Eating the Enemy: Mycoplasma Strategies to Evade Neutrophil Extracellular Traps (NETs) Promoting Bacterial Nucleotides Uptake and Inflammatory Damage.

Neutrophils are effector cells involved in the innate immune response against infection; they kill infectious agents in the intracellular compartment (phagocytosis) or in the extracellular milieu (degranulation). Moreover, neutrophils release neutrophil extracellular traps (NETs), complex structures composed of a scaffold of decondensed DNA associated with histones and antimicrobial compounds; NETs entrap infectious agents, preventing their spread and promoting their clearance. NET formation is triggered by microbial compounds, but many microorganisms have evolved several strategies for NET evasion. In addition, the dysregulated production of NETs is associated with chronic inflammatory diseases. Mycoplasmas are reduced genome bacteria, able to induce chronic infections with recurrent inflammatory symptoms. Mycoplasmas' parasitic lifestyle relies on metabolite uptake from the host. Mycoplasmas induce NET release, but their surface or secreted nucleases digest the NETs' DNA scaffold, allowing them to escape from entrapment and providing essential nucleotide precursors, thus promoting the infection. The presence of Mycoplasma species has been associated with chronic inflammatory disorders, such as systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, Crohn's disease, and cancer. The persistence of mycoplasma infection and prolonged NET release may contribute to the onset of chronic inflammatory diseases and needs further investigation and insights.

Emergence of Anaplasma Species Related to A. phagocytophilum and A. platys in Senegal.

The genus Anaplasma (Anaplasmataceae, Rickettsiales) includes tick-transmitted bacterial species of importance to both veterinary and human medicine. Apart from the traditionally recognized six Anaplasma species (A. phagocytophilum, A. platys, A. bovis, A. ovis, A. centrale, A. marginale), novel strains and candidate species, also of relevance to veterinary and human medicine, are emerging worldwide. Although species related to the zoonotic A. platys and A. phagocytophilum have been reported in several African and European Mediterranean countries, data on the presence of these species in sub-Saharan countries are still lacking. This manuscript reports the investigation of Anaplasma strains related to zoonotic species in ruminants in Senegal by combining different molecular tests and phylogenetic approaches. The results demonstrated a recent introduction of Candidatus (Ca) Anaplasma turritanum, a species related to the pathogenic A. platys, possibly originating by founder effect. Further, novel undetected strains related to Candidatus (Ca) Anaplasma cinensis were detected in cattle. Based on groEL and gltA molecular comparisons, we propose including these latter strains into the Candidatus (Ca) Anaplasma africanum species. Finally, we also report the emergence of Candidatus (Ca) A. boleense in Senegal. Collectively, results confirm that Anaplasma species diversity is greater than expected and should be further investigated, and that Anaplasma routine diagnostic procedures and epidemiological surveillance should take into account specificity issues raised by the presence of these novel strains, suggesting the use of a One Health approach for the management of Anaplasmataceae in sub-Saharan Africa.

Genomic characterization of a novel bat-associated Circovirus detected in European Miniopterus schreibersii bats.

Circoviruses are small circular DNA viruses causing severe pig and poultry disease, recently identified in various bat species worldwide. We report the detection and full-genome molecular characterization of a novel bat-associated Circovirus identified in faecal samples of Miniopterus schreibersii bats (Schreiber's bent-winged bats) from Sardinia, Italy. Full-genomic sequencing revealed a new putative member of Circoviridae family, with a genome size of 2063 nt. Sequencing allowed the characterization of the two major ORFs, inversely arranged, encoding replicase and capsid proteins, as well as the finding of a polythymidine tract within the genome, and highlighted phylogenetic relationships of the novel virus. This is the first report of circovirus in European bats. Giving the high level of genetic diversity of bat circoviruses, it is paramount to further investigate the relationships between these viruses and bats.

MHO_0730 as a Surface-Exposed Calcium-Dependent Nuclease of Mycoplasma hominis Promoting Neutrophil Extracellular Trap Formation and Escape.

