The scene of lung pathology during PRRSV-1 infection.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically important infectious diseases for the pig industry worldwide. The disease was firstly reported in 1987 and became endemic in many countries. Since then, outbreaks caused by strains of high virulence have been reported several times in Asia, America and Europe. Interstitial pneumonia, microscopically characterised by thickened alveolar septa, is the hallmark lesion of PRRS. However, suppurative bronchopneumonia and proliferative and necrotising pneumonia are also observed, particularly when a virulent strain is involved. This raises the question of whether the infection by certain strains results in an overstimulation of the proinflammatory response and whether there is some degree of correlation between the strain involved and a particular pattern of lung injury. Thus, it is of interest to know how the inflammatory response is modulated in these cases due to the interplay between virus and host factors. This review provides an overview of the macroscopic, microscopic, and molecular pathology of PRRSV-1 strains in the lung, emphasising the differences between strains of different virulence.

Inflammation-suppressing cornea-in-a-syringe with anti-viral GF19 peptide promotes regeneration in HSV-1 infected rabbit corneas.

Pathophysiologic inflammation, e.g., from HSV-1 viral infection, can cause tissue destruction resulting in ulceration, perforation, and ultimately blindness. We developed an injectable Cornea-in-a-Syringe (CIS) sealant-filler to treat damaged corneas. CIS comprises linear carboxylated polymers of inflammation-suppressing 2-methacryloyloxyethyl phosphorylcholine, regeneration-promoting collagen-like peptide, and adhesive collagen-citrate glue. We also incorporated GF19, a modified anti-viral host defense peptide that blocked HSV-1 activity in vitro when released from silica nanoparticles (SiNP-GF19). CIS alone suppressed inflammation when tested in a surgically perforated and HSV-1-infected rabbit corneal model, allowing tissue and nerve regeneration. However, at six months post-operation, only regenerated neocorneas previously treated with CIS with SiNP-GF19 had structural and functional features approaching those of normal healthy corneas and were HSV-1 virus-free. We showed that composite injectable biomaterials can be designed to allow regeneration by modulating inflammation and blocking viral activity in an infected tissue. Future iterations could be optimized for clinical application.

Droplet digital PCR as alternative to microbiological culture for Mycobacterium tuberculosis complex detection in bovine lymph node tissue samples.

Introduction: Bovine tuberculosis (bTB) caused by Mycobacterium tuberculosis complex (MTC) remains a significant concern for public health. Direct real-time PCR and droplet digital PCR (ddPCR) are proposed as alternative tools to enhance diagnostic precision and efficiency. This study aims to assess the diagnostic performance of a ddPCR assay targeting IS6110 for the detection of MTC DNA in both microbiological culture and fresh lymph node (LN) tissue samples obtained from cattle, in comparison with the established reference standard, the microbiological culture followed by real-time PCR. Methods: The fresh LNs (N=100) were collected each from a different cattle carcass at the slaughterhouse. The limit of detection of ddPCR-IS6110 was set to 101 copies per 20 µl reaction. Results: DdPCR-IS6110 detected 44 out of 49 reference-standard positive samples and yielded negative results in 47 out of 51 reference-standard negative samples, resulting in adjusted sensitivity (Se) and specificity (Sp) of 90.76% [95% confidence interval (CI): 82.58 - 98.96%)], and 100% (95% CI: 100%) respectively. The estimated adjusted false negative rate (FNR) was 9.23% (95% CI: 1.04 - 17.42%) and the false positive rate (FPR) was 0% (95% CI: 0%). When directly applied from fresh bovine LN tissues, ddPCR-IS6110 identified 47 out of 49 reference-standard positive samples as ddPCR-IS6110-positive and 42 out of 51 reference-standard negative samples as ddPCR-IS6110-negative, resulting in adjusted Se and Sp values of 94.80% [95% (CI): 88.52 - 100%] and 100% (95% CI: 100%), respectively. The adjusted FNR was 5.20% (95% CI: 0 - 11.50%) and the FPR was 0% (95% CI: 0%). Noteworthy, ddPCR-IS6110 disclosed as positive 9 samples negative to reference-standard. Discussion: DdPCR-IS6110 proved to be a rapid, highly sensitive, and specific diagnostic tool as an alternative to reference-standard method.

