Background:

Cryptosporidium parvum (C. parvum) is a tiny protozoal parasite that can cause gastrointestinal illness in a wide variety of mammals, including humans, cattle, sheep, goats, pigs, and horses. It also occurs in various wildlife species such as deer, raccoons, opossums, and rabbits (Fayer et al. and Ungar 1990). In cattle, clinical disease and shedding of the parasite is usually limited to calves under a few months of age (National Animal Health Monitoring System 1994 , Kirkpatrick 1985, Anderson and Hall 1982). Although not confirmed by studies done in the U.S., researchers in England and in Spain have reported the shedding of C. parvum in adult beef cattle (Scott et al. 1994, Lorenzo Lorenzo et al. 1993). In humans, clinical disease and shedding can appear at all ages, but is typically more common among children (Ungar 1990). The predominant clinical sign is profuse, watery diarrhea lasting from a few days to several weeks in normal (immunocompetent) calve s (Kirkpatrick 1985) and humans (Jokipii and Jokipii 1986). While this disease is usually self-limiting in immunocompetent calves and humans, it can be prolonged and life-threatening among immunocompromised people such as AIDS patients. An effective tr eatment for eliminating this parasite from the gastrointestinal track still does not exist (White et al. 1994, Goodgame et al. 1993). A few antibiotics may show some promise in reducing the amount of oocyst shedding in AIDS patients, but further clinica l trials are needed to fully evaluate their efficacy (White et al. 1994, Goodgame et al. 1993). The severity of this disease for the immunosuppressed and the fact that this parasite was implicated in recent large scale water-borne outbreaks of gastroente ritis in humans (MacKenzie et al. 1994, Hayes et al. 1989) has prompted the U.S. Environmental Protection Agency (U.S. EPA), Centers for Disease Control and Prevention, state and local public health agencies and regional water districts to seek ways to r educe surface water contamination of this parasite. Some of this attention has focused on identifying the primary sources of C. parvum in surface water. Cattle are often perceived to be a leading environmental source of water-borne C. parvum. For example, the U.S. EPA has explicitly warned that inclusion of C. parvum into the proposed Enhanced Surface Water Treatment Rule will likely result in new restrictions being placed on the location and management of livestock operations sit uated within watershed regions (U.S. EPA 1994). Presented below is a brief summary of the medical ecology of C. parvum in calves and in humans and the existing scientific evidence that addresses the claim that grazing of cattle on watershed region s puts humans at significant risk for water-borne infection of C. parvum.

Life cycle:

In calves and in humans, transmission occurs when an infected individual fecally sheds oocysts (eggs) of this parasite into the environment and a susceptible individual inadvertently ingests these oocysts either directly or indirec tly through such vectors as contaminated water. The parasite then invades the epithelium of the intestine, replicates, and through sequential reproductive cycles can result, as in the case of calves, in the fecal shedding of up to 10¹⁰ oocysts per day and up to 10⁷ oocysts per gram of feces (Blewett 1989). Shedding of oocysts can last for 3-12 days in calves (Anderson 1981), allowing for heavy concentrations of oocysts to build up in confined operations. A similar pattern is seen h umans, whereby infected individuals can shed up to 10⁵-10⁷ oocysts per gram of feces (Goodgame et al. 1993), with a duration of shedding varying widely from individual to individual and which can range from a few to more than 50 day s (Jokipii and Jokopii 1986). Once shed, these oocysts are immediately infective to another individual, allowing for the rapid spread of this parasite within a group of susceptible individuals.

Oocysts shed from one species of mammal appear to be infectious to other species of mammals. Oocysts from humans have been shown to be infectious to a wide variety of livestock and companion animals (Fayer et al. 1990). Oocysts fr om calves and possibly other mammals appear to be infectious to humans (Dupont et al. 1995, Fayer et al. 1990). People working with diarrheic calves infected with C. parvum have themselves become infected with C. parvum, presumably from the calf. However, working with diarrheic calves is not common for the general public.

The issue:

The critical issue is how would C. parvum from calves gain access to surface waters and end up in drinking water supplies. The essential steps must include calves becoming infected and shedding the oocysts in their feces. T hese oocysts must then enter a surface water supply and remain infective as they journey downstream to water treatment plants and distribution systems.

