Improved Quantitative Estimates of Low Environmental Loading and Sporadic Periparturient Shedding of Cryptosporidium parvum in Adult Beef Cattle

Our primary goal was to generate an accurate estimate of thedaily environmental loading rate of Cryptosporidium parvum oocystsfor adult beef cattle, using immunomagnetic separation coupledwith direct immunofluorescence microscopy for a highly sensitivediagnostic assay. An additional goal was to measure the prevalenceand intensity of fecal shedding of C. parvum oocysts in pre-and postparturient cows as an indicator of their potential toinfect young calves. This diagnostic method could detect witha $>=$ 90% probability oocyst concentrations as lowas 3.2 oocysts g of feces^-1, with a 54% probability of detectingjust one oocyst g of feces^-1. Using this diagnostic method,the overall apparent prevalence of adult beef cattle testingpositive for C. parvum was 7.1% (17 of 240), with 8.3 and 5.8%of cattle shedding oocysts during the pre- and postcalving periods,respectively. The mean intensity of oocyst shedding for test-positivecattle was 3.38 oocysts g of feces^-1. The estimated environmentalloading rate of C. parvum ranged from 3,900 to 9,200 oocystscow^-1 day^-1, which is substantially less than a previous estimateof 1.7 x 10⁵ oocysts cow^-1 day^-1 (range of 7.7 x 10⁴ to 2.3x 10⁵ oocysts cow^-1 day^-1) (B. Hoar, E. R. Atwill, and T. B.Farver, Quant. Microbiol. 2:21-36, 2000). Use of this highlysensitive assay functioned to detect a greater proportion oflow-intensity shedders in our population of cattle, which reducedthe estimated mean intensity of shedding and thereby reducedthe associated environmental loading rate compared to thoseof previous studies.

INTRODUCTION

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Introduction

Cryptosporidium parvum has emerged as a ubiquitous waterbornemicrobial pathogen, with specific genotypes readily transmittedambidirectionally between livestock and humans (7, 9, 12, 25,28, 36). One of the first steps in designing watershed managementprograms for minimizing the occurrence of C. parvum in drinkingwater supplies is to identify significant quantitative sourcesof this parasite. Adult cattle are often considered potentialnonpoint sources of environmental contamination for C. parvum,but there is some disagreement over the relative importancethat adult cattle have in loading watersheds with significantamounts of C. parvum oocysts. In particular, there is a widerange of reported prevalences of fecal shedding of C. parvumfor adult beef and dairy cattle. Numerous investigators havereported mean prevalences of fecal shedding from $~$ 20 to $~$ 70% ingroups of clinically healthy adult cattle (23, 31, 34), yetseveral large cross-sectional epidemiologic surveys have observedprevalences of only 2% or less in asymptomatic adult cattlepopulations (4, 20, 41). Some of this variation in the observedprevalence of fecal shedding can be explained by different investigatorsusing diagnostic assays of differing sensitivity and specificity(11, 13, 15), but much of the variation is the result of studyingdifferent populations of cattle (e.g., beef versus dairy), differentage distributions within those populations, and groups of cattleunder different management practices, especially when only asingle farm or a small sample is examined. For example, we foundin two different studies that calving duration for beef herdswas associated with a three- to sixfold difference in the proportionof cattle shedding C. parvum (5, 20), making interstudy comparisonsof the shedding prevalence for beef cattle potentially confoundedif not adjusted for calving duration.

Determining the importance that adult cattle have in loadingwatersheds with significant amounts of C. parvum oocysts alsorequires an estimate of mean shedding intensity from populationsof cattle. Recent estimates have ranged from 70 to 900 oocystsg of feces^-1 for infected noncalf populations (13, 16, 19, 34),but these values are highly conditional on the underlying agedistribution of the population (16, 39, 41, 44) and the sensitivityof the diagnostic test being employed (11, 15, 19, 21, 42, 43).Increasing the sensitivity of an assay will function to increasinglydetect individuals shedding at low intensities, thereby loweringthe overall estimated mean intensity of shedding for the infectedcohort of a population.

