Retrospective survey of endoparasitism identified in feces of client-owned dogs in North America from 2007 through 2018.

Our main study objective was to determine the prevalence and trend of parasitic infection in client-owned dogs examined at the veterinary parasitology diagnostic laboratory of Oklahoma State University over the past 12 years. All results of centrifugal flotation, saline direct smear, sedimentation, Baermann, acid-fast staining for Cryptosporidium detection, and Giardia antigen examinations on fecal samples from client-owned dogs submitted to the Boren Veterinary Medical Hospital and Oklahoma Animal Disease Diagnostic Laboratory of Oklahoma State University from 2007 through 2018 were included. The impact of sex, age, and seasonality on the prevalence of parasitic infection was also statistically evaluated. A total of 7,409 cases were included for this study. Majority of cases (79.58%; 5,896/7,409) did not include any parasites, eggs, larva, oocysts, or cysts. Approximately 15.41% (1,142/7,409) of client-owned dogs were infected by at least one parasite, and 5.01% (371/7,409) of dogs were infected by multiple parasites. The most common parasite stage observed was Ancylostoma eggs (8.23%; 610/7,409), followed by Cystoisospora oocysts (5.02%; 372/7,409), Giardia cysts/antigen (4.06%; 301/7,409), Trichuris vulpis eggs (2.74%; 203/7,409), Toxocara canis eggs (2.54%; 188/7,409), Dipylidium caninum proglottids/egg packets (0.84%; 62/7,409), taeniid proglottids/eggs (0.47%; 35/7,409), Sarcocystis sporocysts (0.38%; 28/7,409), Cryptosporidium oocysts (0.30%; 22/7,409), Strongyloides stercoralis larvae (0.20%; 15/7,409), Alaria eggs (0.19%; 14/7,409), Toxascaris leonina eggs (0.18%; 13/7,409), Capillaria eggs (0.16%; 12/7,409), Hammondia-like small coccidian oocysts (0.16%; 12/7,409), Uncinaria stenocephala eggs (0.13%; 10/7,409), Spirometra eggs (0.09%; 7/7,409), Physaloptera eggs (0.09%; 7/7,409), Heterobilharzia americana eggs (0.08%; 6/7,409), Nanophyetus salmincola eggs (0.08%; 6/7,409), trichomonads (0.08%; 6/7,409), Mesocestoides proglottids/eggs (0.05%; 4/7,409), Baylisascaris eggs (0.01%; 1/7,409), Macracanthorhynchus eggs (0.01%; 1/7,409), and Paragonimus kellicotti eggs (0.01%; 1/7,409). In addition to endoparasites, some ectoparasites, such as Demodex mites (0.22%; 16/7,409), Otodectes cynotis mites (0.01%; 1/7,409), Rhipicephalus sanguineus ticks (0.01%; 1/7,409), and Sarcoptes scabiei mites (0.01%; 1/7,409), were detected by fecal examinations. Pseudo/spurious parasites were identified in approximately 4.35% of cases (322/7,409). There was no statistically significant difference for parasite prevalence between sexes (p = 0.3231). However, statistically significant differences were observed with certain parasites when compared by age groups, and generally, prevalence of parasitism decreased as age of client-owned dogs increased (p < 0.0001). Statistical analyses also revealed significant differences by months (p = 0.0013). Overall, the prevalence of parasitic infection in client-owned dogs decreased over the past 12 years (p < 0.0001).

Mineralized collagen scaffolds fabricated with amniotic membrane matrix increase osteogenesis under inflammatory conditions.

Defects in craniofacial bones occur congenitally, after high-energy impacts, and during the course of treatment for stroke and cancer. These injuries are difficult to heal due to the overwhelming size of the injury area and the inflammatory environment surrounding the injury. Significant inflammatory response after injury may greatly inhibit regenerative healing. We have developed mineralized collagen scaffolds that can induce osteogenic differentiation and matrix biosynthesis in the absence of osteogenic media or supplemental proteins. The amniotic membrane is derived from placentas and has been recently investigated as an extracellular matrix to prevent chronic inflammation. Herein, we hypothesized that a mineralized collagen-amnion composite scaffold could increase osteogenic activity in the presence of inflammatory cytokines. We report mechanical properties of a mineralized collagen-amnion scaffold and investigated osteogenic differentiation and mineral deposition of porcine adipose-derived stem cells within these scaffolds as a function of inflammatory challenge. Incorporation of amniotic membrane matrix promotes osteogenesis similarly to un-modified mineralized collagen scaffolds, and increases in mineralized collagen-amnion scaffolds under inflammatory challenge. Together, these findings suggest that a mineralized collagen-amnion scaffold may provide a beneficial environment to aid craniomaxillofacial bone repair, especially in the course of defects presenting significant inflammatory complications.

