Human-Related Microsporidian Spores in Farm Chickens from Makeni, Sierra Leone ^†

Abstract

The aim of this paper is to study the presence and temporal variation/distribution of microsporidian species (Enterocytozoon bieneusi and Encephalitozoon spp.) in chickens from different farms in Bombali district (Sierra Leone). Fresh faecal samples were collected from twenty chickens in April 2019, while twelve were collected in Summer 2022. Six faecal samples gave positive results microscopically (spores of Encephalitozoon spp. were observed in five samples, and E. bieneusi in one). Five samples from 2019 were found to be positive by molecular methods: two E. intestinalis and E. hellem, and one E. bieneusi; meanwhile, ten samples from 2022 were positive for spores of the genus Encephalitozoon, specifically from eight chicken.

1. Introduction

Sierra Leone (SL) is facing a number of significant health challenges that demand urgent attention to address its high mortality and morbidity rates, which are the highest globally for maternal and infant groups [1]. Consequently, a series of interventions have been initiated to enhance research and teaching capabilities in various universities in Sierra Leone, since 2019 [2]. These interventions aim to understand and respond to major and emerging human parasites. Thus, in collaboration with the University of Makeni (UniMak, SL), we have detected, for the first time, the presence of microsporidia, an emerging group of enteric opportunistic zoonotic human parasites, in animal faeces and various water environments in Bombali District, one of the largest districts in Sierra Leone. Specifically, we have molecularly detected Enterocytozoon bieneusi and the Encephalitozoon species of E. intestinalis, E. hellem and E. cuniculi in a range of different farm and domestic animals (dog) [3,4]. Furthermore, E. intestinalis has been identified in drinking and open water systems collected from various public spaces throughout this district, including the city of Makeni [5]. This identification was made during a pilot project conducted in April 2019, with the aim of establishing collaboration and enhancing medical parasitology capacity at UniMak.

Microsporidia are a ubiquitous group of obligate intracellular parasites that can infect both vertebrates and invertebrates. To date, seventeen microsporidian species are recognised as human pathogens [6], with E. bieneusi being the most prevalent microsporidian affecting humans, followed by E. intestinalis. The clinical manifestations of microsporidiosis vary, ranging from gastrointestinal disorders and chronic diarrhoea to diffused systemic infections. These conditions can affect both immunocompetent and immunosuppressed individuals [6,7]. The epidemiology of microsporidian infections remains to be elucidated, but most transmissions occur through the oral–faecal route, primarily from contaminated water and food. Furthermore, mounting evidence points to the zoonotic potential of these species [7], as substantiated by the identification of identical genotypes in both humans and animals [8]. Consequently, the presence of these microsporidian species in drinking water and public water bodies, along with their preliminary detection in farm animals from Bombali District, may contribute to the high levels of diarrhoea recorded in Sierra Leone, which is the leading cause of death in this country [1].

Following the preliminary identification of these emerging opportunistic zoonotic parasites in Makeni and the successful capacity building in medical parasitology at UniMak, our multidisciplinary team was provided with further funds from De Montfort University (DMU, UK)’s Quality Research–Global Challenges Research Fund (QR GCRF) to strengthen medical parasitology at the Ernest Bai Koroma University of Science and Technology (EBKUST) and to study whether the presence and circulation of the microsporidian species detected in farm animals would represent a potential risk for the food chain and human health [9]. Thus, the primary objective of this study was to further examine the presence and temporal variation/distribution of the predominant human-related microsporidia species in chickens from various farms in Makeni city and its surroundings (Bombali District, SL).

2. Material and Methods

The present study was conducted with no requirement for ethical approval. The study did not include endangered or protected species. The procurement of faecal samples was authorised by the land and animal’s owners. The acquisition of faecal samples did not result in any harm to animals.

