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Frontiers in Microbiology
Microbial Immunology
Front. Microbiol., 22 September 2015 | https://doi.org/10.3389/fmicb.2015.00985
Salmonella Typhimurium exploits inflammation to its own advantage in piglets
Barbara Chirullo1†, Michele Pesciaroli1,2†, Rosanna Drumo1,3, Jessica Ruggeri4, Elisabetta Razzuoli5, Claudia Pistoia1, Paola Petrucci1, Nicola Martinelli4, Lucilla Cucco6, Livia Moscati6, Massimo Amadori4, Chiara F. Magistrali6, Giovanni L. Alborali4* and Paolo Pasquali1*
1Unit of Prophyilaxis and Control of Bacterial Zoonoses, Department of Food Safety and Veterinary Public Health, Istituto Superiore di Sanità, Rome, Italy
2VISAVET Health Surveillance Centre, Universidad Complutense Madrid, Madrid, Spain
3Department of Comparative Biomedicine and Food Science, Università degli Studi di Padova, Padova, Italy
4Department of Veterinary Diagnostic, Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, Brescia, Italy
5S.S. Genova, Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'Aosta, Genoa, Italy
6Research and Development area, Istituto Zooprofilattico Sperimentale dell'Umbria e delle Marche, Perugia, Italy
Salmonella Typhimurium (S. Typhimurium) is responsible for foodborne zoonotic infections that, in humans, induce self-limiting gastroenteritis. The aim of this study was to evaluate whether the wild-type strain S. Typhimurium (STM14028) is able to exploit inflammation fostering an active infection. Due to the similarity between human and porcine diseases induced by S. Typhimurium, we used piglets as a model for salmonellosis and gastrointestinal research. This study showed that STM14028 is able to efficiently colonize in vitro porcine mono-macrophages and intestinal columnar epithelial (IPEC-J2) cells, and that the colonization significantly increases with LPS pre-treatment. This increase was then reversed by inhibiting the LPS stimulation through LPS antagonist, confirming an active role of LPS stimulation in STM14028-intracellular colonization. Moreover, LPS in vivo treatment increased cytokines blood level and body temperature at 4 h post infection, which is consistent with an acute inflammatory stimulus, capable to influence the colonization of STM14028 in different organs and tissues. The present study proves for the first time that in acute enteric salmonellosis, S. Typhimurium exploits inflammation for its benefit in piglets.
Salmonella enterica serovar Typhimurium (S. Typhimurium) is a pathogenic Gram-negative bacterium of great clinical significance, responsible for foodborne zoonotic infections. The human disease is characterized by self-limiting gastroenteritis that occasionally can cause fever, systemic infection, and severe inflammation of the intestinal mucosal epithelium (Haagsma et al., 2008; Pires et al., 2011).
The architecture of the mucosal epithelium contains several barriers that prevent or block infection by pathogenic bacteria. Mechanisms of protection are exerted by all of these barriers in order to maintain the integrity of the epithelial cell monolayer and limit inflammation-associated damage (Patel and McCormick, 2014). S. Typhimurium, however, is able to overcome these barriers and therefore to colonize the intestinal epithelium inducing inflammation and a marked host immune response. The inflammatory response in the gut is induced by the interaction of S. Typhimurium with host cells including epithelial cells and antigen-presenting cells (APCs), like macrophages and dendritic cells. The inflammation is characterized by the secretion of several cytokines, including interleukin (IL)-23 and IL-18, which in turn stimulates T cells to produce IL-17, and IL-22 in the gut mucosa (Srinivasan et al., 2007; Godinez et al., 2008, 2009; Raffatellu et al., 2008).
S. Typhimurium acquired an evolutionary adaptation to overcome antimicrobial defenses in the lumen of the inflamed intestine and, more importantly, to exploit inflammation in order to outcompete the intestinal microbiota (Lupp et al., 2007; Stecher et al., 2007; Barman et al., 2008; Lawley et al., 2008; Sassone-Corsi and Raffatellu, 2015). The capability of S. Typhimurium to grow in the inflamed mucosal environment relies upon the acquisition of essential nutrients and anaerobically respired tetrathionate to successfully outgrow the resident microbiota (Raffatellu et al., 2009; Winter et al., 2010; Liu et al., 2012; Behnsen et al., 2015).
Most of the current studies about S. Typhimurium infection have been conducted in mice, which naturally do not develop gastroenteritis, but rather a systemic infection. An experimental mouse model using antibiotic treatment in order to eliminate microflora and to induce colitis, has been recently established (Ahmer and Gunn, 2011). However, this model is based on the lack of an intact microbiota, which limits a comprehensive evaluation of the complex interactions of S. Typhimurium within the gastrointestinal environment (Elfenbein et al., 2013). Here, we utilized pigs as model for gastrointestinal research with the aim of evaluating whether S. Typhimurium is able to exploit inflammation favoring an active infection. Our findings provide evidence that the LPS administration induces inflammation that favors a significant increase in colonization of tonsils, cecum, and spleen by S. Typhimurium.
Salmonella spp. Cultures
A wild-type strain of Salmonella Typhimurium ATCC 14028 (STM14028) was used throughout the study. The strain was grown overnight at 37°C in Brain Heart Infusion broth (Oxoid Ltd, UK), harvested by centrifugation at 1500 × g for 10 min and then washed twice in ice-cold (+4°C) phosphate buffer solution (PBS) (Sigma-Aldrich, Italy).
In vitro STM14028 Colonization
Porcine peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by Ficoll centrifugation and resuspended in complete RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS, Gibco-BRL, USA), 2 mM L-glutamine, Gentamicin (100 μg/ml). Mono-macrophage cells were isolated from porcine PBMCs, by a 4 h plastic adherence procedure at 37°C in 5% CO2 atmosphere, followed by extensive washing with PBS (2 times per day for the first 5 days) to eliminate the lymphocyte contamination. After 7–10 days mono-macrophage cells were obtained, the purity of which was ≥90% as determined by FACS (anti-CD14 Mil-2 mAb AbCAM cat. 23919-1, and anti-pig macrophages mAb, AbD Serotec, cat MCA2317F). Cells were then collected, resuspended in complete medium, and transferred into 200 μL per wells of 96-well round-bottom microtiter plates. The IPEC-J2 cell line, porcine intestinal columnar epithelial cells established from normal jejunum of a neonatal unsuckled pig (ACC 701), were grown in Minimum Essential Medium (MEM) (Sigma-Aldrich, St. Louis, MO) enriched with Fetal Calf Serum (FCS, Gibco-BRL, USA) (10% v/v), 2 mM glutamine, and antibiotics (50μg/mL penicillin, 50μg/mL streptomycin, and 10μg/mL neomycin), at 37°C in 5% CO2 atmosphere. Mono-macrophages and IPEC-J2 cell line were employed for in vitro studies. Both types of cells were seeded in 96-well plates at a density of 1 × 105 cells per well and treated overnight with purified lipopolysaccharides (LPS) (1 μM/mL; from Escherichia coli 0111:B4, L4391; Sigma-Aldrich) alone or in combination with a natural antagonist of LPS, the RS-LPS (100 μM/mL; tlrl-prslps, Invivogen, San Diego, USA). The following day, cell cultures were rinsed and STM14028 was diluted in RPMI-10% FBS, added to the cells at a multiplicity of infection (MOI) of 100:1 and incubated for 1 h at 37°C in 5% CO2. After 1 h, the cell cultures were rinsed and incubated in a culture medium containing gentamicin sulfate (100 μg/ml) to kill extracellular bacteria but not the internal ones, and subsequently incubated for 3 and 24 h. Viable intracellular bacteria were recovered by lysing the cells, at both 3 and 24 h post treatment time point, in distilled water with 0.1% of Triton X-100 for 10 min. The quantification of bacteria was performed by plating serial dilutions on agar triptose plates.
