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Prevalence of Giardia sp., Cryptosporidium parvum and Cryptosporidium muris (C. andersoni) in 109 dairy herds in five counties of southeastern New York
Veterinary Parasitology 93 (2000) 1–11
Prevalence of Giardia sp., Cryptosporidium parvum and Cryptosporidium muris (C. andersoni) in 109 dairy herds in five counties of southeastern New York S.E. Wade a,∗ , H.O. Mohammed b , S.L. Schaaf a a b
Parasitology Section, Department of Population Medicine and Diagnostic Science, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA Epidemiology Section, Department of Population Medicine and Diagnostic Science, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA Received 2 May 2000; accepted 12 July 2000
Abstract A cross-sectional study was undertaken to determine the prevalence of Giardia sp. (G. duodenalis group), Cryptosporidium parvum and Cryptosporidium muris (C. andersoni) in dairy cattle in three different age groups, and to evaluate the association of age and season with prevalence. One hundred and nine dairy farms, from a total of 212 farms, in five counties of southeastern New York volunteered to participate. On these farms, 2943 fecal samples were collected from three defined age groups. The farms were randomly assigned for sampling within the four seasons of the year. Each farm was visited once during the study period from March 1993 to June 1994 to collect fecal samples. Demographic data on the study population was collected at the time of sampling by interviewing the farm owner or manager. At collection, fecal samples were scored as diarrheic or non-diarrheic, and each condition was later related to positive or negative infection with these parasites. Fecal samples were processed using a quantitative centrifugation concentration flotation technique and enumerated using bright field and phase contrast microscopy. In this study, the overall population prevalence for Giardia sp. was 8.9%; C. parvum, 0.9%; and C. muris, 1.1%. When considering animals most at the risk of infection (those younger than 6 months of age) Giardia sp. and C. parvum was found in 20.1 and 2.4% of the animals, respectively. Giardia sp. and C. muris were found in all age groups. There was no significant seasonal pattern of infection for any of these parasites. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Giardia sp.; Cryptosporidium parvum; Cryptosporidium muris (C. andersoni); Prevalence; Dairy cattle
∗ Corresponding author. Tel.: +1-607-253-3581; fax: +1-607-253-3943 E-mail address: [email protected] (S.E. Wade).
0304-4017/00/$ – see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 0 0 ) 0 0 3 3 7 - X
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1. Introduction In recent years, there has been increased concern about the presence of two protozoa, Giardia sp. and Cryptosporidium parvum in the environment, especially water, and in animals, including humans (Adam, 1991; Current and Blagburn, 1990; Current and Garcia, 1991; Fayer, 1997; O’Donoghue, 1995; Thompson et al., 1993; Walterspiel and Pickering, 1994). These protozoa are of public health concern because they may cause infection and severe illness in humans. Infections are self-limiting in people with normal immune systems, but infection can be life threatening in people who have compromised immune systems (Current et al., 1983; Smith, 1989). In response to the Federal Safe Drinking Water Act (SDWA) amendments of 1986 (42USC300g-1), and regulations of the Surface Drinking Water Treatment Rule (SDWTR) (54FR27486, June 29, 1989), the USEPA stated that New York city may avoid filtration of its drinking water, if it shows that the source water meets federal and state raw water standards, that adequate disinfection is in place and that an adequate watershed protection program to reduce the risk of waterborne disease is implemented. The NYC Watershed Agricultural Program, created as part of the filtration avoidance, was established in 1993 to maintain the quality of the water supply, and the economic viability of agriculture in the NYC Watershed through whole farm planning. This program focuses on the management of pesticides, sediment, nutrients such as nitrogen and phosphorus, and pathogenic organisms such as Giardia sp. and C. parvum, as they relate to agriculture and water quality. Due to the lack of information concerning the epidemiology of Giardia sp. and C. parvum infections in dairy cattle, generally and specifically in this watershed, this study was initiated. Giardia sp. and C. parvum have both been reported from cattle (Anderson and Hall, 1982; Anderson, 1989; Buret et al., 1990; Harp et al., 1990; Kirkpatrick, 1985; Leek and Fayer, 1984; Lefay et al., 2000; McCluskey, 1992; McCluskey et al., 1995; Moore and Zeman, 1991; O’Handley et al., 1999; Olson et al., 1997a,b; Ongerth and Stibbs, 1989; Ruest et al., 1998; Sobieh et al., 1987; St. Jean et al., 1987; Xiao et al., 1993; Xiao, 1994). The prevalence and clinical significance of Giardia sp. infection in cattle of all ages is not well understood. There have been reports of infection with this parasite in young animals with and without associated clinical illness (Buret et al., 1990; O’Handley et al., 1999; Olson et al., 1997a,b; Ruest et al., 1998; St. Jean et al., 1987; Xiao et al., 1993; Xiao, 1994). Cryptosporidium parvum is known to be associated with diarrheal illness in calves younger than 1 month of age (Anderson and Hall, 1982; Harp et al., 1990; Kirkpatrick, 1985; Leek and Fayer, 1984; Lefay et al., 2000; McCluskey, 1992; McCluskey et al., 1995; Moore and Zeman, 1991; O’Handley et al., 1999; Olson et al., 1997a,b; Ongerth and Stibbs, 1989; Ruest et al., 1998; Sobieh et al., 1987; Xiao et al., 1993). Cryptosporidium muris is also found in cattle in which it may cause gastritis, and is statistically associated with reduced milk production (Anderson, 1989, 1992; Esteban and Anderson, 1995). In any epidemiologic study in cattle, infection with C. muris must be differentiated from infection with C. parvum since the latter species is a known pathogen in humans, while the former is not considered a threat to human health (Fayer, 1997). To determine the extent of the problem in dairy cattle, and to aid in recommending farm management practices relative to Giardia sp. and C. parvum, a cross-sectional study
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was conducted to estimate the prevalence of Giardia sp., C. parvum and C. muris (C. andersoni) in dairy cattle in this geographic area. This study represented the first step towards the development of the recommendations. The associated effects of age and season with prevalence were also evaluated.
