RESEARCH ARTICLE An adjuvant formulation containing Toll-like Receptor 7 agonist stimulates protection against morbidity and mortality due to Anaplasma marginale in a highly endemic region of west Africa James E. FutseID 1*, Songliedong Zumor-Baligi1, Charles N. K. Ashiagbor1, Susan M. NohID 2,3,4, Christopher B. Fox5,6, Guy H. Palmer4,7 1 Animal Disease Biotechnology Laboratory, Department of Animal Science, School of Agriculture, College of Basic and Applied Sciences, University of Ghana, Legon, Accra, Ghana, 2 Animal Diseases Research Unit, USDA-ARS, Pullman, Washington, United States of America, 3 Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington, United States of America, 4 Paul G. Allen School for Global Health, Washington State University, Pullman, Washington, United States of America, 5 Access to Advanced Health Institute, Seattle, Washington, United States of America, 6 Department of Global Health, University of Washington, Seattle, Washington, United States of America, 7 Institute for Tropical Infectious Diseases, University of Nairobi, Nairobi, Kenya * jefutse@ug.edu.gh Abstract Efficient cattle production and provision of animal-sourced foods in much of Africa is con- strained by vector-borne bacterial and protozoal diseases. Effective vaccines are not cur- rently available for most of these infections resulting in a continuous disease burden that limits genetic improvement. We tested whether stimulation of innate immunity using the Toll-like Receptor (TLR) 7 agonist imiquimod, formulated with saponin and water-in-oil emulsion, would protect against morbidity and mortality due to Anaplasma marginale, a tick- borne pathogen of cattle highly endemic in west Africa. In Trial 1, haplotype matched Frie- sian x Sanga (F1) A. marginale negative calves were allocated to either the experimental group (n = 10) and injected with the synthetic TLR 7 agonist/saponin formulation or to an untreated control group (n = 10). TLR7 agonist/saponin injected calves responded with sig- nificantly elevated rectal temperature, enlarged regional lymph nodes, and elevated levels of IL-6 post-injection as compared to control group calves. All calves were then allowed to graze in pasture for natural exposure to tick transmission. All calves in both groups acquired A. marginale, consistent with the high transmission rate in the endemic region. The need for antibiotic treatment, using pre-existing criteria, was significantly lower in the experimental group (odds ratio for not requiring treatment was 9.3, p = 0.03) as compared to the control group. Despite treatment, 6/10 calves in the control group died, reflecting treatment failures that are typical of anaplasmosis in the acute phase, while mortality in the experimental group was 1/10 (odds ratio for survival was 13.5, p = 0.03). The trial was then repeated using 45 Friesian x Sanga calves per group. In Trial 2, the odds ratios for preventing the need for treatment and for mortality in the TLR7 agonist/saponin experimental group versus PLOS ONE PLOS ONE | https://doi.org/10.1371/journal.pone.0306092 August 29, 2024 1 / 12 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Futse JE, Zumor-Baligi S, Ashiagbor CNK, Noh SM, Fox CB, Palmer GH (2024) An adjuvant formulation containing Toll-like Receptor 7 agonist stimulates protection against morbidity and mortality due to Anaplasma marginale in a highly endemic region of west Africa. PLoS ONE 19(8): e0306092. https://doi.org/10.1371/journal. pone.0306092 Editor: Roman R. Ganta, University of Missouri College of Veterinary Medicine, UNITED STATES Received: December 28, 2023 Accepted: June 11, 2024 Published: August 29, 2024 Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Data Availability Statement: All relevant data are within the manuscript and its Supporting information files. Funding: This work was supported by the Bill and Melinda Gates Foundation Grant OPP1110974 provide funds to JEF to prove the concept. The Paul G. Allen School for Global Health at Washington State University provided funds for https://orcid.org/0000-0002-9204-4945 https://orcid.org/0000-0002-8160-9074 https://doi.org/10.1371/journal.pone.0306092 