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Veterinary Parasitology 153 (2008) 126–130
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Cytauxzoon felis infections are present in bobcats
(Lynx rufus) in a region where cytauxzoonosis
is not recognized in domestic cats
Adam J. Birkenheuer a,*, Henry S. Marr a, Camille Warren a, Anne E. Acton a,
Eric M. Mucker b, Jan G. Humphreys c, Melissa D. Tucker a
a
Department of Clinical Sciences, North Carolina State University College of Veterinary Medicine,
4700 Hillsborough Street, Raleigh, NC 27606, United States
b
United States Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Frederick, MD 21702-5011, United States
c
Indiana University of Pennsylvania, 325 Weyandt Hall, Department of Biology, Indiana, PA 15705, United States
Received 3 August 2007; received in revised form 10 January 2008; accepted 11 January 2008
Abstract
This study was performed to determine the prevalence of Cytauxzoon felis (C. felis) infections in bobcats (Lynx rufus) from a
region where C. felis is recognized in domestic cats, North Carolina (NC), and a region where C. felis is not recognized in domestic
cats, Pennsylvania (PA). Samples from NC (n = 32) were obtained post-mortem via cardiac puncture from legally trapped bobcats.
Samples from PA (n = 70) were collected post-mortem onto Nobuto blood collecting strips by the PA Game Commission. Each
sample was tested using a C. felis specific PCR assay as well as a PCR assay targeting host DNA to rule out the presence of PCR
inhibitors. Three samples were excluded due to the presence of PCR inhibitors. Thirty-three percent (10/30) of the samples from NC
and 7% (5/69) of the samples from PA tested positive for the presence of C. felis. The proportion of C. felis positive bobcats from NC
was significantly different than that from PA (P < 0.005). Despite the lower prevalence of C. felis infections in bobcats from PA this
finding is unique and indicates the potential for C. felis infections in domestic cats in the northeastern USA if the appropriate tick
vectors are present. Veterinary practitioners in PA should be on alert for cytauxzoonosis in domestic cats. Further studies about the
epidemiology and transmission of C. felis infections among both domestic cats and bobcats are needed.
# 2008 Elsevier B.V. All rights reserved.
Keywords: Cytauxzoonosis; Piroplasmosis; Cytauxzoon felis; Bobcats; PCR; Tick
1. Introduction
Cytauxzoonosis is an emerging infectious disease of
domestic cats in North America caused by the protozoan
parasite Cytauxzoon felis (C. felis) (Birkenheuer et al.,
2006a). Domestic cats that become infected with C. felis
* Corresponding author. Tel.: +1 919 513 8288;
fax: +1 919 513 6336.
E-mail address: [email protected] (A.J. Birkenheuer).
0304-4017/$ – see front matter # 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetpar.2008.01.020
develop a rapidly progressive febrile illness and succumb
to multi-organ failure secondary to schizont-laden
macrophages occluding small blood vessels. Death
occurred in the majority of infected cats reported in
the literature (Birkenheuer et al., 2006a; Ferris, 1979;
Hoover et al., 1994; Wagner, 1976). Because of this,
domestic cats are typically considered to be an accidental
host for C. felis. They are presumed to become naturally
infected with C. felis via tick vectors and bobcats (Lynx
rufus) are considered to be the reservoir host. The
American dog tick (Dermacentor variabilis) has been
A.J. Birkenheuer et al. / Veterinary Parasitology 153 (2008) 126–130
shown to transmit C. felis to domestic cats from bobcats
under laboratory conditions and is assumed to be the
primary tick vector in nature (Blouin et al., 1984). Unlike
in domestic cats, C. felis appears to undergo a limited
schizogenous phase in bobcats and therefore bobcats
exhibit minimal morbidity and mortality as a result of the
infection (Blouin et al., 1987; Glenn et al., 1983).
Cytauxzoonosis was first reported in Missouri in
1979 and the disease was only recognized in the south
central and southeastern United States for the past 20
years. The geographic distribution of cytauxzoonosis in
domestic cats has been recently documented to extend
east and northeast of its previously recognized range
(Birkenheuer et al., 2006a; Jackson and Fisher, 2006).
Since cytauxzoonosis is not a difficult disease to
diagnose, it seems more likely that these infections
represent a true expansion of the disease distribution
rather than a new recognition of a disease that has been
historically present in these regions at low levels.
