Blood Proteins and Inflammationin the Horse

Mark V. Crisman, DVM, MSa,*,W. Kent Scarratt, DVMa,

Kurt L. Zimmerman, DVM, PhDb

aDepartment of Large Animal Clinical Sciences, Virginia Maryland Regional

College of Veterinary Medicine, Virginia Tech, Blacksburg, VA 24061, USAbDepartment of Biomedical Sciences and Pathobiology, Virginia Maryland Regional

College of Veterinary Medicine, Virginia Tech, Blacksburg, VA 24061, USA

Inflammation is often associated with systemic alterations distant fromthe initial insult that involve many organ systems all designed to eliminatethe offending antigen. Activation of the host response to infection, the‘‘acute-phase response’’ (APR), is a highly organized physiologic reactionthat includes changes in concentrations of plasma proteins termed acute-phase proteins (APPs). The circulating concentrations of these proteinscan provide an objective measure of the severity and extent of the underlyingcondition. The APPs are increasingly being used as markers for prognosisand monitoring response to therapy along with general determinants ofequine health. Use of APPs in veterinary medicine is becoming more wide-spread as more commercial diagnostic kits are being validated. This articlereviews the salient features of APPs and examines their current applicationand potential utility in equine inflammatory disorders.

Vet Clin Equine 24 (2008) 285–297

Acute-phase proteins

A primary challenge in medicine involves the detection and monitoring ofinflammation, which results from myriad disease processes. Inflammation isa complex process involving networks of cellular and humoral events thatare pivotal for the health and survival of all organisms. Early recognitionof systemic inflammation is essential to devise and implement an effectivetreatment plan. This is especially critical if the delicate balance between

* Corresponding author.

E-mail address: farmuse@vt.edu (M.V. Crisman).

0749-0739/08/$ - see front matter � 2008 Elsevier Inc. All rights reserved.

doi:10.1016/j.cveq.2008.03.004 vetequine.theclinics.com

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inflammatory and anti-inflammatory systems malfunctions, resulting in po-tentially fatal sequelae. Inflammation that goes unrecognized or does notdisplay obvious clinical signs may result in subclinical infections that subse-quently impair growth and performance. The resultant clinical deteriorationmay progress to sepsis, multiple organ failure, and death. It is no surprisethat the search for early markers of inflammation has been the focus of hu-man and veterinary medicine over the past several decades. To this end, ef-forts have focused on biochemical identification of APPs as markers for thedegree and time course of inflammation. In response to infection or injury,these proteins are quickly released into the bloodstream and their concentra-tions are directly related to the severity of the underlying condition. In gen-eral, APPs are defined as proteins whose plasma concentration increases ordecreases by at least 25% after an inflammatory stimulus [1]. Quantificationof these proteins can provide valuable diagnostic and prognostic informa-tion and ultimately have a major influence on the outcome of the diseaseprocess.

The APR is a nonspecific, complex, highly orchestrated inflammatory re-sponse designed to minimize tissue damage; enhance the repair process; andrestore homeostasis after infection, trauma, or stress. This response is stim-ulated when injured cells release arachidonic acid metabolites and productsof oxidative stress, followed by elaboration of cytokines, such as interleukin(IL)-1b, IL-6, and tumor necrosis factor-a (TNFa), from macrophages andmonocytes. These cytokines are responsible for many of the cardinal signs ofinflammation, including pyrexia and leukocytosis. Increases in the circula-tion of these proinflammatory mediators (especially IL-6) stimulate the he-patic APR (at the expense of albumin synthesis) [2]. Included among themany roles attributed to APPs are complement activation, coagulation, fi-brinolysis, and inhibition of neutrophil proteases [1]. It is important tonote that within the complex cytokine signaling network, target cells are sel-dom exposed to only a single cytokine. Combinations of cytokines on var-ious target cells may have a stimulatory or suppressive effect. For example,the elaboration of serum amyloid A (SAA) generally requires IL-6 and IL-1or TNFa, whereas IL-1 and TNFa inhibit the induction of fibrinogen (Fb)by IL-6. Additionally, glucocorticoids typically upregulate the stimulatoryeffects of cytokines on the production of APPs, whereas insulin may playan inhibitory role on production of some APPs [1]. Although the APR iscritical in inflammation and healing, it also functions in an ‘‘anti-inflamma-tory’’ capacity that attenuates the inflammatory response to localizedstimuli.

