Introduction
Entamoeba histolytica is an enteric protozoan parasite that causes the disease amebiasis, the second leading cause of death from intestinal parasitic disease worldwide, following cryptosporidiosis (Ali, Reference Ali2015). The exact extent of morbidity and mortality is currently a point of contention; however, estimates suggest that it infects approximately 50 million people worldwide, killing approximately 40 000–100 000 people annually (Inam et al., Reference Inam, Mittal, Rajala, Avecilla and Azam2016). In fact, in 2010, amebiasis was responsible for 55 500 deaths worldwide (Lozano et al., Reference Lozano, Naghavi, Foreman, Lim, Shibuya, Aboyans, Abraham, Adair, Aggarwal, Ahn, Alvarado, Anderson, Anderson, Andrews, Atkinson, Baddour, Barker-Collo, Bartels, Bell, Benjamin, Bennett, Bhalla, Bikbov, Abdulhak, Bin Birbeck, Blyth, Bolliger, Boufous, Bucello, Burch, Burney, Carapetis, Chen, Chou, Chugh, Coffeng, Colan, Colquhoun, Colson, Condon, Connor, Cooper, Corriere, Cortinovis, De Vaccaro, Couser, Cowie, Criqui, Cross, Dabhadkar, Dahodwala, De Leo, Degenhardt, Delossantos, Denenberg, Des Jarlais, Dharmaratne, Dorsey, Driscoll, Duber, Ebel, Erwin, Espindola, Ezzati, Feigin, Flaxman, Forouzanfar, Fowkes, Franklin, Fransen, Freeman, Gabriel, Gakidou, Gaspari, Gillum, Gonzalez-Medina, Halasa, Haring, Harrison, Havmoeller, Hay, Hoen, Hotez, Hoy, Jacobsen, James, Jasrasaria, Jayaraman, Johns, Karthikeyan, Kassebaum, Keren, Khoo, Knowlton, Kobusingye, Koranteng, Krishnamurthi, Lipnick, Lipshultz, Ohno, Mabweijano, MacIntyre, Mallinger, March, Marks, Marks, Matsumori, Matzopoulos, Mayosi, McAnulty, McDermott, McGrath, Memish, Mensah, Merriman, Michaud, Miller, Miller, Mock, Mocumbi, Mokdad, Moran, Mulholland, Nair, Naldi, Narayan, Nasseri, Norman, O'Donnell, Omer, Ortblad, Osborne, Ozgediz, Pahari, Pandian, Rivero, Padilla, Perez-Ruiz, Perico, Phillips, Pierce, Pope, Porrini, Pourmalek, Raju, Ranganathan, Rehm, Rein, Remuzzi, Rivara, Roberts, De León, Rosenfeld, Rushton, Sacco, Salomon, Sampson, Sanman, Schwebel, Segui-Gomez, Shepard, Singh, Singleton, Sliwa, Smith, Steer, Taylor, Thomas, Tleyjeh, Towbin, Truelsen, Undurraga, Venketasubramanian, Vijayakumar, Vos, Wagner, Wang, Wang, Watt, Weinstock, Weintraub, Wilkinson, Woolf, Wulf, Yeh, Yip, Zabetian, Zheng, Lopez and Murray2012). Infection occurs through fecal-oral transmission, wherein the mature cyst of E. histolytica from fecal-contaminated food or water is ingested. Entamoeba histolytica infection is particularly problematic in developing nations due to less capacity for sanitation and hygiene practices (Ashbolt, Reference Ashbolt2004).
