DETECTION AND DIFFERENTIATION OF TRICHURIS IN GIRAFFE (GIRAFFA CAMELOPARDALIS) UNDER HUMAN CARE.

Trichuris spp. are nematode parasites infecting many species, including domestic and wild ruminants in zoological and wildlife parks worldwide. These nematodes cause significant morbidity in giraffes (Giraffa camelopardalis) and other hoofstock. Parasite transmission between ruminant species is well reported; however, relative to domestic species, little is known about Trichuris infections in giraffes under human care. We hypothesized that Trichuris spp. differ between individual giraffes in different US regions, suggesting giraffes are susceptible to Trichuris from other ruminant hosts. The study sites used to assess this hypothesis included The Wilds in Cumberland, Ohio; Fossil Rim Wildlife Center in Glen Rose, Texas; White Oak Conservation in Yulee, Florida; and Binder Park Zoo in Battle Creek, Michigan. Trichuris eggs were collected from the feces of 14 individual giraffes located at the four different study sites and from soil samples from the enclosures where Trichuris-positive giraffes were housed. The eggs were isolated and their genes were amplified by PCR and compared at the molecular level. Trichuris samples from four giraffe hosts and one soil site were sequenced and portions of the cox1 and 18S genes compared. This study found that >12 eggs per gram of fecal-derived Trichuris eggs must be present to amplify Trichuris-specific DNA. The Trichuris spp. found in the majority of giraffes in this study were most similar to T. ovis and T. discolor, and one giraffe sample had greater similarity to T. skrjabini and T. leporis.

Feasibility and comparative analysis of Dirofilaria immitis microfilaria freezing and fixation for student instruction and assessment of clinical parasitology skills.

BACKGROUND: Detection of D. immitis microfilaria (mf) is an important diagnostic skill in veterinary medicine and is critical to Day 1 veterinarians and technicians. Finding a supply of blood containing mf to teach the technique and formalin's adverse environmental effects used in the diagnostic microscopic tests present a challenge. RESULTS: This study evaluated the use of cryopreserved and recently drawn mf-infected blood along with two fixative reagents, acetic acid or formalin for mf detection. The specific aims included determining if veterinary students could 1) detect cryopreserved mf added to fresh blood using routine diagnostic testing and 2) detect morphological differences in the mf. The 236 students were kept blind from the sample status. The ability of the students to identify mf and the mf morphology were compared for the samples and fixatives evaluated. The results demonstrate using a combination of cryopreservation and acetic acid for teaching microfilaria diagnostic techniques is fleasible; however, the quality of the mf morphology is less than optimal when compared to freshly acquired mf containing blood. Compared to reference values, the mf demonstrated a decrease in size with each additional variable evaluated. CONCLUSION: A majority (98.3%) of the 236 students correctly identified the presence of mf. Teaching laboratories could utilize cryopreserved mf-spiked donor blood in lieu of freshly collected mf-containing blood from a naturally or experimentally infected dog. Substitution of less hazardous chemicals for the fixative can be used. Finally, the change in size measurements provides a mechanism to ensure students can correctly measure mf as students are required to do verifiable measurements and cannot copy reference values from a text book since the cryopreservation and fixation methods cause the mf to measure smaller than textbook reference values.

Using the Digital Platform ExamSoft in Veterinary Anatomy and Parasitology Assessments in Written and Laboratory Components.

The Ohio State University College of Veterinary Medicine (CVM), with a class size of 162, is one of the largest in the nation. In an effort to streamline examination procedures, create a consistent assessment format among courses, replace paper exams, track test questions linked to learning objectives, and reduce exam grading time, our DVM program adopted the use of ExamSoft for core courses beginning in the autumn semester 2014. ExamSoft is an electronic assessment application, which provides a secure testing environment and robust reporting features. CVM uses it for high stakes midterm and finals. Although easily adopted into a didactic course format, its application in laboratory-based examinations proved challenging. Designing, setting up and grading exams for Anatomy and Parasitology courses with a laboratory component have always required substantial time investment, and adding a testing application to the process demanded rethinking and restructuring logistics. After two semesters of process refinement and standardization of a testing device to the iPad, faculty teaching in the Anatomy and Parasitology courses were able to implement ExamSoft in a laboratory setting to realize the same assessment and efficiency gains. Here we describe the benefits of ExamSoft testing in the written and laboratory settings and the lessons learned during the 2-year transition.

