Intestinal Lesions in Specific-Pathogen-Free Lambs Associated with a Cryptosporidium from Calves with Diarrhea

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1 Vet. Pathol. 19: (1982) Intestinal Lesions in Specific-Pathogen-Free Lambs Associated with a Cryptosporidium from Calves with Diarrhea K. W. ANGUS, S. TZIPORI and E. W. GRAY Moredun Research Institute, Edinburgh, Scotland, U.K. Abstract. Small and large intestines of seven specific pathogen-free lambs infected with cryptosporidia from calves with diarrhea were examined by scanning and transmission electron microscopy and by light microscopy. The small intestine was infected in all the lambs, and the cecum and colon in three. Small intestinal alterations were severe villous atrophy and dilatation of the crypts of Lieberkiihn. Epithelial cross-bridging between contiguous villi caused much villous fusion. Epithelial cells constituting the bridges were connected by desrnosomal junctions, and were continuous with the epithelial coverings of the associated villi. The lamina propria was heavily infiltrated with neutrophil leukocytes. Infected crypts in cecum and colon were dilated and devoid of mucus-secreting cells, while the ridges between crypts were hypertrophied, and the lamina propria was infiltrated by neutrophils. Cell vegetations with adherent bacteria were present in the surface intestinal epithelium of two lambs infected for 11 and 14 days, respectively. No adherent bacteria were seen in any site in lambs killed up to six days post-inoculation. Cryptosporidia (order Eucoccidiorida, suborder Eimeriorina) are minute coccidian parasites which have been identified in the gastro-intestinal tract of at least 12 species of birds, reptiles and mammals [7]. After sporulation of the oocysts, the various stages of the parasite become attached to the microvillous borders of host enterocytes, and are presumed to derive nourishment from these cells [ 14,201. Because cryptosporidia in domestic animals usually have been found either in association with other enteric organisms [l, 9, 10, 121 or in subjects with inherited immunodeficiencies [14], their role as pathogens has not been determined satisfactorily. Recently, however, an outbreak of diarrhea in calves in the U.K. was reported in which affected calves were excreting Cryptosporidium oocysts in their feces [ 191 although screening tests found no enteric viruses, Salmonellae, or enterotoxigenic Escherichia coli. Oral inoculates of fecal homogenates containing these oocysts infected seven different species experimentally [ 171, and caused severe diarrhea in specific-pathogen-free lambs [ 181. The lesions induced in these lambs are described here. 67

2 68 Angus, Tzipori and Gray Lambs Materials and Methods Seven newborn specific-pathogen-free lambs, maintained in plastic isolators, were used. Details of management, inoculation procedure, and clinical manifestations of the disease produced in lambs are described elsewhere [18]. The initial inoculum used to infect lambs was a homogenate (20% v/v) of intestinal contents from a calf experimentally infected with cryptosporidia from an outbreak of calf diarrhea in which this organism was the only enteropathogen identified [ 191. Inocula and fecal samples taken daily throughout the observation period were examined by electron microscopy and enzyme-linked immunosorbent assay to exclude enteric viruses, and by culture on MacConkey agar, after enrichment in selenite broth, to exclude Salmonella. Colonies of Escherichia coli were tested for production of heat-stable toxin by the infant mouse assay [4] and for K99 antigen by the slide agglutination test [5]. Coccidial oocysts other than Cryptosporidium were not seen in any of the samples. The inoculum was given orally to lambs 1 and 2, and to a litter of newborn specificpathogen-free rats [18]. Lambs 3 and 4 received a homogenate prepared from the gut contents of lamb 2. Lambs 5 and 6 were inoculated with homogenates obtained from the whole intestines of the infected rats. Lamb 7 became infected when placed in a plastic isolator previously occupied by lambs 3 and 4. The lambs were killed at intervals between 2 and 14 days post-inoculation as detailed in table I. Necropsy procedures Lambs were deeply anesthetized with sodium pentobarbitone (Nembutal, CEVA Ltd., Southampton, England) and specimens of small intestine were fixed in 10% Baker s calcium formol [ 181. The sites chosen were at lo%, 25%, 5096, 75% and 90% distances along the total small intestine distally from the pylorus. In lamb 7, pieces of small intestine were taken only at lo%, 50% and 90% distances. Specimens of cecum and colon were obtained from lambs 1 through 5, and lung, liver, abomasum, and mesenteric node from lambs 3 and 4. For electron microscopy, pieces approximately 1 cm long from each intestinal site were immersed in 3% glutaraldehyde in phosphate buffer (ph 7.4). Tissues for light microscopy were processed and embedded in paraffin wax, and sections 5 pm thick were stained routinely with Mayer s hematoxylin and eosin (HE). Other stains used were periodic acid-schiff (PAS), phloxine tartrazine, and Giemsa s method. The remaining tissue samples were trimmed while immersed in glutaraldehyde into 1-mm3 and 5-mm2 pieces for transmission and scanning electron microscopy respectively. All specimens were oost-fixed in osmium tetroxide in phosphate buffer (ph 7.4). Specimens for transmission electron microscopy were dehydrated in graded alcohols and embedded in araldite. Location sections 1 pm thick were stained in 10% Giemsa at 60 C for five minutes. Ultrathin sections stained with saturated aqueous uranyl acetate followed by lead citrate were examined in an electron microscope. Blocks for scanning electron microscopy were dehydrated in graded acetones, critical-point dried and sputter-coated with gold for examination on a scanning microscope. Clinical findings Results Full details of the clinical findings in these lambs have been reported elsewhere [ 181. Briefly, lamb 1 was killed early in the infection (table I) before clinical signs were evident. Lambs 2, 3, 5 and 6 became dejected and anorectic soon after

