Background: Poor disease management and irregular vector control could predispose sheltered animals to disease such as feline Bartonella infection, a vector-borne zoonotic disease primarily caused by Bartonella henselae. Objectives: This study investigated the status of Bartonella infection in cats from eight (n = 8) shelters by molecular and serological approaches, profiling the CD4:CD8 ratio and the risk factors associated with Bartonella infection in shelter cats. Methods: Bartonella deoxyribonucleic acid (DNA) was detected through polymerase chain reaction (PCR) targeting 16S-23S rRNA internal transcribed spacer gene, followed by DNA sequencing. Bartonella IgM and IgG antibody titre, CD4 and CD8 profiles were detected using indirect immunofluorescence assay and flow cytometric analysis, respectively. Results: B. henselae was detected through PCR and sequencing in 1.0% (1/101) oral swab and 2.0% (1/50) cat fleas, while another 3/50 cat fleas carried B. clarridgeiae. Only 18/101 cats were seronegative against B. henselae, whereas 30.7% (31/101) cats were positive for both IgM and IgG, 8% (18/101) cats had IgM, and 33.7% (34/101) cats had IgG antibody only. None of the eight shelters sampled had Bartonella antibody-free cats. Although abnormal CD4:CD8 ratio was observed in 48/83 seropositive cats, flea infestation was the only significant risk factor observed in this study. Conclusions The present study provides the first comparison on the Bartonella spp. antigen, antibody status and CD4:CD8 ratio among shelter cats. The high B. henselae seropositivity among shelter cats presumably due to significant flea infestation triggers an alarm of whether the infection could go undetectable and its potential transmission to humans.

Aims: The diagnosis of cat scratch disease (CSD), a disease caused by Bartonella henselae, is challenging and often hampered by the lack of appropriate laboratory assays in developing countries due to limited resources. Currently, the indirect immunofluorescence assay (IFA) is the mainstay for CSD diagnosis. However, IFA kits are costly as limited samples can be tested on one slide and reading of the immunofluorescence results is subjective. In this study, the sensitivity and specificity of a recombinant B. henselae outer membrane protein (BHp26)-based enzyme-linked immunosorbent assay (ELISA) was assessed for serodiagnosis purposes. Methodology and results: Bartonella henselae outer membrane protein (BHp26) gene was cloned into a pBAD-TOPO expression plasmid and transformed into a TOP10 Escherichia coli host. The recombinant protein BHp26 was purified using an affinity chromatography approach in an AKTA purifier 10 system. The immunogenicity of the purified recombinant protein was evaluated using Western blot (WB). A recombinant outer membrane protein-based enzyme-linked immunosorbent assay (ELISA) was developed for detection against B. henselae antibodies in human sera. The recombinant protein-based ELISA demonstrated 57.7% agreement and 25% sensitivity as compared to IFA. A high specificity (94%) was exhibited when the ELISA was tested against 50 patients’ sera with positive findings to other infectious causes, including dengue, rickettsiae, leptospira, legionella and mycoplasma. Using the ELISA developed in this study, 14% (7/50) of urban blood donors and 9.1% (5/55) of healthy individuals from rural areas had IgG antibodies detected against B. henselae, suggesting previous exposure to the pathogen. Conclusion, significance and impact of study In view of the rising incidence of CSD, the recombinant outer membrane protein-based ELISA will be helpful for screening a large sample size of human sera for serosurveillance study.