Bacterial microbiomes from mucus and breath of southern resident killer whales (Orcinus orca).

Opportunities to assess odontocete health are restricted due to their limited time at the surface, relatively quick movements and large geographic ranges. For endangered populations such as the southern resident killer whales (SKRWs) of the northeast Pacific Ocean, taking advantage of non-invasive samples such as expelled mucus and exhaled breath is appealing. Over the past 12 years, such samples were collected, providing a chance to analyse and assess their bacterial microbiomes using amplicon sequencing. Based on operational taxonomic units, microbiome communities from SRKW and transient killer whales showed little overlap between mucus, breath and seawater from SRKW habitats and six bacterial phyla were prominent in expelled mucus but not in seawater. Mollicutes and Fusobacteria were common and abundant in mucus, but not in breath or seawater, suggesting these bacterial classes may be normal constituents of the SRKW microbiome. Out of 134 bacterial families detected, 24 were unique to breath and mucus, including higher abundances of Burkholderiaceae, Moraxellaceae and Chitinophagaceae. Although there were multiple bacterial genera in breath or mucus that include pathogenic species (e.g. Campylobacter, Hemophilus, Treponema), the presence of these bacteria is not necessarily evidence of disease or infection. Future emphasis on genotyping mucus samples to the individual animal will allow further assessment in the context of that animal's history, including body condition index and prior contaminants burden. This study is the first to examine expelled mucus from cetaceans for microbiomes and demonstrates the value of analysing these types of non-invasive samples.

Hybrid model intermediate between a laboratory and field study: A humane paradigm shift in feline research.

OBJECTIVES: Non-surgical contraceptives are under development to provide accessible, affordable and humane alternatives for the management of free-roaming cat populations. The objective of this project was to develop a research approach for promising non-surgical contraceptives using outbred cats in a simulated free-roaming setting, meeting high standards for both animal welfare and scientific rigor. METHODS: A facility, specially constructed with indoor and outdoor living areas, was approved and regulated as both an animal shelter and a United States Department of Agriculture research facility. Thirty female and five male cats, healthy but at high risk of euthanasia, were recruited from animal shelters and private homes. Guided by a detailed protocol, cats were housed in this facility for up to 18 months after acclimatization. Cats were administered the study product or a placebo, and then entered into a breeding trial. Cats were adopted at the end of the study. A range of methods was used to provide enrichment and balance a natural environment with the need for detailed daily monitoring. RESULTS: Primary study results related to contraceptive safety and efficacy are published separately. Achieving a research model that is an intermediate step between a laboratory and an uncontained free-roaming cat colony was complex. Significant learnings shared in this current publication span: the selection of cats; acclimatization to a simulated colony environment; cat behavioral training during the study and in preparation for adoption; disease management; contract staff and volunteer support; and cat behavior throughout a breeding study. CONCLUSIONS AND RELEVANCE: This model inspires continued movement away from the paradigm of breeding cats for research and instead sources existing cats at risk for euthanasia. The housing and management of the cats elevates research animals' quality of life and provides positive post-study outcomes. While not appropriate for every feline research scenario, this hybrid model (between a laboratory and field study) proved to be a practical, humane and reliable scenario for research requiring a simulated real-world environment.

Capromorelin: a ghrelin receptor agonist and novel therapy for stimulation of appetite in dogs.

Ghrelin is a hormone, secreted from cells in the stomach, which is important in the regulation of appetite and food intake in mammals. It exerts its action by binding to a specific G-protein-coupled receptor, the growth hormone secretagogue receptor 1a (GHS-R1a) which is found in areas of the brain associated with the regulation of food intake. Ghrelin causes a release of growth hormone (GH) through binding to GHS-R1a in the hypothalamus and pituitary gland. A class of compounds known as growth hormone secretagogues, or ghrelin receptor agonists, were developed for therapeutic use in humans for the stimulation of GH in the frail elderly, and have subsequently been studied for their effects on increasing appetite and food intake, increasing body weight, building lean muscle mass, and treating cachexia. Subsequent research has shown that ghrelin has anti-inflammatory and immunomodulatory effects. This article reviews the basic physiology of ghrelin and the ghrelin receptor agonists, including the available evidence of these effects in vitro and in vivo in rodent models, humans, dogs and cats. One of these compounds, capromorelin, has been FDA-approved for the stimulation of appetite in dogs (ENTYCE ®). The data available on the safety and effectiveness of capromorelin is reviewed, along with a discussion of the potential clinical applications for ghrelin receptor agonists in both human and veterinary medicine.