In vivo Studies
Fourteen commercial hybrid pigs aged ~30 days were utilized in the experiment. All the pigs used throughout the study were the offspring of Salmonella-free sows (negative for Salmonella by both serological and bacteriological tests). Before the onset of the experiment, the piglets were proved to be Salmonella-free by culture of feces of each animal. Animals were weighed and randomly allocated to two groups of 6 (A and B) and one group (C) of two pigs. Each group was maintained in separate isolation units under natural day–night rhythm with access to feed and water ad libitum. Groups A and B were intragastrically administered with 20 ml of sodium bicarbonate buffer containing 109 CFU of S. Typhimurium ATCC 14028. At the same time, Group A was intraperitoneally challenged with 12.5 μg/kg BW of lipopolysaccharides from S. enterica serovar Typhimurium (L2262, Sigma Aldrich SRL, Milan, Italy). Group C received only sterile sodium bicarbonate buffer and served as naïve control group. Collection of individual fecal samples (0, 1 day after challenge), blood sampling and registration of rectal temperature (0, 4 h, 1 and 2 days after challenge) were performed as well. Pigs were visually monitored by an independent veterinary officer in charge of the study for 6 h after the inoculum and then twice a day. Two days after the challenge, pigs were weighed again, and then euthanized using a captive bolt pistol and exsanguination. Samples of tonsils, liver, spleen, mesenteric lymph nodes, ileum, cecum, and colon were collected from each pig for the evaluation of bacterial burden.
All the experiments were authorized by national authority and were conducted according to the Italian national regulations enforced at the time of this study (Italian legislative Decree 116/92).
The microbiological analysis of fecal and organ samples were conducted according to the ISO 6579:2002/Amendment 1:2007 protocol. Briefly, samples were weighed and homogenized as 10% suspension in Buffered Peptone Water (BPW) (Oxoid Ltd., UK). This initial solution was then used to perform a decimal dilution series carried out by systematically transferring an aliquot of 1 ml of each successive dilution in 9 ml of BPW. All BPW-diluted samples were incubated at 37°C for 18 ± 3 h. Cultures (0.1 ml) were subsequently seeded on Modified Semisolid Rappaport-Vassiliadis (MSRV) agar plates (Oxoid Ltd., UK) and incubated at 41.5°C for 24 h for the selective enrichment of Salmonella. A loopful of growth from each MSRV plate was streaked onto Xylose-Lysine-Desoxycholate Agar (Oxoid Ltd., UK) and Brilliant Green Agar (Oxoid Ltd., UK) plates and hence incubated at 37°C overnight. Typical colonies were confirmed serologically as Salmonella by polyvalent antiserum (Salmonella Test Serum; Siemens Healthcare Diagnostics, Italy) and API rapid 20 E (Api Rapid 20E; Biomerieux, Italy). This is a semi-quantitative approach that allow the determination of the concentration of Salmonella in a sample within a tenfold band.
Flow Cytometry of Lymph Nodes Cells
Cell treatment was performed according to an established procedure (Razzuoli et al., 2012), with minor modifications. Briefly, frozen lymph nodes cells were thawed at 38°C and washed with FACS-Buffer (0.1% sodium azide + 2% fetal calf serum in PBS). Then, they were divided into aliquots (106 cells each) and reacted with monoclonal antibody (mAb) CD21 (Southern Biotech, cat. 4530-02), Mil-2 (AbCAM, cat. 23919-1), PMN (AbD Serotec, cat. MCA2599F), or FACS buffer only (control) for 30 min at 4°C, respectively. Cells were washed, and again incubated for 30 min at 4°C in FACS buffer containing goat anti-mouse IgG-FITC (Invitrogen, Molecular Probes®, cat: A10683). After washing in FACS buffer, cells were resuspended in 100 μL of the same buffer and 1:4 diluted. Samples were analyzed in a GUAVA MILLIPORE flow cytometer (Millipore Software). The typical forward and side scatter gate was set to exclude dead cells from the analysis. The percentage of positive cells beyond the threshold fluorescence channel was assessed in each sample on 10,000 events and compared between mAb-treated and control cells. For each antibody, results were expressed in terms of net percentage of positive cells.
Analysis of Cytokines
In order to evaluate the serum concentration of IL-1 beta and TNF-alpha ELISA kits were used (cat. N. PLB00B and PTA00 respectively, R&D Systems, Inc. Minneapolis, MN 55413, USA,). These assays employ the quantitative sandwich enzyme immunoassay technique using monoclonal antibodies, specific for porcine IL-1 beta or porcine TNF- alpha, pre-coated onto a microplate. The intensity of the color measured is at 450 nm.
For the in vitro and in vivo assays, the statistical significance of differences between study groups was analyzed using ANOVA and Student's t-test; p < 0.05 was chosen as threshold for significance. These symbols were used to indicate the statistical significance: *P < 0.1; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Salmonella Infection Induces an Innate Immune Response
In order to verify the involvement of the innate immune response during Salmonella infection in piglets, phenotypic analysis in ileo-cecal lymph nodes was performed in piglets orally infected with STM14028 and euthanized 48 h later. As depicted in Figure 1A, it was possible to observe a higher increase in the percentage of CD14+ (mainly monocytes) and polymorphonuclear cells with a minor involvement of CD21+ B cells. Moreover, the STM14028-infection was confirmed by the bacterial count in different organs (Figure 1B). These findings are consistent with a rapid recruitment of neutrophils and monocytes/macrophages toward lymph nodes, crucial for the effective response to lipopolysaccharides stimulus or concomitant bacterial infection. These data suggest a prompt involvement of the innate immune response in the regional lymph nodes draining the gut, after an oral infection with STM14028.
Figure 1. STM14028 infection induces an increase of innate immunity cell compartment and colonizes different organs of piglets orally infected. (A) The prevalence of CD14+, CD21+, and polymorphonuclear (PMN) cells was determined in ileo-cecal lymph nodes, 48 h post-infection with STM14028. The differences were statistically significant (***P ≤ 0.001, multiple comparisons t-test). (B) STM14028 count in ileo-cecal lymph nodes (ICLN), spleen, colon, and cecum of infected piglets. Data represent mean with error bars as SEM of six piglets per group.
LPS Treatment Increases STM14028 Colonization in Isolated Mono-macrophages and IPEC-J2 Cells
In order to assess if STM14028 exploits inflammation in vitro, we used LPS, which is known to stimulate the production of inflammatory molecules either in monocytes/macrophages (Fang et al., 2004) or in IPEC-J2 cells (Razzuoli et al., 2013). Monocytes/macrophages and IPEC-J2 cells were thus primed overnight with purified LPS alone and/or in combination with a natural antagonist of LPS (RS-LPS) capable to inhibit the LPS-stimulation interacting with the TLR-4/MD-2 complex recognized by LPS. Afterwards, the cells were infected with STM14028.
We observed that STM14028 was able to efficiently colonize monocytes/macrophages and IPEC-J2 cells at both 3 and 24 h post STM14028-infection (Figures 2A–D). A previous treatment with purified LPS significantly increased STM14028 colonization in both cell types at 3 and 24 h after infection. When RS-LPS antagonist was used, this markedly inhibited LPS stimulation causing a colonization level similar to the one obtained by STM14028 infection alone (Figures 2A–D). Overall, these results suggest that LPS stimulation can create conditions in which STM14028 is more efficiently phagocytized by cultivated cells, within it can find suitable conditions to multiply.
Figure 2. STM14028 colonization of mono-macrophages and IPEC-J2 cells at 3 and 24 h post infection (A–D). STM14028 colonization increases with LPS pre-treatment and is reduced by RS-LPS antagonist to the values of LPS-untreated cells (*P ≤ 0.1; **P ≤ 0.01, One-Way Anova Turkey's multiple comparisons test, data from one representative experiment out of three with similar results).
LPS Induces Inflammation in Piglets, Which in Turn Favors STM14028 Colonization
To evaluate the influence of inflammation during Salmonella infection in vivo, we established a protocol of inflammation, injecting parenterally LPS in group A and STM14028 by oral route in groups A and B. Piglets in group C were kept untreated and served as control animals. Then, an assessment was made of whether inflammation induced by LPS had favored the progression of Salmonella infection.
LPS was able to induce a rise in body temperature in piglets of group A already at 4 h post STM14028-infection (Figure 3) compared to the control (C) and the STM14028-infected group (B), reaching body temperature similar to those of group A only at 24 and 48 h post infection. No significant differences in body weight were measured among the three groups throughout the 48 h of analysis (data not shown).
