Systemic and local responses of cytokines and tissue histology following intramammary lipopolysaccharide challenge in dairy cows.

During bovine mastitis, immune responses include the release of cytokines and the recruitment of leukocytes, resulting in profound structural and functional changes in the mammary gland. Our aims were to delineate systemic and local cytokine responses and to quantify histological changes in the mammary tissue of lactating cows after acute intramammary lipopolysaccharide (LPS) challenge. Ten multiparous dairy cows were paired to either treatment (TRT) or control (CON) groups. For TRT cows, one side of the udder was randomly assigned to receive treatment with LPS (50 µg in 10 mL of saline, TL) into both the front and rear quarters; the contralateral quarters received saline (10 mL). Udder-halves of CON cows were similarly assigned randomly to receive either saline (10 mL, CS) or no infusion (untreated). Temporal changes in the concentrations of 15 cytokines in the blood (0, 3, 6, 12, and 24 h relative to the LPS infusion) and in mammary tissue (0, 3, and 12 h) were determined, as were concomitant changes in mammary histology. The cytokines IL-6, IL-10, MCP-1, and MIP-1ß showed a systemic response as their concentrations were significantly different in the plasma of TRT cows as compared with CON cows after LPS challenge. The cytokines IL-1α, IL-1ß, IL-6, IL-8, IL-17A, IL-36RA, IP-10, MCP-1, MIP-1α, MIP-1ß, TNF-α, and VEGF-A showed a local response in TL glands, and 8 cytokines, IL-1ß, IL-6, IL-10, IL-17A, IL-36RA, IP-10, MIP-1ß, and VEGF-A showed systemic changes in the nonchallenged mammary glands adjacent to LPS-infused glands. Endotoxin challenge evoked changes in the histology of mammary tissue that included a 5.2- and 7.2-fold increases in the number of neutrophils in alveolar lumens at 3 h and 12 h, respectively. In summary, LPS challenge induced specific local and systemic responses in cytokine induction and elicited neutrophil infiltration in bovine mammary tissue.

Implications of COVID-19 On A Rapidly Growing Thymoma Case.

Introduction: Thymomas are a rare form of slow-growing malignancy that originate from thymic epithelial cells presenting as an anterior mediastinal mass. Although most are asymptomatic, they can have a variety of presentations, such as local thoracic symptoms, superior vena cava syndrome, or paraneoplastic syndromes. Local compressive thoracic symptoms include shortness of breath, chest pain, and cough. Superior vena cava syndrome presents with respiratory, vascular, or neurologic symptoms. Paraneoplastic syndromes, such as myasthenia gravis, are due to abnormal T-cell maturation leading to an increased risk of autoimmune conditions. Case Presentation: We report a case of a 71-year-old White male with multiple comorbidities presenting to the emergency room after a mechanical fall with an incidental finding of a 3.8 cm x 6.0 cm anterior mediastinal mass. The patient had no local compressive symptoms or paraneoplastic syndromes. Due to the coronavirus disease 2019 (COVID-19) pandemic, the patient did not follow through with the discharge recommendations for surgical consultation. Over a year later, the patient presented to the emergency room for congestive heart failure exacerbation, and chest computed tomography revealed the mass had increased in size to 8.2 cm x 7.7 cm. A multidisciplinary approach was used to determine the patient's course of treatment. Due to the patient's debilitated state and concern for local invasion, radical thymectomy with mediastinal lymph node dissection was planned. Despite medical optimization and coordination with a multidisciplinary team, following surgery, the patient became symptomatically bradycardic with acute hypoxic respiratory failure. The patient ultimately passed away after pulseless electrical activity and the family's decision to discontinue resuscitation. Conclusion: It is imperative to consider the negative impacts of the COVID-19 pandemic. Delay in treatment allowed the thymoma to rapidly grow, thus leading to a decreased chance for cure. An extensive surgery increased perioperative risks that led to unforeseen complications resulting in the untimely death of the patient.

Localized and Systemic Inflammatory Mediators in a Murine Acute Mastitis Model.

