sign in sign up
<<
Home , Coccidia, Cryptosporidiosis, Cryptosporidium parvum, Eimeriorina, Toxoplasmosis

MICROBIOLOGICAL REVIEWS, Mar. 1983, p. 84-96 Vol. 47, No. 1 0146-0749/83/010084-13$02.00/0 Copyright © 1983, American Society for Microbiology in and Humans SAUL TZIPORI Attwood Veterinary Research , Westmeadows, Victoria 3047, Australia

INTRODUCTION...... 84 CHARACTERISTICS OF THE ORGANISM ...... 84 Classification ...... 84 Life Cycle ...... 84 Specificity ...... 87 Diagnosis ...... 88 Studies on Oocysts ...... 88 CHARACTERISTICS OF THE ...... 89 The Disease in Calves...... 89 The Disease in Lambs ...... 89 The Disease in ...... 89 The Disease in Humans ...... 89 The in ...... 90 The Infection in Other Species ...... 90 EXPERIMENTAL CRYPTOSPORIDIOSIS ...... 90 Studies with Calf Isolates ...... 90 Studies with Human Isolates...... 91 TREATMENT...... 92 CONCLUSIONS ...... 92 SUMMARY ...... 94 LITERATURE CITED ...... 94

INTRODUCTION CHARACTERISTICS OF THE ORGANISM Classification is a protozoan parasite which completes its life cycle on intestinal and Cryptosporidium is a genus in the family respiratory surface of , Cryptosporidiidae, suborder Eimeriina, order birds, and reptiles (28). The infection, until Eucoccidiida, subclass , class Sporo- recently, was thought to be uncommon and the zoa, (30). At present the organism was thought to be opportunistic and, suborder Eimeriina contains 13 families with like other coccidia, highly host specific. over 1,500 named species. The great majority of Cryptosporidium was first recognized in the these species, however, belong to the genera gastric glands ofthe laboratory mouse by Tyzzer and, to a lesser extent, . Both in 1907 (58). Up to 1975, some 15 reports de- are intracellular parasites which primarily infect scribing the infection in eight species of animals the intestinal tract of . Toxoplasma, a were published; only 5 of them associated cryp- tissue -forming coccidia, is another impor- tosporidiosis with some illness, 3 of which were tant member of the suborder Eimeriina. in calves (36, 42, 52). Since 1975, over 60 Life scientific publications have appeared in the liter- Cycle ature, the great majority being since 1980. Dur- The life cycle of Cryptosporidium has been ing this period our concept of cryptosporidiosis elucidated for a number of representative host was transformed from that of a rare and largely species and generally follows that of other enter- infection to an important cause of ic coccidia (10, 22, 77). However, there remain enterocolitis and in several species, areas of discrepancy among authors regarding including humans. the presence (77) or absence (7, 22, 59) of a In this communication an attempt was made second-generation schizont. It has been suggest- to gather the available information on the orga- ed that the second-generation schizont may sim- nism, to examine the nature of the infection it ply be sporocysts containing four sporozoites produces, and to demonstrate that Cryptospo- (44). However, sporocysts have not been identi- ridium infection can, under certain circum- fied in structures considered to be oocysts (7), stances, cause serious disease in some species of and the genus is not considered to have sporo- animals. by some (29). The morphological charac- 84 VOL. 47, 1983 CRYPTOSPORIDIOSIS IN ANIMALS AND HUMANS 85

FIG. 1. Electron micrograph of a macrogamete with characteristic dense polysaccharide granules and central nucleus (N). Note: (i) the feeder organelle (F) at the attachment zone; (ii) the parasitophore (P) between the inner parasitic plasma membrane and the outer membrane which, like the microvilli of enterocytes, is coated with glycocalyx. Whether the outer membrane is derived from the host cell, therefore making the organism intracellular (10, 22, 77), or is of parasitic origin and therefore extracellular (44, 59) is a point of dispute (x 30,000). teristics and the nature of the parasite-host rela- ever the nature of the host-parasite association, tionship have been studied in some detail in a the physical location of the organism outside the number of representative host species, and the cell boundaries (Fig. 1) distinguishes it from question of whether the parasite ought to be other coccidia which develop and multiply with- regarded as intracellular (7, 10, 22, 77) or extra- in the cytoplasm of the cell. Although some cellular (44, 59, 60) remains unresolved. What- differences in the various morphological studies 86 TZIPORI MICROBIOL. REV. emerge, they presumably reflect differences in surface; oocysts without sporocysts with 4 na- methodology and interpretation rather than spe- ked sporozoites; microgometes without flagella; cies variation. There has been some doubt as to one genus and about 11 named species" (29). the existence of an oocyst (10, 77), but it is now Cryptosporidium has largely been observed in believed that spherical bodies measuring 3 to 4 association with the of the small or ,um (Fig. 2) and containing four naked sporozo- or both of clinically healthy mice ites are the exogenous infective stage (7, 22, 44). (17, 59, 60), rabbits (21, 50, 61), (61), It has been demonstrated in mice recently that geese (47), guinea pigs (24, 76, 77), and (22); these sporozoites sporulate endogenously and or of clinically ill calves (2, 7, 36, 42, 43, 46, 52, therefore are infective when discharged in the 55, 70), lambs (4, 63), humans (2, 27, 35, 41, 49, (49). Figure 3 provides a diagrammatic 54, 57, 66, 78, 79), goats (33, 71), turkeys (20, 48, presentation of an interpretation of the life cycle 53), deer (65), monkeys (13, 26), and immuno- of Cryptosporidium which was adapted from logically deficient foals (56). Iseki (22). The generic character of Cryptospori- Cryptosporidium has also been observed in- dium is described as "Homogeneous: develop- fecting the trachea (20, 32, 48), cloaca (14), mentjust under surface membrane of host cell or bursa of Fabricius (15), and conjunctival sacs within its brush border and not in the cell (32) of birds, the stomach of mice (58) and proper. Oocysts and meronts with a knoblike snakes (11, 34), and the bile ducts of a monkey attachment organelle at some point on their (26) and immunodeficient foals (56).

