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.

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.

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.

Comparative performance of reference laboratory tests and in-clinic tests for Giardia in canine feces.

BACKGROUND: We examined the performance of four in-clinic Giardia diagnostic tests by comparing results to three laboratory methods for detection of Giardia. A set of 177 fecal samples originally submitted to a commercial laboratory by veterinarians for routine ova and parasite (O&P) testing was used. Specimens were examined by direct immunofluorescence assay (DFA) for presence of Giardia cysts which served as the gold standard. Fecal samples were tested using a Giardia-specific cyst wall antigen microtiter plate format enzyme-linked immunosorbent assay (ELISA) and each of the in-clinic assays adhering to the package insert for each kit. RESULTS: Evaluated were four in-clinic antigen test kits: VetScan® Canine Giardia Rapid Test (Abaxis), Anigen® Rapid CPV-CCV-Giardia Antigen Test (BioNote), SNAP® Giardia Test (IDEXX) and Witness® Giardia Test (Zoetis). In the comparison of the in-clinic tests to the DFA standard test sensitivity ranged between 70.0-87.1%, and specificity ranged between 71.1-93.4%. CONCLUSION: Of the tests evaluated here, the SNAP test had the highest sensitivity and specificity. The SNAP test had the highest percent positive and percent negative agreement when compared to the microtiter plate format ELISA and the O&P assay.

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.

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

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

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.

Efficacy of a novel topical combination of fipronil, (S)-methoprene, eprinomectin and praziquantel against induced infections of Ancylostoma spp. nematodes of cats.

Four studies were conducted to examine the efficacy of a novel topical combination of fipronil, (S)-methoprene, eprinomectin, and praziquantel (BROADLINE(®), Merial) against Ancylostoma tubaeforme and Ancylostoma braziliense hookworms of cats. In each study, purpose-bred cats were randomly assigned to treatment groups of 10 or 12 cats per group. In three studies the cats were inoculated with A. tubaeforme and in one study with A. braziliense. The inoculations were undertaken on a schedule which resulted in the hookworms reaching the fourth larval stage in two of the studies, or the adult stage in four of the studies, by the day of treatment. In each study there was also an untreated control and 1 or 2 groups treated with the novel combination. In the two studies where efficacy against the fourth larval stage of A. tubaeforme was tested, the efficacy recorded was 100%. In the three studies where efficacy against the adult stage of A. tubaeforme was tested, efficacy of 100% was also confirmed. In the study where efficacy against the adult stage of A. braziliense was tested efficacy was 99.5%.

First report of Dracunculus insignis in two naturally infected cats from the northeastern USA.

Dracunculiasis is rarely reported in cats, yet over the last few years we have identified two cats with filarioid-like spirurid infections. Case 1 was a 9-year-old cat with pituitary-dependent hyperadrenocorticism from New York tate from which four adult dracunculoid nematodes were isolated from its torso. Based on morphometric characteristics and parasite geographic distribution, the specimens were identified as Dracunculus insignis females; at least one of the females was gravid, suggestive of patent infection. Species identification was confirmed through amplification and sequence analysis of nuclear and mitochondrial loci. Case 2 was a 14-year-old diabetic cat from Massachusetts. Formalin-fixed sections were obtained from a subcutaneous mass excised from the left foreleg. Histopathological examination revealed a large nematode with morphometrical characteristics of Dracunculus, surrounded by lymphocytes and sheets of eosinophils. These two cases appear to be the first published reports of dracunculiasis in domestic cats in the USA, and based on the findings from case 1, D insignis may be the species associated with both infections.

Detecting Aelurostrongylus abstrusus-specific IgG antibody using an immunofluorescence assay.

Diagnosis of feline lungworm, Aelurostrongylus abstrusus, is typically achieved by identifying larvae in feces following concentration through flotation or using the Baermann technique. This work presents observations on the usefulness of an indirect immunofluorescence antibody assay for detection of antibodies to this parasite in the sera of infected cats. Using first-stage larvae of A abstrusus and sera from both experimentally and naturally infected cats, it was determined that the test was fairly sensitive and did not cross-react with serum from an Ancylostoma braziliense (hookworm)-infected cat.

Comparison of Ancylostoma caninum worm counts acquired by endoscopy and necropsy.

