Protecting Human and Animal Health: The Road from Animal Models to New Approach Methods.

Animals and animal models have been invaluable for our current understanding of human and animal biology, including physiology, pharmacology, biochemistry, and disease pathology. However, there are increasing concerns with continued use of animals in basic biomedical, pharmacological, and regulatory research to provide safety assessments for drugs and chemicals. There are concerns that animals do not provide sufficient information on toxicity and/or efficacy to protect the target population, so scientists are utilizing the principles of replacement, reduction, and refinement (the 3Rs) and increasing the development and application of new approach methods (NAMs). NAMs are any technology, methodology, approach, or assay used to understand the effects and mechanisms of drugs or chemicals, with specific focus on applying the 3Rs. Although progress has been made in several areas with NAMs, complete replacement of animal models with NAMs is not yet attainable. The road to NAMs requires additional development, increased use, and, for regulatory decision making, usually formal validation. Moreover, it is likely that replacement of animal models with NAMs will require multiple assays to ensure sufficient biologic coverage. The purpose of this manuscript is to provide a balanced view of the current state of the use of animal models and NAMs as approaches to development, safety, efficacy, and toxicity testing of drugs and chemicals. Animals do not provide all needed information nor do NAMs, but each can elucidate key pieces of the puzzle of human and animal biology and contribute to the goal of protecting human and animal health. SIGNIFICANCE STATEMENT: Data from traditional animal studies have predominantly been used to inform human health safety and efficacy. Although it is unlikely that all animal studies will be able to be replaced, with the continued advancement in new approach methods (NAMs), it is possible that sometime in the future, NAMs will likely be an important component by which the discovery, efficacy, and toxicity testing of drugs and chemicals is conducted and regulatory decisions are made.

A Follow Up to the AARP Age-Friendly Community Survey Through a Focus Group Study.

Introduction: In an effort to help communities plan for their burgeoning aging population, the American Association of Retired Persons (AARP) has facilitated community surveys to enable older adults to rate the current state of their own community for "aging in place." This Focus Group Study extended the findings of the AARP Age-Friendly Community Survey in a small-sized New England City, adding to our knowledge of an older adult population. Aim: In order to elicit the points of view of older adults in one small New England city, six focus groups were conducted via Zoom during the height of the pandemic, from the spring and fall of 2020 on the topic of aging in place. Method: The six focus groups had a total of 32 participants, all of whom were 65 years and older and living in the same New England city. Results: The challenges to aging in place small New England city that the focus group participants described included: knowing where to get complete and reliable information about vital services, the barriers to walkability, and the challenge of transportation when one can no longer safely drive. Conclusion: The Focus Group Study extended the findings of the AARP Age-Friendly Community Survey in a small-sized New England City through the voices of the older adults which led to a more nuanced understanding of what it takes to age in place. The results of the study were utilized by the city in order to write an action plan as a guide to becoming more age-friendly.

Pregnancy-related listeriosis.

About one in seven cases of listeriosis occurs in pregnant women, and, although listeriosis is rare, it is the third leading cause of death from food-borne infections. Pregnancy-related listeriosis increases the risk for fetal and neonatal mortality by approximately 21%. During pregnancy, infections are more likely to occur in the third trimester (66%) than the first trimester (3%). However, fetal and neonatal adverse effects are less common as gestational age increases or with older gestational age at birth. Pregnant women seem highly susceptible in some listeriosis outbreaks making up a large percentage of cases, whereas others contain very few. Whether this results from differences in strains of L. monocytogenes, exposures, or other factors remains to be determined. Food and Agriculture Organization of United Nations/World Health Organization (FAO/WHO) estimates the human lethal dose for 50% (LD50 ) for fetal/neonatal loss is 1.9 × 106 colony forming units (CFUs) L. monocytogenes. Animal models have been developed for pregnancy-related listeriosis showing similar susceptibility and clinical outcomes as in humans. Nonhuman primate and guinea pig animal models have similar (LD50 ) values to the estimated human LD50 . Additional animal studies are needed to understand the pathways leading to fetal and neonatal listeriosis in humans. More information is needed to understand dose response, to model risk for listeriosis at lower concentrations, and to determine why some pregnant women may be more susceptible than others. To better treat listeriosis during pregnancy, biomarkers for early diagnosis of listeriosis are also needed. Last, pregnant women need to be educated about avoiding high-risk foods, like Mexican-style cheese and ready-to-eat meats. Birth Defects Research 109:324-335, 2017.© 2017 Wiley Periodicals, Inc.

The food industry's current and future role in preventing microbial foodborne illness within the United States.