http://crossmark.crossref.org/dialog/?doi=10.1371/journal.pone.0306092&domain=pdf&date_stamp=2024-08-29 http://crossmark.crossref.org/dialog/?doi=10.1371/journal.pone.0306092&domain=pdf&date_stamp=2024-08-29 http://crossmark.crossref.org/dialog/?doi=10.1371/journal.pone.0306092&domain=pdf&date_stamp=2024-08-29 http://crossmark.crossref.org/dialog/?doi=10.1371/journal.pone.0306092&domain=pdf&date_stamp=2024-08-29 http://crossmark.crossref.org/dialog/?doi=10.1371/journal.pone.0306092&domain=pdf&date_stamp=2024-08-29 http://crossmark.crossref.org/dialog/?doi=10.1371/journal.pone.0306092&domain=pdf&date_stamp=2024-08-29 https://doi.org/10.1371/journal.pone.0306092 https://doi.org/10.1371/journal.pone.0306092 https://creativecommons.org/publicdomain/zero/1.0/ https://creativecommons.org/publicdomain/zero/1.0/ the control group were 5.6 (p = 0.0002) and 7.0 (p = 0.004), respectively, reproducing the findings of the initial trial. Together these findings demonstrate that innate immune stimula- tion using a TLR7 agonist formulated with saponin and water-in-oil emulsion provides signifi- cant protection against disease caused by tick borne A. marginale in highly susceptible cross-bred cattle, critically important for their potential to increase productivity for small- holder farmers in Africa. Introduction Improved productivity of cattle in smallholder households in sub-Saharan Africa has been shown to increase milk available for consumption and sale and resulted in increased house- hold expenditures on health and child education [1–3]. However, genetic improvement is lim- ited by the enhanced susceptibility of higher producing cattle breeds to endemic vector-borne infectious disease as compared to lower producing indigenous breeds. As a result, smallholder farmers who lack the resources for disease prevention in improved breeds using acaricides/ insecticides or treatment of sick animals are unable to improve milk production [4]. In west Africa, anaplasmosis, caused by the rickettsial pathogen Anaplasma marginale, is endemic with high prevalence and causes acute, usually mild disease in indigenous breeds but severe, often fatal, disease in improved cattle breeds [5–10]. Notably however, animals that recover from acute disease remain protected from subsequent disease despite repeated expo- sure [11, 12]. We hypothesized that stimulation of an innate immune response in higher pro- ductivity, genetically improved cross-breed cattle would provide protection against severe disease during the acute phase, thus allowing progression to long-term immunity. To test this hypothesis, we inoculated F1 Friesian-Sanga calves with a synthetic Toll-like Receptor (TLR) 7 agonist formulated with saponin and water-in-oil emulsion, assessed the innate immune responses, and then exposed the animals to natural tick-borne challenge within a highly endemic region of Ghana. Major Histocompatibility Locus (MHC) haplotype matched control calves were exposed together with the experimental group. Following confirmation of natu- rally transmitted A. marginale infection in all calves, protection from severe disease that required antibiotic treatment and mortality was assessed and compared between the two groups. The trial was then repeated for reproducibility. Here we report the outcome of these experimental trials and discuss the results in the context of stimulating innate immunity to allow genetic improvement of cattle in sub-Saharan Africa. Materials and methods TLR agonist formulation Synthetic TLR agonist imiquimod stimulates TLR7 [13–16] and has been shown to bind and stimulate the cognate receptor on bovine cells. For our set of experiments, 1 mg of TLR7 ago- nist (imiquimod, Chemagis) and 1 mg of semi-purified saponins from Quillaja saponaria (VetSap, Desert King) were delivered in 1 ml of a water-in-oil emulsion containing 53.5% v/v mineral oil (USP grade, Spectrum Chemical), 6.5% v/v isostearic acid (TCI), 0.97% w/v man- nide monooleate (Sigma Aldrich), 0.2% w/v cholesterol (J.T. Baker), and phosphate buffered saline (Gibco) prepared by high shear mixing followed by gamma irradiation at the Access to Advanced