Cell-cell interactions promote mammary epithelial cell differentiation.

Mammary epithelium differentiates in a stromal milieu of adipocytes and fibroblasts. To investigate cell-cell interactions that may influence mammary epithelial cell differentiation, we developed a co-culture system of murine mammary epithelium and adipocytes and other fibroblasts. Insofar as caseins are specific molecular markers of mammary epithelial differentiation, rat anti-mouse casein monoclonal antibodies were raised against the three major mouse casein components to study this interaction. Mammary epithelium from mid-pregnant mice was plated on confluent irradiated monolayers of 3T3-L1 cells, a subclone of the Swiss 3T3 cell line that differentiates into adipocytes in monolayer culture and other cell monolayers (3T3-C2 cells, Swiss 3T3 cells, and human foreskin fibroblasts). Casein was synthesized by mammary epithelium only in the presence of co-cultured cells and the lactogenic hormone combination of insulin, hydrocortisone, and prolactin. Synthesis and accumulation of alpha-, beta-, and gamma-mouse casein within the epithelium was shown by immunohistochemical staining of cultured cells with anti-casein monoclonal antibodies, and the specificity of the immunohistochemical reaction was demonstrated using immunoblots. A competitive immunoassay was used to measure the amount of casein secreted into the culture medium. In a 24-h period, mammary epithelium co-cultured with 3T3-L1 cells secreted 12-20 micrograms beta-casein per culture dish. There was evidence of specificity in the cell-cell interaction that mediates hormone-dependent casein synthesis. Swiss 3T3 cells, newborn foreskin fibroblasts, substrate-attached material ("extracellular matrix"), and tissue culture plastic did not support casein synthesis, whereas monolayers of 3T3-L1 and 3T3-C2 cells, a subclone of Swiss 3T3 cells that does not undergo adipocyte differentiation, did. We conclude that interaction between mammary epithelium and other specific nonepithelial cells markedly influences the acquisition of hormone sensitivity of the epithelium and hormone-dependent differentiation.

Acute toxicity and tissue distributions of malathion in Ambystoma tigrinum.

The kinetics of the bioaccumulation of malathion (O,O-dimethyl phosphorodithioate of diethyl mercaptosuccinate) and the biological impact of exposure for tiger salamanders, Ambystoma tigrinum, were assessed through exposure to soil surface contaminated with 50 microg/cm(2) or 100 microg/cm(2 )malathion and ingestion of an earthworm exposed to soil contaminated with 200 microg/cm(2) malathion. Malathion and malaoxon burdens in salamanders sampled at different times after exposure(s) were measured by gas chromatography in four tissue/organ subgroups: liver, epaxial muscle, pooled viscera (except the liver and brain), and pooled avisceral carcass (muscle, skin, and bone). The total tiger salamander xenobiotic burdens were calculated from these data. The malathion/malaoxon burden 1 day after exposure was greatest in the avisceral carcass and 2 days after exposure was greatest in the viscera. Bioconcentration and bioaccumulation factors remained less than unity throughout the experiment and did not support the hypothesis of bioaccumulation of malathion in the tiger salamander. Biological impact was assessed with a colorimetric brain cholinesterase microassay. Brain cholinesterase activities in salamanders exposed to malathion-contaminated soil (50 microg/cm(2) or 100 microg/cm(2 )malathion) were suppressed approximately 50-65% and 90%, respectively, compared to unexposed controls. The exposed animals did not exhibit overt clinical signs of malathion toxicosis.

Phylogenetic analysis of spring virema of carp virus reveals distinct subgroups with common origins for recent isolates in North America and the UK.

Genetic relationships between 35 spring viremia of carp virus (SVCV) genogroup Ia isolates were determined based on the nucleotide sequences of the phosphoprotein (P) gene and glycoprotein (G) genes. Phylogenetic analysis based on P gene sequences revealed 2 distinct subgroups within SVCV genogroup Ia, designated SVCV Iai and Iaii, and suggests at least 2 independent introductions of the virus into the USA in 2002. Combined P- and G-sequence data support the emergence of SVCV in Illinois, USA, and in Lake Ontario, Canada, from the initial outbreak in Wisconsin, USA, and demonstrate a close genetic link to viruses isolated during routine import checks on fish brought into the UK from Asia. The data also showed a genetic link between SVCV isolations made in Missouri and Washington, USA, in 2004 and the earlier isolation made in North Carolina, USA, in 2002. However, based on the close relationship to a 2004 UK isolate, the data suggest than the Washington isolate represents a third introduction into the US from a common source, rather than a reemergence from the 2002 isolate. There was strong phylogenetic support for an Asian origin for 9 of 16 UK viruses isolated either from imported fish, or shown to have been in direct contact with fish imported from Asia. In one case, there was 100% nucleotide identity in the G-gene with a virus isolated in China.

