Mercuric Chloride as a Diagnostic Agent for Trypanosomiasis in Camels

CENERAL ARTlcLES.

341

MERCURIC CHLORIDE AS A DIAGNOSTIC AGENT FOR TRYPANOSOMIASIS IN CAMELS.

By S. C. J. BENNETT, B.Sc., M.R.C.V.S., Veterinary Research Officer, Sudan Government, and P. A. C. KENNY, Laboratory Assistant, Veterinary Research Laboratory, Khartoum.

(Received/or publication July 18th, 1928.) I.-INTRODUCTORY. THE routine method of controlling camel trypanosomiasis in the Sudan comprises diagnosis by means of the formol-gel test-with confirmation where possible by microscopic examination of the blood -and treatment of all positive reactors with a single intravenous dose of 10 grammes of Naganol (Bayer 205, Veterinary). This method is based on earlier researches in this country by Knowles," and subsequent observations have confirmed its value. There is no doubt that as a primary" cure for trypanosomiasis in camels Naganol is efficient in nearly 100 per cent. of cases, at any rate where Tryp. soudanense infections are concerned; the efficacy of a system of controlling the disease thereapeutically will therefore depend on the accuracy of diagnosis, so that any new diagnostic method must necessarily be compared with the formol-gel test as at present employed. The formol-gel test largely satisfies the requirements of a diagnostic in that it detects the great majority of infected camels; according to Knowles (I.c.) only two proved infected camels out of his first series of 125 gave negative reactions, one of which was treated at once, while the second developed a "partial reaction" after about three months and, unfortunately, subsequently disappeared. It has been provisionally assumed, however, that infected camels giving a negative formol-gel reaction are probably in an " incubation" stage; they have probably been so recently infected that the property of reacting to the test has not yet been developed, and this assumption is to some extent justified in that of three camels infected by Knowles the" incubation" periods of the test were 37, 44, and 50 days respectively. Even in Knowles' original report, however, there are two cases that do not fit in with this assumption, the one mentioned above where a camel at first" negative " became " partial " and returned to " negative " without treatment, and a second one which when first examined had trypanosomes in the blood and was" positive" but later became " negative" without treatment. It therefore seems not unlikely that infected camels exist that never at any stage of the disease develop a positive reaction to the formol-gel test, and subsequent observations have shown that this is so. The details of these camels are not strictly within the scope of this paper, but it may be mentioned that during the '" KnOWles, R. H. Trypanosomiasis of camels in the Anglo-Egyptian Sudan: Diagnosis, Chemotherapy, Immunity.-Jl. Compo Path. and Therap., 1927, VoL xl, pp . 59-71, 118-143.

342

GENERAL ARTICLES.

last eighteen months one has encountered three artificially infected camels that actually died of trypanosomiasis and two others that were allowed to become almost moribund before treatment without ever developing a positive reaction to the test. A further point in connection with the formol-gel test is that proved non-infected camels may give a" temporary positive" reaction; Knowles records twelve such camels in a batch of 160. This point is of less importance than those already discussed, since in controlling the disease it is more essential that no infected camels be missed than that a few healthy ones receive treatment. The technique of the formol-gel test is still a matter for some concern. In most cases the present standard technique of adding two drops of strong formalin to one cubic centimetre of serum and reading the result after twenty-four hours is satisfactory, but one is frequently puzzled by the so-called" partial" reaction, especially as a " positive" is often recorded if four drops of formalin instead of two be added, or if tbe test be read after forty-eight hours instead of twenty-four hours, and in one case a ",positive" was recorded merely on repeating the test with the same sample of serum according to the standard technique. Another objection to the formol-gel test is the length of time required to carry it out. The inspecting officer has to take samples of blood and allow them to stand for several hours in order to obtain enough serum for the test; actually, with a fairly large batch of camels he will have to wait until the following day. Then, having set up the tests, he must wait another day before reading the results. The former objection could be removed by use of a small centrifuge but the second one cannot. An alternative to the formol-gel test may therefore be better in that: (a) It may detect infected camels in an earlier stage of the disease. (b) It may detect infected camels' that the formol-gel test will always miss. (c) It may not give a positive reaction with so many non-infected camels. (d) The results of the test may be easier to assess. (e) It may take less time. It is not claimed that the test now described fulfils all these requirements, but it shows great promise in the case of all except (c), and in this respect there is not yet sufficient evidence to warrant any conclusions. Even on this point no discrepancy has yet been observed. n.-PRELIMINARY EXPERIMENTS.

