EXPERIMENTAL
38,
PARASITOLOGY
Trypanosoma
153-160
cruzi:
( 1975)
New Foci of Enzootic in California F.
!&ERWIN 614
W.
Shenandoah (Accepted
Street,
Disease
WOOD
Thousand
for publication
Chagas’
Oaks,
September
California
91360
10, 1974)
WOOD, S. F. 1975. Tqpunosoma crmzi: New foci of enzootic Chagas’ disease in California. Experimental Parasitology 38, 153-160. Trypanosoma wuzt was discovered in 2-3rd instar nymphs of Ttiatomu protracta protracta from Paicines, 57 of 257 adults from Mulholland Overcrossing, and 2 males of 27 bugs from Juniper Hills, California. Thirty experimentally infected albino Mus musculus revealed low grade parasitaemias. Of 785 laboratory-raised Triatomu fed on 11 mice with Paicines isolate, 675 T. p. navajoend and 1 T. p. protracta became infected; of 856 T. p. navajoends fed on 13 mice with Mulholland isolate, 792 were positive; and of 137 T. p. navajoensis fed on 6 mice with the Juniper Hills isolate, 32 were infected with Trypanosoma cruzi. Examination of 7599 mm2 area of tissue films for Paicines isolate revealed 31 type b and 15 type c sphaeromastigotes; 5650 mm% for the Mulholland isolate revealed 9 a-, 149 sphaero-, 8 epi-, and 7 trypo-mastigotes; and 15,209 mm% for the Juniper Hills isolate revealed 245 a-, 2865 sphaero-, 42 epi-, and 21 trypo-mastigotes. Field xenodiagnosis of 228 rodents in Juniper Hills revealed 11 Peromyscus trwi montipinoris and 1 Neotomu fuwipes macrotis with Chagas trypanosome. INDEX DESCRIPTORS: Trypanosomu cruzi; Triatomu protracta protracta; T. p. navajoen&; Paicines Isolate; Mulholland Isolate; Jumper Hills Isolate; Mus musculus; Xenodiagnosis; Neotoma fuscipes; Peromyscus tmtei; Amastigotes; Sphaeromastigotes; Epimastigotes; Trypomastigotes; Zoonosis.
monkeys (Wood 1934; Davis 1943) and native mice (Wood 1937, 1941) suggest that the trypanosomes may threaten man with a chronic form of Chagas’ disease. Amastigote development by isolates from Triatoma from Mulholland Overcrossing near Sherman Oaks and from two new locations, Paicines and Juniper Hills, are reported here.
INTR~DUCXT~N
A widespread zoonosis of Trypanosoma cruzi occurs in reservoir mammals and triatomid insect vectors in California. Seventeen of the 24 reported locations in Table I are based on observations of trypanosomes from the enteron of Triatoma protracta protmcta. Amastigote development has been demonstrated in mammal hosts for isolates from Murray Canyon (Kofoid and Donat 1933)) Eaton Canyon (Wood 1941)) Plymouth (Davis 1943)) and GrifIith Park (Mehringer and Wood 1958). The insect vector, T. p. protracta, can produce severe #allergic reactions in man ( Frazier 1969). Experimental infections in
MATERIALS
0 1975 by of reproduction
Academic in any
Press, Inc. form reserved.
METHODS
The triatome cultures were maintained at Los Angeles City College at ambient indoor temperatures. The bugs survived by periodically sucking the blood of laboratory-raised adult albino Mus musculus, 153
Copyright All rights
AND
154
SHERWIN
California Caviary 1 strain, not previously exposed to infected Triatoma or trypanosomes. All experimental mice, unless otherwise specified, were albino Mus musculus. Average weights for males were 18.3 g, range 12.5-28.5 g; females 17.2 g, range 12-21 g. Most conenose bugs used here were the Navajo conenose, Triatoma protracta navajoensis. The inocula used for experimental mice contained one to several voluntary fecal droppings from infected Triatoma deposited on ‘a microscope slide shortly after a blood meal and mixed with 1% saline-citrate solution (0.5 g sodium chloride, 0.5 g sodium citrate, 100 ml H20). Inoculations were made with a l-ml tuberculin syringe and a No. 24 needle. Injections into the gastrocnemius muscle were at right angles and lateral to the long axis of the muscle. The inserted hypodermic needle was pulled back slightly before injection to create a pocket in the tissue and avoid pressure injury to the flagellates. The dark uric acid sphcrules present in both clear and cloudy triatomc feces remained at the depot and marked the site for location at time of excision. The mice to be inoculated were placed in 8-mm mesh hardware cloth cylinders with 6-mm doweling blocking their exit. Each hind leg was withdrawn between the doweling as needed for inoculation. Live-trapping at the Juniper Hills location was facilitated by private ownership of an isolated 5 acres where Sherman live traps could be left outside continuously and reset at weekly intervals from November 1967 through December 1973. The xenodiagnosis of Brumpt (1914) was used to verify the experimental infections and survey the live-trapped mammals from Juniper Hills, This involved feeding uninfected triatomes on the mammals and subsequently examining their feces for the characteristic trypanosomes. For demonstration of amastigote development, three types of slides were prepared from excised, sliced gastrocnemius
F.
WOOD
muscles. For each muscle, one slide was usc,d for temporary storage and slicing, several slides were used for tissue-touch preparations of muscle slices held with fine forceps, and one slide was made from teased muscle slices used for tissue-touch slides. For tissue-touch preparations, the lighest contact possible in a vertical plane produced the best results, i.e., the fewest broken cells. Smudging was prevented by avoiding horizontal movements while lightly touching the slide with the pieces of muscle tissue. Tissue films and blood smears were measured with a millimeter rule to determine area. All counts on stained slides were made with an oil immersion lens and 10~ W. F. eyepieces. Two drops of tail blood under an B-mm circular cover slip were used for live trypanosome counts. Examinations were mainly at weekly intervals. All slides, including tissue films, were air dried and stained with Jenner-Giemsa’s stain. Isolations of trypanosomes were made between 1965 and 1969 from Triatoma collected from the Mulholland, Paicines, and Juniper Hills locations. The terminology used herein for the developmental stages of T. cruzi is that of Hoare (1972). RESULTS
Mulholland
Isolate
Wood and Wood (1964) reported capturing T. p. protracta naturally infected with Trypanosoma cruzi from the Mulholland Drive Overcrossing of the San Diego Freeway in Scpulveda Canyon near Sherman Oaks, Los Angeles County, C&fornia. Since 1964, 83 additional bugs have been collected during early evenings from this site for a total of 297 adults Of 277 (127 males, 150 females) examined, S7 (25 males, 29 females) or 20.5% were positive for Chagas’ trypanosome. Ratios of positive and negative bugs examined for the summer collections were 1960 (46-149). 1961 (l-lo), 1963 (O-2), 1964 (932),
NEW
1965 ( 04),
1966 ( l-13))
FOCI
OF
and 1967 (o-10).
