Effect of different adjuvant formulations on the antibody response of horses to porcine zona pellucida proteins.

Immunization with porcine zona pellucida (PZP) proteins is being used successfully to induce infertility in wildlife including horses. However, widespread adoption of this method to control the growth of horse populations requires further refinement in order to induce long-term infertility, reduce the frequency and severity of injection site reactions, and make the vaccines easier to administer. The next generation of PZP-based vaccines will likely be a controlled-release formulation with different adjuvants from the Freund's adjuvants used in existing vaccines. We evaluated the response of equine peripheral blood mononuclear cells to a cationic nanoparticle adjuvant, Nano-11, alone and with the TLR agonists poly(I:C) and CpG ODN as a screen to develop an adjuvant system suitable for immunization of horses. The secretion of IL-1ß, TNF and CXCL10 were used as readouts. The combination of poly(I:C) with Nano-11 significantly increased the secretion of IL-1ß and TNF in comparison with Nano-11 only, with little effect of further addition of CpG ODN. The efficacy of the Nano-11/poly(I:C) adjuvant to enhance the immune response to native PZP proteins was determined in horses. Horses were immunized twice with the licensed Zonastat-H vaccine or PZP with Nano-11/poly(I:C) emulsified in silicone oil. A third group received PZP with the saponin adjuvant QA-21 emulsified in silicone oil. The horse sera collected monthly after the injections had increased anti-PZP IgG antibodies with the strongest response observed with Zonastat-H. We conclude that Nano-11/poly(I:C) is a potential candidate for the development of a controlled release formulation of a next generation PZP-based immunocontraception.

Fertility Control and the Welfare of Free-Roaming Horses and Burros on U.S. Public Lands: The Need for an Ethical Framing.

To be effective and publicly acceptable, management of free-roaming horses and burros in the United States and elsewhere needs a consistent ethical framing of the animals and the land they occupy. In the U.S., the two laws that largely govern wild horse and burro management, the 1971 Wild Free-Roaming Horse and Burro Act and the 1976 Federal Land Policy and Management Act ("FLPMA"), rest on conflicting foundations, the former based on an ethic of care and the latter on largely utilitarian principles. These conflicts specifically fuel debates over the selection of appropriate fertility control agents for horse and burro management. Because land-use and management decisions are largely controlled by the FLPMA, and because the ethical treatment of animals is typically considered under conditions established by their use, both the larger debate about equids and land management and the specific debate about fertility control are dominated by cost/benefit calculations and avoid broader ethical considerations. In our view, the long-term health and ethical treatment of free-roaming horses and burros, the lands they occupy, and the wildlife and people they share it with will require the replacement of the resource-use model with a more holistic, care-based approach.

Gut microbiota of wild fish as reporters of compromised aquatic environments sleuthed through machine learning.

Human-generated negative impacts on aquatic environments are rising. Despite wild fish playing a key role in aquatic ecologies and comprising a major global food source, physiological consequences of these impacts on them are poorly understood. Here we address the issue through the lens of interrelationship between wild fish and their gut microbiota, hypothesizing that fish microbiota are reporters of the aquatic environs. Two geographically separate teleost wild-fish species were studied (Lake Erie, Ohio, and Caribbean Sea, US Virgin Islands). At each geolocation, fresh fecal samples were collected from fish in areas of presence or absence of known aquatic compromise. Gut microbiota was assessed via microbial 16S-rRNA gene sequencing and represents the first complete report for both fish species. Despite marked differences in geography, climate, water type, fish species, habitat, diet, and gut microbial compositions, the pattern of shifts in microbiota shared by both fish species was nearly identical due to aquatic compromise. Next, these data were subjected to machine learning (ML) to examine reliability of using the fish-gut microbiota as an ecomarker for anthropogenic aquatic impacts. Independent of geolocation, ML predicted aquatic compromise with remarkable accuracy (>90%). Overall, this study represents the first multispecies stress-related comparison of its kind and demonstrates the potential of artificial intelligence via ML as a tool for biomonitoring and detecting compromised aquatic conditions.

Dens evaginatus: the hornet's nest of adolescent orthodontics.