Combination Anthelmintic Treatment for Persistent Ancylostoma caninum Ova Shedding in Greyhounds.

Ancylostoma caninum is a nematode of the canine gastrointestinal tract commonly referred to as hookworm. This study involved eight privately owned adult greyhounds presenting with persistent A. caninum ova shedding despite previous deworming treatments. The dogs received a combination treatment protocol comprising topical moxidectin, followed by pyrantel/febantel/praziquantel within 24 hr. At 7-10 days posttreatment, a fecal examination monitored for parasite ova. Dogs remained on the monthly combination treatment protocol until they ceased shedding detectable ova. The dogs then received only the monthly topical moxidectin maintenance treatment. The dogs remained in the study for 5-14 mo with periodical fecal examinations performed. During the study, three dogs reverted to positive fecal ova status, with two being associated with client noncompliance. Reinstitution of the combination treatment protocol resulted in no detectable ova. Use of monthly doses of combination pyrantel, febantel and moxidectin appears to be an effective treatment for nonresponsive or persistent A. caninum ova shedding. Follow-up fecal examinations were important for verifying the presence or absence of ova shedding despite the use of anthelmintic treatment. Limitations of the current study include small sample size, inclusion of only privately owned greyhounds, and client compliance with fecal collection and animal care.

Efficacy of a Haemonchus contortus vaccine under field conditions in young alpacas.

Anthelmintic resistance to Haemonchus contortus creates increasing management challenges with small ruminants and camelids. The commercial vaccine, Barbervax®, contains H11 and H-gal-GP antigens, derived from gut mucosal membrane enzymes of H. contortus involved in digesting blood. Antibody neutralization of these antigens causes failure of H. contortus to digest blood, resulting in parasite death. H11 and H-gal-GP are considered "hidden" antigens, meaning the host immune system does not encounter these proteins under natural infection. Therefore, repeat immunization is required to maintain protective humoral responses. One previous study evaluated the safety of Barbervax® in camelids but the efficacy could not be assessed due to lack of successful infection in the controls. The objective of the current study was to evaluate clinical parameters of anemia, fecal egg counts (FECs), and humoral immune responses of healthy alpacas after immunizing with Barbervax® compared to non-vaccinated controls, all under natural environmental exposure on parasite-laden pastures. A crossover-like study was performed where twenty alpacas (298 ± 66 days of age) were assigned to be initially vaccinated with Barbervax® (n=10) or receive no treatment (n=10). Three doses of Barbervax® were administered at three-week intervals. Feces and blood were collected on Day -10, 0, 21, 43, 64, 85, 106, and 135 to evaluate FECs, packed cell volume (PCV), and antibody titers. Each group was kept on separate adjacent pastures. Tracer sheep (n=2 per study group) were introduced on Day 43 for a three-week period to ensure parasite acquisition. For the crossover-like component on Day 85, the initial non-vaccinated group was administered Barbervax® with dosing repeated on Day 106 and 135. Results indicated all initially vaccinated alpacas produced antibody titers to vaccine antigen that corresponded to lower mean FECs compared to the initially non-vaccinated group. A reduced mean FEC in the vaccinate group was observed 21 days after peak antibody titers. Similarly, when pooled vaccinate antibody titers were noted to wane on Day 106, an increase in FEC was observed at the following time point (Day 135). Conclusions from our study support the use of Barbervax® to reduce H. contortus burdens in alpacas. Furthermore, a less than 30-day lag time between antibody titer and resultant effect in FECs was observed. Additional studies assessing the ability of Barbervax® to reduce H. contortus burdens during subsequent grazing seasons would provide even greater information regarding the use of Barbervax® within alpaca herds to modulate H. contortus infections, refugia, and anthelmintic use.

Rodent Gut Bacteria Coexisting with an Insect Gut Virus in Parasitic Cysts: Metagenomic Evidence of Microbial Translocation and Co-adaptation in Spatially-Confined Niches.

