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A cross-sectional study was conducted in Colombia to recover Brucella spp. DNA from bovine whole-blood samples through probe-based real-time PCR (Probe-qPCR) and by an SNP-based assay, differentiating vaccine strains from field strains. The associated factors for the presence of Brucella-DNA were reported and evaluated using logistical regression models. A total of 656 random cows from 40 herds were selected. Template DNA was obtained based on a modified salting-out protocol. The Probe- qPCR assay using bcsp31 gene amplification showed an efficiency of 92.35%, with a slope of -3.52 reached in the standard curve. The qPCR assay detected 9.5% (n = 62/656; 95% CI: 7.3,12.0) of the animals with Brucella-DNA presence, and 62.5% (n = 25/40; 95% CI: 45.8,77.3) of the herds with Brucella-DNA presence. Using the SNP-based assay, all positive samples were identified as field Brucella strains. In the final regression model at the animal-level, five variables were associated with Brucella-DNA presence: the use of bulls for mating, recorded history of reproductive problems, pregnant cows, parlor milking, and cows belonging to farms ≤ 200 m from the main road. At the herd-level, two variables were associated with Brucella-DNA presence: recorded history of reproductive problems and bulls' use for mating. Given the fluctuant brucellosis prevalence in endemic areas, updated epidemiological studies are necessary to evaluate the disease dynamic, and if established prevention and control measures have been effective or need to be adjusted. The increase in the prevalence of brucellosis in animal reservoirs creates an important risk of transmission in humans.

This study aims to describe a new detection method of a quantitative real-time polymerase chain reaction (qPCR) targeting the 28 kDa outer membrane protein gene (p28) as well as to compare this method with a conventional PCR (cPCR), which targets the same gene, in order to evaluate the performance of the technique designed in this study in detecting Ehrlichia canis (E. canis). Optimum oligonucleotides concentrations were reached, and the analytical sensitivity and specificity of the qPCR were performed. A total of 218 dogs’ whole blood samples were conventionally collected for this study. The DNA was extracted from each sample. Subsequently, the samples were tested by an established cPCR and the new qPCR to compare each technique’s performances. This new qPCR method for the molecular detection of E. canis presented a detection limit of ten copies of the fragment and was considered specific for E. canis according to analytical specificity analyses performed in vitro and in silico. The standard curve revealed 100% efficiency and a coefficient of determination (R2) equivalent to 99.8%. Among the samples examined by qPCR, 24.31% were considered positive, significantly greater than those detected by cPCR (15.13%). The qPCR technique reached a higher sensitivity than the cPCR when targeting the p28 gene in detecting E. canis. The qPCR standardized in this study is an efficient method for confirming canine monocytic ehrlichiosis (CME) diagnosis and might provide the parasitemia monitoring during the disease treatment.

A cross-sectional study was conducted to determine the associated factors of brucellosis in Colombia's preeminent dairy region declared in quarantine. A total of 656 samples were collected from cows ≥ 2-year-old from 40 herds. Samples were screened by the Rose Bengal Plate Test, and the Fluorescence Polarized Assay test and Competitive ELISA were used as confirmatory tests. A cow was classified as positive if the screening and both confirmatory tests were positive. A herd was classified as positive if at least one cow was seropositive. The factors associated to seropositivity were tested using a logistic regression model with explanatory variables regarding cattle management, zootechnical parameters, and sanitary practices. The seroprevalence at the animal level was 6.6% (43/656) and at herd level 27.5% (11/40). In the model, five variables explained the animal cases: purchase or animal transfer between owner's farms (