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Species of Cryptosporidium detected in weaned cattle on cow–calf operations in the United States
Veterinary Parasitology 170 (2010) 187–192
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Species of Cryptosporidium detected in weaned cattle on cow–calf operations in the United States Ronald Fayer a,∗ , Monica Santín a , David Dargatz b a
United States Department of Agriculture, Agricultural Research Service, Environmental Microbial Food Safety Laboratory, Beltsville, MD 20705, USA United States Department of Agriculture, Animal and Plant Health Inspection Service, Centers for Epidemiology and Animal Health, Fort Collins, CO 80526, USA b
a r t i c l e
i n f o
Article history: Received 23 December 2009 Received in revised form 23 February 2010 Accepted 24 February 2010 Keywords: Cryptosporidiosis Beef cattle Prevalence
a b s t r a c t To determine the species and distribution of Cryptosporidium in weaned beef calves in the United States, fecal specimens were collected from 819 cattle between 6 and 18 months of age from 49 operations in 20 states (Alabama, California, Colorado, Georgia, Idaho, Iowa, Kansas, Louisiana, Mississippi, Missouri, Nebraska, New Mexico, North Dakota, Oklahoma, Oregon, South Dakota, Tennessee, Texas, Virginia, and Wyoming). Fresh feces, collected either from the ground or directly from the rectum of each animal, were sieved and subjected to density gradient centrifugation to remove fecal debris and to concentrate oocysts. DNA extracted from each specimen was subjected to the polymerase chain reaction (PCR) using primers for the SSU rRNA gene. All PCR positive specimens were subjected to sequence analysis. Cryptosporidium was detected in 20.5% of the fecal samples. Cryptosporidium ryanae, C. bovis and C. andersoni were detected in 1.8, 4.8, and 14.0% of the 819 samples, respectively. California (number operations [n] = 2), Iowa (n = 3), and Nebraska (n = 7) had the highest prevalence of infected weaned cattle with 44.4, 41.0 and 40.2% infected, respectively. Cryptosporidium was not detected in any weaned cattle from Alabama (number operations [n] = 1), Georgia (n = 2), New Mexico (n = 1), South Dakota (n = 1), Tennessee (n = 1), or Texas (n = 1). The zoonotic species, C. parvum, was not detected in any samples from 6- to 18-month-old cattle, a finding that parallels reports for dairy cattle of similar age in which less than 1% were found to harbor this species. Published by Elsevier B.V.
1. Introduction Cattle have been reported to be infected with four species of Cryptosporidium: C. parvum (Tyzzer, 1912), C. bovis (Fayer et al., 2005), C. ryanae (Fayer et al., 2008) and C. andersoni (Lindsay et al. (2000). A proposal was made to replace the name C. parvum with C. pestis (Slapeta, 2006). However, the name C. parvum has been used to describe
∗ Corresponding author at: Environmental Microbial Food Safety Laboratory, Animal and Natural Resources Institute Agricultural Research Service, United States Department of Agriculture, Building 173, BARC-East, 10300 Baltimore Avenue, Beltsville, MD 20705, USA. Tel.: +1 301 504 8750; fax: +1 301 504 6608. E-mail addresses: [email protected], [email protected] (R. Fayer). 0304-4017/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.vetpar.2010.02.040
this parasite in cattle for over 20 years in nearly 600 peer reviewed publications and in over 2500 scientific publications on subjects related to public health, water safety, and many other topics (Fayer, 2010). Furthermore, the proposal for a name change did not present solid scientific data or clear rules of nomenclature acceptable to experts working in the field (Xiao et al., 2007a,b). Consequently, virtually all peer reviewed publications after the proposal have continued to use the name C. parvum. Each of these four species presents a different prevalence pattern relative to the age of the cattle (Wyatt et al., 2010). For example, Cryptosporidium parvum, the zoonotic species, has been found in a high numbers of young monogastric calves but in relatively few calves after weaning when they convert to rumenal nutrition. The number of cattle found infected with C. bovis (Fayer et al., 2005) and C. ryanae (Fayer et al., 2008)
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increases immediately after weaning and then decreases steadily as cattle approach maturity. The number of cattle found infected with C. andersoni (Lindsay et al., 2000) has been extremely low in pre-weaned calves and reaches its highest levels in yearling heifers and mature cows. These age-related patterns of Cryptosporidium species have been well documented in dairy cattle from both point prevalence and longitudinal studies (Fayer et al., 2006, 2007; Santín et al., 2008; Xiao et al., 2007a,b). Fewer such studies have been conducted with beef cattle than with dairy cattle. Most beef cattle studies have been associated with cow–calf herds or farmed cattle and most reports have been based on microscopic identification of oocysts in fecal specimens (Kaneta and Nakai, 1998; Hoar et al., 1999; Atwill et al., 1999, 2003; Nakai et al., 2004; McAllister et al., 2005). Some investigators reported screening samples by microscopy and identifying positive specimens or a portion of them using molecular methods (Kvác and Vítovec, 2003; Moriarty et al., 2005; Atwill et al., 2006; Geurden et al., 2006, 2007). Recently, C. bovis and C. ryanae were discovered as species distinct from C. parvum based on gene sequencing. Consequently, reports of C. parvum in beef cattle that were based solely on microscopic identification of oocysts or on restriction fragment length polymorphism (RFLP)/PCR methods that were unable to distinguish C. bovis and C. ryanae from C. parvum now require re-evaluation. Even fewer studies have been conducted to determine the species of Cryptosporidium infecting beef cattle in feedlots (Atwill et al., 2006). The primary objective of the present study was to determine the current species of Cryptosporidium present in cattle from 6 to 18 months of age on cow–calf operations throughout the United States by using molecular methods on all specimens. 