The Tetragonococcus or Tetracoccus versatilis and yellow fever

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					                            THE




                             OK




           -VERSATILIS"hKD
"TETRACOCCUS




                            ET




             CHdRLES        FISLAY,       M.D.




  PP,IXTED    BT   OLIYER   AKD   BOPD,   EDISBURGH.
                                 L
      AIL! PRESIDEXTASD &WI’ li;JIEX,--The ” IIicrococcus tetragenus
  versatilis ” of Dr Sternberg is, as you are aware, the same as the
  one which Dr Delgado and I denominated ” 31. tetragenus febris
  flava2’” when we discovered it in lSSG-Si in our cultures from the
  blood, blister serum, and secretions of yellow fever patients, as well
  as in those from the heads and proboscis of mosquitoes which llad
  been made to sting such patients. At the risk of having it said that
  the ver,;atility of our microbe reveals itself even in the variety of
  its names, I propose that it be given that of I‘ Tetragonococcus,” or
  simply ” Tetracoecus versatilis,” not only for the sake of brevity,
  but    also  because, haring in my recent experiments proved its
 pnthogenicity, I consider that it#snew name harmonizes bet,t.erwith
 those of ot,her knoxn pathogenic microbes, such as the staphylo-
 cocci, streptococci, and diplococci.
     The following is the description of the tetracoccus give11 by DL’
 Sternberg, under the name of 51. letragenns versatilis (E~pwt OIL
 the Etidqy                                         p.      a
                  amL Prerei2tion of Yellow _F~cEI+, 165, \?‘ shington,
 lS90) :-“The       characteristic mode of grouping is shown in Fig. 3,
 Pl. III.     Some bacteriologists would, perhaps, be disposed to place
 it among the sarcin2, but I have never observed any evidence of
division in a third plane, forming packets of eight or more
elernellts, such as are characteristic of’ this genus. I have called
it ’ versatilis,’ because it is very versatile both in the grouping of
the elements and in their dimensions. In the same culture very
wide digerences in size are observed, and at diRerent times and in
different media these variations are very noticeable. The grouping
also varies greatly ; sometimes the greater number of the elements
                                    .T
are arranged in t&ads, or in pail,, in which the large oval elements
are upon the- point of dividing transversely to the line by \vhich
the binary division of a single element is marked. But often tllere
are irregular groups of three or more elements, or there may be a
chain of tetrads which remain attached one to the other.
      In
    Cc a.gar stick cultures, a rather thick and viscid yellow mass is
formed on the surface, about the point of puncture; and in the
                                            4

course of a week or ten days, at a temperature of 20” to 25” C )
this ester& over the entire surface.          The colonr varies frolu
cream yellow to lemon yellow. The gron-tNh          upon potato is siulilal
to that, upon agar.    In stick cultures in mesh-peptone-,nelatine, the
(relatine is liquefied rather slowly near the surface, forming a deep
n
cup-s?laped cavity, as she-ivn in Fig. 7, 1’ . III.
                                            1           Colonies in gela-
tirre roll tubes are at first, pale yeliow, and later, lemon yellow in
colour ; they are opaque arid spherical, and do not usually cause
the liquefaction of the gelatine for several days.
    ” The micro-organisnr has no proper movements, and is a$,obie,
no growth occurring iu au atmosphere of hydrogen.               It is ?lot
pcrthogei~ic            07'
               for mbbilS yi!iilt?Ct-pip”
                                                                  s
     Before taking issue at this last assertion of Dr Sternberg’ , I beg
 to be allowed to point out some details in “ technique,” which may
 be useful to those who desire to repeat my experiments. Young
 colonies in agar seldom present characteristic tetrads; these are
  easily obtained, however, by planting the same colony into broth
 or some other liquid medium. In order to identify our microbe, it
 is not, hovvever, necessary to find it in tetrads ; all that is required
 is to determine the characteristic segmentation of the protoplasm
 of each individual element.     It happens that the same elements
 which with an ordinary magnifying power appear simple cocci, when
 viewed with the strong oil immersion objectives ($%-A of Zeiss
 or Leitz) are found to consist of txo elongated semilunar masses,
 which are well coloureci by fuchsin or methylene blue. At the
 first stages of this segmentation we have the appearance of a semi-
 transparent liue or cleft extending along one of the diameters of
 the coccus, which a co,1~02i*e has aptly compared to a coffee-bean
 \vit.h the furrow dividing its plane surface. As the germination
 proceeds, the cleft, or rather the pale-colonred intermediate
substance, widens, and the two segments become rounded or
lengthened, one or both undergoing, in the meantime, a new
 segmentation in a direction perpendicular to the primary division.
A group is thus formed consisting of four cocci of round, oval, or
 uusymmetrical out.liiie, constituting a. square or tetragon. In some
preparations the group is evidently enclosed in a distinct capsule,
in the interior of which the growth and subdivision of the elements
 may continue for a while ; but it more frequently happens tllat
both the tetrads and the “ coffee beans,” arranged in chaius or
otherriise, are found naked. It is also worthy of notice that the
cleaving of the prot.oplasm is observed in the small as well as in
the large cocci which are seen in the same preparatSion. Witlr
regard to the grouping of the elements, there is another rather
characteristic arrangement which I have often found in the early
stag-esof development, both in solid and liquid media. It consists
in a more or less hexagonal plaque, in the centre of which is seen
a, well-stained coccus,surrounded by a light-coloured zone, dotted
along its margin by five, six, or seven cocci like the centre one1their
                                    5

