Introduction

Horn fly (Haematobia irritans) is an external parasite of cattle that causes discomfort and stress by sucking blood and making sores, which might be infested by screwworms (^{Bruce, 1942}). As all flies that bite intermittently, the horn fly is also a potential disease vector. When flies are chased they temporarily fly away, but they return soon to the animal’s hide (^{Jensen et al., 2004}). Horn fly infestation on grazing cattle decreases weight gain due to blood sucking and energy spend fighting flies (^{Kunz et al., 1984}). In North and South America, horn fly shows two population peaks from spring to autumn (^{Guglielmone et al., 1997}). Horn fly load on cattle is different among breeds (^{Steelman et al., 1991}) and this difference is associated to hair density and amount of skin sebum (^{Steelman et al., 1997}). ^{Presley et al. (1996)} concluded that differences in horn fly load among cattle breeds could be associated to physiological and anatomical attributes of each breed.

Insecticide control of horn fly has been an economic option, as benefits surpass the cost; however, in the long run, continuous insecticide use might bring resistance build-up decreasing control effectiveness (^{Byford et al., 1999}). All along the Gulf of Mexico, central to south Pacific Mexican coast and in north-central Mexico horn fly resistance to some common insecticides has been registered (^{Kunz et al., 1995}; ^{Cruz-Vázquez et al., 2002}; ^{Maldonado et al., 2005}). Resistance patterns vary among these Mexican regions as frequency and intensity of horn fly control programs are not the same either (^{García et al., 2004}).

In addition to resistance, there is food safety, animal welfare and environmental issues that determine pesticide use and management in livestock production operations, demanding innovative external parasite control that complies with policies on these topics (^{Pruett et al., 2003}). ^{Rodríguez and Niemeyer (2005)} stated that the search for alternative pest control strategies should not be based on resistance concern only but also on the need to comply with environmental and food safety protocols, foreseeing the incorporation of bio-pesticides in any pest control program. ^{Khater and Khater (2009)} added that plant extracts should be taken as strong alternatives to synthetic pesticides as plant oils are environmental friendly due to be species specific, of easy and fast degradation and with lower risk of resistance. ^{Choochote et al. (2004)} concluded that bio-insecticides are alternatives to chemical pesticides as they are harmless to non-target organisms and to the environment and they are biodegradable. Integrated pest control should include ecological bases, then bio-pesticides from plant extracts might represent a best option as they have shown minimum negative environmental impact (^{Koul and Walia, 2009}).

Creosote-bush (Larrea tridentata (DC.) Coville) is a shrub of the Zygophyllaceae family, distributed widely in the Sonoran desert with allelopathy potential (^{Brinker, 1993}). ^{Saldívar (2003)} did an extensive and comprehensive review on Creosote-bush leaf and root extract uses, among them: insect repellent, fungus and bacteria growth inhibition, bactericide, fungicide and insecticide. The ability of Creosote-bush to thrive in a very harsh desert environment was associated to the inhibitory impacts this plant might have on other living organisms (^{Barbour, 1969}).

^{Rhoades (1977)} found that Creosote-bush leaves, as it is common in xerophyte species, were coated with a thick resin layer synthesized by glandular trichomes during the development of the leaves, and might represent up to 20 % of the leaf dry weight. ^{Cortez-Rocha et al. (1993)} indicated that ground Creosote-bush leaves might help in preventing insect attack on harvested grains. ^{Viglianco et al. (2006)} found that crude extracts of Creosote-bush showed some insect repellent potential. ^{Arteaga et al. (2005)} concluded that Creosote-bush extracts are useful in the industry and in traditional medicine.

The aim of this study was to determine skin reaction and horn fly load on grazing dairy cattle, sprayed with a solution of Creosote-bush leaf extract, to provide field data of the suitability of this leaf extract as a biological control of horn fly. The hypothesis was that spraying a solution of Creosote-bush leaf extract on grazing dairy cattle would reduce horn fly load and then could be a biological control of this pest.