Macrophage polarization in lymph node granulomas from cattle and pigs naturally infected with Mycobacterium tuberculosis complex.

Tuberculosis in animals is caused by members of the Mycobacterium tuberculosis complex (MTC), with the tuberculous granuloma being the main characteristic lesion. The macrophage is the main cell type involved in the development of the granuloma and presents a wide plasticity ranging from polarization to classically activated or pro-inflammatory macrophages (M1) or to alternatively activated or anti-inflammatory macrophages (M2). Thus, this study aimed to analyze macrophage polarization in granulomas from cattle and pig lymph nodes naturally infected with MTC. Tuberculous granulomas were microscopically categorized into four stages and a panel of myeloid cells (CD172a/calprotectin), M1 macrophage polarization (iNOS/CD68/CD107a), and M2 macrophage polarization (Arg1/CD163) markers were analyzed by immunohistochemistry. CD172a and calprotectin followed the same kinetics, having greater expression in late-stage granulomas in pigs. iNOS and CD68 had higher expression in cattle compared with pigs, and the expression was higher in early-stage granulomas. CD107a immunolabeling was only observed in porcine granulomas, with a higher expression in stage I granulomas. Arg1+ cells were significantly higher in pigs than in cattle, particularly in late-stage granulomas. Quantitative analysis of CD163+ cells showed similar kinetics in both species with a consistent frequency of immunolabeled cells throughout the different stages of the granuloma. Our results indicate that M1 macrophage polarization prevails in cattle during early-stage granulomas (stages I and II), whereas M2 phenotype is observed in later stages. Contrary, and mainly due to the expression of Arg1, M2 macrophage polarization is predominant in pigs in all granuloma stages.

Assessment of the diagnostic performance of intradermal tuberculin test and post-mortem inspection for the diagnosis of bovine tuberculosis according to WOAH guidelines.

Bovine tuberculosis (bTB) constitutes a global challenge for public and animal health with still some deficiencies regarding its diagnosis. This study aimed to estimate the accuracy of the single intradermal tuberculin test (SIT) and post-mortem inspection for different diagnostic objectives following WOAH guidelines. Tissue samples from 59 microbiological culture/PCR-positive and 58 microbiological culture/PCR-negative cattle were evaluated. The diagnostic sensitivity and specificity, the positive and negative probability indices as well as the positive and negative predictive values (PPV and NPV) of each technique were estimated for different pretest probabilities. The SIT with strict interpretation demonstrated moderate precision in confirming the absence of infection in populations historically free of bTB, with a 12.1% rate of false positives, but also detecting positive animals in the early stage of the eradication programs, with a 13.6% rate of false negatives. The diagnostic performance for ruling out bTB was notably high (NPV > 90%) in animals with a pre-test probability (PTP) below 42%. Post-mortem inspection constituted an interesting alternative tool to confirm suspected and positive cases for SIT, particularly in areas with bTB prevalence exceeding 19%, where implementing SIT and eradication measures may be impractical. In these areas, the likelihood that animals with tuberculosis-like lesions are affected by the disease surpasses 90%. Similarly, in herds with a PTP below 25%, the absence of bTB could be confidently ruled out with over 90% certainty. These findings highlight the effectiveness of SIT and post-mortem inspection as valuable techniques for current eradication programs and controlling bTB in high-prevalence areas where molecular techniques may not be feasible.

Monitoring the immune response of macrophages in tuberculous granuloma through the expression of CD68, iNOS and HLA-DR in naturally infected beef cattle.