FIG. 2. Electron micrograph showing an oocyst (left) and a mature macrogamete, attached to the surface epithelium of lamb experimentally infected with Cryptosporidium (x50,000). VOL. 47, 1983 CRYPTOSPORIDIOSIS IN ANIMALS AND HUMANS 87

OUTSIDE HOST

FIG. 3. Diagrammatic representation of the life cycle of Cryptosporidium. (1 to 4) Asexual cycle of the endogenous stage: 1, sporozoite or merozoite invading a microvillus of a small intestinal epithelial cell; 2, a fully grown , 3, a developing schizont with eight nuclei; 4, a mature schizont with eight merozoites. (5 and 6) Sexual cycle: 5, microgametocyte with many nuclei; 6, macrogametocyte. (7) A mature oocyst containing four sporozoites without sporocyst. (8) Oocyst discharged in the feces. a, Merozoite released from mature schizont; b, sporozoite released from mature oocyst. Adapted from Isaki (22).

Species Specificity serially passaged three to four times in mice (unpublished data). The human isolates in our Based on early transmission experiments experience also appear to infect baby mice less Cryptosporidium was thought to be not only readily than do isolates from other species (Tzi- host (22, 58, 76) but site specific (60). More pori, Bhathal, et al., submitted); on the other recent experiments, using specific-- hand, human isolates studied by Reese and co- free (SPF) newborn animals, indicate that Cryp- workers (49) were said to infect adult mice as tosporidium from some species of animals can well as newborns. Tyzzer was able to transmit infect a wide variety of other animals, with or infection between adult mice (60), whereas a without causing illness (64). Thus, isolates from bovine isolate only infected mice younger than calves, humans, deer, goats, and lambs readily 21 days (51). These differences could be attribut- infect other species such as lambs, calves, and ed to either host or parasite factors or both. The piglets, causing diarrhea (37, 62, 64, 67, 72, 75; exact nature and indeed the existence of such S. Tzipori, P. S. Bhathal, M. Smith, and C. strain differences will become clear when more Halpin, submitted for publication), and mice, information is available, including the develop- rats, guinea pigs, chickens, and foals without ment of more sensitive serological tests and the causing illness (49, 51, 64; Tzipori, Bhathal, et availability of techniques for accurately estimat- al., submitted; unpublished data). However, ing the infectious dose in experimental animals. some differences between isolates have been Passage of Cryptosporidium experimentally noted which could indicate "strain" variations. through animals does not appear to alter their For instance, under similar experimental condi- pathogenicity; after 12 serial passages of one calf tions calf isolates studied in Scotland infected isolate in mice, the infection remained asympto- the intestines of chickens (64) and propagated matic for mice and highly pathogenic for lambs serially at least 16 times in SPF mice (51), (51). A calf isolate and a human isolate were whereas Australian isolates consistently infect- passaged some 20 times intermittently through ed the trachea of chickens and could only be SPF calves, pigs, and mice, causing the same 88 TZIPORI MICROBIOL. REV. degree of illness in calves and piglets and sub- acid, sodium hypochlorite, benzylkonium chlo- clinical in mice (unpublished data). ride, sodium hydroxide (12), and two aldehyde- based (5). Infectivity of oocysts Diagnosis was destroyed by ammonia, formol saline (12), The most reliable method for diagnosis is freeze-drying, and exposure to temperatures be- histological demonstration of the endogenous low freezing and above 65°C for 30 min (unpub- stages attached to the brush border of epithelial lished data). Infectivity is also lost over a period cells. Although the existence of oocysts has of 2 to 6 months at 4°C irrespective of the been known for some time, it was only recently method of storage (37, 51). Oocysts can be kept that their presence in feces was utilized for viable for at least 8 to 9 months in containers diagnosis. The method used to identify oocysts where air is excluded. Excystation seems to include the modified zinc sulfate centrifugal flo- occur shortly after exposure to air, presumably tation (22), Giemsa-stained fecal smears (Fig. 4) stimulated by appropriate levels of oxygen and (45, 55), the fecal flotation technique (1), and the carbon dioxide (unpublished data). modified Ziehl-Neelson technique (19). The Few methods have been suggested for quanti- identity of the organism can be confirmed by the tation of infectivity in infectious material for oral inoculation offeces into SPF newborn labo- inoculation. These include flotation and ratory animals in which the endogenous stages counting (22), titration in mice (51), or counting can then be identified histologically (64). Until the number of oocysts present in a 5-,u suspen- recently cryptosporidiosis in humans was diag- sion smeared and stained with Giemsa (75). nosed by intestinal (27, 35, 41, 54, 57, 78, Purification of Cryptosporidium oocysts in 79). In animals that are clinically affected there material intended for animal inoculation was a is a close correlation between illness and excre- problem. Indeed, most animal experiments were tion of oocysts in the feces (2, 4, 37, 45, 55, 63, conducted with preparations which, although 70-73, 75). known to be free of other enteropathogens, contained enteric (6, 37, 49, 62, Studies on Oocysts 64, 67, 72). Recently, however, oocysts were Cryptosporidium oocysts, like those of other successfully purified by treatment of feces with coccidia, are extremely resistant to a variety of alcohol (60% final dilution) before they were disinfectants commonly used in and used for inoculation (74, 75; Tzipori, Bhathal, et hospitals. These include iodophore, cresylic al., submitted).

IMFAgPpI. :q.dii

t ~* .. .

.** 0 + ts 'WI 9 Sd FIG. 4. Giemsa-stained fecal smear taken from a human patient with ; two intact Cryptospori- dium oocysts (arrows) can be seen with some internal structures (x3,000). VOL. 47, 1983 CRYPTOSPORIDIOSIS IN ANIMALS AND HUMANS 89