   protoplasm showing or not. signs of segmentation. The macroscopic
   characters and the size of the colonies of the tetracoccns, as well as
  its chrornogenous properties, awevery variable,-even      more so than
  Dr Stemberg has pointed out: its colow, for instance, may &try
  from a pure w1Gt.eto a_canary yelIo\~.-, viith the intermediate Cuts
  already mentioned by that, author. After several days’ gro&h in
  tubes of brotll, ti;e tetracoecus produces a viscous substance, whicll
  may be drawn out in elastic filamentS,        and is deposited at t’ eh
  bottom of the tube ; but on moving the latter aro~uld its axis the
  viscid mass rises like a dirty yellow string, with its more or less
  wide base attached to the bottom, while the elongated conical
 portion seen in the liquid ends in a point or fringe,--the whole
 presentiug a rather tpyical appearance.
     The cultures of the tetracoecus in broth emit a peculiar
 characteristic odour. With regard to its resistance to beat, I have
 been able to sterilize a culture, a few clays old, by immersion of
 the test tube in water at 65” C. during half an hour ; but this
 pros-ed insufficient to sterilize a balon containing au older culture.
 I therefore suspect that some of the elements are more resisting
 than others ; there may be spores. The most favourable tempera-
 ture for the growth of the microbe is betxeen 35” and 40” C.
    ~111order to obtain the tetracoccus I-ersatilis, I usually employ
 one of the two followtig methods :-One consists in the introcluc-
 tion into a tube of peptonized broth, to n-hich 5 per cent. of glycehe
 has been added, of a droplet of blood from the finger of a yellow
 fever patient at the beginning of the second paroxysm (between
 the third and the fifth day). The following precautions must,
 however, be observed in order to secnre the germination of the
 microbe :-1st.                    -.C-
                   A patiellt n2ubU be chosen whose blood be not
 already sterilized by the use of internal antiseptics, such as the
 benzo-naphthol, especially if the wine be alread~sn~oky ; and, 2m7,
strong germicides, such as mercuric bichloride or carbolic acid,
must, be avoided in the washing of the site of puncture. I was11
 the pulp of the ring finger with soap and water, then xrith pure
water, and finally with absolute alcohol, keeping the skin covered
with cot.ton soaked in alcohol while I prepare the needle, pipette:
and tubes of broth. Eefure pultcturing t,he skin, I dry it with
sterilized cotton. The tubes thus pkmted do not always germinate ;
but, with these precautions, I leave this year obtained the tetra-
coccus in one or both of the two tubes of glycerine broth that
I usually inoculate.
    The other method cousists in applying to a patient (under the
conditions a1waciy mentioned) a mosquito of the dinmal species,
with white rings on its legs (Culex Illoscl”ito-~obiueau                )
                                                              Desvoicl:‘ .
At the end of two or more days the insect is asphyxiated by means
f,f a few drops of ether, and clecapitated-its head and proboscis
being immediately introduced, with all possible cleanliums, iuto a
tube of sterilized broth, which is kept at a temperature of 37” C,
                                        6

    The cultures generally germinate at the end of twenty-four              or
    forty-eight hours ; at times the broth remains sterile.



         111January of this year I determined to ascertain whether D1
                 s
      Sternberg’ assertion that the “ ‘ Tetragenus versatilis ’ is nut
      pathogenic for guinea-pig: or rabbits ” was to be accepted in its
      absolute sense. Ny previous experiments with 1 or 2 c. c. of lix-e
      cultures injected under the skin or into the peritoneum xere in
      accordance with the statement of the distinguished Xorth American
  I bacteriologist ; and, last year, I had not observed any pathogenons
      effects upon a rabbit. after injectin, 0 1 c. c. of live culture into its
     jugular vein. The tetracoccus used in this instance, however, had
      been obtained from blister serum of an immune subject haying
     suffered a severe attack of rellow fever a few weeks before ; it
      was possible, therefore, that the germ, under such circunlstances,
     should have lost any virulence which it might originally have
     possessed,in which case a variability in its pathogenic properties
     would have to be added to the other versatile characters of our
     microbe.
         TPtlxCoccwex &S~:!4ito.-In     the first series of my new esperi-
     ments I employed the tetracoccns obtained from contaminated
     mosquitoes.
         On January 17 I applied one of t,hese insects to a yellow fever
    patient (a fatal case), who occupied bed Ko. 10 in the second x-arc1
    of the Xilitary    Hospital ; on January 19 I made the same mcs-
     quito sting an unacclimated person ~110 demanded a preventive
    inoculation ; on January 22 I killed the insect with a little
     ether, and introduced its head and proboscis into a tube of
     sterilized broth, wlkh Dr Davalos undertook to keep at 37” C.
    in the laboratory of the “ Cronica Medico-Quirugica de la Habana.”
     In the afternoon of January 23 the broth was slightly turbicl,
    and the following day the presence of the ” tetrncoccus versatilis ”
    was verified. It gave yellow colonies in agar and slowly liquefied
    gelatine.      Ko other micro-organism cleveloped in the original
    broth.
        On the 26th of January Dr Davalos planted a balon of pepto-
    nized broth with pure tetracoccus colonies, placing the balon in the
    hot chamber. On Alarch 11, having previously ascertained the
    purity of the culture, it was passeclthrough a Kitasato filter; we
    thus obtained several tubes of transparent tosinee, of a mahogany
    colour, and wit.11the characteristic odour of our microbe. These
    tosines, which we held to he sterile, contained, however, some live
    tetracoccus germs, as m-e were able to determine at a later period.
f
        Guinea-pigs injectBed4h       10 to 20 c. c. of this filtered culture
I slioITed some thermic reaction, but the subsequent symptoms were
    not characteristic, and they all recovered. The result was different,
                                     9

