IMMUNOLOGICAL SURVEY OF ( PEIRCEI) AND IN JACKASS PENGUINS IN SOUTH

GRACZYK T.K.', B1~OSSY J.].", SA DERS M.L. ', D UBEY J.P.···, PLOS A .. ••• & STOSKOPF M. K .. ••••

Sununary : ReSlIlIle: E x-I1V\c n oN l~ lIrIUSATION D'Ar\'"TIGENE DE B ;IB£,'lA PH/Re El EN ELISA ET simoNi,cATIVlTli t'OUR 7 bxo l'l.ASMA GONIJfI DE SI'I-IENICUS was extracted from nucleated erythrocytes Babesia peircei of IJEMIiNSUS EN ArRIQUE D U SUD naturally infected Jackass penguin (Spheniscus demersus) from South Africo (SA). Babesia peircei glycoprotein·enriched fractions Babesia peircei a ele extra it d 'erythrocytes nue/fies p,ovenanl de Sphenicus demersus originoires d 'Afrique du Sud infectes were obto ined by conca navalin A-Sepharose affinity column natulellement. Des fractions de Babesia peircei enrichies en chromatogrophy and separated by sod ium dodecyl sulphate­ glycoproleines onl ele oblenues par chromatographie sur colonne polyacrylam ide gel electrophoresis (SDS-PAGE ). At least d 'alfinite concona valine A-Sephorose et separees par 14 bonds (9, 11, 13, 20, 22, 23, 24, 43, 62, 90, electrophorese en gel de polyacrylamide-dodecylsuJfale de sodium 120, 204, and 205 kDa) were observed, with the major protein (SOS'PAGE) Q uotorze bandes proleiques au minimum ont ete at 25 kDa. samples of 191 adult S. demersus were tes ted observees (9, 1 I, 13, 20, 22, 23, 24, 43, 62, 90, 120, 204, by enzyme-linked immunosorbent assoy (ELISA) utilizing B. peircei et 205 Wa), 10 proleine ma;eure elant de 25 Wo. Des . glycoprotein-enriched frac ti ons to detect anti-B. peircei IgG. Th e prelevements sanguins provenont de 191 S. demersus adultes onl somples originoted from three groups of free-ranging penguins ele lesles par ELISA en utilisont les fractions de B. peircei enrichies (n = 1 10), 1 group of penguins (n = 66) which were rescued en glycoproteines pour deleclel les IgG dirigees con Ire B. peircei. after offshore oil-spill con tominations and rehabilitated ot th e South Les prelevements provenaient de trois groupes de manchols Africon Notional Foundation for the Conservation of Coastal sauvages (n - 1 10), d 'un groupe de manchols (n = 66) ayanl ele (SANCCOB), and the final group from SANCCOB-resident secourus apres avoir ete contamines par une maree noire en mer penguins (n = 151. The overall B. peircei seropreva lence was et soignes d 10 Fondation Nationale Sud Africoine pour Ia 65 %, and the mean seroposi tivity ranged from 60 to 71 % Conservation des Oiseaux Lilforaux (SANCCOB), el d'un demier among the five penguin groups. The ELISA appecred to be specific groupe issu des manchals pensionnaires du SANCCOB (n = 15). for B. peircei IgG as tested against Haemoproleus columbae IgG La prevalence globale pour B. peircei etail de 65 %, et 10 and avian ( relictum, ond P. elongalum) IgG. seropositivile moyenne s'echelonnail de 60 d 7 I % parmi les cinq Toxoplasmo gondii (Ab) were detected by th e d irect graupes de manchols. L'EUSA apparaissail specihque pour les agglutinati on tes t using kill ed T. gondii tachyzoites. All birds were IgG dirigees conlre B. peircei lorsque leslee pour les IgG contre selonegative for T. gonda Ab. The lock of T. gondij-positive columbae el les IgG contre Ie paludisme aviaire penguins wos due to the appropria te so nitary conditi ons and anti­ (, et P. elongatum) . les anlicorps (Ac) diriges Toxoplasma prevention procedures utilized by the SANCCOB. cantre ani ele delecles par Ie lest d 'agglulination directe utilisant des lachyzailes de T. gondii lues. KEY WORDS : Babesia peircei, Jackass penguins, African black·footed penguin , Tous les oiseaux etaient seronegatifs pour les Ac diriges conlre Spheniscus demersus, Toxoplosma gondii, ELISA, SDS·PAGE. T. gondii. I'absence de manchols posilifs pour T. gondii elait due aux condilions son ito ires appropriees et aux methodes de MOTS CLis : Babesia peilcei, manchot, Spheniscus demelSus, Toxoplasma gondii , ELISA, SDS·PAGE. prevenlion con Ire T. gondii ulilisees par Ie SANCCOB.