Mycoplasma lipoproteins play a relevant role in pathogenicity and directly interact with the host immune system. Among human mycoplasmas, Mycoplasma hominis is described as a commensal bacterium that can be associated with a number of genital and extragenital conditions. Mechanisms of M. hominis pathogenicity are still largely obscure, and only a limited number of proteins have been associated with virulence. The current study focused on investigating the role of MHO_0730 as a virulence factor and demonstrated that MHO_0730 is a surface lipoprotein, potentially expressed in vivo during natural infection, acting both as a nuclease with its amino acidic portion and as a potent inducer of Neutrophil extracellular trapsosis with its N-terminal lipid moiety. Evidence for M. hominis neutrophil extracellular trap escape is also presented. Results highlight the relevance of MHO_0730 in promoting infection and modulation and evasion of innate immunity and provide additional knowledge on M. hominis virulence and survival in the host.

Molecular identification of Betacoronavirus in bats from Sardinia (Italy): first detection and phylogeny.

Bats may be natural reservoirs for a large variety of emerging viruses, including mammalian coronaviruses (CoV). The recent emergence of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) in humans, with evidence that these viruses may have their ancestry in bats, highlights the importance of virus surveillance in bat populations. Here, we report the identification and molecular characterization of a bat ß-Coronavirus, detected during a viral survey carried out on different bat species in the island of Sardinia (Italy). Cutaneous, oral swabs, and faecal samples were collected from 46 bats, belonging to 15 different species, and tested for viral presence. Coronavirus RNA was detected in faecal samples from three different species: the greater horseshoe bat (Rhinolophus ferrumequinum), the brown long-eared bat (Plecotus auritus), and the European free-tailed bat (Tadarida teniotis). Phylogenetic analyses based on RNA-dependent RNA polymerase (RdRp) sequences assigned the detected CoV to clade 2b within betacoronaviruses, clustering with SARS-like bat CoVs previously reported. These findings point to the need for continued surveillance of bat CoV circulating in Sardinian bats, and extend the current knowledge on CoV ecology with novel sequences detected in bat species not previously described as ß-Coronavirus hosts.

Felis catus Papillomavirus Types 1, 2, 3, 4, and 5 in Feline Bowenoid in Situ Carcinoma: An In Situ Hybridization Study.

Several studies based on histopathology or molecular investigations suggest a causal relation between Felis catus papillomavirus (FcaPV-2) infection and bowenoid in situ carcinoma (BISC) in cats. Nevertheless, data on distribution of viral DNA for different F. catus papillomavirus types (FcaPV-1, 2, 3, 4, 5) in precancerous skin lesions are lacking. In this study, incisional and excisional skin biopsies from 18 cats with BISC were investigated for the presence of FcaPV DNA by quantitative polymerase chain reaction (qPCR) and chromogenic in situ hybridization (CISH) using specific probes to detect each of the FcaPVs that have been identified so far. By qPCR analysis, 15 of 18 samples were positive for FcaPV-2, 2 were positive for FcaPV-4, and 1 sample was negative for all FcaPVs studied. Two cases were positive for FcaPV-5 by qPCR only. FcaPV-1 and FcaPV-3 were not detected by either method. CISH positivity for FcaPV-2 and FcaPV-4 was 100% concordant with qPCR. FcaPV-2 CISH signal was observed as nuclear dots within grouped neoplastic keratinocytes in 12 BISCs and in the perilesional skin of 9 biopsies. In 3 of these 9 cases, the signal was not observed within the BISC. FcaPV-4 CISH positivity was detected only within BISCs in 2 cases. The overall rate of concordance for FcaPV detection between PCR and CISH was 97.8%. This study suggests that CISH is a reliable method to detect FcaPV-2 and FcaPV-4 infection in cats and provides useful information on the type, rate, and localization of infected cells.

Molecular analysis of carnivore Protoparvovirus detected in white blood cells of naturally infected cats.

BACKGROUND: Cats are susceptible to feline panleukopenia virus (FPV) and canine parvovirus (CPV) variants 2a, 2b and 2c. Detection of FPV and CPV variants in apparently healthy cats and their persistence in white blood cells (WBC) and other tissues when neutralising antibodies are simultaneously present, suggest that parvovirus may persist long-term in the tissues of cats post-infection without causing clinical signs. The aim of this study was to screen a population of 54 cats from Sardinia (Italy) for the presence of both FPV and CPV DNA within buffy coat samples using polymerase chain reaction (PCR). The DNA viral load, genetic diversity, phylogeny and antibody titres against parvoviruses were investigated in the positive cats. RESULTS: Carnivore protoparvovirus 1 DNA was detected in nine cats (16.7%). Viral DNA was reassembled to FPV in four cats and to CPV (CPV-2b and 2c) in four cats; one subject showed an unusually high genetic complexity with mixed infection involving FPV and CPV-2c. Antibodies against parvovirus were detected in all subjects which tested positive to DNA parvoviruses. CONCLUSIONS: The identification of FPV and CPV DNA in the WBC of asymptomatic cats, despite the presence of specific antibodies against parvoviruses, and the high genetic heterogeneity detected in one sample, confirmed the relevant epidemiological role of cats in parvovirus infection.