3 Table I. Intestinal infections and damage in specific-pathogen-free lambs dosed orally with fecal or gut content homogenates containing Cryptosporidium oocysts Killed days post- Lambs Severity of clinical response inoculation 1 2 None Intestinal site (% small intestine) Cecum Colon Anorexia, diarrhea Severe diarrhea C C Intermittent anorexia *5 * and diarrhea 5 4 Anorexia, diarrhea c++ c Anorexia, diarrhea C C 7 11 Diarrhea and gradual C++ NT decline No infection of intestinal changes. Cryptosporidium infection without associated lesions. '' Cryptosporidium infection with extensive villous atrophy and fusion. Cryptosporidium infection with limited villous alterations, typhlitis, or colitis Villous atrophy with bacterial adherence but no cryptosporidia. ' Not taken for histology. C' c++3 C++ C++ - C++ c++ c++ c+4 C C+ C++ C+ C++ c++ c++ C C++ c++ c++ NT~ C++ NT C++ NT - - C+ C+ C NT NT

4 70 Angus, Tzipori and Gray Fig. 1: Jejunal villus of infected lamb. Cryptosporidia appear as dark dots along luminal margins of enterocytes. HE. inoculation, and by day 3 post-inoculation had severe diarrhea. Lamb 4 had intermittent bouts of anorexia and diarrhea for 10 days before it died, while lamb 7 became ill with mild diarrhea four days after being placed in the isolator, and gradually became emaciated and completely anorectic by day 11, when it was killed. Cryptosporidium oocysts were found in fecal smears from day 3 post-inoculation onward, but no other known enteropathogen was identified in feces throughout the observation period [ 181. Lesions All the lambs except lamb 1 were emaciated and appeared dehydrated. The abomasum usually was partly filled with clotted milk, and the intestines, which were distended with fluid, lacked tone. The intestinal mucosa, particularly that of the small intestine, was intensely hyperemic but not hemorrhagic. Intestinal contents usually were yellow and watery. Mesenteric lymph nodes, particularly in lambs 2,4, 6 and 7, were enlarged, congested and edematous. Confirmation by transmission electron microscopy of the presence of cryptosporidia in the lambs, and by scanning electron microscopy of the distribution of the infection, has been reported briefly [17, 181. Cryptosporidia were found in the small intestine of all the lambs, and in the cecum and colon of lambs 3,4 and 5 (table I). Organisms were not seen in the abomasum, mesenteric lymph nodes, intrahepatic bile ducts, or lungs of lambs 3 and 4. The presence of cryptosporidia usually was attended by serious alterations to the intestinal mucosa (table I). In lambs 3 and 6, however, the endogenous cryptosporidia stages were numerous in the proximal intestine, with no associated lesions (fig. 1). Scanning electron microscopy examination of typical affected areas of small intestine (fig. 2) showed severe stunting or

5 Calf Cryptosporidia Transmitted to Lambs 71 Fig. 2: Stunting and fusion of villi in distal small intestine of infected lamb. Numerous cryptosporidia (arrows) on sides of villi. Verrucous formations at villus tips. Bar = 50 pm. Fig. 3 Vertical section of infected intestine: stunted and fused villi. Epithelial cross-bridges (arrows). Straight dilated crypts, some containing dead cells. HE.

