Heartworm disease - Overview, intervention, and industry perspective.

Dirofilaria immitis, also known as heartworm, is a major parasitic threat for dogs and cats around the world. Because of its impact on the health and welfare of companion animals, heartworm disease is of huge veterinary and economic importance especially in North America, Europe, Asia and Australia. Within the animal health market many different heartworm preventive products are available, all of which contain active components of the same drug class, the macrocyclic lactones. In addition to compliance issues, such as under-dosing or irregular treatment intervals, the occurrence of drug-resistant heartworms within the populations in the Mississippi River areas adds to the failure of preventive treatments. The objective of this review is to provide an overview of the disease, summarize the current disease control measures and highlight potential new avenues and best practices for treatment and prevention.

World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) guidelines for studies evaluating the efficacy of parasiticides in reducing the risk of vector-borne pathogen transmission in dogs and cats.

These guidelines are intended to provide an in-depth review of current knowledge and assist the planning and implementation of studies for evaluating the efficacy of parasiticides in reducing transmission of vector-borne pathogens (VBPs) to dogs and cats. At present, the prevention of VBP transmission in companion animals is generally achieved through the administration of products that can repel or rapidly kill arthropods, thus preventing or interrupting feeding before transmission occurs. The present guidelines complement existing guidelines, which focus on efficacy assessment of parasiticides for the treatment, prevention and control of flea and tick infestations, but also give guidance for studies focused on other vectors (i.e. mosquitoes and phlebotomine sand flies). The efficacy of parasiticides in reducing VBP transmission can be evaluated through laboratory or field studies. As such, the present guidelines provide recommendations for these studies, representing a tool for researchers, pharmaceutical companies and authorities involved in the research, development and registration of products with claims for reducing VBP transmission in dogs and cats, respecting the overall principles of the 3Rs (replacement, reduction and refinement). Gaps in our current understanding of VBP transmission times are herein highlighted and the need for further basic research on related topics is briefly discussed.

Where Is the Breakthrough Innovation for Parasite Control?

The need to improve parasite control to overcome drug-resistant parasite populations, and to improve compliance by more convenient drug application methods, is evident. While a number of incremental stepwise improvements are visible, the big disruptive innovation, an iPhone-equivalent breakthrough, has been hard to find. Why?

Blocking transmission of vector-borne diseases.

Vector-borne diseases are responsible for significant health problems in humans, as well as in companion and farm animals. Killing the vectors with ectoparasitic drugs before they have the opportunity to pass on their pathogens could be the ideal way to prevent vector borne diseases. Blocking of transmission might work when transmission is delayed during blood meal, as often happens in ticks. The recently described systemic isoxazolines have been shown to successfully prevent disease transmission under conditions of delayed pathogen transfer. However, if the pathogen is transmitted immediately at bite as it is the case with most insects, blocking transmission becomes only possible if ectoparasiticides prevent the vector from landing on or, at least, from biting the host. Chemical entities exhibiting repellent activity in addition to fast killing, like pyrethroids, could prevent pathogen transmission even in cases of immediate transfer. Successful blocking depends on effective action in the context of the extremely diverse life-cycles of vectors and vector-borne pathogens of medical and veterinary importance which are summarized in this review. This complexity leads to important parameters to consider for ectoparasiticide research and when considering the ideal drug profile for preventing disease transmission.

Macrocyclic lactone resistance in Dirofilaria immitis: Failure of heartworm preventives and investigation of genetic markers for resistance.

