Transcriptomic Profile of Canine DH82 Macrophages Infected by Leishmania infantum Promastigotes with Different Virulence Behavior.

Zoonotic visceral leishmaniosis caused by Leishmania infantum is an endemic disease in the Mediterranean Basin affecting mainly humans and dogs, the main reservoir. The leishmaniosis outbreak declared in the Community of Madrid (Spain) led to a significant increase in human disease incidence without enhancing canine leishmaniosis prevalence, suggesting a better adaptation of the outbreak's isolates by other host species. One of the isolates obtained in the focus, IPER/ES/2012/BOS1FL1 (BOS1FL1), has previously demonstrated a different phenotype than the reference strain MCAN/ES/1996/BCN150 (BCN150), characterized by a lower infectivity when interacting with canine macrophages. Nevertheless, not enough changes in the cell defensive response were found to support their different behavior. Thus, we decided to investigate the molecular mechanisms involved in the interaction of both parasites with DH82 canine macrophages by studying their transcriptomic profiles developed after infection using RNA sequencing. The results showed a common regulation induced by both parasites in the phosphoinositide-3-kinase-protein kinase B/Akt and NOD-like receptor signaling pathways. However, other pathways, such as phagocytosis and signal transduction, including tumor necrosis factor, mitogen-activated kinases and nuclear factor-κB, were only regulated after infection with BOS1FL1. These differences could contribute to the reduced infection ability of the outbreak isolates in canine cells. Our results open a new avenue to investigate the true role of adaptation of L. infantum isolates in their interaction with their different hosts.

Streptococcus ovuberis sp. nov., isolated from a subcutaneous abscess in the udder of a sheep.

One unidentified, Gram-stain-positive, catalase-negative coccus-shaped organism was recovered from a subcutaneous abscess of the udder of a sheep and subjected to a polyphasic taxonomic analysis. Based on cellular morphology and biochemical criteria, the isolate was tentatively assigned to the genus Streptococcus, although the organism did not appear to match any recognized species. 16S rRNA gene sequence comparison studies confirmed its identification as a member of the genus Streptococcus and showed that the nearest phylogenetic relatives of the unknown coccus corresponded to Streptococcus moroccensis and Streptococcus cameli (95.9 % 16S rRNA gene sequence similarity). The sodA sequence analysis showed less than 89.3 % sequence similarity with the currently recognized species of the genus Streptococcus. The novel bacterial isolate was distinguished from close relatives of the genus Streptococcusby using biochemical tests. A mass spectrometry profile was also obtained for the novel isolate using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Based on both phenotypic and phylogenetic findings, it is proposed that the unknown bacterium be classified as a representative of a novel species of the genus Streptococcus, Streptococcus ovuberis sp. nov. The type strain of Streptococcus ovuberissp. nov. is VB15-00779T (=CECT 9179T=CCUG 69612T).

Detection and Antimicrobial Resistance of Enterobacteriaceae other than Escherichia Coli in Raccoons from the Madrid Region of Spain.

Introduction: Raccoons are an invasive alien species widely distributed in the Madrid region of Spain. These animals can carry a variety of enteric bacteria with associated antimicrobial resistance, which can infect humans and livestock. However, to our knowledge, the presence of non-E. coli Enterobacteriaceae in raccoons has not been previously studied. Material and Methods: We conducted a study to examine the species distribution of Enterobacteriaceae isolates other than E. coli, as well as their antimicrobial resistance, in the faeces of 83 raccoons in the Madrid region. Results: We detected 12 Enterobacteriaceae isolates other than E. coli belonging to seven different species: Citrobacter freundii (1 isolate), Citrobacter gillenii (3 isolates), Citrobacter murliniae (1 isolate), Citrobacter portucalensis (2 isolates), Enterobacter hormaechei subsp. hoffmannii (1 isolate), Hafnia paralvei (2 isolates) and Raoultella ornithinolytica (2 isolates). These isolates were found in 7 of the 83 (8.4%) animals studied. To our knowledge, this study is the first report of the presence of non-E. coli Enterobacteriaceae in raccoon faeces. All isolates but one were resistant to at least one of the 14 antimicrobials tested. Resistance to ampicillin (83.3%), amoxicillinclavulanic acid (50%) and cefoxitin (33.3%) was the most frequent. Conclusion: Our study indicates that raccoons are a potential source of infection with Enterobacteriaceae other than E. coli for humans and livestock in the Madrid region.

Properties of virulence emergence of Leishmania infantum isolates from Phlebotomus perniciosus collected during the human leishmaniosis outbreak in Madrid, Spain. Hepatic histopathology and immunological parameters as virulence markers in the mouse model.