Retrospective survey of parasitism identified in feces of client-owned cats in North America from 2007 through 2018.

Ownership of domestic cats in North America has been on the increase; however, there are only a few surveys conducted on the prevalence of parasitism in client-owned cats over years. Our study objective was to evaluate parasite prevalence through statistical analysis of fecal examination results for client-owned cats on samples submitted to the veterinary parasitology diagnostic laboratory of Oklahoma State University over a 12-year period. All results of centrifugal flotation, saline direct smear, sedimentation, and Baermann examinations on fecal samples submitted to the Boren Veterinary Medical Hospital and Oklahoma Animal Disease Diagnostic Laboratory of Oklahoma State University from 2007 through 2018 were included. The impacts of sex, age, and seasonality on the prevalence of infection were also analyzed. A total of 2586 client-owned cat cases were included for this study. Parasites, eggs, oocysts, larvae, or cysts were not detected in the majority of cases (75.5%; 1,953/2586). Approximately 18.8% (485/2586) of client-owned cats were infected by one parasite, and 5.7% (148/2586) of cats were infected by multiple parasites. The most common parasite stage observed was Cystoisospora oocysts (9.4%; 243/2586), followed by Toxocara cati eggs (7.8%; 202/2586), Giardia cysts (4.0%; 104/2586), Alaria eggs (3.5%; 91/2586), Ancylostoma eggs (1.2%; 32/2586), taeniid proglottids/eggs (1.2%; 30/2586), Dipylidium caninum proglottids/egg packets (1.1 %; 29/2586), and Eucoleus aerophilus eggs (0.7%; 18/2586). Less commonly, Physalopetra eggs (0.19%; 5/2586), Toxascaris leonina eggs (0.19%; 5/2586), Tritrichomonas blagburni trophozoites (0.15%; 4/2586), Ollulanus tricuspis larvae/adults (0.12%; 3/2586), Platynosomum fastosum eggs (0.12%; 3/2586), Aelurostrongylus abstrusus larvae (0.08%; 2/2323), Sarcocystis sporocysts (0.08%; 2/2586), Spirometra eggs (0.08%; 2/2586), Mesocestoides proglottids/eggs (0.08%; 2/2586), Trichuris felis eggs (0.08%; 2/2586), Cryptosporidium oocysts (0.04%; 1/2586), and Toxoplasma-like small coccidian oocysts (0.04%; 1/2586) were detected. Additionally, fecal examinations revealed some ectoparasites: Demodex mites (0.9%; 24/2586), Cheyletiella mites (0.15%; 4/2586), and Otodectes cynotis mites (0.04%; 1/2586). There was no statistical significance between different sex groups (p = 0.3316). Age affected the prevalence of Cystoisospora, T. cati, Giardia, and Alaria infections with prevalence decreasing as age increased (p < 0.0001). Statistical analyses also revealed significant differences by months; the higher prevalence of infection occurred from summer through fall (p = 0.0004). Overall, as the number of submittals increased, the prevalence of infection increased over the last 12 years (p < 0.0001). This study supports continuing the current practice of routine broad-spectrum anthelmintic and ectoparasitic treatments for client-owned cats as well as annual/biannual fecal examination.

Shape-fitting collagen-PLA composite promotes osteogenic differentiation of porcine adipose stem cells.

Craniomaxillofacial bone defects can occur as a result of congenital, post-oncologic, and high-energy impact conditions. The scale and irregularity of such defects motivate new biomaterials to promote regeneration of the damaged bone. We have recently described a mineralized collagen scaffold capable of instructing stem cell osteogenic differentiation and new bone infill in the absence of traditional osteogenic supplements. Herein, we report the integration of a millimeter-scale reinforcing poly (lactic acid) frame fabricated via 3D-printing into the mineralized collagen scaffold with micron-scale porosity to form a multi-scale mineralized collagen-PLA composite. We describe modifications to the PLA frame design to increase the compressive strength (Young's Modulus, ultimate stress and strain) of the composite. A critical challenge beyond increasing the compressive strength of the collagen scaffold is addressing challenges inherent with the irregularity of clinical defects. As a result, we examined the potential for modifying the frame architecture to render the composite with increased compressive strength in one axis or radial compressibility and shape-fitting capacity in an orthogonal axis. A library of mineralized collagen-PLA composites was mechanically characterized via compression testing and push-out test to describe mechanical performance and shape-fitting capacity. We also report in vitro comparison of the bioactivity of porcine adipose derived stem cells in the mineralized collagen-PLA composite versus the mineralized collagen scaffold via metabolic activity, gene expression, and functional matrix synthesis. The results suggest that incorporation of the PLA reinforcing frame does not negatively influence the osteoinductive nature of the mineralized collagen scaffold. Together, these findings suggest a strategy to address often competing bioactivity, mechanical strength, and shape-fitting design requirements for biomaterials for craniomaxillofacial bone regeneration.

