FC‐14 Clonality studies of feline cutaneous lymphocytosis

A previous study described cutaneous lymphocytosis (CL) in 23 cats. The process resembles cutaneous pseudolymphoma in humans, a heterogeneous group of benign reactive proliferations of well‐differentiated lymphocytes in the skin of humans. Morphological and immunophenotypic characteristics do not offer reliable criteria to accurately predict the clinical outcome of feline CL or pseudolymphoma in humans. Presence of clonal cell populations is more consistent with a neoplastic process. In a previous study, feline CL lesions (20 cats) were evaluated for clonality using PCR, and only two cats had monoclonal T‐cell populations. Because false‐negative results may occur, the purpose of this study was to repeat the PCR using a revised primer set based on analysis of additional feline T‐cell receptor γ (TCRγ) sequences. DNA was isolated from 29 skin lesions and six internal organs of 20 cats. DNA integrity was assessed by glyceraldehyde‐3‐phosphate dehydrogenase PCR. Polymerase chain reaction clonality was performed using the revised primer set specific for feline TCRγ, and duplicate samples were evaluated. The PCR products were assessed by heteroduplex analysis. Clonal rearrangement of TCRγ was detected in 14 cats (24 of 35 tissues: 21 of 29 skin lesions and three of six internal organs); eight of these cats are still alive and six were euthanized. Monoclonal populations were seen in three of five cats that had involvement of internal organs. These findings indicate that feline CL is best considered as a slowly progressive process which may be reactive, but often evolves into a low‐grade indolent lymphoma. Funding: George H. Muller Fund for Research in Dermatology.     

Histopathologic features, immunophenotyping, clonality, and eubacterial fluorescence in situ hybridization in cats with lymphocytic cholangitis/cholangiohepatitis

Feline lymphocytic cholangitis is a poorly characterized disease complex with respect to histologic lesions, immunophenotype, and etiopathogenesis. Seventy-eight cases of feline lymphocytic cholangitis (n = 51) and feline hepatic lymphoma (n = 27) were reviewed using standardized histopathology, immunophenotyping (B cell and T cell), polymerase chain reaction for T-cell receptor (TCR) gene rearrangement, and fluorescence in situ hybridization (FISH) for eubacteria. Five histopathologic features in cases of lymphocytic cholangitis assisted in its differentiation from hepatic lymphoma: bile duct targeting (n = 32, 62.7%), ductopenia (n = 9, 17.6%), peribiliary fibrosis (n = 37, 72.5%), portal B-cell aggregates (n = 36, 70.6%), and portal lipogranulomas (n = 38, 74.5%). The majority of lymphocytic cholangitis cases (n = 35, 68.6%) were T cell predominant; 15 (29.4%) had an equal mix of B cells and T cells, and 1 (1.9%) had a B cell-predominant infiltrate; 66.6% of hepatic lymphoma cases were T-cell lymphomas. TCR clonality results were unexpected, with 17.1% of cases of lymphocytic cholangitis having clonal or oligoclonal populations and with T-cell lymphomas having variable TCR clonality (63.6% clonal or oligoclonal, 36.3% polyclonal). The majority of lymphocytic cholangitis (n = 32 of 36, 88.8%) and all hepatic lymphoma cases had no detectable eubacteria using FISH. As demonstrated here, bile duct targeting, ductopenia, peribiliary fibrosis, portal B-cell aggregates, and portal lipogranulomas are lymphocytic cholangitis features that, along with polyclonal TCR (83%), help differentiate it from hepatic lymphoma. No strong evidence was found implicating in situ bacterial colonization as an etiopathogenesis of lymphocytic cholangitis.     

Comparison between immunohistochemistry and genetic clonality analysis for cellular lineage determination in feline lymphomas

Clinical application of genetic clonality analysis by PCR has been available in feline lymphoma. However, it is in under dispute whether the analysis can be applied for cellular lineage determination. To evaluate utility of the analysis for lineage determination, the results between immunohistochemistry and the genetic analysis were compared. Based on the result with immunohistochemistry, sensitivity of the primer for IgH was 89% and specificity was 75%. Sensitivity of the primer for TCRγ was 25% and specificity was 100%. Cross-lineage IgH rearrangement was found in 25% of T-cell lymphomas. The present study suggests that the genetic analysis had better be applied as not a definite but an adjunctive tool for lineage determination in feline lymphomas.     