Bovine tuberculosis still represents a universal threat that creates a wider range of public and animal health impacts. One of the most important steps in the pathogenesis of this disease and granuloma formation is the phagocytosis of tuberculous bacilli by macrophages. Mycobacteria replicate in macrophages, which are crucial to the pathophysiology of mycobacterial infections; however, scarce information is available about the dynamics of the granuloma-stage immunological response. Therefore, immunohistochemistry was used in this work to evaluate the expression of CD68, iNOS, and HLA-DR in different stages of TB granulomas from naturally infected cattle with tuberculosis. Two thousand, one hundred and fifty slaughtered beef cattle were examined during the period from September 2020 to March 2022. Sixty of them showed gross tuberculous pulmonary lesions and samples were collected from all of them for histopathological examination, Ziehl-Neelsen (ZN) staining, and bacteriological culturing. Selected samples that yielded a positive result for ZN and mycobacterial culturing were subjected to an immunohistochemical study of CD68, iNOS, and HLA-DR expression by macrophages according to granuloma stages. Immunohistochemical analysis revealed that the immunolabeling of CD68+, iNOS+, and HLA-DR+ macrophages significantly reduced as the stage of granuloma increased from stage I to stage IV (P < 0.003, P < 0.002, and P < 0.002, respectively). The distribution of immunolabeled macrophages was similar for the three markers, with immunolabeled macrophages distributed throughout early-stage granulomas (I, II), and surrounding the necrotic core in late-stage granulomas (III, IV). Our results suggest a polarization to the pro-inflammatory environment and increased expression of CD68+, iNOS+, and HLA-DR+ macrophages in the early stages of granulomas (I, II), which may play a protective role in the immune response of naturally infected beef cattle with tuberculosis.

Organ xenotransplantation acceptance in veterinary students: A multicentric opinion study in Spain.

BACKGROUND: The shortage of available transplant organs has made it necessary to search for alternatives, one of which is xenotransplantation. However, the use of animal organs could face rejection from society and the personnel involved in its implementation. OBJECTIVES: (a) to analyze the attitudes of Veterinary Degree students in six Spanish Universities towards xenotransplantation; and (b) to determine the factors that affect its acceptance. METHODS: Of the 2815 students surveyed in the degree program, 2683 valid surveys were obtained. Attitudes towards organ xenotransplantation were evaluated using a validated questionnaire of organ donation. RESULTS: If xenotransplantation was confirmed as a clinical reality, 93% (n = 2493) of those surveyed would accept a xenotransplanted organ, whilst 7% would not. If the results of xenotransplantation were worse than those obtained with human donors and it entailed more risk, 12% (n = 318) would be in favor. 56% (n = 1497) of the students would accept a xenotransplantation provisionally pending the arrival of a human organ. Attitudes towards xenotransplantation were affected by the academic year in which a student was studying, with more favorable attitudes among students in the last year (88% in first year vs. 95% in fifth year; p < .001). More favorable attitudes are also observed depending on the attitude they have towards organ transplantation, with those students being more in favor of donating their organs when they die (94% vs. 88%; p < .001). CONCLUSION: Veterinary students would have a very favorable attitude toward xenotransplantation if these animal organs functioned as well as human organs. Therefore, these students could play an important role in the future promotion of this technique.

Evaluation of the diagnostic accuracy of the serological test for paratuberculosis in cattle according to tuberculosis status.

BACKGROUND: Enzyme-linked immunosorbent assays (ELISAs) are the most widely used diagnostic tools in bovine paratuberculosis (bPTB) control. However, their diagnostic accuracy may be compromised by bovine tuberculosis (bTB) infection, as both diseases share diagnostic targets. METHODS: The bPTB and bTB infection status of 228 animals was determined using microbiological tissue culture as a reference test. The diagnostic performance (sensitivity, specificity, likelihood ratios and predictive values) of the bPTB-ELISA on blood serum samples, taking into account the bPTB animal-level prevalence of the area and the bTB status of the animals, was evaluated. RESULTS: A sensitivity of 40.7% (95% confidence interval [CI]: 27.5%-53.9%) and a specificity of 94.7% (95% CI: 91.4%-98.0%) were obtained for bPTB-ELISA in all animals. A bPTB-ELISA-positive animal would have a post-test probability of 70% or more of being infected in areas with a bPTB prevalence of 23% or more. A negative bPTB-ELISA result, in areas with a bPTB prevalence of 41% or less, would rule out the disease with more than 70% certainty. In bTB-positive animals, sensitivity increased (94.4% [95% CI: 81.4%-100%] vs. 25.1% [95% CI: 11.8%-38.4%]) and specificity decreased (82.6% [95% CI: 71.8%-93.4%] vs. 99.4% [95% CI: 98.0%-99.9%]). The bPTB-ELISA is a good tool to rule out bPTB co-infection in bTB-positive animals, while in bTB-negative animals, it allows confirmation of disease with more than 70% probability if disease prevalence is 6% or more. LIMITATIONS: The observed differences could be enhanced by the effect of frequent application of the intradermal tuberculin test, which was unknown in the animals studied. CONCLUSIONS: These results provide useful guidance for the application and interpretation of ELISA as a tool for bPTB disease control.

