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Coinfection can markedly alter the response to a pathogen, thereby changing its clinical presentation. For example, non-typhoidal Salmonella (NTS) serotypes are associated with gastroenteritis in immunocompetent individuals. In contrast, individuals with severe pediatric malaria can develop bacteremic infections with NTS, during which symptoms of gastroenteritis are commonly absent. Here we report that, in both a ligated ileal loop model and a mouse colitis model, malaria parasites caused a global suppression of gut inflammatory responses and blunted the neutrophil influx that is characteristic of NTS infection. Further, malaria parasite infection led to increased recovery of Salmonella enterica serotype Typhimurium from the draining mesenteric lymph node (MLN) of mice. In the mouse colitis model, blunted intestinal inflammation during NTS infection was independent of anemia but instead required parasite-induced synthesis of interleukin (IL)-10. Blocking of IL-10 in coinfected mice reduced dissemination of S. Typhimurium to the MLN, suggesting that induction of IL-10 contributes to development of disseminated infection. Thus IL-10 produced during the immune response to malaria in this model contributes to suppression of mucosal inflammatory responses to invasive NTS, which may contribute to differences in the clinical presentation of NTS infection in the setting of malaria.

Gamma interferon (IFN-γ) is an important driver of intestinal inflammation during colitis caused by Salmonella enterica serovar Typhimurium. Here we used the mouse colitis model to investigate the cellular sources of IFN-γ in the cecal mucosa during the acute phase of an S . Typhimurium infection. While IFN-γ staining was detected in T cells, NK cells, and inflammatory monocytes at 2 days after infection, the majority of IFN-γ-positive cells in the cecal mucosa were neutrophils. Furthermore, neutrophil depletion blunted mucosal Ifng expression and reduced the severity of intestinal lesions during S . Typhimurium infection. We conclude that neutrophils are a prominent cellular source of IFN-γ during the innate phase of S . Typhimurium-induced colitis.

E. coli kNOws How to Win The harmonious existence among the various microbial inhabitants of the gut is critical for good health. However, inflammation from injury or inflammatory bowel disease, can disrupt this balance and lead to the outgrowth of particular bacteria. The outgrowth of members of the Enterobacteriaceae family, which includes Escherichia coli , is often observed. Because E. coli are facultative rather an obligate anaerobes, Winter et al. (p. 708 ) postulated that they may be able to use by-products of reactive oxygen and nitrogen species, which are produced during inflammation, for anaerobic respiration, thereby edging out other fermenting bacteria. Indeed, in two mouse models of colitis and in a model of intestinal injury, various E. coli strains were able to use host-derived nitrate as an energy source and outcompete mutant strains unable to do this.

Brucella are facultative intracellular bacteria that cause chronic infections by limiting innate immune recognition. It is currently unknown whether Brucella FliC flagellin, the monomeric subunit of flagellar filament, is sensed by the host during infection. Here, we used two mutants of Brucella melitensis, either lacking or overexpressing flagellin to show that FliC hinders bacterial replication in vivo. The use of cells and mice genetically deficient for different components of inflammasomes suggested that FliC was a target of the cytosolic innate immune receptor NLRC4 in vivo but not in macrophages in vitro where the response to FliC was nevertheless dependent on the cytosolic adaptor ASC, therefore suggesting a new pathway of cytosolic flagellin sensing. However, our work also suggested that the lack of TLR5 activity of Brucella flagellin and the regulation of its synthesis and/or delivery into host cells are both part of the stealthy strategy of Brucella towards the innate immune system. Nevertheless, since a flagellin-deficient mutant of B. melitensis was found to cause histologically demonstrable injuries in the spleen of infected mice, we suggested that recognition of FliC plays a role in the immunologic standoff between Brucella and its host, which is characterized by a persistent infection with limited inflammatory pathology.

Brucella ovis is considered the most important infectious cause of reproductive disorders in sheep. The disease is characterized by epididymitis, subfertility and infertility in rams. B. ovis occasionally results in abortion in ewes, as well. The aim of this study was to evaluate kinetic changes in the reproductive organs of rams experimentally infected with B. ovis. Nine rams were experimentally inoculated intrapreputially with 2mL of a suspension containing 1.2×109CFU (colony-forming units)/mL of B. ovis (strain ATCC25840). In addition, 50μL of a suspension containing 1.2×1010CFU/mL of the same B. ovis strain was inoculated into each conjunctival sac, resulting in 3.6×109CFU total per ram. Six of nine infected rams had developed clinical changes in the tail of the epididymis at 30 days post-infection (dpi), but these changes regressed in 50% of these rams. Ultrasound demonstrated an increase in the area of the tail of the epididymis (P

Conventional wisdom holds that microbes support their growth in vertebrate hosts by exploiting a large variety of nutrients. We show here that use of a specific nutrient (ethanolamine) confers a marked growth advantage on Salmonella enterica serovar Typhimurium ( S. Typhimurium) in the lumen of the inflamed intestine. In the anaerobic environment of the gut, ethanolamine supports little or no growth by fermentation. However, S. Typhimurium is able to use this carbon source by inducing the gut to produce a respiratory electron acceptor (tetrathionate), which supports anaerobic growth on ethanolamine. The gut normally converts ambient hydrogen sulfide to thiosulfate, which it then oxidizes further to tetrathionate during inflammation. Evidence is provided that S. Typhimurium's growth advantage in an inflamed gut is because of its ability to respire ethanolamine, which is released from host tissue, but is not utilizable by competing bacteria. By inducing intestinal inflammation, S. Typhimurium sidesteps nutritional competition and gains the ability to use an abundant simple substrate, ethanolamine, which is provided by the host.