Seventy five years ago, C-reactive protein (CRP) was the first APP recog-nized in human beings, and it has subsequently become an invaluable diag-nostic tool in human medicine to detect and monitor inflammation [3]. Themost frequently measured APPs in equine practice are Fb, SAA, and hapto-globin (Hp) [4]. The APPs are generally classified as ‘‘positive’’ proteins, in-cluding major or moderate, and ‘‘negative’’ proteins, depending on whether

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plasma concentrations increase or decrease in response to the challenge. Thenegative APP in most species is albumin, the most abundant constituent inplasma [5]. During the APR, albumin synthesis is downregulated in favor ofincreasing hepatic synthesis of positive APPs.

The positive major APPs have the following characteristics:

� Low or undetectable concentrations in plasma of healthy individuals� Concentrations increase greater than 10-fold rapidly during APR� Express a large dynamic range� Rapid decrease in concentrations with disease resolution� Relapse or secondary infection results in increased concentrations

Currently, only SAA fulfills the criteria of a positive major APP in horses.The positive moderate APPs have the following characteristics:

� Aways present in the plasma of healthy horses� Concentrations increase 1 to 10 times in response to inflammation orinjury� Response is generally slower (days to weeks) to increase, peak, and re-turn to baseline

Examples of moderate APPs in horses include Hp, Fb, a1-acid glycopro-tein (AGP), and CRP.

In general, a substantial increase in plasma APP concentrations in horseshas been demonstrated with viral and bacterial infections [6,7], surgery [8],colic [9], and experimentally induced arthritis [10]. Moderate changes occurafter strenuous exercise, heatstroke, and parturition. Concentrations of themultiple components of the APR generally increase together, although notall increase uniformly in all horses with the same conditions. The circulatingconcentration of APPs can provide an objective determinant of the health ofan animal, including the severity of any underlying condition, and allowmonitoring of the resolution of disease.

Serum amyloid A protein

Equine SAA is an acute-phase apolipoprotein that increases (O100-fold)rapidly after tissue injury, infection, or inflammation [11]. Produced primar-ily by hepatocytes during the APR, several extrahepatic isoforms of SAA,specifically SAA3, have been identified in horses [10,12,13]. Extrahepatic se-cretion of SAA3 has been demonstrated in the mammary gland (colostrum)and joints (synovial fluid) from horses [13,14]. The physiologic roles of SAAare not completely understood, because various effects have been reported.These include enhancement or inhibition of leukocyte functions, chemotac-tic recruitment of inflammatory cells to the site of infection [15], inhibitionof lymphocyte and endothelial cell proliferation, inhibition of platelet aggre-gation, and phagocytosis. SAA may also inhibit myeloperoxidase releaseand directed migration of phagocytes and modulate connective tissue

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breakdown in normal remodeling. Extrahepatic production of SAA3, al-though speculative, suggests a ‘‘housekeeping’’ role for the protein by pro-viding an immediate defense against tissue injury from inflammatorychallenges. Mammary-associated SAA3 may provide a beneficial functionfor the suckling neonate or maintenance of the mammary gland [13]. Alter-natively, SAA is the primary precursor of amyloid A and has been impli-cated in the pathogenesis of amyloidosis [16].

Reference intervals for plasma SAA concentrations in healthy horseshave been reported to range from less than 0.5 to 20 mg/L [7,14,17]. Thelow constitutive expression of SAA in healthy horses allows straightforwardinterpretation of even moderate increases in SAA concentrations after an in-flammatory stimulus. The short half-life of SAA allows accurate monitoringof disease after therapeutic intervention. Therefore, sequential SAA deter-minations may be potentially useful in patient management and prognosti-cation. The overall diagnostic sensitivity and kinetic profile of SAA(compared with moderate APPs, such as Fb) make it an ideal marker of in-flammation and tissue damage.

