Parasitology (1972) 65 403-425 4 0 3With 9 text-figures

Printed in Great Britain

Taxonomy of trypanosomes (Protozoa Trypanosomatidae)of Spermophilus spp (Rodentia Sciuridae)

BY DONALD F J HILTONf AND JEROME L MAHRT

Department of Zoology University of Alberta Edmonton Alberta Canada

(Received 21 January 1972)

Trypanosoma spp of ground squirrels and most other rodents belong to the3ubgenus Herpetosoma and have been recorded from approximately 80 to 90different species of rodents throughout the world (Davis 1952) They are oftendesignated as leurisi-like after the type species Trypanosoma (Herpetosoma) lewisi(Kent) Laveran amp Mesnil All are very similar in appearance and cannot bedifferentiated with certainty by morphological differences alone since there isgreat variation in body size even within a single host animal (Davis 1952) Thusmost investigators have described as new any trypanosome found in the blood ofa different host species

Trypanosomes have been named from eight species of Spermophilus (= Gitellus)- four in the USSR and four in North America Although Galuzo amp Novinskaya(1968) list nine species of Russian ground squirrels as hosts for trypanosomes manyof their host species are as Ellerman amp Morrison-Scott (1951) and Ognev (1963)show subspecies of more generally accepted species

According to Galuzo amp Novinskaya (1968) trypanosomes from Russian groundsquirrels were first observed by ShalashnikovJ in 1888 from 8 pygmaeus musicusMenetries and 8 suslica guttatus Pallas In 1910 GrjunerJ reported trypanosomesfrom 8 undulatus eversmanni Brant collected at Yakutia Laveran (1911) thengave the name Trypanosoma spermophili to the trypanosomes from these threespecies of hosts In 1913 TartakovskyJ described T schalaschnicovi from 8 suslicaguttatus obtained near Kherson Koltzovf (1914) recorded trypanosomes from8 pygmaeus mugozaricus Lichtenstein and S f fulvus Lichtenstein caught nearUralsk as did NikitinJ (1927) from S s suslica Giildenstaedt obtained at OdessaNeither of these investigators named the trypanosomes Bozhenko (1927) foundtrypanosomes in 8 f fulvus 8 pygmaeus mugozaricus S pygmaeus musicus and8 s suslica trapped in the area between the Urals and the Volga River On thebasis of successful cross-transmission experiments he regarded all four strains asT spermophili Bozhenko was unable to infect white mice guinea-pigs haresskunks grey rats dogs cats and camels ZasukhinJ (1936) observed trypano-

From a portion of a thesis submitted by the senior author in partial fulfilment of therequirements for Doctor of Philosophy

t Present address Imperial College Field Station Ashurst Lodge Aseot BerkshireEngland

We have not seen the original publications of these authors and our interpretation oftheir results is derived from the account presented by Galuzo amp Novinskaya (1968)

26 P A R 65

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404 D F J HILTON AND J L MAHBT

somes in S f fulvus S p pygmaeus Pallas and S s suslica collected from south-west RSFSR (Russian Soviet Federative Socialist Republic) Later Zasukhin(1946) reported trypanosomes in 8 rufescens from the same locality HoweverS rufescens is probably not a valid species since Zasukhin was the first (and only)author to use this specific epithet and it is not listed in either Ellerman amp Morrison-Scott (1951) or Ognev (1963) Zasukhin assigned all these trypanosomes toT spermophili in accordance with the view of Bozhenko Galuzo amp Novinskaya(1968) reported trypanosomes from 8 f fulvus S pygmaeus brevicauda ( = 8 inter-medius) Brandt and 8 p pygmaeus They conducted cross-transmission experi-ments among these three hosts as well as with various species of mammals birdsreptiles and amphibians Only the ground squirrels became infected Galuzo ampNovinskaya followed Bozhenko and considered all three trypanosome strains asT spermophili even though there were minor morphologic differences among them

North American parasitologists have described four species of trypanosomesfrom the genus Spermophilus Wellman amp Wherry (1910) described Trypanozoonotospermophili from 8 (= Otospermophilus) beecheyi (Richardson) in CaliforniaLaveran (1911) assigned this trypanosome to the genus Trypanosoma as T oto-spermophili (Wellman amp Wherry 1910) Laveran 1911 Watson amp Hadwen(1912) described T citelli from 8 richardsonii (Sabine) collected near LethbridgeAlberta Becker amp Roudabush (1934) described T iowensisf from 8 tridecemlineatus(Mitchill) and T hixsoni from 8 franklinii (Sabine) from Iowa They undertooklimited cross-transmission studies (numbers not given) but were unable to infect8 tridecemlineatus with T hixsoni using the infected blood of 8 franklinii How-ever they were later able to infect these same individuals of S tridecemlineatuswith T iowensis These are the only known attempts of cross-transmission oftrypanosomes in ground squirrels in North America

If there is only a single species of trypanosome found in Spermophilus groundsquirrels (as Hoare (1966 1972) believes) then by the law of priority its namewould be T otospermophili If there is one species in North America and anotherin Eurasia the North American would be T otospermophili and the Eurasian oneT spermophili Levine (1965) states that the four North American species probablybelong to one species but in the absence of detailed cross-transmission studiesit is better to retain the names originally assigned to them

The life-cycles and vectors of the trypanosomes in ground squirrels have not beendetermined either in the USSR or North America but fleas are vectors for manyrelated trypanosomes (Hilton 1972 Molyneux 1970)

The major object of our study was to determine the taxonomic status of trypano-somes parasitic in some ground squirrels of the genus Spermophilus in NorthAmerica

We have not seen the original publications of these authors and our interpretation oftheir results is derived from the account presented by Galuzo amp Novinskaya (1968)

t Hoare (1972) states that this species name was incorrectly spelled and should be Tiowense

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Taxonomy of trypanosomes of Spermophilus

MATERIALS AND METHODS

405

Spermophilus columbianus (Ord) S franklinii 8 lateralis tescorum (Hollister)8 richardsonii S tridecemlineatus and S undulatus plesius Osgood groundsquirrels were live-trapped at various localities in Alberta and Yukon Territory(S u plesius only) Canada Prevalence of trypanosomiasis in these hosts isdescribed in another publication (Hilton amp Mahrt 1972) S I tescorum was the onlyspecies not naturally infected

Combinations of statistical analysis of size differences host-specificity intensityand duration of experimental infections and geographical distributions were usedas diagnostic criteria in determining the taxonomic status of the trypanosomespresent in these ground squirrels