Effectiveness of GonaCon as an immunocontraceptive in colony-housed cats.

Objectives Non-surgical contraceptive management of free-roaming cat populations is a global goal for public health and humane reasons. The objectives of this study were to measure the duration of contraception following a single intramuscular injection of a gonadotropin-releasing hormone-based vaccine (GonaCon) and to confirm its safe use in female cats living in colony conditions. Methods GonaCon (0.5 ml/cat) was administered intramuscularly to 20 intact female cats (queens), and saline was administered to 10 queens serving as sham-treated controls. Beginning in late February, 4 months after injection, all cats were housed with fertile male cats in a simulated colony environment. Time to pregnancy, fetal counts and vaccine-elicited injection-site reactions were evaluated. Results All control cats (n = 10/10) and 60% (n = 12/20) of vaccinated cats became pregnant within 4 months of the introduction of males. Two additional vaccinates became pregnant (70%; n = 14/20) within 1 year of treatment. Average fetal counts were significantly lower in vaccinated cats than in control cats. Vaccinates had a significantly longer ( P = 0.0120) median time to conception (212 days) compared with controls (127.5 days). Injection-site reactions ranging from swelling to transient granulomatous masses were observed in 45% (n = 9/20) of vaccinated cats. Conclusions and relevance A single dose of GonaCon provided contraception lasting for a minimum of 1 year in 30% (n = 6/20) of treated cats. The level of contraception induced by this GonaCon dose and vaccine lot was not sufficiently effective to be recommended for use in free-roaming cats.

Changing innovation into a registered product: From concept to regulatory approval.

Innovation in animal health pharmaceuticals is important to address unmet and underserved medical needs, and often comes from products initially developed for human medicine. The purpose of the review is to help readers understand how breakthroughs from human biotechnology may be developed for use in veterinary medicine, while understanding the key drivers to success, the difficulties of regulatory approval, and the realistic risks and rewards of developing applications for animals. The types of human drugs which may be useful for veterinary applications are reviewed, including examples. The regulatory path is discussed, with a review of the various oversight agencies, and the categories of data required to be submitted, including safety, efficacy, manufacturing, environmental impact and human food safety. In conclusion, the cost, development time, and barriers to innovation in veterinary medical pharmaceuticals are discussed.

Respiratory Microbiome of Endangered Southern Resident Killer Whales and Microbiota of Surrounding Sea Surface Microlayer in the Eastern North Pacific.

In the Salish Sea, the endangered Southern Resident Killer Whale (SRKW) is a high trophic indicator of ecosystem health. Three major threats have been identified for this population: reduced prey availability, anthropogenic contaminants, and marine vessel disturbances. These perturbations can culminate in significant morbidity and mortality, usually associated with secondary infections that have a predilection to the respiratory system. To characterize the composition of the respiratory microbiota and identify recognized pathogens of SRKW, exhaled breath samples were collected between 2006-2009 and analyzed for bacteria, fungi and viruses using (1) culture-dependent, targeted PCR-based methodologies and (2) taxonomically broad, non-culture dependent PCR-based methodologies. Results were compared with sea surface microlayer (SML) samples to characterize the respective microbial constituents. An array of bacteria and fungi in breath and SML samples were identified, as well as microorganisms that exhibited resistance to multiple antimicrobial agents. The SML microbes and respiratory microbiota carry a pathogenic risk which we propose as an additional, fourth putative stressor (pathogens), which may adversely impact the endangered SRKW population.

Capromorelin oral solution (ENTYCE®) increases food consumption and body weight when administered for 4 consecutive days to healthy adult Beagle dogs in a randomized, masked, placebo controlled study.