Reducing the incidence or intensity of fecal shedding of C.parvum oocysts by livestock (5, 18, 20, 24, 29) will reducethe likelihood that animal agricultural operations contaminatesurface water with infective C. parvum oocysts. Minimizing infectionin calves is paramount for this strategy to work due to thefact that calves have been shown to be the primary producerof C. parvum oocysts for a herd of beef or dairy cattle (3,4, 15, 16, 26, 35, 41). What remains unresolved is whether pre-or postparturient cows are the primary biological reservoirfor calfhood exposure to C. parvum (3, 13, 15, 34) or, as inthe case of beef cattle grazing open range, whether residentwildlife function to contaminate pastures before or after calving.We have found high levels of fecal shedding for California groundsquirrels that coinhabit rangeland with beef cattle herds, alongwith isolates of C. parvum that are indistinguishable from thoseobtained from infected calves (6).

We previously generated a range of estimates of the maximumpossible environmental loading rate of C. parvum oocysts byCalifornia beef cows, 7.7 x 10⁴ to 2.3 x 10⁵ oocysts cow^-1 day^-1(19). Our primary goal for this project was to generate a moreaccurate estimate of this daily loading rate of C. parvum oocystsfor adult beef cattle based on a highly sensitive diagnosticassay for C. parvum oocysts in cattle feces (11). This improvedmethod for detecting and enumerating C. parvum oocysts is highlyquantitative, can detect one oocyst per gram of feces with aprobability of $~$ 50%, and has a 90% detection threshold (DT₉₀)of approximately four oocysts g of feces^-1. An additional goalwas to measure the prevalence and intensity of fecal sheddingof C. parvum oocysts in pre- and postparturient cows as an indicatorof their potential to infect young calves. Such data would helpguide watershed and herd health programs aimed at minimizingbovine cryptosporidiosis and the associated environmental loadingwith this amphixenotic pathogen.

MATERIALS AND METHODS

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Materials and Methods

Sampling of herds.
Three beef cattle herds were selected from three different geographicalregions of California that were representative of the typicaloak woodland-annual grassland environment commonly utilizedfor cow-calf production in this state. From each herd, fecalsamples were collected from the rectum from a random sampleof 40 adult cows approximately 6 weeks prior to the expectedstart of calving season, and the same animals were resampledapproximately 6 weeks after the start of calving. This provideda diverse set of fecal samples collected from dams ranging from6 weeks to just a few days both prior to and after calving.All samples were collected in separate disposable containers,diluted with an equal volume of 10% formalin, refrigerated,and forwarded to the Veterinary Medical Teaching and ResearchCenter, Tulare, Calif., for enumeration of C. parvum oocysts.

Enumeration of C. parvum oocysts.
Immunomagnetic separation of oocysts coupled with direct immunofluorescencemicroscopy (IMS-DFA) was used to enumerate C. parvum oocystsas described previously by das Graças C. Pereira et al.(11). Briefly, 4 g of the 1:1 feces-formalin mixture (2 g offeces) was resuspended in 40 ml of phosphate-buffered saline,strained through folded two-ply gauze, and centrifuged for 10min at 1,000 x g. The supernatant was discarded, the pelletwas resuspended in 10 ml of sterile, distilled water, and transferredto Leighton tubes for immunomagnetic separation using the anti-CryptosporidiumDynabeads assay as described by the manufacturer (Dynal, Inc.,Lake Success, N.Y.), with oocysts labeled using a fluoresceinisothiocyanate-labeled anti-Cryptosporidium monoclonal antibodyassay (Waterborne, Inc., New Orleans, La.).

