Synergy between fipronil and amitraz in a Rhipicephalus sanguineus tick residual contact test.

Amitraz, a formamidine acaricide, has been reported to act as a synergist of contact insecticides and acaricides in both agriculture and animal health. A laboratory contact test was therefore conducted to determine whether amitraz at 12.5 ppm could improve the contact potency of fipronil on ticks. A controlled glass vial bioassay was used to assess the efficacy of fipronil alone, amitraz alone, and fipronil plus amitraz on unfed adult Rhipicephalus sanguineus. Assessments of lethality were made at 6, 24, and 48 h after the introduction of the ticks to the vials. No significant mortality was observed in the control treatment or in the amitraz alone treatment. Concentration and time dependent mortality rates were observed in ticks exposed to fipronil alone or fipronil plus amitraz, with higher mortality observed in the latter group. Results from this study gave synergistic EC50 ratios between fipronil alone and fipronil plus amitraz of >7.3, 137 and 97 at 6, 24, and 48 h, respectively. A similar response was seen at the EC90 level. These results indicate that fipronil was synergized by amitraz in this adult tick residual contact study. The addition of amitraz to fipronil also provided a significant improvement in the speed of kill.

Brain acetylcholinesterase, acid phosphatase, and 2',3'-cyclic nucleotide-3'-phosphohydrolase and plasma butyrylcholinesterase activities in hens treated with a single dermal neurotoxic dose of S,S,S-tri-n-butyl phosphorotrithioate.

The changes in brain acetylcholinesterase (AChE), acid phosphatase (APase), and 2',3'-cyclic nucleotide-3'-phosphohydrolase (CNP), and plasma butyrylcholinesterase (BuChE) activities were investigated in hens treated with a single, dermal dose (100-1000 mg/kg) of S,S,S-tri-n-butyl phosphorotrithioate (DEF). Three control groups consisted of hens left untreated, given a single, dermal dose of 500 mg/kg tri-o-cresyl phosphate (TOCP, positive control for organophophorous compound-induced delayed neurotoxicity), or 10 mg/kg O,O-diethyl O-4-nitrophenyl phosphorothioate (parathion, negative control). Brain AChE activity, determined 28 days after application, was significantly inhibited in hens given 500-1,000 mg/kg DEF and in TOCP- and parathion-treated hens. In contrast, brain APase and CNP activities were significantly higher in all treatments as compared with those of the untreated hens. Parathion, however, caused the least increase in these enzymatic activities as compared to DEF or TOCP. A single, dermal dose of DEF or TOCP also caused an initial decrease in plasma BuChE activity with maximum depression of enzymatic activity observed 1 to 7 days after administration. This decrease was dose dependent and the enzymatic activity showed partial recovery with time. Hens treated with single, dermal doses of DEF, ranging from 250 to 1000 mg/kg, developed ataxia which progressed to paralysis in some hens. Histopathologic examination revealed axon and myelin degeneration of the spinal cord and peripheral nerves of some hens. The severity and frequency of the neuropathologic lesions were dose dependent. Neurologic dysfunctions and neuropathologic lesions seen in DEF-treated hens were similar to those exhibited in TOCP-treated hens. While parathion produced acute cholinergic effects, it did not cause delayed neurotoxicity. The changes in brain and plasma enzymes are discussed in relation to their role in the pathogenesis of DEF-induced delayed neurotoxicity.

The synergism of n-hexane-induced neurotoxicity by methyl isobutyl ketone following subchronic (90 days) inhalation in hens: induction of hepatic microsomal cytochrome P-450.