6 72 Angus, Tzipori and Gray Fig. 4 Plastic section of two adherent villi, several epithelial bridges. Giemsa. complete atrophy of villi, with widespread fusion and distortion of the usual villous pattern. Fusion of villi was confirmed by examination of paraffin and plastic sections (fig. 3,4), which showed that many contiguous villi were connected along their lateral margins by single or multiple epithelial bridges. Examination of the epithelial bridges by transmission electron microscopy showed that the integrity of the epithelium as a continuous sheet was maintained by the presence of desmosomal junctions between the cells constituting the bridges (fig. 5). Affected villi often were coveied by rounded or cuboidal cells, and aggregates of these cells at the tips of villi formed nodular or verrucous projections (fig. 2). Various stages of the organism, including trophozoites, first and second-generation schizonts, merozoites and gametocytes, were adherent to the microvillous borders of enterocytes in infected sites. Clusters of organisms attached to the lateral epithelia of adjacent villi were seen often, apparently adhering to one another at their free surfaces (fig. 6). No cryptosporidia were seen in the crypts of Lieberkiihn or the lamina propria. Crypts in affected sites were dilated but not elongated, and many contained dead leukocytes. No goblet cells were seen in affected crypts. The lamina propria of affected sites was heavily infiltrated by neutrophils and contained a few macrophages and lymphoid and plasma cells. Peyer s patches were reactivi and contained follicles with active germinal centers.

7 Calf Cryptosporidia Transmitted to Lambs 73 Fig. 5 Epithelial bridges composed of cells united by desmosomal junctions (arrows). Bar = 2 fim. Cecal and colonic infections were characterized by infiltrates of neutrophils, hypertrophy of intercrypt ridges, dilatation of crypts with a neutrophil exudate, loss of goblet cells, and attachment of the various endogenous cryptosporidia stages to enterocytes, both on the surface and in the crypts.

8 14 Angus, Tzipori and Gray Fig. 6 Two adjacent villi infected with cryptosporidia. Electron-dense zone between free surfaces of touching organisms (arrows) suggests adhesion. Bar = 2 pm. Adherent bacteria were numerous in all small intestinal sites in lamb 4, killed on day 14 post-inoculation, and lamb 7, which had been exposed to infection in an isolator for 11 days. Bacteria were adherent to isolated clumps or vegetations of swollen pale-stained epithelial cells at mucosal surfaces (fig. 7) but not to columnar cells of normal appearance and staining affinity. Bacterial infection in the 10% and 25% sites of the small intestine of lamb 4 was associated with villous atrophy, with no concurrent Cryptosporidium infection (table I). More distally, cryptosporidia and foci of adherent bacteria were present in all sites, including the cecum (fig. 8) and colon. By contrast, no adherent bacteria were seen by light microscopy, transmission or scanning electron microscopy, in any site in lambs killed up to day 6 post-inoculation (lambs 1, 2, 3, 5 and 6). The medullary sinuses of mesenteric lymph nodes in lambs 3 and 4 contained numerous neutrophils and macrophages, while blast cells were prominent in medullary cords. No significant lesions were found in abomasa, livers or lungs in these two lambs. Discussion Our study reinforces an earlier paper [18] on the pathogenicity of a calf cryptosporidium for young specific-pathogen-free lambs. Although the initial inoculum used

9 Calf Cryptosporidia Transmitted to Lambs 75 Fig. 7: Anterior jejunum of lamb 4: typical vegetation composed of pale disorganized cells with adherent bacteria. HE. Fig. 8: Bacteria adhering to lurninal margins of cecal enterocytes. Bar = 2 pm.