Beyond the fact that it is probably referable to the more unstable globulins, little is known of the mechanism of the formol-gel reaction, and on this account it was argued that there should be no reason why other agents should not give some kind of globulin reaction. Many chemical compounds have been tried in the Khartoum laboratory, but the first one to give uniformly good results has been mercuric chloride. The broad objective has been to obtain a dilution of a reagent

GENERAL ARTICLES.

343

that will not produce any visible change when mixed with the serum of normal camels, but will produce some noticeable change with the serum of infected camels. Starting with a saturated solution of mercuric chloride and diluting down from this, it was found that a one-thousandth saturated solution gave promising results. This definition is sufficiently vague, but was considered good enough for preliminary efforts at establishing a technique. It is not necessary to describe all the early failures, but the preliminary observations on mixtures of camel serum and the mercuric chloride solution may be summarised as follows ; (i) When a small quantity-say one or two drops--of mercuric chloride solution is added to a relatively larger quantity of camel serum-say one or two cuble centimetres-no change occurs, either in the case of healthy or infected camels. (ii) When the two are mixed in fairly equal proportions a peculiar opalescence develops in all cases. (iii) When a small quantity of serum is added to a relatlvely slightly larger quantity of mercuric chloride solution-say one or two drops to one or two cubic centimetres-a white precipitate is formed in the case of known infected camels but not in the case of normal camels. (iv) If a small quantity of serum be added to a much larger quantity of mercuric chloride solution-say one drop to five or ten cubic centimetres-a white precipitate always occurs with infected and healthy camels alike. Ultimately it was decided that for further preliminary work the standard technique should be to add one drop of serum to one cubic centimetre of mercuric chloride solution, since these seemed to be approximately the best proportions. For several months every camel sent to the Laboratory for examination was thus tested in addition to the routine blood examination and formol-gel test, and before attempting to draw any conclusions it was decided that at least fifty camels should be tested. The actual technique consisted in putting one cubic centimetre of one-thousandth saturated aqueous (distilled water) solution of mercuric chloride in a one quarter or three-eights inch bore test tube; to this one drop of pure serum was added by allowing it to drop cleanly into the solution, and the tube was gently shaken. The results of the test were recorded in terms of opacity at the end of a quarter of an hour. The fifteen minute period was taken as the most convenient, but by leaving the tubes to stand for a longer period the results are very little different; as a rule a precipitate commences to separate out almost immediately if it is going to appear at all, and by leaving the tubes longer one merely finds that the readings have to be somewhat differently assessed. A list of camels tested according to the above method is shown in Table I; the notation " + "in the mercuric chloride column merely denotes a marked precipitate w:ithout reference to degree, while the sign " ± " denotes a faint white haze and not a doubtful opacity. 1'1!

844

GENERAL ARTICLES.

TABLE I. Preliminary tests on camels with mercuric chloride together with the formol-gel test and microscopic blood examination.

Date. 4-12-25 7-12-25

" " "

10-12~25

12-12-25 23-12-25 2-1-26 7-1-26 4-2-26 7-2-26

" 24-2-26

22-3.-26 23-3-26 8-5-26

26-d~26 12-2-27 10-3-27 29-3-27 8-5-27 19-5-27 21-5-27 26-5-27 26-10-27 17-11-27 9-12-27

" "

" " " " " " " "

"

" 17-12-27 20-12-27 " "

" " " " " " " "

"

"

RPjerence No. 1991 L.l L.1474 C.C.457 C.C.437 C.C.1603 2000 2017 MA 2062 C.V.H.7 2135 2155 2156 2220 2275 2276 2283 2284 L.8. 2904 2998 3P27 L.22 3132 3139 3151 L.4 S.S.62 H.5 H.6 H.15 H.23 H.29 H.32 H.40 H.65 H.67 H.70 H.79 H.80 H.81 . S.S.I73 S.S. 87 S.S. 88 8.S.90 S.S. 99 8.S.106 S.S.lI8 8.S.119 S.S.124 S.S.134 8.8.137 S.8.146 S.S.150 W.N. 15

Micro. Exam. -

-

I

Formol-gel

I I

+

-

-

+ + +

+ +

-

--

I

-

-

+

+2 + +

-

-

-

--

-

-

-

-

+ 1

-

-

-

-

Mercuric Chloride.

