Chronic Persistent Infection Research Articles

A Nucleotide Sugar Transporter Involved in Glycosylation of the Toxoplasma Tissue Cyst Wall Is Required for Efficient Persistence of Bradyzoites

Toxoplasma gondii is an intracellular parasite that transitions from acute infection to a chronic infective state in its intermediate host via encystation, which enables the parasite to evade immune detection and clearance. It is widely accepted that the tissue cyst perimeter is highly and specifically decorated with glycan modifications; however, the role of these modifications in the establishment and persistence of chronic infection has not been investigated. Here we identify and biochemically and biologically characterize a Toxoplasma nucleotide-sugar transporter (TgNST1) that is required for cyst wall glycosylation. Toxoplasma strains deleted for the TgNST1 gene (Δnst1) form cyst-like structures in vitro but no longer interact with lectins, suggesting that Δnst1 strains are deficient in the transport and use of sugars for the biosynthesis of cyst-wall structures. In vivo infection experiments demonstrate that the lack of TgNST1 activity does not detectably impact the acute (tachyzoite) stages of an infection or tropism of the parasite for the brain but that Δnst1 parasites are severely defective in persistence during the chronic stages of the infection. These results demonstrate for the first time the critical role of parasite glycoconjugates in the persistence of Toxoplasma tissue cysts.

Bacillus Cereus

Bacillus Cereus

MicroRNA-27a Regulates Lipid Metabolism and Inhibits Hepatitis C Virus Replication in Human Hepatoma Cells

The replication and infectivity of the lipotropic hepatitis C virus (HCV) are regulated by cellular lipid status. Among differentially expressed microRNAs (miRNAs), we found that miR-27a was preferentially expressed in HCV-infected liver over hepatitis B virus (HBV)-infected liver. Gene expression profiling of Huh-7.5 cells showed that miR-27a regulates lipid metabolism by targeting the lipid synthetic transcription factor RXRα and the lipid transporter ATP-binding cassette subfamily A member 1. In addition, miR-27a repressed the expression of many lipid metabolism-related genes, including FASN, SREBP1, SREBP2, PPARα, and PPARγ, as well as ApoA1, ApoB100, and ApoE3, which are essential for the production of infectious viral particles. miR-27a repression increased the cellular lipid content, decreased the buoyant density of HCV particles from 1.13 to 1.08 g/cm(3), and increased viral replication and infectivity. miR-27a overexpression substantially decreased viral infectivity. Furthermore, miR-27a enhanced in vitro interferon (IFN) signaling, and patients who expressed high levels of miR-27a in the liver showed a more favorable response to pegylated IFN and ribavirin combination therapy. Interestingly, the expression of miR-27a was upregulated by HCV infection and lipid overload through the adipocyte differentiation transcription factor C/EBPα. In turn, upregulated miR-27a repressed HCV infection and lipid storage in cells. Thus, this negative feedback mechanism might contribute to the maintenance of a low viral load and would be beneficial to the virus by allowing it to escape host immune surveillance and establish a persistent chronic HCV infection.

T Lymphocytes from Chagasic Patients Are Activated but Lack Proliferative Capacity and Down-Regulate CD28 and CD3ζ

BackgroundChronic persistent infections have been associated with T lymphocytes functional impairment. The aim of this study was to compare the activation status, the proliferative potential and the expression of CD28 and CD3ζ chain on T lymphocytes between chronic chagasic patients and uninfected controls.Methodology/Principal FindingsForty-two chronic chagasic patients, 28 healthy individuals and 32 non-chagasic cardiomyopathy donors were included. Peripheral blood was marked for CD3, CD4, CD8, HLA-DR, CD28, CD38 and intracellular CD3ζ. Peripheral blood mononuclear cells were stained with carboxyfluorescein diacetate succinimidylester and incubated with T. cruzi lysate or phytohemagglutinin for five days. Cells from 3 healthy controls were incubated with T. cruzi trypomastigotes separated with transwells; and the expression of CD3ζ chain and proliferation index was determined. Heart-infiltrating cells from two chronic chagasic patients were tested for the aforementioned cellular markers. Chagasic patients displayed higher frequencies of CD4+/HLA-DR+/CD38+ (8.1%±6.1) and CD8+/HLA-DR+/CD38+ (19.8±8.9) T cells in comparison with healthy (1.6±1.0; 10.6±8.0) and non-chagasic cardiomyopathy donors (2.9±2.9; 5.8±6.8). Furthermore, the percentage of CD4+ activated T cells was higher in chagasic patients with cardiac involvement. CD8+ T cells proliferation index in chagasic donors (1.7±0.3) was lower when compared with healthy (2.3±0.3) and non-chagasic cardiomyopathy individuals (3.1±1.1). The frequencies of CD4+/CD28+ and CD8+/CD28+ T cells, as well as the CD3ζbright/CD3ζdim% ratios in CD4+ and CD8+ were lower in chagasic patients when compared with both control groups. The CD3ζbright/CD3ζdim% ratio and proliferative indexes for CD4+ and CD8+ T lymphocytes decreased gradually in those cells cultivated with parasites and displayed lower values than those incubated with medium alone. Finally, heart-infiltrating T cells from two T. cruzi infected patients also expressed activation markers and down-regulate CD28 and CD3ζ.ConclusionsCD8+ T lymphocytes from chagasic donors displayed reduced proliferative capacity, which might be associated with CD3ζ down-regulation and diminished CD28 expression on CD4 T cells.