These examples verify my opinion that for any one geographical area, at least ten Triatoma should be examined before considering the site negative. On 10 August 1966, one mouse was inoculated in the left gastrocnemius with a 0.05 ml suspension of the rectal contents of a male T. p. protracta naturally infected with T. cruzi collected 3 August 1966, on the Mulholland Overcrossing roadbed. After inoculation, additional diluen! was added to the syringe to suspend the remaining parasites. This fiuid was avidly lapped up by the mouse from the tip of the syringe. Of 98 triatomes fed on this mouse on Days 26, 47, 75, and 99, there were 17-7-1442 positives, respectively. Of 20 T. p. navajoensis fed on the 26th day, 17 (one 4th, eight 3rd, and eight 2nd instar nymphs) were positive and one 4th, one 3rd and one 2nd were negative. Of 78 T. p. protracta fed on Days 47, 75, and 99, there were 63 (1 female, fifteen 5th, twenty-four 4th, thirteen 3rd, and ten 1st) positive and two 5th, eight 4th and five 3rd negative. Subsequent bug-mouse-bug transfers were made on 11 July 1967, with T. p. protracta and 5 August 1969, and 13 July 1971, with T. p. navajoensis. From 1966 through 1971, 14 mice were used to transfer the Mulholland isolate. One mouse was sacrificed for tissue studies (Table II). Ten mice received inoculum in one gastrocnemius, one was fed inoculum, and three, including the tissue study mouse, were injected in both gastrocnemius muscles. Xenodiagnosis was carried out from Days 6 to 341 at weekly intervals. Of 856 T. p. navajoensis fed on these mice, 792, or 92.570, were positive for T. cruzi (1 femaIe, forty-nine 5th, seventy-five 4th, forty-six 3rd, nine 2nd, and six hundred and twelve 1st instar nymphs) and twelve 5th, eighteen 4th, nine 3rd, one 2nd and twenty-four 1st were negative. Most infections were followed to Day 44,
CHAGAS’
DISEASE
155
two mice to Day 55, one to Day 64, one to Day 99, and one to Day 341. Ten mice were positive by xenodiagnosis or blood samples, two were negative by both, and one was positive by xenodiagnosis but negative by blood examinations. Blood trypanosomes totaled 150 from 10 mice in 21 of the 55 samples examined from Days 13 through 75, with a high of 50 counted from one mouse on Day 43. One mouse was inoculated in each gastrocnemius muscle with a 0.05 ml suspension of first voluntary clear (few uric acid spherules) fecal droplets from one male and five female T. p. navajoensis. Xenodiagnosis was positive all days tested as follows: 6-12-21-29-36-74-124-132-139146159-173-201-248-270-279-314, and 341, involving 48 T. p. navajoensis with 42 (twenty-six E&h, seven 4th, and nine 1st) or 87.5% positive. Another mouse was exsanguinated on Day 64 by one hundred and eighteen 1st instar nymphs of T. p. navajoensis all of which became infected. Live weight before inoculation was 18.5 g, dead weight after exsanguination was 25.5 g. For 15 differential leukocyte counts from 7 mice infected with the Mulholland isolate on Days 21(3), 28(3), 29, 34(2), 43( 3), 44, and 50( 2), no significant differences were evident from the normals of Sell (1951). This is compatible with the light parasitaemias noted above and indicates a high resistance of CC1 albino mice to the Mulholland isolate. Another mouse received 0.05 ml inoculum in both gastrocnemius muscles from a 0.1 ml suspension of three clear and six cloudy (many uric acid spherules) voluntary fecal droplets from a previously infected male and female T. p. navajoensis. The mouse was sacrificed on the sixth day and seven slides were prepared from each excised muscle as noted in Table II. All parasites were stages in progressive dcvelopment indicating 149 arising by unrolling or untwisting, probably from spherical pseudocysts and eight by elonga-
156
SHERWIN
tion, possibly from spindle-shaped pseudocysts. The 7 C form trypomastigotes appeared to originate from developing sphaeromastigotes as shown by Wood (1953) and Hoare (1972). No phagocytized parasites were seen and 31 were unclassified due to ruptures, detached flagella, or unclear differentiation. The Mulholland isolate is a weak but persistent parasite for CC1 mice, being more easily recovered by xenodiagnosis than by small blood samples. These data furnish the fifth demonstration of intramuscular development for California isolates of Trypnnosoma cruzi ( Table I ) . Paicines Isolate On 8 August 1965, nine T. p. protracta were collected from a Neotoma brushpile house under some roadside elderberry bushes in the agricultural community of Paicines, 12 miles south of Hollister, San Benito County. There were two 5th, one 4th, five 3rd, and one 2nd instar nymphs. Microscopical examination cf the fecal droplets from 2-3rd instar nymphs revealed epimastigotes and trypomastigotes tYPical of the insect phase of T. cruzi. The other seven nymphs were negative. After 23 days in the laboratory, 0.05 ml of a 1% saline-citrate solution containing one voluntary fecal droplet from a naturally-infected 3rd instar nymph was injected into the gastrocnemius muscle of a mouse. Twenty days after inoculation, four 5th instar nymphs of T. p. navajoensis fed on the mouse. Fifty days later, two of the four bugs showed trypanosomes in their feces. The other two were negative. Subsequently, three similar bug-mousebug transfers were made 1 October 1969, 21 April 1971, and 1X Augllst 1972. Frotlr 1965 through 1973, 13 mice were used to transfer the Paicines isolate, two mice being sacrificed on Days 5 and 6 for tissue studies. Eleven mice were inoculated intramuscularly, one intraperitoneally and one by mouth,
F.
WOOD
Fresh blood samples of 3 of the 11 mice examined from Days 10 to 234 showed from 1 to 7 trypanosomes per sample in 6 of the 54 samples taken from Days 29 to 55. All three received inoculum in one gastrocnemius muscle. Xenodiagnosis revcaled infection in all 11 mice. Positive bug infections were found as early as Day 10 and as late as Day 234 after feeding on the mice. One mouse received via the left gastrocnemius a 0.02 ml suspension of three first voluntary fecal droppings from recently fed 5th( 2) and 4th( 1) instar nymphs of T. p. navajoensis. One hundred twenty-seven of the 128 first instars fed from days 10 through 70 became infected and 201 T. p. navajoensis (thirteen 3rd, seventy-one 2nd, and one hundred and seventeen 1st instar nymphs) fed on Day 234 revealed 76.9% third, 65.1% second, and 53.9% first positive. One mouse was inoculated intraperitoneally with a 0.15 ml suspension of ten first voluntary droppings from recently fed 5th( 1) and 4th( 9) T. p. navajoensis nymphs. One hundred fifty-six of 157 first instars fed from Days 10 through 70 were positive and 104 T. p. navajoensis (9-3rd and 953nd) fed on Day 234 revealed 100% third and 68.4% second instars infected with T. cruzi. Xenodiagnosis was positive for both mice on Days lo-14-19-25-3341~55.~70-831~37-14~5-1~53-161-174-188-21’7, and 234. Total xcnodiagnosis for both mice involved 351 and 283 mostly first instar nymphs of T. p. navajoensis with 79.2% and 84.9% positive, respectively. Total xenodiagnosis for the 11 mice involved 785 bugs; 784 T. p. navajoensis and 1 T. p. protracta. Of these, 51.770 were first instars and 676 or 86.19 became inf~ctcd. ‘I‘hr abundance of infected first instar nymphs would indicate a general high level of blood trypanosomes in the peripheral circulation even though they were mostly undetected by direct examination of tail blood.
