Causes of erroneous white blood cell counts and differentials in clinically healthy young northern elephant seals (Mirounga angustirostris).

From 1993 to 1995, approximately 10% of the clinically healthy northern elephant seals (Mirounga angustirostris) at The Marine Mammal Center in California exhibited a large unexplained increase in their white blood cell (WBC) count. In these animals, WBC counts ranged from 28,780 to 125,000/mm3, with a mean of 50,087/mm3. Significant correlations between the leukocytosis and weight gain and day of admittance were identified, but no correlation existed between leukocytosis and general state of health, sex, length of stay, or diet. Bone marrow contamination of blood samples, erroneous automated leukocyte counts, and leukogram changes consistent with subclinical inflammation were the major factors contributing to the elevated WBC counts in these apparently clinically healthy animals.     

Blood values of juvenile northern elephant seals (Mirounga angustirostris) obtained using a portable clinical analyzer

Background — Sick, injured, or orphaned juvenile northern elephant seals ( Mirounga angustisrostris ) treated at rehabilitation centers frequently present with abnormalities in blood sodium, potassium, chloride, BUN, and glucose concentrations, and HCT. These abnormalities could be detected rapidly using a portable blood analyzer, but the results with this analysis method do not necessarily equate with those obtained using other techniques.
Objective — The objective of this study was to better assess the clinical relevance of blood values obtained from a portable analyzer and to compare the results with values obtained using more common methods of analysis.
Methods — Heparinized whole blood samples were collected from 20 rehabilitated juvenile northern elephant seals. A portable clinical analyzer (i-STAT, i-STAT Corp, East Windsor, NJ, USA) was used to establish baseline values. Serum biochemical values were obtained using an automated chemistry analyzer (Olympus AU5200, Olympus America, Melville, NY, USA). HCT was determined using EDTA whole blood and a cell counter.
Results — Using the portable analyzer, mean (minimum-maximum) values were obtained for sodium, 143 (132–146) mmol/L; potassium, 4.4 (3.9–5.8) mmol/L; chloride, 106 (101–109) mmol/L; BUN, 1.8 (1.1–2.4) mmol/L; glucose, 7.55 (5.99–8.49) mmol/L; and HCT, 0.55 (0.52–0.61) L/L. Average differences between methods were small for potassium (-0.45 mmol/L), BUN (0.1 mmol/L), and HCT (0.037 L/L) but were large for sodium (-6.8 mmol/L), chloride (-6.4 mmol/L), and glucose (-0.56 mmol/L).
Conclusions — These results suggest that the i-STAT portable analyzer could be useful for clinically assessing juvenile elephant seals. However, when making medical decisions, the clinician should be aware of differences associated with various analyzers and sample types.     

The effect of rehabilitation of northern elephant seals (Mirounga angustirostris) on antimicrobial resistance of commensal Escherichia coli

The aim of this study was to determine if antimicrobial drug use increases resistance of commensal gastrointensinal Escherichia coli of wild northern elephant seals (Mirounga angustirostris) treated in rehabilitation, and, if so, identify the risk factors involved. Minimum inhibitory concentration (MIC) levels of twelve antimicrobial drugs were determined for 289 E. coli isolates from 99 seals sampled at admission and 277 isolates obtained at release from rehabilitation using broth microdilution. Prevalence of E. coli antimicrobial resistance, MIC(50), MIC(90), and clustering of MIC values were determined for seals and the data were analyzed using Fisher's exact test, ordinal logistic regression and negative binomial regression. At release from rehabilitation 77.8% of the seals had antimicrobial resistant E. coli compared to 38.4% of the seals at admission. The MIC(90) for amoxicillin-clavulanic acid, chloramphenicol, enrofloxacin, ticarcillin-clavulanic acid, and trimethoprim-saulfamethoxazole were at levels considered to be sensitive at admission but they increased to levels of resistance at release. E. coli were grouped into four clusters by their MIC values, with increasing levels of resistance going from Cluster 1 to 4. A primary risk factor associated with the probability of a seal having E. coli in Clusters 3 and 4 was time in rehabilitation, regardless of whether the animal received treatment with antimicrobial drugs, suggesting nosocomial infection. The results of this study provide evidence that increased levels of hygiene and appropriate use of antimicrobial therapy might be important in the rehabilitation of wild animals to prevent rise in the prevalence of antimicrobial resistant bacteria.     

