Current World Status of Balantidium coli

INTRODUCTION

Protozoan parasitism covers a broad spectrum of diseases. Balantidium coli and balantidiosis, the subjects of this review, barely register among infectious protozoan diseases.

Balantidium is the only ciliated protozoon known to infect humans and is the largest protozoon infecting humans and nonhuman primates. Balantidiosis is a zoonotic disease and is acquired by humans via the fecal-oral route from the normal host, the pig, where it is asymptomatic. Water is the vehicle for most cases of balantidiosis. Human-to-human transmission may also occur. Balantidium's habitats in humans are the cecum and colon. Humans may remain asymptomatic, as does the pig, or may develop dysentery similar to that caused by Entamoeba histolytica. Death is an infrequent consequence of balantidiosis, but in developing countries with undernourished and overparasitized populations, it can make the difference between a healthy life and chronic debilitation.

The organism is cosmopolitan and can be found wherever pigs are found. Disease appears to be a problem mostly of developing countries, where water sources may be contaminated with porcine or human feces. B. coli can become an opportunistic parasite in immunosuppressed hosts living in urban environments, where pigs are not a factor in infection.

Balantidium is an often-neglected pathogen. Research on Balantidium has been sparse. Zaman (80) published an inclusive review on Balantidium 30 years ago, but recently the organism has come to be regarded as an emerging protozoan pathogen and has been reviewed by Garcia (22).

Balantidium has a simple life cycle, as follows: dormant cyst to trophozoite and trophozoite to cyst. Transmission is direct, from a contaminated water or food supply to humans (Fig. 1). No intermediate host, as occurs with many other parasitic species, is needed.

MORPHOLOGY

The trophic ciliate measures 30 to 150 μm in length by 25 to 120 μm in width; the cyst, which may be spherical or slightly ovoid, measures 40 to 60 μm in diameter (41). Size, however, varies, with some balantidia being up to 200 μm in length. The mouth (oral apparatus) is located at the tapering anterior end, and the cytopyge (anus) is at the rounded posterior end (Fig. 2). A sausage-shaped macronucleus and a rounded micronucleus are located in the cytoplasm (Fig. 3). Asexual division occurs as it does in most ciliates. A transverse furrow forms, dividing the mother cell into two asymmetric daughter cells, an anterior (proter) and a posterior (opisthe) cell. The proter retains the oral apparatus, and the opisthe develops a new apparatus. An anterior stomatogenic field appears in the opisthe, leading to formation of the new oral apparatus, but the old oral apparatus may also undergo reorganization (37). The swimming organism exhibits a rotary-type motion by means of its somatic cilia that may facilitate movement through the contents of the colon.

Sexual reproduction as conjugation has been reported for Balantidium, but information is lacking about details of nuclear events. Two successive fissions (meiosis) occur preceded by formation of the conjugants, by either equal or unequal divisions (37). The two conjugants attach at the oral apparatus and exchange micronuclear products of meiosis.

Although the organism lives in an anaerobic environment, Zaman (80) described mitochondrion-like bodies in electron micrographs of Balantidium; in contrast, Entamoeba histolytica and Entamoeba dispar, found in the human colon, are anaerobes and lack mitochondria. Cristae or tubuli, however, were reportedly absent from the mitochondrion-like bodies, raising the possibility that these organelles are hydrogenosomes (80). Hydrogenosomes, relict mitochondria, have been identified in balantidia as well as in other anaerobic ciliates (25, 27). Other cytoplasmic components include endoplasmic reticulum, ribosomes, and numerous vacuoles filled with food particles. A Golgi apparatus was not seen, but vesicles of endoplasmic reticulum function in lieu of Golgi vesicles (49). Mucocysts are also seen (61). Two contractile vacuoles, functioning as osmoregulatory organelles, pulsate at a low rate even though the surrounding environment is isotonic or close to it (79). Undigested residue from food vacuoles is eliminated through the cytopyge.

Balantidium jocularum, from the herbivorous surgeonfish, was found to harbor endosymbiotic bacteria in its macronucleus (26). The bacteria were unusually large, measuring up to 4 μm in length, but apparently without pathogenic potential for the ciliate. They are probably gram-positive organisms because of an endospore-like inclusion within the bacterium. No other reports of endosymbionts from Balantidium have appeared.

