Juan J. Quereda

Virulence and genotype-associated infectivity of interferon-treated macrophages by porcine reproductive and respiratory syndrome viruses.

The polarization into M1 and M2 macrophages (MΦ) is essential to understand MΦ function. Consequently, the aim of this study was to determine the impact of IFN-γ (M1), IL-4 (M2) and IFN-β activation of MΦ on the susceptibility to genotype 1 and 2 porcine reproductive respiratory syndrome (PRRS) virus (PRRSV) strains varying in virulence. To this end, monocyte-derived MΦ were generated by culture during 72h and polarization was induced for another 24h by addition of IFN-γ, IL-4 or IFN-β. MΦ were infected with a collection of PRRSV isolates belonging to genotype 1 and genotype 2. Undifferentiated and M2 MΦ were highly susceptible to all PRRSV isolates. In contrast, M1 and IFN-β activated MΦ were resistant to low pathogenic genotype 1 PRRSV but not or only partially to genotype 2 PRRSV strains. Interestingly, highly virulent PRRSV isolates of both genotypes showed particularly high levels of infection compared with the prototype viruses in both M1 and IFN-β-treated MΦ (P<0.05). This was seen at the level of nucleocapsid expression, viral titres and virus-induced cell death. In conclusion, by using IFN-γ and IFN-β stimulated MΦ it is possible to discriminate between PRRSV varying in genotype and virulence. Genotype 2 PRRSV strains are more efficient at escaping the intrinsic antiviral effects induced by type I and II IFNs. Our in vitro model will help to identify viral genetic elements responsible for virulence, an information important not only to understand PRRS pathogenesis but also for a rational vaccine design. Our results also suggest that monocyte-derived MΦ can be used as a PRRSV infection model instead of alveolar MΦ, avoiding the killing of pigs.

Use of real-time and classic polymerase chain reaction assays for the diagnosis of porcine tuberculosis in formalin-fixed, paraffin-embedded tissues.

The current study was carried out to set up a fast and specific technique for porcine tuberculosis diagnosis in formalin-fixed, paraffin-embedded tissues. A retrospective study was carried out using 54 samples fixed in 10% neutral buffered formalin from 29 slaughtered Iberian pigs. Most of the pigs showed tissue samples positive to immunohistochemical staining (70.4%), and mycobacteria were detected within or near the necrotic cores of the lesions. However, diagnosis by this technique was time-consuming and tedious because of the paucibacillar nature of porcine tuberculous lesions. Classic polymerase chain reaction (PCR) was unsuccessful in mycobacteria genome amplification in all of the examined samples; however, real-time PCR amplified the mycobacteria genome in 23 of 29 examined pigs, identifying the Mycobacterium tuberculosis complex in all but one, which amplified Mycobacterium avium complex. Moreover, when reamplification of the DNA was performed, classic PCR amplified the mycobacteria genome in all the examined pigs (29/29), identifying the M. tuberculosis complex in 28 of 29 studied pigs and M. avium complex in only 1 pig. Results of the current study point out that both real-time and classic PCR assays, with genome reamplification, represent sensitive, fast, and specific diagnostic tools for porcine tuberculosis in formalin-fixed, paraffin-embedded tissues.

Listeriolysin S Is a Streptolysin S-Like Virulence Factor That Targets Exclusively Prokaryotic Cells In Vivo.

