EVJ 06-141 Van Brantegem
09/08/07
4:53 pm
Page 2
418
EQUINE VETERINARY JOURNAL Equine vet. J. (2007) 39 (5) 418-421 doi: 10.2746/042516407X205888
Antibodies to elastin peptides in sera of Belgian Draught horses with chronic progressive lymphoedema L. VAN BRANTEGEM*, H. E. V. and R. DUCATELLE¶
DE
COCK, V. K. AFFOLTER†, L. DUCHATEAU‡, M. K. HOOGEWIJS§, J. GOVAERE§, G. L. FERRARO#
Laboratory of Pathology, Department of Veterinary Medicine, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, Wilrijk, Belgium; †Department of Pathology, Microbiology and Immunology, #Center for Equine Health, School of Veterinary Medicine, University of California, Davis, USA; and ‡Department of Physiology and Biometrics, §Department of Obstetrics, Reproduction and Herd Health, ¶Laboratory of Pathology, Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium. Keywords: horse; Belgian Draught horse; chronic progressive lymphoedema; anti-elastin antibodies; ELISA
Summary
Introduction
Reasons for performing study: Chronic progressive lymphoedema (CPL) is a recently recognised disease of the lymphatic system characterised by lesions in the skin of the lower legs in several draught horse breeds, including the Belgian Draught hourse. Clinical signs slowly progress and result in severe disfigurement of the limbs. Ideally, supportive treatment should be started early in the disease process. However early diagnosis and monitoring progression of CPL is still a challenge. Hypothesis: Elastin changes, characterised by morphological alterations as well as increased desmosine levels, in the skin of the distal limbs of horses affected with CPL are probably associated with a marked release of elastin degradation products, which elicit production of circulating anti-elastin antibodies (AEAbs) in the serum. An enzyme-linked immunosorbent assay (ELISA) for detection of serum AEAbs may document elastin breakdown. Methods: An ELISA technique was used to evaluate levels of AEAbs in sera of 97 affected Belgian Draught horses that were clinically healthy except for possible skin lesions, associated with CPL in their distal limbs. The horses were divided into 5 groups according to the severity of these skin lesions: normal horses (Group 1, n = 36), horses with mild lesions (Group 2, n = 43), horses with moderate lesions (Group 3, n = 8), horses with severe lesions (Group 4, n = 10) and, as a control, healthy Warmblood horses, unaffected by the disease (Group 5, n = 83). Results: Horses with clinical signs of CPL had significantly higher AEAb levels compared to clinically normal Belgian Draught horses and to healthy Warmblood horses. These levels correlated with severity of lesions. Conclusions: CPL in draught horses is associated with an increase of serum AEAbs. Potential relevance: Evaluation of serum levels of AEAbs by ELISA might be a useful diagnostic aid for CPL. Pathological degradation of elastic fibres, resulting in deficient support of the distal lymphatics, is proposed as a contributing factor for CPL in Belgian Draught horses.
Chronic progressive lymphoedema (CPL) is a recently recognised condition in Shires, Clydesdales and Belgian Draught horses characterised by progressive swelling, hyperkeratosis and fibrosis of distal limbs (De Cock et al. 2003; Ferraro 2003). This chronic progressive disease starts at an early age, progresses throughout the horse’s life and often ends in severe disfigurement of the limbs (De Cock et al. 2003; Ferraro 2003). Clinical signs and pathological changes are strikingly similar to chronic lymphoedema or elephantiasis nostra verrucosa in man (Fields et al. 1976; Harwood and Mortimer 1995; Rockson 2001); therefore, the disease has been termed chronic progressive lymphoedema (De Cock et al. 2003). Currently, no successful treatment for lymphoedema in horses is known (Ferraro 2003), which reflects the situation in primary lymphoedema in man (Rockson 2001). Ideally, supportive treatment, such as external compression, massage for lymphatic drainage, appropriate exercise and meticulous skin care should be started at an early stage of the disease, when fibrosis has not yet developed (Sanders 1988; Rockson 2001). Lymphoscintigraphy is the primary technique used to diagnose lymphoedema in man (Partsch 1995; Rockson 2001) and has proved useful in draught horses with CPL, apart from high cost and the requirement for sedation (De Cock et al. 2006a). Early diagnosis and evaluation of progression of equine CPL remains a challenge for the practitioner in the field. Elastin is considered the most resistant protein of the body with a half-life of about 70 years (Frances and Robert 1984; Petersen et al. 2002). Under pathological circumstances elastin is increasingly degraded into peptides (elastin derived peptides: EPs), which are released into the circulation. The immune system initiates an immune response against the released peptides, generating anti-elastin antibodies (AEAbs). In human medicine, an enzyme-linked immunosorbent assay (ELISA) technique for AEAbs has been used extensively to detect and monitor progression of several diseases associated with elastin degradation in skin, lung and blood vessels (Fülöp et al. 1989; Nicoloff et al. 2000; Colburn et al. 2003).
