Criptosporidiosis en serpientes

1. Etiology

Cryptosporidiosis is caused by parasites of the apicomplexan genus Cryptosporidium, which has a worldwide distribution (Xiao et al, 2004). Cryptosporidium serpentis, which is found in the gastric epithelium of snakes (Koudelaand, 1998; Pavlasek, Ryan, 2008; Traversa, 2010 and Slapeta, 2013).

2. Life cycle

The following life cycle is described for Cryptosporidium parvum in a mammalian host. The actual life cycle of C. serpentis has not been described. Infection occurs when the animal ingests a thick-walled, sporulated oocyst that excysts within the lumen of the stomach or intestine, producing four sporozoites, which penetrate the mucosal epithelial cells and develop into trophozoites inside the parasitophorous vacuoles. The trophozoites undergo asexual divisions to form merozoites. The merozoites invade nearby cells and either undergo asexual reproduction of more merozoites or form type II meronts, which in turn form the sexual stages, microgamonts and macrogamonts. After fertilization a zygote is formed and may develop into a thin-walled oocyst that is autoinfective or a thick-walled oocyst that is passed from the host. The cysts are infective immediately on passing from the host (Current, 1999).

3. Transmission

The mode of Cryptosporidium transmission in reptiles is the fecal-oral route from one animal to the next by direct contact or through contact with contaminated objects (Cranfield et al.1999 and Graczyk et al, 2000). In mammals, it is also considered a water-borne disease, with water sources contaminated by C. parvum from cattle or deers. This route is not likely important to reptiles in captivity. In a study at the Baltimore Zoo in which 2000 oocysts were given via stomach tube to healthy snakes, five of nine became fecal positive between 8 to 28 weeks (average, 14 weeks) after infection. Dexamethasone administration at 1 mg / lb for 5 days before and after dosing the snakes increased the rate of infection (Cranfield, 1992).

4. Clinical sings

Subclinical (carrier state)

Healthy snakes may be subclinically infected for years but capable of shedding the organism intermittently.

5. Clinical (Gastric Hyperplasia of the Mucus Secreting Cells)

The usual history in clinical cases of snakes is weight loss with persistent or periodical postprandial regurgitation of undigested mice 3 to 4 day ingestion. These clinically affected snakes may live days to years. Clinically affected snakes have a noticeable swelling in the gastric region, located half the distance between the mouth and the cloaca. In snakes the disease differs from that in mammals and birds in that it appears to affect snakes of all ages and it not acute and self-limiting. (Cranfield et al.1999 and Graczyk et al, 2000).

The clinical signs most commonly observed in infected snakes include chronic postprandial regurgitation occurring one to three days after feeding, together with a firm mid-body swelling associated with hypertrophic gastritis and chronic weight loss. The clinical course is protracted and almost always fatal.

6. Diagnosis

· Examination of Feces or Mucus from Regurgitated Food Items

The acid-fast stain and sugar flotation techniques have been published elsewhere. The use of a commercially available immunofluorescent antibody (IFA) stain (Merifluor, Meridian Bioscience, Cincinnati, OH 45244) increases the sensitivity of the test approximately 16-fold over acid-fast staining for C. serpentis.The Merifluor IFA reacts weakly with the type of Cryptosporidium sp. Typically found in geckos. A positive finding on fecal or gastric wash samples does not necessarily indicate infection with C. serpentis or C. saurophilum. A negative finding may merely indicate lack of shedding (Graczyk et al, 1995).

· Gastric lavage

In reptiles with mild subclinical infections, gastric lavage provides a better sample than feces or mucus. The lavage sample is best performed 3 days after feeding the snake. The increased gastric metabolism after a feeding is thought to increase Cryptosporidium metabolism, resulting in more parasites available for detection (Graczyk et al, 1996).

· Serum Antibody Titers

Snakes take 6 weeks after infection to develop antibodies to Criptosporidium. The presence of serum antibodies in snakes indicates exposure to the organism. Because most infections are long term a positive serum Elisa test represent either exposure or active infection of Cryptosporidium serpenti. The best method is combination of either fecal or gastric lavage with Elisa. If both serum test and fecal test results positive, the animal is infect and actively shedding C. serpentis. If both results are negative then the animal is Cryptosporidium negative.

· Real-time PCR

The real-time PCR is high sensitivity and specificity for C. serpentis; it represents a rapid and specific alternative for diagnosing C. serpentis infection from fecal samples. PCR followed by sequencing is still the method of choice (Da Silva, 2014).

