Surveillance of Ancylostoma caninum in naturally infected dogs in Quebec, Canada, and assessment of benzimidazole anthelmintics reveal a variable efficacy with the presence of a resistant isolate in imported dogs.

Ancylostoma caninum is a widely prevalent parasitic nematode in dogs across the world. There has been a notable increase in reports of anthelmintic resistance in A. caninum within the United States of America in recent years, which has led us to investigate the potential of this scenario in Canada. The study objectives were to assess the prevalence of A. caninum in two different groups, including a colony of rescued dogs in Canada and three imported Greyhound dogs from USA, and to evaluate the efficacy of two benzimidazole (BZ) anthelmintics against A. caninum, complemented with a molecular genetic analysis adapted to low prevalence. Fecal samples were collected at pre- and post-treatment with fenbendazole for the native shelters-origin group, and a combination of anthelmintic formulations, including the pro-BZ febantel for the USA-origin group. The coprology analyses found several genera of internal parasites. Canine ancylostomiasis was the most prevalent parasitosis with 30.77% in the native group and 100% in the USA group, but with overall low average of A. caninum eggs per gram. Through the fecal egg count reduction test (FECRT), applying a cut-off at 90% as baseline of egg reduction for successful efficacy, BZ showed variable efficacy. Furthermore, molecular analysis confirmed the presence of A. caninum in both groups of dogs and found differences in the genetics linked to BZ resistance on the A. caninum ß-tubulin isotype 1 gene. In the isolate from the native group, both codons 167 and 200 were homozygous without the presence of single nucleotide polymorphism (SNP). In contrast, the selected isolate from the USA group, showed a homozygous allele at position 200 and a heterozygous SNP at position 167. The latter was congruent with the low efficacy in FECRT and agrees with the recent findings of USA A. caninum isolate resistant phenotype to the BZ anthelmintics. The limitations of the study include an overall low eggs-per-gram in both canine groups, and the shortage of additional fecal samples from the USA group, restraining the molecular analysis only to one out of the three Greyhounds. This study provided some insights on the efficacy of BZs against A. caninum and revealed the presence of BZ resistant isolates in imported dogs in Quebec, Canada. All this information should be considered, for choosing the best strategy in the control of A. caninum using anthelmintic drugs.

Inactivating Effects of Common Laboratory Disinfectants, Fixatives, and Temperatures on the Eggs of Soil Transmitted Helminths.

Soil-transmitted helminths (STH) are important and widespread intestinal pathogens of humans and animals. It is presently unknown which inactivating procedures may be universally effective for safe transport, preservation, and disinfection of STH-contaminated specimens, and this lack of knowledge may expose laboratory staff to higher risk of laboratory-acquired infections (LAI's). There are limited data on the efficacy of commonly used disinfectants and fecal fixatives for inactivating the eggs of STH. This work tested five disinfectants for surface cleanup, four storage temperature conditions, and six transport/storage fixatives, to inactivate eggs of three species of STH of animal origin (Ascaris suum "roundworm," Trichuris vulpis "whipworm" and Ancylostoma caninum "hookworm") as surrogates for human STH. Among disinfectants, exposure to 10% povidone-iodine for ≥5 min inactivated 100% of the three species tested, while 5 min exposure to 95% ethanol inactivated T. vulpis and A. caninum eggs. All of the fixatives tested had inactivation effects on A. caninum hookworm eggs within 24 h of exposure, except potassium dichromate, which required 48 h. 95% ethanol for ≥48 h inactivated eggs from all three STH species. Freezing at ≤-20°C for ≥24 h inactivated eggs of T. vulpis and A. caninum, but only freezing at -80°C for ≥24 h inactivated >99% eggs, including A. suum. This work provides an evidence base for health and safety guidelines and mitigation strategies for the handling, storage, and disposal of stool samples containing STH eggs in laboratory, health care, childcare, or veterinary settings. IMPORTANCE This study systematically evaluates common laboratory disinfectants and storage conditions for their effectiveness in inactivating the infective stages of soil-transmitted helminths (STH). Animal-infecting proxy species were chosen to represent three major groups of STH that infect humans: roundworms, whipworms, and hookworms. Previously published work in this area typically focuses on a particular inactivation method, either for a single STH species, or on a subset of closely related species. Because prediagnostic fecal specimens must be regarded as potentially infectious with a mix of species, such information may be of limited utility in a working laboratory. We provide a straightforward summary of storage and disinfection methods that can achieve complete inactivation across a range of STH species, which represents a significant advance for clinical, veterinary and research laboratory biosafety.

