Veterinary Parasitology 175 (2011) 2026

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The potential for zoonotic transmission of Giardia duodenalis andCryptosporidium spp. from beef and dairy cattle in Ontario, Canada

Brent Dixona,, Lorna Parringtona, Angela Cookb, Katarina Pintarb,Frank Poa Bureau of Mib C-EnterNet, Lc Department o

a r t i c l

Article history:Received 3 JunReceived in reAccepted 29 S

Keywords:Giardia duodenCryptosporidiuDairy cattleBeef cattleGenotypeZoonotic

CorresponResearch CentOntario, Canad

E-mail add

0304-4017/$ doi:10.1016/j.llarib, David Keltonc, Jeffrey Farbera

crobial Hazards, Health Canada, 251 Sir Frederick Banting Driveway, P.L. 2204E, Ottawa, Ontario, Canada K1A 0K9aboratory for Foodborne Zoonoses, Public Health Agency of Canada, 120-255 Woodlawn Road West, Guelph, Ontario, Canada N1H 8J1f Population Medicine, Ontario Veterinary College, University of Guelph, 50 Stone Road, Guelph, Ontario, Canada N1G 2W1

e i n f o

e 2010vised form 1 September 2010eptember 2010

alism

a b s t r a c t

The objective of this study was to compare the occurrence and the genotypes and speciesof Giardia duodenalis and Cryptosporidium spp. in beef and dairy cattle from farms in theRegional Municipality of Waterloo, Ontario, in an effort to determine the potential forzoonotic transmission from these animals. Pooled manure samples were collected from 45dairy cattle farms and 30 beef cattle farms. The presence of Giardia cysts and Cryptosporid-ium oocysts was determined by immunouorescence microscopy, while nested-PCR andDNA sequencing were used to determine genotypes and species. The overall farm preva-lence was very high for both Giardia and Cryptosporidium, and was similar for dairy cattlefarms (96 and 64%, respectively) and beef cattle farms (97 and 63%, respectively). However,on dairy cattle farms, G. duodenalis and Cryptosporidium spp. were detected in 44% and 6%of total pooled pen manure samples, respectively, with the occurrence of both parasitesbeing generally higher in calves than in older animals. Most Giardia isolates were identiedas either the host-adapted genotype G. duodenalis Assemblage E or the zoonotic Assem-blage B. Cryptosporidium parvum and Cryptosporidium andersoni were the most frequentlyidentied species in dairy cattle, while the non-zoonotic species Cryptosporidium ryanaeand Cryptosporidium bovis were also found. On beef cattle farms, 72% and 27% of the totalpooled pen manure samples were positive for Giardia and Cryptosporidium, respectively,with no obvious correlation with age. All Giardia isolates in beef cattle were identied asG. duodenalis Assemblage E, while all Cryptosporidium isolates were identied by sequenceanalysis as C. andersoni, although microscopic analyses, and subsequent restriction frag-ment length polymorphism analyses, indicated that other Cryptosporidium species werealso present. The results of this study indicate that although Giardia and Cryptosporidiumwere identied in a higher overall percentage of the pooled beef cattle manure samplesthan in dairy cattle, rmly established zoonotic genotypes and species were much morecommon in dairy cattle than in beef cattle in this region. Dairy cattle, and especially dairycalves, may, therefore, pose a greater risk of infection to humans than beef cattle. How-ever, these results may also provide evidence of potential zooanthroponotic transmission(human to animal).

Crown Copyright 2010 Published by Elsevier B.V. All rights reserved.

ding author at: Microbiology Research Division, Bantingre, 251 Sir Frederick Banting Driveway, P.L. 2204E, Ottawa,a K1A 0K9. Tel.: +1 613 957 0904; fax: +1 613 941 0280.ress: Brent.Dixon@hc-sc.gc.ca (B. Dixon).