Clinical applications of serum amyloid A

Several studies have evaluated the application and efficacy of SAA inhealthy and septic neonates [6,17,18]. Neonatal septicemia is one of themost challenging problems encountered by equine veterinarians; thus, rapiddiagnosis and aggressive therapy have a major influence on outcome. Typ-ically, sepsis refers to disseminated gram-negative bacterial infections.Gram-positive bacterial and viral infections, trauma, hypovolemia, andhemorrhage may all activate the proinflammatory pathways, however, re-sulting in the systemic inflammatory response syndrome (SIRS) [19]. Inves-tigations have suggested that SAA is a sensitive indicator of inflammationand may be beneficial in differentiating neonatal weakness, diarrhea, andsepticemia. Increased SAA concentrations have been reported in foalswith various bacterial infections [18], septicemia, localized infections (in-cluding omphalophlebitis), and arthritis [17]. Higher SAA concentrationshave been noted with bacterial infections, whereas viral infections elicita more tempered response [7]. In contrast, noninfectious causes of neonatalweakness (failure of passive transfer, pre- and dysmaturity, maladjustmentsyndrome, and meconium impaction) have been associated with normal[17,18] to slightly increased SAA concentrations [20]. This may be attribut-able to assay variability and sampling technique. Nonetheless, it is generallyagreed that SAA determinations proved superior when compared with clas-sic markers of inflammation (eg, Fb, leukocyte counts) in distinguishing in-fectious from noninfectious causes of SIRS.

Concentrations of SAA in equine respiratory disease have also beenevaluated, specifically equine influenza and Rhodococcus equi pneumonia.A study on equine influenza indicated that SAA concentrations increased

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during the first 48 hours of clinical signs and then returned to baseline over theensuing 11 to 22 days in uncomplicated cases [7]. SAA determinations provedto be a more sensitive indicator of infection than nasal swabs and correlatedwell with disease resolution. A recent study by Cohen and colleagues [6] eval-uated SAA concentration in foals withR equi pneumonia and its utility to dif-ferentiate normal from affected foals. Results indicated that bimonthly SAAdeterminations in foals less than 1 month of age were not a useful screeningtool for R equi infection. This may have been attributable to the nature of thedisease (insidious with walled off pulmonary abscesses) or the long samplinginterval. Regardless, more research is needed in this area to determine conclu-sively the usefulness of SAA in foals with R equi pneumonia.

Concentrations of SAA have been determined in horses with colic result-ing from inflammatory and noninflammatory causes. Horses with colicattributable to inflammatory causes (enteritis, peritonitis, colitis, or abdom-inal abscesses) had significantly higher concentrations of SAA than horseswith noninflammatory causes (displacement or obstruction). Additionally,SAA concentrations were higher in horses that failed to survive the colic ep-isode compared with survivors; however, the difference was not substantialenough to be clinically useful at this time [9].

Studies of SAA response to equine joint disease have been recently per-formed on serum and synovial fluid [21]. SAA concentrations in serum and sy-novial fluid were lower than assay detection limits in healthy horses. Synovialfluid and serum SAA concentrations were significantly elevated in horses withsuspected infectious arthritis and tenovaginitis, suggesting that SAA may bea useful biologicmarker for horses with joint disease. This study corroboratedan earlier project using an experimentally (lipopolysaccharide) induced arthri-tis, in which increases in synovial fluid SAA reflected inflammatory activityand concentrations decreased during stages of clinical improvement [10].

Recently, an excellent review of equine SAA was published detailingmany of the salient features associated with APPs [22].

Analysis of serum amyloid A

Previously, SAA measurements were primarily the domain of researchlaboratories. Several methodologies have been used for determining equineSAA, including ELISA [11], slide-reversed passive latex agglutination [23],and latex agglutination immunoturbidimetric assay [17]. A commercially de-veloped immunoturbidimetric assay for human SAA (LZ test SAA, EIKENLZ-serum amyloid A assay, Mast Group, Merseyside, United Kingdom) hasrecently been evaluated for use in horses [14]. This rapid automated assaydemonstrated good precision and is appropriate for determining equineSAA. A system has been developed in Europe to allow small diagnostic lab-oratories to measure equine SAA in 30 minutes (Equinostic, DN, EVA,Equibnostic, Copenhagen, Denmark). According to company literature,this equine SAA test is rapid and precise.