Many regulatory agencies require that the efficacy of veterinary anthelmintic medications be evaluated by enumerating parasites in treated and untreated animals after necropsy. Current ethical considerations, i.e., the 3 Rs of research, call for the replacement of this method with less invasive techniques that would not require animal sacrifice. This study tested standard gastrointestinal endoscopy as an in vivo method of quantifying the intestinal hookworm, Ancylostoma caninum. Worm counts were compared with those from gold standard necropsy. Thirteen dogs inoculated with third-stage A. caninum larvae underwent endoscopy 4-6 weeks post-infection, just prior to necropsy. Two-thirds of the adult hookworms were located in the middle section of the small intestine that could not be reached for endoscopic examination. Not surprisingly, the total worm counts obtained by endoscopy did not correlate with those from necropsy (R(2)=0.05, p=0.464). One method to increase small intestinal access would be to use specialized balloon or spiral endoscopes developed for this purpose in human gastroenterology. Based on the results of this study, standard endoscopy alone is unsuitable for quantification of A. caninum in the small intestine. Parasites in more accessible sites, such as whipworms in the cecum and colon, might be more appropriate targets for endoscopic counting.

Investigation of an outbreak of besnoitiosis in donkeys in northeastern Pennsylvania.

OBJECTIVE: To describe the clinical, endoscopic, and serologic features of an outbreak of besnoitiosis in 2 donkey operations in northeastern Pennsylvania and to report the outcome of attempted treatment of 1 naturally infected individual. DESIGN: Observational study. ANIMALS: 29 donkeys (Equus asinus) in northeastern Pennsylvania. PROCEDURES: Donkeys were examined for lesions suggestive of besnoitiosis in an outbreak investigation. Information was collected regarding the history and signalment of animals on each premises. Rhinolaryngoscopy was performed to identify nasopharyngeal and laryngeal lesions. Serum samples were collected for immunofluorescent antibody testing and immunoblotting for Besnoitia spp. Skin biopsy samples were obtained from 8 animals with lesions suggestive of besnoitiosis for histologic examination. Quantitative real-time PCR assay for Besnoitia spp was performed on tissue samples from 5 animals. RESULTS: Besnoitiosis was confirmed in 6 of the 8 suspected cases. The most common lesion site was the nares, followed by the skin and sclera. Donkeys with clinical signs of disease had higher serum antibody titers and tested positive for a greater number of immunoblot bands than did donkeys without clinical signs of disease. All animals evaluated by PCR assay tested positive. Putative risk factors for disease included age and sex. Ponazuril was not effective at treating besnoitiosis in a naturally infected donkey. CONCLUSIONS AND CLINICAL RELEVANCE: Knowledge of clinical and serologic features of besnoitiosis in donkeys will assist clinicians in the diagnosis and prevention of this disease in donkey populations. Besnoitiosis may be an emerging disease of donkeys in the United States.

Serological survey of Toxoplasma gondii, Dirofilaria immitis, Feline Immunodeficiency Virus (FIV) and Feline Leukemia Virus (FeLV) infections in pet cats in Bangkok and vicinities, Thailand.

The seroprevalence of Toxoplasma gondii, Dirofilaria immitis (heartworm), feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) infections was examined using serum or plasma samples from 746 pet cats collected between May and July 2009 from clinics and hospitals located in and around Bangkok, Thailand. The samples were tested for heartworm, FIV, and FeLV using a commercial ELISA. Of the 746 samples, 4.6% (34/746) were positive for heartworm antigen, 24.5% (183/746) had circulating FeLV antigen, and 20.1% (150/746) had antibodies against FIV. In addition, the first 348 submitted samples were tested for T. gondii antibodies using a modified agglutination test (MAT, cut off 1:25); 10.1% (35/348) were seropositive. Of the 348 cats sampled for all four pathogens, 11, 10, and 1 were positive for T. gondii antibodies and FIV antibodies, FeLV antigen, or D. immitis antigen, respectively. Of the 35 T. gondii-seropositive cats, 42.9% (15/35) were co-infected with at least one of the other three pathogens. The presence of antibodies to FIV was significantly associated with both age and gender, while FeLV antigen presence was only associated with age. In the case of FIV, males were twice as likely to be infected as females, and cats over 10 years of age were 13.5 times more likely to be infected than cats less than 1 year of age. FeLV antigen was more common in younger cats, with cats over 10 years of age being 10 times less likely to be FeLV positive than cats under 1 year of age. This is the first survey for these four pathogens affecting feline health in Thailand.

Prevalence of fecal-borne parasites detected by centrifugal flotation in feline samples from two shelters in upstate New York.

Over a 3.5-year period, fecal samples from 1322 cats from two shelters and affiliated foster homes in upstate New York were processed for parasite detection by both 1.18spg zinc sulfate and 1.3spg sugar double centrifugal flotation. In 50.9% of the samples at least one parasite was detected. Overall, 18 different parasites ranging in prevalence from 0.2% to 21% were recovered. The most prevalent parasites of foster and shelter cats in this study were Cystoisospora species and Toxocara cati (21% prevalence, each). In order of percentage of positive samples, other findings were: Giardia species (8.9%), Aelurostrongylus abstrusus (6.2%), taeniid eggs (3.9%), Cryptosporidium species (3.8%), Aonchotheca species (3.7%), Eucoleus species (2.3%), Ancylostoma species (2.2%), Cheyletiella species (2.0%), Dipylidium caninum (1.1%), Otodectes species, Toxoplasma-like oocysts and Sarcocystis species (0.8% each), Demodex and Spirometra species (0.4% each), and Alaria species and Felicola subrostratus (0.2% each).

