Sections were clogged immediately with normal goat and rabbit serumand incubated with main antibodies

The samples analyzed using this duplex PCR assay may consist of a pure cell culture isolated on solid medium or of plant extract samples in molecular biology-grade water. The detection of Moko and IIB-4NPB strains may be performed using either symptomatic or latently infected plants, as the concentration within xylem vessels is usually greater than the 105 CFU/mL threshold estimated in this study. Moreover, the new Brazilian Moko-related strains were successfully detected with this duplex PCR assay, which was even able to distinguish Brazilian Moko-related strains from non-Moko strains of sequevars IIB-25 and IIA- 41 and to detect the new sequevar IIA-53. Selectivity assessment showed that no cross-amplification occurred with different banana cultivars, whether Cavendish or plantain. In the fulllength protocol using a semi-selective medium and a specific PCR approach, the detection and characterization of all strains was achieved, providing a high confidence level for the integration of this protocol into official detection schemes to complete or confirm results obtained by other diagnostic technologies. The design of Moko-specific primers first involved a genomic comparison between Moko strains and other Rssc and non-Rssc strains to obtain an optimal gene repertoire for diagnosis. The Moko lineage clusters into seven sequevars belonging to phylotype IIA and IIB: the previously reported Moko sequevars IIA-6, IIA-24, IIB-3, and IIB-4, which have been sequenced, and the newly described sequevars IIA-41, IIA-53, and IIB-25, for which sequenced genomes are not yet available. The paraphyletic nature of the Moko lineage makes the search for specific gene repertoires difficult to manage, as other non-related Moko sequevars could be genetically closer while belonging to another lineage. Unsurprisingly, given the close phylogenetic proximity between IIB-4 Moko strains and IIB-4NPB strains, no locus was found to be specific only to Moko strains; thus, IIB-4NPB strains were added to the genomic search against other Rssc and non-Rssc genomes. This similarity is consistent with the phylogenetic position of the Moko IIB-4 strains as indistinguishable from IIB-4NPB strains. We could then assume that NPB strains share almost all of the genes of the Moko lineage. Nevertheless, the IIB-4NPB strains have a specific SP600125 129-56-6 accessory repertoire of genes, in which a specific IIB-4NPB marker was found. A total of two coding sequences were found to be suitable for primer design. The 93F/R primer set was based on the specific gene repertoire shared by both the Moko and NPB strains, while the 5F/R primer set was based solely on the specific gene repertoire of the IIB-4NPB strains. Ecologically, these two lineages are different but share common traits, such as the ability to invade plantain banana xylem vessels. The clinical manifestations of early Lyme disease are most often characterized by an erythema migrans rash often accompanied by flu-like symptoms. An inflammatory arthritis or neurological dysfunction can be frequent sequelae of untreated infection. Although the majority of patients with Lyme disease can be cured with antibiotics doxycycline or amoxicillin used for 2-4 weeks durations, a subset of patients experience persistent symptoms despite antimicrobial therapy including fatigue, neurocognitive difficulties or musculoskeletal pains. When symptoms last longer than 6 months after antibiotic treatment, this has been proposed as a non-infectious, post-treatment Lyme disease syndrome due to the inability to find viable, remaining organisms and lack of substantial efficacy with longer term monotherapy with ceftriaxone, doxycycline or amoxicillin. It is unclear what mechanisms promulgate this condition in these patients. Concepts raised have included host responses, although slow or ineffective killing of B. burgdorferi persisters has been voiced as a possible explanation though evidence of viable organisms present in PTLDS is lacking. While PTLDS has only subjective symptom complexes, about 10% of patients with late Lyme arthritis have objective, persistent joint swelling despite antibiotic therapy. Though part of this response may include autoimmune mimicry induced by B. burgdorferi in certain hosts, an additional explanation rests on immunological responses driven by continued infection or presence of antigenic debris. The question of whether B. burgdorferi might still persist in some patients with suboptimal immune clearance after antibiotic therapy and further evade host immune clearance has been raised by some but is controversial. In various animal models such as mice, dogs and monkeys, antibiotic therapy with doxycycline, ceftriaxone or tigecycline could not fully eradicate detection of B. burgdorferi including by xenodiagnosis though viable organisms could not be cultured in conventional culture medium.

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