Health Institute (formerly known as the Infectious Diseases Research Institute, Seat- tle, WA USA). Immediately before use, the formulation was resuspended by vigorous shaking. PLOS ONE Toll-like Receptor 7 agonist stimulates protection against morbidity and mortality due to Anaplasma marginale PLOS ONE | https://doi.org/10.1371/journal.pone.0306092 August 29, 2024 2 / 12 visits by GHP to to supervise the work. They were accordingly recognized in the acknowledgement. Competing interests: The authors have declared that no competing interests exist. https://doi.org/10.1371/journal.pone.0306092 Source of crossbred calves and MHC DRB3 haplotypes Three-month old Friesian x Sanga (F1) calves, seronegative for A. marginale by Msp5 CI-E- LISA [17], were obtained from Amrahia Dairy Farm and Animal Research Institute of the Council for Scientific and Industrial Research of Ghana. To control for potential confounding effects of MHC haplotype on the immune responses [18–20], calves were MHC haplotyped and all were confirmed to express DRB3*2404, DRB3*2711, DRB3*2405, DRB3*2406, and DRB3*2407, alleles common within the Sanga breed from Ghana. For trial 1, three-month old Friesian x Sanga calves were allocated randomly to two equal groups. The experimental group (n = 10) was injected with the TLR agonist/saponin emulsion and the control group (n = 10) was left untreated. For trial 2, the experiment was replicated using 45 Friesian x Sanga calves per group. Animal care and use Calves in the experimental group were inoculated subcutaneously with 1 ml of TLR7 agonist formulated with saponin and water-in-oil emulsion. Blood collection by jugular venipuncture from calves in both groups was <10 ml (approximately 0.2% blood volume) per day for eight days pre- and post-injection and then weekly. Neither inoculation or venipuncture requires analgesia or anesthesia. Calves were allowed to graze on pasture by day, during which time there was natural exposure to ticks (i.e. there was no deliberate feeding of ticks on animals nor deliberate infection with A. marginale). All calves were examined daily and oxytetracy- cline treatment was instituted immediately following the onset of a priori defined clinical parameter of anemia in order to relieve suffering. Treated calves were separated from the group and monitored twice daily. No animals were intentionally sacrificed during the study. The cattle used in this study were treated in strict accordance with guidelines set by University of Ghana Institutional Animal Care and Use Committee and the protocol for blood sampling was approved by the Noguchi Memorial Institute for Medical Research (NIACUC protocol number: 2015-01-5X). Measurement of induced innate response The rectal temperature and the sizes of pre-scapular lymph node of each calf were examined daily for four days prior to injection with the TLR agonist/saponin emulsion and for the con- trol group calves to record pre-treatment parameters. Subsequently, 1ml of the agonist prepa- ration was injected subcutaneously into calves (n = 10) in the experimental group. Calves (n = 10) in the control group were not injected. The rectal temperature and sizes of the lymph nodes from individual calves were measured, at same time (08:00 hours), daily for additional four days post-inoculation. Blood was collected daily and serum levels of the proinflammatory cytokine IL-6 were measured by capture cytokine-specific antibody ELISA (NEO Scientific Biolab, Cambridge, Massachusetts) and a standard curve constructed using the SoftMax1 Pro 6 Software. The data were shown as the mean (+/- standard deviation) for all pre- and post- inoculation timepoints for individual calves in each group and reported at the group level as mean (+/- standard error). Significance between the TLR agonist/saponin emulsion injected and control groups was determined by the Student’s t-test with Holm-Sidak correction for multiple comparisons. Detection of infection Screening for A. marginale infection was done using msp5 PCR of peripheral blood and by Msp5 CI-ELISA to detect seroconversion [17, 21–23]. PLOS ONE Toll-like Receptor 7 agonist stimulates protection