Proteomic analysis of extracellular vesicles and extracellular vesicle-depleted excretory-secretory products of Toxocara canis and Toxocara cati larval cultures.

Toxocara canis and Toxocara cati are parasitic nematodes in the order Ascaridida, which inhabit the small intestines of dogs and cats, respectively, as adults. Although often nonpathogenic as adults, nematodes within this genus are capable of causing widespread disease throughout the host while in a larval stage, during which time larvae migrate throughout the body in a process termed larva migrans. Larvae are also capable of surviving within host tissues in an encysted arrested stage, without immune clearance by the host. The ability of larvae to survive within host tissues during migration and encystment may be attributed to immunomodulatory molecules released by the excretory cells of larvae in excretory-secretory (ES) products. ES products of parasites contain a variety of molecules, including proteins, lipids, and extracellular vesicles (EVs). Toxocara excretory-secretory (TES) products have been studied to some degree, with proteomic analysis of TES proteins described previously; however, investigation of the EVs within TES is lacking, despite the suggested role for these molecules in host interaction and potential immunomodulation. To further characterize the protein cargo within EVs in TES, EVs were isolated from larval cultures of T. canis and T. cati via ultrafiltration, with concurrent collection of EV-depleted TES filtrate for additional study. Isolated EVs and EV-depleted TES from both T. canis and T. cati were submitted for proteomic analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS). Proteomic identification results revealed 140 proteins across all samples, with 16 shared by all samples, and 76 total proteins shared between T. canis and T. cati, present within EVs and EV-depleted TES. There were 17 proteins shared exclusively by EV samples, and 15 were shared exclusively between EV-depleted TES samples. Many shared proteins were associated with the host immune response. Several proteins were specific to either T. canis or T. cati, highlighting the potential use of these proteins as diagnostic tools in the differentiation of etiologic agents in cases of toxocariasis. The results of this study build upon previously reported proteomic evaluations of TES, contributing new information in regards to newly identified proteins, EV protein cargo within TES, and potential immunomodulatory functions of these proteins.

Synergistic effects of using sodium hypochlorite (bleach) and desiccation in surface inactivation for Toxocara spp.

Toxocara cati and T. canis are parasitic nematodes found in the intestines of cats and dogs respectively, with a cosmopolitan distribution, and the potential for anthropozoonotic transmission, resulting in human toxocariasis. Spread of Toxocara spp. is primarily through the ingestion of embryonated eggs contaminating surfaces or uncooked food, or through the ingestion of a paratenic host containing a third-stage larva. The Toxocara spp. eggshell is composed of a lipid layer providing a permeability barrier, a chitinous layer providing structural strength, and thin vitelline and uterine layers, which combined create a biologically resistant structure, making the Toxocara spp. egg very hardy, and capable of surviving for years in the natural environment. The use of sodium hypochlorite, household bleach, as a disinfectant for Toxocara spp. eggs has been reported, with results varying from ineffective to limited effectiveness depending on parameters including contact time, concentration, and temperature. Desiccation or humidity levels have also been reported to have an impact on larval development and/or survival of Toxocara spp. eggs. However, to date, after a thorough search of the literature, no relevant publications have been found that evaluated the use of sodium hypochlorite and desiccation in combination. These experiments aim to assess the effects of using a combination of desiccation and 10% bleach solution (0.6% sodium hypochlorite) on fertilized or embryonated eggs of T. cati, T. canis, and T. vitulorum. Results of these experiments highlight the synergistic effects of desiccation and bleach, and demonstrate a relatively simple method for surface inactivation, resulting in a decrease in viability or destruction of T. cati, T. canis and T. vitulorum eggs. Implications for these findings may apply to larger scale elimination of ascarid eggs from both research, veterinary, and farming facilities to mitigate transmission.

Comparison of extracellular vesicle isolation methods for the study of exosome cargo within Toxocara canis and Toxocara cati excretory secretory (TES) products.

