Longitudinal investigation of protozoan parasites in meat lamb farms in southern Western Australia.

Abstract

In this study, 96 faecal samples were collected from pregnant Merino ewes, at two broad-acre, commercial sheep farms in southern Western Australia, on two separate occasions (16 and 2 weeks prior to lambing). Following lambing, 111 (Farm A) and 124 (Farm B) female crossbred lambs (2-6 weeks old), were individually identified using ear tags (a numbered tag and a radio-frequency tag). A total of 1155 faecal samples were collected only from these individually identified lambs on five separate sampling occasions. All samples were screened using PCR to detect Cryptosporidium (18S rRNA and actin loci) and Giardia duodenalis (glutamate dehydrogenase and triosephosphate isomerise loci). The overall prevalences (lambs positive for a parasite on at least one of the five samplings) at Farm A and B were 81.3% and 71.4%, respectively for Cryptosporidium and similarly 67.3% and 60.5% for Giardia, respectively. Cryptosporidium and Giardia prevalences at individual samplings ranged between 18.5 and 42.6% in lambs and were <10% in the ewes. Cryptosporidium xiaoi was the most prevalent species detected at all five samplings and was also isolated from lamb dam water on Farm B. Cryptosporidium ubiquitum was most commonly detected in younger lambs and Cryptosporidium parvum was detected in lambs at all five samplings, typically in older lambs and as part of a mixed species infection with C. xiaoi. A novel, possibly new genotype (sheep genotype I), was identified in six Cryptosporidium isolates from Farm B. Giardia duodenalis assemblage E was the most common genotype detected at all five samplings, with greater proportions of assemblage A and mixed assemblage A and E infections identified in older lambs. This longitudinal study identified high overall prevalences of Cryptosporidium and Giardia in lambs grazed extensively on pastures, while reinforcing that sampling a random selection of animals from a flock/herd on one occasion (point prevalence), underestimates the overall prevalence of these parasites in the flock/herd across an extended time period. Based on these findings, grazing lambs were identified as a low risk source of zoonotic Cryptosporidium and Giardia species/genotypes, with these protozoa detected at all five samplings in some lambs, indicating that these individuals were either unable to clear the naturally acquired protozoan infections or were repeatedly re-infected from their environment or other flock members.

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MURDOCH RESEARCH REPOSITORY
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Sweeny, J.P.A. , Ryan, U.M. , Robertson, I.D. , Yang, R. , Bell, K. and Jacobson, C. (2011) Longitudinal
investigation of protozoan parasites in meat lamb farms in southern Western Australia. Preventive
Veterinary Medicine, 101 (3-4). pp. 192-203.
http://researchrepository.murdoch.edu.au/4881
Copyright © 2011 Elsevier B.V.
It is posted here for your personal use. No further distribution is permitted.

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Longitudinal investigation of protozoan parasites in meat lamb farms in southern
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Western Australia.
2

3
Joshua P. A. Sweenya * U. M. Ryana, I.D. Robertsona, R. Yanga, K. Bella, and C. 4
Jacobsona. 5
6
a School of Veterinary and Biomedical Sciences, Murdoch University, Western Australia, 7
6150, Australia. 8
9
* Corresponding Author:
10
Email: J.Sweeny@murdoch.edu.au 11
Phone: +61 9 9360 2495 12
Fax: +61 9 9360 7390 13

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Abstract
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14
In this study, 96 faecal samples were collected from pregnant Merino ewes, at two broad- 15
acre, commercial sheep farms in southern Western Australia, on two separate occasions 16
(16 and 2 weeks prior to lambing). Following lambing, 111 (Farm A) and 124 (Farm B) 17
female crossbred lambs (2 – 6 weeks old), were individually identified using ear tags (a 18
numbered tag and a radio-frequency tag). A total of 1,155 faecal samples were collected 19
only from these individually identified lambs on five separate sampling occasions. All 20
samples were screened using PCR to detect Cryptosporidium (18S rRNA and actin loci) 21
and Giardia duodenalis (glutamate dehydrogenase and triosephosphate isomerise loci). 22
The overall prevalences (lambs positive for a parasite on at least one of the five 23
samplings) at Farm A and B were 81.3% and 71.4%, respectively for Cryptosporidium and 24
similarly 67.3% and 60.5% for Giardia, respectively. Cryptosporidium and Giardia 25
prevalences at individual samplings ranged between 18.5 – 42.6% in lambs and were 26
<10% in the ewes. Cryptosporidium xiaoi was the most prevalent species detected at all 27
five samplings and was also isolated from lamb dam water on Farm B. Cryptosporidium 28
ubiquitum was most commonly detected in younger lambs and C. parvum was detected in 29
lambs at all five samplings, typically in older lambs and as part of a mixed species infection 30
with C. xiaoi. A novel, possibly new genotype (sheep genotype I), was identified in six 31
Cryptosporidium isolates from Farm B. Giardia duodenalis assemblage E was the most 32
common genotype detected at all five samplings, with greater proportions of assemblage A 33
and mixed assemblage A and E infections identified in older lambs. This longitudinal study 34
identified high overall prevalences of Cryptosporidium and Giardia in lambs grazed 35
extensively on pastures, whilst reinforcing that sampling a random selection of animals 36
from a flock/herd on one occasion (point prevalence), underestimates the overall 37
prevalence of these parasites in the flock/herd across an extended time period. Based on 38
these findings, grazing lambs were identified as a low risk source of zoonotic 39