Amoxicillin pharmacokinetics in harbor seals (Phoca vitulina) and northern elephant seals (Mirounga angustirostris) following single dose intravenous administration: implications for interspecific dose scaling

The pharmacokinetics of sodium amoxicillin after a single intravenous dose of 20 mg/kg were determined in ten harbor seals (Phoca vitulina) and ten northern elephant seals (Mirounga angustirostris). The seals ranged in age from 1 to 6 months and the mean weights were 11.7 kg (range, 9.5-18.5 kg) for harbor seals and 47.1 kg (range, 39.5-61.4 kg) for elephant seals. The median half-life of amoxicillin (quartiles) in harbor seals, 1.5 (1.0-3.1) h. was not statistically different from that of elephant seals, 2.0 (1.4-3.8) h, nor were the differences between the terminal elimination rate constants between the two species. The only statistically significant differences between species were for area-under-the-curve (AUC), and total systemic clearance. The lack of statistical significance for differences in the volume of distribution at steady-state (Vss) may have been due to minor differences in the time frame of data collection and dose administered between the two groups. A true physiologic difference in drug handling, possibly related to renal perfusion or tubal secretory efficiency could affect amoxicillin kinetics in these species, and longer administration intervals may be appropriate for elephant seals as compared to harbor seals when administering multiple dose amoxicillin therapy at 20 mg/kg.     

Urate nephrolithiasis in a northern elephant seal (Mirounga angustirostris) and a California sea lion (Zalophus californianus).

Nephrolithiasis has rarely been reported in marine mammals. During 2004 and 2005, two cases of nephrolithiasis were diagnosed during routine necropsy examination, one in a northern elephant seal (Mirounga angustirostris) and one in a California sea lion (Zalophus californianus). Nephroliths were found throughout both kidneys during necropsy examination, varying in size from 1-10 mm in diameter in the northern elephant seal and from 1-15 mm in diameter in the California sea lion. Necropsy and histopathology revealed nephroliths in association with renal pelvic dilation and pyelonephritis in both animals. In addition, hydronephrosis was noted in the sea lion. Nephroliths were composed of uric acid and ammonium urate in the northern elephant seal and of ammonium urate in the California sea lion. The underlying disease leading to nephrolith formation was not determined; however, it is hypothesized that unknown metabolic derangements due to morphologic or physiologic differences may have played a role. This is the first report of urate nephrolithiasis in the California sea lion and northern elephant seal.     

Comparison of quantitative immunoturbidimetric and semiquantitative latex‐agglutination assays for D‐dimer measurement in canine plasma

Background: D‐dimer measurement in dogs is considered the most reliable test for detecting disseminated intravascular coagulation or thromboembolism. Objectives: The purposes of this study were to compare 2 D‐dimer assays, a quantitative immunoturbidimetric and a semiquantitative latex agglutination assay, and to assess the effect of hemolysis and storage conditions on D‐dimer concentration using the quantitative assay. Methods: The immunoturbidimetric assay was validated using canine citrated plasma samples containing different concentrations of D‐dimer. The effect of storage at various temperatures and times was assessed. Hemolysis was produced by adding lysed RBCs to the samples for a final hemoglobin concentration of 0.35 g/dL. Results: For clinically relevant values (>250 μg/L), intra‐assay and interassay coefficients of variation were 6.8% and 7.2%. The assay was linear (r²=1.00), and the tests had good agreement (κ=0.685, P<.001). Storage at 4 °C and ?20 °C and hemolysis had no significant effect on D‐dimer concentrations. In hemolyzed samples stored at room temperature for ≥48 hours, fine clots were noted and often resulted in falsely increased D‐dimer concentrations. Conclusions: Our findings suggest that the immunoturbidimetric assay validated in this study is reliable and accurate for the measurement of D‐dimer in canine plasma. Samples can be stored for up to 1 month at ?20 °C and moderate hemolysis does not significantly affect the D‐dimer concentration in frozen or refrigerated samples.     

