Characterization by MALDI-TOF MS and 16S rRNA Gene Sequencing of Aerobic Endospore-Forming Bacteria Isolated from Pharmaceutical Facility in Rio de Janeiro, Brazil.

Bacillus and related genera are among the most important contaminants in the pharmaceutical production environment, and the identification of these microorganisms at the species level assists in the investigation of sources of contamination and in preventive and corrective decision making. The aim of this study was to evaluate three methodologies for the characterization of endospore-forming aerobic bacterial strains isolated from a pharmaceutical unit in Rio de Janeiro, Brazil. MALDI-TOF MS was performed using MALDI Biotyper® and VITEK® MS RUO systems, and complete 16S rRNA gene sequencing was performed using the Sanger methodology. The results showed the prevalence of the genera Bacillus (n = 9; 36.0%), Priestia (n = 5; 20.0%), and Paenibacillus (n = 4; 16.0%). Three (20.0%) strains showed <98.7% of DNA sequencing similarity on the EzBioCloud Database, indicating possible new species. In addition, the reclassification of Bacillus pseudoflexus to the genus Priestia as Priestia pseudoflexus sp. nov. is proposed. In conclusion, 16S rRNA and MALDI TOF/MS were not sufficient to identify all strains at the species level, and complementary analyses were necessary.

Myrtucommulones and Related Acylphloroglucinols from Myrtaceae as a Promising Source of Multitarget SARS-CoV-2 Cycle Inhibitors.

The LABEXTRACT plant extract bank, featuring diverse members of the Myrtaceae family from Brazilian hot spot regions, provides a promising avenue for bioprospection. Given the pivotal roles of the Spike protein and 3CLpro and PLpro proteases in SARS-CoV-2 infection, this study delves into the correlations between the Myrtaceae species from the Atlantic Forest and these targets, as well as an antiviral activity through both in vitro and in silico analyses. The results uncovered notable inhibitory effects, with Eugenia prasina and E. mosenii standing out, while E. mosenii proved to be multitarget, presenting inhibition values above 72% in the three targets analyzed. All extracts inhibited viral replication in Calu-3 cells (EC50 was lower than 8.3 µg·mL-1). Chemometric analyses, through LC-MS/MS, encompassing prediction models and molecular networking, identified potential active compounds, such as myrtucommulones, described in the literature for their antiviral activity. Docking analyses showed that one undescribed myrtucommulone (m/z 841 [M - H]-) had a higher fitness score when interacting with the targets of this study, including ACE2, Spike, PLpro and 3CLpro of SARS-CoV-2. Also, the study concludes that Myrtaceae extracts, particularly from E. mosenii and E. prasina, exhibit promising inhibitory effects against crucial stages in SARS-CoV-2 infection. Compounds like myrtucommulones emerge as potential anti-SARS-CoV-2 agents, warranting further exploration.

Cost-effective 3D lung tissue spheroid as a model for SARS-CoV-2 infection and drug screening.

BACKGROUND: The SARS-CoV-2 pandemic has spurred an unparalleled scientific endeavor to elucidate the virus' structure, infection mechanisms, and pathogenesis. Two-dimensional culture systems have been instrumental in shedding light on numerous aspects of COVID-19. However, these in vitro systems lack the physiological complexity to comprehend the infection process and explore treatment options. Three-dimensional (3D) models have been proposed to fill the gap between 2D cultures and in vivo studies. Specifically, spheroids, composed of lung cell types, have been suggested for studying SARS-CoV-2 infection and serving as a drug screening platform. METHODS: 3D lung spheroids were prepared by coculturing human alveolar or bronchial epithelial cells with human lung stromal cells. The morphology, size, and ultrastructure of spheroids before and after SARS-CoV-2 infection were analyzed using optical and electron microscopy. Immunohistochemistry was used to detect spike protein and, thus, the virus presence in the spheroids. Multiplex analysis elucidated the cytokine release after virus infection. RESULTS: The spheroids were stable and kept their size and morphology after SARS-CoV-2 infection despite the presence of multivesicular bodies, endoplasmic reticulum rearrangement, tubular compartment-enclosed vesicles, and the accumulation of viral particles. The spheroid responded to the infection releasing IL-6 and IL-8 cytokines. CONCLUSION: This study demonstrates that coculture spheroids of epithelial and stromal cells can serve as a cost-effective infection model for the SARS-CoV-2 virus. We suggest using this 3D spheroid as a drug screening platform to explore new treatments related to the cytokines released during virus infection, especially for long COVID treatment.