NEW
FOCI
OF
Two mice were used to demonstrate intramuscular development. Each mouse received a 0.01 ml suspension of seven or eight first voluntary fecal deposits from previously infected 4th instar T. p. nauajoensk nymphs in each gastrocnemius muscle, i.e., double inoculations for each mouse. The results are shown in Table II. One type b sphaeromastigote was in fission. This is the sixth demonstration of amastigote development in a California isolate as shown in Table I. Paicines trypanosomes produce a lowgrade persistent, nonlethal parasitaemia with ample blood trypanosomes for continuous seeding of the native triatomid insect vectors. This is the 24th location for recovery of Trypanosoma cruzi in T. p. protmcta in California (Table I). Juniper
Hills Isolate
From 1946 through 1972, examination of fecal droplets from 27 T. p. protracta (14 males, 7 females and two Sth, two 4th, one 3rd, and one 2nd instar nymphs) from Juniper Hills, near Pearblossom, Los Angeles County, California, revealed two males collected 27 July and 6 August 1969, positive for T. cruzi. On 6 August 1969, a mouse was inoculated in the left gastrocnemius with a 0.1 ml suspension of the rectal contents of the T. p. protracta collected on August 6th. Blood examinations were negative on Days 12, 14, and 16, but xenodiagnosis was positive for Days 14 and 16. Of 22 first instar nymphs of T. p. navujoensis fed on the 14th day, 12 were positive and of eleven 2nd and eleven third instar nymphs fed on the 16th day, two 3rd instar nymphs were positive for Chagas’ trypanosome. A bug-mousebug transfer was made on 19 July 1971 from the experimentally infected Triatomn. Isolation by xenodiagnosis from two male and six female Peromyscus truei montipinoris and a female Neotoma fuscipes mucrotis were made in I971 and from one male and two female P. truei montipinoris in 1973.
CHAGAS’
DISEASE
157
From 1969 through 1971, five Mus and a Peromyscus maniculatus received inoculum via the gastrocnemius muscles: three Mus and one Peromyscus in one hind leg and one Mus in both hind legs. Another Mus received inoculum by mouth. Two additional Mus, inoculated in both hind legs, were used for tissue studies being sacrificed on Days 6 and 12, respectively. No blood trypanosomes were seen in any of the six mice (Five Mus, one Peromyscus) examined involving 22 blood samples taken from Days 6 through 44. Xenodiagnosis was positive for the Peromyscus and two Mus, one inoculated in one hind leg and one in both hind legs, All others were negative. Of 137 T. p. navajoensis fed on the six mice, 32 (eighteen 5th, two 3rd, and twelve 1st instar nymphs), or 23.4%, were positive for Chagas’ trypanosome and twenty-five 5th, forty-four 4th, fifteen 3rd, eleven 2nd, and ten 1st were negative. Two Mus inoculated intramuscularly in one hind leg were negative (7 blood samples, 4 xenodiagnoses with 40 bugs). One Mus inoculated by mouth was negative (5 blood samples, 5 xenodiagnoses with 12 bugs). Fifty-four P. municulntus (29 males, 25 females) tested by xenodiagnosis from Juniper Hills were negative. One Mus received 0.05 ml each in the left and right gastrocnemius muscles from a 0.1 ml pool of seven voluntary fecal samples from previously infected adult T. p. navujoensis. The mouse was sacrificed on the sixth day and 20 slides were prepared from the excised muscles. The results shown in Table II revealed 7 type b sphaeromastigotes in fission and 3 S and 18 C form trypomastigotes. All parasites were in progressive development. Of the 542 parasites seen, 91, including 2 in macrophages, were unclassified due to breakage, loose flagella, or unclear differentiation. Another Mus received inocuI,ations similar to the mouse above from a 0.1 ml pool
158
SHERWIN
F.
WOOD
TABLE Progress
in the Discowry
of Trypanosoma
Location Murray Canyon” Eat,on Canyon” Trimmer Springs O’Neals Griffith Parka Plymouth” Fallbrook Three Rivers La Grange Tarzaua Riverside Preston Ranch Knowles Placerville -4lpine Pauma Valley Oroville Kernville Westwood Sherman Oaks Itoscomare Canyon hlr~lholland Overcrossing~~ 16 km E of Redlands Paicinesa Juniper Hills” developmetlt,
demons1
rruzi
in Triaiomes
Arlthor
San I liego Los Angeles Fres1ro AIadera IAN
hgele,s
Amado San 1 )iego Trdare stanis1:m Los Angeles It iverside Madera Madera El Dorado San Diego San Diego Butt)e Kern I,os Angeles 1,o.s .4ngeles T,os Angeles Los Angeles San Bernardino San Benit o IAM Angeles
Jkvelopmcnt and .Jltnipcr
Paicines Paicines Rlulholland Juniper Hills Juniper Hills
.\Iasl
7 238
and 11onat. 1938 1942 1942 1942 c( al. 1943
Wood
1944
1033
1966
16 I5 S7 323 2338
II
Experimentally In/cc/cd with I’aicincs, Jsolntcs o,f Tr!ipanosomrc crr~si - ~ .__~--
igoles
Sphaerot)ype b t,ype
0 0 9
Kofoid Wood Wood Wood Wood 1 )avis
progressive development. Of the 3461 parasites seen, 739 were unclassified because of smudging on the tissue-touch slides. There were 63 parasites in macrophages. Xenodiagnosis of 228 live-trapped mammals in Juniper Hills from November 1967
in .Ilbino Alice Hills, California,
ISOl;tt.~ ‘I-
and date
Wood 19.X Wood 19.3) Wood IO30 Rycakman 1952 Wood and Wood 1964 Wood and Wood 1964 Wood and W(JOd 1964 Wood and Wood 1964 Wood and Wood 1064 Wood atld Wood 1964 Wood and Wood 1964 Wood and Wood 1964 Wood and Wood 1964 Wood alld Wood 1964 Wood and W;ood 1964 Sjogretr alrtl Ryckman 1 Y6.i I Wi!)
