Molecular Characterization of Echinococcus granulosus Cysts in North Indian Patients: Identification of G1, G3, G5 and G6 Genotypes

Background

Cystic echinococcosis (CE) caused by the Echinococcus granulosus, is a major public health problem worldwide, including India. The different genotypes of E. granulosus responsible for human hydatidosis have been reported from endemic areas throughout the world. However, the genetic characterization of E. granulosus infecting the human population in India is lacking. The aim of study was to ascertain the genotype(s) of the parasite responsible for human hydatidosis in North India.

Methodology/Principal Findings

To study the transmission patterns of E. granulosus, genotypic analysis was performed on hydatid cysts obtained from 32 cystic echinococcosis (CE) patients residing in 7 different states of North India. Mitochondrial cytochrome c oxidase subunit1 (cox1) sequencing was done for molecular identification of the isolates. Most of the CE patients (30/32) were found to be infected with hydatid cyst of either G3 (53.1%) or G1 (40.62%) genotype and one each of G5 (cattle strain) and G6 (camel strain) genotype.

Conclusions/Significance

These findings demonstrate the zoonotic potential of G1 (sheep strain) and G3 (buffalo strain) genotypes of E. granulosus as these emerged as predominant genotypes infecting the humans in India. In addition to this, the present study reports the first human CE case infected with G5 genotype (cattle strain) in an Asian country and presence of G6 genotype (camel strain) in India. The results may have important implications in the planning of control strategies for human hydatidosis.     

Alterations of the Ileal and Colonic Mucosal Microbiota in Canine Chronic Enteropathies

Background

The intestinal microbiota is increasingly linked to the pathogenesis of chronic enteropathies (CE) in dogs. While imbalances in duodenal and fecal microbial communities have been associated with mucosal inflammation, relatively little is known about alterations in mucosal bacteria seen with CE involving the ileum and colon.

Aim

To investigate the composition and spatial organization of mucosal microbiota in dogs with CE and controls.

Methods

Tissue sections from endoscopic biopsies of the ileum and colon from 19 dogs with inflammatory bowel disease (IBD), 6 dogs with granulomatous colitis (GC), 12 dogs with intestinal neoplasia, and 15 controls were studied by fluorescence in situ hybridization (FISH) on a quantifiable basis.

Results

The ileal and colonic mucosa of healthy dogs and dogs with CE is predominantly colonized by bacteria localized to free and adherent mucus compartments. CE dogs harbored more (P < 0.05) mucosal bacteria belonging to the Clostridium-coccoides/Eubacterium rectale group, Bacteroides, Enterobacteriaceae, and Escherichia coli versus controls. Within the CE group, IBD dogs had increased (P < 0.05) Enterobacteriaceae and E. coli bacteria attached onto surface epithelia or invading within the intestinal mucosa. Bacterial invasion with E. coli was observed in the ileal and colonic mucosa of dogs with GC (P < 0.05). Dogs with intestinal neoplasia had increased (P < 0.05) adherent (total bacteria, Enterobacteriaceae, E. coli) and invasive (Enterobacteriaceae, E. coli, and Bacteroides) bacteria in biopsy specimens. Increased numbers of total bacteria adherent to the colonic mucosa were associated with clinical disease severity in IBD dogs (P < 0.05).

Conclusion

Pathogenic events in canine CE are associated with different populations of the ileal and colonic mucosal microbiota.     

Polyfunctional Specific Response to Echinococcus Granulosus Associates to the Biological Activity of the Cysts

Background

Cystic echinococcosis (CE) is a complex disease caused by Echinococcus granulosus (E.granulosus), and its immunophatogenesis is still not clearly defined. A peculiar feature of chronic CE is the coexistence of Th1 and Th2 responses. It has been suggested that Th1 cytokines are related to disease resistance, whereas Th2 cytokines are related to disease susceptibility and chronicity. The aim of this study was to evaluate, by multi-parametric flow cytometry (FACS), the presence of CE specific immune signatures.