Evaluation of a new in‐clinic test system to detect feline immunodeficiency virus and feline leukemia virus infection

Background: Many in‐house tests for the diagnosis of feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) infection are licensed for use in veterinary practice. A new test with unknown performance has recently appeared on the market. Objectives: The aims of this study were to define the efficacy of a new in‐clinic test system, the Anigen Rapid FIV Ab/FeLV Ag Test, and to compare it with the current leading in‐clinic test, the SNAP Kombi Plus FeLV Antigen/FIB Antibody Test. Methods: Three‐hundred serum samples from randomly selected healthy and diseased cats presented to the Clinic of Small Animal Medicine at Ludwig Maximilian University were tested using both the Anigen Rapid Test and the SNAP Kombi Plus Test. Diagnostic sensitivity, specificity, and positive and negative predictive values were calculated for both tests using Western blot as the gold standard for verification of FIV infection and PCR as the gold standard for FeLV infection. Results: The presence of antibodies against FIV was confirmed by Western blot in 9/300 samples (prevalence 3%). FeLV DNA was detected by PCR in 15/300 samples (prevalence 5%). For FIV infection the Anigen Rapid Test had a sensitivity of 88.9%, specificity of 99.7%, positive predictive value of 88.9%, and negative predictive value of 99.7%. For FeLV infection, the Anigen Rapid Test had a sensitivity of 40.0%, specificity of 100%, positive predictive value of 100%, and negative predictive value of 96.9%. Diagnostic accuracy was similar to that of the SNAP Kombi Plus Test. Conclusion: The new Anigen Rapid FIV Ab/FeLV Ag Test performed very well and can be recommended for use in veterinary practice.     

Comparison of gel column agglutination with monoclonal antibodies and card agglutination methods for assessing the feline AB group system and a frequency study of feline blood types in northern Italy

Background: A new commercial gel column agglutination system is reported to have high sensitivity in detecting cats with blood type AB. Objectives: The aims of this study were to compare gel column agglutination and card agglutination methods for feline blood‐typing and to determine the frequency distribution of feline blood types in northern Italy. Methods: Blood‐typing was performed on 120 cats using both a commercial gel column containing monoclonal antibodies (ID Gel‐Test Micro Typing System) and a card agglutination method (RapidVet‐H Feline). Results were confirmed with back‐typing. Sensitivity, specificity, positive predictive value, and negative predictive value were calculated for the 2 methods. A second group of 140 Domestic Shorthair (DSH) cats was blood‐typed using the gel column technique to determine the frequency distribution of feline blood types in northern Italy. Results: The card agglutination method demonstrated poor sensitivity in identification of type‐AB cats (61%) and was only 95% specific when identifying type‐B cats. The gel column agglutination technique demonstrated 100% sensitivity and specificity for typing all 3 blood types (A, B, and AB). The frequency distribution study of 140 cats demonstrated that 127 (90.7%) cats were type A, 10 (7.1%) were type B, and 3 (2.1%) were type AB. Conclusion: When blood‐typing cats of breeds with a relatively high frequency of blood types B and AB, methods that use monoclonal antibodies for detection of blood types B and AB are recommended. Alternatively, blood type can be confirmed by more sensitive supplemental testing, such as back‐typing. The high frequency of blood type A in DSH cats in northern Italy was comparable to previously reported frequencies in Italy and world‐wide.     

GeneScan analysis to detect clonality of T-cell receptor γ gene rearrangement in feline lymphoid neoplasms

Lymphoid neoplasms are usually diagnosed on the basis of cytological and histopathological findings. However, in some cases, discrimination of lymphoid neoplasms from reactive lymphoid proliferation is difficult. Polymerase chain reaction (PCR) amplification of the complementarity-determining region (CDR) 3 of the T-cell receptor (TCR) γ gene can be used to assess clonality of T-cell populations as a supportive diagnostic tool for T-cell neoplasms. Because the length variation in the TCRγ CDR3 is relatively small, false positive results may occur in non-neoplastic T-cell populations in the absence of high-resolution analytical methods for PCR products. In the present study, a PCR assay system was developed to detect clonal TCRγ gene rearrangement in feline lymphoid cells using GeneScan analysis. Thirty T-cell neoplasms, 27 B-cell neoplasms, and 34 non-neoplastic tissues were subjected to the newly developed TCRγ gene rearrangement analysis. Clonal TCRγ gene rearrangement was detected in 26 of 30 (87%) T-cell neoplasms, 2 of 27 (7%) B-cell neoplasms, and 1 of 34 (3%) non-neoplastic tissues. To compare GeneScan analysis with conventional PAGE and heteroduplex analysis, 20 clonal and 20 polyclonal samples were subjected to both analyses. Most of the results were concordant between the 2 analyses; however, several clonal peaks (bands) appeared as a single band when analyzed via conventional PAGE with heteroduplex analysis in 4 of the 20 (20%) clonal samples as a result of the difference in resolution. The PCR assay system to detect clonal TCRγ gene rearrangement in feline lymphoid cells, using GeneScan analysis, would be a useful molecular diagnostic tool for feline T-cell neoplasms, with high fidelity.     

