Progress report for GNE21-261
Project Information
Objective 1: Determine prevalence of Salmonella Dublin on dairy farms in Pennsylvania
While the ideal scenario would be to repeatedly sample all PA dairy farms on three separate occasions for accurate determination of the prevalence of S. Dublin, such experimental design is beyond this project’s scope. Thus, we will perform ELISA antibody tests at 900 randomly selected PA dairy farms to estimate the seroprevalence of Salmonella Dublin in the state. This initial screen will yield multiple outcomes. First, it will confirm the presence of Salmonella Dublin in PA dairy operations. The number of positive farms will provide an estimate of prevalence in the state. This knowledge will inform dairy producers of the need to implement risk management intervention strategies as applicable. Finally, fulfilling this objective will allow us to streamline downstream environmental sampling to only those farms seropositive for Salmonella Dublin.
Objective 2: Confirm active presence of Salmonella Dublin on farms with antibody-positive milk
Given the challenging nature of culturing Salmonella Dublin from environmental samples, selective enrichment and PCR-based testing will be used to determine its prevalence on 10 seropositive dairy farms. Based on the work of Cummings et al. 20184 in New York, we expect 0.9% of farms to be seropositive; however, we are in communication with PSU extension team and Penn State Diagnostic laboratory concerning Salmonella Dublin and will target farms that previously tested positive for this pathogen for environmental sampling of manure and calving area. Fulfillment of this objective will confirm active presence of Salmonella Dublin on dairy farms in PA.
Objective 3: Isolate and screen bacteriophages for potential therapeutic intervention
Salmonella Dublin is bovine-adapted and largely, multidrug resistant to antibiotics. In fact, most S. Dublin isolates are resistant to all antimicrobials labeled for treatment of respiratory infections in bovines in the US.2 These characteristics, in combination with general trends toward antimicrobial resistance, justify the need for alternative therapeutic interventions. Phage therapy is increasingly appreciated for its efficacy against multidrug resistant infections in human and animal hosts. Given the resistance of Salmonella Dublin to traditional intervention and the limitations of current preventative measures, phage therapy has the potential to complement existing interventions and improve animal health and production outcomes. We will isolate phages from environmental reservoirs and perform screening for possible candidate phages. This screen will identify phages targeting Salmonella Dublin, providing a phage population that can be further narrowed based on established phage therapy criteria.
The purpose of this project is to estimate the prevalence of Salmonella Dublin in Pennsylvania dairy herds and provide intervention directions to dairy producers facing challenges with this pathogen. Salmonella enterica serovar Dublin (S. Dublin) is a bovine-adapted emerging pathogen in the northeastern United States. The disease presentation is nonclassical in cattle, particularly in calves, and complicates diagnosis.1,2 S. Dublin detection from environmental samples is challenging because the pathogen is often outcompeted by the background flora.3 Due to persistent host colonization, unclassical presentation, detection difficulty, and treatment limitations, maintaining health of dairy herds is largely dependent on preventative risk management.
The epidemiology and effects of S. Dublin on dairy production in the eastern United States are under-researched and, thus, poorly understood. To date, only one study of S. Dublin prevalence among dairy herds in New York has been published.4 Because Pennsylvania is ranked 7th nationally for dairy production5, and the cattle trade spans many northeastern states there is an urgent need to address these knowledge gaps surrounding prevalence and provide data-driven directions for S. Dublin mitigation.
Antimicrobial resistance (AMR) of S. Dublin is also a significant concern, and most farm isolates are multidrug-resistant (MDR),1,6,7,8 creating demand for strategies that prevent infection as well as more sustainable alternative therapeutics.9,10,11 This project will gather preliminary knowledge for the potential development of an alternative to antimicrobials, phage therapy, which is increasingly studied and recognized for its efficacy in treating MDR infections in humans and animals.11,12,13 Because bacteriophages have high host specificity, phage therapy does not apply the broad spectrum selective pressure for AMR of traditional antibiotics. While resistance development is possible, this can be circumvented by applying a combination of phages, known as a phage cocktail, to mitigate resistance development. However, when bacteria develop resistance to phages, there is an evolutionary cost associated with it. For example, in a hypothetical scenario where phage therapy is applied to treat MDR bacteria and phage resistance arises, pathogens usually become susceptible to antimicrobials that they were previously resistant to or become more easily killed by an animal’s immune system.12 Thus, either as a cocktail or in combination with a weaker antibiotic, phage therapy could be effective for treating MDR S. Dublin infections.
This project addresses the emergence of S. Dublin in the northeastern US and investigates potential intervention strategies. The results gathered here will increase awareness among PA farmers concerning the presence of Dublin within their operations and provide preliminary direction for an alternative to antimicrobials. Our results will contribute to reducing animal loss and the need for antimicrobial intervention, leading to a more sustainable dairy production approach. This initial step toward phage therapy is significant because it has the potential to work in tandem with lower dose antibiotics, decreasing their demand and thus relieving some selective pressure toward AMR. The immediate implications of this project are early risk assessment and management, while longer-term implications include potentially reducing animal loss and broad-spectrum antibiotic use in PA dairy production.
