Parelaphostrongylus odocoilei: Life cycle in experimentally infected cervids including the mule deer, Odocoileus h. hemionus

EXPERIAIESTAL PAISASITOLOGY46, 330-338

(19%)

Pareluphosfrongylus odocoilei: Life Cycle in Experimentally Infected Cervids including the Mule Deer, Odocoileus h. hemionus THOMAS R. PLATT AND W. M SAMUEL Department of Zoology, University of Alhertu, Edmonton, Alberta, T6G 2E9 Canada (.4ccepted

for publication

18 September

1978)

PLATT, T. H., AND SAMUEL, W. M. 1978. Pureluphostrongylzlr odocoilei: life cycle in experimentally infected cervids including the n~ule deer, Otlocoileus h. hemionus. Experimental Parasitology 46, 330-338. The life cycle of a metastrongyloid nematode, Purdaphostrongylus odocoilei, \vas successfully completed in three members of the Cervidae: mule deer (Odocoilezrs h. hemior~us), black-tailed deer (0. h. columbianus), and moose ( Alces &es andersoni). The terrestrial gastropod, Triodopsis mrdtilineata, was the experimental intermediate host. White-tailed deer (0. uirginiunus dacotensis) were refractory to infection. The prepatent period of P. odocoilei was significantly shorter in mule deer (X = 53 days) than in the black-tailed deer or moose. There was an inverse relationship between the size of the infective inoculum and the duration of the prepatent period of P. odocoilei in mule deer, but not in black-tailed deer. The duration and intensity of larval production of P. odocoilei were higher in mule deer than in the other hosts. Peak larval production in the feces (approximately 14,000 larvae/g) of mule deer was in excess of previous reports for elaphostrongyline nematodes, regardless of the size of the infective inoculum. Daily larval productiou, estimated at 3 to 4 X 10” larvae/day, was six times higher than estimates for other elaphostrongylines. The duration of patency was not clearly established, but three mule deer and one black-tailed deer passed larvae for 12, 18, 24, and 18 months, respectively. On the basis of the reduced prepatent period and increased length and intensity of larval production, 0. h. hemionus is considered the primary host of P. odocoilei. INDEX DESCRIPTORS: Alces alces undersoni; Odocoileus hemionus columbiunus; Otlocoileus hemionus hemionus; Odocoileus oirginianus; Cervidae; Deer; Nematoda, parasitic; Parekzphostrongylus odocoibi; Life cycle, experimental, prepatent period; Fecundity; Evolution; Gastropoda; Triodopsis multilineota. INTRODUCTION

Pureluphostrongylus odocoilei ( Hobmaier and Hobmaier 1934) is a metastrongyle nemat’ode parasite of mule deer (Odocoileus hemionus hemionus) and Columbian black-tailed deer (0. h. columbiunus). Until the recent identification of this parasite in Alberta, Canada (Platt and Samuel 1978), P. odocoibi was only known from the central Coast Range (Hobmaier and

0014-4894/78/046’2-0330$02.00/0 Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

1934) and the western slopes of the central Sierra Nevada Mountains ( Rrunetti 1g6g ) of California

Hobmaier

The life cycle of P. odocoilei is similar (Platt 1978) in most respects to that described for other elaphostrongylines. The adult worms are found in the dorsal skeletal muscles of mule deer and may or may not be found in conjunction with elements of the circulatory system. Females deposit

Parelaphostrongylus

o&co&i:

LIFE CYCLE

331

of British Columbia. Dr. R. M. F. S. SadIeir of Simon Fraser University, British Columbia, provided black-tailed ,deer fawns from a ,captive herd. All animals were raised and maintained in conditions free of elaphostrongylines at the University of Alberta Vivarimn, Ellerslie, Alberta. The animals were maintained indoors with limited access to an outside pen until weaning. After weaning, animals that had not received infective larvae were placed in outdoor pens (0.14 hectares) that had not been used previously by ruminants. Fawns were bottle fed for approximately 3 months and after weaning they received deer pellets (Northwest Feeds, Edmonton, Alberta), alfalfa pellets, and hay. Moose were given similar foods but also received aspen leaves and branches and ‘other material as browse. Fecal examinations were performed on each animal prior to experimentation. All were initially negative for Iungworm infections. Infective larvae of Pareluphustrongylus odocoilei, used in all trials with the exception of mule deer No. 4 (MD 4), were obt,ained from experimentally infected Triodopsis multilineata ( Say) (Pulmonata: Stylommatophora) as described elsewhere (Platt 1978). First-stage larvae used to infect these snails were ‘obtained from two sources. Mule deer Nos. 1,2, and 3 received L3s that originated from a naturally infected mule deer buck from Jasper, Alberta. The remaining infections were derived from Lls isolated from MD 1. MD 4 received 6 and 8 infective larvae on consecutive days, obtained from two naturally infected Euconulus fulvus (Pulmonata: Stylommatophora), collected in Jasper, Alberta. Infective larvae were .administered to the animals orally, either in milk or saline. All MATERIALSANDMETHODS animals were less than 6 months of age Fawns of mule deer, black-tailed deer, when infected. They were held indoors in and white-tailed deer and calves of moose individual pens with cement %oors. Fecal examinations were initiated apwere obtained as neonates from field perproximately 40 days postexposure and consonnel of the Alberta Fish and Wildlife tinued on a daily basis for 1 to 2 months Division ‘and the Fish and Game Branch

eggs into the circulatory system. The eggs are filtered out in the lungs and initial deveophnent of the first-stage larva ( Ll ) occurs in that organ. The first-stage larvae move up the bronchial tree, are swallowed, and subsequently passed in the feces. Larval development continues to the infective stage (L3) only ‘after the Ll gains entrance into a suit,able molluscan intermediate host. The life cycle is completed when a deer accidentally ingests an infected mollusk while feeding. Basic aspects ,of the life cycle of the elaphostrongylines, such as the length of the prepatent period, duration of patency, ‘and the dynamics of larval production are poorly known, in particular for P. odocoilei. The life cycle of P. odocoilei has been completed experimentally in the Columbian black-tailed ,deer ( Hobmaier and Hobmaier 1934; Brunetti 1969). Prepatent perlods of 2.5 and 4 to 5 months (Brunetti 1969 and Hobmaier and Hobmaier 1934, respectively) have been reported for P. odocoilei in the Columbian black-tailed deer. AIthough neither of these authors quantitatively assessed larval production over time, Brunetti (1969) established a patent period of 6 months for this species in blacktails. The course of infection ‘of P. odocoilei in other species of cervids is unknown. The first objective of the present study is to est,ablish baseline data for the prepatent period iand duration of patency and to quantitatively document larval production of P. oclocoilei in the mule deer. The second objective is to evaluate the ability of P. o&coil& to establish a patent infection in selected cervids and assess the effect of an alternate host on the parameters listed above.

332

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TABLE Results of lhe Experimental

Infed.ions 1leer No.

Mule deer (8)

of Cervids

1 3

14 25 30 100 175 318 322 334

62 54 55 51 .il 50 52 49

26 7 231) 18 15 22

50 50 50 300 300 500

62 72 <39 38 68 60

9 1

200 750

m 72

20 21 14 15 17 5 18 23

10 2.5 60 50

-

60 332 500 750

-

46 6 5b

0 Y

Noose

deer (6)

(2)

White-tailed

deer (8)

wil h I’a~~c~/a~hosf,onyl/l,ts odocoilci

Size of Length of infective prepatent period inocrdr~m (No. of L3) (days) __-__

8 7

Black-tailed

I

a LPG, Larvae per gram of feces wet wt. b Animal still alive and being monitored for larval

postpatency ( PP), then weekly until the animal died ,or was killed. Larval counts were made using the Baermann technique, following the suggestions of Todd et al. (1970). Two methods were employed for the examination of the Baermann fluid. The first, and most commonly used, included drawing off 100 ml of the fluid into a 150-ml beaker. The fluid was bubbled and mixed in an “X7’ motion to assure an even ‘distribution of larvae in solution. A 5-ml aliquot was removed with a pipet, placed in a counting chamber, and allowed to stand for several minutes. Larvae were counted and the process was repeated. If there was a difference of 10% or less between the two counts,

-

-

MaximtIm LPG”