SARS-CoV-2 egress from Vero cells: a morphological approach.

Despite being extensively studied because of the current coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) interactions with mammalian cells are still poorly understood. Furthermore, little is known about this coronavirus cycle within the host cells, particularly the steps that lead to viral egress. This study aimed to shed light on the morphological features of SARS-CoV-2 egress by utilizing transmission and high-resolution scanning electron microscopy, along with serial electron tomography, to describe the route of nascent virions towards the extracellular medium. Electron microscopy revealed that the clusters of viruses in the paracellular space did not seem to result from collective virus release. Instead, virus accumulation was observed on incurved areas of the cell surface, with egress primarily occurring through individual vesicles. Additionally, our findings showed that the emission of long membrane projections, which could facilitate virus surfing in Vero cells infected with SARS-CoV-2, was also observed in non-infected cultures, suggesting that these are constitutive events in this cell lineage.

New azaaurone derivatives as potential multitarget agents in HIV-TB coinfection.

Tuberculosis (TB) disease, caused by Mycobacterium tuberculosis (Mtb) is the leading cause of death among people with human immunodeficiency virus (HIV) infection. No dual-target drug is currently being used to simultaneously treat both infections. This work aimed to obtain new multitarget HIV-TB agents, with the goal of optimizing treatments and preventing this coinfection. These compounds incorporate the structural features of azaaurones as anti-Mtb and zidovudine (AZT) as the antiretroviral moiety. The azaaurone scaffold displayed submicromolar activities against Mtb, and AZT is a potent antiretroviral drug. Six derivatives were synthetically generated, and five were evaluated against both infective agents. Evaluations of anti-HIV activity were carried out in HIV-1-infected MT-4 cells and on endogenous HIV-1 reverse transcriptase (RT) activity. The H37Rv strain was used for anti-Mtb assessments. Most compounds displayed potent antitubercular and moderate anti-HIV activity. (E)-12 exhibited a promising multitarget profile with an MIC90 of 2.82 µM and an IC50 of 1.98 µM in HIV-1-infected T lymphocyte cells, with an 84% inhibition of RT activity. Therefore, (E)-12 could be the first promising compound from a family of multitarget agents used to treat HIV-TB coinfection. In addition, the compound could offer a prototype for the development of new strategies in scientific research to treat this global health issue.

Evaluation of phenotypical and genotypical methods for the identification and typing of Stenotrophomonas maltophilia isolated from a pharmaceutical facility.

AIMS: Evaluate methods for identification and typing of Stenotrophomonas maltophilia isolated from a pharmaceutical facility. METHODS AND RESULTS: From 270 S. maltophilia strains identified by VITEK®2, 40 were selected and submitted to MALDI TOF-MS, 16S and 23S rRNA gene analysis, enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR), and an antimicrobial susceptibility profile. 16S rRNA sequencing was able to identify 39 (97.5%) strains as Stenotrophomonas spp. and one (2.5%) as Luteimonas huabeiensis. MALDI TOF-MS identified 37 (92.5%) strains as S. maltophilia, and three (7.5%) were not identified. PCR targeting 23S rRNA yielded a positive result for 39 (97.5%) strains. However, after sequencing, two strains were identified as Stenotrophomonas rhizophila, showing false-positive results. The confirmed S. maltophilia strains (n = 37) showed 35 distinct ERIC-PCR profiles and exhibited sensitivity to minocycline and levofloxacin, and six (16.3%) showed intermediate resistance to sulfamethoxazole-trimethoprim. CONCLUSION: Matrix-assisted laser desorption lonization-time of flight mass spectrometry (MALDI-TOF MS) was a satisfactory methodology for the identification of S. maltophilia, but expansion of the database is necessary for the identification of other species. 16S rDNA sequencing showed low resolution for Stenotrophomonas species differentiation. PCR targeting 23S rRNA could not differentiate S. maltophilia from S. rhizophila. ERIC-PCR was shown to be a useful tool for the microbial source tracking of S. maltophilia.

