The prevalence of Cryptosporidium, and identification of the Cryptosporidium horse genotype in foals in New York State

Veterinary Parasitology 174 (2010) 139–144

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The prevalence of Cryptosporidium, and identification of the Cryptosporidium horse genotype in foals in New York State A.J. Burton a,∗ , D.V. Nydam a , T.K. Dearen d , K. Mitchell b , D.D. Bowman c , L. Xiao d a

Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA c Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA d Division of Foodborne, Bacterial, and Mycotic Diseases, National Center for Emerging and Zoonotic Infectious Diseases (proposed), Centers for Disease Control and Prevention, Atlanta, GA 30341, USA b

a r t i c l e

i n f o

Article history: Received 20 March 2010 Received in revised form 16 August 2010 Accepted 16 August 2010 Keywords: Cryptosporidium Cryptosporidiosis Genotype Equine Foal Molecular epidemiology

a b s t r a c t To date, little is known about the prevalence, genotypes and zoonotic potential of Cryptosporidium spp. affecting horses, especially in North America. A cross-sectional study was conducted in New York, USA between February 25th and May 1st 2009. Fecal samples were collected from three hundred and forty nine 1–10-week-old foals and their dams on 14 different broodmare farms. All fecal samples were screened for Cryptosporidium spp. using a direct immunofluorescence assay (DFA). DNA extraction and PCR-RFLP analysis of the small-subunit (SSU) rRNA gene were performed on all the foal samples. PCR-positive samples were subtyped by DNA sequencing of the 60-kDa glycoprotein (gp60) gene. On DFA, 13/175 (7.4%) foal samples and 3/174 (1.7%) mare samples were designated positive for Cryptosporidium spp., whereas on SSU rRNA-based PCR, 9/175 (5.1%) foal samples were positive. Cryptosporidium PCR-positive foals were significantly older (13–40 days, median age of 28 days) compared with negative foals (4–67 days, median 18 days, p = 0.02). The number of foals with diarrhea or soft feces was not significantly different between positive and negative foals (p = 0.09). PCR-RFLP analysis of the SSU rRNA gene and DNA sequencing of the gp60 gene identified the parasite as subtype VIaA14G2 of the horse genotype. This is the first report of a group of foals affected with the Cryptosporidium horse genotype, which has recently been detected in humans. As other contemporary molecular studies have identified C. parvum in foals, it seems that equine cryptosporidiosis should be considered a zoonosis. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Cryptosporidium spp. are apicomplexan parasites that infect mammals, birds, reptiles, amphibians, and fish. In recent years, the development of molecular tools to identify morphologically indistinguishable species/genotypes and subtypes has enabled more precise definition of host specificity, zoonotic potential, and transmission pathways

∗ Corresponding author at: Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA. Tel.: +1 706 542 6389; fax: +1 706 542 8335. E-mail addresses: [email protected], [email protected] (A.J. Burton). 0304-4017/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2010.08.019

of Cryptosporidium spp. (Xiao, 2009). Most zoonotic Cryptosporidium infections in humans are caused by C. parvum, often from handling infected cattle (as pre-weaned calves are commonly infected with C. parvum) or through oocyst contamination of food, water, or fomites (Xiao and Feng, 2008; Trotz-Williams et al., 2006; Reif et al., 1989). The species/genotype of Cryptosporidium isolated from infected humans and animals varies geographically and by host species. For example, with respect to human cryptosporidiosis, in Europe and New Zealand, both C. parvum and C. hominis are commonly implicated. In the Middle East, C. parvum predominates, whereas in the rest of the world, including developing nations, C. hominis is the dominant species (Xiao, 2009).