-

-

-

I

-

-

-

I

+

-

-

-

-

-

-

+

-

-

-

-

-

-

-

-

-

-

-

-

+ +

Partial

-

I

-

+ -

+ -

-++ + + + +

+ + +

+ -

-

+ +

-

-

-

-

-

! !

-

-

-

-

-

-

+

-

-

+ +3

-

I

-

I

-

+ +

I

-

±

-

+

-

+

-

I

: I

1

I

1. Camel treated and lost sight of. 2. Trypansomes appeared later in these two camels. 3. Camel treated and became negative to both tests.

+2 + +

-

-

+

+ +

+

+ +

I

--,---

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GENERAL ARTICLES.

A summary of the observations recorded in Table I shows :Positive to all three motheds ... Positive micro. exam. and mercuric chloride only ... Positive formol-gel and mercuric chloride only Positive mercuric chloride only Negative mercuric chloride and positive to any other test Negative to all tests

3 camels 14 " 3 2

nil. 34

"

"

"

Total 56 camels Thus the mercuric chloride test detected infected camels when the other tests failed:Where microscopic examination of the blood failed 5 times Where the formol-gel test failed 16 " Where both failed 2 " It must, however, be mentioned in support of the formol-gel test that fourteen out of the sixteen camels in which it failed were recently infected experimental camels and not natural field cases suspected of trypanosomiasis on clinical grounds. At the same time this observation indicates the probable superiority of the mercuric chloride test in its being able to detect infection at an earlier stage. Its general reliability is in any case beyond doubt, since only one of the twenty-two positive reactors was not proved infected. (Camel No. 1991 ,of December 4th, 1925, was treated with Naganol and lost sight of. It was, however, also positive to the formol-gel test). It may further be objected that there is no proof that some of the" negative" camels were not infected, since no animal inoculations were carried out. This is admitted, but the objection calls for no discussion since the point at issue in these preliminary experiments is merely the relative diagnostic efficacy of the mercuric chloride and formol-gel tests. IlL-DEVELOPMENT OF TECHNQIUE.

The preliminary observations having shown the usefulness of the test, it became necessary to develop a more accurate technique. In the first place, the description of a solution as "one-thousandth saturated" is too vague, but it was estimated that at the average shade temperature of Khartoum it would be about 1-15,000. For the next series of experiments, therefore, a range of accurately prepared solutions of 1-5,000, 1-10,000, 1-15,000, 1-20,000 and 1-25,000 were made in distilled water, and a preliminary test of these, using one drop of serum to one cubic centimetre of solution, was carried out on a known infected and a known clean camel. The result in terms of opacity was as follows : 20-12-27.

Camel Clean W.N.15 Infected 8.8.146

1-5,000

+++ +++

1-10,000

1-15,000

1-20,000

1-25,000

+++

++

+

±

±

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GENERAL ARTICLES.

Again, although earlier rough tests had shown that one drop of serum to one cubic centimetre of solution was a good working proportion it was not certain that it was the best. With one infected and one clean camel, therefore, a second pair of titrations was carried out, the first using varying quantities of serum to the same volume of solution and the second using one drop of serum to varying quantities of solution. In the case of the former test, the readings of which are shown in Table II, fractions of a drop of serum were actually added in the form of one drop of diluted serum. The necessity for this will be explained later. TABLE II. Results obtained by addition of varying quantities of serum to one cubic centimetre of mercuric chloride solution in range of dilutions. 20-12-27 .

I

erum I Camel. drops) I

-I t

1 t

!

- - -,.

1-,5000 i 1-10,000

!

+ + + , + + ! + + ! + i ++ ++ ++ I! ++ + I + + + +++ I ±

Infected Clean

! Infected

--

Clean

Infected Clean

1

Infectl:d Clean

2

Infected Clean

3

Infected Clean

I

I

Mercuric Cyloride Icc

I

1-20,000 , 1-25,000

1-15,000

+ + ++ + ++ -

++ -

i

++ + ++

I!

+ +

+

I

± ±

±

I

-

+

1

-

:-1

--

i

-

±

I --

:

i 1-:>0,0

:

+ +

!

i I

-

,

-

i i I

i

-

Unreadable owin g to opalescence of excess of serum.

_._---

--

In this and subsequent records it is to be noted that the number of pIllS signs is intended to indicate the proportionate opacity, while the sign " ± "indicates a faint haze and not a doubtful reaction, the latter being denoted" ?"