During the past century, the microbiological safety of the US food supply has improved; however, many foodborne illnesses and outbreaks occur annually. Hence, opportunities for the food industry to improve the safety of both domestic and imported food exist through the adoption of risk-based preventive measures. Challenging food safety issues that are on the horizon include demographic changes to a population whose immune system is more susceptible to foodborne and opportunistic pathogens, climate changes that will shift where food is produced, and consumers' preferences for raw and minimally processed foods. Increased environmental and product testing and anonymous data sharing by the food industry with the public health community would aid in identifying system weaknesses and enabling more targeted corrective and preventive actions. Clinicians will continue to play a major role in reducing foodborne illnesses by diagnosing and reporting cases and in helping to educate the consumer about food safety practices.

Dose response of Listeria monocytogenes invasion, fetal morbidity, and fetal mortality after oral challenge in pregnant and nonpregnant Mongolian gerbils.

Listeria monocytogenes is a food-borne pathogen that can result in adverse pregnancy outcomes, such as stillbirth or premature delivery. The Mongolian gerbil was recently proposed as the most appropriate small-animal model of listeriosis due to its susceptibility to the same invasion pathways as humans. The objectives of this study were to investigate invasion and adverse pregnancy outcomes in gerbils orally exposed to L. monocytogenes, to compare the dose-response data to those of other animal models, and to investigate differences in the responses of pregnant versus nonpregnant gerbils. Gerbils were orally exposed to 0 (control), 10(3), 10(5), 10(7), or 10(9) CFU L. monocytogenes in whipping cream. L. monocytogenes was recovered in a dose-dependent manner from fecal samples, adult organs, and pregnancy-associated tissues. Dams exposed to 10(9) CFU had more invaded organs and higher concentrations of L. monocytogenes in almost all organs than nonpregnant animals, though no differences in fecal shedding were seen between the two groups. Adverse pregnancy outcomes occurred only in the dams treated with 10(9) CFU. A 50% infectivity dose (ID50) of 2.60 × 10(6) CFU for fetuses was calculated by fitting the data to a logistic model. Our results suggest that the 50% lethal dose (LD50) falls within the range of 5 × 10(6) to 5 × 10(8) CFU. This range includes the guinea pig and nonhuman primate LD50s, but the observation that L. monocytogenes-induced stillbirths can be seen in guinea pigs and primates exposed to lower doses than those at which stillbirths were seen in gerbils indicates that gerbils are not more sensitive to L. monocytogenes invasion.

Producing timed-pregnant Mongolian gerbils for developmental studies.

The developing fetus is differentially susceptible to chemicals and pathogens depending on the stage of its development. In developmental studies, embryos or fetuses therefore must be exposed to experimental interventions at the same gestational stage. Acquiring sufficient numbers of embryos of the same developmental stage requires the use of timed-pregnant animals. Timed-pregnant Mongolian gerbils currently are not available for purchase. The authors developed a novel method for timed mating of virgin female gerbils. Female gerbils were housed in the same cage as males, but physically separated by a partition, for 3 d in order to expose the females to the males' pheromones before they were allowed to interact. Females were monitored for lordosis to determine sexual receptivity. Lordosis was observed in 10 of 15 females (67%), and 9 of these females (90%) became pregnant. When lordosis was not observed, none of the females became pregnant. These results demonstrate that the timed mating method produces a high rate of mating success and indicate that lordosis is a reliable predictor of sexual receptivity and subsequent successful mating in the Mongolian gerbil.

Metabolomic response of human embryonic stem cell-derived germ-like cells after exposure to steroid hormones.

To assess the potential risks of human exposure to endocrine active compounds (EACs), the mechanisms of toxicity must first be identified and characterized. Currently, there are no robust in vitro models for identifying the mechanisms of toxicity in germ cells resulting from EAC exposure. Human embryonic stem cells can differentiate into numerous functional cell types including germ-like cells (GLCs). These cells possess characteristics indicative of a germ cell state, suggesting they offer a novel system to investigate the consequences of chemical exposure on normal germ cell processes. To characterize these processes, a metabolomic-based approach was employed to determine the response of GLCs following exposure to 0.001, 0.01, 0.1, 1, 10, or 100µM estradiol, testosterone, or progesterone for 48h. Following exposure, cellular extracts underwent gas chromatography coupled with mass spectrometry analysis. Models were then constructed using principal component analysis on acquired spectra to discriminate among steroid hormones as well as doses for each hormone. t-test comparisons generated a preliminary list of metabolites that were statistically significant in GLC's biochemical response to these steroid hormones. Steroid hormone exposures caused fluxes in intracellular pathways such as amino acid synthesis and metabolism, fatty acid synthesis, as well as cholesterol and lipoprotein metabolism. Further pathway analysis, based on these identified metabolites, will aid in modeling the response of GLCs to endogenous steroid hormones and allow for identification of biomarkers delineating germ cell-based developmental and reproductive pathways.

