Antibodies to elastin peptides in sera of Belgian Draught horses with chronic progressive lymphoedema

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.     

Antibodies to elastin peptides in sera of Warmblood horses at different ages

REASONS FOR PERFORMING STUDY: Early diagnosis and monitoring progression of chronic diseases in elastin-rich tissues, such as chronic progressive lymphoedema in draught horses and chronic obstructive pulmonary disease (COPD) is still a real challenge in the horse. Use of an enzyme-linked immunosorbent assay (ELISA) to detect anti-elastin antibody (AEAb) levels might be useful to assess the status of such diseases. Baseline levels, representing physiological breakdown of elastin in normal horses, are not available at present. HYPOTHESIS: Levels of AEAb in healthy horses are generally low and follow the same age-related pattern as found in man. Therefore, elevation of AEAb levels in serum can be used to evaluate pathological elastin breakdown in elastin-rich tissues. METHODS: Sera of 84 clinically healthy Warmblood horses were evaluated for the presence of AEAbs by means of a modified version of an ELISA technique used in man. The horses were divided in 5 age groups: A) < 4 months; B) 4-23 months; C) 2-3 years; D) 4-10 years; and E) > 11 years. RESULTS: Antibodies to elastin were found in all equine serum samples tested. Their levels were lowest in Group A, low in Groups B and E and highest in animals age 2-10 years. CONCLUSIONS: Measuring AEAbs in serum of horses by an ELISA technique proved to be possible and levels were stable during well-defined life stages. POTENTIAL RELEVANCE: Changes in AEAb levels are expected to be useful for early diagnosis and for monitoring progression of diseases that affect elastin-rich tissues, such as chronic progressive lymphoedema and COPD.     

Lymphoscintigraphy of draught horses with chronic progressive lymphoedema

REASONS FOR PERFORMING STUDY: Early diagnosis of chronic progressive lymphoedema (CPL) may result in more effective interventions and provide a basis for further investigation of whether early diagnosis could be used as a means of eliminating potential genetic influences by cessation of breeding from affected individuals. HYPOTHESIS: Lymphoscintigraphy may be useful in draught horses to differentiate early lesions of CPL from other conditions in the pastern region. METHODS: Forelimbs of 2 normal and 5 CPL-affected draught horses were evaluated with lymphoscintigraphy. RESULTS: Lymphoscintigraphy showed clearly the presence of interstitial fluid stasis and delayed lymphatic drainage in the affected extremities of diseased animals in contrast to normal animals of these breeds. The rate of decreased clearance of a particulate radiopharmaceutical from the tissues was related positively to the severity of clinical signs. CONCLUSIONS AND POTENTIAL RELEVANCE: Our findings support the hypothesis that lymph stasis is probably responsible for the progressive swelling and concurrent skin lesions observed in association with CPL in draught horses. Lymphoscintigraphy should also prove useful in diagnosis of CPL in draught horses, even in the mild stages of the disease; such early diagnosis may result in more effective intervention.     

Combined decongestive therapy including equine manual lymph drainage to assist management of chronic progressive lymphoedema in draught horses

Equine chronic progressive lymphoedema (CPL) is a disabling disorder of draught horse breeds. Combined decongestive therapy (CDT) is the treatment of choice for lymphoedema in man and has been adapted for use in horses. Equine CDT, which includes manual lymph drainage (MLD) and subsequent bandaging with short stretch bandages, was expected to improve the signs of CPL in draught horses because CPL resembles primary lymphoedema in man. Five affected horses ‐ Gypsy cob (n = 1), Clydesdale (n = 1), Shires (n = 3) ‐ were included. Lesions were documented pre‐ and post treatment. Percentage volume loss of the distal legs was calculated using the disc model. Initial plans for daily CDT had to be adapted; intermittent treatment of Chorioptes infections required alternating between CDT and MLD in 4/5 horses. Concurrent pyoderma (1/5 horses) was treated throughout the study. Development of unrelated lameness (hoof abscess) allowed limited CDT treatment only in one horse. Marked softening of previously firm tissue indicated the change from ‘brawny’ to pitting oedema in 2/5 horses. Fibrotic nodules and folds in the pasterns became markedly softened and smaller in 2/5 horses. Skin surface notably improved in all horses: hyperkeratosis decreased, erosions and ulcerations healed completely and crusts disappeared. After 2 weeks, a mean volume reduction of 11.25% was seen, ranging from 4.75–21.74% and quality of movement improved. This pilot study documents evidence that CDT assists management of CPL. Current CPL management is limited to palliative treatments of secondary infections. Whilst not a permanent treatment, CDT offers a promising tool to manage horses with CPL, improving their quality of life and potential usefulness. More extensive and prolonged studies with a larger number of horses are warranted to evaluate the full potential of CDT.     