The goal of this study was to morphologically characterize a ligated ileal loop model of Salmonella enterica serotype Typhimurium infection in rhesus macaques ( Macaca mulatta) and to verify the occurrence of Salmonella-induced cell death in vivo. Eight adult healthy male rhesus macaques were used for ligated ileal loop surgery. Four macaques had been intravenously inoculated with simian immunodeficiency virus (SIV) mac251. Ileal ligated loops were inoculated with wild-type (WT) S. Typhimurium strain IR715 (ATCC14028 nalr), an isogenic noninvasive mutant strain (ATCC14028 nalr Δ sipAΔ sopABDE2), or sterile Luria Bertani broth. Loops were surgically removed at 2, 5, and 8 hours post-inoculation (hpi). Intestinal samples were processed for histopathology, immunohistochemistry for detecting Salmonella, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), and transmission electron microscopy. Combined histopathology scores were similar between SIV-infected and control macaques. As expected, the invasion-deficient mutant was less pathogenic than WT S. Typhimurium. Neutrophil infiltrate in the intestinal mucosa correlated with bacterial loads ( r = 0.7148; P < .0001) and fluid accumulation ( r = 0.6019; P < .0001) in the lumen of the intestinal loops. Immunolabeled WT S. Typhimurium was observed in the epithelium and lamina propria at the tip of the villi at 2 hpi, progressing toward deeper lamina propria at 5–8 hpi. Most TUNEL-positive cells localized to the lamina propria, and some had morphological features of macrophages. Ultrastructurally, bacteria were observed intracellularly in the lamina propria as well as within apoptotic bodies. This study provides morphological evidence of Salmonella-induced cell death in vivo in a relevant nonhuman primate model.

Brucellosis is one of the most important zoonotic diseases worldwide, resulting in serious economic losses and public health issues. It is caused by intracellular Gram-negative bacteria of the genus Brucella, which are responsible for a debilitating disease in humans and a chronic infection in domestic animals. The present article considers the pathogenesis of Brucella spp., with the goal to cover clinical aspects of the disease in the different mammalian species along with the target cells used by this pathogen to survive inside the host. Additionally, important molecular mechanisms used by Brucella to invade and persist inside the hosts target cells are also discussed.

Bovine brucellosis is one of the most important zoonotic diseases worldwide, and is of particular significance in developing countries. The disease, which results in serious economic losses due to late term abortion, stillborn and weakly calves, is caused by Gram negative coccobacilli bacteria of the genus Brucella. Lesions consist of necrotic placentitis and interstitial mastitis in pregnant cows, and fibrinous pleuritis with interstitial pneumonia in aborted fetuses and newborn calves. This article considers the pathogenesis of Brucella abortus and reviews the ability of the pathogen to invade phagocytic and non-phagocytic host cells, resist the acidified intraphagosomal environment, and inhibit phagosome-lysosome fusion. Significant aspects of innate and adaptive immunity against brucellosis are also discussed.

Infection with bacteria of the genus Brucella results in major economic and political impact by causing reproductive diseases in a significant number of domestic animal species. Moreover, it has a great social significance, since many species are capable of causing human infection, with severe consequences. Dissemination of knowledge on a specific disease is an essential step for its control. Considering that brucellosis is still the most prevalent zoonosis in the world, information about taxonomy, clinical signs in domestic animals and humans are crucial for attempting to reduce the prevalence of this disease. The recent isolation and characterization of non-classical species of Brucella indicates that a lot remains to be discovered about this genus. Nevertheless, due to the social-economic importance of brucellosis, this review aims to clarify points related to taxonomy of the genus and describe the clinical relevance of infection in humans and domestic animals.

Brucella abortus is an intracellular pathogen that persists within phagocytic cells of the reticuloendothelial system. To identify in vivo interactions between B. abortus and the host that lead to persistent infection, we studied the persistence of B. abortus and an isogenic virB mutant deficient in the VirB type IV secretion system (T4SS) in knockout mice. In contrast to control mice, mice lacking B cells ( Igh6 −/− ) were permissive for infection with the attenuated virB mutant. To determine the basis for this phenotype, we characterized immune functions of Igh6 −/− mice in the context of B. abortus infection. Igh6 −/− mice had greater numbers of extracellular bacteria in the spleen and increased early expression of proinflammatory cytokines during B. abortus infection. Further, a virB mutant, despite its wild-type level of survival, failed to elicit microgranuloma formation in the spleens of Igh6 −/− mice, suggesting a requirement for the T4SS to elicit this pathological change. Passive transfer of immunoglobulin G from naïve mice restored the ability of Igh6 −/− mice to control the persistence of the virB mutant by a complement-independent mechanism. Further, adoptive transfer of CD11b + cells from C57BL/6 mice to Igh6 −/− mice restored the ability of the knockout mice to limit the replication of the virB mutant in the spleen, suggesting that the Igh6 − / − mutation affects phagocyte function and that phagocyte function can be restored by natural antibody.

This report describes a pathological, immunohistochemical and bacteriological study of 42 cows and their progeny (aborted fetuses, weak premature calves, and healthy full-term calves) infected at 6-7 months of gestation by conjunctival inoculation with Brucella abortus. Samples were collected at necropsy within 48 h of abortion or parturition. The most significant lesions were necrotizing and suppurative placentitis and lymphohistiocytic mastitis in cows, and fibrinous pleuritis, fibrinous pericarditis and bronchopneumonia in aborted fetuses. B. abortus was isolated more frequently from milk samples than from mammary tissues, and milk samples from cows with mastitis were often infected. Organisms were often demonstrated immunohistochemically and by culture in tissues showing moderate to severe histological changes.

