Evaluation of the Performance of a Rapid Enzyme-Linked Immunosorbent Assay in the Detection ofAnaplasma phagocytophilumAntibodies in Horses

Abstract

The aim of this study was to evaluate the performance of a commercially available rapid enzyme-linked immonosorbent assay, the Snap® 4Dx test, in the detection of Anaplasma phagocytophilum antibodies in horses. Two hundred apparently healthy horses (asymptomatic) and 244 animals showing clinical symptoms (symptomatic), were tested for A. phagocytophilum immunoglobulin G (IgG) antibodies using both the Snap® 4Dx kit and an indirect fluorescence antibody test (IFAT), with the latter serving as a comparative test. Horses belonging to the symptomatic group were also tested for evidence of active infection with A. phagocytophilum by analysis of IFAT IgM titers and PCR assay amplifying a specific fragment of the 16S rRNA gene. The overall agreement between the results obtained using the two tests, as well as the relative performance exhibited by the Snap® 4Dx test in the two groups, was assessed. Forty of the 45 animals (89%) testing positive for IgG antibodies using IFAT were correctly identified using Snap® 4Dx testing. The agreement between the results of the two tests was very high (k>0.9), with almost identical performances in both symptomatic and asymptomatic animals. Conversely, within the symptomatic group, only 44% (no. 11/25) of Snap® 4Dx positives appeared to be associated with a state of active infection, whereas the remaining 56% (no. 14/25) were related both to not infected animals (no. 1) and to horses whose status of infection needed further evaluations to be confirmed (no. 13/25). This study suggests that the Snap® 4Dx test could represent a valid screening method for use during epidemiological surveys of equine populations. Nevertheless, in-clinic application of the test does not appear to be merited.

Serological and molecular detection of Anaplasma phagocytophilum in horses reared in Korea

“…Numerous studies conducted outside Korea have also reported seropositivity for A. phagocytophilum in horses: 73% (67/90) of horses in the Czech Republic tested positive by IFAT (Praskova et al 2011); 13% (21/162) of horses in Portugal tested positive by IFAT (Ribeiro et al 2013); 13.7% (41/300) and 6.7% (20/300) of horses in Italy tested positive by IFAT and PCR, respectively (Laus et al 2013);16.3% (56/343) of horses in Tunisia tested positive by IFAT (Ben Said et al 2014); and 10.1% (45/444) and 9.0% (40/444) of horses in Italy tested positive by IFAT and ELISA, respectively (Veronesi et al 2014). The prevalence of seropositivity in the present study was lower than that reported in these studies; however, it is consistent with another Korean study that showed 2.2% (2/92) seropositivity for A. phagocytophilum in horses reared on Jeju Island, as determined by IFAT (Chae et al 2009).…”

“…A commercial ELISA kit (SNAP ® 4Dx test; IDEXX Laboratories, USA) was used to detect antibodies against A. phagocytophi- Although the SNAP ® 4Dx test was originally developed to detect canine antibodies against A. phagocytophilum, it can also be used to detect antibodies against A. phagocytophilum in blood samples from horses (Chan et al 2010;Vieira et al 2013;Veronesi et al 2014). The SNAP ® 4Dx test detects antibodies from both active infection and previous exposures to the pathogen.…”

“…The SNAP ® 4Dx test detects antibodies from both active infection and previous exposures to the pathogen. The test has good sensitivity (86.7%) and specificity (99.8%) for blood samples from horses when compared to the indirect fluorescence antibody test (IFAT), which is considered the "gold standard" assay for detecting A. phagocytophilum antibodies (Veronesi et al 2014).…”

Comparison between available serologic tests for detecting antibodies against Anaplasma phagocytophilum and Borrelia burgdorferi in horses in Canada

“…Cross-reactivity is arguably of greater concern in samples with low titers, and, interestingly, a reduced relative sensitivity of the pocELISA in samples with low IFAT titers for antibodies against A. phagocytophilum was previously demonstrated. 24 False-positive results due to nonspecific reactivity with antibodies against flagellin or heat shock proteins may also occur with the B. burgdorferi whole-cell IFAT. 16 In this context, it is interesting to note that Borrelia miyamotoi and Borrelia kurtenbachii have been previously reported to infect I. scapularis in Canada.…”

“…As stated earlier, our study is not suitable to draw any conclusions with regard to sensitivity and specificity of the employed tests in a clinical context. While there may be insufficient information to unequivocally support individual serologic tests as the "gold standard" in horses, previous studies reported high sensitivity and specificity of available serologic tests 7,23,24 when known positive and negative samples were tested. Previous reports in other populations of horses also stated much better agreement between diagnostic tests.…”