CHARACTERISTICS OF THE DISEASE rhea, and 3 subsequently died (71). The affected kids were aged between 5 and 21 days of age and The number of reports which describe crypto- the diarrhea lasted 3 to 7 days. There were seven sporidiosis in calves is by far the greatest and cases of relapse shortly after recovery. Younger suggests that the infection is most important in kids seemed to have suffered for longer periods this species. and were more prone to relapses than older The Disease in Calves animals. Gut sections examined from two dead kids showed organisms attached to the brush The disease is reported to be common in border of enterocytes and mucosal lesions con- North America (9, 39, 45), Europe (18, 40, 46), sistent with cryptosporidiosis seen in calves and and Australia (23). Cryptosporidium infections lambs. in calves occur commonly with other enteric which for a long time made the as- The Disease in Humans sessment of their role as an enteropathogen There have been a dozen or so reports in the uncertain (39, 45, 55). A few recent reports, literature on cases of human cryptosporidiosis. however, have described outbreaks of diarrhea Six of these involved patients who had pro- which could only be attributed to Cryptosporid- longed illness and were shown to be immunolog- ium (2, 9, 18, 70). The clinical picture which ically deficient. Two of them were 6-year-old emerges from field reports is one of mild to patients with congenital immunoglobulin defi- severe diarrhea occurring in calves aged be- ciency who had persistent diarrhea which lasted tween 1 and 4 weeks, with high morbidity and 3 (27) and 6 (54) years, culminating in the death low mortality. The youngest calf reported to be of the latter child. Four other patients had affected with the disease was 4 days (55) and the impaired humoral and cellular immunity as a oldest was 26 days (70). In one study (70) the result of immunosuppressive (35, illness lasted between 2 and 14 days, the average 79) or there was clinical evidence of incompe- being 7 days; relapses occurring after apparent tence (57, 78). One of them (57) died, after a recovery have also been reported. In another persistent diarrhea which lasted 2 years, from report in which a mortality rate of 16% was overwhelming dissemination of . recorded, the clinical signs were said to include However, the underlying cause of death was persistent diarrhea, dehydration, and weakness said to have been severe due to (9). Cryptosporidiosis was also reported to have Cryptosporidium infection of the small intestine. contributed to the etiology of a syndrome, "Ca- One patient recovered within 3 weeks of with- chexia in Veal Calves," described in Holland drawal of immunosuppressive therapy (35). The (16). remaining four reports refer to individuals who The Disease in Lambs were apparently immunologically normal (2, 41, 49, 66). Infection in three of them was diagnosed Two outbreaks of enteritis attributed to cryp- by demonstration of oocyst shedding in the feces tosporidiosis have recently been reported in (2, 49, 66). The diarrhea in these patients lasted lambs (4, 63). The first outbreak occurred in 40 from 1 to 3 weeks and was associated with of 48 artificially reared lambs within 5 to 12 days , , and which of birth, of which 16 subsequently died. Crypto- failed to respond to treatment. In two of these sporidium was detected in 10 of 16 fecal samples reports (2, 49) there was circumstantial evidence examined, and histological sections taken from to suggest that infection had resulted from calf- the ileum of dead lambs confirmed the diagnosis. human transmission. In patients from whom In another outbreak, 200 suckling lambs of a intestinal were examined histologically, total of 532 had diarrhea and 58 subsequently there were varying degrees of mucosal changes died. Lambs between 8 and 12 days of age which included partial villous atrophy, lengthen- developed diarrhea which resulted in either ing of the crypt, low cuboidal surface epitheli- death 2 to 3 days later or recovery after an um, and cellular infiltration of the lamina propria illness of up to 7 days. In lambs, too, there were of the jejunum and ileum. cases of a second bout of diarrhea after apparent A limited serological survey showed that the recovery (4). Two earlier reports (7, 8) which infection was as common among humans as described Cryptosporidium infection in lambs among nine other species of animals tested (68). attributed the diarrhea to concurrent infections Studies on the prevalence of infection in hospital with pathogenic bacteria. patients with gastroenteritis showed that, over the four Australian summer months (February to The Disease in Goats May), 26 of 369 examined (7%) were excreting Cryptosporidium was detected in a dead Cryptosporidium oocysts in the feces; of these, kid with diarrhea (33) and in a herd of 29 only 4 showed evidence of infection with other suckling kids of which 21 suffered severe diar- enteropathogens. For the remainder of the year, 90 TZIPORI MICROBIOL. REV. 10 of 515 patients (2%) were excreting oocysts; stomach and throughout the small and large none of 320 hospital patients without gastroen- bowel, the common bile duct, gall bladder, and teritis (S. Tripori, M. Smith, C. Birch, G. major pancreatic ducts. It was thought that the Barnes, and R. Bishop, submitted for publica- state of in these foals in- tion) did so. The most common clinical manifes- creased susceptibility to the infection. tation was diarrhea, mostly accompanied by Cryptosporidiosis was reported in two juve- vomiting, anorexia, and abdominal pain lasting nile rhesus monkeys, one of which died sudden- from 3 to at least 14 days. The majority of these ly with severe enterocolitis and a second of patients were city dwellers, and in some in- which suffered persistent diarrhea (26). The in- stances there were a few cases which occurred fection was also observed in eight rhesus mon- within one family or a neighborhood, indicating keys which were believed to have died from that transmission may occur among humans. other causes (13). Infection in humans, therefore, does not neces- Illness associated with Cryptosporidium infec- sarily originate from contact with animals as tion was also described in snakes (11, 34), in one some authors believe. This was the first evi- case causing prolonged, severe, chronic hyper- dence that the infection could be an important trophic gastritis in 14 mature reptiles. cause of mild to severe transient diarrhea in Asymptomatic infections were reported to oc- immunologically normal patients and that the cur in mice (17, 58, 59), rabbits (21, 50), cats infection in humans, as in other species, can be (22), guinea pigs (24, 76, 77), and several-week- diagnosed by the presence of oocysts in the old piglets (25, 31). feces. EXPERIMENTAL CRYPTOSPORIDIOSIS The Infection in Birds Experimental disease was studied in calves Turkeys and peacocks are the only reported (37, 44, 45, 74), lambs (6, 62-64, 67, 73), piglets species in which infection was observed in the (37, 64, 72, 75; Tzipori, Bhathal, et al., submit- upper (20, 32, 48). The infection ted), rats, guinea pigs, chicks (64, 76, 77), mice was associated with clinical respiratory signs of (49, 51, 64), monkeys, and foals (unpublished varying