Intradermal vaccination with Porcilis® Begonia can clinically protect against fatal PRV challenge with the highly virulent ZJ01 field strain.

Since pseudorabies (PR) re-emerged and rapidly spread in China at the end of 2011, researchers have focused on effective vaccine strategies to prevent and control pseudorabies virus (PRV) infection in pig herds. Due to the extensive application of an attenuated vaccine based on the Bartha-K61 strain isolated in Hungary in 1961 and the variation of the PRV strain, it has been suggested that traditional vaccines based on the Bartha-K61 strain offer only partial protection against variant strains. It was therefore evaluated whether the Porcilis® Begonia vaccine, which is based on the NIA-3 strain with deletions in the gE and TK genes, is efficacious against experimental infection with the virulent, contemporary Chinese PRV strain ZJ01. In this study, piglets were vaccinated with Porcilis® Begonia through either the intradermal (ID) route or the intramuscular (IM) route and subsequently challenged intranasally with strain ZJ01 at 4 weeks post-vaccination. An unvaccinated challenge group and an unvaccinated/nonchallenged group were also included in the study. All animals were monitored for 14 days after challenge. Vaccinated and negative control pigs stayed healthy during the study, while the unvaccinated control animals developed lesions associated with PRV ZJ01 challenge, and 44% of these pigs died before the end of the experiment. This study demonstrated that ID or IM vaccination of pigs with a vaccine based on the NIA-3 strain Porcilis® Begonia clinically protects against fatal PRV challenge with the ZJ01 strain.

Higher levels of circulating desphospho-uncarboxylated matrix Gla protein over time are associated with worse survival: the prospective Maastricht Intensive Care COVID cohort.

BACKGROUND: Extra-hepatic vitamin K-status, measured by dephosphorylated uncarboxylated matrix Gla protein (dp-ucMGP), maintains vascular health, with high levels reflecting poor vitamin K status. The occurrence of extra-hepatic vitamin K deficiency throughout the disease of COVID-19 and possible associations with pulmonary embolism (PE), and mortality in intensive care unit (ICU) patients has not been studied. The aim of this study was to investigated the association between dp-ucMGP, at endotracheal intubation (ETI) and both ICU and six months mortality. Furthermore, we studied the associations between serially measured dp-ucMGP and both PE and mortality. METHODS: We included 112 ICU patients with confirmed COVID-19. Over the course of 4 weeks after ETI, dp-ucMGP was measured serially. All patients underwent computed tomography pulmonary angiography (CTPA) to rule out PE. Results were adjusted for patient characteristics, disease severity scores, inflammation, renal function, history of coumarin use, and coronary artery calcification (CAC) scores. RESULTS: Per 100 pmol/L dp-ucMGP, at ETI, the odds ratio (OR) was 1.056 (95% CI: 0.977 to 1.141, p = 0.172) for ICU mortality and 1.059 (95% CI: 0.976 to 1.059, p = 0.170) for six months mortality. After adjustments for age, gender, and APACHE II score, the mean difference in plasma dp-ucMGP over time of ICU admission was 167 pmol/L (95% CI: 4 to 332, p = 0.047). After additional adjustments for c-reactive protein, creatinine, and history of coumarin use, the difference was 199 pmol/L (95% CI: 50 to 346, p = 0.010). After additional adjustment for CAC score the difference was 213 pmol/L (95% CI: 3 to 422, p = 0.051) higher in ICU non-survivors compared to the ICU survivors. The regression slope, indicating changes over time, did not differ. Moreover, dp-ucMGP was not associated with PE. CONCLUSION: ICU mortality in COVID-19 patients was associated with higher dp-ucMGP levels over 4 weeks, independent of age, gender, and APACHE II score, and not explained by inflammation, renal function, history of coumarin use, and CAC score. No association with PE was observed. At ETI, higher levels of dp-ucMGP were associated with higher OR for both ICU and six month mortality in crude and adjusted modes, although not statistically significantly.

Quadrivalent neuraminidase RNA particle vaccine protects pigs against homologous and heterologous strains of swine influenza virus infection.

