Z. Parasitenk. 44, 289--298 (1974) © by Springer-Verlag 1974

Cryptosporidium agni sp.n. from Lambs, and Cryptosporidium boris sp.n. from a Calf,

with Observations on the Oocyst

I . K. Barker and P. L. Carbonell

Department of Veterinary Paraelinical Sciences, University of Melbourne, Veterinary Clinical Centre,

Werribee, Australia

Received July 2, 1974

Summary. Cryptosporidium agni sp. n. is described from the small intestine, particularly ileum, of lambs, Ovis aries, and C. bovis sp. n. is described from a similar location in a calf Bos taurus. Ultrastructural observations on trophozoites, schizonts and macrogametes indicated close similarity between the morphology of these species and that of C. wrairi. Oocysts undergoing sporogony, enveloped by an oocyst wall, and containing sporozoites and an oocyst residuum were observed in the calf. The contribution by cryptosporidia to the disease observed in the hosts was undetermined.

Introduction and Preliminary Observations

Cryptospor id ia are minu te p ro tozoan paras i tes inhab i t ing the micro- vil lous border of epi thel ia l cells in the gas t ro in tes t ina l t r ac t of a range of m a m m a l s and birds. The descr ip t ion of the genus has recen t ly been revised and the s ta tus of the n a m e d species reviewed (Vetterl ing, Jervis , Merril l and Sprinz, 1971). Pub l i ca t ion of a r epor t of Cryptosporidium sp. in a calf (Panciera et al., 1971) p r o m p t e d us to review several cases which came to our a t t en t i on in 1970.

The f irst accession consis ted of one three-week-old l amb and two one-week-old l ambs of mixed breeding f rom an ovine milk/cheese farm wi th several hundred mi lk ing ewes. L a m b s were usual ly left on the i r mothers for three days af ter bir th , t hen placed in groups in pens and reared on mi lk replacer. A large p ropor t ion died a t abou t one week of age and the r ema inde r suffered poor growth. Signs were l imi ted to some scouring and conjunct iv i t i s . A Salmonella, T y p e B, had been isola ted from involved lambs by ano ther l abora to ry . The animals r epor ted upon here were born abou t l ive weeks af ter the in i t ia l ou tb reak of disease, by which t ime i t had a b a t e d somewhat in the face of pa ren te ra l and oral an t ib io t ic t he rapy . Some losses and poor g rowth were cont inuing, and resul ted in the submiss ion of these lambs.

290 I. K. Barker and P. L. Carbonell

At necropsy the week-old lambs had intestines devoid of content, congested intestinal mucosa, and prominent Peyers patches. Lesions of other organs were limited to adrenal haemorrhage. Salmonella typhi- murium was isolated on culture of gut content. The three-week-old lamb was emaciated. At post mortem it showed the signs of cachexia, but the gut was nnremarkable on gross examination. In all lambs microscopic examination of Bouins-fixed, paraffin-embedded tissue sections revealed areas of moderate to severe villus atrophy, low surface epithelium, straight dilated intestinal crypts, and a heavy diffuse leukocytic infiltrate in the lamina propria and submucosa. Neutrophils had accumulated in areas of epithelial ulceration and in the lumens of crypts. Many small (1.5 ~m-7 ~m) round haematoxylin-stained orga- nisms were seen in the brush borders of enterocytes in the atrophic areas of gut of all three Iambs, but they were more common in other, more normal intestine and especially in the ileum.

The second case involved a two-week-old calf which had been scouring for two days, and was unresponsive to sulphonamide therapy on the farm. Treatment in the clinic including antibiotics caused the animal to rally, hut it suddenly relapsed and died a day after admission. At necropsy the significant findings were dilation and congestion of a loop of ileum, whieh contained mucoid fluid. No bacteria could be cultured from any organ. Tissues were fixed in 10% formalin, embedded in paraffin, sectioned at 6 ~zm and stained with haematoxylin and eosin. The affected area of gut had tall villi with low eolumnar or cuboidal enteroeytes. Vessels in the lamina propria were congested, hut there was little abnormal eellular reaetion other than a few neutrophils in the lumen of intestinal erypts. Many organisms similar to those seen in the lambs were present in the brush border of enterocytes on the tips, and to a lesser extent, on the sides of villi in the ileum.

