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1.  Molecular Investigation and Phylogeny of Anaplasma spp. in Mediterranean Ruminants Reveal the Presence of Neutrophil-Tropic Strains Closely Related to A. platys 
Few data are available on the prevalence and molecular typing of species belonging to the genus Anaplasma in Mediterranean ruminants. In this study, PCR analysis and sequencing of both 16S rRNA and groEL genes were combined to investigate the presence, prevalence, and molecular traits of Anaplasma spp. in ruminants sampled on the Island of Sardinia, chosen as a subtropical representative area. The results demonstrate a high prevalence of Anaplasma spp. in ruminants, with animals infected by at least four of six Anaplasma species (Anaplasma marginale, A. bovis, A. ovis, and A. phagocytophilum). Moreover, ruminants host a number of neutrophil-tropic strains genetically closely related to the canine pathogen A. platys. The high Anaplasma spp. prevalence and the identification of as-yet-unclassified neutrophil-tropic strains raise concerns about the specificity of serological tests routinely used in ruminants and provide additional background for reconstructing the evolutionary history of species genetically related to A. phagocytophilum.
PMCID: PMC3911010  PMID: 24162569
2.  Mycoplasma agalactiae MAG_5040 is a Mg2+-Dependent, Sugar-Nonspecific SNase Recognised by the Host Humoral Response during Natural Infection 
PLoS ONE  2013;8(2):e57775.
In this study the enzymatic activity of Mycoplasma agalactiae MAG_5040, a magnesium-dependent nuclease homologue to the staphylococcal SNase was characterized and its antigenicity during natural infections was established. A UGA corrected version of MAG_5040, lacking the region encoding the signal peptide, was expressed in Escherichia coli as a GST fusion protein. Recombinant GST-MAG_5040 exhibits nuclease activity similar to typical sugar-nonspecific endo- and exonucleases, with DNA as the preferred substrate and optimal activity in the presence of 20 mM MgCl2 at temperatures ranging from 37 to 45°C. According to in silico analyses, the position of the gene encoding MAG_5040 is consistently located upstream an ABC transporter, in most sequenced mycoplasmas belonging to the Mycoplasma hominis group. In M. agalactiae, MAG_5040 is transcribed in a polycistronic RNA together with the ABC transporter components and with MAG_5030, which is predicted to be a sugar solute binding protein by 3D modeling and homology search. In a natural model of sheep and goats infection, anti-MAG_5040 antibodies were detected up to 9 months post infection. Taking into account its enzymatic activity, MAG_5040 could play a key role in Mycoplasma agalactiae survival into the host, contributing to host pathogenicity. The identification of MAG_5040 opens new perspectives for the development of suitable tools for the control of contagious agalactia in small ruminants.
PMCID: PMC3585158  PMID: 23469065
3.  The liposoluble proteome of Mycoplasma agalactiae: an insight into the minimal protein complement of a bacterial membrane 
BMC Microbiology  2010;10:225.
Mycoplasmas are the simplest bacteria capable of autonomous replication. Their evolution proceeded from gram-positive bacteria, with the loss of many biosynthetic pathways and of the cell wall. In this work, the liposoluble protein complement of Mycoplasma agalactiae, a minimal bacterial pathogen causing mastitis, polyarthritis, keratoconjunctivitis, and abortion in small ruminants, was subjected to systematic characterization in order to gain insights into its membrane proteome composition.
The selective enrichment for M. agalactiae PG2T liposoluble proteins was accomplished by means of Triton X-114 fractionation. Liposoluble proteins were subjected to 2-D PAGE-MS, leading to the identification of 40 unique proteins and to the generation of a reference 2D map of the M. agalactiae liposoluble proteome. Liposoluble proteins from the type strain PG2 and two field isolates were then compared by means of 2D DIGE, revealing reproducible differences in protein expression among isolates. An in-depth analysis was then performed by GeLC-MS/MS in order to achieve a higher coverage of the liposoluble proteome. Using this approach, a total of 194 unique proteins were identified, corresponding to 26% of all M. agalactiae PG2T genes. A gene ontology analysis and classification for localization and function was also carried out on all protein identifications. Interestingly, the 11.5% of expressed membrane proteins derived from putative horizontal gene transfer events.
This study led to the in-depth systematic characterization of the M. agalactiae liposoluble protein component, providing useful insights into its membrane organization.
