Characterization of a plasmid-encoded adhesin of an avian pathogenic Escherichia coli (APEC) strain isolated from a case of swollen head syndrome (SHS)

An avian pathogenic Escherichia coli (APEC) strain designated SHS4, isolated from a chicken with clinical signs of swollen head syndrome (SHS), adhered to but did not invade Hep-2 and tracheal epithelial cells. The PCR amplified fimA, csgA and tsh gene sequences. It produced Ia, Ib, E1, E3, K, and B colicins, but not colicin V and aerobactin. It harboured two plasmids of 60 and 98MDa and was resistant to streptomycin and tetracycline. Conjugation with a nalidixic acid (Na) resistant K-12 recipient strain (MS101) showed that the 98MDa plasmid did not transfer, whereas transfer of the 60MDa plasmid resulted in concomitant transfer of adhesion to Hep-2 and tracheal epithelial cells, production of the colicins Ia, E1, E3, and K, and the tsh-related DNA sequence. Transposon (TnphoA) mutagenesis of strain TR4 gave rise to strain Mut23, which lost its adhesive capacities, but was still able to express the same colicins as did strain TR4. PCR was able to amplify the tsh-related DNA sequence in this strain and a molecular probe based on transposon TnphoA indicated that the transposon was inserted in the 60MDa plasmid. Based on these results, we suggest that the 60MDa plasmid have adhesion genes, which may be responsible for the initial colonization of the upper respiratory tract of chickens.     

Attaching and effacing Escherichia coli isolated from dogs in Brazil: characteristics and serotypic relationship to human enteropathogenic E. coli (EPEC)

Escherichia coli isolates recovered from 182 fecal specimens from dogs up to five months old from the cities of S?o Paulo and Campinas, SP, Brazil, were examined by polymerase chain reaction (PCR) for several virulence factors and properties. The eae gene was found in 23 isolates of E. coli from 22 dogs, 19 of 146 (13%) from dogs with diarrhea and 3 of 36 (8.3%) from dogs with no diarrhea. Two different eae+ isolates were recovered from one dog with diarrhea. Isolates from two dogs with diarrhea harbored the bfpA gene, and none of the isolates possessed genes for enterotoxins, the EAF plasmid or Shiga toxins. PCR showed that, among the 23 isolates, eight were positive for beta intimin, six for gamma, two for, one for alpha, one for kappa, and five showed no amplification with any of the nine pairs of specific intimin primers used. PCR also showed that the LEE (locus of enterocyte effacement) was inserted in selC in four isolates, likely in pheU in seven isolates, and in undetermined sites in twelve isolates. Fifteen isolates adhered to HEp-2 cells and were fluorescence actin staining (FAS) positive. The predominant adherence pattern was the localized adherence-like (LAL) pattern. The eae-positive isolates belonged to a wide diversity of serotypes, including O111:H25, O119:H2 and O142:H6, which are serotypes that are common among human EPEC. These results confirmed the presence of EPEC in dogs (DEPEC) with and without diarrhea. The virulence factors found in these strains were similar to those in human EPEC, leading to the possibility that EPEC may move back and forth among human and canine populations.     

Biological characteristics and pathogenicity of avian Escherichia coli strains.

Fifty avian (chicken) pathogenic Escherichia coli strains (APEC) isolated from individuals suffering from omphalitis, septicaemia and swollen head syndrome, and 30 strains isolated from healthy chickens were studied regarding their biological characteristics such as serogroups, haemolysin, colicin, cytotoxin, toxin and siderophore production, adhesion capacity to in vitro cultivated cells, and absorption of Congo red dye. Serotyping demonstrated that most of the omphalitis and normal strains were untypable, whereas most of the septicaemic strains were either untypable or rough. There was no prevalent serogroup among the pathogenic strains studied. The capacity for adhesion and invasion of in vitro cultured cells (HeLa, HEp-2, KPCC), as well as the agglutination of different types of red blood cells and the LD50 of each strain were also evaluated. No correlation was observed between the biological characteristics and pathogenicity, except that colicin was characteristically produced by swollen head syndrome E. coli strains. No correlation was found between adhesion or haemagglutination patterns and pathogenicity. Only six of the 50 strains revealed invasive capacity and the strain that best invaded the cell lines was the one with the lowest LD50.     

