Elena Grego

Genetic Heterogeneity of Small Ruminant Lentiviruses Involves Immunodominant Epitope of Capsid Antigen and Affects Sensitivity of Single-Strain-Based Immunoassay

ABSTRACT The pol and gag gene fragments of small ruminant lentivirus field isolates collected in the last decade in Italy were amplified, sequenced, and analyzed. Phylogenetic analysis revealed that the majority of ovine isolates form a distinct cluster more similar to caprine lentivirus prototypes than to the visna virus prototype. These findings confirm and extend those reported by Leroux et al. (Arch. Virol., 142:1125-1137, 1997). Moreover, we observed that a variable region of Gag, included in the fragment analyzed, corresponded to one of the three major capsid antigen epitopes, which suggests that the antibody response to this epitope may be type specific. To test this hypothesis, two recombinant peptides, derived from the Icelandic prototype K1514 and this novel genotype, were expressed and used in an enzyme-linked immunosorbent assay to screen a panel of ovine and caprine sera collected from different geographical locations in Italy. Several sera reacted in a type-specific manner, indicating that in a diagnostic setting the combination of at least these two type-specific peptides is necessary to cover a wide range of infections. Additionally, these results support the hypothesis of cross-species transmission based on the phylogenetic analysis described above. This has implications for the control and eradication of small ruminant lentivirus infections.

Further circulation of West Nile and Usutu viruses in wild birds in Italy.

Usutu virus (USUV) and West Nile virus (WNV) are emerging pathogens that can cause neurological disease in humans. From March 2012 to June 2013, a sero-survey on wild birds was carried out to investigate the circulation of both viruses in Northwest (NW) Italy. Samples belonging to 47 different bird species have been collected using a volunteer based network and a wildlife rehabilitation center. Four of 297 serum samples had neutralizing antibodies against USUV (P=1.34%, IC 95% 0.36-3.4), while 10 of 233 samples tested positive for WNV (P=4.29%, IC 95% 2.07-7.75). Neutralizing antibodies for WNV were significantly more prevalent (p<0.001) in trans-Saharan migrants (P=21%, IC 95% 9.55-37.3) than in resident and short-distance birds, but no migratory habit-related differences were found for USUV. Antibodies in resident bird species suggest that both viruses are circulating in NW Italy.

LTR promoter activity of SRLV genotype E, strain Roccaverano

The highly divergent, small ruminant lentivirus (SRLV) genotype E Roccaverano strain has a full genome consisting of 8,418 nucleotides, which lack the entire dUTPase subunit of the pol gene, the vpr-like accessory gene, and the 71-bp repeat of the U3 region within the long terminal repeat (LTR). These deletions affect in reverse transcriptase fidelity in non-dividing cells (dUTPase and vpr-like) and in the regulation of viral replication. Surprisingly, this SRLV strain was able to replicate efficiently in non-dividing cells (i.e., blood-derived macrophages), while replication in fibroblastic-like cells was somewhat restricted. To evaluate whether this observation was due to the presence/absence of specific transcription factors within these fibroblasts, U3 transcriptional activity was measured in the different cell types and revealed that both fibroblasts and macrophages were able to activate the viral promoter in the same manner. Among the transcription factor-binding sites present within the U3 region, the highly conserved Ap4 tandem repeat was shown to be sufficient for LTR promoter activity.

Borrelia lusitaniae OspA Gene Heterogeneity in Mediterranean Basin Area

In this study, Borrelia lusitaniae DNA extracted from ticks and lizards was used to amplify the outer surface protein A (OspA) gene in order to increase knowledge about sequence variability in the Mediterranean basin area, to better understand how Borrelia lusitaniae has evolved and how its distribution has expanded. Phylogenetic trees including Italian and reference sequences showed a clear separation of B. lusitaniae OspA strains in two different major clades. North African isolates form a clade with Portuguese POTIB strains, whereas Italian samples are grouped with German strains and a human Portuguese strain. This subdivision was supported by very high posterior probability values in the trees, by both analysis of molecular variance and selective pressure. These results, based on phylogenetic information contained in the OspA gene sequences, show the presence of two different B. lusitaniae strains circulating in the Mediterranean basin area, suggesting two different evolution paths.

Antigenic variability of ovine lentivirus isolated in Italy

S. Rosati1*, M Profiti1, E. Grego, M.L. Carrozza2, M. Mazzei3 and P. Bandecchi3 1Dipartimento di Produzioni Animali Epidemiologia ed Ecologia, Università di T orino; 2Scuola Normale Superiore, Pisa; 3Dipartimento di Patologia Animale, Università di Pisa *Correspondence: Dipartimento di Produzioni Animali, Epidemiologia ed EcologiaFacoltà di Medicina veterinaria V ia L eonardo da V inci, 44 10095 Grugliasco (TO), Italy e-mail: sergio.rosati@unito.it

Molecular analysis and associated pathology of beak and feather disease virus isolated in Italy from young Congo African grey parrots (Psittacus erithacus) with an “atypical peracute form” of the disease

This study is the first report on the genetic and pathogenic characterization of beak and feather disease virus (BFDV) occurring in Italy. Twenty BFDV strains isolated in Italy from juvenile Congo African grey parrots (Psittacus erithacus) were investigated. Seventeen strains showed an “atypical peracute form” (aPF) of the disease, and three a chronic form (CF). The birds with aPF had been weaned, were independent as far as food and protection were concerned and apparently were without lesions. The gene coding for the putative coat protein was amplified in all isolates while the BFDV genome was sequenced completely in 10 samples, eight of them belonging to aPF affected birds and two from CF of the disease. All full genomes clustered into the J strain of BFDV, where two new subtypes were identified. Recombination analyses showed evidence of genetic exchanges in two BFDV genomes. In addition, a correlation between viral isolate and origin of the breeding material was shown, while an association between the genetic features of the virus and the clinical form was not observed. Histologically, apoptosis was detected frequently in aPF samples and sporadically in CF samples. Interestingly, BFDV antigens were detected in the nuclei and cytoplasm of such apoptotic cells. The data presented here support the hypothesis that, in the absence of a defined BFDV genetic variant accountable for a specific clinical form of psittacine beak and feather disease, differences in the apoptotic rate between aPF and CF are strictly host related.