Evaluation of latex agglutination kits for detection of fibrin(ogen) degradation products and D-dimer in healthy horses and horses with severe colic

Background: Fibrin(ogen) degradation products (FDPs) and D‐dimer are sensitive indicators of excessive fibrinolysis due to disseminated intravascular coagulation (DIC) in dogs. To the authors' knowledge, latex‐agglutination–based plasma FDP and D‐dimer assays have not been validated for use in horses. Objectives: To determine: 1) sensitivity and specificity of latex‐agglutination serum and plasma FDP and D‐dimer assays for diagnosis of DIC; and 2) their prognostic value in horses with severe colic. Methods: At hospital admission and 24 hours later, blood was collected from 30 healthy horses and 20 horses with severe colic. Horses fulfilling predefined laboratory criteria of DIC were enrolled, and their data were subcategorized by survival for analysis. Platelet counts were determined and coagulation panel testing was performed. Serum and plasma FDP concentrations were measured using separate latex agglutination kits. Plasma D‐dimer concentration was measured using 3 latex agglutination kits and a card immunofiltration test. Test sensitivity and specificity results were determined for healthy horses and those with colic. Median test values were compared between colic survivors and nonsurvivors to evaluate the prognostic usefulness of all tests. Results: Performance characteristics varied among assays and kit suppliers. The FDP assays had low sensitivity (<40%), whereas the most accurate D‐dimer kit had 50% sensitivity and 97% specificity. High D‐dimer concentration was the third most common hemostatic abnormality in horses with colic. Median antithrombin (AT) activity was significantly lower and activated partial thromboplastin time (aPTT) was significantly longer in nonsurvivors than survivors. Conclusions: Commercial latex‐agglutination D‐dimer assays might prove useful as adjunctive tests for the diagnosis of DIC in horses with severe colic; however FDP assays are invalid for this purpose. Low AT activity and prolonged aPTT at admission are associated with a poor prognosis in this patient population.     

Systemic mycosis caused by Scedosporium apiospermum in a stranded northern elephant seal (Mirounga angustirostris) undergoing rehabilitation.

A recently weaned, stranded, male northern elephant seal (Mirounga angustirostris) pup that had been undergoing rehabilitation was found severely obtunded with hyponatremia, hypokalemia, hypochloremia, and hypophosphatemia after a history of intermittent regurgitation. The animal was euthanatized, and gross postmortem findings included multifocal abscessation affecting brain, spleen, kidney, muscle, and subcutaneous tissue. Scedosporium apiospermum and mixed bacteria were cultured from brain, kidney, and subcutaneous tissue. Histopathologic examination revealed multiple fungal granulomas of variable size in the kidneys, brain, liver, and skeletal muscle. This is the first report of S. apiospermum infection associated with lesions in a marine mammal.     

Evaluation of plasma antithrombin activity and D-dimer concentration in populations of healthy cats, clinically ill cats, and cats with cardiomyopathy

BACKGROUND: Current coagulation tests lack sensitivity and detect disseminated intravascular coagulation (DIC) only when it is severe. Measurement of antithrombin (AT) activity and D-dimer concentration permits early diagnosis and more precise classification of coagulopathies in some species. OBJECTIVES: The objectives of this study were to validate and determine the diagnostic utility of a chromogenic AT assay and an immunoturbidimetric D-dimer assay for the diagnosis of DIC in cats. METHODS: Citrated plasma samples were collected from 30 healthy cats, 30 ill cats, and 13 cats with cardiomyopathy. Partial thromboplastin time, prothrombin time, fibrin(ogen) degradation products, platelet concentration, and erythrocyte morphology were determined on all samples to document the presence or the absence of DIC. AT activity and D-dimer concentration were then measured. RESULTS: The chromogenic AT assay was linear and precise. Mean AT activity was higher in ill cats and cats with cardiomyopathy compared with healthy cats, but the difference was only significant in ill cats (P = .003). Seven cats met the criteria for DIC. Of the cats with DIC, 2 had decreased AT activity, 1 had increased AT activity, and 4 had AT activities within normal limits. The immunoturbidimetric D-dimer assay did not appear to accurately measure feline D-dimer. CONCLUSIONS: The chromogenic AT assay appeared to measure AT in cats but was not useful for the diagnosis of DIC. AT may be an acute phase reactant in cats. The immunoturbidimetric D-dimer assay was not useful for the diagnosis of DIC in cats.     