Dens evaginatus is a rare dental anomaly that occurs during tooth development and results in an abnormal protrusion from the occlusal surface of the affected tooth, often in the area of the central groove between the buccal and lingual cusps. Of clinical importance to the orthodontist is that these occlusal tubercles fracture easily or can be worn away, resulting in direct pulp exposure in a noncarious tooth. This can cause severe complications, including loss of tooth vitality, facial infection in the form of an abscess or cellulitis, or osteomyelitis of the jaw. If extraction of premolars is indicated for orthodontic treatment after careful diagnosis and treatment planning, it is paramount to establish the health of the premolars that will remain in the dentition before extracting the teeth.

From the pens to the field: real-world wildlife contraception.

There are only two nonlethal approaches with which to manage wildlife populations: remove excess individuals or decrease reproductive rates. In the case of wild horse management, the latter has already been shown to be a more humane and less costly approach. Contraception has been known for many years to be effective in wild horses with regard to both fertility and population alteration. Field testing under real-world management situations is a critical aspect of wildlife contraception assessment. Field testing also requires documentation to justify eventual large-scale use of fertility control in population management. Furthermore, it is likely that full support by the managing agencies and acceptance of fertility control by the public will not occur until success has been demonstrated at the population level in the field. Because the transition from laboratory and captive animal trials to the real world of field research and testing of management potential in free-ranging wildlife is both essential and difficult, we identify considerations for optimizing success in the field, especially for controlling wild horse and white-tailed deer populations. This study identified the following categories for assessment: 1) field-specific logistics and tactics (including access to and identification of horses, censuses, pregnancy testing, and behavior monitoring), 2) training of field personnel and managers (including vaccine biology, preparation and delivery, and basic field methodology), 3) essential field considerations for enabling management (including population variables, environmental variables, and modeling), and 4) pitfalls of field contraception. In this assessment, we will highlight the capability for coincident pursuit of research and management and will explore field considerations applicable to many species where fertility control has potential as a management tool.

Analytic expression for the mean time to absorption for a random walker on the Sierpinski gasket. II. The eigenvalue spectrum.

We continue the study of a particle (atom, molecule) undergoing an unbiased random walk on the Sierpinski gasket, and obtain for the gasket and tower the eigenvalue spectrum of the associated stochastic master equation. Analytic expressions for recurrence relations among the eigenvalues are derived. The recurrence relations obtained are compared with those determined for two Euclidean lattices, the closed chain with an absorbing site and a finite chain with an absorbing site at one end. We check and confirm the internal consistency between the smallest eigenvalue and the mean walklength in each of the cases studied. Attention is drawn to the relevance of the results obtained to a problem of electron transfer in proteins.

Modeling of trabecular bone and lamina dura following selective alveolar decortication in rats.

BACKGROUND: Modifying the balance between resorption and apposition through selectively injuring the cortical plate of the alveolus has been an approach to speed tooth movement and is referred to as periodontally accelerated osteogenic orthodontics. The aim of this study was to investigate the alveolar response to corticotomy as a function of time and proximity to the surgical injury in a rat model. METHODS: Maxillary buccal and lingual cortical plates were injured in 36 healthy adult rats adjacent to the upper left first molars. Twenty-four animals were euthanized at 3, 7, or 11 weeks. In one group, the maxillae were removed and stripped of soft tissues, and histomorphometric analysis was performed to study alveolar spongiosa and periodontal ligament (PDL) modeling dynamics. Catabolic activity was analyzed with tartrate-resistant acid phosphatase-positive osteoclasts and preosteoclasts. Anabolic actions were measured using a fluorescent vital bone stain series followed by sacrifice at 30 and 51 days. To further analyze the new bone formation, a separate group of animals were fed with calcein fluorescent stain and processed for non-decalcified fluorescent stain histology. RESULTS: At 3 weeks, the surgery group had significantly (P <0.05) less calcified spongiosa bone surface, greater periodontal ligament surface, higher osteoclast number, and greater lamina dura apposition width. The catabolic activity (osteoclast count) and anabolic activity (apposition rate) were three-fold greater, calcified spongiosa decreased by two-fold, and PDL surface increased by two-fold. Surgical injury to the alveolus that induced a significant increase in tissue turnover by week 3 dissipated to a steady state by postoperative week 11. The impact of the injury was localized to the area immediately adjacent to the decortication injury. CONCLUSION: Selective alveolar decortication induced increased turnover of alveolar spongiosa, and the activity was localized; dramatic escalation of demineralization-remineralization dynamics is the likely biologic mechanism underlying rapid tooth movement following selective alveolar decortication.