against morbidity and mortality due to Anaplasma marginale PLOS ONE | https://doi.org/10.1371/journal.pone.0306092 August 29, 2024 3 / 12 https://doi.org/10.1371/journal.pone.0306092 Antibiotic treatment All calves were observed clinically twice daily and packed cell volume (PCV) determined. If PCV decreased to� 28, treatment with 20mg/kg long-acting tetracycline was initiated and the animal moved to a separate pen. Treatment was repeated twice at 48-hour intervals. Weight gain Calves in Trial 1 were weighed at day 0, day 28 and 48 to measure effects of stimulation of innate immunity and acute A. marginale infection. A subset of calves from Trials 1 and 2 (n = 10 per group) were maintained on pasture for 13 months to determine if there were any long-term adverse effects on weight gain. Calves were weighed at 28-day intervals in the morn- ing before they were taken out for grazing and the mean monthly gain determined. Results Synthetic TLR7 agonist/saponin emulsion injection stimulates innate immunity of crossbred calves In Trial 1, the rectal temperature from each calf was recorded each day for four days post-inoc- ulation and compared to the same period prior to TLR7 agonist/saponin injection. This con- trols for individual variation among the calves. Similarly, the temperatures were collected on the same days and same time for the non-inoculated control calves. Calves in the TLR7 ago- nist/saponin emulsion experimental group developed significantly increased temperatures fol- lowing injection (increase of 0.98˚C ± 0.08˚C; p = 0.00003) as compared to pre-inoculation temperatures (Table 1 and S1 Table), while there was no significant change for the control calves (0.19˚C ± 0.10˚C; p = 0.49). The mean rectal temperature for the experimental group during the four days following TLR7 agonist/saponin injection was significantly higher than that of the control group over the same period (p = 0.001). Similarly, there were significant increases in the diameter of the pre-scapular lymph node in all experimental calves (Table 2 and S2 Table). The increase was significantly higher over the four days following TLR7 agonist/saponin emulsion injection as compared to the four days prior (p = 0.00001) and significantly greater than the diameter of the lymph nodes of the con- trol calves (p = 0.0001), which did not increase in size (p = 0.28). Consistent with the innate inflammatory response indicated by the increases in rectal tem- perature and lymph node size, IL-6 levels significantly increased (p = 0.00001) over the four- day period in the TLR7 agonist/saponin emulsion group as compared to the control group (Table 3 and S3 Table). Synthetic TLR7 agonist/saponin injection reduces subsequent need for antibiotic treatment and mortality of crossbred calves following A. marginale infection In Trial 1, all calves tested positive by PCR and Msp5 CI-ELISA for A. marginale within 22 days of exposure to field challenge. This was expected given the high levels of transmission in Ghana [7]. Calves were monitored daily and antibiotic treatment was initiated if PCV decreased to 28% or below. Of the control group (n = 10) that did not receive the TLR7 ago- nist/saponin emulsion, 8 calves met the stated criteria for antibiotic treatment while 3 calves in the TLR7 agonist/saponin emulsion injected group required antibiotics (Table 4). The mean low PCV was significantly greater in the experimental group as compared to the control group (Table 4). Despite antibiotic treatment, 6 calves in the control group died as compared to 1 in PLOS ONE Toll-like Receptor 7 agonist stimulates protection against morbidity and mortality due to Anaplasma marginale PLOS ONE | https://doi.org/10.1371/journal.pone.0306092 August 29, 2024 4 / 12 https://doi.org/10.1371/journal.pone.0306092 the experimental group (Table 4). Based on these results, the trial was then independently repeated using larger group sizes, n = 45 for each group. In trial 2, A. marginale infection was again confirmed in all calves within three weeks of nat- ural exposure. Of the 45 calves in the control group, 34 calves (76%) met antibiotic treatment criteria while 16 of 45 (36%) in the TLR7 agonist/saponin injected group required treatment (Table 5). In the control group, 15 calves died, despite treatment, while in the experimental group 3 died (Table 5). IL-6 levels correlate with protection against anemia due to A. marginale infection The mean IL-6 levels were plotted against the mean PCV level of each calf in both groups from Trial 1 (Fig 1 and S4 Table). There was a statistically significant correlation (r = 0.74; p<0.0001) between IL-6 levels and anemia, at the individual calf level, regardless of the group. Table 1. Mean [±SD] rectal temperature of calves 4-days prior to and 4-days after inoculation with the TLR7 agonist/saponin emulsion. Pre-inoculation Post-inoculation Difference Crossbred calf # Control Groupa 5NS24 38.6 [0.05] 38.8 [0.01] 0.2 M9 38.6 [0.07] 38.3 [0.13] -0.3 3NS51 38.7 [0.05] 38.4 [0.04] -0.3 3NS2 38.6 [0.05] 38.7 [0.25] 0.1 3NS53 38.6 [0.10] 38.8 [0.05] 0.2 M3768 38.5 [0.05] 38.8 [0.10] 0.3 M96 38.2 [0.21] 38.5 [0.25] 0.3 N1299 38.1 [0.10] 38.9 [0.15] 0.8 R270 38.2 [0.05] 38.3 [0.03] 0.1 R286 38.4 [0.13] 38.7 [0.02] 0.3 Mean ±SE 0.19 ±0.10 Experimental Group O554 38.7 [0.21] 39.5 [0.05] 0.8 4NS26 38.3 [0.05] 39.7 [0.13] 1.4 4NS22 38.4 [0.1] 39.6 [0.07] 1.2 3777 38.7 [0.04] 39.3 [0.23] 0.6 4NS21 38.6 [0.32] 39.3 [0.2] 0.7 5NS6 38.4 [0.07] 39.4 [0.11 1.0 4533 38.2 [0.05] 39.4 [0.31] 1.2 R287 38.7 [0.08] 39.5 [0.17] 0.8 N39 38.6 [0.2] 39.4 [0.22] 0.8 R288 38.6 [0.05] 39.9 [0.13] 1.3 Mean ±SE 0.98 ±0.08 a Control group calves that did not receive the TLR7 agonist/saponin emulsion were monitored over the same time period. The data shown are the means (+/- SD) of all daily timepoints for individual calves in each group. Calves in the TLR7 agonist/saponin emulsion experimental group developed significantly increased temperatures following injection (p = 0.00003) as compared to pre-inoculation temperatures. There was no significant change in the control group calves over the same period (p = 0.49), The mean rectal temperature for the experimental group during the four days following TLR7 agonist/saponin injection was significantly higher than that of the control group over the same period (p = 0.001). https://doi.org/10.1371/journal.pone.0306092.t001 PLOS ONE Toll-like Receptor 7 agonist stimulates protection against morbidity and mortality due to Anaplasma marginale PLOS ONE | https://doi.org/10.1371/journal.pone.0306092 August 29, 2024 5 / 12 https://doi.org/10.1371/journal.pone.0306092.t001 https://doi.org/10.1371/journal.pone.0306092 Synthetic TLR agonist/saponin emulsion injection does not interfere with the long-term weight gain of calves We also examined whether TLR7 agonist/saponin emulsion injection and short-term stimula- tion of the innate immune response had a long-term impact on weight gain. The average weight gain of calves injected with the TLR7 agonist/saponin in Trials 1 and 2 was 1.72kg ± /month compared with 1.67kg ± /month for the control group. The overall body weight at the end of the trial was not significantly different (p = 0.38) between the two groups. Discussion Anaplasma marginale is highly endemic in Ghana, throughout sub-Saharan Africa, and in most tropical and subtropical countries [7, 21, 22]. Severe morbidity and mortality occur dur- ing the acute phase of infection, when bacteremia levels exceed 109 per ml. Notably, animals that survive acute infection develop life-long immunity [11, 12]. As a result, two strategies have been used to manage disease impact. The first is the use of a live vaccines, which involves Table 2. Mean [±SD] diameter (mm) of the pre-scapular lymph nodes of calves 4-days prior to and 4-days after inoculation with the TLR7 agonist/saponin emulsion. Pre-inoculation Post-inoculation Difference Crossbred calf # Control Groupa 5NS24 93.1 [1.0] 92.5 [0.2] -0.6 M9 80.5 [0.8] 83.3 [0.8] 2.8 3NS51 93 [0.9] 92.6 [0.9] -0.4 3NS2 79.2 [1.7] 80.1 [1.4] 0.9 3NS53 79.3 [1.1] 81.0 [1.3] 1.7 M3768 92.7 [1.3] 96.6 [0.7] 3.9 M96 92.2 [1.2] 92.6 [0.9] 0.4 N1299 78.3 [0.5] 81.5 [1.2] 3.2 R270 79.3 [1.6] 81.2 [0.8] 1.9 R286 79.7 [1.4] 83.3 [0.6] 3.6 Mean ±SE 1.72 ±0.48 Experimental Group O554 89.4 [0.7] 98.7 [2.5] 9.3 