Increased secretion of exopolysaccharide and virulence potential of a mucoid variant of Salmonella enterica serovar Montevideo under environmental stress.

During an investigation to increase the recovery of Salmonella enterica from Oregano, an increased expression of exopolysaccharide was induced in Salmonella serovar Montevideo. The atypical mucoid (SAL242S) and the non-mucoid (SAL242) strains of Montevideo were compared and characterized using various methods. Serotyping analysis demonstrated that both strains are the same serovar Montevideo. Electron microscopy (EM) of cultured SAL242S cells revealed the production of a prominent EPS-like structure enveloping aggregates of cells that are composed of cellulose. Mucoid cells possessed a higher binding affinity for Calcofluor than that of the non-mucoid strain. Genotypic analysis revealed no major genomic differences between these morphotypes, while expression analyses using a DNA microarray shows that the mucoid variant exhibited heightened expression of genes encoding proteins produced by the SPI-1 type III secretion system. This increased expression of SPI1 genes may play a role in protecting Salmonella from environmental stressors. Based on these observations, Salmonella serovar Montevideo mucoid variant under stressful or low-nutrient environments presented atypical growth patterns and phenotypic changes, as well as an upregulated expression of virulence factors. These findings are significant in the understanding of survival abilities of Salmonella in a various food matrices.

Comparison of a PCR serotyping assay, Check&Trace assay for Salmonella, and Luminex Salmonella serotyping assay for the characterization of Salmonella enterica identified from fresh and naturally contaminated cilantro.

Salmonella enterica isolated from fresh cilantro samples collected through the USDA/AMS Microbiological Data Program (MDP) were used to compare a PCR serotyping assay against the Check&Trace assay and the Luminex (BioPlex) Salmonella serotyping assay. The study was conducted to evaluate the effectiveness of the three methods for serotyping Salmonella from both enrichment broth cultures and pure Salmonella cultures. In this investigation, Salmonella spp. serotyping was conducted using 24 h enrichment broth cultures and pure Salmonella cultures from cilantro samples, with the PCR serotyping assay. Conversely, the Check&Trace and Luminex for Salmonella assays required pure cultures for Salmonella serotyping. The cilantro samples contained S. enterica serovar Montevideo, Newport, Saintpaul, and Tennessee, identified by the PCR serotyping assay and Check&Trace for Salmonella, but the Luminex assay only identified two of the four serotypes of the cilantro samples. The anticipated impact from this study is that the PCR serotyping assay provides a time- and cost-effective means for screening, identifying and serotyping Salmonella using DNA extracted from 24 h enrichment cilantro samples.

Multiplex PCR assay targeting a diguanylate cyclase-encoding gene, cgcA, to differentiate species within the genus Cronobacter.

In a comparison to the widely used Cronobacter rpoB PCR assay, a highly specific multiplexed PCR assay based on cgcA, a diguanylate cyclase gene, that identified all of the targeted six species among 305 Cronobacter isolates was designed. This assay will be a valuable tool for identifying suspected Cronobacter isolates from food-borne investigations.

The Pathogen-annotated Tracking Resource Network (PATRN) system: a web-based resource to aid food safety, regulatory science, and investigations of foodborne pathogens and disease.

Investigation of foodborne diseases requires the capture and analysis of time-sensitive information on microbial pathogens that is derived from multiple analytical methods and sources. The web-based Pathogen-annotated Tracking Resource Network (PATRN) system (www.patrn.net) was developed to address the data aggregation, analysis, and communication needs important to the global food safety community for the investigation of foodborne disease. PATRN incorporates a standard vocabulary for describing isolate metadata and provides a representational schema for a prototypic data exchange standard using a novel data loading wizard for aggregation of assay and attribution information. PATRN currently houses expert-curated, high-quality "foundational datasets" consisting of published experimental results from conventional assays and next generation analysis platforms for isolates of Escherichia coli, Listeria monocytogenes, and Salmonella, Shigella, Vibrio and Cronobacter species. A suite of computational tools for data mining, clustering, and graphical representation is available. Within PATRN, the public curated data repository is complemented by a secure private workspace for user-driven analyses, and for sharing data among collaborators. To demonstrate the data curation, loading wizard features, and analytical capabilities of PATRN, three use-case scenarios are presented. Use-case scenario one is a comparison of the distribution and prevalence of plasmid-encoded virulence factor genes among 249 Cronobacter strains with similar attributes to that of nine Cronobacter isolates from recent cases obtained between March and October, 2010-2011. To highlight PATRN's data management and trend finding tools, analysis of datasets, stored in PATRN as part of an ongoing surveillance project to identify the predominant molecular serogroups among Cronobacter sakazakii isolates observed in the USA is shown. Use-case scenario two demonstrates the secure workspace available for private users to upload and analyze sensitive data, and for collating cross-platform datasets to identify and validate congruent datapoints. SNP datasets from WGS assemblies and pan-genome microarrays are analyzed in a combinatorial fashion to determine relatedness of 33 Salmonella enterica strains to six strains collected as part of an outbreak investigation. Use-case scenario three utilizes published surveillance results that describe the incidence and sources of O157:H7 E. coli isolates associated with a produce pre-harvest surveillance study that occurred during 2002-2006. In summary, PATRN is a web-based integrated platform containing tools for the management, analysis and visualization of data about foodborne pathogens.

