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A longitudinal study of cryptosporidiosis in dairy cattle from birth to 2 years of age
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Veterinary Parasitology 155 (2008) 15–23 www.elsevier.com/locate/vetpar
A longitudinal study of cryptosporidiosis in dairy cattle from birth to 2 years of age Mo´nica Santı´n *, James M. Trout, Ronald Fayer Environmental Microbial 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 Received 5 February 2008; received in revised form 27 March 2008; accepted 21 April 2008
Abstract Fecal specimens were collected from 30 calves from birth to 24 months of age at a dairy farm in Maryland to determine the prevalence and age distribution of Cryptosporidium species/genotypes. After centrifugation to remove debris and concentrate oocysts, specimens were examined by immunofluorescence microscopy and polymerase chain reaction (PCR). Fragments of the SSU-rDNA gene amplified by PCR were purified and PCR products were sequenced. All 30 calves shed Cryptosporidium oocysts at some time during the 24 months of the study. Of 990 specimens, 190 were Cryptosporidium-positive (19.2%). The highest prevalence of infection was at 2 weeks of age when 29 of the 30 calves were excreting oocysts. Prevalence was higher in pre-weaned calves (1–8 weeks of age) (45.8%) than in post-weaned calves (3–12 months of age) (18.5%) and heifers (12–24 months of age) (2.2%). Sequence data for 190 PCR-positive specimens identified: C. parvum, C. bovis, the Cryptosporidium deer-like genotype and C. andersoni, with cumulative prevalences of 100, 80, 60, and 3.3%, respectively. C. parvum constituted 97% of infections in preweaned calves but only 4% and 0% of infections in post-weaned calves and heifers, respectively. All C. parvum GP60 nucleotide sequences were subtype IIaA15G2R1. Published by Elsevier B.V. Keywords: Cattle; Cryptosporidium; PCR; C. bovis; Deer-like genotype; C. parvum; C. andersoni; Subtyping
1. Introduction Cryptosporidiosis is a very common infection in cattle worldwide (Santı´n and Trout, 2008). For dairy cattle, Cryptosporidium has become a concern not only because of the direct economic losses associated with the infection, but also from a public health perspective because of the potential for environmental contamination with Cryptosporidium oocysts. Recent molecular studies of cryptosporidiosis in cattle have shown that
* Corresponding author. Tel.: +1 301 504 6774; fax: +1 301 504 6608. E-mail address: [email protected] (M. Santı´n). 0304-4017/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.vetpar.2008.04.018
three species and one genotype of Cryptosporidium are responsible for most cattle infections (C. parvum, C. bovis, and C. andersoni, and Cryptosporidium deer-like genotype) (Lindsay et al., 2000; Santı´n et al., 2004; Fayer et al., 2005, 2006, 2007; Feng et al., 2007). C. parvum is known to infect humans worldwide and is recognized as the major zoonotic Cryptosporidium species, whereas C. andersoni has been reported in humans only once (Leoni et al., 2006). In a series of four sequential point prevalence studies conducted on dairy farms on the East Coast of the United states that included 503 pre-weaned, 468 postweaned, 571 heifers, and 541 milking cows, differences were observed in the prevalence of species and genotypes of Cryptosporidium relative to the age of
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the animals (Santı´n et al., 2004; Fayer et al., 2006, 2007). The most prevalent species were C. parvum in pre-weaned calves, C. bovis and the Cryptosporidium deer-like genotype in post-weaned calves, and C. andersoni in heifers and milking cows. Other molecular-based studies also reported an age-related pattern of Cryptosporidium species in cattle (Langkjær et al., 2006; Feng et al., 2007). Longitudinal studies have been conducted on dairy cattle in Trinidad (Adesiyun et al., 2001), the United Kingdom (Sturdee et al., 2003), Tunisia (Soltane et al., 2007), and New Zealand (Winkworth et al., 2008). However all theses studies identified Cryptosporidium based on Ziehl-Neelsen stained or immune-fluorescent stained oocysts which cannot identify species. Because, there are no data that document the presence of Cryptosporidium species/ genotypes in the same animals over time, the present study is the first long-term longitudinal study to follow the same cattle from birth to breeding age under farm conditions to determine the presence of Cryptosporidium species, genotypes, and subtypes.
2. Material and methods 2.1. Sources and collection of specimens Thirty-three fecal specimens were collected from each of 30 calves from birth until 24 months of age at a dairy farm in Maryland. All were purebred Holstein female calves born over a period of 5 months (November 2004–March 2005). Calves were considered pre-weaned from birth until 8 weeks of age, post-weaned from 3 to 12 months of age, and heifers from 13 to 24 months of age. Calves were individually housed in hutches from birth until they were 3 months of age. From 3 to 24 months of age they were housed in groups in large pens partially covered by a roof. Feces were collected weekly from calves from 1 to 8 weeks of age (8 specimens per calf), biweekly from calves 10–20 weeks of age (6 specimens per calf), and monthly from calves 6–24 months of age (19 specimens per calf), so that a total of 33 fecal specimens were collected from each calf over the 24 months of the study. Feces were collected directly from the rectum of each animal into a plastic cup. Cups were capped, labeled with the calf’s ear tag number, and immediately placed in an insulated container packed with ice or cold packs. Specimens were transported to the USDA laboratory in Beltsville, Maryland and processed within 1–3 days of collection.
