The devil is in the details: emerging insights on the relevance of wastewater surveillance for SARS-CoV-2 to public health

The severe health consequences and global spread of the COVID-19 pandemic have necessitated the rapid development of surveillance programs to inform public health responses. Efforts to support surveillance capacity have included an unprecedented global research response into the use of genetic signals of SARS-CoV-2 in wastewater following the initial demonstration of the virus' detectability in wastewater in early 2020. The confirmation of fecal shedding of SARS-CoV-2 from asymptomatic, infected and recovering individuals further supports the potential for wastewater analysis to augment public health conventional surveillance techniques based on clinical testing of symptomatic individuals. We have reviewed possible capabilities projected for wastewater surveillance to support pandemic management, including independent, objective and cost-effective data generation that complements and addresses attendant limitations of clinical surveillance, early detection (i.e., prior to clinical reporting) of infection, estimation of disease prevalence, tracking of trends as possible indicators of success or failure of public health measures (mask mandates, lockdowns, vaccination, etc.), informing and engaging the public about pandemic trends, an application within sewer networks to identify infection hotspots, monitoring for presence or changes in infections from institutions (e.g., long-term care facilities, prisons, educational institutions and vulnerable industrial plants) and tracking of appearance/progression of viral variants of concern.

Agritourism and Kidding Season: A Large Outbreak of Human Shiga Toxin-Producing Escherichia coli O157 (STEC O157) Infections Linked to a Goat Dairy Farm—Connecticut, 2016

The objective of this study was to determine sources of Shiga toxin-producing Escherichia coli O157 (STEC O157) infection among visitors to Farm X and develop public health recommendations. A case-control study was conducted. Case-patients were defined as the first ill child (aged <18 years) in the household with laboratory-confirmed STEC O157, or physician-diagnosed hemolytic uremic syndrome with laboratory confirmation by serology, who visited Farm X in the 10 days prior to illness. Controls were selected from Farm X visitors aged <18 years, without symptoms during the same time period as case-patients. Environment and animal fecal samples collected from Farm X were cultured; isolates from Farm X were compared with patient isolates using whole genome sequencing (WGS). Case-patients were more likely than controls to have sat on hay bales at the doe barn (adjusted odds ratio: 4.55; 95% confidence interval: 1.41–16.13). No handwashing stations were available; limited hand sanitizer was provided. Overall, 37% (29 of 78) of animal and environmental samples collected were positive for STEC; of these, 62% (18 of 29) yielded STEC O157 highly related by WGS to patient isolates. STEC O157 environmental contamination and fecal shedding by goats at Farm X was extensive. Farms should provide handwashing stations with soap, running water, and disposable towels. Access to animal areas, including animal pens and enclosures, should be limited for young children who are at risk for severe outcomes from STEC O157 infection. National recommendations should be adopted to reduce disease transmission.

Pigs Immunized with the Virus-like Particle Vaccine Are Protected against the Hepatitis E-3 Virus

In this study, we generated the HEV virus-like particle (VLP) vaccine expressing 239 amino acids (367–605 aa) of the HEV-3 ORF2 using the baculovirus expression system. The HEV-3-239-VLP vaccine efficacy was evaluated by dividing 12 pathogen-free pigs into four groups: negative control, positive control, 100 μg VLP-, and 200 μg VLP-vaccinated groups for 10 weeks. The pigs in either of the vaccinated groups were administered the corresponding first and booster doses on weeks 0 and 2. At week 4, the positive control and two vaccinated groups were challenged with 106 HEV-3 genomic equivalent copies; viremia and fecal shedding of the virus were identified in pigs in the positive control and 100 μg VLP-vaccinated pigs showed transient viremia and fecal viral shedding. However, no viruses were detected in the serum or fecal samples of the 200 μg VLP-vaccinated pigs. The 100 and 200 μg VLP-vaccinated pigs had significantly higher (p < 0.01) anti-HEV antibodies than the negative control pigs from weeks 6–10 with normal levels of liver enzymes. The 200 μg VLP-vaccinated pigs showed statistically less liver tissue fibrosis (p < 0.05) than that of the positive control pigs. Thus, the novel baculovirus expression system-generated VLP vaccine dose-dependently protects against HEV-3 challenge and may be useful in other animal species, including humans.