Anthelmintic Activity and Cytotoxic Effects of Compounds Isolated from the Fruits of Ozoroa insignis Del. (Anacardiaceae).

Ozoroa insignis Del. is an ethnobotanical plant widely used in traditional medicine for various ailments, including schistosomiasis, tapeworm, and hookworm infections. From the so far not investigated fruits of Ozoroa insignis, the anthelmintic principles could be isolated through bioassay-guided isolation using Caenorhabditis elegans and identified by NMR spectroscopic analysis and mass spectrometric studies. Isolated 6-[8(Z)-pentadecenyl] anacardic (1), 6-[10(Z)-heptadecenyl] anacardic acid (2), and 3-[7(Z)-pentadecenyl] phenol (3) were evaluated against the 5 parasitic organisms Schistosoma mansoni (adult and newly transformed schistosomula), Strongyloides ratti, Heligmosomoides polygyrus, Necator americanus, and Ancylostoma ceylanicum, which mainly infect humans and other mammals. Compounds 1-3 showed good activity against Schistosoma mansoni, with compound 1 showing the best activity against newly transformed schistosomula with 50% activity at 1µM. The isolated compounds were also evaluated for their cytotoxic properties against PC-3 (human prostate adenocarcinoma) and HT-29 (human colorectal adenocarcinoma) cell lines, whereby compounds 2 and 3 showed antiproliferative activity in both cancer cell lines, while compound 1 exhibited antiproliferative activity only on PC-3 cells. With an IC50 value of 43.2 µM, compound 3 was found to be the most active of the 3 investigated compounds.

Effectiveness of a novel orally administered combination drug product containing milbemycin oxime and lotilaner (Credelio® Plus) for the treatment of larval and immature adult stages of Ancylostoma caninum in experimentally infected dogs.

BACKGROUND: The hookworm, Ancylostoma caninum, is a common and important zoonotic intestinal nematode parasite that infects dogs globally. Both the immature and adult stages of A. caninum ingest large volumes of blood during the feeding process and can cause severe anemia and death in young dogs, even before patent infections can be diagnosed using routine faecal examination methods. Thus, effective treatment of any pre-patent stages of immature hookworms can reduce or eliminate the risk of clinical disease in infected dogs and additionally reduce environmental contamination of eggs and infective larvae. Two randomized, blinded, GCP-compliant, pivotal laboratory dose confirmation studies were conducted to evaluate the effectiveness and safety of a new novel combination of lotilaner and milbemycin oxime tablets (Credelio Plus®) administered orally to dogs experimentally infected with immature (L4 and immature adult [L5]) stages of A. caninum. METHODS: Treatments using the intended global commercial tablet formulation of Credelio Plus were administered in a time frame relative to inoculation with infective larvae so that effectiveness could be assessed against each specific immature stage of A. caninum. In each study, dogs were randomized to one of six (study 1) or four (study 2) treatment groups. Each treatment group contained 8 (study 1) or 10 (study 2) dogs that had been experimentally inoculated with infective A. caninum larvae on day 0 and were dosed once on day 7 or day 11. Enrolled subjects were administered placebo tablets, Credelio Plus tablets, or lotilaner mono tablets to provide minimum dosages of 0.75 mg/kg of milbemycin oxime and 20 mg/kg of lotilaner. All dogs were necropsied 5 days after their respective treatment. All nematodes recovered from the gastrointestinal tract at necropsy were counted by species and stage. RESULTS: For both dose confirmation studies and based on geometric mean worm counts, efficacy of Credelio Plus was ≥ 97.3% against L4 larval stage of A. caninum and ≥ 98.7% against immature adult (L5) A. caninum. CONCLUSIONS: These studies demonstrated that the orally administered Credelio Plus combination tablet was highly efficacious in treating immature (L4 and immature adult [L5]) stages of A. caninum in experimentally infected dogs.

Ketamine can be produced by Pochonia chlamydosporia: an old molecule and a new anthelmintic?

