Summary This study was conducted to determine the electrophoretic pattern of serum protein in goats infected with Babesia ovis. Serum total protein was calculated and serum electrophoresis from 15 goats naturally infected with B. ovis as well as same number of healthy goats was performed. Microscopic examination of Giemsastained peripheral blood smears revealed B. ovis infection. The parasitological diagnosis was confirmed using polymerase chain reaction (PCR) assay by using a pair of B. ovis-specific primers. Mean values total protein and α and γ globulin fractions in infected goats were significantly higher (P&lt;0.05) than those observed in the healthy group

Abtahi

B Esmaeilnejad

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Iranian Journal of Veterinary Research, Shiraz University, 2013, Vol. 14, No. 2, Pages 150-154 
 
 150
Short Paper 
 
Serum protein alterations in goats naturally infected 
with Babesia ovis 
 
Esmaeilnejad, B.1; Tavassoli, M.1*; Asri-Rezaei, S.2; Dalir-Naghadeh, B.2; 
Mardani, K.3; Farhaghpajouh, F.4 and Abtahi, S. M.5 
 
1Department of Pathobiology, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran; 2Department 
of Clinical Sciences, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran; 3Department of Food 
Hygiene and Quality Control, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran; 4MSc in 
Parasitology, Department of Clinical Sciences, Faculty of Veterinary Medicine, Urmia University, Urmia, 
Iran; 5Department of Microbiology, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran 
 
*Correspondence: M. Tavassoli, Department of Pathobiology, Faculty of Veterinary Medicine, Urmia 
University, Urmia, Iran. E-mail: m.tavassoli@urmia.ac.ir 
 
(Received 30 Jan 2012; revised version 26 Jan 2013; accepted 23 Feb 2013) 
 
Summary 
 
 This study was conducted to determine the electrophoretic pattern of serum protein in goats infected with 
Babesia ovis. Serum total protein was calculated and serum electrophoresis from 15 goats naturally infected 
with B. ovis as well as same number of healthy goats was performed. Microscopic examination of Giemsa-
stained peripheral blood smears revealed B. ovis infection. The parasitological diagnosis was confirmed using 
polymerase chain reaction (PCR) assay by using a pair of B. ovis-specific primers. Mean values total protein 
and α and γ globulin fractions in infected goats were significantly higher (P<0.05) than those observed in the 
healthy group. 
 
Key words: Serum protein, Electrophoresis, Babesia ovis, Goats, PCR 
 
Introduction 
 
 Serum protein electrophoresis is a 
valuable tool for evaluating health of body, 
both in diagnosing and clinical monitoring 
of several diseases (Abate et al., 2000). 
Although a specific diagnosis can rarely be 
made with serum protein electrophoresis, 
processes of diseases can be recognized by 
serum protein patterns, and alterations to 
these patterns are associated with a variety 
of different conditions and diseases (Lobetti 
et al., 2000). 
 Blood serum contains two major 
proteins including albumin and globulin. 
Serum protein electrophoresis from healthy 
goats exhibits four fractions: albumin, α, β 
and γ fractions, which, in β-globulin was 
divided to β1 and β2 (Alberghina et al., 
2010). 
 Recent studies have focused on serum 
protein alterations in canine babesiosis 
(Abate et al., 2000; Camacho et al., 2005); 
however, there are no data regarding serum 
protein pattern in goats naturally infected 
with B. ovis. Therefore, this study was 
conducted to evaluate the specific effects of 
Babesia infection on different serum protein 
fractions in goats, as well as to assess its 
application in diagnosis by study of the 
quantitative alterations in serum proteins 
associated to the disease. 
 
Materials and Methods 
 
 Thirty goats of different local breed 
(West-Azerbaijan) were studied and 
classified into two groups. All of the animals 
were more than 1-year-old (≥1 year). The 
infected group was comprised of 15 goats 
(10 female and 5 male) naturally affected by 
B. ovis. As control group, 15 clinically 
healthy goats (8 female and 7 male) were 
also sampled from the same farm. Jugular 
 
Iranian Journal of Veterinary Research, Shiraz University, 2013, Vol. 14, No. 2, Pages 150-154 
 
