Abstract

Cryptosporidiosis is an illness of which very little is known in Mexican wildlife; no reports of this illness in reptiles have been found in our country. This paper describes the first outbreak of cryptosporidiosis in the collection of reptiles in the Guadalajara Zoo. In November of 1996 a Pituophis deppei jani was diagnosed with the illness by analyzing gastric washings. Oocysts of Cryptosporidium spp. were observed using the modified acid-fast stain technique. From that case in November through February 1997, nine deaths caused by this protozoan have occurred. Also, five asymptomatic carriers have been located, two of which have died from other causes although the presence of this protozoan was detected.

Introduction

Cryptosporidiosis is an illness caused by a small protozoan that belongs to the genus Apicomplexa. The Cryptosporidium has been found in birds, mammals, reptiles, amphibians and fish. In reptiles, Cryptosporidium serpentis is a relatively recently identified parasite that causes considerable mortality,^1-5,7,8 and Cryptosporidium infection may last years in a latent or subclinical state. Infection may also cause chronic debilitation of the reptile or shedding of the organism in response to stress or immune suppression.³ Cryptosporidium has sometimes been associated with animals that have recently arrived at an institution. This protozoan has also been detected in wild reptiles but in a much lower percentage than captive animals.¹ In the clinical illness, the parasite infects the stomach causing mucosal cell inflammation, edema and hypertrophy, as well as hemorrhage and focal necrosis. The typical clinical signs are chronic regurgitation, weight loss and emaciation, and secondary bacterial infections.^1,2,4,5 Diagnosis is made by endoscopy, ultrasonography or radiography, and it can be confirmed by gastric biopsy or cytology of gastric washings, regurgitated prey, cloacal/colon washings and feces. Cytology specimens are stained with modified acid alcohol resistant stain and scanned for oocysts.^1,2,4 Recent studies have shown that the use of monoclonal antibody tests for Giardia and human Cryptosporidium have a cross reaction with C. serpentis and may be useful in detecting infection in reptiles.

False negative results with cytology or other ante-mortem tests are not uncommon. Necropsy and histopathological findings can confirm the ante-mortem diagnosis.

There is no known effective treatment. The disease is considered to have zoonotic potential even though no confirmed zoonoses have been seen.^1,2 This is a report of an outbreak of cryptosporidiosis and its epidemiologic implications in the reptile collection at the Guadalajara Zoo. The zoo has maintained a collection of 350–500 specimens of reptiles since its opening in 1988. To our knowledge, this is the first report of cryptosporidiosis in a Mexican zoo.

Case Report

Case 1

In November of 1996 a 2-yr-old snake of the species P. deppei jani, born in this institution, was presented to the zoo clinic. History included regurgitation of 6 wk duration, weight loss and emaciation. The physical examination revealed dehydration and mid-body swelling. Two weeks previously, coproparasitoscopic studies by flotation were negative. A gastric wash was performed and material stained by modified Kinyoun technique. An abundance of acid alcohol resistant oval forms were seen. A tentative diagnosis of cryptosporidiosis was made. The serpent had been housed individually inside a room with 35 other reptiles. The snake was euthanatized 2 days after presentation. The necropsy showed poor nutritional condition and mild gastric mucosa erythema. Histopathologic findings included inflammation of the gastric mucosal cells with hemorrhage and slight necrosis.

Case 2

One day following the euthanasia of the snake in case 1, a Boa constrictor constrictor died. The clinical history included intermittent periods of anorexia and regurgitation for more than a year. The snake had been donated to the zoo from an unknown source. Routine fecal screening for parasites had been previously negative. It had been kept in the same area as the snake from case 1.

Necropsy findings revealed petechial hemorrhages and necrosis of the stomach. Cytology of the stomach contents confirmed the presence of Cryptosporidium using the same stain technique as in case 1. Histopathology showed gastric submucosa edema, mild gastric epithelial degeneration, cellular infiltration of heterophils and eosinophils, atrophy and loss of granular cells, necrosis of the apical surface of the enterocytes and many spherical to ovoid organisms adherent to microvillar borders of surface, pit and glandular epithelium suggestive of Cryptosporidium. Other findings were renal amyloidosis with hydropic and fatty degeneration of the tubular epithelium and severe hepatic lipidosis.

Case 3

In December of 1996 a specimen of Crotalus atrox presented with weight loss and regurgitation of 1 mo duration. Gastric washing found the presence of Cryptosporidium. The animal died 2 wk later. The necropsy and histopathological findings were similar to those in case 2. This snake had been donated to the zoo in 1989. In June of 1995 it had presented with an illness characterized by regurgitation and dysecdysis. Treatment was initiated with metronidazole (Flagyl, Rhone-Poulenc Rorer, Mexico D.F.) p.o., Kaobiotic (The Upjohn Company, Mexico D.F.) p.o., Baytril (Bayer of Mexico, Mexico D.F.) i.m. and fluid therapy. Furthermore, this snake was isolated in the same room in which the snakes in cases 1 and 2 were later isolated. The snake made a clinical recovery by April 1996. In November 1996 regurgitation recurred and the snake was transferred again to the same isolation room.

