Investigation into an outbreak of Border disease virus in pigs in England.

Border disease (BD) was first reported in 1959 in lambs from the border region of England and Wales. The causative virus (BD virus; BDV) has since been identified in several other ruminant species and pigs. The virus is prevalent in sheep flocks of UK, Europe and USA and has potential to inflict substantial economic losses. Natural BDV infection of pigs was first reported in the UK in 1992 from pigs with haemorrhagic lesions and more recently from healthy pigs in Spain and Japan. Here, a persistent problem of poor growth and anaemia in a small proportion of growing pigs on a mixed pig and sheep holding was investigated and tissues were tested in a pan viral microarray. The microarray detected BDV RNA in several tissues which was further confirmed by sequencing, specific BDV PCR and immunohistochemistry. Phylogenetically, the virus clustered with other BDVs in the sub-genotype 1b. This investigation highlights likely interspecies transmission of pestiviruses and their impact on pestivirus detection and eradication programs.

Examining bull semen for residues of Schmallenberg virus RNA.

Schmallenberg orthobunyavirus (SBV) was initially detected in 2011 in Germany from dairy cattle with fever and decreased milk yield. The virus infection is now established in many parts of the world with recurrent epidemics. SBV is transmitted through midges and transplacental. No direct virus transmission including via breeding has ever been demonstrated. In some bulls, however, the virus is detectable transiently, in low to minute quantities, in semen post-infection. While the infection is considered of low impact for the dairy industry, some SBV-free countries have adopted a zero-risk approach requiring bull semen batches to be tested for SBV RNA residues prior to import. This, in turn, obligates a protocol to enable sensitive detection of SBV RNA in semen samples for export purposes. Here, we describe how we established a now ISO/IEC 17025 accredited protocol that can effectively detect minute quantities of SBV RNA in semen and also its application to monitor bull semen during two outbreaks in the United Kingdom in 2012 and 2016. The data demonstrate that only a small number of bulls temporarily shed low amounts of SBV.

A simplified digital workflow for the prosthetic finishing of implant rehabilitations: a case report.

The aim of this article is to describe how, during the provisional and definitive prosthetic phases, using new digital technologies, it is possible to improve the ergonomics of the prosthetist's work and reduce the discomfort of patients, subjecting them to the fewest possible appointments at the dentist. The proposal of a full digital protocol, described by the following case report, for the realization of a definitive prosthetic rehabilitation supported by a reduced number of implants, in fact, allows to considerably reduce the number of appointments and reduce any bias. A 67-year-old male patient presents for the first visit to the Department of Dentistry of the San Raffaele Hospital, wearing a removable upper prosthesis and with the request to heal the aesthetic and functional situation through prosthetics fixed. An initial panoramic radiograph was performed, intra and extra oral photos were taken and also intraoral impressions. A stereo-lithographic models are obtained from intraoral scans, and two total prostheses, upper and lower, were packaged for the provisional post-surgical phase was performed. In accordance with the All-on-4 method 8 implant fixtures were placed. For the final prosthetic phase, the patient underwent only two operative sessions. In the first session, scans were taken with the provisionals in situ, of the patient's mucous membranes and with the Scan-abutments in place. In the second session using specific CADSoftware the matching of the STL files of the three scans were created, the opposing arches of the patient were related on a digital articulator, and the milled titanium bars were immediately constructed and finished with the resin. Finally, the definitive prostheses were delivered to the patient without any other test. Digital technology has allowed a clear reduction in working times and costs and has allowed the reduction of stress for patients who undergo invasive and extensive treatments to recover aesthetics and function, and for clinicians who must manage complex cases with fewer appointments possible.

High genetic variability of Schmallenberg virus M-segment leads to efficient immune escape from neutralizing antibodies.