• Johns H o rkin~ Unive rsity, School of Hygiene and Public Hea lth, The Jackass or Africa n black-footed penguin, Sphe­ De partmen t of Molecut ar Microbio logy and Immuno logy, niscus del17ersus, is an endangered w ith decrea­ Baltimore, Maryland 21205, USA. sing populatio ns in its natural habitat on coasta l islets .. University of Cape Town Medica l School, Obsen'3tolY 7925, of (SA) (Crawford et aI. , 1990). Frequent Der aIlment of Anatomy and Cell Biology, Ca pe Town, South Afri GI. ... US Depa rtment of Agriculrure, Agriculture Resea rch Service, live­ offshore o il-spill contamination pr sents th e most stock and PoultlY Sciences Instit ute, Parasit e Biology and Eride­ devastating haza rds for existing wild p opulations. The miology Ltboratory, Beltsville, Maly land 20705, US A. spills debilitate, injure, and induce disease in penguins .... Percy FitzPatrick tnstitute of African O rnithology, University of (Erasmus el at., 1981; Kerley el al. , 1985). The disa­ Cape Town, Rondebosch 7700, South Africa. ••••• North C. into the wild.

Parasite, ]996, 4, 313-319 L_--1.M~em~02!i!!re!...-_--1------3 13 GRACZYK T.K., BROSSY J.J. , SANDERS M.L., DUBEY JP., PLOS A. & STOSKOPF M.K.

An inrrae rythrocytic piroplasm of free-ra nging (Brossy, 1992), air-dri ed, and stored on filter paper as S. delnersus originally described as Aegyptlanella sp. described by Graczyk et aL. (993). The samples ori­ (Coles, 1941) was reindentified as Babesia sp. (Ben­ ginated from three groups of free-ranging penguins nert et al , ] 992) and named Babesia peircei (Ea rl e from offshore islands (Dassen Island and Robben et al, 1993). Babesia peircei associated I abesiosis is Island) and mainland colony (Boulders, Simons Town), common and e ndemic in w ild S. demersus and contri­ from penguins rescued after offshore oil-spill conta­ butes to illness when associated with minations and rehabilitated at SANCCOB, and from (Brossy, 1992 ; 1993) . The prevalence of B. peircei SANCCOB-reside nt penguins. The penguins were in S. demersus rehabi litated at SANCCOB screened for blood parasites by Giemsa-stained thin vari ed from 11 to 15%, and was 4 % in free-ranging blood smears (Brossy, 1992), and a total of 10 ml of birds (Brossy, 1993). A putative Babesia sp. blood was collected from four penguins with the that ca used regenerative a naemia in ju venil e li ttle pen­ highest B. peil-cei parasitemia. Five thin blood smears guins (Eudyptula minor) in Australia has been reported were prepared and examined (Graczyk et aI. , 1994b) by CI