There are more than 20 species of Entamoeba, with varying pathogenic potential and host specificity (Nozaki and Bhattacharya, Reference Nozaki and Bhattacharya2015). Unfortunately, many of these species of Entamoeba are morphologically indistinguishable from E. histolytica, including the potentially-pathogenic but rare Entamoeba nuttalli (Levecke et al., Reference Levecke, Dorny, Vercammen, Visser, Van Esbroeck, Vercruysse and Verweij2015), as well as several nonpathogenic species of Entamoeba, including Entamoeba dispar, Entamoeba moshkovskii and Entamoeba bangladeshi (Nozaki and Bhattacharya, Reference Nozaki and Bhattacharya2015; Jirku-Pomajbíková et al., Reference Jirku-Pomajbíková, Čepička, Kalousová, Jirku, Stewart, Levecke, Modrý, Piel and Petrželková2016). Consequently, molecular diagnostics are required to differentiate these species. Prevention and control of amebiasis is further complicated by the zoonotic potential of E. histolytica, which is known to infect both human and captive nonhuman primates (NHPs) (Rivera et al., Reference Rivera, Yason and Adao2010). Entamoeba histolytica is of more urgent global health concern as a neglected tropical disease (Ximénez et al., Reference Ximénez, Morán, Rojas, Valadez, Gómez, Ramiro, Cerritos, González, Hernández and Oswaldo2011). Importantly, although E. nuttalli is genetically similar to E. histolytica (Tachibana et al., Reference Tachibana, Yanagi, Pandey, Cheng, Kobayashi, Sherchand and Kanbara2007), it cannot be detected by polymer chain reaction (PCR) based on the gene targeted in the current study (ribosomal small subunit) (Levecke et al., Reference Levecke, Dorny, Vercammen, Visser, Van Esbroeck, Vercruysse and Verweij2015). Despite the clinical significance of E. histolytica, few studies have been conducted on the risks zoonotic transmission pose to public health (Thompson and Smith, Reference Thompson and Smith2011).
Understanding the risk of zoonotic disease transmission is crucial to both human and animal health, especially in systems characterized by high rates of human-animal overlap (Pedersen et al., Reference Pedersen, Altizer, Poss, Cunningham and Nunn2005; Bowden and Drake, Reference Bowden and Drake2013). Anthropogenic habitat change causes humans and NHPs to come into closer and more frequent contact, which leads to an increase in the risk of zoonotic disease transmission (Gillespie et al., Reference Gillespie, Nunn and Leendertz2008; Nunn and Gillespie, Reference Nunn, Gillespie, Wich and Marshall2016). Gombe National Park, Tanzania is home to seven species of NHPs, including endangered eastern chimpanzees (Pan troglodytes schweinfurthii) and olive baboons (Papio anubis) (Gillespie et al., Reference Gillespie, Lonsdorf, Canfield, Meyer, Nadler, Raphael, Pusey, Pond, Pauley, Mlengeya and Travis2010). Many primates are habituated to human presence and live in close proximity to humans, thus human–primate interaction – through means such as primate crop-raiding, shared water sources and human presence in primate habitat – is common (Parsons et al., Reference Parsons, Travis, Lonsdorf, Lipende, Roellig, Kamenya, Zhang, Xiao and Gillespie2015). Additionally, habituation for research and tourism provides opportunities for primates to be exposed to new diseases (Pusey et al., Reference Pusey, Wilson and Anthony Collins2008; Gilardi et al., Reference Gilardi, Gillespie, Leendertz, Macfie, Travis, Whittier and Williamson2015). As Entamoeba spp. have been identified in chimpanzees and baboons, there is a need to investigate whether it is possible for pathogenic species of Entamoeba to transfer to humans in the Greater Gombe Ecosystem (GGE) (Gillespie et al., Reference Gillespie, Lonsdorf, Canfield, Meyer, Nadler, Raphael, Pusey, Pond, Pauley, Mlengeya and Travis2010; Howells et al., Reference Howells, Pruetz and Gillespie2011).
Gombe National Park, established in 1968, is a small (35 km2) forest reserve located on a narrow strip of land between Lake Tanganyika and a rift escarpment that rises from the lakeshore (Pusey et al., Reference Pusey, Wilson and Anthony Collins2008). Since the establishment of the national park following Jane Goodall's research on chimpanzees in 1960 (Goodall, Reference Goodall1986), the woody vegetation and forest cover have increased inside the park (Pusey et al., Reference Pusey, Wilson and Anthony Collins2008). However, rapid human population growth and the dependence of the Tanzanian economy on agriculture, has resulted in deforestation for conversion to farmland in the GGE (Pusey et al., Reference Pusey, Wilson and Anthony Collins2008). Furthermore, desertification and soil degradation from droughts have caused natural resources to decline, leading to fragmented landscapes that increase human–wildlife contact (Parsons et al., Reference Parsons, Gillespie, Lonsdorf, Travis, Lipende, Gilagiza, Kamenya, Pintea and Vazquez-Prokopec2014).