2. Materials and methods 2.1. Cow–calf operation location and sample collection The USDA National Animal Health Monitoring System conducted the Beef 2007–2008 study to characterize the health and management of beef cattle on cow–calf operations in 24 states (USDA, 2008). Participating producers were offered the opportunity to collect fecal samples for analysis for parasite burden. Those producers who elected to participate were sent a sample collection kit and instructions for collecting and shipping the samples via overnight delivery. Up to 20 fecal samples from animals between 6 and 18 months of age were collected either from fresh fecal pats on the ground or directly from the rectum. Samples were collected between March 1 and December 16, 2008. Samples were submitted to one of three designated laboratories for evaluation. When there was sufficient volume of feces remaining after the initial evaluation the remainder was forwarded to the Environmental Microbial and Food Safety Laboratory for detection of Cryptosporidium. 2.2. Detection of Cryptosporidium oocysts by PCR and gene sequencing Oocysts were concentrated from feces as described (Fayer et al., 2000; Santín et al., 2004). Briefly, 15 g of feces
from each specimen bag were mixed with 35 ml deionized water (dH2 O) and passed through a sieve with a 45 m pore size. The filtrate was adjusted to 50 ml with dH2 O and centrifuged at 1800 × g for 15 min. Supernatant was aspirated and the pellet was resuspended in a mixture of 25 ml dH2 O and 25 ml cesium chloride (1.4 g/l) and centrifuged at 300 × g for 20 min. Supernatant, aspirated from each suspension, was washed twice with dH2 O and the final pellet was suspended in 500 l of dH2 O. The suspension was then subjected to total DNA extraction employing a DNeasyTissue Kit (Qiagen, Valencia, CA). The protocol, as described below, utilized the manufacturer’s reagents with slight modification. To 50 l of fecal suspension 180 l of ATL buffer was added and mixed by vortexing. Twenty microliters of proteinase K (20 mg/ml) was added, the sample was mixed and incubated overnight at 55 ◦ C before 200 l of AL buffer was added. The protocol then followed manufacturer’s instructions with the exception that to increase the quantity of recovered DNA, the nucleic acid was eluted in 100 l of AE buffer. A two-step nested PCR protocol was used to amplify an 830 bp segment of the SSU rRNA gene using primers 5 -TTCTAGAGCTAATACATGCG-3 and 5 -CCCATTTCCTTCGAAACAGGA-3 for primary PCR and 5 -GGAAGGGTTGTATTT–ATTAGATAAAG-3 and 5 AAGGAGTAAGGAACAACCTCCA-3 for secondary PCR (Xiao et al., 1999). The primary PCR mixture contained 1× PCR buffer, 3 mM MgCl2 , 0.2 mM dNTP, 2.5 U Taq (Qbiogene, Irvine, CA), 2.5 l bovine serum albumin (0.1 g/10 ml), and 1 M of each forward and reverse primer in a 50 l volume. Of 35 cycles, each consisted of 94 ◦ C for 45 s, 59 ◦ C for 45 s, and 72 ◦ C for 1 min after starting at 94 ◦ C for 3 min and ending at 72 ◦ C for 7 min. The secondary PCR mixture was identical except that the MgCl2 concentration was 1.5 mM. Of 40 cycles, each consisted of 94 ◦ C for 30 s, 58 ◦ C for 90 s, and 72 ◦ C for 2 min after starting at 94 ◦ C for 3 min and ending at 72 ◦ C for 7 min. PCR products placed on 1% agarose gel for electrophoresis were stained with ethidium bromide for detection. Positive PCR products were purified using Exonuclease I/Shrimp Alkaline Phosphatase (Exo-SAPITTM ) (USB Corporation, Cleveland, OH). Purified products were sequenced (using the same PCR primers as in the 10 l reactions) using Big DyeTM chemistries and an ABI 3100 sequencer analyzer (Applied Biosystems, Foster City, CA). Sequence chromatograms of each strand were aligned and examined with Lasergene software (DNASTAR, Inc., Madison, WI). The nucleotide sequences obtained in the present study were deposited in the GenBank database under accession numbers GU831566 (C. ryanae), GU831567 (C. bovis), and GU831568–GU831569 (C. andersoni). 2.3. Sensitivity of detection methods As in previous publications, to test the efficacy of oocyst recovery and sensitivity of our detection methods (Fayer et al., 2006) feces from cattle found negative for the presence of Cryptosporidium oocysts were spiked with oocysts of C. parvum (obtained from an experimentally infected calf) at the rate of 10, 100, or 1000 oocysts/g and subjected to the
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Table 1 Numbers of calf fecal samples examined and species of Cryptosporidium found by state. State
Number of operations
Number of samples per operation (total samples)
Cryptosporidium-positive samples per operation (total positive samples)
Alabama California Colorado Georgia Idaho Iowa Kansas Louisiana Mississippi Missouri Nebraska New Mexico North Dakota Oklahoma Oregon South Dakota Tennessee Texas Virginia Wyoming Total
1 2 2 2 2 3 4 2 1 2 7 1 3 5 2 1 1 1 2 5 49
20 (20) 20, 7 (27) 20, 17 (37) 8, 3 (11) 20, 20 (40) 20, 11, 8 (39) 20, 18, 15, 12 (65) 19, 3 (22) 20 (20) 20, 17 (37) 20, 20, 20, 20, 19, 17, 16 (132) 20 (20) 20, 20, 16 (56) 20, 20, 20, 10, 14 (84) 17, 9 (26) 8 (8) 17 (17) 20 (20) 20, 18 (38) 20, 20, 20, 20, 20 (100) 819
0 (0) 11, 1 (12) 1, 1 (2) 0, 0 (0) 1, 6 (7) 12, 0, 4 (16) 5, 1, 3, 1 (10) 2, 0 (2) 2 (2) 8, 1 (9) 17, 1, 1, 9, 3, 6, 16 (53) 0 (0) 2, 3, 0 (5) 6, 11, 0, 2, 0 (19) 5, 0 (5) 0 (0) 0 (0) 0 (0) 5, 5 (10) 0, 0, 3, 7, 6 (16) 168
Species identified
C.a
C.b
C.r
0 11 0 0 4 3 5 0 0 4 51 0 5 11 5 0 0 0 3 13 115
0 0 1 0 2 13 5 2 0 2 2 0 0 6 0 0 0 0 3 3 39
0 1 1 0 1 1 0 0 2 3 0 0 0 2 0 0 0 0 4 0 15
C.a, Cryptosporidium andersoni; C.b, Cryptosporidium bovis; C.r, Cryptosporidium ryanae.
same concentration and testing procedures used throughout the study. Six 15 g fecal specimens were each spiked with oocysts at each level (10, 100 and 1000 oocysts/g). Positive results were obtained for 6/6 samples with 1000 oocysts/g, 6/6 samples with 100 oocysts/g and 4/6 samples with 10 oocysts/g.