Phenotypical and molecular characterization of Acinetobacter spp. isolated from a pharmaceutical facility.

Characterizing microorganisms according to different criteria is useful when investigating sources of microbiological contamination in the pharmaceutical industry. The aim of this study was to characterize 38 Acinetobacter baumannii complex strains isolated from a biopharmaceutical industry by 16S rRNA sequencing, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS), multilocus sequence typing (MLST), antimicrobial susceptibility profile, biofilm formation, and sensibility to disinfectants. Thirty-three (86.9%) strains were identified by 16S rRNA gene sequencing as A. seifertii/pitti/nosocomialis/lactucae, four (10.5%) as A. baumannii, and one (2.6%) as A. vivianii/courvalini. MALDI-TOF/MS did not identify one strain, and incorrectly identified 30/37 (81.1%) strains as A. baumannii. Strains were assigned to 12 different STs, of which nine were newly defined in this study (STs 2091-2099). Twenty-six (68.4%) strains showed resistance to amikacin and gentamicin. Thirty-three (86.8%) strains were classified as moderately or strongly adherent on polystyrene. Alcohol 70%/15 min and quaternary ammonium 0.08%/20 min were not able to eliminate the biofilm formed, but sodium hypochlorite 0.1%/15 min was efficient. In conclusion, improved methods are needed to improve the identification of Acinetobacter strains in pharmaceutical industries. This organism is of particular concern as it forms recalcitrant biofilms, leading to persistence in the manufacturing environment and increased risk of product contamination.

Evaluation of MALDI-TOF MS, sequencing of D2 LSU rRNA and internal transcribed spacer regions (ITS) for the identification of filamentous fungi isolated from a pharmaceutical facility.

The identification of filamentous fungi through culture characterization may be hampered by phenotypic variability. Information obtained from the identification of microorganisms are important for investigation of sources of contamination of a product or process. The aim of this study was to identify filamentous fungal strains (n = 50) isolated from a pharmaceutical facility by using Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), as well as D2 domain of the large-subunit (LSU) ribosomal RNA gene and internal transcribed spacer regions (ITS) sequencing. MALDI-TOF MS system only identified five strains at the species level, while 45 were not identified. The analysis through GenBank allowed the identification of up to 19 strains at the species level, while MycoBank allowed the identification of up to nine strains at the species level. The databases identified up to 11 genera: Penicillium, Aspergillus, Cladosporium, Chaetomium, Coniochaeta, Curvularia, Diaporthe, Fusarium, Trichoderma, Rhizopus and Microdochium. MALDI-TOF MS showed an insufficient database to identify the species of fungi. DNA sequencing was the best methodology to identify to the genus level but was unable to differentiate between closely related species. Therefore further methods for the identification of filamentous fungi from pharmaceutical areas at species level need to be developed.

Identification of Sutcliffiella horikoshii strains in an immunobiological pharmaceutical industry facility.