Methodology/Principal Findings

We enrolled 54 subjects with suspected CE; 42 of them had a confirmed diagnosis, whereas 12 were classified as NO-CE. Based on the ultrasonography images, CE patients were further categorized as being in "active stages" (25) and "inactive stages" (17). The ability of CD4⁺ T-cells to produce IFN-γ, IL-2, TNF-α, Th2 cytokines or IL-10 was assessed by FACS on antigen-specific T-cells after overnight stimulation with Antigen B (AgB) of E.granulosus. Cytokine profiles were evaluated in all the enrolled subjects. The results show that none of the NO-CE subjects had a detectable AgB-specific response. Among the CE patients, the frequency and proportions of AgB-specific CD4⁺ T-cells producing IL-2⁺TNF-α⁺Th2⁺ or TNF-α⁺Th2⁺ were significantly increased in the “active stages” group compared to the “inactive stages” group. Moreover, an increased proportion of the total polyfunctional subsets, as triple-and double-functional CD4 T-cells, was found in CE patients with active disease. The response to the mitogen, used as a control stimulus to evaluate the immune competence status, was characterized by the same cytokine subsets in all the subjects enrolled, independent of CE.

Conclusions

We demonstrate, for the first time to our knowledge, that polyfunctional T-cell subsets as IL-2⁺TNF-α⁺Th2⁺ triple-positive and TNF-α⁺Th2⁺ double-positive specific T-cells associate with cyst biological activity. These results contribute to increase knowledge of CE immunophatogenesis and the disease outcome in terms of control and persistence.     

Surveillance on the Status of Immune Cells after Echinnococcus granulosus Protoscoleces Infection in Balb/c Mice

Background

Cystic echinococcosis is a global parasitic disease caused by infection with Echinococcus granulosus larvae with potentially life-threatening complications in humans. To date, the status of the immune cells believed to be associated with the pathogenicity of E. granulosus infection has not been demonstrated clearly.

Methodology/Principal Findings

In this study, we developed a multiplex flow cytometry assay to investigate the systemic immune status of innate and adaptive immunity at 30, 180, 360 days post-infection (dpi) in mice infected with E. granulousus. At 30 dpi, an increase in the number of CD11b⁺ and CD11c⁺ antigen-presenting cells (APCs) was observed. This was accompanied by the slight down-regulated expression of the co-stimulatory molecule MHC-II, indicating the impairment of APCs in early infection through the release of secretory-excretory products. In all infected groups, we observed a significant increase in innate immune cells, including APCs and GR-1⁺ cells, and a dramatic increase in the myeloid-derived suppressor cells (MDSC) expressing CD11b⁺/GR-1⁺. Moreover, the upregulation of the activated markers CD69, CD44, CD40L, and the downregulation of CD62L were observed in the CD4⁺ and CD8⁺ T cells following infection. Regulatory T cells expressing CD4⁺/CD25⁺/FoxP₃ ⁺ increased significantly over the course of infection.

Conclusions

Our findings demonstrate that the microenvironment in the peripheral immune system after E. granulosus infection changes in subtle but detectably ways, especially during the persistent period of infection. We found that T cells were activated following infection, but observed that the significant increase of immunosuppressive cells such as MDSC and Treg cells could inhibit T cell response to E. granulosus antigens. We suggest these cells may play a neglected but key role in the downregulation of the immune response in long-term parasitic infection. Understanding the basic functions and temporal interactions of these immunosuppressive cells will pave the way for new strategies of parasite vaccine design.     

Sensitive and Specific Immunohistochemical Diagnosis of Human Alveolar Echinococcosis with the Monoclonal Antibody Em2G11

Background

Alveolar echinococcosis (AE) is caused by the metacestode stage of Echinococcus multilocularis. Differential diagnosis with cystic echinococcosis (CE) caused by E. granulosus and AE is challenging. We aimed at improving diagnosis of AE on paraffin sections of infected human tissue by immunohistochemical testing of a specific antibody.

Methodology/Principal Findings

We have analysed 96 paraffin archived specimens, including 6 cutting needle biopsies and 3 fine needle aspirates, from patients with suspected AE or CE with the monoclonal antibody (mAb) Em2G11 specific for the Em2 antigen of E. multilocularis metacestodes. In human tissue, staining with mAb Em2G11 is highly specific for E. multilocularis metacestodes while no staining is detected in CE lesions. In addition, the antibody detects small particles of E. multilocularis (spems) of less than 1 µm outside the main lesion in necrotic tissue, liver sinusoids and lymphatic tissue most probably caused by shedding of parasitic material. The conventional histological diagnosis based on haematoxylin and eosin and PAS stainings were in accordance with the immunohistological diagnosis using mAb Em2G11 in 90 of 96 samples. In 6 samples conventional subtype diagnosis of echinococcosis had to be adjusted when revised by immunohistology with mAb Em2G11.