Estimating diagnostic test sensitivity.
Naturally infected dairy calves from local commercial dairieswere the source of wild-type C. parvum oocysts. We have determinedpreviously that these oocysts are classified as bovine genotypeA, using the genotyping scheme of Xiao et al. (45). Using anacid-fast procedure to detect oocysts (18), samples having morethan 25 oocysts per microscopic field at a magnification ofx400 were washed through a series of 40-, 100-, 200-, and 270-meshsieves. The resulting suspension was decanted off and centrifugedat 1,000 x g for 10 min. The supernatant was discarded, andthe pellet was washed in Tween water (0.01% Tween 80 in deionizedwater [vol/vol]). A discontinuous sucrose gradient was usedto purify C. parvum oocysts from fecal suspensions (2). Theconcentration of purified oocysts was determined as the arithmeticmean of six separate counts using a phase-contrast hemacytometer(Bright-Line hemacytometer; Hausser Scientific, Horsham, Pa.).

Fecal samples were collected from the rectum from three cowsfrom each of the three herds enrolled in the study and determinedto be free of Cryptosporidium oocysts by IMS-DFA. Aliquots (4.5g) of negative fecal sample were combined with 500 µlof C. parvum oocyst suspension to yield final concentrationsof 10, 50, 100, and 500 oocysts g of adult cow feces^-1. Thespiked fecal samples were then subjected to the processing methoddescribed above (IMS-DFA) for the enumeration of oocysts inperiparturient cow fecal samples.

Percent recovery (r) is defined as r = x/k, where x is the observednumber of oocysts counted for each fecal sample by IMS-DFA andk is the total number of oocysts added to the sample. The kvariable can be further decomposed into cW, where c is the numberof oocysts per gram of feces and W is the mass of fecal materialexamined per assay (e.g., weight of the smear on a slide) (21).In practice, the Poisson distribution tends to underestimatethe variance of r for oocyst count data (3, 19, 37, 38). A flexiblemodel to characterize the variance of r that also allows forcovariates and clustering effects to enter the model is negativebinomial regression, derived as a Poisson gamma mixture distribution(17). With the observed number of oocysts per assay set as theoutcome variable, oocyst concentration was modeled as a continuousvariable, the number of oocysts added per fecal sample set wasmodeled as the offset (or exposure) variable, and cow identification(source of feces) was set as a cluster or group variable toadjust the standard errors for intracow dependencies.

Test sensitivity for a randomly selected sample with c oocystsper gram of feces, S(c), was then defined as the probabilityof detecting one or more oocysts per assay given that oocystswere present. This is equivalent to saying that sensitivityis equal to 1 minus the probability that no oocysts were detected,S(c) = P(X > 0) = 1 - P(X = 0), where X is the number ofoocysts observed per assay. For the negative binomial regressionmodel under consideration (Hardin and Hilbe [17]), the sensitivityequation is

(1)
where the mean of thenegative binomial is given by c_iW_ie^x_i^ß, overdispersionis 1 + ${alpha}$ c_iW_ie^x_i^ß defined above, c_i is the number ofoocysts per gram of feces, W_i is the mass of fecal materialexamined, e^x_i^ß is the percent recovery of the diagnosticassay as a function of the covariates, and ${alpha}$ is an ancillaryparameter for modeling dispersion.

Estimating environmental loading of C. parvum.
For this project, we define environmental loading of C. parvumfrom cattle as the mean number of oocysts produced by an adultcow (age $>=$ 24 months) per day, calculated as thearithmetic mean of kilogram of feces produced per animal perday x arithmetic mean intensity of oocyst shedding for test-positivecattle x prevalence of test-positive cattle. Although a crudeestimate of environmental loading can be calculated from thecattle classified as positive by our IMS-DFA assay, this ignoresthe possibility of any false-negative results in the sample(e.g., infected cattle shedding very low concentrations of oocyststhat were not detected by the IMS-DFA procedure). A more conservativeestimate of the environmental loading rate would be to add thesepotential false-negative cattle to the calculated load (19,21). This is accomplished by using the sensitivity functionS(c) and the specificity of the assay Sp in conjunction withthe binomial distribution to estimate the maximum mean oocystconcentration, c, for a given prevalence, p, of unobserved positivecattle (false-negative results) (3, 19, 21). Specifically, theprobability that a randomly selected sample will test positiveis pS(c) + (1 - p)(1 - Sp), and the probability that z or fewerof the N fecal samples will be positive is given by the followingbinomial distribution:

(2)
To calculatethe maximum oocyst load from test-negative cattle, z is zeroand equation 2 simplifies to

(3)
whereP is fixed at a threshold of, say 5%, S(c) is defined above,Sp is fixed at a justifiable level, N is the sample size oftest-negative cattle, and the right-hand side of the equationis then solved for the maximum oocyst concentration (c) fora given prevalence (p) of false-negative cattle under the givenconstraints of P, S(c), Sp, and N (3, 19, 21). Values for thespecificity Sp tend to be highly constrained when modeling test-negativecattle, given the limited opportunity for false-positive results.

Statistical analysis.
The prevalences of apparent shedding before and after calvingfor the 120 cows enrolled in the study were compared using McNemar'stest, and the intensities of oocyst shedding were compared beforeand after calving by using linear mixed effects regression,with ln(oocyst counts) as the outcome variable, time when samplewas taken (pre- or postparturient) as the covariate, and herdset as the group variable to adjust the standard errors forintraherd dependencies (30).

RESULTS

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Results

The overall prevalence of adult beef cattle testing positivefor C. parvum oocysts was 7.1% (17 of 240) (exact 95% confidenceinterval, 4.2 to 11.1%) (Table 1). For the 17 positive cattle,we observed a crude arithmetic mean (unadjusted for percentrecovery) of 2.706 oocysts per 2 g of fecal sample. The frequencydistribution of the number of oocysts per 2 g of fecal samplefollows: 7 of 17 positive samples had one oocyst each, 2 of17 positive samples had two oocysts each, 3 of 17 positive sampleshad three oocysts each, 3 samples had four oocysts each, andthe last 2 samples had five and nine oocysts, respectively.

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TABLE 1. Observed prevalence and intensity of fecal shedding of C. parvum oocysts by beef cows in California, stratified by herd and calving status

Data from the experiment to estimate percent recovery of thediagnostic assay are presented in Table 2, with the coefficientsand associated P values for the negative binomial regressionmodel presented in Table 3. Percent recovery was significantlyassociated with oocyst concentration (P = 0.005), such thatfor each additional 100 oocysts per g of feces, percent recoverydeclined $~$ 4%. The estimated sensitivity function S(c) (equation1) generated by the coefficients in Table 3 are shown in Fig.1. The concentration of oocysts detected with a 90% probability(DT₉₀) occurred at 3.2 oocysts g of feces^-1, and the probabilityof detecting 1 oocyst g of feces^-1 was 54% (Fig. 1).

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TABLE 2. Percent recovery of IMS-DFA for enumerating C. parvum oocysts in adult beef cattle feces^a

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TABLE 3. Estimated maximum likelihood coefficients of the negative binomial regression model fitted to the recovery data^a

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FIG. 1. Sensitivity of IMS-DFA for detecting C. parvum oocysts in adult beef cattle fecal material. The sensitivity, defined as the probability of detecting one or more oocysts in positive fecal material, was modeled using negative binomial regression.

On the basis of the percent recovery of our diagnostic assay(0.40 at low oocyst concentrations) and an arithmetic mean of2.706 oocysts per positive slide from 2 g of feces, the meanintensity of oocyst shedding was 3.38 oocysts g of feces^-1 fortest-positive cattle and 0.24 oocyst g of feces^-1 for all cattlein the study (Table 1). A crude estimate of the environmentalloading rate of C. parvum per animal can be calculated fromthese values. For example, using a hypothetical 400-kg adultbeef cow producing 16 kg of feces per day (1), we calculate16 kg of feces day^-1 x 240 oocysts kg of feces^-1 = 3,840 oocystscow^-1 day^-1. This estimate of 3,840 oocysts cow^-1 day^-1 ignoresthe possibility of false-negative results among test-negativecattle (i.e., infected cattle shedding very low concentrationsof oocysts that were missed by the IMS-DFA procedure). A profileof feasible scenarios for the prevalence of unobserved shedding(p) by the intensity of oocyst shedding (c) is shown in Fig.2, with p x c scenarios below the threshold of P = 0.05 havinga probability of occurrence of >0.05 and scenarios abovethe threshold having a probability of occurrence of <0.05.On the basis of the constraints for equation 3 [i.e., S(c) constraintsdescribed above, Sp = 1.0, N = 223, and P = 0.05] and allowingthe prevalence of unobserved shedding to range from 2.5 to 97.5%,the maximum intensity of oocyst shedding from our test-negativecattle would be 0.017 to 0.988 oocyst g of feces^-1. ReducingSp to 99% substantially reduces the range of oocyst sheddingto 0.017 to 0.195 oocyst g of feces^-1 (Fig. 2). Collectively,these calculations result in a combined range of estimates forthe intensity of oocyst shedding of 240 to 263 oocysts kg offeces^-1 for all cattle in the study. If we assume that our beefcattle produce between 15 and 35 kg of feces day^-1, the environmentalloading rate for our study population of California beef cowswould range from $~$ 3,600 to $~$ 9,200 oocysts cow^-1 day^-1 (Table 4).