The wild chimpanzee population in Gombe National Park has been studied continuously for over 50 years and bolsters the national economy of Tanzania through tourism (Pusey et al., Reference Pusey, Wilson and Anthony Collins2008). There are three chimpanzee communities: Mitumba, Kasekela and Kalande (Pusey et al., Reference Pusey, Pintea, Wilson, Kamenya and Goodall2007). This study focused on the two habituated communities of chimpanzees that experience different degrees of human encroachment (Pusey et al., Reference Pusey, Pintea, Wilson, Kamenya and Goodall2007). Mitumba, the smaller northern community, is located in proximity to Mwamgongo, a village of approximately 5000 humans and their livestock. Whereas, Kasekela, the larger central chimpanzee community, is located in the less disturbed forest (Parsons et al., Reference Parsons, Travis, Lonsdorf, Lipende, Roellig, Kamenya, Zhang, Xiao and Gillespie2015). Researchers, tourists, park management staff and local field assistants are the only humans allowed to reside inside the park, but the park border is not fenced; therefore, local villagers and their animals have access to the park (Parsons et al., Reference Parsons, Travis, Lonsdorf, Lipende, Roellig, Kamenya, Zhang, Xiao and Gillespie2015). Unlike the densely populated northern and southern borders of the park, the eastern border is less settled because of the high elevation and soil depletion (Pusey et al., Reference Pusey, Pintea, Wilson, Kamenya and Goodall2007). Therefore, the GGE provides a unique setting to study disease transmission among a dense human population and the NHPs they encounter, both directly and indirectly.
Entamoeba histolytica has been identified microscopically in Gombe (Gillespie et al., Reference Gillespie, Lonsdorf, Canfield, Meyer, Nadler, Raphael, Pusey, Pond, Pauley, Mlengeya and Travis2010) but the morphological similarity to other Entamoeba species mandates the molecular confirmation of E. histolytica. While fewer than 10% of those infected with E. histolytica develop invasive amebiasis (Wilson et al., Reference Wilson, Weedall and Hall2012), infection with this intestinal parasite in humans may cause diarrhoea, haemorrhagic dysentery, liver abscesses and death (Ali et al., Reference Ali, Clark and Petri2008). Invasive amebiasis has also been observed in NHPs through necropsy and studies have shown that E. histolytica infection in NHPs mimics human infection (Haq et al., Reference Haq, Sharma, Ahmad, Khan and Khan1985; Verweij et al., Reference Verweij, Vermeer, Brienen, Blotkamp, Laeijendecker, van Lieshout and Polderman2003). Clinical complications of E. histolytica infection for both humans and NHPs may cause detrimental impacts on the human population as well as the endangered chimpanzee population.
The present study aimed to (1) quantify the presence of E. histolytica in a system with human-NHP overlap, and (2) test associations between infection status in humans, chimpanzees and baboons and potential risk factors of enteric disease. We predicted that these primate species would have similar sample prevalence of E. histolytica given that they frequently come into contact, potentially acting as reservoirs for one another. Specifically, we hypothesized that the sample prevalence of E. histolytica would be higher in the Mitumba chimpanzee community, than in Kasekela, because of the natural border it shares with Mwamgongo. To investigate the potential of zoonotic transmission of E. histolytica in this system, we examined patterns of infection with the parasite and assessed risk factors for infection among humans, chimpanzees and baboons in the GGE, Tanzania.
Materials and methods
Study site and sample collection
This study was conducted in the GGE, Kigoma District, Tanzania. Specifically, in Gombe National Park (4°40′S, 29°38′E) and the village of Mwamgongo (4°40′S, 29°34′60′E). Fecal samples were collected between March 2010 and February 2011. Human, chimpanzee and baboon paired fecal samples were collected during the dry (July 1–August 15) and wet (November 1–December 15) seasons. Residents of Mwamgongo (estimated population size ~5000) and residents of Gombe (~100) comprised the human subject pool. Human subjects were chosen by methods described in complementary projects (Parsons et al., Reference Parsons, Gillespie, Lonsdorf, Travis, Lipende, Gilagiza, Kamenya, Pintea and Vazquez-Prokopec2014, Reference Parsons, Travis, Lonsdorf, Lipende, Roellig, Kamenya, Zhang, Xiao and Gillespie2015). Residents of the park consist of Tanzania National Park staff (TANAPA), Jane Goodall Institute (JGI) researchers and members of their families who reside at the park (Mitumba) or JGI (Kasekela). As part of a routine observational monitoring program (Lonsdorf et al., Reference Lonsdorf, Gillespie, Wolf, Lipende, Raphael, Bakuza, Murray, Wilson, Kamenya, Mjungu, Collins, Gilby, Stanton, Terio, Barbian, Li, Ramirez, Krupnick, Seidl, Goodall, Hahn, Pusey and Travis2018), chimpanzees were sampled in Mitumba (71 samples from 27 individuals) and Kasekela (170 samples from 58 individuals) communities at quarterly intervals. Baboons (79 samples from 46 individuals) were opportunistically sampled during the two collection periods.