ched fractions were obtained following the primary and sa mples of 2-wk-old Peking ducklings (A nas jJla(JilhYI1- secondalY elution of the column (Fig. 1). The first and cbos) experim entall y infected with Plasmodium reliC /11 m the second fra ction contained 1.5 and 1.1 pglpl proteins, and P. elongatum, respectively, and bl ood sample from respectively, as determined by the BSA assay. Con A­ pigeon (Columha /ivia) narurall y infec ted with bound fractions of B. peircei AE were incubated for Haemoproteus columhae. Add ili onall y, the we lls we re 5 min at 95° C and separated by SDS-PAGE with a 15 0/0 fill ed with blood samples of two captive adult S. demers/.ls gel Oule Biotechnologies Inc., New Haven, Connec­ naturall y clinically infected with P. ndic/ll11i and P. e/oll­ ticut, USA) run at 100 V constant voltage for 7 h at 2].0 C ga/um, respec tively, and the blood sample from \vild S. in electrode buffer (De Repentigny et al., 1991) and demersus known to be positive for malaria l IgG (G raczyk stained with Coomassie blue (Laemmli, 1970). To deter­ et a/., 1995). All blood sa mples were air-dri ed and stored mine the purity of the B. peircei AE, the RBC extracti on­ on filter paper, eluted in to th e buffer and diluted 1/ 100 derived particles were processed by Con A-Sepharose with PBS. Duckling, pigeon, and captive penguins infec­ 4B affinity column chromatography in the sa me manner. tions \vere determined by ELISA (Graczyk e/ a I. , 1994 c) Prestained broad range molecul ar weight standards (Bio­ and were confirmed by paraS itemia found on the Gie msa­ Rad Laboratories, Hercul es, California, USA) were used. stained thin s. The remainder ELISA protocol fo l­ To further determine purity of the antigen, the glyco­ lowed previous procedures (G ra czyk e/ al., ] 994 c) lItili­ protein enriched fractions of B. peircei AE and RBC were zing bird species-specifi c conjuga tes. separately elecroeluted (Electro-Eluter M422, Bio-Rad To determine the presence of T gOl1dii agglutinating Laboratories, Richmond, California, USA) and , after deter­ antibody CAb), a modifi ed direct agglu tin ation test mination of protein concentration, eluted fractions were with ki lled T gondii tachyzoites was carri ed out (Des­ used to coat the ELISA plate at the same concentration monts & Remington, 1980) In a modified tcst, 2-mer­ (Graczyk & Cranfield , ]995), captoe[hanol was incorporated inl o the anligen instead For the indirect ELISA detection of anti-B. peircei IgG, of into the blood sample to avoid pro longed exposure whole blood, air-dried on filter paper and elu ted into to toxic fu mes (Dubey & Desmonls, ] 987). Positive and buffer, was used (Graczyk et at., 1993). Fi lter paper sto­ NC controls were prepared a de cribed p reviously rage does not diminish IgG binding capacity (Graczyk (Dubey & Desmonts, 1987). All bl ood samples were assayed at 1/ 25, 1/ 50, and 1/ 500 di lu tion with PBS (pl-l et al., 1993). ELISA was performed with Immulon 2 microtitration plates CDynatech Laboratori es Inc. , 7.4) on U-bottom well mi crotitre plates (Dynatech Laboratories Inc., Chantilly, Virgin ia, USA) with Chantilly, Virginia, USA). The pooled Con A-bound T gondii PC and NC incl uded on each pl ate fra ctions were used as an antigen. The optimal antigen concentration for the ELISA with the positi ve control Statistical analys is was ca rri ed out with Amtlyti ca l Soft­ (PC) at a fixed 1/ 100 dilution with PBS and diluted ware Stati sti x 4.1 (Allalytic::d Sofl:\vare, Sr. Paul , Mi n­ 1/1,000 w ith PBS alkaline phosphatase-labell ed ­ nesota, USA). The absorbance valu es of ELI SA-positi ve anti-S. demersus IgG (Gra czyk et at., 1994a) was deter­ penguins were tesled by the approximate Will<-S hapiro mined by serial antigen dilutions. The PC was whole stati stic to determin e if their d istribution confo rms LO a normal distribution. Ana lysis of variance (A NOVA) blood eluted from filter paper of the penguin from was ca rried out to determine the significance of among­ which B. p eircei was extracted. The penguin samples penguin group effects in absorbance va lues. A two­ were assayed in duplicate, eight wells were not coated sampl e t-test was used to compare [he mean absor­ with antigen to determine nonspecific background bance between pe ng uin g roups, a nd the absorbance (NBA), and eight well s were filled with G-heterogeneiry test (Sokal & Hohl r, 198 1) was appli ed each of PC and negative control (NC) . The NC was a blood pool from three, adult captive-reared S. demersus to compare B. p eirce ': seropreva lence among penguin groups. The absorbance refe rence range for B. peil'cei kept indoors. The positive cutoff level was established parasitemi a was established according LO the prolocol as an absorbance greater than the mean ± 3 SD of of LUl1l eij (1994) as mean Cx) 3 SD) of 48 absor­ 8 NC. Sensitivity of the EUSA was determined by tes­ C±. bance va lues obtain ed in th e indirect ELI SA for the ting PC at dilution of 1/ 100, 1/ 200, 11400, 1/ 800, parasitemi c penguin blood. Staristi ca l significa nce was 1/ 1,600, 1/ 3,200, and 116,400. The remainder of the considered to be P < 0.05. ELISA protocol followed previously described proce­ dures (Graczyk et at., 1994 a), and the method of Schwaltz et at. (1991) was used to compare the absor­ bance valu es from different ELISA plates. RESULTS To detelllline the specificity of the indirect ELISA , the plate was coated with B. pein;ei antigen. Following post-coa­ abesia peircei parasitemia in the 10-1111 blood ting with casein blocking'Duffer (G raczyk et at., 1993), the samples va ri ed within th e limits of 78 to well s were filleCl iri 'duplicate with PC and with two blood B 87 %; with a mean C±. SD) = 81.6 ± 4. 1 %.