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Table 1 Cryptosporidium species/genotypes identified in horses worldwide. Location

n

Age group

Genotypes (number horses positive)

Studya

New York state, USA

175

Foals (1–10 weeks)

Present study

Central Italy

150

New Zealand Germany Mid-Wales, UK Czech Republic

67 1 2 2

Foals (0 ≥ 8 weeks) and adults Foals Foal (9 days) Foals (<8 weeks) Prezewalski’s wild horse foals (161 and 12 days)

Cryptosporidium horse genotype (9) C. parvum (12) C. parvum (67) C. parvum (1) C. parvum (2) Cryptosporidium horse genotype (1), C. parvum (1)

Grinberg et al. (2009) Imhasly et al. (2009) Chalmers et al. (2005) Ryan et al. (2003)

a

Veronesi et al. (2009

Only studies where the genotype was confirmed using PCR and sequencing (i.e. not solely a morphologic diagnosis) are included.

There have been two recent molecular epidemiologic studies on Cryptosporidium spp. in groups of horses in New Zealand (Grinberg et al., 2009) and Italy (Veronesi et al., 2009). Nevertheless, relatively little is known about the prevalence, species identity, and public health significance of Cryptosporidium spp. in North American horses. Natural Cryptosporidium infection has been documented in horses, mostly in foals <6 months of age, in Europe (Browning et al., 1991; Majewska et al., 1999; Sturdee et al., 2003; Hajdusek et al., 2004; Chalmers et al., 2005; Imhasly et al., 2009; Veronesi et al., 2009), North America (Cole et al., 1998; Xiao and Herd, 1994), South America (De Souza et al., 2009), the Middle East (Mahdi and Ali, 2002), and New Zealand (Grinberg et al., 2003, 2009). In the few small scale genotyping studies conducted in horses, C. parvum was the most common Cryptosporidium species identified (Table 1) (Grinberg et al., 2003, 2008, 2009; Chalmers et al., 2005; Imhasly et al., 2009; Veronesi et al., 2009). Another Cryptosporidium, the horse genotype, was identified in a Prezewalski’s wild horse foal in the Prague Zoo (Ryan et al., 2003), but the prevalence of this species in domestic horses is not clear. Although the Cryptosporidium horse genotype was initially considered equine-specific (Ryan et al., 2003), it has recently been found in two persons in England and the United States (Robinson et al., 2008; Xiao et al., 2009) and one calf in Northern Ireland (Thompson et al., 2007). Thus, more thorough investigations are needed before we can fully assess zoonotic risk and public health importance of equine Cryptosporidium spp. Here, we conducted a cross-sectional study to assess Cryptosporidium shedding in 349 horses on 14 farms in New York, the distribution of Cryptosporidium species/genotypes in horses, and the public health potential of the infection. 2. Materials and methods 2.1. Sample collection A cross-sectional study was carried out to assess the prevalence of Cryptosporidium spp. in foals in New York. A two-stage design was sampling scheme was employed to select farms and animals enrolled in the study. In the first stage, a group of broodmare farms with large numbers of foals born each year were selected from the New

York Thoroughbred Breeders Association (six farms) and New York Harness Horse Breeders databases (seven farms). The Cornell University Equine Park Warmblood broodmare farm was also used because of convenience. In the second stage, a fixed minimum number of foals and mares from each farm were selected by random sampling. An estimate of the minimum sample size required was calculated based on published data on Cryptosporidium prevalence in horses. It was estimated that approximately 11,250 foals are born per year in New York State (USDA, 2005). The prevalence of Cryptosporidium spp. in non-diarrheic equine populations has been documented to range from 0% to 23.3% (Johnson et al., 1997; Olson et al., 1997; Cole et al., 1998; Majewska et al., 1999; Atwill et al., 2000; Sturdee et al., 2003; Santin et al., 2009; Veronesi et al., 2009; Xiao and Herd, 1994). We used a conservative estimate of 4% for the expected prevalence of Cryptosporidium spp. in foals in New York. Therefore, with an accepted error rate of 3% and a confidence level of 95%, it was determined that the minimum number of fecal samples required for the foals was 169. For each foal sample, a fecal sample was collected from the respective dam, leading to a minimum of 338 total fecal samples. 2.2. Data collection Between February 25th and May 1st 2009, fecal samples were obtained from 14 broodmare farms in various locations in New York State. Rectal fecal samples were taken from foals, whereas rectal or fresh feces from the stall floor were collected for mares. Fecal samples were placed into plain plastic containers, refrigerated for transport and stored at −80 ◦ C prior to diagnostics. For each horse sampled, age, sex and health conditions were documented. A simple fecal score at the time of sampling was recorded for samples from foals, with 1 being normal feces, 2 being soft or loose feces and 3 being overt diarrhea. The study protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at Cornell University. 2.3. Microscopy screening All samples were screened for the presence of Cryptosporidium oocysts by direct immunofluorescence assay (DFA) using the MeriFluor® Cryptosporidium/Giardia kit (Meridian Bioscience, Inc., Cincinnati, OH). A rapid screen-