BACKGROUND: Dogs can suffer from inappetence caused by a variety of medical conditions. This may present as anorexia (complete loss of appetite), hyporexia (decreased appetite) or dysrexia (change in food preferences). A drug with a new mechanism of action, capromorelin, has potential to stimulate appetite in dogs. Capromorelin is a ghrelin receptor agonist, which mimics the action of endogenous ghrelin. It is a member of the growth hormone secretagogue (GHS) class of drugs. Capromorelin oral solution (ENTYCE®) was tested in healthy adult male and female Beagle dogs (n = 6 males and 6 females per group) for its effect on food consumption and body weight. A randomized, masked, placebo controlled study was conducted to measure the effects of a daily 3 mg/kg oral dose given over 4 days. Dogs were observed for clinical signs, physical examinations were completed prior to and at the end of treatment, and blood was drawn before and after treatment for evaluation of serum chemistry and hematology parameters. RESULTS: Capromorelin was well-tolerated, with no abnormalities seen on physical examination or clinical pathology. Some dogs showed increased salivation. Capromorelin treated dogs had increased mean (±SD) food consumption compared to placebo treated dogs (60.55 ± 39.87% versus -11.15 ± 14.23% respectively, P < 0.001). Treated dogs also had increased mean body weights compared to placebo treated dogs (5.96 ± 1.76% versus 0.053 ± 1.14% respectively, P < 0.001). CONCLUSIONS: This study supports the effectiveness of capromorelin oral solution as an appetite stimulant in dogs. Treatment with the oral solution resulted in dramatic increases in appetite, as measured by food consumption, of over 60% compared to placebo. The drug was well tolerated. Capromorelin is the first ghrelin receptor agonist developed for appetite stimulation in any species, and represents a novel mechanism of action for this clinical use.

New approaches to non-surgical sterilization for dogs and cats: Opportunities and challenges.

Over the last 40 years, researchers have explored methods to non-surgically suppress fertility in animals. Immunocontraception has been used to control wildlife populations but does not confer long-term immunity. The gonadotropin-releasing hormone (GnRH) agonist deslorelin, formulated as an implant to provide 6-month to 1-year suppression of fertility in male dogs, is available commercially in some countries. Neither of these approaches provide permanent sterility. A single-dose, permanent treatment would be a valuable tool in dog and cat population control. The Michelson Prize and Grants (MPG) programme was initiated "to eliminate shelter euthanasia of healthy, adoptable companion animals and reduce populations of feral and free-roaming cats and dogs" offering a $25 million US prize for a non-surgical sterilant that is effective as a single treatment in both male and female dogs and cats. Michelson Prize and Grants programme has offered US $50 million in grant money for research and has attracted scientists worldwide. Approaches under study include gene therapy, small interfering RNA to inhibit reproductive targets and delivery of cytotoxins to pituitary gonadotrophs or GnRH producing neurons in the hypothalamus. Research in implant technology that could deliver compounds over an animal's lifetime is also underway. Details of funded grants and results to date can be found at: http://www.michelsonprizeandgrants.org/michelson-grants/research-findings. The next steps are translating the most promising research into products. The Alliance for Contraception of Cats and Dogs (ACC&D) is helping to research practical methods of marking sterilized animals to avoid costly retreatment and population modelling that will help guide field workers in use of resources for sterilization programmes.

Stable isotope-based trophic structure of pelagic fish and jellyfish across natural and anthropogenic landscape gradients in a fjord estuary.