Several titrations with other camels were later carried out on the same lines but all gave similar results, namely, that the only proportions of serum to mercuric chloride solution giving clear-cut results are one drop to one cubic centimetre or " half a drop" to one cubic centimetre. The complementary titrations, using one drop of serum to varying quantities of solution, gave exactly comparable results ; it is hardly necessary to record them in: taublar form, but it can be stated that one drop of serum to two cubic centimetres of solution behaved exactly as " half a drop" to one cubic centimetre, one drop to four cubic centimetres exactly as " quarter of a drop " to one cubic centimetre, and so on in proportion. From these titrations one assumed that the most useful dilutions of mercuric chloride would be from 1-15,000 upwards, and the method of mixing to add (a) half a drop of serum to one cubic centimetre, (b) one drop to one cubic centimetre, or (c) one drop to two cubic

347

GENERAL ARTICLES.

centimetres. Of these the second is the easiest and has since always been used as a routine. At this point one has to draw attention to a very interesting essential in the technique, namely, that whether pure or diluted serum is added to the solution it must be in the form of a drop. For example, in Table II it is recorded that with two or three drops the test cannot be read owing to " opalescence"; this is not strictly accurate, as the true explanation is that ~he first drop produces the usual one-drop precipitate and the addition of further drops alters this in some way but does not clear it up. That it is not merely the proportions of serum and solution can be proved by adding one drop of serum to half or a quarter of a cubic centimetre of solution, whereupon no change occurs. IV.-RANGE OF REACTION IN CLEAN AND INFECTED CAMELS.

The experiments next to be described were designed rather to find the lowest dilution of mercuric chloride in which the serum of normal camels would fail to give a precipitate than to find the highest dilution in which infected sera would react. Several infected camels and an almost unlimited number of clean camels were available, and a series of experiments were carried out at various dates with various techniques. As was already noticed in the preliminary tests, the one drop to one cubic centimetre technique gave the most clean-cut results, and the following readings are those of this technique only. Experiment of 27 -12-27.

Infected canaels :-S.S.88. Artificially infected but trypanosonaes had not yet appeared in the blood streana, fornaol-gel reaction negative. S.S. 124. Trypanosonaes present, fornaol-gel negative. S.S. 146. Trypanosonaes present, fornaol-gel negative. S.S. 173 .. Trypanosonaes had been seen but were absent on day of test, fornaol-gel positive. Clean canaels Nos. 160, 169, 174, 190 and 197. -

----

--.

- - - - - - - - - - - - - - - ' , - - _.. _--'-

I

Camel I No.

,-----,-----'.----_.'.

__. - - -

..

-----------------

.-..

-~~.

Mercuric Chloride I c.c.

i 88 -+'+-1

8.8. 8.8.124 S.s.l46 8.8.173 160 169 174 190 197

-_.

1-5000

1-10,000

++ ++ /+\ I +++ +++1 +++ ? + ? + + ± + ? +

\

I I I

1-15,000

++ ++ +++ +++

1-20,000

\ I

-

-

-

-

I

-

i

-

+ + ++ +

I

-

-

1-25,000

I I I

+ + + ±

-

-

-

I 1-30,000 I 1-50,000 I I

± + + ±

-

-

-

-

-+-

±

-

-

I

348

GENERAL ARTICLES.

Experiment of 5-1-28_

Six known clean camels, with S.S. 173 as infected control. Came! No. 153 157 163 180 196 206 8.8.173

"Mercuric Chloride 1 c.c. 1-10,000

± ± ± ± ? ± +++

I

1-15,000

1-2 0,000

± ? ?

I

-

i i

1-25,000

1-50,000

+

±

[

I

? ?

+++

I

[-

++

I I

-------------.-

Experiment of 11-1-28.

Another batch of six known clean camels, with two infected controls, Nos 8.8. 88 and 8.8 173. Camel No. 167 191 195 204 205 207 8.8. 88 S.S.I73

Mercuric Chloride 1 c.c. 1-10,000

± ± ± ± ± +++ +++

I

1-15,060

II

-

? ? ?

I I

1-20,000 -

I II

I

\

++ ++

I

-

1-25,000

-

I

--

I

± ++ +++

I

-

-

I

1-50,000

i

-

-

-

-

II

+ ++

I

I

± +

--

Experiment of 18-1-28.