Figure 3. LPS-treatment of piglets induces a rise in body temperature 4 h after infection with STM14028. The body temperature was measured at different time points on three different groups of piglets: treated with LPS and infected with STM14028 (group A); only STM14028 infected (group B); naïve control group (group C). At 4 h post infection, group A showed a significant rise in body temperature compared to the B and C groups. Data refer to one out of two separate experiments performed with comparable results. The differences between groups were statistically significant (****P ≤ 0.0001; *P ≤ 0.1; **P ≤ 0.01 multiple comparisons-Fisher's Least Significant Difference test).
Moreover, a remarkable increase of circulating pro-inflammatory cytokines has been found (Figure 4). TNF-alpha was in fact detected in the blood of all animals but it was produced in a higher amount in group A compared to the groups B and C, at 4 h post STM14028-infection, without significant differences within groups at 24 and 48 h post infection.
Figure 4. LPS-treated piglets show an increased inflammation 4 h post STM14028 infection. IL-1beta and TNF-alpha production was measured at different time points on blood samples from three different groups of piglets: treated with LPS and infected with STM14028 (group A); only STM14028 infected (group B); naïve control group (group C). At 4 h post infection, group A showed a significant increase in production of both cytokines compared to the B and C groups. The differences between the groups were statistically significant (*P ≤ 0.1; **P ≤ 0.01, multiple comparisons-Fisher's Least Significant Difference test).
IL-1 beta blood level was higher in group A than in groups B and C at 4 h post STM14028-infection. At 24 h post infection we detected a reduction of the IL-1 beta blood concentration in group A with a concomitant slightly increased level in group B. Finally, the IL-1 beta level completely reversed its trend in groups A and B at 48 h post infection, with higher concentration of IL1-b in group B compared to group A (Figure 4).
These results confirm the induction of a pro-inflammatory status mediated by LPS immediately after its administration. Noteworthy, in group B the level of pro-inflammatory cytokines required 48 h after infection to reach a concentration similar to that measured in group A.
Piglets of the three groups were euthanized 48 h after the treatments, and STM14028 infection was assessed in different organs and tissues in order to evaluate the capability of colonizing either locally in the gut milieu, or systemically. As depicted in Figure 5, piglets treated with LPS and infected with STM14028 showed a significant increase in colonization of tonsils, cecum, and spleen, whereas in mesenteric lymph nodes, colon, ileum and liver no significant difference in STM14028 colonization was observed (Figure 5).
Figure 5. LPS treatment raises STM14028 colonization of tonsils, cecum, and spleen of piglets. Recovery of STM14028 from different organs at 48 h post infection of piglets treated with LPS and infected with STM14028 (LPS+STM group) or only infected with STM14028 (STM group). LPS-treatment increases the colonization of tonsils, cecum, and spleen but does not influence the colonization of mesenteric lymph nodes, colon, ileum, and liver of piglets after STM14028 infection. Data refer to one out of two separate experiments performed with comparable results. The differences between the groups were statistically significant (*P ≤ 0.01, Mann–Whitney unpaired t-test).
On the whole, these findings provide substantial evidence that LPS is able to induce an inflammatory response, which favors STM14028 survival and colonization in the intestinal and systemic compartments.
Intestinal inflammation, induced by both chemical treatments and infectious agents, is known to be associated with a profound dysbiosis of the colonic microbial community structure (Lupp et al., 2007). Many pathogens use inflammation and the accompanying dysbiosis for their advantage in order to overcome colonization resistance (Stecher et al., 2007). In this context, several studies based on the use of the streptomycin-treated mouse colitis model, which is characterized by absence of resident microbiota (Ahmer and Gunn, 2011), highlighted the emerging concept that inflammation of the mucosal epithelium plays a role in environmental fitness of S. Typhimurium. It has been shown, indeed, that unlike avirulent strains, wild-type S. Typhimurium is capable of out-competing commensal microbiota in re-colonization experiments after treatment with antibiotics. Furthermore, S. Typhimurium exploits inflammation to promote its own colonization, out-competing the resident microbiota (Stecher et al., 2007). This model however, despite the important contribution to the study of the pathogens-microbiota interaction in an inflammatory environment, presents several limitations. In particular, the inability of the Salmonella mouse model to reproduce gastroenteritis and, even more importantly, the elimination of the competing flora represent crucial differences with respect to natural Salmonella infection in humans. In this work, an experimental model was used based on piglets infected with a wild type strain of S. Typhimurium, STM14028, representing an ideal animal model potentially capable to overcome the intrinsic limitations of the current streptomycin-treated mouse colitis model.
It was initially observed that, after oral infection with STM14028, the immune response is rapidly activated, involving the innate compartment with a marked increase of polymorphonuclear and mono-macrophage populations in ileo-cecal lymph nodes (Figure 1). This confirms the involvement of the principal populations engaged in the response to Salmonella infection, already known to be relevant for the response to the LPS stimulation. Moreover, these results extend those of recent studies about the increased expression of pro-inflammatory cytokines (Knetter et al., 2015) and raised lymphocytic infiltration of the gut mucosa after S. Typhimurium oral infection of piglets (Gradassi et al., 2013).
It was investigated whether the induction of inflammation by LPS pre-treatment of mono-macrophages and porcine intestinal epithelial IPEC-J2 cells makes these cells more susceptible to STM14028 infection. The results indicate that these cells, primed with LPS, were more prone to the colonization by STM14028 when compared to the LPS-untreated control cells. Otherwise, the use of the RS-LPS antagonist, binding the TLR-4/MD-2 complex, inhibits the LPS stimulation. This significantly reduced the STM14028 intracellular colonization down to the values of LPS-untreated cells (Figure 2).
The higher colonization of STM14028 in LPS-treated cells could be considered unexpected due to the effect of LPS stimulus. It can however be hypothesized that our results are the consequence of high capability of bacterial up-take. It has been well-established, in fact, that for many facultative intracellular pathogens, as well for Salmonella, the key to successful infection lies in the interaction between bacteria and host macrophages. Salmonella is able to mount specific strategies to escape killing and survive within phagocytes (Fields et al., 1986; Groisman and Saier, 1990; Gulig et al., 1998; Ruby et al., 2012). Moreover, recent reports have revealed fascinating insights to explain how Salmonella exploits host response. In particular, the internalization of Salmonella in macrophages via TLR, able to bind Salmonella LPS, is a crucial factor to favor Salmonella virulence in that it facilitates the acidification of the phagosome, which in turn provides a protective niche for Salmonella (Arpaia et al., 2011). In addition, in mouse models, it has been observed that LPS present on live Salmonella provides an essential signal, via functional TLR-4, for macrophages to produce NO and TNF-α (Royle et al., 2003). This may be exploited by Salmonella to modify macrophage functions and promote growth and/or dissemination throughout the host.
Finally, the main effort was to assess whether the inflammation induced LPS-treated piglets was able to influence the colonization of STM14028. Piglets treated with LPS and infected with STM14028 showed a significant increase in body temperature (Figure 3) and the production of IL-1beta and TNF-alpha in the blood (Figure 4) at 4 h post infection. These results indicate that the onset of the LPS-mediated acute inflammation leads to cytokines production and body temperature rise, already at 4 h post infection, promoting significant increase of tonsils, cecum, and spleen STM14028-colonization, compared to the control group (Figure 5).
These results are in line with studies showing that the parenteral administration of dead Gram-negative bacteria or lipopolysaccharide exacerbated the growth of virulent S. enterica in mice (Hormaeche, 1990). It is also possible to envisage that the increased advantage of Salmonella in inflamed systemic environment can also be justified by mechanisms other than a compromised barrier integrity or dysbiosis. Moreover, Foster et al. demonstrated that the intravenous administration of an attenuated Salmonella strain can exacerbate the growth of virulent strains, which involves IL-10 production and requires TLR-4, and its signaling pathways involving the adaptor molecules, the TIR-domain-containing adapter-inducing interferon-β (TRIF), and the Myeloid differentiation primary response gene 88 (MyD88) (Foster et al., 2008). A dysregulated type I IFN response in tissues can affect fundamental regulatory circuits of innate immunity in macrophages, which turns IL-10 into a potent pro-inflammatory cytokine (Sharif et al., 2004). In addition, IL-10 and IFN-γ associated responses may cause a gain of pro-inflammatory activity, as shown in human models of endotoxemia (Lauw et al., 2000).