severity. Histological changes observed data). The most extensively studied isolates in the trachea included thickened mucosa due to were those obtained from calves and humans. infiltration of the lamina propria with histio- cytes, lymphocytes, and heterocytes and flatten- ing of the epithelium. In one case the ceca were Studies with Calf Isolates heavily infected as well. In some cases the Several calf isolates induced enterocolitis and organisms were seen adhering to the mucosa of diarrhea of equal severity in newborn calves, the nasal cavity and the bronchi. The infection SPF lambs, and piglets, except that calves re- was seen in six different flocks within 2 months. mained susceptible to a much older age. There The morbidity and mortality in one instance was no difference in the clinical response or were up to 30 and 20%, respectively (20). pathological findings between calves fed, or One of 30 peacock chicks which died at about deprived of, or whether they were 2 weeks of age from a was infected orally or by contact (74). The disease examined (32). It was depressed and had a was characterized by depression, anorexia, and gurgling respiration, coughing, and sneezing diarrhea. The incubation period varied between with a serous ocular discharge. Organisms were 3 and 5 days and up to 7 days in calves infected observed attached to the surface ofconjunctival, by contact (74). Excretion of Cryptosporidium nasal, sinus, and tracheal epithelium. oocysts in the feces coincided with the duration Other reports in birds include illness with of clinical illness. The small intestine was infect- diarrhea and a low death rate in 10- to 14-day-old ed in all calves, and the cecum and colon were poults due to Cryptosporidium infection infected in more than half (37, 74). Mucosal of the small intestine (53), asymptomatic infec- changes in the small intestine were most severe tion of the cloaca of two Red-lored parrots (14), in the ileum, and the effects most frequently the bursa of Fabricius of three chickens (15), and observed included stunting and swelling of villi the large intestine of one domestic goose (47). with variable degrees of villous fusion. Affected villi were infiltrated by numerous , neutrophils, and eosinophils and were often The Infection in Other Species lined with immature cuboidal cells. The lamina Generalized Cryptosporidium infection was propria of affected areas contained numerous observed in five immunodeficient Arabian foals mononuclear cells, with some eosinophils and a with diarrhea which were thought to have died few neutrophils. Areas in the ileum were hyper- of adenoviral infection (56). In these foals organ- emic and exhibited a focal turgidity, with serum isms were seen adhering to the wall of the protein leakage at the base of the crypts. Small VOL. 47, 1983 CRYPTOSPORIDIOSIS IN ANIMALS AND HUMANS 91 intestinal changes were more severe in calves dation. Lambs inoculated at 30 days of age killed later rather than at the onset of illness. became infected with the organism, but did not Significant pathological changes were absent develop clinical signs of disease or growth retar- from the large intestine even in heavily infected dation (67). areas. Cryptosporidial infections appear to have SPF piglets given a calf isolate became clini- a marked effect on the activity of membrane- cally resistant to Cryptosporidium infection bound enzymes, even in the anterior portion of when 15 days old (75). In piglets aged 3 days or the intestine where infection seldom is heavy less, the entire intestine was extensively infect- and mucosal changes are minimal (Fig 5). There ed and the mucosa was severely damaged, caus- is a strong correlation between the degree of ing serious illness and death. In older piglets infection of the mucosa, extent of mucosal only the lower small and large intestines were changes (Fig. 6), and severity of clinical illness. heavily infected and damaged (72, 75). Infection The distribution of infection in the bowel and the of SPF 4-week-old piglets during weaning failed associated mucosal lesions are essentially simi- to induce post-weaning diarrhea (unpublished lar in all species that are clinically affected under data). either field or experimental conditions. Colostrum-deprived or suckling foals up to 10 The disease in lambs was similar: SPF lambs days old had asymptomatic infections after in- inoculated at less than 6 days of age became oculation with calf Cryptosporidium. The infec- depressed and anorectic and had diarrhea, some tion was sparse with no apparent mucosal le- became moribund, and others died after a pro- sions, and shedding of oocysts in the feces was tracted, intermittent diarrhea and reduced milk very brief (unpublished data). intake which lasted up to 14 days (67, 73). The There was no evidence of infection in two 6- small and large intestines were heavily infected month-old weaned rhesus monkeys after oral with Cryptosporidium, with the terminal portion inoculation with Cryptosporidium, even after a of the ileum being the most severely affected (6). course of treatment with cyclophosphamide; Lambs infected at 5 to 20 days of age had less there was neither evidence of shedding nor severe clinical signs of disease, with intermittent intestinal attachment (unpublished data). diarrhea, reduced milk intake, and growth retar- The infection of mice and chickens has been discussed in the section Species Specificity. Studies with Human Isolates Human Cryptosporidium isolated from a pa- 3 control tient with severe gastroenteritis (66) infected /(n-3) young SPF lambs (62), causing diarrhea which was somewhat milder than that induced by calf w (67, 73) and lamb isolates tested (63). Another human isolate (unpublished data) induced severe enterocolitis and diarrhea in SPF piglets and one calf (Tzipori, Bhathal, et al., submitted for publi- cation), similar in extent to that induced by Cryptosporidium isolated from calves (Fig. 7). In addition to the extensive mucosal damage inflicted in the bowel, infection and associated lesions were also observed in the trachea of one w Cl) infected of two piglets examined. Two other human (n=10) \ isolates induced only subclinical infections, and two additional ones failed to propagate in 6-day- C-) old SPF piglets. .______.The evidence that Cryptosporidium was the l 2 3 4 5 causative agent in the above-mentioned experi- ments is based on the following: (i) calves and SITE SAMPLED piglets developed enteritis and diarrhea when they were infected with inocula containing FIG. 5. Lactase activity (1 IU = 1 smo/min per g, oocysts, which were free of other microorga- wet weiglht) measured at five equally divided sites nisms; (ii) there was a good correlation between along the small intestine of 10 calves infected with Cryptospo ridium (± standard error from sites 1 to 5: clinical symptoms, the extent of intestinal infec- 0.54, 0.56 , 1.33, 0.69, 0.05), compared with three tion, and the degree of mucosal damage ob- uninoculalted control calves (+ standard error: 5.2, served; (iii) examination of fecal samples, gut 3.2, 7.6, 22.9, 0.6). Lactase activity is a quantitative contents, cryostat sections, and histological sec- measure t4 o assess mucosal integrity. tions failed to reveal other known pathogens; 92 TZIPORI MICROBIOL. REV.

