Validation of a simple universal IELISA for the diagnosis of human brucellosis.

The definitive diagnosis of brucellosis requires isolation of the agent, although negative isolation does not rule out the infection. In contrast, serological testing is more sensitive and, therefore, preferred in clinical practice. The majority of reported cases around the world were caused by Brucella melitensis, B. abortus, B. suis and B. canis. The first three species contain O-polysaccharide (OPS) on the cell surface, but B. canis contains no measurable OPS on the rough lipopolysaccharide (R-LPS). A universal indirect enzyme immunoassay for the detection of serum antibody to smooth and rough Brucella spp. in both normal (u-IELISA®) and rapid forms (R-u-IELISA®) has been developed, and, therefore, the potential use of this method was assessed in comparison to cELISA, conventional tests, IELISA and RSAT on a total of 478 sera. The 77 sera from blood donors with no clinical or epidemiological evidence of brucellosis and negative serological tests showed a specificity of 100 % for both u-IELISA® and R-u-IELISA®, with a cut-off value of %P 24 and %P 18, respectively. Sera from 49 culture-positive cases (16 B. suis, 15 B. abortus, 12 B. melitensis and 6 B. canis) yielded a sensitivity of 98 % for u-IELISA® and 95.9 % for R-u-IELISA®. In general, u-IELISA® showed good correlation with cELISA and IELISA for the detection of antibodies to smooth and rough Brucella strains, as well as for monitoring patients during treatment, but R-u-IELISA® seems to need additional optimisation. u-IELISA® is simple to perform and could be a suitable test for field laboratories and hospitals lacking skilled personnel.

Genetic transformation of selected mature cork oak (Quercus suber L.) trees.

A transformation system for selected mature cork oak (Quercus suber L.) trees using Agrobacterium tumefaciens has been established. Embryos obtained from recurrent proliferating embryogenic masses were inoculated with A. tumefaciens strains EHA105, LBA4404 or AGL1 harbouring the plasmid pBINUbiGUSint [carrying the neomycin phosphotransferase II (nptII) and beta-glucuronidase (uidA) genes]. The highest transformation efficiency (4%) was obtained when freshly isolated explants were inoculated with A. tumefaciens strain AGL1. Evidence of stable transgene integration was obtained by PCR for the nptII and uidA genes, Southern blotting and expression of the uidA gene. The transgenic embryos were germinated and successfully transferred to soil.

Vegetative propagation of Quercus suber L. by somatic embryogenesis. II. Plant regeneration from selected cork oak trees.

The regeneration of somatic seedlings from selected 100-year-old cork oak trees is reported. The induction of somatic embryogenesis from leaves of epicormic shoots was significantly affected by genotype, harvesting time and their interaction. Leaves from all five selected trees produced somatic embryos when the segments of branches used as sources of epicormic shoots were collected in May. Genotype, but not the level of photosynthetically active radiation, affected the proliferation of the embryogenic lines and the number of detachable embryos that could be obtained from them. Genotype also affected several steps leading to conversion of somatic embryos, from germination to complete acclimatisation of somatic seedlings. Almost 40% of the somatic embryos from all lines germinated, showing coordinated root and shoot growth. Although the mean percentage of recovery for the whole process was low, plants could be regenerated from four of the five trees tested.

Vegetative propagation of Quercus suber L. by somatic embryogenesis. I. Factors affecting the induction in leaves from mature cork oak trees.

Somatic embryogenesis was induced in expanding leaves from epicormic shoots forced to sprout from segments of branches collected from several hundred-year-old cork oak trees. Following a basic protocol previously defined for leaves taken from seedlings of this species, several factors were studied to improve the response. The induction frequency was significantly higher when the length of exposure to growth regulators was increased from 7 to 30 days. The combined application of NAA and BAP was essential for induction. Although both regulators had a very significant influence, their interaction was not significant, suggesting independent roles. Leaf size had a crucial effect, because beyond a certain threshold, embryogenesis could not be obtained. Embryogenic lines were maintained via repetitive embryogenesis on hormone-free medium for more than 2 years.

Histology of Organogenic and Embryogenic Responses in Cotyledons of Somatic Embryos of Quercus Suber L.

In cork oak (Quercus suber L.), recurrent embryogenesis is produced in vitro through autoembryony without exogenous plant growth regulators (PGRs); secondary embryos appear on the embryo axis but seldom on cotyledons. Focusing mainly on the histological origin of neoformations, we investigated the influence of the embryo axis and exogenous PGRs on the embryogenic potential of somatic embryo cotyledons. Isolated cotyledons of somatic embryos became necrotic when cultured on PGR-free medium but gave secondary embryos when cultured on media containing benzyladenine and naphthaleneacetic acid. Cotyledons of cork oak somatic embryos are competent to give embryogenic responses. Isolated cotyledons without a petiole showed a lower percentage of embryogenic response than did those with a petiole. In petioles, somatic embryos arose from inner parenchyma tissues following a multicellular budding pattern. Joined to the embryo axis, cotyledons did not show morphogenic responses when cultured on PGR-free medium but revealed budlike and phylloid formations when cultured on medium with PGRs. The different morphogenic behavior displayed by somatic cotyledons indicates an influence of the embryo axis and indicates a relationship between organogenic and embryogenic regeneration pathways.