Mycoplasma lipoproteins are major determinants of neutrophil extracellular trap formation.

Neutrophil granulocytes are paramount to innate responses as major effectors of acute inflammation. Among the various strategies enacted by neutrophils to eliminate microbes NETosis is a novel distinct antimicrobial activity in which an interlacement of chromatin fibres rich in granule-derived antimicrobial peptides and enzymes is extruded (NETs, neutrophils extracellular traps ). NETs contribute to the pathogenesis of acute and chronic inflammatory disorders. The interactions of mycoplasmas and innate immune cells, in particular neutrophil granulocytes, are poorly defined. Here, we describe NET formation in vivo in the mammary gland and milk of sheep naturally infected by Mycoplasma agalactiae. Also, we assess the contribution of liposoluble proteins, the most abundant component of the Mycoplasma membrane, in inducing NETosis. We demonstrate that Mycoplasma liposoluble proteins induce NET release at levels comparable to what observed with other stimuli, such as lipopolysaccharides and phorbol 12-myristate 13-acetate. Stimulation of neutrophils with synthetic diacylated lipopeptides based on the M. agalactiae P48, P80, and MAG_1000 proteins, combined in a mix or used individually, suggests that NETosis might not be dependent on a specific lipopeptide sequence. Also, NETosis is partially abolished when TLR2 is blocked with specific antibodies. The results presented in this work provide evidences for the mechanisms underlying NET activation in mycoplasma infections, and on their contribution to pathogenesis of mycoplasmosis.

Ovis aries Papillomavirus 3 in Ovine Cutaneous Squamous Cell Carcinoma.

Squamous cell carcinoma (SCC) is a common malignancy affecting humans and other animals. Papillomaviruses (PVs) are frequently reported as causal agents of cutaneous benign and malignant epithelial lesions in different animal species, but only few studies have investigated their role in ovine SCC. In this study, we explore the possible involvement of the Ovine aries PVs (OaPV1, OaPV2, OaPV3) in cutaneous SCC using an integrated histological and molecular approach. Forty cutaneous SCCs from different anatomical locations of Sardinian sheep and 40 matched non-SCC samples were evaluated histologically and by polymerase chain reaction (PCR) to assess the presence of ovine PVs. In addition, DNA in situ hybridization (ISH) and reverse transcription-polymerase chain reaction (RT-PCR) were carried out to evaluate the cellular localization and viral transcriptional activity, respectively. OaPV3 DNA was detected in 26 of 40 (65%) SCCs and in 12 of 40 (30%) non-SCC samples using PCR. OaPV1 and OaPV2 were not detected. OaPV3 viral DNA was observed by ISH in malignant epithelial squamous cells of 18 of 40 (45%) SCCs. In addition, the viral transcriptional activity was identified in 24 of 40 (60%) SCCs by RT-PCR. Notably, a higher viral positivity was observed in SCCs compared with non-SCC samples. The considerable infection rate of OaPV3 in the most common skin tumor of the sheep suggests that PV could represent a key factor in the onset of ovine SCC.

Bovine papillomavirus type 7 in Italy: complete genomes and sequence variants.

Two novel bovine papillomavirus type 7 (BPV-7) variants have been identified in teat cutaneous papillomas affecting dairy cows in northern Italy. The entire genome sequences of two BPV-7 Italian variants showed major sequence differences in the long control region (LCR) and in the L2 gene compared to the Japanese reference strain. In order to define the stability of these genetic variants, the L2 and LCR sequences of seven further BPV-7 positive isolates were characterized. An insertion of six amino acids in the L2 structural protein has been detected in all samples while different genetic variants have been identified for the LCR. These findings provide new insights on intra-type variability of BPVs and represent a starting point for future studies aimed at establishing the biological role of the different BPV genomic regions and investigating the pathogenic potential of papillomavirus variants.