Similar studies were carried out on Sprague-Dawley white rats and BalB-Cwhite mice infected with the trypanosomes T lewisi (L strain) and T musculirespectively (obtained in 1970 from D R Lincicome of Howard UniversityWashington DC) Both trypanosomes are considered to be valid species and forthis reason comparisons were made among them and the ground squirrel trypano-somes to determine if they were demonstrably different from the latter as regardsmorphology intensity and duration of infection and host specificity

Cross-transmission studies

Blood from infected wild-caught ground squirrels was withdrawn by cardiacpuncture and mixed with an equal volume of mammalian saline (containing 3 sodium citrate by volume as an anticoagulant) Aliquots (usually 0-1-0-4 ml) ofthis blood-saline mixture were injected intraperitoneally into recipient groundsquirrels In order to determine the courses of infection experimentally infectedanimals were blood sampled (by tail clipping) every 1 2 or 3 days until they hadlost their infections (as determined by negative haemocytometer counts for 2consecutive weeks) Blood was drawn to the 0-5 mark in a Thoma white cell blooddiluting pipette then distilled water was sucked up to the 11 mark Distilled waterlysed the red blood cells leaving only white blood cells and trypanosomes (albeitdistorted) which were counted in a bright-lined Neubauer improved haemocyto-meter using phase-contrast microscopy at a magnification of x 200

A supply of known uninfected ground squirrels was obtained by collectingpregnant ground squirrels (S richardsonii and S tridecemlineatus) in April 1971they were dusted with louse powder (containing rotenone and abrasive particles)to remove all ectoparasites and kept in cages supplied with cotton nesting materialOne-half of each litter was experimentally infected (after weaning) with thetrypanosome strain from S columbianus and the other half with the strain fromS richardsonii

Morphological studies

At the time blood was taken for counting a thin smear was also made andstained with Wrights blood stain (Frankel Reitman amp Sonnenwirth 1970)Trypanosomes were drawn at a magnification of x 1000 with the aid of a Wilddrawing tube Measurements of seven trypanosome body regions (total length

26-2

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406 D F J HILTON AND J L MAHRT

Flag lengthmdashvmdashMN-A

mdashvmdashK-MN P-K

v _Length

Fig 1 Generalized drawing of a trypanosome to show major features and measure-ment regions P-K posterior end of body to middle of kintetoplast K-MN middleof kinetoplast to middle of nucleus MN-A middle of nucleus to anterior end ofbody (not including flagellum)

flagellar length nucleus length body width at widest point P-K K-MN andMNmdashA) (see Fig 1) were determined from the drawings using a calibrated mapmeasurer (0-125 in = 3 fim at x 1000) for all strains from both naturally andexperimentally infected ground squirrels Basic statistics calculated for eachmeasurement were number of hosts (NH) number of trypanosomes (NT) meanstandard deviation (sD) of the sample mean estimated standard error (SE) ofthe mean coefficient of variation (cv) expressed as a percentage and rangeVarious comparisons (Tables 3 and 4 are presented as examples) were made amongand within strains (for each body measurement) using the StudentmdashNeuman-Keuls (SNK) analysis of variance test (Rohlf amp Sokal 1969 Sokal amp Rohlf 1969)Since the means were ranked those which are non-significant have been designatedas such by drawing a vertical line beside them (Tables 3 4) as was suggested bySokal amp Rohlf (1969)

Correlation coefficients were computed among the seven measurements todetermine if the size of any particular body region was correlated with that of anyother (s) within any one trypanosome strain All data and programs were FORTRANIV coded on punch cards and computations were performed on an IBM 36067digital computer

RESULTS

Host specificity of trypanosomes

Strains of trypanosomes from four species of ground squirrels were used experi-mentally to inoculate wild-caught individuals of six species of ground squirrels(Table 1) The strain from S franklinii was the only one that failed to produce

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Taxonomy of trypanosomes of Spermophilus 407

Table 1 Results of experimental cross-transmission experiments

Recipient speciesA

I ~

Inoculum source C F L R T U

S columbianus 516f (03)t 01 11 07 (04) 17 (05) 23 (01)Sfranklinii 01(11) 010(110) 02(01) 02(02)Srichardsonii 1122 315 59 2030 55 212S tridecemlineatus 215 (210) 03 (03) 115 (19) 01 (01)

Table lists results of inoculations into wild-caught ground squirrels presumed to benegative Naturally infected animals were used as sources of inoculum

bullf Number of individuals that become infectednumber of individuals inoculated Figures in parentheses are number of hosts that did not develop an infection the first time

but did so after being reinoculated with blood from naturally infected individuals of S richard-sonii (number of individuals that became infectednumber of individuals inoculated)

Note C S columbianus F Sfranklinii L S lateralis R S richardsonii T S tridecem-lineatus U S undulatus

infections The strain from S richardsonii produced infections in all species ofhosts In heterologous recipients the proportion infected varied from 212 S undu-latus to 55 S tridecemlineatus respectively compared with 2030 8 richardsoniithat became infected when inoculated with the trypanosome strain from the samehost species A few (538) squirrels which could not be infected with the strain fromSfranklinii or tridecemlineatus were successfully infected when reinoculated withthe strains from 8 richardsonii This did not occur in thirteen squirrels initiallyinoculated with the strain from S columbianus

Of the juvenile (6-7 weeks old) ground squirrels born and reared in the laboratory(and known never to have been infected) 1622 and 2222 S richardsonii de-veloped infections after inoculation with trypanosome strains from S columbianusand 8 richardsonii respectively All the infections with the strain from S richard-sonii were very heavy (subjective measurement) while those induced by the8 columbianus trypanosome strain were very light Reinoculation (with theS richardsonii trypanosome strain) of the six ground squirrels which had notbecome infected with the strain from 8 columbianus resulted in all six developingheavy infections

Similarly 79 and 99 juvenile (laboratory-reared) S tridecemlineatus developedinfections after inoculations with trypanosome strains from S columbianus andS richardsonii respectively The two 8 tridecemlineatus that did not develop aninfection with the 8 columbianus trypanosome strain were not reinoculatedAlthough infections with the 8 richardsonii trypanosome strain were heavier thanthose with the strain from S columbianus the differences were not as marked asin the S richardsonii juveniles

Ten white rats and ten white mice inoculated with the trypanosome strain fromS richardsonii did not develop parasitaemia Six each of these same rats and micewere later inoculated with T lewisi and T musculi respectively All developedinfections

