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.

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.

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.

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.

In vitro effects of lactoferrin on lipopolysaccharide-induced proliferation, gene expression, and prostanoid production by bovine peripheral blood mononuclear cells.

OBJECTIVE: To evaluate the effect of lactoferrin on lipopolysaccharide (LPS)-induced proliferation of bovine peripheral blood mononuclear cells (PBMCs), gene expression of inflammatory mediators, and production of prostanoids in vitro. SAMPLE POPULATION: PBMCs isolated from 15 Holstein bull calves. PROCEDURES: Mixed populations of PBMCs were isolated by differential centrifugation. Proliferation assays were conducted in 96-well plates designed to allow addition of lactoferrin (200 ng/mL) with and without LPS (1 microg/mL) in a checkerboard design. Incorporation of 3H-thymidine was used to determine proliferation of PBMCs. Prostaglandin E2 production was determined in culture-conditioned medium by use of enzyme immunoassay. Effects of lactoferrin on LPS-induced gene expression of cyclooxygenase (COX)-2 and matrix metalloproteinase (MMP)-9 were monitored by use of PCR assays. RESULTS: Lactoferrin supplementation significantly reduced LPS-induced incorporation of 3H-thymidine and production of prostaglandin E2 by PBMCs. Lactoferrin reduced LPS-induced expression of COX-2 and MMP-9 mRNA. CONCLUSIONS AND CLINICAL RELEVANCE: Lactoferrin reduced LPS-induced cellular proliferation, inflammatory mediator gene expression, and prostaglandin E2 production by bovine PBMCs in vitro. These effects may be beneficial in reducing the impact of endotoxemia in neonates.

Safety and serologic response to a Haemonchus contortus vaccine in alpacas.

Haemonchosis in camelids remains a challenging disease to treat, and prevention has become increasingly problematic due to widespread anthelmintic resistance. Barbervax®is an adjuvanted vaccine containing natural H-11, H-gal-GP antigens obtained from Haemonchus contortus adults via a proprietary process and solubilized in Quil A. This vaccine is approved for use in Australia, after demonstrating its safety and efficacy in sheep and goats. There are no published studies evaluating Barbervax in other ruminants/pseudoruminants such as camelids which can be parasitized with H. contortus. The vaccine utilizes a mixture of the parasite gut mucosal membrane enzymes including H-gal-GP and H11, involved in digesting a blood meal from the host. This study monitored the safety profile of the Barbervax® vaccine in a group of adolescent alpacas. Although designed into the original study of vaccine efficacy, the experimental infection with viable H. contortus third stage larvae could not be completed due to lack of detectable significant variation of infection following experimental challenge. Twelve alpacas (158 + 15 days) were randomized to vaccination with Barbervax® or no treatment. Three doses of Barbervax® were administered at 3 week intervals and investigators involved in animal monitoring and sample collection were blinded to the groupings. Clinical pathologic parameters were evaluated 7 days before vaccination, and 1 and 2 months post-vaccination. Daily clinical observations were made and specific observations regarding the injection site and rectal temperatures were monitored in each alpaca twice daily for 1 week following vaccination. Fecal egg counts, packed cell volume, and total protein were monitored following challenge with 1500 H. contortus larvae on days 42, 46, and 50. An increase in rectal temperature for a duration of 2 days (range 2-4 days) was observed post-vaccination. Vaccinated alpacas were lethargic for 2-3 days following vaccination; however, they maintained an appetite and no visible or palpable injection site reactions were observed. Following the first vaccination, all animals maintained normal clinical pathologic parameters throughout the study period. The vaccinated animals did develop titers to the H. contortus antigen as measured by ELISA. In conclusion, the Barbervax® vaccine demonstrated safety in this small group of young, healthy alpacas, but additional studies are required to evaluate the efficacy of the vaccine under field conditions in protecting alpacas against infection with H. contortus.

Risk of environmental exposure to small coccidia from wild canid feces in rural Ohio.