Research
Objective 1: Determine prevalence of Salmonella Dublin on dairy farms in Pennsylvania
1.1 Participant Recruitment and Sampling
Via collaboration with university and extension partners, we will identify Pennsylvania dairy farms willing to participate. Following communication with the Lancaster DHIA testing labs and milk co-op consent, bulk tank samples collected through the DHIA's routine testing were obtained. Additionally, samples were collected from individual farms that enrolled following communication with Penn State extension partners. Without coordination with DHIA and extension, farms were generally unresponsive to recruitment efforts. As a result, study participation will rely on a convenience sample of any farm willing to participate following communication with extension veterinarians, extension partners, or DHIA.
1.2 Seroprevalence Screen
In accordance with PrioCHECK Salmonella Ab bovine Dublin protocol, 100 mL of each raw milk sample will be used in each of two technical replicates in an indirect enzyme-linked immunosorbent assay (ELISA) against the O antigen of S. Dublin. Results of these colorimetric reactions will be read at 450nm using spectrophotometry. Samples containing antibody against S. Dublin will react in the provided kit reagents, resulting in a color change reflected by OD450 values. These results will be compared to those of provided positive controls and will be corrected using the provided negatived control. Percent positivity will be calculated, with > 35% as the positivity threshold.
1.3 Reporting/Follow-Up
Farms whose samples are seropositive will be contacted concerning additional environmental sampling for study objectives 2 and 3.
Anticipated Objective Outcomes:
Determining estimated S. Dublin seroprevalence among dairy herds in Pennsylvania
Objective 2: Confirm active presence of Salmonella Dublin on farms with antibody-positive milk
2.1 Environmental Sampling
We expect 0.9% of farms to be seropositive based on findings in New York of Cummings et al. 20184. We are in communication with Penn State University Extension and Animal Diagnostic Laboratory about S. Dublin those farms will be targeted in study objective one. Thus, we anticipate a higher number of positives and anticipate identifying 10 seropositive farms.
Environmental samples will be aseptically collected as swabs of different surfaces around the maternity pen, group calf housing, and milkhouse. According to the protocol in Goodman et al.,3 these swabs will be placed in a sterile Whirl-pak bag with ~5 mL of sterile evaporated skim milk and stored on ice or cold packs for transport to maintain bacteria viability.
2.2 Confirmatory Subculturing and Characterization
Using the improved protocol for Salmonella spp. detection by Goodman et al.,3 45 mL of RVS broth will be added to the Whirl-pak bags (2.1) and incubated for 20-24 hours at 40-44°C. As outlined, subculturing will be performed on all samples by plating onto xylose-lysine-deoxycholate (XLD) agar plates and incubated overnight at 33-37°C.11 These isolates will be sequenced in collaboration with FDA GenomeTrakr and analyzed for elements of relatedness and antimicrobial resistance.
2.3 Molecular Diagnostics
To confirm isolates are Salmonella prior to subtyping, invA PCR is used to confirm a putative bacterial isolate as Salmonella. Using the S. Dublin specific PCR primers developed by Afroj et al (2017), isolates were typed as S. Dublin or not. Additionally, we are working with Dr. Nikki Shariat to develop a S. Dublin-specific CRISPR-based qPCR for molecular confirmation.
Anticipated Objective Outcomes:
Confirmation of estimated S. Dublin environmental persistence among dairy herds in Pennsylvania
Objective 3: Isolate and screen bacteriophages for potential therapeutic intervention
3.1 Environmental Sampling
During sampling for objective 2, milkhouse wastewater and manure lagoons, where applicable, will be sampled by a research technician. Using the manual sampling method outlined by the US EPA18, sterile 50 mL conical vials will be dipped into the wastewater source with the mouth facing the current to collect the sample without overfilling. The outside of the sample container will be sanitized with 70% ethanol and stored on ice for transport from the farm to laboratory.
