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Hematozoa in Thin Blood Smears

Article  in  Journal of Wildlife Diseases · August 1995

DOI: 10.7589/0090-3558-31.3.436 · Source: PubMed

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 Journal of Wildlife Diseases, 31(3), 1995, pp. 436-438
© Wildlife Disease Association 1995

LETTER TO THE EDITOR...

Hematozoa in Thin Blood Smears

We have read with interest several re- tial source of variation. Time periods be-
cent papers published in the Journal of tween host death and blood collection can
Wildlife Diseases and elsewhere on avian be extremely variable between host mdi-
hematozoa in thin blood smears. We are viduals sampled in this manner. We have
specifically concerned with the sampling seen birds presented to biologists at check
and quantification procedures used in some stations that ranged from those recently
of these studies, particularly in view of killed (within 15 mm) to those that had
earlier concerns raised by Godfrey et al. been dead for at least 3 hr. Sampling blood
(1987). from these hosts assumes that parasite be-
Workers have examined blood samples havior is similar across the entire range of
collected from both live-trapped and shot time periods (between host death and sam-
birds in several recent studies (Stacey et ple collection) in which hosts are sampled.
al., 1990; Bennett et al., 1991a; Mahrt et Unfortunately, factors associated with he-
al., 1991; Telford et al., 1992; Garvin et matozoan response to host death have not
al., 1993; O’Dell and Robbins, 1994). We been assessed adequately for this assump-
could find no account as to whether these tion to be made unequivocally. We are
two very different sampling procedures aware of only one study (West and Starr,
were tested for possible inherent variation 1940) that anecdotally reported how vary-
in parasitemia prior to pooling and inter- ing time periods between host death and
pretation of the data. Hopefully, this as- sample collection influenced detection of
pect was considered by the authors and Leucocytozoon smithi in domestic turkeys
inadvertently omitted in their papers. Us- (Meleagridis gallopavo). We believe that
ing a mixture of sampling methodologies until there are rigorous studies that can
without regard to inherent variation re- resolve this issue, blood smears taken from
sulting from different techniques seems dead birds should be made within a stan-
precarious. First, it assumes that hemato- dardized time period as soon as possible
zoans are distributed similarly in the blood after host death, and beyond which blood
of both living and dead birds. Second, blood samples are not collected.
often is sampled at a different location in Several workers have presented only
a live host than in a dead host. It does not prevalence data in studies on hematozoa
seem likely that blood obtained via the (Stacey et al., 1990; Bennett et al., 1991a,
brachial or tarsal veins in living birds (sam- b; Mahrt et al., 1991; Telford et al., 1992;
pling peripherally circulating blood) and Forbes et al., 1994; Taft et al., 1994). It is
from the heart or deep core blood in dead uncertain as to what significance can be
birds represents uniformity in sample col- attached to presence and absence data on
lection. We believe that such lack of sam- hematozoans from blood smears. Our view
pling standardization represents poor ex- is based on the following observations. First,
perimental design. Data collected using dif- absence of hematozoans in blood smears
ferent sampling methodologies should be does not necessarily indicate that the host
tested to determine if pooling of the data is not infected. Host immune response and
is appropriate. host-parasite mechanisms associated with
The use of hunter-shot birds (Castle and prepatent, latent, and relapse periods are
Christensen, 1990; Stacey et al., 1990; important factors that influence the prev-
O’Dell and Robbins, 1994) sampled at alence and density of hematozoans found
hunter check stations represents a poten- in circulating blood cells of the host. These

436
 LETTER TO THE EDITOR 437

concepts are reviewed by Atkinson (1991) Statistical analyses of data using deniva-
and Greiner (1991). Thus, low hematozoan tives of actual and estimated quantities
density coupled with short examination seems tenuous. Thus, we question the use-
times of blood smears could result in sub- fulness of presenting partially quantified
stantiah misclassifications, such as, false intensity data that cannot be readily sub-
negatives. Forrester et al. (1974) clearly jected to statistical analyses.
demonstrated how wild turkeys initially We have shown that quantification of
classified negative for Plasmodium sp., Haemoproteus iriaccallumi (=H. colum-
based on blood smears, were positive when hoe n. comb.) in blood smears from mourn-
subinoculation techniques were employed. ing doves (Zenaida macroura) must be
Second, ecological relationships between made by directly counting a sufficient
hosts and their parasites cannot be assessed number of enythnocytes to provide an ac-
adequately using only prevalence data. curate estimate of the number of pana-
Even when density data are available, it sites/number of erythrocytes (Godfrey et
is not always possible to determine periods al., 1987). Application of this methodology
of peak transmission potential or the re- was used to evaluate mntraerythrocytic he-
lation of infection to disease. Certainly, matozoan communities in hosts with high
conclusions regarding these parameters (Godfrey et al., 1990) and low (Fedynich
within host populations based on preva- et al., 1993) parasite densities. We realize
lence data alone are unfounded. Thus, that direct counts of a sufficient number
without intensity data, the ecological and of erythrocytes in some hosts can be te-
clinical significance of hematozoan infec- dious, due to low densities of parasites; it
tion remains obscure. often requires several hours to count 10,000
In reviewing those papers in which in- enythrocytes. However, direct counts of
tensity (number of parasites/number of both parasites and erythrocytes provide a
enythrocytes within infected hosts) was re- reproducible estimate of parasite intensity
ported, we found that intensity often was that can be compared statistically using
calculated by direct counting of parasites various intrinsic and extrinsic factors. Thus,
and estimating the numbers of erythro- until a better methodology is developed,
cytes examined, based on the number of we suggest that researchers directly count
fields of view examined (Apanius and the number of erythnocytes when quan-
Kirkpatrick, 1988; Castle et al., 1988; Allan tifying mntnaenythrocytic hematozoans.
and Mahrt, 1989; Castle and Christensen, Unfortunately, little effort has been di-
1990). We believe that the estimated num- rected toward developing quantification
ber of erythrocytes examined, derived from procedures for hematozoans such as Leu-
extrapolations, is inappropriate for the fol- cocytozoon spp. that may occur in both
lowing reasons. First, due to the extreme enythrocytes and leucocytes. Based on some
variation of erythrocyte densities encoun- data from wild turkeys, L. smithi density
tered on blood smears, estimated total may vary concordantly with erythrocyte
numbers of erythrocytes examined, based densities on blood smears (A. M. Fedynich,
on a subsample directly counted in several unpubl.; 0. E. Rhodes, Jr., pens. comm.).
randomly chosen fields of view, will not We believe that in view of these ideas,
provide reproducible results. Second, when many researchers should upgrade and re-
intensity data are derived from partially vise their experimental design. It is im-
quantified density data (actual number of perative that papers examining hemato-
parasites counted/estimated number of zoans cleanly state how hosts were sam-
erythrocytes examined in each host indi- pled, test for sampling bias if differing col-
vidual), an estimation error is generated lection methods were used, include both
from the original estimated quantities prevalence and intensity data for intraer-
(density values of each individual host). ythnocytic hematozoans, and completely
 438 JOURNAL OF WILDLIFE DISEASES, VOL. 31, NO. 3, JULY 1995

describe
were
how prevalence
determined.
and intensity
Incorporation of
data
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