The cyst of Balantidium is the transmissive stage of the organism. Because of its thickened wall, it is protected from desiccation and other environmental stress (Fig. 4). It survives best in humid surroundings protected from direct sunlight. The trophic ciliate is reportedly unable to survive passage through the stomach because of the low pH of gastric fluid, but Balantidium trophozoites inoculated into the stomach of guinea pigs have been found in the colon (56). The process of encystment begins in the colon and rectum of the host, and cysts are generally found in formed feces (56). Cysts, however, were not produced in cultures of balantidia (32, 80), nor are they produced in cases of acute dysentery. Attempts to simulate in vitro the colorectal environment in which cysts form (resorption of water and increased salt concentrations) were unsuccessful in inducing encystations; overfeeding or starvation was also unsuccessful (32). Loss of the ability to encyst is seen in some other protozoa (e.g., soil amebae) maintained in culture, due in part to less than optimal growth conditions and/or limiting amounts of nutrients essential for encystment. E. histolytica, an agent of amebic dysentery, resembles Balantidium in producing cysts in formed stools but trophozoites in dysenteric stools. In vitro encystment of E. histolytica depends upon a number of factors, including withholding rice starch, the composition of the bacterial flora, and the encystment medium, and may require a complex protocol to induce cyst formation (10).

The diet of the ciliate is made up of bacteria and food particles present or passing through the colon. If extensive damage has been done to the wall of the colon, red blood cells may also be seen in trophic organisms, as well as blood in the stool. The ciliates produce flask-shaped lesions in the submucosa, where they form clusters called nides or nests (4).

TAXONOMY AND MOLECULAR BIOLOGY

Protist taxonomy was recently revised by a committee of the International Society of Protistologists and allied groups, based on the wealth of new information that has accumulated about these organisms since the last “official” taxonomy published in 1980 (1). Since that time, an increased understanding of ultrastructural morphology, biochemical properties and relationships, studies of nuclear and mitochondrial DNAs, and protist ecology has developed. The revised taxonomy does not use the classic categories of phylum, class, order, etc., in order to maximize taxonomic plasticity as additional information on phylogenetic relationships, particularly from genomic studies, becomes available (1).

PHYSIOLOGY

Few studies have examined the energy metabolism of these organisms. Balantidia are equally able to survive under anaerobic as well as aerobic conditions. Carbohydrates are the chief source of energy for the ciliates growing in vivo (80).

In studies of B. coli combining ultrastructure with cytochemistry, peroxisomes were identified in the ciliate. These vesicles contain peroxidase, an enzyme affording protection from the destructive effects of highly oxidative compounds, such as hydrogen peroxide (60). A comparison of the cytoplasm of ciliates from asymptomatic swine and those with acute balantidiosis was made. Peroxisomes were more numerous but smaller (0.6 to 0.8 μm) in balantidia from asymptomatic pigs than in those with acute disease (>0.8 μm). Likewise, nucleic acid contents (particularly RNA, but also DNA) from symptomatic and asymptomatic hogs differ, with the former having more content (62). The difference may depend on the degree of metabolic activity of the ciliates and, in the case of RNA, may be indicative of enhanced protein synthesis. Ciliates with higher nucleic acid content produced more robust cultures, at least in the initial stages of in vitro growth (62).

The enzyme glucose-6-phosphatase was present in small vesicles attached to the endoplasmic reticulum or in the membrane itself (61). Alkaline phosphatase was found in the ciliate cortex, nuclear and ciliary membranes, and kinetosomes as well as in vesicles of the endoplasmic reticulum (61). Phosphatase enzymes are important for their role in making glucose available as an energy source.

Balantidia produce no known toxins, but their ability to produce ulceration of the colon wall is attributed to hyaluronidase, an enzyme that digests hyaluronic acid, a component of the “glue” holding mucosal epithelial cells together (68). The dissolution of group C Streptococcus hyaluronic acid-containing capsules and the breakdown of potassium hyaluronate by living B. coli were taken as evidence of hyaluronidase activity. Entamoeba histolytica, the classic protozoan dysentery agent, attacks the mucosal surface of the colon and was long claimed to possess hyaluronidase activity. Attempts to demonstrate its presence, however, using E. histolytica extracts, did not support the claim (50). In the case of B. coli, other factors may affect the results, including associated bacteria, waning of virulence (see next paragraph) in long-term cultures, up- and downregulation of presumptive virulence genes, and strain differences. Thus, the matter of hyaluronidase production by Balantidium warrants further study. Levine (41) noted that invasion of colonic epithelium by Balantidium might be secondary to damage caused by intestinal bacteria.

The term “virulence” is used here as the degree of pathogenicity of a parasite and involves substances elaborated by a pathogen that facilitate infection and disease (e.g., toxins, enzymes that promote invasiveness, and adhesive properties).