Re-examination of the sections from these cases revealed that the basophilic bodies in the striated borders of enteroeytes were probably eryptosporidia, and a more detailed study was made to confirm the diagnosis and identify the stages of organisms present.

Materials and Methods

Small portions of the blocks of paraffin-embedded tissue were dewaxed, osmi- cated and re-embedded in Epon 812. One micrometre thick sections wert out and stained with toluidine blue for examination by light microscopy and ultra-thin sections were out, stained with lead citrate and uranyl acetate and examined with a Philips EM300 electron microscope.

Cryptosporidium in Lambs and Calf 291

Results

Organisms in Lambs Examinat ion of 1 ~m thick sections of lamb intestine (Fig. 1) revealed

mainly small rounded trophozoites 1.5-3.0 ~tm in diameter, containing a small dark nucleus, in the microvillous border of enterocytes. Some spherical bodies approximately 3-4 [zm in diameter with darker staining areas on their luminal side were interpreted as developing schizonts, and occasionally schizonts containing up to 7 merozoites in section were seen. Bodies thought to be macrogametes were very rarely observed. About 6 [zm in diameter, they had small refractile metachromatic granules around their periphery.

Electron microscopic examination of tissues from lambs revealed numerous cryptosporidia fixed with a characteristic zone of a t tachment to the luminal borders of epithelial cells (Hampton and Rosario, 1966; Vetterling, Takeuchi and Madden, 1971). Trophozoites containing a large nucleus with nucleolus were identifiable (Fig. 2). The outer, host derived, and inner, parasite derived, membranes surrounding the parasite were seen in favourable sections, as was the serration of the host-parasite interface in the zone of a t tachment (Fig. 2). Developing schizonts containing budding merozoites and a large intra-cytoplasmic vacuole were seen (Fig. 3) and occasionally schizonts containing discrete merozoites were observed (Fig. 4). Most mature schizonts contained sections of more than 4 merozoites, the maximum number being 7, but probably not all merozoites could be seen in a single section.

Organisms in Cal/ Intestine

In 1 ~m epon-embedded sections of calf intestine asexual stages similar to those described in the lambs were observed. However, it was noteworthy tha t macrogametes up to 7 [zm in diameter, and densely stained crescent-shaped bodies about 7 ~m from tip to tip were frequently seen in the brush border (Fig. 5).

Electron microscopic examination confirmed the presenee of tropho- zoites and schizonts (Fig. 6). The numerous macrogametes were con- siderably larger than trophozoites, and contained dense round peripheral granules interpreted as wall-forming bodies, as well as electron-lucent areas probably occupied by polysaccharide granules (Fig. 7).

Occasionally, oocysts undergoing sporogony were identified (Fig. 8). A moderately electron-dense thin oocyst wall had formed around the organism within the parasitophorous vacuole, and within it were con- tained several developing sporozoites, and a large oocyst residuum. The oocyst residuum was separated from the a t tachment zone by the

I

Fig. 1. Cryptosporidium agn~ in the microvillous borders of ovine enterocytes. There are trophozoites, schizonts and macrogametes represented as well as a

possible ruptured schizont (arrow). × 3 500

Fig. 2. C. agni trophozoite. The microvillus-derived membrane and parasite pellicle are visible, as is a large nucleus containing a nucleolus. There is a serrated pattern

in the at tachment zone. × 40000

Fig. 3. Schizont of C. agni containing budding merozoites. A large vacuole is seen m the cytoplasm of the schizont. × 28000