~p~a~ra~si~te~,~1~99~6~,_4~,~3 1~3~-3~1~9______-1 ______~AA~e~m~o~l~re~ __ ~-,------315 GRACZYK ToK., BROSSY J.Jo , SANDERS MoL., DUBEYJoPo, PLOS A. & STOSKOPl" M.IC

A B C D ,kOa -205 ,~1~l

-- 50

-- 33 ...... 27

__ 19

Fig. 2. - Two forms (A, 13) of divided meruzuites of liabesia 7 peircei which formed a tetrad (a rrows) in the erythrocytes of natu­ rally infected Jackass penguin (Spbeniscus demersus) rescued after offshore oil-spill contamination and rehabilitated at the Rescue Sta­ tion of the South Africa n National Foundation for the Conselvation Fig. l. - Sodium dodecyl sulrhate-polyacrylamide gel electropho­ of Coastal Birds (SANCCOB) in Milnerton, Cape Town, South Africa . resis (SOS-PAGE) of glycoprotein-enriched fractions obta ined by Giemsa-stained thin blood smear. Scale bar ~ 5 >1m. conca navalin A-Sepharose 4B affinity col umn chromatography with intraelythrocytic sta ges of Ba.besia peit·cei extracted from naturally infected Jackass penguins (Spbeniscus demel'sus) rescued aft er off­ shore oil-spill contamination and rehabilitated at the Rescue Station polar end (Fig. 1). Two forms of the tetrad were of the South African National Foundation for the Conservation of observed in the Giemsa-stained thin blood films Coastal Birds (SANCCOB) in Milnerton, Cape Town, South Africa. (Fig. 1). Extracellular stages of B. peireei were not Babesia peircei antigenic fractions ohtained by the primary (lane B) observed. and secondary elution of the column Clane A). Lane C, supernatant from rh e final srep of extraction. Lane 0, fraction of erythrocytes Analysis of B. peireei protein showed that at least from which th e parasites were ex tracted. Prestained broad range 14 bands ranging from 9 to 205 kDa were seen in SDS­ molecular weight standa rds sta ined with Coomassie blue in kilo­ PAGE and Coomassie blue staining of the first glyco­ d ~ t1ton s (kDa). protein-enriched fraction. The molecular sizes of these glycoproteins were 9, 11 , 13, 20, 22, 23 , 24, 43, 62, 90, 120, 204, and 205 kDa, with the major glycoprotein Two parasi tes per erythrocyte were observed in 38.5- 25 kDa (Fig. 2). All bands > 25 kDa were faint. Ery­ 42.5 % ex, 40.2 .:!: 1.8 %) of examined cells. In the majo­ d1rocyte par1icIes derived from B. pei/'eei extraction pro­ rity o f Singly and doubly infected RBC , the parasites cess displayed two major comigrating bands (10 and were situated on the polar end of the erythrocyte or 12 kDa), and two bands of 26 and 60 kDa. Erythro­ displayed bil ateral polar symmetry of location. The cyte proteins eluted for the gel produced negative round, sphe ri cal, ovoid, and elongated merozoites results when used in the ELISA. were the most common form of the parasite. Divided The reference absorbance range for B. peircei parasi­ elongated merozoites (presented in a low-parasitemia temia was 0.45 to 0.81. Using optimal antigen concen­ sample) which formed tetrads, were rare, occurred tration of 5 l-lg/ ml , it was possible to detect B. peircei singly in erythrocytes, and were usuaIly not on the RBC JgG at the PC dilution of 1/ 3,200. The estimates of