All ground squirrel individuals that had lost their natural or experimental

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408 D F J HILTON AND J L MAHBT

Table 2 Long-term immunity to trypanosome infection

[ost

CLRRRUU

uu

I

Dayssince

collected

+ 6

+11030++++

First inoculation

Dayssince

infectionlast

detected

330

1

1255255255

Inoculumsource Results

FC +RR +R +RFCT

Second inoculationA

Dayssince

infectionlast

detected

300

235200

Inoculumsource Results

R

TT

Plus signs in this column represent ground squirrels which were naturally infected whencollected Thus the first inoculation into these individuals is equivalent to a second inocula-tion as far as interpretation of results is concerned

20 -

E 16

12 -

o

8 -

4 -

30 40 50

Days after inoculation60 70

Fig 2 The three phases of infection that develop during the course of groundsquirrel trypanosomiasis (in this case an infection in S richardsonii)

infections were refractory to reinfection with the same or other trypanosomestrains (Table 2)

Eight naturally infected S undulatus and eight experimentally infectedS richardsonii (two with the S richardsonii and six with the S columbianustrypanosome strains) were reinoculated with the S richardsonii trypanosomestrain once during the course of their infections The times of inoculation varied

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Taxonomy of trypanosomes of Spermophilus 409

5 -

Epound

oX

3oo

4 -

3 -

2 -

1 -

12 -S columbianus

30

Days after inoculation

Fig 3 Intensity and duration of parasitaemia in experimental infections in S lateralisand 8 columbianus All squirrels received aliquots of the same inoculum from anaturally infected S ricliardsonii

from the increase phase until just before termination of the infection (see Fig 2 forinfection stages) No animal developed a detectable super-infection

Intensity and duration of parasitaemia in experimental infections

The prepatent period (as determined by stained thin blood smears) varied from2 to 10 days with the average for each host species as follows S columbianus and8 franklinii (7) S lateralis S richardsonii 8 tridecemlineatus and 8 undulatus(6) These differences are not statistically significant (P = 0-05) Both theT lewisi and T musculi infections of white rats and white mice respectivelybecame patent on the third day after inoculation This difference between groundsquirrels and rats and mice is statistically significant (P mdash 0-05)

Examples of infections produced in some ground squirrels after inoculation withthe trypanosome strain from 8 richardsonii are given in Figs 3 and 4 Generallyinfections in 8 columbianus (Fig 3) S richardsonii (Fig 4) and 8 undulatus couldbe divided into three stages designated as increase decrease and terminal (Fig 2)This is unlike infections in S franklinii S lateralis (Fig 3) and 8 tridecemlineatus(Fig 4) which usually had increase and decrease stages only The intensity andduration of infection that develops in each of these stages depends upon the hostspecies Thus although there were wide individual variations infections inindividuals of all species (except S lateralis) had intensity ranges of 200-3000trypanosomesmm3 Experimental infections in S lateralis were unusual in thatintensities were relatively high (about 40000 trypanosomesmm3) Durations ofinfection in 8 columbianus and 8 richardsonii were 25-60 days compared with

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410 D F J HILTON AND J L MAHRT

72 H

6 0 -

48 -

3 6 -

x 24 -

12 -

o

S tridecemlineatus

bull Died

JmdashT-J-T

15 -

10 -

5 -

i i i

S richardsonii

1 1 I I 112 18 24 30 36 42

Days after inoculation48 54

Fig 4 Intensity and duration of parasitaemia in experimental infections in Srichardsonii and S tridecemlineatus All squirrels received aliquots of the sameinoculum from a naturally infected 8 richardsonii

12-20 days for 8 lateralis and S tridecemlineatus Generalizations about theduration of infection cannot be made for S franklinii or 8 undulatus since therewere wide variations among the three infected individuals of 8 franklinii and therewas only one infected 8 undulatus

White rats infected with the L strain of T letvisi usually developed infections(lethal in 714 rats) with an intensity of about 750000 trypanosomesmm3 and aduration of 15-25 days White mice infected with a strain of T musculi developedaverage parasitaemias of 20000 trypanosomesmm3 which terminated suddenly18 days later

Trypanosomes seem to be completely removed from the hosts body after com-pletion of the terminal infection phase since only 112 ground squirrels splenecto-mized (two of each species except for one 8 tridecemlineatus and three 8 undulatus)at this time developed a recrudescence of parasitaemia This occurred in a naturallyinfected individual of 8 undulatus which had lost its countable infection 16 days

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Taxonomy of trypanosomes of Spermophilus 411

48

47

46

T 45

-5 44boC- 43

pound 41

40

39

38 41iC U R T F L

Ground squirrel species

Fig 5 Comparison of mean total length for trypanosome strains from all natural andexperimental infections bull Natural infections bullgt experimental infections using thetrypanosome strain from S richardsonii as the inoculum source

16

15

14

13

U R T F LGround squirrel species

Fig 6 Comparison of measurement K-MN for trypanosome strains from all naturaland experimental infections bull Natural infections bull experimental infections usingthe trypanosome strain from S richardsonii as the inoculum source

previously but had a positive blood smear on the day of splenectomy (thus theinfection had not terminated) The renewed infection which developed in thisanimal was very short-lived

Size comparisons of trypanosome strains

Of the seven trypanosome body measurements compared length (total) andK-MN generally had coefficients of variation (cv) of about 6 and 10 respec-tively The remaining five measurement regions had coefficients of variation in the

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412 D F J HILTON AND J L MAHRT

Table 3 Comparison of trypanosome size (in im) among the five speciesof naturally infected ground squirrels

Non-significantRange sets

Host mdash-bull v -mdashmdash-~Measurement species N H NT Mean SD SB OV L O Hi 1 2

Length

Width

P-K

K-MN

MN-A

Nucleus

Flagellum

TFTJCRFCTRUFTRCuTFUCRFITTCRTUFCRTFCRU

3312837

3833712

3337812

3312837

3123837

3123837

3383712

15246269117

24691511762

24151176962

15246269117

24621569117

15622469117

15246911762

38-839-547-147-247-6

2-22-52-62-62-7

3-43-44-55-05-5

13-513-615-916-516-6

10-911-311-612-412-7

3-43-43-53-73-8

10-311-713-313-814-4

1-82-43-92-72-7

0-60-50-40-50-5

1-00-51-00-81-1

1-32-01-41-31-3

1-62-21-82-12-4

0-40-90-60-70-8

2-32-52-02-831

0-50-50-50-30-3

0-10-10-1010-1

0-20-10-10-10-1

0-30-40-20-20-1

0-30-30-50-30-2

0-10-10-10-10-1

0-60-50-20-30-4

56866

2721161918

2915221620

1015988

1519161719

1225171921

2221152021

34-532-534-5360390

1-51-51-51-52-0

1-53-03-03030

12-010-011014-013-0

8-07-09-07-06-0

3-030303030

7-08-08-06-08-0

42-043-054-054-055-0

3-04-03-04-04-0

5-04-57-57-07-5

16-017-018-021-019-5

13-516-016-018-021-0

4-06-04-56-06-0

17-016-518-024-019-5

P = 0-01 for Tables 3 and 4 means not significantly different are joined by a verticalline

