Salmonella enterica serovar Choleraesuis derivatives harbouring deletions in rpoS and phoP regulatory genes are attenuated in pigs, and survive and multiply in porcine intestinal macrophages and fibroblasts, respectively

Live attenuated Salmonella enterica strains have been extensively studied as potential vectors for the oral delivery of heterologous antigens. Due to its ability to target immune cells, its specific mechanism for crossing the intestinal barrier, and its swine-restricted tropism, S. enterica subspecies enterica serovar Choleraesuis (S. Choleraesuis) has attracted a great deal of interest for the production of bacterial-based oral carriers specifically adapted to swine. In this study, two mutants of S. Choleraesuis were constructed and their attenuation and intracellular fate analysed with the purpose of engineering new attenuated live strains with improved properties as oral vaccine carriers. Those strains harboured a specific deletion either within the phoP or rpoS genes, which encode virulence-related regulators in S. Typhimurium. In comparison to the wild-type parental S. Choleraesuis, the mutant strains, especially DeltaphoP, were extremely low in virulence in the murine model and in the natural host, the pig. Moreover, when compared with a commercial live vaccine strain, SC-54, the two mutants showed a higher level of attenuation in mice and DeltaphoP also in pigs. In addition, DeltarpoS and DeltaphoP presented a proliferation and survival phenotype within swine intestinal primary fibroblast and macrophage cell cultures, respectively. Collectively, the present results indicate that the DeltarpoS and DeltaphoP strains of S. Choleraesuis gather adequate features to be potential candidates for vaccine vectors for the specific delivery of heterologous antigens adapted to pigs.     

Epidemiologic investigation of an outbreak of goose parvovirus infection in Sweden

An outbreak of goose parvovirus (GPV) infection on a Swedish goose farm in the spring of 2004 increased the mortality rates from 2% in the early unaffected hatches to 90% and 99% respectively in the two hatches following virus introduction and 40% in goslings hatched later in the same breeding season. In this paper we describe the clinical observations, diagnostic procedures, and epidemiologic investigation carried out to elucidate the source of the infection. The diagnosis was confirmed by serology, virus isolation, and sequence analysis of a 493-bp-long fragment of the VP1 gene. Phylogenetically the causative virus was closely related to pathogenic GPV strains isolated in 2003 and 2004 from Poland and the United Kingdom, respectively. The Swedish isolate exhibited less homology with pathogenic strains from Hungary and Asia and with attenuated vaccine strains. The epidemiologic investigation showed that the virus was first introduced to a contract farm (farm A) and then was transferred with newly hatched goslings to the farm that had submitted the birds for necropsy (index farm). The exact time and source of the virus introduction to farm A could not be determined with absolute certainty. Possible sources of the infection included backyard goose eggs that had been delivered to farm A for subcontract incubation and hatching, wild geese that frequented the flock of breeding geese on pasture on farm A, and a clutch of Canada goose eggs (Branta canadensis) that had been produced by wild geese and was hatched in the same machine as the eggs produced by farm A.     

Characterization of the immune response to Mycoplasma bovis lung infection

To better understand the interaction between Mycoplasma bovis and its bovine host, we have characterized the immune response generated during an experimental lung infection with M. bovis. Proliferation ([3H]-thymidine blastogenesis) and Th1/Th2 cytokine production were used to monitor peripheral cellular immune responses. Flow cytometry analysis was used to determine T-cell subset activity by CD25 expression. Humoral immune response was monitored by the identification of antigen-specific IgG1 and IgG2 isotypes over time. Herein, we show that M. bovis antigen stimulates activation of CD4+ and CD8+ cells in vitro in a manner consistent with memory, and that gammadelta-T cells are activated by antigen in a manner consistent with innate immunity. In addition, the percentage of cells producing IFN-gamma during recall response is equal to that of IL-4 producing cells. Serological analysis shows M. bovis stimulates increased production of antigen-specific IgG1 while very little IgG2 is produced. We therefore submit that experimental lung infection of cattle with M. bovis results in a Th2-skewed immune response.     

Validation of an ELISA method for the serological diagnosis of canine brucellosis due to Brucella canis

In the present study, the validation of an enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of canine brucellosis is described. Two different antigenic extracts, obtained by heat or ultrasonic homogenization of microbial antigens from a wild isolate of Brucella canis bacteria, were compared by ELISA and Western blot (WB). A total of 145 canine sera were used to define sensitivity, specificity and accuracy of the ELISA as follows: (1) sera from 34 animals with natural B. canis infection, confirmed by blood culture and PCR, as well as 51 sera samples from healthy dogs with negative results by the agar–gel immunodiffusion (AGID) test for canine brucellosis, were used as the control panel for B. canis infection; and (2) to scrutinize the possibility of cross reactions with other common dog infections in the same geographical area in Brazil, 60 sera samples from dogs harboring known infections by Leptospira sp., Ehrlichia canis, canine distemper virus (CDV), Neospora caninum, Babesia canis and Leishmania chagasi (10 in each group) were included in the study. The ELISA using heat soluble bacterial extract (HE-antigen) as antigen showed the best values of sensitivity (91.18%), specificity (100%) and accuracy (96.47%). In the WB analyses, the HE-antigen showed no cross-reactivity with sera from dogs with different infections, while the B. canis sonicate had various protein bands identified by those sera. The performance of the ELISA standardized with the heat soluble B. canis antigen indicates that this assay can be used as a reliable and practical method to confirm infection by this microorganism, as well as a tool for seroepidemiological studies.     