Optimization of real-time PCR protocols from lymph node bovine tissue for direct detection of Mycobacterium tuberculosis complex.

Bovine tuberculosis (bTB) is a zoonotic disease and a global health problem that is subjected to obligatory eradication programs in the European Union. Microbiological culture is an imperfect technique for bTB diagnosis. This study aims to compare and validate two DNA isolation protocols and three different specific DNA targets, IS6110, IS4, and mpb70, to confirm Mycobacterium tuberculosis complex (MTC) infection by real-time PCR directly from fresh tissue samples. Fresh lymph node samples were collected from 81 cattle carcasses at the slaughterhouse. A comparison of both extraction protocols was performed with IS6110-real-time PCR, showing an adjusted sensitivity (SE) of 78.34% and 95.9% for protocols 1 and 2, respectively, while the specificity (SP) was 100% in both cases. Afterward, the comparison between IS4 and mpb70 targets was performed from the samples extracted with protocol 2, obtaining an adjusted SE of 90.87% and 83.3%, respectively, and an SP of 100% in both cases. The positive likelihood ratio was ∞ for the three targets, and the negative likelihood ratio was 0.04, 0.091, and 0.16 for IS6110, IS4, and mpb70, respectively. Negative predictive values were ≥90%, ≥85%, and ≥80% for real-time PCR targeting IS6110, IS4, and mpb70, respectively, when the true prevalence is ≤60%, and the positive predictive value is 100% in any scenario of true prevalence. According to these results, the DNA extraction protocol 2 and real-time PCR targeting IS6110 or IS4 could be potential first-choice molecular assays to detect MTC directly in fresh bovine tissue samples. IMPORTANCE Bovine tuberculosis (bTB), a chronic infectious and zoonotic disease caused by Mycobacterium tuberculosis complex (MTC), is considered a neglected disease of global importance, causing a detrimental impact on public health, particularly in developing countries where tuberculosis remains a major health problem. However, debate around the efficacy of control measures is still an ongoing matter of concern, with poor diagnostic performance being considered one of the most relevant factors involved in the failure to eradicate the disease since many truly infected animals will be misclassified as bTB-free. This study highlights a DNA extraction protocol and real-time PCR targeting IS6110 or IS4 as potential first-choice molecular assays to detect MTC directly in fresh bovine tissue samples, providing rapid, highly sensitive, and specific diagnostic tools as an alternative to microbiology, which could take up to 3 months to complete, shortening the turnaround time for decision makers to be promptly informed.

Detection of Mycobacterium tuberculosis complex field infections in cattle using fecal volatile organic compound analysis through gas chromatography-ion mobility spectrometry combined with chemometrics.

Bovine tuberculosis is considered a re-emerging disease caused by different species from the Mycobacterium tuberculosis complex (MTC), important not only for the livestock sector but also for public health due to its zoonotic character. Despite the numerous efforts that have been carried out to improve the performance of the current antemortem diagnostic procedures, nowadays, they still pose several drawbacks, such as moderate to low sensitivity, highlighting the necessity to develop alternative and innovative tools to complement control and surveillance frameworks. Volatilome analysis is considered an innovative approach which has been widely employed in animal science, including animal health field and diagnosis, due to the useful and interesting information provided by volatile metabolites. Therefore, this study assesses the potential of gas chromatography coupled to ion mobility spectrometry (GC-IMS) to discriminate cattle naturally infected (field infections) by MTC from non-infected animals. Volatile organic compounds (VOCs) produced from feces were analyzed, employing the subsequent information through chemometrics. After the evaluation of variable importance for the projection of compounds, the final discriminant models achieved a robust performance in cross-validation, as well as high percentages of correct classification (>90%) and optimal data of sensitivity (91.66%) and specificity (99.99%) in external validation. The tentative identification of some VOCs revealed some coincidences with previous studies, although potential new compounds associated with the discrimination of infected and non-infected subjects were also addressed. These results provide strong evidence that a volatilome analysis of feces through GC-IMS coupled to chemometrics could become a valuable methodology to discriminate the infection by MTC in cattle. IMPORTANCE Bovine tuberculosis is endemic in many countries worldwide and poses important concerns for public health because of their zoonotic condition. However, current diagnostic techniques present several hurdles, such as low sensitivity and complexity, among others. In this regard, the development of new approaches to improve the diagnosis and control of this disease is considered crucial. Volatile organic compounds are small molecular mass metabolites which compose volatilome, whose analysis has been widely employed with success in different areas of animal science including animal health. The present study seeks to evaluate the combination of fecal volatilome analysis with chemometrics to detect field infections by bovine tuberculosis (Mycobacterium tuberculosis complex) in cattle. The good robust performance of discriminant models as well as the optimal data of sensitivity and specificity achieved highlight volatilome analysis as an innovative approach with huge potential.

