Differential Gene Expression in Porcine Lung Compartments after Experimental Infection with Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae (Mhyo) is the causative agent of porcine enzootic pneumonia (EP), as well as one of the main pathogens involved in the porcine respiratory disease complex. The host-pathogen interaction between Mhyo and infected pigs is complex and not completely understood; however, improving the understanding of these intricacies is essential for the development of effective control strategies of EP. In order to improve our knowledge about this interaction, laser-capture microdissection was used to collect bronchi, bronchi-associated lymphoid tissue, and lung parenchyma from animals infected with different strains of Mhyo, and mRNA expression levels of different molecules involved in Mhyo infection (ICAM1, IL-8, IL-10, IL-23, IFN-α, IFN-γ, TGF-ß, and TNF-α) were analyzed by qPCR. In addition, the quantification of Mhyo load in the different lung compartments and the scoring of macroscopic and microscopic lung lesions were also performed. Strain-associated differences in virulence were observed, as well as the presence of significant differences in expression levels of cytokines among lung compartments. IL-8 and IL-10 presented the highest upregulation, with limited differences between strains and lung compartments. IFN-α was strongly downregulated in BALT, implying a relevant role for this cytokine in the immunomodulation associated with Mhyo infections. IL-23 was also upregulated in all lung compartments, suggesting the potential involvement of a Th17-mediated immune response in Mhyo infections. Our findings highlight the relevance of Th1 and Th2 immune response in cases of EP, shedding light on the gene expression levels of key cytokines in the lung of pigs at a microscopic level.

Modified surfatron device to improve microwave-plasma-assisted generation of RONS and methylene blue degradation in water.

Microwave-induced plasmas generated at atmospheric pressure are very attractive for a great variety of applications since they have a relatively high electron density and can generate large amounts of reactive species. Argon plasmas can be sustained inside dielectric tubes but are radially contracted and exhibit filamentation effects when the diameter of the tube is not narrow enough (over 1.5 mm). In this work, we describe a new approach for creating microwave (2.45 GHz) plasmas under atmospheric pressure conditions by using a surfatron device and power from 10 W. This modified design of the reactor enables the sustenance of non-filamented argon plasmas. These new plasmas have a higher gas temperature and electron density than the plasma generated in the original surfatron configuration. The new design also allows for the maintenance of plasmas with relatively high proportions of water, resulting in the generation of larger quantities of excited hydroxyl radicals (·OH*). Thus, this novel configuration extends the applicability of microwave-induced plasmas by enabling operation under new conditions. Finally, the degradation of methylene blue (MB) in aqueous solutions has been assessed under different initial dye concentrations and argon flow conditions. The new plasma produces a substantial increase in hydrogen peroxide and nitrate concentrations in water and leads to a noteworthy enhancement in MB degradation efficiency. The introduction of water into the plasma produces a minor additional improvement.

Bioprotection of Olive Trees Against Verticillium Wilt by Pomegranate and Carob Extracts.

Bioprotection using plant extracts is an environmentally friendly strategy in crop protection. Effective control of Verticillium wilt of olive (Olea europaea; VWO), caused by Verticillium dahliae, has proven challenging due to the ineffectiveness of chemicals, which makes it necessary to search for new control tools. Thus, the aim of this study was to evaluate the effect of pomegranate (Punica granatum) and carob (Ceratonia siliqua) extracts against VWO. Extracts derived from pomegranate peels and carob pods and leaves were obtained using ethanol, methanol, or ethyl acetate as solvents. A targeted analysis of their metabolite composition was performed using QTRAP Ultra High-Performance Liquid Chromatography with Mass Spectrometry (QTRAP UHPLC‒MS). Remarkably, gallic acid was detected in all extracts at a high concentration. The effect of the extracts on the mycelial growth and on the germination of conidia and microsclerotia of V. dahliae was evaluated by in vitro sensitivity tests at various doses: 0 (control), 3, 30, 300 and 3,000 mg of extract/liter. Extracts obtained with ethanol or methanol significantly reduced the viability of V. dahliae structures when applied at the highest dose, while those obtained with ethyl acetate were ineffective across all doses. The most effective extracts, as determined in vitro, were then evaluated against the disease in olive plants. Potted plants of cv. Picual were treated by spraying (foliar application) or irrigation (root application) of extracts at 3,000 mg of extract/liter, followed by inoculation with V. dahliae. The results indicated that foliar applications were ineffective, while root treatments with pomegranate peel or carob leaf extracts were more effective in reducing disease severity, regardless of solvent, compared to that of the untreated control.