2. Material and methods The study population consisted of dairy cattle on farms in portions of five counties (Delaware, Greene, Schoharie, Ulster, Sullivan) within the Catskill/Delaware systems of the NYC Watershed in southeastern New York. Based on a reported prevalence of C. parvum in dairy animals (Leek and Fayer, 1984), and by using the formula suggested by Levy and Lemeshow (1980), it was determined that at least 100 farms needed to be sampled. This sample size was computed using the following assumptions: the expected prevalence of C. parvum in calves was 26.5% (Leek and Fayer, 1984); the level of significance to detect this prevalence is 95% (a = 0.05); the power for the estimated prevalence was 90% (b = 0.1); the acceptable difference between the true prevalence and the estimated prevalence was 10%; and the prevalence of Giardia sp. among these herds was equal to the prevalence of C. parvum. A recruitment letter was sent to 212 dairy farms located in the study area. A total of 109 farms volunteered to participate, and were randomly assigned to a season of collection by the response date. Four seasons were identified according to the weather pattern in New York state. These are winter (January–March), spring (April–June), summer (July–September), and fall (October–December). Farms were visited once during the study period from March 1993 to June 1994. The population was stratified into three age groups: 0–6 months of age; older than 6 months -first freshening (about 24 months); and animals older than 24 months. All animals 0–6 months of age were sampled unless more than 20 were present, then 70% of the animals in that age group were sampled. Six animals from the second age group and nine animals from the third age group were sampled. Fecal samples were collected rectally from each animal in the study population and were condition scored as diarrheic or non-diarrheic. Samples were kept refrigerated at 4◦ C from collection to processing and evaluation in the laboratory. All samples were processed within 2 weeks of collection. Laboratory analysis of the fecal samples was conducted using a standard quantitative centrifugation concentration flotation technique (Foreyt, 1989; Georgi and Georgi, 1990; Zajac, 1992). For each sample, one gram of feces was processed using zinc sulfate (sg 1.18) as the flotation medium to recover Giardia sp. cysts, and one gram of feces was processed using sugar (sg 1.33) as the flotation medium to recover Cryptosporidium spp. oocysts. Microscopic examination was carried out using bright field and phase contrast microscopy. An animal was considered positive for the respective parasite if a Giardia cyst or a Cryptosporidium oocyst was detected in the sample with the correct morphology, i.e. optical properties, internal structure, size and shape. Criteria used to identify these parasites were: Giardia cysts measured 12–14 m in length with an axostyle and nuclei; C. parvum oocysts measured 4–6 m, are spherical with a residuum and sporozoites, refract pink in sugar, and have a halo in phase; and C. muris oocysts measure 7–9 m in length, are oval with a
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residuum and sporozoites, refract pink in sugar, and have a halo in phase. The number of oocysts per gram of feces was enumerated for each positive fecal sample. The prevalence of each of the three protozoa in the study population was computed as the proportion of the samples that were positive of all samples examined. Descriptive statistics and other analyzes for the variables in the study were performed using the BMDP statistical software. The association between the age of the animal and the likelihood of infection with either Giardia sp. or C. muris was evaluated using the logistic regression analysis in BMDP (Dixon, 1990). Association between the age of animals younger than 30 days and the likelihood of infection with C. parvum was evaluated using the logistic regression analysis. A similar approach was used for the evaluation of the association between the season of sampling and the likelihood of infection with any of the protozoa (Kelsey et al., 1996). It was assumed that unobserved risk factors were randomly distributed among herds in the study, and the significance of this assumption was evaluated by using a mixed effect logistic regression model (Mohammed et al., 1999; Stiratelli et al., 1984). The mixed effect logistic regression analysis was performed using the EGRET statistical software (Cytel Statistical Software, MA). The significance of this assumption was evaluated by adding the herd as a random effect to the fixed factors in the logistic regression model.
3. Results Fecal samples were collected from a total of 2943 animals on 109 farms in the study population. There were 998 animals older than 24 months of age, 810 animals 6–24 months of age, and 1135 animals younger than 6 months of age. The average size of these farms was 88 animals of all ages. Farm prevalence, the number of farms with one or more infected animals out of the total number of farms sampled, was calculated for each species of protozoa. For Giardia sp. it was 76 of 109 farms (70%); C. parvum, 14 of 109 farms (13%); and C. muris, 20 of 109 farms (18%). None of these parasites was recovered from animals on 28 of 109 farms (26%). Giardia sp. cysts were recovered from animals 5 days of age through adults. C. parvum was recovered only from animals younger than 6 months, specifically from animals 3–30 days of age, and C. muris was recovered from animals 51 days of age through adults. Prevalence of these parasites in the dairy animals was calculated for each of the three age groups, as well as for all animals examined regardless of age (Table 1). There was a significant association between the age of the animal and the risk of infection with Giardia sp. (Table 2). Giardia sp. is 10 times more likely to be recovered from feces of animals younger than 6 months of age when compared to animals between 6–24 months of age. It is Table 1 Animal prevalence (%) of Giardia sp., C. parvum, and C. muris by age group Parasite
All Ages
<6 Months
6–24 Months
>24 Months
Giardia sp. C. parvum C. muris
8.9 0.9 1.1
20.1 2.4 0.5
3.5 0 1.7
0.2 0 1.5
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Table 2 Association between age of animals and the risk of infection with either Giardia sp. or C. muris Factor
Regression Coefficient
Standard Error
Giardia sp Age in months <6 >6–24 >24 Constant
0.0 −1.949 −3.572 −1.381
0.206 0.386 0.074
C. muris Age in months <6 >6–24 >24 Constant
0.0 1.197 0.829 −5.237
0.49 0.50 0.41
Odds Ratio
95% Confidence Interval
1.0 0.14 0.028
0.095–0.21 0.013–0.06
1.0 3.3 2.3
1.3–8.7 0.9–6.1
significantly less common (odds ratio = 0.03) to detect Giardia sp. in adult dairy animals than in animals younger than 6 months of age. Within the group of animals at risk of infection with C. parvum (30 days or less), there was a significant association between the age of the animal and the likelihood of infection (Fig. 1). The relation between the age of the animal and the risk of infection is non-linear where the risk increases with age, peaking at 15 days, and then declines. There was a significant association between the age of the animal and the likelihood of infection with C. muris (Table 2). Animals in the 6–24 month of age group were three times more likely to be infected than animals younger than 6 months of age. Animals that were
Fig. 1. The relationship between age and the risk of infection with C. parvum in dairy calves younger than 30 days of age in southeastern New York state.
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Fig. 2. Prevalence of Giardia sp., C. parvum, and C. murisby month of sampling in dairy cattle in southeastern New York state.
older than 24 months of age were two times more likely to be infected than animals younger than 6 months of age (P value = 0.097). Animals with diarrhea were 36.5 times more likely to be infected with C. parvum (7.3:0.2%) and two times more likely to be infected with Giardia sp. (16.0:8.0%) than animals without diarrhea. Animals without diarrhea were 3.4 times more likely to be infected with C. muris than animals with diarrhea (1.2:0.35%). The number of Giardia sp. cysts recovered from 263 animals ranged from 1 to 85,217 with a mean of 3039 cysts per gram of feces. In the 27 samples positive for C. parvum, the number of oocysts ranged from 1 to 79,040 with a mean of 21,090 oocysts per gram of feces. The number of C. muris oocysts recovered from 32 animals ranged from 1 to 100,000 with a mean of 24,413 oocysts per gram of feces. The relation between the season of sampling and the likelihood of infection with these parasites is shown in Fig. 2. There was no significant association between season and the risk of infection with these parasites. The random effect parameter was not significant in the mixed effect analysis. This result was interpreted that only the fixed effect factor (age) was associated with the risk of infection with these parasites. 4. Discussion This cross-sectional study was designed to address the following objectives: to determine the prevalence of infection of Giardia sp., C. parvum, and C. muris in dairy cattle in the
