The burden of gastrointestinal infections remains a major problem, especially in low-income countries, as worldwide data show that infectious diarrhoeal disorders alone account for nearly 0.8 million deaths in children less than 5 years of age annually [1]. Multiple aetiological agents, including bacteria, viruses, and parasites, contribute to the diarrhoea of infective aetiology in humans and animals [2]. These diarrhoeal agents are most commonly transmitted either through contaminated food and water or through the faecal-oral route. Therefore, lack of access to clean water, insufficient hygiene, and inadequate sanitation conditions in resource-poor settings put the community at high-risk of suffering from diarrhoea [3]. The anthroponotic and zoonotic transmission of diarrhoeal diseases occurs through a wide range of environmental reservoirs contaminated with various diarrhoeagenic pathogens common to both humans and animals [4]. In particular, domestication of livestock and pet practices often contribute to the zoonotic transmission of intestinal pathogens through faecal contamination of bodies of water [5]. The soil might also play a major role in transmission of enteric diseases, but this crucial link to infections has not yet been well studied [6].

Frequent foodborne and waterborne outbreaks of infectious diarrhoea have encouraged microbiologists and epidemiologists to conduct ecological studies to understand the zoonotic and anthroponotic transmission of various diarrhoeal pathogens. In community settings in India, diarrhoeal syndromes are characterized by high faecal shedding, infectivity, growth, persistence, exposure to site-specific environmental conditions, pathogen detection, and faecal contamination [7]. However, only limited information exists regarding the association between domestic exposure and zoonotic transmission of genetically diverse diarrhoeal pathogens in the eastern part of India. A pilot survey was therefore conducted in an eastern coastal province in India in order to investigate the prevalence and possible genetic diversity of major diarrhoeal agents in faecal samples from symptomatic humans, domestic animals, and soil samples from the same locality through microbiological investigations.

A total of 152 of 310 (49.0%) human samples, 67 of 150 (44.7%) animal samples, and 16 of 40 (40.0%) soil samples were found to be positive for at least 1 diarrhoeal pathogen. In the animals, the overall diarrhoeal pathogen detection rate was highest in sheep (41.1%), followed by goats (35.5%) and cattle (33.3%). Diarrhoeagenic Escherichia coli (DEC) was the major enteric pathogen detected in humans (28.7%), animals (38.7%), and soil (32.5%) (Table 1).

In humans, rotavirus was detected in 17.4% of cases, followed by enteropathogenic E. coli (EPEC) (15.5%) Shigella (13.9%), Shiga toxin-producing E. coli (STEC) (9.7%), enterohemorrhagic E. coli (EHEC) and enteroaggregative E. coli (EAEC) (4.5%), Cryptosporidium and adenovirus (3.9%), and Giardia (0.6%) (Table 1). In animals, STEC (28.0%), EPEC (14.7%), and EHEC (14.0%) were the major types of DEC detected, followed by Cryptosporidium (10.0%), adenovirus (4.7%), Shigella (3.3%), and Giardia (0.7%) (Table 1). In the samples, EPEC (30.0%) was the major enteric pathogen detected in soil samples, followed by Shigella (25.0%), STEC (15.0%), Giardia (7.5%) and Cryptosporidium (5.0%) (Table 1).

Sheep were found to be slightly more infected with DEC (41.7%) than goats (35.5%) and cattle (33.3%) (Table 2). Cryptosporidium were more often observed in goats (17.8%), while Shigella infection was predominant in sheep (6.7%). However, cattle were more likely to be positive for adenovirus than sheep and goats (Table 2). The distributions of diarrhoeal pathogens by age in humans and animals are shown in Table 3.

In our study, we observed coinfections with different combinations of bacterial, viral, and protozoan pathogens in both faecal and soil samples. Pathogens were detected simultaneously in 39.5% of human samples, 61.2% of animal samples, and 81.2% of soil samples (Table 4). Multiple pathogens were detected significantly more frequently in soil samples (p=0.009), followed by human samples (p=0.003) and animal samples (p=0.030). In humans, Shigella and STEC (8.5%) was the most common coinfection followed by rotavirus and EPEC (7.2%) and Shigella and EPEC (6.6%) (Table 4). The most frequent combination in animals was EHEC and STEC (23.4%) (Table 4). From all positive animal samples, we observed the highest percentage of coinfections in sheep (70.8%), followed by cattle (50.0%) and goats (37.5%). The combinations of Shigella and EPEC (37.5%) and STEC and EPEC (31.2%) were predominant in soil samples (Table 4).