In medicine, parasitic cysts or cysticerci (fluid-filled cysts, larval stage of tapeworms) are believed to be sterile (no bacteria), and therein, the treatment of cysticerci infestations of deep extra-intestinal tissues (e.g., brain) relies almost exclusively on the use of antiparasitic medications, and rarely antibiotics. To date, however, it is unclear why common post-treatment complications include abscessation. This study quantified the microbial composition of parasitic cyst contents in a higher-order rodent host, using multi-kingdom shotgun metagenomics, to improve our understanding of gut microbial translocation and adaptation strategies in wild environments. Analysis was conducted on DNA from two hepatic parasitic cysts (Hydatigera (Taeenia) taeniaeformis) in an adult vole mouse (Microtus arvalis), and from feces, liver, and peritoneal fluid of three other vole family members living in a vegetable garden in Ohio, USA. Bacterial metagenomics revealed the presence of gut commensal/opportunistic species, including Parabacteroides distasonis, Klebsiella variicola, Enterococcus faecium, and Lactobacillus acidophilus, inhabiting the cysts. Parabacteroides distasonis and other species were also present outside the cyst in the peritoneal fluid. Remarkably, viral metagenomics revealed various murine viral species, but unexpectedly, it detected an insect-origin virus from the army moth (Pseudaletia/Mythimna unipuncta) known as Mythimna unipuncta granulovirus A (MyunGV-A) in both cysts, and in one fecal and one peritoneal sample from two different voles, indicating survival of the insect virus and adaption in voles. Metagenomics also revealed a significantly lower probability of fungal detection in the cysts compared to other samples (peritoneal fluid, p<0.05; and feces p<0.05), with single taxon detection in each cyst for Malassezia and Pseudophaeomoniella oleicola. The samples with a higher probability of fungi were the peritoneal fluid. In conclusion, commensal/pathobiont bacterial species can inhabit parasitic tapeworm cysts, which needs to be considered during therapeutic decisions of cysticerci or other chronic disease scenarios where immune privileged and spatially restricted ecosystems with limited nutrients and minimal presence of immune cells could facilitate microbial adaptation, such as within gut wall cavitating micropathologies in Crohn's disease.

Rodent Gut Bacteria Coexisting with an Insect Gut Virus in Tapeworm Parasitic Cysts: Metagenomic Evidence of Microbial Selection in Extra-Intestinal Clinical Niches.

In medicine, parasitic cysts (e.g., brain cysticerci) are believed to be sterile, and are primarily treated with antiparasitic medications, not antibiotics, which could prevent abscess formation and localized inflammation. This study quantified the microbial composition of parasitic cysts in a wild rodent, using multi-kingdom metagenomics to comprehensively assess if parasitic cysts are sterile, and further understand gut microbial translocation and adaptation in wildlife confined environments, outside the gut. Analysis was conducted on DNA from two hepatic parasitic cysts from a feline tapeworm, Hydatigera (Taenia) taeniaeformis, affecting a wild vole mouse (Microtus pennsylvanicus), and from feces, liver and peritoneal fluid of this and two other concurrent individual wild voles trapped during pest control in one of our university research vegetable gardens. Bacterial metagenomics revealed the presence of gut commensal/opportunistic species, Parabacteroides distasonis, Bacteroides (Bacteroidota); Klebsiella variicola, E. coli (Enterobacteriaceae); Enterococcus faecium and Lactobacillus acidophilus (Bacillota) inhabiting the cysts, and peritoneal fluid. Remarkably, viral metagenomics revealed various murine viral species, and unexpectedly, a virus from the insect armyworm moth (Pseudaletia/Mythimna unipuncta), known as Mythimna unipuncta granulovirus A (MyunGV-A), in both cysts, and in one fecal and one peritoneal sample from the other non-cyst voles, indicating the survival and adaption potential of the insect virus in voles. Metagenomics also revealed a significantly lower probability of fungal detection in cysts compared to that in peritoneal fluid/feces (p < 0.05), with single taxon detection in each cyst (Malassezia and Pseudophaeomoniella oleicola). The peritoneal fluid had the highest probability for fungi. In conclusion, metagenomics revealed that bacteria/viruses/fungi coexist within parasitic cysts supporting the potential therapeutic benefits of antibiotics in cystic diseases, and in inflammatory microniches of chronic diseases, such as Crohn's disease gut wall cavitating micropathologies, from which we recently isolated similar synergistic pathogenic Bacteroidota and Enterobacteriaceae, and Bacillota.