Salmonella populations and prevalence in layer feces from commercial high-rise houses and characterization of the Salmonella isolates by serotyping, antibiotic resistance analysis, and pulsed field gel electrophoresis.

Salmonella species are recognized as a major cause of foodborne illnesses that are closely associated with the consumption of contaminated poultry and egg products. The objectives of this study were to evaluate the Salmonella populations and prevalence in layer feces during the laying cycle and molting of the hen and to characterize the layer fecal Salmonella isolates by serotyping, antibiotic resistance analysis, and pulsed field gel electrophoresis. Fecal samples were collected from a commercial layer complex consisting of 12 houses. Composite fecal samples across each row were collected as a function of bird age [18 wk (at placement), 25 to 28 wk (first peak of production cycle), 66 to 74 wk (molting), and 75 to 78 wk (second peak of production cycle)]. Bird ages and molting practice did not significantly affect (P > 0.05) Salmonella populations with an average of 1.25, 1.27, 1.20, and 1.14 log most probable number/g for the 18-, 25- to 28-, 66- to 74-, and 75- to 7-wk birds, respectively. However, the 18-wk birds had the highest prevalence of Salmonella (55.6%), followed by the 25- to 28-wk birds (41.7%), 75- to 78-wk birds (16.7%), and 66- to 74-wk birds (5.5%). Of the 45 Salmonella isolates characterized, the most predominant serovar was Salmonella Kentucky (62%). Thirty-five percent of the Salmonella isolates were resistant to at least 1 antibiotic. As expected, considerable genetic diversity was observed within and across the different serovars.

Stable Transmission of Borrelia burgdorferi Sensu Stricto on the Outer Banks of North Carolina.

The spirochaete (Borrelia burgdorferi) associated with Lyme disease was detected in questing ticks and rodents during a period of 18 years, 1991-2009, at five locations on the Outer Banks of North Carolina. The black-legged tick (Ixodes scapularis) was collected at varied intervals between 1991 and 2009 and examined for B. burgdorferi. The white-footed mouse (Peromyscus leucopus), house mouse (Mus musculus) marsh rice rat (Oryzomys palustris), marsh rabbit (Sylvilagus palustris), eastern cottontail (Sylvilagus floridanus) and six-lined racerunner (Cnemidophorus sexlineatus) were live-trapped, and their tissues cultured to isolate spirochaetes. Borrelia burgdorferi isolates were obtained from questing adult I. scapularis and engorged I. scapularis removed from P. leucopus, O. palustris and S. floridanus. The prevalence of B. burgdorferi infection was variable at different times and sites ranging from 7 to 14% of examined questing I. scapularis. Mitochondrial (16S) rRNA gene phylogenetic analysis from 65 adult I. scapularis identified 12 haplotypes in two major clades. Nine haplotypes were associated with northern/Midwestern I. scapularis populations and three with southern I. scapularis populations. Sixteen isolates obtained from tick hosts in 2005 were confirmed to be B. burgdorferi by amplifying and sequencing of 16S rRNA and 5S-23S intergenic spacer fragments. The sequences had 98-99% identity to B. burgdorferi sensu stricto strains B31, JD1 and M11p. Taken together, these studies indicate that B. burgdorferi sensu stricto is endemic in questing I. scapularis and mammalian tick hosts on the Outer Banks of North Carolina.

A quantitative real-time RT-PCR assay to measure TGF-beta mRNA and its correlation with hematologic, plasma chemistry and organo-somatic indices responses in triamcinolone-treated Atlantic menhaden, Brevoortia tyrannus.

A quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) assay was developed to measure transforming growth factor-beta (TGF-beta) in Atlantic menhaden (Brevoortia tyrannus), an estuarine-dependent species plagued by ulcerative skin lesions in the estuaries along the eastern United States. Atlantic menhaden were acclimated in a closed system for two weeks prior to initiation of the study. The synthetic glucocorticoid, triamcinolone acetonide (10mg/kg body weight) was administered by intracoelomic injection and its effect on the splenic mononuclear cell TGF-beta mRNA transcription, liver-somatic index, spleno-somatic index, hematology, and plasma chemistry were compared to untreated fish at 48 and 96h post-treatment. Triamcinolone-treated Atlantic menhaden showed suppression of TGF-beta mRNA production, neutrophilia, monocytosis, lymphopenia, and an increase in blood glucose concentrations. The health indices used in this study may help us interpret some of the changes observed during the development of ulcerative skin lesions in wild-caught menhaden.

Cytosolic water-soluble phosphodiester in vertebrate lens.

The water-soluble phosphate profiles determined by 31P nuclear magnetic resonance (NMR) of a broad range of vertebrate lenses are presented. We identify threonine ethanolamine phosphodiester (TEP) in intact lenses and lens extracts by its chemical shift and its physical and chemical characteristics. Amongst the species studied, we give the first characterization of frog lens where not only serine ethanolamine phosphodiesters (SEP) but TEP are found.

Vibrio damsela. A cause of fulminant septicemia.

A previously healthy 70-year-old man presented with a rapidly progressive and fulminant infection due to Vibrio damsela after suffering a knife cut while filleting bluefish at the New Jersey Shore. Despite appropriate antibiotic therapy and localized wound exploration, the patient died. To our knowledge, this is the first reported case of V damsela sepsis with simultaneous isolation of the organism from both blood and wound. We are reporting this case to heighten physicians' awareness of this infection and the importance of early management with antibiotics and surgical consultation.

Cost containment of the second-generation cephalosporins by prospective monitoring at a community teaching hospital.

All patients receiving cefoxitin and cefamandole were prospectively reviewed for appropriate and inappropriate utilization. There were two eight-week survey periods. In period 1, 81 (70%) of 115 patients received cefoxitin appropriately and six (40%) of 15 patients received cefamandole appropriately. In patients receiving antibiotics inappropriately, 12 (35%) of the 34 receiving cefoxitin and eight (89%) of the nine receiving cefamandole had infections that could have been treated with less expensive, equally efficacious antibiotics. Changes in antibiotic therapy were made in 79% of patients based on our recommendations. The estimated annual cost saving for these antibiotics was $40,290. During period 2, 73 (91%) of 80 patients were given cefoxitin appropriately and 14 (61%) of 23 patients received cefamandole appropriately. Forty-three percent of those receiving cefoxitin and 33% of those receiving cefamandole inappropriately could have been treated with a less expensive, equally efficacious antibiotic. In 88% of patients, the attending physicians followed our recommendations.

Mice as reservoirs of the Lyme disease spirochete.

In evaluating the white-footed mouse as a reservoir host for the Lyme disease spirochete, we compared spirochete infection in vector ticks (Ixodes dammini) having different histories of attachment to these mice, estimated their relative importance as hosts for immature I. dammini and compared the seasonality of tick activity and spirochetemia in mice. Infection in trapped white-footed mice appears to be universal. Prevalence of spirochetal infection in I. dammini correlates with frequency of attachment to mice, and in mice, with the season of vector activity. The relative abundance of this mouse makes it numerically the most important host for I. dammini. Most immature I. dammini appear to attach to white-footed mice. Taken together, these considerations demonstrate that the white-footed mouse serves as reservoir for the Lyme disease spirochete in coastal Massachusetts.

Reservoir competence of the southeastern five-lined skink (Eumeces inexpectatus) and the green anole (Anolis carolinensis) for Borrelia burgdorferi.

The reservoir competence of two lizard species, the southeastern five-lined skink (Eumeces inexpectatus) and the green anole (Anolis carolinesis), for Borrelia burgdorferi was evaluated. Skinks and anoles were exposed by needle inoculation or tick bite to B. burgdorferi. Xenodiagnosis with larval Ixodes scapularis and culture of tissues were used to asses infection and the ability of infected lizards to infect attached ticks. Both lizard species were susceptible to B. burgdorferi by both routes of exposure. Xenodiagnostic ticks acquired spirochetes while feeding on both species. One tick that dropped from a skink on the ninth day after exposure was infected. The remainder of xenodiagnostic ticks that acquired spirochetes fed three weeks after exposure of the lizards to the spirochete. Lizards remained infectious to attached ticks for at least five weeks. Overall, more than 20% of xenodiagnostic larvae fed on southeastern five-lined skinks acquired spirochetes. Individual skinks infected up to 34% of attached ticks. A smaller proportion of ticks feeding on green anoles became infected. Borrelia burgdorferi recovered from infected lizards retained their infectivity for mammalian hosts. The ability of the lizards to sustain a Borrelia infection and infect attached ticks suggests that they may play a role in the maintenance of spirochete transmission.