4NS26 78.8 [0.4] 99.4 [2.4] 20.6 4NS22 86.3 [1.5] 98.3 [1.1] 12.0 3777 76.6 [0.8] 90.1 [2.3] 13.5 4NS21 85.6 [0.9] 96.7 [2.2] 11.1 5NS6 84.4 [1.1] 96.6 [0.3] 12.2 4533 72.2 [0.8] 85.4 [1.3] 13.2 R287 75.2 [1.3] 98.1 [2.2] 22.9 N39 72.7 [0.5] 97.1 [2.7] 24.4 R288 71.8 [0.9] 89.0 [3.1] 17.2 Mean ±SE 15.64±1.7 a Control group calves that did not receive the TLR7 agonist/saponin emulsion were monitored over the same time period. The data shown are the means (+/- SD) of all daily timepoints for individual calves in each group. Calves in the TLR7 agonist/saponin emulsion experimental group developed significantly larger lymph nodes following injection (p = 0.00001) as compared to pre-inoculation lymph node diameter. There was no significant change in the control group calves over the same period (p = 0.28), The mean lymph node diameter for the experimental group during the four days following TLR7 agonist/saponin injection was significantly greater than that of the control group over the same period (p = 0.0001). https://doi.org/10.1371/journal.pone.0306092.t002 PLOS ONE Toll-like Receptor 7 agonist stimulates protection against morbidity and mortality due to Anaplasma marginale PLOS ONE | https://doi.org/10.1371/journal.pone.0306092 August 29, 2024 6 / 12 https://doi.org/10.1371/journal.pone.0306092.t002 https://doi.org/10.1371/journal.pone.0306092 deliberate infection of cattle with a less virulent subspecies A. marginale ss. centrale [23–26]. Although generally effective, the widespread use of the live vaccine has been constrained by the requirement for a cold chain based on preservation in liquid nitrogen, which is difficult and expensive to ensure in low-income tropical countries. The second approach has been to Table 3. Mean [±SD] IL-6 levels (log10) in serum of calves of calves 4-days prior to and 4-days after inoculation with the TLR7 agonist/saponin emulsion. Pre-inoculation Post-inoculation Difference Crossbred calf # Control Groupa 5NS24 2.9 [0.2] 3.0 [0.7] 0.1 M9 1.8 [0.5] 2.1 [0.4] 0.3 3NS51 1.9 [0.7] 2.1 [0.7] 0.2 3NS2 2.3 [0.3] 2.5 [0.6] 0.2 3NS53 2.0 [06] 2.2 [0.2] 0.2 M3768 2.5 [0.2] 2.6 [0.8] 0.1 M96 2.1 [0.5] 2.3 [0.3] 0.2 N1299 2.1 [0.1] 2.4 [0.9] 0.3 R270 2.1 [0.8] 2.3 [0.9] 0.2 R286 1.9 [0.4] 2.4 [0.2] 0.5 Mean ±SE 0.23 ±0.04 Experimental Group O554 3.9 [1.2] 9.5 [1.9] 5.6 4NS26 1.6 [0.9] 5.5 [1.7] 3.9 4NS22 1.3 [2.6] 3.6 [0.3] 2.3 3777 3.4 [1.9] 4.1 [1.2] 0.7 4NS21 1.7 [1.9] 4.2 [1.6] 2.5 5NS6 1.1 [2.3] 7.4 [0.7] 6.3 4533 2.9 [1.3] 6.3 [0.7] 3.4 R287 2.8 [2.4] 3.5 [1.1] 0.7 N39 2.3 [0.8] 5.5 [0.8] 3.2 R288 2.5 [2.0] 4.6 [1.5] 2.1 Mean ±SE 3.07±0.58 a Control group calves that did not receive the TLR7 agonist/saponin emulsion were monitored over the same time period. The data shown are the means (+/- SD) of all daily timepoints for individual calves in each group. Calves in the TLR7 agonist/saponin emulsion experimental group had significantly higher IL-6 levels (p = 0.00001) over the four-day post-inoculation period as compared to the control group. https://doi.org/10.1371/journal.pone.0306092.t003 Table 4. Trial 1: Mean PCV, antibiotic treatment, and mortality for stimulated and control calves. Group Mean PCV± SE (95% CI) Antibiotic Treatment Mortality Stimulated (n = 10) 28.9 ± 3.3 (26.5–31.3) 3/10 1/10 Control (n = 10) 24.78 ± 3.4 (22.3–27.2) 8/10 6/10 PCV, packed cell volume; SE, standard error; CI, confidence intervals. The PCV data shown are the means (+/- SE) for all calves in each group. Difference in PCV between stimulated and control group, p<0.0001. Odds ratio for not requiring antibiotic treatment for the stimulated group versus control group was 9.3 (95% confidence interval 1.2–72.9; p = 0.03). Odds ratio for survival was 13.5 (95% confidence interval 1.9–152.2; p = 0.03) for the stimulated group as compared to the control group. https://doi.org/10.1371/journal.pone.0306092.t004 PLOS ONE Toll-like Receptor 7 agonist stimulates protection against morbidity and mortality due to Anaplasma marginale PLOS ONE | https://doi.org/10.1371/journal.pone.0306092 August 29, 2024 7 / 12 https://doi.org/10.1371/journal.pone.0306092.t003 https://doi.org/10.1371/journal.pone.0306092.t004 