Original Article Buffalo Bulletin (April-June 2017) Vol.36 No.2

IN VITRO EFFICACY OF ALPHACYPERMETHRIN ON THE BUFFALO HAEMATOPINUS TUBERCULATUS (BURMEISTER, 1839)

Vincenzo Veneziano1,*, Gianluca Neglia1, Francesco Buono1, Laura Pacifico1, Laura Manna1, Andrea Bassini2, Luca Miotto2, Mario Santoro3 and Cengiz Gokbulut4

ABSTRACT minutes or at the louse death. After 240 minutes the louse vitality was examined each 60 minutes In Italy buffalo farms adopted intensive until 540 minutes. In vitro bioassays revealed that breeding techniques, however the high density of the lousicidal efficacy of ACYP improved as the in intensive breeding favours the diffusion concentration and the exposure time increased. The of ectoparasites, such as louse. results of this in vitro study confirm that ACYP at The aim of this study was to determine the in 1.5% concentration can also be used in buffalo for vitro efficacy of the insecticide alphacypermethrin the control of lice, as already in use in . Further (ACYP) against the buffalo louse, Haematopinus field trials will need to be conducted to confirm the tuberculatus. The study was performed by using safety, the dosage and the in vivo parasitological louse collected from animals in a commercial efficacy of this drug on buffaloes. buffalo farm located in the Campania region of Southern Italy. Lice (adults and nymphs) were Keywords: alphacypermethrin, , collected from highly infested buffaloes. The Bubalus bubalis, louse, Haematopinus tuberculatus ACYP was diluted with physiological solution to different concentrations: 1.5%, 0.75%, 0.37%. A volume of 600 µl of the diluted sample was INTRODUCTION spread evenly over a filter paper held in the lower half of Petri dish. Ten adult lice and ten nymphs The Haematopinus were placed on the top of each filter paper disc. tuberculatus (Burmeister, 1839), Phylum The control groups were treated with physiological Arthropoda, Class Insecta, Order Phthiraptera, solution. Seven replicates were used for each Suborder Anoplura, Family Haematopinidae, is a concentration. The louse vitality was assessed at specific louse of water buffalo (Bubalus bubalis), different time intervals: 1, 2, 4, 8, 10, 15, 20, 30, being the principal ectoparasite which attaches 40, 50, 60 minutes, after every 10 minutes until 240 this species (Bastianetto et al., 2002); it has

1Department of Veterinary Medicine and Productions, University of Naples Federico II, Naples, Italy, *E-mail: [email protected] 2Zoetis, Rome, Italy 3Istituto Zooprofilattico Sperimentale del Mezzogiorno, Portici, Napoli, Italy 4Department of Medical Pharmacology, Faculty of Medicine, Balikesir University, Balikesir, Turkey

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been reported on water buffalo in Asia, Africa, insecticides. Because there is no data on the in Australia, and South America (Meleney and Kim, vitro efficacy of the different group of insecticide 1974). In Europe it has been reported in Albania, on buffalo louse, the aim of this study was to Macedonia, France, England and Italy (Veneziano determine, for the first time, the in vitro efficacy et al., 2003). Cattle, and American bison of the ACYP at different concentrations against the are also susceptible to infection (Chaudhuri and buffalo louse, H. tuberculatus. Kumar, 1961). Louse infestation often leads to skin irritation, anemia, anorexia, restlessness and loss of body condition. The itch caused by H. MATERIALS AND METHODS tuberculatus is responsible for the low milk and meat productivity (Bastianetto and Leite, 2005). The study was performed by using louse The buffalo louse infestation needs to be collected from animals in a commercial buffalo controlled, particularly if the general condition of farm located in the Campania region of Southern animals is affected (Veneziano et al., 2004). On Italy. The buffaloes had naturally lice infestation. water buffalo, several formulations marketed for In order to carry out species identification, 50 louse cattle have been tested in field trials against H. were collected 5 days before the beginning of the tuberculatus in particular macrocyclic lactones trial from 5 randomly selected adult buffaloes. Louse such as ivermectin (Lau and Sing, 1985), specimens were studied by using the following avermectin, doramectin (Bastianetto et al., 2002) routine procedure: in vitro mounting, examination and eprinomectin (Veneziano et al., 2004). under optical and dissection microscopes, and Alphacypermethrin (ACYP) is a synthetic comparison of the resulting morphometric data pyrethroid insecticide, containing more than 90% with those reported in the literature. Species of the most active enantiomer pair of the four cis determination was based on the keys proposed by isomers of cypermethrin as a racemic mixture. Chaudhuri and Kumar (1961) and Meleney and ACYP acts on ’s axons in the peripheral Kim (1974). The buffalo louse H. tuberculatus and central nervous systems by interacting with (nearly 600) was collected by 5 researchers from 5 sodium channels. This compound is effective buffaloes highly infested. against a wide range of pests of many crops and A commercial preparation of ACYP is also used for the control of various veterinary (Renegade™, pour on, 1.5%, w/v, Pfizer Animal and public health , including lice. In Italy Health, Italy) was used to prepare three different ACYP is marketed as a pour-on formulation for working concentrations of ACYP (1,5%, 0.74%, use in cattle, with zero milk-withdrawal time. 0.37%). Therapeutics, such as antiparasitic compounds, are The method used to assess the lousicidal often administered to buffaloes based on dosage activity of ACYP was adapted from the World and intervals recommended for cattle because very Health Organization (WHO, 1981) protocol and few drugs have buffalo-specific label indications according to the methodology from Priestley et (Veneziano et al., 2004). al. (2006). Bioassays were performed at 27°C and This is why the literature lacks information 75% relative humidity (RH). The direct contact on the use and efficacy on buffaloes of most assay was carried out as follows. The ACYP