Conclusions/Significance

Immunohistochemistry with the mAb Em2G11 is a new, highly specific and sensitive diagnostic tool for AE. The staining of small particles of E. multilocularis (spems) outside the main lesion including immunocompetent tissue, such as lymph nodes, suggests a systemic effect on the host.     

Alteration of the Microbiota and Virulence Gene Expression in E. coli O157:H7 in Pig Ligated Intestine with and without AE Lesions

Background

Previously we found that E. coli O157:H7 inoculated into ligated pig intestine formed attaching and effacing (AE) lesions in some pigs but not in others. The present study evaluated changes in the microbial community and in virulence gene expression in E. coli O157:H7 in ligated pig intestine in which the bacteria formed AE lesions or failed to form AE lesions.

Methodology/Principal Findings

The intestinal microbiota was assessed by RNA-based denaturing gradient gel electrophoresis (DGGE) analysis. The DGGE banding patterns showed distinct differences involving two bands which had increased intensity specifically in AE-negative pigs (AE- bands) and several bands which were more abundant in AE-positive pigs. Sequence analysis revealed that the two AE- bands belonged to Veillonella caviae, a species with probiotic properties, and Bacteroides sp. Concurrent with the differences in microbiota, gene expression analysis by quantitative PCR showed that, compared with AE negative pigs, E. coli O157:H7 in AE positive pigs had upregulated genes for putative adhesins, non-LEE encoded nleA and quorum sensing qseF, acid resistance gene ureD, and genes from the locus of enterocyte effacement (LEE).

Conclusions/Significance

The present study demonstrated that AE-positive pigs had reduced activities or populations of Veillonella caviae and Bacterioides sp. compared with AE-negative pigs. Further studies are required to understand how the microbiota was changed and the role of these organisms in the control of E. coli O157:H7.     

Pilot Field Trial of the EG95 Vaccine Against Ovine Cystic Echinococcosis in Rio Negro,Argentina: Second Study of Impact

Background

Cystic echinococcosis (CE) is an important zoonotic disease caused by the cestode parasite Echinococcus granulosus. It occurs in many parts of the world where pastoral activities predominate, including the Rio Negro province of Argentina. Although CE control activities have been undertaken in the western regions of Rio Negro for more than two decades, the disease continues to remain prevalent in both the human and livestock animal populations. Vaccination of animal intermediate hosts of CE with the EG95 vaccine may provide a new opportunity to improve the effectiveness of CE control measures, although data are lacking about field application of the vaccine.

Aims

Evaluate the impact of EG95 vaccination in sheep on the transmission of Echinococcus granulosus in a field environment.

Methodology

Two trial sites were established in western Rio Negro province within indigenous communities. Vaccination of lambs born into one trial site was introduced and continued for 6 years. Prior to initiation of the trial, and at the end of the trial, the prevalence of CE in sheep was determined by necropsy. Weaned lambs received two injections of EG95 vaccine, approximately one month apart, and a single booster injection one year later. Vaccination was not implemented at the second trial site. A total of 2725 animals were vaccinated in the first year. Animals from this cohort as well as age-matched sheep from the control area were evaluated by necropsy.

Key results

Introduction of the vaccine led to a statistically significant in the number and size of hydatid cysts in comparison to the situation prior to the introduction of the vaccine, or compared to CE prevalence in the control area where the vaccine was not applied. The prevalence of infection in the vaccinated area was also significantly reduced by 62% compared to the re-intervention level, being lower than the prevalence seen in the control area, although the difference from the control area after the intervention was not significant possibly due to limitations in the numbers of animals available for necropsy.

Conclusions

Vaccination of sheep with the EG95 vaccine provides a valuable new tool which improves the effectiveness of CE control activities. Vaccination was effective even in a difficult, remote environment where only approximately half the lambs born into the communities were fully vaccinated.     

