ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 1979, p. 372-378 Vol. 15, No. 3 0066-4804/79/03-0372/07$02.00/0 Avermectins, New Family of Potent Anthelmintic Agents: Efficacy of the Bla Component Downloaded from J. R. EGERTON,* D. A. OSTLIND, L. S. BLAIR, C. H. EARY, D. SUHAYDA, S. CIFELLI, R. F. RIEK, AND W. C. CAMPBELL Merck Institute for Therapeutic Research, Rahway, New Jersey 07065 Received for publication 15 November 1978 When given to sheep as a single oral dose at 0.1 mg/kg, the Bia component of http://aac.asm.org/ the avermectins caused a reduction of >95% in the numbers of Haemonchus contortus, Ostertagia circumcincta (including inhibited L4 larvae), Tricho- strongylus axei, Trichostrongylus colubriformis, Cooperia oncophora, and Oe- sophagostomum columbianum. When given to cattle as a single oral dose at 0.1 mg/kg, avermectin B1a was >95% effective in reducing the numbers of Haemon- chus placei, Ostertagia ostertagi (including inhibited L4 larvae), T. axei, T. colubriformis, C. oncophora, Cooperia punctata, Oesophagostomum radiatum, and Dictyocaulus viviparus. Avermectin B1, was similarly effective, with the exception of a detectable loss in activity against adult C. oncophora, when on January 14, 2016 by Washington University in St. Louis administered to cattle as a parenteral injection. Some of these ruminant parasites were fully susceptible to dosages of avermectin Bia at 0.025 mg/kg, e.g., D. viviparus, 0. radiatum, 0. ostertagi, and H. contortus. Avermectin B18 removed 83 to 100% of Ancylostoma caninum from dogs given a single oral dose of 0.003 to 0.005 mg/kg. The poultry nematodes Capillaria obsignata and immature Ascaridia galli were effectively remoyied by avermectin B1i at 0.05 and 0.1 mg/kg, respectively, but 0.1 mg/kg was not effective for Heterakis gallinarum. Thus, the avermectins would appear to have unprecedented potency and spectrum of biological activity. Fermentation-derived natural products which briformis; day 18, 7,500 Trichostrongylus axei and are attributed with anthelmintic activity have 7,500 Cooperia oncophora; day 21, 2,000 Haemonchus been described in the literature. Hygromycin B, contortus. The isolates of T. colubriformis and H. which has been used in treating domestic ani- contortus used to infect sheep were tolerant to benz- was 1958 imidazole anthelmintic treatment, requiring more than mals in practice, described in (8). It the recommended use level of various benzimidazoles was shown to have some effect upon adult swine for high-order efficacy. Numbers of larvae were esti- ascarids when fed to infected pigs (7) but found mated by counting multiple samples of larval suspen- ineffective against the migrating larvae of As- sions in tap water, and each animal was infected per caris suum (6). The principal utility of hygro- os with 5 ml oflarval suspension containing the desired mycin B appears directed against members of number. the nematode order Ascaroidea. In this paper On day 35, when the infections were patent, 15 we report the anthelmintic activity of a natural infected sheep were randomly allotted to five treat- product, the B18 component of the avermectins ment groups of three sheep each. The groups were (3, 9), an a-L-oleandrosyl-a-L-oleandroside ma- randomly allotted to infected control (no treatment, two groups) or treatment with avermectin B,I at 0.1, crocyclic lactone, against nematode parasites of 0.05, or 0.025 mg/kg. Treatments were administered cattle, sheep, dogs, and chickens. as solutions of avermectin B,a in sesame oil at the rate of 0.1 ml/kg of body weight as an oral drench. On day MATERIALS AND METHODS 42, 7 days after treatment, the treated and untreated Sheep. Sheep raised under "parasite-free" condi- infected control sheep were killed, and their residual tions (i.e., management conditions designed to ensure worm burdens were recovered at necropsy. The ana- minimal exposure to helminth infection) and weighing tomical components of the gastrointestinal tract of between 14 and 25 kg at the time of treatment were each sheep were individually separated and slit open, experimentally infected with third-stage infective lar- and the contents were washed into separate con- vae ofsix nematode species, passaged in the laboratory tainers. The mucosal surface was thoroughly washed as pure isolates, according to the following: day 0, 360 under warm tap water, and the washings were added Oesophagostomum columbianum; day 16, 7,500 Oster- to the appropriate container. Formaldehyde solution tagia circumcincta and 7,500 Trichostrongylus colu- was added as a preservative. To recover histotropic 372 VOL. 15, 1979 AVERMECTINS: EFFICACY OF B18 COMPONENT 373 parasitic larvae, the abomasum was placed in 1 liter of groups of three calves each as for experiment 1. The 0.85% (wt/vol) sodium