Macrocyclic lactone (ML) endectocides are used as chemoprophylaxis for heartworm infection (Dirofilaria immitis) in dogs and cats. Claims of loss of efficacy (LOE) of ML heartworm preventives have become common in some locations in the USA. We directly tested whether resistance to MLs exists in LOE isolates of D. immitis and identified genetic markers that are correlated with, and therefore can predict ML resistance. ML controlled studies showed that LOE strains of D. immitis established infections in dogs despite chemoprophylaxis with oral ivermectin or injectable moxidectin. A whole genome approach was used to search for loci associated with the resistance phenotype. Many loci showed highly significant differences between pools of susceptible and LOE D. immitis. Based on 186 potential marker loci, Sequenom(®) SNP frequency analyses were conducted on 663 individual parasites (adult worms and microfilariae) which were phenotypically characterized as susceptible (SUS), confirmed ML treatment survivors/resistant (RES), or suspected resistant/loss of efficacy (LOE) parasites. There was a subset of SNP loci which appears to be promising markers for predicting ML resistance, including SNPs in some genes that have been associated with ML resistance in other parasites. These data provide unequivocal proof of ML resistance in D. immitis and identify genetic markers that could be used to monitor for ML resistance in heartworms.

Vaccination with recombinant paramyosin against the bovine lungworm Dictyocaulus viviparus considerably reduces worm burden and larvae shedding.

BACKGROUND: The lungworm Dictyocaulus viviparus, causing parasitic bronchitis in cattle, induces a temporary protective immunity that prevents clinical disease. A radiation-attenuated larvae based vaccine is commercially available in a few European countries, but has the disadvantages of a live vaccine. As a recombinant subunit vaccine would overcome these disadvantages, the parasite's muscle protein paramyosin (PMY) was tested as a recombinant vaccine antigen. METHODS: D. viviparus-PMY was recombinantly expressed in Escherichia coli as a glutathione-S-transferase (GST)-fused protein. Emulsified in adjuvant Saponin Quil A, the protein was given intramuscularly into calves. Two independent recombinant PMY (rPMY) vaccination trials with negative control groups (first trial: adjuvant only; second trial: non-fused GST) as well as an additional positive control group in the second trial, using the Bovilis Dictol live vaccine to verify vaccination results, were performed. To determine the vaccination success, shedding of larvae as well as worm burden and worm sizes were analyzed. Additionally, ELISA-based determination of development of immunglobulins IgM, IgA, IgE, IgG as well as the subclasses IgG1 and IgG2 was performed. To analyze PMY localization in the bovine lungworm, immunohistochemical staining of adult worms was carried out. RESULTS: Immunohistochemical staining revealed that PMY is part of the bovine lungworm's pharyngeal and body wall muscles. Vaccination with rPMY resulted in 47% [geometric mean: 67%] and 57% (geometric mean: 71%) reduction of larvae shedding in the first and second vaccination trial, respectively. Worm burden was reduced by 54% (geometric mean: 86%) and 31% (geometric mean: 68%), respectively, and worms of rPMY-vaccinated cattle were significantly shorter in both trials. Furthermore, ELISAs showed a clear antibody response towards rPMY with exception of IgE for which titers could not be detected. After challenge infection, rPMY antibodies were only exceptionally elevated among study animals indicating PMY to be a hidden antigen. CONCLUSIONS: Even though vaccination with the attenuated live vaccine was with 94% (geometric mean: 95%) reduction in larvae shedding and 93% (geometric mean: 94%) reduction in worm burden superior to rPMY vaccination, results using the latter are promising and show the potential for further development of a recombinant PMY-based vaccine against the bovine lungworm.

Recent advances in candidate-gene and whole-genome approaches to the discovery of anthelmintic resistance markers and the description of drug/receptor interactions.