Schmallenberg virus (SBV) is the cause of severe fetal malformations when immunologically naïve pregnant ruminants are infected. In those malformed fetuses, a "hot-spot"-region of high genetic variability within the N-terminal region of the viral envelope protein Gc has been observed previously, and this region co-localizes with a known key immunogenic domain. We studied a series of M-segments of those SBV variants from malformed fetuses with point mutations, insertions or large in-frame deletions of up to 612 nucleotides. Furthermore, a unique cell-culture isolate from a malformed fetus with large in-frame deletions within the M-segment was analyzed. Each Gc-protein with amino acid deletions within the "hot spot" of mutations failed to react with any neutralizing anti-SBV monoclonal antibodies or a domain specific antiserum. In addition, in vitro virus replication of the natural deletion variant could not be markedly reduced by neutralizing monoclonal antibodies or antisera from the field. The large-deletion variant of SBV that could be isolated in cell culture was highly attenuated with an impaired in vivo replication following the inoculation of sheep. In conclusion, the observed amino acid sequence mutations within the N-terminal main immunogenic domain of glycoprotein Gc result in an efficient immune evasion from neutralizing antibodies in the special environment of a developing fetus. These SBV-variants were never detected as circulating viruses, and therefore should be considered to be dead-end virus variants, which are not able to spread further. The observations described here may be transferred to other orthobunyaviruses, particularly those of the Simbu serogroup that have been shown to infect fetuses. Importantly, such mutant strains should not be included in attempts to trace the spatial-temporal evolution of orthobunyaviruses in molecular-epidemiolocal approaches during outbreak investigations.

How to dissect the plasticity of antigen-specific immune response: a tissue perspective.

Generation of antigen-specific humoral responses following vaccination or infection requires the maturation and function of highly specialized immune cells in secondary lymphoid organs (SLO), such as lymph nodes or tonsils. Factors that orchestrate the dynamics of these cells are still poorly understood. Currently, experimental approaches that enable a detailed description of the function of the immune system in SLO have been mainly developed and optimized in animal models. Conversely, methodological approaches in humans are mainly based on the use of blood-associated material because of the challenging access to tissues. Indeed, only few studies in humans were able to provide a discrete description of the complex network of cytokines, chemokines and lymphocytes acting in tissues after antigenic challenge. Furthermore, even fewer data are currently available on the interaction occurring within the complex micro-architecture of the SLO. This information is crucial in order to design particular vaccination strategies, especially for patients affected by chronic and immune compromising medical conditions who are under-vaccinated or who respond poorly to immunizations. Analysis of immune cells in different human tissues by high-throughput technologies, able to obtain data ranging from gene signature to protein expression and cell phenotypes, is needed to dissect the peculiarity of each immune cell in a definite human tissue. The main aim of this review is to provide an in-depth description of the current available methodologies, proven evidence and future perspectives in the analysis of immune mechanisms following immunization or infections in SLO.

Timing of oral feeding changes in premature infants who underwent osteopathic manipulative treatment.

BACKGROUND: The delayed transition from gavage-to-nipple feeding is one of the most significant factors that may prolong hospital length of stay (LOS). Osteopathic manipulative treatment (OMT) has been demonstrated to be effective regarding LOS reduction, but no investigations have documented its clinical validity for attaining oral feeding. OBJECTIVES: To assess OMT utility regarding the timing of oral feeding in healthy preterm infants. DESIGN: Preliminary propensity score-matched retrospective cohort study. SETTING: Data were extrapolated from the neonatal intensive care unit (NICU) of Del Ponte Hospital in Varese, Italy, during the period between March 2012 and December 2013. INTERVENTIONS: Two propensity score-matched groups of healthy preterm infants aged 28+0 to 33+6 were compared, observing those supported with OMT until hospital discharge and control subjects. MAIN OUTCOME MEASURES: Days from birth to the attainment of oral feeding was the primary endpoint. Body weight, body length, head circumference and LOS were considered as secondary endpoints. RESULTS: Seventy premature infants were included in the study as the control group (n = 35; body weight (BW) = 1457.9 ± 316.2 g; gestational age (GA) = 31.5 ± 1.73 wk) and the osteopathic group (n = 35; BW = 1509.6 ± 250.8 g; GA = 31.8 ± 1.64 wk). The two groups had analogous characteristics at study entry. In this cohort, we observed a significant reduction in TOF (-5.00 days; p = 0.042) in the osteopathic group with a greater effect in very low birth weight infants. CONCLUSIONS: These data demonstrate the utility and potential efficacy of OMT for the attainment of oral feeding. Further adequately powered clinical trials are recommended.

