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Infectious bovine keratoconjunctivitis (IBK), also known as pinkeye, is one of the most common eye diseases in cattle. Several pathogens have been associated with IBK cases, however, Moraxella bovis, Moraxella bovoculi, Mycoplasma bovis, Mycoplasma bovoculi and bovine herpesvirus type 1 (BHV-1) are most frequently observed. A multiplex real-time PCR assay using two reactions was developed for the detection and differentiation of these five pathogens. Detection sensitivities of the multiplex assays were compared to singleplex reactions testing for the same targets. Correlation coefficients (R2) of >0.99, and PCR efficiencies between 92 and 106% were demonstrated in all singleplex and multiplex real-time PCR reactions. The limits of detection (LOD) of multiplex assays for Moraxella bovis, Moraxella bovoculi, Mycoplasma bovis, Mycoplasma bovoculi and BHV-1 were 19, 23, 25, 24 and 26 copies per reaction, respectively. No cross amplification was observed for specificity testing of 179 IBK positive clinical samples and 55 non-target clinical samples. Percentage of clinical samples positive for Mycoplasma bovoculi, Moraxella bovoculi, Moraxella bovis, BHV-1 and Mycoplasma bovis were 88.8% (159/179), 75.9% (136/179), 60.3% (108/179), 11.7% (21/179) and 10.0% (18/179), respectively. Moraxella bovis, Moraxella bovoculi and Mycoplasma bovoculi were more prevalent than Mycoplasma bovis and BHV-1 in IBK samples collected from animals in this study population. Our data indicates that the multiplex real-time PCR panel assay is highly sensitive and highly specific for the detection and differentiation of the five major pathogens associated with bovine pinkeye., Highlights • A multiplex real-time PCR is developed for the detection of five major IBK pathogens. • Correlation coefficients of all standard curves were >0.99. • PCR amplification efficiencies for the five pathogens were between 92% and 106%. • Limits of detection were between 19 and 26 copies per reaction for the five pathogens. • Moraxella bovoculi, Mycoplasma bovoculi, and Moraxella bovis were more prevalent IBK pathogens.

Infectious respiratory disease is one of the most common diseases in dogs worldwide. Several bacterial and viral pathogens can serve as causative agents of canine infectious respiratory disease (CIRD), including Mycoplasma cynos, Mycoplasma canis, Bordetella bronchiseptica, canine adenovirus type 2 (CAdV-2), canine herpesvirus 1 (CHV-1), canine parainfluenza virus (CPIV), canine distemper virus (CDV), canine influenza virus (CIA) and canine respiratory coronavirus (CRCoV). Since these organisms cause similar clinical symptoms, disease diagnosis based on symptoms alone can be difficult. Therefore, a quick and accurate test is necessary to rapidly identify the presence and relative concentrations of causative CIRD agents. In this study, a multiplex real-time PCR panel assay was developed and composed of three subpanels for detection of the aforementioned pathogens. Correlation coefficients (R

