PCR Multiplex fluorescente para detecção de bactérias em sêmen bovino

Este estudo teve como objetivo avaliar o limiar de detecção da técnica de PCR multiplex fluorescente aliada a eletroforese capilar na detecção de agentes infecciosos em amostras de sêmen experimentalmente contaminadas com concentrações decrescentes das bactérias Brucella abortus, Leptospira interrogans sorovar pomona, Campylobacter fetus e Haemophilus somnus. Amostras de sêmen bovino foram experimentalmente contaminadas com concentrações decrescentes de bactérias obtidas através de diluições seriadas na base 10 de modo a obter-se amostras contendo desde 1 vez até 10-7 bactérias/mL a partir da concentração inicial de Leptospira pomona, Brucella abortus, Campylobacter fetus e Haemophilus somnus. As diluições foram efetuadas individualmente para cada bactéria, bem como nas diferentes concentrações necessárias para a padronização do teste de multiplex PCR. As extrações de DNA de todas as soluções contendo espermatozóides e bactérias analisadas no presente estudo foram realizadas segundo protocolo descrito por Heinemann et al. (2000). Os produtos de PCR multiplex foram avaliados por eletroforese em gel de poliacrilamida 8% e separação eletroforética por sistema capilar em equipamento automático de análise de fragmentos de DNA MegaBace. Observou-se a amplificação de fragmentos de 193pb, 330pb, 400pb e 415pb a partir do DNA de B. abortus, L. pomona, H. somnus, C. fetus, respectivamente. Na análise por eletroforese capilar de produtos da PCR multiplex do DNA para detecção simultânea dos quatro patógenos observou-se a sinal de positividade até a diluição de 10-3 bactérias/mL vezes da concentração inicial da solução estoque de cada bactéria. A técnica de PCR multiplex aliada à eletroforese capilar foi usada pela primeira vez para o diagnóstico direto de quatro bactérias patogênicas no sêmen, demonstrando ser um método rápido na detecção de bactérias causadoras de doenças reprodutivas

Identification of intimin alleles in pathogenic Escherichia coli by PCR-restriction fragment length polymorphism analysis

International audienc

Molecular evolution of psittacine beak and feather disease virus (PBFDV) isolated from african grey parrots (Psittacus erithacus) reared in Italy.

The purpose of this study was to molecularly characterize BFDV strains isolated in Italy from African grey parrots affected by peracute or chronic form of BFDV disease. To our knowledge this is the first survey of BFDV infection in Italy in which, together with the classical sequence comparison analyses, we attempted to associate genetic features of the viruses with clinical forms of the illness. Mixed organs from 8 animals dead displaying peracute form of BFDV disease and blood from 2 animals harbouring chronic signs of this illness were collected. These animals were reared in 8 different breeding facilities located in North and Central Italy. The full‐length genomes of BFDV strains were analyzed using 4 primer sets that amplify overlapping DNA stretches. These fragments were sequenced and aligned with all known BFDV sequences available on GenBank using the program ClustalW (1). Bayesian methods implemented in the computer program MrBayes ver. 3.1.1 (2), were used to draw phylogenetic trees and assess statistical support for clades. All genomes contained the characteristic nonanucleotide circovirus origin of replication sequence located within a stem loop structure and the conserved motifs located within the 2 major open reading frames (ORFs). A peculiar feature of the BFDV viruses we analyzed was the presence of 5 to 6 ORFs, in contrast with published data that ascribed to BFDV genome the presence up to 7 ORFs. ORF6 was always absent. The whole nucleotide sequence identity among our isolates varied from 93.6% to 99.9% and genome sizes extended from 1,997 to 2,001 nucleotides. Full genome analysis showed that the DNA isolated from 8 animals fall into 2 subtypes of the BFDV‐J strain, namely J1 and J2, which share a 98 to 100% intra‐subtype identity, while the sequences of BFDV viruses obtained from other 2 animals, based on the classification system proposed by Varsani et al. (2011), seem to cluster into 2 new subtypes, which we defined as J4 and J5. Finally, by phylogenetic analysis, we observed a correlation among viral strains derived from the same breeding, whereas no association between phylogenetic distribution and clinical symptoms or geographical location of the breeding was noticed. Our study shows that the complete genomes of BFDV strains isolates in Italy grouped into BFDV‐J strain, according to the fact that this is the main strain infecting African grey parrots in Europe. Moreover, even if we were not able to discern an association between genetic feature of the virus and form of the illness, we identify two new circovirus subtypes, namely J4 and J5 characteristic of Italian BFDV viruses

Survey of Infectious Etiologies of Bovine Abortion during Mid- to Late Gestation in Dairy Herds.