Evaluation of FOXC2 as a candidate gene for chronic progressive lymphedema in draft horses

Chronic progressive lymphedema (CPL) is a debilitating condition identified in Clydesdales, Shires and Belgian draft horses and results in progressive swelling of the lower legs associated with the development of thick skin folds, ulcerations, fibrosis and marked hyperkeratosis. The result is severe discomfort and recurrent secondary infection, often requiring euthanasia. Due to the delayed onset, many horses are bred prior to diagnosis. CPL has only been documented in three related draft horse breeds, suggesting a genetic cause. Determining the molecular basis would enable owners to test horses prior to breeding and facilitate the elimination of CPL. Mutations in the FOXC2 gene cause a comparable condition in humans, lymphedema-distichiasis. This gene was sequenced in affected and unaffected draft horses and a control horse. Four single nucleotide polymorphisms (SNPs) were identified in unaffected draft horses and the control horse, indicating that they were not associated with CPL. A fifth SNP was seen in a single affected draft horse and the control horse. Since it was not seen in all affected draft horses, this SNP is not associated with the CPL phenotype.     

Effect of potential therapeutic agents in reducing oxidative stress in pulmonary tissues of recurrent airway obstruction‐affected and clinically healthy horses

Reasons for performing study: To determine and compare the reactive oxygen and nitrogen species (ROS and RNS) in pulmonary tissues of horses affected with recurrent airway obstruction (RAO) and clinically healthy horses, and to evaluate the effectiveness of potential therapeutic agents in reducing ROS and RNS in the tissues of these horses. Objectives: We hypothesised that RAO‐affected horses would have high levels of reactive species and that the test agents would reduce them. The objectives were as follows: 1) to determine the level of ROS and RNS in pulmonary tissues (bronchial and arterial rings) of RAO‐affected and clinically healthy horses; and 2) to determine the ability of pentoxifylline, pyrrolidine‐dithiocarbamate and a combined use of endothelin A and B receptor antagonists (BQ123 and BQ788, respectively) in reducing reactive species. Methods: Arterial and bronchial rings were collected from the diaphragmatic lung lobe of each horse immediately after euthanasia. The levels of ROS and RNS were measured in control tissues and those incubated with test agents, using an electron paramagnetic resonance instrument. Results: The levels of ROS and RNS were significantly greater in arterial and bronchial tissues of RAO‐affected than of clinically healthy horses. Pentoxifylline and endothelin antagonists reduced both ROS and RNS in tissues from RAO‐affected horses. Basal levels of reactive species in clinically healthy horses were not affected by these agents. No difference in the level of reactive species was observed between arterial and bronchial tissues. Conclusions: Horses affected by RAO had higher ROS and RNS than clinically healthy horses. Pentoxifylline and endothelin antagonists effectively reduced ROS and RNS in pulmonary tissues of RAO‐affected horses. Potential relevance: The study suggested a potential use for pentoxifylline and endothelin antagonists in treating RAO‐affected horses. As endothelin is involved in physiological functions, therapeutic use of its antagonists is cautioned.     