Identifying causes of structural ecosystem shifts often requires understanding trophic structure, an important determinant of energy flow in ecological communities. In coastal pelagic ecosystems worldwide, increasing jellyfish (Cnidaria and Ctenophora) at the expense of small fish has been linked to anthropogenic alteration of basal trophic pathways. However, this hypothesis remains untested in part because baseline description of fish-jellyfish trophic dynamics, and the environmental features that influence them are lacking. Using stable isotopes of carbon (δ13C) and nitrogen (δ15N), we examined spatiotemporal patterns of fish and jellyfish trophic structure in greater Puget Sound, an urbanizing fjord estuary in the NW United States. We quantified niche positions of constituent species, niche widths and trophic overlap between fish and jellyfish assemblages, and several community-level trophic diversity metrics (resource diversity, trophic length, and niche widths) of fish and jellyfish combined. We then related assemblage- and community-level measures to landscape gradients of terrestrial-marine connectivity and anthropogenic influence in adjacent catchments. Relative niche positions among species varied considerably and displayed no clear pattern except that fish generally had higher δ15N and lower δ13C relative to jellyfish, which resulted in low assemblage-level trophic overlap. Fish assemblages had larger niche widths than jellyfish in most cases and, along with whole community trophic diversity, exhibited contrasting seasonal patterns across oceanographic basins, which was positively correlated to landscape variation in terrestrial connectivity. In contrast, jellyfish niche widths were unrelated to terrestrial connectivity, but weakly negatively correlated to urban land use in adjacent catchments. Our results indicate that fish-jellyfish trophic structure is highly heterogeneous and that disparate processes may underlie the trophic ecology of these taxa; consequently, they may respond divergently to environmental change. In addition, spatiotemporal variation in ecosystem connectivity, in this case through freshwater influence, may influence trophic structure across heterogeneous landscapes.

Safety and toxicokinetic profiles associated with daily oral administration of grapiprant, a selective antagonist of the prostaglandin E2 EP4 receptor, to cats.

OBJECTIVE To evaluate safety and toxicokinetic profiles associated with daily oral administration of grapiprant, a new analgesic that selectively blocks the prostaglandin E2 EP4 receptor, to cats. ANIMALS 24 healthy domestic shorthair cats (12 males and 12 females). PROCEDURES Cats were randomly assigned (3 of each sex/group) to receive a placebo capsule or grapiprant at 3, 9, or 15 mg/kg, administered PO once daily for 28 days, beginning on day 0. Food consumption and behavior were observed daily, body weight was measured weekly, and clinicopathologic tests were performed on blood and urine samples collected on days -7, 14, and 25. Blood samples for toxicokinetic analyses were collected after treatment on days 0 and 27. Cats were euthanized on day 28, and full necropsies and histologic evaluations were performed. RESULTS Grapiprant rapidly reached peak serum concentrations and maintained substantial concentrations throughout the 28-day period. By day 27, maximum serum concentrations ranged from 683 ng/mL to 4,950 ng/mL, which were attained by 1 to 4 hours after administration. Serum half-lives on day 27 ranged from approximately 2 to 14 hours (median, approx 5 to 6 hours). Grapiprant was well tolerated, and no adverse effects were detected at doses ≤ 15 mg/kg. No significant effects of grapiprant were identified on body weight, food consumption, clinicopathologic variables, or gross or histologic necropsy findings. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested the safety of daily oral administration of grapiprant to cats. Additional studies are needed to evaluate the efficacy of grapiprant for treatment of cats with osteoarthritis.

Grapiprant: an EP4 prostaglandin receptor antagonist and novel therapy for pain and inflammation.

There are five active prostanoid metabolites of arachidonic acid (AA) that have widespread and varied physiologic functions throughout the body, including regulation of gastrointestinal mucosal blood flow, renal haemodynamics and primary haemostasis. Each prostanoid has at least one distinct receptor that mediates its action. Prostaglandin E2 (PGE 2) is a prostanoid that serves important homeostatic functions, yet is also responsible for regulating pain and inflammation. PGE 2 binds to four receptors, of which one, the EP4 receptor, is primarily responsible for the pain and inflammation associated with osteoarthritis (OA). The deleterious and pathologic actions of PGE 2 are inhibited in varying degrees by steroids, aspirin and cyclo-oxygenase inhibiting NSAIDs; however, administration of these drugs causes decreased production of PGE 2, thereby decreasing or eliminating the homeostatic functions of the molecule. By inhibiting just the EP4 receptor, the homeostatic function of PGE 2 is better maintained. This manuscript will introduce a new class of pharmaceuticals known as the piprant class. Piprants are prostaglandin receptor antagonists (PRA). This article will include basic physiology of AA, prostanoids and piprants, will review available evidence for the relevance of EP4 PRAs in rodent models of pain and inflammation, and will reference available data for an EP4 PRA in dogs and cats. Piprants are currently in development for veterinary patients and the purpose of this manuscript is to introduce veterinarians to the class of drugs, with emphasis on an EP4 PRA and its potential role in the control of pain and inflammation associated with OA in dogs and cats.