Ultrastructural,Sensory and Functional Anatomy of the Northern Elephant Seal (Mirounga angustirostris) Facial Vibrissae

Vibrissae (whiskers) play a key role in underwater orientation in foraging phocids through vibrotactile sensation processing. Our aim was to evaluate the structure of northern elephant seal (NES) vibrissae by means of light (LM) and transmission electron microscopy (TEM), in order to elucidate their function. Vibrissal follicles were processed using standardized laboratory methods and LM/TEM techniques. Individual follicular axonal numbers were counted and axonal diameter measured and averaged. NES have mystacial, rhinal, supraorbital and labial vibrissae. The vibrissal follicles are histologically subdivided into a ring, upper and lower cavernous sinuses (LCS). Each vibrissa is innervated by the deep vibrissal nerve. The average number of axons per large mystacial vibrissa is 1804 (±123), rhinal 985 (±241), supraorbital 1,064 (±204) and 374 (±65) in labial vibrissa. The entire vibrissal system carries an estimated 148 573 axons, and mystacial vibrissae alone have 125 323 axons. Axonal conduction velocity for each vibrissal type is 55.26 m/s for labial, 56.58 m/s for rhinal and 35.88 m/s for mystacial vibrissae. TEM and LM revealed a plethora of mechanoreceptors within the vibrissal follicles: Merkel cell‐neurite complexes, lanceolate and pilo‐Ruffini end organs. A vast number of sensory axons projecting from the entire vibrissal system indicate that the vibrissal sensory area takes up a large proportion of phocids’ somatosensory cortex. In conclusion, NES has highly sensitive and finely tuned vibrotactile vibrissal sense organs.     

Thrombin‐activatable fibrinolysis inhibitor activity in healthy and diseased dogs

Background: In people, increased thrombin‐activatable fibrinolysis inhibitor (TAFI) antigen has been associated with increased risk of thrombosis, and decreased TAFI may contribute to bleeding diathesis. TAFI activity in dogs has been described in experimental models, but not in dogs with spontaneous disease. Objective: The aim of this study was to compare TAFI activity in healthy dogs with TAFI activity in dogs with spontaneous disease. Methods: Plasma samples from 20 clinically healthy Beagles and from 35 dogs with various diseases were analyzed using a commercial chromogenic assay that measured TAFI activity relative to activity in standardized pooled human plasma. Results: Median TAFI activity for the 20 Beagles was 46.1% (range 32.2–70.8%) compared with 62.6% (29.1–250%) for the 35 diseased dogs, and 14/35 (40%) had TAFI activities >the upper limit for controls. The highest individual activities (>225%) were in 3 dogs with malignant neoplasms and 1 dog with thrombocytopenia. For data grouped by diagnosis, median TAFI activity was 61.7% for benign neoplasia (n=5), 64.9% for malignant neoplasia (n=8), 75.5% for Angiostrongylus vasorum infection (n=4), 68.8% for bacterial sepsis (n=7), and 58.7% for miscellaneous diseases (n=11). Compared with TAFI activity in control dogs, median TAFI activity was significantly increased only in the group of dogs with bacterial sepsis. Conclusion: Bacterial sepsis was associated with significantly increased TAFI activity, and individual dogs with increased TAFI activities were found in all disease groups. The role of TAFI in the pathogenesis of hemostatic disorders in dogs and its value as a prognostic indicator deserve further investigation.     