Control of cystic echinococcosis in the Middle Atlas,Morocco: Field evaluation of the EG95 vaccine in sheep and cesticide treatment in dogs

BackgroundCystic echinococcosis (CE) is an important cause of human morbidity and mortality worldwide, particularly in Morocco and other North African countries.Methodology/Principal findingsWe investigated the potential of three strategies to reduce Echinococcus granulosus transmission: (1) 4-monthly treatment of dogs with praziquantel, (2) vaccination of sheep with the EG95 vaccine and (3) a combination of both measures. These measures were implemented during four consecutive years in different areas of the Middle Atlas Mountains in Morocco. The outcome of the interventions was assessed through hydatid cyst (viable and non-viable) counts in liver and lungs using necropsy or in vivo ultrasound examination of the liver. A total of 402 lambs were recruited for annual vaccination with the EG95 anti-E. granulosus vaccine and 395 similar lambs were selected as non-vaccinated controls.At approximately four years of age the relative risk (estimated as odds ratio) for vaccinated sheep to have viable hydatid cysts compared with non-vaccinated controls was 3% (9.37% of the vaccinated sheep were found infected while 72.82% of the controls were infected; p = 0.002). The number of viable cysts in vaccinated animals was reduced by approximately 97% (mean counts were 0.28 and 9.18 respectively; p<0.001). An average of 595 owned dogs received 4-monthly treatment during the 44 months trial, corresponding to 91% of the owned dog population. Approximately, 5% of them were examined for E. granulosus adult worms by arecoline purge or eggs in feces (confirmed by PCR). The proportion of infected dogs significantly decreased after treatment (12% versus 35%; p<0.001). Post-treatment incidence of re-infestation corresponded to a monthly risk of 4% (95% CI: 3–6%). Treatment of owned dogs on a 4-monthly basis did not reduce the level of transmission of E. granulosus to sheep, nor did it enhance the level of control generated by vaccination of sheep with EG95, possibly because of unowned dogs and wild canids were not treated.Conclusions/SignificanceThese data suggest that vaccination of sheep with EG95 has the potential to reduce the level of CE in Morocco and in other parts of the world with similar transmission dynamics. Under the epidemiological circumstances existing in the trial area, 4-monthly treatment of owned dogs with praziquantel was insufficient to have a major impact of E. granulosus transmission to sheep.     

Clinical and Demographic Characteristics of Patients with Urinary Tract Hydatid Disease

Background

Human cystic echinococcosis (CE) is caused by flatworm larvae of Echinococcus granulosus and is endemic in many parts of the world. In humans, CE cysts primarily affect the liver and pulmonary system, but can also affect the renal system. However, the clinical manifestations of renal CE can be subtle, so healthcare professionals often overlook renal CE in differential diagnosis. In this study, we examined the clinical and demographic characteristics of patients with urinary tract CE and analyzed the diagnosis and treatment procedures for this disease.

Methods

The records of 19 consecutive renal CE patients who were admitted to the First Affiliated Hospital of Xinjiang Medical University from January 1983 to April 2011 were retrospectively reviewed. In all cases, CE of the urinary tract was confirmed by pathological examination and visual inspection during surgery.

Results

Fifteen patients were males and 4 were females. The most common symptoms were non-specific lower back pain and percussion tenderness on the kidney region. All patients were followed up for 9–180 months after surgery. None of the patients experienced a recurrence of renal CE, but 4 patients experienced non-renal recurrence of hydatid disease.

Conclusions

Hydatid cysts from E. granulosus are structurally similar in the liver and urinary tract. Thus, the treatment regimen for liver CE developed by the World Health Organization/Informal Working Group on Echinococcosis (WHO/IWGE) could also be used for urinary tract CE. In our patients, the use of ultrasound, computed tomography, serology, and clinical characteristics provided a diagnostic accuracy of 66.7% to 92.3%.     

Natural Infection of the Ground Squirrel (Spermophilus spp.) with Echinococcus granulosus in China

Background

Echinococcus granulosus is usually transmitted between canid definitive hosts and ungulate intermediate hosts.

Methodology/Principal Findings

Lesions found in the livers of ground squirrels, Spermophilus dauricus/alashanicus, trapped in Ningxia Hui Autonomous Region, an area in China co-endemic for both E. granulosus and E. multilocularis, were subjected to molecular genotyping for Echinococcus spp. DNA. One of the lesions was shown to be caused by E. granulosus and subsequently by histology to contain viable protoscoleces.

Conclusions/Significance

This is the first report of a natural infection of the ground squirrel with E. granulosus. This does not provide definitive proof of a cycle involving ground squirrels and dogs or foxes, but it is clear that there is active E. granulosus transmission occurring in this area, despite a recent past decline in the dog population in southern Ningxia.     

