Abstract

Therapy based on the protective passive immunity of hyperimmune bovine colostrum (HBC) (raised against Cryptosporidium parvum in dairy cows immunized during gestation) was tested for heterologous efficacy in subclinical and clinical C. serpentis infections of 14 captive snakes, 13 moribund leopard geckos (Eublepharis macularius), 5 Indian star tortoises (Geochelone elegans), and one bog turtle (Clemmys muhlenbergii) infected with Cryptosporidium sp. Six gastric colostrum treatments of 1% body weight at 1-wk intervals each, have histologically cleared C. serpentis in three subclinically infected snakes, and regressed gastric histopathologic changes in one of these snakes. In all snakes, each subsequent colostrum treatment significantly decreased the number of oocysts recovered in gastric lavage eluants. The treatments induced oocyst-negative gastric eluants and stools in all snakes, and improved clinical signs of infection. In geckos, seven gastric HBC treatments at 1-wk intervals each decreased the relative output of Cryptosporidium sp. oocysts and the prevalence of oocyst-positive fecal specimens. Histologically, after 8-wk therapy, 7 of 12 geckos had only single developmental stages of Cryptosporidium sp. in the intestinal epithelium, and 3, 1, and 1 gecko had low, moderate, and high numbers, respectively, of the pathogen developmental stages. Three G. elegans subjected to 5-wk HBC therapy remained positive for Cryptosporidium sp. infection as determined by examination of the feces. As the bovine colostrum treatment was safe and highly efficacious in snakes, it is recommended to administer the bovine hyperimmune colostrum to snakes clinically or subclinically infected with C. serpentis, or to use the colostrum for snake supportive therapy or prophylaxis.

Introduction

Cryptosporidium serpentis-associated reptilian cryptosporidiosis is a common and sometimes life-threatening disease of captive snakes.^3-5 The high morbidity and moderate mortality caused by Cryptosporidium in ophidian collections is due to the lack of an anticryptosporidial compound that could be safely and efficaciously used for prophylaxis or therapy.^2,6,10 Halofuginone, spiramycin, and paromomycin therapeutically used for reptilian cryptosporidiosis, reduced the number of voided oocysts, but did not eliminate infection as determined by histologic sections and oocyst-positive feces.^2,5,6,10,17

Protective passive immunity treatment utilizing hyperimmune bovine colostrum (HBC) (raised against C. parvum in dairy cows during gestation) was safe and efficacious in AIDS patients infected with this pathogen.^1,16 Although HBC has been therapeutically suggested for snake cryptosporidiosis,³ there was no data on the efficacy of any colostrum-derived product against Cryptosporidium infections in reptiles. The purpose of the present study was to determine the therapeutic efficacy of hyperimmune bovine colostrum treatments for Cryptosporidium infections in captive reptiles.

Methods

Hyperimmune bovine colostrum (HBC) therapy was administered to captive snakes, geckos, tortoises, and a turtle. Fourteen snakes infected with C. serpentis were randomly divided into a treatment group of 11 snakes and a control group of 3 snakes. All snakes intermittently voided C. serpentis oocysts as determined by the acid-fast stain (AFS)⁹ and the MERIFLUOR^TM Cryptosporidium/Giardia test kit (IFA)¹² for a 6-mo period prior to initiation of the study^7,8,13. Thirteen leopard geckos (Eublepharis macularius) originating from a private collection and infected with Cryptosporidium sp. were transported to the Baltimore Zoo.¹⁵ All geckos were anorectic and moderately to severely emaciated as noted by severe deficiency of fat in their tails; most of them were lethargic.¹⁵ All geckos had a history of persistent voiding of high number of Cryptosporidium sp. oocysts as determined by AFS.¹⁵ Four Indian star tortoises (Geochelone elegans), and one G. elegans and bog turtle infected with Cryptosporidium sp., originated from the Bronx and Baltimore Zoos, respectively. All reptiles were housed as described previously.^2,7

Hyperimmune bovine colostrum was prepared from equal volumes of first and second milking colostrum from a multiparous cow immunized against C. parvum during gestation as described previously.^13,15 Colostrum was administered to reptiles by gastric intubation six times at weekly intervals; the volume of administered colostrum constituted 1% of reptile body weight (ml to g).^13,15 Gastric lavages¹¹ for detection of Cryptosporidium oocysts in the stomach eluants were carried out for all snakes. The lavages were done 3 days after feeding as the recent meal enhances pathogen reproduction processes and increases number of eluted oocysts.¹¹

Oocyst isolates from snakes were subjected to HBC/IFA competition binding assay to determine the binding efficacy of the HBC immunoglobulins to the oocysts.