1. Introduction

Giardia duodenalis and Cryptosporidium spp. are com-mon protozoan parasites responsible for enteric illnessin humans and animals worldwide. Human transmis-

see front matter. Crown Copyright 2010 Published by Elsevier B.V. All rights reserved.vetpar.2010.09.032/ locate /vetpar

B. Dixon et al. / Veterinary Parasitology 175 (2011) 2026 21

sion is predominantly through the fecaloral route(person-to-person) and contaminated water (drinking andrecreational). However, there has also been consider-able interest and discussion surrounding the potentialfor zoonotlarly from lHunter andOHandley,2008). Trandirect contating zoos, osurface wat

A high phas been remajority ofcalves, inwally been rereporting aand Cryptos2008, 2009reported pr73% for G. dtosporidiumTrotz-Willithe U.S., Troand genotydairy calveswidely varyin pre-weapost-weanebetween 7.in the U.S. (et al., 2005eral studiesinfections ireported aCanada, Gia(Coklin et astudies repspp. in thesin dairy cowranged fromal., 2007).

In beefcumulativepoint prevastrated Gia(Olsonet alMcAllisterprevalencebeef cattle (and Waldnelative Cryptin Alberta, Cshown a relcalves of b1997b; McAand betweeOne study,tosporidiumcalves (Faye

ium in adult beef cattle has been reported to be between0.6and 28.9% in North America (Olson et al., 1997b; Atwill etal., 1999, 2003; McAllister et al., 2005; Gow and Waldner,2006).

ree dibeenhost rd livettle. Aenalis g, butan Ke, 2007erousblage

000; vt et al.,), althorevale, suggprevior prevum, warly, Sthe zos thanimpottle hosporiparvumthe mas it iotherryptosenotyosporian cases of Cc wis andrsoni in, 2004,; Keshe majn studhave

data arstudietypes an bothple, Gof Gia

lgium,on ofs in tho detepeciesdairyaterloowithgens.ic transmission of these pathogens, particu-ivestock (Thompson, 2004; Olson et al., 2004;Thompson, 2005; OHandley and Olson, 2006;2007; Xiao and Feng, 2008; Xiao and Fayer,smission of this type may occur through eitherct in the case of farmers, veterinarians, and pet-r through indirect routes such as contaminateder or foods (Dixon, 2009).revalence of both Giardia and Cryptosporidiumported worldwide in dairy and beef cattle. Thethese studies, however, have involved dairy

hich the prevalence of both parasites has gener-ported to be very high,with a number of studies100% cumulative prevalence of both Giardiaporidium (OHandley et al., 1999; Santn et al.,; Coklin et al., 2010). Studies in Canada haveevalences in dairy calves of between 45.1 anduodenalis, and between 6.2 and 40.6% for Cryp-spp. (Olsonet al., 1997a;OHandley et al., 2000;

ams et al., 2005; Coklin et al., 2007, 2009). Inut et al. (2004, 2005) examined the prevalencepes of G. duodenalis in pre- and post-weanedfrom several states. These authors reported a

ingprevalence of 993%,with an averageof 40%ned calves and an overall prevalence of 52% ind calves. Cryptosporidium spp. prevalences of5% and 49% have been reported in dairy calvesGarber et al., 1994; Santn et al., 2004; Nydam; Starkey et al., 2005; Fayer et al., 2010). Sev-have examined Cryptosporidium and Giardia

n adult dairy cattle in North America, and havegenerally lower prevalence than in calves. Inrdia prevalence in cows was reported as 28.3%l., 2007) and 49% (Uehlinger et al., 2006). Bothorted a complete absence of Cryptosporidiume animals. In the U.S., the prevalence of Giardias ranged from 0 to 64%, while Cryptosporidium5.7 to 12.5% (Fayer et al., 2000, 2007; Trout et

calves, Ralston et al. (2003) reported a 100%prevalence of Giardia in Alberta, Canada, whilelence studies in North America have demon-rdia infection rates of between 22.6 and 37.3%., 1997b; Fayer et al., 2000;Appelbee et al., 2003;et al., 2005; Gow and Waldner, 2006). Giardiarates of 8.7 to 17% have been reported in adultOlson et al., 1997b; McAllister et al., 2005; Gowr, 2006). Ralston et al. (2003) reported a cumu-osporidium prevalence of only 5% in beef calvesanada. Point prevalence studies have similarlyatively low Cryptosporidium prevalence in beefetween 3.1 and 15% in Canada (Olson et al.,llister et al., 2005; Gow and Waldner, 2006),n 0 and 13% in California (Atwill et al., 1999).however, demonstrated a relatively high Cryp-prevalence of 28.8% in 79-month-old beefr et al., 2000). The prevalence of Cryptosporid-