On the whole, these results, using piglets as model, demonstrate for the first time that in acute enteric salmonellosis, S. Typhimurium exploits the inflamed milieu to its own advantage.
BC planned and performed the research, analyzed data and co-wrote the manuscript. PP designed, planned and performed the research, analyzed data and co-wrote the paper. GA designed, planned, and performed the research and analyzed data. MA planned and performed part of the research and analyzed data. RD and MP performed part of the research and contributed to the analysis of the data. ER performed cytofluorimetric analysis. JR and NM performed part of the research. LM performed Immunoenzymatic analysis. CM and LC performed in vivo studies. PP and CP contributed to perform technical experiments.
This work was supported by ISS intramural research funds and by Transnational Research Project EMIDA ERA-NET "HealthyGut - Multi-focal strategies to improve gut health and reduce enteritis in poultry and pigs", MIPAF- DM 27373/7303/2010. The research by Pesciaroli Michele was partly supported by a PICATA postdoctoral contract of the Moncloa Campus of International Excellence (UCM-UPM, Campus Moncloa, VISAVET), "HealthyGut."
Special thanks to Massimiliano Francia and Michele Tentellini for technical assistance.
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Keywords: Salmonella typhimurium, inflammation, immune response, pig, salmonellosis
Citation: Chirullo B, Pesciaroli M, Drumo R, Ruggeri J, Razzuoli E, Pistoia C, Petrucci P, Martinelli N, Cucco L, Moscati L, Amadori M, Magistrali CF, Alborali GL and Pasquali P (2015) Salmonella Typhimurium exploits inflammation to its own advantage in piglets. Front. Microbiol. 6:985. doi: 10.3389/fmicb.2015.00985
Amy Rasley, Lawrence Livermore National Laboratory, USA
Sukanya Narasimhan, Yale University School of Medicine, USA
Hridayesh Prakash, University of Hyderabad, India
Copyright © 2015 Chirullo, Pesciaroli, Drumo, Ruggeri, Razzuoli, Pistoia, Petrucci, Martinelli, Cucco, Moscati, Amadori, Magistrali, Alborali and Pasquali. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Giovanni L. Alborali, Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, Via Bianchi, 9 - 25124 Brescia, Italy, giovanni.alborali@izsler.it;
Paolo Pasquali, Dipartimento di Sanità Pubblica Veterinaria e Sicurezza Alimentare, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy, paolo.pasquali@iss.it
†These authors have contributed equally to this work.

redpajama_set_name: RedPajamaCommonCrawl


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Shira Sussi
July 05, 2019 in 4th Trimester Bodies
Shira Sussi (32), Lior (10 months) and Abe (forever 23 weeks gestation)
Shira shares -
"Lior is my first healthy, living child and my third pregnancy. In January 2017, I had an abortion for medical reasons at 23 weeks. We named him Abe. His birth defects were so severe he wouldn't have been able to breathe or swallow at birth, if he made it to that point. It was the hardest decision I have ever made, and the months following his loss were the darkest and loneliest of my adult life. It took us 7 months to find the courage to try to conceive again and I had a first trimester miscarriage. When I got pregnant with Lior I didn't think I could emotionally make it through his pregnancy. It was an incredibly stressful nine months.
How has parenthood impacted your body image?
The stress of my pregnancies and postpartum has taken more of a toll on my body than anything else. Grief brain is real, as is mom brain so I feel like mentally I've been in a fog for almost 3 years! I look at pre-loss pictures of myself and think, wow I look so beautiful and content. Naivety is bliss. I battle with this more than those 5 extra pounds of baby weight stuck to my thighs.
After losing Abe I really struggled with this idea that my body had betrayed me in creating a sick baby that I wasn't able to save, but since giving birth to Lior I've come to see my body in a new light and appreciate what it's lived through. This is my mother body. It's carried three babies for different lengths of time. It's beautiful and strong, albeit a bit more squishy.
What was your postpartum experience?
Early postpartum has been a lesson in new motherhood – throw your expectations out the door and ride out the beautiful, messy chaos. People say it gets better, but really it gets different. I try to remind myself every phase is temporary, so how can I appreciate what's happening now for what it is knowing it won't last forever. Do I miss waking up every two hours? No, but I do miss all 7 lbs of little Lior falling asleep on my chest at 3 AM after a feeding, breathing in his sweet smell.
Lior's birth was incredibly traumatic. After a short labor I arrived to the hospital ten centimeters dilated and he was born via vacuum-assisted delivery within the hour. Three days later we were in the ER for elevated bilirubin levels. My husband had started a new job a month before Lior's birth so he was back at work after three days at home with us. There was a lot of tears and a lot of just me and L trying to figure things out the two of us. In the thick of early postpartum it was pure survival mode.
Breastfeeding did not come easy – my milk took almost two weeks to fully come in, and even after Lior's tongue tie was corrected it took months to establish a good feeding relationship. I constantly battled with clogged ducts since he couldn't efficiently empty my breasts and was married to the breast pump 8-12x/day. When I imagined what life would be like once Lior arrived earth side it never crossed my mind that I would have such trouble feeding him. Breastfeeding added a whole layer of stress those early weeks, and while I'm grateful I muddled my way through it I now better understand why people choose not to.
I've also struggled with a lot of guilt postpartum. Guilt that Lior is here and healthy; that he will never know his older brother; that I am feeling so much happiness in watching him grow and thrive. I'm still very much working through this.
What is your truth?
I can be grieving and still find joy. For the longest time I kept asking myself, when will the pain go away? The truth is the pain never goes away; it gets softer as time goes on, and it doesn't control my every waking thought like it once did. This took time and patience, trust and acceptance, countless hours of therapy and so much work on my relationship with myself and others. Pregnancy loss robs you of many things - your innocence, a year of "firsts", the ability to say your child's name in normal conversation. But there is so much to be grateful for. I choose to see all the light and beauty. There will always be a void, but my life is SO FULL.
Why did you choose to participate in this movement and share your story?
If my story about ending a wanted pregnancy for medical reasons reaches just one mother who is going through something similar, experiencing the isolation I did back in January 2017 and is the slightest bit comforted knowing she isn't alone then sharing today so publicly will have been worth it.
Tags: Medical Abortion, Abortion, Termination, Stillbirth, Pregnancy After Loss, 4th Trimester, 4th Trimester Bodies, Brain Fog, Birth Trauma, vacuum assistance, Jaunice, Breastfeeding, Tongue Tie, NYC, New York City
Kelsey Mallabar
October 17, 2018 in 4th Trimester Bodies
Kelsey Mallabar (25) Scarlett (Forever one day old), and Iris (3 months)
Kelsey shares -
"I became pregnant during the spring of my senior year of college. Scarlett was diagnosed with ARPKD (Autosomal Recessive Polycystic Kidney Disease) the following September. She died at one day old in November. I became a grieving mother at 22.
At 22 I learned that this person who I love and I will always have a 1:4 chance of having a terribly sick baby. It changed the way I thought about myself. I had barely graduated from college and I was faced with this truth that people three times my age never have to navigate. I had a newly postpartum body and no baby on my hip. I had dealt with my milk coming in, taking over my breasts with no way to relieve them but with cabbage leaves and time. My uterus went from holding a 5lb 10oz baby girl to empty. It wasn't just the physical changes but the mental shift. My very DNA is what made my baby sick. We fell somewhere in between infertility and fertility. Not really fitting the mold of either but somehow relating to both. We were encouraged to try again. I searched for happy endings and they were bountiful.
It didn't take us long to conceive again. We were so hopeful. We tested to see if this baby would also be affected with ARPKD. There is no feeling more crushing anxiety of waiting to find out if your baby is sick with the same disease that took your first child. It's a crawl-out-of-your-skin, hyperventilate type of anxiety. The baby - a boy - tested positive for ARPKD. I never thought I would be someone who terminated a pregnancy but after losing Scarlett we couldn't watch another child suffer like she did. I do not regret our decisions in any way but it still breaks my heart we said goodbye.
I've never come to terms that it happened a third time while we were waiting to start IVF with PGD (Preimplantation Genetic Diagnosis). A surprise but such a welcomed pregnancy. Third time had to be a charm, right? This was the universe giving me a break. But it wasn't. We found ourselves terminating another wanted pregnancy. I have a hard time even talking to my husband about it to this day. I wanted to be a mom so badly and here I am able to conceive but yet somehow we can't make healthy babies. It felt like the universe was mocking me. How could it be so cruel.