At the state level, operations in California, Iowa, Nebraska, and Oklahoma had the highest overall prevalence of positive samples with 44.4, 41.0, 40.2 and 22.6% positive, respectively. Cryptosporidium was not detected in any samples from Alabama, Georgia, New Mexico, South Dakota, Tennessee, or Texas.
3. Results
4. Discussion
Overall, 819 fecal samples were submitted for detection of Cryptosporidium. The fecal samples originated from 49 operations in 20 states. The number of fecal samples per operation ranged from 3 to 20 (Table 1). One or more calf fecal samples were positive for Cryptosporidium on 34 operations from 14 states (Table 1). Of 819 fecal samples examined, 168 (20.5%) were positive for Cryptosporidium. Three species were detected. Cryptosporidium ryanae, C. bovis and C. andersoni were detected in 15 (1.8%), 39 (4.8%), and 115 (14.0%) fecal samples, respectively. Of 115 isolates identified as C. andersoni, 74 showed 100% nucleotide sequence identity to the C. andersoni isolate reported by Koyama et al. (2005) (GenBank Accession number: AB089285), 35 showed 100% nucleotide sequence identity to the C. andersoni isolate reported by Liu et al. (2009) (GenBank Accession number: FJ463187) that contains an insertion of thymine at nucleotide 634, and 6 showed the presence of both sequences of C. andersoni (with and without the thymine insertion) characterized by the presence of overlapping nucleotide peaks in chromatograms, which can be attributed to mixed infections or to allelic sequence heterozygosity of single oocysts. One sample was positive for both C. ryanae and C. bovis. The zoonotic species, C. parvum, was not detected in any fecal sample.
The methods used for oocyst recovery and molecular detection in the present study were the same as in previous studies on dairy cattle (Fayer et al., 2006, 2007; Santín et al., 2008). Spiking studies to determine detection levels conducted herein and in the previous studies have yielded similar results. In the present study, detection at the level of 1000, 100, and 10 oocysts/g was 100, 100 and 67%, respectively, indicating that at levels below 100 oocysts/g some positive samples may not be detected. Therefore the prevalence of positive samples with low numbers of oocysts per gram was likely underestimated. Based on sequence data from the SSU rRNA gene, 3 species of Cryptosporidium were identified among 168 PCR positive fecal samples from the 819 samples evaluated. These included C. andersoni, C. bovis, and C. ryanae (one mixed infection with both C. bovis and C. ryanae) but C. parvum was not found. These results differ from those previously reported for beef cattle or feedlot cattle because of the methods used. In the present study every sample was subjected to PCR and all Cryptosporidium-positive PCR products were subjected to sequence analysis to identify the species. Studies that identified C. parvum by microscopy require re-evaluation because the recently named species C. bovis and C. ryanae have no unique morphological crite-
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ria to clearly distinguish them from C. parvum and therefore species may have been reported as C. parvum inadvertently. Several previous studies investigated Cryptosporidium on cow–calf operations in the United States. In California, of 558 fecal pats collected from the ground from 25 herds 0.18% had oocysts. Specimens collected directly from the rectum had a sixfold higher prevalence of oocysts (Hoar et al., 1999). In another study in California, the point prevalence of Cryptosporidium oocysts (mostly collected per rectum) was determined for 915 calves between 1 and 11 months of age and 484 cattle over 12 months of age (Atwill et al., 1999). Of 1399 samples, 54 (3.9%) contained oocysts. Of 884 cattle over 4 months of age, 6 (2.4%) shed oocysts. A third study in California estimated the daily environmental loading rate of oocysts produced by 40 adult beef cattle per herd in 3 cow–calf production herds approximately 6 weeks before and 6 weeks after calving (Atwill et al., 2003). Of 240 fecal samples, 17 (7.1%) were infected with C. parvum. Although C. parvum was reported in these three previous studies, microscopic methods were used which cannot distinguish C. parvum from C. bovis and C. ryanae. The percentage of samples positive for Cryptosporidium in the California studies were considerably lower than in the present study (20.5% positive overall, 44.4% in California). There are several possible reasons for this difference. The PCR methodology in the present study likely has higher sensitivity for detecting Cryptosporidium, as compared with microscopy. Also, Santín et al. (2008) showed that the prevalence of Cryptosporidium often decreases with age. The cattle in the present study might have been younger than those sampled in one or more of the previous studies. It is also possible that the two California herds participating in the present study had different management practices compared with herds in the previous studies. In western North Dakota, molecular methods were used to determine the prevalence of Cryptosporidium species in 7 beef cow–calf herds in 98 calves 6–8 months of age and 114 cows over 2 years of age (Feltus et al., 2008). Overall, 43 animals were positive but only 5 positives were from cows. C. bovis, the deer-like genotype (C. ryanae) and C. andersoni were identified in 9.4, 6.6 and 1.4% of animals sampled, respectively. In the present study the same species were found. Other studies of beef cattle in the United States investigated Cryptosporidium in feedlot cattle. Atwill et al. (2006) examined 5274 fecal specimens collected from the ground from 22 feedlots in 7 central and western states by DFA microscopy precluding comparison with the present study. Oocysts were detected in only 9 (0.17%) of these specimens and only 4 of the 9 had enough oocysts to enable molecular confirmation as C. parvum. Also in North America, of 20 cow–calf pairs on a ranch north of Calgary, Alberta only 1 calf was positive for Cryptosporidium by fluorescence microscopy (Ralston et al., 2003). Feces were collected per rectum from 669 beef cows 2–14 years of age in southern Ontario and from 192 calves 2–70 days of age in southern British Columbia (McAllister et al., 2005). Based on DFA microscopy, of 669 fecal samples from beef cows 10.6 and 18.4% were diagnosed with C. muris (probably C. andersoni) and C. parvum, respectively, and of 192 calves 13% were diag-
