Howard L. Rhinehart

Bottlenose Dolphins as Marine Ecosystem Sentinels: Developing a Health Monitoring System

Bottlenose dolphins (Tursiops truncatus), as long-lived, long-term residents of bays, sounds, and estuaries, can serve as important sentinels of the health of coastal marine ecosystems. As top-level predators on a wide variety of fishes and squids, they concentrate contaminants through bioaccumulation and integrate broadly across the ecosystem in terms of exposure to environmental impacts. A series of recent large-scale bottlenose dolphin mortality events prompted an effort to develop a proactive approach to evaluating risks by monitoring living dolphin populations rather than waiting for large numbers of carcasses to wash up on the beach. A team of marine mammal veterinarians and biologists worked together to develop an objective, quantitative, replicable means of scoring the health of dolphins, based on comparison of 19 clinically diagnostic blood parameters to normal baseline values. Though the scoring system appears to roughly reflect dolphin health, its general applicability is hampered by interlaboratory variability, a lack of independence between some of the variables, and the possible effects of weighting variables. High score variance seems to indicate that the approach may lack the sensitivity to identify trends over time at the population level. Potential solutions to this problem include adding or replacing health parameters, incorporating only the most sensitive measures, and supplementing these with additional measures of health, body condition, contaminant loads, or biomarkers of contaminants or their effects that can also be replicated from site to site. Other quantitative approaches are also being explored.

Morbillivirus infection in cetaceans of the western Atlantic

We report serologic evidence of morbillivirus infection in eleven of fifteen species of odontocete cetaceans from the western Atlantic since 1986. Blood samples were obtained both from free-ranging and stranded animals. Virus neutralizing titers were higher against porpoise and dolphin morbilliviruses than against peste des petits ruminants virus, phocine distemper virus or canine distemper virus (CDV). Serum from five species, tested in a heterologous immunoprecipitation assay using radiolabelled CDV, precipitated the nucleocapsid (N) protein. Clinical morbillivirus infection may potentially impact already threatened species such as the harbour porpoise and precipitate mass strandings of socially cohesive odontocetes.

Disseminated Mycotic Infection Caused by Colletotrichum acutatum in a Kemp's Ridley Sea Turtle (Lepidochelys kempi)

ABSTRACT Colletotrichum acutatum is a cosmopolitan plant pathogen with a wide host range. While the organisms phytopathogenic potential has been well documented, it has never been reported as an etiologic agent of disease in either animals or humans. In this case, a juvenile Kemps ridley sea turtle, Lepidochelys kempi, probably with immune compromise following cold stunning (extended hypothermia), developed a disseminated mycotic infection in the lungs and kidneys. Prophylactic treatment with oral itraconazole did not prevent or cure the infection. This report of a Colletotrichum acutatum infection in an animal extends the range of disease caused by this organism beyond that of a phytopathogen.

STEADY-STATE PLASMA CONCENTRATIONS OF ITRACONAZOLE AFTER ORAL ADMINISTRATION IN KEMP'S RIDLEY SEA TURTLES, LEPIDOCHELYS KEMPI

Abstract Pharmacokinetic studies of antifungal agents in reptiles are uncommon. Itraconazole, which has been used prophylactically in juvenile sea turtles suffering from hypothermia (cold stunning) on a regular basis, was evaluated for steady-state plasma concentrations. Five Kemps ridley sea turtles (Lepidochelys kempi) receiving itraconazole at several dosages in a rehabilitation program had blood collected within 24 hr to estimate dosing frequency. Subsequently, serial blood samples of Kemps ridley sea turtles that were given itraconazole at several dosages for 30 days to treat cold stunning were collected at various intervals to evaluate itraconazole plasma concentrations. Tissue samples were collected from one Kemps ridley that died during rehabilitation. Plasma concentrations of itraconazole (and of hydroxyitraconazole [OH-ITRA], one of its major bioactive metabolites) were determined using a modified, validated reverse-phase high-performance liquid chromatography technique. Itraconazole concentrations in tissues were determined by bioassay to be far greater than the plasma concentrations measured in any of the turtles. At a 15-mg/kg dosage, the half-life (t1/2) was 75 hr for itraconazole and 55 hr for OH-ITRA. All dosages produced adequate concentrations in some turtles, but consistent therapeutic concentrations were produced only at 15 mg/kg q72hr and 5 mg/kg s.i.d., with the latter producing the highest plasma concentrations.