BACKGROUND: Infection by nematodes is a problem for human health, livestock, and agriculture, as it causes deficits in host health, increases production costs, and incurs a reduced food supply. The control of these parasites is usually done using anthelmintics, which, in most cases, have not been fully effective. Therefore, the search for new molecules with anthelmintic potential is necessary. METHODS: In the present study, we isolated and characterized molecules from the nematophagous fungus Pochonia chlamydosporia and tested these compounds on three nematodes: Caenorhabditis elegans; Ancylostoma ceylanicum; and Ascaris suum. RESULTS: The ethyl acetate extract showed nematicidal activity on the nematode model C. elegans. We identified the major substance present in two sub-fractions of this extract as ketamine. Then, we tested this compound on C. elegans and the parasites A. ceylanicum and A. suum using hamsters and mice as hosts, respectively. We did not find a difference between the animal groups when considering the number of worms recovered from the intestines of animals treated with ketamine (6 mg) and albendazole (P > 0.05). The parasite burden of larvae recovered from the lungs of mice treated with ketamine was similar to those treated with ivermectin. CONCLUSIONS: The results presented here demonstrate the nematicidal activity of ketamine in vitro and in vivo, thus confirming the nematicidal potential of the molecule present in the fungus P. chlamydosporia may consist of a new method of controlling parasites.

Nematicidal and ovicidal activity of Bacillus thuringiensis against the zoonotic nematode Ancylostoma caninum.

Ancylostoma caninum is a gastrointestinal parasite that affect dogs and humans, considered a worldwide public health problem. The control of these parasitosis is increasingly difficult due to the development of multi-drug resistance. Bacillus thuringiensis is an insecticidal, spore forming bacterium, often species specific. The strain GP526 of B. thuringiensis has toxic effect on the cestode Dipylidium caninum and the trematode Centrocestus formosanus, both of them zoonotic parasites. The high degrees of specificity and environment safe make B. thuringiensis suitable for use against pathogen parasites, especially those resistant to synthetic chemical insecticides. The objective of the current work was to evaluate the in vitro effect of B. thuringiensis on Ancylostoma caninum. Spore-crystal mixture of the strains was co-incubated with 120 adult nematodes (males, non-pregnant females and pregnant females) or with 4800 eggs. GP526 showed a nematicidal effect with an LT50 of 35.8 h and an LC50 of 60 µg/ml. It also showed an ovicidal effect with an LC50 of 94.9 µg/ml. Histological analyses showed detachment of the cuticle and of the uterus in adult females, and vacuolization with destruction of the eggs. The effects of GP526 strain were comparable to those of albendazole, allowing us to propose GP526 for A. caninum control, in both, the adult stage at a gastrointestinal level, and in egg stage in the environment. In addition, GP526 can be proposed as a potential broad spectrum antiparasitic drug.

Anthelmintic Activity of Yeast Particle-Encapsulated Terpenes.

Soil-transmitted nematodes (STN) infect 1-2 billion of the poorest people worldwide. Only benzimidazoles are currently used in mass drug administration, with many instances of reduced activity. Terpenes are a class of compounds with anthelmintic activity. Thymol, a natural monoterpene phenol, was used to help eradicate hookworms in the U.S. South circa 1910. However, the use of terpenes as anthelmintics was discontinued because of adverse side effects associated with high doses and premature stomach absorption. Furthermore, the dose-response activity of specific terpenes against STNs has been understudied. Here we used hollow, porous yeast particles (YPs) to efficiently encapsulate (>95%) high levels of terpenes (52% w/w) and evaluated their anthelmintic activity on hookworms (Ancylostoma ceylanicum), a rodent parasite (Nippostrongylus brasiliensis), and whipworm (Trichuris muris). We identified YP-terpenes that were effective against all three parasites. Further, YP-terpenes overcame albendazole-resistant Caenorhabditis elegans. These results demonstrate that terpenes are broad-acting anthelmintics. Terpenes are predicted to be extremely difficult for parasites to resist, and YP encapsulation provides water-suspendable terpene materials without surfactants and sustained terpene release that could lead to the development of formulations for oral delivery that overcome fast absorption in the stomach, thus reducing dosage and toxic side effects.

Multiple drug resistance in the canine hookworm Ancylostoma caninum: an emerging threat?

