Evaluation of adrenocortical function in Florida manatees (Trichechus manatus latirostris)

The study objectives were to determine the predominant manatee glucocorticoid; validate assays to measure this glucocorticoid and adrenocorticotropic hormone (ACTH); determine diagnostic thresholds to distinguish physiological vs. pathological concentrations; identify differences associated with sex, age class, female reproductive status, capture time, and lactate; and determine the best methods for manatee biologists and clinicians to diagnose stress. Cortisol is the predominant manatee glucocorticoid. IMMULITE 1000 assays for cortisol and ACTH were validated. Precision yielded intra‐ and inter‐assay coefficients of variation for serum cortisol: ≤23.5 and ≤16.7%; and ACTH: ≤6.9 and ≤8.5%. Accuracy resulted in a mean adjusted R²≥0.87 for serum cortisol and ≥0.96 for ACTH. Assay analytical sensitivities for cortisol (0.1 µg/dl) and ACTH (10.0 pg/ml) were verified. Methods were highly correlated with another IMMULITE 1000 for serum cortisol (r=0.97) and ACTH (r=0.98). There was no significant variation in cortisol or ACTH with sex or age class and no correlation with female progesterone concentrations. Cortisol concentrations were highest in unhealthy manatees, chronically stressed by disease or injury. ACTH was greatest in healthy free‐ranging or short‐term rehabilitating individuals, peracutely stressed by capture and handling. Cortisol concentrations ≥1.0 µg/dl were diagnostic of chronic stress; ACTH concentrations ≥87.5 pg/ml were diagnostic of peracute stress. In healthy long‐term captive manatees, cortisol (0.4±0.2 µg/dl) and ACTH (47.7±15.9 pg/ml) concentrations were lower than healthy free‐ranging, short‐term rehabilitated or unhealthy manatees. Capture time was not significantly correlated with cortisol; ACTH correlation was borderline significant. Cortisol and ACTH were positively correlated with lactate. Zoo Biol 30:17–31, 2011. © 2010 Wiley‐Liss, Inc.     

Tactile discrimination of textures by Florida manatees (Trichechus manatus latirostris)

Two male Florida manatees (Trichechus manatus latirostris) demonstrated sensitive tactile discrimination in a two‐alternative forced choice task, using a modified staircase method. Stimuli were acrylic plates with vertical gratings of ridges and grooves. The standard stimulus, present on every trial, had 2 mm gratings and the comparison stimuli had wider gratings. The blindfolded subjects were trained to demonstrate discrimination by pressing the target with wider gratings. Discrimination thresholds (75% correct) for the subjects were 2.05 mm and 2.15 mm, corresponding to Weber fractions of 0.025 and 0.075, respectively. These results indicate thresholds on similar stimuli comparable to humans (index finger tasks) and better than harbor seals, Phoca vitulina, and the closely related Antillean manatee, Trichechus manatus manatus. Memory for the tactile task was quite stable for both subjects, over 2 yr in the case of one of the subjects. Video analysis of responses indicated that bristle‐like hairs, perioral bristles, and skin on the oral disk were involved in the discrimination response.     

Seroepidemiology of TmPV1 infection in captive and wild Florida manatees (Trichechus manatus latirostris)

In 1997, cutaneous papillomatosis caused by Florida manatee (Trichechus manatus latirostris [Tm]) papillomavirus 1 (TmPV1) was detected in seven captive manatees at the Homosassa Springs Wildlife State Park, Florida, USA, and, subsequently, in two wild manatees from the adjacent Homosassa River. Since then, papillomatosis has been reported in captive manatees housed in other locations, but not in wild animals. To determine TmPV1 antibody prevalence in captive and wild manatees sampled at various locations throughout Florida coastal regions, virus-like particles, composed of the L1 capsid protein of TmPV1, were generated with a baculovirus expression system and used to measure anti-TmPV1 antibodies in an enzyme-linked immunosorbent assay. Serologic analysis of 156 manatees revealed a TmPV1 antibody prevalence of 26.3%, with no significant difference between captive (n=39) and wild (n=117) manatees (28.2% and 25.6%, respectively). No antibody-positive wild animal showed PV-induced cutaneous lesions, whereas papillomatosis was observed in 72.7% of antibody-positive captive manatees. Our data indicate that Florida manatees living in the wild are naturally infected by TmPV1 but rarely show TmPV1-induced papillomatosis. Hence, it appears that the wild population would not be harmed in a case of contact with captive animals without visible lesions and productive infections, which could be thus released into the wild.     

