José María Sánchez-Carvajal

While several African swine fever virus (ASFV)–encoded proteins potently interfere with the cGAS-STING (cyclic GMP-AMP synthetase–stimulator of interferon genes) pathway at different levels to suppress interferon (IFN) type I production in infected macrophages, systemic IFN-α is induced during the early stages of AFSV infection in pigs. The present study elucidates a mechanism by which such responses can be triggered, at least in vitro. We demonstrate that infection of monocyte-derived macrophages (MDMs) by ASFV genotype 2 strains is highly efficient but immunologically silent with respect to IFN type I, IFN-stimulated gene induction, and tumor necrosis factor production. Additionally, ASFV does not directly activate plasmacytoid dendritic cells (pDCs). However, coculturing pDCs with ASFV-infected MDMs results in a strong pDC response characterized by high levels of IFN-α and tumor necrosis factor. IFN type I, in turn, promoted interleukin-1 receptor antagonist production by macrophages. Similar to the sensing of infected cells by other viruses, pDC activation required integrin-mediated cognate interactions with ASFV-infected MDMs to form an interferogenic synapse. Inhibitor studies indicated that the activation of pDCs requires the STING pathway and the formation of gap junctions. While IL-4–polarized macrophages showed increased susceptibility, IFN-γ–polarized ASFV-infected macrophages induced higher pDC activation. Pretreatment of pDCs with IFN-β and IFN-γ also enhanced IFN-α production in response to ASFV-infected macrophages, highlighting the influence of the immunological microenvironment. These findings suggest that the IFN-α detected during ASFV infection in pigs may be a result of pDC sensing ASFV-infected macrophages.

The non-specific protective effects offered by the concept of “innate immune memory” might represent a promising strategy to tackle early-life threatening infections. Here we tested the potential of an in vitro selected β-glucan in inducing trained immunity using an in vivo porcine model. We assessed the leukocyte transcriptome using blood transcriptomic module (BTM), proinflammatory cytokines, and clinical scoring after a first “training” and a second “stimulation” phase. The possible induction of innate immune memory was tested during a “stimulation” by an LPS-adjuvanted Mycoplasma hyopneumoniae vaccine (Hyogen^®) one day after weaning. Following the “training”, no major group differences were found, with the exception of a plasma TNF that was only induced by Adj and Adj_BG treatment. After vaccination, all groups developed similar antibody responses. A significant induction of plasma TNF and IL-1β was found in groups that received Adj and Adj_BG. However, following vaccination, the expected early innate BTMs were only induced by the PBS group. In conclusion, the adjuvant alone, adjuvant-formulated β-glucan, or orally applied β-glucan were unable to enhance innate immune reactivity but rather appeared to promote innate immune tolerance. Such an immune status could have both positive and negative implications during this phase of the piglet’s life.

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.

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.

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 IS 6110 -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 IS 6110 , 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 IS 6110 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.

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.

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 (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.