Assessment and determination of LC50 of carvacrol and salicylic acid analogues with acaricide activity in larvae and adult ticks of Rhipicephalus (Boophilus) microplus.

Rhipicephalus (Boophilus) microplus is a tick that causes huge economic losses in cattle. The indiscriminate use of acaricides has generated resistance to most compounds present on the market. Carvacrol and salicylic acid have been widely studied for their biological activities and have been evaluated in different strains of Rhipicephalus. In this research the analogues carvacrol and salicylic acid were evaluated in larvae of R. (B.) microplus with data obtained in larval packet test (LPT) and larval immersion test (LIT). A lethal concentration 50 (LC50) was assessed. The most potent compounds were evaluated in the adult ticks since there are no reports of evaluation in the life state of the parasite. From all the tested compounds, the ethyl 2-methoxybenzoate (91.82 ± 1.66%, 0.91 µmol/mL) and ethyl 2,5-dihydroxybenzoate (89.14 ± 1.61%, 2.04 µmol/mL) showed the highest percentage of mortality and the lowest LC50. They were found to be the best candidates for a study in vivo.

Design, synthesis, in vitro evaluation and preliminary SAR studies of N-(2-(heteroaryloxy)propyl)phenothiazines against Rhipicephalus microplus cattle tick.

A family of 15 N-substituted phenothiazines was designed, synthesized and their acaricidal activity against Rhipicephalus microplus was determined in vitro. The synthetic methodology is simple and can be employed in multigram scale. The rationale for the structure-based design of these compounds is the potential for azines and phenothiazine to engage in π-π interactions; these fragments, joined together by a short, flexible alkoxide linker, structurally resemble phenothiazine-based cholinesterase inhibitors, while their weak basicity implies a neutral active form, rather than a cationic one, thus facilitating penetration of the cuticle of ticks. One compound displayed excellent acaricidal activity (LD50=0.58 µg/mL). Preliminary SAR analysis suggests that the activity is influenced by the presence of a weakly basic nitrogen atom, as well as the substitution pattern within the heterocycles.

The effect of the 5-chloro-2-methylthio-6-(1-naphtyloxy)-1H-benzimidazole on the tegument of immature Fasciola hepatica in their natural host.

The damage to the tegument of 3-week-old Fasciola hepatica was evaluated by scanning electron microscopy (SEM) following treatment with the 5-chloro-2-methylthio-6-(1-naphtyloxy)-1H-benzimidazole (called compound alpha) in its natural host. For the present study, flukes were raised in pelibuey sheep infected orally with metacercariae of F. hepatica; the parasites were recovered from the liver of the sacrificed sheep after 6, 12 and 24 h of treatment with compound alpha. At 6 h of treatment, the flukes showed some lesions on the ventral surface of the anterior region, such as a swollen tegument and blebs. At 12 h after treatment, the specimens showed structural disorganization and spine loss in the ventral anterior region. The tegument of the flukes treated for 24 h was completely lost in some areas of the ventral surface, leaving an exposed basal lamina. The tegument of immature F. hepatica might be a target organ for compound alpha to exert its fasciolicide effect.

Tegumental surface changes in adult Fasciola hepatica following treatment in vitro and in vivo with an experimental fasciolicide.

Our objective was to determine by scanning electron microscopy the structural changes in the tegument of adult Fasciola hepatica after treatment with 5-chloro-2-methylthio-6-(1-naphtyloxy)-1 H-benzimidazole, called compound alpha, and its active metabolite sulphoxide, under in vitro and in vivo conditions. For the in vitro studies, flukes from sheep were exposed to 40 mg/l of compound alpha-sulphoxide over different incubation times. Flukes for the in vivo studies were raised in sheep treated orally with compound alpha and killed at different times post-treatment. Non-treated controls were included for each time of incubation. The results showed lesions after 6 h of treatment, such as swelling and furrows. At 12 h, the spines appeared to be surrounded by the tegument. At 24 h the tegument in some areas showed an exposed basal lamina. These changes became more severe as the incubation periods of the treated flukes increased. Compound alpha exerts a significant effect on the tegument of F. hepatica.

Determination of the effective dose of an experimental fasciolicide in naturally and experimentally infected cattle.

The aim of the present study was to determine the effective dose of an experimental fasciolicide called compound alpha or 5-chloro-2-methylthio-6-(1-napthyloxy)1H-benzimidazole in experimentally and naturally infected cattle. In the first experiment, 24 fluke-free heifers were each infected with 800 metacercariae of Fasciola hepatica and re-infected on day 45 with other 600 cysts per animal. On day 75, when the animals had 4- and 10-week-old flukes respectively, they were divided into four groups (G) of six animals each according to fluke egg counts. Groups 1-3 received compound alpha at 10, 12 and 14 mg/kg/p.o., respectively. G4 remained as an untreated control. Twenty days after treatment, the animals were sacrificed for the recovery of flukes. Efficacy was assessed as a percentage of egg or fluke reduction relative to the untreated control. In the second experiment (naturally infected cattle), 24-year-old steers positive to F. hepatica eggs were blocked into four groups of five animals each. Groups 1-3 received compound alpha at 10, 12 and 14 mg/kg/p.o., respectively. Group 4 served as a non-treated control. All procedures to determine efficacy were carried out as mentioned in the first experiment. The results in the first study showed a percentage on egg reduction of 97.3, 100 and 100 and overall fluke reduction of 94.3, 100 and 100 for Groups 1-3, respectively. In the second study, the percentage of egg reduction was of 87.5, 99.1 and 100 and overall efficacy regarding fluke reduction was of 84.2, 99.6, and 100 for Groups 1-3, respectively. It is concluded that the effective dose selected for compound alpha was of 12 mg/kg/p.o. in cattle having an induced or natural F. hepatica infection.

Evaluación de la prueba de Dig-Elisa para la detección de anticuerpos anti-fasciola hepática en ganado bovino / Evaluation of Dig-Elisa test for detection of anti-fasciola hepatica antibodies in bovine cattle

Se implementó la prueba de Dig-Elisa para detectar IgG anti-F. hepática en bovinos, estableciendo la sensibilidad y el valor de corte de ésta. Los promedios de diámetros de la zona de reacción (DZR) ñ desviación estándar en mm, determinados con Dig-Elisa en 2 grupos de sueros de bovinos: negativo (n=335) fueron 2,5 ñ 2,6 y 12,5 ñ 2,2, respectivamente; obteniéndose una sensibilidad de 98,5 por ciento, una especificidad del 100 por ciento y valores predictivos positivo y negativo de 100 por ciento y 91 por ciento, respectivamente, al utilizar un valor de corte de 8 mm. Adicionalmente, se analizaron las variaciones mensuales, durante un año, de IgG anti-F. hepática y de huevos por gramo (hpg) del parásito eliminados en las heces de un hato de bovinos Holstein hembras en producción (n=30), de Tulancingo (zona enzoótica de fasciolosis), Estado de Hidalgo, México. Los DZR obtenidos con los sueros de los bovinos de Tulancingo nunca bajaron de 11 mm, observándose valores máximos en los meses de septiembre de 1991 (15,6 ñ 2,7) y junio de 1992 (14,6 ñ 1,1). Los valores mínimos y máximos de hpg de heces se obtuvieron en septiembre (0,09) y en mayo (28,5) respectivamente. Los valores obtenidos con Dig-Elisa tuvieron menor variación que los del examen coprológico. Se concluye que el Dig-Elisa puede ser de utilidad en estudios epidemiológicos de fasciolosis bovina