We propose prospective mixed-methods cohort research combining quantitative and qualitative methods to assess brucellosis, leptospirosis, and scrub typhus in a peri-urban setting where animal husbandry and agriculture are the primary occupations. Over three years, we will follow 1,293 households, conducting one baseline survey and three follow-up assessments. The study objectives are to establish an integrated surveillance system in a peri-urban setting to

Intervention packages, awareness programs, and public involvement are designed to achieve the objectives and will be discussed in detail later. A detailed flow chart describing the study design and overview is given in Figure 1. The expected outcomes are the prevalence and patterns of zoonotic diseases and their risk factors, as well as developing prevention strategies through behavioural change and WASH practices. The study’s translational value lies in linking community surveillance, laboratory diagnostics, and public health policy.

Balianta and Balipatna, two administrative blocks of the Khordha district in Odisha, were selected as the intervention sites. Their geographic location and the nearest city are presented in Figure 2. According to the latest census in 2011, the population of Balianta was 91,728 from 19,535 households. There were 46,903 males and 44,825 females. The distance between the block and the nearest city (Bhubaneswar) is 9 kilometres. Balipatna block has a total population of 132,796 people and 29,771 households. There are 67,879 males and 64,917 females among them.

Three villages from each block were selected using a community-based participatory approach presented in Figure 3. The villages were chosen based on the population of various domestic livestock (Table 1) and other environmental factors such as proximity to agricultural land, poultry farms, rivers, canals, and closeness to urban centres. We selected the villages with the support of local community networks - community leaders, Panchayati Raj institutions (PRI) members, health care providers, and veterinary professionals. Free-listing and pile-sorting methodologies were used to enumerate and rank the various domestic animals in the selected villages and common animal and human diseases, including reported animal bites from the previous year. All the selected villages are located within a 12-kilometre radius of the nearest city and the highway, ensuring accessibility and a representative peri-urban setting.

After liaison with the local community health centre (CHC) and the sector medical officer (doctor appointed to the primary health centre; PHC), a team of field staff will approach households along with the accredited social health activist (ASHA) to update the household line list, which will be used for calculating the final sample size and participant selection (one adult member from every household) following the inclusion and exclusion criteria given below:

Inclusion criteria

Exclusion criteria

Inclusion criteria

Exclusion criteria

Since we have updated the list of existing households (n = 1,516) and people residing in them, we will select one adult member (age ≥18 years) from each household, using the Kish-Grid method, before going for the survey. Since the number of households is quite close to the calculated sample size, we will target all the households. Using the Kish-Grid method, we shall randomly identify one adult individual from every household and inform our field team to collect quantitative information and blood samples from that person only; one research assistant will be there in the field to supervise this process. If the selected individual is not eligible to be included or does not wish to participate in the survey, the field supervisor will identify the next eligible member and so on. In this way, up to three members within one household will be approached. If participants are still not willing to participate, that household will be eliminated.

To accomplish this method, two field teams (one data entry operator and one laboratory technician in each team, along with one supervisor) will approach households along with multi-purpose health workers (MPHW – male and female) or ASHA workers. Participants who meet the eligibility criteria of the study must sign an ICF. Then, the participant will be allotted a specific Household ID by which the participant gets enrolled in the study. The quantitative information will be collected from the participants regarding socio-demographic characteristics, relevant hygiene practices, exposure history with animals and fever with treatment history. After collecting data from the participants, whole blood will be collected from them.

The Survey tool will cover identifiers and socio-demographic characteristics of the participants, their existing disease profile (especially non-communicable diseases) and previous symptoms, history of hospital visits and hospitalization, food habits and methods of preservation, household animal handling and interaction with animals. Another quantitative tool covers current water, hygiene, and sanitation practices – those could act as preventive measures. To ensure the cultural relevance and clarity of the tool, a pilot test will be conducted in two non-study sites, involving 20 participants from each location, and modifications will be made based on the feedback obtained. Trained field investigators will conduct the face-to-face interviews in the local language, utilising electronic devices for real-time data entry.

All the human samples will be followed up four times, including one baseline survey and three follow-up assessments over three years. At the same time, all eligible animals will be tested at baseline. From the second round onwards, animals will be resampled under the following conditions: (a) Households where the owner is found seropositive (continued testing for disease transmission dynamics). (b) A random subset of animals from seronegative households will be monitored for potential new infections.