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Table 2 Demographic data for the mares and foals on 14 different broodmare farms in New York from which fecal samples were obtained.

Total number of fecal samples Number of fecal samples per farm, median (range) Total number of horses on farm at samplinga Age, median (range) Males Females % With fecal score 1b % With fecal score 2b % With fecal score 3b a b †

Adults

Foals

174 10 (5–38) 119 (25–353) 10 (4–31) years 0 174 – – –

175 10 (5–38) 14 (7–170) 18 (4–67) days 84† 89† 90 7 3

Total 349 20 (10–76) 133 (32–523) – 84 263 – – –

Includes stallions, yearlings, weanlings and horses not involved in the breeding operation Fecal score 1 = normal feces, score 2 = soft or loose feces, score 3 = watery diarrhea. There was no significant difference in the number of male versus female foals (p = 0.5).

ing method developed in the laboratory was employed rather than procedures recommended by the manufacturer. Briefly, a thin fecal smear was made on a glass microscope slide using a sterile cotton swab. Seven microliters of MeriFluor® Detection Reagent (FITC labeled anti-Cryptosporidium and anti-Giardia monoclonal antibodies) was immediately placed on top of the fresh fecal smear and covered with a 22 mm2 cover glass. After 1 min, the slide was examined at 20× magnification under a fluorescent microscope. This method was assessed previously in our laboratory (unpublished data) using spiked calf fecal samples containing known quantities of oocysts and was shown to be comparable in sensitivity and specificity with the conventional procedure. 2.4. Molecular analysis Cryptosporidium DNA was extracted from 500 ␮l of fecal suspension as described previously (Jiang et al., 2005a). Genotyping of Cryptosporidium spp. was carried out using PCR-restriction fragment length polymorphism (RFLP) analysis of the small-subunit (SSU) rRNA (Jiang et al., 2005b). The Cryptosporidium was subtyped by DNA sequencing of the 60-kDa glycoprotein (gp60) gene amplified by a nested PCR (Alves et al., 2003). Two microliters of DNA was used in PCR and replicate analyses at each locus were conducted to confirm the results. 2.5. Data analysis Data were analyzed using Statistix 9.0 software (Analytical Software, Tallahassee, FL). Continuous data were non-normally distributed as assessed by the Shapiro-Wilk test. A Wilcoxon Rank Sum test was used to compare sets of continuous data and Fisher’s Exact Test was used for comparison of dichotomous variables. Alpha was set at 0.05. For the apparent prevalence, the 95% confidence interval (C.I.) was calculated as the exact binomial. 3. Results 3.1. Prevalence of Cryptosporidium spp. A summary of the demographic data pertaining to the 349 mares and foals from which fecal samples were obtained is shown in Table 2. Of the foal fecal samples,