Comparative pathology of experimental pulmonary tuberculosis in animal models.

Research in human tuberculosis (TB) is limited by the availability of human tissues from patients, which is often altered by therapy and treatment. Thus, the use of animal models is a key tool in increasing our understanding of the pathogenesis, disease progression and preclinical evaluation of new therapies and vaccines. The granuloma is the hallmark lesion of pulmonary tuberculosis, regardless of the species or animal model used. Although animal models may not fully replicate all the histopathological characteristics observed in natural, human TB disease, each one brings its own attributes which enable researchers to answer specific questions regarding TB immunopathogenesis. This review delves into the pulmonary pathology induced by Mycobacterium tuberculosis complex (MTBC) bacteria in different animal models (non-human primates, rodents, guinea pigs, rabbits, cattle, goats, and others) and compares how they relate to the pulmonary disease described in humans. Although the described models have demonstrated some histopathological features in common with human pulmonary TB, these data should be considered carefully in the context of this disease. Further research is necessary to establish the most appropriate model for the study of TB, and to carry out a standard characterisation and score of pulmonary lesions.

Characterisation and development of histopathological lesions in a guinea pig model of Mycobacterium tuberculosis infection.

Tuberculosis (TB) remains a very significant infectious disease worldwide. New vaccines and therapies are needed, even more crucially with the increase of multi-drug resistant Mycobacterium tuberculosis strains. Preclinical animal models are very valuable for the development of these new disease control strategies. Guinea pigs are one of the best models of TB, sharing many features with the pathology observed in human TB. Here we describe the development of TB lesions in a guinea pig model of infection. We characterise the granulomatous lesions in four developmental stages (I-IV), using histopathological analysis and immunohistochemical (IHC) techniques to study macrophages, T cells, B cells and granulocytes. The granulomas in the guinea pigs start as aggregations of macrophages and few heterophils, evolving to larger lesions showing central caseous necrosis with mineralisation and abundant acid-fast bacilli, surrounded by a rim of macrophages and lymphocytes in the outer layers of the granuloma. Multinucleated giant cells are very rare and fibrotic capsules are not formed in this animal model.

Spore-FP1 tuberculosis mucosal vaccine candidate is highly protective in guinea pigs but fails to improve on BCG-conferred protection in non-human primates.

Tuberculosis remains a major health threat globally and a more effective vaccine than the current Bacillus Calmette Guerin (BCG) is required, either to replace or boost it. The Spore-FP1 mucosal vaccine candidate is based on the fusion protein of Ag85B-Acr-HBHA/heparin-binding domain, adsorbed on the surface of inactivated Bacillus subtilis spores. The candidate conferred significant protection against Mycobacterium. tuberculosis challenge in naïve guinea pigs and markedly improved protection in the lungs and spleens of animals primed with BCG. We then immunized rhesus macaques with BCG intradermally, and subsequently boosted with one intradermal and one aerosol dose of Spore-FP1, prior to challenge with low dose aerosolized M. tuberculosis Erdman strain. Following vaccination, animals did not show any adverse reactions and displayed higher antigen specific cellular and antibody immune responses compared to BCG alone but this did not translate into significant improvement in disease pathology or bacterial burden in the organs.

Establishment of a Nipah Virus Disease Model in Hamsters, including a Comparison of Intranasal and Intraperitoneal Routes of Challenge.