Thromboelastography in healthy, sick non-septic and septic neonatal foals

Objectives To evaluate citrated recalcified thromboelastography (TEG) in healthy newborn foals, and to determine intra‐assay, inter‐individual and intra‐individual (at 12 h, 24 h and 7 days after birth) variations. Additionally, to compare TEG variables, haematological values and conventional coagulation profiles from healthy, sick non‐septic, and septic foals. Design Prospective study. Methods The study group comprised 18 healthy, 15 sick non‐septic and 17 septic foals. Two citrated (3.2%; 1 : 9 anticoagulant : blood ratio) blood samples were submitted for haemostatic evaluation using a TEG analyser and conventional coagulation profile. TEG values (R time (R), K time (K), angle (α), maximum amplitude (MA) and G value (G)), complete blood count (CBC) and conventional coagulation profile (prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen concentration (Fib) and antithrombin (AT)) were evaluated. Signalment, presenting complaint, sepsis scores, blood culture results and outcome were taken from the medical records of the sick foals. Results Mean values ± SD for TEG variables in healthy neonatal foals were: R = 11.82 ± 5.35 min, K = 3.06 ± 1.34 min, α= 51.19 ± 12.66 degrees, MA = 55.06 ± 6.67 mm and G = 6361 ± 1700 dyn/cm². Mean coefficients of variation for intra‐assay/inter‐individual/intra‐individual in healthy foals were: R = 3.5/45.2/43.1%; K = 5.3/58.7/28.7%; α= 1.5/24.7/11.9%; MA = 0.3/12.1/6.1%; G = 1.6/26.7/14.7%. Septic foals had significantly greater α, MA and G values than sick non‐septic foals, and significantly greater MA and G than healthy foals, changes that are consistent with hypercoagulability. Weak correlations were detected between TEG variables and haematological or haemostatic values. Conclusions TEG could be used to provide additional information about the haemostatic system in equine neonates.     

Association of admission plasma D-dimer concentration with diagnosis and outcome in horses with colic

Background: Coagulopathies detected in horses with gastrointestinal problems seem to be associated with poor outcome. Plasma D‐Dimer concentration is a sensitive test for assessing coagulopathies. Hypothesis: Plasma D‐Dimer concentration tested on admission is related to diagnosis and outcome in horses with colic. Animals: Four hundred and ninety three horses referred for evaluation of abdominal pain. Methods: Prospective observational clinical study. Horses were grouped according to diagnosis (medical and surgical intestinal obstructions, ischemic disorders with and without intestinal resection, enteritis, peritonitis), outcome (survivors, nonsurvivors), and number of coagulopathies (normal profile, 1 or 2 coagulopathies, subclinical disseminated intravascular coagulation [DIC]). Blood samples were collected on admission and plasma D‐Dimer concentration, clotting times (PT and aPTT), and antithrombin activity were determined. Positive likelihood ratios (LR+) were calculated for evaluation of D‐Dimer cut‐off values, which were later tested in a logistic regression model. Results: Horses with enteritis or peritonitis had significantly (P < .001) higher plasma D‐Dimer concentrations and more severe coagulopathies on admission than horses with other diagnoses. Nonsurvivors also had significantly (P < .001) higher plasma D‐Dimer concentrations at presentation than did survivors, and those horses with subclinical DIC on presentation had an odds ratio (OR) 8.6 (95% confidence interval [CI], 3.3–22.5, P < .001) for nonsurvival. Finally, D‐Dimer concentrations >4,000 ng/mL had a LR+ of 5.9 and an OR 8.8 (95% CI, 4.5–17.1, P < .001) for nonsurvival. Conclusion and Clinical Importance: Plasma D‐Dimer concentration measured on admission can be used to facilitate diagnosis and outcome prediction in horses with colic. A potential cut‐off value for nonsurvival was found at approximately 4,000 ng/mL.     

Effects of age,size and condition of elephant seals (Mirounga leonina) on their intravenous anaesthesia with tiletamine and zolazepam

Southern elephant seals (Mirounga leonina) were caught as part of a long-term demographic study on Macquarie Island. Over 18 months, 1033 seals were caught by hand and anaesthetised intravenously with a 1:1 mixture of tiletamine and zolazepam. Assessments were made of the effects of variations in the body condition and age at capture of the seals on the characteristics of their anaesthesia, including induction time and weighted recovery time. The size and condition of the seals were assessed by morphometric and ultrasound measurements. Weighted recovery times decreased as the body condition and age of the seals increased, but there were no residual effects of sex. There were no fatalities, and no periods of apnoea longer than five minutes were recorded. In individual seals there was a significant increase in weighted recovery time with successive captures.