Consenting human participants received specimen cups and instructions on how to collect the sample. Chimpanzee and baboon specimens were non-invasively collected from identified individuals immediately after defecation and transferred to a screw cap plastic vial containing a 2.5% potassium dichromate solution (Fisher Scientific, Pittsburgh, PA). For chimpanzee and baboon samples, care was taken to avoid contamination from the ground by transferring only the interior and topmost portion of feces to the vial using a sterile wooden spatula or swab and avoiding the collection of soil, foliage, or water contaminants. Each vial was labelled with a unique identification number and date of collection. Information such as observer name, location and the animal name was also recorded. Samples were sealed with Parafilm (Pechiney Plastic Packaging, Chicago, IL), stored at 4 °C and shipped in ice to Atlanta, GA, USA.
DNA extraction and molecular detection
Nucleic acid was extracted from 587 human, chimpanzee and baboon (267, 241 and 79, respectively) fecal samples preserved in 2.5% potassium dichromate solution using the FastDNA® SPIN Kit for Soil (MP Biomedicals, LLC, Solon, OH) following the protocol outlined in da Silva et al. (Reference da Silva, Bornay-Llinares, Moura, Slemenda, Tuttle and Pieniazek1999). DNA extracts were subsequently tested using a polymerase chain reaction (PCR) assay adapted from Foo et al. (Reference Foo, Chan, See Too, Tan, Wong, Lalitha and Lim2012). First, a segment (~748 bp) of the Entamoeba spp. small subunit ribosomal ribonucleic acid (SSU-rRNA) gene was amplified by PCR. For specimens positive for Entamoeba spp., a segment (~301 bp) of E. histolytica SSU-rRNA gene was also amplified by PCR. PCR was performed by addition of 2 µL DNA template to a tube containing 18 µL of mastermix (10 µL of Taq DNA Polymerase (QIAGEN, Germantown, MD), 6 µL of distilled water and 1 µL of each primer). Appropriate positive and negative controls were used for all analyses. For detection of Entamoeba spp., Entamoeba spp. forward and Entamoeba common reverse primers were used. For detection of E. histolytica, E. histolytica forward conserved and Entamoeba common reverse primers were used. All primers used are from Foo et al. (Reference Foo, Chan, See Too, Tan, Wong, Lalitha and Lim2012). PCR amplification cycles were performed in an Eppendorf Mastercycler pro (Hamburg, Germany) thermal cycler. PCR amplification cycles for Entamoeba spp. and E. histolytica, independently, were optimized from those described (Foo et al., Reference Foo, Chan, See Too, Tan, Wong, Lalitha and Lim2012). For Entamoeba spp., thermal cycler settings were 95 °C for 5 min; 35 cycles of 95 °C for 30 s, 55.8 °C for 30 s and 72 °C for 30 s; 72 °C for 10 min, and 4 °C ∞. For E. histolytica., thermal cycler settings were 95 °C for 5 min; 35 cycles of 95 °C for 30 s, 52.1 °C for 30 s and 72 °C for 30 s; 72 °C for 10 min and 4 °C ∞. The amplified PCR products were resolved by 1.0% agarose gel electrophoresis stained with Invitrogen™ UltraPure™ Ethidium Bromide (Fisher Scientific, Pittsburgh, PA) and run for 30 min at 80 v. DNA bands were visualized under UV illumination and photographed using a Molecular Imager Gel Doc™ XR System (BIO-RAD, Hercules, CA).
Human risk factor survey and NHP health-monitoring program
A cross-sectional survey was administered by trained local field assistants in the national language (Swahili) to minimize response bias. Human subjects were selected by methods described in complementary projects (Parsons et al., Reference Parsons, Gillespie, Lonsdorf, Travis, Lipende, Gilagiza, Kamenya, Pintea and Vazquez-Prokopec2014, Reference Parsons, Travis, Lonsdorf, Lipende, Roellig, Kamenya, Zhang, Xiao and Gillespie2015) and enrollment was facilitated by a health officer. Each human subject was surveyed on topics related to acquiring enteric diseases. Data were first recorded on paper forms and then entered into spreadsheets in Microsoft Excel (Redmond, WA).
