Abstract

West Indian or Florida manatees (Trichechus manatus latirostris) are commonly afflicted with inflammatory and infectious disease secondary to human interaction, such as boat strike and entanglement, as well as "cold stress syndrome". White blood cell count and fever, primary indicators of systemic inflammation in most species, are insensitive in diagnosing inflammatory disease in manatees. Acute phase response (APR) proteins, such as haptoglobin and serum amyloid A (SAA), have proven to be sensitive measures of internal inflammation/infection in domestic large animal species. This study assessed diagnosis of generalized inflammatory disease by different methods including CBC, albumin:globulin ratio (A:G ratio), gel electrophoresis analysis, C-reactive protein (CRP), alpha1 acid glycoprotein, haptoglobin, fibrinogen, and SAA. Samples were collected from 71 normal and 27 abnormal animals during diagnostic medical examination. Serum amyloid A (measured by ELISA), followed by A:G ratio (measured by plasma gel electrophoresis), were most sensitive in diagnosing inflammatory disease with each test having diagnostic sensitivity and specificity of approximately 90%. The reference interval for SAA was <10-50 µg/ml with an equivocal interval of 50-70 µg/ml. The reference interval for A:G ratio by plasma gel electrophoresis was 0.7-1.1. Albumin:globulin ratio, calculated using biochemical techniques, was not accurate due to overestimation of albumin by bromcresol green dye-binding methodology (Table 1). Albumin:globulin ratio, measured by serum gel electrophoresis, had a low sensitivity of 15% due to the lack of fibrinogen in the sample. Haptoglobin, measured by hemoglobin titration, had a reference interval of 0.4-2.4 mg/ml, a diagnostic sensitivity of 60%, and a diagnostic specificity of 93%. Fibrinogen, measured by heat precipitation, had a reference interval of 100-400 mg/dl, a diagnostic sensitivity of 40%, and a diagnostic specificity of 95%.

Further investigation of other potential diagnostically useful proteins that currently cannot be assessed with commercially available assays was performed. Two dimensional gel electrophoresis was performed on two healthy manatees and one manatee with subacute injury. The gels were overlayed and four different proteins unique to the sick animal were identified for identification by Edmann sequencing.

Table 1. Methods comparison for albumin.

Hitachi analyzer, using a nonspecific bromcresol green methodology, yields results that are approximately 30% higher. Since globulin and A:G ratio calculations are based on albumin concentrations, inaccurate albumin concentrations cause inaccurate globulin concentration and A:G ratio.