2.2. Oocysts concentrated from feces Oocysts were concentrated from feces as previously described (Fayer et al., 2000; Santı´n et al., 2004). Briefly, 15 g of feces from each specimen cup were mixed with 35-ml of distilled water (dH2O). The suspension was passed through a sieve with a 45 mm pore size screen. The filtrate volume was adjusted to 50 ml with dH2O and centrifuged at 1800 g for 15 min. The pellet was resuspended in a mixture of 25 ml dH2O and 25 ml CsCl (1.4 g/l) and centrifuged at 300 g for 20 min. Supernatant, aspirated from each suspension, was washed twice with dH2O and the final pellet was suspended in 500 ml of dH2O. The suspension was examined by microscopy and molecular methods as described below. 2.3. IFA A 100 ml aliquot of fecal suspension was transferred to a microcentrifuge tube and washed once with dH2O. The pellet was resuspended in 50 ml of premixed Merifluor reagent (Meridian Diagnostics, Cincinnati, Ohio) and 2 ml of suspension was transferred to a well (11 mm diameter) of a 3-well glass microscope slide. The slide was covered with a 24 mm 50 mm coverslip and the entire well area was examined by fluorescence microscopy at 400 using a Zeiss Axioskop equipped with epifluorescence and an FITC-Texas RedTM dual wavelength filter. 2.4. DNA extraction Total DNA was extracted from each CsCl-cleaned fecal sample using a DNeasyTissue Kit (Qiagen, Valencia, California) with a slightly modified protocol. The protocol, described below, utilized reagents provided by the manufacturer. A total of 50 ml of processed feces were suspended in 180 ml of ATL buffer and thoroughly mixed by vortexing. To this suspension, 20 ml of proteinase K (20 mg/ml) was added, and the sample was thoroughly mixed. Following an overnight incubation of the mixture at 55 C, 200 ml of AL buffer was added. The remaining protocol followed manufacturer’s instructions with one exception. To increase the quantity of recovered DNA, the nucleic acid was eluted in 100 ml of AE buffer. 2.5. Gene amplification and sequencing A two-step nested PCR protocol was used to amplify an 830 bp fragment of the 18S rRNA gene using primers
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50 -TTCTAGAGCTAATACATGCG-30 and 50 -CCCATTTCCTTCGAAACAGGA-30 for primary PCR and 50 -GGAAGGGTTGTATTT-ATTAGATAAAG-30 and 50 -AAGGAGTAAGGAACAACCTCCA-30 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, California), 2.5 ml BSA (0.1 g/10 ml), and 1 mM of each forward and reverse primer in a 50 ml reaction volume. Each of 35 cycles consisted of 94 C for 45 s, 59 C for 45 s, and 72 C for 1 min after an initial hot start at 94 C for 3 min and ending with 72 C for 7 min. The secondary PCR mixture was identical except that the MgCl2 concentration was 1.5 mM. Each of 40 cycles consisted of 94 C for 30 s, 58 C for 90 s, and 72 C for 2 min after an initial hot start at 94 C for 3 min and ending with 72 C for 7 min. To subtype C. parvum, all specimens obtained from calves up to 8 weeks of age (n = 240) were also analyzed by GP60 PCR irrespective of the 18S rRNA genotyping results. Subtyping of C. parvum isolates was performed using a nested PCR protocol to amplify a 450 bp fragment of the 60 kDa glycoprotein (GP60) gene using primers 50 -ATAGTCTCCGCTGTATTC-30 and 50 -GAGATATATCTTGGTGCG-30 for primary PCR and 50 0 0 TCCGCTGTATTCTCAGCC-3 and 5 -CGAACCACATTACAAATGAAGT-30 for secondary PCR (Sulaiman et al., 2005). For both primary and secondary the PCR mixture contained 1 PCR buffer, 3 mM MgCl2, 0.2 mM dNTP, 2.5 U Taq (Qbiogene, Irvine, California), 2.5 ml BSA (0.1 g/ 10 ml), and 1 mM of each forward and reverse primer in a 50 ml reaction volume. Each of 35 cycles consisted of 94 C for 45 s, 50 C for 45 s, and 72 C for 1 min after an initial hot start at 94 C for 5 min and ending with 72 C for 10 min. Subtypes were determined and named based on both the number of trinucleotide repeats and mutations in the nonrepeat regions (Sulaiman et al., 2005). PCR products were analyzed on 1% agarose gel and visualized after ethidium bromide staining. Positive PCR products, purified using Exonuclease I/Shrimp Alkaline Phosphatase (Exo-SAP-ITTM) (USB Corporation, Cleveland, Ohio), were sequenced in both directions using the same PCR primers in 10 ml reactions, Big DyeTM chemistries, and an ABI 3100 sequencer analyzer (Applied Biosystems, Foster City, California). Sequence chromatograms of each strand were aligned and examined with Lasergene software (DNASTAR, Inc., Madison, Wisconsin).
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2.6. Statistical analysis The prevalence of Cryptosporidium infection determined by IFA and molecular methods was compared among age groups (pre-weaned calves, post-weaned calves, and heifers). The Chi-square Test for Independence was used to analyze the data and differences were considered very highly significant when p = < 0.0001. 3. Results 3.1. Prevalence of Cryptosporidium More specimens were found positive for Cryptosporidium by PCR than by IFA. Of the 990 specimens collected 190 (19.2%) were positive by PCR and 128 (12.9%) by IFA. More specimens were found positive by PCR in all three age groups but the differences between both methods were more obvious in post-weaned calves specimens with twofold more positives by PCR (18.5%) than by IFA (9.2%) (Fig. 1). The highest prevalence was observed in pre-weaned calves specimens and prevalence significantly decreased with the age of the animals as determined by both methods ( p < 0.0001) (Fig. 1). The prevalence of Cryptosporidium as determined at weekly, biweekly, and monthly age intervals up to 24 months of age is shown in Fig. 2. The highest prevalence of Cryptosporidium was observed at 2 weeks of age when 29 of the 30 calves were positive (96.6%). A second peak, with lower prevalence, was observed at 18 weeks of age when 14 of the 30 calves were found to be infected (46.7%). 3.2. Molecular characterization of Cryptosporidium by 18S rDNA PCR All 190 Cryptosporidium PCR-positive specimens were sequenced, and four Cryptosporidium species/
Fig. 1. Prevalence of Cryptosporidium-positive specimens detected by IFA and in PCR in pre-weaned calves, post-weaned calves, and heifers.
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Fig. 2. Prevalence of Cryptosporidium spp., C. parvum, C. bovis, C. andersoni, and Cryptosporidium deer-like genotype in calves from 1 week to 24 months of age by PCR.
genotypes were identified: C. parvum, C. bovis, C. andersoni, and the deer-like genotype in 110, 45, 33, and 2 specimens, respectively. Of the 110 C. parvum positive samples, 48 had 100% similarity with C. parvum nucleotide sequence AF093490, 21 had 100% similarity with C. parvum nucleotide sequence AF308600, and in 41 samples a combination of both nucleotide sequences (AF093490 and AF308600) was observed. C. parvum, C. bovis, deer-like genotype, and C. andersoni constituted 11.1%, 4.5%, 3.3%, and 0.2%, respectively, of the 990 samples. The cumulative prevalence for the Cryptosporidium species/genotypes identified in the study were 100%, 80%, 60%, and 3.3% for C. parvum, C. bovis, deer-like genotype, and C. andersoni, respectively. Each species and genotype of Cryptosporidium displayed a different prevalence pattern relative to age of the cattle (Fig. 2). C. parvum was detected in 24 of the 30 calves examined at 1 week of age (80%), and by 2 weeks of age was detected in 29 of the calves (96.6%). After 10 weeks of age, only two calves were infected with C. parvum: one calf at 16 weeks of age and another at 6 months of age. The initial detection of C. bovis and the Cryptosporidium deer-like genotype was at 4 and 10 weeks of age, respectively. Prevalence of C. bovis and the Cryptosporidium deer-like genotype peaked at 16 and 18 weeks of age, respectively. Most C. bovis infections were observed among calves from 12 weeks
of age to 14 months of age, with only three animals infected before and one after that interval. Most Cryptosporidium deer-like genotype infections were observed among calves from 14 weeks to 6 months of age, with only three animals infected after that interval; C. andersoni was not found until the 19th months of the study and it was identified in the same animal for two consecutive monthly examinations. The percentage that each species/genotypes of Cryptosporidium represented among the 190 Cryptosporidium-positive specimens in the three age categories is presented in Fig. 3. C. parvum constituted 97% of the species in pre-weaned calves. In post-weaned calves and heifers the majority of infections were C. bovis and the Cryptosporidium deer-like genotype. C. andersoni was found only in heifers and constituted 25% of the infections in this age category. 3.3. Subtyping of C. parvum by GP60 PCR To determine C. parvum subtypes all 240 specimens collected from pre-weaned calves were subjected to a nested PCR that amplified a fragment of the GP60 gene and 91 were found positive. DNA sequencing indicated that all GP60 nucleotide sequences were identical to each other and had a 100% similarity with C. parvum subtype IIaA15G2R1 (GenBank accession number DQ630518).
