Division of Comparative Pathology, Department of Pathology & Laboratory Medicine, Miller School of Medicine, University of Miami, Miami, FL, USA
Abstract
Tracking the acute phase response has long been a goal of protein electrophoresis. The use of immunoassays to quantitate specific acute phase proteins has been well described in companion and large animals and rodents. In more recent years, commercial reagents have been studied for their reactivity to avian and exotic species and nondomestic mammals. There are clear applications for these biomarkers in health assessments and prognosis. The how, what, and why of acute phase protein testing continues to be a work in progress for many species.
Introduction
The acute phase response (APR) is an integral part of the innate immune system.5,14,20 The APR is a complex systematic inflammatory process that begins with a local stimulus, such as a response to injury or infection, and then the production of cytokines that signal the liver to initiate or upregulate the expression of acute phase proteins (APP). It has been estimated that more than 200 APP may be produced during the APR.20 APP have unique roles, including opsonization, complement activation, and enhancement of phagocytosis (C-reactive protein, CRP); enhancement of chemotaxis and aid to tissue repair (serum amyloid A, SAA); and binding hemoglobin to minimize oxidative activity (haptoglobin, HP).5 These proteins have been found to be highly conserved with observations of these or similar proteins from mollusks to turtles to mammals.
APP are classified as positive or negative.5,11,14 Major APP can increase ten- to a thousandfold. The increase is rapid rising from near negligible levels and occurs within 16–24 hours. In most companion and large animals, SAA and CRP act as major APP. Mild and moderate APP are present in detectable levels in normal animals. Increases may be two- to tenfold for moderate APP and less than twofold for minor APP. Expression is often delayed to 4–6 days after insult, and elevated levels will persist after resolution of the inflammatory process. HP is often defined as a minor to moderate APP in most species.
Albumin is classified as a negative APP, as it decreases in ongoing APR. In some mammals, transferrin has also been classified as a negative APP. Transferrin has been defined as a positive APP in avian species.
Value of Tracking the Acute Phase Response
APP greatly contrast the sensitivity of other traditional measures of inflammation, including white blood cell count, neutrophil count, and the A/G ratio. These independent measures have been conducted in dogs and horses. In dogs with various inflammatory processes (n=900), a correlation of r=0.44 between WBC counts and CRP was reported.5 In horses, the correlation between WBC counts and SAA was weak (r=0.11).1 In the latter study, SAA had the highest diagnostic accuracy for inflammation versus the other measures at 75%.1 In a large study of clinically ill cattle, animals were classified as experiencing either acute or chronic inflammation. SAA levels were found to have 100% sensitivity, and HP levels showed 76% specificity. Neutrophil counts showed 30% sensitivity and 7% specificity. It should be noted that these comparisons are biased by the different timelines of expression of each measure.
When strong agents of inflammation, such as turpentine oil, are injected into laboratory animals, there can be a mild transient increase in WBC; however, a true leukocytosis is not observed until 4–7 days later. Serum albumin will decrease but will not reach minimum concentrations until day 5. In contrast, CRP and SAA increase several hundredfold by 48 hours. An impressive characteristic of major APP is the rapid decrease in levels as the insulting stimuli is addressed. With a short half-life and negative feedback, the levels can drop within a few days. The rapid increase and rapid decrease provide key value to the use of APP as prognostic indicators.
In some cases, elevated APP levels can aid in a differential diagnosis. In horses with serious enteritis, colitis, or peritonitis, fiftyfold higher SAA levels were observed versus horses with obstruction, perforation, or ulcers.5 In a large study of horses with various infections, the highest SAA levels were consistently observed with bacterial infections, including bacterial pneumonia.1 In the aforementioned study on clinically ill cattle, acute illnesses were associated with SAA expression, whereas the slower-forming HP response was associated with chronic processes.11
As the origin of the APP stimulus may be infection, inflammation, trauma, stress, or neoplasia, APP are rarely solely diagnostic for any one disease or etiology.5
Measuring Acute Phase Proteins
An ongoing APR can be reflected in fraction changes in protein electrophoresis.20 Classic changes have been observed with feline infectious peritonitis, ehrlichiosis, and myeloma in companion animals. Newer automated and semi-automated methodologies, including enzyme-linked immunosorbent assay, immunoturbidity, and colorimetry have been employed to quantitate specific APP. Many of these reagents are used in assays to measure human APP and have variable cross-reactivity in animals. In the antibody-based protocols, methods and reagents must be individually validated for each species.21 If reagents are identified as cross-reactive based on a sample set of clinically normal and abnormal animals, traditional studies of coefficient of variation analysis and linearity under dilution must be undertaken. From that point, the study can then include a large number of samples to determine reference intervals and assess the clinical impact of using these biomarkers with patients. In limited instances, there are species-specific reagents; these often are in ELISA format.
Birds
Studies in chickens have dominated the literature on APP expression in birds.3,24 With injection of gold standard inflammatory agents, changes in SAA, transferrin, PIT-54 (analog of HP), and alpha-1 acid glycoprotein have all been documented. In the author’s laboratory experience, finding reagents with wide avian species cross-reactivity has been difficult, although the colorimetric reagents used to quantitate HP appear to be valid. Increased SAA expression has been documented in falcons with aspergillosis.2,15 It appears that this APP is a major APP in this species.