Anthelmintic resistance has a great impact on livestock production systems worldwide, is an emerging concern in companion animal medicine, and represents a threat to our ongoing ability to control human soil-transmitted helminths. The Consortium for Anthelmintic Resistance and Susceptibility (CARS) provides a forum for scientists to meet and discuss the latest developments in the search for molecular markers of anthelmintic resistance. Such markers are important for detecting drug resistant worm populations, and indicating the likely impact of the resistance on drug efficacy. The molecular basis of resistance is also important for understanding how anthelmintics work, and how drug resistant populations arise. Changes to target receptors, drug efflux and other biological processes can be involved. This paper reports on the CARS group meeting held in August 2013 in Perth, Australia. The latest knowledge on the development of molecular markers for resistance to each of the principal classes of anthelmintics is reviewed. The molecular basis of resistance is best understood for the benzimidazole group of compounds, and we examine recent work to translate this knowledge into useful diagnostics for field use. We examine recent candidate-gene and whole-genome approaches to understanding anthelmintic resistance and identify markers. We also look at drug transporters in terms of providing both useful markers for resistance, as well as opportunities to overcome resistance through the targeting of the transporters themselves with inhibitors. Finally, we describe the tools available for the application of the newest high-throughput sequencing technologies to the study of anthelmintic resistance.

Worms--a "license to kill".

Worm infections can cause severe harm and death to both humans and numerous domestic and wild animals. Despite the fact that there are many beneficial worm species, veterinarians, physicians and parasitologists have multiple reasons to combat parasitic worms. The pros and cons of various approaches for the discovery of new control methods are discussed, including novel anthelmintics, vaccines and genetic approaches to identify novel drug and vaccine targets. Currently, the mainstay of worm control remains chemotherapy and prophylaxis. The importance of knowledgeable and wise use of the available anthelmintics is highlighted.

New advancement in anthelmintic drugs in veterinary medicine.

Anthelmintic treatment of nematode infections remains the mainstay of worm control in farm and companion animals. However, control is threatened by the occurrence of drug resistant nematodes. In recent years, three new anthelmintics have been introduced to the market. Here, we describe the main features including mode of action, availability, spectrum, dose, tolerability, safety, and resistance of emodepside, monepantel, and derquantel.

The genome of the heartworm, Dirofilaria immitis, reveals drug and vaccine targets.

The heartworm Dirofilaria immitis is an important parasite of dogs. Transmitted by mosquitoes in warmer climatic zones, it is spreading across southern Europe and the Americas at an alarming pace. There is no vaccine, and chemotherapy is prone to complications. To learn more about this parasite, we have sequenced the genomes of D. immitis and its endosymbiont Wolbachia. We predict 10,179 protein coding genes in the 84.2 Mb of the nuclear genome, and 823 genes in the 0.9-Mb Wolbachia genome. The D. immitis genome harbors neither DNA transposons nor active retrotransposons, and there is very little genetic variation between two sequenced isolates from Europe and the United States. The differential presence of anabolic pathways such as heme and nucleotide biosynthesis hints at the intricate metabolic interrelationship between the heartworm and Wolbachia. Comparing the proteome of D. immitis with other nematodes and with mammalian hosts, we identify families of potential drug targets, immune modulators, and vaccine candidates. This genome sequence will support the development of new tools against dirofilariasis and aid efforts to combat related human pathogens, the causative agents of lymphatic filariasis and river blindness.

Efficacy of monepantel, derquantel and abamectin against adult stages of a multi-resistant Haemonchus contortus isolate.

Drug resistance in gastrointestinal nematodes is a severe problem for sheep farmers. With the recent introduction of monepantel (Zolvix®) and of derquantel plus abamectin (Startect®) in New Zealand, two new anthelmintic classes will be available to control gastrointestinal nematodes. While monepantel covers a broad spectrum of nematodes, the efficacy of derquantel is mid-spectrum and limited to a smaller number of species and stages. The combination of derquantel and abamectin allows to enlarge the spectrum and to cover most parasitic nematodes in sheep. However, the question remained open, if the efficacy of the new anthelmintics can be maintained in the presence of severe anthelmintic resistance. The present study investigated the efficacy against adult stages of a multi-resistant Haemonchus contortus isolate. While monepantel resulted in 100 % elimination, derquantel in combination with abamectin resulted in efficacies <95 % (faecal egg counts and worm counts).

Differences in efficacy of monepantel, derquantel and abamectin against multi-resistant nematodes of sheep.

