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

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

Observations on Antricola ticks: small Nymphs feed on mammalian hosts and have a salivary gland structure similar to Ixodid ticks

Ticks use bloodmeals as a source of nutrients and energy to molt and survive until the next meal and to oviposit, in the case of females. However, only the larvae of some tick species are known to feed upon bats; females are obligatorily autogenous, and nymphal stages are believed to not feed. We investigated the presence of blood in a natural population of nymphal Antricola delacruzi ticks collected from bat guano; their ability to feed upon laboratory hosts; and the microscopic structure of both salivary glands and gut. DNA amplification of gut contents of freshly collected material was positive for a mammal in 4 of 11 first instar nymphs, but we were unsuccesful in the amplification of host bloodmeal DNA from late instar nymphs. All early nymphal stages (n = 10) fed on rabbits, and host DNA was detected and sequenced from gut contents. However, all the large nymphs (n = 10) rejected feeding, and host DNA remained undetected in these ticks. All stages of A. delacruzi have salivary glands similar in morphology to the ixodid agranular Type I salivary gland acini and to granular Type II or Type B acini. All stages of A. delacruzi had a similar gut structure, consisting of digestive cells in the basal portion that contained hematin granules. Neither regenerative nor secretory cell traces were observed in the sections of gu

Observations on Antricola ticks: Small nymphs feed on mammalian hosts and have a salivary gland structure similar to ixodid ticks

Ticks use bloodmeals as a Source of nutrients and energy to molt and survive until the next meal and to oviposit, in the case of females. However, only the larvae of some tick species are known to feed upon bats females are obligatorily autogenous, and nymphal stages are believed to not feed. We investigated the presence of blood ill a natural population of nymphal Antricola delacruzi ticks collected from bat guano; their ability to feed upon laboratory hosts: and the microscopic structure of both salivary glands and gut. DNA amplification of gut contents of freshly collected material was positive for a mammal in 4 of 11 first instar nymphs, but we were unsuccesful in the amplification of host bloodmeal DNA from late instar nymphs. All early nymphal stages (n = 10) fed oil rabbits. and host DNA was detected and sequenced from gut contents. However, all the large nymphs (n = 10) rejected feeding, and host DNA remained undetected in these ticks. All stages of A. delacruzi have salivary glands similar in morphology to the ixodid agranular Type I salivary gland acini and to granular Type II or Type B acini. All stages of A. delacruzi had a similar gut structure. consisting of digestive cells in the basal portion that contained hematin granules. Neither regenerative nor secretory cell traces were observed in the sections Of gut