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NYC Watershed in southeastern New York state; to determine any correlation between the prevalence of infection and age of the animals; and to determine any seasonality of infection. Such a design is suitable for problem identification in a large population and for generating hypotheses regarding the factors that are related to the infection of animals (Kelsey et al., 1996). Central to these cross-sectional studies are the sampling design and sampling frame. Fifty percent of the dairies in the target population of this study were sampled, while some other studies either sampled a smaller percentage of a larger population (Garber et al., 1994; Olson et al., 1997b; Ruest et al., 1998), or a convenient sample (Anderson and Hall, 1982; McCluskey, 1992; Moore and Zeman, 1991; Olson et al., 1997a; Xiao et al., 1993). The majority of these studies had a targeted sampling design in which only pre-weaned calves were sampled (Anderson and Hall, 1982; Leek and Fayer, 1984; McCluskey, 1992; McCluskey et al., 1995; Moore and Zeman, 1991; Ongerth and Stibbs, 1989; Sobieh et al., 1987; Xiao et al., 1993). In this study, the total population was sampled to determine whether there were age differences related to possible environmental contamination. The sampling design and number of herds to be sampled was based on an expected animal prevalence of C. parvum of 26.5% (Leek and Fayer, 1984). The true prevalence in this study in calves younger than 6 months of age is much lower (2.4%) than other reports. Other studies reported prevalences ranging from 5.6 (Xiao, 1994) to 94% (McCluskey, 1992). The differences in the prevalence can be attributed to several factors: sampling design as explained above, the size of the population, and diagnostic methods employed. In addition, this study was stratified to include animals younger than 6 months of age. The primary objective was to estimate the prevalence in dairy cattle that overshadowed the prevalence of animals in the high-risk group (animals 30 days of age). If the targeted animals had been younger than 30 days of age, the prevalence of C. parvum would have been higher. In one study (Garber et al., 1994), 49% of the herds fell under the category of small (less than 100 milking animals), while in this study 98% of the farms were in this category (average size was 88 animals of all ages). Farms with less than 100 milking animals are representative of this geographical area and calving patterns show a definite seasonal trend, late summer and early fall. This meant that some of the farms did not have animals at risk of infection when fecal samples were collected. In larger herds, animals in the high risk category are expected to be present throughout the year. A quantitative centrifugation concentration flotation technique was used for diagnosis of infections because it is cost effective for screening a large number of samples, and has a higher probability of picking up low level infections than do methods, such as direct fecal smears, which do not concentrate the sample (Foreyt, 1989; Georgi and Georgi, 1990; McCluskey et al., 1995; Zajac, 1992). This is true even when the sample is stained (Garber et al., 1994; Kirkpatrick, 1985). In this study, C. parvum was recovered only from calves younger than 30 days of age. This finding is consistent with other studies (O’Handley et al., 1999; Olson et al., 1997a), where cattle of different ages were tested. Since this is the population most at risk, any efforts designed to control C. parvum infection must be directed towards this age group. The presence of Cryptotosporidium in cattle were reported over a greater age range in some studies (Garber et al., 1994; Olson et al., 1997b; Ruest et al., 1998; Xiao et al., 1993), although most infected animals were pre-weaned calves. This wider range of ages can be explained by using a diagnostic technique that does not differentiate between C. parvum
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and C. muris. Studies from Europe and Africa (Henriksen and Krogh, 1985; Mtambo et al., 1997; Quilez et al., 1996) reported a wider age range of animals positive for C. parvum. It is not clear whether this represents a true age difference, geographical variation, or is due to lack of discrimination between the two species of Cryptosporidium found in cattle. No association was found between the season of the year when animals were sampled and the likelihood of infection with C. parvum. This is consistent with one previous report (Ongerth and Stibbs, 1989), although a significant seasonal association with C. parvum infection has been reported by others (Garber et al., 1994; Moore and Zeman, 1991). C. muris was recovered in 1.1% of the animals sampled in this study. This prevalence is similar to those reported (Anderson, 1989, 1992; Esteban and Anderson, 1995; Olson et al., 1997a), but is much less than one report (Esteban and Anderson, 1995). This difference in the latter prevalence could be attributed to the fact that this was a convenience sample in one commercial dairy. Oocysts of C. muris were recovered in animals over a broad age range (51 days of age through adults). It appears that C. muris is more likely to be found in animals older than 6 months of age (Table 2). Since some studies have not used diagnostic methods which allow precise identification of the species of Cryptosporidium in cattle, or have not differentiated the species found, reports of C. parvum in animals older than 3 months must be viewed skeptically (Garber et al., 1994; Olson et al., 1997b). Based on the results of this study, reports of Cryptosporidium in animals older than 30 days of age most likely are C. muris. Previous studies have indicated a higher number of cattle, especially young animals, positive for Giardia sp and C. parvum, when diarrhea was present (Garber et al., 1994; O’Handley et al., 1999; Olson et al., 1997a; St. Jean et al., 1987; Xiao et al., 1993). This was found to be true in this study also. Animals with diarrhea were 36.5 times more likely to be infected with C. parvum (7.3:0.2%) and two times more likely to be infected with Giardia sp. (16.0:8.0%) than animals without diarrhea. On the other hand, when cattle are infected with C. muris, diarrhea was usually not seen. In this study, animals without diarrhea were 3.5 times more likely to be infected with C. muris than animals with diarrhea (1.2:0.35%). This agreed with a study of calves in British Columbia (Olson et al., 1997a). Quantifying the number of protozoan oocysts per gram of feces gives an idea of the intensity of possible environmental contamination due to the infected animals. The number of Giardia sp. cysts recovered from 263 animals ranged from 1 to 85,217 with a mean of 3039 cysts/gm feces. In 27 samples positive for C. parvum, the number of oocysts ranged from 1 to 79,040 with a mean of 21,090 oocysts/gm feces. The number of C. muris oocysts recovered from 32 animals ranged from 1 to 100,000 with a mean of 24,413 oocysts/gm feces. One study reported a greater range of Giardia cysts and C. parvum oocysts, but a much lower range of C. muris oocysts recovered. However, all the mean number of cysts were much lower (Olson et al., 1997a). To our knowledge, no comparable cross-sectional studies for the prevalence of Giardia sp. in cattle have been reported. There are several case reports on a smaller non-randomized study population (Buret et al., 1990; Deshpande and Shastri, 1981; Diaz et al., 1996; O’Handley et al., 1999; Olson et al., 1997a; Quilez et al., 1996; St. Jean et al., 1987; Xiao et al., 1993; Xiao, 1994). Most of these studies reported higher prevalence of infection in younger animals (Buret et al., 1990; Deshpande and Shastri, 1981; O’Handley et al., 1999; Olson et al., 1997a; Xiao, 1994), which agrees with our findings. The Giardia sp. posi-
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tive animals in this study occurred most often at 2–5 months of age. Overall prevalence of Giardia infections has been reported to range from 0.16 (Diaz et al., 1996) to 73% (Olson et al., 1997a) in cattle. The overall prevalence found in this study (8.9%) falls in the lower end of that range. No seasonal variation was seen in the occurrence of Giardia sp. in cattle. The farm prevalence for Giardia sp. of 69.7% fell about half way between reports of 100 (Olson et al., 1997a,b) and 45.7% (Ruest et al., 1998), respectively. The prevalence of 12.8% for C. parvum was much lower than reported values which ranged from 56 to 100% (Anderson and Hall, 1982; Olson et al., 1997a,b; Ruest et al., 1998). The 18.3% prevalence for C. muris was also much lower than one report of 40% (Olson et al., 1997a). In conclusion, although the present study has demonstrated the prevalence of Giardia sp., C. parvum, and C. muris in dairy herds, more analytical studies are needed to identify factors that may contribute to their presence in these animals. For example, geographical location including data on temperature, rainfall and soil type, herd size and management practices related to animal husbandry all may play important roles in the development and perpetuation of parasite infections in cattle.
Acknowledgements The project was supported financially by New York city through the Department of Environmental Protection and the Watershed Agricultural Council as part of the New York City Watershed Agricultural Program. The authors wish to thank all of the farmers who cooperated with them in this project. The technical support of Michelle Burley, Dorothy Crane, Lena Gutberlet, Curtis Kretz, and Angela Struble is gratefully acknowledged.
