Four captive-raised coyote pups consumed tissues from Neospora caninum-infected calves. Feces were examined from 4 days before to 28 days after infection. One pup shed N. caninum-like oocysts, which tested positive for N. caninum and negative for Hammondia heydorni using PCR tests. Coyotes are the second discovered definitive host of N. caninum, after dogs. In North America, the expanding coyote ranges and population increase the probability of contact with domestic livestock. To reduce the risk of transmission of N. caninum to intensively farmed cattle, we recommend protection of feedstuffs using canid-proof fences, and careful disposal of dead stock

Gondim, Luis F.P.

McAllister, Milton M.

Pitt, William C.

Zemlicka, Doris E.

English

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February 2004 
Coyotes (Canis latrans) are definitive hosts of Neospora caninum 
Luis F.P. Gondim 
University of Illinois, Urbana, IL 
Milton M. McAllister 
University of Illinois, Urbana, IL 
William C. Pitt 
USDA/APHIS/WS, National Wildlife Research Center 
Doris E. Zemlicka 
USDA/APHIS/WS, National Wildlife Research Center 
Follow this and additional works at: https://digitalcommons.unl.edu/icwdm_usdanwrc 
 Part of the Environmental Sciences Commons 
Gondim, Luis F.P.; McAllister, Milton M.; Pitt, William C.; and Zemlicka, Doris E., "Coyotes (Canis latrans) 
are definitive hosts of Neospora caninum" (2004). USDA National Wildlife Research Center - Staff 
Publications. 378. 
https://digitalcommons.unl.edu/icwdm_usdanwrc/378 
This Article is brought to you for free and open access by the U.S. Department of Agriculture: Animal and Plant Health 
Inspection Service at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in USDA 
National Wildlife Research Center - Staff Publications by an authorized administrator of DigitalCommons@University 
of Nebraska - Lincoln. 
Rapid communication
Coyotes (Canis latrans) are definitive hosts of Neospora caninum
Luis F.P. Gondima, Milton M. McAllistera,*, William C. Pittb, Doris E. Zemlickac
aDepartment of Veterinary Pathobiology, College of Veterinary Medicine, University of Illinois, 2001 South Lincoln Avenue, Urbana, IL 61802, USA
bUSDA/APHIS/WS, National Wildlife Research Center, Hilo Field Station, P.O. Box 10880, Hilo, HI 96721, USA
cUSDA/APHIS/WS, National Wildlife Research Center, Logan Field Station, Utah State University, Logan, UT 84322-5295, USA
Received 18 November 2003; received in revised form 18 December 2003; accepted 19 December 2003
Abstract
Four captive-raised coyote pups consumed tissues from Neospora caninum-infected calves. Faeces were examined from 4 days before to
28 days after infection. One pup shed N. caninum-like oocysts, which tested positive for N. caninum and negative for Hammondia heydorni
using PCR tests. Coyotes are the second discovered definitive host of N. caninum, after dogs. In North America, the expanding coyote ranges
and population increase the probability of contact with domestic livestock. To reduce the risk of transmission of N. caninum to intensively
farmed cattle, we recommend protection of feedstuffs using canid-proof fences, and careful disposal of dead stock.
q 2004 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.
Keywords: Neospora caninum; Coyote; Canis latrans; Canid; Oocyst; Definitive host
Neosporosis, caused by the protozoan parasite Neospora
caninum, is a frequent cause of bovine abortion worldwide
(Dubey, 2003). Dogs are a definitive host of the parasite
(McAllister et al., 1998). When identifying definitive hosts
of N. caninum, it is essential to avoid confusion with the
closely related organism Hammondia heydorni (Ellis et al.,
1999). Besides dogs, other canids have been considered to be
potential definitive hosts of N. caninum (McAllister, 1999).
Antibodies to N. caninum have been found in North
American coyotes (Canis latrans), British red foxes (Vulpes
vulpes), and Australian dingoes (Canis familiaris dingo;
Lindsay et al., 1996; Barber et al., 1997; Buxton et al., 1997).
In Texas, Barling et al. (2000) performed a spatial analysis
study and found statistical associations among the density of
cattle, seropositivity for N. caninum, and abundance of
coyotes and grey foxes (Urocyon cinereoargenteus).