Toxocara is a genus of nematodes, which infects a variety of hosts, principally dogs and cats, with potential zoonotic risks to humans. Toxocara spp. larvae are capable of migrating throughout the host tissues, eliciting eosinophilic and granulomatous reactions, while surviving for extended periods of time, unchanged, in the host. It is postulated that larvae are capable of altering the host's immune response through the release of excretory-secretory products, containing both proteins and extracellular vesicles (EVs). The study of EVs has increased exponentially in recent years, largely due to their potential use as a diagnostic tool, and in molecular therapy. To this end, there have been multiple isolation methods described for the study of EVs. Here, we use nanoparticle tracking to compare the yield, size distribution, and % labelling of EV samples acquired through various reported methods, from larval cultures of Toxocara canis and T. cati containing Toxocara excretory-secretory products (TES). The methods tested include ultracentrifugation, polymer precipitation, magnetic immunoprecipitation, size exclusion chromatography, and ultrafiltration. Based on these findings, ultrafiltration produces the best results in terms of yield, expected particle size, and % labelling of sample. Transmission electron microscopy confirmed the presence of EVs with characteristic cup-shaped morphology. These findings can serve as a guide for those investigating EVs, particularly those released from multicellular organisms, such as helminths, for which few comparative analyses have been performed.

Multicenter evaluation of the Vetscan Imagyst system using Ocus 40 and EasyScan One scanners to detect gastrointestinal parasites in feces of dogs and cats.

The Vetscan Imagyst system (Zoetis) is a novel, artificial intelligence-driven detection tool that can assist veterinarians in the identification of enteric parasites in dogs and cats. This system consists of a sample preparation device, an automated digital microscope scanner, and a deep-learning algorithm. The EasyScan One scanner (Motic) has had good diagnostic performance compared with manual examinations by experts; however, there are drawbacks when used in veterinary practices in which space for equipment is often limited. To improve the usability of this system, we evaluated an additional scanner, the Ocus 40 (Grundium). Our objectives were to 1) qualitatively evaluate the performance of the Vetscan Imagyst system with the Ocus 40 scanner for identifying Ancylostoma, Toxocara, and Trichuris eggs, Cystoisospora oocysts, and Giardia cysts in canine and feline fecal samples, and 2) expand the assessment of the performance of the Vetscan Imagyst system paired with either the Ocus 40 or EasyScan One scanner to include a larger dataset of 2,191 fecal samples obtained from 4 geographic regions of the United States. When tested with 852 canine and feline fecal samples collected from different geographic regions, the performance of the Vetscan Imagyst system combined with the Ocus 40 scanner was correlated closely with manual evaluations by experts. Sensitivities were 80.0‒97.0% and specificities were 93.7‒100.0% across the targeted parasites. When tested with 1,339 fecal samples, the Vetscan Imagyst system paired with the EasyScan One scanner successfully identified the targeted parasite stages; sensitivities were 73.6‒96.4% and specificities were 79.7‒100.0%.

Elimination of probable praziquantel-resistant Dipylidium caninum with nitroscanate in a mixed-breed dog: a case report.

BACKGROUND: Praziquantel is the drug of choice for treating the tapeworm Dipylidium caninum in dogs; however, resistance is possible, and regular, non-targeted administration of praziquantel may select for anthelminthic-resistant populations. METHODS: The zinc sulfate fecal floatation procedure was conducted. Gross visualization was used to identify Dipylidium spp. segments, and capsule endoscopy was used to visualize adult tapeworms within the intestinal tract. RESULTS: An 18-month-old spayed female terrier mix was presented due to diarrhea, hematochezia and weight loss. The dog received appropriate anthelmintic therapy for Giardia spp., Ancylostoma spp. and Dipylidium spp. The dog's clinical signs resolved, and elimination of Ancylostoma spp. was confirmed by subsequent fecal analysis. However, Dipylidium spp. segments were repeatedly present in the stool. Observation of the segments confirmed the presence of adult Dipylidium spp in feces. Treatment with praziquantel and epsiprantel were unsuccessful in eliminating the organism but was apparently successful in flea prevention. A single dose of nitrosconate was administered and eliminated Dipylidium spp. infection in the dog. CONCLUSIONS: Nitrosconate can be an effective treatment for praziquantel-resistant dipylidiasis in dogs. The novel application of capsule endoscopy confirmed the anthelmintic efficacy of this treatment.

Vector-borne and other pathogens of potential relevance disseminated by relocated cats.

