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Revista Scientia Parasitologica 2004, 5(1-2):71-74
EFFECT OF HAEMOGREGARINA STEPANOVI DANILEWSKY, 1885
(APICOMPLEXA:HAEMOGREGARINIDAE) ON ERYTHROCYTE
MORPHOLOGY IN THE EUROPEAN POND TURTLE, EMYS
ORBICULARIS (LINNAEUS, 1758) BLANFORD, 1876
(TESTUDINES:EMYDIDAE)
Mihalca A.D., Cozma V., Gherman C., Achelăriţei D.
University of Agricultural Sciences and Veterinary Medicine, Faculty of Vererinary Medicine,
Department of Parasitology and Parasitic Diseases, str. Mănăştur 3, 400372, Cluj-Napoca, Romania,
email: amihalca@usamvcluj.ro
Key words: Haemogregarina, Emys, erythrocyte morphology
Abstract. Changes in erythrocyte morphology were studied in five european pond turtles
(Emys orbicularis) infected with microgametoytes and trophozoites of Haemogregarina stepanovi.
Shape alterations were mor pronounced in erythrocytes infected with trophozoites. The more severely
affected morphometrical parameters were shape index of erythrocytes and shape index of the nucleus.
INTRODUCTION
Haemogregarina stepanovi has been described by Danilewsky in 1885 and has been
reported by many authors in the European pond turtle, Emys orbicularis. Hahn (1909)
described in detail the stages in the turtle. Reichenow (1910) published a detailed review on
the species. Mihalca (2002) described the effect of the infection on the differential blood
count of Emys orbicularis.
The intraerythrocytic stages in turtles include trophozoites, macroschizonts,
macromerozoites,
microschizonts,
micromerozoites,
microgametocytes
and
macrogametocytes. Part of these stages and transformations take place in the bone marrow,
only a few stages being seen in the peripheric blood. In this paper we present the results of the
study of the effect of infection with Haemogregarina stepanovi trophozoites and
microgametcoytes on erythrocytic morphology in turtles.
MATERIAL AND METHOD
Five turtles, infected with Haemogregarina stepanovi, collected from Drăgăşani
(Vâlcea County, Romania) were used in this study. Blood was collected from the coccigian
vein using heparin as anticoagulant. Smears were stained using Dia Panoptic ® (Reagens Kft;
Diagon Kft, Hungary) method and examined with the immersion objective of an Olympus
BX41® microscope. Pictures were taken with an Olympus DP10 ® digital camera and
measured using Adobe Photoshop 6.0. From each turtle, 60 erythrocytes were measured (20
uninfected, 20 infected with trophozoites and 20 infected with microgametocytes). Four
parameters were taken in account: length of erythrocyte (L), width of erythrocyte (W), length
of nucleus (Ln) and width of nucleus (Wn). Four indexes were calculated: L/W, Ln/Wn, L/Ln
and W/Wn. Some staining properties and cytomorphology aspects are also commented.
RESULTS AND DISCUSSIONS
In erythrocytes infected with trophozoites, the nucleus was displaced either in
marginal position (figure 1) or in polar position (figure 2) with normal or elongated shape. In
both cases, the nucleus was stained more intensly than in normal erythrocytes. In some cases,
a vacuole can be noticed around the trophozoite (figure 3). In other cases, the erythrocytes can
take abnormal shapes with expansions (figure 4). Sometimes the erythrocytes can be ruptured
and trophozoites seen free, outside the cell (figure 5).
In erythrocytes infected with microgametocytes, usually the nucleus preserves its
position. Some nuclei preserve their shape (figure 6) while others have irregular shapes
(figure 7). Severe cell shape alteration can occure in erythrocites infected with
microgametocytes (figure 8). Double infections with microgametocytes rarely occure (figure
9).
The morphometric values of normal and infected erythrocytes is shown in table 1. The
ratios between morphometric parameters are shown in table 2.