ABSTRACT Streptolysin S (SLS)-like virulence factors from clinically relevant Gram-positive pathogens have been proposed to behave as potent cytotoxins, playing key roles in tissue infection. Listeriolysin S (LLS) is an SLS-like hemolysin/bacteriocin present among Listeria monocytogenes strains responsible for human listeriosis outbreaks. As LLS cytotoxic activity has been associated with virulence, we investigated the LLS-specific contribution to host tissue infection. Surprisingly, we first show that LLS causes only weak red blood cell (RBC) hemolysis in vitro and neither confers resistance to phagocytic killing nor favors survival of L. monocytogenes within the blood cells or in the extracellular space (in the plasma). We reveal that LLS does not elicit specific immune responses, is not cytotoxic for eukaryotic cells, and does not impact cell infection by L. monocytogenes. Using in vitro cell infection systems and a murine intravenous infection model, we actually demonstrate that LLS expression is undetectable during infection of cells and murine inner organs. Importantly, upon intravenous animal inoculation, L. monocytogenes is found in the gastrointestinal system, and only in this environment LLS expression is detected in vivo. Finally, we confirm that LLS production is associated with destruction of target bacteria. Our results demonstrate therefore that LLS does not contribute to L. monocytogenes tissue injury and virulence in inner host organs as previously reported. Moreover, we describe that LlsB, a putative posttranslational modification enzyme encoded in the LLS operon, is necessary for murine inner organ colonization. Overall, we demonstrate that LLS is the first SLS-like virulence factor targeting exclusively prokaryotic cells during in vivo infections. IMPORTANCE The most severe human listeriosis outbreaks are caused by L. monocytogenes strains harboring listeriolysin S (LLS), previously described as a cytotoxin that plays a critical role in host inner tissue infection. Cytotoxic activities have been proposed as a general mode of action for streptolysin S (SLS)-like toxins, including clostridiolysin S and LLS. We now challenge this dogma by demonstrating that LLS does not contribute to virulence in vivo once the intestinal barrier has been crossed. Importantly, we show that intravenous L. monocytogenes inoculation leads to bacterial translocation to the gastrointestinal system, where LLS is specifically expressed, targeting the host gut microbiota. Our study highlights the heterogeneous modes of action of SLS-like toxins, and we demonstrate for the first time a further level of complexity for SLS-like biosynthetic clusters as we reveal that the putative posttranslational modification enzyme LlsB is actually required for inner organ colonization, independently of the LLS activity. IMPORTANCE The most severe human listeriosis outbreaks are caused by L. monocytogenes strains harboring listeriolysin S (LLS), previously described as a cytotoxin that plays a critical role in host inner tissue infection. Cytotoxic activities have been proposed as a general mode of action for streptolysin S (SLS)-like toxins, including clostridiolysin S and LLS. We now challenge this dogma by demonstrating that LLS does not contribute to virulence in vivo once the intestinal barrier has been crossed. Importantly, we show that intravenous L. monocytogenes inoculation leads to bacterial translocation to the gastrointestinal system, where LLS is specifically expressed, targeting the host gut microbiota. Our study highlights the heterogeneous modes of action of SLS-like toxins, and we demonstrate for the first time a further level of complexity for SLS-like biosynthetic clusters as we reveal that the putative posttranslational modification enzyme LlsB is actually required for inner organ colonization, independently of the LLS activity.

Regulating Bacterial Virulence with RNA

Noncoding RNAs (ncRNAs) regulating virulence have been identified in most pathogens. This review discusses RNA-mediated mechanisms exploited by bacterial pathogens to successfully infect and colonize their hosts. It discusses the most representative RNA-mediated regulatory mechanisms employed by two intracellular [Listeria monocytogenes and Salmonella enterica serovar Typhimurium (S. Typhimurium)] and two extracellular (Vibrio cholerae and Staphylococcus aureus) bacterial pathogens. We review the RNA-mediated regulators (e.g., thermosensors, riboswitches, cis- and trans-encoded RNAs) used for adaptation to the specific niches colonized by these bacteria (intestine, blood, or the intracellular environment, for example) in the framework of the specific pathophysiological aspects of the diseases caused by these microorganisms. A critical discussion of the newest findings in the field of bacterial ncRNAs shows how examples in model pathogens could pave the way for the discovery of new mechanisms in other medically important bacterial pathogens.

