Outbreaks of typhoid fever in the United States, 1960-99.

Although the incidence of typhoid fever in the United States has been low since the 1940s, Salmonella Typhi continues to cause outbreaks. We reviewed reported outbreaks of typhoid fever from 1960 to 1999. There were 60 outbreaks; in 54, exposure occurred within the United States. These 54 outbreaks accounted for 957 total cases (median 10) and 4 deaths. In 36 (67%) outbreaks the route of transmission was identified, and in 16 (62%) of the 26 foodborne outbreaks an asymptomatic carrier was identified by culture or serology. The median incubation period was 2 weeks. Isolates from 10 (40%) of 25 outbreaks were phage type E1. The average frequency of outbreaks decreased from 1.85/year during 1960-79 to 0.85/year during 1980-99 (P=0.0001). S. Typhi outbreaks in the United States are generally small in size but can cause significant morbidity, and are often foodborne, warranting thorough investigation.

Specific localization of transcription factors in the chromatin of mouse mature spermatozoa.

We previously characterized a nuclease-hypersensitive fraction of mouse sperm chromatin, which is organized in a typical nucleosomal structure. A partial genomic library was constructed with the DNA from the nuclease-hypersensitive chromatin, which revealed a high content in retroposon/retroviral DNA sequences. Here we report that the cloned nuclease-hypersensitive DNA also contains clusters of potential sites for transcription factors: among those, binding sites for Oct-1, Oct-4, TBP, Ets-1, and C/EBP are most abundant. This observation prompted us to ask whether mature spermatozoa contain the corresponding protein factors. Indirect immunofluorescence experiments show that all analyzed factors are indeed present in the sperm heads. Moreover, transcription factors are associated with the nuclease-hypersensitive chromatin of spermatozoa, as endogenous nucleases that degrade the hypersensitive fraction also cause the concomitant release of transcription factors from sperm cells into the medium. Band-shift assays with proteins extracted from the supernatant, and immunofluorescence analysis of sperm pellets, indicate that transcription factors are largely recovered in the supernatant while being absent or poorly retained in spermatozoa. The possible involvement of these factors in early embryogenesis is discussed.

E2F transcription factors are differentially expressed in murine gametes and early embryos.

We have examined the murine genes encoding transcription factors E2F1, -3, -5 and -6 in gametes and early embryos. All genes are expressed as maternal transcripts and all are efficiently transcribed after the blastocyst stage. Between those two stages, each E2F mRNA is transcribed with a distinctive and unique pattern. E2F proteins are also differentially expressed and compartmentalized in pre-implantation embryos.

Nucleosomal domains of mouse spermatozoa chromatin as potential sites for retroposition and foreign DNA integration.

Exogenous DNA molecules are spontaneously taken up by sperm cells, internalized in nuclei, and eventually integrated in the sperm genome. The actual occurrence of the integration suggests that the sperm chromosomal DNA is not uniformly and tightly packed with protamines, implying the existence of genomic sites where the chromosomal DNA is accessible to foreign molecules. We have characterized a hypersensitive, nucleosomal subfraction of mouse sperm chromatin that is highly enriched in unmethylated retroposon DNA from a variety of families. Here we propose that both the integration of exogenous DNA molecules, and the endogenous retroposition activity, occur in the same site(s) of sperm chromatin.

DNA dose and sequence dependence in sperm-mediated gene transfer.

We have tested three parameters in sperm-mediated gene transfer assays with mice and pigs: (i) the epididymal versus ejaculated origin of sperm cells, (ii) the primary structure, and (iii) the amount of the challenging foreign DNA. We have found that the pVLCNhGH construct, of retrotransposon origin, causes a massive embryo lethality and yet increases the yield of genetic transformation among born animals of both species compared to viral constructs. Arrest of embryonic development is a DNA dose-dependent effect, which is observed with high DNA doses, while lower doses are compatible with development. Finally, the overall efficiency of sperm-mediated gene transfer is higher when ejaculated, versus epididymal, spermatozoa are used. We suggest that this difference is related to the highly efficient apoptotic response in epididymal compared to ejaculated spermatozoa, triggered by the interaction of exogenous DNA molecules with the sperm membrane.

Reverse transcriptase activity in mature spermatozoa of mouse.

We show here that a reverse transcriptase (RT) activity is present in murine epididymal spermatozoa. Sperm cells incubated with human poliovirus RNA can take up exogenous RNA molecules and internalize them in nuclei. Direct PCR amplification of DNA extracted from RNA-incubated spermatozoa indicate that poliovirus RNA is reverse-transcribed in cDNA fragments. PCR analysis of two-cell embryos shows that poliovirus RNA-challenged spermatozoa transfer retrotranscribed cDNA molecules into eggs during in vitro fertilization. Finally, RT molecules can be visualized on sperm nuclear scaffolds by immunogold electron microscopy. These results, therefore, reveal a novel metabolic function in spermatozoa, which may play a role during early embryonic development.

A fraction of mouse sperm chromatin is organized in nucleosomal hypersensitive domains enriched in retroposon DNA.