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Cryptosporidium and Giardia species/genotypes, with these protozoa detected at all five
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40
samplings in some lambs, indicating that these individuals were either unable to clear the 41
naturally acquired protozoan infections or were repeatedly re-infected from their 42
environment or other flock members. 43
Keywords: Sheep; Cryptosporidium; Giardia; water; zoonotic; prevalence. 44
45

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1. Introduction
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The intestinal protozoan parasites, Cryptosporidium and Giardia, have the capacity 47
to infect a wide variety of animals, including humans. There is an increasing interest in the 48
prevalence of these parasites in ruminant livestock industries, due to the potential risk they 49
present with respect to the contamination of human drinking water (Zhou et al., 2003; Goh 50
et al., 2004; McCarthy et al., 2008) and their possible impacts on productivity of infected 51
animals. 52
Several studies in sheep have determined point prevalence of these protozoa at a 53
single time period by sampling a random selection of lambs or sheep within a flock at a 54
specific time (Geurden et al., 2008b; Gómez-Muñoz et al., 2009; Yang et al., 2009; Nolan 55
et al., 2010; Robertson et al., 2010; Wang et al., 2010), in diarrhoeic lambs (Aloisio et al., 56
2006) and sheep in lairage (livestock holding facilities before slaughter) (Ryan et al., 57
2005). However, determination of prevalence at one sampling, may not provide a true 58
indication of the overall prevalence in flocks over an extended period of time. Reported 59
sporadic (oo)cyst excretion by these protozoa, requires multiple faecal samples to be 60
collected for an accurate diagnosis (O'Handley et al., 1999; Thompson et al., 2008), 61
therefore overall prevalence is potentially underestimated with only one sampling. 62
Despite this knowledge of sporadic (oo)cyst excretion, there has been little research 63
into overall protozoa prevalences and varying species/genotypes demographics in 64
individual sheep over an extended time period. Santin et al., (2007) sampled 2–6 year old 65
ewes (n=32) and their lambs (n=31) over one month in a barn environment. Robertson et 66
al., (2010) described individual prevalences and different species/genotypes of these 67
protozoa isolated from randomly sampled lambs, from 6 Norwegian sheep flocks at two 68
samplings. However, there is no information available regarding Cryptosporidium and 69
Giardia overall prevalences (tracking individually identified lambs across more than two 70

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separate samplings) in young lambs reared under extensive, broad-acre grazing
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conditions (agriculture practiced on a large scale, whereby extensive areas of land are 72
used for the production of crops or the grazing of livestock at low stocking densities for 73
meat, wool or milk production, with animals not housed indoors). 74
The aim of this longitudinal study was to investigate the overall prevalences and 75
variation in species/genotype demographics of Cryptosporidium and Giardia in pregnant 76
Merino ewes and their cross-bred lambs. These parasites were detected by screening 77
individual faecal DNA extracts from ewe and lamb faecal samples using PCR. All sheep 78
and lambs were managed under extensive, broad-acre grazing conditions at two separate 79
commercial sheep enterprises in southern Western Australia. 80
81
2. Materials and Methods
82
2.1 Study sites, animals and experimental protocol 83
This experiment was approved and supervised by the Murdoch University Animal 84
Ethics Committee (permit R2236/09). The two sheep farms in this study were located at 85
Pingelly (Farm A) and Arthur River (Farm B), both approximately 200–250km south-east of 86
Perth and separated from one another by approximately 150km. These sheep farms are 87
located in southern Western Australia and experience a Mediterranean environment (hot, 88
dry summers and cool, wet winters) with a predominantly winter rainfall pattern (Hill et al., 89
2004; Moeller et al., 2008). Both farms have similar average annual rainfall, ranging 90
between 450–500mm, with winter stocking rates measured using dry sheep equivalents 91
per hectare (DSE/ha) (McLaren, 1997), as shown in Table 1. A general overview of all 92
lamb samplings and time progression throughout the study for each farm is shown in Table 93
2. 94

