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Journal of Experimental Medicine

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ETIOLOGY OF OROYA FEVER.
I I . VIABILITY OF BARTONELLA BACILLIFORMIS IN CITLTURES AND
IN TH~ PRESERVED BLOOD AND AN EXCISEDNODULE OF MACACUS RHESUS.
BX HIDEYO NOGTJCHI, M.D.

(From the Laboratories of The Rockefeller Institute for Medical Researck.)(Received for publication, June 4, 1926.)

Pure cultures of Bartonella bacilliformis were first obtained from aspecimen of human blood in citrate solution, transported from Lima,Peru, to New York City in the refrigerator. I The first successful
cultures were made on October 3, and the second on October 20, 1925,
the blood being 28 and 43 days old, respectively, when used for thepurpose of cultivation. The fact that pure cultures were obtained on
both occasions indicated a surprising ability of Bartonella bacilliformis
to exist under modified conditions and suggested experiments on theviability of the organism.

Viability of Bartonella bacilliformis in Infective Blood.
Table I shows the results of viability tests made with citrated bloodof infected monkeys (M. rhesus) kept constantly at 4°C. All of thefifteen specimens, which were derived from seven monkeys infectedwith cultures of Bartonella bacilliformis or monkey passage strains,yielded cultures of the organism when tested after periods of refrigeration varying from 24 to 152 days following withdrawal from the animals. In most instances the number of organisms was considerably
reduced, as is evident from the fact that none of the preserved specimens yielded growth in dilutions higher than 1:10, whereas the titerin some instances had been 1 : 100,000 at the time of withdrawal.
In testing the effect of room temperature upon similar material,
two specimens of citrated blood, derived from M. rhesus 85 and M.
x Noguchi, H., and Battistini, T. S., Y. Exp. Med., 1926, xliii, 851.
2 Noguchi, H., J. Exp. Med., 1926, xliv (in press).
533

534

ETIOLOGY OF OROYA :FEVER.

IIrhesus 12, 8 were employed (Table II). T h e temperature of the labor a t o r y where the samples of blood were kept varied from 15 ° to 22°C.,
and the tall test-tubes serving as containers were stoppered with cottonplugs and were not protected from the light. T h e r e was still a traceof what appeared to be hemoglobin in the s u p e r n a t a n t portion of theplasma after 45 days, but the general tone had become brownish

TABLE I.
Viability Tests of Bartonella bacilliformis in Citrated Blood Kept at 4°C.
Source of bloodof test
Date of withdrawal onResult
Jan. 29, 1926

Rhesus 1

Nov. 11, 1925
Dec. 8, "" 18, "
Jan. 4, 1926

"~2cc

"

3~ccc

"

4"
~5ic"

6"

7"

8

Days after
withdrawal

Result of teston Apr. 12,1926w

Days after
withdrawal

7932
4225+-.1"+

152
104
114
97978674
114
110
89
104++++++"

25, "
6, "
Dec. 18, 1925
" 22, "
Jan. 12, 1926
Dee. 28, 1925
Jan. 4, 1926++Feb.

Dec. 18, 1925+++++++

25
24
42

TABLE II.
Viability of Bartonella bacilliformis in Citraled Blood Kept at Room Temperature.
Source of blood

Rhesus 8"12Date of withdrawal

Feb. 3, 1925

Titer of freshblood

Result of teston Mar. 20,1926 (after 45days)

Titer of blood on
Apr. 12, 1926 (after67 days)

1:10
1:10++> 1:10,000,000>1:10,0o0,000

when examination was m a d e 67 days after withdrawal of the blood.
As the table shows, the titer of the preserved blood specimens was atleast 1 : 10,000,000, whereas the titer of the fresh blood in each instance
had been only 1 : 10, t h a t is, multiplication had taken place at room
temperature. Film preparations made from the specimens showed
8 Nogucln, H., J. Exp. Med., 1926, xliv (in press).

HIDE¥0 NOGUCm535

the organisms to be in small clumps scattered among the corpuscles.
There was no special tendency to localization within or around theerythrocytes, such as is evident in the blood of human beings suffering from Oroya fever.

Viability of Bartonella bacilliformis in the Nodule from an Animal
Experimentally Infected.
Bartonella badUiformis as it occurs in the local lesions of infectedanimals is exclusively an intracellular parasite, being found usuallyin the cytoplasm, occasionally within the nuclei, of proliferating endothelial cells (dasmatocytes). In actively growing nodules the numberof microorganisms is very large.
TABLE III.

Viability of Bartonella bacilliformis in the Excised Nodule and Emulsion at 4°C. andat Room Temperature.
Unground tissueEmulsion

Nodule excised on Mar. 1, 1926
Mar. 7 ( 7 d a y s ) .
" 14 (14 " )" 28 (28 " )Apr.
"4(3525 (56" )" )4°C.

Room4°C.

Room++++++++++++++++The suspension made by triturating with citrate solution a portionof the large subcutaneous nodule that developed on the abdominal
wall of M. rhesus 14~ following the injection of a mixture of culture
and passage strain yielded a culture of BartoneUa bacilliformis in a dilution of 1:100,000. This material was used for determining the viability of the organism in the tissue of the excised nodule. The results,
which are recorded in Table III, indicate that Bartonella bacilliformis
survives in an untriturated piece of excised nodule or in the suspension of the tissue for more than 56 days at 4°C. At room temperature,on the other hand, the organisms had died out after a period of 35days, though growth had been obtained from the suspension after 28days. The autolysis of tissue taking place at the higher temperature
may have been detrimental to the organism, or there may have been

536

ETIOLOGYOF

OROYA FEVER. II

an exhaustion of nutrient substances. It was found, however, thatthe addition, at the beginning of the experiment, of about 10 per centof fresh rabbit or horse serum and a truce of hemoglobin to the tubes
containing the suspension not only prevented the death of the organism but actually induced growth.