 151
blood samples were obtained from infected 
and control goats. Blood serum was then 
separated by centrifugation at 750 g for 15 
min and stored at -20°C until measurements 
were done. Also, blood samples from ear 
vein were obtained for the preparation of 
thin blood smears, stained with Giemsa, for 
the detection of Babesia piroplasms. PCR 
was performed not only to detect B. ovis in 
blood of infected animals but also to rule out 
the presence of Babesia spp. infection in 
both healthy and infected animals. DNA was 
extracted using a DNA purification kit 
(Fermentas, Germany) according to the 
manufacturer’s instructions. One pair of 
primers, P1 (5'-CACAGGGAGGTAGTGA 
CAAG-3') and P2 (5'-AAGAATTTCACCT 
ATGACAG-3'), specific for amplification of 
Babesia species 18S ssu RNA (Shayan et 
al., 2007), were used in PCR assay as a 
positive quality control for the Babesia 
species. Seminested PCR of the PCR 
products of B. crassa with primer pair of P2 
(5'-AAGAATTTCACCTATGACAG-3') 
and P4 (5'-GTTATGGCCCGTTGGCTTAT-
3') and B. motasi with primers P2 (5'-
AAGAATTTCACCTATGACAG-3') and P5 
(5'-CGCGATTCCGTTATTGGAG-3') were 
conducted as well (Shayan et al., 2007). In 
addition, amplification of B. ovis was 
performed by species-specific primer pair 
previously reported and used to amplify a 
fragment of 549 bp. The sequences of 
primers were as follows: 
 
Bbo-F, 5'-TGGGCAGGACCTTGGTTGTTCT-3' 
Bbo-R, 5'-CCGCGTAGCGCCGGCTAAATA-3' 
 
 PCR was performed in 50 µl total 
reaction volume containing 5 µl of 10 × 
PCR buffer, 2 mM MgCl2, 250 µM of each 
of the four deoxynucleotide triphosphate, 
1.25 U Taq DNA polymerase (Fermentas, 
Germany), 50 pmol of each primer and 50 
ng of extracted DNA. For negative control, 
distilled water was added instead of nucleic 
acid. PCR reaction was performed in a DNA 
thermocycler (Model CP2-003; Corbett 
Research, Australia). Cycling condition was 
95°C for 5 min, followed by 45 cycles at 
94°C for 45 sec, 63°C for 45 sec and 72°C 
for 1 min with a final extension step of 72°C 
for 10 min. Amplified products were 
electrophoresed on 2% agarose gel and 
stained with ethidium bromide to visualize 
the amplified DNA fragments under 
ultraviolet light. Serum total protein 
concentrations were determined by Biuret 
method and spectrophotometer (Perkin-
Elimer, Junior model 35). Albumin and 
globulins were separated by cellulose acetate 
(Helena, France) electrophoresis in barbital 
buffer (Helena, France), pH = 8.6 at 180 V, 
4 mA, for 15 min using Sebia system (Sebia, 
France). The separated proteins were stained 
with Panceau S (Sigma, Germany), and then 
destained. After plate drying at 50-60°C, the 
relative levels of separated proteins were 
scanned by densitometer at 525 nm. Data 
was checked graphically (histograms, q-q 
plots and box plots) for the outliers and 
normality. No extreme outlier was detected 
and the assumptions were satisfied. 
Independent samples t-test was used to 
compare the differences between groups. 
The level of significance was set at P<0.05. 
Statistical analyses were carried out using 
SPSS (Release 17, SPSS Inc., and Chicago, 
Illinois). Results are presented as 
means±SEM. 
 
Results 
 
 All of the infected animals were positive 
by PCR with B. ovis-specific primers (549 
bp). On the contrary, there was no 
amplification of B. ovis DNA in the control 
animals (Fig. 1). 
 Serum protein electrophoresis of 
samples exhibited four fractions: albumin, α, 
β and γ fractions in goats which, in the 
samples, α globulin band was divided into α1 
and α2. Electrophoretic pattern of serum 
from healthy and infected goats is shown in 
Fig. 2. 
 Concentrations (mean±SEM) of serum 
total protein, albumin and globulins of 
healthy and affected animals are presented 
in Table 1. 
 
Discussion 
 
 Although evaluation of serum proteins 
and their electrophoretic profiles have been 
widely used in human medicine since the 
1950s, in veterinary medicine the mentioned 
technique is not used as a common 
diagnostic method for the evaluation of 
 
Iranian Journal of Veterinary Research, Shiraz University, 2013, Vol. 14, No. 2, Pages 150-154 
 
 152
dysproteinemias (Attaelmannan and 
Levinson, 2000). Many researchers stated 
that fractions display important differences 
among all domestic animals between healthy 
and infected animals (Kean and Doxey, 
1981). As far as goats are concerned, values 
of serum protein fractions in healthy animals 
disagreed with a previous study (Mendes 
Ahid and Soto-Blanco, 2010). 
 
