Presence of Torque teno sus virus 1 and 2 in porcine circovirus 3‐positive pigs

In this study, the co‐infection of Torque teno sus virus (TTS uV) and porcine circovirus type 3 (PCV 3) was reported. One hundred and ten of 132 (83.3%) PCV 3‐positive samples were co‐infected with Torque teno sus virus 1 (TTS uV1). Ninety‐four of 132 (71.2%) PCV 3‐positive samples were co‐infected with Torque teno sus virus 2 (TTS uV2). Sixty‐six of 132 (50.0%) of PCV 3‐positive samples were co‐infected with both TTS uV1 and TTS uV2. There were no clinical signs of infection in pigs that were both PCV 3‐positive and PCV 2‐negative, in either multiparous sows or live‐born infants. The high co‐infection rate provides valuable information for the further study of the pathological correlation between PCV 3 and TTS uVs.     

High detection rates of Torque teno sus virus in co‐infection with important viral pathogens in porcine kidneys on St. Kitts Island,Lesser Antilles

We report here high rates of detection (50.8%, 31/61 pigs) of Torque teno sus virus (TTS uV) in kidneys of slaughter‐age, apparently healthy pigs on St. Kitts island, Lesser Antilles. TTS uV1 and TTS uVk2a were detected in 23 (37.7%) and 13 (21.3%) pigs, respectively, including mixed infection in five animals. By nucleotide sequence identities and phylogenetic analysis, significant genetic diversity was observed among both TTS uV1 and TTS uVk2a on St. Kitts, with TTS uVk2a showing higher genetic diversity than TTS uV1. Fourteen (45.2%) and 10 (32.2%) of the TTS uV infected pigs tested positive for porcine circovirus type 2 (PCV 2) and porcine parvovirus (PPV ), respectively, revealing high rates of co‐infection of TTS uV with PCV 2 and PPV . This is the first report on detection and genetic diversity of TTS uV from the Lesser Antilles. Also, PCV 2 and PPV were detected for the first time in the Lesser Antilles. Considering the impact of pig farming on the regional livestock economy, the increasing demand for local pork and lack of information on emerging and re‐emerging porcine viruses in the Lesser Antilles, the present findings have important implications on swine health.     

Similar frequency of Porcine circovirus 3 (PCV‐3) detection in serum samples of pigs affected by digestive or respiratory disorders and age‐matched clinically healthy pigs

Porcine circovirus 3 (PCV‐3) has been identified in pigs affected by different disease conditions, although its pathogenicity remains unclear. The objective of the present study was to assess the frequency of PCV‐3 infection in serum samples from animals suffering from post‐weaning respiratory or digestive disorders as well as in healthy animals. A total of 315 swine serum samples were analysed for PCV‐3 DNA detection by conventional PCR; positive samples were further assayed with a quantitative PCR and partially sequenced. Sera were obtained from 4 week‐ to 4 month‐old pigs clinically diagnosed with respiratory (n = 129) or digestive (n = 126) disorders. Serum samples of age‐matched healthy animals (n = 60) served as negative control. Pigs with clinical respiratory signs had a wide variety of pulmonary lesions including suppurative bronchopneumonia, interstitial pneumonia, fibrinous‐necrotizing pneumonia and/or pleuritis. Animals with enteric signs displayed histopathological findings like villus atrophy and fusion, catarrhal enteritis and/or catarrhal colitis. Overall, PCV‐3 DNA was detected in 19 out of 315 analysed samples (6.0%). Among the diseased animals, PCV‐3 was found in 6.2% (8 out of 129) and 5.6% (7 out of 126) of pigs with respiratory and digestive disorders, respectively. The frequency of PCV‐3 PCR positive samples among healthy pigs was 6.7% (4 out of 60). No apparent association was observed between PCR positive cases and any type of histopathological lesion. The phylogenetic analysis of the partial genome sequences obtained showed high identity among viruses from the three groups of animals studied. In conclusion, PCV‐3 was present in the serum of diseased and healthy pigs to similar percentages, suggesting that this virus does not seem to be causally associated with respiratory or enteric disorders.     