Comparison of Diagnostic Techniques for Helicobacter cetorum Infection in Wild Atlantic Bottlenose Dolphins (Tursiops truncatus)

ABSTRACT Helicobacter cetorum sp. nov. has been cultured from the stomach of Atlantic white-sided dolphins (Lagenorhynchus acutus) and the feces of Pacific white-sided (L. obliquidens) and Atlantic bottlenose (Tursiops truncatus) dolphins and a beluga whale (Delphinapterus leucas). H. cetorum has high homology to Helicobacter pylori as shown by 16S rRNA sequencing, and H. cetorum infection has been associated with gastritis and clinical signs in cetaceans. Because the prevalence of H. cetorum in wild populations is unknown, minimally invasive techniques for detecting H. cetorum were compared for 20 wild bottlenose dolphins sampled as part of a long-term health study. Fecal samples were tested for helicobacter by culture, Southern blotting, and PCR using genus-specific and H. cetorum-specific primers. An enzyme-linked immunosorbent assay (ELISA) was developed to measure H. cetorum immunoglobulin G (IgG). H. cetorum was cultured from 4 of 20 fecal samples, 7 samples were positive using Helicobacter sp. PCR, and 8 samples were positive for H. cetorum using species-specific primers. Two additional fecal samples were positive by Helicobacter sp. Southern blotting, suggesting infection with another helicobacter. All 20 sera contained high levels of IgG antibodies to H. cetorum that were significantly lowered by preabsorption of the sera with whole-cell suspensions of H. cetorum (P < 0.02). Until the specificity of the serum ELISA can be determined by testing sera from dolphins confirmed to be uninfected, PCR and Southern blot screenings of feces are the most sensitive techniques for detection of H. cetorum, and results indicate there is at least a 50% prevalence of H. cetorum infection in these dolphins.

An Approach to the Rehabilitation of Kogia spp.

Abstract Pygmy (Kogia breviceps) and dwarf (K. sima) sperm whales are rarely seen in the wild, but often seem to live-strand, particularly in cow-calf pairs. The rehabilitation of live-stranded individuals of both species has proven to be exceedingly diffi-cult. The few released animals might not have been completely healthy, an alternative chosen due to their poor survival in captivity. The rehabilitation challenges for Kogia are numerous because limited knowledge exists regarding even the basic biology of both species. This report provides information derived from the rehabilitation of 13 live-stranded K. breviceps and K. sima (including five calves) over the last decade at the Dolphin and Whale Hospital at Mote Marine Laboratory and Aquarium in Sarasota, Florida. One K. breviceps calf survived for almost 21 months in captivity and one K. sima survived for over 15 months, both apparent world-wide records. From these cases we learned that it is critical to provide supplemental fluids in addition to solid food to maintain continuous activity of the intestinal tract, especially if maintained in chlorine-treated water, and that digestibility of squid species typically fed to captive marine mammals was poor. Both species appear to be susceptible to adverse reactions to a number of the drugs commonly used during rehabilitation. In addition, an artificial calf formula was developed to provide adequate nutri-tion for young calves. Finally, gastric and intesti-nal stasis appears to lead to death in many of these whales in captivity.Key Words: Rehabilitation, pygmy sperm whale, dwarf sperm whale, calf formula, orphan calves, survival, captivity, Kogia breviceps, Kogia sima

PHARMACOKINETICS OF TICARCILLIN IN THE LOGGERHEAD SEA TURTLE (CARETTA CARETTA) AFTER SINGLE INTRAVENOUS AND INTRAMUSCULAR INJECTIONS