BACKGROUND: The canine hookworm, Ancylostoma caninum is the most prevalent and important intestinal nematode parasite of dogs in the USA. Hookworms are typically well controlled by treatment with all commonly used anthelmintics that are approved for this use in dogs. However, in the past few years, cases of recurrent/persistent canine hookworm infections appear to have dramatically increased, suggesting that anthelmintic resistance (AR) may have evolved in this parasite. These cases are highly overrepresented by greyhounds, but multiple other breeds are also represented. The aim of this study was to characterize several of these suspected resistant isolates using in vitro, genetic and clinical testing to determine if these cases represent true anthelmintic resistance in A. caninum. METHODS: Fecal samples containing hookworm eggs from three cases of persistent hookworm infections; one from a greyhound, one from a miniature schnauzer and one from a hound-mix, were received by our laboratory. These were then used to establish infections in laboratory dogs and to perform egg hatch assays (EHA) and larval development assays (LDA) for detecting resistance to benzimidazoles and macrocyclic lactones, respectively. Additional EHA and LDA were performed on eggs recovered from the laboratory-induced infections. Fecal egg count reduction tests were performed to detect resistance to pyrantel. Deep amplicon sequencing assays were developed to measure the frequency of non-synonymous single nucleotide polymorphisms (SNP) at codons 167, 198 and 200 of the A. caninum isotype-1 ß-tubulin gene. RESULTS: Resistance ratios for the three A. caninum isolates tested ranged from 6.0 to > 100 and 5.5 to 69.8 for the EHA and LDA, respectively. Following treatment with pyrantel, reduction in faecal egg counts was negative or 0%. Deep amplicon sequencing of the isotype-1 ß-tubulin gene identified a high frequency of resistance-associated SNPs at codon 167 in all three resistant isolates and in two additional clinical cases. CONCLUSIONS: These data conclusively demonstrate multiple anthelmintic resistance in multiple independent isolates of A. caninum, strongly suggesting that this is an emerging problem in the USA. Furthermore, evidence suggest that these resistant hookworms originate from racing greyhound farms and kennels, though additional research is needed to confirm this.

Efficacy of single topical treatment of Selamectin (Revolution®) against Ancylostoma ceylanicum in experimentally infected cats.

Ancylostoma ceylanicum, a species of hookworm, is a common parasite of dogs and cats in the Asia-Pacific region. The objective of this study was to determine the efficacy of a single topical treatment of selamectin (Revolution®) against A. ceylanicum in experimentally infected cats. 12 kittens were injected with 300 infective stage larvae (L3) of A. ceylanicum by the subcutaneous route. Faecal samples were examined daily (days 7-15) for the presence of hookworm eggs. On day 18 kittens were stratified by faecal egg count and randomly allocated in equal numbers to control (n = 6) and treatment (n = 6) groups. Faecal egg counts were then performed daily (days 14-27) or every 3 days (days 28-51) until the end of the study and compared between the treated and control groups. Kittens in the treatment group were administered a single topical dose of selamectin (Revolution®), per label recommendations (6 mg/kg), on day 19. Kittens in the control group were not treated. At 4 days post-treatment, no hookworm eggs were detected in the treated group. Faecal samples from this group remained negative throughout the study, the treatment efficacy was 100% egg reduction (P < .0001). Average faecal egg counts remained high (558 ±â€¯231 eggs per gram) in the untreated control group until the end of the study period. In conclusion, a single topical treatment of selamectin (Revolution®) at the recommended dose was highly efficacious against infection with A. ceylanicum in cats.

Drug Screening for Discovery of Broad-spectrum Agents for Soil-transmitted Nematodes.

Soil-transmitted nematodes (STNs), namely hookworms, whipworms, and ascarids, are extremely common parasites, infecting 1-2 billion of the poorest people worldwide. Two benzimidazoles, albendazole and mebendazole, are currently used in STN mass drug administration, with many instances of low/reduced activity reported. New drugs against STNs are urgently needed. We tested various models for STN drug screening with the aim of identifying the most effective tactics for the discovery of potent, safe and broad-spectrum agents. We screened a 1280-compound library of approved drugs to completion against late larval/adult stages and egg/larval stages of both the human hookworm parasite Ancylostoma ceylanicum and the free-living nematode Caenorhabditis elegans, which is often used as a surrogate for STNs in screens. The quality of positives was further evaluated based on cheminformatics/data mining analyses and activity against evolutionarily distant Trichuris muris whipworm adults. From these data, two pairs of positives, sulconazole/econazole and pararosaniline/cetylpyridinium, predicted to target nematode CYP-450 and HSP-90 respectively, were prioritized for in vivo evaluation against A. ceylanicum infections in hamsters. One of these positives, pararosaniline, showed a significant impact on hookworm fecundity in vivo. Taken together, our results suggest that anthelmintic screening with A. ceylanicum larval stages is superior to C. elegans based on both reduced false negative rate and superior overall quality of actives. Our results also highlight two potentially important targets for the discovery of broad-spectrum human STN drugs.

Development and optimization of a high-throughput screening method utilizing Ancylostoma ceylanicum egg hatching to identify novel anthelmintics.