All the collected human blood samples will be sent through a cold chain to the Indian Council of Medical Research – Regional Medical Research Centre (ICMR-RMRC), Bhubaneswar for further laboratory testing. Blood vials will be left undisturbed at room temperature for 30 min to allow clot formation. Then, it will be centrifuged at 2000 rpm for 5 min in a centrifuge instrument (Rotanta 460, Hettich) for serum separation. Enzyme-linked immunosorbent assay (ELISA) will be performed to test serum IgG and IgM antibodies against human brucellosis, leptospirosis, and scrub typhus infection using commercial kits (Supplementary Table S2). The final reading (optical density) will be measured in a 96-well plate reader platform (Lisascan EM, Erba Lachema) and analysed as per the manufacturer’s instructions. Internal quality assurance of the laboratory tests will be performed, which includes a daily run of quality control, periodic blind re-testing of processed and fresh samples, and weekly maintenance of the instruments. External quality control will be corroborated by a nationally accredited laboratory at ICAR-NIVEDI, Bengaluru.

Animal blood samples will be collected with the help of trained veterinarians, and serum will be separated at ADRI, Cuttack. Serum samples will then be sent to ICAR-NIVEDI for further laboratory investigations of brucellosis and leptospirosis. This laboratory is a WOAH-designated reference laboratory for leptospirosis, which participates in an annual proficiency test to ensure the quality assurance of employed serovars. Confirmation of brucellosis will be performed using the Rose Bengal plate test and ELISA. The microscopic agglutination test (MAT) will be performed for leptospirosis utilizing around 20 live serovars representing 17 serogroups, with results interpreted at the serogroup level. All samples will be screened at a 1:100 dilution, and MAT titers will be determined following WOAH guidelines (33). All serum samples will be stored at −20 °C for subsequent analysis.

We will conduct geospatial mapping of the water bodies having a possibility of human-animal interactions (direct or indirect) using a GPS-enabled tablet in the six study villages. To capture the seasonal variation, the samples will be collected at six-month intervals from multiple sources, including ponds, community wells, and other water bodies with frequent human or animal contact. The quality of water from a subset will be assessed by evaluating the presence of indicator organisms by the most probable number (MPN) method and culturing on endo and eosin methylene blue agar.

This study will use the principles of the Theory of Change (ToC) to build and test the complex intervention model for the objectives (Figure 4). Theory of change involves a thorough illustration and description of how and why desired changes are expected to happen in a specific context. It helps to connect the dots between what a programme does and the goals it wants to achieve. The construction of a ToC typically occurs through a consultative process, requiring stakeholders to reflect on how their programmes can bring about change. The following interventions will be developed to achieve the goal:

Open data kit (ODK ver. 2023.3.6) will be used to make online data entries and to link data from all the waves. For sample tracing from the field, as well as to maintain the flow of samples within the laboratory, unique identities and bar codes will be employed. Electronic instruments like tablets and voice recorders will be used for data entry in both qualitative and quantitative studies. Survey staff will enter the lab results into the electronic database. All survey data will be stored on password-protected tablets or computers, with access limited to authorized staff. Only the data monitoring members and the principal investigator will have access to the entered data. FGDs and IDIs will be translated and transcribed to facilitate analysis using NVivo (v14). To ensure intercoder reliability, two independent researchers will code the transcripts, develop an initial codebook, refine it through comparisons and resolve the discrepancies through discussions. Various themes and sub-themes will be generated from participants’ narratives using the inductive approach, without imposing existing theories, and will be analysed by the thematic framework. Audio recordings, transcripts, and translations will be stored securely, and encrypted backups will be maintained to prevent data loss.

We shall establish a surveillance system for the three target zoonotic diseases, in collaboration with human and veterinary health systems. For this initiative, a fever screening tool will be developed and piloted in real-time in the first year of the study, utilising the ODK toolkit. Issues associated with the tool will be iterated based on observations from the field visits. At the end of the second year, a digital application named JEEVOne app (Joint Effort for Epidemiological Vigilance for One Health) will be developed based on a fever-tracking algorithm in collaboration with ICMR, Dibrugarh, and NESAC, Shillong, which will be provided to primary-level health workers.

In the final year of the study digital application will be subjected to a formal piloting process in selected sites to measure its usability, operational feasibility, and general acceptability. Field testing will involve close observation of real-time usage, user interviews, and monitoring of system performance under different conditions, such as where connectivity is limited. Before rolling out the app in all sites, recommendations from the users will be taken regarding the app interface and operational challenges. In addition to this, the content validity of the application will be assessed through expert reviews to ensure that the content (symptoms and exposures) included in the app to capture the disease is relevant and evidence-based. In accordance with the Digital Personal Data Protection (DPDP) Act of 2023 in India, the app will be developed with stringent user privacy protections and data security procedures, ensuring that all health and personal data is anonymized, encrypted, and accessible only by authorized staff. The app will be integrated with existing human and veterinary workflows to ensure that data collection, reporting, and follow-up align with the system, thereby facilitating adoption and sustainability.

