Veterinary Parasitology 177 (2011) 13–19

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Veterinary Parasitology

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Tunicamycins, a class of nucleoside antibiotics similar to corynetoxinsof the Rathayibacter toxicus, increase susceptibility of mice to Neosporacaninum

Lili Caoa,b, Xichen Zhanga, Wenbin Tuob,∗

a College of Animal Science and Veterinary Medicine, Jilin University, Changchun, Jilin Province 130062, Chinab Animal Parasitic Diseases Laboratory, USDA/ARS, Beltsville, MD 20705, United States of America

a r t i c l e i n f o

Article history:Received 9 September 2010Received in revised form23 November 2010Accepted 24 November 2010

Keywords:Neospora caninumCattleAbortionMouseAnnual ryegrass toxicityCorynetoxinsTunicamycins

a b s t r a c t

Neosporosis is the leading cause of abortion in cattle. Neospora caninum-associated abor-tion may exhibit both endemic and epidemic patterns. It was reported that the epidemicoutbreaks took place in the form of “abortion storms” and were not significantly correlatedwith seasonal changes or consumption of any particular feeds; and thus, the mechanismsby which the epidemic “abortion storms” are triggered remain unclear. Annual ryegrasstoxicity (ARGT) is a severe or fatal neurological disorder of livestock of Australia and SouthAfrica. This disorder is caused by the ingestion of several plant genera (Lolium, Polypogon,and Agrostis) colonized by a nematode (Anguina sp.) and a bacterium, Rathayibacter toxicus.Corynetoxins (CTs) produced by R. toxicus contaminate plants which are in turn ingestedby sheep and cattle, causing severe or often fatal hepatocerebral disorders in affected ani-mals. We hypothesize that N. caninum-associated fetal deaths and abortions in cattle maybe potentiated by pre-exposure of sublethal levels of CT-contaminated plants prior to N.caninum infection or reactivation of a latent infection. The exposure of sublethal CTs maynot cause clinical diseases, but may increase susceptibility to pathogens such as N. caninum.Indeed, CT poisoning surviving pregnant sheep can have up to 10% abortions. The presentstudy investigated whether animals pre-exposed to tunicamycins (TMs), a functional sub-stitute for CTs, had a lowered resistance to sublethal experimental infection by N. caninumtachyzoites in mice. The results showed that sublethal doses of TMs or N. caninum alonedid not cause significant deaths. Sublethal doses of N. caninum induced high mortality inTM-treated mice in a dose dependent manner. When mice were treated with a low dose ofTMs (5 �g/mouse), as few as 2.5 × 106 tachyzoites were needed to induce more than 30% ofmortality which is equivalent to the mortality rate caused by 40 × 106 tachyzoites. Spleencells of mice treated with TMs had reduced (p < 0.05) Neospora antigen-induced interferon-

gamma (IFN-g) production and Con A-stimulated proliferation. The results suggest that CTcontaminated plants, when exposed to animals under natural conditions, may contribute tolowered host resistance and increased N. caninum-associated fetal morbidity and mortalityin affected animals.

∗ Corresponding author at: Animal Parasitic Diseases Laboratory,USDA/ARS, BARC/ANRI/APDL, Building 1040, Room 208, Beltsville, MD20705, United States. Tel.: +1 3015048258.

E-mail address: wenbin.tuo@ars.usda.gov (W. Tuo).

0304-4017/$ – see front matter. Published by Elsevier B.V.doi:10.1016/j.vetpar.2010.11.037

Published by Elsevier B.V.

1. Introduction

Neospora caninum is an intracellular protozoan para-site which infects numerous mammalian hosts, but causesclinical neosporosis only in cattle and dogs (Dubey et al.,2007). It has been recognized that neosporosis is a leading

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14 L. Cao et al. / Veterinary

cause of abortion in cattle worldwide (Dubey et al., 2007).The dog and other canine species serve as definitive hostsand shed oocysts in feces. Cattle may be infected by theingestion of environmental oocytes and the infected damsmay also transplacentally transmit the parasite to theiroffspring, resulting in fetal deaths, abortion, still birth, orbirth of infected calves with or without clinical symptoms.Most congenitally infected cows are clinically normal untilthey are pregnant where recrudescence of latent infectionsduring gestation may occur and cause abortion (Trees andWilliams, 2005; Wouda et al., 1999b). N. caninum associ-ated abortion in cattle is endemic in many parts of theworld, it can also occur in an epidemic pattern. Althoughintensive research has been conducted worldwide since thefirst description of neosporosis (Dubey et al., 1988; Bjerkaset al., 1984), it still remains unknown about what mighthave triggered the epidemic “abortion storms” caused byN. caninum. Immunosuppression or ingestion of toxic feedshas been speculated as causes for the reactivation of latentinfection but none was confirmed (Bartels et al., 1999,2005; Pfeiffer et al., 2002; Wouda et al., 1999a).