Thhavewidehoofein caduodrangetle (vet al.NumAssemal., 2Trou2006ble pcattlethanhigheBelgiSimilwithcalvebe an

CaCryptiumto besion,threeandClike gCrypthumspecispecicalveandeet al.2007

ThzatiocattleLessa fewgenospp. iexamlenceinBemissicalvewas tand sbothof Wriskspathofferent molecular assemblages of G. duodenalisreported in cattle. Assemblage A, which has aange including humans, and the host adaptedstock Assemblage E, are frequently recognizedssemblage B, which represents the second G.enotype found in humans, also has a wide host

has only more recently been identied in cat-ulen et al., 2002; Lalle et al., 2005; Mendonca; Coklin et al., 2007; Winkworth et al., 2008).studies have reported a higher prevalence ofE in cattle than Assemblage A (OHandley et

an Keulen et al., 2002; Appelbee et al., 2003;2004, 2005, 2006, 2007; OHandley and Olson,ugh Uehlinger et al. (2006) reported compara-nce rates for both Assemblages in adult dairyesting a greater risk of zoonotic transmissionusly thought. Geurden et al. (2008) reported aalence of Assemblage A than E in dairy calves inhile Assemblage E predominated in beef calves.antn et al. (2009) demonstrated that infectionsonotic Assemblage A are more common in dairypreviously reported, suggesting that calvesmayrtant source of human infection.ave been reported as primary hosts for fourdium species (Fayer et al., 2008). Cryptosporid-

has a very wide host range and is thoughtost important in terms of zoonotic transmis-

s frequently identied in human cases. Of thespecies found in cattle, Cryptosporidium bovisporidium ryanae (previously knownas the deer-pe) are host specic and non-zoonotic, whiledium andersoni has been reported in only foures (Leoni et al., 2006; Morse et al., 2007). Theryptosporidium found in dairy cattle are age-

th C. parvum highly prevalent in pre-weanedbeing replaced with C. bovis, C. ryanae and C.post-weaned calves and older animals (Santn2008; Fayer et al., 2006, 2007; Thompson et al.,avarz et al., 2009).ority of prevalence and molecular characteri-ies of G. duodenalis and Cryptosporidium spp. ininvolved dairy cattle, and particularly calves.e available with regard to beef cattle, and onlys have directly compared the prevalence, or thend species ofG. duodenalis and Cryptosporidiumdairy and beef cattle from the same region. Foreurden et al. (2008) reported a higher preva-rdia infection in beef calves than in dairy calvesandKvc et al. (2006) lookedat sourcesof trans-Cryptosporidium in both dairy calves and beefe Czech Republic. The objective of this studyrmine the occurrence, as well as the genotypes, of G. duodenalis and Cryptosporidium spp. inand beef cattle from the Regional Municipality, Ontario, in order to determine their relative

regards to the zoonotic transmission of theseThiswork is part of on-going surveillance by the

22 B. Dixon et al. / Veterinary Parasitology 175 (2011) 2026

C-EnterNet Program of the Public Health Agency of Canadathat monitors human enteric illness and enteric pathogenexposure through food animals, retail foods, and water atCanadian sentinel sites.

2. Materia

2.1. Sample

A total ofrom 45 daWaterloo,Othree freshent age groustored man112 pooledcattle farmsMarch 2008and 25 storcollected inthen cappetainers pacshipped tothey were rdays of coll

2.2. Sample

The suc(2006) wasof feces, 35Triton X-10was thorousion was thpreparationied at thidivided int1M sucrose50ml Falcofor 5min. Fupper layerclean tube,and re-centwas decantPBS and usetion.

2.3. Micros

Foreacha microcenlabeledmona-Glo andLouisiana)texed. Thefor 45minbody was wcentrifuginthen pipettre-suspendscope slide,

Eclipse E600 epiuorescence microscope (Nikon CanadaInc. Instruments,Mississauga,Ontario).Whereverpossible,Giardia cysts and Cryptosporidium oocysts were conrmedusing differential interference contrast (DIC) microscopy.