Validation of a Commercial Enzyme Immunoassay for Detection of Clostridium difficile Toxins in Feces of Horses with Acute Diarrhea

Background: Clostridium difficile infection (CDI) is a recognized cause of colitis in the horse. Identification of its toxins is important for management of individual cases and for prevention of transmission and zoonosis. In humans, CDI diagnosis is performed with enzyme immunoassays, none of which have been validated for horses. Hypothesis/Objectives: (1) Establish which test for CDI diagnosis was more frequently used by diagnostic laboratories, (2) determine the identified test's performance, sensitivity, and specificity, and (3) validate its use in diarrheic horses. Animals: Samples were obtained from 72 horses presented with acute diarrhea and hospitalized at the Ontario Veterinary College, University of Guelph. Methods: A survey was conducted to establish which of the tests for CDI diagnosis in horses is most commonly used throughout North America. A questionnaire was sent to all laboratories registered in the Veterinary Infection Control Society and the American Association of Veterinary Laboratory Diagnosticians. The performance of the test was evaluated by comparison to a cell cytotoxicity assay (CTA), the accepted Gold Standard for C. difficile toxin detection. Results: The Techlab C. difficile Tox A/B II ELISA was the most frequently used test. Compared with the CTA, no significant difference was observed, and a good level of agreement (93%) was obtained. The diagnostic performance of the ELISA test was adequate (84% sensitivity and 96% specificity). Conclusions and Clinical Importance: Results demonstrate that the Techlab C. difficile Tox A/B II ELISA is a reliable, adequate, and practical tool for identification of C. difficile toxins in horse feces.     

Serum protein concentrations from clinically healthy horses determined by agarose gel electrophoresis

Background: Serum protein electrophoresis is a useful screening test in equine laboratory medicine. The method can provide valuable information about changes in the concentrations of albumin and α‐, β‐, and γ‐globulins and thereby help characterize dysproteinemias in equine patients. Reference values for horses using agarose gel as a support medium have not been reported. Objectives: The purpose of this study was to establish reference intervals for serum protein concentrations in adult horses using agarose gel electrophoresis and to assess differences between warm‐blooded and heavy draught horses. In addition, the precision of electrophoresis for determining fraction percentages and the detection limit were determined. Methods: Blood samples were obtained from 126 clinically healthy horses, including 105 Thoroughbreds and 21 heavy draught horses of both sexes and ranging from 2 to 20 years of age. The total protein concentration was determined by an automated biuret method. Serum protein electrophoresis was performed using a semi‐automated agarose gel electrophoresis system. Coefficients of variation (CVs) were calculated for within‐run and within‐assay precision. Data from warm‐blooded and draught horses were compared using the Mann–Whitney U test. Results: Within‐run and within‐assay CVs were <5% for all protein fractions. No significant difference was found between warm‐blooded and heavy draught horses and so combined reference intervals (2.5–97.5%) were calculated for total protein (51.0–72.0 g/L), albumin (29.6–38.5 g/L), α₁‐globulin (1.9–3.1 g/L), α₂‐globulin (5.3–8.7 g/L), β₁‐globulin (2.8–7.3g/L), β₂‐globulin (2.2–6.0 g/L), and γ‐globulin (5.8–12.7 g/L) concentrations, and albumin/globulin ratio (0.93–1.65). Conclusion: Using agarose gel as the supporting matrix for serum protein electrophoresis in horses resulted in excellent resolution and accurate results that facilitated standardization into 6 protein fractions.     

IgE-bearing cells in bronchoalveolar lavage fluid and allergen-specific IgE levels in sera from RAO-affected horses

Recurrent airway obstruction (RAO) is a common condition in stabled horses characterized by small airway inflammation, airway neutrophilia and obstruction following exposure of susceptible horses to mouldy hay and straw and is thus regarded as a hypersensitivity reaction to mould spores. However, the role of immunoglobulin E antibodies (IgE) in the pathogenesis of RAO is unclear. We hypothesized that the number of cells with receptor-bound IgE in bronchoalveolar lavage fluid (BALF) and IgE levels in serum would be higher in RAO-affected than in healthy horses living in the same environment. Therefore, IgE-positive (+) cells were identified by immunocytochemistry on cytospins from BALF and counted. IgE levels against the mould extracts Aspergillus fumigatus (Asp. f.) and Alternaria alternata (Alt. a.) and the recombinant mould allergen Aspergillus fumigatus 8 (rAsp f 8) were measured by enzyme-linked immunosorbent assay (ELISA) in the sera of seven RAO-affected and 22 clinically healthy mature horses housed in the same conventional stable environment. After correcting for the number of neutrophils, there were no significant differences in IgE+ cells on cytospins from BALF between both groups of horses (5% versus 7%, P > 0.1). Serum IgE levels against the mould extracts were significantly higher in RAO-affected than in clinically healthy horses [median = 119 versus 66 relative ELISA units (REU), P < 0.05]. Furthermore, significantly more RAO-affected than healthy horses had detectable serum IgE against the recombinant allergen rAsp f 8 (4/7 and 3/22, respectively, P < 0.05). Age had no significant effect on BALF cell ratios or on specific serum IgE levels. These results show that high IgE levels against mould antigens are associated with RAO under controlled environmental conditions but ranges of mould-specific serum IgE levels overlapped too much between diseased and clinically healthy animals to be of any diagnostic value. Further studies are needed to assess whether IgE-mediated reactions contribute to the pathogenesis of RAO.     