Hookworms remain a major health burden in the developing world, with hundreds of millions currently afflicted by these blood-feeding parasites. There exists a vital need for the discovery of novel drugs and identification of parasite drug targets for the development of chemotherapies. New drug development requires the identification of compounds that target molecules essential to parasite survival and preclinical testing in robust, standardized animal models of human disease. This process can prove costly and time consuming using conventional, low-throughput methods. We have developed a novel high-throughput screen (HTS) to identify anthelmintics for the treatment of soil-transmitted helminths. Our high-throughput, plate reader-based assay was used to rapidly assess compound toxicity to Ancylostoma ceylanicum L1 larva. Using this method, we screened 39,568 compounds from several small molecule screening libraries at 10 µM and identified 830 bioactive compounds that inhibit egg hatching of the human hookworm A. ceylanicum by >50%. Of these, 132 compounds inhibited hookworm egg hatching by >90% compared to controls. The nematicidal activities of 268 compounds were verified by retesting in the egg hatching assay and were also tested for toxicity against the human HeLa cell line at 10 µM. Fifty-nine compounds were verified to inhibit A. ceylanicum egg hatching by >80% and were <20% toxic to HeLa cells. Half-maximal inhibitory concentration (IC50) values were determined for the 59 hit compounds and ranged from 0.05-8.94 µM. This stringent advancement of compounds was designed to 1) systematically assess the nematicidal activity of novel compounds against the egg stage of A. ceylanicum hookworms in culture and 2) define their chemotherapeutic potential in vivo by evaluating their toxicity to human cells. Information gained from these experiments may directly contribute to the development of new drugs for the treatment of human hookworm disease.

Autochthonous Cutaneous Larva Migrans in France and Europe.

Cutaneous larva migrans is a dermatitis, typically acquired in warm tropical or sub-tropical countries, caused by migration of the larvae of nematodes (hookworm; mainly Ancylostoma braziliense and, occasionally, Ancylostoma caninum or Uncinaria stenocephala), which are parasitic on animals such as cats and dogs, into the patient's skin. The larvae penetrate the skin after contact with infected soil and cause a typical creeping eruption. Patients with cutaneous larva migrans seen in Europe have usually acquired the disease following a stay in a tropical or sub-tropical area. How-ever, some cases of cutaneous larval migrans are acquired in Europe. We report here 5 autochthonous cases in France and give an overview of European autochthonous cases.

Ovicidal and larvicidal effects of extracts from leaves of Andrographis paniculata (Burm. f.) Wall.ex Nees against field isolates of human hookworm (Ancylostoma duodenale).

ETHNOPHARMACOLOGICAL RELEVANCE: The whole plant of Andrographis paniculata (Burm. f.) Wall.ex Nees is used traditionally in different forms by the local people of Asian countries owing to its myriad medicinal properties. Its use as an anthelmintic has been mentioned in literature but has not been well elucidated. AIM OF THE STUDY: To determine anthelmintic effects of extracts from leaves of A.paniculata against human hookworm species based on a standard assay system and to establish the effects of major active compounds responsible for the effects. MATERIALS AND METHODS: Ovicidal and larvicidal activities of extracts of leaves of A.paniculata in different solvents ethanol (Et), methanol (Met), ethyl acetate (EA) and petroleum ether (PE) was studied against field isolates of Ancylostoma duodenale collected and cultivated from hookworm infected human stool samples by egg hatch and larval motility assays. Major active compounds namely andrographolide (AP1), neoandrographolide (AP2) and andrograpanin (AP3) were estimated quantitatively in all the extracts by high-performance liquid chromatography (HPLC) and mass spectrometry (MS) analysis. Anthelmintic effects (ED50, LC50) and presence of the marker compounds in each extract was statistically analyzed by principal component analysis (PCA). Further, biological activities of pure compounds of AP1, AP2, AP3 were assessed to validate the results of the study. RESULTS: Extracts in ethanol and methanol showed highest activity in inhibition of egg hatching with lowest ED50 values (0.017 and 0.02 mg/mL respectively) while ethyl acetate extract had the highest activity against larval motility (0.001 mg/mL) followed by ethanol (0.019 mg/mL). On HPLC analysis, andrographolide content (%), the major diterpene compound, in Met and Et was 0.85 and 1.43 respectively. On PCA, andrographolide component in the extracts was associated with significant inhibitory effects both on egg hatching and larval motility. Pure compound AP1 also showed significant ovicidal and larvicidal activities at concentrations 0.125 µg/mL and 0.019 mg/mL respectively. CONCLUSION: Andrographolide is one of the main phytochemical responsible for significant ovicidal and larvicidal activity against field isolates of A.duodenale from human infections and can be developed as a potential therapeutic choice.

