Introduction

Zoonotic diseases are considered a significant threat to public health, social stability and the world economy [1, 2]. It is estimated by the World Health Organization (WHO) that every year, approximately one billion illnesses and millions of human fatalities emerge globally due to zoonotic diseases [3]. Over 60% of the newly emerging infectious diseases reported globally stem from zoonotic sources, and about 75% of the most recent 30 emerging human pathogens have their roots in animal populations [1]. Zoonotic infections resulting from parasite transmission are widespread and common. Parasitic zoonotic infections are transmitted through the consumption of contaminated food and water, close contact with animals, and exposure to infected soil. Despite imposing a significant global health burden on individuals and exerting substantial financial pressure on the livestock sector, most parasitic zoonoses are considered neglected diseases [4]. In India, many such parasitic zoonoses are endemic and reemerge in regular outbreaks [5, 6]. Since ancient times, swine have been implicated as hosts of zoonotic parasites, including T. gondii and Trichinella spp. [7].

T. gondii is an obligate coccidian intracellular parasite with the capability to infect a broad spectrum of warm-blooded vertebrates, including humans, and continues to be a major public health concern. Antibodies against this parasite are carried by more than one-third of the world’s human population [8, 9]. It has been reported that approximately 24% of all deaths attributed to foodborne pathogens are caused by T. gondii alone [10]. Cats serve as key definitive hosts shedding resistant oocysts in their faeces [9]. Pigs play a crucial role as an intermediate host, acquiring infections through contaminated feed, water, and rodent carcasses that may contain viable tissue cysts of T. gondii.

Trichinella spp. is a foodborne zoonotic helminth infecting human and a wide range of animals, including domestic pigs, wild boars, rats, horses, etc [11]. This disease is recognized to affect approximately ten thousand individuals annually, with a mortality rate of around 0.2% [12]. Trichinellosis holds significance not only for its zoonotic potential but also for its substantial influence on swine production, food safety and international trade [13]. Omnivores and carnivores, including those with predating, scavenging, and cannibalistic tendencies, can serve as primary reservoirs for Trichinella spp. Pigs, in particular, have a significant role as a reservoir and are a common source of human trichinellosis. As a result, human Trichinella infections are typically concentrated in regions where pork consumption is widespread [11].

In the present study, the seroprevalence of T. gondii and Trichinella spp. in pigs reared by tribal communities and smallholder farmers in five states (Arunachal Pradesh, Meghalaya, Manipur, Nagaland, and Mizoram) in northeastern India was aimed to be determined. This region houses 46.8% of the pig population of India and shares porous international borders with four countries. Furthermore, the risk factors associated with seroprevalence were analysed. The data and knowledge generated from this investigation are considered crucial for developing and implementing evidence-based surveillance systems and effective one health approach-based control programs for T. gondii and Trichinella spp. in the pig and human populations of the region. Additionally, this survey is recognized as the first, to our knowledge, to record the seroprevalence of T. gondii in backyard pigs in Northeastern India.