During the following 2 mo nine animals died with the presence of Cryptosporidium: B. constrictor constrictor (n=1), Pituophis deppei jani (n=1), Trimeresurus gramineus (n=1), Sistrurus ravus ravus (n=2), C. atrox (n=1), Crotalus molosus nigrensis (n=1), Crotalus aquillus (n=1), and Mastticophis flagellus (n=1). Two other animals that died of other causes were found to be infected with Cryptosporidium. Three asymptomatic carriers have been found in a group of five rattlesnakes. The protozoan has now been found in all areas of the serpentarium.

Discussion

This is the first finding of Cryptosporidium in the Guadalajara Zoo. There are no reports of necropsy filed prior to 1994. Previously no tests for Cryptosporidium were done in quarantine since there had been no reports of disease in Mexico.

The two factors that are necessary for the onset of cryptosporidiosis are the reproduction of the organism and a state of stress or immune suppression in the host. The snake in case 3 likely reproduced and disseminated the protozoan during the illness it suffered in 1995. The source of the organism in this outbreak is unknown but it is probable that Cryptosporidium is widely distributed in Mexico. Most of the cases of Cryptosporidium at our zoo have occurred in the two places occupied by the snake of case 3.

Besides the fact that they are in captivity which in itself offers some stress, there are other factors in our serpentarium that affect the immune status of the animals.

The temperature and humidity in the shelters of some of these snakes is inadequate. Some are not provided with ultraviolet light of sun baths. However, the factor that we consider the most important in predilection for stress and infection is obesity. The snakes in our collection tend to be overfed with varying degrees of obesity. The presence of fatty livers on necropsies has been a frequent finding on necropsies since 1994 (in some occasions the only finding). A few months before case 1, the bioterium, (the area of the zoo that provides rodents to the serpentarium) began substituting commercial dog food with a high fat content for the rodents instead of the previous rodent food. This added to the overfeeding of the reptiles, may have caused some hepatic lipidosis in the snakes. Vitamin E deficiency may have played a role as well as the unsaturated fats of the rodents competed for this essential antioxidant. These factors may have decreased the general state of health of the reptiles, diminished their immune status making them susceptible to illness including cryptosporidiosis.

Preventative measures have been initiated to inhibit the spread of parasites. Quality, quantity and frequency of food items has been altered. Vitamin E supplements are also being used. Hygiene procedures are being used in the serpentarium to prevent dissemination of Cryptosporidium. Quarantine procedures have been reinforced and testing for Cryptosporidium during quarantine initiated. Carriers or sick animals are isolated and evaluated so that in the case of a severe illness they may be euthanatized. The control of temperature and humidity has been improved. Testing of animals from other areas of the country will aid us in understanding the state of Cryptosporidium in Mexico. To this date no treatment has been initiated for sick species. Special care is taken against the risk of zoonosis for the workers as well as disinfection of utensils and work areas.

Acknowledgments

We thank Dr. Francisco Rodriguez Herrejon, Director of the Guadalajara Zoo; Dr. Pablo Varela, Head, Veterinarian Services of the Guadalajara Zoo; the staff of Area Tecnica and Herpetario of the Guadalajara Zoo; Dr. Oscar H. Sanchez Molgado, Pathologist, for his assistance; and especially Mrs. Barbara Keenan for the translation of the manuscript.

Literature Cited

1.  Agnew DW. 1992. Cryptosporidiosis in reptiles - A review. Infectious Disease Reviews. American Association of Zoo Veterinarians.

2.  Cranfield MR, Graczyk TK. 1995. An update on ophidian cryptosporidiosis. Proc Joint Conf Am Assoc Zoo Vet/Wildl Dis Assoc/Am Assoc Wildl Vet. Pp. 225–230.

3.  Brownstein DG, Strandberg JD, Montali RJ, et al. 1977. Cryptosporidium in snakes with hypertrophic gastritis. Vet Pathol. 14:606.

4.  Cranfield MR, Graczyk TK. 1996. Cryptosporidiosis. In: Mader DR. Reptile Medicine and Surgery. WB Saunders Company. Pp. 359–363.

5.  Frost DF, Nichols DK, Citino SB. 1994. Gastric cryptosporidiosis in two ocellated Lacertas (Lacerta lepida). J Zoo Wildl Med. 25(1):138–142.

6.  Graczyk TK, Cranfield MR, Fayer R. 1995. A comparative assessment of direct fluorescent antibody, modified acid fast stain, and sucrose flotation techniques for detection of Cryptosporidium serpentis oocysts in snake fecal specimens. J Zoo Wildl Med. 26(3):396–402.

7.  Klingenberg RJ. 1996. Enteric cryptosporidiosis in a colony of indigo snakes, a panther chamaeleon, and a savannah monitor. Bull Assoc Reptilian and Amphibian Vet. 6(1):5–9.

8.  Tilley M, Upton SJ, Freed PS. 1990. A comparative study on the biology of Cryptosporidium serpentis and Cryptosporidium parvum. J Zoo Wildl Med. 21(4)L 463–467.