CULTIVATION

Balantidia are available from fresh pig feces, particularly from animals with evidence of acute balantidiosis, and from slaughterhouses. An insulated bottle is used for transport of porcine intestinal contents to the laboratory. There were a number of early attempts at maintenance or cultivation in vitro. Gastric mucin media were developed for Balantidium and maintained growth for as long as 30 months. Calf serum and rice starch or rice powder were also required. Starch grains, if present in culture medium, are ingested and serve as a carbohydrate source (80). The addition of soluble sugars to growth media encourages overgrowth of accompanying bacteria, whereas starch particles are efficiently ingested and digested by the phagotrophic ciliates (10). A defined medium with cysteine HCl and i-inositol was used for short-term physiologic experiments with the organisms, but little in the way of results appeared in the literature (35). Trophic ciliates, however, did not survive agitation in attempts at manometric studies.

Biphasic media in tubes were often used with an agar, inspissated egg yolk, or serum butt overlaid with nutrient medium. Bacteria present in the sample can overgrow in an enriched medium, requiring the addition of antibiotics (e.g., penicillin-streptomycin) to suppress bacterial proliferation. When growing in tubes, balantidia favor the bottom of the tube, where conditions are microaerophilic or anaerobic. Zaman maintained monoxenic cultures of B. coli with Escherichia coli, using antibiotics to control bacterial overgrowth (80). Diamond's TYSGM medium for Entamoeba and other enteric parasites will also support the xenic (with bacteria) growth of balantidia (10). The medium contains Trypticase (casein digest), yeast extract, serum, and porcine gastric mucin. Starch powder is added to tubes at the time of inoculation of medium. Zaman (80) noted that B. suis from the pig is not as readily cultured as B. coli from humans. Others apparently encountered no difficulties with cultivation of B. suis (62). Among the variables involved in cultivation are the growth medium and pH (optimal range, 5.4 to 8.0) (32), associated undefined bacterial flora, strain differences, and differences in nutritional requirements between ciliates from different hosts.

In the case of many pathogens maintained in vitro, prolonged cultivation attenuates virulence, and animal passage may be required to restore it. The difficulty in infecting laboratory animals (e.g., the guinea pig) and the absence of an animal model of disease are obstacles in attempts to study the pathogenicity of the ciliate.

HOST-PARASITE INTERACTIONS

In pigs, as in some humans and other mammals, Balantidium infects but causes no serious disease of the gastrointestinal tract. It can thrive there in balance with its host without causing dysenteric symptoms, such as severe diarrhea and bloody stools. The problem with this détente is that malnutrition, alcoholism, or a compromised immune system (as in human immunodeficiency virus [HIV]/AIDS, etc.) can tip the balance in favor of the ciliate, leading to disease (3, 20, 70). In acute disease, explosive diarrhea may occur as often as every 20 min (38). Perforation of the colon may also occur in acute disease, with life-threatening consequences.

Hosts vary in their susceptibility to Balantidium, and attempts at infecting humans have been disappointing (76). This may be due to virulence of the particular strain used for infection, the intrinsic health of the host, and/or the dosage of the ciliate administered to the host. There is no evidence that other intestinal flora of humans, whether bacteria, protozoa, or viruses, render the host more susceptible to infection. It is known, however, that the presence of pathogenic bacteria (e.g., Salmonella) in the intestine can worsen an infection by invading colonic lesions caused by balantidia (41, 57, 59, 60).

Nonhuman primates, particularly the great apes and Old World monkeys, can develop Balantidium infections. This is of concern since some of these animals are endangered species or close to it because of disease, encroachment by humans on their natural habitats, and resultant density-dependent changes in their populations. Orangutans, for example, are less likely to be infected by Balantidium in their natural setting, while those in “rehabilitation” centers for injured or orphaned animals have a higher prevalence of the ciliate (34). Reasons for this include overcrowding at the centers, with increased stress on individuals; contacts with humans and other animal species and their associated bacterial, viral, and protozoan organisms; and water sources contaminated by fecal material in confined areas (34). In the wild, the orangutan population density is 2 individuals km⁻² (34).

DISEASE POTENTIAL

Balantidiosis has a range of mild to severe clinical presentations. The following three clinical manifestations of balantidiosis can occur (71): (i) asymptomatic hosts who are carriers of disease and serve as reservoirs of infection in the community; (ii) chronic infection that presents with nonbloody diarrhea, cramping, halitosis, and abdominal pain secondary to trophozoite invasion of the large intestine (71, 75); and (iii) patients with fulminating balantidiosis passing mucoid, bloody stools.