Note NH number of hosts NT number of trypanosomes SD standard deviation SEestimated standard error cv coefficient of variation expressed in per cent

range of 15-25 depending upon the measurement trypanosome strain and typeof comparison Thus when comparisons were made among and within trypanosomestrains using only total length and K-MN there was a better chance of findingsignificant differences However many comparisons made with the other bodymeasurements confirmed the results obtained with length and K-MN

In the comparisons of trypanosome size among the five species of naturally

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Taxonomy of trypanosomes of Spermophilus 413

Table 4 Comparison of trypanosome size inim) among all species of ground squirrelsexperimentally infected with the trypanosome strain from S richardsonii) white ratsinfected with T lewisi) and white mice infected with T musculi)

Measure-ment

Length

Width

P - K

K-MN

MN-A

Nucleus

Flagellum

Hostspecies

TXMLFUCRLFCTURXMTFURLCXMMTXFLUCRTXLFU

cRMCXFMUTLRMXTLRCFU

N H NT Mean SD SE CV

3 673 10010 1465 160

668183

3289 227

16066836781

9 2273 10010 146

3 673 662 819 2275 1608 833 10010 146

10 1463 673 1003 665 1602 81

839 227

67100160668183

9 22710 146

8 833 1003 6610 1462 813 675 1609 22710 1463 1003 675 1609 227

836681Note

39-340-341-943-044-045-045-045-4

2-22-52-52-62-72-73-23-6

3-63-83-83-94-04-04-68-0

10-213-815-015-115-315-515-616-2

9-39-410-110-610-911-211-413-6

3-63-73-94-14-14-44-44-510-111-412-713-714014-214-614-8

2-4 0-34-4 0-44-2 0-43-2 0-32-5 0-32-3 0-330 0-32-6 0-2

0-5 0-10-5 0-10-5 0-1

0-7 0-10-8 0-10-9 010-7 0-10-8 0-10-8 0-10-8 0-10-9 0-1

1-5 0-11-3 0-2

1-4 0-21-5 0-11-5 0-21-6 0-21-3 01

0-70-70-70-60-71-00-80-9

2-32-42-32-72-42-52-52-5

0000000-1010-20-20-30-20-20-30-30-3

111076576

0-5 0-0 240-5 0-1 21

211918

0-5 0-0 182323251920212022

1-8 0-2 402-5 0-2 32

159

1-8 0-2 129910108

1-8 0-2 191-9 0-2 202-4 0-2 241-9 0-2 181-9 0-2 171-9 0-22-2 0-12-4 0-2

161918

1819191517241919

2321181917181717

32-522-530031-537-037-537-0350

1-51-51-51-52-01-52-0202-02-02-02-02-02-02-0306-0

12-07-5

12-012-010-012012-0

5-03 04-06-06-07-04-58-02-020303-03-0302-0304-06-07-06-07-06-07-57-5

44-551-051-050-048-050-052-051-0

303-03-03-04-03-06-06-06 06-06-06-06-07-0

12-013-013-516-019-519-519-019019-020-013-513-516-015-015-016-518-019-55-05-06-06-0606-07-07-0

17-022-018-020-024019-519-020-0

M white mice X white rats

Non-significantsets

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414 D F J HILTON AND J L MAHBT

infected ground squirrels (Table 3 Figs 5 6) it is evident that the five trypanosomestrains fall into two groups Spermophilus franklinii and S tridecemlineatus strainsform one group which were not significantly different from one another (but lengthand K-MN were lowest in trypanosomes from S tridecemlineatus) they did differsignificantly from strains of the other group 8 columbianus S richardsonii andS undulatus Trypanosomes from the latter group did not differ significantly fromone another except for P-K and K-MN even in these measurements the threespecies were much closer to each other than they were to the 8 franklinii and8 tridecemlineatus group

As in natural infections the experimental infections in 8 columbianus S richard-sonii and S undulatus were very closely associated (even if size differences weresometimes significant) Similarly infections in S franklinii and 8 tridecemlineatushad lower mean values for length and KmdashMN than did those in the other threespecies Unlike natural infections though experimental infections in individuals of8 franklinii more closely resembled (as far as size is concerned) those in S columbi-anus S richardsonii and 8 undulatus than those in 8 tridecemlineatus

Trypanosome strains of T lewisi (from white rats) and T musculi (from whitemice) were similar in size (for length and K-MN) to trypanosomes from experi-mental infections in 8 tridecemlineatus (Table 4) However the strains of T lewisiand T musculi were usually significantly different from one another It does notappear that populations of T lewisi and T musculi are readily separable (by sizealone) from the trypanosome populations of ground squirrels

Trypanosomes from naturally infected ground squirrels were usually larger insize (sometimes significantly) than those from experimental infections in the samehost species (Figs 5 6) Exceptions to this occurred in 8 franklinii and 8 tri-decemlineatus in which no matter what the inoculum source trypanosomes fromnatural infections were usually smaller than those from experimental infectionsbut not significantly different from them except for those in 8 franklinii

The trypanosomes from males of a ground squirrel species were often smallerthan those from females (though not significantly so except for 8 franklinii) Withoccasional exceptions this same trend occurred in both naturally and experi-mentally infected ground squirrels In contrast T lewisi and T musculi from malewhite rats and white mice respectively were usually larger (not significantly) thanthose from females both experimentally infected

The trypanosome populations from two species of ground squirrels experimentallyinfected with aliquots of the same inoculum (Figs 7 8) exhibited similar sizedifferences Thus the lengths of trypanosomes from 8 richardsonii and S tri-decemlineatus (Fig 7) and 8 columbianus and 8 tridecemlineatus (Fig 8) often weresignificantly different from each other In contrast trypanosome populations fromthe following pairs of host species usually did not differ significantly in size8 columbianus against S lateralis (Fig 7) 8 franklinii against S undulatus (Fig 8)and 8 columbianus against 8 franklinii (Fig 8) When several trypanosome strainswere inoculated into different individuals of the same host species the trypano-somes from these experimental infections within one host species were not signifi-cantly different from one another in length (Fig 8)