Drug resistance has become a global phenomenon in gastrointestinal nematodes of sheep, particularly resistance to macrocyclic lactones. New anthelmintics are urgently needed for both the control of infections with multi-resistant nematodes in areas where classical anthelmintics are no longer effective, and the prevention of the spread of resistance in areas where the problem is not as severe. Recently, two new active ingredients became commercially available for the treatment of nematode infections in sheep, monepantel (Zolvix®) and derquantel, the latter used only in a formulated combination with the macrocyclic lactone, abamectin (Startect®). In order to assess the potential of the new actives for the control and prevention of spread of anthelmintic resistance, two characterized multi-resistant field isolates from Australia were used in a GLP (good laboratory practice) conducted efficacy study in sheep. Eight infected sheep in each group were treated orally according to the product labels with 2.5 mg/kg body weight monepantel, 0.2 mg/kg abamectin, or with the combination of 2.0 mg/kg derquantel and 0.2 mg/kg abamectin. The results demonstrate that monepantel was fully effective against multi-resistant species, Trichostrongylus colubriformis and Haemonchus contortus (99.9%). In contrast, the combination of derquantel and abamectin was effective against T. colubriformis (99.9%), but was not effective against larval stages of the barber's pole worm H. contortus (18.3%).

Phylogenomics of ligand-gated ion channels predicts monepantel effect.

The recently launched veterinary anthelmintic drench for sheep (Novartis Animal Health Inc., Switzerland) containing the nematocide monepantel represents a new class of anthelmintics: the amino-acetonitrile derivatives (AADs), much needed in view of widespread resistance to the classical drugs. Recently, it was shown that the ACR-23 protein in Caenorhabditis elegans and a homologous protein, MPTL-1 in Haemonchus contortus, are potential targets for AAD action. Both proteins belong to the DEG-3 subfamily of acetylcholine receptors, which are thought to be nematode-specific, and different from those targeted by the imidazothiazoles (e.g. levamisole). Here we provide further evidence that Cel-ACR-23 and Hco-MPTL-1-like subunits are involved in the monepantel-sensitive phenotype. We performed comparative genomics of ligand-gated ion channel genes from several nematodes and subsequently assessed their sensitivity to anthelmintics. The nematode species in the Caenorhabditis genus, equipped with ACR-23/MPTL-1-like receptor subunits, are sensitive to monepantel (EC(50)<1.25 µM), whereas the related nematodes Pristionchus pacificus and Strongyloides ratti, which lack an ACR-23/MPTL-1 homolog, are insensitive (EC(50)>43 µM). Genome sequence information has long been used to identify putative targets for therapeutic intervention. We show how comparative genomics can be applied to predict drug sensitivity when molecular targets of a compound are known or suspected.

Efficacy, safety and pharmacokinetics of 1,2,4-trioxolane OZ78 against an experimental infection with Fasciola hepatica in sheep.

The synthetic peroxide OZ78 is an effective flukicide in the rodent model, but the potential of OZ78 in target animals has not been studied to date. In the present study, OZ78 was administered at 50mg/kg orally and subcutaneously to sheep harbouring an experimental Fasciola hepatica infection and the efficacy, tolerability and pharmacokinetic profiles were monitored. OZ78 given orally or subcutaneously revealed no effect neither on faecal egg counts nor on worm burdens. Apart from significant subcutaneous swelling at the injection sites of most of the treated animals, no other treatment related adverse events occurred. OZ78 had no significant effect on any haematological, coagulation or clinical chemistry variables tested. Following oral administration, a mean C(max) of 45.8±13 µg/ml was reached after 1h. An estimated elimination half-life of 1.0 h and a mean AUC of 116.2±47 µg min/ml was calculated for the oral administration. Following subcutaneous treatment with OZ78 C(max) and t(max) were 13.7±6.1µg/ml and 0.9±0.4h, respectively. The α and ß half-lives were 4.5±4.3 h and 56.5±36 h, respectively and the mean AUC was 219.1±74 µg min/ml. Further studies are needed to determine whether the excellent activity observed with OZ78 in the rat model can be translated into efficacy in larger mammals.