The aim of our study was to determine if coyotes are a
definitive host of N. caninum, by feeding them infected
bovine tissues and then examining their faeces.
Three newborn dairy bull calves (1, 2, and 3) seronegative
for N. caninum at 1:25 dilution by indirect fluorescent
antibody test (IFAT), were acquired from the University of
Illinois Dairy. The calves were kept indoors and were
exclusively fed milk replacer without antibiotics. The three
calves were infected during the first week of life with
N. caninum. Calf 1 was injected intravenously with 5 £ 105
tachyzoites (NC-Illinois strain; Gondim et al., 2002), calf 2
was injected intravenously with a mixture of 1 £ 106
tachyzoites of NC-beef strain (McAllister et al., 1998,
2000) and NC-Illinois strain (5 £ 105 tachyzoites from each
strain), and calf 3 was orally administered 29,000 oocysts of
the NC-beef strain via an oesophageal feeder. Calves were
euthanised 6–12 weeks after infection. At the time of
euthanasia the three calves had N. caninum antibody titres
$1:800 by IFAT. The entire brain and spinal cord, as well as
a portion of the heart, tongue, diaphragm, other skeletal
muscles and kidney, were cut into pieces of approximately
3 cm3, mixed, and shipped on ice to the Logan Field Station
of the National Wildlife Research Center, in Logan, UT.
Four female coyote puppies were used in the experiment.
Two littermates (A and B) were infected when they were 8
weeks old, and the other two pups (C and D), from different
litters, were infected when they were 12 weeks old. They
were born and raised in captivity at the Logan Field Station.
The puppies were housed in outdoor pens with their
littermates and mothers, prior to study initiation, and were
fed a commercially prepared carnivore diet (Fur Breeders
Agriculture Cooperative, Sandy, UT). The pups were
negative for antibodies against N. caninum at a 1:25 dilution,
as determined using an IFAT (Dubey et al., 1988). During the
study, animals were housed individually; coyotes A and B
0020-7519/$30.00 q 2004 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.ijpara.2004.01.001
International Journal for Parasitology 34 (2004) 159–161
www.parasitology-online.com
* Corresponding author. Tel.: þ1-217-333-2449; fax: þ1-217-244-7421.
E-mail address: mmmcalli@uiuc.edu (M.M. McAllister).
in indoor kennels, and C and D in outdoor pens. Pups A and B
each consumed 1 kg of tissue from calf 1, within a 2-day
period (time offirst exposure, day 0). Pups C and D refused to
eat the infected tissue and were difficult to handle; therefore
it was necessary to induce general anaesthesia (using sodium
thiopental) on two successive days, in order to administer
blended tissues mixed with water, via an oesophageal tube.
Pup C was administered a total of 1 kg of mixed tissues from
calf 3, and pup D was administered a total of 0.8 kg of mixed
tissues from calf 2.
Faecal samples were collected for 4 days before the
coyotes were infected with calf tissues, and for 28 days after
infection. Faeces were examined by a standard sucrose
flotation technique as described earlier (Gondim et al.,
2002). No N. caninum-like oocysts were observed in any
specimens prior to consuming the infected tissues, although
Isospora spp. oocysts were observed in the faeces of 2/4
coyotes before and after infection. Isospora spp. have a
direct faecal-oral life cycle (Baek et al., 1993) and were
presumed to have been transmitted from the pups’ mothers.
One of the four coyotes (C) shed approximately 500
Neospora-like oocysts between 8 and 10 days after infection
(Fig. 1A). The unsporulated oocysts were spherical to
subspherical and measured 10 mm in diameter ðn ¼ 10Þ:
They were aerated in 2% H2SO4 as previously described
(Gondim et al., 2002). After aeration, sporulated oocysts
contained two sporocysts, each with sporozoites (Fig. 1B).