INTRODUCTION: Milk depression is the major driver of economic loss due to mastitis in dairy animals. The aim of this study was to identify potential mediators of milk depression by investigating the local and systemic changes in gene expression or cytokine production during endotoxin challenge of the mammary gland in a mouse model. METHODS: The left and right sides of the 4th pair of mouse mammary glands were alternatively injected with either lipopolysaccharide (LPS, Escherichia coli 055: B5, 50 µL of 0.4 mg/mL) or sterile PBS through the teat meatus 3 days postpartum (n = 9). The 4th glands were individually collected 12 h after LPS injection and analyzed to identify gene expression changes by RNA sequencing and real-time PCR, and the plasma was collected before and after LPS challenge and analyzed to determine the levels of 32 cytokines. RESULTS: Transcriptome analysis showed that in addition to strong pro-inflammatory responses, which included granulocyte and monocyte migration and cytokine production and signaling, the LPS-treated glands exhibited strong ubiquitin-mediated and immune-mediated proteasome activation and an increase in nitric oxide-mediated oxidative stress. Furthermore, LPS induced a down-regulation in vesicle membrane, vesicle-mediated trafficking, and metabolic processes of amino acids and other organic molecules in the mammary gland. Of the 32 cytokines analyzed, the levels of 24 (mainly IL-6, G-CSF, MCP-1, RANTES, MIG, MIP-1b, KC, MIP-2, IP-10, and TNFα) were increased or tended to increase in the blood after LPS treatment, and only the levels of IL-9 were decreased. In the mammary gland after LPS challenge, the levels of IL-5, IL-6, IP-10, LIF, MCP-1, MIP-2, and TNFα were significantly increased, and the levels of INFΥ, IL-2, IL-4, IL-10, and IL-12 (p40) were decreased. DISCUSSION: These observations provide potential markers and targets for further studies on the prevention and treatment of gram-negative bacteria-induced mastitis.

Comparison of Primary Virus Isolation in Pulmonary Alveolar Macrophages and Four Different Continuous Cell Lines for Type 1 and Type 2 Porcine Reproductive and Respiratory Syndrome Virus.

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) has a highly restricted cellular tropism. In vivo, the virus primarily infects tissue-specific macrophages in the nose, lungs, tonsils, and pharyngeal lymphoid tissues. In vitro however, the MARC-145 cell line is one of the few PRRSV susceptible cell lines that are routinely used for in vitro propagation. Previously, several PRRSV non-permissive cell lines were shown to become susceptible to PRRSV infection upon expression of recombinant entry receptors (e.g., PK15Sn-CD163, PK15S10-CD163). In the present study, we examined the suitability of different cell lines as a possible replacement of primary pulmonary alveolar macrophages (PAM) cells for isolation and growth of PRRSV. The susceptibility of four different cell lines (PK15Sn-CD163, PK15S10-CD163, MARC-145, and MARC-145Sn) for the primary isolation of PRRSV from PCR positive sera (both PRRSV1 and PRRSV2) was compared with that of PAM. To find possible correlations between the cell tropism and the viral genotype, 54 field samples were sequenced, and amino acid residues potentially associated with the cell tropism were identified. Regarding the virus titers obtained with the five different cell types, PAM gave the highest mean virus titers followed by PK15Sn-CD163, PK15S10-CD163, MARC-145Sn, and MARC-145. The titers in PK15Sn-CD163 and PK15S10-CD163 cells were significantly correlated with virus titers in PAM for both PRRSV1 (p < 0.001) and PRRSV2 (p < 0.001) compared with MARC-145Sn (PRRSV1: p = 0.22 and PRRSV2: p = 0.03) and MARC-145 (PRRSV1: p = 0.04 and PRRSV2: p = 0.12). Further, a possible correlation between cell tropism and viral genotype was assessed using PRRSV whole genome sequences in a Genome-Wide-Association Study (GWAS). The structural protein residues GP2:187L and N:28R within PRRSV2 sequences were associated with their growth in MARC-145. The GP5:78I residue for PRRSV2 and the Nsp11:155F residue for PRRSV1 was linked to a higher replication on PAM. In conclusion, PK15Sn-CD163 and PK15S10-CD163 cells are phenotypically closely related to the in vivo target macrophages and are more suitable for virus isolation and titration than MARC-145/MARC-145Sn cells. The residues of PRRSV proteins that are potentially related with cell tropism will be further investigated in the future.

Porcine reproductive and respiratory syndrome virus whole-genome sequencing efficacy with field clinical samples using a poly(A)-tail viral genome purification method.