Activity patterns and spatial use of facility by a group of captive female manatees (Trichechus manatus latirostris)

As a means to compare and contrast to free‐ranging Florida manatees, we examined the behavior and movement patterns of nine adult‐female captive manatees at Homosassa Springs Wildlife State Park (HSWSP). HSWSP has a unique manatee exhibit that consists of a natural river and a man‐made pool. Because the manatees are exposed to a natural environment including natural food resources, the study of various aspects of behavioral and physiological parameters of the captive manatees is especially valuable with this group. We determined activity pattern and spatial use of the facility by manatees over three allotted periods of a day (noon, mid‐afternoon, and late‐afternoon) and three seasons (winter, spring, and summer). Behavioral strategies of the manatees were: 1) to remain “inactive,” probably to conserve energy, during the day while the park provisioned food; 2) to alter locations during the course of the afternoon, apparently with the direction of the sun, possibly to assist with thermoregulation; and 3) to change location seasonally where and when food resources became abundant, showing a strong interest in natural vegetation as it became available. Spatial use and activity pattern of the HSWSP captive manatees apparently were influenced by energy constraints and nutrient intake by provisioned food availability over the day and natural vegetation over the study period. The behavior of the female group of manatees may have been affected by the single‐sex living arrangement. In addition, these manatees are subject to a largely invariable temperature (ca. 23°C) that may have created thermal stress and immunological suppression over time. This study demonstrated that for these captive manatees the activity patterns and spatial use were comparable to that of free‐ranging manatees in relation to the availability of food resources. Zoo Biol 0:1–17, 2006. © 2006 Wiley‐Liss, Inc.     

Characterization of highly informative cross-species microsatellite panels for the Australian dugong (Dugong dugon) and Florida manatee (Trichechus manatus latirostris) including five novel primers

The Australian dugong (Dugong dugon) and Florida manatee (Trichechus manatus latirostris) are threatened species of aquatic mammals in the order Sirenia. Sirenian conservation and management actions would benefit from a more complete understanding of genetic diversity and population structure. Generally, species-specific microsatellite markers are employed in conservation genetic studies; however, robust markers can be difficult and costly to isolate. To increase the number of available markers, dugong and manatee microsatellite primers were evaluated for cross-species amplification. Furthermore, one manatee and four dugong novel primers are reported. After polymerase chain reaction optimization, 23 (92%) manatee primers successfully amplified dugong DNA, of which 11 (48%) were polymorphic. Of the 32 dugong primers tested, 27 (84%) yielded product in the manatee, of which 17 (63%) were polymorphic. Dugong and manatee primers were compared and the most informative markers were selected to create robust and informative marker-panels for each species. These cross-species microsatellite marker-panels can be employed to assess other sirenian populations and can provide beneficial information for the protection and management of these unique mammals.     