995. A Murine Model of Klebsiella pneumoniae Gastrointestinal Colonization with Parenteral Vancomycin Administration

Background As a leading cause of nosocomial infections, Klebsiella pneumoniae poses a significant threat due to its propensity to acquire resistance to many classes of antibiotics, including carbapenems. Gastrointestinal (GI) colonization by K. pneumoniae is a risk factor for subsequent infection as well as transmission to other patients. To study this crucial step in pathogenesis, we developed a mouse model of K. pneumoniae GI colonization using a clinically relevant parenteral antibiotic regimen. Methods To improve the clinical relevance of our model, we elected to use intraperitoneal injections of vancomycin, one of the most highly utilized antibiotics in the United States. Results To optimize dosage in C57bl/6 mice, we injected 20mg/kg, 350mg/kg, or vehicle (PBS) for three days prior to gastric gavage with 108 colony forming units (CFU) of a low-resistance strain of K. pneumoniae. The mice who received 350mg/kg (a mouse equivalent of a human dose of 1g/day calculated through the FDA guidelines for estimating safe dosing) shed about 107 CFU/g of feces at Day 7 while those receiving the lower dose or vehicle shed 104 CFU/g. Next, we compared 3- or 5-day pre-treatments with vancomycin prior to inoculation with an ST258 (epidemic carbapenem-resistant) strain. At Day 7 post-inoculation, mice who received 5 days shed 1010 CFU/g feces while those who received vancomycin for 3 days or vehicle for 5 days (PBS) shed 106 or 104 CFU/g feces respectively. Thus, we chose 5 days of 350mg/kg vancomycin injection as our regimen for inducing robust GI colonization in mice. Finally, we tested the durability of colonization by following fecal shedding in mice up to Day 60 post-inoculation with a second ST258 strain. Shedding during the first 7 days occurs at about 1010 CFU/g feces, and from day 14 to day 60 fecal loads are stable around 107 CFU/g feces. Results are comparable between male and female mice. Conclusion In conclusion, we have developed a mouse model of robust, prolonged GI colonization with multiple strains of K. pneumoniae using controlled dosing of a clinically relevant antibiotic. This model may be used to study a key step in K. pneumoniae pathogenesis and infection prevention in the future. Disclosures All Authors: No reported disclosures

Implication and challenges of direct-fed microbial supplementation to improve ruminant production and health

Direct-fed microbials (DFMs) are feed additives containing live naturally existing microbes that can benefit animals’ health and production performance. Due to the banned or strictly limited prophylactic and growth promoting usage of antibiotics, DFMs have been considered as one of antimicrobial alternatives in livestock industry. Microorganisms used as DFMs for ruminants usually consist of bacteria including lactic acid producing bacteria, lactic acid utilizing bacteria and other bacterial groups, and fungi containing Saccharomyces and Aspergillus. To date, the available DFMs for ruminants have been largely based on their effects on improving the feed efficiency and ruminant productivity through enhancing the rumen function such as stabilizing ruminal pH, promoting ruminal fermentation and feed digestion. Recent research has shown emerging evidence that the DFMs may improve performance and health in young ruminants, however, these positive outcomes were not consistent among studies and the modes of action have not been clearly defined. This review summarizes the DFM studies conducted in ruminants in the last decade, aiming to provide the new knowledge on DFM supplementation strategies for various ruminant production stages, and to identify what are the potential barriers and challenges for current ruminant industry to adopt the DFMs. Overall literature research indicates that DFMs have the potential to mitigate ruminal acidosis, improve immune response and gut health, increase productivity (growth and milk production), and reduce methane emissions or fecal shedding of pathogens. More research is needed to explore the mode of action of specific DFMs in the gut of ruminants, and the optimal supplementation strategies to promote the development and efficiency of DFM products for ruminants.

