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.

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.

Sequencing and Analysis of Lumpy Skin Disease Virus Whole Genomes Reveals a New Viral Subgroup in West and Central Africa.

Lumpy skin disease virus (LSDV) is a member of the capripoxvirus (CPPV) genus of the Poxviridae family. LSDV is a rapidly emerging, high-consequence pathogen of cattle, recently spreading from Africa and the Middle East into Europe and Asia. We have sequenced the whole genome of historical LSDV isolates from the Pirbright Institute virus archive, and field isolates from recent disease outbreaks in Sri Lanka, Mongolia, Nigeria and Ethiopia. These genome sequences were compared to published genomes and classified into different subgroups. Two subgroups contained vaccine or vaccine-like samples ("Neethling-like" clade 1.1 and "Kenya-like" subgroup, clade 1.2.2). One subgroup was associated with outbreaks of LSD in the Middle East/Europe (clade 1.2.1) and a previously unreported subgroup originated from cases of LSD in west and central Africa (clade 1.2.3). Isolates were also identified that contained a mix of genes from both wildtype and vaccine samples (vaccine-like recombinants, grouped in clade 2). Whole genome sequencing and analysis of LSDV strains isolated from different regions of Africa, Europe and Asia have provided new knowledge of the drivers of LSDV emergence, and will inform future disease control strategies.

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.

A field study evaluating the humoral immune response in Mongolian sheep vaccinated against sheeppox virus.

Sheeppox is a transboundary disease of small ruminants caused by infection with the capripoxvirus sheeppox virus. Sheeppox is found in Africa, the Middle East and Asia and is characterized by fever, multifocal cutaneous raised lesions and death. Vaccination with live attenuated capripoxvirus (CPPV) strains is an effective and widely used strategy to contol sheeppox outbreaks; however, there are few reports of post-vaccination field surveillance studies. This study used a commercially available enzyme-linked immunosorbent assay (ELISA) to examine quantitative and temporal features of the humoral response of sheep vaccinated with a live-attenuated CPPV strain in Mongolia. Four hundred samples were tested using the ELISA commercial kit, and a subset of 45 samples were also tested with a virus neutralization test (VNT). There was substantial agreement between the VNT and ELISA tests. Antibodies to CPPV were detected between 40 and 262 days post-vaccination. There was no significant difference between serological status (positive/negative) and sex or age; however, an inverse correlation was found between the length of time since vaccination and serological status. Animals between 90 and 180 days post-vaccination were more likely to be positive than animals greater than 180 days post-vaccination. Our results show that a commercial CPPV ELISA kit is a robust and reliable assay for post-CPPV vaccination surveillance in resource-restricted settings and provide temporal parameters to be considered when planning sheeppox post-vaccination monitoring programmes.

Madin-Darby bovine kidney (MDBK) cells are a suitable cell line for the propagation and study of the bovine poxvirus lumpy skin disease virus.

Lumpy skin disease virus (LSDV) is a poxvirus that causes systemic disease in cattle, resulting in substantial economic loss to affected communities. LSDV is a rapidly emerging pathogen of growing global concern that recently spread from Africa and the Middle East into Europe and Asia, impacting the cattle population in these regions. An increase in research efforts into LSDV is required to address key knowledge gaps, however this is hampered by lack of suitable cell lines on which to propagate and study the virus. In this work we describe the replication and spread of LSDV on Madin-Darby bovine kidney (MDBK) cells, and the formation of foci-type poxvirus plaques by LSDV on MDBK cells. Methods utilising MDBK cells to quantify neutralising antibodies to LSDV, and to purify LSDV genomic DNA suitable for short read sequencing are described. These research methods broaden the tools available for LSDV researchers and will facilitate the gathering of evidence to underpin the development of LSD control and prevention programmes.

Leopardus wiedii Papillomavirus type 1, a novel papillomavirus species in the tree ocelot, suggests Felidae Lambdapapillomavirus polyphyletic origin and host-independent evolution.

The limited knowledge on Papillomavirus diversity (particularly in wild animal species) influences the accuracy of PVs phylogeny and their evolutionary history, and hinders the comprehension of PVs pathogenicity, especially the mechanism of virus - related cancer progression. This study reports the identification of Leopardus wiedii Papillomavirus type 1 (LwiePV1), the first PV type within Lambdapapillomavirus in a Leopardus host. LwiePV1 full genome sequencing allowed the investigation of its taxonomic position and phylogeny. Based on results, LwiePV1 should be assigned to a novel PV species providing evidence for a polyphyletic origin of feline lambda PVs, and representing an exception to codivergence between feline lambda PVs and their hosts. Results improve our knowledge on PV diversity and pave the way to future studies investigating biological and evolutionary features of animal PVs.