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Taxonomy of trypanosomes of Spermophilus 415

30 35

Total length (fim)

40 45 50 55 60

20

30 35 40 45 50 55 60Fig 7 Variations in total length of trypanosomes from different host individuals andspecies All infections are sublines derived from a naturally infected S richardsoniiRecipient squirrels in each group received aliquots of the same inoculum Note1 number of trypanosomes measured are listed to left of range line 2 letter toright of range line denotes host species 3 horizontal line represents range verticalline represents mean standard deviation of mean is represented by black andwhite bars on either side of mean two estimated standard errors are representedby black bar on either side of mean

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416 D F J HILTON AND J L MAHET

30 35Total length (ltm)

40 45 50 55 60

gt laquobullbull

75 J=plusmn

c bullbull

Fig 8 Variations in total length of trypanosomes from infections produced inground squirrels of different species Recipient squirrels in each group receivedaliquots of the same inoculum Note symbolism employed is the same as that inFig 7

Each of the experimental infections compared in Table 4 have been divided intoincrease decrease and terminal stages (see Fig 2) and comparisons made amongthese stages for each host species There were usually no significant size differencesamong the stages from experimentally infected ground squirrels However inwhite rats experimentally infected with the trypanosome T lewisi trypanosomesfrom the increase and decrease phases were much more highly variable (ie largecv) than the terminal phase even though the size differences among them werenot statistically significant The same was true for the differences between theincrease and decrease phases from white mice infected with the trypanosomeT musculi except that there was no terminal phase

With the exception of total length body measurements of trypanosomes fromnaturally and experimentally infected ground squirrels and from white rats andmice experimentally infected with the trypanosomes T lewisi and T musculi

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Taxonomy of trypanosomes of Spermophilus 417

respectively showed no significant correlations Since P-K K-MN MN-A andflagellar length together make up total length they were usually positivelycorrelated with it

DISCUSSION

Host specificity of ground squirrel trypanosomes

The fact that white rats and white mice could not be infected with the trypano-some strain from naturally infected 8 richardsonii corroborates the related findingsof Becker amp Roudabush (1934) Bozhenko (1927) Culbertson (1941) Davis (1952)Dorney (1967) Galuzo amp Novinskaya (1968) Grewal (1957) Molyneux (1969a b)Quay (1955) and Wood (1936) These workers all found it was impossible to infecthosts from different genera or higher taxonomic categories

We were able to infect ground squirrels of six species with the trypanosomestrains from three of the same six species but were unable to do so with thetrypanosome strain from Sfranklinii (Table 1) In our cross-transmission experi-ments the species of ground squirrel and number of individuals of each speciesthat could be successfully infected depended upon the trypanosome strain inocu-lated Thus varying numbers of individuals of all six host species could be infectedwith the trypanosome strain from S richardsonii whereas only small numbers ofindividuals of 24 species of squirrels could be infected when the trypanosomestrain from S tridecemlineatus was used Probably the failure to infect moreground squirrels with the strain from S columbianus was a result of immuneanimals being used as recipients none of those which resisted infection could laterbe infected with the strain from 8 richardsonii Some of these failures may havebeen due to inadvertent insertion of the needle into the stomach or intestinallumen during inoculation (Simmons Cunningham van Hoeve amp Lumsden 1963)

There is a certain amount of host specificity involved Thus 2222 8 richardsoniiand 99 8 tridecemlineatus individuals (all laboratory reared) were heavily infectedwith the 8 richardsonii trypanosome strain whereas of the litter mates only 16228 richardsonii and 79 S tridecemlineatus developed light infections when inocu-lated with the S columbianus trypanosome strain The six 8 richardsonii in-dividuals which failed to develop infections with the S columbianus strain laterbecame heavily infected when inoculated with the strain from S richardsonii

Once a ground squirrel had lost its natural infection it could not be reinfectedwith the same or a different strain of trypanosome (Table 2) We were also unableto produce super-infections in ground squirrels using the same or different trypano-some strains This suggests there are basic antigens common to all these trypano-some strains and once ground squirrels develop antibodies to these antigensreinfection cannot normally be effected Similar results have been obtained withthe T brucei group of pathogenic African trypanosomes (Zuckerman amp Ristic1968) in which there are both shared and variant-specific antigens

Intensity and duration of parasitaemia in experimental infectionsThe course of an experimental infection was determined by both individual and

species differences among hosts (Figs 2-4) 8 columbianus Sfranklinii 8 richard-sonii and S undulatus could be placed in one group since all have similar infections

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418 D F J HILTON AND J L MAHBT

while 8 later alls and 8 tridecemlineatus were different from one another as well asfrom the 8 columbianusmdash8 frankliniimdashS richardsonii-S undulatus group

All the infections in ground squirrels were different from those produced inwhite rats and white mice by the trypanosomes T lewisi and T musculi respec-tively This emphasizes that both the host and trypanosome species determine theintensity and duration of experimental infections

Following termination of parasitaemia in ground squirrels it appears that theimmunity which has developed is sterile (ie trypanosomes are no longer presentin the hosts body) and of very long duration possibly a life time (Table 2) Splen-ectomy did not result in recrudescence of infections This is similar to immunity inwhite rats against T lewisi (Corradetti 1963)

Size comparisons of trypanosome strains

Host individuals exert an effect upon trypanosome size (Figs 7 8) If allowancesare made for this variation the five trypanosome strains obtained from naturallyinfected ground squirrels (Figs 5 6 Table 3) can be divided into two groups Thestrains from S columbianus 8 richardsonii and S undulatus form one group whilethose from 8 franlclinii and 8 tridecemlineatus comprise the second

The differences in trypanosome size between natural and experimental infectionswere probably not the result of experimental infections having been initiated withthe trypanosome strain from S richardsonii Even within this host speciestrypanosomes from the natural and experimental infections were significantlydifferent in size One reason for these size differences may have been that cyclicaldevelopment is required in the arthropod vector(s) before the strain sizes shown inTable 3 can be attained