Through IVF with PGD we were beyond blessed with a healthy baby girl this past May. I love her so much it hurts. I look at her and I literally feel endorphins releasing. Pregnancy with her was scary. I expected the other shoe to drop at any time. After three pregnancies of loss I knew there was no "safe" point. Towards the end I obsessed over getting her here safely. I constantly thought she was going to die. I was induced partially because I wanted to be, partially due to high fluid. Her birth was difficult, much harder than Scarlett's and ended assisted with the vacuum. She had a large hematoma on her head that made her cry constantly for the first week. We couldn't put her down because she sounded like she was in pain each time we tried. Grief resurfaced hard in the first few weeks. Life with Iris reminded me of everything I missed with Scarlett.
Physically Iris was a larger baby than Scarlett and my body shows that. My hips are a little wider and the stretch marks her left on my body are more prominent. I look in the mirror and I don't quite recognize my reflection. I love it and hate it all at once.
My identity as a mother has ebbed and flowed in its definition. For years I felt like a mother, biologically I was a mother, but to the world I wasn't a mother. I had no baby to show for it. I sat on the sidelines of my dream and watched everyone else participate in the game. I became more isolated. I protected my heart inside the four walls of my home. Everywhere else was filled with pregnant women and babies. It made my heart bleed. I only found solace in the loss community on social media. Those mamas are some of the most badass mamas I've ever known.
Now I am just as much a mother but somehow more of one at the same time. I birthed a baby but she stayed with me this time. I don't just carry her in my heart but I also carry her in my arms. I love her hard. I look at her in amazement every time I see her. I also know her mortality and I think that's most difficult. Loving her is easy but knowing she can be taken away in an instant is a difficult reality to live with. I do wish I wasn't so familiar with these dark truths. They've made me stronger, but sometimes I don't want to be strong.
My journey to motherhood has been an uphill battle. So many times the universe said no but somehow we have come out the other side. My body has been through so much and now in my postpartum journey I am trying to learn to love it. I hope taking pictures with BOTH my babies helps this evolve.
I also think it's important in today's political climate to hear stories. Access to reproductive health is being jeopardized. If I did not have the ability to exercise my right to chose to carry out a pregnancy my life and mental health could be at a very different state. It's not my place to tell people what to believe but it is to share my story and bring awareness. Life is not black and white."
Tags: Autosomal Recessive Polycystic Kidney Disease, Child Loss, Termination, IVF, Hematoma, Preimplantation Genetic Diagnosis, Burlington, 4th Trimester Bodies, 4th Trimester, vacuum assistance
Shannon Fuscaldo
Shannon Fuscaldo (37). Mother to Jaden (17) and Tilly (21 months - pictured)
Fort Wayne, Indiana | Portland, Oregon
Shannon shares -
"I had my first child at age 19, when I was just coming into my body as a woman. After he was born, I felt like everything quickly returned to its prior shape except my breasts and skin on my lower belly. I thought maybe after some time and more exercise they would bounce back and tighten up, but no amount of exercise did the trick. I grew very self-conscious of these areas and even consulted with a plastic surgeon about breast implants. Even though I didn't love these parts of myself, I so badly wanted to embrace all of the markings that parenthood gave me and decided against surgery.
Now, 17 years later, I still struggle with accepting the parts of myself that do not live up to this unattainable expectation I have, but I am working to change the thought patterns in my head. I am so fortunate to have a belly that expanded enough to grow two babies to term and breasts that have nourished them for the beginning of their lives. Also, I feel a tremendous responsibility to be a body positive role model to my daughter. My consciousness around the way I talk about my body or anyone's body or physical appearance in general in front of my daughter causes me to be more conscious about these thoughts even when she isn't around.
My son was born in a hospital where I had an epidural after I realized that labor is no joke, an episiotomy, and after pushing for several hours, my son was helped out with a vacuum. Days later when my milk came in, my rock-hard breasts were about five times their original size and my nipples could barely withstand the pain from still figuring out our latch. We did eventually figured it out and I healed up just fine. I did have a lot of family support, but I may have experienced a little postpartum depression, and definitely some loneliness and isolation because of being in such a different place from my peer group.
My daughter was born in a birthing center sixteen years later. In the throes of labor, I remembered why I opted to have an epidural the first time around! However, I did feel like a badass superwoman and I still feel a sense of strength when I remember what my body is capable of. I ended up with a small tear and chose not to get stitches (I couldn't bear the thought of being sewn up down there) so was put on bed rest for the first week postpartum. I got to spend an entire week holding her and nursing. I didn't think I'd have any trouble nursing since I'd done it before, but I still needed the guidance of a lactation consultant to get it down.
After an entire week in our house my body was feeling really good so we walked around the neighborhood and I started bleeding more heavily. My body was telling me to take it easy. We continued this dialogue where I would be a little too active and my body would respond by bleeding for six weeks postpartum. Eventually I did learn to listen to my body and slowly worked up to the level of activity that feels right for me.
I wanted to participate in this project because of the way it showcases the beauty of all mothers, regardless of their shape.
I would tell my former self or a new parent to not be afraid to accept help from others. If someone offers to hold your baby for 15 minutes so you can take a shower, let them! If a friend wants to bring dinner so you can bond with your family instead of working in the kitchen, graciously accept. If people offer to help, it's because they want to. You can pay it forward later.
Tags: Portland OR, 4th Trimester Bodies, 4th Trimester, Teen Mom, Epidural, vacuum assistance, Breastfeeding, Hospital Birth, Birth Center, Vaginal Tearing, Bed Rest, Lactation Consultant
Hannah Holdsworth
January 20, 2017 in 4th Trimester Bodies
Hannah Holdsworth, Walter (5), and Hugo (18 months)
When Hannah was young she was told that she might have a hard time getting pregnant because of the tilt of her uterus. She's since learned that this is a very common thing and is not sure why the doctor would have said this to her and it has proved to be the opposite of true. She became pregnant unexpectedly before she had the wherewithal to take care of a child and chose to terminate the pregnancy. "The thought of it makes me sad but I can't say I regret it. Knowing how challenging being a mother can be I am not sure how I would have taken care of a child then".
She has always wanted to be a mother but it's not something she spent a lot of time thinking about or planning for. She got pregnant the first month they tried and was really hoping to have an unmedicated natural birth with her first. She labored naturally for about 20 hours but found being in the hospital really tough and was not comfortable at all. She panicked once she hit transition and though her doula told her it wouldn't be much more time, she didn't believe her. Her contractions were right on top of each other leaving her no space to catch her breath. The doctor administering the epidural was very rude and lectured her about how this was, "good medicine," when she expressed her disappointment at needing it. He was so rude the nurses got cross with him and he eventually left.
Less than an hour later she was fully dilated and ready to push. She pushed for 3 hours but was so tired. "Wally was born with a vacuum assist. He had a bruise on his head but other than that a perfect little Christmas elf (he was born on Christmas day). The nurses in the hospital were amazing and everyone worked with us so we could leave the hospital after just 1 day instead of 2". Hannah bonded with Wally without problems but also developed a fair amount of anxiety postpartum. She met with a therapist and just hearing how normal everything she was experiencing was helped a ton. Breastfeeding went really well and she was even able to donate a lot of milk to another baby in Brooklyn. Wally weaned (for the most part) when she was pregnant with Hugo of his own accord right around his 3rd birthday.
Pregnancy with Hugo was a surprise. "It was wonderful because it took the question out of finding the perfect time to have another. My son knew I was pregnant and asked to see the baby inside me before we had told anyone. I am kind of convinced he willed his little brother into existence". Hannah decided to have a home birth with Hugo because she knew the hospital had made her uncomfortable the first time. Labor with Hugo lasted all day but with strong contractions about 10/15 minutes apart. They got closer together at about 8:30pm and he was born at 10:06pm. "It was the most wonderful, manageable labor anyone could ask for. And I was so happy to be in my bedroom with my husband that night and to have Wally asleep in the room next door. It was an incredibly cozy experience and one that felt very healing to me. Breastfeeding came easily again. I still have anxiety but no where near as much as after having my first".