Fig. 4. Schizonts of C. agni. On the top is an immature schizont containing two nuclei, while on the bottom is a mature schizont containing merozoites with

numerous dense granules. × 18000

Cryptosporidium in Lambs and Calf 293

Fig. 5. C. boris in microvillous border of cells in calf intestine. Trophozoites, macrogametes and oocysts are visible, x 4000

Fig. 6. C. bovis late trophozoite (right) with nucleus and endoplasmic reticulum, and schizont (left) containing budding merozoites, x 29000

294 I. K. Barker and P. L. Carbonell

7

|vi

8

Fig. 7. C. bovi~ macrogamete containing endoplasmic reticulum, wall forming bodies and polysaccharide granules. × 38000

Fig. 8. C. bovis oocyst undergoing sporogony. The oocyst wall completely surrounds the organism within the parasitophorous vacuole, even in the region of the attach- ment zone. Within the oocyst wall are sporozoites, and a large oocyst residuum containing numerous polysaccharide granules in a fine granular matrix. × 31000

Cryptosporidium in Lambs and Calf 295

Fig. 9. C. bovis oocyst, partia|ly collapsed, probably artefactually, corresponding to crescent-shaped bodies in Fig. 5. × 40000

Fig. 10. Membranes surrounding oocyst of C. bovis. The outer layer is composed of microvillar and inverted microvillar membranes fused, and containing the parasitophorous vacuole. The oocyst wall has developed within the outer membrane

of the parasite pellicle. × 120000

296 I. K. Barker and P. L. Carbonell

oocyst wall, and contained polysaccharide granules interspersed in a matrix containing fine electro-dense granules. The ereseent-shaped bodies seen in 1 ~m thick sections were oocysts whieh had shrunk, probably as a processing artefact (Fig. 9).

The eomplex of membranes surrounding the oocyst had a superficial layer of fused outer and inverted mierovillus-derived membranes (Vetterling, Takeuchi and Madden, 1971), separated from the macro- gamete pellicle by the parasitophorous vacuole. Within the parasite pellicle was the thieker oocyst wall, which eompletely surrounded the oocyst contents (Fig. 10).

No microgametocytes eould be identified with confidence, nor any schizonts with only 4 mature merozoites.

Discussion

The finding of cryptosporidia in the guts of three lambs extends the host range of the genus to include sheep. The prevalence of infeetion in lambs is unknown, but presumably the organism is rare or usually causes clinically inapparent infeetion. I t has not been seen since during detailed histological examination of the intestines of a further group of older lambs from the same farm, used for studies of nematode parasi- tism by one of us (I. K. B.).

Although the state of tissue preservation was less than ideal, it is obvious that mõrphologically, the organisms from lambs and calves closely resemble C. wrairi from guinea pigs (Vetterling, Takeuehi and Madden, 1971), althöugh oocysts, not previously described in erypto- sporidia, were found in the calf. There can be little doubt that these bodies are oocysts undergoing sporõgony, since the oocyst wall lies between sporozoites and parasitophorous vacuole, whereas merozoites develop directly within the parasitophorous vacuole (Vetterling, Takeuehi and Madden, 1971). Furthermore, the oocyst wall isolates the oocyst residuum from the attachment zone at the base of the organism, while in mature schizonts the at taehment zone composes part of the schizont residuum (Hampton and Rosario, 1966; Vetterling, Takeuchi and Madden, 1971).

Although transfer of infeetion probably occurs via the faeces, the infective stage of Cryptosporidium has yet to be positively demonstrated, and the only experimental oral passages of infection probably involved transfer of merozoites in intestinal scrapings, although clean in-contact guinea pigs could be infected by 40 days exposure to infected animals (Vetterling, Jervis, Merrill and Sprinz, 1971). The failure thus rar to find cryptosporidial oocysts in tissue sections from infected animals excepting

Cryptosporidium in Lambs and Calf 297

the calf reported here is not readily explained. I t is not known whether oocysts are discharged entire into the gut contents, or release naked sporozoites independently into the lumen, possibly causing superinfection. The relatively delicate nature of the oocyst wall may indicate tha t if passed entire, oocysts would not be highly resistant to adverse environ- mental conditions.

