Time Series Transcriptomic Analysis of Bronchoalveolar Lavage Cells from Piglets Infected with Virulent or Low-Virulent Porcine Reproductive and Respiratory Syndrome Virus 1.

Porcine reproductive and respiratory syndrome virus (PRRSV) has evolved to escape the immune surveillance for a survival advantage leading to a strong modulation of host's immune responses and favoring secondary bacterial infections. However, limited data are available on how the immunological and transcriptional responses elicited by virulent and low-virulent PRRSV-1 strains are comparable and how they are conserved during the infection. To explore the kinetic transcriptional signature associated with the modulation of host immune response at lung level, a time-series transcriptomic analysis was performed in bronchoalveolar lavage cells upon experimental in vivo infection with two PRRSV-1 strains of different virulence, virulent subtype 3 Lena strain or the low-virulent subtype 1 3249 strain. The time-series analysis revealed overlapping patterns of dysregulated genes enriched in T-cell signaling pathways among both virulent and low-virulent strains, highlighting an upregulation of co-stimulatory and co-inhibitory immune checkpoints that were disclosed as Hub genes. On the other hand, virulent Lena infection induced an early and more marked "negative regulation of immune system process" with an overexpression of co-inhibitory receptors genes related to T-cell and NK cell functions, in association with more severe lung lesion, lung viral load, and BAL cell kinetics. These results underline a complex network of molecular mechanisms governing PRRSV-1 immunopathogenesis at lung level, revealing a pivotal role of co-inhibitory and co-stimulatory immune checkpoints in the pulmonary disease, which may have an impact on T-cell activation and related pathways. These immune checkpoints, together with the regulation of cytokine-signaling pathways, modulated in a virulence-dependent fashion, orchestrate an interplay among pro- and anti-inflammatory responses. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the major threats to swine health and global production, causing substantial economic losses. We explore the mechanisms involved in the modulation of host immune response at lung level performing a time-series transcriptomic analysis upon experimental infection with two PRRSV-1 strains of different virulence. A complex network of molecular mechanisms was revealed to control the immunopathogenesis of PRRSV-1 infection, highlighting an interplay among pro- and anti-inflammatory responses as a potential mechanism to restrict inflammation-induced lung injury. Moreover, a pivotal role of co-inhibitory and co-stimulatory immune checkpoints was evidenced, which may lead to progressive dysfunction of T cells, impairing viral clearance and leading to persistent infection, favoring as well secondary bacterial infections or viral rebound. However, further studies should be conducted to evaluate the functional role of immune checkpoints in advanced stages of PRRSV infection and explore a possible T-cell exhaustion state.

The jigsaw of PRRSV virulence.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of the, probably, most economically important disease for the pig industry worldwide. This disease, characterised by producing reproductive failure in sows and respiratory problems in growing pigs, appeared in the late 1980s in the United States and Canada. Since its appearance, strains capable of producing higher mortality rates as well as greater severity in clinical signs and lesions than classical strains have been identified. However, since the first reports of these "virulent" PRRSV outbreaks, no homogeneity and consensus in their description have been established. Moreover, to the authors' knowledge, there is no published information related to the criteria that a PRRSV strain should fulfil to be considered as a "virulent" strain. In this review, we revise the terminology used and gather the information related to the main characteristics and differences in clinical signs, lesions, viral replication and tropism as well as immunological parameters between virulent and classical PRRSV strains and propose a first approximation to the criteria to define a virulent PRRSV strain.

Prevalence of mycoplasma-like lung lesions in pigs from commercial farms from Spain and Portugal.