Chimpanzee and baboon behavioural and observational data were provided by the Gombe Ecosystem Health Program (Lonsdorf et al., Reference Lonsdorf, Gillespie, Wolf, Lipende, Raphael, Bakuza, Murray, Wilson, Kamenya, Mjungu, Collins, Gilby, Stanton, Terio, Barbian, Li, Ramirez, Krupnick, Seidl, Goodall, Hahn, Pusey and Travis2018). Continuous behavioural and observational data, such as primate behaviour and fecal condition, were non-invasively collected. The long-term behavioural record of these primates allows for the inclusion of demographic data such as age and sex.
Statistical analyses and control for sample bias
PCR results were manually recorded and entered into Microsoft Excel (Redmond, WA). Chimpanzee and baboon individuals can have uneven sampling because of the degree of habituation, distance of travel after defecation, or other factors, thus we calculated sample prevalence using the number of individuals instead of the number of samples (Gillespie et al., Reference Gillespie, Lonsdorf, Canfield, Meyer, Nadler, Raphael, Pusey, Pond, Pauley, Mlengeya and Travis2010). To control for sample bias, we calculated sample prevalence as the proportion of individuals in each group positive for E. histolytica divided by the total number of individuals in each group examined. If a sample was negative for Entamoeba spp., the sample was considered negative for E. histolytica. If any single individual sample was confirmed positive for E. histolytica, the subject was considered positive for the collection period.
Statistical analyses were performed in SAS 9.4 (SAS Institute Inc., Cary, NC). Associations between human survey responses and infection status were compared using logistic regression and odds ratios (OR) with 95% confidence intervals (CI) were calculated. A generalized estimating equation (GEE) with exchangeable working correlation structure was used to account for repeat sampling of individuals. Associations between available chimpanzee and baboon demographic and observational health data and infection status were also compared using the same statistical methods.
Results
In total, 587 fecal samples were screened for Entamoeba spp., including 267 human, 241 chimpanzee and 79 baboon specimens (Table 1); representing approximately 2% of Mwamgongo, 94% of park residents, 100% of Mitumba chimpanzee community, 89% of Kasekela chimpanzee community and 23% of all baboons. Overall, Entamoeba spp. (including E. histolytica) were detected by PCR from 389 (66.3%) fecal samples and E. histolytica was detected by PCR from 69 (11.8%) fecal samples. Fecal samples represent all samples collected, which include paired fecal samples collected during the dry and wet seasons.
Detection of Entamoeba spp. and E. histolytica in three primate species (total samples) in and around Gombe National Park, Tanzania
a Some individuals have repeat sampling due to collection during both wet and dry seasons.
More than half of the samples collected from each primate species were positive for Entamoeba spp. (Table 1). Approximately 60% of human samples screened were positive for Entamoeba spp. One-hundred of the 152 Mwamgongo samples (65.8%) and 61 of the 115 park resident samples (53%) were positive for Entamoeba spp. For all chimpanzees sampled, 65.6% of samples were positive for Entamoeba spp., 71.2% of Kasekela chimpanzees and 52.1% of Mitumba chimpanzees. Almost 90% of baboon samples were positive for Entamoeba spp.
Sample prevalence, representing individual infection, was calculated for individuals confirmed positive for E. histolytica (Table 2). The sample prevalence of E. histolytica was higher in chimpanzees (34.1%) than humans (12.1%) or baboons (10.9%). Of the 23 detections of E. histolytica in humans, 14 (60.9%) resided in Mwamgongo and nine (39.1%) lived inside of the park (seven in Kasekela camp and two in Mitumba camp). Humans living in Kasekela camp had more positive individuals (7/61) than Mitumba camp (2/33). Similarly, Kasekela chimpanzees had more positive individuals (21/58) compared with Mitumba chimpanzees (8/27).
Sample prevalence of E. histolytica detected by species and location in and around Gombe National Park, Tanzania
Chimpanzees had a significantly higher sample prevalence among individuals positive for E. histolytica than either humans and baboons (Fisher's exact test P = 0.0326 and P = 0.0369, respectively). No significant difference in sample prevalence of positive individuals was observed between humans and baboons (Fisher's exact test P = 0.4989) or between the two chimpanzee communities (Fisher's exact test P = 0.4433). There were also no significant differences in sample prevalence observed between humans living inside and outside the park (Fisher's exact test P = 0.8337) or between the two camps located inside the park (Fisher's exact test P = 0.4907).