13/175 were positive for Cryptosporidium spp. by DFA and 9/175 positive by SSU rRNA PCR (Table 1). Of the mare fecal samples, 3/174 were positive for Cryptosporidium spp. by DFA and 0/174 positive by SSU rRNA PCR. The apparent prevalence of Cryptosporidium spp. on 14 broodmare farms in New York is summarized in Table 3 and also shown in Table 4. Only one foal fecal sample was PCR positive but DFA negative. SSU rRNA PCR was performed on the 3 DFA positive mare fecal samples and yielded negative results. Thus, based on the results from the foals (i.e. most SSU rRNA PCR-positive samples were DFA positive also), SSU rRNA PCR was not pursued on the rest of the DFA negative mare samples. All PCR-positive samples were from foals on one farm. Cryptosporidium PCR-positive foals were significantly older (13–40 days, median age of 28 days) compared with negative foals (4–67 days, median 18 days, p = 0.02). The number of foals with diarrhea or soft feces (fecal score 2 or 3) was not significantly different between positive (3/9, 33.3%) and negative (13/127, 10.2%) foals (p = 0.09). However, there was a trend towards a greater number of fecal scores 2 and 3 in Cryptosporidium positive foals versus negative foals. 3.2. Identity of Cryptosporidium in horses RFLP analysis of the SSU rRNA PCR products from all nine PCR-positive samples produced an SspI and VspI banding suggestive of the presence of the Cryptosporidium horse genotype (Table 1), which differed from the banding pattern of C. parvum by having a smaller upper VspI band (∼500 bp versus greater than 600 bp; Fig. 1). DNA sequencing of two SSU rRNA PCR products each from four positive samples produced ∼800 bp sequences that were identical to those previously obtained from the Cryptosporidium horse genotype. The sequences were identical to sequences obtained from specimens from a Prezewalski’s wild horse in the Czech Republic (FJ435963), a calf in Northern Ireland (FJ435964), a human in New Mexico (FJ435962), a human in England (EU437418), and raw wastewater in Shanghai (FJ153238), with the only exception of the presence of the previously observed “AG” to “GA” or “GG” substitutions (Xiao et al., 2009) in one of the samples. PCR amplified the gp60 gene in 7/9 SSU rRNA PCR positive samples. DNA sequencing of the gp60 gene produced ∼830 bp sequences that were identical to each other. They

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Table 3 Prevalence of Cryptosporidium spp. in mares and foals on 14 different broodmare farms in New York.

Number of horses positive by DFA % (95% C.I.) of horses positive by DFA Number of horses positives by PCR % (95% C.I.) of horses positive by PCR Number of farms with positives by DFA (total farms = 14) Number of farms with positives by PCR (total farms = 14)

Adults (n = 174)

Foals (n = 175)

Total (n = 349)

3 1.7 (0.4–5) 0 0 (0–2.1) 2 0

13 7.4 (4–12.4) 9 5.1 (2.4–9.5) 3 1

16 4.6 (2.6–7.3) 9 2.6 (1.2–4.8) 3 1

were also almost identical to those obtained from the VIa subtype family of the Cryptosporidium horse genotype found in a Prezewalski’s wild horse in the Czech Republic (FJ435960) and a calf in Northern Ireland (DQ648547), with one nucleotide substitution each in the non-repeat region. The sequences obtained in this study had 14 copies of the TCA repeat and two copies of the TCG repeat, thus was named the VIaA14G2 subtype, comparing to the VIaA14G4 in the calf isolate and VIaA11G3 in the Prezewalski’s wild horse. In contrast, the two human isolates from England (Robinson et al., 2008) and New Mexico (Xiao et al., 2009) belonged to the same subtype, VIbA13, of a different subtype family of the Cryptosporidium horse genotype, VIb. 4. Discussion The prevalence of Cryptosporidium spp. in foals (sample population predominantly Standardbreds and Thoroughbreds) in New York State appears to be relatively low (5.1%). Table 4 summarizes the prevalence and age patterns of Cryptosporidium spp. in other published studies of various equine populations. It is difficult to draw firm conclusions regarding the most susceptible age, as the studies often targeted different age groups. However, foals are generally more frequently infected than adults, in agreement with our findings. It should be noted that all the PCRpositive samples in our study originated from one farm,