References Adam, R.D., 1991. The biology of Giardia spp. Microbiol. Rev. 55, 706–732. Anderson, B.C., Hall, R.F., 1982. Cryptosporidial infection in Idaho dairy calves. J. Am. Vet. Med. Assoc. 181, 484–485. Anderson, B.C., 1989. Cryptosporidium spp in cattle. In: Angus, K.W., Blewett, D.A. (Eds.), Proceedings of 1st International Workshop on Cryptosporidiosis. Edinburgh Moredun Res. Inst., Edinburgh, September 7–8, 1988, pp 55–63. Anderson, B.C., 1992. Bovine gastric and intestinal cryptosporidiosis: present situation. Bovine Proc. 24, 15–24. Buret, A., denHollander, N., Wallis, P.M., Befus, D., Olson, M.E., 1990. Zoonotic potential of giardiasis in domestic ruminants. J. Infect. Dis. 162, 231–237. Current, W.L., Garcia, L.S., 1991. Cryptosporidiosis. Clin. Microbiol. Rev. 4, 325–358. Current, W.L., Blagburn, B.L., 1990. Cryptosporidium: infections in man and domesticated animals. In: Long, P.L. (Ed.), Coccidiosis of Man and Domesticated Animals. CRC Press, Boston, pp. 55-185. Current, W.L., Reese, N.C., Ernst, J.V., Bailey, W.S., Heyman, M.B., Weinstein, W.M., 1983. Human cryptosporidiosis in immunocompetent and immunodeficient persons. New Eng. J. Med. 308, 1252–1257. Dixon, W.J., 1990. BMDP Statistical Software Manual. University of California Press, Berkeley, CA., pp. 1105–1144. Deshpande, P.D., Shastri, U.V., 1981. Incidence of Giardia infection in calves in Maharashtra state. India. Trop. Anim. Hlth. Prod. 13, 34. Diaz, V., Campos, M., Lozano, J., Manas, I., Gonzalez, J., 1996. Aspects of animal giardiosis in Granada province (southern Spain). Vet. Parasitol. 64, 171–176.
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Esteban, E., Anderson, B.C., 1995. Cryptosporidium muris: prevalence, persistency, and detrimental effect on milk production in a drylot dairy. J. Dairy Sci. 78, 1068–1072. Fayer, R. (Ed.), 1997. Cryptosporidium and Cryptosporidiosis. CRC Press, New York, NY. Foreyt, W.J., 1989. Diagnostic Parasitology. Vet. Clinic. North Am. Small Anim. Pract. 19, 979–1000. Garber, L.P., Salman, M.D., Hurd, H.S., Keefe, T., Schlater, J.L., 1994. Potential risk factors for Cryptosporidium infection in dairy calves. J. Am. Vet. Med. Assoc. 205, 86–91. Georgi, J.R., Georgi, M.E., 1990. Parasitology for Veterinarians. 5th Edition. W.B. Saunders Co., Philadelphia, PA., pp. 297–301. Harp, J.A., Woodmansee, D.B., Moon, H.W., 1990. Resistance of calves to Cryptosporidium parvum: effects of age and previous exposure. Infect. Immun. 58, 2237–2240. Henriksen, S.A., Krogh, H.V., 1985. Bovine cryptosporidiosis in Denmark. 1. Prevalence, age distribution, and seasonal variation. Nord. Vet. Med. 37, 34–41. Kelsey, J.L., Thompson, W.D., Evans, A.S., 1996. Methods in Observational Epidemiology. 2nd Edition. Oxford University Press, New York, NY. Kirkpatrick, C.E., 1985. Cryptosporidium infection as a cause of calf diarrhea. Vet. Clinic North Am. Food Anim. Pract. 1, 515–528. Leek, R.G., Fayer, R., 1984. Prevalence of Cryptosporidium infections, and their relation to diarrhea in calves on 12 dairy farms in Maryland, and their relation to diarrhea in calves on 12 dairy farms in Maryland. Proc. Helminthol. Soc. Wash. 51, 360–361. Lefay, D., Naciri, M., Poirer, P., Chermette, R., 2000. Prevalence of Cryptosporidium infection in calves in France. Vet. Parasitol. 89, 1–9. Levy, P.S., Lemeshow, S., 1980. Sampling for Health Professionals. Life-Time Learning Publications, Belmont, CA., pp. 37–61. McCluskey, B.J., 1992. Cryptosporidium parvum oocyst shedding in Florida dairy calves. Animal Health Insight, pp. 1–5. McCluskey, B.J., Greiner, E.C., Donovan, G.A., 1995. Patterns of Cryptosporidium oocyst shedding in calves and a comparison of two diagnostic methods. Vet. Parasitol. 60, 185–190. Mohammed, H.O., Wade, S.E., Schaaf, S., 1999. Risk factors associated with Cryptosporidium parvum infection in dairy cattle in southeastern New York state. Vet. Parasitol. 83, 1–13. Moore, D.A., Zeman, D.H., 1991. Cryptosporidiosis in neonatal calves: 277 cases (1986-1987). J. Am. Vet. Med. Assoc. 198, 1969–1971. Mtambo, M.M.A., Scbatwale, J.B., Kambarage, D.M., 1997. Prevalence of Cryptosporidium spp. oocysts in cattle and wildlife in Morogoro region. Tanzania. Prev. Vet. Med. 31, 185–190. O’Donoghue, P.J., 1995. Cryptosporidium and cryptosporidiosis in man and animals. Int. J. Parasit. 25, 139–195. Ongerth, J.E., Stibbs, H.H., 1989. Prevalence of Cryptosporidium infection in dairy calves in western Washington. Am. J. Vet. Res. 50, 1069–1070. O’Handley, R.M., Cockwill, C., McAllister, T.A., Jelinski, M., Morck, D.W., Olson, M.E., 1999. Duration of naturally acquired giardiosis and cryptosporidiosis in dairy calves and their association with diarrhea. J. Am. Vet. Med. Assoc. 214, 391–396. Olson, M.E., Guselle, N.J., O’Handley, R.M., Swift, M.L., McAllister, T.A., Jelinski, M.D., Morck, D.W., 1997a. Giardia and Cryptosporidium in dairy calves in British Columbia. Can. Vet. J. 38, 703–706. Olson, M.E., Thorlakson, C.L., Deselliers, L., Morck, D.W., McAllister, T.A., 1997b. Giardia and Cryptosporidium in Canadian farm animals. Vet. Parasitol. 68, 375–381. Quilez, J., Sanchez-Acedo, C., del Cacho, E., Clavel, A., Causape, A.C., 1996. Prevalence of Cryptosporidium and Giardia infections in cattle in Aragon (northeastern Spain). Vet. Parasitol. 66, 139–146. Ruest, N., Faubert, G.M., Couture, Y., 1998. Prevalence and geographical distribution of Giardia spp. and Cryptosporidium spp. in dairy farms in Quebec. Can. Vet. J. 39, 697–700. St. Jean, G., Couture, Y., Dubreuil, P., Frechette, J.L., 1987. Diagnosis of Giardia infection in 14 calves. J. Am. Vet. Med. Assoc. 191, 831–832. Smith, P.D., 1989. Giardia lamblia. In: Walzer, P.D., Genta, R.M. (Eds.), Parasitic Infections in the Compromised Host. Marcel Dekker, New York, NY, pp. 343–384. Sobieh, M., Tacal Jr., J.V., Wilcke Jr., B.W., Lawrence, W., El-Ahraf, A., 1987. Investigation of cryptosporidial infection in calves in San Bernardino County, California. J. Am. Vet. Med. Assoc. 191, 816–818.
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Stiratelli, R., Laird, N.M., Ware, J.H., 1984. Random-effect models for serial observations with binary response. Biometrics 4, 961–971. Thompson, R.C.A., Reynoldson, J.A., Mendis, A.H.W., 1993. Giardia and giardiasis. Adv. Parasitol. 32, 71–160. Walterspiel, J.N., Pickering, L.K., 1994. Giardia and giardiasis, In: Sun, T. (Ed.), Progress in Clinical Parasitology. Vol. 4, CRC Press, London, pp. 1–26. Xiao, L., Herd, R.P., Rings, D.M., 1993. Concurrent infections of Giardia and Cryptosporidium on two Ohio farms with calf diarrhea. Vet. Parasitol. 51, 41–48. Xiao, L., 1994. Giardia infection in farm animals. Parasitol. Today 10, 436–438. Zajac, A.M., 1992. Giardiasis. Compend. Contin. Edu. Prac. Vet. 14, 604–611.