The most severe presentation of B. coli occurs with weight loss, tenesmus, and bloody stools (71). Intestinal hemorrhage and perforation can also occur and are mediated by the production of B. coli proteolytic enzymes (3). Direct evidence for the presence of proteolytic enzymes is lacking, but proteolysis is generally assumed to be a factor in digesting the mucous coating of the colon and facilitating tissue invasion, abscess formation, ulceration, and perforation of the intestine (3). Entamoeba histolytica, which has a similar pattern of pathogenicity, has been shown to possess and secrete cysteine, serine, aspartic, and metallo-proteases, some of which can target the mucus layer of the intestinal wall and aid in penetration of the underlying tissue (43, 69).

Hemorrhage and perforation were reported for fatal cases of B. coli infections (17). Fulminating balantidiosis has a case fatality rate of 30% (19). Vasquez and Vidal (71) described the case of a 60-year-old pig farmer with pancolonic damage and microperforation who died despite antiparasitic treatment. Another fatal case of balantidiosis occurred in a 63-year-old pig farmer, who died of dysentery and subsequent hemorrhage after 8 days; an autopsy revealed ulcerative colitis (54). Fatal cases of balantidiosis have also been associated with sepsis secondary to intestinal disease (51, 58). A malnourished 2-year-old girl with an anorectal malformation who was diagnosed with balantidiosis developed septic shock and died despite antimicrobial treatment with ampicillin and amikacin for sepsis and metronidazole for balantidiosis (7).

Although the intestine is the most common site of B. coli disease, there are extraintestinal sites of infection. These include the appendix but rarely the liver (16). Dodd (16) reported a case of a 16-year-old who presented with abdominal pain, fever, and elevated white blood cell count and who had a gangrenous appendix. Pathological examination of the appendix revealed inflammation, ulceration, necrosis, and B. coli trophozoites (16). Genitourinary sites of infection, including uterine infection, vaginitis, and cystitis, are thought to occur via direct spread from the anal area or secondary to rectovaginal fistulas created from infection with B. coli (58). Lung infections with Balantidium are infrequent but noteworthy. A necrotizing lung infection was reported for a 42-year-old organic farmer who routinely used pig manure to fertilize his vegetables, probably as a result of aerosolizing the manure and inhaling airborne cysts (58). Airborne transmission of cysts is unlikely. Cysts of Balantidium are large and would not be carried over great distances, either on air currents or in water droplets. Thus, infection by inhalation would require direct contact with aerosol droplets.

EPIDEMIOLOGY

B. coli infection is uncommon in humans despite its potential for worldwide distribution. The organism, though pathogenic, is of low virulence. The worldwide prevalence is estimated at 0.02 to 1% (19), but it varies widely by geographic location (3).

Areas of high prevalence include regions of Latin America, the Philippines, Papua New Guinea and West Irian, and areas of the Middle East (63, 75). In New Guinea, the rate of infection among pig farmers is as high as 28% (53), and in the Altiplano area of Bolivia, balantidiosis rates range between 6 and 29% (19).

The major factors leading to human balantidiosis include (i) close contact between pigs and humans, (ii) a lack of appropriate waste disposal such that swine and human excrement contaminate drinking water sources (e.g., wells and streams) and food, and (iii) subtropical and/or tropical climatic conditions (e.g., warmth and humidity) favoring survival of cysts. Balantidia, however, are adaptive to less than ideal conditions, as evidenced by their ability to infect compromised hosts living in urban settings and to survive in hogs in decidedly nontropical locations, such as Denmark (31) and Poland (62). Human-to-human spread by the fecal-oral route can take place as with other enteric diseases.

