Detection of Aspergillus-specific antibodies by agar gel double immunodiffusion and IgG ELISA in feline upper respiratory tract aspergillosis

https://doi.org/10.1016/j.tvjl.2014.12.020Get rights and content

Highlights

  • Feline antibodies against cryptic Aspergillus spp. cross react with an aspergillin containing A. fumigatus antigens.

  • Brachycephalic cats are prone to upper respiratory tract aspergillosis (URTA).

  • The agar gel immunodiffusion (AGID) assay has low sensitivity for diagnosis of URTA.

  • IgG ELISA has high sensitivity and specificity for diagnosis of URTA.

  • This study provides evidence that cats with URTA are systemically immunocompetent.

Abstract

Feline upper respiratory tract aspergillosis (URTA) is an emerging infectious disease. The aims of this study were: (1) to assess the diagnostic value of detection of Aspergillus-specific antibodies using an agar gel double immunodiffusion (AGID) assay and an indirect immunoglobulin G (IgG) ELISA; and (2) to determine if an aspergillin derived from mycelia of Aspergillus fumigatus, Aspergillus niger and Aspergillus flavus can be used to detect serum antibodies against cryptic Aspergillus spp. in Aspergillus section Fumigati. Sera from cats with URTA (group 1: n = 21) and two control groups (group 2: cats with other upper respiratory tract diseases, n = 25; group 3: healthy cats and cats with non-respiratory, non-fungal illness, n = 84) were tested. Isolates from cats with URTA comprised A. fumigatus (n = 5), A. flavus (n = 1) and four cryptic species: Aspergillus felis (n = 12), Aspergillus thermomutatus (Neosartorya pseudofischeri, n = 1), Aspergillus lentulus (n = 1) and Aspergillus udagawae (n = 1).

Brachycephalic purebred cats were significantly more likely to develop URTA than other breeds (P = 0.013). The sensitivity (Se) of the AGID was 43% and the specificity (Sp) was 100%. At a cut-off value of 6 ELISA units/mL, the Se of the IgG ELISA was 95.2% and the Sp was 92% and 92.9% for groups 2 and 3 cats, respectively. Aspergillus-specific antibodies against all four cryptic species were detected in one or both assays. Assay Se was not associated with species identity. Detection of Aspergillus-specific antibodies by IgG ELISA has high Se and Sp for diagnosis of feline URTA.

Introduction

Feline upper respiratory tract aspergillosis (URTA) is increasingly being recognised (Barrs and Talbot, 2014). There are two anatomical forms of disease, sino-nasal aspergillosis (SNA) and sino-orbital aspergillosis (SOA) (Barrs et al, 2012, Barrs et al, 2014). A strong association has been identified between the infecting fungal species and the anatomical form of disease; SNA is most commonly caused by Aspergillus fumigatus, while Aspergillus felis, a recently discovered ‘cryptic’ species in Aspergillus section Fumigati (Aspergillus viridinutans complex), is the most common cause of SOA (Barrs et al, 2013, Barrs et al, 2014, Barrs, Talbot, 2014). So-called cryptic species are indistinguishable on morphological features from A. fumigatus sensu stricto.

Similar to SNA in dogs, feline SNA is usually non-invasive, such that fungal hyphae do not penetrate the respiratory mucosa (Whitney et al., 2005); in contrast, in SOA fungal hyphae invade sino-nasal and paranasal tissues. Invasive mycoses typically occur in immunocompromised hosts. However, systemic immunodeficiency has not been detected in most cats with URTA (Barrs et al., 2012), one exception being a cat with feline leukaemia virus (FeLV) infection (Goodall et al., 1984).

