Combination of thymol and eugenol for the control of Rhipicephalus sanguineus sensu lato: Evaluation of synergism on immature stages and formulation development.

The aim of this study was to investigate whether the combination of thymol with eugenol has a synergistic effect on the immature life stages of Rhipicephalus sanguineus sensu lato (s.l.), to evaluate the cost-benefit ratio of using these compounds in combination, and to develop a formulation combining thymol with eugenol with activity on immature stages of R. sanguineus s.l. To evaluate synergism, thymol and eugenol, combined (ratio 1:1) or not, were tested at concentrations of 2.5, 5.0, 10.0, 15.0 and 20.0 mg/mL on unfed larvae and nymphs using a larval packet test, and 0.625, 1.25, 2.5, 5.0 and 10 mg/mL on engorged larvae and nymphs using an immersion test. A cost estimate was calculated to produce 1 L of a solution containing a concentration of thymol and eugenol, combined or not, that could cause a tick mortality rate greater than 95 %. Finally, a formulation was developed, consisting of a micellar dispersion containing polymers (MDP), with thymol + eugenol (1:1), at concentrations of 2.5, 5.0, 10.0, 15.0 and 20.0 mg/mL, and the activity was evaluated on unfed and engorged larvae and nymphs. For unfed larvae and nymphs, concentrations of 2.5 and 5.0 mg/mL and 2.5, 5.0 and 10.0 mg/mL, respectively, presented synergistic effects. In tests with engorged larvae and nymphs, respective concentrations of 0.625, 1.25 and 2.5 mg/mL and 2.5 and 5.0 mg/mL had synergistic effects. The estimated costs for producing a solution of 1 L with efficacy greater than 95 % was $5.97 using only thymol (15 mg/mL), $ 5.93 using only eugenol (15 mg/mL), and $ 3.97 using thymol + eugenol (1:1 - 5,0 mg/mL). In tests with MDP, the combination of thymol + eugenol resulted in a mortality rate higher than 95 % at concentration of 10 mg/mL for unfed and engorged larvae and nymphs. Thus, the combination of thymol + eugenol, depending on the concentration, has synergistic effects and this combination lowers the cost for the active ingredients thymol and eugenol. The combination of thymol + eugenol in MDP had acaricidal activity against immature life stages of R. sanguineus s.l.

Assessment of carvacrol for control of avian aspergillosis in intratracheally challenged chickens in comparison to voriconazole with a reference on economic impact.

AIM: This study was designed to investigate the efficacy of essential oils as an alternative prophylaxis and treatment for avian aspergillosis. METHODS AND RESULTS: The in vitro susceptibility of Aspergillus fumigatus strains to antifungal drugs and carvacrol, thymol, eugenol, thymoquinone and cinnamon was determined using the macrodiffusion and microdilution methods. Carvacrol has antifungal activity in comparison to voriconazole (VCZ) (MIC 0·5, 0·25 µg ml-1 respectively). While cinnamon, euganol, thymol and thymoquinone displayed moderate to weak inhibitory activity. For the efficacy study, five groups of 10-day-old chicks (n = 48) were infected intratracheally either with A. fumigatus conidia or saline (negative control). Chicks in carvacrol prophylactic and treatment (CRPT) group were fed for 10 days beginning from hatch with carvacrol (200 mg kg-1 per diet) supplemented diets. VCZ (VCZT:20 mg kg-1 body weight (BW)), carvacrol treatment (CRT, CRPT) was started upon appearance of the first clinical signs and continued for 10 days. Birds were monitored for an additional 15 days following treatment. Fungal burden and therapeutic efficacy were assessed by survival, BW, quantitative (q) culture (CFU), quantitative real-time PCR (qPCR) and histopathological changes at several time points. Serum biochemical changes were also assessed. VCZT, CRPT, CRT in comparison to the sham-treated (SHAM) group have prolonged survival (87·5, 83·4, 79·2, 41·7% respectively). In VCZT and CRPT, a significant reduction in clinical signs, lesions, CFU and qPCR counts to the limit of detection were observed. CRPT has the lowest BW reduction, economic losses and significant low total cholesterol levels. CONCLUSIONS: Carvacrol has a promising potential to be used as a prophylactic and treatment against A. fumigatus. SIGNIFICANCE AND IMPACT OF THE STUDY: Prognosis of avian aspergillosis is often poor due to delayed diagnosis and treatment failure. However, the widespread uses of azole prophylaxis in birds are thought to be the major driver of azole resistance. These findings create a possibility to develop an effective drug-free alternative strategy for control of avian aspergillosis.

Assessment of losses in honey yield due to the chalkbrood disease, with reference to the determination of its economic injury levels in Egypt.

In Egypt, the chalk brood (CHB) disease caused by the fungus, Ascosphaera apis Maassen started again infesting the honeybee, Apis melifera L (Hymenoptera: Apidae) colonies after a cessation pause of seven to nine years. For the first time, an attempt has been made to look into the problem of the CHB disease from the view point of assessing losses in both clover (Trifolium alexandrinum) and chinus (Chinus molus) honey yields. In this regard, two techniques were adopted. Under the natural conditions of the experimental apiary (first technique), loss in clover honey was 18.412 +/- 0.663%, on average, whereas the average loss in chinus honey was 18.332 +/- 1.536%. In the second technique of loss appraisal (artificial infection). The tested honeybee colonies were artificially infected with four sources of A. apis infection, I.e. black mummies, white mummies, pollen grains and water, in addition to controls to create the so-called "different levels of infection". The mean percentages of losses in clover honey were 30.06 +/- 1.807, 27.95 +/- 1.062; 21.13 +/- 0.987; 16.96 +/- 0.672 and 0.00 +/- 0.00 for black mummies, white mummies, pollen grains, water and control, respectively. Taking into account the relationship between number of resulted mummies in each source of infection and % loss in clover honey, it could be concluded that as the resulted mummies increased, the corresponding clover honey yield decreased in each source of infection. It was noticed that the % loss in clover honey differed as the used technique differed. For example, the percentage loss in clover honey produced from colonies exposed to the natural conditions was relatively less than that of the artificially infected ones. This has been discussed in the text. However, the causative pathogen of CHB disease resulted in serious decrease in honey production. Loss appraisal is a perquisite step for the determination of the economic injury levels (EILs). By regressing % loss in clover honey yield against the total number of fallen mummies, the linear equation was worked out to be: Y = 0.242 + 0.040 x (r2 = 0.99, F = 62408.865). Depending on the total costs of controlling CHB disease, the EIL values were 31.875 and 11.250 mummies/colony for cedar oil and thymol granules, subsequently. As to the chinus honey the corresponding EILs were 18.940 and 6.683 mummies/colony for cedar oil and thymol granules, in respect, as a result of adopting the regression formula: Y = -6.762 + 0.101 X. As for the clover honey (artificial infection) the EIL values were 39.844 and 14.063 mummies/ colony in case of using cedar oil and thymol grains, subsequently. These values were worked out using the formula Y = 5.871 + 0.032 X. From the practical point of view, apiarists should not use thymol or cedar oil as fallen mummies don't reach the suggested EILs values to minimize control costs as much as possible.