Streptococcus equi subsp. equi is a Gram positive bacterial pathogen commonly associated with strangles in horses, a respiratory disease characterized by abscessation of submandibular and retropharyngeal lymph nodes which can lead to obstruction of the airway. Several real-time PCR (qPCR) assays have been developed for detection of S. equi from horses with many targeting conserved regions of the S. equi cell wall-associated M-protein (SeM), a major virulence factor and immunogen of S. equi. Our objective was to develop a nested PCR (nPCR) targeting SeM and an 18S rRNA internal control gene for detection of S. equi from horses with potential improvement in detection sensitivity compared to a qPCR. Primers and probes from the Kansas State Veterinary Diagnostic Laboratory (KSVDL) S. equi clinical testing assay were utilized for all qPCR testing. Primers flanking the SeM qPCR target region were selected for an initial end-point PCR step of the nested assay; PCR product from the end-point reaction then served as template for the qPCR reaction step of the nested assay. Sample nucleic acid was also tested directly with qPCR to allow for assay comparison. Nucleic acid from clinical specimens (n = 188) submitted to KSVDL were tested in parallel with each assay. The nPCR and qPCR assays identified 22.9% (43/188) and 13.3% (25/188) of samples positive for S. equi, respectively. None of the samples positive by qPCR were negative by nPCR. The PCR products from all positive samples were submitted for DNA sequencing. Each of the 25 samples positive by both assays had a high nucleotide identity match (>96%) to the SeM gene. Among the samples positive by nPCR but negative by qPCR, 17 of 18 were sequence confirmed for SeM at greater than 96% nucleotide identity. Based on the nPCR Ct (37.8) of the one sequence un-confirmed case, it is likely that the S. equi bacterial load in this sample was below the necessary concentration for successful sequencing. Limit of detection (LOD) for the nPCR was established at a Ct of 37, and based both on the LOD of the qPCR assay (Ct of 37), as determined by standard curve data, and on the highest nPCR Cts (~37) of clinical samples able to result in SeM sequence-confirmation. As demonstrated by sequencing confirmation, the nPCR assay targeting the SeM gene is highly specific to S. equi. The increased sensitivity of the nPCR, compared to the qPCR, may reduce the number of false negative sample results in clinical testing and provide a superior detection method during low bacterial shedding periods.

A multiplex PCR procedure that detects six major virulence genes, fliC, stx1, stx2, eae, rfbE, and hlyA, in Escherichia coli O157:H7 was developed. Analyses of the available sequences of the six major virulence genes and the published primers allowed us to develop the six-gene, multiplex PCR protocol that maintained the specificity of each primer pair. The resulting six bands for fliC, stx1, stx2, eae, rfbE, and hlyA were even and distinct with product sizes of 949, 655, 477, 375, 296, and 199 bp, respectively. The procedure was validated with a total of 221 E. coli strains that included 4 ATCC, 84 cattle, and 57 human E. coli O157:H7 strains as well as 76 non-O157 cattle and human E. coli strains. The results of all 221 strains were similar to the results generated by established multiplex PCR methods that involved two separate reactions to detect five virulence genes (stx1, stx2, eae, fliC, and hlyA). Specificity of the O antigen was indicated by amplification of only O157, and not O25, O26, O55, O78, O103, O111, O127, and O145 E. coli serotypes. Sensitivity tests showed that the procedure amplified genes from a fecal sample spiked with a minimum of 104 CFU/g (10 cells/reaction) of E. coli O157. After a 6-h enrichment of E. coli O157-spiked samples, a sensitivity level of 10 CFU/g was achieved.

Rapid diagnosis and treatment of disease is often based on the identification and characterization of causative agents derived from phenotypic characteristics. Current methods can be laborious and time-consuming, often requiring many skilled personnel and a large amount of lab space. The objective of our study was to develop a spotted microarray for rapid identification and characterization of bacterial pathogens and their antimicrobial resistance genes. Our spotted microarray consists of 489 70mer probes that detect 40 bacterial pathogens of medical, veterinary and zoonotic importance (including 15 NIAID Category A, B and C pathogens); associated genes that encode resistance for antimicrobial and metal resistance; and DNA elements that are important for horizontal gene transfer among bacteria. High specificity and reliability of the microarray was achieved for bacterial pathogens of animal and human importance by validating MDR pathogenic bacteria as pure cultures or by following their inoculation in complex and highly organic sample matrices, such as soil and manure.

Successful printing and hybridization is essential for efficient and reliable data acquisition in a spotted microarray experiments. In this study we demonstrated that printing a 25 mer (printed 25 mer) with a standard 70 mer probe in each spot followed by the use of a fluorescently labeled 25 mer complement in the hybridization mixture ensures monitoring overall printing quality of the chip. This system can also be used as a control to evaluate adequate hybridization, washing, and alignment of spots to position the tracking grids during scanning. A print correction value incorporated in data analysis enhances consistency and reliability of results.