Effects of a 28-day dietary co-exposure to melamine and cyanuric acid on the levels of serum microRNAs in male and female Fisher 344 rats.

We showed previously that a 28-day combined dietary exposure to melamine and cyanuric acid (MEL&CYA) induced kidney lesions in NCTR Fisher 344 (F344) rats. Histopathological changes were significant in females dosed with ≥240 ppm MEL&CYA and in males dosed with ≥180 ppm MEL&CYA; however, the nephrotoxicity biomarkers blood urea nitrogen (BUN) and serum creatinine (SCr) were increased only by ≥240 ppm MEL&CYA. The serum miRNome has been reported to reflect toxicity of several organs, including the kidney. Here, we compared the dose-response of alterations in serum miRNAs to those of BUN, SCr, and kidney histopathology in rats co-exposed to MEL&CYA. The serum miRNome of male F344 rats dosed with 0, 180, or 240 ppm MEL&CYA was screened using quantitative real-time RT-PCR (qRT-PCR) and the levels of selected serum miRNAs were analyzed further in both sexes over the full dose range. The levels of several miRNAs were significantly reduced in rats treated with 240 ppm MEL&CYA versus control. In addition, miR-128-3p and miR-210-3p were decreased in males treated with 180pm MEL&CYA, a dose at which the levels of BUN and SCr were not yet affected by treatment. These data suggest that the serum miRNome is affected by nephrotoxic doses of MEL&CYA in male and female rats.

Highlights from the United States Food and Drug Administration's public workshop on the development of animal models of pregnancy to address medical countermeasures in an "at-risk" population of pregnant women: Influenza as a case study.

The U.S. Food and Drug Administration (FDA) and other federal agencies partner to ensure that medical countermeasures (e.g., drug therapies and vaccines) are available for public health emergencies (FDA, 2014). Despite continuing progress, providing medical countermeasures and treatment guidelines for certain populations (e.g., pregnant women) is challenging due to the lack of clinical and/or animal data. Thus, a workshop was convened to discuss animal models of pregnancy for the evaluation of disease progression and medical countermeasures.

Susceptibility to Cronobacter sakazakii decreases with increasing age in neonatal CD-1 mice.

Neonatal, premature, or very low birth weight infants fed reconstituted powdered infant formula contaminated with Cronobacter (Enterobacter sakazakii) may develop infections resulting in severe outcomes such as septicemia, necrotizing enterocolitis, meningitis, or death. Infants who recover from infection may have morbidities such as hydrocephalus, mental retardation, or developmental delays. Although increasing age appears to reduce susceptibility to Cronobacter infection, it is not known at what age or why these infants become less susceptible. Our study objectives were to compare the susceptibilities of neonatal mice of different ages to Cronobacter sakazakii infection. Timed-pregnant CD-1 mice were allowed to give birth naturally. Neonatal mice were orally gavaged at postnatal days (PNDs) 1.5, 5.5, and 9.5 with a single dose of vehicle or 10(3), 10(7), or 10(10) CFU/ml C. sakazakii strain MNW2 in reconstituted powdered infant formula. Pups were euthanized 7 days after challenge. Brains, livers, and ceca were excised and analyzed for C. sakazakii invasion, and blood was collected for serum amyloid A analysis as a biomarker of infection. C. sakazakii invasion was age dependent; the pathogen was isolated from brains, livers, and ceca of neonatal mice treated at PNDs 1.5 and 5.5 but not from those of pups treated at PND 9.5. C. sakazakii was more invasive at PND 1.5 in brains than in livers and ceca and was isolated from 22, 14, and 18% of these tissue samples, respectively. Serum amyloid A was detected in only one treated neonate. Mortality was observed only in neonates treated at PND 1.5. In conclusion, neonatal mice had a time-dependent susceptibility to C. sakazakii infection, with resistance increasing with increasing age.

Time course of fetal tissue invasion by Listeria monocytogenes following an oral inoculation in pregnant guinea pigs.

Pregnant women are 20 times more likely to develop listeriosis than are members of the general population, and infection can result in abortion, stillbirth, or neonatal illness. The objective of this study was to orally challenge pregnant guinea pigs with Listeria monocytogenes to assess maternal and fetal tissue invasion at postinoculation days 2, 6, 9, and 21. The time course of invasion was followed by fluorescence microscopy and a traditional culture method. Guinea pigs were treated on gestation day 35 with L. monocytogenes doses ranging from 10(4) to 10(8) CFU. L. monocytogenes was isolated and viewed in maternal and fetal tissues as early as 2 days postinoculation. L. monocytogenes was isolated from placentas, fetal livers and brains, and maternal spleens at similar rates, suggesting that invasion of the spleen could be indicative of fetal invasion. When comparing fecal shedding, all animals treated with 10(4) CFU were shedding L. monocytogenes by postinoculation day 7, and all animals treated with the higher doses (10(6) or 10(8) CFU) were shedding L. monocytogenes by postinoculation day 5. These data suggest that L. monocytogenes crosses the fetoplacental barrier and invades the fetus by day 2 after maternal ingestion. When comparing the sensitivities of microscopy and culture, neither method consistently detected L. monocytogenes at a higher rate. However, detection in individual tissues differed. Microscopy was significantly more sensitive with fetal liver (P<0.001) and brain (P<0.001) at the highest dose of 10(8) CFU, but at the lowest dose of 10(4) CFU culture was significantly more sensitive with maternal spleen (P=0.04).