TABLE Tissue
in California
rated.
of six voluntary fecal samples from adult T. p. navajoensis. Ten slides were prepared from the right and nine from the left gastrocnemius muscles on the 12th day after inoculation (Table II). Fiftyseven of the type b sphaeromastigotes were in fission and all parasites were in
Com.parativc
and Mammals
c0llnt.y ~___
~-~.
~1Amastigote
I
Epi-
Typo-
c 7
0
0
x
0
0
62
8
7
100
0
21 0
84
42
Tisslle film area il, mm2
;Il7tlhollan~l
L)R> sacxrificed
No. of micro. slides
5th 6th 6th
IS 12 14
7792
6th
20
7417
12th
I9
4136 3463 5650
NEW
FOCI
OF
through December 1973, involved 814 triatomes (592 T. p. navajoensis and 222 T. p. protracta) with I5 positive 5th instar T. p. nauajoensis, 11 in 1971, and 4 in 1973. Twelve rodents, or 5.3%, were positive for Trypanosoma cruzi. There were 11 Peromyscus truei montipinoris (3 adult males, 4 adult females, 4 immature females) and one adult female Neotoma fuscipes mucrotis harboring Chagas’ zoonosis. Locations of capture for 228 rodents centered around a triangle of home-garage-canyon habitats approximately 300 m on a side, the garage downhill and NW and the canyon uphill and SW of the residence. Of the 12 infected rodents, one Neotoma and six Peromyscus were live-trapped along the south side of the home. Two Peromyscus were caught in a water heater cabinet attached to the south wall, two inside the garage and one in the nearby canyon near wood rat houses and a dry streambed. Although three male Peromyscus (1 P. truei, 2 P. maniculatus) were trapped inside the home, none was positive for Chagas’ trypanosome. Thirteen rodents from nearby locations, negative by xenodiagnosis, were captured within I.2 km of the primary location. Trypanosoma peromysci is the most common blood parasite in Juniper Hills native mice. Two female P. truei, one adult captured on 3 October 1971, and one immature on 18 February 1973, harbored both T. peromysci and T. cruzi at the time sampled. Total yearly xenodisagnoses for T. cruzi numbered 7-48-5-12-84-44 and 28 for native rodents from 1967 through 1973 in Juniper Hills. A summary of xenodiagnoses by quarters revealed 33 (3 positive) for Winter (December, January, February), 73 (all negative) Spring (March, April May), 56 (8 positive) Summer (June, July, August) and 66 (1 positive) Fall (September, October, November). There were 152 Peromyscus truei (77 males, 75 females) and 19 Neotoma fuscipes (3 males, 16 females)
CHAGAS’
159
DISEASE
trapped with 11, or 7.2%, of the former and 1 or 5.2%, of the latter positive by xenodiagnosis for T. cruzi. DISCUSSION
In previous attemps to demonstrate carriers of Chagas’ zoonosis nearer sea level, as in Griffith Park, Los Angeles, the temperature stress of winter appeared to trigger more positive findings (Wood and Wood 1967). In Juniper Hills at 1663 m (4800 ft ) where eight positives were captured during a populsation irruption during the summer of 1971, food availability may be more important, Hunger stress during the population irruption may have resulted in more naturally infected Triatoma being consumed, thus explaining the higher number of positives at this location. Both Peromyscus and Neotoma will eat Triatoma in the laboratory, and probably also in their native habitats. I believe that the prediction of Kofoid and Donat (1933) that Chagas’ zoonosis is widespread in California wherever Neotoma fuscipes and naturaIIy infected Triatoma protracta occur is correct. For 40 yr, Chagas’ zoonosis was known in California coastal localities ‘and bordering the inland San Joaquin Valley. The present report is the first demonstration of its presence on the eastern edge of the San Gabriel Mountains in southern California where several hundred miles of desert separate it from the nearest naturally infected vectors demonstrated in Arizona and Utah. This report from Juniper Hills constitutes the 25th location for recovery of infected insect vectors and the seventh demonstration of intramuscular development of Trypanosoma crud Chagas for California (Table I). Uninfected CC1 albino Mus frequently developed tumors similar to mammary gland carcinomas (Snell Fig. 88). None of these tumors was seen in any of the mice infected with the California isolates
SHERWIN
160
of T. cruxi even though their weights at death were often double or more the weight at time of inoculation. The significance of this is yet to be investigated. REFERENCES
BRUMPT, E. 1914. Le xknodiagnostic. Application au diagnostic de quelques infections parasitaires et en particulier a la trypanosomose de Chagas. Bulletin de la Soci&te’ de Pathologie Erotique 7, 706-710. DAVIS, D. J. 1943. Infection in monkeys with strains of Trypanosoma cruzi isolated in the United States. Public Health Reports, United States Public Heal& Service 58, 775-777. FRAZIER, C. A. 1969. Insect Allergy. W. H. Green, Inc., St. Louis, Missouri. HOARE, C. A., 1972. The Trypanosomes of Mammals. F. A. Davis Co., Philadelphia, Pennsylvania. KOFOID, C. A., AND F. DOXAT. 1933. South American trypanosomiasis of the human type-occurrence in mammals in the United States. California and Western Medicine 38, 245-249. MEHRINGER, P. J., JR., AND S. F. WOOD. 1958. A resampling of wood rat houses and human habitations in Griffith Park, Los Angeles, for Triatoma protracta and Trypanosoma cruzi. Bulletin of the Southern CaZifornia Academy of Sciences 57, 39-46. SJOGREN, R. D., AND R. E. RYCKMAN. 1966.
F.
WOOD
Epizootiology of Trypanosoma cruzi in southwestern North America. VIII. Nocturnal flights of Triatoma protracta (Uhler) as indicated by collections at black light traps ( Hemiptera: Reduviidae; Triatominae) Journal of Medical Entomology 3, 81-92. SNHLL, G. D. (ed). 1941. Biology of the Laborutory Mouse. Dover Publications, Inc., New York. WOOD, F. D. 1934. Experimental studies on Trypanosoma cruzi in California. Proceedings of the Society for Experimental Biology and Medicine 32, 61-62. \vOOD, S. F. 1937. Cytological variations in the blood and blood-forming organs of whitefooted mice experimentally infected with Trypanosoma cruzi. Vniver.yity of Californ’a Publications in Zoology 41, 389-418. WOOD, S. F. 1941. New localities for Trypanosoma cruzi Chagas in southwestern United States. American Journal of Hygiene 34, l-13. WOOD, S. F. 1953. Hematologic differentiation of the intramuscular developmental forms of Trypanosoma cruzi Chagas. American Journal of Tropical Medicine and Hygiene 2, 1015-1035. WOOD, S. F., AND F. 1). Woou. 1964. New locations for Chagas’ trypanosome in California. Bulletin of the Southern California Academy of Sciences 62, 104-111. WOOD, S. F., AND F. D. WOOD. 1967. Ecological relationships of Triatoma p. protracta (Uhler) in Griffith Park, Los Angeles, Calif. Pacific Insects 9, 537-550.