BACKGROUND: Mycoplasma hyopneumoniae causes a chronic respiratory disease that produces important economic losses due to poor productive performance, increased mortality and costs for several control strategies. The prevalence of mycoplasma-like lesions (MLL) at abattoir has been widely studied in different countries, making use of different scoring systems. However, most of them are difficult to apply in abattoirs with high number of pigs sacrificed per hour. For that reason, it is necessary to adapt the scoring system to the reality of the modern abattoir, even if there is a loss of accuracy. Our purpose was to investigate the prevalence and severity of MLL at abattoirs in Spain and Portugal using a 0 to 5 scoring system adapted to abattoirs with high number of sacrificed pigs per hour and to highlight the histopathological diagnosis as confirmatory method to identify patterns of pneumonia correlated to gross lesions. RESULTS: Cranioventral pulmonary consolidation, a typical MLL, was the most frequent lung lesion (30.97 %) detected at the abattoir, followed by dorsocaudal infarcts with pleurisy (12.51 %) and pleurisy alone (6.26 %). The average score for all examined lungs at abattoir was 1.99 out of 5 points. The histopathological study revealed that the 78.17 % of the randomly selected lungs with MLL presented microscopic lesions compatible with M. hyopneumoniae infection. Most bronchointerstitial and interstitial pneumonia lesions had a chronic course while most suppurative and fibrinous bronchopneumonia lesions had an acute course and a higher degree of severity. The combination of microscopic lesions more frequently observed was bronchointerstitial pneumonia + interstitial pneumonia + suppurative bronchopneumonia. CONCLUSIONS: The prevalence of MLL at abattoir was 30.97 %, however, after microscopic examination the real prevalence of lungs with lesions compatible with M. hyopneumoniae infection was reduced up to 24.21 %. The six more prevalent combinations of lesions in the microscopic study involved the 66.13 % of examined lungs, and in all of them, microscopic lesions characteristic of M. hyopneumoniae infection were found, what supports the importance of M. hyopneumoniae as a primary pathogen in cases of PRDC.

Activation of pro- and anti-inflammatory responses in lung tissue injury during the acute phase of PRRSV-1 infection with the virulent strain Lena.

Porcine reproductive and respiratory syndrome virus (PRRSV) plays a key role in porcine respiratory disease complex modulating the host immune response and favouring secondary bacterial infections. Pulmonary alveolar macrophages (PAMs) are the main cells supporting PRRSV replication, with CD163 as the essential receptor for viral infection. Although interstitial pneumonia is by far the representative lung lesion, suppurative bronchopneumonia is described for PRRSV virulent strains. This research explores the role of several immune markers potentially involved in the regulation of the inflammatory response and sensitisation of lung to secondary bacterial infections by PRRSV-1 strains of different virulence. Conventional pigs were intranasally inoculated with the virulent subtype 3 Lena strain or the low virulent subtype 1 3249 strain and euthanised at 1, 3, 6 and 8 dpi. Lena-infected pigs exhibited more severe clinical signs, macroscopic lung score and viraemia associated with an increase of IL-6 and IFN-γ in sera compared to 3249-infected pigs. Extensive areas of lung consolidation corresponding with suppurative bronchopneumonia were observed in Lena-infected pigs. Lung viral load and PRRSV-N-protein+ cells were always higher in Lena-infected animals. PRRSV-N-protein+ cells were linked to a marked drop of CD163+ macrophages. The number of CD14+ and iNOS+ cells gradually increased along PRRSV-1 infection, being more evident in Lena-infected pigs. The frequency of CD200R1+ and FoxP3+ cells peaked late in both PRRSV-1 strains, with a strong correlation between CD200R1+ cells and lung injury in Lena-infected pigs. These results highlight the role of molecules involved in the earlier and higher extent of lung lesions in piglets infected with the virulent Lena strain, pointing out the activation of routes potentially involved in the restraint of the local inflammatory response.

Reassessing the role of internalin B in Listeria monocytogenes virulence using the epidemic strain F2365.

OBJECTIVES: To investigate the contribution to virulence of the surface protein internalin B (InlB) in the Listeria monocytogenes lineage I strain F2365, which caused a deadly listeriosis outbreak in California in 1985. METHODS: The F2365 strain displays a point mutation that hampers expression of InlB. We rescued the expression of InlB in the L. monocytogenes lineage I strain F2365 by introducing a point mutation in the codon 34 (TAA to CAA). We investigated its importance for bacterial virulence using in vitro cell infection systems and a murine intravenous infection model. RESULTS: In HeLa and JEG-3 cells, the F2365 InlB+ strain expressing InlB was ≈9-fold and ≈1.5-fold more invasive than F2365, respectively. In livers and spleens of infected mice at 72 hours after infection, bacterial counts for F2365 InlB+ were significantly higher compared to the F2365 strain (≈1 log more), and histopathologic assessment showed that the F2365 strain displayed a reduced number of necrotic foci compared to the F2365 InlB+ strain (Mann-Whitney test). CONCLUSIONS: InlB plays a critical role during infection of nonpregnant animals by a L. monocytogenes strain from lineage I. A spontaneous mutation in InlB could have prevented more severe human morbidity and mortality during the 1985 California listeriosis outbreak.