*Author to whom correspondence should be addressed. [Paper received for publication 11.10.06; Accepted 30.01.07]
EVJ 06-141 Van Brantegem
09/08/07
4:53 pm
Page 3
L. Van Brantegem et al.
419
In the present study, an established ELISA test for detection of AEAbs in horses (Van Brantegem et al. 2007) was used to detect serum AEAb levels in Belgian Draught horses affected with CPL. Serum AEAb levels of affected horses were compared to values of AEAbs in clinically normal Belgian Draught horses. Mean AEAb levels in healthy Warmblood horses, of an unaffected breed, were used as reference values.
with moderate lesions or of horses with severe lesions (n = 10). The control Group 5 consisted of healthy Warmblood horses (n = 83). Post partum serum from a foal, collected prior to colostrum administration, served as a negative control. Positive control serum was collected from a pony with severe chronic obstructive pulmonary disease. All serum samples were frozen immediately after collection and stored at -20°C until analysed.
Materials and methods
Enzyme-linked immunosorbent assay
Animals and sera
An ELISA technique for detection of serum AEAbs is described by Van Brantegem et al. (2007). All serum samples were tested in triplicate. Where one result differed by >10% from the mean value of all 3 results, the test was repeated. Reproducibility of the test procedure was determined by intra- (CV1) and interassay (CV2) coefficients of variance (CV = s.d./mean) by measuring 6 replicates of the positive and negative controls in a single plate and subsequently in 11 separate assays. To correct for day-to-day variations results were expressed as corrected optical densities (COD) obtained as follows:
Ninety-seven privately owned Belgian Draught horses were selected, all in good health and body condition except for the lesions on the distal limbs associated with CPL. These lesions were evaluated clinically by palpation of the limbs: mild lesions consisted of a slight swelling of the limb and 1 or 2 small skin folds in the palmar/plantar pastern region; moderate lesions of a distinct swelling of the limb, several thick skin folds and, occasionally, small firm nodules in the palmar/plantar pastern region; and severe lesions of a severe swelling of the limb, several thick skin folds and firm nodules in the palmar/plantar pastern region (Fig 1). When measuring AEAbs, all 4 limbs were considered and not each limb individually. Therefore, horses were divided into 4 clinical groups based on the evaluation of all 4 limbs: Group 1, clinically normal horses (n = 36); Group 2, 1–4 limbs with mild lesions (n = 43); Group 3, ≤2 limbs with moderate and no severe lesions (n = 8); and Group 4, 3 or 4 limbs
COD =
ODsample - ODnegative control ODpositive control - ODnegative control
Statistical analysis Analysis of variance was used first to compare the log-transformed COD between the 2 breeds at a global significance level of 5%. The log-transformed COD values were used because they were distributed normally (according to Shapiro-Wilks test). Multiple comparisons between the clinically normal horses and the 3 other clinical categories of the Belgian Draught horse were based on Dunnett’s adjustment technique. Results
Fig 1: Severely affected limb of a draught horse with CPL. Note the diffuse swelling of the limb, several thick skin folds in the palmar region and numerous small ulcers.