Comparative analysis of adaptive immune responses following experimental infections of cattle with bovine viral diarrhoea virus-1 and an Asiatic atypical ruminant pestivirus.

Atypical ruminant pestiviruses are closely related to the two bovine viral diarrhoea virus (BVDV) species, BVDV-1 and BVDV-2. While there is evidence of cross-protective immune responses between BVDV-1 and BVDV-2, despite antigenic differences, there is little information on the antigenic cross-reactivity with atypical ruminant pestiviruses. The aim of this study was therefore to assess the specificity of antibody and T cell responses induced by experimental infection of calves with BVDV-1 strain Ho916, Th/04_KhonKaen (TKK), an Asiatic atypical ruminant pestivirus, or co-infection with both viruses. Homologous virus neutralization was observed in sera from both single virus infected and co-infected groups, while cross-neutralization was only observed in the TKK infected group. T cell IFN-γ responses to both viruses were observed in the TKK infected animals, whereas Ho916 infected calves responded better to homologous virus. Specifically, IFN-γ responses to viral non-structural protein, NS3, were observed in all infected groups while responses to viral glycoprotein, E2, were virus-specific. Broader antigen-specific cytokine responses were observed with similar trends between inoculation groups and virus species. The limited T cell and antibody immune reactivity of Ho916 inoculated animals to TKK suggests that animals vaccinated with current BVDV-1-based vaccines may not be protected against atypical ruminant pestiviruses.

Incursion of Schmallenberg virus into Great Britain in 2011 and emergence of variant sequences in 2016.

Schmallenberg virus (SBV) is a vector-borne orthobunyavirus in the family Bunyaviridae, first identified in Germany before rapidly spreading throughout Europe. To investigate the events surrounding the incursion of this virus into Great Britain (GB) and its subsequent spread, archived sheep serum samples from an unrelated field survey in 2011 were analysed for the presence of SBV specific antibodies, to determine the earliest date of seroconversion. This serological study, along with analysis of the spatial spread of the sources of samples submitted for SBV analysis after January 2012, suggests that SBV entered GB on more than one occasion and in more than one location. Phylogenetic analysis of SBV sequences from 2012 ovine samples, from a variety of counties and dates, demonstrated a non-linear evolution of the virus, i.e. there was no distinct clustering between host species, geographical locations or during the outbreak. This also supports the notion of multiple viruses entering GB, rather than a single virus incursion. Premature termination signals were present in several non-structural putative protein sequences. One SBV sequence exhibited large deletions in the M segment of the genome. After the first outbreak in 2011-2012, interest in SBV in GB waned and continuous surveillance was not upheld. The re-emergence of SBV in 2016 has raised renewed concern and ended speculation that SBV might have been eradicated permanently from GB. When SBV sequences from 2012 were compared with those from the re-emergence in 2016-2017, a second distinct clade of SBV was identified that separates recent strains from those observed during the first outbreak.

Establishment of a Bovine Viral Diarrhea Virus Type 2 Intranasal Challenge Model for Assessing Vaccine Efficacy.

The objective of this study was to develop a bovine viral diarrhea virus type 2 (BVDV-2) challenge model suitable for evaluation of efficacy of BVDV vaccines; a model that mimics natural infection and induces clear leukopenia and viremia. Clinical, hematological and virological parameters were evaluated after infection of two age groups of calves (3 and 9 months) with two BVDV-2 strains (1362727 and 502643). Calves became pyrexic between 8 and 9 days post inoculation and exhibited symptoms, such as nasal discharge, mild depression, cough, and inappetence. Leukopenia with associated lymphopenia and neutropenia was evident in all groups with lowest leukocyte and lymphocyte counts reached 8 dpi and granulocyte counts between 11 and 16 dpi, dependent on the strain and age of the calves. A more severe thrombocytopenia was seen in those animals inoculated with strain 1362727. Leukocyte and nasal swab samples were positive by virus isolation, as early as 3 dpi and 2 dpi respectively, independent of the inocula used. All calves seroconverted with high levels of BVDV-2 neutralizing antibodies. BVDV RNA was evident as late as 90 dpi and provides the first evidence of the presence of replicating virus long after recovery from BVDV-2 experimental infection. In summary, moderate disease can be induced after experimental infection of calves with a low titer of virulent BVDV-2, with leukopenia, thrombocytopenia, viremia, and virus shedding. These strains represent an attractive model to assess the protective efficacy of existing and new vaccines against BVDV-2.

