Abstract

Entamoeba invadens is an important gastrointestinal pathogen in large reptile collections and may cause large losses in short periods of time. Turtles, crocodilians and some species of snakes and lizards have been identified as carriers and should not be housed with susceptible species. Disinfection of tools between exhibits is important in reducing the likelihood of an epizootic, and for controlling a current epizootic. Detection of Entamoeba cysts and trophozoites in carrier animals can be difficult due to low numbers of organisms passed in the feces. It is also difficult to differentiate Entamoeba trophozoites from other reptilian intestinal amoebas. Treatment options currently recommended in the literature are based on clinical experience rather than scientific data. The current research project at the Baltimore Zoo is directed towards improving efficiency of detection of carrier and subclinical cases via improved culture and sampling methods, and determining safety and efficacy of three different drugs (metronidazole, diloxanide furoate and iodoquinol) at three doses in snakes and turtles.

Introduction

Many species of amoebae can be found in the gastrointestinal tract of reptiles. Some may be commensals and others are considered pathogens. Cases of reptilian amebiasis with morbidity and mortality are usually attributed to the organism, Entamoeba invadens, although there are multiple species of reptilian Entamoeba.^6,9 Some of the described Entamoeba sp. are morphologically distinct from Entamoeba invadens, while others are very similar.³ Amebiasis can be a serious and devastating disease in a large reptile collection due to its ability to infect reptiles across taxonomic orders, unlike most viral diseases.² Although morbidity is variable, the organism is difficult to eradicate from the environment or the carrier animals once it enters a collection, and losses may continue for long periods of time. Detection of amebic cysts or trophozoites in fecal samples from clinically affected animals is considered diagnostic. Detection of carrier animals is difficult with currently available microbiologic techniques. Snakes and lizards are considered to be the most susceptible to fatal amebiasis. Although turtles, crocodilians and reptile-eating snakes and lizards are considered to be resistant to the disease and potential carriers, it is also possible for them to develop clinical disease.

Amebiasis causes morbidity and mortality by invading the epithelium of the gastrointestinal tract, especially the colon, and lysing the cells. The extent of damage to the intestinal mucosa is variable, but may result in thickening or obstruction of the gastrointestinal tract, or perforation. Secondary bacterial infection is usually concurrent, worsening the clinical course of disease. Trophozoites may also enter the mesenteric circulation and invade the liver, causing abscesses or other extra-intestinal signs of amebiasis. Thrombic emboli may form causing necrosis of large portions of the liver or gastrointestinal tract. Reptiles may show no clinical signs, and be found unexpectedly dead, or may become anorectic and dehydrated. Mucous and or blood may be seen in the feces, but it is not consistent.

The organism has a direct life cycle and the mode of transmission is fecal-oral contamination with cysts from enclosure mates or from fomites brought in from other enclosures. The reptile ingests a cyst which excysts within the lumen of the gut and becomes a trophozoite, the replicating phase. The reproducing trophozoites invade the gut wall or encyst and are passed out in the feces. Trophozoites are not thought to be infective as they desiccate rapidly in the environment. It may be difficult for technicians to detect the trophozoites or the cysts on routine fecal examinations in carrier animals. It is somewhat more obvious in clinically affected cases. Culture techniques^1,8 may improve detection compared to direct and flotation fecal examinations. The most important method of preventing an epizootic within a collection is to avoid housing probable carrier animals in mixed species exhibits with susceptible animals. Quarantine procedures, properly followed, and appropriate disinfection of tools and equipment between exhibits are also very important.

The quadrinucleate cysts of E. invadens (Ei) are morphologically similar to those of the human amoebas E. histolytica (Eh, pathogenic) and E. dispar (Ed, non-pathogenic) and can be distinguished in vitro by organism temperature preferences in culture. Ei grows best at 25°C and will not grow above 33°C^.7 In human medicine, Eh and Ed can be distinguished from each other by the presence of anti-amebic antibodies (elicited by Eh only), the presence of occult blood in feces (Eh), or the presence of ingested erythrocytes within the trophozoites (Eh). In light of this information regarding the difficulty in differentiating human Entamoeba sp., there is no reason to assume that Ei is the only pathogenic species of reptilian ameba, that all cases of amebiasis in reptiles are caused by Ei, or that all strains of pathogenic ameba are equally pathogenic in all species of reptiles. Other morphologically indistinguishable species may be present, whether causing disease or not.