We have characterized a nuclease hypersensitive chromatin fraction from murine spermatozoa. Endogenous nuclease activity can be induced in mouse epididymal spermatozoa by appropriate stimuli and cause the localized degradation of chromosomal DNA. Based on these observations, we have isolated nuclease hypersensitive chromatin regions released from spermatozoa in the supernatant of pelleted sperm cells, and have cloned and characterized the DNA. Gel electrophoresis of end-labelled released DNA fragments showed a typical nucleosomal distribution. Peripherally distributed nucleohistones were visualized by immunofluorescence in sperm nuclei, and histones were identified by western blot in sperm chromatin. Moreover, the released DNA is enriched in retroposon DNA from a variety of families. FISH and immunofluorescence analysis showed that retroposon DNA and nucleohistone chromatin co-localize and are both peripherically distributed in nuclei of spermatozoa. In contrast, a major satellite DNA probe, used for control, co-localizes with highly condensed chromatin in the central region of sperm nuclei. The nuclear Ran and RCC1 proteins were also visualized in the dorsal margin of sperm nuclei, and were abundantly released with the hypersensitive chromatin fraction. Together, these results indicate that nucleohistone chromatin fraction(s) with typical features of 'active' chromatin are present in murine spermatozoa, are hypersensitive to nuclease cleavage, enriched in retroposon DNA and organized in nucleosomal domains. These observations suggest that nucleohistone domains identify a fraction of the sperm genome which may be functional during early embryogenesis.

Sperm/DNA interaction: integration of foreign DNA sequences in the mouse sperm genome.

Foreign DNA is spontaneously taken up by mouse epididymal sperm cells and is further internalized into nuclei. The interaction and/or internalization of the exogenous DNA triggers the activation of sperm endogenous nucleases which mediate rearrangements of the internalized DNA. Foreign DNA sequences are found to be tightly bound to the sperm nuclear scaffold, and to undergo a recombination process with the sperm chromosomal DNA. Sequence analysis of randomly selected clones from a library of sperm genomic DNA transformed with pSV2CAT plasmid showed that foreign sequences were integrated in a unique site of the sperm genome. Preliminary results suggest that the integration process is mediated by a retrotranscription step.

Submicroscopic mathematical evaluation of spermatozoa in assisted reproduction. 4. The bovine fertilization (Notulae seminologicae 10).

In this paper we apply a modification of the formula of Baccetti et al. (1995) in the evaluation of submicroscopical characteristics of bull spermatozoa used in assisted reproduction. In the present experiment sperm quality is proposed as a useful parameter in predicting the success of fertilization. Our results demonstrate that the percentage of spermatozoa devoid of submicroscopic defects, according to the particular Bayesan formula proposed by us, is clearly correlated with the result of artificial insemination. In fact, the parameter concerning sperm quality obtained in variously successful donors shows a large correlation with fertility power. The synthetic parameters observed are therefore a good tool in the prediction of sperm power in artificial fertilization. The evaluation is mainly concerned with the quality of the acrosomal characters, the status of the chromatin, the shape of mitochondria, the position of the postacrosomal sheath, the perinuclear space and the axonemal pattern. All these characters are expressed with different means in ejaculates. All these data confirm that submicroscopic-mathematical evaluation offers a convincing and reliable diagnosis based upon sperm structure and functions such as acrosomal reaction and cell motility. It has been also demonstrated that sperm quality is a major factor in the success of artificial insemination and it is clearly revealed in the integrity of most of sperm organelles.

Meat grinders and molecular epidemiology: two supermarket outbreaks of Escherichia coli O157:H7 infection.

Between 23 June and 15 July 1994, 21 cases (19 primary and 2 secondary) of Escherichia coli O157:H7 infection were identified in the Bethel, Connecticut, area. Three pulsed-field gel electrophoresis (PFGE) patterns from 15 isolates (I, n = 13; II, n = 2; and III, n = 1) were observed. A case-control study that excluded secondary cases and patients with PFGE II and III patterns (n = 16) demonstrated that consumption of food from one supermarket was associated with illness (15/16 cases vs. 31/47 geographically matched controls, odds ratio [OR] undefined, lower 95% confidence interval OR = 1.45, P = .018). No one food was associated with illness. Inspection of the supermarket revealed deficiencies in hygiene and meat handling practices. The 2 cases with PFGE II ate raw beef and raw lamb from a second supermarket. These outbreaks demonstrate the value of PFGE in supporting epidemiologic investigations and the potential for outbreaks arising from retail outlets.

Morphogenesis of the decapitated and decaudated sperm defect in two brothers.

The "decapitated sperm" defect, found in both of two sterile brothers, may be assumed to have a genetic origin. The present material suggests that the term "decapitated spermatozoa" is not exact, because detached heads and tails were found in the brothers' ejaculate that could be regarded as "decapitated tails" and "decaudated heads." The present report describes frequent, more or less advanced stages of detachment. Both heads and tails showed a normal structure in which only the postnuclear region was deficient, lacking basal plate and implantation fossa. A break at a different level of the midpiece, and therefore three kinds of separation, were observed. The defect, according to the present research, must originate in the testicular region, whereas the detachment occurs in the epididymis.

Crater defect in human spermatozoa.

This report describes the "crater defect" in human spermatozoa, a malformation that consists of a nuclear and acrosomal invagination present in 100% of the cells, whereas tail structure and motility are fairly normal. The defect occurs during spermiogenesis. A possible concomitance with abnormalities in the microtubular apparatus involved in the sperm molding is discussed.

Immunocytochemistry and sperm pathology.

In this paper the Authors describe the localization of some significative proteins (acrosin, actin, tubulin, vimentin) detected by immunocytochemical methods in human infertile spermatozoa and correlate their distribution with the ultrastructural characteristics of the same spermatozoa, in order to clarify the structural bases of infertility.