Development and validation of a multiplex electrochemiluminescence immunoassay to evaluate dry eye disease in rat tear fluids.

Dry eye disease (DED) is a challenge in ophthalmology. Rat models represent valuable tools to study the pathophysiology and to develop novel treatments. A major challenge in DED research is detecting multiple biomarkers in a low tear volume sample. Multiplex immunoassays for DED rat research are missing. We have developed a multiplex electrochemiluminescence immunoassay (ECLIA) to detect three biomarkers for DED: MMP-9, IL-17 and ICAM-1. Tears, used as matrix, were collected from six healthy Wistar rats. Assays were run based on the U-Plex Meso Scale Diagnostics (MSD) platform, by two independent operators according to the EMA guideline on bioanalytical method validation. Linear mixed, regression models were fit to perform the statistical analysis on the range of concentrations for the chosen analytes. During optimization, it has observed that incubation time, temperature and agitation affected the robustness of the protocol. ECLIA optimum conditions include the use of antibodies at 0.5 µg/ml concentration and 1 h incubation at room temperature with shaking. Precision met the acceptance criteria in the chosen range: 1062-133 pg/ml for ICAM-1, 275-34.4 pg/ml for IL-17, 1750-219 pg/ml for MMP-9. Accuracy and linearity were acceptable for a broader range. This is the first report of a validated ECLIA that allows measurements of three relevant DED biomarkers in rat tear fluids.

Retrospective Biomolecular Investigation of Coxiella burnetii and Leptospira spp. DNA in Cases of Abortion, Stillbirth and Neonatal Mortality in Dogs and Cats.

Abortion and neonatal mortality are events that can occur in breeding bitches and queens. It has been reported that up to 55% and 33% of these cases remain without a known cause, respectively, in canine and feline pregnancies. Unusual abortigenic and potentially zoonotic agents, including Coxiella burnetii and Leptospira spp., may be involved in these cases. C. burnetii is able to cause reproductive disorders in cattle, sheep and goats, and cases of abortion have been observed in dogs and cats. Moreover, several outbreaks of C. burnetii infection in humans have been caused by delivering bitches and queens, and some of these animals experienced abortion. Leptospira interrogans sensu lato is able to cause abortion or stillbirth in several animal species and its abortigenic role has occasionally been described in bitches and queens. The aim of this study was to search for C. burnetii and Leptospira spp. DNA in a retrospective series of 103 cases of canine and feline abortion, stillbirth, and neonatal mortality submitted for the identification of possible infectious agents. One hundred and fifty-one specimens were tested using PCR assays and found negative for C. burnetii and Leptospira DNA. However, in 49 samples (47.6%) other infectious causes of abortion, stillbirth, and neonatal mortality were identified. These results showed that C. burnetii and Leptospira spp. are probably not common abortigenic agents or causes of neonatal deaths in dogs. However, given the potential abortigentic and zoonotic role of these agents, surveillance of canine and feline abortion, stillbirth, and neonatal mortality could be advisable for a systematic investigation of these events.