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FIG. 2. Profile of feasible scenarios for the prevalence of unobserved shedding (p) by the intensity of oocyst shedding (c), with p x c scenarios below the threshold of P = 0.05 having a probability of occurrence of >0.05 and scenarios above the threshold having a probability of occurrence of <0.05. The specificity of the diagnostic assay, IMS-DFA, was set at 99% (----) and 100% (—).

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TABLE 4. Estimated daily environmental loading rate of C. parvum oocysts by adult beef cattle in California

The overall prevalences of apparent fecal shedding during thepre- and postcalving periods were 8.3% (10 of 120) and 5.8%(7 of 120), respectively (Table 1). This difference of 2.5%in the prevalence of fecal shedding between the pre- and postcalvingperiods was not significantly different (P = 0.61). None ofthe cows that tested positive during the precalving period testedpositive during the postcalving period. The apparent intensityof oocyst shedding was not significantly different for cowsin the precalving versus postcalving period (P = 0.22).

DISCUSSION

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Discussion

We determined that the environmental loading rate of C. parvumoocysts from adult beef cattle in California ranged from $~$ 3,900to $~$ 9,200 oocysts cow^-1 day^-1, depending on whether an individualanimal produces 15 to 35 kg of feces day^-1, respectively. Thisrange of values for oocyst loading should be quite robust inthat these values are based not only on apparent or observablefecal shedding of oocysts (test-positive cattle) but also includea wide range of assumed prevalences of unobserved fecal sheddingin test-negative beef cattle (2.5 to 97.5% of test-negativecattle shedding undetectable levels of oocysts). Furthermore,if we assume that our specificity is less than 100%, then estimatesof environmental loading are likewise reduced due to the occurrenceof false-positive cattle in the loading estimate (Table 4).To put these values of oocyst loading into a broader perspective,we have calculated that a typical California ground squirrel(Spermophilus beecheyi) weighing 550 g sheds on average 113,000C. parvum oocysts animal^-1 day^-1 (6), or approximately 12 to29 times the amount of an adult beef cow in our study population.Population densities of these squirrels in areas grazed by beefcattle range from 8 to 94 adults ha^-1 in California (8, 27,32), resulting in oocyst loading rates from ground squirrelpopulations of 9.0 x 10⁵ oocysts ha^-1 day^-1 for low-densitypopulations to 1.1 x 10⁷ oocysts ha^-1 day^-1 for high-densitypopulations (6). In contrast, population densities of beef cattleherds, when calculated as the number of adult cattle per totalarea of access per year, typically vary from 0.1 to 0.25 adultcow ha^-1 for the annual grassland-oak woodland complexes ofcentral California. Assuming an individual cow sheds about 6,500oocysts day^-1, this results in oocyst loading rates of 650 oocystsha^-1 day^-1 for low-density cow populations to 1,625 oocystsha^-1 day^-1 for high-density populations, or $<=$ 0.002 times theoocyst loading rate produced by colonies of ground squirrels.Alternatively, an infected dairy calf can produce far in excessof 10⁶ oocysts animal^-1 day^-1 (14, 22, 39, 44), or $>=$ 100times the amount of a mature beef cow in our study population.