GeographicaJ origin of penguins Penguins Absorbance

Tota l Pos itive· number number (%) Range Mean ± SO

•• Oassen Island 33° 26'S 18°04'E 30 18 (60) 0. 23-0.89 0.38 ± 0. 15 .. Robben Island 33° 49'S 18°22'E 46 29 (63) 0.27-1.00 0.53 ± 0.19 .. Mainland 34° 14'S 18°26'10 34 22 (65) 0.23-0.77 0.44 ± 0. 15 ... SANCCOB 34° 14'S 18°26'E 66 47 (71) 0.20-1.00 0.47 ± 0.1 9 .... SANCCOB 34° 14'S 18°26'E 15 9 (60) 0.41-0.93 0.61 ± 0.19

• Above th e cutoff level of 0.] 6 . .. Free- ranging pengUins. ••• Penguins rescued after offshore oil·spill contamination . .... ReS ident pengu in population.

Table I. - Seroprevalencc for Babesia peircei IgG determined by the indirect enzyme-linked immunosorbent assay (EU SA) of adult Jac­ kass penguins (Spbeniscus demercus) at rh e Rescue Station of th e South Africa n National Foundation for the Conservation of Coastal Birds (SANCCOB) in Milnerton, Cape Town, South Africa.

316 ------r---N\r,~;frE?""'7T""lf------'Parasite,- 1996,-'-- 4, 313-319- IMMUNOLOGICAL SURVEY OF BABESIOSIS (BADESlA PEI1?CEI) AND TOXOPLASMOSIS IN JACKASS PENGUINS IN Soum AFRICA

