Listeria monocytogenes is among the most important food-borne pathogens and is well adapted to persist in the environment. To gain insight into the genetic relatedness and potential virulence of L. monocytogenes strains causing central nervous system (CNS) infections, we used multilocus variable-number tandem-repeat analysis (MLVA) to subtype 183 L. monocytogenes isolates, most from ruminant rhombencephalitis and some from human patients, food, and the environment. Allelic-profile-based comparisons grouped L. monocytogenes strains mainly into three clonal complexes and linked single-locus variants (SLVs). Clonal complex A essentially consisted of isolates from human and ruminant brain samples. All but one rhombencephalitis isolate from cattle were located in clonal complex A. In contrast, food and environmental isolates mainly clustered into clonal complex C, and none was classified as clonal complex A. Isolates of the two main clonal complexes (A and C) obtained by MLVA were analyzed by PCR for the presence of 11 virulence-associated genes (prfA, actA, inlA, inlB, inlC, inlD, inlE, inlF, inlG, inlJ, and inlC2H). Virulence gene analysis revealed significant differences in the actA, inlF, inlG, and inlJ allelic profiles between clinical isolates (complex A) and nonclinical isolates (complex C). The association of particular alleles of actA, inlF, and newly described alleles of inlJ with isolates from CNS infections (particularly rhombencephalitis) suggests that these virulence genes participate in neurovirulence of L. monocytogenes. The overall absence of inlG in clinical complex A and its presence in complex C isolates suggests that the InlG protein is more relevant for the survival of L. monocytogenes in the environment.Listeria monocytogenes is a food-and feed-borne pathogen of major concern with regard to public and animal health. This Gram-positive bacterium affects a wide range of mammalian species, most commonly humans and domestic ruminants (15). Clinical manifestations are similar in all susceptible hosts and include septicemia, abortion, severe gastroenteritis, and central nervous system (CNS) infections, such as meningitis, meningoencephalitis, and rhombencephalitis (2, 55). CNS involvement is a characteristic feature in humans and ruminants and accounts for the high fatality rates (20 to 50%) associated with listeriosis compared to other food-borne infections (9, 43, 55). Rhombencephalitis is the most commonly encountered form of listeriosis in farm ruminants and is of importance not only because it results in significant economic losses in livestock production due to morbidity and high mortality in animals but also because of its impact on food safety and public health, as infected animals represent a possible reservoir for human infection (4,38,44,61). Clinically affected animals and healthy carriers may shed the bacteria in their feces and milk, thus contaminating pastures, vegetables, surface water, and milk products (5,14,28,62).L. monocytogenes is widespread in the environment due to its eurythe...