FIG. 6. Electron micrograph of lamb ileum experimentally infected with Cryptosporidium. A degenerating enterocyte, surrounded by organisms attached to the brush border, is about to slough into the lumen. Cells below are electron dense. Stages of the life cycle observed include: macrogametes (M) (note the endoplasmic reticular [ER] formation in the more mature macrogametes), a breaking up schizont with eight merozoites (S), and (T), some with nucleus and nucleolus (x7,500).

and (iv) in some cases the inocula were propa- some direct or circumstantial evidence to asso- gated in SPF several times before being ciate infection with illness. Some species, al- used for experimental studies. though readily infected, appear to have an innate resistance; in rats, mice, and guinea pigs, for TREATMENT instance, the infection is asymptomatic. Rumi- Over 40 antimicrobial agents, including cocci- nants, on the other hand, are susceptible and can diostats and other antiprotozoa compounds, become ill if infected at an early age. The nature broad-spectrum , and even anthelmin- of the infection in other species requires further tics, have been tested against Cryptosporidium, investigation. Humans are probably susceptible either in the course of treatment of infections in to disease. There is little doubt that cryptospori- humans (54, 57, 78) and calves (55) or experi- diosis can have serious consequences in immu- mentally in calves (38) and mice (69). None nologically compromised individuals, and some appear to have been effective (Table 1). Lasalo- evidence exists to suggest that it may also be a cid was said to be effective at a dose which was cause of transient, mild, or acute diarrheal ill- toxic to calves (38). ness in immunologically normal individuals. Several human cases suggest that the disease may be acquired from infected animals, which CONCLUSIONS substantiates an earlier proposal that crypto- Cryptosporidium is a common infection sporidiosis is indeed a zoonotic disease (64). The which, to date, has been described in 16 different significance of cryptosporidiosis in humans, species of animal; in at least 10 of them there is however, depends on the severity of the disease VOL. 47, 1983 CRYPTOSPORIDIOSIS IN ANIMALS AND HUMANS 93 adult humans with congenital immunodeficiency (27, 54), undergoing chemotherapy with immu- nosuppressive drugs (35, 79), concurrently in- fected with another organism such as toxoplas- ma (57), or subjected to some other forms of stress (e.g., traveling [travelers diarrhea; Tzi- pori, Smith, et al., submitted] infection), under such circumstances, could probably contract the illness. It is worth indicating here that the rela- tionship between immunosuppression induced by chemotherapy and cryptosporidiosis is prob- ably not a simple one. We have inoculated young adult and rhesus monkeys treated with high doses ofcyclophosphamide with Cryp- tosporidium without ill effect (unpublished data). This is not surprising considering that there are more human patients treated with immunosuppressive drugs than there are report- ed cases of human cryptosporidiosis. The pathogenesis of the infection requires investigation. It is not clear whether damage to the mucosa is mechanical, mediated by destruc- tion of cells by liberation of parasite metabolites or toxins, or a hypersensitivity reaction of the mucosa to parasite . In animals that are clinically affected the changes in the mucosa are FIG. 7. Calf ileum infected with Cryptosporidium more dramatic, including pronounced inflamma- isolated from a human patient with gastroenteritis. The tory reaction, often unrelated to the number of villi are shortened, cellular, and coated with low organisms attached to the surface. cuboidal, immature enterocytes. Note small spherical bodies (2 to 4 ,um) embedded in the brush border Whereas the evidence against host specificity (x225). is strong, some differences between animal iso- lates do exist, the extent of which is not fully understood. Some strains of Cryptosporidium it produces and the incidence in the population, probably propagate in some species, and organs both of which are unknown. within a given species, more readily than in Normal adults of susceptible species appear to others. For instance, it has been shown that the be resistant to clinical illness. However, individ- mouse, guinea pig, and isolates may propa- uals with impaired immunocompetence, as in gate more readily in their respective adult host

TABLE 1. List of antimicrobial agents reported to be ineffective against Cryptosporidium infection Drugs used to treat human cryptosporidiosis Drugs tested experimentally in: Weinstein et al., Sloper et al., 1982 Stemmermann et Calves (Moon et Mice (Tzipori et 1981 (78) (54) al., 1980 (57) al., 1982 [38]) al., 1982 [691) Sulfisoxazole Amprolium Ethopabate Colistin Sulfamethoxazole Sulfadimidine Nicarbazin Metronidazole Oxytetracycline Trimethoprim Trimethoprim- Sulfaquinoxaline Metronidazole Pyrimethamine Furaltadone Piperazine Sulfadiazine Dimetridazole Enterolyte-N Thiobendazole Levamisol Metronidazole Sulfamethazine Pentemidine Ampicillin Amphotericin B Ipronidazole Trinamide Sulfathalidine Erythromycin Cholestyramine Quinacrine Amprol Penicillin Monensin Phenamidine Ampicillin Lasalocid Zoaquin Septrin Halofuginone Gentamicin Salinomycin Cloxacillin Monensin Carbenicillin Emtryl Apprinocid Amprolium 94 TZIPORI MICROBIOL. REV. and in a particular organ. They presumably Therefore, it is surprising that in the decades would also infect, and may even induce, entero- since its discovery the ability of the organism to colitis ofvarying degrees in newborn susceptible cause disease has not received more attention. hosts and asymptomatic infection in newborn laboratory animals. A limited serial passage SUMMARY through one host does not appear to alter their The protozoan Cryptosporidium (family Cryp- affinity to that host; it may require much greater tospiridiidae), which was identified in 1907, has numbers of passages to do so. The nature of only recently been shown to be an important differences between isolates has wide implica- cause of enterocolitis and diarrhea in a nuber of tions in terms of our understanding of compara- mammalian species. The disease in humans was tive medicine, natural transmission between initially reported to occur in immunologically species, and the of infection in compromised individuals, but a recent study general. among hospital patients with gastroenteritis indi- The difference in the incubation period be- cated that the infection is common in immuno- tween susceptible and resistant species may be a logically normal patients. The organism lacks reflection of variation in the life cycle of the host specificity, a characteristic uncommon organism which, in turn, influences the outcome among other enteric coccidia, and is therefore a of the infection. For instance, the number of potential . It is extremely resistant to generations of schizonts before the sexual stage the action of common laboratory disinfectants and the role, if it indeed exists, of autoinfection and to antimicrobial agents tested so far. A can influence both the length ofthe life cycle and preliminary serological survey indicates that the the outcome of infection. infection is prevalent among and within popula- The organism appears to be resistant to a great tions of 10 mammalian species examined. Field variety of drugs that have been used successful- outbreaks of diarrhea attributed to Cryptospori- ly for treatment against other protozoan infec- dium infection have so far been reported in tions. It is anticipated that effective drugs may calves, lambs, deer, and goats and the disease be those that are taken up by infected cells. had been reproduced experimentally in lambs, Cryptosporidiosis caused more severe clinical calves, and piglets. Sporadic cases of illness in illness in lambs and calves and in older animals other species have also been reported, and in than did , enterotoxigenic Escherich- birds it has been shown to cause upper respira- ia coli or both (73, 74; unpublished data). Pig- tory tract infection. The organism infects the lets, on the other hand, became resistant to the entire bowel but most commonly the lower small infection at a much younger age than they did to intestine, where extensive mucosal changes oc- rotavirus or enteropathogenic E. coli (77). The cur. The organism completes its life cycle on the disease causes prolonged brush border maldi- mucosal lining by adhering to the brush border gestion and, to a greater extent, malabsorption of enterocytes. Cryptosporidium causes partial resulting from altered mucosal architecture of atrophy, fusion, and distortion of villi, resulting the lower small bowel. Death resulting from in maldigestion in the brush border and malab- dehydration and acidosis is less common. sorption. Cryptosporidium strains primarily infect the alimentary tract and the upper respiratory tract ACKNOWLEDGMENTS of some birds, but in one instance infection of I am grateful to the staff of the Moredun Research Institute, the trachea in a piglet experimentally inoculated Edinburgh, U.K., where my involvement with this area of with a human isolate was also noted. The fre- research began, for their enthusiastic support; in particular, I quency and significance of respiratory tract in- thank Kenneth W. Angus. fection in mammals are not clear and may imply ADDENDUM IN PROOF that transmission ofinfection may be by aerosol, Cryptosporidium infection has recently been de- as well as fecal. Indeed, in our experimental scribed in a gray squirrel (J. P. Sundberg, D. Hill, and inoculation of chickens, where only the trachea M. J. Ryan, J. Am. Vet. Med. Assoc. 181:1420-1422, was apparently infected, no oocysts were de- 1982) and in a raccoon (B. L. Carlson and S. W. tected in the feces (unpublished data). The in- Nielson, J. Am. Vet. Med. Assoc. 181:1405-1406, volvement ofthe respiratory tract in an infection 1982). Additional information regarding natural and may also jeopardize the effectiveness of future, experimental cryptosporidiosis in lambs (B. C. Ander- nonsystemic, oral chemotherapy. son, J. Am. Vet. Med. Assoc. 181:151-153, 1982), If our assumption of a single species genus experimental fecal transmission of Cryptosporidium between humans and piglets (H. W. Moon, A. (64), in addition to the diversity of tissue tro- Schwartz, M. J. Welch, P. P. McCann, and P. L. phism, is correct and the preliminary serological Runnels, Vet. Pathol. 19:700-707, 1982), and a chronic studies (69) are a true reflection of prevalence, diarrhea and wasting in a cat (K. B. Pooncha and C. then Cryptosporidium must be one of the most Pippin, Vet. Pathol. 19:708-710, 1982) is now avail- successful and adaptable parasites known. able. VOL. 47, 1983 CRYPTOSPORIDIOSIS IN ANIMALS AND HUMANS 95

