Strain-level variations of Dirofilaria immitis microfilariae in two biochemical assays.

BACKGROUND: The increase in reports of resistance to macrocyclic lactones in the canine heartworm, Dirofilaria immitis is alarming. While DNA based tests have been well-validated, they can be expensive. In a previous study, we showed that two biochemical tests adapted to a 96- well plate format and read in a spectrophotometer could detect differences among lab validated D. immitis isolates. The two tests- Resazurin reduction and Hoechst 33342 efflux-detect metabolism and P-glycoprotein activity respectively in microfilariae isolated from infected dog blood. METHODS: Our objective was to optimize the two assays further by testing various assay parameters in D. immitis isolates not tested previously. We tested microfilarial seeding density, incubation time and the effect of in vitro treatment with ivermectin and doxycycline in five other D. immitis isolates-JYD-34, Big Head, Berkeley, Georgia III and LOL. All assays were performed in 3 technical replicates and 2-4 biological replicates. To understand the molecular basis of the assays, we also performed qPCR for selected drug metabolism and elimination associated genes of the ABC transporter and cytochrome P450 gene families. RESULTS: Metabolism and ABC transporter activity as detected by these assays varied between strains. Anthelmintic status (resistant or susceptible) did not correlate with metabolism or P-gp efflux. Basal transcriptional variations were found between strains in ABC transporter and cytochrome P450 genes. CONCLUSIONS: These assays provide a greater understanding of the biochemical variation among isolates of D. immitis, which can be exploited in the future to develop in vitro diagnostic tests capable of differentiating susceptible and resistant isolates.

The nematode (Ascaris suum) intestine is a location of synergistic anthelmintic effects of Cry5B and levamisole.

A novel group of biocidal compounds are the Crystal 3D (Cry) and Cytolytic (Cyt) proteins produced by Bacillus thuringiensis (Bt). Some Bt Cry proteins have a selective nematocidal activity, with Cry5B being the most studied. Cry5B kills nematode parasites by binding selectively to membrane glycosphingolipids, then forming pores in the cell membranes of the intestine leading to damage. Cry5B selectively targets multiple species of nematodes from different clades and has no effect against mammalian hosts. Levamisole is a cholinergic anthelmintic that acts by selectively opening L-subtype nicotinic acetylcholine receptor ion-channels (L-AChRs) that have been found on muscles of nematodes. A synergistic nematocidal interaction between levamisole and Cry5B at the whole-worm level has been described previously, but the location, mechanism and time-course of this synergism is not known. In this study we follow the timeline of the effects of levamisole and Cry5B on the Ca2+ levels in enterocyte cells in the intestine of Ascaris suum using fluorescence imaging. The peak Ca2+ responses to levamisole were observed after approximately 10 minutes while the peak responses to activated Cry5B were observed after approximately 80 minutes. When levamisole and Cry5B were applied simultaneously, we observed that the responses to Cry5B were bigger and occurred sooner than when it was applied by itself. It is proposed that the synergism is due to the cytoplasmic Ca2+ overload that is induced by the combination of levamisole opening Ca2+ permeable L-subtype nAChRs and the Ca2+ permeable Cry5B toxin pores produced in the enterocyte plasma membranes. The effect of levamisole potentiates and speeds the actions of Cry5B that gives rise to bigger Ca2+ overloads that accelerates cell-death of the enterocytes.

The nematode (Ascaris suum) intestine is a location of synergistic anthelmintic effects of Cry5B and levamisole.