How common is it for calves with access to surface water to shed this parasite? Few studies have been done in beef calves, with most research conducted on dairy calves. Dr. Atwill, University of California-Davis, has been testing cattle from rangeland cow-calf operations from throughout California and has found that most infections are limited to calves under 5 months of age (See Figure 1) (unpublished data). One of the largest surveys to date on shedding of C. parvum in b eef calves was conducted by the USDA's National Animal Health Monitoring System in cooperation with USDA's National Veterinary Services Laboratories. Twenty percent of diarrheic calves (n=391) and 11% of non-diarrheic calves (n=1,053) from a total of 210 operations were found to be shedding C. parvum oocysts at the time of sampling (National Animal Health Monitoring System 1994). Around 40% of these 210 operations had one or more calves shedding C. parvum oocysts at the time of sampling. In this same study shedding of oocysts was documented in 9% of asymptomatic calves between 3 and 6 months of age, indicating that shedding can occur in these older age groups and without clinical signs. In Manitoba, Canada, 22% of beef calves from 148 h erds known to have problems with neonatal diarrhea were found to shed C. parvum (Mann et al. 1986). In England, 36-39% of diarrheic beef calves tested positive for C. parvum while only 8% of healthy beef and dairy calves tested positive (R eynolds et al. 1986). From across the United States, 22% of 7,369 dairy calves tested positive for this parasite (Garber et al. 1994).

Wildlife considerations:

We are just beginning to study the prevalence of shedding among wildlife species with access to surface waters, but much more research is needed in order to fully understand what contribution wildlife make to surface water contamin ation. In a survey of 100 raccoons, 13 juveniles had oocysts in their feces (Snyder 1988). Cryptosporidial infection has been confirmed in grey squirrels (Sundberg et al. 1982) and in a large variety of neonatal captive deer, including mule deer (Heusc hele et al. 1986). Thirty percent (35/115) of wild mice trapped at a dairy shed oocysts (Klesius et al. 1986). Oocysts obtained from these mice were shown to be infective to calves, perhaps indicating a mouse-calf cycle. Sixty three percent (46/73) of w ild brown rats trapped on rural farms were shedding C. parvum (Webster and MacDonald, 1995). Atwill et al., 1997, found that 12 (5.4%) of 221 California feral pigs were shedding C. parvum oocysts. Younger pigs (£8 months) and pigs from high density populations (>2.0 feral pigs/km²) were significantly more likely to shed oocysts compared to older pigs (> 8 months) and pigs from low density populations (£> 1.9 feral pigs/km²). Given the propensity for feral pigs to focus their activity in riparian areas, feral pigs may serve as a source of protozoal contamination for surface water.

Human considerations:

The prevalence of shedding among groups of people is highly dependent on which country, which population, and can range from 0-60%, with the higher proportion of shedding among diarrheic individuals (Ungar 1990). The Centers for Di sease Control and Prevention estimates that the overall background prevalence of shedding in the United States is around 0.5-1.0%, but the relationship between shedding in humans and levels of viable C. parvum oocysts in surface water contaminatio n remains unknown. Outbreaks of human cryptosporidiosis have been linked to swimming in pools (Bell et al. 1993). Also unknown is what proportion of cryptosporidiosis in humans is due to water-borne infection as opposed to human-to-human direct infection .

Survivability:

How long do C. parvum oocysts survive in the environment once they are shed in feces? Oocysts became non-viable after several hours of in-door drying at room temperature (Robertson et al. 1992). Oocysts recovered from calf f ecal patties which had been kept inside a barn (summer) or inside an unheated shed (winter) became non-infective for 3-7 day old mice in 1-4 days (Anderson 1986). If fecal material thoroughly dries before reaching water, the oocysts would presumably bec ome non-infectious for animals and humans. Ten or more days of freezing at -22^oC caused over 90% of oocysts to become non-viable (Robertson et al. 1992). Using mice to determine infectivity, as few as 24 hours of freezing at -20^o C or as few as 7 days of freezing at -15^oC appeared to render the oocysts non-infective (Fayer and Nerad, 1996). This would suggest that oocysts shed by calves during winter conditions may not survive through the season. Oocysts in distilled w ater became non-infective if heated to 72.4^oC or higher for 1 minute or if heated to 64.2^oC or higher for 2 minutes (Fayer 1994). What if fecal material is deposited directly in a stream? One study found that after 33 days in rive r water, an estimated 40-44% of purified oocysts were incapable of excystation. After 176 days, 89-99% were estimated to be incapable of excystation (Robertson et al. 1992). Approximately 67-88% of oocysts were still viable after 33 days when kept in ma nure at 4^oC (Jenkins et al., 1997; Robertson et al. 1992). Environmental survival of C. parvum is an area of active research and we should have considerably more information in the next few years regarding how quickly oocysts die in ou r western climates.