Identification and pathogenicity of Neophysopella species associated with Asian grapevine leaf rust in Brazil

Asian grapevine leaf rust (AGLR) causes severe crop losses in Brazilian viticulture, mainly in latitudes <25°S. The purpose of this study was to identify the pathogen(s) involved with AGLR in Brazil, based on phylogenetic and morphological analysis and pathogenicity tests. In total, 56 monouredinial isolates from six Brazilian states were identified using the internal transcribed spacer 2 and the large subunit rRNA gene D1/D2 regions. All 50 isolates from the south-central region were classified as Neophysopella tropicalis, and the other six isolates from the north-east region as Neophysopella meliosmae-myrianthae. This result provides evidence that two pathogen introductions from different sources may have occurred in the country. For both species, paraphyses were cylindrical, incurved, aseptate, and hyaline, while urediniospores were short-pedicellate, obovoid or obovoid-ellipsoid, with the wall colourless or pale yellowish, evenly echinulate. Representative isolates from both species caused typical AGLR symptoms on Vitis vinifera 'Merlot' and V. labrusca 'Niagara Rosada'. Overall, regardless of the Neophysopella species, isolates caused similar leaf disease severities. Higher disease severity was observed in Niagara Rosada (average of 40.3% of diseased leaf area) compared to Merlot (20.5%). This study reports, for the first time, the characterization of Neophysopella species associated with AGLR in Brazil.     

Sensitivity of Lasiodiplodia theobromae from Brazilian papaya orchards to MBC and DMI fungicides

Stem rot caused by Lasiodiplodia theobromae is an important postharvest disease of papaya in Brazil, responsible for reducing the quality and quantity of fruits. Fungicide use is one of the main disease management measures. However, there are no estimates available of pathogen sensitivity to commonly employed fungicides. Therefore, the EC₅₀ from 120 isolates of L. theobromae from northeastern Brazil, representative of six populations of the pathogen, was estimated in vitro for fungicides of the methyl benzimidazole carbamates—MBC (benomyl and thiabendazole) and demethylation-inhibiting—DMI (imazalil, prochloraz, tebuconazole) groups. Mycelial growth on fungicide-free media and virulence on papaya fruits of the MBC-sensitive and non-sensitive isolates were compared. For MBCs, 8.4% of isolates were non-sensitive to fungicides. For the remaining 91.6%, the mean EC₅₀ ranged from 0.002 to 0.13 μg ml⁻¹ and 0.36 to 1.27 μg ml⁻¹ for benomyl and thiabendazole, respectively. For DMIs, the mean EC₅₀ range for imazalil was 0.001 to 2.27 μg ml⁻¹, 0.04 to 1.75 μg ml⁻¹ for prochloraz, and 0.14 to 4.05 μg ml⁻¹ for tebuconazole. The EC₅₀ values of non-sensitive isolates were significantly (P≤0.05) higher those for the sensitive isolates for each of the DMI fungicides. Differences (P≤0.05) were found in the levels of sensitivity to DMI fungicides among the isolate populations associated with orchards. The populations from two orchards were less sensitive to DMIs. No solid evidence was found for fitness costs relating to MBC non-sensitive isolates because mycelial growth in fungicide-free media and virulence on papaya fruits were similar to those of sensitive isolates.     

Factors involved in Agrobacterium tumefaciens-mediated gene transfer into Pinus nigra Arn. ssp. salzmannii (Dunal) Franco

Cotyledons from dissected sterile embryos of salgareño pine (Pinus nigra Arn. ssp. salzmannii (Dunal) Franco) were inoculated with different disarmed Agrobacterium tumefaciens strains harbouring the binary vector p35SGUSint. The transient expression of a β-glucuronidase gene (uidA) was studied, using a histochemical staining procedure. Nineteen days after inoculation, the activity of β-glucuronidase was detected in epidermal and subepidermal layers of cotyledonary explants. The EHA105 strain harbouring a disarmed agropine-type Ti-plasmid (pTiBO542) was the most effective for gene transfer of the uidA gene. The effects of exudates and extracts from 0-day-old embryos on induction of vir gene expression in A. tumefaciens were also examined. The results of this study showed that salgarño pine embryo exudates contain a substance(s) that induce vir gene expression, in similar way to that observed with 100 μM acetosyringone (AS).All these findings suggest that T-DNA processing and transfer might take place when Agrobacterium infects suitable tissues of salgareño pine.