Detection of Echinococcus multilocularis in carnivores in Razavi Khorasan province, Iran using mitochondrial DNA

Background

Echinococcus multilocularis is the source of alveolar echinococcosis, a potentially fatal zoonotic disease. This investigation assessed the presence of E. multilocularis infection in definitive hosts in the Chenaran region of Razavi Khorasan Province, northeastern Iran.

Methodology/Principal Findings

Fecal samples from 77 domestic and stray dogs and 14 wild carnivores were examined using the flotation/sieving method followed by multiplex PCR of mitochondrial genes. The intestinal scraping technique (IST) and the sedimentation and counting technique (SCT) revealed adult Echinococcus in the intestines of five of 10 jackals and of the single wolf examined. Three jackals were infected only with E. multilocularis but two, and the wolf, were infected with both E. multilocularis and E. granulosus. Multiplex PCR revealed E. multilocularis, E. granulosus, and Taenia spp. in 19, 24, and 28 fecal samples, respectively. Echinococcus multilocularis infection was detected in the feces of all wild carnivores sampled including nine jackals, three foxes, one wolf, one hyena, and five dogs (6.5%). Echinococcus granulosus was found in the fecal samples of 16.9% of dogs, 66.7% of jackals, and all of the foxes, the wolf, and the hyena. The feces of 16 (21.8%) dogs, 7 of 9 (77.8%) jackals, and all three foxes, one wolf and one hyena were infected with Taenia spp.

Conclusions/Significance

The prevalence of E. multilocularis in wild carnivores of rural areas of the Chenaran region is high, indicating that the life cycle is being maintained in northeastern Iran with the red fox, jackal, wolf, hyena, and dog as definitive hosts.     

The Effect of Probiotic Treatment on Patients Infected with the H7N9 Influenza Virus

Background

A novel avian-origin influenza A (H7N9) virus emerged and spread among humans in Eastern China in 2013. Prophylactic treatment with antibiotics and probiotics for secondary infection is as important as antiviral treatment. This study aims to assess the ability of probiotic treatment to restore internal homeostasis under antibiotic pressure and to reduce/ameliorate the risk of secondary infections resulting from infection with the H7N9 virus.

Methods

This is a retrospective study in archival samples. Between April 1 and May 10, 2013, 113 stool, sputum, and blood specimens were collected and analyzed by denaturing gradient gel electrophoresis (DGGE) to determine the composition of the patient microbiomes. Microbial diversity was calculated using Gel-Pro analyzer and Past software. Cluster analysis of DGGE pattern profiles was employed to create a phylogenetic tree for each patient, and multidimensional scaling (MDS) and principal component analysis (PCA) were performed to visualize relationships between individual lanes.

Results

Five patients had secondary infections, including Klebsiella pneumonia, Acinetobacter baumanii and Candida albicans infection. The DGGE profiles of fecal samples obtained at different time points from the same individual were clearly different, particularly for patients with secondary infections. Shannon’s diversity index and evenness index were lower in all infected groups compared to the control group. After B. subtilis and E. faecium or C. butyricum administration, the fecal bacterial profiles of patients who had not been treated with antibiotics displayed a trend of increasing diversity and evenness. C. butyricum failed to reduce/ameliorate secondary infection in H7N9-infected patients, but administration of B. subtilis and E. faecium appeared to reduce/ameliorate secondary infection in one patient.

Conclusion

H7N9 infection might decrease intestinal microbial diversity and species richness in humans. C. butyricum failed to reduce/ameliorate secondary infection in H7N9-infected patients. B. subtilis and E. faecium may also play a role in reducing/ameliorating secondary infection in these patients.     

Urinary Proteins,Vitamin D and Genetic Polymorphisms as Risk Factors for Febrile Urinary Tract Infection and Relation with Bacteremia: A Case Control Study

Objective/Purpose

Febrile urinary tract infection (UTI) is a common bacterial disease that may lead to substantial morbidity and mortality especially among the elderly. Little is known about biomarkers that predict a complicated course. Our aim was to determine the role of certain urinary cytokines or antimicrobial proteins, plasma vitamin D level, and genetic variation in host defense of febrile UTI and its relation with bacteremia.

