International Journal for Parasitology: Parasites and Wildlife 16 (2021) 199–207

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International Journal for Parasitology: Parasites and Wildlife

journal homepage: www.elsevier.com/locate/ijppaw

Prevalence and gross pathology of liver fluke in macropods cohabiting

livestock farms in north eastern NSW, Australia, and diagnosis using cELISA
Jane Lamb a, *, Emma Doyle a, Jamie Barwick a, b, Michael Chambers c, Lewis Kahn a
a
University of New England, Armidale, NSW, 2351, Australia
b
Precision Agricultural Research Group, University of New England, Armidale, NSW, 2351, Australia
c
Invetus Pty Ltd, Locked Bag 6865, West Armidale, NSW, 2350, Australia

A R T I C L E I N F O A B S T R A C T

Keywords: Liver fluke (Fasciola hepatica) is a parasite of herbivores including wildlife. Macropods, such as Eastern grey
Fasciola hepatica kangaroo (Macropus giganteus) and Common wallaroo (Osphranter robustus), are frequently observed sharing
Prevalence grazing sites with domestic livestock. The impact of Macropods, as reservoirs of infection, on livestock pro­
Macropods
duction and risks to cross-species transmission are largely unknown. In Phase 1 of this study, liver and faecal
Livestock production
Australia
samples were collected from 245 Macropods (181 Eastern grey kangaroos, 64 Common wallaroos) cohabiting
livestock farms (n = 7) in the Northern Tablelands regions of New South Wales. Total fluke (TFC) and fluke eggs
(FEC) were counted in the liver and faeces, respectively, to assess prevalence. Faecal antigens were also
measured using the commercial Bio-X Diagnostic Monoscreen AgELISA Fasciola hepatica kit (cELISA) to assess
suitability as a diagnostic tool. In Phase 2, Macropod faecal samples were collected from 60 livestock farms to
conduct FEC and assess prevalence by region. Liver fluke was prevalent in 22% of Eastern grey kangaroo and
20% of Common wallaroos with prevalence as high as 45% in the Eastern grey kangaroo. Fluke burdens ranged
from 1 to 122 flukes (mean = 9 flukes) with a FEC range of 0–195 eggs per gram (epg) of faeces (mean = 18 epg).
Evidence of dead and live flukes trapped within fibrotic capsules confirms the ability of Macropods to resolve
infections. cELISA proved highly specific (100%) and sensitive (98%) in liver fluke detection however fibrotic
capsules observed in the liver may reduce the correlation of coproantigens with fluke burden. Phase 2 revealed
that 27% of livestock farms had Macropods infected with liver fluke. Overall, this study confirmed Eastern grey
kangaroo and Common wallaroo are susceptible hosts and potential reservoirs for liver fluke and, monitoring
infections in Macropods would assist in livestock disease management.

1. Introduction risk.
The liver fluke intermediate snail host, Austropeplea spp. (Boray,
Australian Macropods (i.e. kangaroos, wallabies, tree kangaroos and 1978; Boray et al., 1985), is predominately found in higher rainfall areas
pademelons) harbour a diverse range of parasites however of these, only (>600 mm annually) of eastern Australia (Boray, 1964; Barger et al.,
Fasciola hepatica (liver fluke) and Echinoccocus granulosus (hydatid 1978). Throughout this region, the Eastern grey kangaroo and Common
tapeworm) are known to infect domestic livestock (Beveridge et al., wallaroo (Osphranter robustus) are also widespread (NSW Government
1998; Beveridge and Emery, 2015). Macropods are herbivores and are Department of Planning, Industry and Environment, 2021). In the
frequently observed sharing grazing sites with livestock (Caughley et al., Northern Tablelands region of New South Wales (NSW), a region of
1987; Taylor, 1983) yet the impact of Macropods, as reservoirs of eastern Australia and where this study was conducted, the Common
infection, on livestock production is largely unknown as is the risk of wallaroo (136 km ˉ2) and Eastern grey kangaroo (79 km ˉ2) have been
cross-species transmission. In an earlier study, Eastern grey kangaroo detected in higher density on improved agricultural pastures compared
(Macropus giganteus) cohabiting with livestock were reported with a to unimproved areas (32 kmˉ2 and 37 kmˉ2 respectively) (Taylor, 1984).
higher liver fluke prevalence (73%) than those originating from forest Macropods cohabiting livestock farms, within liver fluke endemic areas,
areas (24%) (Spratt and Presidente, 1981) highlighting the potential may facilitate the liver fluke life cycle and act as reservoirs of infection