Phylogenetic analysis was done to investigate the genetic relatedness and evolutionary dynamics of the strains circulating between humans and animals in the study region. Phylogenetic trees were constructed separately for each group of pathogens (Supplementary Materials 2-5). Among the DEC strains, STEC, EPEC, EHEC, and EAEC clustered in individual nodes isolated from humans and animals were found close to each other (Supplementary Material 2). Similar patterns were observed in Shigella isolates from humans and animals (Supplementary Material 3). Cryptosporidium and adenovirus isolates showed close relatedness with other strains that were isolated from domestic animals, birds, or environmental samples (Supplementary Materials 4 and 5).

Based on principal component analysis, 3 different clusters were generated for the human, animal, and soil samples representing the patterns of infectious agents at the genus level in the study region (Figure 2). All 3 groups shared a large portion of genera, revealing that the distribution of infections in human, animal, and soil samples was comparable. Samples collected from 4 different zones showed very similar patterns of distribution of infectious agents. All detected pathogens were distributed throughout the study area.

Many resource-poor or developing countries have limited sanitary infrastructure, accompanied by a lack of awareness among communities that are suspected to be deprived of adequate education and awareness. Irrespective of focal urbanization and development of sanitation facilities, communities in underdeveloped pockets in and around urban areas are commonly reported to have higher rates of infections, particularly those associated with enteric pathogens [11]. This also places urban populations at a higher risk of acquiring infections because of their dependence on the communities living in the outskirts. In the present study, at least 1 diarrhoeal agent was detected in 40–50% of environmental samples and samples from animals and humans. When comparing the isolates from these 3 sources, we observed genetic similarities among the isolates, indicating the possibility of circulation of these microbial agents among humans, animals, and the environment in the study region.

Pathogenic E. coli was present in 32.5% of the soil samples. The detection of pathogenic E. coli in soil was also reported in another recent study from Kenya [12]. Hence, it is important to evaluate the environmental sources (soil and water) that might play an important role in retaining diarrhoeal pathogenic agents and act as a source of infection transmission, affecting both humans and animals.

Cattle and other ruminant animals might serve as reservoirs of STEC strains that are potentially pathogenic in humans [13,14]. In our study, STEC was detected most frequently in cattle (26.7%), followed by soil (15.0%) and human samples (9.7%). Similar patterns were observed in a previous study from Tanzania, although the prevalence rates were lower (cattle, 9.0%; humans, 3.2%; and soil, 0.8%) [15].

Open defecation by animals and humans is a major contributor to microbial shedding into the environment (soil and water). Thus, various microbial agents from the soil can contaminate nearby community bodies of water, thereby exposing both humans and animals. Therefore, the soil can be a potential mode of transmission of diarrhoeal pathogens in low-income countries; Pickering et al. [16] were able to isolate pathogenic E. coli, enterovirus, rotaviruses, and human Bacteriodales from soil samples. In our study, we detected Shigella, EPEC, STEC, Cryptosporidium, and Giardia isolates in soil samples from the locality, while animal and human diarrhoeal cases were also found to harbour similar aetiological agents. Sequencing of the human EPEC and EHEC virulence genes eaeA and aggR showed similarities with the corresponding animal isolates. In contrast, the Cryptosporidium 18s rRNA sequence and adenovirus hexon gene sequence were similar to other Cryptosporidium parvum and adenovirus strains, respectively, that were isolated from domestic animals, birds, or environmental samples [17,18].

Molecular epidemiological studies of pathogenic E. coli have suggested that cattle, sheep, and goats are potential sources of diarrhoeagenic EPEC, EHEC, and STEC [5,19-21]. In our study, the frequency of detection of STEC, EPEC, and EHEC was higher in sheep than in goats and cattle. Similar results were reported in another study from Turkey, where the isolation rate of STEC, EPEC, and EHEC was higher in sheep and goats [22]. STEC have been found to be closely related genetically when isolates from cattle [23] and sheep [24] were compared. Our study showed the presence of STEC in both humans and animals, suggesting possible zoonotic transmission of this pathogenic strain of E. coli.

According to the results of the present study, Shigella was the third most common aetiological agent detected in symptomatic humans and animals in the study region. Cryptosporidium and Giardia were the other major enteric pathogens detected in all 3 sources, while adenovirus was detected in humans and animals only. Odagiri et al. [25] reported that adenovirus, Giardia, and Cryptosporidium were found in rural India with a higher prevalence than in our study.