Prevalence of Giardia sp., Cryptosporidium parvum and Cryptosporidium muris (C. andersoni) in 109 dairy herds in five counties of southeastern New York S.E. Wade a,∗ , H.O. Mohammed b , S.L. Schaaf a a b
Parasitology Section, Department of Population Medicine and Diagnostic Science, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA Epidemiology Section, Department of Population Medicine and Diagnostic Science, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA Received 2 May 2000; accepted 12 July 2000
Abstract A cross-sectional study was undertaken to determine the prevalence of Giardia sp. (G. duodenalis group), Cryptosporidium parvum and Cryptosporidium muris (C. andersoni) in dairy cattle in three different age groups, and to evaluate the association of age and season with prevalence. One hundred and nine dairy farms, from a total of 212 farms, in five counties of southeastern New York volunteered to participate. On these farms, 2943 fecal samples were collected from three defined age groups. The farms were randomly assigned for sampling within the four seasons of the year. Each farm was visited once during the study period from March 1993 to June 1994 to collect fecal samples. Demographic data on the study population was collected at the time of sampling by interviewing the farm owner or manager. At collection, fecal samples were scored as diarrheic or non-diarrheic, and each condition was later related to positive or negative infection with these parasites. Fecal samples were processed using a quantitative centrifugation concentration flotation technique and enumerated using bright field and phase contrast microscopy. In this study, the overall population prevalence for Giardia sp. was 8.9%; C. parvum, 0.9%; and C. muris, 1.1%. When considering animals most at the risk of infection (those younger than 6 months of age) Giardia sp. and C. parvum was found in 20.1 and 2.4% of the animals, respectively. Giardia sp. and C. muris were found in all age groups. There was no significant seasonal pattern of infection for any of these parasites. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Giardia sp.; Cryptosporidium parvum; Cryptosporidium muris (C. andersoni); Prevalence; Dairy cattle
∗ Corresponding author. Tel.: +1-607-253-3581; fax: +1-607-253-3943 E-mail address: [email protected] (S.E. Wade).
0304-4017/00/$ – see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 0 0 ) 0 0 3 3 7 - X
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1. Introduction In recent years, there has been increased concern about the presence of two protozoa, Giardia sp. and Cryptosporidium parvum in the environment, especially water, and in animals, including humans (Adam, 1991; Current and Blagburn, 1990; Current and Garcia, 1991; Fayer, 1997; O’Donoghue, 1995; Thompson et al., 1993; Walterspiel and Pickering, 1994). These protozoa are of public health concern because they may cause infection and severe illness in humans. Infections are self-limiting in people with normal immune systems, but infection can be life threatening in people who have compromised immune systems (Current et al., 1983; Smith, 1989). In response to the Federal Safe Drinking Water Act (SDWA) amendments of 1986 (42USC300g-1), and regulations of the Surface Drinking Water Treatment Rule (SDWTR) (54FR27486, June 29, 1989), the USEPA stated that New York city may avoid filtration of its drinking water, if it shows that the source water meets federal and state raw water standards, that adequate disinfection is in place and that an adequate watershed protection program to reduce the risk of waterborne disease is implemented. The NYC Watershed Agricultural Program, created as part of the filtration avoidance, was established in 1993 to maintain the quality of the water supply, and the economic viability of agriculture in the NYC Watershed through whole farm planning. This program focuses on the management of pesticides, sediment, nutrients such as nitrogen and phosphorus, and pathogenic organisms such as Giardia sp. and C. parvum, as they relate to agriculture and water quality. Due to the lack of information concerning the epidemiology of Giardia sp. and C. parvum infections in dairy cattle, generally and specifically in this watershed, this study was initiated. Giardia sp. and C. parvum have both been reported from cattle (Anderson and Hall, 1982; Anderson, 1989; Buret et al., 1990; Harp et al., 1990; Kirkpatrick, 1985; Leek and Fayer, 1984; Lefay et al., 2000; McCluskey, 1992; McCluskey et al., 1995; Moore and Zeman, 1991; O’Handley et al., 1999; Olson et al., 1997a,b; Ongerth and Stibbs, 1989; Ruest et al., 1998; Sobieh et al., 1987; St. Jean et al., 1987; Xiao et al., 1993; Xiao, 1994). The prevalence and clinical significance of Giardia sp. infection in cattle of all ages is not well understood. There have been reports of infection with this parasite in young animals with and without associated clinical illness (Buret et al., 1990; O’Handley et al., 1999; Olson et al., 1997a,b; Ruest et al., 1998; St. Jean et al., 1987; Xiao et al., 1993; Xiao, 1994). Cryptosporidium parvum is known to be associated with diarrheal illness in calves younger than 1 month of age (Anderson and Hall, 1982; Harp et al., 1990; Kirkpatrick, 1985; Leek and Fayer, 1984; Lefay et al., 2000; McCluskey, 1992; McCluskey et al., 1995; Moore and Zeman, 1991; O’Handley et al., 1999; Olson et al., 1997a,b; Ongerth and Stibbs, 1989; Ruest et al., 1998; Sobieh et al., 1987; Xiao et al., 1993). Cryptosporidium muris is also found in cattle in which it may cause gastritis, and is statistically associated with reduced milk production (Anderson, 1989, 1992; Esteban and Anderson, 1995). In any epidemiologic study in cattle, infection with C. muris must be differentiated from infection with C. parvum since the latter species is a known pathogen in humans, while the former is not considered a threat to human health (Fayer, 1997). To determine the extent of the problem in dairy cattle, and to aid in recommending farm management practices relative to Giardia sp. and C. parvum, a cross-sectional study
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was conducted to estimate the prevalence of Giardia sp., C. parvum and C. muris (C. andersoni) in dairy cattle in this geographic area. This study represented the first step towards the development of the recommendations. The associated effects of age and season with prevalence were also evaluated.