Isolation and characterization of a naturally occurring multidrug-resistant strain of the canine hookworm, Ancylostoma caninum.

Soil-transmitted nematodes infect over a billion people and place several billion more at risk of infection. Hookworm disease is the most significant of these soil-transmitted nematodes, with over 500 million people infected. Hookworm infection can result in debilitating and sometimes fatal iron-deficiency anemia, which is particularly devastating in children and pregnant women. Currently, hookworms and other soil-transmitted nematodes are controlled by administration of a single dose of a benzimidazole to targeted populations in endemic areas. While effective, people are quickly re-infected, necessitating frequent treatment. Widespread exposure to anthelmintic drugs can place significant selective pressure on parasitic nematodes to generate resistance, which has severely compromised benzimidazole anthelmintics for control of livestock nematodes in many areas of the world. Here we report, to our knowledge, the first naturally occurring multidrug-resistant strain of the canine hookworm Ancylostoma caninum. We reveal that this isolate is resistant to fenbendazole at the clinical dosage of 50 mg/kg for 3 days. Our data shows that this strain harbors a fixed, single base pair mutation at amino acid 167 of the ß-tubulin isotype 1 gene, and by using CRISPR/Cas9 we demonstrate that introduction of this mutation into the corresponding amino acid in the orthologous ß-tubulin gene of Caenorhabditis elegans confers a similar level of resistance to thiabendazole. We also show that the isolate is resistant to the macrocyclic lactone anthelmintic ivermectin. Understanding the mechanism of anthelmintic resistance is important for rational design of control strategies to maintain the usefulness of current drugs, and to monitor the emergence of resistance. The isolate we describe represents the first multidrug-resistant strain of A. caninum reported, and our data reveal a resistance marker that can emerge naturally in response to heavy anthelminthic treatment.

Albendazole resistance induced in Ancylostoma ceylanicum is not due to single-nucleotide polymorphisms (SNPs) at codons 167, 198, or 200 of the beta-tubulin gene, indicating another resistance mechanism.

Mass drug administration has been implicated as the major cause of drug resistance in nematodes of ruminants. Single-nucleotide polymorphisms (SNPs) at codons 167, 198, and 200 of the beta-tubulin isotype 1 gene are associated with albendazole resistance mechanisms. Although drug resistance is suspected to occur in nematodes of the same order, at present, there is no evidence of a strong correlation between these canonical SNPs and albendazole resistance in hookworms. In the absence of a hookworm strain that is naturally resistant to albendazole, we produced an albendazole-resistant Ancylostoma ceylanicum strain by selective drug pressure. Restriction fragment length polymorphism-PCR (RFLP-PCR) was employed to identify the presence of SNPs previously associated with drug resistance in other nematodes. However, none of the benzimidazole resistance-associated SNPs known in other nematodes were found. A beta-tubulin isotype 1 gene mini-cDNA library was constructed to obtain the complete cDNA gene sequence for the analysis of the entire gene to identify distinct SNPs associated with resistance. Some SNPs were found, but the resulting sequences were not reproducibly detected among the different clones, preventing their association with the resistance mechanism. The parasitological and hematological parameters of the albendazole-resistant strain were characterized and compared to those of the sensitive strain. Although the albendazole-resistant strain was less adapted to its host, with fewer worms recovered, all other parameters analyzed were similar between both strains. The results of the present study indicate that the mechanism of albendazole resistance of the resistant strain described herein must differ from those that have previously been characterized. Thus, new mechanistic studies are needed in the future.

Efficacy and safety of a new topical formulation of selamectin plus sarolaner in the treatment and control of natural infections of Ancylostoma tubaeforme and Toxocara cati in cats presented as veterinary patients in the United States.

