Development of Elisa for detecting antibody against Bartonella spp. specific protein in cats and its application in natural infection

Abstract

This study aims to evaluate the risk factor and an association between Bartonella spp. infection and immune status by using retroviral-infected cats as an immunocompromised model and to develop the recombinant B. henselae specific antigen protein (17kDa and GroEL)-based ELISA test for antibody detection against Bartonella spp. infection in cats. From 2017 to 2020, 161 client-owned clinical healthy cats at veterinary clinics and hospitals in the Bangkok metropolitan area were recruited and tested for Bartonella spp. infection statuses (PCR and IFA serology), blood profiles, feline retroviral statuses (FIV and FeLV), and T lymphocyte subsets (CD4+, CD8+, and CD4+ to CD8+ ratio). In this investigation, the prevalence of Bartonella spp. polymerase chain reaction at the veterinary clinics and hospitals in the Bangkok metropolitan area was 16.1% and the seroprevalence was 94.9%. Cats older than one year were more at risk of being seropositive than cats younger than one-year-old (OR 4.296; 95%CI: 1.010 - 18.275). The CD8+ percentage was significantly higher in seropositive cats (p = 0.026). There was a significant reduction of CD4+ to CD8+ ratio between cats and concurrent Bartonella spp. and retrovirus-infected cats (p = 0.041). Regarding diagnostic tools, it is necessary to develop a sensitive diagnostic test that is specially developed for veterinary use to screen for bartonellosis in cats. This study used 17kDa and GroEL, B. henselae specific proteins (BSP), have been identified as immunodominant antigens and proposed as new diagnostic targets. These two genes, 17kDa and groEL were cloned in pET28b and pH6HTC vectors and expressed under IPTG induction. The recombinant proteins were purified by affinity chromatography using HisTrap matrix. Both purified proteins showed the immunoreactivity to seropositive cat serum by immunoblot assay. The 17-kDa and GroEL recombinant antigen proteins were also deployed to develop the antibody detection tool against Bartonella spp. infection in cats by indirect ELISA assay. The optimum concentrations of recombinant antigens, cat serum, and conjugated antibody (Goat anti-cat IgG) dilutions were 1.25 µg/ ml, 1:200, and 1: 12,000 for both newly developed ELISAs. The 17-kDa and GroEL-based ELISA were also tested in selected field cat sera (12 seropositive and 7 seronegative sera) and showed 75 % and 83.33 % sensitivity and 57.14% and 71.43% specificity in the IgG antibody detection. Moreover, the combination of positive results for at least one protein indicates satisfactory sensitivity and specificity (91.67 and 42.86%). In summary, this study showed a higher risk of seropositivity against Bartonella spp. in cats older than one-year-old and cats that were immunocompromised or retrovirus-infected may debilitate Bartonella spp. infection in cats. Our study also indicates that the recombinant 17-kDa and GroEL proteins are promising candidates for the development of serological detection tests of Bartonella spp. infection in cats.