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.

RNA interference in the cat flea, Ctenocephalides felis: Approaches for sustained gene knockdown and evidence of involvement of Dicer-2 and Argonaute2.

Effective RNA interference (RNAi) methods have been developed in many pest species, enabling exploration of gene function. Until now RNAi had not been attempted in the cat flea, Ctenocephalides felis, although the development of RNAi approaches would open up potential avenues for control of this important pest. This study aimed to establish if an RNAi response occurs in adult C. felis upon exposure to double-stranded RNA (dsRNA), which administration methods for dsRNA delivery could bring about effective gene knockdown and to investigate dynamics of any RNAi response. Knockdown of 80% of GSTσ was achieved by intrahaemoceolic microinjection of dsGSTσ but this invasive technique was associated with relatively high mortality rates. Immersing C. felis in dsGSTσ or dsDicer-2 overnight resulted in 65% knockdown of GSTσ or 60% of Dicer-2, respectively, and the degree of knockdown was not improved by increasing the dsRNA concentration in the bathing solution. Unexpectedly, the greatest degree of knockdown was achieved with the continuous administration of dsRNA in whole blood via a membrane feeding system, resulting in 96% knockdown of GSTσ within 2 days and sustained up to, at least, 7 days. Thus, unlike in many other species, the gut nucleases do not impair the RNAi response to ingested dsRNA in C. felis. A modest, but significant, upregulation of Dicer-2 and Argonaute2 was detectable 3 h after exposure to exogenous dsRNA, implicating the short-interfering RNA pathway. To our knowledge this study represents the first demonstration of experimentally induced RNAi in the cat flea as well as giving insight into how the gene knockdown response progresses.

Reference gene selection and RNA preservation protocol in the cat flea, Ctenocephalides felis, for gene expression studies.

The cat flea, Ctenocephalides felis, is a major pest species on companion animals thus of significant importance to the animal health industry. The aim of this study was to develop sampling and storage protocols and identify stable reference genes for gene expression studies to fully utilize the growing body of molecular knowledge of C. felis. RNA integrity was assessed in adult and larvae samples, which were either pierced or not pierced and stored in RNAlater at ambient temperature. RNA quality was maintained best in pierced samples, with negligible degradation evident after 10 days. RNA quality from non-pierced samples was poor within 3 days. Ten candidate reference genes were evaluated for their stability across four group comparisons (developmental stages, genders, feeding statuses and insecticide-treatment statuses). Glyceraldehyde 3 phosphate dehydrogenase (GAPDH), 60S ribosomal protein L19 (RPL19) and elongation factor-1α (Ef) were ranked highly in all stability comparisons, thus are recommended as reference genes under similar conditions. Employing just two of these three stable reference genes was sufficient for accurate normalization. Our results make a significant contribution to the future of gene expression studies in C. felis, describing validated sample preparation procedures and reference genes for use in this common pest.

Discovery of sarolaner: A novel, orally administered, broad-spectrum, isoxazoline ectoparasiticide for dogs.

The novel isoxazoline ectoparasiticide, sarolaner, was identified during a lead optimization program for an orally-active compound with efficacy against fleas and ticks on dogs. The aim of the discovery program was to identify a novel isoxazoline specifically for use in companion animals, beginning with de novo synthesis in the Zoetis research laboratories. The sarolaner molecule has unique structural features important for its potency and pharmacokinetic (PK) properties, including spiroazetidine and sulfone moieties. The flea and tick activity resides in the chirally pure S-enantiomer, which was purified to alleviate potential off-target effects from the inactive enantiomer. The mechanism of action was established in electrophysiology assays using CHO-K1 cell lines stably expressing cat flea (Ctenocephalides felis) RDL (resistance-to-dieldrin) genes for assessment of GABA-gated chloride channel (GABACls) pharmacology. As expected, sarolaner inhibited GABA-elicited currents at both susceptible (CfRDL-A285) and resistant (CfRDL-S285) flea GABACls with similar potency. Initial whole organism screening was conducted in vitro using a blood feeding assay against C. felis. Compounds which demonstrated robust activity in the flea feed assay were subsequently tested in an in vitro ingestion assay against the soft tick, Ornithodoros turicata. Efficacious compounds which were confirmed safe in rodents at doses up to 30mg/kg were progressed to safety, PK and efficacy studies in dogs. In vitro sarolaner demonstrated an LC80 of 0.3µg/mL against C. felis and an LC100 of 0.003µg/mL against O. turicata. In a head-to-head comparative in vitro assay with both afoxolaner and fluralaner, sarolaner demonstrated superior flea and tick potency. In exploratory safety studies in dogs, sarolaner demonstrated safety in dogs≥8 weeks of age upon repeated monthly dosing at up to 20mg/kg. Sarolaner was rapidly and well absorbed following oral dosing. Time to maximum plasma concentration occurred within the first day post-dose. Bioavailability for sarolaner was calculated at >85% and the compound was highly protein bound (>99.9%). The half-life for sarolaner was calculated at 11-12 days. Sarolaner plasma concentrations indicated dose proportionality over the range 1.25-5mg/kg, and these same doses provided robust efficacy (>99%) for ≥35days against both fleas (C. felis) and multiple species of ticks (Rhipicephalus sanguineus, Ixodes ricinus and Dermacentor reticulatus) after oral administration to dogs. As a result of these exploratory investigations, sarolaner was progressed for development as an oral monthly dose for treatment and control of fleas and ticks on dogs.

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.

The discovery of isoxazoline oxime ethers as a new class of ectoparasiticides for the control of Haematobia irritans (horn fly) in cattle.

Haematobia irritans (horn fly) infestation in cattle is responsible for over a billion dollars a year in global economic loss due to decreased milk production and lower feed conversion. There is significant need for new insecticidal agents since current treatments such as organophosphates and pyrethroids suffer from field resistance. Isoxazoline oxime ethers represent a new class of γ-aminobutyric acid (GABA) receptor channel blockers which show good activity (LD(90) = 1.0 µg/mL) against horn flies in an in vitro feed assay and have demonstrated efficacy (>90% reduction at 1.0mg/kg) as a topical treatment in a field study.

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.