Recent reports indicate that Leishmania chagasi has tropism to the male canine genital system, which is associated with shedding of the organism in the semen, supporting the hypothesis of venereal transmission. The aim of this study was to describe the lesions and assess parasite load in the genital system of bitches with canine visceral leishmaniasis (CanL). Symptomatic (n = 5) and asymptomatic (n = 5) bitches seropositive for CanL were randomly selected at the Center for Zoonosis Control (Belo Horizonte, State of Minas Gerais, Brazil). Five serologically negative, healthy, adult bitches also from the CZC were used as controls. Samples from genital organs (vulva, vagina, cervix, uterine body, uterine horns, uterine tubes, and ovaries), liver, and spleen were histologically evaluated and processed for immunodetection of Leishmania sp., and PCR. The most significant histological change was a mild to moderate vulvar dermatitis, characterized by a histio-plasma-lymphocytic infiltrate. This change was detected in all asymptomatic, four symptomatic, and three uninfected control bitches. In one symptomatic and one asymptomatic bitch intracytoplasmic amastigotes were observed within macrophages in the inflammatory infiltrate. Samples from all the segments of the genital tract were positive in at least one infected animal, in the absence of detectable amastigotes in the tissue. These findings support the notion that L. chagasi does not have genital tropism in the bitch, which is in contrast to our previous findings in naturally infected male intact dogs.

Brucella ovis is a major cause of epididymitis in sexually mature rams, resulting in subfertility, infertility, and economic losses for the sheep industry worldwide. The aim of this study was to develop an indirect ELISA (iELISA) using recombinant proteins, namely rBoP59 and rBP26, as antigens for serological diagnosis of B. ovisinfection. The BoP59 and BP26 recombinant proteins were expressed in E. coli and purified by affinity chromatography. Antigenicity was tested by Western blot and iELISA. Standardization of iELISA was performed with 500ng and 1µg BoP59 and rBP26 per well, testing serum from uninfected and experimentally infected rams. rBP26 was effective in distinguishing positive from negative rams. The rBP26 iELISA developed in this study is the first to use a completely purified rBP26 as antigen resulting in high sensitivity (100%) and specificity (90.2%), and an overall accuracy equal to 1.0. Brucella ovis é uma das principais causas de epididimite em carneiros sexualmente maduros, resultando em subfertilidade e infertilidade e consequentes perdas econômicas para a ovinocultura em todo o mundo. O objetivo deste estudo foi desenvolver ELISA indireto (ELISAi), utilizando como antígeno proteínas recombinantes BoP59r e BP26r para diagnóstico da infecção por B. ovis. BoP59r e BP26r foram expressas em E. colie purificadas por cromatografia de afinidade e a antigenicidade testada por Western blot e ELISAi. A padronização do ELISAi foi realizada testando 500 ng e 1 µg de BoP59r e BP26r por poço e soros de carneiros infectados e não infectados. A BP26r foi eficiente em diferenciar ovinos negativos de positivos. O ELISAi com BP26 desenvolvido neste estudo foi o primeiro a utilizar BP26 completamente purificada como antígeno, resultando em elevada sensibilidade (100%) e sensibilidade (90,2%), com acurácia igual a 1,0.

© 2014 Silva et al. Brucella ovis is a major cause of reproductive failure in rams and it is one of the few well-described Brucella species that is not zoonotic. Previous work showed that a B. ovis mutant lacking a species-specific ABC transporter (ΔabcBA) was attenuated in mice and was unable to survive in macrophages. The aim of this study was to evaluate the role of this ABC transporter during intracellular survival of B. ovis. In HeLa cells, B. ovis WT was able to survive and replicate at later time point (48 hpi), whereas an ΔabcBA mutant was attenuated at 24 hpi. The reduced survival of the ΔabcBA mutant was associated with a decreased ability to exclude the lysosomal marker LAMP1 from its vacuolar membrane, suggesting a failure to establish a replicative niche. The ΔabcBA mutant showed a reduced abundance of the Type IV secretion system (T4SS) proteins VirB8 and VirB11 in both rich and acid media, when compared to WT B. ovis. However, mRNA levels of virB1, virB8, hutC, and vjbR were similar in both strains. These results support the notion that the ABC transporter encoded by abcEDCBA or its transported substrate acts at a post-transcriptional level to promote the optimal expression of the B. ovis T4SS within infected host cells.

Treatment with streptomycin enhances the growth of human commensal Escherichia coli isolates in the mouse intestine, suggesting that the resident microbial community (microbiota) can inhibit the growth of invading microbes, a phenomenon known as “colonization resistance.” However, the precise mechanisms by which streptomycin treatment lowers colonization resistance remain obscure. Here we show that streptomycin treatment rendered mice more susceptible to the development of chemically induced colitis, raising the possibility that the antibiotic might lower colonization resistance by changing mucosal immune responses rather than by preventing microbe-microbe interactions. Investigation of the underlying mechanism revealed a mild inflammatory infiltrate in the cecal mucosa of streptomycin-treated mice, which was accompanied by elevated expression of Nos2, the gene that encodes inducible nitric oxide synthase. In turn, this inflammatory response enhanced the luminal growth of E. coli by nitrate respiration in a Nos2-dependent fashion. These data identify low-level intestinal inflammation as one of the factors responsible for the loss of resistance to E. coli colonization after streptomycin treatment., IMPORTANCE Our intestine is host to a complex microbial community that confers benefits by educating the immune system and providing niche protection. Perturbation of intestinal communities by streptomycin treatment lowers “colonization resistance” through unknown mechanisms. Here we show that streptomycin increases the inflammatory tone of the intestinal mucosa, thereby making the bowel more susceptible to dextran sulfate sodium treatment and boosting the Nos2-dependent growth of commensal Escherichia coli by nitrate respiration. These data point to the generation of alternative electron acceptors as a by-product of the inflammatory host response as an important factor responsible for lowering resistance to colonization by facultative anaerobic bacteria such as E. coli.