Verrucous hemangioma with pseudoepitheliomatous epidermal hyperplasia in an adult horse.

Verrucous hemangiomas are a rare specific variant of equine skin tumors not well described in the literature. An 8-year-old gelding presented a unilateral lesion on the pastern. Macroscopically, the mass showed a warty and verrucous surface with focal ulcerations. The histology showed a dermal proliferation of endothelial-layered capillaries and venules separated by a delicate stroma of scant fibroblasts and collagen deposition, with pseudoepitheliomatous hyperplasia (exuberant reactive irregular epithelial hyperplasia with tongue-like projections extending into the dermis, mimicking downgrowth of squamous cell carcinoma) and orthokeratotic hyperkeratosis of the overlying epidermis. The immunohistochemical study confirmed the endothelial origin of the tumor, and a final diagnosis of verrucous hemangioma with pseudoepitheliomatous hyperplasia was confirmed. To the knowledge of the authors, this is the first detailed description of this entity in adult horses. Moreover, the clinical progression and epidermal changes have not been previously reported, emphasizing the importance of a pathological study of any epithelial preneoplastic lesion to rule out an underlying dermal neoplasia.

Type 2 Porcine Reproductive and Respiratory Syndrome Virus infection mediated apoptosis in B- and T-cell areas in lymphoid organs of experimentally infected pigs.

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) infection is characterized by persisting in lungs and lymphoid tissue, resulting in systemic lymphoid depletion. The aim of this study was to correlate the histological changes, viral antigen expression and apoptosis phenomena in tonsil, medial retropharyngeal and mediastinal lymph nodes of 12 pigs inoculated with a type 2 PRRSV isolate (Chilean strain 2402). Apoptosis phenomena were observed mainly in lymphocytes and secondly in macrophages of lymph nodes and tonsils of inoculated animals, showing a peak of both apoptotic cells and viral antigen expression at the end of the study (21 dpi). However, the number of apoptotic cells was higher than the number of PRRSV-positive cells at the end of the study. This finding together with the location of apoptotic cells and PRRSV-positive cells in different structures of lymphoid organs supports the hypothesis that PRRSV-positive macrophages might modulate the apoptosis phenomena in other cells, mainly lymphocytes, by means of an indirect mechanism. Furthermore, apoptotic cells were detected both in B- and T-cell areas of lymphoid organs, suggesting that apoptosis phenomena may play a role in the impairment of the host immune response during PRRS.

Downregulation of antigen-presenting cells in tonsil and lymph nodes of porcine reproductive and respiratory syndrome virus-infected pigs.

Porcine reproductive and respiratory syndrome virus (PRRSV) can persist in different organs of infected pigs, which suggests a failure in the immune response. Antigen-presenting cells (APCs) play a pivotal role in the induction of effective T- and B-cell responses. In this study, we investigated the changes in the different APC subpopulations and T- and B-cell counts in the tonsil, retropharyngeal and mediastinal lymph nodes of pigs experimentally infected with a European PRRSV field isolate. Our results demonstrated that the expression of S100, SWC3, HLA-DR molecule and CD3 was diminished in the studied organs throughout the study, observing a significant negative correlation between viral antigen and HLA-DR expression in both retropharyngeal and mediastinal lymph nodes. In contrast, λ-light chains showed an increase during the study. Taking all into account, after PRRSV infection, no enhancement in the number of APCs and T cells was observed, suggesting an impairment of the immune function which may allow the persistence of PRRSV into the organism.

Immunohistochemical expression of IL-12, IL-10, IFN-α and IFN-γ in lymphoid organs of porcine reproductive and respiratory syndrome virus-infected pigs.