OBJECTIVE To determine the extent of environmental exposure to heteroxenous coccidia from wild canid feces in southeastern Ohio. SAMPLE 285 presumed wild canid fecal samples collected across an ecological system in southeastern Ohio. PROCEDURES Morphological classification and molecular analysis were used to determine the canid genus for collected fecal samples. Microscopic and molecular analysis were used to detect coccidian oocysts and DNA. Several variables were analyzed for associations with coccidian DNA detection or prevalence. RESULTS Coccidian DNA was detected in 51 of 285 (17.9%) fecal samples. Of those positive samples, 1% (95% confidence interval, 0.4% to 3%) had positive results for Hammondia heydorni and none had positive results for Neospora caninum, for an estimated environmental N caninum prevalence of 0% (95% confidence interval, 0% to 7%)/1-km2 hexagonal area evaluated. Morphological classification revealed that 78.9% (225/285) of fecal samples were from coyotes and 17.2% (49/285) were from foxes. No difference in proportions of coccidian DNA-positive fecal samples was identified among canid species. Environmental temperature and fecal freshness were associated with coccidian DNA detection. Land use type, relative canid density, and cattle density were not associated with the prevalence of coccidian DNA-positive samples. CONCLUSIONS AND CLINICAL RELEVANCE The low prevalence of coccidia shed in wild canid feces in this study, including the estimated 0% environmental prevalence of N caninum, suggested that the role of the oocyst environmental phase in coccidia transmission to ruminants is likely minor in rural southeastern Ohio.

Biochemical and enzymatic characterization of purified covalent complexes of matrix metalloproteinase-9 and haptoglobin released by bovine granulocytes in vitro.

OBJECTIVE: To characterize and purify covalent complexes of matrix metalloproteinase-9 (MMP-9) and haptoglobin released by bovine granulocytes in vitro. SAMPLE POPULATION: Blood samples obtained from healthy cows and cows with acute and chronic inflammation to obtain WBCs and sera. PROCEDURES: WBCs were isolated by differential centrifugation, hypotonic lysis of RBCs, and degranulated by stimulation with phorbol ester (20 ng/mL). Cell-conditioned medium was subjected to affinity and gel chromatography and purified proteins subjected to SDS- PAGE gelatin zymography, western blot analysis, Coomassie blue staining, and peptide mass spectrometry for protein identification. Sera of cows hospitalized for acute and chronic septic conditions and of clinically normal cows were analyzed with similar methods. RESULTS: Matrix metalloproteinase-9 was released from neutrophils in vitro and migrated to a molecular mass of approximately 220 kd (prodimer), approximately 105 kd (promonomer), and > 220 kd (high-molecular mass complexes). These high-molecular mass complexes were composed of alpha- and beta-haptoglobin and MMP-9 (ratio13:13:1). Complexes of MMP-9 and haptoglobin had biochemical properties of both its protein constituents (i.e., enzymatic activity toward gelatin and hemoglobin binding). Complexes of MMP-9 and haptoglobin were also detected in sera of cows with acute inflammation, but not in clinically normal cows or cows with chronic disease. CONCLUSIONS AND CLINICAL RELEVANCE: A fraction of neutrophil MMP-9 is released in complex with haptoglobin. The complex is present in granules and retains biological activity of its components. Detection of the complex in serum may provide an indicator of acute inflammation.

Small sarcocysts can be a feature of experimental infections with Sarcocystis neurona merozoites.

Several reports indicate the presence of small tissue cysts associated with Sarcocystis neurona infections. Several failed attempts to develop tissue cysts in potential intermediate host using in vitro derived parasites originally isolated from horses with equine protozoal myeloencephalitis suggest that the experimental methods to achieve bradyzoites with those isolates was not possible. Those prior studies reported the lack of detectable sarcocysts based on histology and in vivo feeding trials. A recent report of successful production and detection of small sarcocysts triggered us to review archived tissues from earlier experimental infection studies. The retrospective review sought to determine if small sized sarcocysts were not detected due to their relatively smaller size and infrequency as compared to larger sized sarcocysts produced with other isolates in these experimental inoculation trials. Tissues from two prior in vivo inoculation studies, involving in vitro-produced parasites inoculated into laboratory-reared cats and raccoons, were re-examined by immunohistochemistry staining to more easily detect the tissue cysts. In the experimental cat study no small tissue cysts were seen, consistent with the original publication results. However, in the experimental raccoon study, one raccoon inoculated with an EPM-derived isolate, SN-UCD1, had small sarcocysts not reported in the original publication. This retrospective study suggests that much closer scrutiny of tissues, including the use of immunohistochemistry on tissue sections is required to detect the smaller S. neurona sarcocysts associated with the experimental inoculations of the isolates originally derived from horses with EPM.