Journal of Helminthology (2008) 82, 85–88 doi: 10.

1017/S0022149X07874232

The relationship between normocytic,

hypochromic anaemia and iron
concentration together with hepatic enzyme
activities in cattle infected with Fasciola
hepatica
S. Lotfollahzadeh1*, M. Mohri2, Sh. Ranjbar Bahadori3,
M.R. Mokhber Dezfouly4 and P. Tajik4
1
Department of Clinical Sciences, Faculty of Veterinary Medicine,
University of Tehran, Tehran, Iran: 2Department of Clinical Sciences,
School of Veterinary Medicine, Ferdowsi University of Mashhad, Mashad,
Iran: 3Department of Parasitology, School of Veterinary Medicine, Garmsar
azad University, Garmsar, Iran: 4Department of Clinical Sciences, School of
Veterinary Medicine, University of Tehran, Tehran, Iran

Abstract

Erythrograms determined from whole blood analyses and serum analyses for
aspartate aminotransferase (AST), g-glutamyl transpeptidase (GGT) and
alkaline phosphatase (ALP) activities, and iron concentration, were used in
infected and uninfected cattle to determine the type of anaemia and degree of
hepatic damage caused by Fasciola hepatica. Blood samples from 86 infected and
30 uninfected cattle were taken at slaughter. Haematological analyses revealed
decreased levels of packed cell volume (PCV), haemoglobin concentration, mean
corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concen-
tration (MCHC) in infected compared with uninfected cattle (P , 0.05).
A decrease in the concentration of serum iron was also observed in infected
cattle compared with uninfected cattle (P , 0.05). Significant increases in AST,
GGT and ALP activities were observed in cattle infected with F. hepatica when
compared with uninfected cattle (P , 0.05). It was concluded that the anaemia
observed in cattle infected with F. hepatica is a normocytic, hypochromic anaemia
and the most important aetiology of the anaemia is the chronic blood loss due to
the blood-sucking activity of the adult flukes and leakage of blood from the bile
duct to the intestine, which results in iron deficiency. The increased activities of
serum enzymes indicated chronic hepatic and bile duct injuries associated with
chronic infection with F. hepatica.

