Genetic characterization of phenicol-resistant Escherichia coli and role of wild-type repressor/regulator gene (acrR) on phenicol resistance.

The genetic basis for phenicol resistance was examined in 38 phenicol-resistant clinical Escherichia coli isolates from poultry. Out of 62 isolates, 38 showed resistance for chloramphenicol and nine for florfenicol, respectively. Each strain also demonstrated resistance to a variety of other antibiotics. Molecular detection revealed that the incidence rates of the cat1, cat2, flo, flo-R, cmlA, and cmlB were 32, 29, 18, 13, 0, and 0%, respectively. Nineteen strains were tolerant to organic solvents. PCR amplification of the complete acrR (regulator/repressor) gene of five isolates revealed the amino acid changes in four isolates. DNA sequencing showed the non-synonymous mutations which change the amino acid, silent mutation, and nucleotide deletion in four isolates. MY09C10 showed neither deletion nor mutation in nucleotide. The AcrA protein of the AcrAB multidrug efflux pump was overexpressed in these strains. Complementation with a plasmid-borne wild-type acrR gene reduced the expression level of AcrA protein in the mutants and partially restored antibiotic susceptibility one- to fourfold. This study shows that mutations in acrR are an additional genetic basis for phenicol resistance.

Contribution of Salmonella Enteritidis virulence factors to intestinal colonization and systemic dissemination in 1-day-old chickens.

Salmonella enterica serovar Enteritidis is one of the most common serovars associated with poultry and poultry product contamination in the United States. We previously identified 14 mutant strains of Salmonella Enteritidis phage type 4 (PT4) with significantly reduced invasiveness in human intestinal epithelial cells (Caco-2), chicken macrophages (HD-11), and chicken hepatocellular epithelial cells (LMH). These included Salmonella Enteritidis mutants with transposon insertions in 6 newly identified Salmonella Enteritidis-specific genes (pegD and SEN1393), and genes or genomic islands common to most other Salmonella serovars (SEN0803, SEN0034, SEN2278, and SEN3503) along with 8 genes previously known to contribute to enteric infection (hilA, pipA, fliH, fljB, csgB, spvR, and rfbMN). We hypothesized that Salmonella Enteritidis employs both common Salmonella enterica colonization factors and Salmonella Enteritidis-specific traits to establish infection in chickens. Four Salmonella Enteritidis mutants (SEN0034::Tn5, fliH::Tn5, SEN1393::Tn5, and spvR::Tn5) were indistinguishable from the isogenic wild-type strain when orally inoculated in 1-d-old chickens, whereas 2 mutants (CsgB::Tn5 and PegD::Tn5) were defective for intestinal colonization (P < 0.05) and 8 mutants (hilA::Tn5, SEN3503::Tn5, SEN0803::Tn5, SEN2278::Tn5, fljB::Tn5, rfbM::Tn5, rfbN::Tn5, and pipA::Tn5) showed significant in vivo attenuation in more than one organ (P < 0.05). Complementation studies confirmed the role of rfbN and SEN3503 during infection. This study should contribute to a better understanding of the mechanisms involved in Salmonella Enteritidis pathogenesis, and the target genes identified here could potentially serve as targets for the development of live-attenuated or subunit vaccine.

VscO, a putative T3SS chaperone escort of Vibrio alginolyticus, contributes to virulence in fish and is a target for vaccine development.

Type III secretion system (T3SS) in Vibrio alginolyticus is essential for its pathogenesis. VscO's homologous proteins FliJ, InvI and YscO have been suggested to be putative chaperone escorts although its function in V. alginolyticus is unclear. To investigate the physiological role of VscO, a mutant strain of V. alginolyticus with an in-frame deletion of the vscO gene was constructed in the present study. One finding was that the mRNA expression levels of SycD, VopB and VopD proteins decreased in the ΔvscO mutant. In addition, the ΔvscO mutant showed an attenuated swarming ability and a ten-fold decrease in the virulence to fish. However, the ΔvscO mutant showed no difference in the biofilm formation and ECPase activity. Complementation of the mutant strain with the vscO gene could restore the phenotypes of the wild-type strain. Finally, the recombinant VscO protein caused a high antibody titer and an effective protection against lethal challenge with the wild-type strain V. alginolyticus. These results indicated that VscO protein has a specific role in the pathogenesis of V. alginolyticus and it may be a candidate antigen for development of a subunit vaccine against vibriosis.