Time of patency of mnximum LPG

720 713 2260 95,s 13,250

25 17 10 19 30

14,580 11,096

34 I>ayx 25 Days

464 113 667 235 5 235 4 11 -

20 10 17 12 6 21

Weeks Ilays Weeks Days Days

Days Days Days Dngs Weeks Days

10 Weeks 15 Weeks -

-

production.

they were averaged and the mean was used to calculate the number of larvae per gram of feces. If the counts differed by more than lo%, a third aliquot was taken and the mean of all three counts was used in subsequent calculations. In heavy infections, the procedure was modified so that a l-ml aliquot was used. A second, more sensitive, method was a modification of the first. The 100 ml of fluid were divided between two 50-ml centrifuge tubes and centrifuged at 1500 rpm for 6 to 8 min. The top 45 ml of supernatant were discarded and the remaining 5 ml were examined for larvae as previously described. This method was employed prior to and in the early stages of patency. In addition, it was used if the first

Parelaphostrongylus

I I 123456

1

I

I

WEEKS

I OF

I 7

I 6

I 9

Od4XOiki:

I I 1011

PATENCY

FIG. 1. Larval production of Parekzphostrongylus odocoilei in mule deer (Odocoileus h. he&onus) that received a moderate ( 100334 L3s) inoculum.

method failed to detect larvae in an animal that was previously positive. Results are expressed as the number of larvae per gram of feces wet weight (LPG ). RESULTS

Mule Deer Duration of the prepatent period varied from 49 to 52 days in five fawns that received from 100 to 334 infective larvae of

5

10

15

20

LIFE

333

CYCLE

Parelaphostrongylus odocoilei (Table I ) , Nettles and Prestwood (1976) considered that 300 L3.s of P. andersoni in white-tailed deer, the normal definitive host, was a “moderate dose.” Larval prmoduction of P. odocoilei increased logarithmically for the first 3 to 4 weeks of patency (Fig. 1). MD 3 was killed 1 month PP, following the log phase of larval production, and was examined for adult worms. MD 7 was sacrificed on 27 August 1978 in the 12th month of patency; numbers of larvae produced varied from
25

WEEKS

FIG. 2. Larval production of Parelaphostrongylus hemionus) that received a low ( ~50 L3s) inoculum.

OF

30

40

45

50

in mule

deer

35 PATENCY

odocoilei

(Odocoileus

h.

334

PLATT

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SAMUEL

MD 5, which received 50 L3s, until 35 weeks PP. Both animals were producing low numbers of larvae 18 and 24 months after the initial infection. MD 4 will continue to be monitored on a long-term basis. Since the 50th week of patency (Fig. 2), its larval production has varied from 2 LPG (Week 101) to 363 LPG (Week 75). MD 6, which received 25 L3s as an infective inoculum, had values of larval output iutermediate (713 LPG at 17 days PP) between MD 4 and 5 during the first 7 weeks of patency. Black-Tailed

1

45 WEEKS

OF

PATENCY

FIG. 3. Larval production of Pareluphostrongylus odocoilei in black-tailed deer (Odocoileus h. columbianus). (A) BT 2 and 22 received 50 and 500 L3s, respectively. (B) BT 7, 18, and 23D received 50, 300, and 50 L3s, respectively.

ceived from 14 to 50 L3s (Table I). Larval production increased logarithmically for the first 3 to 4 weeks of patency (Fig. 2). Peak production, however, was considerably below the levels of moderately infected animals. Larval production in MD 4, which received 14 L3s, remained below that of

Deer

The prepatent period of P. odmoilei in 0. h. columbianus ranged from 58 to 72 days in six animals that received from 50 to 500 infective larvae (Table I). Larval production in blacktails increased logarithmically for the first 2 to 3 weeks of patency and then declined sharply from 4 to 7 weeks (Fig. 3b). Larval production in BT 7, 18, and 23D fell to less than 1 LPG by 4 to 7 weeks PP, with occasional recurrences of larval production, interspersed with periods of reduced larval output (Fig. 3b). BT 2 and 22, although having drops in larval output at approximately 1 month PP (Fig. 3a), maintained a relatively high, though erratic, level of larval production for the duration of the observation period.