Approximately 150 oocysts were concentrated by
sucrose, washed thrice in water, and a final volume of
200 ml of sediment containing oocysts was obtained. The
sediment was suspended with 500 ml of PBS and ground for
10 min by vortexing in a 1.5 ml tube with 500 ml of glass
beads (500 mm in diameter). The solution was transferred to
a new tube, mixed with 600 ml of a digestion buffer [100 mM
NaCl, 10 mM Tris–HCl (pH 8.0), 25 mM ethylenediami-
netetraacetic acid, 0.5% sodium dodecyl sulphate], 5 ml of
proteinase K (20 mg/ml), and incubated at 65 8C for
2 h. DNA was extracted by standard phenol–chloroform
and 2-propanol precipitation. The DNA was suspended in
water, but the final solution was yellow as a consequence of
the large amount of sediment in the sample. This DNA
sample was further purified using a DNA extraction kit
(SoilMastere DNA Extraction Kit, Epicentre, Madison,
WI), according to the manufacturer’s protocol.
DNA extracted from these oocysts were tested by PCR
using the N. caninum-specific primer pair Np6/Np21
(Yamage et al., 1996); the PCR conditions comprised an
initial denaturing step at 94 8C for 1 min, followed by
40 cycles at 94 8C for 1 min, 50 8C for 1 min, 72 8C for 2 min,
with a final extension step at 72 8C for 2 min. A positive
control (N. caninum DNA) and a negative control (Vero cell
DNA) were included in the test. PCR to detect H. heydorni
using the coyote oocysts was conducted using the specific
primers (JS4/JS5) and conditions reported by Slapeta et al.
(2002). DNA from H. heydorni (kindly provided by Dr John
Ellis) and Vero cell DNA were used as positive and negative
controls, respectively. The oocysts shed by coyote C tested
positively for N. caninum by PCR; in contrast, PCR for
H. heydorni yielded negative results (Fig. 2).
Blood samples were collected from the coyotes 28 days
p.i. All developed antibody titres for N. caninum (A, 1:800;
B, 1:800; C, 1:800; D, 1:1600). Their sera were also tested
for Toxoplasma gondii by IFAT, and were negative at 1:50.
This study demonstrates that coyotes are a definitive host
of N. caninum. One coyote pup clearly produced N. caninum
oocysts, as demonstrated by morphologic and genetic
techniques. This is the second discovered definitive host
of N. caninum, following dogs (C. familiaris). It is pertinent
to note that wolves (Canis lupus) are more closely related to
dogs than are coyotes (C. latrans; Vilà et al., 1999), and thus
probably are also definitive hosts of this protozoan.
The minimum infectious dose of N. caninum oocysts
needed to infect cattle is not known, but it is no more than 300
oocysts (Gondim et al., 2002). In the present study
using coyotes, one 12-week-old pup shed approximately
500 oocysts. If such slight shedding of oocysts is typical of
coyotes, then they could not be an efficient definitive host of
this organism. However, the first experimental protocols in
Fig. 1. Neospora caninum oocysts shed by a coyote (Canis latrans). Bars
10 mm. (A) Unsporulated oocyst. (B) Sporulated oocyst containing two
sporocysts.
Fig. 2. PCR for Neospora caninum and Hammondia heydorni using DNA
extracted from oocysts shed by a coyote (Canis latrans). (1) Vero cell DNA
using specific primers to N. caninum (negative control), (2) N. caninum
DNA (positive control) showing prominent band at approximately 328 bp,
(3) DNA extracted from the coyote oocysts using specific primers to N.
caninum, (4) Vero cell DNA using specific primers to H. heydorni (negative
control), (5) H. heydorni DNA (positive control) showing prominent band
at approximately 270 bp, (6) DNA extracted from the coyote oocysts using
specific primers to H. heydorni, (7) F £ 174 DNA marker (Invitrogen).
L.F.P. Gondim et al. / International Journal for Parasitology 34 (2004) 159–161160
dogs also induced scant production of oocysts (McAllister,
1999), similar to the present initial finding in coyotes. Using a
more recently reported protocol, hound pups were induced to
shed a mean of 160,000 oocysts (enough to infect .500
cattle per dog) after consuming tissues from freshly killed N.
caninum-infected calves (Gondim et al., 2002). The current
study was hampered by several technical problems that may
have mitigated production of oocysts by the coyotes. The
availability of captive-raised coyotes is highly limited.
Unlike previous studies, in which dogs consumed infected
tissues immediately after euthanasia of intermediate hosts, in
the present experiment tissues were chilled and shipped by
overnight courier from the University of Illinois to the Logan
Field Station in Utah; it is unknown if this reduced the
viability of the protozoan’s tissue stages. Coyotes are
seasonal breeders, which created scheduling difficulties and
resulted in the use of pups that were younger than preferred.