Characterization of catalytic and non-catalytic activities of EgGST2-3, a heterodimeric glutathione transferase from Echinococcus granulosus.

Glutathione transferases (GSTs) perform several catalytic and non-catalytic roles in the defense against toxicities of electrophile compounds and oxidative stress, and therefore are involved in stress-response and cell detoxification. Previously, we have provided evidence indicating that EgGST2 and EgGST3, two phylogenetically distant Echinococcus granulosus GSTs, can naturally form a heterodimeric structure (EgGST2-3). In the present work, the recombinant heterodimer GST (rEgGST2-3) is characterized. Hence, rEgGST2-3 was able to conjugate GSH to three substrates: 1-chloro-2,4-dinitrobenzene (CDNB, general substrate for GSTs), 1,2-dichloro-4-nitrobenzene (specific substrate for mammalian Mu class) and trans,trans-deca-2,4-dienal (reactive carbonyl). The canonical activity was considerably reduced by all the conventional inhibitors (cybacron blue, triphenylthin chloride and bromosulfophthalein) and by other inhibitors (ellagic acid, alizarin and chenodeoxycholic acid). Besides this, rEgGST2-3 activity was inhibited by a number of anthelmintic drugs, where the halogenated phenolic drugs (mainly bithionol and hexachlorophene) acted as stronger inhibitors, suggesting they may bind to the EgGST2-3. Moreover, rEgGST2-3 exhibited glutathione-peroxidase activity, and its specific constant (kcat/KM) was calculated. Finally, rEgGST2-3 displayed the ability to bind non-substrate molecules, particularly anthelmintic drugs, suggesting that ligandin activity may have potential to act as a passive protection parasite mechanism. Overall, the rEgGST2-3 behavior was shown to be both complementary and redundant to that reported for rEgGST1, another characterized GST from E. granulosus. It would be appropriate that different enzymes in the same organism do not have exactly the same functional properties to develop a better adaptation to life in the host.

First molecular characterization of canine parvovirus strains in Sardinia, Italy.

Canine parvovirus type 2 (CPV-2) is responsible of acute hemorrhagic gastroenteritis in young dogs. CPV-2 emerged in 1978 in the USA, but new antigenic types, CPV-2a, 2b and 2c, have completely replaced the original type. In this study, we analyzed 81 animals collected in Sardinia, Italy. The VP2 sequence analysis of 27 positive samples showed that all antigenic CPV-2 types are circulating. CPV-2b seems to be the most widespread variant, followed by CPV-2a. Furthermore, 12 CPV-2b strains displayed further amino acid substitutions and formed a separate cluster in a phylogenetic tree, indicating regional genetic variation.

Proteomic changes in the ileum of sheep infected with Mycobacterium avium subspecies paratuberculosis.

Johne's disease (JD) is a chronic enteritis of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). To identify the processes activated in the sheep intestine during natural MAP infection, and to provide a panel of differential host and pathogen proteins with diagnostic and prognostic potential, a differential shotgun proteomics workflow, including mass spectrometry, label-free quantisation and pathway analysis, was applied to ileal tissues of ewes with and without JD. Out of 2889 total proteins identified, 384 were differentially expressed and 341 were expressed at a higher level in JD. On the basis of Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) analysis, these proteins were involved in numerous relevant biological networks and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, including inhibition of phagosome acidification (such as V-ATPase), bacterial invasion, leucocyte recruitment and activation, and antimicrobial activity (such as haptoglobin, lactoferrin, cathelicidins, calgranulins and interleukins). A total of 28 MAP proteins were identified, including bacterioferritin, ß-lactamase and heparin-binding haemagglutinin (HBHA), a mycobacterial adhesin crucial for dissemination of infection.

A retrospective study detects a novel variant of porcine epidemic diarrhea virus in England in archived material from the year 2000.