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Fig. 3. Percentage that C. parvum, C. bovis, C. andersoni, and Cryptosporidium deer-like genotype represent in Cryptosporidiumpositive pre-weaned calves, post-weaned calves and heifers.
4. Discussion Nine hundred and ninety fecal specimens collected from the same 30 dairy calves from birth to 2 years of age were subjected to IFA and PCR to estimate the presence of Cryptosporidium spp. The number of Cryptosporidium-positive specimens obtained by PCR was significantly higher ( p = 0.0002) than the number obtained by IFA (19.2% vs 12.9%). Although the number of Cryptosporidium-positive specimens obtained by PCR was 19.2% the cumulative prevalence was 100%, indicating that oocysts were shed by all 30 calves at some time during the study. By examining calves for the presence of Cryptosporidium at short intervals over time the present study provides clarity to point prevalence studies that report a wide range of prevalence of infection, probably due to differences in the ages of the animals from which feces were obtained, the time of fecal collection (before, during or after patency). The time and frequency of collection and examination of feces in the present study (weekly, biweekly, and monthly intervals) were selected based on data obtained in point prevalence studies that indicated the presence of different species and genotypes at specific ages (Santı´n et al., 2004; Fayer et al., 2006, 2007). The detection method used in the present study was identical to that used previously for detection of Cryptosporidium in dairy cattle (Santı´n et al., 2004; Fayer et al., 2006, 2007). This method had been tested for sensitivity by spiking 25 fecal specimens each with 10, 50, and 100 oocysts per gram and finding that 24, 56 and 84% were positive by PCR, respectively (Santı´n et al., 2004). Although not every week in the first 2 years of theses calves lives were examined and some change species prevalence might have been missed this study is the most complete, longest term
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study yet conducted on any host species and provides a unique picture of the trend of parasitism in a herd over time. Cryptosporidium was detected in calves from 1 week to 20 months of age. The highest prevalence of infection was found in pre-weaned calves (45.8%), followed by post-weaned calves (18.5%), and then heifers (2.2%). Similar results have been observed in cattle in point prevalence studies worldwide (Xiao and Herd, 1994; Sischo et al., 2000; Wade et al., 2000; Huetink et al., 2001; Sturdee et al., 2003; Olson et al., 2004; Kvac et al., 2006; Langkjær et al., 2006). In Denmark, the prevalence was 96, 84, and 14% in young calves, older calves, and cows, respectively (Maddox-Hyttel et al., 2006). In Portugal, a higher prevalence was reported in calves than in cows (25.4 vs 4.5%) (Mendonca et al., 2007). In the USA, the prevalence in a series of point prevalence studies was 41, 26, 12, and 5.7% in preweaned calves, post-weaned calves, heifers, and adult cows, respectively (Santı´n et al., 2004; Fayer et al., 2006, 2007). Although calves in the present study were removed from their dams within minutes to an hour after birth and direct physical contact between pre-weaned calves was not possible, Cryptosporidium was found in all calves by 3 weeks of age. These findings confirm point prevalence studies that have concluded that cryptosporidiosis in calves is generally established during the first 2 weeks of life (Xiao and Herd, 1994; Quilez et al., 1996; Uga et al., 2000; Wade et al., 2000; Huetink et al., 2001; Castro-Hermida et al., 2002; Sturdee et al., 2003; Trotz-Williams et al., 2007). On commercial dairy farms fecal contamination from cattle of all ages is present in fields, pens, water supplies, buildings, tools, and on animals themselves. Previous studies have found that contact surfaces, flies, and birds, can serve as mechanical vectors of Cryptosporidium (Conn et al., 2007). Animal handlers could also mechanically transport infective oocysts in feces on shoes and clothing. With regard to bovine cryptosporidiosis, results of the present study indicate: (1) if only one fecal sample is collected per animal (‘‘point prevalence’’), the prevalence data will underestimate the actual number of infected animals; (2) prevalence can vary remarkably depending of the age of the calves; and (3) the detection method can influence the prevalence data because molecular methods are more sensitive and accurate than microscopic methods. Molecular characterization of Cryptosporidium using 18S rRNA indicated the presence of C. parvum, C. bovis, C. andersoni, and the Cryptosporidium deer-like genotype in agreement
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with point prevalence studies of cattle (Lindsay et al., 2000; Santı´n et al., 2004; Fayer et al., 2006, 2007; Feng et al., 2007; Geurden et al., 2007; Feltus et al., 2008). In the present study all 30 calves became infected with C. parvum (cumulative prevalence of 100%), whereas C. bovis and Cryptosporidium deer-like genotype had cumulative prevalences of 80 and 60%, respectively, and C. andersoni was observed in only 1 animal (cumulative prevalence of 3.3%). In earlier reports from point prevalence studies, C. parvum and C. andersoni were the two species most commonly reported in cattle worldwide (Xiao and Herd, 1994; Fayer et al., 2000; Wade et al., 2000; Huetink et al., 2001; Enemark et al., 2002; Peng et al., 2003). The application of molecular methods has resulted in the identification of oocysts of an additional species and genotype from cattle. C. bovis (previously named as Bovine B genotype; Fayer et al., 2005) and the Cryptosporidium deer-like genotype, not reported until 2002 and 2004, respectively (Xiao et al., 2002; Santı´n et al., 2004; Fayer et al., 2005), are prevalent in dairy cattle worldwide. C. bovis has been reported in Belgium, Brazil, Canada, China, Denmark, India, Northern Ireland Zambia, United Kingdom, and in the United States (Xiao et al., 2002; Santı´n et al., 2004; Fayer et al., 2006, 2007; Geurden et al., 2006, 2007; Langkjær et al., 2006; Coklin et al., 2007; Feng et al., 2007; Siwila et al., 2007; Thomaz et al., 2007; Thompson et al., 2007; Brook et al., 2008; Feltus et al., 2008), and the Cryptosporidium deer-like genotype has been reported in China, Denmark, Hungary, Kenya, Malaysia, Northern Ireland, Zambia, United Kingdom, and in the United States (Santı´n et al., 2004; Fayer et al., 2006, 2007; Langkjær et al., 2006; Feng et al., 2007; Plutzer and Karanis, 2007; Siwila et al., 2007; Thompson et al., 2007; Brook et al., 2008; Feltus et al., 2008; Halim et al., 2008; Szonyi et al., 2008;). Infections with C. hominis, C. suis, C. suis-like, C. felis, C. canis, and Cryptosporidium pig genotype II have also been reported for cattle (Bornay-Llinares et al., 1999; Fayer et al., 2001, 2006; Smith et al., 2005; Geurden et al., 2006, 2007; Langkjær et al., 2006; Park et al., 2006). These are reports of experimental infections or occasional findings and none have cattle as a primary host. The host age-related susceptibility to C. parvum, C. bovis, C. andersoni, and the Cryptosporidium deer-like genotype observed in the current study has been described previously (Santı´n et al., 2004; Fayer et al., 2006, 2007; Langkjær et al., 2006; Thompson et al., 2007). Virtually all infections in calves 8 weeks of age and younger were caused by C. parvum. However, C.