Molecular detection and groEL typing of Rickettsia aeschlimannii in Sardinian ticks.

Rickettsia aeschlimannii is an emerging tick-borne pathogen of the spotted fever group Rickettsiae with considerable impact on both human and animal health. This study reports the molecular detection and groEL characterization of R. aeschlimannii in ticks collected from birds and ruminants in a typical Mediterranean environment. Phylogeny of R. aeschlimannii and species representative of the spotted fever and typhus groups based on the groEL gene is reconstructed for the first time. Results expand the knowledge on distribution and typing of emerging human tick-borne diseases in Sardinia and pave the way for future molecular epidemiology studies of zoonotic Rickettsiae.

Neutrophil extracellular traps in sheep mastitis.

Neutrophil extracellular traps (NETs) are structures composed of DNA, histones, and antimicrobial proteins that are released extracellularly by neutrophils and other immune cells as a means for trapping and killing invading pathogens. Here, we describe NET formation in milk and in mammary alveoli of mastitic sheep, and provide a dataset of proteins found in association to these structures. Nucleic acid staining, immunomicroscopy and fluorescent in-situ hybridization of mastitic mammary tissue from sheep infected with Streptococcus uberis demonstrated the presence of extranuclear DNA colocalizing with antimicrobial proteins, histones, and bacteria. Then, proteomic analysis by LTQ-Orbitrap Velos mass spectrometry provided detailed information on protein abundance changes occurring in milk upon infection. As a result, 1095 unique proteins were identified, of which 287 being significantly more abundant in mastitic milk. Upon protein ontology classification, the most represented localization classes for upregulated proteins were the cytoplasmic granule, the nucleus, and the mitochondrion, while function classes were mostly related to immune defence and inflammation pathways. All known NET markers were massively increased, including histones, granule proteases, and antimicrobial proteins. Of note was the detection of protein arginine deiminases (PAD3 and PAD4). These enzymes are responsible for citrullination, the post-translational modification that is known to trigger NET formation by inducing chromatin decondensation and extracellular release of NETs. As a further observation, citrullinated residues were detected by tandem mass spectrometry in histones of samples from mastitic animals. In conclusion, this work provides novel microscopic and proteomic information on NETs formed in vivo in the mammary gland, and reports the most complete database of proteins increased in milk upon bacterial mastitis.

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.

Clinicopathological and molecular findings in a case of canine Anaplasma phagocytophilum infection in Northern Italy.

A documented case of canine granulocytic anaplasmosis coupled with the molecular characterization of the etiological agent is reported for the first time in Northern Italy. The patient showed nonspecific clinical signs such as fever and weight loss. The most relevant clinicopathological findings were thrombocytopenia, hypoalbuminemia, and normal azotemic proteinuria consistent with glomerular diseases. Blood smear examination revealed the presence of intracytoplasmatic inclusions in neutrophils associated with high positive serology for Anaplasma phagocytophilum. PCR analysis and sequencing of the amplicon confirm serological diagnosis of A. phagocytophilum. Phylogenetic analysis evidenced that the detected bacterial strain belongs to the A. phagocytophilum Europe 1 lineage. Data indicates that A. phagocytophilum circulates in natural environments of Emilia-Romagna region (Northern Italy) and its prevalence in dogs could be underestimated because the clinical signs are frequently nonspecific and a certain diagnosis requires the combination of clinicopathological and molecular assays. Pets living in this area should be regularly monitored and treated for ectoparasites to minimize health risks for humans and pets. Also, surveillance of A. phagocytophilum should be improved in Northern Italy and canine anaplasmosis should be considered in differential diagnosis of persistent proteinuria.

First Gammaherpesvirus detection in a free-living Mediterranean bottlenose dolphin.

Recently, herpes viruses have been detected in different cetacean species from the Atlantic and in Mediterranean striped dolphins (Stenella coeruleoalba). While pathogens such as cetacean morbillivirus have been widely studied following recent epizootics, herpesvirus (HV) distribution and pathogenic effects in cetaceans are still understudied. This study reports the first molecular identification of a Gammaherpesvirus in the genital mucosa of a free-living Mediterranean bottlenose dolphin (Tursiops truncatus) stranded off the coast of central Italy. Sequenced herpesviral PCR product was closely related to other HVs recently isolated in the genital mucosa of various cetacean species.

Ovine papillomaviruses: Diversity, pathogenicity, and evolution.