Vancomycin: a review.

This article reviewed the past experience and updated the present data on vancomycin. In recent years, with the release of more purified preparations of vancomycin and new clinical problems facing the clinician (that is, increasing prevalence of serious methicillin-resistant staphylococcal infections; use of hemodialysis and CAPD; widespread use of prosthetic devices; aggressive chemotherapy), the indication for vancomycin use has increased dramatically. More information is needed on the distribution of vancomycin in body tissues and the incidence and mechanisms of toxicity. Close surveillance of in vitro susceptibility patterns will be necessary as widespread use of vancomycin continues to increase.

Bunyavirus infections in North Carolina white-tailed deer (Odocoileus virginianus).

We analyzed serum from white-tailed deer (Odocoileus virginianus) collected in southeastern North Carolina in 1991 for neutralizing antibodies to six mosquito-borne bunyaviruses (Lacrosse, Jamestown Canyon, Keystone,Cache Valley, Potosi, and Tensaw), including several of public health importance. Evidence was found for all six to be locally transmitted, although greatest seroprevalence was found for Potosi, Jamestown Canyon, and Cache Valley viruses.

Comparison of an enzyme-linked immunosorbent assay to an indirect immunofluorescence assay for the detection of antibodies to Borrelia burgdorferi in the dog.

An enzyme-linked immunosorbent assay (ELISA) was compared to an indirect immunofluorescence assay (IFA) for detection of IgG antibodies to Borrelia burgdorferi in dog sera. The concordance of the two tests was 93.5% for sera from dogs from Maryland (n = 93), 98.0% for sera from dogs from North Carolina (n = 446), and 97.2% for the combined sample groups (n = 539). Twenty-five of the 27 samples with discordant or low positive results were tested, and showed immunoblot reactions to 1 to 10 different bands. Reaction patterns and intensity of the bands were quite variable, and did not explain a reason for the discordance.

Shared flagellar epitopes of Borrelia burgdorferi and Borrelia anserina.

Antigenic cross-reactivity between Borrelia burgdorferi and Borrelia anserina was studied using mouse immune sera and monoclonal antibodies. With immune sera, significant cross-reactivity between B. burgdorferi and B. anserina was demonstrated by indirect immunofluorescent assay. In immunoblots, most of the cross-reactivity was shown to be associated with the periplasmic flagella. Using monoclonal antibodies in immunoblots, it was shown that B. burgdorferi and B. anserina shared at least two flagellar epitopes, one of which was not shared with Borrelia hermsii or Borrelia coriaceae. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of whole cell lysates and the use of a species-specific monoclonal antibody (H5332) which reacts with a major outer surface protein (Osp A) of B. burgdorferi readily differentiated the two species at the molecular level.

Borrelia burgdorferi in ticks (Acari: Ixodidae) from coastal Virginia.

Ixodid ticks removed from hosts and from vegetation during March-November 1987 at sites in coastal Virginia and North Carolina were examined for Borrelia burgdorferi. B. burgdorferi was evident in nine (22%) Ixodes cookei Packard removed from rice rats (Oryzomys palustris), a white-footed mouse (Peromyscus leucopus), and raccoons (Procyon lotor); four (6%) Amblyomma americanum (L.) removed from raccoons; and two (3%) Dermacentor variabilis (Say) removed from a raccoon and a rice rat. B. burgdorferi was also detected in Ixodes dentatus Marx removed from a brown thrasher (Toxostoma rufum), a Carolina wren (Thryothoros ludovicianus), and a towhee (Piplio erythrophthalamus); and in Haemaphysalis leporispalustris (Packard) removed from a brown thrasher and a white-throated sparrow (Zonotrichia albicollis) netted at Kiptopeke Beach, Va. Two Ixodes dammini Spielman, Clifford, Piesman & Corwin were collected on Parramore Island; one specimen was examined for spirochetes, and it was infected with B. burgdorferi. No spirochetes were detected in host-seeking A. americanum and Amblyomma maculatum Koch removed from vegetation. The plasma of one P. leucopus and sera obtained from two P. lotor contained antibodies to B. burgdorferi. All infected ticks and the seroreactive hosts were collected from the Eastern Shore of Virginia.