Diagnostic performance of faecal and tissue multiplex qPCR IS900/F57 for the detection of Mycobacterium avium subspecies paratuberculosis in cattle.

Mycobacterium avium subspecies paratuberculosis (MAP) is responsible for bovine-paratuberculosis (bPTB), which causes high production losses in cattle. A cross-sectional study was conducted in 228 cattle to evaluate the validity and diagnostic utility of a multiplex real-time PCR (qPCR) on faecal and intestinal samples [ileocaecal valve (ICV) and ileocaecal lymph nodes (ICLN)], using intestinal tissue culture as a reference test. Based on the sensitivity, specificity, and likelihood ratios (LR) obtained, the diagnostic value of faecal qPCR for confirming MAP infection was moderate (sensitivity 50.3%, specificity 93.5%, positive LR 7.8), and low to rule it out (negative LR 0.5). In areas with a prevalence of >23% the credibility of positive results was higher than 70%. In the case of negative results, their credibility was higher than 90% in herds with an infection rate below 19%, so faecal qPCR would be very useful in these areas to certify the absence of infection. For post-mortem diagnosis, qPCR on ICV samples showed good diagnostic accuracy to confirm the disease (sensitivity 71.7%, specificity 93.3%, positive LR 10.8), with a credibility higher than 70% in animals from areas or herds with a prevalence of infection greater than or equal to 18%. The best strategy to rule out the disease was the parallel combination of both tissues (ICV + ICLN) (sensitivity 81.3%, specificity 89.5%, negative LR 0.2) with a credibility of over 95% in animals from areas with an infection prevalence of 0-20%. Faecal and tissues qPCR techniques can be used to monitor bPTB, the interpretation of results, according to epidemiological situation of the herd or area, are shown.

Impact of supplementation with dihydroxylated vitamin D3 on performance parameters and gut health in weaned Iberian piglets under indoor/outdoor conditions.

BACKGROUND: Vitamin D may improve innate antimicrobial response and the integrity of the intestinal mucosal barrier representing an alternative to antibiotics for improving pig health. Therefore, benefits of dietary supplementation with a product based on vitamin D3 metabolite-rich plant extracts were assessed in 252 purebred Iberian piglets for a period of 60 days. The study group received 1,25 dihydroxyvitamin D (1,25(OH)2D) (100 ppm) in the conventional feed, which already included vitamin D (2000 IU in the starter and 1000 IU in the adaptation diets, respectively). Average daily gain (ADG), feed conversion ratio (FCR) and coefficient of variation of body weight (CV-BW) were assessed along the study. Blood samples, from 18 animals of the study group and 14 animals of the control group, were collected at selected time points to determine white blood cell count, concentration of vitamin D3 and its metabolites, and IgA and IgG in serum. Histopathology, morphometry, and immunohistochemistry (IgA and FoxP3) from small intestine samples were performed on days 30 and 60 of the study from 3 animals per group and time point. RESULTS: The ADG (493 vs 444 g/day) and FCR (2.3 vs 3.02) showed an improved performance in the supplemented animals. Moreover, the lower CV-BW indicated a greater homogeneity in the treated batches (13.17 vs 26.23%). Furthermore, a mild increase of IgA and in the number of regulatory T cells in the small intestine were observed in treated pigs. CONCLUSIONS: These results highlight the benefits of this supplementation and encourage to develop further studies along other production stages.

The Role of Histopathology as a Complementary Diagnostic Tool in the Monitoring of Bovine Tuberculosis.