Large populations of unowned cats constitute an animal welfare, ecological, societal and public health issue worldwide. Their relocation and homing are currently carried out in many parts of the world with the intention of relieving suffering and social problems, while contributing to ethical and humane population control in these cat populations. An understanding of an individual cat's lifestyle and disease status by veterinary team professionals and those working with cat charities can help to prevent severe cat stress and the spread of feline pathogens, especially vector-borne pathogens, which can be overlooked in cats. In this article, we discuss the issue of relocation and homing of unowned cats from a global perspective. We also review zoonotic and non-zoonotic infectious agents of cats and give a list of practical recommendations for veterinary team professionals dealing with homing cats. Finally, we present a consensus statement consolidated at the 15th Symposium of the Companion Vector-Borne Diseases (CVBD) World Forum in 2020, ultimately to help veterinary team professionals understand the problem and the role they have in helping to prevent and manage vector-borne and other pathogens in relocated cats.

First paleoparasitological evidence of Muellerius sp. in sheep or goat dated to the Sasanian Empire (500 CE) in ancient Iran.

A fecal pellet was recovered in an ancient salt mine in Chehrabad located in western Iran (36.55° N, 47.51° E). Based on prior publications showing the success of the salt mine's environment to preserve various life forms, it was decided to try and ascertain whether this faex contained any parasites of paleoparasitologic interest. The rehydration involved placing the pellet in an aqueous solution of 0.5% trisodium phosphate for a week, followed by the examination of aliquots of the entire rehydrated sample on a total of 153 microscope slides. The examination of the rehydrated material revealed the presence of two larval nematodes; there were no eggs or oocysts recovered. The larvae were photographed and measured, and the decision was made not to submit the two larvae to grinding and DNA extraction for molecular diagnostics due to their excellent state of preservation. The larvae were identified as first-stage larvae that appear to represent the genus Muellerius. Herein are reported the finding of two first-stage larvae of Muellerius nematodes (Metastrongyloidea, Protostrongylidae) from the rehydrated fecal pellet collected in a Chehrabad salt mine from the era of the Sasanian Empire.

Further data on the distribution of Dirofilaria spp. in the Czech Republic in dogs.

Based on previously published data, the Czech Republic is regarded an endemic country of the onchocercid nematodes Dirofilaria immitis (Leidy, 1856) and Dirofilaria repens Railliet et Henry, 1911. Nevertheless, while cases of D. repens are commonly reported from dogs in South Moravia, no recent records of D. immitis are available. Therefore, the present study was performed to clarify the occurrence of both species of Dirofilaria Railliet et Henry, 1910. Blood samples of 551 dogs sampled during 2015 and 2016 were analysed microscopically for presence of microfilariae and blood sera were examined by IDEXX SNAP® 4Dx® test (IDEXX, USA). DNA from blood of microscopically positive dogs was extracted and PCR protocol amplifying fragment of cytochrome c oxidase I (COI) gene was performed; PCR products were then sequenced. All dogs from the Bohemian part of the Czech Republic were negative. The prevalence of D. repens in the Moravian region was 5.7 % (27/476). BLAST analyses of obtained sequences confirmed the presence of D. repens (99-100% identical to KX265049). All sampled animals showed a negative result for D. immitis antigen in IDEXX SNAP® 4Dx® test. Our study confirmed the previously reported occurrence of D. repens in South Moravia and revealed its spreading from the epicentre to the north and west. PCR with subsequent sequencing together with negative results for D. immitis antigen in IDEXX SNAP® 4Dx® test revealed only D. repens infection. A previously published autochthonous infection of dogs with D. immitis in South Moravia was not confirmed.

Feline ophthalmomyiasis externa caused by Cuterebra larvae: four cases (2005-2020).

CASE SERIES SUMMARY: Described are four cats diagnosed with ophthalmomyiasis externa caused by Cuterebra larvae. Medical records were retrospectively reviewed to identify cats with ophthalmomyiasis externa between 2005 and 2020 at Cornell University Hospital for Animals. Signalment, history, clinical and diagnostic findings, treatment and outcome were recorded. All cats were young (< 3 years of age), had outdoor access and were initially examined during the summer months. All cases had unilateral disease with the right eye affected. Two cases had nictitating membrane lesions and two had orbital disease. Concurrent superficial corneal ulceration was present in three cats. Two cats suffered from pyrexia, suspected secondary to inflammation from the larval infestation. Successful larval removal was performed in all cats, which resulted in improvement of discomfort and clinical signs. A corneal ulcer persisted in one cat, which was lost to follow-up prior to ulcer resolution. Parasite identification confirmed Cuterebra species infestation in all cases. RELEVANCE AND NOVEL INFORMATION: To the authors' knowledge, this is the first report of feline ophthalmomyiasis externa caused by Cuterebra species. Parasite removal was successful in restoring comfort and resolving clinical signs in all cats with adequate follow-up information.