Figures: 1. Marginal displacement of the nucleus. 2. Apical displacement of the nucleus. 3. Vacuole around the
trophozoite. 4. Abnormal shape of eythrocyte. 5. Free trophozoite. 6. Nucleus with normal shape apical
displacement. 7. Irregular shape of nucleus. 8. Altered shape of erythrocytes. 9. Double infection with
microgametocytes
Table 1
Morphometric values of normal erythrocytes (N) compared to erythrocytes infected with microgametcoytes (M)
and trophozoites (T) of Haemogregarina stepanovi
L
N
M
T
19,99 19,92 20,68
±
±
±
Emo1
1,23
0,85
1,56
20,36 19,51 22.62
±
±
±
Emo2
1,26
0,81
0,95
21,89 19,15 22,13
±
±
±
Emo3
1,35
0,49
1,43
20,01 19,92 22,93
±
±
±
Emo4
1.30
1,63
1,76
19,93 19,63 22,13
±
±
±
Emo6
1,13
1,13
1,51
Mean 20.44 19.63 22.10
St dev
0.83
0.32
0.86
* - values outside normal ranges
Turtle
N
12,39
±
0,74
12,45
±
0,96
13,67
±
0,77
12.77
±
0,78
12,78
±
0,95
12.92
0.45
W
M
11,87
±
1,10
12,01
±
1,20
12,05
±
1,77
11,43
±
1,23
11,95
±
0,74
11.86
0.25
T
11,39
±
1,23
11,84
±
0,70
13,21
±
1,22
11.07
±
2,28
13,13
±
1,60
12.13
0.99
N
6,65
±
0,50
7,20
±
0,71
6,71
±
0,71
6,25
±
0,65
7,11
±
0,66
6.78
0.38
Ln
M
6,19
±
0,59
6,62
±
0,91
6,33
±
1,23
6,25
±
0,56
6,75
±
0,72
6.43
0.24
T
7,97
±
0,78
7,93
±
0,78
8,32
±
0,98
8,00
±
0,48
8,20
±
0,28
8.08
0.17
N
4,20
±
0,35
4,90
±
0,47
4,55
±
0,43
4,73
±
0,46
4,85
±
0,40
4.65*
0.28
Wn
M
3,55
±
1,10
3,78
±
0,89
3,73
±
0,75
3,82
±
0,54
4,42
±
0,60
3.86*
0.33
T
2,86
±
0,28
3,13
±
0,50
3,10
±
0,93
3,24
±
0,34
3,40
±
0,28
3.15*
0.20
Table 2
Morphometric ratios of normal erythrocytes (N) compared to erythrocytes infected with microgametcoytes (M)
and trophozoites (T) of Haemogregarina stepanovi
L/W
N
M
T
1,62
1,69
1,84
±
±
±
Emo1
0,14
0,11
0,28
1,64
1,62
1,92
±
±
±
Emo2
0,11
0,12
0,17
1,61
1,60
1,69
±
±
±
Emo3
0,12
0,19
0,20
1,57
1,76
2,16
±
±
±
Emo4
0,13
0,26
0,61
1,57
1,65
1,69
±
±
±
Emo6
0,13
0,12
0,09
Mean
1.60
1.66
1.86
St dev
0.03
0.06
0.19
* - values outside normal ranges
Turtle
N
1,59
±
0,16
1,48
±
0,19
1,49
±
0,25
1,34
±
0,20
1,47
±
0,16
1.47
0.09
Ln/Wn
M
1,94
±
0,82
1,75
±
0,44
1,77
±
0,69
1,68
±0,35
1,55
±
0,27
1.74
0.14
T
2,81
±
0,32
2,61
±
0,56
2,94
±
1,02
2,47
±
0,12
2,42
±
0,12
2.65*
0.22
N
3,02
±
0,29
2,85
±
0,30
3,29
±
0,31
3,24
±
0,44
2,82
±
0,18
3.04
0.22
L/Ln
M
3,24
±
0,31
2,95
±
0.32
3,07
±
0,52
3,21
±
0,38
2,93
±
0,31
3.08
0.14
T
2,62
±
0,32
2,88
±
0,39
2,70
±
0,36
2,88
±
0,34
2,70
±
0,09
2.76
0.12
N
2,96
±
0,24
2,56
±
0,25
3,03
±
0,30
2,72
±
0,24
2,64
±
0,20
2.78
0.20
W/Wn
M
3,66
±
1,34
3,17
±
0,87
3,34
±
1,15
3,07
±
0,63
2,75
±
0,41
3.20
0.34
T
4,02
±
0,57
3,87
±
0,61
4,67
±
1,63
3,41
±
0,63
3,86
±
0,15
3.97
0.46
As shown in the tables above, the infection with Haemogregarina stepanovi induces
different erythocytic changes, depending on the infective stage. The more marked changes
occur in the case of infection with trophozoites, because of their large size. The erythrocytes
are longer and slightly thinner with a higher L/W ratio. The length of the nucleus is also
increased but its width is reduced giving a flattened aspect to the nucleus. In erythrocytes
infected with microgametocytes, the changes are less evident. The cells are slightly smaller,
with a smaller and flattened nucleus.
Normal morphometric values of erythrocytes in Emys orbicularis was studied by
Uğurtaş et al. (2003) in Turkey. They found the following ranges: L = 19.52-23.18; W =
10.98-14.64; L/W = 1.50-2.05; Ln = 6.10-8.54; Wn = 4.88-6.71; Ln/Wn = 0.90-1.75.
According to these data, in our turtles, all morphometric values for uninfected erythrocytes
are within these ranges except the width of the nucleus which is slightly narrower than in
turtles from Turkey. In the infected erythrocytes, there are values which do not fit in the
normal ranges presented by the turkish authors. Thus, the nucleus of the infected erythrocytes
(with microgametocytes or with trophozoites) is more narrow and nucleus shape index is also
higher in erythrocytes infected with trophozoites.
We also have to consider the possibility that in infected turtles, the uninfeced
erythrocytes could suffer morphological changes but further studies need to be done.
BIBLIOGRAPHY
1.
2.
3.
4.
Hahn C.W., 1909, The stages of Haemogregarina stepanovi Danilewsky found in the blood of turtles
with especial reference to changes in the nucleus, Archiv für Protistenkunde 17:307-376
Mihalca A.D., Achelăriţei D., Popescu P., 2002, Haemoparasites of the genus Haemogregarina in a
population of european pond turtles (Emys orbicularis) from Drăgăşani, Vâlcea county, Romania,
Revista Scientia Parasitologica 3(2):22-27
Reichenow E., 1910, Haemogregarina stepanowi. Die Entwicklungsgeschichte einer Hämogregarine,
Archiv für Protistenkunde 20:251-350
Uğurtaş I.H., Sevinç M., Yildirimhan H.S., 2003, Erythrocyte size and morphology os some tortoises
and turtles from Turkey, Zoological Studies 42(1):173-178