It has been well-characterized that several plant species(Lolium, Polypogon and Agrostis) are parasitized by a nema-tode (Anguina sp.) which can be infected by a bacteriumRathayibacter toxicus. The R. toxicus, a nematode-vectoredGram-positive coryneform bacterium, is responsible forARGT by producing as many as 16 toxins collectivelyreferred to as CTs (Finnie, 2006; Riley et al., 2003; Eckardt,1983; Vogel et al., 1981). The CTs are glycolipid in nature,belonging to the tunicaminyluracil family of nucleosideantibiotics (Finnie, 2006; McKay and Riley, 1993; Eckardt,1983; Vogel et al., 1981). In 1981, CTs were first isolatedby HPLC from galled seedheads of annual ryegrass andpartially characterized, and the purified fractions wereconfirmed to be highly toxic to rats (Vogel et al., 1981).Corynetoxins (CTs) cause severe neurological disordersknown as annual ryegrass (Lolium rigidum) toxicity (ARGT),often resulting in death in grazing livestock includingsheep, cattle and horses in Australia and South Africa(Finnie, 2006). A similar disease was also seen in sheep andcattle in the state of Oregon, USA, and found to be causedby a CT-related compound from a fescue (Festula nigrescens)colonized by A. agrestis and a Rathayibacter-like organism.This observation was consistent with the reported presenceof Rathayibacter sp. in orchard grass seed and R. toxicus inchewing’s fescue seed in Oregon (Riley et al., 2003).

CTs are structurally and functionally similar to thetunicamycins (TMs) that are produced by the bac-terium Streptomyces iysosuperficus (Takatsuki et al., 1971;Takatsuki and Tamura, 1971a,b). Both CTs and TMs sharea common N-acetylglucosamine tunicaminyluracil corestructure and vary in length, terminal branching formationand hydroxylation states of the fatty acid chains (Eckardt,1983). The structural and functional similarities betweenthe CTs and TMs allows the use of TMs as a substitute inevaluating CT functions in vitro and in vivo. Indeed, many

effects of CTs on cells and animals were obtained usingTMs as a substitute, and numerous reports confirmed thatthe effects of CTs and TMs are indistinguishable (Finnie,2006). Interestingly, previous reports showed that TMsincreased neuroinvasion and encephalitis in mice infected

logy 177 (2011) 13–19

with Venezuelan equine encephalitis virus (Steele et al.,2006; Maheshwari et al., 1983). In these reports, it wasshown that TM treatment increased viral-associated mor-tality by possibly increasing viral titers in the brain withoutaltering viremia profiles or viral replication, suggestingenhanced neuroinvasion by the toxin.

The intracellular protozoan parasite N. caninum, sim-ilar to Toxoplasma gondii, also preferentially invades theneuronal cells and becomes resident in those cells inimmunocompetent animals. We hypothesize that pre-exposure to CTs (or TMs as an experimental substitute) willpredispose the animals to N. caninum-associated mortal-ity by increasing susceptibility. One possible mechanismfor increased susceptibility by CTs or TMs may be throughsuppressing the immune responses of the dam (Iraharaet al., 1987; Sun et al., 2006). This may occur under nat-ural conditions where animals ingest plants contaminatedwith CTs from Rathayibacter-like organisms. The objectiveof the present study was to determine if pre-treatment ofmice by TMs would increase the mortality rate followingexperimental infection by N. caninum. The results showedthat the injection of mice with sub-lethal doses of TMs dra-matically enhanced N. caninum associated mortality. Thisis the first study demonstrating that animals exposed tosublethal levels of CTs will have increased susceptibility toinfectious diseases such as neosporosis.

2. Materials and methods

2.1. Animals

Five weeks old BALB/C mice of both males and femaleswere injected intraperitoneally (i.p.) with TMs and/orN. caninum tachyzoites in 0.5 ml RPMI-1640. N. caninumtachyzoites were given 6 h after mice were treated withTMs. Mice were observed daily for clinical symptomsfollowing treatments. Mice suffered from severe clinicalsymptoms affecting eating, drinking and movement weresubjected to euthanasia. Animal use and care was approvedby institutional animal care and use committee.