* Corresponding author. School of Environmental & Rural , University of New England, Armidale, NSW, 2351, Australia.
E-mail address: [email protected] (J. Lamb).

https://doi.org/10.1016/j.ijppaw.2021.10.006
Received 6 September 2021; Received in revised form 10 October 2021; Accepted 11 October 2021
Available online 13 October 2021
2213-2244/© 2021 The Authors. Published by Elsevier Ltd on behalf of Australian Society for Parasitology. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
J. Lamb et al. International Journal for Parasitology: Parasites and Wildlife 16 (2021) 199–207

thus reducing the effectiveness of integrated parasite management sensitivity (Palmer et al., 2014). An investigation of liver fluke in Mac­
strategies for liver fluke control. ropods offers the opportunity to examine the suitability of cELISA as a
Fasciolosis in livestock requires diagnosis as clinical signs of infec­ diagnostic tool for use in these species given they harbour a diverse
tion are nonspecific or often not apparent. Whilst fluke egg counts (FEC) range of parasites which are often in high numbers (Beveridge and
provide a simple diagnostic test for liver fluke detection, FEC can only Arundel, 1979; Brown et al., 2014).
detect patent infections from 10 to 12 weeks post infection (wpi) with In order to ascertain the potential of Macropods as reservoirs of liver
sensitivity ranging from 30 to 81% (Mazeri et al., 2016; Woodgate et al., fluke infection in livestock production, Macropods cohabiting livestock
2016). An alternative test, measuring faecal F. hepatica antigens using farms in the Northern Tablelands region of NSW were examined to
the BIOK201-2 coproantigen enzyme linked immunosorbent assay confirm: (i) liver fluke prevalence and; (ii) pathogenicity. Additionally,
(Bio-X Diagnostic, Belgium) has higher sensitivity and specificity (Mezo the BIOK201-2 coproantigen ELISA kit was assessed as a diagnostic tool
et al., 2004; Kajugu et al., 2012, 2015; Brockwell, 2013), detecting liver for liver fluke detection in Macropods.
fluke earlier from 5 to 6 wpi (Flanagan et al., 2011; Brockwell et al.,
2013; George et al., 2017). In wild red deer (Cervus elaphus) (French 2. Material and methods
et al., 2016) and boars (Sus scrofa) (Mezo et al., 2013), cELISA demon­
strated higher sensitivity than FEC and the assay was considered a 2.1. Experimental design, sampling site and animals
practical diagnostic test to monitor disease in wildlife. In horses (Equus
caballus) however, cELISA was considered unsuitable having low The study consisted of two separate experimental phases. In Phase 1,

Fig. 1. Geographical location of livestock farms (A–G) surveyed in the Northern Tablelands region of NSW, Australia, to assess liver fluke prevalence in Macropods
(ArcGIS 10.4.1 software, 2018).

