Generating super-shedders: co-infection increases bacterial load and egg production of a gastrointestinal helminth

Lass, Sandra; Hudson, Peter J.; Thakar, Juilee; Saric, Jasmina; Harvill, Eric; Albert, Réka; Perkins, Sarah E.

Mice were placed in groups of up to three, within their treatment group inside an IVIS 50 (Caliper Life Sciences, Hopkinton, MA, USA). Mice were anaesthetized using a continuous flow of 5 per cent isoflurane mixed with oxygen for 5 min to allow acquisition of an image quantifying the light emission in vivo from B. bronchiseptica lux þ . Bacterial load measurements were obtained from three regions of the mouse: (i) whole body, within a square of standard size 3.0 cm width and 5.0 cm height, (ii) head, including nose and trachea using an elliptical region of size 2.3 cm width and 2.0 cm height, and (iii) lungs using a standardized square region of 3.0 cm width and 3.0 cm height. Mice were imaged at approximately the same time of day, from day zero ( just prior to inoculation) until day 22. An image was acquired over a 5 minute period and the software LIVING IMAGE (v. 2.6.1, Xenogen Corporation, Almeda, TX, USA) was used to convert the photons emitted from B. bronchiseptica lux þ within the host into relative light units (RLUs). The spatial location of infection in the mouse was overlaid on a photographic image using a pseudo-colour to quantify the RLUs. Previous studies have shown the light output (RLUs) from self-bioluminescent bacteria to correlate positively with viable counts of bacteria in vivo [30 -33] , thereby giving a real-time quantification rsif.royalsocietypublishing.org J R Soc Interface 10: 20120588 of bacterial load. To validate the relationship between B. bronchiseptica in vivo and in vitro, comparisons were made between viable counts of bacteria from a parallel experiment where animals were sacrificed at set time points (see §2. 1