Viability of Bartonella bacilliformis in Cultures.
There are two culture media on which BartoneUa baciIliformis
grows well, 1 the so called leptospira medium and blood agar slants.
The hydrogen ion concentration most suitable for growth is pH 7.8
to 8, and it is advisable to adjust the medium to this reaction. It has
been shown also, through the cooperation of Dr. J. H. Bauer, that theT A B L E IV.

Viability of Bartonella bacilliformis in Culture on Leptospira Medium.
No. of tube--Temperature

No. of days

Result oftransplant

Result ofNo. of tube

TemperatureNo. of days

transplant

93737373737373737373747
47
5O
606265+++---V---c.

12345678: 25252525
25
25
25
25

5357
53

95
120+lO
111213141516+

substitution of Huntoon's hormone broth or Meyer's peptic digest
broth for the ordinary meat infusion broth as the basis of these media
has a decidedly favorable influence upon the growth of the organism.
One of the peculiarities of Bartonella bacilliformis in culture is thatit soon reaches the limit of growth at 37°C., though multiplicationcontinues steadily at 25°C. On leptospira medium the organisms
remain viable longer than on blood agar slants, growth progressing fora month or more in the former case, while in the latter the maximum
seems to be reached in about 14 days at 25°C., and then only when theevaporation of the medium is retarded by soaking the cotton plugs
with paraffin or closing the tubes with rubber stoppers. As a rule
Bartonella bacilliformis remains motile on blood agar for 10 days, occa-HIDE¥O

N0 GUCHI537

sionally as long as 14 days, but it is usually non-motile when grown on
the semisolid medium. Microscopic examination alone is not sufficient for the detection of viability, therefore; cultural tests must also
be made.
Many culture tubes having been preserved from the time of theisolation of Bartonella bacilliformis in October, 1925, it was possible to
make a series of viability tests, the results of which are recorded inTable IV. In order to retard evaporation, the cultures on leptospiramedium intended for preservation were covered with a layer 2 cm. deepof sterile paraffin oil.
As the table shows, a culture of Bartonella bacilliformis on leptospira medium at 25°C. remained transferable for a period of at least
120 days, while tubes kept at 37°C. longer than 50 days no longer gave
growths on new medium. Culture tubes removed to a refrigerator at
4°C. after their maximum growth had been reached (28 days at 25°C.)
were found to be still viable at the end of 4 months.
The viability of the organism on blood agar slants is very inconstant.
If the plug closing the tube is not practically impermeable the surface
quickly dries, and the organisms cease to multiply and degenerate
within a fortnight. On a slant at 25°C. containing sufficient condensation water and closed with a nearly air-tight stopper the organisms
remain motile for about 2 weeks and transferable for a month ormore. The stopper should not be made absolutely air-tight, because
Bartonella bacilliformis does not grow in the absence of oxygen. 1
As already stated, a slant culture kept at 37°C. dies out in about 10days.
S~AR¥,Fifteen specimens of citrated blood from seven monkeys infectedwith Bartonella bacilliformis were kept at 4°C. for periods of 24 to152 days, and at the end of each period were tested for viability by thecultural method. All yielded cultures, although there was a considerable reduction in the number of living organisms, as shown bytitration.
Two specimens of citrated blood, from infected monkeys, which had
been kept for 45 days at room temperature yielded growth when adrop of each was inoculated into leptospira medium; while after 67

538

ETIOLOGY OF OROYA FEVER.

IIdays at the same temperature 0.1 cc. of a 1:10,000,000 dilution ofeach specimen was sufficient to yield growth. Since the original titerof the blood had been only 1:10, it is evident that Bartonella bacilliformis had multiplied considerably under the conditions. Smearsof the specimens showed dumps of organisms among the corpuscles
but no intracorpuscular multiplication.
Bartonella bacilliformis survived in the excised nodule from amonkey for at least 56 days at 4°C., and for 28 days at room temperature, when a piece of the tissue was covered with citrate-saline solution
or ground up in it. The death of Bartonella bacilliformis at roomtemperature under these conditions m a y be due to the effect of autolysis of the tissue or to a lack of nutrient substances. The suspension
alone is not a suitable culture medium, but a trace of hemoglobin
and about 10 per cent of fresh horse or rabbit serum make it a favorable
one.
The viability of Bartonella bacilliformis was tested in cultures
kept at 25 °, 37 °, and 4°C. for varying periods. At 25°C. cultures on
leptospira medium remained transferable after 120 days, and whenplaced in the refrigerator at the time of maximum growth (after 28days at 25°C.) they were still viable at the end of 4 months. Theviability of cultures on blood agar slants depends to a considerable
extent upon the care with which the surface of the medium is protectedfrom drying. Under favorable conditions and at 25°C. the organisms
remain motile for about 2 weeks and transferable for a month orlonger. Cultures on either of these media die out after 50 days at37°C.
The data presented suggest that it may be fruitful to make a study
by cultural methods of pathological material brought from distantparts of the world, even when many days or weeks have elapsed
since it was procured.