Despite the numerous studies carried out, the mechanisms used by porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) to impair the host immune response are not yet clear. The aim of this study was to determine the expression of IL-12, IL-10, IFN-α and IFN-γ in lymphoid organs of PRRSV experimentally-infected pigs. Twenty eight piglets were inoculated with PRRSV field isolate 2982 and killed in batches of four at 3, 7, 10, 14, 17, 21 and 24 days post-inoculation (dpi). Control animals were mock-inoculated and killed at the end of the study. Samples from mediastinal and retropharyngeal lymph nodes and tonsil were collected and fixed for histopathological and immunohistochemical analyses. PRRSV antigen was mainly detected in the cytoplasm of macrophages, displaying a bimodal expression with a first peak at 3-7 dpi and a second peak at 14 dpi. The expression of IFN-α showed an early enhancement at 3 dpi, and both IL-12 and IFN-γ displayed a similar trend in all the lymphoid organs analysed, showing an increase at 3-7 dpi and at 14-17 dpi. On the other hand, the expression of IL-10 was lower than the one observed for the other cytokines. The expression of IL-10 compared with the higher expression of IL-12, IFN-α and IFN-γ detected in this study, indicates that other mechanisms besides the expression of IL-10 play a role in the inducement of an erratic host immune response. Taking into account the enhanced expression of IFNs together with the detection of PRRSV antigen until the end of the study in the examined lymphoid organs, further studies are being conducted to rule out a down-regulation in IFN signalling pathway.

Malignant mesenchymoma of the heart base in a dog with infiltration of the pericardium and metastasis to the lung.

A 9-year-old male rottweiler was presented with abdominal distension, ascites and respiratory distress and marked bulging in the perineal region. At necropsy examination the animal had profuse ascites and hydropericardium and a multinodular mass in the right auricle of the heart infiltrating the epicardium and pericardium and metastasizing to the caudal lobe of the left lung. Microscopically and immunohistochemically the tumour was composed of neoplastic cells with muscular, cartilaginous and adipose differentiation. A diagnosis of malignant mesenchymoma with leiomyosarcomatous (≈ 50%), rhabdomyosarcomatous (≈ 30%), chondrosarcomatous (25%) and liposarcomatous (5%) components was made. Metastatic malignant mesenchymoma has not been reported previously at this site in the dog.

Differential expression of proinflammatory cytokines in the lymphoid organs of porcine reproductive and respiratory syndrome virus-infected pigs.

Porcine reproductive and respiratory syndrome (PRRS) is considered one of the most important diseases in the swine industry. Although several studies have been carried out to elucidate the host immune response evoked against PRRS virus (PRRSV), there are several aspects that still remain unclear. The aim of this study was to determine the expression of IL-1α, IL-6 and TNF-α in the lymphoid organs (mediastinal and retropharyngeal lymph nodes and tonsil) of PRRSV-infected pigs and to determine their correlation with the expression of PRRSV antigen. Proinflammatory cytokine expression was different depending on the body compartment examined. Thus, whereas IL-1α and TNF-α were the main cytokines expressed in the mediastinal lymph node, IL-6 was the most highly expressed cytokine in the retropharyngeal lymph node, and no expression of proinflammatory cytokines was observed in the tonsil. These findings may be related to the impairment of the host immune response evoked after PRRSV infection. Therefore, lymphoid organs and proinflammatory cytokines represent an important target of study for clarifying the immunopathogenesis of PRRS.

Tuberculosis in alpacas (Lama pacos) caused by Mycobacterium bovis.

We report three cases of tuberculosis in alpacas from Spain caused by Mycobacterium bovis. The animals revealed two different lesional patterns. Mycobacterial culture and PCR assay yielded positive results for M. bovis. Molecular typing of the isolates identified spoligotype SB0295 and identical variable-number tandem repeat (VNTR) allele sizes.

Cytokine expression by macrophages in the lung of pigs infected with the porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome (PRRS) is caused by a virus that predominantly replicates in alveolar macrophages. The aim of the present study was to characterize the production of cytokines by subpopulations of pulmonary macrophages in pigs infected by the PRRS virus (PRRSV). Expression of interleukin (IL) 1alpha, IL-6 and tumour necrosis factor (TNF)-alpha correlated with the severity of pulmonary pathology and the numbers of pulmonary macrophages. Significant correlations were observed between PRRSV infection and the expression of IL-10, between the expression of IL-12p40 and interferon (IFN)-gamma, and between the expression of TNF-alpha and IFN-gamma. These findings suggest that PRRSV modulates the immune response by the up-regulation of IL-10, which may in turn reduce expression of cytokines involved in viral clearance (e.g. IFN-alpha, IFN-gamma, IL-12p40 and TNF-alpha). The results also suggest that expression of IFN-gamma is stimulated by IL-12p40 and TNF-alpha, but not by IFN-alpha. All of these cytokines were expressed mainly by septal macrophages with weaker expression by alveolar macrophages, lymphocytes and neutrophils. There appears to be differential activation of septal and alveolar macrophages in PRRSV infection, with septal macrophages being the major source of cytokines.