Materials and methods

Results

Descriptive analysis and features of the study population

An equal number of samples (n = 80) were collected from all five states in northeastern India, totalling 400 samples, covering 400 households. The mean age of the pig population was ≈11.1 months, with ages ranging from 0 to over 24 months. To provide a broad overview of the age distribution within the study population, age was categorized into four groups: 0–6 months, 7–12 months, 13–24 months, and over 24 months. A significant majority fell into the "young" category, with pigs aged 0–6 months accounting for 23.5% (94/400) of the total sample. Pigs in the 7–12 months age group represented 43.75% (175/400) of the population. Those in the 13–24 months age group constituted 22.5% (90/400) of the sample, while pigs over 24 months old made up 10.25% (41/400) of the total. The population consisted of 57.5% female pigs and 42.5% male pigs. Crossbred pigs were the most common, comprising 78.75% (315 out of 400), while exotic breeds accounted for only 2%, and local pigs constituted 19.25%. The majority of households raised fewer than 10 pigs (77.75%), while 15.5% raised between 11 and 20 pigs, and only 6.75% raised more than 20 pigs. Farm sizes were categorized as small (1 to 10), medium (11 to 20), and large (more than 20) for analysis. Fattening purposes, following the all-in-all-out system, accounted for 49% of pigs, while 32.5% were raised for fattening purposes following the continuous cycle system, and 18.5% were raised for breeding purposes to produce piglets. Out of the 400 households participating in the serological survey, only 0.5% (2 out of 400) reported a history of abortion, stillbirth, or ocular disease in the family. The majority of pigs (63.25%) were reared using the confined housing system, as opposed to pigs reared in semi-confined conditions (17.5%) and non-confined conditions (19.25%). Concrete floors were present in 55.5% (222/400) of farms, while non-concrete floors were found in 44.5% (178/400) of farms. Regular deworming of pigs and disinfection of enclosures were reported by 40% (160/400) and 62.25% (249/400) respondents, respectively. Exposure to cats was recorded in 43.75% (175 out of 400) households, exposure to rodents was recorded in 43.75% (175 out of 400) households, and exposure to birds was recorded in 64.25% (257 out of 400) households. Concerning feed sources, 64.75% (259 out of 400) of households fed their pigs with commercial and household feed, 5.75% (23 out of 400) fed their pigs exclusively with household kitchen waste, while 29.5% (118 out of 400) fed their pigs only with swill feed obtained from restaurants and bakeries. Out of the 400 households that participated in the serological survey, 22.75% (91 out of 400) did not have lavatories, while 77.25% (309 out of 400) of the sampled animals belonged to households with lavatories. Table 1 presents a summary of the different demographic characteristics and seroprevalence data.

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Table 1. Univariate analysis of associated risk factors for seropositivity of anti-T. gondii antibodies.

https://doi.org/10.1371/journal.pone.0298357.t001

Potential risk factors

Twenty variables including animal and farm characteristics, management practices and disease conditions in farmers, were examined through univariate analysis. Few variables demonstrated a significantly strong association with the occurrence of T. gondii antibodies (p ≤ 0.05). For multivariate analysis, all variables were included, revealing that four variables were significantly associated with the seroconversion outcome (p ≤ 0.05). The odds ratios (OR) with CI and p values for determining potential risk factors for T. gondii seropositivity using univariate and multivariate logistic regression models are presented in Tables 1 and 2, respectively. In univariate analysis, significant variables at the animal level included age, where animals aged between 13 to 24 months (OR = 2.24, 95% CI 1.23–4.12) and over 24 months (OR = 13.07, 95% CI 5.27–37.69) showed a significant association with T. gondii seropositivity. Additionally, local or non-descript breeds were more likely to be seropositive (OR = 1.90, 95% CI 1.15–3.17). Farm characteristics such as production methods revealed that farms operating for breeding purposes (OR = 5.60, 95% CI 3.01–11.04), were strongly associated with higher odds of T. gondii seropositivity (p < 0.0001) compared to fattening farms following all-in-all-out and continuous cycles. Farms with a history of abortion and stillbirth in pigs (OR = 1.90, 95% CI 1.12–3.25) had significantly higher odds of T. gondii infection than those without such history. Farms that purchased piglets from live markets had significantly lower odds of T. gondii infection compared to farms using piglets from their own farm (OR = 1.02, 95% CI 0.62–1.68). The final multivariate logistic regression model (Table 2) revealed two variables, namely age and exposure to cats, with a significantly stronger association with T. gondii infection. Pigs aged between 13 to 24 months (OR = 2.58, 95% CI 1.22–5.57) and pigs older than 24 months (OR = 7.20, 95% CI 2.45–23.71) demonstrated a significantly higher likelihood of T. gondii antibodies. Similarly, farms with exposure to cats (OR = 5.87, 95% CI 2.55–14.05) were significantly more likely to have T. gondii seropositivity compared to farms with no cat exposure. Univariate and multivariate analyses for Trichinella spp. infection were not conducted due to the low seropositivity rate of only 0.75% (3/400) recorded in the present study.

