Study of host-microbe interactions in zebrafish.

All animals are ecosystems, home to diverse microbial populations. Animal-associated microbes play important roles in the normal development and physiology of their hosts, but can also be agents of infectious disease. Traditionally, mice have been used to study pathogenic and beneficial associations between microbes and vertebrate animals. The zebrafish is emerging as a valuable new model system for host-microbe interaction studies, affording researchers with the opportunity to survey large populations of hosts and to visualize microbe-host associations at a cellular level in living animals. This chapter provides detailed protocols for the analysis of zebrafish-associated microbial communities, the derivation and husbandry of germ-free zebrafish, and the modeling of infectious disease in different stages of zebrafish development via different routes of inoculation. These protocols offer a starting point for researchers to address a multitude of questions about animals' coexistence with microorganisms.

In vivo deficiency of both C/EBPß and C/EBPε results in highly defective myeloid differentiation and lack of cytokine response.

The CCAAT/enhancer binding proteins (C/EBPs) are transcription factors involved in hematopoietic cell development and induction of several inflammatory mediators. Here, we generated C/EBPß and C/EBPε double-knockout (bbee) mice and compared their phenotypes to those of single deficient (bbEE and BBee) and wild-type (BBEE) mice. The bbee mice were highly susceptible to fatal infections and died within 2-3 months. Morphologically, their neutrophils were blocked at the myelocytes/metamyelocytes stage, and clonogenic assays of bone marrow cells indicated a significant decrease in the number of myeloid colonies of the bbee mice. In addition, the proportion of hematopoietic progenitor cells [Lin(-)Sca1(+)c-Kit(+)] in the bone marrow of the bbee mice was significantly increased, reflecting the defective differentiation of the myeloid compartment. Furthermore, microarray expression analysis of LPS- and IFNγ-activated bone marrow-derived macrophages from bbee compared to single knockout mice revealed decreased expression of essential immune response-related genes and networks, including some direct C/EBP-targets such as Marco and Clec4e. Overall, the phenotype of the bbee mice is distinct from either the bbEE or BBee mice, demonstrating that both transcription factors are crucial for the maturation of neutrophils and macrophages, as well as the innate immune system, and can at least in part compensate for each other in the single knockout mice.

Fetal meconium peritonitis complicated with bacterial infection.

Fetal meconium peritonitis complicated by bacterial infection is extremely rare. We report a case of fetal ascites at 21 weeks of gestation with subsequent development of loculation, encapsulation, and calcification at 25 weeks. Paracentesis of loculated ascitic fluid at 28 weeks of gestation showed a purulent appearance with the presence of cocci bacteria, increase in white cell count, and a low glucose level, which were suggestive of bacterial infection. However, no sources of maternal infection could be identified. The total bilirubin level of the ascitic fluid was normal (21 micromol/L). A healthy baby was delivered at 37 weeks. CT scan revealed normal bowel without any sign of perforation. We postulate that when ascitic fluid becomes loculated, a normal bilirubin level on paracentesis indicates spontaneous closure of a previous bowel perforation.

Trans-shell infection by pathogenic micro-organisms reduces the shelf life of non-incubated bird's eggs: a constraint on the onset of incubation?

Many birds initiate incubation before clutch completion, which results in asynchronous hatching. The ensuing within-brood size disparity often places later-hatched nestlings at a developmental disadvantage, but the functional significance of the timing of the onset of incubation is poorly understood. Early incubation may serve to maintain the viability of early-laid eggs, which declines over time owing to the putative effects of ambient temperature. An unexplored risk to egg viability is trans-shell infection by micro-organisms. We experimentally investigated the rate and magnitude of microbial trans-shell infection of the egg, and the relative effects of ambient temperature and micro-organisms on hatching success. We show that infection of egg contents is prevalent and occurs within the time required to lay a clutch. The probability of infection depends on the climatic conditions, the exposure period and the phylogenetic composition of the eggshell microbiota. We also demonstrate that microbial infection and ambient temperature act independently to reduce egg viability considerably. Our results suggest that these two factors could affect the onset of avian incubation in a wide range of environments.

[Procalcitonin as a marker of perinatal infection in newborn--preliminary data]. / Prokalcytonina jako parametr pomocny w rozpoznawaniu zakazenia wrodzonego.

UNLABELLED: Intrauterine and intrapartum infections in newborn infants are still difficult to recognise. The newborn does not manifest the classic clinical signs of infection usually observed in children and adults and up to now there is no good laboratory marker. In the last few years, procalcitonin (PCT) has been found to increase during different inflammatory processes, especially bacterial ones. In this study we analysed the clinical value of PTC in parturient, umbilical cord and newborn blood for predicting perinatal infection. MATERIAL AND METHODS: Thirty parturients with symptoms of intrauterine infection were classified for this study. Blood samples were obtained from the mother, the umbilical cord and the newborn on the second day of life. Serum was stored at -70 degrees C and thawed at the time of analysis. Among the newborns there were 21 infants without and 9 with symptoms and signs of infection. PCT concentration was measured by immunoluminometric assay--LUMI test PCT (BRAHMS). RESULTS: Statistically significant results were found on the second day of life: 5.83 (4.70) ng/ml in ill, 1.41 (0.68) ng/ml in healthy (p < 0.0005). We observed a significant correlation between PCT concentration in mother and umbilical cord blood (y = 0.40x + 1.06; p < 0.05), as well as between umbilical cord blood and venous blood on the second day of life in newborns (y = 0.16x 1.21; p < 0.01). CONCLUSIONS: Measurement of PCT concentration in perinatal period in the mother and in umbilical cord blood of the newborn may be useful for early diagnosis and monitoring of infectious complications in neonates. We need more data on reference ranges of PCT concentration in pregnant women, parturients and umbilical cord blood.

Laboratory diagnosis of fetal infections.

In utero infections of the fetus can lead to significant morbidity and mortality in the newborn child. The signs and symptoms of clinical disease, however, do not always suggest a given pathogen. The laboratory must be able to provide an early and accurate diagnosis of the causative agent so that prompt and appropriate antimicrobial therapy and medical care can be initiated. The scope of this article includes the methods employed by the laboratory to assist in the diagnosis of bacterial, fungal, parasitic, and viral infections of the fetus. Where appropriate, detection methods were addressed for the diagnosis of the major pathogens responsible for infection during the birth process.