Introduction

In many species, leukocyte count and fever are sensitive indicators of internal inflammation and infection. In a few species, such as the cow, leukocyte counts do not become elevated and left shifted unless disease is severe and then may proceed to drop precipitously, as consumption of leukocytes exceeds production by the bone marrow. For this reason, leukocyte count, though occasionally useful in bovine species, is not a sensitive indicator of inflammation. Clinical reports from veterinarians indicate that manatees' leukocyte response seems to be similar to cows. Additionally, adequately assessing the core temperature of a 2,000-pound aquatic animal can be inaccurate at best. Acute phase response (APR) proteins, such as haptoglobin, are used as a primary indicator in these species because APR proteins have proven to be sensitive measures of internal inflammation/infection. In Europe, appropriate acute phase proteins are used as mandatory screening tests for inflammatory disease in livestock. There is a distinct possibility that acute phase proteins could be a valuable screening diagnostic for inflammation in manatees as they are in cows and other large domestic animals. Currently, there is no published literature on acute phase proteins in manatee species.

There are several practical factors to consider when establishing and validating an APR protein for manatees: for instance, physiologic activity of the protein within manatees, stability of the protein over time during transport with potential temperature change, accuracy of the methodology employed in identifying the protein in this species, and clinical use in different disease states specific to manatees. All of these factors must be evaluated prior to clinical use as a diagnostic test. The purpose of this study was to identify APR protein methodologies that can be applied to manatees and to validate these methodologies.

Acute Phase Proteins

The acute phase reaction (APR) is a nonspecific response to inflammation (infection, autoimmune disease, etc.) or tissue damage (trauma, surgery, or tumors). Positive APR proteins (haptoglobin, C-reactive protein, fibrinogen, serum amyloid A, alpha1 acid glycoprotein, and others) are produced by the liver during APR in response to cytokines released at the site of injury to either protect the body or to combat a potential pathogen. In humans, APR proteins are nonspecific indicators of disease similar to fever or leukocyte counts.¹ Plasma levels of the individual proteins change at different rates after the initial insult, thus providing useful information not only about the inflammation, but also about the duration of disease. Sequential measurement can also aid in assessment of response to treatment.

Haptoglobin

Haptoglobin (Hp) is an alpha2-glycoprotein that binds hemoglobin (Hb) irreversibly. Hp-Hb complexes are large enough to prevent or greatly reduce renal loss of Hb and its iron. The complexes are removed rapidly by hepatocytes, which degrade the proteins, and iron and amino acids are reutilized. The Hp-Hb complex is also a peroxidase, capable of hydrolyzing peroxides released by neutrophils at sites of inflammation. Hp also functions as a natural bacteriostatic agent for iron-requiring bacteria by preventing the utilization of hemoglobin iron by these organisms.

Haptoglobin is quickly consumed in hemolytic syndromes, and severe hepatocellular disease also results in decreased synthesis of haptoglobin. It is increased during the acute phase response. In humans, it is also increased in response to exogenous steroid and NSAID administration, during some protein losing syndromes such as nephrotic syndrome, and during severe biliary obstruction.¹ Haptoglobin is undetectable in the blood of healthy cows. In cows with inflammation or infection, such as mastitis, metritis, pyometra, traumatic reticulitis, abomasal displacement, bacterial nephritis, and hepatic lipidosis, haptoglobin levels increase markedly.^2-5 It has proven to be a sensitive indicator of inflammatory disease in cows.

C-reactive Protein

C-reactive protein (CRP) has been useful as a marker for numerous inflammatory disease states in humans. In humans, dogs, and rabbits, CRP is one of the first APR proteins to become elevated in inflammatory disease, and it also exhibits a dramatic increase in concentration. It is clinically useful for screening for organic disease, assessing the activity of inflammatory diseases, detecting concurrent infections in systemic lupus erythematosus, in leukemia or after surgery, and managing neonatal septicemia and meningitis.^1,6,7

In the presence of calcium, CRP binds not only polysaccharides present in many bacteria, fungi, and protozoal parasites, but also phosphorylcholine, phosphatidylcholine and polyanions (such as nucleic acids). In the absence of calcium, CRP binds polycations, such as histones. When bound, CRP activates the classic complement pathway starting at C1q. Like antibodies, CRP initiates opsonization, phagocytosis, and lysis of invading organisms including bacteria and viruses. CRP also binds toxic autogenous substances released from damaged tissue and aids in their clearance from blood.

Fibrinogen

Fibrinogen was the first APR protein recognized. Increased production by the liver results in elevated levels in inflammatory states as well as pregnancy.¹ It is integral in the aggregation of platelets. In the coagulation cascade, it is broken down by thrombin to form fibrin, the backbone of the thrombus. Though, elevations are mild in many species, it has proven very useful in detecting inflammation in ruminant species, specifically cows. Low concentrations of fibrinogen can occur in disseminated intravascular coagulation, liver failure, and cachexia.