Data from the cross-sectional survey were used to identify potential risk factors for E. histolytica infection. Logistic regression models were specified to include possible confounding variables within the survey results. After checking for confounding effects through comparisons of unadjusted and adjusted ORs among all the variables, age and sex were controlled for in the models which had more than a 10% difference between unadjusted and adjusted ORs. Therefore, adjusted ORs are reported where applicable (Table 3). Persons who experienced gastrointestinal symptoms had approximately twice the odds of E. histolytica infection when controlling for age and sex (OR = 2.2723; 95% CI 1.0318–5.0043; P = 0.0416; Table 3). When reviewing survey data for E. histolytica positive persons, 7/23 reported cramping; four reported having diarrhea; one sought treatment at the village clinic (Metronidazole); and three had a watery or bloody stool. Seven of the 23 individuals positive for E. histolytica lived in a household with at least one other E. histolytica positive person. Chimpanzee and baboon demographic factors such as age and sex were not risk factors for E. histolytica infection in this sample (Tables 4 and 5). Diarrhea was also not a reliable predictor of E. histolytica infection in chimpanzees (Table 4). No significant association with the season was observed in humans, chimpanzees, or baboons (Tables 3–5). Human samples showed a smaller amount of infected samples during the wet season (36%); whereas, chimpanzee and baboon samples contained a higher amount of infection during the wet season (72% and 80%, respectively).
Risk factors for E. histolytica infection in humans living in and around Gombe National Park, Tanzania
* Significant at α = 0.05.
a Controlling for age.
b Controlling for age and sex.
c Controlling for sex.
Risk factors for E. histolytica infection in chimpanzees in Gombe National Park, Tanzania
Risk factors for E. histolytica infection in baboons in Gombe National Park, Tanzania
Discussion
This was the first study to molecularly confirm the presence of E. histolytica in the GGE. We sampled humans, chimpanzees and baboons, resulting in 587 samples overall. E. histolytica was found in all communities sampled. High sample prevalence identified among all species could indicate significance, as death from infectious diseases is the leading cause of mortality for Gombe chimpanzees (Lonsdorf et al., Reference Lonsdorf, Gillespie, Wolf, Lipende, Raphael, Bakuza, Murray, Wilson, Kamenya, Mjungu, Collins, Gilby, Stanton, Terio, Barbian, Li, Ramirez, Krupnick, Seidl, Goodall, Hahn, Pusey and Travis2018). However, chimpanzees had significantly higher sample prevalence than both humans and baboons, yet diarrhoea was not a risk factor for E. histolytica presence in chimpanzees. Therefore, chimpanzees with E. histolytica may not be symptomatic; however, our methods do not confirm current infection, so we cannot confirm this. Furthermore, we did not have baboon fecal consistency data. Further investigations into whether the NHPs of the GGE are symptomatic is necessary. Interestingly, Rivera et al. (Reference Rivera, Yason and Adao2010) confirmed E. histolytica in NHPs, but also noted that they did not exhibit typical symptoms of amebiasis. NHPs could be more resistant against infection from E. histolytica than humans.
Our methodology was 2-fold: first screen all samples for Entamoeba spp., then identify E. histolytica in the Entamoeba spp. positive samples. This is important because pathogenic and non-pathogenic species are morphologically similar. The recent separation of E. histolytica, E. dispar and E. nuttalli adds to the complexity of the epidemiology of Entamoeba spp. In 1993, E. histolytica was re-described as separated from the non-pathogenic E. dispar, which was first introduced by Brumpt in 1925 (Diamond and Clark, Reference Diamond and Clark1993). The name E. nuttalli was revived in 2007 for another pathogenic Entamoeba spp. strain that is similar to E. histolytica, but phylogenetically between E. histolytica and E. dispar (Tachibana et al., Reference Tachibana, Yanagi, Pandey, Cheng, Kobayashi, Sherchand and Kanbara2007). Most studies to date have only identified E. nuttalli in NHPs (Tachibana et al., Reference Tachibana, Yanagi, Pandey, Cheng, Kobayashi, Sherchand and Kanbara2007, Reference Tachibana, Yanagi, Lama, Pandey, Feng, Kobayashi and Sherchand2013; Levecke et al., Reference Levecke, Dreesen, Dorny, Verweij, Vercammen, Casaert, Vercruysse and Geldhof2010; Rivera et al., Reference Rivera, Yason and Adao2010); however, it has been detected in human zoo caretakers who were in contact with E. nuttalli positive NHPs (Levecke et al., Reference Levecke, Dorny, Vercammen, Visser, Van Esbroeck, Vercruysse and Verweij2015), suggesting that transmission of E. nuttalli from NHPs to humans might also be possible.