Prevalence of the Novel Torque Teno Sus Virus Species k2b from Pigs in the United States and Lack of Association with Post‐Weaning Multisystemic Wasting Syndrome or Mulberry Heart Disease

The family Anelloviridae includes a number of viruses infecting humans (Torque teno viruses, TTV) and other animals including swine (Torque teno sus viruses, TTSuV). Two genetically distinct TTSuV species have been identified from swine thus far (TTSuV1 and TTSuVk2), although their definitive association with disease remains debatable. In 2012, a novel TTSuV species was identified from commercial swine serum and classified in the genus Kappatorquevirus as TTSuVk2b. The other Kappatorquevirus species, TTSuVk2a, has been associated with post‐weaning multisystemic wasting syndrome (PMWS) when coinfected with porcine circovirus type 2 (PCV2). Therefore, in this study, we initially amplified a portion of TTSuVk2b ORF1 and, subsequently, assessed the molecular prevalence of the virus in pigs in the United States. A total of 127 serum and 115 tissue samples were obtained from pigs with PMWS or mulberry heart disease (MHD) in six states and tested by PCR for the presence of TTSuVk2b DNA. Approximately 27.6% of the serum and 21.7% of tissue samples tested positive for TTSuVk2b DNA, and the positive products were confirmed by sequencing. However, we did not detect a correlation between TTSuVk2b infection and PMWS or MHD. The near full‐length genomic sequence of US TTSuVk2b was determined, and sequence analysis revealed that the US TTSuVk2b isolates were 95% identical to the TTSuVk2b isolate from Spain, with most of the variations clustering in ORF1. We conclude that the novel TTSuVk2b species is present in pigs in the United States and its potential association with a disease warrants further investigation.     

Evidence of porcine circovirus‐like virus P1 in piglets with an unusual congenital tremor

Outbreaks of trembling and shaking were reported among pigs at two pig farms in Jiangsu Province, China. Serum and tissue samples tested positive for porcine circovirus‐like virus P1 and negative for classical swine fever virus, porcine circovirus type 2, astrovirus and porcine pestivirus using PCR/RT‐PCR and immunohistochemical techniques. High P1 viral genome loads were identified in sera, brain and lymph node tissue samples by qPCR. In addition, one of the most notable pathological changes was dissolution of the nucleus in Purkinje cells. The results of this study provide molecular evidence of an association between congenital tremor in pigs and P1 virus.     

Herd‐level prevalence and incidence of porcine epidemic diarrhoea virus (PEDV) and porcine deltacoronavirus (PDCoV) in swine herds in Ontario,Canada

Porcine epidemic diarrhoea virus (PEDV ) and porcine deltacoronavirus (PDC oV) were first identified in Canada in 2014. Surveillance efforts have been instrumental in controlling both diseases. In this study, we provide an overview of surveillance components for the two diseases in Ontario (Canada), as well as PEDV and PDC oV incidence and prevalence measures. Swine herds located in the Province of Ontario, of any type, whose owners agreed to participate in a voluntary industry‐led disease control programme (DCP ) and with associated diagnostic or epidemiological information about the two swine coronaviruses, were eligible to be included for calculation of disease frequency at the provincial level. PEDV and PDC oV data stored in the industry DCP database were imported into the R statistical software and analysed to produce weekly frequency of incidence counts and prevalence counts, in addition to yearly herd‐level incidence risk and prevalence between 2014 and 2016. The yearly herd‐level incidence risk of PEDV , based on industry data, was 13.5%, 3.0% and 1.4% (95% CI : 11.1–16.2, 2.0–4.2, 0.8–2.3), while the yearly herd‐level incidence risk of PDC oV was 1.1%, 0.3%, and 0.1% (95% CI : 0.5–2.2, 0.1–0.9, 0.0–0.5), for 2014, 2015 and 2016, respectively. Herd‐level prevalence estimates for PEDV in the last week of 2014, 2015 and 2016 were 4.4%, 2.3% and 1.4%, respectively (95% CI : 3.1–6.0, 1.5–3.3, 0.8–2.2), while herd‐level prevalence estimates for PDC oV in the last week of 2014, 2015 and 2016 were 0.5%, 0.2% and 0.2%, respectively (95% CI : 0.1–1.2, 0.0–0.6, 0.0–0.6). Collectively, our results point to low and decreasing incidence risk and prevalence for PEDV and PDC oV in Ontario, making both diseases possible candidates for disease elimination at the provincial level.     