Using suitable descriptive and summary measures, quantitative and qualitative data will be evaluated and aggregated to describe the One Health concept. We will use STATA (StataCorp LLC, TX, USA) and an open-source geographic information system (GIS) software QGIS for quantitative data analysis, plotting the incidence cases and establishing causal relations between the incidence and the environment. According to the degree and pattern of missingness, missing data will either be dropped or imputed using statistical techniques such as multiple imputation or complete case analysis. Individuals who did not take part in the entire study will not be included in the analysis. Attrition rates will be calculated to assess differential dropout, and baseline traits of completers and non-completers will be compared. Depending on the variable type, comparisons will be made using chi-square tests/t-tests. Additionally, we will investigate the variations in outcome variables using relevant statistical techniques, such as regression models and time series analysis. Finally, all quantitative data will be combined to create a ‘One Health’ model in which relationships between illness patterns, health-seeking behaviour, prescribing, and resistance from various sources – human, animal, and water will be linked and tracked over time. Univariate descriptive statistics and odds ratios will be calculated. Potential confounders will be handled by including them in the multivariable models after testing for collinearity. Multivariable, stepwise conditional logistic regression will be performed to determine independent risk factors for each disease condition. In addition to this, we will employ Generalised Estimating Equations to assess the population average relationship between exposures and disease outcomes.

Integrating quantitative and qualitative data by specific mechanisms such as data transformation, joint display, convergent design, sequential exploratory design, and data triangulation would enhance the robustness of the study. A comprehensive strategy will be developed for disseminating study findings to relevant stakeholders on a local and global scale, taking into account diverse communication channels such as peer-reviewed publications, conference presentations and involvement with mainstream and social media. In addition, focused outreach efforts will be made to policymakers, healthcare professionals, and community groups to ensure that the findings are accurate. The study will be reported according to the STROBE (Strengthening the Reporting of Observational Studies in Epidemiology) guidelines to ensure transparency and completeness of the data.

The study protocol has been approved by the Institutional Ethics Committee of the ICMR-RMRC, Bhubaneswar (Ref No: ICMR-RMRC/IHEC-2022/128) and will follow the National Ethical Guidelines for Biomedical and Health Research Involving Human Participants by the ICMR, New Delhi. The State Ethics and Review Committee, Department of Health and Family Welfare, Government of Odisha also approved this project proposal (Ref No. 25588 dated 25/10/2023). We will ensure prior permission from the local administration for fieldwork. All participants will be briefed on the study’s objective, and a written ICF will be obtained before the interview. The participation was purely voluntary, and the respondent could withdraw from the interview at any point in time without citing any reason. Written informed consent will be obtained from participants before they participate in the study, and they will be provided with a participant information sheet (PIS) for their information. For domestic animals, written informed consent will be obtained from the owners. To ensure confidentiality, personal identifiers will be removed during data entry and analysis.

The serological data from both humans and animals will elucidate the prevalence, distribution pattern and transmission dynamics of brucellosis, leptospirosis, and scrub typhus. It will also identify the associated risk factors based on socio-economic conditions, perceptions, and knowledge of zoonoses, availability of essential amenities, daily lifestyle, and other environmental factors. The spatial data will help in mapping zoonotic disease hotspots, identifying risk zones, and predicting disease emergence through integrating multisectoral data. This One Health approach will lead to enhanced stewardship, compliance, and interspecies equity (balanced health benefits across species), enabling humans to benefit in the long term from their domestic animals and ecosystems. The conceptual framework will predict disease onset more efficiently, with improved community awareness and robust communication among responsible stakeholders that facilitate rapid action on illness. This epidemiological data will aid in the development of evidence-based policy through the processes of identifying vulnerable populations, policy experimentation, policy evaluation, implementation optimisation, and long-term impact assessment.

The primary purpose of using this application will be to facilitate the early screening of individuals with probable and specific symptoms of brucellosis, leptospirosis, and scrub typhus. Once someone is identified as a probable case, both the human and veterinary health systems will be informed to take immediate action for diagnosis. It will also include questions that help identify the source of infection, especially if it originated from an animal. Additionally, it will help to determine how many people may have been exposed to the infected source. This digital application-based integrated surveillance system will assist in:

Outbreak preparedness

The study outcome will enhance the capacity of community members and key stakeholders by convening and consulting experts from diverse fields to effectively assess a peri-urban cohort for addressing zoonotic diseases with a One Health approach. The findings will directly inform the formulation and adaptation of intervention strategies like animal vaccination, WASH improvements or behavioural changes. The intervention packages, based on the ToC approach, will be tailored to local contexts and will address specific environmental, veterinary, and behaviour-related issues. The active participation of various experts: clinicians, veterinarians, and public health professionals will help ensure that the interventions are evidence-based, culturally appropriate, and feasible to implement. The policy decisions are expected to be useful for district- and state-level governance by presenting viable models for interdisciplinary disease surveillance and prevention. By anchoring the interventions in locally relevant socio-ecological realities while aligning them with national health priorities, the study aims to bridge existing gaps between research, practice, and governance.