The diagnosis of bovine tuberculosis (bTB) is based on the single intradermal tuberculin test (SIT), interferon gamma, and compulsory slaughter of reactor animals. Culture and PCR from fresh tissue are regarded as gold standard techniques for post-mortem confirmation, with the former being time-consuming and presenting moderate to low sensitivity and the latter presenting promising results. Histopathology has the advantage to identify and categorize lesions in both reactor and non-reactor animals. Therefore, this study aims to highlight the role of histopathology in the systematic diagnosis of bTB to shorten the time to disclose positive animals. Blood (212) and lymph node (681) samples were collected for serological, bacteriological, and histopathological analyses from a total of 230 cattle subjected to the Spanish bTB eradication program. Seventy-one lymph nodes and 59 cattle yielded a positive result to bacteriology, with 59 lymph nodes and 48 cattle presenting a positive result in real-time PCR from fresh tissue. Roughly 19% (40/212) of sera samples gave a positive result to ELISA. Tuberculosis-like lesions (TBLs) were observed in 11.9% (81/681) of the lymph nodes and 30.9% (71/230) of cattle. Noteworthy, TBLs were evidenced in 18 out of 83 SIT- and real-time PCR and bacteriology negative animals, with 11/18 disclosing a positive result to Ziehl-Neelsen technique and two of them to ddPCR from paraffin blocks targeting IS6110. Six out of these 11 ZN+ corresponded with mesenteric LN and were confirmed positive to paratuberculosis. Histopathology yielded a sensitivity of 91.3% (CI95 83.2-99.4%) and a specificity of 84.4% (CI95 78.6-89.3%) with good agreement (κ = 0.626) when compared with real-time PCR. Our results confirm that histopathology allows a rapid confirmation of real-time PCR and bacteriology, emphasizing its contribution to bTB control and monitoring.

Porcine reproductive and respiratory syndrome virus impacts on gut microbiome in a strain virulence-dependent fashion.

Porcine reproductive and respiratory syndrome (PRRS) is a viral disease defined by reproductive problems, respiratory distress and a negative impact on growth rate and general condition. Virulent PRRS virus (PRRSV) strains have emerged in the last years with evident knowledge gaps in their impact on the host immune response. Thus, the present study examines the impact of acute PRRS virus (PRRSV) infection, with two strains of different virulence, on selected immune parameters and on the gut microbiota composition of infected pigs using 16S rRNA compositional sequencing. Pigs were infected with a low virulent (PRRS_3249) or a virulent (Lena) PRRSV-1 strain and euthanized at 1, 3, 6, 8 or 13 days post-inoculation (dpi). Faeces were collected from each animal at the necropsy time-point. Alpha and beta diversity analyses demonstrated that infection, particularly with the Lena strain, impacted the microbiome composition from 6 dpi onwards. Taxonomic differences revealed that infected pigs had higher abundance of Treponema and Methanobrevibacter (FDR < 0.05). Differences were more considerable for Lena- than for PRRS_3249-infected pigs, showing the impact of strain virulence in the intestinal changes. Lena-infected pigs had reduced abundancies of anaerobic commensals such as Roseburia, Anaerostipes, Butyricicoccus and Prevotella (P < 0.05). The depletion of these desirable commensals was significantly correlated to infection severity measured by viraemia, clinical signs, lung lesions and immune parameters (IL-6, IFN-γ and Hp serum levels). Altogether, the results from this study demonstrate the indirect impact of PRRSV infection on gut microbiome composition in a strain virulence-dependent fashion and its association with selected immune markers.

PRRSV-1 induced lung lesion is associated with an imbalance between costimulatory and coinhibitory immune checkpoints.

Porcine reproductive and respiratory syndrome virus (PRRSV) induces a dysregulation on the innate and adaptive immune responses. T-cell activation requires a proper interaction and precise balance between costimulatory and coinhibitory molecules, commonly known as immune checkpoints. This study aims to evaluate the expression of immune checkpoints in lung and tracheobronchial lymph node from piglets infected with two PRRSV-1 strains of different virulence during the early stage of infection. Seventy 4-week-old piglets were grouped into three experimental groups: (i) control, (ii) 3249-infected group (low virulent strain), and (iii) Lena-infected group (virulent strain) and were euthanized at 1, 3, 6, 8, and 13 days post-infection (dpi). Lung and tracheobronchial lymph node were collected to evaluate histopathological findings, PRRSV viral load and mRNA expression of costimulatory (CD28, CD226, TNFRSF9, SELL, ICOS, and CD40) and coinhibitory (CTLA4, TIGIT, PD1/PDL1, TIM3, LAG3, and IDO1) molecules through RT-qPCR. Our findings highlight a mild increase of costimulatory molecules together with an earlier and stronger up-regulation of coinhibitory molecules in both organs from PRRSV-1-infected animals, especially in the lung from virulent Lena-infected animals. The simultaneous expression of coinhibitory immune checkpoints could work in synergy to control and limit the inflammation-induced tissue damage. Further studies should be addressed to determine the role of these molecules in later stages of PRRSV infection.