Thymoma‐associated lymphocytosis in a dog

A 9‐year‐old female spayed English Springer Spaniel was evaluated for a cranial mediastinal mass and lymphocytosis. Flow cytometric immunophenotyping of peripheral blood lymphocytes revealed 97% as CD3 positive, confirming a T‐cell lineage. Additionally, T‐cell subset assessment showed 53.2% to be double‐negative T‐lymphocytes, expressing neither CD4 nor CD8 surface markers. The number of double‐negative lymphocytes in circulation coincided with the number of T‐cell receptor (TCR) γδ‐expressing T‐cells in circulation. Molecular T‐cell clonality analysis of TCR Gamma (TCRG) gene rearrangement showed a polyclonal expansion of T‐lymphocytes. Histopathology confirmed the mass to be a thymoma, supporting the diagnosis of thymoma‐associated T‐cell lymphocytosis. Resolution of the lymphocytosis after removal of the thymoma provided further evidence for this diagnosis. To the authors' knowledge, this case is only the second report of thymoma‐associated peripheral lymphocytosis in the veterinary literature, and is the first to report a confirmed thymoma‐associated peripheral γδ T‐cell lymphocytosis in a dog.     

Measurement of feline lipase activity using a dry-chemistry assay with a triolein substrate and comparison with pancreas-specific lipase (Spec fPLTM)

Pancreatic lipase immunoreactivity (Spec fPL) is currently considered to be the most accurate blood test for the diagnosis of feline pancreatitis. In this study, we measured lipase activity in cats using a newer catalytic lipase assay of dry-chemistry system (FDC-v-LIP) to determine the reference range and compared the results with those for Spec fPL. Based on the results of healthy cats, the reference range of FDC-v-LIP was determined to be less than 30 U/l. FDC-v-lip did not show a strong correlation with Spec fPL in cats with various diseases, which resulted in the low sensitivity and positive predictive value. However, the relatively high (>90%) specificity and negative predictive value indicated that FDC-v-LIP could be a useful patient-side screening test for the exclusion of feline pancreatitis.     

Ability of a 5‐Minute Electrocardiography (ECG) for Predicting Arrhythmias in Doberman Pinschers with Cardiomyopathy in Comparison with a 24‐Hour Ambulatory ECG

Background: Ventricular premature contractions (VPCs) are common in the occult stage of cardiomyopathy in Doberman Pinschers. Although the gold standard for detecting arrhythmia is the 24‐hour ambulatory electrocardiography (ECG) (Holter), this method is more expensive, time‐consuming and often not as readily available as common ECG. Objectives: Comparison of 5‐minute ECGs with Holter examinations. Animals: Eight hundred and seventy‐five 5‐minute ECGs and Holter examinations of 431 Doberman Pinschers. Methods: Each examination included a 5‐minute ECG and Holter examination. A cut‐off value of >100 VPCs/24 hours using Holter was considered diagnostic for the presence of cardiomyopathy. Statistical evaluation included calculation of sensitivity, specificity, positive predictive value, and negative predictive value. Results: Holter examinations revealed >100 VPCs/24 hours in 204/875 examinations. At least 1 VPC during a 5‐minute ECG was detected in 131 (64.2%) of these 204 examinations. No VPCs were found in the 5‐minute ECG in 73 (35.8%) examinations of affected Doberman Pinschers. A 5‐minute ECG with at least 1 VPC as cut‐off had a sensitivity of 64.2%, a specificity of 96.7%, a positive predictive value of 85.6% and a negative predictive value of 89.9% for the presence of >100 VPCs/24 hours. Conclusions and Clinical Importance: A 5‐minute ECG is a rather insensitive method for detecting arrhythmias in Doberman Pinschers. However, the occurrence of at least 1 VPC in 5 minutes strongly warrants further examination of the dog, because specificity (96.7%) and positive predictive value (85.6%) are high and could suggest occult cardiomyopathy.     