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245 Macropods (181 Eastern grey kangaroo and 64 Common wallaroo) repeated until eggs were clean of bile. Fluke eggs were counted and
cohabiting on seven livestock farms were examined for the presence of recorded for each gall bladder using a stereo microscope (Nikon
liver fluke via total fluke (TFC) and fluke egg counts (FEC), cELISA and SMZ800N) at 40× magnification. Fluke eggs (100 eggs) were incubated
gross pathology. Macropods were humanely euthanised by commercial in water at 25 ◦ C for 14 days then exposed to 2 h of artificial light to
harvesters or by landholders with a NSW Government Licence to Harm confirm hatching to viable miracidia. Confirmation of egg development
Kangaroos. Permission was obtained from the NSW National Parks and and hatching were based on those described by Fairweather et al.
Wildlife Service (NPWS) to opportunistically collect samples after (2012).
euthanasia (NSW NPWS Office of Environment & Heritage Scientific
Licence, SL102153). This study was conducted with approval from the 3.2. Coproantigen ELISA
University of New England Animal Ethics Committee (AEC18-101).
In Phase 2, Macropod faecal samples were collected from 60 farms Coproantigens were measured in triplicate on faecal samples (0.5 g)
(distinct from those in Phase 1) to assess liver fluke prevalence via fluke collected from all Macropods in Phase 1 that were confirmed positive for
egg counts (FEC). liver fluke by TFC or FEC (n = 52), and randomly selected from 30
Macropods with no liver fluke. The commercial cELISA kit (BIOK201-2
2.2. Sampling sites Monoscreen AgELISA Fasciola hepatica, Bio-X Diagnostic, Belgium) was
used to measure coproantigens with the manufacturer’s guidelines
Phase 1 and 2 samples were collected from Macropods cohabiting modified to include an overnight soak of faecal samples at room tem­
livestock farms within a 105 km radius of Armidale, NSW (30.5016◦ S, perature in the supplied dilution buffer prior to centrifugation (Brock­
151.6662◦ E) (Fig. 1). Phase 1 samples (liver and faecal) were collected well et al., 2013). Samples were considered positive for F. hepatica if
from seven farms (A-F) throughout November 2018–October 2020. mean optical density was >8% at 450 nm. Coproantigens were
Farms were classified by risk of liver fluke infection based on the number measured to assess sensitivity and specificity of cELISA for use in Mac­
of freshwater springs, identified from farm inspections, that were ropods with whole liver examinations used as a reference standard
capable of supporting the habitat of the intermediate snail host; (French et al., 2016).