Dietary exposure to harmful algal bloom (HAB) toxins in the endangered manatee (Trichechus manatus latirostris) and green sea turtle (Chelonia mydas) in Florida,USA

Florida is a hotspot for cyano- and microalgal harmful algal blooms (HABs) with annual red-tide events off-shore and blooms of Lyngbya spp. commonly observed in both marine and freshwater environments. This region also provides extensive foraging habitat for large populations of herbivorous green turtles (Chelonia mydas) and manatees (Trichechus manatus latirostris). The exposure of aquatic organisms to HAB toxins is not well known and whilst acute exposures are better understood, the vulnerability of aquatic animals to chronic exposure from multiple HAB toxins over prolonged periods has rarely been addressed. This study aimed to identify the presence of toxic compounds produced by HAB species commonly found in Florida (brevetoxins, okadaic acid, saxitoxins and Lyngbya toxins) in tissues and gut samples from manatee and green sea turtles that stranded in Florida, USA. Muscle, liver and alimentary tract samples were opportunistically collected from 14 manatees and 13 green turtles that stranded on the Florida shoreline between December 2003 and February 2006. Samples from each animal were assessed for the presence of brevetoxin, okadaic acid, lyngbyatoxin-A and saxitoxin. Nine (64%) manatees and 11 (85%) turtles were found to have been exposed to one or more of the HAB toxins. Okadaic acid and saxitoxin were only found in alimentary tract samples, whereas brevetoxin was more widely distributed. No lyngbyatoxin-A was observed in any tissue samples. The majority of turtles (13) stranded on the Atlantic coast between St. Johns and Monroe counties, with one on the Gulf coast at Bay County, whereas nine manatees were stranded on the Gulf coast between Levy and Lee counties, with the remaining five between Volusia and Brevard counties on the Atlantic coast. This HAB toxin screen has identified that a large proportion of a random sample of turtles and manatees that stranded in Florida in 2003–2006 were exposed to HAB toxins. Most of the concentrations measured were low, and the toxins were directly linked to the death of only three of these animals. However, the presence of these compounds, and in some cases the presence of multiple HAB toxins in individual animals, indicates that turtles and manatees in Florida are exposed to deleterious compounds at sub-lethal levels in their environment, which could ultimately compromise their health.     

Purification and characterization of the major whey proteins from the milks of the bottlenose dolphin (Tursiops truncatus), the Florida manatee (Trichechus manatus latirostris), and the beagle (Canis familiaris)

The major whey proteins of the milks of the dolphin, manatee, and beagle were purified by gel filtration and ion exchange chromatography and characterized and identified by molecular weight determination, amino acid analysis, N-terminal sequencing, and activity measurements. The major whey protein components from all three species were found to be monomeric beta-lactoglobulins. These proteins were all active in binding retinol. Dolphin milk contained two beta-lactoglobulins (designated 1 and 2) which showed a slight difference in molecular weight and considerably divergent N-terminal sequences, whereas the other milks only contained a single form of beta-lactoglobulin. alpha-Lactalbumins were purified from dolphin and dog milks and were active in promoting lactose synthesis by bovine galactosyltransferase. The dolphin protein had an N-terminal sequence more similar to ruminant alpha-lactalbumins than to those known from other species. Although alpha-lactalbumin activity has been detected in manatee milk at low levels, the corresponding protein was not isolated. In addition, dog milk was found to contain high levels of lysozyme (greater than 1.0 mg/ml), which were identified by activity and sequencing. The functional and evolutionary implications of these results are discussed.     

The Immune Response of the Tasmanian Devil (Sarcophilus harrisii) and Devil Facial Tumour Disease

One of the most remarkable aspects of Devil Facial Tumour Disease (DFTD) is its infectious nature, and for successful transmission it must avoid detection by the devil’s immune system. For this to occur, the devil either is severely immunosuppressed or factors produced by the tumor contribute to its avoidance of immune detection. An analysis of the devil’s immune system revealed the presence of normal-looking lymphoid organs and lymphoid cells. At a functional level the lymphocytes proliferated in response to mitogen stimulation. Subcutaneous injection of a cellular antigen produced a strong antibody response, providing compelling evidence that the devil has a competent immune system. Tumor cell analysis demonstrated that the tumor expresses the genes of the major histocompatibility complex; however, there was a limited diversity. Therefore, the most likely explanation for devil-to-devil transmission of DFTD is that the tumor is not recognized by the devil as “non-self” because of the limited genetic diversity. With its consistent morphology and relatively stable genome, this tumor would provide a reasonable target for a vaccine approach, provided the immune system can be coaxed into recognizing the tumor as “non-self.”     