Influenza A virus in swine (IAV-S) continues to cause significant negative impact to both sows and growing pigs. The viral hemagglutinin (HA) and neuraminidase (NA) genes continue to evolve with HA diversifying at a faster rate than NA. Depending on country, whole inactivated virus (WIV) commercial and autogenous vaccines, as well as veterinary prescription vaccines targeting HA, are currently available. The use of these vaccines is focused on reducing virus and clinical signs in sows and to provide HA-specific maternally derived antibodies (MDA) to their suckling pigs. The deficiency in this strategy is that HA-MDA does not persist long enough to protect pigs through their growing phase from infection, and HA-MDA can interfere with effective pig immunization. This study evaluated the immunogenicity and efficacy of an adjuvanted, quadrivalent RNA Particle vaccine (Sequivity NA), currently licensed as Sequivity® IAV-S NA. This vaccine was formulated based on four NA antigens representing the major NA clades of IAV subtypes H1N1, H1N2 and H3N2 circulating in swine herds in the United States. In a series of trials, pigs were vaccinated twice, at three days and three weeks of age (WOA), followed by challenge with either homologous or heterologous IAV strains at 8 or 15 WOA. The Sequivity NA vaccine induced robust serum NA inhibition (NI) antibody and protected against IAV-S strains with homologous and heterologous NA to that of the vaccine. The magnitude and duration of nasal shedding was reduced in vaccinated-pigs challenged with either homologous or heterologous virus within the same NA clade. This NA-based RNA Particle vaccine avoids the known impact of HA-MDA on pig vaccination and provides a new tool to successfully reduce IAV-induced disease in the pig population.

Regional peak flow as a novel approach to assess regional pulmonary mechanics by electrical impedance tomography: an observational validation study.

Background: Spontaneous breathing efforts during mechanical ventilation are a widely accepted weaning approach for acute respiratory distress syndrome (ARDS) patients. These efforts can be too vigorous, possibly inflicting lung and diaphragm damage. Higher positive end expiratory pressure (PEEP) levels can be used to lower the magnitude of vigorous breathing efforts. Nevertheless, PEEP titrating tools are lacking in spontaneous mechanical ventilation (SMV). Therefore, the aim is to develop an electrical impedance tomography (EIT) algorithm for quantifying regional lung mechanics independent from a stable plateau pressure phase based on regional peak flow (RPF) by EIT, which is hypothetically applicable in SMV and to validate this algorithm in patients on controlled mechanical ventilation (CMV). Methods: The RPF algorithm quantifies a cumulative overdistension (ODRPF) and collapse (CLRPF) rate and is validated in a prospective cohort of mechanically ventilated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) patients on CMV. ODRPF and CLRPF are compared with compliance-based cumulative overdistension (ODP500) and collapse (CLP500) rates from the Pulmovista 500 EIT device at multiple PEEP levels (PEEP 10 cmH2O to PEEP 24 cmH2O) in EIT measurements from CMV patients by linear mixed models, Bland-Altman analysis and intraclass correlation coefficient (ICC). Results: Seventy-eight patients were included. Linear mixed models revealed an association between ODRPF and ODP500 of 1.02 (0.98-1.07, P<0.001) and between CLRPF and CLP500 of 0.93 (0.80-1.05, P<0.001). ICC values ranged from 0.78 to 0.86 (P<0.001) for ODRPF and ODP500 and from 0.70 to 0.85 (P<0.001) for CLRPF and CLP500 (PEEP 10 to PEEP 24). The mean bias between ODRPF and ODP500 in these PEEP levels ranged from 0.80% to 4.19% and from -1.31% to 0.13% between CLRPF and CLP500. Conclusions: A RPF approach for quantifying regional lung mechanics showed a moderate to good agreement in coronavirus disease 2019 (COVID-19) related ARDS patients on CMV compared to the compliance-based approach. This, in addition to being independent of a plateau pressure phase, indicates that the RPF approach is a valid method to explore for quantifying regional lung mechanics in SMV.

Pulmonary pathophysiology development of COVID-19 assessed by serial Electrical Impedance Tomography in the MaastrICCht cohort.