Monepantel allosterically activates DEG-3/DES-2 channels of the gastrointestinal nematode Haemonchus contortus.

Monepantel is the first drug of a new family of anthelmintics, the amino acetonitrile derivatives (AAD), presently used to treat ruminants infected with gastrointestinal nematodes such as Haemonchus contortus. Monepantel shows an excellent tolerability in mammals and is active against multidrug-resistant parasites, indicating that its molecular target is absent or inaccessible in the host and is different from those of the classic anthelmintics. Genetic approaches with mutant nematodes have suggested acetylcholine receptors of the DEG-3 subfamily as the targets of AADs, an enigmatic clade of ligand-gated ion channels that is specific to nematodes and does not occur in mammals. Here we demonstrate direct interaction of monepantel, its major active metabolite monepantel sulfone, and other AADs with potential targets of the DEG-3 subfamily of acetylcholine receptors. H. contortus DEG-3/DES-2 receptors were functionally expressed in Xenopus laevis oocytes and were found to be preferentially activated by choline, to permeate monovalent cations, and to a smaller extent, calcium ions. Although monepantel and monepantel sulfone did not activate the channels by themselves, they substantially enhanced the late currents after activation of the channels with choline, indicating that these AADs are type II positive allosteric modulators of H. contortus DEG-3/DES-2 channels. It is noteworthy that the R-enantiomer of monepantel, which is inactive as an anthelmintic, inhibited the late currents after stimulation of H. contortus DEG-3/DES-2 receptors with choline. In summary, we present the first direct evidence for interaction of AADs with DEG-3-type acetylcholine receptors and discuss these findings in the context of anthelmintic action of AADs.

Quarantine treatment of sheep with monepantel--rapidity of fecal egg count reduction.

In the presence of multiple anthelmintic resistance, effective quarantine treatment of sheep entering a farm has become a crucial tool in nematode management. In the present work, the use of the amino-acetonitrile derivative, monepantel as a quarantine treatment was investigated. Of special interest was the rapidity of decrease in fecal egg counts (FEC) compared to other anthelmintics used in sheep. In three different studies, sheep were either infected with Haemonchus contortus of known resistance status or with a panel of susceptible and multi-resistant gastro-intestinal nematodes. Fecal egg counts were determined from several hours up to 14 days after treatment with monepantel, benzimidazoles, levamisole, macrocyclic lactones or combinations of the latter three classes. The treatment of susceptible isolates with effective anthelmintics, either as single or combined applications caused a reduction of FEC to 0 within 3-4 days. The status of the resistant nematodes could be confirmed, as treatment with the affected classical anthelmintics never resulted in complete absence of parasite eggs in all samples analyzed. In the case of multi-resistant isolates, only monepantel was able to stop egg excretion, while all other treatments resulted in only a partial reduction of FEC. The resistance status of the nematodes did not influence the rapid decline of egg counts after treatment with monepantel.

The effect of sheep breed, age, and gender on the pharmacokinetics and efficacy of monepantel, an amino-acetonitrile derivative.

