Many rough mutants selected from isogenic smooth virulent and avirulent strains of Shigella flexneri were examined for virulence, using tissue culture infection and Sereny tests. Many of the rough mutants isolated from a virulent smooth strain were capable of penetrating tissue culture cells but incapable of producing a positive Sereny test. In contrast, we could not obtain from smooth avirulent strains any rough mutants capable of penetrating HeLa cells. Chemical analysis of lipopolysaccharide of some representative rough strains showed several patterns of sugar composition with a range of from Ra to Re chemotypes. There was no correlation between HeLa cell invasiveness and chemotypes of lipopolysaccharides, thus indicating little significance of oligosaccharides of the rough core as well as O antigens in the ability of S. flexneri to penetrate HeLa cells. When these invasive rough strains were given O antigen genes from a smooth avirulent Shigella Hfr strain, most of the transconjugants that expressed O antigens regained the ability to evoke keratoconjunctivitis in guinea pigs. We also examined the chromosomal loci of HeLa cell invasion by transferring carbohydrate fermentation genes of Escherichia coli K-12 Hfr and found two chromosomal loci, the rha and lac-gal regions, which control the ability to penetrate HeLa cells. These results suggested that O antigens and ability to penetrate tissue culture cells are independent and prerequisite attributes of virulence in Shigella flexneri to evoke keratoconjunctivitis in guinea pigs.
ABSTRACT. A neonatal calf developed nervous symptoms followed by diarrhea. The principal pathological changes were fibrinopurulent meningitis with necrosis of the cerebral parenchyma, and attaching and effacing lesions in the intestine. Cerebral necrosis was frequently associated with vascular changes. Enterobacter cloacae was isolated from the brain and Escherichia coli (O128) from the intestinal content. These suggest that cerebral and intestinal lesions were caused by the isolated organisms, and that most necrosis of the cerebral parenchyma might be infarctive. A neonatal calf was affected on a dairy farm raising 25 Holstein-Friesian cows or calves in Iwate Prefecture. The calf was born at full term after dystocia in September 1999, and had made a little attempt to suck colostrum from the dam. Since birth, the animal had shown anorexia, depression, and difficulty in standing and walking, and impaired vision was indicated by the frequency of bumping its head against the wall. The body temperature was not examined. After worsening day by day, the animal developed swollen cubital joints and excreted loose feces with mucus at the age of eight days, and was euthanatized two days later. There was no medical treatment attempted during the disease course.At necropsy, the principal lesions were confined to the CNS and digestive tract. There was an increased amount of cloudy cerebrospinal fluid along with dilation of the ventricles and mesencephalic aqueduct, especially both of the lateral ventricles. The meninges of the brain and spinal cord were congestive and cloudy with petechiae. On frontal sections of the CNS, necrotic foci ranging from 2 to 10 mm in diameter were scattered throughout the corpus striatum and thalamus, and occasionally in the cerebral parietal lobe. The small and large intestinal walls were flaccid with small amounts of fluid in the lumina. Mucus was found on the colonic and rectal mucosa, and blood-tinged mucus was present on the congestive cecal mucosa. Other findings were fibrinous exudate on the peritoneal surface, an increased quantity of cloudy synovial fluid in many limb joints, especially the cubital joints, and atrophic thymus.Tissue blocks collected throughout the body were fixed in 10% neutral buffered formalin and embedded in paraffin wax. Sections of tissue were stained with hematoxylin and eosin (HE). Selected sections of the CNS and intestine were stained with Gram (Brown and Brenn method), phosphotungstic acid hematoxylin (PTAH), periodic acid Schiff (PAS), azan, Masson trichrome, and Weigert's fibrin as well as the streptavidin-biotin-peroxidase (SAB) method. The SAB method was applied using a SAB kit (Nichirei Co., Tokyo) and rabbit hyperimmune sera against E. coli O128 (Denka Seiken Co., Tokyo) or against E. cloacae [11]. Specimens from the ileum fixed in 10% buffered formalin were postfixed in 1% osmium tetroxide-phosphate buffer, embedded in epoxy resin, sectioned, and stained with uranyl acetate and lead citrate for transmission electron microscopy. The specimens of osmium fi...
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