Recent anthropic activity related to the construction of the Bosquesur Green Park in a large urban setting in Madrid (Spain) has resulted in the largest reported community outbreak of human leishmaniosis in Europe. Previous phylogenetic and molecular-typing studies of parasite isolates have implicated the Leishmania infantum ITS-Lombardi genotype in this outbreak. In an unusual scenario, visceral leishmaniosis (VL) is affecting a significant number of individuals, suggesting that an increase in parasite virulence has occurred. In this work, using an in vivo BALB/c model of VL, we aimed to investigate the properties of emergent virulence of the L. infantum POL2FL7 and BOS1FL1 isolates obtained from Phlebotomus perniciosus collected in the outbreak area and compare them with those of the well-characterized strain BCN150 MON-1 isolated from a dog. The P. perniciosus specimens were collected during an entomological survey conducted in the transmission season of 2012. We observed a range of virulence phenotypes from moderately to highly aggressive after 5 weeks of infection. IV challenge of mice with outbreak isolates from sand flies induced higher splenic and liver parasite burdens, higher serological titres of specific anti-Leishmania antibodies and impaired capacities to control infection, as revealed by the arginine metabolism and low ratios of Th1/Th2 cytokine profiles analysed, compared with the corresponding measures evaluated in mice infected with the BCN150 strain. The BOS1FL1 isolate showed the highest degree of virulence among the isolates, superior to that of POL2FL7, as evidenced by the analysed biomarkers and the histopathological severity of liver lesions. These results provide insight into how L. infantum isolates from sand flies collected in the outbreak area have been able to affect not only immunosuppressed patients but also middle-aged people with normal immunocompetence in the largest human VL outbreak in Europe.

A further investigation of the leishmaniosis outbreak in Madrid (Spain): low-infectivity phenotype of the Leishmania infantum BOS1FL1 isolate to establish infection in canine cells.

Human leishmaniosis caused by Leishmania infantum is a zoonotic disease, with dogs as the main reservoir in Mediterranean Basin countries. The largest European outbreak of human leishmaniosis declared in the southwestern Madrid region (Spain) is characterized by unusual epidemiological and clinical features, such as the emergence of new wild reservoirs (hares and rabbits), whereas the seroprevalence, infection, and severity of canine leishmaniosis have not substantially changed since the first studies conducted in Madrid before the outbreak. Previous studies reported that L. infantum isolates from the Madrid leishmaniosis focus displayed elevated virulence in in vivo models of infection and increased infectivity in murine target cells. With the aim of studying whether changes in the host-parasite interaction and virulence profile have developed, we first assessed the behaviour of one circulating isolate of the outbreak, IPER/ES/2012/BOS1FL1 (BOS1FL1), compared to that of a well-characterized strain from canine leishmaniosis, MCAN/ES/1996/BCN150 (BCN150), in terms of infection capacity (percentage of infected cells, representing infectivity, and number of amastigotes per infected cell, representing the intensity of infection) in canine monocytes and macrophages. BCN150 displayed significantly higher infectivity (76.82 ±â€¯4.40 vs 38.58 ±â€¯2.19; P <  0.0001) and intensity of infection (3.64 ± 0.13 vs 1.83 ±â€¯0.12; P <  0.0001) than BOS1FL1 when interacting with canine cells. Our ROS induction results did not differ significantly between the two isolates or with the responses previously described for other L. infantum isolates. Paradoxically, increased resilience to hydrogen peroxide exposure was observed for BOS1FL1 (% viability 40.62 ±â€¯5.54 vs 26.37 ±â€¯2.93; P = 0.039). Finally, we demonstrated that a decreased intracellular load of BOS1FL1 was associated with increased IFN-γ (261.21 ±â€¯26.29 vs 69.80 ±â€¯9.02; P = 0.0151) and decreased IL-10 production (165.06 ±â€¯23.87 vs 264.41 ±â€¯30.58; P = 0.0002). In this study, we provide the first detailed insight into the differences between the isolate BOS1FL1 from the outbreak in Madrid and the well-characterized strain BCN150 MON-1 obtained from a dog in their response to interacting with canine cells. However, further studies are necessary to shed light on the immune mechanisms resulting in BOS1FL1 exhibiting less virulent behaviour in canine cells than in cells derived from other host species.

Salmonella enterica serovar Choleraesuis derivatives harbouring deletions in rpoS and phoP regulatory genes are attenuated in pigs, and survive and multiply in porcine intestinal macrophages and fibroblasts, respectively.

Live attenuated Salmonella enterica strains have been extensively studied as potential vectors for the oral delivery of heterologous antigens. Due to its ability to target immune cells, its specific mechanism for crossing the intestinal barrier, and its swine-restricted tropism, S. enterica subspecies enterica serovar Choleraesuis (S. Choleraesuis) has attracted a great deal of interest for the production of bacterial-based oral carriers specifically adapted to swine. In this study, two mutants of S. Choleraesuis were constructed and their attenuation and intracellular fate analysed with the purpose of engineering new attenuated live strains with improved properties as oral vaccine carriers. Those strains harboured a specific deletion either within the phoP or rpoS genes, which encode virulence-related regulators in S. Typhimurium. In comparison to the wild-type parental S. Choleraesuis, the mutant strains, especially DeltaphoP, were extremely low in virulence in the murine model and in the natural host, the pig. Moreover, when compared with a commercial live vaccine strain, SC-54, the two mutants showed a higher level of attenuation in mice and DeltaphoP also in pigs. In addition, DeltarpoS and DeltaphoP presented a proliferation and survival phenotype within swine intestinal primary fibroblast and macrophage cell cultures, respectively. Collectively, the present results indicate that the DeltarpoS and DeltaphoP strains of S. Choleraesuis gather adequate features to be potential candidates for vaccine vectors for the specific delivery of heterologous antigens adapted to pigs.