This analysis investigated the influence of breed and gender on the pharmacokinetics of monepantel, and influence of breed, age, and gender on its efficacy against gastrointestinal nematodes of sheep. In a comparison of pharmacokinetic profiles from two studies, Merino lambs had significantly greater maximum concentrations of monepantel and monepantel sulfone, and faster times to reach these concentrations than Dorset cross lambs. Males had a statistically greater area under the curve (0-504 h) than females for monepantel sulfone. The biological relevance of these relatively small differences is unclear because efficacy was not evaluated in these studies. For efficacy, a breed effect existed for some nematodes when sheep were treated at a sub-optimum dose (1.25 mg/kg). There were no gender effects between sheep infected with adult parasites and treated at 1.25 mg/kg but there were differences between females and males treated at this dose when infected with fourth-stage larvae of Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus colubriformis, and Cooperia curticei. There were no breed or gender differences for sheep treated at the recommended dose (2.5 mg/kg). There was a potential trend for declining efficacy with increasing animal age for fourth-stage Trichostrongylus axei. This analysis demonstrated that, similarly to what is observed with other anthelmintics, the pharmacokinetics and efficacy of monepantel can vary with factors like breed, age, and gender. Identifying these covariates is important for understanding inter-individual variability in drug response. While further investigation is warranted, correctly treating sheep at the recommended dose of 2.5 mg/kg appears to mitigate any associated risk.

A pooled analysis of the efficacy of monepantel, an amino-acetonitrile derivative against gastrointestinal nematodes of sheep.

Monepantel is the first compound from the amino-acetonitrile derivative class of anthelmintics to be developed for the control of gastrointestinal nematodes of sheep. An analysis of pooled data from a series of controlled studies is reported providing a single point of efficacy (+/- 95% confidence interval) for each gastrointestinal nematode tested at the fourth larval and/or adult stages. For most nematode species, the pooled efficacy was greater than 99%, and for the remaining few species, efficacy was greater than 90%. These data are well supported by field studies conducted across five countries, where the pooled efficacy (on the basis of fecal worm egg count reduction) was in most cases, greater than 99% (depending on the calculation used). Monepantel is highly effective when administered to sheep at 2.5 mg/kg, and its introduction as a new anthelmintic for sheep is timely, given the problems with anthelmintic resistance that the world's sheep farmers are now experiencing.

In vitro selection of Haemonchus contortus for benzimidazole resistance reveals a mutation at amino acid 198 of beta-tubulin.

Benzimidazoles were the first broad-spectrum anthelmintics and are still in use today against gastro-intestinal nematodes of ruminants such as Haemonchus contortus. Benzimidazoles block the polymerization of nematode microtubules. However, their efficacy is jeopardized by the spread of drug-resistant parasites that carry point mutations in beta-tubulin. Here we use a novel in vitro selection-in vivo propagation protocol to breed drug-resistant H. contortus. After 8 generations of selection with thiabendazole an in vitro resistance factor of 1000 was reached that was also relevant in vivo in infected sheep. The same procedure carried out with ivermectin produced only a moderate resistance phenotype that was not apparent in sheep. Cloning and sequencing of the beta-tubulin genes from the thiabendazole-resistant H. contortus mutants revealed all of the isotype 1 alleles, and part of the isotype 2 alleles, to carry the mutation glutamate(198) to alanine (E198A). An allele-specific PCR was developed, which may be helpful in monitoring the prevalence of alanine(198) encoding alleles in the beta-tubulin isotype 1 gene pool of H. contortus in the field.

Standardization of the egg hatch test for the detection of benzimidazole resistance in parasitic nematodes.

The ability to reliably detect anthelmintic resistance is a crucial part of resistance management. If data between countries are to be compared, the same test should give the same results in each laboratory. As the egg hatch test for benzimidazole resistance is used for both research and surveys, the ability of different laboratories to obtain similar results was studied through testing of known isolates of cyathostomins, Haemonchus contortus, Ostertagia ostertagi, and Cooperia oncophora in programs supported by the EU (Cost B16 and FP6-PARASOL). Initial results showed difficulties in obtaining reproducible and similar data within and between laboratories. A series of ring tests, i.e., simultaneous and coordinated rounds of testing of nematode isolates in different laboratories was subsequently performed. By adopting identical protocols, especially the use of deionized water and making dilutions of thiabendazole in dimethyl sulfoxide in the final ring test, laboratories correctly identified both susceptible and resistant isolates. The protocols for the test and preparation of solutions of thiabendazole are described.