A novel group of biocidal compounds are the Crystal 3D (Cry) and Cytolytic (Cyt) proteins produced by Bacillus thuringiensis (Bt). Some Bt Cry proteins have a selective nematocidal activity, with Cry5B being the most studied. Cry5B kills nematode parasites by binding selectively to membrane glycosphingolipids, then forming pores in the cell membranes of the intestine leading to damage. Cry5B selectively targets multiple species of nematodes from different clades and has no effect against mammalian hosts. Levamisole is a cholinomimetic anthelmintic that acts by selectively opening L-subtype nicotinic acetylcholine receptor ion-channels (L-AChRs) that have been found on muscles of nematodes. A synergistic nematocidal interaction between levamisole and Cry5B has been described previously, but the location, mechanism and time-course of this synergism is not known. In this study we follow the timeline of the effects of levamisole and Cry5B on the Ca2+ levels in enterocyte cells from the intestine of Ascaris suum using fluorescence imaging. The peak Ca2+ responses to levamisole were observed after approximately 10 minutes while the peak responses to activated Cry5B were observed after approximately 80 minutes. When levamisole and Cry5B were applied simultaneously, we observed that the responses to Cry5B were bigger and occurred sooner than when it was applied by itself. It is proposed that there is an irreversible cytoplasmic Ca2+ overload that leads to necrotic cell-death in the enterocyte that is induced by levamisole opening Ca2+ permeable L-subtype nAChRs and the development of Ca2+ permeable Cry5B toxin pores in enterocyte plasma membranes. The effects of levamisole potentiate and speed the actions of Cry5B.

Assessment of in vitro efficacy for common surface disinfectants and antiseptics against Tritrichomonas foetus trophozoites.

The protozoan Tritrichomonas foetus causes early embryonic death in cattle, there are no legal options for treating this parasite in the United States, and there are few developed protocols for cleaning veterinary and obstetrical equipment that may have been contaminated with trophozoites. In this study, we evaluated bleach, ethanol, acetic acid, chlorhexidine gluconate, and hydrogen peroxide solutions for the ability to kill trophozoites in vitro. Our findings suggested that ethanol and bleach could adequately disinfect tools and equipment. Acetic acid, chlorhexidine, and hydrogen peroxide had applications as surface disinfectants in addition to potential as local topical treatments due to their past uses in veterinary theriogenology. Chlorhexidine gluconate demonstrated trophocidal effects by damaging parasite cell membranes and had the lowest effective concentration 50 (EC50) of any compound tested and was in the micromolar range. These findings, in conjunction with accepted clinical uses of chlorhexidine gluconate suggest that this is a convenient agent for disinfecting equipment. In addition, topical use of chlorhexidine is relatively common, setting the stage for further investigation of this compound as a topical therapeutic option for bovine trichomonosis.

Haplotypic analysis of cox1 from Toxocara canis demonstrates five distinct clades that are not geographically defined.

BACKGROUND: Toxocara canis is a cosmopolitan parasite of dogs that is transmitted transplacentally to puppies resulting in widespread shedding of eggs in the environment. However, it is not clear if there are dominant parasite genotypes that are more common, pathogenic, or likely to be zoonotic. METHODS/PRINCIPLE FINDINGS: Sequences of mitochondrial cox1 gene from adult worms were used to compare parasites from the United States with submitted sequences from parasites isolated from dogs in different countries. Our analysis revealed at least 55 haplotypes. While we expected the North American worms to form a distinct cluster, we found haplotypes of T. canis reported elsewhere existing in this population. Interestingly, combining the sequence data from our study with the available GenBank data, analysis of cox1 sequences results in five distinct clades that are not geographically defined. CONCLUSIONS: The five clades of T. canis revealed in this study potentially have unique life histories, traits, or host preferences. Additional investigation is needed to see if these distinct clades represent cryptic species with clinically useful attributes or genotypes with taxonomic value. Evaluation of common mitochondrial genes may reveal distinct populations of zoonotic T. canis.

Repertoire of P-glycoprotein drug transporters in the zoonotic nematode Toxocara canis.

Toxocara canis has a complex lifecycle including larval stages in the somatic tissue of dogs that tolerate macrocyclic lactones. In this study, we investigated T. canis permeability glycoproteins (P-gps, ABCB1) with a putative role in drug tolerance. Motility experiments demonstrated that while ivermectin failed to abrogate larval movement, the combination of ivermectin and the P-gp inhibitor verapamil induced larval paralysis. Whole organism assays revealed functional P-gp activity in larvae which were capable of effluxing the P-gp substrate Hoechst 33342 (H33342). Further investigation of H33342 efflux demonstrated a unique rank order of potency for known mammalian P-gp inhibitors, suggesting that one or more of the T. canis transporters has nematode-specific pharmacological properties. Analysis of the T. canis draft genome resulted in the identification of 13 annotated P-gp genes, enabling revision of predicted gene names and identification of putative paralogs. Quantitative PCR was used to measure P-gp mRNA expression in adult worms, hatched larvae, and somatic larvae. At least 10 of the predicted genes were expressed in adults and hatched larvae, and at least 8 were expressed in somatic larvae. However, treatment of larvae with macrocyclic lactones failed to significantly increase P-gp expression as measured by qPCR. Further studies are needed to understand the role of individual P-gps with possible contributions to macrocyclic lactone tolerance in T. canis.