Nipah virus (NiV) is an emerging pathogen that can cause severe respiratory illness and encephalitis in humans. The main reservoir is fruit bats, distributed across a large geographical area that includes Australia, Southeast Asia, and Africa. Incursion into humans is widely reported through exposure of infected pigs, ingestion of contaminated food, or through contact with an infected person. With no approved treatments or vaccines, NiV poses a threat to human public health and has epidemic potential. To aid with the assessment of emerging interventions being developed, an expansion of preclinical testing capability is required. Given variations in the model parameters observed in different sites during establishment, optimisation of challenge routes and doses is required. Upon evaluating the hamster model, an intranasal route of challenge was compared with intraperitoneal delivery, demonstrating a more rapid dissemination to wider tissues in the latter. A dose effect was observed between those causing respiratory illness and those resulting in neurological disease. The data demonstrate the successful establishment of the hamster model of NiV disease for subsequent use in the evaluation of vaccines and antivirals.

OVX033, a nucleocapsid-based vaccine candidate, provides broad-spectrum protection against SARS-CoV-2 variants in a hamster challenge model.

Spike-based COVID-19 vaccines induce potent neutralizing antibodies but their efficacy against SARS-CoV-2 variants decreases. OVX033 is a recombinant protein composed of the full-length nucleocapsid (N) protein of SARS-CoV-2 genetically fused to oligoDOM®, a self-assembling domain which improves antigen immunogenicity. OVX033 including N as an antigenic target is proposed as new vaccine candidate providing broad-spectrum protection against sarbecoviruses. OVX033 demonstrated its ability to trigger cross-reactive T cell responses and cross-protection against three variants of SARS-CoV-2 (B.1 Europe, Delta B.1.617.2, and Omicron B.1.1.529) in a hamster challenge model, as evidenced by lower weight loss, lower lung viral loads, and reduced lung histopathological lesions.

Tumour Necrosis Factor-α, Chemokines, and Leukocyte Infiltrate Are Biomarkers for Pathology in the Brains of Venezuelan Equine Encephalitis (VEEV)-Infected Mice.

Venezuelan equine encephalitis virus (VEEV) is a disease typically confined to South and Central America, whereby human disease is characterised by a transient systemic infection and occasionally severe encephalitis, which is associated with lethality. Using an established mouse model of VEEV infection, the encephalitic aspects of the disease were analysed to identify biomarkers associated with inflammation. Sequential sampling of lethally challenged mice (infected subcutaneously) confirmed a rapid onset systemic infection with subsequent spread to the brain within 24 h of the challenge. Changes in inflammatory biomarkers (TNF-α, CCL-2, and CCL-5) and CD45+ cell counts were found to correlate strongly to pathology (R>0.9) and present previously unproven biomarkers for disease severity in the model, more so than viral titre. The greatest level of pathology was observed within the olfactory bulb and midbrain/thalamus. The virus was distributed throughout the brain/encephalon, often in areas not associated with pathology. The principal component analysis identified five principal factors across two independent experiments, with the first two describing almost half of the data: (1) confirmation of a systemic Th1-biased inflammatory response to VEEV infection, and (2) a clear correlation between specific inflammation of the brain and clinical signs of disease. Targeting strongly associated biomarkers of deleterious inflammation may ameliorate or even eliminate the encephalitic syndrome of this disease.

Effect of mucosal adjuvant IL-1ß on heterotypic immunity in a pig influenza model.

T cell responses directed against highly conserved viral proteins contribute to the clearance of the influenza virus and confer broadly cross-reactive and protective immune responses against a range of influenza viruses in mice and ferrets. We examined the protective efficacy of mucosal delivery of adenoviral vectors expressing hemagglutinin (HA) and nucleoprotein (NP) from the H1N1 virus against heterologous H3N2 challenge in pigs. We also evaluated the effect of mucosal co-delivery of IL-1ß, which significantly increased antibody and T cell responses in inbred Babraham pigs. Another group of outbred pigs was first exposed to pH1N1 as an alternative means of inducing heterosubtypic immunity and were subsequently challenged with H3N2. Although both prior infection and adenoviral vector immunization induced strong T-cell responses against the conserved NP protein, none of the treatment groups demonstrated increased protection against the heterologous H3N2 challenge. Ad-HA/NP+Ad-IL-1ß immunization increased lung pathology, although viral load was unchanged. These data indicate that heterotypic immunity may be difficult to achieve in pigs and the immunological mechanisms may differ from those in small animal models. Caution should be applied in extrapolating from a single model to humans.