2. Material and methods The study population consisted of dairy cattle on farms in portions of five counties (Delaware, Greene, Schoharie, Ulster, Sullivan) within the Catskill/Delaware systems of the NYC Watershed in southeastern New York. Based on a reported prevalence of C. parvum in dairy animals (Leek and Fayer, 1984), and by using the formula suggested by Levy and Lemeshow (1980), it was determined that at least 100 farms needed to be sampled. This sample size was computed using the following assumptions: the expected prevalence of C. parvum in calves was 26.5% (Leek and Fayer, 1984); the level of significance to detect this prevalence is 95% (a = 0.05); the power for the estimated prevalence was 90% (b = 0.1); the acceptable difference between the true prevalence and the estimated prevalence was 10%; and the prevalence of Giardia sp. among these herds was equal to the prevalence of C. parvum. A recruitment letter was sent to 212 dairy farms located in the study area. A total of 109 farms volunteered to participate, and were randomly assigned to a season of collection by the response date. Four seasons were identified according to the weather pattern in New York state. These are winter (January–March), spring (April–June), summer (July–September), and fall (October–December). Farms were visited once during the study period from March 1993 to June 1994. The population was stratified into three age groups: 0–6 months of age; older than 6 months -first freshening (about 24 months); and animals older than 24 months. All animals 0–6 months of age were sampled unless more than 20 were present, then 70% of the animals in that age group were sampled. Six animals from the second age group and nine animals from the third age group were sampled. Fecal samples were collected rectally from each animal in the study population and were condition scored as diarrheic or non-diarrheic. Samples were kept refrigerated at 4◦ C from collection to processing and evaluation in the laboratory. All samples were processed within 2 weeks of collection. Laboratory analysis of the fecal samples was conducted using a standard quantitative centrifugation concentration flotation technique (Foreyt, 1989; Georgi and Georgi, 1990; Zajac, 1992). For each sample, one gram of feces was processed using zinc sulfate (sg 1.18) as the flotation medium to recover Giardia sp. cysts, and one gram of feces was processed using sugar (sg 1.33) as the flotation medium to recover Cryptosporidium spp. oocysts. Microscopic examination was carried out using bright field and phase contrast microscopy. An animal was considered positive for the respective parasite if a Giardia cyst or a Cryptosporidium oocyst was detected in the sample with the correct morphology, i.e. optical properties, internal structure, size and shape. Criteria used to identify these parasites were: Giardia cysts measured 12–14 m in length with an axostyle and nuclei; C. parvum oocysts measured 4–6 m, are spherical with a residuum and sporozoites, refract pink in sugar, and have a halo in phase; and C. muris oocysts measure 7–9 m in length, are oval with a
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residuum and sporozoites, refract pink in sugar, and have a halo in phase. The number of oocysts per gram of feces was enumerated for each positive fecal sample. The prevalence of each of the three protozoa in the study population was computed as the proportion of the samples that were positive of all samples examined. Descriptive statistics and other analyzes for the variables in the study were performed using the BMDP statistical software. The association between the age of the animal and the likelihood of infection with either Giardia sp. or C. muris was evaluated using the logistic regression analysis in BMDP (Dixon, 1990). Association between the age of animals younger than 30 days and the likelihood of infection with C. parvum was evaluated using the logistic regression analysis. A similar approach was used for the evaluation of the association between the season of sampling and the likelihood of infection with any of the protozoa (Kelsey et al., 1996). It was assumed that unobserved risk factors were randomly distributed among herds in the study, and the significance of this assumption was evaluated by using a mixed effect logistic regression model (Mohammed et al., 1999; Stiratelli et al., 1984). The mixed effect logistic regression analysis was performed using the EGRET statistical software (Cytel Statistical Software, MA). The significance of this assumption was evaluated by adding the herd as a random effect to the fixed factors in the logistic regression model.
3. Results Fecal samples were collected from a total of 2943 animals on 109 farms in the study population. There were 998 animals older than 24 months of age, 810 animals 6–24 months of age, and 1135 animals younger than 6 months of age. The average size of these farms was 88 animals of all ages. Farm prevalence, the number of farms with one or more infected animals out of the total number of farms sampled, was calculated for each species of protozoa. For Giardia sp. it was 76 of 109 farms (70%); C. parvum, 14 of 109 farms (13%); and C. muris, 20 of 109 farms (18%). None of these parasites was recovered from animals on 28 of 109 farms (26%). Giardia sp. cysts were recovered from animals 5 days of age through adults. C. parvum was recovered only from animals younger than 6 months, specifically from animals 3–30 days of age, and C. muris was recovered from animals 51 days of age through adults. Prevalence of these parasites in the dairy animals was calculated for each of the three age groups, as well as for all animals examined regardless of age (Table 1). There was a significant association between the age of the animal and the risk of infection with Giardia sp. (Table 2). Giardia sp. is 10 times more likely to be recovered from feces of animals younger than 6 months of age when compared to animals between 6–24 months of age. It is Table 1 Animal prevalence (%) of Giardia sp., C. parvum, and C. muris by age group Parasite
All Ages
<6 Months
6–24 Months
>24 Months
Giardia sp. C. parvum C. muris
8.9 0.9 1.1
20.1 2.4 0.5
3.5 0 1.7
0.2 0 1.5
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Table 2 Association between age of animals and the risk of infection with either Giardia sp. or C. muris Factor
Regression Coefficient
Standard Error
Giardia sp Age in months <6 >6–24 >24 Constant
0.0 −1.949 −3.572 −1.381
0.206 0.386 0.074
C. muris Age in months <6 >6–24 >24 Constant
0.0 1.197 0.829 −5.237
0.49 0.50 0.41
Odds Ratio
95% Confidence Interval
1.0 0.14 0.028
0.095–0.21 0.013–0.06
1.0 3.3 2.3
1.3–8.7 0.9–6.1
significantly less common (odds ratio = 0.03) to detect Giardia sp. in adult dairy animals than in animals younger than 6 months of age. Within the group of animals at risk of infection with C. parvum (30 days or less), there was a significant association between the age of the animal and the likelihood of infection (Fig. 1). The relation between the age of the animal and the risk of infection is non-linear where the risk increases with age, peaking at 15 days, and then declines. There was a significant association between the age of the animal and the likelihood of infection with C. muris (Table 2). Animals in the 6–24 month of age group were three times more likely to be infected than animals younger than 6 months of age. Animals that were
Fig. 1. The relationship between age and the risk of infection with C. parvum in dairy calves younger than 30 days of age in southeastern New York state.
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Fig. 2. Prevalence of Giardia sp., C. parvum, and C. murisby month of sampling in dairy cattle in southeastern New York state.
older than 24 months of age were two times more likely to be infected than animals younger than 6 months of age (P value = 0.097). Animals with diarrhea were 36.5 times more likely to be infected with C. parvum (7.3:0.2%) and two times more likely to be infected with Giardia sp. (16.0:8.0%) than animals without diarrhea. Animals without diarrhea were 3.4 times more likely to be infected with C. muris than animals with diarrhea (1.2:0.35%). The number of Giardia sp. cysts recovered from 263 animals ranged from 1 to 85,217 with a mean of 3039 cysts per gram of feces. In the 27 samples positive for C. parvum, the number of oocysts ranged from 1 to 79,040 with a mean of 21,090 oocysts per gram of feces. The number of C. muris oocysts recovered from 32 animals ranged from 1 to 100,000 with a mean of 24,413 oocysts per gram of feces. The relation between the season of sampling and the likelihood of infection with these parasites is shown in Fig. 2. There was no significant association between season and the risk of infection with these parasites. The random effect parameter was not significant in the mixed effect analysis. This result was interpreted that only the fixed effect factor (age) was associated with the risk of infection with these parasites. 4. Discussion This cross-sectional study was designed to address the following objectives: to determine the prevalence of infection of Giardia sp., C. parvum, and C. muris in dairy cattle in the
