Accepted Manuscript

Comparison of serum amyloid A in horses with infectious and non-infectious

respiratory diseases

Molly Viner, Melissa Mazan, Daniela Bedenice, Samantha Mapes, Nicola Pusterla

PII: S0737-0806(16)30249-0
DOI: 10.1016/j.jevs.2016.09.005
Reference: YJEVS 2184

To appear in: Journal of Equine Veterinary Science

Received Date: 13 May 2016

Revised Date: 8 August 2016
Accepted Date: 13 September 2016

Please cite this article as: Viner M, Mazan M, Bedenice D, Mapes S, Pusterla N, Comparison of serum
amyloid A in horses with infectious and non-infectious respiratory diseases, Journal of Equine Veterinary
Science (2016), doi: 10.1016/j.jevs.2016.09.005.

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1 Short Communication Journal of Equine Veterinary Science

3 Comparison of serum amyloid A in horses with infectious and non-infectious respiratory

4 diseases

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6 Molly Vinera, Melissa Mazanb, Daniela Bedeniceb, Samantha Mapesa, Nicola Pusterlaa,*

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8 Department of Medicine and Epidemiology, School of Veterinary Medicine, University of

9 California, Davis, CA, USA

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Department of Clinical Sciences, Cummings School of Veterinary Medicine at Tufts University,
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11 N. Grafton, MA, USA
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13 * Corresponding author at: Nicola Pusterla, Department of Medicine and Epidemiology, School
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14 of Veterinary Medicine, University of California, One Shields Avenue, Davis, CA 95616.

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15 E-mail address:

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24 Abstract

25 The acute phase protein serum amyloid A (SAA) has been shown to be a useful inflammatory

26 parameter in the horse, but studies showing SAA responses to specific respiratory disease

27 etiologies is limited. The goal of this study was to evaluate SAA responses in horses with

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28 infectious and non-infectious respiratory diseases as well as healthy, control horses. Two

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29 hundred and seven horses were grouped into the following categories: equine influenza virus

30 (EIV), equine herpesvirus-4 (EHV-4), Streptococcus equi subspecies equi (S. equi ss equi),

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31 inflammatory airway disease (IAD), and healthy controls. SAA concentrations were determined

32 for all horses on serum using a stall-side lateral flow immunoassay test. SAA levels were found

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to be significantly higher for infectious respiratory diseases (EIV, EHV-4, S. equi ss equi) and
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34 horses with IAD when compared to control horses. There was a significant difference between

35 viral and bacterial infections and IAD. Although SAA values from horses with S. equi ss equi
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36 were significantly higher when compared to horses with viral infections (EIV/EHV-4), the wide
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37 range of SAA values precluded accurate classification of the infectious cases. In conclusion,
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38 SAA is more reliably elevated with infections of the respiratory tract rather than non-infectious

39 airway conditions. This can facilitate early detection of respiratory infections, help track disease
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40 progression, and aid practitioners in making recommendations about proper biosecurity and

41 isolation of potentially contagious horses.

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43 Keywords: Serum amyloid A, Infectious respiratory diseases, Inflammatory airway disease,

44 Healthy horses, Horses

45 1. Introduction
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46 Serum amyloid A (SAA) is a major acute phase protein (APP) identified in horses that

47 maintains very low levels in health and undergoes as much as 5000-10,000 mg/L in animals with

48 severe inflammation [1,2]. It has become a useful maker for inflammation, as it rapidly increases

49 in concentration over 6-12 hours, much faster than the greater than 72 hour response time

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50 identified for fibrinogen, a minor APP [3]. In addition, SAA has a short half-life, which results in

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51 a more rapid decrease in concentration and an ability to assess response to treatment and changes

52 in disease process [3]. SAA was studied as a marker for inflammation in horses affected with

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53 equine influenza and found to peak during the acute stages of infection and did not return to

54 baseline until 11-22 days later, for uncomplicated cases [4]. Conversely, SAA was studied in