Wilk-Shapiro statistics for absorbance values (0.74, is probably a transmitting agent of B. peircei. The mean ti ck load was 1.0 .:!:. 1.64 per adult S. demersus 0.96, 0.94, 0.89, and 0.84) for the five penguin gro ups and 2.0 (range 1-50) per nestling on the Marcus lsland, listed in Table I, respectively, showed that the absor­ SA (Duffy, 1991). The low rick infestation in that study bance values were normally distributed. The seropre­ was explai ned by (he presence of ants (Duffy , 1991), valence of B. peircei IgG ranged from 60 to 71 % but weather patterns play an important role in ti ck pre­ among the five penguin groups (Table I), with an ove­ rall seroprevalence of 65 % 025 of 191). The G­ va lence. heterogeneity test showed a nonsignificant effect in the The ELISA results in our studies in dicated (hal the sero­ prevalences of B. p eircei seropositivity among these prevalence of B. p eircei was uniformly distributed among the five populations of Jackass penguins. If the groups (G = 1.07, P > 0.05). The absorbance va lues of positive penguins vari ed within the limits of 0.20 to bovine/equine model (Spickett & Fivaz, 1992; Fivaz 1.00, with the total mean absorbance C± SD) of 0.48 & De Waal, 1993) of babesiosis is applicable to free­ ± 0.19. The absorbance val ues varied nonsignificantly ranging penguins, (he observed uniformity of sero­ among the three groups of free-ranging S. demersus conversion may refl ect the uniformity in the exposure CANOVA; F = 1.89, P> 0.05). The absorbance values to the . of 59 of 191 (31 %) penguins exceeded the 100ver refe­ Glycoproteins of a number of the protozoans parasi­ rence range of 0.45 established for B. peircei parasi­ tizing erythrocytes have been shown to be parasite temia. Those were: 3 of 18 (17 %) of Dassen Island, stage-specific, and to be the most immunogenic frac­ 18 of 29 (62 %) of Robben Island, 11 of 22 (50 %) of tion of th e parasite surface proteins (M itc he ll & Anders, Boulders penguins, and 19 of 48 (40 %) of oiled birds, 1982). The penguin RBC-derived glycoproteins in this and 8 of 9 (89 %) of penguin-resident at SANCCOB. study showed two major comigrating bands 00 and The G-heterogeneity test showed a significant effect 12 kDa) of close molecul ar weight to the minor gly­ among five penguin groups in the percentage of birds coproteins of the pathogen a fraction (9, 11 , and having absorbance values exceeding the reference 13 kDa). Therefore, B. peircei extract should be assayed by SDS-PAGE before use in the ELISA in order to deter­ range of B. peircei parasitemia (C = 10.63, P < 0.5) mine if it is contaminated with fo reign glycoproteins. The blood samples of C. livia naturally infected with H. co/wnhae, Peking ducklings experimentally infected The antigenic extract contained th e proteins of at least with P. relictum and P. elongatum, and S. demersus nine intraerythrocytic sta ges of B. peirce; (Ea rl €: et a l. , naturally infected with avian malaria gave negative 1993), and IgG responses against other avia n bl ood results in the indirect ELISA utilizing B. p eircei antigen. parasitiC (H. columhae, P. relic/um and P. elonga/um) were not specific to B. p eircei, as deter­ Toxoplasma gondii Ab were not detected in penguin mined by the ELISA. We conclude that th e humoral plasmas. responses were directed against blood stages of B. peircei. However, except in the penguins from which B. peircei was extracted, the relati onship bet­ DISCUSSION ween IgG magnitude and parasitemia level remain unknown. Serological studies on captive S. demersus ore in depth immunologic studies o n avian naturally infected with avian malaria show thar IgG res­ babesiosis are needed to interpret the pre­ ponses do not correlate with parasitemia (Graczyk M valence range of B . peircei seroconversion et al., 1994 d). Further studies are necessary to in ves­ of Jackass penguins. A comprehensive review of pen­ tigate if this is also the case fo r B. peircei infecti ons. guin diseases and parasites (Clarke & Knowles, 1993) [n non-oil ed penguins, are primarily around the listed six species of and three species of Omi­ , at the commissures of the mouth, on the webs thodoros ticks from 14 species of penguins; however, between the roes, and around the cloaca (Clarke & only Omithodoros capel1sis was reported from free-ran­ Knowles, 1993). Thus, we do not feel oil-impaired fea­ ging S. demersus CDaturi, 1986 ; Duffy & Daturi, 1987 ; thers make Jackass penguins more vulnerable ro ti cks Brossy, 1993). It was documented that pengu ins that feeding. However, oil ed Jackass penguins can develop reuse (he same colony and nest sites for generations fata l infections witb Iyphimurium and Sta­ are particularly vulnerable to ticks and tick-transmitted phylococcus mweus (Westphal & Rowan, 1970), and pathogens (Duffy, 1991). Ixodes UTiae, implica ted as a mount weaker Ab responses agai nst avian malaria for B. peircei (Ea rle et al. , 1993) is distributed (Graczyk et aI., 1995). Interestingly, B. peircei was worldwide h av ing even been re ported fro m the described as a new species frol11 oiled Jackass penguins Antarctic Peninsula (Clarke & Knowles, 1993) . (Ea rle et al., 1993). However, only O. capensis is present in the natural Toxoplasmosis is always a concern in bird centers habitat of Jackass penguins (Daturi, 1986; Duffy & because high density of birds favor transmission of the Daturi, 1987; Brossy, 1993) indicating that o. capensis

~p~arn~si~t e~,~19~9~6~,_4~, ~31~3~-3_J ~9 ______-L ______~AA~e~'m~0~ir~e______Jr------______31 7 GRACZYK T.K., BROSSY J.J. , SANDERS M.L., DUBEY J.P., PLOS A. & STOSKOPF M.K.