The coyote pups were approximately half the size of the
previously mentioned hound pups, and they were only able to
consume about one-third as much infected tissue. Further
investigation is needed to determine factors that may
potentiate oocyst production by coyotes, to compare the
relative efficiency of dogs and coyotes as definitive hosts of
N. caninum, and to estimate the potential risk of N. caninum
transmission that coyotes pose to livestock.
Historically, coyotes were confined to the prairie areas of
North America, but during the last 100 years the coyotes’
range has expanded to include the entire continental US,
much of Canada, and Central America (Moore and Parker,
1992). The expanding range and population of these animals
increase the probability of contact with domestic animals,
and this increases the risk of N. caninum transmission
between coyotes and livestock.
Based upon the finding that coyotes are a definitive host
of N. caninum, we suggest that reasonable steps be taken to
reduce the risk of transmission of this organism between
coyotes and ruminant livestock. For intensively farmed
cattle, such measures could include erection of canid-proof
fences around silage piles, baled hay, and other feedstuffs
that are kept outdoors. Dead livestock and the offal from
home slaughter should be disposed off in a manner that
prevents consumption by canids. Coyotes and dogs should
be discouraged from denning adjacent to barns or pens that
house livestock or where feeds are stored. An interesting
consideration is that, in some circumstances, dogs may help
to repel coyotes, although dogs themselves can transmit
N. caninum to cattle (De Marez et al., 1999; Gondim et al.,
2002). We do not recommend a policy of coyote eradica-
tion, because of this animal’s ability to adapt to changing
conditions, to rapidly rebound from reduced population
density, and also because of public sentiment.
Acknowledgements
The authors thank John Shivik for support with the study
protocol, and Stacey Brummer, Patrick Darrow, and Jared
Hedelius for handling, feeding, and collecting samples from
the coyotes. We also thank Susan Steenbergen for her
assistance with DNA purification techniques.This study was
supported by the University of Illinois and by the National
Wildlife Research Center’s Logan Field Station. Luis F. P.
Gondim is the recipient of a fellowship from CNPq, Brazil.
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McAllister, M.M., 1999. Uncovering the biology and epidemiology of
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McAllister, M.M., Dubey, J.P., Lindsay, D.S., Jolley, W.R., Wills, R.A.,
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L.F.P. Gondim et al. / International Journal for Parasitology 34 (2004) 159–161 161


Coyotes (\u3ci\u3eCanis latrans\u3c/i\u3e) are definitive hosts of \u3ci\u3eNeospora caninum\u3c/i\u3e

DigitalCommons@University of Nebraska

University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln USDA National Wildlife Research Center - Staff Publications U.S. Department of Agriculture: Animal and Plant Health Inspection Service February 2004 Coyotes (Canis latrans) are definitive hosts of Neospora caninum Luis F.P. Gondim University of Illinois, Urbana, IL Milton M. McAllister University of Illinois, Urbana, IL William C. Pitt USDA/APHIS/WS, National Wildlife Research Center Doris E. Zemlicka USDA/APHIS/WS, National Wildlife Research Center Follow this and additional works at: https://digitalcommons.unl.edu/icwdm_usdanwrc  Part of the Environmental Sciences Commons Gondim, Luis F.P.; McAllister, Milton M.; Pitt, William C.; and Zemlicka, Doris E., "Coyotes (Canis latrans) are definitive hosts of Neospora caninum" (2004). USDA National Wildlife Research Center - Staff Publications. 