Morphological analysis and effect of selection for conformation in the Noriker draught horse population

To evaluate the breeding program in the Austrian Noriker draught horse population, which is mostly based on conformation, 31 body measurements from 497 horses in seven breeding areas of Austria were recorded. In addition, the data of 2376 horses (the current breeding population) from the studbook, containing 10 scored conformation traits and 4 body measurements per individual, were analysed. We assumed breeding regions, coat colour strains and breeding classes assigned according to conformation evaluation to be the main factors in phenotypic diversity of this Austrian draught horse breed. Significant differences and distances were found for all of these factors. Whereas differences between breeding areas mostly are due to housing conditions and feeding strategies, the breeding classes provide a very distinct picture of the current trends in Noriker breeding. Breeding organisations favour a long and high Noriker draught horse, a tendency that is the same for all departments. Heritability estimates for morphological traits vary from 0 to 0.67, and from 0.08 to 0.37 for linear type traits.     

Chronic obstructive pulmonary disease (COPD) in horses: aetiological studies: responses to intradermal and inhalation antigenic challenge.

Micropolyspora faeni and Aspergillus fumigatus were identified as common causes of respiratory hypersensitivity in horses affected with chronic obstructive pulmonary disease (COPD). Rye grass pollen and an Actinomycete evoked respiratory allergy in a few horses. Not infrequently, individual horses were found to have respiratory hypersensitivity to two or more antigens. The methods used to examine for allergy were intradermal testing and inhalation challenge with environmental antigens. An intradermal test using an M faeni extract was demonstrated to be suitable for diagnostic use in horses previously accurately diagnosed as suffering from COPD. In contrast, the A fumigatus antigen used proved unsatisfactory for such a purpose. Skin reaction to M faeni and A fumigatus extracts by horses affected with COPD indicated that the hypersensitivity was a dual one--a weak response shortly after injection followed by an Arthus-like response 4 to 8 hours later. As a parameter for monitoring responses to inhalation challenge, maximum intrathoracic pressure change (max delta Ppl) proved satisfactory, whereas changes in partial pressure of arterial oxygen (PaO2) did not.     

Suspected clinical Lyme disease in horses: Serological and antigen testing differences between clinically ill and clinically normal horses from an endemic region

Equine Lyme disease is difficult to diagnose because of its nonspecific clinical signs and the high incidence of subclinical infection in endemic regions. In this study we compared serology, antigen presence, hematology, blood chemistries and clinical presentation of 22 horses from a highly endemic region that were clinically diagnosed with Lyme disease to that of 21 clinically normal horses from the same region. We found that horses clinically diagnosed with Lyme disease were more likely to have Borrelia burgdorferi spirochetal DNA in their blood and urine, have a higher percentage of positive immunoblots containing antibodies to certain B. burgdorferi proteins, and tend to have higher ELISA titers than healthy horses from the same region. These results may help to improve diagnostic testing for equine Lyme disease.     