10 76 Angus, Tzipori and Gray in the first pair of lambs probably was contaminated with fecal organisms other than those excluded by preliminary screening, the successful passage of the protozoon through suckling rats to a pair of specific-pathogen-free lambs is good circumstantial evidence for its pathogenicity. Furthermore mucosal damage associated with heavy infections of cryptosporidia was established in four lambs by day 6 post-inoculation without the presence of adherent bacteria, although these were seen in both small and large intestines in more prolonged infections. Even if non-enterotoxigenic but adherent strains of E. coli [3], or other organisms with a similar capability for adherence and cytotoxic damage to enterocytes, were included with Cryptosporidium in the inoculum, our experimental evidence suggests that the intestinal damage was caused by the cryptosporidia in advance of any bacterial effects. This apparent temporal relationship also agrees with the alternative hypothesis that organisms normally regarded as non-pathogenic might adhere to the luminal margins of enterocytes previously damaged by cryptosporidia. These questions probably will be resolved when a bacteria-free inoculum of Cryptosporidium is available. Cryptosporidia infected both small and large intestines in three lambs, and in all except the lamb killed on day 2 post-inoculation there was severe damage to the mucosa of the distal small intestine by day 6 post-inoculation. The extent and severity of the small intestinal damage was consistent with the clinical findings, as shown in table I. An earlier report records villous atrophy with dilated, straight crypts in the small intestine of a lamb infected with cryptosporidia and salmonellae [I], while another report 121 describes shortening of villi and increased crypt mitosis in the ileum of diarrheic lambs infected with the protozoon. Villous atrophy with fusion has also been seen in the ileum of Iambs in a field outbreak in Scotland [16], in which both small and large intestines were infected with cryptosporidia. Thus, our experimental results appear to corroborate observations in naturally-occurring outbreaks of Cryptosporidium infection with diarrhea in lambs. The small intestinal changes were much more extensive and severe than those described in gnotobiotic lambs experimentally infected with rotavirus [ 1 11, astrovirus [13] or enterotoxigenic E. coli, either alone or in combination with rotavirus (S. Tzipori, unpublished data). Whereas the effect on the large intestine is difficult to assess on morphological grounds alone, the stunting and fusion of villi and epithelial cross-bridging in the small intestine must reduce the capacity of the intestine for absorption of fluids and nutrients. In addition, distortion of the upper mucosa presumably obstructed the openings of the crypts, causing crypt distension and diminished secretion of mucin. The lack of muscle tone and stasis accompanying these mucosal alterations may have contributed to the adherence of opportunistic bacteria to enterocytes in both small and large intestines in the lambs with prolonged infections. A combination of all these phenomena could explain the intractability of diarrheas in which cryptosporidia have been positively implicated [S, 10, 121. Fusion of villi and epithelial cross-bridging were seen, in which the bridges were composed of continuous sheets of epithelial cells with desmosomal junctions linking

11 Calf Cryptosporidia Transmitted to Lambs 77 adjacent villi. A possible mechanism of fusion is illustrated in fig. 6, where groups of organisms attached to contiguous villi appear to be mutually adherent at their free surfaces. Shedding of such clusters of organisms and loss of the underlying enterocytes could be followed by extrusion and fusion of replacement cells from each villus. Such a mechanism has been suggested to explain the villous fusion in experimental bacillary dysentery in guinea pigs [15]. Sloughing of groups of villous epithelial cells with adherent bacteria could occur in the same way, with subsequent fusion of extruded replacement cells. Cryptosporidia once were thought to be site-specific, inhabiting the distal small intestine [20]. Cecal infections in lambs in our experiment were associated with morphologic and inflammatory changes, while the colon was affected more mildly. Attachment of endogenous stages of the parasite to large-intestinal and smallintestinal enterocytes was identical. One point of difference from the infection in the small intestine was that cryptosporidia were found in the crypts in both cecum and colon. Since cryptosporidia have been found in guinea pig [6] and calf cecum [8], and the large intestine of lambs, red deer and experimentally infected laboratory animals and piglets (unpublished data), histological examination of the large intestine should be included in diagnosis of natural infections. The role of Cryptosporidium in naturally occurring diarrheic diseases is uncertain, because of the frequent association of other agents. Experimental infections with ileal scrapings containing the organism with no other known enteropathogens, however, caused diarrhea in calves [9], and our study indicates that severe intestinal damage can occur in experimentally infected lambs. Thus cryptosporidia may be important primary pathogens in the neonatal period. Acknowledgements The authors gratefully acknowledge the helpful advice of Dr. R.M. Barlow in the preparation of the paper, the skilled technical assistance of Glynnis Hutchison, and B.J. Easter and A. Inglis for the photographs. References 1 BARKER, I.K.; CARBONELL, P.L.: Cryptosporidium agni sp. n. from lambs and Cryptosporidium bovis sp. n. from a calf, with observations on the oocyst. Z Parasitenkd 44: , BERG, 1.E.; PETERSON, A.C.; FREEMAN, T.P.: Ovine cryptosporidiosis. J Am Vet Med Assoc 172: , CANTEY, J.R.; BLAKE, R.K.: Diarrhea due to Escherichia coli in the rabbit: a novel mechanism. J Infect Dis , DEAN, G.A.; CHING, Y.C.; WILLIAMS, R.G.; HARDEN, L.B.: Test for Escherichia coli enterotoxin using infant mice. Application in a study of diarrhoea of children in Honolulu. J Infect Dis , GUINEE, P.A.M.; VELDKAMP, J.; JANSEN, W.H.: Improved Minca method for the detection of K99 antigen in calf enterotoxigenic strains of Escherichia coli. Infect Immun 15: , 1977