We previously estimated that the maximum environmental loadingrate of C. parvum per adult beef cow defecating 16 kg of fecesday^-1 was $~$ 1.7 x 10⁵ oocysts cow^-1 day^-1 (range of 7.7 x 10⁴to 2.3 x 10⁵ oocysts cow^-1 day^-1), which used a similar statisticalapproach but relied on a substantially less sensitive assay(DT₉₀ = 755 oocysts g of feces^-1) (19). For comparison purposes,we can likewise assume that we have 400-kg beef cows defecatingon average 16 kg of feces day^-1 (1). Therefore, 16 kg of fecesday^-1 multiplied by 263 oocysts kg of feces^-1 equals 4,208 oocystscow^-1 day^-1. This estimate of 4,208 oocysts cow^-1 day^-1 is 2.5%of our previous estimate, or a $~$ 1.6 log₁₀ reduction in the calculatedloading rate. It is important to note that this reduced estimatefor maximum oocyst loading by adult beef cattle can be directlyattributed to the highly sensitive assay of IMS-DFA. To ourknowledge, this diagnostic method has the lowest DT₉₀ (3.2 oocysts/gof feces) published thus far for detecting and enumerating oocystsin bovine feces. Quantitative estimates for environmental loadingof fecal-oral pathogens from host species are often overestimatedby using less sensitive tests, not underestimated. While theprevalence of test-positive animals is certainly biased downwardby the use of insensitive diagnostic assays, estimates for theintensity of pathogen shedding can be biased upward for insensitivetests, particularly if the population of interest sheds belowthe DT₉₀ of the assay, as is the case for many asymptomaticinfections in healthy adult vertebrate species. The use of highlysensitive assays in such populations will function to increasinglydetect low-intensity shedders, reducing the calculated meanintensity of shedding and thereby reducing the associated environmentalloading rate.

We found an apparent prevalence of $~$ 7% for periparturient beefcows shedding on average 3.38 oocysts g of feces^-1 (Table 1).This finding of a low prevalence of fecal shedding for C. parvumin beef cattle is consistent with previous work on beef cattlepopulations in California (4, 20). More generally, the reportedprevalence of fecal shedding of C. parvum oocysts in adult beefand dairy cattle varies widely in the literature, ranging froma low of 0 to 10% prevalence (3, 5, 15, 19, 26, 35, 40, 41)to highs of 20 to 70% (16, 23, 34). This variation occurs inpart due to different investigators studying different populationsof cattle using diagnostic methods of differing sensitivityand specificity, but it is also likely that a systematic differencein the medical ecology of asymptomatic adult shedding of bovineC. parvum exists between these populations with low and highprevalence of fecal shedding of C. parvum oocysts. For example,a hypothesis test for a significant difference in the prevalenceof oocyst shedding reported in this study for adult beef cattlein California (7.1% [17 of 240]) to the prevalence reportedfor adult beef cattle in Scotland (62.4% [345 of 553]) (34)has an exact P value of 0.002 (95% confidence interval for differencein prevalence, 0.49 to 0.61), indicating that the underlyingprevalence is likely different in the two populations.

One explanation for not finding a higher prevalence of C. parvumoocysts in periparturient beef cattle is that we used an insensitivemethod to detect oocysts. This speculation is likely to be incorrectgiven the fact that the IMS-DFA method is highly sensitive (11),with the probability of detecting just one oocyst g of feces^-1being 54% (Fig. 1). If we assume that our sample of periparturientbeef cattle were shedding oocysts similar to the mean 500 oocystsg of feces^-1 reported for dairy cattle in Quebec, Canada (13),then the probability that our diagnostic method failed to detectone or more oocysts from such a cow would be P < 10^-10. Intensitiesof fecal shedding have been reported for cows and heifers inPennsylvania (range of 90 to 371 oocysts g of feces^-1) and from6- to 10-year-old cattle in Scotland (range of 25 to 1.8 x10⁴oocysts g of feces^-1) (16, 33). The probability of our diagnosticmethod failing to detect one or more oocysts from similarlyinfected animals would range from P = 1.2 x 10^-5 for cattleshedding 25 oocysts g of feces^-1 to P < 1 x 10^-10 for cattleshedding 18,000 oocysts g of feces^-1. If cattle are sheddingmoderate to high intensities of oocysts ( $>=$ 25 oocystsg of feces^-1) in our study population, then the cattle are presentat a low prevalence.