The use of chosen serological diagnostic methods in Lyme disease in horses. Part II. Western blot

In this investigation the Western blot test was treated as a method verifying results of the IFA, commercial ELISA and standardized ELISA tests (described in Part I). The verifying investigations were performed on 82 serum samples, which in the commercial ELISA were positive in 36 cases, dubious in 31 cases and negative in 15 cases as well as on 5 serum samples obtained from horses infected with Leptospira spp., which in the ELISA commercial were dubious (total of 87 sera samples). The antigens, against which the immunological response in horses was directed, were also established. The Milenia--Blot--Borrelia IgG test (MIDBO IgG-Kit 30 Tests: DPC Bierman GmbH) was used in the investigation. In view of species differences, rabbit anti-horse IgG (whole molecule) alkaline phosphatase conjugate, no A6063 SIGMA-ALDRICH was used interchangeably. Also the control sera were substituted with the horse control sera. It was demonstrated that the Western blot test is the most reliable in the serological diagnosis of B. burgdorferi infection in horses. The commercial ELISA and standardized ELISA tests represent a lower diagnostic value than the Western blot test, although similar to each other, while the value of the IFA is minimal. In the Western blot test antigens were established against which the immunological response in horses in mostly directed. In the sera evaluated in this test as positive the presence of antibodies, mainly against antigens with the following molecular weights: 41 kDa, 62/60 kDa, 93 kDa, 72 kDa, 34 kDa (OspB), 66 kDa was noted. At the same time, antibodies contained in the sera accepted as negative, in 55.5% cases also reacted with the antigen of 41 kDa. It points to its minimal specificity. On the basis of the results obtained it is recommended that serological examination of horses should be with the ELISA and that positive or dubious results should be verified with the Western blot test.     

Clinical and pathological findings in a HERDA-affected foal for 1.5 years of life

A Quarter horse filly bred from two horses affected with HERDA (hereditary equine regional dermal asthenia) was observed clinically and its skin histologically for the 1.5 years of its life. Severe signs of the disease did not manifest until 1.5 years of age, and were not temporally related to saddling. Histological comparison to an age-, breed- and sex-matched control did not show any consistent diagnostic features. Monitoring of the proband substantiated previous reports of (i) the autosomal recessive nature of the disease, (ii) mares affected with HERDA being able to foal without damage to the skin or reproductive tract, (iii) HERDA foals appearing phenotypically normal throughout the first year of life, and (iv) demonstrated that histological interpretation of skin specimens from grossly normal skin may be insufficient to differentiate HERDA-affected horses from controls.     

Effect of flunixin meglumine on selected physiologic and performance parameters of athletically conditioned thoroughbred horses subjected to an incremental exercise stress test

Twelve clinically sound, healthy, athletically conditioned Thoroughbred horses were subjected to an incremental exercise stress test to determine the effects and period of detection of a single dose of flunixin meglumine (1.1 mg/kg by intravenous injection) in serum and urine by ELISA. Flunixin concentrations, performance, and hematologic and clinical chemical parameters were measured. All horses were rotated through four treatment groups of a Latin-square design providing for each horse to serve as its own control. Flunixin meglumine reduced prostaglandin F(1alpha) and thromboxane concentrations that had been increased by intense exercise. Performance parameters did not improve and prostaglandin concentrations did not significantly correlate with total run time. Exercise did not change the flunixin elimination profile in either serum or urine, and concentrations were found to be below the detection limit of the ELISA test within 36 hours in serum and 120 hours in urine.     

A histamine release assay to identify sensitization to Culicoides allergens in horses with skin hypersensitivity

Skin hypersensitivity is an allergic disease induced in horses by allergens of Culicoides midges. The condition is typically diagnosed by clinical signs and in some horses in combination with allergy testing such as intradermal skin testing or serological allergen-specific IgE determination. Here, we describe an alternative method for allergy testing: a histamine release assay (HRA) that combines the functional aspects of skin testing with the convenience of submitting a blood sample. The assay is based on the principle that crosslinking of allergen-specific IgE bound via high-affinity IgE receptors to the surfaces of mast cells and basophils induces the release of inflammatory mediators. One of these mediators is histamine. The histamine was then detected by a colorimetric enzyme-linked immunosorbent assay. The histamine assay was used to test 33 horses with skin hypersensitivity and 20 clinically healthy control animals for histamine release from their peripheral blood basophils after stimulation with Culicoides allergen extract or monoclonal anti-IgE antibody. An increased histamine release was observed in the horses with skin hypersensitivity compared to the control group after allergen-specific stimulation with Culicoides extract (p=0.023). In contrast, stimulation with anti-IgE induced similar amounts of released histamine in both groups (p=0.46). For further evaluation of the HRA, we prepared a receiver operating-characteristic (ROC) curve and performed a likelihood-ratio analysis for assay interpretation. Our results suggested that the assay is a valuable diagnostic tool to identify sensitization to Culicoides allergens in horses. Because some of the clinically healthy horses also showed sensitization to Culicoides extract, the assay cannot be used to distinguish allergic from non-allergic animals. The observation that sensitization is sometimes detectable in non-affected animals suggested that clinically healthy horses use immune mechanisms to control the reaction to Culicoides allergens that are different or absent in allergic horses.     