A fresh look at the SarcoFluor antibody test for the detection of specific antibodies to Sarcocystis neurona for the diagnosis of equine protozoal myeloencephalitis.

Equine protozoal myeloencephalitis (EPM) is a challenging disease to diagnose in horses with neurological signs. To optimize contemporary diagnostic testing, including the use of serum:CSF antibody ratios, the SarcoFluor antibody test for Sarcocystis neurona requires revalidation. The SarcoFluor, a previously validated immunofluorescent antibody test (IFAT) for the detection of antibodies specific to S. neurona in serum and cerebrospinal fluid (CSF) of naturally infected horses was analyzed using recent data and considering a serum:CSF antibody ratio threshold. Utilization of serum and CSF phosphorylated neurofilament heavy protein (pNfH) concentrations in support of an EPM diagnosis was also evaluated. 172 horses were divided into three groups: EPM-positive horses (EPM+, n=42), neurological non-EPM horses (n=74) confirmed with non-EPM neurological diseases (cervical vertebral compressive myelopathy, equine neuroaxonal dystrophy/equine degenerative myeloencephalopathy), and control horses (control, n=56) without neurological signs and neurological abnormalities on histology. Logistic regression was used to compare EPM diagnostic regimens. Specifically, EPM+ horses were compared with neurological non-EPM horses showing neurological signs. To consider diagnostic utility, post-test probabilities were calculated by titer. When differentiating between EPM and other neurological diseases, the combination of serum and CSF SarcoFluor testing added more information to the model accuracy than either test alone. Using serum and CSF for pNfH in support of an EPM diagnosis did not identify cutoffs with statistically significant odds ratios but increased the overall model accuracy when used with the IFAT. Utilization of IFAT titers against S. neurona in serum and CSF result in a high post-test probability of detecting EPM+ horses in a clinical setting.

Reflecting on the past and fast forwarding to present day anthelmintic resistant Ancylostoma caninum-A critical issue we neglected to forecast.

Reports of anthelmintic resistance in Ancylostoma caninum are increasing in frequency in the United States of America (USA). In the last few years in vitro and in vivo studies characterized individual isolates, demonstrating multiple anthelmintic drug resistance (MADR). In 2021, the American Association of Veterinary Parasitologists initiated a hookworm task force to address this issue. The first report of drug resistant A. caninum occurred in 1987 in Australian racing Greyhounds. In the last five years multiple case reports and investigations show drug resistant A. caninum is becoming a much greater problem in the USA and now extends beyond racing Greyhounds into the general companion animal dog population. The literature, regarding drug resistance in livestock and equine nematodes, provides helpful guidance along with diagnostic methods to better understand the evolution and selection of canine MADR hookworms; however, there are limitations and caveats due to A. caninum's unique biology and zoonotic potential. Mass drug administration (MDA) of anthelminthic drugs to humans to reduce morbidity associated with human hookworms (Necator americanus) should consider the factors that contributed to the development of MADR A. caninum. Finally, as Greyhound racing undergoes termination in some regions and the retired dogs undergo subsequent rehoming, drug resistant parasites, if present, are carried with them. Drug resistant A. caninum requires greater recognition by the veterinary community, and small animal practitioners need to be aware of the spread into current pet dog populations. The current understanding of anthelmintic resistance, available treatments, and environmental mitigation for these drug resistant A. caninum isolates must be monitored for horizontal spread. A major goal in this emerging problem is to prevent continued dissemination.

Detection of Zoonotic Bacteria and Paragonimus kellicotti in Red Swamp Crayfish (Procambarus clarkii) and the Assessment of Traditional Crayfish Boils.