    however, with tlte young rabbits, weighing less than 1000 gtmmttes,
     which I inoculated ott Xarch 14 and 23 Kit11 tile same culture.
    Thefirst rabljit, a male (like all the others), weighed S40 gratnmes,
    and had been brought from the country two clays before. I in-
    jected into its peritoneal cavity 5 c. e. of the filtered culture, and
    anotlter 5 c. c. under the ski11 01 the abdomen. The secotld day its
    temperature had risen to 40”$ C. ; the third to 40” ; the fourth
                                       *
    to 39O.S; the fifth it fell to 3S’ S ; on the sixth a second paroxysm
    set itt with 41’ ; the seventh day the temperature oscillated between
       .
    JO’ 7 and Al”*1 ; on the eighth between AOom3        and 40”.7 ; on the
    ninth clay there was a defersescence to 38O.3, the rabbit dying S$
    clays after tile inoculation, with a loss of a fourth of its weight. A
    supeA&J autopsy shoxved itttense venous congestiou, the right
    heart being distended with blood, injection of’ the rnesenteric and
    peritoneal vessels, congestion of the liver and kidneys ; the bladder
   cotitaiued a fair amount of urine, with a trace of albutnen. At the
   site of the inoculation there was neither redness, nor suppuration,
   nor oxiema. Hardened in alcohol, sections of the liver and kidneys
   showed small hemorrhagic infarcti, small punctuate hxmorrltat,aes
   being visible in the regions of the glotneruli, and streaks of blood
   in the pelvis of the kidneys. X0 cultures were made.
      Tlte second rabbit weighed 1000 gratnmes before inoculation on
  AIaxh 23.         I iujeeted 10 c. c. of the fikered culture into the
  peritoneal cavity, and 9 c. c. under the skin. The temperature was
   ttot tdiell  during the first eighteen hours. On the second day it
  oscillated between 39” and 39’ 4 ; tlte third between 3S”*5 and
                                        ~
  39”.4; the fourth between 39’        5 and 39”*7 ; the fifth it kept at
  38”% ; the sixth, in the morning it fell to 3S”*2, the second paroxysm
  settittg in in the afternoon with 39”.S ; the seventh day the tem-
  perature ranged front 3ZP.6 to :j9”% ; the eighth between 3S”.S and
  39”*6 ; the ninth it was at 39”% in the morning, but fell to 3s” in
  the afternoon, the animal dyin g at S A.X. the folloxvittg day--S2
 clays after the inoculation.       The autopsy gave the same findings as
 in the first rabbit, with less venous stasis and no albumen itt the
 urine. Xo cultures were made.
      On Xarch 27 Dr Davalos planted a second balon of yepton-
 ized broth with colonies of the yellow tetracoccus which had served
 to verify the purity of the previous culture. At the end of eighteen
                                  9
 days we passed the contc?nt, through the Kitasato filter, with the
 same result as before; for we Eere able to ascertain, a few clays
later, that some live germs had passed through the filter. On the
 15th of April I inoculated txo adult rabbits; one weighed 13%
and the other EGO gramtnes. The first received 20 c. c. of
filtered culture in the peritoneal cavity, the other had the same
amount injected under its skin. Tile acute itttosication observed
in the ~oittig rabbits did not occur, but in its place a state of
chronic tnarasritus lx-asproduced, with progressive loss of flesh till
the weight was twluced by one-third, death occurrittg at the end
                                   s
offive  or six w eeks, when no longer under strict surveillnnce. The
one itlocuiatecl subcutaneously was more severely affected tllan the
0  ther.
     These txo inoculations convinced me that, like other n-ell-studied
micrococci (E.g., the staphglococcus pyogenes), the tetracoccus is
specially pa_thogenic for young rabbits, and much less for full-
groan ones. Hence in my other inoculations I only used rabbits
betweeu 500-1000 gramlmes \I-eight, males, of the ordinary race of
this country, recen:Iy brought iuto tolx-n, and either white or black,
like the tlvo first in the series. I dso suppressed the preliininary
filtration, in view of the w~reliability of this operat,ioll for excluding
live gerlns.
     I shall not xearg you x-it11 a deta.iled xcount of all the tn-else
young rabbits inoculated x-iti1 the “ Tetragellus es mosquito,” the
clinical results of their observation being given in the acco~npany-
i:lg Chart, nuder the letters A and L. These rabbits were inoculated
with successive generations of the germs originally obtained front
the mosquito which on January 17 llstcl stung the Tellow fever
patient at the Military Xospital, although in rabbits D, E, F, G,
H the tetracoccus injected had passed through rabbits C, D, or E,
having been recovered from these with blood ccllectecl from the
 live animals’ ears, or from the right auricle of the heart after death.
 I inoculated cultures of different -ages and in variable closes,iu
 order to appreciate 110~ those cox~ilt~ons    might modify the ensuing
 pathogenic effects.
     Xy txo first rabbits appear in the Chart with letters d and E ;
 their weights were only noted 2-t the begiuniug and end ; all the
 others x-ere n-eighecl each day shortly before feeding time, in order
 to obtain their minimum daily weight.            The usefulness of the
  weight tracings for t.he fuller comprehwsion of the clinical history
 is best shoxn in the case of rabbit C ; its weight ~-as not aflt’ ctede
  (luring the first fort,night,, but from this date forward the loss of
   l
 i’ esh progressively increased until it reached one-fourth of its
  primitive weight, this loss coinciding with the acute seven days’
  fever, similar to that of d and E, which preceded its death. The
  first fifteen clans that followed the inoculation must therefore be
  considered as a true incubation.         This is the ody one that has
  presented such a prolonged incubation, perhaps ii1 solne measure
  due to the cixumstance that the tetraeoccns had in this case been
  developed in broth to which 1 per cent,. of caue sugar had been
  ddecl ; for there ]~a,5also sugar in the old cultures injected into
  rabbits I, K, L, xho~e tracing suggests the occurrence of an incttba-
  tiou peiiocl of two or three da!-5
      Cultures macle from blood of the ear during the sickness of
  rabbits C, D, and E all gave the tetracoccus versatilis, either pure
  or contaminated, the disinfection of the skin having heen insdi-
  cient. The microbe was, however, carefully isolatd before l;eillg
  inoculated into D, E, and I?. Esceptillg ill rabbits X al:cl 33,
  cultures were tiade from the heart blood of all the rest beloqin,a
  to this series, the tetracoccus being recovered each time, either
  alone (in C, D, E, J, and L) or associated with some bacillus, not
  alwa,ys the same (iu I?, G, H, I, and Ii>. Remarkable difYerences
  were observed in the colour of the colonlea of the recovered tetra-
  coccus, differing at tirues in those that appeared in one same agar
 trtbe.
     AI1 the rabbits inoculated with the mosquito tehacoccus died in
 consequence of tLmt inoculation.      In most of them there was a rise
  of temperature between 40’ and 41” C. in the first six to twelve
  hours, this immediate reaction marking in some cases the com-
  mencement of the infectious fever, whilst in others several days of
 incubatiou were interposed. The characteristic febrile movement
 consisted of two paroxysms separated by a remission of one or two
 clays, during which the temperature fell lower than in the preced-
 ing or following clays. Death took place, as a rule, between the
 fifth and ninth day of the attack.
     Two of the rabbits (J and K> of the present series, as also three
 of the nest @I, Q, R), whose symptoms I was able to watch
 during the last hours before their cleath, have presentecl a very
 remarkable set of s~mptonis, El&h I find myself justified in
 describing at length ; all the more so, as I suspect that several of
 the other animals died in a similar manner.
     Beyond a certain amount of depression when the fever was high,
 and the great loss of flesh, the animals dicl not appear to be in
 danger of death until the qmptoms which I refer to began to show
 themselves. They had no diarrhcea, no paralysis nor ciyspucea;
 notwithstanding their comparative xwakuess they moved about,
 skipping and taking their food ; but a few hours before death the
 rabbit forsakes its companions and food, clraws itself up close to a
 wall or corner, doubled up, with its head drooping, and seems
 drowq-, but is easily roused by any sound or contact ; sometimes
 it shop-s signs of hallucinations by certain brusque nlo\-ements, by
 a sudden jump or run across the stable without apparent motive ;
                                                         o
 at times t,here is some emission of urine or faxes. n’ w and then
 contraction of the frontal muscles or of the elevators of the licls,
 some oscillations of the eyeball, and convulsive twitches of the body
 and extremities, may be seen. These disturbances may be uni-
 lateral, or more marked on one side than on the other. The ears
                                  or
 are likewise nlovecl one >~rag t,he other by spasmodic twitches.
 Certain muscles may momentarily give way, but there is no per-
 manent paralpsis nor contraction. Then, suddenly, the limbs of
 one side or of both relax, and the animal drops either 011 its side
or on its stomach, the legs remaiGng for a while inert, alld a real
eclaniptic seizure follows. The head is drawn back ; the legs
rigiclly estended ; the upper lid clrawn up, at first ; then clonic
convulsions, with fomwd and backward nioverneuts of the less ;
thorxic con~ul~ious, some momentary winking or iiystNagluus,aud,
                                   10