Eradication of persistent intracellular bacterial pathogens with antibiotic therapy is often slow or incomplete. However, strategies to augment antibiotics are hampered by our poor understanding of the nutritional environment that sustains chronic infection. Here we show that the intracellular pathogen Brucella abortus survives and replicates preferentially in alternatively activated macrophages (AAMs), which are more abundant during chronic infection. A metabolic shift induced by peroxisome proliferator-activated receptor γ (PPARγ), which increases intracellular glucose availability, is identified as a causal mechanism promoting enhanced bacterial survival in AAMs. Glucose uptake was crucial for increased replication of B. abortus in AAMs, and for chronic infection, as inactivation of the bacterial glucose transporter gluP reduced both intracellular survival in AAMs and persistence in mice. Thus, a shift in intracellular nutrient availability induced by PPARγ promotes chronic persistence of B. abortus within AAMs, and targeting this pathway may aid in eradicating chronic infection. © 2013 Elsevier Inc.

Expression of capsular polysaccharides is a variable trait often associated with more-virulent forms of a bacterial species. For example, typhoid fever is caused by the capsulated Salmonella enterica serovar Typhi, while nontyphoidal Salmonella serovars associated with gastroenteritis are noncapsulated. Here we show that optimization of the immune evasive properties conferred by the virulence-associated (Vi) capsular polysaccharide involved an additional alteration to the cell envelope of S. Typhi, namely inactivation of the fepE gene, encoding the regulator of very-long O-antigen chains. Introduction of the capsule-encoding viaB locus into the nontyphoidal S. enterica serovar Typhimurium reduced complement deposition in vitro and intestinal inflammation in a mouse colitis model. However, both phenotypes were markedly enhanced when the viaB locus was introduced into an S. Typhimurium fepE mutant, which lacks very-long O-antigen chains. Collectively, these data suggest that during the evolution of the S. Typhi lineage, loss of very-long O-antigen chains by pseudogene formation was an adaptation to maximize the anti-inflammatory properties of the Vi capsular polysaccharide., IMPORTANCE Genomic comparison illustrates that acquisition of virulence factors by horizontal gene transfer is an important contributor to the evolution of enteric pathogens. Acquisition of complex virulence traits commonly involves horizontal transfer of a large gene cluster, and integration of the gene cluster into the host genome results in the formation of a pathogenicity island. Acquisition of the virulence-associated (Vi) capsular polysaccharide encoded by SPI7 (Salmonella pathogenicity island 7) was accompanied in the human-adapted Salmonella enterica serovar Typhi by inactivation of the fepE gene, encoding the regulator of very-long O-antigen chains. We show that the resulting loss of very-long O-antigen chains was an important mechanism for maximizing immune evasion mediated by the Vi capsular polysaccharide. These data suggest that successful incorporation of a capsular polysaccharide requires changes in the cell envelope of the hosting pathogen.

Our innate immune system distinguishes microbes from self by detecting conserved pathogen-associated molecular patterns. However, these are produced by all microbes, regardless of their pathogenic potential. To distinguish virulent microbes from those with lower disease-causing potential the innate immune system detects conserved pathogen-induced processes, such as the presence of microbial products in the host cytosol, by mechanisms that are not fully resolved. Here we show that NOD1 senses cytosolic microbial products by monitoring the activation state of small Rho GTPases. Activation of RAC1 and CDC42 by bacterial delivery or ectopic expression of SopE, a virulence factor of the enteric pathogen Salmonella, triggered the NOD1 signalling pathway, with consequent RIP2 (also known as RIPK2)-mediated induction of NF-κB-dependent inflammatory responses. Similarly, activation of the NOD1 signalling pathway by peptidoglycan required RAC1 activity. Furthermore, constitutively active forms of RAC1, CDC42 and RHOA activated the NOD1 signalling pathway. Our data identify the activation of small Rho GTPases as a pathogen-induced process sensed through the NOD1 signalling pathway.

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2013-08-22T18:56:41Z No. of bitstreams: 1 S0102-09352013000100009.pdf: 421172 bytes, checksum: be75be660de26891c6ce20f0f68ab9a8 (MD5) Made available in DSpace on 2013-08-22T18:56:41Z (GMT). No. of bitstreams: 1 S0102-09352013000100009.pdf: 421172 bytes, checksum: be75be660de26891c6ce20f0f68ab9a8 (MD5) Previous issue date: 2013-02-01 Made available in DSpace on 2013-09-30T19:50:53Z (GMT). No. of bitstreams: 2 S0102-09352013000100009.pdf: 421172 bytes, checksum: be75be660de26891c6ce20f0f68ab9a8 (MD5) S0102-09352013000100009.pdf.txt: 22674 bytes, checksum: 5a81fad632fecba20a8f9315b047a820 (MD5) Previous issue date: 2013-02-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:39:51Z No. of bitstreams: 2 S0102-09352013000100009.pdf: 421172 bytes, checksum: be75be660de26891c6ce20f0f68ab9a8 (MD5) S0102-09352013000100009.pdf.txt: 22674 bytes, checksum: 5a81fad632fecba20a8f9315b047a820 (MD5) Made available in DSpace on 2014-05-20T13:39:51Z (GMT). No. of bitstreams: 2 S0102-09352013000100009.pdf: 421172 bytes, checksum: be75be660de26891c6ce20f0f68ab9a8 (MD5) S0102-09352013000100009.pdf.txt: 22674 bytes, checksum: 5a81fad632fecba20a8f9315b047a820 (MD5) Previous issue date: 2013-02-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) O presente estudo objetivou avaliar uma técnica de nested PCR espécie-específica delineada a partir de PCR espécie-específica descrita anteriormente para detecção de B. ovis em sêmen e urina de carneiros infectados experimentalmente. O desempenho da nested PCR espécie-específica foi comparado com os resultados de uma PCR gênero-específica. Quatorze carneiros foram infectados experimentalmente com Brucella ovis REO 198 e amostras de sêmen e de urina foram colhidas semanalmente até 180 dias após a infecção. de 83 amostras de sêmen, 42 (50,6%) foram positivas pela nested PCR espécie-específica, e 23 (27,7%) foram positivas pela PCR gênero-específica. de 75 amostras de urina, 49 (65,3%) foram positivas pela nested PCR espécie-específica, enquanto 11 (14,6%) foram positivas em PCR gênero-específica. A técnica de nested PCR espécie-específica foi significativamente mais sensível (P