• Low risk - no or few freshwater springs (<2); farms A and B. 4. Liver assessments
• Medium risk - freshwater springs (1–10) encompassing up to 1% of
the area of the property; farms E, D and G. 4.1. Total fluke counts
• High risk: numerous freshwater springs (>10) encompassing 5% or
more of the property; farms C and F. TFCs were conducted according to Wood et al. (1995) on thawed
livers. The liver was sliced to 0.5–1.0 cm wide strips with each strip
Phase 2 faecal samples were collected from 60 farms throughout examined and bile ducts squeezed to release fluke. Liver slices were
December 2018–June 2021, randomly selected within the 105 km radius subsequently soaked overnight in warm saline (9.0 g NaCl/L H₂0), then
of Armidale from respondents to a pre-trial grazier survey. washed with tap water over a 300 μm mesh sieve to collect any residual
fluke. Fluke were counted based on the number of whole fluke and heads
2.3. Sample collection recovered from each liver. Adult fluke were distinguished from imma­
ture fluke based on the presence of reproductive organs (ovary and
Macropods were identified by location (A-G) and number within testis) and fluke eggs (Valero et al., 2001, 2005). Other parasites
farm. The species, sex and number of pouch young were recorded for cohabiting the liver of Macropods were identified (Presidente and Bev­
each individual and the tail and foot length measured using a metric eridge, 1978; Beveridge and Emery, 2015) and recorded.
measuring tape (Poole et al., 1982). The chest cavity was subsequently
opened to collect: liver with intact gall bladder, kidneys with sur­ 4.2. Liver pathology score
rounding adipose tissue, and a faecal sample. Livers were then weighed,
photographed and the gall bladder removed prior to freezing (- 20 ◦ C) Gross pathology of the liver was assessed and scored (0–5 scale)
the liver pending TFC and gross pathology assessment. The gall bladder based on the methodology described by Sargent et al. (2009).
and contents were assessed on the day of collection for liver fluke and
their eggs. The kidneys were separated from surrounding adipose tissue 5. Kidney fat index
and weighed separately within 24 h of collection to measure kidney fat
index (KFI). Faecal samples were stored at 4 ◦ C and FECs conducted Kidney fat index (KFI) was used to assess body condition. The fat and
within seven days of collection. A sub-sample of each faecal sample was kidneys were weighed separately to calculate the mean weight for each
stored frozen (- 20 ◦ C) for the later measurement of coproantigens. individual. KFI was calculated by dividing the mean weight of the fat by
At each farm in Phase 2, up to 20 fresh Macropod faecal samples (as the mean weight of the kidney and multiplied by 100 (Finger et al.,
available) were collected from the ground. Fresh Macropod faeces was 1981).
identified based on colour, shape and texture (in accordance with
Catchpole, 2007). Samples were stored at 4 ◦ C and FEC conducted 6. Statistical analysis
within 7 days of collection.
Data were checked for normality and the homogeneity of variance
3. Faecal assessments assumption confirmed using Levene’s test. The statistical software
JMP®16 was used to calculate least squares means (lsm) ± standard
3.1. Fluke egg counts error (s.e.). Analyses were based on the effects of species, sex, risk rating,
season and their possible interactions.
FEC were conducted on duplicate faecal samples (Phase 1: 3 g; Phase Chi-square tests (x2) were used to assess the association between
2: 6 g) as described by Lamb et al. (2021). Fluke eggs were recovered prevalence of F. hepatica and species, sex, risk site and sampling season.
from the gall bladder by washing the bile through a 90 μm sieve to a 500 Correlations conducted on parametric data used linear regression or
ml conical flask. After eggs were left to sediment for 30–60 min, the Pearson’s correlation whilst correlations on non-parametric data used
supernatant was reduced to 100 ml using a vacuum suction pump. The Spearman’s correlation.
flask was then re-filled with tap water and the sedimentation process Tabulated non-parametric data were presented as back-transformed

