The search for Ixodes dammini and Borrelia burgdorferi in Nova Scotia

Twenty-four Ixodes dammini ticks (23 adults and one nymph) have been recovered in Nova Scotia since 1984. There has not been a systematic search for larvae and none has been identified. The recovery of the nymph from a road-killed yellow throat bird, Geothypis trichas, in late May 1990 supports the contention that migrating birds are bringing deer ticks into the province every spring. In March and April 1991, four adult deer ticks were identified, suggesting that these ticks had overwintered. These deer tick specimens indicate that it is possible that I dammini is becoming established in Nova Scotia, if it is not already established. There has been no evidence for the existence of Borrelia burgdorferi in the province. The spirochete was not cultured from 650 Dermacentor variabilis ticks, nor were antibodies detected in a small sample of feral rodents using an indirect fluorescent antibody test. A survey of 137 dog sera samples, analyzed by enzyme-linked immunosorbent assay, also proved negative. There has been no confirmed indigenous case of Lyme disease in Nova Scotia to date.

1984.Les larves n•onl pas etc syslcmatiquement recherchees el aucune n•a ele identifiees.La decouverte d'une nymphe chez une fauvelle masquee (Geothypis trichas) luee sur Ia route.a Ia fin mai 1990 appuie l'hypoU1ese selon laquellc les oiseaux migraleurs apporlenl Ia tique clu wapiti clans Ia province chaque an nee.En mars el avril 1991.qualre liques clu wapiti adulles onl ete iclentifiees.suggerant que ces liques avaient hiverne el que I dammini est en voie cle s•etablir en Nouvelle-Ecosse-si ce n•esl deja fait.Rien ne semble incl iquer Ia presence clc Borrelia burgdorjeri clans Ia province.Aucun spirochete n•a ete mis en cu lture a partir cle 650 Dennacentor variabilis (lique clu chien) el aucun anlicorps n•a ete decele par immunofluorescence indirecle dans un petit echanlillon de rongcurs sauvages.L'analyse de 137 serums de chien effectuee par Ia methode enzymo-immunologique a donne elle aussi des resultats negalifs.Jusqu•a ce jour.aucun cas indigene cle borreliose cle Lyme n•a etc confirme en Nouvelle-Ecosse.T ICK-BORNE LYME DISEASE WAS RECOGNIZED AND C HARAC- terized in North America in the early 1970s (1 .2).There have now been thousands of cases reported throughout the contiguous United Stales which have contributed to the presen t awareness of the e tiology and epidemiology of the d isease in the United Stales (3.4).Information on the disease in Canada is nol as complete.
The first case of Lyme disease in Canada was diagnosed in 1977 (5), and at a sympos ium on Lyme d isease in Canada conducted at the Un iversity of Guelph in January 1991 (6). it was revealed U1al 65 indigenous cases of th e d isease had been reported to the Laboratory Centre for Disease Control in the previous seven years (personal communication).Fifty-four of U1ese cases were limited to Ontario.with only one case occurring in the Maritimes, in New Bru nswick (7).However.there has been some concern expressed by health professionals about the potential for the disease in Nova Scotia largely because of the proximity to disease foci in the state of Maine ( 8) and the possibility that migrating birds could be introducing infected Ixodes CAN J INFECT DIS VOL 3 No 5 SEPTEMBER/OCTOBER 1992 dammini ticks into the province each spring (9).Certainly because of the very la rge numbers of Dermacen• tor variabiLis ticks in Nova Scotia (10).introduced at the turn of the century.U1ere is palpable concern an1ong the general public.These factors p rovided the impetus in 1984 for this continuing search for the Lyme disease agent.BorreLia burgdor:feri.and the vector.I dammini. in Nova Scotia.Approximately 100 to 130 traps (Sh enna n 9 in ch live tra ps : HB Shem1an, Florida ) were set each ni <Jht a t each s ite for four nights to give a n a verage of 4 50 tra p n ights pe r s ite an d 4 960 Lra p-n ight.s for all sites for lhe entire season (Wes t.Pa ra dise was s ampled for U1ree weeks ).