Bovine abortion of unknown infectious etiology still remains a major economic problem. Thus, we investigated whether Brucella spp., Listeria monocytogenes, Salmonella spp., Campylobacter spp. and Coxiella burnetii are associated with abortion and/or stillbirth in Tunisian dairy cattle. Using a pan-Chlamydiales PCR, we also investigated the role of Chlamydiaceae, Waddlia chondrophila, Parachlamydia acanthamoebae and other members of the Chlamydiales order in this setting. Veterinary samples taken from mid to late-term abortions from twenty dairy herds were tested. From a total of 150 abortion cases collected, infectious agents were detected by PCR in 73 (48.66%) cases, 13 (8.66%) of which represented co-infections with two infectious agents. Detected pathogens include Brucella spp (31.3%), Chlamydiaceae (4.66%), Waddlia chondrophila (8%), Parachlamydia acanthamoebae (5.33%), Listeria monocytogenes (4.66%) and Salmonella spp. (3.33%). In contrast, Campylobacter spp. and Coxiella burnetii DNA were not detected among the investigated veterinary samples. This demonstrates that different bacterial agents may cause bovine abortion in Tunisia. This is the first report suggesting the role of Parachlamydia acanthamoebae in bovine abortion in Africa. Further studies with a larger number of samples are necessary to confirm whether this emerging pathogen is directly linked to abortion in cattle

Pharmacokinetic and pharmacodynamic evaluations of levofloxacin in broiler chickens

Title: Pharmacokinetic and pharmacodynamic evaluations of levofloxacin in broiler chickens Objective: To evaluate the PK/PD and residues of levofloxacin (L) after IV and oral administrations in broiler chickens. Materials & Methods: 65 healthy broiler chickens were randomly divided into 4 groups (A n=20, B n=20, C n=20 and D n=5). Groups A, B and C received 5 mg/kg of L (A and B oral, C IV) while D was the control group. Blood samples were withdrawn at different scheduled times for the group A and C, while animals in group B were sub-divided in to 5 sub-groups (n=4) and euthanized for organ (liver, kidney, muscle, lung) collections at different times (1, 6, 10, 24, 48 h). L concentrations in blood and tissues were quantified by validated HPLC-FL method. PK data of L was fitted using a non-compartment model. As PD studies, two field E. coli isolates were collected from cloacal swabs. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) against E.coli (isolated in clinical broilers) were determined in broth and in serum obtained from the healthy animals using the microdilution method according to Clinical and Laboratory Standards Institute (CLSI) guidelines. In vitro and ex vivo anti-bacterial effects of L were determined using time killing curve. Solutions of L in serum (0.0, 0.03, 0.06, 0.125, 0.25, 0.5 and 1 μg/ml) and serum samples (10, 30 min, 1, 4, 6, 10, 24 and 48 h) were used for the determination of in vitro and ex vivo antibacterial effects against E. coli. In vivo Cmax/MIC and AUC24h/MIC, were calculated by linking PK data with PD index (MIC) for serum after oral administration of L in clinically isolated E. coli. Ex vivo AUC24h/MIC were obtained after 24h incubation from ex vivo time killing curve and fitted using the sigmoidal inhibitory Emax model. Using PK-PD modelling, the optimal oral dose in broilers were predicted. Results & Conclusion: The pharmacokinetic profiles of L after oral and IV administrations were almost overlapping in the elimination phase. The oral F% was 124±39. All the tested organs contained quantifiable L concentrations up to 48 hours. The liver was the most contaminated organ then in descending order kidney, lung and muscle. The results of ex vivo growth inhibition curves were consistent with the in vitro time-kill study. Plasma concentration above 8xMIC led to eradication of E.coli. The MIC and MBC of L in MHB and serum were 0.03, 0.06 and 0.125 μg/mL, respectively. The pharmacokinetic profiles of levofloxacin were similar with those in previous studies in chickens and calves. Levofloxacin showed plasma concentration dependent antibacterial activities against clinical E.coli. A dose of 5 mg/kg of L appears to be able to kill E.coli. Keywords: broiler chickens, levofloxacin, pharmacokinetics, pharmacodynamics, residues References: 1. Giorgi, M., Rota, S., Giorgi, T., Capasso, M., Briganti, A., 2013. Journal of Exotic Pet Medicine 22, 192-199