Methods

A case-control study. Out of a cohort of consecutive adults with febrile UTI (n = 787) included in a multi-center observational cohort study, 46 cases with bacteremic E.coli UTI and 45 cases with non-bacteremic E.coli UTI were randomly selected and compared to 46 controls. Urinary IL-6, IL-8, LL37, β-defensin 2 and uromodulin as well as plasma 25-hydroxyvitamin D were measured. In 440 controls and 707 UTI patients polymorphisms were genotyped in the genes CXCR1, DEFA4, DEFB1, IL6, IL8, MYD88, UMOD, TIRAP, TLR1, TLR2, TLR5 and TNF.

Results

IL-6, IL-8, and LL37 are different between controls and UTI patients, although these proteins do not distinguish between patients with and without bacteremia. While uromodulin did not differ between groups, inability to produce uromodulin is more common in patients with bacteremia. Most participants in the study, including the controls, had insufficient vitamin D and, at least in winter, UTI patients have lower vitamin D than controls. Associations were found between the CC genotype of IL6 SNP rs1800795 and occurrence of bacteremia and between TLR5 SNP rs5744168 and protection from UTI. The rare GG genotype of IL6 SNP rs1800795 was associated with higher β-defensin 2 production.

Conclusion

Although no biomarker was able to distinguish between UTI with or without bacteremia, two risk factors for bacteremia were identified. These were inability to produce uromodulin and an IL6 rs1800795 genotype.     

Infection by Intestinal Parasites,Stunting and Anemia in School-Aged Children from Southern Angola

Introduction

Intestinal parasites are responsible for morbidity in children worldwide, especially in low income countries. In the present study we determine the prevalence of intestinal parasites and explore its association with anemia and stunting in school-aged children.

Methods

A cross-sectional study was conducted from September to October 2010 enrolling 328 children attending the primary school in Lubango, the second largest city after the capital Luanda. Stool samples were collected for parasite detection through microscopy and molecular identification of Entamoeba histolytica and Entamoeba dispar. Stunting was assessed using the z-scores of height for age and hemoglobin concentration was determined using a portable hemoglobin analyzing system.

Results

The global prevalence of pathogenic intestinal parasites was 44.2%, the most common being Ascaris lumbricoides (22.0%), Giardia lamblia (20.1%) and Hymenolepis nana (8.8%). Molecular detection revealed that 13.1% of the children carried E. dispar and 0.3% were infected with E. histolytica. The prevalence of stunting (mild to severe) was 41.5%. Stunting was more frequent in older children (p = 0.006, OR = 1.886), while anemia was more frequent in younger children (p = 0.005, OR = 2.210). The prevalence of anemia was 21.6%, and we found a significant association with infection by H. nana (p = 0.031, OR = 2.449).

Conclusions

This is one of the few published studies reporting intestinal parasites infection, nutritional status and anemia in children from Angola. Furthermore, the present work highlights the importance of regular intestinal parasites screening in children.     

A Systematic Review of the Epidemiology of Echinococcosis in Domestic and Wild Animals

Background

Human echinococcosis is a neglected zoonosis caused by parasites of the genus Echinococcus. The most frequent clinical forms of echinococcosis, cystic echinococcosis (CE) and alveolar echinococcosis (AE), are responsible for a substantial health and economic burden, particularly to low-income societies. Quantitative epidemiology can provide important information to improve the understanding of parasite transmission and hence is an important part of efforts to control this disease. The purpose of this review is to give an insight on factors associated with echinococcosis in animal hosts by summarising significant results reported from epidemiological studies identified through a systematic search.

Methodology and Principal Findings

The systematic search was conducted mainly in electronic databases but a few additional records were obtained from other sources. Retrieved entries were examined in order to identify available peer-reviewed epidemiological studies that found significant risk factors for infection using associative statistical methods. One hundred studies met the eligibility criteria and were suitable for data extraction. Epidemiological factors associated with increased risk of E. granulosus infection in dogs included feeding with raw viscera, possibility of scavenging dead animals, lack of anthelmintic treatment and owners'' poor health education and indicators of poverty. Key factors associated with E. granulosus infection in intermediate hosts were related to the hosts'' age and the intensity of environmental contamination with parasite eggs. E. multilocularis transmission dynamics in animal hosts depended on the interaction of several ecological factors, such as hosts'' population densities, host-prey interactions, landscape characteristics, climate conditions and human-related activities.