Fecal survey of parasites in free-roaming cats in northcentral Oklahoma, United States.

Cats can be infected by various intestinal parasites, some that are zoonotic. Although surveys of parasite prevalence in owned and shelter cats have been published, none addressed free-roaming, wild-trapped, domestic cat (Felis catus) populations. An opportunity to determine the prevalence of intestinal parasites in wild-trapped, free-roaming cats in northcentral Oklahoma, United States occurred through a trap-neuter-return (TNR) program conducted at Oklahoma State University, Boren Veterinary Medical Hospital, between February 2015 and April 2016. Approximately 1 g to 5 g of feces was collected from 846 free-roaming cats either from cage traps, when available, or rectally using disposable fecal loops and examined by centrifugal fecal flotation tests with 33% zinc sulfate solution. Parasite infections were confirmed by microscopic detection of eggs, cysts, or oocysts and visual detection of proglottids. Approximately 63.9% (541/846) of free-roaming cats were infected by at least one parasite, and 24.9% (211/846) of cats were infected by multiple parasites. The most common intestinal parasite infections detected were: Toxocara cati (44.6%; 377/846), followed by Alaria (13.4%; 113/846), Ancylostoma (11.2%; 95/846), Cystoisospora (9.7%; 82/846), taeniids (7.7%; 65/846), Dipylidium caninum (4.5%; 38/846), Physaloptera (2.2%; 19/846), Eucoleus aerophilus (1.4%; 12/846), Giardia (1.2%; 10/846), and a small (10-12 µm in diameter) Toxoplasma-like oocyst (0.1%; 1/846). A few ectoparasites, Demodex gatoi (0.5%; 4/846) and Cheyletiella (0.1%; 1/846), were also detected by fecal flotation. Our findings indicate that a higher prevalence of parasite infections occurs in free-roaming cats in Oklahoma than in owned cats, and these free-roaming cats contribute to contamination of the environment with several zoonotic parasites. Regional data on the prevalence of parasites in free-roaming cats can aid in the justification for parasite control programs in owned cats since both can share the same environment and supports the current practice of routine broad-spectrum anthelmintic and ectoparasite treatments for owned cats.

Comprehensive Audiometric Analysis of Hearing Impairment and Tinnitus After Cisplatin-Based Chemotherapy in Survivors of Adult-Onset Cancer.

PURPOSE: Cisplatin is widely used but highly ototoxic. Effects of cumulative cisplatin dose on hearing loss have not been comprehensively evaluated in survivors of adult-onset cancer. PATIENTS AND METHODS: Comprehensive audiological measures were conducted on 488 North American male germ cell tumor (GCT) survivors in relation to cumulative cisplatin dose, including audiograms (0.25 to 12 kHz), tests of middle ear function, and tinnitus. American Speech-Language-Hearing Association criteria defined hearing loss severity. The geometric mean of hearing thresholds (0.25 to 12 kHz) summarized overall hearing status consistent with audiometric guidelines. Patients were sorted into quartiles of hearing thresholds of age- and sex-matched controls. RESULTS: Increasing cumulative cisplatin dose (median, 400 mg/m(2); range, 200 to 800 mg/m(2)) was significantly related to hearing loss at 4, 6, 8, 10, and 12 kHz (P trends, .021 to < .001): every 100 mg/m(2) increase resulted in a 3.2-dB impairment in age-adjusted overall hearing threshold (4 to 12 kHz; P < .001). Cumulative cisplatin doses > 300 mg/m(2) were associated with greater American Speech-Language-Hearing Association-defined hearing loss severity (odds ratio, 1.59; P = .0066) and worse normative-matched quartiles (odds ratio, 1.33; P = .093) compared with smaller doses. Almost one in five (18%) patients had severe to profound hearing loss. Tinnitus (40% patients) was significantly correlated with reduced hearing at each frequency (P < .001). Noise-induced damage (10% patients) was unaffected by cisplatin dose (P = .59). Hypertension was significantly related (P = .0066) to overall hearing threshold (4 to 12 kHz) in age- and cisplatin dose-adjusted analyses. Middle ear deficits occurred in 22.3% of patients but, as expected, were not related to cytotoxic drug dosage. CONCLUSION: Follow-up of adult-onset cancer survivors given cisplatin should include routine inquiry for hearing status and tinnitus, referral to audiologists as clinically indicated, and hypertension control. Patients should be urged to avoid noise exposure, ototoxic drugs, and other factors that further damage hearing.

