We gained substantial and seemingly important information relative to the aims of this study. We were able to obtain the initial set of samples on April 26, 2018 so we had a short delay in starting this study. Subsequently, the COVID epidemic and loss of research personal resulted in repeated interruptions in our research efforts. After completing all Year I study aims, we continued to work on improvements in immunohistochemistry and FISH localization of Bartonella organisms within various cell types.
Both of these study aims proved to be substantially more challenging than anticipated. One unanticipated complication arose when we found out that the mouse monoclonal antibody was no longer being made and sold commercially. Thus, we initiated work with a commercial entity to generate a new monoclonal antibody for future IHC evaluation of HSA cases and testing of other tissues where Bartonella infection is suspected or confirmed by DNA testing. B. henselae specific FISH probes were designed, after which optimization of the FISH probes resulted in our ability to visualize the bacteria in controlled cell culture infection studies.
As we had earlier documented that detection of Bartonella spp. antibodies using current IFA and WB serology assays was very insensitive in dogs with hemangiosarcoma, we subsequently confirmed that molecular diagnostic testing targeting both tumor and non-tumor tissues was needed to assess the prevalence of Bartonella spp. infection. These findings are of relevance to individual dogs and epidemiological studies of hemangiosarcoma in dogs.
We have published several manuscripts as a result of this research funding. One manuscript in the Journal of Clinical Microbiology, representing focused research funding from our AKC-CHF Grant #02287, allowed us to define the Western Blotting (WB) criteria for serodiagnosis of bartonellosis in dogs. That work required a substantial amount of time and research effort to validate WB testing criteria. Although WB marginally improved diagnostic sensitivity, it, like IFA serology, resulted in numerous false negative results in dogs with hemangiosarcoma, dogs with PCR confirmed Bartonella infection.
In contrast, we are very excited by the qPCR and ddPCR results obtained from the fresh frozen hemangiosarcoma tissues provided by the NIH-CCOGC. Those results strongly support a role for Bartonella spp. in the etiopathogenesis of hemangiosarcoma in dogs. The extent to which infection with Bartonella spp. is causative or only an association with hemangiosarcoma awaits the results of multiple future studies.
The regional study PCR results were disappointing as formalin fixation negatively affected both qPCR and ddPCR amplification of Bartonella spp. DNA. All three of the regions identified, collected and shipped all necessary samples from their region. Those samples were tested by ddPCR, which required months of validation.
Validation of the ddPCR methods were published in the Journal of Microbiological Methods and in the journal Pathogens. To assist clinicians and researchers with information related to diagnostic test selection, we published a manuscript in PLOS One describing the comparative sensitivity, specificity and diagnostic utility of IFA and WB serology, as compared to qPCR and ddPCR DNA amplification from blood and tissues of dogs with hemangiosarcoma.