Identification and characterization of Cronobacter iron acquisition systems.

Cronobacter spp. are emerging pathogens that cause severe infantile meningitis, septicemia, or necrotizing enterocolitis. Contaminated powdered infant formula has been implicated as the source of Cronobacter spp. in most cases, but questions still remain regarding the natural habitat and virulence potential for each strain. The iron acquisition systems in 231 Cronobacter strains isolated from different sources were identified and characterized. All Cronobacter spp. have both the Feo and Efe systems for acquisition of ferrous iron, and all plasmid-harboring strains (98%) have the aerobactin-like siderophore, cronobactin, for transport of ferric iron. All Cronobacter spp. have the genes encoding an enterobactin-like siderophore, although it was not functional under the conditions tested. Furthermore, all Cronobacter spp. have genes encoding five receptors for heterologous siderophores. A ferric dicitrate transport system (fec system) is encoded specifically by a subset of Cronobacter sakazakii and C. malonaticus strains, of which a high percentage were isolated from clinical samples. Phylogenetic analysis confirmed that the fec system is most closely related to orthologous genes present in human-pathogenic bacterial strains. Moreover, all strains of C. dublinensis and C. muytjensii encode two receptors, FcuA and Fct, for heterologous siderophores produced by plant pathogens. Identification of putative Fur boxes and expression of the genes under iron-depleted conditions revealed which genes and operons are components of the Fur regulon. Taken together, these results support the proposition that C. sakazakii and C. malonaticus may be more associated with the human host and C. dublinensis and C. muytjensii with plants.

Cpa, the outer membrane protease of Cronobacter sakazakii, activates plasminogen and mediates resistance to serum bactericidal activity.

Cronobacter spp. are emerging neonatal pathogens in humans, associated with outbreaks of meningitis and sepsis. To cause disease, they must survive in blood and invade the central nervous system by penetrating the blood-brain barrier. C. sakazakii BAA-894 possesses an ~131-kb plasmid (pESA3) that encodes an outer membrane protease (Cpa) that has significant identity to proteins that belong to the Pla subfamily of omptins. Members of this subfamily of proteins degrade a number of serum proteins, including circulating complement, providing protection from the complement-dependent serum killing. Moreover, proteins of the Pla subfamily can cause uncontrolled plasmin activity by converting plasminogen to plasmin and inactivating the plasmin inhibitor α2-antiplasmin (α2-AP). These reactions enhance the spread and invasion of bacteria in the host. In this study, we found that an isogenic cpa mutant showed reduced resistance to serum in comparison to its parent C. sakazakii BAA-894 strain. Overexpression of Cpa in C. sakazakii or Escherichia coli DH5α showed that Cpa proteolytically cleaved complement components C3, C3a, and C4b. Furthermore, a strain of C. sakazakii overexpressing Cpa caused a rapid activation of plasminogen and inactivation of α2-AP. These results strongly suggest that Cpa may be an important virulence factor involved in serum resistance, as well as in the spread and invasion of C. sakazakii.

Characterization of putative virulence genes on the related RepFIB plasmids harbored by Cronobacter spp.