LITERATURE CITED Aust. Vet. J. 57:434-435. 1. Anderson, B. C. 1981. Patterns of shedding ofcryptospori- 24. Jervis, H. B., T. G. Merrill, and H. Sprinz. 1966. Coccidi- dial oocysts in calves. J. Am. Vet. Med. Assoc. osis in the guinea pig small intestine due to a Cryptospori- 178:982-984. dium. Am. J. Vet. Res. 27:408-414. 2. Anderson, B. C., and M. S. Bulgin. 1981. Enteritis caused 25. Kennedy, G. A., G. L. Kreitner, and A. C. Strafuss. 1977. by Cryptosporidium in calves. Vet. Med. Small Anim. Cryptosporidiosis in three pigs. J. Am. Vet. Med. Assoc. Clin. 76:865-868. 170:348-350. 3. Anderson, B. C., T. Donndelinger, R. M. Wilkins, and J. 26. Kovatch, R. M., and J. D. White. 1972. Cryptosporidiosis Smith. 1982. Cryptosporidiosis in a veterinary student. J. in two juvenile Rhesus monkeys. Vet. Pathol. 9:426-440. Am. Vet. Med. Assoc. 180:408-409. 27. Lasser, K. H., K. J. Lewin, and F. W. Ryning. 1979. 4. Angus, K. W., W. T. Appleyard, J. D. Menzies, I. Camp- Cryptosporidial enteritis in a patient with congenital hypo- bell, and D. Sherwood. 1982. An outbreak of diarrhoea gammaglobulinaemia. Hum. Pathol. 10:234-240. associated with cryptosporidiosis in naturally reared 28. Levine, N. D. 1973. Protozoan parasites of domestic lambs. Vet. Rec. 110:129-130. animals and man, p. 229-230, 2nd ed. Burgess Publica- 5. Angus, K. W., D. Sherwood, G. Hutchison, and I. Camp- tions, Minneapolis. bell. 1982. Evaluation of two aldehyde-based disinfectants 29. Levine, N. D. 1980. Some corrections of coccidian (Api- on the infectivity of faecal cryptosporidia for mice. Res. complexa:) nomenclature. J. Parasitol. 66:830- Vet. Sci., 33:379-381. 834. 6. Angus, K. W., S. Tzipori, and E. W. Gray. 1982. Intestinal 30. Levine, N. D., J. 0. Corliss, F. E. G. Cos, G. Deroux, J. lesions in specific-pathogen-free lambs associated with Grain, B. M. Honigberg, G. F. Leedale, A. R. Loeblich Cryptosporidium from calves with diarrhoea. Vet. Pathol. III, J. Lom, D. Lynn, E. G. Merinfeld, F. C. Page, G. 19:67-78. Poljansky, V. Sprague, J. Vavra, and F. G. Wallace. 1980. 7. Barker, I. K., and P. L. Carbonell. 1974. Cryptosporidium A newly revised classification of the protozoa. J. Proto- agni sp. n. from lambs, and Cryptosporidium bovis sp. n. zool. 27:37-58. from a calf, with observations on the oocysts. Z. Parasi- 31. Links, I. J. 1982. Cryptosporidial infection of piglets. tenkd. 44:489-498. Aust. Vet. J. 58:60-62. 8. Berg, I. E., A. C. Peterson, and T. P. Freeman. 1978. 32. Mason, R. W., and W. J. Hartley. 1980. Respiratory Ovine cryptosporidiosis. J. Am. Vet. Med. Assoc. cryptosporidiosis in a peacock chick. Avian Dis. 24:771- 173:1586-1587. 776. 9. Bergeland, M. E., D. D. Johnson, and H. Shave. 1979. 33. Mason, R. W., W. J. Hartley, and L. Tilt. 1981. Intestinal Bovine cryptosporidiosis in the north-central United cryptosporidiosis in a kid goat. Aust. Vet. J. 57:386-388. States, p. 131-138. In 22nd Annual Proceedings, Ameri- 34. McKenzie, R. A., P. E. Green, W. J. Hartley, and C. C. can Association of Veterinary Laboratory Diagnosticians. Pollitt. 1978. Cryptosporidium in a red-bellied black snake 10. , R. G., and M. D. Smith. 1980. Cryptosporidiosis in (Pseudechis porphyriacus). Aust. Vet. J. 54:365-366. man: parasite life cycle and fine structural pathology. J. 35. Meisel, J. L., D. R. Perera, C. Meligro, and C. E. Rubin. Pathol. 132:217-233. 1976. Overwhelming watery diarrhoea associated with a 11. Brownstein, D. G., J. D. Strandberg, R. J. Montali, M. Cryptosporidium in an immunosuppressed patient. Gas- Bush, and J. Fortner. 1977. Cryptosporidium in snakes troenterology 70:1156-1160. with hypertrophic gastritis. Vet. Pathol. 14:606-617. 36. Meuten, D. J., H. J. van Kruiningen, and D. H. Lein. 1975. 12. Campbell, I., S. Tzipori, G. Hutchison, and K. W. Angus. Cryptosporidiosis in a calf. J. Am. Vet. Med. Assoc. 1982. The effect of disinfectants on survival of Crypto- 165:917. sporidium oocysts. Vet. Rec., 111:414-415. 37. Moon, H. W., and W. K. Bemrick. 1981. Faecal transmis- 13. Cockerell, B. Y., M. G. Valerio, and F. M. Garner. 1974. sion of calf cryptosporidia between calves and pigs. Vet. Cryptosporidiosis in the intestines of Rhesus monkeys Pathol. 18:248-255. (Macaca mulatta). Lab. Anim. Sci. 24:881-887. 