Reservoir competence of the rice rat (Rodentia: Cricetidae) for Borrelia burgdorferi.

The reservoir competence of the rice rat, Oryzomys palustris, for Borrelia burgdorferi is described. Infected Ixodes scapularis Say (I. dammini, Spielman, Clifford, Piesman & Corwin) nymphs were used to infect animals. Borrelia infection was diagnosed by xenodiagnostic feeding of noninfected I. scapularis larvae and by reisolation of the spirochetes from blood and other tissues. Rice rats acquired B. burgdorferi and maintained spirochete infection for 5-9 wk. B. burgdorferi were cultured from samples of skin and urinary bladders from all animals killed on day 21 (three rats), 35 (three rats), or 56 (three rats) after infection. The spirochetes were also detected in blood samples obtained 1 and 2 wk after exposure. Spirochetes that persisted for 5 wk in rice rats did not lose their infectivity for golden Syrian hamsters. The prepatent period for infecting xenodiagnostic ticks was 1 wk. Overall, 75.6% of I. scapularis larvae (n = 694) that fed on infected rice rats acquired B. burgdorferi. Prevalence of infection reached 83% in ticks that fed on tick-exposed animals during the 2nd-4th wk, 68% during the 5th wk, and 17.4% during the 9th wk. The duration of rice rat infectivity for ticks exceeded 2 mo. I. scapularis nymphs infected as larvae on rice rats transmitted B. burgdorferi. Taken together, these studies confirm the reservoir competence of the rice rat for B. burgdorferi.

Lizards as hosts for immature Ixodes scapularis (Acari: Ixodidae) in North Carolina.

Previously archived museum specimens of lizards collected throughout North Carolina were examined for Ixodes scapularis (Say). Lizards (n = 1,349) collected in 80 of North Carolina's 100 counties were examined. Lizards with ticks were collected in 23 (29%) of the 80 counties from which lizards were examined. I. scapularis was detected on 8.7% (n = 117) of the lizards and was the sole species of tick obtained from lizards. Immature ticks were most frequently found on the southeastern five-lined skink, Eumeces inexpectatus, and the eastern glass lizard, Ophisaurus ventralis. Larvae were most frequently found on the six-lined racerunner, Cnemidophorus sexlineatus. One C. sexlineatus harbored 177 larvae and 2 nymphs. Nymphs were most frequently observed on E. inexpectatus. The majority of counties (chi 2, P < 0.01) where ticks were found on lizards were in the Coastal Plain.

Attempted Ehrlichia risticii transmission with Dermacentor variabilis (Acari: Ixodidae).

Larval Dermacentor variabilis (Say) (n = 327) were fed on Balb/C mice inoculated with Ehrlichia risticii, the etiologic agent of equine monocytic ehrlichiosis (Potomac horse fever). All mice displayed clinical signs of E. risticii infection at the time of feeding. After molting, resulting nymphs (n = 74) were fed on susceptible mice. No clinical signs were observed, and the mice remained seronegative for 6 wk after feeding.

Canine exposure to Borrelia burgdorferi and prevalence of Ixodes dammini (Acari: Ixodidae) on deer as a measure of Lyme disease risk in the northeastern United States.

Surveillance programs that identify areas where both the vector (Ixodes dammini) and etiologic agent (Borrelia burgdorferi) are present may identify the risk of Lyme disease and its spread earlier and more accurately than do programs relying on any single method, particularly human case reports. Hunter-killed deer (n = 1,204) from 22 counties in Maryland, Massachusetts, New Hampshire, New York, and Pennsylvania were examined in fall 1989 and all ectoparasites were identified. The following spring, canine sera (n = 884) were obtained from these sites, which included known endemic areas and those where Lyme disease is uncommon, and tested by enzyme-linked immunosorbent assay. Regression analysis of canine seroprevalence versus tick prevalence on deer from the same counties demonstrate a significant positive linear relationship. Sites were designated as low-, moderate-, or high-risk counties based upon their position on the regression curve. The geographic distribution of the sites correlated well with the distribution of known Lyme disease endemic and nonendemic areas. Locations were also identified where Lyme disease may be emerging. The positive relationship between measures of vector and pathogen abundance determined in this study permits public health workers to identify endemic and potentially endemic areas independently of human case reports.