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racehorses with inflammatory airway disease (IAD) and was not increased when compared to
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56 healthy controls [5]. Infectious and non-infectious conditions of the upper respiratory tract of

57 horses can present similarly, with poor performance, exercise intolerance, coughing, and nasal
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58 discharge. The presence of fever and multiple horses affected on a premise are criteria often used
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59 to gage the infectious and contagious nature of an upper airway disease. It is therefore imperative
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60 that practitioners use clinical and laboratory information to minimize spread of infectious

61 respiratory viruses and bacteria The purpose of this study was to evaluate the response of SAA
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62 with inflammatory-infectious (EIV, EHV-4, and S. equi ss equi) and inflammatory/non-

63 infectious (IAD) respiratory diseases using a stall-side lateral flow immunoassay test.
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65 2. Materials and Methods

66 For this study, convenience serum samples collected from 207 horses of varying ages,

67 breeds, and genders were used (Table 1). The study horses were selected from various repository

68 sample collections and assigned to an infectious respiratory disease group, an inflammatory

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69 airway disease (IAD) group, or a control group based on the following inclusion criteria. Serum

70 samples were all collected within 24 months of analysis and kept frozen at 80 C prior

71 thawing. For this study only horses with infectious respiratory disease were included if they had

72 a fever of 38.5 C and respiratory signs (cough and/or nasal discharge) and a positive qPCR

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73 result of nasal secretions for EIV (n=42), EHV-4 (n=43), or S. equi ss equi (n=44). Horses were

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74 assigned to the IAD group (n=38) based on clinical signs (chronic, intermittent cough, increased

75 mucoid airway secretions and decreased performance), imaging findings (bronchial pattern),

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76 pulmonary function testing and cytological analysis of broncho-alveolar lavage fluid (increased

77 total nucleated cell count with increased neutrophils, lymphocytes, monocytes, eosinophils

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and/or mast cells) in line with the ACVIM consensus statement by Coutil and colleagues [6].
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79 Control horses (40) were considered healthy with a normal physical evaluation and qPCR
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80 negative results for common respiratory pathogens (EIV, EHV-1, EHV-4 and S. equi ss equi).

81 Serum samples from all horses were tested for SAA levels using a commercially
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82 available stall-side lateral flow immunoassay test (StableLab Equine Blood Analysis Kit, Sligo,
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83 Ireland). The stall-side test has been validated by the manufacturer and the precision and

84 accuracy was determined to be 98.6% and 95.6% at concentrations ranging between 50 and
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85 2,000 mg/L. In a recent preliminary validation of the analytical accuracy of the stall-side lateral

86 flow immunoassay test, this assay showed good linearity between 0 to 2,000 mg/L with good
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87 agreement with a turbidimetric immunoassay (Eiken, Tokio, Japan). The intra-assay CVs
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88 ranged between 13 and 18% for low, intermediate and high SAA concentrations. Further,

89 SAA concentrations in whole blood and serum/plasma were positively correlated (Diana

90 Schwartz, personal communication). The detection range for the test is 0 to 3,000 mg/L.
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91 Samples with SAA concentrations >3,000 mg/L were not diluted to obtain an absolute

92 concentration and were reported as 3,000 mg/L.

93 Differences in SAA values amongst the groups were determined using the exact Kruskal-

94 Wallis test. P<0.05 was considered statistically significant for all analyses.

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96 3. Results

97 The majority of the healthy horses had undetectable SAA (minimum 0 g/ml, maximum

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98 2 mg/L, median 0 mg/L; Figure 1). Six horses with IAD had detectable SAA up to 586 mg/L

99 (minimum 0 mg/L, maximum 586 mg/L, median 0 mg/L). The SAA for EIV qPCR positive