3.2 Isolation
Adhering to the methods outlined by Carey-Smith et al.,19 wastewater will be diluted 1:10 in SM buffer and incubated at 4°C overnight. Following centrifugation at 1600g for 25 minutes, samples will be filtered through a disposable 0.22mM pore-size filter. 100mL of this filtrate will be added to a soft agar overlay containing 100mL of a confirmed S. Dublin isolate in the exponential growth phase. Once the overlays are poured and solidified, plates will be incubated at 37°C for 24 hours and then assessed for plaque formation.19 Plaques will be purified through subsequent serial dilution and plating in soft agar overlays as outlined previously.20
Once purified, phages will be serially diluted in SM buffer. Each dilution will be plated three times and the dilution exhibiting confluent lysis will be chosen for phage recovery and stock creation. To recover phages from the soft-agar layer, 5 mL of SM buffer will be added to each plate and left to incubate at room temperature for a minimum of 60 minutes, swirling the plates regularly. In addition to this liquid, the soft-agar layer will also be removed and added to a conical vial containing 20mL of SM buffer. Once shaken for 30 minutes, the vials will be centrifuged at 1300g for 10 minutes. Once the supernatant is filtered through a disposable 0.22 mM pore-size filter, 0.2% chloroform will be added.19
3.3 In vitro assessment of host specificity
To determine the host specificity of the phages isolated in 3.2, 1:10 dilutions of the phage stock will be plated in soft-agar overlays inoculated with one of the following bacteria: S. Dublin, Salmonella Enteritidis,21 Salmonella Typhimirium,21,22 Salmonella Heidelberg,23 and Escherichia coli. E. coli will act as one of two negative controls. Overlay inoculation with E. coli and the isolated phages should not yield plaques. Additionally, the use of a known E. coli specific bacteriophage in overlays with Salmonella spp. will serve as a phage negative control. An overlay containing both E. coli bacteria and E. coli bacteriophage will confirm the viability of the E. coli bacteriophage and serve as the positive control as noted in the attached figure. These overlays will be created in triplicate for each isolated phage and incubated overnight at 37°C for 24 hours before inspection of plaque formation. Plaque formation in the overlay containing the environmental phage isolates and S. Dublin and the absence of such plaques in overlays containing the same phage isolate but other bacteria inoculum will confirm the host specificity of the isolate phage stock against S. Dublin.
Anticipated Objective Outcomes:
Isolation and confirmed host specificity of a bacteriophage targeted S. Dublin
Objective 1 Results (Working):
As of January 2023, we have identified two S. Dublin positive herds which have been sampled. Also as of January 2023, through DHIA milk testing, we have identified 6 putative S. Dublin positive farms that will be contacted regarding herd health history and environmental sampling scheduling. In total, 72 different farms have enrolled in the study and recruitment is ongoing on a rolling basis.
Objective 2 Results (Working):
To date environmental sampling was conducted at 2 dairies on which S. Dublin has become endemic. Sampling has been refined to include only maternity pen, group calf housing, and milkhouse locations as these consistently yield Salmonella. Of the 23 Salmonella isolates cultured from environmental samples, none were PCR-positive as S. Dublin.
Objective 3 Results (Working):
Research activities for Objective 3 are planned for spring 2023.
Education & Outreach Activities and Participation Summary
Participation Summary:
A primary short-term goal of this proposal is increase awareness of S. Dublin significant threat to herd health. This will be accomplished through writing a bulletin to be disseminated by Penn State University Extension. In addition to our findings, the bulletin will include information concerning health risk, infection presentation, potential reservoirs, and risk management strategies.
We will develop three 5-minute “Learn Now Videos”: The first video will explain the particularities of S. Dublin infections in dairy herds and how to spot a potential case. A second video will cover potential management strategies that can be applied by herds to prevent the introduction of S. Dublin into a herd, including biosecurity measures. The third video will focus educating farmers that may be facing issues with S. Dublin on their herd on a previously developed risk assessment tool and how it can be used to improve management and mitigate the effect of S. Dublin infection in positive herds. All videos will be developed by the graduate student in consultation with Penn State Extension personnel (see letters of collaboration) and made publicly available on The Pennsylvania State University extension website to facilitate easy learning.
Given our longer-term goal of increasing the sustainability of dairy cattle by improving herd health, our findings will be presented at relevant regional and national conferences such as the Penn State Dairy Cattle Nutrition Workshop and the American Dairy Science Association annual meeting. The results will also be submitted for peer-review publication in a dairy or veterinary-oriented journal, providing more science-based information for Pennsylvania dairy producers on AMR reduction among their herds. The results of this project will lay out the foundation for a broader effort in quantifying the prevalence of S. Dublin in the Northeastern US, and the application of a multi-pronged approach to mitigate the impact of this pathogen in the dairy industry.
Drs. Hovingh and Springer, who work in collaboration with the USDA-ARS Environmental Microbial and Food Safety Laboratory, hold extension roles and anticipate integrating these results in farm programs related to antimicrobial resistance, antimicrobial stewardship, and promotion of judicious antimicrobial use in calf rearing.
Combined, these strategies will inform farmers of actions they can be taking to mitigate potential harm to their herds and business as well as inform researchers of the need for continued surveillance and improved strategies for combating antimicrobial resistant pathogens that pose significant threats to livestock health, dairy production sustainability, and food security.
Progress to-date (January 2023):
An informational article regarding S. Dublin prevention and management was developed and published in Penn State Extension's Dairy Digest Newsletter in July 2022. Informational pamphlets were developed and distributed at Penn State Ag Progress Days in Fall 2022. Also at Penn State Ag Progress Days, an information talk about S. Dublin prevention and management was given.