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Table 2. Multivariate logistic regression model for the analysis of risk factors associated with T. gondii infection in pigs.

https://doi.org/10.1371/journal.pone.0298357.t002

Discussion

In India, the seroprevalence of T. gondii has been investigated in various animals, including livestock, poultry, and captive wild species such as cattle [19, 20], chicken [21], Mithun [22], sheep [23], goats [24], as well as Asiatic lions and Royal Bengal tigers [25]. Thakur et al. [16] reported a seroprevalence of 48.3% (73/151) in pigs reared in Punjab. In a recent study, a seroprevalence of 24.7% was reported among pigs raised in various agroclimatic zones of the North Indian state of Haryana [26]. This study represents the first large-scale investigation covering five northeastern states of India. Moreover, the reported seroprevalence of T. gondii in our study (45%) falls within the range of previous reports [21, 26]. In this study, a T. gondii seroprevalence of 45% in pigs was observed. Notably, comparable results have been documented in studies employing the PrioCHECK^® ELISA kit. For instance, Basso et al. [27] reported a seroprevalence of 46.1% in pig sera in Switzerland using the PrioCHECK^® ELISA. Similarly, Macaluso et al. [28] conducted a study involving 115 naturally exposed and 12 experimentally infected pig sera, revealing a seroprevalence of 51.9% with the same kit. Additionally, a study in Italy by Pipia et al. [29] documented a T. gondii seroprevalence of 52.9% using the PrioCHECK^® ELISA. Furthermore, a study in Spain by Castillo-Cuenca et al. [30] revealed a seroprevalence of 25.9% in fattening pigs and sows. The utilization of the same ELISA kit in these findings contributes to the consistency and reliability of the reported seroprevalence rates across diverse geographical locations. Recent reports from neighboring countries of northeastern India, namely China and Myanmar, which share international boundaries with India, reveal seroprevalences of T. gondii in pigs as 20.85% [31] and 18.4% [32], respectively. In India, T. gondii is circulating among the human population, including blood donors and pregnant women, with multiple reports available in the public domain [5, 33, 34]. Additionally, three reports document the seroprevalence of T. gondii in veterinarians, slaughterhouse workers, pet keepers, and farmers in Northern India [35], Central India [36], and Northeastern India [37]. This emphasizes the significance of T. gondii as a zoonotic pathogen and highlights the occupational risks faced by animal rearers and handlers.

Similarly, reports of Trichinella spp. in the livestock and wildlife populations in India, including Northeastern India, are limited [17, 38, 39]. In Central India, Kumar et al. [17] documented a larval prevalence of 0.69% for Trichinella spp. in the diaphragms of slaughtered pigs in an abattoir. In Assam (Northeastern India), Konwar et al [38] reported a seroprevalence of 2.87% for Trichinella spp. Acheenta et al. [40] found anti-Trichinella antibodies in porcine sera from Assam, Meghalaya, Arunachal Pradesh, Mizoram, and Tripura at rates 0.27%, 0.41%, 0%, 0%, and 0%, respectively. Consistent with these earlier reports from India, the present study also revealed a seroprevalence of 0.75% for Trichinella spp. in pigs from Northeastern India. Specifically in Manipur and Arunachal Pradesh, 2.5% and 1.25% of the samples, respectively, tested positive for Trichinella antibodies. No seropositive samples were identified in Meghalaya, Mizoram, and Nagaland. Multiple human case reports of trichinellosis causing pyomyositis and osteomyelitis have been documented across India [41, 42]. In Uttarakhand, a northern state of India, multiple outbreaks of trichinellosis occurred, with 70 suspected cases and 11 mortalities reported following the consumption of undercooked or raw wild boar meat [43]. Nehra et al. [39] also described the molecular detection of T. spiralis in a deceased leopard in Uttarakhand. Therefore, although a rare zoonosis, trichinellosis poses a threat to the Indian population, especially among those who regularly consume pork and bush meat.