The heat precipitation method can be used as a quick estimate of fibrinogen concentration. More accurate methods are the modifications of the Ratnoff-Menzie assay, the measurement of clot weight, and quantification of immunoprecipitate formed with specific antifibrinogen antisera.

Serum Amyloid-A

Serum amyloid-A (SAA) is produced by the liver in inflammatory states and circulates complexed to a lipoprotein. It is also elevated in many autoimmune states, polyarthritis, granulomatous disease, and neoplasia. Deposits of amyloid-A protein (AA) are most often found in the kidneys, liver, and spleen in chronic disease states, but may be found in any organ. In horses, SAA has been reported to be a very sensitive indicator of inflammation.^8,9 In equine species, SAA is found in only trace amounts in healthy animals and increases dramatically in nonspecific inflammatory states, especially bacterial and viral infection.

Alpha1 Acid Glycoprotein

Alpha1 acid glycoprotein (AAG), also known as orosomucoid, contains a high percentage of carbohydrate with a large number of sialic acid residues, is highly water soluble, and is the major constituent of the seromucoid fraction of plasma. AAG's true physiological role is still unknown; however, it has been shown to bind to and inactivate basic and lipophilic hormones, including progesterone and several drugs, including the benzodiazepines.¹⁰ AAG increases during the APR, especially with gastrointestinal inflammation and neoplasia and with the administration of steroids and non-steroidal anti-inflammatory drugs.¹ Levels are low in protein losing syndromes due to loss, and in women with increased estrogen concentrations through decreased production.

Materials and methods

Briefly, two methodologies were investigated for haptoglobin: a turbidometric test involving acid precipitation of hemoglobin (Tri-Delta Diagnostics, Inc. Morris Plains, NJ) and an anti-human haptoglobin-based ELISA (Tina-quant Haptoglobin, Roche Diagnostics Corporation, Indianapolis, IN). Two methodologies were investigated for C-reactive protein: an anti-porcine CRP ELISA (Tri-Delta Diagnostics, Inc., Morris Plains, NJ) and an anti-human CRP turbidometric assay (Tina-quant C-reactive protein--high sensitivity, Roche Diagnostics Corporation, Indianapolis, IN). The heat precipitation method and plasma gel electrophoresis (Becton Dickinson and Co, Franklin Lakes, NJ) were used to quantitate fibrinogen. Additionally, a thrombin clotting time assay was evaluated. One serum amyloid-A assay was investigated: an anti-bovine SAA ELISA (Tri-Delta Diagnostics, Inc., Morris Plains, NJ). One alpha1 acid glycoprotein methodology was investigated: an anti-bovine radial immuno-diffusion assay (Tri-Delta Diagnostics, Inc., Morris Plains, NJ).

SDS PAGE will be used to ascertain that proteins measured using the above methodologies are identified correctly. Percent capture of the protein as well as inappropriate cross reactivity will be assessed. Healthy and sick manatees will be compared using SDS PAGE to identify proteins only present in sick manatees that may be used to develop future diagnostics.

Results

The monoclonal antibodies against human C-reactive protein and human haptoglobin do not cross-react with these proteins in manatees. The polyclonal antibody against porcine C-reactive protein does not cross-react with manatee C-reactive protein.

The monoclonal antibody against serum amyloid-A does cross-react with manatee protein. Those manatees that have been struck by boats do contain measurable levels of serum amyloid-A, while other more healthy manatees contain only trace levels of SAA. Preliminary data is currently being analyzed to begin to determine sensitivity and specificity in diagnosis of inflammatory disease. SDS PAGE gels are currently being run to ascertain that the protein identified in this ELISA is indeed SAA.

Haptoglobin can be measured in manatees using an acid denaturation assay that is not antibody-dependent. One animal struck by a boat is definitively positive. Reference intervals for haptoglobin vary widely between species. A reference interval must be defined in a population of apparently healthy manatees to separate abnormal animals. This work is currently being performed. Preliminary data from this year will be used to begin to ascertain sensitivity and specificity.

The normal reference interval for fibrinogen in manatees appears to be 100-400mg/dl. We have seen increased fibrinogen in one boat struck manatee using the heat precipitation technique. Gel electrophoresis data and the thrombin test are currently being analyzed for accuracy and diagnostic utility.

Acknowledgements

This work was supported by a grant from Florida Fish and Wildlife Conservation Commission (s018) under IACUC#A726. The authors would like to acknowledge Dr. David Murphy and the staff at Lowry Park Zoo, Drs. Beth Chittick and Mike Walsh and the staff at Sea World, Orlando, Drs. Elsa Haubold, Mark Sweat and Lucy Keith and the staff at Florida Marine Research Institute, and Robert Bonde at United States Geologic Survey for sample collection and collaboration in assessing the health of these animals. The authors would also like to acknowledge the technical support of Melanie Pate, Tina Conrad, Maxine Sacher, Lavonne Williams and Pat Kindland without whom the project could not have been completed.

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