38. Moon, H. W., G. N. Woode, and F. A. Ahrens. 1982. 14. Doster, A. R., E. A. Mahaffey, and J. R. McClearen. Attempted chemoprophylaxis of cryptosporidiosis in Cryptosporidium in the cloacal coprodeum of Red-lored calves. Vet. Rec. 110:181. parrots (Amazona autumnalis). Avian Dis. 23:654-661. 39. Morin, M., S. Lariviere, and R. Lallier. 1976. Pathological 15. Fletcher, 0. J., J. F. Munnell, and R. K. Page. 1975. and microbiological observations made on spontaneous Cryptosporidiosis of the Bursa of Fabricius of chickens. cases of acute neonatal calf diarrhoea. Can. J. Comp. Avian Dis. 19:630-639. Med. 40:228-240. 16. Hage-Noordam, A. W., J. M. A. Pol, and P. W. de Leeuw. 40. Nagy, B., A. Antal, and F. Ratz. 1979. Occurrence of 1982. Cryptosporidium in veal calves affected with ca- bovine cryptosporidiosis in Hungary. Magy. Allatorv. chexia. Tijdschr. Diergeneeskd. 107:497-502. Lapja 34:585-588. 17. Hampton, J. C., and B. Rosario. 1966. The attachment of 41. Nime, F. A., J. D. Burek, D. L. Page, M. A. Holscher, and protozoan parasites to intestinal epithelial cells of the J. H. Yardley. 1976. Acute enterocolitis in a human being mouse. J. Parasitol. 52:939-949. infected with the protozoan Cryptosporidium. Gastroen- 18. Heiene, J. von, and J. Boch. 1981. Kryptosporidien- terology 70:592-598. Infektionen beim Kalb. Nachweis, Vorkommen und ex- 42. Panciera, R. J., R. W. Thomassen, and F. M. Garner. perimentelle Ubertragung. Berl. Muenich. Tieraerztl. 1971. Cryptosporidial infection in a calf. Vet. Pathol. Wochenschr. 94:289-292. 8:479-484. 19. Henriksen, S. A., and J. F. L. Pohlenz. 1981. Staining of 43. Pearson, G. R., and E. F. Logan. 1978. Demonstration of cryptosporidia by a modified Zeihl-Neelsen technique. Cryptosporidia in the small intestine of a calf by light Acta Vet. Scand. 22:594-596. transmission and scanning electron . Vet. Rec. 20. Hoerr, F. J., F. M. Ranck, and T. F. Hastings. 1978. 103:212-213. Respiratory cryptosporidiosis in turkeys. J. Am. Vet. 44. Pohlenz, J., W. J. Bemrick, H. W. Moon, and N. F. Med. Assoc. 173:1591-1593. Cheville. 1978. Bovine cryptosporidiosis: a transmission 21. Inman, L. R., and A. Takeuchi. 1979. Spontaneous cryp- and scanning electron microscopic study of stages in the tosporidiosis in an adult female rabbit. Vet. Pathol. 16:89- life cycle and of the host-parasite relationship. Vet. 95. Pathol. 15:417-427. 22. Iseki, M. 1979. Cryptosporidium felis sp. n. (Protozoa: 45. Pohlenz, J., H. W. Moon, N. F. Cheville, and W. J. ) from the domestic cat. Jpn. J. Parasitol. Bemrick. 1978. Cryptosporidiosis as a probable factor in 28:285-307. neonatal diarrhoea of calves. J. Am. Vet. Med. Assoc. 23. Jerrett, I. V., and D. R. Snodgrass. 1981. Cryptosporidia 172:452-457. associated with outbreaks of neonatal calf diarrhoea. 46. Pol, J. M. A., B. E. C. Schreuder, G. J. Kok, and P. W. de 96 TZIPORI MICROBIOL. REV. Leeuw. 1982. Cryptosporidium: a new factor in the aetiol- 64. Tzipori, S., K. W. Angus, I. Campbell, and E. W. Gray. ogy of neonatal diarrhoea in calves. Tijdschr. Diergen- 1980. Cryptosporidium: evidence for a single species eeskd. 107:503-510. genus. Infect. Immun. 30:884-886. 47. Proctor, S. J., and R. L. Kemp. 1974. Cryptosporidium 65. Tzipori, S., K. W. Angus, I. Campbell, and D. Sherwood. anserinum sp. n. (sporozoa) in a domestic goose Anser 1981. Diarrhoea in young Red Deer associated with infec- anser L. from Iowa. J. Protozool. 21:664-666. tion with Cryptosporidium. J. Infect. Dis. 144:170-175. 48. Ranck, F. M. 1979. Cryptosporidiosis-a possible cocci- 66. Tzipori, S., K. W. Angus, E. W. Gray, and I. Campbell. dial respiratory pathogen in turkeys, p. 49-51. In Proceed- 1980. Vomiting and diarrhoea associated with cryptospor- ings of the 28th Western Poultry Disease Conference and idial infection. N. Engl. J. Med. 303:818. 13th Poultry Health Symposium. 67. Tzipori, S., K. W. Angus, E. W. Gray, L. Campbell, and F. 49. Reese, N. C., W. L. Current, J. V. Ernst, and W. S. Allan. 1981. Diarrhoea in lambs experimentally infected Bailey. 1982. Cryptosporidiosis of man and calf: a case with Cryptosporidium isolated from calves. Am. J. Vet. report and results of experimental infections in mice and Res. 42:1400-1404. rats. Am. J. Trop. Med. Hyg. 31:226-229. 