Genetic diversity of Hepatozoon spp. in coyotes from the south-central United States.

To better define the strains and species of Hepatozoon that infect coyotes in the south-central United States, whole blood and muscle samples were collected from 44 coyotes from 6 locations in Oklahoma and Texas. Samples were evaluated by a nested polymerase chain reaction (PCR) using primers amplifying a variable region of the apicomplexan 18S rRNA gene as well as histopathology (muscle only) for presence of tissue cysts. Hepatozoon spp. infections were identified in 79.5% (35/44) of coyotes tested including 27 of 44 (61.4%) whole blood samples and 17 of 44 (38.6%) muscle samples tested by PCR and 23 of 44 (52.3%) muscle samples evaluated by histological examination. Analysis revealed 19 distinct sequences comprising 3 major clusters of Hepatozoon spp., i.e., 1 most closely related to Hepatozoon americanum, another most closely related to Hepatozoon canis , and the third an intermediate between the 2 groups. The diversity of Hepatozoon spp. in wild canids appears greater than previously recognized and warrants further investigation.

Fecal egg counts after anthelmintic administration to aged horses and horses with pituitary pars intermedia dysfunction.

OBJECTIVE: To determine effects of pituitary pars intermedia dysfunction (Cushing's disease) and age on fecal egg count and time to egg reappearance after anthelmintic treatment in horses residing in similar environments. DESIGN: Cross-sectional study. ANIMALS: 29 healthy horses (4 to 35 years old) and 13 horses with PPID (13 to 33 years old). PROCEDURES: Fecal egg counts were performed by use of a modified Wisconsin flotation method at 2-week intervals before and after ivermectin treatment. RESULTS: Horses with PPID had higher fecal egg counts before and 8, 10, and 12 weeks after ivermectin treatment, compared with counts for site-matched healthy horses. There was no difference in the period for < 90% reduction in fecal egg counts between the 2 groups. Age did not affect fecal egg counts at any time point. CONCLUSIONS AND CLINICAL RELEVANCE: For similar environmental conditions, horses with PPID were more likely to have higher fecal egg counts than were healthy horses. Therefore, horses with PPID may need to have a more aggressive parasite prevention program than do healthy horses. Age did not affect fecal egg counts or time to egg reappearance after anthelmintic treatment, which suggested age alone does not likely require special consideration when designing a parasite control program for adult horses.

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.

Experimental transmission of Hepatozoon americanum to New Zealand White rabbits (Oryctolagus cuniculus) and infectivity of cystozoites for a dog.

Inflammatory lesions containing parasitic cystozoites developed in multiple organs and tissues of laboratory-raised Oryctolagus cuniculus that were administered approximately 100 sporulated oocysts of Hepatozoon americanum (Oklahoma isolate, GenBank accession AF176836) orally. The predominantly granulomatous inflammatory lesions were detected histologically 8 weeks after exposure to oocysts. Cystozoites, recognized by cresent-shaped, uninucleated bodies surrounded by an accumulation of globular, PAS-positive polysaccharide material, were evident within macrophages as monozoic and dizoic cysts. Neither meronts nor gamonts were detected in any of the laboratory-raised lagomorphs during the 24-week observation period. Nested PCR assay of rabbit tissues for a 488 bp fragment of the 18S rRNA Hepatozoon spp. gene was positive at 8 and 24 weeks post-exposure. The sequence was 97.1% similar with sequence from the H. americanum carrier used to infect ticks. A Hepatozoon-free dog fed tissues from the 24-week post-exposure rabbit principal developed American canine hepatozoonosis. Gamonts were first detected 5 weeks after the dog ingested the rabbit tissues containing cystozoites. PCR assay of blood from the dog was positive for the Hepatozoon spp. gene fragment. Sequencing confirmed that the parasite in the dog was H. americanum.

New developments in canine hepatozoonosis in North America: a review.

