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.

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.

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.

Canine Leishmaniasis Control in the Context of One Health.

Dogs are the main reservoir of Leishmania infantum and in some countries have been regularly culled as part of government policy to control visceral leishmaniasis. At the 13th Symposium of the Companion Vector-Borne Diseases World Forum in Windsor, UK, March 19-22, 2018, we consolidated a consensus statement regarding the usefulness of dog culling as a means of controlling visceral leishmaniasis. The statement highlighted the futility of culling infected dogs, whether healthy or sick, as a measure to control the domestic reservoir of L. infantum and reduce the risk for visceral leishmaniasis.

Inactivation of Ascaris Eggs in Human Fecal Material Through In Situ Production of Carboxylic Acids.

Discovering new ways to inactivate pathogens in human waste is critical for the improvement of worldwide access to sanitation and for the reduction of the environmental impact of conventional waste treatment processes. Here, we utilized the carboxylate platform and chain elongation to produce n-butyric acid and n-caproic acid via the anaerobic fermentation of human fecal material. Then, we inactivated Ascaris eggs through exposure to these carboxylic acids. Using batch experiments with human fecal material as substrate, we accumulated n-butyric acid and n-caproic acid at total concentrations (uncharged acid plus conjugate base) of 257 and 27.1 mM, respectively. We then showed that carboxylic acids at these concentrations inactivated Ascaris eggs when the pH was below the pKa for the acids, causing them to exist primarily in the uncharged forms. We observed that uncharged carboxylic acids affected viability rather than the pH itself or conjugate bases. In addition, we modeled the viability of Ascaris eggs as a function of uncharged carboxylic acid concentration for n-butyric acid and n-caproic acid at exposure times of 2, 6, 12, and 20 days. The results presented here indicate that in situ biological production of carboxylic acids in HFM provides a promising method of pathogen inactivation and may lead to new developments in sanitation technology and treatment of fecal sludge.

Explaining premunition with Kin selection using Haemonchus contortus.

Premunition is the state in a disease where an existing infection protects the host from reinfection with the same species. The cause of premunition is not clearly understood. In this study, we hypothesized that kin-selection might be a contributing factor in premunition. To test this theory, sheep were infected either once with a linguiform or smooth vulval morphotype of Haemonchos contortus, twice with the same morphotype or twice with different morphotypes. All infections resulted in a similar number of adult parasites. However, there were differences in the morphotypes recovered providing potential evidence of kin selection. Negative interference competition might also contribute to the reduction of the incoming population. Allelopathic or physical interactions between the parasites may be the mechanism behind the observed phenomena.

Conjunctival habronemiasis in a square-lipped rhinoceros (Ceratotherium simum).

A captive female square-lipped rhinoceros born in 1993 had been showing intermittent signs of bilateral conjunctivitis and conjunctival proliferation since 1998. Periodic improvement was noted, especially in winter, but overall the condition had deteriorated over the years. Treatment with various topical, intralesional, and systemic antibiotics and glucocorticosteroids was largely ineffective, as were repeated dewormings. No primary cause for these lesions was found in biopsies taken in 2000 and 2006, although a severe infiltrate of numerous eosinophils was observed in the latter. As the condition worsened, secondary corneal changes were noted, and eventually vision was lost due to proliferative conjunctival tissue. Aggressive resection of the proliferating tissue in 2013 restored vision and submitted biopsies yielded a diagnosis of severe allergic conjunctivitis, eosinophilic granuloma, and habronematid (Habronema or Draschia) larval infection. As no other rhinoceros in the herd was affected, including two calves born to the patient who were in close contact with their mother, it was concluded the presentation was most likely due to a hypersensitivity reaction to the dead or dying larvae. Fly repellent is now regularly applied around the eye of this rhinoceros, and a protective face mask has been fitted. Ongoing periodic relapses are treated with oral ivermectin, topical antibiotics, and steroids.

AAVP Recommendations for Core Competency Standards Relating to Parasitological Knowledge and Skills.

As part of the accreditation process, the American Veterinary Medical Association Council on Education has defined nine broad areas of core competencies that must be met by graduating students earning a Doctor of Veterinary Medicine degree. To define competencies in veterinary parasitology, the American Association of Veterinary Parasitologists (AAVP) has developed a detailed list of knowledge and skills that are recommended for inclusion in professional curricula. These recommendations were developed by instructors from colleges/schools of veterinary medicine in the US, Canada, and the Caribbean, and were reviewed and endorsed following AAVP guidelines.

Canine Infections with Onchocerca lupi Nematodes, United States, 2011-2014.

Infections with Onchocerca lupi nematodes are diagnosed sporadically in the United States. We report 8 cases of canine onchocercosis in Minnesota, New Mexico, Colorado, and Florida. Identification of 1 cytochrome c oxidase subunit 1 gene haplotype identical to 1 of 5 from Europe suggests recent introduction of this nematode into the United States.

Laboratory Evaluation of the Efficacy of 10 % Imidacloprid + 2.5 % Moxidectin Topical Solution (Advantage® Multi, Advocate®) for the Treatment of Dirofilaria immitis Circulating Microfilariae in Dogs.