Diethylcarbamazine, TRP channels and Ca2+ signaling in cells of the Ascaris intestine.

The nematode parasite intestine absorbs nutrients, is involved in innate immunity, can metabolize xenobiotics and as we show here, is also a site of action of the anthelmintic, diethylcarbamazine. Diethylcarbamazine (DEC) is used to treat lymphatic filariasis and activates TRP-2, GON-2 & CED-11 TRP channels in Brugia malayi muscle cells producing spastic paralysis. DEC also has stimulatory effects on ascarid nematode parasites. Using PCR techniques, we detected, in Ascaris suum intestine, message for: Asu-trp-2, Asu-gon-2, Asu-ced-11, Asu-ocr-1, Asu-osm-9 and Asu-trpa-1. Comparison of amino-acid sequences of the TRP channels of B. malayi, and A. suum revealed noteworthy similarity, suggesting that the intestine of Ascaris will also be sensitive to DEC. We used Fluo-3AM as a Ca2+ indicator and observed characteristic unsteady time-dependent increases in the Ca2+ signal in the intestine in response to DEC. Application of La3+ and the TRP channel inhibitors, 2-APB or SKF 96365, inhibited DEC mediated increases in intracellular Ca2+. These observations are important because they emphasize that the nematode intestine, in addition to muscle, is a site of action of DEC as well as other anthelmintics. DEC may also enhance the Ca2+ toxicity effects of other anthelmintics acting on the intestine or, increase the effects of other anthelmintics that are metabolized and excreted by the nematode intestine.

Characterization of a P-glycoprotein drug transporter from Toxocara canis with a novel pharmacological profile.

P-glycoproteins from the ATP-binding cassette transporter family are responsible for drug evasion by bacterial pathogens and neoplastic cells. More recently, these multidrug resistance transporters have been investigated for contributions to drug resistance in nematode parasites. In this study, we cloned and characterized the P-glycoprotein Tca-Pgp-11.1 from Toxocara canis, the canine intestinal ascarid. Large numbers of Tca-Pgp-11 transcripts were observed in the intestine of adult male and female worms. Heterologous expression studies confirmed sensitivity to known P-glycoprotein inhibitors. Interestingly, the competitive inhibitor verapamil had lower IC50 values than newer generation inhibitors that are designed to allosterically modulate mammalian P-glycoprotein. Consistent with other nematode P-glycoproteins, Tca-Pgp-11.1 was sensitive to ivermectin and selamectin but not moxidectin. Taken together, our data suggests that T. canis P-glycoproteins represent nematode-specific drug targets that could be exploited to enhance efficacy of existing anthelmintics.

Bovine Trichomonosis Cases in the United States 2015-2019.

Tritrichomonas foetus is a sexually-transmitted protozoan parasite that causes early embryonic death in cattle. Tritrichomonas foetus is enzootic in the United States but is not a reportable disease at the national level. Thus, it is difficult to understand the prevalence and relative distribution of the disease for the purpose of developing appropriate control measures. In this study, a survey of state veterinarians was used to determine the number of reported cases in each state from 2015 to 2019. Our investigation revealed infections in 25 different states and a total of 3,817 reported cases nationwide. Infections occurred throughout different regions of the country, and numbers of cases were only weakly correlated with total number of cattle in each state. Tritrichomonas foetus is a significant pathogen in the United States and understanding the relative distribution of the parasite is useful for prioritizing surveillance and intervention strategies going forward.

Global prevalence of Mesocestoides infections in animals - A systematic review and meta-analysis.