Daily rhythms of leukocytes populations,biochemical and enzymatic blood parameters in a carnivorous freshwater catfish (Lophiosilurus alexandri)

The aim of this study was to investigate the daily rhythms of hematological, biochemical and enzymatic parameters of the blood of a nocturnal model of fish (Lophiosilurus alexandri) bred in the laboratory (F1). Thirty-six juveniles were stocked in six tanks of a recirculation aquaculture system for 20 days. The fish were exposed to a light:dark cycle of 12:12 h and were fed 1% of biomass twice a day with commercial diet. The daily rhythms of hematological, biochemical and enzymatic parameters were then measured at six sampling times “zeitgeber time = ZT” at four-hour intervals under light:dark 12:12 h (lights on = ZT0, at 8.00 a.m). No differences were observed to alkaline phosphatase, glucose, cortisol, aspartate aminotransferase, superoxide dismutase, total protein and hematocrit (p > 0.05). However, white blood cell count, Lymphocytes (LYN), Neutrophils (NEU), Eosinophil and Neutrophils to Lymphocytes ratio were significant different between sample times (p < 0.05). Also, a significant difference in alanine transaminase was observed, with a peak of production at nighttime. In contrast, glutathione peroxidase peaked at 8:00. Uric acid, magnesium and Calcium (Ca⁺⁺) showed statistically significant differences (p < 0.05). A significant difference was observed (p < 0.05), with a peak of albumin at 08:00 and triglycerides at 12:00, while cholesterol was low (p < 0.05) at 08:00 and higher from 12:00 to 04:00. Cosinor analysis revealed also rhythmicity to SOD, UA, Mg and Ca⁺⁺, ALB and CHO (p < 0.05). In conclusion, the time of day must be considered a key factor when using blood parameters as biomarkers for disease, health and welfare in the L. alexandri aquaculture.     

Monitoring of blood gas parameters and acid-base balance of pregnant and non-pregnant rabbits (Oryctolagus cuniculus) in routine experimental conditions.

Blood gas parameters and acid-base balance values were determined in adult pregnant New Zealand rabbits (Oryctolagus cuniculus) in standard laboratory housing conditions and during anaesthesia with an association of ketamine-chlorpromazine, administered before surgical procedures. All the variables were also studied in adult non-pregnant female, used as controls. No differences in pH, sO2c, O2Hb, COHb, sO2m and a-vDO2 were found between pregnant and non-pregnant rabbits in physiological conditions and during anaesthesia. Ketamine-chlorpromazine and pregnancy seemed to change the other parameters used to assess the acid-base balance and the oxygenation conditions. Anaesthesia affected only Hb, O2Ct, O2Cap, CcO2 and P50. The additive effect of pregnancy and anaesthesia modified pCO2, pO2, HCO3-, TCO2, BEb, SBC, BEecf, A-aDO2, RI, MetHb, RHb, CaO2 and CvO2. The patterns described are close to those of other species, suggesting the New Zealand rabbit might be a reliable animal model for monitoring selected variables.     

The clinical effect of Prednisone in combination with Mycophenolate mofetil on idiopathic thrombocytopenic purpura (ITP) and its influence on the level of peripheral blood T lymphocytes and NK lymphocytes