It may be that most oocysts do not remain infective as they journey from infected calves to surface water to water treatment plant to human consumption. Although there are severe environmental pressures for oocysts to remain infect ive when excreted on land, apparently only a few oocysts would need to remain viable in order to pose a risk to humans. Experimental studies in healthy humans determined that the infectious dose at which 50% of subjects acquire infection (ID₅₀ ) was 132 calf-derived oocysts, with as few as 30 oocysts sufficient to induce cryptosporidiosis (Dupont et al. 1995).

Cattle and Cryptosporidium in water:

What evidence directly links the presence of C. parvum in surface water supplies to livestock production? In attempting to answer this question, one must test samples of water which is a procedure with some limitations. Envi ronmental studies to date have had a difficult time determining if the Cryptosporidium found in surface water is C. parvum or some other Cryptosporidium species not infectious to humans, potentially not shed by cattle, yet detected b y one of the laboratory assays used for environmental testing. For example, it appears that C. muris which is shed by cattle, rodents, and other mammals, C. meleagridis which is shed by turkeys, C. serpentis which is shed by snakes, and various other isolates of Cryptosporidium found in lizards can cross-react to some degree with the Merifluor monoclonal antibody produced by Meridian Diagnostics, Inc. (Graczyk et al., 1996; Smith and Rose 1990). All of these species o f Cryptosporidium are not known to be infectious to humans. Dr. Atwill has confirmed in his laboratory that isolates of C. muris obtained from adult dairy cattle from the central San Joaquin Valley in California can cross react with the Me rifluor monoclonal antibody. Hence, the possibility of false positives is very likely when testing water for C. parvum. On the other hand, the recovery efficiency for testing raw water samples, which is the estimated proportion of oocysts recovere d out of all oocysts initially present, can vary from below 10% to as high as 60% (Smith and Rose 1990). Hence, water samples with lower concentrations of oocysts could be erroneously classified as negative. With these limitations in mind (likelihood of false positives or false negatives), C. parvum oocysts are quite common throughout the surface water supplies of the United States. For example, 50-60% of raw water samples from primarily Midwest and East Coast surface water sources were positive for Cryptosporidium oocysts. The geometric mean of detectable levels of Cryptosporidium oocysts was 2.4-2.7 oocysts/Liter, with a range between 0.07 and 484 oocysts/Liter (LeChevallier and Norton 1995, LeChevallier et al. 1991). Rose, 1988, detected Cryptosporidium oocysts in 51% of 111 raw surface water samples from 13 states. Large west coast rivers in Washington and in California were found to have concentration of Cryptosporidium oocysts ranging from 2 to 112 oocysts/Lite r, with a mean of 25 oocysts/Liter (Ongerth and Stibbs 1987). Yet, the link between beef cattle grazing and elevated levels of Cryptosporidium oocysts in surface water is not very clear. For example, one study found little difference in the conce ntration of Cryptosporidium oocysts from protected surface waters (0.3-4.0 oocysts/Liter) as compared to surface waters subject to agricultural run-off (0.1-2.0 oocysts/Liter) (LeChevallier et al. 1991). Moreover, 68% of these oocysts had become n on-viable. Another study measured 5,800 oocysts/Liter in irrigation canal water running through agricultural acreage with cattle pastures (beef or dairy not specified), compared to 127 oocysts/Liter in river water subject to human recreation and 0.8 oocy sts/Liter for stream water exposed to ranch land runoff (Madore et al. 1987). One of the most compelling studies to date was conducted on grazed watersheds in British Columbia. They found concentrations of Cryptosporidium oocysts in river water t o be higher just below a cattle ranch (13.3 oocysts/100 Liters) compared to the just above the ranch (5.6 oocysts/100 Liters) (Ong et al, 1996). These differences appeared to be limited to the period when young calves were present on the watershed and pr esumably rainfall events were occurring (spring). It would be interesting to know whether increases of waterborne Cryptosporidium would have occurred in the absence of young calves or rainfall. Unfortunately, the species of Cryptosporidium was not specified, but was most likely C. parvum since the increase in waterborne Cryptosporidium was most dramatic during and just following the calving season. Cryptosporidium oocysts from pristine surface waters have been found to contain 0.005-18 oocysts/Liter, indicating that this organism occurs naturally in pristine watersheds (Madore et al. 1987). This would suggest that wildlife need to be carefully examined for their role in contaminating surface water with this parasite.