Cronobacter spp. are emerging neonatal pathogens that cause meningitis, sepsis, and necrotizing enterocolitis. The genus Chronobacter consists of six species: C. sakazakii, C. malonaticus, C. muytjensii, C. turicensis, C. dublinensis, and Cronobacter genomospecies group 1. Whole-genome sequencing of C. sakazakii BAA-894 and C. turicensis z3032 revealed that they harbor similarly sized plasmids identified as pESA3 (131 kb) and pCTU1 (138 kb), respectively. In silico analysis showed that both plasmids encode a single RepFIB-like origin of replication gene, repA, as well as two iron acquisition systems (eitCBAD and iucABCD/iutA). In a chrome azurol S agar diffusion assay, it was demonstrated that siderophore activity was associated with the presence of pESA3 or pCTU1. Additionally, pESA3 contains a cpa (Cronobacter plasminogen activator) gene and a 17-kb type 6 secretion system (T6SS) locus, while pCTU1 contains a 27-kb region encoding a filamentous hemagglutinin gene (fhaB), its specifc transporter gene (fhaC), and associated putative adhesins (FHA locus), suggesting that these are virulence plasmids. In a repA-targeted PCR assay, 97% of 229 Cronobacter species isolates were found to possess a homologous RepFIB plasmid. All repA PCR-positive strains were also positive for the eitCBAD and iucABCD/iutA iron acquisition systems. However, the presence of cpa, T6SS, and FHA loci depended on species, demonstrating a strong correlation with the presence of virulence traits, plasmid type, and species. These results support the hypothesis that these plasmids have evolved from a single archetypical plasmid backbone through the cointegration, or deletion, of specific virulence traits in each species.

Molecular characterization of Cronobacter lipopolysaccharide O-antigen gene clusters and development of serotype-specific PCR assays.

Cronobacter (formerly Enterobacter sakazakii) is a recently defined genus consisting of six species, C. sakazakii, C. malonaticus, C. dublinensis, C. muytjensii, C. turicensis, and Cronobacter genomospecies 1. In this study, MboII restriction fragment length polymorphism (RFLP) patterns of O-antigen gene clusters, located between galF and gnd, were used to identify serotypes in Cronobacter spp. Seven O-antigen RFLP clusters were generated, including three C. sakazakii clusters, previously identified as serotypes O1, O2, and O3. The O-antigen regions of six strains with unique RFLP patterns, including two C. sakazakii strains, two C. malonaticus strains, one C. turicensis strain, and one C. muytjensii strain, revealed three O-antigen gene clusters shared among Cronobacter species. PCR assays were developed, targeting the wzx O-antigen polymerase gene, and used to screen 231 Cronobacter strains to determine the frequency of these newly identified serotypes.

A Child with Enlarged Extremities - A Case of Macrodystrophia Lipomatosa.

Macrodystrophia lipomatosa (ML) is a rare, non-hereditary, developmental anomaly that occurs because of the progressive proliferation of all mesenchymal elements of single or multiple digits or entire extremity, with a disproportionate increase in fibroadipose tissue. Commonly one or few digits of an extremity will be enlarged and present as macrodactyly or as enlarged limb. Lower limb involvement is more common and frequently unilateral. The diagnosis of ML is made by accurate clinical assessment and imaging modalities, such as plain X-ray, computed tomography scan, magnetic resonance imaging, and confirmed by histopathological study. In this case, we described a 10-year-old child who was brought to us with enlarged upper and lower extremities and was diagnosed as a case of ML with the help of clinico-radiological studies and presented here because of focal gigantism involving all four limbs, which is very rare.

A hybrid reference-guided de novo assembly approach for generating Cyclospora mitochondrion genomes.

Cyclospora cayetanensis is a coccidian parasite associated with large and complex foodborne outbreaks worldwide. Linking samples from cyclosporiasis patients during foodborne outbreaks with suspected contaminated food sources, using conventional epidemiological methods, has been a persistent challenge. To address this issue, development of new methods based on potential genomically-derived markers for strain-level identification has been a priority for the food safety research community. The absence of reference genomes to identify nucleotide and structural variants with a high degree of confidence has limited the application of using sequencing data for source tracking during outbreak investigations. In this work, we determined the quality of a high resolution, curated, public mitochondrial genome assembly to be used as a reference genome by applying bioinformatic analyses. Using this reference genome, three new mitochondrial genome assemblies were built starting with metagenomic reads generated by sequencing DNA extracted from oocysts present in stool samples from cyclosporiasis patients. Nucleotide variants were identified in the new and other publicly available genomes in comparison with the mitochondrial reference genome. A consolidated workflow, presented here, to generate new mitochondrion genomes using our reference-guided de novo assembly approach could be useful in facilitating the generation of other mitochondrion sequences, and in their application for subtyping C. cayetanensis strains during foodborne outbreak investigations.