Treatment of pseudomonas and Staphylococcus bronchopulmonary infection in patients with cystic fibrosis.

The optimal antibiotic regimen is unclear in management of pulmonary infections due to pseudomonas and staphylococcus in cystic fibrosis (CF). We systematically searched all the published literature that has considered the evidence for antimicrobial therapies in CF till June 2013. The key findings were as follows: inhaled antipseudomonal antibiotic improves lung function, and probably the safest/most effective therapy; antistaphylococcal antibiotic prophylaxis increases the risk of acquiring P. aeruginosa; azithromycin significantly improves respiratory function after 6 months of treatment; a 28-day treatment with aztreonam or tobramycin significantly improves respiratory symptoms and pulmonary function; aztreonam lysine might be superior to tobramycin inhaled solution in chronic P. aeruginosa infection; oral ciprofloxacin does not produce additional benefit in those with chronic persistent pseudomonas infection but may have a role in early or first infection. As it is difficult to establish a firm recommendation based on the available evidence, the following factors must be considered for the choice of treatment for each patient: antibiotic related (e.g., safety and efficacy and ease of administration/delivery) and patient related (e.g., age, clinical status, prior use of antibiotics, coinfection by other organisms, and associated comorbidities ones).

CD4 T Cell Responses in Latent and Chronic Viral Infections

The spectrum of tasks which is fulfilled by CD4 T cells in the setting of viral infections is large, ranging from support of CD8 T cells and humoral immunity to exertion of direct antiviral effector functions. While our knowledge about the differentiation pathways, plasticity, and memory of CD4 T cell responses upon acute infections or immunizations has significantly increased during the past years, much less is still known about CD4 T cell differentiation and their beneficial or pathological functions during persistent viral infections. In this review we summarize current knowledge about the differentiation, direct or indirect antiviral effector functions, and the regulation of virus-specific CD4 T cells in the setting of persistent latent or active chronic viral infections with a particular emphasis on herpes virus infections for the former and chronic lymphocytic choriomeningitis virus infection for the latter.

Clinical, radiological and histological diagnoses of periapical periodontitis spreading to the adjacent tooth: A case of endodontic failure

Aims: This article describes the apical infection in endodontically treated tooth 4.5 that spread to adjacent tooth 4.4. Case Report: A 52-year-old woman was referred for the presence of radiolucency extending from tooth 4.5 and mental foramen. Spontaneous symptoms were present. Tooth 4.5 showed poor-quality endodontics. The vitality of tooth 4.4 was negative, even though no mechanical trauma had been reported, nor was caries present. Both teeth were sensitive to percussion. Endodontic re-treatment of 4.5 and endodontic treatment of 4.4 were performed in a single visit. A large amount of endodontic sealer squeezed mesially from the root of tooth 4.5, where a partial horizontal root fracture was hypothesized. 6-, 12-, and 18-month radiographic follow-ups, by both periapical and cone-beam computed tomography (CBCT) analyses, showed incomplete osseous healing. CBCT excluded root fracture on 4.5. Local symptoms were still present. Clinical and radiological conditions led to extractions, and a cystic lesion was enucleated for histopathologic analyses. Histopathologic diagnosis was a periapical cyst. The supposed partial horizontal root fracture of 4.5 was actually a large lateral canal. Although the root canal treatments followed high standards in terms of quality, a persistent chronic infection developed histologically. The cystic lesion was one consistent reason for the unsuccessful healing of 4.5.