ObjectiveTo explore he curative effect and safety of Prednisone in combination with Mycophenolate in treating ITP and its influence on the level of peripheral blood T lymphocytes and NK lymphocytes.Method93 cases of ITP patients were divided into the observation group and the control group by the Random Number Table method, 48 cases for the observation group, 45 for another. Patients in the control group orally took 0.5 mg/kg Prednisone Acetate tablets daily, two times each in the morning and evening. And the observation group, based on the treatment of the control group, orally took Mycophenolate Mofetil Dispersible tablets twice a day, 1 g each time. According to patients’ conditions, 3 to 5 courses were set for treatment with 3 weeks a course. Compared PLT amount and the changing situation of inflammatory factors, CD3⁺ and CD3⁺CD95L⁺ before and after the treatment, the level of CD3⁺Caspase-3⁺ and CD3⁺Caspase-8⁺, NK⁺, NK⁺ CD95L⁺, NK⁺Caspase-3⁺, NK⁺Caspase-8, the curative effect and adverse events.ResultAfter treatment, PLT amount in both groups increased, and the increase in the observation group was much higher than that of the control group, the difference had statistical significance (P < 0.05). The time needed for PLT amount in the control group to reach the normal and peak values was longer than that of the observation group, whose PLT peak value was higher than another group. The difference had statistical significance (P < 0.05). After the treatment, the levels of TNF-α and IL-6 were lowered, and the value of the observation group was lower than that of another. The difference between and within the group has statistical significance. After the treatment, the level of CD3⁺, CD3⁺CD95L⁺ and CD3⁺Caspase-8⁺ is much higher and CD3⁺Caspase-3⁺ level lower than that before the treatment. The difference has statistical significance (P < 0.05). After the treatment, the level of NK⁺ and NK⁺ CD95L⁺ is higher and the level of NK⁺Caspase-8⁺ lower than that before the treatment. The difference has statistical significance (P < 0.05). After the treatment, the total effective rate 91.67% of the observation group is much higher than that 75.56% of another. The difference has statistical significance (P < 0.05). After the treatment, the incidence rate of adverse events in the control group is 11.11% (5/45), while 4.17% (2/48) in the observation group. The difference between groups has statistical significance (χ² = 3.890, P < 0.05).ConclusionThe curative effect of Prednisone in combination with Mycophenolate on ITP patients is better than orally taking Prednisone tablets. Moreover, when it comes to Prednisone in combination with Mycophenolate, both the PLT amount and immunocompetence are improved without much adverse reaction, and the molecules of peripheral blood T lymphocytes and NK lymphocytes can be effectively adjusted to relieve the symptoms. So the method is trustworthy to be popularized for clinical practices.     

Identification of chemically modified peptide from poly(D,L-lactide-co-glycolide) microspheres under in vitro release conditions

The purpose of this research was to study the chemical reactivity of a somatostatin analogue octreotide acetate, formulated in microspheres with polymers of varying molecular weight and co-monomer ratio under in vitro testing conditions. Poly(D,L-lactide-co-glycolide) (PLGA) and poly(D,L-lactide) (PLA) microspheres were prepared by a solvent extraction/evaporation method. The microspheres were characterized for drug load, impurity content, and particle size. Further, the microspheres were subjected to in vitro release testing in acetate buffer (pH 4.0) and phosphate buffered saline (PBS) (pH 7.2). In acetate buffer, 3 microsphere batches composed of low molecular weight PLGA 50∶50, PLGA 85∶15, and PLA polymers (≤10 kDa) showed 100% release with minimal impurity formation (<10%). The high molecular weight PLGA 50∶50 microspheres (28 kDa) displayed only 70% cumulative release in acetate buffer with significant impurity formation (∼24%). In PBS (pH 7.4), on the other hand, only 50% release was observed with the same low molecular weight batches (PLGA 50∶50, PLGA 85∶15, and PLA) with higher percentages of hydrophobic impurity formation (ie, 40%, 26%, and 10%, respectively). In addition, in PBS, the high molecular weight PLGA 50∶50 microspheres showed only 20% drug release with ∼60% mean impurity content. The chemically modified peptide impurities inside microspheres were structurally confirmed through Fourier transform-mass spectrometry (FT-MS) and liquid chromatography/mass spectrometry (LC-MS/MS) analyses after extraction procedures. The adduct compounds were identified as covalently modified conjugates of octreotide with lactic and glycolic acid monomers within polymeric microspheres. The data suggest that due to steric hindrance factors, polymers with greater lactide content were less amenable to the formation of adduct impurities compared with PLGA 50∶50 copolymers.     