12 78 Angus, Tzipori and Gray 6 JERVIS, H.R.; MERRILL, T.G.; SPRINZ, H.L.: Coccidiosis in the guinea pig small intestine due to cryptosporidium. Am J Vet Res , LEVINE, N.D.: Protozoon Parasites of Domestic Animals and Man, 2nd ed., pp Burgess, Minneapolis, MORIN, M.; LARIVIERE, S.; LALLIER, R.: Pathological and microbiological observations made on spontaneous cases of acute neonatal calf diarrhoea. Can J Comp Med , NAGY, B.; ANTAL, A.; LAKNER, J.: Significance of intestinal cryptosporidiosis in calf diarrhoea. Proc. 2nd Int Symp Vet Lab Diagnosticians, pp Lucerne, Switzerland, POHLENZ, J.; MOON, H.W.; CHEVILLE, N.F.; BEMRICK, W.J.: Cryptosporidiosis as a probable factor in neonatal diarrhoea of calves. J Am Vet Med Assoc , SNODGRASS, D.R.; ANGUS, K.W.; GRAY, E.W.: Rotavirus infection in lambs: pathogenesis and pathology. Arch Virol55: , SNODGRASS, D.R.; ANGUS, K.W.; GRAY, E.W.; KEIR, W.A.; CLERIHEW, L.W.: Cryptosporidia associated with rotavirus and an Escherichiu coli in an outbreak of calf scour. Vet Rec , SNODGRASS, D.R.; ANGUS, K.W.; GRAY, E.W.; MENZIES, J.; PAUL, G.: Pathogenesis of diarrhoea caused by astrovirus infections in lambs. Arch Virol , SNYDER, S.P.; ENGLAND, J.J.; MCCHESNEY, A.E.: Cryptosporidiosis in immunodeficient Arabian foals. Vet Pathol , TAKEUCHI, A.; SPRINZ, H.; LABREC, E.H.; FORMAL, S.P.: Experimental bacillary dysentery. An electron microscopic study of the response of the intestinal mucosa to bacterial invasion. Am J Path , TZIPORI, S.; ANGUS, K.W.; CAMPBELL, I.; CLERIHEW, L.W.: Diarrhea due to Cryptosporidium infection in artificially reared lambs. J Clin Microbiol , TZIPORI, S.; ANGUS, K.W.; CAMPBELL, I.; GRAY, E.W.: Cryptosporidium: a possible singlespecies genus. Infect Immun , TZIPORI, S.; ANGUS, K.W.; GRAY, E.W.; CAMPBELL, I.: Diarrhoea in lambs experimentally infected with cryptosporidium isolated from calves. Am J Vet Res , TZIPORI, S.; CAMPBELL, I.; SHERWOOD, D.; SNODGRASS, D.R.; WHITELAW, A.: An outbreak of calf diarrhoea attributed to cryptosporidia. Vet Rec , VETTERLING, J.M.; JERVIS, H.R.; MERRILL, T.G.; SPRINZ, H.: Cryptosporidium wruiri sp. n. from the guinea pig cuviuporcellus, with an emendation of the genus. J Protozool , 1971 Request reprints from Kenneth W. Angus, Moredun Research Institute, 408 Gilmerton Road, Edinburgh EH17 7JH, Scotland, (UK).