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Introduction

Snakes are often considered to most impressive exhibits in zoological collections, with the majority of establishments maintaining and breeding a large population. Recently however, interest in reptiles has spread, many novice herpetologists keeping and breeding snakes as pets, and consequently the veterinary surgeon is increasingly likely to be approached to provide advice and treatment in various areas of reptilian husbandry and medicine. It is therefore vital that the clinician is as informed as possible, especially where a perilous disease such as amoebiasis is a distinct possibility.

The following review of Entamoeba invadens, a protozoan parasite causing disease in captive snakes, aims to provide the clinician with the information required to successfully recognise, understand and treat this highly pathogenic infection, termed amoebiasis.

SUMMARY

Of the thirty-one different species of nonhaemic protozoa isolated from the tissues and/or faeces of reptiles, most are ubiquitous and non- pathogenic, several forming a beneficial component of the normal microflora of many reptiles. However, one of the most significant protozoan parasites of snakes in captivity is Entamoeba invadens .

The protozoan Entamoeba invadens belongs to the subphylum Sporozoa, class Rhizopodea and order Amoebida. According to Keymer (1981) and Frye (1991) Entamoeba invadens interacts non-pathogenically with 'many species of herbivorous and omnivorous reptiles belonging to the orders Chelonia (tortoises and terrapins), Squamata (lizards, skinks, geckos and slow worms) and Crocodilia (crocodiles).' Such observations not only illustrate the well developed, commensal host-parasite relationships that naturally exist between Entamoeba spp. and certain reptiles, but also that the possibility exists for healthy carriers to shed Entamoeba invadens cysts which may provide reservoirs of infection for the more susceptible snakes.

There is little point in compiling a list of genera containing susceptible species of snake, because all totally carnivorous reptiles are potential though inappropriate, hosts for E. invadens and are highly susceptible to the pathogenic effects of this protozoan parasite.

AMOEBIASIS IN SNAKES

Parasitic disease in snakes due to Entamoeba invadens was first recorded by Ratcliffe and Geiman (1934) and there have been a considerable number of further reports of the infection, a particularly good description of the disease being that by Donaldson, Heyneman, Dempster and Garcia (1975). The organism has been the subject of detailed research, initially by medical protozoologists because of its close relationship to the human pathogen Entamoeba histolytica .

Host Specificity and Species

There does not appear to be any particular host-susceptibility or resistance with reference to the genus of the snake host, and although boas and pythons seem to predominate in clinical outbreaks, this may simply be a reflection of the popularity of these snakes in captivity.

The pathogenicity of E. invadens is a result of its poor adaptation to the gastro-intestinal tract of the purely carnivorous snakes. In particular, it appears that suitable conditions for intestinal commensalism only exist in herbivorous or partly herbivorous reptiles, since polysaccharides of plant origin are essential if the amoebae are to accumulate particulate plant carbohydrate which is a prerequisite for mass encystation (refer to Biology and Life Cycle and Clinical Signs and Pathology) in vitro and in vivo.

Distribution and Incidence

E. invadens appears to have a world-wide distribution among reptiles in captivity with numerous reports of epizootic outbreaks demonstrating very high rates of morbidity and mortality. Such epidemiological and pathological characteristics ensure that E. invadens is considered the most dangerous protozoan disease of captive snakes, indeed it is probably the most important of all reptilian infectious diseases.

Biology and Life Cycle

The life cycle of E. invadens is direct without intermediate hosts. Fully-developed cysts with four nuclei (Figure 1) are the infective stage and are ingested with contaminated water and, more rarely, with food. The cysts may be actively transported by various arthropods commonly found in zoological exhibitions. Cysts ingested by cockroaches, flies, ants and crickets, pass unaffected through the insect digestive tract, and result in viable, infective cysts being deposited with the insect’s faeces in another location. In such circumstances insects may, as transport hosts, play an important role in the epidemiology of the disease, especially in large zoological collections. Cysts may also be inadvertently transported from one snake to another by the keeper’s contaminated hands, footwear, and utensils.

When ingested by a snake, the cysts, measuring 9-24um, pass through the stomach and start to encyst in the small intestine. From each cyst a single trophozoite (9- 38.6um) emerges as a quadrinucleate amoeba. The four nuclei and I cytoplasm within the trophozoite divide to form eight small uninucleate amoebae (3.5-7.3um) which pass into the colon and mature to form fully developed trophozoites. The trophozoites multiply by binary fission in the colon and, after repeated cell divisions, attempt to encyst. Encystation cannot occur normally in the colonic lumen, and can only be accomplished by penetration and invasion of the intestinal mucosa, with the subsequently produced cysts expelled in the faeces. Cysts undergo no further development until ingested by another susceptible host where the life-cycle is repeated.

McConnachie (1975) discovered that cysts could survive for at least 14 days at 8°C but for less than 7 days at 37°C, thereby providing additional testimony to the adaptation of E. invadens to the poikilothermic reptiles, compared with E. histolytica in mammals which display an almost reversed temperature preference.