 
 
Fig. 1: PCR products amplified using B. ovis 
specific primers. Lane 1: 50 bp molecular 
ladder, Lane 2: positive blood sample (positive 
control), Lane 3: distilled water (negative 
control), and Lane 4-6: PCR products from 
infected goats 
 
 Earlier studies showed that protein 
fractions display important differences 
among all domestic animals between healthy 
and Babesia-infected animals (Barrera et al., 
2010). 
 In our study, the mean value obtained 
for serum total protein in the affected 
animals was significantly higher (P<0.05) 
than that obtained in the healthy ones. 
Hyperproteinemia in babesiosis is usually 
caused by dehydration, resulting from 
lethargy and anorexia associated with the 
disease (Divers, 1998). The hydration leads 
to hemconcentration through reduction in 
fluid volume and consequent hyper-
proteinemia (Kaneko et al., 2008). 
 
Table 1: Concentration (means±SEM) of 
serum total protein and protein fractions 
(g/dl) in healthy and infected goats 
Parameter Control (n=15) Infected (n=15) 
Tp† 7.05 ± 0.01   7.50 ± 0.09*
Alb‡ 2.70 ± 0.06   2.22 ± 0.01** 
α1 0.85 ± 0.02   1.31 ± 0.07
** 
α2 1.00 ± 0.03   1.04 ± 0.02
NS 
β 0.58 ± 0.02   0.63 ± 0.01NS 
γ 1.90 ± 0.03   2.20 ± 0.04** 
Asterisks indicate the presence of the 
significance between the two groups, * P<0.01 
and ** P<0.05. NS: Non-statistically significant. 
Tp†: Total protein, and Alb‡: Albumin 
 
 The mean concentration of albumin in 
healthy animals was significantly higher 
(P<0.05) than that obtained in diseased 
animals. The hypoalbuminemia observed in 
goats in this study is in agreement with the 
findings of Apaydin and Dede (2010) and 
Esmaeilnejad et al. (2012) in ovine 
babesiosis. The most likely explanation for 
this finding would be hepatopathy that may 
develop during the disease (Colville, 2002), 
to the symptomatic anorexia state (Kaneko 
et al., 2008) and urinary loss of albumin 
associated with renal failure (Camacho et
 
 
 
Fig. 2: Representative serum protein patterns of healthy (a) and infected (b) goats 
 
Iranian Journal of Veterinary Research, Shiraz University, 2013, Vol. 14, No. 2, Pages 150-154 
 
 153
al., 2005). 
 The proteins located in α1-globulin band 
are significantly increased (P<0.05) in 
diseased animals compared with the healthy 
ones. These results are in accordance with 
the findings of Camacho et al. (2005). The 
most likely reason for this elevation is the 
release of acute phase reactants, such as α1-
antitrypsin, α1-acid glycoprotein, α2-
macroglobulin and haptoglobin in response 
to inflammation process (Barrera et al., 
2010). 
 When the result of β-globulin sub-
fraction in goats with babesiosis was 
compared with those obtained in the control 
group, no significant difference was 
observed (Table 1). An increase in its 
concentration related to acute hepatic 
damage has been described as a 
consequence of the increase in transferrin 
concentration (Kaneko et al., 2008). This 
protein also contributes to the β-globulin 
decrease if the hepatic disease is chronic 
(Barrera et al., 2010). Increase in the 
plasminogen concentration, which is 
included in the β-globulin fraction, due to 
intra-vascular coagulation may be explained 
by elevation of β-globulin concentration 
(Kaneko et al., 2008). However, further 
studies are needed to precisely assess the 
acute phase response in caprine babesiosis. 
Finally, concentration of β-globulin fraction 
may be related to the albumin fraction in 
cases of extra-vascular liquid exudation 
(Barrera et al., 2010). This fact, namely 
hypoalbuminemia and lower β-globulin was 
observed in present study. 
 The immunity mechanisms in babesiosis 
are mainly of humoral nature (Rahbari et al., 
2008), although the protozoa can activate a 
cellular-type immune response (Tizard, 
1996). In accordance with the findings of 
other studies (Camacho et al., 2005), our 
results indicated that the mean value 
obtained for the γ-globulin fraction in 
infected goats was significantly higher 
(P<0.05) than the one obtained in healthy 
goats. This increase, observed in the serum 
γ-globulins concentration is related to the 
humoral immunity activation by parasitation 
of the animals studied (Barrera et al., 2010). 
 The present study revealed that the 
increase of total protein, associated with the 
increase of α and β globulin fractions, is a 
common finding in serum electrophoresis of 
goats suffering from babesiosis, which can 
be useful for diagnosis and prognosis of 
caprine babesiosis as a supplementary 
analysis in combination with clinical, 
parasitological and molecular findings of 
this disease. 
 
Acknowledgement 
 
 The positive control for B. ovis was 
kindly provided by Prof. Rahbari (Faculty of 
Veterinary Medicine, University of Tehran, 
Iran). 
 
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Serum protein alterations in goats naturally infected with Babesia ovis

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Serum protein alterations in goats naturally infected with Babesia ovis

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