bovis was identified for the first time in two 4-week-old calves, and the Cryptosporidium deer-like genotype was identified in a 10-week-old calf. In the present study, as in previous studies, infection with the Cryptosporidium deer-like genotype was found in the same age group as C. bovis, but, with a lower prevalence of infection. In Belgium, as in the present study, C. bovis was more prevalent in older calves compared to neonatal calves (Geurden et al., 2007). However, as suggested by Feng et al. (2007), infections with C. bovis and the deer-like genotype in younger calves might be concealed by an overwhelming C. parvum infection. It was shown previously that PCR tools with broad specificity, such as the 18S rDNA PCR, selectively amplify the predominant species of Cryptosporidium (Cama et al., 2006). The prevalence of C. andersoni was very low, with this species detected in only 0.2% of the samples examined. A low prevalence (1.1%) of C. andersoni in cattle has been reported previously (Wade et al., 2000). Conversely, a high prevalence of C. andersoni was found in cattle during two separate visits to a beef farm in Denmark (18% and 28%) (Enemark et al., 2002). During a survey of 2943 fecal specimens, C. andersoni was identified in dairy cattle from 51 days of age to adults, but most was found in animals older than 6 months of age (Wade et al., 2000). Two peaks in prevalence were observed in the present study; the first peak, observed in calves between birth and 3 week of age, was attributable to C. parvum, whereas, C. bovis and the Cryptosporidium deer-like genotype contributed to the second peak, which was observed in calves 16–20 weeks of age. Similar findings were reported in a point prevalence study conducted in 7 states on the east coast of the U.S. (Santı´n et al., 2004). To determine C. parvum subtypes all specimens from pre-weaned calves, were subjected to a nested PCR to amplify a fragment of the GP60 gene. Of the 240 specimens analyzed, 91 were GP60 positive. DNA sequencing showed that all 91 GP60 positive specimens had the C. parvum subtype IIaA15G2R1. This is the most common subtype found in calves and humans; it has been widely reported in cattle in Belgium, Canada, India, Ireland, United Kingdom, and the United States, and (Feng et al., 2007; Geurden et al., 2007; O’Brien et al., 2008; Brook et al., 2008) and in humans in Australia, Canada, India, Japan, Kuwait, Northern Ireland, Portugal, Slovenia, and the United States (Strong et al., 2000; Glaberman et al., 2002; Alves et al., 2003, 2006; Peng et al., 2003; Stantic-Pavlinic et al., 2003; Wu et al., 2003; Chalmers et al., 2005; Sulaiman et al., 2005; Abe et al., 2006; Trotz-Williams et al., 2006).
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5. Conclusions To our knowledge, there are no comparable longitudinal studies for the prevalence of Cryptosporidium in cattle that include molecular characterization of all Cryptosporidium-positive samples. The present study documents for the first time a sequential association of species of Cryptosporidium in 30 calves observed over a 24 months study period. It is crucial to know which species and/or genotypes are actually present in Cryptosporidium-positive samples to determine the zoonotic implications. Consequently, studies in which C. parvum-like oocysts have been detected in cattle should be interpreted with caution if molecular characterization has not been performed. Based on our results, C. parvum infects virtually all pre-weaned calves in their first 3 weeks of life. Previous studies have indicated that humans working closely with calves are at risk of becoming infected with C. parvum (Lengerich et al., 1993; Siwila et al., 2007). Individuals in the presence of young calves should exercise care to protect themselves from acquiring C. parvum infection and thus becoming a source of infection for others. Acknowledgements The authors thank Brooke Reich, Kristin Cameron, and Brandon Hall for technical services in support of this study. References Abe, N., Matsubayashi, M., Kimata, I., Iseki, M., 2006. Subgenotype analysis of Cryptosporidium parvum isolates from humans and animals in Japan using the 60-kDa glycoprotein gene sequences. Parasitol. Res. 99, 303–305. Adesiyun, A.A., Kaminjolo, J.S., Ngeleka, M., Mutani, A., Borde, G., Harewood, W., Harper, W., 2001. A longitudinal study on enteropathogenic infections of livestock in Trinidad. Rev. Soc. Bras. . Med. Trop. 34, 29–35. Alves, M., Xiao, L., Sulaiman, I., Lal, A.A., Matos, O., Antunes, F., 2003. Subgenotype analysis of Cryptosporidium isolates from humans, cattle, and zoo ruminants in Portugal. J. Clin. Microbiol. 41, 2744–2747. Alves, M., Xiao, L., Antunes, F., Matos, O., 2006. Distribution of Cryptosporidium subtypes in humans and domestic and wild ruminants in Portugal. Parasitol. Res. 99, 287–292. Bornay-Llinares, F.J., da Silva, A.J., Moura, I.N.S., Przemyslaw, M., Pietkiewicz, H., Kruminis-Lozowska, W., Graczyk, T.K., Pieniazek, N.J., 1999. Identification of Cryptosporidium felis in a cow by morphologic and molecular methods. Appl. Environ. Microbiol. 65, 1455–1458. Brook, E.J., Anthony Hart, C., French, N.P., Christley, R.M., 2008. Molecular epidemiology of Cryptosporidium subtypes in cattle in England. Vet. J., doi:10.1016/j.tvjl.2007.10.023
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Veterinary Parasitology 155 (2008) 15–23 www.elsevier.com/locate/vetpar