Reactome: a knowledgebase of biological pathways.

Reactome, located at http://www.reactome.org is a curated, peer-reviewed resource of human biological processes. Given the genetic makeup of an organism, the complete set of possible reactions constitutes its reactome. The basic unit of the Reactome database is a reaction; reactions are then grouped into causal chains to form pathways. The Reactome data model allows us to represent many diverse processes in the human system, including the pathways of intermediary metabolism, regulatory pathways, and signal transduction, and high-level processes, such as the cell cycle. Reactome provides a qualitative framework, on which quantitative data can be superimposed. Tools have been developed to facilitate custom data entry and annotation by expert biologists, and to allow visualization and exploration of the finished dataset as an interactive process map. Although our primary curational domain is pathways from Homo sapiens, we regularly create electronic projections of human pathways onto other organisms via putative orthologs, thus making Reactome relevant to model organism research communities. The database is publicly available under open source terms, which allows both its content and its software infrastructure to be freely used and redistributed.

Genome Sequences of Malonate-Positive Cronobacter sakazakii Serogroup O:2, Sequence Type 64 Strains CDC 1121-73 and GK1025, Isolated from Human Bronchial Wash and a Powdered Infant Formula Manufacturing Plant.

We introduce draft genome sequences of strains CDC1121-73 (human bronchial wash isolate) and GK1025 (powdered infant formula manufacturing facility isolate), which are both malonate-positive Cronobacter sakazakii serogroup O:2, sequence type 64. Assemblies for these strains have sizes of 4,442,307 and 4,599,266 bp and % G+C contents of 56.9 and 56.7, respectively.

Is low serum tocopherol in Wilson's disease a significant symptom?

BACKGROUND: Free radical mediated injury is increasingly recognized in many metabolic diseases including Wilson's disease (WD). Use of antioxidants as an adjunctive therapy in WD may have therapeutic significance. AIM: The aim of the study was to correlate serum levels of tocopherols with serum copper and ceruloplasmin and clinical status of these patients. METHODS: Serum levels of tocopherol of were measured spectrophotometrically using the Emmerie-Engel reaction in 34 patients from a large cohort of WD being followed up at a tertiary care center. RESULTS: Majority of patients were male (M/F=23:11). The mean serum copper was 43.6+/-26.2 microg/dl (range=10-121 microg/dl) and serum ceruloplasmin was 5.6+/-5.5 mg/dl (range=0-30 mg/dl). The mean serum tocopherol level was 0.68+/-0.18 mg/dl (range=0.23-1.14 mg/dl) and compared to the control (1.07+/-0.17 mg/dl), nearly 59% of patients had decreased levels (p<0.001). No significant correlation was noted between low serum tocopherol levels and serum copper levels, Mini Mental Status Examination (MMSE) scores and CHU staging. However, serum tocopherol levels were lower in patients with relatively short duration of treatment (7.8 years vs. 12.4 years). CONCLUSION: Decreased levels of serum tocopherol were detected in 59% of patients compared to controls. However, low tocopherol levels did not correlate with clinical status or biochemical parameters of WD, except for relatively shorter duration of treatment. Further studies, especially in newly diagnosed patients, need to be done to validate the role of low tocopherol levels in Wilson's disease.

Undernutrition and the developing cerebellar cortex in the rat.

Undernutrition of the newborn rats, produced during the first 3 weeks by increasing the litter size and restricting the mother's diet, resulted in reduction of the body and brain weights of the experimental animals. One group of undernourished animals showed especially severe reduction of body and cerebellar weights. These animals, on the 10th postnatal day, had an immature cerebellar cortex corresponding to that of the 7th day postnatal control animals. The external granular layer persisted in the cerebellar cortex of the underweight animals until the 23rd day, while it disappeared by 20th day in the control animals. Mitotic activity was evident until the 21st postnatal day in these animals while it stopped in the normal animal by 16th postnatal day. There was no marked difference in the fine structure of the various cell types in the control and undernourished animals. Midsagittal tracings of the cerebellar cortex showed a reduced surface area in the undernourished animals, while the thickness of the external granular layer and molecular layer did not show any significant difference when compared to that of the control animals, thus showing a reduction in total cell number, but not per unit area. The normal morphological appearance of the cerebellar cortex in the underfed animals of higher weight probably indicates that these animals are adequately nourished in spite of the reduction in weight when compared to the control animals, which probably are overfed.

