International Journal for Parasitology 17 "0887# 0362Ð0367

RAPID COMMUNICATION are de_nitive hosts of caninum Milton M[ McAllistera\ \ J[ P[ Dubeyb\ David S[ Lindsayc\ William R[ Jolleya\ Rebecca A[ Willsa\ Angela M[ McGuirea

aUniversity of Wyoming\ College of Agriculture\ Department of Veterinary Sciences\ 0063 Snowy Range Rd[\ Laramie\ WY 71969\ U[S[A[ bUnited States Department of Agriculture\ Agriculture Research Service\ and Poultry Sciences Institute\ and Parasite Biology and Epidemiology Laboratory\ BARC!East\ Bldg[ 0990\ Beltsville\ MD 19694!1249\ U[S[A[ cCenter for Molecular Medicine and Infectious Diseases\ Department of Biomedical Sciences and Pathobiology\ VirginiaÐMaryland Regional College of Veterinary Medicine\ Virginia Tech\ 0309 Prices Ford Rd[\ Blacksburg\ VA 13950!9231\ U[S[A[

"Received 04 July 0887^ accepted 19 July 0887#

Abstract

Dogs were investigated to determine if they are de_nitive hosts of Neospora caninum[ Four dogs were fed N[ caninum tissue cysts in infected mouse tissue\ and two negative control dogs were fed uninfected mouse tissue[ faeces were examined daily for 29 days using a sucrose ~otation technique[ Three challenged dogs shed spherical to subspherical unsporulated oocysts\ measuring 09 to 00 mm in diameter[ Oocysts sporulated within 2 days and contained two sporocysts\ each with four sporozoites[ Outbred\ inbred\ and gamma!interferon knockout mice were inoculated with canine faecal extracts and monitored for evidence of neosporosis using a variety of morphologic\ immunohistologic\ serologic\ and genetic analyses[ Mice that received faeces from each dog observed to shed oocysts were demonstrated to have neosporosis by two or more techniques[ One mouse was demonstrated to be infected with N[ caninum by immunohistochemistry\ ultrastructural analysis\ and a species!speci_c PCR test[ No evidence of neosporosis was observed in control animals[ Based on this study\ dogs are a de_nitive host of Neospora caninum[ Þ 0887 Australian Society for Parasitology[ Published by Elsevier Science Ltd[ All rights reserved[

Keywords] Neospora caninum^ Neosporosis^ Life cycle^ De_nitive host^ Dog^ Canine^ Oocyst[

Neospora caninum "] # 3\ 4Ł[ The parasite exists on at least six continents is a protozoan that infects domestic and wild canids\ ð0Ð2Ł[ To date\ N[ caninum is the only recognised \ and horses ð0Ð2Ł[ Neosporosis is econ! species in the genus^ canine and bovine isolates omically important in cattle\ and is the most com! appear to be identical ð5Ł[ monly diagnosed cause of bovine abortion in Prior to its description in 0877\ N[ caninum was California\ the Netherlands\ and New Zealand ð0\ frequently misidenti_ed as ð6Ł[ Toxoplasma!like parasites have a heteroxenous life cycle with sexual stages occurring in the intestine of a de_nitive host ð7Ł[ Until now\ the de_nitive host Present address of corresponding author] University of Illi! nois\ Department of Veterinary Pathobiology\ 1411 VMBSB\ of N[ caninum was unknown[ Cats\ dogs\ \ 1990 S[ Lincoln Ave[\ Urbana\ IL 50791\ U[S[A[ Tel[] 106!222! and several species of carnivorous birds have been 1338^ fax] 106!133!6310^ e!mail] mcallisterÝcvm[uiuc[edu[ tested with negative results ð0\ 8Ð00Ł[ The number