Identifying copy number variation of the dominant virulence factors msa and p22 within genomes of the fish pathogen Renibacterium salmoninarum.

Renibacterium salmoninarum is the causative agent of bacterial kidney disease, an important disease of farmed and wild salmonid fish worldwide. Despite the wide spatiotemporal distribution of this disease and habitat pressures ranging from the natural environment to aquaculture and rivers to marine environments, little variation has been observed in the R. salmoninarum genome. Here we use the coverage depth from genomic sequencing corroborated by real-time quantitative PCR to detect copy number variation (CNV) among the genes of R. salmoninarum. CNV was primarily limited to the known dominant virulence factors msa and p22. Among 68 isolates representing the UK, Norway and North America, the msa gene ranged from two to five identical copies and the p22 gene ranged from one to five copies. CNV for these two genes co-occurred, suggesting they may be functionally linked. Isolates carrying CNV were phylogenetically restricted and originated predominantly from sites in North America, rather than the UK or Norway. Although both phylogenetic relationship and geographical origin were found to correlate with CNV status, geographical origin was a much stronger predictor than phylogeny, suggesting a role for local selection pressures in the repeated emergence and maintenance of this trait.

Widespread detection of human- and ruminant-origin Bacteroidales markers in subtidal waters of the Salish Sea in Washington State.

Rising populations around coastal systems are increasing the threats to marine water quality. To assess anthropogenic fecal influence, subtidal waters were examined monthly for human- and ruminant-sourced Bacteroidales markers at 80 sites across six oceanographic basins of the Salish Sea (Washington State) from April through October, 2011. In the basins containing cities with individual populations>190,000, >50% of sites were positive for the human marker, while in the basins with high densities of dairy and cattle operations, ∼30% of sites were positive for the ruminant marker. Marker prevalence was elevated in spring (April and May) and fall (October) and reduced during summer (June through September), corresponding with seasonal precipitation. By logistic regression, the odds of human marker detection increased with percentage of adjacent catchment impervious surface, dissolved nitrate concentration, and abundance of low nucleic acid bacteria, but decreased with salinity and chlorophyll fluorescence. The odds of ruminant marker detection increased with dissolved ammonium concentration, mean flow rate for the nearest river, and adjacent shoreline length. These relationships are consistent with terrestrial to marine water flow as a transport mechanism. Thus, Bacteroidales markers traditionally used for identifying nearby sources can be used for assessing anthropogenic fecal inputs to regional marine ecosystems.

Evaluation of the safety of long-term, daily oral administration of grapiprant, a novel drug for treatment of osteoarthritic pain and inflammation, in healthy dogs.

OBJECTIVE: To investigate the safety of daily oral administration of grapiprant to dogs. ANIMALS: Thirty-six 9-month-old Beagles of both sexes. PROCEDURES: Dogs were randomly assigned to groups that received grapiprant via oral gavage at 0, 1, 6, or 50 mg/kg (total volume, 5 mL/kg), q 24 h for 9 months. Each group contained 4 dogs of each sex (ie, 8 dogs/group), except for the 50 mg/kg group, which included 4 additional dogs that were monitored for an additional 30 days after treatment concluded (recovery period). All dogs received ophthalmologic, ECG, and laboratory evaluations before treatment began (baseline) and periodically afterward. All dogs were observed daily. Dogs were euthanized at the end of the study for necropsy and histologic evaluation. RESULTS: All dogs remained clinically normal during treatment, with no apparent changes in appetite or demeanor. Emesis and soft or mucoid feces that occasionally contained blood were observed in all groups, although these findings were more common in dogs that received grapiprant. In general, clinicopathologic findings remained within baseline ranges. Drug-related changes in serum total protein and albumin concentrations were detected, but differences were small and resolved during recovery. No drug-related gross or microscopic pathological changes were detected in tissue samples except mild mucosal regeneration in the ileum of 1 dog in the 50 mg/kg group. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested the safety of long-term oral administration of grapiprant to dogs. Efficacy of grapiprant in the treatment of dogs with osteoarthritis needs to be evaluated in other studies.