chloride containing 600,000 U calves were exposed to infection as described for sheep of procaine penicillin G plus 0.75 g of dihydrostrepto- as follows: day 0, 1,000 0. radiatum; day 11, 2,000 D. mycin sulfate per liter and held at 37°C for 18 to 20 h. viviparus; day 16, 5,000 H. placei, 15,000 0. ostertagi, The saline was then poured onto a 200-mesh screen and 15,000 C. oncophora; day 18, 10,000 T. axei and (0.074-mm aperture), and the mucosal surface was 10,000 T. colubriformis. On days 42 to 44, calves were Downloaded from thoroughly washed under warm running tap water treated with avermectin B18 at dosages of 0.1, 0.05, or onto the screen. The material caught on the screen 0.025 mg/kg administered as subcutaneous or intra- was washed onto a container and preserved with form- muscular injections of solutions in isopropyl myristate aldehyde solution. at the rate of 0.025 ml/kg. Injection sites were in the Estimates of the residual worm burdens were made neck midway between the jaw and shoulder and on the basis of microscopic examination of 10% of the slightly above the cervical vertebrae. The treated and individual preserved abomasal content, abomasal untreated infected control calves were killed 7 days soak, and small intestinal content. The 10% samples after treatment (days 49 to 51) and examined for for each gut portion were obtained by suspending the residual burdens of gastrointestinal nematodes and total preserved material in 2 liters of tap water by lungworm as described above. http://aac.asm.org/ mechanical agitation and withdrawing four 50-ml sam- (iii) Experiment 3: oral activity against im- ples. The total content and washings of the large mature nematodes of cattle. Sixteen Jersey calves intestine and cecum were examined for worms. All raised parasite-free and weighing between 75 and 125 nematodes were identified as to species and stage of kg were allotted to five groups of three calves each development and enumerated as they were encoun- (the extra median calf was allotted at random to one tered. group designated as untreated infected control) by Cattle. (i) Experiment 1: oral treatment for restricted randomization based on body weight. On adult nematodes in cattle. Twelve Jersey calves day 0, each calf was exposed to infection by oral raised under parasite-free conditions, weighing be- administration of 5 ml of tap water containing 7,500 tween 80 and 150 kg at time time of treatment, were H. placei, 15,000 0. ostertagi, 10,000 T. axei, 10,000 T. on January 14, 2016 by Washington University in St. Louis experimentally infected with third-stage infective lar- colubriformis, 10,000 C. oncophora, 10,000 Cooperia vae, derived from pure isolates of seven species of punctata, 1,000 0. radiatum, and 2,000 D. viviparus nematode parasites of cattle. The calves were allotted infective third-stage larvae. At 8 days postinfection, to four groups of three calves each by restricted ran- two randomly selected groups of three calves each domization based on body weight at the time of initial were treated with avermectin B18 as a single oral dose infection, and each was exposed to infection, as de- of a solution in sesame oil at 0.022 or 0.011 mg/kg at scribed for sheep, as follows: day 0, 1,000 Oesophago- a volume of 0.1 ml of solution per kg. At 15 days stomum radiatum; day 18, 2,000 Dictyocaulus vivi- postinfection, the remaining two groups ofthree calves parus; day 22, 14,000 Ostertagia ostertagi, 15,000 C. each were treated similarly. At 35 days postinfection, oncophora, and 4,000 Haemonchus placei; day 24, the calves were killed and examined for residual worm 10,000 Trichostrongylus axei and 10,000 T. colubrifor- burdens as previously described. mis. On days 49 to 51, one calf from each of the four Dogs. Purebred beagle dogs, weighing 4 to 8 kg, groups was randomly selected for treatment with av- were inoculated subcutaneously with an estimated 413 ermectin B18 (three calves per day) or retained as an Ancylostoma caninum larvae each. Treatment was untreated infected control (one calf per day). Aver- administered at 2 months after inoculation, the mat- mectin Bi. was administered as a single oral dose as a uration of the hookworms having first been demon- solution in sesame oil, 0.1 ml/kg of body weight, at strated in all dogs by the finding of hookworm eggs in dosage levels of 0.1, 0.05, and 0.025 mg/kg, each to one the feces. Treatment was given as a single oral dose calf for each treatment day. On days 56 to 58, at 7 days and consisted of avermectin B18 dissolved in a mixture after treatment, the calves were killed and examined of two parts of dimethylsulfoxide and one part of for residual worm burdens as described for sheep, polyethylene glycol in a volume of 0.1 ml/kg of body except that the abomasa were soaked in 2 liters of weight.