Even with this expanded distribution, cytauxzoonosis in domestic cats does not appear to correlate with the
natural distribution of the presumed reservoir host, L.
rufus, which includes most of North America and
extends as far south as southern Mexico (Long, 2003).
The cause or causes for this discrepancy are unknown.
Some possibilities include a lack of C. felis in bobcats
throughout their entire range or the absence of
competent vectors that can transmit C. felis from
bobcats to domestic cats in areas where cytauxzoonosis
is not recognized. Studies investigating the prevalence
of C. felis in bobcats are limited to the southcentral
region of the US. These studies found that C. felis could
be detected by examination of stained blood smears in
30–50% of wild-trapped bobcats (Glenn et al., 1982;
Kocan et al., 1985). To the authors’ knowledge, studies
examining the prevalence of C. felis infections in
bobcats from areas of the US where cytauxzoonosis is
not recognized in domestic cats have not been
performed. The purpose of this study was to determine
and compare the proportion of bobcats that were
infected with C. felis from a region where cytauxzoonosis is recognized in domestic cats to that where it is
not recognized.
2. Materials and methods
Bobcats were sampled from a state where cytauxzoonosis is recognized in domestic cats, North Carolina
(NC) and another state where cytauxzoonosis in
domestic cats has never been reported, Pennsylvania
(PA). All bobcats were legally trapped by licensed
personnel. All samples were obtained post-mortem and
127
the carcasses were either fresh or had been frozen at
20 8C and thawed immediately prior to sample
collection. Blood samples from NC (n = 32) were
obtained via cardiac puncture. All samples were frozen
at 20 8C until processing. Total deoxyribonucleic acid
(DNA) was extracted from 100 mg of clotted whole
blood samples using a commercially available kit
(QIAamp DNA blood mini kit, Qiagen, Valencia, CA).
Blood samples from bobcats from PA (n = 70) were
collected directly onto Nobuto blood collecting strips
(Advantec, Toyo Roshi Kaisha, Japan) as previously
described (Mucker et al., 2006). Samples from PA were
collected between October and February of 2002.
Samples from NC were collected in January and
February during 2004, 2005 and 2006. Total DNA was
extracted from dried blood strips using a commercially
available kit (DNeasy tissue kit, Qiagen, Valencia, CA).
Each sample was tested using a C. felis specific
polymerase chain reaction assay designed to amplify a
284 base pair fragment of the 18S rRNA gene that is
able to detect as few as ten copies of the target DNA per
microliter of sample with 100% sensitivity (Birkenheuer et al., 2006b). The assay did not produce
amplicons when feline DNA, Babesia canis vogeli,
B. c. canis, B. c. rossi, B. gibsoni (Asian genotype), B.
gibsoni (CA genotype), B. annae, Ehrlichia canis, E.
ewingii, E. chafeensis, Neorickettsia risticii, Anaplasma
phagocytophilum, A. platys, Mycoplasma haemofelis,
M. haemominutm, Rickettsia rickettsii, Leishmania
infantum, Bartonella henselae or Bartonella vinsonii
ssp. berkhoffii were used as templates (Birkenheuer
et al., 2006b). Positive controls consisted of DNA
extracted from the blood of C. felis infected domestic
cats. The C. felis 18S rRNA genes from these positive
control cats have been characterized previously
(Birkenheuer et al., 2006b). Negative (i.e. no DNA)
water controls were included with each amplification
run. To demonstrate that the extracted DNA was
amenable to PCR testing, each sample was tested using
an assay designed to amplify a fragment of a
glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
pseudogene from most mammals as previously
described (Birkenheuer et al., 2003). Amplicons were
visualized by ethidium bromide staining and ultraviolet
trans-illumination after electrophoresis in a 2% agarose
gel. Randomly selected amplicons (n = 5) were purified
using a commercially available kit (Qiaquick PCR
purification kit, Qiagen, Valencia, CA) and sequenced
directly (Davis Sequencing, Davis, CA). These
sequences were aligned with C. felis 18S rRNA
sequences obtained from domestic cats with cytauxzoonosis (ClustalW, http://www.mbio.ncsu.edu/BioE-
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A.J. Birkenheuer et al. / Veterinary Parasitology 153 (2008) 126–130
dit/bioedit.html). The proportions of C. felis infected
bobcats from each region were compared using a
Fisher’s exact test.