Conclusions/Significance

Results derived from epidemiological studies provide a better understanding of the behavioural, biological and ecological factors involved in the transmission of this parasite and hence can aid in the design of more effective control strategies.     

Cholesteryl Esters Are Elevated in the Lipid Fraction of Bronchoalveolar Lavage Fluid Collected from Pediatric Cystic Fibrosis Patients

Background

Host-derived lipids including cholesteryl esters (CEs) such as cholesteryl linoleate have emerged as important antibacterial effectors of innate immunity in the airways and cholesteryl linoleate has been found elevated in the context of inflammation. Cystic fibrosis (CF) patients suffer from chronic infection and severe inflammation in the airways. Here, we identified and quantified CEs in bronchoalveolar lavage fluid (BALF) from CF patients and non-CF disease controls, and tested whether CE concentrations are linked to the disease.

Materials and Methods

CEs in BALF from 6 pediatric subjects with CF and 7 pediatric subjects with non-CF chronic lung disease were quantified by mass spectral analysis using liquid chromatography coupled with tandem mass spectrometry and multiple reaction monitoring. BALFs were also examined for total lipid, total protein, albumin, and, as a marker for inflammation, human neutrophil peptide (HNP) 1–3 concentrations. Statistical analysis was conducted after log 10 transformation of the data.

Results

Total lipid/protein ratio was reduced in CF BALF (p = 0.018) but the concentrations of CEs, including cholesteryl linoleate, were elevated in the total lipid fraction in CF BALF compared to non-CF disease controls (p < 0.050). In addition, the concentrations of CEs and HNP1-3 correlated with one another (p < 0.050).

Conclusions

The data suggests that the lipid composition of BALF is altered in CF with less total lipid relative to protein but with increased CE concentrations in the lipid fraction, likely contributed by inflammation. Future longitudinal studies may reveal the suitability of CEs as a novel biomarker for CF disease activity which may provide new information on the lipid mediated pathophysiology of the disease.     

Using the Electronic Nose to Identify Airway Infection during COPD Exacerbations

Background

The electronic nose (e-nose) detects volatile organic compounds (VOCs) in exhaled air. We hypothesized that the exhaled VOCs print is different in stable vs. exacerbated patients with chronic obstructive pulmonary disease (COPD), particularly if the latter is associated with airway bacterial infection, and that the e-nose can distinguish them.

Methods

Smell-prints of the bacteria most commonly involved in exacerbations of COPD (ECOPD) were identified in vitro. Subsequently, we tested our hypothesis in 93 patients with ECOPD, 19 of them with pneumonia, 50 with stable COPD and 30 healthy controls in a cross-sectional case-controlled study. Secondly, ECOPD patients were re-studied after 2 months if clinically stable. Exhaled air was collected within a Tedlar bag and processed by a Cynarose 320 e-nose. Breath-prints were analyzed by Linear Discriminant Analysis (LDA) with “One Out” technique and Sensor logic Relations (SLR). Sputum samples were collected for culture.

Results

ECOPD with evidence of infection were significantly distinguishable from non-infected ECOPD (p = 0.018), with better accuracy when ECOPD was associated to pneumonia. The same patients with ECOPD were significantly distinguishable from stable COPD during follow-up (p = 0.018), unless the patient was colonized. Additionally, breath-prints from COPD patients were significantly distinguished from healthy controls. Various bacteria species were identified in culture but the e-nose was unable to identify accurately the bacteria smell-print in infected patients.

Conclusion

E-nose can identify ECOPD, especially if associated with airway bacterial infection or pneumonia.     

Comparative Genomics and Characterization of Hybrid Shigatoxigenic and Enterotoxigenic Escherichia coli (STEC/ETEC) Strains

Background

Shigatoxigenic Escherichia coli (STEC) and enterotoxigenic E. coli (ETEC) cause serious foodborne infections in humans. These two pathogroups are defined based on the pathogroup-associated virulence genes: stx encoding Shiga toxin (Stx) for STEC and elt encoding heat-labile and/or est encoding heat-stable enterotoxin (ST) for ETEC. The study investigated the genomics of STEC/ETEC hybrid strains to determine their phylogenetic position among E. coli and to define the virulence genes they harbor.