Characterization of tuberculous lesions in naturally infected African buffalo (Syncerus caffer)

Tuberculosis pathology was studied on 19 African buffalo (Syncerus caffer) from a herd in the Hluhluwe-iMfolozi Park in South Africa. The animals tested positive with the comparative intradermal tuberculin test and were euthanized during a test-and-cull operation to decrease prevalence of bovine tuberculosis (bTB) in the park. The lymph nodes and lungs were examined grossly for presence of tuberculous lesions, which were scored on a 0–5 scale for macroscopic changes. The gross lesions were examined histologically and classified into grade I, II, III, or IV according to a grading system used for bTB lesions in domestic cattle. Macroscopic lesions were limited to the retropharyngeal, bronchial, and mediastinal lymph nodes and the lungs. The most frequently affected lymph nodes were the bronchial (in 16 animals) and mediastinal (in 11 animals). All four grades of microscopic lesions were observed, grade II lesions were the most frequent. Mycobacterium bovis was detected by PCR in 8 out of 19 animals, and acid-fast bacilli were seen in 7 out of 19 animals, together both techniques identified mycobacteria in 5 out of 19 animals. Lesions were paucibacillary, as acid-fast bacilli were only rarely observed. The absence of lesions in the mesenteric lymph nodes and the high frequency of lesions in respiratory tract associated lymph nodes suggest that the main route of M. bovis infection in African buffalo is by inhalation.

Listeriolysin S: A bacteriocin from epidemic Listeria monocytogenes strains that targets the gut microbiota

ABSTRACT Listeria monocytogenes is a Gram-positive food-borne pathogen that in humans may traverse the intestinal, placental and blood/brain barriers, causing gastroenteritis, abortions and meningitis. Crossing of these barriers is dependent on the bacterial ability to enter host cells, and several L. monocytogenes surface and secreted virulence factors are known to facilitate entry and the intracellular lifecycle. The study of L. monocytogenes strains associated to human listeriosis epidemics has revealed the presence of novel virulence factors. One such factor is Listeriolysin S, a thiazole/oxazole modified microcin that displays bactericidal activity and modifies the host microbiota during infection. Our recent results therefore highlight the interaction of L. monocytogenes with gut microbes as a crucial step in epidemic listeriosis. In this article, we will discuss novel implications for this family of toxins in the pathogenesis of diverse medically relevant microorganisms.

Porcine endogenous retrovirus copy number in different pig breeds is not related to genetic diversity.

The risk of zoonoses is a major obstacle to xenotransplantation. Porcine endogenous retrovirus (PERV) poses a potential risk of zoonotic infection, and its control is a prerequisite for the development of clinical xenotransplantation. The copy number of PERV varies among different breeds, and it has been suggested that the PERV integrations number is increased by inbreeding. The purpose of this study was (i) to examine the copy number of PERV in different Spanish pig breeds, Spanish wild boar and commercial cross‐bred pigs from five different farms and genetic background (CCP1–CCP5) and (ii) to investigate the correlation between PERV copy number and the genetic background of the pigs in order to improve the selection of pigs for xenotransplantation. PERV copy number was determined by quantitative, real‐time polymerase chain reactions. Thirty‐four microsatellite markers were genotyped to describe the genetic diversity within populations (observed and expected heterozygosities, Ho and He, respectively) and the inbreeding coefficient (F). Pearson′s correlation coefficient was used to determine the relationship between PERV copy number and Ho, He and F. The copy number of PERV among different pig breeds was estimated to range between three (CCP1) and 43 copies (Iberian Pig). Statistical differences were found among the studied populations concerning PERV copy number. No correlation was found between the PERV copy number and the heterozygosity (calculated at an individual level or at a population level) or the inbreeding coefficient of each population. Our data suggest that pigs inbreeding does not increase PERV copy number and support the idea that careful selection of pigs for organ donation with reduced PERV copy number will minimize the risk of retrovirus transmission to the human receptor.

