Detection of virulence-associated genes in pathogenic and commensal avian Escherichia coli isolates

Poultry colibacillosis due to Avian Pathogenic Escherichia coli (APEC) is responsible for several extra-intestinal pathological conditions, leading to serious economic damage in poultry production. The most commonly associated pathologies are airsacculitis, colisepticemia, and cellulitis in broiler chickens, and salpingitis and peritonitis in broiler breeders. In this work a total of 66 strains isolated from dead broiler breeders affected with colibacillosis and 61 strains from healthy broilers were studied. Strains from broiler breeders were typified with serogroups O2, O18, and O78, which are mainly associated with disease. The serogroup O78 was the most prevalent (58%). All the strains were checked for the presence of 11 virulence genes: 1) arginine succinyltransferase A (astA); ii) E. coli hemeutilization protein A (chuA); iii) colicin V A/B (cvaA/B); iv) fimbriae mannose-binding type 1 (fimC); v) ferric yersiniabactin uptake A (fyuA); vi) iron-repressible high-molecular-weight proteins 2 (irp2); vii) increased serum survival (iss); viii) iron-uptake systems of E. coli D (iucD); ix) pielonefritis associated to pili C (papC); x) temperature sensitive haemaglutinin (tsh), and xi) vacuolating autotransporter toxin (vat), by Multiplex-PCR. The results showed that all genes are present in both commensal and pathogenic E. coli strains. The iron uptake-related genes and the serum survival gene were more prevalent among APEC. The adhesin genes, except tsh, and the toxin genes, except astA, were also more prevalent among APEC isolates. Except for astA and tsh, APEC strains harbored the majority of the virulence-associated genes studied and fimC was the most prevalent gene, detected in 96.97 and 88.52% of APEC and AFEC strains, respectively. Possession of more than one iron transport system seems to play an important role on APEC survival.

Dual layer solid phase extraction based on molecular imprinting technology: Seeking a route to enhance selectivity for trace analysis of pesticide residues in olive oil.

Aiming to introduce a multiresidue analysis for the trace detection of pesticide residues belonging to organophosphorus and triazine classes from olive oil samples, a new sample preparation methodology comprising the use of a dual layer of “tailor-made” molecularly imprinted polymers (MIPs) SPE for the simultaneous extraction of both pesticides in a single procedure has been attempted. This work has focused on the implementation of a dual MIP-layer SPE procedure (DL-MISPE) encompassing the use of two MIP layers as specific sorbents. In order to achieve higher recovery rates, the amount of MIP layers has been optimized as well as the influence of MIP packaging order. The optimized DL-MISPE approach has been used in the preconcentration of spiked organic olive oil samples with concentrations of dimethoate and terbuthylazine similar to the maximum residue limits and further quantification by HPLC. High recovery rates for dimethoate (95%) and terbuthylazine (94%) have been achieved with good accuracy and precision. Overall, this work constitutes the first attempt on the development of a dual pesticide residue methodology for the trace analysis of pesticide residues based on molecular imprinting technology. Thus, DL-MISPE constitutes a reliable, robust, and sensitive sample preparation methodology that enables preconcentration of the target pesticides in complex olive oil samples, even at levels similar to the maximum residue limits enforced by the legislation.

A real time Taqman RT-PCR for the detection of rabbit hemorrhagic disease virus 2 (RHDV2)

As key prey, the wild rabbit downsize constitutes a major drawback on the endangered Iberian lynx (Lynx pardinus) re-introduction in the Iberia. Several captive breeding units mostly located in Alentejo, endeavour the wild rabbit repopulation of depleted areas assigned for the lynx re-introduction. Here we report an RHDV2 outbreak that occurred in early 2016 in a wild rabbit captive breeding unit located in Barrancos municipality. The estimated mortality rate between March and April 2016 was approximately 8.67%. Anatomopathologic examination was carried out for 13 victimized rabbits. Molecular characterization was based on the complete vp60 capsid gene. The 13 rabbit carcasses investigated showed typical macroscopic RHD lesions testing positive to RHDV2- RNA. Comparison of the vp60 nucleotide sequences obtained from two specimens with others publically available disclosed similarities below 98.22% with RHDV2 strains originated in the Iberia and Azores and revealed that the two identical strains from Barrancos-2016 contain six unique single synonymous nucleotide polymorphisms. In the phylogenetic analysis performed, the Barrancos-2016 strains clustered apart from other known strains, meaning they may represent new evolutionary RHDV2 lineages. No clear epidemiological link could be traced for this outbreak where the mortalities were lower compared with previous years. Yet, network analysis suggested a possible connection between the missing intermediates from which the strains from Barrancos 2013, 2014 and 2016 have derived. It is therefore possible that RHDV2 has circulated endemically in the region since 2012, with periodic epizootic occurrences. Still, six years after its emergence in wild rabbits, RHDV2 continues to pose difficulties to the establishment of natural wild rabbit populations that are crucial for the self-sustainability of the local ecosystems.

Challenges in the rabbit haemorrhagic disease 2 (RHDV2) molecular diagnosis of vaccinated rabbits

Molecular methods are fundamental tools for the diagnosis of viral infections. While interpretation of results is straightforward for unvaccinated animals, where positivity represents ongoing or past infections, the presence of vaccine virus in the tissues of recently vaccinated animals may mislead diagnosis. In this study, we investigated the interference of RHDV2 vaccination in the results of a RT-qPCR for RHDV2 detection, and possible associations between mean Cq values of five animal groups differing in age, vaccination status and origin (domestic/wild). Viral sequences from vaccinated rabbits that died of RHDV2 infection (n = 14) were compared with the sequences from the commercial vaccines used in those animals. Group Cq means were compared through Independent t-test and One-way ANOVA. We proved that RHDV2 vaccine-RNA is not detected by the RT-qPCR as early as 15 days post- vaccination, an important fact in assisting results interpretation for diagnosis. Cq values of vaccinated and non-vaccinated infected domestic adults showed a statistically significant difference (p < 0.05), demonstrating that vaccination-induced immunity reduces viral loads and delays disease progression. Contrarily, in vaccinated young rabbits higher viral loads were registered compared to non-vaccinated kittens. No significant variation (p = 0.3824) was observed between viral loads of non- vaccinated domestic and wild RHDV2-victimised rabbits. Although the reduced number of vaccinated young animals analysed hampered a robust statistical analysis, this occurrence suggests that passively acquired maternal antibodies may inhibit the active immune response to vaccination, delaying protection and favouring disease progression. Our finding emphasises the importance of adapting kitten RHDV2 vaccination schedules to circumvent this interference phenomenon.

First detection of Bursaphelenchus luxuriosae associated with Pinus pinaster in Portugal

During a field survey carried out on symptomatic maritime pine trees (Pinus pinaster) in Góis, central Portugal, Bursaphelenchus luxuriosae was isolated for the first time in Portugal, and in Europe. Identification of the nematodes was based on morphological characters and molecular analyses for this species. The general morphology of both females and males is in agreement with the original description for B. luxuriosae, namely the typical morphology of the male spicules and the conspicuous morphology of female tail. Species identification was confirmed through sequencing of the ITS rDNA region and the fragment spanning the D2/D3 domain of the 28S rDNA gene. This species belongs to the xylophilus-group and is the third species in this group known in Portugal. The nematodes were retrieved in small numbers (<100 nematodes/100 g dry wood), and no insect vector association could be established.