pathogen and the fatal cases, including p en g uins, were CliANDRAWATHANI P., TSUJ I N., KAwA ZU S. , ISH IKAWA M. & FUJI­ reported from s u ch centers (Ra tcliff & Worth, 1951; SAKI K. Seroepidemiological studies of bovine babeSiosis Hubbard et at. , ]986; H artley & Dubey, 1991). Ratcliff caused by Babesia oua/a, B. higemina, and B. bovts in & Worth (1951) reported fatal toxoplasmosis in four Peninsular Malaysia . j ournal oj Veterinary Medical SCience, 1994, 56, 929-932. S. humboLdti, two S. magellanicus, and seven S. demersus at the Philad e lphia Zoo (USA). The seven CL>\RKE]. R. & KNOWLES It K. Diseases and parasites of pen­ S. demel"sus were healthy but becam e ill o ne week afte r g uins. Kurean j ournal oj Polar Reseal-c/) , 1993, 4, 79-96. arrival at the Zoo; it is most like ly that p e n g uins COLES ].D.W.A. An epizootiC in seabirds: A visit to Dassen acquired toxoplasmosis at the Zoo. and Malgas Islands. joumal oj tbe South African Veteri­ nmy Medical Association, 1941 12, 23-30. In the presenr study, Ab to T gondii in diluted pen­ guin sera were tested by the direct agglutinatio n lest. CMWFORD R.].M ., WILLIAMS A.]. , RANDALL ItM., BEIUWTI A. & Hoss G.].B. Recent populalion trends of Jackass penguins U nlike th e Sabin-Feldman dye test, the direct aggluti­ off Southern Africa. Biological Comeruation, 1990, 52, nation test has proved to be highly sensitive for the 229-243. detection of T gondii Ab in m a ny avian sp ecies inclu­ DATUIU A. A preliminalY study of tick populations in Jackass ding , quails, turkeys, , pheasants, a nd penguin nests on Marcus Island, South Africa. Ostrich, partridges exp e rime nta ll y infected with T gondii 1986,57, 95-1 00. (Dubey et a i. , 1995), Many avian sp ecies including chic­ DE REPENTIGNY 1. , Kn.ANOWS KI E., PE DENAU LT L. & BOUSHIRA M. k e n s and turkeys do not d evelo p a ppropriate Ab for Immunoblot analyses of the serologic response [0 Asper­ the dye test (Frenke l, 1981). gillus Jumigatu.s antigens in experimental invasive asper­ The negative o utcom e in o ur Toxoplasma survey indi­ g ill OSiS. The jou.rnal oj Infectious Dl~'eases, 1991, 163, cates that n o n e of the p e nguins h ad contracte d 1305-131l. T gondii tachyzoites at the time o f blood sample col­ D ESMONT G. & REM INGTON ].S. Direct agglutination test fo r the lection. The lack of T gondii-p ositive p e n g uins in diagnosis of Toxoplasma infection: method for increasing SANCCOB facilities indicates a ppropriate sanitary sensitivity and specificily. j ournal oj Clinical MiC1"obiology, conditio ns and su ccessful anti- Toxoplasma m a nage­ 1980, 11, 562-568. m ent prevenrion procedures used by the cente r. D UBEY J.P. & DESMONT G. Serological responses of equids fed Toxoplasma gondii oocysts. Equine Veterina1Ji j oul'nal, 1987, 19, 337-339. ACKNOWlEDG EMENTS D UBEY].P. & BEATTIE c.P. Toxoplasmosis o f animals and man. CRS Press, Boca Raton, Florida, 1988, 220 p. e tha nk the South African Nationa l Foun­ D UBEY J.P. , GOODWI N M.A., RUfF M.D., SliEN S.K. , KWOK datio n for the Conservation of Coastal O.C.H., W ILKI NS G .L. & TH ULLfE Z P. Experimental toxo­ W Birds ( Cape T own , South Africa), the plasmosis in Chukar partridges (Alectons gmeca). Avian Nati o n a l Park Board o f South Africa, the West Cape Patbology, 1995, 24, 95-107. Nature Conservation, and the South African Correc­ DUFFY D.C. Ants, ticks, and nesting seabirds: dynamiC tional Services located o n Robben Island (South Africa) interactio ns? Tn: J.E. Loye & M. Zuk (eds.) Bird-parasite for facilitating collection of the p enguin blood samples. interactions, ecology, evolutio n, and behaviour. 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