No surgery required: the future of feline sterilization: An overview of the Michelson Prize & Grants in Reproductive Biology.

OVERVIEW: For many years, researchers have been studying reproduction of cats and dogs, including approaches to non-surgical sterilization, but scant funding has been available for this work. Recognizing the need to fund research and to attract researchers from the biomedical community to apply their expertise to this area, the Michelson Prize & Grants (MPG) in Reproductive Biology program was founded. Since 2009, it has funded 34 research projects in seven countries toward discovery of a safe single-administration lifetime non-surgical sterilant in male and female cats and dogs. GOAL: The goal of the MPG program is the reduction or elimination of the approximately 2.7 million deaths of healthy shelter cats and dogs in the US every year. The successful product is expected to be a single-dose injectable product approved by the US Food and Drug Administration as a veterinary prescription item. The most optimistic prediction is that such a product will reach the hands of practicing veterinarians within the next decade. AREAS OF RESEARCH: Active research is in progress using approaches such as immunocontraception with a single-administration vaccine against gonadotropin releasing hormone (GnRH). Long-term therapy with GnRH agonists such as deslorelin administered in controlled-release devices is also being studied. Other scientists are targeting cells in the brain or gonads with cytotoxins, such as are used in cancer chemotherapy. Gene therapy expressing proteins that suppress reproduction and gene silencing of peptides essential to reproduction are further avenues of research. Findings are available at www.michelsonprizeandgrants.org/michelson-grants/research-findings.

Put a label (claim) on it: Getting non-surgical contraceptives approved for use in cats and dogs.

RELEVANCE: Non-surgical contraceptives or sterilants need regulatory approval to be sold for that use. This approval process gives veterinarians the information required to assess the benefits and risks of each product, and to provide comprehensive information on the required dose, method and duration of use, safety and effectiveness. AIM: This article reviews the information that must be developed and provided to regulatory agencies worldwide, with a focus on the European Union and the United States, in order to achieve regulatory approval. PROCESSES: The main components of developing a drug include developing extensive information on the safety and effectiveness of the product, and also the safety to the environment and to humans handling and administering the drug. Most importantly, a robust method of manufacturing both the drug itself and the formulated drug product (pill, liquid implant or injection) must be developed to assure quality and consistency in each batch. This information is then compiled and submitted to regulatory agencies; in the United States, this includes the Food and Drug Administration, the United States Department of Agriculture and the Environmental Protection Agency, and, in Europe, the European Medicines Agency. CHALLENGES: Because of the unique nature of non-surgical contraceptives for use in cats and dogs, particularly the desire to have these products last over multiple years, there are special challenges to their regulatory approval that are discussed in this review.

Microevolution of Renibacterium salmoninarum: evidence for intercontinental dissemination associated with fish movements.

Renibacterium salmoninarum is the causative agent of bacterial kidney disease, a major pathogen of salmonid fish species worldwide. Very low levels of intra-species genetic diversity have hampered efforts to understand the transmission dynamics and recent evolutionary history of this Gram-positive bacterium. We exploited recent advances in the next-generation sequencing technology to generate genome-wide single-nucleotide polymorphism (SNP) data from 68 diverse R. salmoninarum isolates representing broad geographical and temporal ranges and different host species. Phylogenetic analysis robustly delineated two lineages (lineage 1 and lineage 2); futhermore, dating analysis estimated that the time to the most recent ancestor of all the isolates is 1239 years ago (95% credible interval (CI) 444-2720 years ago). Our data reveal the intercontinental spread of lineage 1 over the last century, concurrent with anthropogenic movement of live fish, feed and ova for aquaculture purposes and stocking of recreational fisheries, whilst lineage 2 appears to have been endemic in wild Eastern Atlantic salmonid stocks before commercial activity. The high resolution of the SNP-based analyses allowed us to separate closely related isolates linked to neighboring fish farms, indicating that they formed part of single outbreaks. We were able to demonstrate that the main lineage 1 subgroup of R. salmoninarum isolated from Norway and the UK likely represent an introduction to these areas ~40 years ago. This study demonstrates the promise of this technology for analysis of micro and medium scale evolutionary relationships in veterinary and environmental microorganisms, as well as human pathogens.