The pharmaceutical industry must comply with the requirements for good manufacturing practices to reduce inherent contamination risks in the production process. Bacillus and related genera are among the main bacterial isolated from clean areas, raw material, and products in the pharmaceutical industries, but the correct identification of these species is still a challenge. The aim of this study was to characterize by phenotyping, protein profiling, and 16S rRNA gene sequencing Sutcliffiellahorikoshii strains (n = 6) isolated from an immunobiological pharmaceutical facility, and to propose the reclassification of Bacillus tianshenii to the genus Sutcliffiella, and Sutcliffiella tianshenii sp. nov. The strains were characterized by VITEK®2, matrix-assisted laser desorption ionization-time of flight/mass spectrometry (MALDI-TOF/MS) using VITEK®MS, and 16S rRNA gene sequencing analysis. MALDI-TOF/MS did not identify any strains that were identified by 16S rRNA as S. horikoshii. VITEK®2 showed false-positive results, with misidentification as B. sporothermodurans (reclassified as Heyndrickxia sporothermodurans) and Geobacillus thermoleovorans. After MALDI-TOF/MS database expansion, with the creation of SuperSpectrum, the strains were correctly identified as S. horikoshii. This study is the first report of isolation of S. horikoshii strains from a pharmaceutical industry. More studies are necessary to better understand the ability of S. horikoshii to contaminate the environment and products.

A SARS-CoV-2 Negative Antigen Rapid Diagnostic in RT-qPCR Positive Samples Correlates With a Low Likelihood of Infectious Viruses in the Nasopharynx.

Severe acute respiratory syndrome-related coronavirus (SARS-CoV-2) transmission occurs even among fully vaccinated individuals; thus, prompt identification of infected patients is central to control viral circulation. Antigen rapid diagnostic tests (Ag-RDTs) are highly specific, but sensitivity is variable. Discordant RT-qPCR vs. Ag-RDT results are reported, raising the question of whether negative Ag-RDT in positive RT-qPCR samples could imply the absence of infectious viruses. To study the relationship between negative Ag-RDT results with virological, molecular, and serological parameters, we selected a cross-sectional and a follow-up dataset and analyzed virus culture, subgenomic RNA quantification, and sequencing to determine infectious viruses and mutations. We demonstrated that RT-qPCR positive while SARS-CoV-2 Ag-RDT negative discordant results correlate with the absence of infectious virus in nasopharyngeal samples. A decrease in sgRNA detection together with an expected increase in detectable anti-S and anti-N IgGs was also verified in these samples. The data clearly demonstrate that a negative Ag-RDT sample is less likely to harbor infectious SARS-CoV-2 and, consequently, has a lower transmissible potential.

Evaluation of the Panbio COVID-19 Antigen Rapid Diagnostic Test in Subjects Infected with Omicron Using Different Specimens.

Community testing is a crucial tool for the early identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and transmission control. The emergence of the highly mutated Omicron variant (B.1.1.529) raised concerns about its primary site of replication, impacting sample collection and its detectability by rapid antigen tests. We tested the performance of the Panbio antigen rapid diagnostic test (Ag-RDT) using nasal and oral specimens for COVID-19 diagnosis in 192 symptomatic individuals, with quantitative reverse transcription-PCR (RT-qPCR) of nasopharyngeal samples as a control. Variant of concern (VOC) investigation was performed with the 4Plex SARS-CoV-2 screening kit. The SARS-CoV-2 positivity rate was 66.2%, with 99% of the positive samples showing an amplification profile consistent with that of the Omicron variant. Nasal Ag-RDT showed higher sensitivity (89%) than oral (12.6%) Ag-RDT. Our data showed good performance of the Ag-RDT in a pandemic scenario dominated by the Omicron VOC. Furthermore, our data also demonstrated that the Panbio COVID-19 antigen rapid diagnostic test does not provide good sensitivity with oral swabs for Omicron Ag-RDT detection. IMPORTANCE This study showed that the antigen rapid test for COVID19 worked fine using nasal swabs when it was utilized in patients infected with the Omicron variant, showing a concordance with PCR in 93% of patients tested. The nasal swab yielded more reliable results than the oral swab when an antigen rapid diagnosis test (the Panbio COVID-19 antigen rapid diagnostic test) was used in patients infected with the Omicron variant.

Brucella activates the host RIDD pathway to subvert BLOS1-directed immune defense.