PMCID: PMC2941501  PMID: 20738845
4.  A Paradigm for Virus–Host Coevolution: Sequential Counter-Adaptations between Endogenous and Exogenous Retroviruses 
PLoS Pathogens  2007;3(11):e170.
Endogenous retroviruses (ERVs) are remnants of ancient retroviral infections of the host germline transmitted vertically from generation to generation. It is hypothesized that some ERVs are used by the host as restriction factors to block the infection of pathogenic retroviruses. Indeed, some ERVs efficiently interfere with the replication of related exogenous retroviruses. However, data suggesting that these mechanisms have influenced the coevolution of endogenous and/or exogenous retroviruses and their hosts have been more difficult to obtain. Sheep are an interesting model system to study retrovirus-host coevolution because of the coexistence in this animal species of two exogenous (i.e., horizontally transmitted) oncogenic retroviruses, Jaagsiekte sheep retrovirus and Enzootic nasal tumor virus, with highly related and biologically active endogenous retroviruses (enJSRVs). Here, we isolated and characterized the evolutionary history and molecular virology of 27 enJSRV proviruses. enJSRVs have been integrating in the host genome for the last 5–7 million y. Two enJSRV proviruses (enJS56A1 and enJSRV-20), which entered the host genome within the last 3 million y (before and during speciation within the genus Ovis), acquired in two temporally distinct events a defective Gag polyprotein resulting in a transdominant phenotype able to block late replication steps of related exogenous retroviruses. Both transdominant proviruses became fixed in the host genome before or around sheep domestication (∼ 9,000 y ago). Interestingly, a provirus escaping the transdominant enJSRVs has emerged very recently, most likely within the last 200 y. Thus, we determined sequentially distinct events during evolution that are indicative of an evolutionary antagonism between endogenous and exogenous retroviruses. This study strongly suggests that endogenization and selection of ERVs acting as restriction factors is a mechanism used by the host to fight retroviral infections.
Author Summary
The genome of all vertebrates is heavily colonized by “endogenous” retroviruses (ERVs). ERVs derive from retrovirus infections of the germ cells of the host during evolution, leading to permanent integration of the viral genome into the host DNA. Because ERVs are integrated in the host genome, they are transmitted to subsequent generations like any other host gene. The function of endogenous retroviruses is not completely clear, but some ERVs can block the replication cycle of horizontally transmitted “exogenous” pathogenic retroviruses. These observations lead to the hypothesis that ERVs have protected the host during evolution against incoming pathogenic retroviruses. Here, by characterizing the evolutionary history and molecular virology of a particular group of endogenous betaretroviruses of sheep (enJSRVs) we show a fascinating series of events unveiling the endless struggle between host and retroviruses. In particular, we discovered that: (i) two enJSRV loci that entered the host genome before speciation within the genus Ovis (∼ 3 million y ago) acquired, after their integration, a mutated defective viral protein capable of blocking exogenous related retroviruses; (ii) both these transdominant enJSRV loci became fixed in the host genome before or around sheep domestication (∼ 10,000 y ago); (iii) the invasion of the sheep genome by ERVs of the JSRV/enJSRVs group is still in progress; and (iv) new viruses have recently emerged (less than 200 y ago) that can escape the transdominant enJSRV loci. This study strongly suggests that endogenization and selection of ERVs acting as restriction factors is a mechanism used by the host to fight retroviral infections.
PMCID: PMC2065879  PMID: 17997604
5.  Equine and Canine Anaplasma phagocytophilum Strains Isolated on the Island of Sardinia (Italy) Are Phylogenetically Related to Pathogenic Strains from the United States 
Applied and Environmental Microbiology  2005;71(10):6418-6422.
The presence of Anaplasma phagocytophilum, a tick-transmitted zoonotic pathogen, was investigated in Sardinia using a molecular approach. Phylogenetic analysis revealed that Sardinian strains are genetically distinct from the two lineages previously described in Europe and are closely related to strains isolated in different areas of the United States.
PMCID: PMC1265917  PMID: 16204571
6.  Anaplasma phagocytophilum, Sardinia, Italy 
Emerging Infectious Diseases  2005;11(8):1322-1324.
PMCID: PMC3320504  PMID: 16110587
Keywords: zoonosis; groEL; tick-borne diseases; Anaplasma phagocytophilum; molecular diagnosis

Results 1-6 (6)