nosed with Cryptosporidium. In cows and calves during the calving season in western Canadian cow–calf herds fresh fecal samples were collected from 560 beef cows and 605 calves (Gow and Waldner, 2006). Feces collected from mature cows on 59 farms and from calves on 100 farms were examined for the presence of Cryptosporidium using a quantitative sucrose gradient immunoflourescent antibody test. Only 1.1% of the cows and 3.1% of the calves were positive for Cryptosporidium spp. Again, the oocyst recovery and microscopic methods used in these studies cannot be fairly compared with those of the present study. Several studies investigated the presence of Cryptosporidium in beef cattle in Europe, Africa and Asia. In Scotland two beef herds of breeding animals over 1 year of age were sampled (Scott et al., 1995). Of 553 fecal samples examined, 62.4% had oocysts, reported as C. parvum, in direct smears observed microscopically. On farm 1, 268 of 437 (61.3%) cattle were positive and on farm 2, 77 of 116 (66.4%) cattle were positive. In Bohemia, in a long-term study of beef cattle the prevalence of C. andersoni highly exceeded the prevalence reported in other studies (Kvác and Vítovec, 2003). Of 96 cows 43.8% were positive and at 27 weeks of age 92.9% of 42 calves were positive for C. andersoni as determined by oocyst measurements. Mean oocyst size was 8.48 × 6.41 l and molecular characterization of a partial sequence of the 18S rDNA was consistent with C. andersoni. In Ireland, feces in 21 of 288 beef cattle at slaughter had oocysts detected by DFA microscopy (Moriarty et al., 2005). Molecular testing of positive feces identified 54.5% as C. andersoni and 45.5% as C. parvum. In Belgium, feces were collected from a total of 499 dairy and 333 beef calves less than 10 weeks of age and examined by DFA microscopy for the presence of Cryptosporidium oocysts and 11 specimens from beef calves were examined by molecular methods to determine species (Geurden et al., 2007). The overall prevalence was 37 and 12%, respectively in these young dairy and beef calves and 7 of the 11 samples from beef calves tested by molecular methods were C. parvum. In Northern Portugal, feces from dairy and beef cattle (Mendonc¸a et al., 2007) were tested by PCR followed by sequencing of the hsp70 and 18S rRNA genes. Of 176 adult cattle 8 (4.5%) were positive. Six had C. parvum, one had C. andersoni, and one had C. meleagridis. In Zambia, feces from 238 beef calves, 250 dairy calves, and 256 calves on “traditional” farms were frozen and tested for Cryptosporidium by a commercial ELISA test (Geurden et al., 2006). All calves were 3 months of age or younger. Beef calves (breeds included Brahman, Hereford, Boran, Simmental or cross breeds) had a mean age of 24 days and 8.0% were positive. Of eight beef calves tested by molecular methods seven were reported to have C. parvum. In Japan, after detecting C. muris (most likely C. andersoni) oocysts in 24 (4.7%) of 512 beef cattle in an abattoir in Miyagi Prefecture (Kaneta and Nakai, 1998) a survey was conducted of grazing cattle that detected C. muris type oocysts in 21 (4.1%) of 516 beef cattle on a farm (Nakai et al., 2004). Nakai et al. (2004) cited earlier reports (in Japanese) of C. muris-like oocysts from cattle in slaugh-
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terhouses throughout Japan including 1.2% in Fukushima, 1.2% in Saitama, 3.9% in Kanagawa, and 3.4% in Tokushima prefectures. Age-related similarities appear between beef cattle in the present study and dairy cattle with regard to the presence of Cryptosporidium species. Based on molecular methods, of 571 12–24-month-old dairy heifers, 68 were found to be infected with Cryptosporidium (Fayer et al., 2006). By gene sequencing, 1 (0.02%), 4 (0.07%), 10 (1.8%), 24 (4.2%), and 29 (5.1%) of these calves were found to be infected with C. suis, C. parvum, C. ryanae, C. bovis, and C. andersoni, respectively. Other reports also indicate that less than 1% of postweaned and adult dairy cattle were found to excrete C. parvum (Fayer et al., 2007; Santín et al., 2008; Uehlinger et al., 2006). The finding of C. andersoni, C. bovis, and C. ryanae in 6–18month-old cattle in the present study and absence of C. parvum parallels the findings in dairy cattle of similar ages. 5. Conclusions Although C. parvum was not detected in any of the fecal specimens in the present study it might have been present in numbers below the level of detection (<10 oocysts/g of feces). The fact that it was not detected suggests that 6–18-month-old cattle on cow–calf operations do not significantly contribute to environmental contamination with this zoonotic species, contrary to earlier microscopy based reports. Acknowledgments We thank Judy Rodriguez for support in managing the data collection and analysis for this project and Julie Headley and Meghan Heffron for technical support. References Atwill, E.R., das Gracas, C., Pereira, M., Alonso, L.H., Elmi, C., Epperson, W.B., Smith, R., Riggs, W., Carpenter, L.V., Dargatz, D.A., Hoar, B., 2006. Environmental load of Cryptosporidium parvum oocysts from cattle manure in feedlots from the central and western United States. J. Environ. Qual. 35, 200–206. Atwill, E.R., Hoar, B., das Gracas, C., Pereira, M., Tate, K.W., Rulofson, F., Nader, G., 2003. Improved quantitative estimates of low environmental loading and sporadic periparturient shedding of Cryptosporidium parvum in adult beef cattle. Appl. Environ. Microbiol. 69, 4604–4610. Atwill, E.R., Johnson, E., Klingborg, D.J., Veserat, G.M., Markegard, G., Jensen, W.A., Pratt, D.W., Delmas, R.E., George, H.A., Forero, L.C., Philips, R.L., Barry, S.J., McDougald, N.K., Gildersleeve, R.R., Frost, W.E., 1999. Age, geographic, and temporal distribution of fecal shedding of Cryptosporidium parvum oocysts in cow–calf herds. Am. J. Vet. Res. 60, 420–425. Fayer, R., 2010. Taxonomy and species delimitation in Cryptosporidium. Exp. Parasitol. 124, 90–97. Fayer, R., Santin, M., Trout, J.M., 2007. Prevalence of Cryptosporidium species and genotypes in mature dairy cattle on farms in eastern United States compared with younger cattle from the same locations. Vet. Parasitol. 145, 260–266. Fayer, R., Santín, M., Trout, J.M., 2008. Cryptosporidium ryanae n. sp. (Apicomplexa: Cryptosporidiidae) in cattle (Bos taurus). Vet. Parasitol. 156, 191–198. Fayer, R., Santín, M., Trout, J.M., Greiner, E., 2006. Prevalence of species and genotypes of Cryptosporidium found in 1–2-year-old dairy cattle in the eastern United States. Vet. Parasitol. 135, 105–112.