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Haptoglobin

Hp is classified as a moderate APP, demonstrating an increase of 1 to10 times greater than the reference interval in horses during the APR (ref-erence interval: 2–10 g/L). Hp is classified as a major APP in ruminantsand has been proved to be an effective marker for the presence and sever-ity of such diseases as mastitis, pneumonia, and endocarditis in cattle [24].Produced primarily by hepatocytes, Hp is an a2-globulin that primarilyfunctions to prevent the loss of iron by the formation of stable complexeswith free hemoglobin (Hb) in the blood. Hp synthesis is stimulated by theHb concentration in plasma, and the resultant Hp-Hb complex providesan efficient means for collection of free Hb, which prevents externalleak or loss of iron and ameliorates the oxidative damage to tissues asso-ciated with free Hb (from hemolysis). Additionally, the Hp-Hb complexesare large enough to reduce renal filtration of free Hb and iron substan-tially from plasma. These complexes are removed by hepatocytes, allowingreutilization of iron and amino acids. Although several functions havebeen ascribed to Hp, it is believed to have a bacteriostatic effect by lim-iting the availability of iron, which is essential for bacterial growth. Hpmay also have anti-inflammatory actions by protecting against reactiveoxygen species and inhibiting granulocyte chemotaxis and phagocytosis[24]. Hp is also reported to aid in wound repair by stimulating angiogen-esis [25].

Clinical application of haptoglobin

As an APP, Hp concentrations increase during any inflammatory process(eg, infection, stress, trauma, allergy). Increased serum Hp concentrations inhorses have been observed after surgery [26], noninfectious arthritis [4], andcarbohydrate-induced laminitis [27]. Horses with colic did not demonstratean increase in Hp concentrations [28]. Serum concentrations of Hp can beinfluenced by factors other than the APR, however. Increased concentra-tions of free Hb in serum (ie, acute hemolytic event) are followed by a sub-stantial decline in concentration of free Hp, because it is quickly consumedduring such hemolytic syndromes. Therefore, serum Hp concentration maybe a sensitive indicator of intra- or extravascular hemolysis and infection orinflammation in horses [26,29].

Analysis of haptoglobin

Currently, techniques used to determine equine Hp concentrations arefairly laborious and generally restricted to research laboratories. Techniquesinclude single radial immunodiffusion (SRID) [30], serum protein electro-phoresis (SPE; increased a2-globulin fraction) [31], Hb-Hp binding capacityassay [4], and immunoturbidimetry [32]. A method for estimation of serumHp using capillary zone electrophoresis has also been described [33].

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Fibrinogen

Fb was one of the earliest recognized APPs. Fb, a soluble plasma glyco-protein synthesized by the liver, is considered a moderate APP with concen-trations increasing 1- to 10-fold over 24 to 72 hours after the induction ofinflammation. The relatively wide reference interval for Fb concentrationsin healthy horses (200–400 mg/dL, 2–4 g/L) and lengthy response period af-ter an inflammatory stimulus have rendered Fb a fairly insensitive APP. Sev-eral functions have been ascribed to Fb, including providing a substrate forfibrin formation in tissue repair and providing a matrix for migration of in-flammatory-related cells. Fb binds to cell surface integrins (CD11/CD18) ofphagocytes, initiating a cascade of intracellular signals promoting the en-hancement of degranulation, phagocytosis, and antibody-dependent cyto-toxicity. Over the past several decades, Fb has been used to diagnose andmonitor various inflammatory conditions in horses. A recent study evaluat-ing serum iron and plasma Fb concentrations in systemic inflammatory dis-eases in horses concluded that an increase in Fb concentration wasassociated with a poor prognosis. Hypoferremia was a more accurate reflec-tion of acute, subacute, and chronic inflammation in sick horses older than2 months of age, however [34]. Plasma Fb concentrations have been used todetect and monitor R equi pneumonia in foals. Measurement of Fb concen-trations and leukocyte counts proved useful for early identification ofR equi–infected foals, although leukocyte counts proved superior under fieldconditions [35]. Another study evaluated SAA and Fb concentrations inhealthy horses experimentally infected with Streptococcus zooepidemicusand monitored the progression of pneumonia. Results indicated that SAAresponded more rapidly than Fb to changes in clinical signs of pneumonia[36]. Together, these studies suggest that an alteration in Fb concentrationis not necessarily in agreement with actual disease detection or progression.Although determination of plasma Fb concentration has long been used fordetecting inflammatory diseases in horses, its relatively slow APR after aninflammatory insult seriously hampers its clinical utility. Nevertheless, Fbmeasurements are relatively easy and inexpensive, and this fact has likely se-cured its continued wide use in veterinary medicine.