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On each farming property, flocks of 2–4 year old Merino ewes were joined with 95
Suffolk rams in January/February 2009. On each of the two farms, 48 faecal samples were 96
collected from the same pregnant ewes on two occasions, firstly within four months of the 97
commencement of lambing and secondly within two weeks of the commencement of 98
lambing (total 192 samples; 96 from each farm). 99
When lambs were approximately 2–6 weeks old (day 0 of study), 111 female lambs 100
from Farm A and 124 female lambs from Farm B were randomly selected and identified 101
with a uniquely numbered ear tag and a radio-frequency ear tag. Faeces were collected 102
directly from the rectum of only these identified female lambs, such that faecal samples 103
were collected from the same lambs at five separate samplings, between the first (2–6 104
weeks of age) and final (7–8 months of age) samplings (Table 2). A total 107 and 119 105
lambs from Farm A and B respectively, were sampled at all five samplings and overall 106
parasite prevalences were determined from only these lambs. Each flock was mustered 107
from their paddock into nearby yards for faecal sampling, except for the final sampling, 108
which took place in lairage following the transport of lambs by road to the abattoir from 109
their respective farms. 110
Faecal samples were collected from only the identified lambs in each flock using 111
fresh latex gloves to prevent cross contamination between faecal samples. All faecal 112
samples were placed in individually labelled, airtight 70mL containers and transported to 113
the laboratory within six hours of collection. Faecal samples were stored at 2–4oC and 114
genomic DNA was extracted from each sample within seven days of collection. The 115
transport and storage practices utilised in this study were consistent with other similar 116
studies that used PCR to detect these parasites (Yang et al., 2009; Ng et al., 2010b; 117
Robertson et al., 2010). Lambs on Farm A were consigned for slaughter in two separate 118

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groups, the first on day 199 and the second group on day 240 of the study. Lambs from
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119
Farm B were consigned for slaughter as a single group on day 188 of the study. 120
On each farm, lambs were born and raised on one annual pasture paddock 121
consisting primarily of annual rye-grass (Lolium spp.) and sub-terraneum clover (Trifolium 122
subterraneum). Water was supplied ad libitum via a creek (Farm A), dam (Farm B) or by 123
troughs filled from one of these water sources. Approximately 100g/head/day of a 124
supplementary feed grain mixture (comprising of 35% lupins and 65% oats) was given to 125
each lamb flock after they had been weaned. On each farm lambs were treated with 12mg 126
Abamectin and 6mg selenium (Virbamec Oral Plus Selenium, Virbac Australia) before 127
weaning, on days 39 and 73 of the study for Farm A and B respectively (Table 2 and 3). 128
2.2 Genomic DNA extraction 129
A total of 1,347 faecal samples (192 faecal samples from pregnant ewes and 1,155 130
rectal faecal samples from tagged identified lambs) were collected. Genomic DNA was 131
extracted from 250–300mg of each faecal sample using a Power Soil DNA Kit (MolBio, 132
West Carlsbad, California, USA). Minor modifications to the manufacturer’s protocol were
133
made. Briefly, samples were subjected to five cycles of freeze-thaw (freezing each sample 134
with liquid nitrogen for 4 minutes and then thawing samples at 90oC for 4 minutes). The 135
final elution volume of C6 solution (MolBio) was adjusted to 50µL from the recommended 136
100µL, in order to increase the final DNA concentration. After elution, DNA was stored at –
137
20oC until use. A negative control (no faecal sample) and positive control (faecal sample 138
spiked with Cryptosporidium and Giardia (oo)cysts) were used in each faecal extraction 139
group. 140
A 10L water sample was collected from each flock’s drinking water source on the 141
second and third (pre-weaning I and II) samplings. Flock A drank water from a flowing 142