Introduction Andrews, 1999; Rapsch et al., 2006). Anaemia is one of the

Fascioliasis is an economically important disease of
domestic livestock, in particular cattle and sheep; and is
caused by digenean trematodes of the genus Fasciola,
commonly referred to as liver flukes (Soulsby, 1986;
*Fax: þ 98 2166279080
E-mail: samadz@yahoo.com
most important clinical and clinical pathological findings
in animals infected with Fasciola hepatica (Holmes et al.,
1968; Berry & Dargie, 1976; Jennings, 1976). Profound
anaemia and changes in serum proteins are found in
sheep, and these are much less marked in cattle (Soulsby,
1986). The primary cause of anaemia in the case of
infection of animals with F. hepatica is thought to be the
loss of blood from intrahepatic haemorrhages caused by
fluke migration and from damage to the walls of biliary
86 S. Lotfollahzadeh et al.
tract caused by the presence of the flukes and their
feeding on blood (Holmes et al., 1968; Berry & Dargie,
1976, 1978). Berry & Dargie (1978) demonstrated that
early packed cell volume (PCV) reduction during the first
7 weeks of an experimental infection with F. hepatica in
sheep is due to marked hypervolaemia and further
haemodilution. However, 8 – 9 weeks after infection,
intrabiliary haemorrhage and consequent loss of red
blood cells occurs into the intestine, producing anaemia
due to blood loss (Berry & Dargie, 1978). The type of
anaemia in fascioliasis is a matter of controversy and
there are different opinions on this subject (Behm &
Sangster, 1999; Radostitis et al., 2007). Other biochemical
changes that occur in infected animals are increased
activities of the serum enzymes g-glutamyl transpepti-
dase (GGT) and aspartate aminotransferase (AST) caused
by liver injury due to F. hepatica infection (Anderson et al.,
1977; Wensvoort & Over, 1982; Wycoff & Bradley, 1985;
Yang et al., 1998; Bossaert et al., 1999; Gajewswka et al.,
2005). However, enzyme analyses in other studies have
shown no significant increases in AST, GGT and alkaline
phosphatase (ALP) activities as compared to those of
uninfected animals (Simensen & Nansen, 1974; Bulgin
et al., 1984). In the present study, the effects of chronic
infection of cattle with F. hepatica on haematological
indices, serum iron status and hepatic enzyme activities
were investigated, in order to elucidate the underlying
aetiology of anaemia in infected animals.
Materials and methods
Blood samples were taken at slaughterhouse in the city
of Ghaemshahr (Mazandaran province, northern Iran),
where fluke infection is quite common. Blood samples
were taken randomly, by jugular venepuncture using
vacutainers (Pars Khavar Co., Qazvin, Iran), from cows
going to be slaughtered. Samples were taken into
heparinized and plain tubes. Infection was confirmed in
animals by observing adult flukes in the bile ducts of
slaughtered animals following transfer to the laboratory.
Blood samples from uninfected cattle, as a control group,
were also taken in the same manner. Serum samples for
biochemical analyses were removed by centrifugation
shortly after bleeding. Haematological analyses were
performed on the same day as the blood was sampled,
whereas serum samples were frozen at 2 208C. Eighty-six
cattle infected with F. hepatica and 30 uninfected cattle were
studied. Blood parameters of samples, including PCV,
RBC (red blood cells), Hb (haemoglobin), MCV (mean
corpuscular volume), MCH (mean corpuscular haemo-
globin) and MCHC (mean corpuscular haemoglubin
Table 1. Mean values of packed cell volume (PCV), red blood cells (RBC) and haemoglobin
(Hb) concentrations in F. hepatica-infected and uninfected cattle (mean ^ SE).
Groups Number
Infected 86
Uninfected 30
Reference rangea – 24–46
* Significantly different (P , 0.05).
a
Taken from Radostitis et al. (2007).
concentration), were measured in this study. RBC
parameters and indices were determined by an auto-
mated Coulter counter (Sysmex, Germany). Enzyme
assays were carried out on the serum samples for the
measurement of activities of AST, ALP and GGT, using
commercial kits. Activities were expressed in units/litre
of serum. The iron concentration of serum samples was
also determined in infected and non-infected animals.
Commercial reagent kits, based on spectrophotometric
methods, were used to assay these parameters (Pars
Azmoon Co., Tehran, Iran). Our results were compared
with reference values reported previously for cattle
(Radostitis et al., 2007).
Statistical analyses
Mean values of PCV, RBC and Hb as well as MCV,
MCH and MCHC, and also the mean activities of three
serum enzymes and the mean concentration of serum
iron, in infected and uninfected cattle, were analysed by
using Student’s t-test, and a value of P , 0.05 was
considered to be significant.
Results
Table 1 shows the mean values of PCV, RBC and Hb
concentrations in sampled cattle and reference values in
cattle for the mentioned parameters. The mean values of
RBC parameters in infected cows were significantly lower
than in uninfected cattle (P , 0.05) and also lower than
the minimum amount reported for cattle in references.
Table 2 shows the mean values of MCV, MCH and MCHC
of the infected and uninfected groups. Mean values of
RBC indices for infected cattle were also lower than those
of the control group, but the difference between the MCH
and MCHC values of two groups was statistically
significant (P , 0.05). As can be seen in table 2, the
MCHC values of infected cattle in the present study were
lower than the reference values reported for cattle.
Results of the biochemical analyses in the present study
are shown in the table 3. The serum iron concentration in
infected cattle (43.97 ^ 5.89 mg/dl) in the present study
was significantly lower than those in uninfected cattle
(121.2 ^ 15.26 mg/dl) (P , 0.05) and lower than reference
values in cattle (57 –162 mg/dl). The mean serum activity
of GGT for fluke-infected animals was 39.12 ^ 5.26 IU/l,
compared with an average value of 13.12 ^ 0.87 IU/l in
animals with fluke-free livers and a range of 0 –31 IU/l
reported for various cattle species. The corresponding
average AST values were 161.54 ^ 1.91 IU/l for infected
animals, 86.7 ^ 3.7 IU/l for uninfected animals and a range
PCV (%) RBC (1012/l) Hb (g/dl)
20.87 ^ 0.46* 4.53 ^ 0.13* 5.17 ^ 0.36*
33.42 ^ 1.11 6.51 ^ 0.18 10.86 ^ 0.55
5–10 8–15
 Anaemia and hepatic enzyme activities in F. hepatica-infected cattle 87