The effect of methyl isobutyl ketone (MiBK) on n-hexane-induced neurotoxicity was investigated via inhalation in seven groups of five hens each for 90 days followed by a 30-day observation period. One group was exposed to vapors containing 1000 ppm n-hexane and another group to vapors having 1000 ppm MiBK. Four groups were exposed simultaneously to 1000 ppm of n-hexane and 100, 250, 500, or 1000 ppm MiBK. Another group was exposed similarly to ambient air in an exposure chamber and used as a control. Hens continuously exposed to 1000 ppm MiBK developed leg weakness with subsequent recovery, while inhalation of the same concentration of n-hexane produced mild ataxia. Hens exposed to mixtures of n-hexane and MiBK developed clinical signs of neurotoxicity, the severity of which depended on the MiBK concentration. Thus, all hens exposed to 1000 ppm n-hexane in combination with 250, 500, or 1000 ppm MiBK progressed to paralysis. Hens continuously exposed to 1000/100 n-hexane/MiBK showed severe ataxia which did not change during the observation period. The neurologic dysfunction in hens exposed simultaneously to n-hexane and MiBK was accompanied by large swollen axons and degeneration of the axon and myelin of the spinal cord and peripheral nerves. The results indicate that the nonneurotoxic chemical MiBK synergized the neurotoxic action of the weak neurotoxicant n-hexane since the coneurotoxicity coefficient for joint exposure was more than two times the additive effect of each treatment alone. In another experiment, to investigate the mechanism of MiBK synergism of n-hexane neurotoxicity, continuous inhalation for 50 days of 1000 ppm n-hexane had no effect on hen hepatic microsomal enzymes, whereas inhalation of 1000 ppm MiBK for 50 days or a mixture of 1000 ppm of each of n-hexane and MiBK for 30 days significantly induced aniline hydroxylase activity and cytochrome P-450 contents in hen liver microsomes. Liver microsomal proteins from these hens and from hens treated with beta-naphthoflavone (beta-NF) and phenobarbital (PB) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. While beta-NF increased the 55-kDa band (1408%), PB, MiBK, and MiBK/n-hexane increased the protein band (49 kDa) (258, 335, and 253%, respectively), indicating that MiBK induces chicken hepatic cytochrome P-450. The results suggest that the synergistic action of MiBK on n-hexane neurotoxicity may be related to its ability to induce liver microsomal cytochrome P-450, resulting in increased metabolic activation of n-hexane to more potent neurotoxic metabolites.

Heinz body production and hematological changes in the hen after administration of a single oral dose of n-butyl mercaptan and n-butyl disulfide.

n-Butyl mercaptan (nBM) is a breakdown product of S,S,S,-tri-n-butyl phosphorotrithioate (DEF) and S,S,S-tri-n-butyl phosphorotrithioite (merphos) in hens and in the environment. n-Butyl disulfide (nBD) is an oxidation product of nBM. A single 500 mg/kg dose of nBM and nBD was administered in gelatin capsules to groups of five 12-month old laying hens. A third group (five hens) was given gelatin capsules. One day after administration, the hens exhibited weakness which progressed to unsteadiness and inability to stand by the third day. These signs were accompanied by a pale comb 18--24 hr after dosing, which changed to dark color at 48 hr. Treated hens improved with time. Heinz bodies and extensive erythrocyte deformation and lysis were observed in blood smears taken from hens 24 and 48 hr after treatment. Hemoglobin concentration, packed cell volume, erythrocytes, and glucose-6-phosphate dehydrogenase activity were significantly lower than controls, while methemoglobin was significantly higher. As the clinical condition of these hens improved, these hematologic changes disappeared. nBM caused an initial increase in plasma butyrylcholinesterase activity which was dose-dependent and returned to normal by the end of the 28-day experiment. Also, brain acetylcholinesterase activity was not different from that of the control at termination.

Neurotoxicity of continuous (90 days) inhalation of technical grade methyl butyl ketone in hens.

Neurotoxicity was produced in 1-yr-old hens (five hens per treatment) by continual 90-d exposure in inhalation chambers to atmospheres containing 50, 100, 200, or 400 ppm technical grade methyl butyl ketone (MBK) containing 70% methyl n-butyl ketone (MnBK) and 30% methyl isobutyl ketone (MiBK). A 30-d observation period followed. Severity of clinical condition and progression or improvement of neurological deficit signs were dependent on the concentration of MBK and duration of exposure. Hens exposed to the two highest levels developed ataxia and paralysis; they died or were sacrificed before the designated exposure period ended. The intermediate level of MBK (100 ppm) caused severe ataxia; most treated hens showed no change in clinical condition during the observation period. Hens exposed to 50 ppm exhibited gross ataxia, with most demonstrating partial regression of neurological deficit after the exposure ceased. Hens exposed to the lowest tested level (10 ppm) remained normal. Only hens exposed to 400 or 200 ppm showed significant weight loss. Some hens from the 50-400 ppm treatment groups showed unequivocal histopathologic changes in the spinal cord and peripheral nerves. Severity of histopathologic changes depended on the level and duration of MBK exposure. These changes were characterized by excessive swelling, phagocytosis, degeneration, and demyelination of the axons.