38,
PARASITOLOGY
Trypanosoma
153-160
cruzi:
( 1975)
New Foci of Enzootic in California F.
!&ERWIN 614
W.
Shenandoah (Accepted
Street,
Disease
WOOD
Thousand
for publication
Chagas’
Oaks,
September
California
91360
10, 1974)
WOOD, S. F. 1975. Tqpunosoma crmzi: New foci of enzootic Chagas’ disease in California. Experimental Parasitology 38, 153-160. Trypanosoma wuzt was discovered in 2-3rd instar nymphs of Ttiatomu protracta protracta from Paicines, 57 of 257 adults from Mulholland Overcrossing, and 2 males of 27 bugs from Juniper Hills, California. Thirty experimentally infected albino Mus musculus revealed low grade parasitaemias. Of 785 laboratory-raised Triatomu fed on 11 mice with Paicines isolate, 675 T. p. navajoend and 1 T. p. protracta became infected; of 856 T. p. navajoends fed on 13 mice with Mulholland isolate, 792 were positive; and of 137 T. p. navajoensis fed on 6 mice with the Juniper Hills isolate, 32 were infected with Trypanosoma cruzi. Examination of 7599 mm2 area of tissue films for Paicines isolate revealed 31 type b and 15 type c sphaeromastigotes; 5650 mm% for the Mulholland isolate revealed 9 a-, 149 sphaero-, 8 epi-, and 7 trypo-mastigotes; and 15,209 mm% for the Juniper Hills isolate revealed 245 a-, 2865 sphaero-, 42 epi-, and 21 trypo-mastigotes. Field xenodiagnosis of 228 rodents in Juniper Hills revealed 11 Peromyscus trwi montipinoris and 1 Neotomu fuwipes macrotis with Chagas trypanosome. INDEX DESCRIPTORS: Trypanosomu cruzi; Triatomu protracta protracta; T. p. navajoen&; Paicines Isolate; Mulholland Isolate; Jumper Hills Isolate; Mus musculus; Xenodiagnosis; Neotoma fuscipes; Peromyscus tmtei; Amastigotes; Sphaeromastigotes; Epimastigotes; Trypomastigotes; Zoonosis.
monkeys (Wood 1934; Davis 1943) and native mice (Wood 1937, 1941) suggest that the trypanosomes may threaten man with a chronic form of Chagas’ disease. Amastigote development by isolates from Triatoma from Mulholland Overcrossing near Sherman Oaks and from two new locations, Paicines and Juniper Hills, are reported here.
INTR~DUCXT~N
A widespread zoonosis of Trypanosoma cruzi occurs in reservoir mammals and triatomid insect vectors in California. Seventeen of the 24 reported locations in Table I are based on observations of trypanosomes from the enteron of Triatoma protracta protmcta. Amastigote development has been demonstrated in mammal hosts for isolates from Murray Canyon (Kofoid and Donat 1933)) Eaton Canyon (Wood 1941)) Plymouth (Davis 1943)) and GrifIith Park (Mehringer and Wood 1958). The insect vector, T. p. protracta, can produce severe #allergic reactions in man ( Frazier 1969). Experimental infections in
MATERIALS
0 1975 by of reproduction
Academic in any
Press, Inc. form reserved.
METHODS
The triatome cultures were maintained at Los Angeles City College at ambient indoor temperatures. The bugs survived by periodically sucking the blood of laboratory-raised adult albino Mus musculus, 153
Copyright All rights
AND
154
SHERWIN
California Caviary 1 strain, not previously exposed to infected Triatoma or trypanosomes. All experimental mice, unless otherwise specified, were albino Mus musculus. Average weights for males were 18.3 g, range 12.5-28.5 g; females 17.2 g, range 12-21 g. Most conenose bugs used here were the Navajo conenose, Triatoma protracta navajoensis. The inocula used for experimental mice contained one to several voluntary fecal droppings from infected Triatoma deposited on ‘a microscope slide shortly after a blood meal and mixed with 1% saline-citrate solution (0.5 g sodium chloride, 0.5 g sodium citrate, 100 ml H20). Inoculations were made with a l-ml tuberculin syringe and a No. 24 needle. Injections into the gastrocnemius muscle were at right angles and lateral to the long axis of the muscle. The inserted hypodermic needle was pulled back slightly before injection to create a pocket in the tissue and avoid pressure injury to the flagellates. The dark uric acid sphcrules present in both clear and cloudy triatomc feces remained at the depot and marked the site for location at time of excision. The mice to be inoculated were placed in 8-mm mesh hardware cloth cylinders with 6-mm doweling blocking their exit. Each hind leg was withdrawn between the doweling as needed for inoculation. Live-trapping at the Juniper Hills location was facilitated by private ownership of an isolated 5 acres where Sherman live traps could be left outside continuously and reset at weekly intervals from November 1967 through December 1973. The xenodiagnosis of Brumpt (1914) was used to verify the experimental infections and survey the live-trapped mammals from Juniper Hills, This involved feeding uninfected triatomes on the mammals and subsequently examining their feces for the characteristic trypanosomes. For demonstration of amastigote development, three types of slides were prepared from excised, sliced gastrocnemius
F.
WOOD
muscles. For each muscle, one slide was usc,d for temporary storage and slicing, several slides were used for tissue-touch preparations of muscle slices held with fine forceps, and one slide was made from teased muscle slices used for tissue-touch slides. For tissue-touch preparations, the lighest contact possible in a vertical plane produced the best results, i.e., the fewest broken cells. Smudging was prevented by avoiding horizontal movements while lightly touching the slide with the pieces of muscle tissue. Tissue films and blood smears were measured with a millimeter rule to determine area. All counts on stained slides were made with an oil immersion lens and 10~ W. F. eyepieces. Two drops of tail blood under an B-mm circular cover slip were used for live trypanosome counts. Examinations were mainly at weekly intervals. All slides, including tissue films, were air dried and stained with Jenner-Giemsa’s stain. Isolations of trypanosomes were made between 1965 and 1969 from Triatoma collected from the Mulholland, Paicines, and Juniper Hills locations. The terminology used herein for the developmental stages of T. cruzi is that of Hoare (1972). RESULTS
Mulholland
Isolate
Wood and Wood (1964) reported capturing T. p. protracta naturally infected with Trypanosoma cruzi from the Mulholland Drive Overcrossing of the San Diego Freeway in Scpulveda Canyon near Sherman Oaks, Los Angeles County, C&fornia. Since 1964, 83 additional bugs have been collected during early evenings from this site for a total of 297 adults Of 277 (127 males, 150 females) examined, S7 (25 males, 29 females) or 20.5% were positive for Chagas’ trypanosome. Ratios of positive and negative bugs examined for the summer collections were 1960 (46-149). 1961 (l-lo), 1963 (O-2), 1964 (932),
NEW
1965 ( 04),
1966 ( l-13))
FOCI
OF
and 1967 (o-10).