The sensitivity (Se) of serological tests for detection of fungal antigens or Aspergillus-specific antibodies in aspergillosis depends on the systemic immunocompetence of the host as reflected by the ability to clear fungal antigen from the circulation and to mount an antibody response. An ELISA to detect a fungal cell wall antigen, galactomannan (GM), in serum (Platelia Aspergillus EIA, Bio-Rad) has a Se of up to 90% in immunocompromised patients, including neutropenic human patients with pulmonary aspergillosis and dogs with disseminated invasive aspergillosis (DIA) (Maertens et al, 1999, Garcia et al, 2012). However, the Se of this test is <30% in non-neutropenic human patients with aspergillosis, in immunocompetent dogs with SNA and in cats with URTA (Billen et al, 2009, Kitasato et al, 2009, Whitney et al, 2013).

Conversely, detection of serum Aspergillus-specific antibodies by agar gel double immunodiffusion (AGID) or by immunoglobulin G (IgG) ELISA has a high test Se in immunocompetent patients, including dogs with SNA (67–88%) and humans with chronic pulmonary aspergillosis (74–94%) (Pomrantz et al, 2007, Billen et al, 2009, Guitard et al, 2012, Ohba et al, 2012). A detectable antibody response is mounted in <30% of neutropenic humans with aspergillosis and dogs with DIA (Day et al, 1985, Hope et al, 2005, Schultz et al, 2008).

We hypothesised that Aspergillus-specific antibodies would be detectable in the majority of cats with URTA, since most cats with URTA are not, as far as it is possible to currently evaluate, systemically immunocompromised. The aims of this study were: (1) to assess the diagnostic value of detection of Aspergillus-specific antibodies using an AGID assay and an indirect IgG ELISA; and (2) to determine if a commercial aspergillin derived from mycelia of A. fumigatus, Aspergillus niger and Aspergillus flavus can be used to detect serum antibodies against cryptic Aspergillus spp. in Aspergillus section Fumigati.

Section snippets

Materials and methods

Signalment data and serum (1–2 mL per cat) were collected prospectively from cats diagnosed with URTA (group 1), cats with upper respiratory tract (URT) signs not attributable to aspergillosis (group 2), and from cats without respiratory or fungal disease (group 3). Samples were collected with informed consent according to the guidelines of the Animal Ethics Committee of the University of Sydney (approval number N00/9–2012/5774, date of approval 22 June 2012). Serum samples were collected at

Cats

In group 1, one cat (cat 5) was determined to be FIV-infected on the basis of a positive FIV antibody response and no history of FIV vaccination; the other 20 cats in group 1 tested negative for FIV and FeLV (Table 1). The mean age of cats in group 1 (6.3 years) was significantly different from that of cats in group 2 (9.8 years; P< 0.01) and group 3 (i) (4.0 years, P< 0.01), but not from the combined group 3 (i and ii: 8.2 years, P= 0.1) or a combined control group (groups 2 + 3: 8.7 years;

Discussion

In this study, we demonstrated that antibodies against four cryptic species of Aspergillus (A. felis, A. udagawae, A. lentulus and A. thermomutatus) can be detected in feline serum with assays utilising a commercial aspergillin derived from A. fumigatus, A. niger and A. flavus. Although this result was not unexpected given the close phylogenetic relationship of these cryptic species to A. fumigatus (Barrs et al, 2013, Novakova et al, 2014), it is important to demonstrate this cross reactivity,

Conclusion

Detection of Aspergillus-specific IgG by AGID and ELISA was highly specific for the diagnosis of aspergillosis in cats. The Se of IgG detection by ELISA was high, whereas the Se of detection using AGID was low. Depending on the cut-off value used, the ELISA has good discriminatory power to distinguish between presumed environmental exposure, which increases with age, and that induced by colonisation and infection. This study provides further evidence that feline URTA affects systemically

Conflict of interest statement

None of the authors has any other financial or personal relationships that could inappropriately influence or bias the content of the paper.

Acknowledgements

This study was funded by an Australian Companion Animal Health Foundation grant (015/2013). The sponsors were not involved in any aspect of the study or in the decision to publish this manuscript. The authors thank many colleagues for contributions of clinical samples for this study.

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