-I MOOSE MOOSE

1 9

i I

I

WEEKS

OF

PATENCY

FIG. 4. Larval production of Parelaphostrongylus odocoilei Moose 1 and 9 received 750 and 200 L3s, respectively.

in moose (Alces

alces andersoni).

Parelaphostrongylus

odocoih?i:

335

LIFE CYCLE

BT 2 is in the 18th month of patency; since Week 45 (Fig. 3), larval production has remained less than 3 LPG. BT 15 represents an anomaly. Larval production was severely reduced ( < 1 LPG) during most of the patent period and never rose above 5 LPG during more than 2 months of patency. Moose The prepatent period of P. odocoilei was 68 to 72 days in Moose 9 and 1, respectively (Fig. 4). Larval ‘output in Moose 1 was low initially (<1 LPG) and fluctuated irregularly until the animal was killed 15 weeks PP. Larval production in Moose 9 was also low but was not as erratic as Moose 1. This moose died with clinical signs similar to those of MD 1 and 2. White-Tailed

Deer

I

I

1 1

INOCULUM

* (log)

FIG. 5. Regressionanalysis of the size of the infective inoculum (log) and the duration of the prepatent period (days) of Parelaphostrongylus odocoilei in the mule deer, Odocoileus h. hemionus.

argued that this reflects strain differences as the worms used in this study were of Eight white-tailed deer were given from mule deer origin. However, experimental 10 to 750 infective larvae of P. oducoilei infections of 0. h. columbianus with P. odo(Table I). Fecal examinations were concoiki of blacktail origin have also resulted tinued for 6 months past the initial exin extended prepatent periods (Brunetti posure except for WTD 20, 21, and 23 1969; Samuel and Bartlett unpublished) which have been monitored for SO, 80, and and reduced larval output (Samuel unpub137 days postexposure. None of these trials lished). Additiona research is necessary to resulted in a patent infection. Pathology clarify this point. studies of all species of hosts are in progress. The prepatent period of elaphostrongyline nematodes appears to vary within DISCUSSION relatively narrow limits. Anderson (1965) The mean prepatent period, 63 days for reported a prepatent period for P. tends in Parelaphostrongylus odocoilei in Odocoileus white-tailed deer of 83 to 92 days. Nettles h. columbianus, confirms in part the pre- and Prestwood (1976) established a period vious report of a prepatent period of ‘ap- of 56 to 67 days for P. andersoni, also in proximately 2.5 months for this worm in white-tailed deer. As shown above, the prethe same host (Brunetti 1969). A prepatent patent period of P. odocoilei in ,mule deer period of 4 to 5 months for P. odocoilei, as demonstrates a similar variation. reported by Hobmaier and Hobmaier The prepatent period of P. odocoilei in ( 1934), must be viewed with some SLLS- mule deer shows a significant negative relapicion. tionship with the size of the infective inocuThe mean prepatent period of P. odo- lum (Fig. 5). This is evidently indicative coilei in 0. h. hemionus (It = 53 days) is of a logistics problem encountered by dioecious parasites at low densities (Kennedy significantly shorter than that in black1976). A threshold of approximately 100 tailed deer (P < 0.05; to.05 [7] = 3.499; t, = 3.919) and indicates the mule deer is the infective larvae is necessary to obtain the preferred host of this parasite. It may be minimum prepatent period (Table I). This

336

PLATT

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relationship does not exist for P. odocoiki in black-tailed deer (P > 0.5; F, = 0.14). No comparison of this phenomenon can be made with other elaphostrongylines because the number ‘of infective larvae administered in other studies is unknown (Anderson 1963) or the inocula are uniformly high (Nettles and Prestwood 1976) or low (Prestwood and Nettles 1977). The variation in the prepatent period of other species must be attributed to differences in larval viability and/or differential establishment rates in an individual host. The prepatent period of P. odocoilei is similar to that of P. andersoni in the whitetailed deer. The prepatent periods of the other elaphostrongylines, that are known to include a migration in the central nervous system (CNS): P. tenuis, 83 to 92 days 1965) and Elaphostrongylus (Anderson cervi 3 to 5 months ( Mitskevich 1964), are considerably longer. The migratory routes of the muscleworm species (P. odocoilei and P. andersoni) in their respective definitive hosts are unknown and at this time there is no reason to postulate a sojourn in the CNS for these species. None of the cervids experimentally infected with P. odocoilei iI1 this study displayed symptoms characteristic of CNS involvement as described by Anderson (1965, 1971) for cervids infected with P. tenuis. The longer prepatent period of P. tenuis and E. cervi may be the result of the CNS migration, which has either been lost or was never acquired in the evolution of the muscleworm species (Platt 1978). Larval production by P. odocoilei in mule deer is similar in many respects to that recorded for P. andersoni in white-tailed deer (Nettles and Prestwood 1976). Larval output for both species shows an initial logarithmic increase, followed by a plateau phase and an irregular, but slow, decline of long duration (i.e., 1 year). A similar situation was described (Panin 1964) for Elaphostrongylus panticola ( = E. cervi panticoZa) in the maral deer (Cervus