https://doi.org/10.1371/journal.pone.0306092 rely on indigenous breeds of cattle which, although equally susceptible to infection, are signifi- cantly more resistant to developing severe morbidity and mortality [27–33]. Although effec- tive, the inability to introduce higher productivity traits into indigenous breeds by crossbreeding without increased susceptibility to severe morbidity and mortality has resulted in lower productivity and decreased economic gains for smallholder farmers. Relevant to the cross-bred cattle used in this current study, F1 Sanga x Friesian cows provide approximately twice the daily milk yield as compared to Sanga cows raised under the same pasture conditions Table 5. Trial 2: Mean PCV, antibiotic treatment, and mortality for stimulated and control calves. Group Mean PCV± SE (95% CI) Antibiotic Treatment Mortality Stimulated (n = 45) 27.1 ± 4.7 (23.7–30.5) 16/45 3/45 Control (n = 45) 23.7 ± 3.7 (21.1–26.3) 34/45 15/45 PCV, packed cell volume; SE, standard error; CI, confidence intervals. The PCV data shown are the means (+/- SE) for all calves in each group. Difference in PCV between stimulated and control group, p<0.0001. Odds ratio for not requiring antibiotic treatment for the stimulated group versus control group was 5.6 (95% confidence interval 2.2–14; p = 0.0002). Odds ratio for survival was 7.0 (95% confidence interval 1.9–26.3; p = 0.004) for the stimulated group as compared to the control group. https://doi.org/10.1371/journal.pone.0306092.t005 Fig 1. Correlation between IL-6 levels and PCV of calves. The X values represent the mean percent packed cell volume (% PCV) of all 20 calves (n = 10 for control and 10 for calves injected with the TLR7 agonist/saponin emulsion. The Y values represent the corresponding mean log IL-6 (pg/ml) of individual calves. R is: 0.742; P = 0.0001; R2 = 0.55. https://doi.org/10.1371/journal.pone.0306092.g001 PLOS ONE Toll-like Receptor 7 agonist stimulates protection against morbidity and mortality due to Anaplasma marginale PLOS ONE | https://doi.org/10.1371/journal.pone.0306092 August 29, 2024 8 / 12 https://doi.org/10.1371/journal.pone.0306092.t005 https://doi.org/10.1371/journal.pone.0306092.g001 https://doi.org/10.1371/journal.pone.0306092 [34–36]. Even minor increases in milk production have been shown to translate into increased household income and expenditures on additional food, health care, and education [2, 37]. Based on the concept that there is an energetic trade-off between resources dedicated to innate immunity versus productivity, we tested the hypothesis that direct stimulation of the innate immune system would provide sufficient short-term protection in crossbred cattle to avoid severe morbidity and mortality. The selection of the synthetic TLR7 agonist imiquimod and its incorporation into a slow-release water-in-oil emulsion was based on the evidence that bovine TLRs 7 and 8 responded to stimulation with cytokine release [13, 14, 16, 38, 39]. The confirma- tion that F1 Sanga x Friesian crossbred cattle responded with significantly increased tempera- tures, enlarged regional lymph nodes, and IL-6 levels over the first four days post-injection indicated innate immune stimulation. The subsequent significant reduction in need for antibi- otic treatment and reduced mortality, confirmed in two independent trials with a total of 55 animals per group, support the efficacy of this approach. Notably, tracking subsequent weight gain of animals in the second trial group revealed no significant difference between the TLR7 agonist/saponin emulsion injected and control cattle, indicating that any energetic trade-off between innate immunity and productivity was transient. This latter finding is important as the overall goal is to increase productivity for the smallholder farmer. This study is a proof of concept that supports an overall approach to managing severe dis- ease due to A. marginale infection. There are several important caveats. The first is that although there was a statistically significant reduction in the need for antibiotic treatment and in mortality, multiple animals in the TLR7 agonist/saponin injected groups