ABSTRACT: Studies of red swamp crayfish (Procambarus clarkii) outside of the United States confirm the presence of a variety of zoonotic pathogens, but it is unknown whether these same pathogens occur in P. clarkii in the United States. The U.S. commercial crayfish industry generates $200 million yearly, underscoring the need to evaluate this consumer commodity. The study objectives were to evaluate specific zoonotic pathogens present on P. clarkii from Alabama and Louisiana, states in the southeastern United States, and to determine the effectiveness of traditional food preparation methods to reduce pathogens. Experiment A evaluated the presence of Escherichia coli, Salmonella, Staphylococcus aureus, and Vibrio spp. in crayfish and environmental samples over a 2-month collection period (May to June 2021). Crayfish sampling consisted of swabbing the cephalothorax region; 15 samples were tested for E. coli, Salmonella, and S. aureus, and an additional 15 samples for Vibrio spp. Additionally, crayfish shipping materials were sampled. In experiment B, 92 crayfish were evaluated for Paragonimus kellicotti. Experiment C compared live and boiled crayfish for the presence of Vibrio spp. In experiments A and B, all 60 (100%) crayfish samples and 13 (81.25%) of 16 environmental samples showed growth characteristic of Vibrio spp. Three (5%) of 60 samples showed E. coli growth, with no statistical difference (P = 0.5536) between farms. P. kellicotti, Salmonella, and S. aureus were not recovered from any samples. In experiment C, all 10 (100%) of the live preboiled crayfish samples showed characteristic growth, whereas 1 (10%) of 10 samples of crayfish boiled in unseasoned water showed Vibrio growth (P < 0.0001). These results confirm that Vibrio spp. and E. coli may be present on U.S. commercial crayfish and that care should be taken when handling any materials that come into contact with live crayfish because they can potentially be contaminated.

Equine Protozoal Myeloencephalitis associated with Neospora caninum in a USA captive bred zebra (Equus zebra).

A 6-year-old female captive zebra (Equus zebra) had a three-year history of slow progressive neurologic signs that recently worsened with hind limb ataxia, head tilt, and circling. Gross examination including the brain and spinal cord were unremarkable. On histopathology, the brain and brainstem had multiple random areas of severe lymphoplasmacytic meningoencephalitis associated with numerous 15-25 µm in diameter protozoal cysts with a discernible outer wall containing numerous 2 × 4 µm oval to crescent-shaped organisms. Immunohistochemistry and PCR identified the presence of Neospora organisms associated with the lesions. Equine protozoal myeloencephalitis (EPM) is generally associated with Sarcocystis neurona or less commonly Neospora hughesi. Molecular characterization revealed the first case of EPM associated with Neospora caninum in an equid as confirmed by DNA analysis.

Detection of Trichuris eggs in feces and soil from giraffe (Giraffa camelopardalis) and other hoofstock enclosures under human care in the USA.

Trichuris spp. are nematode parasites infecting wild ruminants in zoological institutions worldwide. These helminths cause significant morbidity in giraffe (Giraffa camelopardalis) and other hoofstock located in zoological institutions throughout the United States. Historically, studies and institutions have used a variety of nematode detection methods with various flotation solutions. Optimization of Trichuris egg detection is necessary for monitoring collections. Fecal and soil optimized protocols were generated in this study using samples containing Trichuris eggs from multiple semi free-ranging zoological institutions. First, Sheather's sugar (specific gravity (SG) 1.27), sucrose (SG 1.40), magnesium sulfate (SG 1.26), and zinc sulfate (SG 1.18) were compared as flotation solutions by quantitative eggs per gram using a modified Stoll method. Then a soil recovery method was optimized comparing Tween 20, sodium hydroxide, Dawn™ (Procter and Gamble) detergent, and sodium chloride as liberating solutions to free eggs from the soil. We found that Sheather's sugar and sucrose solutions were the most effective for Trichuris egg detection, and either sodium hydroxide or sodium chloride liberated eggs from soil.

Insights From Veterinary Disciplinary Actions in California 2017-2019.

There is increasing concern within the veterinary medical community (veterinarians and veterinary students) that disgruntled clients are unfairly leveraging various legal tools against veterinarians. Clinical veterinarians and veterinary students should be aware of the most common types of problems arising within the clinic and how they can lead to formal consumer complaints. The study describes and categorizes with greater detail the types of violations or "causes for discipline" that occur, as well as specific sanctions imposed on veterinarians formally disciplined for standard of care-related violations between 2017 and 2019, for California. In addition, the study calculated the frequency of disciplinary actions and their basic summary statistics regarding the temporal aspect of how lawsuits typically unfold. Using public documents from California, the study describes the analysis and trends for the purpose of providing contextual evidence to inform and guide potential veterinary educational interventions. Although specific to California, this study can serve as a template methodology for comparisons to other states.