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creek with water soaks constructed to trap water, while Flock B drank from a clay dam.
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143
Using Envirochek filters (Pall Life Sciences, New South Wales, Australia), filtration of the 144
water samples was performed in accordance with the manufacturer’s instructions, except
145
for some slight modifications as described by Wohlsen et al., (2004). The eluate was 146
subjected to the same genomic DNA extraction method detailed above. 147
2.3 PCR amplification 148
A spike analysis was performed for all PCRs detailed below, in order to determine if 149
there were any PCR inhibitors present in the faecal DNA extracts, which would prevent 150
accurate detection of the protozoan parasites. This was conducted by spiking PCR 151
reactions with 0.5µL of the respective positive controls, with no inhibition detected in any of 152
the samples screened by PCR in this present study. All faecal and genomic livestock water 153
DNA extracts were screened for Cryptosporidium and Giardia, with positive samples 154
sequenced to determine species/genotypes present. 155
For Cryptosporidium, all faecal and livestock water DNA extracts were screened at 156
the 18S rRNA locus for Cryptosporidium and positives were genotyped by sequencing. A 157
two-step nested PCR protocol was used to amplify the 18S rRNA locus of Cryptosporidium 158
previously described by Ryan et al., (2003), producing a product of ~540bp. All 159
Cryptosporidium positive samples at the 18S rRNA locus were confirmed by another two- 160
step nested PCR protocol, conducted to amplify a product of ~830bp at the actin gene of 161
Cryptosporidium, as described by Ng et al., (2006). This identified if mixed infections 162
existed (i.e. one species amplified at the 18S locus and a different species identified at the 163
actin locus). To confirm C. parvum positives detected at both the 18S rRNA and actin 164
genes, a two-step nested PCR was used to sub-genotype C. parvum positives at the 165
60kDa glycoprotein (gp60) gene, which amplified a fragment of ~832bp (Strong et al., 166
2000; Sulaiman et al., 2005). 167

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All samples positive for C. parvum only (positive for C. parvum at both the 18S 168
rRNA and actin genes) or C. parvum as part of a mixed infection (e.g. C. xiaoi sequenced 169
at 18S rRNA locus and C. parvum at actin locus) were further screened using a C. parvum 170
qPCR at a unique Cryptosporidium specific protein coding locus, previously described by 171
Yang et al., (2009), to confirm that C. parvum was present in each of the samples. 172
All samples were screened for Giardia at the gdh (glutamate dehydrogenase) gene 173
as previously described by Read et al., (2004), producing a product of ~480bp. All 174
samples which tested positive for Giardia at the gdh gene, were also screened at the 175
triosephosphate isomerise (tpi) gene with a two-step nested PCR protocol. The primary 176
PCR was performed as described by Sulaiman et al., (2003). For the second round 177
reaction, assemblage-specific primers and conditions for assemblage A (product ~332bp) 178
and E (product ~388bp) were used as previously described (Geurden et al., 2008a; 2009). 179
Each of the positive samples at gdh gene were screened for both G. duodenalis 180
assemblage E and assemblage A to confirm the assemblage detected at the gdh gene 181
and secondly to determine if there were mixed G. duodenalis assemblage infections 182
present (i.e. samples positive for different assemblages at the two loci). 183
2.4 Sequence and phylogenetic analysis 184
Positive Cryptosporidium (18S rRNA, actin and gp60) and Giardia (gdh) PCR 185
products isolated were purified using an UltraCleanTM
DNA Purification Kit (MolBio, West 186
Carlsbad, California, USA) and sequenced using an ABI PrismTM
Terminator Cycle 187
Sequencing Kit (Applied Biosystems, Foster City, California, USA) on an Applied 188
Biosystem 3730 DNA Analyzer. Sequence searches were conducted using BLAST 189
(http://blast.ncbi.nlm.nih.gov/Blast.cgi) and nucleotide sequences were analysed using 190
Chromas lite version 2.0 (http://www.technelysium.com.au) and aligned with reference 191
genotypes from GenBank using Clustal W (http://www.clustalw.genome.jp). Phylogenetic 192