The two monitoring studies were performed as follows: Fresh faecal samples, apparently normal and with no signs of intestinal disorders, were collected from twenty chickens (Gallus gallus) from Lion Poultry, a chicken farm on the outskirts of Makeni, in April 2019, using a sterile spatula and gloves, and placed into labelled sterile Falcon tubes, following the methods previously described by Galván-Díaz et al. [7]. In a similar manner, twelve additional faecal samples from chickens were collected in Summer 2022, from Lion Poultry and other private family farms across Bombali District, in conjunction with 25 water, 20 food and 3 leaf samples that the chickens were using for eating/drinking, following similar methods.

Faecal samples collected in 2019 were microscopically screened for microsporidia spores at a magnification of 1000×, following staining of thin smears with a chromotropic stain (chromotrope 2R), Fast Green and phosphotungstic acid (all from Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) in accordance with the procedure described by Weber et al. [10]. Samples that exhibited spore-compatible structures, ovoid and refractile structures, stained pink-red, were considered positive [8]. Furthermore, all 2019 samples were molecularly processed for the simultaneous detection of E. bieneusi and Encephalitozoon spp. [E. intestinalis, E. hellem and E. cuniculi] after the removal of potential inhibitors by washing faecal samples with phosphate-buffered saline solution with ethylenediaminetetraacetic acid (PBS-EDTA) and centrifuging them at 2500 rpm for 15 min [11]. DNA was then extracted from each sample using with Fast-Prep for Soil^® kit (MP Biomedicals, Solon, OH, USA), following the manufacturer’s instructions, and the homogeniser FastPrep-24^TM 5G (MP Biomedicals, Solon, OH, USA), as described by Da Silva et al. [12]. The QIAquick PCR kit (QIAGEN, Chastsworth, CA, USA) was then utilised to remove any residual polymerase chain reaction (PCR) inhibitors, followed by SYBR Green real-time PCR according to the methods described by Polley et al. [13] and modified by Andreu-Ballester et al. [14]. This molecular assay partially amplifies the small subunit ribosomal ribonucleic acid (SSU-rRNA), allowing it to distinguish microsporidian species based on the melting temperature (Tm) of the amplicons, as follows [14]: 82.85–83.9 °C E. hellem/E. intestinalis, 84.45 ± 0.4 °C E. cuniculi, and 82.35 ± 0.4 °C E. bieneusi. Appropriate positive controls for E. bieneusi, E. intestinalis and E. cuniculi were also utilised in all the essays as previously indicated in Peña-Fernández et al. [15].

Our group has started to process the most recent samples collected in 2022. To date, all 2022 samples have undergone appropriate concentration and pretreatment processes prior to testing for Encephalitozoon species using an Immunofluorescence Antibody Test (IFAT). This test employs a specific monoclonal antibody (Mab) of murine origin, IgG2a, which has been patented as a diagnostic tool by the Parasitology group at the University of San Pablo CEU (Spain) [16]. This monoclonal antibody, designated as MAb 2C2, has been shown to recognise the wall of the exospore of the Encephalitozoon species as well as the developing stages of the parasite [17]. The slides were prepared by adding this antibody to each well (imprint) and incubating at 37 °C for a period of 60 min in a wet chamber. Subsequent to this, slides were washed three times in distilled water and then were air-dried at room temperature. The slides were then subjected to a second incubation with a secondary antibody, conjugated with fluorescein isothiocyanate (Sigma, St. Louis, MO, USA), at 37 °C for a duration of 60 min. This was followed by three washes in deionised water and subsequent air-drying at room temperature in the dark, as previously described by Redondo et al. [17]. The examination of the slides was conducted at a magnification of 40× using a Nikon immunofluorescent microscope. Positive controls were also included.

3. Results and Discussion

Six faecal samples yielded positive results microscopically (spores of Encephalitozoon spp. were observed in five samples, and E. bieneusi in one). Four of these samples were subsequently confirmed through molecular means, although five exhibited positive results for the following species: two for E. intestinalis and E. hellem, and one for E. bieneusi (Figure 1A,B). Meanwhile, five samples were found to be molecularly positive.