While we recognize that the absence of DNA sequencing is a limitation of this study, we targeted the SSU-rRNA gene of E. histolytica, which is distinct from E. nuttalli (Levecke et al., Reference Levecke, Dreesen, Dorny, Verweij, Vercammen, Casaert, Vercruysse and Geldhof2010, Reference Levecke, Dorny, Vercammen, Visser, Van Esbroeck, Vercruysse and Verweij2015). Thus, the reported sample prevalence for E. histolytica represent only E. histolytica; however, our approach does not rule out the presence of E. nuttalli in the GGE. A subset of the Entamoeba spp. positive specimens may represent E. nuttalli.
Entamoeba spp. were detected in more than 60% of all samples collected in the GGE, further indicating the importance of understanding the distribution of Entamoeba in this system. Sample prevalence ranging from 6 to 15% in humans and 30–36% in chimpanzees suggest there is a risk for zoonotic transmission. The sample prevalence of E. histolytica was expected to be higher in the Mitumba chimpanzee community due to the natural border it shares with Mwamgongo, assuming greater human-NHP contact. However, surprisingly, E. histolytica sample prevalence did not differ significantly between the two chimpanzee communities.
Water is a possible source for transmission of E. histolytica, as the cysts of this parasite are very resistant and can survive for several months in water (Hemmati et al., Reference Hemmati, Hooshmand and Hosseini2015). In Gombe, humans drink from the same stream as NHPs and use the same lake for bathing and washing clothes and utensils that baboons consume (Wallis and Lee, Reference Wallis and Lee1999). Despite contacting Lake Tanganyika more often than chimpanzees (Murray et al., Reference Murray, Stem, Boudreau and Goodall2000), baboons had a significantly lower sample prevalence than chimpanzees. Therefore, future studies using comprehensive water sampling to investigate E. histolytica contamination in water sources used by humans and NHPs in the GGE would be beneficial to the understanding of E. histolytica transmission in this system.
Another potential factor involved with the transmission of E. histolytica is seasonality. Although we did not find a significant association between infection status and season among the three primate species, there is often an association between diarrheal disease and season (Haque et al., Reference Haque, Mondal, Duggal, Roy, Farr, Sack, Kabir and Petri2006). Studies have shown that E. histolytica infection in humans does have marked seasonality, with peaks during the wet season (Mukherjee et al., Reference Mukherjee, Das, Bhattacharya, Nozaki and Ganguly2010). However, other studies have also found no relationship between E. histolytica infection and season (Haque et al., Reference Haque, Mondal, Duggal, Roy, Farr, Sack, Kabir and Petri2006; Mukherjee et al., Reference Mukherjee, Das, Bhattacharya, Nozaki and Ganguly2010). In this study, humans had higher sample prevalence during the dry season; in contrast, chimpanzees and baboons had higher sample prevalence during the wet season. Therefore, a more in depth investigation into the seasonal relationship of E. histolytica infection could increase knowledge of the transmission dynamics of E. histolytica in this system.