 at times, such violent contractions that the body rolls over once or
 twice. During the seizure the lips are cyanotic, the pupils dilated,
 and there occur certain movements of the head and neck which
 remind one of hiccough or the effort of sK_allowing, and once I
 have seen the animal extend its neck, opening and shutt.ing its
 mouth t,hree or four times, as if atttempting to bite. After the first
 attacks the labbit rises on its legs, and at times, during a lxett;-
 long interval, it appears to have completely recovered, being anxious
 to eat, and moving about as if nothitlg had occurred. Later, tl:e
 seizures are repeated at shorter intervals, till, finally, 8 state of
 stupor or coma sets in, in Lx-hich one only perceives that the
 animal is not dead by the persistence of respiration. Tllis finally
 ceases,and death takes @tee, lx-ith the head thrown back and the
 legs stretched out or drawn close to the body. It1 one of the
 rabbits there had heeu dyspncea before and during the attack,
 and the autopsy showed markedly congested 1u11gs,but no
 hepatization.
    In describing at such length the death of these rabbits inoculated
 with the tetracoccus, my object has been to place beyoncl a doubt.
the eclamptic nature of their last symptoms. The intimate rela-
tion between eclampsia and uremia, on the one hand, and the fact
tllat in the great majority of fatal yellow fever casesthe immediate
cause of death is attributed to uremia, will espIain my dwelling so
nluch on this point. The examination of the cranial and meclullary
cavities would have been of interest, as well as the histological
analysis of the extirpated viscera, but I had no time to make these
investigations.