In comparison to a single pathogen, the presence of multiple diarrhoeal pathogens might cause more severe diarrhoea and disease pathogenesis [26]. In one of our previous studies, we detected multiple diarrhoeal pathogens, similar to the findings of other studies [27]. In the present study, multiple diarrhoeal pathogens were detected significantly more often in humans, animals, and soil samples than single pathogens. The most common pairs of concurrent pathogens in this study were Shigella and STEC in humans, EHEC and STEC in animals, and STEC and EPEC in soil samples. Given the paucity of data on the rates of specific coinfections with multiple diarrhoeal pathogens in human, animal, and soil samples, it is difficult to say whether our data are within the expected range. This is certainly an area that needs further investigation to obtain a better understanding of patterns of coinfection and their associations with disease transmission dynamics.

In order to understand the genetic relatedness between the isolated pathogenic strains, sequencing was carried out and a phylogenetic tree was constructed. The phylogenetic tree showed similarities between the human, animal, and soil isolates. We observed that few DEC strains, such as EPEC, EAEC and EHEC, and Shigella shared similar genetic sequences and clustered under the same branch. This suggests the possible zoonotic transmission of DEC and Shigella between humans and domesticated animals in the study area. A phylogenetic tree analysis of Cryptosporidium and adenovirus found that these isolates showed sequence similarities with previously isolated human and animal strains.

Livestock is an important reservoir for a number of enteric pathogens that can affect human and animal health. A recent study suggested that 15 major enteric pathogens are responsible for zoonotic transmission in low-income and middle-income countries (LMICs), of which 5 enteric pathogens cause approximately 1 million annual deaths [28]. Systematic reviews have demonstrated that after the introduction of improved sanitation in LMICs, a 30-40% decrease in childhood diarrhoea occurred [29,30]. Interventional sanitation efforts may reduce the quantity of human excreta in the environment, but animals are still often present in the domestic environment in LMICs, and people in these countries may have frequent contact with them [31-33]. Thus, contamination from animal faeces may still contribute to a substantial burden of disease in humans. This study demonstrated the coexistence of potential diarrhoeal enteric pathogens in human, animal, and soil samples in the study region, suggesting the possibility of zoonotic and anthroponotic transmission.

Successive federal government programmes have emphasised building toilets to end open defecation. The current programme, the Swachh Bharat Mission, aims to provide sanitation to all households to end open defecation by October 2019. Prior to the launch of the cleanliness campaign, the coverage of sanitation in the state of Odisha was a mere 10.9%, and after all the efforts of the last 3 years sanitation coverage reached upto 70%, Odisha remains amongst the lowest-performing states, with nearly one-third of the population still not having access to toilets [34,35]. The present study area included many underdeveloped pockets in and around the city of Bhubaneswar, where access to improved sanitation is poor. This results in the practice of open defecation by large segments of the population. Direct dispersal of animal excreta into the environment is also common throughout the study region. This increases the potential risk of transmission of faecal pathogens in exposed communities. The present findings provide preliminary evidence of the diversity of potential possible transmission patterns of bacterial, viral, and protozoal diarrhoeal pathogens and provide an improved understanding of the distribution of these pathogens in humans, animals, and the shared environment (soil). Overall, this study will be helpful for expanding our knowledge of disease transmission in this region, so that transmissible diseases of concern can be controlled, thereby enhancing quality of life for the community.

Although the present observational study deployed a unique approach to study both animal and human pathogens from the same locality, there are still some limitations, particularly in the choice of environmental samples and the sample numbers. As discussed previously, only soil samples near ponds or paddy fields were investigated based on the assumption that these are the most preferred sites for open defecation, as locations where both human and animals visit frequently for various daily activities. The initial plan to include similar numbers of human/animal faecal and soil samples could not be fulfilled due to various reasons, including but not limited to funding, time constraints, and the exclusion of a few samples due to unavoidable technical errors during transportation from the field to the analysing laboratory. To better explore the role of zoonotic transmission, pairing of human faecal, animal faecal, and soil samples collected from the same locality would be valuable, as would information on animal ownership and contact with livestock; however, these factors went beyond the scope of this study. Therefore, larger and longitudinal cohort studies of infants, children, adults, animals, soil, and community water sources would provide improved estimates of the prevalence of these diarrhoeal pathogens and their transmission in the community.

The present study provides an improved understanding of the distribution of major enteric pathogens coexisting in humans and animals in the region, thereby suggesting a high potential for transmission among livestock and communities residing in the studied locality via contaminated soil and/or water (Figure 3). Future research on zoonotic and anthroponotic transmission of faecal contaminants should involve host-specific markers to determine the precise pathways of pathogen transmission in the region.