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Wyatt, C.R., Riggs, M.W., Fayer, R., 2010. Cryptosporidiosis in the neonatal calf. Vet Clin. N. Am. Food Anim. Pract. 26, 89–103. Xiao, L., Escalante, L., Yang, C., Sulaiman, I., Escalante, A.A., Montali, R.J., Fayer, R., Lal, A.A., 1999. Phylogenetic analysis of Cryptosporidium parasites based on the small-subunit rRNA gene locus. Appl. Environ. Microbiol. 65, 1578–1583.
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Species of Cryptosporidium detected in weaned cattle on cow–calf operations in the United States Ronald Fayer a,∗ , Monica Santín a , David Dargatz b a
United States Department of Agriculture, Agricultural Research Service, Environmental Microbial Food Safety Laboratory, Beltsville, MD 20705, USA United States Department of Agriculture, Animal and Plant Health Inspection Service, Centers for Epidemiology and Animal Health, Fort Collins, CO 80526, USA b
a r t i c l e
i n f o
Article history: Received 23 December 2009 Received in revised form 23 February 2010 Accepted 24 February 2010 Keywords: Cryptosporidiosis Beef cattle Prevalence
a b s t r a c t To determine the species and distribution of Cryptosporidium in weaned beef calves in the United States, fecal specimens were collected from 819 cattle between 6 and 18 months of age from 49 operations in 20 states (Alabama, California, Colorado, Georgia, Idaho, Iowa, Kansas, Louisiana, Mississippi, Missouri, Nebraska, New Mexico, North Dakota, Oklahoma, Oregon, South Dakota, Tennessee, Texas, Virginia, and Wyoming). Fresh feces, collected either from the ground or directly from the rectum of each animal, were sieved and subjected to density gradient centrifugation to remove fecal debris and to concentrate oocysts. DNA extracted from each specimen was subjected to the polymerase chain reaction (PCR) using primers for the SSU rRNA gene. All PCR positive specimens were subjected to sequence analysis. Cryptosporidium was detected in 20.5% of the fecal samples. Cryptosporidium ryanae, C. bovis and C. andersoni were detected in 1.8, 4.8, and 14.0% of the 819 samples, respectively. California (number operations [n] = 2), Iowa (n = 3), and Nebraska (n = 7) had the highest prevalence of infected weaned cattle with 44.4, 41.0 and 40.2% infected, respectively. Cryptosporidium was not detected in any weaned cattle from Alabama (number operations [n] = 1), Georgia (n = 2), New Mexico (n = 1), South Dakota (n = 1), Tennessee (n = 1), or Texas (n = 1). The zoonotic species, C. parvum, was not detected in any samples from 6- to 18-month-old cattle, a finding that parallels reports for dairy cattle of similar age in which less than 1% were found to harbor this species. Published by Elsevier B.V.
1. Introduction Cattle have been reported to be infected with four species of Cryptosporidium: C. parvum (Tyzzer, 1912), C. bovis (Fayer et al., 2005), C. ryanae (Fayer et al., 2008) and C. andersoni (Lindsay et al. (2000). A proposal was made to replace the name C. parvum with C. pestis (Slapeta, 2006). However, the name C. parvum has been used to describe
∗ Corresponding author at: Environmental Microbial Food Safety Laboratory, Animal and Natural Resources Institute Agricultural Research Service, United States Department of Agriculture, Building 173, BARC-East, 10300 Baltimore Avenue, Beltsville, MD 20705, USA. Tel.: +1 301 504 8750; fax: +1 301 504 6608. E-mail addresses: [email protected], [email protected] (R. Fayer). 0304-4017/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.vetpar.2010.02.040
this parasite in cattle for over 20 years in nearly 600 peer reviewed publications and in over 2500 scientific publications on subjects related to public health, water safety, and many other topics (Fayer, 2010). Furthermore, the proposal for a name change did not present solid scientific data or clear rules of nomenclature acceptable to experts working in the field (Xiao et al., 2007a,b). Consequently, virtually all peer reviewed publications after the proposal have continued to use the name C. parvum. Each of these four species presents a different prevalence pattern relative to the age of the cattle (Wyatt et al., 2010). For example, Cryptosporidium parvum, the zoonotic species, has been found in a high numbers of young monogastric calves but in relatively few calves after weaning when they convert to rumenal nutrition. The number of cattle found infected with C. bovis (Fayer et al., 2005) and C. ryanae (Fayer et al., 2008)
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increases immediately after weaning and then decreases steadily as cattle approach maturity. The number of cattle found infected with C. andersoni (Lindsay et al., 2000) has been extremely low in pre-weaned calves and reaches its highest levels in yearling heifers and mature cows. These age-related patterns of Cryptosporidium species have been well documented in dairy cattle from both point prevalence and longitudinal studies (Fayer et al., 2006, 2007; Santín et al., 2008; Xiao et al., 2007a,b). Fewer such studies have been conducted with beef cattle than with dairy cattle. Most beef cattle studies have been associated with cow–calf herds or farmed cattle and most reports have been based on microscopic identification of oocysts in fecal specimens (Kaneta and Nakai, 1998; Hoar et al., 1999; Atwill et al., 1999, 2003; Nakai et al., 2004; McAllister et al., 2005). Some investigators reported screening samples by microscopy and identifying positive specimens or a portion of them using molecular methods (Kvác and Vítovec, 2003; Moriarty et al., 2005; Atwill et al., 2006; Geurden et al., 2006, 2007). Recently, C. bovis and C. ryanae were discovered as species distinct from C. parvum based on gene sequencing. Consequently, reports of C. parvum in beef cattle that were based solely on microscopic identification of oocysts or on restriction fragment length polymorphism (RFLP)/PCR methods that were unable to distinguish C. bovis and C. ryanae from C. parvum now require re-evaluation. Even fewer studies have been conducted to determine the species of Cryptosporidium infecting beef cattle in feedlots (Atwill et al., 2006). The primary objective of the present study was to determine the current species of Cryptosporidium present in cattle from 6 to 18 months of age on cow–calf operations throughout the United States by using molecular methods on all specimens. 