    In the month of June I determiued to obtain the tetracoccus
 directly from yellow fever patients, in order to compare its patho-
 genic effects with those of the same micro-organism obtained by
 means of the mosquito.         On June 27 I planted two tubes of
 glycerine broth with the finger blood of a patient in the fourth
 day of melano-alburninnric yellow fever, who occupied bed So. 204
              s
 of Dr Scull’ ward in the Nercedes Hospital.            Both tubes gave
pure cultures of the yellow tetracoccus. An inoculation n-it11
 16 c. c. of a six days’ culture proceeding from pure colonies of this
                                                     u
sample into rabbit 31, produced a rise of temperat’ re of 40”.7 within
nine hours, followed by a tsro daj x’ incubation ; the regular febrile
attack declaring itself on the fifth clay after the inoculation.   This
fever showed a remission on the fourth day, and the anilnal died on
the seventh or eighth (eleventh since tlleinoeulat.ioll), with dyspncea,
eelampsia, and coma. The autopq shoivecl hxmorrliages over the
heart and over the surface of the great vessels at their origin ;
haxnorrhagic infar& in the tissues of the liver, kiclneJs, and
1ungs ; hyperzmic and punctate haxnorrhages upon the gastric
mucous membrane ; venous stasis ; and the urine clrawn from the
       blaclcler was free of a1tm1nen.    Two tubes planted with heart blood I ’
       gave pure cultures of the tetracoccus.
           On July 1 I planted two tubes of glycerine broth with blood
        of a patient (U. V., female) in the fourth day of yellow fever
        (typical non-albuminuric form). She was a wet nurse. 011 the
                                                 o
       same day I introduced into an agar tu’ e some of her milk pressed
       directly from the nipple, which I had previously washed with soap
       atld water and wiLh absolute alcohol. The tubes plan ted with blood
       C~ermiiiated, giving only straw-colonred tetracoceus, and the agar
       n
       tube presented, all along the track followed by the milk, an abuud-
       ant growth of n-hite tetracoccus colonies, and no others.         Rabbit
       P, inoculated with 7& c. c. of this white tetracoccus from human
       milk, in a five days’ culture produced a reaction of 41” C. on the
                                            7s
       same day, followed by two claJ without fever, alld inr-a.sion of
       the regular attacli on the fourth day after tile inoculation.        Ih
                                                                            ‘’ e
       fever showed a remission on the third day, and defervescence on
       the eighth ; the animal continued, however, losing flesh, and died
       on the fifteentit day after the inoculation.      No autopsy nor cul- I
      tures were made.
           On July 2 I p!anted ti5r-o tube s of g1ycerine broth with fingel
      blood of a yellow fever patient (a simple albuminuric case) in the
      fourth clay of the disease, occupying bed Xo. 93 of Dr Scull’ wards
      in the Mercedes Hospital. Both tulles germinated, giving the straw-
      coloured tetracoccus. On the 6th a11d 10th of the same month
     rabbits X and 0 were i~loxlated-the          first with 9 c. c. of a forty-
     tiTo hours’ culture, atld the second with 10 c. c. of a fifty-six hours
i    culture of the tetracoccus from this -source, N showed a febrile
                        n
     reaction of 4O’ i C., followed by a two-paroxysm fever, with a
     remission below 39” C. on the sixth day, but without notable loss
     in \Jreiglit. Dc!fervescence took place on the fourteenth day; tile
     animal recovering definitely.        At the end of twenty-seven days
     the original w-eight of ScjO grammea bad increased to 9S0, beirlg
     the only one of my inoculated rabbits which did not s~~cc~~~k~l~ to
     the inoculation.     Lh
                          ‘ ’ e ot8herrabbit, 0, which had received a larger
     alnount of a more developed culture, showed a febrile reaction of
     40”.6 at the etld of seven hours, followed by a fever of the same
                  s    th
     type as S’ , wvi a. remission from the fifth to the sixth day ; but
    the loss of flesh became g     (jradually more marked, evolving into a
    chronic process which caused death at the end of twenty-three days,
                                         7
    the loss of weight being from ‘ 50 to 5i5 grammes. No autopsy
    nor cultures were made.
          Finally, on the 9th of August, I planted two tubes ivitll the
    fitlger blood of a yellow fever patient (severe albuminuric type)
    in the fourth day of the illness. Both tubes germinated, develop-
    ing pure cultures of yellow tetracoccus, more iii tensely Coloured
    and more virulent than the precediq ones. Tell c. e. of a foul
    days’ culture of this tetracoccus inoculated illto rubbits Q alld P,
    produced on the first and second daJs a klxile reactiun of 4(jG.;l
and 10' C ; Q dj ing in the first paroxysm of this fever thirty-
eight hours after i~~oeulation ; while R presented a remission on
tlie third day, and died in the second paroxysm of this fever seventy-
two hours after inoculation.     Both these rabbits presented before
death the eclamptic qmptoms described above.              The autopsy
                           a
showed h2emorrliagic int’ rcti in the liver, kidlieys, and lnugs ; there
was a trace of albumen in the urine of Q, but nolie in that of R. ’
Cultures from heart blood gave the pure straw-coloured tetracoccus.
    Tile facts developed in the present series show tile pathogeni-
city of the yellow fever tetracoccus, and its similarity or ideutitj
in kind with that of the mosquito t,etracoccus; attention being
at the same time called to the circumstauce that the various intell-
sitiea observed in its virulence were in keeping with the severity
of the disease iu the patients from whose blood or milk the germs
had been obtained.



    The greater part of niy inoculated rabbits of the mosquito series
awl sonle of the blood tetracoccus series were kept at the Labora-
 tory of tlie Cronica Xedieo-Qilirugica ; the remainder were kept
at my house, in order to exclude any influence of locality.      This
circnrnstauce enabled me to witness an esa:uple of contagion in a
rabbit of a foreign race, with a thin skin and long grey hair. It
 was 3 male, and weighed 650 grammes on being brought from the
coutltry on March 23, at which time other rabbits were being               7
o’ sex&
  u          The grey one appeared healthy, and received no inoeu-
lation, but was left in the same portion of the stable to,oether
with the inoculated rabbits. At the end of eight days it was
found   dead, with its head throFu back, as in the case of rabbits
which had died flom the inoculation ; and cultures from its heart
Llood developed pure cultures of yellow tetracoccus.
   On June 26 eleven clays had passed wiLllout there being any
rabbits at my honse. iA_ Spanisli wet nurse (E. V.) came lvitll one
of my relations to speud some time there. She had suffered no
febrile disturbance since her arrival in Cuba (November 1994), not
even during her confinement, Khicli took place in April of this year.
On Juue 29 she awoke with fever and symptoms of a probable
attack of yellow fever, uild was transferred to a large n-ell-venti-
lated roan; in dcosta Street, where I attended her during her ill-
ness. IIers was a typical case of non-albuminuric yellow fever, if
vie except a slight opalescence in a single specimen of her urine of
                                             s
the third day, on testing it Cth EaLach’ solution, and boiling.
                                                              ;
The acme of the fever during the first paroxysm was 339”.5 there
was a remission to %“.S ancl 3$“*1 between the fourth and fifth
days, a second paroxysm, with 3F.S and 39”*2, on t,lie fifth and
sixth, and defervescence to 36”*5 on the seventh. No quinine nor
                                        ll
other antipyretica had beet1 used. ‘ ’ le urine wiis scanty ; the
wn~s bled a little, and some bloody phlegm was spat up between
b

