Abstract
The early identification and staging of inflammatory disease in animals has been enhanced by knowledge of acute phase protein responses in various species. Fibrinogen and haptoglobin have been identified as useful indicators of inflammation in domestic ruminants and cervids.1,12 Major acute phase proteins (APP) should either have low concentrations or be nonexistent in serum or plasma of healthy animals.5,8,12 This study was conducted to ascertain normal values of fibrinogen and haptoglobin in wapiti. Serum and plasma samples were taken from 41 wapiti ranging from 8 mo to several years of age.
Introduction
Acute phase proteins are a group of primarily glycoproteins that are produced by the liver in response to inflammation.2,6-8 They have been further defined as plasma proteins that increase in concentration by 25% or more in the first 7 days following tissue damage.4 Their production is directly stimulated by mediators produced by leucocytes and macrophages during episodes of infection or inflammation.6 These mediators include interferon, and cytokines such as interleukin-1, interleukin-6, and tumor necrosis factor.2-4 Major APPs of various animal species quickly reach high circulating values after inflammatory disease commences.6 Their degree of response is directly related to the amount of tissue damage.5,6 Likewise, they decline rapidly when the disease process diminishes.6
Species differences are evident with regard to the magnitude of production of each APP.2,5,12 An example of this is the prevalence of haptoglobin in bovine and equine inflammatory diseases.12 It is an excellent indicator of pulmonary inflammation in domestic cattle and appears to be the major APP in all ruminants.6,9 However, it is a poor indicator of respiratory disease in horses.13 In contrast, fibrinogen is recognized as the most universal APP in animal species.6 It has been used in combination with haptoglobin to improve the detection of tuberculosis in deer.6 This emphasizes the point that APP combinations are superior to single proteins in identifying inflammation and its various stages.3,12 Acute phase proteins used in combination may indicate the magnitude of tissue damage, the length of time during which inflammation continues, and the response to treatment.
Methods
The wapiti sampled in this project were deemed physically healthy by visual examination and by limited physical examination while in a drop-floor restraint chute. Blood was collected from a jugular vein of each animal during physical restraint and placed in CaEDTA and clot tubes. Fibrinogen is a component of blood coagulation and is removed from plasma during the clotting process. For this reason, fibrinogen samples were collected in CaEDTA tubes.
Plasma fibrinogen was evaluated using the heat precipitation method. In this method, whole blood is centrifuged in paired capillary tubes. One tube is used for plasma protein quantification with a refractometer. The other capillary tube is immersed in a water bath at 56°C. This process causes coagulation of fibrinogen. Fibrinogen is the only plasma protein that coagulates at this relatively low temperature. This tube is centrifuged once again, separating the coagulated fibrinogen from the remaining plasma protein. The plasma protein is again determined with a refractometer and this value is subtracted from the first plasma protein value. The result of this subtraction is the fibrinogen value.10
Haptoglobin was determined by using an indirect assay, rather than using an immunological method.2,7,9,11 This avoided the necessity of having to use a species-specific antibody to haptoglobin.4,5 The assay was run on an Abbott Series II automated biochemical analyzer at 37°C, using primary and secondary wavelengths of 404 and 380 nm. This procedure makes use of the ability of haptoglobin-bound cyanmethemoglobin to resist the effects of incubation in an acid environment. For this reason, it is important to use unhemolyzed serum samples. However, hemolyzed samples containing up to 200 mg/dL of hemoglobin can be successfully analyzed.
Results
Forty-one samples were evaluated for haptoglobin levels. The mean value was 7.7 mg/dL with a range of 31 mg/dL. Only 37% (3/8) of the adult wapiti had circulating levels of haptoglobin. Most of the wapiti 1-yr-old and under had haptoglobin present in their serum (31/33; 94%). The mean haptoglobin for adults was 1.25 mg/dL as compared to 9.18 mg/dL in animals 1-yr-old and younger. Twenty animals less than 1-yr-old had a mean haptoglobin value of 11.7 mg/dL.
Forty-one samples were evaluated for fibrinogen levels. The mean value for all animals was 165 mg/dL with a range of 400 mg/dL. Only two wapiti in this study, both yearling animals, had no plasma fibrinogen levels (2/40; 5%). Animals 1-yr-old and under averaged 169 mg/dL while adults averaged 150 mg/dL. It is noteworthy that 19 wapiti under the age of 1-yr had a mean fibrinogen value of 221 mg/dL.
Discussion
It was evident from this study that both haptoglobin and fibrinogen are present in wapiti and are found in low blood concentrations in healthy animals. This research also revealed some significant age-related information. Normal haptoglobin levels were higher in juvenile wapiti (11.7 mg/dL) that adults (1.25 mg/dL). Similarly, juvenile wapiti had higher resting levels of fibrinogen (221 mg/dL) than adult animals (150 mg/dL).
Haptoglobin and fibrinogen may individually be useful in identifying inflammatory processes in wapiti. It is also likely that tandem use of these acute phase proteins may assist the staging of inflammatory processes in this species as has been seen in domestic animals. These proteins should be further evaluated in wapiti by using cytokines to induce inflammatory events and then measuring the blood concentration and duration of response.
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