Activation of T-bet, FOXP3, and EOMES in Target Organs From Piglets Infected With the Virulent PRRSV-1 Lena Strain.

Transcription factors (TFs) modulate genes involved in cell-type-specific proliferative and migratory properties, metabolic features, and effector functions. Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogen agents in the porcine industry; however, TFs have been poorly studied during the course of this disease. Therefore, we aimed to evaluate the expressions of the TFs T-bet, GATA3, FOXP3, and Eomesodermin (EOMES) in target organs (the lung, tracheobronchial lymph node, and thymus) and those of different effector cytokines (IFNG, TNFA, and IL10) and the Fas ligand (FASL) during the early phase of infection with PRRSV-1 strains of different virulence. Target organs from mock-, virulent Lena-, and low virulent 3249-infected animals humanely euthanized at 1, 3, 6, 8, and 13 days post-infection (dpi) were collected to analyze the PRRSV viral load, histopathological lesions, and relative quantification through reverse transcription quantitative PCR (RT-qPCR) of the TFs and cytokines. Animals belonging to both infected groups, but mainly those infected with the virulent Lena strain, showed upregulation of the TFs T-bet, EOMES, and FOXP3, together with an increase of the cytokine IFN-γ in target organs at the end of the study (approximately 2 weeks post-infection). These results are suggestive of a stronger polarization to Th1 cells and regulatory T cells (Tregs), but also CD4+ cytotoxic T lymphocytes (CTLs), effector CD8+ T cells, and γδT cells in virulent PRRSV-1-infected animals; however, their biological functionality should be the object of further studies.

Development of an antigen Enzyme-Linked AptaSorbent Assay (ELASA) for the detection of swine influenza virus in field samples.

Influenza viruses are highly variable pathogens that infect a wide range of mammalian and avian species. According to the internal conserved proteins (nucleoprotein: NP, and matrix proteins: M), these viruses are classified into type A, B, C, and D. Influenza A virus in swine is of significant importance to the industry since it is responsible for endemic infections that lead to high economic loses derived from poor weight gain, reproductive disorders, and the role it plays in Porcine Respiratory Disease Complex (PRDC). To date, swine influenza virus (SIV) diagnosis continues to be based in complex and expensive technologies such as RT-qPCR. In this study, we aimed to improve actual tools by the implementation of aptamers as capture molecules. First, three different aptamers have been selected using as target the recombinant NP of Influenza A virus expressed in insect cells. Then, these molecules have been used for the development of an Enzyme-Linked AptaSorbent Assay (ELASA) in combination with specific monoclonal antibodies for Influenza A detection. A total of 171 field samples (nasal swabs) have been evaluated with the newly developed assay obtaining a 79.7% and 98.1% sensitivity and specificity respectively, using real time RT-PCR as standard assay. These results suggest that the assay is a promising method that could be used for Influenza A detection in analysis laboratories facilitating surveillance labours.

Application of Volatilome Analysis to the Diagnosis of Mycobacteria Infection in Livestock.

Volatile organic compounds (VOCs) are small molecular mass metabolites which compose the volatilome, whose analysis has been widely employed in different areas. This innovative approach has emerged in research as a diagnostic alternative to different diseases in human and veterinary medicine, which still present constraints regarding analytical and diagnostic sensitivity. Such is the case of the infection by mycobacteria responsible for tuberculosis and paratuberculosis in livestock. Although eradication and control programs have been partly managed with success in many countries worldwide, the often low sensitivity of the current diagnostic techniques against Mycobacterium bovis (as well as other mycobacteria from Mycobacterium tuberculosis complex) and Mycobacterium avium subsp. paratuberculosis together with other hurdles such as low mycobacteria loads in samples, a tedious process of microbiological culture, inhibition by many variables, or intermittent shedding of the mycobacteria highlight the importance of evaluating new techniques that open different options and complement the diagnostic paradigm. In this sense, volatilome analysis stands as a potential option because it fulfills part of the mycobacterial diagnosis requirements. The aim of the present review is to compile the information related to the diagnosis of tuberculosis and paratuberculosis in livestock through the analysis of VOCs by using different biological matrices. The analytical techniques used for the evaluation of VOCs are discussed focusing on the advantages and drawbacks offered compared with the routine diagnostic tools. In addition, the differences described in the literature among in vivo and in vitro assays, natural and experimental infections, and the use of specific VOCs (targeted analysis) and complete VOC pattern (non-targeted analysis) are highlighted. This review emphasizes how this methodology could be useful in the problematic diagnosis of tuberculosis and paratuberculosis in livestock and poses challenges to be addressed in future research.