the fourth and sixth dais. 011 the third day I planted two tubes
of broth with blood from the finger, and also an agar tube with
milk from the Lreast-,as before explained. Both tubes planted Kith
blood gave the pale yellow tetracoccus, and the agar tube planted
 with illilk gave an abundant germination of white tetracoccus
cwlonies, which were inoculated, as already stated, illto rab5it P,
producir~g death in fifteen days.
                                                           e
   011 July l-1 the hUsiJaIlC1 (E. S.) of the wet nurse f’ il sick with
an attack of non-albuminuric         yellow feTer. Tile temperature
                                                          s
 curt-e, though lower; was ot’ the same tx)e as his wife’ . On July
 18 1 planted two tubes wi[h his finger blood. One of the tU\Jes
remained steriie, the other gave a pure culture of straw-coloured
tetracoccus.
   On July 30 a sister (C. V.) of the Ket nurse fell sick with a
siulilar attack J she lml bee11 staying   w:‘h
                                            t  her  since July 23. Ko
albumen ; deferveseence on the 7th of August.
   On August .5, in the morning, a female friend (C. IL) of tllis
family, who ha(_l been with them since July 20, fell ill with a
serious attack of albuminuric yellow fever, the albumeu being
specially abunclant ou the fifth, sixth, and seventll clays, having
lasted from the third uiltil the eleventh day. She had repentecl
epistasis, vomiting, without blood or black matter, during’ the
second paroqsui ; defervescence set iu on the eighth clay. Two
tubes of glycerine brotll planted from her finger blood 011the tllird
day both gave pure cultures of the yellow tetracoccus, which proved
so virulent ou being inocul:tted into rabbits Q and IL.
   On August 5, in the evening, another sister of the wet nurse,
who had been livil!g with her since July 20, was attacked with
simple nll~i~n~ii~uric yellow fever. The albumen was not very
abunclant, but lasted from the third to the ninth day ; renlissiou
occurred on the fifth, and defervescence on the seventh day ; the
meuses    were anticipated, and the g-runsbled a little.  Of tR-0 tubes
                    l
of glyceri lie Lrot’ l planted with her finger blood, one remained
sterile, while the other gave the tetracoccus in pure culture.
   In this remarkable series of five consecutive cases of yellow
fever, with or without albuminuria, I adwit that it is anything but
proved that the wet nurse acquired the disease in my house. I
must mention, however, that during the whole time that the inocu-
lated rabbits had been kept in it mosquitoes had been unusually
nbuuclant, and that the woman had declared from the first that she
llacl never been so much tormented by them since her arrival in
Cuba. It is well to keep this in mind, in view of a possible trans-
mission of the disease from animaIs to man. With regard to the
four subsequerlt cases des-eloped in the room of Acosta Street, I
do not think it can be doubted that the infection was brought
by the xet nurse, and that the o  ~ernls proceeded from her n;hich in
successive generatio; iuwcdecl the otller~.
    By comparing the result s of the above-mentioned experiments
with the comrnnnieations presented by Dr De&do and myself to
tllis learned Society ii1 the course of the last iiine years, it must
be admitted that we were not far wrong in the three conclusiolis
we came to in a paper wlikh n.as published in _Ln 2Zi~‘lciclOpedic of
                                                         i
Dr Go:lzaiez (February lS77), viz. :-
   “ 1. That the micrococcus tetragenus febris flavz is the charac-
teristic form of the Illicrobe of yellow fever.
   “ 2. That the c&x mosquito is a 1mon-n agent through which
the disease may be transxittecl.
   “ 3. That, judgin,o by our experiments (ISSYj, it is possil~le to
protect non-acclimated sulrjects against severe yellow fever by
means of our mosquito inoculations.”
   The third of these coxcluaions does not properly belong to the
subject under coilsideration, though I 1001; upon     it as faYourablJ
decided by the fact that of 012ehz~m~i& presumably susceptible
persons who have received the mosquito inoculation during the
last fourteen years, Only three have SLdJSeqLmltl~’     died  Of yellow
fever.
   111order to prose the second proposition, it would be sufficient
to demonstrate the first,-i.e. that the “ tetracoccus veraatilis” is
the germ of yellow fever ; the identity of the Iuoaquito tetixoccua
and the yellow fever tetracoccus having been sllown.
   We must now consider if our germ fulfils the conditions
demanded by Koch for the acceptance of a micro-organism as tile
cause of a disease, viz.-(I.)   That it be found in the liquids alltl
tissues of those attacked vAth the disease; (2) tllat it be isolated iu
pure cultures; (3) that the disease be reproduced by inoculation ;
(4) that the microbe be recovered from the inoculat,ed subjects.
   The first of these conditions implie s the presence of the tetra-
coccus in yellow fever patients. From the examples cited in this
paper can readily be seen the facility with which the tetracoecus
may be obtained from a tiny drop of blood drawn from the finger of
a patient, under the conditions which I have pcinted out. To this
the opponents of my former ” tekagenus ” will probably object
that my SteAlization of the skin is insufficient, and that the germ
obtained proceeds from the skin and not from the blood ; but if
this myere  true, it would Le extremely unlikely that ally pure cul-
ture of a given microbe could be thus obtained, and when the
tet,racoccusdid not develop, some other microbe would liave done
so ; ivhereas my tubes plauted rrit,h finger blood have -generally
given pure tetracoccus cultures, and fhose in wliicl1 tlils has not
germinated have remained completely sterile. Dr Sternberg him-
self is satisfied that he has found l:ly “ tetragenus ” upon the skin
of the generality of C,
                    Y    \-ellow fever patiellk esaruinecl by him in
   Vera Cruz and in Havana, and I remember that ill one of 111s’      agree-
   able “ causeries ” wit.11this distinguished lxxteriologist, he employed
   against its significance the following specious argument : if the
   tetragenus that is found upon the skin of yellow fever patients
   were the cause of yellow ferer, $0~1would expect the disease to be
   emiuentl~ contagions by mere contact for susceptible persons,
   whereas Ellis is Bnow~ not to be the case. In this argument, ho\r-
   ever, tn-o capital points are overlooked : one is tllat, according to
   my ideas, the germ of yellow fever is only- pathogenons wllen
   introduced by inoculation; and the other, tliat the presence of the
   tetracoccus on the skin of the patients should not be considered
   as the cause, but as the result of the infection, aud I plausibly
   attribute it to an elimination of the microbe by the cutaneons
   secretions as happens with the “ staphylococcus aureus,” as A.
                                           s
   Preto probed in lS92 (Baumgarten’ Jidmsbcs~idt, lS92, vol. viii.
   p. 44). The fact of finding the tetracoccus ill the milk of one 0E
   my yellow fever patients certainly confirms tliis view. On the
   other hand, it is of no importance that the same micro-organism
   be found on the skin of acclimated patients, for there is no reason
  why    it sl~ould not penetrate into t&e system and be eliminated
  with their secretions without occasioning a disease against which
  those persons are immune ; and it may be furtller postulate11 that
  the tetracocci developed in the t,issues of such immune subjvcts
  will probably have lost their virulence, or will have it more or
                                       a
  less attenuated. Dr SternLerg’ investigations have shown that
  after death from yellow fever the blood and tissues are foullcl
  invaded by other bacteria besides the specific germ of the disease.
  It is very probable. therefore, that such associations, retarding or
  impeding (as I have wit,nessed) the growth of tile tetracoccus,
  may account for the only- exceptional development of the latter
 ill the culkres frO:li heart blood or from liver and kidney juices
 obtained by Dr Sternberg, 2nd also once by Dr Gibier, and by our
 worthy member Dr TamaJ-o.
     I consider, therefore, the presence of the tetracoccus in yellow
 fever patients as sufficiently proved.
     The second condition, -that the tetracoccus shonld be isolated
 in pure cultures, offers no difticnlty.
     The third condition is the reproduction of the disease in healtlly
 susceptible sul3jects. This, for obvious reasons, could not be
 attempted iii man with so serious a disease as yellow fever ;
 nevertheless, I can almost be said to ha\-e achievecl it in sollle of my
 mosquito inoculations, having obtained the tetracoccus from tile
 llend and proboscis ot’ the insect after the ilioculation, alld tile
illocnlatec! person f:tllill, 0 ill with a mild alb~~niinuric yeltow fever.
To-day bacteriologists are conteut witlt proving t1la.t t!lle micro-
organism is pathoge~lous for animals, witikout esact*illjr tllat t,lke
disease be reproduced in them nith the same synlptoms observed
in man. With how much more reason may not I consider that
                                   16