These examples verify my opinion that for any one geographical area, at least ten Triatoma should be examined before considering the site negative. On 10 August 1966, one mouse was inoculated in the left gastrocnemius with a 0.05 ml suspension of the rectal contents of a male T. p. protracta naturally infected with T. cruzi collected 3 August 1966, on the Mulholland Overcrossing roadbed. After inoculation, additional diluen! was added to the syringe to suspend the remaining parasites. This fiuid was avidly lapped up by the mouse from the tip of the syringe. Of 98 triatomes fed on this mouse on Days 26, 47, 75, and 99, there were 17-7-1442 positives, respectively. Of 20 T. p. navajoensis fed on the 26th day, 17 (one 4th, eight 3rd, and eight 2nd instar nymphs) were positive and one 4th, one 3rd and one 2nd were negative. Of 78 T. p. protracta fed on Days 47, 75, and 99, there were 63 (1 female, fifteen 5th, twenty-four 4th, thirteen 3rd, and ten 1st) positive and two 5th, eight 4th and five 3rd negative. Subsequent bug-mouse-bug transfers were made on 11 July 1967, with T. p. protracta and 5 August 1969, and 13 July 1971, with T. p. navajoensis. From 1966 through 1971, 14 mice were used to transfer the Mulholland isolate. One mouse was sacrificed for tissue studies (Table II). Ten mice received inoculum in one gastrocnemius, one was fed inoculum, and three, including the tissue study mouse, were injected in both gastrocnemius muscles. Xenodiagnosis was carried out from Days 6 to 341 at weekly intervals. Of 856 T. p. navajoensis fed on these mice, 792, or 92.570, were positive for T. cruzi (1 femaIe, forty-nine 5th, seventy-five 4th, forty-six 3rd, nine 2nd, and six hundred and twelve 1st instar nymphs) and twelve 5th, eighteen 4th, nine 3rd, one 2nd and twenty-four 1st were negative. Most infections were followed to Day 44,
CHAGAS’
DISEASE
155
two mice to Day 55, one to Day 64, one to Day 99, and one to Day 341. Ten mice were positive by xenodiagnosis or blood samples, two were negative by both, and one was positive by xenodiagnosis but negative by blood examinations. Blood trypanosomes totaled 150 from 10 mice in 21 of the 55 samples examined from Days 13 through 75, with a high of 50 counted from one mouse on Day 43. One mouse was inoculated in each gastrocnemius muscle with a 0.05 ml suspension of first voluntary clear (few uric acid spherules) fecal droplets from one male and five female T. p. navajoensis. Xenodiagnosis was positive all days tested as follows: 6-12-21-29-36-74-124-132-139146159-173-201-248-270-279-314, and 341, involving 48 T. p. navajoensis with 42 (twenty-six E&h, seven 4th, and nine 1st) or 87.5% positive. Another mouse was exsanguinated on Day 64 by one hundred and eighteen 1st instar nymphs of T. p. navajoensis all of which became infected. Live weight before inoculation was 18.5 g, dead weight after exsanguination was 25.5 g. For 15 differential leukocyte counts from 7 mice infected with the Mulholland isolate on Days 21(3), 28(3), 29, 34(2), 43( 3), 44, and 50( 2), no significant differences were evident from the normals of Sell (1951). This is compatible with the light parasitaemias noted above and indicates a high resistance of CC1 albino mice to the Mulholland isolate. Another mouse received 0.05 ml inoculum in both gastrocnemius muscles from a 0.1 ml suspension of three clear and six cloudy (many uric acid spherules) voluntary fecal droplets from a previously infected male and female T. p. navajoensis. The mouse was sacrificed on the sixth day and seven slides were prepared from each excised muscle as noted in Table II. All parasites were stages in progressive dcvelopment indicating 149 arising by unrolling or untwisting, probably from spherical pseudocysts and eight by elonga-
156
SHERWIN
tion, possibly from spindle-shaped pseudocysts. The 7 C form trypomastigotes appeared to originate from developing sphaeromastigotes as shown by Wood (1953) and Hoare (1972). No phagocytized parasites were seen and 31 were unclassified due to ruptures, detached flagella, or unclear differentiation. The Mulholland isolate is a weak but persistent parasite for CC1 mice, being more easily recovered by xenodiagnosis than by small blood samples. These data furnish the fifth demonstration of intramuscular development for California isolates of Trypnnosoma cruzi ( Table I ) . Paicines Isolate On 8 August 1965, nine T. p. protracta were collected from a Neotoma brushpile house under some roadside elderberry bushes in the agricultural community of Paicines, 12 miles south of Hollister, San Benito County. There were two 5th, one 4th, five 3rd, and one 2nd instar nymphs. Microscopical examination cf the fecal droplets from 2-3rd instar nymphs revealed epimastigotes and trypomastigotes tYPical of the insect phase of T. cruzi. The other seven nymphs were negative. After 23 days in the laboratory, 0.05 ml of a 1% saline-citrate solution containing one voluntary fecal droplet from a naturally-infected 3rd instar nymph was injected into the gastrocnemius muscle of a mouse. Twenty days after inoculation, four 5th instar nymphs of T. p. navajoensis fed on the mouse. Fifty days later, two of the four bugs showed trypanosomes in their feces. The other two were negative. Subsequently, three similar bug-mousebug transfers were made 1 October 1969, 21 April 1971, and 1X Augllst 1972. Frotlr 1965 through 1973, 13 mice were used to transfer the Paicines isolate, two mice being sacrificed on Days 5 and 6 for tissue studies. Eleven mice were inoculated intramuscularly, one intraperitoneally and one by mouth,
F.
WOOD
Fresh blood samples of 3 of the 11 mice examined from Days 10 to 234 showed from 1 to 7 trypanosomes per sample in 6 of the 54 samples taken from Days 29 to 55. All three received inoculum in one gastrocnemius muscle. Xenodiagnosis revcaled infection in all 11 mice. Positive bug infections were found as early as Day 10 and as late as Day 234 after feeding on the mice. One mouse received via the left gastrocnemius a 0.02 ml suspension of three first voluntary fecal droppings from recently fed 5th( 2) and 4th( 1) instar nymphs of T. p. navajoensis. One hundred twenty-seven of the 128 first instars fed from days 10 through 70 became infected and 201 T. p. navajoensis (thirteen 3rd, seventy-one 2nd, and one hundred and seventeen 1st instar nymphs) fed on Day 234 revealed 76.9% third, 65.1% second, and 53.9% first positive. One mouse was inoculated intraperitoneally with a 0.15 ml suspension of ten first voluntary droppings from recently fed 5th( 1) and 4th( 9) T. p. navajoensis nymphs. One hundred fifty-six of 157 first instars fed from Days 10 through 70 were positive and 104 T. p. navajoensis (9-3rd and 953nd) fed on Day 234 revealed 100% third and 68.4% second instars infected with T. cruzi. Xenodiagnosis was positive for both mice on Days lo-14-19-25-3341~55.~70-831~37-14~5-1~53-161-174-188-21’7, and 234. Total xcnodiagnosis for both mice involved 351 and 283 mostly first instar nymphs of T. p. navajoensis with 79.2% and 84.9% positive, respectively. Total xenodiagnosis for the 11 mice involved 785 bugs; 784 T. p. navajoensis and 1 T. p. protracta. Of these, 51.770 were first instars and 676 or 86.19 became inf~ctcd. ‘I‘hr abundance of infected first instar nymphs would indicate a general high level of blood trypanosomes in the peripheral circulation even though they were mostly undetected by direct examination of tail blood.
