Cryogenic preservation of Dirofilaria immitis microfilariae, reactivation and completion of the life-cycle in the mosquito and vertebrate hosts.

BACKGROUND: The cryopreservation of filarial nematodes has been studied for nearly 70 years. Largely, these studies examined the effectiveness of cryopreservation methods by using the post-thaw survival of microfilariae (mf) and the development to third-stage larvae (L3s) following inoculation into a competent insect vector. Only one study reported complete reestablishment of a filarial nematode (Brugia malayi) life-cycle in a competent vertebrate host from cryopreserved stock. Expanding on this previous research, a cryopreservation method was developed to cryopreserve the mf of the dog heartworm, Dirofilaria immitis. METHODS: A combination of cryoprotectants, dimethyl sulfoxide (DMSO) and polyvinyl pyrolidone (PVP) at 6% and 4 mM, respectively, provided acceptable post-thaw survival of mf that developed into L3s in Aedes aegypti. L3s developed from cryopreserved and freshly collected mf in mosquitoes were inoculated into ferrets and dogs and were assessed after a sufficient duration post-inoculation for development into adult heartworms. RESULTS: Fewer adult heartworms derived from cryopreserved stocks of mf were recovered from ferrets compared to adult heartworms derived from freshly collected mf, and the former were smaller by weight and length. The onset of patency (circulating mf) occurred at similar post-inoculation time points and at similar mf densities in dogs infected with L3s sourced from cryopreserved stocks or freshly collected mf. Adults derived from cryopreserved mf have survived and produced viable mf for more than 3 years in dogs. Approximately 60% of inoculated L3s were recovered as adults from dogs at 2 and 3.5 years post-inoculation. CONCLUSIONS: The results from these direct comparisons demonstrate that cryopreserved mf can develop into L3s in vector mosquitoes and that these L3s are infective to both dogs and ferrets, where they undergo normal development into adult worms. These worms are able to mate and produce viable mf and complete the heartworm lifecycle in dog.

Design and synthesis of sarolaner, a novel, once-a-month, oral isoxazoline for the control of fleas and ticks on dogs.

Over the last decade, the isoxazoline motif has become the intense focus of crop protection and animal health companies in their search for novel pesticides and ectoparasiticides. Herein we report the discovery of sarolaner, a proprietary, optimized-for-animal health use isoxazoline, for once-a-month oral treatment of flea and tick infestation on dogs.

Anthelmintic paraherquamides are cholinergic antagonists in gastrointestinal nematodes and mammals.

Oxindole alkaloids in the paraherquamide/marcfortine family exhibit broad-spectrum anthelmintic activity that includes drug-resistant strains of nematodes. Paraherquamide (PHQ), 2-deoxoparaherquamide (2DPHQ), and close structural analogs of these compounds rapidly induce flaccid paralysis in parasitic nematodes in vitro, without affecting adenosine triphosphate (ATP) levels. The mechanism of action of this anthelmintic class was investigated using muscle tension and microelectrode recording techniques in isolated body wall segments of Ascaris suum. None of the compounds altered A. suum muscle tension or membrane potential. However, PHQ blocked (when applied before) or reversed (when applied after) depolarizing contractions induced by acetylcholine (ACh) and the nicotinic agonists levamisole and morantel. These effects were mimicked by the nicotinic ganglionic blocker mecamylamine, suggesting that the anthelmintic activity of PHQ and marcfortines is due to blockade of cholinergic neuromuscular transmission. The effects of these compounds were also examined on subtypes of human nicotinic ACh receptors expressed in mammalian cells with a Ca2+ flux assay. 2DPHQ blocked nicotinic stimulation of cells expressing alpha3 ganglionic (IC50 approximately 9 microm) and muscle-type (IC50 approximately 3 microm) nicotinic cholinergic receptors, but was inactive at 100 microm vs. the alpha7 CNS subtype. PHQ anthelmintics are nicotinic cholinergic antagonists in both nematodes and mammals, and this mechanism appears to underlie both their efficacy and toxicity.

Marcfortine and paraherquamide class of anthelmintics: discovery of PNU-141962.

Three distinct chemical classes for the control of gastrointestinal nematodes are available: benzimidazoles, imidazothiazoles, and macrocyclic lactones. The relentless development of drug resistance has severely limited the usefulness of such drugs and the search for a new class of compounds preferably with a different mode of action is an important endeavor. Marcfortine A (1), a metabolite of Penicillium roqueforti, is structurally related to paraherquamide A (2), originally isolated from Penicillium paraherquei. Chemically the two compounds differ only in one ring; in marcfortine A, ring G is six-membered and carries no substituents, while in paraherquamide A, ring G is five-membered with methyl and hydroxyl substituents at C14. Paraherquamide A (2) is superior to marcfortine A as a nematocide. 2-Desoxoparaherquamide A (PNU-141962, 53) has excellent nematocidal activity, a superior safely profile, and is the first semi-synthetic member of this totally new class of nematocides that is a legitimate candidate for development. This review describes the chemistry, efficacy and mode of action of PNU-141962.