  the above condition is fulfilled ~heu in my inoculations of pure
  tetracoccus cultures into young rabbits I have produced a fever
 of a special trpe, presenting, like sellow fever, txo parosysms,
 death att,end.rd with eclampsia, and post-mortem evidence ot
 iufnrcti, such as Crewaus found in 50 per cent. of the yellow fever
 cadavers examined IJs llilu ill ~rel~ch GLla3_xna(ddiirt?.s dti 11&d.
 f17amI~, lsi7, tome xxviii. 1). 2%).
     I
     ‘ he fourth condition 1~ been sat,isfied every time I have
 attempted it upon my inoculated rabbits; always recovering the
 tetracoccus. QIoreover, in the case of mild albuminuric yellow
 fever which resulted after a mosquito inoculation (as mentioned
in the preceding paragraph), I n-as able to recover the tetracoccus
from the head and proboscis of a mosquito which I had made sting
the patient during the attack.
    111view of the facts and arguments set forth in this paper, I
consider myself justified in regardin,(r the pathogenicity of the
tetracoccus versatilis as esperimentally proven, and in assuming
that this microbe is the specific germ of yellow fever, a concln-
sion already foreseen ever since my previous investigations in
collaboration with Dr De&ado in lSS7.
    Before fiuishing, I beg to express my sincere thanks to our
distinguished colleagues, Dr Santos Fernandez, fL!r the l,aluable
resources he has kindly placed at my disposal in the Laboratory of
the Cronica-~ledico-Quirugica, and to Dr Davalos aucl to Dr
Acosta, for their effectual assistance.