The statistical methods described herein allow one to calculatethe probability of detecting one or more oocysts for specificcombinations of prevalence (p) by shedding intensity (numberof oocysts per gram of feces) in order to ascertain whethercertain p x c scenarios are plausible (3, 19). Setting a thresholdof P < 0.05 to classify scenarios of p x c as unlikely, givenour sample size of 240 fecal samples, sensitivity function S(c)as described above, and Sp equal to $>=$ 99%, scenariosabove the thresholds outlined in Fig. 2 are unlikely (P <0.05). Scenarios below the threshold of P = 0.05 cannot be excludedfrom occurring in our population of beef cattle. Hence, givenour sample size of 240 fecal samples and given our S(c) as shownin Fig. 1 and Sp of $>=$ 99%, it is highly unlikelythat we have more than 1.4% of periparturient beef cattle sheddingmoderate to high concentrations of undetectable oocysts in ourstudy population. Using a different diagnostic method and adifferent sample of beef cattle, we calculated a similar valueof $<=$ 2% for these potential moderate-to-high intensity yet undetectedshedders in California beef cows (19). In contrast, 26% of dairycows and heifers were found to shed an average of 70 oocystsg of feces^-1 in Pennsylvania (16), and 70% (92 of 131) of asymptomaticadult cattle tested positive for C. parvum oocysts in Spain(23). These higher prevalences and intensities of oocyst sheddingcompared to our data suggest that there are substantial andsystematic differences in the medical ecology of genotypes ofC. parvum that infect adult cattle in different geographicalregions in the United States and the world.

We found no evidence of elevated levels of C. parvum sheddingfrom beef cows in the postparturient period than in the preparturientperiod. With respect to the biological reservoir of C. parvumoocysts for newborn beef calves, it is noteworthy that 7% ofour periparturient cattle were shedding on average 3.4 oocystsg of feces^-1. Accurate information on the 50% infective doseof bovine-derived C. parvum for young beef calves of mixed geneticorigin is not available at this time, but presumably the dailycontact between an infected dam and a susceptible beef calf,such as during nursing behavior in combination with fecallycontaminated udders, would cause C. parvum to be transmittedto the calf eventually. Once a set of index calves become infectedwithin the herd, C. parvum could be readily transmitted to theremaining susceptible calves. This pattern of intraherd transmissionof C. parvum within beef calf populations (i.e., a propagatingepidemic) would also be created by a wildlife reservoir seedingpastures or rangeland with C. parvum. For example, we have foundthat California ground squirrels (Spermophilus beecheyi) thatinhabit the annual grassland-oak woodland complexes of Californiacan shed substantial numbers of oocysts during spring and summer,with genotypes of C. parvum similar to those obtained from beefcalves (6). Similar speculations have been made previously forpopulations of mice and voles at an agricultural site in Warwickshire,United Kingdom (10). A more detailed longitudinal study wouldneed to be conducted in order to resolve which, if either ofthese hosts of C. parvum, function as the primary source ofC. parvum for beef calves located on rangeland.

ACKNOWLEDGMENTS

This work was supported in part by grants from the Divisionof Agriculture and Natural Resources, University of California.

FOOTNOTES

* Corresponding author. Mailing address: Veterinary Medicine Teaching and Research Center, School of Veterinary Medicine, University of California—Davis, Tulare, CA 93274. Phone: (559) 688-1731. Fax: (559) 686-4231. E-mail: ratwill{at}vmtrc.ucdavis.edu.

REFERENCES

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