The family Papillomaviridae includes a plethora of viral species infecting virtually all vertebrates excluding amphibians, with astonishing impact on human and animal health. Although more than 250 species have been described in humans, the total number of papillomaviruses (PVs) discovered in animals does not reach up to this number. In animals, PV infections are mostly asymptomatic or can cause variable clinical conditions ranging from self-limiting papillomas and other cutaneous and mucosal benign lesions to cancer. Most of animal PV types have been discovered in cattle, dogs, horses, and cats with other farm host species remaining overlooked. In particular, the number of PV types so far identified in sheep is limited. This paper comprehensively reviews ovine PVs features, including viral taxonomy and evolution; genome organization; viral tropism and pathogenesis; macroscopical features and histopathological patterns, as well as available diagnostics tools. Data are critically presented and discussed in terms of impact on veterinary and public health. The development of future dedicated research is also discussed.

Ovine papillomavirus type 3 virus-like particle-based tools for diagnosis and detection of infection.

The design of prophylactic and diagnostic tools specific to animal papillomaviruses is hampered by the difficulties of viral in vitro manipulation and by the scarce availability of dedicated biotechnological tools. This paper reports the production of Ovine Papillomavirus 3 (OaPV3)-based virus-like particles (OaPV3-VLPs) in the baculovirus system and their use to investigate host humoral immune response through the establishment of an indirect ELISA test., Polyclonal sera and monoclonal antibodies were generated against OaPV3-VLPs, and their isotype and reactivity were determined. Additionally, antibodies allowed OaPV3 detection in ovine squamous cell carcinoma (SCC) samples by immunohistochemistry. Results encourage the standardization of OaPV3-specific prophylactic and serological diagnostic tools, and open new perspectives for the study of host-viral interaction and SCC development.

Molecular typing of bovine papillomaviruses in Costa Rica.

Bovine papillomaviruses are related to cause fibroepithelial proliferations in the skin and mucosae and are associated with economic loss mainly related to poor body condition and reduced milk production. This study aimed to investigate the presence and types of bovine papillomaviruses (BPVs) in cattle sampled in different areas of Costa Rica using molecular techniques. A descriptive study with a non-probability convenience sampling was carried out. A total of 99 papillomatous lesions were collected from 63 animals in 32 farms, and analyzed by polymerase chain reaction, rolling circle amplification (RCA), sequencing, and restriction enzymes digestion. Seven bovine papillomavirus types (BPV1, BPV2, BPV4, BPV6, BPV7, BPV10, BPV11) and two putative novel viral variants (BPV-CR1 and BPV-CR2) were identified for the first time in Costa Rica. BPV6 was the most frequently detected virus in lesions (31.2%), followed by BPV2 (25%) and BPV1 (25%). BPV1 and BPV2 were the most widely distributed in the Country. Coinfections were recorded in two animals (BPV1 / BPV2 and BPV4 / BPV6). Restriction analyses allowed differentiating BPV1 from BPV2, BPV4, and BPV7, but failed to identify BPV6, BPV10, and BPV11. Results suggest that a great PVs diversity is harbored by bovines in Costa Rica and indicate the need for further investigations aimed to uncover PV diversity at the full genomic level.

Development of immunodiagnostic tools for in situ investigation of Ovis aries papillomavirus 3 (OaPV3).

Cutaneous squamous cell carcinoma (cSCC) is a malignant lesion characterized by proliferation and transformation of keratinocytes in the epidermis and infiltrating derma. cSCC is reported in domestic and wild animal species, worldwide. The occurrence and development of cSCC rely on synergic multifactorial conditions, most importantly sunlight exposure and Papillomavirus (PV) infection. In sheep, the development of such lesions represents a threat both to animal welfare and milk production. Ovis aries papillomavirus 3 (OaPV3) is the main cSCC viral determinant and oncogenic properties of viral E6 and E7 proteins were preliminarily investigated. However, E6 and E7 role and mechanisms resulting in cSCC have not been fully clarified, mainly due to the lack specific immunological tools, such as antibodies for in situ detection of ovine papillomavirus. This paper reports the development of specific serological tools for the investigation of OaPV3 pathogenicity, and their preliminary use to screen 4 ovine cSSC formalin-fixed paraffin embedded tissues. Relevance of immunological tools to investigation of viral biological properties and diagnosis are also discussed.