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trees were constructed for Cryptosporidium isolates at 18S rRNA and actin loci, with
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193
additional isolates obtained from GenBank. Distance estimation was performed, based on 194
evolutionary distances calculation with the Kimura 2-paramter model and grouped firstly 195
using TREECON software to conduct Neighbour-Joining analysis and secondly using 196
Mega 5 software to conduct maximum-parsimony analysis. The confidence of groupings 197
from both analyses was assessed by bootstrapping, using 1000 replicates. A percentage 198
bootstrap support of >50% was used for each phylogenetic tree (Figures 1 and 2). 199
The nucleotide sequences of sheep genotype I 18S rRNA and actin sequences 200
have been deposited in GenBank under the accession numbers HQ317903 and 201
HQ317904 respectively. 202
2.5 Statistical analysis 203
Statistical analysis was performed using SPSS Statistics 17.0 (Statistical Package 204
for the Social Sciences) for Windows (SPSS inc. Chicago, USA). Overall and individual 205
sampling protozoan prevalences (including 95% confidence intervals) were calculated. 206
McNemars chi squared test for two-sided significance was used to determine if significant 207
differences existed between parasite prevalences within each farming property at different 208
samplings and also between both farms on the same sampling occasion. 209
210
3. Results
211
3.1 Cryptosporidium prevalence 212
Cryptosporidium was detected on both farms at all samplings, both in the pregnant 213
ewes (Table 3) and their lambs (Tables 4, 5 and 6). From all five samplings, the number of 214
times each lamb tested positive for Cryptosporidium ranged from 0–5 (Table 4). 215

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Cryptosporidium prevalence in the ewes 4 months and 2 weeks before lambing 216
commenced was the same on Farm A and B (Table 3). On both farms, for lambs that were 217
faecal sampled at all five sampling occasions, the overall prevalence for Cryptosporidium 218
was 81.3% (87/107) for Farm A and 71.4% (85/119) for Farm B, with lambs classified 219
positive for Cryptosporidium, when at least one positive result was identified for this 220
parasite out of the five samplings. 221
The prevalences of Cryptosporidium in the lambs at each of the five samplings on 222
Farm A ranged from a low of 26.6% at the fourth (post-weaning) sampling, to a high of 223
42.6% at the third (pre-weaning II) sampling (Table 5). Highest Cryptosporidium 224
prevalences were found in unweaned lambs between 2–4 months old (Table 5). 225
Prevalence of Cryptosporidium decreased between the third (pre-weaning II) sampling 226
(42.6%) and the fourth (post-weaning) sampling (26.6%) (P=0.014 McNemar chi squared 227
test). Cryptosporidium prevalence was found to have increased between the fourth (post- 228
weaning) prevalence of 26.6% and the final (lairage) prevalence of 36.4% (P=0.002). No 229
other significant differences between Cryptosporidium prevalences were observed. 230
Across the five samplings, Cryptosporidium prevalences Farm B lambs ranged from 231
a low of 18.5% at the first (marking) sampling, to a high of 42.0% at the final (lairage) 232
sampling (Table 5). Cryptosporidium prevalence on Farm B at the first (marking) sampling 233
was 18.5% and this was significantly lower than all subsequent sampling prevalences 234
(P<0.001). Cryptosporidium prevalence of 29.2% at the second (pre-weaning I) was 235
different to the prevalence of 39.0% at the third (pre-weaning II) sampling (P=0.08) and 236
42.0% at the final (lairage) sampling (P=0.002). 237
At the first (marking) sampling, there was a difference (P<0.001) between the 238
Cryptosporidium prevalence on Farm A (31.5%) and B (18.5%). This was the only 239
difference in Cryptosporidium prevalences between farms, within each sampling. 240

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3.2 Cryptosporidium species and genotypes
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The Cryptosporidium genotypes identified are shown in Tables 6 and 7. A combined 242
total of 387 isolates were identified as positive for Cryptosporidium at the 18S rRNA and 243
actin loci. From the pregnant ewes on both farms, all isolates were identified as C. xiaoi at 244
the 18S rRNA locus and this was confirmed at the actin locus. 245
On Farm A, C. xiaoi was the most common Cryptosporidium species isolated from 246
lambs with 134/189 (70.9%) positive isolates. At each of the last three samplings, the 247
proportion of Cryptosporidium positive isolates identified as C. xiaoi was 78.3% (36/46), 248
75.9% (22/29) and 82.0% (32/39) respectively (Table 6). 249
Cryptosporidium ubiquitum was most commonly isolated in young, unweaned lambs 250
on Farm A at the first two samplings. At the first (marking) and second (pre-weaning I) 251
samplings, 37.1% (13/35) and 17.5% (7/40) of Cryptosporidium positive isolates were 252
identified as C. ubiquitum, respectively. Cryptosporidium ubiquitum was also detected at 253
each of the last three samplings, but only in 1–2 isolates at each sampling (Table 6). 254
Cryptosporidium parvum was identified in a total of 18 isolates from Farm A lambs 255
across the five samplings, which were sub-genotyped at the gp60 locus and identified as 256
the C. parvum genotype IIdA20G1. The highest number of C. parvum isolates was six and 257
this was identified at the third (pre-weaning II) sampling, although the highest proportion of 258
Cryptosporidium positive isolates identified as C. parvum, was 13.8% (4/29) at the fourth 259
(post-weaning) sampling. 260
A total of 12 mixed Cryptosporidium species infections of C. xiaoi and C. parvum 261
were identified in lambs from Farm A, whereby C. xiaoi was isolated at the 18S rRNA 262
locus and C. parvum isolated at the actin locus. This was confirmed by qPCR at the 263
diagnostic locus for C. parvum. Four mixed Cryptosporidium species infections were 264

