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.

Boar semen proteomics and sperm preservation.

Recently numerous proteomic approaches have been undertaken to identify sperm and seminal plasma (SP) proteins that can be used as potential biomarkers for sperm function, including fertilization ability. This review aims firstly to briefly introduce the proteomic technologies and workflows that can be successfully applied for sperm and SP proteomic analysis. Secondly, we summarize the current knowledge about boar SP and the sperm proteome, focusing mainly on its relevance to sperm preservation procedures (liquid storage or cryopreservation) and their outcomes in terms of sperm function and fertility.

Profile and reproductive roles of seminal plasma melatonin of boar ejaculates used in artificial insemination programs.

Melatonin (MLT) is present in seminal plasma (SP) of mammalian species, including pigs, and it is credited with antioxidant properties. This study aims to identify the sources of variation and the role of boar SP MLT on sperm quality and functionality and in vivo fertilizing ability of liquid-stored semen doses used in AI programs. The SP MLT was measured using an ELISA kit in a total of 219 ejaculates collected from 76 boars, and reproductive records of 5,318 AI sows were recorded. Sperm quality was assessed according to motility (computer-aided sperm analysis) and viability (cytometry evaluation). Sperm functionality was assessed according to the cytometric determination of intracellular HO generation, total and mitochondrial O production, and lipid peroxidation in liquid AI semen samples stored at 17°C over 144 h. The concentration of SP MLT differed among seasons ( < 0.01) and day length periods ( < 0.001) of the year, demonstrating that the ejaculates collected during the increasing day length period (9.80 ± 1.38 pg/mL, range: 2.75-21.94) had lower SP MLT concentrations than those collected during the decreasing day length period (16.32 ± 1.67 pg/mL, range: 5.02-35.61). The SP MLT also differed ( < 0.001) among boars, among ejaculates within boar, and among portions within the ejaculate, demonstrating that SP from the first 10 mL of sperm-rich ejaculate fraction (SRF) exhibited lower MLT concentrations than post-SRF. The SP MLT was negatively related ( < 0.001) to mitochondrial O production in viable sperm. The SP MLT did not differ among AI boars ( = 14) hierarchically grouped according to high and low fertility outcomes. In conclusion, SP MLT concentration in AI boars varies depending on the season of ejaculate collection and differs among boars, ejaculates within boar, and portions within ejaculate. The SP MLT may act at the mitochondrial level of sperm by reducing the generation of O. However, this antioxidant role of SP MLT was not reflected in sperm quality or in vivo fertility outcomes of AI semen doses.

The activity of paraoxonase type 1 (PON-1) in boar seminal plasma and its relationship with sperm quality, functionality, and in vivo fertility.

Paraoxonase 1 (PON-1) is a hydrolytic enzyme present in body fluids, capable of protecting cells against oxidative stress. The hypothesis was hereby to test that PON-1, present in seminal plasma (SP), acts protecting boar spermatozoa when showing a reasonable high activity in the ejaculate. SP-PON-1 activity differed (p < 0.001) among boars (from 0.10 to 0.29 IU/mL). Intra-boar variability was also observed (p < 0.05), but only in two of the 15 boars. SP-PON-1 activity differed among ejaculate portions, showing the spermatozoa-peak portion of spermatozoa-rich ejaculate fraction the highest levels (0.35 ± 0.03 IU/mL, ranging from 0.12 to 0.69) and the post-sperm ejaculate fraction the lowest levels (0.12 ± 0.01 IU/mL, ranging from 0.03 to 0.21). SP-PON-1 activity was positively correlated with the percentage of spermatozoa with rapid and progressive movement (p < 0.01) and negatively correlated with the generation of intracellular reactive oxygen species (p < 0.01) in semen samples after 72 h of liquid storage. SP-PON-1 activity was highest (p < 0.01) in boars with highest farrowing rates. In conclusion, SP-PON-1 activity differed among boars and ejaculate fractions/portions. SP-PON-1 activity was positively correlated with sperm quality and functionality of liquid-stored semen samples and it evidenced a positive association with in vivo fertility.

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.

Improvement of boar sperm cryosurvival by using single-layer colloid centrifugation prior freezing.

The aim of this experimental study was to evaluate the effectiveness of sperm selection using single-layer centrifugation (SLC) prior to freezing on the sperm cryosurvival of boar ejaculates. Twenty-four sperm rich ejaculate fractions (SREF), collected from 24 boars (one per boar), were divided into two groups according to their initial semen traits: standard (n = 15) and substandard (n = 9). Semen samples from each SREF were split in two aliquots, one remained untreated (control samples) and the other was single-layer centrifuged (500 g for 20 min) using 15 mL of Androcoll-P Large (SLC samples). The yield of total, motile (assessed by CASA) and viable (cytometrically evaluated after staining with H-42, propidium iodide (PI) and FITC-PNA) sperm after SLC was higher (P < 0.05) in standard than substandard semen samples. The semen samples were cryopreserved using a standard 0.5-mL straw freezing protocol. Post-thaw sperm motility and viability (assessed at 30 and 150 min post-thawing) were higher (P < 0.05) in SLC than in control samples, regardless of the initial semen traits of the ejaculates. Additionally, thawed spermatozoa from SLC samples were more resistant (P < 0.05) to lipid peroxidation (BIOXYTECH MDA-586 Assay Kit) than those from control samples, regardless of the initial semen traits of the ejaculates. The SLC-treatment also influenced the functionality of thawed spermatozoa undergoing an in vitro capacitation process. The percentage of viable sperm showing high membrane fluidity (assessed with merocyanine 540) was lower (P < 0.05) in the SLC than in the control samples, regardless of the initial semen traits of the ejaculates. Thawed viable spermatozoa of SLC samples generated less (P < 0.05) reactive oxygen species (assessed with CM-H(2)DCFDA) than those of control samples in the substandard ejaculates. These findings indicate that the sperm selection before freezing using SLC improves the freezability of boar sperm.

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.

Acute phase response in porcine reproductive and respiratory syndrome virus infection.

This study was focused on the changes observed in the serum concentration of haptoglobin (Hp), C-reactive protein (CRP), serum amyloid A (SAA) and Pig-major acute protein (Pig-MAP), during experimental porcine reproductive and respiratory syndrome virus (PRRSV) infection and in their relationship with the expression of interleukin 1ß (IL-1ß), interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α). Hp and Pig-MAP serum levels were increased at 10 dpi, but CRP and SAA showed a delayed and highly variable increase. All three proinflammatory cytokines were poorly expressed, and only a mild increase in IL-1ß was observed at 7 dpi. The increased expression of Hp coincided with the light enhancement observed in both IL-6 and TNF-α, and might be related with an increased expression of IL-10. The low expression of TNF-α might point to a possible mechanism of viral evasion of host-immune response. This issue and the delayed expression of CRP and SAA should be taken into account in future studies about modulation of the immune response by PRRSV infection.

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.

[Compulsory vaccination programs in the light of legislation]. / Los programas de vacunación obligatoria a la luz de la legislación.

The adequate development of immunization programs deals with the problem of obtaining a good vaccination coverage, an essential aspect for the control and eradication of the disease in the population. One way of facing this deals with the implementation of coercion measures. The present paper analyzes Spanish legislation in force with regards to the possibility of applying compulsory vaccination programme in the general population and in specific subgroups.