Results

Gastric eluants of all snakes prior to the initiation of HBC therapy were positive for C. serpentis oocysts and they contained progressively fewer oocysts as the therapy progressed. Stomach eluants of one snake become oocyst-negative after the first colostrum treatment, five snakes had negative gastric eluants after the fourth treatment, and three snakes were negative after the sixth treatment. No Cryptosporidium oocysts were found in the fourth collection of gastric eluants of all treated snakes. Three of five colostrum-treated and euthanatized snakes, subclinically infected prior to the therapy, did not have developmental stages of Cryptosporidium in histologic sections of the gastric region (Table 1). Two other euthanatized snakes had a small number of Cryptosporidium developmental stages on the surface of gastric epithelium (Table 1).

Table 1. Results of therapy based on the protective passive immunity of HBC

The results of therapy based on the protective passive immunity of hyperimmune bovine colostrum raised against Cryptosporidium parvum in dairy cows during gestation applied to captive snakes clinically and subclinically infected with Cryptosporidium serpentis and euthanatized after the therapy. Colostrum administered six times with 1-wk-interval. Control snakes treated with saline.
^aAcid-fast stain
^bImmunofluorescent antibody
^cSevere clinical infection
^dControl snakes

In geckos, no significant differences were observed in intensity of Cryptosporidium sp. infections, decrease of oocyst output, or prevalence of oocyst-positive fecal specimens. All geckos were positive for developmental stages of Cryptosporidium sp. which occurred exclusively in the small intestine.

Three G. elegans subjected to 5-wk HBC therapy remained positive for Cryptosporidium sp. infection as determined by examination of the feces. The feces of one G. elegans and bog turtle became oocyst-negative after HBC therapy.

Discussion

Cryptosporidiosis in reptiles is a potentially devastating disease that threatens large collections and the search for prophylaxis and therapy represents a most vital and urgent need.^3,4,6 To accurately evaluate the therapeutic efficacy of colostrum treatment, we recommend using the most advantageous diagnostic techniques developed in our laboratories specifically for snake cryptosporidiosis in its subclinical phase (i.e., gastric lavage)¹¹ and immunofluorescent antibody (IFA),^9,12 together with histologic detection of the pathogen developmental stages. Also, we recommend performing the HBC/IFA competition binding assay to determine the binding efficacy of the HBC immunoglobulins to Cryptosporidium isolates infecting reptiles.

In all C. serpentis-infected snakes, HBC therapy based on six treatments at 1-wk-intervals resulted in oocyst-negative stools after the fourth treatment.¹³ The stomach eluants obtained 3 days after feeding of the snakes, when the oocyst numbers should be at their peak¹¹, showed decreased oocyst numbers and were negative in all snakes after the sixth treatment¹³. Considering the above facts, we conclude that in snakes the therapeutic efficacy of the HBC treatment was high.¹³

We recommend gastric delivery of colostrum which is relatively simple to perform¹¹, safe for snakes, and assures an efficient use of the costly medication^11,13. As Cryptosporidium in snakes is confined to the gastric region,^2-4 stomach administration delivers immunoglobulin-rich medium directly into the area occupied by the pathogen. Colostrum immunoglobulins that play an essential anticryptosporidial therapeutic role¹³ are labile, and therefore colostrum has to be properly stored and handled to preserve its immunologic activity.

In geckos, the efficacy of HBC was lower than observed in C. serpentis-infected snakes;^13,15 which may be due to the intestinal location of Cryptosporidium sp. infection in these geckos. Besides anticryptosporidial activity of immunoglobulins present in the HBC, the colostrum itself is rich in easy-digestible proteins which when delivered orally may be beneficial for geckos with significant weight lost.¹⁴

Hyperimmune bovine colostrum therapy was the least efficacious in tortoises, which again may be due to the intestinal location of Cryptosporidium sp. infection in tortoises.

Acknowledgments

The study was supported by the Morris Animal Foundation, Englewood, Colorado, grant 98ZO-28.