"Even though I find myself critical of my own body, I feel very strongly that a woman's body should be celebrated. I think we should do away with the concept and phrase, "bouncing back." I think a woman is forever changed after pregnancy and/or giving birth so there is no real going back - and any emphasis should be on her emotional wellbeing and not on her body! I think a lot of women have little idea what to expect after giving birth and the more we all share the better. So many feelings and emotions are normal and if we don't all talk about them it makes it so much harder".
Tags: New York City, Hospital Birth, Homebirth, Breastfeeding, Milk Sharing, Termination, vacuum assistance
Jenna Mitchell
Jenna Mitchell and Andrew Joseph (21 months) Jenna also miscarried a pregnancy at 7 weeks in September 2016.
Jenna says that she has wanted to be a mother since a very young age, and was thrilled when she found out she was pregnant just 2 months after her wedding, two cycles after going off the birth control pill. Her pregnancy was fairly uneventful, with only a bout of carpel tunnel syndrome from 20 weeks until the end of the pregnancy. Jenna's due date came and went and she saw her doctor for a routine appointment at 40 weeks 4 days where he stripped her membranes.
At 5am the following day, her water broke and they headed to the hospital. Jenna had fully intended to have a drug and intervention free birth, however, once in labor and delivery they immediately started Pitocin despite her requests. The contractions became so strong and so fast that she decided to have an epidural at 4pm just to get some relief and was able to rest comfortably but was too excited to sleep. She started to push at 1:30am and there was very little progress. She pushed for three hours before they had her stop for an hour to let the baby get further into the birth canal. By this point, the epidural was no longer working and she was in so much discomfort.
After 23 hours in labor, she was a mess. They suggested the vacuum and she was warned of the risk of needing a cesarean but she was so exhausted that she just needed the baby to come out so she went for it. Finally, Andrew Joseph entered the world at 8 pounds 12 ounces. Jenna tore on her own and felt every stitch sewing her back up. She had every intention to breast feed, and tried for the first two weeks. Andrew wasn't gaining weight so we went to see a lactation consultant and she was diagnosed with hypoplasia/insufficient glandular tissue. She was on a very strict feed and pump schedule for a week and it made life very difficult. "I felt as though we were so consumed by ounces and formula and pumping, which was made even more difficult by a sleepy baby and non-existent milk production. It was putting a lot of stress on both my relationship with my son and my relationship with my husband. We finally made the decision at 15 days old to exclusively formula feed. I had a lot of guilt but I knew it was the best thing for our family". Andrew started gaining weight and he was a happy, healthy and alert baby.
When he turned 6 weeks old, Jenna became overwhelmed with sadness. She thought constantly about how her maternity leave was half way over and how much she was going to miss him once she went back to work. Once she went back to work she was still so consumed by sadness and dread. She finally realized that she was suffering from postpartum depression. "I worked with my therapist and started on an anti-depressant. I could feel my mood lighten and joy renter my life. I worked with my therapist and 6 months later I was able to stop taking the medication. We found our groove with working and drop offs and pick ups and our routines were going strong".
When Andrew was 17 months old they found out they were pregnant again. The symptoms weren't as strong as with her first, but she just chalked it up to every pregnancy being different. At 7 weeks she started bleeding and had an ultrasound where they found an empty sac. Within the next week everything worked its way out and the pregnancy was over. She was shocked by the intense hormonal shift but feels strengthened by the experience.
"I have had weight and body image issues throughout my whole life, suffering from disordered eating patterns throughout high school and college and even into adulthood. Throughout pregnancy and into motherhood, I have made peace with my body. I have found that since following this project they aren't as intense as they once were, and for that I am grateful. I am proud of my body. I want to promote body positivity and encourage others to be proud of who they are".
Tags: New York City, Miscarraige, Induction, Epidural, vacuum assistance, Formula Feeding, Insufficient Glandular Tissue, mammary hypoplasia
Celeste Johnson
November 10, 2016 in 4th Trimester Bodies
The stunning Celeste Johnson, Lily (5), and Mila (3)
Celeste says that she had dreamed for years of birthing a daughter that looked exactly like her father, long before she even considered getting pregnant. She feels incredibly lucky to have conceived on the very first try without any protection and never doubted that it was the girl she had been dreaming about.
Her name was Lily Echo and she was born at 40 weeks 4 days after Celeste was induced with Pitocin and had her water broken manually. Labor was 17.5 hrs; long and stressful. Her body kept stalling and she had to push for 2 long hours. She was determined to let her body tear instead of having a episiotomy but her body wouldn't tear! "I clearly remember my OB telling me we could keep going another hour or more or let her cut me and we'd have a baby in under 10 minutes. I consented because I felt my body couldn't take anymore". Lily was born a few minutes later with the assistance of the episiotomy and a vacuum. She came out with the cord around her neck and was unresponsive due to being in the birth canal too long. She took several minutes to come around and it was found she had low oxygen and blood sugar.
While the birth was difficult, Celeste says that her biggest heartbreak was breastfeeding. "I thought it would be easy for me but Lily wasn't interested. After a day of refusing to nurse I sobbed my way to the store to buy formula. It was more important to make sure she ate so we had formula until I could pump enough. I pumped exclusively until she was 4 months then we went back to formula. I was working full time and couldn't pump enough to meet her needs".
27 months later, Mila Rebecca was born and it was such an amazing and empowering birth compared to her first. Her water broke at home and she decided to shower and get her older daughter to day care before going into the hospital. She was able to walk, use the birth ball and feel in control of getting her body ready.
Unfortunately, things moved slowly and the contractions stopped and started even with Pitocin. She became so exhausted they stopped everything until she could get some rest. Mila was born after 24.5 hrs with vacuum assistance and an episiotomy. She was hell-bent to breastfeed this time and Mila was a champ. They breastfed until she was 17 months and even now she's still obsessed with her breasts.
"Parenthood is exhausting and endlessly rewarding. I loathe the mommy wars and do my best to support other mother's and not judge. I work full time and while I love my job I miss kids. I'm torn between feeling guilty for not wanting to be with them 24-7 and relief at the break and fulfillment I get from working".
"I have severe body issues and spend a lot of time hating myself for the way I look. I don't want to pass that to my girls. I think the project is amazing and a good way to get some catharsis - hopefully."
Tags: Raleigh, Episiotomy, vacuum assistance, Breastfeeding, Formula Feeding, Vaginal Birth
Lacey Schmidt
The lovely Lacey Schmidt, Miles (4.5) and Jack (almost 3).
Lacey is a solo parent of two beautiful, amazing boys. She had a difficult first pregnancy that was not planned and was very scary for her. Miles arrived weeks late and presented in a posterior position. She had planned to birth at home, but after hours of pushing had to transfer to the hospital where she ultimately delivered via cesarean. "It was my worst nightmare and I was ill prepared how to recover from it especially on my own. I remember it used to take me up to ten or fifteen minutes to rollover in the middle of the night to nurse my baby and for awhile I was so crazed I stopped taking my pain meds and ended up in the ER. I thought something was wrong but I was really in so much pain I was half out of my mind".
Miles was a high needs baby which became increasingly difficult for Lacey to cope with. And her body image was a constant struggle. She gained a lot of weigt during pregnancy and it didn't come of easily. "I was appalled by my body, even as it nourished my baby. I was overwhelmed, exhausted - and I realize now, seriously depressed. I told almost no one. I used to take him outside to play because it was the only place I knew we were safe, and I wouldn't cry because the neighbors were all around us and would often stop by or acknowledge us with a wave. He never took a bottle. He slept poorly. I was back at work part time at 6 weeks and full time at 10. His dad came maybe 2 or 3 weekends a month. I thought every day we might die".
Lacey conceived again unexpectedly just after Miles' first birthday. Her children's dad was thrilled but she was devastated. Lacey had started physical therapy just prior to conceiving again in hopes of understanding her birth and her body. She was finally willing to touch her c-section scar. "When asked about my birth, I would say - and still do - that my baby was cut out of my body. And now, I was pregnant again". She focused on eliminating most of the carbs from her diet in hopes of gaining less weight as her providers thought she may have had undiagnosed gestational diabetes in her first pregnancy. She hoped that in growing a smaller baby she could have a successful VBAC, which she was approved to pursue through her midwife group.