3. Results
Three samples, two from NC and one from PA, had
to be excluded from the analyses because the GAPDH
pseudogene fragment could not be amplified. C. felis
specific amplicons were detected in 33% (10/30) of the
bobcat samples from NC, the region where cytauxzoonosis is recognized in domestic cats. In contrast, C. felis
DNA was detected by PCR from only 7% (5/69) of the
bobcat samples from PA, the region where domestic cat
cytauxzoonosis is not recognized. Amplicons were not
detected in the negative controls (i.e. no DNA)
throughout the study. The sequences (n = 5) of the
randomly selected amplicons, three from PA and two
Fig. 1. (A) The approximate distribution of bobcats (Lynx rufus) in the United States (shaded area) (Long, 2003). (B) The states in which
cytauxzoonosis has been documented in domestic cats (shaded area) (Birkenheuer et al., 2006a; Greene, 2006; Jackson and Fisher, 2006). (C) The
approximate distribution of Dermacentor variabilis in the United States (shaded area) (redrawn based on the distributions reported by the centers for
disease control: http://www.cdc.gov). (D) The approximate distribution of Amblyomma americanum in the United States (shaded area) (redrawn
based on the distributions reported by the centers for disease control: http://www.cdc.gov). (E) The approximate distribution of Ixodes scapularis in
the United States (shaded area) (redrawn based on the distributions reported by the centers for disease control: http://www.cdc.gov).
A.J. Birkenheuer et al. / Veterinary Parasitology 153 (2008) 126–130
from NC, were 100% identical to the C. felis 18S rRNA
sequences from North America deposited in GenBank
(Accession numbers L19080, AY679105, AY531524,
and AF399930). The proportion of C. felis infected
bobcats from PA was significantly lower (P < 0.005)
than that of bobcats from NC.
4. Discussion
The geographic distribution of bobcats (Fig. 1A)
does not correlate well with recognized distribution of
cytauxzoonosis in domestic cats (Fig. 1B). The
findings of this study suggest that the lack of
recognition of cytauxzoonosis in domestic cats in
PA is not due to the absence of C. felis infections in
bobcats in that state. Assuming that the prevalence of
C. felis infections in the PA bobcats tested in this
study is representative of the prevalence of C. felis in
all PA bobcats, this could contribute to a decreased
reservoir capacity with a subsequent lower prevalence, or absence, of cytauxzoonosis in domestic cats.
The possibility that C. felis infections in PA bobcats
are a recent occurrence and that cytauxzoonosis in
domestic cats may occur there in the future must also
be considered. Ultimately, the presence of C. felis
infections in PA bobcats mandates that veterinary
clinicians in PA should be vigilant for cytauxzoonosis
in domestic cats.
The presence of C. felis in PA bobcats along with the
apparent absence of cytauxzoonosis in PA domestic
cats may suggest that the routes of C. felis infection
among bobcats and domestic cats differ. Perhaps
similar to other piroplasma, C. felis infections can be
transmitted transplacentally and can be perpetuated in
bobcats in the absence of a tick vector. Alternatively
intra-species and inter-species transmission of C. felis
may occur via different tick species. While D.
variabilis has been demonstrated to have vector
competence for both inter- and intra-species transmission of C. felis from bobcats (Blouin et al., 1984, 1987),
the geographic distribution of D. variabilis (Fig. 1C)
does not appear to correlate well with the recognized
distribution of cytauxzoonosis in domestic cats. In fact
the distribution of cytauxzoonosis in domestic cats
appears to correspond best with the distribution of the
lonestar tick (Amblyomma americanum) (Fig. 1D) and
the black-legged tick (Ixodes scapularis) (Fig. 1E). In
North Carolina, A. americanum is frequently detected
feeding on domestic cats that have access to outdoors
(Birkenheuer, unpublished data). Potential vector
competence of A. americanum is at least minimally
supported by the detection of C. felis DNA by PCR
129
from A. americanum ticks (Bondy et al., 2005). Based
on these findings, further studies are indicated
evaluating the vector competence and vectorial
capacity of A. americanum and perhaps I. scapularis
for the transmission of C. felis.
This study represents the first to screen large
numbers of bobcats for C. felis infections by PCR,
and the first to screen bobcats from an area where
cytauxzoonosis is not recognized in domestic cats. Very
little is known about the epidemiology of C. felis
infections and further studies are clearly indicated.
Studies evaluating the reservoir competence and
capacity of bobcats as well as the vector competence
and capacity of several ixodid tick species for the
maintenance and transmission of C. felis infections are
needed.
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