Ticks and mammals we re re turned lo Acadia Univers ity each wee k for ide ntifica tion and proces s ing.Tic ks were identified wi.th th e k eys ofKiera n s and Litw a k (1 2).Kie ran s a nd Clifford (1 3) a nd Sone n s h in e (14) .1988 Cultivatio n of B burgdoiferi from ticks a n d small mammals and seroconversi o n: Adu lt licks from each s ite were pooled into se ts of five by sex.Afte r s urface sterilization with 70% volume/ volum e e thano l. 10% volum e / volume hydrogen peroxide a nd t-in s ing vvith s terile wa te r. the licks were homogeni zed in a sterile mortar and pestle wilh 2.0 mL phos ph a te buffe red s alin e (PBS , pH 7.4) .
Mammals were a n esU1e tized .exsangu in a ted by cardi ac punc ture a nd lh e spleen a nd kidney excised a nd h omogenized in a Po tter Elvehj em lissu e grinde r in 2 .0m L PBS.Blood samples we re al lowed to clot.by in cuba-lion a t 3 7°C for 20 m ins: s era were the n s ep a rated by centrifuga lion a nd s tored a t -70°C.
Sa mp les (50 pL) or lick and mammal organ homogen a te we re inoc u lated , in duplicate, into 8 mL Barbour-Sloenne r-Ke lly (BSK II) medium with antib iotics (15) a nd inc ubated for up to three monlhs a t 33°C.Posilive contro ls we re ru n by inoculation wilh B burgdorj eTi ATCC 3 5210.Th e presen ce of spiroc hetes in the m edium wa s det ermined after at least two months in cubalion by e pilluorescence microscopy wilh a cridine ora nge (16. 17) .
Antibodies to B burgdorjeri in Lh e Peromyscus L eucopu.sspecimen s were determined usin g U1e indirect fluorescent antibody (IFA) Lest.des cribed by Wilkinson (18), with s light.modifica tions by Arts ob (pe rs onal communication) u s ing fluores cein isothiocyanale-conj ugaled ra bbit.a nlimou se polyclonal s era (Cedarlane Labora tories Ltd).1990 Canine ser opr evalence testing: Antibodies to B burgdorj eri in domestic dogs were tested in s era collec ted in 1990 with U1e a ssistance of three ve terinary clinics in U1e province .Thes e we re: Bayview Animal Clinic , Dig by (Dr N PoU1ier); TriCount.yVeter inary Servi ces .Arcadia.n ear Yarmouth (Dr T O'Brien): a nd Ca pe Bre ton Ve lerinary S ervices.Sydn ey (D r B Buick).The loca tion of th ese cl in ics is s hov.rn in Figure 2 .The ve te rina ri a n s were asked lo bleed up to 50 dogs from each clinic .The dogs h a d lo be ov er s ix m onU1s old a nd t.o h a ve always lived in Nova Scalia .If the dogs ha d vis ited a n a rea endemic for Lym e diseas e the own ers we re asked to indicate lh is on a n accompanying qu eslionnai re .Sampling began a t U1e beginning of May 1990 a nd continued until U1 e beginn in g of Nove mber .whe n U1 e via ls we re collected .Each clog was bled once only, a nd a ll s e ra we re kept fro zen a fte r sepa ra tion.
This phase of U1e proj ect was pe rformed in colla boralion with U1e Main e Medical Cente r in Portla nd , Ma ine.which supplied all blood s a mplin g equ ipment a nd a rra n ged for a nalysis a t U1e Veterina ry Diagnos ti c Labora lOJy of Tufts University.The sera we re s hipped frozen to Boston and a n alyzed for seroconvers io n by enzymelinked immunos or bent a s say (19).

RESU LTS Occurrence of B burg d oife ri with D variabilis:
A lo tal of 650 ticks (405 male.245 female) were collected from the 1988 sampling programme.Examination of BSK li media did nol reveal spirochetes in any of U1e lubes.although Ulere wa heavy growUl of rods and cocci.Tubes with filamentous structures suggestive of spirochetes were sh ipped to RA Wirtz of Ule Waller Reed Army Medical Center in Wash in gton DC.Indirect fluorescent antibody testing wiU1 both species-specific monoclonal H5332 and genus -specific H9724 confirmed Lhal they a ll were negative.Control lubes produced positive growth of B burgdo1Jeri.