Table 2. Mean values of mean corpuscular volume (MCV) (fl), mean corpuscular
haemoglobin (MCH) (pg) and mean corpuscular haemoglobin concentration (MCHC) (%)
in infected and uninfected groups (mean ^ SE).

Groups Number MCV (fl) MCH (pg) MCHC (%)

Infected 86 48.12 ^ 1.46 14.17 ^ 0.49* 27.74 ^ 0.51*

Uninfected 30 50.07 ^ 0.9 16.55 ^ 0.4 32.94 ^ 0.54
Reference rangea – 40 –60 11–17 30–36

* Significantly different (P , 0.05).

a
Taken from Radostitis et al. (2007).

of 60–150 IU/l for cattle species. Mean value of ALP activity
in the serum of infected cattle was 377.98 ^ 2.76 IU/l
compared with a mean value of 170.28 ^ 2.4 IU/l in
animals with fluke-free livers. Statistical analyses for mean
values showed significant increases in activities of GGT,
AST and ALP in sera of the infected group as compared
with the uninfected group (P , 0.05).
Discussion
Anaemia is probably the single most important factor
contributing to host morbidity and mortality in F. hepatica
infection (Jennings, 1976; Berry & Dargie, 1978; Spengler
& Isseroff, 1981; Soulsby, 1986; Behm & Sangster, 1999). Its
aetiology has long been debated, but it is now widely
accepted that it is a combination of haemorrhagic
anaemia and the release of proline from the worms,
which suppresses bone marrow activity of infected
animals (Holmes et al., 1968; Berry & Dargie, 1976;
Spengler & Isseroff, 1981; Soulsby, 1986). The main cause
of anaemia in fascioliasis is the passage of red blood cells
into the gastrointestinal tract, presumably via flukes and
the bile duct. It has been estimated that blood is lost at the
rate of 0.2 – 0.5 ml/day per fluke (Holmes et al., 1968;
Jennings, 1976). In the present study, the results of
haematological analyses showed severe anaemia in cattle
infected with F. hepatica compared with an uninfected
group and the reference values reported for cattle (tables
1 and 2). With regard to the results shown in tables 1 and
2, it can be observed that the anaemia encountered in
infected cattle is a normocytic, hypochromic anaemia
which is different from that described in other studies.
In the subacute and chronic form of F. hepatica a severe
hypochromic, macrocytic anaemia has been reported
(Radostitis et al., 2007). In another study that was carried
out on rats to determine whether or not F. hepatica could
produce an anaemia in rats when haematophagia was
prevented, haematological analyses showed significant
decreases in erythrocyte counts and haemoglobin
concentration and significant increases were observed in
mean corpuscular volume of red blood cells (Spengler &
Isseroff, 1981). However, Behm & Sangster (1999)
suggested: ‘There is still some mystery why the anaemia
in fascioliasis is normochromic and normocytic’. Normo-
cytic, hypochromic anaemia occurs in typical forms of
iron deficiency (Jain, 1993). A significant decrease in the
level of serum iron was observed in the current study for
cattle infected with F. hepatica (43.97 ^ 5.89 mg/dl)
compared with uninfected cattle (121.2 ^ 15.26 mg/dl)
and also reference values reported for cattle (57 – 162 mg/
dl) (Radostitis et al., 2007) (P , 0.05). Symonds et al.
(1983) showed that 7– 8 weeks after experimental
infection with F. hepatica the rate of loss of iron in bile
increased rapidly and after 10 weeks bile flow increased.
The iron causing an increase in iron excretion was
predominantly from red blood cells, with only insignif-
icant quantities derived from plasma (Symonds et al.,
1983). Berry & Dargie (1978) found a progressive
reduction in iron concentration between the 6th and
10th weeks after experimental infection of sheep. On the
basis of the results of the present study (tables 1, 2 and 3)
it has been found that anaemia in cattle infected naturally
with F. hepatica is a normocytic, hypochromic anaemia,
which is brought about by chronic blood loss, resulting in
iron deficiency, caused by the blood-sucking activity of
adult flukes and by the leakage of blood from the liver to
the intestines.
Increased serum activities of the enzymes AST, GGT
and ALP in infected cattle, compared with the uninfected
group and reference values reported for cattle, have been
shown in this study (table 3). AST, GGT and ALP activities
in the present study were similar to those reported by
others for F. hepatica infection in sheep and cattle. Wycoff
& Bradley (1985) found significantly higher AST activity