A longitudinal study of cryptosporidiosis in dairy cattle from birth to 2 years of age Mo´nica Santı´n *, James M. Trout, Ronald Fayer Environmental Microbial 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 Received 5 February 2008; received in revised form 27 March 2008; accepted 21 April 2008
Abstract Fecal specimens were collected from 30 calves from birth to 24 months of age at a dairy farm in Maryland to determine the prevalence and age distribution of Cryptosporidium species/genotypes. After centrifugation to remove debris and concentrate oocysts, specimens were examined by immunofluorescence microscopy and polymerase chain reaction (PCR). Fragments of the SSU-rDNA gene amplified by PCR were purified and PCR products were sequenced. All 30 calves shed Cryptosporidium oocysts at some time during the 24 months of the study. Of 990 specimens, 190 were Cryptosporidium-positive (19.2%). The highest prevalence of infection was at 2 weeks of age when 29 of the 30 calves were excreting oocysts. Prevalence was higher in pre-weaned calves (1–8 weeks of age) (45.8%) than in post-weaned calves (3–12 months of age) (18.5%) and heifers (12–24 months of age) (2.2%). Sequence data for 190 PCR-positive specimens identified: C. parvum, C. bovis, the Cryptosporidium deer-like genotype and C. andersoni, with cumulative prevalences of 100, 80, 60, and 3.3%, respectively. C. parvum constituted 97% of infections in preweaned calves but only 4% and 0% of infections in post-weaned calves and heifers, respectively. All C. parvum GP60 nucleotide sequences were subtype IIaA15G2R1. Published by Elsevier B.V. Keywords: Cattle; Cryptosporidium; PCR; C. bovis; Deer-like genotype; C. parvum; C. andersoni; Subtyping
1. Introduction Cryptosporidiosis is a very common infection in cattle worldwide (Santı´n and Trout, 2008). For dairy cattle, Cryptosporidium has become a concern not only because of the direct economic losses associated with the infection, but also from a public health perspective because of the potential for environmental contamination with Cryptosporidium oocysts. Recent molecular studies of cryptosporidiosis in cattle have shown that
* Corresponding author. Tel.: +1 301 504 6774; fax: +1 301 504 6608. E-mail address: [email protected] (M. Santı´n). 0304-4017/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.vetpar.2008.04.018
three species and one genotype of Cryptosporidium are responsible for most cattle infections (C. parvum, C. bovis, and C. andersoni, and Cryptosporidium deer-like genotype) (Lindsay et al., 2000; Santı´n et al., 2004; Fayer et al., 2005, 2006, 2007; Feng et al., 2007). C. parvum is known to infect humans worldwide and is recognized as the major zoonotic Cryptosporidium species, whereas C. andersoni has been reported in humans only once (Leoni et al., 2006). In a series of four sequential point prevalence studies conducted on dairy farms on the East Coast of the United states that included 503 pre-weaned, 468 postweaned, 571 heifers, and 541 milking cows, differences were observed in the prevalence of species and genotypes of Cryptosporidium relative to the age of
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the animals (Santı´n et al., 2004; Fayer et al., 2006, 2007). The most prevalent species were C. parvum in pre-weaned calves, C. bovis and the Cryptosporidium deer-like genotype in post-weaned calves, and C. andersoni in heifers and milking cows. Other molecular-based studies also reported an age-related pattern of Cryptosporidium species in cattle (Langkjær et al., 2006; Feng et al., 2007). Longitudinal studies have been conducted on dairy cattle in Trinidad (Adesiyun et al., 2001), the United Kingdom (Sturdee et al., 2003), Tunisia (Soltane et al., 2007), and New Zealand (Winkworth et al., 2008). However all theses studies identified Cryptosporidium based on Ziehl-Neelsen stained or immune-fluorescent stained oocysts which cannot identify species. Because, there are no data that document the presence of Cryptosporidium species/ genotypes in the same animals over time, the present study is the first long-term longitudinal study to follow the same cattle from birth to breeding age under farm conditions to determine the presence of Cryptosporidium species, genotypes, and subtypes.
2. Material and methods 2.1. Sources and collection of specimens Thirty-three fecal specimens were collected from each of 30 calves from birth until 24 months of age at a dairy farm in Maryland. All were purebred Holstein female calves born over a period of 5 months (November 2004–March 2005). Calves were considered pre-weaned from birth until 8 weeks of age, post-weaned from 3 to 12 months of age, and heifers from 13 to 24 months of age. Calves were individually housed in hutches from birth until they were 3 months of age. From 3 to 24 months of age they were housed in groups in large pens partially covered by a roof. Feces were collected weekly from calves from 1 to 8 weeks of age (8 specimens per calf), biweekly from calves 10–20 weeks of age (6 specimens per calf), and monthly from calves 6–24 months of age (19 specimens per calf), so that a total of 33 fecal specimens were collected from each calf over the 24 months of the study. Feces were collected directly from the rectum of each animal into a plastic cup. Cups were capped, labeled with the calf’s ear tag number, and immediately placed in an insulated container packed with ice or cold packs. Specimens were transported to the USDA laboratory in Beltsville, Maryland and processed within 1–3 days of collection.