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least square means. Risk site and pathology score were assessed as The livers of Eastern grey kangaroo were significantly (p = 0.001)
ordinal traits. As only one Common wallaroo was examined at the low heavier than for the Common wallaroo (lsm 430.0 g ± 10.4 and 359.2 g
risk site (Phase 1), this animal was excluded from analysis when ± 17.9 respectively). Livers weights were also significantly (p < 0.001)
assessing prevalence by risk site. heavier for male Macropods than for females (lsm 487.6 g ± 16.9 and
301.7 g ± 12.0 respectively). Liver weight had a weak positive corre­
7. Results lation with TFC (R2 = 0.3, p < 0.001) and pathology score (R2 = 0.3, p <
0.001). Liver fluke infections in Macropods are summarised in Table 1.
7.1. Phase 1 – liver fluke prevalence The mean fluke egg hatch rate of eggs recovered from the gall
bladder of Macropods was 37.4 ± 4.4% (range 0–96%).
7.1.1. Macropod species
Overall, 39 of 181 Eastern grey kangaroos (22%) and 13 of 64 7.2.2. Gross pathology
Common wallaroos (20%) were infected with liver fluke (x2 = 0.02, p = Significant differences in pathology score (x2 = 22.2, p < 0.001)
0.879). No significant effects or interactions for fluke prevalence were were observed by species with Eastern grey kangaroo having greater
observed between species and sex (x2 = 2.67, p = 0.446). pathology than the Common wallaroo. No significant interaction was
observed between the effects of species and sex for pathology score.
7.1.2. Risk site Liver pathology score had a strong positive correlation with TFC (r =
Liver fluke prevalence differed significantly by risk site (x2 = 31.3, p 0.9, p < 0.001) and FEC (r = 0.7, p < 0.001).
< 0.001) and was 33.1%, 12.6% and 2.6% for high, medium and low risk Liver fluke were found throughout all areas of the liver. Macropods
sites respectively (Fig. 2). There was a strong suggestion that liver fluke with low infections (<12 flukes) had pathological lesions limited to
prevalence differed by species across risk sites (x2 = 3.64, p = 0.06) with small areas and livers were dark in colour with regular formation
Eastern grey kangaroo appearing more sensitive than Common wallaroo (Fig. 4A and B). Pathological lesions consisted of partial or complete
to risk rating (Fig. 2). A significant interaction between risk site and sex fibrotic capsule formations, encapsulating dead and live flukes.
(x2 = 10.6, p = 0.001) was also observed with a higher fluke prevalence When liver fluke burdens were higher (≥12 flukes) or immature
in female Macropods across the medium and high risk sites. fluke were present, pathological lesions were more extensive with hae­
morrhagic lesions, necrotic migratory tracks, bile duct hyperplasia,
7.1.3. Season cholangitis and fibrotic lesions (Fig. 4C, D, E). Livers ranged from dark to
Rainfall and temperature data recorded at Armidale airport by the pale in colour with irregular form. Gross pathology extended to the
Bureau of Meteorology throughout the sample period are detailed in mucosal lining of the gall bladder (hyperplasia) in heavy infestations.
Fig. 3. For both species, liver fluke prevalence was unaffected by season
(x2 = 4.96, p = 0.174).
7.3. Phase 1 — other parasites
7.2. Phase 1 — pathogenicity
Progamotaenia festiva (Kangaroo tapeworm) were also found in the
7.2.1. Total fluke count and fluke egg counts liver and in higher prevalence in Eastern grey kangaroo (70%) than
TFC ranged from 0 to 122 flukes in the Eastern grey kangaroo (lsm Common wallaroo (34%). Infections were detected across all farms but
0.2 ± 0.03) with a FEC range of 0–195 epg (lsm 0.16 ± 0.03). Lower in lower prevalence at sites where liver fluke were detected. In Macro­
burdens (all <12 flukes) were recovered from the Common wallaroo pods with liver fluke, 6% (Common wallaroo) and 10% (Eastern grey
(lsm 0.1 ± 0.05) with FEC range of 0–82 epg (lsm 0.12 ± 0.06). No ef­ kangaroo) were also co-infected with Kangaroo tapeworm. Gross pa­
fects or interactions of species, sex or risk site in TFC or FEC were sig­ thology attributed to kangaroo tapeworm was considered mild with
nificant. A strong positive correlation was observed between TFC and slight enlargement of bile ducts walls and cholangitis.
FEC (R2 = 0.9, p < 0.001). Infections were of mixed age however 92% of Echinococcus granulosus (Hydatid tapeworm) was only found in fe­
Macropods infected with liver fluke had adult fluke whilst the remaining male Eastern grey kangaroo at the medium and high risk sites and all
Macropods had immature fluke which were identified throughout all were co-infected with liver fluke. Livers presented with multiple cysts,
seasons. ranging up to 50 mm wide, hepatomegaly and disfigurement.

Fig. 2. Liver fluke prevalence in Macropods (infected/total sampled) cohabiting farms in the Northern Tablelands region of NSW, Australia. Number of farms by risk
site: low – 2 farms, medium – 3 farms, high – 2 farms.

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Fig. 3. Rainfall and temperature data throughout 2019–2020 recorded at the Armidale airport NSW, Australia (Australian Government of Bureau of Meteorology,
2019, 2020).