Risk factors and impact of COVID-19-related clinic closures on the detection of gastrointestinal parasites in dogs, a cross-sectional study.

Infections with endoparasites, especially gastrointestinal helminths, are a common finding in client-owned dogs. The Community Practice section at the Ohio State University Veterinary Medical Center (OSU-VMC) follows Companion Animal Parasite Council, American Animal Hospital Association, and American Veterinary Medical Association guidelines for parasitology by recommending annual fecal analyses for dogs and prescribing year-round, broad-spectrum parasite preventatives. There is increasing interest in determining if parasite occurrence is changing in client-owned dogs. Therefore, a retrospective study was designed to examine risk factors associated with the detection of parasites in samples submitted to the OSU-VMC Clinical Veterinary Parasitology Diagnostic Laboratory. Of the 1198 canine fecal samples, 254 (21.2%) of these samples had a positive fecal analysis for gastrointestinal (GI) parasites. The age of the dog, time of year, reproductive status, purpose of fecal examination, GI signs, and type of parasite preventatives were assessed as potential risk factors for GI parasite infection in dogs. To determine if Coronavirus Disease 2019 (COVID-19)-associated hospital restrictions impacted the number of fecal examinations performed, data from January to December 2020, was compared to the same period in 2019. There was nearly a 50% reduction in canine fecal samples submitted to the OSU-VMC Clinical Veterinary Parasitology Diagnostic Laboratory in March 2020 compared to March 2019. At least one canine GI parasite was found in over 20% of all 1198 canine fecal samples (21.2%, 254/1198). The most commonly detected canine GI parasite in all the fecal samples was hookworm at 9.6% (115/1198), followed by Giardia sp. at 7.6% (91/1198). Age, use of parasite preventatives, breed, and reproductive status were found to be associated with parasite occurrence in the dog samples. Identifying such risk factors in dogs will guide veterinarians to advise annual fecal examinations more strongly to clients with high-risk dogs or when routine health visits are postponed for an extended period.

The effects of protein supplementation and pasture maintenance on the growth, parasite burden, and economic return of pasture-raised lambs.

The objective of this experiment was to evaluate the impact of protein supplementation and pasture contamination with gastrointestinal nematodes on the mitigation of parasitic infection in grazing lambs. We hypothesized that there would be no difference between protein supplementation and newly sown pasture in evaluating lamb growth and health parameters associated with parasitism. Furthermore, we questioned if there would be an interaction between protein supplementation and pasture type. A total of 192, 60-d-old lambs (28.3 ± 5.1 kg) were randomly assigned to one of four treatments: 1) new pasture without supplementation (NN); 2) new pasture with supplementation (NS); 3) established pasture without supplementation (EN); and 4) established pasture with supplementation (ES) and grazed for 112 d. Lambs were supplemented at a rate of 1% body weight/d. Supplemented lambs had greater body weight (BW) and average daily gain (ADG) when compared with non-supplemented lambs (P < 0.04). Additionally, lambs on newly sown pasture demonstrated greater BW and ADG when compared with lambs grazing on established pasture (P < 0.05). For lamb health, lambs in the EN treatment group had the greatest FAMACHA eye scores and lowest packed cell volume (PCV) over the course of the 112-d grazing period (P < 0.05). Moreover, NS and ES treatment lambs demonstrated similar FAMACHA eye scores when compared with NN treatment lambs; however, NN treatment lambs showed lower PCV when compared with NS and ES treatment lambs (P < 0.05). In evaluating fecal egg counts (FEC), lambs on new pasture or given supplement demonstrated lesser FEC when compared with those lambs on established pasture or not given supplement (P < 0.05). Sixty-four lambs were harvested to evaluate total abomasum nematode counts which demonstrated that Haemonchus contortus represented approximately 80% of total nematodes. Furthermore, based upon gross margin analysis, lambs given a protein rich supplement on pasture had a 9.3 kg increase in lamb BW whereas newly sown pasture had a 1.3 kg increase in lamb BW. A protein rich supplement given to lambs grazing pastures contaminated primarily with H. contortus or placing lambs on newly sown pasture increases lamb BW and improves parasite resiliency. Selection of parasite management strategies may be influenced by cost of production and market opportunities.