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was diluted in physiological solution to different considered significant. concentrations: 1.5%, 0.74%, 0.37%. Three drops of Tween 80 were added as emulsifier. A volume of 600 µl of each concentration was properly RESULTS AND DISCUSSION distributed over a 9 cm diameter filter paper held in the lower half of a 9 cm glass Petri dish. The The morphometric data regarding the liquid was allowed to evenly spread for 15 minutes lice collected during this study were closely until the filter paper was totally soaked with the corresponded to those reported for H. tuberculatus insecticide. (Figure 1). Ten adult lice and ten nymphs of H. The results demonstrated that ACYP was tuberculatus were placed in two Petri dishes for each effective against both adults and nymphs at all concentration. Additional adult and nymphs stages concentration levels. In vitro bioassays revealed treated with the same volume of physiological a higher efficacy of ACYP on lice with increasing solution and Tween 80 only served as untreated concentrations and exposure times. The results control and were placed in two separate plates. have been summarized in Figure 2 and Figure 3, Seven replicates were used for each concentration. showing the knockdown activity of the ACYP Louse vitality was assessed by against H. tuberculatus. The Table 1 shows the stereomicroscopy (Leica EZ4 HD) at different time efficacy values of all concentrations at different intervals: 1, 2, 4, 8, 10, 15, 20, 30, 40, 50, 60 minutes time intervals, expressed as percent reduction. and then every 10 minutes until 240 minutes or at ACYP was effective against the buffalo the louse death. After 240 minutes, the vitality of louse at all concentration levels. The 1.5% lice has been measured at 60 minutes intervals concentration has shown the fastest parasiticidal up to 540 minutes, i.e. the time when all treated effect on adults lice, with a complete knockdown parasites were found to be dead. Lice showing no activity achieved within 70 minutes. On the movements of legs and intestine and unresponsive contrary, while a parasiticidal activity was when stimulated with an entomological pin were observed at concentrations 0.75% and 0.37%, the considered as dead (Priestley et al., 2006). death of all lice occurred at 200 and 540 minnutes, The efficacy (%) of ACYP was calculated respectively. Moreover, a significant difference at 1, 2, 4, 8, 10, 15, 20, 30, 40, 50, 60, 70 to 240 (P<0.05) between 1.5% and lower concentrations minute (at ten minutes’ interval) and 240 to 540 was reported as early as 4 minutes post-treatment, minutes (at sixty minutes’ interval), by using a indicating the important role of drug dilution. At modified Abbot’s formula (1925): lower concentrations (0.75 to 0.37%), no significant differences were observed, although the death of Efficacy=100 x [n live lice in control plate - n live lice in treated plate] n live lice in control plate lice occurred more rapidly at 0.75%. The nymph stages have shown higher susceptibility to ACYP Differences in louse count were analysed than adults, with a overall knockdown activity using an ANOVA test for comparison of the being achieved earlier. The 1.5% concentration of treated with the control group. P-values<0.05 were ACYP displayed a complete knockdown activity

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Figure 1. Haematopinus tuberculatus at different stages.

Figure 2. Knockdown activity of Alphacypermethrin on adult stages of Haematopinus tuberculatus at different

concentrations.