The genomic surveillance of porcine reproductive and respiratory syndrome virus (PRRSV) is based on sequencing of the ORF5 gene of the virus, which covers only 4% of the entire viral genome. It is expected that PRRSV whole-genome sequencing (WGS) will improve PRRSV genomic data and allow better understanding of clinical discrepancies observed in the field when using ORF5 sequencing. Our main objective was to implement an efficient method for WGS of PRRSV from clinical samples. The viral genome was purified using a poly(A)-tail viral genome purification method and sequenced using Illumina technology. We tested 149 PRRSV-positive samples: 80 sera, 33 lungs, 33 pools of tissues, 2 oral fluids, and 1 processing fluid (i.e., castration liquid). Overall, WGS of 67.1% of PRRSV-positive cases was successful. The viral load, in particular for tissues, had a major impact on the PRRSV WGS success rate. Serum was the most efficient type of sample to conduct PRRSV WGS poly(A)-tail assays, with a success rate of 76.3%, and this result can be explained by improved sequencing reads dispersion matching throughout the entire viral genome. WGS was unsuccessful for all pools of tissue and lung samples with Cq values > 26.5, whereas it could still be successful with sera at Cq ≤ 34.1. Evaluation of results of highly qualified personnel confirmed that laboratory skills could affect PRRSV WGS efficiency. Oral fluid samples seem very promising and merit further investigation because, with only 2 samples of low viral load (Cq = 28.8, 32.8), PRRSV WGS was successful.

Whole-Genome Sequencing of Porcine Reproductive and Respiratory Syndrome Virus from Field Clinical Samples Improves the Genomic Surveillance of the Virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major economic concern worldwide. There are currently large data sets available about the ORF5 gene of the virus, with thousands of sequences available, but little data are currently available on the full-length genome of PRRSV. We hypothesized that whole-genome sequencing (WGS) of the PRRSV genome would allow better epidemiological monitoring than ORF5 gene sequencing. PRRSV PCR-positive serum, oral fluid, and tissue clinical samples submitted to the diagnostic laboratory for routine surveillance or diagnosis of PRRSV infection in Québec, Canada, swine herds were used. The PRRSV reverse transcription-quantitative PCR Cq values of the processed samples varied between 11.5 and 34.34. PRRSV strain genomes were isolated using a poly (A)-tail method and were sequenced with a MiSeq Illumina sequencer. Ninety-two full-length PRRSV genomes were obtained from 88 clinical samples out of 132 tested samples, resulting in a PRRSV WGS success rate of 66.67%. Three important deletions in ORF1a were found in most wild-type (i.e., not vaccine-like) strains. The importance of these deletions remains undetermined. Two different full-length PRRSV genomes were found in four different samples (three serum samples and one pool of tissues), suggesting a 4.55% PRRSV strain coinfection prevalence in swine. Moreover, six PRRSV whole genomes (6.52% of PRRSV strains) were found to cluster differently than they did under the ORF5 classification method. Overall, WGS of PRRSV enables better strain classification and/or interpretation of results in 9.10% of clinical samples than ORF5 sequencing, as well as allowing interesting research avenues.

WHOLE GENOME SEQUENCING OF AN AVIPOXVIRUS ASSOCIATED WITH INFECTIONS IN A GROUP OF AVIARY-HOUSED SNOW BUNTINGS (PLECTROPHENAX NIVALIS).

Avipoxvirus infections have been reported in both free-ranging and domestic birds worldwide. Fowlpox and canarypox viruses belong to the genus Avipoxvirus among the virus family Poxviridae. They cause cutaneous lesions with proliferative growths on the unfeathered parts of the skin and/or diphtheritic lesions generally associated with necrosis in the upper respiratory and digestive tracts. In this study, a poxvirus has been identified in wild-caught snow buntings (Plectrophenax nivalis) housed in an outdoor aviary in the region of Rimouski, Quebec. During the falls and winters of 2015 and 2016, eight snow buntings affected by this infection were examined. Macroscopic and microscopic lesions observed were characteristic of an avipoxvirus infection. Electron microscopy imaging of an ultrathin section of the histopathological lesions of two birds confirmed the presence of the poxvirus. Afterward, the presence of the poxvirus was confirmed in three birds by a specific polymerase chain reaction assay that amplified a segment of the gene encoding the fowlpox virus 4b core protein. A 576-nucleotide amplicon was obtained from one of them and sequenced. The analyses revealed a 99% homology to other previously described avipoxviruses. Using high-throughput sequencing, almost the entire viral genome of this avipoxvirus was revealed and found to possess a 359,853-nucleotide sequence in length. Bioinformatic analyses revealed that the virus was genetically related to canarypox virus. To our knowledge, this is the first confirmed case and full description of a poxviral infection in this species. This episode suggests a high susceptibility of this northern species of passerine to avipoxviruses circulating in southeastern Canada during the summer months. Even if the source of the viral infections remains undetermined, transmission by local biological vectors is suspected. Management of poxviral infections in snow buntings housed outdoors in southeastern Canada could rely on the control of biting insects.