Spread of anthelmintic resistance in intestinal helminths of dogs and cats is currently less pronounced than in ruminants and horses - Yet it is of major concern.

Anthelmintic resistance (AR) has thus far only rarely been reported for intestinal helminths of dogs and cats, in contrast to parasites of livestock and horses. We highlight possible reasons for this striking and important discrepancy, including ecological, biological and genetic factors and/or intervention regimens of key intestinal helminths concerning both host groups. In view of the current knowledge related to the genetics, mechanisms and principles of AR development, we point at issues which in our view contribute to a comparatively lower risk of AR development in intestinal helminths of dogs and cats. Finally, we specify research needs and provide recommendations by which, based on the available information about AR in ruminant and equine helminths, the development of AR in dog and cat helminths may best be documented, prevented or at least postponed.

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.

Effectiveness of Credelio® Plus, a novel chewable tablet containing milbemycin oxime and lotilaner for the treatment of larval and immature adult stages of Toxocara canis in experimentally infected dogs.

BACKGROUND: The ascarid, Toxocara canis, is a common and important zoonotic intestinal nematode parasite that infects dogs globally. An effective treatment that kills any pre-patent stages of immature T. canis could additionally reduce or eliminate the development of patent infections that can result in clinical disease in infected dogs and would further reduce environmental contamination of eggs. Two randomized, blinded, GCP-compliant, pivotal laboratory dose confirmation studies were conducted to assess the effectiveness and safety of a new novel combination of lotilaner and milbemycin oxime tablets (Credelio Plus) administered orally to dogs that were experimentally infected with immature (L4 or immature adult [L5]) stages of T. canis. METHODS: The commercial tablet formulation of Credelio Plus® was administered in a time frame relative to inoculation with infective eggs. This allowed for effectiveness to be assessed against each specific immature stage of T. canis. In each study, dogs were randomized and allocated to one of four treatment groups. Each treatment group contained ten dogs that had been experimentally inoculated on Day 0 with infective T. canis eggs and then were dosed once on Day 14 or Day 24 using either placebo tablets or Credelio Plus tablets (IP) to provide minimum dosages of 0.75 mg/kg of milbemycin oxime and 20 mg/kg of lotilaner. All dogs were necropsied 5 or 6 days after their respective treatment. At necropsy, all nematodes recovered from the gastrointestinal tract were counted by species and stage. RESULTS: In both dose confirmation studies using geometric mean worm counts, effectiveness of Credelio Plus was ≥ 98.6% and ≥ 96.8% against L4 larval stage T. canis and immature adult [L5] T. canis in both studies, respectively. CONCLUSIONS: These studies demonstrated that the Credelio Plus combination tablet administered orally to dogs was highly efficacious against experimental infections with L4 and immature adult [L5] stages of T. canis.

Ascaris and Toxocara as foodborne and waterborne pathogens.

This is a review on the current status of Ascaris and Toxocara as agents of disease as a foodborne pathogen that is often transmitted onto produce via contaminated water. The paper introduces the parasites, their biology, disease caused, and mode of transmission. It summarizes recent research on methods for the detection of the parasites on crops, their prevalence in recent surveys of ready to eat plants in different parts of the world, and potential aspects of their means for remediation on vegetable crops. Also discussed is the ubiquitous nature of the presence of these eggs in the environment due to the many humans infected and means currently underway to control these parasites though improve water quality, sanitation, and hygiene.

Selected advanced imaging techniques were unable to quantify in vivo parasitic burden in heartworm-infested dogs.

This prospective exploratory study aimed to determine whether certain noninvasive advanced imaging techniques could estimate parasitic burden in heartworm-infested dogs; a noninvasive method is needed for ethical considerations and permitting longitudinal drug studies. Three cardiac-gated and respiratory-gated 3T MRI techniques and CT pulmonary angiography (CTPA) were performed in three healthy beagles to optimize imaging techniques. Once the imaging techniques were established, a pilot study was performed to determine which one of the MRI techniques would be used in an observer comparison study. Ultimately, spoiled gradient recalled (SPGR)-cine-MRI and CTPA were performed in four and five heartworm-infested dogs, respectively. Heartworms were detected in the pulmonary arteries in all dogs during SPGR-cine-MRI and in no dog during CTPA. However, counting the number of worms was unsuccessful. In conclusion, CTPA and SPGR-cine-MRI were unable to replace necropsy for quantifying parasitic burden in heartworm-infested dogs.

