Using the Comamonas-specific probe, we were able to demonstrate a specific signal in the gut epithelium of S. lupi larvae (Figure 4). The localization of the present Comamonas bacterium

in the nematode’s gut epithelium, and the phylogenetic proximity to other Comamonas spp. detected in blood-feeding insect hosts, may suggest that this novel Comamonas sp. plays a role in blood digestion or degradation within S. lupi, which feeds on its vertebrate hosts’ blood and tissues [9]. In addition, the FISH result, combined with the detection of Comamonas sp. in all the tested developmental stages of S. lupi using PCR, as described above, are in support of a stable, non- axenic infection of S. lupi by this bacterium. Figure 4 Comamonas sp. is restricted to the gut epithelium of Spirocerca lupi L3 larva. Images of fluorescence in-situ hybridization

analysis selleck compound of S. lupi L3 larva stained with Comamonas-specific probe (green), detected using confocal microscopy. (a) No-probe control; (b) Intact L3; (c, d) Ruptured L3; (e) Enlargement of (d), showing specific signal in the LY2874455 larval gut; (f) One optical section showing a specific signal only in the gut epithelium region. The arrow points to a specific focal point. All images but (f) are combined optical Z sections, overlaid on a bright-field image. Detection of S. lupi-derived Comamonas sp. in blood samples of infected dogs DNA detection from the S. lupi-derived Comamonas sp. in infected dogs may potentially be important in understanding the pathogenesis and promoting the diagnosis of spirocercosis. Recently, the symbiotic bacterium Wolbachia was detected in blood samples of dogs infected by the heartworm Dirofilaria immitis [26]. In the present study, we used a diagnostic semi-nested PCR with Comamonas-specific primers

on DNA extracted from blood samples of dogs definitely diagnosed with spirocercosis and of negative control dogs. Comamonas sp. DNA was detected in 9/18 (50%) samples obtained from dogs with spirocercosis, but in none of 11 negative control samples (Figure 5). The rather low detection rate of Comamonas sp. in the dogs infected with the nematode may be due to several reasons; an unavailable bacterial template; improper storage of blood samples, resulting in insufficient DNA preparation, next or an undetectable symbiont template in standard PCR due to unknown PCR inhibitors on a low concentration of Comamonas DNA in the blood. Alternatively, detection of the symbiont in blood samples may depend on the specific interactions between the bacterium and the nematode within the definitive canine host. It may be speculated that bacteria are only released from the nematode upon its death and disintegration, or within a limited specific time-point during infection within the definitive canine host. Further studies are warranted, to assess the optimal blood storage protocols and DNA extraction methods of canine samples, along with spiking experiments with Comamonas sp.