S9919!6408:87:,08[99 Þ 0887 Australian Society for Parasitology[ Published by Elsevier Science Ltd[ All rights reserved[ PII] S9919!6408"87#99027!5 0363 M[M[ McAllister et al[ : International Journal for Parasitolo`y 17 "0887# 0362Ð0367 of tissue cysts consumed\ the number of animals\ Bioassays were performed in gamma!interferon and other aspects of the experimental design varied knockout mice "BALB:c!Ifngtm0Ts\ Jackson Lab! widely among these investigations[ We wished to oratories\ Bar Harbor\ Maine# and in cort! further test the hypothesis that dogs are a de_nitive icosteroid!treated outbred mice[ Mice that became host of N[ caninum[ ill were euthanatised and attempts were made to Four\ 7!week!old beagle dogs "Harlan Sprague isolate protozoa in tissue culture from liver\ lung\ Dawley\ Indianapolis\ Indiana# were housed in sep! and spleen\ and touch preparations of these organs arate cages[ Two age!matched\ same!source beagles were stained with Giemsa and examined micro! were similarly housed in a separate room\ to serve scopically for tachyzoites[ In addition\ tissues from as negative controls[ Dogs had never consumed the _rst knockout mouse to die were examined his! uncooked meat or meat by!products[ Dogs were tologically\ immunohistologically\ ultrastructur! fed dry dog food and were released into the room ally\ and by a PCR procedure[ Specimens of lung\ for several h each day to permit socialisation and liver\ and spleen were _xed in 09) neutral bu}ered exercise[ Blood samples were obtained prior to and formalin prior to routine histologic processing\ and 26 days after consuming infected mice\ and IFAT 4 mm thick sections were prepared[ Some histologic serology was performed using N[ caninum tachy! sections were stained with H and E[ Other sections zoites as antigen ð01Ł[ were immunohistochemically stained using N[ can! Tissue cysts of the NC!1 ð02Ł\ NC!Liverpool ð03Ł\ inum!speci_c mAb 5G6 or with N[ caninum anti! and NC!beef strains of N[ caninum were produced serum\ using reagents and methods previously in outbred white mice that were immunosuppressed described ð08\ 19Ł^ the speci_city of these reagents with corticosteroids according to previously has been tested extensively[ Portions of spleen were described protocols ð04\ 05Ł[ The NC!beef strain\ processed for EM by _xing in 2) glutaraldehyde previously undocumented\ was isolated in vitro in in Millonig|s phosphate bu}er "pH 6[3#\ post!_xing our laboratories from a naturally!infected beef calf\ in 0) osmium tetroxide\ dehydrating in a series of approximately 0 month prior to inoculating the ethanols\ passing through two changes of propylene mice used in the present study[ After 0 or more oxide\ and embedding in Poly:Bed 701 "Poly! months\ mice were killed by cervical dislocation\ sciences Inc[\ Pennsylvania#[ Thin sections were blood was collected for IFAT serology\ and tissue stained with uranyl acetate and lead citrate and cysts were enumerated by examination of brain examined with a JEOL!099CX II TEM operating squashes ð06Ł[ Portions of brain homogenates were at 79 KV[ A previously described PCR procedure inoculated onto tissue cultures in order to verify was used to detect an N[ caninum!speci_c DNA viability of organisms\ after digestion in an acidÐ sequence ð10Ł in an extract of splenic tissue[ pepsin solution ð06Ł[ Brains and the skinned\ evis! In addition to the bioassay methods already men! cerated\ blended carcasses of infected mice were fed tioned\ six seronegative\ corticosteroid!treated to four dogs on three consecutive days "days p[i[ BALB:c mice "Harlan Sprague Dawley# per dog 9\ 0\ and 1#[ Seronegative\ uninfected mice were were inoculated orally with aerated faecal extracts\ processed identically and fed to two dogs on the collected between 7 and 03 days after dogs con! same dates\ as a negative control[ sumed infected mice[ Blood samples were collected The faeces in each dog|s cage were collected daily 03 days later\ and sera were tested for N[ caninum and examined using a sucrose ~otation procedure antibodies at a 0]49 dilution using the IFAT[ described for Toxoplasma gondii oocysts ð07Ł\ modi! The results of examination of the oral inocula fed _ed to permit ~otation of the entire faecal mass[ The to dogs\ parasitologic examinations of dog faeces\ ~otation supernatant was diluted in approximately serology of dogs\ and an interpretive summary of _ve volumes of water and centrifuged for 09 min at mouse bioassays are presented in Table 0[ Unsporu!