To the best of our knowledge, this is the first report of the presence of emerging human-related microsporidian species in farm chickens in Bombali District, which could highlight a potential risk to human health. Previous reports have found a specific E. bieneusi ribosomal internal transcribed spacer (ITS) genotype “J” in chickens, for which the lack of a transmission barrier to humans has been suggested [18]. Similar studies in free-range chickens living on small farms with other animals, including cattle, pigs and dogs, have shown the presence of human pathogenic genotypes of E. bieneusi in their faeces [19]. Despite the absence of E. cuniculi in the 2019 samples examined, further PCR-based studies utilising a more extensive sample size are needed, given the documented presence of this species in chickens raised in both domestic and commercial farms across various locations in Behera province, Egypt [20]. The prevalences detected in this study are likely to be similar or higher than those reported in similar studies conducted on chickens [21].

Furthermore, the circulation of these emerging pathogens in the monitored area is supported by our observations on our most recent samples, as ten 2022 samples were positive for spores of the genus Encephalitozoon, specifically eight from chickens and two from water (Figure 1C,D). It is noteworthy that none of the food samples were found to be positive, although it is important to consider that our results are preliminary. This is due to the fact that recent reviews have reported the presence of human-related microsporidia spores in various food products, including vegetables [22]. Moreover, structures that are compatible with Encephalitozoon spp. were observed in one chicken sample and three water samples. These findings serve to confirm our previous screenings of drinking water samples in Makeni city and its surroundings. The identification of spores of the genus Encephalitozoon in water suggests it can play a role in the transmission of this parasite for both animals and humans [19]. Microsporidia have been described in surface water and can survive in water up to a year at low temperatures [23]. The spores of these parasites are highly resistant to external environmental conditions and to many physical and chemical disinfection methods, which are traditionally used in drinking water treatment plants, swimming pools, and irrigation systems [24]. Thus, human pathogenic microsporidian spores have also been isolated in influent water samples from drinking water treatment plants (DWTPs) [25], indicating the potential role of water in the transmission of these parasites.

The results also suggest a moderate circulation of Encephalitozoon spp. in chickens and farms, which could represent a risk for Sierra Leonean individuals living in close proximity to these farms or a food risk through consumption of contaminated water and/or these farm animals. Previous reports have suggested that E. cuniculi may be transmitted naturally to the chicken egg, and the chicken production units could be easily contaminated with this species due to the inapparent infections that they can cause in chickens [26]. Studies reporting on the humoral response in chickens infected with E. hellem, one of the common human microsporidian species, have shown that the birds were infected with a moderate course of infection and all animals remained asymptomatic [27].

Although the zoonotic transfer of microsporidia from farm animals to humans is rare, the implementation of appropriate cleaning and food processing protocols at farms is required, due to the high presence of microsporidia spores found, in conjunction with hygiene awareness with regard to the processing of poultry meat, particularly for sensitive population such as Ebola survivors. Our recommendation aligns with those recently outlined following comprehensive systematic reviews, which call for the formulation of suitable strategies for the control and prevention of microsporidia infections in birds, with a view to safeguarding human health [28].

The presence of Encephalitozoon spp. in the water supply on the monitored farms could be indicative of environmental contamination by their spores via sewage from poultry farms or slaughterhouses. Further investigation is recommended to prevent the spread of the contamination due to the small size of microsporidian spores in surface and ground water or the food chain.

4. Conclusions

To the best of our knowledge, this is the first study reporting the presence and circulation of E. bieneusi and Encephalitozoon species (E. intestinalis, E. hellem) in chickens and water in Makeni city and its surroundings. The moderate circulation of Encephalitozoon spp. in chickens and on farms could pose a risk to individuals residing in close proximity to these farms, or a food safety risk through the consumption of contaminated water, chicken meat, or chicken eggs. This underscores the necessity for effective management of poultry waste, in conjunction with heightened food hygiene awareness, particularly in view of the high prevalence of immunocompromised individuals living in Sierra Leone.