"I went into PT, I went to ICAN and birth circle meetings. I did Hypnobabies. I prepared my birth space, grew my community of mama's. I prepared to do this on my own, and everything hinged on a VBAC". She carried three days post dates and was in dial that she was laboring. Her body took over and by time her midwife arrived it was clear to everyone but her that she was ready to push. He was also malpositioned and she pushed hard for hours. Her vulva began to swell and she transferred to the hospital. The pain was intense and they called in a team recommending a repeat cesarean, citing that the baby wasn't descending as far as her midwife had thought. She spoke up for herself and ultimately decided to push with vacuum assistance. "I ended up pushing for almost 20 minutes, while my entire birth team - midwife, doula, birth photographer, mom - cheered for me like I was in the biggest event of my life. I was so tired and remember thinking as he crowned forever - "can they still section him and pulled him out if i just quit?" I didn't. He came out calm and perfect".
This birth was difficult on her low abs and pelvic floor and she's still healing today. Lacey went through serious PPD but this time, knew to get help. "I still don't know how we made it, that first year". She returned to work quickly and while it was a struggle is so grateful to have such great support from her extended family. She eventually took a new job and moved across the country away from her friends and family but has been making it work.
"Motherhood hasn't come easy to me - it's been a lot of hard work, and our circumstances have made it seem almost impossible most of the time to do anything but my career and mother my children. I know I need me in there too and we are finally getting to the point where I feel like I can care for myself better - so I'm doing more yoga, physical therapy so I can run again without feeling like my uterus is falling out, and doing some things i enjoy, like reading and traveling. The boys are always by my side and I've learned it's about integration, not balance, or sacrifice. What's happened has made me question so much about gender and perception of working women and my own ability and power. Dating after some of the comments that were made to me, and after two children so close and are still so young has also been really hard. I'm still nursing my youngest. My body still isn't mine. All that I thought this would be like...it hasn't been. At the same time it's all been better and bigger and I've grown so much as a person and as a woman than I ever thought possible. I'm so grateful to my boys for providing that".
"I wanted the opportunity to define my story, and come to some terms about how I have transformed -including physically. I'm so proud of what my body has and continues to do to care for my little family. It's such a different body than what it was - and so much better. My life is like that too. I feel so compelled to also start to share my story - and be proud of who I am, what we've become and continue to grow to be. Solo parenting is no joke; it's the hardest, sometimes crushing and always absolutely amazing and exhilarating work I've ever done. I want other women to also know and see what's possible. If your partner disappears that's ok. Your baby doesn't take a bottle? It's ok. You lose the weight, gain the weight, things tighten back up or you don't have time for that - it's all okay. Better than ok. I'd rather be out in this world with this body and these little men than to go back to the tight abs and perky breasts I used to have. Someday, I hope they see our participation here - and really in the world at large - and I hope they are super proud of me, and of themselves, and their role in transforming me, and maybe even helping transform someone else in this world. Women are so often belittled and diminished and made to feel like what we have to say, what we have to do, and what we look like isn't enough. In fact, we have all the power in the world. We are more than enough. We just have to be the ones who acknowledge that and put that forth in the world. Participating in this project is part of mine."
Tags: Albuquerque, Cesarean Birth, VBAC, Single Mom, Breastfeeding, vacuum assistance
Irene Barajas
April 11, 2016 in 4th Trimester Bodies
The beautiful Irene Barajas, Bella Mae (5), Devyn Kay (3), Kylee Ann (13 months).
Irene's first pregnancy was wonderful and the entire time her OB was on board with her plans and wishes. Her water broke about 12 days prior to her due date and she went into the hospital without labor really starting. Her family all came to visit and while she was progressing, it wasn't fast enough for her doctor. After about 16 hours, even though both she and baby were doing well the doctor pushed Pitocin. Irene began having back labor that was both intense and constant rather than the waves that contractions usually bring. She had an epidural which provided some relief but they kept the blood pressure cuff on her which meant that every 15 minutes she was woken up.
She continued to progress in the morning and eventually she was told she was ready to push even though she wasn't feeling any urge to do so. Irene pushed for 4 hours in extreme discomfort. Her doctor kept trying to stretch her perineum despite her asking for her to stop and attempted vacuum extraction three times unsuccessfully. Every time the vacuum came off Bella's head with a loud pop, Irene screamed at the thought of her babies head coming with it. It wasn't until recently that she realized how upset she still is about her experience.
Eventually, Irene was told that she was going to have a cesarean if she wasn't able to get her out baby out immediately and she says she found the strength somewhere in her pinkie toe to push her out. This birth experience informed her decisions after and she knew she wanted to birth differently with her future children.
Both Devyn and Kylee were born at home with the same midwife after very short labors. She woke up in labor with Devyn which was quite shocking after not experiencing the natural onset of labor with Bella. Devyn was born in the water and Bella was immediately bonded with her sister. Kylee was the only of her daughters to arrive after her due date and she had a smooth and uncomplicated labor. As she was pushing in the water, she pooped - as you do - and Bella came over to reassure her by saying, "It's okay mommy, I poop in the bathtub sometimes too!"
Irene hopes to create an environment in which her daughters can grow up confident in themselves and their bodies with a strong foundation unshakeable by the hang-ups our society perpetuates.
Tags: Portland OR, hospital birth, Homebirth, vacuum assistance, Pitocin, Epidural, Vaginal Birth
Laura Clark
The lovely Laura Clark and Leona (14 months). Laura has always had an innate feeling that she's meant to be a mother. She watched 'The Business of Being Born' long before she was ready to have her own children and it strongly informed what she desired for her own pregnancy and birth experience.
Laura had her IUD removed and was able to conceive after having just one cycle. Her pregnancy was amazing and she loved every second of it - she would do it a million times if she could. So much so, that she hopes to become a surrogate/gestational carrier after she's done having children of her own.
Laura knew she wanted to deliver at a free-standing birth center and had all of her prenatal care there. She took hypnobabies courses and read all the books she could to prepare for natural childbirth. Her water broke at 40 weeks 5 days but labor never really ramped up on it's own. A non-stress test revealed that Leona was doing well and her midwives gave her a cotton root tincture to try to get things going. After returning home to labor, Laura went back to the birthing center the following morning in hopes that the transition would help things pick up. She was only 2-3cms, too early for the birth center to allow her to stay there, so she went to a hotel and ultimately did everything they could to get things going - more tincture, inversion, nipple stimulations but nothing was helping. 36 hours after her water broke she had reached the maximum time the birth center would allow her to labor so she was transferred to hospital.
Laura went through transition while on the ride from the birthing center to the hospital and was nearly 8cms when she checked in. While it was what was needed, at the time it was a shock to suddenly be in a hands on sterile environment. She decided to get an epidural and cried tears of joy that she could finally relax and breathe. After about 4 more hours she was fully dilated but the baby's heart rate wasn't responding through contractions so they wanted to stimulate and once they stimulated by touching her head her heart rate was through the roof. Laura pushed for four hours and ended up having a vacuum assisted delivery. There was meconium present and Leona had an infection when she arrived earth side.
At the time, Laura was so caught up in the joy of her daughters arrival she didn't realize until the next day that nothing had happened the way she had dreamed for so long. For 4 or 5 months she found herself up in the middle of the night crying and unable to sleep thinking about how she could have done things differently. In hindsight, she realizes she may have had some postpartum depression. She feels lucky, thought that Leona has been an amazingly happy baby and joy to be with even though her birthing dreams weren't what she had hoped for.
Laura is a teacher and has been around kids her whole life so she thought motherhood would be a breeze. It's been quite the humbling experience learning that it's so much harder than she ever imagined but also that it's more amazing, she never knew you could love someone so much it almost hurts.
She thought breastfeeding would also be easy - "It's just what you do!" - and she didn't realize how hard it could be until she started. Leona had a very severe lip tie that led to a lot of pain with breastfeeding, nipple injuries and anxiety due to the pain. A lactation consultant helped her learn how to use a nipple shield which she continued using for her first three months. She is grateful it allowed her nipples to heal but it made feeding, and even more so feeding in public much more of a chore. Leona had two lip tie revisions before she was able to nurse without discomfort.