549 g[ The sediment was mixed with 1) H1SO3 and lated\ spherical to subspherical oocysts\ measuring aerated on a shaker for 2 to 6 days[ For bioassay\ 09 to 00 mm in diameter and containing a central acidi_ed faecal extracts were neutralised with 2[2) sporont\ were observed to be shed by three dogs NaOH and inoculated orally and s[c[ into mice ð07Ł[ that consumed N[ caninum!infected mice\ but not by M[M[ McAllister et al[ : International Journal for Parasitolo`y 17 "0887# 0362Ð0367 0364

Table 0 Summary of Neospora caninum oral inoculation of dogs and bioassay in mice

N[ caninum strain Number of tissue Tissue culture Dog Ab titre at 26 Days p[i[ oocysts Evidence of in mice fed to cysts fed to dogs results of mouse days p[i[ observed in dog neosporosis in Dog ID dogs brains fed to dogs "reciprocal# faeces mouse bioassay

A NC!1 299 positive 799 7Ð16 yes B NC!beef 049 negative ³49 02Ð19 yes C NC!1 049 positive r0599 02Ð12 yes ý NC!beef 14 negative ýýý ýNC!Liverpool 64 positive ýýý D NC!1 099 positive r0599 none observed no NC!Liverpool 14 positive ýýý E none 9 negative ³49 none observed no F none 9 negative ³49 none observed no

either dog that consumed uninfected mice[ Oocysts icosteroid!treated outbred mouse and three sporulated within 2 days and contained two spo! gamma!interferon knockout mice died or were rocysts\ each with four sporozoites[ The oocysts euthanatised with evidence of systemic neosporosis\ were morphologically similar to T[ gondii\ Ham! as revealed by the presence of tachyzoites in touch mondia hammondi\ and Hammondia heydorni "Fig[ preparations\ and in one case by recovery of tachy! 0 A\ B# ð11Ł[ zoites in tissue culture[ One of these mice correlated The _rst mouse that died in the bioassay was a with Dog A "NC!1#\ two mice correlated with Dog gamma!interferon knockout mouse inoculated s[c[ B "NC!beef#\ and one mouse correlated with Dog with a faecal extract containing oocysts from dog C "mixed inoculum#[ One of these mice had been B\ which had consumed NC!beef!infected mice[ inoculated by gavage only[ The organism isolated Touch preparations revealed large numbers of in tissue culture was successfully transferred to tachyzoites in liver\ spleen\ and lung that were mor! uninfected cells\ unlike those of H[ hammondia or phologically identical to those of N[ caninum "Fig[ H[ heydorni\ which cannot be passaged ð11Ł[ 0 C#[ Histology revealed numerous tachyzoites in Fourteen days after consuming faecal extracts these organs\ with multifocal hepatic necrosis and from dogs that had consumed N[ caninum!infected minimal associated in~ammation "Fig[ 0 D#[ Immu! mice\ two of 13 BALB:c mice developed N[ caninum nohistology demonstrated positive staining of these antibody titres of r0]49[ One of these mice cor! organisms with N[ caninum!speci_c mAb 5G6 and related with Dog A and the other with Dog C[ polyclonal antisera "Fig[ 0 E#[ Intracellular and Twelve mice\ correlating with the negative control extracellular tachyzoites were observed using EM dogs\ did not seroconvert[ "Fig[ 0 F#[ Intracellular tachyzoites were usually Dogs are a de_nitive host of N[ caninum[ After in a parasitophorous vacuole[ They contained a consumption of tissue cysts in infected mouse car! conoid\ micronemes\ dense granules\ a vesicular casses\ three of four dogs shed oocysts resembling nucleus\ and numerous electron dense rhoptries\ those of T[ gondii and Hammondia spp[ Protozoal but they lacked the prominent anterior vacuole tachyzoites were observed in organs of mice used characteristic of H[ heydorni tachyzoites ð12Ł[ in bioassays of faeces from each of these dogs[ Mice Analysis of a splenic extract using PCR resulted in inoculated with faecal extracts from two of these ampli_cation of a N[ caninum!speci_c DNA dogs developed N[ caninum antibody titres\ and a sequence "Fig[ 1#[ mouse inoculated with faecal extracts from the third Within the _rst 1 weeks of inoculation with faecal dog was proven to develop neosporosis by immuno! extracts containing unidenti_ed oocysts\ one cort! histochemistry\ ultrastructural examination\ and 0365 M[M[ McAllister et al[ : International Journal for Parasitolo`y 17 "0887# 0362Ð0367