In this study, T. gondii seropositivity in the tested pigs was strongly correlated with certain risk factors. Significantly higher seropositivity was observed in pigs sampled from three states, namely Nagaland (OR 16.23, 95% CI 7.66–36.65), Mizoram (OR 5.17, 95% CI 2.63–10.50), and Meghalaya (OR 2.68, 95% CI 1.36–5.40). Similar regional variations were noted in a prior study in Spain, possibly influenced by factors such as climate and cultural practices [44]. Age emerged as a significant factor in seropositivity, with pigs aged between 13–24 months (OR 2.24, 95% CI 1.23–4.12) and those above 24 months (OR 13.07, 95% CI 5.27–37.69) showing a higher likelihood of seropositivity. Similar age-related trends have been observed in earlier studies, indicating prolonged exposure to oocysts and carcasses of other animals and rodents in older pigs [45–48]. Farms maintaining local or non-descript pig breeds (OR 1.90, 95% CI 1.15–3.17) exhibited higher seropositivity compared to farms maintaining crossbred and exotic pigs. This difference might be attributed to better hygienic management practices and disinfection on farms with improved breeds. Additionally, farms operating for breeding purposes (OR 5.60, 95% CI 3.01–11.04) were significantly associated with high seropositivity, aligning with similar observations in earlier studies where breeding farms and farms not following an all-in-all-out production system displayed higher T. gondii seroprevalence [44, 49]. Although not statistically significant, higher seropositivity was noticed among sows compared to boars, a common scenario in breeding farms. This could be attributed to increased age-related contact and the lifelong persistence of antibodies [44, 47]. Farms with a history of abortion and stillbirth (OR 1.90, 95% CI 1.12–3.25) exhibited a statistically significant correlation with T. gondii seropositivity, likely due to the persistence of T. gondii oocysts in the farm premises. In the final multivariate regression model, T. gondii seroprevalence was strongly associated with age and exposure to cats. The presence of cats in farm premises has consistently been identified as a significant risk factor in numerous previous studies [44, 50, 51]. Given that cats are the definitive hosts for T. gondii and can shed over 20 million oocysts during a three-week infection cycle, they substantially contribute to the environmental contamination of pig farms [48].

The present study has a few limitations. Notably, genetic characterization or diversity analysis of T. gondii was not conducted, as the haplotypes circulating in the Indian livestock population remains unknown. Additionally, the limited number of serum samples possessing Trichinella spp. antibodies (n = 3) hindered the ability to conduct risk factor analysis using univariate and multivariate logistic regression. Addressing these limitations would require a larger sample size, and these considerations should guide future studies.

Conclusions

This study offers detailed insight into the seroepidemiological status of T. gondii and Trichinella spp. in pigs in Northeastern India. While toxoplasmosis can be considered highly prevalent, Trichinella spp. infection was found to be of low prevalence (below 1%). Significant risk factors associated with the seroprevalence of T. gondii included age above 24 months, exposure to cats, and farms operating for breeding purposes. The high prevalence of toxoplasmosis in pigs may play a crucial role in disease transmission to humans, particularly among farmers, abattoir workers, and pork consumers through direct and indirect contact with infected pigs and meat. Advocating for public education and awareness campaigns regarding the risks of toxoplasmosis and preventive measures is crucial for preventing and controlling the spread of the disease. Generating baseline data on the seroprevalence of neglected zoonotic diseases can be helpful to livestock, human, and environmental health authorities. In the One Health era, knowledge sharing between sectors becomes paramount to curbing the burden of emerging infectious diseases through informed planning and evidence-based programmes. The study’s coverage of the pig population in five Northeastern Indian states, sharing porous international boundaries with China, Myanmar, Bhutan, and Bangladesh, provides findings that may be valuable to their authorities, given the porous nature of the borders and the common clandestine movement of pigs and pig products.

Supporting information

Acknowledgments

The authors acknowledge ICAR Research Complex for NEH Region, Meghalaya for providing the facilities, and support for the successful completion of this study.