which suggests the possible occurrence of an outbreak of cryptosporidiosis on that particular farm. Whether this outbreak was of clinical significance is unknown, especially as there was no association between Cryptosporidium status and fecal score. Minor discrepancies between DFA and PCR detection were observed. It is difficult to determine whether the fecal samples that were diagnosed positive by DFA but not by PCR were false negatives on PCR or false positives on DFA. However, it should be noted that the oocyst counts in the DFA positive, PCR negative samples were all very low (<5 per field of view). Therefore, other structures might have been mistaken for oocysts when no counter stain was used, even though our modified DFA was of similar sensitivity and specificity to the method detailed in the MeriFluor® Cryptosporidium/Giardia kit (Meridian Bioscience, Inc., Cincinnati, OH) instructions. The pathogenicity of Cryptosporidium spp. in foals is not clear. Some studies, including the present one, have found no association between oocyst shedding and diarrhea in foals (Xiao and Herd, 1994; Veronesi et al., 2009). Equine cryptosporidial diarrhea was first documented in immunodeficient foals (Snyder et al., 1978) and has traditionally been associated with this syndrome (Mair et al., 1990; Bjorneby et al., 1991; Perryman and Bjorneby, 1991). However, one attempt to produce disease in neonatal normal, and colostrum deprived foals using Cryptosporidium spp. isolates from calves was unsuccessful (Tzipori, 1983).

Table 4 Reported prevalence of Cryptosporidium spp. in different equine populations. Location

n

Age range

Prevalence (%)a

Studyb

New York state, USA New York State, USA Central Italy, Europe Central Italy, Europe Central Italy, Europe Brazil, S. America W. Poland, Europe Warwickshire, UK Iraq, Middle East Sierra Nevada, USA Texas, USA Texas, USA Alberta, Ontario and Yukon, Canada Alberta, Ontario and Yukon, Canada California, USA Kentucky, USA Kentucky, USA Ohio, USA Ohio, USA Louisiana, USA

175 174 90 30 30 396 318 80 25 305 366 40 10 24 90 74 50 42 55 57

Foals, 1–10 weeks Adults (mares) Foals, 0–8 weeks Foals >8 weeks Adults (mares) Adults Adults Adults Adults Adults Adults Foals, 10–21 days Adults Foals <6 months Adults Adults >1 year Foals and weanlings Adults >1 year Foals and weanlings Foals

2.5 0–2.1 4.4 23.3 3.3 0.8 3.5 6.4 12 0–2.3 0–10.4 1–15 10 21 0–3.2 1.3 18 0 10.9 14

Present study Present study Veronesi et al. (2009) Veronesi et al. (2009) Veronesi et al. (2009) De Souza et al. (2009) Majewska et al. (1999) Sturdee et al. (2003) Mahdi and Ali (2002) Atwill et al. (2000) Cole et al. (1998) Cole et al. (1998) Olson et al. (1997) Olson et al. (1997) Johnson et al. (1997) Xiao and Herd (1994) Xiao and Herd (1994) Xiao and Herd (1994) Xiao and Herd (1994) Coleman et al. (1989)

a

Reported prevalence varies between apparent prevalence (AP) and true prevalence (TP) depending on the individual study methodology. Studies that only examined diarrheic animals are excluded as these do not represent a ‘normal’ equine population and are therefore not comparable to our study. b

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Fig. 1. Differentiation of the Cryptosporidium horse genotype and C. parvum by RFLP analysis of the PCR products of the SSU rRNA gene using restriction enzymes SspI (left panel) and VspI (right panel). Lanes 1 and 2, the Cryptosporidium horse genotype from samples; lane 3, C. parvum as positive control; lane 4, 100-bp molecular markers.