Abstract Three captive loggerhead sea turtles, Caretta caretta, were used in four trials, one i.v. and three i.m., to determine the pharmacokinetic properties of a single dose of ticarcillin. For the i.v. study, each turtle received a single 50 mg/kg dose and blood samples were collected at 0, 0.5, 1, 2, 4, 6, 8, and 12 hr and at 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, and 14 days after administration. For the i.m. study, each turtle received one of three dosages (25, 50, or 100 mg/kg) in a randomized complete block design and blood samples were collected at the same time intervals. Each trial was separated by a minimum of 28 days to allow for complete drug clearance. Drug concentration in plasma was determined by a validated liquid chromatography–mass spectrometry assay. For the i.v. study, the elimination half-life was 5.0 hr. The apparent volume of distribution and plasma clearance were 0.17 L/kg and 0.0218 L/hr/kg, respectively. For the i.m. study, mean time to maximum plasma concentrations ranged from 1.7 (±0.58) hr in the 50 mg/kg group to 3.7 (±2.5) hr in the 100 mg/kg group. Mean bioavailability ranged from 45% (±15%) in the 50 mg/kg group to 58% (±12%) in the 100 mg/kg group, and the mean residence time ranged from 7.5 (±2.6) hr in the 25 mg/kg group to 16 (±6.8) hr in the 100 mg/kg group. Two turtles had slight alanine aminotransferase elevations that were not clinically apparent at two different dosages, but otherwise, blood chemistries were unaffected. Possible i.m. dosage regimens for loggerhead sea turtles are 50 mg/kg q24 hr or 100 mg/kg q48 hr. Liver enzymes should be monitored during treatment.

Use of human recombinant erythropoietin for the treatment of nonregenerative anemia in a rough-toothed dolphin (Steno bredanensis).

Abstract Erythropoietin, a glycoprotein growth hormone that is produced primarily in the kidneys, promotes mitosis and survival of erythroid progenitors. The recent synthesis of the human form of the hormone by recombinant technology has provided a new therapeutic option, which is being used in both human and veterinary medicine for treatment of various anemias. A mature male rough-toothed dolphin, Steno bredanensis, was treated with human recombinant erythropoietin in an attempt to resolve a nonregenerative anemia. Two i.m. injections 48 hr apart were associated with an almost immediate increase in circulating immature reticulocytes, total reticulocytes, and nucleated erythrocytes. Over the next several weeks, the hematocrit, hemoglobin, and erythrocyte counts returned to normal, and the animal was subsequently released back into the wild. Endogenous erythropoietin concentrations were determined for this animal as well as three other conspecifics by an enzyme-linked immunosorbent assay for human erythropoietin. These measurements showed circulating erythropoietin concentrations (5–20+ mU/ml) similar to those of most other mammals. This study suggests that human recombinant erythropoietin can be safely and effectively used in this species and may have applicability to other cetacean species for the treatment of nonregenerative anemia. Caution should be exercised during long-term use because production of antibodies to human recombinant and endogenous erythropoietin may lead to potentially serious side effects.

SUBACUTE ATROPINE TOXICITY IN A PYGMY SPERM WHALE, KOGIA BREVICEPS

Abstract Atropine, an anticholinergic agent commonly used in human and veterinary medicine, is reported to cause toxicity associated with its antimuscarinic action. A juvenile pygmy sperm whale, Kogia breviceps, was treated with atropine in an attempt to relieve symptoms similar to pyloric stenosis, as has been used in humans. Two doses of 0.01 mg/kg were given i.m., 12 hr apart, followed by three doses of 0.005 mg/kg i.m. s.i.d. over the next 3 days. Symptoms associated with atropine toxicity developed gradually and included hyperexcitability, a generalized ascending paralysis of body musculature, shallow, rapid respiration, vomiting, aspiration of seawater, and pulmonary edema. Treatment with physostigmine salicylate (two doses of 2 mg i.m., 1 hr apart) was effective in counteracting the paralysis, as well as other symptoms, beginning in as little as 17 min after the first dose, and the whale was back to swimming on its own after 8 hr. All overt symptoms of atropine toxicity were gone in about 24 hr, but there were other possible sequella that lasted much longer.

Use of Human Granulocyte Colony-Stimulating Factor in a Green Sea Turtle, Chelonia mydas

ABSTRACT Trauma of undetermined cause resulted in the massive injury, infection, and subsequent stranding of a juvenile green sea turtle, Chelonia mydas. With an initial calculated total leukocyte count of zero cells/μl and no mature circulating heterophils on the differential, the turtle was treated with antibiotics and recombinant human granulocyte colony-stimulating factor, (hG-CSF, filgrastim), in an attempt to increase heterophil production and possibly activation. Three daily doses o f hG-CSF at 6.7 mcg/kg given subcutaneously resulted in a rapid increase in acidophilic progranulocytes, which subsequently declined over the next three days. A second regimen, consisting of a repeat of the first three-dose daily regimen followed by continued dosing every 48 hr for an additional nine days, maintained a white blood cell count of 11,600 – 24,700 cells/μl. Three weeks after initiating therapy, mature heterophils began to appear in the peripheral blood and the hG-CSF was discontinued. Finally, after the tur...