SAMUEL

ekphus mural). The initial rise in larval production was not as rapid as has been documented for Parelaphostrongylus spp.; however, the patent period appeared to be of similar duration. This prolonged period of larval production, a common characteristic of protostrongylid nematodes (e.g., Kassai 1962), is a K-selected feature in organisms generally considered to be r-selected (see Esch et al. 1977 for a review). Prolonged larval production in parasitic organisms has recently been interpreted as a natural response to an extremely favorable and stable habitat (Jennings and Calow 1975) rather than a response to the “perils of parasitism.” A precise determination of the length of the patent period has not been accomplished. A minimum life span for P. odocoibi in mule deer and black-tailed deer is 18 months. A number of animals (BT 2 and MD 4) are continuing to pass larvae of P. odocoilei and will be monitored on a long term basis. Peak larval output for P. odocoilei in mule deer that received approximately 300 L3s was six times higher than P. andersoni or E. cervi in their natural definitive hosts (Nettles and Prestwood 1976; Panin 1964) receiving similar numbers of L3s as an infective inoculum. Nettles and Prestwood (1976) estimated that a white-tailed deer heavily infected with P. andersoni passed 7.5 x 10” larvae per day. Applying their estimate of fecal production (300 g/day) to MD 1 of the present study, which had the highest number of larvae per gram of feces (14,580), results in an estimate of 4.2 x 10F larvae/day! Reasons for a higher level of larval production of P. odocoilei are unknown. This may be related to host age, sex, immune condition, or other nongenetic factors (Kennedy 1976) rather than a high innate fecundity of P. odocoilei. Peak larval output, as a measure of fecundity, was higher in mule deer that received identical or fewer infective larvae than Columbian black-tailed deer. Although

Parelaphostrongylus it is difficult to evaluate differences in the duration ~oflarval output between the two subspecies of host, the intensity of sustained larval production was low in 0. h. columbianus, occasionally dropping to zero (Fig. 3b). It is generally recognized that parasitic organisms have a shorter prepatent period and higher reproductive potential in the preferred host. Increased length ‘of the prepatent period, and reduced larval production, both peak and sustained, of P. odocoilei in blacktails and moose indicates a reduction in host-parasite ,compatibility and points to 0. h. hemionus as the primary definitive host of this parasite, The failure to infect white-tailed deer with P. odocoilei, while establishing a patent infection in moose, is perplexing. Establishment of a patent infection is indicative of minimal conditions required by the parasite for reproduction; therefore the absence of Lls in the lungs or feces of whitetailed deer suggests the absence of the proper biochemical stimuli for establishment or an inhibitor to P. odocoilei in white-tailed deer. Successful infection of MD 4 using infective larvae from naturally infected Euconulus fulvus raises the possibility that more than one species of elaphostrongyline is involved; that is, one species of Jasper, Alberta origin in Triodopsis multilineata Reand one of similar origin in E. f&us. covery of a few (
odocoibi:

LIFE CYCLE

337

tional Research Council of Canada (Operating Grant A-6603 to W-MS.), the Boreal Institute for Northern Studies, and the University of Alberta (Teaching Assistantship and Dissertation Fellowship to T.R.P.). D. Hebert, British Columbia Fish and Wildlife Branch, provided black-tailed deer fawns, materials, and personnel. P. Lewis, University of Lethbridge, D. Ashley, University of Nebraska, and A. K. Prestwood, University of Georgia, graciously supplied the authors with Triodopsis spp. We thank the staff of the University of Alberta Vivarium, in particular D. Mackay, J. Bouvier, and K. Taylor, for assistance in maintenance of experimental animals and collections of fecal samples. The following people assisted in the tedious job of rearing young deer and moose or locating adult nematodes: M. Barker, M. Barrett, C. Bartlett, D. Colwell, R. Hobbs, A. Kennedy, S. Knispel, S. Ramalingam, S. Rickabaugh, A. Shostak, and M. Stock.