2.2. Preparation of TMs and N. caninum tachyzoites

The stock solution of TMs (Calbiochem) was made bydissolving TMs in DMSO (Fisher Scientific) at 1 mg/ml. Priorto use, the stock solution was further diluted in RPMI-1640medium (pH 10.0) to prepare working stocks containing 30,25, 20, 15, 10, or 5 �g/ml TMs. N. caninum tachyzoites werecultured in RPMI-1640 complete medium supplementedwith 2% bovine calf serum, 25 mM glutamine and 50 �g/mlgentamicin maintained using African green monkey fibro-blast cells (CV1 cells, ATCC) as described previously (Tuoet al., 2005). In brief, prior to host cell lysis by the parasite,

the parasite/host cell suspension was collected and passedthrough 20 G and 27 G needles. The mixture was dilutedin serum free RPMI-1640 at 20 × 106, 10 × 106, or 5 × 106

tachyzoites per ml and placed on ice until injected i.p. at0.5 ml/mouse.

Parasitology 177 (2011) 13–19 15

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2

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3

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Table 1Mortality of mice treated with TMsa.

TMs(�g/mouse)b

Number of deaths/total animals per group

30 25 20 15 10 5 0

Exp. 1 10/10 11/11 0/7 0/7 0/7 0/7 0/7

L. Cao et al. / Veterinary

.3. IFN-g production and T cell proliferation by spleenells

Following treatment i.p. with TMs (15 �g/mouse) for2 h, mice were euthanized by CO2 and cervical disloca-ion and spleen removed for the preparation of spleenells. In brief, the whole spleen was forced through a metalesh using a syringe plunger into a petri dish contain-

ng RPMI-1640–EDTA. Cell suspension was pipetted upnd down to break up cell clumps and underplayed withhe Histopaque-1088 (Sigma). Following centrifugation at00 × g for 30 min at 4 ◦C, cells at the interface were col-

ected and washed 3 times with RMPI-1640–EDTA andesuspended with complete medium (RPMI-1640 supple-ented with 10% FBS, 2.5 mM GLN, 5 �g/ml gentamicin).

pleen cells were seeded in 24-well plates at 2 × 106 cellser well in 1 ml complete medium in duplicate and treatedith increasing concentrations (0, 1, or 5 �g/ml) of N. can-

num tachyzoite total soluble antigen (NcAg). Plates werencubated at 37 ◦C in the presence of 5% CO2 and 95% airor 24 h and supernatants were collected, centrifuged andtored at −20 ◦C until assayed for cytokines by mouse IFN-gLISA.

For T cell proliferation, spleen cells were seeded in6-well opaque-walled plates at 2 × 105 cells per well

n 100 �l complete medium in triplicate and co-culturedith 0, 0.5 and 5 �g/ml Concanavalin A (Con A, Sigma) for

8 h. CellTiter-Glo (Promega) solution of equal volume wasdded and mixed on an orbital shaker for 10 min at roomemperature. The plates were then read by a luminescencelate reader (FlouSrtar Optima). The data are expressed astimulation index (mean ± stdev) which was calculated byividing counts of treatment wells by counts of the back-round wells.

.4. Statistical analysis

Mortality rates of various N. caninum infection doses atach fixed dose of TMs were analyzed by one-way ANOVAith a multiple comparison test. The probability of p < 0.05as considered significant.

. Results

Sublethal doses of TMs and N. caninum tachyzoites inALB/C mice. BALB/C mice have been used as a model

or neosporosis. However, the mortality and morbidity ofhese mice caused by experimental treatment of TMs haveever been reported. The purpose of this experiment waso determine sublethal doses of TMs at which experimentalnoculation of N. caninum at different doses may be intro-uced. The results showed that TMs at 25 or 30 �g perouse was lethal and all mice were dead after 2–5 days

f injection, whereas mice received TMs at 5, 10, 15, or0 �g per mouse survived for at least 40 days followingreatment (Table 1). Sporadic deaths were observed in mice

eceived 20 �g/mouse TMs and some animals did appear toave rough hair coat and decreased appetite, but majorityf the sick mice recovered in 3–5 days (data not shown).he doses of 5, 10, 15 and 20 �g per mouse were selectedn the subsequent experiments to determine the lethality