PREVALENCE OF BALANTIDIUM COLI

LABORATORY DIAGNOSIS

Because of their large size and spiraling motility, balantidia can readily be recognized in wet mount slide preparations, even at a low magnification (×100). This is the case with freshly collected diarrheic stool samples, which are likely to contain actively swimming trophic ciliates, as well as bronchoalveolar wash fluid. Stool samples for examination should be collected over several days because excretion of parasites can be erratic. Cyst stages are more common in formed stools. To search for cysts, a portion of formed stool is broken up in phosphate-buffered saline or fixative (10% phosphate-buffered formalin or polyvinyl alcohol) and coarsely filtered through gauze or a sieve to remove large pieces of debris. The resulting fluid can be examined microscopically for cysts in formed stools or for trophozoites in diarrheic stools. A phase-contrast microscope is helpful for viewing internal structures of unstained living or fixed ciliates. Staining can be done using Lugol's iodine (1:5 to 1:100 dilutions), but the stain concentrates progressively in the cytoplasm, obscuring details such as the macronucleus. The same is true for permanent stains, such as hematoxylin-eosin, as cells can take up excess stain, obscuring all internal detail. Heavily stained cysts can be mistaken for helminth ova, leading to misdiagnosis. Biopsy of the colon, if performed, followed by hematoxylin-eosin staining of sections may be useful in evaluating the extent of damage to the wall (Fig. 4). Methods for concentration of parasites from stool samples, making them easier to find, include sedimentation and flotation (21). Since balantidiosis is a rare disease in developed countries, most technicians would not normally be looking for trophozoites or cysts of Balantidium in examining stool samples. Thus, it is particularly important that balantidiosis be considered a possibility for patients from areas of endemicity and travelers returning from such areas. The number of balantidia in a stool sample may be high; 1,230 organisms g⁻¹ feces was reported for the stool of a chimpanzee in Japan (49). A Danish study of pigs at a research farm found an average of 865 cysts g⁻¹ feces from pigs of 28 to >52 weeks of age (31).

PREVENTION OF INFECTION

The best means of protecting human populations from balantidiosis is by providing a source of clean, uncontaminated water for drinking and other purposes. Chlorine, at the concentrations normally used for ensuring water safety, is not effective against cysts of Balantidium. Pigs should not be allowed to roam in and around feeder streams or rivers that empty into reservoirs that are used for providing municipal water supplies (29). Likewise, spreading of sludge from sewage processing as fertilizer can lead to contamination of produce or water sources with cysts of balantidia (2). Pigs should not have access to areas where crops are being raised. Judging from the occurrence of balantidiosis in immunosuppressed individuals living in urban settings, there are additional sources of infection besides pig-to-human transmission. Raising Balantidium-free pigs is an unrealistic goal. Piglets become infected from their mother or, if not that, through coprophagy.

ANTIMICROBIAL THERAPY

Tetracyclines and metronidazole are treatments of choice for human Balantidium infection. A number of different dosage regimens and treatment durations exist in the literature (3, 20, 23, 75). For metronidazole (Flagyl), the treatment is typically 5 days (adult dosage, 750 mg three times a day; pediatric dosage, 35 to 50 mg kg of body weight⁻¹ day⁻¹ in three doses [maximum dosage, 2 g]), in contrast to tetracycline (adult dosage, 500 mg four times a day; pediatric dosage, 40 mg kg⁻¹ dose⁻¹ in four doses) treatment over 10 days. Iodoquinol, for a 20-day treatment course (adult dosage, 650 mg three times a day; pediatric dosage, 40 mg kg⁻¹ dose⁻¹ in three doses), and doxycycline are alternatives (3, 58). There is some evidence that nitazoxanide (Alinia), a broad-spectrum antiparasitic and antihelminthic drug, may be another treatment for balantidiosis (52). The reader may wish to consult The Medical Letter on Drugs and Therapeutics, Drugs for Parasitic Infections (46). Dosages given here are from the 2004 edition but are unchanged in the latest edition (2007).

CONCLUSIONS

Balantidium coli is a cosmopolitan parasitic-opportunistic pathogen that can be found throughout the world. Its reservoir host is the pig, and humans become infected through direct or indirect contact with pigs. In rural areas and in some developing countries where pig and human fecal matter contaminates the water supply, there is a greater likelihood that balantidiosis may develop in humans. The infection may be subclinical in humans, as it mostly is in pigs, or may develop as a fulminant infection with bloody and mucus-containing diarrhea; this can lead to perforation of the colon. The disease responds to treatment with tetracycline or metronidazole.

Balantidiosis is a disease that need never exist given access to clean water and a public health infrastructure that monitors the water supply and tracks infections. Its spread can be limited by sanitary measures and personal hygiene, but it is a disease that will be around as long as there are pigs. Immunocompromised individuals have developed balantidiosis without any direct contact with pigs, perhaps with rats or contaminated produce as a possible source of infection. For the clinician, balanatidiosis should be included in the differential diagnosis for persistent diarrhea in travelers to or from Southeast Asia, the Western Pacific islands, rural South America, or communities where close contact with domestic swine occurs.

Warming of the earth's surface may provide a more favorable environment, even in the now temperate areas of the world, for survival of trophic and cystic stages of Balantidium, and its prevalence may increase. Effective sanitation and uncontaminated water are the most useful weapons against infection. Fortunately, balantidiosis responds to antimicrobial therapy, and there have been no reports of resistance to the drugs of choice.