Retrospective study of canine endoparasites diagnosed by fecal flotation methods analyzed across veterinary parasitology diagnostic laboratories, United States, 2018.

BACKGROUND: Companion animal endoparasites play a substantial role in both veterinary medicine and public health. Updated epidemiological studies are necessary to identify trends in occurrence and distribution of these parasites, and their associated risk factors. This study aimed to assess the occurrence of canine endoparasites  retrospectively, using fecal flotation  test data available through participating academic veterinary parasitology diagnostic laboratories across the United States of America (USA). METHODS: Canine fecal flotation records from ten veterinary diagnostic laboratories located in nine states in the USA acquired from January 1, 2018, to December 31, 2018, were included. RESULTS: A total of 4692 fecal flotation test results were obtained, with a majority comprised of client-owned dogs (3262; 69.52%), followed by research dogs (375; 8.00%), and shelter dogs (122; 2.60%). Samples from 976 (20.80%) dogs were positive for at least one parasite, and co-infections of two or more parasites were found in 3.82% (179/4692) of the samples. The five most commonly detected parasites were: Giardia sp., (8.33%; 391/4692), Ancylostomatidae (5.63%; 264/4692), Cystoisospora spp. (4.35%; 204/4692), Toxocara canis (2.49%;117/4692), and Trichuris vulpis (2.43%; 114/4692). Various other internal parasites, including gastrointestinal and respiratory nematodes, cestodes, trematodes, and protozoans were detected in less than 1% of samples. CONCLUSIONS: These data illustrate the importance of parasite prevention, routine fecal screening, and treatment of pet dogs. Additionally, pet owners should be educated about general parasite prevalence, prevention, and anthelmintic treatment regimens to reduce the risks of environmental contamination and zoonotic transmission.

Evidence for Unknown Sarcocystis-Like Infection in Stranded Striped Dolphins (Stenella coeruleoalba) from the Ligurian Sea, Italy.

Two striped dolphins (SD1, SD2), stranded along the Ligurian coast of Italy, were diagnosed with a nonsuppurative meningoencephalitis associated with previously undescribed protozoan tissue cysts. As tissue cysts were morphologically different from those of Toxoplasma gondii, additional histopathological, immunohistochemical, ultrastructural, and biomolecular investigations were performed, aiming to fully characterize the organism. Histopathology revealed the presence of large Sarcocystis-like tissue cysts, associated with limited inflammatory lesions in all CNS areas studied. IHC was inconclusive, as positive staining with polyclonal antisera did not preclude cross-reaction with other Sarcocystidae coccidia. Applied to each animal, 11 different PCR protocols precluded a neural infection by Sarcocystis neurona, Sarcocystis falcatula, Hammondia hammondi, and Neospora caninum. T. gondii coinfection was confirmed only in dolphin SD2. Sarcocystis sp. sequences, showing the highest homology to species infecting the Bovidae family, were amplified from SD1 myocardium and SD2 skeletal muscle. The present study represents the first report of Sarcocystis-like tissue cysts in the brain of stranded cetaceans along with the first description of Sarcocystis sp. infection in muscle tissue of dolphins from the Mediterranean basin.

Comparative H-gal-GP and H11 specific antibody binding in equine cyathostomins.