Literature Cited

1.  Blagburn BL, R Soave. 1997. Prophylaxis and chemotherapy of human and animals. In: R Fayer, ed. Cryptosporidium and Cryptosporidiosis. CRC Press, Boca Raton, FL, pp. 111–128.

2.  Cranfield MR, TK Graczyk. 1994. Experimental infection of elaphid snakes with Cryptosporidium serpentis (Apicomplexa: Cryptosporidiidae). J. Parasitol. 80: 823–826.

3.  Cranfield MR, TK Graczyk. 1996. Cryptosporidiosis. In: DR Mader, ed. Manual of Reptile Medicine and Surgery. W.B. Saunders Company, Philadelphia, PA, pp. 359–363.

4.  Cranfield MR, TK Graczyk. 1999. Cryptosporidia in reptiles. In: N Anderson, D Meloni, JD Bonagura, eds. Current Veterinary Therapy. W.B. Saunders Company, Philadelphia, PA (in press).

5.  Cranfield MR, R Noranbrock, M Skjoldager, DM Ialeggio, S Stahl. 1991. Cryptosporidiosis. In: DR Mader, ed. Proceedings of the 7th Avian and Exotic Animal Medicine Symposium. University of California, Davis Schalm Hall Press, Davis, CA, pp. 183–193.

6.  Cranfield MR, TK Graczyk. 1995. An update on ophidean cryptosporidiosis. In: RE Junge, ed. Proceedings of the American Association of Zoo Veterinarians, Wildlife Disease Association, and American Association of Wildlife Veterinarians. 1995, East Lansing, Michigan, USA, 225–230.

7.  Graczyk TK, MR Cranfield. 1996. Assessment of the conventional detection of fecal Cryptosporidium serpentis oocysts of subclinically infected captive snakes. Vet. Res. 27: 185–192.

8.  Graczyk TK, MR Cranfield. 1997. Detection of Cryptosporidium-specific immunoglobulins in captive snakes by a polyclonal antibody in the indirect ELISA. Vet. Res. 28: 131–142.

9.  Graczyk TK, MR Cranfield, R Fayer. 1995. 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: 396–402.

10.  Graczyk TK, MR Cranfield, SL Hill. 1996a. Therapeutical efficacy of spiramycin and halofuginone treatment against Cryptosporidium serpentis (Apicomplexa: Cryptosporiidiidae) infections in captive snakes. Parasitol. Res. 82: 143–148.

11.  Graczyk TK, R Owens, MR Cranfield. 1996b. Diagnosis of subclinical cryptosporidiosis in captive snakes based on stomach lavage and cloacal sampling. Vet. Parasitol. 67: 143–151.

12.  Graczyk TK, MR Cranfield, R Fayer. 1996c. Evaluation of commercial enzyme immunoassay (EIA) and immunofluorescent antibody (IFA) tests kits for detection of Cryptosporidium oocysts other than Cryptosporidium parvum. Am. J. Trop. Med. Hyg. 53: 274–279.

13.  Graczyk TK, MR Cranfield, P Helmer, R Fayer, EF Bostwick. 1998. Therapeutical efficacy of hyperimmune bovine colostrum treatment against clinical and subclinical Cryptosporidium serpentis infections in captive snakes. Vet. Parasitol. 74: 123–132.

14.  Graczyk TK, MR Cranfield, J Mann, JD Strandberg. 1998. Intestinal Cryptosporidium sp. infection in Egyptian tortoise (Testudo kleinmanni). Intl. J. Parasitol. 28: 1885–1888.

15.  Graczyk TK, MR Cranfield, EF Bostwick. 1999. Hyperimmune bovine colostrum treatment of moribund Leopard geckos (Eublepharis macularius) infected with Cryptosporidium sp. Vet. Res. 30:(in press).

16.  Greenberg PD, JP Cello. 1996. Treatment of severe diarrhea caused by Cryptosporidium parvum with oral bovine immunoglobulin concentrate in patients with AIDS. J. AIDS Human Retrov. 13: 348–354.

17.  Pare JA, JR Barta. 1997. Treatment of cryptosporidiosis in Gila monsters (Heloderma suspectum) with paromomycin. In: MW Frahm, ed. Proceedings of the Fourth Annual Conference of the Association of Reptilian and Amphibian Veterinarians. 1997, Houston, Texas, 23.