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horses ranged from 0 to 3,000 mg/L (median 731 mg/L, 5 samples with SAA 3,000 mg/L).
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101 The SAA values for EHV-4 qPCR positive horses ranged from 0 to 3,000 mg/L (median 1,173
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102 mg/L, 1 sample with SAA 3,000 mg/L). The SAA for S. equi ss equi qPCR positive horses

103 ranged from 0 to 3,000 mg/L (median 1,953 mg/L, 5 sample with SAA 3,000 mg/L). There
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104 were significant differences between the control group and the various inflammatory disease
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105 groups (P = 0.0001). There was also a significant difference between horses from the IAD group

106 and the three infectious groups (P = 0.0001). There was a significant difference between the
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107 EHV-4 group and the S. equi ss equi group (P = 0.002), but not between the EHV-4 group and

108 the EIV group (P = 0.096) and not between the EIV group and the S. equi ss equi group (P =
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109 0.169). When both viral disease groups were combined and compared to the S. equi ss equi
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110 group, there was a significant difference (P = 0.0095).

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112 4. Discussion
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113 Past studies have looked at the SAA levels in horses with EIV infection and IAD, but

114 have not compared non-infectious with infectious respiratory diseases [4,5]. Collectively, these

115 studies showed that SAA increases with acute inflammation from an infectious respiratory

116 disease. A commercially available stall-side SAA lateral flow immunoassay test was recently

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117 introduced into the equine market and gives practitioners the ability to rapidly measure an

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118 important marker of inflammation.

119 To the authors knowledge, this is the first report comparing SAA levels in natural

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120 infections with viral and bacterial infections of the upper respiratory tract, and inflammatory,

121 non-infectious etiologies. This study confirms the diagnostic hypothesis that SAA will more

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reliably be elevated with infections of the respiratory tract rather than non-infectious airway
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123 conditions. The population used in this study represents horses of various ages and breeds with

124 acute onset of respiratory signs. Furthermore, all horses with infectious and non-infectious
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125 respiratory diseases presented with coughing and nasal discharge of varying degrees. Early in the
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126 course of the disease process, equine practitioners may be faced with the challenge of
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127 differentiating disease etiologies with similar clinical pictures. Equine practitioners can use SAA

128 during the acute onset of disease to help differentiate respiratory infections from inflammatory,
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129 non-infectious respiratory diseases showing similar clinical signs. Also, it has been shown that

130 SAA levels remain undetectable in horses that, despite being in contact with transmissible
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131 pathogens, did not contract the disease [7]. Measuring SAA values in horses can be especially
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132 useful to track disease progression and make recommendations about biosecurity and isolation.

133 Although there is a significant difference in SAA values between bacterial and viral infectious

134 respiratory diseases, there is a lot of overlap in SAA values between these two groups. Therefore,

135 it is not recommended that SAA levels alone be used to distinguish between bacterial and viral
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136 respiratory diseases. Future studies may look to grade horses with varying clinical disease and

137 give scores based on severity of clinical signs and compare severity of inflammatory response

138 and SAA levels to infectious etiologies.

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140 Acknowledgements

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141 This study was supported by the School of Veterinary Medicine, University of California, Davis.

142 StableLab kindly provided the testing material.

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143

144 References

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[1] Kjelgaard-Hansen M, Jacobsen S. Assay validation and diagnostic applications of major
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146 acute-phase protein testing in companion animals. Clin Lab Med 2011;31:51-70.

147 [2] Belgrave RL, Dickey MM, Arheart KL, Cray C. Assessment of serum amyloid A testing of
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148 horses and its clinical application in a specialized equine practice. J Am Vet Med Assoc
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149 2013;243:113-9.
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150 [3] Jacobsen S, Anderson PH. Tutorial Article: The acute phase protein serum amyloid A (SAA)

151 as a maker of inflammation in horses. Equine Vet Educ 2007;19:38-46.