Table 1
Back transformed least square means ± s.e. (range) describing liver fluke infections in the Eastern grey kangaroo and Common wallaroo cohabiting livestock farms in
the Northern Tablelands region of NSW.
Species No. Sex (F/ Kidney fat No. Liver pathology Liver weight Total fluke Fluke egg count (gall Fluke Egg
M)a Index (%) score (0–5) (g) count bladder) count (epg)b
with with

liver fluke pouch

(prevalence) young

Eastern grey 39 of 181 (22%) 22 F 18.0 ± 3.4 19 of 22 1.8 ± 0.3 401.3 6.0 454.2 7.0
kangaroo (86%) ±26.1 ±1.3 ±1.9 ±1.4
(224–777) (1–122) (0–94,400) (0–195)
17 M 11.8 ± 4.1 N/A 1.3 ± 0.3 647.8 4.0 404.5 3.7
±29.7 ±1.3 ±2.0 ±1.4
(398–890) (1–26) (0–83,200) (0–87)
Common 13 of 64 (20%) 7F 38.0 ± 6.0 6 of 7 1.0 ± 0.5 292.8 2.6 289.2 5.5
wallaroo (86%) ±46.3 ±1.5 ±3.0 ±1.8
(194–338) (1–7) (3–13,500) (0–82)
6M 14.0 ± 6.5 N/A 1.4 ± 0.5 549.6 3.4 6960 2.4
±50.0 ±1.5 ±3.3 ±1.8
(417–692) (1–12) (355–73,080) (0–19)
p-value (species x sex) 0.091 – 0.174 0.897 0.349 0.087 0.833
a
F = female, M = male.
b
epg = eggs per gram faeces.

7.4. Phase 1 — morphometric measurements moderate positive correlation was observed between cELISA; and TFC
(Spearman’s p = 0.55, p < 0.001) and FEC (Spearman’s p = 0.61, p <
Macropod tail length had a strong positive correlation (r = 0.8, p < 0.001). No significant differences (p = 0.304) were observed in cop­
0.001) with foot length. TFC also had a positive correlation with tail roantigens by species (Eastern grey kangaroo lsm 1.2 ± 0.1, Common
length (r = 0.2, p = 0.018) and foot length (r = 0.1, p < 0.05). wallaroo lsm 1.0 ± 0.2) or by sex (p = 0.962) and no significant dif­
ferences were observed in the interaction between the effects of species
7.5. Phase 1 — kidney fat index and sex. Coproantigens had a weak positive correlation with pathology
score (R2 = 0.2, p = 0.54).
No significant differences (p = 0.09) in KFI were detected between One cELISA result which was positive for coproantigens (Optical
Macropod species (Eastern grey kangaroo lsm 12.2 ± 1.1, Common density at 450 nm = 1.0) had no liver flukes in the liver however fluke
wallaroo lsm 16.0 ± 1.9) however KFI was higher (p < 0.001) in female eggs were detected in the gall bladder (496 eggs) and faeces (0.2 epg)
Macropods (lsm 18.3 ± 1.2 female, 9.9 ± 1.8 male). In Macropods with inferring an active infection.
liver fluke, Eastern grey kangaroo had a significantly lower (p = 0.04) Of the four Macropods which were negative for coproantigens, three
KFI than Common wallaroo (lsm 14.9 ± 2.7, 26.0 ± 4.4). No significant Macropods had resolved infections (no or dead liver flukes) with mini­
differences were observed between the interaction of season and sex (p mal fluke eggs in the gall bladder (1, 8 and 137 eggs) and faeces (0, 0 and
= 0.08) for KFI. 0.5 epg). The fourth Macropod was a false negative having 14 flukes
encapsulated within two fibrotic capsules and fluke eggs in the gall
bladder (144 eggs) and faeces (6.5 epg).
7.6. Phase 1 — cELISA
In those Macropods with mixed age infections (adult and immature
fluke), all were positive for coproantigens including one Macropod with
In those Macropods positive for liver fluke by TFC or FEC, 48 of 52
only immature fluke (Optical density at 450 nm = 0.4, TFC = 1, FEC =
Macropods (92%) were also positive for coproantigens (Fig. 5). A

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Fig. 4. A. Common wallaroo liver (visceral surface) with prominent fibrotic capsules. B. Liver cross-section of fibrous capsules. C. Eastern grey kangaroo liver
(visceral surface) with irregular form, hepatomegaly, fibrotic lesions and bile duct hyperplasia. D. Necrotic tracks generated by immature fluke. E. Immature
fluke (mm).