378. https://digitalcommons.unl.edu/icwdm_usdanwrc/378 This Article is brought to you for free and open access by the U.S. Department of Agriculture: Animal and Plant Health Inspection Service at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in USDA National Wildlife Research Center - Staff Publications by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Rapid communicationCoyotes (Canis latrans) are definitive hosts of Neospora caninumLuis F.P. Gondima, Milton M. McAllistera,*, William C. Pittb, Doris E. ZemlickacaDepartment of Veterinary Pathobiology, College of Veterinary Medicine, University of Illinois, 2001 South Lincoln Avenue, Urbana, IL 61802, USAbUSDA/APHIS/WS, National Wildlife Research Center, Hilo Field Station, P.O. Box 10880, Hilo, HI 96721, USAcUSDA/APHIS/WS, National Wildlife Research Center, Logan Field Station, Utah State University, Logan, UT 84322-5295, USAReceived 18 November 2003; received in revised form 18 December 2003; accepted 19 December 2003AbstractFour captive-raised coyote pups consumed tissues from Neospora caninum-infected calves. Faeces were examined from 4 days before to28 days after infection. One pup shed N. caninum-like oocysts, which tested positive for N. caninum and negative for Hammondia heydorniusing PCR tests. Coyotes are the second discovered definitive host of N. caninum, after dogs. In North America, the expanding coyote rangesand population increase the probability of contact with domestic livestock. To reduce the risk of transmission of N. caninum to intensivelyfarmed cattle, we recommend protection of feedstuffs using canid-proof fences, and careful disposal of dead stock.q 2004 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.Keywords: Neospora caninum; Coyote; Canis latrans; Canid; Oocyst; Definitive hostNeosporosis, caused by the protozoan parasite Neosporacaninum, is a frequent cause of bovine abortion worldwide(Dubey, 2003). Dogs are a definitive host of the parasite(McAllister et al., 1998). When identifying definitive hostsof N. caninum, it is essential to avoid confusion with theclosely related organism Hammondia heydorni (Ellis et al.,1999). Besides dogs, other canids have been considered to bepotential definitive hosts of N. caninum (McAllister, 1999).Antibodies to N. caninum have been found in NorthAmerican coyotes (Canis latrans), British red foxes (Vulpesvulpes), and Australian dingoes (Canis familiaris dingo;Lindsay et al., 1996; Barber et al., 1997; Buxton et al., 1997).In Texas, Barling et al. (2000) performed a spatial analysisstudy and found statistical associations among the density ofcattle, seropositivity for N. caninum, and abundance ofcoyotes and grey foxes (Urocyon cinereoargenteus).The aim of our study was to determine if coyotes are adefinitive host of N. caninum, by feeding them infectedbovine tissues and then examining their faeces.Three newborn dairy bull calves (1, 2, and 3) seronegativefor N. caninum at 1:25 dilution by indirect fluorescentantibody test (IFAT), were acquired from the University ofIllinois Dairy. The calves were kept indoors and wereexclusively fed milk replacer without antibiotics. The threecalves were infected during the first week of life withN. caninum. Calf 1 was injected intravenously with 5 £ 105tachyzoites (NC-Illinois strain; Gondim et al., 2002), calf 2was injected intravenously with a mixture of 1 £ 106tachyzoites of NC-beef strain (McAllister et al., 1998,2000) and NC-Illinois strain (5 £ 105 tachyzoites from eachstrain), and calf 3 was orally administered 29,000 oocysts ofthe NC-beef strain via an oesophageal feeder. Calves wereeuthanised 6–12 weeks after infection. At the time ofeuthanasia the three calves had N. caninum antibody titres$1:800 by IFAT. The entire brain and spinal cord, as well asa portion of the heart, tongue, diaphragm, other skeletalmuscles and kidney, were cut into pieces of approximately3 cm3, mixed, and shipped on ice to the Logan Field Stationof the National Wildlife Research Center, in Logan, UT.Four female coyote puppies were used in the experiment.Two littermates (A and B) were infected when they were 8weeks old, and the other two pups (C and D), from differentlitters, were infected when they were 12 weeks old. Theywere born and raised in captivity at the Logan Field Station.The puppies were housed in outdoor pens with theirlittermates and mothers, prior to study initiation, and werefed a commercially prepared carnivore diet (Fur BreedersAgriculture Cooperative, Sandy, UT). The pups werenegative for antibodies against N. caninum at a 1:25 dilution,as determined using an IFAT (Dubey et al., 1988). During