Comp Haematol Int (1994) 4:17%179
© 1994 Springer-Verlag London Limited
Original Article
Hepatozoon canis: Size Measurement of the Gametocyte Using Image
Analysis Technology
T. Waner 1, G. Baneth 2, A. Z u c k e r m a n 3 and A. Nyska 4
1Life Science Research Israel, Ness Ziona; 2Koret School of Veterinary Medicine, Hebrew University, Beit Dagan; 3Israel Institute of
Biological Research, Ness Ziona; and 4Department of Pathology, Kimron Veterinary Institute, Beit Dagan, Israel
Abstract. This study presents definitive dimensions of
the gametocyte Hepatozoon canis using image analysis
technology. The mean length and width of the gameto-
cyte was found to be 11.42 #m and 5.39/~m, respecti-
vely. The mean area of the gametocyte was found to be
45.88 #m 2. The average perimeter of the gametocyte
was 28.92 /~m. The results compare well with some
previous measurements using light-microscopic tech-
niques.
Keywords: Dogs; Hepatozoon canis; Morphology;
Neutrophil; Protozoa
Introduction
Hepatozoon canis, a protozoan with a coccidian-like life
cycle was first described in dogs by James in 1905 (Laird
1959). This haemogregine inhabits the neutrophils of
dogs in the form of a gametocyte (Craig et al. 1978;
Ibrahim et al. 1989). The organism is transmitted in
dogs and other carnivores by ingestion of the brown dog
tick, Rhipicephalus sanuineus. Neutrophils become
infected by phagocytosis or direct penetration of micro-
merozoites, which develop into gametocytes (Swango et
al. 1989). This is the form of H. canis found in the
leucocytes of infected dogs.
The first report of the size of the gametocyte of H.
canis was made by Perak in 1906 in Malaya. He reported
its dimensions as 11-12 /~m by 4.2-5.2 /zm (Laird
1959). Mathis and Leger stated in 1911 that the average
Correspondence and offprint requests to: Dr Trevor Waner, Life
Science Research Israel, PO Box 139, Ness Ziona 70451, Israel.
COMPARATIVE
HAEMATOLOGY
INTERNATIONAL
measurements of examples from Tonkin were 10.5 by 5
/~m, and Porter reported in 1919 that the gametocyte
was in the range 8-12/tm by 4.5-6/~m in Transvaal,
South Africa (Laird 1959). Soulsby (1968) states that
the gametocytes measure 6 x 3/~m. These measure-
ments were made microscopically using a micrometer
measuring a representative number of organisms. This
technique is not regarded as accurate, and the results
revealed values that vary widely with large limits. The
technique of image analysis has not been previously
used to measure physical characteristics of protozoa,
and to the best of our knowledge has not been used to
measure the gametocyte of H. canis in dogs.
This report documents some of the physical size
characteristics of H. canis from a number of naturally
infected dogs in Israel. Variations in physical size
characteristics of H. canis between dogs and within dogs
is presented. We propose this technique as a standard
and accurate method as an aid in the classification of
organisms such as H. canis.
Materials and Methods
Three cases of dogs naturally infected with H. canis
were used in this study. Peripheral blood smears were
prepared from blood in EDTA anticoagulant. The
smears were fixed in alcohol and stained with a May-
Grunwald-Giemsa stain (Mercer and Craig 1993)
Image analysis was carried out using the Galai Super-
Cue 3/ScanArray system. This consisted of an Olympus
BH2-MJLT microscope with a motorised stage and
auto-focus controller attached to a video camera (Sony
RGB CCD model). The image was projected onto an
image monitor through Colour Frame Grabber (CFG)
software in a personal computer (AT386). The Cue-3
178 T. Waner et al.