NA ex

tal DNntrate

Missisal of 2as adight a

oteinait. Theand g

e Kit foNA, thr.

iardia

ested-PRNA gon of fscribes of th et aloresisium br

rypto

ested-PRNA gfor CryRNA g010). P2% (w/stainin) analon ofestrict; FengSP-70007). Rrophor

NA se

Aseqenoma 373CalifoMultiSdirectimplitic Com10.3, M1999).ls and methods

collection

f 179 pooled manure samples were collectediry cattle farms in the Regional Municipality ofntario, fromMay toOctober, 2006. At eachvisit,pooled manure samples (n=138), from differ-ps or different areas of the farm, as well as oneure sample (n=41), were collected. Similarly,manure samples were collected from 30 beefin the same region between February 2007 and, with a total of 87 fresh pen manure samplesed manure samples. All manure samples weresterile pre-labeled plastic cups, which were

d and immediately placed into insulated con-ked with ice or cold packs. The samples wereOttawa, Ontario, by overnight courier whereefrigerated and processed within one to threeection.

preparation

rose otation technique of Uehlinger et al.used with the following modication. To 20gml of phosphate buffered saline with 0.01%0 (PBS-TX) pH 7.4 was added, and the slurryghly mixed with an applicator. The suspen-en passed through four layers of gauze. Samplefor the beef manure samples was further mod-s point as follows; ltered suspension waso equal volumes, and layered over 15ml ofsolution (specic gravity 1.13) in two conical

n tubes. Samples were centrifuged at 800 gollowing centrifugation, the interface and theof liquid from each tube was transferred to a

the volume was adjusted to 45ml with PBS-TXrifuged at 1500 g for 10min. The supernatanted and the pellet was re-suspended in 1mld for microscopy and molecular characteriza-

copy

sample, 200l of suspensionwas transferred totrifuge tube. Fluorescein isothiocyanate (FITC)oclonal antibodysolution (50l eachofGiardi-Crypt-a-Glo, Waterborne Inc., New Orleans,was added to the tube, which was then vor-tube was then incubated at room temperaturein the dark. After incubation, the excess anti-ashed by adding 1ml of PBS, vortexing, and

g at 10,000 g for 10min. The supernatant wased off, leaving 100l which was then used tothe pellet. Twenty l was added to a micro-coverslipped, andexaminedat 200onaNikon

2.4. D

ToconceInc.,A totple wovernof prsue Ktube,Tissuthe Dbuffe

2.5. G

N16S rcatias dementCaccitrophethid

2.6. C

N18S rgene18S ral. (2on 1.mide(RFLPcatithe r1999the Hal. (2elect

2.7. D

DNand GusingCity,porebothinal aGenesionHall,traction

A was extracted from each sucrose otationd sample using the DNeasy Tissue Kit (Qiagen

sauga, ON), using a slightly modied protocol.00l of sucrose otation concentrated sam-

ded to 1.5ml microcentrifuge tubes and lysedt 56 C using 180l of lysis buffer and 20lse K (20mg/ml) supplied with the DNeasy Tis-

subsequent lysate was transferred to a newenomic DNA was isolated using the DNeasyllowing manufacturers instructions. To purifye nucleic acid was eluted in 150l of elution

PCR

CR was performed to amplify fragments of theene and the -giardin gene for Giardia. Ampli-ragments of the 16S rRNA gene was performedd in Coklin et al. (2007). Amplication of frag-e-giardin gene was performed as described in. (2002). PCR products were separated by elec-on 1.2% (w/v) agarose gels, and visualized byomide staining.

sporidium PCR

CR was performed to amplify fragments of theene and the heat shock protein-70 (HSP-70)ptosporidium. Amplication of fragments of theene was performed as described in Coklin etCR products were separated by electrophoresisv) agarose gels, and visualized by ethidium bro-g. A restriction fragment lengthpolymorphism

ysis of 18S rRNA-PCR amplicons for the identi-Cryptosporidium species was performed usingion enzymes SspI, VspI, and MboII (Xiao et al.,et al., 2007). RFLP amplication of fragments ofgene was performed as described in Coklin etFLP banding patterns were observed followingesis on 2% (w/v) agarose gels.