Fig[ 0[ Neospora caninum stages in dogs and mice[ A[ Unsporulated oocyst with a central mass[ B[ Sporulated oocyst with two sporocysts "arrow# and with sporozoites "arrowheads#[ C[ Smear of liver from a knockout mouse inoculated with dog faeces\ showing individual tachyzoites "arrowheads# and a zoite dividing by endodyogeny "arrow#[ Giemsa stain[ D[ Tachyzoites "arrows# in a focus of necrosis in a section of liver from the mouse in Fig[ 0 C[ H and E stain[ E[ Section from the same liver as in Fig[ 0 C\ showing individual "arrowheads# and a group "arrow# of tachyzoites[ Immunohistochemical stain with N[ caninum!speci_c monoclonal antibody[ F[EM of tachyzoites in liver of mouse in Fig[ 0 C[ Note conoid "c#\ micronemes "m#\ and electron!dense rhoptries "r#[ M[M[ McAllister et al[ : International Journal for Parasitolo`y 17 "0887# 0362Ð0367 0366

Acknowledgements*This research was supported in part by USDA!NRICGP grant 85!91421[ The authors thank Jennifer D[ Tranas\ S[ K[ Shen\ O[ C[ H[ Kwok\ Diane Hawkins!Cooper\ Virginia Viers\ Kathy Lowe and Jill Songers for technical assist! ance\ and Catherine A[ Jones and Wendi R[ Wilson for animal care[