2.2. Oocysts concentrated from feces Oocysts were concentrated from feces as previously described (Fayer et al., 2000; Santı´n et al., 2004). Briefly, 15 g of feces from each specimen cup were mixed with 35-ml of distilled water (dH2O). The suspension was passed through a sieve with a 45 mm pore size screen. The filtrate volume was adjusted to 50 ml with dH2O and centrifuged at 1800 g for 15 min. The pellet was resuspended in a mixture of 25 ml dH2O and 25 ml CsCl (1.4 g/l) and centrifuged at 300 g for 20 min. Supernatant, aspirated from each suspension, was washed twice with dH2O and the final pellet was suspended in 500 ml of dH2O. The suspension was examined by microscopy and molecular methods as described below. 2.3. IFA A 100 ml aliquot of fecal suspension was transferred to a microcentrifuge tube and washed once with dH2O. The pellet was resuspended in 50 ml of premixed Merifluor reagent (Meridian Diagnostics, Cincinnati, Ohio) and 2 ml of suspension was transferred to a well (11 mm diameter) of a 3-well glass microscope slide. The slide was covered with a 24 mm 50 mm coverslip and the entire well area was examined by fluorescence microscopy at 400 using a Zeiss Axioskop equipped with epifluorescence and an FITC-Texas RedTM dual wavelength filter. 2.4. DNA extraction Total DNA was extracted from each CsCl-cleaned fecal sample using a DNeasyTissue Kit (Qiagen, Valencia, California) with a slightly modified protocol. The protocol, described below, utilized reagents provided by the manufacturer. A total of 50 ml of processed feces were suspended in 180 ml of ATL buffer and thoroughly mixed by vortexing. To this suspension, 20 ml of proteinase K (20 mg/ml) was added, and the sample was thoroughly mixed. Following an overnight incubation of the mixture at 55 C, 200 ml of AL buffer was added. The remaining protocol followed manufacturer’s instructions with one exception. To increase the quantity of recovered DNA, the nucleic acid was eluted in 100 ml of AE buffer. 2.5. Gene amplification and sequencing A two-step nested PCR protocol was used to amplify an 830 bp fragment of the 18S rRNA gene using primers
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50 -TTCTAGAGCTAATACATGCG-30 and 50 -CCCATTTCCTTCGAAACAGGA-30 for primary PCR and 50 -GGAAGGGTTGTATTT-ATTAGATAAAG-30 and 50 -AAGGAGTAAGGAACAACCTCCA-30 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, California), 2.5 ml BSA (0.1 g/10 ml), and 1 mM of each forward and reverse primer in a 50 ml reaction volume. Each of 35 cycles consisted of 94 C for 45 s, 59 C for 45 s, and 72 C for 1 min after an initial hot start at 94 C for 3 min and ending with 72 C for 7 min. The secondary PCR mixture was identical except that the MgCl2 concentration was 1.5 mM. Each of 40 cycles consisted of 94 C for 30 s, 58 C for 90 s, and 72 C for 2 min after an initial hot start at 94 C for 3 min and ending with 72 C for 7 min. To subtype C. parvum, all specimens obtained from calves up to 8 weeks of age (n = 240) were also analyzed by GP60 PCR irrespective of the 18S rRNA genotyping results. Subtyping of C. parvum isolates was performed using a nested PCR protocol to amplify a 450 bp fragment of the 60 kDa glycoprotein (GP60) gene using primers 50 -ATAGTCTCCGCTGTATTC-30 and 50 -GAGATATATCTTGGTGCG-30 for primary PCR and 50 0 0 TCCGCTGTATTCTCAGCC-3 and 5 -CGAACCACATTACAAATGAAGT-30 for secondary PCR (Sulaiman et al., 2005). For both primary and secondary the PCR mixture contained 1 PCR buffer, 3 mM MgCl2, 0.2 mM dNTP, 2.5 U Taq (Qbiogene, Irvine, California), 2.5 ml BSA (0.1 g/ 10 ml), and 1 mM of each forward and reverse primer in a 50 ml reaction volume. Each of 35 cycles consisted of 94 C for 45 s, 50 C for 45 s, and 72 C for 1 min after an initial hot start at 94 C for 5 min and ending with 72 C for 10 min. Subtypes were determined and named based on both the number of trinucleotide repeats and mutations in the nonrepeat regions (Sulaiman et al., 2005). PCR products were analyzed on 1% agarose gel and visualized after ethidium bromide staining. Positive PCR products, purified using Exonuclease I/Shrimp Alkaline Phosphatase (Exo-SAP-ITTM) (USB Corporation, Cleveland, Ohio), were sequenced in both directions using the same PCR primers in 10 ml reactions, Big DyeTM chemistries, and an ABI 3100 sequencer analyzer (Applied Biosystems, Foster City, California). Sequence chromatograms of each strand were aligned and examined with Lasergene software (DNASTAR, Inc., Madison, Wisconsin).
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2.6. Statistical analysis The prevalence of Cryptosporidium infection determined by IFA and molecular methods was compared among age groups (pre-weaned calves, post-weaned calves, and heifers). The Chi-square Test for Independence was used to analyze the data and differences were considered very highly significant when p = < 0.0001. 3. Results 3.1. Prevalence of Cryptosporidium More specimens were found positive for Cryptosporidium by PCR than by IFA. Of the 990 specimens collected 190 (19.2%) were positive by PCR and 128 (12.9%) by IFA. More specimens were found positive by PCR in all three age groups but the differences between both methods were more obvious in post-weaned calves specimens with twofold more positives by PCR (18.5%) than by IFA (9.2%) (Fig. 1). The highest prevalence was observed in pre-weaned calves specimens and prevalence significantly decreased with the age of the animals as determined by both methods ( p < 0.0001) (Fig. 1). The prevalence of Cryptosporidium as determined at weekly, biweekly, and monthly age intervals up to 24 months of age is shown in Fig. 2. The highest prevalence of Cryptosporidium was observed at 2 weeks of age when 29 of the 30 calves were positive (96.6%). A second peak, with lower prevalence, was observed at 18 weeks of age when 14 of the 30 calves were found to be infected (46.7%). 3.2. Molecular characterization of Cryptosporidium by 18S rDNA PCR All 190 Cryptosporidium PCR-positive specimens were sequenced, and four Cryptosporidium species/
Fig. 1. Prevalence of Cryptosporidium-positive specimens detected by IFA and in PCR in pre-weaned calves, post-weaned calves, and heifers.
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Fig. 2. Prevalence of Cryptosporidium spp., C. parvum, C. bovis, C. andersoni, and Cryptosporidium deer-like genotype in calves from 1 week to 24 months of age by PCR.
genotypes were identified: C. parvum, C. bovis, C. andersoni, and the deer-like genotype in 110, 45, 33, and 2 specimens, respectively. Of the 110 C. parvum positive samples, 48 had 100% similarity with C. parvum nucleotide sequence AF093490, 21 had 100% similarity with C. parvum nucleotide sequence AF308600, and in 41 samples a combination of both nucleotide sequences (AF093490 and AF308600) was observed. C. parvum, C. bovis, deer-like genotype, and C. andersoni constituted 11.1%, 4.5%, 3.3%, and 0.2%, respectively, of the 990 samples. The cumulative prevalence for the Cryptosporidium species/genotypes identified in the study were 100%, 80%, 60%, and 3.3% for C. parvum, C. bovis, deer-like genotype, and C. andersoni, respectively. Each species and genotype of Cryptosporidium displayed a different prevalence pattern relative to age of the cattle (Fig. 2). C. parvum was detected in 24 of the 30 calves examined at 1 week of age (80%), and by 2 weeks of age was detected in 29 of the calves (96.6%). After 10 weeks of age, only two calves were infected with C. parvum: one calf at 16 weeks of age and another at 6 months of age. The initial detection of C. bovis and the Cryptosporidium deer-like genotype was at 4 and 10 weeks of age, respectively. Prevalence of C. bovis and the Cryptosporidium deer-like genotype peaked at 16 and 18 weeks of age, respectively. Most C. bovis infections were observed among calves from 12 weeks
of age to 14 months of age, with only three animals infected before and one after that interval. Most Cryptosporidium deer-like genotype infections were observed among calves from 14 weeks to 6 months of age, with only three animals infected after that interval; C. andersoni was not found until the 19th months of the study and it was identified in the same animal for two consecutive monthly examinations. The percentage that each species/genotypes of Cryptosporidium represented among the 190 Cryptosporidium-positive specimens in the three age categories is presented in Fig. 3. C. parvum constituted 97% of the species in pre-weaned calves. In post-weaned calves and heifers the majority of infections were C. bovis and the Cryptosporidium deer-like genotype. C. andersoni was found only in heifers and constituted 25% of the infections in this age category. 3.3. Subtyping of C. parvum by GP60 PCR To determine C. parvum subtypes all 240 specimens collected from pre-weaned calves were subjected to a nested PCR that amplified a fragment of the GP60 gene and 91 were found positive. DNA sequencing indicated that all GP60 nucleotide sequences were identical to each other and had a 100% similarity with C. parvum subtype IIaA15G2R1 (GenBank accession number DQ630518).