· Gastric biopsy

It is not possible to determine whether cysts obtained from feces or gastric lavage are nonreptilian Cryptosporidium sp. cysts passing through the gastrointestinal (GI) tract, and therefore not infective to the host, or whether the cysts are C. serpentis or C. saurophilum, and thus represent an infection. Histopathology samples collected by gastric biopsy indicating that the gut epithelial cells are infected are necessary to prove the snake has cryptosporidiosis. Gastric biopsy may be performed using endoscopy or surgery (Denver, 2008).

· Necropsy

The pathologic changes seen grossly have been covered extensively in the literature and include increase in the diameter of the stomach, edematous rugae, thickening of the gastric mucosa, decreased lumen size, mucosal petechiae, and focal necrosis in predominantly gastric cases and mild to severe enteritis in other cases (Brower, 2001 and Brownstein, 1977).

· Histopathology

Histologically, in infected animals, the organisms are seen on the brush border of the epithelial cells. There may be little damage to the architecture in subclinical cases, or substantial changes may occur, including loss of the brush border, hyperplasia and hypertrophy of gastric glands, proliferation of gastric mucous cells, edema and inflammation of the submucosa and lamina propria, reduced luminal diameter, and inflammation of the mucosal layer (Brower, 2001 and Brownstein, 1977).

7. Differential diagnoses

Regurgitation due to husbandry related issues such as low ambient temperature and overhandling, Neoplasia, metazoan parasitism, viral gastroenteritis and abscessation.

8. Treatment

Cryptosporidiosis is extremely difficult to eradicate. Anticoccidial drugs are not effective at eliminating Cryptosporidium at nontoxic doses in humans or animals. Some pharmacologic agents may reduce the number of organisms or eliminate fecal shedding, but the organism is still present in the GI tract (Denver, 2008).

No commercially available effective treatments to eliminate the parasite in reptiles are documented in the literature. Trimethoprim sulfa (SMA-TMP Biocraft Laboratories Elmwood Park, NJ; 30 mg/kg) once a day for 14 days and then one to three times weekly for several months, spirarnycin (160 mg/kg) for 7 days and then twice a week for 3 months (author's experience), and paromomycin (100 mg /kg) for 7 days and then twice a week for 3 months were effective at reducing clinical signs and reducing or eliminating oocyte shedding. Although spiramycin and paromomycin treatment produced negative fecal results, postmortem results revealed cryptosporidial organisms still present on gastric mucosa. Necropsies were not done after the other treatment regime, although gastric biopsies collected after trimethoprim sulfa treatment had negative results (Graczyk, 1996).

In each of these treatment regimes, the snakes were given extensive supportive treatment, such as high environmental temperatures (80°F), subcutaneous fluids, and regular stomach tubing with highly digestible foods. These added treatments may have influenced the course of the disease or been synergistic with treatment. (Cranfield and Graczyk, 2006)

The most promising treatment for reptilian cryptosporidiosis to date has been the use of hyperimmune bovine colostrum (HBC); however, this product is not commercially available. HBC eliminated shedding but did not eliminate the organisms from the stomachs of snakes or the intestines of geckos within a 6-week treatment period. However, a longer treatment period may have succeeded in eliminating the organisms. HBC did eliminate infection in savanna monitors. (Graczyk, et al., 1998; Graczyk, et al., 1999 and Graczyk, et al., 2000).

9. Control

The safest way to eliminate cryptosporidiosis from a collection is through strict quarantine protocols with rigorous testing procedures and elimination of infected individuals. Reptiles with subclinical infections may appear healthy but serve as a source of infection for other reptiles for many years (Denver, 2008).

Infectivity of oocysts was neutralized with exposure to moist heat between 45°C and 60°C for 5 to 9 minutes. Because oocysts stored at 4°C remain infective for 2 to 6 months, cleanliness and removal of organic matter should be emphasized. (Campbell, 1982). Crates, pens, feeding bottles, and utensils should be thoroughly cleaned with an ammonia solution and allowed to dry for a period of at least 3 days (Cranfield et al.1999 and Graczyk et al, 2000).

Endangered reptiles with cryptosporidiosis that are not considered candidates for euthanasia, even if infected, should be kept strictly isolated from noninfected reptiles(Denver, 2008).

Periodic testing and treatment when shedding may help prolong the quality of life and breeding potential of these endangered reptiles and may reduce transmission to the rest of the collection (Denver, 2008).

No efficacious cornmercially available treatment for cryptosporidiosis in reptiles has been found, although prornising results from supportive care and treatment with spiramycin or paromomycin in reptiles have been obtained. Hyperirnmune bovine colostrum treatment on the basis of passive irnmune transfer has been shown to be the best treatment in snakes and certain species of lizards. Hyperimmune bovine colostrum treatment may become cornmercially available in the near future to treat reptilian cryptosporidiosis.

