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.

Quantitative counts of self-bioluminescent bacteria were made in vivo (the bacterial load), whereas the number of eggs shed by helminths and their development to infective stages (L3 larvae) were used as a proxy measure of helminth infectiousness. Daily measurement of the bacterial load was carried out in vivo until equilibrium densities were reached, whereby a persistent nasal infection was observed for more than 5 days in a row (a total of 22 days for bacteria) or no eggs were shed (365 days for helminths). A controlled laboratory setting was used to eliminate confounding factors known to cause variation in parasite dynamics, including environment, nutrition, prior parasite exposure, host genetics, sex and age [25] [26] [27] [28] . All mice used were females, aged six to eight weeks caged individually in randomized locations within a single room in an animal facility for the duration of the experiment and provided with food ad libitum. A total of 40 female BALB/c mice (The Jackson Laboratory, Bar Harbor, ME, USA) were randomly allocated to one of four treatment groups (10 in each group): (i) inoculation with the respiratory pathogen B. bronchiseptica lux þ ; (ii) inoculation with the helminth H. polygyrus; (iii) simultaneous inoculation with B. bronchiseptica lux þ and H. polygyrus; (iv) a control group that received sham inocula of culture medium. We used B. bronchiseptica strain RB50, which had been rendered self-bioluminescent by the chromosomal insertion of a plasmid-pSS4266 to produce B. bronchiseptica lux þ [29] . The lux operon is driven by the fha promoter and is constitutively expressed such that bacteria are self-bioluminescent (i.e. do not require addition of a substrate), and the light emitted was quantified in vivo over time using BLI. Bacterial doses were confirmed by plating dilutions and carrying out colony counts prior to inoculation. Mice were intra-nasally inoculated under light anaesthesia (continuous flow 5% isoflurane in oxygen) with a 50 ml droplet of B. bronchiseptica lux þ in PBS þ 1 per cent Stainer -Scholte medium (ca 10 4 bacterial cells). The helminth-only treatment group received an intranasal sham-inoculation of 50 ml PBS þ 1 per cent Stainer -Scholte medium. Mice assigned to a co-infection or helminth-only treatment were simultaneously inoculated with 180 + 30 H. polygyrus infective L3 larvae in 20 ml distilled water, administered via oral gavage. The mean number of larvae in each inoculum was estimated from 10 direct counts of larvae in 20 ml of water prior to gavaging. The bacteria only and control treatment groups received a sham-inoculation of 20 ml distilled water, administered via oral gavage. Inoculation of animals was carried out in a random order on day zero of the experiment.