Characterization of the upper and lower respiratory tract microbiota in Piedmontese calves

BackgroundThe microbiota of the bovine upper respiratory tract has been recently characterized, but no data for the lower respiratory tract are available. A major health problem in bovine medicine is infectious bronchopneumonia, the most common respiratory syndrome affecting cattle. With this study, we used 16S rRNA gene sequencing to characterize and compare the microbial community composition of the upper and lower respiratory tracts in calves.ResultsThe microbiota of the upper (nasal swab [NS]) and the lower (trans-tracheal aspiration [TTA]) respiratory tracts of 19 post-weaned Piedmontese calves with (8/19) and without (11/19) clinical signs of respiratory disease, coming from six different farms, was characterized by 16S rRNA gene metabarcoding. A total of 29 phyla (29 in NS, 21 in TTA) and 305 genera (289 in NS, 182 in TTA) were identified. Mycoplasma (60.8%) was the most abundant genus identified in both the NS (27.3%) and TTA (76.7%) samples, followed by Moraxella (16.6%) in the NS and Pasteurella (7.3%) in the TTA samples. Pasteurella multocida (7.3% of total operational taxonomic units [OTUs]) was the most abundant species in the TTA and Psychrobacter sanguinis (1.1% of total OTUs) in the NS samples. Statistically significant differences between the NS and the TTA samples were found for both alpha (Shannon index, observed species, Chao1 index, and Simpson index; P = 0.001) and beta (Adonis; P = 0.001) diversity. Comparison of the NS and TTA samples by farm origin and clinical signs revealed no statistical difference (P > 0.05), except for farm origin for the NS samples when compared by the unweighted UniFrac metric (P = 0.05).ConclusionsUsing 16S rRNA gene sequencing, we characterized the microbiota of the upper and lower respiratory tracts of calves, both healthy individuals and those with clinical signs of respiratory disease. Our results suggest that environmental factors may influence the composition of the upper airway microbiota in cattle. While the two microbial communities (upper and lower airways) differed in microbial composition, they shared several OTUs, suggesting that the lung microbiota may be a self-sustaining, more homogeneous ecosystem, influenced by the upper respiratory tract microbiota.

Use of small rodents for the surveillance of agents and vectors of tick-borne zoonoses in the northern Apennines, Italy

Vector borne zoonoses are emerging threats in Europe. Data collection from animals may be useful to evaluate their occurrence and intensity of transmission, and to detect their introduction into previously free geographic areas. Indeed, vertebrates serve as hosts for pathogens and for arthropod vectors, although their ecological role can vary according to the diseases. Data collection on small rodents was used to study the eco-epidemiology of two tick-borne pathogens, Rickettsia slovaca (agent of tick-borne lymphadenopathy, transmitted by Dermacentor marginatus) and Borrelia burgdorferi sensu lato (agent of Lyme Borreliosis, transmitted by Ixodes ricinus), in the Apennine mountains, Tuscany (Italy), where human cases of tick-borne diseases were reported. Small rodents are preferential hosts for the immature stages of the two tick vectors and are involved in the transmission cycle of both diseases. In the summers from 2009 to 2012, we live trapped Apodemus spp. and Myodes glareolus from 1100 to 1650 m above the sea level (a.s.l.). Rodents were found infested by immature I. ricinus and D. marginatus. The monthly activities of these two tick species on the same hosts were different, reflecting differences in their life cycles. Although few individuals were co-infested, both tick species tended to aggregate on the same Apodemus spp. males. R. slovaca and B. burgdorferi s.l. were detected in rodent ear biopsies and attached ticks up to 1650 m a.s.l. In our study area, rodents might play a role as amplifiers of R. slovaca infection; even in the absence of the hosts systemic infection, tick aggregation on the same individuals might favour the transmission of the pathogen through co-feeding. While D. marginatus had been found at the same location in studies carried out in 1994, I.ricinus was very rare or absent. Data collection on small rodents thus highlighted the recent range expansion of I. ricinus and B. burgdorferi s.l. in a previously unoccupied area. Major land use changes, the increased abundance of wildlife populations, as well as a general climate warming in the Mediterranean area, might be interacting factors affecting the altitudinal range expansion of I. ricinus in the Northern Apennines, and in Italy in general.

Small ruminant lentiviruses in Jordan: evaluation of sheep and goat serological response using recombinant and peptide antigens

Small ruminant lentiviruses infect sheep and goats worldwide, causing chronic progressive diseases and relevant economic losses. Disease eradication and prevention is mostly based on serological testing. The goal of this research was to investigate the presence of the small ruminant lentiviruses (SRLVs) in Jordan and to characterize the serological response in sheep and goat populations. A panel of sera were collected from flocks located in Northern Jordan and Jordan Valley. The samples were tested using three ELISA assays: a commercially available ELISA based on p25 recombinant protein and transmembrane peptide derived from British maedi–visna virus (MVV) EV1 strain, an ELISA based on P16-P25 recombinant protein derived from two Italian strains representative of MVV- and caprine arthritis encephalitis virus (CAEV)-like SRLVs, and an ELISA based on SU5 peptide from the same two Italian isolates. The results indicate that both MVV- and CAEV-like strains are present in Jordan and that the majority of the viruses circulating among sheep and goat populations belong to the MVV-like genotype.