Conclusion:

Until we have more detailed studies which provide a causal link between grazing practices and elevated levels of infective C. parvum in nearby surface water across different watersheds, different ranching operations and acro ss different seasons, it would be premature to claim that cattle production is a leading environmental source of infective C. parvum for water. The presence of young calves in close proximity to surface water during conditions of rainfall may lead to C. parvum contamination of surface water, but these conditions (young calves and rain) are not present year round on our western watersheds. Instead, much of our private and public grazing land enjoys only seasonal rain and calving seasons are in many cases not coincident with this rainfall season. As such, contaminating water with bovine C. parvum would be very difficult when young calves are not present or when rainfall is not occurring, unless of course young calves are allowed to d efecate directly into streams, rivers, reservoirs or lakes. Lastly, if in the words of US EPA (1994) we are to "minimize the potential for source water contamination" by C. parvum, then we must first identify the primary quantitative source(s) of this parasite in the environment, be it livestock, wildlife, humans, companion animals, or human-associated sewage effluent, and continue to unravel the medical ecology of this parasite.

References:

Anderson, B.C. 1981. Patterns of shedding of cryptosporidial oocysts in Idaho calves. J. Am. Vet. Med. Assoc. 178:982-984.

Anderson, B.C. and R.F. Hall. 1982. Cryptosporidial infection in Idaho dairy calves. J. Am. Vet. Med. Assoc. 181:484-485.

Anderson, B.C. 1986. Effect of drying on the infectivity of cryptosporidia-laden calf feces for 3- to 7-day old mice. Am. J. Vet. Res. 47:2272-2273.

Atwill, E.R., R.A. Sweitzer, M. Das Graças C. Pereira, et al. 1997. Prevalence of and associated risk factors for shedding Cryptosporidium parvum and Giardia within feral pig populations in California. Appl. En viron. Microb. 63: 3946-3949.

Bell, A., R. Guasparini, D. Meeds, et al. 1993. A swimming pool-associated outbreak of cryptosporidiosis in British Columbia. Can. J. Public .Health. 84:344-7.

Blewett, D.A. 1989. Quantitative techniques in Cryptosporidium research, p. 85-95. In: K.W. Angus and D.A. Blewett (eds.), Proc. 1st Int. Workshop. Cryptosporidiosis Edinburgh, Scotland.

DuPont, H.L., C.L. Chappell, C.R. Sterling, et al. 1995. The infectivity of Cryptosporidium parvum in healthy volunteers. N. Engl. J. Med. 332:855-859.

Fayer, R. and T. Nerad. Effects of low temperatures on viability of Cryptosporidium parvum oocysts. Appl. Environ. Microbiol. 62:1431-33.

Fayer, R. 1994. Effect of high temperature on infectivity of Cryptosporidium parvum oocysts in water. Appl. Environ. Microbiol. 60:2732-35.

Fayer, R., C.A. Speer and J.P. Dubey. 1990. General biology of Cryptosporidium, p. 2-29. In: J.P. Dubey, C.A. Speer and R. Fayer (eds.), Cryptosporidiosis of man and animals. CRC Press, Boca Raton, Fla.

Garber, L.P., M.D. Salman, H.S. Hurd, et al. 1994. Potential risk factors for Cryptosporidium infection in dairy calves. J. Am. Vet. Med. Assoc. 205:86-91.

Goodgame, R.W., R.M. Genta, A.C. White, et al. 1993. Intensity of infection in AIDS-associated cryptosporidiosis. J. Inf. Dis. 167:704-709.

Graczyk, T. K., M. R. Cranfield, and R. Fayer. 1996. Evaluation of commercial immunoassay (EIA) and immunofluorescent antibody (IFA) test kits for detection of Cryptosporidium oocysts of species other than Cryptosporidium parvum. Am. J. Trop. Med. Hyg. 54:274-279.

Hayes, E.B., T.D. Matte, T.R. O'Brien, et al. 1989. Large community outbreak of cryptosporidiosis due to contamination of a filtered public water supply. N. Engl. J. Med. 320:1372-1376.

Heuschele, W.P., J. Oosterhuis, D. Janssen, et al. 1986. Cryptosporidial infections in captive wild animals. J. Wild. Dis. 22:493-495.

Kirkpatrick, C.E. 1985. Cryptosporidium infection as a cause of calf diarrhoea. Vet. Clin. North Am. Food Anim. Pract. 1:515:528.