A strong influence is exerted on trypanosome size by the host species Thus whendifferent individuals of S columbianus and 8 tridecemlineatus were inoculatedwith the S columbianus S richardsonii and S tridecemlineatus trypanosomestrains and the 8 columbianus and 8 richardsonii strains respectively trypano-somes from almost all experimental infections produced in either one of these twohost species did not differ in size Conversely when aliquots of the same inoculumwere injected into individuals of 8 tridecemlineatus and ground squirrels of anotherspecies the size differences between trypanosomes from the two infections thatwere produced were often significantly different

Body measurements with a high coefficient of variation (cv) during theincrease phase of a trypanosome infection are an indication of trypanosome repro-duction in the hosts bloodstream This has been shown for infections of T lewisiin white rats (Taliaferro 1923 Taliaferro amp Taliaferro 1922) Demonstration ofreproducing forms in blood smears is confirmation that such reproduction occursThe decrease and terminal phases of infection have lower cv values due to theremoval of all young dividing (and therefore variable) trypanosomes by the first-crisis antibody leaving the monomorphic adult forms (Ormerod 1963 Taliaferroamp Taliaferro 1922) A similar situation exists in T musculi infections of white miceexcept that differences in cv are not as great (DAlesandro 1970)

Trypanosomes of ground squirrels do not have a reproductive stage in the

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Taxonomy of trypanosomes of Spermophilus 419

Increase

Decrease

Terminal

24 m

Fig 9 Morphology of ground squirrel trypanosomes during the increase decreaseand terminal phases of infection Drawings were made from blood smears obtainedfrom S richardsonii individuals experimentally infected with the trypanosomestrain from the same host species

bloodstream This was shown by the similar coefficients of variation within eachmeasurement region and by the monomorphic trypanosomes present in all threeinfection phases (Fig 9) This means there must be a tissue stage of reproductionfrom which trypomastigotes are released into the blood stream following a pre-patent period of 6-7 days (as determined by stained blood smears) Reproductionof this type has been found to occur in the rodent trypanosomes T evotomysT microti T nabiasi and T zapi (Molyneux 1970)

The body measurements we determined for trypanosomes from naturallyinfected ground squirrels were considerably larger than those recorded by otherinvestigators (although our measurements showed similar differences betweenstrains) Thus Becker amp Roudabush (1934) found total length of the trypanosomesT hixsoni (from S franklinii) and T iowensis (from S tridecemlineatus) to be 32-0and 26-3 on respectively In contrast our results for the strains from these twohost species were 39-5 and 38-8 im respectively Watson amp Hadwen (1912) onlymeasured a few (numbers not stated) trypanosomes of T citelli from S richardsoniiand recorded the mean length as 35-0 tm This compared with a mean length of

27 P A R 65

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420 D F J H I L T O N AND J L MAHRT

47-6 fim which we obtained for trypanosomes from the same host species Sincetrypanosomes from 8 columbianus and S undulatus (in North America) have notpreviously been reported no comparative measurements are available Davis (1952)and Noble amp Shipman (1958) recorded total length for two different populations ofT otospermophili from 8 beecheyi as 30-7 and 25-2 fim respectively Galuzo ampNovinskaya (1968) do not give measurements for the populations of trypanosomesthey described However Bozhenko (1927) determined mean total length fortrypanosomes from S f fulvus 8 pygmaeus mugozaricus and 8 p musicus to be32-5 32-4 and 33-0 fim respectively

Most of the above authors do not state what measuring devices they used butin the past it has usually been calipers or a series of short straight lines drawnalong the central axis of the trypanosome Since we used a calibrated map measurerwe were able to follow every curve in the trypanosomes body and our measurementswould be greater than could be obtained with either calipers or straight lines

Size discrepancies may also be due to differences in optical equipment fixingand staining methods as well as strain variability (Taliaferro 1923)

Natural transmission of trypanosomes

Hilton amp Mahrt (1971) have shown that of the ectoparasites present on threespecies of ground squirrels in Alberta only fleas and lice could serve as efficientvectors since they are the only ectoparasites which are abundant and widelydistributed on ground squirrels Of these two groups fleas are the more likelyvectors because (unlike lice) they are active and transfer readily from individualto individual of a host species (Hopkins 1949 1957) In all species thoroughlystudied (Hilton 1972 Molyneux 1970) fleas have been the biological vectorsof lewisi-like trypanosomes of rodents even though lice can be mechanicalvectors (providing they contain infected undigested blood) if they are ingestedby a susceptible host (Wenyon 1926) We found developmental stages of try-panosomes in the hmdguts of 48 fleas that were allowed to feed on an infected 8richardsonii However these fleas (though laboratory reared) were not reared fromisolated eggs in a sterile medium and thus we cannot prove that fleas are vectorsof the trypanosomes of ground squirrels since fleas have their own monogenetictrypanosome parasites (Wallace 1966 Wenyon 1926)

Although we have been able to infect ground squirrels of different species withtrypanosome strains from other species of ground squirrels we do not knowwhether this transmission occurs in nature When we blood-sampled two popula-tions of 8 franklinii and S richardsonii (separated by 400 m) at weekly intervalsfrom April to August 1971 we found that natural cross-transmission did not appearto take place between individuals of S richardsonii and those of S franklinii eventhough 22-2 (836) of the S richardsonii were infected (none (021) of the8 franklinii was ever infected) It is possible that there was insufficient movementof ground squirrels between the two areas for the exchange of infected ectoparasitesnecessary to produce infection Ectoparasite exchange does occasionally occurbetween host species because fleas of the species Opisocrostis bruneri normallyspecific for S franklinii have been recorded from S richardsonii and S tridecem-

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Taxonomy of trypanosomes of Spermophilus 421

lineatus (Holland 1949) Such exchange could easily occur if individuals of onespecies of ground squirrel enter the (abandoned or temporarily vacant) burrowsof another This would provide an opportunity for fleas (which are nest parasites)to transfer to a different host species

In Alberta Canada the ranges of the following ground squirrels overlap8 franklinii 8 richardsonii and S tridecernlineatus S colunibianus 8 richard-sonii and 8 tridecernlineatus and 8 columbianus and 8 lateralis Within theseoverlapping ranges we have often seen individuals of two or three species livingwithin a few metres of one another In these situations natural cross transmissionmight occur

TAXONOMIC STATUS OF GROUND SQUIRREL TRYPANOSOMES

Although the trypanosomes of ground squirrels are not distinguishable fromT lewisi and T musculi by size alone (Table 4) they can be distinguished becausethe latter have (1) variable body size during the increase stage of infection (2) adifferent type of reproduction in the vertebrate host (3) rigid host specificityand (4) large differences in duration and intensity of infections These characterstaken together serve to define T lewisi and T musculi as distinct species readilyseparable from the trypanosomes of ground squirrels