The phagocytosis and destruction of pathogens in lysosomes constitute central elements of innate immune defense. Here, we show that Brucella, the causative agent of brucellosis, the most prevalent bacterial zoonosis globally, subverts this immune defense pathway by activating regulated IRE1α-dependent decay (RIDD) of Bloc1s1 mRNA encoding BLOS1, a protein that promotes endosome-lysosome fusion. RIDD-deficient cells and mice harboring a RIDD-incompetent variant of IRE1α were resistant to infection. Inactivation of the Bloc1s1 gene impaired the ability to assemble BLOC-1-related complex (BORC), resulting in differential recruitment of BORC-related lysosome trafficking components, perinuclear trafficking of Brucella-containing vacuoles (BCVs), and enhanced susceptibility to infection. The RIDD-resistant Bloc1s1 variant maintains the integrity of BORC and a higher-level association of BORC-related components that promote centrifugal lysosome trafficking, resulting in enhanced BCV peripheral trafficking and lysosomal destruction, and resistance to infection. These findings demonstrate that host RIDD activity on BLOS1 regulates Brucella intracellular parasitism by disrupting BORC-directed lysosomal trafficking. Notably, coronavirus murine hepatitis virus also subverted the RIDD-BLOS1 axis to promote intracellular replication. Our work establishes BLOS1 as a novel immune defense factor whose activity is hijacked by diverse pathogens.

Boosting SARS-CoV-2 detection combining pooling and multiplex strategies.

RT-qPCR is the gold standard technique available for SARS-CoV-2 detection. However, the long test run time and costs associated with this type of molecular testing are a challenge in a pandemic scenario. Due to high testing demand, especially for monitoring highly vaccinated populations facing the emergence of new SARS-CoV-2 variants, strategies that allow the increase in testing capacity and cost savings are needed. We evaluated a RT-qPCR pooling strategy either as a simplex and multiplex assay, as well as performed in-silico statistical modeling analysis validated with specimen samples obtained from a mass testing program of Industry Federation of the State of Rio de Janeiro (Brazil). Although the sensitivity reduction in samples pooled with 32 individuals in a simplex assay was observed, the high-test sensitivity was maintained even when 16 and 8 samples were pooled. This data was validated with the results obtained in our mass testing program with a cost saving of 51.5% already considering the expenditures with pool sampling that were analyzed individually. We also demonstrated that the pooling approach using 4 or 8 samples tested with a triplex combination in RT-qPCR is feasible to be applied without sensitivity loss, mainly combining Nucleocapsid (N) and Envelope (E) gene targets. Our data shows that the combination of pooling in a RT-qPCR multiplex assay could strongly contribute to mass testing programs with high-cost savings and low-reagent consumption while maintaining test sensitivity. In addition, the test capacity is predicted to be considerably increased which is fundamental for the control of the virus spread in the actual pandemic scenario.

Autophagy Modulators in Coronavirus Diseases: A Double Strike in Viral Burden and Inflammation.

Coronaviruses are the etiologic agents of several diseases. Coronaviruses of critical medical importance are characterized by highly inflammatory pathophysiology, involving severe pulmonary impairment and infection of multiple cell types within the body. Here, we discuss the interplay between coronaviruses and autophagy regarding virus life cycle, cell resistance, and inflammation, highlighting distinct mechanisms by which autophagy restrains inflammatory responses, especially those involved in coronavirus pathogenesis. We also address different autophagy modulators available and the rationale for drug repurposing as an attractive adjunctive therapy. We focused on pharmaceuticals being tested in clinical trials with distinct mechanisms but with autophagy as a common target. These autophagy modulators act in cell resistance to virus infection and immunomodulation, providing a double-strike to prevent or treat severe disease development and death from coronaviruses diseases.

Identification and characterisation of SARS-CoV-2 and Human alphaherpesvirus 1 from a productive coinfection in a fatal COVID-19 case.