Comparison of virulence of three strains of Cronobacter sakazakii in neonatal CD-1 mice.

Cronobacter sakazakii (Enterobacter sakazakii) is an emerging pathogen that has been isolated from powdered infant formula and associated with outbreaks of infection in infants in neonatal intensive care units. In a previous study, we observed that neonatal CD-1 mice are susceptible to C. sakazakii infection and that the pathogen invades brain, liver, and cecum tissues. The study objective was to compare the virulence of three strains of C. sakazakii in neonatal CD-1 mice. The strains tested were MNW2 (a food isolate), SK81 (a clinical isolate), and 3290 (a clinical isolate). Timed-pregnant CD-1 mice were allowed to give birth on gestation day 19 or 20. Neonatal mice were sexed and culled to 10 per litter, each having five males and five females. Neonates were orally gavaged with C. sakazakii strains MNW2, SK81, or 3290 at doses ranging from 10(2.8) to 10(10.5) CFU on postnatal day 3.5. Pups surviving to postnatal day 10.5 were euthanized, and brain, liver, and cecum tissues were excised. C. sakazakii was isolated from all three tissues in mice treated with C. sakazakii, regardless of strain. C. sakazakii strain 3290 was significantly more invasive in brains (42.1% of mice) than were strains MNW2 (6.7%) and SK81 (15.9%). Mortality was observed for all strains of C. sakazakii tested, with SK81 being significantly more lethal (5.6%) than MNW2 (1.2%) or 3290 (0.6%). Our findings suggest that invasiveness does not necessarily correlate with mortality among different strains of C. sakazakii, and the clinical isolates are more virulent than the food isolate.

Risk of fetal mortality after exposure to Listeria monocytogenes based on dose-response data from pregnant guinea pigs and primates.

One-third of the annual cases of listeriosis in the United States occur during pregnancy and can lead to miscarriage or stillbirth, premature delivery, or infection of the newborn. Previous risk assessments completed by the Food and Drug Administration/the Food Safety Inspection Service of the U.S. Department of Agriculture/the Centers for Disease Control and Prevention (FDA/USDA/CDC) and Food and Agricultural Organization/the World Health Organization (FAO/WHO) were based on dose-response data from mice. Recent animal studies using nonhuman primates and guinea pigs have both estimated LD(50)s of approximately 10(7) Listeria monocytogenes colony forming units (cfu). The FAO/WHO estimated a human LD(50) of 1.9 x 10(6) cfu based on data from a pregnant woman consuming contaminated soft cheese. We reevaluated risk based on dose-response curves from pregnant rhesus monkeys and guinea pigs. Using standard risk assessment methodology including hazard identification, exposure assessment, hazard characterization, and risk characterization, risk was calculated based on the new dose-response information. To compare models, we looked at mortality rate per serving at predicted doses ranging from 10(-4) to 10(12) L. monocytogenes cfu. Based on a serving of 10(6) L. monocytogenes cfu, the primate model predicts a death rate of 5.9 x 10(-1) compared to the FDA/USDA/CDC (fig. IV-12) predicted rate of 1.3 x 10(-7). Based on the guinea pig and primate models, the mortality rate calculated by the FDA/USDA/CDC is underestimated for this susceptible population.

Immunological and pathological changes in the placenta during infection with Listeria monocytogenes in pregnant guinea pigs.

Exposure to Listeria monocytogenes during pregnancy can result in spontaneous abortion and stillbirths; however, the mechanisms are unknown. Our objective was to determine the effects of infection on specific inflammatory and anti-inflammatory cytokine mRNA expression and apoptosis in the placenta after infection with L. monocytogenes. Pregnant guinea pigs were treated on gestation day (gd) 35 with 10(8) colony forming units L. monocytogenes and sacrificed on gd 37, 41, 44, or 55. At gd 41, IFN-gamma and IL-2 mRNA expression was significantly decreased in placentas from treated dams (0.0012-fold and 0.131-fold, respectively). At gd 55, TNF-alpha mRNA expression was significantly decreased (0.19-fold), while IFN-gamma mRNA expression was significantly increased (32-fold), and apoptosis was detected in 100% of placentas from treated dams. In conclusion, inflammatory cytokine mRNA expression is altered and apoptosis is increased in the placenta after treatment with L. monocytogenes, and these changes may contribute to fetal death.