Exp. 2 10/10 10/10 1/16 0/10 0/10 0/10 0/10

a Mortality was monitored daily for 40 days.b TMs in 0.5 ml RPMI-1640 (pH 10) was injected intraperitoneally.

of N. caninum under the influence of TMs. It appeared thatolder mice were more tolerant than the younger one to TMtoxicity (data now shown). Sublethal doses of N. caninumtachyzoites were also determined in mice. The BALB/C miceappeared to be rather tolerant to i.p. N. caninum tachyzoiteinfection in that parasite doses only at 4 × 107 or higherwere lethal to adult mice, which was consistent to thosereported previously (Penarete-Vargas et al., 2010) (data notshown). Sublethal doses of N. caninum tachyzoites weredetermined empirically and those of 2.5 × 106, 5 × 106, and10 × 106 were chosen to infect animals.

Dose-dependent lethality of N. caninum in mice pre-treated with various sublethal doses of TMs. Mice are muchlike other mammals such as cattle, they can be infected byN. caninum, but adult mice are rather resistant to killing bythis parasite. This experiment was designed to determineif sublethal doses of N. caninum tachyzoites cause death inBALB/C mice that were pre-treated with sublethal doses ofTMs for 6 h. This experimental design was to mimic a natu-ral condition where animals may have experienced eitheran endogenous or an exogenous exposure to CTs and N.caninum almost simultaneously. As expected, TMs alone atthe highest dose of 20 �g/ml did not cause any deaths byday 40 post treatment. No mortality was seen 40 days postinoculation in mice injected with 2.5–10 million N. can-inum tachyzoites, however, clinical illness was obvious insome mice received N. caninum tachyzoite alone at the doseof 10 × 106 per animal, but all recovered within 3–5 days(data not shown). At each TM dose between 5 and 20 �g permouse, N. caninum tachyzoites at all doses caused deathsin a dose-dependent manner (Fig. 1). Likewise, at each N.caninum tachyzoite dose, TMs at all doses used inducedmortality in a dose-dependent fashion. In general, mostdeaths occurred within 15 day post inoculation, it was par-ticularly so in the highest TMs and N. caninum combinations(Fig. 2).

Disease progression in mice treated with N. caninumtachyzoites, TMs, or TMs plus tachyzoites. To determinethe sublethal doses of Neospora tachyzoites, 106, 10 × 106,20 × 106, 40 × 106, 60 × 106, 80 × 106, or 100 × 106 permouse were used for infection. Mice received 106 tachy-zoites did not show any clinical symptoms. However,10–20 × 106 doses of tachyzoites caused illnesses charac-terized by rough hair coat, depression, loss of appetite andslight dehydration, but recovered within 3–7 days. Animalsreceived 40 × 106 or higher doses all showed similar but

severe symptoms and died within 2 weeks. To determinethe sublethal doses of TMs, mice were i.p. injected with5, 10, 15, 20, 25, or 30 �g per mouse. All animals treatedwith 25 or 30 �g doses had severe illnesses showing rough

16 L. Cao et al. / Veterinary Parasitology 177 (2011) 13–19

0 10 20 30 400

20

40

60

80

100

Day post inoculation0 10 20 40

0

20

40

60

80

100

Day post inoculation

Perc

ent s

urvi

val

0 10 20 30 400

20

40

60

80

100

NcTz aloneTM alone

10 x 1065 x 1062.5 x 106

0 10 20 30 400

20

40

60

80

100

Perc

ent s

urvi

val 51=MT02=MT

TM=10 TM=5TM=5

num tacninumas moni

Fig. 1. Survival rate of mice treated with TMs and infected with N. cani6 h before inoculation (i.p.) of N. caninum tachyzoites. Each TMs and N. caRPMI-1640 alone, TMs alone, or N. caninum tachyzoites alone. Mortality w

hair coat, loss of appetite, dehydration, depression, occa-sional aggression, and died within 3–5 days. Mice received5–20 �g doses of TMs all appeared normal except for the20 �g dose where animals showed slight depression, lossof appetite, and rough hair coat, but recovered within 3–5days. Based on the clinical appearance of mice treated withTMs and N. caninum, doses for TMs at 5, 20, 15 and 20 �gper mouse and 2.5 × 106, 5 × 106, and 10 × 106 were cho-sen for the synergistic effects of TMs and N. caninum. Micereceived all combinations of TMs and N. caninum showedclinical symptoms similar to those of the high doses of N.caninum tachyzoites alone. The apparent disease severityappeared to be correlated to dosages of TMs and N. caninumtachyzoites.