Fig. 5. Scatter plot of Fasciola hepatica coproantigen concentration (optical density, 450 nm) and total fluke count in Macropods.

0 epg). 7.7. Phase 2 — liver fluke prevalence on-farm

Macropods (n = 30) which were confirmed negative for liver fluke by
TFC and FEC, were also negative for F. hepatica coproantigens. Of the 60 farms surveyed, 16 farms (27%) had Macropods infected
with liver fluke (FEC range 6–22 epg). Liver fluke were detected in
Macropods cohabiting farms in Guyra, Uralla, Walcha and Armidale

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the density of Eastern grey kangaroo (26.4 kmˉ2) in the Northern Ta­
blelands region of NSW is higher than Common wallaroo (7.4 kmˉ2)
(Cairns et al., 2020) which would further increase grazing pressure
within their habitat during periods of low rainfall and pasture growth.
The prevalence of liver fluke was also higher in female Macropods.
Although reasons for this are unclear, age, immune response (Siddle
et al., 2010), nutritional stress (Miller, 1987; Brandimarti et al., 2021),
high energy requirements whilst supporting young (Stannard et al.,
2020) and grazing behaviour (Cripps et al., 2011) may all be contrib­
uting factors. Female macropods are also philopatric, preferring to stay
or return to their place of birth (Best et al., 2013; King and Goldizen,
2016), which may increase competition for food and frequency of
grazing ‘fluky’ habitats where they exist.
Macropods with longer morphometric measurements also had higher
liver fluke prevalence. As morphometric measurements increase with
age (Poole et al., 1982), results suggest either that older Macropods are
more susceptible to liver fluke or that they have accumulated greater
exposure time to fluke infection. Younger Macropods would have a
shorter accumulation period and be grazing less as they are not weaned
until 13–18 months of age (King and Goldizen, 2016).
The prevalence of liver fluke on farms in Phase 2 (27%) was com­
parable to levels of Phase 1 (21%). Liver fluke were detected in Mac­
Fig. 6. Livestock farms in the Northern Tablelands region of NSW, Australia, ropods throughout Guyra, Walcha, Uralla and Armidale regions. These
with Macropods harbouring liver fluke infections (December 2018–June 2021). areas have high rainfall, basalt soils with freshwater springs (“black
springs”) and soil pHCaCl2 > 4.5 which are all attributes considered
(Fig. 6). No significant differences were detected in prevalence by season suitable for snail habitats (Boray, 1964; Upjohn et al., 2005). The Dor­
or year across farms. rigo region also has high rainfall but soil pH is comparatively acidic
(pHCaCl2 < 4.5) which may deter snail establishment (Boray 1964).
8. Discussion Seasonality patterns of liver fluke prevalence typically observed in
livestock (Sissay et al., 2007; Ali et al., 2011; Hernández-Guzmán et al.,
This study confirmed liver fluke in Eastern grey kangaroo (22%) and 2021) were not apparent in Macropods. Without anthelmintic inter­
Common wallaroos (20%) cohabiting livestock farms in the Northern vention, Macropods may be harbouring persistent infections spanning a
Tablelands region of NSW with prevalence as high as 45% in Eastern number of seasons or years, limiting the effectiveness of control pro­
grey kangaroo across high risk sites. Macropods harbouring low fluke grammes. Immature fluke were also detected in Macropods year-round
burdens (<12 flukes) had pathological lesions limited to small areas of demonstrating susceptibility in all seasons and the ability of (at least
the liver and flukes were frequently found encapsulated in fibrous some) metacercariae to survive winters (Caminade et al., 2019), espe­
capsules. Extensive lesions were observed in Macropods with higher cially the warmer winter of 2019.
fluke burdens ranging from 12 to 122 flukes. The detection of liver fluke Macropods with 1–122 flukes generated a FEC range of 1–195 epg