Cervical thymoma originating in ectopic thymic tissue in a cat

Abstract: An 11‐year‐old female spayed domestic shorthair cat was referred to The Ohio State University Veterinary Teaching Hospital (OSU‐VTH) for evaluation of a 6 × 4 × 3.5 cm mass in the left midcervical region causing increased respiratory sounds and lateral deviation of the trachea. A fine needle aspirate of the mass was obtained before referral and the cytology results were compatible with a reactive lymph node. Immunocytochemistry showed increased numbers of CD3+ T lymphocytes and small numbers of CD20+ and CD79a+ medium to large lymphocytes. Differential diagnoses from the referral pathologist were T‐cell‐rich B‐cell lymphoma and feline Hodgkin's‐like lymphoma. A subsequent fine needle aspirate performed at the OSU‐VTH showed similar results. On flow cytometry the majority of cells were CD3+ T lymphocytes that were double positive for CD4 and CD8 (73%), compatible with either a double‐positive (CD4+CD8+) T‐cell lymphoma or lymphocytes from ectopic thymic tissue. The mass was surgically removed. Histopathology and immunohistochemistry of the mass revealed a predominant population of CD3+ small lymphocytes and small numbers of medium to large lymphocytes with moderate anisocytosis and anysokaryosis. A population of cytokeratin‐positive epithelial cells surrounded small microcystic structures filled with eosinophilic material and structures interpreted as Hassall's corpuscles. These findings were consistent with thymic tissue and a diagnosis of ectopic thymoma was made. PCR results for lymphocyte antigen receptor rearrangement (PARR) were negative. The cat had no evidence of disease 16 months after removal of the mass. To our knowledge this is the first report of an ectopic cervical thymoma in a cat. The clinical and diagnostic features of this unusual case will be useful in helping veterinarians and pathologists obtain a presurgical diagnosis and establish a prognosis for similar lesions.     

Comparison of postmortem techniques for the detection of Mycobacterium bovis in white-tailed deer (Odocoileus virginianus).

A retrospective study of various diagnostic postmortem techniques used in a 4-year surveillance program for detection of Mycobacterium bovis infection in wild white-tailed deer (Odocoileus virginianus) was conducted. The tests evaluated were routine histopathology, acid-fast staining, detection of acid-fast bacilli in culture, and an M. tuberculosis group-specific genetic probe applied to pure cultures. Each of these techniques were compared with a reference or "gold standard" of mycobacterial culture and identification. Histopathology, the most rapid form of testing for M. bovis infection in white-tailed deer samples, had a sensitivity of 98% and a specificity of 87%, resulting in a positive predictive value of 94%. The detection of acid-fast bacilli by staining was less sensitive than histopathology (90%), but its higher specificity (97%) resulted in a positive predictive value of 99%. The detection of acid-fast bacilli on culture was both highly specific (93%) and sensitive (100%). The group-specific genetic probe had the highest sensitivity and specificity and produced results in complete agreement with those of mycobacterial culture, suggesting that this technique could be used as the new "gold standard" for this particular wildlife tuberculosis surveillance program.     

Proteomic identification and profiling of canine lymphoma patients*

This study employed proteomic and bioinformatic approaches to identify serum biomarkers in canine lymphoma patients. Chilled serum samples derived from non‐lymphoma (n = 92) and lymphoma (n = 87) patients were shipped from first opinion veterinary practices, subjected to ion exchange chromatography and analysed by surface‐enhanced laser desorption ionization mass spectrometry. Nineteen serum protein peaks were identified between the two groups as being significantly different (P < 0.05) based upon their normalized ion intensities. Two biomarkers were identified that were capable of differentiating lymphoma and non‐lymphoma patients. Analysis of the test data provided a positive predictive value (PPV) of 82%. A clinical follow‐up study was carried out on 96 canine patients suspected of having lymphoma. Evaluation of this data gave a specificity value of 91%, sensitivity of 75%, PPV of 80% and negative predictive value of 88%. In conclusion, the expression pattern of two serum biomarkers has enabled serum samples to be classified into either lymphoma or non‐lymphoma categories.     