Patients with SARS-CoV-2 infection present with different lung compliance and progression of disease differs. Measures of lung mechanics in SARS-CoV-2 patients may unravel different pathophysiologic mechanisms during mechanical ventilation. The objective of this prospective observational study is to describe whether Electrical Impedance Tomography (EIT) guided positive end-expiratory pressure (PEEP) levels unravel changes in EIT-derived parameters over time and whether the changes differ between survivors and non-survivors. Serial EIT-measurements of alveolar overdistension, collapse, and compliance change in ventilated SARS-CoV-2 patients were analysed. In 80 out of 94 patients, we took 283 EIT measurements (93 from day 1-3 after intubation, 66 from day 4-6, and 124 from day 7 and beyond). Fifty-one patients (64%) survived the ICU. At admission mean PaO2/FiO2-ratio was 184.3 (SD 61.4) vs. 151.3 (SD 54.4) mmHg, (p = 0.017) and PEEP was 11.8 (SD 2.8) cmH2O vs. 11.3 (SD 3.4) cmH2O, (p = 0.475), for ICU survivors and non-survivors. At day 1-3, compliance was ~ 55 mL/cmH2O vs. ~ 45 mL/cmH2O in survivors vs. non-survivors. The intersection of overdistension and collapse curves appeared similar at a PEEP of ~ 12-13 cmH2O. At day 4-6 compliance changed to ~ 50 mL/cmH2O vs. ~ 38 mL/cmH2O. At day 7 and beyond, compliance was ~ 38 mL/cmH2O with the intersection at a PEEP of ~ 9 cmH2O vs. ~ 25 mL/cmH2O with overdistension intersecting at collapse curves at a PEEP of ~ 7 cmH2O. Surviving SARS-CoV-2 patients show more favourable EIT-derived parameters and a higher compliance compared to non-survivors over time. This knowledge is valuable for discovering the different groups.

Efficacy of Simultaneous Intradermal Vaccination of Swine against Porcine Circovirus 2, Porcine Reproductive and Respiratory Syndrome Virus, Mycoplasma hyopneumoniae and Lawsonia intracellularis.

The combined application of vaccines in swine offers many benefits, including reduced time and labour costs, and improved animal welfare, due to fewer injections and manipulations. This study investigated if simultaneous intradermal vaccinations against porcine circovirus 2, porcine reproductive and respiratory syndrome virus, Mycoplasma hyopneumoniae, and Lawsonia intracellularis, using a specialised needle-free applicator would confer comparable protection against experimental infection compared to the single vaccines. In all cases, the administration of the vaccines together was as efficacious as the administration of the vaccines alone, significantly reducing clinical signs associated with each of the four pathogens.

Assessing the Protective Dose of a Candidate DIVA Vaccine against Classical Swine Fever.

Classical swine fever is a highly contagious and deadly disease in swine. The disease can be controlled effectively by vaccination with an attenuated virus known as the "Chinese" (C)-strain. A single vaccination with the C-strain provides complete protection against highly virulent isolates within days after vaccination, making it one of the most efficacious veterinary vaccines ever developed. A disadvantage of the C-strain is that vaccinated animals cannot be serologically differentiated from animals that are infected with wild-type Classical swine fever virus. Previously, a C-strain-based vaccine with a stable deletion in the E2 structural glycoprotein was developed, which allows for differentiation between infected and vaccinated animals (DIVA). The resulting vaccine, which we named C-DIVA, is compatible with a commercial E2 ELISA, modified to render it suitable as a DIVA test. In the present work, three groups of eight piglets were vaccinated with escalating doses of the C-DIVA vaccine and challenged two weeks after vaccination. One group of four unvaccinated piglets served as controls. Piglets were monitored for clinical signs until three weeks after challenge and blood samples were collected to monitor viremia, leukocyte and thrombocyte levels, and antibody responses. The presence of challenge virus RNA in oropharyngeal swabs was investigated to first gain insight into the potential of C-DIVA to prevent shedding. The results demonstrate that a single vaccination with 70 infectious virus particles of C-DIVA protects pigs from the highly virulent Brescia strain.

An octavalent vaccine provides pregnant gilts protection against a highly virulent porcine parvovirus strain.