Infection with Seasonal H1N1 Influenza Results in Comparable Disease Kinetics and Host Immune Responses in Ferrets and Golden Syrian Hamsters.

Animal models of influenza are important in preclinical research for the study of influenza infection and the assessment of vaccines, drugs and therapeutics. Here, we show that Golden Syrian hamsters (Mesocricetus auratus) inoculated via the intranasal route with high dose of influenza H1N1 display comparable disease kinetics and immune responses to the 'gold standard' ferret (Mustela furo) model. We demonstrate that both the hamster and ferret models have measurable disease endpoints of weight loss, temperature change, viral shedding from the upper respiratory tract and increased lung pathology. We also characterised both the humoral and cellular immune responses to infection in both models. The comparability of these data supports the Golden Syrian hamster model being useful in preclinical evaluation studies to explore the efficacy of countermeasures against influenza.

The Omicron Sub-Variant BA.4 Displays a Remarkable Lack of Clinical Signs in a Golden Syrian Hamster Model of SARS-CoV-2 Infection.

The ongoing emergence of SARS-CoV-2 virus variants remains a source of concern because it is accompanied by the potential for increased virulence as well as evasion of immunity. Here we show that, although having an almost identical spike gene sequence as another Omicron variant (BA.5.2.1), a BA.4 isolate lacked all the typical disease characteristics of other isolates seen in the Golden Syrian hamster model despite replicating almost as effectively. Animals infected with BA.4 had similar viral shedding profiles to those seen with BA.5.2.1 (up to day 6 post-infection), but they all failed to lose weight or present with any other significant clinical signs. We hypothesize that this lack of detectable signs of disease during infection with BA.4 was due to a small (nine nucleotide) deletion (∆686-694) in the viral genome (ORF1ab) responsible for the production of non-structural protein 1, which resulted in the loss of three amino acids (aa 141-143).

Pathological features of African horse sickness virus infection in IFNAR-/- mice.

African Horse Sickness (AHS) is a vector-borne viral disease of equids. The disease can be highly lethal with mortality rates of up to 90% in non-immune equine populations. The clinical presentation in the equine host varies, but the pathogenesis underlying this variation remains incompletely understood. Various small animal models of AHS have been developed over the years to overcome the financial, bio-safety and logistical constraints of studying the pathology of this disease in the target species. One of the most successful small animal models is based on the use of interferon-alpha gene knock-out (IFNAR-/-) mice. In order to increase our understanding of African Horse Sickness virus (AHSV) pathogenesis, we characterised the pathology lesions of AHSV infection in IFNAR-/- mice using a strain of AHSV serotype 4 (AHSV-4). We found AHSV-4 infection was correlated with lesions in various organs; necrosis in the spleen and lymphoid tissues, inflammatory infiltration in the liver and brain, and pneumonia. Significant viral antigen staining was only detected in the spleen and brain, however. Together these results confirm the value of the IFNAR-/- mouse model for the study of the immuno-biology of AHSV infections in this particular in vivo system, and its usefulness for evaluating protective efficacy of candidate vaccines in preclinical studies.

Syrian hamster convalescence from prototype SARS-CoV-2 confers measurable protection against the attenuated disease caused by the Omicron variant.