in Macropods by cELISA proved to be a reliable assessment with high demonstrating their ability to contaminate pastures grazed by livestock.
specificity (100%) and sensitivity (98%), detecting low fluke burdens The fecundity of liver fluke in Macropods however appears lower than in
(1–2 flukes) of mixed aged however pathology in the liver may limit sheep, which are capable of shedding 20,000–50,000 eggs per day
correlation of coproantigens with fluke burden. (Happich and Boray, 1969), suggesting Macropods may be less suitable
Liver fluke prevalence in Macropods would largely reflect their fre­ hosts for liver fluke despite their susceptibility.
quency of grazing the habitats of the intermediate snail host. The home To date, there have been mixed reports concerning the pathogenicity
range of Macropods is estimated to be 20–300 ha (Caughley et al., 1987; of liver fluke in Macropods. For example, Eastern grey kangaroo har­
Hill, 1982; Jarman and Taylor, 1983; Grice et al., 1988; Croft, 1991; bouring up to 95 flukes were reported with no clinical signs of infection
Viggers and Hearn, 2005). Free-roaming Macropods with unrestricted (Presidente and Beveridge, 1978; Spratt and Presidente, 1981) whilst
access to wetlands, supporting green pastures, would preferentially Portas and Taylor (2015) reported two Eastern grey kangaroo with 18
graze these areas when forage is limited or when rainfall is low as and 36 flukes having severe clinical signs of infection requiring eutha­
experienced in 2019 (40% below the long term average annual rainfall). nasia. In the present study, liver pathology correlated strongly with
Within liver fluke endemic regions, Macropods may pose risks to live­ fluke burden. Some Macropods however had fluke encapsulated in
stock production by maintaining the liver fluke life cycle in the grazing fibrous capsules of the liver, attaining a lower pathology score. Evidence
environment and limiting fluke control. Moreover, Macropods cohab­ of dead decaying flukes within these capsules confirms their inherent
iting farms with anthelmintic resistance may also be vectors (Walker ability to resolve infections. Similar capsule formations, described as
et al., 2011; Chintoan-Uta et al., 2014) for dispersal of resistant strains to “pockets” or “cyst-like lesions”, have been identified in red (Cervus ela­
neighbouring farms. phus) (French et al., 2016) and fallow (Dama dama) deer (Jenkins et al.,
Results suggest Eastern grey kangaroo had a higher susceptibility to 2020; Lamb et al., 2021) but also elk (Cervus canadensis) infected with
infection. These results are consistent with earlier findings by Spratt and Fascioloides magna (Pybus et al., 2015). Having the ability to isolate and
Presidente (1981) who reported Eastern grey kangaroo with the highest restrict fluke migration minimises damage in the liver and would reduce
liver fluke prevalence (59%) compared to all other mammals examined. clinical signs of infection. Moreover, the life span of liver fluke in
Habitat and behaviour may account for species susceptibility. Eastern Macropods may be shorter than the life span in sheep where flukes are
grey kangaroo inhabit a diverse range of habitats including open pas­ capable of surviving up to 11 years (Andrews, 1999).
tures on livestock farms (Taylor, 1984). The Common wallaroo are Detection of liver fluke by cELISA demonstrated high specificity and
comparatively sedentary within their habitat (Croft, 1991) preferring sensitivity, detecting low infections (1 fluke) as well as immature fluke.
mountain and rocky landscapes (Taylor, 1984) which are areas less These results are comparable to that observed in sheep and cattle, also
likely to support the habitat of the intermediate snail host. Furthermore, detecting infections of just 1–2 fluke (Mezo et al., 2004). One false

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