Ageing changes in the transplants of fetal substantia nigra grafted to striatum of adult rat.

Fetal nigral neurons from 16 and 17 gestational days were transplanted into the intact striatum of adult rat. On different post-transplantation days (30-360 days), the structural and immunohistochemical details of the transplants were studied. The grafted neurons matured and showed phenotypical characteristics comparable to that of normal nigral neurons in adult rats until 180 days. Tyrosine hydroxylase-positive neurons were seen not only in the transplant but also in the adjacent host striatum. Tyrosine hydroxylase-positive fibres were also seen extending for a short distance into the host striatum. A large number of synapses in the transplants were of asymmetric type, containing clear round vesicles. These synapses resembled the few intrinsic type present in the normal substantia nigra. On the other hand, the predominant type 2 synapses with pleomorphic vesicles in the normal nigra were infrequently encountered in the transplants. On the 300th day, the cytoplasm of a few of the neurons showed ageing changes in the form of clear spaces, paucity of organelles especially rough endoplasmic reticulum, membrane-bound vacuoles and increase in the lipofuscin population. In addition, localized thickening of the soma and the dendrites were seen in relation to randomly distributed neurons. By 360 days, more than one quarter (26%) of the total neurons showed these changes indicating ageing. The number per unit volume of normal neurons decreased significantly when compared to the transplants on 60 and 90 days. In the substantia nigra of age-matched control, except for an increase in the lysosomal population, other ageing changes were not detectable. The neurons of intact substantia nigra of the host rat, chronologically 4-8 months older than the transplanted neurons, also appeared normal but for lipofuscin granules. The present study provides morphological evidence for rapid ageing of neurons in the long term nigral transplants. These observations raise fresh doubts regarding permanent survival of grafted neurons in the host brain. Studies so far conducted are after prior nigral lesions. Trophic factors following lesions of the host tissue may have influenced the long term survival of the transplanted neurons. On the other hand, such changes may have been missed since no detailed morphological investigations of the long term transplants have been done so far.

Cell surface molecules (NCAM and L1) in intrastriatal transplants of embryonic mesencephalon in rats.

Cell surface molecules, NCAM and L1, reported to have a role in synaptogenesis, growth and fasciculation of the neurites in the brain, were traced in the embryonic nigral transplants in the host striatum of adult rats. Substantia nigra of five, 15 and 25 postnatal days were also examined for the same molecules. Tyrosine hydroxylase label was used as a marker to localize the nigral neurons and glial fibrillary acidic protein to detect if glial scar present. In the control as well as transplants large neurons had expressed tyrosine hydroxylase. By 15th postnatal day tyrosine hydroxylase neurons appeared mature and were scattered, suggesting a well-formed neuropil. NCAM and L1 reaction was seen as a peripheral rim in most of the cells on the fifth postnatal day. The reaction was mainly in relation to the large cells and more extensive on the 15th day. Thereafter on the 25th day, activity was negligible. Large neurons demonstrated strong reactivity for NCAM and L1 during early post-transplantation days. After 30 days only smaller cells were reactive, many of which could be identified as neurons. Strong reaction for these molecules was present only until 60 days, though faint reaction could be detected even on the 90th day. These observations indicate that the growth promoting molecules, the type seen in the neonatal period, can be detected normally only until the neurons mature. Prolonged expression of these molecules by the grafted neurons indicate delay in the maturation of these cells due to absence of adequate target sites for synaptic connections. Some of the smaller cells expressing these molecules after 30 days of transplantation could be astroglia, either proliferating or reactive.

Identification of rat mammary tumor-1 gene (RMT-1), which is highly expressed in rat mammary tumors.