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Fig. 3. Percentage that C. parvum, C. bovis, C. andersoni, and Cryptosporidium deer-like genotype represent in Cryptosporidiumpositive pre-weaned calves, post-weaned calves and heifers.
4. Discussion Nine hundred and ninety fecal specimens collected from the same 30 dairy calves from birth to 2 years of age were subjected to IFA and PCR to estimate the presence of Cryptosporidium spp. The number of Cryptosporidium-positive specimens obtained by PCR was significantly higher ( p = 0.0002) than the number obtained by IFA (19.2% vs 12.9%). Although the number of Cryptosporidium-positive specimens obtained by PCR was 19.2% the cumulative prevalence was 100%, indicating that oocysts were shed by all 30 calves at some time during the study. By examining calves for the presence of Cryptosporidium at short intervals over time the present study provides clarity to point prevalence studies that report a wide range of prevalence of infection, probably due to differences in the ages of the animals from which feces were obtained, the time of fecal collection (before, during or after patency). The time and frequency of collection and examination of feces in the present study (weekly, biweekly, and monthly intervals) were selected based on data obtained in point prevalence studies that indicated the presence of different species and genotypes at specific ages (Santı´n et al., 2004; Fayer et al., 2006, 2007). The detection method used in the present study was identical to that used previously for detection of Cryptosporidium in dairy cattle (Santı´n et al., 2004; Fayer et al., 2006, 2007). This method had been tested for sensitivity by spiking 25 fecal specimens each with 10, 50, and 100 oocysts per gram and finding that 24, 56 and 84% were positive by PCR, respectively (Santı´n et al., 2004). Although not every week in the first 2 years of theses calves lives were examined and some change species prevalence might have been missed this study is the most complete, longest term
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study yet conducted on any host species and provides a unique picture of the trend of parasitism in a herd over time. Cryptosporidium was detected in calves from 1 week to 20 months of age. The highest prevalence of infection was found in pre-weaned calves (45.8%), followed by post-weaned calves (18.5%), and then heifers (2.2%). Similar results have been observed in cattle in point prevalence studies worldwide (Xiao and Herd, 1994; Sischo et al., 2000; Wade et al., 2000; Huetink et al., 2001; Sturdee et al., 2003; Olson et al., 2004; Kvac et al., 2006; Langkjær et al., 2006). In Denmark, the prevalence was 96, 84, and 14% in young calves, older calves, and cows, respectively (Maddox-Hyttel et al., 2006). In Portugal, a higher prevalence was reported in calves than in cows (25.4 vs 4.5%) (Mendonca et al., 2007). In the USA, the prevalence in a series of point prevalence studies was 41, 26, 12, and 5.7% in preweaned calves, post-weaned calves, heifers, and adult cows, respectively (Santı´n et al., 2004; Fayer et al., 2006, 2007). Although calves in the present study were removed from their dams within minutes to an hour after birth and direct physical contact between pre-weaned calves was not possible, Cryptosporidium was found in all calves by 3 weeks of age. These findings confirm point prevalence studies that have concluded that cryptosporidiosis in calves is generally established during the first 2 weeks of life (Xiao and Herd, 1994; Quilez et al., 1996; Uga et al., 2000; Wade et al., 2000; Huetink et al., 2001; Castro-Hermida et al., 2002; Sturdee et al., 2003; Trotz-Williams et al., 2007). On commercial dairy farms fecal contamination from cattle of all ages is present in fields, pens, water supplies, buildings, tools, and on animals themselves. Previous studies have found that contact surfaces, flies, and birds, can serve as mechanical vectors of Cryptosporidium (Conn et al., 2007). Animal handlers could also mechanically transport infective oocysts in feces on shoes and clothing. With regard to bovine cryptosporidiosis, results of the present study indicate: (1) if only one fecal sample is collected per animal (‘‘point prevalence’’), the prevalence data will underestimate the actual number of infected animals; (2) prevalence can vary remarkably depending of the age of the calves; and (3) the detection method can influence the prevalence data because molecular methods are more sensitive and accurate than microscopic methods. Molecular characterization of Cryptosporidium using 18S rRNA indicated the presence of C. parvum, C. bovis, C. andersoni, and the Cryptosporidium deer-like genotype in agreement
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with point prevalence studies of cattle (Lindsay et al., 2000; Santı´n et al., 2004; Fayer et al., 2006, 2007; Feng et al., 2007; Geurden et al., 2007; Feltus et al., 2008). In the present study all 30 calves became infected with C. parvum (cumulative prevalence of 100%), whereas C. bovis and Cryptosporidium deer-like genotype had cumulative prevalences of 80 and 60%, respectively, and C. andersoni was observed in only 1 animal (cumulative prevalence of 3.3%). In earlier reports from point prevalence studies, C. parvum and C. andersoni were the two species most commonly reported in cattle worldwide (Xiao and Herd, 1994; Fayer et al., 2000; Wade et al., 2000; Huetink et al., 2001; Enemark et al., 2002; Peng et al., 2003). The application of molecular methods has resulted in the identification of oocysts of an additional species and genotype from cattle. C. bovis (previously named as Bovine B genotype; Fayer et al., 2005) and the Cryptosporidium deer-like genotype, not reported until 2002 and 2004, respectively (Xiao et al., 2002; Santı´n et al., 2004; Fayer et al., 2005), are prevalent in dairy cattle worldwide. C. bovis has been reported in Belgium, Brazil, Canada, China, Denmark, India, Northern Ireland Zambia, United Kingdom, and in the United States (Xiao et al., 2002; Santı´n et al., 2004; Fayer et al., 2006, 2007; Geurden et al., 2006, 2007; Langkjær et al., 2006; Coklin et al., 2007; Feng et al., 2007; Siwila et al., 2007; Thomaz et al., 2007; Thompson et al., 2007; Brook et al., 2008; Feltus et al., 2008), and the Cryptosporidium deer-like genotype has been reported in China, Denmark, Hungary, Kenya, Malaysia, Northern Ireland, Zambia, United Kingdom, and in the United States (Santı´n et al., 2004; Fayer et al., 2006, 2007; Langkjær et al., 2006; Feng et al., 2007; Plutzer and Karanis, 2007; Siwila et al., 2007; Thompson et al., 2007; Brook et al., 2008; Feltus et al., 2008; Halim et al., 2008; Szonyi et al., 2008;). Infections with C. hominis, C. suis, C. suis-like, C. felis, C. canis, and Cryptosporidium pig genotype II have also been reported for cattle (Bornay-Llinares et al., 1999; Fayer et al., 2001, 2006; Smith et al., 2005; Geurden et al., 2006, 2007; Langkjær et al., 2006; Park et al., 2006). These are reports of experimental infections or occasional findings and none have cattle as a primary host. The host age-related susceptibility to C. parvum, C. bovis, C. andersoni, and the Cryptosporidium deer-like genotype observed in the current study has been described previously (Santı´n et al., 2004; Fayer et al., 2006, 2007; Langkjær et al., 2006; Thompson et al., 2007). Virtually all infections in calves 8 weeks of age and younger were caused by C. parvum. However, C.