required treatment and 7% died (versus 38% in the controls). We did not include a control group of age-matched Sanga cattle that would have allowed us to determine if the TLR agonist/saponin induced a similar level of protection as fully indigenous cattle, which would be a relevant benchmark. Secondly, the selection of agonist was largely empirical based on the ability of TLR 7 agonist/ saponin stimulation to increase CD4+ T cell secretion of IFN-γ [40], previously show to associ- ate with immune protection against A. marginale challenge [41]. Similarly, the dose and for- mulation were also empirical and unlikely to be optimal. However, the correlation between protection from anemia as a measure of disease severity and IL-6 levels suggests that IL-6 is a measurable parameter to optimize agonist, dose and formulation. It is notable that the compo- nents employed in the formulation are low cost and commercially available, and the processing requires minimal equipment. Additional or different agonists, doses, or slow-release formula- tion should be tested with the goal of reducing antibiotic treatment and mortality to less than or equal to that of the fully indigenous Sanga. In addition, the duration of innate immunity and protection induced by the TLR7 agonist/saponin is unknown. In the current study con- ducted in an endemic region with a high level of transmission, challenge occurred within three weeks following natural exposure for which short-term protection may be sufficient. In regions with lower transmission or where transmission is seasonal, the duration of protection becomes a key question and requires additional experimentation. Finally, efficacy was only tested against acute A. marginale infection. However, there are multiple infectious diseases of tropical and subtropical livestock, including babesiosis and East Coast Fever, that have a simi- lar course of acute disease followed by life-long antigen specific immunity; the approach of short-term stimulation of innate immunity may be an effective strategy for protection against multiple pathogens. Supporting information S1 Table. Rectal temperature of calves. The rectal temperatures of individual calves in the control and experimental (TLR agonist) groups four days prior to and four days following PLOS ONE Toll-like Receptor 7 agonist stimulates protection against morbidity and mortality due to Anaplasma marginale PLOS ONE | https://doi.org/10.1371/journal.pone.0306092 August 29, 2024 9 / 12 http://www.plosone.org/article/fetchSingleRepresentation.action?uri=info:doi/10.1371/journal.pone.0306092.s001 https://doi.org/10.1371/journal.pone.0306092 injection. (DOCX) S2 Table. Lymph node sizes. The diameter of prescapular lymph nodes of individual calves in the control and experimental (TLR agonist) groups four days prior to and four days following injection. (DOCX) S3 Table. IL-6 levels in individual calves. The IL6 levels (pg/ml) of individual calves in the control and experimental (TLR agonist) groups 24, 48, and 72 hours following injection. (DOCX) S4 Table. Mean IL-6 levels and mean PCV in individual calves. The mean IL6 levels (pg/ml) and the mean PCV (%) of individual calves in the control and experimental (TLR agonist) groups. (DOCX) Acknowledgments We wish to thank Mr Abdulai Mamah for the care and maintenance of calves. We also acknowledge Mr Jonathan Quaye for his technical support and Dr Francis Dogodzi for his vet- erinary assistance. Author Contributions Conceptualization: James E. Futse, Susan M. Noh, Guy H. Palmer. Data curation: James E. Futse, Songliedong Zumor-Baligi, Charles N. K. Ashiagbor, Susan M. Noh, Christopher B. Fox, Guy H. Palmer. Formal analysis: James E. Futse, Songliedong Zumor-Baligi, Charles N. K. Ashiagbor, Susan M. Noh, Guy H. Palmer. Funding acquisition: James E. Futse, Guy H. Palmer. Investigation: James E. Futse, Songliedong Zumor-Baligi, Susan M. Noh, Guy H. Palmer. Methodology: James E. Futse, Songliedong Zumor-Baligi, Charles N. K. Ashiagbor, Susan M. Noh, Christopher B. Fox, Guy H. Palmer. Project administration: James E. Futse, Susan M. Noh, Guy H. 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