Antigenic diversity in pathogenic microbes can be a result of at least three different processes: diversifying selection by acquired immunity, host-pathogen coevolution and/or host specialization. Here, we investigate whether host specialization drives diversity at ospC (which encodes an immunodominant surface protein) in the tick-transmitted bacterium Borrelia afzelii. We determined prevalence and infection intensity of ospC strains in naturally infected wild mammals (rodents and shrews) by 454 amplicon sequencing in combination with qPCR. Neither prevalence nor infection intensity of specific ospC strains varied in a species-specific manner (i.e. there were no significant ospC × host species interactions). Rankings of ospC prevalences were strongly positively correlated across host species. Rankings of ospC infection intensities were correlated more weakly, but only in one case significantly < 1. ospC prevalences in the studied mammals were similar to those in ticks sampled at the study site, indicating that we did not miss any mammal species that are important hosts for specific ospC strains. Based on this, we conclude that there is at best limited host specialization in B. afzelii and that other processes are likely the main drivers of ospC diversity.

The tick-borne bacterium 'Candidatus Neoehrlichia mikurensis' has recently been recognized as a human pathogen in Europe and appears to be the second most common pathogenic bacterium in Ixodes ricinus ticks in central Europe, second to Borrelia afzelii. Here, we investigate the prevalence of 'Candidatus N. mikurensis' in host-seeking ticks in southern Sweden and the rate of co-infection with B. afzelii. We developed a real-time qPCR assay targeting the groEL gene of 'Candidatus N. mikurensis' and applied this assay to 949 I. ricinus ticks collected at several locations over 2 years. We found an overall prevalence of 6.0%, which means that Candidatus N. mikurensis is one of the most common tick-transmitted zoonotic agents in this area. Co-infections with both 'Candidatus N. mikurensis' and B. afzelii occurred in 2.1% of the ticks, which is significantly more than expected under random co-occurrence. The infection intensity (number of bacterial cells) of 'Candidatus N. mikurensis' was not affected by co-infection with B. afzelii, and vice versa. We conclude that there is a risk for simultaneous transmission of these 2 tick-borne pathogens. The potential medical consequences of this require further investigation.

We show how to use free open-source CellProfiler for droplet microfluidic image analysis.

Recent development of imaging tools has facilitated studies of pathogen infections in vivo in real time. This trend can be exemplified by advances in bioluminescence imaging (BLI), an approach that helps to visualize dissemination of pathogens within the same animal over several time points. Here, we employ bacterial BLI for examining routes of entry and spread of Aeromonas salmonicida susbp. salmonicida in rainbow trout. A virulent Danish A. salmonicida strain was tagged with pAKgfplux1, a dual-labelled plasmid vector containing the mutated gfpmut3a gene from Aequorea victoria and the luxCDABE genes from the bacterium Photorhabdus luminescens. The resulting A. salmonicida transformant exhibited growth properties and virulence identical to the wild-type A. salmonicida, which made it suitable for an experimental infection, mimicking natural conditions. Fish were infected with pAKgfplux1 tagged A. salmonicida via immersion bath. Colonization and subsequent tissue dissemination was followed over a 24-h period using the IVIS spectrum imaging workstation. Results suggest the pathogen's colonization sites are the dorsal and pectoral fin and the gills, followed by a progression through the internal organs and an ensuing exit via the anal opening. This study provides a tool for visualizing colonization of A. salmonicida and other bacterial pathogens in fish.

Furunculosis, a septicaemic infection caused by the bacterium Aeromonas salmonicida subsp. salmonicida, currently causes problems in Danish seawater rainbow trout production. Detection has mainly been achieved by bacterial culture, but more rapid and sensitive methods are needed. A previously developed real-time PCR assay targeting the plasmid encoded aopP gene of A. salmonicida was, in parallel with culturing, used for the examination of five organs of 40 fish from Danish freshwater and seawater farms. Real-time PCR showed overall a higher frequency of positives than culturing (65% of positive fish by real-time PCR compared to 30% by a culture approach). Also, no real-time PCR-negative samples were found positive by culturing. A. salmonicida was detected by real-time PCR, though not by culturing, in freshwater fish showing no signs of furunculosis, indicating possible presence of carrier fish. In seawater fish examined after an outbreak and antibiotics treatment, real-time PCR showed the presence of the bacterium in all examined organs (1-482 genomic units mg ). With a limit of detection of 40 target copies (1-2 genomic units) per reaction, a high reproducibility and an excellent efficiency, the present real-time PCR assay provides a sensitive tool for the detection of A. salmonicida.