Bacterial Biofilm Formation Induces Strong Shifts in Lipid Composition Resulting in Increased Resistance Towards Antimicrobial Peptide Activity

Bacteria that interact with surfaces under hydrated conditions can form aggregated structures known as biofilms. Biofilms are characterized by having increased resistance to a variety of antibacterial agents. This resistance is responsible for the generation of persistent chronic infections, and represents a serious threat to human health. Several antimicrobial agents, including hydrolytic enzymes such as PLA2-IIA and antimicrobial peptides (AMPs) such as Magainin-2, act by disrupting bilayer membrane integrity. Since these antimicrobial agents require physical disruption of the bilayer membrane, their activity is likely to be sensitive to lipid packing.. In this work we show, by measuring generalized polarization of Laurdan incorporated into lipid extracts, drastic changes in the level of lipid packing in Staphylococcus aureus during biofilm formation. When analyzing lipid composition we find a significant reduction in the level of branched lipids in the biofilm membranes. A strong reduction in the level of carotenoids is also observed during biofilm formation. Additionally, we present evidence that this shift in the melting temperature modulates resistance towards magainin-2 at 37C where bacterial lipids are in the liquid-crystalline phase. These results point to a mechanism by which bacterial membranes can generate resistance towards membrane active antibacterial agents through the modulation of lipid composition during biofilm formation.

Implications of anti-cytokine therapy in colorectal cancer and autoimmune diseases

Up to 20% of all cancers have been linked to chronic inflammation and persistent infections. However, almost all solid tumours contain immune infiltrates, and tumour-associated inflammatory cells play broad roles...

Hepatitis E Virus: A Novel Opportunistic Pathogen in Recipients of Allogeneic Hematopoietic Stem Cell Transplantation

AbstractAbstract 4137Hepatitis E virus (HEV) is an emerging pathogen in developed countries. It may cause an acute, most times self-limiting viral hepatitis in healthy individuals. However, persistent chronic infection and cirrhosis in immunocompromised patients, such as recipients of solid organ transplantation, have recently been reported. Sofar, the incidence and sequela of hepatitis due to HEV is unknown in recipients of allogenic stem cell transplantation (alloHSCT). Following two recent cases, we set out to evaluate the point prevalence and clinical sequela of HEV infection in a recent cohort of alloHSCT recipients. 328 patients who received an alloHSCT between January 2006 and July 2011 were included. Recent follow-up plasma samples taken after transplantation were screened for the presence of HEV RNA by real-time PCR. In addition, available plasma samples from all episodes with CTC grade 2–4 liver function abnormalities were retrospectively screened for HEV RNA. Chronic HEV infection was defined as having HEV RNA in serum or plasma lasting for more than 6 months. Furthermore, phylogenetic analysis was performed to determine genotype and to exclude a common source of infection and to examine potential HEV reactivation.The cohort included 178 male and 150 female patients with a median age at transplantation of 50 (range: 17–66) years. Stem cell sources and donors included sibling donors (n=149), unrelated donors (n=141), and 48 umbilical cord blood (UCB) grafts. 207 episodes of liver function abnormalities were evaluated in addition to the cross-sectional RT-PCR analysis. In total, eight cases (2.4%) of HEV were found, of which six were found in cross-sectional RT-PCR analysis and two by evaluation of abnormal liver functions. Furthermore, HEV specific IgG could be detected prior to alloHSCT in 12.9% of the patients. Two patients (0.6%) were IgM positive, though HEV viremia could not be detected by PCR. Phylogenetic analyses did not reveal a common viral strain in these patients. The median age of HEV infected patients was 56 (range 39–66) months, with 5 males and 3 females. All eight patients were transplanted with grafts from an alternative donor, including MUD (n=5) and UCB (n=3). The median time from alloHSCT to infection was 4.6 (range: 2–18) months and the median duration of HEV infection was 6.4 (range: 2–42) months. The median of maximum PT (ALAT) values at infection was 289 (range: 138–1507) U/l, and median of maximum ferritin levels prior to infection was 1842 (range: 104–5049) ug/l. At the time of infection, six patients were receiving intensive immunosuppression, prescribed for prevention of GVHD (n=2) or for GVHD treatment (n=4). HEV infected patients were mistakenly diagnosed before as hepatic graft-versus-host disease (GVHD) (n=5), or drug-toxicity (n=3). Remarkably, one patient presented with two episodes of viremia with negative HEV PCR in between, characterized as viral reactivation by identical HEV ORF2 sequences, while no HEV specific IgG or IgM antibodies were developed. Five patients failed to rapidly clear HEV and developed chronic HEV infection. Four patients died with HEV viremia and signs of ongoing hepatitis, whereas four other patients cleared HEV within a median period of 8.8 (range: 2–42) months, containing two patients with chronic hepatitis and fibrosis diagnosed with liver biopsy.In conclusion, although HEV is a relatively infrequent opportunistic pathogen after alloHSCT, HEV may be associated with persistent viremia and the development of chronic active hepatitis, especially in recipients of alternative donor alloHSCT. In addition, recipients of alloHSCT with positive serology for hepatitis E prior transplantation may develop viral reactivation leading to hepatitis. Given the relative frequent diagnosis of hepatic GVHD and/or drug associated liver toxicity, a differential diagnosis including hepatitis E is mandatory. Future alloHSCT recipients from endemic area's should be screened for hepatitis E prior to transplantation and may be monitored during episodes of intensive immunnosuppressive therapy. Disclosures:No relevant conflicts of interest to declare.