Canine hepatozoonosis is caused by Hepatozoon canis and Hepatozoon americanum, apicomplexan parasites transmitted to dogs by ingestion of infectious stages. Although the two agents are phylogenetically related, specific aspects, including characteristics of clinical disease and the natural history of the parasites themselves, differ between the two species. Until recently, H. canis infections had not been clearly documented in North America, and autochthonous infection with H. americanum has yet to be reported outside of the southern United States. However, recent reports demonstrate H. canis is present in areas of North America where its vector tick, Rhipicephalus sanguineus, has long been endemic, and that the range of H. americanum is likely expanding along with that of its vector tick, Amblyomma maculatum; co-infections with the two organisms have also been identified. Significant intraspecific variation has been reported in the 18S rRNA gene sequence of both Hepatozoon spp.-infecting dogs, suggesting that each species may represent a complex of related genogroups rather than well-defined species. Transmission of H. americanum to dogs via ingestion of cystozoites in muscle of infected vertebrates was recently demonstrated, supporting the concept of predation as a means of natural transmission. Although several exciting advances have occurred in recent years, much remains to be learned about patterns of infection and the nature of clinical disease caused by the agents of canine hepatozoonosis in North America.

Infectivity of Hepatozoon americanum cystozoites for a dog.

Hepatozoon americanum cystozoites from experimentally infected, laboratory-raised rodents were fed to a Hepatozoon-free dog. Gamonts were detected by examination of blood smear 42 and 56 days post-exposure. PCR analysis of blood was positive for the 18S rRNA Hepatozoon gene on days gamonts were demonstrated. Meronts were detected histologically in a skeletal muscle biopsy 90 days after ingestion of cystozoites. Sequencing confirmed that the parasite in the dog was H. americanum. Xenodiagnosis was conducted by replete feeding of Ambylomma maculatum larvae on the dog; 40 days after detachment, sporulated oocysts were recovered from recently molted nymphs.

Experimental transmission of Hepatozoon americanum to rodents.

Laboratory-raised cotton rats (Sigmodon hispidus), outbred white mice (Mus musculus), and C57BL/6J-Lystbg-J/J mice (M. musculus) that were administered approximately 50 sporulated oocysts of Hepatozoon americanum (AF176836) by gavage developed inflammatory lesions containing parasitic cystozoites in cardiac and skeletal muscle, kidney, and lung. Sprague-Dawley rats (Rattus norvegicus) similarly exposed showed no evidence of infection. Cystozoites were first detected by histopathologic examination four weeks after exposure to oocysts. Globular, PAS-positive material accumulated around the cystozoites as the duration of infection lengthened. Nested PCR analysis of tissues collected 16 weeks post-exposure was positive for the 18S rRNA Hepatozoon sp. gene and the DNA sequence of the fragment amplified was 99.6% and 99.8% identical to H. americanum sequences previously reported from naturally-infected dogs (AF176836 and AY864676, respectively). Merogonous and gamontogonous stages of the parasite were not detected in any of the cystozoite-infected rodents.

A retrospective study of 11 cases of lungworm (Didelphostrongylus hayesi) infection in opossums (Didelphis virginiana).

A juvenile, female North American opossum (Didelphis virginiana) died of verminous pneumonia caused by Didelphostrongylus haysei despite aggressive treatment with oral fenbendazole, corticosteroids, and antibiotics. This prompted a retrospective study of lungworm infection in opossums, during which 19 additional necropsy reports from opossums were reviewed. Including the subject of this report, a total of 11 (55%) of these cases included a diagnosis of lungworm infection. This diagnosis was considered to have contributed to death in eight out of the 11 cases (73%). Histologically, 10 of the 11 (91%) opossums had granulomatous bronchopneumonia with small to moderate numbers of adult nematodes in the airways and parenchyma. Four of the 11 (36%) opossums had free larvae within the parenchyma or terminal airways. Inflammation was usually associated with larvae, degenerating parasites, and nonintact adult nematodes. Superimposed bacterial pneumonia was evident in three animals, and sections of lung examined from all the opossums were characterized by moderate to severe smooth-muscle hyperplasia in airways, including terminal respiratory bronchioles and alveolar ducts. Nine animals had prominent medial smooth-muscle hyperplasia in small- and medium-sized arterioles. Lesions in other organs, particularly in liver, heart, and gastrointestinal tract, were frequently identified. Three animals had concomitant septicemia or bacterial bronchopneumonia (or both), which contributed to the cause of death. Seven animals had gastric nematodosis (Physaloptera sp.), although three of them had been treated with a 14-day course of fenbendazole.