Mesocestoides spp. are zoonotic cestodes found as adults in carnivorous domestic and wild definitive hosts and as metacestodes in several taxa of intermediate hosts. Although several regional studies record its occurrence in different host populations, the global prevalence and patterns of occurrence of Mesocestoides spp. are not fully understood. The objective of this study was to conduct a systematic review and meta-analysis of published literature to estimate the global prevalence of Mesocestoides spp. in major definitive and intermediate host taxa. Records published in English were collected from NCBI PubMed, Science Direct, Web of Science and Google Scholar databases, with 364 papers being included in the meta-analysis. The overall pooled prevalence estimates show that 21.72 % (95 % CI: 18.49-25.14) of terrestrial carnivore definitive hosts and 7.09 % (95 % CI: 5.79-8.51) of intermediate hosts are infected. Among definitive hosts, opossums and foxes were most commonly infected with pooled global prevalence of 48.16 % (95 % CI: 14.62 - 82.69) and 35.97 % (295 % CI: 9.54 - 42.66) respectively. Pooled global prevalence in domestic dogs and cats were 7.97 % (95 % CI: 5.67 - 10.63) and 8.32 % (95 % CI: 3.78 - 14.41) respectively. Among intermediate hosts, birds and snakes were most commonly infected with pooled global prevalence of 16.19 % (95 %CI: 5.9 - 30.31) and 15.74 % (95 % CI: 10.59 - 21.69) respectively. Our analysis demonstrates that prevalence of Mesocestoides spp. is variable across the world. The sylvatic cycle in wild hosts is likely to be more important than the domestic cycle for the maintenance of Mesocestoides spp. globally. Currently available genetic data at the mitochondrial COI locus was also phylogenetically analyzed. The genetic data supports the taxonomic distinctiveness of only a few of the numerous morphologically described Mesocestoides spp.

Contracaeciasis in an American white pelican.

An American white pelican migrating through Iowa, USA exhibited regurgitation and anorexia. At the time of necropsy, numerous nematodes were observed in the crop and proventriculus with evidence of proventriculitis. Nematodes were identified as Contracaecum spp. based on morphological features of the adult worms and eggs. Species level identification of C. fagerholmi were made using nucleotide sequence analysis of the partial cox2 gene. Contracaecum infections are highly prevalent in piscivorous birds that acquire the infection by ingesting fish infected with larval stages of the parasite. Considering the possible zoonotic nature of Contracaecum, humans whose diets include uncooked fresh-water and/or marine fish should handle fresh fish with care, as these may harbor immature stages of Contracaecum spp.

Syngamus asphyxiation in a captive ring-necked pheasant.

A deceased ring-necked pheasant (Phasianus colchicus) presented for necropsy following a history of chronic wasting. Necropsy revealed nematodes consistent with the genus Syngamus partially obstructing the trachea. Phylogentic analysis failed to reveal conclusive results regarding the species. Syngamus spp. can cause obstruction of the trachea in several different hosts. Additional genetic data from this taxon would aid in the more precise identification of diagnostic specimens.

Afoxolaner as a Treatment for a Novel Sarcoptes scabiei Infestation in a Juvenile Potbelly Pig.

A 2 months old female Vietnamese potbellied pig presented to a veterinary teaching hospital with a referring complaint of pruritus. A human caretaker of the pig had recently been diagnosed with a Sarcoptes spp. dermatitis. Microscopic examination of the skin scrape samples and BLAST analysis confirmed the species of the mite as most closely related to Sarcoptes scabiei var. canis (AY493391). The pig was treated with afoxolaner as previous treatment with ivermectin was not efficacious. Recheck examinations and follow up revealed the pig to be non-pruritic and resolving. Afoxolaner may be a therapeutic option when treating Sarcoptes spp. infections in companion pigs.

Laboratory assays reveal diverse phenotypes among microfilariae of Dirofilaria immitis isolates with known macrocyclic lactone susceptibility status.