thestudy, animals were housed individually; coyotes A and B0020-7519/$30.00 q 2004 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.doi:10.1016/j.ijpara.2004.01.001International Journal for Parasitology 34 (2004) 159–161www.parasitology-online.com* Corresponding author. Tel.: þ1-217-333-2449; fax: þ1-217-244-7421.E-mail address: mmmcalli@uiuc.edu (M.M. McAllister).in indoor kennels, and C and D in outdoor pens. Pups A and Beach consumed 1 kg of tissue from calf 1, within a 2-dayperiod (time offirst exposure, day 0). Pups C and D refused toeat the infected tissue and were difficult to handle; thereforeit was necessary to induce general anaesthesia (using sodiumthiopental) on two successive days, in order to administerblended tissues mixed with water, via an oesophageal tube.Pup C was administered a total of 1 kg of mixed tissues fromcalf 3, and pup D was administered a total of 0.8 kg of mixedtissues from calf 2.Faecal samples were collected for 4 days before thecoyotes were infected with calf tissues, and for 28 days afterinfection. Faeces were examined by a standard sucroseflotation technique as described earlier (Gondim et al.,2002). No N. caninum-like oocysts were observed in anyspecimens prior to consuming the infected tissues, althoughIsospora spp. oocysts were observed in the faeces of 2/4coyotes before and after infection. Isospora spp. have adirect faecal-oral life cycle (Baek et al., 1993) and werepresumed to have been transmitted from the pups’ mothers.One of the four coyotes (C) shed approximately 500Neospora-like oocysts between 8 and 10 days after infection(Fig. 1A). The unsporulated oocysts were spherical tosubspherical and measured 10 mm in diameter ðn ¼ 10Þ:They were aerated in 2% H2SO4 as previously described(Gondim et al., 2002). After aeration, sporulated oocystscontained two sporocysts, each with sporozoites (Fig. 1B).Approximately 150 oocysts were concentrated bysucrose, washed thrice in water, and a final volume of200 ml of sediment containing oocysts was obtained. Thesediment was suspended with 500 ml of PBS and ground for10 min by vortexing in a 1.5 ml tube with 500 ml of glassbeads (500 mm in diameter). The solution was transferred toa new tube, mixed with 600 ml of a digestion buffer [100 mMNaCl, 10 mM Tris–HCl (pH 8.0), 25 mM ethylenediami-netetraacetic acid, 0.5% sodium dodecyl sulphate], 5 ml ofproteinase K (20 mg/ml), and incubated at 65 8C for2 h. DNA was extracted by standard phenol–chloroformand 2-propanol precipitation. The DNA was suspended inwater, but the final solution was yellow as a consequence ofthe large amount of sediment in the sample. This DNAsample was further purified using a DNA extraction kit(SoilMastere DNA Extraction Kit, Epicentre, Madison,WI), according to the manufacturer’s protocol.DNA extracted from these oocysts were tested by PCRusing the N. caninum-specific primer pair Np6/Np21(Yamage et al., 1996); the PCR conditions comprised aninitial denaturing step at 94 8C for 1 min, followed by40 cycles at 94 8C for 1 min, 50 8C for 1 min, 72 8C for 2 min,with a final extension step at 72 8C for 2 min. A positivecontrol (N. caninum DNA) and a negative control (Vero cellDNA) were included in the test. PCR to detect H. heydorniusing the coyote oocysts was conducted using the specificprimers (JS4/JS5) and conditions reported by Slapeta et al.(2002). DNA from H. heydorni (kindly provided by Dr JohnEllis) and Vero cell DNA were used as positive and negativecontrols, respectively. The oocysts shed by coyote C testedpositively for N. caninum by PCR; in contrast, PCR forH. heydorni yielded negative results (Fig. 2).Blood samples were collected from the coyotes 28 daysp.i. All developed antibody titres for N. caninum (A, 1:800;B, 1:800; C, 1:800; D, 1:1600). Their sera were also testedfor Toxoplasma gondii by IFAT, and were negative at 1:50.This study demonstrates that coyotes are a definitive hostof N. caninum. One coyote pup clearly produced N. caninumoocysts, as demonstrated by morphologic and genetictechniques. This is the second discovered definitive hostof N. caninum, following dogs (C. familiaris). It is pertinentto note that wolves (Canis lupus) are more closely related todogs than are coyotes (C. latrans; Vilà et al., 1999), and thusprobably are also definitive hosts of this protozoan.The minimum infectious dose of N. caninum oocystsneeded to infect cattle is not known, but it is no more than 300oocysts (Gondim et al., 2002). In the present studyusing coyotes, one 12-week-old pup shed approximately500 oocysts. If such slight shedding of oocysts is typical ofcoyotes, then they could not be an efficient definitive host ofthis organism. However, the first experimental protocols inFig. 