Ex vivotranscriptional profiling reveals a common set of genes important for the adaptation ofPseudomonas aeruginosato chronically infected host sites

The opportunistic bacterium Pseudomonas aeruginosa is a major nosocomial pathogen causing both devastating acute and chronic persistent infections. During the course of an infection, P. aeruginosa rapidly adapts to the specific conditions within the host. In the present study, we aimed at the identification of genes that are highly expressed during biofilm infections such as in chronically infected lungs of patients with cystic fibrosis (CF), burn wounds and subcutaneous mouse tumours. We found a common subset of differentially regulated genes in all three in vivo habitats and evaluated whether their inactivation impacts on the bacterial capability to form biofilms in vitro and to establish biofilm-associated infections in a murine model. Additive effects on biofilm formation and host colonization were discovered by the combined inactivation of several highly expressed genes. However, even combined inactivation was not sufficient to abolish the establishment of an infection completely. These findings can be interpreted as evidence that either redundant traits encode functions that are essential for in vivo survival and chronic biofilm infections and/or bacterial adaptation is considerably achieved independently of transcription levels. Supplemental screens, will have to be applied in order to identify the minimal set of key genes essential for the establishment of chronic infectious diseases.

Stenotrophomonas maltophilia

Stenotrophomonas maltophilia can cause persistent airway colonization and chronic infection in cystic fibrosis (CF) patients. The clinical effect of chronic S. maltophilia infection is still a matter of debate. The purpose of the review is to summarize studies published during the last 1.5 years with a focus on whether S. maltophilia should be regarded as a 'true' CF pathogen. Studies concerning the microbiological characteristics of this microorganism with special focus on identification, virulence factors, cross-infection and resistance pattern, and studies on clinical effects of chronic infection, specifically serological responses to this infection, negative effects on the clinical status and possible effects of antibiotic treatment have been reviewed. S. maltophilia can be identified with certainty, has well-described virulence factors, is capable of cross-infection and often shows multiresistance. Although CF patients with chronic S. maltophilia infection have lower lung function parameters compared with other CF patients and experience more exacerbations, chronic S. maltophilia infection in itself was not associated with a worse clinical outcome. True, pathogenic effects of S. maltophilia could not be found, but the presence of chronic S. maltophilia could be a marker of more pronounced lung disease in affected patients.

HTLV‐1‐associated infective dermatitis: updates on the pathogenesis

HTLV-1-associated infective dermatitis (HAID) is the main paediatric manifestation of human T-cell lymphotropic virus type 1 (HTLV-1). It is characterised by a chronic exudative eczematous eruption and persistent infection with Staphylococcus aureus (SA) and beta-haemolytic streptococci (BHS). Prevalence is highest in the Caribbean and Brazil; however, cases have been reported in other HTLV-1 endemic regions. Approximately 20 million people worldwide are infected with HTLV-1 and only 5-10% suffer from disease. Other manifestations include adult T-cell leukaemia/lymphoma (ATLL) and tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM). HAID may also progress to ATLL or TSP/HAM. Treatment options are limited to prolonged antibiotic therapy. The aim of this paper is to review existing evidence and propose new theories on the pathogenesis of HAID. The current view is that HTLV-1 infection is required and in susceptible individuals leads to immune dysregulation with subsequent immunosuppression and superinfection with SA and BHS. Evidence suggests that host, environment and genetic factors may play a causative role. Genetic factors within ethnic groups determine host immune response and carrier state or disease manifestation of HTLV-1 infection. Increased IgE levels may contribute to the SA and BHS superinfection in HAID. Additionally, the possible impact of filaggrin, skin proteinase dysregulation, Langerhans cell dysfunction and TH2 chemokines is highlighted. More than 45years since the discovery of HAID, the exact pathogenesis is still not fully understood. Further research is still needed to clearly elucidate the exact pathogenic mechanism of HAID.