Since the trypanosome strains (so far examined) from ground squirrels are allmorphologically identical they cannot be separated into different species on thebasis of morphological criteria There are sometimes differences between strains inmensural variations intensity and duration of infections host specificity andgeographical restriction However these are all characters which when takenseparately could (at the most) be used only in distinguishing subspecies notspecies (Corliss 1962 Hoare 1966 Sonneborn 1957)

The trypanosome strains from different species of ground squirrels should notbe designated as subspecies because (1) strain differences are host influenced and(2) geographical isolation of 8 undulatus from all other North American groundsquirrels has not resulted in appreciable differences between the trypanosomestrain in S undulatus and those in the other host species The fact that straindifferences are host influenced is further emphasized by noting that trypanosomestrains from ground squirrels of the subgenus Spermophilus (ie 8 columbianusS richardsonii and S undulatus) are all similar to one another (as regards size andtypes of infection) but different from the strains found in 8 (Poliocitellus) frank-linii and 8 (Ictidomys) tridecemlineatus Trypanosome strains from ground squirrelsof the latter subgenera show some differences from each other (size in experimentalinfections levels of host specificity duration of infections) as well as somesimilarities (size in natural infections intensity of infections)

Hoares (1966 1967) suggested application of the term xenodeme to host-restricted trypanosome populations does not really apply to trypanosome strainsfrom ground squirrels since in these latter cases host specificity is not absoluteIn addition geographical ranges of many ground squirrels overlap (Hall amp Kelson1959) and it has been shown (Holland 1949) that exchange of fleas can occuramong ground squirrels of different species thus perhaps providing opportunities

27-2

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422 D F J HILTON AND J L MAHRT

for natural cross-transmission (assuming fleas are vectors) Gilmour amp Heslop-Harrisons (1955) term plastodeme is partially applicable to trypanosome strainsfrom ground squirrels since it denotes populations differing phenotypically How-ever even plastodeme is not entirely satisfactory because it indicates only thatthere are differences without specifying what types

As most of the differences among trypanosome strains of ground squirrels arehost influenced it would be advantageous to have a collective term which denotesthis variation Consequently we believe it is useful to use two descriptive prefixesin front of the root (deme) as has been done by Gilmour amp Heslop-Harrison (1955)Thus populations of trypanosomes from different species of ground squirrelswould be termed plastoxenodemes ie populations whose differences are host-influenced

Since most differences among trypanosome strains of ground squirrels resultfrom host influences (and host specificity is not absolute) there is no basis forpreserving the specific status of T citelli T hixsoni and T iowensis (describedfrom 8 richardsonii 8 franklinii and S tridecemlineatus respectively) Althoughwe have no comparable information for the trypanosome strain (described asT otospermophili) from S beecheyi in California there are at least four reasons forbelieving that it is also a plastoxenodeme (1) 8 beecheyi is not geographicallyisolated and has overlapping distributions with 8 beldingi Merriam 8 lateralis andS mohavensis Merriam These latter three species also have overlapping distribu-tions with one or more other North American ground squirrels thus providing acontinuum with the five species of ground squirrels we worked with in AlbertaThis would (in theory at least) provide opportunities for natural cross-transmission(2) Even though S undulatus in North America is geographically isolated from allother North American species the trypanosomes in S undulatus are very similarto those from S columbianus and 8 richardsonii as regards size and types ofinfection produced (3) Similarly to our work Bozhenko (1927) and Galuzo ampNovinskaya (1968) determined (on the basis of complete cross-transmission) thatthere is only one species of trypanosome (T spermophili) present in Eurasianground squirrels (4) Trypanosomes from S beecheyi are identical in morphology toall other ground squirrel trypanosomes as well as being similar in size to theplastoxenodemes from 8 franklinii and 8 tridecemlineatus

As a result of the foregoing we believe that only one species of trypanosome(T otospermophili) is present in ground squirrels (Spermophilus spp) in NorthAmerica It is probable that T otospermophili and T spermophili are the samespecies based on our work the cross-transmission experiments of Bozhenko (1927)and Galuzo amp Novinskaya (1968) and the fact that 8 undulatus has a Holarcticdistribution which overlaps with the distribution of other species of groundsquirrels in south-central Asia However until more conclusive evidence isobtained the two species should remain separate Hoare (1972) treats both formsas subspecies (ie T o otospermophili and T o spermophili) because of theirgeographical separation

A revised description of T otospermophili (using mensural data from the litera-ture as well as our own) its synonyms and recorded hosts are given below

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Taxonomy of trypanosomes of Spermophilus 423

Trypanosoma (Herpetosoma) otospermophili (Wellman amp Wherry 1910) Laveran1911 (Figure 9)

SYNONYMS Trypanosoma citelli Watson amp Hadwen 1912 T hixsoni Becker ampRoudabush 1934 T iowensis Becker amp Roudabush 1934

DESCRIPTION Characters as for the genus in addition to being lewisi-ike (ieresembling the trypomastigote stage of T lewisi) in general appearance bodymeasurements very variable depending upon host sex and species Total length25-2-55-0tm (mean 39-4tm) width at widest point (not including undulatingmembrane) 1-5-4-0tm (mean 2-4an) posterior end of body to kinetoplast1-5-7-5tin (mean 3-9an) kinetoplast to middle of nucleus 8-3-21-0tm (mean13-4 on) middle of nucleus to anterior end of body 6-0-21-0 tm (mean 10-9 tm)length of free flagellum 6-0mdash24-0tm (mean 11-2tm) nucleus length 2-4-6-0tm(mean 3-2tm) Kinetoplast round to triangular occupying entire body widthnucleus elongate ovoid with long axis oriented parallel to body length not usuallyoccupying entire body width Kinetoplast and nucleus stain medium to deeppurple with Giemsas or Wrights blood stains while body cytoplasm stains paleblue or pale violet

RECORDED HOSTS Spermophilus beecheyi 8 columbianus Sfranklinii S richard-sonii S tridecemlineatus S undulatus plesius In addition S lateralis tescorumhas been experimentally infected

SUMMARY

(1) Spermophilus columbianus Sfranklinii S lateralis tescorum S richardsoniiS tridecemlineatus and S undulatus plesius (ground squirrels) were collected fromAlberta and Yukon Territory (S u plesius only) Canada Trypanosome infectionswere detected in all but S I tescorum