Listeria monocytogenes infection in pregnant guinea pigs is associated with maternal liver necrosis, a decrease in maternal serum TNF-alpha concentrations, and an increase in placental apoptosis.

Stillbirths and spontaneous abortions can result when pregnant women are exposed to the food borne pathogen, Listeria monocytogenes. Fetuses and neonates account for one-third of the 2500 cases annually. The objectives were to determine the dose dependent trends of immunological and pathological effects in pregnant guinea pigs after infection with L. monocytogenes. Timed pregnant guinea pigs were treated on gestation day (gd) 35 with doses of 10(4) to 10(8) colony forming units (CFUs) and sacrificed on gd 56. Hepatic lesions were found in dams treated with >or=10(5)CFUs. Apoptosis was detected in significantly more placentas from dams treated with >or=10(6)CFUs compared to controls. Maternal serum TNF-alpha concentrations were significantly decreased in all dose groups compared to controls. In conclusion, increases in premature delivery, maternal hepatic effects and placental apoptosis along with a decrease in TNF-alpha concentrations were associated with L. monocytogenes infection in pregnant guinea pigs.

A processing plant persistent strain of Listeria monocytogenes crosses the fetoplacental barrier in a pregnant guinea pig model.

The foodborne pathogen Listeria monocytogenes can cause infection in immunocompromised humans and in the fetuses of pregnant women. We have demonstrated that one group of genetically similar L. monocytogenes strains (random amplified polymorphic DNA [RAPD] type 9) dominate and persist in several independent fish processing plants. The purpose of the present study was to determine the virulence potential of one RAPD type 9 strain (La111), one human clinical strain (Scott A), and one monkey clinical strain (12443) in a pregnant guinea pig model. Animals were orally exposed to 10(8) CFU of L. monocytogenes in whipping cream on gestation day (GD) 36 and euthanized on GD 42, 45, or 56. Strains 12443 and Scott A were shed from treated animals for 20 days, whereas La111 was shed only in the first 10 days. Strains 12443 and Scott A were recovered from maternal liver, spleen, and gallbladder on all 3 days of euthanization, whereas La111 was recovered only at GD 45 and 56. Scott A was not isolated from any placentas or fetuses. For dams treated with 12443, 22% of the fetuses were positive for L. monocytogenes, and surprisingly, treatment of dams with La111 resulted in 56% infected fetuses. L. monocytogenes was isolated from 16 and 20% of placentas for 12443 and La111, respectively. The study demonstrates that a food processing plant persistent strain of L. monocytogenes is able to cross the fetoplacental barrier in pregnant guinea pigs. Furthermore, we demonstrate that although information can be gained from model virulence assays, assessment of the virulence potential of a strain may require more complex hosts.

Dose-response of Listeria monocytogenes after oral exposure in pregnant guinea pigs.

Listeriosis, a severe disease that results from exposure to the foodborne pathogen Listeria monocytogenes, is responsible for approximately 2500 illnesses and 500 deaths in the United States each year. Pregnant women are 20 times more likely to develop listeriosis than the general population, with adverse pregnancy outcomes that include spontaneous abortions, stillbirths, and neonatal meningitis. The objective of this study was to determine an infective dose that resulted in stillbirths and infectivity of selected tissues in pregnant guinea pigs. Pregnant guinea pigs were exposed orally on gestation day 35 to 10(4) to 10(8) L. monocytogenes CFU in sterile whipping cream. L. monocytogenes was recovered at 64, 73, 90, and 100% from the livers of animals infected with 10(5), 10(6), 10(7), and 10(8) CFU, respectively. In dams exposed to > or =10(6) CFU, L. monocytogenes was cultured from 50% of the spleen samples and 33% of the gallbladder samples. Eleven of 34 dams infected with > or =10(6) CFU delivered stillborn pups. L. monocytogenes was cultured from the placenta, liver, and brain tissue of all stillbirths. Dams that delivered nonviable fetuses after treatment with > or =10(7) L. monocytogenes CFU had fecal samples positive for L. monocytogenes at every collection posttreatment. On the basis of a log-logistic model, the dose that adversely affected 50% of the pregnancies was approximately 10(7) L. monocytogenes CFU compared with that estimated from a human outbreak of 106 CFU. Listeriosis in pregnant guinea pigs can result in stillbirths, and the overall disease is similar to that described in nonhuman primates and in humans.