BACKGROUND: Porcilis® Ery+Parvo+Lepto is an octavalent inactivated ready-to-use vaccine that contains Erysipelothrix rhusiopathiae (Ery), porcine parvovirus (PPV), and six serogroups of Leptospira (Lepto). The efficacy of Porcilis® Ery + Parvo+Lepto against reproductive problems associated with porcine parvovirus (PPV) infection was evaluated in pregnant gilts. For this, a group of ninegilts was vaccinated twice (at 5 and 6 months old) with Porcilis® Ery + Parvo+Lepto (Group 1), while a group of eight gilts was included as unvaccinated controls (Group 2). All pigs were artificially inseminated 4 weeks after the second vaccination. They were challenged during early gestation with PPV-27a, a virulent cluster D strain, and euthanized to collect their offspring by hysterectomy around day 90 in pregnancy. Antibody responses against PPV in gilts were measured, and the presence of PPV in progeny was also determined. RESULTS: No clinical signs were observed after vaccination. After PPV challenge, all foetuses from the vaccinated gilts were alive (132/132), while in the unvaccinated group only 41% were alive (46/112), 19.6% were dead and 39.4% of the offspring (44/112) were mummified. PPV could be detected by qPCR in 14% of the progeny from vaccinated gilts at an average of 4.7 log10/ml, whereas this was significantly higher in the control group, where 90% of the progeny were PPV positive, with titres of 9.8 log10/ml on average. CONCLUSIONS: The present study demonstrates that vaccination of gilts with Porcilis® Ery + Parvo+Lepto was safe and induced an immune response sufficient to protect progeny against PPV by reducing transplacental infection.

Method to grow Actinobacillus pleuropneumoniae biofilm on a biotic surface.

BACKGROUND: Actinobacillus pleuropneumoniae is a Gram-negative bacterium and a member of the Pasteurellaceae family. This bacterium is the causative agent of porcine pleuropneumonia, which is a highly contagious respiratory disease causing important economical losses to the worldwide pig industry. It has been shown that A. pleuropneumoniae can form biofilms on abiotic surfaces (plastic and glass). Although in vitro models are extremely useful to gain information on biofilm formation, these models may not be representative of the conditions found at the mucosal surface of the host, which is the natural niche of A. pleuropneumoniae. RESULTS: In this paper, we describe a method to grow A. pleuropneumoniae biofilms on the SJPL cell line, which represents a biotic surface. A non-hemolytic, non-cytotoxic mutant of A. pleuropneumoniae was used in our assays and this allowed the SJPL cell monolayers to be exposed to A. pleuropneumoniae for longer periods. This resulted in the formation of biofilms on the cell monolayer after incubations of 24 and 48 h. The biofilms can be stained with fluorescent probes, such as a lectin against the polymer of N-acetyl-D-glucosamine present in the biofilm matrix, and easily observed by confocal laser scanning microscopy. CONCLUSIONS: This is the first protocol that describes the formation of an A. pleuropneumoniae biofilm on a biotic surface. The advantage of this protocol is that it can be used to study biofilm formation in a context of host-pathogen interactions. The protocol could also be adapted to evaluate biofilm inhibitors or the efficacy of antibiotics in the presence of biofilms.

Postexposure subunit vaccination against chronic enteric mycobacterial infection in a natural host.

The control of chronic bacterial diseases with high prevalence in areas of endemicity would strongly benefit from availability of postexposure vaccines. The development of these vaccines against mycobacterial infections, such as (para)tuberculosis, is hampered by lack of experience in natural hosts. Paratuberculosis in cattle is both a mycobacterial disease of worldwide importance and a natural host model for mycobacterial infections in general. The present study showed beneficial effects of therapeutic heat shock protein 70 (Hsp70) vaccination in cattle with naturally acquired chronic infection with Mycobacterium avium subsp. paratuberculosis. Vaccination-induced protection was associated with antibody responses, rather than with induction of specific T helper 1 cells. Targeted therapeutic postexposure vaccination complementary to selective use of antibiotics could be an effective approach for control of chronic mycobacterial infections.