Table 3. The mean activities of three serum enzymes and the mean concentration of serum iron in F. hepatica-
infected and uninfected cattle (mean ^ SE).

Groups Number AST (IU/l) GGT (IU/l) ALP (IU/l) Fe (mg/dl)

Infected 86 161.54 ^ 1.91* 39.12 ^ 5.26* 377.98 ^ 2.76* 43.97 ^ 5.89*

Uninfected 30 86.7 ^ 3.7 13.12 ^ 0.87 170.28 ^ 2.4 121.2 ^ 15.26
Reference rangea – 60–150 0–31 35–350 57–162

AST, aspartate aminotransferase; GGT, g-glutamyl transpeptidase; ALP, alkaline phosphatase; Fe, iron.
* Significantly different (P , 0.05).
a
Taken from Radostitis et al. (2007).
88 S. Lotfollahzadeh et al.
in experimentally infected calves as compared with that
in a control group. They also observed severely increased
GGT activity in sera of infected calves, which coincided
with the establishment of adult flukes in the bile ducts
(Wycoff & Bradley, 1985). Anderson et al. (1977) reported
increased plasma activities of AST, GGT and ALP in calves
infected with 500 metacercaria of F. hepatica. In their
research, they found that the maximal mean activities of
GGT and AST of infected calves were nearly 13 and 2.1
times greater than the means of the control group
(Anderson et al., 1977), whereas in the present study the
mean activities of GGT, AST and ALP of infected cattle
were 2.98, 1.86 and 2.21 times greater than the means of
the control group. Bulgin et al. (1984) reported that
experimental infection of calves with flukes did not result
in an increase of serum ALP and serum AST values;
measurement of these enzymes would not be useful in
assessing the degree of fluke infection. In contrast, the
serum values of GGT increased after infection (Bulgin
et al., 1984). In another study in Denmark, analyses of
GGT and AST activities in infected animals revealed no
significant increase in either (Simensen & Nansen, 1974).
To explain this difference, Simensen & Nansen (1974)
proposed that: (1) the infected cows in the study were
adult cattle, which may have grazed in wet, lowland areas
with snail habitats at least twice, or usually for several
seasons, before slaughter; or (2) there were decreased
numbers of established parasites following successive
grazing seasons because the cows had a considerable
resistance to infection.
The results of the present study have shown that
anaemia in cattle naturally infected with F. hepatica is
normocytic and hypochromic due to iron deficiency.
References
Anderson, P.H., Berrett, S., Brush, P.J., Hebert, C.N.,
Parfitt, J.W. & Patterson, D.S.P. (1977) Biochemical
indicators of liver injury in calves with experimental
fascioliosis. Veterinary Record 15, 43 – 45.
Andrews, S.J. (1999) The life cycle of Fasciola hepatica.
pp. 1– 27 in Dalton, J.P. (Ed.) Fasciolosis. Wallingford,
UK, CABI Publishing.
Behm, C.A. & Sangster, N.C. (1999) Pathology, patho-
physiology, and clinical aspects of fasciolosis. pp. 160–224
in Dalton, J.P. (Ed.) Fasciolosis. Wallingford, UK, CABI
Publishing.
Berry, C.I. & Dargie, J. (1976) The role of host nutrition in
the pathogenesis of ovine fascioliosis. Veterinary
Parasitology 2, 317– 332.
Berry, C.I. & Dargie, J.D. (1978) Pathophysiology of ovine