Treatment recommendations for reptiles have been based on human treatment regimens, of which metronidazole is a cornerstone of chemotherapeutics for invasive amebiasis. Metronidazole dosages and frequencies for reptiles that have been recommended in the literature range from 275 mg/kg orally once, to 50 mg/kg orally s.i.d. for 5 days and many dosages and frequencies in between. Metronidazole is most active against the trophozoites but does not eliminate all cysts from the gut lumen. Metronidazole at high doses can cause hepatotoxicity with neurologic signs. Dimetridazole is also reported to be effective but is not currently available in the United States. Tetracyclines or erythromycin may be used in patients that are intolerant of metronidazole, but are less effective than metronidazole and are rarely used in reptilian medicine for that reason. Emetine class drugs were used historically to treat invasive amebiasis in humans and reptiles, however, significant cardiotoxic side effects and neuromuscular toxicity have virtually eliminated the use of this group of drugs.¹⁰

In human clinical cases, metronidazole is often given concurrently with or immediately followed by lumen active agents: diloxanide furoate, paromomycin or iodoquinol (diiodohydroxyquin) to reduce cyst passage in the feces.^4,10 There are anecdotal reports of the use of these agents for the treatment of amebiasis in reptiles although no clinical studies have been performed determining safety or efficacy of these drugs. Diloxanide furoate is the drug of choice for eradication of Eh from the intestinal lumen (95% clearance rate in humans), however, it is not readily available in the United States. Use of diloxanide furoate in reptiles at 0.5 g/kg as a single oral dose has been published,⁵ but no clinical studies have been cited to support this dosage. Paromomycin is an oral aminoglycoside which may cause mild gastric irritation and fungal overgrowth. It is only used in mild or asymptomatic infections in humans. Iodoquinol is probably as effective as diloxanide furoate, but requires twice as long a treatment period and has been reported to cause optic nerve atrophy.¹⁰

Study Design

The Baltimore Zoo Medical and Herpetology Departments have undertaken a multifaceted research project involving early diagnosis and treatment of pathogenic amebiasis in reptiles. The first phase of the study is designed to improve diagnostic techniques to detect carriers and subclinical cases of Entamoeba sp. Direct microscopic examination of samples obtained by cloacal flushing with saline was compared to culture techniques for detection of cysts and trophozoites in samples. Single sampling events were compared to multiple sampling events for detection of cysts and trophozoites. Organisms were then transferred into susceptible hosts (juvenile corn snakes, Elaphe guttata) in order to identify pathogenic strains. Once pathogenic strains are identified, molecular techniques will be utilized to find markers to attempt to differentiate between pathogenic and non-pathogenic strains or species of Entamoeba. Pathogenic strains will be transferred into susceptible species and efficacy of metronidazole, diloxanide furoate and iodoquinol will be evaluated in infected reptiles. Metronidazole, diloxanide furoate and iodoquinol have also been evaluated at several doses in healthy reptiles to determine safety. The three drugs will also be evaluated for efficacy and safety in box turtles and wood turtles which are infected with Entamoeba of unknown species or pathogenicity.

The safety trials consisted of evaluating metronidazole at 50 mg/kg s.i.d. for 5 days, repeated twice 2 wk apart; metronidazole at 250 and 500 mg/kg p.o. every 2 wk for three treatments. The two lower doses are based on reports in the reptile literature. Iodoquinol was evaluated at 30, 60 and 120 mg/kg p.o., s.i.d. for 14 days. The lowest dose is the recommended human pediatric dose. The length of treatment was based on a recommendation from Barbara Bonner, DVM (pers. comm.). Diloxanide furoate was evaluated at 20, 40 and 80 mg/kg p.o., s.i.d. for 10 days. Again, the lowest dose is based on the human pediatric dose. Length of treatment is the same as that recommended for humans. The higher doses of all drugs were evaluated for toxicity purposes. Juvenile black rat snakes (Elaphe obsoleta) were used to evaluate safety as some of our adult black rat snakes had developed hepatotoxicity with neurologic signs when treated with metronidazole at 250 mg/kg every 2 wk for three treatments. Three snakes were evaluated per drug dose. Three control snakes were given saline at 1% body weight for 10 days.

Results

Multiple sampling events are better for the detection of carrier animals. Results of the diagnostic evaluations to date indicate that 24°C is the best temperature at which to culture the organism, that no benefit is gained from culturing for more than 3 days and that samples deteriorate over time after 3 days. It is best to centrifuge the tubes before removing a sample for microscopic analysis for detection or for transfer to susceptible hosts in order to have the greatest chance of detecting or transferring cysts. If the culture tubes are not centrifuged, there is a high degree of variability within the culture medium of where the cysts are most likely to grow. Inconsistent sampling technique introduces a number of variables into detection of oocysts. We have been unable to detect a pathogenic strain to evaluate efficacy of the drugs.

No snakes developed clinical signs of toxicity during the study. At the end of the treatment period, the snakes were euthanatized and necropsied. No gross abnormalities were noted. Histopathology is pending.

Conclusion

Entamoeba spp. can be a devastating disease in a reptile collection. Proper separation of carrier animals from susceptible animals reduces the likelihood of an epizootic in a collection. Sanitation and proper tool disinfection are equally important. Early detection of cysts and trophozoites prior to presentation of clinical signs could save many animals lives. Culture techniques may offer increased frequency of detection over standard fecal smears or floatation in carrier or subclinical animals. Metronidazole, iodoquinol and diloxanide furoate tested at three different dosages in black rat snakes appear to be safe in that species.

Literature Cited

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