68. Tzipori, S., and I. Campbell. 1981. Prevalence of Crypto- 50. Rehg, J. E., G. W. Lawson, and S. P. Pakes. 1979. sporidium in ten species of animals. J. Clin. Cryptosporidium cuniculus in the rabbit (Oryctolagus Microbiol. 14:455-456. cuniculus). Lab. Anim. Sci. 29:656-660. 69. Tzipori, S., I. Campbell, and K. W. Angus. 1982. The 51. Sherwood, D., K. W. Angus, D. R. Snodgrass, and S. therapeutic effect of 16 antimicrobial agents on Crypto- Tzipori. 1982. Experimental cryptosporidiosis in labora- sporidium infection in mice. Aust. J. Exp. Biol. Med. Sci. tory mice. Infect. Immun. 38:471-475. 60:187-190. 52. Shmitz, J. A., and D. H. Smith. 1975. Cryptosporidium 70. Tzipori, S., I. Campbell, D. Sherwood, D. R. Snodgrass, infection in a calf. J. Am. Vet. Med. Assoc. 167:731-732. and A. Whitelaw. 1980. An outbreak of calf diarrhoea 53. Slavin, D. 1955. Cryptosporidium meleagridis (sp. nov.). attributed to cryptosporidial infection. Vet. Rec. 107:579- J. Comp. Pathol. 65:262-266. 580. 54. Sloper, K. S., R. R. Dourmashkin, R. B. Bird, G. Slavin, 71. Tzipori, S., J. Larsen, M. Smith, and R. Lugfl. 1982. and A. D. B. Webster. 1982. Chronic malabsorption due Diarrhoea in goat kids attributed to Cryptosporidium to cryptosporidiosis in a child with immunoglobulin defi- infection. Vet. Rec. 111:35-36. ciency. Gut 23:80-82. 72. Tzipori, S., E. McCartney, G. H. K. Lawson, A. C. Row- 55. Snodgrass, D. R., K. W. Angus, E. W. Gray, W. A. Keir, land, and I. Campbell. 1981. Experimental infection of and L. W. Clerihew. 1980. Cryptosporidia associated with piglets with Cryptosporidium. Res. Vet. Sci. 31:358-368. rotavirus and an Escherichia coli in an outbreak of calf 73. Tzipori, S., D. Sherwood, K. W. Angus, I. Campbell, and scour. Vet. Rec. 106:458-459. M. Gordon. 1983. Diarrhea in lambs: experimental infec- 56. Snyder, S. P., J. J. England, and A. E. McChesney. 1978. tions with enterotoxigenic Escherichia coli, rotavirus, and Cryptosporidiosis in immunodeficient arabian foals. Vet. Cryptosporidium. Infect. Immun. 33:401-406. Pathol. 15:12-17. 74. Tzipori, S., M. Smith, C. Halpin, K. W. Angus, D. Sher- 57. Stemmermann, G. N., T. Hayashi, G. A. Glober, N. Oishi, wood, and I. Campbell. 1983. Experimental Cryptospori- and R. I. Frankel. 1980. Cryptosporidiosis, report of a dium in calves; clinical manifestations and pathological fatal case complicated by disseminated toxoplasmosis. findings. Vet. Rec., in press. Am. J. Med. 69:637-642. 75. Tzipori, S., M. Smith, T. Makin, and C. Halpin. 1982. 58. Tyzzer, E. E. 1907. A sporozoan found in the peptic glands Enterocolitis in piglets caused by Cryptosporidium puri- of the common mouse. Proc. Soc. Exp. Biol. Med. 5:12- fied from calf faeces. Vet. Parasitol., 11:121-126. 13. 76. Vetterling, J. M., H. R. Jervis, T. G. Merrill, and H. 59. Tyzzer, E. E. 1910. An extracellular coccidium Crypto- Sprinz. 1971. Cryptosporidium wrairi sp. n. from the sporidium muris (gen. et sp. nov.) of the gastric glands of guinea pig Cavea porcellus, with an emendation of the the common mouse. J. Med. Res. 23:487-509. genus. J. Protozool. 18:243-247. 60. Tyzzer, E. E. 1912. (sp. nov.) a 77. Vetterling, J. M., A. Takeuchi, and P. A. Madden. 1971. coccidium found in the small intestine of the common Ultrastructure of Cryptosporidium wrairi from the guinea mouse. Arch. Protistenkd. 26:394-412. pig. J. Protozool. 18:248-260. 61. Tyzzer, E. E. 1929. in gallinaceous birds. Am. 78. Weinstein, L., S. M. Edelstein, J. L. Madara, K. R. Fal- J. Hyg. 10:269-383. chuk, B. M. McManus, and J. R. Trier. 1981. Intestinal 62. Tzipori, S., K. W. Angus, and I. Campbell. 1982. Experi- cryptosporidiosis complicated by disseminated cytomega- mental infection of lambs with Cryptosporidium isolated lovirus infection. Gastroenterology 81:584-591. from a human patient with diarrhoea. Gut 23:71-74. 79. Welsburger, W. R., D. F. Hutcheon, J. H. Yardley, J. C. 63. Tzipori, S., K. W. Angus, I. Campbell, and L. W. Cleri- Roche, W. D. Hills, and P. Charache. 1979. Cryptospori- hew. 1981. Diarrhea due to Cryptosporidium infection in diosis in an immunosuppressed renal transplant recipient artificially reared lambs. J. Clin. Microbiol. 14:100-105. with IgA deficiency. Am. J. Clin. Pathol. 72:473-378.