Social and reproductive physiology and behavior of the Neotropical cichlid fish Cichlasoma dimerus under laboratory conditions

In this work we describe for the first time the social and reproductive behavior of the Neotropical fish Cichlasoma dimerus (Heckel, 1840) [Perciformes: Cichlidae], endemic to the Paraná River basin, using a comprehensive-integral approach, including morphological and physiological features. This substrate breeding fish has biparental care of the fry and presents a dominance hierarchy that determines access to breeding territories among males, and to males with territories among females. Gregarious behavior associated with a pale body color, was observed before reproductive behaviors started. Afterwards, a dominance hierarchy was established through aggressive interactions. Territorial individuals had bright body color patterns and non territorial an opaque grey one. Black ventral coloration was associated with reproductive individuals. Courtship displays, which were similar to threatening displays, had the common effect of increasing the visible area of the individual. The dominant male was always the largest one suggesting that size is probably a major factor determining the hierarchy establishment and that these intra-sexually selected traits may have been reinforced by inter-sexual selection. Reproductive males had higher pituitary levels of β-follicle stimulating hormone (β-FSH) and somatolactin (SL) than non reproductive ones, while no differences were found among females. No differences were found among male gonadosomatic indexes. Non reproductive individuals had higher plasma cortisol levels for both sexes. It is possible that dominant reproductive individuals may be inhibiting reproduction of subordinate fish through physical contact, increasing their cortisol levels and diminishing FSH and SL pituitary content. However, this was not reflected as an inhibition at the gonadal level in our experimental design.

En este trabajo se describen por primera vez el comportamiento social y reproductivo del pez cíclido neotropical Cichlasoma dimerus (Heckel, 1840) [Perciformes: Cichlidae], endémico de la cuenca del Paraná, desde un enfoque integral y abarcador, incluyendo características morfológicas y fisiológicas. Éste pez incubador de substrato, tiene cuidado biparental de las crías y presenta una jerarquía de dominancia que determina el acceso a territorios reproductivos entre los machos, y a machos con territorios entre las hembras. Se observó un comportamiento gregario con una coloración corporal pálida característica, antes que comenzaran los comportamientos reproductivos. Luego, una jerarquía de dominancia se estableció a través de interacciones agresivas. Los individuos territoriales presentaron patrones de coloración corporal brillantes y los individuos no territoriales uno gris opaco. Una coloración ventral oscura fue observada asociada a individuos reproductivos. Los despliegues de cortejo fueron similares a los de amenaza y tuvieron la característica común de aumentar el área visible de los peces. El macho dominante fue siempre el más grande, sugiriendo que probablemente la fuerza (tamaño) es un factor preponderante determinando el establecimiento de las jerarquías y que éstas características seleccionadas intrasexualmente pueden haber sido reforzadas por selección intersexual. Los machos reproductivos presentaron un mayor contenido hipofisario de β-FSH y SL que aquellos no reproductivos, mientras que no se encontraron diferencias entre las hembras. No se encontraron diferencias entre los índices gonadosomáticos de los machos. Los individuos no reproductivos presentaron niveles plasmáticos mayores de cortisol para ambos sexos. Aunque los individuos reproductivos dominantes podrían estar inhibiendo la reproducción de los peces menos dominantes a través de interacciones de contacto físico, aumentando sus niveles de cortisol y disminuyendo el contenido hipofisario de FSH y SL, esto no se vería reflejado a nivel gonadal en nuestro diseño experimental.

Identification of novel attenuated Salmonella Enteritidis mutants.

Salmonella Enteritidis is a major food-borne pathogen that causes nontyphoidal diarrhoea in humans. Infection of adult egg-laying hens usually results in symptomless carriage but in young chicks it may cause paratyphoid disease. It is not known whether S. Enteritidis requires genes additional to known virulence genes for systemic infection of young chickens. A transposon insertion library was created using S. Enteritidis 10/02, which yielded 1246 mutants. Of 384 mutants screened in chickens for attenuation (30.8% of insertion library), 12 (3.1%) had a 50% lethal dose at least 100 times that of the parental strain. Sequencing revealed insertions in genes involved in the biosynthesis of lipopolysaccharide, cell membrane, ATP biosynthesis, transcriptional regulation of virulence and the yhbC gene, which has an unknown function. Evaluation of in vitro virulence characteristics of a Delta yhbC mutant revealed that its ability to invade HeLa cells and survive within a chicken macrophage cell line (HD11) was significantly reduced. It was also less resistant to reactive oxygen and nitrogen intermediates and had a retarded growth rate. Chickens challenged with the Delta yhbC mutant cleared the organism from the liver and spleen 1 week faster than the parental strain and were able to develop specific serum IgG antibodies against the Delta yhbC mutant.

Characterization of the type I secretion system of the RTX toxin ApxII in "Actinobacillus porcitonsillarum".