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NYC Watershed in southeastern New York state; to determine any correlation between the prevalence of infection and age of the animals; and to determine any seasonality of infection. Such a design is suitable for problem identification in a large population and for generating hypotheses regarding the factors that are related to the infection of animals (Kelsey et al., 1996). Central to these cross-sectional studies are the sampling design and sampling frame. Fifty percent of the dairies in the target population of this study were sampled, while some other studies either sampled a smaller percentage of a larger population (Garber et al., 1994; Olson et al., 1997b; Ruest et al., 1998), or a convenient sample (Anderson and Hall, 1982; McCluskey, 1992; Moore and Zeman, 1991; Olson et al., 1997a; Xiao et al., 1993). The majority of these studies had a targeted sampling design in which only pre-weaned calves were sampled (Anderson and Hall, 1982; Leek and Fayer, 1984; McCluskey, 1992; McCluskey et al., 1995; Moore and Zeman, 1991; Ongerth and Stibbs, 1989; Sobieh et al., 1987; Xiao et al., 1993). In this study, the total population was sampled to determine whether there were age differences related to possible environmental contamination. The sampling design and number of herds to be sampled was based on an expected animal prevalence of C. parvum of 26.5% (Leek and Fayer, 1984). The true prevalence in this study in calves younger than 6 months of age is much lower (2.4%) than other reports. Other studies reported prevalences ranging from 5.6 (Xiao, 1994) to 94% (McCluskey, 1992). The differences in the prevalence can be attributed to several factors: sampling design as explained above, the size of the population, and diagnostic methods employed. In addition, this study was stratified to include animals younger than 6 months of age. The primary objective was to estimate the prevalence in dairy cattle that overshadowed the prevalence of animals in the high-risk group (animals 30 days of age). If the targeted animals had been younger than 30 days of age, the prevalence of C. parvum would have been higher. In one study (Garber et al., 1994), 49% of the herds fell under the category of small (less than 100 milking animals), while in this study 98% of the farms were in this category (average size was 88 animals of all ages). Farms with less than 100 milking animals are representative of this geographical area and calving patterns show a definite seasonal trend, late summer and early fall. This meant that some of the farms did not have animals at risk of infection when fecal samples were collected. In larger herds, animals in the high risk category are expected to be present throughout the year. A quantitative centrifugation concentration flotation technique was used for diagnosis of infections because it is cost effective for screening a large number of samples, and has a higher probability of picking up low level infections than do methods, such as direct fecal smears, which do not concentrate the sample (Foreyt, 1989; Georgi and Georgi, 1990; McCluskey et al., 1995; Zajac, 1992). This is true even when the sample is stained (Garber et al., 1994; Kirkpatrick, 1985). In this study, C. parvum was recovered only from calves younger than 30 days of age. This finding is consistent with other studies (O’Handley et al., 1999; Olson et al., 1997a), where cattle of different ages were tested. Since this is the population most at risk, any efforts designed to control C. parvum infection must be directed towards this age group. The presence of Cryptotosporidium in cattle were reported over a greater age range in some studies (Garber et al., 1994; Olson et al., 1997b; Ruest et al., 1998; Xiao et al., 1993), although most infected animals were pre-weaned calves. This wider range of ages can be explained by using a diagnostic technique that does not differentiate between C. parvum
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and C. muris. Studies from Europe and Africa (Henriksen and Krogh, 1985; Mtambo et al., 1997; Quilez et al., 1996) reported a wider age range of animals positive for C. parvum. It is not clear whether this represents a true age difference, geographical variation, or is due to lack of discrimination between the two species of Cryptosporidium found in cattle. No association was found between the season of the year when animals were sampled and the likelihood of infection with C. parvum. This is consistent with one previous report (Ongerth and Stibbs, 1989), although a significant seasonal association with C. parvum infection has been reported by others (Garber et al., 1994; Moore and Zeman, 1991). C. muris was recovered in 1.1% of the animals sampled in this study. This prevalence is similar to those reported (Anderson, 1989, 1992; Esteban and Anderson, 1995; Olson et al., 1997a), but is much less than one report (Esteban and Anderson, 1995). This difference in the latter prevalence could be attributed to the fact that this was a convenience sample in one commercial dairy. Oocysts of C. muris were recovered in animals over a broad age range (51 days of age through adults). It appears that C. muris is more likely to be found in animals older than 6 months of age (Table 2). Since some studies have not used diagnostic methods which allow precise identification of the species of Cryptosporidium in cattle, or have not differentiated the species found, reports of C. parvum in animals older than 3 months must be viewed skeptically (Garber et al., 1994; Olson et al., 1997b). Based on the results of this study, reports of Cryptosporidium in animals older than 30 days of age most likely are C. muris. Previous studies have indicated a higher number of cattle, especially young animals, positive for Giardia sp and C. parvum, when diarrhea was present (Garber et al., 1994; O’Handley et al., 1999; Olson et al., 1997a; St. Jean et al., 1987; Xiao et al., 1993). This was found to be true in this study also. Animals with diarrhea were 36.5 times more likely to be infected with C. parvum (7.3:0.2%) and two times more likely to be infected with Giardia sp. (16.0:8.0%) than animals without diarrhea. On the other hand, when cattle are infected with C. muris, diarrhea was usually not seen. In this study, animals without diarrhea were 3.5 times more likely to be infected with C. muris than animals with diarrhea (1.2:0.35%). This agreed with a study of calves in British Columbia (Olson et al., 1997a). Quantifying the number of protozoan oocysts per gram of feces gives an idea of the intensity of possible environmental contamination due to the infected animals. The number of Giardia sp. cysts recovered from 263 animals ranged from 1 to 85,217 with a mean of 3039 cysts/gm feces. In 27 samples positive for C. parvum, the number of oocysts ranged from 1 to 79,040 with a mean of 21,090 oocysts/gm feces. The number of C. muris oocysts recovered from 32 animals ranged from 1 to 100,000 with a mean of 24,413 oocysts/gm feces. One study reported a greater range of Giardia cysts and C. parvum oocysts, but a much lower range of C. muris oocysts recovered. However, all the mean number of cysts were much lower (Olson et al., 1997a). To our knowledge, no comparable cross-sectional studies for the prevalence of Giardia sp. in cattle have been reported. There are several case reports on a smaller non-randomized study population (Buret et al., 1990; Deshpande and Shastri, 1981; Diaz et al., 1996; O’Handley et al., 1999; Olson et al., 1997a; Quilez et al., 1996; St. Jean et al., 1987; Xiao et al., 1993; Xiao, 1994). Most of these studies reported higher prevalence of infection in younger animals (Buret et al., 1990; Deshpande and Shastri, 1981; O’Handley et al., 1999; Olson et al., 1997a; Xiao, 1994), which agrees with our findings. The Giardia sp. posi-
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tive animals in this study occurred most often at 2–5 months of age. Overall prevalence of Giardia infections has been reported to range from 0.16 (Diaz et al., 1996) to 73% (Olson et al., 1997a) in cattle. The overall prevalence found in this study (8.9%) falls in the lower end of that range. No seasonal variation was seen in the occurrence of Giardia sp. in cattle. The farm prevalence for Giardia sp. of 69.7% fell about half way between reports of 100 (Olson et al., 1997a,b) and 45.7% (Ruest et al., 1998), respectively. The prevalence of 12.8% for C. parvum was much lower than reported values which ranged from 56 to 100% (Anderson and Hall, 1982; Olson et al., 1997a,b; Ruest et al., 1998). The 18.3% prevalence for C. muris was also much lower than one report of 40% (Olson et al., 1997a). In conclusion, although the present study has demonstrated the prevalence of Giardia sp., C. parvum, and C. muris in dairy herds, more analytical studies are needed to identify factors that may contribute to their presence in these animals. For example, geographical location including data on temperature, rainfall and soil type, herd size and management practices related to animal husbandry all may play important roles in the development and perpetuation of parasite infections in cattle.
Acknowledgements The project was supported financially by New York city through the Department of Environmental Protection and the Watershed Agricultural Council as part of the New York City Watershed Agricultural Program. The authors wish to thank all of the farmers who cooperated with them in this project. The technical support of Michelle Burley, Dorothy Crane, Lena Gutberlet, Curtis Kretz, and Angela Struble is gratefully acknowledged.
References Adam, R.D., 1991. The biology of Giardia spp. Microbiol. Rev. 55, 706–732. Anderson, B.C., Hall, R.F., 1982. Cryptosporidial infection in Idaho dairy calves. J. Am. Vet. Med. Assoc. 181, 484–485. Anderson, B.C., 1989. Cryptosporidium spp in cattle. In: Angus, K.W., Blewett, D.A. (Eds.), Proceedings of 1st International Workshop on Cryptosporidiosis. Edinburgh Moredun Res. Inst., Edinburgh, September 7–8, 1988, pp 55–63. Anderson, B.C., 1992. Bovine gastric and intestinal cryptosporidiosis: present situation. Bovine Proc. 24, 15–24. Buret, A., denHollander, N., Wallis, P.M., Befus, D., Olson, M.E., 1990. Zoonotic potential of giardiasis in domestic ruminants. J. Infect. Dis. 162, 231–237. Current, W.L., Garcia, L.S., 1991. Cryptosporidiosis. Clin. Microbiol. Rev. 4, 325–358. Current, W.L., Blagburn, B.L., 1990. Cryptosporidium: infections in man and domesticated animals. In: Long, P.L. (Ed.), Coccidiosis of Man and Domesticated Animals. CRC Press, Boston, pp. 55-185. Current, W.L., Reese, N.C., Ernst, J.V., Bailey, W.S., Heyman, M.B., Weinstein, W.M., 1983. Human cryptosporidiosis in immunocompetent and immunodeficient persons. New Eng. J. Med. 308, 1252–1257. Dixon, W.J., 1990. BMDP Statistical Software Manual. University of California Press, Berkeley, CA., pp. 1105–1144. Deshpande, P.D., Shastri, U.V., 1981. Incidence of Giardia infection in calves in Maharashtra state. India. Trop. Anim. Hlth. Prod. 13, 34. Diaz, V., Campos, M., Lozano, J., Manas, I., Gonzalez, J., 1996. Aspects of animal giardiosis in Granada province (southern Spain). Vet. Parasitol. 64, 171–176.