Transforming properties of ovine papillomaviruses E6 and E7 oncogenes.

An increasing number of studies suggest that cutaneous papillomaviruses (PVs) might be involved in skin carcinogenesis. However, only a few animal PVs have been investigated regard to their transformation properties. Here, we investigate and compare the oncogenic potential of 2 ovine Delta and Dyokappa PVs, isolated from ovine skin lesions, in vitro and ex vivo. We demonstrate that both OaPV4 (Delta) and OaPV3 (Dyokappa) E6 and E7 immortalize primary sheep keratinocytes and efficiently deregulate pRb pathway, although they seem unable to alter p53 activity. Moreover, OaPV3 and OaPV4-E6E7 expressing cells show different shape, doubling time, and clonogenic activities, providing evidence for a stronger transforming potential of OaPV3 respect to OaPV4. Also, similarly to high-risk mucosal and cutaneous PVs, the OaPV3-E7 protein, constantly expressed in sheep squamous cell carcinomas, binds pRb with higher affinity compared to the E7 encoded by OaPV4, a virus associated to fibropapilloma. Finally, we found that OaPV3 and OaPV4-E6E7 determine upregulation of the pro-proliferative proteins cyclin A and cdk1 in both human and ovine primary keratinocytes. Collectively, results provide evidence for implication of ovine PVs in cutaneous proliferative lesions and skin cancer progression, and indicate sheep as a possible animal model for the study of cutaneous lesions and malignancies.

Host cell tropism, genome characterization, and evolutionary features of OaPV4, a novel Deltapapillomavirus identified in sheep fibropapilloma.

Investigating papillomavirus (PV) diversity is crucial to fully comprehend pathogenicity, genetic features, and evolution of taxa hosted by domestic and wild animal species. This study reports the identification of OaPV4, a novel ovine PV type within Deltapapillomaviruses 3. The study of OaPV4 genomic features combined to in situ hybridization and immunohistochemistry investigations allowed extrapolating several general biological features of ovine PVs, such as their cellular tropism, pathogenicity, and evolutionary history. Based on results, ovine PVs can be grouped into a polyphyletic ancient group of viruses, which splits in two main subgroups having peculiar cellular tropism and pathogenicity. Results add up to animal PV diversity and are crucial to future studies aimed to investigate the correlation between animal PV and cutaneous benign and malign proliferations.

A life-threatening brainstem compression by cerebral Echinococcus granulosus.

Cystic echinococcosis (CE) is a zoonotic disease caused by Echinococcus granulosus. It is of worldwide importance, and is widespread in the Mediterranean region and Middle East. This tapeworm shows great intraspecific variation in relation to host specificity, epidemiology and morphology. This variability led in previous years to the identification of ten (G1-G10) different genotypes of the parasite. Cerebral localization of E. granulosus is not common: it especially affects children and is more frequently located in the supratentorial region. It can be life-threatening due to its localization in eloquent areas especially in the posterior fossa. Despite the benign nature of hydatid cyst, invasion of critical areas may cause significant mortality and morbidity in some patients. Urgent surgical decompression and adjuvant medical treatment must be employed as soon as possible in these patients. We present a clinical case of life-threatening brainstem compression in a child due to a rare form of CE which was confirmed with biomolecular techniques. She presented with respiratory distress and progressive quadriparesis. All cysts were removed by microsurgical technique and albendazole was given postoperatively for one year with regular follow-ups.

Molecular detection and identification of Rickettsiales pathogens in dog ticks from Costa Rica.

Although vector-borne diseases are globally widespread with considerable impact on animal production and on public health, few reports document their presence in Central America. This study focuses on the detection and molecular identification of species belonging to selected bacterial genera (Ehrlichia, Anaplasma and Rickettsia) in ticks sampled from dogs in Costa Rica by targeting several genes: 16S rRNA/dsb genes for Ehrlichia; 16S rRNA/groEL genes for Anaplasma, and ompA/gltA/groEL genes for Rickettsia. PCR and sequence analyses provides evidences of Ehrlichia canis, Anaplasma platys, and Anaplasma phagocytophilum infection in Rhipicephalus sanguineus s.l ticks, and allow establishing the presence of Rickettsia monacensis in Ixodes boliviensis. Furthermore, the presence of recently discovered Mediterranean A. platys-like strains is reported for the first time in Central America. Results provide new background on geographical distribution of selected tick-transmitted bacterial pathogens in Costa Rica and on their molecular epidemiology, and are pivotal to the development of effective and reliable diagnostic tools in Central America.

Mycoplasma agalactiae MAG_5040 is a Mg2+-dependent, sugar-nonspecific SNase recognised by the host humoral response during natural infection.

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.