Neurological Complications of Hepatitis C Infection

Though well-known as a cause of liver disease, Hepatitis C virus infection is emerging as a cause of a variety of peripheral and central nervous system disorders. The virus causes chronic persistent infection with complex immune responses in the majority of individuals. Viral infection may have the potential to generate neurological illness through direct infection of neural cells or through immune-mediated mechanisms, including enhancement of autoimmune responses. Moreover, the mainstay of antiviral treatment of hepatitis C infection, interferon-alpha, is itself associated with neurological morbidity. Thus neurologists are increasingly faced with diagnosing or even predicting a wide spectrum of neurological complications of hepatitis C infection and/or its treatment.

Use of ATP analogs to inhibit HIV-1 transcription

Human immunodeficiency virus type 1 (HIV-1) is the etiological agent of AIDS. Chronic persistent infection is an important reason for the presence of “latent cell populations” even after Anti-Retroviral Therapy (ART). We have analyzed the effect of ATP analogs in inhibiting cdk9/T1 complex in infected cells. A third generation drug named CR8#13 is an effective inhibitor of Tat activated transcription. Following drug treatment, we observed a decreased loading of cdk9 onto the HIV-1 DNA. We found multiple novel cdk9/T1 complexes present in infected and uninfected cells with one complex being unique to infected cells. This complex is sensitive to CR8#13 in kinase assays. Treatment of PBMC with CR8#13 does not kill infected cells as compared to Flavopiridol. Interestingly, there is a difference in sensitivity of various clades to these analogs. Collectively, these results point to targeting novel complexes for inhibition of cellular proteins that are unique to infected cells.

Tim-3 Negatively Regulates Cytotoxicity in Exhausted CD8+ T Cells in HIV Infection

Cytotoxic CD8+ T cells (CTLs) contain virus infections through the release of granules containing both perforin and granzymes. T cell ‘exhaustion’ is a hallmark of chronic persistent viral infections including HIV. The inhibitory regulatory molecule, T cell Immunoglobulin and Mucin domain containing 3 (Tim-3) is induced on HIV-specific T cells in chronic progressive infection. These Tim-3 expressing T cells are dysfunctional in terms of their capacities to proliferate or to produce cytokines. In this study, we evaluated the effect of Tim-3 expression on the cytotoxic capabilities of CD8+ T cells in the context of HIV infection. We investigated the cytotoxic capacity of Tim-3 expressing T cells by examining 1) the ability of Tim-3+ CD8+ T cells to make perforin and 2) the direct ability of Tim-3+ CD8+ T cells to kill autologous HIV infected CD4+ target cells. Surprisingly, Tim-3+ CD8+ T cells maintain higher levels of perforin, which was mainly in a granule-associated (stored) conformation, as well as express high levels of T-bet. However, these cells were also defective in their ability to degranulate. Blocking the Tim-3 signalling pathway enhanced the cytotoxic capabilities of HIV specific CD8+ T cells from chronic progressors by increasing; a) their degranulation capacity, b) their ability to release perforin, c) their ability to target activated granzyme B to HIV antigen expressing CD4+ T cells and d) their ability to suppress HIV infection of CD4+ T cells. In this latter effect, blocking the Tim-3 pathway enhances the cytotoxcity of CD8+ T cells from chronic progressors to the level very close to that of T cells from viral controllers. Thus, the Tim-3 receptor, in addition to acting as a terminator for cytokine producing and proliferative functions of CTLs, can also down-regulate the CD8+ T cell cytotoxic function through inhibition of degranulation and perforin and granzyme secretion.