10
S.E. Wade et al. / Veterinary Parasitology 93 (2000) 1–11
Esteban, E., Anderson, B.C., 1995. Cryptosporidium muris: prevalence, persistency, and detrimental effect on milk production in a drylot dairy. J. Dairy Sci. 78, 1068–1072. Fayer, R. (Ed.), 1997. Cryptosporidium and Cryptosporidiosis. CRC Press, New York, NY. Foreyt, W.J., 1989. Diagnostic Parasitology. Vet. Clinic. North Am. Small Anim. Pract. 19, 979–1000. Garber, L.P., Salman, M.D., Hurd, H.S., Keefe, T., Schlater, J.L., 1994. Potential risk factors for Cryptosporidium infection in dairy calves. J. Am. Vet. Med. Assoc. 205, 86–91. Georgi, J.R., Georgi, M.E., 1990. Parasitology for Veterinarians. 5th Edition. W.B. Saunders Co., Philadelphia, PA., pp. 297–301. Harp, J.A., Woodmansee, D.B., Moon, H.W., 1990. Resistance of calves to Cryptosporidium parvum: effects of age and previous exposure. Infect. Immun. 58, 2237–2240. Henriksen, S.A., Krogh, H.V., 1985. Bovine cryptosporidiosis in Denmark. 1. Prevalence, age distribution, and seasonal variation. Nord. Vet. Med. 37, 34–41. Kelsey, J.L., Thompson, W.D., Evans, A.S., 1996. Methods in Observational Epidemiology. 2nd Edition. Oxford University Press, New York, NY. Kirkpatrick, C.E., 1985. Cryptosporidium infection as a cause of calf diarrhea. Vet. Clinic North Am. Food Anim. Pract. 1, 515–528. Leek, R.G., Fayer, R., 1984. Prevalence of Cryptosporidium infections, and their relation to diarrhea in calves on 12 dairy farms in Maryland, and their relation to diarrhea in calves on 12 dairy farms in Maryland. Proc. Helminthol. Soc. Wash. 51, 360–361. Lefay, D., Naciri, M., Poirer, P., Chermette, R., 2000. Prevalence of Cryptosporidium infection in calves in France. Vet. Parasitol. 89, 1–9. Levy, P.S., Lemeshow, S., 1980. Sampling for Health Professionals. Life-Time Learning Publications, Belmont, CA., pp. 37–61. McCluskey, B.J., 1992. Cryptosporidium parvum oocyst shedding in Florida dairy calves. Animal Health Insight, pp. 1–5. McCluskey, B.J., Greiner, E.C., Donovan, G.A., 1995. Patterns of Cryptosporidium oocyst shedding in calves and a comparison of two diagnostic methods. Vet. Parasitol. 60, 185–190. Mohammed, H.O., Wade, S.E., Schaaf, S., 1999. Risk factors associated with Cryptosporidium parvum infection in dairy cattle in southeastern New York state. Vet. Parasitol. 83, 1–13. Moore, D.A., Zeman, D.H., 1991. Cryptosporidiosis in neonatal calves: 277 cases (1986-1987). J. Am. Vet. Med. Assoc. 198, 1969–1971. Mtambo, M.M.A., Scbatwale, J.B., Kambarage, D.M., 1997. Prevalence of Cryptosporidium spp. oocysts in cattle and wildlife in Morogoro region. Tanzania. Prev. Vet. Med. 31, 185–190. O’Donoghue, P.J., 1995. Cryptosporidium and cryptosporidiosis in man and animals. Int. J. Parasit. 25, 139–195. Ongerth, J.E., Stibbs, H.H., 1989. Prevalence of Cryptosporidium infection in dairy calves in western Washington. Am. J. Vet. Res. 50, 1069–1070. O’Handley, R.M., Cockwill, C., McAllister, T.A., Jelinski, M., Morck, D.W., Olson, M.E., 1999. Duration of naturally acquired giardiosis and cryptosporidiosis in dairy calves and their association with diarrhea. J. Am. Vet. Med. Assoc. 214, 391–396. Olson, M.E., Guselle, N.J., O’Handley, R.M., Swift, M.L., McAllister, T.A., Jelinski, M.D., Morck, D.W., 1997a. Giardia and Cryptosporidium in dairy calves in British Columbia. Can. Vet. J. 38, 703–706. Olson, M.E., Thorlakson, C.L., Deselliers, L., Morck, D.W., McAllister, T.A., 1997b. Giardia and Cryptosporidium in Canadian farm animals. Vet. Parasitol. 68, 375–381. Quilez, J., Sanchez-Acedo, C., del Cacho, E., Clavel, A., Causape, A.C., 1996. Prevalence of Cryptosporidium and Giardia infections in cattle in Aragon (northeastern Spain). Vet. Parasitol. 66, 139–146. Ruest, N., Faubert, G.M., Couture, Y., 1998. Prevalence and geographical distribution of Giardia spp. and Cryptosporidium spp. in dairy farms in Quebec. Can. Vet. J. 39, 697–700. St. Jean, G., Couture, Y., Dubreuil, P., Frechette, J.L., 1987. Diagnosis of Giardia infection in 14 calves. J. Am. Vet. Med. Assoc. 191, 831–832. Smith, P.D., 1989. Giardia lamblia. In: Walzer, P.D., Genta, R.M. (Eds.), Parasitic Infections in the Compromised Host. Marcel Dekker, New York, NY, pp. 343–384. Sobieh, M., Tacal Jr., J.V., Wilcke Jr., B.W., Lawrence, W., El-Ahraf, A., 1987. Investigation of cryptosporidial infection in calves in San Bernardino County, California. J. Am. Vet. Med. Assoc. 191, 816–818.
S.E. Wade et al. / Veterinary Parasitology 93 (2000) 1–11
11
Stiratelli, R., Laird, N.M., Ware, J.H., 1984. Random-effect models for serial observations with binary response. Biometrics 4, 961–971. Thompson, R.C.A., Reynoldson, J.A., Mendis, A.H.W., 1993. Giardia and giardiasis. Adv. Parasitol. 32, 71–160. Walterspiel, J.N., Pickering, L.K., 1994. Giardia and giardiasis, In: Sun, T. (Ed.), Progress in Clinical Parasitology. Vol. 4, CRC Press, London, pp. 1–26. Xiao, L., Herd, R.P., Rings, D.M., 1993. Concurrent infections of Giardia and Cryptosporidium on two Ohio farms with calf diarrhea. Vet. Parasitol. 51, 41–48. Xiao, L., 1994. Giardia infection in farm animals. Parasitol. Today 10, 436–438. Zajac, A.M., 1992. Giardiasis. Compend. Contin. Edu. Prac. Vet. 14, 604–611.