Outbreaks of porcine epidemic diarrhea (PED) were first recorded in England in the 1970s and continued to be confirmed until 2002. Retrospective analysis of archived material from one of the last confirmed cases in England in the year 2000 demonstrates the previous existence of a very diverse PED virus strain. Following the outbreaks of PED in North America in 2013, there has been renewed interest in phylogenetic analysis of sequences from PEDV strains worldwide. There is a gap in the available sequence data between the mid 1980s and the mid 2000s. This work is an example of how this gap can be at least partially filled by the examination of archived material.

Exposure of Asian Elephants and Other Exotic Ungulates to Schmallenberg Virus.

Schmallenberg virus (SBV) is an emerging Orthobunyavirus, first described in 2011 in cattle in Germany and subsequently spread throughout Europe, affecting mainly ruminant livestock through the induction of foetal malformations. To gain a better understanding of the spectrum of susceptible species and to assess the value of current SBV serological assays, screening of serum samples from exotic artiodactyls and perissodactyls collected at the Living Collections from the Zoological Society of London (Whipsnade and London Zoos) and Chester Zoo was carried out. There was compelling evidence of SBV infection in both zoological collections. The competitive ELISA has proved to be applicable for the detection of SBV in exotic Bovidae, Cervidae, Suidae, Giraffidae and most notably in endangered Asian elephants (Elephas maximus), but unreliable for the screening of Camelidae, for which the plaque reduction neutralisation test was considered the assay of choice.

"Phthisiophobia": the difficult recognition of transmission of tuberculosis to health care workers.

BACKGROUND: Even if the contagious nature of tuberculosis was universally accepted during the nineteenth century, its transmission to health care workers (HCWs) was initially denied by the scientific community. Working among TB patients was not considered dangerous for healthy adults, so the potential risks for HCWs were branded as unwarranted "phthisiophobia" (fear of contracting tuberculosis). OBJECTIVES: This study aims at analyzing the problem of tuberculosis transmission among health care workers from an historical perspective, particularly highlighting the contribution made by the Italian Occupational Medicine community. METHODS: Scientific literature and historical sources on different theories regarding tuberculosis transmission were investigated, specially focusing on the period at the turn of the 19th and 20th centuries. RESULTS: At the beginning of the twentieth century, Luigi Devoto (1864-1936), an Italian pioneer in the field of Occupational Medicine, was one of the first scientists to conduct research on the transmission of tuberculosis among nurses. Since the 1920s several studies, conducted mainly on medical and nursing students, confirmed the risk for HCWs. However an international consensus on this issue was only achieved during the 1950s, when the institution of mandatory chest radiographs on admission for all patients significantly decreased the cases of tuberculosis among HCWs. CONCLUSIONS: Devoto was one of the first scholars who postulated the transmission of tuberculosis to HCWs. He also theorized that hospital personnel with active disease could also be a source of contagion to patients. Nowadays, "third party risk" and latent tuberculosis infection pose a new challenge for occupational physicians in hospitals.

Detection of Schmallenberg virus serum neutralising antibodies.

Schmallenberg virus (SBV) emerged in continental Europe in late 2011, and further work is required to assess the prevalence of SBV throughout Europe. Since its detection in Germany, SBV has now been detected in other European countries, including the United Kingdom. Infection with SBV can cause mild clinical signs in ruminants, including diarrhoea and reduced milk yield. However, the virus can have a devastating effect on the developing foetus leading to malformation in newborn offspring. This is a feature shared by other members of the Simbu group of orthobunyaviruses. Since disease in adult animals can be inapparent, serology offers the best method for monitoring for the presence of SBV and assisting in livestock management. This protocol describes a method for initial titration of SBV on African Green Monkey kidney (Vero) cells, and a plaque reduction neutralisation test (PRNT) for the detection of neutralising antibodies against SBV in cattle and sheep sera. This assay can be used to screen ruminant sera in order to confirm exposure to the virus, and the results obtained are comparable to a recently developed commercial enzyme linked immunosorbent assay (ELISA). Thus, these two assays constitute an effective diagnostic tool-box for providing confirmation of exposure to SBV.

The first case of a bull persistently infected with Border disease virus in New Zealand.