quence analysis

uencingwasperformedat theMcGillUniversitye Quebec Innovation Centre, Montreal, Quebec,0xl DNA Analyser (Applied Biosystems, Fosterrnia). PCR products were puried with Milli-creenHTSPCR96-well plates, and sequenced inons using the same PCR primers as for the orig-cations. DNA sequences were aligned using theputer Group sequence analysis package (ver-adison, Wisconsin) or Bioedit (version 7.0.9,

B. Dixon et al. / Veterinary Parasitology 175 (2011) 2026 23

Table 1G. duodenalis and Cryptosporidium spp. in pooled dairy cattle manure.

Sample type (n) Microscopy PCR

Giardia positive (%) Cryptosporidium positive (%) Giardia positive (%) Cryptosporidium positive (%)

Calves (11) 7 (64) 4 (36) 10 (91) 5 (46)Heifers (48) 31 (65) 3 (6) 28 (58) 9 (19)Cows (79) 23 (29) 1 (1) 31 (39) 10 (13)Stored manure (41) 11 (27) 6 (15) 19 (46) 15 (37)Total pens (138) 61 (44) 8 (6) 69 (50) 24 (17)

Table 2G. duodenalis and Cryptosporidium spp. in pooled beef cattle manure.

Sample type

Calves (10)Heifers (18)Steers (47)Cows (4)Bulls (1)Cow/calf (5)Stored manuTotal pens (8

3. Results

Of the 4rdia cysts woocysts wefound to betle farms, GCryptosporionly one faFarm prevapen manuremicroscopy

The ovetosporidiumsamples temicroscopyof G. duodedeterminedtively. Whethe animalsally highescows (Tablefrom the bfor G. duodby microsc(Table 2). Wand Cryptosin dairy capooled man

osporir thans, thereef catved w

ber ofber ofNA seqpooledive poost G

adapteblagezoon

bly, Aslves, wheiferm (50

Table 3Molecular cha

Sample type

Calves (8)Heifers (8)Cows (19)Total (35)(n) Microscopy

Giardia positive (%) Cryptosporidium positive (%)

7 (70) 1 (10)13 (72) 8 (44)38 (81) 14 (30)

0 01 (100) 02 (40) 0

re (25) 10 (40) 4 (16)5) 61 (72) 23 (27)

5 dairy cattle farms sampled in this study, Gia-ere detected on 43 (96%), and Cryptosporidiumre detected on 29 (64%). Only two farms werenegative for both parasites. Of the 30 beef cat-iardia cysts were detected on 29 (97%), and

dium oocysts were detected on 19 (63%), andrm was found to be negative for both parasites.lence was based on the total number of positiveand stored manure samples detected by eitheror PCR.

rall occurrence of G. duodenalis cysts and Cryp-spp. oocysts in the 138 pooled pen manure

sted from the dairy farms, determined by, was 44% and 6%, respectively. The occurrencenalis cysts and Cryptosporidium spp. oocystsbyPCRwas50%and17%of the samples, respec-

CryptOthecalvethe bobsernumnum

Ditiveposittle. Mhost-AssemwhileNotain cabothparvun data were analysed according to the age of, the occurrence of both parasites was gener-

t in calves, followed by heifers and then adult1).Of the85pooledpenmanure samples testedeef cattle farms, 72% and 27% were positiveenalis and Cryptosporidium spp., respectively,opy, and 77% and 28%, respectively, by PCRhile the total pen prevalence for both Giardiaporidium was much higher in beef cattle thanttle, a considerably smaller proportion of theure samples from beef calves were positive for

frequently ipredominaheifers andbovis (13%animals on

Based onpen manurpooled penbeef cattlewhile all Crsequence a

racterization of G. duodenalis in dairy cattle.

