Response to Letter to the Editor on “ Coxiella burnetii in Infertile Dairy Cattle With Chronic Endometritis”

In her Letter to Editor, Dr. Egan expressed concerns about the images of double-color immunofluorescence presented in the article “Coxiella burnetii in Infertile Dairy Cattle With Chronic Endometritis,” ³ affirming that autofluorescence is responsible for the colocalization between anti–C. burnetii and anti-CD68 antibodies. Dr. Egan also asserted that the article does not mention important information such as the positive or negative controls employed for the study and the methods used for the analysis of the immunofluorescence signal. The presence of intrinsic biomolecules in cells and tissues acting as endogenous fluorophores results in autofluorescence (AF). ² There are several endogenous fluorophores recurring in cells and tissues: lipofuscins, ⁴ lipofuscin-like lipopigments, flavins, nicotinamide adenine dinucleotide (NADH), fatty acids, vitamin A, porphyrins, and collagen/elastin, ² each one with a representative spectral profile. As Dr. Egan stated, AF may represent a complication because its signal can often result in a background hindering the specific detection of the exogenous marker emission. In their remarkable work, Croce and Bottiroli ² summarized the different strategies to address this nuisance, for instance, avoiding or removing it by proper optical filtering, by bleaching cells or tissues through preirradiation, or by using specific computer software or exogenous fluorophores with excitation/emission ranges longer than the blue region (which is the predominant one for most endogenous fluorophores). Interestingly, several authors are taking advantage of AF for research and diagnostic purposes by evaluating the changes in the emission properties of endogenous fluorochromes in response to specific morphological and metabolic conditions of cells and tissues. ²

We strongly value the application of a correct scientific methodology. ¹ We clearly stated in our article that negative controls were used for immunohistochemical analysis to identify nonspecific labeling: “for each tissue section, 2 controls were performed incubating 1 section with PBS alone and the other with Negative Control Mouse IgG1.” The same approach was used for immunofluorescence controls, incubating 2 sections with PBS alone in order to verify the presence of nonspecific labeling or autofluorescence in our samples. Negative controls data for immunofluorescence are included as supplemental figures to this response letter. We did not use a specific software to measure the immunofluorescence signal, hence the missing information. A photo editing program (Adobe Photoshop CC; Adobe, San Jose, CA) was used to improve the image by the adjustments of contrast, color balance, and sharpness and also limiting the autofluorescent background. In the article, 3 figures from different stains (both histochemical and immunohistochemical) of the same polymerase chain reaction C. burnetii–positive endometrial biopsy were used to consistently demonstrate the bacteria within the macrophages. We believe that the colocalization between anti–C. burnetii and CD68 antibodies provided useful additional information to the article. Since C. burnetii is an obligate intracellular bacterium that infects cells of the macrophage lineage, such colocalization was actually expected rather than surprising. We hope that this response will dispel all doubts and concerns that Dr. Egan and other colleagues and readers may have regarding our work.

Supplemental Material

Supplemental Material, DS1_VET_10.1177_0300985818823781 - Response to Letter to the Editor on “Coxiella burnetii in Infertile Dairy Cattle With Chronic Endometritis”

Supplemental Material, DS1_VET_10.1177_0300985818823781 for Response to Letter to the Editor on “Coxiella burnetii in Infertile Dairy Cattle With Chronic Endometritis” by Davide De Biase and Orlando Paciello in Veterinary Pathology