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after 50 minutes post-treatment, and mortality in treatment by using some essential oils. While the nymphs was significantly higher (P<0.05) than in vitro treatment of H. tuberculatus with different in adults as early as 8 minutes post-treatment. concentrations of d-phenothrin determined the Similarly, mortality in nymphs was found higher 100% mortality within 20 minutes. (P<0.05) than adults also at concentration 0.75% In relation with the lousicidal efficacy, as early as 8 minutes from the start of treatment. At according to WAAVP guidelines (Holdsworth et 0.37%, no increase in mortality has been observed al., 2006), an in vivo ectoparasiticide drug should for the nymphs as compared to adults. provide a reduction in the parasitic population A full knockdown activity of ACYP at of at least 95% to demonstrate its efficacy in different concentrations has been indicated on louse control. Highly efficacy values have been nymphs after 50 minutes at 1.5%, and after 190 recorded within 60 minutes on adults and from 30 and 360 minutes at 0.75% and 0.37%, respectively. minutes on nymphs at 1.5% concentration. The Although 1.5% was statistically the most effective concentration of 0.75% the efficacy value ≥ 95% concentration as compared to lower dilutions (as has been achieved starting 190 and 180 minutes early as 2 minutes post-treatment) also on nymphs, on the adults and on the nymphs, respectively. The 0.75% concentration was more effective than times were longer at the concentration 0.37%, 220 0.37% from 4 to 110 minutes (P<0.05). Thereafter, minutes and 190 minutes on adults and on nymphs, these two concentration levels induced similar respectively. reductions of lice. The preliminary results of this in vitro There are limited data available on in vitro study confirm that ACYP may be used in buffaloes, efficacy of drugs against the buffalo louse, the as well as in cattle, for the control of sucking lice at results observed in the present study are comparable a 1.5% concentration. only to data reported by Khater et al. (2009). The This is the first study to evaluate the in authors showed, in a similar in vitro bioassay, that vitro efficacy of ACYP against the buffalo louse all treated lice were killed within a minute after H. tuberculatus. Further field trials are required to

Figure 3. Knockdown activity of Alphacypermethrin on nymph stages of Haematopinus tuberculatus at different concentrations.

331 Buffalo Bulletin (April-June 2017) Vol.36 No.2 - - - - 7.1 7.1 2.9 7.1 7.1 2.9 (%) 13.0 13.0 17.1 22.9 47.1 57.1 67.1 82.9 82.9 84.3 87.1 88.6 92.9 92.9 Efficacy 9 8 5 5 T 11 61 65 65 68 65 65 61 68 70 70 70 58 54 37 30 23 12 12 70 N° C 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 Nymphs 0.37% ------2.9 (%) 20.0 12.9 37.1 42.9 47.1 60.0 70.0 72.9 80.0 87.1 87.1 92.9 92.9 92.9 92.9 Efficacy 9 9 5 5 5 5 T 56 61 70 70 70 68 44 70 70 70 70 40 37 28 21 19 14 70 C 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 N° Adults - - 7.1 2.9 (%) 50.0 47.1 20.0 27.1 27.1 37.1 52.9 20.0 57.1 60.0 82.3 82.3 82.3 82.3 87.1 90.0 90.0 90.0 92.9 97.1 Efficacy 9 7 7 7 5 2 T 35 37 56 51 51 44 33 56 65 68 30 28 12 12 12 12 70 70 N° C Nymphs 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 0.75% ------2.9 2.9 (%) 12.9 10.0 10.0 40.0 42.9 45.7 60.0 62.9 67.1 72.3 72.3 80.0 87.1 87.1 92.9 94.3 Efficacy 9 9 5 4 T 61 63 68 68 63 42 70 40 38 28 26 23 19 19 14 70 70 70 70 70 C 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 N° Adults - 1.4 (%) 98.6 85.7 92.9 95.7 70.0 54.3 27.1 14.3 100.0 Efficacy 0 1 5 3 T 10 21 32 51 60 69 70 N° Nymphs C 70 70 70 70 70 70 70 70 70 70 70 1.5% - - 4.3 (%) 97.1 91.4 85.7 45.1 57.1 74.3 37.1 24.3 17.1 100.0 Efficacy 2 6 0 T 10 38 30 18 44 53 58 67 70 70 C 70 70 70 70 70 70 70 70 70 70 70 70 70 N° Adults 8 4 2 1 0 60 50 40 15 20 30 70 10 80 90 110 100 120 130 140 150 160 170 180 Time (minutes) Concentrations Table 1. Lousicidal efficacy of different concentrations of Alphacypermetrin at different time intervals. Alphacypermetrin at different concentrations of 1. Lousicidal efficacy of different Table Treated = T C = Control