2. Materials and methods 2.1. Cow–calf operation location and sample collection The USDA National Animal Health Monitoring System conducted the Beef 2007–2008 study to characterize the health and management of beef cattle on cow–calf operations in 24 states (USDA, 2008). Participating producers were offered the opportunity to collect fecal samples for analysis for parasite burden. Those producers who elected to participate were sent a sample collection kit and instructions for collecting and shipping the samples via overnight delivery. Up to 20 fecal samples from animals between 6 and 18 months of age were collected either from fresh fecal pats on the ground or directly from the rectum. Samples were collected between March 1 and December 16, 2008. Samples were submitted to one of three designated laboratories for evaluation. When there was sufficient volume of feces remaining after the initial evaluation the remainder was forwarded to the Environmental Microbial and Food Safety Laboratory for detection of Cryptosporidium. 2.2. Detection of Cryptosporidium oocysts by PCR and gene sequencing Oocysts were concentrated from feces as described (Fayer et al., 2000; Santín et al., 2004). Briefly, 15 g of feces
from each specimen bag were mixed with 35 ml deionized water (dH2 O) and passed through a sieve with a 45 m pore size. The filtrate was adjusted to 50 ml with dH2 O and centrifuged at 1800 × g for 15 min. Supernatant was aspirated and the pellet was resuspended in a mixture of 25 ml dH2 O and 25 ml cesium chloride (1.4 g/l) and centrifuged at 300 × g for 20 min. Supernatant, aspirated from each suspension, was washed twice with dH2 O and the final pellet was suspended in 500 l of dH2 O. The suspension was then subjected to total DNA extraction employing a DNeasyTissue Kit (Qiagen, Valencia, CA). The protocol, as described below, utilized the manufacturer’s reagents with slight modification. To 50 l of fecal suspension 180 l of ATL buffer was added and mixed by vortexing. Twenty microliters of proteinase K (20 mg/ml) was added, the sample was mixed and incubated overnight at 55 ◦ C before 200 l of AL buffer was added. The protocol then followed manufacturer’s instructions with the exception that to increase the quantity of recovered DNA, the nucleic acid was eluted in 100 l of AE buffer. A two-step nested PCR protocol was used to amplify an 830 bp segment of the SSU rRNA gene using primers 5 -TTCTAGAGCTAATACATGCG-3 and 5 -CCCATTTCCTTCGAAACAGGA-3 for primary PCR and 5 -GGAAGGGTTGTATTT–ATTAGATAAAG-3 and 5 AAGGAGTAAGGAACAACCTCCA-3 for secondary PCR (Xiao et al., 1999). The primary PCR mixture contained 1× PCR buffer, 3 mM MgCl2 , 0.2 mM dNTP, 2.5 U Taq (Qbiogene, Irvine, CA), 2.5 l bovine serum albumin (0.1 g/10 ml), and 1 M of each forward and reverse primer in a 50 l volume. Of 35 cycles, each consisted of 94 ◦ C for 45 s, 59 ◦ C for 45 s, and 72 ◦ C for 1 min after starting at 94 ◦ C for 3 min and ending at 72 ◦ C for 7 min. The secondary PCR mixture was identical except that the MgCl2 concentration was 1.5 mM. Of 40 cycles, each consisted of 94 ◦ C for 30 s, 58 ◦ C for 90 s, and 72 ◦ C for 2 min after starting at 94 ◦ C for 3 min and ending at 72 ◦ C for 7 min. PCR products placed on 1% agarose gel for electrophoresis were stained with ethidium bromide for detection. Positive PCR products were purified using Exonuclease I/Shrimp Alkaline Phosphatase (Exo-SAPITTM ) (USB Corporation, Cleveland, OH). Purified products were sequenced (using the same PCR primers as in the 10 l reactions) using Big DyeTM chemistries and an ABI 3100 sequencer analyzer (Applied Biosystems, Foster City, CA). Sequence chromatograms of each strand were aligned and examined with Lasergene software (DNASTAR, Inc., Madison, WI). The nucleotide sequences obtained in the present study were deposited in the GenBank database under accession numbers GU831566 (C. ryanae), GU831567 (C. bovis), and GU831568–GU831569 (C. andersoni). 2.3. Sensitivity of detection methods As in previous publications, to test the efficacy of oocyst recovery and sensitivity of our detection methods (Fayer et al., 2006) feces from cattle found negative for the presence of Cryptosporidium oocysts were spiked with oocysts of C. parvum (obtained from an experimentally infected calf) at the rate of 10, 100, or 1000 oocysts/g and subjected to the
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Table 1 Numbers of calf fecal samples examined and species of Cryptosporidium found by state. State
Number of operations
Number of samples per operation (total samples)
Cryptosporidium-positive samples per operation (total positive samples)
Alabama California Colorado Georgia Idaho Iowa Kansas Louisiana Mississippi Missouri Nebraska New Mexico North Dakota Oklahoma Oregon South Dakota Tennessee Texas Virginia Wyoming Total
1 2 2 2 2 3 4 2 1 2 7 1 3 5 2 1 1 1 2 5 49
20 (20) 20, 7 (27) 20, 17 (37) 8, 3 (11) 20, 20 (40) 20, 11, 8 (39) 20, 18, 15, 12 (65) 19, 3 (22) 20 (20) 20, 17 (37) 20, 20, 20, 20, 19, 17, 16 (132) 20 (20) 20, 20, 16 (56) 20, 20, 20, 10, 14 (84) 17, 9 (26) 8 (8) 17 (17) 20 (20) 20, 18 (38) 20, 20, 20, 20, 20 (100) 819
0 (0) 11, 1 (12) 1, 1 (2) 0, 0 (0) 1, 6 (7) 12, 0, 4 (16) 5, 1, 3, 1 (10) 2, 0 (2) 2 (2) 8, 1 (9) 17, 1, 1, 9, 3, 6, 16 (53) 0 (0) 2, 3, 0 (5) 6, 11, 0, 2, 0 (19) 5, 0 (5) 0 (0) 0 (0) 0 (0) 5, 5 (10) 0, 0, 3, 7, 6 (16) 168
Species identified
C.a
C.b
C.r
0 11 0 0 4 3 5 0 0 4 51 0 5 11 5 0 0 0 3 13 115
0 0 1 0 2 13 5 2 0 2 2 0 0 6 0 0 0 0 3 3 39
0 1 1 0 1 1 0 0 2 3 0 0 0 2 0 0 0 0 4 0 15
C.a, Cryptosporidium andersoni; C.b, Cryptosporidium bovis; C.r, Cryptosporidium ryanae.
same concentration and testing procedures used throughout the study. Six 15 g fecal specimens were each spiked with oocysts at each level (10, 100 and 1000 oocysts/g). Positive results were obtained for 6/6 samples with 1000 oocysts/g, 6/6 samples with 100 oocysts/g and 4/6 samples with 10 oocysts/g.