CASE HISTORY: Poor reproductive performance was observed in 62 dairy heifers, with a pregnancy rate of 23% following 57 days mating with one 3-year-old and two 2-year old Belted Galloway bulls that were sourced from separate sheep and beef farms. CLINICAL FINDINGS: The 3-year-old bull was small for its age with small testes. This bull was seropositive for bovine viral diarrhoea virus type I (BVDV 1) using an Ag-ELISA, and positive on PCR for border disease virus (BDV). DIAGNOSTIC INVESTIGATION: Phylogenetic analysis of the BDV isolate from the affected bull indicated that it was part of the BDV 1 group. For 40 of the heifers exposed to the bull that were tested, all of them had a positive VNT (virus neutralisation test) titre to both BDV (titre≥1:4) and BVDV 1 (titre>1:4). On the farm of origin of the affected bull there was no evidence of BDV circulating between cattle. DIAGNOSIS: Persistent infection of a bull with BDV. CLINICAL RELEVANCE: Cattle persistently infected with BDV can act as a source of virus for infection of other cattle. The benefit of testing cattle for bovine viral diarrhoea could be enhanced by using tests that also detect BDV.

Characterisation of vaccine-induced, broadly cross-reactive IFN-γ secreting T cell responses that correlate with rapid protection against classical swine fever virus.

Live attenuated C-strain classical swine fever viruses (CSFV) provide a rapid onset of protection, but the lack of a serological test that can differentiate vaccinated from infected animals limits their application in CSF outbreaks. Since immunity may precede antibody responses, we examined the kinetics and specificity of peripheral blood T cell responses from pigs vaccinated with a C-strain vaccine and challenged after five days with a genotypically divergent CSFV isolate. Vaccinated animals displayed virus-specific IFN-γ responses from day 3 post-challenge, whereas, unvaccinated challenge control animals failed to mount a detectable response. Both CD4(+) and cytotoxic CD8(+) T cells were identified as the cellular source of IFN-γ. IFN-γ responses showed extensive cross-reactivity when T cells were stimulated with CSFV isolates spanning the major genotypes. To determine the specificity of these responses, T cells were stimulated with recombinant CSFV proteins and a proteome-wide peptide library from a related virus, BVDV. Major cross-reactive peptides were mapped on the E2 and NS3 proteins. Finally, IFN-γ was shown to exert potent antiviral effects on CSFV in vitro. These data support the involvement of broadly cross-reactive T cell IFN-γ responses in the rapid protection conferred by the C-strain vaccine and this information should aid the development of the next generation of CSFV vaccines.

[Vaccinal prophylaxis against tetanus and hepatitis B: difficulties in application of law and actual status of workers' protection in Lombardy among workers of building sector]. / La profilassi vaccinale antitetanica ed antiepatite B: problematiche applicative della norma e state attuale della copertura tra i lavoratori del Comparto Edile in Lombardia.

Vaccinal prophylaxis is a preventive action which is compulsory, if available and useful for health protection of workers exposed to biological work risk. Definition andrealization of compulsory vaccinal campaigns is assigned by law to Occupational Health Doctor. We report a research carried out in 135 firms of building sector on 2158 workers exposed to biological work risk and concerning their vaccinal status against Tetanus and Hepatitis B. We found that only in a part of firms a plan of vaccination has been activated among workers exposed to biological work risk and, if activated, these plans frequently were partial and incomplete. Vaccinal protections among workers therefore resulted insufficient especially among foreigners workers and among workers active in first aid and emergency work teams. We think that a more active role of Occupational Health Doctors in promotion and realization of vaccinal campaigns is necessary for medico-legal and scientific reasons.

[Work-related stress risk assessment in a home care agency]. / La valutazione del rischio stress lavoro-correlato in una cooperativa di assistenza domiciliare.

The workload, the quality of professional relationships and emotional involvement have a significant impact on distress and burnout in health care-workers; this impact has an hight variability among the different environments and different care facilities (hospital, erderly nursing homes, home care). The risk assessment of work-related stress performed in 2010 in a homecare agency highlighted organizational problems related to the content/context of work and risk factors for health and safety. High turn-over is evidenced as critical among the "sentinel events". The level of job-strain was moderate, even if some critical issues were evidenced especially in the group of physiotherapists; nurses were configured as a homogeneous group with a low level of job-strain. In informative meetings the workers identified the discomfort related to the time for transferring patients from their homes in a high-traffic metropolitan area was identified as the most critical aspect.