Clinical Signs and Pathology

During the terminal phase of the ingestion, the multiplying trophozoites attempt to encyst in the colon. In vitro and in vivo studies by McConnachie (1955) and Meerovitch (1957) demonstrated that the presence of plant polysaccharides in the large intestine of herbivorous or partly herbivorous reptiles permitted the acquisition of particulate carbohydrate matter by the amoebae, and hence facilitated mass encystation within the colonic lumen. In the intestine of totally carnivorous reptiles, the snakes, no such polysaccharides are readily available and therefore the amoebae, being compulsory phagotrophs, resort to feeding on the mucus secretions of the intestinal epithelium and invasion of the tissues in order to fulfil the prerequisite of encystation and perpetuate their life cycle. It is this fact that accounts for the severe pathology and subsequent high morbidity and mortality associated with amoebiasis in snakes.

E. invadens cysts are most often ingested by snakes as a result of water contaminated with the faecal material from either healthy carriers or previously infected snakes. This protozoan subsequently induces a fulminating enteritis, hepatitis and, occasionally, nephritis.

There are very few specific or pathognomonic signs attributable to amoebiasis in reptiles. Clinical signs are most often observed during the terminal phase of the disease and are related to lethargy, regurgitation of undigested food and severe diarrhoea, often accompanied with blood or bile-tinged green mucus, and/or remnants of intestinal mucosa. Food is often refused and water intake may significantly increase, indicating dehydration with hepatic and renal insufficiency. On clinical examination, the cloacal region may be swollen and firm, with the inflamed colon clearly palpable within the live snake.

At necropsy, the classical lesions occur in the colon and lower small intestine and are characterized by inflammation, erosion, ulceration, haemorrhage and foci of necrosis, with congestion apparent during the more acute stage. As the disease progresses, the ulceration leads to granulomatous thickening of the intestinal ‘mucosa, which may involve the entire colon and lead to occlusion of the lumen. The foci of necrosis coalesce and the sloughed epithelium combines with the fibrinous exudate to form a fibrinonecrotic pseudomembrane. In very severe infections, intestinal ulceration may extend from the epithelium to the serosal surface, resulting in perforation of the intestine and subsequent acute peritonitis. Gastric lesions are often observed, consisting of congestion of the epithelium, ulceration and haemorrhage. However, these lesions are rarely as severe as those affecting the small intestine and colon. The liver may also be affected being swollen and mottled in appearance. Multiple, discrete and partly confluent areas of liver necrosis are often observed, these varying enormously depending upon the intensity of infection.

Histopathologically, the characteristic lesions attributable to E. invadens are initiated by a circumscribed desquamation of the epithelial lining of the colon leading to a necrotic colitis. Donaldson et aI (1975) reported multiple necrotic ulcers extending through the mucosa and submucosa and occasionally deep into the muscularis, with hyperaemia present throughout the affected intestine. The necrotic foci within the liver varied in size, with lesions extending out from the portal vessels. Central areas of hepatic necrosis also occurred, surrounded by areas of fatty degeneration where the parenchymal cells had been destroyed and only the stroma remained. Nonspecific microscopic changes were also observed in the kidneys.

Donaldson et al (1975) confirmed the pathogenesis of E. invadens by culturing E. invadens in vitro and experimentally infecting a healthy juvenile boa (Boa constrictor constrictor). The snake exhibited the characteristic clinical signs and shed E. invadens cysts before dying 3 days after inoculation. Necropsy revealed an acute enteritis and hepatitis, consistent with the account given above.

Diagnosis

The disease can only be accurately diagnosed by identification of the parasite in the faeces. Both trophozoites an cysts may be found, although the former can only be recognised in fresh faecal samples when mixed with normal saline and viewed microscopically at 37°C. Cysts may be found either by direct microscopic examination, especially in heavy infections, or after floatation/concentration procedures.

The cysts can be stained with double-strength Lugol’s iodine solution (4% potassium iodide plus 2% iodine in distilled water).

Such coprological procedures will readily identify cysts shed in the faeces, but, differentiating between other protozoan parasites of the class Rhizopodea, such as Acanthamoeba, Endolimax, Hartmanella and Naegferia, may present further complications. Modern immunofluorescent antibody techniques and electrophoretic iso-enzyme patterns are specialized laboratory procedures which enable an accurate, species-specific, identification of the protozoan in question. However, such diagnostic practices are not normally available to the majority of veterinary clinicians. Nevertheless, since E. invadens is considered the sole, truly pathogenic, amoeba of snakes, differential diagnosis is only a problem while attempting to trace healthy carriers, when it is essential to distinguish it from other species of amoebae.

From the clinical perspective, any snake excreting Amoebida cysts and exhibiting clinical signs compatible with those already discussed should, most definitely, be considered as infected with Entamoeba invadens , especially as morbidity and mortality rates of 100% are common if intervention and treatment are not immediately instituted.