fascioliosis: The influence of dietary protein and iron on
the erythrokinetics of sheep experimentally infected
with Fasciola hepatica. Veterinary Parasitology 4, 327– 339.
Bossaert, K., Lonneux, J.F., Godeau, J.M. & Losson, B.
(1999) Serological and biochemical follow-up in cattle
naturally infected with Fasciola hepatica, and compari-
son with a climate model for predicting risks of
fascioliosis. Veterinary Research 30, 615– 628.
Bulgin, M.S., Anderson, B.C., Hall, R.F. & Lang, B.Z.
(1984) Serum gamma glutamyl transpeptidase activity
in cattle with induced fascioliasis. Research in Veterinary
Science 37, 167– 171.
Gajewswka, A., Smaq-Kozlowska, K. & Wisniewski, M.
(2005) Pathological changes of liver in infection of
Fasciola hepatica. Wiadomosci Parazytologiczne 51,
115 – 123.
Holmes, P.H., Dargie, D.J., MacLean, J.M. & Mulligan,
W. (1968) The anaemia in fascioliosis: studies with
51
Cr-labeled red cells. Journal of Comparative Pathology
78, 415– 420.
Jain, N.C. (1993) Essential of veterinary hematology. 125 pp.
Philadelphia, Lea & Febiger.
Jennings, F.W. (1976) The anaemia of parasitic infections.
pp. 41 – 67 in Soulsby, E.J.L. (Ed.) Pathophysiology of
parasitic infection. New York, Academic Press.
Radostitis, O.M., Gay, C.C., Hinchcliff, K.W. & Constable,
P.D. (2007) Veterinary medicine. 10th edn. pp. 2047–2050.
Edinburgh, Saunders Co.
Rapsch, C., Schewizer, G., Grimm, F., Kohler, L., Bauer,
C., Deplazer, P. & Braun, U. (2006) Estimating the true
prevalence of Fasciola hepatica in cattle slaughtered in
Switzerland in the absence of an absolute diagnostic
test. International Journal of Parasitology 36, 1153– 1158.
Simensen, M.G. & Nansen, P. (1974) Serum-g-glutamyl-
transpeptidase (g-GT) and aspartate-aminotransferase
(AspAT) activities in adult cattle with chronic Fasciola
hepatica infection. Acta Veterinary Scandinavia 15,
239– 243.
Soulsby, E.J.L. (1986) Helminthes, arthropods and protozoa of
domesticated animals. 7th edn. 40 pp. London, Baillière
Tindall.
Spengler, R.N. & Isseroff, H. (1981) Facioliasis: is the
anaemia caused by hematophagia? Journal of Parasitology
67, 886–892.
Symonds, H.W., Mather, D.L., Mallinson, C.B. &
Hughes, D.L. (1983) Bile flow, bile salt secretion and
excretion of iron, copper, zinc and manganese in the
bile of calves infected with Fasciola hepatica. Research in
Veterinary Science 35, 69 – 74.
Wensvoort, P. & Over, H.J. (1982) Cellular proliferation of
bile ductules and gamma-glutamyl transpeptidase in
livers and sera of young cattle following a single
infection with Fasciola hepatica. Veterinary Quarterly 4,
161– 172.
Wycoff, J.H. III & Bradley, R.E. (1985) Diagnosis of
Fasciola hepatica infection in beef calves by plasma
enzyme analyses. American Journal of Veterinary
Research 46, 1015– 1019.
Yang, Q., Mao, H.Y.W., Ferre, I., Bayon, J.E., Mao, X.Z. &
Gonzalez-Gallego, J. (1998) Plasma aspartate amino-
transferase (AST), glutamate dehydrogenase (GLDH)
and gamma-glutamyl transpeptidase (GGT) activities
in water buffaloes with experimental subclinical
fasciolosis. Veterinary Parasitology 78, 129– 136.
(Accepted 25 September 2007)
First Published Online 14 January 2008
q 2008 Cambridge University Press