A fourth batch of six clean camels, with No. S.8. 173 as infected control. Camel No. 154 179 192 198 208 209 S.8.173

"Mercuric Chloride. 1-10,000

1-15,000

± ± ? ± ± ± +++

?

1-20,000 -

-

-

?

-

-

+++

-

-

++

1-25,000

-

-

--- ------±. ----

A review of these four experiments shows that, of twenty-three clean camels tested, in no case was a precipitate produced by mercuric chloride in a higher dilution than 1-15,000 and in twelve of these the 1-10,000 dilution was the highest; whereas with the serum of infected camels precipitates were produced in dilutions up to 1-50,000, and this is not necessarily the end titre, as will be shown in the next section. At this point it appears safe to regard a camel as infected if its titre is 1-20,000 or over. The test, moreover, appears to be at least as reliable as the formol-gel test and has the additional virtue that a result is obtained in fifteen minutes instead of twenty-four hours.

349

GENERAL ARTICLES.

V.-INCUBATION PERIOD AND ULTIMATE TITRE IN INFECTED CAMELS.

It has been shown that camels may develop a positive reaction to the mercuric chloride test before they do so to the formol-gel test, but no details have been mentioned of the actual length of time required. In this connection one has to consider at least two aspects. (a) In the case of a camel that ultimately becomes positive to the formol-gel test, how much sooner, if at all, does it develop ability to react to the mercuric chloride test? (b) In the case of a camel that never becomes positive to the formolgel test, will a reaction to the mercuric chloride test develop? Each of these aspects must be considered relatively to at least three main types of infection, namely, the acute progressive infection, the infection that is at first acute and later becomes chronic, and the infection that is chronic ab initio, where trypanosomes are probably never to be seen in the blood stream. Information on all these points is at present scanty, but records have been made of at least one camel presenting each of the abovementioned aspects. The question of ultimate titre is subsidiary but is most easily considered here. The following are the records of approximately weekly examinations of the individual camels. Case No. J. Acute progressive infection. Camel No. 60, used as a control in an infection experiment. 1 c.c. camel blood containing Tryp. soudanense on 10-4-28.

- - - - - , - - - - - - - - - , - - - - - - - - ---

Micro. Exam.

Date. 10-4-28 16-4-28 19-4-28 22-4-28

Formal Gel.

Injected with

-----------

-

Mercuric Chloride.

1-15,000

1-2~00

1-20,000

11-5:00

ttl

+ +

Died with trypanosomes swarming.

It is not certain that trypanosomiasis was the sole cause of death, but a post-mortem examination revealed no other possible cause. With regard to the extreme rapidity of death, it has been noticed in several experimental camels during the last year that hyperacute symptoms often develop within a fortnight. Case No.2. Acute reverting to chronic infection. Camel No. 8.8.173, kept in the laboratory as a reservoir of Tryp. soudanense. Infected on 10-12-27 .

.

Date.

----

10-12-27 17-12-27 : 20-12-27 27-12-27 4-1-28 11-1-28 I 18-1'-28 25-1-28 i

Micro. Exam.

-

+ + +

-

-

-

- - .--.~--~

I

Formol Gel. ! 1-15,000

-

+ + + + +

-

+ ++ +++ +++ +++ +++ +++

...

-.---~-

Mercuric Chloride.

I 1-20,000 i

-

+ I ++ ++ ! ++ ++ I +++

1-25,000

I 1-50,000

-

I

±

I

-

+ + + + +

-

± ± + +

±

I

I

..

-

1-100,000

-

-

± ± ±

350

GENERAL ARTICLES.

It is not necessary to give full details of the subsequent observations on No. 8 .S. 173. From · 25-1-28 the reaction to the formol-gel test remained positive and the mercuric chloride titre was over 1-100,000 (actually a precipitate occurred in plain distilled water). The camel continued to lose condition and trypanosomes were rarely present in the blood. On 11-4-28, when apparently at the point of death , a dose of Naganol was given and the camel recovered.

Case. No.3. Chronic infection throughout. soudanense, on 16-12-28.

Camel No. S.S. 88, infected with Tryp.

_._-

Formol Gel .

Micro. E xam .

Date.

Mercuric Chloride.