The control population consisted of 83 horses age 4 months to 22 years, and the Belgian Draught horse population of 97 horses, including 50 mares and 47 stallions, with the same age distribution as the control population. According to the clinical evaluation, these draught horses were categorised into 4 different groups: Group 1, 36 clinically normal horses, Group 2, 43 with mild lesions, Group 3, 8 horses with moderate lesions and Group 4, 10 with severe lesions. The raw OD values of the positive and negative control sera were 0.991 (range 0.760–1.235) and 0.100 (range 0.093–0.109), respectively. The CV1 was 4.91% and 2.42% for the positive and negative controls, respectively, while the CV2 was 9.73% and 4.35%. The mean ± s.e. COD of the AEAb levels in the different groups are shown in Table 1. The levels were low in the
TABLE 1: Mean ± s.e. corrected optical density (COD) and median of anti-elastin antibodies (AEAbs) in Belgian Draught horses with chronic progressive lymphoedema
Number of horses Mean COD ± s.e. Median
Group 1 No lesions
Group 2 Mild lesions
Group 3 Moderate lesions
Group 4 Severe lesions
36 0.43 ± 0.05 0.38
43 0.72 ± 0.07* 0.56
8 0.87 ± 0.22* 0.70
10 0.99 ± 0.21* 0.76
*Statistically significant (P<0.05), compared to Group 1.
Group 5 Control population (Warmblood horses) 83 0.36 ± 0.02 0.37
EVJ 06-141 Van Brantegem
09/08/07
4:53 pm
Page 4
420
Warmblood horses (Group 5), as well as in the clinically normal draught horses (Group 1), but increased gradually in the affected draught horses (Group 2 < Group 3 < Group 4), corresponding with severity of clinically observed lesions. There was no statistically significant difference (P>0.05) between AEAb levels of clinically normal Belgian Draught horses (Group 1) and the control population of Warmblood horses (Group 5). Multiple comparisons between clinically normal draught horses (Group 1) and the 3 categories of affected draught horses (Groups 2, 3 and 4), showed statistically significant differences (P = 0.0004, P = 0.0075 and P = 0.0008, respectively). Discussion Chronic progressive lymphoedema is a devastating disorder that affects draught breeds including Shires, Clydesdales and Belgian Draught horses (De Cock et al. 2003). Early diagnosis of the disease would significantly increase the effectiveness of clinical management of affected horses. This study documents the use of an ELISA test for evaluation of increased levels of circulating serum AEAbs in Belgian Draught horses with CPL and the correlation of severity of clinical lesions with the levels of the serum AEAbs measured. Under pathological circumstances, elastin is increasingly degraded into EPs which are released into the circulation. The immune system initiates an immune response against the released EPs, generating AEAbs. In human medicine, the ELISA technique to detect circulating AEAbs has been optimised and used in the diagnosis and prognosis of pathological conditions affecting elastin-rich tissue such as skin, lung and blood vessels (Fülöp et al. 1989; Nicoloff et al. 2000; Colburn et al. 2003). A modified ELISA technique for detection of circulating AEAbs in equine sera has been established (Van Brantegem et al. 2007). This study reveals that serum levels of AEAbs in healthy horses are not breed dependant. Clinically normal Belgian Draught horses had serum AEAb levels comparable to serum levels in healthy warmblood horses. This indicates that healthy horses of different breeds appear to have the same physiological elastin turnover. In addition, the study reveals that the ELISA technique used to identify serum levels of AEAbs is a valuable tool for the diagnosis of CPL. A statistically significant difference in mean COD values was found between normal and affected draught horses. The mean COD (0.86) of all clinically affected Belgian Draught horses (Groups 2, 3 and 4) were twice the mean COD of clinically normal Belgian Draught horses (0.43) (Group 1) and healthy warmblood horses (0.36) (Group 5). Hence, monitoring serum levels of AEAbs may be used to assess progression of CPL in a particular horse. Therefore, evaluation of AEAbs and clinical progression during a longterm follow-up study is warranted. This technique has several advantages compared to lymphoscintigraphy. Firstly, there is no need for transportation to an equipped veterinary practice or sedation of the horse. Secondly, lymphoscintigraphy is expensive and time-consuming as only one horse at a time can be examined and this may take about 2.5 h (De Cock et al. 2006a). In comparison, with the ELISA technique, the veterinarian needs only to collect blood samples in situ. Increased serum levels of AEAbs may, however, also be due to degenerative changes of other origin, in particular in elastinrich tissue such as the skin, lungs and blood vessels (Fülöp et al. 1989; Nicoloff et al. 2000; Colburn et al. 2003); and, there is no