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.

Preimplantation exposure to bisphenol A (BPA) affects embryo transport, preimplantation embryo development, and uterine receptivity in mice.

To investigate the effects of bisphenol A (BPA) on embryo and uterine factors in embryo implantation, timed pregnant C57BL6 females were treated subcutaneously with 0, 0.025, 0.5, 10, 40, and 100mg/kg/day BPA from gestation days 0.5-3.5. In 100mg/kg/day BPA-treated females, no implantation sites were detected on day 4.5 but retention of embryos in the oviduct and delayed embryo development were detected on day 3.5. When untreated healthy embryos were transferred to pseudopregnant females treated with 100mg/kg/day BPA, no implantation sites were detected on day 4.5. In 40 mg/kg/day BPA-treated females, delayed implantation and increased perinatal lethality of their offspring were observed. Implantation seemed normal in the rest BPA-treated groups or the female offspring from 40 mg/kg/day BPA-treated group. These data demonstrate the adverse effects of high doses of BPA on processes critical for embryo implantation: embryo transport, preimplantation embryo development, and establishment of uterine receptivity.

Chicken intestine microbiota following the administration of lupulone, a hop-based antimicrobial.

The use of antibiotic growth promotants in poultry rearing is a public health concern due to antibiotic resistance in bacteria and the harborage of resistance genes. Lupulone, a hop ß-acid from Humulus lupulus, has been considered as a potential feed additive growth promotant. Here, the effect of lupulone was evaluated for its effect on the microbiota of the chicken intestine. The intestinal microbiota of broilers was quantified after the addition of 125 mg L(-1) lupulone to water and challenge with Clostridium perfringens. Microbial DNA was extracted from the broiler midgut and cecal sections and bacterial groups were quantified using real-time PCR. The predominant cecal bacterial groups were Clostridium leptum subgroup 16S rRNA gene Cluster IV, Clostridium coccoides subgroup 16S rRNA gene Clusters XIVa and XIVb and Bacteroides, whereas Lactobacillus, the Enterobacteriaceae family and Enterococcus dominated the midgut. Lupulone at 125 mg L(-1) significantly decreased the C. perfringens subgroup 16S rRNA gene Cluster I, which contains several pathogenic species, in both the midgut and the cecum and Lactobacillus in the midgut. No significant changes were noted in the overall microbiota for the cecum or the midgut. Lupulone warrants further evaluation as a botanical agent to mitigate C. perfringens overgrowth in antibiotic-free reared poultry.

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.

Neonatal mice as models for Cronobacter sakazakii infection in infants.

Cronobacter sakazakii is an opportunistic pathogen that has been isolated from powdered infant formulas. C. sakazakii infection can result in serious illnesses such as bacteremia, septicemia, meningitis, and death in at-risk infants who are orally fed contaminated reconstituted powdered infant formulas. The objective of this study was to compare the susceptibilities of BALB/c, C57BL/6, and CD-1 mice to C. sakazakii strain MNW2. We acquired timed-pregnant CD-1 mice and allowed them to give birth naturally. On postnatal day 3.5, each pup was administered a total dose of approximately 10(2) to 10(11) CFU C. sakazakii strain MNW2 in reconstituted powdered infant formula. Mice were observed twice daily for morbidity and mortality. At postnatal day 10.5, the remaining pups were euthanized, and brain, liver, and cecum were excised and analyzed for the presence of C. sakazakii. C. sakazakii was isolated from brains, livers, and ceca in all three mouse strains. The CD-1 mouse strain was the most susceptible of the three, with the lowest infectious dose (10(2) CFU) and the lowest lethal dose (also 10(2) CFU).

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.

The Key Events Dose-Response Framework: its potential for application to foodborne pathogenic microorganisms.

The Key Events Dose-Response Framework (KEDRF) is an analytical approach that facilitates the use of currently available data to gain insight regarding dose-response relationships. The use of the KEDRF also helps identify critical knowledge gaps that once filled, will reduce reliance on assumptions. The present study considers how the KEDRF might be applied to pathogenic microorganisms, using fetal listeriosis resulting from maternal ingestion of food contaminated with L. monocytogenes as an initial example. Major biological events along the pathway between food ingestion and the endpoint of concern are systematically considered with regard to dose (i.e., number of organisms), pathogen factors (e.g., virulence), and protective host mechanisms (e.g., immune response or other homeostatic mechanisms). It is concluded that the KEDRF provides a useful structure for systematically evaluating the complex array of host and pathogen factors that influence the dose-response relationship. In particular, the KEDRF supports efforts to specify and quantify the sources of variability, a prerequisite to strengthening the scientific basis for food safety decision making.

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.