First detection of novel enterovirus G recombining a torovirus papain‐like protease gene associated with diarrhoea in swine in South Korea

Enterovirus species G (EV‐G) comprises a highly diversity of 20 genotypes that is prevalent in pig populations, with or without diarrhoea. In the present study, a novel EV‐G strain (KOR/KNU‐1811/2018) that resulted from cross‐order recombination was discovered in diagnostic faecal samples from neonatal pigs with diarrhoea that were negative for swine enteric coronaviruses and rotavirus. The recombinant EV‐G genome possessed an exogenous 594‐nucleotide (198‐amino acid) sequence, flanked by two viral 3C^pro cleavage sites at the 5′ and 3′ ends in its 2C/3A junction region. This insertion encoded a predicted protease similar to the porcine torovirus papain‐like cysteine protease (PLCP), which was recently found in the EV‐G1, ‐G2, and ‐G17 genomes. The complete KNU‐1811 genome shared 73.7% nucleotide identity with a prototype EV‐G1 strain, but had 83.9%–86.7% sequence homology with the global EV‐G1‐PLCP strains. Genetic and phylogenetic analyses demonstrated that the Korean recombinant EV‐G's own VP1 and inserted foreign PLCP genes are most closely related independently to contemporary chimeric G1‐PLCP and G17‐PLCP strains respectively. These results implied that the torovirus‐derived PLCP gene might have undergone continuous nucleotide mutations in the respective EV‐G genome following its independent acquisition through naturally occurring recombination. Our results advance the understanding of the genetic evolution of EV‐G driven by infrequent viral recombination events, by which EV‐G populations laterally gain an exotic gene encoding a virulence factor from heterogeneous virus families, thereby causing clinical disease in swine.     

Distinction between intact and antibiotic‐inactivated bacteria by real‐time PCR after treatment with propidium monoazide

One limitation to the use of the polymerase chain reaction (PCR) to identify orthopedic infections has been apparent false‐positive results, possibly due to the detection of dead bacteria. We recently showed that the use of DNA‐binding agent propidium monoazide (PMA) could distinguish viable from heat‐inactivated bacteria, and, in this study, we investigated whether the same technique can be applied to bacteria killed by two antibiotics with distinctly different mechanisms of action, a test of greater clinical relevance than thermal inactivation. Staphylococcus aureus and S. epidermidis were inactivated by vancomycin and gentamicin and treated with PMA or left untreated before DNA extraction. The threshold cycle difference of antibiotic‐treated bacteria with and without PMA pretreatment was investigated with PCR primers for the 16S rDNA and tuf genes. Our results indicated that PMA effectively inhibited detection by PCR of bacteria, which had been inactivated by either vancomycin or gentamicin. The effect was statistically significant at 24 h after treatment (C_t difference consistently >3; p < 0.05) and after 10 days of treatment (C_t difference >4; p < 0.01), when compared to viable cells (C_t difference 1–2). Vancomycin had a stronger effect on the C_t value than gentamicin, reflecting the different mechanism of action of each antibiotic. Techniques of this type may help reduce clinically false‐positive PCR results caused by the detection of dead bacteria, and may be especially useful in patients who have received antibiotics, such as patients undergoing the second stage of a two‐stage revision for infected arthroplasty. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1245–1251, 2010     

Development of a novel robotic platform with controllable stiffness manipulation arms for laparoendoscopic single‐site surgery (LESS)

Background

For current LESS robotic systems, the trade‐off between dexterity and payload capability is always present. This paper presents a novel LESS robotic platform equipped with controllable stiffness manipulation arms.

Methods

Each manipulation arm with an articulated section and a controllable stiffness continuum section (CSCS) can be switched between a 7‐DoF compliant status and 5‐DoF rigid status according to the operation requirement. Screw theory and product exponential formula are used to quantify the kinematic performance.

Results

The stiffness of the manipulation arm promotes 3.03 to 4.12 times from compliant to rigid CSCS with maximum payload of 10 N in rigid status. The shortest rigid/compliant switching time is 5 s. The precision of a tracking test and an ex vivo procedure verified the accuracy and effectiveness of the controllable stiffness manipulation arms.

Conclusions

This robot could potentially improve the surgical performance and further expand robotic LESS procedures.     