Prevention of canine heartworm disease caused by Dirofilaria immitis relies on chemoprophylaxis with macrocyclic lactone anthelmintics. Alarmingly, there are increased reports of D. immitis isolates with resistance to macrocyclic lactones and the ability to break through prophylaxis. Yet, there is not a well-established laboratory assay that can utilize biochemical phenotypes of microfilariae to predict drug resistance status. In this study we evaluated laboratory assays measuring cell permeability, metabolism, and P-glycoprotein-mediated efflux. Our assays revealed that trypan blue, propidium iodide staining, and resazurin metabolism could detect differences among D. immitis isolates but none of these approaches could accurately predict drug susceptibility status for all resistant isolates tested. P-glycoprotein assays suggested that the repertoire of P-gp expression is likely to vary among isolates, and investigation of pharmacological differences among different P-gp genes is warranted. Further research is needed to investigate and optimize laboratory assays for D. immitis microfilariae, and caution should be applied when adapting cell death assays to drug screening studies for nematode parasites.

Repurposing the open access malaria box reveals compounds with activity against Tritrichomonas foetus trophozoites.

The protozoan parasite Tritrichomonas foetus causes early embryonic death in cattle which results in severe economic loss. In the United States, there are no drugs are approved for treatment of this pathogen. In this study, we evaluated in vitro anti-protozoal effects of compounds from an open access chemical library against T. foetus trophozoites. An initial high-throughput screen identified 16 compounds of interest. Further investigation revealed 12 compounds that inhibited parasite growth and 4 compounds with lethal effects. For lethal compounds, dose-response curves were constructed and the LD50 was calculated for laboratory and field strains of T. foetus. Our experiments revealed chemical scaffolds that were parasiticidal in the micromolar range, and these scaffolds provide a starting point for drug discovery efforts. Further investigation is still needed to investigate suitability of these scaffolds and related compounds in food animals. Importantly, open access chemical libraries can be useful for identifying compounds with activity against protozoan pathogens of veterinary importance.

Room Decontamination Using Ionized Hydrogen Peroxide Fog and Mist Reduces Hatching Rates of Syphacia obvelata Ova.

This study evaluated the efficacy of ionized hydrogen peroxide (iHP) fog and mist for environmental and surface decontaminationof Syphacia obvelata ova in rodent rooms. Ova were collected by perianal tape impression from S. obvelata infectedmice. In experiment 1, ova were exposed to iHP using a whole-room fogging decontamination system with a 15 min initialfog application cycle in unoccupied rodent rooms. Ova were removed from the fogged environment after a 15 min, 30 min, 90min, or 240 min iHP exposure time. In experiment 2, a second cohort of ova were exposed to iHP using the whole-room foggingdecontamination system. Ova were removed after 3, 4 or 6 continuous fog application cycles with 45 min dwelling timebetween each cycle and 15 h dwelling time for the last time point. In experiment 3, a third set of ova was exposed to an iHPsurface misting unit with 1, 2, or 3 iHP mist applications. A 7 min contact time followed each application. After exposure, ovawere incubated in a hatching medium for 6 h. Control ova were maintained at room temperature without iHP exposure beforeincubation in the hatching medium. After incubation, the number of ova hatched was assessed by microscopic examination.For experiment 1, results ranged from 46% to 57% of exposed ova hatched. For experiment 2, results ranged from 43% to 49%of ova hatched. For experiment 3, 37% to 46% of exposed ova hatched. Conversely, for the control groups above 80% of ovahatched for all 3 experiments. These data suggest that exposure to iHP fog and mist has variable effectiveness in reducingviability of S. obvelata ova at the time points tracked. Further studies are needed to identify iHP exposures that will furtherreduce or eliminate the hatching of rodent pinworm ova.

Pharmacological characterization of a homomeric nicotinic acetylcholine receptor formed by Ancylostoma caninum ACR-16.