Analysis of fibrinogen

Aheat precipitationmethod is used as a quick estimate of Fb concentration[37]. More accurate methods include modifications of the Ratnoff-Menzie assay, measurement of clot weight, and quantification of immunopre-cipitate formed with specific anti-Fb antiserum.

a1-acid glycoprotein

AGP is a highly glycosylated protein synthesized and secreted primarilyby hepatocytes. It is considered a moderate APP in most species and is

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more likely to be associated with chronic conditions rather than acute in-flammation. Local (extrahepatic) AGP production has been confirmedand is believed to contribute to the general maintenance of homeostasisby reducing tissue damage associated with inflammation, particularly in ep-ithelial and endothelial cells [38]. Two major functions have been attributedto AGP, namely, drug binding and immunomodulation. Similar to albumin,AGP is capable of binding to endogenous or exogenous substances, such asheparin, histamine, serotonin, and steroids [38]. This critical function maykeep total drug-binding levels constant during the APR, whereas albumin,a negative APP, decreases in total concentration. AGP has been reportedto inhibit neutrophil activation, increase secretion of IL-1 receptor antago-nist by macrophages, and enhance clearance of lipopolysaccharide by di-rectly binding and neutralizing the latter [38,39].

Although AGP has been proved to be a useful APP in other species, in-cluding pigs [40] and cattle [41,42], little work has been done in horses. Onestudy reported increased concentrations of AGP (as determined by SRID)in colts 2 to 3 days after castration and in adult horses after jejunojejunos-tomy and return to baseline values 14 to 28 days later [43]. Another studyevaluating a carbohydrate overload model of laminitis in ponies reported in-creased concentrations of AGP 4 hours after administration of carbohy-drate (24 hours before the onset of clinical lameness) [27].

C-reactive protein

CRP has been well documented as an APP in human beings, ruminants,dogs, and, to a lesser degree, horses [1,24]. It is considered to be a moderateAPP in horses, with a two- to threefold increase over several days. CRP hasseveral proinflammatory effects, including activation of the complement cas-cade, induction of inflammatory cytokines, and phagocytosis. CRP also hassignificant anti-inflammatory effects, such as inhibiting chemotaxis and thegeneration of superoxide by neutrophils and preventing the adhesion of neu-trophils to endothelial cells. Studies conducted in the early 1990s suggestedthat high CRP concentrations occurred in horses with pneumonia, enteritis,and arthritis [44].

Serum protein electrophoresis

Serum proteins consist of albumin and globulins, which usually are quan-titated on a standard biochemical profile. In contrast to albumin, which isa single type of protein, globulins are a mixture of proteins that tend to mi-grate in groups on SPE [31]. These groups are known as a-globulins, b-glob-ulins, and g-globulins. The first step to investigate an increase or decrease intotal serum globulins, as noted on the biochemical profile, is to perform SPE[31]. SPE is the current standard method for the fractionation of serum

293BLOOD PROTEINS AND INFLAMMATION IN THE HORSE

proteins, and the results can be a useful diagnostic aid to the clinician. Thereare, however, only a few diseases for which the pattern of SPE is pathogno-monic [31].

The principle of the electrophoretic separation of serum proteins is basedon the migration of charged proteins in an electric field [31]. The directionand rate of migration of a protein are based on the type of charge (anionor cation) and size of the protein. A ‘‘normal’’ equine SPE consists of sixfractions, including albumin, a1-globulin, a2-globulin, b1-globulin, b2-glob-ulin, and g-globulin.

The electrophoretogram is stained, and a densitometer is used to deter-mine the proportion of proteins in these fractions, which are then used inconjunction with the total serum protein concentration to determine specificconcentrations of the fractions. Reference values for these fractions on SPEof the adult horse are albumin (26–37 g/L), a1-globulin (0.6–7 g/L), a2-glob-ulin (3–13 g/L), b1-globulin (4–16 g/L), b2-globulin (3–9 g/L), g-globulin(6–19 g/L), and total serum protein (52–79 g/L) [31].

Albumin is the most prominent of the normal serum proteins on SPE andconstitutes approximately 50% of the total serum protein [31]. The albuminfraction migrates closest to the anode and is the most homogeneous fractionon SPE [31,45]. Equine serum often has a minor postalbumin fraction,which appears as a shoulder on the cathodal side of the albumin peak.This shoulder often becomes more prominent with hypoalbuminemia [31].

The a-globulin fraction is the most rapidly migrating fraction of the glob-ulins and migrates as a1- (fast) and a2- (slow) globulin fractions [31]. The a1-and a2-globulin fractions are identified as the first two peaks after albuminon SPE. Important a1- and a2-globulins include antitrypsin, high-density li-poprotein, very-low-density lipoprotein, macroglobulin, ceruloplasmin, andHp [31].