References Fig[ 1[ Electrophoretic separation of DNA products obtained by polymerase chain reaction using Neospora caninum!speci_c ð0Ł Dubey JP\ Lindsay DS[ A review of Neospora caninum and primers ð10Ł[ Lane 0] marker in 012 base pair increments[ Lanes neosporosis[ Vet Parasitol 0885^56]0Ð48[ 1 and 2] positive controls consisting of in vitro cultured N[ ð1Ł Barber JS\ Gasser RB\ Ellis J\ Reichel MP\ McMillan S\ caninum tachyzoites[ Lane 3] splenic extract from a mouse that Trees AJ[ Prevalence of antibodies to Neospora caninum in had been inoculated with faeces from a dog that consumed N[ di}erent canid populations[ J Parasitol 0886^72]0945Ð0947[ caninum tissue cysts and was shedding unidenti_ed oocysts[ Lane ð2Ł Huong LT\ Ljungstrom BL\ Uggla A\ BjoÃrkman C[ Preva! 4] negative control "water#[ The 217 bp band represents the N[ lence to Neospora caninum and Toxoplasma gondii in cattle caninum DNA sequence[ The primer band represents residual and water bu}aloes in southern Vietnam[ Vet Parasitol primers\ primerÐdimers\ or nonspeci_c ampli_cation product[ 0887^64]42Ð46[ ð3Ł Anderson ML\ Palmer CW\ Thurmond MC\ Picanso JP\ Blanchard PC\ Breitmeyer RE\ Layton AW\ McAllister M\ Daft B\ Kinde H\ Read DH\ Dubey JP\ Conrad PA\ Barr PCR[ Although this study demonstrates that N[ BC[ Evaluation of abortions in cattle attributable to neo! caninum can be transmitted in dog faeces\ the ident! sporosis in selected dairies in California[ J Am Vet Med ity of the oocyst is presumptive and requires con! Assoc 0884^196]0195Ð0109[ _rmation[ ð4Ł Thornton RN\ Thompson EJ\ Dubey JP[ Neospora abor! The concentration of oocysts in faeces was low tion in New Zealand cattle[ N Zeal Vet J 0880^28]018Ð022[ ð5Ł Holmdahl J\ BjoÃrkman C\ Stenlund S\ Uggla A\ Dubey JP[ and appeared to be roughly in proportion to the Bovine Neospora and Neospora caninum] one and the same[ number of tissue cysts consumed by the dogs[ Parasitol Today 0886^02]39Ð30[ Maximum oocyst shedding for Dog B was _ve per ð6Ł Dubey JP\ Carpenter JL\ Speer CA\ Topper MJ\ Uggla A[ coverslip^ this dog was fed 049 tissue cysts[ It is Newly recognized fatal protozoan disease of dogs[ J Am possible that the number of tissue cysts fed to dogs Vet Med Assoc 0877^081]0158Ð0172[ ð7Ł Current WL\ Upton SJ\ Long PL[ Taxonomy and life and coyotes in previous experiments was too low cycles[ In] Long PL\ editor[ of man and dom! to induce detectable shedding of oocysts[ Presence estic animals[ Boston] CRC Press\ 0889]0Ð05[ or absence of seroconversion of test subjects may ð8Ł McAllister MM\ Jolley WR\ Wills RA\ Lindsay DS\ not be an indicator of an adequate or inadequate McGuire AM\ Tranas JD[ Oral inoculation of cats with challenge inoculum "Table 0#\ and perhaps is in~u! tissue cysts of Neospora caninum[ Am J Vet Res 0887^48]330Ð333[ enced by the parasite strain[ Future testing of wild ð09Ł Lindsay DS\ Kelly EJ\ McKown RD\ Stein FJ\ Plozer J\ canids is warranted to ascertain if they are also Herman J\ Blagburn BL\ Dubey JP[ Prevalence of Neospora de_nitive hosts of this parasite[ Without enu! caninum and Toxoplasma gondii antibodies in coyotes meration of tissue cysts in the oral inocula fed to "Canis latrans# and experimental infections of coyotes with test subjects\ negative results should be viewed Neospora caninum[ J Parasitol 0885^71]546Ð548[ skeptically[ ð00Ł Baker DG\ Morishita TY\ Brooks DL\ Shen SK\ Lindsay DS\ Dubey JP[ Experimental oral infections in birds to Neosporosis abortion outbreaks ð13Ł may occur evaluate potential de_nitive hosts of Neospora caninum[J in cattle herds fed mixed rations if the feed is con! Parasitol 0884^70]672Ð674[ taminated[ Farmers should consider taking steps to ð01Ł Dubey JP\ Hattel AL\ Lindsay DS\ Topper MJ[ Neonatal limit opportunities for dogs to defaecate in feed Neospora caninum infection in dogs] Isolation of the causa! intended for breeding cattle or goats[ In many tive agent and experimental transmission[ J Am Vet Med Assoc 0877^082]0148Ð0152[ instances\ this could be accomplished by fencing ð02Ł Hay WH\ Shell LG\ Lindsay DS\ Dubey JP[ Diagnosis and around open!faced buildings used to store feed\ treatment of Neospora caninum infection in a dog[ J Am uncovered silage piles\ and hay[ Vet Med Assoc 0889^086]76Ð78[ 0367 M[M[ McAllister et al[ : International Journal for Parasitolo`y 17 "0887# 0362Ð0367

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