At the state level, operations in California, Iowa, Nebraska, and Oklahoma had the highest overall prevalence of positive samples with 44.4, 41.0, 40.2 and 22.6% positive, respectively. Cryptosporidium was not detected in any samples from Alabama, Georgia, New Mexico, South Dakota, Tennessee, or Texas.
3. Results
4. Discussion
Overall, 819 fecal samples were submitted for detection of Cryptosporidium. The fecal samples originated from 49 operations in 20 states. The number of fecal samples per operation ranged from 3 to 20 (Table 1). One or more calf fecal samples were positive for Cryptosporidium on 34 operations from 14 states (Table 1). Of 819 fecal samples examined, 168 (20.5%) were positive for Cryptosporidium. Three species were detected. Cryptosporidium ryanae, C. bovis and C. andersoni were detected in 15 (1.8%), 39 (4.8%), and 115 (14.0%) fecal samples, respectively. Of 115 isolates identified as C. andersoni, 74 showed 100% nucleotide sequence identity to the C. andersoni isolate reported by Koyama et al. (2005) (GenBank Accession number: AB089285), 35 showed 100% nucleotide sequence identity to the C. andersoni isolate reported by Liu et al. (2009) (GenBank Accession number: FJ463187) that contains an insertion of thymine at nucleotide 634, and 6 showed the presence of both sequences of C. andersoni (with and without the thymine insertion) characterized by the presence of overlapping nucleotide peaks in chromatograms, which can be attributed to mixed infections or to allelic sequence heterozygosity of single oocysts. One sample was positive for both C. ryanae and C. bovis. The zoonotic species, C. parvum, was not detected in any fecal sample.
The methods used for oocyst recovery and molecular detection in the present study were the same as in previous studies on dairy cattle (Fayer et al., 2006, 2007; Santín et al., 2008). Spiking studies to determine detection levels conducted herein and in the previous studies have yielded similar results. In the present study, detection at the level of 1000, 100, and 10 oocysts/g was 100, 100 and 67%, respectively, indicating that at levels below 100 oocysts/g some positive samples may not be detected. Therefore the prevalence of positive samples with low numbers of oocysts per gram was likely underestimated. Based on sequence data from the SSU rRNA gene, 3 species of Cryptosporidium were identified among 168 PCR positive fecal samples from the 819 samples evaluated. These included C. andersoni, C. bovis, and C. ryanae (one mixed infection with both C. bovis and C. ryanae) but C. parvum was not found. These results differ from those previously reported for beef cattle or feedlot cattle because of the methods used. In the present study every sample was subjected to PCR and all Cryptosporidium-positive PCR products were subjected to sequence analysis to identify the species. Studies that identified C. parvum by microscopy require re-evaluation because the recently named species C. bovis and C. ryanae have no unique morphological crite-
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ria to clearly distinguish them from C. parvum and therefore species may have been reported as C. parvum inadvertently. Several previous studies investigated Cryptosporidium on cow–calf operations in the United States. In California, of 558 fecal pats collected from the ground from 25 herds 0.18% had oocysts. Specimens collected directly from the rectum had a sixfold higher prevalence of oocysts (Hoar et al., 1999). In another study in California, the point prevalence of Cryptosporidium oocysts (mostly collected per rectum) was determined for 915 calves between 1 and 11 months of age and 484 cattle over 12 months of age (Atwill et al., 1999). Of 1399 samples, 54 (3.9%) contained oocysts. Of 884 cattle over 4 months of age, 6 (2.4%) shed oocysts. A third study in California estimated the daily environmental loading rate of oocysts produced by 40 adult beef cattle per herd in 3 cow–calf production herds approximately 6 weeks before and 6 weeks after calving (Atwill et al., 2003). Of 240 fecal samples, 17 (7.1%) were infected with C. parvum. Although C. parvum was reported in these three previous studies, microscopic methods were used which cannot distinguish C. parvum from C. bovis and C. ryanae. The percentage of samples positive for Cryptosporidium in the California studies were considerably lower than in the present study (20.5% positive overall, 44.4% in California). There are several possible reasons for this difference. The PCR methodology in the present study likely has higher sensitivity for detecting Cryptosporidium, as compared with microscopy. Also, Santín et al. (2008) showed that the prevalence of Cryptosporidium often decreases with age. The cattle in the present study might have been younger than those sampled in one or more of the previous studies. It is also possible that the two California herds participating in the present study had different management practices compared with herds in the previous studies. In western North Dakota, molecular methods were used to determine the prevalence of Cryptosporidium species in 7 beef cow–calf herds in 98 calves 6–8 months of age and 114 cows over 2 years of age (Feltus et al., 2008). Overall, 43 animals were positive but only 5 positives were from cows. C. bovis, the deer-like genotype (C. ryanae) and C. andersoni were identified in 9.4, 6.6 and 1.4% of animals sampled, respectively. In the present study the same species were found. Other studies of beef cattle in the United States investigated Cryptosporidium in feedlot cattle. Atwill et al. (2006) examined 5274 fecal specimens collected from the ground from 22 feedlots in 7 central and western states by DFA microscopy precluding comparison with the present study. Oocysts were detected in only 9 (0.17%) of these specimens and only 4 of the 9 had enough oocysts to enable molecular confirmation as C. parvum. Also in North America, of 20 cow–calf pairs on a ranch north of Calgary, Alberta only 1 calf was positive for Cryptosporidium by fluorescence microscopy (Ralston et al., 2003). Feces were collected per rectum from 669 beef cows 2–14 years of age in southern Ontario and from 192 calves 2–70 days of age in southern British Columbia (McAllister et al., 2005). Based on DFA microscopy, of 669 fecal samples from beef cows 10.6 and 18.4% were diagnosed with C. muris (probably C. andersoni) and C. parvum, respectively, and of 192 calves 13% were diag-