1. Neospora caninum oocysts shed by a coyote (Canis latrans). Bars10 mm. (A) Unsporulated oocyst. (B) Sporulated oocyst containing twosporocysts.Fig. 2. PCR for Neospora caninum and Hammondia heydorni using DNAextracted from oocysts shed by a coyote (Canis latrans). (1) Vero cell DNAusing specific primers to N. caninum (negative control), (2) N. caninumDNA (positive control) showing prominent band at approximately 328 bp,(3) DNA extracted from the coyote oocysts using specific primers to N.caninum, (4) Vero cell DNA using specific primers to H. heydorni (negativecontrol), (5) H. heydorni DNA (positive control) showing prominent bandat approximately 270 bp, (6) DNA extracted from the coyote oocysts usingspecific primers to H. heydorni, (7) F £ 174 DNA marker (Invitrogen).L.F.P. Gondim et al. / International Journal for Parasitology 34 (2004) 159–161160dogs also induced scant production of oocysts (McAllister,1999), similar to the present initial finding in coyotes. Using amore recently reported protocol, hound pups were induced toshed a mean of 160,000 oocysts (enough to infect .500cattle per dog) after consuming tissues from freshly killed N.caninum-infected calves (Gondim et al., 2002). The currentstudy was hampered by several technical problems that mayhave mitigated production of oocysts by the coyotes. Theavailability of captive-raised coyotes is highly limited.Unlike previous studies, in which dogs consumed infectedtissues immediately after euthanasia of intermediate hosts, inthe present experiment tissues were chilled and shipped byovernight courier from the University of Illinois to the LoganField Station in Utah; it is unknown if this reduced theviability of the protozoan’s tissue stages. Coyotes areseasonal breeders, which created scheduling difficulties andresulted in the use of pups that were younger than preferred.The coyote pups were approximately half the size of thepreviously mentioned hound pups, and they were only able toconsume about one-third as much infected tissue. Furtherinvestigation is needed to determine factors that maypotentiate oocyst production by coyotes, to compare therelative efficiency of dogs and coyotes as definitive hosts ofN. caninum, and to estimate the potential risk of N. caninumtransmission that coyotes pose to livestock.Historically, coyotes were confined to the prairie areas ofNorth America, but during the last 100 years the coyotes’range has expanded to include the entire continental US,much of Canada, and Central America (Moore and Parker,1992). The expanding range and population of these animalsincrease the probability of contact with domestic animals,and this increases the risk of N. caninum transmissionbetween coyotes and livestock.Based upon the finding that coyotes are a definitive hostof N. caninum, we suggest that reasonable steps be taken toreduce the risk of transmission of this organism betweencoyotes and ruminant livestock. For intensively farmedcattle, such measures could include erection of canid-prooffences around silage piles, baled hay, and other feedstuffsthat are kept outdoors. Dead livestock and the offal fromhome slaughter should be disposed off in a manner thatprevents consumption by canids. Coyotes and dogs shouldbe discouraged from denning adjacent to barns or pens thathouse livestock or where feeds are stored. An interestingconsideration is that, in some circumstances, dogs may helpto repel coyotes, although dogs themselves can transmitN. caninum to cattle (De Marez et al., 1999; Gondim et al.,2002). We do not recommend a policy of coyote eradica-tion, because of this animal’s ability to adapt to changingconditions, to rapidly rebound from reduced populationdensity, and also because