Methods

The whole genomes of three STEC/ETEC strains possessing both stx and est genes were sequenced using PacBio RS sequencer. Two of the strains were isolated from the patients, one with hemolytic uremic syndrome, and one with diarrhea. The third strain was of bovine origin. Core genome analysis of the shared chromosomal genes and comparison with E. coli and Shigella spp. reference genomes was performed to determine the phylogenetic position of the STEC/ETEC strains. In addition, a set of virulence genes and ETEC colonization factors were extracted from the genomes. The production of Stx and ST were studied.

Results

The human STEC/ETEC strains clustered with strains representing ETEC, STEC, enteroaggregative E. coli, and commensal and laboratory-adapted E. coli. However, the bovine STEC/ETEC strain formed a remote cluster with two STECs of bovine origin. All three STEC/ETEC strains harbored several other virulence genes, apart from stx and est, and lacked ETEC colonization factors. Two STEC/ETEC strains produced both toxins and one strain Stx only.

Conclusions

This study shows that pathogroup-associated virulence genes of different E. coli can co-exist in strains originating from different phylogenetic lineages. The possibility of virulence genes to be associated with several E. coli pathogroups should be taken into account in strain typing and in epidemiological surveillance. Development of novel hybrid E. coli strains may cause a new public health risk, which challenges the traditional diagnostics of E. coli infections.     

Gene Expression Profile in the Liver of BALB/c Mice Infected with Fasciola hepatica

Background

Fasciola hepatica infection still remains one of the helminthic neglected tropical diseases (NTDs). It has a huge worldwide distribution, affecting mainly cattle and, sometimes, human beings. In addition to data reported about the immunological response induced by helminthic infections and that induced by Fasciola hepatica, little is known about the gene expression profile in its organ target, the liver, which is where adult worms are established and live for long periods of time, causing its characteristic pathology. In the present work, we study both the early and late gene expression profiles in the livers of mice infected with F. hepatica metacercariae using a microarray-based methodology.

Methodology

A total of 9 female-6-week-old BALB/c mice (Charles River Laboratories, Barcelona, Spain) weighing 20 to 35 g were used for the experiments. Two groups of BALB/c mice were orally infected with seven F. hepatica metacercariae, and the other group remained untreated and served as a control. Mice were humanely euthanized and necropsied for liver recovery, histological assessment of hepatic damage, RNA isolation, microarray design and gene expression analysis on the day of infection (t0), seven days post-infection (t7) and twenty-one days post-infection (t21).

Results

We found that F. hepatica infection induces the differential expression of 128 genes in the liver in the early stage of infection and 308 genes in the late stage, and most of them are up-regulated. The Ingenuity Pathway Analysis revealed significant changes in the pathways related to metabolism, biosynthesis and signaling as well as genes implicated in inducing liver-toxicity, injury and death.

Conclusion

The present study provides us insights at the molecular level about the underlying mechanisms used by F. hepatica, leading to liver damage and its subsequent pathophysiology. The expression pattern obtained here could also be used to explain the lack of association between infection with F. hepatica and cholangiocarcinoma. However, more studies should be performed to confirm this hypothesis.     

Genome-Wide Specific Selection in Three Domestic Sheep Breeds

Background

Commercial sheep raised for mutton grow faster than traditional Chinese sheep breeds. Here, we aimed to evaluate genetic selection among three different types of sheep breed: two well-known commercial mutton breeds and one indigenous Chinese breed.

Results

We first combined locus-specific branch lengths and d_i statistical methods to detect candidate regions targeted by selection in the three different populations. The results showed that the genetic distances reached at least medium divergence for each pairwise combination. We found these two methods were highly correlated, and identified many growth-related candidate genes undergoing artificial selection. For production traits, APOBR and FTO are associated with body mass index. For meat traits, ALDOA, STK32B and FAM190A are related to marbling. For reproduction traits, CCNB2 and SLC8A3 affect oocyte development. We also found two well-known genes, GHR (which affects meat production and quality) and EDAR (associated with hair thickness) were associated with German mutton merino sheep. Furthermore, four genes (POL, RPL7, MSL1 and SHISA9) were associated with pre-weaning gain in our previous genome-wide association study.

Conclusions

Our results indicated that combine locus-specific branch lengths and d_i statistical approaches can reduce the searching ranges for specific selection. And we got many credible candidate genes which not only confirm the results of previous reports, but also provide a suite of novel candidate genes in defined breeds to guide hybridization breeding.