NEW
FOCI
OF
Two mice were used to demonstrate intramuscular development. Each mouse received a 0.01 ml suspension of seven or eight first voluntary fecal deposits from previously infected 4th instar T. p. nauajoensk nymphs in each gastrocnemius muscle, i.e., double inoculations for each mouse. The results are shown in Table II. One type b sphaeromastigote was in fission. This is the sixth demonstration of amastigote development in a California isolate as shown in Table I. Paicines trypanosomes produce a lowgrade persistent, nonlethal parasitaemia with ample blood trypanosomes for continuous seeding of the native triatomid insect vectors. This is the 24th location for recovery of Trypanosoma cruzi in T. p. protmcta in California (Table I). Juniper
Hills Isolate
From 1946 through 1972, examination of fecal droplets from 27 T. p. protracta (14 males, 7 females and two Sth, two 4th, one 3rd, and one 2nd instar nymphs) from Juniper Hills, near Pearblossom, Los Angeles County, California, revealed two males collected 27 July and 6 August 1969, positive for T. cruzi. On 6 August 1969, a mouse was inoculated in the left gastrocnemius with a 0.1 ml suspension of the rectal contents of the T. p. protracta collected on August 6th. Blood examinations were negative on Days 12, 14, and 16, but xenodiagnosis was positive for Days 14 and 16. Of 22 first instar nymphs of T. p. navujoensis fed on the 14th day, 12 were positive and of eleven 2nd and eleven third instar nymphs fed on the 16th day, two 3rd instar nymphs were positive for Chagas’ trypanosome. A bug-mousebug transfer was made on 19 July 1971 from the experimentally infected Triatomn. Isolation by xenodiagnosis from two male and six female Peromyscus truei montipinoris and a female Neotoma fuscipes mucrotis were made in I971 and from one male and two female P. truei montipinoris in 1973.
CHAGAS’
DISEASE
157
From 1969 through 1971, five Mus and a Peromyscus maniculatus received inoculum via the gastrocnemius muscles: three Mus and one Peromyscus in one hind leg and one Mus in both hind legs. Another Mus received inoculum by mouth. Two additional Mus, inoculated in both hind legs, were used for tissue studies being sacrificed on Days 6 and 12, respectively. No blood trypanosomes were seen in any of the six mice (Five Mus, one Peromyscus) examined involving 22 blood samples taken from Days 6 through 44. Xenodiagnosis was positive for the Peromyscus and two Mus, one inoculated in one hind leg and one in both hind legs, All others were negative. Of 137 T. p. navajoensis fed on the six mice, 32 (eighteen 5th, two 3rd, and twelve 1st instar nymphs), or 23.4%, were positive for Chagas’ trypanosome and twenty-five 5th, forty-four 4th, fifteen 3rd, eleven 2nd, and ten 1st were negative. Two Mus inoculated intramuscularly in one hind leg were negative (7 blood samples, 4 xenodiagnoses with 40 bugs). One Mus inoculated by mouth was negative (5 blood samples, 5 xenodiagnoses with 12 bugs). Fifty-four P. municulntus (29 males, 25 females) tested by xenodiagnosis from Juniper Hills were negative. One Mus received 0.05 ml each in the left and right gastrocnemius muscles from a 0.1 ml pool of seven voluntary fecal samples from previously infected adult T. p. navujoensis. The mouse was sacrificed on the sixth day and 20 slides were prepared from the excised muscles. The results shown in Table II revealed 7 type b sphaeromastigotes in fission and 3 S and 18 C form trypomastigotes. All parasites were in progressive development. Of the 542 parasites seen, 91, including 2 in macrophages, were unclassified due to breakage, loose flagella, or unclear differentiation. Another Mus received inocuI,ations similar to the mouse above from a 0.1 ml pool
158
SHERWIN
F.
WOOD
TABLE Progress
in the Discowry
of Trypanosoma
Location Murray Canyon” Eat,on Canyon” Trimmer Springs O’Neals Griffith Parka Plymouth” Fallbrook Three Rivers La Grange Tarzaua Riverside Preston Ranch Knowles Placerville -4lpine Pauma Valley Oroville Kernville Westwood Sherman Oaks Itoscomare Canyon hlr~lholland Overcrossing~~ 16 km E of Redlands Paicinesa Juniper Hills” developmetlt,
demons1
rruzi
in Triaiomes
Arlthor
San I liego Los Angeles Fres1ro AIadera IAN
hgele,s
Amado San 1 )iego Trdare stanis1:m Los Angeles It iverside Madera Madera El Dorado San Diego San Diego Butt)e Kern I,os Angeles 1,o.s .4ngeles T,os Angeles Los Angeles San Bernardino San Benit o IAM Angeles
Jkvelopmcnt and .Jltnipcr
Paicines Paicines Rlulholland Juniper Hills Juniper Hills
.\Iasl
7 238
and 11onat. 1938 1942 1942 1942 c( al. 1943
Wood
1944
1033
1966
16 I5 S7 323 2338
II
Experimentally In/cc/cd with I’aicincs, Jsolntcs o,f Tr!ipanosomrc crr~si - ~ .__~--
igoles
Sphaerot)ype b t,ype
0 0 9
Kofoid Wood Wood Wood Wood 1 )avis
progressive development. Of the 3461 parasites seen, 739 were unclassified because of smudging on the tissue-touch slides. There were 63 parasites in macrophages. Xenodiagnosis of 228 live-trapped mammals in Juniper Hills from November 1967
in .Ilbino Alice Hills, California,
ISOl;tt.~ ‘I-
and date
Wood 19.X Wood 19.3) Wood IO30 Rycakman 1952 Wood and Wood 1964 Wood and Wood 1964 Wood and W(JOd 1964 Wood and Wood 1964 Wood and Wood 1064 Wood atld Wood 1964 Wood and Wood 1964 Wood and Wood 1964 Wood and Wood 1964 Wood alld Wood 1964 Wood and W;ood 1964 Sjogretr alrtl Ryckman 1 Y6.i I Wi!)