Genetic parameters for chronic progressive lymphedema in Belgian Draught Horses

Genetic parameters for chronic progressive lymphedema (CPL)‐associated traits in Belgian Draught Horses were estimated, using a multitrait animal model. Clinical scores of CPL in the four limbs/horse (CPL_clin), skinfold thickness and hair samples (hair diameter) were studied. Due to CPL_clin uncertainty in younger horses (progressive CPL character), a restricted data set (D_3+) was formed, excluding records from horses under 3 years from the complete data set (D_full). Age, gender, coat colour and limb hair pigmentation were included as fixed, permanent environment and date of recording as random effects. Higher CPL_clin certainty (D_3+) increased heritability coefficients of, and genetic correlations between traits, with CPL_clin heritabilities (SE) for the respective data sets: 0.11 (0.06) and 0.26 (0.05). A large proportion of the CPL_clin variance was attributed to the permanent environmental effect in D_full, but less in D_3+. Date of recording explained a proportion of variance from 0.09 ± 0.03 to 0.61 ± 0.08. Additive genetic correlations between CPL_clin and both skinfold thickness and hair diameter showed the latter two traits cannot be used as a direct diagnostic aid for CPL. Due to the relatively low heritability of CPL_clin, selection should focus on estimated breeding values (from repeated clinical examinations) to reduce CPL occurrence in the Belgian Draught Horse.     

Equine dysautonomia (grass sickness) is associated with altered plasma amino acid levels and depletion of plasma sulphur amino acids

To determine whether equine dysautonomia (ED) is associated with alterations in plasma amino acid metabolism, plasma amino acid profiles were determined for horses with acute (n = 10), subacute (n = 6) and chronic (n = 7) ED and for healthy cograzing horses (n = 6) and control horses (n = 10). Horses with acute ED had perturbations in plasma amino acid profiles resembling those of severe protein malnutrition. In addition, horses with ED and cograzing healthy horses had depletion of the plasma sulphur amino acids cyst(e)ine and methionine. As similar plasma amino acid perturbations occur in subacute/chronic cyanide toxicity, the role of cyanogenic glycosides in the aetiology of ED warrants further study. Unfortunately, amino acid analysis cannot be used as a definitive premortem diagnostic test for ED, since there was overlap in the individual amino acid levels of control, cograzing and ED horses.     

An enzyme-linked immunosorbent assay for the detection of vesicular stomatitis virus antibodies

A liquid-phase enzyme-linked immunosorbent assay (ELISA) was developed for the detection of vesicular stomatitis virus (VSV) types New Jersey (VSV-NJ) and Indiana subtype Indiana I (VSV-IND₁) antibodies in the sera of naturally and experimentally infected cattle, horses and swine. Four different VSV preparations were compared for use as antigen in the ELISA: virus used in neutralization tests, complement-fixation antigen, immunodiffusion ager gel antigen and viral glycoprotein. Comparative antibody titration results of virus neutralization (VN) and ELISA showed no statistically significant difference between serum titers obtained with the four antigens to VSV-NJ (P=0.21) and VSV-IND₁ (P=0.14). The viral glycoprotein antigen was incorporated in the ELISA system because it is non-infectious and induces neutralizing antibodies. The reliability and variability of the ELISA was determined by testing 516 bovine, equine and swine sera which originated from areas free of vesicular stomatitis, and by testing 186 sera from areas where outbreaks occur. ELISA and VN results were correlated (P < 0.001 for both serotypes), and no statiscafically difference was found between replications of the tests. The ELISA allows the testing of a larger number of sera in a shorter time than is possible with the VN test and it can be used in diagnostic laboratories in VSV-free areas for the support of epidemiological surveillance programs.