Seroprevalence in dogs:
A lola! of 137 serum samp les were collected from the three veterinary clinics (50 from Digby.46 from Arcadia and 41 from Sydney).Analyses al Tufts University revealed that a ll were negative -in facl.well below the critical optical density of 0.170.which represents a value three standard deviations above lhal of sera from clogs from nonendemic a reas .Occurrence of I dammini: Since 1984 a total of 23 adu lt and one nymphal I dammini have been recovered from Nova Scalia (Table 1).all U1rough submissions from U1e O"eneral public.The distribution of Ulese ticks did nol follow any obvious pattern (Figure 2).Prior lo 1991 U1e majority of adult deer ticks were submitted in the fall.the earliest submission occurring in June 1991 has been exceptional in that four adult licks were recovered in March and April .
The one nymph was recovered from a recenUy migrated yellow throat bird.Geothypis trichas (Linnaeus).which was killed after colliding \Aiith the windshield of a car in Windsor.

DISCUSSION
The distribution of D variabilis in Canada is in southern Ontario.southern Manitoba.souU1eastern Saskatchewan and southwestern Nova Scotia (5.20).
There was initial concern that this lick could act as a vector for Lyme disease because specimens found in the field were infected \Aiiih B burgdoiferi (21 ,22).Evidence has since accumulated which suggests that D variabilis may not be a competent vector for the disease largely because of the tick's inability for transtadial transmission (23 .24).Thus the inJected D variabilis found by Anderson et al (21) and Barker et.al (22) presumably reflect ticks which have recenUy fed on hosts infected \AiiU1 the spirochete but will not maintain U1e bacterium long enough to represent a major transmission route to humans.
The data presented here on the zero infection of 650 D variabilis specimens from Nova Scotia is in keeping \AiiU1 this idea.It is also in keeping \AiiU1 more recent findings from Ontario where D variabilis licks san1pled outside the endemic area of Long Point have never shown infection \Aiith spirochetes (personal communication).and also from Manitoba.where a large survey of D variabilis produced negative results (25.26).
The recovery of 24 I dammini from various locations around the province is more intriguing and relevant to potential Lyme disease.This coll ection of 24 deer licks represents the highest recovery in Canada.excluding Long Point (27).Larvae have not been observed in the province (see addendum) so these specin1ens cannot be considered members of an indigenous population.A statistically valid search for larvae has never bee n conducted and is needed.in light of Lhe number of adult specimens recovered.
The deer licks have been submitt.edfrom most regions of the province \Aiith the exception of Cape Breton Island (see addendum).Such a dispersion is probably the result of ticks dropping off birds as U1ey alight on their northward migration.Certainly the infestation of birds in the New England states has been well documented (9.28.29).and the recove1y of U1e nymph on a Yellow throat in U1e first week of June 1990 supports U1is contention.The 19 adu lt.I dammini collected in years prior to 1991 cou ld then represent nymphs introduced on birds in the same years.IL was believed that such populations posed lit.Ue threat to U1e establishment of an indigenous population because these licks would probably perish during U1e \A/inter.
The recovery of four adult I dammini (the three females were all engorged) in early spring 1991 is an interesting development.It is likely U1at these ticks had over\Aiintered: the spring bird migration had not yet begun, and there are no records of adult I dammini infesting birds (30).Of course.even U1ese over\Aiinlered ticks may have originated from nymphs introduced by birds the previous summer; it nonetheless raises U1e possibiHty of a spring breeding season in addition to a fall season (31).The provision of two breeding seasons increases the chance of successful mating which is recognized as U1e most serious impediment to U1e completion of U1e three-host life cycle of the deer tick.
I dammini have been recorded in U1e slate of Maine as far north as Machias (32).\A!iU1 indigenous populations documented further south (8,33.34).Some of these more isolated deer tick populations.such as U1e one on Monhegan Island.may have been introduced by birds (personal communication).Generally.U1e dist.I.ibulion of deer ticks in the state follows a 32 km strip next to U1e coast, which could reflect the moderatin,.influence of U1e ocean on temperature.Nova Scotia has a similar oceanic climate: indeed.there are pockets such as the Annapolis Valley where U1e climate provides a warm summer (35).which could be an1enable to the establishment of I dammini.Certainly the province appears no more inhospitable to I dammini than U1e norlliern slates of Maine or Wisconsin \AiiU1 their established foci (36.37).
If I dammini is moving into Nova Scotia.there is no evidence U1at B burgdorjeri is present..In addition to the negative results reported here on the cultivation of the spirochete from small mammals.seroconversion in small man1mals, and seroconversion in dogs .there has been no confirmed case of Lyme disease in U1e human population (personal communication).Magnarelli et al (38.39) found high Utres of B burgdorferi in 20.5% of the rodent population in endemic areas of New England.and Anderson et al (40) were ab le to cu lture spirochetes from 75% of the small mammal population in Connecticut.Obviously Nova Scotia does not compare to U1ese high incidence areas.
The canine serosurvey represents more persuasive evidence on the low incidence in Nova Scotia.The data presented here do not suffer from the quoted problems of cross-reactivity and false positives (41) as it is exceptional for its lack of reactivity.Lindenmayer et al (19) after a comparison of llie enzyme-linked immunosorbenl assay used in the present study reported a maximum specificity of 93.5% (18).A false positive percent.a<1e of 6.5% did not apply to lliese data.The 41 samples la.ken from Sydney were designed as negative controls because of the lack of licks D vwiabilis and I dammini on Cape Breton Island.The questionnaire completed by U1e owners confirmed mat U1is was U1e case.ln contrast.the dogs san1pled in Di,.by and Yarmouth Counties e>..1Jerienced a considerable tick load.The exceptionally low level of reactivity in all three populations suggests thal Lyme disease is nol yet present in the province's dogs.
The comp lete lack of evidence of B burgdo1jeri in Nova Scotia is reassuring news for the general public.Perhaps it is not too surprising considering the limited number of the classic vector.I dammini.and the confirmation that the ab undant tick.D variabilis, is not a competent vector.The situation deserves car eful monitoring in U1e years al1ead.The deer ticks found in Nova Scotia over the past seven years could represent small pockets of established populations or may indicate adventitious populations attempting to establish themselves.If U1e latter.then lhe millions of birds migrating through llie province every year and bringing nymphs with lliem constitute a formidable •auempt" each spring which shou ld not be ignored.Weisbrod and Johnson (42) demonstrated lliat approximately 1% of llie birds in U1 e central Dyway were infested with I dammini.and of these d eer ticks about 20% we re infected wiU1 B burgdorjeri.
Maine is now considered an e merging area for Lyme disease .with 26 documented cases (14 acquired in the slate) and ind igenous populations of I dammini in U1e soulli of the slate (32) .The vector is crucial to U1e epidemiology of U1e disease (43).and in order to predict if Nova Scotia could become an emerging area for Lyme disease in Atlantic Canada.further clarification of the status of I dammini in U1e province is required.