TABLE Tissue
in California
rated.
of six voluntary fecal samples from adult T. p. navajoensis. Ten slides were prepared from the right and nine from the left gastrocnemius muscles on the 12th day after inoculation (Table II). Fiftyseven of the type b sphaeromastigotes were in fission and all parasites were in
Com.parativc
and Mammals
c0llnt.y ~___
~-~.
~1Amastigote
I
Epi-
Typo-
c 7
0
0
x
0
0
62
8
7
100
0
21 0
84
42
Tisslle film area il, mm2
;Il7tlhollan~l
L)R> sacxrificed
No. of micro. slides
5th 6th 6th
IS 12 14
7792
6th
20
7417
12th
I9
4136 3463 5650
NEW
FOCI
OF
through December 1973, involved 814 triatomes (592 T. p. navajoensis and 222 T. p. protracta) with I5 positive 5th instar T. p. nauajoensis, 11 in 1971, and 4 in 1973. Twelve rodents, or 5.3%, were positive for Trypanosoma cruzi. There were 11 Peromyscus truei montipinoris (3 adult males, 4 adult females, 4 immature females) and one adult female Neotoma fuscipes mucrotis harboring Chagas’ zoonosis. Locations of capture for 228 rodents centered around a triangle of home-garage-canyon habitats approximately 300 m on a side, the garage downhill and NW and the canyon uphill and SW of the residence. Of the 12 infected rodents, one Neotoma and six Peromyscus were live-trapped along the south side of the home. Two Peromyscus were caught in a water heater cabinet attached to the south wall, two inside the garage and one in the nearby canyon near wood rat houses and a dry streambed. Although three male Peromyscus (1 P. truei, 2 P. maniculatus) were trapped inside the home, none was positive for Chagas’ trypanosome. Thirteen rodents from nearby locations, negative by xenodiagnosis, were captured within I.2 km of the primary location. Trypanosoma peromysci is the most common blood parasite in Juniper Hills native mice. Two female P. truei, one adult captured on 3 October 1971, and one immature on 18 February 1973, harbored both T. peromysci and T. cruzi at the time sampled. Total yearly xenodisagnoses for T. cruzi numbered 7-48-5-12-84-44 and 28 for native rodents from 1967 through 1973 in Juniper Hills. A summary of xenodiagnoses by quarters revealed 33 (3 positive) for Winter (December, January, February), 73 (all negative) Spring (March, April May), 56 (8 positive) Summer (June, July, August) and 66 (1 positive) Fall (September, October, November). There were 152 Peromyscus truei (77 males, 75 females) and 19 Neotoma fuscipes (3 males, 16 females)
CHAGAS’
159
DISEASE
trapped with 11, or 7.2%, of the former and 1 or 5.2%, of the latter positive by xenodiagnosis for T. cruzi. DISCUSSION
In previous attemps to demonstrate carriers of Chagas’ zoonosis nearer sea level, as in Griffith Park, Los Angeles, the temperature stress of winter appeared to trigger more positive findings (Wood and Wood 1967). In Juniper Hills at 1663 m (4800 ft ) where eight positives were captured during a populsation irruption during the summer of 1971, food availability may be more important, Hunger stress during the population irruption may have resulted in more naturally infected Triatoma being consumed, thus explaining the higher number of positives at this location. Both Peromyscus and Neotoma will eat Triatoma in the laboratory, and probably also in their native habitats. I believe that the prediction of Kofoid and Donat (1933) that Chagas’ zoonosis is widespread in California wherever Neotoma fuscipes and naturaIIy infected Triatoma protracta occur is correct. For 40 yr, Chagas’ zoonosis was known in California coastal localities ‘and bordering the inland San Joaquin Valley. The present report is the first demonstration of its presence on the eastern edge of the San Gabriel Mountains in southern California where several hundred miles of desert separate it from the nearest naturally infected vectors demonstrated in Arizona and Utah. This report from Juniper Hills constitutes the 25th location for recovery of infected insect vectors and the seventh demonstration of intramuscular development of Trypanosoma crud Chagas for California (Table I). Uninfected CC1 albino Mus frequently developed tumors similar to mammary gland carcinomas (Snell Fig. 88). None of these tumors was seen in any of the mice infected with the California isolates
SHERWIN
160
of T. cruxi even though their weights at death were often double or more the weight at time of inoculation. The significance of this is yet to be investigated. REFERENCES
BRUMPT, E. 1914. Le xknodiagnostic. Application au diagnostic de quelques infections parasitaires et en particulier a la trypanosomose de Chagas. Bulletin de la Soci&te’ de Pathologie Erotique 7, 706-710. DAVIS, D. J. 1943. Infection in monkeys with strains of Trypanosoma cruzi isolated in the United States. Public Health Reports, United States Public Heal& Service 58, 775-777. FRAZIER, C. A. 1969. Insect Allergy. W. H. Green, Inc., St. Louis, Missouri. HOARE, C. A., 1972. The Trypanosomes of Mammals. F. A. Davis Co., Philadelphia, Pennsylvania. KOFOID, C. A., AND F. DOXAT. 1933. South American trypanosomiasis of the human type-occurrence in mammals in the United States. California and Western Medicine 38, 245-249. MEHRINGER, P. J., JR., AND S. F. WOOD. 1958. A resampling of wood rat houses and human habitations in Griffith Park, Los Angeles, for Triatoma protracta and Trypanosoma cruzi. Bulletin of the Southern CaZifornia Academy of Sciences 57, 39-46. SJOGREN, R. D., AND R. E. RYCKMAN. 1966.
F.
WOOD
Epizootiology of Trypanosoma cruzi in southwestern North America. VIII. Nocturnal flights of Triatoma protracta (Uhler) as indicated by collections at black light traps ( Hemiptera: Reduviidae; Triatominae) Journal of Medical Entomology 3, 81-92. SNHLL, G. D. (ed). 1941. Biology of the Laborutory Mouse. Dover Publications, Inc., New York. WOOD, F. D. 1934. Experimental studies on Trypanosoma cruzi in California. Proceedings of the Society for Experimental Biology and Medicine 32, 61-62. \vOOD, S. F. 1937. Cytological variations in the blood and blood-forming organs of whitefooted mice experimentally infected with Trypanosoma cruzi. Vniver.yity of Californ’a Publications in Zoology 41, 389-418. WOOD, S. F. 1941. New localities for Trypanosoma cruzi Chagas in southwestern United States. American Journal of Hygiene 34, l-13. WOOD, S. F. 1953. Hematologic differentiation of the intramuscular developmental forms of Trypanosoma cruzi Chagas. American Journal of Tropical Medicine and Hygiene 2, 1015-1035. WOOD, S. F., AND F. 1). Woou. 1964. New locations for Chagas’ trypanosome in California. Bulletin of the Southern California Academy of Sciences 62, 104-111. WOOD, S. F., AND F. D. WOOD. 1967. Ecological relationships of Triatoma p. protracta (Uhler) in Griffith Park, Los Angeles, Calif. Pacific Insects 9, 537-550.