Acanthamoeba sclerokeratitis in a cat.

CASE DESCRIPTION: A 12-year-old neutered male domestic shorthair cat with chronic anterior uveitis and secondary glaucoma of the right eye was examined for persistent blepharospasm 2 weeks after corneal debridement and grid keratotomy for nonhealing superficial ulcerative keratitis. CLINICAL FINDINGS: Examination of the right eye revealed a central superficial corneal ulcer associated with corneal epithelial and subepithelial infiltrates and mild aqueous flare. Structures consistent with amoeboid cysts and trophozoites were detected in the cornea by in vivo confocal microscopy. Suppurative keratitis was identified cytologically. An Acanthamoeba spp was isolated through culture and identified by a PCR assay of corneal specimens. TREATMENT AND OUTCOME: Symptomatic and antiamoebic (polyhexamethylene biguanide 0.02% ophthalmic solution) treatments were instituted. Over the following 6 weeks, the cat lost vision in the affected eye and lesions progressed to nonulcerative stromal keratitis associated with a dense paracentral corneal stroma ring infiltrate and anterior lens luxation. The globe was enucleated, and lymphoplasmacytic sclerokeratitis, anterior uveitis, and retinal detachment were noted. Acanthamoeba organisms were detected within the corneal stroma and anterior sclera with histologic and immunohistochemical stains. The amoebae were classified to the Acanthamoeba T4 genotype by DNA sequencing. The cat had no medical problems attributed to Acanthamoeba infection over 36 months after enucleation, until the cat was lost to follow-up. CLINICAL RELEVANCE: Naturally acquired Acanthamoeba sclerokeratitis is described in a cat for the first time. Acanthamoeba infection should be considered for cats with superficial corneal disease refractory to appropriate treatments and especially occurring after ocular trauma, including keratotomy.

Parasites and vector-borne diseases disseminated by rehomed dogs.

The Companion Vector-Borne Diseases (CVBD) World Forum is a working group of leading international experts who meet annually to evaluate current scientific findings and future trends concerning the distribution, pathogenesis, clinical presentation, diagnosis and prevention of vector-borne infections of dogs and cats. At the 14th Symposium of the CVBD World Forum in Trieste, Italy (March 25-28, 2019), we identified the need to (i) bring attention to the potential spread of parasites and vectors with relocated dogs, and (ii) provide advice to the veterinary profession regarding the importance of surveillance and treatment for parasites and vector-borne infections when rehoming dogs. This letter shares a consensus statement from the CVBD World Forum as well as a summary of the problem faced, including the role of veterinary professionals in parasite surveillance, causal issues, and the importance of interdisciplinary cooperation in addressing the problem. To limit opportunities for dissemination of parasites and vectors, whenever possible, underlying problems creating the need for dog rehoming should be addressed. However, when it is necessary to rehome dogs, this should ideally take place in the country and national region of origin. When geographically distant relocation occurs, veterinary professionals have a vital role to play in public education, vigilance for detection of exotic vectors and infections, and alerting the medical community to the risk(s) for pathogen spread. With appropriate veterinary intervention, dog welfare needs can be met without inadvertently allowing global spread of parasites and their vectors.

History of Toxocara and the associated larva migrans.

This review covers the systematics and nomenclature of the Ascaridoid genus toxocara, and more specifically the species Toxocara canis and Toxocara cati. Also discussed is the discovery of the persistence of these larvae in the tissues of paratenic hosts, and the role that other species of this genus might or could play in other such hosts; including those where the life cycle has been described, i.e., Toxocara vitulorum, Toxocara pteropodis, Toxocara mackerrasae, and Toxocara tanuki. Also examined is the work that led to the realization that the larval stage leaving the egg actually being a third rather than a second stage larva. Also discussed on the work showing that the larvae can persist in paratenic host with remarkable longevity without undergoing any morphological change for years and that these larvae can be transmitted from one paratenic host to another by ingestion. People are usually infected by the ingestion of eggs containing third-stage larvae, but infections also occur on occasions from the ingestion of uncooked paratenic hosts.