This study examined the efficacy of 10 % imidacloprid + 2.5 % moxidectin topical solution (Advantage ® Multi, Advocate®, Bayer) for the treatment of circulating microfilariae from dogs naturally infected with Dirofilaria immitis. The study included two groups of 11 dogs each that consisted of two replicates. Replicate 1 contained 12 dogs (6 treated and 6 controls) and replicate 2 contained 10 dogs (5 treated and 5 controls). Six of the 10 dogs in replicate 2 were the controls from replicate 1. All dogs entering the study completed a physical examination including chest radiographs, blood collections for examination of Dirofilaria immitis circulating microfilariae, serum chemistry, complete blood counts and urinalysis. To qualify for the study each dog was required to have a geometric mean ≥ 300 microfilariae per ml of blood from 3 consecutive samples collected during the 8 day acclimation period and a heartworm disease classification of 1 or 2. Dogs were treated on study days 0 and 28. Post-treatment microfilarial counts were performed on study days 1, 2, 3, 7, 14, 21, 28, 29, 35, and 42. Percent microfilarial reduction was determined by comparing the geometric mean number of circulating microfilaria remaining in treated dogs with those remaining in the control dogs post-treatment. Seven days after the first treatment, the geometric mean microfilarial counts in treated dogs were reduced by > 99 % compared to the control dogs. Reduction remained at > 99 % through the end of the study at 42 days after the first treatment (14 days after the second treatment). The results of this study demonstrated that Advantage® Multi for dogs is efficacious for treatment of circulating D. immitis microfilariae in naturally infected heartworm-positive dogs with no treatment-related adverse events observed.

Ophthalmomyiasis interna anterior in a dog: keratotomy and extraction of a Cuterebra sp. larva.

A 1-year-old female spayed mixed-breed dog was presented for evaluation of acute onset of lethargy and blepharospasm OD. Slit-lamp biomicroscopy revealed a segmented larva embedded in the inferonasal iris. Moderate anterior uveitis was present. After pharmacologic dilation, mild vitreal hemorrhage and chorioretinal migration tracks were identified. The dog was treated empirically for uveitis for 5 days, after which a keratotomy and larval foreign body extraction was performed. The dog recovered uneventfully from intraocular surgery and remained visual and comfortable. Parasite evaluation confirmed the larva to be a Cuterebra sp. To the authors' knowledge, this is the first report of canine ophthalmomyiasis interna anterior, and the first report of successful surgical removal from the anterior segment in a dog with preservation of vision.

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%.

Description of fecal shedding of Cryptosporidium parvum oocysts in experimentally challenged dairy calves.

The objective was to describe the probability of Cryptosporidium parvum fecal oocyst shedding at different magnitudes of exposure, the pattern of fecal shedding over time, and factors affecting fecal shedding in dairy calves. Within the first 24 h of life, 36 calves were experimentally challenged with C. parvum oocysts at one of four possible magnitudes of oral exposure (1 × 10(3), 1 × 10(4), 1 × 10(5), and 1 × 10(6) oocysts), and 7 control calves were sham dosed. Fecal shedding occurred in 33 (91.7 %) experimentally challenged calves and in none of the control calves. There was a difference in the log-total number of oocysts counted per gram of feces dry weight among the four exposure groups; calves with the lowest magnitude of exposure (1 × 10(3) oocysts) shed less than the other three groups. At higher magnitudes of exposure, there was more variability in the range of fecal oocyst shedding. There was an inverse relationship between the log-total amount of oocysts counted per gram of feces dry weight and the number of days to the onset of fecal shedding per calf, i.e., the more time that elapsed to the onset of fecal shedding, the fewer oocysts that were shed. The pattern of fecal shedding over time for all calves shedding oocysts was curvilinear; the number of oocysts increased with time, reached a peak, and declined. Therefore, the dynamics of oocyst shedding can be influenced in part by limiting exposure among calves and delaying the onset of fecal oocyst shedding.

Cryptosporidium tyzzeri and Cryptosporidium pestis: which name is valid?

The dispute on the validity of Cryptosporidium pestis and Cryptosporidium tyzzeri origins from the uncertainty on the identity of Cryptosporidium parvum described by Tyzzer in 1912 and the interpretation of the Principal of Priority of the International Code of Zoological Nomenclature (ICZN). Using a rigid interpretation of the Principal of Priority, one researcher proposed to rename C. parvum as C. pestis and retain C. parvum for Cryptosporidium mouse genotype I on the basis that Tyzzer was probably describing mouse genotype I. However, the ICZN clearly states that the Principle of Priority is to be used to promote stability and is not intended to upset a long-accepted name. Because mice are known to be naturally infected with C. parvum, and the 1985 taxonomic re-description of C. parvum for bovine and human isolates is accepted by almost all Cryptosporidium researchers, the prevailing name C. parvum for the species infective to calves and humans must be retained to avoid confusion. The designation of C. tyzzeri for the mouse genotype I brings further clarity to the taxonomy of Cryptosporidium spp. in humans, cattle, and domestic mice.

Protozoan parasites in group-living primates: testing the biological island hypothesis.

A series of articles by W.J. Freeland published in the 1970s proposed that social organization and behavioral processes were heavily influenced by parasitic infections, which led to a number of intriguing hypotheses concerning how natural selection might act on social factors because of the benefits of avoiding parasite infections. For example, Freeland [1979] showed that all individuals within a given group harbored identical gastrointestinal protozoan faunas, which led him to postulate that social groups were akin to "biological islands" and suggest how this isolation could select specific types of ranging and dispersal patterns. Here, we reexamine the biological island hypothesis by quantifying the protozoan faunas of the same primate species examined by Freeland in the same location; our results do not support this hypothesis. In contrast, we quantified two general changes in protozoan parasite community of primates in the study area of Kibale National Park, Uganda, over the nearly 35 years between sample collections: (1) the colobines found free of parasites in the early 1970s are now infected with numerous intestinal protozoan parasites and (2) groups are no longer biological islands in terms of their protozoan parasites. Whatever the ultimate explanation for these changes, our findings have implications for studies proposing selective forces shaping primate behavior and social organization.

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.

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.

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.

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.

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.