Fig. 1. The gametocyte, Hepatozoon canis.

image analysis software (Version 5.1) was obtained gametocyte are presented in Tables 1-4. The mean
from Galei Production Ltd, Israel. length and width of 26 gametocytes examined from the
Calibration of the system was carried out for each lens dogs was found to be 11.42/~m and 5.39/zm respecti-
of the microscope using a reference stage graticule of vely. The range of the length of the gametocyte was
the National Physical Laboratory (Middlesex, UK). 10.66/~m to 12.99/zm, and the width 4.74 #m to 8.22
The procedure for measuring the gametocyte was as ~m. No differences among the three dogs examined
follows. The gametocyte was first identified on the were found regarding these parameters of the gameto-
monitor (Fig. 1). The 'object' to be measured was cyte.
defined for the entire gametocyte in the 'subarea mode'. The mean area of the gametocyte was found to be
The 'object' is defined as an image demarcated by a 45.88 #m 2. No differences between the gametocytes
specific wavelength in which all the pixels (smallest
spatially digitised unit of an image) are connected, and Table 1. Length of gametocyte of Hepatozoon canis as measured by
image analysis technology
which is completely surrounded by pixels of an alternate
state (Joyce Loebl, 1989).
The measurements made were as follows. Dog 1 Dog 2 Dog 3 Overall ANOVA
P
1. A r e a - the number of pixels in the 'object' multiplied
by the calibrated area of a pixel; Mean (#m) 11.44 11.71 11.22 11.42 0.1826
2. Perimeter- total length of the border of the 'object'; Standard
3. Length - Feret's maximum diameter; deviation (/~m) 0.67 0.37 0.34 0.51
4. Width - Feret's minimum diameter. Coefficient of
variance (%) 5.81 3.18 2.99 4.50
(Feret measurement of diameter is defined as the Maximum (gin) 12.99 12.04 11.88 12.99
distance between two parallel tangents which are on the Minimum (/~m) 10.66 11.03 10.86 10.66
opposite side of the 'object' (Joyce and Loebl, 1989).) Number of
Measurements were made 36 times about the object in gametocytes 10 6 10 26
order to determine the maximum and minimum
diameters.
Table 2. Width of gametocyte of Hepatozoon canis as measured by
image analysis technology
Statistical Analysis
Dog 1 Dog 2 Dog 3 Overall ANOVA
The mean, standard deviation and coefficient of vari- P
ance were calculated for each parameter and analysed
by the analysis of variance (ANOVA). All statistical Mean (/zm) 5.10 5.94 5.36 5.39 0.1689
procedures were carried out using SAS software (SAS Standard
Institute Inc., Cary, NC). deviation (#m) 0.25 1.09 1.03 0.86
Coefficient of
variance (%) 4.93 18.30 19.17 16.01
Results Maximum (/~m) 5.60 7.79 8.22 8.22
Minimum (/~m) 4.81 5.06 4.74 4.74
Statistical parameters (mean, standard deviation, coef- Number of
ficient of variance, maximum and minimum values) gametocytes 10 6 10 26
describing the length, width, perimeter and area of the
Size Measurements of the Gametocyte, Hepatozoon canis 179

Table 3. Area of gametocyte of Hepatozoon canis as measured by measured by a number of researchers using microscopic
image analysis technology techniques. We propose the use of image analysis
techniques for the accurate measurement of the organ-
Dog 1 Dog 2 Dog 3 Overall ANOVA ism.
P The measurement of the gametocyte of H. canis in the
neutrophils of naturally infected dogs, can be regarded
Mean Gum2) 44.89 47.94 45.64 45.88 0.2642 as accurate and definitive due to the technology used.
Standard The values previously presented in the literature (Laird
deviation (/~m2) 3.90 3.77 3.04 3.62 1959; Soulsby 1968), were within the range of the
Coefficient of measurements found in this study, although those
variance (%) 8.69 7.96 6.66 7.88
reported by Soulsby (1986) were markedly lower.
Maximum (~um2) 49.80 53.31 50.87 53.31
No previous studies have reported the area and
Minimum (#m2) 38.46 41.72 41.00 38.46
perimeter of the H. canis gametocyte. The reason for
Number of
gametocytes 10 6 10 26 the statistical differences found between the perimeter
measurements of the gametocytes of the three dogs
examined is not understood, although it should be noted
that the differences between the values for the three
Table 4. Perimeter of gametocyte of Hepatozoon canis as measured by dogs was very small.
image analysis technology
In conclusion, this study presents definitive
dimensions of the gametocyte H. can& using image
Dog 1 Dog 2 Dog 3 Overall ANOVA analysis technology. The results compare well with
P some of the previous measurements using light-micro-
scopic techniques.
Mean (/~m) 30.52 28.89 27.33 28.92 0.0008
Standard
deviation (/~m) 2.11 1.46 0.98 2.10
Coefficient of
variance (%) 6.91 5.04 3.59 7.26
References
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