Cryptosporidiosis and giardiasis in dogs and cats: veterinary and public health importance.

Dogs and cats are the only domestic animals that still routinely reside in the same domicile as their owners around the world, and hence the interest in their role as reservoirs of potentially zoonotic agents. In the case of cryptosporidiosis and giardiasis, current data suggests that dogs and cats do not routinely share their infections with healthy people. Dogs are hosts of Cryptosporidiumcanis and Giardiaduodenalis Assemblages C and D. Cats are hosts to Cryptosporidiumfelis and G. duodenalis Assemblage F. Dogs and cats (and other animals) are sometimes infected with sub-Assemblage AI, an Assemblage also found in people, but people usually have sub-Assemblage AII. Unfortunately, severely immunocompromised individuals and malnourished children can be made ill by infections with C. canis and C. felis. People should practice good sanitation and hygiene to minimize environmental contamination and contact with the infectious (oo)cysts that may be shed by their pets.

Heartworm biology, treatment, and control.

This article is a review of the systematics, taxonomy, biology, prevention, control, and treatment of the canine heartworm, Dirofilaria immitus. This filarioid parasite remains one of the most important and dangerous diseases of the dog throughout the United States. The geographic range of the parasite is expanding, and in many parts of the country it has emerged as a threat to canine welfare only in the last 50 or so years. The article also discusses the pathophysiological mechanisms behind the disease induced, the means for diagnosing the disease, and the means of assessing the success of therapy. The treatment of potential complications of heartworm infection, such as post-adulticide thromboembolism, eosinophilic granulomatous pneumonitis, and caval syndrome, is also discussed.

Prevalence of intestinal parasites in pet dogs in the United States.

To determine the national, regional, and age-related prevalence of intestinal parasites in dogs presenting to veterinarians in the United States, we reviewed the results of examination via zinc sulfate centrifugal flotation of 1,199,293 canine fecal samples submitted to Antech Diagnostics in 2006. The most commonly identified intestinal parasites were ascarids (2.2%), hookworms (2.5%), whipworms (1.2%), Giardia (4.0%), and Cystoisospora (4.4%). With the exception of whipworms, intestinal parasites were more commonly identified in dogs less than 6 months of age (29.6% positive) as compared to those greater than 1 year of age (6.1% positive) although infections with each parasite considered were identified in all age classes of dogs. Hookworm eggs were most commonly identified in fecal samples submitted from dogs from the South (4.0% positive), whereas ascarid eggs and Giardia cysts were most commonly seen in samples from dogs from the West (2.8% and 6.3% positive, respectively). When compared to previous data from shelter dogs, the prevalence of intestinal helminths, particularly ascarids and hookworms, was greatly suppressed in pet dogs in the southern United States (90-91% reduction) and much less so in dogs in the West (52-78% reduction), perhaps due in part to the routine year-round use of monthly anthelmintics effective at controlling both heartworm infection and intestinal helminths in dogs in the South. Taken together these data indicate that intestinal parasites remain a common, important finding in dogs presenting to veterinary practices although in most of the country infection rates in pet dogs appear to be greatly reduced from the level reported from dogs in animal shelters.

Treatment of naturally occurring, asymptomatic Giardia sp. in dogs with Drontal Plus flavour tablets.

The administration of three consecutive daily doses of the recommended 1x dose of Drontal Plus flavour tablets (Bayer) was examined for its effect on Giardia sp. cyst-shedding in 7 treated and 7 untreated random-source dogs. Dogs were treated on study days 0, 1 and 2. Cysts were quantified using direct immunofluorescent labelling on days -7, -5, -3 and -2, and daily from day 1 through 11. Three treated dogs never shed cysts again during the study, one shed again only on day 4, and the remaining three dogs started to shed again on days 8, 9 and 11. The mean numbers of cysts per gramme in the faeces of the treated dogs were significantly reduced (t-tests using log(10)(counts)) on days 1 and 2 (geometric means: controls = 447,000; treated = 1,050; p = 0.004) and days 3 to 8 (geometric means: controls = 23,400; treated 5.0; p < 0.001). Four controls that had been consistently positive, changed to negative status on day 11, and thus, on the final day of the trial, there were only three positive control and three positive treated dogs. Three consecutive days of treatment with Drontal Plus flavour tablets halted Giardia sp. cyst shedding by dogs. But starting six days post third treatment, some of the dogs started shedding cysts again. Since the prepatent period of Giardia sp. can be as short as 4 days, shedding of Giardia sp. cysts 6 days after treatment could be caused by a reinfection.