            ~Io~Qu~L’
                    o-TETRXCOCC~S
                                SEKIES (A TO L).                           i

A. Black rabbit-S&O grammes. Z_rch 14, lS95-filtered                  47
      clays’ culture of iilosyuito-tetrac.occus : 5 c. c. in peritoneum,
      and 5 c. c. subcutaneously (=&).           Died iu the night of
      Jlnrch 23, weighing 630 gr.
B. Black and white rabbit - IO00 grammes.                  Uarch 22 -
      filtered A7 days’ culture : 19 c. c. in peritoneum
      c. c. subcutaneously (= A).              J       1     31 S _ .&.
                                            Died Maich . , ‘( m:illVg;
      weighed 740 gr.
C. Black rabbit - 760 gramines.           April 36, 2 Px. - 10 c. c.
                           Ji
      ( =&), live 49 da\ s’ culture subcutan., 15 days’ incubation ;
      invasion 3lav II.       Died Xay 17, 1 P.U. ; weigllscl 590 gr.
D. Wliite ralbit--%5G       grammcs.      &ly    M, 10 AN. - s c. c.
      (=A),     live 6 days’ culture tetracoccus from ear of C
      (Xny 1). Died Xay 25, 2 P.M. ; weighed A90 gr.
E. Black ancl white rabbit-i50        gramrnes. 1fa.y 26, 10 AX-
      10 c. c. (==+$), 4S hours’ culture tetracoecus from ear of D
      (Xay 21). Died June 2, night; weighed 565 gr.
 .
$‘ Black rabbit-590     grammes. June 6, :3 P.M.---~ c. c. (=&-),
      25 hours culture tetracoccus from heart blood of E. D&cl.
      June 15, 3 P.X. ; weighed 4:3Q gr.
3
F
iz
W
G. Black rabbit-590       ,araulnles. June 6, 3 P.M.----~ c. c. (z&),
       25 hours culture tetracoecm from heart blood of E. Died
       June 14, 9 mr. ; weighed 4135gr.
H. Black and white rabbit--6.5          grammes.      June 6, 3 P.M.-
       i c. c. (=+)     of another 25 hours’ crllture from heart ljl~ocl
       of E. Dikd June lS, 1 P.M.; \yeigllecl 440 gr,
 I. Wllite rabbit - 665 ~ramnles.           August   4, 5 P.U.-q      c. c!.
       (=+g,     o!il 4 inonths’ culture of ori~innl tetracoccus. Died
       August 12, night; weighed Z-10 sr.
 J. BIack and white rabbit-TSO         ~mnmes.     August   4, 5 P.x.-
       II c. c. (=A),      old 4 months culture.       Died August S,
       9 P.M.; \vei,nhed 610 gr.
I<, Yellow rc?bl,it--TSc3 ~ramnles.       August 4, .5 P.M.- S$ c. c,
       (=A),     old 4 nionths’ culture. Died August 12, 1 p.31.          J
       wei~rhed 360 gr.




  .
31’ Black and n-bite rabbit-96.5         gramnes.     July 6, 3 P.x.-
        16 c. c. (=A),    6 days’ culture tetracoceus from finger blood
       of melano-al t~uminnric yellow fever. Died July IT, 7 i~.~~.       ;
       weighed 730 gr.
3. Black rabbit-S60                           ;
                            gratnmes. July ‘ , 3 P.~~.--9 c. c. (= A),
       42 hours culture tetracoccns fYoni finger blood of simkjle
       alburninuric y. f. Recovered after 10 or 12 days fever ;
       now wei& 9% gr.
                     T
0. Black rabl>it-‘ .50 grarumes.          July 10, IO AX--lo          c.c.
       (=A)> 36 h ours culture tetracoccus ot’ finger l~lootl of
       same simple albuminuric y. f. Died Augnst            3,  4   P.M. ;
       weight (1).
P. Black rabbit-820          ~wxnmes.     July IO, 10 m.--‘ + ;       c. c.
       (=&),       5 clays’ culture of white tetracoccus from mill< of
       wet nur~it: with non-albumiliu~ie y. f.          Died July 24,
       Ilight; weighed 575 8’      .
Q. Black mcd white rabl~it-66,s         gramnnes. Angust 19, 3 P.M.
      10 c. c. (=A),             ‘
                          4 da>3’ culture of fi qer blood tetmcocc77~
      from severe all,umiI?uric y. f. Died August 21st, 7 ~.hl. ;
      weighed 590 gr.
                                                                           I

                                   1s

                             APPENDIX.
     Since the above KG in type, Dr Felkin has received from Dr
 Finlay the following letter :-
     “ I send nine additional tracings to be added to the Chart.
 They are tracings of yellow ferer temperatures ol~tai:led frown
 my peimital observations.        They are marked with the letters
 FA. (’ fiebre amarilla, being the- Spanish for yellow feverj.      I
 also send the esplmlation of these tracings (I. to IX.).
    “ My observations have received a further confirmation frOlll the
 fact that I have obtained a pure culture of the tetracoccus from
 finger blood of a child eight months old, born in Havma, and
 ivllo was attacked v;ith a fever of eight days’ duration, with two
parox~~ns and black vomit on the fourth day. The child had
 never left this city, ancl had aim-nys lived in a hotel where it was
born and xvhere, at the time of its illness, a Spanish officer w-as
just recovering from a severe attack of yellow fever.
    “A culture of this tetracoccus injected into a rabbit has proved
pathoge~ions. I have therel’ re every reason to believe that it is
                               o



    I.   E. V.-con-albuminuric         yellow fever, iI1 t.he wet nurse
                    alluded to in t&e text, and whose milk gave the
                    white tetracoccus with which rabbit P n-as
                    inoculated.
    II. P. PL.-Soil-albulnilinric      TellomT fever in the person who
                    was inocnla.ted xith the n>osyuito from which the
                   primitive tetracoccus was obtained and used in the
                                                           h
                   rabbit inoculations (A, B, C, etc.). l’ e attack of
                   yellow fever diednot occur, however, until fifty-six
                   days after the mosquito had been applied.
  III.  C.T7.---Simple albuminwie yellow fever ill a sister of E. V.,
                   only seen on the third day of the attack.
  IV. R. C.-Severe albumil~uric yellolv fever in the friend of E. V.
                   referred to in the text, and from whose finger
                   blood the yellow tetracoccus was obtained and
                   inoculated illto rabbits Q and PL.
    V. P. H.---Simple albuminuric yellow fever-albumen           ratllel
                   abundant. From the finger blood of this patient
                   the tetracoccus was obtained associated with a
                   bacillus.
  -VT.     R.--Fatal case of melano-albuminuric yellow fever, lSS4.
VII.    PA.-Fatal       case of ruelano-albuminuric yellow fever, lS9-3.
WIT.    Y.D.--Fh a t a1 case of melano-albu~in~~~ic yellow fever, lS94.
 IX.    P. I.--Fatal    caseof albuminuric yellow fever, uramia, lS93.


                                            IIDIKECRGH.

				
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