332 Buffalo Bulletin (April-June 2017) Vol.36 No.2 (%) 98.5 98.5 97.1 97.1 97.1 97.1 95.6 100.0 Efficacy 0 1 1 2 2 2 2 3 T N° C 68 68 68 68 68 68 68 68 Nymphs 0.37% (%) 98.6 98.6 98.6 97.1 97.1 97.1 95.7 94.3 94.3 94.3 100.0 Efficacy 1 1 0 1 2 2 2 3 4 4 4 T C 70 69 69 70 70 70 70 70 70 70 70 N° Adults (%) 100.0 Efficacy 0 T N° C Nymphs 70 0.75% (%) 97.1 100.0 Efficacy 2 0 T C 70 70 N° Adults (%) Efficacy T N° Nymphs C 1.5% (%) Efficacy T C N° Adults 420 480 540 360 300 240 230 220 210 190 200 Time (minutes) Concentrations Table 1. Lousicidal efficacy of different concentrations of Alphacypermetrin at different time intervals. (Cont.) Alphacypermetrin at different concentrations of 1. Lousicidal efficacy of different Table Treated = T C = Control

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confirm safety, dosage and in vivo parasitological efficacy of some essential oils against lice efficacy of this insecticide on water buffalo. and flies infesting water buffaloes in Egypt. Vet. Parasitol., 164: 257-266. Lau, H.D. and N.P. Singh. 1985. Efficacy ACKNOWLEDGMENT of ivermectin in control of louse (Haematopinus tuberculatus) in buffaloes, The authors would like to thank Zoetis, p. 47. In Proceedings of the 11th International Italy for technical support. Conference of World Association for the Advancement of Veterinary Parasitology. Meleney, W.P. and K.C. Kim. 1974. A comparative REFERENCES study of cattle-infesting Haematopinus, with redescription of H. quadripertusus Abbott, W.S. 1925. A method of computing the Fahrenholz, 1919 (Anoplura: effectiveness of an insecticide. J. Econ. Haematopinidae). J. Parasitol., 60: 507- Entomol., 18: 265-267. 522. Bastianetto, E., V.M. Barbosa and R.C. Leite. 2002. Priestley, C.M., I.F. Burgess and E.M. Williamson. Evaluation of the different avermectin bases 2006. Lethality of essential oils constituents in the control of Haematopinus tuberculatus. towards the human louse Pediculus p. 357-359. In Proceedings of 1st Buffalo humanus, and its eggs. Fitoterapia, 77: 303- Symposium of Americas. 309. Bastianetto, E. and R.C. Leite. 2005. Control of the Veneziano, V., L. Rinaldi, S. Giannetto and louse (Haematopinus tuberculatus) in herds G. Cringoli. 2003. The first record of of water buffalo (Bubalus bubalis) raised Haematopinus tuberculatus on Bubalus for milk and meat. Rev. Bras. Reprod. Anim. bubalis (water buffalo) in Italy. Bubalus 29: 118-121. Bubalis, 9(3): 69-75. Chaudhuri, R.P. and P. Kumar. 1961. The life Veneziano, V., L. Rinaldi, C. Grassi, G. Neglia, history and habits of the buffalo louse, G. Campanile and G. Cringoli. 2004. Haematopinus tuberculatus (Burmeister) Efficacy of eprinomectin pour-on against Lucas. Indian J. Vet. Sci., 31: 275-287. Haematopinus tuberculatus infestation in Holdsworth, P.A., J. Vercruysse, S. Rehbein, Italian Mediterranean buffalo (Bubalus R.J. Peter, T. Letonja and P. Green. 2006. bubalis) and influence of the treatment on World Association for the Advancement milk production. Bubalus Bubalis, 2: 56-65. of Veterinary Parasitology (W.A.A.V.P.) World Health Organization. 1981. Instructions for guidelines for evaluating the efficacy of determining the susceptibility or resistance ectoparasiticides against biting lice, sucking of body lice and head lice to insecticides. lice and sheep keds on ruminants. Vet. World Health Organization, 81: 5-55. Parasitol., 136(1): 45-54. Khater, H.F., M.Y. Ramadan and R.S. El-Madawy. 2009. Lousicidal, ovicidal and repellent

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