Evaluation of Praziquantel effects on Echinostoma paraensei ultrastructure

Echinostomiasis is a food-borne, intestinal, zoonotic, snail-mediated helminthiasis caused by digenean trematodes of the family Echinostomatidae with seven species of the genus Echinostoma infecting humans or domestic and wildlife animals. Echinostoma paraensei is a peristomic 37-collar-spined echinostome belonging to the “revolutum group”.     

Temperature-dependent development rates of <Emphasis Type="Italic">Bracon vulgaris,</Emphasis> a parasitoid of boll weevil

The duration of development of Bracon vulgaris Ashmead, parasitoid of the boll weevil Anthonomus grandis Boheman, was determined at nine constant temperatures between 18°C and 38°C. Nonlinear regression analysis was used to test the fit of temperature-dependent development rates to the Sharpe and DeMichele and Lactin et al. models. At the highest tested temperature (38°C) all the parasitoid eggs died before hatching and no evidence of development was observed. The high values of R ² for the models of Sharpe and DeMichele (0.8432 to 0.9834), and Lactin et al. (0.9071 to 0.9795) indicated that these models are suitable to estimate the development rate of B. vulgaris as a function of temperature. B. vulgaris showed tolerance to high temperature which is represented by the high value of H _H (change in enthalpy associated with high-temperature inactivation of the enzyme) for the prepupa stage of this insect obtained with the Sharpe and DeMichele model. According to that model, B. vulgaris exhibits thermal stress at 35.7°C, which indicates that maximum thermal stress estimated by this model was close to the real one.     

The expression of plasmid mediated afimbrial adhesin genes in an avian septicemic Escherichia coli strain

An Escherichia coli strain (SEPT13) isolated from the liver of a hen presenting clinical signs of septicaemia had a LD₅₀ of 4.0 × 10⁵ CFU/ml in one-day-old chickens, expressed Ia, Ib, E1, E3, K and B colicins and aerobactin. The strain was ampicillin and streptomycin resistant, and found to have fimA, csgA and tsh DNA related sequences; it could adhere to and invade HEp-2 and tracheal epithelial cells, expressed fimbriae (observed by electron microscopy), and had five plasmids of 2.7, 4.7, 43, 56, and 88 MDa. Transposon mutagenesis of strain SEPT13, with transposon TnphoA, resulted in a mutant strain named ST16 that had a LD₅₀ of 1.2 × 10¹² CFU/ml. All other biological characteristics of strain ST16 were the same as those detected for strain SEPT13 except for the migration of an 88 MDa plasmid to the 93 MDa position indicating the insertion of the transposon into the 88 MDa plasmid. The 93 MDa plasmid of strain ST16 was transferred, by electroporation assay, to non-pathogenic receptor strains (E. coli strains K12 MS101 and HB101), resulting in transformant strains A and B, respectively. These strains exhibited adhesion properties to in vitro cultivated HEp-2 cells but did not have the capacity for invasion. The adherence occurred despite the absence of fimbriae; this finding suggests that the 88 MDa plasmid has afimbrial adhesin genes.     

Genotype-environment interaction in Cordia trichotoma (Vell.)Arráb.Ex Steud.progenies in two different soil conditions

Investment in silvicultural techniques is notice-ably lacking,especially in breeding programs for non-con-ventional wood species.Studying genotype × environment interaction (G × E) is essential to the development of breed-ing programs.Thus,this study aimed to estimate genetic diversity of and the effects of G × E interaction on two prog-eny tests of Cordia trichotoma,including the estimation of genetic gain and genetic diversity after selection.For the experiment,30 progenies of C.trichotoma were tested at two sites with differing soil textures.Diameter at breast height (1.30 m above soil surface,dbh-cm),total height,diameter at 30 cm from the soil,first branch height,and survival were all monitored for four years.Statistical devi-ance,best linear unbiased estimator,and harmonic mean of relative performance of genetic values (MHPRVG) were all calculated to predict breeding values,estimate genetic parameters,and analyze deviance.All quantified traits varied significantly among progenies by soil type,with greatest variation recorded for genetic variability.Heritability of the progenies led to predictions of genetic gain,ranging from 7.73 to 15.45％,for dbh at four years of age.The calculated decrease in genetic diversity after selection showed that this parameter should be monitored in subsequent breed-ing cycles.G × E was low for all tests.The best-performing progenies proved most stable and best adapted to the differ-ent environmental conditions tested.     