Ivacaftor, vismodegib, and ingenol mebutate

Ivacaftor, vismodegib, and ingenol mebutate

Relationship between HLA-DP gene polymorphisms and clearance of chronic hepatitis B virus infections: Case–control study and meta-analysis

Hepatitis B virus (HBV) infection is a serious public health problem worldwide. Two common genetic variants (rs3077 and rs9277535) of human leukocyte antigen DP (HLA-DP) have been reported to be associated with persistent HBV infection in populations of Japan and Thailand. To confirm whether the association can be replicated in Chinese populations, an independent case–control study were conducted, and two polymorphisms (rs3077 and rs9277535) were genotyped using the TaqMan SNP genotyping assay in 282 persistent chronic HBV carriers and 64 spontaneously HBV recovered carriers. To provide a more definitive conclusion, a meta-analysis combining and summarizing five studies was performed by random-effects model using the DerSimonian and Laird’s method. By using logistic regression analysis with adjustment for covariates, including age, sex, and alcohol consumption, the results of our independent case–control study showed that the minor allele’s homozygote (AA genotype) of rs3077 and rs9277535 was significantly associated with decreasing risk/protection of HBV persistent chronic infection (for rs3077: P=0.0017, OR=0.29, 95% CI=0.13–0.62; for rs9277535, P=0.0004, OR=0.26, 95% CI=0.12–0.54). The results of meta-analysis pooling all eligible studies also showed that rs3077-A and rs9277535-A alleles were associated with an increased clearance rate to HBV infection (rs3077: OR=0.57, 95% CI=0.44–0.75; rs9277535: OR=0.56, 95% CI=0.47–0.63). These results further confirmed the strong influence of HLA-DP gene variants on risk of spontaneous HBV clearance from persistent HBV infection. Both A alleles of HLA-DP SNP rs3077 and rs9277535 showed strong protective effects for spontaneous HBV clearance from persistent HBV infection in the Han Chinese population.

Targeting Persisters for Tuberculosis Control

Mycobacterial persisters, the survivors from antibiotic exposure, necessitate the lengthy treatment of tuberculosis (TB) and pose a significant challenge for our control of the disease. We suggest that persisters in TB are heterogeneous in nature and comprise various proportions of the population depending on the circumstances; the mechanisms of their formation are complex and may be related to those required for persistence in chronic infection. Results from recent studies implicate multiple pathways for persister formation, including energy production, the stringent response, global regulators, the trans-translation pathway, proteasomal protein degradation, toxin-antitoxin modules, and transporter or efflux mechanisms. A combination of specifically persister-targeted approaches, such as catching them when active and susceptible either by stimulating them to "wake up" or by intermittent drug dosing, the development of new drugs, the use of appropriate drug combinations, and combined chemotherapy and immunotherapy, may be needed for more effective elimination of persisters and better treatment of TB. Variations in levels of persister formation and in host genetics can play a role in the outcome of clinical treatment, and thus, these may entail personalized treatment regimens.

Infected Dendritic Cells Facilitate Systemic Dissemination and Transplacental Passage of the Obligate Intracellular Parasite Neospora caninum in Mice

The obligate intracellular parasite Neospora caninum disseminates across the placenta and the blood-brain barrier, to reach sites where it causes severe pathology or establishes chronic persistent infections. The mechanisms used by N. caninum to breach restrictive biological barriers remain elusive. To examine the cellular basis of these processes, migration of different N. caninum isolates (Nc-1, Nc-Liverpool, Nc-SweB1 and the Spanish isolates: Nc-Spain 3H, Nc-Spain 4H, Nc-Spain 6, Nc-Spain 7 and Nc-Spain 9) was studied in an in vitro model based on a placental trophoblast-derived BeWo cell line. Here, we describe that infection of dendritic cells (DC) by N. caninum tachyzoites potentiated translocation of parasites across polarized cellular monolayers. In addition, powered by the parasite's own gliding motility, extracellular N. caninum tachyzoites were able to transmigrate across cellular monolayers. Altogether, the presented data provides evidence of two putative complementary pathways utilized by N. caninum, in an isolate-specific fashion, for passage of restrictive cellular barriers. Interestingly, adoptive transfer of tachyzoite-infected DC in mice resulted in increased parasitic loads in various organs, e.g. the central nervous system, compared to infections with free parasites. Inoculation of pregnant mice with infected DC resulted in an accentuated vertical transmission to the offspring with increased parasitic loads and neonatal mortality. These findings reveal that N. caninum exploits the natural cell trafficking pathways in the host to cross cellular barriers and disseminate to deep tissues. The findings are indicative of conserved dissemination strategies among coccidian apicomplexan parasites.