Antibodies to elastin peptides of Belgian Draught horses with CPL
information available about changes of AEAb levels in serum of horses with degenerative changes in these tissues. To exclude false positives, it is of importance that a sufficient veterinary examination is carried out before the horse is tested. As all animals in this study were in good health and body condition, changes in AEAbs were considered due to CPL and not to another pathological condition. Lymphoedema is characterised by regional accumulation of excessive amounts of interstitial protein-rich fluid as a result of an imbalance between the demand for and capacity of the lymphatic circulation (Rockson 2001). Elastic fibres are critical to the mechanism of dermal lymphatic drainage: not only as a ‘guide rail’, presenting a low resistance pathway for the transinterstitial transport of fluid (Skobe and Detmar 2000) but also as an ‘elastic sleeve’ around the dermal lymphatics (Ryan and de Becker 1995). The whole elastic network is believed to stretch open the lymphatic channels when interstitial fluid accumulates, constituting an elastic device aiding propulsion of the lymph. As nearly 50% of the collecting lymphatics in horses consist of elastin (Harland et al. 2004), degradation could be a very important contributing factor for lymphatic failure in horses, resulting in lymphoedema. In man, chronic lymphoedema is known to be accompanied by disruption of elastic fibres (Gerli et al. 1990; Daróczy 1995; Ryan and de Becker 1995; Rockson 2005). Fragmentation and disorganisation of cutaneous and subcutaneous elastic fibres, including the fibres around lymphatics, has also been documented in draught horses affected with CPL (De Cock et al. 2003). Moreover, measurement of elastin levels by identifying tissue desmosine, indicates the influence of an altered elastin metabolism in horses with CPL (De Cock et al. 2006b). The presence of increased serum AEAbs in affected horses confirms the presence of pathological elastin degradation. In conclusion, AEAbs in sera of draught horses affected with CPL reflect the severity of the skin lesions and, therefore, are a useful aid in the diagnosis of CPL. Further research should be directed towards evaluation of this technique in subclinically affected animals as an early diagnostic tool. As in man, this would contribute significantly to better clinical management of the disease, as conservative treatment can begin at an earlier stage (Sanders 1988). Acknowledgements The authors thankfully acknowledge the excellent technical assistance of Karolien Hermy, Marleen Foubert, Nathalie van Rysselberghe and Sarah Loomans; all veterinarians who collected blood samples; and all horse owners for their cooperation. We are grateful to the Center for Equine Health, School of Veterinary Medicine, University of California, Davis, for providing us with financial support. References Colburn, K.K., Langga-Shariffi, E., Kelly, G.T., Malto, M.C., Sandberg, L.B., Baydanoff, S. and Green, L.M. (2003) Abnormalities of serum antielastin antibodies in connective tissue diseases. J. Investig. Med. 51, 104-109. Daróczy, J. (1995) Pathology of lymphedema. Clin. Dermatol. 13, 433-444. De Cock, H.E.V., Affolter, V.K., Wisner, E.R., Ferraro, G.L. and MacLachlan, N.J. (2003) Progressive swelling, hyperkeratosis, and fibrosis of distal limbs in Clydesdales, Shires, and Belgian Draft Horses, suggestive of primary lymphedema. Lymphat. Res. Biol. 1, 191-199. De Cock, H.E.V., Affolter, V.K., Wisner, E.R., Larson, R.F. and Ferraro, G.L. (2006a) Lymphoscintigraphy of draught horses with chronic progressive lymphoedema. Equine vet. J. 38, 148-151.