bovis was identified for the first time in two 4-week-old calves, and the Cryptosporidium deer-like genotype was identified in a 10-week-old calf. In the present study, as in previous studies, infection with the Cryptosporidium deer-like genotype was found in the same age group as C. bovis, but, with a lower prevalence of infection. In Belgium, as in the present study, C. bovis was more prevalent in older calves compared to neonatal calves (Geurden et al., 2007). However, as suggested by Feng et al. (2007), infections with C. bovis and the deer-like genotype in younger calves might be concealed by an overwhelming C. parvum infection. It was shown previously that PCR tools with broad specificity, such as the 18S rDNA PCR, selectively amplify the predominant species of Cryptosporidium (Cama et al., 2006). The prevalence of C. andersoni was very low, with this species detected in only 0.2% of the samples examined. A low prevalence (1.1%) of C. andersoni in cattle has been reported previously (Wade et al., 2000). Conversely, a high prevalence of C. andersoni was found in cattle during two separate visits to a beef farm in Denmark (18% and 28%) (Enemark et al., 2002). During a survey of 2943 fecal specimens, C. andersoni was identified in dairy cattle from 51 days of age to adults, but most was found in animals older than 6 months of age (Wade et al., 2000). Two peaks in prevalence were observed in the present study; the first peak, observed in calves between birth and 3 week of age, was attributable to C. parvum, whereas, C. bovis and the Cryptosporidium deer-like genotype contributed to the second peak, which was observed in calves 16–20 weeks of age. Similar findings were reported in a point prevalence study conducted in 7 states on the east coast of the U.S. (Santı´n et al., 2004). To determine C. parvum subtypes all specimens from pre-weaned calves, were subjected to a nested PCR to amplify a fragment of the GP60 gene. Of the 240 specimens analyzed, 91 were GP60 positive. DNA sequencing showed that all 91 GP60 positive specimens had the C. parvum subtype IIaA15G2R1. This is the most common subtype found in calves and humans; it has been widely reported in cattle in Belgium, Canada, India, Ireland, United Kingdom, and the United States, and (Feng et al., 2007; Geurden et al., 2007; O’Brien et al., 2008; Brook et al., 2008) and in humans in Australia, Canada, India, Japan, Kuwait, Northern Ireland, Portugal, Slovenia, and the United States (Strong et al., 2000; Glaberman et al., 2002; Alves et al., 2003, 2006; Peng et al., 2003; Stantic-Pavlinic et al., 2003; Wu et al., 2003; Chalmers et al., 2005; Sulaiman et al., 2005; Abe et al., 2006; Trotz-Williams et al., 2006).
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5. Conclusions To our knowledge, there are no comparable longitudinal studies for the prevalence of Cryptosporidium in cattle that include molecular characterization of all Cryptosporidium-positive samples. The present study documents for the first time a sequential association of species of Cryptosporidium in 30 calves observed over a 24 months study period. It is crucial to know which species and/or genotypes are actually present in Cryptosporidium-positive samples to determine the zoonotic implications. Consequently, studies in which C. parvum-like oocysts have been detected in cattle should be interpreted with caution if molecular characterization has not been performed. Based on our results, C. parvum infects virtually all pre-weaned calves in their first 3 weeks of life. Previous studies have indicated that humans working closely with calves are at risk of becoming infected with C. parvum (Lengerich et al., 1993; Siwila et al., 2007). Individuals in the presence of young calves should exercise care to protect themselves from acquiring C. parvum infection and thus becoming a source of infection for others. Acknowledgements The authors thank Brooke Reich, Kristin Cameron, and Brandon Hall for technical services in support of this study. References Abe, N., Matsubayashi, M., Kimata, I., Iseki, M., 2006. Subgenotype analysis of Cryptosporidium parvum isolates from humans and animals in Japan using the 60-kDa glycoprotein gene sequences. Parasitol. Res. 99, 303–305. Adesiyun, A.A., Kaminjolo, J.S., Ngeleka, M., Mutani, A., Borde, G., Harewood, W., Harper, W., 2001. A longitudinal study on enteropathogenic infections of livestock in Trinidad. Rev. Soc. Bras. . Med. Trop. 34, 29–35. Alves, M., Xiao, L., Sulaiman, I., Lal, A.A., Matos, O., Antunes, F., 2003. Subgenotype analysis of Cryptosporidium isolates from humans, cattle, and zoo ruminants in Portugal. J. Clin. Microbiol. 41, 2744–2747. Alves, M., Xiao, L., Antunes, F., Matos, O., 2006. Distribution of Cryptosporidium subtypes in humans and domestic and wild ruminants in Portugal. Parasitol. Res. 99, 287–292. Bornay-Llinares, F.J., da Silva, A.J., Moura, I.N.S., Przemyslaw, M., Pietkiewicz, H., Kruminis-Lozowska, W., Graczyk, T.K., Pieniazek, N.J., 1999. Identification of Cryptosporidium felis in a cow by morphologic and molecular methods. Appl. Environ. Microbiol. 65, 1455–1458. Brook, E.J., Anthony Hart, C., French, N.P., Christley, R.M., 2008. Molecular epidemiology of Cryptosporidium subtypes in cattle in England. Vet. J., doi:10.1016/j.tvjl.2007.10.023
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