(number sequenced) Assemblage E number (%) Assemb

3 (38) 4 (50)5 (63) 3 (38)

12 (63) 5 (26)20 (57) 12 (34)PCR

Giardia positive (%) Cryptosporidium positive (%)

7 (70) 012 (67) 8 (44)42 (89) 16 (34)2 (50) 0

1 (100) 01 (20) 0

10 (40) 7 (28)65 (77) 24 (28)

dium than the pooledmanure fromdairy calves.this lower occurrence ofCryptosporidium in beefe was no obvious correlation between age oftle and the occurrence of either parasite, as wasith the dairy cattle, possibly due to the greaterage categories of beef cattle, and the smallersamples in each.uence data were available for 35 Giardia pos-pen manure samples and 15 Cryptosporidium

oled pen manure samples from the dairy cat-iardia isolates were identied as either thed hoofed livestock genotype G. duodenalisE (57%) or the zoonotic Assemblage B (34%),

otic Assemblage A was less common (9%).semblage B was the most common genotypehile Assemblage E was the most common ins and cows (Table 3). The zoonotic species C.%), as well as C. andersoni (31%), were the most

dentied species in dairy cattle, with C. parvumting in calves, and C. andersoni only present incows. The non-zoonotic species C. ryanae and C.and 6%, respectively), were present in youngerly (Table 4).DNA sequencing of 63 Giardia positive pooled

e samples and 17 Cryptosporidium positivemanure samples, all G. duodenalis isolates inwere identied as Assemblage E (Table 5),yptosporidium spp. isolates were identied bynalysis as C. andersoni (Table 6). Microscopical

lage B number (%) Assemblage A number (%)

1 (13)0

2 (11)3 (9)

24 B. Dixon et al. / Veterinary Parasitology 175 (2011) 2026

Table 4Molecular characterization of Cryptosporidium spp. in dairy cattle.

Sample type (number sequenced) C. parvum number (%) C. andersoni number (%) C. ryanae number (%) C. bovis number (%)

Calves (5) 4 (80) 0 1 (20) 0Heifers (4) a

Cows (6)Total (15)

a One poole

Table 5Molecular cha

Sample type Assemb

Calves (7) 0Heifers (11) 0Steers (41) 0Cows (2) 0Bulls (1) 0Cow/calf (1) 0Total (63) 0

Table 6Molecular cha

Sample type umber (

Calves (1)Heifers (5)Steers (11)Total (17)

a There wer .b C. parvumc C. bovis wa

analysis, hothan C. andsmaller ooc18S rRNA-Pthat, in addthree, and C

PCRbaseunit (16S an-giardin gnalis and Crand beef cacattle manugene identithat the 16thanonege

4. Discussi

The prescomparingG. duodenacattle in thof G. duodedairy cattletosporidiumbut were sfarms. Highin a numbeet al., 19982005; McA

ner, 20, 2009ge into1 (25) 2 (50)3 (50) 3 (50)8 (50) 5 (31)

d heifer sample showed both C. bovis by HSP-70 and C. ryanae by 18S.

racterization of G. duodenalis in beef cattle.

(number sequenced) Assemblage E number (%)

7 (100)11 (100)41 (100)2 (100)1 (100)1 (100)

63 (100)

racterization of Cryptosporidium spp. in beef cattle.

a (number sequenced) C. parvum number (%) C. andersoni n

0 1 (100)0 5 (100)0b 11 (100)0 17 (100)

e no Cryptosporidium positives in samples from cows, bulls or cow/calveswas identied in 3 steers by PCR-RFLP.s identied in 1 steer by PCR-RFLP.

wever, revealed the presence of species otherersoni in some of these samples based on theiryst size. Subsequent PCR-RFLP analysis on the

Waldet al.ing aCR amplicons of eight such samples indicatedition to C. andersoni, C. parvum was present in. bovis was present in one (all steers).d on amplication of a portion of the small sub-d 18S) rRNA genes was more sensitive than theene or the HSP-70 gene for detecting G. duode-yptosporidium spp., respectively, in both dairyttle manure. However, in both dairy and beefre, PCR based on amplication of the -giardined a small number of G. duodenalis positivesS rRNA gene did not, justifying the use of morene in thePCR-baseddetectionof theseparasites.