EVJ 06-141 Van Brantegem
09/08/07
4:53 pm
Page 5
L. Van Brantegem et al.
De Cock, H.E.V., Affolter, V.K., Farver, T.B., Van Brantegem, L., Scheuch, B. and Ferraro, G.L. (2006b) Measurements of skin desmosine as an indicator of altered cutaneous elastin in draft horses with chronic progressive lymphedema. Lymphat. Res. Biol. 4, 67-72. Ferraro, G. (2003) Chronic progressive lymphedema in draft horses. J. equine vet. Sci. 23, 189-190. Fields, G.S., Howard, I. and Stuart, M. (1976) Elephantiasis nostras. A case report and review of the subject. J. Am. Podiatry Ass. 66, 28-31. Frances, C. and Robert, L. (1984) Elastin and elastic fibers in normal and pathological skin. Int. J. Dermatol. 3, 166-179. FĂźlĂśp, T.Jr., Jacob, M.P. and Robert, L. (1989) Determination of anti-elastin peptide antibodies in normal and arteriosclerotic human sera by ELISA. J. Clin. Lab. Immunol. 30, 69-74. Gerli, R., Ibba, L. and Fruschelli, C. (1990) A fibrillar elastic apparatus around human lymph capillaries. Anat. Embryol. 181, 281-286. Harland, M.M., Fedele, C. and Berens v. Rautenfeld, D. (2004) The presence of myofibroblasts, smooth muscle cells and elastic fibers in the lymphatic collectors of horses. Lymphology 37, 190-198. Harwood, C.A. and Mortimer, P.S. (1995) Causes and clinical manifestations of lymphatic failure. Clin. Dermatol. 13, 459-471. Nicoloff, G., Baydanoff, S., Stanimorova, N., Petrova, C. and Cristova, P. (2000) Relationship between elastin-derived peptides and the development of microvascular complications: a longitudinal study in children with Type 1 (insulin-dependent) diabetes mellitus. Gen. Pharmacol. 35, 59-64. Partsch, H. (1995) Assessment of abnormal lymph drainage for the diagnosis of lymphedema by isotopic lymphangiography and by indirect lymphography. Clin. Dermatol. 13, 445-450.
421
Petersen, E., Wagberg, F. and Angquist, K.A. (2002) Serum concentrations of elastinderived peptides in patients with specific manifestations of atherosclerotic disease. Eur. J. Vasc. Endovasc. Surg. 24, 440-444. Rockson, S.G. (2001) Lymphedema. Am. J. Med. 110, 288-295. Rockson, S.G. (2005) Lymphedema therapy in the vascular anomaly patient: therapeutics for the forgotten circulation. Lymphat. Res. Biol. 3, 253-255. Ryan, T.J. and de Berker, D. (1995) The interstitium, the connective tissue environment of the lymphatic, and angiogenesis in human skin. Clin. Dermatol. 13, 451-458. Sanders, L.J., Slomsky, J.M. and Burger-Caplan, C. (1988) Elephantiasis nostras: an eight-year observation of progressive nonfilarial elephantiasis of the lower extremity. Cutis 42, 406-411. Skobe, M. and Detmar, M. (2000) Structure, function and molecular control of the skin lymphatic system. J. Investig. Dermatol. 5, 14-19. Van Brantegem, L., De Cock, H.E.V., Affolter, V.K., Duchateau, L., Govaere, J., Ferraro, G.L. and Ducatelle, R. (2007) Antibodies to elastin peptides in sera of Warmblood horses at different ages. Equine vet. J. 39, 414-416.
Author contributions The initiation, conception and planning of this study were by L.V.B., H.D.C., V.K.A. and R.D. Its execution was by L.V.B. and H.D.C., pathology by L.V.B., V.K.A., M.K.H. and G.L.F., and statistics by H.D.C. and L.D. All authors contributed to the writing.