Early infection dynamics after experimental challenge with Mycobacterium avium subspecies paratuberculosis in calves reveal limited calf-to-calf transmission and no impact of Hsp70 vaccination.

Efficient control of bovine paratuberculosis is hampered by lack of a vaccine. The purpose of this study was to evaluate efficacy of a candidate vaccine, consisting of recombinant Mycobacterium avium subspecies paratuberculosis (MAP) Hsp70 with DDA adjuvant, in calves experimentally infected with MAP. Four groups of 14 animals each were used. Animals in group 1 and 2 were all vaccinated with Hsp70/DDA at day 0, 84, 168 and 357, and those in group 3 and 4 were non-vaccinated controls. In each group half (n=7) of the animals were challenged and the remaining half served as contacts. Blood and fecal samples were collected at three week intervals until day 588, and subsequently all animals were subjected to necropsy. The primary outcomes assessed were fecal culture (FC) of MAP, tissue colonization of MAP, and transmission of infection to contact animals. The kinetics of MAP shedding in feces of challenged animals showed a peak around 130 days post-challenge, irrespective of vaccination status. At necropsy no differences in the level of tissue colonization between vaccinated animals and controls were observed in the challenged groups. Only one contact animal (non-vaccinated) was positive at necropsy, indicating limited to no transmission within groups. These findings indicate that Hsp70/DDA vaccination does not influence early infection dynamics after experimental infection. However, early shedding of MAP in calves did not result in efficient transmission of infection to contact animals. The latter implies that introduction of an infected calf in a cohort of susceptibles has limited consequences for spread of infection.

The complete genome sequence of Campylobacter jejuni strain 81116 (NCTC11828).

Campylobacter jejuni is a major human enteric pathogen that displays genetic variability via genomic reorganization and phase variation. This variability can adversely affect the outcomes and reproducibility of experiments. C. jejuni strain 81116 (NCTC11828) has been suggested to be a genetically stable strain (G. Manning, B. Duim, T. Wassenaar, J. A. Wagenaar, A. Ridley, and D. G. Newell, Appl. Environ. Microbiol. 67:1185-1189, 2001), is amenable to genetic manipulation, and is infective for chickens. Here we report the finished annotated genome sequence of C. jejuni strain 81116.

Characterization of apxIVA, a new RTX determinant of Actinobacillus pleuropneumoniae.

A fourth type of RTX determinant was identified in Actinobacillus pleuropneumoniae and was designated apxIVA. When expressed in Escherichia coli, recombinant ApxIVA showed a weak haemolytic activity and co-haemolytic synergy with the sphingomyelinase (beta-toxin) of Staphylococcus aureus. These activities required the presence of an additional gene, ORF1, that is located immediately upstream of apxIVA. The apxIVA gene product could not be detected in A. pleuropneumoniae cultures grown under various conditions in vitro; however, pigs experimentally infected with A. pleuropneumoniae serotypes 1, 5 and 7 started to produce antibodies that reacted with recombinant ApxIVA 14 d post-infection, indicating that apxIVA is expressed in vivo. In addition, sera from pigs naturally and experimentally infected with any of the serotypes all reacted with recombinant ApxIVA. The apxIVA gene from the serotype 1 A. pleuropneumoniae type strain Shope 4074T encodes a protein with a predicted molecular mass of 202 kDa which has typical features of RTX proteins including hydrophobic domains in the N-terminal half and 24 glycine-rich nonapeptides in the C-terminal half that bind Ca2+. The glycine-rich nonapeptides are arranged in a modular structure and there is some variability in the number of modules in the ApxIVA proteins of different serotypes of A. pleuropneumoniae. The deduced amino acid sequences of the ApxIVA proteins have significant similarity with the Neisseria meningitidis iron-regulated RTX proteins FrpA and FrpC, and to a much lesser extent with other RTX proteins. The apxIVA gene could be detected in all A. pleuropneumoniae serotypes and seems to be species-specific. Although the precise role of this new RTX determinant in pathogenesis of porcine pleuropneumonia needs to be determined, apxIVA is the first in vivo induced toxin gene that has been described in A. pleuropneumoniae.