Materials and Methods

The study was conducted in the grazing dairy production unit of the experimental farm of the University of Chapingo (19° 29’ N, 98° 53’ W, altitude of 2250 m, climate is sub-humid temperate, mean annual temperature of 18 °C, mild winters, six months (spring and summer) of rainy season and 645 mm of annual rainfall) (^{CONAGUA, 2015}). Handling and treatment of experimental animals complied Mexican regulation for humanitarian treatment and animal welfare (^{DOF, 2001}). Two independent experiments were carried out, one was to test skin reaction to the Creosote-bush leaf extract and the other to monitor horn fly load during the warmest part of summer on grazing dairy cows sprayed with Creosote-bush leaf extract. The extract was obtained from oven-dried and ground (1mm mesh) leaves (250 g) that were set into ethyl alcohol (1 L) for 48 h, and the infusion was placed in a rotatory-evaporator to get the extract at 90-95 % concentration.

In the skin reaction, experiment two treatments were compared: control and 20 % Creosote-bush leaf extract solution spray. Control treatment was plain water spray. On the skin of a 10-month heifer, six areas were marked, three per side (left and right) on the neck, middle and rear of the animal; each area was divided into two circles of 63.6 cm² each. A graduated handheld one-nozzle sprayer was used, a spray hood made out of a pet bottle was fitted to the nozzle to avoid spray drift and to assure a homogeneous spray of the whole surface of the targeted circle, 10 mL of water or leaf extract solution were sprayed each time. A total of five sprayings at five-day interval were done, spraying was always done at 10:00 h. Minutes before each application of water or leaf extract two persons independent from each other filled out a checklist to define their appreciation of some hair and skin changes within the sprayed areas. Checklist items were: hair colour change, brittle hair, hair loss, signs of pain when pressing with fingertip on sprayed skin surface, spots or change in skin colour, reddish skin, abraded or lacerated skin and swollen spots. Each circle was a replicate, and statistical analysis was based on frequency of responses (^{Saris and Gallhofer, 2007}). At the end of the skin reaction experiment and once it was found that 20 % Creosote-bush leaf extract solution was not harmful to skin and hair of cattle, the second experiment was set up.

In the second experiment treatments were: spraying or not (control) of a water solution with 20 % Creosote-bush leaf extract. Experimental design was a completely random with seven and six replicates for the sprayed and control treatments, respectively. Experimental unit was a Holstein mature lactating cow grazing alfalfa-orchard grass pastures. The experiment lasted from June to July, the warmest months of summer, when horn fly load on cattle tends to be the highest (^{Maldonado et al., 2006}). Cows in the control treatment were sprayed with plain water; from 50 to 60 ml of water (control) or leaf extract solution were sprayed on each cow’s side, two graduated handheld one nozzle sprayers were used, one for the control and the other for the leaf extract solution treatments. Spraying was parallel to the top line of the cow’s body and from ear to tail implantations. Five sprayings were done at a week interval, starting on June 18^th, 2011.

The response variable was horn fly count per cow, two persons one from each side of the cow were recording with a mechanical counter the number of horn flies present on the surface of the skin of the cow (^{Maldonado et al., 2006}). Horn fly load was recorded nine times, first count was a week before the first spraying, the second four days after the first spraying, thereafter fly count was done two times between consecutive sprayings: just before the cow was sprayed again and four days after being sprayed. Horn fly counts were compared by t Test (^{Witte and Witte, 2015}), an independent analysis was done for each time fly count was measured.

Results and Discussion

From the third spraying on, both observers indicated that in all leaf extract sprayed circles, hair tips showed a green colour, implying that the extract adhered to the outer part of hairs; however, no other changes were registered by any of the two persons in any of the five sprayings done. ^{Arteaga et al. (2005)} agree in not finding any toxicity or harmful effect on large animals with external application of Creosote-bush extracts at different concentration, they pointed out that toxicity might develop with forced ingestion of Creosote-bush extract above therapeutic dosages used by traditional medicine. ^{Saldivar (2003)} in his review found that topical application of a cream containing Creosote-bush extract was not harmful or toxic to humans and other large animals.

As the adherence of some Creosote-bush leaf extract solution to hairs’ outer end showed no harmful effects on cows’ wellbeing, a 20 % Creosote-bush leaf extract solution might be taken as a safe and therapeutic dosage.