While a recent study detected no E. histolytica in a group of chimpanzees living in the Issa Valley in Tanzania (Jirku-Pomajbíková et al., Reference Jirku-Pomajbíková, Čepička, Kalousová, Jirku, Stewart, Levecke, Modrý, Piel and Petrželková2016), approximately 100 km east of the Gombe chimpanzee population, the Gombe chimpanzees showed a significantly higher sample prevalence of E. histolytica infection than both humans and baboons. Unlike the Gombe chimpanzees, the Issa Valley chimpanzees do not come into regular contact with humans, aside from researchers (Jirku-Pomajbíková et al., Reference Jirku-Pomajbíková, Čepička, Kalousová, Jirku, Stewart, Levecke, Modrý, Piel and Petrželková2016). This suggests that close proximity to humans could be an important factor in potential E. histolytica infection in chimpanzees and vice versa. Chimpanzees could also be at higher risk because they consume red colobus monkeys (Colobus badius tephrosceles) and bushpigs (Potamochoerus larvatus) (Gilby, Reference Gilby2006). Entamoeba histolytica has been found in colobus monkeys (Gillespie et al., Reference Gillespie, Greiner and Chapman2005) and in non-primate mammals, such as dogs (Alam et al., Reference Alam, Maqbool, Nazir, Lateef, Khan and Lindsay2015); therefore, transmission could occur through consumption of cysts from infected animals. Reports show that the Issa Valley chimpanzees consume blue duiker (Philantomba monticola) meat, but not red colobus monkeys or bushpigs, even though they are both present in the habitat (Ramirez-Amaya et al., Reference Ramirez-Amaya, Stewart and Piel2015). This further supports the possibility that the Gombe chimpanzees become infected by ingesting cysts from the colonic content of their prey. Additional investigations into the presence of E. histolytica in other wildlife and domestic animals in the GGE could shed further light into the transmission of E. histolytica in this system. Moreover, lower E. histolytica sample prevalence in humans could be a result of improved hygiene and sanitation in the region due to the implementation of interventions such as the JGI's TACARE program (Mavanza and Grossman, Reference Mavanza and Grossman2007) and protocols intended to improve sanitation in staff quarters in Gombe (Gillespie et al., Reference Gillespie, Lonsdorf, Canfield, Meyer, Nadler, Raphael, Pusey, Pond, Pauley, Mlengeya and Travis2010).
Persons experiencing gastrointestinal symptoms, which included diarrhoea and stomach cramping, were more likely to be infected with E. histolytica than those who were not experiencing gastrointestinal symptoms. This suggests that individuals may have been suffering from symptomatic E. histolytica, which emphasizes the importance of controlling E. histolytica infection in this region. The role of NHPs in E. histolytica transmission has not yet been elucidated. Our findings highlight the potential for zoonotic transmission of Entamoeba spp. and stress the need for further phylogenetic studies to determine cross-species transmission of both E. nuttalli and E. histolytica at the human–NHP interface. Interventions targeting better sanitation and hygiene practices for humans living in and around Gombe National Park can help prevent E. histolytica infection in humans, while also protecting the endangered chimpanzees and other primates in this region.
Acknowledgments
We are grateful to Deus Mjungu, Juma Baranyikwa and the field assistants of the Jane Goodall Institute's Gombe Stream Research Centre for assisting in the collection of chimpanzee and baboon demographic and health data and nonhuman primate fecal specimens. We thank the Kigoma district health officers for assistance with human survey and sampling and the Tanzanian Commission for Science and Technology, Tanzania National Parks and Tanzania Wildlife Research Institute for permission to conduct the research. We thank J. Bodager, K. Cross, M. Hensley, S. Kuthyar, L. Rautman and D. Ryu for laboratory assistance.
Financial support
This work was supported by the Morris Animal Foundation (MAF D09ZO-041 and MAF D09ZO-634), the Emory University Global Health Institute, the Arcus Foundation, the Leo S. Guthman Fund, and the National Institutes of Health (R01 AI58715).
Conflict of interest
None.
Ethical standards
This project was reviewed and approved by the Emory University Institutional Review Board (approval #: IRB00018856) under the Expedited review process per 45 CFR 46.110(3), Title 45 CFR Subpart D section 46.404, one parent consent and 21 CFR 56.110 with deferral from CDC Institutional Review Board, and the Tanzanian National Institute for Medical Research Institute, Dar es Salaam, Tanzania, which approved oral consent due to low literacy rates. All adult subjects provided informed consent and a parent or guardian of any child participant provided informed consent on their behalf. Oral informed consent was obtained by trained local field assistants and documented by witnessed notation on IRB-approved enrollment forms. All animal use followed the guidelines of the Weatherall Report and the NIH Guide for the Care and Use of Laboratory Animals on the use of NHPs in research, and was approved by the Tanzania Wildlife Research Institute and Tanzania Commission for Science and Technology (permit number 2009-279-NA-2009-184), and the Emory University Animal Care and Use Committee (protocol ID 087-2009). Approval was also obtained from Tanzania National Parks (Permit number TNP/HQ/C10/13) to collect samples from wild primates.