PREVENTION and TREATMENT

Meticulous attention to hygiene and the prevention of faecal contamination of food and water is essential for the prevention of amoebiasis. All personnel associated with reptiles should thoroughly disinfect their footwear and utensils, and wash their hands when moving from one cage to another.

The phenomenon of arthropod transport hosts is another means of transmission which should be addressed. Effective control by the use of insecticides to reduce insect populations is a practical option. Only insecticides that are known to be relatively non-toxic in the vicinity of snakes, such as Nuvan Staykil spray (iodofenphos 2.0% w/w, 0.2% dichlorvos) or Vapona strips (plastic impregnated with dichlorvos 4.5g) should be used. Nevertheless, great care must always be taken when using insecticides in the presence of reptiles. These procedures are particularly important in large zoological collections if epizootic outbreaks, such as that describe by Donaldson et al (1975), are to be avoided.

Various therapeutics, such as dimetridazole, chloraquine, and oxytetracycline, have been used to treat amoebiasis in snakes, To date, however, the most effective and reliable amoebicide with fewest side effects is metronidazole. This drug possesses the additional advantages of low cost, and at the dosage recommended by Donaldson et al (1975) of a single oral treatment of 275 mg/kg body weight, the required volume can be administered via a lubricated gastric tube. However, Frye (1991) found that a single oral dose of 125mg/kg was fully effective, while Keymer (1981) reported success using 1 60mg/kg daily for three consecutive days. Lawton (1991) has recently commented that the repeated use of small doses of metronidazole may produce neurological signs, and subsequently recommended using a single high dose as soon as the disease is detected or suspected. On account of the seriousness of amoebiasis in snakes, the prophylactic use of metronidazole at 125 mg/kg (Donaldson et al 1975) should be advocated, especially when considering valuable individuals (Figure 2) and the danger of an epizootic outbreak in a large population.

Concern over the use of oral metronidazole has been raised concerning neurological and carcinogenic side-effects. For example, in an attempt to eradicate amoebiasis from the Institute for Herpetological Research, California, the prophylactic use of metronidazole resulted in adenocarcinoma of the colon in three pythons (Ross and Marzec, 1990). Despite these occasional and often unsubstantiated reports of adverse effects or lack of efficacy, the majority of cases respond very well to metronidazole, which remains the drug of choice for amoebiasis.

In addition to metronidazole therapy, supportive medical care should also be provided in the form of fluids and multivitamins, specifically to alleviate the problems of dehydration and prevent visceral gout.

CONCLUSION

Arnoebiasis is a parasitic disease of captive snakes that can spread through an entire collection with dire consequences. However, by understanding the biology of E. invadens , preventative measures can be established, but should these fail, the successful recognition and treatment of the disease with metronidazole (125-275mg/kg per Os) and supportive therapy will reduce the levels of morbidity and mortality and quickly resolve the predicament.


REFERENCES

Donaldson, M., Heyneman, D., Dempster, R. and Garcia, L. (1975). Epizootic of Fatal Amoebiasis Among Exhibited Snakes: Epidemiologic, Pathologic, and Chemotherapeutic Considerations. American Journal of Veterinary Research, 36: 807-81 7.
Frank, W. (1984). Non-Hemoparasitic Protozoans, In: Diseases of Amphibians and Reptiles (Hoff, G.L., Frye, FL. and Jacobson, E.R., Eds) New York, Plenum; 259-384.
Frye, FL. (1991). Applied Clinical Nonhemic Parasitology of Reptiles. In: Reptile Care: An Atlas of Diseases and Treatments Volume 1 (Frye. FL., Ed) New Jersey, TFH Publications, 278-325.
Keymer, IF. (1991). Protozoa. In: Diseases of the Reptilia Volume 1 (Cooper, J.E. and Jackson, OF., Eds) New York, Academic Press; 233-290.
Lawton, M.P.C. (1991). Reptiles-Part Two Lizards and Snakes. In: Manual of Exotic Pets (Beynon, PH. and Cooper, J.E., Eds) DSAVA Publication; 244-260.
McConnachie, E.W. (1955). Studies of Entamoeba invadens Rodham, 1934, in vitro, and its Relationship to Some Other Species of Entamoeba Parasitology, 45; 452-481.
Meerovitch, E. (1957). On the Relation of the Biology of Entamoeba invadens to its Pathogenicity in Snakes. Journal of Parasitology, 43: Suppl, 41 no.108.
Ratcliffe, H.L. and Gieman, Q.M. (1934). Amoebiasis in Reptiles. Science, 79: 324-325.
Ross, R.A. and Marzec, G. (1990). Chapter 2 Reproductive Husbandry. In: The Reproductive Husbandry of Pythons and Boas (Ross, R.A. and Marzec, G., Eds) Institute of Herpetological Research, California; 73


Reproduced with Kind permission of the Reptilian Magazine.
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