Power of treatment success definitions when the Canine Brief Pain Inventory is used to evaluate carprofen treatment for the control of pain and inflammation in dogs with osteoarthritis.

OBJECTIVE: To determine the optimal method for use of the Canine Brief Pain Inventory (CBPI) to quantitate responses of dogs with osteoarthritis to treatment with carprofen or placebo. ANIMALS: 150 dogs with osteoarthritis. PROCEDURES: Data were analyzed from 2 studies with identical protocols in which owner-completed CBPIs were used. Treatment for each dog was classified as a success or failure by comparing the pain severity score (PSS) and pain interference score (PIS) on day 0 (baseline) with those on day 14. Treatment success or failure was defined on the basis of various combinations of reduction in the 2 scores when inclusion criteria were set as a PSS and PIS ≥ 1, 2, or 3 at baseline. Statistical analyses were performed to select the definition of treatment success that had the greatest statistical power to detect differences between carprofen and placebo treatments. RESULTS: Defining treatment success as a reduction of ≥ 1 in PSS and ≥ 2 in PIS in each dog had consistently robust power. Power was 62.8% in the population that included only dogs with baseline scores ≥ 2 and 64.7% in the population that included only dogs with baseline scores ≥ 3. CONCLUSIONS AND CLINICAL RELEVANCE: The CBPI had robust statistical power to evaluate the treatment effect of carprofen in dogs with osteoarthritis when protocol success criteria were predefined as a reduction ≥ 1 in PIS and ≥ 2 in PSS. Results indicated the CBPI can be used as an outcome measure in clinical trials to evaluate new pain treatments when it is desirable to evaluate success in individual dogs rather than overall mean or median scores in a test population.

Recurrent die-offs of adult coho salmon returning to spawn in Puget Sound lowland urban streams.

Several Seattle-area streams in Puget Sound were the focus of habitat restoration projects in the 1990s. Post-project effectiveness monitoring surveys revealed anomalous behaviors among adult coho salmon returning to spawn in restored reaches. These included erratic surface swimming, gaping, fin splaying, and loss of orientation and equilibrium. Affected fish died within hours, and female carcasses generally showed high rates (>90%) of egg retention. Beginning in the fall of 2002, systematic spawner surveys were conducted to 1) assess the severity of the adult die-offs, 2) compare spawner mortality in urban vs. non-urban streams, and 3) identify water quality and spawner condition factors that might be associated with the recurrent fish kills. The forensic investigation focused on conventional water quality parameters (e.g., dissolved oxygen, temperature, ammonia), fish condition, pathogen exposure and disease status, and exposures to metals, polycyclic aromatic hydrocarbons, and current use pesticides. Daily surveys of a representative urban stream (Longfellow Creek) from 2002-2009 revealed premature spawner mortality rates that ranged from 60-100% of each fall run. The comparable rate in a non-urban stream was <1% (Fortson Creek, surveyed in 2002). Conventional water quality, pesticide exposure, disease, and spawner condition showed no relationship to the syndrome. Coho salmon did show evidence of exposure to metals and petroleum hydrocarbons, both of which commonly originate from motor vehicles in urban landscapes. The weight of evidence suggests that freshwater-transitional coho are particularly vulnerable to an as-yet unidentified toxic contaminant (or contaminant mixture) in urban runoff. Stormwater may therefore place important constraints on efforts to conserve and recover coho populations in urban and urbanizing watersheds throughout the western United States.