Background Infectious ovine epididymitis results in substantial economic losses worldwide due to reproductive failure and culling of breeders. The most common causative agents of these infections are Brucella ovis, Actinobacillus seminis, and Histophilus somni. The aim of this study was to develop a multiplex PCR assay for simultaneous detection of Brucella ovis, Actinobacillus seminis, and Histophilus somni with species-specific primers applied to biological samples for molecular diagnosis of these infections. Results The multiplex assay was capable of detecting B. ovis, A. seminis, and H. somni DNA simultaneously from genomic bacterial DNA samples and pool of semen samples from experimentally infected rams. The method was highly specific since it did not amplify DNA from other bacterial species that can potentially cause epididymitis in rams as well as species phylogenetically related to B. ovis. All negative control samples were negative in PCR multiplex assay. Urine can be used as an alternative to semen samples. Conclusions The species-specific multiplex PCR assay developed in this study can be successfully used for the detection of three of the most common bacterial causes of ovine epididymitis.

Evasion of host immune responses is a prerequisite for chronic bacterial diseases; however, the underlying mechanisms are not fully understood. Here, we show that the persistent intracellular pathogen Brucella abortus prevents immune activation of macrophages by inducing CD4+CD25+ T cells to produce the anti-inflammatory cytokine interleukin-10 (IL-10) early during infection. IL-10 receptor (IL-10R) blockage in macrophages resulted in significantly higher NF-kB activation as well as decreased bacterial intracellular survival associated with an inability of B. abortus to escape the late endosome compartment in vitro. Moreover, either a lack of IL-10 production by T cells or a lack of macrophage responsiveness to this cytokine resulted in an increased ability of mice to control B. abortus infection, while inducing elevated production of pro-inflammatory cytokines, which led to severe pathology in liver and spleen of infected mice. Collectively, our results suggest that early IL-10 production by CD25+CD4+ T cells modulates macrophage function and contributes to an initial balance between pro-inflammatory and anti-inflammatory cytokines that is beneficial to the pathogen, thereby promoting enhanced bacterial survival and persistent infection., Author Summary Brucella spp. are pathogens causing chronic intracellular infections that evade detection by pattern recognition receptors of the innate immune system. In this work, we tested the hypothesis that, in addition to eliciting a weak proinflammatory response during infection, induction of an immunoregulatory environment early during infection could promote persistent infection. Our results show that IL-10 produced at early time points is important for blunting inflammatory responses to B. abortus in infected tissues. CD4+ T cells are an important source of this cytokine, since mice lacking T cell-derived IL-10 exhibited increased inflammatory pathology and also were better able to control B. abortus infection. A target of this CD4 T cell-derived IL-10 is macrophages, since treatment of these cells with IL-10 in vitro supported intracellular replication of B. abortus, while blocking IL-10 restricted the ability of B. abortus to exit the phagolysosomal compartment and replicate intracellularly. Further, mice conditionally deficient for IL-10 receptor on macrophages were better able to control infection with B. abortus. Taken together, our results support a model in which IL-10 production by T cells promotes chronic infection by rendering macrophages more permissive for intracellular replication of B. abortus.

Brucella are facultative intracellular bacteria that cause chronic infections by limiting innate immune recognition. It is currently unknown whether Brucella FliC flagellin, the monomeric subunit of flagellar filament, is sensed by the host during infection. Here, we used two mutants of Brucella melitensis, either lacking or overexpressing flagellin to show that FliC hinders bacterial replication in vivo. The use of cells and mice genetically deficient for different components of inflammasomes suggested that FliC was a target of the cytosolic innate immune receptor NLRC4 in vivo but not in macrophages in vitro where the response to FliC was nevertheless dependent on the cytosolic adaptor ASC, therefore suggesting a new pathway of cytosolic flagellin sensing. However, our work also suggested that the lack of TLR5 activity of Brucella flagellin and the regulation of its synthesis and/or delivery into host cells are both part of the stealthy strategy of Brucella towards the innate immune system. Nevertheless, since a flagellin-deficient mutant of B. melitensis was found to cause histologically demonstrable injuries in the spleen of infected mice, we suggested that recognition of FliC plays a role in the immunologic standoff between Brucella and its host, which is characterized by a persistent infection with limited inflammatory pathology.

Intestinal inflammation changes the luminal habitat for microbes through mechanisms that have not been fully resolved. We noticed that the FepE regulator of very long O-antigen chain assembly in the enteric pathogen Salmonella enterica serotype Typhimurium (S. Typhimurium) conferred a luminal fitness advantage in the mouse colitis model. However, a fepE mutant was not defective for survival in tissue, resistance to complement or resistance to polymyxin B. We performed metabolite profiling to identify changes in the luminal habitat that accompany S. Typhimurium-induced colitis. This analysis suggested that S. Typhimurium-induced colitis increased the luminal concentrations of total bile acids. A mutation in fepE significantly reduced the minimal inhibitory concentration (MIC) of S. Typhimurium for bile acids in vitro. Oral administration of the bile acid sequestrant cholestyramine resin lowered the concentrations of total bile acids in colon contents during S. Typhimurium infection and significantly reduced the luminal fitness advantage conferred by the fepE gene in the mouse colitis model. Collectively, these data suggested that very long O-antigen chains function in bile acid resistance of S. Typhimurium, a property conferring a fitness advantage during luminal growth in the inflamed intestine., Author Summary Microbial communities inhabiting the intestinal tract have been characterized using high-throughput sequencing approaches, but little is known about factors that change their luminal habitat. Using metabolite profiling we discovered that luminal concentrations of total bile acids become significantly elevated during Salmonella-induced colitis. Very long O-antigen chains of Salmonella enterica serotype Typhimurium (S. Typhimurium) were required for luminal growth in the presence of such elevated bile concentrations during colitis. In contrast, very long O-antigen chains were dispensable for luminal growth in the presence of normal concentrations of bile acids or for growth in organs of mice. Thus, elevated bile concentrations during Salmonella-induced colitis change the luminal habitat and microbial growth in this environment requires increased bile resistance, which in case of S. Typhimurium is conferred by very long O-antigen chains.