Up-Regulation of Immune Checkpoints in the Thymus of PRRSV-1-Infected Piglets in a Virulence-Dependent Fashion.

Virulent porcine reproductive and respiratory syndrome virus (PRRSV) strains, such as the Lena strain, have demonstrated a higher thymus tropism than low virulent strains. Virulent PRRSV strains lead to severe thymus atrophy, which could be related to marked immune dysregulation. Impairment of T-cell functions through immune checkpoints has been postulated as a strategy executed by PRRSV to subvert the immune response, however, its role in the thymus, a primary lymphoid organ, has not been studied yet. Therefore, the goal of this study was to evaluate the expression of selected immune checkpoints (PD1/PDL1, CTLA4, TIM3, LAG3, CD200R1 and IDO1) in the thymus of piglets infected with two different PRRSV-1 strains. Thymus samples from piglets infected with the low virulent 3249 strain, the virulent Lena strain and mock-infected were collected at 1, 3, 6, 8 and 13 days post-infection (dpi) to analyze PRRSV viral load, relative quantification and immunohistochemical staining of immune checkpoints. PD1/PDL1, CTLA4, TIM3, LAG3 and IDO1 immune checkpoints were significantly up-regulated in the thymus of PRRSV infected piglets, especially in those infected with the virulent Lena strain from 6 dpi onwards. This up-regulation was associated with disease progression, high viral load and cell death. Co-expression of these molecules can affect T-cell development, maturation and selection, negatively regulating the host immune response against PRRSV.

Real-Time PCR Validation for Mycobacterium tuberculosis Complex Detection Targeting IS6110 Directly From Bovine Lymph Nodes.

Rapid and accurate diagnostic tools, such as Real-Time PCR (qPCR), need to be implemented as a confirmatory test in the framework of bovine tuberculosis (bTB) surveillance and control programs, shortening the turnaround time to confirm bTB infection. The present study aimed to evaluate a direct qPCR from fresh tissue samples targeting the insertion sequence IS6110 using individually homogenized bovine lymph nodes compared with microbiological culture. Retropharyngeal, tracheobronchial, and mesenteric lymph nodes fresh tissue samples (n = 687) were collected from 230 different cattle carcasses at the slaughterhouse. Only 23 of the 230 examined animals showed tuberculosis-like lesions, with 62 of 230 considered as positive. Among these 62 animals, 61 resulted as culture-positive, whereas 48 were qPCR-positive. Thus, this qPCR targeting IS6110 showed an apparent diagnostic sensitivity and specificity values of 77.1% [95% confidence interval (CI): 66.5-87.6%] and 99.4% (95% CI: 98.3-100.6%), respectively, and a positive predictive value of 97.9% (95% CI: 93.9-102.0%) and negative predictive value of 92.3% (95% CI: 88.4-96.2%). Positive and negative likelihood ratios were 130.2 and 0.2, respectively, and the agreement between microbiological culture and this qPCR was almost perfect (κ = 0.82). These results highlight this qPCR targeting IS6110 as a suitable complementary method to confirm bTB in animals with either tuberculosis-like lesions or non-tuberculosis-like lesions, decreasing the number of samples subjected to microbiological culture and, hence, its overall associated costs and the turnaround time (under 48 h) to confirm bTB infection. Besides, sampling mesenteric lymph node, which is uncommonly sampled, together with tracheobronchial and retropharyngeal ones, is advisable during postmortem inspection in bTB surveillance programs at the slaughterhouse, especially in areas with a low bTB prevalence scenario.