The biological-based vaccine (Barbervax®) generates effective antibodies against the biologically essential H-gal-GP and H11 protein complex of the ruminant parasite Haemonchus contortus to target and kill the parasites after taking a blood meal. A comparative analysis of several parasite genera was performed to determine if a similar protein complex or one that is recognized by H-gal-GP and H11 specific antibodies was present. If so, it suggests the vaccine could be effective for other nematode parasites. Ancylostoma caninum, H. contortus, equine cyathostomins, bovine Bunostomum phlebotomum, Dracunculus lutrae, Parascaris sp., Ixodes scapularis, Amblyomma americanum, Dirofilaria immitis and Brugia malayi were evaluated for specific antibody binding using hyperimmunized antibodies against H-gal-GP and H11 native proteins. Of the parasites evaluated, specific and reproducible staining was observed in H. contortus and adult and encysted cyathostomins only. To further evaluate the similar reactivities between cyathostomins and H. contortus, cross-reactivity of equine serum with antibodies to cyathostomins on a H. contortus adult histology cross-section was observed using immunofluorescence. These findings pave the way for future studies on the safety and efficacy of H-gal-GP and H11 protein complex as a potential control for cyathostomins.

Strains of Sarcocystis neurona exhibit differences in their surface antigens, including the absence of the major surface antigen SnSAG1.

A gene family of surface antigens is expressed by merozoites of Sarcocystis neurona, the primary cause of equine protozoal myeloencephalitis (EPM). These surface proteins, designated SnSAGs, are immunodominant and therefore excellent candidates for development of EPM diagnostics or vaccines. Prior work had identified an EPM isolate lacking the major surface antigen SnSAG1, thus suggesting there may be some diversity in the SnSAGs expressed by different S. neurona isolates. Therefore, a bioinformatic, molecular and immunological study was conducted to assess conservation of the SnSAGs. Examination of an expressed sequence tag (EST) database revealed several notable SnSAG polymorphisms. In particular, the EST information implied that the EPM strain SN4 lacked the major surface antigen SnSAG1. The absence of this surface antigen from the SN4 strain was confirmed by both Western blot and Southern blot. To evaluate SnSAG polymorphisms in the S. neurona population, 14 strains were examined by Western blots using monospecific polyclonal antibodies against the four described SnSAGs. The results of these analyses demonstrated that SnSAG2, SnSAG3, and SnSAG4 are present in all 14 S. neurona strains tested, although some variance in SnSAG4 was observed. Importantly, SnSAG1 was not detected in seven of the strains, which included isolates from four cases of EPM and a case of fatal meningoencephalitis in a sea otter. Genetic analyses by PCR using gene-specific primers confirmed the absence of the SnSAG1 locus in six of these seven strains. Collectively, the data indicated that there is heterogeneity in the surface antigen composition of different S. neurona isolates, which is an important consideration for development of serological tests and prospective vaccines for EPM. Furthermore, the diversity reported herein likely extends to other phenotypes, such as strain virulence, and may have implications for the phylogeny of the various Sarcocystis spp. that undergo sexual stages of their life cycle in opossums.

SnSAG5 is an alternative surface antigen of Sarcocystis neurona strains that is mutually exclusive to SnSAG1.

Sarcocystis neurona is an obligate intracellular parasite that causes equine protozoal myeloencephalitis (EPM). Previous work has identified a gene family of paralogous surface antigens in S. neurona called SnSAGs. These surface proteins are immunogenic in their host animals, and are therefore candidate molecules for development of diagnostics and vaccines. However, SnSAG diversity exists in strains of S. neurona, including the absence of the major surface antigen gene SnSAG1. Instead, sequence for an alternative SnSAG has been revealed in two of the SnSAG1-deficient strains. Herein, we present data characterizing this new surface protein, which we have designated SnSAG5. The results indicated that the protein encoded by the SnSAG5 sequence is indeed a surface-associated molecule that has characteristics consistent with the other SAGs identified in S. neurona and related parasites. Importantly, Western blot analyses of a collection of S. neurona strains demonstrated that 6 of 13 parasite isolates express SnSAG5 as a dominant surface protein instead of SnSAG1. Conversely, SnSAG5 was not detected in SnSAG1-positive strains. One strain, which was isolated from the brain of a sea otter, did not express either SnSAG1 or SnSAG5. Genetic analysis with SnSAG5-specific primers confirmed the presence of the SnSAG5 gene in Western blot-positive strains, while also suggesting the presence of a novel SnSAG sequence in the SnSAG1-deficient, SnSAG5-deficient otter isolate. The findings provide further indication of S. neurona strain diversity, which has implications for diagnostic testing and development of vaccines against EPM as well as the population biology of Sarcocystis cycling in the opossum definitive host.