Eradication of osteosarcoma by fluorescence‐guided surgery with tumor labeling by a killer‐reporter adenovirus

In a previous study, we developed fluorescence‐guided surgery (FGS) for osteosarcoma using an orthotopic model with 143B human osteosarcoma cells expressing red fluorescent protein (RFP) implanted into the intramedullary cavity of the tibia in nude mice. The FGS‐treated mice had a significantly higher disease‐free survival (DFS) rate than the bright‐light surgery (BLS). However, although FGS significantly reduced the recurrence of the primary tumor, it did not reduce lung metastasis. In the present study, we utilized the OBP‐401 telomerase‐dependent killer‐reporter adenovirus, carrying green fluorescent protein (GFP), to label human osteosarcoma in situ in orthotopic mouse models. OBP‐401‐illuminated human osteosarcoma cell lines, 143B and MNNG/HOS cells in vitro and in vivo. OBP‐401 tumor illumination enabled effective FGS of the 143B‐derived orthotopic mouse model of human osteosarcoma model as well as FGS eradication of residual cancer cells after BLS. OBP‐401‐assisted FGS significantly inhibited local recurrence and lung metastasis after surgery, thereby prolonging DFS and overall survival (OS), achieving a very important improvement of therapeutic outcomes over our previously reported FGS study. These therapeutic benefits of FGS were demonstrated using a clinically‐viable methodology of direct labeling of human osteosarcoma in situ with the OBP‐401 killer‐reporter adenovirus in contrast with previous reports, which used genetically engineered labeled cells or antibody‐based fluorescent labels for FGS. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:836–844, 2016.     

Passive immunization with anti‐glucosaminidase monoclonal antibodies protects mice from implant‐associated osteomyelitis by mediating opsonophagocytosis of Staphylococcus aureus megaclusters

Towards the development of a methicillin‐resistant Staphylococcus aureus (MRSA) vaccine we evaluated a neutralizing anti‐glucosaminidase (Gmd) monoclonal antibody (1C11) in a murine model of implant‐associated osteomyelitis, and compared its effects on LAC USA300 MRSA versus a placebo and a Gmd‐deficient isogenic strain (ΔGmd). 1C11 significantly reduced infection severity, as determined by bioluminescent imaging of bacteria, micro‐CT assessment of osteolysis, and histomorphometry of abscess numbers (p < 0.05). Histology also revealed infiltrating macrophages, and the complete lack of staphylococcal abscess communities (SAC), in marrow abscesses of 1C11 treated mice. In vitro, 1C11 had no direct effects on proliferation, but electron microscopy demonstrated that 1C11 treatment phenocopies ΔGmd defects in binary fission. Moreover, addition of 1C11 to MRSA cultures induced the formation of large bacterial aggregates (megaclusters) that sedimented out of solution, which was not observed in ΔGmd cultures or 1C11 treated cultures of a protein A‐deficient strain (ΔSpa), suggesting that the combined effects of Gmd inhibition and antibody‐mediated agglutination are required. Finally, we demonstrated that macrophage opsonophagocytosis of MRSA and megaclusters is significantly increased by 1C11 (p < 0.01). Collectively, these results suggest that the primary mechanism of anti‐Gmd humoral immunity against MRSA osteomyelitis is macrophage invasion of Staphylococcal abscess communities (SAC) and opsonophagocytosis of megaclusters. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1389–1396, 2014.     

Immunotherapy synergizes with debridement and antibiotic therapy in a murine 1‐stage exchange model of MRSA implant‐associated osteomyelitis

Methicillin‐resistant Staphylococcus aureus (MRSA) reinfection following revision surgery remains a major orthopaedic problem. Toward the development of immunotherapy with anti‐glucosaminidase monoclonal antibodies (anti‐Gmd), we aimed to: (i) develop a murine 1‐stage exchange model of bioluminescent MRSA (USA300LAC::lux) contaminated femoral implants; and (ii) utilize this model to demonstrate the synergistic effects of combination vancomycin and anti‐Gmd therapy on reinfection and bone healing. Following an infection surgery, the original plate and two screws were removed on day 7, and exchanged with sterile implants. Mice were randomized to five groups: (i) no infection control; (ii) infected placebo; (iii) anti‐Gmd; (iv) vancomycin; and (v) combination therapy. Bioluminescent imaging (BLI) was performed on days 0, 1, 3, 5, 7, 8, 10, 12, and 14. Mice were euthanized on day 14 (day 7 post‐revision), and efficacy was assessed via colony forming units (CFU) on explanted hardware, micro‐CT, and histology. As monotherapies, anti‐Gmd inhibited Staphylococcus abscess communities, and vancomycin reduced CFU on the implants. However, only combination therapy prevented increased BLI post‐revision surgery, with a significant 6.5‐fold reduction on day 10 (p < 0.05 vs. placebo), and achieved sterile implant levels by day 12. Synergistic effects were also apparent from reduced osteolysis and increased new bone formation around the screws only observed following combination therapy. Taken together, we find that: (i) this murine femoral plate 1‐stage revision model can efficiently evaluate therapies to prevent reinfection; and (ii) immunotherapy plays a distinct role from antibiotics to reduce reinfection following revision surgery, such that synergy to achieve osseointegration is possible. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1590–1598, 2018.