10. Bibliography

· Jacobson ER. Parasites and Parasitic Diseases of Reptiles. In: Elliott R. Jacobson (ed.), Infectious Diseases and Pathology of Reptiles; CRC press, 2007: 575_578

· Cranfield, M. and Craczyk, T. Cryptosporidiosis. In: Mader DR (ed.), Reptile Medicine and Surgery, 2nd ed., St Louis: Saunders Elsevier, 2006: 756-761.

· Denver, M. Reptile Protozoa: In Fowler, M. and Miller, E. Zoo and Wild Animal Medicine Current Therapy. Edition 6. 2008. Saunders. Missouri, pp- 154-159.

· Brower AI, Cranfield MR. Cryptosporidium sp.–associated enteritis without gastritis in rough green snakes (Opheodrys aestivus) and a common garter snake (Thamnophis sirtalis), J Zoo Wildl Med 32(1):101-105, 2001.

· Graczyk TK, Cranfield MR, Fayer R: A comparative assessment of direct fluorescence 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, 1995.

· Current WL: Cryptosporidium species. In Fowler ME, Miller RE, editors: Zoo and wild animal medicine: current therapy, ed 4, Philadelphia, 1999, Saunders, pp 121-131.

· Koudela, B., Modr´y, D., 1998. New species of Cryptosporidium (Apicom-plexa: Cryptosporidiidae) from lizards. Folia Parasitol. (Praha) 45,93–100.

· Pavlasek, I., Ryan, U. 2008. Cryptosporidium varanii takes precedence overC. saurophilum. Exp. Parasitol. 118, 434–437.

· Slapeta, J., 2013. Cryptosporidiosis and Cryptosporidium species in ani-mals and humans: a thirty colour rainbow? Int. J. Parasitol. 43,957–970.

· Traversa, D., 2010. Evidence for a new species of Cryptosporidium infectingtortoises: Cryptosporidium ducismarci. Parasit. Vectors. 3, 21.

· Pavlasek, I., Ryan, U., 2008. Cryptosporidium varanii takes precedence overC. saurophilum. Exp. Parasitol. 118, 434–437.

· Xiao L, Ryan UM, Graczyk TK, et al: Genetic diversity of Cryptosporidium spp. in captive reptiles, Appl Environ Microbiol 70(2):891-899, 2004.

· Brownstein DG, Strandber JD, Montali RJ, et al: Cryptosporidium in snakes with hypertrophic gastritis, Vet PathoI14:606, 1977.

· Graczyk TK, Crruúield MR: Cryptosporidiurn serpentis oocysts and microsporidian spores in stools of captive snakes, JParasitol 86(2):413, 2000.

· Klingenberg, R. (2007) Understanding Reptile Parasites: From the Experts at Advanced Vivarium Systems.Irvine.CA.

· Cranfield MR, Graczyk TK, Wright K. Cryptosporidiosis, Bull Assoc. Reptl Amphib Vet 9(3):15, 1999.

· Cranfield MR, Ialeggio DM, Noranbrook R. Cryptosporidiosis, Proc 7th Avian Exotie Animal Medicine Symposiurn, Davis, Calif, 1992.

· Graczyk TK, Owens R, Cranfield MR. Diagnosis of subclinical cryptosporidiosis in captive snakes based on stomach lavage and cloacal sampling, Vet Parasitol 67(3-4):143, 1996.

· Da Silvaa, D.; Philipp Paivab, R.; Nakamuraa, A. Homema, C.; Sato, M.; Fernandes, K.; Vasconcelos, G. The detection of Cryptosporidium serpentis in snake fecal samples by real-time PCR. Veterinary Parasitology 204 (2014) 134–138.

· Graczyk TK, Cranfield MR, Hill SL. Therapeutical efficacy of piramycin and Halofuginone treatment against Cryptosporidizl1lt serpentis (Apicomplexa: Cryptosporüdiidae) infections in captive snakes, Parasitol Res 82(2):143, 1996.

· Graczyk TK, Cranfield MR, Bostwick EF. Hyperimmune bovine colostrum treatment of moribund leopard geckos (Eublepharis macularius) infected with Cryptosporidium sp., Vet Res 30:377-382, 1999.

· Graczyk TK, Cranfield MR, Bostwick EF. Successful hyperimmune bovine colostrum treatment of Savanna monitors (Varanus exanthematicus) infectedwith Cryptosporidium sp., J Parasitol 86(3):631-632, 2000.

· Graczyk TK, Cranfield MR, Helmer P. Therapeutic efficacy of hyperimmune bovine colostrum treatment against clinical and subclinical Cryptosporidium serpentis infections in captive snakes, Vet Parasitol 74(2-4):123-132, 1998.