ADDENDUM
A gravid I dammini adult was discovered on Cape Breton Island in llie summer of 1991.The eggs ultimately hatched into larvae.Full details have been submitted to Canada Communicable Disease Report.

\F i eld s ite 7 Fi e ld site 6 Field site 3 I dammini and 8 Figure 1 )
Figure 1) Distribution qf Dermacentor variabilis and the location of lhe 1988 field s ites in sotllhwestern Nova Scotia.(So lid circles represent 1985 data)

Figure 2 )
Figure 2) Location of Ixodes dammini recovered in Nova Scotia and the three veterinary clinics participating in tile canine serosurvey

TABLE 1
Ixodes dammini recovered in Nova Scotia small mammals: The 1988 live trapping captured 87 animals consisting of one Blarina brevicauda.one Napaeozapus insiguis .two Tamiasciurus hudsonicus, three Tamias st1iaLus.36 Peromyscus leucopus and 44 Inocu lation of spleen and kidn ey homogenales in BSK II for spirochetes all proved negative.Indirect fluorescent antibody testing performed on 20 of the P leucopus serum samples all proved negative (titres less Ulan 1 :8).The remaining 16 specimens cou ld nol be tested because of inadequate blood recovery.The 1990 trapping program caplu red 19 mice representing two species (18 Zapus hudsonius and one P leucopus) and 27 shrews representing two species.all of which died upon captu re.No I dammini were found on any of U1ese animals.