Chemotaxis enhances the fitness of Salmonella enterica serotype Typhimurium (S. Typhimurium) during colitis. However, the chemotaxis receptors conferring this fitness advantage and their cognate signals generated during inflammation remain unknown. Here we identify respiratory electron acceptors that are generated in the intestinal lumen as by-products of the host inflammatory response as in vivo signals for methyl-accepting chemotaxis proteins (MCPs). Three MCPs, including Trg, Tsr and Aer, enhanced the fitness of S. Typhimurium in a mouse colitis model. Aer mediated chemotaxis towards electron acceptors (energy taxis) in vitro and required tetrathionate respiration to confer a fitness advantage in vivo. Tsr mediated energy taxis towards nitrate but not towards tetrathionate in vitro and required nitrate respiration to confer a fitness advantage in vivo. These data suggest that the energy taxis receptors Tsr and Aer respond to distinct in vivo signals to confer a fitness advantage upon S. Typhimurium during inflammation by enabling this facultative anaerobic pathogen to seek out favorable spatial niches containing host-derived electron acceptors that boost its luminal growth., Author Summary The ability to thrive in the inflamed gut is crucial for Salmonella serotypes to ensure their transmission to a new host. Salmonella serotypes can use alternative electron acceptors that become available during colitis to support their growth by anaerobic respiration, therefore giving the pathogen an advantage over the fermenting microbiota. While it is known that S. Typhimurium benefits from chemotaxis and motility during colitis, the chemotaxis signals the pathogen detects in vivo have not been elucidated. Here we demonstrate that during intestinal inflammation, S. Typhimurium is able to seek energetically favorable niches by detecting the presence of nitrate and tetrathionate, two respiratory electron acceptors that are generated as by-products of the host inflammatory response. Through this mechanism, energy taxis provides a fitness advantage for S. Typhimurium, and likely other facultative anaerobic bacteria, during in vivo growth in the intestinal lumen.

Information on how emerging pathogens can invade and persist and spread within host populations remains sparse. In the 1980s, a multidrug-resistant Salmonella enterica serotype Typhimurium clone lysogenized by a bacteriophage carrying the sopE virulence gene caused an epidemic among cattle and humans in Europe. Here we show that phage-mediated horizontal transfer of the sopE gene enhances the production of host-derived nitrate, an energetically highly valuable electron acceptor, in a mouse colitis model. In turn, nitrate fuels a bloom of S. Typhimurium in the gut lumen through anaerobic nitrate respiration while suppressing genes for the utilization of energetically inferior electron acceptors such as tetrathionate. Through this mechanism, horizontal transfer of sopE can enhance the fitness of S. Typhimurium, resulting in its significantly increased abundance in the feces., IMPORTANCE During gastroenteritis, Salmonella enterica serotype Typhimurium can use tetrathionate respiration to edge out competing microbes in the gut lumen. However, the concept that tetrathionate respiration confers a growth benefit in the inflamed gut is not broadly applicable to other host-pathogen combinations because tetrathionate respiration is a signature trait used to differentiate Salmonella serotypes from most other members of the family Enterobacteriaceae. Here we show that by acquiring the phage-carried sopE gene, S. Typhimurium can drive the host to generate an additional respiratory electron acceptor, nitrate. Nitrate suppresses genes for the utilization of energetically inferior electron acceptors such as tetrathionate while enhancing the luminal growth of S. Typhimurium through anaerobic nitrate respiration. Pathways for anaerobic nitrate respiration are widely conserved among members of the family Enterobacteriaceae, thereby making our observations relevant to other enteric pathogens whose relative abundance in the intestinal lumen increases during infection.

The invasion-associated type III secretion system (T3SS-1) of Salmonella enterica serotype Typhimurium (S. Typhimurium) activates the transcription factor NF-κB in tissue culture cells and induces inflammatory responses in animal models through unknown mechanisms. Here we show that bacterial delivery or ectopic expression of SipA, a T3SS-1-translocated protein, led to the activation of the NOD1/NOD2 signaling pathway and consequent RIP2-mediated induction of NF-κB-dependent inflammatory responses. SipA-mediated activation of NOD1/NOD2 signaling was independent of bacterial invasion in vitro but required an intact T3SS-1. In the mouse colitis model, SipA triggered mucosal inflammation in wild-type mice but not in NOD1/NOD2-deficient mice. These findings implicate SipA-driven activation of the NOD1/NOD2 signaling pathway as a mechanism by which the T3SS-1 induces inflammatory responses in vitro and in vivo., IMPORTANCE Salmonella enterica serotype Typhimurium (S. Typhimurium) deploys a type III secretion system (T3SS-1) to induce intestinal inflammation and benefits from the ensuing host response, which enhances growth of the pathogen in the intestinal lumen. However, the mechanisms by which the T3SS-1 triggers inflammatory responses have not been resolved. Here we show that the T3SS-1 effector protein SipA induces NF-κB activation and intestinal inflammation by activating the NOD1/NOD2 signaling pathway. These data suggest that the T3SS-1 escalates innate responses through a SipA-mediated activation of pattern recognition receptors in the host cell cytosol.