I 1-15 ,000

1-20,000

1-25,000

1-50,000

(j-12-27 13- 12-27 20-12-27 ± ± 27-12-27 i + + ++ 4-1-28 + + ++ ± 11-1-28 ++ . ++ + + 11<- 1-28" ++ + + ++ 25-1-28 -!- -+~ ++ + + j. ] - 2-28 + ++ ++ + + + _L 8- 2- 28 ++-l.L ++ + 15- 2. 2S ++ + + ++ ± 22- 228. + + ++ 2!l-2.28 ±. -r + :1-: 7.3-28 ± + :::l~ I 14-3-28 + ± 21·3-28 I ± ± 28.::\.28 + ++ :i: 4-4-28 + ++ ± I 11-4-28 ::±+ ± 18-4- 28 ± + 25-4-27 + ± ± 2-5-28 + ± ------ ---_._ . - - -•..- -•..---.--.- .- - - --• Two white rats inoculated and became infected with Tryp . soudanense.

__ L

-

On May 2nd, 1928, Camel No. SS.88 had reached a clinical condition making treatment necessary; he was given a dose of Naganol and recovered. Case No.4. A subacute case. Camel No. 218, a laboratory reservoir of Tryp. soudanense, infected on 10-4-28. I

lHicro. Exam.

Date. ]0-4·28 18-4.- 28 25.4-28 2-5-28 10. 5-28 16-5-28 2:1 -5-28 26 ..5-28 8- 6-28 14-6-28 21-6-28

:

I

i

II

-

-

+

-

-

-

+ +

I

I

Formol gel .

-

partial partial partial partial

+ + + +

Mercuric Chloride.

I

1-15,000 -

± ± ++ ++ ++ ++ ++ + +++ +++ ++ +

I I

1-20,000

-

± ± ++ ++ ++ ++ +++ +++ +++ ++

I

-----

:

1-25,000

-

1-50,000

I

-

+ + + + ++ ++ ± +

[

Camel still under obsenation.

-

+ + + + + + ± ±

GENERAL ARTICLES.

351

A study of the records of these four cases shows the value of the mercuric chloride test. To summarise each case separately : Case No.1. Very acute progressive trypanosomiasis. The case could certainly have been diagnosed microscopically, but a reaction to the mercuric chloride test (i.e. opacity in a dilution of at least 1-20,000) developed one week after infection, whereas a reaction to theformol-gel test never developed. Case No.2. Acute infection reverting to chronic. The camel became positive to the mercuric chloride test one week after infection, and before tyrpanosomes appeared in the blo()d stream. A positive reaction to the formolgel test did not develop until a week later (a relatively early development of this reaction). Case No.3. A chronic case throughout. A reaction to the mercuric chloride test developed two weeks after infection, and although the camel was allowed to become almost moribund trypanosomes were never seen in the blood and no reaction to the formol-gel test developed in a period of approximately five months. A most.interesting feature of this case is the gradual tendency of the reaction with mercuric chloride to disappear; it is not, however, desirable to discuss this point until more records are available for study. In any case, this is a type of infection that is probably not rare, and with earlier methods of diagnosis would almost certainly be mis3ed, since animal inoculation is not a practicable routine procedure in field work. Case No.4. A subacute case, of which one of the puzzling features with earlier methods would have been the " partial reaction" to the formol-gel test accompanied by the infrequent appearance of trypanosomes in the blood stream. A positive reaction to the mercuric chloride test developed in about a week, whereas trypanosomes were seen for the first time after four weeks and the formol-gel reaction was not fully positive until the seventh week.

The four camels ·mentioned above are the only ones that have been observed untreated over a considerable period, but in confirmation of the earlier development of a reaction to the mercuric chloride test it is permissible to mention two other camels which were infected in the course of another experiment; these two developed a positive reaction by the end of the second week. Thus, of six untreated camels one developed a positive reaction in one week and the remaining five within two weeks. Regarding the point of secondary importance, namely, the ultimate titre, there is lttle information of interest. In the first place, the ultimate titre does not matter very much; the important issue is to define the lowest positive titre, and it seems that one is safe in accepting this as 1-20,000, since of twenty-four clean camels hitherto recorded in no case has the titre been above 1-15,000 while all of the nine infected camels examined have gone above 1-20,000. The ultimate titre is of interest, however, when pursued to infinity; it is found that after a dilution of about 1-100,000 mercuric chloride the presence of the salt is not necessary and a faint haze will appear if the serum of an infected camel be dropped into pure distilled water. This phenomenon has not been sufficiently studied to promote any discussion, but it tends to support the a priori conception that the whole phenomenon is referable to the globulin fractions of the serum. The mechanism of

352

GENERAL ARTICLES.

the reaction is still under consideration and does not call for further discussion at this stage. VI.-DISCUSSION.