REFERENCES ANDERSON, R. C. 1963. The incidence, development and experimental transmission of Pneumostrong&s tenuis Dougherty ( Metastrongyloidea: Protostrongylidae) of the meninges of the whitetailed deer (Odocoileus virginianus borealis) in Ontario. Canadian Journal of Zoology 41, 775791. ANDERSON, R. C. 1965. Cerebrospinal nematodiasis (Pneumostrongylus ten&s) in North American cervids. Transactions of the North American Wildlife and Natural Resources Conference 30, 155-167. ANDERSON, R. C. 1971. Neurologic disease in reindeer (Rangifer tarandus) introduced in Ontario. Canadian Journal of Zoology 49, 159-166. BRUNETTI, 0. A. 1969. Redescription of Parelaphostrongylus (Boev and Schult’s, 1950) in California deer with studies on its life history and pathoIogy. California Fish and Game 55, 307316. ESCH, G. W., HAZEN, T. C., AND AHO, J. M. 1977. Parasitism and r- and K-selection. In “Regulation of Parasite Populations” (G. W. Esch, ed.), pp. 9-62. Academic Press, New York. HOBMAIER, A., AND HOBMAIER, M. 1934. EEaphostrongylus odocoilei n. sp., a new lungworm in black-tail deer ( Odocoileus columbianus) . Description and life history. Proceedings of the Society of Experimental Biology and Medicine 31, 509-514. JENNINGS, J. B., AND CALOW, P. 1975. The relationship between high fecundity and the evolution of entoparasitism. OecoZogia 21, 109-115. KASSAI, T. 1962. A protostrongylidak ellettart a maral. Magyar Allatorvosok Lapja 17, 262-264.

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KENNEDY, C. R. 1976. Reproduction and dispersal. In “Ecological Parasitology” (C. R. Kennedy, ed.), pp. 143-160. North-Holland, Amsterdam. MITSKEVICH, V. I. 1964 [Cycle of development of Eluphostrongylus ~anglferi Miz. 19581. In [“Parasites of Farm Animals in Kazakhstan”] (S. N. Boev, ed. ), pp. 49-60. Izdatel’stvo Akademia Nauk Kazakhstan, Alma-Ata, SSR. [In Russian] NEITLES, V. F., AND PRESTWOOD, A. K. 1976. Experimental Parelaphostrongylus anaersoni infections in white-tailed deer. Veterinary Pathology 13,381-393. PANIN, V. I. 1964. [Life cycle of Eluphostrongylus pantida Liubimov, 19451. In [“Parasites of Farm Animals in Kazakhstan”] (S. N. Boev, ed.), pp. 34-48. Izdatel’stvo Akademia Nauk Kazakhstan, Alma-Ata, SSR. [In Russian] PLATT, T. R. 1978. “The life cycle and systematics

SAMUEL

of Pareluphostrongylus odocoilei (Nematoda: Metastrongyloidea), a parasite of the mule deer (Odocoileus h. hemionus), with special reference to the molluscan intermediate host.” Ph.D. thesis, University of Alberta. PLATT, T. R., AND SAMUEL, W. M. 1978. A redescription and neotype designation for Pareluphostrongylus odocoilei (Nematoda: Metastrongyloidea). Journal of Parasitology 64, 226-232. PRESTWOOD, A. K., AND NETTLES, V. F. 1977. Repeated low-level infection of white-tailed deer with Parelaphostrongylus andersoni. Journal of Parasitology 63, 974-978. TODD, K. S., LEVINE, N. D., AND ANDERSON, F. L. 1970. An evaluation of the Baermann technique using infective larvae of Haemonchus contortus. Proceedings of the Helminthological Society of Washington 37, 57-63.