(2) All six species of squirrels were experimentally inoculated with trypanosomesfrom naturally infected S columbianus S franklinii S richardsonii and S tride-cemlineatus The strains from S columbianus S richardsonii and S tridecemlineatusproduced infections in some or all of the other host species whereas the strain fromS franklinii did not

(3) Natural transmission of trypanosomes has not been demonstrated but itseems likely that fleas act as vectors

(4) Intensities and durations of experimental infections were variable anddepended upon the trypanosome strain host species and individual Infectionswere non-pathogenic

(5) Previously infected ground squirrels could not be reinfected with the sameor a different trypanosome strain and immunity appeared to be lifelong

(6) Size comparisons among and within trypanosome strains from naturallyand experimentally infected ground squirrels revealed that the strains fromSfranklinii and S tridecemlineatus were similar to one another but different fromthe other three trypanosome strains (those from S columbianus S richardsoniiand S undulatus)

(7) It is suggested that there is only one species of trypanosome Trypanosomaotospermophili in the genus Spermophilus in North America and that differences

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424 D F J HILTON AND J L MAHRT

in size and types of infection are due to host-induced variations (ie they areplastoxenodemes) T spermophili (present in Eurasian Spermophilus spp) mightbe synonymous with T otospermophili

The encouragement help and advice of Dr J C Holmes is gratefully acknow-ledged Financial support was received from the National Research Council(Canada) through a Postgraduate Scholarship to D F J H and grants to J L M

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CULBERTSON J T (1941) Trypanosomiasis in the Florida cotton rat Sigmodon hispiduslittoralis Journal of Parasitology 27 45-52

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DAVIS B S (1952) Studies on the trypanosomes of some California mammals University ofCalifornia Publications in Zoology 57 145-250

DOBNEY R S (1967) Incidence taxonomic relationships and development of lewisi-liketrypanosomes in Wisconsin Sciuridae Journal of Protozoology 14 425-8

ELLERMAN J R amp MORRISON-SCOTT T C S (1951) Check-list of Palaearctic and IndianMammals 1758 to 1946 London British Museum (Natural History)

FHANXEL S REITMAN S amp SONNENWTRTH A C (1970) GradwoMs Clinical LaboratoryMethods and Diagnosis vol i St Louis C V Mosby Co

GALUZO I G amp NOVINSKAYA V F (1968) Trypanosomes of the animals of KazakhstanII Trypanosomes of rodents In Natural Nidality of Diseases and Questions of Parasitology(ed N D Levine) Urbana University of Illinois Press

GILMOUR J S L amp HESLOP-HARRISON J (1955) The deme terminology and the units ofmicro-evolutionary change Oenetica 27 147-62

GREWAL M S (1957) The life-cycle of the British rabbit trypanosome Trypanosoma nabiasiRailliet 1895 Parasitology 47 100-18

HALL E R amp KELSON K R (1959) The Mammals of North America New York RonaldPress

HILTON D F J (1972) Developmental sites of Trypanosoma (Herpetosoma) spp in theirflea vectors Transactions of the Royal Society of Tropical Medicine and Hygiene 66 357-9

HILTON D F J amp MAHRT J L (1971) Ectoparasites from three species of Spermophilus(Rodentia Sciuridae) in Alberta Canadian Journal of Zoology 49 1501-4

HILTON D F J amp MAHRT J L (1972) Prevalence of Trypanosoma otospermophili (ProtozoaTrypanosomatidae) in five species of Spermophilus (Rodentia Sciuridae) Parasitology65 427-32

HOABE C A (1966) The classification of mammalian trypanosomes Ergebnisse der Mikro-biologie Immunitaetsforschung und Experimentellen Therapie 39 43-57

HOARE C A (1967) Evolutionary trends in mammalian trypanosomes Advances in Para-sitology 5 47-91

HOARE C A (1972) The Trypanosomes of Mammals Oxford BlackwellHOLLAND G P (1949) The Siphonaptera of Canada Publ no 817 Ottawa Canada Depart-

ment of Agriculture

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HOPKINS G H E (1949) The host-associations of the lice of mammals Proceedings of theZoological Society of London 119 388-604

HOPKINS G H E (1957) Host-associations of Siphonaptera International Union BiologicalScience Ser B 32 64-87

LAVBBAN A (1911) Identification et essai de classification des trypanosomes de mammiferesAnnales de VInstitut Pasteur 25 497-517

LEVTNE N D (1965) Trypanosomes and Haemobartonella in wild rodents in Illinois Journalof Protozoology 12 225-8

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MOLYNEUX D H (19696) The morphology and biology of Trypanosoma (Herpetosoma)evotomys of the bank-vole Clethrionomys glareolus Parasitology 59 843-57

MOLYNEUX D H (1970) Developmental patterns in trypanosomes of the subgenus Herpeto-soma Annales de la Societe Beige de Medecine Tropicale 50 229-38

NOBLE E B amp SHIPMAN D (1958) Trypanosomes in American ground squirrels Journal ofProtozoology 5 247-9

OONEV S I (1963) Mammals of the USSR and Adjacent Countries Vol v RodentsJerusalem Israel Program for Scientific Translations

ORMEROD W E (1963) The initial stages of infection with Trypanosoma lewisi control ofparasitaemia by the host In Immunity to Protozoa (ed P C C Gamham et al) OxfordBlackwell

QUAY W B (1955) Trypanosomiasis in the collared lemming Dicrostonyx torquatus(Rodentia) Journal of Parasitology 41 562-5

ROHLF F J amp SOKAL R R (1969) Statistical Tables San Francisco W H Freeman and CoSIMMONS V CUNNINGHAM M P VAN HOEVE K amp LUMSDEN W H R (1963) Investiga-

tions concerning the destination of intraperitoneal inocula in mice East African Trypano-somiasis Research Organization Report (January 1962 to June 1963) p 25

SOKAL R R amp ROHLF F J (1969) Biometry The Principles and Practice of Statistics inBiological Research San Francisco W H Freeman and Co

SONNEBOEN T M (1957) Breeding systems reproductive methods and species problems inProtozoa In The Species Problem (ed E Mayr) Washington DC American Associationfor the Advancement of Science

TALIAFERRO W H (1923) A study of size and variability throughout the course of pureline infections with Trypanosoma lewisi Journal of Experimental Zoology 37 127-67

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WALLACE F G (1966) The trypanosomatid parasites of insects and arachnids ExperimentalParasitology 18 124-93

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