478 Dietary Butyrate and Valerate Glycerides: Effects on Growth and Systemic Immunity of Weanling Piglets Coinfected with F4 and F18 Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) F4 and F18 are the two most dominant pathogenic strains in weaned pigs. The objective of this experiment was to test the effects of dietary monobutyrin and monovalerin on performance and systemic immunity of weanling piglets coinfected with F4/F18 ETEC. Twenty weaned pigs (8.21 ± 1.23 kg) were individually housed and were randomly allotted to one of three diets: control (n = 6), 0.1% monobutyrin (n = 7), or 0.1% monovalerin (n = 7). The experiment was conducted 14 days, including 7 days’ adaption and 7 days post-inoculation (PI). On d 0, d 1, and d 2 PI, pigs were inoculated with 0.5 × 109 CFU/1.5 mL each of F4 and F18 ETEC for three consecutive days. Diarrhea score was recorded daily to determine frequency of diarrhea. Piglets and feeders were weighed throughout the trial to analyze growth performance. Fecal cultures from pigs on d 0, 2, and 4 PI were inspected to identify the absence or presence of hemolytic coliforms. Blood was collected on d 0, 4, and 7 PI for complete blood cells count. All data were analyzed by the Proc Mixed of SAS with randomized complete block design. Pigs supplemented with monovalerin and monobutyrin had numerically higher ADG (249 and 282 g/day) from d 0 to d 7 PI than pigs in control (198 g/day). Supplementation of monovalerin reduced (P &lt; 0.05) frequency of diarrhea throughout the experiment. Pigs fed monovalerin had lower (P &lt; 0.05) neutrophil counts on d 4 PI compared with control. Hemolytic coliforms were observed in all fecal cultures from d 2 and d 4 PI, confirming fecal shedding of ETEC. Results of this study indicate the potential benefits of monovalerin supplementation on performance and disease resistance of weaned pigs coinfected with F4 and F8 ETEC.

Tracking the temporal variation of COVID-19 surges through wastewater-based epidemiology during the peak of the pandemic: a six-month long study in Charlotte, North Carolina

The global spread of SARS-CoV-2 has continued to be a serious concern after WHO declared the virus the causative agent of the coronavirus disease 2019 (COVID-19) a global pandemic. Monitoring of wastewater is a useful tool for assessing community prevalence given that fecal shedding of SARS-CoV-2 occurs in high concentrations by infected individuals, regardless of whether they are asymptomatic or symptomatic. Using tools that are part of the wastewater-based epidemiology (WBE) approach, combined with molecular analyses, wastewater monitoring becomes a key piece of information used to assess trends and quantify the scale and dynamics of COVID-19 infection in a specific community, municipality, or area of service. This study investigates a six-month long SARS-CoV-2 RNA quantification in influent wastewater from four municipal wastewater treatment plants (WWTP) serving the Charlotte region of North Carolina (NC) using both RT-qPCR and RT-ddPCR platforms. Influent wastewater was analyzed for the nucleocapsid (N) genes N1 and N2. Both RT-qPCR and RT-ddPCR performed well for detection and quantification of SARS-CoV-2 using the N1 target, while for the N2 target RT-ddPCR was more sensitive. SARS-CoV-2 concentration ranged from 103 to105 copies/L for all four plants. Both RT-qPCR and RT-ddPCR showed a significant moderate to a strong positive correlation between SARS-CoV-2 concentrations and the 7-day rolling average of clinically reported COVID-19 cases using a lag that ranged from 7 to 12 days. A major finding of this study is that despite small differences, both RT-qPCR and RT-ddPCR performed well for tracking the SARS-CoV-2 virus across WWTP of a range of sizes and metropolitan service functions.