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152 [4] Hultn C, Sandgren B, Skildebrand E, Klingeborn B, Marhaug G, Forsberg M. The acute

153 phase protein serum amyloid A (SAA) as an inflammatory marker in equine influenza virus
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154 infection. Acta Vet Scand 1990;40:323-33.

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155 [5] Leclere M, Lavoie-Lamoureux A, Lavoie JP. Acute phase proteins in racehorses with

156 inflammatory airway disease. J Vet Intern Med 2015;29:940-5.

157 [6] Coutil, LL, Hoffman AM, Hodgson J, Buechner-Maxwell V, Viel L, Wood JLN, et al.

158 Inflammatory airway diseaserevised consensus statement. J Vet Intern Med 2007;21:356-61.
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159 [7] Pepys MB, Baltz ML, Tennent GA, Kent J, Ousey J, Rossdale PD. Serum amyloid A protein

160 (SAA) in horses: objective measurement of the acute phase response. Equine Vet J 1989;21:106-

161 9.

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163 Legend to Figures

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164 Figure 1: Distribution of SAA values in healthy control horses, horses with IAD and horses with

165 EIV, EHV-4 and S. equi ss equi infection. The horizontal lines represent median SAA values.

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166 Table 1

167 Signalment and clinical signs of healthy horses and horses with infectious and non-infectious respiratory diseases.

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EHV-4 EIV Streptococcus equi ss equi IAD Control
Sample Size 43 horses 42 horses 44 horses 38 horses 40 horses
Age (years) Range = 0.3-13 Range = 0.1-20 Range = 0.5-29 Range = 3-25 Range = 5 -21 years

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Median = 2 Median = 4 Median = 8 Median = 10 Median = 11.5 years
Unknown = 3 Unknown = 4 Unknown = 1 Unknown = 0 Unknown = 0
Sex Female = 18 Female = 21 Female = 12 Female = 9 Female = 37

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Male = 24 Male = 18 Male = 29 Male = 29 Male = 3
Unknown =1 Unknown = 3 Unknown = 3 Unknown = 0 Unknown = 0
Breed Quarter Horse = 16 Quarter Horse = 16 Quarter Horse = 27 Warmblood = 11 Quarter Horse = 22

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Thoroughbred = 7 Welsh Pony = 3 Thoroughbred = 3 Quarter Horse = 7 Thoroughbred = 10
Arabian = 6 Saddlebred = 3 Saddlebred = 3 Welsh Pony = 4 Warmblood = 8

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Paint Horse = 5 Percheron = 3 Arabian = 3 Friesian = 2 Unknown = 0
Friesian = 2 Warmblood = 2 Haflinger = 2 Paint Horse = 2
Other = 4 Thoroughbred = 2 Other = 6 Thoroughbred = 2

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Unknown = 3 Friesian = 2 Unknown = 0 Gypsy Vanner = 2
Other = 8 Other = 8
Unknown = 3 Unknown = 0

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Rectal temperature Range = 38.5 41.7 Range = 38.5 40.6 Range = 38.5 41.1 All afebrile (<38.5) All afebrile (<38.5)
(C) Median = 39.7 Median = 39.6 Median = 39.6

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Cough 43/43 had cough 42/42 had cough 44/44 had cough ranging 30/38 had cough 0/40 had cough
ranging from ranging from from occasional to frequent
occasional to frequent occasional to frequent
Nasal Discharge 43/43 had nasal 42/42 had nasal 44/44 had nasal discharge 21/38 had nasal 0/40 had nasal
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discharge ranging discharge ranging ranging from mild to discharge discharge
from mild to severe from mild to severe severe
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Highlights

Horses with infectious upper airway diseases have higher SAA concentrations compared to horses with

inflammatory airway disease and healthy control horses

Although there is a significant difference in SAA values between bacterial and viral infectious

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respiratory diseases, there is a lot of overlap in SAA values between these two groups

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Equine practitioners can use the stall-side SAA LFIA to distinguish between infectious and non-

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infectious respiratory diseases when clinical signs are equivocal

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