The cutaneous manifestations of atypical complete DiGeorge syndrome: a histopathologic and immunohistochemical study.

DiGeorge syndrome is a congenital anomaly with a constellation of findings that includes thymic hypoplasia. Only a small subset of patients with DiGeorge syndrome has complete athymia, classified as complete DiGeorge anomaly; one third of these patients show an eczematous dermatitis, oligoclonal T-cells and lymphadenopathy, known as atypical complete DiGeorge anomaly. Six biopsies from six patients with the distinctive clinical phenotype of atypical complete DiGeorge anomaly were studied. Every biopsy showed exocytosis (100%), parakeratosis, often confluent and spongiosis (100%). Neutrophilic abscesses (50%), dyskeratosis (67%) and satellite cell necrosis (50%) were seen. Perieccrine and perivascular inflammation were seen in half of the cases. Eosinophils were identified (83%); most commonly in both the epidermis and dermis. All of lymphocytes were CD3 positive. Most (83%) of cases contained T-cell intracellular antigen 1 (TIA-1) positive cells. Special testing of the selected patients using spectratyping identified oligoclonal T-cell populations. The presence of dyskeratotic keratinocytes, satellite cell necrosis and parakeratotic scale with neutrophils characterizes the cutaneous rash seen in this subset of complete DiGeorge syndrome patients. Such skin lesions from patients with DiGeorge anomaly should alert the pathologist to the potential diagnosis of atypical complete DiGeorge anomaly. The pathophysiologic role of the oligoclonal T-cells in this entity requires additional study.

Melanocytic nevi with nonsurgical trauma: a histopathologic study.

There is a belief among dermatopathologists that benign melanocytic nevi (BMN) may display atypical histologic characteristics when traumatized. However, to our knowledge, a systematic study of nonsurgically traumatized melanocytic nevi (TMN) has not been published. We studied a series of 92 TMN. Cases were analyzed for histologic evidence of architectural and cytologic criteria associated with atypia. Of the patients, 54 were female and 37 were male. The mean age was 38 years old (range 8-74 years old). Nevi were present, in order of frequency, on the extremities, trunk, and head/neck, but there were no acral sites. Histologic findings of trauma were as follows: parakeratosis (92%), dermal telangiectasias (61%), ulceration (51%), dermal inflammation (49%), melanin within stratum corneum (24%), and dermal fibrosis (25%). Pagetoid spread of melanocytes was limited to the site of trauma in 20% of cases and was identified away from areas of trauma in 8% of cases. Melanocytic atypia was seen in three cases. Dermal mitoses were rare (one mitotic figure in three cases). Pagetoid spread under a traumatized epidermis was relatively frequent and, in isolation, is compatible with a benign TMN. Any traumatized melanocytic lesion that displays cytologic atypia, pagetoid spread outside of the area of the traumatized epidermis, or dermal mitoses should be treated with caution because these findings were rarely seen in TMN.

Use of mucosal immunization with porcine zona pellucida (PZP) in mice and rabbits.

Rabbits (Oryctolagus cuniculus) and two strains of mice (Mus musculus, one inbred and one outbred) were immunized against porcine zona pellucida (PZP) antigen. Alginate microspheres or cholera toxin B were used alone or in combination when mucosal immunization routes were used. Serum antibody responses and fertility were assessed. Neither rabbit or mouse groups immunized by mucosal routes generated significant antibody responses to PZP as compared to parenteral immunization (ANOVA, P > 0.05). The study shows that porcine zona pellucida is not an effective mucosal antigen in small mammals.

Fecal corticosterone assessment in the epaulette shark, Hemiscyllium ocellatum.

The present study examined the feasibility of measuring the steroid hormone corticosterone in fecal extracts of epaulette sharks, Hemiscyllium ocellatum. Six immature, captive-raised epaulette sharks (four females and two males) were obtained from two different zoos and were maintained in a closed-system, 530-liter aquarium. After a one-month adaptation, fecal samples were collected daily from each animal for 33 days. Five-day sets of samples were pooled within animals to insure sufficient material for analysis. Fecal hormone extraction was achieved using repeated cycles of dichloromethane and aqueous washes. The levels of corticosterone were measured by reverse-phase high-performance liquid chromatography (HPLC). Corticosterone presence in HPLC eluent peaks from fecal extracts was determined by comparison of the elution pattern of corticosterone standard with the elution patterns of fecal extracts with and without the addition of tritiated corticosterone or exogenous, unlabeled corticosterone. Exclusive presence of corticosterone in HPLC eluent peaks presumed to be corticosterone was determined by nuclear magnetic resonance mass spectrometry. Corticosterone levels, calculated from a 10-point standard curve, ranged from 1.2 to 20.9 ng/g feces across all sharks, with 92.3% of values being < or =13.5 ng/g. Within individuals, the lowest average for corticosterone levels across 33 days was 2.6+/-0.4 ng/g feces, and the highest average was 8.4+/-2.2 ng/g feces. This study demonstrated that corticosterone was extractable from and reliably measurable in fecal extracts of epaulette sharks. This is the first evidence of this hormone in epaulette sharks and the first report of fecal corticosterone in elasmobranchs.