A new topical formulation of selamectin plus sarolaner (Revolution® Plus/Stronghold® Plus, Zoetis) was evaluated in the treatment and control of naturally occurring infections of Ancylostoma tubaeforme and Toxocara cati in cats presented as veterinary patients in the United States. Three thousand three hundred three (3303) cats were screened in 25 veterinary practices in 15 states and 153 hookworm-positive cats (A. tubaeforme and/or A. braziliense), mainly from Alabama, Mississippi, Texas, and Hawaii, were identified; 135 cats met all the criteria for enrollment and were included on study. The cats were randomly assigned to treatment with Revolution® (at the label dosage, to provide a minimum dosage of 6 mg/kg selamectin) or selamectin plus sarolaner (at a dosage of 6-12 mg/kg plus 1-2 mg/kg, respectively). Treatments were administered at the time of enrollment and repeated 30 days later. Fecal samples were collected for differential fecal egg count prior to the first treatment (Day 0), prior to the second treatment (Day 30), and approximately 30 days later (Day 60). Efficacy was based on the percentage reductions in geometric mean fecal egg count for A. tubaeforme on Day 30 and Day 60 compared with Day 0. Where cats were co-infected with T. cati, efficacy against this species was also evaluated. Efficacy data were evaluated for A. tubaeforme for 40 cats on both Day 30 and Day 60 for the group treated with the selamectin/sarolaner combination and reductions in geometric mean fecal egg counts of 99.4% and 99.7% were demonstrated for Day 30 and Day 60, respectively. For the group treated with selamectin alone, 44 and 40 cats were evaluated and percent reductions for Day 30 and Day 60 were 99.5% and 99.9%, respectively. For T. cati, 14 cats were evaluated in the selamectin/sarolaner-treated group for Day 30 and for Day 60, and the reduction in geometric mean fecal egg count was 100% for both days. There were 11 and 9 cats evaluated for Day 30 and Day 60, respectively, for the selamectin-treated group and the reduction was again 100% for both days. The geometric mean fecal egg counts post-treatment were significantly lower than pre-treatment for both A. tubaeforme and T. cati, for both treatments, and for both periods of interest (P < 0.0001). No serious adverse events related to treatment with either product occurred during the study. Thus, both selamectin alone and the combination product of selamectin/sarolaner were safe and effective when administered on a monthly basis for the treatment and control of natural infections of A. tubaeforme and T. cati. The addition of sarolaner to the formulation did not interfere with the efficacy of selamectin against these nematodes.

Standardization and application of a modified RFLP-PCR methodology for analysis of polymorphisms linked to treatment resistance in Ancylostoma braziliense.

BACKGROUND: Single nucleotide polymorphisms (SNPs) in codons 167, 198 and 200 of the beta-tubulin isotype 1 gene are associated with benzimidazoles resistance in many helminths. Codon 167 mutation has never been described in hookworms; however, polymorphisms in codons 198 and 200 have been described for Ancylostoma caninum and Necator americanus. These mutations have never been investigated in Ancylostoma braziliense; therefore, it is not known if they are present in this species and whether they are correlated with treatment resistance. The RFLP-PCR technique has been used to analyze these polymorphisms in some nematodes, but depending on the species, these alterations do not create or eliminate any restriction enzyme cleavage site, making it impossible to use this technique. Here, we describe the standardization and application of a modified RFLP-PCR technique for detecting polymorphisms in individual A. braziliense worms recovered from naturally infected dogs in two Brazilian states. RESULTS: The molecular techniques used were sensitive, specific, and easy to apply. To our knowledge, we report for the first time the presence of a polymorphism at codon 198 of the beta-tubulin gene of A. braziliense (1/81; 95% CI: 0-3.69%). CONCLUSIONS: It is not known whether the presence of the mutation in codon 198 of the beta-tubulin gene of A. braziliense has importance for this parasite. However, based on studies of other helminths, it is possible that this polymorphism is directly related to the resistance to benzimidazoles. This may be a major concern, since this nematode has considerable relevance as a parasite of canids and felids and as one of the agents of cutaneous larva migrans in humans. Standardized methodologies will be useful for screening for polymorphisms in the beta-tubulin gene of canine hookworms in a broader population. The method could also be adapted for the analysis of other SNPs in other nematode species.

Bacillus thuringiensis Cry5B protein as a new pan-hookworm cure.

