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Evidence of Salmonella Enteritidis (SE) in internal organs of White Leghorns once they are inoculated via the oral (OR) or intracloacal (IC) route has not been consistently demonstrated. The aim of the current study was to evaluate OR or IC inoculation route of a nalidixic acid (Nal) resistant SE (SENAR) on the SE colonization of ceca and the invasion of internal organs in mature White Leghorns. Five experiments were conducted, and hens were inoculated with 108 colony-forming units (cfu) of SENAR. Hens were euthanized at 7 and 14 d post inoculation (dpi), and the ceca, spleen, liver with gall bladder (L/GB), and ovaries were collected for bacteriological analyses. The recovery of SENAR in ceca was 100% at 7 dpi. Recovery from the ovaries was lower than the other organs for both routes of inoculation. The SE recovery of L/GB, spleen, and ovaries at 7 dpi was not different between the two routes. By 14 dpi, all organs approached negative, and the recovery rate was similar between OR and IC. Fecal shedding was 100% positive at 3 dpi and reduced to almost 0% by 14 dpi. Mature hens were colonized by SENAR with either OR or IC inoculation when using a larger volume and a higher cfu/mL (0.1 mL OR in experiment 1 vs. 1.0 mL OR and IC in the rest). SENAR showed some translocation into other organs, to a greater extent with IC. The colonization did not persist either in ceca or the internal organs at 14 dpi.