The mutation profile of the SARS-CoV-2 Omicron (lineage BA.1) variant posed a concern for naturally acquired and vaccine-induced immunity. We investigated the ability of prior infection with an early SARS-CoV-2 ancestral isolate (Australia/VIC01/2020, VIC01) to protect against disease caused by BA.1. We established that BA.1 infection in naïve Syrian hamsters resulted in a less severe disease than a comparable dose of the ancestral virus, with fewer clinical signs including less weight loss. We present data to show that these clinical observations were almost absent in convalescent hamsters challenged with the same dose of BA.1 50 days after an initial infection with ancestral virus. These data provide evidence that convalescent immunity against ancestral SARS-CoV-2 is protective against BA.1 in the Syrian hamster model of infection. Comparison with published pre-clinical and clinical data supports consistency of the model and its predictive value for the outcome in humans. Further, the ability to detect protection against the less severe disease caused by BA.1 demonstrates continued value of the Syrian hamster model for evaluation of BA.1-specific countermeasures.

Carboxymethylcellulose/Hydrotalcite Bionanocomposites as Paraben Sorbents.

In this work, we synthesized several bionanocomposites of hydrotalcites containing carboxymethylcellulose as interlayer anion (HT-CMC) to be used as sorbents for parabens, a family of emergent pollutants (specifically, for 4-methyl-, 4-propyl- and 4-benzylparaben). Bionanocomposites were obtained by ultrasound-assisted coprecipitation and characterized by X-ray diffraction analysis, fourier transform infrared and raman spectroscopies, elemental and thermogravimetric analysis, scanning and transmission electron microscopies and X-ray fluorescence. All materials proved to be efficient sorbents for parabens through a process conforming to a pseudo second-order kinetics. The experimental adsorption data fitted the Freundlich model very closely and were also highly correlated with the Temkin model. The effects of pH, adsorbate concentration, amount of sorbent and temperature on the adsorption process was evaluated, obtaining the best results for methylparaben adsorption at pH 7, 25 mg of adsorbent and 348 K. The sorbent, HT-CMC-3, showed the highest adsorption capacity (>70%) for methylparaben. Furthermore, a reusability study showed that the bionanocomposite is reusable after its regeneration with methanol. The sorbent still retained its adsorption capacity for up to 5 times with a little loss of efficiency (<5%).

Mycolactone causes catastrophic Sec61-dependent loss of the endothelial glycocalyx and basement membrane: a new indirect mechanism driving tissue necrosis in Mycobacterium ulcerans infection.

The drivers of tissue necrosis in Mycobacterium ulcerans infection (Buruli ulcer disease) have historically been ascribed solely to the directly cytotoxic action of the diffusible exotoxin, mycolactone. However, its role in the clinically-evident vascular component of disease aetiology remains poorly explained. We have now dissected mycolactone's effects on primary vascular endothelial cells in vitro and in vivo. We show that mycolactone-induced changes in endothelial morphology, adhesion, migration, and permeability are dependent on its action at the Sec61 translocon. Unbiased quantitative proteomics identified a profound effect on proteoglycans, driven by rapid loss of type II transmembrane proteins of the Golgi, including enzymes required for glycosaminoglycan (GAG) synthesis, combined with a reduction in the core proteins themselves. Loss of the glycocalyx is likely to be of particular mechanistic importance, since knockdown of galactosyltransferase II (beta-1,3-galactotransferase 6; B3Galt6), the GAG linker-building enzyme, phenocopied the permeability and phenotypic changes induced by mycolactone. Additionally, mycolactone depleted many secreted basement membrane components and microvascular basement membranes were disrupted in vivo. Remarkably, exogenous addition of laminin-511 reduced endothelial cell rounding, restored cell attachment and reversed the defective migration caused by mycolactone. Hence supplementing mycolactone-depleted extracellular matrix may be a future therapeutic avenue, to improve wound healing rates.

SARS-CoV-2 Disease Severity in the Golden Syrian Hamster Model of Infection Is Related to the Volume of Intranasal Inoculum.