Are Veterinary Students in Favour of Xenotransplantation? Comparative Opinion Study in a Brazilian and a Spanish University

BACKGROUND The shortage of organs has made it necessary to search for new alternatives such as xenotransplantation. However, the use of animal organs could be opposed by society and the personnel involved in its implementation. This study aimed to analyze the attitude of veterinary degree students in a Brazilian university towards xenotransplantation, to determine factors that affect its acceptance, and to compare the attitudes among a control group of veterinary degree students in a Spanish university. METHODS Of the 422 students registered for a veterinary course from 2010 to 2011, 374 were surveyed with a questionnaire completion rate of 89%. Attitudes were evaluated using a validated questionnaire that was self-administered administered anonymously. The process was coordinated by an independent health care worker. We applied the student t and the chi-squared-tests for statistical analysis. RESULTS If xenotransplantation was confirmed as a clinical reality, 90% (n = 338) of Brazilian students would accept the use of a xenotransplanted organ; 94% (n = 350), tissue; and 97% (n = 360), cell xenotransplantation. Attitudes toward xenotransplantation were not determined by the academic year, any psychosocial variable, or attitudes toward deceased human organ donation (P = .167). However, the attitudes would be affected by a belief that the transplanted animal organ would not change anything (P = .001). Interaction with other people was also related to more favorable attitudes (P = .015). Subjects who expressed a more favorable attitude tended to more readily accept cell (P = .000) or tissue xenotransplantation (P = .000). In Spain (control group), the results were similar: 91% (n = 436) would accept a xenotransplantation; 95% (n = 457) tissue; and 97% (n = 467), cell xenotransplantation. Also, this attitude was not affected by the academic year, any psychosocial variable, or attitude toward organ donation (P = .779). CONCLUSION Both Brazilian and Spanish veterinary students had favorable attitudes toward xenotransplantation.

Acute phase proteins as a tool for differential diagnosis of wasting diseases in growing pigs

The concentrations of haptoglobin (Hp), C–reactive protein (CRP) and serum amyloid A (SAA) were measured in wasted pigs, first to evaluate their usefulness in the diagnosis of infectious, wasting diseases in pigs, and second, to evaluate whether their concentrations can distinguish the lymphoid depletion score in the lymph tissues of wasted affected pigs. Fifty–three wasted pigs and seven specific pathogen free (SPF) pigs were postmortem examined. Gross lesions were evaluated and samples for histopathological, immunohistochemical, molecular biology and microbiological analysis were taken. Thirty–one pigs were diagnosed as postweaning multisystemic wasting syndrome (PMWS) and 22 as porcine respiratory disease complex (PRDC). Lymphoid depletion degree in lymph tissues of PMWS and PRDC affected pigs was determined. Serum Hp was significantly higher in pigs with PRDC in comparison with the PMWS affected pigs. Serum CRP concentration was significantly lower in pigs with PRDC than in PMWS affected pigs (P<0.001). CRP and SAA levels increased with the lymphoid depletion score, presenting statistical differences between pigs with no depletion and pigs with low, moderate or severe lymphoid depletion (P<0.05, P<0.05 and P<0.001 for CRP and P<0.01, P<0.01 and P<0.01 for SAA, respectively). Hp was higher in pigs with no or low depletion compared with the pigs suffering severe lymphoid depletion (P<0.001 and P<0.05, respectively).

Validation of a Quantitative Polymerase Chain Reaction Method for Human Alu Gene Detection in Microchimeric Pigs Used as Donors for Xenotransplantation

This work was undertaken to evaluate whether a real-time quantitative polymerase chain reaction (qPCR) is as an adequate method for detection and quantification of human-specific DNA elements (Alu gene) in tissues and blood samples of pigs in which human stem cells were engrafted. Real-time qPCR quantification was performed with the use of previously described primers. The human DNA was mixed with different quantities of porcine DNA. The primer concentration and specificity, the qPCR efficiency, the quantification variations due to different porcine DNA concentrations, and the dissociation curve produced by the assay were evaluated. The qPCR proved to be specific, robust, with a reproducible and specific bimodal melting curve. High porcine DNA concentration produced subquantification, especially with low human DNA quantity. However, the assay proved to be useful for the detection of chimeric piglets produced by human cells injected in utero, because the effect caused by the porcine DNA interference was corrected in quantification of human DNA from piglets.