growth of PRMs . In this study, we investigated the efficacy of ivermectin alone and in combination with allicin against PRMs. We applied 1 mg/mL ivermectin via two different methods, namely, dropping and spraying. We found that dropping was better than spraying, and the acaricidal ability of ivermectin and allicin gradually increased after treatment (Table 1). The combination TA B L E 1 Individual and combination effects of ivermectin and allicin in the extermination of poultry red mites. Values are means ± standard deviation of 3 samples. *Statistical significance (p < 0.05) among solvent control and treated samples. suggesting that allicin is safe (Salehi et al., 2019). The combination of ivermectin and allicin showed synergistic and promising results, which indicated that it can be used as a novel treatment option against PRMs.
Ivermectin can efficiently kill human head lice, a parasite with physiological properties similar to those of PRMs (Chosidow et al., 2010).
Ivermectin targets glutamate-gated chloride channel receptors found on neurons and muscle cells of organisms, causing paralysis and death (Atif et al., 2017). The potential efficacy of ivermectin in this study suggested that it may affect vital physiological processes of PRMs, such as the functioning of chloride channels ( Values are means ± standard deviation of 3 samples. *Statistical significance (p < 0.05) between I0.25+A1+P1 and I0.25+A1. I, ivermectin; A, alicin; P, picrotoxin.
be optimised for clinical use. Nevertheless, the development of a suitable dosage of 0.25 mg/mL ivermectin and 1.00 mg/mL allicin combination would be effective for its rapid application and transport.