Control and leaf extract sprayed cows started the experiment with the same (p ≥ 0.05) horn fly load a week before the first spraying, an overall mean of 32.5 flies per cow. Both groups of cows showed a similar general seasonal pattern of horn fly load, there was a steady increment in horn fly load as the summer went by up to a peak, then at the end of the season there was a drop in horn fly count (Figure 1). Season long trend in leaf extract sprayed cows was to show a lower horn fly count than control cows; however, after the first spraying, in four out of the eight times horn fly load was registered, leaf extract sprayed cows showed a significant (p ≤ 0.05) lower amount of horn flies than control cows. On these occasions, the reductions in horn fly count went from 9.5 to 68 % relative to the amount of horn flies found on control cows (Figure 1).

Figure 1 Horn fly count on grazing dairy cows with (treated) and without (control) spraying of a 20 % Creosote-bush leaf extract solution. Fly counts with different letter were different (p ≤ 0.05). 

The seasonal trend in horn fly load shown by both groups of cows (control and leaf extract sprayed) is similar to that observed by ^{Maldonado et al. (2006)}, they explained this seasonal trend on basis of increasing favourable climatic conditions as air temperature and relative humidity, for the growth and dispersion of horn fly individuals. The Creosote-bush leaf extract application decreased horn fly load on cows, however the seasonal pattern of horn fly population on cows was the same.

Replications were few and the length of the study was short, however the reduction range of 9.5 to 68 % of horn fly and the trend to lower efficacy after some weeks of application were also found by ^{Madzimure et al. (2013)} using other plant extracts. These authors suggested that standard changes in the population of the specific pathogen and washing off of plant extracts by environmental factors as rains might explain this response pattern. ^{Malik et al. (2007)} suggested that the commercial viability of any bio-pesticide should not be ruled out if the population reduction is within the range of 30 to 85 % considering the easy availability of plant material and the eco-friendliness of the resultant product. ^{Oyarzún et al. (2008)} and later on ^{Khater and Khater (2009)} emphasized that in the evaluation of plant extracts as bio-pesticides, environment degradation readiness, susceptibility to resistance development and extent to be species specific should be included in addition to control extent of the pest.

Crude leaf neem extract (^{Miller and Chamberlain, 1989}; ^{Singh and Kaur, 2016}) was proposed as an alternative bio-pesticide based on its biodegradable attributes and minimal pollution potential, in addition to its toxicity to H. irritans larvae and myiasis-causing Chrysomya bezziana larvae. ^{Koul et al. (2008)} registered that the efficacy of bio-pesticides could fall short when compared to synthetic pesticides; ^{Rodríguez and Niemeyer (2005)} added that bio-pesticides should be taken as one component of an integrated pest management among others like the strategic use of chemical pesticides.

^{Saldivar (2003)} indicated that there were some in vitro evidences that Creosote-bush leaf extract showed direct effect inhibiting insect, bacteria and fungus proliferation; however, ^{Cortez-Rocha et al. (1993)} and ^{Viglianco et al. (2006)} observed that Creosote-bush leaf powder and extract showed an adult insect repellent and antifeedant action rather than a biocide one. Then, in our study, the reduction of horn fly load on cows sprayed with Creosote-bush leaf extract could be explained on basis of having an adult insect repellent and/or antifeedant action. Besides, horn fly larvae develop away from the host animal (^{Mendes and Linhares, 1999}) so the Creosote-bush leaf extract applied to cows could not have any biocide action on larvae. Creosote-bush leaf extract seems to be a promising bio-pesticide as some plant essential oils (^{Malik et al., 2007}; ^{Palacios et al., 2009}) and extract of Larrea cuenifolia from South America (^{Batallán et al., 2013}) a component in the development and implementation of new strategies against horn fly.

Conclusions

Creosote-bush (L. tridentata (D.C.) Coville) crude leaf extract sprayed on grazing dairy cows could be a suitable biological control of horn fly Haematobia irritans load on cattle, without skin side-effects, and a component of an integrated pest management with sound ecological basis.