Contrastingly, there have also been reports of diarrhea with Cryptosporidium spp. infection in apparently immunocompetant foals (Grinberg et al., 2003; Netherwood et al., 1996). In the light of these conflicting reports and accumulating molecular epidemiological data, it seems likely that as with many organisms, the pathogenicity of Cryptosporidium spp. depends both on the genetic background and immune status of the animals and the virulence of the specific genotypes and subtypes involved. In addition, as with calves, Cryptosporidium infection when present as a co-infection with other enteric pathogens such as rotavirus, Salmonella or Clostridium perfringens may well compound the severity of gastrointestinal damage, inflammation and diarrhea. Although the most important zoonotic Cryptosporidium species, C. parvum, was not found in horses in this study, another human-pathogenic Cryptosporidium, the horse genotype, was shown to be present on one farm. The Cryptosporidium horse genotype has recently been found in one person with diarrhea in rural SW England (Robinson et al., 2008) and one severely ill pet store worker in New Mexico (Xiao et al., 2009), suggesting that the Cryptosporidium horse genotype may be virulent in humans. Both patients were immunocompetant adults (30 and 18 years of age, respectively), and at least one had no contact with horses. The Cryptosporidium horse genotype is known to infect horses, however, one human patient had contact with other animals but not horses, hence the genotype may have broader host specificity than the name implies. Therefore, this parasite may present a public health threat in areas where it is prevalent in animals and when environment favors its transport and dispersion. Because two human-pathogenic species have been seen in horses, Cryptosporidium shedding in equids (be it due to the Cryptosporidium horse genotype or C. parvum) should be considered a zoonotic risk.

Acknowledgements This study was supported in part by the Cornell University Clinical Fellowship (A.J. Burton). The authors wish to thank G. Perkins, L. Bookbinder, A. Prentice, T. Linden and E. Johnson for assistance with sample collection and the broodmare farms in New York State for access to the animals. The findings and conclusions in this manuscript are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention. References 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. Atwill, E.R., McDonald, N.K., Perea, L., 2000. Cross-sectional study of faecal shedding of Giardia duodenalis and Cryptosporidium parvum among packstock in the Sierra Nevada Range. Equine Vet. J. 32, 247–252. Bjorneby, J.M., Leach, D.R., Perryman, L.E., 1991. Persistent cryptosporidiosis in horses with severe combined immunodeficiency. Infect. Immun. 59, 3823–3826. Browning, G.F., Chalmers, R.M., Snodgrass, D.R., Batt, R.M., Hart, C.A., Ormarod, S.E., Leadon, D., Stoneham, S.J., Rossdale, P.D., 1991. The prevalence of enteric pathogens in diarrhoeic thoroughbred foals in Britain and Ireland. Equine Vet. J. 23, 405–409. Chalmers, R.M., Thomas, A.L., Butler, B.A., Morel, M.C., 2005. Identification of Cryptosporidium parvum genotype 2 in domestic horses. Vet. Rec. 156, 49–50. Cole, D.J., Cohen, N.D., Snowden, K., Smith, R., 1998. Prevalence of and risk factors for fecal shedding of Cryptosporidium parvum oocysts in horses. J. Am. Vet. Med. Assoc. 213, 1296–1302. Coleman, S.U., Klei, T.R., French, D.D., Chapman, M.R., Corstvet, R.E., 1989. Prevalence of Cryptosporidium sp in equids in Louisiana. Am. J. Vet. Res. 50, 575–577. De Souza, P.N., Bomfim, T.C., Huber, F., Abboud, L.C., Gomes, R.S., 2009. Natural infection by Cryptosporidium sp., Giardia sp. and Eimeria leuckarti in three groups of equines with different handlings in Rio de Janeiro, Brazil. Vet. Parasitol. 160, 327–333. Grinberg, A., Pomroy, W.E., Carslake, H.B., Shi, Y., Gibson, I.R., Drayton, B.M., 2009. A study of neonatal cryptosporidiosis of foals in New Zealand. N.Z. Vet. J. 57, 284–289.

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