The b-globulin fraction trails the a-globulin fraction on SPE and mi-grates as b1- (fast) and b2- (slow) globulin fractions [31]. The b1- and b2-globulin fractions are identified as the third and fourth peaks after albuminon SPE. Important b-globulins include complement (C3, C4), transferrin,ferritin, and CRP. Some of the immunoglobulins (IgM and IgA) can mi-grate in the b-globulin region [31].

The g-globulin fraction trails the b-globulin fraction on SPE and includesIgG, IgA, IgM, and IgG subclass T (IgG [T]). The concentrations of theseimmunoglobulins in horses have also been quantitated by SRID [46].

Interpretation of serum protein electrophoresis

The profile of SPE and the absolute values of the individual fractions oc-casionally can be used to make a diagnosis but are often used to direct ad-ditional diagnostic tests. The profile of SPE in an individual animal isrelatively constant but may be influenced by age, hormones, pregnancy,and lactation [31]. A deficiency of dietary protein, hypothermia,

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hyperthermia, and inflammation also can influence the profile of SPE [31].Common abnormalities identified on SPE include hypoalbuminemia, hyper-globulinemia, and hypoglobulinemia.

Hypoalbuminemia is caused by a decreased synthesis or an increased lossof albumin. Albumin is synthesized in the liver, and hypoalbuminemia isa feature of chronic diffuse liver disease [31,45]. A prominent postalbuminfraction on SPE, with or without hypoalbuminemia, has been consideredpathognomonic for liver disease in the horse. An increased loss of albuminmay be caused by renal or gastrointestinal disease and accumulation withinthe thoracic or abdominal cavity [31,45].

Hyperglobulinemia is caused by an increase in the a-, b-, or g-globulinfractions and occurs in a variety of disorders. An increase in the a-globulinfraction occurs in acute inflammatory disorders, because the APPs, includ-ing SAA and macroglobulin, migrate in the a-globulin fraction [31]. An in-crease in the b-globulin fraction occurs in active liver disease, becausetransferrin and IgM migrate in the b-globulin fraction [31]. An increase inthe beta and gamma globulin fractions (beta-gamma bridging) on SPE isnoted when there is no clear separation between the beta-2 and gamma glob-ulin fractions. Beta-gamma bridging may be caused by an increase in IgM orIgA, chronic active hepatitis, or lymphosarcoma [31,45]. Experimental infec-tions of the intestinal tract with Strongylus vulgaris larvae have been asso-ciated with an increased concentration of IgG (T) [47,48].

Hypergammaglobulinemia may be caused by a broad increase (poly-clonal gammopathy) or a sharp increase (monoclonal gammopathy) ingamma globulins. The broad increase in gamma globulins that characterizesa polyclonal gammopathy is caused by the heterogeneity of clones of plasmacells, which produce a heterogeneous mix of immunoglobulins. Any or all ofthe immunoglobulin groups can be increased. A polyclonal gammopathyoften is associated with a chronic inflammatory disease, such as hepatitis,pleuropneumonia, immune-mediated disease, neoplasia, or a chronic suppu-rative disorder [31,45].

A monoclonal gammopathy is characterized by a sharp increase (orspike) in one of the immunoglobulins. The monoclonal spike is causedby a single clone of plasma cells that produces a single class of immuno-globulin or an immunoglobulin fragment (referred to as a paraprotein, Mprotein, or M component), which can be identified by the results ofelectrophoresis, immunoelectrophoresis, or immunodiffusion [31,49,50].Monoclonal gammopathy occurs infrequently in the horse and has beenassociated with plasma cell myeloma [49], malignant lymphoma [51] andidiopathic causes [52].

The diagnostic and prognostic value of SPE in horses with chronic diar-rhea was reported [49]. Horses with larval cyathostomiasis had significantlyhigher levels of beta-1 globulin. A normal concentration of beta-1 globulinwas not a reliable indicator of the absence of larval cyathostomiasis, how-ever. Horses with chronic diarrhea that did not survive were more likely

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to have a lower concentration of albumin and a higher concentration ofalpha-2 globulin [53].

Hypoglobulinemia may be caused by a reduction in the alpha, beta, orgamma globulin fractions and occurs in a variety of disorders. Failure ofpassive transfer of immunity in foals is associated with a deficiency ofgamma globulin. A horse with a protein-losing gastroenteropathy oftenhas hypoglobulinemia and hypoalbuminemia.

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