nosed with Cryptosporidium. In cows and calves during the calving season in western Canadian cow–calf herds fresh fecal samples were collected from 560 beef cows and 605 calves (Gow and Waldner, 2006). Feces collected from mature cows on 59 farms and from calves on 100 farms were examined for the presence of Cryptosporidium using a quantitative sucrose gradient immunoflourescent antibody test. Only 1.1% of the cows and 3.1% of the calves were positive for Cryptosporidium spp. Again, the oocyst recovery and microscopic methods used in these studies cannot be fairly compared with those of the present study. Several studies investigated the presence of Cryptosporidium in beef cattle in Europe, Africa and Asia. In Scotland two beef herds of breeding animals over 1 year of age were sampled (Scott et al., 1995). Of 553 fecal samples examined, 62.4% had oocysts, reported as C. parvum, in direct smears observed microscopically. On farm 1, 268 of 437 (61.3%) cattle were positive and on farm 2, 77 of 116 (66.4%) cattle were positive. In Bohemia, in a long-term study of beef cattle the prevalence of C. andersoni highly exceeded the prevalence reported in other studies (Kvác and Vítovec, 2003). Of 96 cows 43.8% were positive and at 27 weeks of age 92.9% of 42 calves were positive for C. andersoni as determined by oocyst measurements. Mean oocyst size was 8.48 × 6.41 l and molecular characterization of a partial sequence of the 18S rDNA was consistent with C. andersoni. In Ireland, feces in 21 of 288 beef cattle at slaughter had oocysts detected by DFA microscopy (Moriarty et al., 2005). Molecular testing of positive feces identified 54.5% as C. andersoni and 45.5% as C. parvum. In Belgium, feces were collected from a total of 499 dairy and 333 beef calves less than 10 weeks of age and examined by DFA microscopy for the presence of Cryptosporidium oocysts and 11 specimens from beef calves were examined by molecular methods to determine species (Geurden et al., 2007). The overall prevalence was 37 and 12%, respectively in these young dairy and beef calves and 7 of the 11 samples from beef calves tested by molecular methods were C. parvum. In Northern Portugal, feces from dairy and beef cattle (Mendonc¸a et al., 2007) were tested by PCR followed by sequencing of the hsp70 and 18S rRNA genes. Of 176 adult cattle 8 (4.5%) were positive. Six had C. parvum, one had C. andersoni, and one had C. meleagridis. In Zambia, feces from 238 beef calves, 250 dairy calves, and 256 calves on “traditional” farms were frozen and tested for Cryptosporidium by a commercial ELISA test (Geurden et al., 2006). All calves were 3 months of age or younger. Beef calves (breeds included Brahman, Hereford, Boran, Simmental or cross breeds) had a mean age of 24 days and 8.0% were positive. Of eight beef calves tested by molecular methods seven were reported to have C. parvum. In Japan, after detecting C. muris (most likely C. andersoni) oocysts in 24 (4.7%) of 512 beef cattle in an abattoir in Miyagi Prefecture (Kaneta and Nakai, 1998) a survey was conducted of grazing cattle that detected C. muris type oocysts in 21 (4.1%) of 516 beef cattle on a farm (Nakai et al., 2004). Nakai et al. (2004) cited earlier reports (in Japanese) of C. muris-like oocysts from cattle in slaugh-
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terhouses throughout Japan including 1.2% in Fukushima, 1.2% in Saitama, 3.9% in Kanagawa, and 3.4% in Tokushima prefectures. Age-related similarities appear between beef cattle in the present study and dairy cattle with regard to the presence of Cryptosporidium species. Based on molecular methods, of 571 12–24-month-old dairy heifers, 68 were found to be infected with Cryptosporidium (Fayer et al., 2006). By gene sequencing, 1 (0.02%), 4 (0.07%), 10 (1.8%), 24 (4.2%), and 29 (5.1%) of these calves were found to be infected with C. suis, C. parvum, C. ryanae, C. bovis, and C. andersoni, respectively. Other reports also indicate that less than 1% of postweaned and adult dairy cattle were found to excrete C. parvum (Fayer et al., 2007; Santín et al., 2008; Uehlinger et al., 2006). The finding of C. andersoni, C. bovis, and C. ryanae in 6–18month-old cattle in the present study and absence of C. parvum parallels the findings in dairy cattle of similar ages. 5. Conclusions Although C. parvum was not detected in any of the fecal specimens in the present study it might have been present in numbers below the level of detection (<10 oocysts/g of feces). The fact that it was not detected suggests that 6–18-month-old cattle on cow–calf operations do not significantly contribute to environmental contamination with this zoonotic species, contrary to earlier microscopy based reports. Acknowledgments We thank Judy Rodriguez for support in managing the data collection and analysis for this project and Julie Headley and Meghan Heffron for technical support. References Atwill, E.R., das Gracas, C., Pereira, M., Alonso, L.H., Elmi, C., Epperson, W.B., Smith, R., Riggs, W., Carpenter, L.V., Dargatz, D.A., Hoar, B., 2006. Environmental load of Cryptosporidium parvum oocysts from cattle manure in feedlots from the central and western United States. J. Environ. Qual. 35, 200–206. Atwill, E.R., Hoar, B., das Gracas, C., Pereira, M., Tate, K.W., Rulofson, F., Nader, G., 2003. Improved quantitative estimates of low environmental loading and sporadic periparturient shedding of Cryptosporidium parvum in adult beef cattle. Appl. Environ. Microbiol. 69, 4604–4610. Atwill, E.R., Johnson, E., Klingborg, D.J., Veserat, G.M., Markegard, G., Jensen, W.A., Pratt, D.W., Delmas, R.E., George, H.A., Forero, L.C., Philips, R.L., Barry, S.J., McDougald, N.K., Gildersleeve, R.R., Frost, W.E., 1999. Age, geographic, and temporal distribution of fecal shedding of Cryptosporidium parvum oocysts in cow–calf herds. Am. J. Vet. Res. 60, 420–425. Fayer, R., 2010. Taxonomy and species delimitation in Cryptosporidium. Exp. Parasitol. 124, 90–97. Fayer, R., Santin, M., Trout, J.M., 2007. Prevalence of Cryptosporidium species and genotypes in mature dairy cattle on farms in eastern United States compared with younger cattle from the same locations. Vet. Parasitol. 145, 260–266. Fayer, R., Santín, M., Trout, J.M., 2008. Cryptosporidium ryanae n. sp. (Apicomplexa: Cryptosporidiidae) in cattle (Bos taurus). Vet. Parasitol. 156, 191–198. Fayer, R., Santín, M., Trout, J.M., Greiner, E., 2006. Prevalence of species and genotypes of Cryptosporidium found in 1–2-year-old dairy cattle in the eastern United States. Vet. Parasitol. 135, 105–112.
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