     

EBV‐Associated Leukoencephalopathy with Late Onset of Central Nervous System Lymphoma in a Kidney Transplant Recipient

Central nervous system (CNS) lymphoma is a rare posttransplant lymphoproliferative disorder (PTLD), which usually has a poor outcome. To date, no specific conditions predisposing to this complication have been identified. We here describe the case of a renal transplant patient who was initially diagnosed as having Epstein‐Barr virus (EBV)‐associated leukoencephalopathy and ultimately developed EBV‐positive CNS lymphoma. The patient was a young lady who, 2 years after transplantation, presented with focal neurological and electroencephalographic abnormalities and diffuse white matter lesions on brain magnetic resonance imaging. EBV‐DNA was detected in the cerebrospinal fluid (CSF) by polymerase chain reaction. After acyclovir therapy and immunosuppressive drug tapering, the symptoms and electroencephalographic abnormalities subsided, and EBV‐DNA disappeared from the CSF. Ten years later, a bulky cerebral mass was found. After excision, a diagnosis of EBV‐positive, Hodgkin‐like monomorphic B‐cell PTLD was made. This case illustrates the potential pathophysiological relationships between EBV infection, leukoencephalopathy and CNS lymphoma; although a long time elapsed from the initial neurological illness to CNS lymphoma, a link between these two conditions cannot be excluded. Therefore, a careful long‐term follow‐up of EBV‐related encephalopathy is advisable.     

Very low CD19+ B‐lymphocyte percentage is associated with high levels of academic stress among healthy graduate students

Elevated chronic psychological stress is associated with weakened immune response to vaccines. B‐lymphocyte development may provide a pathway by which psychological stress can weaken immune response to vaccines. The current study examined the effect of chronic psychological stress on B‐ and T‐lymphocytes among doctoral students, after their qualifying exams, and matched community controls. Blood was drawn from 10 doctoral students immediately after their 3‐day qualifying exams and from 10 age‐ and gender‐matched community controls. B‐ (CD19+) and T‐ (CD3+) lymphocyte percentages were enumerated with flow cytometry. Psychological stress was measured with the Perceived Stress Scale (PSS). The mean PSS score was higher for the graduate students compared with the control group (p < 0.01). Mean CD19+ lymphocyte percentage for the students (5.82 per cent) was dramatically lower than the control group (15.32 per cent, p < 0.0001). CD3+ lymphocyte percentage did not differ between the two groups. There was a significant negative correlation between CD19+ lymphocyte percentage and PSS score (r = ?0.60, p = 0.003). Chronic psychological stress is associated with significant reductions in B‐lymphocytes among doctoral students undergoing qualifying exams and community participants. B‐lymphocyte decrements are a potential mechanism linking psychological stress and reduced antibody response to vaccines. Copyright © 2008 John Wiley & Sons, Ltd.     

Long‐term follow‐up of beta cell replacement therapy in 10 HIV‐infected patients with renal failure secondary to type 1 diabetes mellitus

The approach to transplantation in human immunodeficiency virus (HIV)‐positive patients has been conservative due to fear of exacerbating an immunocompromised condition. As a result, HIV‐positive patients with diabetes were initially excluded from beta cell replacement therapy. Early reports of pancreas transplant in patients with HIV described high rates of early graft loss with limited follow‐up. We report long‐term follow‐up of islet or pancreas transplantation in HIV‐positive type 1 diabetic patients who received a kidney transplant concurrently or had previously undergone kidney transplantation. Although 4 patients developed polyoma viremia, highly active antiretroviral therapy and adequate infectious prophylaxis were successful in providing protection until CD4+ counts recovered. Coordination with HIV providers is critical to reduce the risk of rejection by minimizing drug‐drug interactions. Also, protocols for prophylaxis of opportunistic infections and strategies for monitoring and treating BK viremia are important given the degree of immunosuppression required. This series demonstrates that type 1 diabetic patients with well‐controlled HIV and renal failure can be appropriate candidates for beta cell replacement, with a low rate of infectious complications, early graft loss, and rejection, so excellent long‐term graft survival is possible. Additionally, patients with HIV and cardiovascular contraindications can undergo islet infusion.