on

ent study represents one of very few directlythe occurrence and the zoonotic potential oflis and Cryptosporidium spp. in dairy and beefe same region. The overall farm prevalencenalis was very high in this study, with bothand beef cattle farms approaching 100%. Cryp-spp. were less prevalent than G. duodenalis,

till present on the majority of both types offarm prevalences have similarly been reportedr of other studies (Olson et al., 1997a,b; Ruest; Appelbee et al., 2003; Trotz-Williams et al.,llister et al., 2005; Kvc et al., 2006; Gow and

Cryptosporipen manurethose fromto the highcattle. A higthan in daiviously (GeCryptosporiin the pres(OHandleyof reasonshigher prevcalves (Atwal., 2005; O

ZoonoticCryptosporicattle thanparticular,was the mozoonotic spG. duodenalAssemblagepredominanand concludthreat. Simbeef cattleas non-zoo1 (25) 1 (25)0 0

2 (13) 1 (6)

lage B number (%) Assemblage A number (%)

0000000

%) C. ryanae number (%) C. bovis number (%)

0 00 00 0c

0 0

06; Trout et al., 2007; Fayer et al., 2007; Coklin). Based on total pen results, and without tak-

account, the occurrence of G. duodenalis and

dium spp.was considerably higher in thepooledsamples collected from beef cattle farms than

dairy cattle farms, although thiswas due largelyer occurrence of these parasites in older beefher prevalence of G. duodenalis in beef calvesry calves has only rarely been reported pre-urden et al., 2008). The higher occurrence ofdium spp. in dairy calves than in beef calvesent study is in agreement with other studiesand Olson, 2006; Kvc et al., 2006). A varietyhave been put forth to explain this generallyalence of Giardia and Cryptosporidium in dairyill et al., 1999; Olson et al., 2004; McAllister etHandley, 2007).genotypes and species of G. duodenalis and

dium spp. were much more common in dairyin beef cattle in this region. Dairy calves, inare of concern as the zoonotic Assemblage Bst common genotype of G. duodenalis, and theecies, C. parvum, predominated. In contrast, allis isolates in beef cattle were the non-zoonoticE. Appelbee et al. (2003) similarly reported ace of Assemblage E in beef calves in Alberta,ed that these animals pose a minimal zoonoticilarly, all Cryptosporidium spp. isolates frommanure in the present study were identiednotic. The presence of other species, includ-

B. Dixon et al. / Veterinary Parasitology 175 (2011) 2026 25

ing C. parvum, detected in a small number of samplesby microscopy and PCR-RFLP analysis may have been theresult of the preferential PCR amplication of the predom-inant species, C. andersoni, as has been recently reportedin cattle intion and met al. (2009sion of OHof a risk ofnot commosoni has beet al., 2006;pose someparticular rC. andersonsurface watReports, 20prevalencethe presentas an imposources. Hothese sameidentied t

The occspp. in poothose fromof zoonoticbe a risk of tor petting zgeneral humor oocyst ccultural runto crop lannated wateand speciesdence for tevidence folivestock regenotypes aponotic traconsiderabltosporidiumet al., 19942000; Fayeal., 2003; Bhigh prevalC. parvum,of infectionCoklin et atle may bethrough sewthrough direvidence oonly infectwhereas dahuman conhowever, ifthroponoticthese genoamong thecharacteriz

tosporidium in fecal samples from veterinarians, farmersand other animal handlers would be benecial in provid-ing better insight on the possible transmission dynamics ofthese parasites.

e resuand Creef caidenticattletypes athantherefbeef crovidon, ania and

owled

e autkulurds, Hs also

er verio eldle collgriculgri-Fo

ences

bee, A.J.ce andnada. V, E.R., Josen, Wilips, R.L99. Ageyptospor0425., E.R., Ho03. Impd sporault beefr, K.A., R04. Molns in dstralia., S.M., Dgiardinstrictionodenalis23103, T., Farbar charadairy ca, T., Uehxon, B.Rsporidiuand, Can, T., Farb10. Temardia dulves at a1846., B.R., 20ardia duerres,Mds.), GiaternatioIndia (Paul et al., 2009). Preferential amplica-ixed infections have been discussed by Santn). The results of this study support the conclu-andley (2007) that beef calves may pose lesszoonotic transmission than dairy calves. Whilenly associated with human infections, C. ander-en reported in a small number of cases (LeoniMorse et al., 2007), suggesting that it may alsozoonotic risk. This is of some concern in thisegion of Ontario, as studies have indicated thati is, in fact, the predominant species detected iners (Government of Canada, C-EnterNet Annual062008). This nding corresponds to the highof C. andersoni in both dairy and beef cattle instudy, and provides further evidence for cattle