Proteomic profiling using SELDI‐TOF mass spectrometry: a diagnostic tool for canine B‐cell lymphoma

Introduction: Surface enhanced laser desorption ionization time of flight (SELDI‐TOF) mass spectrometry is a powerful new tool for biomarker discovery and diagnostic test development. In this process, proteins in biological samples are selectively bound to chip surfaces that have various chemistries and then subjected to mass spectrometry. Selectively bound proteins are separated by mass and the relative quantities of each protein compared among samples. Unlike conventional diagnostic test formats that commonly use single biomarkers, SELDI‐TOF technology allows multiple biomarkers to be used in a single assay to improve sensitivity and specificity. This technology has been used to improve diagnostic tests for human prostate and ovarian cancers. The objective of this study is to investigate the utility of this technology for the proteomic profiling of canine B‐cell lymphoma. Developing a diagnostic serum screening test for B‐cell lymphoma in dogs would be valuable as early diagnosis and treatment may confer a more favorable outcome. Methods: Serum samples were collected from 26 dogs diagnosed with B‐cell lymphoma prior to treatment and from 26 apparently healthy dogs that were similar in age, breed, and sex to the test group. Serum proteins were selectively bound to weak cationic, strong anionic, and nickel chelating chip surface chemistries under optimized buffer conditions, and subjected to mass spectrometry. The protein profiles were then compared using Biomarker Wizard and classification trees developed using Biomarker Patterns software from Ciphergen Biosystems, Inc (Fremont, CA). Results: To date several putative biomarkers have been identified. These putative biomarkers have been used to build classification tree models that predict disease in test samples. Preliminary results using cross‐validation of the test and control sample sets indicate that trees built with two or more biomarkers have greater than 90% sensitivity and specificity. Conclusions: Proteomic profiling using SELDI‐TOF technology in canine serum is feasible. Further testing is planned to confirm these initial results.     

Canine and feline retinal lymphoma: a retrospective review of 12 cases

This retrospective study identified 12 cases (6 canine and 6 feline) of ocular lymphoma with extensive retinal involvement and relative sparing of other ocular tissues. Our objectives were to describe the morphologic and immunohistochemical features of retinal lymphoma, assess the degree of correlation to the human counterpart, assign subtypes based on the veterinary‐adapted WHO classification system, and promote accurate reporting of retinal involvement in cases of intraocular lymphoma. Our findings suggest that a distinct retinal tropism is quite rare, representing approximately 1% of all cases of canine and feline ocular lymphoma. No breed or sex predispositions were identified. The mean age of the affected animal was 7 years (range 4–10) and 11 years (range 6–19) for dogs and cats, respectively. Nine cases (5 canine and 4 feline) were classified as diffuse large B‐cell lymphoma (DLBCL) subtype. The remaining cases were classified as peripheral T‐cell lymphoma (PTCL).     

Evaluation of Pax5 expression and comparison with BLA.36 and CD79αcy in feline non‐Hodgkin lymphoma

Paired box gene 5 (Pax5) is a widely used B‐cell marker for human and canine non‐Hodgkin's lymphoma (nHL); however, in the literature there is only one case report using Pax5 in a cat B‐cell lymphoma. The purposes of this study were to investigate the expression and detection of B‐cell specific activator protein (BSAP) using a monoclonal anti‐Pax5 antibody in feline nHL (FnHL) tissue samples to evaluate its diagnostic relevance as a B‐cell marker. A total of 45 FnHL samples in 45 cats were evaluated. B‐cell lymphoma was the most common immunophenotype (51.1%) for all the samples and T‐cell the most common immunophenotype (64.3%) for the gastrointestinal (GI) form. Pax5 stained 82.6% of all B‐cell lymphomas and no expression was found in any of the T‐cell lymphomas. Anti‐Pax5 antibody staining in FnHL is similar to that reported in human and canine counterparts and may offer an excellent B‐cell marker in cats.     

The Utility of Flow Cytometry for the Diagnosis of Cranial Mediastinal Malignancy in Canine Patients

Introduction:  The most common neoplasms located in the anterior mediastinum in the canine are thymoma and lymphoma. Distinguishing between the two is a diagnostic challenge. Treatment and prognosis for these diseases differs significantly. Thymomas contain a population of normally developing T cells. The majority of these T cells exhibit an immature phenotype, characterized by co‐expression of CD4 and CD8. This phenotype is rarely seen on neoplastic lymphocytes. The purpose of this study was to determine if analysis by flow cytometry could discriminate thymoma from lymphoma based on these cell surface markers.
Methods:  Fine needle aspirates were obtained from ten canine patients with mediastinal masses. Cells were analyzed by flow cytometry using a panel of T and B cell markers.
Results:  Six cases with 10% or greater CD4 + CD8+ cells were diagnosed with thymoma and were confirmed by histopathology. Four cases had fewer than 5% CD4 + CD8+ cells, having lymphocytes expressing CD4 only (3 cases) or CD21, a B cell marker(1 case). These were confirmed as lymphoma by cytology and/or a clonality assay. The sensitivity and specificity of this assay when used in the diagnostic work‐up for suspected thymoma was 100%.
Conclusion:  Flow cytometry may provide important, complementary information in the diagnostic work‐up of the canine patient with a mediastinal mass.     