BACKGROUND: During routine Coronavirus disease 2019 (COVID-19) diagnosis, an unusually high viral load was detected by reverse transcription real-time polymerase chain reaction (RT-qPCR) in a nasopharyngeal swab sample collected from a patient with respiratory and neurological symptoms who rapidly succumbed to the disease. Therefore we sought to characterise the infection. OBJECTIVES: We aimed to determine and characterise the etiological agent responsible for the poor outcome. METHODS: Classical virological methods, such as plaque assay and plaque reduction neutralisation test combined with amplicon-based sequencing, as well as a viral metagenomic approach, were performed to characterise the etiological agents of the infection. FINDINGS: Plaque assay revealed two distinct plaque phenotypes, suggesting either the presence of two severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains or a productive coinfection of two different species of virus. Amplicon-based sequencing did not support the presence of any SARS-CoV-2 genetic variants that would explain the high viral load and suggested the presence of a single SARS-CoV-2 strain. Nonetheless, the viral metagenomic analysis revealed that Coronaviridae and Herpesviridae were the predominant virus families within the sample. This finding was confirmed by a plaque reduction neutralisation test and PCR. MAIN CONCLUSIONS: We characterised a productive coinfection of SARS-CoV-2 and Herpes simplex virus 1 (HSV-1) in a patient with severe symptoms that succumbed to the disease. Although we cannot establish the causal relationship between the coinfection and the severity of the clinical case, this work serves as a warning for future studies focused on the interplay between SARS-CoV-2 and HSV-1 coinfection and COVID-19 severity.

Polyclonal F(ab')2 fragments of equine antibodies raised against the spike protein neutralize SARS-CoV-2 variants with high potency.

We used the recombinant trimeric spike (S) glycoprotein in the prefusion conformation to immunize horses for the production of hyperimmune globulins against SARS-CoV-2. Serum antibody titers measured by ELISA were above 1:106, and the neutralizing antibody titer against authentic virus (WT) was 1:14,604 (average PRNT90). Plasma from immunized animals was pepsin digested to remove the Fc portion and purified, yielding an F(ab')2 preparation with PRNT90 titers 150-fold higher than the neutralizing titers in human convalescent plasma. Challenge studies were carried out in hamsters and showed the in vivo ability of equine F(ab')2 to reduce viral load in the pulmonary tissues and significant clinical improvement determined by weight gain. The neutralization curve by F(ab')2 was similar against the WT and P.2 variants, but displaced to higher concentrations by 0.39 log units against the P.1 (Gamma) variant. These results support the possibility of using equine F(ab')2 preparation for the clinical treatment of COVID patients.

Prolonged SARS-CoV-2 Positivity in Immunocompetent Patients: Virus Isolation, Genomic Integrity, and Transmission Risk.

Current guidelines for patient isolation in COVID-19 cases recommend a symptom-based approach, averting the use of control real-time reverse transcription PCR (rRT-PCR) testing. However, we hypothesized that patients with persistently positive results by RT-PCR for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could be potentially infectious for a prolonged time, even if immunocompetent and asymptomatic, which would demand a longer social isolation period than presently recommended. To test this hypothesis, 72 samples from 51 mildly symptomatic immunocompetent patients with long-lasting positive rRT-PCR results for SARS-CoV-2 were tested for their infectiousness in cell culture. The serological response of samples from those patients and virus genomic integrity were also analyzed. Infectious viruses were successfully isolated from 34.38% (22/64) of nasopharynx samples obtained 14 days or longer after symptom onset. Indeed, we observed successful virus isolation up to 128 days. Complete SARS-COV-2 genome integrity was demonstrated, suggesting the presence of replication-competent viruses. No correlation was found between the isolation of infectious viruses and rRT-PCR cycle threshold values or the humoral immune response. These findings call attention to the need to review current isolation guidelines, particularly in scenarios involving high-risk individuals. IMPORTANCE In this study, we evaluated mildly symptomatic immunocompetent patients with long-lasting positive rRT-PCR results for SARS-CoV-2. Infectious viruses were successfully isolated in cell cultures from nasopharynx samples obtained 14 days or longer after symptom onset. Indeed, we observed successful virus isolation for up to 128 days. Moreover, SARS-CoV-2 genome integrity was demonstrated by sequencing, suggesting the presence of replication-competent viruses. These data point out the risk of continuous SARS-CoV-2 transmission from patients with prolonged detection of SARS-CoV-2 in the upper respiratory tract, which has important implications for current precaution guidelines, particularly in settings where vulnerable individuals may be exposed (e.g., nursing homes and hospitals).