A new type of pterocarpanquinone that affects Toxoplasma gondii tachyzoites in vitro

Toxoplasma gondii, the agent of Toxoplasmosis, is an obligate intracellular protozoan able to infect a wide range of vertebrate cells, including nonprofessional and professional phagocytes. Therefore, drugs must have intracellular activities in order to control this parasite. The most common therapy for Toxoplasmosis is the combination of sulfadiazine and pyrimethamine. This treatment is associated with adverse reactions, thus, the development of new drugs is necessary. In previous studies, naphthoquinone derivatives showed anti-cancer activity functioning as agents capable of acting on groups of DNA, preventing cancer cells duplication. These derivatives also display anti-parasitic activity against Plasmodium falciparum and Leishmania amazonensis. The derivative pterocarpanquinone tested in this work resulted from the molecular hybridization between pterocarpans and naphtoquinone that presents anti-tumoral and anti-parasitic activities of lapachol. The aim of this work was to determine if this derivative is able to change T. gondii growth within LLC-MK2 cells. The drug did not arrest host cell growth, but was able to decrease the infection index of T. gondii with an IC(50) of 2.5 μM. Scanning and transmission electron microscopy analysis showed morphological changes of parasites including membrane damage. The parasite that survived tended to encyst as seen by Dolichos biflorus lectin staining and Bag-1 expression. These results suggest that pterocarpanquinones are drugs potentially important for the killing and encystment of T. gondii.     

Light and scanning electron microscopy of sporocysts of Eurytrema coelomaticum (Giard et Billet, 1892) Looss, 1907

CD154 is a cell surface molecule expressed by activated T cells. CD40 and CD154 interaction is critically important in regulating humoral and cell-mediated immune responses. In this study we have investigated whether a DNA vaccine encoding rhoptry protein 1 (ROP1) of Toxoplasma gondii, and encoding ovine CD154 induces an enhanced ROP1-specific immune response in sheep. Two groups of twelve animals received two intramuscular injections, of a DNA plasmid encoding T. gondii ROP1 antigen (group 1) or an ROP1 antigen fused to ovine CD154 (group 2). There were two control groups of sheep. One was injected with an empty vector (group 3) and the other received no injections at all (group 4). The injection of the plasmid containing ROP1 (group 1) at weeks 0 and 4 induced a significant IgG2 response at week 2 which was amplified at week 4 after the booster injection and persisted to week 8 compared to the control animals in groups 3 and 4. For IgG1, significant differences from the control animals were only observed from week 5 onwards. The fusion of CD154 and ROP1 elicited significant IgG1 and IgG2 responses from week 1 which were amplified from weeks 5 to 8 compared to the control animals in groups 3 and 4. The IgG1 response was significantly higher in group 2 animals receiving pROP1-CD154 compared to group 1 receiving pROP1 only. There was no significant difference in IgG2 responses between groups 1 and 2. Significant differences in IFN-γ levels were only observed in treatment group 1 at week 2 and treatment group 2 at weeks 1 and 2 compared to the control animals. The results demonstrated that an intramuscular injection of pROP1-CD154 gene to sheep significantly enhanced their immune response and induced a mixed Th1/Th2 response while the intramuscular injection of pROP1 only induced a Th1-specific immune response.     

A new species of Trichuris from Thrichomys apereoides (Rodentia: Echimyidae) in Brazil: Morphological and histological studies

Trichuris thrichomysi n. sp., recovered from the cecum of the wild rodent Thrichomys apereoides from a transition zone between the Atlantic Forest and Cerrado morfoclimatic domains, and its life cycle observed under experimental conditions are described. This new species is closely related to Trichuris travassosi, Trichuris chiliensis and Trichuris fulvi, but can be distinguished from them mainly by differences in the posterior end of males. Details of the surface such as the bacillary gland, cuticular inflations and several morphological details obtained by scanning electron microscopy and field emission scanning electron microscopy confirmed the characteristics that differentiate the new species. The histopathology of the intestinal wall of naturally infected rodents is also reported. The present study extends the geographical distribution of T. thrichomysi n. sp to the Pantanal ecosystem and reports a new host, Thrichomys pachiurus.