Parasitic nematode infections are treated using anthelmintic drugs, some of which target nicotinic acetylcholine receptors (nAChRs) located in different parasite tissues. The limited arsenal of anthelmintic agents and the prevalence of drug resistance imply that future defense against parasitic infections will depend on the discovery of novel targets and therapeutics. Previous studies have suggested that Ascaris suum ACR-16 nAChRs are a suitable target for the development of antinematodal drugs. In this study, we characterized the pharmacology of the Ancylostoma caninum ACR-16 receptor using two-electrode voltage-clamp electrophysiology. This technique allowed us to study the effects of cholinergic agonists and antagonists on the nematode nAChRs expressed in Xenopus laevis oocytes. Aca-ACR-16 was not sensitive to many of the existing cholinomimetic anthelmintics (levamisole, oxantel, pyrantel, and tribendimidine). 3-Bromocytisine was the most potent agonist (> 130% of the control acetylcholine current) on the Aca-ACR-16 nAChR but, unlike Asu-ACR-16, oxantel did not activate the receptor. The mean time constants of desensitization for agonists on Aca-ACR-16 were longer than the rates observed in Asu-ACR-16. In contrast to Asu-ACR-16, the A. caninum receptor was completely inhibited by DHßE and moderately inhibited by α-BTX. In conclusion, we have successfully reconstituted a fully functional homomeric nAChR, ACR-16, from A. caninum, a model for human hookworm infections. The pharmacology of the receptor is distinct from levamisole-sensitive nematode receptors. The ACR-16 homologue also displayed some pharmacological differences from Asu-ACR-16. Hence, A. caninum ACR-16 may be a valid target site for the development of anthelmintics against hookworm infections.

Detection and quantification of Parascaris P-glycoprotein drug transporter expression with a novel mRNA hybridization technique.

Macrocyclic lactone-resistant Parascaris have been reported throughout the world. In part, the drug resistant phenotype is hypothesized to be associated with ATP-binding cassette transporters known as P-glycoproteins. In many systems, P-glycoproteins efflux drugs out of cells thereby precluding drug binding to target receptors. Parascaris may evade macrocyclic lactone-mediated death by effluxing drugs away from target receptors in the nervous system. Alternatively, P-glycoprotein expression in the gut or body wall could prevent penetration of drugs into the body of the parasite altogether. In the present study, we evaluate expression of Peq-pgp-11 and Peq-pgp-16 using a novel multiple nucleic acid hybridization method. This method allowed for visualization of individual mRNA transcripts within fixed tissue sections of Parascaris adults. Our investigation revealed expression of Peq-pgp-11 and Peq-pgp-16 in the intestine, body wall, nerves, lateral cords, and reproductive tissues of male and female parasites. These results suggest that Pgp could efflux drugs locally at the level of parasite neuronal tissue as well as at sites of entry for drugs such as the hypodermis and intestine. The multiple nucleic acid hybridization method could be useful for providing tissue context for gene expression in a variety of nematode parasites.

Use of praziquantel to control platyhelminth parasites of fish.

Fish are common definitive and intermediate hosts for a variety of parasitic flatworms. In unstressed wild populations, parasitic infections often go unnoticed and are perceived to represent a lesser threat to fish health. In contrast, platyhelminth parasitism of captive fish often results in decreased weight gain and increased mortality which often necessitates chemotherapeutic treatment. The presence of platyhelminth parasites in fish tissues is not only unappealing but in some cases also represents a threat to human health. In veterinary medicine, one of the most commonly used agents with anti-flatworm activity is praziquantel; yet, no praziquantel products are labeled for use in fish in the United States. Veterinarians may use praziquantel preparations approved for other vertebrate species under the Animal Medicinal Drug Use Clarification Act (AMDUCA). However, such extra-label use should be informed by scientific evidence including efficacy and tissue residue studies. Herein, we review studies testing the efficacy of praziquantel for treatment of platyhelminthes along with an assessment of routes of administration, pharmacokinetics, and toxicity information.

Praziquantel Resistance in the Zoonotic Cestode Dipylidium caninum.

Dipylidium caninum is a cosmopolitan cestode infecting dogs, cats, and humans. Praziquantel is a highly effective cestocidal drug and resistance in adult cestodes has not been reported. From 2016 to 2018, a population of dogs with cestode infections that could not be eliminated despite multiple treatments with praziquantel or epsiprantel was identified. Cases of D. caninum were clinically resistant to praziquantel and could not be resolved despite increasing the dose, frequency, and duration of treatment. Resistant isolates were identified and characterized by sequencing the 28S, 12S, and voltage-gated calcium channel beta subunit genes. Cases were only resolved following treatment with nitroscanate or a compounded pyrantel/praziquantel/oxantel product. Clinicians should be aware of this alarming development as treatment options for cestodes are limited in both human and veterinary medicine.