Occurrence and Molecular Analysis of Balantidium coli in Mountain Gorilla (Gorilla beringei beringei) in the Volcanoes National Park, Rwanda

Balantidium coli is the only species of trichostome ciliate currently considered to be pathogenic (Ponce-Gordo et al., 2011). Balantidium coli is a cosmopolitan parasite with a direct lifecycle and a wide host range including pigs (Sus scrofa; the natural reservoir host), rodents, and primates (Schuster and Ramirez-Avila, 2008). While the prevalence of balantidiasis is high in captive populations of great apes and old-world simians (between 16–71% [Pomajbíková et al., 2010]), this has not been mirrored in free-ranging great apes, with the organism scarcely recorded in wild populations of western lowland gorilla (Gorilla gorilla gorilla) and chimpanzee (Pan troglodytes; Lilly et al., 2002; Gillespie et al., 2010).

The taxonomic validity of species belonging to the genus Balantidium remains controversial, although advances in molecular characterization now support phylogenetic differentiation (Ponce-Gordo et al., 2008). Ponce-Gordo et al. (2011) identified two distinct “sequence variants” of B. coli from four different hosts, including captive lowland gorilla, but found insufficient evidence to separate the organisms into distinct species. To date B. coli has never been described in mountain gorilla (Gorilla beringei beringei).

Mountain gorilla feces were collected as part of a study investigating the potential for anthropozoonotic parasitic disease transmission in the Volcanoes National Park (VNP), Rwanda (Hogan et al., 2013). Fecal samples (n = 130; each collected from the night-nest of an individual mountain gorilla belonging to a family group that were all sampled on a single occasion) were processed in the Mountain Gorilla Veterinary Project's (MGVP) regional laboratory. Feces were prepared as previously described (Hogan et al., 2013). Fecal flotation was performed using 40% w/v sodium nitrate solution (NaNO₃) solution. Samples were centrifuged (1,000 × G, 6 min) and coverslips were placed on slides and examined under a light microscope. Morphologic features allowed identification of “Balantidium-like” cysts in one sample (1/130), although the feces of the “positive” gorilla were well formed and not characteristic of an animal suffering from clinical balantidiasis. All samples were shipped to the Royal Veterinary College in 70% ethanol at a ratio of 3∶1 (feces∶ethanol) where repeat flotation confirmed morphologic B. coli identification from the same individual (Fig. 1). Genomic DNA was extracted for confirmatory PCR and sequencing of the internal transcribed spacer (ITS)-1-5.8S rRNA-ITS2 region using the primers B5D and B5RC as described by Ponce-Gordo et al. (2011). Duplicate sample PCR, electrophoresis, purification, cloning, and sequencing were completed as previously described (Barkway et al., 2011). Five clones were processed per PCR yielding 10 sequences. ClustalX alignment (Thompson et al., 1997) identified two distinct sequences (GenBank HF545448 [6/10] and HF545449 [4/10]). Sequence similarity analysis found HF545448 to be most closely comparable to B. coli sequence variant A0 (Ponce-Gordo et al., 2011; NCBI BLASTn suite-2sequences, E value 0.0, query coverage 100%, maximum identity 98%). Sequence HF545449 was identical to B. coli sequence variant B1 (0.0, 100%, 100%). Although nonquantitative, the A0∶B1 sequence ratio suggests either approximately equal proportions of genetically distinct B. coli isolates, or more likely, two sequence variants within a single strain. Sequences of the A0 and B1 types have been reported in B. coli isolated from captive gorilla in Cameroon, the Czech Republic, and the UK (A0), and in Spain (B1), although this is the first report of an A0/B1 combination in a single sample probe (Ponce-Gordo et al., 2011).

Gastrointestinal disease represents a low percentage (4%) of mortality in mountain gorillas even though rates of gastrointestinal parasitism are relatively high (Sleeman et al., 2000; Rothman and Bowman, 2003; Rothman et al., 2008). Fatalities attributed to balantidiasis are rare in captive great apes and, although little is known of the disease-causing potential of B. coli in wild populations, one may expect the course of disease following tissue-invasive trophozoite infections to mimic the effects seen in captivity and in humans (Lankester et al., 2008).

The low prevalence of Balantidium in this study presents two epidemiologic scenarios, 1) that Balantidium infection exists endemically in the gorilla population (but below the detection limits of this study), or 2) that gorillas are sporadically infected from an independent reservoir host(s). Although horizontal transmission between people has been implicated in the past, and there is evidence to suggest that the direct fecal-oral passage of trophozoites between great apes is the primary cause of balantidiasis outbreaks in zoological collections, a review of coprologic studies on free-ranging African great apes reveals how scarce B. coli infection is (Rothman and Bowman, 2003; Pomajbíková et al., 2010). This, coupled with the historical absence of cysts and trophozoites in histopathologic sections or feces of this population of mountain gorilla, suggests that they are unlikely to be an indigenous host for the parasite. While the cross-sectional design of this project, coupled with omission of sedimentation as a diagnostic technique, may have led to an underestimation of prevalence, an endemic infection in these highly social, group-orientated animals could lead to a considerably higher overall prevalence of infection. As such, sporadic transmission from a reservoir appears a more likely source of infection.

Routes of disease transmission at the human-wildlife interface have been studied in both the VNP and Bwindi, with cattle and humans implicated as sources for Cryptosporidium, Giardia, and microsporidia infections in wild mountain gorillas (Nizeyi et al., 2002; Hogan et al., 2013). Prevalence of Balantidium in communities bordering the VNP is unknown, but poor sanitary conditions coupled with high numbers of domestic swine may provide favorable conditions for Balantidium spp. to persist and pose a risk of infection for mountain gorillas crossing the park boundary. Additional routes of anthroponotic transmission may include park staff and tourists visiting the gorillas, but a comprehensive employee health program run by MGVP has not detected B. coli infection in members of staff working within the park (Lukusa, pers. comm.). Advances in subgenotyping have allowed the molecular epidemiology of these infections to be charted, providing a tool to investigate local transmission pathways (Xiao, 2010; Hogan et al., 2013). The ITS1 sequence variants identified in this study have been found to define B. coli obtained from captive gorilla but are not known to be host species-specific (Ponce-Gordo et al., 2011). The collation and genetic analysis of more isolates, and from a broader range of locations, is required in order to elucidate the genetic diversity of this parasite species.

This study is the first to describe B. coli in mountain gorillas, albeit at low prevalence. Further investigation of B. coli prevalence in people, livestock, and wildlife associated with the park is warranted to assess whether the potential for transmission pathways and a reservoir of infection exist on the park boundary.