The current impression is that protein electrophoresis provides very good sensitivity for ongoing APR. It provides a broad impression of different globulin changes and specifically quantitates albumin, which is an important and sensitive negative APP in birds.10 In a comparison of total WBC counts versus EPH abnormalities in African grey parrots (judged by a change in the A/G ratio), a marginal correlation was observed between significantly elevated WBC counts and significantly decreased A/G ratio (C. Cray, personal communication, April 2017). In 49% of the cases with a decreased A/G ratio, an elevated WBC count was found. Notably, 43% of the cases showed a normal WBC count when a significant decrease in the A/G ratio was present. Similarly, the majority of the cases showed a normal WBC count when a mild to moderate decrease in the A/G ratio was present. This data indicates a differential sensitivity between these two methods and supports the use of electrophoresis in routine clinical pathology investigations of avian species.
Reptiles
SAA expression (at the RNA level) was found to increase a thousandfold in softshell turtles experimentally infected with gram-negative bacteria.28 To date, attempts to validate commercially available SAA antibodies in most reptiles have not been successful. Hemoglobin binding activity, quantitated by the colorimetric assay for HP, has been demonstrated in box turtles, loggerhead turtles, inland bearded dragons, and rattlesnakes (C. Cray, personal communication, April 2017).13,16 As additional reagents for major APP become available, studies should consider the relative sensitivity of protein electrophoresis vs. APP assays in these species.
Small Exotic Mammals
Rats and mice have interesting differences in APP expression versus companion animals; to date, analysis is best performed using ELISA methods.11 There has been little to no description of APP expression in guinea pigs, hamsters, and gerbils.
CRP expression has been described in rabbits with suspected Encephalitozoon cuniculi infection.8 Elevated CRP levels acted as an adjunct diagnostic test to improve the specificity of serologic titers against this organism and also served as a prognostic indicator.6 Recent studies indicate also the utility of newly described VET-SAA reagents (C. Cray, personal communication, April 2017).
Ferret SAA was cloned and found to be similar to other mammalian SAA. In studies from the author’s laboratory, SAA was found to be increased two- to fivefold in clinically abnormal ferrets.26 A tenfold increase in HP was observed in a ferret with myeloma.
Fish and Sharks
APP expression has been well documented at the RNA level in several species of fish. Attempts to document expression in a trauma model in koi were not successful and may have been related to lack of reagent cross-reactivity and experimental design.4 CRP has been validated for use in bonnethead sharks.18 A 2.5-fold mean increase was found in clinically abnormal sharks, which included cases of bacterial septicemia and suspected Fusarium infection. CRP also has been validated in sand tiger and sandbar shark species (C. Cray, personal communication, April 2017).
Marine Mammals
SAA expression and use in health assessments were first described in the manatee by Harr et al.17 The use of an automated SAA assay was described in 2013.7 Approximately thirtyfold higher mean SAA levels were quantitated in manatees with cold stress or trauma. The sensitivity was 93% and specificity was 98%, which were superior to total white blood count and albumin quantitation. Additional studies have been completed in elephant seals and dolphins. The latter utilizes a dolphin specific reagent.
Primates
APP expression has been documented in many species of nonhuman primates. SAA, CRP, and HP testing was validated in rhesus macaques, and more than a two hundredfold increase in CRP was observed in animals with chronic active inflammation associated with Mycobacterium infection.22 SAA was also described to be increased in macaques with hepatic amyloidosis.23 SAA and CRP have been found to be increased in the orangutan with various infectious and inflammatory diseases (C. Cray, personal communication, April 2017).
Other Nondomestic Mammals
APP expression has been well documented in the cheetah. SAA tests have been validated, and significant increases were reported in clinically abnormal animals.12 SAA testing has also been validated for use in lions, tigers, and leopards (C. Cray, personal communication, April 2017).
SAA and HP testing has been validated in elephants.19,27 Significant increases in SAA were observed in elephants with >10,000 virus genome copies/ml EEHV-1 in blood. This biomarker appeared to have excellent prognostic value in elephants with this infectious disease.27 In addition, increases in SAA were observed in Asian elephants with pododermatitis and traumatic injuries.19
There are considerable other publications in a wide variety of species including alpaca, antelope, capybara, and water buffalo. Caution should be used when submitting for APP testing, as the published and nonpublished/anecdotal reports will widely vary with reagent cross-reactivity. Refer to the website of the University of Miami Acute Phase Protein Laboratory (www.cpl.med.miami.edu/acute-phase-protein) for a full list of lab-validated species. Contact the lab if you have interest in submitting samples for other species, as basic testing may already be completed.
Concluding Remarks
The field of proteomics is rapidly growing, so it is expected many more biomarkers will be identified to gauge the acute phase response and assist in the diagnosis of specific diseases. In a spectroscopy study of samples from falcons with aspergillosis, 3-hydroxybutytrate is significantly altered versus in normal falcons.25 Lipoprotein fractions were also found to be significantly altered in clinically abnormal cownose rays.9 These observations underlie the opportunities for further study in these areas in other species.
Reagent availability has increased in recent years, making it possible to test possible cross-reactivity in relatively easy fashion. Still, APP expression in some species has defied further elucidation and may necessitate a basic science approach. Of note, point-of-care options for testing are and will continue to become more commonplace. While the applications are especially exciting, these reagents (like those used at the reference laboratory) need to be validated species by species.
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