Protease Inhibitors as Promising Weapons against COVID-19: Focus on Repurposing of Drugs used to Treat HIV and HCV Infections.

As a part of the efforts to quickly develop pharmaceutical treatments for COVID-19 through repurposing existing drugs, some researchers around the world have combined the recently released crystal structure of SARS-CoV-2 Mpro in complex with a covalently bonded inhibitor with virtual screening procedures employing molecular docking approaches. In this context, protease inhibitors (PIs) clinically available and currently used to treat infectious diseases, particularly viral ones, are relevant sources of promising drug candidates to inhibit the SARS-CoV-2 Mpro, a key viral enzyme involved in crucial events during its life cycle. In the present perspective, we summarized the published studies showing the promising use of HIV and HCV PIs as potential repurposing drugs against the SARS-CoV-2 Mpro.

Association of neurologic signs with high-field MRI findings in 100 dogs with osseous-associated cervical spondylomyelopathy.

Although osseous-associated cervical spondylomyelopathy (OA-CSM) findings have been well described using magnetic resonance imaging (MRI), there are no large-scale published studies on the associations between dog size, age, high-field MRI and neurologic findings. Using a retrospective, observational study design, we aimed to investigate an association between neurologic and high-field MRI characteristics in OA-CSM. Records were reviewed for dogs diagnosed with OA-CSM using high-field MRI. One-hundred dogs were included: 73/100 (73%) were giant breeds, 27/100 (27%) large breeds. Mean and median ages were, respectively, 3.1 and 2 years (0.3-9.75 years), with 2.6 and 2 years for giant-breed; and 4.4 and 4 years for large-breed dogs. The majority of dogs were male (75%) with chronic presentation (89%), more than one site of spinal cord compression (78%) and foraminal stenosis (91%). Dogs with multiples sites of spinal cord compression were more likely to have severe spinal cord compression (p < 0.001), severe foraminal stenosis (p < 0.001) and ligamentum flavum/soft tissue proliferation (p = 0.03) than those with a single compressive site. There was a weak correlation between neurologic grade and severity of spinal cord compression (r = 0.27; p = 0.007), number of affected sites (r = 0.24; p = 0.0183) and spinal cord T2W hyperintensity (r = 0.24; p = 0.0152). Intervertebral disc degeneration was seen in 80% of dogs. Age did not appear to have a prominent role in the manifestation of OA-CSM. This study showed that OA-CSM affects a sizeable proportion of young large-breed, in addition to giant-breed dogs.

Milk lymphocyte profile and macrophage functions: new insights into the immunity of the mammary gland in quarters infected with Corynebacterium bovis.

BACKGROUNDS: The present study explored the viability of bovine milk macrophages, their intracellular production of reactive oxygen and nitrogen species (RONS), and their phagocytosis of Staphylococcus aureus, as well as the profile of lymphocytes, from healthy udder quarters and udder quarters infected by Corynebacterium bovis. The study included 28 healthy udder quarters from 12 dairy cows and 20 udder quarters infected by C. bovis from 10 dairy cows. The percentages of macrophages and lymphocytes were identified by flow cytometry using monoclonal antibodies. Macrophage viability, RONS production, and S. aureus phagocytosis were evaluated by flow cytometry. RESULTS: Milk samples from quarters infected with C. bovis showed a lower percentage of macrophages but an increased number of milk macrophages per mL and a higher percentage of macrophages that produced intracellular RONS and phagocytosed S. aureus. No effect of C. bovis infection on macrophage viability was found. Udder quarters infected by C. bovis showed a higher percentage of T cells and CD4+ T lymphocytes, but no effect was found on the percentage of CD8+ CD4- T, CD8- CD4- T, or B lymphocytes. CONCLUSIONS: Thus, our results corroborate, at least in part, the finding that intramammary infections by C. bovis may offer protection against intramammary infections by major pathogens.