of public sentiment.AcknowledgementsThe authors thank John Shivik for support with the studyprotocol, and Stacey Brummer, Patrick Darrow, and JaredHedelius for handling, feeding, and collecting samples fromthe coyotes. We also thank Susan Steenbergen for herassistance with DNA purification techniques.This study wassupported by the University of Illinois and by the NationalWildlife Research Center’s Logan Field Station. Luis F. P.Gondim is the recipient of a fellowship from CNPq, Brazil.ReferencesBaek, B.K., Kim, C.S., Kim, J.H., Han, K.S., Kim, Y.G., 1993. Studies onisosporosis in dogs. I. Isolation and sporulation of Isospora ohioensis.Korean J. Parasitol. 31, 201–206.Barber, J.S., Gasser, R.B., Ellis, J., Reichel, M.P., McMillan, D., Trees,A.J., 1997. Prevalence of antibodies to Neospora caninum in differentcanid populations. J. Parasitol. 83, 1056–1058.Barling, K.S., Sherman, M., Peterson, M.J., Thompson, J.A., McNeill, J.W.,Craig, T.M., Adams, L.G., 2000. Spatial associations among density ofcattle, abundance of wild canids, and seroprevalence to Neosporacaninum in a population of beef calves. J. Am. Vet. Med. Assoc. 217,1361–1365.Buxton, D., Maley, S.W., Pastoret, P.P., Brochier, B., Innes, E.A., 1997.Examination of red foxes (Vulpes vulpes) from Belgium for antibody toNeospora caninum and Toxoplasma gondii. Vet. Rec. 141, 308–309.De Marez, T., Liddell, S., Dubey, J.P., Jenkins, M.C., Gasbarre, L., 1999.Oral infection of calves with Neospora caninum oocysts from dogs:humoral and cellular immune responses. Int. J. Parasitol. 29, 1647–1657.Dubey, J.P., 2003. Review of Neospora caninum and neosporosis inanimals. Korean J. Parasitol. 41, 1–16.Dubey, J.P., Hattel, A.L., Lindsay, D.S., Topper, M.J., 1988. NeonatalNeospora caninum infection in dogs: isolation of the causative agent andexperimental transmission. J. Am. Vet. Med. Assoc. 193, 1259–1263.Ellis, J.T., Morrison, D.A., Liddell, S., Jenkins, M.C., Mohammed, O.B.,Ryce, C., Dubey, J.P., 1999. The genus Hammondia is paraphyletic.Parasitology 118, 357–362.Gondim, L.F.P., Gao, L., McAllister, M.M., 2002. Improved production ofNeospora caninum oocysts, cyclical oral transmission between dogs andcattle, and in vitro isolation from oocysts. J. Parasitol. 88, 1159–1163.Lindsay, D.S., Kelly, E.J., McKown, R.D., Stein, F.J., Plozer, J., Herman,J., Blagburn, B.L., Dubey, J.P., 1996. Prevalence of Neospora caninumand Toxoplasma gondii antibodies in coyotes (Canis latrans) andexperimental infections of coyotes with Neospora caninum. J. Parasitol.82, 657–659.McAllister, M.M., 1999. Uncovering the biology and epidemiology ofNeospora caninum. Parasitol. Today 15, 216–217.McAllister, M.M., Dubey, J.P., Lindsay, D.S., Jolley, W.R., Wills, R.A.,McGuire, A.M., 1998. Dogs are definitive hosts of Neospora caninum.Int. J. Parasitol. 28, 1473–1478.McAllister, M.M., Björkman, C., Anderson-Sprecher, R., Rogers, D.G.,2000. Evidence of point-source exposure to Neospora caninum andprotective immunity in a herd of beef cows. J. Am. Vet. Med. Assoc.217, 881–887.Moore, G.C., Parker, G.R., 1992. Colonization by the eastern coyote (Canislatrans). In: Boer, A., (Ed.), Ecology and Management of the EasternCoyote, Wildlife Research Unit, University of New Brunswick,Fredericton, pp. 23–37.Slapeta, J.R., Koudela, B., Votypka, J., Modry, D., Horejs, R., Lukes, J.,2002. Coprodiagnosis of Hammondia heydorni in dogs by PCR basedamplification of ITS 1 rRNA: differentiation from morphologicallyindistinguishable oocysts of Neospora caninum. Vet. J. 163, 147–154.Vilà, C., Maldonado, J.E., Wayne, R.W., 1999. Phylogenetic relationships,evolution, and genetic diversity of the domestic dog. J. Hered. 90, 71–77.Yamage, M., Flechtner, O., Gottstein, B., 1996. Neospora caninum: specificoligonucleotide primers for the detection of brain cyst DNA ofexperimentally infected nude mice by the polymerase chain reaction(PCR). J. Parasitol. 82, 272–279.L.F.P. Gondim et al. / International Journal for Parasitology 34 (2004) 159–161 161

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Coyotes (\u3ci\u3eCanis latrans\u3c/i\u3e) are definitive hosts of \u3ci\u3eNeospora caninum\u3c/i\u3e

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