Klesius, P.H., T.B. Hayes and L.K. Malo. 1986. Infectivity of Cryptosporidium sp isolated from wild mice for calves and mice. J. Am. Vet. Med. Assoc. 189:192-193.

Jenkins, M.B., L.J. Anguish, D.D. Bowman, et al. 1997. Assessment of a dye permeability assay for determination of inactivation rates of Cryptosporidium parvum oocysts. Appl. Environ. Microbiol. 63:3844-50.

Jokipii, L. and M.M. Jokipii. 1986. Timing of symptoms and oocyst excretion in human cryptosporidiosis. N. Engl. J. Med. 315:1643-46.

LeChevallier, M.W., W.D. Norton and R.G. Lee. 1991. Occurrence of Giardia and Cryptosporidium spp. in surface water supplies. Appl. Environ. Microbiol. 57:2610-16.

LeChevallier, M.W. and W.D. Norton. 1995. Giardia and Cryptosporidium in raw and finished water. J. Am. Water. Works. Assoc. 87:54-68.

Lorenzo Lorenzo, M.J., E. Ares-Mazás and I. Villacorta Martinez de Maturana. 1993. Detection of oocysts and IgG antibodies to Cryptosporidium parvum in asymptomatic adult cattle. Vet. Parasitol. 47: 9-15.

MacKenzie, W.R., N.J. Hoxie, M.E. Proctor, et al. 1994. A massive outbreak in Milwaukee of Cryptosporidium infection transmitted through the public water supply. N. Engl. J. Med. 331:161-167.

Madore, M.S., J.B. Rose, C.P. Gerba, et al. 1987. Occurrence of Cryptosporidium oocysts in sewage effluents and selected surface waters. J. Parasit. 73:702-5.

Mann, E.D., L.H. Sekla, G.P.S. Nayar, et al. 1986. Infection with Cryptosporidium spp. in humans and cattle in Manitoba. Can. J. Vet. Res. 50:174-8.

National Animal Health Monitoring System Staff. 1994. Cryptosporidium and Giardia in beef calves. VS, APHIS, USDA, Fort Collins, Colo.

Ong, C., W. Moorehead, A. Ross, and J. Isaac-Renton. 1996. Studies of Giardia spp. and Cryptosporidium spp. in two adjacent watersheds. Appl. Environ. Microbiol. 62:2798-2805.

Ongerth, J.E. and H.H. Stibbs. 1987. Identification of Cryptosporidium oocysts in river water. Appl. Environ. Microbiol. 53:672-6.

Reynolds, D.J., J.H. Morgan, N. Chanter, et al. 1986. Microbiology of calf diarrhoea in southern Britain. Vet. Rec .119:34-9.

Robertson, L.J., A.T. Campbell and H.V. Smith. 1992. Survival of Cryptosporidium parvum oocysts under various environmental pressures. Appl. Environ. Microbiol. 58:3494-3500.

Rose, J. 1988 Occurrence and significance of Cryptosporidium in water. J. Am. Water. Works. Assoc. 2:53-58.

Scott, C.A., H.V. Smith and H.A. Gibbs. 1994. Excretion of Cryptosporidium parvum oocysts by a herd of beef suckler cows. Vet. Rec. 134:172.

Smith, H.V. and J.B. Rose. 1990. Waterborne cryptosporidiosis. Parasitol. Today 6:8-12.

Snyder, D.E. 1988. Indirect immunofluorescent detection of oocysts of Cryptosporidium parvum in the feces of naturally infected racoons (Procyon lotor). J. Parasitol . 74:1050-55.

Sundberg, J.P., D. Hill D and M.J. Ryan. 1982. Cryptosporidiosis in a gray squirrel. J. Am. Vet. Med. Assoc. 181:1401-2.

Ungar, B.L.P. 1990. Cryptosporidiosis in humans (Homo sapiens), p. 59-82. In: J.P. Dubey, C.A. Speer and R. Fayer (eds.), Cryptosporidiosis of man and animals. CRC Press, Boca Raton, Fla.

U.S. EPA. 1994. National Primary Drinking Water Regulations: Enhanced Surface Water Treatment Requirements. Fed. Register 59:38832-38858.

Webster, J.P. and D.W. MacDonald. Cryptosporidiosis reservoir in wild brown rates (Rattus norvegicus) in the UK. Epidemiol. Infect. 115:207-209.

White, A.C., C.I. Chappell, C.S. Haya, et al. 1994. Paromycin for cryptosporidiosis in AIDS: a prospective, double-blind trial. J. Infect. Dis. 70:419-24.