Brucellosis is a zoonotic infection caused primarily by the bacterial pathogens Brucella melitensis and B. abortus. It is acquired by consumption of unpasteurized dairy products or by contact with infected animals. Globally, it is one of the most widespread zoonoses, with 500,000 new cases reported each year. In endemic areas, Brucella infections represent a serious public health problem that results in significant morbidity and economic losses. An important feature of the disease is persistent bacterial colonization of the reticuloendothelial system. In this review we discuss recent insights into mechanisms of intracellular survival and immune evasion that contribute to systemic persistence by the pathogenic Brucella species.

The development of T helper 17 (T H 17) cells is a well-established adaptive mechanism for the production of interleukin-17A (IL-17A), a cytokine involved in neutrophil recruitment. However, pathways contributing to mucosal expression of IL-17A during the initial phase of a bacterial infection have received less attention. Here we used the mouse colitis model of Salmonella enterica serotype Typhimurium infection to investigate the contribution of myeloid differentiation primary response protein 88 (MyD88) to inflammation and mucosal IL-17A expression. Expression of IL-23 in the cecal mucosa during S. Typhimurium colitis was dependent on the presence of MyD88. Furthermore, initial expression of IL-17A at 24 h after S. Typhimurium infection was dependent on MyD88 and the receptor for IL-1β. IL-23 and IL-1β synergized in inducing expression of IL-17A in splenic T cells in vitro . In the intestinal mucosa, IL-17A was produced by three distinct T cell populations, including δγ T cells, T H 17 cells, and CD4 − CD8 − T cells. The absence of IL-1β signaling or IL-17 signaling reduced CXC chemokine expression but did not alter the overall severity of pathological lesions in the cecal mucosa. In contrast, cecal pathology and neutrophil recruitment were markedly reduced in Myd88-deficient mice during the initial phases of S. Typhimurium infection. Collectively, these data demonstrate that MyD88-dependent mechanisms, including an initial expression of IL-17A, are important for orchestrating early inflammatory responses during S. Typhimurium colitis.

Brucella ovis is a major cause of reproductive failure in sheep, which is associated with epididymitis and infertility in rams. Importantly, B. ovis is one of the few Brucella species that is not zoonotic. Due to the scarcity of studies on B. ovis infection, a murine model of infection was developed. The roles of B. ovis genes encoding a putative hemagglutinin and an ABC transporter were investigated in the mouse model. The kinetics of B. ovis infection were similar in BALB/c and C57BL/6 mice, and both strains of mice developed multifocal microgranulomas in the liver and spleen, but only minimal colonization and histopathological changes were observed in the genital tract. Therefore, the mouse was considered a suitable infection model for B. ovis but not for B. ovis -induced genital disease. Two mutant strains were generated in this study (the Δ abcAB and Δ hmg strains). The B. ovis Δ abcAB strain was attenuated in the spleens and livers of BALB/c mice compared to the wild-type (WT) strain ( P < 0.001). Conversely, the Δ hmg strain infected mice at the same level as WT B. ovis , suggesting that a putative hemagglutinin is not required for B. ovis pathogenesis. Additionally, the Δ abcAB strain did not survive in peritoneal macrophages, extracellularly in the peritoneal cavity, or in RAW 264.7 macrophages. Moreover, infection with the Δ abcAB strain was not lethal for male regulatory factor 1-knockout mice, whereas infection with the B. ovis WT strain was 100% lethal within 14 days postinfection. These results confirm that the predicted ABC transporter is required for the full virulence and survival of B. ovis in vivo .

The objective was to determine the effectiveness of various antimicrobial agents added to semen extender for inactivation of B. ovis or A. seminis in ovine semen after cryopreservation. In Experiment 1, 20 ejaculates from a crossbred ram infected with B. ovis were cryopreserved in Tris-based extenders with various antimicrobial agents: (I) control without antibiotics, (II) with penicillin and streptomycin (1000 IU/mL and 1 mg/mL, respectively), (III) lincomycin (0.15 mg/mL), (IV) sulphadiazine (0.60 mg/mL), and (V) gentamicin sulphate (0.25 mg/mL). Semen was stored in 0.25 mL straws at a final concentration of 150 × 10(6) spermatozoa/mL. After thawing (37 °C for 30 s), sperm total motility (TM), sperm morphology, integrity of sperm membranes, and bacterial growth were assessed. In Experiment 2, six B. ovis isolates were separately inoculated into aliquots of a fresh ejaculate from a B. ovis-free ram. Mock inoculated semen was processed for cryopreservation using the five extenders described above, and bacteriologically evaluated after thawing. In Experiment 3, sensitivity of A. seminis to the same antimicrobial agents was evaluated by inoculating an ejaculate from an A. seminis and B. ovis-free ram. There were no significant differences among treatments in post-thawing sperm parameters. B. ovis was isolated from 100% (20/20), 0% (0/20), 95% (19/20), 100% (20/20), and 5% (1/20) of semen samples diluted in tris-based extender of untreated (I) and treated semen samples with antimicrobial agents II, III, IV, and V, respectively. Frequencies of isolation from samples treated with antimicrobial agent II and V were significantly lower than untreated ones (P0.05). There were no significant differences in the profile of antimicrobial resistance of different B. ovis isolates. A. seminis had a similar sensitivity to the antimicrobial agents. We concluded that addition of a combination of penicillin and streptomycin or gentamicin alone to ram semen cryo-extenders inactivated B. ovis and A. seminis.