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identified at the first (marking) sampling and two identified at each of the other four
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265
samplings. In addition, there was one mixed Cryptosporidium species infection of C. 266
ubiquitum and C. parvum identified at the first (marking) sampling (Table 6). 267
Farm B had similar species/genotype demographics to Farm A. Cryptosporidium 268
xiaoi was the most common species detected in lambs and was detected in 76.3% 269
(151/198) of the Cryptosporidium positive isolates. Cryptosporidium xiaoi was the most 270
common species isolated in older lambs (3–7 months of age) with 77.1% (37/48), 82.9% 271
(34/41) and 84.0% (42/50) of Cryptosporidium positive isolates recorded for the last three 272
samplings respectively (Table 6). 273
Cryptosporidium ubiquitum was most commonly isolated in young lambs from Farm 274
B, identified in 13.0% (3/23) and 19.4% (7/36) of Cryptosporidium positive isolates at the 275
first two samplings. Cryptosporidium ubiquitum was identified at the last three samplings 276
but at low proportions (only 1 to 2 isolates per sampling). Cryptosporidium parvum was 277
detected only at the last two samplings (post weaning and lairage) (Table 6). 278
A novel Cryptosporidium genotype, hereafter referred to as sheep genotype I, was 279
identified in six isolates from Farm B, all of which were genetically distinct at both the 18S 280
and actin loci. At the 18S locus, multiple mutations were identified at positions 150, 153, 281
181, 197, 308, 311, 345, 359, 361, 365 – 368, 390 and 392. There were also significant 282
deletions in the sheep genotype I isolates when compared to C. ubiquitum calf and goat 283
sequences, which occurred between positions 380 – 389 and 394 – 417. At the actin 284
locus, base pair differences were detected at positions 250, 253, 279 – 283 and 87 – 88. 285
There was also one T insertion at bp position 255 in the novel genotype isolates. This 286
resulted in sheep genotype I having 13 different amino acids coded at the end of the actin 287
sequence (250 – 289bp), when compared to C. ubiquitum goat and calf isolates. 288

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Mixed Cryptosporidium species infections of C. xiaoi and C. parvum were identified 289
in lambs from Farm B at all five samplings, with the highest proportion of Cryptosporidium 290
positive isolates found as 21.7% (5/23) detected at the first (marking) sampling. 291
Two, one and one isolates of C. andersoni were identified at the 18S rRNA and 292
actin loci respectively at the third (pre-weaning II), fourth (post-weaning) and final (lairage) 293
samplings for Farm B. 294
On Farm A, all creek water samples tested negative for Cryptosporidium, however 295
both dam water samples collected from Farm B tested positive for C. xiaoi at the 18S 296
rRNA and actin loci. 297
3.3 Cryptosporidium phylogenetic analysis 298
At the 18S locus, phylogenetic analysis confirmed the six sheep genotype I isolates 299
from Farm B, as genetically distinct, sharing only a 94% similarity to calf and goat C. 300
ubiquitum sequences, as reported by Nichols et al., (2010) across 420 bp (Fig. 1). At the 301
actin locus, sheep genotype I shared only a 95% similarity to actin calf and goat C. 302
ubiquitum sequences, as reported by Nichols et al., (2010) across 289 base pairs (Fig. 2). 303
3.4 Giardia prevalence 304
Giardia was detected in both flocks at all samplings in the ewes (Table 3) and 305
lambs (Tables 4, 5 and 6). Over the five samplings for the lambs, the number of times 306
each lamb tested positive to Giardia ranged from 0 – 5 (Tables 4). 307
The Giardia prevalences in the ewes 4 months and 2 weeks before lambing 308
commenced were the same on Farm A and B (Table 3). 309
On both farms, for lambs that were faecal sampled at all five samplings, the overall 310
prevalence for Giardia was 67.3% (72/107) for Farm A and 60.5% (72/119) for Farm B, 311