Measurement of fecal glucocorticoids in parrotfishes to assess stress.

Coral reefs are in decline worldwide from a combination of natural and human forces. The environmental compromises faced by coral reef habitats and their associated fishes are potentially stressful, and in this study we examined the potential for assessing stress levels in coral reef fish. We determined the feasibility of using fecal casts from parrotfishes for remote assessment of stress-related hormones (cortisol and corticosterone), and the response of these hormones to the stress of restraint and hypoxia. Measurement of these hormones in fecal extracts by high performance liquid chromatography (HPLC) was validated using mass spectrometry, chemical derivitization, and radioactive tracer methods. In aquarium-adapted parrotfish, baseline levels of cortisol and corticosterone averaged 3.4+/-1.1 and 14.8+/-2.8 ng/g feces, respectively, across 32 days. During 13 days of periodic stress these hormones, respectively, average 10.8-fold and 3.2-fold greater than baseline, with a return to near baseline during a 23-day follow-up. Testosterone was also measured as a reference hormone which is not part of the stress-response axis. Levels of this hormone were similar across the study. These fecal hormones were also measured in a field study of parrotfish in 10 fringing coral reef areas around the Caribbean Island of St. John, US Virgin Islands. Extracts of remotely collected fecal casts of three parrotfish species revealed no difference in respective average hormone levels among these species. Also, there was no difference in respective hormone levels between aquarium and field environments. However, levels of both cortisol and corticosterone, but not testosterone, were elevated in two of the 10 reef sites surveyed. This study demonstrates that parrotfish fecals can be collected in aquarium and field conditions and that steroid hormones in these fecals can be extracted and reliably measured. The study also demonstrates that cortisol and corticosterone in parrotfish fecals can be used as an indicator of the stress-response which is unlikely to be masked by intrinsic variability in the sample source, environment or methodology.

Remote assessment of stress in white rhinoceros (Ceratotherium simum) and black rhinoceros (Diceros bicornis) by measurement of adrenal steroids in feces.

This study monitored fecal cortisol and corticosterone levels in 14 black rhinoceroses (Diceros bicornis) and in seven white rhinoceroses (Certotherium simum) under various conditions of captivity, including translocation. Free cortisol and free corticosterone were measured in methylene chloride extracts of feces, using high-performance liquid chromatography (HPLC). The extraction-assay method was validated for quantitative measurement of these hormones by mass spectroscopy analysis, chemical derivitization, and radiolabel tracking and recovery. Both cortisol and corticosterone were extractable from feces and routinely detectable by HPLC. In three nonstressed, captivity-adapted white rhinoceroses monitored across 21 days of routine activity, fecal cortisol ranged from 2.0 to 7.3 ng/g dry feces and corticosterone from 4.0 to 10.8 ng/g dry feces, with no observable trend. Matched plasma, urine, and fecal samples in these rhinoceroses yielded corticosterone:cortisol ratios of 2.0:1.0, 2.7:1.0, and 2.2:1.0, respectively. Both black rhinoceroses (n = 5) and white rhinoceroses (n = 4) exhibited higher fecal cortisol (6.9- to 10.0-fold) and corticosterone (3.2- to 4.5-fold) levels in association with restraint-translocation than in limited free-roaming conditions. In five black rhinoceroses monitored across 6 wk after release from translocation, fecal levels of both cortisol and corticosterone decreased significantly between week 1 and weeks 4-6. In general, cortisol and corticosterone paralleled each other, with cortisol exhibiting a greater range of response. Measurement of either hormone in feces appears to be reliable for adrenal axis monitoring in the white and the black rhinoceroses.