Studies on internal exposure doses received by the cuban population due to the intake of radionuclides from the environmental sources.

Studies for the determination of radionuclide concentrations in foodstuffs, water and air were carried out in Cuba for the estimation of annual committed effective doses to members of the public as a result of environmental radionuclides via ingestion and inhalation. As a result of these studies, it was possible to determine the concentrations of 226Ra, 210Pb, 210Po, 232Th, 90Sr and 137Cs in different food groups that constitute the diet of the Cuban population, as well as the 222Rn concentrations in air. Based on these results and using previously obtained results for doses due to the 40K body content, the annual committed effective doses due to the intake of studied radionuclides were estimated. An average value of 120+/-4 microSv y-1 was obtained for doses due to ingestion of food and water and the obtained value for 222Rn inhalation was 240+/-1 microSv y-1. Using the representative value obtained previously for 40K (150+/-40 microSv y-1) and assuming a dose of 50+/-50 microSv y-1 for the probable contribution of 220Rn by inhalation, a representative value of 560+/-20 microSv was estimated for the average annual committed effective doses due to ingestion and inhalation of radionuclides for the Cuban population. Obtained values are consistent with the expected results, taking into account the characteristics of Cuban exposure scenarios, with low-activity concentration levels in environmental objects and high air exchange rates in dwellings: These results are in the same order of magnitude as results obtained by other authors and the reference values established by the USNCEAR.

GH response to GH-releasing hormone (GHRH) in Alzheimer and vascular dementia. Relation with somatostatin cerebrospinal levels.

OBJECTIVE: To clarify the functional state of the somatotropinergic system in patients with Alzheimer's disease (AD) and in patients with vascular dementia (VD) in relation with somatostatin cerebrospinal levels. DESIGN We studied the GH response to GHRH in controls and in AD and VD patients and correlated their responses with somatostatin cerebrospinal levels. PATIENTS: Twelve control subjects and 56 patients, 28 with AD and 28 with VD, were studied. A GHRH test was performed in every case. One hundred mcg of GHRH was administered as a intravenous bolus and blood samples were drawn at -15, 0, 30, 60, 90 and 120 minutes. Cerebrospinal fluid was obtained after lumbar puncture. Measurements of GH and somatostatin were determined by specific radioimmunoassay. RESULTS: The GH response to GHRH exhibited no difference between AD and VD patients (12.2 +/- 2 mcg/l vs 9.9 +/- 1.8). There were 17 AD patients and 16 VD patients with exaggerated GH response to GHRH. There was no difference between cerebrospinal levels of somatostatin (30 +/- 2.1 pg/mL in AD patients vs 42 +/- 2.4 in VD patients), and there was no correlation among GH response to GHRH and cerebrospinal levels. CONCLUSION: In our study we observed no difference between the AD and VD patients in GH response to GHRH and no difference in somatostatin cerebrospinal levels in these patients.

Clinical evaluation of an intraoral device for the controlled release of fluoride.

An intraoral-releasing device designed to release 0.5 mg of fluoride per day was evaluated in a one-month trial. The results showed that the 11 men who wore the fluoride-releasing device on their maxillary first molars had significantly elevated levels of fluoride in their saliva and plaque compared with baseline levels. No significant changes were observed in mean serum or urine fluoride levels or in the gingival or plaque indexes during the study. The prevalence of S mutans in whole saliva did not change during the study and the relative proportion of S. mutans, S. sanguis, and A viscosus and A naeslundii in plaque also remained relatively stable. The elevated fluoride levels in saliva and plaque are presumptive evidence that the intraoral fluoride-releasing device could exert a cariostatic effect in humans. However, long-term clinical trials are needed to determine the cariostatic potential of this fluoride releasing system.