Full-term pregnancy early in life results in a permanent reduction in lifetime breast cancer risk in women. Parous rats and mice are also refractory to chemical carcinogenesis. Therefore, investigation of the differences between mammary glands from virgin and parous rats would provide valuable information regarding the protective effects of early full-term pregnancy. In this report, we examined the gene expression patterns in mammary glands from virgin and parous Lewis rats. Using differential display technology, a novel 4.2 kb cDNA, designated rat mammary tumor-1 (RMT-1) was isolated. Northern blot analysis of RMT-1 showed that RMT-1 expression was higher in the pre-pubertal and pubertal stages during rat mammary gland development while it was down-regulated in mammary glands from mature virgin and parous rats. RMT-1 expression was highest in rat mammary cancers compared with either the mammary glands of virgin or parous rats. At the Northern blot sensitivity level, RMT-1 expression was found only in the mammary gland. Northern blot analysis also showed that the expression of this gene was found in 74% of N-methyl-nitrosourea (MNU)-induced mammary cancers while it was not found in MNU-induced cancers from other organs. The examination of the RMT-1 gene structure revealed that it consists of five exons spanning 5.9 kb. Using fluorescence in situ hybridization, the gene was localized on rat chromosome 1 band q 43-51. The present data show that there is a correlation between high RMT-1 expression and rat mammary carcinogenesis or decreased RMT-1 expression and parity associated refractoriness to chemically induced mammary carcinogenesis. However, whether or not RMT-1 gene has a functional role in these processes remains to be investigated.

Recovery of sleep after fetal preoptic transplantation in medial preoptic area-lesioned rats.

Changes in sleep after fetal preoptic (POA) tissue transplantation were studied in rats which had been made insomniac by a medial preoptic area (mPOA) lesion. Two days after the N-methyl D-aspartic acid (NMDA) lesion of the mPOA, fetal POA tissues (obtained from 14- to 17-day-old fetuses) were transplanted into the lesioned mPOA. Insomnia was less marked in these animals, as compared to nontransplanted lesioned rats, even on the 4th day after transplantation. The quantum of sleep nearly attained the prelesion level by the 20th day. Body weight also showed recovery after transplantation. Rectal temperature, which was increased by the lesion of the mPOA, remained unaltered even after the transplantation. These results suggest that the recovery of sleep and rectal temperature may follow different time courses. Surviving transplanted neurons were seen at the site of lesion on postmortem examination. Humoral interaction between the host and the transplant may be responsible for the early recovery of sleep, though the establishment of neural connections between the host and transplant might have contributed to the later recovery. This is the first study to show the recovery of sleep function in insomniac animals after fetal preoptic tissue transplantation. However, the specificity of the POA fetal tissue, in comparison with other neural tissues to promote sleep recovery, remains to be established.

Recovery of preoptic-anterior hypothalamic functions after transplantation.

Kainic acid lesion of the medial preoptic area resulted in the impairment of the thermoregulation and disruption of the reproductive cycle in female rats. The preoptic-anterior hypothalamic area from fetal donor was transplanted into the lesioned area ofthe host. After transplantation, these animals showed signs of estrus cyclicity and a recovery in basal temperature towards normal. They also showed a reduced shift of body temperature on exposure to a hot environment. Thus the female rats, which received transplantation, showed some degree of recovery of functions which were disrupted by kainic acid lesions.

Ultrastructure of developing substantia nigra in humans.

Electron microscopy of the maturing neurons and developing and maturing synapses in the substantia nigra of 14 human embryos/foetuses of 8-24 weeks of gestation are reported. At 8 weeks, cells were immature with very little cytoplasm and cellular organelles. Contact sites of processes appeared more electron dense than the other areas. At 12 weeks, many of the cells had acquired more cytoplasm and cellular organelles and could be identified as neurons. Asymmetric synapses with clear, round synaptic vesicles also were identifiable at this age. Such synapses, first to appear in the developing substantia nigra, are reported to be formed by recurrent collateral nigro-striatal fibres. Substance P fibres from the striatum also are contributing to this type of synapse. At 15-16 weeks, not only was the number of such synapses increased, but many appeared morphologically mature. Symmetric synapses having clear round vesicles along with a few dense core vesicles also appeared at this stage, suggesting striatal input. By 24 weeks of gestation, most of the neurons had cytological features comparable to that of the mature neurons. There was an increase in the total number of synapses and the individual variety from 15 to 24 weeks of gestation. The present study indicates that synaptogenesis starts at 8 weeks and continues beyond 24 weeks of gestation.