Reduced immune responses in mice treated with TMs.Robust IFN-g production by murine naïve spleen cells inresponse to Neospora antigen was shown previously (Tuoet al., 2005; Feng et al., 2010). This experiment was toshow if host IFN-g production elicited by Neospora antigenwas impaired by TM treatment. The results showed thatspleen cells of TM-treated mice had a significant (p < 0.05)decrease in IFN-g production in response to Neospora anti-gen stimulation (Fig. 3A). Furthermore, spleen cells fromTM-treated mice responded poorly (p < 0.05) to a T cellmitogen, Con A, in comparison to spleen cells isolated fromuntreated mice (Fig. 3B).

4. Discussion

N. caninum-associated abortion in cattle is endemicworldwide, which can also occur in epidemic patterns.Since the first recognition and description of this pathogen(Dubey et al., 1988; Bjerkas et al., 1984), it is still largelyunknown about what triggers the epidemic “abortion

hyzoites. Mice were treated (i.p.) with TMs at 5, 10, 15 or 20 �g/ml fortachyzoite dose combination had a total of 15–23 mice. Controls includetored daily for 40 days. The results represent 5 independent experiments.

storms” caused by N. caninum. If epidemic abortion is due toexposure to environmental oocysts, the variable abortionrisk may be attributed to factors including pathogenicityof different parasite strains, infection dose, or susceptibil-ity of the dams with regard to immunologic status andstage of pregnancy (Gondim et al., 2004; Quinn et al.,2004; Innes et al., 2002). A number of probable causesfor the outbreaks have been speculated, including clini-cal immunosuppression or ingestion of toxic feeds whichmay induce the reactivation of latent infection, however,none of these was confirmed (Pfeiffer et al., 2002; Bartelset al., 1999; Wouda et al., 1999a). Thus, it is of importanceto understand how the epidemic outbreaks are triggeredand what may be the mediators of reactivation of latentinfections when an “abortion storm” is to occur. The CTpoisoning is a highly severe and often lethal neurologicaldisorder of livestock in Australia and South Africa (Finnie,2006). A disease similar to CT poisoning was also reportedin sheep and cattle in OR, USA, and confirmed to be causedby a CT-related compound from a fescue (Festula nigrescens)colonized by A. agrestis and a Rathayibacter-like organism.These results were consistent with the reports that Rathay-ibacter sp. in orchard grass seed and R. toxicus in chewing’sfescue seed were present in Oregon (Riley et al., 2003).There is no doubt that animals exposed to lethal dosesof CTs will result in death as shown in sheep (Hendrickset al., 1988; Berry et al., 1976; McIntosh and Thomas, 1967),the reproductive performance will be affected and abor-tion can occur in up to 10% of pregnant sheep that survived

the CT poisoning (Finnie, 2006). Most importantly, it willbe critical to know if exposures to sublethal doses of CTsor doses that do not even produce clinical symptoms inaffected animals increase susceptibility to opportunisticpathogens.

L. Cao et al. / Veterinary Parasitology 177 (2011) 13–19 17

F ection bi nimals bi d by TMt

pbpittfWishscwtT

ig. 2. Cumulative survival rates of mice treated with TMs followed by infnoculation. Mortality rate is calculated by dividing the number of dead anduced deaths in the dose-dependent manner (p < 0.05) in mice treateunicamycins; M, million.

N. caninum may be one of the most typical opportunisticathogens, which causes no clinical symptoms in infectedulls or non-pregnant cows. It has been speculated thatregnancy, as a physiological state that may induce a slight

mmunosuppression in the uterus, will trigger the reac-ivation of a latent infection or lower the host resistanceo an exogenous infection, leading to the clinical mani-estation of fetal loss in some infected cows (Trees and

illiams, 2005). Evidence indicates that not all N. caninum-nfected cows will abort and in fact, abortion occurs to aubset of pregnant cows, particularly in herds with veryigh seroprevalence (see review Dubey et al., 2007). It may

uggest that most immunocompetent, pregnant animalsan suppress the parasite to a latent state if the animalsere infected previously, or have the parasite under con-

rol rapidly if the animals are infected during pregnancy.his further implies that epidemic outbreaks may be in part

y different doses of N. caninum tachyzoites on days 20 (A) and 40 (B) posty the number of all animals in a particular group. N. caninum tachyzoites at each dose. The results represent 5 independent experiments. TM,

provoked by factors other than those associated with preg-nancy. With a possible global distribution of the R. toxicusdue to seed export and unrestricted vector nematodes forR. toxicus, toxins such as CTs contaminating cattle feed andpastures in some regions of the world should be of greatimportance (Riley and McKay, 1990; Riley, 1992; Riley et al.,2003).