The golden Syrian hamster (Mesocricetus auratus) is now commonly used in preclinical research for the study of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and the assessment of vaccines, drugs and therapeutics. Here, we show that hamsters inoculated via the intranasal route with the same infectious virus dose of prototypical SARS-CoV-2 administered in a different volume present with different clinical signs, weight loss and viral shedding, with a reduced volume resulting in reduced severity of disease similar to that obtained by a 500-fold reduction in the challenge dose. The tissue burden of the virus and the severity of pulmonary pathology were also significantly affected by different challenge inoculum volumes. These findings suggest that a direct comparison between the severity of SARS-CoV-2 variants or studies assessing the efficacy of treatments determined by hamster studies cannot be made unless both the challenge dose and inoculation volume are matched when using the intranasal route. Additionally, analysis of sub-genomic and total genomic RNA PCR data demonstrated no link between sub-genomic and live viral titres and that sub-genomic analyses do not provide any information beyond that provided by more sensitive total genomic PCR.

The P323L substitution in the SARS-CoV-2 polymerase (NSP12) confers a selective advantage during infection.

BACKGROUND: The mutational landscape of SARS-CoV-2 varies at the dominant viral genome sequence and minor genomic variant population. During the COVID-19 pandemic, an early substitution in the genome was the D614G change in the spike protein, associated with an increase in transmissibility. Genomes with D614G are accompanied by a P323L substitution in the viral polymerase (NSP12). However, P323L is not thought to be under strong selective pressure. RESULTS: Investigation of P323L/D614G substitutions in the population shows rapid emergence during the containment phase and early surge phase during the first wave. These substitutions emerge from minor genomic variants which become dominant viral genome sequence. This is investigated in vivo and in vitro using SARS-CoV-2 with P323 and D614 in the dominant genome sequence and L323 and G614 in the minor variant population. During infection, there is rapid selection of L323 into the dominant viral genome sequence but not G614. Reverse genetics is used to create two viruses (either P323 or L323) with the same genetic background. L323 shows greater abundance of viral RNA and proteins and a smaller plaque morphology than P323. CONCLUSIONS: These data suggest that P323L is an important contribution in the emergence of variants with transmission advantages. Sequence analysis of viral populations suggests it may be possible to predict the emergence of a new variant based on tracking the frequency of minor variant genomes. The ability to predict an emerging variant of SARS-CoV-2 in the global landscape may aid in the evaluation of medical countermeasures and non-pharmaceutical interventions.

Immunization with matrix-, nucleoprotein and neuraminidase protects against H3N2 influenza challenge in pH1N1 pre-exposed pigs.

There is an urgent need for influenza vaccines providing broader protection that may decrease the need for annual immunization of the human population. We investigated the efficacy of heterologous prime boost immunization with chimpanzee adenovirus (ChAdOx2) and modified vaccinia Ankara (MVA) vectored vaccines, expressing conserved influenza virus nucleoprotein (NP), matrix protein 1 (M1) and neuraminidase (NA) in H1N1pdm09 pre-exposed pigs. We compared the efficacy of intra-nasal, aerosol and intra-muscular vaccine delivery against H3N2 influenza challenge. Aerosol prime boost immunization induced strong local lung T cell and antibody responses and abrogated viral shedding and lung pathology following H3N2 challenge. In contrast, intramuscular immunization induced powerful systemic responses and weak local lung responses but also abolished lung pathology and reduced viral shedding. These results provide valuable insights into the development of a broadly protective influenza vaccine in a highly relevant large animal model and will inform future vaccine and clinical trial design.

Solar driven CO2 reduction with a molecularly engineered periodic mesoporous organosilica containing cobalt phthalocyanine.

A molecular cobalt phthalocyanine (CoPc) catalyst has been integrated in an ethylene-bridged periodic mesoporous organosilica (PMO) to fabricate a hybrid material, CoPc-PMO, that catalyses CO2 reduction to CO in a photocatalytic system using [Ru(bpy)3]2+ (bpy = 2,2'-bipyridine) as a photosensitizer and 1,3-dimethyl-2-phenyl-2,3-dihydro-1H-benzo[d]imidazole (BIH) as an electron donor. CoPc-PMO displays a Co-based turnover number (TONCO) of >6000 for CO evolution with >70% CO-selectivity after 4 h irradiation with UV-filtered simulated solar light, and a quantum yield of 1.95% at 467 nm towards CO. This system demonstrates a benchmark TONCO for immobilised CoPc-based catalysts towards visible light-driven CO2 reduction.