The significance of Cryptosporidium as a cause of disease is unknown. I t has been previously associated with diarrhoea and moderate villus a trophy in calves (Panciera et al., 1971), diarrhoea in turkey poults (Slavin, 1955), and villus a trophy and mononuclear infiltration of the intestinal lamina propria of guinea pigs (Jervis et al., 1966), but these reactions are common to a wide variety of insults. I t s association with diseased calves and lambs may be merely fortuitous, clinically normal infected hosts passing unnoticed. The lambs described here almost certainly had salmonellosis, or its sequelae, which brought them to laboratory attention, and histological lesions in the intestine of lambs and the calf were not necessarily related to the distribution of crypto- sporidia.

The organisms described conform to the criteria for inclusion in the genus Cryptosporidium (Vetterling, Jervis, Merrill and Sprinz, 1971), and since cryptosporidia are probably quite host specific they are considered to merit specifie designation. The previously unnamed organism from the small intestine of the calf (Bos taurus) observed in the U.S.A. by Panciera et al. (1971) and A. M. Kelly and E. J. L. Soulsby (Vetterling, pers. com., 1973) and reported here from Vietoria, Australia, we propose to call Cryptosporidium boris. That from lambs, described from the small intestine of the sheep, Ovis aries, near Melbourne, Victoria, Australia, gains the appellation Cryptosporidium agni.

Key to Abbreviations used on Figures

A polysaccharide granules Oo AZ attachment zone OoW D dense granules P ER endoplasmic reticulum Pom Ma macrogamete PV 1V[V microvillar membrane S MVi microvillar membrane reflexed Sz Mz merozoite T N nucleus V Nu nucleolus WB

oocyst oocyst wall parasite pellicle outer membrane of parasite pellicle parasitophorous vacuole schizont sporozoite trophozoite membrane bound vesicle wall forming body

298 I. K. Barker and P. L. Carbonell

Acknowledgements. The generous advice offered by Dr. J. M. Vetterling during the preparation of this manuscript is acknowledged with gratitude. Drs. J. Mvula and P. T. Hooper were involved in the initial diagnostic work on several of the animals reported. The electron microscope facility of the Dept. of Pathology, Monash University was utilized. I. K. B. was a Fellow of the Medical Research Council of Canada during the course of this work.

Rderences

Hampton, J. C., Rosario, B. : The attachment of protozoan parasites to intestinal epithelial cells of the mouse. J . Parasit. 52, 939-949 (1966)

Jervis, H. R., Merrill, T. G., Sprinz, H. : Coccidiosis in the guinea pig small intestine due to a Cryptosporidium. Amer. J . ver. Res. 27, 408-414 (1966)

Panciera, R. J. , Thomasson, R. W., Garner, F. M. : Cryptosporidial infection in a calf. Ver. Path. 8, 479-484 (1971)

Slavin, D. : Cryptosporidium meleagridi« (sp. nov.) J. comp. Path. 65, 262-266 (1955) Vetterling, J. M., Jervis, H. R., Merrill, T. G., Sprinz, H. : Cryptosporidium wrairi

sp. n. from the guinea pig Cavia porcellus with an emendation of the genus. J. Protozool. 18, 243-247 (1971)

Vetterling, J. M., Takeuchi, A., Madden, P. A. : Ultrastructure of Cryptosporidium wrairi from the guinea pig. J. Protozool. 18, 248-260 (1971)

I. K. Barker P. L. Carbonell Department of Veterinary Paraclinical Sciences University of Melbourne Veterinary Clinical Centre Werribee Victoria 3030, Australia