rtant source of contamination of surface waterwever, in a limited number of samples fromsurface waters, only Giardia microti have been

o date.urrence of G. duodenalis and Cryptosporidiumled dairy cattle manure samples, particularlyyoung animals, along with the high prevalencegenotypes and species, suggests that there mayransmission to farmers, veterinarians, and farmoovisitors bymeans of direct contact, and to thean population in the region through the cyst

ontamination of surface water (through agri-off) or produce (through manure application

ds, or irrigation and processing with contami-r). However, the presence of similar genotypesin cattle and humans is not necessarily evi-

ransmission from cattle to humans, and directr zoonotic transmission from cattle and othermains rather scant (Dixon, 2009). These similarnd speciesmay also be indicative of zooanthro-nsmission (human to animal). While there ise evidence for transmissionofGiardia andCryp-from cow-to-calf or from calf-to-calf (Garber

; OHandley et al., 1999; Faubert and Litvinsky,r et al., 2000; Huetink et al., 2001; Ralston etecher et al., 2004; Gow and Waldner, 2006), aence of zoonoticG. duodenalisAssemblages, andin cattle is also suggestive of a human sourcein these animals (OHandley and Olson, 2006;l., 2007; Geurden et al., 2008), whereby cat-exposed to cysts and oocysts of human originage contaminated drinking water or feed, or

ect contact with infected animal handlers. Asf this, in the present study, beef cattle wereed with non-zoonotic genotypes and species,iry cattle, which generally have much moretact, also had zoonotic isolates. It is not clear,these results are evidence for on-going zooan-transmission or whether, once established,

types and species have continued to circulatese animals. Prevalence testing and molecularation, including subtyping of Giardia and Cryp-

Thnalisand bwerebeefgenocattlemay,thanalso pmissiGiard

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DixonGiPi(EInlts of this study indicate that both G. duode-yptosporidium spp. are very common on dairyttle farms in Ontario. Although both parasitesed in a higher percentage of the total pooledmanure samples than in dairy cattle, zoonoticnd species were much more common in dairy

in beef cattle. Dairy cattle, and especially calves,ore, pose a greater risk of infection to humansattle in this region. However, these results maye evidence of potential zooanthroponotic trans-d further work on the transmission patterns ofCryptosporidium in cattle is warranted.

gements

hors wish to thank Tatjana Coklin, Thembaand Oksana Mykytczuk, Bureau of Microbialealth Canada, for technical assistance. Ourgo to Mollie Campbell, University of Guelph,

cation of the sample collection spreadsheets,technicians from the University of Guelph, forection. The C-EnterNet program was funded byture Policy Framework initiative of Agricultureod Canada.

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, geographic, and temporal distribution of fecal shedding ofidium parvum oocysts in cow-calf herds. Am. J. Vet. Res. 60,

ar, B., Pereira, M.D.G.C., Tate, K.W., Rulofson, F., Nader, G.,roved quantitative estimates of low environmental loadingdic periparturient shedding of Cryptosporidium parvum incattle. Appl. Environ. Microbiol. 69, 46044610.obertson, I.D., Fraser, D.M., Palmer, D.G., Thompson, R.C.A.,ecular epidemiology of Giardia and Cryptosporidium infec-airy calves originating from three sources in WesternVet. Parasitol. 123, 19.e Giacomo, M., Pozio, E., 2002. Sequence analysis of thegene and development of a polymerase chain reaction-fragment length polymorphism assay to genotype Giardiacysts from human faecal samples. Int. J. Parasitol. 32,

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The potential for zoonotic transmission of Giardia duodenalis and Cryptosporidium spp. from beef and dairy cattle in Ontar...IntroductionMaterials and methodsSample collectionSample preparationMicroscopyDNA extractionGiardia PCRCryptosporidium PCRDNA sequence analysis

ResultsDiscussionAcknowledgementsReferences