Brucella ovis infection is a major cause of epididymitis and infertility in rams, resulting in reproductive failure and significant economic losses worldwide. The goal of this study was to develop a PCR test targeting specific B. ovis genomic sequences. Specific primer pairs were designed targeting 12 of those ORFs. Samples of blood, serum, semen, urine, and preputial wash were collected from experimentally infected rams ( n = 9) every other week up to 180 days post infection (dpi), when tissue samples were obtained. Blood, serum, semen, urine, and preputial wash samples were obtained, in weekly intervals for 1 month, from eight rams belonging to a B. ovis -free flock. Semen samples were also obtained from rams belonging to naturally infected flocks ( n = 40). The limit of detection of this PCR protocol was 100, 10, and 1 CFU/mL for semen, urine and prepucial wash samples, respectively. Sensitivity and specificity values obtained with this PCR method were similar to that of bacteriology when evaluating biological samples. Agreement between PCR and bacteriology results was greater than 90%. These results clearly indicate that this species-specific PCR method is highly efficient for the diagnosis of B. ovis infection in semen, urine, preputial wash and tissue samples from infected rams.

Visceral leishmaniasis (VL) is a chronic and often fatal protozoal disease that is endemic in Belo Horizonte (State of Minas Gerais, Brazil). Leishmania sp. is an intracellular obligatory parasite of macrophages that can naturally infect several mammalian species. Non-human primates (NHP) have been used as experimental models for infection with Leishmania of the donovani complex. The present report describes a case of visceral leishmaniasis in a black-fronted titi. Among 41 primates kept in captivity in a zoo in Belo Horizonte (State of Minas Gerais, Brazil), one animal, a black-fronted titi (Callicebus nigrifrons), was positive for Leishmania chagasi infection by PCR and immunohistochemistry, and developed a fatal disease with clinical signs and lesions compatible with VL. Other 17 NHP, including six black-fronted titis (C. nigrifrons), one howler monkey (Alouatta guariba), three golden-bellied capuchins (Cebus xanthosternos), one golden-headed lion tamarin (Leontopithecus crysomelas), one black-headed owl monkey (Aotus nigriceps), two Rio Tapajos sakis (Pithecia irrorata) and three emperor tamarins (Saguinus imperator) had blood samples that tested positive for amplification of Leishmania kDNA by PCR, although these NPH had no clinical signs of the disease.

Infectious abortion is a significant cause of reproductive failure and economic losses in cattle. The goal of this study was to detect nucleic acids of several infectious agents known to cause abortion including Arcanobacterium pyogenes, Bovine Herpesvirus 1, Brucella abortus, Campylobacter fetus subsp. venerealis, Chlamydophila abortus, Leptospira sp., Listeria monocytogenes, Salmonella sp., Mycoplasma bovis, Mycoplasma bovigenitalium, Neospora caninum, and Tritrichomonas foetus. Tissue homogenates from 42 fetuses and paraffin-embedded tissues from 28 fetuses and 14 placentas/endometrium were included in this study. Brucella abortus was detected in 14.2% (12/84) of the samples. Salmonella sp. DNA was amplified from 2 fetuses, and there was one positive for Neospora caninum, and another for Listeria monocytogenes. This PCR-based approach resulted in identification of the etiology in 19% of samples, or 20% if considered fetal tissues only.

Leishmania chagasi, the agent of visceral leishmaniasis in dogs in the Americas has a tropism to the male genital system, particularly the epididymis, prepuce, and glans penis, resulting in shedding of Leishmania in the semen. The goal of this study was to verify the possibility of venereal transmission of L. chagasi. Twelve Leishmania-free bitches, housed in the absence of the insect vector, copulated with multiple naturally infected dogs that were shedding Leishmania in the semen. PCR analysis of serially collected ejaculates indicated that shedding of Leishmania in the semen is intermittent. Three bitches seroconverted, and six were PCR positive by the end of the experimental period (165 days after the last copulation). These data support the notion that L. chagasi may be sexually transmitted from naturally infected dogs to susceptible bitches in the absence of the biological insect vector.

Non-typhoidal Salmonella serotypes (NTS) cause a self-limited gastroenteritis in immunocompetent individuals, while children with severe Plasmodium falciparum malaria can develop a life-threatening disseminated infection. This co-infection is a major source of child mortality in sub-Saharan Africa. However, the mechanisms by which malaria contributes to increased risk of NTS bacteremia are incompletely understood. Here, we report that in a mouse co-infection model, malaria parasite infection blunts inflammatory responses to NTS, leading to decreased inflammatory pathology and increased systemic bacterial colonization. Blunting of NTS-induced inflammatory responses required induction of IL-10 by the parasites. In the absence of malaria parasite infection, administration of recombinant IL-10 together with induction of anemia had an additive effect on systemic bacterial colonization. Mice that were conditionally deficient for either myeloid cell IL-10 production or myeloid cell expression of IL-10 receptor were better able to control systemic Salmonella infection, suggesting that phagocytic cells are both producers and targets of malaria parasite-induced IL-10. Thus, IL-10 produced during the immune response to malaria increases susceptibility to disseminated NTS infection by suppressing the ability of myeloid cells, most likely macrophages, to control bacterial infection., Author Summary Non-typhoidal Salmonella serotypes (NTS) most frequently cause diarrheal disease, which is self-limiting. However, in sub-Saharan Africa, NTS is one of the most common causes of life-threatening bloodstream infections. Individuals with these bloodstream infections frequently have an underlying condition such as HIV in adults or malaria in children. We used a mouse model to investigate why malaria predisposes children to invasive NTS infections. Our results implicate an anti-inflammatory response induced by malaria parasites via the cytokine IL-10 in promoting increased growth of bacteria that have disseminated from the intestine to other organs of the body. This response is beneficial in that it prevents death from malaria, but has an adverse effect on phagocytic cells, blocking their ability to control growth of bacteria that have disseminated from the intestine to other organs of the body.