Evaluation of an immunofluorescence test on direct smears of conjunctival and urogenital swabs taken from koalas for the detection of Chlamydia psittaci

The Chlamydia-Cel Vet IF Test (CCVIT), a commercially available immunofluorescence test for use on direct smears of clinical specimens, was evaluated in a colony of 43 captive koalas. The test is based on a monoclonal antibody directed against the chlamydial common group specific lipopolysaccharide antigen. Swabs were taken from conjuncitva and penis or urogenital sinus and used for direct smear evaluation and cell culture isolation. Compared with isolation of the organism in cell culture, the CCVIT on direct smears of conjunctival swabs presented a sensitivity of 88%, a specificity of 100%, a positive predictive value (PV+) of 100% and a negative predictive value (PV-) of 97%. The CCVIT on direct smears of urogenital swabs presented a sensitivity of 90%, a specificity of 84%, a PV+ of 86% and a PV- of 89%. The overall sensitivity was 89% (95% confidence interval [CI] of 71% to 97%), the specificity 94% (95% CI of 84% to 98%), the PV+ 89% and the PV- 94%. It was concluded that the CCVIT on direct smears was suitable as a diagnostic screening test for the detection of Chlamydia psittaci in koalas.     

A Comparative Study of a New Rapid and One‐Step Test for the Detection of Parvovirus in Faeces from Dogs,Cats and Mink

A one‐step immunochromatographic test, based on the use of monoclonal antibodies, was developed for the detection of canine parvovirus (CPV) in dog faeces. In addition to canine parvovirus the test can also be used for the diagnosis of infections with viruses causing parvovirus enteritis in cats (feline panleukopenia virus) and mink (mink enteritis virus). Four hundred and forty‐three faecal samples were evaluated by comparative testing between this one‐step test and three different enzyme‐linked immunosorbent assays (ELISA) in Sweden, Denmark and The Netherlands. The result of the evaluation showed an overall relative sensitivity and specificity of 95.8 and 99.7 %, respectively. Furthermore, the comparative testing of 83 dog samples in Germany between the one‐step test and an immune electron microscopy (IEM) agreed to 85.5 %. The sensitivity and specificity were 83.9 and 88.9 %, respectively. These results show that the one‐step test is a rapid, simple, reproducible and sensitive diagnostic test for the detection of parvovirus in faecal samples of dogs, cats and mink.     

Clinical Evaluation of a Peptide‐ELISA based upon N‐terminal B‐cell Epitope of the VapA Protein for Diagnosis of Rhodococcus equi Pneumonia in Foals

A total of 227 field samples from naturally exposed foals aged between 3 weeks and 6 months were used in an evaluation of a peptide‐based enzyme‐linked immunosorbent assay (ELISA) for diagnosis of Rhodococcus equi infection. A biotinylated peptide derived from the virulence‐associated protein A (VapA) of R. equi, a horse pathogen, was synthesized and designated as PN11‐14. The peptide corresponds to the N‐terminal B‐cell epitope TSLNLQKDEPNGRASDTAGQ of the VapA protein. Based upon a serum immunoglobulin (Ig)G titre of 512 as a positive cut‐off value for the R. equi infection, the ELISA provided the overall sensitivity of 47.62%, specificity of 69.67% and an accuracy of 59.47% with a positive predictive value of 57.47% for true R. equi pneumonia. The assay was improved by detecting VapA‐specific IgGb antibodies against N‐terminal B‐cell epitope of the VapA protein rather than IgG antibodies. The VapA‐IgGb ELISA showed the overall sensitivity of 70.47%, specificity of 72.13% and accuracy of 71.36% with a positive predictive value of 68.52%. Diagnosis of R. equi disease in 6‐week‐old foals showed that the VapA‐IgGb ELISA provided an increasing trend (P = 0.0572) in sensitivity of 82.4% in comparison with the VapA‐IgG ELISA which showed the sensitivity of 58.8%. However, differences in specificity of both tests were statistically insignificant (P = 0.357) as analysed by the McNemar test. These results indicated that detection of VapA‐specific IgGb antibodies may be a better predictor of R. equi disease in foals.