Single chain antibodies against ‘Ca. Liberibacter asiaticus’

Antibodies are widely used as microbiological reagents, but antibodies that recognize ‘Ca. Liberibacter asiaticus’ are generally lacking.  We have developed and applied immunization and affinity screening methods to create a primary library of recombinant single chain variable fragment (scFv) antibodies in an M13 vector, pKM19.  The antibody population is enriched for antibodies that bind antigens of ‘Ca. Liberibacter asiaticus’ and Diaphorina citri.  The primary library has more than 107 unique antibodies and the genes that encode them.  We have screened this library of antibodies for antibodies that bind to specifically chosen proteins that are present on the surface of ‘Ca. Liberibacter asiaticus’.  These proteins were used as ‘bait’ for affinity-based selection of scFvs that bind to the major outer membrane protein, OmpA; the polysaccharide capsule expressing protein KpsF; a protein component of the type IV pilus (CapF); and two flagellar proteins FlhA and FlgI.  These scFvs have been used in ELISA and dot blot assays against purified protein antigens and ‘Ca. Liberibacter asiaticus’ infected plant extracts.  We also have isolated scFv that bind to surface exposed portions of the TolC proteins and of a protein called InvA.  These proteins may have critical roles in pathogenicity.  Thus far, screening of these scFvs is more efficient when using phage bound, rather than soluble scFvs.  We have demonstrated a technology to produce antibodies and select at will and against any protein target encoded by ‘Ca. Liberibacter asiaticus’.  Future applications will include advanced diagnostic methods for huanglongbing and the development of immune labeling reagents for in planta applications

Identification of a non-host plant of Xylella fastidiosa to rear healthy sharpshooter vectors Identificação de uma planta não-hospedeira de Xylella fastidiosa para criação de insetos vetores sadios

Rearing leafhopper (Hemiptera: Cicadellidae) vectors free of Xylella fastidiosa is a requirement for studies of various aspects of vector-pathogen interactions. The selection of a plant that allows vector development but not bacterial multiplication is desirable to produce healthy vectors. In this study, two leafhopper hosts, Vernonia condensata ('boldo') and Aloysia virgata ('lixeira') were needle inoculated with citrus and coffee strains of X. fastidiosa to evaluate if these plants support pathogen colonization. The inoculated plants did not present symptoms and the pathogen was not detected by culture and PCR tests, neither soon after inoculation (7-14 days) nor later, at 1, 4, 6 and 12 months after inoculation. To obtain healthy adults of the leafhopper vectors Acrogonia citrina, Bucephalogonia xanthophis, Dilobopterus costalimai, Homalodisca ignorata and Oncometopia facialis, early-instar nymphs were reared on V. condensata. X. fastidiosa was not detected in any of 175 adults obtained. V. condensata and A. virgata are nonpropagative hosts of X. fastidiosa and enable the production of healthy leafhoppers for vector studies.<br>A obtenção de cigarrinhas (Hemiptera: Cicadellidae) livres de Xylella fastidiosa é importante para estudos de interação entre essa bactéria e seus vetores, sendo desejável a seleção de uma planta que permita a criação desses insetos, mas não a multiplicação da bactéria. Neste estudo, duas plantas hospedeiras de cigarrinhas, Vernonia condensata (boldo) e Aloysia virgata (lixeira), foram inoculadas por agulha com as estirpes de citros e de cafeeiro de X. fastidiosa, para avaliar a possibilidade deste patógeno colonizá-las. Não foram observados sintomas, nem se detectou a bactéria por isolamento em meio de cultura e/ou PCR em períodos curtos (7 e 14 dias) ou longos (1, 4, 6 e 12 meses) após a inoculação. Para obtenção de adultos sadios das cigarrinhas vetoras, Acrogonia citrina, Bucephalogonia xanthophis, Dilobopterus costalimai, Homalodisca ignorata e Oncometopia facialis, ninfas de primeiros ínstares foram criadas em plantas de boldo. Não foi detectada X. fastidiosa em nenhum de 175 adultos obtidos da criação. V. condensata e A. virgata não permitem a colonização de X. fastidiosa, possibilitando assim a obtenção de cigarrinhas sadias para estudos com vetores