Strains of Actinobacillus porcitonsillarum are regularly isolated from the tonsils of healthy pigs. A. porcitonsillarum is non pathogenic but phenotypically it strongly resembles the pathogenic species Actinobacillus pleuropneumoniae, thereby interfering with the diagnosis of the latter. A. porcitonsillarum is hemolytic but unlike A. pleuropneumoniae, it contains only apxII genes and not apxI or apxIII genes. In contrast to the truncated apxII operon of A. pleuropneumoniae, which lacks the type I secretion genes BD, characterization of the apxII operon in A. porcitonsillarum revealed that it contains an intact and complete apxII operon. This shows a typical RTX operon structure with the gene arrangement apxIICABD. The region upstream of the apxII operon is also different from that in A. pleuropneumoniae and contains an additional gene, aspC, encoding a putative aspartate aminotransferase. Trans-complementation experiments in Escherichia coli and A. pleuropneumoniae indicated that the entire apxII operon of A. porcitonsillarum is sufficient to express and secrete the ApxIIA toxin and that the ApxIIA toxin of A. pleuropneumoniae can be secreted by the type I secretion system encoded by apxIIBD. These findings suggest that the complete apxII operon found in A. porcitonsillarum might be an ancestor of the truncated homologue found in A. pleuropneumoniae. The genetic context of the apxII locus in A. porcitonsillarum and A. pleuropneumoniae suggests that in the latter, the contemporary truncated operon is the result of a recombination event within the species, rather than a horizontal transfer of an incomplete operon.

Re-examination of the role of the Brucella melitensis HtrA stress response protease in virulence in pregnant goats.

Based on previously reported studies describing the experimental infection of pregnant goats with B. melitensis strain RWP5, we proposed that the HtrA protease plays an important role in the virulence of B. melitensis in its natural ruminant host. Subsequent studies, however, have shown that RWP5 is actually an htrA cycL double mutant. In order to definitively evaluate the role of the B. melitensis htrA in virulence, we constructed an authentic htrA mutant and examined this strain in pregnant goats. The findings of these studies indicate that the contribution of the htrA gene product to the virulence of B. melitensis in its natural host is not as great as was previously proposed.

A 14-3-3 protein homologue is expressed in feline enteroepithelial-stages of Toxoplasma gondii.

Fourteen cDNA clones encoding epitopes of proteins of Toxoplasma gondii feline enteroepithelial-stages parasites were isolated and expressed in Escherichia coli in an effort to determine the antigenecity of the parasites. Sequence analysis showed that four of the cDNA clones had a 930-bp open-reading frame encoding a product showing similarity to the 14-3-3 protein mRNA sequence.(1) Southern hybridization of DIG-labeled positive clone with T. gondii genomic DNA cleaved with EcoRI, BamHI and HindIII resulted in one or two bands in each case. In an immunofluorescence assay, polyclonal and monoclonal antibodies raised against the expressed protein showed strong reactivity with feline enteroepithelial-stages parasites and sporozoites. In a complementation assay in which a plasmid carrying the protein-coding region of the isolated cDNA was introduced into a Saccharomyces cerevisiae mutant, strain DS9-22, the expressed protein showed complementation of the function of the 14-3-3 protein in yeast transformants. These findings suggest that T. gondii parasites produce a protein showing partial homology with members of the 14-3-3 protein family and this protein is expressed in feline enteroepithelial-stages parasites.

New gene assignments using a complete, characterized sheep-hamster somatic cell hybrid panel.

The generation and characterization of new sheep-hamster cell hybrids is reported from the fusion of sheep white blood cells with six different hamster auxotrophs. Selection from these and previously generated cell hybrids has led to the production of a panel of 30 hybrids covering the complete sheep genome of 28 chromosomes. Over half of the cell hybrids in this panel contain single sheep chromosomes. By complementation, the following new assignments have been made using the panel: phosphoribosyl N-formylglycinamide amidotransferase (PRFGA) to sheep chromosome (chr) 11; adenylosuccinate synthetase (ADSS) to sheep chr 12; adenylosuccinate lyase (ADSL) to sheep chr 3q; 3-hydroxy-3-methylglutaryl-coenzyme A synthase (HMGCS) to sheep chr 16; dihydrofolate reductase (DHFR) to sheep chr 5; and adenine phosphoribosyltransferase (APRT) to sheep chr 14. The gene phosphoribosylaminoinidazole-carboxamide formyltransferase/Inosinicase (PRACFT) has now been regionally assigned to chr 2q. By isozyme analysis, phosphogluconate dehydrogenase (PGD) was assigned to sheep chr 12, anchoring the sheep syntenic group U1 to this chromosome, and mannose phosphate isomerase (MPI) was assigned to sheep chr 18. Furthermore, the chromosomal assignment of 110 microsatellites was confirmed using this cell panel.