Hookworms are intestinal nematode parasites that infect nearly half a billion people and are globally one of the most important contributors to iron-deficiency anemia. These parasites have significant impacts in developing children, pregnant women and working adults. Of all the soil-transmitted helminths or nematodes (STNs), hookworms are by far the most important, with disease burdens conservatively estimated at four million DALYs (Disability-Adjusted Life Years) and with productivity losses of up to US$139 billion annually. To date, mainly one drug, albendazole is used for hookworm therapy in mass drug administration, which has on average ∼80% cure rate that is lower (<40%) in some places. Given the massive numbers of people needing treatment, the threat of parasite resistance, and the inadequacy of current treatments, new and better cures against hookworms are urgently needed. Cry5B, a pore-forming protein produced by the soil bacterium Bacillus thuringiensis (Bt) has demonstrated good efficacy against Ancylostoma ceylanicum hookworm infections in hamsters. Here we broaden studies of Cry5B to include tests against infections of Ancylostoma caninum hookworms in dogs and against infections of the dominant human hookworm, Necator americanus, in hamsters. We show that Cry5B is highly effective against all hookworm parasites tested in all models. Neutralization of stomach acid improves Cry5B efficacy, which will aid in practical application of Cry5B significantly. Importantly, we also demonstrate that the anti-nematode therapeutic efficacy of Cry5B is independent of the host immune system and is not itself negated by repeated dosing. This study indicates that Bt Cry5B is a pan-hookworm anthelmintic with excellent properties for use in humans and other animals.

In vivo and in vitro studies of Cry5B and nicotinic acetylcholine receptor agonist anthelmintics reveal a powerful and unique combination therapy against intestinal nematode parasites.

BACKGROUND: The soil-transmitted nematodes (STNs) or helminths (hookworms, whipworms, large roundworms) infect the intestines of ~1.5 billion of the poorest peoples and are leading causes of morbidity worldwide. Only one class of anthelmintic or anti-nematode drugs, the benzimidazoles, is currently used in mass drug administrations, which is a dangerous situation. New anti-nematode drugs are urgently needed. Bacillus thuringiensis crystal protein Cry5B is a powerful, promising new candidate. Drug combinations, when properly made, are ideal for treating infectious diseases. Although there are some clinical trials using drug combinations against STNs, little quantitative and systemic work has been performed to define the characteristics of these combinations in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Working with the hookworm Ancylostoma ceylanicum-hamster infection system, we establish a laboratory paradigm for studying anti-nematode combinations in vivo using Cry5B and the nicotinic acetylcholine receptor (nAChR) agonists tribendimidine and pyrantel pamoate. We demonstrate that Cry5B strongly synergizes in vivo with both tribendimidine and pyrantel at specific dose ratios against hookworm infections. For example, whereas 1 mg/kg Cry5B and 1 mg/kg tribendimidine individually resulted in only a 0%-6% reduction in hookworm burdens, the combination of the two resulted in a 41% reduction (P = 0.020). Furthermore, when mixed at synergistic ratios, these combinations eradicate hookworm infections at doses where the individual doses do not. Using cyathostomin nematode parasites of horses, we find based on inhibitory concentration 50% values that a strongylid parasite population doubly resistant to nAChR agonists and benzimidazoles is more susceptible or "hypersusceptible" to Cry5B than a cyathostomin population not resistant to nAChR agonists, consistent with previous Caenhorhabditis elegans results. CONCLUSIONS/SIGNIFICANCE: Our study provides a powerful means by which anthelmintic combination therapies can be examined in vivo in the laboratory. In addition, we demonstrate that Cry5B and nAChR agonists have excellent combinatorial properties-Cry5B combined with nAChR agonists gives rise to potent cures that are predicted to be recalcitrant to the development of parasite resistance. These drug combinations highlight bright spots in new anthelmintic development for human and veterinary animal intestinal nematode infections.

Sertraline, Paroxetine, and Chlorpromazine Are Rapidly Acting Anthelmintic Drugs Capable of Clinical Repurposing.

Parasitic helminths infect over 1 billion people worldwide, while current treatments rely on a limited arsenal of drugs. To expedite drug discovery, we screened a small-molecule library of compounds with histories of use in human clinical trials for anthelmintic activity against the soil nematode Caenorhabditis elegans. From this screen, we found that the neuromodulatory drugs sertraline, paroxetine, and chlorpromazine kill C. elegans at multiple life stages including embryos, developing larvae and gravid adults. These drugs act rapidly to inhibit C. elegans feeding within minutes of exposure. Sertraline, paroxetine, and chlorpromazine also decrease motility of adult Trichuris muris whipworms, prevent hatching and development of Ancylostoma caninum hookworms and kill Schistosoma mansoni flatworms, three widely divergent parasitic helminth species. C. elegans mutants with resistance to known anthelmintic drugs such as ivermectin are equally or more susceptible to these three drugs, suggesting that they may act on novel targets to kill worms. Sertraline, paroxetine, and chlorpromazine have long histories of use clinically as antidepressant or antipsychotic medicines. They may represent new classes of anthelmintic drug that could be used in combination with existing front-line drugs to boost effectiveness of anti-parasite treatment as well as offset the development of parasite drug resistance.