Effects of acute hypoxic stress on biochemical parameters,immune regulation and metabolic capacity of the blood in genetically improved farmed tilapia (GIFT,Oreochromis niloticus)

Dissolved oxygen (DO) in the culture water is an important environmental factor in fish farming. This study tested whether genetically improved farmed tilapia (GIFT, Oreochromis niloticus) challenged with a hypoxic episode would show inhibited growth, increased metabolic stress and a reduced immune response. GIFT subjected to 72 hr of semi‐lethal hypoxia (determined by linear regression as DO = 0.64 mg/L) showed changes in blood biochemistry, energy metabolism and related immune responses. In the early stages of stress, GIFT showed increased anaerobic respiration and increased lactic acid accumulation. Compared with a control group, white and red blood cell counts, hematocrit and blood hemoglobin concentration in GIFT exposed to 4 hr of semi‐lethal hypoxic stress were not significantly changed. However, the levels of glutamic‐pyruvic transaminase, glucose, cholesterol and cortisol were significantly higher under hypoxic stress at 2 and 4 hr. In the later stages of stress, GIFT displayed increased oxygen utilization and increased aerobic respiration associated with increased red blood cell count, hematocrit and hemoglobin concentration. However, the white blood cell count, serum lysozyme and complement C3 activities began to decline, associated with increased mortality. Reverting to a normal DO environment (5.0 mg/L) for an additional 72 hr, GIFT showed strong recovery, with respiratory metabolic enzymes, immune indicators and associated energy metabolites restored to near normal levels. Our observations contribute to a better understanding of the mechanisms of energy and immune regulation in fish, and will help reduce the damage caused by hypoxic stress during culture.     

Protracted release of the LHRH agonist avorelin (MF 6001) from two depot formulations in dogs and men

Avorelin is a new superagonist of naturalluteinizing-hormone-releasing-hormone. Avorelin hasbeen formulated in high molecular weight polylactic glycolic acid to afford protracted andcontinuous release of the peptide from subcutaneousimplants. Two different formulations (10 and 15 mg)were tested first in dogs and then in men during aclinical phase II trial. Chemical castration wasmaintained for at least 6 months in dogs withboth formulations. A similar duration of activity(approximately 6 months) was observed in men.     

Protracted release of the LHRH agonist avorelin (MF 6001) from two depot formulations in dogs and men

Summary Avorelin is a new superagonist of natural luteinizing-hormone-releasing-hormone. Avorelin has been formulated in high molecular weight polylactic glycolic acid to afford protracted and continuous release of the peptide from subcutaneous implants. Two different formulations (10 and 15 mg) were tested first in dogs and then in men during a clinical phase II trial. Chemical castration was maintained for at least 6 months in dogs with both formulations. A similar duration of activity (approximately 6 months) was observed in men.     

An in vitro study of the stereoselective dissolution of (rac)-verapamil from two sustained release formulations

The stereoselectivity of the in vitro dissolution of two commercially available sustained release formulations of rac-verapamil (rac-VER) has been investigated. The studies were carried out using a single-tablet continuous-flow apparatus and the concentrations of R- and S-VER released from the formulations were measured using enantioselective chromatography on a high performance liquid chromatography (HPLC) chiral stationary phase containing immobilized α₁-acid glycoprotein (Chiral AGP-column). The data from this study demonstrates that the two formulations have different dissolution profiles and that the amount of drug dissolved was highly dependent on pH. In addition, between pH 3 and 8, the total cumulative amount of R-VER released was greater than the amount of S-VER and a statistically significant difference (P < 0.01) was detected at pH 6. The results of this study indicate that bioavailability and bioequivalency studies should consider the possibility of enantioselective dissolution when racemic compounds are present in the formulations. © 1993 Wiley-Liss, Inc.