The observation that the serum of camels infected with Trypanosoma soudanense yields a precipitate with solutions of mercuric chloride of 1-20,000 dilution and upwards within a few minutes of mixture will probably be of considerable importance in the control of camel trypanosomiasis in the field. The method at present employed in the Sudan of using the formol-gel test for diagnosis has been shown to require at least two days for completion, and in practice this period usually extends to three days because at least one cubic centimetre of clear serum must separate out from the clotted sample of blood, and this quantity is not always expressed within a few hours; after setting up the test a further twenty-four hours must elapse before reading the result. In the case of the mercuric chloride test only one drop of serum is required and this will be obtainable after a very short time-at any rate necessarily in a much shorter time than one cubic centimetre-while after setting up the test the result can be read off within a quarter of an hour. In point of accuracy it must be admitted that no very large number of camels have been tested; in this paper records are, however, given of 87 camels, in 86 of which the results have been coincident with presence or absence of trypanosome infection, while in the remaining case there has been no proof that a positive reacting camel was infected except that it was also positive to the formol-gel test at the time of testing. A further important point is the rapidity with which an infected animal can be detected after infection; from the cases observed it seems that the majority can be detected within about a fortnight, but on this point a greater number of observations is required. In any case, with the actual camels recorded, few as they are, the mercuric chloride test was always able to detect infection at an earlier stage than the formol-gel test, and in two cases it detected infection where a reaction to the latter test did not develop. The result of the test is, moreover, more easy to read off; in the case of the formol-gel test one of the difficulties has been to assess the " partial reaction," whereas in the mercuric chloride test the resultant solution is either water clear or it is not. In cases of very early infection the precipitate is often very light, but this objection is of little importance. There must necessarily be a short interval during which a camel is developing a positive reaction to any form of test, and in the case of the mercuric chlori!1e test it is agreeably short. SUMMARY.

It is not intended to formulate any general conclusions at this stage, since the results of further work may modify one's present con-

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353

ceptions. Within the limits of the experiments already carried out, however, it is permissible to state that : (i) Dilute solutions of mercuric chloride are reliable for detecting Trypanosoma soudanensf! infection in camels when employed according to the technique described, namely, one drop of serum is added to one cubic centimetre of 1-20,000 or greater dilution. A positive reaction is denoted by an opacity and a negative reaction by no change at the end of a quarter of an hour. This form of test appears to be more reliable than the formol-gel test. (ii) The test detects infected camels in a shorter time after original infection than the formol-gel test, and may do so before trypanosomes first appear in the blood stream. (iii) The test is more easily and rapidly carried out than the formol-gel test. (iv) The results are more easy to assess than those of the formolgel test.

I.-TRANSMISSION OF FOOT-AND-MOUTH DISEASE IN RODENTS BY CONTACT. II. -THE SPREAD OF FOOT-AND-MOUTH DISEASE BY RATS, RABBITS, AND BIRDS.

By J. M. BEATTIE, ZAKI MaRCOS, and D. PEDEN. From the Bacteriological Department, University of Liverpool.

1. IT has been shewn by Burbury* that spontaneous infection may occur in rabbits which have been left in close contact with infected animals. Two out of eight rabbits kept in contact with eight others which had been inoculated into the tongue developed vesicles on the tongue and mucous membrane of the mouth. These two animals had wounded surfaces-one having been bitten on the neck and lips by an infected rabbit, and the other having recently lost several clawsand it is suggested that the infection occurred through their wounds. In the course of our work we have noted a considerable number of" contact infections" and have carried out a series of experiments to ascertain if this method of infection was common. Our method was to place the animals in a cage with an infected animal, or with the feet of killed infected animals, or to place them in cages which had previously been occupied by infected animals and not cleaned. Series I.-In our earlier experiments we found that guinea-pigs in contact with infected animals from twelve hours to twenty days shewed definite ulceration of the mucous membrane of the mouth, and in a few cases the feet and the alae of the nose were also involved. Guinea-pig 10 was a contact for twelve hours. It was killed thirteen days

*

Burbury, Jour. Compar. Path. and Therap., 1927, Vol. xl., p. 34.