"Our bodies are so capable and amazing to create these little humans but our bodies are so changed in the process". It is a myth that breastfeeding causes the weight to melt away, that hasn't happened for me and sometimes I want my body back, but this body is the one that has grown and nourished her and I need to respect that".
Tags: Denver, birthing center, Birth Center, hospital birth, Epidural, vacuum assistance, Meconium, Interventions, Postpartum Depression, breastfeeding complications, breastfeeding, Lip Tie, Lactation Consultant, nipple shield
Jenn Gray
June 12, 2015 in 4th Trimester Bodies
The gorgeous Jenn Gray with her daughter Emme (4) and son Finn (17 months). Jenn had healthy pregnancies with each of her children and conception was not a concern either time. Her birth with Emme was pretty straight forward and things progressed fairly quickly. She labored at home for a long time and when she arrived at the hospital they were a bit upset she'd waited so long to come in. She labored down but when they check her they discovered Emme had her arm up by her face and she needed a bit of vacuum assistance to help her out. They were able to have a wonderful nursing relationship and she continued to nurse into her pregnancy with Finn. Jenn's second pregnancy was a bit more challenging in that she had a toddler at home as well. Labor started and progressed very well again this time but Finn was 10lbs 12oz and when she pushed his head out his shoulders became stuck and he also needed some assistance to be pulled out. She and Finn also have a wonderful, ongoing nursing relationship.
While Jenn is grateful that both of her children were born healthy she has continued to struggle with the fact that she needed some help getting them out. As a nurse and a mother she struggles with the question of whether she could have done more or been better education. On the other hand she knows that there isn't anything she could have done differently and hopes that taking part of this and sharing her story helps her to truly believe that.
Tags: Boise, Boise Idaho, hospital birth, vacuum assistance, breastfeeding, Vaginal Birth


This delicious and nourishing Instant Pot beef recipe makes a perfect carefree dinner!
Instant Pot Creamy Beef with Mushroom, Cream cheese, and Broccoli – This tender and nourishing Instant Pot beef recipe will feed a family of four and makes a perfect carefree dinner. Serve on top of mashed cauliflower and you have a hearty Keto/low carb meal that cooks super fast in the pressure cooker. While the list seems like a lot of ingredients, it is mostly pantry items you should have on hand. Enjoy!
1. Melt 2 tablespoons butter in the insert of your Instant Pot and brown beef on all sides for 2 to 3 minutes on Saute setting.
2. Add the onion, garlic, sliced mushroom, and broccolini. Season with black pepper, then add beef stock or water and cream cheese to the pot.
3. Add the pouch of the gravy to 1 cup of hot water and dissolve. Pour over the meat and other ingredients in the Instant Pot.
4. Set your Instant Pot to the meat/stew setting and cook on high pressure for 20 minutes. Do a quick release and you'll be ready to serve.
5. In the meantime, nuke cauliflower rice or florets in a large shallow plate with 1/2 cup water for 7 minutes, covered. Transfer to a food processor and puree until smooth. Serve with the creamy beef and mushroom stew. Enjoy!
If you'd like to make this recipe in your crock pot, sear the meat in a skillet. Brown the onions and mushrooms in the same skillet and add the gravy mix to your crock and cook on high 4 hours or low 8 to 10 hours.
If you like crunchier broccolini, you can add them to the pot after pressure cooking and keep in reheat mode for 5 minutes, or until broccolini is done to your liking.


Kaitlin Henninger joined The Nelson Company as Creative Director in 2012. Her professional experience includes marketing, sales, database development and inventory management. She has held positions at many established institutions including Towson University, University of Baltimore, the Walters Art Museum, and several private art galleries along the East Coast. She has a Bachelor of Arts and Masters Degree in Art and Art History from Towson University in Maryland.
At The Nelson Company, Kaitlin oversees creative operations unique to design development and overall branding strategy. She manages the social media team with an emphasis on audience engagement through blogging. She also oversees the company's cloud-based asset management system. One of her major roles at Nelson is designer and editor of a monthly newsletter that unifies Nelson's employees and locations in seven different states.
Kaitlin looks forward to showcasing The Nelson Company's creative capabilities while creating effective and efficient processes that will allow for growth and brand expansion.
Is flooding an issue? Plastic pallets to the rescue!
Happy Earth Day! Let's Dispel Wood Pallet Myths!


text:
Rentje Frederik (Renger) Witkamp (Vlaardingen, 9 maart 1959) is een Nederlands bioloog en farmacoloog.

Sinds 15 mei 2006 is hij hoogleraar "Voeding en Farmacologie" aan de Wageningen Universiteit en Researchcentrum, een vakgebied dat hij zelf omschrijft als "het wetenschapsgebied dat zich bezighoudt met het bestuderen van effecten van biologisch actieve stoffen in de voeding, met als doel een gezondheidsbevorderend of genezend effect te bewerkstelligen".

Voeding en Farmacologie werd bij de aanstelling van Renger Witkamp als nieuwe leerstoel ingesteld aan de Wageningen Universiteit (afdeling Humane Voeding).

Witkamp heeft biologie (1977-1980) en farmacie (1980-1986) gestudeerd in Utrecht. Zijn promotieonderzoek (1988-1992) vond plaats aan de faculteit Diergeneeskunde van de Universiteit Utrecht en had als onderwerp: "Effecten van hormonen op omzetting van geneesmiddelen". Hij was daar tussen 1996 en 2000 Universitair hoofddocent bij de afdeling Veterinaire Farmacologie (0,2 fte).

Van april 1996 tot eind 2010 werkte hij bij TNO Pharma (Zeist en Leiden) en heeft daar verschillende functies bekleed. Tot 1 mei 1996 gaf hij leiding aan TNO Pharma. Hij is sinds eind 2010 voltijds in dienst bij Wageningen University.

Persoonlijk
Witkamp is opgegroeid in Apeldoorn, is getrouwd en heeft twee dochters.

Farmacoloog
Nederlands bioloog
Voedingswetenschapper
Hoogleraar aan Wageningen University

redpajama_set_name: RedPajamaWikipedia


Welcome to our Ninaxu.com "Virus" Android removal instructions. They should work on most Android devices.
Why are there annoying pop-ups all over my smartphone's screen?
If you too are experiencing the same problem, where al sorts of annoying online ads start appearing on your screen as soon as you attempt to use your smartphone, then you've come to the right place. Here we will give you all the information you need to know about what is causing these obstructive adverts, as well as how you can effectively get rid of them and prevent the issue from reoccurring.
What's the purpose of adware programs?
Intrusive online ads are a very common complaint from both PC and android users. In both cases the problem is caused by a specific type of software called adware. Programs of that type exist for the sole purpose of generating those all sorts of pop-up, banners, box messages, etc and making it nearly impossible to use your device without interacting with them. Ninaxu.com "Virus" is an adware, that has been specifically developed to get on android devices and annoy its users with the various online ads it displays. The reason why those adverts are so incredibly annoying is because for each click/tap they receive, the developer of Ninaxu.com "Virus" gains revenue. This is method is called pay-per-click.
How do they get on my device and are they dangerous?
It is important to note that, as annoying as they are, adware programs/apps are not dangerous to your device. In most cases, the biggest problem with them is that they heavily obstruct your regular working process by displaying more and more adverts that sometimes cover your whole screen. The only way to get rid of those rage-inducing pop-ups adverts is to have the adware removed from your smartphone. To do that, you will need to know what has gotten you the unwanted piece of software in the first place. When it comes to android smartphones, the possible ways via which an adware might get on your device are limited. Usually Ninaxu.com "Virus" gets installed on your phone by being bundled with another application. In fact, in most cases the adware is a built-in feature to that other app, rather than a separate piece of programming. Therefore, the only way to fully remove Ninaxu.com "Virus" from your device, is to have the application that contains it uninstalled. Under this article, we have prepared a detailed guide on how to do that, so if you're currently dealing with an adware, go ahead and give ur guide a try.
Lastly, we ought to inform you that even though adware on itself is not dangerous for yous smartphone, the ads it displays may sometimes contain potential security hazards. Therefore, it is essential to remember to avoid tapping on them no matter how intrusive they may get. In most cases the ads are okay and won't threaten your device's safety, but there's still no need to needlessly expose your phone to potential dangers.