The present study determined whether sublethal dosesof TMs, a structural and functional substitute for CTs,could increase the susceptibility of adult mice to infectionby sublethal doses of N. caninum tachyzoites. Our resultsshowed that mice developed clinical symptoms when they

received the highest sublethal doses of TMs or N. can-inum tachyzoites, but most animals recovered within 3–5days. However, at each TM dose, N. caninum tachyzoitescaused deaths in mice in a dose-dependent manner. AtTMs 20 �g/mouse, most mice were killed by 15 days post

18 L. Cao et al. / Veterinary Parasito

NcAg(ug/ml)

Con A (ug/ml)

*

*

*

*

A

B

*

*

*

*

Stim

ulat

ion

inde

xIF

N-g

(pg/

ml)

0

250

500

750

1000

0

40

80

120

510

50.50

ControlTM treated

ControlTM treated

Fig. 3. Spleen cell IFN-g production (A) and proliferation (B) in responseto Neospora total soluble antigen (NcAg) and Con A, respectively, in micetreated with or without TMs. Mice were treated i.p. with or without15 �g/mouse TMs for 12 h and spleen cells were isolated and co-culturedwith NcAg for 24 h in 24-well plates and supernatants were collected andmeasured for IFN-g production by ELISA. For proliferation studies, spleencells were stimulated by Con A for 48 h and proliferation was determinedusing the CellTiter-Glo Luminescent Cell Viability Assay kit (Promega).

Results are expressed as stimulation index (mean ± standard deviation)that was calculated by dividing the counts of treatment wells by counts ofthe background wells. NcAg, Neospora total soluble antigen. “*” indicatesstatistical difference (p < 0.05) among treatments.

inoculation. The results clearly indicate that the presenceof TMs (a substitute for natural CTs) at doses that did noteven cause any clinical symptoms when given alone wouldpotentiate the lethal effects of N. caninum tachyzoites at allsublethal doses tested.

The mechanisms by which TMs potentiate N. can-inum lethality are yet to be determined. Previous reportsshowed that TMs inhibit N-linked glycosylation of keymolecules mediating immune responses and suppressmixed lymphocyte reaction and mitogen-stimulated lym-

phocyte proliferation (Sun et al., 2006; Finnie and O’Shea,1990; Irahara et al., 1987; Heyman et al., 1985). Our resultsdemonstrated that TM treated mice had reduced IFN-gproduction and T cell proliferation by spleen cells, suggest-ing that TMs may render the host susceptible to Neospora

logy 177 (2011) 13–19

infection by inducing a state of systemic immunosuppres-sion. Thus, it may be speculated that systemically loweredhost resistance such as systemic immunosuppression in thedam would be sufficient to allow for the parasite to repli-cate and cause pathology in the pregnant uterus, with orwithout the toxins crossing the placenta. To our knowl-edge, this may be one of the first studies to determine theeffect of CTs on N. caninum virulence or host resistance.The outbreaks of CT poisoning have been reported in sev-eral countries and its clinical impact in animal death hasbeen well recognized. Yet, the impact of sublethal expo-sure of CTs in feed or on pastures to animals has not beenfully determined. The toxic effect of low levels of CTs alonemay be easily overlooked because it was only monitoredby the number of death in sheep or other affected animals(Finnie, 2006), its effect to decrease host resistance maynot nonetheless be ignored as shown in this study. Fur-ther investigation is warranted to identify how sublethallevels of CTs or other natural plant-associated toxins maycontribute to fetal death in N. caninum-infected cows. Theresults of this study may facilitate the development of on-farm management strategies to prevent neosporosis.

Acknowledgments

Lili Cao is a visiting student from the College of Ani-mal Science and Veterinary Medicine, Jilin University,Changchun, China and supported by the China ScholarshipCouncil. Wenbin Tuo is an adjunct faculty and OverseasCultural and Educational Expert of the College of Ani-mal Science and Veterinary Medicine, Jilin University,Changchun, China.

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